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Volume 34, Number s4 April 2016
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Recent Developments in LC Column Technology: Impact on a World of Disciplines . . . . . . . . . . . . . . . 8David S. BellA brief introduction of the articles presented in this supplement.
The Impact of Superficially Porous Particles and New Stationary-Phase Chemistries on the LC–MS Determination of Mycotoxins in Food and Feed . . . . . . . . . . . . . . . . . . . 10Andreas BreidbachThis fit-for-purpose LC–MS-based method provides fast analysis of four mycotoxins using
standard HPLC equipment with a pentafluorophenyl SPP column.
The Synthetic Cannabinoid Chemical Arms Race and Its Effect on Pain Medication Monitoring . . . . . 15Sheng Feng, Brandi Bridgewater, Gregory L. McIntire, and Jeffrey R. EndersAn investigation of C18 and phenyl-hexyl column chemistries for definitive identification
of 13 synthetic cannabinoid metabolites in patient samples.
HPLC Column Technology in a Bioanalytical Contract Research Organization . . . . . . . . . . . . . . . . . . 24Ryan Collins and Shane NeedhamWhen presented with a new analyte, a bioanalytical CRO must quickly develop a robust method with good chromatographic
resolution, repeatable results, and a quick run time. Recent developments in LC column technology make that possible.
Characterizing SEC Columns for the Investigation of Higher-Order Monoclonal Antibody Aggregates . . . 28Ronald E. Majors and Linda L. LloydWhen selecting the optimum phase for SEC separations, several key column parameters must be considered carefully.
Positive Impacts of HPLC Innovations on Clinical Diagnostic Analysis . . . . . . . . . . . . . . . . . . . . . . . 37Michael J.P. Wright and Sophie HepburnAs clinical diagnostic assays move to LC–MS-MS, the emphasis has turned to emerging stationary phases that
use alternative mechanisms of retention to separate the analyte–interference critical pairs.
Latest Advances in Environmental Chiral Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42Denise WallworthRecent advances in chiral stationary phases have enabled higher efficiency and faster separations in studies of the differing
enantiomeric activity of pesticides, their environmental transformation, and the degradation of pollutants in general.
Cover Imagemore Co, Ltd./Stocktrek Images/Andrew Brookes/Liz Pedersen/EyeEm/GIPhotoStock/Andy Sacks/Arne Pastoor/Getty Images
Articles
Apr i l 2016
Volume 34 Number s4
6 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY APRIL 2016
Recent Developments in
LC Column
Technology
Recent Developments in
LC Column
Technology
www.chromatographyonline.com
SPP speed. USLC® resolution.A new species of column.
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8 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY APRIL 2016 www.chromatographyonline.com
FROM the GUEST EDITOR
Recent Developments in LC Column Technology: Impact on a World of Disciplines
There have been many advances in liquid chromatography (LC) during the past
decade. Much attention has been paid to the development of new and improved
particle designs to achieve higher efficiency and there have been many new
developments in the surface treatments of these particles that impact retention and
selectivity. Novel particle designs such as sub-2-μm and superficially porous media
have vastly improved the speed and efficiency of separation tasks. Newly developed
chemical modifications and their implementation using these modern particle archi-
tectures have greatly expanded their utility. The underlying theme for this special
supplement edition was to bring together articles that discuss how these innovations
have impacted analysis across a wide variety of disciplines.
Andreas Breidbach from the European Commission, Joint Research Center at the
Institute for Reference Materials and Measurements provides insight on how mod-
ern technologies have impacted the liquid chromatography–mass spectrometry (LC–
MS) analysis of mycotoxins in food and feed. The work demonstrates the increased
efficiency garnered from the use of superficially porous particles as well as added
selectivity through modern surface chemistry modifications. Sheng Feng and col-
leagues from Ameritox provide examples of similar achievements for the analysis of an
ever-growing number of synthetic cannabinoids for toxicology and forensic analyses.
Again, superficially porous particles combined with alternative surface chemistries
has enabled rapid, selective, and sensitive LC–MS-MS identification of 13 synthetic
cannabinoids in patient urine samples. Collins and Needham from Alturas Analytics
discuss the impact of recent column technology advancements and emerging devel-
opments in microflow LC technologies with respect to improving productivity in
the bioanalytical contract research realm. The authors note that these technologies
facilitate the development of robust and reliable methods, which may lead to lowering
the cost of complex biotherapeutics. Continuing with the theme of bioanalysis, Lloyd
and Majors discuss the importance of particle architecture and surface treatments
with respect to current needs in size-exclusion chromatography (SEC). The growing
attention of the pharmaceutical market on biotherapeutics has necessitated the imple-
mentation of many modes of chromatography to fully characterize these complex
systems. The authors point out the importance of particle pore size (and distribu-
tion), pore volume, and surface chemistry treatments as it pertains to modern SEC
requirements. From the world of clinical diagnostics and testing, Wright and Hep-
burn provide examples of how modern particle technologies, surface modifications,
and multiple-channel high performance liquid chromatography (HPLC) instruments
have enabled faster analyses for various disease states and patient types. This is a
crucial step toward providing high-quality health care. Lastly, Wallworth highlights
some of the recent advances in chiral stationary phases (CSP) and how they impact
important environmental concerns. Chirality plays a significant role in the study of
pollutants, agrochemical usage, and pharmaceutical waste on our environment. The
author anticipates that recent applications of CSPs on modern particle designs will
positively impact research in this arena.
In applications ranging from food to pharma and biotherapeutics to biomes,
advances in liquid chromatography are playing a critical role. Modern particle designs
and surface chemistry treatments are continually being adopted in a variety of dis-
ciplines. As exemplified by the articles within this supplement, developments in our
craft are improving the quality of life around the world. Enjoy!
David S. Bell
LCGC “Column Watch” editor
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10 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY APRIL 2016 www.chromatographyonline.com
Andreas Breidbach
The Impact of Superficially Porous Particles and New Stationary-Phase Chemistries on the LC–MS Determination of Mycotoxins in Food and Feed
Superficially porous particles with their favorable chromatographic
properties were a great advance for liquid chromatography (LC).
Analytical LC columns packed with those particles allow for much
faster separations even with standard LC equipment rated at a
maximum pressure of 400 bar. This speed is exemplified by a LC–mass
spectrometry (MS) method of analysis for four mycotoxins, spanning log
P values from -0.7 to 3.6, with an analysis time of just over 8 min and
excellent performance. Another issue is the separation of closely related
mycotoxins, like 3- and 15-acetyldeoxynivalenol. With the common C18
chemistries, they are coeluted and identification and quantification can
only be achieved through differing MS-MS signals. Now, with the newer
pentafluorophenyl chemistries these two mycotoxins can be separated
by LC and MS quantification of them has become much more precise.
In 2006, high performance liquid
chromatography (HPLC) columns
packed with superficially porous
particles (SPP) (also known as porous-
shell, core–shell, and solid-core parti-
cles) were introduced to the market. In
performance rivaling sub-2-μm technol-
ogy, SPP packed columns have enabled
highly efficient separations to be car-
ried out with standard HPLC systems
because of the much lower back pres-
sure they generate (1). This favorable
characteristic has also been exploited
for the determination of mycotoxins in
food and feed.
Mycotoxins are secondary metabo-
lites of certain fungi whose occurrence
in food and feed is difficult to avoid.
Therefore, many countries have regu-
lated this occurrence of mycotoxins
(2,3). A wealth of methods of analysis
to enforce these regulations exist (4)
and among them liquid chromatog-
raphy–mass spectrometry (LC–MS)-
based detection is gaining momentum.
LC–MS is primarily gaining momen-
tum for two reasons: sample preparation
requirements can be relaxed because of
the high specificity and sensitivity of
MS detection, and multiple mycotoxins
can be determined in one go. Both of
these reasons are of particular interest
to official control laboratories since they
will lead to higher throughput compared
to traditional one analyte per prepara-
tion and run approaches with extensive
cleanup. This higher throughput has
been shown for traditional HPLC equip-
ment with an analytical column packed
with fully porous particles by Biselli
and colleagues (5). Using a 150 mm ×
2.1 mm column with 3-μm particles at
1-mL/min f low, 18 mycotoxins could
be detected during a 15-min analytical
run. With those settings, deoxyniva-
lenol (DON) eluted at 3.80 min and
zearalenone (ZON) at 7.38 min. To stay
within the operational envelope of their
electrospray ionization (ESI) source the
column eff luent was split 1:5. Using a
sub-2-μm fully porous particle packed
column of 100 mm × 2.1 mm dimen-
sions, Varga and colleagues (6) were able
to show a multimycotoxin separation
APRIL 2016 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY 11www.chromatographyonline.com
in which DON eluted at 1.45 min and
ZON at 6.44 min with a total run time
of 11.5 min. To perform this separation,
an ultrahigh-pressure liquid chromatog-
raphy (UHPLC) system capable of deliv-
ering flows at pressures as high as 1200
bar was used.
With the desire to determine mul-
tiple mycotoxins in one run, the
necessity arose to be able to separate
closely related mycotoxins. One such
example would be DON and its two
acetylated relatives, 3- and 15-acetyl-
deoxynivalenol (AcDON). Although
DON can be separated from the two
AcDONs on a C18 column, the two
AcDONs are coeluted. Because of dif-
ferent fragmentation behavior it is still
possible to obtain individual quanti-
tative data using MS-MS detection,
but with lesser confidence than with a
full chromatographic separation (5). A
more recent stationary phase chemis-
try capable of separating such isomers
is the so-called pentaf luorophenyl
(PFP, F5) modified silica. The pentaf-
luorphenyl system is electron deficient
and can interact with the analyte in
multiple ways: π-π, dipole-dipole, and
charge-transfer interactions. Because
of these multiple interactions, struc-
tural isomers can often be separated.
This article presents a fit-for-purpose
LC–MS-based method of analysis for
the four mycotoxins DON, HT-2 toxin,
T-2 toxin, and ZON utilizing standard
HPLC equipment with an SPP column.
Performance characteristics in unpro-
cessed cereals, as determined in-house
and verified through a collaborative
trial, were in line with traditional single
analyte methods with a short analysis
time of under 9 min. The article also
shows how the F5 stationary phase
chemistry enables the separation of
the closely related mycotoxins 3- and
15-acetyldeoxynivalenol.
Experimental
Chemicals and Materials
All chemicals were purchased from either
Sigma-Aldrich or VWR and were of at
least analytical grade. For the mobile
phase LC–MS Chromasolv-grade (Fluka,
Sigma-Aldrich) water and methanol
were used. Deionized water was gener-
ated by a MilliQ system (Millipore). All
tested materials came from the material
pool of the European Union Reference
Laboratory (EURL) for mycotoxins at
the Institute for Reference Materials
and Measurements (IRMM) of the Joint
Research Centres (JRC) of the European
Commission (EC).
The mycotoxins DON, HT-2, T-2,
ZON, 3-AcDON, and 15-AcDON,
and the isotopologues 13C15-DON, 13C22-HT2, 13C24-T2, and 13C18-ZON
were purchased from Biopure (Romer
Labs) as either solids or ready-to-use
solutions. From these, a stock solution
of 3.2-μg/mL DON, 0.5-μg/mL HT-2
toxin, 0.3-μg/mL T-2 toxin, and 0.3-μg/
mL ZON in neat acetonitrile was pre-
pared and stored. This stock solution
was freshly diluted for every calibration
task. An internal standard solution with
the same concentrations of the respec-
tive 13C-isotopologues in neat acetoni-
trile was also prepared and used undi-
luted. These solutions were stable for at
least three months in the dark at 2–8 °C.
Equipment
Measurements were performed on
an LC–MS system consisting of two
LC‐20AD pumps (Shimadzu, high-
pressure binary gradient), an Accela
autosampler (Thermo Scientific), and
a TSQ Quantum Ultra triple-quadru-
pole mass spectrometer with an Ion-
Max HESI2 interface (both Thermo
Scientif ic). For analytical columns
either an Ascentis Express C18 (75
mm × 2.1 mm, 2.7-μm particle size,
Supelco, Sigma-Aldrich), a Kinetex
C18, or a Kinetex PFP (both 100 mm
× 2.1 mm, 2.6-μm particle size, Phe-
nomenex) were used. The gradient con-
ditions with the Ascentis Express C18
column were as follows: 0 min, 8% B;
2 min, 57% B; 6 min, 61% B; 6.1 min,
95% B; 7.6 min, 95% B; 7.7 min, 8%
B; 8.7 min, 8% B with mobile-phase A
consisting of 999:1 (v/v) water–formic
acid and mobile-phase B consisting of
999:1 (v/v) methanol–formic acid at
a f low rate of 0.3 mL/min. The col-
umn was maintained at 40 °C during
analysis. This nonintuitive gradient
was designed with optimal resolution
and shortest analysis time for just the
four mycotoxins in mind. For the two
Kinetex columns more-generic gradi-
ent conditions were used: 0 min, 8%
B; 8 min, 95% B; 8.1 min, 8% B; 10
min, 8% B at a column temperature
of 50 °C. The mobile phases and f low
Table I: MS source and analyzer settings. (The segment run times relate to
the Ascentis Express C18 column; for the Kinetex columns they were adjust-
ed to the respective retention times of the analytes.)
Item Segment 1 Segment 2 Segment 4
Run time (min) 0–2.6 2.6–4.9 4.9–8.7
Analyte DON + AcDON +
13C15-DON
HT2 + 13C22-HT2,
T2 + 13C24-T2
ZON + 13C18-ZON
Adduct Protonated Sodium Deprotonated
Transitions (collision energy [eV])
297A231 (16),297A249 (13),339A213 (20),339A261 (20),312A263 (9),312A276 (9)
447A285 (22),447A345 (20),469A300 (19),469A362 (18),489A245 (30),489A327 (25),513A260 (26),513A344 (23)
317A131 (25),317A175 (22),335A185 (26),335A290 (21)
Tube lens (V) 80 110 80
Polarity Pos Pos Neg
Spray voltage (V) 2800 2800 2000
Vaporizer temperature (°C) 350
Sheath gas pressure (arbitrary units)
30
Auxiliary gas pressure (arbitrary units)
10
Transfer capillary temperature (°C)
320
12 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY APRIL 2016 www.chromatographyonline.com
rate were as stated above. The MS sys-
tem settings can be found in Table I.
The data acquisition was segmented to
limit the number of acquired transi-
tions and enable longer dwell times
per segment.
Sample Preparation
In an appropriately sized tube, 2 g of
unprocessed cereal (comminuted to
<500 μm particle size) was fully sus-
pended in 8 mL of water. Then 16 mL
of ethyl acetate was added and after a
brief, hard shake the mixture was soni-
cated for 30 min. After sonication 8 g of
sodium sulphate was added. The mix-
ture was again shaken hard and then left
for 10 to 20 min to allow the sodium
sulfate to crystallize. To settle particu-
late matter and aid phase separation the
tube was centrifuged at a relative cen-
trifugal force of 3000g for at least 1 min.
Next, 500 μL of clear supernatant was
transferred to a silylated autosampler
vial (2 mL, Supelco, Sigma-Aldrich), 25
μL of internal standard mix was added,
and the contents of the vial were evapo-
rated to dryness with a stream of dry
nitrogen (boil-off ) at 60 °C. The dry
residue was reconstituted with 250 μL of
mobile-phase B and 250 μL of mobile-
phase A, in that order. Initial recon-
stitution with the pure organic mobile
phase significantly improved the dis-
solution of the more hydrophobic ana-
lytes. Finally, 5 μL of this solution was
injected without further treatment. Tur-
bidity of the injection solutions, often
seen in these reconstituted extracts, did
not negatively affect column lifetime in
our experience.
Method Validation
To validate the method, the cereals
maize, wheat, oat, and rice but also
soy and a cereal-based compound feed
were investigated. Among the char-
acteristics determined were matrix
effects, method recovery, repeatabil-
ity, and intermediate precision. For
matrix effect and method recovery
determination, different amounts of
the analytes were spiked into materi-
als free of the analytes before extrac-
tion (set A) and after extraction of the
analyte-free materials (set B). After
regression, analysis of the slopes of the
signals of the sets A and B were then
compared with the slopes of a cali-
bration done in neat solvent (set C).
Comparing slopes A and C indicated
method recovery, while comparing
slopes B and C determined the extent
of matrix effects (7). For repeatabil-
ity and intermediate precision, natu-
rally contaminated cereal mixes were
prepared and measured 20 times on
the same day (repeatability) and once
each on a total of eight days by three
different operators (intermediate pre-
cision). A detailed validation report
is available on-line (8). The method
was then further validated through a
collaborative trial (9). Currently, this
method and the results of the collab-
orative trial are in the process of being
published by the European committee
for standardization (CEN).
Results and Discussion
The performance characteristics of this
method are very satisfactory. Matrix
effects that can have a significant inf lu-
ence on results in LC–MS were found
to be negligible for all four analytes in
all six tested materials. The absence of
significant matrix effects allows for the
use of calibration solutions in neat sol-
vent. This can be attributed to the use
of the stable isotopologues. To keep the
total usage of isotopologues low, and
with that the expense per test, they
were added after extraction to only
an aliquot of the extract. So instead
of having to add the equivalent of
2 g of test material, only the equivalent
of 0.125 g had to be spiked. Because
this setup does not account for any loss
of analytes during extraction, method
recovery had to be determined. In
this context, method recovery equals
extraction efficiency, which has shown
to be stable for a given extraction sol-
vent–analyte system across different
cereal matrices.
The HT-2, T-2, and ZON recoveries
in all six test materials were not signifi-
cantly different from 1. Only DON with
an average recovery of 0.83 was different.
This is not very surprising given that the
log P of DON is -0.7 and ethyl acetate
is not the most polar solvent; however,
this method recovery is well within the
commonly accepted ranges. Compared
to more-traditional acetonitrile–water
extracts, the ethyl acetate extracts
seemed to cause, in general, less of a
matrix effect for the analysis of these
four mycotoxins. It is also less hazardous
and expensive than acetonitrile.
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100 100
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60
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00 1 2 3 4
RT: 1.65
RT: 3.62
RT: 4.53 RT: 1.65
RT: 3.63
RT: 4.53
RT: 5.50
0.54
RT: 5.51
5 6 7 8 0 1 2 3 4 5 6 7 8
Time (min)
(b)(a)
Figure 1: (a) A total ion current chromatogram and (b) extracted ion current chromato-grams (top to bottom: DON, HT-2, T-2, ZON) of a QC sample with circa 90-μg/kg DON (RT 1.65), 30-μg/kg HT-2 toxin (RT 3.62), 10-μg/kg T-2 toxin (RT 4.53), and 10-μg/kg ZON (RT 5.51); the peak areas in (a) are mostly representing the 13C-labeled isotopologues.
APRIL 2016 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY 13www.chromatographyonline.com
Repeatability was determined with
naturally contaminated materials at
three different contamination levels.
Near the low end of the calibration
range, the relative repeatability stan-
dard deviations (RSDr) were between
11% and 18% for the four analytes.
Toward higher contamination lev-
els, which were smaller than exist-
ing (DON and ZON) or anticipated
(HT-2 and T-2) legislative limits in
the European Union (EU), these val-
ues improved to ≤9%. Two of those
materials, the lowest and the high-
est contaminated, were also tested on
eight different days by three different
operators to determine intermediate
precision, or within laboratory repro-
ducibility. For the low contaminated
material relative intermediate preci-
sions (RSDi) were between 13% and
25% for the four analytes. For the
high contaminated material they were
between 11% and 17%. All of these
f indings were comparable with the
results of the collaborative trial (9).
As already mentioned, these per-
formance characteristics are quite
satisfactory considering the analysis
time is only 8.7 min. This is signifi-
cantly shorter than the analysis times
reported by Biselli (5) or Varga (6).
Figure 1 shows a typical chromato-
gram of the four analytes, which span
log P values from -0.7 (DON) to 3.6
(ZON). The narrow peaks with a
baseline width of ≤0.2 min attest to
the high efficiency of the SPP parti-
cles packed in a 75-mm column. Even
though a mobile phase with metha-
nol–water was used, the back pressure
during analysis never exceeded 230
bar, which is well below the maximum
pressure of standard HPLC equipment.
Compared to this, analysis time of the
same material in a different labora-
tory during the collaborative trial on
a 150-mm column packed with fully
porous particles takes more than twice
as long (20 min) with larger baseline
peak widths between 0.4 and 0.9 min
(Figure 2). Thus, the SPP column
provides superior resolution at shorter
analysis times.
The benefits of short analysis times
are obvious: higher throughput and
lower solvent consumption. Benefits
of the better resolution might not be
so obvious. Matrix effects in LC–MS
measurements inf luence ionization
eff iciency caused by, amongst other
things, coeluted compounds. Because
of the high specificity of MS, particu-
larly MS-MS, coeluted compounds,
more likely than not, will be unde-
tected. Better resolution will limit
possible coelution and, therefore, min-
imize inf luences on ionization eff i-
ciencies and maximize the ability of
unbiased determination. Furthermore,
in our case, the better resolution comes
from narrower and, hence, taller peaks,
which has a positive effect on limit of
detection and quantification.
To show how a stationary phase chem-
istry change helps in obtaining better
and more confident results, a maize
sample highly contaminated with DON,
AcDONs, and ZON was analyzed with
two columns with identical SPPs but
different chemistries, namely the Kine-
tex C18 and PFP columns. Figure 3
shows the two total ion chromatograms
(TICs). Even though the two AcDONs
were not separated with the C18 chem-
istry, they were with the PFP chemistry.
Retention for all analytes was slightly
higher on the PFP column. Because of
the different fragmentation behavior
of the two AcDONs in MS-MS the
contamination level of the individual
AcDONs can be estimated even from
peaks 2 and 3 in Figure 3b. But because
of significant overlap of the product ions,
this estimation comes with an increased
uncertainty. It goes without saying that
a separation as shown in Figure 3a is
absolutely preferable.
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Time (min)
RT: 11.92
RT: 10.29
RT: 14.09
18.009.108.385.643.052.69 16.2514.61
RT: 5.97
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
00 2 4 6 8 10 12 14 16 18
Figure 2: Total ion current of the same QC sample as in Figure 1. Run times: DON, 5.97 min; HT-2, 10.29 min; T-2, 11.92 min; ZON, 14.09 min. Column: 150 mm × 2 mm, 4-μm dp Synergi Hydro-RP (Phenomenex).
14 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY APRIL 2016 www.chromatographyonline.com
Conclusions
Through the use of an SPP packed col-
umn, a short method of analysis for four
mycotoxins in cereals was developed that
is fit for the purpose of official food and
feed control. The total run time was 8.7
min for the mycotoxins DON, HT-2,
T-2, and ZON spanning log P values
from -0.7 to 3.6. Despite the short run
time, excellent resolution was obtained
with very satisfactory performance char-
acteristics. Method recoveries were indis-
tinguishable from 1 for HT-2, T-2, and
ZON. For DON a recovery of 0.83 was
determined and results for DON should
be corrected for this recovery level. Val-
ues of RSDr were 18% or smaller for
low contamination levels and improved
to 9% or smaller toward higher levels,
which were still below existing or antici-
pated EU legislative limits. Because of
the intelligent use of stable isotopologues,
matrix effects were negligible at a mini-
mal cost per sample.
Changing the stationary-phase chem-
istry from C18 to pentaf luorophenyl
enabled the separation of the structural
isomers 3- and 15-acetyldeoxynivalenol
as well as DON and ZON in a natu-
rally contaminated maize sample. This
stands to show that SPP-packed col-
umns and new stationary-phase chemis-
tries have advanced mycotoxin analysis
in food and feed.
Acknowledgments
The author would like to thank Katrien
Bouten, Kati Kröger, and Karsten
Mischke for their excellent technical
support during method validation and
the collaborative study. The highly con-
taminated maize was a courtesy of the
Austrian National Reference Laboratory
for mycotoxins (AGES, Linz, Austria).
Disclaimer
Any trade names, trademarks, prod-
uct names, and suppliers named above
are only named for the convenience
of the reader of this publication and
their mentioning does not constitute an
endorsement by IRMM, JRC, or EC of
the products named. Equivalent prod-
ucts may lead to the same results.
References
(1) J.J. Kirkland, S.A. Schuster, W.L. John-
son, and B.E. Boyes, J. Pharm. Anal. 3(5),
303–312 (2013).
(2) Food Quality and Standards Service
(ESNS). Worldwide regulations for myco-
toxins in food and feed in 2003. 2004;
Avai lable from: http://www.fao.org/
docrep/007/y5499e/y5499e00.htm.
(3) European Commission, Commission Reg-
ulation (EC) No 1881/2006 of 19 Decem-
ber 2006 setting maximum levels for cer-
tain contaminants in foodstuffs (Text with
EEA relevance). Official Journal of the
European Union, 2006. L 364: p. 5–24.
(4) F. Berthiller et al., World Mycotoxin J. 8(1),
5–35 (2015).
(5) S. Biselli, L. Hartig, H. Wegner, and
C. Hummert, LCGC Europe Special Edi-
tion: Recent Applications in LC-MS 17(11a),
25–31 (2004).
(6) E. Varga et al., Anal. Bioanal. Chem.
402(9), 2675–2686 (2012).
(7) B.K. Matuszewski, J. Chromatogr. B
830(2), 293–300 (2006).
(8) A. Breidbach, Validation of an Analyti-
cal Method for the Simultaneous Deter-
mination of Deoxynivalenol, Zearalenone,
T-2 and HT-2 Toxins in Unprocessed
Cereals - Validation Report. 2011; Avail-
able from: http://skp.jrc.cec.eu.int/skp/
download?documentId=51161.
(9) A. Breidbach, K. Bouten, K. Kröger, J.
Stroka, and F. Ulberth, LC-MS Based
Method of Analysis for the Simultaneous
Determination of Four Mycotoxins in Cere-
als and Feed: Results of a Collaborative
Study (Publications Office of the European
Union, 2013). Available at: http://publica-
tions.jrc.ec.europa.eu/repository/bitstream/
JRC80176/la-na-25853-en-n.pdf.
Andreas Breidbach is with the European
Commission, Joint Research Centre, at
the Institute for Reference Materials
and Measurements in Geel, Belgium.
Direct correspondence to:
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Time (min)
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2,3
3
4
(a) (b)
4
2.477.19 2.31
6.56
3.81
4.18
4.29
100
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90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
0
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ce
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85
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50
45
40
35
30
25
20
15
10
5
00 1 2 3 4 5 6 7 8 9 10
Time (min)
0 1 2 3 4 5 6 7 8 9
Figure 3: Total ion current of a maize sample highly contaminated with DON, AcDONs, and ZON; sample extract was diluted eight times; separation with (a) Kinetex PFP and (b) Kinetex C18 columns; Peaks: 1 = DON, 2 = 15-AcDON, 3 = 3-AcDON, 4 = ZON.
APRIL 2016 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY 15www.chromatographyonline.com
Sheng Feng, Brandi
Bridgewater, Gregory L.
McIntire, and Jeffrey R. Enders
The Synthetic Cannabinoid Chemical Arms Race and Its Effect on Pain Medication Monitoring
In recent years, synthetic cannabinoids (“K2” or “spice”) have
experienced a boom in popularity. The negative health effects of these
drugs coupled with their increasing popularity led to placement onto
Schedule I by the Drug Enforcement Administration (DEA). In response,
the chemists behind these illicit compounds frequently invent new
compounds to circumvent the law. Thus, new classes and new examples
within classes of “spice” continue to become available for illicit use. In
this paper, we examine the use of two column chemistries (C18 and
phenyl-hexyl) in an effort to definitively identify synthetic cannabinoid
compounds in patient samples. Distinct synthetic cannabinoid
compounds interact differently with specific stationary phases and the
hope is that this extra dimension of data will help to rule out similar
interferent compounds that would otherwise cause false-positive results.
Synthetic cannabinoids, com-
monly known as “K2,” “spice,” or
“synthetic marijuana,” are often
sprayed onto or mixed with dried plant
materials and sold in convenience stores,
gas stations, smoke shops, and on the
internet. This ready availability causes
confusion about their safety and legality
(1). In recent years, synthetic cannabi-
noids have become increasingly popular
among adolescents and young adults as
one of several frequently abused sub-
stances. These synthetic drugs mimic
delta-9-tetrahydrocannabinol (THC),
but can be much more potent, which
results in psychoactive doses less than
1 mg (2). In fact, synthetic cannabi-
noids, which have a similar psychoactive
effect as cannabis, have strong addictive
properties often coupled with unknown
physiological impacts on users. A recent
study indicates that the use of synthetic
cannabinoids can be a cause of death (3).
Because of the high abuse potential
and lack of medical knowledge or usage,
these synthetic cannabinoids have been
added to the Schedule I list by the United
States Drug Enforcement Administra-
tion (DEA), as “necessary to avoid immi-
nent hazard to the public safety” (4). In
response, the chemists instigating this
illegal proliferation have synthesized
many new K2 analogs by slightly altering
chemical structures (5). Therefore, com-
pared with the relatively stagnant pool of
other compounds, such as opiates, that
most pain medication monitoring labo-
ratories deal with, the number of agents
on the list of synthetic cannabinoids has
been and continues to be increasing (6).
Testing for synthetic cannabinoids has
become a routine demand among pain
treatment clinics.
There are various types of synthetic
cannabinoids with different modifica-
tions on the core structure. The first
THC analogs, including HU-210 (7)
and CP-47, 497 (8), were synthesized in
the 1980s. Their inventions allowed the
discovery of G protein-coupled recep-
tors, CB1 and CB2 (9). Later on, a struc-
turally different analog, WIN55, 212-
2, was reported. Surprisingly, WIN55,
212-2 has higher affinity toward CB1
16 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY APRIL 2016 www.chromatographyonline.com
and CB2 than THC does (10). Subse-
quently, John W. Huffman developed
a series of “JWH compounds” by sim-
ply replacing the aminoalkyl group in
WIN55, 212-2 with simple alkyl chains
(11). JWH-018 has become the proto-
typical JWH compound. Synthetic can-
nabinoids have also been developed by
generating f luoro-derivatives of JWH
compounds. For example, AM-2201
and MAM-2201 are f luoro-derivatives
of JWH 018 and JWH 122, respec-
tively (12). By replacing the ketone in
the 3-indole position of JWH-018 with
an ester linkage, PB-22 and BB-22
compounds have been synthesized (13).
Furthermore, another class of synthetic
cannabinoids contains the tetrameth-
ylcyclopropyl ketone indoles, such as
UR-144 and its f luoro-derivative, XLR-
11 (14). Both UR-144 and XLR-11 have
cyclopropyl rings, and are therefore
likely to exhibit similar retention times
in liquid chromatography (LC).
The increasing number of sophisti-
cated reversed-phase LC separations has
led to the need for optimized stationary
phases to offer improved selectivity and
efficiency (15). In the present work, we
investigate C18 and phenyl-hexyl col-
umn chemistries for definitively identify-
ing 13 synthetic cannabinoid metabolites
in standards and patient samples.
Materials and Methods
Chemicals
Reference standards of AKB48
5-hydroxypentyl metabolite, AKB48
pentanoic acid metabolite, AM2201
4-hydroxypentyl metabolite, BB-22
3-carboxyindole metabolite, JWH-018
pentanoic acid metabolite, JWH-073
butanoic acid metabolite, JWH-122
5-hydroxypentyl metabolite, MAM-
2201 4-hydroxypentyl metabolite,
PB-22 3-carboxyindole metabo-
lite, PB-22 pentanoic acid metabolite,
UR-144 5-hydroxypentyl metabolite,
UR-144 pentanoic acid metabolite, and
XLR11 4-hydroxypentyl metabolite
were purchased from Cayman Chemi-
cal Company. Reference standards of
11-nor-9-Carboxy-Δ9-THC (THCA),
THCA glucuronide, and THCA-D9
were purchased from Cerilliant Cor-
poration. Solvents including methanol
(optima grade), acetonitrile (optima
grade), and formic acid (88%) were
purchased from VWR. Dimethylsulf-
oxide (DMSO) (HPLC grade), ethyl
acetate (optima grade), and ammonium
hydroxide (A.C.S. Plus) were purchased
from Fisher Scientific. Recombinant
β-glucuronidase enzyme was purchased
from IMCS. Drug-free normal human
urine (NHU) was purchased from
UTAK Laboratories, Inc. Deionized
(DI) water was obtained in-house from a
Thermo Scientific Barnstead Nanopure
water purification system.
HU-210
JWH-018 AM-2201 JWH-122 MAM-2201
PB-22 BB-22 UR-144 XLR-11
OH OH
OHOHH
O
O
O
O O OO
O
O OO
O
O
O
H
N
N
N
N NN
N
N
N
N N F
F
F
NH3C
H3C H3C
H3CH3C
H3CH3CH3C
CH3CH3
CH3
CH3
CH3
CH3 CH3
CH3
CH3
CH3 CH3
CH3
CH3CH3
CP-47, 497 WIN55, 212-2
XLR11 N-(4-hydroxypentyl) metabolite
UR-144 N-pentanoic acid metabolite
UR-144 N-(5-hydroxypentyl) metabolite
%B solvent
1 2 1 2
100
03 4 5
1 2 1 2 3 4 5
Time (min)Time (min)
C18
Rela
tive in
ten
sity
100
0
%B
%B
Phenylhexyl
Figure 1: Chemical structures of recent synthetic cannabinoids.
Figure 2: Total ion chromatography of 100 ng/mL calibrator in C18 and phenyl-hexyl columns with 2.5-min or 5-min methods. Red, blue, and green peaks represent XLR11 N-(4-hydroxypentyl), UR-144 N-pentanoic acid, and UR-144 N-(5-hydroxypentyl), re-spectively. Blue dashed lines indicate solvent gradients.
APRIL 2016 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY 17www.chromatographyonline.com
Sample Preparation
Reference standards not already in solution were dissolved in
DMSO. Solutions of reference standards were aliquoted, dried,
and reconstituted with NHU to make a low calibrator concen-
tration at 1 ng/mL for all analytes except BB-22 3-carboxyin-
dole metabolite and THCA with low calibrator levels at 5 ng/
mL and 10 ng/mL, respectively. A high calibrator concentration
of 100 ng/mL in NHU was used for all analytes. An 18.5-ng/
mL THCA glucuronide hydrolysis–negative control (HNEG)
and a 20-ng/mL positive control (20CON) were similarly pre-
pared in NHU. This protocol uses THCA glucuronide as a
hydrolysis control. Accordingly, every curve and patient batch
has a hydrolysis control that contains 18.5 ng/mL of THCA
glucuronide. For this control to be considered passing, it must
return the expected THCA (parent) concentration within 30%.
Into 13 mm × 10 mm borosilicate glass tubes, 800 μL of
calibrators, controls, and samples were each aliquoted and com-
bined with 200 μL of THCA-D9 (2.5 μg/mL)/recombinant
β-glucuronidase (1000 enzyme units/mL) solution in 25:25:50
methanol–DI water–pH 7.5 phosphate buffer. All samples were
vortexed, transferred to SPEware CEREX PSAX 3 mL/35 mg
extraction columns in sample racks by SPEware, and heated
in a VWR Symphony oven for 15 min at 60 °C. Samples were
cooled for 5 min and placed on an automated liquid dispens-
ing-II (ALD-II) system for extraction. A light positive pressure
was applied to push the samples onto the solid-phase extraction
(SPE) packing. The ALD-II system then washed columns with
85:14:1 DI water–acetonitrile–ammonium hydroxide, washed
with 30:70 DI water–methanol, and finally eluted samples into
1800-μL amber autosampler vials using 98:2 ethyl acetate–for-
mic acid. Samples were dried under nitrogen for ~35 min at
25 °C in a SPEware Cerex sample concentrator, then each
reconstituted with 400 μL of 50:50 DI water–methanol. Sam-
ples were capped, vortexed for 20 s, and spun for 5 min at 4000
rpm on a Sorvall ST 40 centrifuge.
Patient Sample Collection
Patient urine specimens were collected at clinics and shipped to
Ameritox Ltd. These de-identified patient samples were treated
similarly to standards, that is, they were diluted, extracted, and
subjected to liquid chromatography–tandem mass spectrom-
etry (LC–MS-MS). Patient samples were selected for this study
Columnchemistry
C18
Co
un
tsC
ou
nts
Co
un
tsC
ou
nts
Pati
en
t 01
Pati
en
t 02
C18
Phenyl-hexyl
Phenyl-hexyl
JWH-018 N-pentanoic acidmetabolite qual372.2 → 126.9
1.8E4
1E4
0
1.6E4
0.8E4
0
1.2E4
0.6
0
9E3
4E3
0
1.8E4
1E4
0
0.4
1 1.2 1.4 1.6 1.8 1 1.2 1.4 1.6 1.8
1 1.2 1.4 1.6 1.8 1 1.2 1.4 1.6 1.8
0.6 0.8 1 1.2 1.4 0.6 0.8 1 1.2 1.4
1.2E4
0.6
0
1.2E4
0.6
0
Time (min) Time (min)
0.4 0.6 0.8 1 1.2 1.4 0.6 0.8 1 1.2 1.4Time (min) Time (min)
JWH-018 N-pentanoic acidmetabolite quant
372.2 → 155.1
IR fail5.4 ng/mL
IR pass14.5 ng/mL
IR fail5.4 ng/mL
IR pass14.5 ng/mL
Figure 3: Comparison of suspected JWH-018 pentanoic acid patient samples. The gray areas are integrated peaks. The dashed lines indicate the expected retention time based on the calibrators.
Columnchemistry
C18
Co
un
ts
Pati
en
t 02
Phenyl-hexyl
Time (min) Time (min)
MAM2201 N-(4-hydroxypentyl)metabolite quant
390.1 → 169.0
MAM2201 N-(4-hydroxypentyl)metabolite qual390.1 → 141.0
1E3
5E2
0
Co
un
ts
58
50
42
80
65
50
IR fail1.2 ng/mL
IR fail0 ng/mL
3.5E2
2.0E2
0.5E2
1.2
0.6 0.8 1 1.2 1.4 1.6 0.8 1 1.2 1.4 1.6
1.4 1.6 1.8 2 1.2 1.4 1.6 1.8 2
Figure 4: Comparison of suspected MAM-2201 metabolite pa-tient samples. The gray areas are integrated peaks. The dashed lines indicate the expected retention time based on the cali-brators.
( Keeping quality control under control. )
Amino acid analysis in accordance toEuropean Pharmacopeia 8.0
www.pickeringlabs.com
CATALYST FOR SUCCESS
PINNACLE PCX
18 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY APRIL 2016 www.chromatographyonline.com
Table I: Mass spectrometry conditions for all methods in this study. The retention times coordinate with the 2.5 min
C18 and phenyl-hexyl method.
Compound NamePrecursor
IonProduct Ion
Fragmentation
(V)
Collision
Energy (V)
Cell Accelerator
(V)
C18 RT
(min)
Phenyl-hexyl
RT (min)
AKB-48 5-hydroxy-pentyl
382.11
107.00 380 52 2 1.99 1.24
92.90 380 60 2 1.99 1.24
135.10 380 10 5 1.99 1.24
AF4–MALS–dRI 396.1193.00 380 60 3 1.94 1.22
135.10 380 10 5 1.94 1.22
AM-2201 4-hy-droxypentyl
376.11
143.80 380 40 3 1.3 0.88
127.10 380 56 2 1.3 0.88
155.10 380 25 3 1.3 0.88
BB-22 3-carboxy-indole
258.01
118.00 380 24 5 1.8 0.97
54.90 380 36 2 1.8 0.97
175.90 380 10 7 1.8 0.97
JWH-018 N-penta-noic acid
372.21
126.90 380 60 2 1.36 0.94
55.00 380 56 2 1.36 0.94
155.10 380 25 3 1.36 0.94
JWH-073 butanoic acid
358.21
127.20 380 60 2 1.26 0.84
43.30 380 48 2 1.26 0.84
155.10 380 45 3 1.26 0.84
JWH-122 5-hydroxy-pentyl
372.11
115.10 380 72 4 1.65 1.11
169.10 380 21 4 1.65 1.11
141.00 380 55 4 1.65 1.11
THCA 345.20
327.20 380 18 2 2.1 1.31
299.20 380 18 6 2.1 1.31
193.20 380 18 2 2.1 1.31
MAM-2201 N-(4-hydroxypentyl)
390.11141.00 380 48 2 1.53 1.04
169.00 380 10 7 1.53 1.04
PB-22 3-carboxy-indole
232.01
118.00 380 16 2 1.53 0.75
43.10 380 24 2 1.53 0.75
132.00 380 10 7 1.53 0.75
PB-22 pentanoic acid
389.31
144.00 380 36 3 1.14 0.73
54.90 380 56 4 1.14 0.73
244.00 380 10 3 1.14 0.73
UR-144 5-hydroxy-pentyl
328.1155.00 380 44 2 1.74 0.93
125.00 380 10 3 1.74 0.93
UR-144 N-pentano-ic acid
342.11
125.00 380 20 3 1.68 0.92
54.90 380 48 4 1.68 0.92
244.00 380 10 4 1.68 0.92
XLR-11 4-hydroxy-pentyl
346.11143.90 380 44 3 1.49 0.79
248.00 380 20 2 1.49 0.79
THCA-d9 (internal standard)
354.10 336.10 380 13 5 2.09 1.29
APRIL 2016 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY 19www.chromatographyonline.com
that were deemed positive by the current
method’s criteria, but were then deemed
negative upon closer manual inspection.
Instrumentation
All analyses were conducted by LC–
MS-MS on an Agilent 6490 triple-
quadrupole system run in electrospray
ionization (ESI) positive mode using
an Agilent 1290 chromatographic sys-
tem (1290 Inifinity binary pump, 1290
TCC, 1290 autosampler, and 1290 ther-
mostat) with a 100 mm × 2.1 mm, 2.7-
μm dp Agilent Poroshell 120 EC-C18 or
50 mm × 2.1 mm Phenomenex Kinetex
2.6 μm Phenyl-Hexyl column. Source
conditions were optimized with a
250 °C gas temperature, gas f low at
19 L/min, nebulizer set to 45 psi, sheath
gas heater at 300 °C, sheath gas f low
at 11 L/min, capillary voltage at 3.5
kV, and charging voltage at 2 kV. The
run time for this method is 2.21 min
with a cycle time of approximately
2.5 min. A longer chromatographic
method (roughly 5 min) was also used
in this study to help resolve question-
able interferences. All of these assays
monitor two or three transitions for
each of the following 14 analytes:
AKB48 5-hydroxypentyl metabolite,
AKB48 pentanoic acid metabolite,
AM2201 4-hydroxypentyl metabolite,
BB-22 3-carboxyindole metabolite,
JWH 018 pentanoic acid metabolite,
JWH 073 butanoic acid metabolite,
JWH 122 5-hydroxypentyl metabolite,
MAM2201 4-hydroxypentyl metabo-
lite, PB-22 3-carboxyindole metabo-
lite, PB-22 pentanoic acid metabolite,
UR-144 5-hydroxypentyl metabolite,
UR-144 pentanoic acid metabolite,
XLR11 4-hydroxypentyl, and THCA;
and one transition for one internal stan-
dard, THCA-D9. THCA is analyzed
by the mass spectrometer, but it is not
actively monitored in patient samples.
MS method parameters are shown in
Table I. The chromatographic start-
ing conditions are 40% mobile-phase
A (0.1% formic acid in 90:10 water–
methanol) and 60% mobile-phase B
(0.1% formic acid in methanol) with a
Table II: Gradient properties of the 2.5-min method
StepFlow Rate
(mL/min)
Time
(min)
%A (0.1% Formic Acid in
90:10 Water–Methanol)
%B (0.1% Formic
Acid in Methanol)
0 0.5 Initial 40 60
1 0.5 0.80 30 70
2 0.5 1.60 5 95
3 0.5 2.20 5 95
4 0.5 2.21 40 60
5 0.5 2.50 40 60
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0.5-mL/min f low throughout (Tables
II and III). The 2.5-min phenyl-hexyl
method was validated according to a
previously published procedure (16).
Results and Discussion
Various methods including colorimet-
ric detections (17), immunochemical
assays (18), nuclear magnetic resonance
(NMR) (19), gas chromatography–mass
spectrometry (GC–MS) (20), and LC–
MS-MS (21), have been developed for the
analysis of synthetic cannabinoids. With
those methods, many synthetic cannabi-
noids have been successfully analyzed in
different samples such as plant materi-
als, human hair, saliva, serum, and urine.
Several analytical reviews have summa-
rized the identification and quantifica-
tion techniques for synthetic cannabi-
noids that are currently popular (22,23).
Among those methods, LC–MS-MS
has clear advantages of ease and speed
of sample preparation and the capabil-
ity of automation. However, most of the
current methods only focus on a few
synthetic cannabinoids, or need a very
long chromatographic gradient to affect
resolution of spice compounds of inter-
est (usually longer than 10 min, see Table
IV). To improve the analysis of synthetic
cannabinoids, we developed new LC–
MS-MS methods with two different col-
umn chemistries (C18 and phenyl-hexyl),
which take either 2.5 min or 5 min for
each sample to achieve optimal resolu-
tion. These methods were applied to the
analysis of 13 synthetic cannabinoids.
We have analyzed a 100-ng/mL
synthetic cannabinoid calibrator that
includes all the K2 and spice com-
pounds of interest to this work with
both the 2.5-min or 5-min methods
in two different columns. Most of
the compounds were eluted in similar
order in the different columns, though
the elution time changed. Overall, the
compounds in the phenyl-hexyl column
are eluted earlier compared with ones
in the C18 column under both the 2.5-
min and 5-min methods, which may be
solely due to the shorter length of the
column or a combination of length and
selectivity. In addition, the three com-
pounds that share the tetramethylcyclo-
propyl ketone indole structural moiety
(that is, XLR11 N-[4-hydroxypentyl],
UR-144 N-pentanoic acid, and UR-144
N-[5-hydroxypentyl]) exhibit changed
elution order in the two different col-
umns. In both the 2.5-min and 5-min
methods, those three compounds were
eluted much earlier in order with the
phenyl-hexyl column compared to the
C18 column. This change in elution
order is not because of the change in the
column length. However, it might be
Table III: Gradient properties of the 5-min method
StepFlow Rate
(mL/min)Time (min)
%A (0.1% Formic Acid in
90:10 Water–Methanol)
%B (0.1% Formic
Acid in Methanol)
0 0.5 Initial 65 35
1 0.5 0.90 40 60
2 0.5 1.70 35 65
3 0.5 2.50 32 68
4 0.5 4.00 5 95
5 0.5 4.30 5 95
6 0.5 4.31 65 35
7 0.5 5.00 65 35
Table IV: LC–MS-MS conditions for synthetic cannabinoids in urine samples in
selected studies
Targets Purification ColumnTime of
Gradient
LOD
(ng/mL)Reference
Metabolites of JWH-018 and JWH-073
Dilution (hydrolysis)
Zorbax Eclipse XDB-C18 (150 mm × 4.6 mm, 5 μm)
10 min <2.0 (24)
Metabolites of JWH-018 and JWH-073
SPE (hydrolysis)
Zorbax Eclipse XDB-C18 (150 mm × 4.6 mm, 5 μm)
10 min <0.1 (25)
Metabolites of 8 synthetic cannabinoids
LLE (hydrolysis)
AQUASIL C18 (100 mm × 2.1 mm, 5 μm) (Thermo Scientific)
14 min 0.1 (26)
Metabolites of JWH-018 and JWH-073
LLE (hydrolysis)
Acquity UPLC HSS T3 (100 mm × 2.1 mm, 1.8 μm) (Waters)
More than 3.2
min(27)
Metabolites of 7 synthetic cannabinoids
LLE (hydrolysis)
Luna C18 (150 mm × 2 mm, 5 μm) (Phenomenex)
15 min (28)
Metabolites of UR-144 and its pyrolysis product
LLE (hydrolysis)
Zorbax Eclipse XDB-C18 (150 mm × 2.1 mm, 3.5 μm) (Agilent)
19 min (29)
9 synthetic cannabinoids, 20 metabo-lites
PP (hydrolysis)
XB-C18 (50 mm × 3.0 mm, 2.6 μm) (Kinetex)
10 min 0.5–10 (21)
15 indole derivative synthetic cannabinoids
LLE (hydrolysis)
Ascentis C18 (150 mm × 2.1 mm, 5 μm) (Supelco)
16 min 0.1–0.5 (30)
17 me-tabolites of synthetic cannabinoids
LLE (hydrolysis)
Phenomenex Gemini C18 (150 mm × 4.6 mm, 3.0 μm)
12 min 0.01–0.5 (31)
SPE = solid-phase extraction; LLE = liquid-liquid extraction; PP = protein precipitation
APRIL 2016 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY 21www.chromatographyonline.com
due to their tetramethylcyclopropyl structure having a higher
affinity toward the C18 column than for the phenyl-hexyl
column. Although this observation may seem trivial, it helps
illustrate the breadth of chemical components inherent in a
synthetic cannabinoid method. This challenge of chemical
breadth can be used as an advantage, however, if one con-
siders that synthetic cannabinoids with different chemical
structures will have different elution behaviors in two dis-
tinct column chemistries. In most cases, newly invented spice
compounds only slightly change the side chains of the banned
chemicals. It is possible that evaluating potential patient posi-
tives for this class of compounds using two different column
chemistries might help better separate compounds with simi-
lar chemical structures, thereby improving the detection of
novel compounds from existing agents.
These new methods for analyzing synthetic cannabinoids
were applied to suspected patient positive samples identified
from a production method. When the urine sample of patient
01, positive for JWH-018 pentanoic acid metabolite, was ana-
lyzed using both C18 and phenyl-hexyl columns, both quanti-
fier (quant) and qualifier (qual) peaks for JWH-018 pentanoic
acid metabolite came out earlier than expected based on cali-
brators (Figure 3). However, the ion ratio failed in the analysis
on the C18 column because of a missing qual peak, whereas the
ion ratio passed in the analysis with the phenyl-hexyl column.
Regardless of column chemistry, a human reviewer would likely
review this sample as negative or “unable to confirm” since
retention times do not perfectly line up. However, with the
phenyl-hexyl column data the peaks that passed the ion ratio
criteria were not all that far off with regards to retention time.
On a production floor it is not unreasonable for peaks to drift
0.3 min (18 s) over a given day or week, especially if this instru-
ment is used to run two different methods that may or may not
use different columns and solvents.
Meanwhile, in the test of patient 02, also potentially pos-
itive for JWH-018 pentanoic acid, all peaks showed up at
the expected retention times. The ion ratios passed on the
phenyl-hexyl column, but failed on the C18 column, which
is consistent with the result of patient 01. The data suggests
the phenyl-hexyl column significantly improved the detec-
tion of JWH-018 pentanoic acid metabolite in our methods
compared to the C18 column. The fact that this patient sam-
ple fails ion ratio (IR) on the C18 column and passes on the
phenyl-hexyl possibly indicates that an interferent coeluted
with one or both of the C18 peaks, thereby throwing off the
ion ratio. Cannabinoids (synthetic or otherwise), due to their
chemical makeup, are generally fat soluble and by extension
they also tend to be chromatographically coeluted with any
lipid content that may be in a sample. It is possible that this
interferent, which is throwing off the ion ratio in the C18 sam-
ple, is a lipid component that was able to survive the hydrolysis
and extraction protocol to be coeluted on the C18 column, but
on the phenyl-hexyl column it is sufficiently separated. It is
also possible that the compound from the patient sample is
isobaric with JWH-018 pentanoic acid and possesses the same
multiple reaction monitoring (MRM) transitions as JWH-018,
but at different ratios than the true calibrator compound. This
is possible if a small change in side chain configuration is envi-
sioned (for example, straight chain versus branched chain). The
technical and ethical issues associated with making a positive
call on such samples are not trivial.
Next, for a suspected MAM-2201 N-(4-hydroxypentyl)
metabolite, we found that patient sample 02 showed an
interfering peak, with slightly incorrect retention time, on
the C18 column. The chemistry of this interferent seems to
be drastically different compared to the MAM-2201 N-(4-
hydroxypentyl) metabolite, since it was not observed in the
Columnchemistry
C18
Pati
en
t 03
Phenyl-hexyl
Time (min)
UR-144 N-pentanoic acidmetabolite quant
342.1 → 125.0
UR-144 N-pentanoic acidmetabolite qual342.1 → 244.0
Co
un
tsC
ou
nts
7E3
3E3
0
5.0E3
2.5E3
0
IR fail5.2 ng/mL
IR fail4.9 ng/mL
4E4
2E4
0
3.0E4
1.5E4
0
1.2 1.4 1.6 1.8 2 1.2 1.4 1.6 1.8 2
0.6 0.8 1 1.2 1.4
Time (min)
0.60.4 0.8 1 1.2 1.4
Figure 5: Comparison of suspected UR-144 N-pentanoic acid patient samples. The gray areas are integrated peaks. The dashed lines indicate the expected retention time based on the calibrators. In this particular patient sample (when run on the C18 column), the actual qualifier peak was visible and chro-matographically separated; however, the integration software (under reasonable integration conditions) incorrectly selected the interferent for integration.
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window for the phenyl-hexyl column.
These types of interferences are ram-
pant among positive and questionably
positive synthetic cannabinoid patient
samples.
Patient 03 had a very strong well sepa-
rated quant peak for UR-144 N-penta-
noic acid, but the qual peak showed an
interferent just a few seconds away from
the targeted retention time. This inter-
ferent made detection of the qual peak
of interest very difficult. The qual peak
is still visible in the C18 separation;
however, the software (under reasonable
integration conditions) incorrectly inte-
grated the interferent. With the phenyl-
hexyl column chemistry, the qualifier
peak has coalesced into the interfer-
ent peak entirely and is not able to be
resolved, even with manual integration
intervention. The fact that this interfer-
ent moved proportionally with reference
to the expected UR-144 N-pentanoic
acid retention time indicates that this
interferent might share some chemical
functionality as discussed above.
Conclusions
A rapid, selective, and sensitive LC–
MS-MS method identifying 13 syn-
thetic cannabinoids in patient urine
samples has been described. Two dif-
ferent column chemistries (that is, C18
and phenyl-hexyl) have been applied
using this method. Three compounds,
including XLR-11 N-(4-hydroxylpen-
tyl), UR-144 N-pentanoic acid, and
UR-144 N-(5-hydroxylpentyl) metabo-
lites, demonstrate the different order
of elution on a phenyl-hexyl column
compared to the C18 column, while
most of the compounds maintain their
elution order. The fact that newly
invented synthetic cannabinoids often
only slightly change the side chains of
the banned drugs makes the detection
of those compounds more difficult. At
our laboratory, synthetic cannabinoids
are requested in roughly 20% of our
total samples and therefore should not
be written off as a fringe interest in
the pain medication monitoring arena
in spite of the very low positivity rate.
Using a second LC–MS-MS method to
confirm patient positives (as illustrated
here) is potentially useful for large scale
laboratories on a daily basis because of
the low positivity rates observed.
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Sheng Feng, Brandi Bridgewater,
Gregory L. McIntire, and Jeffrey R.
Enders are with Ameritox Ltd., in Greens-boro, North Carolina.
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APRIL 2016 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY 25www.chromatographyonline.com
ciency necessary to function as a CRO in
today’s bioanalytical world is the develop-
ment of column technology over the past
few decades. The reliable repeatability of
columns on the market today, combined
with the plethora of unique column types
that can be implemented, allow for the effi-
cient development of an HPLC–MS-MS
method for high-throughput analysis.
Because all bioanalytical work depends
on high-throughput analysis, many of the
trends in emerging technologies in the
bioanalytical market are directly related to
increasing on-instrument productivity and
reducing costs. This includes smaller par-
ticle size in columns coupled with ultrahigh
pressure liquid chromatography (UHPLC),
superficially porous shell column technol-
ogy, and microflow HPLC. This article
presents a quick background into the details
of developing an HPLC–MS-MS method
from the perspective of a CRO in relation
to column choice. It also focuses on recent
column technologies, the instrumentation
surrounding them, and their benefits in a
CRO environment.
Method Development
High-throughput bioanalysis CROs are usu-
ally a fast-paced environment, where it is nec-
essary to create a productive, rugged method
from the ground up for what is often times
an unknown novel therapeutic. A large part
of a CRO’s efficiency stems from its ability
to quickly develop a rugged method that
will repeatedly hold up to rigorous indus-
try and regulatory standards. As efficiency
can often be derived from simplicity, when
developing a new method the simplest solu-
tion is always the first approach. This is why,
despite the plethora of columns available for
use, it is almost always best to start with a
C18 or C8 column. One of the most versatile
and widely used columns, the C18 column
has been in use in one form or another for
decades. Comprising a simple octadecyl
carbon chain bonded silica-based stationary
phase, the C18 column is the go-to column
of choice for a large majority of molecules
analyzed by HPLC–MS-MS. C18 columns
have proven to provide good retention and
resolution for a vast array of small molecules.
With a proven track record of negligible
lot-to-lot and column-to-column variabil-
ity, there is minimal concern of anomalous
behavior throughout the life of a method
on a C18. C18 columns also tend to be very
rugged, with the average lifespan lasting for
upwards of thousands of injections. This is
a very important point in the development
of any method; if a seemingly scientifically
sound method has been developed, but the
column only lasts a few hundred injections
before peak deterioration, then the method
probably isn’t rugged or productive enough
to be feasible. A large benefit in the flex-
ibility of the C18 is that it allows for the
standardization of many HPLC–MS-MS
methods, which greatly increases the pro-
ductivity of high-throughput analysis.
With multiple standardized methods rely-
ing on one type of column and identical
mobile phases for an array of molecules,
it is possible to keep instruments running
continuously without interruption. This is
crucial to the high-volume requirement in
the bioanalytical CRO world.
However, there are always going to be
analytes that do not work on a C18 column.
For multiple analytes, resolution (Rs) and
chromatographic selectivity (α) will play
a role. However, here we focus on method
development of one analyte. Whether due
to poor retention (tR), poor asymmetry fac-
tor (AF), or poor repeatability, decisions
Is it a chiral molecule?
Is it a mobile phasemismatch?
Polarendcapped
column
Look at the functional groups andselect specialty column
Is it a mobile phasemismatch?
Chiral column
No
C18
Yes
No No
F5
C18
Ion pairing HILIC
Yes Yes
NonpolarPolar
Good tR
and good AF Good tR
and poor AFPoor tR
and good AF
Key
tR
= Retention time
AF = Asymmetry Factor
Poor tR
and poor AF
2-μm solid core
0.5-μm shell (3 μm total) 3-μm fully porous particle
Figure 1: Representative column method development flowchart.
Figure 2: Representative structure of SPP and fully porous particles.
26 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY APRIL 2016 www.chromatographyonline.com
can then be made on what type of specialty
column to look at. This process can quickly
become overwhelming given the plethora of
columns and column types on the market
today. Having an approach to address the
most common column-based issues during
method development, as seen in the flow-
chart in Figure 1, is an important aspect
in maintaining efficiency during method
development. Once it has become apparent
that a method will not be adequately devel-
oped on a C18 column, the next step is typi-
cally to evaluate the polar moieties and func-
tional groups exhibited by the molecule. For
a polar molecule, some of the more common
approaches available are to choose a polar
endcapped column or to implement an ion-
pairing reagent (where an ion-pairing reagent
such as heptafluorobutyric acid [HFBA] is
added to the mobile phases or extraction
solvents). When presented with a particu-
larly small, polar molecule, another option
available is to choose a column such as an F5
column (a pentafluorophenylpropyl station-
ary phase) or to use a hydrophilic-interaction
chromatography (HILIC) method. HILIC
methods use gradients with a high percent-
age of organic content coupled either with
an unmodified silica column, an amino col-
umn, a zwitterionic column, or any one of
a number of columns made specifically for
HILIC methods.
Recent Column Advancements
Although efficiency in method development
is paramount to being cost effective in a bio-
analytical CRO environment, this efficiency
would amount to nothing if the actual
methods themselves were not productive in
the long run. Even if all the scientific bench-
marks may have been met during develop-
ment, the overall costs of performing the
method determine whether it will actually
be feasible. The costs of a method are largely
determined based on two factors: the over-
all costs of disposable supplies (for example,
extraction supplies, solvents, and columns)
and time. With this in mind, it is no surprise
that many of the emerging technologies in
the industry seek to minimize both of these
aspects of HPLC–MS-MS.
One such way to increase HPLC–MS-MS
productivity that has been developed and
implemented in the past decade is the decrease
in column packing particle size. Traditionally,
the packing in LC columns has been made
up of fully porous particles ranging in size
from 3 to 10 μm. However, by decreasing
the particle size below the previous standards
to sub-2-μm particles, there is an increase
in chromatographic efficiency leading to an
increase in theoretical plate counts and, thus,
greater resolution (1). However, one of the
side effects of decreasing the particle size is a
fairly large increase in pressure, which limited
the widespread use and commercial viability
of sub-2-μm columns until fairly recently. To
withstand the back pressures involved with
using sub-2-μm columns, new instrumenta-
tion was devised; thus, UHPLC was born
(2,3). Using an UHPLC system available
from various vendors, it is possible to success-
fully implement smaller particle size columns
and run at pressures as high as ~20,000 psi
(4). These UHPLC systems have proven to
be robust enough for high-throughput bio-
analysis work and have been implemented
throughout the industry.
However, since cost effectiveness is an
overall goal of a bioanalytical CRO, it may
not be the most practical option to purchase
an entirely new HPLC system to attain
what may amount to only a slight increase
in method productivity and decrease in run
time. For laboratories already in existence and
set up with traditional HPLC instruments
rather than UHPLC, it is much more desir-
able to find a smaller-scale solution to increase
method productivity. Another recent advent
to the column market in the last decade,
superficially porous particle (SPP) columns
take the idea of smaller column particles to
the next, albeit somewhat divergent, step.
Rather than decreasing the size of fully porous
particles in the columns, the idea behind SPP
is a small, solid inner core (which generally
range from 1.3 to 5 μm) surrounded by a per-
meable shell of porous silica. While the outer
shell of the particles are similar in materials
and function as a conventional fully porous
column particle, the inner core is imperme-
able (hence the term superficially porous), as
can be seen in Figure 2. Although the idea
of shell-based stationary phases have been
around since the late 1960s, with the use of
larger (~50 μm) pellicular particles (5), it is
only recently that the particle sizes have been
reduced down to conventional standards.
With the combination of the small diameter
of the inner core and the porous nature of the
shell, SPPs provide the benefits of sub-2-μm
fully porous particles while eliminating many
of the back pressure issues (6,7). Although
the theory behind the increase in efficiency
attributed to SPP columns is not discussed in
detail here, Figure 3 shows a rough represen-
tation of how the rate of diffusion is increased
throughout an SPP column as opposed to a
column containing fully porous particles of
comparable size. This increased rate of diffu-
sion relates to quicker, more efficient separa-
tions than were previously possible with fully
porous columns, and results in a tighter peak
shape as the shorter path reduces the diffu-
sion of the analytes (8).
Emerging Technologies
Yet another approach of reducing costs and
increasing efficiency in bioanalytical analy-
sis is the implementation of microflow LC
coupled with a mass spectrometer. As com-
pared to the standard high flow of HPLC–
MS-MS, which generally uses around a
700-μL/min flow rate, microflow LC–
MS-MS employs the use of pumps that
can accurately deliver a flow rate of well
2-μm solid core
0.5-μm shell (3 μm total) 3-μm fully porous particle
Path of analyte
Path of analyte
Figure 3: Representation of possible analyte paths between SPP and fully porous particles.
APRIL 2016 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY 27www.chromatographyonline.com
below 100 μL/min, greatly reducing the
consumption of solvents. This reduction in
solvent use directly translates to a cost sav-
ings in the purchasing of solvents, disposal
of solvent waste, and the labor of solvent
preparation—none of which are insignifi-
cant expenses in a high-throughput labora-
tory that is virtually running continuously.
The drastically lower flow rates associated
with microflow LC–MS-MS also translate
to less solvent flowing through the electro-
spray ionization (ESI) source. This means a
cleaner MS system and a lower cost associ-
ated with MS maintenance.
Microflow LC–MS-MS employs col-
umns with drastically decreased inter-
nal column diameter. While standard
HPLC–MS-MS may use columns with
internal diameters ranging from 2 to 4.6
mm, microflow LC–MS-MS uses columns
with internal diameters ranging from 0.2
to 0.3 mm (micro) down to <0.2 mm
(nano), which can be used with flow rates
of 10 and 0.3 μL/min, respectively. Solvent
consumption and savings aside, microflow
LC–MS-MS has also been documented to
increase ESI response (9) while reducing
matrix effects (10) and increasing ionization
efficiency (11). Early works on ESI response
demonstrated that as the mobile-phase flow
rate of ESI is reduced, there is an increase in
proportional MS signal-to-noise ratio (12).
Some of the challenges in the integra-
tion of microflow LC–MS-MS into the
high-throughput bioanalysis world are
longer run times, dead volumes in fittings
and connections having a greater impact
on chromatography, and a perceived lack
of robustness of microflow instrumentation.
To address some of these challenges, work
has been performed by multiple vendors
on implementing an integrated, in-source
column. By integrating a column directly
into the source, many of the dead volume
issues related to microflow LC–MS-MS are
resolved. The idea behind the application is
to simplify instrument setup by minimizing
connections and reducing the length of tub-
ing required between the LC injector and
the MS, and thus minimizing the impact of
pre-column and post-column volumes. As
shown in the corresponding chromatogram
in Figure 4, the combination of a micro
internal diameter column integrated into the
source coupled with microflow LC–MS-MS
provides a greatly increased signal as com-
pared to HPLC–MS-MS; in addition, the
system maintains a run time of less than 5
min. With the possibility of a system that is
generating higher sensitivity (among other
chromatographic benefits) coupled with
lower flow rates leading to lower solvent
consumption, microflow LC–MS-MS com-
bined with integrated, in-source columns
seems to be a highly promising direction for
high-throughput bioanalysis.
Conclusion
With the advancements in column and
other LC technology in recent years, devel-
oping robust methods for novel therapeu-
tics has become a more reliable process
than ever. It is possible to efficiently create
productive methods for molecules of ever-
increasing complexity. This will become
more important in years to come as
HPLC–MS-MS is increasingly looked to as
the solution for analysis of large molecules
including peptides, proteins, and biomark-
ers. Increasing efficiency and productivity
on both the front end (method develop-
ment) and back end (sample analysis) will
be made continuously possible with further
advancements such as SPP columns and
microflow LC–MS-MS. Looking to the
future, the expectation for the pharmaceu-
tical and biotech industries will be to supply
the global community with therapeutics at
a reasonable cost. Thus, the highest levels
of productivity and efficiency will be para-
mount to meet this goal.
References
(1) J.E. MacNair, K.C. Lewis, and J.W. Jorgenson,
Anal. Chem. 69, 983–989 (1997).
(2) J.E. MacNair, K.D. Patel, and J.W. Jorgenson,
Anal. Chem. 71, 700–708 (1999).
(3) N. Wu, J.A. Lippert, and M.L. Lee, J. Chro-
matogr. A 911, 1–12 (2001).
(4) “In the News,” Trends Anal. Chem. 61, iv–x
(2014).
(5) C. Horváth, B.A. Preiss, and S.R. Lipsky, Anal.
Chem. 39, 1422 (1967).
(6) J.J. DeStefano, T.J. Langlois, and J.J Kirkland, J.
Chrom. Sci. 46, 254–260 (2008).
(7) D.V. McCalley, J. Chromatogr. A 1218,
2887−2897 (2011).
(8) G. Guiochon and F. Gritti, J. Chromatogr. A
1218, 1915–1938 (2011).
(9) G. Valaskovic and N. Kelleher, Curr. Top. Med.
Chem. 2(1), 1–12 (2002).
(10) E. Gang, M. Annan, N. Spooner, and P. Vou-
ros, Anal. Chem. 73(23), 5635–5644 (2001).
(11) R. Juraschek, T. Dulcks, and M. Karas, J. Am.
Soc. Mass Spectrom. 10, 300–308 (1999).
(12) P. Kebarle and L. Tang, Anal. Chem. 65,
972A–986A (1993).
Ryan Collins and Shane Needham are
with Alturus Analytics, Inc., in Moscow,
Idaho. Direct correspondence to:
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8
Time (min)
0.0
1.0e4
2.0e4
3.0e4
4.0e4
5.0e4
6.0e4
7.0e4
8.0e4
9.0e4
1.0e5
1.1e5
1.2e5
1.3e5
1.4e5
1.5e5
1.6e5
1.7e5
1.8e5
1.9e5
2.0e5
Inte
nsi
ty (
cps)
7 μL/min, 0.2 mm i.d.
44 μL/min, 0.5 mm i.d.
700 μL/min, 2.0 mm i.d.
Figure 4: Comparative chromatogram between HPLC–MS-MS (red trace), microflow LC–MS-MS (blue trace), and in-source column PicoFuze (green trace) from the analysis of a sur-rogate peptide from MAOB from human plasma using a gradient of acetonitrile and water with 1% formic acid on C18 columns. Stationary phase for all analyses: Prontosil, 3 μm.
28 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY APRIL 2016 www.chromatographyonline.com
Ronald E. Majors
and Linda L. Lloyd
Characterizing SEC Columns for the Investigation of Higher-Order Monoclonal Antibody Aggregates
With many new biopharmaceuticals now being developed, robust
analytical methods are needed to ensure that these protein-based
drugs are of high purity and safe with a minimum amount of
side effects. Size-exclusion chromatography (SEC) is an important
technique for investigating purity and is useful to identify and monitor
protein aggregation, which can have economic and immunogenicity
effects. This article discusses those column parameters that are most
important in the selection of the optimum phase for SEC separations.
The development of biological-based
pharmaceuticals is growing. In 2012,
of the top selling 200 drug products
in the United States 25% were based on a
biological entity (1). It is anticipated that
by 2020 52% of all top selling drugs will
fit into this category (2). These continuing
trends will have strong implications for the
analytical techniques used to characterize
these large-molecule products. Chromato-
graphic separations will still play a key role
not only in the purification of these biolog-
ics, but also in the analysis from the early
phases of product development to the final
quality control of formulations. Continued
improvements in liquid chromatography
(LC) column materials to cope with higher-
molecular-weight biopharmaceuticals
will be needed. Many of the attributes for
optimized chromatographic packings that
have been developed for small-molecule
drugs will not always directly extrapolate
to those needed for these biological-based
drugs. For example, LC separations requir-
ing nondenaturing conditions will not tol-
erate high concentrations of organic mobile
phases or, when LC coupled to mass spec-
trometry (MS) is used, high amounts of
nonvolatile salt buffers. New workflows
may be required to ensure that the analysis
conditions do not cause degradation of sen-
sitive biomolecules. The complexity of new
biological drugs may require much greater
levels of resolution than was required for
well characterized small-molecule drugs.
Two-dimensional (2D) chromatographic
separations may become the norm for some
of these drugs, especially when biosimilars
are undergoing characterization.
Monoclonal Antibodies
and Aggregation
Monoclonal antibodies (mAbs) are in favor
since they are highly specific and often
bind to a single antigen target. The cellular
processes to produce mAbs are complex,
however, and multistep purification pro-
cedures subject the protein to numerous
changes in their environment. Like many
recombinant proteins that are inherently
unstable, the increased degree of handling
of the mAbs may cause conformational
changes and increased levels of aggrega-
tion with the possibility of visible precipi-
tation and invisible soluble aggregates. At
the molecular level, the process of mAb
aggregation is complex with a possible loss
of its three-dimensional (3D) structure by
interacting with other protein molecules.
Aggregation can be reversible or irreversible
APRIL 2016 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY 29www.chromatographyonline.com
and, in some cases, the protein can become
irreversibly denatured thereby losing its
bioactivity. There are many mechanical
stress and chemical conditions that can
cause or change aggregation including
storage, interactions with surfaces or solids,
flow or agitation, and temperature changes.
An earlier paper (3) provided more details
of the upstream and downstream processes
that can affect mAb aggregation including
the method of analysis.
The impact of aggregation on the
process economics (product yield), effi-
cacy (decreased bioactivity), and immu-
nogenicity (recipient immune system
response) are considerable, thus, reliable
and accurate methods of analysis and
quantitation are required. Although there
are a number of traditional methods
commonly used to measure aggregation
(see Figure 1), the technique of size-exclu-
sion chromatography (SEC) is a required
technique for soluble aggregation analysis
and quantitation.
Size-Exclusion Chromatography
Unlike all other modes of high perfor-
mance liquid chromatography (HPLC),
pure SEC involves absolutely no interac-
tion between the analyte and the pack-
ing material. The molecules are separated
based on differences of size in solution,
their hydrodynamic volumes. Figure 2
shows a schematic of the differential flow
paths as a function of molecular size along
with the associated chromatogram. The
SEC packing material consists of neutral,
porous, spherical particles with a defined
pore size. The “fit” of the molecule into
the porous structure will determine its
residence time inside of the packing. The
largest molecule, depicted in green in Fig-
ure 2, will not permeate very far into the
pore, if at all, and it will move down the
packed bed virtually unretained and will
be eluted first from the column. The blue
molecule, being smaller in size, will per-
meate further into the pore, spend more
time inside the packing, and will be eluted
from the column after the largest green
molecule. The red molecule, being the
smallest in size, will permeate well into the
porous packing and spend the most resi-
dence time there and will be eluted after
the blue molecule. Thus, as depicted in
the chromatogram of the right hand side
of Figure 2, the order of elution is green
(large), blue (intermediate), and red (small).
The pore size of an SEC column will
define the molecular sizes that can be
resolved—anything that is bigger than
the pore opening will be excluded and all
molecules equal to or larger than the pore
will be eluted at the exclusion volume (Ve),
sometimes referred to as the interstitial vol-
ume (Vi), of the column and the smallest
molecules that permeate all of the pore vol-
ume will be eluted at the total permeation
volume (V0). These two volumes define
the elution volume–resolving range of the
column and all separation must take place
within these two volumes. Thus, SEC is
quite different from the other LC modes
that can have the separation take place over
Dynamic light scattering
Static light scattering
Aggregates Particles
AUC
SEC Microscope
Counter principle
Flow imaging microscopy
Light microscopy
Monomersoligomers
Visible particlesSubvisible particles
10 100 10 100 10 100mmnm μm cm
Visual inspection
FFF-MALS
Figure 1: Classical techniques for aggregate determination—SEC is used for the quantitation of the soluble aggregates that are typically less than 80 nm in size.
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Shiseido
HPLC Columns
30 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY APRIL 2016 www.chromatographyonline.com
many column volumes. Also, for SEC, the
elution order is unlike other LC modes
such as reversed-phase chromatography
where the larger, more-hydrophobic mol-
ecules are eluted last and the smaller, more-
hydrophilic molecules are eluted first.
How is the SEC data used? Most fre-
quently, a calibration curve is first generated
(see Figure 3). In this plot, the log of the
molecular weight (MW) of known protein–
peptide standards are plotted versus reten-
tion time (or elution volume). In Figure 3,
a protein–peptide standard mix consisting
of various known molecular weight com-
pounds was used to make up this plot. The
proteins, peptides, and their respective MWs
are identified in the figure caption. The cali-
bration plots are generally-fitted to a polyno-
mial. Because the SEC separation is based on
the hydrodynamic volume of a molecule in
solution and not solely on MW, any extrapo-
lated MW is referred to as apparent MW. In
this example, an unknown sample solution
of protein containing a small amount of
its dimer was injected onto a modern SEC
column containing a packing with a 300-Å
pore size. By noting the retention time on
the calibration curve of 8.6 min, the appar-
ent MW of the main compound was deter-
mined to be 18.4 kDa, which coincides with
the MW of β-lactoglobulin. The small peak
eluted just before the major peak has a reten-
tion time of 8.3 min, which from the calibra-
tion curve is determined to have an apparent
MW of 37 kD and thus was estimated to
be the β-lactoglobulin dimer. By measuring
the relative peak areas one could estimate the
level of dimer in the original solution. For
an absolute MW, another method beyond
ultraviolet (UV) or refractive index detec-
tion must be used. Most often a light scatter-
ing (LS) detector is used to provide absolute
MW and also provides an increased sensitiv-
ity for the higher MW aggregates. A mass
spectrometer can also be used to measure an
absolute MW and provide structural identi-
fication for unknown impurity peaks.
Characterization of SEC
Columns for mAb Analysis
Now that we have introduced the concept
of SEC and how the separation mode can
be used to separate biomacromolecles and
higher order earlier eluted aggregates, we
would like to look at those characteristics
of packed SEC columns that can be used
to optimize their ability to provide the best
resolution of mAb monomers from higher
order aggregates in the shortest possible time.
The overall desire is for the SEC column to
deliver accurate separation and precise quan-
titation. A typical SEC column has notable
parameters that define its separation charac-
teristics, some unique to the SEC mode and
some that are well known chromatographic
principles. Table I provides typical column
parameters that are useful for comparison
with advantages and disadvantages listed.
Out of this large number of parameters, we
shall now look at the more important ones
Retention time (min)
Log
(M
W)
4.002.00
2.50
3.00
3.50
4.00
4.50
5.00
5.50
6.00
6.50
5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
Unknown is β-lactoglobulinMW 18.4 kD
β-Lactoglobulin dimer, apparentMW 37 kD
Ovalbumin, 44.3 kD
8.24
1
7.84
2
10.0
25
Myoglobin, 17 kD
Figure 3: Determination of equivalent molecular weight of an unknown protein. A calibration curve is constructed using proteins and peptides of known molecu-lar weight and as small molecule, uracil. By plotting the retention time against mo-lecular weight the polynominal fit equation can be used to calculate the equiva-lent molecular weight from the retention time of the unknown. Column: 300 mm × 7.8 mm, 2.7-μm dp AdvanceBio SEC, 300 Å (Agilent Technologies); eluent: 150 mM sodium phosphate, pH 7.0; flow rate: 1.0 mL/min. Molecules for calibration, left to right: thyroglobulin dimer (Ve marker), 1340 kDa; thyroglobulin, 670 kDa; IgG dimer, 300 kDa; IgG, 150 kDa; ovalbumin dimer, 88.6 kDa; ovalbumin, 44.3 kDa; myoglobin, 17 kDa; aprotinin, 6.5 kDa; neurotensin, 1.7 kDa; angiotensin II, 1.05 kDa; uridine (V0
marker) 0.24 kDa.
Figure 2: Mechanism of SEC in the separation of different sizes of molecules. Mol-ecules can permeate the pores of the stationary phase to different extents depending on their size in solution. The largest molecules (green circles) cannot permeate the pores and are eluted first, the small molecules (red circles) can permeate all of the pore structure and are eluted last. Molecules that have a size between these two (blue circles) will partially permeate the pores and will be eluted between these two limits.
APRIL 2016 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY 31www.chromatographyonline.com
Table I: Important parameters in the characterization of an SEC column
Column
ParameterInfluences Advantages Disadvantages Comments
Particle pore size
Defines molecular sizes that can be resolved (separa-tion range)
Large pore sizes allow sepa-ration of larger macromol-ecules; small pore sizes for smaller biomolecules
One pore size column may not resolve both large and small biomolecules in same sample; may require columns in series with different pore sizes
Calibration curves provide guidance for separation range of SEC column; typical pore sizes for SEC are 100, 200, 300, 450, and 500 Å
Pore size distribution (PSD)
Separating range Narrow PSD columns will pro-vide higher resolution over a narrow range of molecular sizes
Narrow PSD will provide lower resolution over a wide range of molecular sizes
The alternative wider PSD will provide wider fractionation range but calibration curve will have a steeper slow
Pore volume of packing
Resolution Larger pore volume extends useful range of calibration curve giving higher resolu-tion and accuracy
Small pore volume may not allow resolution of close molecular sizes
Difficult to make stable silica-based particles with large pore volume
Pore volume of column
Resolution Longer columns or multiple columns extend separation range by increasing total pore volume
Additional columns slow down separation and in-crease the cost
Multipore columns have been developed but single multipore columns have limited resolution
Particle size Column efficiency Smaller particles provide narrower peaks and there-fore better resolution and better sensitivity than larger particles
Smaller particles give rise to higher back pressure, may generate frictional heating; may require lower band dis-persion and higher pressure rated instrument to get full efficiency gains
Small particles used with high flow rates may induce shear degradation of large biomolecules and are more likely to clog with large mol-ecules; typical particle sizes are 1.7, 2.7, 5, and 10 μm
Column length Resolution Longer or multiple columns give better resolution
Longer or multiple columns give longer analysis times, greater pressure drop and cost more
Typical modern SEC columns are 150 or 300 mm in length
Column internal diameter
Speed and sensitivity
Narrow internal diameter columns have greater sensi-tivity and are suitable for use with MS detection
Wide internal diameter columns are more robust and less impacted by instrument dispersion; larger sample capacity for LS detectors
Typical modern column internal diameters are 4.6 and 7.8 mm
Nonspecific interactions
Resolution, sensitivity
No significant advantages for pure SEC size separations
May cause peak tailing, peak loss, low recovery, peak elution outside of operating range of SEC column, and loss of sensitivity
Overall quantitation, accura-cy, and reproducibility is af-fected; surface deactivation procedure with hydrophilic properties is paramount
Flow rate Speed and efficiency
High flow rates decrease analysis time, may affect ef-ficiency and raise pressure
Low flow rates increase anal-ysis time, increase efficiency, and lower pressure
Compromise must be made just like any chromatograph-ic experiment
Particle stability Column lifetime and performance
Robust silica-based particles stand up to UHPLC conditions and allow higher pressure operation
Unstable particles create voids, give higher back pres-sure as they break down, and create problems with LS and MS detectors.
Modern particles are gener-ally engineered to withstand UHPLC conditions; older particles may not handle as well
Column stability
Replacement costs Longer lifetime and higher number of injections result in overall savings; allow higher flow rates and pressures
Long lifetimes present no disadvantages as long as separation persists
Modern HPLC and UHPLC columns should provide a minimum of 1000 injections, often more with good labo-ratory practice
Batch-to-batch and column-to-column repro-ducibility
Data reproducibil-ity and quantita-tion
Reproducible batches of packings and packed col-umns provide data integrity and eliminate unnecessary revalidation
Nonreproducibility of pack-ing and columns provides nonrugged methods and lots of rework
Manufacturers should ensure that their products meet the performance needs of their customers.
32 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY APRIL 2016 www.chromatographyonline.com
and see if these parameters can be tested to
meet the separation requirements.
Effect of Pore Size on SEC Resolution
One must select the proper pore size to
allow adequate resolution for molecules
of interest. Figure 4 shows the results of
a calibration curve of the standard pro-
tein mixture on two different columns
with the same dimensions but with dif-
ferent pore sizes, 130 Å and 300 Å. The
molecular weights of the compounds in
the protein–peptide test mixture goes
from the thyroglobulin dimer (MW
1340 kDa) to the V0 marker uridine (MW
0.24 kDa). For the smaller-pore-size col-
umn, the largest molecules IgG dimer,
thyroglobulin, and the thyroglobulin
dimer are totally excluded from all the
pores of the packing and are eluted in a
single volume (Figure 4a). Other more
moderate sized proteins and dimers are
separated nicely on this column. For
the larger-pore-size column (300 Å), the
entire range of proteins and peptides can
be adequately resolved and it would be
the column of choice if a large range of
proteins and peptides were encountered
(Figure 4b). In addition, the larger-pore-
size column also possesses a larger pore
volume, which allows for better resolution
throughout the chromatogram. Some-
times one can achieve improvements in
resolving range by coupling two columns
with different pore sizes in series—say a
200-Å column and a 450-Å column—but
run times are increased as is added expense
in purchasing two columns instead of one.
For increased resolving power, one can
also add additional columns of the same
Time (min)
Time (min)Ab
so
rban
ce (
mA
U)
Ab
so
rban
ce (
mA
U)
Monomer
DimerTrimerHigher orderaggregates
Mobile phase: 150 mM sodium phosphate, pH 7.0Flow rate: 0.5mL/minSample: lgG 19640
; 1.0mL/min ; 1.5mL/min
140
120
100
80
60
40
20
0
140
120
100
80
60
40
20
0
0 2 4 6 8 10 12 14
2 4 6 8 10 12 14
Figure 6: Further increasing the speed of analysis by increasing flow rate when using a 150-mm-long column. Inset shows all three chromatograms overlaid indicating no or little loss in resolution with flow rate.
300 mm x 4.6 mm
Time (min)
Ab
so
rban
ce (
mA
U)
Flow rate: 0.35 mL/minInjection volume: 2 μL
300 mm x 7.8 mm
Flow rate: 1.0 mL/minInjection volume: 6 μL
200
175
150
125
100
75
50
25
0
Ab
so
rban
ce (
mA
U) 200
175
150
125
100
75
50
25
2 4 6 8 10 12 14
2 4 6 8 10 12 14
0
Rs 1.82
Rs 2.12
Rs 1.91
Rs 2.23
Figure 5: Separation of protein standards on 7.8-mm and 4.6-mm i.d. columns. Upper chro-matogram: 300 mm × 4.6 mm, 2.7-μm dp AdvanceBio SEC 300 Å, 0.35 mL/min. Lower chro-matogram: 300 mm × 7.8 mm, 2.7-μm dp AdvanceBio SEC 300 Å, 1.0 mL/min. Eluent: 150 mM sodium phosphate, pH 7.0.
Figure 4: Example calibration curves for 130-Å and 300-Å pore size SEC columns: (a) 300 mm × 7.8 mm, 2.7-μm dp AdvanceBio SEC 130 Å; (b) 300 mm × 7.8 mm, 2.7-μm dp AdvanceBio SEC 300 Å. Eluent: 150 mM sodium phosphate, pH 7.0; flow rate: 1.0 mL/min. Compounds used to construct calibration curve are the same as in Figure 3.
Retention time (min)
4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
Thyroglobulin
lgG dimer
dimer
Ovalbumin
Ovalbumin
Myoglobin
Aprotinin
Neurotensin
Angiotensin II
Uridine
lgG
Thyroglobulindimer
Log
(M
W)
2.00
2.50
3.00
3.50
4.00
4.50
5.00
5.50
6.00
6.50(a)
Retention time (min)
4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
Thyroglobulin
lgG dimer
dimerOvalbumin
Ovalbumin
Myoglobin
Aprotinin
NeurotensinAngiotensin II
Uridine
lgG
Thyroglobulindimer
(b)
Log
(M
W)
2.00
2.50
3.00
3.50
4.00
4.50
5.00
5.50
6.00
6.50
APRIL 2016 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY 33www.chromatographyonline.com
pore size to increase the total pore volume
and hence resolution between peaks.
Effect of Column Dimensions
and Flow Rate on SEC Separations
In recent years, faster separations became
the name of the game. As the number of
samples increase and laboratory personnel
are pushed for higher productivity, every-
body wants to do things faster. In the past,
SEC columns were considered somewhat
fragile, especially when the soft gels were
used in low-pressure columns. Most labo-
ratories practicing HPLC and ultrahigh-
pressure liquid chromatography (UHPLC)
have high-pressure systems available that
can achieve fast separations in a matter of
minutes. Although SEC has some limita-
tions of column dimensions (smaller col-
umn lengths and volumes mean lower reso-
lution because of decreased pore volume
availability), there has been a tendency to
shift from the standard 7.8-mm i.d. SEC
columns to those diameters more popular
in HPLC, such as 4.6 mm. Figure 5 shows
a separation of protein standards on 300-Å
columns 300 mm in length, but with 7.8-
mm and 4.6-mm internal diameters. The
7.8-mm column run at 1.0 mL/min gave
a separation time of just under 12 min as
did the 4.6-mm column run at same lin-
ear velocity (0.35 mL/min). Compared to
the 7.8-mm i.d. column, the overall resolu-
tion for the 4.6-mm i.d. column was barely
impacted for these proteins. However, the
amount of injected sample required for a
4.6-mm i.d. column is smaller so in sample-
limited situations, a 4.6-mm i.d. column
would be preferred. The injected volume
is adjusted downward based on the inverse
square of the column radii. In addition, a
lower flow rate for the 4.6-mm i.d. col-
umn saves mobile phase. For applications
requiring the use of less-sensitive detectors
including light scattering and refractive
index detectors and longer UV detector
wavelengths (when using mobile-phase elu-
ents that have a high background at lower
wavelengths, for example), then 7.8-mm
i.d. columns offer the capability to handle
much larger sample volumes.
Newer SEC packings that are more rigid
and robust can withstand higher operating
pressures. Thus, separation times can be
shortened even further by using higher flow
rates. Figure 6 shows results using a 4.6-mm
i.d. column with an even shorter column
length of 150 mm, which in itself allows for
a decrease of 50% of the run time observed
with the popular 300 mm columns. A series
of chromatograms of an IgG sample con-
taining dimers, trimers, and higher order
aggregates was generated at three flow rates:
0.5, 1.0, and 1.5 mL/min; the total run
times were determined to be 12, 6, and 3
min, respectively. The inset chromatogram
included in Figure 6 shows that all three
chromatograms—when normalized for
time and aligned—gave virtually complete
overlap without any sacrifice in resolution.
Thus, an increase in sample throughput of a
factor of three was achieved while the chro-
1
1
1
1
2
2
2
2
2
2
2
2
1
1
1
1
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
6
6
6
6
6
6
6
6
750
500
250
0
5 10 15
750
500
250
0
Vendor A, 5 μm, 250 A
Time (min)
5 10 15Time (min)
5 10 15Time (min)
5 10 15Time (min)
5 10 15Time (min)
5 10 15Time (min)
4 62 8 10 12 14Time (min)
4 62 8 10 12 14Time (min)
Vendor A, 4 μm, 250 A
Vendor B, 3.5 μm, 200 A
Vendor B, 3.5 μm, 450 A
Vendor A, 3 μm, 300 A
Vendor C, 2.7 μm, 300 A
Vendor B, 2.5 μm, 450 A
Vendor B, 1.7 μm, 200 A
Ab
sorb
an
ce (
mA
U)
Ab
sorb
an
ce (
mA
U)
750
500
250
0Ab
sorb
an
ce (
mA
U)
750
500
250
0Ab
sorb
an
ce (
mA
U)
750
500
250
0Ab
sorb
an
ce (
mA
U)
750
500
250
0Ab
sorb
an
ce (
mA
U)
750
500
250
0Ab
sorb
an
ce (
mA
U)
750
500
250
0Ab
sorb
an
ce (
mA
U)
Figure 7: Comparison of various commercial SEC columns of varying particle size and pore size. The sample consists of the same standards used in Figure 5.
34 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY APRIL 2016 www.chromatographyonline.com
matographic resolution was maintained.
Particle Size of SEC Packing
As with any form of chromatography, the
particle size is an important parameter. In
aqueous SEC, sometimes referred to as gel
filtration chromatography, original particles
were quite large (in the tens of micrometers)
and quite soft (for example, polydextran and
agarose). Modern SEC packings are closer
to the range of other HPLC packings, with
5 μm having been the standard diameter for
many years. More recently, particles in the
3-μm range have become more popular and
even a few sub-2-μm packings have been
introduced. The larger-pore silica-based
SEC packings (>300 Å) become more frag-
ile and sub-2-μm particles generate too high
of a pressure drop for long-term stability, so
most manufacturers have settled on particle
diameters of 2.5–3 μm for these products.
Of course, in SEC, particle size is only
part of the equation. The pore size comes
into play to a greater extent than in other
LC modes. This can be clearly seen in Fig-
ure 7 where several popular commercial
products of different particle sizes and pore
sizes are compared. The sample was a stan-
dard protein mixture. Figure 7 is organized
by the largest particle size at the top and
the smallest currently available particle size
at the bottom. The pore sizes are shown
next to each chromatogram. The column
dimensions were 300 mm × 7.8 mm,
except for the two bottom chromatograms
that were obtained using columns with
smaller internal diameters, 4.6 mm, which
are especially designed for more-sensitive
methods. Although, the particle size of the
smallest SEC packing is 1.7 μm, the pore
size (200 Å) is not sufficiently large enough
to resolve the thyroglobulin dimer from
thyroglobulin and thus for the purposes of
this study, a larger-pore-size column would
be required. To resolve the monomer and
dimer, one would have to resort to a larger-
pore-size packing (300 Å or 450 Å) with
a larger particle size. It is readily apparent,
as one scans down the figure for the vari-
ous columns, particle size appears to have
a minimal influence of resolution for this
test mix while pore size is more influential.
Batch-to-Batch and
Column-to-Column Reproducibility
For validated methods, it is imperative that
each batch of column packing behave like
its predecessors. As part of any ruggedness
test protocol, most biochromatographers
are required to investigate multiple batches
(at least three) and multiple columns to
ensure that the method can be reproduced
over a long period of time. Figures 8 and
9 show four chromatograms indicating the
reproducibility of four batches of manufac-
tured material. Batches were tested with a
standard protein mix (Figure 8) as well as
a test of the target analytes that are higher-
order aggregates from the monomeric mAb
(Figure 9). The resolution of the myoglobin–
ovalbumin pair was used for batch-to-batch
comparison (Figure 8), while the resolution
of the mAb dimer and mAb monomer was
used for the target analyte test (Figure 9).
Rather than testing each column with a
series of proteins and mAb aggregate sam-
ples, for quality control purposes, an inert
small molecule is used to ensure that the
column is packed according to specification.
Therefore, users can be assured that the col-
umn that is received has not seen any pro-
tein sample. In addition, to prevent any pos-
sibility of bacterial growth during shipping
or storage, most SEC columns are shipped
and stored in a solvent such as a 0.02%
sodium azide or a solvent rich in organic
solvent. Before use, columns from any ven-
dor should be thoroughly rinsed with the
mobile phase that will be used for SEC.
Particle, Phase, and Column Stability
SEC columns are expensive, and all precau-
tions taken with any HPLC or UHPLC
column should also be observed with SEC
columns. Most of the aqueous SEC columns
used for protein–peptide size separations are
based on spherical silica gel, which has been
produced by any number of synthesis pro-
cedures. Silica gel is a more rugged packing
than the soft gels of yesteryear, but neverthe-
less does require some care in its use. SEC
columns do have defined pH limits, upper
pressure limits, upper temperature limits,
and so on—the biochromatographer should
be familiar with these attributes before use.
To cut down or eliminate nonspecific surface
Ab
sorb
an
ce (
mA
U)
150
Batch 6273369
Rs = 2.12
Batch 6273380
Rs = 2.17
Batch 6279525
Rs = 2.12
Batch 6277528
Rs = 2.12
Ovalbumin Myoglobin
100
50
0
Ab
sorb
an
ce (
mA
U)
150
100
50
0
Ab
sorb
an
ce (
mA
U)
150
100
50
0
Ab
sorb
an
ce (
mA
U)
150
100
50
0
2 4
4.6
30
5.1
07
4.6
04
4.5
76
4.9
81
5.4
63
6.3
02
7.0
29
7.9
13
8.5
62
11.1
65
11.3
228
.878
8.2
50
6.6
64
7.3
78
5.7
84
5.2
60
4.7
38
5.0
82
5.5
74
6.4
33
7.1
53
8.0
34
8.6
72
11
.21
2
5.6
23
6.4
98
7.2
28
8.4
16
8.7
62
11
.32
3
6 8 10 12 14Time (min)
2 4 6 8 10 12 14Time (min)
2 4 6 8 10 12 14Time (min)
2 4 6 8 10 12 14Time (min)
Figure 8: Batch-to-batch reproducibility of SEC columns for protein standards. Col-umn: 300 mm × 7.8 mm, 2.7-μm dp AdvanceBio SEC 300 Å; mobile phase: 150 mM so-dium phosphate, pH 7.0; flow rate: 1.0 mL/min. Protein standards: 1 = thyroglobu-lin dimer, 2 = thyroglobulin, 3 = IgA, 4 = IgG, 5 = ovalbumin dimer, 6 = ovalbumin, 7 = myoglobin, and 8 = vitamin B12 (marker).
APRIL 2016 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY 35www.chromatographyonline.com
interactions, silica gel SEC columns require
some surface deactivation by bonding, coat-
ing, or building into the phase strong hydro-
philic characteristics. The bonding of the
diol functionality appears to be the favored
approach to provide a hydrophilic surface,
but newer approaches such as bonding with
a hydrophilic polymer may prove to be more
successful. If proteins are allowed to interact
with the silica surface, sample integrity may
be compromised. Tailing, irreversible adsorp-
tion with subsequent low recovery and non-
reproducible separations are signs of possible
nonspecific interactions. Repetitive injections
of a test protein sample should be performed.
Peak areas should be reproducible with less
than a 1% relative standard deviation.
Column lifetime is another parameter of
great interest. Besides the expense of replac-
ing a dead column, the time to re-equilibrate
and recalibrate the column and running
necessary blanks should also be taken into
account. Modern SEC columns, if properly
treated, should provide at least 1000 injec-
tions. Lifetimes can be even further extended
by the use of guard columns, which are
a lot less expensive to replace and, as long
as connections are minimized to prevent
band broadening, should have no effect
on the separation. To test an SEC column,
a series of 1200 injections of a mAb con-
taining dimers and higher order aggregates
(depicted as aggs in the figure) was made over
a period of 10 days. Although not shown, the
peaks were well resolved and the resolution
of the monomer–dimer changed about 3%
over the time period. Figure 10 shows that
the quantitation for monomer, dimer, and
aggregates was still reproducible after 1200
injections, and the quantitation is consistent
over the lifetime of the column.
Application of Optimized SEC
Column to a Stressed Monoclonal
Antibody and Biosimilar—A Typical
Biopharma Application
To test an SEC column on a real sample,
the innovator drug rituximab, a medica-
tion to treat non-Hodgkin’s lymphoma or
chronic lymphocytic leukemia and the first
monoclonal approved by the United States
Food and Drug Administration (FDA)
in 1997, and a biosimilar were subjected
to forced degradation studies. The result-
ing breakdown products were separated by
SEC. Samples of the mAbs were prepared
by first diluting them in mobile phase and
then a pH stress test was performed by add-
ing hydrochloric acid to the sample solu-
tions to adjust the pH to 1.0, then adding
sodium hydroxide to adjust the pH to 10.0,
and finally getting the pH back to 6.0 by
the addition of hydrochloric acid (4). The
resulting solution was incubated at 60 °C for
Mo
no
mer
are
a %
Ag
gs
an
d d
imer
are
a %
100.0 30.0
25.0
20.0
15.0
10.0
5.0
0.0
95.0
90.0
85.0
80.0
75.0
70.0
65.0
60.0200 400 600 800 1000 12000
Injection number
Sample changed
Monomer% Aggs% Dimer%
Figure 10: Column lifetime study of mAb and its dimer and higher order aggregates. In this study, a use-case scenario was simulated by running a series of mAb samples with a protein standard mix and a small molecule before and after each mAbs se-quence. After each sequence was completed the flow was stopped before starting the next sequence. Column: 300 mm × 4.6 mm, 2.7-μm dp AdvanceBio SEC 300 Å; mo-bile phase: 150 mM sodium phosphate, pH 7; flow rate: 0.35 mL/min.
Ab
sorb
an
ce (
mA
U) 80
60
40
20
0
Ab
sorb
an
ce (
mA
U) 80
60
40
20
0
Ab
sorb
an
ce (
mA
U) 80
60
40
20
0
Ab
sorb
an
ce (
mA
U) 80
60
40
20
0
4.5
89
4.5
56
5.1
51
5.5
82
6.4
36
7.7
72
11
.80
911.8
34
11.8
53
7.9
97
6.6
66
5.7
98
5.3
50
4.6
98
7.6
41
6.3
04
5.4
74
5.0
62
4.5
43
5.1
98
5.6
29
6.4
92
7.8
47
11
.90
8
Batch 6273369
Rs = 1.92
Batch 6273380
Rs = 1.99
Batch 6279525
Rs = 1.90
Batch 6277528
Rs = 1.96
mAb dimer mAb monomer
2 4 6 8 10 12 14Time (min)
2 4 6 8 10 12 14Time (min)
2 4 6 8 10 12 14Time (min)
2 4 6 8 10 12 14Time (min)
Figure 9: Batch-to-batch reproducibility of SEC columns for target analytes. Column: 300 mm × 7.8 mm, 2.7-μm dp AdvanceBio SEC 300 Å; mobile phase: 150 mM sodium phosphate, pH 7; flow rate: 1.0 mL/min; sample: mAb and its dimer.
36 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY APRIL 2016 www.chromatographyonline.com
60 min. Figures 11a (innovator drug, red
trace) and 11b (biosimilar drug, red trace)
show the initial profile of each drug deter-
mined by SEC and both were found to give
a single, fairly symmetrical peak showing no
indication of aggregation or degradation.
After the pH and heat-stressed experi-
ments were carried out, the SEC profiles
were dramatically changed. The innovator
drug (Figure 11a, blue trace) showed evi-
dence of aggregate formation as can be seen
by the small higher-MW aggregate peaks
eluted before the monomer. Additional
lower-MW degradation peaks were observed
after the elution of the monomer. For the
rituximab biosimilar (Figure 11b, blue trace)
no evidence of higher-order aggregates was
found but lower-MW fragments could be
observed in the SEC profile. In both cases, a
relative decrease in the main mAb peak was
observed, indicating a molecular breakdown
caused by the stress experiments. More infor-
mation about these drugs and quantitative
results can be found in reference 5. This
series of experiments shows that SEC can
be very helpful in the process of mAb-based
product development especially for the quan-
titation of dimer and higher-order aggregates.
Conclusion
As the shift in pharmaceutical drug devel-
opment toward biological-based entities
continues, HPLC and UHPLC column
technology will have to shift with the mar-
ket demands. Columns that were suited
for small molecules will not necessarily be
useful for the larger biomolecules, and older
biocolumns that have been used for years
may not have the proper characteristics to
meet the demands required for treatment
of newer biopharmaceuticals. In this article,
we have tried to show the important char-
acteristics that impact the performance of
an aqueous SEC column, particularly one
that is suited for the separation and quanti-
tation of a monoclonal antibody and higher
aggregates such as dimers, trimers, and other
high-molecular-weight species. Some of the
characteristics are familiar chromatographic
principles (such as column length, particle
size, and flow rate) but others are unique to
SEC (for example, pore size, pore volume,
and nonspecific interactions). It is anticipated
that further developments in SEC columns
for biomolecules will come about in future
years with further research and development
for smaller particles and tuned inert porous
surfaces underway. It should be noted that,
because of the fixed retention mechanism
of SEC, a single column and mobile phase
can be used for multiple types of samples
requiring a size separation including frag-
ment analysis, separation of antibody-drug
conjugates, PEGylated proteins, and general
protein and peptide separations.
References
(1) http://cbc.arizona.edu/njardarson/group/top-
pharmaceuticals-poster.
(2) World Preview Outlook to 2020, EvaluatePha-
rma (2014).
(3) L. Lloyd, LCGC North Am. 32(s4), 30–35
(2014).
(4) B. Basak Kukrer, V. Filipe, E. van Duijn, P.T.
Klasper, R.J. Vreeken, A.J.R. Heck, and W. Jis-
koot, Pharm. Res. 27, 2197–2204 (2010).
(5) M.S. Palaniswamy, “Separate and Quantify
Rituximab Aggregates and Fragments with
High-Resolution SEC,” Agilent Technologies,
Application Note 5991-6304EN, October, 2015.
Disclaimer
For research use only. Not for diagnostic
purposes. This information is subject to
change without notice.
Ronald E. Majors is Column Editor Emeritus
for LCGC, and an analytical consultant in
West Chester, Pennsylvania. Linda L. Lloyd
is with Agilent Technologies in Church
Stretton, Shropshire, UK. Direct correspon-
dence to: [email protected] ◾
Ab
sorb
an
ce (
mA
U)
Ab
sorb
an
ce (
mA
U)
(a)
(b)
160
140
120
100
80
60
40
20
04 6 8 10 12 14
160
140
120
100
80
60
40
20
0
Monomer8.286
Aggregates
Fragments
10.293 14.494
14.494
14.494
11.985
8.401
8.292
Fragments
Monomer
5.668 6.945
11.990
12.760
Time (min)
42 6 8 10 12 14
Time (min)
Figure 11: Application of SEC to characterize a commercial mAb and its biosimilar: intact and stressed conditions. SEC Chromatograms of (a) intact ribuximab innova-tor (red trace) overlaid with pH and heat-stressed sample (blue trace) and (b) intact rituximab biosimilar (red trace) overlaid with stressed sample (blue trace). Chromato-graphic conditions: Column: 300 mm × 7.8 mm, 2.7-μm dp AdvanceBio SEC 300 Å; mo-bile phase: phosphate buffered saline (PBS), 50 mM sodium phosphate containing 150 mM sodium chloride, pH: 7.4; temperature: ambient; injection volume: 10 μL; flow rate: 0.8 mL/min; detection: UV absorbance at 220 and 280 nm.
APRIL 2016 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY 37www.chromatographyonline.com
Michael J.P. Wright
and Sophie Hepburn
Positive Impacts of HPLC Innovations on Clinical Diagnostic Analysis
The last decade has seen a series of advances in the field of liquid
chromatography that have resulted in improvements for many clinical
diagnostic services. These innovations have included the expansion
of superficially porous particle columns, new or improved stationary
phase options, and user-friendly multiple-channel high performance
liquid chromatography (HPLC) instrument options that allow sequential
analysis—a boon for low- and moderate-throughput laboratories with
limited hardware. As a result, diagnostic services are able to offer faster
turnaround times and measure analytes in patient types and disease
states that were previously problematic. This article presents examples of
the impact these innovations have had in a number of hospital settings.
Chromatographic techniques (includ-
ing liquid, gas, and thin-layer chro-
matography), have been used for
decades in specialist clinical laboratories for
the separation and (semi-) quantitation of
established biomarkers. High performance
liquid chromatography (HPLC) emerged as
the most useful technique in the clinical field
and has been commonplace for biospecimen
analysis since the 1970s (1). Over the last
20 years there has been a shift toward mass
spectrometry (MS) detection rather than
conventional detection methods (ultraviolet
[UV], fluorescence, and electrochemical) (2).
This shift was driven by the uptake of liq-
uid chromatography coupled to tandem MS
(LC–MS-MS) in newborn screening and
therapeutic drug monitoring laboratories
where the advantages of reduced turnaround
times and a simplified workflow were para-
mount. In recent years, LC–MS-MS has
also started to replace gas chromatography
(GC)–MS and immunoassay methods for
vitamins, hormones, and metabolites. This
trend is mainly due to the superior selectiv-
ity and adequate sensitivity of LC–MS-MS
compared to immunoassays (3) and the
higher throughput and capacity compared
to GC–MS. The result is that, today, many
specialist clinical laboratories continue to
use a dwindling number of well-established
GC, GC–MS, HPLC–UV, HPLC–elec-
trochemical detection (ECD), and HPLC–
fluorescence detection (FD) methods and
an ever-increasing number of LC–MS-MS
methods.
Diagnostic clinical chemistry depart-
ments, particularly those within the public
health system, usually receive budgetary
funding on a reimbursement per result basis.
In these laboratories, the majority of sample
analysis is performed on large, fully auto-
mated colorimetric, nephelometric–turbidi-
metric, or ligand binding assay instruments,
which are often acquired from vendors via
reagent rental or cost-per-assay agreements.
This means that the vendor provides the
instrument in exchange for a guaranteed
purchase of reagents per year, or alternatively,
the clinical laboratory must pay the vendor a
specified amount per test processed. These
arrangements work well because they are
suitable for the budget model and remove
the considerable capital outlay of purchasing
equipment. In addition, because the average
contract expires after five years, laboratories
can keep up with technological advances via
the tendering process. This remuneration
scenario is not the norm for chromatogra-
phy and MS equipment vendors supplying
38 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY APRIL 2016 www.chromatographyonline.com
instrumentation to specialist clinical chem-
istry laboratories. As a result, obtaining the
capital funds to purchase LC–MS-MS
instrumentation often requires detailed busi-
ness cases demonstrating cost-effectiveness
to accompany the predicted clinical benefit
of using this technology.
Despite a history spanning more than 40
years, chromatographic challenges are com-
monplace in clinical diagnostic analysis, even
with the improved selectivity offered by MS
detection. Interferences that prevent result
reporting can arise from specific patient
groups because of age, sex, diet, disease states,
and drug regimens. The complexity of sam-
ples from various patient populations can
necessitate more than one chromatographic
method for the analysis of a target biomarker
to enable measurement across all patient sub-
groups and allow for confirmatory testing
when unusual interferents are present. This
need places a great deal of emphasis on sta-
tionary-phase options that explore different
retention mechanisms, and emerging phases
on the market are met with great enthusiasm.
Rapid and Robust Analysis
without the Back Pressure
Many HPLC services provided in specialist
centers employ established assays using older
instrumentation with back-pressure limits
of 5000 psi or less. In the past, the relatively
long chromatographic run times were not
of concern because sample numbers were
low. However, the ever-increasing workload
(typically ≥10% expansion year on year) has
meant that chromatographic run time has
become a limiting factor. Unfortunately,
the costs of replacing instrumentation with
ultrahigh-pressure liquid chromatography
(UHPLC) equipment capable of recogniz-
ing the gains of sub-2-μm dp columns can
be prohibitive.
An important development in column
technology was the emergence of >2-μm
superficially porous silica particles (SPPs)
that can provide increased efficiency with-
out the same back-pressure gains as those
seen with sub-2-μm fully porous particle
columns. For established clinical HPLC
assays, for example, serum vitamins A and
E (HPLC–UV) and urine catecholamines,
metanephrines, and 5-hydroxyindoleace-
tic acid (HPLC-ECD), the introduction
of higher efficiency SPPs enabled the use
of shorter columns to produce very similar
chromatographic separation with greatly
reduced run times and minimal changes
2.5Time (min)
4.0
Resp
on
se (
nA
)R
esp
on
se (
nA
)
10Time (min)
2.5
0
0
3.23
3.87 5.60
9.01
0.93
1.071.48
2.28(b)
(a)1
2 3
4
1
2 3
4
BP = 1810 psi
BP = 1880 psi
50 mm x 2.1 mm, 1.6-μm dp SPP C18
Back
pre
ssu
re (
psi
)
8000
N = 1700
Injection count
N = 2257
N = 1720
7000
6000
5000
4000
3000
2000
1000
00 201 401 601 801 1001 1201 1401 1601 1801 2001 2201 2401 2601 2801 3001
50 mm x 2.1 mm, 1.8-μm dp C18
50 mm x 2.1 mm, 2.7-μm dp SPP C18
Back pressure limit of HPLC instruments in the laboratory
Figure 1: Accelerated chromatography with superficially porous particles provides faster patient sample turn-around times without requiring UHPLC instrumentation: (a) Urine catecholamine screen performed by HPLC–ECD with a 20-μL injection onto a 150 mm × 4.6 mm, 5-μm dp fully porous particle C18 column at a flow rate of 1.2 mL/min; (b) 5 μL of the same sample injected onto a 50 mm × 4.6 mm, 2.7-μm dp SPP C18 column at a flow rate of 1.5 mL/min. Peaks: 1 = noradrenaline, 2 = adrenaline, 3 = DHBA (internal standard), 4 = dopamine.
Figure 2: Back-pressure and robustness advantages of 2.7-μm particle columns ver-sus sub-2-μm columns. Protein precipitated serum samples were injected onto three columns fitted into a column oven. After each batch of 200 sample injections the column was switched to the next in line and the same samples were re-injected. The back pressure was recorded at the beginning of each batch. The red dotted line indi-cates the back-pressure limit of the standard HPLC systems in the laboratory.
APRIL 2016 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY 39www.chromatographyonline.com
in back pressure (Figure 1). In many cases,
this increased the capacity of existing HPLC
instrumentation 3–4-fold. Interestingly, the
main limitation in accelerating the chroma-
tography further is no longer the back pres-
sure, but rather a combination of other fac-
tors including autosampler operating speed,
detector cell volume, and the data collection
rate limit of the detector.
Accelerated chromatography can cer-
tainly aid in improving patient sample turn-
around times; however, ideally it should not
come at the expense of robustness of the
method. System blockages cause delays in
patient result reporting while the instrument
undergoes troubleshooting and repairs. One
trade-off seen with UHPLC systems fitted
with sub-2-μm dp columns, when compared
to a HPLC system fitted with a larger dp col-
umn, is the requirement for cleaner, particu-
late-free mobile phases and more exhaustive
sample preparations before injection to pre-
vent blockages. In a high-throughput clini-
cal laboratory these extra sample preparation
requirements can be an additional burden
on staffing and workflow. SPP columns
with >2-μm particles provide an intermedi-
ate solution.
To assess robustness we performed the fol-
lowing experiment: 100 μL of serum sam-
ples were protein precipitated by the addition
of 25 μL of 0.2 mol/L zinc sulfate followed
by 200 μL of methanol. The resulting solu-
tion was passed through a 0.45-μm 96-well
Multiscreen Solvinert filter plate (Millipore)
before being loaded onto a Nexera series
UHPLC system (Shimadzu) coupled to
an API 6500QTRAP mass spectrometer
(Sciex). The three columns under evalua-
tion were fitted into the column oven using
multiport selection valves. Next, 20 μL of
each prepared sample was injected onto the
column and after each batch of 200 sample
injections the column was switched to the
next in line and the process was repeated.
Mobile-phase A was 0.1% formic acid in
water and B was 0.1% formic acid in metha-
nol. A rapid gradient of 50–100% B was per-
formed over 1 min at 0.4 mL/min, followed
by 100% B at 1 mL/min for 1 min, and then
50% B at 1 mL/min for 0.5 min. The back
pressure was recorded at the beginning of
each batch and column efficiency was deter-
mined by an injection of a system suitabil-
ity test solution containing testosterone. As
expected, the sub-2-μm dp columns initially
generated a higher back pressure than the 2.7
μm dp column; however, the efficiency (N)
was dependent on column type rather than
purely the particle size (Figure 2). Back pres-
sure increased for all columns as the injec-
tion number increased, but this elevation
was more marked in those with sub-2-μm dp
particles. Unlike the UHPLC system used in
this study, a number of the HPLC systems in
the laboratory have a back-pressure limit of
5000 psi, so for this example only the 2.7-μm
dp column would be suitable for all instru-
ments over a large number of injections.
Innovations in
Stationary Phases
The move toward LC–MS-MS analysis
in clinical chemistry has provided many
benefits; however, challenges involving
chromatographic separation of similar com-
pounds remain. With the number of solvents
and additives now limited to those that are
considered “MS friendly” (that is, volatile,
proton donating, or accepting, do not form
unwanted adducts), the selection of station-
ary phase has taken on increased importance
(4). Chromatography is required not only for
the removal of interferents causing ion sup-
pression, particularly salts and phospholipids
present in blood and urine, but also for the
separation of isobaric compounds that share
the mass transitions used for quantitation.
Alkyl-bonded stationary phases have been the
traditional mainstay of clinical HPLC separa-
tions with mobile-phase buffers and ion-pair
reagents providing the additional selectivity
required. As assays move to LC–MS-MS, the
emphasis has turned to emerging stationary
phases that use alternative mechanisms of
retention to separate the analyte–interference
critical pairs.
Serum 25OH vitamin D3 measurement
has seen substantial growth in clinical chem-
istry laboratories over the past 10 years; with
test requests having increased approximately
twofold per year, every year. One of the
challenges presented by the measurement
of this biomarker with LC–MS-MS is sepa-
rating the C3-epimeric forms often found
in samples from infants. The 3-epi-25OH
form of vitamin D3 is thought to be an inac-
tive or possibly a suppressing form of 25OH
vitamin D3 with the general consensus that
it should be separated for measurement of
25OH vitamin D3 in infant patient samples
(5). Because the C3-epimeric forms share
the same precursor–product ion mass spec-
tra they need to be separated before arrival
at the mass spectrometer ion source. Many
LC–MS-MS methods in the literature were
designed for older patients and used C8 or
C18 stationary phases that were unable to
resolve this critical pair. In the past, chro-
matography using cyano stationary phases
were utilized but limited selectivity of the
phase resulted in run times of 18–45 min,
6.0Time (min)
Inte
nsi
ty (
cps)
Time (min)
1.9e5
Inte
nsi
ty (
cps)
2.52
(b)
5.3e4
(a)
25OHD3 3epi -25OHD3
3epi -25OHD3
4.594.84
25OHD3 and
4.0
HO
OH
HO
OH
Figure 3: Serum 25OH vitamin D3 analysis in adults and infants: (a) A rapid on-line solid-phase extraction (SPE) method for 25OH vitamin D3 measurement in serum from adult patients. Serum is protein precipitated and filtered before injection onto a 20 mm × 2 mm, 20-μm dp C8 extraction cartridge followed by elution (reverse elu-tion time point indicated by the dotted line) onto a 50 mm × 2.1 mm, 2.7-μm dp C8 column. This phase does not separate the 3-epi-25OH vitamin D3 form sometimes found in infants. (b) The same on-line SPE method as above but eluted onto a 100 mm × 2.1 mm, 2.7-μm dp pentafluorophenyl phase column resolves the 3-epimers.
40 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY APRIL 2016 www.chromatographyonline.com
which were too long to be feasible for the
volume of test requests (6). The develop-
ment of pentafluorophenyl (PFP) phases
has shown improved selectivity for 25OH
vitamin D3 and 3-epi-25OH vitamin D3,
enabling quantitation of the target analyte
in this patient group (Figure 3), either as a
secondary method for infant samples or, if
time permits, as a front-line analysis for all
samples (7).
Issues with isobaric interferences in clini-
cal LC–MS-MS can be exacerbated when
the compounds are extremely polar and
difficult to separate by most hydrophilic-
interaction chromatography (HILIC) phases.
The measurement of Kreb cycle compounds
demonstrates this scenario where the criti-
cal pairs of isocitrate–citrate and succinate–
methylmalonic acid need to be resolved. The
evolution and diversification of phases that
incorporate an embedded polar group (EPG)
with an alkyl ligand has produced station-
ary phases with a wide range of chromato-
graphic properties that are capable of oper-
ating at 100% aqueous mobile phases (8).
In this example, an EPG stationary phase,
operated under aqueous conditions, enabled
resolution of these critical pairs (Figure 4a).
For the measurement of serum ste-
roids such as cortisol, testosterone, or
17-hydroxyprogesterone, the target analyte
is part of a large group of closely related
endogenous compounds that share a fused
ring system of three cyclohexanes and one
cyclopentane and where isobaric interfer-
ences are common. To further complicate
matters, because of their structural similar-
ity, steroids often have similar fragmenta-
tion patterns and can also be present at sup-
raphysiological concentrations, for example,
when used for treatment. Thus, even for
compounds that have slightly different pre-
cursor mass-to-charge ratios (m/z), the pos-
sibility of interference from naturally occur-
ring isotopes of steroids with smaller m/z
values have to be taken into consideration.
This is particularly true when developing
LC–MS-MS methods for the analysis of
samples from certain patient groups, such
as those with steroidogenesis defects. The
emergence of biphenyl phases introduces
separation mechanisms such as shape selec-
tivity and π-π interactions while providing
a greater amount of hydrophobic retention
than seen with traditional phenyl phases.
The use of a biphenyl phase enables the sep-
aration of common isobaric steroid interfer-
ences, such as those outlined in Figure 4b.
Leveraging the Most
Out of Instrumentation
For small to moderate-sized specialist clini-
cal laboratories (500–5000 patient samples/
week) the LC–MS-MS workflow consists of
numerous applications where small batches
of patient samples are run on a regular
basis (daily or weekly). Often these applica-
tions rely on different mobile and station-
ary phases to achieve the chromatographic
selectivity required. To leverage the capacity
of the LC–MS-MS instruments to achieve
favorable cost-effectiveness, the systems
should run continuously with an automated
process for changing from one method to
another without intervention by staff.
HPLC systems that incorporate selec-
tion-valve configurations allowing multiple
mobile phase and column combinations
to be run simultaneously have existed for
some time in research and assay develop-
ment laboratories. However, because of reli-
ability issues, complicated software, con-
cerns regarding service-support, and a lack
of experienced operators in the laboratories
themselves, these instruments were not
regularly promoted to clinical diagnostic
laboratories. This situation has changed in
recent years with a number of LC–MS-MS
vendors providing simpler instrument set-
ups with numerous on-board mobile phases
and columns easily controlled via instru-
ment software.
Figure 5a illustrates a system where
binary pumps have solvent-selection valves
attached allowing multiple mobile phases
(typically, four or six) to each pump. In our
laboratory, one of the lines running to each
pump is reserved for a “cleaning solvent” of
50% methanol. The column oven houses
two seven-port, six-position selection valves
allowing availability of up to six columns
without system reconfiguration. In this
setup, one column option is sacrificed for a
direct line for use during conditioning steps.
For sequential analysis of multiple assays,
all sample preparation is completed during
normal working hours and the prepared
samples are loaded into the autosampler by
the end of the day. The batches of samples
are submitted together with conditioning
batches introduced between different assays
to run overnight as follows:
t� The first batch is run using Method 1:
column 1 and mobile-phase A1 and B1.
t� A “conditioning batch” is run using
blank samples injected utilizing Gen-
eral Conditioning Method*: direct
line (no column) and mobile-phase A4
and B4 (cleaning solvent)—this step
purges the system with 50% metha-
nol, removing the mobile phases from
Method 1 and thus preventing the pos-
1.65
1.37
Time (min)
Inte
nsit
y (
cp
s)
3.0
1.37
1.01 3.71
3.58
3.71
4.41
2.75
2.352.26
Time (min)
Inte
nsit
y (
cp
s)
6.0
(b)(a)
1
2
3
4
5 6 7
8
9
10 11
Figure 4: Separation of target analytes from isobaric interferences for clinical diagnostic LC–MS analysis using embedded polar group and biphenyl phases: (a) MRM transitions of hydrophilic Kreb cycle metabolites on a 100 mm × 2.1 mm, 2.7-μm dp reversed-phase amide column with isocratic 100% aqueous mobile phase containing 0.4% formic acid; (b) separation of glucocorticoids and sex steroids on a 50 mm × 2.1 mm, 1.7-μm dp biphe-nyl column with mobile-phase A consisting of 0.1% formic acid in water and B consisting of 0.1% formic acid in methanol; a 40–100% B gradient over 4.5 min was used. Peaks: 1 = isocitrate, 2 = citrate, 3 = succinate, 4 = methylmalonic acid, 5 = predniolone, 6 = cortisol, 7 = cortisone, 8 = epi-testosterone, 9 = testosterone, 10 = 17OH-hydroxyprogesterone, 11 = 11-deoxycorticosterone. Note: The MRM transition for peaks 5 and 7 in (b) is de-tecting the naturally occurring M+2 isotope.
APRIL 2016 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY 41www.chromatographyonline.com
sibility of incompatible mobile phases
mixing from two different methods.
t� A second conditioning batch is run with
blank samples injected utilizing Method
2 Conditioning Method*: direct line
(no column) and mobile-phase A2 and
B2 (Method 2 mobile phases)—This
primes the system with the correct
mobile phases for Method 2 in prepara-
tion for the next column to be switched
on-line.
t� The second batch is run using Method
2: column 2 and mobile-phase A2 and
B2.
t� The “conditioning batch” is run again
with blank samples injected using Gen-
eral Conditioning Method*: direct
line (no column) and mobile-phase A4
and B4 (cleaning solvent).
t� A third conditioning batch is run with
blank samples injected using Method 3
Conditioning Method*: direct line (no
column) and mobile-phase A3 and B3 . . .
and so on, with up to five different clini-
cal assay methods running without user
intervention.
t� *The liquid flow during the various
conditioning batches is diverted to waste
immediately before the mass spectrom-
eter to prevent fouling of the ion source.
On-line solid phase extraction (SPE) is
a popular technique in clinical diagnostic
laboratories because of the labor and cost
savings it represents compared to off-line
SPE. Adding multiple on-line SPE stations
to a system (Figure 5b) can be achieved by
the introduction of a second set of pumps,
fitted with solvent selection valves, and a
high-pressure selection valve to direct the
flow from these pumps to the switching
valve fitted with the on-line SPE cartridge.
Again, one mobile-phase channel for each
pump is reserved for a “cleaning solvent” of
50% methanol and one option from the
selection valve is sent to waste for use dur-
ing conditioning steps. As with the example
described previously, general conditioning
and method-specific conditioning batches
are submitted between batches to prepare
the system with the correct mobile phases,
column oven temperatures, and mass spec-
trometer ion source conditions for the subse-
quent sample batch.
A clear advantage in running assays
sequentially on a single system (or even par-
allelism in larger laboratories) is that of opera-
tion time (uptime) allowing laboratories to
provide a 24-h service to users. Priority is
obviously given to urgent tests, which can be
run during the daytime with rapid reporting
of results. However, routine batch tests can
run throughout the night to minimize sam-
ple congestion during working hours where
instruments would be better used for assay
development and improvement processes. It
also allows LC–MS-MS methods to com-
pete with routine immunoassay analyzers
that are common to core testing facilities that
often perform 50+ reactions concurrently.
Conclusion
HPLC continues to have widespread
applications in clinical laboratories, and,
when coupled to MS, it is the preferred
method for the measurement of many
low-concentration endogenous biomark-
ers. The advent of SPP introduced higher
efficiencies on existing HPLC equipment,
enabling the faster run-times required
to match the growing sample numbers
without the need to purchase expensive
UHPLC equipment. In addition, the
introduction and expansion of a greater
variety of stationary phases to the market
has helped to solve many of the chromato-
graphic challenges facing clinical labora-
tories moving to LC–MS-MS technology.
Throughput limitations for LC–MS-MS
platforms have been resolved by multiple-
channel systems that enable programming
of sequential analyses over a 24-h period.
Adding on-line SPE to these platforms
further streamlines the workflow and
introduces cost-savings in a busy clinical
setting. These innovations allow several
assays to run back to back with a variety of
stationary and mobile phases without the
need for expensive, and often unfavorable,
night-shift schedules for highly skilled
staff while also delivering value for money
from expensive LC–MS-MS platforms.
References
(1) C.A. Burtis, J. Chromatogr. 52, 97–106 (1970).
(2) S.K.G. Grebe and R.J. Singh, Clin. Biochem.
Rev. 32, 5–31 (2011).
(3) V.M. Carvalho, J. Chromatogr. B 883–884,
50–58 (2012).
(4) S.R. Needham, P.R. Brown, K. Duff, and D.
Bell, J. Chromatogr. A 869, 159–170 (2000).
(5) A. De La Hunty, A.M. Wallace, S. Gibson,
H. Viljakainen, C. Lamberg-Allardt, and M.
Ashwell, Br. J. Nutr. 104(4), 612–619 (2010).
(6) K.W. Phinney, M. Bedner, S.S. Tai, V.V.
Vamathevan, L.C. Sander, K.E. Sharpless, S.A.
Wise, J.H. Yen, R.L. Schleicher, M. Chaud-
hary-Webb, C.M. Pfeiffer, J.M. Betz, P.M.
Coates, and M.F. Picciano, Anal. Chem. 84(2),
956–962 (2012).
(7) C.R. Aurand, D.S. Bell, and M. Wright, Bio-
analysis 4(22), 2681–2691 (2012).
(8) M.R. Euerby and P. Petersson, J. Chromatogr.
A 1088, 1–15 (2005).
Michael J.P. Wright and Sophie
Hepburn are with the SEALS Depart-
ment of Clinical Chemistry and Endocri-
nology at the Prince of Wales Hospital
in Sydney, Australia. Direct correspon-
dence to: [email protected] or
(b)
(a)
Mass
spectrometer
Mass
spectrometer
Column
oven
Autosampler
Solvent
selection valve
On-line SPE cartridge Two-position divert valve
Six-position selection valve
Analytical column
Autosampler
Pumps 2Waste
Pumps 1
Pumps 1
Column
oven
Figure 5: LC–MS systems designed for automated sequential method transfer: (a) System containing solvent selector valves, allowing multiple solvents to the pumps, and six-position high-pressure selection valves allowing multiple column se-lection; (b) system with a second binary pump, two-position high-pressure divert valves, and another six-position high-pressure selection valve, enabling on-line-SPE to be added to the automated sequential method transfer system.
42 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY APRIL 2016 www.chromatographyonline.com
Denise Wallworth
Latest Advances in Environmental Chiral Applications
This article provides a brief overview of some of the chiral
environmental studies carried out recently that cover the differing
enantiomeric activity of pesticides, their environmental transformation,
and the degradation of pollutants in general. It highlights some of the
recent advances in chiral stationary phases that have enabled higher
efficiency and faster separations than previously seen in this area.
While in earlier years environ-
mental applications concen-
trated mainly on agrochemi-
cals in use, it is now a much broader field
covering not just preferential enantiose-
lective activity, but also the influence of
microbial population on selective degra-
dation. The impact of excess and metab-
olized pharmaceuticals is also widely
studied along with persistent organic
pollutants (POPs), that includes a range
of both pesticides and fluorinated organ-
ics. There is increasing alarm about these
POPs potentially not being fully extracted
in waste-treatment plants, affecting both
human and fish populations. Monitoring
for banned pesticides is also a key activity.
Recent developments in chiral sta-
tionary-phase (CSP) technology have
principally concerned major advances in
smaller particle technology to support
these needs. The result is vastly improved
chiral column efficiency and selectivity
enabling many new applications along
with the potential for more comprehen-
sive multicomponent screening. A signifi-
cant number of applications have used
3-μm particle size versions of both the
immobilized and coated polysaccharide
CSPs to enable much faster separations.
There has been much interest in the utili-
zation of sub-2-μm ultrahigh-pressure liq-
uid chromatography (UHPLC) particles
for chiral separations, mostly on brush
type and cellulosic CSPs. A recent paper
by Gasparrini (1) demonstrated the ben-
efits of both UHPLC and supercritical
fluid chromatography (SFC) of the high
efficiencies obtained by bonding onto
Whelk-O-1 onto both 1.7-μm porous sil-
ica and superficially porous particle (SPP,
core-shell) silica. The next phase of new
CSP development for this year, however,
is very likely to use the recent introduc-
tion of a 1.9-μm monodisperse totally
porous particle (TPP) (2) that appears
to provide new opportunities to increase
CSP efficiencies even further. These par-
ticles, when bonded with C18 exhibited
an extremely low reduced plate height, h,
of 1.7 in narrow-bore (2.1 mm i.d.) col-
umns, extremely low when compared to
classical porous particles. Gasparrini (3)
demonstrated extremely high efficiencies
obtained by bonding teicoplanin to TPPs
and carried out extensive fundamental
studies of the CSP, reporting efficien-
cies of 200,000–250,000 plates/m at the
optimum flow rate. Additionally, Arm-
strong and coworkers (4,5) reported ultra-
fast separations by bonding cyclofructan,
cyclodextrin, and all the macrocyclic
chiral selectors to TPPs. Separations in
seconds were demonstrated that could,
for instance, provide on-line chiral moni-
toring of asymmetric synthesis. The very
significant increase in efficiency should
enable the separation of far more complex
mixes in addition to being used for two
dimensional (2D)-UHPLC for the sepa-
W
APRIL 2016 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY 43www.chromatographyonline.com
ration of multichiral center pesticides in
the near future (4).
Environmental Applications
The fast growth in agrochemicals has
fueled much research into their environ-
mental impact. It is estimated that 28%
of agrochemicals are currently chiral (6),
a growth in part because of advances in
asymmetric synthesis and process scale
simulated moving bed chromatography
that has significantly reduced the com-
mercial cost for multitonnage agricultural
requirements. Despite this, some 24%
of these are applied as a racemate (7),
resulting in the potential release of inac-
tive products into the environment. Of
the $223 billion global pesticide indus-
try, more than 40% of the products are
used in China and the largest proportion
of papers published in the last two years
reflects this, especially where related to
their impact on the important tea produc-
tion industry.
The fate of pesticides in the environ-
ment is expected to be subject to enan-
tioselective biodegradation by microor-
ganisms, possibly in quite a different way
0 2 4 6 8 10 12 14
Cl Cl
Cl
Cl
Cl
Cl
ClCl
Cl
ClClCl
Mirrorα-HCH (peaks 1 and 2)
CCl2
Cl Cl
Cl
Cl
Cl
Cl
cis-Chlordane (peaks 3 and 4)
Time (min)
Figure 1: An example of chiral GC separations of organochlorine pesticides: chlor-dane and α-HCH on Chiraldex G-BP, 10 m × 0.25 mm, at 170 °C with helium as carrier gas. (Taken from G005050, SiAL source.)
HILICpak VG-50
A polymer based tertiary amino columnfor phosphorylated saccharide HILIC analysis
44 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY APRIL 2016 www.chromatographyonline.com
compared to microorganisms present in
water because of residue matrix bind-
ing effects. The differing enantiomeric
activities of chiral pesticides, the effect
of various microorganisms giving differ-
ing modes of degradation, and the result-
ing unbalancing of the microbiological
makeup of the environment are all of
great interest. Pharmacologically active
compounds—drugs, their metabolites,
and illicit drugs—are routinely tested
in wastewater, but could also be present
in solid waste and sludge providing an
additional source of bioavailable uptake.
Overall, there are many reasons for
increased environmental testing.
Chiral Method Development
for Environmental Applications
While chiral separations in environmental
applications have generally not kept pace
with those for pharmaceutical products,
the number of publications has grown
considerably in recent years due, in part,
to these increasing environmental con-
cerns. CSPs used for environmental sepa-
rations need to be capable of separating
relatively polar molecules. Fortunately,
high performance liquid chromatography
(HPLC), gas chromatography (GC), and
SFC have all proven to be useful. SFC was
used for the enantiomeric analysis of the
triazole fungicide flutriafol in vegetables,
fruits, and soils in 3.5 min using a 3-μm
bonded amylose tris(3,5-dimethylphenyl-
carbamate) CSP in carbon dioxide–meth-
anol. Formic acid in methanol was added
post column to enhance mass spectrom-
etry (MS) ionization. Using QuEChERS
(quick, easy, cheap, effective, rugged, and
safe) for sample preparation, this method
provided a limit of quantitation (LOQ)
down to 0.41 μg/kg, making it useful for
both environmental and food analysis
(8). This separation has also been carried
out by LC–MS using a cellulosic tris(3-
chloro-4-methyl phenyl carbamate) phase
in 40:60 (v/v) acetonitrile–water giving
a limit of detection (LOD) of 15 μg/kg
(9). This was found to be a much faster
method than using a cellulosic phase
under normal-phase conditions. Elution
order and configuration were assigned
using electronic circular dichroism (ECD)
and found to be (R)-(-) for the first elut-
ing enantiomer and (S)-(+) for the second.
Linearity and precision was checked in
seven different matrices, in preparation
for future environmental and food studies.
Chiral GC was used for a haloxyfop study
(10,11), separating these herbicides as
their methyl esters using a custom-made
OV170 GC column coated with 15% w/w
permethylated beta cyclodextrin (0.1-μm
film thickness). The levels of organochlo-
rine pesticides in air and surface water in
the Indian Ocean were measured using
chiral GC–MS (12) employing EI detec-
tion in multiple reaction monitoring
(MRM) mode and a 20% tert-butyldi-
methylsilyl-beta-cyclodextrin CSP dis-
solved in 15% phenyl-, 85% methylpoly-
siloxane. Significant decreases in α-HCH
and γ-HCH but increases in p,p′-DDT,
o,p′-DDT and cis- and trans-chlordane
were observed. An example separation of
chlordane and HCH is shown in Figure 1.
For the monitoring of active pharma-
ceuticals in wastewater, a method for the
simultaneous enantioselective determina-
tion of ibuprofen, naproxen, and keto-
profen was developed using LC–MS-MS.
The method used a single-step sample
treatment based on microextraction with
a supramolecular solvent that provided
low method detection limits of 0.5–
1.2 ng/L. This was optimized and the
analytical method validated on a van-
comycin bonded 5-μm CSP (13). The
method was reported as suitable for using
the enantiomeric fraction of ibuprofen as
an indicator of the discharge of untreated
or poorly treated wastewaters. In contrast,
a (nonchromatographic) 14C isotope trac-
ing MS-MS method was used to inves-
tigate the fate of the four isomers of IPP,
a novel, broad spectrum neonicotinoid
insecticide (7). Stereoselective soil bind-
ing and the microbial influence on epi-
mer-selective degradation were reported.
Enantioselective Activity
As is well known for chiral pharmaceu-
tical products in biological systems, if a
pesticide is a chiral molecule, it is com-
mon that one enantiomer carries greater
activity than its pair. A great example is
deltamethrin, where only one of the eight
enantiomers (αS,1R,3R′-) has the desired
insecticidal activity, the other seven being
0
20
40
60
80
100
120
0 1 2 3 4
Rela
tive c
on
ce
ntr
ati
on
(%
)
Incubation time (d)
Sum of enantiomers
R-Ha-acid
S-Ha-acid
Figure 2: Microbial chiral inversion of S-haloxyfop through incubation of rac-haloxy-fop acid in soil. (Reproduced with permission reference 10.)
APRIL 2016 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY 45www.chromatographyonline.com
nonactive or less active. Only one of the
enantiomers of the herbicides dichlorprop
and mecoprop is responsible for their
activity—the (S)-isomer is completely
inactive in each case. Another in this
class is haloxyfop-methyl, a chiral herbi-
cide that was first introduced as racemate
but later replaced by its R-enantiomer,
responsible for the herbicidal action (10).
The first study of the enantioselective
biodegradation and activity of difenocon-
azole was carried out using a coated 5-μm
tris(4-methylbenzoate) cellulosic CSP. It
was used to conclude that the (2R,4S)-
enantiomer would be the better choice
rather than the stereoisomers to maximize
bioactivity and reduce environmental
damage (14). Although there have been
health concerns for acute exposure to
acephate related to its more toxic metabo-
lite, methamidophos (which was banned
in the European Union [EU] in 2015,
although at the time of writing this article
it was still in use in the US), the effect of
chirality had not previously been studied.
Recently, it was found that enantioselec-
tive enrichment depended on soil type
and was shown to be microbially activated
(15). GC–MS-MS was used, employing
a heptakis(2,3-di-O-methyl-6-O-tert-
butyldimethylsilyl)-β-cyclodextrin chiral
GC capillary column. Sample preparation
used a modified QuEChERS method,
followed by drying with anhydrous mag-
nesium sulfate to protect the chiral GC
column. Relative enantioselective bioac-
tivity of the enantiomers of acephate and
methamphos is in this case, however, not
so clear as it appears to depend on the
species it is applied to. This study investi-
gated the different enantioselective degra-
dation rates under various soil conditions
as a possible cause and concluded that dif-
fering microbial populations could play a
significant role.
Enantiomeric Degradation
Environmental biodegradation of chiral
pesticides and herbicides is frequently
enantioselective. As in any guest-host
interaction in biological systems, the
interaction of such molecules with micro-
organisms in the environment is chiral
and can result in differing metabolism
(microbial transformation), causing pos-
sible selective accumulation of one iso-
mer over the other. Many recent studies
provide evidence of such microbial trans-
formation by comparing transformation
profiles in sterile and nonsterile soils.
In the case of haloxyfop and haloxyfop
methyl, a study was carried out using chi-
ral GC–MS employing a custom made
permethylated beta cyclodextrin phase
(OV 1701 with 15% (w/w) permethyl-
β-cyclodextrin and a film thickness of
0.1 μm) (10). Haloxyfop was derivatized
as the ethyl ester to enable simultaneous
separation of haloxyfop and haloxyfop
methyl, and the derivatization procedure
was shown to be nonenantioselective. It
was shown that rapid degradation by
cleavage of the ester group occurred in
three different types of soils studied, but
was not observed in sterile soils, possibly
explained by the presence of microbial
carboxy esterases. Further, chiral inver-
sion occurred, with rapid conversion of
the S-enantiomer to the R-enantiomer
in nonsterile soils (Figure 2), reaching a
steady state when the R-enantiomer level
was about 10 times that of the S-enan-
tiomer. Interestingly, faster inversion was
observed for the acid when originally
applied as haloxyfop methyl.
Individual enantiomers were isolated
for the study using a cellulose tricin-
namate CSP in 95:5:0.1 heptane–isopro-
panol–acetic acid, purifiying 2 mg from
a total of 10 injections (20 min per injec-
tion) and confirmed with >99% enan-
tiomeric purity by chiral GC–MS as the
methyl ester. Analytically, 85:5:10 hep-
tane–isopropanol–methanol provided
a separation in under 10 min with the
same column. If the herbicide is applied
to the soil for root update, then this rapid
interconversion to the active R-enantio-
mer results in independence of herbicidal
activity from the enantiomeric composi-
tion applied. Any difference because of
the mode of application to the growing
plant was also studied, using the same
GC–MS method, and found that, when
applied to the leaves, no interconversion
takes place such that the effect of apply-
ing individual enantiomers directly to the
plant will be very different and only the
R-enantiomer of haloxyfop effective (11).
A newly developed antiviral agent,
dufulin, used widely in China to prevent
disease in rice, tobacco, and vegetables
was found to degrade 6–8 times faster in
nonsterile soils (16,17), providing confir-
mation of its degradation by soil micro-
bial action but in this case without any
chiral inversion. After extraction of the
soil samples with acetonitrile, the chiral
separation was carried out in normal
phase on immobilized amylosic tris(3,5-
dimethylphenylcarbamate). ECD was
used to determine the absolute configu-
rations of the two dufulin enantiomers,
confirmed as the S-(+)-enantiomer for
the first eluting enantiomer, and R-(−)-
enantiomer as the second one.
0 2 4Time (min)
O
F
F
F
NHCH3
Figure 3: Separation of fluoxetine enantiomers on Chirobiotic V2, 10 mm × 2.1 mm, in the polar ionic mode, 13 mM ammonium acetate in methanol. (Adapted with per-mission from Sigma-Aldrich.) (Taken from G004476, SiAL source.)
46 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY APRIL 2016 www.chromatographyonline.com
It is also now established that there
is enantioselective toxicity from many
pharmaceutically active compounds and
illicit drugs to freshwater species, espe-
cially through adsorption on sediments
and suspended solids. For example, S-(+)-
fluoxetine and S-(-)-atenolol significantly
inhibit the growth of a freshwater proto-
zoan, Tetrahymena thermophilia, compared
to the opposite enantiomer (18). This study
aimed to develop a comprehensive screen-
ing protocol for multiresidue identification.
After microwave assisted extraction and
solid-phase extraction (SPE), separations
were carried out on a 5-μm cellobiohydro-
lase CSP in reversed-phase mode for the
amphetamines and, for all other analytes,
on a vancomycin bonded 5-μm silica CSP
(in the polar ionic mode, using methanol,
4 mM ammonium acetate and 0.005% for-
mic acid) (see, for example, Figure 3). This
method was used to investigate stereoselec-
tive effects in sludge treatment processes.
In another study, microbial degradation of
the chiral fungicide, benalaxyl (BX), was
investigated in water, sediment, and water–
sediment environments (19). A separation
of the enantiomers of both the parent com-
pound and its acid metabolite was achieved
using a tris(3,5-dimethylphenylcarbamate)
coated cellulosic CSP in a mobile phase of
n-hexane and 2-propanol (91:9, v/v). Elu-
tion order, determined using a polarimet-
ric detector at 426 nm, was (-)-BX, (+)-BX,
(-)-BX acid, and (+)-BX acid. Sediment
microbial populations were found to be
responsible for enrichment of the more
toxic (+)-enantiomer, causing higher risk
in aquatic environments. Additionally, the
(-)-enantiomer was preferentially degraded,
enriching the presence of the persistent (up
to 70 days) benalaxyl acid, of concern to the
aquatic environment.
Enantioselective Transformation
A study of indoxacarb on immobilized
amylosic tris(3,5-dimethylphenylcarba-
mate) in normal phase reported no inter-
conversion but degradation of each isomer
depended on soil pH and its microbial
activity (20). Many studies have been car-
ried out over the years on polychlorinated
biphenyls (PCBs) and a recent study
looked at the transfer of PCBs 95, 132,
135, and 149 into chickens via soil and
chicken feed (21). The results indicated
enantioselective metabolism, but nonse-
lective maternal transfer to chicks and it
was found that enantiomeric enrichment
of PCBs 95, 132, and 149 and intercon-
version of PCB 135 later occurred in the
chick resulting in different toxicity com-
pared to the adult.
Interestingly, the unexpected appear-
ance of the banned antibiotic chloram-
phenicol in animal feed has, for the first
time, been traced back to its production
naturally by bacterial activity in soils.
Uptake into animal feed crops was stud-
ied by chiral LC–MS using an α1-acid
glycoprotein CSP and found to be related
to its bioavailability (22).
Summary
Stereoselective investigations need to con-
tinue to play a significant role in the study
of the environmental impact of agrochem-
icals, POPs and pharmaceutical products.
Apart from their impact on living organ-
isms, a critical outcome of their presence
is the disruption of the natural microbial
status resulting from stereospecific trans-
formation of these molecules, as well as the
potential for their enantioselective persis-
tence in the environment. The majority of
applications reported used either polysac-
charide CSPs or derivatized cyclodextrin-
based capillary GC columns. Although
there have not been any new developments
for the latter, or for protein-based CSPs,
these phases retain their usefulness in this
area. The advent of smaller particle CSPs
for the polysaccharide CSPs has increased
both speed and selectivity, enabling more
complex and difficult separations to be
developed, while the future of ultraeffi-
cient TPP-based CSPs bonded with a wide
range of chiral selectors is set to transform
chiral HPLC separations yet again.
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Denise Wallworth is with Sigma-
Aldrich UK, a subsidiary of Merck, in
Poole, Dorset, UK.
Direct correspondence to:
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