pg. 3Headspace SPME-GC/MS Analysis of
Terpenes in Hops and Cannabis
Applications Newsletter Volume 34.4
pg. 14LC/MS/MS METHOD FOR DETERMINATION
OF GLYPHOSATE, AMPA, AND GLUFOSINATE IN CEREALS
pg. 26REACHING THE “PEAK” OF RECOVERY:
IMPROVING ANTIBODY SEPARATIONS WITH THE INCLUSION OF ORGANIC ALCOHOLS
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Reporter 34.4 |2 EnvironmentalReporterVolume 34.4
Can You Test It? Yes You Cannabis!
Dear Colleague,
Legalized marijuana, a thought for some, a dream for several, and a concern for others, has now become a reality in various areas of the United States. Whether for medicinal purposes or for recreational use, the constant release of new legislation on cannabis is a continuous process, varying drastically state by state, and sometimes region by region within each respective state. As legal implications become less and less stringent, most people believe that the American cannabis conflict is gradually being resolved. However, cannabis legalization has been leading to even more questions and concerns, which in turn, will eventually give rise to additional legislation. The majority of these concerns are centered around two main areas: safety and quality. In both cases, analytical testing may be used to provide the answers to questions like: Are the levels of residual pesticides on this bud harmful? Is this cannabis pure, or is it adulterated with hops? Is the amount of THC in this gummy bear safe for consumption? Does this marijuana contain artificial flavors or does the flavor come from the natural cannabis terpenes? Are the effects of this brownie caused by natural or synthetic cannabinoids? Could there be mycotoxins present on this bud?
Earlier this year, we highlighted applications focusing on the aspect of cannabis safety. By coupling innovative sample preparation technologies with analysis techniques, these articles provided viable approaches for pesticide residue, mycotoxin, and residual solvent analysis in cannabis and similar/related matrices.
In this current issue, we touch upon ways in which the quality of various cannabis products may be measured. For instance, the article entitled Headspace SPME-GC/MS Analysis of Terpenes in Hops and Cannabis highlights the use of solid phase microextraction (SPME) in combination with GC/MS as a non-destructive method to identify terpenes, compounds responsible for flavors, aromas, and therapeutic properties (in some cases). The article Analysis of Active Cannabis Compounds in Edible Food Products: Gummy Bears and Brownies focuses on the use of a quick extraction technique, coupled with separation of the cannabinoids on a novel Ascentis® Express Biphenyl HPLC column. Cannabinoids, such as psychoactive tetrahydrocannabinols (THC), indicate potency. In this case, this method is applicable to both safety and quality aspects of edible products containing cannabis.
For more applications and products used for cannabis testing, visit sigma-aldrich.com/cannabis
Sincerely,
Jennifer E. Claus Product Manager Food and Environmental Sample Preparation
sigma-aldrich.com/analytical
Visit us on the web at sigma-aldrich.com/thereporter
Cover Photo: Although most people associate marijuana with the iconic leaf, the most widely used part of the cannabis plant is the bud, as shown in this picture..
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Table of Contents
Food and Beverage Analysis
Headspace SPME-GC/MS Analysis of Terpenes in Hops and Cannabis . . . . . . . . . 3
Analysis of Active Cannabis Compounds in Edible Food Products: Gummy Bears and Brownies . . . . . . . . . . . . . . . 7
Improved Recoveries and Lower Background for GC/MS/MS and LC/MS/MS Analysis of Pesticides in Green Tea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
LC/MS/MS Method for Determination of Glyphosate, AMPA, and Glufosinate in Cereals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Analysis of Bisphenol A and Analogous Compounds in Infant Formula . . . . . . . . . . . . 17
Pharmaceutical
Analysis of Human Plasma Lipids Using sub-2 µm C18 UHPLC Column with MS Detection . . . . . . . . . . . . . . . . . . . . . . . . 20
Rapid Determination of Protein Binding Affinity Using Solid Phase Microextraction. . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Biochromatography
Reaching the “Peak” of Recovery: Improving Antibody Separations with the Inclusion of Organic Alcohols . . . . . . . . . 26
Robustness of the Discovery® HS F5 HPLC Column in the Study of Metabolomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Jennifer E. Claus
Product Manager Food and Environmental Sample Preparation
3Order: 800-325-3010 (U.S.) 814-359-3441 (Global)
Headspace SPME-GC/MS Analysis of Terpenes in Hops and Cannabis
Katherine K. Stenerson, Principal [email protected]
In this application, headspace-SPME combined with GC/MS was used to analyze some of the terpenes present in both common hops and cannabis.
Terpenes are small molecules synthesized by some plants. The name terpene is derived from turpentine, which contains high concentrations of these compounds. Terpene molecules are constructed from the joining of isoprene units in a head-to-tail configuration (Figure 1). Classification is then done according to the number of these isoprene units in the structure (Table 1). The configurations of terpenes can be cyclic or open, and can include double bonds, and hydroxyl, carbonyl or other functional groups. If the terpene contains elements other than C and H, it is referred to as a terpenoid.1
Figure 1. Isoprene Unit
Terpenes are present in essential oils derived from plants and often impart characteristic aromas to the plant or its oil. For example, d-Limonene, which is found in lemon, orange, caraway and other plant oils, has a lemon-like odor. Essential oils, with their subsequent terpenes and terpenoids, have been applied in therapeutic use known as aromatherapy to aid in the relief of conditions such as anxiety, depression, and insomnia.2 This has led to the use of plants whichcontain these compounds in preparations such as oils, teas, and tonics.
Using Terpene Profile for Plant IdentificationThe cannabis sativa (cannabis or marijuana) plant contains over 100 different terpenes and terpenoids, including mono, sesqui, di, and tri, as well as other miscellaneous compounds of terpenoid orgin.3 Although the terpene profile does not necessarily indicate geographic origin of a cannabis sample, it can be used in forensic applications to determine the common source of different samples.4 In addition,
different cannabis strains have been developed which have distinct aromas and flavors; a result of the differing amounts of specific terpenes present.5 Humulus lupulus (common hops) and cannabis are both members of the family Cannabaceae.6 Consequently, there are similarities in the terpenes each contains. Terpenes give both plant commodities characteristic organoleptic properties and, in the case of cannabis, produce characteristic aromas when the buds are heated or vaporized.7
ExperimentalDried cannabis sample was obtained courtesy of Dr. Hari H. Singh, Program Director at the Chemistry & Physiological Systems Research Branch of the United States National Institute on Drug Abuse at the National Institute of Health. The extract strain of the sample was not known. Hop flowers of an unknown variety were purchased from an on-line source. Pelletized of Cascade and US Golding hop varieties were purchased at a local home-brew supply shop. Chromatographic separation was performed on an Equity®-1 capillary GC column, and identification was done using retention indices and spectral library match. Final analytical conditions appear in the figures.
SPME Method OptimizationThe SPME method was developed using a sample of dried hops flowers (0.2 g in 10 mL vial). The initial SPME parameters were based on previously published work.8 The GC/MS results of this analysis are shown in Figure 2. This initial set of parameters used the 100 µm PDMS fiber, a 1 g sample size, and 60 minute equilibration at room temperature prior to extraction. The sample size was then scaled down to 0.2 g, and the equilibration temperature increased to 40 °C. This increased temperature allowed the equilibration time to be decreased from 60 to 30 minutes without a loss in sensitivity (Figures 3 and 4). The initial extraction time used was 20 min, and a shorter extraction time of 10 minutes was evaluated. However a loss in sensitivity was noted, thus extraction time was maintained at 20 minutes. The DVB/CAR/PDMS fiber was then evaluated (Figure 5). As expected, this fiber extracted more of the lighter compounds, which by MS spectral match, were identified as short chain alcohols and acids.
Identification of Terpenes Using GC/MSUsing the DVB/CAR/PDMS fiber, samples of hops and cannabis were analyzed using the optimized SPME method. Peak identifications were assigned using MS spectral matching against reference spectra in the Wiley and NIST libraries. Confirmatory identification was done based on retention index. Retention indices were calculated for the compounds identified in each sample using an n-alkane standard analyzed under the same GC conditions. This data was compared with published values (Tables 2 and 3), and final identifications were assigned, as shown in Figures 6 and 7.
tail
head
Table 1. Classification of Terpenes
Classification Number of Isoprene UnitsMonoterpene 2
Sesquiterpene 3
Diterpene 4
Triterpene 6
Tetraterpene 8
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Food and Beverage Analysis
Figure 2. Headspace SPME-GC/MS Analysis of Dried Hops Flowers (100 µm PDMS Fiber, 1 g Sample)
sample/matrix: 1 g ground hop flowersSPME fiber: 100 µm PDMS (57341-U)
sample equilibration: 60 min, room temperatureextraction: 20 min, headspace, 40 °C
desorption process: 3 min, 270 °Cfiber post bake: 3 min, 270 °C
column: Equity-1, 60 m x 0.25 mm I.D., 0.25 µm (28047-U)oven: 60 °C (2 min), 5 °C/min to 275 °C (5 min)
inj. temp.: 270 °Cdetector: MSD
MSD interface: 300 °Cscan range: full scan, m/z 50-500carrier gas: helium, 1 mL/min constant flow
liner: 0.75 mm ID SPME
10 20 30Time (min)
0.00
E+00
2.00
E+07
Abu
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ce
Figure 3. Headspace SPME-GC/MS Analysis of Dried Hops Flowers (100 µm PDMS Fiber, 0.2 g Sample)
Conditions same as Figure 2 except:
sample/matrix: 0.2 g ground hop flowers
10 20 30Time (min)
0.00
E+00
2.00
E+07
Abu
danc
e
Figure 4. Headspace SPME-GC/MS Analysis of Dried Hops Flowers, Increased Sample Equilibration Temperature (100 µm PDMS Fiber, 0.2 g Sample) Conditions same as Figure 2 except:
sample/matrix: 0.2 g ground hop flowers sample equilibration: 30 min, 40 °C
10 20 30
Time (min)
0.00
E+00
2.00
E+07
4.00
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Abu
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Figure 5. Headspace SPME-GC/MS Analysis of Dried Hops Flowers, Increased Sample Equilibration Temperature (DVB/CAR/PDMS Fiber, 0.2 g Sample)
Conditions same as Figure 2 except:
sample/matrix: 0.2 g ground hop flowers
SPME fiber: 50/30 µm DVB/CAR/PDMS (57298-U)
sample equilibration: 30 min, 40 °C
10 20 30Time (min)
0.00
E+00
2.00
E+07
4.00
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6.00
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Small chain acids and alcohols
ß-myrcene should elute hereHumulene should elute here
Caryophyllene
Table 2. Terpenes in Hops Pellets Identified by MS Spectral Library Match and Retention IndexPeak No.
RT (min) Name
RI (calculated)
RI (literature) Reference
1 8.58 Hexanal — 780 112 12.84 α-Pinene 939 942 113 13.28 Camphene 953 954 114 13.71 6-Methyl-5-hepten-
2-one966 968 11
5 14.1 β-Pinene 979 981 116 14.41 β-Myrcene 988 986 117 15.32 Cymene 1018 1020 118 15.65 d-Limonene 1030 1030 119 15.98 β-Ocimene 1041 1038 11
10 16.72 cis-Linalool oxide 1066 1068 1111 17.49 Linalool 1089 1092 1112 21.86 Geraniol 1239 1243 1113 25.28 Geranyl acetate 1363 1364 1114 25.85 α-Ylangene 1384 1373 815 25.97 α-Copaene 1388 1398 1116 27.22 Caryophyllene 1437 1428 1117 27.4 trans-α-Bergamotene +
unknown1445 1443 12
18 17.63 trans-β-Farnesene 1454 1450 819 28.11 Humulene 1473 1465 1120 28.41 γ-Muurolene 1484 1475 1121 28.45 γ-Selinene 1486 1472 1222 28.68 Geranyl isobutyrate 1495 1493 1123 28.79 β-Selinene 1499 1487 824 28.94 α-Muurolene 1505 1500 1125 28.97 α-Selinene 1507 1501 1226 29.31 γ-Cadinene 1521 1518 1127 29.37 Calamenene 1524 1518 1128 29.45 Δ-Cadinene 1527 1524 1129 30.93 Caryophyllene oxide 1590 1584 830 31.5 Humulene oxide 1614 1599 12
5Order: 800-325-3010 (U.S.) 814-359-3441 (Global)
Table 3. Terpenes in Dried Cannabis Identified by MS Spectral Library Match and Retention Index
Peak No.
RT (min) Name
RI (calculated)
RI (literature) Reference
1 8.57 Hexanal — — —
2 10.05 Hexene-1-ol — — —
3 10.89 2-Heptanone — — —
4 12.56 α-Thujene 928 938 11
5 12.86 α-Pinene + unknown
939 942 11
6 13.27 Camphene 953 954 11
7 13.69 6-Methyl-5-hepten-2-one
966 968 11
8 14.09 β-Pinene 979 981 11
9 14.27 β-Myrcene 984 986 11
10 15.09 Δ-3-Carene 1010 1015 12
11 15.2 α-Terpinene 1014 1012 12
12 15.29 Cymene 1018 1020 11
13 15.6 d-Limonene 1028 1030 11
14 16.42 γ-Terpinene 1056 1057 11
15 16.6 trans-Sabinene hydrate
1062 1078 11
16 16.72 cis-Linalool oxide 1066 1068 11
17 17.43 Linalool 1087 1092 11
18 18.04 d-Fenchyl alcohol 1107 1110 11
19 18.82 trans-Pinocarveol 1135 1134 12
20 19.59 Borneol L 1161 1164 11
21 19.81 1,8-Methandien-4-ol 1168 1173 8
22 19.81 p-Cymen-8-ol 1168 1172 12
23 19.92 Terpinene-4-ol 1172 1185 11
24 20.22 α-Terpineol 1181 1185 11
25 24.2 Piperitenone 1322 1320 12
26 24.76 Piperitenone oxide 1344 1352 12
27 25.85 α-Ylangene 1384 1373 8
28 25.97 α-Copaene 1388 1398 11
29 26.76 γ-Caryophyllene 1419 1403 12
30 27.01 α-Santalene 1429 1428 12
31 27.16 Caryophyllene 1435 1428 11
32 27.36 trans-α-Bergamotene + unknown
1443 1443 12
33 27.49 α-Guaiene 1448 1441 8
34 27.56 trans-β-Farnesene 1451 1446 12
35 27.98 Humulene 1467 1465 11
36 28.17 Alloaromadendrene 1475 1478 11
37 28.25 α-Curcumene 1478 1479 12
38 28.75 β-Selinene 1497 1487 8
39 28.97 α-Selinene 1507 1497 8
40 28.97 β-Bisobolene 1507 1506 8
41 29.13 α-Bulnesene 1514 1513 12
42 30.12 Selina-3,7(11)-diene 1556 1542 12
43 30.94 Caryophyllene oxide 1590 1595 12
44 31.5 Humulene oxide 1614 1599 12
45 32.48 Caryophylla-3, 8(13)- dien-5-ol A
1658 1656 12
Figure 6. Headspace SPME-GC/MS Analysis of Hops Pellets Using Final Optimized Method
The peak elution order is listed in Table 2.Conditions same as Figure 2 except:
sample/matrix: 0.5 g ground hop flowers (hops pellets)SPME fiber: 50/30 µm DVB/CAR/PDMS (57298-U)
sample equilibration: 30 min, 40 °C
Min10 20 30
1
2
3
4
5
6
7
8
9
10
11
1213
14
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12
15
14
17
20, 21
22
23
242526
27
30
28
Min
b. US Golding (Ground pellets)
a. Cascade (Ground pellets)
Figure 7. Headspace SPME-GC/MS Analysis of Dried Cannabis Using Final Optimized Method
The peak elution order is listed in Table 3. Same as Figure 2 except:
sample/matrix: 0.5 g dried, ground cannabisSPME fiber: 50/30 µm DVB/CAR/PDMS (57298-U)
sample equilibration: 30 min, 40 °C
Time (min)
0 10 20 30
1
2 3 4
5
6
78
9
1011
12
13
1415
16
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19 20
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23
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2526
2728
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30
31
3233
3435
36
37
3839 ,4 041
4243
44 45
(continued on next page)
6 Reporter 33.4 |
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Terpenes in Hops SamplesFor the dried hop flower sample (Figure 5), the terpene profile should have shown a predominance of β-myrcene, humulene, and caryophyllene, which are typical aroma compounds in hops and hop oil.9 While caryophyllene was identified, both β-myrcene and humulene were not present at levels high enough to be detected by a library search. This may be due to the condition of sample or the actual variety of hops analyzed since terpene profiles are known to vary between different hop varieties10. The variety of the hop flowers analyzed is unknown, as the identity was not indicated on the packaging. For comparison, samples of two different varieties of pelletized hops were analyzed after grinding. These samples appeared green in color, and had a much more characteristic hops-like odor than the dried flowers. Analysis of these samples showed a characteristic terpene profile, with high levels of β-myrcene, caryophyllene, and humulene present in both (Figure 6). The SPME method was able to detect differences in the terpene profiles between the two hops varieties. For example, farnesene (peak 18) was identified in the Cascade hops, but was too low to be confirmed in the US Goldings sample. The level of farnesene in Cascade hops is expected to be 3-7% of total oils, while in US Goldings the level should be <1%.13
Terpenes in Cannabis SampleThe terpenes identified in the cannabis sample (Figure 7) are indicated in Table 3. The profile was similar to those found previously in the analysis of dried cannabis.4,8 Peaks 1-27 in Figure 7 (with the exception of peak 7) were monoterpenes and monoterpenoids. The later eluting peaks consisted of sequiterpenes and caryophyllene oxide, which is a sequiterpenoid. The most abundant terpene was caryophyllene. The predominance of this compound could be due to the specific strain of cannabis tested, and/or the nature of the sample tested, which was dried. Previous studies have shown the level of this compound to increase significantly relative to other terepenes and terpenoids with drying.4 Consequently, the levels of the more volatile monoterpenes and terpenoids would be expected to be less, and this was observed to some degree. Among the monoterpenes and terpenoids the most abundant were α-pinene and d-Limonene.
ConclusionA simple headspace SPME-GC/MS method was used in the analysis of the terpene/terpenoid profiles of both hops and cannabis. The method was able to detect the characteristic terpenes and terpenoids of both, and to distinguish between different hops varieties.
References1. Fessenden, R.J; Fessenden, J.S. Lipids and Related Natural Products. Organic
Chemistry, 2nd Edition; Willard Grant Press: Boston, MA, 1979; pp 897-904.
2. Bauer, B., M.D. What are the Benefits of Aromatherapy? mayoclinic.org/healthy-lifestyle, accessed 9/22/15.
3. Turner, C.E.; Elsohly, E.G.; Boeren, E.G. Constituents of Cannabis Sativa L., XVIII. A Review of the Natural Constituents. J. Nat. Prod., 1980, 43, 169-234.
4. Hood, L.V.S.; Barry, G.T. Headspace Volatiles of Marihuana and Hashish: Gas Chromatographic Analysis of Samples of Different Geographic Origin. J. Chrom. A., 1978, 166, 499-506.
5. Rahn, B. Terpenes: The Flavors of Cannabis Aromatherapy. 2/12/2014. www.leafly.com/news, accessed September 18, 2015.
6. Plants Database. www.usda.gov, accessed September 14, 2015.
7. Kemsley, J. Cannabis Safety. CEN, 2015, April 20, 27-28.
8. Marchini, M.; Charvoz, C.; Dujourdy, L.; Baldovini, N. Multidimensional Analysis of Cannabis Volatile Constituents: Identification of 5,5-dimethyl-1-vinylbicyclo[1.1.1]hexane as a Volatile Marker of Hashish, the Resin of Cannabis Sativa L. J. Chrom A., 2014, 1370, 200-215.
9. Wolfe, Peter H. MS Thesis, Oregon State University, 2012.
10. Hop varieties. BarthHaasGroup.com, September 15, 2015.
11. Jennings, W.; Shibamoto, T. Qualitative Analysis of Flavor and Fragrance Volatiles by Glass Capillary Gas Chromatography, Academic Press, 1980.
12. NIST Chemistry WebBook. www.webbook.nist.gov/chemistry.
13. Variety Manual USA Hops. Hop Growers of America, www.USAhops.org, accessed September 22, 2015.
Description Cat. No.Capillary GC columnEquity®-1, 60 m × 0.25 mm I.D., 0.25 µm 28047SPME Fibers and AccessoriesSPME fiber assembly Divinylbenzene/Carboxen/ Polydimethylsiloxane (DVB/CAR/PDMS), df 50/30 µm, needle size 23 ga, StableFlex™, for use with autosampler, pk of 3
57298-U
SPME fiber assembly Polydimethylsiloxane (PDMS), df 100 μm (nonbonded phase), needle size 23 ga, for use with autosampler, pk of 3
57341-U
SPME fiber holder for CTC autosampler 57347-USPME fiber holder for manual sampling 57330-UAccessoriesInlet Liner, Direct (SPME) Type, Straight Design, 0.75 mm I.D. for Agilent GC
2637501
Molded Thermogreen® LB-2 Septa, with injection hole, 11 mm, pk of 50
28336-U
Headspace vial, screw top, rounded bottom, 10 mL, clear glass, pk of 100
SU860099
Magnetic Screw Cap for Headspace Vials, PTFE/silicone septum, pk of 100
SU860103
Featured Products
Food and Beverage Analysis
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Analysis of Active Cannabis Compounds in Edible Food Products: Gummy Bears and Brownies
Olga Shimelis,¹ Principle R&D Scientist; Kathy Stenerson,¹ Principle R&D Scientist; Margaret Wesley,² 2016 R&D Summer Intern¹ MilliporeSigma, Bellefonte, PA² Pennsylvania State University, University Park, State College, PA
IntroductionPotency testing in marijuana-infused edibles is an important problem that analytical labs are facing due to the complexity of the involved matrices. Among the most popular matrices are gummy bear candies and brownies. According to one laboratory site, the concentration of active ingredients in the edibles can range from a few parts per million to 3.5 parts per thousand.¹ In this application, a procedure was developed to extract active cannabinoid compounds from gummy bears and brownies. The procedure included a simple and fast extraction of the active compounds from the studied foods, and analysis by HPLC-UV using a biphenyl stationary phase chemistry.
ExperimentalCannabinoid standards from Cerilliant®, available as 1 mg/mL solutions in either methanol or acetonitrile, were used for this experiment. The concentration of cannabinoids allowed for the spiking of both gummy extract and brownies at about 40 ppm with all compounds. The following compounds were included in this study: cannabidivarinic acid (CBDVA), cannabidivarin (CBDV), cannabigerolic acid (CBGA), cannabigerol (CBG), cannabidiolic acid (CBDA), cannabidiol (CBD), tetrahydrocannabivarin (THCV), cannabinol (CBN), (-)-Δ9-Tetrahydrocanabinol (Δ9-THC), (-)-Δ8-Tetrahydrocanabinol (Δ8-THC), and (-)-Δ9-Tetrahydrocanabinolic acid (THCAA). This list of 11 different cannabinoids includes several acidic forms; thus HPLC analysis was used in order to quantitate these in their native forms.
The HPLC column used was Ascentis® Express Biphenyl, 2.7 µm particle size, which gave the best separation of all 11 compounds in under 13 minutes. The use of this column with superficially porous particle architecture resulted in low back-pressure, thus, a standard pressure HPLC system could be used during this experiment.
Sample Preparation
One gummy bear candy, non-spiked, (2.3 g) was dissolved in 20 mL of warm water. This solution was then spiked with cannabinoids and extracted using a QuEChERS procedure. The average spiking level in each gummy bear was 45 ppm for each compound. Bears of four different colors were tested – orange, yellow, red, and green. After spiking, the water/candy solution was transferred to 50-mL plastic
QuEChERS extraction tube (55248-U). Acetonitrile (10 mL) was added, and the tube was shaken for one minute by hand. Supel™ QuE non-buffered salts (55295-U) were added, and the samples were shaken for 5 min on an automated QuEChERS shaker. Post-shaking, the samples were centrifuged for 5 minutes at 5000 rpm. The top layer was collected and injected directly into the HPLC.
For brownies, a 2.5 g sample of a non-spiked brownie with frosting was weighted into the QuEChERS extraction tube. This sample was spiked with cannabinoids and allowed to sit for 30 min prior to extraction. The average spiking level for the brownies was 40 ppm. The QuEChERS extraction was performed as described above for gummy bears. Post-extraction, the top acetonitrile layer was collected into a vial and kept under refrigeration for a minimum of 3 hours prior to HPLC analysis.
A calibration curve was constructed in acetonitrile bracketing the expected concentration of 10 µg/mL in the final extracts. The following calibration points were included: 2 µg/mL, 5 µg/mL, 10 µg/mL, 20 µg/mL and 25 µg/mL.
Results and DiscussionFor the gummy bear samples, it was found that neither the red, yellow, nor green color interfered with detection of cannabinoids at 220 nm. The red color was partially extracted into acetonitrile, while the green and yellow colors stayed in the aqueous layer upon extraction. However, the orange color from the gummy bear, when extracted into acetonitrile, was found to have an interfering peak that co-eluted with CBDVA. Thus, for the orange gummy bear, quantitation of CBDVA was done at 280 nm, where CBDVA has significant absorbance free of interference. Quantitation was done at 220 nm for the rest of compounds in this study.
While no cleanup was required for gummy bear samples post-extraction, the co-extractives in the brownie were found to decrease the recoveries of the analytes if the brownie extract was injected into HPLC without further processing. The brownie extract was cleaned by refrigeration to precipitate the co-extracted fats.
The ruggedness of the method for brownies was tested by injecting the brownie extract multiple times followed by the injection of the 10 µg/mL standard. After 7 injections of the brownie extract, it was found that the peak retention times were not affected, indicating that the column was being thoroughly cleaned between injections. The peak areas for the standards showed a slight decrease of 4%.
Excellent recovery values of above 90% for gummies and above 80% for brownies were achieved with good accuracies (Table 1).
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Peak No. Compound
Yellow Gummy
Orange Gummy
Red Gummy
Average Gummy and RSD
Average Brownie and RSD
1 CBDVA 90% 92% * 92% 91% (2%) 91% (1%) 2 CBDV 93% 100% 100% 98% (3%) 93% (5%) 3 THCV 87% 93% 90% 90% (3%) 87% (1%) 4 CBDA 94% 90% 95% 94% (3%) 95% (1%) 5 CBGA 87% 91% 89% 91% (4%) 90% (2%) 6 CBD 95% 100% 98% 97% (3%) 89% (5%) 7 CBG 93% 99% 98% 96% (4%) 91% (5%) 8 CBN 88% 95% 97% 95% (6%) 84% (4%) 9 Delta-9-THC 93% 99% 100% 97% (3%) 82% (4%)10 Delta-8-THC 91% 97% 98% 95%(3%) 80% (4%)11 THCAA 89% 89% 89% 92% (7%) 91% (2%)
Table 1. Recoveries From Spiked Gummy Bears and Brownies
*The orange gummy was done at 280 nm due to the interfering background peak quantitation.
Figure 1. HPLC Chromatogram of Orange Gummy Bear Extract at (a) 220 nm and (b) 280 nm The peak elution order is listed in Table 1. column: Ascentis Express Biphenyl, 10 cm × 2.1 mm I. D., 2.7 µm
particles (64065-U) mobile phase: (A) 0.1% TFA in water; (B) 0.1% TFA in acetonitrile gradient: at 47% B, to 50% B in 13 minutes, to 100% B in 0.1 min,
100% B for 3 minutes, to 47% B in 0.1 min, at 47% B for 2.5 minutes
flow rate: 0.70 mL/min column temp.: 35 °C detector: UV, 220 nm and 280 nm injection: 5 µL pressure: 340 bar instrument: Agilent 1200, with UV detector
0 2
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(b)
Figure 2. HPLC of a Brownie Extract at 220 nm
The peak elution order is listed in Table 1. Conditions same as Figure 1.
0 2
1
2
3 45
67
8 910
11
1
2
3 45
67
8 910 11
1
2 34 5
6 7
8
9 10
11
4 6 8 10 12Min
0 2 4 6 8 10 12Min
0 2 4 6 8 10 12Min
(a)
(b)
Reference1. Analytical 360, Test Results, Sour Gummy Bears. http://analytical360.com/m/
archived/216628, (accessed July 2016).
Description Cat. No.Supel™ QuE QuEChERS ProductsNon-buffered Extraction Tube 2, 12 mL, pk of 50 55295-UEmpty Centrifuge Tube, 50 mL, pk of 50 55248-UAscentis® Express Biphenyl HPLC Column10 cm × 2.1 mm I.D., 2.7 µm particle size 64065-UAnalytical Standards and ReagentsCannabidivarinic acid (CBDVA), 1 mg/mL in acetonitrile, CRM C-152Cannabidivarin (CBDV), 1 mg/mL in methanol C-140Cannabigerolic acid (CBGA), 1mg/mL in acetonitrile C-142Cannabigerol (CBG), 1 mg/mL in methanol C141Cannabidiolic acid (CBDA), 1 mg/mL in acetonitrile C-144Cannabidiol (CBD), 1 mg/mL in methanol C-045Tetrahydrocannabivarin (THCV), 1 mg/mL in methanol T-094Cannabinol (CBN), 1 mg/mL in methanol C-046(-)-Δ9-Tetrahydrocanabinol (Δ9-THC), 1 mg/mL in methanol T-005(-)-Δ8-Tetrahydrocanabinol (Δ8-THC), 1 mg/mL in methanol T-032(-)-Δ9-Tetrahydrocanabinolicacid (THCAA), 1 mg/mL in acetonitrile D3415AccessoriesQuEChERS Shaker and Rack Starter Kit, USA compatible plug 55278-UQuEChERS Shaker and Rack Starter Kit, EU Schuko plug 55438-UCertified Vial Kit, Low Adsorption (LA), 2 mL, pk of 100 29653-U
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ConclusionA method was presented for analysis of active cannabinoid compounds in both brownies and gummy bears. The extraction procedure involved a salting out step into acetonitrile, and did not require intensive cleanup. The separation of eleven compounds was achieved on a biphenyl stationary phase, and was completed in 13 minutes. The active compound CRM standards are available through sigmaaldrich.com from Cerilliant.
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Improved Recoveries and Lower Background for GC/MS/MS and LC/MS/MS Analysis of Pesticides in Green Tea
Katherine K. Stenerson, Principal [email protected]
IntroductionTea is second only to water as the most popular beverage worldwide. All teas come from the leaves of the same plant, camellia sinensis. The various types of tea arise from differences in how and where the plants are grown, and how the leaves are processed. Green tea is produced by a quick drying of leaves after they are harvested. This stops the oxidation process that starts once they are picked. By comparison, black tea leaves are allowed to oxidize fully, producing a beverage with more robust flavor and higher caffeine content.1 The consumption of green tea is associated with health benefits such as reduced risk of heart disease and several types of cancers. This is due primarily to its antioxidant properties, which are a result of catechins. These compounds have been found to be more effective than other anti-oxidants such as vitamins C and E at stopping oxidative cell damage.2
Owing to its popularity, tea is considered to be a major agricultural commodity around the world, with 4.8 million tons produced in 2012.3 The mass production of tea often uses pesticides to maximize crop yield, making exposure to these chemicals a concern for those who consume it. Thus, testing for pesticide residues is required by many countries which import tea. These countries include the United States, Canada, Australia, Japan, and those in the European Union (EU). The requirements for maximum residue limits (MRLs) are different for each of these countries, with those established by the EU the lowest among tea importing countries; ranging from 0.05 to 50 mg/kg, depending on the pesticide.3 Meeting these MRLs creates a challenge for analytical laboratories doing pesticide residue testing of tea. This is due to the high levels of background that is co-extracted along with the pesticides. These compounds can include pigments, sugars, polyphenols, and alkaloids; and these can interfere with chromatographic analysis. For tea extracts, common cleanups include the use of QuEChERS with primary-secondary amine (PSA), C18 and graphitized carbon black (GCB), and SPE with dual-layer cartridges containing GCB.4,5,6 GCB effectively removes green pigments, but will also retain any target pesticides with planar structures. In the case of QuEChERS cleanup with GCB, recoveries of these planar pesticides are often reduced. If cleanup is done using a dual-layer SPE cartridge, toluene can be added to the elution solvent in order to recover these compounds. While having toluene present in the final extract is compatible with injection into a GC system, a solvent exchange must be done prior to HPLC analysis.
In this application, a new SPE cartridge, the Supelclean™ Ultra 2400, was used in the cleanup of QuEChERS extracts of green tea. Supelclean Ultra 2400 is a small volume dual-layer SPE cartridge
designed for cleanup of high background samples such as dry commodities (tea, coffee and spices). It contains a mixture of carbon/PSA/C18 in the top layer, and Z-Sep in the bottom layer. The carbon, Graphsphere™ 2031, is engineered to reduce pigmentation and increase recovery of planar pesticides without the need for toluene in the elution solvent. The Z-Sep in the bottom layer provides removal of oils and fatty components, and also provides additional reduction in pigmentation. The small size of the cartridge (which is available in 1 and 3 mL) was designed to provide sufficient cleanup while reducing solvent usage.
In this study, green tea was spiked at 5 and 50 ng/g and extracted using QuEChERS. Cleanup using a 1 mL Supelclean Ultra 2400 cartridge was then compared with QuEChERS cleanup using PSA/C18/GCB. The final extracts were analyzed by LC/MS/MS and GC/MS/MS. Performance of the cleanups was compared with regards to background and pesticide recoveries.
ExperimentalOrganic green tea was obtained from a local grocery store and ground into a fine powder prior to use. Samples were spiked at 5 ng/g and 50 ng/g and extracted using the procedure shown in Figure 1. Samples were cleaned using the Supelclean Ultra 2400 SPE cartridge and PSA/C18/ GCB, as indicated in Figure 2.
Figure 1. QuEChERS Extraction Procedure Used for Green Tea
2 g ground sample + 10 mL water. Allow to hydrate for 60 min
Add 10 mL of acetonitrile. Shake for 10 min at 2500 rpm
Add contents of Supel™ QuE Citrate extraction tube (55227-U) and shake for 1 min
Centrifuge at 5000 rpm for 5 min
Draw off supernatant for cleanup
(continued on next page)
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Condition: 1 mL acetonitrile
Load: 300 µL extract Shake 1 min
Elute: 2 mL 80:20 methanol:acetonitrile
w/2 mM ammonium formate
Concentrate: FV 300 µL
Dispense 1 mL of extract into 2 mL tube with sorbents
Centrifuge at 5000 rpm for 3 min
Remove supernatant
1 mL Ultra 2400 SPE PSA/C18/GCB
Figure 2. Cleanups of Green Tea QuEChERS Extracts
Spiked replicates and blanks were prepared using each cleanup. Samples were analyzed against matrix-matched 5-point calibration curves from 0.5-10 ng/mL with separate sets prepared for each cleanup. For both cleanups, the same sample extracts were analyzed by both LC/MS/MS and GC/MS/MS. The analysis conditions are shown in Tables 1 and 2, as well as 3 and 4, respectively.
Table 1. LC/MS/MS Analysis Conditions
column: Ascentis® Express RP-Amide, 10 cm × 2.1 mm I.D., 2 µm (51576-U)
mobile phase: (A) 5 mM ammonium formate, 0.1% formic acid in water; (B) 5 mM ammonium formate, 0.1% formic acid in 95:5 acetonitrile:water
gradient: 90% A, 10% B held for 1 min; to 100% B in 13 min; held for 2 min; to 90% A in 0.5 min; held at 90% A for 3 min
flow rate: 0.4 mL/min column temp.: 30 ºC detector: MRM (see Table 2) injection: 5 µL
Table 2. Pesticides Analyzed by LC/MS/MS, MRMs
PesticideRet. Time
(min) MRM Frag (V) CE (V)Acephate 1.2 184/143 70 0
Boscalid (Nicobifen) 8.88 343/307.1 145 16
Carbaryl 7.18 202.1/145.1 65 4
Diazinon (Dimpylate) 9.75 305.1/97 105 40
Etoxazole 11.43 360.2/141 120 26
Fenpyroximate (E) 11.3 422.21/366.2 135 12
Hexythiazox 11.27 353.1/227.9 90 8
Imazalil (Enilconazole) 6.14 297.1/159 115 20
Imidacloprid 4.32 256/208.9 80 12
Malathion 8.92 331/126.9 80 5
Metalaxyl 6.83 280.2/160.1 95 20
Myclobutanil 8.45 289.1/125.1 110 32
Table 3. GC/MS/MS Analysis Conditions
column: SLB®-5ms, 20 m × 0.18 mm I.D., 0.18 µm (28564-U)
oven: 50 °C (2 min), 8 °C/min to 325 °C (5 min) inj. temp: 250 °C carrier gas: helium, 1.4 mL/min, constant flow detector: MRM (see Table 4) MSD interface: 325 °C injection: 1 µL pulsed splitless (50 until 0.75 min, splitter
on at 0.75 min) liner: 4 mm I.D. FocusLiner™ with taper
PesticideRT
(min) MRM 1 CE MRM 2 CEBifenazate 26.6 184/156.2 10 199/184.2 10
Bifenthrin 26.46 181.2/166.2 10 181.2/165.2 25
Captan 14.66 151/80 5 149/79.1 10
Chlorpyrifos 21.16 181.2/152.1 15 196.9/169 15
Cyfluthrin isomers I-IV
29.30, 29.42, 29.49, 29.55
162.9/90.9 15 162.9/127 5
Cypermethrin isomers I-IV
29.68, 29.81, 29.88, 29.93
163/91 10 163/127 5
Dichlorvos 10.67 109/79 5 184.9/93 10
Fenoxycarb 26.56 255.2/186.2 10 186.2/158.2 5
Fipronil 22.19 350.8/254.8 15 366.8/212.8 25
Cyhalothrin (Gamma)
27.68 181.2/152.1 25 197.0/141.1 10
Methoxychlor 26.6 227/169.1 25 227/141.1 40
Paclobutrazol 22.81 236/125.1 10 125.1/89 20
Permethrin I, II 28.61, 28.77
183/168 10 182.9/165.1 10
Propargite 20.58 135/107.1 10 149.9/135.1 5
Tetramethrins, I, II
26.33, 26.53
164/107.1 10 164/77.1 25
Table 4. Pesticides Analyzed by GC/MS/MS, MRMs
PesticideRet. Time
(min) MRM Frag (V) CE (V)Phosmet (Imidan) 8.48 317.99/160 70 8Piperonyl butoxide 10.55 356.2/119.1 80 40Propoxur 6.35 210.11/153.1 55 0Pyraclostrobin 10.05 388.11/193.8 95 8Quinoxyfen 10.87 308/196.9 115 36Spinosyn A 8.12 732.5/142.1 155 28Spinosyn D 8.5 746.5/142.1 145 35Spirodiclofen 11.94 411.1/313 110 5Spiromesifen 11.73 388/273 110 10Spirotetramat 8.15 374.2/216.1 120 36Tebuconazole 8.95 308.1/70 100 40Tetrachlorvinphos (Dietreen T)
9.03 364.9/127 120 16
Trifloxystrobin 10.37 409.1/186 110 12
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Results and DiscussionBackground – After cleanup, extracts cleaned with Supelclean™ Ultra 2400 had slightly less color than those cleaned with PSA/C18/GCB (Figure 3). GC/MS scan runs of each extract (Figure 4) show the least amount of background present in the sample cleaned with Ultra 2400. The Ultra cartridge removed more polyphenols (peaks eluting between 10–20 min) than the PSA/C18/GCB; however, caffeine (large peak at 20 min) was still present in both extracts. At the 5 ng/g spiking level, background was more prevalent, and more interference was noted in the PSA/C18/GCB than the Ultra 2400 cleaned samples, especially in GC/MS/MS analysis. An example of this is shown in Figure 5 for γ-cyhalothrin.
Figure 3. QuEChERS Extracts of Green Tea (Undiluted), Before and After Cleanup
No Cleanup PSA/C18/GCBSupelclean Ultra 2400
Figure 5. GC/MS/MS Analysis of γ-Cyhalothrin at 5 ng/g in Green Tea: MRM 181.2/152.1, (a) PSA/C18/GCB Cleanup, (b) Supelclean Ultra 2400 Cleanup
27.6 27.8
Time (min)
27.6 27.8Time (min)
(a) (b)PSA/C18/GCB Ultra 2400
Pesticide Recovery – A summary and comparison of the pesticide recoveries and reproducibilities obtained at both spiking levels is presented in Table 5 and Figure 6. Overall, at both spiking levels, performance was better using Ultra 2400 than PSA/C18/GCB for cleanup. Using Ultra 2400 cleanup, a greater number of pesticides could be quantitated, and were within 70–120% recovery with RSD <20% for spiked replicates.
Figure 6. Comparison of Cleanup Performance for Pesticide Residue Analysis from Spiked Green Tea (a total of 41 pesticides were analyzed)
120%
70-120% Recovery 70-120% Recovery and RSD < 20%
100%
80%
60%
40%
20%
0%RSD < 20%
Perc
enta
ge o
f pes
ticid
es s
pike
d (4
1 to
tal)
Ultra 2400, 50 ng/g
PSA/C18/GCB, 5 ng/g PSA/C18/GCB, 50 ng/g
Ultra 2400, 5 ng/g
At 5 ng/g, acephate and spirodiclofen could not be detected after either cleanup. As indicated in Table 5, several pesticides could not be quantitated after PSA/C18/GCB cleanup due to matrix. Captan could not be analyzed at either 5 or 50 ng/g due to matrix. The tetronic acid and tetramic pesticides (spirodiclofen, spiromesifen, spirotetramat), and the spinosads (spinosyn A&D) had lower recoveries from PSA/C18/GCB at both spiking levels compared to Ultra 2400. It is suspected that this is due to retention of these compounds on the GCB, since several of these compounds have planar components to their structures and/or are very large in size. Quinoxyfen, another pesticide with a planar element to its structure, had >70% recovery at both spiking levels after cleanup with Ultra 2400, but recovery of < 60% using PSA/C18/GCB.
Figure 4. GC/MS Scan Analyses of Green Tea Extracts, (a) No Cleanup, (b) Cleanup with Supelclean Ultra (1 mL), (c) Cleanup with PSA/C18/GCB
10 20 30 40Time (min)
0.00
E+00
2.00
E+09
ca�eine
(a)
(b)
10 20 30 40
Time (min)
0.00
E+00
2.00
E+09
(c)
Time (min)
10 20 30 400.00
E+00
2.00
E+09
(continued on next page)
Reporter 34.4 |12
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Table 5. Average Recoveries and (%RSD) Values for Pesticides from Green Tea; QuEChERS Extraction Followed by Indicated Cleanup
n=3 Spiking Level 5 ng/g 50 ng/gCleanup Ultra 2400 PSA/C18/GCB Ultra 2400* PSA/C18/GCB
Pesticide Analysis Acephate LC/MS/MS ND ND 90 (9) 79 (6)
Bifenazate GC/MS/MS 45 (11) matrix 94 (1) 104 (1)
Bifenthrin GC/MS/MS 81 (12) matrix 77 (1) 69 (3)
Boscalid LC/MS/MS 115 (11) 72 (5) 93 (17) 89 (2)
Captan GC/MS/MS 19 (40) matrix 85 (6) matrix
Carbaryl LC/MS/MS 108 (6) 78 (2) 95 (11) 96 (1)
Chloropyrifos GC/MS/MS 94 (8) 39 (13) 81 (10) 93 (8)
Avg. Cyfluthrins (4 isomers)
GC/MS/MS 90 (10) 55 (24) 81 (15) 96 (6)
Avg. Cypermethrins (4 isomers)
GC/MS/MS 92 (10) 47 (55) 81 (6) 91 (12)
Diazinon LC/MS/MS 106 (4) 63 (4) 78 (11) 89 (1)
Dichlorvos GC/MS/MS 38 (45) 30 (43) 34 (58) 51 (26)
Avg. Dimethomorphs (2 isomers)
LC/MS/MS 121 (10) 58 (13) 92 (12) 87 (2)
Etoxazole LC/MS/MS 109 (5) 51 (5) 91 (12) 80 (2)
Fenoxycarb GC/MS/MS 78 (15) 31 (22) 88 (16) 95 (2)
Fenpyroximate LC/MS/MS 108 (6) 54 (5) 96 (10) 79 (2)
Fipronil GC/MS/MS 92 (8) 31 (8) 113 (4) 108 (6)
Cyhalothrin-γ GC/MS/MS 69 (6) 20 (61) 85 (20) 93 (6)
Hexythiazox LC/MS/MS 111 (12) 61 (8) 105 (12) 76 (4)
Imazalil LC/MS/MS 87 (6) 44 (4) 83 (11) 70 (2)
Imidacloprid LC/MS/MS 120 (8) 52 (2) 87 (14) 77 (1)
Malathion LC/MS/MS 124 (8) 71 (4) 102 (17) 97 (1)
Metalaxyl LC/MS/MS 112 (4) 65 (1) 97 (7) 93 (1)
Methoxychlor GC/MS/MS 88 (14) 41 (10) 104 (3) 104 (4)
Myclobutanil LC/MS/MS 121 (8) 80 (5) 101 (7) 91 (1)
Paclobutrazol GC/MS/MS 100 (5) 88 (37) 112 (0.4) 108 (7)
Avg. Permethrins (2 isomers)
GC/MS/MS 87 (8) matrix 74 (12) 76 (9)
Phosmet LC/MS/MS 124 (6) 84 (8) 99 (3) 98 (3)
Piperonyl butoxide LC/MS/MS 111 (6) 59 (5) 99 (8) 90 (2)
Propargite GC/MS/MS 145 (26) 35 (69) 93 (7) 103 (2)
Propoxur LC/MS/MS 110 (4) 85 (6) 89 (3) 99 (2)
Pyraclostrobin LC/MS/MS 115 (13) 60 (5) 116 (3) 91 (2)
Quinoxyfen LC/MS/MS 110 (11) 49 (12) 95 (2) 58 (2)
Spinosyn A LC/MS/MS 107 (6) 36 (8) 102 (9) 61 (4)
Spinosyn D LC/MS/MS 113 (6) 36 (10) 104 (7) 54 (5)
Spirodiclofen LC/MS/MS ND ND 98 (15) 76 (13)
Spiromesifen LC/MS/MS 103 (12) 51 (8) 92 (11) 78 (3)
Spirotetramat LC/MS/MS 120 (3) 43 (19) 84 (14) 56 (17)
Tebuconazole LC/MS/MS 108 (9) 63 (9) 105 (4) 86 (1)
Tetrachlorvinphos LC/MS/MS 108 (20) 70 (8) 106 (11) 95 (2)
Avg. Tetramethrins (2 isomers)
GC/MS/MS 86 (8) 36 (16) 94 (5) 105 (7)
Trifloxystrobin LC/MS/MS 118 (7) 65 (4) 108 (5) 96 (2)
*avg. of 2 replicates
ND: not detected
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Featured Products
Description Cat. No.Solid Phase Extraction CartridgeSupelclean Ultra 2400, 1 mL, pk of 108 52779-USupel QuE QuEChERS ProductsEmpty Centrifuge Tube, 50 mL, pk of 50 55248-USupel QuE Citrate Extraction Tube, 12 mL, pk of 50 55227-UPSA/C18/ENVI-Carb™ Tube, 2 mL, pk of 100 55289-UCapillary GC ColumnSLB®-5ms, 20 m × 0.18 mm I.D., 0.18 µm 28564-UHPLC ColumnAscentis® Express RP-Amide, 10 cm × 2.1 mm I.D., 2 µm 51576-UAccessoriesQuEChERS Shaker and Rack Starter Kit, USA compatible plug 55278-UQuEChERS Shaker and Rack Starter Kit, EU Schuko plug 55438-UVisiprep™ DL SPE Vacuum Manifold, 12-port model 57044Disposable Liners for Visiprep DL, PTFE, pk of 100 57059
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Description Cat. No.AccessoriesInlet Liner, Split/Splitless Type Single Taper FocusLiner™ Design (wool packed)
2879901-U
Molded Thermogreen® LB-2 Septa, solid discs, pk of 50 28676-UTherm-O-Ring™ Inlet Liner O-Ring, pk of 10 21003-UGold-Plated Inlet Seal (Straight Design), pk of 2 23318-UCapillary Column Nut for Agilent® MS, pk of 5 28034-UMillex® syringe filter units, disposable, PTFE Z227544-100eaVialsCertified Vial Kit, Low Adsorption (LA) QsertVial™ 0.3 mL w/PTFE silicone septa, pk of 100
29663-U
Certified Vial Kit, Low Adsorption (LA) QsertVial™ (amber) 0.3 mL w/PTFE silicone septa (slit), pk of 100
29664-U
ConclusionsUsing QuEChERS for extraction of pesticide residues from green tea produces an extract with high background which contains pigments, polyphenolics, and caffeine. For cleanup of this sample, Ultra 2400 SPE resulted in lower background than QuEChERS cleanup using PSA/C18/GCB. This was evidenced by extract color, GC/MS-scan background, and GC/MS/MS data. While neither cleanup was able to retain caffeine, the Ultra 2400 cartridge removed as much pigment and more polyphenolics than PSA/C18/GCB. For pesticide recovery, Ultra 2400 performed better than PSA/C18/GCB, especially for the 5 ng/g spiking level, at which 73% of the 41 pesticides tested had recoveries in the range of 70–120%, compared to 20% for PSA/C18/GCB. At a higher spiking level of 50 ng/g, the Ultra 2400 cleanup still yielded better results than PSA/C18/GCB, with 95% of the pesticides within acceptable recovery and reproducibility ranges, vs. 83% for the later cleanup.
In summary, in the cleanup of green tea extracts, Supelclean™ Ultra 2400 SPE was found to provide lower background than QuEChERS cleanup using PSA/C18/GCB. This allowed for the analysis of more pesticides at lower levels. The small size of the cartridge offers an advantage over larger, 6 mL dual-layer cartridges containing GCB in that it uses less solvent, and does not require the use of toluene in the elution solvent.
References1. Tea Source. teasource.com (accessed Aug 2016)
2. Benefit of drinking green tea. Harvard Health Publications. health.harvard.edu (accessed Aug 2016)
3. Chang, K., Atici, C. Implications of Maximum Residue Levels (MRLs) on Tea Trade; Food and Agriculture Organization of the United Nations, Market and Policy Analyses of Raw Materials, Horticulture and Tropical Products Team: Rome, 2015.
4. Lozano, A.; Rajski, Ł.; Belmonte-Valles, N.; Uclés, A.; Uclés, S.; Mezcua, M.; Fernández-Alba, A.R. Pesticide analysis in teas and chamomile by liquid chromatography and gas chromatography tandem mass spectrometry using a modified QuEChERS method: Validation and pilot survey in real samples. J. Chrom. A, 2012, 1268, 102–122.
5. Guan, Y.; Tang, H.; Chen, D.; Xu, T.; Lei, L. Modified QuEChERS method for the analysis of 11 pesticides residues in tea by liquid chromatography-tandem mass spectrometry. Analytical Methods, 2013, 5, 3056–3067.
6. Huang, Z.; Zhang, Y.; Wang, L.; Ding, L.; Wang, M.; Yan, H.; Li, Y.; Zhu, S. Simultaneous determination of 103 pesticides in residues in tea samples by LC-MS/MS. J. Sep Sci., 2009, 32, 1294–1301.
High Capacity Gas Purifier Starter KitThe best purifier choice for removing moisture and oxygen from carrier gas is Supelco’s High Capacity Gas Purifier. This starter kit includes a clam shell oven, two elements (installed) and a converter tube.
Note: Not for use with hydrogen gas.
For more information, visit sigma-aldrich.com/hcgp
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LC/MS/MS Method for Determination of Glyphosate, AMPA, and Glufosinate in Cereals
Olga I. Shimelis, Principal R&D Scientist
IntroductionGlyphosate is one of the most commonly used herbicides in the world. Its usage has significantly increased after the introduction of genetically modified glyphosate tolerant crops such as corn, soybeans, and cotton. Currently, more than 1.4 billion pounds of glyphosate are applied to fields per year.1 US EPA regulation document CFR-title 40-volume 24 set the tolerance levels for the occurrence of glyphosate in food commodities and produce.2
EPA tolerance levels for glyphosate residues in cereal grains (also called crop group 15) are set at 30 ppm. These exclude rice, soy, and corn. In rice the tolerance is 0.1 ppm, whereas in sweet corn it is 3.5 ppm.2 For glufosinate, a herbicide that is often included with glyphosate in analytical methods, the tolerance values are 0.4 ppm for cereal grains and 1.0 ppm for rice. These tolerance values include metabolites and degradants. Therefore, a glyphosate metabolite (aminomethyl)-phosphonic acid (AMPA), was also included in this study (Figure 1).
Oats were also spiked to contain 100 ppb of AMPA. Organic corn flour was also purchased and tested for glyphosate and was found to be glyphosate-free. The same methodology can also be applied for testing corn flour and corn products.
For screening studies, the following matrices used the developed methods and were tested for glyphosate: white flour, instant oatmeal, infant rice cereal, infant oat cereal, and infant mixed grain cereal.
Sample Pre-treatment
The extraction method was based on the QuPPe (Quick Polar Pesticides Method) methodology developed in the European Union (EU) for fruits and vegetables, and used water:methanol (50:50) containing formic acid as the final extraction solvent.5 A five-gram sample of homogenized grain or cereal was weighed into a 50 mL centrifuge tube. Water (10 mL) and 100 µL of an internal standard solution (20 µg/mL of each analyte in water) were added. The samples were then left to stand for 30 minutes to two hours. After that, 10 mL of methanol containing 1% v/v formic acid were added. The samples were mixed for 15 minutes on a laboratory shaker and centrifuged. Two samples used during validation, oats and wheat, gave significantly different extracts. While oats produce a clear, yellow extract, the wheat extract was cloudy and difficult to filter. As a result, two different sample cleanup procedures were used for the different grain samples.
Sample Cleanup Using SPE
For the samples that did not have particulates after extraction and centrifugation, a solid phase extraction (SPE) cleanup using Supel™-Select HLB cartridge was applied, similar to a method reported by Chamkasem and Harmon.6 The HLB cartridges were conditioned using 100% methanol followed by water:methanol (50:50) containing 0.5% v/v formic acid. For 1 mL SPE cartridges, 0.5 mL of the sample extract was used to further condition the cartridge. The eluate from this conditioning step was discarded. A second aliquot of the sample extract (0.5 mL) was loaded into the HLB cartridge. This eluate was collected and filtered through 0.2 micron polypropylene membrane filter vials.
Sample Cleanup Using Ultrafiltration
Ultrafiltration devices were used for cleanup of sample extracts, such as wheat, that had particulates after the centrifugation step. Polyethersulfone membranes at 3 kDa molecular weight cutoff (MWCO) were used in the present work. The membranes were pre-conditioned by passing 0.5 mL of the extraction solvent through by centrifugation at 4000 rpm for 5 minutes in order to keep the retention time of glyphosate consistent. This pass-through solvent was discarded and 1 mL of the sample extract was loaded. The ultrafiltration step was performed by centrifugation for 45 minutes at 4000 rpm. It was also determined that ultrafiltration devices containing regenerated cellulose membrane, such as Amicon™ Ultra centrifugal filters with 3kDa MWCO, can be used in this step without pre-conditioning. The clear sample that passed through the membrane was collected and analyzed. For analysis, low-absorption vials were used to prevent loss of analytes on the glass surface.
Figure 1. Structures of Glyphosate, AMPA and Glufosinate
Glyphosate AMPA Glufosinate
HOOH
HN
O
OHP
OH2N
OOH
OHP H3C
OPOH NH2
ONH4
O
Since glyphosate is widely used with production of soybeans and corn, it was expected to be found in these commodities. In this application we focused on exploring the presence of glyphosate in other grains (oats and wheat) used to make breakfast cereals, including infant cereal products.
Various methods for glyphosate analysis were developed over the last 30 years. Some required derivatization of analytes for HPLC with fluorescence detection with o-phthalaldehyde.3 A method with glyphosate derivatization using fluorenylmethyloxycarbonyl chloride (FMOC) and fluorescence detection has been proposed and used by some laboratories.4 Recently, with the advent of modern, more sensitive and rugged LC/MS/MS instruments, it has become possible to analyze glyphosate and its metabolites without derivatization. We used direct analysis of glyphosate by MS/MS in this work.
ExperimentalIn order to confirm method performance, organic instant oatmeal and organic whole wheat flour were used. These foods were scanned for the presence of glyphosate and none was found. Whole wheat flour was used as is and the quick oats were ground prior to use. For the method performance study, both matrices were spiked to contain 100 ppb of glyphosate and 100 ppb of glufosinate.
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Sample Preparation for Beer
Beer samples could be analyzed using the same methodology. First, a thorough degassing of beer was undertaken by placing the beer sample in the ultrasonic bath for 15 minutes. Then 5 mL of the beer sample was mixed with 5 mL of methanol with 1% formic acid and internal standard. This sample was briefly mixed and cleaned using the SPE procedure.
LC/MS/MS Method
The HPLC column used for this analysis was the polymer-based apHera™ NH2 column, which provides stable and robust LC separations from pH 2 to 13. The mobile phase gradient used water and ammonium carbonate at pH 9. This mobile phase ensured the proper ionization of glyphosate, which has a phosphate group in its structure, with detection under negative ESI conditions. In addition, ammonium carbonate buffer is volatile and is fully compatible with LC/MS instrumentation. Table 1 lists the MS conditions for all analytes and Figure 2 presents a chromatogram of a standard injection. Analysis was performed using an AB Sciex QTrap 3200 instrument. This limited method sensitivity. However, as can be seen below, the method was able to quantify glyphosate contamination in most samples in this study.
ResultsMethod Performance
Both the SPE and ultrafiltration cleanup methods provided good sample cleanup, and were acceptable for LC/MS analysis. In oatmeal, all three analytes were detected and quantified at 100 ppb. In wheat flour, glyphosate and glufosinate were quantified at 100 ppb. The results of method tests are shown in Table 2. The method for wheat samples produced slightly higher uncertainties, up to 19% RSD. The wheat method did not use SPE cleanup and the resulting signals were found to have higher ion suppression in comparison to the samples that were cleaned using SPE, such as oats. In general, various degrees of ion suppression, from 50% to 80%, were present in all samples, and thus it was important to employ internal standards for accurate quantification.
Compound Q1 Q3 DP EPGlyphosate Quant 167.8 63 -30 -5.5
Qual 167.8 79 -30 -5.5Glyphosate-2-13C,15N Quant 19.8 62.9 -30 -4.5AMPA Quant 110 63 -45 -10
Qual 110 81 -40 -10AMPA-13C,15N,D2 Quant 113.9 63 -40 -5Glufosinate Quant 180 63 -35 -6
Qual 180 136 -40 -4Glufosinate-D3 Quant 182.9 63 -30 -7.5
Table 1. MS Parameters for Glyphosate and Analogs
Figure 2. LC/MS Analysis of Glyphosate, AMPA, and Glufosinate on apHera NH2
(Quantitative transitions are shown for each analyte.) column: apHera NH2, 15 cm × 4.6 mm I.D., 5 µm particles
(56401AST) mobile phase: [A] water; [B] 20 mM ammonium carbonate, pH 9;
[C] methanol:water (50:50) gradient: 100% A for 2 min, to 90% B, 10% C in 0.1 min, held until
10 min, to 100% A in 0.1 min, held for 5 min flow rate: 0.5 mL/min column temp.: 35 °C detector: MS/MS, ESI negative, multiple transitions injection: 60 µL sample: each compound, 20 ng/mL in methanol:water (1:1)
containing 0.5% formic acid pressure: 220 bar instrument: Agilent® 1200 stacked with AB Sciex QTrap 3200
5 6 7 8
Min
Glyphosate
AMPAGlufosinate
9 10
Analyte/Matrix Glyphosate AMPA GlufosinateN=6 Recovery
(%)RSD (% )
Recovery (%)
RSD (% )
Recovery (%)
RSD (% )
Oatmeal 125 5 118 15 105 5
Whole wheat 125 19 — — 105 16
Beer 118 21 — — 112 7
Table 2. Validation Results at 100 ppb Spiking Level in Cereal/Grains and 50 ppb Spiking Level in Beer
Glyphosate in Beer
No analytes were found in the tested beer. Thus, the beer sample was spiked at 50 ppb with glyphosate and glufosinate and analyzed. The results are presented in Table 2. In this work we did not attempt further screening of beer.
Identification and Quantitation of Glyphosate in Cereals
The results of glyphosate quantitation in cereals are presented in Table 3. The samples of instant oatmeal (Figure 3) and white flour contained significant amounts of glyphosate, 1.2 and 0.8 ppm respectively. The organic oatmeal sample did not contain detectable amounts of glyphosate (Figure 4). Infant rice cereal had very low levels of glyphosate. The levels were close to the limit of instrument sensitivity in that the matrix and the resulting RSD was high. Infant oat cereal had glyphosate at 1.1 ppm and infant mixed cereal was found to contain glyphosate at 0.25 ppm. None of the infant cereals were labelled organic. Glufosinate was not found in any cereal products. AMPA was found only in instant oatmeal at low levels.
Analyte/Matrix Glyphosate AMPA GlufosinateN=3 Found, ppm % RSD Found, ppm % RSD Found, ppmOatmeal 1.2 8 0.04 27 ND
White wheat flour 0.8 12 NT — ND
Infant rice cereal 0.06 12 ND — ND
Infant oat cereal 1.1 4 NT — ND
Infant mixed cereal 0.25 5 NT — ND
Beer (Lager) ND — ND — ND
Table 3. Glyphosate Quantitation Results in Grains and Cereals
NT=not tested, ND=not detected.(continued on next page)
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ConclusionsThe proposed method developed for glyphosate and related compounds uses LC/MS/MS detection and an ion-exchange polymer-based apHera NH2 column that is stable under higher pH conditions. The sample preparation methodology was developed to extract glyphosate out of cereal food products. The methodology included cleanup using polymeric SPE, which was successfully applied to samples of cereals including oatmeal and infant cereal products. Cleanup that included ultrafiltration was developed for wheat-containing products and was successfully applied to wheat flour samples. Use of isotopically labelled standards resulted in better accuracy for glyphosate determination and allowed the use of solvent-based calibration curves by compensating for ionization effects present in samples. The method proved itself to be rugged and performed well, even when run on older and less sensitive instrumentation.
Figure 3. Glyphosate in Instant Oatmeal
Two MRM transitions are shown: 168/63 in blue and 168/79 in red.
Conditions same as Figure 2.
5 6 7 8Min
9 10
168/63
168/79
Figure 4. Organic Oatmeal Sample, Blank and Spiked at 100 ppb GlyphosateMRM transition 168/63 is shown.Conditions same as Figure 2.
5 6 7 8Min
9 10
Spiked
Blank
Description Cat. No.StandardsGlyphosate, PESTANAL®, Analytical Standard, 45521Glyphosate-2-13C,15N 90479(Aminomethyl)phosphonic acid, Analytical Standard 05164Aminomethylphosphonic acid-13C,15N,D2 Solution CDNLM-6786Glufosinate ammonium, PESTANAL®, Analytical Standard 45520ColumnapHera NH2 HPLC column, 15 cm × 4.6 mm, 5 µm particle size
56401AST
Sample PreparationEmpty Centrifuge Tube, 50 mL, pk of 50 55248-USupel-Select HLB SPE tube, 30 mg/1 mL , pk of 100 54181-UAmicon™ Ultra Centrifugal 0.5 mL Filter, 3kDa MWCO 3kDa Z677094AccessoriesQuEChERS Shaker and Rack Starter Kit, USA compatible plug, AC input 115 V
55278-U
QuEChERS Shaker and Rack Starter Kit, EU compatible Schuko plug, AC input 230 V
55438-U
Certified Vial Kit, Low Adsorption (LA), 2 mL, pk of 100 29653-U
Featured Products
References1. What Do We Really Know About Roundup Weed Killer?
http://news.nationalgeographic.com/2015/04/150422-glyphosate-roundup-herbicide-weeds/ (accessed October 10, 2016).
2. Glyphosate; tolerances for residues. https://www.gpo.gov/fdsys/pkg/ CFR-2014-title40-vol24/xml/CFR-2014-title40-vol24-part180.xml#seqnum180.364 (accessed October 7, 2016).
3. Glyphosate, Environmental Chemistry Method. https://www.epa.gov/ sites/production/files/2014-12/documents/408816-01-w.pdf (accessed October 7, 2016).
4. Ehling, S.; Reddy, T.M. Total Residue Analysis of Glyphosate and Three Metabolites in Soy and Milk-based Nutritional Ingredients by Derivatization with Fluorenylmethyloxycarbonyl Chloride and Liquid Chromatography/Mass Spectrometry. Poster in Proceedings of ASMS 2016, San Antonio, TX.
5. Quick Polar Pesticides Method. Quppe.eu (accessed October 7, 2016).
6. Chamkasem,. N.; Harmon, T. Direct determination of glyphosate, glufosinate, and AMPA in soybean and corn by liquid chromatography/tandem mass spectrometry. Anal. Bioanal. Chem. 2016, 408(18), 4995-5004.
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Analysis of Bisphenol A and Analogous Compounds in Infant Formula Using Molecularly Imprinted Polymer Solid Phase Extraction and HPLC with Fluorescence Detection
Compound Full Name CAS NumberBPA 2,2-Bis(4-hydroxyphenyl)propane 80-05-7
BPC 2,2-Bis(4-hydroxy-3-methylphenyl)propane 79-97-0
BPG 2,2-Bis(4-hydroxy-3-isopropylphenyl)propane 127-54-8
BPF 4,4'-Methylenediphenol 620-92-8
BADGE 2,2-Bis[4-(glycidyloxy)phenyl]propane 1675-54-3
Olga I. Shimelis, Principal R&D Scientist; K. G. Espenschied, R&D Technician; and Jennifer Claus, Non-Bio Sample Preparation Product Manager
IntroductionBisphenol A (BPA) monomer is widely used for manufacturing plastics and epoxies. Such plastic materials are used to line food containers and metal cans. The monomer is never fully reacted, and detectable levels of BPA are known to leach from these materials into foodstuffs that contact them. Therefore, the presence of BPA in canned goods using these materials is presumed.
BPA levels in foodstuffs used for human consumption are regulated in the US and internationally but the regulations are not uniform. For example, some agencies may, and do hold that no risk to infant health is posed by using baby bottles made with plastics containing BPA. Others have moved to legally ban the use of BPA containing materials in any products used for infants and children.1,2 Acceptable levels of BPA contamination in products used by adult consumers are also subject to regulations. The overall result is testing protocols and concentration standards pertaining to BPA in food products and packaging vary accordingly; and BPA is a major analyte of interest in many testing laboratories.
There is, however, a trend to curtail the use of BPA in plastics associated with foods. In order to manufacture plastics without BPA, manufacturers are turning to other monomers with similar structures as substitutes for BPA. Among these compounds are Bisphenol S (BPS) and Bisphenol F (BPF). Scientific work indicates these compounds may be similar to BPA in terms of adverse human health effects. For this reason, the EU currently sets limits for Bisphenol S migration in food contact articles.3, 4 A need exists, therefore, for analytical methods capable of detecting and quantifying a range of compounds.
In this work, we explored the use of a SupelMIP® SPE - Bisphenol A cartridge for the extraction of five related compounds from infant formula. Molecularly imprinted polymers (MIPs) are designed for the selective extraction of specific compounds or classes of compounds from various matrices. The polymeric sorbents in these SPE cartridges contain areas of functionality created during the polymerization process designed to retain a molecule with a specific chemical structure. In the case of compounds with highly similar structures, cross-selectivity is possible. An SPE cartridge designed to select for BPA may also specifically bind analogous compounds such as Bisphenol F and others.
ExperimentalFive compounds were chosen for this work: BPA, BADGE, BPC, BPF, BPG. Chemical nomenclature and CAS numbers are included in Table 1.
Table 1. BPA and Analogous Compounds Studied in Infant Formula
SPE Tube SupelMIP SPE – Bisphenol A, 100 mg/3 mL (53775-U)
Conditioning 3 mL 2% formic acid in methanol, 3 mL acetonitrile, 3 mL water
Sample Addition 6 mL of prepared sample
Washing 9 mL of water, 6 mL of 40:60 acetonitrile:water; vacuum was pulled for 3 min
Elution 3 mL of 75:25 methanol:acetonitrile
Eluate post-treatment
evaporate to dryness under nitrogen
Table 2. Solid Phase Extraction (SPE) Procedure for the Extraction of BPA and Analogs
The premixed liquid infant formula sample was spiked with a mixture of BPA compounds at 10 ppb. A 5 mL volume of sample was mixed with 5.5 mL of acetonitrile and centrifuged at 4500 rpm for 10 min in a polypropylene centrifuge tube. The top layer was collected and 3 mL of the top layer was diluted with 3 mL of water for use as the SPE loading sample.
The SupelMIP SPE - Bisphenol A method was modified to ensure complete recovery of all five compounds. Our work indicated it was necessary to change the elution solvent from 100% methanol to 3 mL of 3:1 methanol:acetonitrile (v/v) in order to improve recoveries for all compounds. The full description of the SPE method used is presented in Table 2.
The samples were reconstituted into 0.5 mL of 40:60 acetonitrile:water. All samples were filtered through Durapore® filter units prior to analysis. The HPLC method incorporated a Titan™ UHPLC column that provided good retention and peak separation for all tested compounds (Figure 1).
(continued on next page)
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Results and Discussion
Analysis of BPA and Analogues – Elimination of Laboratory Background
Laboratory environments include a large variety of plastic materials. As a result, BPA contamination can be an issue when preparing and analyzing samples, especially when performing trace analyses. During our studies it became apparent that storage containers, auto-sampler vials, and caps can contribute to contamination, and even chromatography solvents can be contaminated. Our experience with BPA background when doing trace analysis resulted in the implementation of the following precautions as a means to reduce or eliminate environment BPA contamination:
• Reagent solutions and samples were prepared and stored in glass vials and containers
• For trace levels of studies, LC/MS grade solvents were used for sample preparation and analysis
• Vials and caps were screened for BPA prior to use
• Mixtures were gently swirled when possible, versus being vigorously shaken or vortexed to avoid contact of the liquid with the cap plastic liners
• Time during which the samples were in contact with any plastic vessels or tubes was minimized
• Sample analyses were conducted immediately after sample preparation
Significant BPA background arising from SPE tubes containing the sorbent was not observed. Plastic centrifuge tubes were used during the extraction step, and blank extracts did contain small amounts of BPA and BPG, possibly originating from the tubes (Figure 2.) The final results were corrected for these background values.
Figure 1. Injection of 10 ng/mL BPA and Analogous Compound Standard Mix column: Titan™ C18, 10 cm × 2.1 mm, 1.9 µm particles (577124-U)
mobile phase: (A) water (B) acetonitrile
flow rate: 0.25 - 0.40 mL/min
column temp.: 35 °C
detector: FLD, ex 230 nm, em 315 nm
injection: 5 µL
pressure : 270 bar
instrument: Agilent® 1290-Infinity II with Fluoresence detector:
gradient:
0 21 3 5 74 6 8
Min
1 23
45
Figure 2. Unspiked Blank Extracted Infant Formula Samples column: Titan C18, 10 cm × 2.1 mm, 1.9 µm particles (577124-U)
mobile phase: (A) water (B) acetonitrile
flow rate: 0.25 - 0.40 mL/min
column temp.: 35 °C
detector: FLD, ex 230 nm, em 315 nm
injection: 5 µL
pressure : 270 bar
instrument: Agilent 1290-Infinity II with Fluoresence detector
gradient:
0 21 3 5 74 6 8
Min
12
1. BPF2. BPA3. BPC4. BADGE5. BPG
1. BPA2. BPG
Recovery for BPA CompoundsThe SPE procedure was first developed and tested using spiked solvent solutions. The developed method was then applied to infant formula. Most compounds showed acceptable recovery from both as seen in Table 3. BPF recovery was low from infant formula at only 41%. It was found that this compound required more polar conditions during the extraction step than the rest of the compounds. The ratio of infant formula to the acetonitrile (5 mL to 5.5 mL) provided a good compromise for accepted recoveries of BPF and BADGE, the latter requiring less polar extraction conditions. All other compounds demonstrated recovery values of 70% or higher using the developed SPE method.
n=3 In Infant Formula In 20% AcetonitrileCompound Recovery RSD Recovery RSDBPA 70% 11% 91% 3%
BPC 76% 14% 82% 6%
BPF 41% 14% 91% 3%
BPG 70% 13% 91% 3%
BADGE 72% 10% 88% 3%
Table 3. Recoveries for BPA and Related Compounds Spiked Into Infant Formula and From Buffer at 10 ng/mL
Time (min) %A %B Flow (mL/min)0.00 60 40 0.252.50 20 80 0.257.00 20 80 0.257.10 0 100 0.409.50 0 100 0.409.60 60 40 0.25
12.50 60 40 0.25
Time (min) %A %B Flow (mL/min)0.00 60 40 0.252.50 20 80 0.257.00 20 80 0.257.10 0 100 0.409.50 0 100 0.409.60 60 40 0.25
12.50 60 40 0.25
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Figure 3. LC-Fluorescence Chromatogram of Extracted Infant Formula Sample, Spiked at 10 ppb column: Titan™ C18, 10 cm × 2.1 mm, 1.9 µm particles (577124-U)
mobile phase: (A) water (B) acetonitrile
flow rate: 0.25 - 0.40 mL/min
column temp.: 35 °C
detector: FLD, ex 230 nm, em 315 nm
injection: 5 µL
pressure : 270 bar
instrument: Agilent® 1290-Infinity II with Fluoresence detector
gradient:
0 21 3 5 74 6 8
Min
12
3 4 5
Description Cat. No.SupelMIP® Bisphenol A SPE Cartridge3 mL, pk of 50 52775-UDurapore® Membrane FilterMillex® syringe filter units, disposable, Durapore® PVDF, pk of 1000
Z227447
Titan C18 UHPLC Column10 cm × 2.1 mm I.D., 1.9 µm particle size 577124-UAnalytical Standards and ReagentsBisphenol A 239658BPG 50953BPF B47006BADGE D3415VialsABC Vial™, 2 mL (vial only), 9 mm thread, pk of 100 27330Screw cap (open-top), 9 mm thread, with liner, pk of 100 27326
Featured Products
Related Information
Visit our “Food & Beverage Analysis” web portal at sigma-aldrich.com/food
ConclusionBisphenol A (BPA) and four structurally related compounds were extracted from infant formula using SupelMIP SPE-Bisphenol A. The results demonstrated that the same molecularly imprinted polymer (MIP) phase developed for BPA also works to extract compounds of similar chemical structure. The analysis was performed using a Titan C18 UHPLC column with fluorescence detection which gave baseline resolution for all five peaks. The samples were spiked at 10 ppb, and, with the exception of BPF, recoveries for all compounds were above 70%.
1. BPF2. BPA3. BPC4. BADGE5. BPG
Time (min) %A %B Flow (mL/min)0.00 60 40 0.252.50 20 80 0.257.00 20 80 0.257.10 0 100 0.409.50 0 100 0.409.60 60 40 0.25
12.50 60 40 0.25
References1. INFOSAN Information Note No. 5/2009 - Bisphenol A. Bisphenol A .BISPHENOL A
(BPA) - Current State of Knowledge and Future Actions by WHO and FAO, November 2009, http://www.who.int/foodsafety/publications/fs_management/No_05_Bisphenol_A_Nov09_en.pdf (accessed April 2016)
2. Rubin, B.S. Bisphenol A: an endocrine disruptor with widespread exposure and multiple effects. J. Steroid Biochem, 2011, 127, 27-34.
3. Eladak, S.; Grisin, T.; Moison, D.; Guerquin, M-J.; N'Tumba-Byn, T.; Pozzi-Gaudin, S.; Benachi, A.; Livera, G.; Rouiller-Fabre, V.; Habert, R. A new chapter in the bisphenol A story: bisphenol S and bisphenol F are not safe alternatives to this compound. Fertility and Sterility, 2015, 103, 11-21.
4. Food Packaging Forum: Bisphenol S. http://www.foodpackagingforum.org/food-packaging-health/bisphenol-s (accessed April 2016).
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Results and Discussion
UHPLC-qMS Method
When a large number of samples is analyzed, as in clinical cohorts, the total run time for analytical analysis plays an important role. A compromise is needed to maximize the sample throughput and the lipidome coverage, shorten the analysis time, limiting the peak capacity decreasing and maintaining the high quality MS information. The most suitable choice to achieve this compromise is the employment of a UHPLC system capable of operations at high back pressures up to 1200 bar. Using a UHPLC system, columns with sub-2 μm, like the Titan™ C18 column used here, particles can be operated at high mobile phase flow rates, thus reducing the analysis time without loss of resolution under optimized conditions.2 Until 2014, 65% of lipidomics studies developed reported analysis run time of about 30 minutes.3 Different solvents in combination with water were tested to optimize the UHPLC method. The water level in the mobile phase is particularly critical at the beginning of the gradient in reversed phase (RP) LC, affecting the chromatographic resolution of polar lipids. Higher concentration of water significantly improves not only the elution profile, but also the signal-to-noise ratio.4 The use of methanol or acetonitrile as solvent B, independently from the gradient applied, required a rather long separation time (> 30 minutes) to elute TAGs (triacylglycerols) and CEs (cholesterol esters).
Better results were achieved by using water containing 20 mM ammonium formate as solvent A and a mixture of isopropanol:acetonitrile:20 mM of ammonium formate (60:36:4 v/v/v) containing formic acid 0.1% as solvent B. The relatively low pH, achieved by using formic acid, allowed minimal tailing of FFAs (free fatty acids) due to the interaction of an ionized carboxyl function with free silanol sites on the LC column packing. The chromatographic pattern obtained partially fitted to the well-known model used for TAGs identification in RP-LC, where the retention of lipids increases proportionally to their Equivalent Carbon Number (ECN).4-6 The chromatographic LC method proposed was suitable, without any adjustments, to be used with both ESI and APCI interfaces. In such a way, two chromatographic profiles, perfectly equivalent in terms of retention times, were obtained. Complementary MS information can be extrapolated for a comprehensive and detailed characterization of each single sample.
The lipid extract from human plasma was analyzed by using the described UHPLC-ESI/APCI-qMS method. Starting from polar lipids, free fatty acid (FFA), and lysophospholipid (LPL) species, to non-polar lipids, triacylglycerols (TAG), and cholesterol esters (CE), all lipids elute within a 20 minute window. Figure 1 shows TIC (+) chromatogram by RP-UHPLC-APCI-MS of a human plasma sample. Figure 2 shows the mass spectra of PC-18:2/16:0 (FAs in phospholipids follow the nomenclature previously proposed for TAGs, not considering sn-position) from a human plasma sample using ESI and APCI interfaces in both positive and negative ionization modes.6
Analysis of Human Plasma Lipids Using sub-2 µm C18 UHPLC Column with MS Detection
Luigi Mondello, Chromaleont s.r.l., c/o “Scienze del Farmaco e Prodotti per la Salute” Department, University of Messina, viale Annunziata, 98168 Messina, Italy
IntroductionLipidomics is a branch of metabolomics that, through an in-depth characterization, investigates the structures, functions and dynamic changes of lipids in cells, tissues, or bodily fluids. Moreover, lipidomics usually studies the correlation between the lipid profiles of biological samples and the health status of the human organism. The high level of structural diversity in lipid classes present in biological samples makes their characterization a very challenging task. The aim of this work was to develop a simple, fast, and versatile UHPLC-MS method. The method developed was suitable for both ESI and APCI MS-interfaces, for untargeted lipid profile characterization of human plasma.
Experimental
Sample and Sample Preparation
The lipid fraction was extracted according to a modified Folch method.1 Briefly, 1 mL of plasma was placed in a centrifuge tube with 9 mL of chloroform:methanol (2:1 v/v), extracted for three times and centrifuged for 20 minutes at 3,000 rpm. To facilitate the separation between the phases, 500 µL of water was added to the previous mixture. The lower lipid containing organic phases were combined, dried with anhydrous Na2SO4, filtered on filter paper, and then dried using a rotary evaporator. Final extracts were dissolved in 500 µL of isopropanol:methanol (1:1 v/v) and injected into the LC/MS system.
LC/MS Instrumentation
The analyses were performed on a Shimadzu Ultra High Performance Liquid Chromatograph-Nexera system. The UHPLC system was coupled to an LC-MS-2020 quadrupole mass spectrometer equipped with both ESI and APCI interfaces and coupled to an LCMS-IT-TOF equipped with an ESI interface. The samples were simultaneously analyzed in full scan mode and under selected-ion monitoring (SIM) acquisition modes.
MS Parameters: Full-scan LC/MS chromatograms were obtained by scanning from m/z 350-1250, with a scan speed of 5000 amu/sec and an event time of 0.2 sec, in positive mode both for APCI and ESI, and from m/z 150-1250 and 160-1250 with a scan speed of 6000 amu/sec and an event time of 0.2 sec, in negative mode for APCI and ESI, respectively. ESI parameters were as follows: nebulizing gas (N2) flow rate: 2 L/min; drying gas (N2) flow: 15 L/min; detector voltage: 1.5 kV; interface voltage: 4.5 kV; desolvation line (DL) temperature: 250 °C; heat block temperature: 200 °C. APCI parameters were as follows: nebulizing gas (N2) flow rate: 3 L/min; drying gas (N2) flow: 15 L/min; detector voltage: 1.5 kV; interface voltage: 4.5 kV; interface temperature: 450 °C; DL temperature: 250 °C; heat block temperature: 200 °C.
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Figure 1. TIC (+) Chromatogram of Human Plasma Sample by UHPLC-APCI-qMS
column: Titan C18, 10 cm × 2.1 mm I.D., 1.9 µm particles (577124-U) mobile phase: [A] 20 mM ammonium formate; [B] 0.1% formic acid in isopropanol:acetonitrile:20 mM ammonium formate (60:36:4), v/v) gradient: 80 to 100% B in 6 min, held for 16 min, to 80% B in 0.1 min, held for 3 min flow rate: 0.4 mL/min column temp.: 40 °C detector: MS/MS, m/z 350-1250, multiple transitions injection: 1 µL sample: human plasma extract in isopropanol:methanol (1:1 v/v)
×100,000,000
1.50
1.25
1.00
0.75
0.50
0.25
0.002.5 5.0 7.5 10.0 12.5 15.0 17.5 19.0
Min
LPLsFFAs
CholPLsSMsDAGs
TAGsCEs
1:TIC(+)
Figure 2. MS Spectra of Phosphocholine C34:2 (PC-C18:2-C16:0)
Conditions same as Figure 1.
[M+Na]+, 780
400 500 600 700 800 900 m/z
100
75
50
25
0
Inte
n. %
(+)
[M+H]+, 758
[M+H]+, 758
Inte
n. %
(+)
[M+H-183]+, 575
[M+H-59]+, 699
[M+H-41]+, 717
[M-CH2+H]+, 744100
75
50
25
0400 500 600 700 800 900
m/z
782
897
Inte
n. %
(-)
776
[M+HCOO]-, 802
870
m/z
100
75
50
25
0200 300 400 500 600 700 800 900
768
Inte
n. %
(-)
m/z
[C16:0-H]-, 255
[M-C18:2-86]-, 391
[M-(C17:2=C=O)-86]-, 407
[M-C16:0-86]-, 415
[M-(C15:0=C=O)-86]-, 431
[M-86]-, 671
[M-72]-, 685
[M-60]-, 697
[M-CH3]-, 742100
75
50
25
0200 300 400 500 600 700 800 900
[C18:2-H]-, 279
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References1. Folch, J.; Lees, M.; Sloane Stanley, G. H. A simple method for the isolation
and purification of total lipids from animal tissues. J. Biol. Chem., 1957, 226, 497–509.
2. Chen, S. I.; Hoene, M.; Li, J.; Li, Y. J.; Zhao, X. J.; Haring, H. U. Simultaneous extraction of metabolome and lipidome with methyl tert.butyl ether from a small tissue for ultra high performace liquid chromatography/mass spectrometry. J. Chromatogr. A., 2013, 1298, 9-16.
3. Cajka, T.; Fiehn, O. Comprehensive analysis of lipids in biological systems by liquid chromatography-mass spectrometry. Trends in Analytical Chemistry, 2014, 61, 192–206.
4. Lisa, M.; Cifkova, E.; Holkapek, M. Lipidomic profiling of biological tissues using off-line two-dimensional high-performance liquid chromatography-mass spectrometry. J. Chromatogr. A, 2011, 1218, 5146–5156.
5. Holčapek, M.; Lísa, M.; Jandera, P.; Kabátová, N. Quantitation of triacylglycerols in plant oils using HPLC with APCI-MS, evaporative light-scattering, and UV detection. J. Sep. Sci., 2015, 28, ,1315–1333.
6. Beccaria, M.; Costa, R.; Sullini, G.; Grasso, E.; Cacciola, F.; Dugo, P.; Mondello, L. Determination of the triacylglycerol fraction in fish oil by comprehensive liquid chromatography techniques with the support of gas chromatography and mass spectrometry data. Anal. Bioanal. Chem., 2015, 407, 5211–5225.
Featured Product
Description Cat. No.Titan C18, 10 cm x 2.1 mm I.D., 1.9 µm particles 577124-U
While ESI shows related molecular ions, both in positive and negative ion modes, APCI shows as base peak, in positive ion mode, the diacylglycerol specie due to the loss of head polar group [M-head polar group]+ (head polar group: 183 m/z), followed by different related molecular ions [M+H]+, [M+H-CH2]+, and [M+Na-CH2]+. In negative ion mode, a set of four parent ions [M-CH3]-, [M-60]- (loss of trimethylamine group), [M-86]- (loss of choline residue group), and an undefined ion, [M-72]-, related to a partial choline fragmentation, were observed. The simultaneous loss of choline residue (86 m/z) and a fatty acid from the backbone of glycerol allowed the characterization of fatty acids profile into PCs species.
ConclusionsThe proposed splitless method can be a comprehensive platform for lipidomics studies. It can be applied without any modification in chromatographic conditions (mobile phase composition and/or flow rate), coupled to both ESI and APCI interfaces. Such a chromatographic method can be easily reproduced. Although the sensitivity of APCI-MS is usually less than ESI-MS when a buffer is added to the mobile phase, the structural information to be gleaned from the fragmentation is well worth the trade off in sensitivity. However, in some cases, ESI is an indispensable complement to APCI when lipids containing oxygen functional groups are investigated, or for confirming related molecular ions that are generally less expressed by APCI. LC-qMS can be a valid, simple, and economical technique for lipidomics studies, and can provide similar information compared to more sophisticated techniques.
Pharmaceutical
Low Adsorption Vials
Supelco’s Low Adsorption, Center Drain (CD™), MRQ30, and QsertVial™ products offer the benefit of maximum sample extraction without the worry of trace analytes being adsorbed by the vial surface.
• Maintains sample integrity during storage
• Minimizes pH shifts in the sample
• Reduces metal contamination in the sample
• Compatible with most autosamplers
For more information, visit sigma-aldrich.com/lavials
23Order: 800-325-3010 (U.S.) 814-359-3441 (Global)
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Rapid Determination of Protein Binding Affinity Using Solid Phase Microextraction
Emily R. Barrey, Senior Scientist, R&D and Craig Aurand, Innovation Manager, Advanced Analytical
Determination of free circulating drug concentration is important in establishing pharmacokinetic activity. In most cases, drug-protein complexes could be formed which affect the active level of circulating drugs in the body. Techniques used for determining drug-protein binding levels consist of ultrafiltration, ultracentrifugation, and microdialysis. Automation can be used in the case of microdialysis, but processing may be greater than 6 hours due to the need to reach equilibrium. In this study, a novel biocompatible microextraction device is evaluated as a rapid means of extracting the non-bound fraction of drugs from plasma. Initial studies demonstrate that drug binding affinities can be determined in less than 60 minutes using this microextraction technique. A model set of protein binding drugs was selected and their free concentrations were compared using the SPME LC (BioSPME) approach and the equilibrium dialysis technique (rapid equilibrium dialysis device-RED device). Drugs with reference binding affinities ranging from 20%-99% were selected for comparison of the sampling devices.
The BioSPME technique enables direct analysis of biological samples without the need for protein precipitation, centrifugation, or digestion. The BioSPME technique allows for isolation of target analytes while minimizing coextraction of sample matrix, allowing for more sensitive and robust analysis. The extraction mechanism for the SPME LC utilizes differential migration that is dependent upon the affinity of the analyte for the phase coating on the fiber to the affinity for the matrix. In the case of the SPME LC fibers, the polymeric binder used to adhere the phase onto the fiber core acts as a shield that prevents large molecular weight molecules (i.e. proteins) from absorbing onto the fiber, thus allowing for only the free fraction (unbound) analyte to be extracted by the fiber coating. Figure 1 depicts a representation of the extraction mechanism for the SPME LC fibers.
ExperimentalCustom standard mixes containing codeine, diazepam, diclofenac, propranolol, warfarin, and quinidine were prepared. Labeled internal standards were also obtained for all of the compounds except quinidine. Rat plasma stabilized with K2EDTA (BioReclamation, IVT, Hicksville, NY USA) was spiked at 200 ng/mL of binding analytes and allowed to equilibrate for 3 hours at 37 °C prior to extraction studies. Phosphate buffered saline (PBS, pH 7.4) was prepared with the following concentrations: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4. PBS was spiked at 200 ng/mL with the analytes of interest and this solution was used for the SPME extraction studies. Blank PBS was used for the RED device binding studies. Samples were analyzed using an Agilent 1290 Infinity II UPLC with a 6460 QQQ mass spectrometer. The mass spectrometer parameters are listed in Table 1.
Compound Name Precursor Ion Product Ion Fragmentor Collision EnergyCodeine 300.2 152.1 125 76
Codeine-d3 303 152.1 125 76
Diazepam 285.1 193.1 166 32
Diazepam-d5 290.11 198 145 32
Diclofenac 296 214 90 32
Diclofenac-d6 343.15 289.1 80 8
Propranolol 260.2 56.2 120 28
Propranolol-d7 267.2 56.2 120 28
Quinidine 325.2 81.2 150 36
Warfarin 309.1 163 100 8
Warfarin-d5 314.15 163 105 8
Table 1. LC/MS Parameters Used for Detection of Drugs of Abuse and the Concentration in Standard Solution Shown in Figure 2.
Metal Core
Bio�uid
Well
Extraction Time
Embedded Functionalized Particles
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lyte
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Figure 1. Extraction Mechanism for SPME LC Fibers
SPME LC C18 Preparation
Fibers were conditioned by soaking in methanol for 10 minutes. Fibers were then equilibrated by soaking in water for 10 minutes. The samples were prepared by placing 800 µL of plasma and buffer samples into a 2.0 mL Nunc® 96-well plate. The SPME extraction was conducted by placing the SPME C18 fibers directly into 800 µL of plasma or buffer and agitated for 30 minutes at 500 rpm. Five sample replicates were prepared. Fibers were then transferred from the samples and placed directly into a 600 µL conical 96-well plate that had been prefilled with 300 µL of internal standard desorption solvent (50 ng/mL in acetonitrile). The samples were desorbed in the well plate by agitating for 10 minutes at 500 rpm. The SPME fibers were removed, and the well plate was capped, vortexed, and analyzed directly.
Reporter 34.4 |24
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RED Device Preparation
The RED Device samples were prepared by placing 200 µL of plasma into the plasma compartment of the RED device and 350 µL of buffer into the buffer compartment. The RED device was capped with a sealing mat and equilibrated at 37 °C at 250 rpm for 4 hours. After the equilibration step, 50 µL aliquots were taken from each compartment, 50 µL of plasma was added to the buffer compartment aliquot, and 50 µL of the buffer was added to the plasma compartment aliquot. After mixing, 300 µL of ice cold acetonitrile with 50 ng/mL internal standard were added. Samples were vortexed at 1200 rpm for ~ 5 minutes and then centrifuged at 15,000 rpm for 10 minutes. The supernatants were decanted into a glass HPLC vial for analysis.
ResultsThe total SPME processing time took ~ 40 minutes, whereas the total processing time for the RED Device was ~ 5 hours. A typical chromatogram and calibration curve are shown in Figures 2 and 3. Analyte binding concentrations were calculated with the following equations:
Analyte Binding Calculation for SPME LC Technique
% protein bound = (concentration PBS – concentration plasma) × 100
concentration PBS
Analyte Binding Calculation for RED Equilibrium Dialysis Technique
% protein bound = 100% - concentration PBS chamber
× 100% concentration plasma chamber
Figure 2. Chromatogram of Analytes Used for Protein Binding column: Ascentis Express C18, 5 cm × 2.1 mm I.D., 2.7 µm
particles (53822-U) mobile phase A: 5 mM ammonium formate mobile phase B: 5 mM ammonium formate in 90:10 acetonitrile:water flow rate: 500 µL/min column temp.: 40 °C detector: MS/MS, ESI (+), MRM transitions injection: 2 µL gradient: Min %A %B 0 95 5 3 30 70 3.1 10 90 4 10 90 4.1 95 5 6 95 5 pressure: 380 bar
instrument: Agilent 1290 Infinity II with Agilent 6460 QQQ
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2. Quinidine 1.68
3. Propranolol 1.95
4. Warfarin 2.42
5. Diclofenac 2.50
6. Diazepam 2.78
Figure 3. Representative Calibration Curve in Solvent for Propranolol
-0.5 0.5
00.20.40.60.8
11.21.41.6
Propranolol - 6 Levels, 6 Levels Used, 12 Points Used, 2 QCs
y = 1.610827 * x + 0.004609R^2 = 0.99953636Type: Linear, Orgin: Ignore, Weight: 1/x
x101
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Codeine 0.9987 1.6
Quinidine 0.9959 748.8
Propranolol 0.9995 1.61
Warfarin 0.994 57.9
Diclofenac 0.9961 5.12
Diazepam 0.9985 1.09
Pharmaceutical
Table 2. Binding Affinity Comparison
0.0
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Codeine Quinidine Propranolol Warfarin Diclofenac Diazepam
Protein Binding Levels
SPME LC Tips RED Device Ref
25Order: 800-325-3010 (U.S.) 814-359-3441 (Global)
ConclusionsIn the case of the SPME LC analysis, the free fraction of the analyte is measured in both the reference (PBS) and the plasma sample. This technique simplifies the calculation for determining the protein binding level. The SPME LC approach allows for direct sampling of the plasma sample, eliminating the need for protein precipitation as in the equilibrium dialysis device. The ability to concentrate analytes onto the fiber and desorb them into smaller volumes enables lower detection limits. This approach will be beneficial especially for those analytes that have high binding affinities and low MS sensitivity. This direct sampling approach also minimizes concern associated with matrix interference. Plasma protein precipitation will not remove matrix interferences like phospholipids, whereas the phospholipids will not be extracted onto the SPME fiber.
Protein binding affinities for both the SPME LC and the equilibrium dialysis devices closely matched the referenced range for all analytes. By utilizing the SPME approach, binding affinities correlating to the reference values were obtained in a significantly shorter time span compared to membrane dialysis techniques.
References1. Banker et al., J Pharm Sci, 2003, 92, 967-974.
2. Edwards et al., J Pharm Sci, 1984, 73, 1264-1267.
3. Moffat et al., Clarke’s Isolation and Identification of drugs in pharmaceuticals, body fluids, and post-mortem material. 2nd ed. London: The pharmaceutical press, p. 490-491.
4. Waters, et.al., J Pharm Sci, 2008, 97, 4586-4595.
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Description Cat. No.SPME-LC Pipette Tips, 96-tip array, C18 coating, pk of 96 57234-UAscentis Express C18, 5 cm × 2.1 mm I.D., 2.7 µm particles 53822-U
Description Cat. No.SPME LC Pipette Tips, 96-tip array, PDMS/DVB coating, pk of 96 57248-USPME-LC Fiber Needle Probe, C18 coating, pk of 5 57281-U
Supelco® Introduces SFC Columns from ES Industries
Supelco is pleased to announce the introduction of GreenSep™ SFC (Supercritical Fluid Chromatography) columns, as well as ChromegaChiral™ columns for HPLC and SFC, from ES Industries.
SFC – A "Green" Chromatographic TechniqueGreenSep
• The right choice for achiral purification
ChromegaChiral
• An innovative chiral HPLC stationary phase
To view product information and availability for GreenSep and ChomegaChiral SFC columns, visit
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Biochromatography
Reaching the “Peak” of Recovery: Improving Antibody Separations with the Inclusion of Organic Alcohols
Hillel Brandes, Supervisor, Principal R&D Chemist and Cory E. Muraco, Senior R&D Scientist
Monoclonal antibodies (mAbs) are a promising class of biologics for the treatment of several autoimmune diseases and cancers. An additional application of mAbs, however, is when a cytotoxic payload (i.e., drug) is attached to the mAb, allowing the mAb to target a certain cell type or tissue and deliver the payload to the specific target. This combination of mAb plus cytotoxic drug, connected through an organic linker, is known as an antibody-drug conjugate (ADC). As of July 2016, there are approximately 60 ADCs in the pharmaceutical pipeline; of those, 25% are in late Phase II or Phase III clinical trials.10
One downside of mAb-based drugs, however, is that, due to their structural complexity, there is significant heterogeneity. This heterogeneity can arise due to the presence of charge variants, glycosylation variants, phosphorylation variants, and/or payload variants, that arise by nature of the biological and chemical production processes.
Several different chromatographic strategies are applied to investigate and resolve the different structural and chemical variants of mAbs. Size exclusion chromatography is used as one method to assess the aggregation of a mAb sample. Ion-exchange chromatography is a method to conduct charge variant analysis. Both of these chromatographic modes can present issues of compatibility with electrospray ionization mass spectrometry (ESI-MS), which is routinely used for protein characterization. Hydrophilic interaction chromatography is routinely employed for analysis and characterization of protein glycans. Reversed-phase chromatography (RPC) has long been a method of choice for analyzing proteins due to its high resolution and compatibility with MS. RPC of proteins, however, has its own issues. Of primary significance is that protein structures can be flexible in comparison to structures of small organic molecules. This fact may present a chromatographic challenge as various structural conformations may differentially interact with the stationary phase. With proteins, peak shape in RPC is generally enhanced by parameters that stabilize a single denatured state.1-3 Temperature is one parameter that can dramatically affect the tertiary and quaternary structure of proteins and thus a “denatured state”.
Another aspect of protein and peptide reversed-phase chromatography is that, for most applications, elution must be by utilization of a solvent strength gradient. This requirement is due to at least two reasons: 1) polypeptides are generally polyionic, and, therefore, can present problems of secondary interactions with the silica surface, potentially causing issues of peak tailing and 2) partitioning of polypeptide analytes between the mobile and stationary phase occurs over a narrow window of solvent strengths (as compared to most small molecules), therefore exhibiting much more of an on-off adsorption phenomenon. With the requirement
for gradient elution comes the requirement for column re-equilibration prior to injection of a sample. Column re-equilibration can be shorten by reducing changes to the solvation state of the silica surface as has been shown by inclusion of low levels of small, primary alcohols in the mobile phase.4,5 How this mechanistically takes place has not been defined, but computer modelling of short, primary alcohols in binary mobile phase systems is consistent with intercalation of the alcohol into the stationary phase, with the hydroxyl hydrogen-bonding to the surface silanols or an adsorbed water layer.6 This phenomenon may have additional benefits in masking silanols, therefore improving peak shape. Scott & Simpson reported that 1-butanol can form a simple monolayer on a C18-bonded silica surface;7 this too fits with a model of a small, primary alcohol hydrogen-bonding to the surface silanols.
High temperature has been shown to be necessary in achieving optimal recovery and peak shape in RPC of mAbs.8 This fact has been confirmed to be the case, irrespective of the column used or the specific mAb sample.9 Additionally, Fekete et al. have shown that inclusion of low levels of 1-butanol reduced the temperature optimum for the RPC of the mAb.8 Thus, the mechanism of any conferred benefits of inclusion of low levels of primary alcohols in the mobile phase is not entirely clear. These previously published data suggest a primary mechanism of masking of the silica surface; another possibility might be imagined to explain the effects on antibody chromatography in which the intercalated 1-butanol is oriented with the hydroxyl facing the bulk mobile phase, thus lending some polarity to the environment at the antibody-stationary phase interface. Such models might be elucidated by inspecting results with analogs of 1-butanol.
This hypothesis was investigated further using a mAb standard, SiLu™ Lite SigmaMAb, catalog number MSQC4. Initially, the goal was to at least confirm previous reports on the chromatographic effects of 1-butanol on the RP chromatography of mAbs. Two primary alcohols, 1-propanol and 1-butanol, were investigated. Figure 1 shows chromatographic results of the recovery of SigmaMAb at varying percentages of 1-butanol at 55 °C.
As noted in Figure 1, the peak area and height of the analyte increased as the concentration of 1-butanol increased. In addition, as the concentration of 1-butanol increased, one can begin to resolve impurities from the main analyte peak. This phenomenon was further investigated by looking at how temperature played a role in the recovery of SigmaMAb. The results of this analysis are displayed in Figure 2.
As can be seen in Figure 2, it should become obvious that one of the main advantages of including 1-butanol in the mobile phase is the much lower temperature required to achieve maximum recovery of the analyte. The data, however, cannot differentiate effects due to possible mitigation against thermal degradation or impacts on the actual chromatography of the mAb. The experiment was repeated, this time with 1-propanol as the mobile phase modifier. Figure 3 shows the results of this analysis.
8070605040
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27Order: 800-325-3010 (U.S.) 814-359-3441 (Global)
Figure 1. Analysis of SigmaMAb by RPC with Varying Amounts of 1-Butanol
column: BIOshell A400 Protein C4, 10 cm × 4.6 mm I.D., 3.4 µm
mobile phase: [A] 70:30, (0.1% TFA in water):(0.1% TFA in acetonitrile); [B] 60:40, (0.1% TFA in water):(0.1% TFA in acetonitrile); [C] 70:25:5, (0.1% TFA in water):(0.1% TFA in acetonitrile): (0.1% TFA in alcohol); [D] 60:35:5, (0.1% TFA in water): (0.1% TFA in acetonitrile):(0.1% TFA in alcohol)
gradient: [(100-x)% A, 0% B, x% C, 0% D] to [0% A, (100-x)% B, 0% C, x% D] in 15 min, where x = 0, 20, 40, 60, 80, or 100
flow rate: 1.0 mL/min
column temp.: as indicated
detector: UV, 215 nm
injection: 3.0 µL
sample: SigmaMAb (catalog number MSQC4), 1 g/L, 0.05% TFA in water
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Figure 2. Recovery, Measured by Peak Area, as a Function of Temperature and 1-Butanol ConcentrationNote that the maximum recovery of the analyte occurs at much lower temperatures as the concentration of 1-butanol increases. Chromatographic conditions are the same as those described in Figure 1.
Figure 4. Recovery, Measured by Peak Area, as a Function of Temperature and 2-Butanol ConcentrationConditions the same in Figure 1.
Figure 3. Recovery, Measured by Peak Area, as a Function of Temperature and 1-Propanol ConcentrationNote the much reduced recovery at 50 °C with 1-propanol versus 1-butanol. Chromatographic conditions were the same as those described in Figure 1.
Figure 5. Recovery, Measured by Peak Area, as a Function of Temperature and 1,4-Butanediol ConcentrationNote that the concentration of 1,4-butanediol does not appear to have an effect on recovery.
Reporter 34.4 |28 Biochromatography
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As shown in Figure 3, the temperature required to achieve the maximum recovery of the analyte was much higher (around 80 °C) in comparison to 1-butanol. While 1-propanol has been shown to have similar benefits as 1-butanol for keeping the silica surface solvated,4 and shielding surface silanols,5 it clearly did not provide the same chromatographic benefits in this case, as compared to 1-butanol.
Continuing the investigation, the effect of type of alcohol (primary, secondary, etc.) on analyte recovery was examined. As noted in Figure 4, the secondary alcohol elicits good recovery of the antibody albeit not as good as 1-butanol. It could be inferred that, due to steric effects, 2-butanol should be not as effective as primary alcohols in hydrogen-bonding with surface silanols or an adsorbed water layer. However, 2-butanol achieved higher recovery of the analyte at low to moderate temperatures than with 1-propanol, suggesting that the mechanistic explanation is not as simple as hydrogen-bonding to the silica surface.
A final test was to try 1,4-butanediol. The idea is that this alcohol could possibly play a dual role of hydrogen-bonding to the silica surface as well contributing additional polarity to the interface where protein adsorption takes place on the stationary phase. The results are shown in Figure 5.
As can be deduced from Figure 5, there appears to be no advantage in adding 1,4-butanediol to the mobile phase. Perhaps the two terminal hydroxyls render it sufficiently polar such that it no longer readily distributes within the stationary phase to provide any performance benefit to the reversed-phase chromatography.
Despite these seemingly, conflicting results, what emerges is a picture in which the role of the alcohol in conferring a chromatographic benefit to the RPC of mAbs (or maybe most any other IgG molecule) is not as simple as has been suggested from other chemical and computer modeling studies – that something other than masking the silica surface is at play here. Perhaps there is a separate effect on the thermal stability of the mAb in this common mobile phase. Nevertheless, as shown in Figures 1 and 2, the addition of 1-butanol to the mobile phase can elicit excellent recovery of an antibody standard at far lower temperatures than in its absence and can serve as a general method for RPC of antibodies, or perhaps any other proteins that exhibit poor peak shape at moderate temperatures.
References1. Nugent, K. D., W. G. Burton, T. K. Slattery, B. F. Johnson, and L. R. Snyder.
Separation of Proteins by Reversed-Phase High-Performance Liquid Chromatography: II. Optimizing Sample Pretreatment and Mobile Phase Conditions. Journal Chromatogr. A 1988. 443 : 381–97.
2. Benedek, K., S. Dong, and B. L. Karger. Kinetics of Unfolding of Proteins on Hydrophobic Surfaces in Reversed-Phase Liquid Chromatography. Journal Chromatogr. 1984. 317: 227–43.
3. Lu, X. M., K. Benedek, and B. L. Karger. Conformational Effects in the High-Performance Liquid Chromatography of Proteins Further Studies of the Reversed-Phase Chromatographic Behavior of Ribonuclease A. Journal Chromatogr. A 1986 359: 19–29.
4. Cole, Lynn A., and John G. Dorsey. Reduction of Reequilibration Time Following Gradient Elution Reversed-Phase Liquid Chromatography. Analytical Chemistry 1990. 62: 16–21.
5. Schellinger, Adam P., Dwight R. Stoll, and Peter W. Carr. High Speed Gradient Elution Reversed Phase Liquid Chromatography of Bases in Buffered Eluents: Part II. Full Equilibrium. Journal Chromatogr. A 2008. 1192 (1): 54–61.
6. Rafferty, Jake L., Ling Zhang, J. Ilja Siepmann, and Mark R. Schure. Retention Mechanism in Reversed-Phase Liquid Chromatography: A Molecular Perspective. Analytical Chem 2007. 79 (17): 6551–58.
7. Scott, R. P. W., and C. F. Simpson. Solute-Solvent Interactions on the Surface of Reverse Phases. Interactive Characteristics of Some Short-Chain Aliphatic Moderators Having Different Functional Groups. Faraday Symp. Chem. Soc. 1980. 15: 69–82.
8. Fekete, Szabolcs, Serge Rudaz, Jean-Luc Veuthey, and Davy Guillarme. Impact of Mobile Phase Temperature on Recovery and Stability of Monoclonal Antibodies Using Recent Reversed-Phase Stationary Phases. Journal Sep Sci 2012. 35 (22): 3113–23.
9. Eksteen, R.; Bell, D.; Brandes, H.; Using Temperature to Improve Peak Shape of Hydrophobic Proteins in Reversed-Phase HPLC. HPLC 2014 Poster. Supelco/Sigma-Aldrich. T414073.
10. Beck, A., G. Terral, F. Debaene, E. Wagner-Rousset, J. Marcoux, M-C. J. Bussat, O. Colas, A. Van Dorsselaer and S. Cianféranib. Cutting-edge mass spectrometry methods for the multi-level structural characterization of antibody-drug conjugates. Expert Rev Proteomics 2016. 13(2): 157-183.
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Description Cat. No.BIOshell A400 Protein C4, 10 cm × 4.6 mm I.D., 3.4 µm 66828-U
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Robustness of the Discovery® HS F5 HPLC Column in the Study of Metabolomics
Nathaly Reyes-Garcés, Barbara Bojko, Janusz Pawliszyn, Department of Chemistry, University of Waterloo, Ontario, Canada N2L 3G1
IntroductionThe performance of the Discovery HS F5 HPLC column was monitored during the metabolomics study executed on a liquid chromatograph – high resolution mass spectrometer (LC/MS) platform. The comparison of chromatograms of selected compounds was done before and after 170, 450, 730, 1125, and 1525 injections of tissue extracts obtained with solid phase microextraction. The results showed excellent robustness, stability, and reproducibility of the column in combination with highly efficient sample clean-up offered by SPME.
Metabolomics is currently one of fastest developing areas of bioanalysis. Its main goal is to characterize the full profile of small molecules in the system under study. In order to cover analytes with a very wide range of different physical and chemical properties, the elements of an analytical protocol should be carefully optimized and validated. High method precision is required in order to avoid false positive results and enhance proper identification of the extracted analytes. Herein, the performance of the Discovery HS F5 HPLC column after using solid phase microextraction as a sample preparation tool will be evaluated for use in the study of metabolomics.
Experimental Human brain tumors and piglet lung were sampled (30 min extraction time) with 7 mm mix-mode SPME fibers in vitro and in vivo, respectively. The extracted analytes were subsequently desorbed in acetonitrile:water, 1:1 v/v and extracts were analyzed via LC/MS containing the following components: Accela autosampler with cooled system tray, Accela LC Pumps, and Exactive Orbitrap mass spectrometer (Thermo, San Jose, CA, US). The MS experiments were performed in both positive and negative ionization mode. A 10 cm × 2.1 mm, 3 µm particle size PFP column connected to a Discovery HS F5 guard column cartridge (2 cm × 2.1 mm, 3 µm) were used in the study.
For analysis in positive ionization mode, mobile phase A consisted of water:formic acid, 99.9:0.1, v/v and mobile phase B consisted of acetonitrile:formic acid (99.9:0.1, v/v). For analysis in negative ionization mode, mobile phase A consisted of a 1 mM NH4F solution and mobile phase B consisted of acetonitrile. The flow rate of the mobile phase was 300 µL/min. The following gradient elution was used: 100% A from 0 to 3.0 min, followed by a linear gradient to 10% A from 3.0 to 25.0 min, and an isocratic hold at 10% A until 34.0 min. The total run time was 40 min per sample, including a 6 min column re-equilibration time. The injection volume was 10 μL.
The QC used for monitoring of column performance in ESI+ consisted of tranexemic acid, trans-4-(Aminomethyl)cyclohexanecarboxylic acid (ACHCA), phenylalanine, phenylalanine-d5, tryptophan and progesterone. Taurocholid acid, cholic acid, and chenodeoxycholic acid-d4 in ESI-.
ResultsSolid phase microextraction is a sample preparation method, which combines sampling, sample preparation, and extraction in one step. The biocompatibility of the mix-mode extraction phase achieved by the use of polyacrylonitrile as a binding glue prevents occlusion of the sample matrix constituents other than small molecules thus providing excellent sample clean up. This, in turn, counteracts phospholipid buildup of compounds on the column, which is common issue for solvent-based extraction methods, and prolongs column lifetime. Monitoring of QC samples containing standards which cover abroad range of retention times, during the long-term use of the Discovery HS F5 column confirmed this finding and demonstrated the robustness of the column when operated under optimum conditions for compounds of different physical and chemical properties.
Figure 1. Chromatogram of Selected Compounds Obtained in the First Set of QC Sample Injections in Negative Ionization Mode
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NL: 1.10E6m/z= 395.3093-395.3133MS QC_5
Taurocholic acid
Cholic acid
Chenodeoxycholic acid-d4
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Biochromatography
Figure 2. Chromatogram of Selected Compounds Obtained in the First Set of QC Sample Injections in Positive Ionization Mode
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0
50
100 NL: 5.85E5m/z= 144.1010- 144.1024 MS QC_10
NL:1.01E6m/z= 158158.1181 .1165-MS QC_10
NL: 4.95E5m/z= 171.1165-171.1183 MS QC_10
NL: 4.97E5m/z= 166.0853- 166.0869MS QC_10
NL: 3.87E5m/z= 205.0959-205.0979 MS QC_10
NL: 1.81E6m/z= 315.2294-315.2326MS QC_10
Tranexemic acid
(ACHCA) acid
Phenylalanine-d5
Progesterone
Tryptophan
Phenylalanine
Figure 4. Chromatogram of Selected Compounds Present in QC Sample After 730 Injections of Tissue Extracts Obtained with SPME (ESI+)
Min
(ACHCA) acid
Tranexemic acid
Phenylalanine-d5
Phenylalanine
Progesterone
Tryptophan
RT: 0.00 - 40.03
0 5 10 15 20 25 30 35 400
50
1000
50
1000
50
1000
50
1000
50
1000
50
100 NL: 1.70E6m/z= 144.1010-144.1024MSQC_inst_18
NL: 2.13E6m/z= 158.1165- 158.1181MSQC_inst_18
NL: 9.24E5m/z= 171.1165-171.1183MSQC_inst_18
NL: 9.57E5m/z= 166.0853-166.0869MSQC_inst_18
NL: 6.99E5m/z= 205.0959-205.0979MSQC_inst_18
NL: 2.78E6m/z= 315.2294-315.2326MSQC_inst_18
Figure 5. Chromatogram of Selected Compounds Present in QC Sample After 1525 Injections of Tissue Extracts Obtained with SPME (ESI+)
Min
RT: 0.00 - 40.03
0 5 10 15 20 25 30 35 400
50
1000
50
1000
50
1000
50
1000
50
1000
50
100 NL: 6.29E5m/z= 144.1010-144.1024MSQCinst_20
NL:7.91E5m/z= 158.1165-158.1181MSQCinst_20
NL: 5.56E5m/z= 171.1165-171.1183MSQCinst_20
NL: 5.62E5m/z= 166.0853-166.0869MSQCinst_20
NL: 4.01E5m/z=205.0959-205.0979MSQCinst_20
NL: 1.81E6m/z= 315.2294-315.2326MSQCinst_20
(ACHCA) acid
Tranexemic acid
Phenylalanine-d5
Phenylalanine
Progesterone
Tryptophan
Figure 3. Chromatogram of Selected Compounds Present in QC Sample After 450 Injections of Tissue Extracts Obtained with SPME (ESI-)
Min
Taurocholic acid
Cholic acid
Chenodeoxycholic acid-d4
RT: 0.00 - 40.03
0 5 10 15 20 25 30 35 400
20
40
60
80
100
Rela
tive
Abu
ndan
ce
0
20
40
60
80
100
Rela
tive
Abu
ndan
ce
0
20
40
60
80
100
Rela
tive
Abu
ndan
ce NL: 6.16E5m/z= 514.2829-514.2881MSQC_inst_15
NL: 6.75E5m/z= 407.2792-407.2832MSQC_inst_15
NL: 8.99E5m/z= 395.3093-395.3133MSQC_inst_15
Description Cat. No.Discovery HS F5 HPLC Column, 10 cm x 2.1 mm I.D., 3 µm 567502-U
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SHM84615/T216004
1116
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