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Syagen Technology, Inc.1411 Warner AvenueTustin, CA 92780www.syagen.com
APPI-LC/MS Analysis of APPI-LC/MS Analysis of AcylglycerolsAcylglycerols
Sheng-Suan Cai, Luke Short, and Jack SyageSyagen Technology, Inc.
Jonathan CurtisOcean Nutrition Canada
Solvent (S) Analyte (A)
En
erg
y [e
V]
IP
IP
AA++
SS
[A[A--m]m]++ + m+ m
Fragmentation
Photoionization
Benefits of Photoionization Ionizes wide range of compounds (e.g.,
non-polars, electronegative cpds, etc.) Predominantly parent ion signal Minimum fragmentation Minimum solvent signal Minimum ion suppression Signal linear with concentration
H2O, CO2, O2, N2
Ionization potential
Drugs
Chemicalweapons
Aromatics
MeOH, AcCN, chloro-solvents
Solvent (S) Analyte (A)
En
erg
y [
eV
]
IP
IP
AA++
SS
[A-m][A-m]++ + m + m
Fragmentation
pump
to MS
~~~
~~
~ ~
LC eluent /injection
cone
probe
VUV lamp
APPI Source
Direct APPI vs. Dopant-assisted APPI
Direct APPI
M + hv M+ + e-
M+ + S MH+ + S[-H]
Dopant APPI
D + hv D+ + e-
D+ + M MH+ + D[-H]D+ + M M+ + D
Analyte molecule M is ionized to a molecular radical ion M+. (If analyte ionization potential is below photon energy)
In the presence of protic solvents, M+ may abstract a hydrogen atom to form MH+.
A photoionizable dopant is delivered in large concentration to yield many D+ ions.
D+ ionizes analyte M by proton or electron transfer.
This is PI-initiated APCI.
Published APPI Literature
Literature Articles
04 5
7
14
21
31
11
0
5
10
15
20
25
30
35
40
45
1999 2000 2001 2002 2003 2004 2005 2006
Art
icle
s P
ub
lish
ed
Publications in 2006 are through March,2006.
Open box (2006) represents projected publications for 2006.
ASMS Abstract Count
0
6
1411
15
28
38
0
10
20
30
40
1999 2000 2001 2002 2003 2004 2005
Ab
stra
cts
Pre
sen
ted
Over 1000 APPI sources in users hands since introduction in 2001
Bibliography available on www.syagen.com
Objectives
Developed improved method relative to conventional methods
GC or GC/MS requires tedious sample prep and analyte derivatization
Conventional LC (i.e., with UV or ELSD) lacks sensitivity and specificity
Difficulties in analyzing nonpolar lipids by reversed phase LC/MS due to low solubility of analytes in reversed phase solvent systems (i.e., MeOH:H2O or CH3CN:H2O)
Normal phase LC/MS may be better choice
To investigate the advantage of using APPI over APCI and ESI for analysis of nonpolar lipids by comparing
Mass spectra Dynamic linear range Sensitivity
Selected Target Analytes
Four individual non-polar lipid standards were tested
EPA and EPA methyl ester (fatty acid group)
Monoarachidin (saturated monoglyceride, C20:0)
Diarachidin (saturated diglyceride, C20:0)
Trielaidin (monounsaturated triglyceride, C18:1)
Trielaidin EPA
S.- S. Cai and J. A. Syage, Anal. Chem. 78, 1191-1199 (2006).
S.- S. Cai and J. A. Syage, J. Chromatogr. A, 1110, 15-26 (2006).
EPA Methyl Ester (MW = 316) Mass Spectra
APPI and APCI mobile phase was hexane, ESI mobile phase was 1:1 isooctane/IPA without or with 10 mM ammonium formate
m/z150 200 250 300 350 400
%
0
100
Lipid18MAY05_01 165 (0.833) Scan AP+ 5.99e5317.3
315.4285.3
211.2
318.4
APCI+ [M+H]+5.99e5
m/z150 200 250 300 350 400
%
0
100
Lipid11APR05_17 826 (4.171) Scan APPI+ 9.44e5317.3
285.3161.0
267.3
318.4
[M+H]+APPI+9.44e5
m/z150 200 250 300 350 400
%
0
100
Lipid23MAY05_07 276 (1.532) Scan ES+ 1.71e5371.2
163.2 339.2261.2
181.1 217.2 259.2
317.2
305.3
291.1
385.2
389.2
[M+H]+[M+Na]+ESI+ 1.71e5
m/z300 310 320 330 340 350
%0
100
Lipid14JUN05_20 100 (0.958) Scan ES+ 9.36e5317
315
313
303 311
331
329327
339
334
344
347349
ESI+[M+H]+ [M+NH4]+
[M+Na]+
9.36e5
Comparison of APPI, APCI, and ESI
Monoarachidin Linearity Plots. Mobile phase: 1:1 isooctane/IPA (APPI & APCI). 10:15:1 isooctane/IPA/water with 15.4 mM sodium acetate (ESI sodium adduct) and 1:1 isooctane:IPA with 10 mM ammonium formate (ESI ammonium adduct).
ESI+ [M+NH4]+
y = -0.23x2 + 966.37x + 36689
R2 = 0.9622
0.E+00
4.E+05
8.E+05
1.E+06
0 500 1000 1500 2000 2500
Inj. Amount (ng)
Pea
k A
rea
R2 = 0.8501
0.E+00
1.E+03
2.E+03
0 0.1 0.2 0.3
APPI+ [M+H-H2O]+
y = 2333.8x
R2 = 0.9986
0.E+00
2.E+06
4.E+06
6.E+06
0 500 1000 1500 2000 2500
Inj. Am ount (ng)
Pe
ak
Are
aR2 = 0.9994
0
200
400
600
0 0.1 0.2 0.3
APCI+ [M+H-H2O]+
y = 699.11x
R2 = 0.9995
0.E+00
1.E+06
2.E+06
0 500 1000 1500 2000 2500
Inj. Amount (ng)
Pea
k A
rea
R2 = 0.9992
0
100
200
300
400
0 0.2 0.4 0.6
ESI+ [M+Na]+
y = 98.201x
R2 = 0.9786
0.0E+00
5.0E+04
1.0E+05
1.5E+05
2.0E+05
2.5E+05
0 500 1000 1500 2000 2500
Inj. Amount (ng)
Pea
k A
rea
R2 = 0.90530
20406080
100
0 0.1 0.2 0.3
Monoarachidin
Peak Smoothness, Area Count and S/N Ratio
Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50
%
1
Lipid02MAY05_19 SIR of 1 Channel APPI+ 317.3
8.21e3S/N:PtP=137.87
APPI+Area=983S/N Ratio = 138
Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50
%
2
Lipid20MAY05_24 SIR of 1 Channel AP+ 317.3
4.04e3S/N:PtP=46.14
APCI+Area = 445S/N Ratio = 46
Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
%
13
Lipid23MAY05_05 SIR of 1 Channel ES+ 317.3
1.39e4S/N:PtP=35.39
ESI+Area = 1718S/N Ratio = 35
EPA Methyl Ester [M+H]+, 1000 pg
0.E+00
4.E+02
8.E+02
1.E+03
2.E+03
2.E+03
APPI+ APCI+ ESI+
Peak A
rea
0.E+00
5.E+01
1.E+02
2.E+02
APPI+ APCI+ ESI+
S/N
Rati
o
High area count does not necessarily mean high S/N ratio
Comparison of Detection Limits
ESI [M+Na]+ signal unstable,
NaOAc causes source fouling,
ESI [M+NH4]+ poor linearity, nonlinear or extremely narrow linear range
Trielaidin
0
5
10
15
20
APPI+ APCI+ ESI+
DL
(p
g)
[M+NH4]+
Monoarachidin
0
10
20
30
40
APPI+ APCI+ ESI+ ESI+ ESI+
DL
(p
g)
[M+Na]+
[M+Na]+
[M+NH4]+
Day1
Day2
ESI Signal Nonlinear
ESI Linear upto only 5 ng
[M+NH4]+
Diarachidin
0
20
40
60
80
100
120
APPI+ APCI+ ESI+
DL
(p
g)
ESI Linear upto only 10 ng
[M+NH4]+
Triacylglycerol (TAG) Analytes
Name Abbrev. TAG Type MW
Trilinolenin LnLnLn C18:3/C18:3/C18:3 873.34
Trilinolein LLL C18:2/C18:2/C18:2 879.38
1,2-dilinoleoyl-3-oleoyl-rac-glycerol LLO C18:2/C18:2/C18:1 881.4
Triolein OOO C18:1/C18:1/C18:1 885.43
1,2-distearoyl-3-oleoyl-rac-glycerol SSO C18:0/C18:0/C18:1 889.46
Tristearin SSS C18:0/C18:0/C18/0 891.48
Chemical Structures of TAG Analytes
LnLnLn, C18:3/C18:3/C18:3
LLL, C18:2/C18:2/C18:2
OOO, C18:1/C18:1/C18:1
LLO, C18:2/C18:2/C18:1
SSO, C18:0/C18:0/C18:1
SSS, C18:0/C18:0/C18:0
APPI Full Scan Mass Spectra of TAGs
500 600 700 800 900 1000m/z0
100
%
607.6
608.6
913.9609.7
[M+Na]+
[M-C18:0]+
SSS, C18:0/C18:0/C18:0
500 600 700 800 900 1000m/z0
100
%
607.6
608.7
911.9663.5 685.5
[M-C18:1]+
[M-C18:0]+
[M+Na]+
SSO, C18:0/C18:0/C18:1
500 600 700 800 900 1000m/z0
100
%
881.8
601.6 879.8
685.5 723.6 877.7
882.8935.7
[M-C18:2]+
[M-C18:1]+
[M+H]+
LLO, C18:2/C18:2/C18:1
500 600 700 800 900 1000m/z0
100
%
603.6
604.6 907.8
685.5 885.9
OOO, C18:1/C18:1/C18:1
[M-C18:1]+
[M+H]+
[M+Na]+
500 600 700 800 900 1000m/z0
100
%
879.7
599.6
880.7
901.7[M-C18:2]+
[M+H]+
LLL, C18:2/C18:2/C18:2
500 600 700 800 900 1000m/z0
100
%
873.7
595.5 723.6
874.7
875.8
[M+H]+
[M-C18:3]+
LnLnLn, C18:3/C18:3/C18:3
As degree of unsaturation increases, [M+H]+ intensity increases
Strategies for Establishments of NA-RP Mobile Phases by Gradient Elution
Six possible combinations as binary mobile phase:
MeOH:IPA, MeOH: CH2Cl2, MeOH:CHCl3
CH3CN:IPA, CH3CN:CH2Cl2, CH3CN:CHCl3
MeOH or CH3CN IPA or CH2Cl2 or CHCl3 or ……
Mobile Phase A
Weak Solvent Strength Strong Solvent Strength
Mobile Phase B
Poor solubility
Good solubility
Nonaqueous RP-LC Separations of TAGs
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00Time0
100
%
0
100
%
0
100
%
0
100
%
0
100
%
0
100
%MeOH:IPA, 9:1 for 0.25 min, linear gradient to 4:6 in 4 min and hold
CH3CN:IPA, 9:1 for 0.25 min, linear gradient to 3:7 in 4 min and hold
MeOH:CHCl3, 9:1 for 0.25 min, linear gradient to 6:4 in 4 min and hold
CH3CN:CHCl3, 9:1 for 0.25 min, linear gradient to 5:5 in 4 min and hold
MeOH:CH2Cl2, 9:1 for 0.25 min, linear gradient to 6:4 in 4 min and hold
CH3CN:CH2Cl2, 9:1 for 0.25 min, linear gradient to 5:5 in 4 min and hold
LnLnLn LLL LLO OOO SSO SSS
Waters ZQ APPI-LC/MS. Gemini C18 Column, 150 x 2 mm. Mobile phase flow rate 0.2 mL/min, dopant flow rate 0.04 mL/min. 10 ng each.
No dopant
Dopant acetone
Dopant acetone
Dopant acetone
Dopant acetone
Dopant acetone
Mobile Phase: MeOH/IPAMeOH/IPA, Peak Area
0
5000
10000
15000
20000
25000
LnLnLn LLL LLO OOO SSO SSS
Are
a
No Dopant
Acetone
Toluene
MeOH/IPA, S/N Ratio
0
100
200
300
400
500
600
700
800
LnLnLn LLL LLO OOO SSO SSS
S/N
Rat
io
No Dopant
Acetone
Toluene
10.00 12.00 14.00 16.00Time0
100
%
No Dopant
Acetone
Toluene
Dopants do not enhance overall sensitivity
Peak Area
S/N Ratio
Mobile Phase: MeOH/CHCl3
MeOH/CHCl3, Peak Area
0
5000
10000
15000
20000
25000
LnLnLn LLL LLO OOO SSO SSS
Pe
ak A
rea
No Dopant
Acetone
Toluene
MeOH/CHCl3, S/N Ratio
0
100
200
300
400
500
600
LnLnLn LLL LLO OOO SSO SSS
S/N
Rat
io No Dopant
Acetone
Toluene
Dopants enhance performance and acetone wins due to lower baseline noise than toluene
8.00 10.00 12.00Time0
100
%No dopant
Acetone
Toluene
Peak Area
S/N Ratio
Summary and Conclusions
Triacylglycerols in free acid and methyl ester forms in standards and in fish oils were studied by LC/MS using APPI, APCI, and ESI
APPI and APCI offer comparable linear range (i.e., 4-5 decades) APPI is 2-4x more sensitive than APCI and much more sensitive than
ESI w/o mobile phase additives. ESI sensitivity dramatically enhanced by mobile phase modifiers, but
at much reduced linear range. Flow injection LODs <10 pg, and overall on-column LODs are 25 –
200 pg for a wide range of solvent conditions Use “APPI-Friendly” solvents such as IPA or MeOH for high sensitivity
w/o dopants Use CH3CN or CHCl3 for lower column backpressure and better
resolution, but dopants needed Acetone outperforms toluene as a dopant by not increasing and
sometimes even suppressing baseline noise
We acknowledge partial funding from NIH
Estimated On-Column Limits of Detection
0
100
200
300
400
500
600
700
800
900
Ln
Ln
Ln
LL
L
LL
O
OO
O
SS
O
SS
S
Ln
Ln
Ln
LL
L
LL
O
OO
O
SS
O
SS
S
Ln
Ln
Ln
LL
L
LL
O
OO
O
SS
O
SS
S
Ln
Ln
Ln
LL
L
LL
O
OO
O
SS
O
SS
S
Ln
Ln
Ln
LL
L
LL
O
OO
O
SS
O
SS
S
Ln
Ln
Ln
LL
L
LL
O
OO
O
SS
O
SS
S
Lim
its
of
Det
ect
ion
(p
g)
Most of LODs fall below 200 pg levels.
Estimated from injections of 1 ng/µL mixed standard with 10 µL injection volume. LODs equivalent to the amount at S/N = 3.
MeOH/IPA CH3CN/IPA MeOH/CHCl3 CH3CN/CHCl3 MeOH/CH2Cl2 CH3CN/CH2Cl2 No dopant Acetone Acetone Acetone Acetone Acetone