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