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APPI-LC/MS Analysis of Acylglycerols. Sheng-Suan Cai, Luke Short, and Jack Syage Syagen Technology, Inc. Jonathan Curtis Ocean Nutrition Canada. Syagen Technology, Inc. 1411 Warner Avenue Tustin, CA 92780 www.syagen.com. Photoionization. [A-m] + + m. S. Fragmentation. A +. IP.

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Syagen technology inc 1411 warner avenue tustin ca 92780 www syagen com l.jpg

APPI-LC/MS Analysis of Acylglycerols

Sheng-Suan Cai, Luke Short, and Jack Syage

Syagen Technology, Inc.

Jonathan Curtis

Ocean Nutrition Canada

Syagen Technology, Inc.

1411 Warner Avenue

Tustin, CA 92780

www.syagen.com


Photoionization l.jpg

Photoionization

[A-m]+ + m

S

Fragmentation

A+

IP

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

IP

Energy [eV]

Solvent (S)

Analyte (A)


Slide3 l.jpg

LC eluent /

injection

probe

cone

~

~

~

~

~

~

~

VUV lamp

APPI Source

pump

to MS


Direct appi vs dopant assisted appi l.jpg

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 l.jpg

Published APPI Literature

Over 1000 APPI sources in users hands since introduction in 2001

Bibliography available on www.syagen.com


Objectives l.jpg

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 l.jpg

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 l.jpg

9.44e5

ESI+

APPI+

1.71e5

[M+H]+

[M+Na]+

[M+H]+

[M+NH4]+

9.36e5

[M+H]+

5.99e5

[M+H]+

APCI+

ESI+

[M+Na]+

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


Comparison of appi apci and esi l.jpg

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).


Peak smoothness area count and s n ratio l.jpg

Peak Smoothness, Area Count and S/N Ratio

EPA Methyl Ester

[M+H]+, 1000 pg

APPI+

Area=983

S/N Ratio = 138

APCI+

Area = 445

S/N Ratio = 46

ESI+

Area = 1718

S/N Ratio = 35

High area count does not necessarily mean high S/N ratio


Comparison of detection limits l.jpg

Monoarachidin

40

ESI Linear up

to only 5 ng

ESI Linear up

to only 10 ng

Diarachidin

ESI Signal Nonlinear

30

120

100

Day2

DL (pg)

80

20

DL (pg)

[M+Na]+

60

[M+NH4]+

[M+NH4]+

40

Day1

20

10

[M+NH4]+

[M+Na]+

0

APPI+

APCI+

ESI+

0

APPI+

APCI+

ESI+

ESI+

ESI+

[M+NH4]+

Comparison of Detection Limits

  • ESI [M+Na]+ signal unstable,

  • NaOAc causes source fouling,

  • ESI [M+NH4]+ poor linearity, nonlinear or extremely narrow linear range


Triacylglycerol tag analytes l.jpg

Triacylglycerol (TAG) Analytes


Chemical structures of tag analytes l.jpg

LLO, C18:2/C18:2/C18:1

LnLnLn, C18:3/C18:3/C18:3

SSO, C18:0/C18:0/C18:1

LLL, C18:2/C18:2/C18:2

SSS, C18:0/C18:0/C18:0

OOO, C18:1/C18:1/C18:1

Chemical Structures of TAG Analytes


Appi full scan mass spectra of tags l.jpg

[M+H]+

[M-C18:0]+

[M-C18:2]+

[M+Na]+

[M-C18:1]+

SSS, C18:0/C18:0/C18:0

LLO, C18:2/C18:2/C18:1

[M+H]+

[M-C18:1]+

[M-C18:0]+

[M-C18:2]+

[M+Na]+

LLL, C18:2/C18:2/C18:2

SSO, C18:0/C18:0/C18:1

[M-C18:1]+

[M+H]+

[M+Na]+

[M-C18:3]+

[M+H]+

OOO, C18:1/C18:1/C18:1

LnLnLn, C18:3/C18:3/C18:3

APPI Full Scan Mass Spectra of TAGs

As degree of unsaturation increases, [M+H]+ intensity increases


Strategies for establishments of na rp mobile phases by gradient elution l.jpg

Mobile Phase B

Mobile Phase A

MeOHorCH3CN

IPAorCH2Cl2orCHCl3or ……

StrongSolvent Strength

Weak Solvent Strength

Good solubility

Poor solubility

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


Nonaqueous rp lc separations of tags l.jpg

Nonaqueous RP-LC Separations of TAGs

LnLnLn LLL LLO OOO SSO SSS

MeOH:IPA, 9:1 for 0.25 min,

linear gradient to 4:6 in 4 min and hold

No dopant

CH3CN:IPA, 9:1 for 0.25 min,

linear gradient to 3:7 in 4 min and hold

Dopant acetone

MeOH:CHCl3, 9:1 for 0.25 min,

linear gradient to 6:4 in 4 min and hold

Dopant acetone

CH3CN:CHCl3, 9:1 for 0.25 min,

linear gradient to 5:5 in 4 min and hold

Dopant acetone

MeOH:CH2Cl2, 9:1 for 0.25 min,

linear gradient to 6:4 in 4 min and hold

Dopant acetone

CH3CN:CH2Cl2, 9:1 for 0.25 min,

linear gradient to 5:5 in 4 min and hold

Dopant acetone

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.


Mobile phase meoh ipa l.jpg

Mobile Phase: MeOH/IPA

Peak Area

Toluene

Acetone

No Dopant

S/N Ratio

Dopants do not enhance overall sensitivity


Mobile phase meoh chcl 3 l.jpg

Toluene

Acetone

No dopant

Mobile Phase: MeOH/CHCl3

Peak Area

S/N Ratio

Dopants enhance performance and acetone wins due to lower baseline noise than toluene


Summary and conclusions l.jpg

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 l.jpg

Estimated On-Column Limits of Detection

MeOH/IPA CH3CN/IPA MeOH/CHCl3 CH3CN/CHCl3 MeOH/CH2Cl2 CH3CN/CH2Cl2

No dopant Acetone Acetone Acetone Acetone Acetone

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.


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