Syagen Technology, Inc Warner Avenue Tustin, CA APPI-LC/MS Analysis of Acylglycerols Sheng-Suan Cai, Luke Short, and Jack Syage Syagen Technology, Inc. Jonathan Curtis Ocean Nutrition Canada

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 Solvent (S)Analyte (A) Energy [eV] IP A+A+A+A+ S [A-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 Over 1000 APPI sources in users hands since introduction in 2001 Bibliography available on

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:H 2 O or CH 3 CN:H 2 O)  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, (2006). S.- S. Cai and J. A. Syage, J. Chromatogr. A, 1110, (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 APCI+ [M+H] e5 [M+H] + APPI+ 9.44e5 [M+H] + [M+Na] + ESI+ 1.71e5 ESI+ [M+H] + [ M+NH 4 ] + [M+Na] e5

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 APPI+ Area=983 S/N Ratio = 138 APCI+ Area = 445 S/N Ratio = 46 ESI+ Area = 1718 S/N Ratio = 35 EPA Methyl Ester [M+H] +, 1000 pg 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+NH 4 ] + poor linearity, nonlinear or extremely narrow linear range [M+NH 4 ] + Monoarachidin APPI+APCI+ESI+ DL (pg) [M+Na] + [M+NH 4 ] + Day1 Day2 ESI Signal Nonlinear ESI Linear up to only 5 ng [M+NH 4 ] + Diarachidin APPI+APCI+ESI+ DL (pg) ESI Linear up to only 10 ng [M+NH 4 ] +

Triacylglycerol (TAG) Analytes

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 [M+Na] + [M-C18:0] + SSS, C18:0/C18:0/C18:0 [M-C18:1] + [M-C18:0] + [M+Na] + SSO, C18:0/C18:0/C18:1 [M-C18:2] + [M-C18:1] + [M+H] + LLO, C18:2/C18:2/C18:1 OOO, C18:1/C18:1/C18:1 [M-C18:1] + [M+H] + [M+Na] + [M-C18:2] + [M+H] + LLL, C18:2/C18:2/C18:2 [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 : CH 2 Cl 2, MeOH : CHCl 3 CH 3 CN : IPA, CH 3 CN : CH 2 Cl 2, CH 3 CN : CHCl 3 MeOH or CH 3 CN IPA or CH 2 Cl 2 or CHCl 3 or …… Mobile Phase A Weak Solvent Strength Strong Solvent Strength Mobile Phase B Poor solubility Good solubility

Nonaqueous RP-LC Separations of TAGs MeOH:IPA, 9:1 for 0.25 min, linear gradient to 4:6 in 4 min and hold CH 3 CN:IPA, 9:1 for 0.25 min, linear gradient to 3:7 in 4 min and hold MeOH:CHCl 3, 9:1 for 0.25 min, linear gradient to 6:4 in 4 min and hold CH 3 CN:CHCl 3, 9:1 for 0.25 min, linear gradient to 5:5 in 4 min and hold MeOH:CH 2 Cl 2, 9:1 for 0.25 min, linear gradient to 6:4 in 4 min and hold CH 3 CN:CH 2 Cl 2, 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 C 18 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

Mobile Phase: MeOH/IPA No Dopant Acetone Toluene Dopants do not enhance overall sensitivity Peak Area S/N Ratio

Mobile Phase: MeOH/CHCl 3 Dopants enhance performance and acetone wins due to lower baseline noise than toluene 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 CH 3 CN or CHCl 3 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 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