selected reaction monitoring during gas chromatography/mass spectrometry of eicosanoids

BIOMEDICAL AND ENVIRONMENTAL MASS SPECTROMETRY, VOL. 16, 409413 (1988)

Selected Reaction Monitoring During Gas Chromatography/Mass Spectrometry of Eicosanoids

Helen Hughes, Jean Nowlin and Simon J. Gaskelli Center for Experimental Therapeutics, Baylor College of Medicine, Houston, Texas 77030, USA

Victor C. Parr VG Tritech Ltd, Wythenshawe, Manchester, UK

Gas chromatography/electron capture negative ion mass spectrometry of eicosanoids, as pentafluorobenzyl (PFB) ester, methyl oxime (where applicable), trimethylsilyl (TMS) ether derivatives, is reported using a double-focusing instrument of trisector configuration. Sub-picogram detection limits were observed for prostaglandins El, E, and F,, during selected ion monitoring (SIM) of [M -PFBJ- ions. Selected reaction monitoring (SRM) of [M - PFBl- + [M - PFB - TMSOHI-, occurring in the first field-free region, was of modest sensitivity, reflect- ing the stability of the (M - PFBJ- ions. The leukotriene B, (LTB,) derivative was successfully analyzed by SIM at the low-picogram level. In this instance, the fragmentation [M - PFBl- + [M - PFB - TMSOH] occurred in high yield in the first field-free region. The advantageous improvement in selectivity of detection that may be achieved with SRM was evident during the analysis of a serum extract for LTB, .

INTRODUCTION

The determination of eicosanoids (arachidonic acid metabolites formed via the cyclooxygenase or lipoxygenase pathways) remains one of the most challenging biomedical problems of trace analysis by mass spectrometry. The analyses generally require high sensitivities, to give detection limits at least at the low-picogram level, and high selectivities, to permit the analysis of complex biological extracts. Impressive sensitivities of detection have been achieved by analysis of pentafluorobenzyl (PFB) ester derivatives, using gas chromatography/electron capture negative ion mass spectrometry, with selected ion monitoring of [M - PFB] - ions.'-' The fluorinated derivative confers strongly electron-capturing properties, and sub- sequent loss of the PFB group affords the stable carboxylate ion, which frequently represents the only peak of significant intensity in the negative ion mass

The use of the electron capture negative ion mode may also provide some enhancement of selectivity by comparison with electron impact (EI) mass spectrometry but further improvements may be required for the most demanding trace analyses. One general approach to the improvement of selectivity is the use of selected reaction monitoring (SRM). Gas chromatography/mass spectrometry (GC/MS) with the monitoring of reactions occurring in the first field-free region (1st FFR) of a double-focusing mass spectrometer was introduced for the analysis of steroids' and has

t Author to whom correspondence should be addressed.

0 8 8 7 4 1 3 4 / 8 8 / 2 4 5 $05.00 0 1988 by John Wiley & Sons, Ltd.

since been applied to a variety of analytes, such as can- nabinoids," drug metabolites' ' and cyclic hydrocar- bons.I2 The principal limitation to this variant of the SRM technique is the restricted resolution achieved, particularly the effective resolution of parent ions. Strife and Simms13 have described SRM of prostaglandins using a reverse-geometry instrument, with detection of collisionally activated decompositions occurring in the 2nd FFR. In this instance, parent ion resolution is good but daughter ion resolution, achieved by the electric sector, is poor. The simultaneous achievement of unit resolution, or better, of parent and daughter ions during SRM requires the use of multiple analyzer instruments. Extensive use has been made of triple quadrupoles for SRM analyses (see, for example, Ref. 14). Further improvements in selectivity, where these are required, may be obtained on hybrid tandem instruments where increased parent ion resolution can be achieved.'

Several recent reports have described analyses of cyclooxygenase metabolites of arachidonic acid by GC/ MS/SRM using triple-quadrupole instr~ments.'~-'' Analyses have been performed in both EI16*' 7 ~ 1 9 and electron capture modes.' 7*18

In this brief communication, we describe GC/MS analyses of several eicosanoids using electron capture ionization and both conventional SIM and SRM detection. The analyses were carried out on a new instrument of trisector geometry incorporating an air-cored electromagnet20*21 (Fig. 1). SRM was performed of decompositions occurring in the 1st FFR. Effective parent and daughter ion resolutions are equivalent to those obtained with a conventional double-focusing mass spectrometer.

410 H. HUGHES ET AL.

S O U R C E L - I n - ' I U ESA 1

FIRST FIELD-FREE REGION

P'+Qf+ N

Figure 1. Configuration of the VG TS250 instrument, showing the decomposition region relevant to SRM analyses.

EXPERIMENTAL

Materials

Eicosanoid standards were purchased from Cayman Chemical Co. (Ann Arbor, Michigan). Pentafluoro- benzyl (PFB) bromide and methoxyamine hydrochloride (2% solution in pyridine) were from Pierce Chemical Co. (Rockford, Illinois). (N,O-bistrimethyl- sily1)trifluoroacetamide (BSTFA)/trimethylchlorosilane (TMCS) 99 + 1) was obtained from Supelco (Bellefonte, Pennsylvania) and tert-butyldimethylchlorosilane (TBDMCS)/imidazole (1 M and 2.5 M, respectively, in dimethylformamide) was from Alltech Associates/ Applied Science Laboratories (Deerfield, Illinois).

For GC/EI mass spectrometry of leukotriene B, (LTB,), the methyl ester bis-tert-butyldimethylsilyl ether derivative was used. All GC/electron capture mass spec- trometric analyses used the PFB ester, methyl oxime (where applicable), TMS ether derivatives.

Methyl esters were prepared by reaction with ethereal diazomethane at room temperature. PFB esters were prepared by reaction with PFB bromide/ diisopropylethylamine/acetonitrile (2 + 2.5 + 100; 100 pl) at 40 "C for 30 min. The reagents were evaporated to dryness and the residue was dissolved in hexane/ dichloromethane (1 + 1; 1 ml); the resulting solution was washed with water (0.5 ml). The organic phase was recovered and taken to dryness. Where applicable, methyl oximes were prepared by overnight reaction at room temperature with methoxyamine hydrochloride in pyridine (50 pl). Following the reaction, the solvent was removed under a stream of nitrogen, water (50 pl) added, and the product extracted into ethyl acetate (300 pl). Trimethylsilyl ethers were prepared by reaction (1 h; 60°C) with BSTFA/TMCS. The reagent was removed under nitrogen and the product redissolved in hexane for GC/MS analysis. Tert-butyldimethylsilyl ethers were prepared by reaction (overnight at room temperature) with the TBDMCS reagent (50 pl). The

reaction mixture was taken to minimum volume under nitrogen, water (200 pl) was added, and the derivative was extracted into hexane (400 pl).

Mass spectrometry

All analyses were performed using an HP5890A gas chromatograph (Hewlett-Packard, Avondale, Penn- sylvania) coupled to a VG TS250 mass spectrometer (VG Tritech, Manchester, UK). Separations were achieved on an open-tubular fused silica capillary column (30 m x 0.32 mm, i.d.) of the DB-5 bonded- phase type (J&W Scientific, Rancho Cordova, California), with temperature programming, 250-300 "C, 18" min-I. The carrier gas was helium. Sample introduction was via an all-glass, falling needle device (Allen Scientific, Boulder, Colorado). Ionization was by EI (70 eV) or electron capture, using methane as the moder- ator gas. The TS250 instrument incorporates a VG 11/250 data system for control of data acquisition and processing.

Extraction of LTB, from blood serum

Whole blood (5 ml) was incubated for 1 h at 20", with or without addition of the Ca2+ ionophore, A23187. Serum was recovered by centrifugation and a 1 ml aliquot was acidified to pH 3 by addition of hydro- chloric acid (2 M). The serum was applied to a C,, Sep-Pak cartridge (Water Chromatography Division of Millipore, Milford, Massachusetts), previously treated with methanol and water. The Sep-Pak was subse- quently washed with 10 ml each of water, methanol/ water (1/3), water and hexane prior to collection of an ethyl acetate eluate (7 ml). The extract was taken to dryness under nitrogen and converted to the PFB ester as described and redissolved in high-performance liquid chromatography (HPLC) mobile phase. The PFB ester derivative was purified by straight-phase HPLC using an Econosphere 5 pm silica column (250 x 4.6 mm;

SELECTED REACTION MONITORING OF EICOSANOIDS 41 1

Alltech Associates/Applied Science Laboratories), with the mobile phase, hexane/isopropanol/acetic acid (96/4/ O.l), at a flow rate of 2 ml min-’. The fraction corres- ponding to LTB, PFB ester was collected, taken to dryness and converted to the bis-TMS ether as described above.

a)

RESULTS AND DISCUSSION

GC/electron capture mass spectrometry of prostaglandins El, E, and FZa, as PFB ester, TMS ether, methyl oxime (where applicable) derivatives, with SIM of [M - PFBI- ions gave sub-picogram detection limits, consistent with earlier reports.’ Linked scanning (with B/E constant) to give spectra of daughter ions derived by decomposition of [M - PFBI- ions in the 1st FFR indicated that, for each analyte, no daughters were formed with relative abundances which exceeded 0.5% of that of the transmitted parent. No useful increase in daughter ion signal was observed when helium was introduced into the collision cell (Fig. 1). These findings confirm the stability of the carboxylate anions formed under electron capture conditions. Clearly, this stability is beneficial for conventional SIM but problematic for the SRM approach. There may nevertheless be some value to the use of SRM in some biological applications where selectivity is important but sensitivity is not limit- ing. Analyses of authentic PGE, and PGE,, as the PFB, methyl oxime, TMS derivatives, with SRM of the transition, [M - PFBI- + [M - PFB - TMSOHI-, indicated detection limits of approximately 100 pg.

Successful GC/MS analyses for derivatives of LTB, (an arachidonic acid metabolite via the 5-lipoxygenase pathway) depend on the quality of the gas chromatograph column and on the use of an all-glass injection system to avoid losses via degradation or irreversible adsorption.” Problems arising from the lability of LTB, derivatives during gas-phase analysis may be overcome by prior hydr~genation,’~ but this approach has the disadvantage of removing the distinction, during GC/MS analysis, between several possible unsaturated precursors. Recent ~ o r k , ’ ~ , ’ ~ however, has indicated that successful analyses of LTB, derivatives, without prior hydrogenation, may be performed at the picogram level and our data confirm this. Thus, during GC/EI impact mass spectrometry of the methyl ester, bis- TBDMS derivative, with SIM of [M - C4H9] + ions, a linear relationship between response and amount injected was observed for the range lOo(r10 pg (the lowest amount analyzed).

Optimal sensitivities are again observed during GC/ electron capture mass spectrometry of the PFB ester of LTB, , with conversion of hydroxyl groups to alkylsilyl ethers.22~24~zs The mass spectrum of the PFB, TMS derivative includes prominent ions attributable to [M - PFBI- (m/z 479), [M - PFB - TMSOHl- (m/z 389) and [M - PFB - 2(TMSOH)]- (m/z 299); the extent of loss of TMSOH appears to depend on the temperature of analysis. Figure 2(a) and (b) show GC/MS analyses of 25 pg LTB, PFB, TMS with SIM of [M - PFBI- (m/z 479) and

b) n C )

3 28 4 88 4 48 5 28 6 88

mlnutei Figure 2. GC/MS analyses of LTB, as the PFB, TMS derivative. (a) SIM of [M-PFBI - ; 25 pg. (b) SIM of [M-PFB -TMSOH]-; 25 pg. (c) SRM of [M - PFBI- -+ [M - PFB -TMSOH]-; 125 pg.

[M - PFB - TMSOHl- (m/z 389), and indicate a low- picogram detection limit.

The facile loss of TMSOH groups from the [M - PFBI- ion is confirmed by observation of decompositions occurring in the 1st FFR. Figure 2(c) shows the GC/MS analysis of 125 pg LTB, PFB, TMS with SRM of the m/z 479 + 389 transition. The sensitivity of detection during SRM indicates a remarkably high efficiency of fragmentation of the parent ion. The analyses were performed without the addition of gas to the collision cell in the 1st FFR. It is very likely, however, that a high proportion of the daughter ions observed were nevertheless the result of collisionally activated decomposition since a relatively high pressure will prevail in the 1st FFR region adjacent to the source exit.26

The selectivity advantage which may be achieved with SRM is evident during analyses of a serum extract for LTB,. The total serum extract was fractionated by HPLC of the PFB derivative, prior to conversion to the TMS ether and GC/MS analysis. Figure 3(a) shows an

H. HUGHES ET AL. 412

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48

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4 8 8 5 8 8 6 8 8 198 8 9 8 ! E L 4 8 8 5 0 0 608 1 9 9 8 8 8 3 0 0 4 8 8 588 6 8 8 7 8 8 8 0 8 363

MINUTES

Figure 3. GC/MS of a serum extract after purification by HPLC and conversion to the PFB, TMS derivative. (a) SIM of m/z 389. (b) SRM of m/z 479 + 389. (c) Repeat of (b l with coiniection of authentic LTB, PFB, TMS. The arrows indicate the retention time of authentic LTB, PFB, TMS.

SIM analysis; a prominent chromatographic peak appears with a retention time similar, but not identical, to that of the LTB, derivative and thus obscures detection of the intended analyte. During SRM analysis (Fig. 3(b)), an apparently homogeneous peak was detected with a retention time precisely correct for the LTB, derivative. Co-injection with authentic standard led to enhancement of peak intensity. Approximate quantifica- tion of LTB, indicated a concentration of 200 pg ml-', but it is not at present clear the extent to which this represents ex uiuo production during sampling and serum preparation. Intentional ex uiuo stimulation of leukocytes, by addition of ionophore, gave a more than ten-fold increase in the concentration of LTB, .

The significant increase in selectivity during SRM of LTB, PFB, TMS has been achieved despite the limitation of effective parent ion resolution achieved with the double-focusing instrument. Furthermore, the frag-

mentation monitored (loss of TMSOH) is characteristic of the derivative rather than the analyte structure itself. Strife and Simms13 have argued a preference for the monitoring of 'backbone-specific' fragmentations and this is clearly desirable on the grounds of specificity alone. The highest yields of daughter ions, however, are frequently obtained from derivative-directed fragmentations so that sensitivity considerations may dictate the monitoring of such reactions. Recent report~'~.' ' have described the SRM of fragmentations of the eicosanoid moiety during analyses of prostaglandin derivatives in the EI mode, using a triple-quadrupole instrument. Further study is required to establish whether, during the analysis of biological extracts, the improvement in selectivity which may be expected (by comparison with SRM of derivative-specific cleavages) will offset the probable sacrifice in basic sensitivity associated with the detection of positive, rather than negative ions.

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selected reaction monitoring during gas chromatography/mass spectrometry of eicosanoids

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