mass spectrometric quantification of cysteine-containing leukotrienes in rat bile using 13c-labeled...

BIOLOGICAL MASS SPECTROMETRY, VOL. 21, 509-516 (1992)

Mass Spectrometric Quantification of Cysteine-containing Leukotrienes in Rat Bile Using 3C-Labeled Internal Standards

Mark J. Raftery,? Ulla Justesen, Hartmut Jaeschke and Simon J. Caskell$ Center for Experimental Therapeutics, Baylor College of Medicine, Houston, Texas 77030, USA

Leukotriene C, and N-acetyl leukotriene E, were determined in rat bile using a procedure based on high- performance liquid chromatographic fractionation, hydrogenation to 5-hydroxyeicosanoic acid, and gas chomato- graphiclmass spectrometric selected ion monitoring analysis of the pentatluorobenzyl ester, trimethylsilyl ether derivatives. "C,-Labeled analogs of the leukotrienes were synthesized and used as internal standards. The concentrations of both leukotrienes in rat bile were markedly elevated after administration of endotoxin to anesthetized animals; N-acetyl leukotriene E4 was the more abundant product. The presence of leukotriene C, in rat bile after endotoxin challenge was coofirmed by fast atom bombardment/tandem mass spectrometry witb precursor ion scanning. Quantitative determination of leukotriene C, using the tandem mass spectrometric procedure was consistent wth the gas chromatographic/mass spectrometric data but the latter procedure gave a substantially lower detection limit.

INTRODUCTION

Cysteine-containing leukotrienes (LTs) are formed from arachidonic acid via the 5-lipoxygenase pathway and conjugation with glutathione, yielding LTC, . Removal of the y-glutamyl moiety yields LTD, and subsequent loss of the glycyl group results in the formation of LTE,, which may be further metabolized (in the rat) to N-acetyl LTE,. Synthesis of LTs by a number of cell types implies their involvement in a variety of physiological and pathological processes. Investigation of the biological roles of LTs has been hampered, however, by the difficulty of their quantitative determination; this is attributable to their chemical and metabolic lability and the low concentrations at which their biological effects are apparent.

LTs are commonly quantified in biological extracts using a combination of high-performance liquid chromatography (HPLC) and immunoassay.' In general, the chromatographic step is required to achieve separations both from other endogeneous components and of the cysteine-containing LTs themselves, since the available antisera generally do not distinguish between them. Both radioimmunoassay' and en~ymeimmunoassay~ have been used. It remains attractive, however, to employ physicochemical techniques for the determination of leukotrienes, either to assess the validity of more routine methods or to establish baseline data in a novel physiological setting.

Prior to the introduction of fast atom bombardment (FA B) ionization, mass spectrometric analyses of intact cysteine-containing LTs were generally problematic.

Present address : Department of Organic Chemistry, University of Adelaide, Adelaide, South Australia 5001, Australia. $ Author to whom correspondence should be addressed.

1052-~9306/92/100509-08 $09.00 (0 1992 by John Wiley & Sons, Ltd.

Some structural information was revealed by the electron ionization spectrum of LTD, as the trimethylsilyl ether, N-acetyl, methyl ester but the molecular ion was weak.4 Abundant ions retaining the intact molecule were observed by ammonia desorption chemical ionization of derivatives of LTC, and LTE,'. Molecular mass information has also been obtained by thermospray mass spectrometry.6 Abundant protonated and depro- tonated molecular ions were generated by FAB;'-'' recent work in this laboratory" and by Murphy and coworkers" has established that significant structural information may be obtained by collisional activation of [M + H] + and [M - H] - ions during tandem mass spectrometric analyses. We have additionally suggested' ' that FAB tandem mass spectrometry (MS/MS) provides the basis for quantitative determination of cysteine-containing LTs and a preliminary report of this approach has been pre~ented.'~

An alternative strategy for mass spectrometric determination of cysteine-containing LTs was introduced by Balazy and Murphy', and refined by Murphy and Sa1a.l' Hydrogenation and thioether cleavage yielded 5- hydroxyeicosanoic acid as a common product of all the cysteine-containing LTs (Scheme 1). Conversion to the pentafluorobenzyl (PFB) ester, trimethylsilyl (TMS) ether gave a derivative with excellent gas chromatographic properties that was detected with high sensitivity during electron capture mass spectrometry with negative ion detection. Separate determination of indi- vidual cysteine-containing LTs required their chromatographic separation prior to the hydrogenation/ thioether cleavage step. In the original method,', (' 802)5-hydroxyeicosatetraenoic acid ((l80,)5-HETE) was added prior to hydrogenation, derivatization, and gas chromatography/mass spectrometry (GC/MS), to compensate for losses during these stages of the analytical procedure. ( 1802)5-HETE is readily prepared' whereas the '80-labeled cysteine-containing LTs are

Received I April 1992 Revised 26 May 1992

510 M. J. RAFTERY, U. JUSTESEN, H. JAESCHKE A N D S. J. GASKELL

5-hydroxyeicosanoic acid

Scheme 1

reportedly’ difficult to synthesize with routine success. Compensation for incomplete extraction of LTs and for losses during chromatographic separation (prior to the hydrogenation step) was made by the addition of 3H- labeled LTs to the biological sample.’’ ’H-Labeled analogs of the LTs, in which deuterium is incorporated in vinylic or allylic positions, are unsuitable as internal standards since loss of label is expected during hydrogenation.’ ’

Keppler and colleagues have studied the production of cysteine-containing LTs following endotoxin administration to the rat and have postulated a role for these mediators in eliciting the cardiovascular effects charac- teristic of endot~xemia.”-~~ On the basis of HPLC iso- lations and radioimmunoassay, N-aceyl LTE, was determined to be the principal cysteine-containing LT present in rat bile following surgical trauma and endotoxin admini~tration.”,’~ In the present study, we have re-examined this model by using mass spectrometric procedures to determine the concentrations of LTC, and N-acetyl LTE, in rat bile following endotoxin administration. We have utilized an adaptation of the GC/MS procedure described by Murphy and col- l e a g u e ~ , ’ ~ , ~ ~ and have additionally used FAB MS/MS to substantiate the characterization of LTC, . Critically, we have incorporated the use of 13C-labeled analogs of the cysteine-containing LTsZ1 as internal standards to achieve true isotope dilution assays with mass spectrometric determination.

EXPERIMENTAL

All solvents were of HPLC grade (Baxter, Houston, Texas) and were used as received. LTC, , (8,9,10,11- 13C,)LTC,, N-acetyl-LTE, and N-acetyl-(8,9,10,11- I3C,)LTE4 were prepared in this laboratory as described previously.’ l v Z 1 The isotopic purities of the I3C,-labeled analogs exceeded 97%. Sources of reagents were as follows: diisopropylethylamine, Sigma (St Louis, Missouri); PFB bromide, Pierce (Rockford, Illinois); N,O-bis-TMS trifluoroacetamide/trimethyl-

chlorosilane (99 : l), Supelco (Bellfonte, California); rhodium black, Alpha (Wardhill, Massachusetts). All glassware was surface deactivated by rinsing with a solution of dichlorodimethylsilane (10% by volume) in toluene followed by three rinses with methanol.

In vivo dosing experiments

Male Fischer-344 rats (200-250 g) were purchased from Harlan Sprague Dawley Inc. (Houston, Texas) and allowed free access to food (Purina 5001 rodent chow) and tap water. Fed animals were used in all experiments. Each animal was anesthetized with pentobarbital (60 mg kg-’) and the common bile duct was cannulated with PE-10 tubing.” After an equilibration period of 10 min, bile was collected for 30 min before endotoxin and for 30 min thereafter. Bile collection was made into pre-weighed tubes containing 100 pg of the anti-oxidant 4-hydroxy-2,2,2,6-tetramethylpiperidene-l-oxyl (4-OH TEMPO ; Aldrich, Milwaukee, Wisconsin); tubes were stored on ice during collection and were subsequently stored at - 70 “C prior to analysis. Salmonella enteritidis endotoxin (Sigma) was dissolved in saline (2.5 mg ml-’) and injected via the dorsal penile vein (5 mg kg-’ body weight).

Fractionation of bile

Each aliquot of bile (25 pl) was supplemented with 10 p1 each of methanol solutions of (8,9,10,1 1-13C,)LTC4 (0.4 or 10 ng total) and N-a~ety1-(8,9,10,11-~~C,)LTE, (0.85 or 23 ng total). (Later experiments employed the higher levels of internal standards after initial analyses established the relatively high concentrations of these leukotrienes in rat bile following endotoxin challenge.) Each total sample was then fractionated by HPLC on a Novapak octadecylsilyl silica (4 pm particle size; 80 A pore size) reverse-phase column (300 x 3.9 mm; Waters, Milford, Massachusetts). The solvent system comprised acetonitrile (component A) and water containing 0.1 YO acetic acid, buffered to pH 5.6 with ammonium hydrox- ide (component B). The solvent gradient during analysis

QUANTIFICATION OF LEUKOTRIENES 5 1 1

was 10% A to 100% A in 45 min. The flow rate was 1 ml min-'.

The fractions between 20.0 and 21.1 min (for LTC,) and between 21.8 and 22.8 min (for N-acetyl LTE,) were separately collected into screw-cap tubes (13 x 100 mm). The fractions were reduced to dryness under vacuum. Methanol was added to each fraction for subsequent application of the hydrogenation and derivatization procedure.

For selected samples, an alternative extraction and fractionation procedure was applied to precede detection of LTC, by FAB MS/MS. A larger sample of bile (0.5-1.0 ml, supplemented with 100 ng (13C4)LTC,) was reduced to a volume of 150-200 pl under vacuum. Each total sample was then fractionated by HPLC on a p Bondapak octadecylsilyl silica (10 pm particle size; 125 A pore size) reverse-phase column (300 x 3.9 mm; Waters). A linear solvent gradient was applied over 45 min between acetonitrile/l mM ammonium acetate in water (1 : 9) and 100% acetonitrile. The flow rate was 1.5 ml min-'. The fraction corresponding to LTC, was collected, reduced to a volume of 50 p1 and further fractionated by HPLC on the same reverse-phase column. In this instance, the solvent system comprised a linear gradient over 19 min from methanol/water (containing 0.02% acetic acid, buffered to pH 5.7 with ammonium acetate) (40: 60) to 100% methanol. The flow rate was 1 ml min- '. The fraction corresponding to LTC, was collected and reduced to a volume of 50 pl. Final purifi- cation was achieved by reverse-phase HPLC using a Nova-pak octadecylsilyl silica (4 pm particle size; 60-80 A pore size) column (300 x 4.6 mm; Waters); the solvent gradient was the same as that applied in the first stage of HPLC.

Hydrogenation and derivatization

For detection of the analytes by combined GC/MS, the HPLC fractions corresponding to cysteine-containing LTs were hydrogenated using a modification of the procedure of Murphy and coworker^.^^,^^ Approximately 1 mg rhodium black was added to the methanol solution (1 ml) of each fraction and the samples were placed in a glass vessel. The vessel was twice evacuated and flushed with hydrogen (while the samples were stirred). The samples were left for 30 min, after which the methanol was removed under vacuum. Alternatively, approximately 1 mg of rhodium black was added to a methanol solution (300 pl) of the dry HPLC fraction in a screw-capped tube. The tube was flushed with hydrogen and the solution was stirred for 10 min. The procedure was repeated twice. (The latter hydrogenation method was found the more experimentally convenient and was adopted for all later analyses.) The methanol was removed under a stream of nitrogen. (Removal of the rhodium black was not required before ester- ification.) To each sample was added acetonitrile (95 pl), diisopropylethylamine (5 pl) and pentafluorobenzyl bromide (5 pl) and the solution was heated to 40°C for 30 min. The samples were reduced to minimum volume under nitrogen and redissolved in hexane (80 pl). The total sample was separated by normal-phase HPLC on a Vydac silica (5 pm particle size, 300 A pore size)

column (250 x 4.6 mm; the Separations Group, Hes- peria, California). Separations were, performed iso- cratically using hexane/isopropanol (99 : 1) at 1.5 ml min-l.

For each sample, the appropriate fraction (3.7-5.2 min, as judged from GC/MS analyses of derivatized fractions from the HPLC of standard samples) was collected and taken to dryness under nitrogen. N,O-bis- TMS trifluoroacetamide/trimethylchlorosilane (99 : 1 ; 50p1) was added and the sample heated to 65°C for 30min. The reagent was removed under a stream of nitrogen and the sample was dissolved in hexane (50 pl) for GC/MS analysis. Typically, a 5 p1 aliquot was used.

GCWS

GC/MS analyses were performed using a Hewlett Packard 5890 gas chromatograph coupled to a VG Trio-1000 bench-top quadrupole mass spectrometer. Separations were achieved using a fused-silica capillary column (30 m x 0.32 mm) of the DB-1 or DB-5 type, with film thicknesses of 0.25 pm (J&W Scientific, Folsom, California). (Equivalent results were obtained using the two column types.) The column temperature was programmed, 220-320" C, 5" C min '. Samples were introduced using an all-glass falling needle injector (Allen Scientific, Boulder, Colorado). Negative ions were generated by electron capture using methane as moder- ator gas. The electron energy was set to 70 eV and the emission current to 300 pA. The ion source temperature was 200 "C and the gas chromatograph/mass spectrometer interface temperature was 250 "C. All tuning vari- ables were optimized daily by reference to the ion of m/z 452 derived from the volatile standard FC-43 (perfluorotributylamine) which was introduced into the ion source via the reference inlet. Ions of m/z 399 and 403 were detected during dual selected ion monitoring (SIM) analyses. Quantification was based on a standard curve derived from analyses of known mixtures of LTC, (0-1.3 ng or 0-10 ng) and (I3C,)LTC, (0.4 or 10 ng), and of N-acetyl LTE, (0-2.7 ng or 0-40 ng) and N- acetyl (13C4)LTE, (0.85 or 23 ng); response ratio calcu- lations were based on peak areas. The standard mixtures were subjected to the same reduction and derivatization procedure applied to the bile extracts.

FAB MSWS

LT standards and some fractions obtained by reverse- phase HPLC of biological extracts were analyzed by FAB MS/MS using a VG ZAB-SEQ hybrid instrument (with the geometry, magnetic sector/electric sector/radio frequency (r.f.)-only quadrupole/quadrupole mass filter). The liquid matrix was a mixture (1 : 1 by volume) of thioglycerol and 2,2'-dithiodiethanol, saturated with oxalic acid. A solution (1-3 pl) of the sample in methanol was added to the matrix on the sample probe. Ion- ization followed FAB using xenon at 8 keV. Precursor ions were subjected to collisional activation in the r.f.- only quadrupole. The collision energy was 7 eV (in the laboratory frame of reference) and the collision gas was argon at an estimated pressure of 2 x lo-, mbar. LTC,

512 M. J. RAFTERY, U. JUSTESEN, H. JAESCHKE AND S. J. GASKELL

was detected using a narrow mass range (m/z 618-630) scan of precursors of m/z 308. Thus, the second quadrupole was set to transmit m/z 308 and the magnet was scanned.

RESULTS

Mass spectrometric detection of cysteine-containing LTs

FAB ionization of intact cysteine-containing LTs pro- duces abundant [M + HI+ ions which fragment to give a series of informative product ions following low- energy (CAD). As previously reported,'' narrow mass range scans during MSJMS may be used for selective detection of the cysteine-containing leukotrienes. Thus, for example, a scan of precursors of m/z 308 (corresponding to protonated glutathione) detects LTC4.11 Figure 1 shows the application of this approach to the analyses of synthetic LTC, and (l3C4)LTC4. The analysis of LTC, by scanning of precursors of m/z 308 (Fig. l(a)) showed an isotope pattern reflecting the natural isotope composition of the neutral fragment lost (C,,H3,03 , derived biosynthetically from arachidonic acid). Lower-mass precursor ions (m/z 625 and 624), observed at low relative abundance, presum- ably resulted from oxidation processes taking place in the FAB matrix. The corresponding analysis of (I3C4)LTC, (Fig. l(b) indicated the high degree of incorporation of the isotopic label in the C,, moiety.

In routine operation, the sensitivity of detection achieved by these approaches was limited; typically 5-10 ng was required to obtain satisfactory precision in quantitative analyses of LTC, . Nevertheless MS/MS

200 400 MH1 m/z

Figure 2. Electron capture negative ion mass spectrum of (8,9,10, 11 -'3C4)5-hydroxyeicosanoic acid as the PFB ester, TMS ether derivative.

was used here to confirm the presence of LTC, in rat bile (see below).

More sensitive detection of cysteine-containing LTs was achieved by GC/MS of derivatives obtained following reduction to the common product, 5- hydroxyeicosanoic acid (Scheme 1). Figure 2 shows the electron capture negative ion mass spectrum of the PFB ester, TMS ether derivative of (8,9,10,11- '3C,)5-hydroxyeicosanoic acid (obtained by reduction of (13C,)LTC4). As previously reported by others,15 the carboxylate anion derived by loss of the PFB group (m/z 399 for the unlabeled isotope abundance analyte and m/z 403 for the '3C,-labeled analog) was prominent together with an ion derived from subsequent loss of trimethylsilanol (m/z 309 and 313, respectively). SIM of m/z 399 during negative ion GC/MS of 5- hydroxyeicosanoic acid PFB ester, TMS ether (or of m/z 403 for the labeled analog) achieved routine detection limits in the low picogram range, more than adequate for the determination of the cysteine-containing LTs in the biological samples that were the subject of this investigation. Thus, for example, GC/MS SIM analysis of a 10% aliquot of the product of hydrogenation of a mixture of 500 pg LTC, and 10 ng (l3C,)LTC4 (representing the lowest point on the standard curve used for quantitative analyses) gave a signal/ background ratio of approximately 5 : 1 for detection of the unlabeled analyte.

1 I .TC~

626 - > 308 626 - > 308 I .TC~

B l

Determination of LTs in rat bile

m/z (precursor)

Figure 1. MS/MS analyses of synthetic (a) LTC, and ( b ) (8,9, 10.1 1 -'3C,)LTC4 : narrow range scans of precursors of product ions of m/z 308 formed in the quadrupole decomposition region of a hybrid mass spectrometer.

Portions (25 pl) of rat bile were supplemented with internal standards and fractionated directly by reverse- phase HPLC. Figures 3 illustrates the separations achieved. Figure 3(a) shows the analysis of authentic LTC, and N-acetyl LTE,, indicating a separation of -1.5 min. In separate analyses, the retention times of authentic LTD, and LTE, were found to exceed that of N-acetyl LTE, by 0.6 min and approximately 3 min, respectively. Figure 3(b) shows the fractionation of bile taken from a rat before administration of endotoxin; a similar analysis with co-injection of synthetic LTC, and N-acetyl LTE, is shown in Fig. 3(c). Fractions of approximately 1 min duration were collected for hydrogenation, derivatization and GC/MS analysis. Accord- ingly, complete separation of LTC, and N-acetyl LTE, fractions was achieved, and collection of these fractions

QUANTIFICATION OF LEUKOTRIENES

I I

513

- u v

A

LTC4

/--

N-acetyl LTE4

min - 10 20 10 20 10 20

Figure 3. Reverse-phase HPLC separations of (a) synthetic LTC, and N-acetyl-LTE, (40 ng of each); (b) an aliquot (25~11) of rat bile; (c) an aliquot (25 P I ) of rat bile co-injected with synthetic standards (40 ng). Detection was by ultraviolet absorption at 280 nm. For details of the chromatographic separation see the Experimental section.

preceded the elution of any LTD, and LTE, (the pos- sible presence of which was not evaluated in the current analyses).

Incorporation of l3C-1abeled analogs of the cysteine- containing LTs as internal standards for quantitative analyses mitigates the requirement for high yields in the hydrogenation/desulfurization step to precede deriva- tion and GC/MS analysis. In our hands, however, hydrogenation using rhodium-on-alumina as catalyst (as originally described by Murphy and colleague^'^) gave extremably variable yields. Substitution of rhodium black gave reproducibly satisfactory recov- eries.

Figure 4 shows SIM traces for the detection of LTC, and (13C4)LTC, in extracts of rat bile sampled before and after endotoxin administration. In all cases, an excellent signal-to-background ratio was observed, per- mit ting precise quantification. Equivalent data for N - acetyl LTE, are shown in Fig. 5 ; the quality of the traces obtained was again high.

The performance of the analytical method for the determination of N-acetyl LTE, and LTC, was assess- ed by analyses of bile samples (from an animal not subject to endotoxin challenge) to which known quan- tities of synthetic LTs (labeled and unlabeled) were added. Data were compared with the results of direct analyses (hydrogenation, derivatization and GC/MS) of the same standard mixtures of labeled and unlabeled LTs. Table 1 shows the results of linear regression analyses of response ratio (analyte/internal standard) on quantity of analyte. Comparison of the data for spiked bile extracts and standard mixtures indicated that the performance of the quantitative determinations was satisfactory in the context of the marked elevations in leukotriene concentrations observed in rat bile following endotoxin administration (see below).

Figure 6(a) and (b) shows the biliary excretions of LTC, and N-acetyl LTE, , respectively, for four animals before and after endotoxin administration. LT production has been expressed as pg min-' g-' liver to allow for variations in bile flow. When expressed as

A I - 1

I

Figure 4. Negative ion GC/MS analyses for LTC, in rat bile extracts following hydrogenation and derivatization of appropriate HPLC fractions: SIM of mJz 399 (derived from endogenous LTC,) and m/z 403 (derived from (13C,)LTC, internal standard). Each trace is independently normalized; relative responses during the analysis of each sample are indicated on the y-axes. (a) Bile collected in the pre-administration period (&30 min after insertion of the cannula); (b) bile collected 0-30 min after endotoxin administration. For these analyses, the lower amounts of internal standards were employed (see Experimental section).


1 1 ''----------_I

Figure 5. Negative ion GC/MS analyses for N-acetyl LTE, in rat bile extracts following hydrogenation and derivatization of appropriate HPLC fractions: SIM of m/z 399 (derived from endogeneous N-acetyl LTE,) and m/z 403 (derived from N-acetyl (l3C4)LTE4 internal standard). Each trace is independently normalized; relative responses during the analysis of each sample are indicated on the y-axes. (a) Bile collected during the pre- administration period (C30 min after insertion of the cannula); (b) bile collected 0-30 rnin after endotoxin administration. For these analyses, the lower amounts of internal standards were employed (see Experimental section).

biliary concentrations, LTC, increased from an average of approximately 25 pg pl- pre-dose to approximately 110 pg p1-I after endotoxin. The equivalent figures for N-acetyl LTE, were 65 pg p1-I and 1330 pg p1-l. The analytical data for bile collected for the 30 min period following cannula insertion (and prior to endotoxin administration) may have reflected to some degree an elevation of LT production following surgery but in all cases a substantial increase in biliary concentrations was observed following endotoxin administration.

Further evidence for the presence of LTC, in rat bile was obtained by FAB MS/MS detection of the intact analyte. 'Conventional ' mass spectrometric detection

(a) 275

250

50

25

0 r r

. 2 3 4

Figure 6. Effect of endotoxin administration on the biliary excre- tion of (a) LTC, and (b) N-acetyl LTE, in four rats. The biliary excretions are expressed as pg min-' g- ' liver to allow for variations in bile flow. Hatched blocks correspond to analyses for LTs in bile samples collected during a 30 rnin period between cannula insertion and endotoxin administration. Open blocks represent data for LT analyses in bile samples collected for 30 rnin post- endotoxin. The standard deviations of the average (AVG) values are indicated. LT concentrations in bile were determined using the GC/MS procedure.

Table 1. Quantitative analyses of LTC, and N-acetyl LTE, using the GCWS procedure. Linear regression analyses" of response ratio (analyte/internal standard) on quantity of analyte: comparison of data for spiked bile extracts and standard mixtures.

r rn C

N-Acetyl LTE, Standard mixtureb 0.999 0.044 f 0.003 -0.020 0.065 Spiked bile 0.995 0.049 f 0.009 -0.023 f 0.1 68

LTC, Standard mixture" 0.997 0.090 f 0.01 3 -0.024 f 0.066 Spiked bile 0.988 0.081 f 0.023 0.1 06 f 0.1 14

" y = mx + c, where y = response ratio (analyte/internal standard), x = quantity (ng) of analyte; 95% confidence limits are quoted for values of m and c;23 r = correlation coefficient. &40 ng N-acetyl LTE,, 23 ng N-acetyl ("C,) LTE, . C10 ng LTC,, 10 ng (13C,) LTC, .

(that is, a narrow mass range scan of the spectrum of source-formed ions) was of insufficient specificity for confident identification or satisfactory quantification of the LT. In contrast, Fig. 7 shows the MS/MS analysis of LTC, in an extract of bile (1.0 ml) following additional HPLC fractionation (see Experimental section). Scan- ning of precursors of product ions of m/z 308, formed in the r.f.-only quadrupole decomposition region of the hybrid instrument, permitted detection of endogenous LTC, and allowed quantification by determination of the response ratio, LTC,/(13C,)LTC, (Fig. 7). In the example shown (of an extract of bile sampled after endotoxin administration), the approximate concentra- tion of LTC, was found to be consistent with the levels of post-endotoxin LTC, determined using the GC/MS procedure.

QUANTIFICATION OF LEUKOTRIENES 515

626 --> 308 1

m f z (precursor)

Figure 7. FAB MS/MS analysis for LTC, of an HPLC fraction obtained from the bile (1 ml aliquot) of an animal administered endotoxin: limited mass range scan of precursor ions fragmenting in the r.f.-only quadrupole of the tandem instrument to give m/z 308, corresponding to protonated glutathione.

DISCUSSION

The rigorous quantitative determination of cysteine- containing LTs remains a significant analytical challenge, due in large part to the lability of these compounds. Precise and accurate analyses are accord- ingly strongly dependent on rigorous internal standardization. Particular significance therefore attaches to the introduction, in the present work, of '3C-labelled analogs as internal standards for the mass spectrometric analysis of cysteine-containing LTs. I3C,-labeled internal standards are ideal owing to (i) the non-lability of the isotopic label, (ii) chromatographic properties essen- tially identical to the unlabeled analogues, and (iii) a mass separation sufficient to eliminate interference between detection of labeled and unlabeled species during mass spectrometric analysis. 'H-Labeled analogs of the LTs, in which deuterium is incorporated in vinylic or allylic positions, are unsuitable as internal standards when the analytical procedure includes a hydrogenation step prior to derivatization and GC/MS, since loss of label is expected during hydr~genation. '~ The preparation of "O,-labeled cysteine-containing LTs has been reported to be difficult." o-Trideuterated

cysteine-containing LTs, the synthesis of which has been reported by Prakash et d.,' may represent a satisfactory alternative to the (I3C4)LTs for the purpose of internal standardization.

The present studies have confirmed that negative ion GC/MS of hydrogenated and derivatized products rep- resents a highly sensitive method for the determination of cysteine-containing LTs. The sensitivity was more than adequate for the detection of LTC, and N-acetyl LTE, in rat bile, where the concentrations of these analytes was in the range 06-35 ng per 25 p1 sample. Detection by FAB MS/MS has the advantage of direct detection of the intact cysteine-containing LTs but is much less sensitive than the GC/MS procedure.

Analyses of bile taken from rats before and after injection of endotoxin established that concentrations of both LTC, and N-acetyl LTE, were elevated several- fold after administration of the toxin; N-acetyl LTE, was observed at higher concentrations than LTC, . Keppler and coworkers" have previously reported N- acetyl LTE, as the major cysteine-containing LT in rat bile following endotoxin administration; quantitative analyses were based on HPLC separation and radioimmunoassay. No data were reported for LTC,. The concentrations of N-acetyl LTE, determined in the present study were approximately fifty times higher than those reported by Keppler et d.'* Exact compari- sons between the data are, however, difficult. The earlier study used a different strain of rat (Wistar). Further- more, no compensation was made in that study for losses occurring during sample preparation. The principal advantage of the methodology described in the present paper is the high selectivity of detection of the cysteine-containing LTs combined with the use of stable isotope labeled internal standards. Application of these techniques to the further study of the rat model of endo- toxemia will be described elsewhere.

Acknowledgements

This work was supported in part by the National Institutes of Health (A1 26916 to S . J. G., G M 42957 to H. J.) and by the CIBA-GEIGY Corporation. We thank Fisons/VG Instruments (Manchester, UK) for the loan of the Trio-1000 gas chromatograph/mass spectrometer, and Dr R. M. Caprioli (University of Texas Medical School, Houston, Texas) for making available HPLC equipment.

REFERENCES

1.

2.

3.

4.

5.

6.

J. Y. Westcott, K. Johnston, R. A. Batt, S. E. Wenzel and N. F. Voelkel, J. Appl. Physiol. 68, 2640 (1 990). R. A. Lewis and K. F. Austen, J. Allergy Clin. Immunol. 74,

7.

8. 396 (1 983). P. Pradelles. J. Grassi and J. Maclouf, Anal. Chem. 57, 1170 (1985). 9.

Nature 285, 104 (1 980). H. R. Morris, G. W. Taylor, P. J. Piper and J. R. Tippins,

10. R. L. Maas, J. A. Lawson, A. R. Brash and J. A. Oates, Advances in Prostaglandin. Thromboxane and Leukotriene 11. Research, Vol. 11, ed. by 6. Samuelsson, R. Paoletti and P.

12. G . W. Taylor, C. G. Chappell, S. R. Clarke, D. J. Heavey, R. Richmond, N. C. Turner, D. Watson and C. T. Dollery, The 13. beukotrienes: Their Biological Significance, ed. by P. J. Piper, p. 67. Raven Press, New York (1 986).

Ramwell. p. 229, Raven Press, New York (1 983).

R. C. Murphy, W. R. Mathews, J. Rokach and C. Fenselau, Prostaglandins 23, 201 (1 982). G. W. Taylor, H. R. Morris, B. Beaubien and P. M. Clinton, Leukotrienes and Other Lipoxygenase Products. ed. by P. J. Piper, p. 277. Wiley, Chichester (1983). J. Y. Westcott, K. L. Clgy and R. C. Murphy, J. Allergy Clin. lmmunol. 74, 363 (1 984). C. Prakash, B. J. Sweetman, A. Sivakumar, I. M. Taffer, R. E. Zipkin and I. A. Blair, Prostaglandins 37, 251 (1989). M. J. Raftery, G. C. Thorne, R. S. Orkiszewski and S. J. Gaskell, Biomed. Enwiron. Mass Spectrorn. 19. 465 (1 990). A. Sala, K. Kayganich, J. A. Zirrolli and R. C. Murphy, J. Am. SOC. Mass. Spectrom. 2, 31 4 (1 991 ). M. J. Raftery, H. Jaeschke and S. J. Gaskell, Proceedings of the 38th Annual Conference on Mass Spectrometry and Allied Topics, June 3-8,1990, Tucson, Arizona. p. 1395.


14. M. Balazy and R. C. Murphy. Anal. Chem. 58, 1098 (1 986). 15. R. C. Murphy and A. Sala, in Methods in Enzymology, Vol.

187: Arachidonate Related Lipid Mediators, ed. by R. C. Murphy and F. A. Fitzpatrick, p. 90. Academic Press, San Diego (1990).

16. R. C. Murphy and K. L. Clay, in Methods in Enzymology, Vol. 86: Prostaglandins and Arachidonate Metabolites, ed. by W. E. M. Lands and W. L. Smith, p. 547. Academic Press, San Francisco, (1 990).

17. W. Hagmann, C. Denzlinger and D. Keppler, FEBS Lett. 180, 309 (1985).

18. W. Hagmann, C. Denzlinger, S. Rapp, G. Weckbecker and D. Keppler, Prostaglandins 31, 239 (1 986).

19. W. Hagmann, C. Denzlinger and D. Keppler, Circulafory Shock 14, 223 (1984).

20. D. Keppler, W. Hagmann and S. Rapp, Rev. Infect. Dis. 9. S580 (1 987).

21. M. J. Raftery and S. J. Gaskell, J. Label. Cornpds Radiopharrn. 29,314 (1991).

22. H. Jaeschke, H. Krell and E. Pfaff, Gastroenterology 85, 808 (1983).

23. J. C. Miller and J. N. Miller, Statistics forAnalytical Chemistry, 2nd edn. Ellis Horwood, Chichester (1988).

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