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[11] Q u a n t i t a t i o n o f S u l f i d o p e p t i d e L e u k o t r i e n e s in Biological Fluids by Gas Chromatography-Mass Spectrometry

By ROBERT C. MURPHY and ANGELO SALA Sulfidopeptide leukotrienes can be quantitated in biological fluids by several means. First, the biological activity of sulfidopeptide leukotrienes can be used as a basis for quantitative analysis. These assays which have been described previously I typically involve contraction of a smooth muscle and the response pharmacologically quantitated. However, following the structure elucidation of slow-reacting substance of anaphylaxis (SRS-A), more specific assays are typically employed. The second general analytical protocol involves the use of specific antibody clones to recognize metabolites of arachidonic acid. Such immunoassay techniques can involve the use of radiolabeled tracers (radioimmunoassay)2 or enzyme tracers (enzyme immunoassays). 3 The analytical protocols developed using these techniques are highly sensitive (for example, a few picograms can be detected with some immunoassays), however, cross-reactivity of known or unknown molecules can compromise interpretation of results.4 Third, analytical techniques based on physicochemical properties of these molecules have been developed for quantitative analysis. The anal~,sis of sulfidopeptide leukotrienes by HPLC separation with on-line UV detection can quantitate these substances when a few nanograms are present. 5 Mass spectrometry can also be adapted for quantitative analysis of these molecules. Such procedures can be sensitive (for example, detect a few picograms), quite precise, and accurate. A major drawback in the direct analysis by mass spectrometry is the fact that the sulfidopeptide leukotrienes are not volatile substances natively, nor do simple derivatives of the carboxyl, amino, and hydroxyl moieties of these molecules impart appreciable volativity. 6'7 Thus, in order to use combined gas chroC. W. Parker, M. M. Huber, and S. F. Falkenhein, this series, Vol. 86, p. 655. 2 F. A. Fitzpatrick, this series, Vol. 86, p. 286. 3 p. Pradelles, J. Grassi, and J. Maclouf. Anal. Chem. 57, 1170 (1985). 4 j. y . Westcott, S. Chang, M. Balazy, D. O. Stene, P. Pradelles, J. Maclouf, N. F. Voelkel, and R. C. Murphy, Prostaglandins 32, 857 (1986). 5 W. R. Mathews, J. Rokach, and R. C. Murphy, Anal. Biochem. 118, 96 (1981). 6 R. C. Murphy, S. Hammarstrom, and B. Samuelsson, Proc. Natl. Acad. Sci. U.S.A. 76, 4275 (1979). 7 R. C. Murphy, W. R. Mathews, J. Rokach, and C. Fenselau, Prostaglandins 23, 201 (1982).

METHODS IN ENZYMOLOGY, VOL. 187

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GC-MS OF SULFIDOPEPTIDELEUKOTRIENES

91

matography-mass spectrometry (GC-MS) for the analysis of sulfidopeptide leukotrienes it is necessary to change substantially the chemical structure of these molecules into derivatives that can be made volatile. 8 The overall procedure described here is one which relies on the facile cleavage of the carbon-sulfur bond by noble metal catalysts in the presence of hydrogen gas (hydrogenolysis).

Reagents Rhodium on alumina, Rh/AI203 (Aldrich Chemical Co., Milwaukee, WI), rhodium content, 5% Diisopropylethylamine (Aldrich, WI) Bis(trimethylsilyl)trifluoroacetamide (BSTFA) (Supelco Chemical Co., Bellefonte, PA) Pentafluorobenzyl bromide (PFB-Br) (Supelco) Leukotriene C4, D4, E4 (Biomol Research Labs., Plymouth Meeting, PA) [3H]LTC4, [3H]LTD4, and [3H]LTE4 (>20 mCi/mmol) (New England Nuclear Research Products, Boston, MA 5-Hydroxyeicosatetraenoic acid (5-HETE) (Cayman Chemical Co., Ann Arbor, MI) Sep-Pak, reversed-phase solid extraction cartridges (Waters Associates, Milford, MA) H2mO (Isotec Inc., Miamisburg, OH) Methanol, dichloromethane, acetonitrile (HPLC-grade, Fisher Chemical Co.) Principle of the Method. The assay for the sulfidopeptide leukotrienes described below is based on stable isotope dilution with quantitative analysis by selected-ion monitoring GC-MS. The steps (shown in Fig. 1) are as follows: I. Addition of [3H]leukotriene to the biological fluid as initial recovery internal standard; 2. solid-phase extraction of leukotrienes from biological fluid using reversed-phase Sep-Pak, or optional reversed-phase HPLC purification to separate LTC4, LTD4, and LTE4; 3. recovery of [3H]leukotriene determined in Sep-Pak methanol fraction; 4. addition of [~80215-HETE as mass spectrometric internal standard; 5. catalytic desulfuration and reduction with Rh/AI203 ; 6. extraction of 5-HEA; 7. conversion to the pentafluorobenzyl ester, trimethylsilyl ether derivative; and 8. quantitative analysis of the ratio of unlabeled to oxygen-18 internal standard by selected-ion monitoring GC-MS (m/z 399 and 403).

8 M. Balazy and R. C. Murphy, Anal. Chem. 58, 1098 (1986).

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Biological Matrix

[3H|LTs Sep-Pak (HPLC) Purified Leukotrienes

1180215-HETE Rh/AI203; H2 Extract 5-Hydroxyeicosanoic Acid

OH

"0

PFB-Br BSTFA GC-MS

FIG. 1. Protocol for the quantitation of sulfidopeptide leukotrienes by negative-ion electron capture-mass spectrometry. The structures of the sulfidopeptide leukotrienes and the various internal standards (radiolabeled sultidopeptide leukotrienes and ~SO-labeled 5HETE) are indicated along with the chemical modifications during the process.

[11]

GC-MS OF SULFIDOPEPTIDELEUKOTRIENES

93

Internal Standards The quantitative analysis of leukotrienes by stable isotope dilution techniques requires the availability of an appropriate stable isotopically labeled species of the analyte to be quantitated. The commercially available deuterium-labeled compounds, in which the deuterium atoms are present either on a vinylic or allylic position, are unsuitable for use in this assay. This is due to the fact that hydrogen atoms in such positions are completely lost during the catalytic reduction process because of exchange with hydrogen gas at the catalytic surface. The formation of zr-allyl intermediate at a coordination site on the catalyst surface facilitates both hydrogenolysis and deuterium exchange. 9 For this reason, the 1SO-labeled 5-HETE is employed which has stable isotopes (180) that cannot be lost during the catalytic reduction step. Synthesis of 180-labeled intact leukotriene C4, D4, or E4 has been difficult to achieve routinely. The synthesis of [1802]5-HETE has been described in a previous volume of this series, lo The [180215-HETE has two oxygen-18 atoms in the carboxylic acid moiety and after reduction yields the identical compound, 5-hydroxyeicosanoic acid, as that obtained from any sulfidopeptide leukotriene except that it has a molecular weight four units higher. Since this internal standard is structurally different from sulfidopeptide leukotrienes, it is added to the sample just prior to the reduction step and thus cannot account for any loss of the leukotrienes prior to this conversion step. In order to circumvent this problem, tritium-labeled sulfidopeptide leukotrienes must be added to the biological fluid to account for recovery prior to this reduction step. Of critical concern is the isotopic purity of the [180215-HETE and the absence of any back-exchange during the assay protocol. The standard curve becomes a critical step in the assessment of these points.

Assay of Sulfidopeptide Leukotrienes in Biological Fluids Step 1. To an aliquot of 1 to 5 ml of physiological fluid is added [3H]LTC4 (or [3H]LTD4, [3H]LTE4 depending on the exact species to quantitate) at a precisely known level (e.g., 20,000 dpm). The mixture is vigorously shaken for 2 min to effect mixing. Steps 2 and 3. The biological fluid is passed through a Sep-Pak which had been previously washed with 10 ml of methanol, 5 ml 0.1 mM EDTA, and 10 ml water. The Sep-Pak is then eluted with 10 ml distilled water 9 H. O. House, "Modern Synthetic Reactions," p. 23. W. A. Benjamin, Menlo Park, CA, 1972. 10R. C. Murphy and K. L. Clay, this series, Vol. 86, p. 547.

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followed by 10 ml of hexane and 10 ml ethyl acetate containing 1% methanol (v/v) to remove prostaglandins and dihydroxyleukotrienes as well as other hydroxy acids including any 5-hydroxyeicosatetraenoic acid in the biological system, i~ Finally, the Sep-Pak is eluted with 5 ml of 80% methanol/water and the radioactivity recovery determined in an aliquot of this fraction. A modification of the above protocol is to separate each sulfidopeptide leukotriene prior to reduction. This involves addition of all three radiolabeled internal standards [3H]LTC4, [3H]LTD4, and [3H]LTE4 followed by separation of the sulfidopeptide leukotrienes by reversed-phase HPLC using methanol/water (65/35/0.02 acetic acid), pH 5.7. The HPLC fractions corresponding to each sulfidopeptide leukotriene are analyzed separately by adding [I802]5-HETE and carrying out the catalytic reduction. Even though the identical molecules are quantitated in this modified protocol, namely 5-HEA and [lso2]5-HEA, the prior separation and use of radiolabeled sulfidopeptide leukotriene to correct for recoveries of the separation step, allows quantitation of each sulfidopeptide leukotriene. Steps 4 and5. A precise amount of [180215-HETE (typically 0.5-5.0 ng) is added to the methanol Sep-Pak eluate. The sample is adjusted to 50% methanol/water (v/v) either by drying the sample and dissolving it in this solvent system or by adding water to the eluate fraction. Reduction and desulfurization of leukotrienes is carried out at roqm temperature by adding 2-5 mg of Rh/AIz03 and slowly bubbling hydrogen into the sample for 25 min. Special attention is paid to the contact between catalyst and hydrogen which is necessary for maximum desulfurization. The rate of hydrogen addition is maintained as that necessary for suspension of the catalyst. Step 6. The solution is then made basic with 50/.d of 1 N KOH, in order to release the product completely from the catalyst surface. This step destroys a portion of the catalytic surface itself and any chemisorption of 5-HEA is eliminated. The catalyst is removed by centrifugation in a microcentrifuge (12,000 g, 5 min) and the supernatant removed. The supernatant is acidified with 10/zl formic acid and then extracted with 2 volumes of dichloromethane. The two phases are separated after centrifugation and the lower phase, which contains the 5-HEA, is dried under a stream of nitrogen. Step 7. The extracted 5-HEA is derivatized first into the pentafluorobenzyl ester by the addition of 25 /xl 10% diisopropylethylamine in acetonitrile followed by 25 ttl of a 10% solution (v/v) of pentafluorobenzyl bromide (PFB-Br) in acetonitrile. Esterification proceeds for 25 min at room temperature. Excess reagents are then removed by a stream of H W. S. Powell, this series, Vol. 86, p. 467.

[ 11]

G C - M S OF SULFIDOI'EPTIDE LEUKOTRIENES

95

399 7 O-TMS

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m# FIG. 2. (A) Mass spectrum of 5-hydroxyeicosanoic acid (5-HEA) as a trimethylsilyl ether, pentafluorobenzyl ester using electron capture (CH4) and negative-ion detection. (B) The mass spectrum of the oxygen- 18 internal standard of 5-HEA. The two ions at m/z 401 and 403 indicate one and two oxygen-I 8 atoms incorporated in the carboxy moiety. These derivatives were analyzed by capillary GC-MS.

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nitrogen. The hydroxy group in 5-HEA is then converted to the trimethylsilyl ether derivative by the addition of 50/zl of BSTFA and 50 p.l of acetonitrile. The reaction is heated to 60 ° for 1 hr. This final solution can be directly injected into the gas chromatograph or alternatively to effect concentration, the sample can be dried under a stream of nitrogen and dissolved in a smaller volume of hexane containing 5% BSTFA. Step 8. The gas chromatographic separation of the pentafluorobenzyl ester, trimethylsilyl ether of 5-hydroxyeicosanoic acid is carried out on a gas chromatograph equipped with a 10 m x 0.25 mm id capillary column having a 25/zm film thickness of DB 1 (J & W Scientific, Rancho Cordova, CA). The gas chromatographic column is programmed from 150° to 300 ° at a linear rate of 15°/min using helium as carrier gas (50 cm/sec). This column is directly interfaced to a quadrupole mass spectrometer (Nermag 1010C, Paris, France) operated in the negative-ion electron-capture mode using methane as moderating gas at a source pressure 0.1 Torr. Source temperature is maintained at 180° and electron energy typically at 80 eV. Selected-ion monitoring of negative ions is performed at m/z 399 corresponding to the carboxylate anion of endogenous 5-HEA from the sulfidopeptide leukotriene and m/z 403 corresponding to the added []802]5HETE. The resultant mass spectra for these derivatives is shown in Fig. 2.

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FIG. 3. (A) Selected-ion recording of m/z 399 indicating the presence of a sulfidopeptide leukotriene as analyzed as the 5-HEA-TMS, pentafluorobenzyl ester. The sulfidopeptide leukotriene was isolated from a murine mast cell preparation passively sensitized with IgE and challenged with DNP-albumin. (B) Selected-ion recording trace of m/z 403 derived from 0.5 ng of [JaO2]5-HETE added as internal standardto the leukotriene extract before reduction and derivatization.

tO

[11]

GC-MS OF SULFIDOPEPTIDELEUKOTRIENES

4

=

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5 LTC4 (rig)

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FIG. 4. Standard curve used in the quantitativeanalysisof sulfidopeptideleukotrienes with [~sOz]5-HETE(0.5 ng) as internal standard. The protocolfor preparation of the sample for analysisis illustratedin Fig. 1.

Retention time of the 5-HEA derivative in the conditions listed above is approximately 8 min as seen in Fig. 3. Preparation of Standard Curve For each batch of samples, a standard curve is generated where a fixed amount of [I802]5-HETE (1 ng) is added to known concentrations of the sulfidopeptide leukotriene (typically 0.2, 0.5, 1, 5, 10 ng). The volume of internal standard solution ([180215-HETE) used for this curve is precisely that used for the biological samples. These standard solutions, made in 50% methanol/water (v/v), are treated with Rh/AI203 starting at step 6 described above. The resulting ratio of m/z 399 to 403 is then plotted against the sulfidopeptide leukotriene concentration as seen in Fig. 4. Five different quantities of sulfidopeptide leukotrienes are typically used for such standard curves. The Y-intercept corresponds to the isotopic purity of the [lSO2]5-HETE and such curves should be quite linear. Quantitation of Sulfidopeptide Leukotrienes from Mast Cells The protocol described above was used to quantitate LTC4 from murine-transformed mast cells passively sensitized with monoclonal mouse IgE anti dinitrophenol (DNP) conjugate, then challenged with DNP-albumin. Following challenge, the cell supernatant was taken for analysis as in Fig. 1. As seen in Fig. 3 the selected-ion recording trace for the ions corresponding to the sulfidopeptide leukotriene (5-HEA deriv-

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ative) with 1 ng of [lSo2]5-HETE internal standard indicated production of 2.8 ng/ml LTC4 eq./106 mast cells following immunological challenge. This assay can be used to separately quantitate sulfidopeptide leukotrienes if one institutes a prior separation of each sulfidopeptide leukotriene typically by reversed-phase HPLC. Acknowledgement This workwas supportedby a grantfromthe NationalInstitutesof Health(HL25785).

[ 12] A u t o m a t e d O n - L i n e E x t r a c t i o n a n d Profiling of L i p o x y g e n a s e P r o d u c t s of A r a c h i d o n i c Acid by HighPerformance Liquid Chromatography

By PIERRE BORGEAT, SERGE PICARD, PIERRE VALLERAND, SYLVAIN BOURGOIN, ABDULRAHMAN ODEIMAT, PIERRE SIROIS, a n d PATRICE E. POUBELLE Because of the potential importance of the lipoxygenase products in several human diseases, substantial effort has been put into the development of methods for their assay. Immunoassays and mass spectrometric (MS) assays enabling detection of picogram amounts of material are now available for some compounds. Reversed-phase high-performance liquid chromatography (RP-HPLC) is also widely used for analysis of lipoxygenase products; the present popularity of reversed-phase HPLC for the analysis of leukotrienes (LTs) and related compounds arises from the fact that most lipoxygenase products carry a UV chromophore (Fig. 1) which enables their measurement by photometry. Materials and Methods Standards of the lipoxygenase products (5S,12R)-dihydroxy-(6Z,8E, 10E, 14Z)-eicosatetraenoic acid (LTB4), 20-hydroxy-LTB4 (20-OH-LTB4), 20-carboxy-LTB4 (20-COOH-LTB4), (5S)-hydroxy-(6R)-S-glutathionyl(7E,gE, 11Z, 14Z)-eicosatetraenoic acid (LTC4), (5S)-hydroxy-(6R)-S-cysteinylglycine-(7E,9E,11Z,14Z)-eicosatetraenoic acid (LTD4), (5S)-hydroxy-(6R)-S-cysteinyl-(7E,9E,11Z,14Z)-eicosatetraenoic acid (LTE4), the N-acetyl derivatives of LTC4, ])4, E4, the sulfoxide forms of LTC4, D4, E4 (LTC4SO,LTD4SO,LTE4SO), (5S)-hydroxy-(6E,SZ, 11Z, 14Z)-eicosatetraenoic acid (5-HETE), (12S)-hydroxy-(5Z,8Z,10E, 14Z)-eicosatetraMETHODSIN ENZYMOLOGY,VOL. 187

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