High-performance liquid chromatography—thermospray mass spectrometry of prostaglandin and thromboxane acetyl derivatives

Journal of Chromatography, 568 (1991) 11 24 Biomedical Applications Elsevier Science Publishers B.V., Amsterdam CHROMB10. 5902

High-performance liquid chromatography-thermospray mass spectrometry of prostaglandin and thromhoxane acetyl derivatives MOTOTERU YAMANE* and AKIHISA ABE

Department of Biochemistry, Tokyo Medical College, 6-1-1 Shinjuku, Shin[uku-ku, Tokyo (Japan) (First received October 24th, 1990; revised manuscript received March 13th, 1991) ABSTRACT A quantitative analysis of prostaglandin-related substances has been developed. Hydroxyl groups of prostaglandin and thromboxane were acetylated by acetic anhydride, the mixture was partially purified on a Sep-Pak C1~ cartridge and analysed by high-performance liquid chromatography combined with thermospray mass spectrometry. Using this method, twenty kinds of prostaglandin derivative could be detected simultaneously within 11 min on a selected-ion monitoring detection chromatogram without a gradient system. Generally, the base ion, [M + H - n(60)] +, is produced through elimination of acetic acid (n = number of the hydroxyl group of prostaglandin or thromboxane). The detection limit for these derivatives was ca. 0.2 pmol at the levels of prostaglandin-related substances prior to derivatization. They could be analysed in the range 0.5-10 pmol. The assay was successfully applied to prostaglandin-related substances in human seminal fluid and rat brain.

INTRODUCTION

Prostaglandins (PGs) and thromboxanes (TXs) have been assayed by highperformance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS) and radioimmunoassay (RIA). With HPLC, quantitation is generally conducted following pre-column fluorescence derivatization using 9anthryldiazomethane [1,2]. However, with this technique, disturbance of fluorescence caused by the by-product produced from 9-anthryldiazomethane is usually a problem that is difficult to eliminate. At present, GC-MS is the most reliable method, although its operation is complicated because carboxyl, hydroxyl and carbonyl groups all have to be derivatized [3-6]. RIA methods still suffer from problems of cross-reaction with other sample components and, in most cases, a commercial antibody is not available, particularly in the cases of new or more unstable metabolites [7-9]. High-performance liquid chromatographythermospray mass spectrometry (HPLC-TSP-MS) has recently come to be used for the analysis of trace compounds. Analysis of arachidonic acid metabolites using this technique has been reported [10-12]. However, this procedure is not 0378-4347/91/$03.50

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1991 Elsevier Science Publishers B.V.

12

M. YAMANE, A. ABE

satisfactory owing to the considerable time required lbr HPLC and quantification using a deuterium-labelled internal standard (I.S.). This paper describes a newly developed method in which the time for HPLC is only 11 min, owing to use of a single mobile phase and acetylation of PG-related substances. During this short period, twenty kinds of PG-related substance can be detected simultaneously by selected-ion monitoring (SIM). A TSP interface of high temperature and a mobile phase composed of acidic buffer are used, resulting in greatly improved sensitivity. The single mobile phase facilitates quantitative analysis, in which a deuterium-labelled I.S. is used. EXPERIMENTAL

Standards and reagents PG or TX standards (PGA1, PGA2, PGB1, PGB2, PGD2, PGE1, PGE2, PGFI,, 6-keto-PGF~, and TXB2) were obtained from Sigma (St. Louis, MO, USA). Other PG or TX standards (PGA3, PGE3, PGF2,, PGF3~, PGJ2, 6-ketoPGEa, 15-keto-PGE2, 15-keto-PGF~,, 15-keto-PGF2~, 13,14-dihydro-15-ketoPGA2, 13,14-dihydro- 15-keto-PGE2, 13,14-dihydro- 15-keto-PGD2, 1l-dehydroTXB/ and [3,3,4,4-2H2]PGF2,) were obtained from Cayman (Ann Arbor, MI, USA). The water for the HPLC eluent was of Milli-Q grade (Waters Assoc., Milford, MA, USA), that for preparing 15% ethanol, 5% acetonitrile, 2 M HC1 and 10% acetic acid, and for washing and equilibrating the Sep-Pak Cls cartridge (Waters Assoc.) was prepurified by a Sep-Pak C18 cartridge. The other solvents and reagents were of analytical-reagent or chromatographic grade. Extraction from biological samples To each 100 mg of biological sample were added 139 pmol of [3,3,4,4-2H2]PGF2~, as the I.S., and 1 ml of ethanol cooled to -20°C. After homogenization for 1 rain at 0°C with a Physcotron homogenizer (Nichion, Tokyo, Japan), the mixture was centrifuged at 1500 g for 10 rain at 4°C. The supernatant was evaporated under reduced pressure, and 1.0 ml of 15% ethanol and 5 /A of 2 M HC1 were added to the residue. The mixture was adjusted to ca. pH 3 and applied to a Sep-Pak Cls cartridge equilibrated with water. The cartridge was washed successively with 5 ml of 5% acetonitrile, 10 ml of water and 5 ml of n-hexane. PGs and TXs in the cartridge were eluted with 6 ml of acetonitrile, and the eluent was evaporated to dryness under reduced pressure. Derivatization PGs and TXs were derivatized using acetic anhydride in pyridine to obtain the acetic ester. PGs and TXs were dissolved in 0.2 ml of pyridine, and 0.08 ml of acetic anhydride was added. The reaction mixture was left overnight under argon at 5°C. The mixture was acidified to pH 4-5 by addition of 3.5 ml of 10% acetic acid and 0.5 ml of acetonitrile. The mixture was then immediately applied to a

HPLC-TSP-MS OF PG AND TX DERIVATIVES

13

Sep-Pak C18 cartridge equilibrated with water. The cartridge was washed successively with 3 ml of 5% acetonitrile and 10 ml of water, and the acetyl derivatives of PGs and TXs in the cartridge were eluted with 5 ml of acetonitrile. The eluent containing the PG and TX derivatives was stored at - 2 0 ° C until analysis by HPLC-TSP-MS. HPLC-TSP-MS

A Shimadzu (Kyoto, Japan) LC/GC/MS-QPI000S provided with Vestec (Houston, TX, USA) Model 750B HPLC-TSP-MS interface, a Shimadzu LC-9A HPLC pump and a Rheodyne injector fitted with a 20-gl loop were used. HPLC separation was carried out on a Nucleosil 100-5C~8 (5 pm particle size, Macherey-Nagel, Duren, Germany) 150 mm x 4.6 mm I.D. column, with a mobile phase of 0. I M ammonium acetate-0. I M acetic acid-acetonitrile (4:6:15, v/v) at a flow-rate of 1.0 ml/min. The TSP interface temperature was optimized for maximum detection sensitivity for the acetyl derivatives. In the positive-ion mode, the optimal vaporizer control, vaporizer tip, vapour, block and tip heater temperatures were maintained at 163, 302, 325, 349 and 352°C, respectively, on an electron beam-off or electrical discharge-off. RESULTS AND DISCUSSION

The MS patterns of the derivatives showed a characteristic base ion (Fig. 1). As shown in Table I, the observed base ion is [M + H - n ( 6 0 ) ] ÷, based on the elimination of acetic acid (60 mass units) from the molecular ion. However, this was not noted for 15-keto-PGF2~, 19R-hydroxy-PGE1 or 19R-hydroxy-PGE2. With 19R-hydroxy-PGE1 or 19R-hydroxy-PGE2, the base ion became [M + H ( n - 1) • 60] +, possibly owing to the stability of acetyl groups introduced at the 19 position. The relationship between the TSP interface temperature and the [M + H - 60] + ion intensity of the PGB2 acetyl derivative is shown in Fig. 2. The best temperature conditions for detecting the base ion were 165°C (control), 303°C (tip) and 325°C (vapour). Chemical ionization due to an electric beam (filament on 150/~A) or electrical discharge caused no increase in the ion intensity of the PG acetyl derivatives (Fig. 2, data for electrical discharge not shown). Most acetyl derivatives of PGs and TXs could be easily detected on the SIM detection chromatogram at ca. 5 pmol of PG-related substances prior to derivatization (Fig. 3). The derivatives of TXB2, PGJ2 and 1l-dehydro-TXB2 showed lower sensitivity than the others, possibly owing to the low yields of their acetylation, but this was not confirmed. In our method, most PGE-type derivatives were converted into the corresponding PGA-type derivatives during acetylation and gave the same base ions (Table I). The dehydration of PGE type may be prevented by methylation of the

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c a r b o x y l a t e g r o u p [12], but this has yet to be confirmed. 15-Keto-PGF2~ and 6 - k e t o - P G F l , , which overlap in the S I M detection c h r o m a t o g r a m using ion 317, could be detected by the [M + H - 60] + ion (m/z 377, 100%) and [M + NH+] + ion (m/z 514, 33%), respectively. Fig. 4 shows the S I M detection c h r o m a t o g r a m of P G - r e l a t e d substances prior to derivatization o f ca. 0.2 pmol. This a m o u n t is considered the limit o f detection. The relationship between the p e a k area on S1M detection and the a m o u n t o f P G - r e l a t e d substances prior to derivatization is given in Fig. 5. D e t e r m i n a t i o n was carried out f r o m 0.5 to 10 p m o l o f P G - r e l a t e d substances prior to derivatization.

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SIM detection chromatograms for the acetyl derivatives of human seminal fluid extract containing some PGs are shown in Fig. 6. On the chromatograms, peaks due to the derivatives of PGF2~, PGF1,, PGE2, PGA2, PGD2, 15-ketoPGF1,, P G A t , PGE~, 19R-hydroxy-PGE~ and 19R-hydroxy-PGE2 can be seen. Similarly, PGE2, PGD2, 6-keto-PGE1 and 1 1-dehydro-TXB2 from rat brain were noted and are shown in Fig. 6. However, no other peaks could be identified. H P L C - T S P - M S techniques have been used previously for the determination of PG-related substances, but the sensitivity was not adequate because different fragment ions appeared following elimination of hydroxy groups [ 10]. The proton affinity (PA) of PG-related substances is less than that of the conjugate base of the reagent ion. PG-related substances are thus not efficiently ionized through chemical ionization or in the presence of ammonium acetate. Methods that change the PA or the polarity of compounds by diethylaminoethylation or meth-

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ylation, thereby improving the sensitivity, have been reported [11,12]. In these methods, since PG-related substances or derivatives have various hydroxy groups and are characterized by a wide range of polarities, gradient elution is used. To minimize overlap in the SIM detection chromatogram, measurement requires as long as 20-30 rain. However, in our method, the hydroxy groups of PG-related substances are acetylated and the derivatives are characterized by a limited range of polarities. Consequently, the retention time required for HPLC of the derivatives of PGrelated substances is only 11 rain when a single mobile phase is used and detection is by TSP-MS. Also, c a . twenty kinds of PG-related substance can be detected simultaneously by SIM of these base ions. In quantitation using MS, the deuterium-labelled compound of a sample is best as the I.S., but not all deuterium-labelled analogues of PG-related substances

HPLC-TSP-MS OF PG AND TX DERIVATIVES

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are available. To determine PG-related substances corresponding to unavailable deuterium-labelled compounds, some other deuterium-labelled compounds must be used. In HPLC-TSP-MS methods using a gradient elution, the composition of the mobile phase on the TSP interface changes owing to differences in the retention times of samples and the I.S., and such changes affect the sensitivity. Consequently, reproducibility is a problem in quantitative analysis. However, in our method, the composition of the mobile phase on the TSP interface is fixed because a single mobile phase is used, thus lessening the variation in sensitivity and improving the reproducibility. At present, our method is being used for monitoring the biosynthesis of PGrelated substances from various unsaturated fatty acids in homogenates of rat organs, and significant results have been obtained (unpublished data). This method is thus useful for studying the biosynthesis or metabolism of PG-related substances.

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Time (min)

Fig. 6. SIM detection chromatograms of the acetyl derivative of extracts from biological samples. (A) Human seminal fluid; (B) rat brain. Derivatization, HPLC and TSP conditions as described in Experimental.

REFERENCES 1 2 3 4

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