Quantitative analysis of prostaglandins in cell culture medium by high-resolution gas chromatography with electron-capture detection

Prostaglandins have been shown to be important modulators of haemostatis , immune responses, and growth of normal and neoplastic cells. In order to investigate the cell origin and metabolic profile of the endogenous prostaglandins in human tumours, a convenient extraction and gas chromatographic method for measuring the various classes of prostaglandins was developed. Infiltrating macrophages from human tumours were isolated using adherence to plastic. Macrophage-enriched and macrophage-depleted cell populations were then cultured in vitro and the media supernatant was studied for the presence of prostaglandins E1, E2, F2 alpha, and 6-keto-F1 alpha (the spontaneous breakdown product of prostacyclin, PGI2). Routinely, 1 ml of medium containing 10(6) cells was studied. The eicosanoids were extracted using commercially available octadecylsilyl silica reversed-phase columns prior to derivatization. Standards and samples were prepared as pentafluorobenzyl ester (methoxime) trimethylsilyl ether derivatives for analysis on an OV-101 (25 m X 0.2 mm) fused-silica capillary column. Recovery of standards ranged from 93% to 37%, with linear recovery in all instances (regression coefficients greater than 0.98). Detection limits were 20 pg for each of the prostaglandins. Analysis of cell subpopulations from six human tumours revealed that infiltrating macrophages produce various prostaglandin profiles and are largely responsible for the prostaglandin production in human cancer. The described analytical method is the first application of high-resolution gas chromatography with electron-capture detection to the quantitative profiling of prostaglandins from human cell culture.     

The simultaneous determination of the prostaglandins by chemospecific deuteration with separation and quantification by combined gas chromatography — chemical ionization mass spectrometry

A new gas chromatography/mass spectrometry (g.c./m.s.) procedure is described which simultaneously quantifies eight prostaglandins using one internal standard. The method could easily be extended to include more prostaglandins. The basis of the new method derives from two developments; 1) the discovery of quantitative chemospecific deuteration conditions for the C₅–C₆ double bonds of the prostaglandins (PG₂), and 2) the development of special computer software to solve the analytical problem. Eight prostaglandins, present in the 1–100 ppm range, were determined with errors only occasionally over 10%. Substantially better performance may be expected with a more stable mass spectrometer. Samples with only 2 prostaglandins showed errors only occasionally over 1%.     

Prostacyclin and Synthetic Analogues

The prostanoids continue to attract a great deal of attention, particularly in the pharmacological sector. Many of the numerous biological studies on prostaglandins deal with their cardiovascular effects. The pronounced antihypertensive and platelet aggregation-inhibiting properties of prostacyclin (PGI₂) resulted in world-wide efforts to synthesize acid-stable analogues of the natural product. Whereas refined analytical procedures disproved the hypothesis that PGI₂ is a circulating hormone, exploratory human trials with PGI₂ confirmed the pharmacological findings obtained from animal experiments. Clinical studies of PGI₂ analogues have been started. In vitro studies on isolated vessels revealed that potent cardiovascular drugs stimulate prostacyclin synthesis; however, it has not as yet been possible to demonstrate whether these effects are also of therapeutic significance. A survey of the PGI₂ analogues synthesized so far is presented, and, with the aid of a few examples, the problems encountered during their synthesis are outlined; structure-activity relationships are also discussed.     

Determination of prostaglandins and thromboxane in whole blood by high-performance liquid chromatography with fluorimetric detection

A highly sensitive and specific assay for the quantitation of prostaglandins (PGs) such as PGE1, PGE2, PGF1 alpha, PGF2 alpha, 6-keto-PGF1 alpha, and including thromboxane B2, is described. The method involves the addition of PGF1 alpha and PGE1 as the internal standards, extraction from whole blood and purification by silica gel column chromatography. Following conversion into the methoximes, purification by reversed-phase chromatography and esterification with panacyl bromide, samples are analysed by high-performance liquid chromatography with fluorimetric detection. The lower limit of detection of the eicosanoids 6-keto-PGF1 alpha, thromboxane B2 and PGF2 alpha in blood is ca. 50 pg/ml and that of PGE2 is 100 pg/ml. Assay linearity is demonstrated over a range from 60 pg to 60 ng of eicosanoid injected. The method allows simultaneous assessment of prostaglandins and thromboxane extracted from complex biological fluids at picogram levels.     

Glass capillary column chromatography of mixed derivatives of primary prostaglandins, 6-keto prostaglandin F1α and thromboxane B2

This work describes the use of glass capillary columns (GCC) in the rapid concurrent analysis of primary prostaglandins (PGs) (e. g. PGE₂, PGE₂, PGF_2α) and other functionally significant metabolites of arachidonic acid (AA) such as TXB₂ and 6-keto PGF_1α. The use of a new multistep mixed derivatization approach that generates the methyl esters of n-butylboronate, pentafluorobenzyloxime, trimethylsilyl ether derivatives of these compounds remarkably simplifies the GC profiling of the three main pathways of AA metabolism (PGs, thromboxane, and prostacyclin). Furthermore, isomeric species giving very similar or identical mass spectral patterns can be easily identified by their relative retention times on high efficiency capillary columns.     

Purification and quantitative analysis of urinary prostanoids in human and in rat by packed and capillary gas chromatography-negative-ion chemical-ionization mass spectrometry

We describe a method for the quantitative analysis of prostaglandin (PG) E2 and the major urinary metabolites PGI2 and thromboxane (Tx) A2 in human and in rat by combined gas chromatography and negative-ion chemical-ionization mass spectrometry. The procedure is based on the sequential use of small columns with distinct properties combined with a thin-layer chromatography step, for the extraction and the purification of urinary prostaglandins. The compounds are then analysed as their pentafluorobenzyl ester-O-methyloxime-trimethylsilyl ether derivatives, using either packed or capillary columns. Deuterated analogues are used as internal standards. The method was established by using tritiated prostaglandins covering the extremes of polarity in order to optimize the recovery of prostanoids as well as the quality of the chromatograms and spectra. The overall recovery was 24%. Standard curves were obtained by the same procedure and found to be reproducible, with a maximal day-to-day variation of +/- 5%. The relatively simple approach required for the sequential extraction and purification of prostaglandins on small columns of distinct properties, combined with the highly specific and highly sensitive method of detection, places this procedure among the most reliable method for measuring urinary prostanoids in both humans and animals. In addition, the procedure is faster than classical approaches and necessitates smaller amounts of samples and solvents.     

Solid-phase extraction of prostanoids using an automatic sample preparation system.

A commercial automated solid-phase extraction system for cyclooxygenase arachidonic acid metabolites in urine samples has been evaluated. Comparison of manual and automatic batch (36 samples) extraction procedures for tritium labelled prostanoids added as tracers to urine samples has shown equivalent results with recoveries greater than 90% for prostaglandins E2, F2alpha and 6-keto prostaglandin F1alpha as well as thromboxane B2. Analyte stability is not affected by the automated procedure, which uses less solvents and has a faster overall processing time than the manual method. The automated system has been applied to the extraction of prostanoids in urine samples from workers exposed to dichloroethane.     

Simultaneous determination of prostaglandins E1, A1, and B1 by reversed-phase high-performance liquid chromatography for the kinetic studies of prostaglandin E1 in solution.

A method for the simultaneous determination of prostaglandins E1, A1 and B1 (PGE1, PGA1 and PGB1) in solution has been developed by reversed-phase high-performance liquid chromatography using a 3 microns C18 column. The mobile phase consisted of 35% acetonitrile in 0.002 M phosphate buffer (pH 3.5) and its flow-rate was 1.5 ml/min. Quantitative measurement was performed using a photodiode array detector system at 190, 220, and 280 nm for PGE1, PGA1 and PGB1, respectively. The method has been applied to the primary kinetic studies for reaction profile for PGE1----PGA1----PGB1 at 60 degrees C in pH 2.0, 7.2, 10.0 and 12.0 buffer solutions.     

Determination of 2,3-dinor-6-ketoprostaglandin F1 alpha in urine samples by liquid chromatography and radioimmunoassay

A method for 2,3-dinor-6-ketoprostaglandin F1 alpha quantification based on high-performance liquid chromatography-radioimmunoassay is described. Samples are acidified to pH 3 and processed through C18 disposable cartridges. The prostanoids are eluted with methyl formate and further separated on a reversed-phase column using acetonitrile-acetic acid-triethylamine buffer (32:68). Studies of the effect of eluent pH were performed in order to optimize resolution and separation of 2,3-dinor-6-keto-PGF1 alpha from other prostanoids. Eluates were collected and assayed by radioimmunoassay using a heterologous system, with 6-keto-PGF1 alpha as radioligand and an antiserum with high cross-reactivity for 2,3-dinor-6-keto-PGF1 alpha. Sensitivity, precision and accuracy of the assay procedure are reported together with the validation of its specificity. The proposed method has been applied to the determination of this prostacyclin metabolite in human urine.     

Rapid Separation of Prostaglandins by Linear High Performance Thin Layer Chromatography

Abstract

A continuous development technique using silica gel linear high performance TLC plates is described for the separation of prostaglandins 6-keto-F_1α, F_2α, E₂, 13,14-dihydro-15-keto-F_2α, 13–14-dihydro-15-keto-E₂, and thromboxane B₂. Complete separation of all six prostaglandins was achieved with a solvent system of ethyl acetate/acetone/acetic acid (90:5:1). The method is simple, rapid and provides excellent resolution of plasma prostaglandins prior to quantitation by gas chromatography-mass spectrometry.     

The synthesis of prostaglandin E1 and related substances

The authors describe in detail the conversion of 4 isometric exo-substituted bicyclo (3.1.0) hexanones 1a, 1b, 2a and 2b (R=CH3) to 4 prostaglandins of the E series, including crystalline dl-prostaglandin E1 (PGE1). Oxidative solvolysis of the keto esters 1a and 2a and of the desired keto acids 1a and 2a (R=H) according to the method of Just and Simonovitch resulted in a good yield of the mixture of vic-glycols of unrearranged carbon skeleton. The presence of dl-PGE1, or dl-8-iso PGE1 or their methyl esters was not detected in either case. Analysis of the unalkylated ketones 5a and 5b under the same and other conditions yielded unrearranged vic-glycols 6 as essentially the only product. A reaction sequence produced dl-8-isoprostaglandin E1, the 15-epimers of these prostaglandins, and dl-PGA1 and dl-PGB1 methyl esters.     

Direct analysis of the major human seminal prostaglandins by thermospray high-performance liquid chromatography-mass spectrometry

Thermospray high-performance liquid chromatography-mass spectrometry (HPLC-MS) can be a powerful tool for characterizing eicosanoids in complex biological samples. The positive ion spectra obtained from primary prostaglandins such as PGE1 PGE2, 19-OHPGE1, 19-OHPGE2, PGF2 alpha, PGD2, 6-keto-PGF1 alpha and from leukotriene B4 are very simple, with base peaks corresponding to ions arising from the loss of H2O from the (M + H)+ and (M + NH4)+ ions, except for PGB2 and PGF2, where the latter two ions predominate. The application of this technique to the concurrent determination of the E1 and E2 prostaglandins and their 19-hydroxylated derivatives in human semen is described. The technique affords a moderate level of sensitivity (5-20 ng on-column) and excellent specificity so that virtually no sample manipulation is required other than dilution in acetone and centrifugation. The clear supernatant is injected directly into the HPLC-MS system. A similar analysis by either gas chromatography (GC) or GC-MS would need multi-step derivatization, thus increasing the sample manipulation required and the total analysis time.     

Ultra-pressure liquid chromatography/tandem mass spectrometry targeted profiling of arachidonic acid and eicosanoids in human colorectal cancer

Cumulative evidence shows that eicosanoids such as prostaglandins, leukotrienes, thromboxanes and hydroxy eicosatetraenoic acids play an important role in associating inflammation with human colorectal cancer (CRC). In this study an ultra‐pressure liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) method was developed and validated for the targeted profiling of eight relevant eicosanoids and the major metabolic precursor, arachidonic acid (AA), in human colon. Multiple reaction monitoring (MRM) experiments were performed in negative electrospray ionization mode. The metabolites were separated using a C₁₈ column consisting of 1.7 µm ethylene‐bridged hybrid particles (100 × 2.1 mm i.d.) and gradient elution (50 to 95% of solvent B) with a mobile phase comprising water (0.1% formic acid) [solvent A] and acetonitrile (0.1% formic acid) [solvent B] at a flow rate of 0.4 mL/min. The analysis time for each sample was 5.5 min. Our UPLC/MS/MS method demonstrated satisfactory validation results in terms of selectivity, sensitivity, matrix effect, linearity, extraction efficiency, intra‐ and inter‐day precision, accuracy and autosampler stability. The method was applied for the clinical profiling of matched pairs of cancerous and normal colon mucosae obtained from eight colorectal cancer patients. Endogenous levels of AA and selected eicosanoids such as prostaglandin E₂ (PGE₂), prostacyclin (PGI₂) [assayed as its stable hydrolytic product 6‐keto‐prostaglandin_1α (6‐k PGF_1α)] and 12‐hydroxy‐5Z,8Z,10E,14Z‐eicosatetraenoic acid (12‐HETE) were found to be significantly different (p <0.05; paired t‐test) between cancerous and normal mucosae. Copyright © 2011 John Wiley & Sons, Ltd.     

Low molecular weight heparin enhances prostacyclin production by cultured human endothelial cells.

Confluent cultures of vascular endothelial cells derived from the human umbilical vein were incubated in a serum-free medium in the presence of low molecular weight heparin (LMWH) with molecular weights of 4000-6000 dalton (Da), or of unfractionated heparin (UFH) with average molecular weight 12,000 Da, and prostacyclin production was determined by radioimmunoassay for 6-keto-prostaglandin F1 alpha, the stable metabolite of prostacyclin. LMWH at 1 U/ml as anti-factor Xa activity significantly increased prostacyclin production after 6h or longer; however, UFH at 1 USP U/ml did not induce such a significant change. The LMWH-induced increase in prostacyclin production occurred at 0.1 U/ml and above after 6 h of treatment. Since prostacyclin is both a potent inhibitor of platelet aggregation and a vasodilator, it was suggested that the increased endothelial cell prostacyclin production induced by LMWH may be a component of the anticoagulant activity of the drug.     

Determination of leukotrienes and prostaglandins in [14C] arachidonic acid labelled human lung tissue by high-performance liquid chromatography and radioimmunoassay

A liquid chromatographic method for the determination of 14C-labelled prostaglandins, leukotrienes and other lipoxygenase products formed by human lung tissue is described. In this paper we report our problems identifying these substances when 3H- or 14C-labelled compounds are compared with measurements of the mass by absorption or radioimmunoassay. Furthermore, some preliminary results of [14C] arachidonic acid labelled human lung tissue, stimulated by the Ca-ionophore A23187, show that, of the lipoxygenase products, mostly leukotriene B4 like compounds are formed and less leukotriene C4, E4 and D4. Relatively large amounts of hydroxyeicosatetraenoic acids are present. The main cyclooxygenase products are thromboxane B2, 6-ketoprostaglandin F1 alpha and prostaglandin D2.     

O-(2,3,4,5,6-pentafluorophenyl)methylhydroxylamine hydrochloride: a versatile reagent for the determination of carbonyl-containing compounds.

A review on the use of O-(2,3,4,5,6-pentafluorophenyl)methylhydroxylamine hydrochloride (PFBHA) for the determination of carbonyl-containing compounds is presented. PFBHA has been used in the determination of such diverse compounds as thromboxane B2, prostaglandins, amygdalin and a variety of other aldehydes, ketones and acids. PFBHA has been used for the determination of these compounds found in water, blood, urine, air and even clothing. The review covers literature referenced in Chemical Abstracts from 1975, when PFBHA was first synthesized, through March 1992.     

A New Approach to the Extraction of Prostaglandins 6-Keto-F1α, F2α, E2, and E1 from Gastric Mucosa with Quantitative Analysis by Reverse Phase Liquid Chromatography

Abstract

A simple and reliable method is presented for the extraction of Prostaglandins 6-Keto-F₁ α, F₂ α, E₂ and E₁ from gastric mucosa with quantitation by Reverse Phase Liquid Chromatography. Extraction is accomplished without the need for the harsh chemicals normally associated with tissue extraction. The efficiency of prostaglandin extraction is demonstrated utilizing radiolabeled prostaglandins. Quantitation of these compounds is compared to measurement by radioimmunoasay.     

Separation of Prostaglandins on a Hydrophobic Sephadex Derivative

Abstract

Liquid-gel chromatography on a hydrophobic Sephadex derivative has been used for the separation of prostaglandins. Solvent systems are given for separation of PGE and PGF compounds either as free acids or as methyl esters, using a Sephadex derivative containing 12% hydroxyalkyl groups. The columns are simple to prepare and can be used repeatedly over long periods of time. The recovery is high and the technique should be a useful complement to other methods used for the isolation of prostaglandins.     

A simple procedure for the chromatographic isolation and determination of prostaglandins from human seminal plasma

Summary A relatively simple procedure for the isolation and determination of the prostaglandins present in human seminal fluid is described. It involves preliminary chromatographic purification of these compounds from the major non-prostaglandin impurities followed by their total elution in one solvent (one-step elution). The prostaglandins thus obtained were almost free from other lipids and were further resolved into prostaglandin-groups and individual prostaglandins by repeated thin-layer chromatography. Data are also presented for prostaglandin contents of fresh semen samples from five individuals and results compared with those from the stored samples.

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Einfaches Verfahren zur chromatographischen Isolierung und Bestimmung von Prostaglandinen aus menschlichem Sperma

Zusammenfassung Das Verfahren umfaßt eine chromatographische Abtrennung der Verbindungen von den hauptsächlichsten Verunreinigungen und die Gesamtelution mit einem Lösungsmittel. Die von anderen Lipiden fast völlig freien Prostaglandine werden durch wiederholte Dünnschicht-Chromatographie in Gruppen und Einzelverbindungen getrennt. Werte werden angegeben über die Prostaglandingehalte von frischem im Vergleich zu gelagertem Sperma.

     

A convenient TLC schedule for differential separation and quantification of prostaglandins,thromboxanes, and hydroxy fatty acids formed from [1-14C]arachidonic acid in human blood platelets

Eleven TLC solvent systems were evaluated for the separation of prostaglandins F_2α, E₂ and D₂, thromboxane B₂, 12-OH-5,8,10-heptadecatrienoic acid (HHT), 12-OH-5,8,10,14-eicosatetraenoic acid (HETE), and arachidonic acid (AA) from each other. A three-solvent development TLC procedure, which can conveniently be used for routine resolution of mixtures of these metabolites on a single TLC plate, is reported. The method was used for quantification of the metabolites formed from ¹⁴C-labeled AA by washed human platelets.