Determination of Taxotere in human plasma by a semi-automated high-performance liquid chromatographic method.

A rapid, selective and reproducible high-performance liquid chromatographic (HPLC) method with ultraviolet detection was developed for the determination of the anti-cancer agent Taxotere in biological fluids. The method involves a solid-phase extraction step (C2 ethyl microcolumns) using a Varian Advanced Automated Sample Processor (AASP) followed by reversed-phase HPLC. The validated quantitation range of the method is 10-2500 ng/ml in plasma with coefficients of variation < or = 11%. The method is also suitable for the determination of Taxotere in urine samples under the same conditions. The method was applied in a phase I tolerance study of Taxotere in cancer patients, allowing the pharmacokinetic profile of Taxotere to be established.     

Chemical conversion of arachidonic acid to slow reacting substances

The synthesis of slow reacting substances, leukotrienes C, D, and E, can be accomplished conveniently by a stereoselective biomimetic route. Details are provided for the conversion of 5-HPETE methyl ester 4 to leukotriene methyl ester (2) and thence to leukotrienes C and D.     

Automatic determination of urinary 4-hydroxy-3-methoxymandelic (vanillylmandelic) acid by liquid chromatography with electrochemical detection

An automated liquid chromatographic method for the determination of urinary concentrations of 4-hydroxy-3-methoxymandelic acid (VMA) is described. Urine samples are purified by solid-phase extraction on an anion-exchange cartridge and automated on-line chromatographic elution is carried out using a Varian AASP (advanced automated sample processor) system. The column effluent is monitored with an electrochemical detector using a glassy carbon working electrode. The method allows the determination of VMA in 0.05 ml of normal urine with a relative standard deviation of less than 3%. The analysis time can be shortened by use of back-flushing technique, and the correlation with a classical (but non-automated) VMA analysis method is excellent.     

Determination of CGP 6140 and its N-oxide metabolite,CGP 13 231, in plasma and urine by columns or cartridge switching HPLC

Summary Two methods were developed for the determination of CGP 6140 and its N-oxide metabolite. The first method was based on trace enrichment, rinsing on a first column (LiChroprep RP-2) andbackflush on the analytical column (Spherisorb ODS 1). The second method was based on trace enrichment onto a cartridge andforwardflush on the analytical column using the Advanced Automated Sample Processor (AASP). CGP 6140 and its N-oxide metabolite are spectrophotometrically detected at 405nm. The reproducibility and accuracy of the methods were convenient. The limits of quantitation (50nmol/l in plasma and 250 nmol/l in urine) were sufficient to follow the concentrations of CGP 6140 and its N-oxide metabolite after a 100mg single oral dose of CGP 6140 to volunteers.     

Rapid method for automated on-line extraction and fractionation of plasma leukotrienes and 12-hydroxy-5,8,10,14-eicosatetraenoic acids by reversed-phase high-performance liquid chromatography.

A method is described for automated on-line extraction and fractionation of plasma leukotrienes (LTs) and (5Z,8Z,10E,14Z)-(12S)-hydroxy-5,8,10,14-eicosate traenoic acid [12(S)-HETE] by reversed-phase high-performance liquid chromatography (RP-HPLC). This method was utilized to assess the accuracy of leukotriene B4 (LTB4) and leukotriene C4 (LTC4) determinations obtained by direct immunoassay of guinea pig plasma. Plasma LTB4 levels were significantly higher (p less than 0.01) and plasma LTC4 levels were unchanged when immunoassays were performed post versus pre RP-HPLC fractionation. Rapid separation, high recovery and baseline separation of LTB4, LTC4 and 12(S)-HETE, and minimal hardware requirements clearly demonstrate the general utility of this method.     

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.     

Total synthesis of slow reacting substances (SRS). “Leukotriene C-2” (11-trans-leukotriene C) (3) and leukotriene D (4)

Syntheses are described for the “slow reacting substances” 11-$\text{trans}$-leukotriene C (3) (previously known as leukotriene C-2) and leukotriene D (4), the cys-gly analog of leukotriene C (2). The synthesized leukotrienes 3 and 4 were instrumental in the assignment of structure to these members of the family of naturally occuring slow reacting substances which includes also 2.     

Single-step procedure for the extraction and purification of leukotrienes B4, C4 and D4

A rapid and simple method for the on-line concentration and high-performance liquid chromatographic (HPLC) purification of the leukotrienes in good yield from biological fluids is described. Readily available antisera are used in conjunction with this system to give a specific and sensitive assay for leukotrienes B4, C4 and D4 with sub-nanogram limits of detection. Tritium-labelled leukotrienes are used as internal standards, both to locate the leukotrienes post-HPLC and to accurately determine recoveries.     

Reversed-Liquid Phase Chromatographic Determination of Cefpodoxime in Human Plasma

Abstract

The reversed-phase liquid chromatographic determination of cefpodoxime in human plasma is described. Plasma samples were acidified with 1 mL of phosphoric acid solution containing internal standard, and adsorbed onto a C8 solid phase cassette designed specifically for use with a Varian Advanced Automated Sample Processor. Each cartridge on the cassette was washed with 1 mL of phosphoric acid containing 5% methanol and then was placed into the automated sample processor for direct elution of the retained drugs into the chromatographic system. Chromato-graphy was free from interferences in the analyte and internal standard windows, and yielded excellent linearity, accuracy, and precision in studies performed in this laboratory.     

Comparative study of solid phase extraction techniques for isolation of leukotrienes from plasma

We have compared five commercially available absorbent materials (i.e. C18 Sep-Pak, C18 J.T. Baker, Amberlites XAD-7, XAD-2 and XAD-4) for their applicability as effective tools for extraction of leukotrienes from plasma samples. Leukotriene C4 (LTC4) and B4 (LTB4) were selected as representatives of peptidic and non-peptidic leukotrienes, respectively. These leukotrienes were added to 1 ml of plasma and passed through columns containing the above described adsorbent materials. The recovery was determined for each material using different combinations of solvents. XAD-4 gave the highest recovery for LTB4 (90%), whereas XAD-4 and XAD-2 gave identical recoveries for LTC4 (90%) when an eluting solvent mixture of pyridine-water--dimethylformamide (50:45:5) was used. The efficiency of the other three solid adsorbent materials for leukotriene extraction were in order of decreasing magnitude, C18 J.T. Baker greater than XAD-7 greater than C18 Sep-Pak. XAD-7 was shown to be more efficient for LTB4 than for LTC4, whereas the octadecylsilane C18 materials gave approximately similar recoveries for both of the leukotrienes. In addition to very good extraction properties of XAD-4 and XAD-2 as compared to octadecylsilane silica, these solid adsorbent materials retained less plasma impurities than the C18 materials, giving cleaner chromatograms for leukotrienes extracted from plasma. Therefore, XAD-4 or XAD-2 are the best overall choice for extraction of leukotrienes from plasma for reversed-phase high-performance liquid chromatographic analysis.     

Application of reversed phase high performance liquid chromatography to the analysis of sulphidopeptide leukotrienes in pig bile

Reversed phase HPLC methodology has been developed for separation of peptide leukotrienes and indomethacin in porcine bile. Reproducible recoveries were obtained using radioactive leukotrienes ([3H]LTC4, 57.1 +/- 2.5%; [3H]LTE4, 62.7 +/- 1.9%; [3H]LTD4, 54.3 +/- 2.7%). Radioimmunoassay of column eluant demonstrated that as little as 300 pg of exogenous leukotrienes could be measured in bile fluids, with similar recoveries. Analysis of bile sampled 60-90 min after initiation of experimental endotoxic shock in indomethacin treated pigs revealed a leukotriene concentration of 5.24 +/- 1.16 ng/mL(LTD4). This was significantly greater (p less than 0.05, n = 3) than that observed in samples collected prior to endotoxin (0.42 +/- 0.23 ng/mL), or from untreated animals (0.85 +/- 0.51 ng/mL). This method is thus applicable to investigation of the role of 5-lipoxygenase products in porcine models of human disease, including shock conditions such as endotoxaemia, during cyclooxygenase inhibition by indomethacin.     

Direct injection method for quantitation of endogenous leukotriene E4 in human urine by liquid chromatography/electrospray ionization tandem mass spectrometry with a column-switching technique.

A new analytical method employing liquid chromatography/tandem mass spectrometry (LC/MS/MS) with a column-switching system was developed for quantitative determination of leukotriene E4 (LTE4) in human urine. A column-switching system using a trapping column, which concentrates the analyte and removes salts and other water-soluble contaminants, allowed direct injection of human urine. Because simultaneously eluted endogenous contaminants suppressed the ionization efficiency of LTE4, good liquid chromatographic separation was very important for establishing this method, notwithstanding the high selectivity of MS/MS. The calibration curve was linear over the range from 10 to 3000 pg/mL, and the method showed good accuracy and precision. This method should therefore be very useful for determination of LTE4 amounts in human urine in studies on leukotriene metabolism and the efficacy of antileukotriene drugs.     

High-performance liquid chromatographic analysis of arachidonic acid metabolites by pre-column derivatization using 9-anthryldiazomethane

Arachidonic acid (AA) metabolites produced by washed human platelets and rat macrophages were analyzed by high-performance liquid chromatography (HPLC) using a pre-column derivatization method. The reagent, 9-anthryldiazomethane, used in this study and AA metabolites derivatized by the reagent were purified by gel permeation chromatography (PG-pak C column), prior to normal-phase HPLC analysis. A sample containing eleven derivatives (12-, 15- and 5-hydroxyeicosatetetraenoic acid, 12-L-heptadecatrienoic acid, leukotriene B4, prostaglandins B2, D2, E2 and F2 alpha, thromboxane B2 and 6-keto-prostaglandin F1 alpha) was separated on a normal-phase column (PG-pak B); the detection limit is better than 100 pg for all components.     

A sensitive high performance liquid chromatography microassay for bemoradan, a novel cardiotonic agent, in plasma/serum using a semi-automatic liquid/solid extraction sample preparation system--AASP

A high performance liquid chromatographic method for the measurement of bemoradan levels in plasma/serum is described. This method uses Varian's AASP (Varian Associates, Sunnyvale, CA, USA), a semi-automatic liquid/solid extraction sample preparation system. It requires only small volumes of plasma/serum samples (0.2-1 mL) and needs no organic solvent for sample preparation. The mean recovery of bemoradan at plasma or serum concentrations of 0.5-100 ng/mL is 82%. The assay has a detection limit of 0.5 ng/mL (when 1 mL of plasma/serum is used) and is linear in the concentration range 0.5-500 ng/mL.     

Cyclooxygenase and lipoxygenase arachidonic acid metabolism by monocytes from human immune deficiency virus-infected drug users.

Prostaglandin E2, thromboxane B2 and leukotriene B4 monocyte production have been determined in human immune deficiency virus (HIV)-infected drug users (n = 36) and healthy subjects (n = 29). Eicosanoids were extracted from the incubates using C18 solid-phase cartridges and determined by radioimmunoassay. An enhanced production of prostaglandin E2 and thromboxane B2 was detected in monocytes from HIV-positive drug users whether or not they had been previously stimulated with zymosan. Concomitant leukotriene B4 increases were not observed. The results reported in this paper indicate that altered cyclooxygenase arachidonic acid metabolism in monocytes from HIV-infected drug users is associated with the severe cellular immunodysfunction characteristic of AIDS. In contrast, leukotriene B4 does not seem to play a role in AIDS-associated immunosuppression.     

Improved high-performance liquid chromatographic method for analysis of cyclosporin A using an automated sample processor

Transplant patients receiving the immunosuppressive drug cyclosporin A require regular monitoring to maintain levels within a narrow therapeutic range. A stable, accurate and reproducible high-performance liquid chromatographic method for analysis of cyclosporin A in whole blood has been developed using the Varian Advanced Automated Sample Processor. Starting with 200 microliters of blood, absolute recovery of both cyclosporin A and the internal standard was 81% with a detection limit of 12.5 ng/ml. The assay is perfectly linear over the range 0-1000 ng/ml (r2 = 1.0). At a concentration of 250 ng/ml, the coefficient of variation, both between samples and between assays, is 1.87%. Chromatographic cycle time is 10.2 min per sample. Up to eighty samples can be processed by one person in a working day, with final results within 16 h.     

Enzymic preparation of dioxygen-18 labelled leukotriene E4 and its use in quantitative gas chromatography-mass spectrometry.

A simple and rapid method is described for the preparation of a stable isotope oxygen-18 labelled leukotriene E4 (LTE4). Oxygen-18 labelling of LTE4 methyl ester in oxygen-18 water catalysed by a pig liver esterase resulted in the incorporation of two oxygen-18 atoms in the carboxylic group of LTE4 to the extent of 89.8% ([18O2]LTE4) and one oxygen-18 atom to the extent of 9.4% ([16O18O]LTE4), with only 0.7% remaining unchanged ([16O2]LTE4). [18O2]LTE4 was found not to back-exchange following incubation in acidified urine (pH 4.0) at 4 degrees C for up to 20 h. [18O2]LTE4 was demonstrated to be a useful internal standard in a method for the quantitative determination of LTE4 in human urine involving high-performance liquid chromatography and gas chromatography with negative-ion chemical ionization tandem mass spectrometry: the concentration of LTE4 in a 24-h urine sample of a healthy subject was determined to be 68.1 pg/ml.     

Measurement of urinary estriol glucuronides during the menstrual cycle by high-performance liquid chromatography

A simple high-performance liquid chromatographic (HPLC) method for measuring estriol-3-glucuronide (E3-3-G) and estriol-16-glucuronide (E3-16-G) in the urine of non-pregnant women is described. Estriol conjugates were extracted from 4 ml of urine with a small cartridge of graphitized carbon black (Carbopack B). After washing, E3-3-G and E3-16-G were desorbed separately by a two-step elution system. After solvent removal, the two glucuronides were quantified by isocratic ion-suppression HPLC with fluorimetric detection. The analytical recovery of the two estriol metabolites was about 95%. The detection limit of the method was 0.6 ng/ml for both analytes in urine, which is well below the concentrations of clinical interest, and the method is not susceptible to substantial interferences. Data relative to urinary levels of E3-3-G and E3-16-G measured by this method on a daily basis in early morning samples from nine women during their menstrual cycles were compared with those reported in the literature and obtained by radioimmunoassay techniques. Moreover, the potential use of defined changes in the concentrations of the two conjugates for predicting the fertile period of women was assessed.     

Determination of four carboxylic acid metabolites of felodipine in plasma by high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatography method with ultraviolet detection at 220 nm was developed to determine four carboxylic acid metabolites in plasma following therapeutic doses of the calcium antagonist felodipine. After the addition of an internal standard the analytes were isolated by liquid-liquid and solid-phase extraction. The metabolites were applied to a C2 cartridge in their free acid form, but they were transformed and retained as ion pairs with tetrabutylammonium during a wash with phosphate buffer (pH 7), prior to automated elution and injection by the Varian AASP system onto the analytical C18 column. Using a sample volume of 1 ml of plasma, the lower limit of determination for the metabolites was about 20 nmol/l. The influence of the pH of the mobile phase on the retention time of the metabolites and the structural requirements for the internal standard were studied. The method was applied to plasma samples from four dogs collected after an oral dose of felodipine. The plasma concentration-time profiles of the metabolites gave useful information about the mechanisms by which they were formed and eliminated.     

Structural analysis of leukotriene C<Subscript>4</Subscript> isomers using collisional activation and 157 nm photodissociation

The fragmentation of 5-hydroxy-6-glutathionyl-7,9,11,14-eicosatetraenoic acid [leukotriene C4 or LTC4 (5, 6)] and its isomeric counterpart LTC4 (14, 15) were studied by low and high-energy collisional induced dissociation (CID) and 157 nm photofragmentation. For singly charged protonated LTC4 precursors, photodissociation significantly enhances the signal intensities of informative fragment ions that are very important to distinguish the two LTC4 isomers and generates a few additional fragment ions that are not usually observed in CID experiments. The ion trap enables MSn experiments on the fragment ions generated by photodissociation. Photofragmentation is found to be suitable for the structural identification and isomeric differentiation of cysteinyl leukotrienes and is more informative than low or high-energy CID. We describe for the first time the structural characterization of the LTC4 (14, 15) isomer by mass spectrometry using CID and 157 nm light activation methods.