Production of 19-hydroxy-11-deoxycorticosterone and 19-oxo-11-deoxycorticosterone from 11-deoxycorticosterone by cytochrome P-450(11)beta

Incubation of 11-deoxycorticosterone with a cytochrome P-450(11)beta-reconstituted system yielded, in addition to corticosterone and 18-hydroxy-11-deoxycorticosterone, a new steroid product. The retention time of the new product was identical with that of authentic 19-hydroxy-11-deoxycorticosterone on high performance liquid chromatography (HPLC). The turnover number of 19-hydroxy-11-deoxycorticosterone formation was 7.0 mol/min/mol P-450. When a large amount of cytochrome P-450(11)beta was used for the reaction and the products were analyzed by HPLC, the 19-hydroxy-11-deoxycorticosterone peak disappeared from the chromatogram and concomitantly new unidentified peaks appeared. These results suggest that 19-hydroxy-11-deoxycorticosterone was further metabolized to other steroids by cytochrome P-450(11)beta. Therefore, we next incubated 19-hydroxy-11-deoxycorticosterone with cytochrome P-450(11)beta and analyzed the reaction products by HPLC. The above-mentioned unidentified peaks appeared again in the chromatogram. The retention time of one of the peaks coincided with that of authentic 19-oxo-11-deoxycorticosterone. This peak substance was purified by repeated HPLC and subjected to mass spectrometry and 1H NMR analyses. Its field desorption mass spectrum (FD-MS) showed a M+ peak at m/e 344. The 1H NMR spectrum showed the signal of an aldehyde proton instead of those of hydroxymethyl protons at the C-19 position. These results suggest that cytochrome P-450(11)beta can catalyze the 19-hydroxylation of 11-deoxycorticosterone, and the 19-hydroxy-11-deoxycorticosterone produced is further oxidized at the C-19 position to 19-oxo-11-deoxycorticosterone.     

Production of 19-oic-11-deoxycorticosterone from 19-oxo-11-deoxycorticosterone by cytochrome P-450(11)beta and nonenzymatic production of 19-nor-11-deoxycorticosterone from 19-oic-11-deoxycorticosterone

19-Oxo-11-deoxycorticosterone was incubated with a cytochrome P-450(11)beta-reconstituted system, and the metabolites were analyzed by high performance liquid chromatography(HPLC). The main product found after chromatography was collected and treated with diazomethane. HPLC and 1H-NMR analysis of the methylated derivative indicated that it was 19-oic-11-deoxycorticosterone methyl ester. When 19-oic-11-deoxycorticosterone was stored at -20 degrees C for 1 month, it was spontaneously converted to other steroids. Structural analysis of the main degradation product indicated that it was 19-nor-11-deoxycorticosterone. These results suggest that the conversion of 19-oxo-11-deoxycorticosterone to 19-oic-11-deoxycorticosterone occurs through the P-450(11)beta-catalyzed reaction, and that the 19-oic-11-deoxycorticosterone thus formed is nonenzymatically converted into 19-nor-11-deoxycorticosterone.     

Enantiospecific gas chromatographic-mass spectrometric analysis of urinary methylphenidate: implications for phenotyping

A chiral derivatization gas chromatographic-mass spectrometric (GC-MS) method for urine methylphenidate (MPH) analysis was developed and validated to investigate preliminary findings regarding a novel MPH poor metabolizer (PM). Detection was by electron impact (EI) ionization-selected ion monitoring of the N-trifluoroacetylprolylpiperidinium fragments from MPH and the piperidine-deuterated MPH internal standard. The PM eliminated approximately 70 times more l-MPH in urine (9% of the dose over 0-10h), and approximately 5 times more of the d-isomer (10% of the dose), than the mean values determined from 10 normal metabolizers of MPH. Only minor amounts of the metabolite p-hydroxy-MPH were found in the urine of both the PM and normal metabolizers, while the concentration of MPH lactam was not high enough to be detectable. The described method indirectly gauges the functional carboxylesterase-1 status of patients receiving MPH based on the evaluation of relative urine concentrations of d-MPH:l-MPH. Clinical implications concerning rational drug selection for an identified or suspected MPH PM are discussed.     

A rapid gas chromatographic-mass spectrometric method for prostaglandin analysis at picomole levels

A rapid method for the qualitative and quantitative determinations of PGE₁, E₂, F_1α, and F_2α is described. Tris-TMS-PGF and mono-TMS-PGB methyl esters are obtained by the reaction of the corresponding PGF and PGE methyl esters with N-trimethylsilylimidazole (TSIM) and piperidine. The reaction is instantaneous, and a single derivative with excellent gas chromatographic properties is obtained for each prostaglandin tested. The presence of very prominent peaks in the mass spectra of the derivatives allows the determination of prostaglandins at picomole levels using multiple ion detection (MID).     

A gas chromatographic-mass spectrometric analysis of the esterifying alcohols of pumpkin seed protochlorophylls

The esterifying alcohols of protochlorophyll a and 4-vinyl-(4-desethyl)-protochlorophyll a (purified as the respective pheophytins) from pumpkin seeds were examined by gas chromatography-mass spectrometry. The results of the analysis suggested that pumpkin seed protochlorophyll a is esterified with all possible C₂₀ isoprenoid alcohols between and including geranylgeraniol and phytol, phytol comprising 90% or more of the mixture of esterifying alcohols, and that the 4-vinyl-(4-desethyl)-protochlorophyll a is esterified with farnesol and all possible C₂₀ isoprenoid alcohols between and including geranylgeranoid and phytanol, phytol comprising 50% or more of the mixture of esterifying alcohols. The 4-vinyl-(4-desethyl)-protochlorophyll a from a sample of older mature pumpkin seeds was found to be richer in esterifying alcohols corresponding to isoprenoid precursors of phytol then was the 4-vinyl-(4-desethyl)-protochlorophyll a from a sample of younger mature seeds. Other isoprenoid alcohols may have been present in very minor quantities in the mixtures of esterifying alcohols from the pumpkin seed protochlorophylls but were not looked for in this study. These results are discussed in terms of a biosynthetic accumulation of 4-vinyl-(4-desethyl)-protochlorophyll a in pumpkin inner seed-coat tissue.     

Liquid chromatographic-mass spectrometric method for the determination of alpha-,beta-arteether in rat serum

This study reports the development and validation of a sensitive and selective assay method for the determination of alpha-,beta-arteether in rat serum by liquid chromatography-mass spectrometry. The mobile phase was composed of methanol-0.1 mM sodium acetate (pH 5) (80:20%) at a flow-rate of 1 ml min(-1) and chromatographic separations were achieved on a Ultracarb, 5 ODS 20, Phenomenex column (5 micrometer, 30 mmx4.6 mm I.D.). The total effluent from the column was split so that one-tenth was injected into the electrospray LC-MS interface. ESI-MS analysis was carried out using a Micromass Quattro II Triple Quadrupole Mass Spectrometer equipped with an electrospray source. The MS analysis was carried out at a cone voltage of 52 V with a scan range of 100-400 Da. The analytes were quantified from the [M+Na](+) ion chromatograms of alpha-,beta-arteether at m/z 335 and artemisinin at m/z 305. A simple liquid-liquid extraction with 2x2 ml n-hexane was used to isolate alpha-,beta-arteether from rat serum. The method was validated in terms of recovery, linearity, accuracy and precision (within- and between-assay variation). The recovery from spiked control samples ranged from 88.41 to 96.17% with a maximum CV of 10.8% for alpha-arteether and 69.83-79.69% with a maximum CV of 17.06% for beta-arteether. Linearity in serum was observed over the range 20-320 ng ml(-1). Percent bias (accuracy) was well within the acceptable range. Within- and between-assay precision were less than 15%. The assay method described here is being applied to study the pharmacokinetics of CDRI developed intramuscular formulation Emal (alpha-/beta-arteether in the ratio of 30:70) in rats. The method is sensitive enough to monitor alpha-,beta-arteether up to 24 h after a single 30 mg kg(-1) i.m. dose.     

Rapid gas chromatographic-mass spectrometric diagnosis of dihydropyrimidine dehydrogenase deficiency and dihydropyrimidinase deficiency

A rapid yet reliable chemical diagnosis for dihydropyrimidine dehydrogenase (DHPD) deficiency, and possibly dihydropyrimidinase (DHP) deficiency in cancer patients, prior to therapy with pyrimidine analogues such as 5-fluorouracil, is desired for prevention of severe side-effects by these drugs. We have reported the basic separation and quantitation technology for pyrimidine metabolites using gas chromatography-mass spectrometry. A proposal to use the number (n) of standard deviations (SD) above the normal mean, as the index of the excessive urinary excretion of the metabolites appears not to be commonly used. When used, the values were too small, such as two or three, even in genetic disorders. Here, we applied the method to 11 urine specimens from proven cases including two DHP carriers and proved how specific the method is, because "n"-values were markedly large for thymine (T), uracil (U) and/or dihydrothymine (DHT) and dihydrouracil (DHU). In three cases with DHPD deficiency, two were siblings, one with symptoms and the other without, n was 12 for T and 5.9 for U, and 5-hydroxymethyluracil was distinctly detected. These values indicate that the nature of genetic mutation relates closely to the degree of metabolite accumulation in pyrimidine disorders. In six patients with DHP deficiency, n was 8.4-12 for DHT and 7.2-11 for DHU. Many mutations are known for both genes and the assay of residual enzyme activity may be time-consuming or invasive especially for those with DHP deficiency. Thus, this noninvasive yet comprehensive urinalysis has great value for those without a family history, as the first trial, before DNA or the enzyme assay. Our findings again raise the question whether the metabolic block really causes the symptoms found in pyrimidine disorders.     

The measurement of 11 beta-hydroxy-4-pregnene-3,20-dione (21-deoxycorticosterone) by radioimmunoassay in human plasma

A specific radioimmunoassay (RIA) method is described for the determination of 21-deoxycorticosterone (21 DB) in human plasma. 21-Deoxycorticosterone-3-(O-carboxymethyl) oxime-bovine serum albumin conjugate was used to generate antisera in rabbits. Steroids which reacted significantly with the antisera were found to be progesterone, pregnenolone, corticosterone and 11-oxo progesterone. However, after extraction of plasma and column chromatography on Celite, all these steroids were separated from 21-deoxycorticosterone and consequently did not interfere with the radioimmunoassay. The intra- and interassays coefficients of variation were 8% and 11% respectively. Mean plasma 21-deoxycorticosterone level for healthy subjects was very low: 17.8 +/- 14.8 pmol/l (mean +/- SD) with no statistical difference between males and females. During the ACTH stimulation test, the 21-deoxycorticosterone levels of healthy subjects increased to 84.7 +/- 26.3 pmol/l (mean +/- SD) for males and 79.3 +/- 31.6 pmol/l (mean +/- SD) for females. Consequently high levels of plasma 21-deoxycorticosterone were found in treated patients suffering from congenital adrenal hyperplasia (CAH) with 21-hydroxylase deficiency, particularly in CAH salt-losers with high plasma renin activity (PRA), where the plasma level reached 40,545 pmol/l. Thus, 21-deoxycorticosterone may be a new marker for adrenal 21-hydroxylase deficiency.     

Adrenal and liver metabolites of 18-hydroxy-11-deoxycorticosterone in the rat. Identification of reduced compounds (18-OH-TH-DOC)

The presence of reduced metabolites of 18-hydroxy-11-deoxycorticosterone has been investigated in the adrenals of 23 day-old and adult rats and in the liver of adult rats. By thin-layer chromatography a fraction of the adrenal steroid extract migrating like tetrahydro-corticosterone has been isolated. By gas chromatography-mass spectrometry several isomers of 3,18,21-trihydroxy-pregnan-20-one (18-OH-TH-DOC) have been separated in this fraction and identified by comparison with authentic samples which have been chemically and enzymatically synthesized. The major tetrahydrogenated metabolite in the adult and prepuberal rat adrenals is 3β,18,21-trihydroxy-5α-pregnan-20-one (18-OH-TH-DOC II). The 3α,18,21-trihydroxy-5β-pregnan-20-one has been found only in the prepuberal rat adrenal. A third tetrahydrogenated isomer has been tentatively identified as 3α,18,21-trihydroxy-5α-pregnan-20-one. Quantitative measurements by mass fragmentography show that adrenal reductase activity on 18-hydroxy-11-deoxycorticosterone is higher than on corticosterone. The 18-OH-TH-DOC II has been identified in the liver of adult male rat.     

Unified gas chromatographic-mass spectrometric method for quantitating tyrosine metabolites in urine and plasma

Tyrosine and many of its catabolites play significant roles in the in the toxicity associated with acquired and congenital forms of hypertyrosinemia. We now report a specific and sensitive GC/MS method for the simultaneous determination of tyrosine metabolites maleylacetone (MA), fumarylacetone (FA), succinylacetone (SA), fumarate and acetoacetate in urine and plasma. Tyrosine metabolites and an internal standard, 2-oxohexanoic acid (OHA), in urine or plasma samples were derivatized to their methyl esters with a 12% boron trifluoride-methanol complex (12%BF3-MeOH). The reaction mixture was extracted with methylene chloride and analyzed by GC/MS, using a selected ion monitoring (SIM) mode. The detection limits were in the range of 0.08-0.4 ng and the quantitation limits were 0.2-2 ng. Most of the intraday and interday coefficients of variation for three concentrations (low, medium and high) of the analytes were below 10%. Sensitivity and selectivity are superior to existing HPLC or enzymatic methods and derivatization of samples is simpler than the traditional silylation of organic acids used for analysis by GC/MS or derivatization to oximes, followed by silylation in the case of the ketoacids, such as SA. Furthermore, the current procedure can be performed in aqueous solution, which results in a high percentage yield without appreciable analyte degradation or formation of side products. Thus far, the method has been successfully applied in the analysis of over 5000 urine and plasma samples from humans and rodents.     

Sensitive and simple gas chromatographic-mass spectrometric determination for amphetamine in microdialysate and ultrafiltrate samples

A gas chromatographic-mass spectrometric (GC-MS) method is described for the measurement of amphetamine (AMP) using negative chemical ionization (NCI) mode. Without prior extraction AMP was derivatized with 2,3,4,5,6-pentafluorobenzoyl chloride (PFBC) and simultaneously extracted into toluene. The toluene extract was injected directly into GC-MS equipped with a HP-1 capillary column. The method is simple and more sensitive than most of the previously published methods. The limit of quantification of amphetamine is 25pg (1.4pg on column) with a very limited sample volume (25microl). The within-day precision was from 1.7 to 5.1% and between-day precision was from 2.2 to 7.3%. The method has been used for the measurement of several thousand microdialysate and ultrafiltrate samples and proven reliable.     

Gas chromatographic-mass spectrometric analysis of lipoxygenase products in post-ischemic rabbit myocardium

Leukotriene B4 (LTB4) is a potent chemotactic compound for neutrophils and is thought to be an important mediator of myocardial ischemia-reflow injury. We have measured LTB4 in rabbit cardiac tissue following ischemia-reflow using a sensitive and specific gas chromatographic-mass spectrometric (GC-MS) assay. The concentration of LTB4 in rabbit myocardium following 45 min ischemia and 3 h reflow was 48.7 +/- 12.5 pg/g, significantly higher than in non-ischemic tissue from the same animal (17.5 +/- 3.9 pg/g). These concentrations were at least an order of magnitude lower than previously reported values assessed by radioimmunoassay (RIA). Compared with the GC-MS method, RIA greatly overestimated LTB4 concentrations in cardiac tissue. The capacity of post-ischemic myocardium to produce lipoxygenase products, LTB4, 5-, 12- and 15-HETEs was also assessed following incubation of myocardium ex vivo with calcium ionophore. In all animals ischemic cardiac tissue produced greater amounts of LTB4, 5-, and 12-HETEs than non-ischemic myocardium and 12-HETE was the major product. Neutrophils that have accumulated in the injured tissue may be a major source of these products. However, in contrast to cardiac tissue, isolated rabbit neutrophils stimulated with A23187 produced 5-HETE as the major product with very little 12-HETE formed. These latter findings suggest that cells other than neutrophils may contribute to the production of lipoxygenase products during myocardial ischemia-reflow injury.     

Liquid chromatographic-mass spectrometric analysis of phospholipids. Chromatography, ionization and quantification

This review deals with the LC-MS analysis of phospholipids. The advantages of including liquid chromatography in phospholipids are highlighted. Special attention is paid to the most-used ionization methods and the role of solvents in chromatography and ionization. Difficulties associated with different quantification strategies are discussed.     

Headspace solid-phase microextraction and capillary gas chromatographic-mass spectrometric determination of rivastigmine in canine plasma samples

A simple, rapid and sensitive method for determination of rivastigmine in plasma samples was developed using headspace solid-phase microextraction (HS-SPME) and gas chromatography with mass spectrometry (GC-MS). The optimum conditions for the SPME procedure were: headspace extraction on a 65-microm polydimethylsiloxane/divinylbenzene (PDMS/DVB) fiber; 0.5 ml of plasma modified with 1.0 ml of sodium hydroxide-sodium carbonate solution (0.7 M:0.5M); extraction temperature of 100 degrees C, with stirring at 2000 rpm for 30 min. The calibration curve showed linearity in the range from 0.2 to 80 ng/ml with regression coefficient corresponding to 0.9965 and coefficient of the variation of the points of the calibration curve lower than 10%. The quantification limit for rivastigmine in plasma was 0.2 ng/ml. The method was applied to determination of rivastigmine in canine plasma samples from animals after a single oral administration.     

On-column gas chromatographic-mass spectrometric assay for metabolic profiling of valproate in brain tissue and serum

A sensitive capillary gas chromatographic-mass spectrometric method for the determination of valproic acid and at least twelve of its metabolites in serum based on tert.-butyldimethylsilyl (tBDMS) derivatives is described. Low detection limits are achieved by using a direct on-column injection technique. The addition of dry pyridine during the derivatization step now leads to uniform formation of 3-keto-VPA di-tBDMS derivatives and thereby avoids the necessity of a deuterated internal standard. A novel extraction procedure for metabolic profiling of valproate in brain tissue samples is presented. Using this method, (Z)-2-en-VPA was determined in rat brain tissue for the first time.     

Gas chromatographic-mass spectrometric method to characterise the transfer of dietary odorous compounds into plasma and milk

The flavours contained in a mammalian mother's milk can exert a marked influence on her offspring's proximate suckling behaviour and later preferences. The aim of this study was to establish a reliable analytical procedure to characterise the mammary transfer of selected volatile constituents of maternal food from non-pregnant and recently parturient ewes. Six known volatile compounds, most representative of cumin aroma (α-pinene, γ-terpinene, cuminaldehyde, p-cymene, limonene and cineole), were traced in the blood and milk of ewes fed with cumin seeds, using liquid-liquid extraction combined with gas chromatography-specific ion monitoring mass spectrometry. Among the six cumin odour markers, only one, p-cymene, was transfered in quantifiable amounts into the venous plasma. The other cumin markers could only be detected as traces corresponding to amounts lower that the limit of quantification. In milk, four of the cumin markers could be detected, and two of these were quantified.     

Sensitive biomonitoring of monoterpene exposure by gas chromatographic-mass spectrometric measurement of hydroxy terpenes in urine

An isocratic reversed-phase high-performance liquid chromatographic method with ultraviolet detection at 205 nm has been validated for the determination of indinavir, ritonavir and lopinavir (ABT 378) in human plasma. The ritonavir analogue A-86093.0 was used as internal standard. Good chromatographic separation was achieved using a stainless steel column packed with 5 microm Phenomenex phenyl hexyl material operated at 40 degrees C, and a mobile phase consisting of acetonitrile-10 mM potassium phosphate buffer (50:50, v/v). The calibration curve for indinavir was linear over the range of 50 to 1000 microg/l while the ritonavir and lopinavir calibration curves were linear over the range of 100 to 15,000 microg/l. The lower limit of quantitations for indinavir, ritonavir and lopinavir were 50, 100 and 100 microg/l, respectively, using 500 microl of human plasma. The validation data showed that the assay is sensitive, specific and reproducible for determination of indinavir, ritonavir and lopinavir. This method is being used in a therapeutic drug monitoring service to quantitate these therapeutic agents in patients infected with human immunodeficiency virus.