Simultaneous determination of [2-15N]- and [5-15N]glutamine with gas chromatography-mass spectroscopy: applications to nitrogen metabolic studies

A gas chromatographic-mass spectrometric method for analysis of L-[2-15N]- and L-[5-15N]glutamine is described. The method is based on direct acylation of glutamine with trifluoroacetic anhydride and the formation of the N,N-bis-trifluoroacetyl-L-glutamine derivative. This simple and sensitive method is capable of detecting approximately 0.5 atom% excess 15N in as little as 10 microliter of plasma with a mean coefficient of variance of 11.6%. The method was applied to determine the appearance of 15N enrichment in plasma amino-N and amide-N of glutamine in a healthy adult volunteer during a constant infusion of 15NH4Cl. A plateau level of 3.7 and 2.6 atom% excess was observed in amide-N and amino-N, respectively, at 1 and 2 h after 15NH4Cl infusion was started.     

Hypercholesterolemia impairs basal nitric oxide synthase turnover rate: a study investigating the conversion of L-[guanidino-(15)N(2)]-arginine to (15)N-labeled nitrate by gas chromatography--mass spectrometry.

Endothelial function is impaired in hypercholesterolemia and atherosclerosis, which is probably due to reduced biological activity of endothelium-derived nitric oxide (NO). NO is synthesized in functionally intact endothelium by oxidation of the terminal guanidino nitrogen atom(s) of the amino acid precursor, L-arginine. We applied stable isotope dilution techniques and gas chromatographic-mass spectrometric approaches to investigate metabolism of L-[guanidino-(15)N(2)]-arginine to (15)N-labeled nitrate in hypercholesterolemic rabbits and controls. After 4 weeks on control or 1% cholesterol-enriched diet, rabbits received 267 +/- 6 micromol of L-[guanidino-(15)N(2)]-arginine/kg of body weight via gastric cannulation. (15)N-isotope content of L-arginine in plasma and in platelet lysates increased 2h later in both groups, and almost returned to baseline until 24h. (15)N-isotope content of plasma nitrite and nitrate also increased in both groups at 2h, and had almost returned to natural content 24h later. (15)N-isotope content of urinary nitrate was significantly increased in control animals in urines collected from 0 to 12, 12 to 24, and had returned to baseline in the urine sample collected from 24 to 48 h. In the cholesterol group only a slight, insignificant elevation of (15)N-isotope content was observed for urinary nitrate. The extent of conversion of L-[guanidino-(15)N(2)]-arginine to (15)N-labeled nitrate was strongly and inversely correlated to plasma concentration of the endogenous NO synthase inhibitor, asymmetric dimethylarginine (ADMA), which was elevated in cholesterol-fed rabbits (R=0.77; p < 0.05). Our data show that baseline NO synthase turnover rate is reduced in rabbits during early hypercholesterolemia. Our study gives evidence that the mechanism of the impaired conversion of L-[guanidino-(15)N(2)]-arginine to (15)N-labeled nitrate most likely involves inhibition of NO synthase by ADMA, which is present in elevated concentrations in hypercholesterolemia.     

Tracer priming in human protein turnover studies with [15N]glycine

Sixty-three studies in healthy normal volunteers (n = 29), malnourished cancer (n = 8) or non-cancer patients (n = 9), and postoperative radical cystectomy patients (n = 17) were conducted to evaluate the primed constant infusion labeling technique for the estimation of whole-body protein turnover under a variety of dietary conditions. [15N]Glycine was used as the tracer with a prime to infusion ratio of 1300 to 3300 min and a continuous-infusion rate of 0.11 to 0.33 micrograms 15N . kg-1 . min-1 for 24 to 36 hr. The isotopic steady-state enrichment was reached in all subjects both in urinary urea and ammonia between 10 and 26 hr (mean 18 +/- 2). During protein calorie fasting the attainment of isotopic steady state is much quicker (10 to 18 hr) with a primed constant infusion than with a constant infusion alone (approximately 38 hr). A P/I ratio greater or less than 1800 (min) usually resulted in a delay of plateau attainment without affecting the protein turnover values. Reliable estimates of protein kinetics in humans can be made in clinical conditions with a 26-hr infusion of glycine at the rate of 0.28 microgram 15N . kg-1 . min-1 with a P/I ratio of 1800 min, collecting six urine samples every 2 hr from 16 hr and analyzing for both urinary urea and ammonia enrichments.     

Rapid and sensitive determination of nalmefene in human plasma by gas chromatography-mass spectrometry

A rapid gas chromatography-mass spectrometric method for the determination of nalmefene in human plasma is described. The procedure involves protein precipitation, extraction with ethanol-chloroform mixture and derivatization with pentafluropropionic anhydride. The deuterated analog of nalmefene, 6beta-naltrexol-d(7), was used as the internal standard. Quantitation was achieved on a HP-1 column (12 mx0.2 mm I.D.) with negative chemical ionization (NCI) using methane:ammonia (95:5) as the reagent gas. The standard curves were fitted using a quadratic equation with the curve encompassing a range of 0.5 to 200 ng/ml, and the intra- and inter-assay variations for three different nalmefene levels were less than 10% throughout. The limit of quantitation was found to be 0.5 ng/ml. The method described is highly specific and reproducible, and could also be applied for the determination of naltrexone and 6beta-naltrexol. Application of the method to actual human plasma samples is demonstrated.     

Rapid and sensitive determination of sertraline in human plasma using gas chromatography-mass spectrometry

A method for the determination of sertraline in human plasma using gas chromatography-mass spectrometry (GC-MS), with the selected ion-monitoring (SIM) mode, was described. The following was used in this study: (1) single liquid-liquid extraction at alkaline pH after deproteinization of plasma protein and (2) perfluoroacylation with HFBA, which has higher sensitivity (about 10-fold) compared with previous reported derivatization. The detection limit for the SIM of sertraline as an N-HFB derivative was 0.1 ng/ml, and its recovery was 80-85%. The linear response was obtained in the range of 0.2-10.0 ng/ml with a correlation coefficient of 0.999. The coefficient of variation (C.V.%) was less than 12.1% in the 1-30 ng/ml, and less than 18.2% at 0.2 ng/ml, and the accuracy was less than 10% at all of the concentration range. These findings indicate that this assay method has adequate precision and accuracy to determine the amount of sertraline in human plasma. After pharmacokinetics was performed with this assay method following oral administration of sertraline hydrochloride in man, moment analysis revealed that pharmacokinetic parameters for sertraline (Cmax, 10.3 ng/ml; Tmax, 8.0 h; T(1/2) 28.6 h) were similar to previously reported results. These results indicate that this simple and sensitive assay method is readily applicable to the pharmacokinetic studies of sertraline.     

Analysis of plasma hippurate in humans using gas chromatography-mass spectrometry: concentration and incorporation of infused [15N]glycine

To allow in vivo determination of synthetic rates for individual proteins, physiological incorporation of infused [15N]glycine into urinary hippuric acid has been used as an indicator of intrahepatic tracer dilution. Although the kidneys might contribute to hippurate production, the relationship between hepatic, plasma, and urinary hippurate has not yet been established in humans. To further investigate these issues we developed a fast, sensitive, and reliable method for measuring simultaneously hippurate concentrations and in vivo tracer incorporation into hippurate in plasma and urine using stable isotopes and gas chromatography-mass spectrometry. We then tested this assay under several experimental conditions. Reference compounds [( 15N]- and [ring-2H5]hippurate) were synthesized and gave linear standard curves. Postabsorptive hippurate plasma levels in healthy subjects ranged from 1.2 to 10.5 microM and protein binding was 79 +/- 6% (mean +/- SD). Following a bolus dose of [15N]glycine tracer appeared in plasma hippurate; enrichment in hippurate was indistinguishable from that in glycine after an equilibration period of 20 min, indicating a close relationship between intracellular glycine and plasma hippurate. A 16-h infusion of [15N]glycine resulted in identical enrichment levels in urinary and plasma hippurate; glycine enrichment in a hepatic export protein (VLDL-ApoB) was approaching plasma hippurate but not plasma free glycine enrichment. The ability to monitor plasma hippurate is of practical advantage compared to the sampling of urine. Furthermore it allows the monitoring of rapid events in the intrahepatic dilution of an infused glycine tracer. This assay may, therefore, become an important tool in the study of hepatic protein metabolism.     

[15N]aspartate metabolism in cultured astrocytes. Studies with gas chromatography-mass spectrometry.

The metabolism of 2.5 mM-[15N]aspartate in cultured astrocytes was studied with gas chromatography-mass spectrometry. Three primary metabolic pathways of aspartate nitrogen disposition were identified: transamination with 2-oxoglutarate to form [15N]glutamate, the nitrogen of which subsequently was transferred to glutamine, alanine, serine and ornithine; condensation with IMP in the first step of the purine nucleotide cycle, the aspartate nitrogen appearing as [6-amino-15N]adenine nucleotides; condensation with citrulline to form argininosuccinate, which is cleaved to yield [15N]arginine. Of these three pathways, the formation of arginine was quantitatively the most important, and net nitrogen flux to arginine was greater than flux to other amino acids, including glutamine. Notwithstanding the large amount of [15N]arginine produced, essentially no [15N]urea was measured. Addition of NaH13CO3 to the astrocyte culture medium was associated with the formation of [13C]citrulline, thus confirming that these cells are capable of citrulline synthesis de novo. When astrocytes were incubated with a lower (0.05 mM) concentration of [15N]aspartate, most 15N was recovered in alanine, glutamine and arginine. Formation of [6-amino-15N]adenine nucleotides was diminished markedly compared with results obtained in the presence of 2.5 mM-[15N]aspartate.     

The roles of insulin and glucagon in the regulation of amino acid turnover rate and pool size: in vivo study with [15N]glycine and gas chromatography--mass spectrometry

Gas chromatography--mass spectrometry analysis of plasma amino acid derivatives has been used to determine the 15N enrichment time decay curves of plasma glycine following a single dose administration of [15N]glycine in untreated and insulins-, glucagon-, and cycloheximide-treated rabbits. The present study indicated the following: (a) Increases of 80 and 50% in plasma glycine disappearance rate constants occurred in insulin- and glucagon-treated rabbits as compared with control postabsorptive rabbits; (b) The hormones in the intact rabbits caused a significant depletion in glycine pool size, which led to a moderate reduction in the fluxes of glycine. (c) A significant reduction in glycine turnover rate constants and pool size was noted at 3 and 24 hr following the administration of a sublethal dose of cycloheximide and a restoration towards control postabsorptive values was observed 48 hr after cycloheximide administration. (d) Sublethal doses of cycloheximide inhibited by 60 and 90% the stimulatory action of insulin and glucagon on plasma glycine disappearance, respectively. The present data suggest that both insulin and glucagon may act directly on plasma glycine disappearance rates. The stimulatory action of insulin differs from the action of glucagon in that it is not completely blocked by cycloheximide. Presumably glucagon and insulin modify the glycine transport system at different sites or by a different mechanism.     

Gas chromatography-mass spectrometry determination of [15N]ammonia enrichment in blood and urine

A rapid gas chromatography-mass spectrometry method for [¹⁵N]ammonia analysis is deseribed which is based on the formation of [¹⁵N]glutamic acid from ammonia and analysis of isotopic abundance in the N-trifluoroacetyl-n-butylester glutamate derivative. Mean recovery of [¹⁵N]ammonia added to either plasma or urine was greater than 99% with a relative standard deviation of less than 10%. The method can be applied to the determination of extremely low levels of ammonia through an isotope dilution technique. The [¹⁵N]ammonia abundance of blood and urine was determined in an adult following on oral dose (500 mg) of ¹⁵NH₄Cl. A peak isotopic abundance of 13 atoms% excess was reached by 30 min. Urinary excretion of [¹⁵N]ammonia during the first 4 h after administration of the isotope amounted to 4.1% of the isotope administered.     

Rapid determination of acetone in human plasma by gas chromatography-mass spectrometry and solid-phase microextraction with on-fiber derivatization

Acetone is an important volatile disease marker. Due to its nature of activity and volatility, it is a difficult task to measure the concentration of acetone in biological samples with accuracy. In this paper, we developed a novel method for determination of trace amount acetone in human plasma by solid-phase microextraction technique with on-fiber derivatization. In this method, the poly(dimethylsiloxane)/divinylbenzene (PDMS/DVB) fiber was used and O-2,3,4,5,6-(pentafluorobenzyl) hydroxylamine hydrochloride (PFBHA) was first loaded on the fiber. Acetone in plasma sample was agitated into headspace and extracted by solid-phase microextraction (SPME) fiber and subsequently derivatized with PFBHA on the fiber. Acetone oxime was analyzed by gas chromatography-mass spectrometry (GC-MS). Quantitative analysis of acetone in plasma was carried out by using external standard method. The SPME conditions (extraction temperature and time) and the method validation were studied. The present method was tested by determination of acetone in diabetes plasma and normal plasma. Acetone concentration in diabetes plasma was found to be higher than 1.8mM, while in normal plasma was lower than 0.017 mM. The results show that the present method is a potential tool for diagnosis of diabetes.     

Rapid micromethod for determination of 15N enrichment in plasma lysine: application to measurement of whole body protein turnover.

A simple, sensitive method, requiring less than 1 ml of blood, is deseribed for measurement of stable isotope enrichment in plasma lysine with a relative precision of better than ±0.5% of the observed value. A measurement of whole body protein turnover obtained by this method was virtually identical to values obtained by more tedious macromethods. The speed of the reported procedure allows its application to routine clinical studies which generate a large number of samples.     

Simultaneous determination of dehydroepiandrosterone and its 7-oxygenated metabolites in human serum by high-resolution gas chromatography--mass spectrometry

A highly sensitive and specific method has been developed for the simultaneous measurement of free (unconjugated) or sulfate-conjugated forms of dehydroepiandrosterone (DHEA), 7alpha-hydroxy-DHEA (7alpha-OH-DHEA), 7beta-hydroxy-DHEA (7beta-OH-DHEA), and 7-oxo-DHEA (7-oxo-DHEA) in human serum. This method is based upon a stable isotope-dilution technique by gas chromatography-selected-ion monitoring mass spectrometry. Free steroids were extracted from serum with an organic solvent and the sulfate-conjugated steroids remained in aqueous phase. Free steroids were purified by solid-phase extraction, while sulfate-conjugated steroids were hydrolyzed by sulfatase and deconjugated steroids were purified by solid-phase extractions. The extracts were treated with O-methylhydroxylamine hydrochloride and were subsequently dimethylisopropylsilylated. The resulting methyloxime-dimethylisopropylsilyl (MO-DMIPS) ether derivatives were quantified by gas chromatography-selected-ion monitoring mass spectrometry in a high-resolution mode. The detection limits of MO-DMIPS ether derivatives of DHEA, 7alpha-OH-DHEA, 7beta-OH-DHEA and 7-oxo-DHEA were 1.0, 0.5, 0.5 and 2.0pg, respectively. Coefficients of variation between samples ranged from 10.6 to 22.9% for free 7-oxygenated DHEA to less than 10% for DHEA and sulfate-conjugated 7-oxygenated DHEA. The concentrations of these steroids were measured in 18 sera samples from healthy volunteers (9 males and 9 females; aged 23-78 years). Free DHEA, 7alpha-OH-DHEA, 7beta-OH-DHEA and 7-oxo-DHEA levels ranged between 0.21-3.55, 0.001-0.194, 0.003-0.481, and 0.000-0.077ng/ml, respectively, and the sulfate-conjugated steroid levels of these metabolites ranged between 253-4681, 0.082-3.001, 0.008-0.903, and 0.107-0.803ng/ml, respectively. The free DHEA-related steroid concentrations were much lower than those previously measured by RIA and low-resolution GC-MS. The present method made it possible to determine simultaneously serum DHEA-related steroid levels with sufficient sensitivity and accuracy.     

Microbial production of L-[15N]glutamic acid and its gas chromatography-mass spectrometry analysis

L-[15N]Glutamic acid was prepared in high yields via a fermentative process. Brevibacterium lactofermentum, growing on a medium containing 97% enriched 15NH4Cl as a sole isotopic precursor, excreted mostly L-[15N]glutamic acid. The L-[15N]glutamic acid was purified and identified. Gas chromatography-mass spectrometry analysis was performed to demonstrate its usefulness in clinical studies.     

Quantitative determination of nitroglycerol in human plasma by gas chromatography negative ion mass spectrometry using 15N labelled nitroglycerol as internal standard

Negative ionization resulted in the simplification of a previously published method. The new method permits the determination of 0.25 nmol l-1 nitroglycerol in plasma with a coefficient of variation of 9.1%.     

Microbial preparation of L-[15N]tyrosine and [15N]tyramine and their gas chromatographic-mass spectrometric analyses

The preparation of L-[15N]tyrosine and [15N]tyramine by microbial synthesis is described. Immobilized Erwinia herbicola cells were added to a reaction mixture containing phenol, pyruvic acid, and 15NH4Cl. The reaction was driven by excess nonlabeled pyruvate and phenol. Under these denaturing concentrations of phenol, immobilized cells were more effective than free ones. Gram quantities of L-[15N]tyrosine were obtained without label dilution. The conversion of this L-[15N]tyrosine into [15N]tyramine by Streptococcus faecalis was performed at maximal efficiency. Gas chromatographic-mass spectrometric studies and 1H and 15N NMR analyses of the labeled compounds are reported.     

Validation of the determination of oxymetholone in human plasma analysis using gas chromatography-mass spectrometry. Application to pharmacokinetic studies

A simple and rapid procedure for extraction of oxymetholone in human plasma using gas chromatography coupled with quadrupole mass spectrometric was evaluated. The method involves analyte extraction with tert.-butylmethylether after alkalinization of the plasma and derivatization with MSTFA-NH(4)I-2-mercaptoethanol before the high resolution gas chromatographic-mass spectrometry separation. Methyltestosterone was used as internal standard. The calibration curves were linear, with typical r(2) values >0.995 and F(table)>F(calculated) (alpha=0.05). Recovery from plasma proved to be more than 70%. The method was accurate and reproducible, and was successfully applied to determine the pharmacokinetic parameters of oxymetholone for healthy volunteers after oral administration of 50 mg of the compound. The (C(max)) and (T(max)) were 18.8 +/- 0.4 ng/ml and 210 +/- 42.4 min, respectively.     

Rapid and sensitive determination of vinorelbine in human plasma by liquid chromatography-tandem mass spectrometry and its pharmacokinetic application

Vinorelbine is a semi-synthetic vinca alkaloid with demonstrated high activities against various types of advanced cancer. To support a clinical pharmacokinetic study, a simple, rapid and sensitive method to determine vinorelbine in human plasma was developed using reversed phase liquid chromatography (LC) coupled with electrospray ionization mass spectrometry/mass spectrometry (ESI-MS/MS). Vinorelbine and vinblastine (the internal standard) were extracted from human plasma by one-step liquid-liquid extraction (LLE) with methyl-t-butyl ether. The chromatographic separation was achieved on a Spursil polar-modified C(18) column (50 mm×2.1 mm, 3 μm, Dikma Technologies) with an isocratic mobile phase of a 75:25 (v/v) acetonitrile-4 mmol/L ammonium formate (pH 3.0) mixture at a flow-rate of 0.4 mL/min. The MS/MS detection was performed in the positive ion multiple reaction monitoring (MRM) mode by monitoring the precursor→product ion transitions at m/z 779.4→122.0 and m/z 811.3→224.2 for vinorelbine and the internal standard, respectively. The assay was validated in the range 0.1-200 ng/mL (r>0.997), the lowest level of this range being the lower limit of quantification (LLOQ) based on 50 μL of plasma. The intra- and inter-day precisions were within 6.0%, while the accuracy was within ±4.7% of nominal values. Detection and quantification of both analytes within 2 min make this method suitable for high-throughput analyses. The method was successfully applied to evaluate the systemic pharmacokinetics of vinorelbine after a 20-min intravenous infusion of 25 mg/m(2) of vinorelbine to patients with metastatic breast cancer.