Gas chromatographic-mass spectrometric analysis of erythrocyte 3-deoxyglucosone in hemodialysis patients.

The erythrocyte levels of 3-deoxyglucosone (3-DG) were measured by a selected ion monitoring method of gas chromatography-chemical ionization mass spectrometry using [13C6]-3-DG as an internal standard. Because the erythrocyte levels of 3-DG measured after deproteinization using ethanol were 18 times higher than those using ultrafiltration, we used ethanol deproteinization for measurement of total 3-DG in the erythrocytes. The concentration of 3-DG was significantly elevated in hemodialysis (HD) patients compared with healthy subjects. Although HD treatment could remove the erythrocyte 3-DG efficiently, its post-HD levels were still elevated compared with the healthy subjects.     

Gas chromatographic-mass spectrometric analysis of urinary sugar and sugar alcohols during pregnancy.

A refined and simplified method has been developed for the simultaneous analysis of urinary sugar and sugar alcohols after urease treatment by using capillary gas chromatography-mass spectrometry (GC-MS). Since carbohydrate metabolism during pregnancy is considered to be diabetogenic, our interest has been concentrated on understanding the mechanism of the metabolic deviation by assessing the glucose excursion and glucose fluxes. The present study suggests that changes of the levels of glucose, sorbitol, fructose, myo-inositol, and 1,5-anhydro-D-glucitol (1,5-AG) may reflect a mild alteration in carbohydrate metabolism that goes undetected by conventional diabetic indicators.     

Gas chromatographic-mass spectrometric determination of plasma saturated fatty acids using pentafluorophenyldimethylsilyl derivatization

An improved method for the detection of 11 saturated fatty acids (SFAs) including C12:0-C26:0 (even numbers only), C17:0, C19:0 and C23:0 in human plasma by gas chromatography-mass spectrometry (GC-MS) with a stable isotope internal standard as d3-stearic acid is described. This procedure was based on acidic treatment, liquid-liquid extraction, and chemical derivatization prior to instrumental analysis. Eleven pentafluorophenyldimethylsilyl-SFA derivatives were well separated without any interfering peaks in plasma samples. The characteristic ions at M-15, constituting the base peaks in the electron impact mass spectra for 11 SFAs, permitted their sensitive detection by GC-MS in the selected ion monitoring (SIM) mode. The SIM responses were linear with correlation coefficients varying from 0.993 to 0.999 in the concentration range of 0.05 to approximately 50 microg/ml for the 11 SFAs. The detection limits for SIM of the SFAs varied in the range of 0.05 to approximately 10.0 pg. When applied to the plasma samples of normal subjects and patients with X-linked adenoleukodystrophy, which is one of the hereditary peroxisomal disorders, the present method enabled us to determine the SFAs with good sensitivity and good overall precision and accuracy within the concentration ranges of 0.14 to approximately 82.35 micromol/l.     

Gas chromatographic-mass spectrometric analysis of stable isotopes of cysteine and glutathione in biological samples.

A gas chromatographic-mass spectrometric (GC-MS) procedure for the determination of stable isotope labelled glutathione has been applied to animal and human samples. The method, based on preparation of the N,S-ethoxycarbonyl methyl ester derivative of the intact peptide, is rapid and requires little or minor tissue treatment. The same method was applied to cysteine. The method was found to be reliable in terms of within-day and between-day precision, accuracy and linearity. The procedure was applied in humans and animals to determine in vivo the glutathione fractional synthesis rate using labelled cysteine infusion. The glutathione fractional synthesis rate was found to be 22.5%/day in blood from a healthy volunteer and 337+/-29%/day in rat liver.     

Gas chromatographic-mass spectrometric analysis of dichlorobenzene isomers in human blood with headspace solid-phase microextraction.

Headspace solid-phase microextraction (HS-SPME) was utilized for the determination of three dichlorobenzene isomers (DCBs) in human blood. In the headspace at 30 degrees C, DCBs were absorbed for 15 min by a 100-micron polydimethylsiloxane (PDMS) fiber. They were then analyzed by capillary column gas chromatography-mass spectrometry (GC-MS). By setting the initial column oven temperature at 20 degrees C, the three isomers were resolved at the baseline level. p-Xylene-d10 was used as the internal standard (I.S.). For quantitation, the molecular ion at m/z 146 for each isomer and the molecular ion at m/z 116 for I.S. were selected. For day-to-day precision, relative standard deviations in the range 3.2-10.7% were found at blood concentrations of 1.0 and 10 micrograms/ml. Each compound was detectable at a level of at least 0.02 microgram per 1 g of whole blood (by full mass scanning). HS-SPME-GC-MS, when performed at relatively low temperatures, was found to be feasible in toxicological laboratories. Using this method, the plasma levels of one patient who had drunk a pesticide-like material were measured.     

Metabolism of anabolic steroids by recombinant human cytochrome P450 enzymes. Gas chromatographic-mass spectrometric determination of metabolites

Metabolism of steroid hormones with anabolic properties was studied in vitro using human recombinant CYP3A4, CYP2C9 and 2B6 enzymes. The enzyme formats used for CYP3A4 and CYP2C9 were insect cell microsomes expressing human CYP enzymes and purified recombinant human CYP enzymes in a reconstituted system. CYP3A4 enzyme formats incubated with anabolic steroids, testosterone, 17alpha-methyltestosterone, metandienone, boldenone and 4-chloro-1,2-dehydro-17alpha-methyltestosterone, produced 6beta-hydroxyl metabolites identified as trimethylsilyl (TMS)-ethers by a gas chromatography-mass spectrometry (GC-MS) method. When the same formats of CYP2C9 were incubated with the anabolic steroids, no 6beta-hydroxyl metabolites were formed. Human lymphoblast cell microsomes expressing human CYP2B6 incubated with the steroids investigated produced traces of 6beta-hydroxyl metabolites with testosterone and 17alpha-methyltestosterone only. We suggest that the electronic effects of the 3-keto-4-ene structural moiety contribute to the selectivity within the active site of CYP3A4 enzyme resulting in selective 6beta-hydroxylation.     

Gas chromatographic-mass spectrometric method for quantitation of phenylalanine and tyrosine in serum.

A validated gas chromatographic-mass spectrometric method for quantitation of phenylalanine and tyrosine in serum is described. Quantitation of phenylalanine and tyrosine with a non-labelled non-endogenous internal standard, L-2-chlorophenylalanine, compared favourably with isotope dilution mass spectrometric quantitation. The 95% reference ranges for phenylalanine. tyrosine and the phenylalanine-tyrosine molar ratio in neonate cord blood serum were determined by isotope dilution mass spectrometry and were found to be 77.1-144.7, 33.3-109.3 micromol/l and 1.1-3.0, respectively.     

Gas chromatographic-mass spectrometric analysis of veterinary tranquillizers in urine: evaluation of method performance

A method for analysis of veterinary tranquillizers in urine using gas chromatography-mass spectrometry (GC-MS) is described. Detection limits are 5 microg/l for ketamine, azaperone and the phenothiazines (chlor-, aceto- and propionylpromazine), 10 microg/l for haloperidol, 20 microg/l for xylazine and 50 microg/l for azaperol, recoveries for all analytes were higher than 70%. Method performance in terms of within-batch, between-days and between-analysts reproducibility was studied and found to be acceptable. Compliance with European Union criteria for confirmation of GC-MS "positive" results is evaluated and discussed.     

Gas chromatographic-mass spectrometric determination of total homocysteine in human plasma by stable isotope dilution: method and clinical applications.

The detection and quantitation of slight increases of plasma homocysteine levels is of growing interest. This has prompted us to develop a highly sensitive and accurate capillary gas chromatography-mass spectrometry (GC-MS) method. The method proved to be highly sensitive (DL=0.17 micromol/l) with between- and within-run precision less than 6% and 7%, respectively. Reference values of plasma total homocysteine have been determined for men (n=39) and women (n=36), showing a significant difference (P=0.003) between gender. Preliminary results in cerebrovascular accidents and in venous thrombosis are presented.     

Gas chromatographic-mass spectrometric method for quantitative determination of ketotifen in human plasma after enzyme hydrolysis of conjugated ketotifen.

A validated method for determination of total amount of ketotifen (unchanged and conjugated) in human plasma has been presented. An enzyme hydrolysis of conjugated ketotifen was conducted with combination of beta-glucuronidase and arylsulfatase. After the enzyme hydrolysis a solid-phase extraction was applied as a cleaning step. The quantitative determination by gas chromatography with mass-spectrometry detection (GC-MS) was performed. Pizotifen has been used as an internal standard. A reliable hydrolysis as well as a satisfactory accuracy, improved precision in the linear region from 0.500 to 10.0 ng/ml plasma, limit of detection of 0.010 ng/ml and prolonged capillary column life have been achieved.     

Gas chromatographic-mass spectrometric confirmation of atractyloside in a patient poisoned with Callilepis laureola.

The South African traditional remedy Impila (Callilepis laureola) contains the mitochondrial toxin atractyloside. The plant is sold widely and continues to lead to fatalities in patients. We describe, for the first time, a simple GC-MS procedure for the identification of atractyloside, which we have applied to the gastric washing from a poisoned patient and to extracts of Impila tuber.     

Gas chromatographic-mass spectrometric assay for rocuronium with potential for quantifying its metabolite, 17-desacetylrocuronium, in human plasma.

A rapid, sensitive and selective method has been developed for the quantification of plasma concentrations of neuromuscular blocking drug, rocuronium, using gas chromatography with mass spectrometric detection. 3-Desacetylvecuronium served as the internal standard. The method involved iodide ion pair formation and a single-step liquid-liquid extraction with dicholoromethane. This method also permits simultaneous determination of its putative metabolite, 17-desacetylrocuronium, although the high detection limit for the metabolite limits the practical application of this method in pharmacokinetic study of the metabolite. The extraction efficiency was approximately 75% for rocuronium and approximately 50% for 17-desacetylrocuronium. The limit of quantification was 26 ng/ml for rocuronium and 870 ng/ml for its metabolite. The assay was used successfully in a patient undergoing liver transplantation and receiving rocuronium as a constant rate infusion and in a patient undergoing general elective surgery receiving the drug as an intravenous bolus. This assay is a time-saving alternative to published gas or liquid chromatographic methods for assaying rocuronium.     

Gas chromatographic-mass spectrometric determination of 4-nonylphenols and 4-tert-octylphenol in biological samples

A simple and rapid method is described for the GC-MS determination of 4-nonylphenols (NOs) and 4-tert-octylphenol (OC) in biological samples. The NOs and OC in the sample are extracted with acetonitrile and the lipid in the sample extract is eliminated by partitioning between hexane and acetonitrile. After Florisil PR column clean-up, the sample extract is analyzed by GC-MS in the selected ion monitoring (SIM) mode. Average recoveries in pale chub (fish) and corbicula (shellfish) are 86.0 and 93.4% for NOs, and 95.8 and 96.4% for OC, respectively, spiked at the levels of 1.0 microg of NOs and 0.1 microg of OC per 5 g of fish and shellfish samples. The detection limits are 20 ng/g for NOs and 2 ng/g for OC.     

Modified gas chromatographic-mass spectrometric assay for the determination of gacyclidine enantiomers in human plasma.

A modified method for the determination of gacyclidine enantiomers in human plasma by GC-MS with selected-ion monitoring using the deuterated derivative of gacyclidine (d3-gacyclidine) as internal standard was developed. Following a single-step liquid-liquid extraction with hexane, drug enantiomers were separated on a chiral fused-silica capillary column (CP-Chirasil-Dex; Chrompack). The fragment ion, m/z 266, was selected for monitoring d3-gacyclidine (retention times of 35.2 and 35.6 min for the (+)- and (-)-enantiomer, respectively) whereas the fragment ion, m/z 263, was selected for quantitation of gacyclidine (retention times of 35.4 and 35.9 min for the (+)- and (-)-enantiomer, respectively). The limit of quantitation for each enantiomer was 0.3 ng/ml, using 1 ml of sample, with a relative standard deviation (RSD) < 14% and a signal-to-noise ratio of 5. The extraction recovery of both gacyclidine enantiomers from human plasma was about 75%. The calibration curves were linear (r2 > 0.996) over the working range of 0.312 to 20 ng/ml. Within- and between-day RSD were < 9% at 5, 10 and 20 ng/ml, and < 16% at 0.312, 0.625, 1.25 and 2.5 ng/ml. Intraday and interday bias were less than 11% for both enantiomers. The chromatographic behavior of d3-gacyclidine remained satisfactory even after more than 500 injections. Applicability of this specific and stereoselective assay is demonstrated for a clinical pharmacokinetic study with racemic gacyclidine.     

Gas chromatographic-mass spectrometric analysis of hydroxylamine for monitoring the metabolic hydrolysis of metalloprotease inhibitors in rat and human liver microsomes

A gas chromatographic-mass spectrometric (GC-MS) method was developed for the analysis of hydroxylamine (HA) in supernatants obtained from liver microsomes. HA monitoring was used to determine the metabolic hydrolysis of two hydroxamic acid-based matrix metalloprotease inhibitors in rat and human liver microsomes. The hydrolysis of the hydroxamic acids to their corresponding carboxylic acids releases HA as a common metabolic product. HA was derivatized to acetone oxime by addition of acetone to the liver microsomal supernatant, followed by direct injection of the supernatant into the GC-MS, with detection of the oxime by selected-ion-monitoring. The method is simple, reproducible, and sensitive for the determination of the hydrolysis of hydroxamic acid compounds, where hydrolysis is the major metabolic pathway. The methodology can be used for rank ordering and selecting hydroxamic acid analogs based on their susceptibility to hydrolysis.     

Fast liquid chromatographic-mass spectrometric determination of pharmaceutical compounds.

We present fast LC-MS-MS analyses of multicomponent mixtures containing flavones, sulfonamides, benzodiazepines and tricyclic amines. Using a short microbore HPLC column with small particle size, five to eight compounds were partially resolved within 15 to 30 s. TurboIonSpray and atmospheric pressure chemical ionization interfaces were well suited to tolerate the higher eluent flow-rates of 1.2 to 2 ml/min. The methods were applied to biological sample matrices after clean-up using solid-phase or liquid-liquid extraction. Good precision and accuracy (average 8.9 and 97.7%, respectively) were achieved for the determination of tricyclic amines in human plasma. Benzodiazepines were determined in human urine with average precision of 9% and average accuracy of 95% for intra- and inter-assay. Detection limits in the low ng/ml range were obtained. An example for 240 injections per hour of demonstrated the feasibility of rapid LC-MS-MS analysis.     

Liquid chromatographic-mass spectrometric determination of itraconazole and its major metabolite, hydroxyitraconazole, in dog plasma

A fast, reliable and sensitive liquid chromatography-mass spectrometry (LC-MS) assay for the determination of itraconazole and hydroxyitraconazole in dog plasma has been developed. The analysis involves a simple liquid-liquid extraction followed by LC-MS analysis using electrospray ionization in the positive mode. Total separation of itraconazole, hydroxyitraconazole and the internal standard, miconazole, was achieved on a C18 column in 3.5 min using an isocratic mixture of acetonitrile and 10 mM ammonium acetate. The response was linear over four-orders of magnitude, allowing reliable quantification of each species. This paper describes the development of the method and its validation.     

Gas chromatographic-mass spectrometric determination of alpha-ketoisocaproic acid and [2H7]alpha-ketoisocaproic acid in plasma after derivatization with N-phenyl-1,2-phenylenediamine

A method for determination of alpha-ketoisocaproic acid (KIC) and [4,5,5,5,6,6,6-2H7]alpha-ketoisocaproic acid ([2H7]KIC) in rat plasma was developed using gas chromatography-mass spectrometry-selected ion monitoring (GC-MS-SIM). [5,5,5-2H3]alpha-Ketoisocaproic acid ([2H3]KIC) was used as an analytical internal standard to account for losses associated with the extraction, derivatization and chromatography. The keto acids were extracted by cation-exchange chromatography using BondElut SCX cartridge and derivatized with N-phenyl-1,2-phenylenediamine to form N-phenylquinoxalinone derivatives. Quantitation was performed by SIM of the respective molecular ions at m/z 278, 281 and 285 for the derivatives of KIC, [2H3]KIC and [2H7]KIC on the electron impact method. The limit of detection was found to be 70 fmol per injection (S/N=3) and the limit of quantitation for [2H7]KIC was around 50 nM in rat plasma. Endogenous KIC concentrations in 50 microl of rat plasma were measured with relative intra- and inter-day precision of 4.0% and 3.3%, respectively. The intra- and inter-day precision for [2H7]KIC spiked to rat plasma in the range of 0.1 to 10 microM gave good reproducibility with relative standard deviation (RSD) of 6.5% and 5.4%, respectively. The intra- and inter-day relative errors (RE) for [2H7]KIC were less than 6.4% and 3.8%, respectively. The method was applied to determine the plasma concentration of [2H7]KIC after an intravenous administration of [2H7]KIC in rat.     

Automated sample preparation and gas chromatographic-mass spectrometric analysis of urinary androgenic anabolic steroids

This paper presents an automated method for extracting anabolic agents from urine samples for their GC-MS analysis by selected-ion monitoring. The sample preparation was carried out in a Hewlett-Packard 7686 SPE PrepStation system. Each 0.6-ml aliquot was hydrolyzed, extracted, dried and trimethylsilyl (TMS) derivatized in a 2-ml vial without any hands-on labor. When sample preparation was finished 2 microl of the extract was injected into the gas chromatograph by split (1:10) mode. Due to the small amount of free space in the 2-ml vials for handling the sample, parameters like time of hydrolysis, type of shaking, number of extractions and some TMS derivatization parameters had to be adjusted to achieve the best recovery for all of the compounds in the screening. Manual and automated sample preparation schemes were compared in terms of linearity, precision, accuracy, limit of detection and recovery data. When large concentrations were analyzed using the automated method no carry-over effect was observed.