Gas chromatographic analysis of ketamine and norketamine in plasma and urine: Nitrogen-sensitive detection

A sensitive gas chromatographic method for quantitative analysis of ketamine and norketamine in human and animal biological fluids is described. The nitrogen-sensitive detection procedure used is more stable than electron-capture detection and reduced analysis time. The method used bromo-ketamine as an internal standard for quantitation and is linear from 10–25,000 ng/ml. No interferences were shown with drugs commonly associated with cardiac surgery with cardiopulmonary by-pass. This assay is sensitive, specific, using either native or derivatized drugs and can be used for routine analysis of ketamine and norketamine in plasma or urine.     

Quantitative microdetermination of DNA by two-dimensional electron capture gas chromatography of the thymine constituent

A highly sensitive and specific two-dimensional electron-capture gas chromatographic method has been developed for determining small amounts of DNA by measuring its thymine content. The method can also be used to measure RNA based on uracil content. The nucleic acids were hydrolyzed and released constituents were separated and detected as their chloromethyldimethylsilyl ethers. The minimal amount detected was 5 pg of each base. Standard curves were linear from 5 to 200 pg. This method allowed quantitative determination of 2 ng of DNA (routinely detectable quantity) after hydrolysis of biological material in formic acid, even in the presence of large amounts of RNA and/or protein. For example, this method has been shown to be successful in determination of the DNA contents of manually isolated nucleic such as from amphibian oocytes. Besides being accurate, the procedure was rapid: after maximal hydrolysis (usually about 45 min) the derivatization and gas chromatographic analysis was completed in another 15 min. The procedure described represents a direct biochemical alternative to cytophotometric estimation of nuclear contents and has the advantage of providing values for absolute DNA content per nucleus.     

Purification of rat liver asparagine synthetase by affinity chromatography on reactive blue 2-agarose

Studies on analysis of free animo acids using a support-coated, open-tube capillary column, and electron-capture detection or selective ion monitoring have been performed on samples from biological microenvironments. For most amino acids the detection limit was found to be less than 1 pg. The preparation of the support-coated open-tube capillary column is described as well as the gas-chromatographic conditions for direct injection and temperature-programmed separation of the N-heptafluorobutyryl iso butyl ester derivatives. Electron-capture detection and selected ion monitoring are compared with respect to linearity and sensitivity and the bases for the greater sensitivity of electron-capture detection compared with flame-ionization detection using halogenated derivatives is discussed. Applications of the gas-chromatographic method for analysis of free amino acids in environments deliberately chosen very small are demonstrated.     

Analysis of malondialdehyde in biological matrices by capillary gas chromatography with electron-capture detection and mass spectrometry

A gas chromatographic method is described for the quantification of free and total malondialdehyde (MDA) in biological materials. The procedure involves derivatization of the analyte with 2,4,6-trichlorophenylhydrazine, extraction with n-hexane, and separation of the cyclic derivatization product on a OV-5 gas chromatographic column. Concentration of the derivatization reagent, pH, reaction time, and temperature were investigated to determine the optimal derivatization conditions. Under these conditions, the method allows for the selective detection of free and total MDA at femtomole levels in several biological materials without any interferences. The procedure yields relative standard deviation values for the intra- and interassays in the range 3.3 and 3.9%, respectively, for the electron-capture and mass-selective (SIM mode) detection systems. Recoveries of MDA from spiked matrices reached 96%. The present method offers the advantage of the alternative use of either electron-capture or mass-selective detection. Furthermore it avoids overestimation of MDA since it employs mild conditions for sample processing and there is no need for preventing protein separation for the assessment of free MDA.     

An electron-capture gas–liquid-chromatographic method for the determination of prostaglandin F2α in biological fluids

A sensitive electron-capture gas-liquid-chromatographic method for the determination of sub-nanogram quantities of prostaglandin F(2alpha) was developed. The method is based on the sub-microgram scale conversion of the prostaglandin into the electron-capturing pentafluorobenzyl ester, and analysis of the latter as the tris-trimethylsilyl ether. The lower limit of detection was 12.5pg of the ester injected ;on-column' as the silylated product. The method was successfully applied to the determination of prostaglandin F(2alpha) in monkey plasma. The specificity of the analytical procedure was increased by incorporating a thin-layer chromatographic fractionation before gas-liquid chromatography. The utility of the analytical methodology developed was demonstrated by its application to the determination of plasma concentrations of intact prostaglandin F(2alpha) in a Rhesus monkey, after subcutaneous administration of a single dose of prostaglandin F(2alpha). The electron-capture gas-liquid-chromatographic assay is compared with the radioimmunoassay and the gas-liquid-chromatographic-mass-spectrometry assay for the determination of prostaglandin F(2alpha).     

Determination of ritodrine in biological fluids of the pregnant sheep by fused-silica capillary gas chromatography using electron-capture detection

A sensitive and selective gas chromatographic assay method employing splitless injection, fused-silica capillary columns and electron-capture detection is reported for the quantitation of the tocolytic drug, ritodrine, in a variety of biological fluids obtained from the pregnant ewe and fetus. This method has improved sensitivity and selectivity over previously published assay procedures. A 25 m × 0.31 mm I.D., cross-linked 5% phenylmethylsilicone, fused-silica capillary column was employed for all analyses. Linearity of response was observed over the range 2.5–75 ng of ritodrine base per 0.05–0.5 ml of biological fluid, representing ≈ 1–75 pg at the detector. The coefficient of variation was less than 10% over the range 2.5–75 ng of added ritodrine. The minimum quantifiable amounts is ≈ 2.5 ng from a 0.5-ml biological fluid sample. Applicability of this method to biological fluids, obtained from ovine subjects, is demonstrated by the analysis of samples obtained during the course of ritodrine placental transfer studies.     

Quantitative gas-liquid chromatographic determination of ftorafur and 5-fluorouracil in biological specimens

A method developed for measuring the antitumor agent ftorafur and its biotransformation product 5-fluorouracil was applied to biological specimens. After extraction with ethylacetate, ftorafur, 5-fluorouracil, and the internal standard 2-methyl-4-hydroxy-6-chloromethylpyrimidine are converted to their chloromethyldimethylsilyl derivatives and assayed by glc, using either an electron-capture or a flame ionization detector. The minimum detectable amount is 100 pg/injection for ftorafur and 50 pg/injection for 5-fluorouracil employing electron-capture detection. Linearity was found up to microgram amounts of both substances, without any interference from endogenous substrates. Preliminary data are reported on the comparative serum kinetics of ftorafur and 5-fluorouracil in mice.     

Determination of phenolic flame-retardants in human plasma using solid-phase extraction and gas chromatography–electron-capture mass spectrometry

A method for determination of phenolic flame-retardants in human plasma utilizing solid-phase extraction (SPE) and gas chromatography with electron-capture mass spectrometric detection (GC–ECMS), has been developed. The plasma lipids were decomposed by application of concentrated sulphuric acid directly on the polystyrene–divinylbenzene SPE column. The method has been validated for 2,4,6-tribromophenol (TriBP), pentabromophenol (PeBP), tetrachlorobisphenol-A (TCBP-A) and tetrabromobisphenol-A (TBBP-A) in the concentration range 1.2–25, 0.4–40, 4–200 and 4–200 pg g⁻¹ plasma, respectively. The average absolute recovery of the analytes ranged from 51 to 85%. Tetrabromo-o-cresol and chlorotribromobisphenol-A were found suitable as internal standards, and the average recovery of the analytes relative to the internal standards was in the range 93–107%. The repeatability of the method was in the range 4–30% relative standard deviation. The estimated detection limits of TriBP, PeBP, TCBP-A and TBBP-A were 0.3, 0.4, 3.0 and 0.8 pg g⁻¹ plasma, respectively. The method has been used for analysis of plasma samples from potentially occupationally exposed human individuals.     

Determination of plasma, urine, and bovine serum albumin low-molecular-weight carbonyl levels by capillary gas chromatography with electron-capture and mass-selective detection

Peroxidation of lipids produces low-molecular-weight carbonyl compounds, which are reactive with biological nucleophiles. The analysis of these compounds is often difficult. A multicomponent method for the determination of 11 of them in biological samples is reported. The samples are subjected to a pretreatment-derivatization procedure followed by gas chromatographic analysis with either electron-capture detection (ECD) or mass-selective detection (MSD) in the selected-ion monitoring mode. The procedure involves derivatization of the analyte with 2,4,6-trichlorophenylhydrazine, extraction with n-hexane, and separation of the derivatization products on a nonpolar gas chromatographic column. The concentration of the derivatization reagent, pH, reaction time, temperature, and presence of extraneous ions were investigated to determine the optimal derivatization conditions. Under these conditions, the method allows for the selective detection of low-molecular-weight carbonyl compounds at femtomole levels in several biological materials such as plasma, urine, and bovine serum albumin without interferences. The limits of detection were in the ranges 0.01-0.2 microM for ECD and 0.15-1.5 microM for MSD. The mean procedural recoveries obtained during the method validation were within the range 85-95% and the intra- and interassay standard deviations do not exceed 4.6 and 6.1%, respectively.     

Determination of peptido-aminobenzophenone (2-o-chlorobenzoyl-4-chloro-N-methyl-N′-glycyl-glycinanilide) by electron-capture gas—liquid chromatography

A gas—liquid chromatographic method for the determination of peptido-aminobenzophenone (2-o-chlorobenzoyl-4-chloro-N-mehtyl-N′-glycyl-glycinanilide, I) in dog plasma was developed. Decomposition of compound I was observed during chromatography. In alkaline medium, compound I in plasma was submitted to ring closure to yield 3-amino-6-chloro-5-(2-chlorophenyl)-1-methylquinolin-2-one (aminoquinolone), and the hexane extract was assayed by gas—liquid chromatography using electron-capture detection. The concentration range of compound I studied was 10–90 ng per 0.5 ml of plasma. Interference from both endogenous and exogenous sources was negligible. The method could be applied to the determination of the plasma level of compound I in dogs following oral administration of a single 5 mg/kg dose.     

Determination of 2,5-hexanedione,a metabolite of n-hexane,in urine: evaluation and application of three analytical methods

Three methods for the determination of 2,5-hexanedione (2,5-HD) in urine were compared in order to assess their applicability for toxicokinetic studies and biological monitoring of occupational exposure to n-hexane. Two of them were based on derivatization, followed by gas chromatography and electron-capture detection. of these two, one is a modification of the other, already published, method. The third one involves direct extraction of 2,5-HD followed by gas chromatography and flame-ionization detection. To determine 2,5-HD in urine of workers occupationally exposed to n-hexane, the most straightforward method, direct extraction of 2,5-HD from urine, has been proven to be the most suitable. However, in case of very low concentrations of 2,5-HD in urine, or analysis of small samples of blood, e.g. in kinetic studies, it is necessary to use a more sensitive procedure. The sensitivity of the methods based on the derivation of 2,5-HD followed by electron-capture detection, was, as expected, much higher in terms of analytical reliability. By using these methods, however, precautions are necessary to avoid a matrix effect.     

Gas—liquid chromatographic determination of flurazepam and its major metabolites in plasma with electron-capture detection

A sensitive and selective gas—liquid chromatographic method, using the electron-capture detector for the quantitative determination of flurazepam and its major blood metabolites is described. After extraction and back-extraction steps, flurazepam (I) is well separated from its main metabolites, N-1-hydroxyethylflurazepam (metabolite II) and N-1-desalkylflurazepam (metabolite III). Metabolite II is quantitated after forming its stable tert-butyldimethylsilyl derivative by reaction with tert-butyldimethylchlorosilane—imidazole reagent. The procedure permits the rapid and selective routine determination of flurazepam and its metabolites (II and III) in plasma with a detection limit of 3 ng/ml for flurazepam (I), 1 ng/ml for metabolite II and 0.6 ng/ml for metabolite III. The procedure is linear over the range of concentrations encountered after administration of a single oral therapeutic dose. No interference from the biological matrix is apparent. The suitability of the method for the analysis of biological samples was tested by studying the variation with time of flurazepam and its metabolites' plasma concentrations in normal human volunteers after a single, therapeutic 30-mg oral dose of flurazepam.     

Sensitive gas chromatographic method for determination of mercapturic acids in human urine

A method was developed for sensitive determination of the specific benzene metabolite S-phenylmercapturic acid and the corresponding toluene metabolite S-benzylmercapturic acid in human urine for non-occupational and occupational exposure. The sample preparation procedure consists of liquid extraction of urine samples followed by precolumn derivatization and a clean-up by normal-phase HPLC. Determination of analytes occurs by gas chromatography with electron-capture detection. With this highly sensitive method (detection limits 60 and 65 ng/l, respectively) urinary S-phenylmercapturic and S-benzylmercapturic acid concentrations for non-occupationally exposed persons (e.g. non-smokers) can be measured precisely in one chromatographic run. Validation of the method occured by comparison with a HPLC method we have published recently.     

Gas chromatographic—mass spectrometric determination of ethambutol in human plasma

A quantitative gas chromatographic—mass spectrometric assay has been developed for the determination of ethambutol (EMB) in human plasma. Plasma samples were taken from a patient after oral administration of EMB (with proven tuberculosis infection). Deuterated EMB and a non-deuterated analogue of EMB were synthesized and used as internal standards in this procedure; both gave excellent agreement in the analysis. The derivatizing agent used was trifluoroacetic anhydride (TFAA) and quantitative derivatization was complete in one hour, forming EMB-(TFA). Selective ion monitoring was utilized to monitor the gas chromatographic effluent. Ions were generated by electron impact at 70 eV. The limit of detection was 36 ng EMB per ml plasma. This method is compared with the electron-capture gas chromatographic procedure of Lee and Benet.     

Determination of paroxetine levels in human plasma using gas chromatography with electron-capture detection

A simple, rapid and sensitive procedure using gas chromatography with electron-capture detection to measure paroxetine levels in human plasma has been developed. The analyte was extracted from plasma with ethyl acetate after basification of the plasma and then derivatized with heptafluorobutyric anhydride before gas chromatographic separation. The calibration curves were linear, with typical r² values >0.99. The assay was highly reproducible and gave peaks with excellent chromatographic properties.     

Determination of acetaldehyde in biological samples by gas chromatography with electron-capture detection

A simple specific assay was developed for the determination of acetaldehyde in biological samples. Acetaldehyde was derivatized to 2,4-dinitrophenylhydrazone, which was determined by gas chromatography with electron-capture detection. The use of this detection method is an important device to which no one drew notice. This procedure was very simple and so sensitive that as little as 500 fmol of acetaldehyde could be measured in aqueous solution. The calibration curve of acetaldehyde was linear at least up to 40 μM. Its recoveries from human plasma and rat liver homogenate were 96.5 and 95.7%, respectively.     

Routine determination of plasma catecholamines using reversed-phase,ion-pair high-performance liquid chromatography with electrochemical detection

A procedure is described for the determination of plasma catecholamines using reversed-phase, ion-pair high-performance liquid chromatography coupled with electrochemical detection. Optimisation of chromatographic conditions with respect to detector performance and adherence to procedures and precautions described, render the method applicable to both neurochemical research and routine clinical analysis. The limit of quantitative detection of the method was found to be approximately 30 pg per injection for individual catecholamines. A single chromatographic run, providing adequate resolution of each component, could be completed in approximately 12 min.     

Detection of metabolites of lysergic acid diethylamide (LSD) in human urine specimens: 2-oxo-3-hydroxy-LSD,a prevalent metabolite of LSD

Seventy-four urine specimens previously found to contain lysergic acid diethylamide (LSD) by gas chromatography–mass spectrometry (GC–MS) were analyzed by a new procedure for the LSD metabolite 2-oxo-3-hydroxy-LSD (O-H-LSD) using a Finnigan LC–MS–MS system. This procedure proved to be less complex, shorter to perform and provides cleaner chromatographic characteristics than the method currently utilized by the Navy Drug Screening Laboratories for the extraction of LSD from urine by GC–MS. All of the specimens used in the study screened positive for LSD by radioimmunoassay (Roche Abuscreen^®). Analysis by GC–MS revealed detectable amounts of LSD in all of the specimens. In addition, isolysergic diethylamide (iso-LSD), a byproduct of LSD synthesis, was quantitated in 64 of the specimens. Utilizing the new LC–MS–MS method, low levels of N-desmethyl-LSD (nor-LSD), another identified LSD metabolite, were detected in some of the specimens. However, all 74 specimens contained O-H-LSD at significantly higher concentrations than LSD, iso-LSD, or nor-LSD alone. The O-H-LSD concentration ranged from 732 to 112 831 pg/ml (mean, 16 340 pg/ml) by quantification with an internal standard. The ratio of O-H-LSD to LSD ranged from 1.1 to 778.1 (mean, 42.9). The presence of O-H-LSD at substantially higher concentrations than LSD suggests that the analysis for O-H-LSD as the target analyte by employing LC–MS–MS will provide a much longer window of detection for the use of LSD than the analysis of the parent compound, LSD.     

Determination of a novel xanthine derivative, MKS 213–492, in plasma by high-performance liquid chromatography with electrochemical detection

A reversed-phase liquid chromatographic method with coulometric detection has been developed for the determination of free MKS 213–492 (a novel xanthine derivative) in plasma. This method is based on a simple and rapid plasma extraction procedure using precolumn-switching. The oxidation of this pharmacologically active xanthine derivative was optimized with respect to applied potential, pH of mobile and rinsing phase and rinsing time. The detection limit for MKS 213–492 was found to be 54 pg/injection.     

Packed-column supercritical fluid chromatography of artemisinin (qinghaosu) with electron-capture detection

In this preliminary report, a supercritical fluid chromatographic method is described for the determination of artemisinin in whole blood. The chromatography is carried out on a 20 cm × 1 mm I.D. Deltabond cyano supercritical fluid chromatographic column with detection of the artemisinin via an electron-capture detector. The sample work-up uses a liquid-liquid extraction with hexane, giving a recovery of 82%. The current limit of detection using 1 ml of blood is 20 ng/ml. We speculate that the endoperoxide moiety accounts for the response to the electron-capture detector and thus provides a new approach by which this class of compounds may be analyzed.