Development and validation of a method for measuring the glycine and taurine conjugates of bile acids in bile by high-performance liquid chromatography

We developed and validated a simple method for measuring the individual glycine and taurine conjugates of bile acids in bile by high-performance liquid chromatography with a C₁₈ reversed-phase column using an isocratic solvent system of acidified methanol—potassium phosphate. Without preliminary derivatization or purification, complete separation of the ten major conjugated bile acids present in bile could be achieved in 65 min. Total bile acid concentrations were identical when measured enzymatically and by summing the individual bile acids determined by high-performance liquid chromatography. Bile acid composition determined by gas—liquid chromatography correlated with results by high-performance liquid chromatography. Finally, measurements of individual glycine and taurine conjugates in human bile and in mixtures of bile acid standards by high-performance liquid chromatography and thin-layer chromatography gave similar results. This high-performance liquid chromatographic system permits simultaneous quantification of total and individual bile acids and their glycine and taurine conjugates in bile.     

Simultaneous determination of triazolo-benzophenone [2′,5-dichloro-2-(3-glycylaminomethyl-5-methyl-4H-1,2,4-triazol-4-yl)-benzophenone] and its major blood metabolite, triazolam, in monkey plasma by electron-capture gas—liquid chromatography

A gas—liquid chromatographic method for the simultaneous determination of triazolobenzophenone [2′,5-dichloro-2-(3-glycylaminomethyl-5-methyl-4H-1,2,4-triazol-4-yl)-benzophenone, TB] and its major blood metabolite, triazolam, 8-chloro-6-(o-chlorophenyl)-1-methyl-4H-s-triazolo[4,3-a][1,4]benzodiazepine (TZ), in monkey plasma was developed. Decomposition of TB was observed during gas—liquid chromatography. In alkaline medium, TB in plasma was submitted to ring closure reaction to yield triazolo-aminoquinoline, [4-amino-7-chloro-5-(2-chlorophenyl)-1-methyl-4H-s-triazolo[4,3-a]quinoline (TAQ), while TZ remained unaffected, and TAQ and TZ in the benzene extract were assayed by gas—liquid chromatography using an electron-capture detector. The concentration ranges studied were from 5 to 40 ng of TB per 0.5 ml of plasma and from 2 to 20 ng of TZ per 0.5 ml of plasma. This method could be applied to the determination of the plasma levels of TB and TZ in monkeys following intravenous administration of a single 0.2 mg/kg dose of TB.     

Modification of the simultaneous determination of alditol acetates of neutral and aminosugars by gas—liquid chromatography : Application to the fractionation of sialoglycoproteins from bone

A modification of a gas—liquid chromatographic method is described that allows better simultaneous separations of the neutral and aminosugar alditol acetate derivatives as single peaks. Using 3% SP-2340 on 100–200 mesh Supelcoport, retention times were relatively short and baseline separation between glucose and galactose was achieved. The method is particularly suitable for monitoring the fractionation of complex mixtures of glycoproteins and glycosaminoglycans, and its application is illustrated in the fractionation of bone matrix extracts subjected to ion-exchange chromatography. A convenient procedure allowing the separation and estimation of sialic acid in the same aliquot is also described and evaluated.     

Reliable routine method for determination of perazine in serum by thin-layer chromatography with an internal standard

The use of a high-performance thin-layer chromatography linear chamber and of thioridazine as internal standard increases the performance of thin-layer chromatography (TLC) with direct densitometric scanning, and allows the rapid determination of serum levels of the neuroleptic drug perazine under usual therapeutic conditions. TLC is superior to gas—liquid chromatography in so far as the main metabolite desmethylperazine can be easily separated and detected by the same procedure.     

New electron-capture gas-liquid chromatographic method for the determination of mexiletine plasma levels in man

A method for the determination of mexiletine in human plasma by gas—liquid chromatography with electron-capture detection is described. Plasma samples are extracted at pH 12 with dichloromethane after addition of the internal standard, the 2,4-methyl analogue of mexiletine. A derivative is obtained using heptafluorobutyric anhydride; according to gas chromatography—mass spectrometry it is a monoheptafluorobutyryl compound. The minimum detectable amount of mexiletine is 5 pg. Accurate determinations of human plasma levels were performed after oral or intravenous treatment.     

Simultaneous determination of blood concentrations of methohexital and its hydroxy metabolite by gas chromatography and identification of 4′-hydroxymethohexital by combined gas—liquid chromatography—mass spectrometry

A simple, sensitive and selective method is described for the simultaneous determination of low concentrations (less than 50 ng/ml) of underivatized methohexital and its hydroxy metabolite in small (0.1 ml) samples of human and rat plasma or whole blood by gas chromatography with nitrogen-selective detection.Moreover, the main metabolite in rat and man was identified as 4′-hydroxymethohexital by comparison of chromatograms from gas—liquid chromatography (GLC) with data obtained from GLC—mass spectrometry and ¹H-nuclear magnetic resonance spectrometry of this metabolite, produced both by incubating methohexital with isolated rat liver microsomes and by isolating this metabolite from rat urine.     

Analysis of profiles of conjugated steroids in urine by ion-exchange separation and gas chromatography—mass spectrometry

A simplified, flexible method for the analysis of metabolic profiles of steroids in urine is described. Solid extraction with Amberlite XAD-2 or Sep-Pak C_{1 g} cartridges is followed by group fractionation of unconjugated neutral and phenolic steroids, monoglucuronides, monosulphates and disulphates on the lipophilic strong anion exchanger triethylaminohydroxyproply Sephadex LH-20 (TEAP-LH-20). Following brief enzymatic hydrolysis or solvolysis the steroids are purified on TEAP-LH-20. O-Methyloxime and trimethylsilyl ether derivatives are prepared and purified by filtration through Lipidex 5000, and are then analyzed by glass capillary column gas—liquid chromatography and gas chromatography—mass spectrometry.Between 2 and 5 ml of urine are used for a comprehensive analysis. Unconjugated neutral and phenolic steroids are isolated in half a day, corresponding steroids in the conjugate fractions in two days. No fraction containing steroids is discarded, but the analysis can be limited to a selected fraction.     

Improved selective ion monitoring mass-spectrometric assay for the determination of n,n-dimethyltryptamine in human blood utilizing capillary column gas chromatography

A gas—liquid chromatographic—mass spectrometric procedure is described for the assay of dimethyltryptamine (DMT) in whole blood. The use of a glass capillary column in combination with selective ion monitoring results in an assay with a high degree of specificity and sensitivity. 5-Methoxy-DMT is used as an internal standard and carrier in the isolation procedure. The superior resolving characteristics of the capillary column (as compared to previously employed packed columns) allows monitoring of the intense m/e 58 ion arising from the DMT side-chain. A sensitivity limit of 10 pg/ml blood is realized with a 10-ml blood sample.     

Determination of urinary amino acids by means of glass capillary gas—liquid chromatography with alkali-flame ionisation detection and flame ionisation detection

The determination of amino acids as their N(O)-heptafluorobutyryl-isobutyl ester derivatives by glass capillary gas—liquid chromatography has been studied. Separations of amino acids obtained from insulin hydrolysate and human urine analysed with a flame ionisation detector, an alkali-flame ionisation detector and an electron-capture detector are shown. The quantitative results of urinary amino acids of one deep-sea diver are presented.     

High-performance liquid chromatographic and gas—liquid chromatographic determination of diazepam and nordiazepam in plasma

A one-step method for extraction of diazepam, nordiazepam, and internal standard into toluene is followed by chromatographic separation and detection with either dual-wavelength high-performance liquid chromatography or electron-capture gas—liquid chromatography. Agreement between the two methods was excellent for diazepam (r = 0.99, n = 38) and good for nordiazepam (r = 0.96, n = 79) over a concentration range that included subtherapeutic, therapeutic, and toxic plasma levels.     

Rapid determination of diazepam and nordiazepam in plasma by electron capture gas—liquid chromatography : Application in clinical pharmacokinetic studies

A rapid method was developed for the determination of diazepam and nordiazepam (N-desmethyldiazepam) in human plasma using electron capture gas—liquid chromatography (GLC—ECD). The concentration of diazepam and nordiazepam is determined using 0.5 ml of plasma extracted with 1.0 ml of benzene containing 25 ng/ml of methylnitrazepam as the internal standard. The benzene extract is removed and an aliquot is subjected to automated GLC—ECD analysis. The method has a sensitivity limit of 5 ng diazepam and 10 ng nordiazepam per milliliter of plasma. The method was used to determine the plasma levels in man following the first 5-mg diazepam dose, as well as during chronic oral administration of 5 mg diazepam three times daily and 15 mg diazepam once a day.     

Pre-column derivatization of free bile acids for high-performance liquid chromatographic and gas chromatographic—mass spectrometric analysis

Pentachlorophenyl (PCP) esters of five free bile acids (FBA) were obtained by reacting the FBA and Kovacs' complex (KC) in a 1:8 molar ratio in acetone at 65°C, and were purified by column chromatography on silica gel. The esters were crystallized from benzene—hexane, derivatized as trimethylsilyl ethers for gas chromatography on a DB-1 capillary column and for gas chromatography—mass spectrometry with a DB-5 column, and mass spectrometry (MS) in the electron-impact (EI) positive-ion mode at 70 eV. The reaction is specific for FBA even in the presence of glycine and taurine conjugates of bile acids. The PCP esters were treated with benzylamine in chloroform or methanol to produce N-benzyl derivatives of FBA. The N-benzylamides were separated by high-performance liquid chromatography (HPLC) on a 4-μm Nova-Pak C₁₈ column, studied by thermospray—LC—MS, and in the direct insertion probe—EI positive-ion mode.     

An approach to the systematic analysis of urinary steroids

1. Human urine, its extracts, extracts of urine pretreated with enzyme preparations containing β-glucuronidase and steroid sulphatase or β-glucuronidase alone, and products derived from the specific solvolysis of urinary steroid sulphates, were submitted to the following sequence of operations: reduction with borohydride; oxidation with a glycol-cleaving agent (bismuthate or periodate); separation of the products into ketones and others; oxidation of each fraction with tert.-butyl chromate, resolution of the end products by means of paper chromatography or gas–liquid chromatography or both. 2. Qualitative experiments indicated the kind of information the method and some of its modifications can provide. Quantitative experiments were restricted to the direct treatment of urine by the basic procedure outlined. It was partly shown and partly argued that the quantitative results were probably as informative about the composition of the major neutral urinary steroids (and certainly about their presumptive secretory precursors) as those obtained by a number of established analytical procedures. 3. A possible extension of the scope of the reported method was indicated. 4. A simple technique was introduced for the quantitative deposition of a solid sample on to a gas–liquid-chromatographic column.     

Recent methods for the determination of volatile and non-volatile organic compounds in natural and purified drinking water

Four analytical protocols for the extraction and preconcentration of organic residues in natural or purified drinking water were investigated and compared: closed loop stripping analysis; simultaneous extraction—distillation; purge and trap analysis; continuous liquid—liquid extraction. Organic extracts were submitted to a variety of separation and identification techniques. Volatiles were determined by conventional capillary column gas chromatography with tandem mass spectrometry, using triple-stage quadrupole instruments. Non-volatile and thermally labile molecules were investigated by several different techniques (high-temperature gas chromatography, capillary column supercritical fluid chromatography, pyrolysis gas chromatography—mass spectrometry, thermospray liquid chromatography with tandem mass spectrometry and conventional fast-atom bombardment with tandem mass spectrometry). Several samples recently examined in the laboratory provide examples of this multitechnique approach for a more complete knowledge of the organic carbon distribution in water-dissolved organic matter, taking into account organic substances with widely different volatilities, polarities and thermal stabilities.     

Absence of carbohydrate in crystalline Fraction I protein isolated from tobacco leaves

By means of gas—liquid chromatography—mass spectra analyses, three-times recrystallized Fraction I protein from tobacco leaved contains less than 0.0005% carbohydrate and is therefore different from other species of protein which contain up to 5% carbohydrate.     

Determination of carbimide in plasma by gas—liquid chromatography

A sensitive and selective method for the measurement of carbimide, the hydrolytic product of calcium carbimide, in plasma is described. The procedure involves extraction with ethyl acetate, derivatization with heptafluorobutyric anhydride and analysis by gas—liquid chromatography with electron-capture detection. The lower limit of sensitivity of the assay is 5.0 ng/ml carbimide in plasma. The overall accuracy of the procedure is 96.1% with a coefficient of variation not exceeding 8.7%. This assay has been used to investigate the time-course of plasma carbimide concentration in the rat following oral administration of calcium carbimide.     

Quantitative analysis of 6,11-dihydro-11-oxo-dibenz[b,e] oxepin-2-acetic acid (isoxepac) in plasma and urine by gas-liquid chromatography

A method is described for the quantitative analysis of 6,11-dihydro-11-oxo-dibenz[b,e] - oxepin-2-acetic acid (isoxepac) in plasma and urine. Isoxepac and internal standard are extracted from acidified plasma and urine, converted to the corresponding methyl esters and analysed by gas—liquid chromatography using a flame ionization detector. The method is accurate and precise over the range 0.1–30 μg/ml. The method has been applied to the analysis of plasma and urine from both healthy volunteers and patients receiving therapeutic oral doses of isoxepac.     

High-performance liquid chromatographic determination of naproxen, ibuprofen and diclofenac in plasma and synovial fluid in man

High-performance liquid chromatographic assay procedures have been developed for naproxen, ibuprofen and diclofenac in human plasma and synovial fluid samples. A single liquid—liquid extraction procedure was used to isolate each compound from acidified biological matrix prior to the quantitative analysis. A Spherisorb ODS column (12.5 cm × 4.6 mm I.D.) was used for all the chromatography. Naproxen was eluted with a mobile phase of methanol—Sörensen's buffer at pH 7 (37:63, v/v). Ibuprofen and diclofenac were eluted using mobile phases of methanol—water at pH 3.3 (65:35, v/v and 63:37, v/v, respectively). Diphenylacetic acid was used as the internal standard for the assay of naproxen and flurbiprofen was used in the analysis of ibuprofen and diclofenac. Inter- and intra-day coefficients of variation were less than 7%. The assays were used in clinical studies of the three drugs in osteo- and rheumatoid arthritis patients.     

Prostaglandins E and F, and 19-hydroxylated E and F (series I and II) in semen of fertile men : Gas and liquid chromatographic separation with selected ion detection

Low concentrations of prostaglandins (PG) could be related to male clinical infertility although relevant experimental data are scarce. The aim of this work is to establish reliable seminal PG levels in fertile men by rigorous sample control, to prevent degradation, and by rapid and simple extraction and assay procedures.Single semen samples from healthy fertile men were immediately centrifuged (within 30 min of ejaculation) adding PGF_2α D₄ to the seminal plasma as internal standard. The samples were next ultrafiltered and the PGs in the ultrafiltrate were derivatized with a mixture of N,O-bis(trimethylsilyl)trifluoroacetamide and piperidine (1:1) at 60° for 30 min. Optimum gas—liquid chromatographic separation of all of the peaks of interest was achieved on 4 m × 1/4 in. I.D. Dexsil 300 packed columns at 280°. The detection and quantitation of all the peaks of interest depends on the selected ion monitoring of specific masses. The values obtained (in μg/ml, range in parentheses) were: PGEs, 63.5 ± 49.3 (9–164); PGFs, 2.6 ± 1.92 (0.95–6.63); 19-OH PGEs, 592.6 ± 312.5 (142.1–1047); and 19-OH PGFs, 12.66 ± 5.21 (4–19). Individual values for members of both series I and II are also presented.The sample collection and extraction procedures were further checked by high-performance liquid chromatography on a μPorasil column, with individual isolation and collection of all of the PGs, including the 19-OH PGs not previously separated by liquid chromatography.