Sampling of solid spent agar media for gas chromatography of diffused acidic fermentation products

Abstract Sampling and processing of solid agar media for gas chromatographic analysis is described. Small pieces of agar are cut from a 'sterile' area of the plate leaving the actual bacterial growth undisturbed for further tests. It is shown that diffused fermentation acids are adequately represented in these samples to permit their use for phenotypic and taxonomic characterization of isolates. Because of the established qualitative and quantitative correspondence of fermentation patterns from agar-grown and from broth-grown bacteria, respectively, the method is offered as a rapid alternative for the sampling of spent culture broths. In addition, the sampling of spent agar plugs seems to hold promise for the study of defined mixed cultures and of mixed growth from primary plates.     

Quantitative determination of histamine metabolites by capillary gas chromatography

A method using capillary gas chromatography is described for the determination of histamine and eight of its basic and acid metabolites in a single biological sample of serum, urine, or gastric juice. Ion-exchange chromatography and extraction with organic solvents are used for isolation and purification, and gas chromatography for identification and quantitation. The heptafluorobutyryl derivatives of histamine and some basic metabolites are compatible with nitrogen-phosphorus and electron capture detection modes and offer an excellent sensitivity (detection limit 0.1 pmol with electron capture). The acid metabolites are quantitated after esterification. The linearity range, the sensitivity, a partial study of reproducibility and typical chromatograms show that the method is adaptable to a variety of applications.     

Simultaneous determination of phenol,cresol, xylenol isomers and napthols in urine by capillary gas chromatography

An attempt was made to establish a method for the simultaneous determination of urinary concentrations of phenol, o-, p- and m-cresols, 1 and 2-naphthol and xylenol isomers by capillary gas chromatography. Urine samples were extracted after acid hydrolysis of glucuronides and sulfates by solid-phase extraction. The ten substances were separated gas chromatographically using a capillary column (Ultra 2) of cross-linked 5% phenylmethyl silicone. Calibration graphs were linear for 5–100 μg/ml of all the phenols determined. The corresponding detection limits for phenolic compounds varied from 0.1 to 0.2 μg/ml. The relative standard deviations for samples in urine were in the range 2.6–16.6% and the accuracy was in the range 1.4–25%. Recoveries were generally over 80%.     

Simultaneous determination of norethisterone and six metabolites in human plasma by capillary gas chromatography with mass-selective detection

A method for the simultaneous determination of norethisterone (NET) and six metabolites in human plasma by capillary gas chromatography-mass-selective detection (GC-MS) is described. The compounds are determined in plasma after enzymatic hydrolysis. After addition of norgestrel as the internal standard, the compounds are extracted from plasma at pH 5 using an Extrelut column and elution with dichloromethane. After evaporation, the compounds are converted into bistrimethylsilyl derivatives which are determined by gas chromatography using a mass-selective detector at m/z 429 for the two dihydro-NET (5β-NET and 5α-NET), m/z 431 for the four tetrahydro-NET (3α,5α-NET, 3α,5β-NET, 3β,5β-NET and 3β,5α-NET), m/z 442 for NET and m/z 456 for the internal standard. The reproducibility and accuracy of the method were found suitable over the range of concentrations between 0.50 and 8 ng/ml for NET, and metabolites except for 5α-dihydro-NET (between 1 and 8 ng/ml). The method was applied to clinical samples.     

Simultaneous determination of imipramine and its metabolite desipramine in human plasma by capillary gas chromatography with mass-selective detection

An analytical method for the simultaneous determination of imipramine (IMI) and its N-desmethyl metabolite, desipramine (DIMI) in human plasma by capillary gas chromatography–mass selective detection (GC–MS), with D4-imipramine (D4-IMI) and D4-desipramine (D4-DIMI) as internal standards, was developed and validated. After addition of the internal standards, the compounds were extracted from plasma at basic pH into n-heptane–isoamyl alcohol (99:1, v/v), back-extracted into acidic aqueous solution and re-extracted at basic pH into toluene. Desipramine and D4-desipramine were converted into their pentafluoropropionyl derivatives. The compounds were determined by gas chromatography using a mass selective detector at m/z 234 for IMI, m/z 238 for D4-IMI, m/z 412 for DIMI and m/z 416 for D4-DIMI. The method was applied to clinical samples.     

Simultaneous determination of nicotine and cotinine in plasma using capillary column gas chromatography with nitrogen-sensitive detection

A rapid and sensitive method is described for the simultaneous determination of nicotine and its principal metabolite, cotinine, in plasma. A one-step extraction procedure is employed and the quantitative analyses are performed by capillary column gas chromatography using a thermionic specific detector. Other special measures to avoid contamination from external sources such as atmosphere, solvents and laboratory equipment, which constitutes the major limiting factor of nicotine assay, were also undertaken. The structural analogues of nicotine and cotinine, N-methylanabasine and N-ethylnorcotinine, are used as internal standards. Moreover, a micromethod, which requires only 0.1 ml of plasma and found to be suitable for analysis of cotinine in finger-tip samples of blood, is described. Linearity over the concentration ranges 5–100 ng of nicotine per ml of plasma and 5–500 ng of cotinine per ml of plasma is demonstrated. The precision of the method has been investigated by determining the reproducibility at different levels of nicotine and cotinine within the working ranges, for both 1-ml and 0.1-ml samples of plasma.     

Simultaneous profile analysis of plasma amino and organic acids by capillary gas chromatography

A simultaneous GC analysis of more than 20 amino and nearly 30 non-amino organic acids abundant in plasma is for the first time possible. Isolation of the analytes from the plasma matrix is not necessary, keto acids do not require a preliminary oximation. An instantaneous derivatization of the acids with ethyl chloroformate takes place directly in the medium after deproteinization. Less than 30 min are required to prepare a plasma sample for the GC analysis.     

Simultaneous determination of isosorbide dinitrate and its mononitrate metabolites in human plasma by capillary gas chromatography with electron-capture detection

A method for the simultaneous determination of isosorbide dinitrate (ISDN) and its mononitrate metabolites (2- and 5-ISMN) in human plasma by capillary gas chromatography with electron-capture detection was developed. Two internal standards were used: isomannide dinitrate (IMDN) for the determination of ISDN and isomannide mononitrate (IMMN) for the determinations of 2- and 5-ISMN. After addition of the internal standards, the compounds were isolated from plasma by solid-liquid extraction. They were determined by gas chromatography using an electron-capture detector. The reproducibility and accuracy of the method were found suitable in the range of concentrations 2.5–83 ng/ml for ISDN, 2.6–208 ng/ml for 2-ISMN and 2.3–1010 ng/ml for 5-ISMN. The limit of quantitation (LOQ) was about 2.5 ng/ml for each compound. The method was applied to clinical samples.     

Simultaneous determination of nitrazepam and its metabolites in urine by micellar electrokinetic capillary chromatography

We applied micellar electrokinetic capillary chromatography to simultaneous separation and determination of nitrazepam and its major metabolites, 7-aminonitrazepam and 7-acetamidonitrazepam, in spiked urine. Prior to electrophoresis, the three compounds were successfully extracted from the spiked urine with commercial disposable solid-phase cartridges. The optimum running buffer for the separation was prepared by combining 85 parts of 60 mM sodium dodecyl sulphate—6 mM phosphate—borate, adjusted to pH 8.5, with 15 parts of methanol. The separation order, completed within 25 min, was 7-aminonitrazepam > 7-acetamidonitrazepam > nitrazepam, at an applied potential of 20 kV. We obtained reproducible electropherograms in successive repetitions, and few other peaks or interferences appeared in the electropherogram. The detection limits of the three compounds were 50–100 pg (0.1–0.2 μg/ml of analyte in spiked urine), and the recoveries were 78.9–100.8% for 1 μg/ml and 84.1–100.3% for 5 μg/ml. The application of this method to forensic or clinical samples is demonstrated.     

Simultaneous determination of glucuronides of trimetozine in human urine by gas chromatography

A gas chromatographic method for the simultaneous determination of four glucuronides (metabolites) of trimetozine excreted in human urine is described. The methodinvolves pretreatment of the urine specimen [i.e. removal of interfering substances by solvent extraction, desalting on an ion-exchange (Amberlite XAD-2) column], and permethylation of glucuronides by reaction with methylsulfinyl carbanion and methyl iodide. The permethylated derivatives were submitted to gas chromatographic separation on an OV-17 column, and their structures were investigated by subsequent gas chromatographic—mass spectrometric analysis. The minimum detectable concentration of each glucuronide is 5 μg/ml when 1 ml of urine is used. The utility of the present method is successfully demonstrated by determining the urinary concentration of four glucuronides following oral administration of trimetozine to human subjects.     

Simultaneous determination of griseofulvin and 6-desmethylgriseofulvin in plasma by electron-capture gas chromatography

Two methods have been developed for the simultaneous determination of griseofulvin and its major metabolite 6-desmethylgriseofulvin in plasma using electron-capture gas chromatography. The first method was based on the quantitative reversion of the 6-desmethyl metabolite to griseofulvin by diazomethane. Plasma extract was chromatographed both before and after treatment with diazomethane, the former being the measure of griseofulvin and the latter representing the sum of the two compounds. In the second method, plasma extract was treated with diazobutane and griseofulvin and the butylated metabolite were separated by gas chromatography. The sensitivity for griseofulvin was 20 ng/ml by both methods and that for the metabolite was 20 ng/ml and 40 ng/ml by the first and the second method, respectively. The concentrations of the metabolite as well as griseofulvin were determined in dog and human plasma after oral administration of griseofulvin.     

Simultaneous determination of cyclandelate and its metabolite in human plasma by capillary column gas-liquid chromatography

A method was developed for the simultaneous determination of cyclandelate and mandelic acid concentrations in plasma, involving extraction from plasma followed by trimethylsilylation and chromatography of the derivatives on a glass capillary column with hydrogen flame-ionization detection. Calibration graphs were linear down to at least 20 μg/ml for each substance. The precision was excellent with a pooled relative standard deviation of 6.3% and 6.4% for cyclandelate and mandelic acid serum samples, respectively. Concentrations below 500 ng/ml of each substance could be detected in human plasma. The method was developed for use in bioavailability and metabolism studies.     

The use of capillary gas chromatography/mass spectrometry for the determination of acidic dopamine metabolites in human brain

A capillary gas chromatographic/mass spectrometric method was developed for the determination of homovanillic acid and 3,4-dihydroxyphenylacetic acid in discrete areas of human brain known to contain only small amounts of dopamine metabolites. The electron impact mass spectra of the trimethylsilyl derivatives of homovanillic acid and 3,4-dihydroxyphenylacetic acid and their deuterated isotopic variants were used for their identification and quantification.     

Nitrate analysis by capillary gas chromatography

A microanalytical gas chromatographic (GC) method for the analysis of nitrate in rat urine is described. The method involves the conversion of nitrate to nitromesitylene and quantitation using 3,4-dimethylnitrobenzene as an internal standard. The nitroaromatics were separated on a wide-bore capillary column and detected with a nitrogen-phosphorus thermionic detector. The method exhibited linearity over the range of 1.0 to 1000 microM nitrate, and the detection limit was 0.5 microM nitrate (200-microliters sample). The coefficient of variation (CV) range for intra-day precision was 2.2 to 5.8% (20 microM level) and 3.1 to 6.5% (200 microM level). Inter-day CVs ranged from 2.0 to 6.1% for the samples tested. The average recovery was 77% (20 microM level) and 80% (20 microM level). The accuracy of the GC method compared favorably with results obtained from a standard colorimetric nitrate assay. Interference by urinary chloride was eliminated by pretreatment of samples with saturated silver acetate. Both processed and unprocessed samples were stable for at least 60 days at -15 degrees C. The procedure was used to measure urinary nitrate in rats fed a custom low-nitrate diet.     

Preparation of extracts of culture liquids for gas-chromatographic determination of acidic fermentation products

An extraction method coupled with gas-chromatographic analysis has been devised for the determination of small amounts of C₁-C₇ branched and straight-chain saturated fatty acids as well as 18 mono-, di- and tricarboxylic acids and related compounds, the latter being analyzed either as their methylesters or as their trimethylsilyl-derivatives. Methods for extracting non-volatile acids or both volatile and non-volatile acids are described for large and small volumes of culture media. The proposed methods have been successfully applied to the determination of non-acidic fermentation products, known amounts of alcohols, and to volatile and non-volatile acids in bacterial fermentation solutions, namely a batch culture ofLactobacillus casei subsp.rhamnosus ATCC 7469. A carbon balance sheet of this organism's metabolic behaviour throughout growth is proposed.     

Headspace gas chromatography with capillary column for urine alcohol determination

A headspace gas chromatographic method using a fused-silica capillary column Poraplot Q has been developed and validated for the detection and quantification of ethanol in urine. Under optimized conditions, ethanol was properly separated from acetaldehyde, acetone, isopropanol, methanol and n-propanol. Limits of detection (LODs) and quantification (LOQs) were 0.008 and 0.010 g/l, respectively. The precision studies within-run and between-run, using spiked urine samples (0.08, 0.8 and 2.0 g/l) showed maximum coefficients of variation 5.9 and 6.5%, respectively. Results of ethanol recovery varied from 91.6±0.8 to 103.3±1.8% over the concentration range from 0.01 to 3.20 g/l. The method was appropriate for the detection of ethanol in urine samples. This matrix can be used for monitoring alcohol abuse in the workplace and used in alcohol rehabilitation programs.     

Simultaneous determination of paracetamol, caffeine and propyphenazone in ternary mixtures by micellar electrokinetic capillary chromatography

A new micellar electrokinetic capillary chromatographic method has been developed to analyze the pharmaceutical preparations containing ternary combination of paracetamol (PAR), caffeine (CAF) and propyphenazone (PRO). Best results were obtained by using 20mM pH 9.0 borate buffer containing 30mM sodiumdodecylsulphate as the background electrolyte. Diflunisal (DIF) was used as internal standard (IS). The separation was performed through a fused silica capillary (50microm internal diameter, 44cm total length, 35.5cm effective length) at 25 degrees C with the application of 3s of hydrodynamic injection at 50mbar pressure and a potential of 29kV. Detection wavelength was 200nm. Under these conditions, the migration times were found to be 5.174min for PAR, 5.513min for CAF, 7.195min for DIF, and 9.366min for PRO. Linearity ranges for the method were determined as 2-200microgmL(-1) for PAR and CAF and 3-200microgmL(-1) for PRO. Limit of detections were found as 0.6microgmL(-1) for PAR and CAF and 0.8microgmL(-1) for PRO. According to the validation study, the developed method was proved to be accurate, precise, sensitive, specific, rugged and robust. Three pharmaceutical preparations, which are produced by different drug companies in Turkey, were analyzed by the developed method. One of the same preparations was also analyzed by the derivative ratio spectro zero-crossing spectrophotometric method reported in literature. No significant differences were found statistically.