Headspace gas chromatographic determination of beta-galactosidase activity using electron-capture detection

A headspace gas chromatographic method for the determination of beta-galactosidase (E.C. 3.2.1.23) activity is described. The method, in which 2,2,2-trichloroethyl beta-D-galactopyranoside (beta-TCG) is used as substrate, involves determination of the liberated 2,2,2-trichloroethanol by gas chromatography with electron capture detection. The preparation of beta-TCG and of 2,2,2-trichloroethyl alpha-D-galactopyranoside is described. A Km = 0.80 mM was found for the enzymatic hydrolysis of beta-TCG employing beta-galactosidase from Escherichia coli. The assay has been applied to the quantitative determination of E. coli bacteria.     

Gas chromatographic determination of mefloquine in human and dog plasma using electron-capture detection

A sensitive and selective gas-liquid chromatographic method for the determination of plasma levels of mefloquine in human and dog plasma is described. The drug and internal standard were extracted from plasma at pH 9.0 into isopropyl acetate. After evaporation of the solvent, the residue was taken up in toluene and derivatised with heptafluorobutyrylimidazole. The derivative was quantified by gas-liquid chromatography on a 3% GC GE-SE30 column with electron-capture detection. The limit of detection for mefloquine in plasma was 10 ng/ml. The mean overall recovery from plasma was 102.7 +/- 3.3%. The method was shown to be specific for mefloquine without any interference from endogenous compounds in plasma or from the drugs pyrimethamine and sulfadoxine (compounds often administered in combination with mefloquine). The assay described was successfully applied to the determination of plasma levels of mefloquine in man and dog following oral and intravenous administration, respectively.     

Enantioselective gas chromatographic assay with electron-capture detection for dl-ritalinic acid in plasma

Enantioselective gas chromatographic assays for the quantitation of methylphenidate and its major metabolite ritalinic acid in plasma are described. The procedures involved the extraction of methylphenidate enantiomers from alkanised plasma. The plasma was then washed to ensure complete removal of methylphenidate before saturation with sodium carbonate to promote the extraction of ritalinic acid enantiomers with ethyl acetate-isopropanol (60:40) solvent mixture. Subsequently, ritalinic acid enantiomers were converted back into methylphenidate enantiomers by Fisher-Speier esterification. N-Heptafluorobutyryl-L-prolyl chloride, a chiral acylating reagent, was used to convert the enantiomers of methylphenidate into their corresponding diastereomeric amide derivatives, which were separated cleanly on an achiral capillary column (OV-225) and quantitated with electron-capture detection. The assays were sensitive, reliable and reproducible.     

Rapid micromethod for the analysis of mitotane and its metabolite in plasma by gas chromatography with electron-capture detection

A rapid micromethod for 50-microliter samples is described for the analysis in plasma of mitotane (o,p'-DDD) and its metabolite (o,p'-DDE), using p,p'-DDD as internal standard. The compounds are extracted with heptane without sample pretreatment, and an aliquot is directly analysed by gas chromatography with electron-capture detection. Absolute recoveries for all three compounds were ca. 87% and coefficients of variation were less than 5%. The method is being used successfully for monitoring patients with Cushing's syndrome who receive chronic oral mitotane therapy.     

High-performance liquid chromatographic method with ultraviolet detection for the determination of dapsone and its hydroxylated metabolite in human plasma

A validated high-performance liquid chromatographic method with ultraviolet detection for the quantitative determination of dapsone (4,4'-diaminodifenyl sulfone, DDS) and a metabolite, hydroxylaminodapsone (4-amino-4-hydroxylaminodiphenyl sulfone, DDS-NOH), in human plasma is described. Human plasma was deproteinized with acetone and the clear supernatant solution after centrifugation was evaporated to dryness under a gentle stream of nitrogen at 70 degrees C. The residue was dissolved in a mixture of HPLC eluent and acetone (18:5 v/v) and an aliquot of this solution (50 microL) was injected onto the HPLC column. Dapsone, hydroxylaminodapsone and diazoxide as internal standard, were separated within 10 min by isocratic elution with water:acetonitrile:glacial acetic acid:triethylamine (80:20:1.0:0.5 by volume) as eluent. Detection was by ultraviolet at the wavelength of 295 nm. The within-day repeatability coefficients of variation were 3-5% for dapsone (0.301-20.0 mg/L, n = 5) and 3-5% for hydroxylaminodapsone (0.0948-6.32 mg/L, n = 5), whereas the between-day repeatability coefficients of variation were 3-8% (0.301-20.0 mg/L, n = 5) for dapsone and 4-10% for hydroxylaminodapsone (0.0948-6.32 mg/L, n = 5). The mean recoveries -were 92-107% (0.301-20.0 mg/L, n = 2), 80-82% (0.0948-6.32 mg/L, n = 2) and 88% (0.0200 mg/mL, n = 5), for dapsone, hydroxylaminodapsone and diazoxide, respectively. The average correlation coefficient of the calibration curve was 0.99988 (n = 5) for dapsone at a concentration range of 0.301-20.0 mg/L, whereas the average correlation coefficient of the hydroxylaminodapsone calibration curve was 0.99981 (n = 5) at a concentration range of 0.0948-6.32 mg/L. The limits of detection were 0.00200 and 0.0470 mg/L for dapsone and hydroxylaminodapsone, respectively. The method is suitable for drug level monitoring and for pharmacokinetic studies.     

Rapid and sensitive electron-capture gas chromatographic method for the determination of pinazepam and its metabolites in human plasma, urine and milk.

A rapid, sensitive and specific gas-liquid chromatographic method for the measurement of pinazepam and its metabolites in biological fluids is reported. After a single extraction of the sample with toluene, the organic phase is concentrated and, after chromatography on a 3% OV-17 column, measured with an electron-capture detector. The sensitivity was 1.0 ng/ml for pinazepam and 5.0 ng/ml for its metabolites. Plasma levels and urinary excretion in human volunteers and plasma and milk levels in women suffering from anxiety during breastfeeding are reported.     

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.     

Micro-scale liquid chromatographic method for the determination of bamifylline and its major metabolite in human plasma

A sensitive micro-scale method based on isocratic elution reversed-phase ion-pair chromatography for the determination of bamifylline and its major metabolite (AC 119) in human plasma is described. The method is based on a liquid-liquid extraction clean-up followed by analysis on an LC-Packings Fusica (Delta Pak, RP-18, 5 microns, 300 A) column (15 cm x 330 microns. I.D.) with 0.03 M heptanesulphonate (pH 3.5)-acetonitrile (7:3, v/v) as the mobile phase. Data with respect to recovery, reproducibility and limits of detection are reported and discussed.     

Automated high-performance liquid chromatographic method for the simultaneous determination of cefotiam and delta 3-cefotiam in human plasma using column switching.

An automated high-performance liquid chromatographic method using column switching was established for the simultaneous determination of cefotiam (I) and delta 3-cefotiam (II) in human plasma after oral administration of cefotiam hexetil dihydrochloride. The method allowed the determination of analytes in plasma by the direct injection of diluted specimen with phosphate buffer. The analytes were enriched onto the C18 short pretreatment column by 0.05 M phosphate buffer (pH 7.7), while proteins and endogenous hydrophilic substances in plasma were washed off to waste. The enriched analytes were then back-flushed onto the analytical C18 column, separated by a mixture of 0.05 M phosphate buffer (pH 7.7)-acetonitrile (88:12, v/v) and detected by the ultraviolet absorbance at 254 nm. Recoveries from spiked plasma were quantitative, and the coefficients of variation were below 4%. The lower detection limits in plasma were 10 ng/ml for both I and II. Concentrations of I and II in plasma determined by the present method were in good agreement with those obtained by the conventional deproteinization method.     

High-performance liquid chromatographic method for the simultaneous determination of the enantiomers of carvedilol and its O-desmethyl metabolite in human plasma after chiral derivatization

Quantitative methodology for the simultaneous high-performance liquid chromatographic (HPLC) resolution and determination of the enantiomers of carvedilol, a new multiple-action antihypertensive agent exhibiting both vasodilator and beta-blocking activity, and its active metabolite, O-desmethylcarvedilol, in human plasma is described. The method involves reversed-phase solid-phase extraction of the analytes, followed by derivatization of the extract with the chiral reagent, 2,3,4,6,-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate and injection of the resultant diastereoisomers onto a reversed-phase HPLC column coupled to a fluorescence detector. Both pairs of diastereoisomers formed are completely resolved within 12 min (resolution for the respective pairs is 2.26 and 3.32) and the baseline is clean and free from extraneous peaks. The assay is linear over the range 0.6-80 ng/ml of human plasma with a lower limit of detection of approximately 100 pg on-column for each of the enantiomers. The method can be adapted for a number of structural analogues of carvedilol and is currently applied in support of preclinical and clinical studies of the drug.