Gas chromatographic determination of the hypoglycaemic agent gliclazide in plasma

A gas chromatographic method has been developed that permits the accurate and specific determination of the hypoglycaemic agent gliclazide in plasma. Gliclazide is extracted with chloroform and, after clean-up, derivatized with diazomethane followed by heptafluorobutyric anhydride to form N-methyl-N′-heptafluorobutyrylgliclazide, which is assayed on a gas chromatograph equipped with a flame ionization detector, an electron-capture detector or a nitrogen—phosphorus sensitive detector.Accurate determinations are possible with flame ionization detection over a concentration range of 1–15 μg/ml of gliclazide in plasma with a relative standard deviation of 5.2%. The minimum detectable concentration with electron-capture detection is 0.02 μg per sample. Plasma levels of gliclazide in dogs following single oral administration (40 mg per dog) have also been determined.     

Gas—liquid chromatographic method for the measurement of plasma levels of d-7,8-dimethoxy-3-methyl-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine acid maleate (SCH-12679) and its major metabolites in aggressive mental retardates

A sensitive gas—liquid chromatographic technique for the quantitative analysis of SCH-12679 (d-7,8-dimethoxy-3-methyl-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine acid maleate) and its major metabolites in plasma of aggressive mental retardates receiving therapeutic doses of the medicament has been developed. The lower limits of detection are 20 ng/ml for SCH-12679, 0.5 ng/ml for 3-desmethyl SCH-12679 and 0.4 ng/ml for 7-desmethyl plus 8-desmethyl SCH-12679. SCH-12679 is estimated with a flame ionization detector. Its metabolites are quantitated using an electron-capture detector after conversion of the compounds to their heptafluorobutyryl derivatives by reaction with the appropriate anhydride. Data on plasma levels of unchanged SCH-12679, 3-desmethyl SCH-12679 and a combination of 7-desmethyl and 8-desmethyl SCH-12679 in fifteen patients treated with the medicament are presented.     

Electron-capture—gas chromatographic determination of atenolol in plasma and urine, using a simplified procedure with improved selectivity

A sensitive gas chromatographic method for the quantitative analysis of atenolol in human plasma and urine is described. Atenolol is extracted with dichloromethane containing heptafluorobutanol to improve the extraction ability. Derivatization with trifluoroacetic anhydride in diethyl ether gives a bistrifluoroacetyl derivative which is more selectively detected by an electron-capture detector than is the corresponding heptafluorobutyryl derivative. The method allows determination down to 20 nmol/1 (5 ng/ml) in 1 ml of sample with a relative standard deviation below 10%.     

Separation and trace estimation of benzidine and its macromolecular adducts using supercritical fluid chromatography

A sensitive, rapid, selective and reproducible method has been developed to measure blood plasma levels of benzidine (BZ) and its acetylated metabolite, N-OH-N,N'-diacetylbenzidine (N-OH-DABZ), using supercritical fluid chromatography (SFC) for the first time. Benzidine and N-OH-N,N'-diacetylbenzidine were extracted from the plasma using ether. Separation was done on a Nucleosil (250 mm x 4.6 mm) 10 microm, Nucleosil-RP-C18 column with 7.4% (v/v) methanol-modified supercritical fluid carbon dioxide (2.5 ml min(-1)) as mobile phase. The column temperature was 45 degrees C and the outlet pressure was set at 8.83 MPa. The detection was done using a UV-Vis detector set at 280 nm. The limit of quantification was 0.10 ng ml(-1) (BZ) and 0.14 ng ml(-1) (N-OH-diacetylbenzidine) using 1 ml plasma specimen. The mean extraction recovery of BZ was found to be 98.6%. The SFC method was directly compared to a published HPLC-UV method. With respect to speed, organic solvent usage, sensitivity, specificity and accuracy, SFC was found to be superior. The method has been successfully used to estimate the BZ, N-OH-diacetylbenzidine levels in blood plasma of the animals who were administered 15 microg kg(-1) body weight of benzidine.Further, this method has been also applied for the detection and quantification of benzidine DNA and hemoglobin adducts from the blood and tissue samples of the benzidine dosed animals.     

Assay of timolol in human plasma using gas chromatography with electron-capture detection

A simple and sensitive gas chromatographic method has been developed for the determination of timolol in plasma using electron-capture detection and propranolol as internal standard. Timolol was extracted using butyl chloride and derivatized using trifluoroacetic anhydride in butyl acetate. The lower detection limit for the assay was found to be 1 ng/ml from 1 ml of plasma. Extracted standards gave within-day precision of 12.55, 9.68 and 3.78% for 1, 20 and 100 ng/ml plasma samples, respectively. A recovery of at least 80% of timolol was found using the extraction method described. The assay was used in a randomized cross-over bioequivalence trial using an oral administration of 20 mg of timolol. Pharmacokinetic parameters compare favourably with other literature values.     

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.     

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.     

Supercritical fluid extraction and high-performance liquid chromatography-diode array-electrochemical detection of signature redox compounds from sand and soil samples

A supercritical fluid extraction procedure and a chromatographic separation/detection method were developed for the detection of Earth-based microorganisms. After microbes in a sand or a soil sample were hydrolyzed in a diluted NH(4)OH/acetone solution, several redox compounds from bacteria could be effectively extracted with trimethylamine-modified supercritical CO(2) at 35 degrees C and 300 atm. These signature redox-active compounds were separated by a reversed-phase HPLC column in an ion-pair mode and then monitored with a diode array detector and an electrochemical detector. The analytical results demonstrated the feasibility of using the reported techniques to detect the chemical signature of life in barren desert sand samples.     

Gas chromatographic determination of zopiclone in plasma after solid-phase extraction

A gas chromatographic technique for determining zopiclone based on a solid-phase extraction procedure with C₁₈ cartridge for sample clean-up is presented. Quantification can be achieved with 1 ml of plasma. The method uses prazepam as internal standard. Zopiclone is separated on a 5% phenyl methyl silicone analytical column and detected with an electron-capture detector, which consequently allows a limit of quantitation of 2 μg/l. It is thus simple, rapid, sensitive and linear over the range 5–2000 μg/l.     

Improved gas chromatographic procedure for the determination of clonazepam levels in plasma using a nitrogen-sensitive detector

A gas—liquid chromatographic procedure (GLC) is described for the determination of clonazepam in plasma. The drug is extracted from buffered plasma at pH 9.0 with diethyl ether and then back-extracted into 6 N hydrochloric acid—6 N sulfuric acid (95:5) and hydrolyzed at 100°C to convert the drug into its benzophenone derivative. The benzophenone derivative of flurazepam is added to plasma as an internal reference standard. Drug derivatives are finally extracted from the neutralized aqueous phase and assayed by GLC. The present procedure makes use of a nitrogen-sensitive detector which is more stable and selective than the commonly employed electron-capture procedure. The sensitivity of the detector for clonazepam is 1 ng/ml.     

Determination of prostaglandins and thromboxane as their pentafluorobenzyl-trimethylsilyl derivatives by electron-capture gas chromatography

The optimization of the parameters affecting the chromatographic properties and separation of prostaglandin pentafluorobenzyl derivatives by gas chromatography using electron-capture detection is described. The effects of composition and flow-rate of carrier gas, temperatures of detector and column, and nature of stationary phases on the detector response to different pentafluoroebenzyl (both oxime and ester) trimethylsilyl ether derivatives of prostaglandins were systematically examined. The stability of some selected prostaglandin derivatives at ?20°C was also determined. After standardizing these parameters, prostaglandins and related compounds from biological samples, e.g. semen, rat aorta, dog serum and trout gill were successfully analyzed. Identification of prostaglandins was confirmed by gas chromatography—mass spectrometry.     

Rapid determination of artemisinin and related analogues using high-performance liquid chromatography and an evaporative light scattering detector

Artemisinin and its analogues are a class of compounds of current interest in the treatment of drug-resistant malaria. These antimalarials are preferentially taken up into malaria infected erythrocytes as compared to uninfected erythrocytes, a fact that may represent an important parameter in drug potency. Numerous methods for the analysis of specific artemisinin analogues have been developed, but most are not widely adaptable to a large range of analogues. In this paper we describe a high-performance liquid chromatographic method developed and validated for artemisinin and several analogues of artemisinin using a readily available evaporative light scattering detector. This quantitation method was found to be straight forward, rapid, inexpensive and reproducible. Standard calibration curves constructed for six artemisinin compounds were linear with the detection limit determined between 6 and 60 ng. The intra- and inter-day accuracy were found to be 2.75% and 4.15%, respectively with less than 3% variation in precision. The validated assay was applied to a mixture of artemisinin derivatives, where they were easily separated and quantitated.     

Simultaneous Measurement of Fluoroquinolones in Eggs by a Combination of Supercritical Fluid Extraction and High Pressure Liquid Chromatography

Simultaneous detection of the fluoroquinolone antibiotics ciprofloxacin, enrofloxacin, ofloxacin, and norfloxacin in eggs by a combination of supercritical fluid extraction (SFE) and high pressure liquid chromatography (HPLC) was studied. Lipid matrices that have been considered to result in poor extraction and isolation of fluoroquinolones in eggs were removed first by SFE with supercritical CO₂ alone, and then the fluoroquinolones were extracted by SFE with supercritical CO₂ containing 20% (v/v) methanol for HPLC analysis. A time-course study of the extraction of lipid matrices of eggs suggested that the SFE method successfully removed the matrices within 20 min. When the fluoroquinolones added to control eggs were extracted by SFE, the extraction efficiency was similar to that by the solvent extraction method, giving the recovery percentages from 83 to 96% in a 40 min-extraction time. The fluoroquinolones extracted from eggs by SFE were analyzed simultaneously by HPLC equipped with a fluorescence detector with detection sensitivity at about 10 ppb for the detection limit. The standard calibration profiles of fluoroquinolones showed linear responses to HPLC, showing more than 0.995 for the mean r ² value. This is the first report of the simultaneous measurement of fluoroquinolones in eggs by a combination of SFE and HPLC. Using the SFE method allowed us to avoid extensive sample preparation such as solvent extraction and chromatographic cleanup that are basically required in extraction of fluoroquinolones.     

Quantitative analysis of trifluoroacetic acid in body fluids of patients treated with halothane

A simple procedure for the quantitative analysis of trifluoroscetic acid (TFA) in urine and serum from patients narcotized with halothane is described. This involves addition of sodium hydroxide to the body fluid, evaporation of the aqueous phase and esterification of TFA in concentrated sulphuric acid with 2,2,2-trichloroethanol. The gaseous phases above the reaction mixture were then analyzed by gas chromatography with a nickel-63 electron-capture detector. The detection limit was 1 μg of TFA per mililitre of body fluid (200 μg of body fluid are analysed) and the relative standard deviation was ±6%. Patients treated with ethrane, another commercial ansesthetic, did not produce any detectable TFA.     

Solid-phase extraction of isosorbide dinitrate and two of its metabolites from plasma for gas chromatographic analysis

A rapid, accurate and selective method for the determination of isosorbide dinitrate and its 2- and 5-isosorbide mononitrate metabolites in 1.0 ml of human plasma has been developed. Before chromatographic quantitation by gas-liquid chromatography with electron-capture detection, the compounds are subjected to solid-phase extraction, using ENVI 18 cartridges (Supelco). The intra-day and inter-day coefficients of variation are less than 10%, except the inter-day coefficient of variation for the assay of 5-isosorbide dinitrate which is less than 15%. Limits of quantitation are 10, 10 and 20 ng/ml for isosorbide dinitrate, 2-isosorbide mononitrate and 5-isosorbide mononitrate, respectively. Recoveries are in excess of 90% for isosorbide dinitrate and 70% for its two metabolites.     

The analysis of arildone in plasma,urine and feces by gas—liquid chromatography with electron-capture detection

The analysis of arildone in plasma, urine and feces by gas—liquid chromatography with electron-capture detection is described. O-(2,3,4,5,6-Pentafluorohenzyl)hydroxylamine is the derivatizing agent for the plasma and urine analysis; 3-nitrophenylhydrazine is utilized for fecal analysis. The mean (± S.E.) minimum quantifiable level of arildone was 1.4 (± 0.2) ng/ml in urine, 6.4 (± 0.1) ng/ml in plasma, and 12.6 (± 1.0) ng/g in feces. The chromatographic response was linear in the range of 0 and 10–120 ng/ml for plasma, 0 and 2.5–20 ng/ml for urine and 0 and 25–250 ng/g for feces. The estimated overall precision of the assay was 5.5%, 6.4% and 8.9% in urine, plasma and feces, respectively.     

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.     

High-performance liquid chromatographic assay for laudanosine in biological fluids and tissue for neurotoxic studies in rats

A procedure for the determination of laudanosine, the central nervous system active metabolite of the neuromuscular blocking drug atracurium, in serum, cerebrospinal fluid and brain is described. The method uses a readily available internal standard, ethavrine, and a single-step protein precipitation with acetonitrile followed by high-performance liquid chromatographic separation with ultraviolet detection. Norlaudanosine, the major metabolite of laudanosine, can also be quantified. Linearity of detector response was obtained between 1 and 25 μg/ml or μg/g and the method is suitable for determining neurotoxic concentrations of laudanosine in experimental animals.     

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 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.