Ultra-sensitive method for determination of ethanol in whole blood by headspace capillary gas chromatography with cryogenic oven trapping

We have established an ultra-sensitive method for determination of ethanol in whole blood by headspace capillary gas chromatography (GC) with cryogenic oven trapping. After heating a blood sample containing ethanol and isobutyl alcohol (internal standard, IS) in a 7.0-ml vial at 55°C for 15 min, 5 ml of the headspace vapor was drawn into a glass syringe and injected into a GC port. All vapor was introduced into an Rtx-BAC2 wide-bore capillary column in the splitless mode at −60°C oven temperature to trap entire analytes, and then the oven temperature was programmed up to 240°C for GC measurements with flame ionization detection. The present method gave sharp peaks of ethanol and IS, and low background noise for whole blood samples. The mean partition into the gaseous phase for ethanol and IS was 3.06±0.733 and 8.33±2.19%, respectively. The calibration curves showed linearity in the range 0.02–5.0 μg/ml whole blood. The detection limit was estimated to be 0.01 μg/ml. The coefficients of intra-day and inter-day variation for spiked ethanol were 8.72 and 9.47%, respectively. Because of the extremely high sensitivity, we could measure low levels of endogenous ethanol in whole blood of subjects without drinking. The concentration of endogenous ethanol measured for 10 subjects under uncontrolled conditions varied from 0 to 0.377 μg/ml (mean, 0.180 μg/ml). Data on the diurnal changes of endogenous ethanol in whole blood of five subjects under strict food control are also presented; they are in accordance with the idea that endogenous blood ethanol is of enteric bacterial origin.     

Determination of methanol in whole blood by capillary gas chromatography with direct on-column injection

A gas chromatographic procedure was developed for the determination of methanol in small-volume whole blood samples. Samples (100–200 μl) were prepared by protein precipitation, with direct injection of the supernatant on a wide-bore capillary column. The recovery of methanol and acetonitrile (the internal standard) was approximately 90% and did not vary with sample volume. The assay was linear from 2 μg/ml (the limit of detection) through 1000 μg/ml and was highly reproducible (intra-day coefficient of variation <2.5%). Assay performance was assessed following exposure of rats to methanol. The results indicate that the present procedure is suitable for studies of methanol disposition in small rodent species.     

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.     

Determination of remifentanil in human blood by capillary gas chromatography with nitrogen-selective detection

A validated method for the determination of remifentanil in human blood, applicable to all therapeutic concentrations, using capillary GC with nitrogen-specific detection and fentanyl as the internal standard has been developed. Citrated whole blood samples were extracted into 1-chlorobutane following precipitation of proteins with methanol. The drugs were back extracted into 10 mM HCl and re-extracted into methanol-1-chlorobutane. The extracts were reconstituted in methanol and injected onto a 25-m BPX-5 column. The lower limit of quantitation was 0.2 ng/ml with within- and between-day coefficients of variation of less than 15%.     

Determination of paraldehyde by gas chromatography in whole blood from children

A rapid, sensitive and selective gas chromatographic method with flame ionization detection was developed for the determination of paraldehyde in small blood samples taken from children. Whole blood samples (300 microl) collected in a 3 ml Wheaton glass sample vial were spiked with acetone (internal standard: 15 ng) followed by addition of concentrated hydrochloric acid. The mixture was heated in the sealed airtight sample vial in a water bath (96 Celsius; 5 min) to depolymerize paraldehyde to acetaldehyde. A 2 ml aliquot of the headspace was analyzed by gas chromatography with flame ionization detector using a stainless steel column (3 m x 4 mm i.d.) packed with 10% Carbowax 20 M/ 2% KOH on 80/100 Chromosorb WAW. Calibration curves were linear from 1.0-20 microg (r2>0.99). The limit of detection was 1.5 microg/ml, while relative mean recoveries at 2 and 18 microg were 105.6 +/- 8.4 and 101.2 +/- 5.9%, respectively (n = 10 for each level). Intra- and inter-assay relative standard deviations at 2, 10 and 18 microg were <15%. There was no interference from other drugs concurrently used in children with severe malaria, such as anticonvulsants (diazepam, phenytoin, phenobarbitone), antipyretics/analgesics (paracetamol and salicylate), antibiotics (gentamicin, chloramphenicol, benzyl penicillin) and antimalarials (chloroquine, quinine, proguanil, cycloguanil, pyrimethamine and sulfadoxine). The method was successfully applied for pharmacokinetic studies of paraldehyde in children with convulsions associated with severe malaria.     

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.     

Rapid quantitation of cyanide in whole blood by automated headspace gas chromatography

Cyanide (CN), a chemical asphyxiant, is a rapidly acting and powerful poison. We have developed a sensitive, rapid, simple, and fully automated method for measuring CN in whole blood. The assay is based on the use of gas chromatography (GC) with nitrogen-phosphorus detection and acetonitrile as an internal reference. Following the automated addition of phosphoric acid to the blood sample, the released hydrogen cyanide is analyzed using a fully automated headspace GC system. The assay, validated on human blood samples spiked with potassium cyanide and on clinical samples from fire victims who had smoke inhalation injury, can detect CN at a wide range of concentrations (30-6000 microg/l) in about 17 min (including incubation and GC run time, and <2 min for manual sample preparation). This automated, high-throughput, simple, and sensitive method is suitable for the rapid diagnosis of CN in clinical and forensic specimens.     

Simple determination of cyclosporine in human whole blood by high-performance liquid chromatography

A simple and reproducible high-performance liquid chromatography (HPLC) method was developed for determination of cyclosporine (CyA, also known as cyclosporin A) in human whole blood. The method entailed direct injection of the blood samples after deproteination using acetonitrile. Chromatography was carried out using an ODS column under isocratic elution with acetonitrile-5mM disodium hydrogen phosphate (75:25, v/v), pH 5.1 at 70 degrees C and a detector set at 210 nm. The mean absolute recovery of cyclosporine from blood was 97%, and the linearity was assessed in the range of 100-3000 ng/ml blood, with a correlation coefficient of greater than 0.999. The limit of quantification and detection of the present method were 100 and 50 ng/ml, respectively. This method has been used to analyze several hundred human blood samples for bioavailability studies.     

Sensitive determination of bromazepam in human tissues using capillary gas chromatography-mass spectrometry

A reliable and sensitive gas chromatographic-mass spectrometric method was devised to determine the levels of bromazepam in human tissues. Bromazepam was extracted from body tissues using a three-step solvent extraction procedure. N-Desmethyldiazepam served as the internal standard. Selected ion monitoring withm/z 317 for bromazepam andm/z 270 for internal standard was used for quantitation. Calibration curves in all body tissues were linear over the concentration range from 50–500 ng/g. The lower detection limit in body tissues was 2–5 ng/g and the absolute recovery in body tissues was 27.8–68.0%. This method was used to determine the levels of bromazepam in tissues of an autopsied individual who had been prescribed psychotropic drugs and who was found dead in a car.     

Simultaneous determination of benzene, toluene, ethylbenzene, and xylenes in urine by thermal desorption–gas chromatography

The determination of metabolites of benzene, toluene, ethylbenzene, and xylenes in urine has been used to assess human exposure to these compounds. The analyses of urine samples for these metabolites are tedious and time consuming. The determination of unmetabolized individual compounds in urine has been studied previously with some success. A simultaneous determination of several unmetabolized VOC compounds in urine by thermal desorption–gas chromatography was conducted to assess the exposure of smokers and nonsmokers to these compounds. The method of thermal desorption–GC was sensitive enough to detect a significant difference in exposure levels due to the contribution of light smoking in the environmentally-exposed group.     

Fluorometric determination of total vitamin C in whole blood by high-performance liquid chromatography with pre-column derivatization

A reliable and semi-automated high-performance liquid chromatographic (HPLC) method is described for the determination of total vitamin C in whole blood. After deproteinization of whole blood and enzymatic oxidation of l-ascorbic acid to dehydro-l-ascorbic acid, the latter is condensed with o-phenylenediamine to its quinoxaline derivative. This derivative is separated on a reversed-phase HPLC column and detected fluorometrically. Total vitamin C in whole blood can be determined in concentrations as low as 0.2 μmol/l.Special attention was paid to the stability of vitamin C in whole blood and of its quinoxaline derivative in the extract. Results of our investigations showed that total vitamin C in whole blood is stable for eight days at −20°C, provided ethyleneglycol-bis-(β-amino-ethyl ether)-N,N,N′,N′-tetraacetic acid and glutathione are immediately added to the blood sample. The quinoxaline derivative of vitamin C in the blood extract is stable for at least 24 h if stored in the dark at 4°C.Routine vitamin C determinations can be carried out in a series of 100 samples within 48 h. The within-assay and between-assay coefficients of variation were 3.7% and 4.6%, respectively. The between-assay analytical recovery of l-ascorbic acid added to whole blood samples was 97.0 ± 7.0% (mean ± S.D.). Reference values of vitamin C in whole blood of normal healthy Dutch adults were found in the range 20–80 μmol/l.     

Sensitive and selective method for the determination of chlormezanone in plasma by electron-capture gas chromatography

A sensitive and selective determination method of chlormezanone in plasma has been divised. Chlormezanone in plasma was extracted with toluene at pH 4.5, and converted into p-chlorobenzaldehyde in 0.1 N NaOH. Using p-bromobenzaldehyde as an internal standard, the hydrolysis product and the internal standard were extracted with n-hexane, and the extract was concentrated in vacuo in the presence of isoamyl alcohol to prepare the sample solution. The sample solution was submitted to electron-capture gas chromatography. Chlormezanone was determined by use of the peak height ratio of p-chlorobenzaldehyde against the internal standard. The method was utilized successfully for pharmacokinetic studies of chlormezanone in plasma.     

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.     

Sensitive capillary chromatography mass spectrometric methods for the determination of salcatonin in human biological matrices

New methods employing capillary liquid chromatography in combination with time-of-flight mass spectrometry (microLC-TOF/MS) were developed for the rapid determination of salcatonin in human urine and plasma. The present approaches utilize (13)C(6)-leucine (19)-labeled salcatonin as internal standard, small matrix volumes and simple sample preparation procedures. They allow TOF/MS to be used as a highly selective detector for providing accurate quantitation of salcatonin. Data acquisition was performed in enhanced mode optimizing the signal for the triply charged species of salcatonin and its internal standard. We demonstrate that the determination of salcatonin is straightforward and reliable and can be performed with excellent linearity (R(2)>0.999), precision and accuracy over the concentration ranges of 2.9-290 pmol/mL in human urine, and 7.3-730 pmol/mL in human plasma.     

Simultaneous determination of volatile and non-volatile acidic fermentation products of anaerobes by capillary gas chromatography

A method previously used for the analysis of organic acids in silage has been applied to the detection and quantification of acidic fermentation products (C₁ to C₆ volatile fatty acids, lactic and succinic acid) of rumen bacteria and anaerobic fungi grown in pure culture. The acids were converted to tertiary butyldimethylsilyl derivatives prior to separation on a 30 m DB1 capillary gas chromatographic column. The quantitative recoveries of formic and succinic acids were found to be comparable to the recoveries of other acids reported in the original study. The quantitative recovery of lactic acid was found to be dependent on storage of the samples at ambient temperature for at least 24 h following derivatization. The simultaneous determination of a wide range of volatile and non-volatile acidic products is an important feature of this method.     

Solvent-modified solid-phase microextraction for the determination of diazepam in human plasma samples by capillary gas chromatography

This paper describes microextraction and gas chromatographic analysis of diazepam from human plasma. The method was based on immobilisation of 1.5 μl of 1-octanol on a polyacrylate-coated fiber designed for solid-phase microextraction. The solvent-modified fibre was used to extract diazepam from the samples. The plasma sample was pre-treated to release diazepam from the protein binding. The fibre was inserted into the modified plasma sample, adjusted to pH 5.5, an internal standard was added and the mixture was carefully stirred for 4 min. The fibre with the immobilised solvent and the enriched analytes was injected into the capillary gas chromatograph. The solvent and the extracted analytes were evaporated at 300°C in the split-splitless injection port of the gas chromatograph, separated on a methylsilicon capillary column and detected with a nitrogen-phosphorus detector. The method was shown to be reproducible with a detection limit of 0.10 nmol/ml in human plasma.     

Determination of chloral hydrate and its metabolites in blood plasma by capillary gas chromatography with electron capture detection

A sensitive, accurate, and reliable method is described for the quantitative determination of chloral hydrate (CH) and its metabolites in blood plasma of mice and rats. Metabolites of CH include trichloroacetic acid (TCA), trichloroethanol (TCE), and trichloroethanol glucuronide (TCE-Glu). This new method uses capillary gas chromatography with electron-capture detection (GC/ECD). Procedures for improving sample stability and quality assurance are also described that were not mentioned in previous literature. Rat or mouse plasma (50 microl) is acidified (or treated enzymatically for TCE-Glu determination) and extracted with peroxide free methyl t-butyl ether. Distilled diazomethane (CH(2)N(2)) is added to derivatize TCA to its methyl ester. Detection limits were estimated at 0.2 microg/ml for CH and TCE, and 0.1 microg/ml for TCA. Detector response to TCA and TCE were shown to be linear in the range of 3.125-200 microg/ml (r> or =0.9996). For CH, the response fits a second-order equation in this same range (r=0.99994)