Analysis of human breath samples with a multi-bed sorption trap and comprehensive two-dimensional gas chromatography (GCxGC)

A multi-bed sorption trap designed to quantitatively collect volatile organic compounds from large-volume vapor samples and inject them into a gas chromatograph is combined with a comprehensive two-dimensional gas chromatograph (GCxGC) for the analysis of organic compounds in human breath samples. The first-column effluent of the GCxGC is modulated by a single-stage, resistively-heated and air-cooled segment of 0.18-mm i.d. stainless steel column using the same stationary phase as the first column. Cooling gas is provided by a two-stage conventional refrigeration system, and thus no consumables other than carrier gas and electric power are required. The sorption trap uses four discreet beds, three containing different grades of graphitized carbon and one containing a carbon molecular sieve. The ordering of the beds in the trap tube is from the weakest to strongest adsorbent during sample collection. Breath samples are collected in gas sampling bags, and samples are passed through the trap at a flow rate of about 50 cm3/min. After sample collection, hydrogen carrier gas flow is initiated in the direction opposite to the sample collection flow, and the metal trap tube is resistively heated to inject a sample plug into the GCxGC. Performance data for the combined GCxGC/sorption-trap instrument is described, and human breath-sample chromatograms are presented.     

The measurement of nanogram amounts of acetylcholine in tissues by pyrolysis gas chromatography

Abstract— A method previously described for measuring ACh in biological effluents has been simplified and extended for use with tissues. The tissue is homogenized in acetonitrile containing propionylcholine as the internal standard and after centrifugation the acetonitrile is removed by shaking with toluene. To the aqueous solution is added a solution of KI-I₂ to precipitate the quaternary compounds. The precipitate is dissolved in aqueous acetonitrile and then drawn through a small column of ion-exchange resin to convert the periodides of the quaternary compounds to chlorides which are then simultaneously pyrolysed and gas chromatographed. On the column the pyrolytic product of choline has a slower retention time than that of acetylcholine; under these circumstances the choline present in tissues does not obscure the measurement of acetylcholine. Specificity was demonstrated by several procedures including mass spectroscopy. The method can measure 25 ng (171 pmoles) of acetylcholine in extracts of brain, simply, and with high reproducibility. With the usual gas chromatograph, 16 samples can be run in a working day. The content of acetylcholine in rat brain was 26.4 nmol/g or almost precisely the values found with other gas chromatographic methods. The pyrolytic method was shown to be applicable to the detection of biologically interesting substances other than choline esters, including betaine, carnitine and the non- quaternary compound, ?-aminobutyric acid, which is readily converted to a volatile compound (probably its methyl ester) when pyrolysed in the presence of tetramethylammonium hydroxide. Of additional general interest is the demonstration of the advantages of acetonitrile as a solvent for extracting water-soluble compounds from tissues.     

Ethanol analysis from biological samples by dual rail robotic autosampler

Detection, identification, and quantitation of ethanol and other low molecular weight volatile compounds in liquid matrices by headspace gas chromatography-flame ionization detection (HS-GC-FID) and headspace gas chromatography-mass spectrometry (HS-GC-MS) are becoming commonly used practices in forensic laboratories. Although it is one of the most frequently utilized procedures, sample preparation is usually done manually. Implementing the use of a dual-rail, programmable autosampler can minimize many of the manual steps in sample preparation. The autosampler is configured so that one rail is used for sample preparation and the other rail is used as a traditional autosampler for sample introduction into the gas chromatograph inlet. The sample preparation rail draws up and sequentially adds a saturated sodium chloride solution and internal standard (0.08%, w/v acetonitrile) to a headspace vial containing a biological sample, a calibrator, or a control. Then, the analytical rail moves the sample to the agitator for incubation, followed by sampling of the headspace for analysis. Using DB-624 capillary columns, the method was validated on a GC-FID and confirmed with a GC-MS. The analytes (ethanol, acetonitrile) and possible interferences (acetaldehyde, methanol, pentane, diethyl ether, acetone, isopropanol, methylene chloride, n-propanol, and isovaleraldehyde) were baseline resolved for both the GC-FID and GC-MS methods. This method demonstrated acceptable linearity from 0 to 1500 mg/dL. The lower limit of quantitation (LOQ) was determined to be 17 mg/dL and the limit of detection was 5 mg/dL.     

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.     

Measurement of volatile organic compounds in exhaled breath as collected in evacuated electropolished canisters

A set of three complementary analytical methods were developed specifically for exhaled breath as collected in evacuated stainless steel canisters using gas chromatographic-mass spectrometric detection. The first is a screening method to quantify the carbon dioxide component (generally at 4–5% concentration), the second method measures the very volatile high-level endogenous compounds [e.g. acetone and isoprene at 500–1000 parts per billion by volume (ppbv), methanol, ethanol, dimethylsulfide at 2–10 ppbv], and the third method is designed to measure trace-level environmental contaminants and other endogenous volatile organic compounds (VOCs) (sub-ppbv) in breath. The canister-based sample format allows all three methods to be applied to each individual sample for complete constituent characterization. Application of these methods is shown to be useful in the following ways: analysis of CO₂ levels indicates the approximate quantity of alveolar breath collected (as opposed to whole breath) in a sample; levels of major endogenous compounds are shown to be influenced by physical activities and subsequent recovery periods; and environmental exposures to xenobiotic VOCs can be characterized by assessment of post-exposure breath elimination curves. The instrumentation and methodology are described and example chromatograms and quantitative data plots demonstrating the utility of the methods are presented.     

Gas-liquid chromatographic analysis of volatile short chain fatty acids in fecal samples as pentafluorobenzyl esters.

A protocol was developed for the analysis of volatile short chain fatty acids in microsamples of feces, short chain fatty acid (SCFA) extraction was from fecal samples using ethanol incorporating n-hexanoic acid as an internal standard. The SCFAs were converted to pentafluorobenzyl esters with alpha-2,3,4,5,6-pentafluorotoluene and analyzed on a gas-liquid chromatograph equipped with an electron capture detector. One hundred milligrams of sample was routinely used but analysis could be carried out on 20 mg of sample.     

Automatic on-line fermentation headspace gas analysis using a computer-controlled gas chromatograph

A computer-controlled headspace gas chromatograph was used to monitor the progress of ethanol production from both aerobic batch and anaerobic continuous fermentations. Using an automatic, electropneumatic sampling system, aliquots of fermentation headspace gas were injected directly onto the column for quantitative ethanol determinations every six minutes. A sample volume of 1 mL permitted liquid ethanol concentrations from 2 to 100 g/L to be measured with better than 3% standard deviation on five repeated injections. Provided fermenter liquid temperature and ionic strength were maintained constant, the signal-tohyphen;concentration ratio remained linear to 80 g/L ethanol. This quantitative gas chromatographic (GC) method is suitable for accurate, precise analysis of multiple solvent fermentations, and is limited only by the elution rate and separating capacity of the GC column.     

Simultaneous determination of oxprenolol and 2H6-labelled oxprenolol in human plasma by gas chromatography/mass spectrometry

A gas chromatographic/mass spectrometric method for the specific determination of oxprenolol and 2H6-labelled oxprenolol when both are present in the same sample is described. After addition of 13C3-labelled oxprenolol as internal standard, plasma is alkalized and extracted by a mixture of dichloromethane and diethyl ether. The residue following evaporation of the organic phase is derivatized with heptafluorobutyric anhydride. Negative ion detection with N2O as reagent gas is used for the measurements at m/z 488, 491 and 494 for oxprenolol, the 13C3-labelled internal standard and 2H6-labelled oxprenolol, respectively. The precision and accuracy of the analytical method were investigated using samples containing both unlabelled and 2H6-labelled oxprenolol. The overall mean recovery (% +/- SD, n = 70) in the concentration range 20-1500 nmol l-1 (around 6-450 ng ml-1 of the hydrochloride salts) was 100.6 +/- 3.3 and 101.0 +/- 3.5 for oxprenolol and 2H6-labelled oxprenolol, respectively. The limit of quantification was around 20 nmol l-1 for both compounds.     

Volatile compounds detected in blood of drunk drivers by headspace/capillary gas chromatography/ion trap mass spectrometry.

As shown by others, ethanol and methanol appear in the breath of normals, and endogenous methanol becomes detectable also in the blood after intake of ethanol. In this study I have investigated whether low-molecular-weight volatile organics, other than methanol, arise in the blood of drunk drivers who had imbibed alcoholic beverages. To this end a method for searching for such compounds in the blood is described. It was based on headspace extraction, gas chromatographic separation on a DB-WAX capillary, and ion trap detection in the mass range 29-99 u. Detection limits, as defined by the analyte concentration that gives a signal equal to three times the standard deviation of the baseline noise, were estimated for the different mass numbers used in the substance search. Given the detection limits, presented as mmoles per litre (numbers within parentheses), in every drunk driver's blood with more than 10 mmol l-1 of ethanol between seven and nine different volatile substances were spotted. These were ethanol (0.15), 2-propanone (0.015), ethyl acetate (0.0005), 2-butanone (0.006), methanol (1.5), 2-propanol (0.06), ethanol (0.7), 2-butanol (0.03), and 1-propanol (0.03).     

Improved method for the analysis of furosemide in plasma by high-performance liquid chromatography

Modifications of existing rapid high-performance liquid chromatographic procedures for the determination of furosemide in plasma were made in order to achieve greater sensitivity. To a small volume of plasma was added an internal standard structurally related to furosemide. Then, following previously described procedures, acetonitrile was added to precipitate the proteins and the clear supernatant was separated. However prior to injection of the supernatant the pH and composition of the sample were adjusted. This modification of the sample enabled an injection volume of up to 300 μl of the supernatant to be injected onto the chromatographic column. The effluent was monitored spectrofluorimetrically. A standard linear calibration curve with a mean precision of ± 4.4% was obtained for plasma samples containing 20–900 ng/ml of furosemide. Two structurally related compounds were used as internal standards in the furosemide assay.     

Determination of pentose sugars in fermentation solutions by a simple gas chromatographic procedure

Summary Pentoses in aqueous acidic solutions were analysed by conversion to furfurals in a gas chromatograph containing a porous polymer packed column at 200°C. Hexoses, volatile fatty acids and fermentation media did not interfere with the measurement of pentoses.     

Determination of 4-(4-chlorophenyl)-4-hydroxypiperidine, a metabolite of haloperidol, by gas chromatography with electron-capture detection

An electron-capture gas chromatographic procedure was developed for the analysis of 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP), a metabolite of haloperidol. The assay involved basic extraction of this metabolite from the biological samples, followed by back-extraction with HCl. After basification of the acid phase, extractive derivatization with pentafluorobenzoyl chloride in toluene was conducted. The pentafluorobenzoyl derivative was quantified on a gas chromatograph equipped with a fused-silica capillary column, an electron-capture detector and a printer-integrator. N-(3-Trifluoromethylphenyl)piperazine was carried through the procedure as an internal standard and calibration curves were determined for each assay run. The procedure was demonstrated to be linear and reproducible and was utilized to detect and quantify CPHP in urine, plasma, brain and liver samples from rats treated with haloperidol. The structure of the derivatized metabolite was confirmed by gas chromatography-mass spectrometry.     

Gas chromatography column as an ambient‐temperature volatile trap

Open‐tube volatile traps have largely been shunned in favor of solid adsorbent containing traps for the collection of volatile pheromones and attractants. Solid adsorbents require large solvent rinses and glass capillaries can be difficult to maneuver for the collection of volatiles from small or hard‐to‐reach odor sources. A gas chromatograph (GC) column (DB‐1), an open‐tube glass capillary, and a SuperQ^®‐containing capillary were compared for their collection efficiencies from rubber septa and live calling insects. All three traps captured similar ratios of test compounds from septa at airflows >10 ml per min. Eluting analytes from a packed adsorbent, SuperQ, required at least 30× more solvent than was required to collect all the pheromone from the open‐tube glass capillaries, and the GC column enjoyed an additional three‐fold reduced solvent volume compared to the glass capillary. Thus, analytes could be eluted from the GC‐column trap and directly analyzed on GC without solvent evaporation. We placed glass wool ‘plugs’ in both GC columns and glass capillaries and found no volatiles in these plugs, indicating that breakthrough did not occur during 1‐h collections at 25 ml per min. We demonstrate here that at ambient laboratory temperatures, a DB‐1 GC column effectively collects Oriental fruit moth sex pheromone volatiles from a rubber septum and live pheromone‐releasing moths. Release ratios of pheromone from rubber septa are consistent with earlier reports from static air systems, whereas the release ratio of the (Z)‐8‐dodecenyl alcohol (Z8‐12:OH) from female Grapholita molesta Busck (Lepidoptera: Tortricidae) differed from published results and is likely due to different collection methods or moth‐strain origin.     

A column chromatographic method for determination of plasma and erythrocyte levels of inosine and hypoxanthine

A column chromatographic separation of inosine and hypoxanthine in plasma and erythrocytic samples after deproteination by ultrafiltration, adsorption of the compounds onto charcoal, elution in pyridine/ethanol solution, and filtration by celite gel is described. Sephadex G-10 was used to separate the compounds with small but different molecular weights. Inosine and hypoxanthine values were determined by enzymatic spectrophotometric assay. In arterial, coronary venous, and erythrocyte samples the mean ± SD values for inosine were calculated as 1365 ± 560 nmol/liter of plasma, 915 ± 310 nmol/liter of plasma, and 8925 ± 6720 nmol/liter of blood, respectively. The corresponding values for hypoxanthine were 2525 ± 1950 nmol/liter, 1835 ± 1315 nmol/liter, and 11090 ± 7600 nmol/liter, respectively.     

Simultaneous determination of amiodarone and its major metabolite desethylamiodarone in plasma, urine and tissues by high-performance liquid chromatography

A simple and sensitive high-performance liquid chromatographic method for the simultaneous assay of amiodarone and desethylarniodarone in plasma, urine and tissues has been developed. The method for plasma samples and tissue samples after homogenizing with 50% ethanol, involves deproteinization with acetonitrile containing the internal standard followed by centrifugation and direct injection of the supernatant into the liquid chromatograph. The method for urine specimens includes extraction with a diisopropyl ether—acetonitrile (95:5, v/v) mixture at pH 7.0 using disposable Clin-Elut 1003 columns, followed by evaporation of the eluate, reconstitution of the residue in methanol—acetonitrile (1:2, v/v) mixture and injection into the chromatograph. Separation was obtained using a Radial-Pak C₁₈ column operating in combination with a radial compression separation unit and a methanol–25% ammonia (99.3:0.7, v/v) mobile phase. A wavelength of 242 nm was used to monitor amiodarone, desethylamiodarone and the internal standard. The influence of the ammonia concentration in the mobile phase on the capacity factors of amiodarone, desethylamiodarone and two other potential metabolites, monoiodoamiodarone (L6355) and desiodoamiodarone (L3937) were investigated. Endogenous substances or a variety of drugs concomitantly used in amiodarone therapy did not interfere with the assay.The limit of sensitivity of the assay was 0.025 μg/ml with a precision of ± 17%. The inter- and intra-day coefficient of variation for replicate analyses of spiked plasma samples was less than 6%. This method has been demonstrated to be suitable for pharmacokinetic and metabolism studies of amiodarone in man.     

Rapid determination of dipicolinic acid in the spores of Clostridium species by gas-liquid chromatography.

A gas-liquid chromatographic procedure has been developed to quantitate dipicolinic acid in bacterial spores. The culture, washed from a plate, was hydrolyzed with acid containing the internal standard, pyridine-2,4-dicarboxylate, and then extracted into methyl isobutyl ketone. The internal standard and dipicolinic acid were then extracted into a small volume of trimethylphenylammonium hydroxide. Injection of the resultant quaternary ammonium salts into a gas chromatograph yielded, via thermal decomposition, the methyl ester derivatives of the dipicolinic acid and the internal standard. The amount of dipicolinic acid in the sample was determined from a standard curve. The method was sensitive to 100 ng of dipicolinic acid per sample and was 1,000 to 5,000 times more sensitive than the commonly used methods. Preparation of the sample required less than 1.5 h and less than 15 min of the analyst's time.     

Determination of clofilium, a new antifibrillatory agent, in plasma by gas chromatography—mass spectrometry

A sensitive and selective method for the assay of the new quaternary amine antifibrillatory agent clofilium is described. Plasma samples were extracted with dichloromethane (98.5 ± 0.2% recovery) and analyzed by gas chromatography—mass spectrometry operating in the electron-impact mode. The method involves a Hofmann elimination of an N-alkyl radical from clofilium and the internal standard in the presence of a strong nucleophile in the injector of the gas chromatograph. The resulting tertiary amines are chromatographed and detected by selective ion monitoring. The ratio of the clofilium base peak (m/z 224) to the internal standard peak (m/z 210) was linear relative to the plasma clofilium concentration over the range of 25–1000 ng/ml plasma.     

Quantitative determination of nefopam in human plasma,saliva and cerebrospinal fluid by gas—liquid chromatography using a nitrogen-selective detector

A sensitive and selective gas—liquid chromatographic method for the determination of nefopam in human plasma, saliva and cerebrospinal fluid has been developed. The method includes the selective extraction of nefopam and the internal standard, orphenadrine, from biological fluids by a double extraction procedure. The extracted nefopam and internal standard are analyzed by a gas chromatograph equipped with a 3% OV-17 glass column and a nitrogen—phosphorus flame ionization detector (NPFID) operated in the nitrogen mode. The detector provides the needed high sensitivity and also selectivity due to the inherent characteristics of NPFID to discriminate against non-nitrogen containing materials. Five nanograms nefopam per ml plasma or saliva are routinely quantitated with a 1-ml sample or as little as 2 ng per ml cerebrospinal fluid with a 3-ml sample. The intra-day reproducibilities, expressed as the relative standard deviation, are 5, 2 and 3% at 10, 35 and 75 ng/ml plasma levels, respectively. The accuracies expressed by relative error at these levels are 12, ?4 and ?2%, respectively. The inter-day reproducibility is demonstrated by the small relative standard deviation, 2%, of the slopes from ten plasma standard curves run on ten different days. In various clinical studies in humans the method has been successfully applied to the study of single-dose pharmacokinetics of nefopam and the monitoring of nefopam concentrations in saliva and cerebrospinal fluids.     

The specific nature of plant cell wall polysaccharides

Polysaccharide compositions of cell walls were assessed by quantitative analyses of the component sugars. Cell walls were hydrolyzed in 2 n trifluoroacetic acid and the liberated sugars reduced to their respective alditols. The alditols were acetylated and the resulting alditol acetates separated by gas chromatography. Quantitative assay of the alditol acetates was accomplished by electronically integrating the detector output of the gas chromatograph. Myo-inositol, introduced into the sample prior to hydrolysis, served as an internal standard.     

Determination of diclofenac in plasma using a fully automated analytical system combining liquid—solid extraction with liquid chromatography

A fully automated analytical system based on liquid—solid extraction combined with column liquid chromatography is described for the determination of diclofenac in plasma. After addition of pH 5 buffer and the internal standard solution to the plasma sample, both sample preparation via a C₁₈ disposable extraction column and injection were performed by a Gilson ASPEC system. Diclofenac and the internal standard were separated on a reversed-phase column, using methanol—pH 7.2 phosphate buffer (56:44, v/v) as mobile phase at a flow-rate of 0.4 ml/min. The reproducibility and accuracy of the method were acceptable over the concentration range 31–3140 nmol/l in plasma.