Gas-liquid Chromatographic Procedure Research Articles

Routine detection and identification in urine of stimulants, analgesics, antihistamines, local anaesthetics and other drugs, some of which may be used to modify performance in sport.

Abstract MANY sporting authorities throughout the world proscribe, in addition to the stimulants, other classes of drugs, e.g. the narcotics and phenothiazine tranquillizers. Present gas-liquid chromatographic procedures for the routine detection in urine of all these classes of drugs suffer from the disadvantage of using a variety of operating conditions (see for example Kazyak & Knoblock, 1963, who listed retention time data for 59 compounds using a single SE-30 column and temperatures from 115–250°, and also Street, 1967). The work of Beckett, Tucker & Moffat (1967) on stimulants is therefore now extended, using two isothermal GLC systems, to obtain retention data for 74 additional compounds. This allows the analysis of 116 compounds to be made using only three isothermal GLC systems. Drugs, other than those which may be used to modify performance in sport, are included since sportsmen may use them for medical reasons, e.g. phenacetin in analgesic preparations or a local anaesthetic for injury, and these may interfere with the dope control test.

Measurement of serum lamotrigine by high performance liquid chromatography using a phenyltriazine as internal standard

Lamotrigine, an anti-epileptic drug with a phenyltriazine molecular structure, is commonly measured for therapeutic drug monitoring purposes by high performance liquid chromatography (HPLC) or gas liquid chromatography (GLC). A convenient internal standard is the structurally related phenyltriazine compound BWA725C previously obtainable from the Wellcome Foundation, UK. Irsogladine is also structurally similar to lamotrigine and was therefore tested as a possible replacement for BWA725C. A GLC procedure with thermionic detection (NPD) has been utilized routinely for lamotrigine in our drug monitoring facility. Irsogladine was unsuitable, however, because the retention times of irsogladine and a co-prescribed drug, carbamazepine, were very similar. An HPLC method utilizing a Prodigy Phenomenex ODS3 column performed well using either of the internal standards. The pH of the mobile phase had a distinct impact on the spectra of lamotrigine and BWA725C. A mobile phase at pH 3, with detection at 225 nm was required to effectively resolve lamotrigine from sulthiame and irsogladine from phenobarbitone. Comparison of the HPLC and the existing GLC method with routine patient specimens (n = 43) gave an equation, y = 0.9382x + 0.8238, R(2) = 0.9862. Irsogladine was found to be a suitable internal standard for an HPLC analysis of lamotrigine.

Recent Advances in Chromatographic Methods to Detect Drugs of Abuse in Alternative Biological Matrices

In recent years, many studies have been developed with the aim of improving drug detection in both conventional specimens and alternative biological matrices. A large number of drug abuse studies, forensic toxicology analyses, drugs in the workplace and even in doping control in sports activities related to drug detection in biological samples have been reported in the literature. The interest in the development and optimization of analytical techniques to detect drugs of abuse in different specimens is explained by the several possibilities and information that they can provide. Conventional samples such as urine and blood and more recently, saliva and sweat, are of fundamental importance whenever recent exposure to drugs is under investigation. Human keratinized tissues such as hair and nails are especially important for obtaining data of chronic long-term exposure with the great advantage of being collected in a non-invasive way. Meconium can be a useful biological sample to evaluate fetal drug exposure following maternal drug use. This paper reviews chromatographic procedures for determination of amphetamines, cannabinoids, opiates, nicotine, cocaine and alcohol in alternative biological matrices. Gas chromatographic and liquid chromatographic procedures with different detectors (including mass spectrometry) and sample preparation techniques such as liquid-liquid extraction (LLE), solid phase extraction (SPE) and solid phase microextraction (SPME) were considered. Keywords: Alternative biological matrices, Hair, Sweat, Nails, Saliva, Meconium, Chromatographic Techniques, Sample preparation for drug analysis

A phytosterol database: Fatty foods consumed in Sweden and the Netherlands

The aim of this study was to quantify seven specific dietary phytosterols (PS) (campesterol, β-sitosterol, stigmasterol, brassicasterol, 5-avenasterol, β-sitostanol and campestanol) in fatty foods. Gas–liquid chromatography procedure was applied to 87 items of Swedish and Dutch origin. In general, β-sitosterol was the dominating phytosterol (60% represented the median total concentration), followed by campesterol (24%), stigmasterol (7%) and 5-avenasterol (5%), brassicasterol (3%), β-sitostanol (1%) and campestanol (<0.1%). The median total content of PS for the 42 fat spreads and margarines was 204 mg/100 g with minimum 73 mg/100 g in a low-fat margarine and maximum 775 mg/100 g in a polyunsaturated fat spread. Among 19 plant oils, median total PS content was 348 mg/100 g, with minimum 39 mg/100 g in palm oil and maximum 978/100 g in corn oil. Median value for 12 nuts and seeds was 144 mg/100 g, with minimum 68 mg/100 g in coconut rasps and maximum 404 mg/100 g in sesame seeds. Median value for total PS was 100 mg/100 g among 14 miscellaneous fatty foods with minimum 11 mg/100 g in Dutch non-dairy ice cream and maximum 484 mg/100 g in mayonnaise. The results are discussed against a background of methodological aspects with the chosen analytical procedure and the importance of setting up valid PS databases for epidemiological studies.

Development of a Gas−Liquid Chromatographic Method for the Analysis of Fatty Acid Tryptamides in Cocoa Products

The determination of the occurrence and level of cocoa shells in cocoa products and chocolate is an important analytical issue. The recent European Union directive on cocoa and chocolate products (2000/36/EC) has not retained the former limit of a maximum amount of 5% of cocoa shells in cocoa nibs (based on fat-free dry matter), previously authorized for the elaboration of cocoa products such as cocoa mass. In the present study, we report a reliable gas-liquid chromatography procedure suitable for the determination of the occurrence of cocoa shells in cocoa products by detection of fatty acid tryptamides (FATs). The precision of the method was evaluated by analyzing nine different samples (cocoa liquors with different ranges of shells) six times (replicate repeatability). The variations of the robust coefficient of variation of the repeatability demonstrated that FAT(C22), FAT(C24), and total FATs are good markers for the detection of shells in cocoa products. The trueness of the method was evaluated by determining the FAT content in two spiked matrices (cocoa liquors and cocoa shells) at different levels (from 1 to 50 mg/100 g). A good relation was found between the results obtained and the spiking (recovery varied between 90 and 130%), and the linearity range was established between 1 and 50 mg/100 g in cocoa products. For total FAT contents of cocoa liquor containing 5% shells, the measurement uncertainty allows us to conclude that FAT is equal to 4.01 +/- 0.8 mg/100 g. This validated method is perfectly suitable to determine shell contents in cocoa products using FAT(C22), FAT(C24), and total FATs as markers. The results also confirmed that cocoa shells contain FAT(C24) and FAT(C22) in a constant ratio of nearly 2:1.

Capillary gas chromatography determination of benzaldehyde arising from benzyl alcohol used as preservative in injectable formulations

A simple, precise and accurate capillary gas–liquid chromatographic procedure has been developed to determine benzaldehyde, the toxic oxidation product of the widely used preservative and co-solvent benzyl alcohol, in injectable formulations of the non-steroidal anti-inflammatory drugs, diclofenac and piroxicam, as well as in Vitamin B-complex injection solutions. Following liquid–liquid extraction with chloroform, separation and quantification are achieved on a fused silica capillary column (25 m×0.53 mm i.d.) coated with 0.5 μm film of OV-101. 3-Chlorobenzaldehyde was used as internal standard with flame-ionization as the detection mode. The ability of the system to resolve benzaldehyde peak from interfering components is good. The method displays excellent linearity over the concentration range 0.5–100 μg/ml of benzaldehyde and a precision of better than 2.5% from intra- and inter-day analyses. The quantification limit for benzaldehyde is 0.4 μg/ml. Levels of benzaldehyde in generic diclofenac and piroxicam injection formulations were found to be seven to 15 times higher than in reference formulations, and double in generic Vitamin B-complex injection formulations.

Effects of Dieldrin on Growth and Accumulation in Tissues of &lt;i&gt;Clarias gariepinus&lt;/i&gt;, Under Laboratory Conditions

Mature adult, Clarias gariepinus were obtained at the ABRU hatchery in Sonning (UK), were they had been bred and reared for several years. These were exposed to two concentrations of dieldrin in water (2.4 µg.l-1 and 4.0 µg.l-1) for one month. Samples of liver, brain and muscles weighing 250 mg from both exposed and control fish were taken and residue analysis of dieldrin these tissues was carried out using gas liquid chromatographic analytical procedures. The results indicated significantly (P Key words : Dieldrin, chronic toxicity, Clarias gariepinus. Journal of Aquatic Sciences Vol.18(1) 2003: 35-38

The Phytosterol Content of Some Cereal Foods Commonly Consumed in Sweden and in the Netherlands

The aim of this report was to quantify five specific dietary phytosterols and phytostanols (campesterol, β-sitosterol, stigmasterol, β-sitostanol, and campestanol) in cereal foods and to study the effect of boiling on sterol content. A capillary column gas liquid chromatography procedure was used to analyse 76 cereal food items of Swedish and Dutch origin: 19 various flours, grains and germs, 31 processed cereals, including bran, flakes, cereal grains and pasta, nine breads, and 17 biscuits, cakes, crackers, cookies and sweet breads. The median total phytosterol concentration was 49 (range 4.1–344) mg/100 g edible portion (e.p.). Flours, grains and germs had a median concentration of 52 (17–344) mg/100 g e.p., which was similar to breads, 54 (29–89) mg/100 g e.p., and different types of miscellaneous cakes and cookies, 52 (27–112) mg/100 g e.p. Processed cereal products had slightly lower concentrations with a median value of 39 (4.1–200) mg/100 g e.p. In general, β-sitosterol was the dominant phytosterol (62% of the total concentration), followed by campesterol (21%), while there were only small amounts of stigmasterol (4%), β-sitostanol (4%) and campestanol (2%). Boiling of cereal products only influenced the phytosterol concentration through change of water content.

Optimization of Synthesis of Mono- O -methylglycerol Isomers

Parameters were refined for synthesis of glycerol 1-monomethyl ether by methylation of glycerol with dimethyl sulfate in the presence of alkali; a gas-liquid chromatographic procedure was proposed for identification of structural isomers of mono-O-methylglycerol.

A novel sphingophosphonolipid head group 1-hydroxy-2-aminoethyl phosphonate in Bdellovibrio stolpii.

Members of the bacterial genus Bdellovibrio include strains that are free-living, whereas others are known to invade and parasitize larger Gram-negative bacteria. The bacterium can synthesize several sphingophospholipid compounds including those with phosphoryl bonds as well as phosphonyl bonds. In the present study, the dominant sphingophosphonolipid component was isolated by column chromatography, and the long-chain bases, fatty acids, and polar head groups were identified by thin-layer and gas-liquid chromatographic procedures. The definitive structural identity of the sphingolipid was established by nuclear magnetic resonance and mass spectrometry of hydrolysis products and the intact compound. The compound was identified as N-2'-hydroxypentadecanoyl-2-amino-3,4-dihydroxyheptadecan-1-phosphono-(1-hydroxy-2-aminoethane).

A simple, rapid gas-liquid chromatographic procedure for the determination of valproic acid in serum.

A simple, rapid method for the gas-liquid chromatographic (GLC) determination of valproic acid in serum without prior derivatization on a Nukol wide-bore capillary column is presented. The method was determined to have a lower limit of detection (LOD) of 5 mg/L and a lower limit of quantitation (LOQ) of 10 mg/L; the method was linear up to 6000 mg/L. Within-run precision (expressed as percent coefficient of variation [CV]) for control specimens containing 60 mg/L and 120 mg/L was 4.3% CV (n = 10) and 3.0% CV (n = 10), respectively. The between-run precision of control sera analyzed over four week yielded 8% at 25 mg/L, 5% at 60 mg/L, and 3% at 120 mg/L. The absolute, uncorrected, analytical recoveries of valproic acid at 10, 32, and 120 mg/L were 97% (n = 5), 101% (n = 5), and 84% (n = 5), respectively. The absolute recovery of the internal standard was 97% (n = 9). Drugs commonly indicated for therapeutic monitoring and other serum constituents were found not to interfere with the procedure. The results of an intermethod comparison study of serum specimens analyzed by TDx immunoassay versus the presented GLC method demonstrated good correlation. At 95% confidence limits, no statistically significant differences were observed between results (p < 0.05). The method is particularly advantageous to toxicology laboratories because chromatography is performed on a polar GLC column that can be modified readily for the routine clinical analysis of other polar compounds such as ethylene glycol and other glycols.

Headspace Gas–Liquid Chromatographic Determination of Dithiocarbamate Residues in Fruits and Vegetables with Confirmation by Conversion to Ethylenethiourea

Ethylene-bis-dithiocarbamate (EBDC) residues were determined as carbon disulfide (CS2) by an improved headspace gas-liquid chromatographic (GLC) procedure. Among 837 samples of 30 agricultural commodities tested, 43% contained residues above the detection limit for the method: 100% of broccoli samples; 80% of cabbage, kiwifruit, and grape samples; and 71% of cucumber samples. Most of the residues in kiwifruit were on or near the skin. Elimination of false-positive detections by the improved method was confirmed with kiwifruit in 2 independent procedures. Whole fruits were washed with 5% EDTA to remove surface residues of EBDC. Analysis of washes for CS2 by the headspace procedure after treatment with acidic stannous chloride and for ethylene thiourea by LC after prolonged treatment at 60 degrees C qualitatively identified the EDTA-soluble residues as EBDC. Although the improved method does not produce false-positive results, peak area responses of CS2 and the internal standard, thiophene, are influenced by chemical composition of the matrix. With matrixes high in sugar or lipids, the apparent CS2 content may be overestimated by 4-fold. Accurate determination of EBDC residues in these sample types requires appropriate adjustments to matrixes.

Chromatographic procedures for the determination of felbamate in serum.

A gas-liquid chromatographic procedure and a high-performance liquid chromatographic (HPLC) procedure for the quantitative determination of the anticonvulsant drug felbamate in serum are presented. Both methods employ methyl felbamate as the internal standard. The efficiencies of felbamate extraction by both C18 solid-phase extraction (SPE) followed by HPLC and liquid-liquid extraction followed by gas chromatography (GC) were evaluated. The mean absolute recovery of felbamate over a serum concentration range of 10-100 mg/L for SPE and liquid-liquid extraction were 89.4 and 84.4%, respectively. Calibration curves for the GC and HPLC procedures were linear from 5 to 200 mg/L felbamate. The overall within-run precision of liquid-liquid extraction followed by GC analysis yielded coefficients of variation Os) of 7.3% at 16 mg/L (n = 10) and 5.8% at 100 mg/L (n = 10). The overall between-run precision for this method calculated by three determinations on a single day for three successive weeks yielded CVs of 7.8% at 16 mg/L (n = 45) and 6.3% at 100 mg/L (n = IO). The overall within-run precision of SPE followed by HPLC analysis yielded a CV of 2.9 % at 50 mg/L (n = 10), and the overall between-run precision for this method calculated by three determinations on a single day for three successive weeks yielded CVs of 3.3% at 25 mg/L (n = 40) and 3.5% at 50 mg/L (n = 37). The SPE-HPLC procedure was found to yield less variable results than the liquid-liquid-GC procedure. Limits of quantitation and detection of both methods were set at 5 mg/L felbamate. Popular anticonvulsant and acid or neutral extractable drugs did not interfere with either the GC or HPLC methods. The results of an interlaboratory, intermethod comparison study performed on patient serum specimens analyzed in one laboratory by the SPE-HPLC procedure and in another laboratory by the liquid-liquid extraction-GC procedure correlated well (r2 = 0.967; n = 41). Both methods provide clinically useful and comparable results.

Determination of atropine and obidoxime in automatic injection devices used as antidotes against nerve agent intoxication

A capillary gas-liquid chromatographic (GLC) and an ion-pair high performance liquid chromatographic (HPLC) method were developed for the assay of atropine sulphate and obidoxime chloride from a parenteral solution in commercial automatic injection devices. The injectors are aimed for the emergency treatment of poisoning by nerve agents. The two-step GLC method consists of extraction of atropine as a free base prior to GLC analysis using scopolamine as an internal standard. Obidoxime is determined directly in a diluted sample solution by reversed-phase HPLC using sodium 1-heptanesulphonate as a counter ion in the mobile phase. The relative standard deviation (R.S.D.) was 1.81% for the GLC procedure with injectors containing only atropine and 2.37% for the GLC of atropine in atropine-obidoxime injectors. The R.S.D. for the HPLC procedure of obidoxime in atropine-obidoxime injectors was 0.82%. The corresponding R.S.D.s for the sampling of atropine-obidoxime injectors were 0.36% and 0.27%. The coefficient of determination ( r 2) was 1.000 for both methods. The recoveries at the target concentration averaged 101.0% and 98.7% with a standard error of the mean of 0.30 for both methods. The retention times for atropine and obidoxime were 6.27 and 4.29 min, respectively.

Determination of SCH 39304 by megabore capillary gas-liquid chromatography.

A gas-liquid chromatographic procedure for determination of SCH 39304 at low nanogram concentrations in serum, cerebrospinal fluid, and urine is presented. The methodology combines a high selectivity and sensitivity nitrogen-specific detector, a gas chromatograph equipped with a capillary "megabore" column, and an internal standard that is very similar in chemical structure to the drug being assayed. This method is suitable for both pharmacokinetic studies as well as for monitoring drug levels in patients receiving SCH 39304 for antifungal treatment.

A rapid GLC method for the direct analysis of plasma medium chain fatty acids

A new, rapid, gas-liquid-chromatographic (GLC) method for the direct determination of plasma medium-chain fatty acids (MCFA) (from C8 to C12) which does not require derivatization procedures is described. Analysis of underivatized MCFA by GLC showed a detection limit ranging from 0.50 to 0.217 μg depending on chain length: the longer the chain, the higher the detection limit. Compared with the HPLC and GLC methods currently described in the literature, this GLC procedure appears to be easy, adequately sensitive, and well reproducible for those MCFA, like n-octanoic acids, present in plasma after p.o. or i.v. MCT administration and it requires a short time (about 30 min) for complete processing.

Gas-Liquid Chromatographic Determination of Almitrine in Rat Intestinal Fluid Using Flame Ionization Detection

Abstract A simple gas liquid chromatographic procedure using flame ionization detection is described for the determination of almitrine bismesylate in intestinal fluid in the presence of 2-allylamino, 4-amino, 6-(4′, 4″-difluoro 1 benzhydryl 4-piperazinyl (s-triazine bismesylate as internal standard (IST). A linear relationship between peak area ratio (almitrine bismesylate/IST) and almitrine bismesylate concentration is found in the range of 4–400 μg/ml. The least squares regression line is Y = 3.2X + 0.01 with a correlation coefficient of 0.9994. The percent coefficient of variation of the assay within days is estimated at 1.3% for 500 μg/ml and 2.0% at 50 μg/ml.

Analytical methods for identifying and quantitating deamidated sialic acid (2-keto-3-deoxy- d-glycero- d-galactonononic acid) and α2 → 8-linked poly(oligo)nonulosonate residues in glycoconjugates

In 1986 we reported the natural occurrence of deaminated neuraminic acid (2-keto-3-deoxy- d-glycero- d-galactonononic acid, KDN) in fish egg glycoprotein. Subsequently, we have shown that many types of sialic acid as well as KDN occur in polymeric chains, poly(oligo)-Sia and poly(oligo)KDN in nature. In this study we demonstrate that the conventional colorimetric and gas-liquid chromatographic methods used in the analysis of sialic acid can be applied to analysis of these new nonulosonate and poly(oligo)nonulosonates. We report that the thiobarbituric acid reaction can be used to analyze both free and bound KDN, but gives lower extinction values when applied to poly(oligo)KDN without prior hydrolysis. Further, the published hydrolytic and/or methanolytic procedures are suitable to release the terminal sialic acid residues, but are not appropriate for quantitative release of the nonulosonic acids from poly(oligo)nonulosonates. A new gas-liquid chromatographic procedure for the identification-quantitation of nonulosonates in poly(oligo)meric forms is described.

Assay of glyceryl trinitrate, isosorbide dinitrate, and their metabolites in plasma by large‐bore capillary column gas‐liquid chromatography

Two large-bore capillary columns, one with dimethyl polysiloxane (HP-1) as the stationary phase and the other with phenyl (50 per cent) methyl (50 per cent) polysiloxane (DB-17), were used to develop gas-liquid chromatographic (GLC) assays for measuring isosorbide dinitrate (ISDN), glyceryl trinitrate (GTN), and their metabolites. ISDN, isosorbide-2-mononitrate (2-ISMN), and isosorbide-5-mononitrate (5-ISMN) in plasma, ranging in concentration from 1 to 300 nM, and GTN, glyceryl-1,2-dinitrate (1,2-GDN), and glyceryl-1,3-dinitrate (1,3-GDN), ranging in concentration from 3 to 60 nM in plasma, were analysed on both columns. GLC analysis yielded baseline resolution of the analytes. The method using the dimethyl polysiloxane column gave a lower limit of detectability for GTN of 0.75 nM (signal/noise (s/n) = 2), and the procedure using the phenyl-methyl column provided a lower limit of detectability for ISDN of 81 pM (s/n = 2). The large-bore column GLC procedures exhibited shorter retention times for both ISDN and GTN than those previously reported for capillary-column assays. The chromatographic resolution of analytes and column efficiency of the large-bore capillary columns were comparable to the results previously found using capillary-column GC. The assays for ISDN and GTN have been shown to be appropriate for pharmacokinetic studies in volunteers and patients. We determined that the HP-1 column is appropriate for the analysis of GTN and metabolites, and the DB-17 column is suitable for analysis of ISDN and its metabolites. We conclude that the use of large-bore capillary columns provides rapid and reliable GLC assays for organic nitrates.

Gas chromatographic method for the assay of aliphatic and aromatic sulphonates as their tert.-butyldimethylsilyl derivatives

A gas—liquid chromatographic procedure is described which permicts analysis of aliphatic and aromatic sulphonates as their tert.-butyldimethylsilyl dervatives. The tert.-butyldimethylsilylation of sulphonic acids in their free or sodium salt form is accomplished in a single derivatization step with N-methyl-N-( tert.-butyldimethylsilyl) trifluoroacetamide and tert.-butyldimethylchlorosilane in acetonitrile. Stablity results and mass spectral analysis of all tert.-butyldimethylsilyl sulphonates are presented. Each derivative displays a prominent and characteristic [M—57] fragment ion in its mass spectrum.