Rapid and sensitive determination of nalmefene in human plasma by gas chromatography-mass spectrometry

A rapid gas chromatography-mass spectrometric method for the determination of nalmefene in human plasma is described. The procedure involves protein precipitation, extraction with ethanol-chloroform mixture and derivatization with pentafluropropionic anhydride. The deuterated analog of nalmefene, 6beta-naltrexol-d(7), was used as the internal standard. Quantitation was achieved on a HP-1 column (12 mx0.2 mm I.D.) with negative chemical ionization (NCI) using methane:ammonia (95:5) as the reagent gas. The standard curves were fitted using a quadratic equation with the curve encompassing a range of 0.5 to 200 ng/ml, and the intra- and inter-assay variations for three different nalmefene levels were less than 10% throughout. The limit of quantitation was found to be 0.5 ng/ml. The method described is highly specific and reproducible, and could also be applied for the determination of naltrexone and 6beta-naltrexol. Application of the method to actual human plasma samples is demonstrated.     

Rapid and sensitive determination of sertraline in human plasma using gas chromatography-mass spectrometry

A method for the determination of sertraline in human plasma using gas chromatography-mass spectrometry (GC-MS), with the selected ion-monitoring (SIM) mode, was described. The following was used in this study: (1) single liquid-liquid extraction at alkaline pH after deproteinization of plasma protein and (2) perfluoroacylation with HFBA, which has higher sensitivity (about 10-fold) compared with previous reported derivatization. The detection limit for the SIM of sertraline as an N-HFB derivative was 0.1 ng/ml, and its recovery was 80-85%. The linear response was obtained in the range of 0.2-10.0 ng/ml with a correlation coefficient of 0.999. The coefficient of variation (C.V.%) was less than 12.1% in the 1-30 ng/ml, and less than 18.2% at 0.2 ng/ml, and the accuracy was less than 10% at all of the concentration range. These findings indicate that this assay method has adequate precision and accuracy to determine the amount of sertraline in human plasma. After pharmacokinetics was performed with this assay method following oral administration of sertraline hydrochloride in man, moment analysis revealed that pharmacokinetic parameters for sertraline (Cmax, 10.3 ng/ml; Tmax, 8.0 h; T(1/2) 28.6 h) were similar to previously reported results. These results indicate that this simple and sensitive assay method is readily applicable to the pharmacokinetic studies of sertraline.     

A sensitive method for 4-hydroxybutyric acid in urine using gas chromatography-mass spectrometry

4-Hydroxybutyric acid (4HB) was analyzed by gas chromatography-mass spectrometry. Under acidified conditions, 4HB is difficult to detect due to lactonization. Using a urine sample containing 0.01 mg creatinine, we performed trimethylsilyl derivatization without extraction, only adding dimethylsuccinic acid as an internal standard and 10 microl of 0.1 N NaOH methanol solution with adequate evaporation. Urine 4HB levels in a patient with 4-hydroxybutyric aciduria was determined to be 1258 mmol/mol Cr (control, 0.28-2.81 mmol/mol Cr) in this method. Direct derivatization of samples without extraction showed good reproducibility and linearity. Only a small sample of urine was required. Alkalinization by NaOH prevented not only lactonization of 4HB, but also loss of the compounds during evaporation.     

Sensitive headspace gas chromatography-mass spectrometry determination of trihalomethanes in urine

A sensitive and straightforward method for the determination of trihalomethanes (THMs) in urine by using headspace extraction technique has been developed. Chemical and instrumental variables were studied in order to optimize the method for sensitivity: an excess of KCl (4 g per 12 ml of urine), an oven temperature of 85 degrees C and an equilibration time of 30 min were selected. The use of the mass spectrometer in selected ion monitoring mode allows achieving linear ranges between 10 and 5000 ng/l and detection limits from 3 to 10 ng/l, for 12 ml of urine. The stability of the urine sample during storage at 4 and -20 degrees C was also evaluated: THMs remained stable for up to 2 days and 2 months, respectively. Finally, the method was successfully applied to study the THM uptake from swimmers of an indoor swimming pool, as well as non-swimmers. This study revealed that the concentrations of THMs in urine increased approximately three times for chloroform and bromodichloromethane after swimming activity. In addition, THMs in unchanged form were mainly excreted within 2-3h after the end of exposure.     

Rapid and sensitive method for prenatal diagnosis of propionic acidemia using stable isotope dilution gas chromatography-mass spectrometry and urease pretreatment

Propionic acidemia is one of the most frequent inborn errors of metabolism caused by a deficiency of propionyl-CoA carboxylase. Methylcitric acid, a key indicator of this disorder, is increased in amniotic fluid when a fetus is affected. Therefore, the direct chemical analysis of cell-free amniotic fluid for methylcitric acid, using stable isotope dilution gas chromatography-mass spectrometry, was carried out for the prenatal diagnosis of propionic acidemia. We developed a simple, highly sensitive, and accurate method for quantitation of this polar methylcitric acid in amniotic fluids by applying a simplified urease pretreatment which we devised earlier for urine. As the recovery of methylcitric acid from amniotic fluid was as high as 91% with a coefficient of variation lower than 3% in this procedure, only 0.02 ml of sample was required for the analysis of the affected fetus. This new procedure takes 1 h for sample pretreatment, including derivatization, and 15 min for GC-MS measurement and provides final results within 1.5 h.     

A sensitive method for the quantification of the mass of inositol phosphates using gas chromatography-mass spectrometry

A sensitive method to directly measure the mass of inositol phosphates from biologic samples is described. The procedure uses ammonium sulfate gradient elution anion exchange column chromatography to isolate inositol monophosphate, bisphosphate, trisphosphate, and tetrakisphosphate. The isolated fractions are dephosphorylated and subsequently desalted by a novel approach using solid barium hydroxide in a 1:1 stoichiometric ratio to the amount of ammonium sulfate present in the dephosphorylated sample. The myo-inositol derived from each inositol phosphate species was quantified by stable isotope dilution gas chromatography-mass spectrometry of the hexakis(trimethylsilyl) derivative using hexadeutero-myo-inositol as the internal standard. The applicability and sensitivity of this method are illustrated by measuring the mass of individual inositol phosphates in isolated adult canine cardiac myocytes.     

Rapid screening procedure based on headspace solid-phase microextraction and gas chromatography-mass spectrometry for the detection of many recreational drugs in hair

An increasing number of synthetic drugs are appearing on the illicit market and on the scene of drug use by youngsters. Official figures are underestimated. In addition, immunochemical tests are blind to many of these drugs and appropriate analytical procedures for routine clinical and epidemiological purposes are lacking. Therefore, the perceived increasing abuse of recreational drugs has not been proved yet. In a previous paper, we proposed a procedure for the preliminary screening of several recreational substances in hair and other biological matrices. Unfortunately, this procedure cannot apply to cocaine. Consequently, we performed a new headspace solid-phase microextraction and gas chromatography-mass spectrometry (HS-SPME-GC-MS) procedure for the simultaneous detection of cocaine, amphetamine (A), methamphetamine (MA), methylen-dioxyamphetamine (MDA), methylen-dioxymethamphetamine (MDMA), methylen-dioxyethamphetamine (MDE), N-methyl-1-(1,3-benzodioxol-5-yl)-2-butanamine (MBDB), ketamine, and methadone in human hair. Hair was washed with water and acetone in an ultrasonic bath. A short acid extraction with 1M hydrochloric acid was needed; the fiber was exposed to a 5 min absorption at 90 degrees C and thermal desorption was performed at 250 degrees C for 3 min. The procedure was simple, rapid, required small quantities of sample and no derivatization. Good linearity was obtained over the 0.1-20.0 ng/mg range for the target compounds. Sensitivity was good enough: limits of detection (LOD) were 0.7 ng/mg of hair for the majority of substances. The intra-day precision ranged between 7 and 20%. This paper deals with the analytical performance of this procedure and its preliminary application to hair samples obtained on a voluntary basis from 183 young people (138 males and 45 females) in the Rome area.     

Rapid and sensitive procedure for the separation and quantitation of - and -serine in rat brain using gas chromatography-mass spectrometry

A very simple and rapid GC-MS procedure for the separation and quantitation of - and -serine has been developed utilizing a conventional bonded-phase capillary column. The procedure involves initial esterification with isobutanol followed by acylation with the chiral derivatizing reagent S-(−)-N-(heptafluorobutyryl)prolyl chloride (HPC). This procedure requires neither extraction nor clean-up steps and is sensitive to 50 pg on-column. Total time of the procedure is under 3 h and derivatives are stable at room temperature for at least 5 days, making this procedure ideal for automated injections. A simple, one-day synthesis of HPC is described which yields >99.9% optical purity.     

Field gas chromatography-mass spectrometry for fast analysis

The objective of this presentation is to demonstrate the original device and procedure for fast gas chromatography-mass spectrometry (GC-MS) analysis of gaseous and liquid samples and to discuss its features and capabilities. The concept was developed in order to expand the range of compounds suitable for GC separation and to reduce the time of analysis. Field GC-MS, consisting of original "concentrator-thermodesorber" (CTD) unit, multiple module GC system and compact magnetic mass spectrometer with powerful two-stage vacuum system and multicollector ion detector, is represented. The whole weight of the device is 90 kg. Power consumption is 250 W. The device and analytical procedures allow high speed screening of toxic substances in air and extracts within 100 s per sample. The examples of applications are described, including fast screening of tributyl phosphate (TBP) in air at low ppt level at the rate 1 sample/min.     

On-line fermenter headspace gas analysis of methanol and ethanol by capillary inlet mass spectrometry

Summary A new capillary inlet system was used with a magnetic sector mass spectrometer to analyse headspace gas from air-sparged aqueous solutions of methanol and ethanol. The system responded to pulse additions within 2 minutes and gave 90% of equilibrium response after 10 minutes. No memory effects or hysteresis were observed. Signal to concentration ratio was linear with alcohol concentrations up to 5 g/L. Liquid ethanol concentration in aerobic yeast fermentation was followed successfully by on-line headspace gas analysis.     

Mercury determination in blood by gas chromatography-mass spectrometry

A stable isotope dilution gas chromatography-mass spectrometry method using¹⁹⁶Hg as an internal standard is described for determining Hg in blood. In this method, the blood samples are not subjected to any digestion to avoid the loss of Hg. A solution of 0.6M HCl is used to free Hg present in blood from proteins. The pH of the solution is adjusted to 9 using borate buffer and Hg chelated using lithiumbis(trifluoroethyl)dithiocarbamate. All isotope ratio measurements are made using an organic mass spectrometer. Overall precision values for the five major Hg isotopes relative to²⁰²Hg are 1.6–2.3% when 10 ng samples of chelated Hg are analyzed. No appreciable memory or carryover effect is observed when two synthetic mixtures differing in¹⁹⁶Hg/²⁰²Hg ratios by a factor of 30 are sequentially analyzed. The method is validated by determining Hg in blood samples using isotope dilution GC-MS.     

Simultaneous detection of amphetamine-like drugs with headspace solid-phase microextraction and gas chromatography-mass spectrometry

A headspace solid-phase microextraction and gas chromatography-mass spectrometry (HS-SPME-GC-MS) procedure for the simultaneous detection of methylen-dioxyamphetamine (MDA), methylen-dioxymethamphetamine (MDMA), methylen-dioxyethamphetamine (MDE) and N-methyl-1-(1,3-benzodioxol-5-yl)-2-butanamine (MBDB) in hair has been developed. This method is suitable for the separation of primary and secondary amines, is reproducible, is not time consuming, requires small quantities of sample and does not require any derivatization. It provides sufficient sensitivity and specificity, with limits of detection (LOD) and limits of quantitation (LOQ) for each substance of <0.7 and 1.90 ng/mg, respectively. Intra- and inter-day precision were within 2 and 10%, respectively. This method is suitable for routine clinical, epidemiological and forensic purposes and can be used for the preliminary screening of many other substances (amphetamine, methamphetamine, ketamine, ephedrine, nicotine, phencyclidine, methadone) in hair and other biological matrices such as saliva, urine and blood. We also describe the first application of this HS-SPME-GC-MS procedure to the analysis of hair and saliva samples from young people attending a disco in the Rome area. All positive hair samples were confirmed by the gas chromatography-mass-mass (GC-MS(2)) technique in positive chemical ionization (PCI) mode. Some examples of the use of the method in detecting different drugs are reported.     

A gas chromatography-mass spectrometry method for the quantitative analysis of melatonin in plasma and cerebrospinal fluid

A method has been developed for the quantitative analysis of melatonin in human plasma and cerebrospinal fluid. The technique involves a simple one-step extraction with chloroform and conversion of the melatonin in the extract to its N-trimethylsilyl derivative, which is then measured by means of high-sensitivity gas chromatography—mass spectrometry. Intra-assay precision for triplicate samples at the 20-pg/ml level was better than 20%, while the interassay precision for separate determinations made over the course of a week was better than 18%. In a series of human plasma samples taken over several times during a 24-hr period, a significant increase in melatonin level was noted during the hours of darkness.     

Determination of metaldehyde in human serum by headspace solid-phase microextraction and gas chromatography-mass spectrometry

A rapid headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) method has been developed for the determination of metaldehyde in human serum samples. Metaldehyde is extensively used as a molluscicide for the control of slugs and snails, and cases of metaldehyde poisoning have been reported. Metaldehyde was headspace-extracted on a polydimethylsiloxane (PDMS) fiber at 70 degrees C for 25 min, desorbed, and analyzed rapidly by GC-MS. The method was validated for limit of detection (LOD), linearity, precision, and recovery. Although the recovery of the sample was very low, the method itself was rapid with a low detection limit of 0.25 microg/ml, R.S.D. value 12.6%, and linearity range 0.5-25.0 microg/ml (r(2)=0.999). The results demonstrated that the SPME-GC-MS method for the analysis of metaldehyde is simple, rapid, solvent-free, and does not require any pre-analysis conversions.     

Gas chromatography-mass spectrometry with headspace for the analysis of volatile organic compounds in waste water

Headspace analysis combined with high-resolution gas chromatography and detection by mass spectrometry was evaluated for the analysis of 53 volatile organic compounds (VOCs) in river waters, waste waters and treated water samples down to 0.1 microgl(-1) concentration levels. The conditions optimised included sample thermostatting time and temperature, autosampler parameters and the nature of salt, added to the sample. The pollutions origin and their seasonal rippling have been done. It was shown that the content of VOCs in river water mainly correlates to the content of these compounds in waste waters, which shows the anthropogenic character of the pollutions.     

Validation of a gas chromatography-mass spectrometry method for the analysis of sterol oxidation products in serum

A validated gas chromatography-mass spectrometry (GC-MS) detection method for the quantitative analysis of sterol oxidation products (SOPs) in serum is described. After a lipid extraction procedure with chloroform-methanol, a cold saponification and purification by solid phase extraction, oxysterols were derivatized to form trimethyl-sylil-ethers which were subjected to GC-MS analysis. Calibration curves for cholesterol oxidation products showed determination coefficient (R(2)) of 1.0, with low values for the coefficient of variation of the response factors (< 1%). Detection and quantification limits were below 5 ng/mL and 10 ng/mL, respectively. Recovery data were between 77.65% and 110.29% (CV < 10% for all compounds). Good results were obtained for within- and between-day repeatability, with values below 10%. In conclusion, the method performed is suitable for the determination and quantification of SOPs in serum.     

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.     

Rapid diagnosis of phenylketonuria and other aminoacidemias by quantitative analysis of amino acids in neonatal blood spots by gas chromatography-mass spectrometry

A reversed-phase HPLC method compatible with evaporative light scattering (ELS) and electrospray mass spectrometric (ES-MS) detection was developed for separation of phosphatidylserine (PS) molecular species. The method was optimised for separation of three disaturated synthetic species: dipalmitoyl glycerophosphoserine, palmitoyl-stearoyl glycerophosphoserine and distearoyl glycerophosphoserine using isocratic elution with a mixture of 2-propanol, tetrahydrofuran and ammonium formate. Baseline separation was obtained on three different columns: one polystyrene/divinylbenzene (PS/DVB) column and two silica based C(18) and C(30) columns. The best chromatographic resolution was achieved with the C(30) column. The limit of detection for DPPS was 5 microg/ml (S/N=3) with ELS detection and 0.1 microg/ml (S/N=3) with negative ion ES-MS in the single ion monitoring mode. Baseline separation of the five main species in a biological PS sample, bovine brain PS, was obtained with the PS/DVB column. Species identification was done by using the retention times of the intact PS species and their corresponding carboxylate anion fragments obtained by in-source fragmentation. Data have shown that individual PS species can be identified by their retention times using direct ELS detection in a mixture of disaturated PS species. However, for the bovine brain PS electrospray-MS detection was necessary for species identification due to the many possible fatty acid combinations in biological PS.     

Simple, rapid and sensitive assay method for simultaneous quantification of urinary nicotine and cotinine using gas chromatography-mass spectrometry

Nicotine is a major addictive compound in cigarette. Its smoke is rapidly and extensively metabolized to several metabolites in human. Cotinine as a major metabolite of nicotine is commonly used as a biomarker to determine active and passive smokers. Cotinine has a longer half-life ( approximately 20 h) compared to nicotine ( approximately 2h). A simple, sensitive, rapid and high throughput GC-MS method was developed for simultaneous quantification of urinary nicotine and cotinine in passive and active smokers. In the sample preparation method, the analytes and internal standard were first basified and followed by liquid-liquid extraction. Upon completion, anhydrous sodium sulphate was added to the solvent mixture to trap moistures. The clear extract obtained was directly injected into GC-MS, operating under selective ion monitoring (SIM) mode. Calibration curves in the range of 0.5-5000 ng/mL of the analytes in urine matrix were established with linear correlation coefficients (r(2)) greater than 0.997. The limit of detection for both nicotine and cotinine were 0.20 ng/mL. The mean recoveries for nicotine and cotinine were 93.0 and 100.4%, respectively. The within- and between-assay accuracies were between 2.1 and 7.9% for nicotine and between 0.7 and 11.1% for cotinine. Within- and between-assay precisions of 3.3-9.5% for nicotine and 3.4-9.8% for cotinine were also achieved. The method can be used in routine assessment and monitoring of active smoking and exposure to environmental tobacco smoke. The applicability of the assay was demonstrated in a small-scale comparison study between smokers and non-smokers.     

Human blood and environmental media screening method for pesticides and polychlorinated biphenyl compounds using liquid extraction and gas chromatography-mass spectrometry analysis

Screening assessment methods have been developed for semi- and non-volatile persistent organic pollutants (POPs) for human blood and solid environmental media. The specific methodology is developed for measuring the presence of "native" compounds, specifically, a variety of organochlorine pesticides (OCPs), organophosphate pesticides (OPPs), and for polychlorinated biphenyls (PCBs). The method is demonstrated on anonymous Red Cross blood samples as well as two potential environmental sources, tracked in soil and dog hair. This work is based on previously developed methods for semi-volatile hydrocarbon exposure from fuels usage and similarly employs liquid solvent extraction, evaporative volume reduction. and subsequent specialized gas chromatography-mass spectrometry analysis (GC-MS). Standard curves, estimates of recovery efficiency, and specific GC-MS SIM quantification methods were developed for common pesticides including diazinon. aldrin, chlorpyrifos, malathion, dieldrin, DDT, permethrin, cyhalothrin, and cypermethrin, and for seven selected PCBs. Trace levels of certain PCBs and pesticides such as permethrin, dieldrin, malathion, lindane, diazinon, and chlorpyrifos were tentatively identified in anonymous blood samples as well as in two potential environmental sources. tracked in soil and dog hair. The method provides a simple screening procedure for various media and a variety of common organic pollutants without extensive sample preparation. It is meant to complement and augment data from more specific or complex methodology, to provide initial broad spectrum guidance for designing targeted experiments, and to provide confirmatory evidence for the usual metabolic biomarker measurements made to assess human exposure.