Gas Chromatographic-mass Spectrometric Procedure Research Articles

From Plant Extracts to Historical Textiles: Characterization of Dyestuffs by GC–MS

A gas chromatographic–mass spectrometric procedure for the analysis of dyes from plant extracts was optimized and applied for the detection of components in extracts from 12 dyeing plants native from all over the world. The main marker molecules in each of the dyestuffs, as well as accessory compounds, were successfully separated and identified by means of their electron impact mass spectra, thus demonstrating that a single GC–MS procedure can be conveniently applied to the detection of natural dyes such as flavonoids, neoflavonoids, anthraquinones and tannins. Other constituents of plant extracts, such as organic acids, oils and sugars, that hydrolyse during the extraction process, were also detected and recognized in the same chromatographic run. The GC–MS method was tested on woollen references dyed according to traditional recipes, and on historical wool samples taken from a tapestry of the sixteenth century, and the main dyestuffs used for colouring could be traced even with the low amounts usually available with the archaeometric samples. Besides some limitations, that are addressed, the results show that the GC analysis is a useful tool for quick assessment and control of natural extracts and the application of the technique for the characterization of dyes in historical textiles, in addition or in place of the more widely used procedures that employ liquid chromatography, is encouraged.

Determination of Tuaminoheptane in Doping Control Urine Samples

Since January 2007, the list of prohibited substances established by the World Anti-Doping Agency includes the sympathomimetic compound tuaminoheptane (1-methyl-hexylamine, 2-heptylamine). Primarily used as nasal decongestant drug it has been considered relevant for sports drug testing due to its stimulating properties. A confirmatory gas chromatographic-mass spectrometric procedure was developed including liquid-liquid extraction and imine formation of tuaminoheptane employing various aldehydes and ketones such as formaldehyde, acetaldehyde, benzaldehyde and acetone. Extraction and derivatisation conditions were optimised for utmost efficiency, and characteristic fragment ions obtained after electron ionisation allowed for a sensitive and selective analytical assay, which was validated with regard to recovery (50%), lower limit of detection (20 ng mL(-1)) as well as interday- and intraday precision (<15%). The applicability to authentic urine samples was demonstrated using administration study specimens obtained from two male persons using Rhinofluimucil (tuaminoheptane hemisulfate) for intranasal application. The administered drug was detected up to 46 h after repeated topical instillation of a total of approximately 3 mg.

Cathinone (Khat) and methcathinone (CAT) in urine specimens: a gas chromatographic-mass spectrometric detection procedure.

A gas chromatographic-mass spectrometric procedure for detection of cathinone (Khat) and methcathinone (CAT) in urine was developed. The compounds were detected as 4-carboethoxyhexafluorobutyryl derivatives. Three ions for the drugs and two ions for the internal standards were monitored. The drugs were identified by comparing retention times and ion ratios with that of reference compounds. The concentrations were measured by using amphetamine-d6 as internal standard for cathinone and methamphetamine-d9 as internal standard for methcathinone, and were linear over the range of 25-5000 ng/mL for cathinone and 12.5-5000 ng/mL for methcathinone. The overall recoveries of cathinone and methcathinone were 86 and 78%, respectively. Intrarun and inter-run variations were < 20%. To verify that the drugs are not metabolites of over-the-counter medications, cathinone and methcathinone were tested in urine specimens collected from individuals who ingested phenylpropanolamine and pseudoephedrine. None of the specimens showed the keto-amines as the metabolic products. When the procedure was applied to test 66 amphetamine-immunoassay-positive specimens containing no amphetamine or methamphetamine, two specimens were found positive for cathinone (118 and 3266 ng/mL) and six specimens were found positive for methcathinone (13-91 ng/mL).

Simple gas chromatographic–mass spectrometric procedure for diagnosing pyrimidine degradation defects for prevention of severe anticancer side effects

Inborn errors of pyrimidine degradation, dihydropyrimidine dehydrogenase deficiency and dihydropyrimidinase deficiency, are less rare than has generally been assumed. Many asymptomatic cases have been reported, and in patients with symptoms, the clinical abnormalities are variable and nonspecific. Withdrawal of pyrimidine analogues such as 5-fluorouracil (5FU), a commonly used anticancer drug, from the cancer chemotherapy regimens of patients with pyrimidine degradation deficiencies, however, is critical because 5FU is degraded in vivo by pyrimidine-degradative enzymes. Patients with these deficiencies suffer from severe neurotoxicity, sometimes leading to death, following administration of 5FU, and even otherwise asymptomatic homozygotes or heterozygotes may develop severe clinical symptoms upon administration of such medication. Therefore, a rapid and specific method for identifying cancer patients with these enzyme deficiencies prior to treatment with 5FU is critical. To address this problem, we established methods for highly sensitive yet specific determinations of thymine, uracil, dihydrothymine, dihydrouracil, orotate and creatinine simultaneously in 0.1-ml liquid urine or filter-paper urine. This method involves stable isotope dilution, a simplified urease treatment previously described and gas chromatography–mass spectrometry without prior fractionation. The high recovery and low C.V. values were obtained and healthy control values were also determined for these metabolites. Using artificially prepared urine specimens simulating these disorders, the chemical diagnosis can be made clearly, and no further analysis appears to be required for differential chemical diagnosis.

Simultaneous GC-MS Analysis of meta- and para-Hydroxybenzoylecgonine and Norbenzoylecgonine: A Secondary Method to Corroborate Cocaine Ingestion Using Nonhydrolytic Metabolites

Positive benzoylecgonine (BZE) urinalysis results are sometimes challenged in legal and administrative proceedings on the grounds that the presence of BZE is due to the addition of cocaine to the urine sample with subsequent in vitro hydrolysis to BZE. Consequently, counsel for the respondent or defendant may move that an ecgonine methyl ester (EME) analysis be preformed because EME is presumed to be solely an in vivo cocaine metabolite. For these reasons, a sensitive and rapid gas chromatographic-mass spectrometric procedure was developed for the simultaneous analysis of m-hydroxybenzoylecgonine (m-OHBZE), p-hydroxybenzoylecgonine (p-OHBZE), and N-desmethyl benzoyl ecgonine (norBZE), all of which are cocaine metabolites believed to arise exclusively via in vivo metabolism. Analysis of human urine specimens previously reported positive for BZE using GC-MS at the Department of Defense cutoff of 100 ng/mL demonstrated that at least one of the three metabolites was present in 79 of the 82 specimens studied (96.3%). Thus, the simultaneous analysis of r-OHBZE, p-OHBZE, and norBZE could be used to substantiate that the presence of BZE in urine specimens is the result of cocaine ingestion. Additionally, the premise that EME is a "true" in vivo cocaine metabolite was investigated by assessing the stability of cocaine in unpreserved urine samples at several pHs ranging from 5.0 to 9.0.

The Hemodynamic and Adrenergic Effects of Perioperative Dexmedetomidine Infusion after Vascular Surgery

We tested dexmedetomidine, an alpha(2) agonist that decreases heart rate, blood pressure, and plasma norepinephrine concentration, for its ability to attenuate stress responses during emergence from anesthesia after major vascular operations. Patients scheduled for vascular surgery received either dexmedetomidine (n = 22) or placebo (n = 19) IV beginning 20 min before the induction of anesthesia and continuing until 48 h after the end of surgery. All patients received standardized anesthesia. Heart rate and arterial blood pressure were kept within predetermined limits by varying anesthetic level and using vasoactive medications. Heart rate, arterial blood pressure, and inhaled anesthetic concentration were monitored continuously; additional measurements included plasma and urine catecholamines. During emergence from anesthesia, heart rate was slower with dexmedetomidine (73 +/- 11 bpm) than placebo (83 +/- 20 bpm) (P = 0.006), and the percentage of time the heart rate was within the predetermined hemodynamic limits was more frequent with dexmedetomidine (P < 0.05). Plasma norepinephrine levels increased only in the placebo group and were significantly lower for the dexmedetomidine group during the immediate postoperative period (P = 0.0002). We conclude that dexmedetomidine attenuates increases in heart rate and plasma norepinephrine concentrations during emergence from anesthesia. The alpha(2) agonist, dexmedetomidine, attenuates increases in heart rate and plasma norepinephrine concentrations during emergence from anesthesia in vascular surgery patients.

Simple and effective gas chromatographic mass spectrometric procedure for the speciation analysis of organotin compounds in specimens of marine mussels. An evaluation of the organotin pollution of the lagoon of venice.

This paper describes a simple, effective analytical procedure, based on a gas chromatographic mass spectrometric technique, for the speciation analysis of organotin compounds (OTC) in mussel samples. The direct alkylation reaction of the organotin chlorides in the aqueous digestion solution by NaBEt(4) allowed a short analysis time and a good recovery. The evaluation of the yield of each step constituting the analytical procedure indicated that the alkylation step is the most critical one. The proposed method was advantageously utilised to monitor the level of OTC pollution of the Lagoon of Venice. All the sites examined, both near to and far from anthropogenic activities, revealed significant levels of OTC pollution.

Gas chromatographic–mass spectrometric procedures for determination of the catechol- O-methyltransferase (COMT) activity and for detection of unstable catecholic metabolites in human and rat liver preparations after COMT catalyzed in statu nascendi derivatization using S-adenosylmethionine

A procedure is presented for determination of the catechol- O-methyltransferase (COMT) activity in liver cytosolic preparations using 3,4-dihydroxyphenethylamine as substrate and by quantifying the product 3-methoxy-4-hydroxyphenethylamine (3-MHP). For quantification of 3-MHP in liver cytosolic preparations a gas chromatographic–mass spectrometric procedure after liquid–liquid extraction and acetylation was established and validated. The intra- and inter-day accuracy and precision were better than 15% and 20%, respectively. Extraction efficiency and selectivity were also sufficient. For in statu nascendi derivatization of unstable catecholic metabolites in liver microsome preparations, cytosolic preparations with COMT activities of at least 1 nmol product/min/mg protein were used after addition of S-adenosylmethionine. Such catecholic metabolites, which are claimed to be responsible for toxic effects in vivo, e.g., neurotoxicity or carcinogenesis, must not be overlooked in in vitro metabolism studies. Using this trick, gas chromatography–mass spectrometry (GC–MS) was suitable for the determination of catecholic metabolites in human and rat liver preparations after the same sample preparation as for 3-MHP quantification. The applicability was exemplified for the antidepressant paroxetine.

Distinction among eight opiate drugs in urine by gas chromatography-mass spectrometry

Opiates are commonly abused substances, and forensic urine drug-testing for them requires gas chromatographic-mass spectrometric (GC-MS) confirmation. There are also medical reasons to test urine for opiates, and confirmation procedures other than GC-MS are often used for medical drug-testing. A thin-layer chromatographic (TLC) method distinguishes morphine, acetylmorphine, hydromorphone, oxymorphone, codeine, dihydrocodeine, hydrocodone, and oxycodone in clinical specimens. In certain clinical circumstances, GC-MS confirmation is requested for opiates identified by TLC, but, to our knowledge, no previous report examines all of the above opiates in a single GC-MS procedure. We find that they can be distinguished by GC-MS analyses of trimethylsilyl (TMS) ether derivatives, and identities of 6-keto opiates can be further confirmed by GC-MS analysis of methoxime (MO)-TMS derivatives. Inclusion of deuterium-labeled internal standards permits identification of the opiates in urine at concentrations below the TLC cutoff level of 600 ng/ml, and the GC-MS assay is linear over a concentration range that spans that level. This GC-MS procedure has proved useful as a third-stage identification step in a medical drug-testing sequence involving prior immunoassay and TLC.

Sensitive gas chromatographic-mass spectrometric method for the determination of gacyclidine in rat plasma and spinal cord dialyzates.

A sensitive gas chromatographic-mass spectrometric (GC-MS) procedure is described for the selective determination of gacyclidine (a non-competitive N-methyl-D-aspartate antagonist) in rat plasma and spinal cord dialyzates. It involves a single-step liquid-liquid extraction of plasma samples and dialyzates with hexane (pH 8.0) and the use of phencyclidine as an internal standard. The compounds were separated on a GC capillary column and specifically detected by MS in the selected-ion monitoring mode. Gacyclidine and its internal standard were monitored by using the fragment ions at m/z 206 and 200, respectively. The method was accurate and reproducible (intra- and inter-day reproducibility < 12%) with a limit of quantification of 1.6 ng ml-1 using 100 microliters plasma of dialyzate samples. The calibration curves for rat plasma and Ringer's solution were linear (r2 > 0.996) over a range from 1.6 to 200 ng ml-1. The extraction efficiency was close to 100%. This simple and rapid assay (total run time < 10 min) was validated for a pilot pharmacokinetic study in healthy rats after intravenous injection of a bolus dose of gacyclidine (2.5 mg kg-1).

A sensitive GC-MS procedure for the analysis of flunitrazepam and its metabolites in urine.

Analysis of urine specimens collected from individuals ingesting 2 and 4 mg flunitrazepam (FN) showed positive results by OnLine and OnTrak immunoassays for up to 60 h. Gas chromatographic-mass spectrometric (GC-MS) analysis of these specimens for FN, 3-OH-FN, 7-amino-FN, 7-amino-3-OH-FN, desmethyl-FN, and 3-OH-desmethyl-FN after glucuronidase treatment showed only low levels of 7-amino-FN with almost none of the other metabolites. These levels were far below the expected results based on the immunoassay data. This study reports on a GC-MS procedure for FN and the previously listed metabolites. The method is based on acid hydrolysis of the urine specimens, which converts FN and all its metabolites described previously to one of four amino-benzophenone derivatives (1-4) with oxazepam-d5 as the internal standard. Under the experimental conditions, the internal standard is converted to 2-amino-5-chloro-benzophenone-d5. The limit of detection for 7-amino-FN and 7-amino-desmethyl-FN and their 3-OH derivatives was less than 1 ng/mL. Analysis of urine specimens collected for 72-h postingestion of 1, 2, or 4 mg FN showed appreciable levels of benzophenone 3 (product of 7-amino-FN and 7-amino-3-OH-FN) and lower levels of benzophenone 4 (product of 7-amino-desmethyl-FN and 7-amino-3-OH-desmethyl-FN) with no detectable levels of benzophenones 1 and 2. The method makes it possible to confirm the presence of FN metabolites in urine at least 72-h postingestion of small doses of the drug.

Gas chromatographic–mass spectrometric quantitation of urinary buprenorphine and norbuprenorphine after derivatization by direct extractive alkylation

A gas chromatographic–mass spectrometric procedure for the quantitation of buprenorphine and norbuprenorphine has been developed in which the analytes were converted, after enzyme hydrolysis, to their methyl derivatives by direct extractive alkylation using tetrahexylammonium hydrogen sulphate phase transfer reagent and iodomethane dissolved in tert.-butylmethyl ether. The procedure utilised a sample volume of 2 ml and gave a detection limit of 0.2 ng ml −1 for buprenorphine and norbuprenorphine. The buprenorphine and norbuprenorphine standard curves were linear in the concentration range of 1–100 ng ml −1 with r=0.999. The coefficients of variation for the intra-run precision were 1.3% for buprenorphine and 8.8% for norbuprenorphine ( n=10). The coefficients of variation for the inter-run precision were 7.7% for buprenorphine and 10.1% for norbuprenorphine ( n=5). The method recovery was 92% (C.V.=3.3%) for buprenorphine and 104% (C.V.=2.9%) for norbuprenorphine ( n=10).

Detection of non-steroidal anti-inflammatory drugs in equine plasma and urine by gas chromatography-mass spectrometry

A gas chromatographic-mass spectrometric (GC-MS) procedure for the detection of seventeen non-steroidal anti-inflammatory drugs (NSAIDs) in equine plasma and urine samples is described. The extraction of the compounds from the biological matrix was performed at acidic pH (2–3) with diethyl ether. Ethereal extracts were washed with a saturated solution of sodium hydrogencarbonate (urine) or treated with a solid mixture of sodium carbonate and sodium hydrogencarbonate (plasma). The ethereal extracts were dried and derivatized by incubation at 60°C with methyl iodide in acetone in the presence of solid potassium carbonate. Mono- or bismethyl derivatives of the NSAIDs were obtained. After derivatization kinetic studies, 90 min was the incubation time finally chosen for screening purposes for adequate methylation of all the compounds under study. For individual confirmation analyses, shorter incubation times can be used. The chromatographic analysis of the derivatives was accomplished by GC-MS with a run time of 13 min. In general, extraction recoveries ranged from 23.3 to 100% in plasma and from 37.5 to 83.8% in urine samples. Detection limits from less than 5 to 25 ng/ml were obtained for both plasma and urine samples using selected-ion monitoring. The procedure was applied to the screening and confirmation of NSAIDs in routine doping control of equine samples.

Purge-and-Trap Gas Chromatographic-Mass Spectrometric Determination of Benzene in Denture Adhesives

A purge-and-trap gas chromatographic-mass spectrometric procedure is presented for determination of trace levels of benzene in denture adhesives and their raw materials. The limit of quantitation is 10 ppb, reproducibility is +/- 10%, and recoveries of d6-benzene are 70-85%. Results of a nationwide survey of 109 finished products and their raw materials representing 32 brand name products from 17 manufacturers are reported.

Gas chromatographic-mass spectrometric determination of chlorophenols in the urine of sawmill workers with past use of chlorophenol-containing anti-stain agents.

The aim of this work was to establish whether workers at sawmills where chlorophenol-containing anti-stain agents had been previously used were still exposed to chlorophenols. A gas chromatographic-mass spectrometric (GC-MS) procedure for the determination of chlorophenols in urine was developed. Chlorophenols were determined from hydrolysed urine as acetylated derivatives and determined by GC-MS using selected ion monitoring (SIM). Isolation of the chlorophenols with toluene gave the best extraction efficiencies (65-119%). Stability tests at -20 degrees C showed no degradation of the chlorophenols studied during 6 months. The limit of quantification was 3.6 ng mg-1 creatinine for 2,4,6-trichlorophenol, 5.5 ng mg-1 for 2,3,4,6-tetrachlorophenol and 3.6 ng mg-1 for pentachlorophenol. Traces of 2,4,6-trichlorophenol, 2,3,4,6-tetrachlorophenol and pentachlorophenol were found both in previously exposed workers and in non-exposed controls, the urinary concentrations ranging from 1.1 to 15.9 ng mg-1 creatinine. The concentrations of urinary chlorophenols in previously exposed workers were of the same magnitude as those found in non-exposed controls and in the general population. The results show that it is unlikely that sawmill workers would be currently occupationally exposed to chlorophenols or polychlorinated dibenzo-p-dioxins and dibenzofurans via a contaminated work environment.

GC-MS determination of phenobarbitone entrapped in poly-ϵ-caprolactone nanocapsules

In order to evaluate the concentration of a hydrophilic drug, phenobarbitone, in a suspension of poly-ϵ-caprolactone nanocapsules, a gas chromatographic-mass spectrometric procedure, performed after methylation of the drug, was developed and validated. Free phenobarbitone (in solution in the liquid phase), released phenobarbitone (after opening the nanocapsules with ethyl acetate) and total entrapped phenobarbitone (after extraction with methylene chloride), were measured. Experimental results for four lots with various concentrations showed that the highest preparation of entrapped drug (80%) was obtained for a total concentration of 3.64 mg ml − in the nanocapsule suspension.

Screening of non-steroidal anti-inflammatory drugs, barbiturates and methyl xanthines in equine urine by gas chromatography-mass spectrometry.

A gas chromatographic-mass spectrometric (GC-MS) screening procedure for 23 acidic drugs in equine urine is described. With the GC-MS method fifteen anti-inflammatory drugs, five barbiturates and three methyl xanthines can be detected with good sensitivity and selectivity. The method consists of alkaline hydrolysis, extraction with organic solvent using salting-out, clean-up extraction, methylation and screening with GC-MS in selected-ion monitoring mode. The limit of detection is 10 micrograms 1(-1) or lower, for most drugs.

Determination of plasma propofol levels using gas chromatography-mass spectrometry with selected-ion monitoring.

Propofol (2,6-diisopropylphenol, I.C.I. 35,868) is a rapid-acting, intravenous anesthetic agent recently introduced for the induction and maintenance of general anesthesia. This paper describes a gas chromatographic-mass spectrometric procedure using selected-ion monitoring for the determination of plasma propofol levels. The drug and the internal standard (thymol) were extracted from plasma into diethyl ether-pentane, and derivatized to their trimethylsilyl derivatives before analysis. The reproducibility of the daily standard curves had coefficients of variation ranging from 2.7% to 10.2%. The precision of the assay yielded a coefficient of variation ranging from 4.5% to 5.6%, and the concentration means for the seeded control samples were found to be within -1.6% to +0.6% of the theoretical values for propofol. No interfering peaks have been observed in application of this procedure to either normal volunteer or patient samples. The minimum detectable level under the conditions described was 0.20 ng propofol/ml plasma. This assay and a high-performance liquid chromatographic assay with fluorescence detection were both used to measure plasma propofol concentrations in 89 human plasma samples, and the correlation between the two methods was excellent.

Gas chromatographic—mass spectrometric quantitation of urinary 11-nor-Δ 9-tetrahydrocannabinol-9-carboxylic acid after derivatization by direct extractive alkylation

A gas chromatographic—mass spectrometric procedure for the quantitation of urinary 11-nor-Δ 9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) has been developed in which the THC-COOH was derivatized to its corresponding methyl ester—methyl ether derivative by direct extractive alkylation using tetrahexylammonium (THA +) counter-ion and iodomethane dissolved in toluene. The procedure utilised a sample volume of 2 ml and gave a detection limit of 0.25 ng/ml. The inter-run and intra-run coefficients of variation were 7.0% and 4.8%, respectively. The inter-day standard curves were linear in the concentration range 0–300 ng/ml with a mean r = 0.9997 ( n = 4).

Enantiospecific gas chromatographic—mass spectrometric procedure for the determination of ketoprofen and ibuprofen in synovial fluid and plasma: application to protein binding studies

A method for the enantiospecific quantitation of two commonly prescribed non-steroidal anti-inflammatory drugs (ketoprofen and ibuprofen) is described. The method involves formation of a mixed anhydride of the drug with ethylchloroformate and subsequent conversion to an amide by reaction with optically active amphetamine. The subsequently formed diastereomers are separated by gas chromatography—mass spectrometry using selected-ion monitoring. The assay is capable of quantifying ketoprofen (2 ng/ml) and ibuprofen (3 ng/ml) enantiomers from a 200-μl sample of synovial fluid or plasma and is particularly suitable for protein binding studies.