Gas Chromatography-tandem Mass Spectrometry Analysis Research Articles

An automated sample preparation for detection of 72 doping‐related substances

Automation of sample preparation procedures in a doping control laboratory is of great interest due to the large number of samples that have to be analyzed, especially in large events where a high throughput protocol is required to process samples over 24 h. The automation of such protocols requires specific equipment capable of carrying out the diverse mechanical tasks required for accomplishing these analytical methodologies, which include pipetting, shaking, heating, or crimping. An automated sample preparation procedure for the determination of doping-related substances by gas chromatography-mass spectrometry (GC-MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS) analysis, including enzymatic hydrolysis, liquid-phase extraction and derivatization steps, was developed by using an automated liquid handling system. This paper presents a description of the equipment, together with the validation data for 72 doping-related compounds including extraction efficiency, evaluation of carry-over, interferences, and robustness. Validation was approached as a comparison between the results obtained using the manual protocol and the transferred automated one. The described methodology can be applied for sample preparation in routine anti-doping analysis with high sample throughput and suitable performance.

Gas chromatography–tandem mass spectrometry analysis of 52 monohydroxylated metabolites of polycyclic aromatic hydrocarbons in hairs of rats after controlled exposure

A method based on gas chromatography-tandem mass spectrometry after derivatization with N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide was developed for the analysis of monohydroxylated polycyclic aromatic hydrocarbons (OH-PAHs) in hair. The method focused on 52 target compounds corresponding to two- to six-ring monohydroxylated metabolites of polycyclic aromatic hydrocarbons (PAHs). The limits of quantification ranged from 0.2 to 50 pg mg(-1). The method was then applied to the analysis of hair samples collected from rats exposed to 12 PAHs at 0.01, 0.1, and 1 mg kg(-1), by intraperitoneal injection, for 28 days. The results of this study confirm that these metabolites can be incorporated in hair after intraperitoneal administration of the corresponding parent compound. Only 20 of the 52 metabolites were actually detected in hair samples and corresponded to nine parent PAHs. The mean concentrations of OH-PAHs in rat hair samples exposed to PAHs at 1 mg kg(-1) ranged from 0.6 ± 0.2 pg mg(-1) for 8-hydroxybenzo[b]fluoranthene to 6.7 ± 1.0 pg mg(-1) for 1-hydroxypyrene. The results also demonstrated that hair pigmentation has no influence on the concentration of most OH-PAHs. This animal experiment confirmed the incorporation of PAH metabolites in hair and demonstrated that the method was sufficiently sensitive to detect low levels of exposure to PAHs. These results confirmed the usefulness of hair analysis in the biomonitoring of human exposure to PAHs.

A low-density DNA microchip for the detection of (anti-)estrogenic compounds and their relative potencies

In the current study, a set of 12 reference compounds was tested in a low-density DNA microchip that contains probes for 11 different estrogen-responsive marker genes. Our results show that the seven most informative marker genes on the chip resulted in fingerprints that correctly predicted the (anti-)estrogenic activity of the model compounds except that of the negative control testosterone. Two marker genes, myeloid leukemia factor-1 interacting protein and ubiquitin-conjugating enzyme E2C, were even capable of correctly predicting the estrogenic potency of all five estrogen receptor (ER) agonists tested and correlated well with the potencies as determined in the MCF-7/BOS proliferation assay and the in vivo uterotrophic assay. In addition, it was demonstrated that the estrogenic responses of testosterone, both in the array tube assay and in the proliferation assay, were partially due to the conversion of testosterone into 17β-estradiol by aromatase but also due to formation of other estrogenic metabolites, the presence and estrogenic potency of which were confirmed by gas chromatography–tandem mass spectrometry analysis and a yeast-based reporter gene assay, respectively. It is concluded that low-density DNA microchip-based fingerprinting in MCF-7/BOS cells for estrogenicity marker genes provides a faster in vitro alternative to the current MCF-7/BOS cell proliferation assay (E-screen).

Endocrine-Disrupting Effects of Thioxanthone Photoinitiators

Photoinitiators used in food packaging ink, such as 2-isopropylthioxanthone (2-ITX), have been shown to migrate into food and beverages. Recently, several studies indicated that 2-ITX might be an endocrine-disrupting chemical. In this work, the effects of 2-ITX, 4-isopropylthioxanthone (4-ITX), 2,4-diethylthio xanthone (2,4-diethyl-TX), 2-chlorothioxanthone (2-chloro-TX), and 1-chloro-4-propoxythioxanthone (1-chloro-4-propoxy-TX) on steroidogenesis and androgen and estrogen receptor-mediated transcription activation have been studied using human H295R adrenocarcinoma cells and yeast hormone bioassays, respectively. None of the compounds showed androgenic or estrogenic activities, but clear antiandrogenic and antiestrogenic activities were observed for 2-ITX, 4-ITX, and 2,4-diethyl-TX, whereas 2-chloro-TX showed only antiandrogenic activity. In an adapted version of the H295R steroidogenesis assay, using gas chromatography-tandem mass spectrometry analysis of H295R media, all five compounds increased levels of 17ß-estradiol and estrone. H295R cells incubated with 2-ITX also showed significantly reduced androgen and increased pregnenolone and progesterone levels. Expression of particular steroidogenic genes, including the one encoding for aromatase (CYP19A1), was significantly upregulated after incubation of H295R cells with 2-ITX, 4-ITX, and 2,4-diethyl-TX. In line with the increased CYP19A1 mRNA expression, 2-ITX increased catalytic activity of aromatase in H295R cells as measured by cognate aromatase assays. The results indicate that thioxanthone derivatives can act as potential endocrine disruptors both at the level of nuclear receptor signaling and steroid hormone production.

Mass spectrometric description of novel oxymetholone and desoxymethyltestosterone metabolites identified in human urine and their importance for doping control

The metabolism of two anabolic steroids - oxymetholone and desoxymethyltestosterone - was reinvestigated to identify new targets potentially valuable for the antidoping analysis. Excretion urine samples from the laboratory reference collection were used in this study. Following fractionation of the urinary extract by means of high performance liquid chromatography (HPLC), each fraction was subjected to gas chromatography-mass spectrometry (GC-MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS) analysis after trimethylsilylation. About 20 metabolites were found for desoxymethyltestosterone and more than 40 for oxymetholone, with many of them being isomeric compounds. In addition to the well-known reduced and hydroxylated metabolites, 18-nor-17,17-dimethyl and 18-nor-17-hydroxymethyl-17-methyl steroids were also identified. Having evaluated all the metabolites in terms of how long they could be detected, we suggest that 18-nor-2ξ,17β-hydroxymethyl-17α-methyl-5α-androst-13-en-3α-ol is an important marker of oxymetholone abuse. In case of desoxymethyltestosterone, better detectability could be achieved if 18-nor-17,17-dimethyl-5α-androst-13-en-2ξ,3α-diol is monitored. These novel metabolites could be detected using GC-MS/MS at least for 14 days after administration of these anabolic steroids compared to 5-7 days for previously reported metabolites.

Streamlining sample preparation and gas chromatography–tandem mass spectrometry analysis of multiple pesticide residues in tea

In this work, a new rapid method for the determination of 135 pesticide residues in green and black dry tea leaves and stalks employing gas chromatography coupled to tandem mass spectrometry (GC–MS/MS) with a triple quadrupole was developed and validated. A substantial simplification of sample processing prior to the quantification step was achieved: after addition of water to a homogenised sample, transfer of analytes into an acetonitrile layer was aided by the addition of inorganic salts. Bulk co-extracts, contained in the crude organic extract obtained by partition, were subsequently removed by liquid–liquid extraction using hexane with the assistance of added 20% (w/w) aqueous NaCl solution. The importance of matrix hydration prior to the extraction for achieving good recoveries was demonstrated on tea samples with incurred pesticide residues. For most of the analytes, recoveries in the acceptable range of 70–120% and repeatabilities (relative standard deviations, RSDs) ≤20% were achieved for both matrices at spiking levels of 0.01, 0.1 and 1mgkg−1. Under optimised GC–MS/MS conditions, most of the analytes gave lowest calibration level ≤0.01mgkg−1, permitting the control at the maximum residue levels (MRLs) laid down in Regulation (EC) No 396/2005. The developed method was successfully applied to the determination of pesticide residues in real tea samples.

Antibacterial effect of silver nanoparticles on Staphylococcus aureus

Antibacterial activity of silver nanoparticles (AgNPs) was investigated using Staphylococcus aureus PTCC1431 as a model of Gram-positive bacteria. The mechanism of antibacterial activity of AgNPs was then studied by analyzing the growth, morphology, and molecular variations in the cell wall. Experimental data showed that AgNPs at a concentration of 4 μg/ml completely inhibited bacterial growth. Transmission electron microscopy results confirmed cell wall damage produced by AgNPs as well as accumulation of AgNPs in the bacterial membrane. Meanwhile, the AgNP-treated bacteria were monitored by circular dichroism to reveal peptidoglycan variations. Some degree of variation in the α-helix position of the peptide chain was observed. Moreover, increasing the AgNP concentration to 8 μg/ml resulted in release of muramic acid (MA) into the medium, which could be attributed to cell wall distraction. A gas chromatography-tandem mass spectrometry analysis and release of MA, as a bacterial indicator, showed that glycan strands may also be decomposed as a result of AgNP treatment.

Validation of an Analytical Biomarker Approach for the Detection of Nandrolone Abuse in the Porcine

The use of anabolic steroids as growth promoting agents in food production is prohibited under European Union legislation, but there is currently no internationally accepted method for detecting the abuse of the anabolic steroid nandrolone in the porcine. Therefore, an analytical biomarker approach based on gas chromatography-tandem mass spectrometry (GC-MS-MS) analysis of the major urinary free fraction nandrolone metabolite 19-noretiocholanolone was developed and validated. The lower and upper limits of quantification of the assay were 25 and 3,000 pg mL−1 respectively. The limit of detection was calculated as 13.2 pg mL−1, which is significantly lower than previously reported methods. When applied to a population of untreated animals, 19-noretiocholanolone distributions in boars and gilt were bimodal, with a small number of concentrations in each sex at around the 1,000 pg mL−1 region and the majority of concentrations closer to the lower end of the calibration range. Statistical analysis of the data was carried out in order to suggest screening and confirmatory threshold approaches for this steroid in the urine of boars and gilts. The adopting of particular screening thresholds would be at the discretion of the individual regulating authorities, but at a false non-compliance rate of 1 in 10,000 of the normal population, the suggested confirmatory thresholds (7,501.6 pg mL−1 for boars and 19,200.4 pg mL−1 in gilts) are able to detect the abuse of nandrolone for several weeks following administration of this steroid.

Sensitive determination of 2,4,6-trichloroanisole in water samples by ultrasound assisted emulsification microextraction prior to gas chromatography–tandem mass spectrometry analysis

A novel application of an ultrasound assisted emulsification microextraction (USAEME) technique is proposed for the extraction and preconcentration of 2,4,6-trichloroanisole (2,4,6-TCA) from water samples prior to its determination by gas chromatography-tandem mass spectrometry (GC-MS/MS). USAEME employs a non-polar high-density solvent (extractant solvent), which forms an oil-in-water emulsion (O/W) in the aqueous sample bulk assisted by ultrasonic radiation. Several factors including, solvent type and volume, extraction time, extraction temperature, shaking mode and matrix modifiers were studied and optimized over the relative recovery of the target analyte. An aliquot of 5mL water sample was conditioned by adding 150microL 6.15molL(-1) sodium chloride and 300microL 0.05molL(-1) phosphate buffer (pH 6), and finally extracted with 40microL chloroform by using USAEME technique. Under the optimal experimental conditions 2,4,6-TCA was quantitatively extracted achieving an enrichment factor (EF) of 555. The detection limit (LOD), calculated as three times the signal-to-noise ratio (S/N), was 0.2ngL(-1) and the RSD was 6.3% (n=5) when 1ngL(-1) 2,4,6-TCA standard mixture was analyzed. The coefficients of estimation of the calibration curves obtained following the proposed methodology was >or=0.997 and the linear working range was 1-5000ngL(-1). Finally, the proposed technique was successfully applied for extraction and determination of the 2,4,6-TCA in water samples. Recovery studies lead values >or=94%, which showed a successfully robustness of the analytical methodology for determination of nanogram per liter of 2,4,6-TCA in water samples.

Occurrence of pharmaceuticals and personal care products in fish: Results of a national pilot study in the united states

Pharmaceuticals and personal care products are being increasingly reported in a variety of biological matrices, including fish tissue; however, screening studies have presently not encompassed broad geographical areas. A national pilot study was initiated in the United States to assess the accumulation of pharmaceuticals and personal care products in fish sampled from five effluent-dominated rivers that receive direct discharge from wastewater treatment facilities in Chicago, Illinois; Dallas, Texas; Orlando, Florida; Phoenix, Arizona; and West Chester, Pennsylvania, USA. Fish were also collected from the Gila River, New Mexico, USA, as a reference condition expected to be minimally impacted by anthropogenic influence. High performance liquid chromatography-tandem mass spectrometry analysis of pharmaceuticals revealed the presence of norfluoxetine, sertraline, diphenhydramine, diltiazem, and carbamazepine at nanogram-per-gram concentrations in fillet composites from effluent-dominated sampling locations; the additional presence of fluoxetine and gemfibrozil was confirmed in liver tissue. Sertraline was detected at concentrations as high as 19 and 545 ng/g in fillet and liver tissue, respectively. Gas chromatography-tandem mass spectrometry analysis of personal care products in fillet composites revealed the presence of galaxolide and tonalide at maximum concentrations of 2,100 and 290 ng/g, respectively, and trace levels of triclosan. In general, more pharmaceuticals were detected at higher concentrations and with greater frequency in liver than in fillet tissues. Higher lipid content in liver tissue could not account for this discrepancy as no significant positive correlations were found between accumulated pharmaceutical concentrations and lipid content for either tissue type from any sampling site. In contrast, accumulation of the personal care products galaxolide and tonalide was significantly related to lipid content. Results suggest that the detection of pharmaceuticals and personal care products was dependent on the degree of wastewater treatment employed.

A Rapid, Cost-Effective Method for Analyzing Organophosphorus Pesticide Metabolites in Human Urine for Counter-Terrorism Response

Organophosphorus (OP) pesticides are used as insecticides in agriculture and pest control and are often called "junior strength" nerve agents because they share the same mechanism of toxicity. OP pesticides are metabolized to dialkylphosphates and other metabolites, which are excreted in urine. In case of a terrorism incident involving widely available OP pesticides, an occurrence that may be likely given their widespread availability, a rapid, accurate, and cost-effective method for detecting exposure is required. We have evaluated several analytical methods to determine the most reliable and cost-effective methods for incident response. Our comparisons have included different internal standards (isotopically labeled standards versus chemically similar surrogate standards), different isolation techniques (some of which are automatable), and different analysis platforms. We found that isotopically labeled standards were a necessity to provide accurate quantification; the chemically similar surrogate was not suitable as an internal standard. The most sensitive and precise method uses isotopically labeled standards with gas chromatography-tandem mass spectrometry analysis. However, the most cost-effective method employed isotopically labeled standards with gas chromatography-single quadrupole-mass spectrometry using a less expensive mass selective detector. Because this method is lower in cost, it may be a more viable option for equipping multiple laboratories with chemical-terrorism response capabilities.

Gas Chromatography-Tandem Mass Spectrometry Analysis of Red Blood Cells from Göttingen Minipig® following Whole-Body Vapor Exposure to VX

A method to detect fluoride ion generated O-ethyl methylphosphonofluoridate (VX-G) in Göttingen minipig red blood cells (RBC) following whole-body exposure to VX vapor utilizing a gas chromatograph-tandem mass spectrometer (GC-MS-MS) has been developed. Dose-response curves for VX exposure were generated after applying the fluoride ion reactivation assay to the RBC fraction of serially collected whole blood samples that were taken after whole-body exposures that varied in both duration and concentration. GC-MS-MS analysis of minipig RBC samples following 180-min exposures at two different concentrations was a more precise indicator for severity of exposure than the analysis of acetylcholinesterase (AChE) inhibition for the same samples. AChE enzyme activity recovered faster than indicated by the apparent elimination rate of VX-G. GC-MS-MS analyses of RBC samples following VX exposure demonstrate this technique has both adequate sensitivity and specificity to indicate the severity of exposure.

Detection of THCA in Oral Fluid by GC-MS-MS

A major marijuana metabolite, 11-nor-9-carboxy-tetrahydrocannabinol (THCA), has been identified in oral fluids from donors that previously tested positive for Delta(9)-tetrahydrocannabinol (THC). The method consisted of solid-phase extraction of the oral fluid samples followed by gas chromatography-tandem mass spectrometry analysis of the extracts. Testing for THCA was performed on 223 oral fluid samples previously analyzed for THC. The THCA assay was linear from 10 to 240 pg/mL. The mean recovery of spiked THCA in oral fluid was 104%, and precision was 4% at 20 pg/mL using fortified negative samples. This method was rugged and robust, providing detection and quantification of THCA in oral fluids at levels not previously reported. Results of this study showed that THCA was detectable in 21 of 26 oral fluid samples previously reported positive for THC. The range of concentrations from these samples was from 10 pg/mL up to 142 pg/mL THCA.

Analysis of beta-lyase metabolites of sulfur mustard in urine by electrospray liquid chromatography-tandem mass spectrometry.

A method is described for the analysis of b-lyase metabolites of sulfur mustard, 1-methylsulfinyl-2- [2-(methylthio)ethylsulfonyl]ethane and 1,1'-sulfonylbis [2-(methylsulfinyl)ethane], in human urine. The analytes were concentrated from urine on an ENV+ solid-phase extraction cartridge and analyzed by liquid chromatography-positive ion electrospray-tandem mass spectrometry in the selected reaction monitoring mode. Quantitation was performed against deuterated internal standards. Limits of detection were 0.1-0.5 ng/mL. The metabolites were detected in samples of urine from human casualties of sulfur mustard poisoning. The method provides a simpler alternative to gas chromatography-tandem mass spectrometry analysis, avoiding the need for reduction to less polar analytes.

The use of nonendcapped C18 columns in the cleanup of clenbuterol and a new adrenergic agonist from bovine liver by gas chromatography-tandem mass spectrometry analysis.

More specific official methodology is needed to survey the illegal use of clenbuterol in animal production plus the synthesis of new compounds that currently elude routine analytical methods. The identification of a new adrenergic agonist, N1-(2-(4-amino-3,5-dichlorophenyl)-2-hydroxyethyl)-N1-isopropyl-propanamide (known as compound A) in animal feed has prompted studies to verify if the existing cleanup procedures developed for clenbuterol are really effective. This study considers the ion-exchange mechanism on cyanopropyl (CN), sulfonic cation exchange (SCX), mixed phase (MPH) (C8 + SCX), and nonendcapped C18 (C18NE) solid-phase extraction (SPE) columns. Results indicate that compound A (by contrast with clenbuterol) is not efficiently retained on the CN, SCX, and MPH SPE columns (recovery <10%). This finding thus leads to the development of a gas chromatography-tandem mass spectrometry procedure based on C18NE SPE that is able to purify both agonists from bovine livers spiked at 0.5, 1.0, and 2.0 ppb with a mean recovery of 93% for clenbuterol and 92% for compound A.

House dust induces IL-6 and IL-8 response in A549 epithelial cells.

The in vitro potency of house dust to induce cytokine response in A549 lung epithelial cells was studied. Dusts collected from carpet, bed, shelf and floor of a villa and an apartment by vacuuming were found to trigger the production of interleukin-8 (IL-8) and interleukin-6 (IL-6) in a dose-dependent manner, and the interleukin production was several-fold higher than of swine dust (used as a positive control). The IL-8 and IL-6 production of pure Escherichia coli lipopolysaccharide was significantly lower than of the dusts and a peptidoglycan-polysaccharide complex did not show any stimulatory effect at all. The lipopolysaccharide and peptidoglycan contents of the samples were determined by gas chromatography-tandem mass spectrometry analysis of, respectively, 3-hydroxy fatty acids and muramic acid; in addition, ergosterol was monitored for fungal biomass. The inflammatory properties of house dust upon inhalation may be reflected in its high potency to induce cytokine response in lung epithelial cells.

Quantitative determination of LSD and a major metabolite, 2-oxo-3-hydroxy-LSD, in human urine by solid-phase extraction and gas chromatography-tandem mass spectrometry.

An assay has been developed for quantitative determination of lysergic acid diethylamide (LSD) and a major metabolite of LSD in human urine at concentrations as low as 10 pg/mL. In most LSD-positive urine samples the metabolite, 2-oxo-3-hydroxy-LSD, is present at higher concentrations than LSD and can be detected for a longer time than LSD after ingestion of the drug. Urine samples are extracted using Varian Bond Elut Certify extraction cartridges. Confirmatory identification is accomplished by trimethylsilylation of LSD and 2-oxo-3-hydroxy-LSD, followed by gas chromatography-tandem mass spectrometry analysis using positive ion chemical ionization and selected reaction monitoring. Commercially available lysergic acid methylpropylamide and 2-oxo-3-hydroxy-LAMPA are used as internal standards. With selected reaction monitoring, both compounds gave linear calibration curves from 10 pg/mL to 5000 pg/mL. Forty-nine human urine samples that had previously been shown to contain LSD were reanalyzed by the new method. These samples showed an average LSD concentration of 357 pg/mL and an average 2-oxo-3-hydroxy-LSD concentration of 3470 pg/mL. Additional experiments using clinical samples in which two subjects were dosed with LSD support the conclusion that analysis for 2-oxo-3-hydroxy-LSD can permit identification of LSD users for a longer period following ingestion than analysis for the parent drug.