Simona Nicoara

Detection of Mycobacterium tuberculosis in Sputum by Gas Chromatography-Mass Spectrometry of Methyl Mycocerosates Released by Thermochemolysis

Tuberculosis requires rapid diagnosis to prevent further transmission and allow prompt administration of treatment. Current methods for diagnosing pulmonary tuberculosis lack sensitivity are expensive or are extremely slow. The identification of lipids using gas chromatography- electron impact mass spectrometry (GC-EI/MS) could provide an alternative solution. We have studied mycocerosic acid components of the phthiocerol dimycocerosate (PDIM) family of lipids using thermochemolysis GC-EI/MS. To facilitate use of the technology in a routine diagnostic laboratory a simple extraction procedure was employed where PDIMs were extracted from sputum using petroleum ether, a solvent of low polarity. We also investigated a method using methanolic tetramethylammonium hydroxide, which facilitates direct transesterification of acidic components to methyl esters in the inlet of the GC-MS system. This eliminates conventional chemical manipulations allowing rapid and convenient analysis of samples. When applied to an initial set of 40 sputum samples, interpretable results were obtained for 35 samples with a sensitivity relative to culture of 94% (95%CI: 69.2,100) and a specificity of 100% (95%CI: 78.1,100). However, blinded testing of a larger set of 395 sputum samples found the assay to have a sensitivity of 61.3% (95%CI: 54.9,67.3) and a specificity of 70.6% (95%CI: 62.3,77.8) when compared to culture. Using the results obtained we developed an improved set of classification criteria, which when applied in a blinded re-analysis increased the sensitivity and specificity of the assay to 64.9% (95%CI: 58.6,70.8) and 76.2% (95%CI: 68.2,82.8) respectively. Highly variable levels of background signal were observed from individual sputum samples that inhibited interpretation of the data. The diagnostic potential of using thermochemolytic GC-EI/MS of PDIM biomarkers for diagnosis of tuberculosis in sputum has been established; however, further refinements in sample processing are required to enhance the sensitivity and robustness of the test.

Volatile Organic Chemical Pollutants in Laboratory Indoor Air

A quantitative method for measuring volatile organic compounds by pre-con centration on Tenax GC followed by gas chromatography analysis is de scribed. The method showed good linearity, precision and reproducibility for the organic compounds investigated. The limit of detection permits the deter mination of 0.1-1 μg, depending on the compound. Measurements were used to determine the airborne chemical pollutants in a mass spectrometry labora tory at our institute.

Development and optimization of a gas chromatography/mass spectrometry method for the analysis of thermochemolytic degradation products of phthiocerol dimycocerosate waxes found in Mycobacterium tuberculosis

RATIONALE: The phthiocerol dimycocerosates (PDIMs) are certain stable and hydrophobic waxes found in the cell membrane of Mycobacterium tuberculosis, bacteria that cause an infectious disease of growing concern worldwide. Previous studies report the analysis of derivatives of the hydrolysed PDIMs from biological samples, following complex extraction and offline derivatization of PDIMs biomarkers, prior to their analysis by gas chromatography/mass spectrometry (GC/MS). METHODS: We developed and optimized a GC/MS method based on selected ion monitoring (SIM) to detect the derivatives produced via the thermally assisted hydrolysis and methylation (THM) of the PDIMs from the cell membrane of M. tuberculosis. The extraction of PDIMs from culture is simple, and their thermochemolysis is carried out automatically online, thus avoiding the time-consuming derivatization steps of hydrolysis and esterification, usually performed offline. RESULTS: For standard PDIMs in petroleum ether, our optimized method gave an excellent linearity (R2 = 0.99) at concentrations between 0.172 and 27.5 ng/mL, a good precision (RSD = 11.42 %), and a limit of detection (LOD) of 100 pg/mL. For the PDIMs extracted from dilutions of M. tuberculosis culture, the method gave good linearity (R2 = 0.9685) and an estimated LOD of 400 CFU/mL (CFU = colony forming units) in sterile distilled water. CONCLUSIONS: A GC/MS(SIM) method is presented for the rapid and quantitative detection of M. tuberculosis, based on the online thermochemolysis of lipidic biomarkers extracted from the bacterial culture. The method has the potential to be applied in human and veterinary clinical laboratories for the rapid diagnosis of tuberculosis in infected biological samples.

Exposure Assessment of Nicotine and Cotinine by GC-MS

Environmental tobacco smoke exposure of human subjects in different public houses was evaluated. The levels of airborne nicotine from environmental tobacco smoke and changes in urinary cotinine and nicotine levels between smoking and non-smoking subjects were compared. A gas chromatography-mass spectrometric (GC-MS) quantitative method for nicotine and cotinine levels in the indoor air and in urine was developed. A Thermo Quest Trace DSQ GC-MS equipped with a Rtx-5MS capillary column, 15m 0.25mm, 0.25 m film thickness, was used in a temperature programme starting from 50°C, for 2min, then increased at 8°C·min-1 to 250°C and held for 10min. The method was validated and gave good linearity, precision, accuracy and limit of detection. A significant correlation between urinary cotinine increase and airborne nicotine is presented.

Development of sample clean up methods for the analysis of Mycobacterium tuberculosis methyl mycocerosate biomarkers in sputum extracts by gas chromatography–mass spectrometry

Highlights • We developed a sample clean-up method to detect tuberculosis from sputum by GC–MS.• Biomarkers recovered: 64–70% (standards solution), and 36–68% (sputum extracts).• Cholesterol removed: 93–98% (standards solution) and 62–92% (sputum extracts).• Less cholesterol in the filtered extracts avoids overloading of the analytical system.• Analyzing large sample batches will need fewer interruptions for system cleaning.

Mass spectra interpretation of some thiophosphororganic compounds

Abstract The fragmentation pattern of six thiophosphonic-amyd-aryl-sulphonimido derivatives is presented. The electron impact mass spectra were recorded at 70 eV. High resolution mass measurements and metastable analyses were performed to elucidate the ions composition and the fragmentation processes.

Study of amino acid transmembranar transport in human red cell and rat hepatocyte

Abstract A study of amino acid transmembranar transport in human red cells, using isotope dilution GC/MS technique, compared with radioisotopical transport measurements in rat hepatocyte is presented. A stable isotopically labeled amino acid, 15 N-glycine was used. Small influx was detected for glycine in the two types of cells studied. Incubation of the cell in the presence of some ingredients shows increased amino acid influx.

Determining the levels of volatile organic pollutants in urban air using a gas chromatography-mass spectrometry method.

The paper presents the application of a method based on coupled gas chromatography-mass spectrometry, using an isotopically labelled internal standard for the quantitative analysis of benzene (B), toluene (T), ethyl benzene (E), and o-, m-, p-xylenes (X). Their atmospheric concentrations were determined based on short-term sampling, in different sites of Cluj-Napoca, a highly populated urban centre in N-W Romania, with numerous and diversified road vehicles with internal combustion engines. The method is relatively inexpensive and simple and shows good precision and linearity in the ranges of 7–60 μg/m3 (B), 13–90 μg/m3 (T), 7–50 μg/m3 (E), 10–70 μg/m3 (X-m,p), and 20–130 μg/m3 (X-o). The limits of quantitation/detection of the method LOQ/LOD are of 10/5 μg/m3 (Xo), 5/3 μg/m3 (B, E, X-m,p), and of 3/1 μg/m3 (T), respectively.

Selected Ion Monitoring – Gas Chromatography/Mass Spectrometry Determination of Halothane Fumes in Operating Theatres

Halothane vapour, used as an inhaled anaesthetic for narcosis, is released during surgical operations causing occupational exposure to medical staff and nurses. The current work continues the controversial issues related to the biological side effects of halothane, and gives a simple method for sampling and determining the air concentration of the anaesthetic in different rooms used for surgical operations. Air contaminants were collected on active charcoal, using a portable pump. Liquid extraction with toluene was followed by injection of the eluate onto a GC capillary column coupled to a quadrupole mass detector. The method was validated with respect to linearity, precision and accuracy. The concentration of halothane in the air was found to depend on room volume, air ventilation rate inside the theatres, and on the technical condition of the vaporiser and anaesthesia devices. Introduction

Gas Chromatographic Determination of Halothane Levels in Hospital Operating Theatres

A simple, rapid, and sensitive method for the analysis of halothane in the air of operating theatres has been developed. The procedure involves the pre-concentration of halothane on active charcoal in an adsorption tube, then desorption in toluene, followed by capillary gas chromatography (GC) analysis using a flame ionisation detector (FID). The recovery from air samples after pre-concentration was 67%. The linearity was established with standards over a concentration range between 0 and 3 μL mL−1 of halothane in toluene with an excellent coefficient of correlation (0.99) and a limit of detection of 1 μL halothane per cubic metre of air. The time for analysis was approximately 10 min with an adsorption step of 60-90 min per sample collected on site. The procedure was used for occupational exposure assessment, by quantitation of halothane in two different hospital operating theatres, during a single general anaesthesia for a routine orthopaedic surgery and for a routine internal surgery, respectively.