Chromatography-triple Quadrupole Tandem Mass Spectrometry Research Articles

Gas chromatography–triple quadrupole tandem mass spectrometry: a powerful tool for the (ultra)trace analysis of multiclass environmental contaminants in fish and fish feed

A new method for rapid determination of 73 target organic environmental contaminants including 18 polychlorinated biphenyls, 16 organochlorinated pesticides, 14 brominated flame retardants and 25 polycyclic aromatic hydrocarbons in fish and fish feed using gas chromatography coupled with triple quadrupole tandem mass spectrometry (GC-MS/MS) was developed and validated. GC-MS/MS in electron ionization mode was shown to be a powerful tool for the (ultra)trace analysis of multiclass environmental contaminants in complex matrices, providing measurements with high selectivity and sensitivity. Another positive aspect characterizing the newly developed method is a substantial simplification of the sample preparation, which was achieved by an ethyl acetate QuEChERS (quick, easy, cheap, effective, rugged and safe) based extraction followed by silica minicolumn clean-up. With use of this sample preparation approach the sample laboratory throughput was increased not only because six samples may be prepared in approximately 1 h, but also because all the above-mentioned groups of contaminants can be determined in a single GC-MS/MS run. Under the optimized conditions, the recoveries of all target analytes in both matrices were within the range from 70 to 120% and the repeatabilities were 20% or less. The method quantification limits were in the range from 0.005 to 1 μg kg(-1) and from 0.05 to 10 μg kg(-1) for fish muscle tissue and fish feed, respectively. The developed method was successfully applied to the determination of halogenated persistent organic pollutants and polycyclic aromatic hydrocarbons in fish and fish feed samples.

Multi-residual Pesticide Monitoring in Commercial Chinese Herbal Medicines by Gas Chromatography–Triple Quadrupole Tandem Mass Spectrometry

To investigate multi-residual pesticide monitoring data in commercial Chinese herbal medicines on major markets, an easy, rapid, and selective gas chromatography with mass spectrometry (GC/MS/MS) method was established for simultaneously determining multi-residual pesticides including organochlorine, pyrethroid, carbamate, and organophosphorus pesticides in Chinese herbal medicines. The analytical method was based on an efficient extraction procedure and further cleanup steps by solid-phase extraction columns, yielding recovery rates in the range of 70.0–120.0 % for the majority of pesticides, except for hexachlorobenze, diazinon, β-HCH, δ-HCH, and omethoate, with precision values expressed as relative standard deviation of 0.1–14.7 %. The limits of detection of the established GC/MS/MS method for all investigated pesticides ranged from 0.01 to 3.6 μg kg−1 and limits of quantification from 0.03 to 11.88 μg kg−1. With this validated method, multi-residual pesticides of 132 Chinese herbal medicine samples were analyzed. The monitoring results indicated that pesticide residue was found in 74 samples. In total, 51 pesticides were found with detection rate ranging from 0.76 to 18.94 %. An 82.3 % of positive pesticides were found in less than 6 % of samples. Hexachlorobenzene was found in 25 samples, quintozene in 15 samples, and acephate and simazine in 13 samples. Concentrations of pesticide residue from monitoring data obtained ranged from 0.5 to 203.5 μg kg−1. The simple and rapid method can be used as routine analysis method in multi-residual pesticide monitoring of Chinese herbal medicines.

Determination of congo red in beef by high performance liquid chromatography-tandem quadrupole time of flight mass spectrometry and ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry

A method was developed for the determination of congo red in beef. The analyte was identified by high performance liquid chromatography-tandem quadrupole time of flight mass spectrometry (LC-QTOF MS) and quantitatively determined by ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry. After purified by liquid-liquid extraction, the congo red in the beef sample was separated on an Agilent ZORBAX Eclipse Plus C18 Rapid Resolution HD UPLC column (50 mm x 2.1 mm, 1.8 microm) HPLC , using 95% (volume percentage) methanol as the mobile phase at 0.2 mL/min. The detection was performed on an AB 4000 + triple quadrupole mass spectrometer utilizing electrospray ionization (ESI) interface operated in negative ion mode and multiple-reaction monitoring (MRM) mode. The results showed that the linear range of congo red mass concentration was 0.03 - 1 mg/L with the correlation coefficient of 0.999 8. The method had a good precision with the RSDs lower than 5% and the recoveries ranging from 88% to 91%. The limit of detection (LOD) of congo red was 0.01 mg/L. With good reproducibility, the method is simple, fast and effective for the determination of the illegally added congo red in beef and other meat products.

Simultaneous determination of ethyl carbamate and chloropropanols in flavorings by gas chromatography- triple quadrupole tandem mass spectrometry

A simultaneous determination method for ethyl carbamate (EC) and chloropropanols (3-monochloropropane-1, 2-diol (3-MCPD) and 2-monochloropropane-1, 3-diol (2-MCPD)) in flavorings was developed by gas chromatography-triple quadrupole tandem mass spectrometry (GC-MS/MS). After spiked with internal standard, the sample was extracted by matrix solid-phase dispersion extraction technique with an Extrelut NT column. Hexane was used to wash the fat soluble matrix interferences and then an ethyl acetate-ethyl ether (20: 80, v/v) mixture was added to elute the analytes. The concentrated extract was detected by GC-MS/MS in multiple reaction monitoring (MRM) mode. The limits of detection (LODs) were 2, 5 and 5 microg/kg for EC, 3-MCPD and 2-MCPD, respectively. The linear ranges were 5 - 1 000 microg/kg (r = 0.9997), 10-1000 microg/kg (r = 0.999 1) and 10-1000 microg/kg (r = 0.999 5) for EC, 3-MCPD and 2-MCPD, respectively. In soy sauce, yellow rice wine, salami sauce and flavoring of instant noodle matrices, the recoveries (RSDs, n = 7) in MRM mode at the levels of 20, 100 and 400 microg/kg were 87.7%-104% (4.3%-10.7%), 90.1%-109% (2.6%-10.2%), and 90.9%-103% (3.0%-9.5%), respectively. EC, 3-MCPD and 2-MCPD were found in some real samples of the soy sauce, wine and flavoring of instant noodle. EC or 3-MCPD was found in some of the salami samples. The method is accurate, fast and suitable for the simultaneous determination of EC, 3-MCPD and 2-MCPD in flavorings.

Comparative assessment of three extraction procedures for determination of emerging Fusarium mycotoxins in pasta by LC–MS/MS

A new rapid, sensitive, reproducible and reliable method was developed for the quantitative determination of enniatins A, A1, B and B1, beauvericin and fusaproliferin in dry and fresh pasta by liquid chromatography-triple quadrupole-tandem mass spectrometry. A comparative study of different rapid and economical extraction procedures was performed for the extraction of these mycotoxins in pasta. For this purpose, three different approaches were studied during the extraction step (Ultra-Turrax, ultrasonic bath and microwave). Optimal extraction conditions were reached using Ultra-Turrax with acetonitrile for 3 min without purification step. The chromatographic separation of the six mycotoxins was accomplished in 15 min. The method was extensively validated with satisfactory results: recovery rates ranged from 86 to 112% and the relative standard deviations were lower than 15%. Limits of detection ranged from 0.02 to 0.15 μg kg−1. The applicability of the method was assessed with the analysis of 30 samples of dry and fresh pasta.

Detection of Clenbuterol at Trace Levels in Doping Analysis Using Different Gas Chromatographic–Mass Spectrometric Techniques

This study demonstrates the development of a gas chromatography-triple quadrupole tandem mass spectrometry (GC-MS-MS) assay to detect clenbuterol in human urine and the comparison of this method with GC-MS techniques and gas chromatography-high resolution mass spectrometry (GC-HRMS) techniques. Urine samples were hydrolyzed with β-glucuronidase, extracted with methyl tert-butyl ether and dried under nitrogen. The derivative reagent was N-methyl-N-(trimethylsilyl)-trifluoroacetamide with NH4I and was analyzed by GC-MS, GC-MS-MS and GC-HRMS. A validation study was conducted by GC-MS-MS. The analyses of clenbuterol using different mass spectrometric techniques were compared. The limit of detection (LOD) for clenbuterol in human urine was 2 ng/mL by GC-MS (selected ion monitoring mode: SIM mode), 0.06 ng/mL by GC-HRMS and 0.03 ng/mL by GC-MS-MS, respectively, while the LOD by GC-HRMS was 0.06. With GC-MS-MS, the intra-assay and inter-assay precisions were less than 15%, the recoveries were 86 to 112% and the linear range was 0.06 to 8.0 ng/mL. The GC-MS under SIM mode can be used as a screening tool to detect clenbuterol at trace levels in human urine. The GC-MS-MS and GC-HRMS methods can confirm clenbuterol when its concentration is below 2 ng/mL. The results demonstrate that the GC-MS-MS method is quite sensitive, specific and reliable for the detection of clenbuterol in doping analysis.

Simultaneous Determination of 15 Plant Growth Regulators in Bean Sprout and Tomato with Liquid Chromatography–Triple Quadrupole Tandem Mass Spectrometry

The present paper describes the development and validation of a new reversed-phase liquid chromatography–electrospray ionization tandem mass spectrometric method for determination of 15 plant growth regulators in bean sprout and tomato. Plant growth regulators were extracted with mixed solvents methanol/acetonitrile/water/acetic acid (v/v/v/v, 40/40/20/1) from 10 g vegetable and analyzed directly without further cleanup. The identification of each analyte was established by chromatographic retention time, analyte-specific fragmentation patterns, and relative peak area ratios of precursor/product ion pairs. The recoveries of 15 plant growth regulators in bean sprout and tomato were 70.3–110.0 %. The repeatability of this method is excellent, and relative standard deviation value is less than 10 %.

Analytical performance of a triple quadrupole mass spectrometer compared to a high resolution mass spectrometer for the analysis of polybrominated diphenyl ethers in fish

Polybrominated diphenyl ethers (PBDEs) are a class of flame retardants used globally in many consumer products and industrial applications. Traditionally, gas chromatography–high resolution mass spectrometry (GC–HR-MS) is the method of choice for analysis of PBDEs in environmental samples because it offers high sensitivity and selectivity, resulting in less interferences. However, the specificity offered by gas chromatography-triple quadrupole tandem mass spectrometry (GC–QQQ-MS/MS), operated in selected reaction monitoring mode, provides a more affordable alternative to GC–HR-MS for the analysis of PBDEs in complex environmental samples. In this study, an analytical method was developed for the analysis of 41 PBDE congeners in fish using GC–QQQ-MS/MS. Results from the analysis of three fish species [lake trout (Salvelinus namaycush), yellow perch (Perca flavescens), and round goby (Neogobius melanostomus)] using GC–QQQ-MS/MS were compared with those obtained by GC–HR-MS. These species were selected because they represent varying levels of lipid-rich matrix and contaminant loads. Instrumental limits of detection for the GC–QQQ-MS/MS ranged from 0.04pg to 41pg, whereas those for the GC–HR-MS ranged from 5pg to 85pg. The PBDE values obtained from these two methods were highly correlated, R2 values >0.7, for all three fish species, supporting the suitability of GC–QQQ-MS/MS for analysis of PBDEs in fish with varying fat content.

Validation of a rapid lateral flow test for the simultaneous determination of β-lactam drugs and flunixin in raw milk.

β-Lactam antibiotics are the most commonly used drugs on dairy farms. β-Lactam residues in milk are kept out of the human milk supply with good agricultural practices and mandatory truck screening performed by the dairy industry under Appendix N of the Pasteurized Milk Ordinance. Flunixin, a nonsteroidal and anti-inflammatory drug, appears in dairy cattle tissue residues with a frequency similar to the occurrence of penicillin G. This creates concern that flunixin residues could be in milk and would go undetected under current milk screening programs. A single test that combines mandatory β-lactam screening with voluntary flunixin screening is an economical approach for monitoring and controlling for potential flunixin or 5-hydroxyflunixin, the primary flunixin metabolite marker in milk. The objective of this study was to validate a β-lactam and flunixin rapid lateral flow test (LFT) and compare the results obtained with a liquid chromatography-triple quadrupole tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of flunixin and 5-hydroxyflunixin in raw milk with a limit of detection of , 1 ppb, equivalent to 1 ng/ml. Using the LFT, three combined manufactured lots of test strips detected penicillin G at 2.0 ppb, ampicillin at 6.8 ppb, amoxicillin at 5.9 ppb, cephapirin at 13.4 ppb, ceftiofur (total metabolites) at 63 ppb, and 5-hydroxyflunixin at 1.9 ppb at least 90% of the time with 95% confidence. The LFT also detected incurred flunixin milk samples that were analyzed with the LC-MS/MS and diluted to tolerance in raw milk. The detection levels for the LFT are lower than the U.S. safe levels or tolerances and qualify the test to be used in compliance with U.S. milk screening programs.

Determination of the Nicotine Content in Solanaceae Vegetables by Solid-Phase Extraction Coupled with Ultra High-Performance Liquid Chromatography–Tandem Mass Spectrometry

A validated method based on solid-phase extraction and ultra high-performance liquid chromatography–triple quadrupole tandem mass spectrometry, with electrospray ionization operated in the positive ion mode and multiple reaction monitoring, was developed for the determination of nicotine in Solanaceae vegetables. Sample preparation involved liquid–solid extraction, centrifugation, filtration, and solid-phase extraction. Two kinds of solid-phase extraction adsorbents, based on different retention mechanisms, were investigated. Relatively higher recoveries were obtained with a hydrophilic–lipophilic-balanced cartridge. A deuterated internal standard was used for quantification. The limit of quantification (LOQ) of nicotine in different vegetables was found to be between 0.07 and 0.5 μg/kg. The nicotine levels in the vegetable samples were above the LOQs. The method described here is thus suitable for the analysis of large sample batches, because it provides accurate quantification, high sensitivity and rapid chromatographic separation with facile preparation. The solid-phase extraction cartridges and organic solvents used in this work are easy to obtain, enabling the application of this method in most analytical laboratories.

Multiresidue pesticide analysis in Korean ginseng by gas chromatography–triple quadrupole tandem mass spectrometry

In this study, a new analytical method was developed based on gas chromatography-triple quadrupole tandem mass spectrometry (GC-MS/MS) and used to determine 32 multiclass pesticides in ginseng products. The analytical method was validated, yielding recovery rates in the range of 55.2-108.3%, with precision values expressed as relative standard deviation (RSD) lower or equal to 12% at the spiking levels of 30, 100, and 1000 μg/kg. Correlation coefficients and LOQs (limit of quantification) were in the range 0.9801-0.9989 and 0.15-70 g/kg, respectively. With these validation data and this method, multiresidue pesticides of ginseng samples (fresh ginseng (n=118), red ginseng (n=24), dried ginseng (n=10)) were analysed. Among them, the most frequently detected pesticide was tolclofos-methyl. Tolclofos-methyl was detected in 86.4% of fresh ginseng (18.25-404.5 μg/kg), 91.7% of red ginseng (13.14-119.4 μg/kg), and 87.5% of dried ginseng (23.15-3673 μg/kg).

Determination of thiophanate-methyl and carbendazim in cucumber and soil by QuEChERS-high performance liquid chromatography-triple quadrupole tandem mass spectrometry

A method was developed for the determination of thiophanate-methyl and carbendazim residues in cucumber and soil by using QuEChERS-high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). The residues in the samples were extracted by acetonitrile, cleaned up by developed QuEChERS method, and then analyzed by using LC-MS/MS in multiple reaction monitoring (MRM) mode via positive electrospray ionization with an Agilent ZORBAX SB-C, column (30 mm x 2.1 mm, 5 microm) as the analytical column. The recoveries of thiophanate-methyl spiked at four levels of 0.01, 0.05, 0.1 and 1.0 mg/kg were from 87.3% to 96.0% with the relative standard deviations (RSDs) of 8.0%-9.3% in cucumber, from 88.8% to 93.4% with the RSDs of 5.3%-9.9% in soil; the recoveries of carbendazim spiked at the same levels as those of thiophanate-methyl were from 87.1% to 92.3% with the RSDs of 5.2%-7.5% in cucumber, from 85.8% to 90.9% with the RSDs of 5.3%-13.2% in soil. The method is simple, rapid and characterized with acceptable sensitivity and accuracy to meet the requirements of the pesticide residue analysis. This method is applicable to confirm the residues of thiophanate-methyl and carbendazim

Immunochemical-based zilpaterol measurement and validation in urine and tissues

Because of abuse potential of the feed-additive zilpaterol, a need exists for rapid, sensitive and specific analyses. Polyclonal and monoclonal antibody-based enzyme-linked immunosorbent assays (ELISAs) were developed and their usefulness for agricultural applications explored. Immunobiosensor formats were developed for both monoclonal and polyclonal zilpaterol antibodies. Zilpaterol ELISA and immunobiosensor were tested by measuring tissue and urinary concentrations from sheep treated with zilpaterol for 10 days. The study demonstrated that sheep eliminated zilpaterol rapidly. A zilpaterol study in horses demonstrated that urinary zilpaterol in horses was initially much higher than in other species and that urinary zilpaterol depleted in a biphasic manner. Zilpaterol was detectable using either ELISA or ultra-high performance liquid chromatography-triple quadrupole-tandem mass spectrometry (UHPLC-TQ-MS/MS) after 21 days of withdrawal in horses. These studies demonstrated that the ELISA procedure was rapid and was in good agreement with instrumental methods while the biosensor method provided greater precision than the ELISA procedure.

Determination of aflatoxins in cereals and oils by liquid chromatography-triple quadrupole tandem mass spectrometry

The high performance liquid chromatography (HPLC)-triple quadrupole tandem mass spectrometry was applied for the determination of the aflatoxins: B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2), in cereals and oils. The samples were first extracted by ultrasonic method. The optimized conditions of ultrasonic extraction were as follows: temperature of 50 degrees C, extraction time of 3 min, methanol-water (containing 40 g/L NaCl) (80: 20, v/v) solution as the medium and a ratio of sample to solvent of 1 : 3 (g: mL). The extracts were then purified using an immunoaffinity column. The separation was performed on a C18 column with mobile phases of 10 mmol/L ammonium acetate and methanol in gradient elution. The sensitive detection of the four AFT compounds by electrospray ionization mass spectrometry (ESI-MS) was carried out in selected reaction monitoring (SRM) mode with aflatoxin M1 (AFM1) as the internal standard. Under the optimized conditions, the limits of detection of AFB1, AFB2, AFG1 and AFG2 were 0.002, 0.004, 0.004 and 0.012 microg/kg, respectively. The recoveries of aflatoxins in different spiked cereals and oils were in the range from 87% to 111%. The intra-day and inter-day precisions were not more than 6.7% and 5.6%, respectively. In comparison with the external standard method, this method can effectively inhibit the matrix effects, and greatly improve the accuracy.

One-year monitoring of aflatoxins and ochratoxin A in tiger-nuts and their beverages

A sensitive and selective liquid chromatography–triple quadrupole–tandem mass spectrometry (LC–ESI–MS–MS) method was developed for the routine analysis of aflatoxins (AFB1, AFB2, AFG1 and AFG2) and ochratoxin A (OTA) in tiger nuts and tiger-nut beverage (horchata). A matrix solid phase dispersion was adapted to eliminate lipidic interferences. The solid support was C18, while the elution solvent was acetonitrile. Mean recoveries obtained at two fortification levels were 72–83% and 71–81% for horchata and tiger nut respectively with relative standard deviations (RSDs) <13% and 15% respectively. The LC–MS–MS method allowed quantification and identification at low levels in two matrices. The method was applied for the routine analysis of tiger-nuts and horchata samples collected from different supermarkets of Valencia (Spain) during one year (March 2009–March 2010). A total of 238 samples were analysed and 32 samples were found positives for OTA, AFB1, AFB2 and AFG2.

Multi-residue method for the determination of organochlorine pesticides in fish feed based on a cleanup approach followed by gas chromatography–triple quadrupole tandem mass spectrometry

A multi-residue method for the determination of organochlorine pesticides in fish feed samples was developed and optimized. The method is based on a cleanup step of the extracted fat, carried out by liquid–liquid extraction on diatomaceous earth cartridge with n-hexane/acetonitrile (80/20, v/v) followed by solid phase extraction (SPE) with silica gel–SCX cartridge, before the identification and quantification of the residues by gas chromatography–triple quadrupole tandem spectrometry (GC–MS/MS). Performance characteristics, such as accuracy, precision, linear range, limits of detection (LOD) and quantification (LOQ), for each pesticide were determined. Instrumental LODs ranged from 0.01 to 0.11 μg L −1, LOQs were in the range of 0.02–0.35 μg L −1, and calibration curves were linear ( r 2 > 0.999) in the whole range of explored concentrations (5–100 μg L −1). Repeatability values were in the range of 3–15%, evaluated from the relative standard deviation of six samples spiked at 100 μg kg −1 of fat, and in compliance with that derived by the Horwitz's equation. No matrix effects or interfering substances were observed in fish feed analyses. The proposed method allowed high recoveries (92–116%) of spiked extracted fat samples at 100 μg kg −1, and very low LODs (between 0.02 and 0.63 μg kg −1) and LOQs (between 0.05 and 2.09 μg kg −1) determined in fish feed samples.

Determination of Polycyclic Aromatic Hydrocarbons in Airborne Particulate Matter by Gas ChromatographyTriple Quadrupole Tandem Mass Spectrometry

An accurate and sensitive method for the identification and the quantification of 24 polycyclic aromatic hydrocarbons (PAHs) in airborne particulate matter has been developed and validated using GC/MS/MS with a triple quadrupole analyzer. Ultrasonic extraction (USE) and pressurized liquid extraction (PLE) were evaluated. PLE was selected because it provided better recoveries and reduced treatment time and use of solvents compared to USE. Cleanup of PLE extracts was not necessary. The accuracy of the optimized method was tested using a standard reference material of urban dust (SRM 1649a). PAH recoveries were between 70 and 99% (except for naphthalene, which was lower than 20%) with RSDs ranging from 2.6 to 15.3%. Linearity in the range 5-500 ng/mL provided R2 values higher than 0.99 for all compounds. LODs for PAHs ranged from 0.001 to 0.435 ng/mL and LOQs ranged from 0.003 to 0.757 ng/mL. The proposed method was applied to the analysis of real samples collected in the city of Almeria, Spain, where some PAHs were found at levels lower than the legislated limit.

Development of a multi-residue screening method for the determination of pesticides in cereals and dry animal feed using gas chromatography–triple quadrupole tandem mass spectrometry

A multi-residue screening method for simultaneous analysis of 122 gas chromatography amenable pesticides in dry matrices such as cereal grain and certain feedingstuffs was developed. The method entails a simple extraction of re-hydrated sample with acetonitrile followed by a dispersive solid phase extraction (dispersive-SPE) clean-up step prior to the final determination by gas chromatography/triple quadrupole tandem mass spectrometry (GC–MS/MS). Due to complexity of analyzed matrices, two MS/MS transitions were set for each pesticide to eliminate the need for re-analysis of potentially positive samples, and provide unequivocal identification of detected pesticides in accordance with recent guidelines, in a single analytical run. Thus, in the developed GC–MS/MS acquisition method, a total of 216 different multiple reactions monitoring (MRM) transitions were monitored in one set of experimental conditions. To evaluate performance of the method, validation experiments were carried out on wheat grain at three spiking levels (0.01, 0.02 and 0.05 mg kg −1). Additional recovery tests at 0.05 mg kg −1 were carried out on several other matrices. The recoveries ranged between 73 and 129% with associated relative standard deviations between 1 and 29% for the majority of pesticides. Limits of detection were less or equal to 0.01 mg kg −1 for approximately 68% of pesticides. The applicability of the proposed method to detect and quantify pesticide residues has been demonstrated in the analysis of 136 real samples. Additionally, the method was favorably compared with an acetone extraction method (accepted as a reference method by some of European and U.S. authorities) in the analysis of real samples known to contain pesticide residues.

Scission of the Lactyl Ether Bond of N-Acetylmuramic Acid by Escherichia coli “Etherase”

The ubiquitous bacterial cell wall sugar N-acetylmuramic acid (MurNAc) carries a unique D-lactyl ether substituent at the C3 position. Recently, we proposed an etherase capable of cleaving this lactyl ether to be part of the novel bacterial MurNAc dissimilation pathway (Dahl, U., Jaeger, T., Nguyen, B. T., Sattler, J. M., Mayer, C. (2004) J. Bacteriol. 186, 2385-2392). Here, we report the identification of the first known MurNAc etherase. The encoding gene murQ is located at 55 min on the Escherichia coli chromosome adjacent to murP, the MurNAc-specific phosphotransferase system. A murQ deletion mutant could not grow on MurNAc as the sole source of carbon and energy but could be complemented by expressing murQ from a plasmid. The mutant had no obvious phenotype when grown on different carbon sources but accumulated MurNAc 6-phosphate at millimolar concentrations from externally supplied MurNAc. Purified MurQ-His6 fusion protein and extracts of cells expressing murQ both catalyze the cleavage of MurNAc 6-phosphate, with GlcNAc 6-phosphate and D-lactate being the primary products. The 18O label from enriched water is incorporated into the sugar molecule, showing that the C3-O bond is cleaved and reformed by the enzyme. Moreover, an intermediate was detected and identified as an unsaturated sugar molecule. Based on this observation, we suggested a lyase-type mechanism (beta-elimination/hydration) for the cleavage of the lactyl ether bond of MurNAc 6-phosphate. Close homologs of murQ were found on the chromosome of several bacteria, and amino acid sequence similarity with the N-terminal domain of human glucokinase-regulatory protein (GckR or GKRP) was recognized.

Determination and pharmacokinetic profile of pyrazinamide in rat blood, brain and bile using microdialysis coupled with high-performance liquid chromatography and verified by tandem mass spectrometry

This study demonstrates a rapid and sensitive method for the concurrent determination of unbound pyrazinamide in rat blood, brain and bile using microdialysis coupled with high-performance liquid chromatography (HPLC) for further pharmacokinetic study. Microdialysis probes were simultaneously inserted into the jugular vein toward right atrium, the striatum of brain and the bile duct of male Sprague–Dawley rats for biological fluid sampling after the administration of pyrazinamide (10, 30 or 100 mg kg −1) through the femoral vein. Pyrazinamide was separated from various dialysates using a reversed-phase C18 column maintained at ambient temperature. The mobile phase consisted of acetonitrile −10 mM monosodium phosphate (10:90, v/v) for blood and brain dialysates, and methanol–10 mM monosodium phosphate (10:90, v/v) for bile dialysate. The UV detector wavelength was set at 268 nm and the detection limit of pyrazinamide was 100 ng ml −1. The presence of pyrazinamide in biological fluid samples was verified with liquid chromatography–triple quadrupole tandem mass spectrometry using electrospray ionization. The results suggest that microdialysis sampling followed by LC separation with UV detection is a viable approach for the measurement of free pyrazinamide in rat blood, brain and bile. The decline of unbound pyrazinamide in the blood, brain striatum and bile fluid indicates that there was rapid exchange and equilibration between the compartments of the peripheral and central nervous systems. In addition, the results demonstrate that pyrazinamide was able to penetrate the blood–brain barrier and undergo hepatobiliary excretion.