Supramolecular Solvent Extraction-Gas Chromatography-Tandem Mass Spectrometry for Detection of Benzodiazepines in Urines / 法医学杂志

Objective To establish a method using supramolecular solvent and gas chromatography-tandem mass spectrometry （GC-MS/MS） to analyze 9 benzodiazepines in urines. Methods Urine samples containing 9 benzodiazepines reference substance were subjected to liquid-liquid extractions with supramolecular solvent, which consisted of tetrahydrofuran and 1-hexanol. The solvent layer was evaporated to dryness by stream of nitrogen. The residue was reconstituted with methanol, and GC-MS/MS analysis was performed on it. The way of data collection was multiple reaction monitoring （MRM） mode; internal standard method was employed for quantification. Results In urine samples, when the range of mass concentration was 1-100 ng/mL for diazepam, midazolam, flunitrazepam and clozapine, 5-100 ng/mL for lorazepam and alprazolam, 2-100 ng/mL for nitrazepam and clonazepam, and 0.2-100 ng/mL for estazolam, respectively, good linearities were obtained, correlation coefficients were 0.999 1-0.999 9, the lower limits of the quantifications ranged from 0.2 to 5 ng/mL, the extraction recovery rates were 81.12%-99.52%. The intra-day precision ［relative standard deviation （RSD）］ and accuracy （bias） were lower than 9.86% and 9.51%, respectively; the inter-day precision （RSD） and accuracy （bias） were lower than 8.74% and 9.98%, respectively. Nine drugs in urine samples showed good stability at ambient temperature and -20 ℃ within 15 days. The mass concentrations of alprazolam in urine samples obtained from 8 volunteers who took alprazolam tablets orally within 8-72 h after ingestions ranged from 6.54 to 88.28 ng/mL. Conclusion The supramolecular solvent extraction GC-MS/MS method for analysis of 9 benzodiazepines in urines provided by this study is simple, fast, accurate and sensitive, which can provide technical support for monitoring of poisoning by benzodiazepines for clinical treatment and judicial identification.

Effect of diesel exhaust exposure on 8-hydroxy-2' deoxyguanosine level in biological samples of automobile manufacturing workers / 中国职业医学

OBJECTIVE: To investigate the feasibility of using 8-hydroxy-2'deoxyguanosine(8-OHdG) in blood and urine samples as biomarkers for the evaluation of human DNA oxidative damage caused by diesel exhaust(DE). METHODS: A convenient sampling method was used to select 56 male workers exposed to DE in a car manufacturing factory as exposure group, and 52 male workers without exposure to DE were selected as the control group.Urine samples and blood samples were collected from workers in the 2 groups 8 hours after work, and the levels of 8-OHdG in urine and plasma were measured by ultra-high performance liquid chromatography tandem mass spectrometer. RESULTS: The median level of urinary 8-OHdG in the exposure group was higher than that of control group(2.54 vs 2.03 μg/g Cr, P<0.05). The median levels of plasma 8-OHdG in the exposure group and control group showed no statistical significance(32.20 vs 31.40 ng/L, P>0.05). CONCLUSION: The urinary 8-OHdG can be used as a biomarker for evaluating the oxidative damage induced by DE exposure.

Evaluation on measurement uncertainty of detecting procedure of amino acids and carnitines by using non-derivatized tandem mass spectrometry method / 国际检验医学杂志

Objective To evaluate the measurement uncertainty of the detecting procedure of amino acids and carnitines by the Waters ACQUITY TQD tandem mass spectrometer and PerkinElmer NeoBaseTM non‐derivatized MSMS kit ,and to discuss the meaning of evaluation .Methods According to the method provided by the Medical Laboratories‐Evaluation and Expression of Measurement Uncertainty ,the internal quality control was detected by using two different batch codes of kit for 20 d ,once before and after the routine sample detection on each working day ,the detector were randomly changed ,and then the measurement uncer‐tainty introduced by measurement reproducibility was calculated;the amino acid external assessment quality control data in 20 times of neonatal hereditary metabolic disease tandem mass spectrometry screening provided by the National Center for Clinical Laborato‐ry in 2014 and 2015 ,and the same and 16 times of carnitines external assessment quality control data were performed the statistics , and then the measurement uncertainty introduced by bias was calculated .Next the relative standard uncertainty and the relative ex‐panded uncertainty according to the measurement uncertainty introduced by bias and measurement reproducibility were calculated . The allowed total error indicators of amino acid and carnitines external quality assessment in the neonatal hereditary metabolic dis‐ease tandem mass spectrometry screening by the National Center for Clinical Laboratory were used as the target expanded uncer‐tainty .Results The relative expanded uncertainties of citrulline ,methionine ,phenylalanine ,propionyl carnitine ,octanoyl carnitine , dodecanoyl carnitine ,palmitoyl carnitine and octadecanoyl carnitine were 19 .1% -26 .1% (k=2) ,which were smaller than the tar‐get uncertainty .The relative expanded uncertainties of leucine ,tyrosine ,valine ,free carnitine were 31 .0% -43 .3% (k=2) ,which were greater than the target uncertainty .The uncertainty of isovaleryl carnitine needed to be estimated separately .Conclusion As‐sessing the measurement uncertainty of the detecting procedure of amino acids and carnitines by the non‐derivatized tandem mass spectrometer method can not only provide an opportunity for continuously improving the detection quality ,but also can help the ex‐perimental technique staffs to interpret the test data correctly and the clinician to use the detection reports correctly .

Determination of PA-824 in Rat Plasma by HPLC-MS/MS and Application in Pharmaco-kinetic Study / 医药导报

Objective To develop an HPLC-MS/ MS method for quantitative determination of PA-824 in rat plasma and to study the pharmacokinetics of PA-824 in rat after oral administration. Methods An HPLC-MS/ MS method was developed and validated for determination of PA-824 in rat plasma using metronidazole as internal standard.The proteins in plasma samples were precipitated with methanol,and PA-824 was enriched for analysis by HPLC-MS/ MS.An Inertsil? ODS3 C18 column (150 mm×4.6 mm,5 μm) was applied with mobile phase composed of methanol- 0.03% triethylamine (TEA) in water (90:10) ,at a flow rate of 0. 5 mL ? min-1 and column temperature of 30 ℃ . Quantitation was performed on a triple quadrupole mass spectrometer applying electrospray ionization technique and operating in multiple reaction monitoring (MRM) and positive ion mode with transitions at 360.1/ 175.0 for PA-824 and 172.0/ 128.0 for metronidazole.The concentration of PA-824 in plasma was tested after oral administration at various time points and the data were processed with software DAS.2.0. Results The standard calibration curve for spiked rat plasma containing PA-824 was linear over the range of 0. 1 - 10. 0 μg?mL-1 . The recoveries obtained for PA-824 were greater than 92.13%.Intra-day and inter-day coefficient of variation were less than 6.6%.After oral administration,the main pharmacokinetic parameters were AUC(0-t) : ( 3 297. 503 ± 320. 958) mg ? L-1 ? min-1 , AUC(0-∞ ) :(3 558.315±338.860)mg?L-1?min-1 ,tmax:(360.000±64.143)min,Cmax:(3.5±0.3)μg?mL-1 . Conclusion The method is rapid,accurate,simple,and successfully applied in a pharmacokinetic study of fixed dose oral administration of PA-824 in rats.

Determination of thirty antihypertensive chemical medicines adulterateds in traditional Chinese patent medicines and health foods by UPLC-MS/MS / 中国药学杂志

OBJECTIVE: To establish a method for determination of 30 antihypertensive medicines adulterated in traditional Chinese patent medicines and health foods by UPLC triple-quadrupole tandem mass spectrometer. METHODS: Multi-reactions monitoring (MRM) technology was used. The separation was performed on a Waters ACQUTTY UPLC™ BEHC18 column with a gradient mobile phase of methanol - 0.1% formic acid at flow rate of 0.2 mL · min-1. The ionization mode was ESI+. RESULTS: The standard curves of the 30 chemical medicines showed good linearity over the concentration ranges of 10 - 1000.0, 0 - 400.0 and 2.0 - 400.0 μg · L-1 (r = 0.9903-0.9998). The detection limits of the 30 chemical medicines were between 0.01 - 0.83 μg · L-1 and the average recoveries were 72.2% - 123.4% at 3 levels. CONCLUSION: The method is rapid and sensitive, and suitable for the determination of 30 chemical medicines in traditional Chinese patent medicines and health foods. Copyright 2012 by the Chinese Pharmaceutical Association.

Identification and determination of the major constituents in traditional Chinese medicine Longdan Xiegan Pill by HPLC-DAD-ESI-MS / 药物分析学报

A novel and sensitive HPLC-UV method has been developed for the simultaneous determination of twelve major compounds in Longdan Xiegan Pill.The chemical profile of the twelve compounds,including geniposidic acid（1）,geniposide（2）,gentiopicroside（3）,liquiritin（4）,crocin（5）,baicalin（6）,wogonoside（7）,baicalein（8）,glycyrrhizic acid（9）,wogonin（10）,oroxylin A（11）and aristolochic acid A（12）,was acquired using high-performance liquid chromatography-diode array detector coupled with an electrospray tandem mass spectrometer（HPLC-DAD-ESI-MS）.The analysis was performed on a Dikma Platisil ODS C18 column（250 mm×4.6 mm,5 μm）with a gradient solvent system of acetonitrile-0.1% aqueous formic acid.The validation was carried out and the linearities（r〉0.9996）,repeatability（RSD〈1.8%）,intra-and inter-day precision（RSD〈1.3%）,and recoveries（ranging from 96.6% to 103.4%）were acceptable.The limits of detection（LOD）of these compounds ranged from 0.29 to 4.17 ng.Aristolochic acid A,which is the toxic ingredient,was not detected in all the batches of Longdan Xiegan Pill.Furthermore,hierarchical cluster analysis was used to evaluate the variation of the herbal prescription.The proposed method is simple,effective and suitable for the quality control of this traditional Chinese medicine（TCM）.

Identification of homoisoflavonoids in Ophiopogon japonicus alcohol extract by an LC-MS based on precise mass and tandem mass spectrometer / 中草药

Objective: To develop an LC/MS approach based on precise mass and tandem mass spectrometer to identify homoisoflavonoids from Ophiopogon japonicus alcohol extract. Methods: Separation was performed on an Alltima C18 reverse-phase column (250 mm x 4.6 mm, 5 μm) and an Alltech pre-column. The mobile phase consisted of water containing 1 mmol/L ammonium acetate-acetonitrile containing 0.01% acetic acid was used as gradient elution. The flow rate was 1.0 mL/min. The detection wavelength was 298 nm. A combination of time-of-flight mass spectrometer (TOF/MS) and ion trap tandem mass spectrometer (Trap/MSn) was applied for qualitative analysis under negative ion mode. Results: From the alcohol extract of O. japonicus, the molecular formulae of 11 components were screened by TOF/MS and 11 chemical components were identified by Trap/MSn. All of the peaks were attributed. Conclusion: This method can be used to identify medicinal ingredients quickly and provide an effective and reliable analytical model, as well as other complex system of traditional Chinese medicine compound.

Identification and determination of the major constituents in traditional Chinese medicine Longdan Xiegan Pill by HPLC-DAD-ESI-MS / 药物分析学报

A novel and sensitive HPLC-UV method has been developed for the simultaneous determination of twelve major compounds in Longdan Xiegan Pill.The chemical profile of the twelve compounds,including geniposidic acid (1),geniposide(2),gentiopicroside(3),liquiritin(4),crocin(5),baicalin(6),wogonoside(7),baicalein(8),glycyrrhizic acid (9),wogonin (10),oroxylin A ( 11 ) and aristolochic acid A (12),was acquired using high-performance liquid chromatography-diode array detector coupled with an electrospray tandem mass spectrometer (HPLC-DAD-ESI-MS).The analysis was performed on a Dikma Platisil ODS C18 column (250 mm × 4.6 mm,5 μm ) with a gradient solvent system of acetonitrile-0.1％ aqueous formic acid.The validation was carried out and the linearities ( r ＞ 0.9996),repeatability (RSD＜1.8％),intra- and inter-day precision (RSD＜1.3％),and recoveries (ranging from 96.6％ to 103.4％ ) were acceptable.The limits of detection (LOD) of these compounds ranged from 0.29 to 4.17 ng.Aristolochic acid A,which is the toxic ingredient,was not detected in all the batches of Longdan Xiegan Pill.Furthermore,hierarchical cluster analysis was used to evaluate the variation of the herbal prescription.The proposed method is simple,effective and suitable for the quality control of this traditional Chinese medicine (TCM).

Analysis of Low Molecular Weight Proteome from H. pylori Cell Extract Using the High Performance Liquid Chromatography

Low molecular proteins (LMPs) which are smaller than 20 kDa are difficult to visible on a standard two-dimensional SDS-polyacrylamide gel electrophoresis (2-D SDS-PAGE) map. LMPs must be enriched appropriately to be analyzed. We isolated LMPs of Helicobacter pylori 26695 from 1-D polyacrylamide gel and digested by pepsin. Pepsin-digested LMPs were separated by HPLC and each fraction was analyzed by hybrid tandem mass spectrometer. Seventy nine peptides, representing 27 genes, including copper ion binding protein (CopP, 7 kDa), thioredoxin (TrxA, 11.9 kDa) and ribosomal protein L23 (Rpl23, 10.5 kDa) were identified. Some proteins larger than 40 kDa including Omp2, Omp21, Omp27, Omp30, Omp32, catalase and HP1083 were also identified. This work may give researchers a useful way to analyse the expressed LMPs which could not be identified on the conventional 2-D SDS-PAGE.

Study on Fragmentation Pathways of Amoxicillin by Electrospray Ionization Multi-stage Tandem Mass Spectrometry / 中国药房

OBJECTIVE: To investigate fragmentation pathways of amoxicillin using electrospray ionization multi-stage tandem mass spectrometry,and to provide reference for quality and quantity analysis of amoxicillin.METHODS: Amoxicillin was further purified by high performance liquid chromatography,and the fragmentation mechanisms were investigated by electrospray ionization multi-stage tandem mass spectrometer.RESULTS: The ?-lactam ring of amoxicillin has three different opening modes;2 kinds of different m/z:114 ion fragments are obtained by two different fragmentation pathways;m/z:349 ion fragments can be a characteristic fragment of amoxicillin for quality and quantity analysis of amoxicillin.CONCLUSION: The method is sensitive and accurate for further study of transformation and metabolism of amoxicillin and its quality and quantity analysis.

Identification of multi-component in Danshen Dropping Pills by using a combinative LC/MS approach / 中成药

AIM: To develop a combinative LC/MS for the identification of multi-component in Danshen Dro-(pping) Pills(Radix et Rhizoma Salviae miltiorrhizale,Radix et Rhizoma Notoginseng,Borneolum Syntheticum,etc.). METHODS: Separation was performed on a Phenomenex Luna C_(18) reverse-phase column(250 mm?4.6 mm,5 ?m).The mobile phase consisted of water containing 0.2% formic acid and acetonitrile was used as gradient elute.The flow rate was 1 mL/min.A combination of time-of-flight mass spectrometer(TOF-MS) and ion trap tandem mass spectrometer(Trap-MS~n) was applied for qualitative analysis under negative ion mode. RESULTS: Under optimized LC/MS condition,the molecular formulae of 14 components were screened by TOF-MS and 13 of them were then identified by Trap-MS~n.All of the components were surveyed and classified according to their medicinal materials derivation. CONCLUSION: The established combinative LC/MS method can thus be considered as an effective and credible pattern for the analysis of Chinese multi-herb remedy,as well as other complex systems.