Determination of urine catecholamines and metanephrines by reversed-phase liquid chromatography-tandem mass spectrometry

The measurement of urine catecholamine and metanephrine concentrations is important for biochemical screening and diagnosis of pheochromocytoma.The goal of this work was to develop a simple liquid chromatography-tandem mass spectrometry(LC-MS/MS)method for determining catecholamines and metanephrines in urine to replace an existing liquid chromatographic method using electrochemical detection.Urine samples were prepared using Oasis weak-cation-exchange cartridges.The eluate was analyzed on an Agilent ZORBAX Eclipse Plus Phenyl-Hexyl column in 3 min.Adrenaline,noradrenaline,dopamine,metanephrine,normetanephrine,and their deuterated internal standards were monitored in positive electrospray ionization mode by multiple reaction monitoring(MRM).No evidence of ion suppression was observed.The assay was linear up to 5μmol/L for adrenaline,5μmol/L for noradrenaline,6.1μmol/L for dopamine,5.6μmol/L for metanephrine,and 34.6μmol/L for normetanephrine,with lower limits of quantification of 5,5,12,6 and 7nmol/L,respectively.The intra-day and inter-day precisions for all analytes ranged from 0.59%to 4.64%and1.98%to 4.80%,respectively.External quality assurance samples were assayed and showed excellent agreement with the target values.This simple method provides an improved assay for determining urine catecholamines and metanephrines.

Stable Isotope Dilution Analysis of Gibberellin Residues in Tomato Paste by Liquid Chromatography-Tandem Mass Spectrometry

An accurate and sensitive method for the simultaneous determination of gibberellic acid（GA3）, gibberellin A4（GA4） and gibberellin A7（GA7） residues in tomato paste was developed by coupling solid phase extraction to high performance liquid chromatography-tandem mass spectrometry（LC-MS/MS） with electrospray ionization based stable isotope dilution analysis（SIDA）. The isotope labeled internal standard can compensate for the losses during the extraction and cleanup steps and for discrimination due to ion suppression. After extraction from methanol, hydrophile lipophilic balance（HLB） solid phase extraction（SPE） column was tested for the capacity of the cleanup of the tomato paste in compared with C18 SPE column which is the common way to the detection of GAs, and the former gained better result. Spiked experiments were performed in the non-contaminated tomato pastes and the recoveries of GA3, GA4 and GA7 were 42.6%―75.0% in external standard method（ESM） and 91.1%―103.8% in internal standard method（ISM） respectively. The validities of this method were investigated and good analytical performance for the three GAs was obtained, including low limits of method detection（2 ng/g for GA3 and GA4, 0.3 ng/g for GA7）, excellent linear dynamic ranges（5―500 ng/g for GA3 and GA4, 1―100 ng/g for GA7） and good relative standard deviation ranges（4.8%―9.4% for the intra-day test and 3.5%―11.9% for the inter-day test）.

Liquid Chromatography-Tandem Mass Spectrometry Assay to Detect Ethyl Glucuronide in Human Fingernail: Comparison to Hair and Gender Differences

Over the past decade, the use of hair specimens for the long-term detection of the alcohol biomarker ethyl glucuronide has been increasing in popularity and usage. We evaluated the usefulness of fingernail clippings as a suitable alterna-tive to hair for ethyl glucuronide detection. A liquid chromatography-tandem mass spectrometry method for the detection of ethyl glucuronide in fingernail clippings was fully validated and used to analyze the hair and/or fingernail specimens of 606 college-aged study participants. The limit of detection was 2 pg/mg, the limit of quantitation was 8 pg/mg and the method was linear from 8 to 2000 pg/mg. Intra- and inter-assay imprecision studies at three different concentrations (20, 40, 200 pg/mg) were all within 7.8% and all intra- and inter-assay bias studies at these levels were within 115.1% of target concentration. Ethyl glucuronide levels in fingernail (mean = 29.1 ± 55.6 pg/mg) were higher than ethyl glucuronide levels in hair (mean = 9.48 ± 22.3 pg/mg) and a correlation of the matched pairs was observed (r = 0.552, P < 0.01, n = 529). Evaluating each gender separately revealed that the correlation of male fingernail to male hair was large and significant (r = 0.782, P < 0.01, n = 195) while female hair to female fingernail was small yet sig-nificant (r = 0.249, P < 0.01, n = 334). The study results demonstrated that fingernail may be a suitable alternative to hair for ethyl glucuronide detection and may be the preferred sample type due to the lack of a gender bias.

Rapid and Sensitive Liquid Chromatography-Tandem Mass Spectrometry for Quantification of Glycyrrhetic Acid in Human Plasma

A simple, rapid and sensitive liquid chromatography-tandem mass spectrometry（LC-MS/MS） for the determination of glycyrrhetic acid in human plasma with ginsenoside Rh2 as internal standard was developed and validated. The plasma samples were prepared via liquid-liquid extraction with ethyl acetate. Chromatographic separation was accomplished on a Venusil MP-C18（50 mm×2.1 mm, 5 μm i.d.） column at 25 °C. The mobile phase consisted of acetonitrile/5 mmol？L-1 ammonium acetate（10：90, volume ratio） at a flow rate of 0.4 mL/min. Negative electrospray ionization was utilized as the ionization source. Glycyrrhetic acid and internal standard were determined via the mutiple reaction monitoring of precursor→production ion transitions at m/z 469→425, 409 and m/z 621→161, respectively. Each sample was chromatographed within 2.5 min. The lower limit of quantification was 0.50 ng/mL for 200 μL of plasma sample and the linear range was from 0.50 ng/mL to 800 ng/mL. The intra- and inter-day precisions were less than 8.76% in terms of relative standard deviation（RSD）, and the accuracy was within a range of -3.25%-1.32% in terms of relative error（RE）. The method was successfully applied to the pharmacokinetic studies of glycyrrhetic acid in healthy male Chinese volunteers after a single oral administration of 75 mg of glycyrrhizin.

Simultaneous quantification of several classes of antibiotics in water, sediments, and fish muscles by liquid chromatography-tandem mass spectrometry

Precise and sensitive methods for the simultaneous determination of different classes of antibiotics, including sulphonamides, fluoroquinolones, macrolides, tetracyclines, and trimethoprim in surface water, sediments, and fish muscles were developed. In water samples, drugs were extracted with solid-phase extraction （SPE） by passing 1000 mL of water through hydrophilic lipophilic balanced （HLB） SPE cartridges. Sediment samples were solvent-extracted, followed by tandem SPE （strong anion exchange （SAX） ＋ HLB） clean-ups. Fish muscles were extracted by a mixture of acetonitrile and citric buffer （80：20, v/v） solution, and cleaned by SPE. Liquid chromatography-tandem mass spectrometry （LC-MS/ MS） with multiple reaction monitoring （MRM） detection was employed to quantify all compounds. The recoveries for the antibiotics in the spiked water, sediment, and fish samples were 60.2%-95.8%, 48.1%-105.3%, and 59.8%- 103.4%, respectively. The methods were applied to samples taken from Dianchi Lake, China. It showed that concentrations of the detected antibiotics ranged from limits of quantification （LOQ） to 713.6 ng- L1 （ofloxacin） in surface water and from less than LOQ to 344.8 μg·kg-1 （sulphamethoxazole） in sediments. The number of detected antibiotics and the overall antibiotic concentrations were higher in the urban area than the rural area, indicating the probable role of livestock and human activities as important sources of antibiotic contamination. In fish muscles, the concentration of norfioxacin was the highest （up to 38.5 μg·kg-1）, but tetracyclines and macrolides were relatively low. Results showed that the methods were rapid and sensitive, and capable of determining several classes of antibiotics from each of the water, sediment, and fish matrices in a single run.

Therapeutic Drug Monitoring of Vancomycin and Voriconazole by Liquid Chromatography-Tandem Mass Spectrometric Method

A reliable liquid chromatography-tandem mass spectrometric（LC-MS/MS） method was developed and va- lidated for the simultaneous determination of vancomycin and voriconazole in human plasma. The analytes and internal standard 10-hydroxycarbazepine were separated at a flow rate of 0.9 mL/min using a Zorbax SB-C18 column （50 min×4.6 mm, 2.7 μm）. Positive ion electrospray ionization was used to detect vancomycin, voriconazole and intrenal standard（IS） 10-hydroxycarbazepine followed by multiple reaction monitoring（MRM） of the transition at m/z 725.5→144.2, 350.3→281.0 and 253.1→208.0, respectively. The total run time for both vancomycin and voriconazole samples was 5 min; 0.30μg/mL was the lower limit of quantification. The precision of intraday and interday was no more than 12.4%. The method was successfully and resoundingly applied in therapeutic drug monitoring of 156 patients treated with vancomycin and voriconazole.

Rapid Quantification of Astilbin in Rat Plasma by Liquid Chromatography-tandem Mass Spectrometry and Its Application to Pharmacokinetic Study

Astilbin is a potential immunosuppressive agent with minor cytotoxicity. Its oral bioavailability is supposed to be rather low and therefore a sensitive analytical method is required for its pharmacokinetic study after oral administration. A simple, sensitive and rapid liquid chromatography-tandem mass spectrometry（LC-MS/MS） method was developed and validated for the determination of astilbin in rat plasma. Plasma samples were subjected to liquid-liquid extraction with ethyl acetate and separated by reversed phase high performance liquid chromatography（HPLC） with methanol-0.01%（volume fraction） formic acid（50：50, volume ratio） as mobile phase. Quantitive determination was achieved on negative LC-MS/MS by a multiple reaction moitoring method with transitions m/z 449.1→150.9（quantifier） and m/z 449.1→284.9（qualifier） for astilbin and m/z 128.9→42.0 for internal standard（IS）. A lower limit of quantification（LLOQ） of ng/mL was achieved within a short cycle time of 3.4 min. The method was successfully applied to a pharmacokinetic study involving oral and intravenous administrations of 6 mg/kg astilbin to six rats.

Exploring Chemovar-Specific Cannabis Extracts Quantification and Evaluation of Cytotoxic Compounds for Targeting Glioblastoma Multiforme

Glioblastoma Multiforme (GBM) represents one of the most aggressive and metastatic brain tumors, with a dismal success rate of less than three percent after five years, particularly in tumors with active immune checkpoints. This necessitates the development of targeted endogenous agents for precise GBM treatment. Previous experiments utilizing Chemovar Specific Cannabis Extractions (CSCEs), fractionated with polar solvents and quantified using Liquid and Gas Column Chromatography combined with Mass Spectrometry (LC/GCMS), have shown reduced viability and motility in human GBM cell lines. However, the complexity of the botanical substance has hindered the personalization of standard cannabis medicines for GBM due to unknown synergistic effects of multiple compounds. To address this limitation, our study focuses on exposing AM251 cells to chemovar fractions extracted using a non-polar solvent, thereby isolating a broader spectrum of constituents. By employing LC/GCMS in conjunction with Nuclear Magnetic Resonance (NMR), we have identified and quantified nine* compounds present in the non-polar CSCE that exhibit significant efficacy (0.1 μM) in inducing cytotoxicity* in GBM tumor cells. Conversely, the polar fraction in our experiment did not demonstrate efficacy against UM251 cells. The quantification of individual compounds within a cannabis extraction that selectively induces cell death in brain tumors holds promise for guiding future research and facilitating the development of a standardized CSCE for GBM therapy.

高效液相色谱-串联质谱法测定比格犬血浆中的莫诺苷浓度及其药代动力学

运用高效液相色谱-串联质谱联用技术，建立了简单、快速、灵敏的比格犬灌胃莫诺苷后血药浓度的检测方法。血浆样品采用蛋白质沉淀法处理，以芍药苷作为内标，色谱柱为 Inertsil ODS-SP 色谱柱（50 mm ×2.1 mm，5μm），流动相为水（含1 mmol / L 甲酸钠）-乙腈，梯度洗脱，流速0.4 mL / min。采用电喷雾离子源（ ESI），正离子多反应监测（MRM）模式。绘制血药浓度-时间曲线，并采用 DAS 2.0软件计算药代动力学参数。方法学实验结果表明内源性杂质不干扰莫诺苷和内标的测定，线性范围为2～5000μg / L（ r =0.9966），定量限为2μg / L。方法精密度、准确度、回收率和基质效应均符合生物样品测定的要求，适合比格犬血浆中莫诺苷浓度的测定，可以应用该方法进行莫诺苷的药代动力学研究。比格犬灌胃莫诺苷3个剂量（5、15、45 mg / kg）后的血药浓度-时间曲线下面积（AUC（0-∞））分别为（1631.20±238.50）、（3984.05±750.38）、（10397.64±3156.34）μg / L＆#183;h，与给药剂量之间呈现良好的线性关系。

废弃电路板环氧树脂真空热解及产物分析

在真空条件下,应用程序升温的管式炉反应器对废弃电路板中环氧树脂热解规律进行研究,考察不同的热解终温、升温速率、真空度(压力)及保温时间等各种因素对产物产率的影响。此外,利用傅里叶红外(FT-IR)和气质联用(GC/MS)技术对热解油产物进行表征分析。实验结果表明:温度对产物产率的影响最大,升温速率、真空度及保温时间对热解产物产率也有重要影响。选择适当的热解温度(400～550℃)、升温速率(15～20℃/min)、真空度(压力15kPa)及保温时间(30min)有利于提高热解液体产品的产率;热解油的主要成分是酚类物质,其总含量为84.08%,其中,含溴化合物含量为15.34%。

在线固相萃取/液相色谱-串联质谱法快速检测全血及尿液中15种杀鼠药

建立了同时测定血液和尿液样品中15种杀鼠药的在线固相萃取/液相色谱-三重四极杆串联质谱(On-line SPE/LC-MS/MS)分析方法。用乙腈沉淀样品中的蛋白质,经稀释、离心、过滤后直接进样。通过在线固相萃取柱HLB富集纯化,以ZORBAX Eclipse Plus C18柱为分析柱,乙腈-0.01 mol/L乙酸铵水溶液为流动相进行梯度洗脱。采用电喷雾电离(ESI+/ESI-)快速正负切换模式,动态多反应监测模式(DMRM)扫描,外标法定量。结果表明,15种杀鼠药采用二次方程拟合时线性关系良好,血液中r^2≥0.997 8,尿液r^2≥0.996 5,方法的检出限和定量下限分别为0.10~5.00μg/L和0.50~10.0μg/L,3个添加水平下的回收率为81.8%~109.6%,日内相对标准偏差(RSD)为0.3%~3.6%,日间RSD为0.3%~3.8%(n=6)。该方法简单方便,灵敏度高,能够用以血液和尿液样品中15种杀鼠药的快速筛查和准确定量。

同位素稀释高压液相色谱-串联质谱法测定鱼虾中丙酸睾酮残留量

目的：采用高压液相色谱-串联质谱(high pressure liquid chromatography-tandem mass spectrometry, HPLC-MS/MS)快速检测鱼虾等水产品中丙酸睾酮残留。方法向捣碎匀浆后的样品中加入内标诺龙-D3混匀后,叔丁基甲醚超声提取,-80℃冷冻30 min后12000 r/min离心净化。以Eclipse Plus C18为色谱分离柱,以乙腈-0.1%甲酸水溶液为流动相,流速为0.3 mL/min,采用三重四极杆质谱在正离子模式下进行选择反应离子监测。结果丙酸睾酮在0.5~100 ng/mL范围内呈线性关系,相关系数为0.9989；方法定量限为0.5μg/kg。当添加水平为0.5~10.0μg/kg时,平均回收率为71.2%~104.5%,相对标准偏差为2.26%~5.65%。结论该方法操作简单、快速、灵敏度高,适用于快速检测水产品中丙酸睾酮的残留量。

加速溶剂萃取-液相色谱-串联质谱法分析烟叶中的92种农药残留

建立了一种同时检测烟叶中92种农药残留的加速溶剂萃取-液相色谱-串联质谱方法.通过在加速溶剂萃取池底部放置净化剂,将烟叶样品的提取、净化一步完成,之后采用液相色谱-串联质谱（LC-MS/MS）的多反应监测模式（MRM）进行分析.结果表明：①最优实验条件为：提取溶剂乙腈,净化剂4 9弗罗里硅土（florisil）和40 mg石墨化炭黑（GCB）,提取温度120 ℃,提取压力10 MPa（1500 psi）,静态提取时间6 min,冲洗体积50％,静态循环2次.②目标物基质匹配标准曲线线性良好（r2＞0.99）,0.1,0.5和1 mg/kg 3种加标水平的回收率在70％～124％之间,相对标准偏差（RSD）不大于20％.该方法准确度和精密度好,适合用于烟草样品中92种农药残留的检测.

超高压液相色谱-串联质谱法同时测定人乳中的硫胺素、核黄素、烟酰胺、泛酸和吡哆醛

目的建立一种快速、灵敏的同时测定人乳中多种B族维生素的超高压液相色谱-串联质谱法。方法样品经甲醇沉淀蛋白、乙醚去除非极性杂质,UPLC HSS T3色谱柱分离后采用保留时间和多反应离子检测(MRM)定性、定量,同位素内标校正。结果维生素B1(硫胺素)、维生素B2(核黄素)和维生素B6(吡哆醛)的定量下限(LOQ)均为0.2μg/100 g,维生素B3(烟酰胺)和维生素B5(泛酸)的LOQ均为0.3μg/100 g。标准加入法测定所有被测物的高、中、低三个浓度加标水平的回收率在93.1%~101.1%之间,相对标准偏差(RSD)在2.8%~6.2%之间。5种水溶性维生素在各自浓度范围内线性良好,R2均大于0.998。结论本文建立了同时测定人乳中5种B族水溶性维生素的超高压液相色谱-串联质谱法。方法前处理简单、快速,样品需要量少;采用质谱检测器同位素内标一对一校正定量,检测结果准确、灵敏。方法可以为人乳中维生素含量的检测、调查、研究提供技术支持。

高效液相色谱-串联质谱法测定食品添加剂中蛇床子素含量

To establish HPLC electrospray ionization mass spectrometry (HPLC-ESI MS) method for determination of osthole in food additive.The samples were dipped in methanol overnight and extracted with it by ultrasonic instrument,then the supernatant was send to sampler for detection.Sample was chromatographed using 0.4%HAc solution-MeOH (35:65,by volume) mobile phase on an Xterra MS C18 column.Analyte determination was performed by ESI MS/MS in the multiple reaction monitoring(MRM)mode.The established method of LC-MS/MS determining the concentration of osthole in food additive has the characteristic of strong specificity,high sensitivity and good reproducibility.The linear range of papaverine was 0.001-10 μg/mL,method recovery was 97.42%,RSD was 8.37%,the limit of detection was 1 ng/mL and the limit of quantitation was 0.001 μg/mL.

First observation of domoic acid and its isomers in shellfish samples from Shandong Province,China

A total of 133 shellfish samples were collected in seven cities of Shandong Province,China,from May to October,2019.The domoic acid(DA)concentrations were determined by liquid chromatographytandem mass spectrometry(LC-MS/MS),and their distribution characteristics were investigated.DA concentration was detected high in over 1/3(36.1%)of the samples of four kinds of shellfish in all three seasons in range from 0 to 102μg/kg.The highest DA concentrations were 102,101,36.7,and 10.2μg/kg in Crassostrea gigas,Chlamys farreri,Mactra veneriformis,and Mytilus edulis,respectively.Geographically,Yantai(22.0μg/kg)and Weihai(16.9μg/kg)showed relatively high concentrations of DA,whereas Rizhao and Dongying presented only 0.85-and 1.76-μg/kg DA,respectively.DA concentrations in the shellfish samples were strongly related to seasonal changes,being significantly higher in autumn and summer than that in spring.The DA risk exposure assessments indicate that dietary seafood consumption did not pose a health threat to general human population.In addition,three isomers(isoA,isoD,isoE)and 5′-epimer DA were detected in 3.00%-15.80%of the shellfish samples.This study is the first to observe DA and its isomers in shellfish samples of Shandong Province.The results demonstrate that DA contamination is very common and should be continuously monitored.

Characterization of a Highly Potent Insecticidal Lectin from <i>Colocasia esculenta</i>Tuber and Cloning of Its Coding Sequence

Hemipteran insects are the most devastating pest for different crops of high economic value. Colocasia esculenta tuber agglutinin (CEA), a mannose binding monocot lectin from araceae family was previously reported by the present group to be effective against some members of this class of pests. In the present study, efficacy of this potent lectin has been extended to cotton aphid (Aphis gossypii) which is becoming a highly damaging pest of cotton in recent days. Because, like other aphids, A. gossypii not only extracts the phloem fluid but also transmit disease causing viruses and add to the high degree of yield loss. Efficacy of the lectin on cotton aphid as well as other hemipteran insects prompted us further to clone the protein coding gene. Very little sequence information of this gene was available in the database. Hence, attempt had been made to study the protein through liquid chromatography-tandem mass spectrometry (LC-MS/MS) to have the detailed peptide information. On the basis of the peptide homology information obtained from LC-MS/MS the complete coding sequence of CEA was determined. The coding sequence corresponding to CEA was cloned further using primers designed on the basis of above information and genome walk technology for its potential utilisation in insect management programme.

Targeted Metabolomics Based on LC-MS/MS Revealing Alteration of Bile Acids in Male Migraine Patients

Migraine is an episodic neurological disorder and the second most disabling disease with unclear pathogenesis.Since dietary adjustment and probiotics supplement can improve the symptoms of migraine,the intestinal flora metabolites of bile acids(BAs)attract attentions in this work.21 BAs,including cholic acid(CA),chenodeoxycholic acid(CDCA),deoxycholic acid(DCA),lithocholic acid(LCA),ursodeoxycholic acid(UDCA),hyocholic acid(HCA),hyodeoxycholic acid(HDCA)and their glycine-and taurine-conjugated species,were compared in serum of migraine patients and healthy controls using liquid chromatography-tandem mass spectrometry(LC-MS/MS),which is the first study about the correlation between BAs and migraine.Two secondary BAs,DCA and LCA as well as their glycine-and taurine-conjugated forms,were demonstrated with significant difference between male patients and male controls,while no obvious difference was found in the two female groups.The result indicated that the variation of BAs might be gender-related when referred to migraine,which would emphasize the importance of gender-stratified analysis for the disease with varying morbidity in male and female.Five differential metabolites may serve as potential serum biomarkers for the male migraine patients,providing a new sight for the understanding and biomarker exploring of the migraine in male.

LC-MS/MS Method for the Quantitation of Cefotetan in Human Plasma and Its Application to Pharmacokinetic Study

A rapid and sensitive liquid chromatography-tandem mass spectrometry（LC-MS/MS） method for the de- termination of cefotetan in human plasma was developed and validated. After the protein precipitation of sample with acetonitrile, the analyte and internal standard（IS）, tramadol, were separated on a Zorbax XDB C8 column using ace- tonitrile/1%（volume fraction） formic acid（volume ratio 35：65, pH=2.5） as mobile phase at a flow rate of 1.0 mL/min with a 1 ： 1 split. The detection was performed by electrospray ionization with positive ion mode, followed by multiple reaction monitoring of the transitions for cefotetan at m/z 576.3→460.2（quantifier） and m/z 576.3→432.2（qualifier） and for IS at m/z 264.1→58.1. Cefotetan and IS were eluted at 1.86 and 1.87 rain, respectively. The assay was linear over the concentration range of 0.1-100 gg/mL for 20 μL of human plasma only with intra- and inter-day preci- sions（expressed as the relative standard deviation） of less than 6.62% and accuracies（as relative error） of ＋1.31%. The method was applied to the pharmacokinetic study of a l-h intravenous infusion of 1.0 g of cefotetan disodium for human volunteers（n=6）.

Quantitative Analysis of Retinoic Acid Combining 2-Nitrophenylhydrazine Derivatization with LC-MS/MS

All-trans-retinoic acid(atRA)plays an essential role in organ formation and differentiation,tissue cell proliferation,and apoptosis,and is closely related to the occurrence and development of various diseases.Liquid chromatography-tandem mass spectrometry(LC-MS/MS)has become a powerful tool for metabolic biological analysis because of the high sensitivity,specificity and throughput.We have developed a new method for the quantification of atRA by chemical derivatization with 2-nitrophe-nylhydrazine(2-NPH)for the first time,which reduced the interference of detection by changing the ion mass of the molecule.atRA from its endogenous geometric isomers,such as,9-cis-RA and 13-cis-RA,has been baseline resolved within 15 minute under the optimized chromatographic conditions.The method was applied to measure atRA in mouse serum and tissues including liver,kidney,brain,pancreas,and subcutaneous tissue.The method is easy to operate,highly sensitive and well selective,and can be used for accurate and rapid determination of at RA in biological samples.