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.

Calcium-chelating peptides from rabbit bone collagen:characterization,identification and mechanism elucidation

This study aimed to characterize and identify calcium-chelating peptides from rabbit bone collagen and explore the underlying chelating mechanism.Collagen peptides and calcium were extracted from rabbit bone by instant ejection steam explosion(ICSE)combined with enzymatic hydrolysis,followed by chelation reaction to prepare rabbit bone peptide-calcium chelate(RBCP-Ca).The chelating sites were further analyzed by liquid chromatography-tandem mass(LC-MS/MS)spectrometry while the chelating mechanism and binding modes were investigated.The structural characterization revealed that RBCP successfully chelated with calcium ions.Furthermore,LC-MS/MS analysis indicated that the binding sites included both acidic amino acids(Asp and Glu)and basic amino acids(Lys and Arg),Interestingly,three binding modes,namely Inter-Linking,Loop-Linking and Mono-Linking were for the first time found,while Inter-Linking mode accounted for the highest proportion(75.1%),suggesting that chelation of calcium ions frequently occurred between two peptides.Overall,this study provides a theoretical basis for the elucidation of chelation mechanism of calcium-chelating peptides.

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.

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。

超高效液相色谱串联四极杆质谱联用分析番茄酱中苏丹红Ⅰ～Ⅳ含量

The method of simultaneous analysis of Sudan Red Ⅰ-Ⅳ residues in ketchup was developed with an ultra performance liquid chromatography-tandem mass spectrum.Acetonitrile was used to extract the substance from the ketchup,and the upper layer was detected by the UPLC-MS/MS after refrigeration and centrifuge.The sample was separated on a Waters Acquity BHE C18 column,and detected by the MRM mode of the mass spectrum.The LOD was 2 ng/mL and the LOQ was 5 ng/mL all of the four substances.The average recoveries were between 67.42%-84.59% of the 100,200,300 μg/kg three spiked concentration levels and the RSD values were between 2.87%-9.57%.

超高效液相色谱串联四极杆质谱联用分析动物血样中乌头类生物碱

An ultra performance liquid chromatography-tandem mass spectrum method has been developed for the determination of three aconitum alkaloids (aconitine,hypaconitine and mesaconitine).The three alkaloids were analyzed simultaneously with an Waters Acquity BHE C18 column by gradient elution using 0.05% aqueous ammonia-acetonitrile as mobile phase and detected by the MRM mode of the mass spectrum.The recoveries of the SPE method were between 68.79%-95.65% (RSD<7.94%,n=4),and all the alkaloids showed good linearity (r>0.998) in a relatively wide concentration range.The LOD reached 0.0516,0.0640,0.0744 ng/mL of aconitine,hypaconitine and mesaconitine,respectively.The analysis time was within 3 min which could meet the high-throughput detection.The results indicated that contents of alkaloids in animal blood can be well detected;and this method can be used in quality control of aconitum drugs and pharmacokinetics study.

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.

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.

Application of polypyrrole nanofibers in the selective extraction of methotrexate and its polyglutamate metabolites from hospital effluents

A novel solid-phase extraction(SPE)strategy based on polypyrrole(Ppy)nanofibers was developed for the determination of trace methotrexate and its polyglutamate metabolites(MTXs)in hospital effluents.Ppy was coated on the surface of electrospun polystyrene nanofibers by in situ oxidative polymerization to form Ppy electrospun nanofibers.The mechanism of adsorption on MTXs was explored through static adsorption studies.The MTX contents,after extraction,were determined by liquid chromatography-tandem mass spectrometry analysis.Results show that the physical/chemical adsorption of targets occurs on the surface of Ppy nanofibers,which is most likely dominated by multiple adsorptions and heterogeneous adsorption sites.Ppy nanofibers exhibit satisfying extraction performance.The content of targets detected in medical wastewater samples ranges from 21 to 2908 ng/L.The novel strategy based on Ppy nanofiber SPE can extract trace MTXs effectively,guarantee analytical accuracy,and circumvent the storage and transportation of water samples during on-site sampling operations.