Analysis of semicarbazide in baby food by liquid chromatography tandem mass spectrometry (LC-MS-MS)--in-house method validation

A method for detection of semicarbazide (SEM) in baby food was validated. SEM was extracted with hydrochloric acid and derivatised with 2-nitrobenzaldehyde, using [15N2,13C] semicarbazide as internal standard. The extract was neutralised, purified on a solid phase extraction cartridge and SEM was determined by reversed phase LC-MS-MS. Linearity was demonstrated in the ranges from 0.1 ng ml(-1) to 1 ng ml(-1) and from 2 ng ml(-1) to 80 ng ml(-1). Matrix effects were non significant for meat-based and significant for apple and rice-based baby foods, in both ranges. Mean recoveries ranged from 87.8% to 107.2% with relative standard deviation from 0.2% to 9.1%, considering both ranges. Limits of detection and quantification were 0.1 microg kg(-1) and 0.25 microg kg(-1), respectively. The results of the validation process demonstrated the method suitability for use in food control.     

A liquid chromatography/positive ion tandem mass spectrometry method for the determination of cilazapril and cilazaprilat in human plasma

A simple, rapid, and sensitive high-performance liquid chromatography (HPLC)-electrospray ionization (ESI) tandem mass spectrometric method (LC-MS/MS) has been developed for simultaneous determination of cilazapril levels and its active metabolite, cilazaprilat, in human plasma using enalapril as internal standard. The acquisition was performed in the multiple reaction monitoring mode; monitoring the transitions: m/z 418.4 > 211.1 for cilazapril and m/z 390.3 > 211.1 for cilazaprilat. The method involves a simple single-step liquid-liquid extraction with ethyl acetate. The analyte was chromatographed on an YMC C₈ reversed-phase chromatographic column by isocratic elution with 10 mM ammonium formate buffer-methanol (10:90, v/v; pH 3.2 with formic acid). Numerous compounds did not interfere with specific multiple reaction monitoring in tandem mass spectrometric detection following C₈ reversed-phase chromatographic separation under conditions that eluted cilazapril, cilazaprilat, and enalapril within 2 min. This method was validated over 0.1-500 ng ml⁻¹ of cilazapril and 0.5-50 ng ml⁻¹ of cilazaprilat. Cilazapril and cilazaprilat were stable in standard solution and in plasma samples under typical storage and processing conditions. The assay was successfully applied to a pharmacokinetic study of cilazapril given as a single oral dose (5 mg) to healthy volunteers.     

Method validation of microcystins in water and tissue by enhanced liquid chromatography tandem mass spectrometry

A liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI–MS/MS) method has been developed and validated to identify and quantify trace levels of cyanotoxins or microcystins (MC) in water, bivalves and fish tissue with enhanced sensitivity and specificity. The method enables confirmation and quantification of six MCs (MC-LA, LF, LR, LW, RR and YR) with a single chromatographic run. The applied chromatography also allows determination of certain MC metabolites (Desmethyl-LR and -RR). By using LC-ESI–MS/MS in multiple reaction monitoring (MRM) mode, the limit of detection and quantitation for the microcystins studied, were determined to be between 0.2 and 1 pg on column (5:1 S/N ratio). These values are below the 2 pg detection limits found in the available literature.     

Analysis of urinary aromatic acids by liquid chromatography tandem mass spectrometry

The separation and detection of 11 urinary aromatic acids was developed using HPLC-MS/MS. The method features a simple sample preparation involving a single-step dilution with internal standard and a rapid 8 min chromatographic separation. The accuracy was evaluated by the recovery of known spikes between 87 and 110%. Inter- and intra-assay precision (CV) was below 11% in all cases and the analytes were observed to be stable for up to 8 weeks when stored at -20 degrees C. The method was validated based upon linearity, accuracy, precision and stability and was used to establish reference intervals for children and adults.     

Dissipation of carbendazim and its metabolites in cucumber using liquid chromatography tandem mass spectrometry

Currently, there is growing interest in the degradation pathways of organic contaminants such as pesticides. In the case of pesticides, the determination of metabolites in agricultural products and environment is necessary as some of them could present similar toxicity to or even higher toxicity than the parent compound. The development of analytical methodology for the identification and quantification of carbendazim fungicide and its metabolites in cucumber was studied. Cucumber (cucumis sativus) is a global food in terms of economic importance and nutritional quality. Careful optimisation of the liquid chromatography–mass spectrometry (LC-MS)/MS parameters was achieved in order to attain a fast separation with the best sensitivity. The detection was carried out on an Ion-Trap tandem mass spectrometer (MS/MS) by electrospray ionisation in positive ion mode (ESI+) with multiple reaction monitoring (MRM).     

Multi-residue confirmatory method for the determination of twelve coccidiostats in chicken liver using liquid chromatography tandem mass spectrometry

The confirmatory LC-MS/MS method for the determination of residues for twelve coccidiostats including ionophore antibiotics (lasalocid, maduramycin, monensin, narasin, salinomycin, semduramycin) and chemical coccidiostats (clazuril, decoquinate, diclazuril, halofuginone, nicarbazin and robenidine) in poultry liver has been developed. The sample preparation was based on extraction with acetonitrile, defatting with Alumina columns and clean-up on Oasis HLB spe. The separation of analytes was performed on PhenylHexyl column with a gradient of acetonitrile, methanol and the ammonium formate pH 4.0. For all analytes, at least 2 diagnostic fragmentation ions were monitored. The validation, performed according to the CD 2002/657/EC, proved the suitability of the method for the confirmatory analysis of the coccidiostats. For lasalocid, however, low reproducibility was observed and the proper quantification could not be performed with this method.     

Rapid confirmatory method for the determination of 11 nitroimidazoles in egg using liquid chromatography tandem mass spectrometry

A rapid confirmatory method has been developed and validated for the simultaneous identification, confirmation and quantitation of 11 nitroimidazoles in eggs by liquid chromatography tandem mass spectrometry (LC–MS/MS). The method is validated in accordance with Commission Decision 2002/657/EC and is capable of analysing metronidazole (MNZ), dimetridazole (DMZ), ronidazole (RNZ), ipronidazole (IPZ) and their hydroxy metabolites MNZ-OH, HMMNI (hydroxymethyl, methyl nitroimidazole), IPZ-OH. The method is also capable of analysing carnidazole (CRZ), ornidazole (ORZ), tinidazole (TNZ) and ternidazole (TRZ). MNZ, DMZ and RNZ have been assigned a recommended level (RL) of 3 μg kg⁻¹ by the Community Reference Laboratory (CRL) in Berlin. The developed method described in this study is easily able to detect all the nitroimidazole compounds investigated at this level and below. Egg samples are extracted with acetonitrile, and NaCl is added to help remove matrix contaminants. The acetonitrile extract undergoes a liquid–liquid wash step with hexane; it is then evaporated and reconstituted in mobile phase. The reconstituted samples are analysed by liquid chromatography tandem mass spectrometry (LC–MS/MS). The decision limits (CCα) range from 0.33 to 1.26 μg kg⁻¹ and the detection capabilities (CCβ), range from 0.56 to 2.15 μg kg⁻¹. The results of the inter-assay study, which was performed by fortifying hen egg samples (n = 18) on three separate days, show the accuracy calculated for the various analytes to range between 87.2 and 106.2%. The precision of the method, expressed as %CV values for the inter-assay variation of each analyte at the three levels of fortification (3, 4.5 and 6.0 μg kg⁻¹), ranged between 3.7 and 11.3%. A Day 4 analysis was carried out to examine species variances in eggs from different birds such as duck and quail and investigating differences in various battery and free range hen eggs.     

Determination of triapine,a ribonucleotide reductase inhibitor,in human plasma by liquid chromatography tandem mass spectrometry

Triapine is an inhibitor of ribonucleotide reductase (RNR). Studies have shown that triapine significantly decreases the activity of RNR and enhanced the radiation‐mediated cytotoxicity in cervical and colon cancer. In this work, we have developed and validated a selective and sensitive LC‐MS/MS method for the determination of triapine in human plasma. In this method, 2‐[(3‐fluoro‐2‐pyridinyl)methylene] hydrazinecarbothioamide (NSC 266749) was used as the internal standard (IS); plasma samples were prepared by deproteinization with acetonitrile; tripaine and the IS were separated on a Waters Xbridge Shield RP₁₈ column (3.5 µm; 2.1 × 50 mm) using a mobile phase containing 25.0% methanol and 75.0% ammonium bicarbonate buffer (10.0 mm , pH 8.50; v/v); column eluate was monitored by positive turbo‐ionspray tandem mass spectrometry; and quantitation of triapine was carried out in multiple‐reaction‐monitoring mode. The method developed had a linear calibration range of 0.250–50.0 ng/mL with correlation coefficient of 0.999 for triapine in human plasma. The IS‐normalized recovery and the IS‐normalized matrix factor of triapine were 101–104% and 0.89–1.05, respectively. The accuracy expressed as percentage error and precision expressed as coefficient of variation were ≤±6 and ≤8%, respectively. The validated LC‐MS/MS method was applied to the measurement of triapine in patient samples from a phase I clinical trial. Copyright © 2015 John Wiley & Sons, Ltd.     

Simultaneous determination of ramipril, ramiprilat and telmisartan in human plasma using liquid chromatography tandem mass spectrometry

A rapid and sensitive liquid chromatography tandem mass spectrometry method has been developed and validated for the simultaneous determination of ramipril, ramiprilat and telmisartan in human plasma. The solid-phase extraction technique was used for the extraction of ramipril, ramiprilat and telmisartan from human plasma. Trandolaprilat and hydrochlorothiazide were used as the internal standards (ISs). Chromatography was performed on a Hypurity C18, 5 μm, 50 mm × 4.6 mm column, with the mobile phase consisting of ammonium acetate and acetonitrile (in a 20:80 ratio), followed by detection using mass spectrometry. The method involves a simple reversed isocratic chromatography condition and mass spectrometry detection, which enables detection at sub-nanogram levels. The method was validated and the lower limit of quantification for ramipril, ramiprilat and telmisartan was found to be 0.1 ng mL⁻¹, 0.1 ng mL⁻¹ and 2 ng mL⁻¹, respectively. The mean recovery for ramipril, ramiprilat and telmisartan ranged from 90.1 to 104.1%. This method increased the sensitivity and selectivity; resulting in high-throughput analysis of ramipril, ramiprilat and telmisartan using two different ISs in a single experiment for bioequivalence studies, with a chromatographic run time of 1.5 min only.     

高效液相色谱-串联质谱法快速测定6类食品中增效醚的残留

建立了水果、蔬菜、粮谷、油料、动植物脂类、动物源性食品等6类食品中增效醚残留量快速测定的液相色谱-质谱/质谱(LC-MS/MS)方法。样品盐析并除水后,用三氯甲烷提取,氟罗里硅土填料固相萃取柱净化,丙酮-三氯甲烷混合溶剂洗脱,以乙腈-0.1%甲酸为流动相,在Zobax SB C18液相色谱柱完成分离,并于电喷雾正离子多反应监测模式下质谱测定。考察了提取溶剂、净化方法、柱容量、仪器条件、基质效应对分析结果的影响。增效醚质量浓度在0.5～100μg/L范围内,线性相关系数(r)为0.9976,方法定量限(LOQ)为10μg/kg。17种食品基质添加LOQ、低MRL、2倍MRL、高MRL 4个不同浓度水平时,回收率在80.3%～96.3%之间,相对标准偏差为2.7%～14%,方法可以满足多种食品基质中增效醚残留量的定性和定量检测要求。     

Rapid simultaneous determination of 14 sulfonamides in wastewater by liquid chromatography tandem mass spectrometry

This paper describes a rapid method for the determination of 14 kinds of sulfonamides (SAs) in wastewater using SPE, and LC-MS/MS with positive ESI (ESI(+)) and selected reaction monitoring (SRM) mode. The SPE was performed on an Oasis hydrophilic-lipophilic-balanced (HLB) cartridge. Chromatographic separation on a C18 column was achieved using a binary eluent containing methanol and water with 0.2% formic acid. Typical recoveries of the analytes ranged from 22.3 to 87.0% at a fortification level of 100 ng/L. The LODs in wastewater except sulfathiazole (3 ng/L) could be detected and quantified at levels as low as 1 ng/L. Finally, the method was applied to water from the municipal outlet and the aquaculture wastewater effluent. Sulfamethazine (SM(2)), sulfamethoxypyridazine (SMP), and sulfamethoxazole (SMZ) were most frequently found in wastewater in a concentration range between 1.2 and 31.7 ng/L.     

On-line extraction coupled with liquid chromatography tandem mass spectrometry for quantitation of pravastatin and its metabolite in human serum

A high-throughput bioanalytical method for simultaneous quantitation of pravastatin and its metabolite (M1) in human serum was developed and validated using on-line extraction following liquid chromatography tandem mass spectrometry (LC-MS/MS). The on-line extraction was accomplished by the direct injection of a 50 microL serum sample, mixed 4:1 with an aqueous internal standard solution, into one of the extraction columns with aqueous 1 mm formic acid at flow rate of 3 mL/min. The separation and analysis were achieved by back-eluting the analytes from the extraction column and the analytical column to the mass spectrometer with an isocratic mobile phase consisting of 62% aqueous 1 mm formic acid and 38% acetonitrile at a flow rate of 0.8 mL/min. The second extraction column was being equilibrated while the first column was being used for analysis, and vice versa. The standard curve range was 0.500-100 ng/mL for pravastatin and M1. The lower limit of quantitation, 0.500 ng/mL for all the analytes, was achieved when 50 microL of human serum was used. The intra- and inter-day precisions were within 7.4%, and the accuracy was between 95 and 103%. The on-line extraction was finished in 0.5 min and total analysis time was 2.5 min per sample.     

Determination of tetracycline residues in soil by pressurized liquid extraction and liquid chromatography tandem mass spectrometry

An optimized extraction and cleanup method for the analysis of chlortetracycline (CTC), doxycycline (DC), oxytetracycline (OTC) and tetracycline (TC) in soil is presented. Soil extraction in a pressurized liquid extraction system, followed by extract clean up using solid-phase extraction (SPE) and tetracycline determination by liquid chromatography tandem mass spectrometry (LC-MS/MS) provided appropriate efficiency and reproducibility. Different dispersing agents and solvents for soil extraction and several SPE cartridges for cleanup were compared. The best extraction results were obtained using ethylenediamine tetraacetic acid-treated sand as dispersing agent, and water at 70 °C. The most effective cleanup was obtained using Strata-X^TM sorbent in combination with a strong anion exchange cartridge. Recoveries ranged from 71% to 96% and precision, as indicated by the relative standard deviations, was within the range of 8–15%. The limits of quantification (LOQs) by using LC-MS/MS, based on signal-to-noise ratio (S/N) of 10, ranged from 1 μg kg⁻¹ for TC to 5 μg kg⁻¹ for CTC. These results pointed out that this technique is appropriate to determine tetracyclines in soils. Analysis of 100 samples taken in the Valencian Community revealed that, in soil, up to 5 μg kg⁻¹ CTC, 15 μg kg⁻¹ OTC, 18 μg kg⁻¹ TC, and 12 μg kg⁻¹ DC could be detected. Detection of the analytes in several samples, which typify great part of the Spanish agricultural soils, should be outlined as most important result of this study. Electronic supplementary material  The online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

Sensitive and rapid liquid chromatography/tandem mass spectrometry assay for the quantification of amlodipine in human plasma

A simple, sensitive and rapid high-performance liquid chromatography/electrospray ionization tandem mass spectrometry method was developed and validated for the assay of amlodipine in human plasma. Following liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a reverse-phase C(18) column and analyzed by MS in the multiple reaction monitoring mode using the respective [M+H]+ ions, m/z 409/238 for amlodipine and m/z 409/228 for the IS. The assay exhibited a linear dynamic range of 50-10,000 pg/mL for amlodipine in human plasma. The lower limit of quantification was 50 pg/mL with a relative standard deviation of less than 8%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The average absolute recoveries of amlodipine and the IS from spiked plasma samples were 74.7 +/- 4.6 and 72.1 +/- 2.0%, respectively. A run time of 1.5 min for each sample made it possible to analyze more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies. The observed maximum plasma concentration (Cmax) of amlodipine (2.5 mg oral dose) was 1425 pg/mL, time to observed maximum plasma concentration (Tmax) was 8.1 h and elimination half-life (T(1/2)) was 50.1 h.     

Dodging matrix effects in liquid chromatography tandem mass spectrometric assays—compilation of key learnings and perspectives

Triple quad liquid chromatography mass spectrometric assays (LC/MS/MS) have revolutionized the analysis of drug(s)/metabolite(s) with exceptional speed, sensitivity and selectivity features. From inception to date, several new and innovative features have been regularly proposed by researchers to further enhance the value in the applicability of this analytical tool. However, owing to such compressed run times and scanty sample preparation procedures, LC/MS/MS assays that are not fully optimized generally have issues of matrix effects, where ionization potential is either suppressed or enhanced due to the presence of other materials (endogenous/exogenous) in the matrix. By definition, even co‐medications, isomeric or isobaric impurities, and drug excipients used in dosing solutions could also potentially contribute to matrix effects. This article captures some of the interesting work carried out by researchers to understand and handle matrix effects. Additionally, it provides perspectives to effectively deal with matrix effects. Copyright © 2008 John Wiley & Sons, Ltd.     

A rapid determination of propiverine and its N-oxide metabolite in human plasma by high performance liquid chromatography-electrospray ionization tandem mass spectrometry

A simple, fast and sensitive high-performance liquid chromatography (HPLC)-electrospray ionization (ESI) tandem mass spectrometric method (LC-MS/MS) has been developed for determination of propiverine and propiverine N-oxide metabolite in human plasma using oxybutynin as internal standard. Instead of extracting propiverine from plasma using organic solvents, which should be separated from the aqueous phase and evaporated before injecting the sample into the chromatograph, plasma sample containing propiverine and N-oxide was directly injected after precipitating proteins with acetonitrile. Numerous compounds in the plasma did not interfere with the highly specific multiple reaction monitoring in tandem mass spectrometric detection following C₈ reversed-phase chromatographic separation under conditions that eluted propiverine, N-oxide and oxybutynin within 2 min (0.1% formic acid in water/acetonitrile, 25:75, v/v). The LC-MS/MS method and an alternative LC-MS method, using methyl-t-butyl ether extraction and selected ion monitoring, were validated over 1-250 ng ml⁻¹ of propiverine and 2 to 500 ng ml⁻¹ of N-oxide, and successfully applied in a pharmacokinetic study. The lower limit of quantitation was 1 ng ml⁻¹ for propiverine and 2 ng ml⁻¹ for N-oxide in both methods.     

液相色谱-串联质谱法检测贝类产品中的原多甲藻酸贝类毒素

建立了一种贝类组织中原多甲藻酸(azaspiracid, AZA)贝类毒素主要成分AZA1的高效液相色谱-串联质谱检测方法。本方法采用甲醇-水(80:20, v/v)溶液对贝类组织中AZA1进行提取,并用MAX阴离子交换固相萃取(SPE)柱富集净化,使用Atlantis dC18(150 mm×4.6 mm, 5.0 μm)色谱柱分离,以含有50 mmol/L甲酸和2 mmol/L甲酸铵的乙腈-水溶液(80:20, v/v)为流动相进行等度洗脱,质谱采用选择反应监测(SRM)模式。AZA1在5 min内获得完全分离,且在48.85～2 442 ng/L范围内线性良好,相关系数为0.998 1。该方法检出限(S/N=3)为11.00 pg/g,添加水平为36.64、73.27、146.54 pg/g时的平均回收率为75.8%～82.5%(n=6),相对标准偏差小于10%。利用该方法对采自大连、青岛、广州水产品市场上的112个贝类样品进行了分析,发现采自大连和广州的部分贝类样品中含有AZA1。结果表明,该方法具有简单、快速、灵敏度高等特点,能充分满足贝类中AZA1检测的要求。     

Multi-mycotoxin analysis in eggs using a QuEChERS-based extraction procedure and ultra-high-pressure liquid chromatography coupled to triple quadrupole mass spectrometry

A reliable and rapid method has been developed for the determination of 10 mycotoxins (beauvericin, enniatin A, A1, B1, citrinin, aflatoxin B1, B2, G1, G2 and ochratoxin A) in eggs at trace levels. Ultra-high-pressure liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) has been used for the analysis of these compounds in less than 7 min. Mycotoxins have been extracted from egg samples using a QuEChERS-based extraction procedure (Quick, Easy, Cheap, Effective, Rugged and Safe) without applying any further clean-up step. Extraction, chromatographic and detection conditions were optimised in order to increase sample throughput and sensitivity. Matrix-matched calibration was used for quantification. Blank samples were fortified at 10, 25, 50 and 100 μg kg(-1), and recoveries ranged from 70% to 110%, except for ochratoxin A and aflatoxin G1 at 10 μg kg(-1), and aflatoxin G2 at 50 μg kg(-1). Relative standard deviations were lower than 25% in all the cases. Limits of detection ranged from 0.5 μg kg(-1) (for aflatoxins B1, B2 and G1) to 5 μg kg(-1) (for enniatin A, citrinin and ochratoxin A) and limits of quantification ranged from 1 μg kg(-1) (for aflatoxins B1, B2 and G1) to 10 μg kg(-1) (for enniatin A, citrinin and ochratoxin A). Seven samples were analyzed and aflatoxins B1, B2, G1, G2, and beauvericin were detected at trace levels.     

4-Methylamino antipyrine determination in human plasma by high-performance liquid chromatography tandem mass spectrometry

In the present study, a simple and rapid method for metamizole metabolite 4-methylamino antipyrine (MAA) determination in human plasma was developed, validated and successfully applied to a clinical trial. Chromatographic separation was achieved in HILIC mode on a YMC-Pack SIL column (100 × 2.0 mm; S-5 μm, 30 nm), with a mobile phase consisting of acetonitrile, water and formic acid. Protein precipitation of a small plasma volume using acetonitrile was selected for sample preparation. The multiple reaction monitoring transitions in the positive ionization mode were m/z 218.2 → 56.2 for MAA and m/z 221.2 → 56.2 for MAA-d3 (IS, internal standard). Concentration levels of MAA calibration standards were in the range of 0.100–20 μg/ml. Metamizole conversion into MAA in both water and organic media was investigated, and the level of the conversion in commercially available injection solutions was estimated.     

Quantitative determination of uridine in rabbit plasma and urine by liquid chromatography coupled to a tandem mass spectrometry

Recently a pyrimidine nucleoside, uridine, has been show to have a protective effect on cultured human corneal epithelial cells, and on dry eye animal model and patients. In this study, we introduce a sensitive liquid chromatography/tandem mass spectrometry method for the determination of uridine in rabbit plasma and urine. After protein precipitation with methanol including methaqualone (internal standard), the analyte was chromatographed on a reversed-phase column with a mobile phase of 0.1% formic acid aqueous solution and methanol (1:4, v/v). The accuracy and precision of the assay were in accordance with Food and Drug Administration regulations for the validation of bioanalytical methods. This method was used to measure the concentrations of uridine in plasma and urine after a single oral administration of 450 mg/kg uridine in rabbits.