Validation of direct injection electrospray LC-MS/MS for confirmation of opiates in urine drug testing

A method based on the direct injection of diluted urine for the identification and quantification of morphine, morphine-3-glucuronide, morphine-6-glucuronide, codeine, codeine-6-glucuronide, ethylmorphine, ethylmorphine-6-glucuronide and 6-acetylmorphine (6AM) in human urine by electrospray ionisation liquid chromatography-tandem mass spectrometry was validated for use as a confirmation procedure in urine drug testing. Four deuterium labelled analogues were used as internal standards: morphine-3-glucuronide-D3, morphine-D3, codeine-D3 and 6AM-D3. Twenty microlitre aliquots of urine were mixed with 80 mul of the internal standard solution in autosampler vials and 10 mul was injected. The chromatographic system consisted of a 2.0 x 100 mm C18 column and the gradient elution buffers used acetonitrile and 25 mmol/l formic acid. Two product ions produced from the protonated molecular ions were monitored in the selected reaction monitoring mode. The intra- and inter-assay variability (coefficient of variation) was below 10% at higher levels for all analytes, but at the reporting limits the variation was above 20% for 6AM, morphine-3-glucuronide and codeine-6-glucuronide. Ion suppression occurred early after injection but did not affect the identification and quantification of the analytes in authentic samples. The method was further validated by comparison with a reference gas chromatographic-mass spectrometric method using authentic urine samples. The two methods agreed almost completely (99%) regarding the identified analytes, but for the quantitative results there were slightly lower levels when measuring glucuronides directly as compared to total determination after hydrolysis by gas chromatography-mass spectrometry.We conclude that the presented liquid chromatographic-tandem mass spectrometric method is robust and reliable, and suitable for use as a confirmation method in urine drug testing for opiates     

Analysis of anthocyanins in commercial fruit juices by using nano-liquid chromatography-electrospray-mass spectrometry and high-performance liquid chromatography with UV-vis detector

Nano-LC and conventional HPLC techniques were applied for the analysis of anthocyanins present in commercial fruit juices using a capillary column of 100 μm id and a 2.1 mm id narrow-bore C(18) column. Analytes were detected by UV-Vis at 518 nm and ESI-ion trap MS with HPLC and nano-LC, respectively. Commercial blueberry juice (14 anthocyanins detected) was used to optimize chromatographic separation of analytes and other analysis parameters. Qualitative identification of anthocyanins was performed by comparing the recorded mass spectral data with those of published papers. The use of the same mobile phase composition in both techniques revealed that the miniaturized method exhibited shorter analysis time and higher sensitivity than narrow-bore chromatography. Good intra-day and day-to-day precision of retention time was obtained in both methods with values of RSD less than 3.4 and 0.8% for nano-LC and HPLC, respectively. Quantitative analysis was performed by external standard curve calibration of cyanidin-3-O-glucoside standard. Calibration curves were linear in the concentration ranges studied, 0.1-50 and 6-50 μg/mL for HPLC-UV/Vis and nano-LC-MS, respectively. LOD and LOQ values were good for both methods. In addition to commercial blueberry juice, qualitative and quantitative analysis of other juices (e.g. raspberry, sweet cherry and pomegranate) was performed. The optimized nano-LC-MS method allowed an easy and selective identification and quantification of anthocyanins in commercial fruit juices; it offered good results, shorter analysis time and reduced mobile phase volume with respect to narrow-bore HPLC.     

Ultra‐trace level determination of diquat and paraquat residues in surface and drinking water using ion‐pair liquid chromatography with tandem mass spectrometry: A comparison of direct injection and solid‐phase extraction methods

Direct injection and solid‐phase extraction methods for the determination of diquat and paraquat in surface and drinking water were developed using liquid chromatography with tandem mass spectrometry. The signal intensities of analytes based on six ion‐pairing reagents were compared with each other, and 12.5 mM nonafluoropentanoic acid was selected as the best suited amongst them. A clean‐up method was developed using Oasis hydrophilic–lipophilic balance; this was compared to the direct injection method, with respect to limits of detection, interference, precision, and accuracy. Limits of quantification of diquat and paraquat were 0.03 and 0.01 μg/L using the direct injection method, and 0.002 and 0.001 μg/L using the hydrophilic–lipophilic balance method. When the hydrophilic–lipophilic balance method was used to analyze target compounds in 114 surface water and 30 drinking water samples, paraquat and diquat were detected within a concentration range of 0.001–0.12 and 0.002–0.038 μg/L in surface water, respectively. When the direct injection method was used to analyze target compounds in the same samples, the detected concentrations of paraquat and diquat were within 25% in samples being >0.015 μg/L using the hydrophilic–lipophilic balance method. The liquid chromatography with tandem mass spectrometry method using direct injection can thus be used for routine monitoring of paraquat and diquat in surface and drinking water.     

A review of direct liquid introduction interfacing for LC/MS Part I: Instrumental aspects

Summary Considerable progress has been made in the coupling of liquid chromatography and mass spectrometry over the past ten years. Three interfaces tend to dominate the LC/MS market: transport systems, direct liquid introduction, and the thermospray interface. In this paper the developments in direct liquid introduction interfacing for LC/MS will be reviewed. The paper will be published in two parts. Mass spectrometry and applications will be discussed in the second part. This first part of the review concentrates on the various instrumental aspects of direct liquid introduction, such as the design of vacuum systems, the interface probes and the desolvation chambers.     

Multiclass analysis of illicit drugs in plasma and oral fluids by LC-MS/MS

An analytical procedure for the simultaneous determination in human plasma and oral fluids of several illicit drugs belonging to different chemical and toxicological classes is presented. Amphetamine, methamphetamine, morphine, 6-monoacetylmorphine, methylenedioxyamphetamine, methylenedioxyethylamphetamine, methylenedioxymethamphetamine, cocaine, benzoylecgonine, tetrahydrocannabinol, carboxytetrahydrocannabinol, ketamine, and phencyclidine have been quantified in real samples using a very rapid sample treatment, basically a protein precipitation. The quantitative analysis was performed by liquid chromatography–tandem mass spectrometry and has been fully validated. All the analytes were detected in positive ionization mode using a TurboIonSpray source, except carboxytetrahydrocannabinol, which was detected in negative ionization mode. The use of a diverter valve between the column and the mass spectrometer allows the preservation of the ion source performances for high-throughput analysis. Figure Diverter system     

防晒类化妆品中2种帕地马酯的高效液相色谱检测及质谱确证

建立了同时测定防晒类化妆品中帕地马酯A和帕地马酯O的高效液相色谱分析方法及质谱确证方法。化妆品样品以甲醇为提取溶剂进行超声提取,提取液离心处理,取上清液经微孔滤膜过滤后测定。采用XTerra MS C18柱(250×4.6mm,5μm)分离,外标法定量。结果表明防晒类化妆品中帕地马酯A和帕地马酯O的方法定量限均为0.5mg/kg,在低、中、高3个添加水平范围内平均回收率为89.1%~112.5%,相对标准偏差为2.0%~7.7%。对于疑似阳性样品,进一步采用液相色谱-串联质谱法进行确证分析。该方法准确、快速、灵敏度高,适用于防晒类化妆品的实际检验工作。     

A review of direct liquid introduction interfacing for LC/MS Part II: Mass spectrometry and applications

Summary The coupling liquid chromatography and mass spectrometry is still growing in popularity. Direct liquid introduction has become one of the most important interfaces in LC/MS coupling. The various aspects of the interface have been investigated by several groups. This paper is the second part of a review of the developments in direct liquid introduction. Instrumental aspects of direct liquid introduction interfacing were discussed in the first part. This part will deal with the mass spectrometric aspects, i.e. chemical ionization and the influence of the various experimental conditions on the spectra. The applications of direct liquid introduction will also be reviewed briefly.     

Trace-level determination of pharmaceutical residues by LC-MS/MS in natural and treated waters. A pilot-survey study

A pilot-survey study was performed by collecting samples (influent and effluent wastewaters, rivers and tap waters) from different locations in Europe (Spain, Belgium, Germany and Slovenia). A solid-phase extraction (SPE) followed by liquid chromatography–tandem mass spectrometry method was applied for the determination of pharmaceuticals (ibuprofen, naproxen, ketoprofen, diclofenac and clofibric acid). Method detection limits and method quantification limits were at the parts-per-trillion level (7.5–75 ng/L). The recovery rates of the SPE from deionized water and effluent wastewater samples spiked at 100- and 1,000-ng/L levels ranged from 87 to 95%. Identification criteria in compliance with the EU regulation for confirmatory methods of organic residues were applied. A detailed study of signal suppression evaluation for analysis of pharmaceutical residues in effluent wastewaters is presented.     

On-line solid phase extraction LC-MS/MS analysis of pharmaceutical indicators in water: A green alternative to conventional methods

A method using automated on-line solid phase extraction (SPE) directly coupled to liquid chromatography/tandem mass spectrometry (LC-MS/MS) has been developed for the analysis of six pharmaceuticals by isotope dilution. These selected pharmaceuticals were chosen as representative indicator compounds and were used to evaluate the performance of the on-line SPE method in four distinct water matrices. Method reporting limits (MRLs) ranged from 10 to 25 ng/L, based on a 1 mL extraction volume. Matrix spike recoveries ranged from 88 to 118% for all matrices investigated, including finished drinking water, surface water, wastewater effluent and septic tank influent. Precision tests were performed at 50 and 1000 ng/L with relative standard deviations (RSDs) between 1.3 and 5.7%. A variety of samples were also extracted using a traditional off-line automated SPE method for comparison. Results for both extraction methods were in good agreement; however, on-line SPE used approximately 98% less solvent and less time. On-line SPE coupled to LC-MS/MS analysis for selected indicators offers an alternative, more environmentally friendly, method for pharmaceutical analysis in water by saving time and costs while reducing hazardous waste and potential environmental pollution as compared with off-line SPE methods.     

Optimisation of a liquid chromatography-tandem mass spectrometric method for the determination of acrylamide in foods

A robust and fitted routine method resulting from an analytical optimisation has been applied for the determination of acrylamide in several foods including mainly potato and cereal products. For the sample treatment, different materials were evaluated for filtration and purification of the extract. To increase the performances in terms of sensitivity, a preconcentration to small volume was introduced before liquid chromatography coupled to tandem mass spectrometry analysis on a μ-Bondapak C₁₈ column using d₃-acrylamide as internal standard. For identification, relative retention time and two diagnostic ions were monitored. A limit of detection of 10 μg kg⁻¹, a limit of quantitation of 20 μg kg⁻¹, mean recoveries ranging from 100 to 115%, coefficients of variation from 1.36 to 8.06% for repeatability and from 3.3 to 18.2% for reproducibility within the laboratory and a measurement uncertainty of 42% were obtained during an in-house validation procedure. Results of tests, validation data and Z-score obtained during participation to proficiency studies are presented.     

Quantitation of ribavirin in human plasma and red blood cells using LC–MS/MS

LC–MS/MS has been applied for the rapid determination of the nucleoside analogue ribavirin in human plasma and red blood cells. The incorporation of ribavirin to the erythrocytes has been assayed after in vitro incubation of the cells at different concentrations of the antiviral drug. After protein precipitation, samples were injected into a C₈ column, achieving a complete separation of ribavirin from the endogenous isobaric compound uridine. Calibration ranges varied from 10 to 10 000 ng/mL in plasma and from 0.2 to 200 ng/cell pellet in red blood cells. Precision and accuracy values were always below 10 and 13%, respectively, in all assayed matrices. Ribavirin was demonstrated to remain unchanged after short and long time storage. No matrix effects could be assessed for the analyzed matrices. The developed method has been fully validated. Monitoring of ribavirin concentration in red blood cells in addition to the classic plasma monitoring of the drug could help to explain its efficacy and safety profiles in patients.     

Pesticide residues analysis in honey using ethyl acetate extraction method: validation and pilot survey in real samples

This paper presents a cost-effective and validated multi residue confirmatory method for the determination of 167 chemically different pesticides and a survey study on Cyprus honey samples. This method uses ethyl acetate for the extraction of pesticides from honey and the determination is performed with liquid chromatography (LC) coupled to mass spectrometry (MS) operating in tandem mode (MS/MS) and with GC–ECD (gas chromatography with electron capture detector) analysis. The LC-MS/MS analytical system is especially important in the analysis of polar and non-volatile pesticides. For the validation of the method, blank honey samples were spiked with 146 pesticides (organophosphorous, carbamates, triazoles, amides, neonicodinoids, strobilurines, phenylureas, bendimidazoles and others) for the LC-MS/MS analysis at three levels: 0.01, 0.05 and 0.1 mg kg^?1 and with 21 pesticides for the GC-ECD analysis at two levels: 0.01 and 0.05 mg kg^?1for organochlorines and 0.05 and 0.2 mg kg^?1for the pyrethroids. As blank sample, a sample of honey which did not contain detectable levels of the analytes sought was used. The validation study was in accordance to the DG SANCO guidelines. The scope of validation included recovery, linearity, limits of quantification and precision. Linearity is demonstrated all along the range of concentration that was investigated with correlation coefficients ≥0.98. Recoveries of the majority of compounds were in the 70%–120% range and were characterised by precision lower or equal to 20%. The validated method was used for a survey of 36 samples of honey produced in different areas of Cyprus and this is the first work on Cypriot honey samples investigating a broad range of pesticides. Only coumaphos was detected at concentrations higher than 0.01 mg kg^?1 in the 58.6% of the honey samples analysed for Coumaphos. The results were evaluated in accordance to the provisions of the Commission Regulation (EU) No 37/2010 on pharmacologically active substances and their classification regarding maximum residue limits (MRLs) in foodstuffs of animal origin. The concentrations of coumaphos in all positive samples were at levels much lower than the MRL.     

Fast multiresidue screening of 300 pesticides in water for human consumption by LC-MS/MS

The study tested the determination of 300 pesticides in mineral water at levels of 0.1 and 1.0 μg/L. Measurements were conducted by direct sample injection into a liquid chromatograph coupled to a tandem mass spectrometer without any sample enrichment and/or cleanup. Two separate injections enabled the recording of two transitions per analyte (600 selected reaction monitoring transitions in total). For 285 analytes the sensitivity of direct sample injection (100 μL) was sufficient to quantify residues at 0.1 μg/L. All remaining pesticides were detected at 1.0 μg/L. Calibration functions were linear for more than 80% of analytes. Signal suppression or enhancement compared with signals in high-performance liquid chromatography water was equal to or smaller than 20% for 240 analytes. Even the largest matrix-induced suppression did not result in the disappearance of peaks. Combining the results of seven mineral waters, the relative standard deviation of “recovery” was 20% or less for 87% of the substances. A second transition for confirmatory purposes was often available. Consequently, the proposed direct injection of samples without any sample enrichment and/or cleanup is suitable for screening of many pesticides in mineral and drinking water.     

Practical determination of methotrexate in serum of rheumatic patients by LC-MS/MS

A rapid and simple liquid chromatography tandem mass spectrometry method for determination of methotrexate (MTX) in rheumatic patients' serum is described. Serum spiked with pterin as an internal standard was deproteinized with methanol. The separation of MTX from interfering peaks in matrix was achieved on a Luna 3 µm C₁₈ (100 × 4.6 mm i.d.) column with a mixture of 1% acetic acid and acetonitrile (88:12, v/v) within 5 min. Multiple reaction monitoring transitions monitored for MTX were m/z 455.2–308.1. The calibration curve of MTX in serum showed a good linearity (r = 0.999). Limits of detection and quantification of MTX at a signal‐to‐noise ratio of 3 and 10 were 3.0 n m (4.4 fmol/injection) and 10.0 n m (14.5 fmol/injection), respectively. The accuracy and precision for intra‐ and inter‐day assays were 94.6–106.5% and <5.5 and <5.1%, respectively. Furthermore, the proposed method was successfully applied to the sera nine rheumatic patients receiving MTX treatment. Copyright © 2012 John Wiley & Sons, Ltd.     

基质校正固相萃取液相色谱-串联质谱法测定食用菌中22种农药残留

选择氨基柱为固相萃取净化柱,以电喷雾(ESI)为离子源,正负离子切换多级反应离子检测(MRM)方式,建立了基质校正固相萃取液相色谱-串联质谱法(LC-MS/MS)测定食用菌中22种农药残留的分析方法。研究了不同固相萃取柱和洗脱剂对农药保留行为的影响,优化了净化方法、色谱分离条件和质谱条件。研究了平菇、香菇、杏鲍菇和蟹味菇的基质效应。22种农药在0.05～100μg/L(或0.1～200μg/L)范围内线性相关,相关系数为0.9874～0.9997,方法定量限为0.031～1.76μg/kg。除甲基硫菌灵和克螨特外,20种农药在4种食用菌中3个添加浓度的回收率在54.6%～128.0%,RSD为0.3%～29.0%之间,方法灵敏度高,选择性强。     

Determination of fungicides in wine by mixed-mode solid phase extraction and liquid chromatography coupled to tandem mass spectrometry

A novel procedure for the determination of nine selected fungicides (metalaxyl-M, azoxystrobin, myclobutanil, flusilazole, penconazole, tebuconazole, propiconazole, diniconazole and difenoconazole) in wine samples is presented. Sample enrichment and purification is simultaneously performed using mixed-mode, anion exchange and reversed-phase, OASIS MAX solid-phase extraction (SPE) cartridges. Analytes were determined by liquid chromatography coupled to tandem mass spectrometry using atmospheric pressure electrospray ionization (LC-ESI-MS/MS). Parameters affecting the chromatographic determination and the extraction-purification processes were thoroughly investigated. Under optimized conditions, 10 mL of wine were firstly diluted 1:1 with ultrapure water and then passed through the mixed-mode SPE cartridge at a flow of ca. 5 mLmin(-1). After a washing step with 5 mL of an aqueous NH(4)OH solution (5%, w:v), analytes were recovered with just 1 mL of methanol and injected in the LC-MS/MS system without any additional purification. The selective extraction process avoided significant changes in the ionization efficiency for red and white wine extracts in comparison with pure standards in methanol. Performance of the method was good in terms of precision (RSDs<11%) and accuracy (absolute recoveries>72%, determined against pure standards in methanol) reporting method LOQs in the range of 0.01-0.79 ngmL(-1) for target compounds, which are far below the EU maxima residue levels (MRLs) for fungicides in vinification grapes and wine. Several commercial wines from different geographic areas in Spain were analyzed. In most samples, metalaxyl-M and azoxystrobin were found at concentrations up to several ngmL(-1).     

脑啡肽的固相合成与LC-MS/MS分离鉴定

多肽在生命过程中扮演着重要的角色,对其生理生化功能的研究与应用,离不开对单一多肽物质的需求,而化学合成法是获取目标多肽的最有效方法之一。对合成产物的分离与鉴定,是优化合成条件,以得到高产率的重要保证。以两种内源性神经肽亮氨酸脑啡肽和甲硫氨酸脑啡肽为模型,利用Fmoc固相多肽合成策略对其进行合成,并建立了HPLC-ESI-MS/MS新方法用于所制备的亮氨酸脑啡肽和甲硫氨酸脑啡肽的分离与结构鉴定。研究结果显示,主要合成产物均为目标多肽,副产物主要包括C端丢失1个氨基酸所形成的四肽,以及由于甲硫氨酸残基氧化而形成的含甲硫氨酸亚砜的多肽。该研究为高效合成含敏感氨基酸的生理活性多肽提供了新信息。     

Study of different atmospheric-pressure interfaces for LC-MS/MS determination of acrylamide in water at sub-ppb levels

A rapid, sensitive and selective method based on LC-MS/MS has been developed for the direct determination of acrylamide residues in water in compliance with the current European Union (EU) 98/83 Drinking Water Directive. Given the high polarity of acrylamide, the application of a rapid on-line solid phase extraction step, commonly used for preconcentrating low analyte levels, was not found to be completely satisfactory. Therefore, an alternative approach based on the use of direct large-volume injection into the LC-MS/MS system has been used. Three atmospheric-pressure interfaces (ESI, APCI and Ion Sabre APCI) were checked to reach the required sensitivity (0.1 microg/l). All three interfaces were tested by analysis of six different water samples (surface water, groundwater, drinking water and three treated water samples) spiked at three concentration levels each (0.1, 1 and 10 microg/l). When using ESI, poor sensitivity and high matrix effects were observed. This situation improved when APCI was used as the interface because no matrix effect was found, although sensitivity was not completely satisfactory. The best results were obtained by interfacing the Ion Sabre APCI; its higher sensitivity for acrylamide (LOD 0.03 microg/l) and the absence of matrix effects recommended its selection. Using this approach, satisfactory recoveries (90-97%) and precision (<12%) were obtained for all water samples studied. Besides, the acquisition of two different MS/MS transitions allowed not only the quantification but also the confirmation of acrylamide in water at concentration levels around 0.1 microg/l.     

Development and Application of an LC-MS/MS Untargeted Exposomics Method with a Separated Pooled Quality Control Strategy

Pooled quality controls (QCs) are usually implemented within untargeted methods to improve the quality of datasets by removing features either not detected or not reproducible. However, this approach can be limiting in exposomics studies conducted on groups of exposed and nonexposed subjects, as compounds present at low levels only in exposed subjects can be diluted and thus not detected in the pooled QC. The aim of this work is to develop and apply an untargeted workflow for human biomonitoring in urine samples, implementing a novel separated approach for preparing pooled quality controls. An LC-MS/MS workflow was developed and applied to a case study of smoking and non-smoking subjects. Three different pooled quality controls were prepared: mixing an aliquot from every sample (QC-T), only from non-smokers (QC-NS), and only from smokers (QC-S). The feature tables were filtered using QC-T (T-feature list), QC-S, and QC-NS, separately. The last two feature lists were merged (SNS-feature list). A higher number of features was obtained with the SNS-feature list than the T-feature list, resulting in identification of a higher number of biologically significant compounds. The separated pooled QC strategy implemented can improve the nontargeted human biomonitoring for groups of exposed and nonexposed subjects.     

GC–MS/MS method for the determination and pharmacokinetic analysis of borneol and muscone in rat after the intravenous administration of Xingnaojing injection

A simple and sensitive gas chromatography with tandem mass spectrometry method was developed and validated for the simultaneous determination of borneol and muscone in rat plasma. The analytes and internal standard, naphthalene, were extracted using a convenient one‐step liquid–liquid extraction method with ethyl acetate. The chromatographic separation was realized on a HP‐5MS capillary column and detected in multiple reaction monitoring mode. Excellent linearity (R ² ≥ 0.996) was shown over 10.0–5000 ng/mL for borneol and 2.5–250 ng/mL for muscone. The lower limit of quantitation was 10 and 2.5 ng/mL for borneol and muscone, respectively. The intra‐ and interday precisions were less than 7.52%, and the accuracy values were between  −8.03 and 14.52%. The extraction recovery, matrix effect, and stability were sufficient to meet the Food and Drug Administration criteria. Meanwhile, the assay was successfully applied to the preclinical pharmacokinetic study of borneol and muscone following intravenous administration of Xingnaojing injection, a modern Chinese herbal medicine preparation.