Matrix-matched Calibration Curves Research Articles

Determination of polypeptide antibiotics in animal tissues using liquid chromatography tandem mass spectrometry based on in-line molecularly imprinted solid-phase extraction

Effective purification and enrichment of polypeptide antibiotics in animal tissues is always a challenge, due to the co-extraction of other endogenous peptides which usually interfere their final determination. In this study, a molecularly imprinted column was prepared by packing polymyxin E-imprinted particles into a 100 mm × 4.6 mm i.d. HPLC column. The as-prepared imprinted columns were able to tolerate 100% aqueous phase and exhibited good stability and high column efficiency. Polypeptides antibiotics with similar molecular size or spatial structure to polymyxin E were well retained by the imprinted column, suggesting class selectivity. After optimization of mobile phase conditions of imprinted column, polypeptide antibiotics in animal tissue extracts were enriched and cleaned up by in-line molecularly imprinted solid-phase extraction, allowing the screening of target analytes in complex samples at low concentration levels by UV detection. Eluate fraction from the imprinted column was collected, and further dried and re-dissolved with methanol-0.5% formic acid aqueous solution (80:20, v/v) for final LC-MS/MS analysis. Analysis was accomplished using multiple reaction monitoring (MRM) in positive electrospray ionization mode and analytes quantified using the matrix-matched external calibration curves. The results showed high correlation coefficients for target analytes in the linear range of 2 ∼ 200 μg kg−1. At four different concentration levels (limit of quantification, 50, 100 and 200 μg kg−1), recoveries of four polypeptide antibiotics in swine, cattle and chicken muscles ranged from 66.7 to 94.5% with relative standard deviations lower than 16.0%. The limits of detection (LOD) were 2.0 ∼ 4.0 μg/kg, depending upon the analyte and sample. Compared with a conventional pretreatment method, the imprinted column was able to remove more impurities and to significantly reduce matrix effects, allowing the accurate analysis of polypeptide antibiotics.

Determination of 43 pesticide residues in intact grape berries (Vitis Vinifera L.) by using an ultrasound-assisted acetonitrile extraction method followed by LC–MS/MS

An ultrasound-assisted acetonitrile extraction method was applied for the determination of 43 pesticide residues in intact grape berries (Vitis Vinifera L.) by liquid chromatography-tandem mass spectrometry (LC–MS/MS). The results showed that ultrasound-assisted extraction from intact grape berries exhibited better efficiency than extraction from homogenized grape berries. Matrix-matched calibration curves were applied from 0.01 to 200 μg/L and determination coefficients (R2) were greater than 0.99. The recovery values were 70.6%–119.7% with a relative standard deviation (RSD) range of 0.02%–9.92%. The limits of detection (LODs) and limits of quantification (LOQs) ranged between 0.005 μg/kg and 0.2 μg/kg, and between 0.10 μg/kg and 10 μg/kg, respectively. The method was applied in the analysis of wine grape samples from three different regions of China. A total of 10 pesticides were detected in samples during fruit ripeness.

Response boosting-based approach for absolute quantification of gelatin peptides using LC-MS/MS

Response-boosting of MS signal was observed in gelatin samples due to abundant Glycine residues produced by collagen enzymolysis. In this work, a new strategy utilizing response-boosting to enhance detection sensitivity was developed for absolute quantification of Asini Corii Colla, a kind of gelatin commonly used as food therapy products in Asia, by high performance liquid chromatography coupled to tandem mass spectrometry. Peptidomics analysis was used to evaluate the similarity between eight different protein matrices, and deer-hide gelatin was selected as the appropriate simulated matrix. Isotope-labelled internal standard was used to compensate the matrix effect and construct matrix-matched calibration curves. The established method showed reliability in absolute quantification of three species-specific gelatin peptides with good linearity (r2 > 0.997), precision (RSD < 8.5%), repeatability (RSD < 8.9%), accuracy (recovery 89.4%∼106.5%) and sensitivity (LOD 0.02 ∼ 0.98 ng/mL). Thus, the present response-boosting based protocol provides a promising application in quality control of food rich in gelatins.

High-resolution mass spectrometry-based strategies for the target analysis and suspect screening of per- and polyfluoroalkyl substances in aqueous matrices

Per- and polyfluoroalkyl substances (PFASs) are a man-made diverse group of highly fluorinated aliphatic molecules, entailing risks to the ecosystems and human health. Herein, a streamlined, single-shot, HRMS-based analytical workflow for the quantitation and suspect screening of various PFAS was developed. Sample preparation was based on weak-anion exchange solid phase extraction, while instrumental analysis was performed by highly sensitive liquid chromatography Orbitrap–high-resolution mass spectrometry. The target method was optimized and validated for drinking water, surface water, and wastewater, while it was also modified to be efficient for leachates. Method performance was excellent in terms of linearity (R2 > 0.99), detection/quantification limits (reaching even at pg L−1 scale), recoveries (70–108 %, for most of the compounds), and precision (<20%). Isotopically labelled internal standards and matrix-matched calibration curves were used for quantification. The suspect analysis performed on a single-stage HRMS method operating in data-dependent ‘discovery’ mode. Thus, content-rich spectra were acquired and once combined with suspect screening features such as peak picking, feature alignment, and compound annotation enabled the putative annotation of the tested PFAS with high confidence. After evaluation, the presented fully elaborated workflow was applied to real samples to explore the putative annotation of the tested PFAS with high confidence for the first time in Greece for five different matrices, revealing maximum concentrations for PFOS 1.5 ng L−1 in tap water and 394.4 ng L−1 in effluent wastewater, and PFHxA 4284.8 ng L−1 in leachates.

Microscale assessment of iodate fortificant in food-grade salt using the potassium iodide/polyvinyl alcohol colorimetric reagent

A rapid and straightforward spectrophotometric measurement of iodate in food-grade sodium chloride was developed based on the formation of a purple-red polyvinyl alcohol-iodine complex monitored at 490 nm. The conditions that influence the colorimetric reactions were optimized using a univariate approach. The method exhibited optimum results using 2% polyvinyl alcohol, 10 mM KI, and 0.5 M phosphoric acid. A matrix-matched calibration curve was prepared to eliminate interference due to the presence of NaCl and to provide a direct reading of iodate concentration in salt from the absorbance reading. Under optimum conditions, a linear calibration plot was observed from 5 to 140 mg iodate per kilogram (r = 0.9931). The method exhibited a sensitivity of 6.8 × 10−3 (mg iodate per kg of salt)−1 with a detection limit of 0.61 mg kg−1. The method was applied in actual samples, and the results agreed well with the titration method at a 95% confidence level using a paired t-test. The method supports the green analytical method as evaluated using Green Analytical Procedure Index (GAPI) and Analytical Eco-scale.

Simultaneous Determination of Levamisole, Mebendazole, and the Two Metabolite Residues of Mebendazole in Poultry Eggs by High-Performance Liquid Chromatography–Tandem Mass Spectrometry

The quantitative determination of levamisole (LMS), mebendazole (MBZ), and the two metabolites of MBZ, 5-hydroxymebendazole (HMBZ) and 2-amino-5-benzoylbenzimidazole (AMBZ), in poultry eggs (hen, duck, and goose) was achieved with high-performance liquid chromatography–tandem triple quadrupole mass spectrometry (HPLC–MS/MS). Samples were pretreated by liquid–liquid extraction and solid-phase extraction (LLE–SPE) to extract the target compounds, and an Oasis MCX SPE column was used for purification. Determination was performed on an Xbridge C18 column with 0.1% formic acid aqueous solution and acetonitrile as mobile phases. LMS, MBZ, HMBZ, and AMBZ were detected in a triple-quadrupole mass spectrometer with ESI in positive mode and quantified with an external standard. In blank eggs, the target analyte concentrations were within the limits of quantification (LOQs)—25 μg/kg (LMS) and 150 μg/kg (MBZ, HMBZ, and AMBZ)—and the matrix-matched calibration curves had good linearity (R2 ≥ 0.9990). In the same concentration range, the average recoveries of the target analytes were 85.98–97.38% (n = 6); the relative standard deviation (RSD), intraday RSD, and interday RSD ranged from 2.06 to 4.22%, 1.40 to 5.85%, and 2.34 to 6.32%, respectively. The limits of detection (LODs) ranged from 0.03 to 0.33 µg/kg, and the LOQs ranged from 0.08 to 1.00 µg/kg. Experimental verification showed that the HPLC–MS/MS method exhibited high specificity and sensitivity for quantitative analyses of egg samples. This study provides a rapid, efficient, and sensitive method for the simultaneous detection of LMS, MBZ, HMBZ, and AMBZ residues in foods of animal origin.

Simultaneous Determination of Pyridate, Quizalofop-ethyl, and Cyhalofop-butyl Residues in Agricultural Products Using Liquid Chromatography-Tandem Mass Spectrometry.

An analytical method was developed to simultaneously determine pyridate, quizalofop-ethyl, and cyhalofop-butyl in brown rice, soybean, potato, pepper, and mandarin using LC-MS/MS. Purification was optimized using various sorbents: primary–secondary amine, octadecyl (C18) silica gel, graphitized carbon black, zirconium dioxide-modified silica particles, zirconium dioxide-modified silica particles (Z-SEP), and multi-walled carbon nanotubes (MWCNTs). Three versions of QuECHERS methods were then tested using the optimal purification agent. Finally, samples were extracted using acetonitrile and QuEChERS EN salts and purified using the Z-SEP sorbent. A six-point matrix-matched external calibration curve was constructed for the analytes. Good linearity was achieved with a determination coefficient ≥0.999. The limits of detection and quantification were 0.0075 mg/kg and 0.01 mg/kg, respectively. The method was validated after fortifying the target standards to the blank matrices at three concentration levels with five replicates for each concentration. The average recovery was within an acceptable range (70–120%), with a relative standard deviation <20%. The applicability of the developed method was evaluated with real-world market samples, all of which tested negative for these three herbicide residues. Therefore, this method can be used for the routine analysis of pyridate, quizalofop-ethyl, and cyhalofop-butyl in agricultural products.

Method Development and Validation of Gas Chromatography Methods using Nitrogen Phosphorus Detector (GC-NPD) and Isotope Dilution Triple Quadrupole Mass Spectrometry (GC-IDMS/MS) for the Determination of Chlorpyrifos in Mango

Pesticides residue determination in fruits like chlorpyrifos is important due to its health effects on humans. Thus, there is a need for sensitive and accurate methods for better quantification. In this study, the performance of two gas chromatographic detection methods, nitrogen phosphorus detector (GC-NPD) and isotope dilution triple quadrupole mass spectrometer (GC-IDMS/MS), for the determination of chlorpyrifos in mango was evaluated to be used for the development of candidate reference material. Other than liquid-liquid extraction (LLE), Quick, Easy, Cheap, Efficient, Rugged and Safe (QuEChERS) sample extraction and dispersive solid phase extraction (dSPE) clean-up procedures were optimized to extract chlorpyrifos from the sample matrix. Comprehensive method validations were performed in GC-NPD and GC-IDMS/MS with HP-5 capillary column to establish linearity, the limit of detection (LOD), the limit of quantification (LOQ), accuracy, repeatability, and intermediate precision using freeze-dried mango samples. The LOD and LOQ were 2.35 and 3.47 µg kg-1 for GC-NPD, and 1.23 and 1.85 µg kg-1 for GC-IDMS/MS, respectively. Matrix-matched calibration curves showed excellent linearity of r2=0.999 for both GC-NPD and GC-IDMS/MS protocols. Acceptable repeatability was obtained for three spike concentrations of 10, 30, and 60 µg kg-1 (n=10) and intermediate precision for 0.5-, 1- and 3-months in the same three-spike concentrations (n=3) expressed as relative standard deviation (RSD) ranging from 0.34-5.61 % for GC-NPD and 0.46-4.22 % for GC-IDMS/MS. Satisfactory mean recoveries (n=10) were achieved: 88.48-109.81% in GC-NPD and 97.61-108.40% in GC-IDMS/MS. GC-IDMS/MS and GC-NPD methods were fit for the purpose of quantifying chlorpyrifos in Philippine mango accurately with the use of isotope dilution technique and traceability to international standards by the application of gravimetric sample preparation.

Carcinogenic and non-carcinogenic risk assessment induced by pesticide residues in honey of Iran based on Monte Carlo simulation

A multi-residue method was developed for 45 pesticides from different groups, encompassing the most widely used pesticides. 30 samples of honey were randomly collected from different regions of Iran and extraction was performed using validated QuEChERS method. Next, pesticide residues were identified and measured using optimized UHPLC-MS/MS and GC-MS analysis. All validation assays were performed with a blank sample. For the evaluation of linearity, matrix-matched calibration curves were prepared with spiked samples at seven concentration levels, ranging from 0.005 to 0.5 mg L−1 for pesticides in UHPLC-MS/MS and from 0.025 to 2 mg L−1 for GC-MS analysis. Recovery and precision were certified through extraction of spiked samples at concentration levels of 0.1 and 0.25 mg L−1. The limit of quantification (LOQ) was fixed as the lowest concentration level of the calibration curve, which presented adequate recovery and precision. Results indicated that the observed pesticides in honey samples were approximately 44.4% of the total studied pesticides, of which 90% belonged to the group of insecticides. Eventually, the human health risk assessment was performed for children and adult consumers based on Monte Carlo simulation. The rank order of pesticides based on HQ was lindane > diazinon > trichlorfon > permethrin for honey samples. In our study, the calculated hazard index (HI) for adults (0.18) and for children (0.57) lower than 1, suggested no potential health risks to the honey consumers. All pesticides except for lindane did not pose a cancer risk to humans, unfortunately due to lindane residue as a Persistent Organic Pollutants (POPs), CR (2.5E-5) was higher than 1.0E-6 and controlling plans should be conducted to decrease the concentration of this pesticide in Iran.

Rapid and simultaneous determination of glyphosate, glufosinate, and their metabolites in soil by high performance liquid chromatography-tandem mass spectrometry

建立了快速同时测定土壤中草甘膦(GLY)、草铵膦(GLUF)及其代谢物的高效液相色谱-串联质谱(HPLC-MS/MS)分析方法。分别对前处理和色谱-质谱条件进行优化,样品采用0.5 mol/L氨水作为溶剂振荡提取,离心,上清液过滤膜后,直接采用HPLC-MS/MS测定,电喷雾离子源(ESI-),多反应监测(MRM)模式下检测,外标法定量。结果表明,采用Dikma Polyamino HILIC色谱柱(150 mm×2.0 mm, 5 μm)进行分离,目标化合物峰形良好,响应值高。GLY及其代谢物氨甲基膦酸(AMPA)在5.0~500 μg/L范围内线性关系良好;GLUF及其代谢物N-乙酰基-草铵膦(NAG)、3-(甲基膦基)丙酸(MPP)在2.0~500 μg/L范围内线性关系良好;5种化合物的相关系数(r2)均大于0.998,可以满足定量分析的要求。GLY、GLUF及其代谢物的方法检出限(LOD)在2.0~4.0 μg/kg之间,方法定量限(LOQ)在6.7~13.3 μg/kg之间。在空白土壤样品中添加0.02、0.05、0.2 mg/kg 3个水平的5种化合物混合标准溶液考察回收率,结果表明,低有机质含量土壤中5种化合物的平均加标回收率在74.2%~101%之间,相对标准偏差(RSD)在0.93%~6.8%之间;高有机质含量土壤中5种化合物的平均加标回收率在90.8%~116%之间,RSD在0.40%~7.1%之间。采用建立的方法对实际土壤样品进行测定,AMPA、GLY、MPP、GLUF、NAG的检出率分别为45%、25%、10%、5%和5%,最大残留量分别为147、35.2、154、21.6和11.0 μg/kg,说明土壤中GLY、GLUF及其代谢物残留需要引起一定的关注。该方法简化了前处理步骤,具有简单、快速、绿色环保、成本低、准确度和灵敏度高、重现性好等优点,适用于大批量不同有机质含量土壤样品的检测,为研究其在土壤中的残留状况和环境行为提供了可靠的技术支持。

Investigation of tetracycline residues in poultry meat samples from Dhaka city by high-performance liquid chromatography

The objective of the study was to quantify tetracycline (TCs) i.e., oxytetracycline (OTC), tetracycline (TC) and chlortetracycline (CTC) residues in thirty poultry meat samples (n = 30) collected from the local market and super shop around the Dhaka University campus during May 2019 to January 2020. Three samples were collected from each of ten locations. All samples were extracted by Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) method. Samples were analyzed by reversed-phase High-Performance Liquid Chromatograph equipped with Photo Diode Array Detector (LC-PDA) and a reported method was validated with good linear correlation coefficients of standards and matrix-matched calibration curves with r2=1.00, 0.99, 0.99 and r2= 0.99, 0.99,0.99 in the linearity range of 0-10 µg/kg for OTC, TC and CTC, respectively. The limit of detection (LOD) and limit of quantification (LOQ) for OTC, TC, and CTC were 1.05, 1.17, and 1.09 μg/kg and 3.15, 3.51 and 3.27 μg/kg, respectively. Accuracy that is expressed by the recovery percentages were calculated at two different concentrations (2.5 and 5 μg/kg) were 91 and 100%, 102 and 100%, and 106 and 100% for OTC, TC, and CTC, respectively. Intra-day (n=3) and inter-day (n=9, 3 days) precision data were under 10% for all sample matrixes. Standard deviations were calculated ±0.06, ±0.11 and ±0.03 and precision (expressed by RSD%) were found 5.57, 9.14 and 2.35%, respectively for OTC, TC and CTC. The HPLC-PDA method is affordable for screening of large number meat samples for residual antibiotics in biological matrices by any laboratories. The method is also cheaper in comparison with LC-MSMS. Analysis of real 30 poultry meat samples showed that the tetracyclines residues were below the quantification limit in all samples.

Targeted proteomics for rapid and robust peanut allergen quantification

This study improves LC-MS-based trace level peanut allergen quantification in processed food by refining method robustness, total analysis time and method sensitivity. Extraction buffer (six compared) and peptide choice were optimised and found to profoundly affect method robustness. A rapid extraction and in-solution digestion method was developed omitting subsequent reduction, alkylation and sample clean-up steps effectively reducing total analysis time from the previously reported ∼5.5–20 h to ∼2.5 h. For the three best performing peptides, accurate quantification (CVs < 15%) with matrix-matched calibration curves (R2 = 0.99–0.97) was achieved for peanut muffin and ice-cream with excellent linearity (0.25–1000 mg kg−1). The best performing peptide enabled excellent recovery rates in ice-cream (106.0 ± 15.1%) and peanut muffin (72.7 ± 13.4%). Sensitivity (LOD = 0.25–0.5 mg kg−1; LOQ = 0.5–1.0 mg kg−1) was 2- to 20-fold improved compared to previous methods depending on the peptide. These methodological improvements contribute to robust peanut detection in food and can be translated to additional food-borne allergens.

Multi residue determination of 96 veterinary drug residues in domestic livestock and fishery products in South Korea

This study was to develop a multi-residue quantification method for determination of 96 veterinary drugs in livestock products (beef, pork, chicken, milk, egg) and fishery products (flatfish, eel, and shrimp) using liquid chromatography-tandem mass spectrometry. In addition, the purpose of this study was to verify the identification of veterinary drug residues by applying this analytical method to livestock and aquaculture animals. The coefficient of determination (r2) was more than 0.98 at the matrix-matched calibration curve. The proposed method was validated according to the CODEX guidelines (CAC/GL-71-2009), and most target compounds were found to be in acceptable quantities under the requirements of the validation guidelines. The limit of detection and limit of quantification ranged from 0.3 to 19.7 μg kg−1 and 1 to 65 μg kg−1. Samples (n = 581) were collected and analyzed to investigate the residues in target animal derived foods and 131 samples were detected (22.5%). In addition, only one rockfish sample exceeded the Korean MRL for sulfadiazine (523 μg kg−1; MRL: 100 μg kg−1) and trimethoprim (195 μg kg−1; MRL: 50 μg kg−1). The most frequently detected veterinary drugs were flumequine followed by oxytetracycline, enrofloxacin, trimethoprim, ciprofloxacin, and mebendazole. These results showed that we developed an acceptable analytical method of 96 veterinary drugs in animal derived foods and it will be useful for the safety management of veterinary drugs in livestock products, fish, and shrimp.

LC-HRMS-based targeted metabolomics for high-throughput and quantitative analysis of 21 growth inhibition-related metabolites in Chinese hamster ovary cell fed-batch cultures.

Chinese hamster ovary (CHO) cells have been widely used in the biopharmaceutical industry for production of therapeutic proteins. CHO cells in fed-batch cultures produce various amino acid-derived intermediate metabolites. These small molecule metabolic byproducts have proven to be critical to cell growth, culture performance, and, more interestingly, antibody drug productivity. Herein, we developed an LC-HRMS-based targeted metabolomics approach for comprehensive quantification of total 21 growth inhibition-related metabolites generated from 14 different amino acids in CHO cell fed-batch cultures. High throughput derivatization procedures, matrix-matched calibration curves, stable isotope-labeled internal standards, and accurate mass full MS scan were utilized to achieve our goal for a wide range of metabolite screening as well as validity and reliability of metabolite quantification. We further present a novel analytical strategy for extending the assay's dynamic range by utilizing naturally occurring isotope M + 1 ion as a quantification analog in the circumstances where the principal M ion is beyond its calibration range. The integrated method was qualified for selectivity, sensitivity, linearity, accuracy, precision, isotope analysis, and other analytical aspects to demonstrate assay robustness. We then applied this metabolomics approach to characterize metabolites of interest in a CHO cell-based monoclonal antibody (mAb) production process with fed-batch bioreactor culture mode. Absolute quantification combined with multivariate statistical analysis illustrated that our target analytes derived from amino acids, especially from branched-chain amino acids, closely correlated with cell viability and significantly differentiated cellular stages in production process.

Determination of nine food-borne stimulant drug residues in pork, egg, and milk by ultra-performance liquid chromatography-tandem mass spectrometry

β-受体激动剂、β-阻断剂、蛋白同化制剂属于兴奋剂类药物,在动物饲养和屠宰过程中的违禁使用成为食源性兴奋剂类药物残留的来源,危害人类健康。目前β-受体激动剂、蛋白同化制剂的检测较多,β-阻断剂检测报道较少,动物源食品中β-阻断剂检测尚无标准方法。该文建立了超高效液相色谱-串联质谱测定猪肉、鸡蛋、牛奶中β-受体激动剂、β-阻断剂、蛋白同化制剂三大类共9种食源性兴奋剂类药物残留的方法。样品中加入乙酸铵缓冲液(pH 5.2)和β-葡萄糖醛酸酶/芳基硫酸酯酶,在37 ℃条件下酶解16 h,酶解后的试样冷却至室温,NaOH溶液调节pH 9.5后用乙腈提取,提取液盐析分层,经增强型脂质去除净化管(EMR-Lipid)净化,无水硫酸镁除水,氮吹浓缩至近干,残渣用1 mL乙腈-0.1%甲酸水溶液(1:9, v/v)溶解。以甲醇-0.1%甲酸水溶液作为流动相进行梯度洗脱,采用ACQUITY UPLC HSS T3色谱柱(100 mm×2.1 mm, 1.8 μm)分离,电喷雾正离子(ESI+)电离,多反应监测(MRM)模式下测定,基质匹配标准曲线内标法定量。实验优化了前处理过程中提取溶剂和pH对提取效率的影响,讨论了仪器分析过程中色谱柱、流动相、定容液等影响因素。结果表明:9种兴奋剂在0.5~20 μg/L浓度范围内线性关系良好,相关系数(r2)均大于0.99,方法检出限为0.3~0.6 μg/kg,定量限为1.0~2.0 μg/kg,在1、2、5倍定量限添加水平下,平均回收率为65.2%~117.0%,相对标准偏差(RSD)为1.3%~14.4%。应用建立的方法对市场上购入的猪肉、鸡蛋、牛奶三类动物源性食品进行测定,9种兴奋剂类药物残留均未检出。该方法快速、灵敏、准确、稳定,适用于猪肉、鸡蛋、牛奶等样品中9种食源性兴奋剂类药物残留的检测。

Development and Validation of a Reliable UHPLC-MS/MS Method for Simultaneous Quantification of Macrocyclic Lactones in Bovine Plasma.

A fast, accurate and reliable ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) method was developed for simultaneous quantification of ivermectin (IVER), doramectin (DORA), and moxidectin (MOXI) in bovine plasma. A priority for sample preparation was the eradication of possible infectious diseases to avoid travel restrictions. The sample preparation was based on protein precipitation using 1% formic acid in acetonitrile, followed by Ostro® 96-well plate pass-through sample clean-up. The simple and straightforward procedure, along with the short analysis time, makes the current method unique and suitable for a large set of sample analyses per day for PK studies. Chromatographic separation was performed using an Acquity UPLC HSS-T3 column, with 0.01% acetic acid in water and methanol, on an Acquity H-Class ultra-high performance liquid chromatograph (UHPLC) system. The MS/MS instrument was a Xevo TQ-S® mass spectrometer, operating in the positive electrospray ionization mode and two multiple reaction monitoring (MRM) transitions were monitored per component. The MRM transitions of m/z 897.50 > 753.4 for IVER, m/z 921.70 > 777.40 for DORA and m/z 640.40 > 123.10 for MOXI were used for quantification. The method validation was performed using matrix-matched calibration curves in a concentration range of 1 to 500 ng/mL. Calibration curves fitted a quadratic regression model with 1/x2 weighting (r ≥ 0.998 and GoF ≤ 4.85%). Limits of quantification (LOQ) values of 1 ng/mL were obtained for all the analytes, while the limits of detection (LOD) were 0.02 ng/mL for IVER, 0.03 ng/mL for DORA, and 0.58 ng/mL for MOXI. The results of within-day (RSD < 6.50%) and between-day (RSD < 8.10%) precision and accuracies fell within acceptance ranges. No carry-over and no peak were detected in the UHPLC-MS/MS chromatogram of blank samples showing good specificity of the method. The applicability of the developed method was proved by an analysis of the field PK samples.

Determination of disulfoton and its metabolites in agricultural products by dispersive soild phase extraction-ultra high performance liquid chromatography-tandem mass spectrometry

基于超高效液相色谱-串联质谱(UHPLC-MS/MS)建立定量分析乙拌磷及其5个代谢物(乙拌磷砜、乙拌磷亚砜、内吸磷-S、内吸磷-S-砜、内吸磷-S-亚砜)的方法,应用该方法分析其在农产品(豌豆、芦笋、小麦、咖啡豆和花生)中的残留量。对样品前处理及色谱条件进行优化,样品经过乙腈涡旋提取(小麦、咖啡豆和花生先加水润湿),经盐析分层,取上清液经50 mg C18、50 mg PSA和50 mg NH2混合分散固相萃取净化后,经Thermo Syncronis C18色谱柱(150 mm×2.1 mm, 5 μm)分离,柱温40 ℃,进样量2 μL,以水和乙腈为流动相梯度洗脱后,采用ESI源,在正离子扫描和多反应监测(MRM)模式下检测,外标法定量。结果表明,乙拌磷及其代谢物在2.0~200.0 μg/L范围内线性关系良好,相关系数(R2)≥0.9981。方法的检出限(LOD)为0.02~2.0 μg/kg,并以最小添加水平5 μg/kg为定量限(LOQ)。乙拌磷及其代谢物在豌豆、芦笋、小麦、咖啡豆和花生中5、100、1000 μg/kg 3个添加水平下的平均回收率为75.0%~110.0%,相对标准偏差(RSD)为0.7%~14.9%,方法的准确度和精密度符合农药残留测定。应用建立的方法对市售的40份小麦样品和40份花生样品进行检测,均低于方法的检出限。该方法具有操作简单、快速、灵敏、准确的特点,适用于谷物、油料、蔬菜等多种农产品中乙拌磷及其代谢物的残留检测。

Combination of fabric phase sorptive extraction with UHPLC-ESI-MS/MS for the determination of adamantine analogues in human urine

Herein, fabric phase sorptive extraction – a member of sorbent-based microextraction family – is utilized for the rapid isolation of selected antiviral drugs namely amantadine, memantine and rimantadine from human urine prior to their analysis with UHPLC-MS/MS. Fabric phase sorptive extraction membrane coated with sol–gel sorbent coated poly(propylene glycol)-poly(ethylene glycol)-poly(propylene glycol) sorbent was identified as the optimum extraction membrane for the target analytes. Due to the engineered affinity of the fabric phase sorptive extraction towards the analytes, the extraction proceeds in relatively short time (20 min) while its high permeability permits the extraction of biological samples without any other pretreatment. Following selection of the appropriate membrane, significant parameters including extraction time, sample volume, membrane size, elution solvent, etc were optimized. The drugs were analyzed under positive electrospray ionization using multi-reaction monitoring mode. The method was linear in the range of 5 – 100 ng mL−1 by constructing weighted matrix-matched calibration curves. The intra-day and inter-day accuracy were ranged between – 18.7 to + 18.1% and – 18.3 to + 16.1%, respectively. The precision of the method (intra-day, inter-day) were less than 18.5 % in all cases. The method limits of detection were in the range of 0.2 – 0.8 ng mL−1 while the lower limit of quantitation was set at 5 ng mL−1 for all analytes. The developed analytical scheme was efficiently applied to the analysis of the target drugs in human urine and could be adopted as a rapid and robust green analytical tool for clinical applications.

Quantification of allantoin and other metabolites of the purine degradation pathway in human plasma samples using a newly developed HILIC-LC-MS/MS method.

The development of a simple HILIC-LC-MS/MS method to quantify the plasma levels of allantoin, inosine, hypoxanthine, and adenosine, using stripped plasma for the bioanalytical method validation, was the purpose of this study. Chromatographic separation conducted using an XBridge BEH Amide column (2.1 × 150mm,3.5 μm) was achieved under gradient elution with two mobile phases: 0.1% formic acid-ACN (5:95) and 0.1% formic acid-ACN (50:50). Multiple reaction monitoring MS detection was performed using a triple quadrupole. The method validation experiments were performed according to the European Medicines Agency and the U.S. Food and Drug Administration guidelines. The lower LOQ was 50nM, 5nM, 20nM, and 2nM for allantoin, inosine, hypoxanthine, and adenosine, respectively. The recovery was repeatable and stable. The intraday precision ranged from 1.6% to 6.5%, while the interday precision ranged from 3.4% to 58.7%. Therefore, it is necessary to make a matrix-matched calibration curve each day to overcome this issue. Since the quality control samples' stability did not always comply with the guidelines, the samples need to be analyzed soon after collection.

Determination of phthalic acid esters and di(2-ethylhexyl) adipate in fish and squid using the ammonium formate version of the QuEChERS method combined with gas chromatography mass spectrometry

In the present study, the ammonium formate version of the QuEChERS method, considered highly advantageous in relation to instrument maintenance and other issues, was applied for the first time to extract a group of twelve phthalic acid esters (PAEs, i.e. dipropyl phthalate, DPP; diisobutyl phthalate, DIBP; dibutyl phthalate, DBP; diisopentyl phthalate, DIPP; di-n-pentyl phthalate, DNPP; dihexyl phthalate, DHP; butyl benzyl phthalate, BBP; dicyclohexyl phthalate, DCHP; di(2-ethylhexyl) phthalate, DEHP; di-n-octyl phthalate, DNOP; diisononyl phthalate, DINP; and diisodecyl phthalate, DIDP) and one adipate (di(2-ethylhexyl) adipate, DEHA) from two species of fish (Scomber colias and Katsuwonus pelamis) and one of squid (Loligo gahi). The method was validated in terms of linearity, trueness and matrix effects. Determination coefficients (R2) for matrix-matched calibration curves were higher than 0.99 in all cases, being the lowest calibration levels in the range 0.5–10 ng/g. Mean recovery values were between 70 and 117% with relative standard deviation values ≤20%. Matrix effects were soft (between −20 and +20%) for most analytes and matrices, except in squid samples, which was mostly medium with a moderate ion suppression. The analysis of 10 samples of each type showed the presence of DIBP, DBP and DEHP at concentrations up to 44.2 ± 2.1 ng/g of wet weight in some of the samples and species, still not representing concerning values when considering the daily intake of such species of seafood in the human diet (tolerable daily intake -TDI- values were not exceeded). Results demonstrated that the ammonium formate version of the QuEChERS method can be applied with success for the extraction and determination of the selected PAEs and DEHA in fish and squid samples.