Surveillance study of a number of synthetic and natural growth promoters in bovine muscle samples using liquid chromatography-tandem mass spectrometry

A simple, new method permitting the simultaneous determination and confirmation of trace residues of 24 different growth promoters and metabolites using liquid chromatography-mass spectrometry was developed and validated. The compounds were extracted from bovine tissue using acetonitrile; sodium sulphate was also added at this stage to aid with purification. The resulting mixture was then evaporated to approximately 1 ml and subsequently centrifuged at high speed and an aliquot injected onto the LC-MS/MS system. The calculated CCα values ranged between 0.11 and 0.46 μg kg(-1); calculated CCβ were in the range 0.19-0.79 μg kg(-1). Accuracy, measurement of uncertainty, repeatability and linearity were also determined for each analyte. The analytical method was applied to a number of bovine tissue samples imported into Ireland from third countries. Levels of progesterone were found in a number of samples at concentrations ranging between 0.28 and 30.30 μg kg(-1). Levels of alpha- and beta-testosterone were also found in a number of samples at concentrations ranging between 0.22 and 8.63 μg kg(-1) and between 0.16 and 2.08 μg kg(-1) respectively.     

Confirmatory method for the determination of streptomycin and dihydrostreptomycin in honey by LC-MS/MS

A method was specifically developed for the determination and confirmation of streptomycin and dihydrostreptomycin in different types of honey. The method is simple, rapid, sensitive and was validated for streptomycin and dihydrostreptomycin in accordance with Commission Decision 2002/657/EC. After extraction with phosphate buffer and a pH change, clean-up was performed via SPE with polymeric phase. LC-MS/MS analysis was carried out using two different HILIC columns for the separation of the analytes and using a triple quadrupole mass spectrometer in positive ESI mode to measure the transitions of the substances in MRM mode. For the quantification of both substances, matrix calibration curves in a linear range of 5-80 g kg(-1) were used. The validation parameters established for streptomycin and dihydrostreptomycin, CCα (11.8 and 11.5 μg kg(-1), respectively), CCβ (18.9 and 19.9 μg kg(-1), respectively), recovery (97 and 101%, respectively) and the relative within-laboratory reproducibility RSD(wR) (16.4 and 20.8%, respectively) at the recommended concentration of 40 μg kg(-1), fulfil the requirements of Commission Decision 2002/657/EC.     

Validation of an LC-MS/MS method for malachite green (MG), leucomalachite green (LMG), crystal violet (CV) and leucocrystal violet (LCV) residues in fish and shrimp

A quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous analyses of malachite green (MG), crystal violet (CV) and its major metabolites, leucomalachite green (LMG) and leucocrystal violet (LCV) residues in fish and shrimp samples has been validated. Fish and shrimp samples were extracted with citrate buffer/acetonitrile, and the extracts were purified on strong cation-exchange (SCX) solid-phase extraction (SPE) cartridge. After conversion of LMG into MG using a post column oxidation reactor containing lead (IV) oxide (PbO(2)), the effluents were analysed. Residues were analysed using positive-ion electrospray ionisation (ESI). Identification and quantification of analytes were based on the ion transitions monitored by multiple reaction monitoring (MRM). Validation of the method was carried out in accordance with the Decision 2002/657/EC, which establishes criteria and procedures for the validation of methods. The following parameters were determined: decision limit (CCα), detection capability (CCβ), linearity, accuracy, precision, selectivity, specificity and matrix effect. The decision limits (CCα) for MG, LMG, CV and LCV were 0.164, 0.161, 0.248 and 0.860 μg kg(-1). The respective detection capabilities (CCβ) were 0.222, 0.218, 0.355 and 1.162 μg kg(-1). Typical recoveries (intermediate precision) in shrimp, for MG, CV, LMG and LCV for 2.0 μg kg(-1) level fortified samples using the optimised procedure were in the range 69%, 97%, 80.3% and 71.8%, respectively. The findings demonstrate the suitability of the method to detect simultaneously MG, CV and its metabolite (LMG and LCV) in fish and shrimp.     

Validation of a quantitative and confirmatory method for residue analysis of aminoglycoside antibiotics in poultry, bovine, equine and swine kidney through liquid chromatography-tandem mass spectrometry

The use of aminoglycoside antibiotics in food animals is approved in Brazil. Accordingly, Brazilian food safety legislation sets maximum levels for these drugs in tissues from these animals in an effort to guarantee that food safety is not compromised. Aiming to monitor the levels of these drugs in tissues from food animals, the validation of a quantitative, confirmatory method for the detection of residues of 10 aminoglycosides antibiotics in poultry, swine, equine and bovine kidney, with extraction using a solid phase and detection and quantification by LC-MS/MS was performed. The procedure is an adaptation of the US Department of Agriculture, Food Safety and Inspection Service (USDA-FSIS) qualitative method, with the inclusion of additional clean-up and quantification at lower levels, which proved more efficient. Extraction was performed using a phosphate buffer containing trifluoroacetic acid followed by neutralization, purification on a cationic exchange SPE cartridge, with elution with methanol/acetic acid, evaporation, and dilution in ion-pair solvent. The method was validated according to the criteria and requirements of the European Commission Decision 2002/657/EC, showing selectivity with no matrix interference. Linearity was established for all analytes using the method of weighted minimum squares. CCα and CCβ varied between 1036 and 12,293 μg kg(-1), and between 1073 and 14,588 μg kg(-1), respectively. The limits of quantification varied between 27 and 688 μg kg(-1). The values of recovery for all analytes in poultry kidney, fortified in the range of 500-1500 μg kg(-1), were higher than 90%, and the relative standard deviations were lower than 15%, except spectinomycin (21.8%). Uncertainty was estimated using a simplified methodology of 'bottom-up' and 'top-down' strategies. The results showed that this method is effective for the quantification and confirmation of aminoglycoside residues and could be used by the Brazilian programme of residue control.     

Determination and confirmation of chloramphenicol in honey, fish and prawns by liquid chromatography-tandem mass spectrometry with minimum sample preparation: validation according to 2002/657/EC Directive

A reliable, simple and sensitive liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/MS) confirmation method has been developed for chloramphenicol (CAP) determination in honey, fish and prawns. For honey, samples were extracted with ethyl acetate, an aliquot was evaporated to dryness and re-dissolved in mobile phase. For fish and prawns, tissues were extracted with acetonitrile and chloroform. The organic layer was evaporated to dryness and the residue was re-constituted with water: acetonitrile (90:10). LC separation was achieved on a C18 column with gradient elution using a mobile phase of acetonitrile and water. Analysis was carried out on a triple-quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode via electrospray interface operated in negative ionisation mode, with deuterated chloramphenicol-d(5) (d(5)-CAP) as internal standard. Method validation was performed according to the criteria of Commission Decision 2002/657/EC. Four identification points were obtained for CAP with one precursor ion and two product ions. The limit of detection (LOD) was 0.02 μg kg(-1). Linear calibration curves were obtained over concentration ranges of 0.1-1.0 μg kg(-1) in tissues. Mean recoveries ranged from 85.5% to 115.6%, with the corresponding intra- and inter-day variation ranging from 1.0% to 22.5%, depending on matrix type and level of concentration. The decision limit (CCα) and detection capability (CCβ) of the method were obtained for all matrices: 0.04 and 0.06 μg kg(-1), respectively, for prawns and fish and 0.05 and 0.09 μg kg(-1) for honey.     

Confirmatory analysis of steroids in muscle using liquid chromatography–tandem mass spectrometry

A method is described for screening and confirmation of synthetic and endogenous steroids in muscle tissue. The method is sensitive, selective, and rapid and the consumption of organic solvents is low, compared to previously published methods. The procedure involves hydrolysis, defattening with heptane and final clean up with SPE using C₁₈ cartridge. After filtration, the analytes are analysed by LC/MS/MS and quantification is performed using deuterated internal standards. Decision limits (CCα) varied from 0.02 to 0.33?µg?kg^?1 and the detection capabilities (CCβ) were <0.50?µg?kg^?1. The mean within-laboratory reproducibility ranged 5–22% (%RSD_IR). Endogenous steroids (e.g. testosterone, epitestosterone and androstenedione) have been included in the method, to provide an insight into their levels, as the presence of these steroids was detected several times during analysis of imported meat.     

Determination of (fluoro)quinolones in eggs by liquid chromatography with fluorescence detection and confirmation by liquid chromatography-tandem mass spectrometry

A multiresidue analytical procedure for determination of seven fluoroquinolones (marbofloxacin, norfloxacin as internal standard, ciprofloxacin, danofloxacin, enrofloxacin, sarafloxacin and difloxacin), and three quinolones (oxolinic acid, nalidixic acid and flumequine) in eggs is presented. The procedure is based on dispersive solid-phase extraction technique with acetonitrile as extractant. Norfloxacin and ciprofloxacin - d8 were used as internal standards to quantify the (fluoro)quinolones. Analyses were realised by LC-FLD for screening and LC-MS/MS for confirmatory purposes. The whole procedure was evaluated according to the Commission Decision 2002/657/EC. Specificity, decision limit (CCα), detection capacity (CCβ), recovery (absolute and relative), precision (repeatability and reproducibility) were determined during validation process. Recoveries (relative) for the LC-FLD screening determination ranged from 85% to 93%, repeatability and reproducibility were in the range of 5-9% to 9-16%, respectively. CCα and CCβ were 13-37 and 17-43 μg/kg pending on analite. For the LC-MS/MS confirmatory method, the relative recoveries were satisfactory (92-99%) with repeatability and reproducibility in the range of 4-7% to 6-12%, respectively. CCα and CCβ were 3-7 and 7-11 μg/kg depending on the analite. The results of both prepared methods showed these analytical procedures simple, rapid, sensitive and suitable for routine control of eggs.     

Immunoaffinity chromatography purification and ultra-high-performance liquid chromatography-tandem mass spectrometry determination of four β-agonists in beef

A highly selective and sensitive method was developed for the simultaneous determination of four β-agonists (clenbuterol, salbutamol, ractopamine and terbutaline) in beef by immunoaffinity chromatography purification coupled to ultra-high-performance LC-MS/MS. The MS/MS conditions, ultra-high-performance LC mobile phase, injection solution, sample purification process and matrix effect were studied to optimise the operation conditions. The limits of detection (LODs) of the instrument for the studied β-agonists ranged from 0.20 to 0.25 μg l(-1), and the LODs of the method for the studied β-agonists ranged from 0.20 to 3.00 μg kg(-1) for beef. Calibration curves were constructed using a standard solution diluted with blank beef matrix. The linear ranges of the calibration curves ranged from 5 to 100 μg kg(-1) and the coefficients of determination were >0.9942 (n = 10) for all four β-agonists. Samples spiked at 5, 10 and 50 μg kg(-1) showed recoveries >72% and RSDs <6.6%. The method is suitable for the simultaneous detection of four β-agonists at trace levels in beef.     

Validation of a commercial receptor kit Sulfasensor Honey for the screening of sulfonamides in honey according to Commission Decision 2002/657/EC

The Sulfasensor Honey kit is a receptor test dedicated to the screening of sulphonamide residues respectively in different matrices. The aim of this project was to evaluate and validate this kit according to the Community Reference Laboratory (CRL) guideline for the validation of screening methods to achieve the French control plan for honey. The test is robust, quick (90 min for 40 samples), easy to perform and easy to read. The false-positive rate was estimated to be 12.5%. The detection capabilities CCβ of the kit were lower than or equal to 25 μg kg(-1) for sulfamethazine, sulfamerazine, sulfathiazole and sulfapyridine, and between 25 and 50 μg kg(-1) for sulfadiazine and sulfadimethoxine, 150 μg kg(-1) for sulfaquinoxaline, and 1000 μg kg(-1) for sulfamethoxazole and sulfamethizole. Sulfanilamide was not detected by the kit. The kit was applicable to a wide variety of honeys (different floral and geographical origins, liquid or solid). This kit was used to implement the French control plan for the detection of antibiotic residues in honey in 2010 in parallel with an HPLC method. However, in 2011 the kit was replaced by an LC-MS/MS method for the screening and confirmation of sulfonamide residues in honey, which detects all the sulfonamides of interest.     

In-house validation of PremiTest, a microbiological screening test with solvent extraction, for the detection of antimicrobial residues in poultry muscles

PremiTest, a microbial inhibition test for the screening of antimicrobial residues, was validated according to the criteria established by Decision 2002/657/EC. Sensitivity, detection capability (CCβ), specificity, selectivity, robustness and applicability were evaluated. The methodology involves the technique of solvent extraction, which increases the detection capability of the test for a wider range of antibiotics. The following CCβ values in poultry muscle were found: penicillin G ≤ 12.5 μg kg(-1), total sulfonamides ≤ 75 μg kg(-1), erythromycin 75 μg kg(-1) and lincomycin 50 μg kg(-1). The detection capability of chlortetracycline was equal to its maximum residue limit (100 μg kg(-1)) and the method did not detect gentamicin (1000 μg kg(-1)), for which no MRL is established in poultry muscle. Specificity evaluated in relation to different analytes and matrices did not detect any interferences in the tests results; whilst the robustness showed that the pH neutralisation point of the extract affects the analytical results and the kits' performance. Only the screening of tetracyclines requires the analysis of extracts without pH neutralisation. The results of the validation process showed that this method is acceptable for screening β-lactam, sulfonamide and macrolide antimicrobial groups in the National Residues and Contaminants Control Programme (PNCRC), and that for this it is fit for purpose.     

Validation of a rapid and sensitive routine method for determination of chloramphenicol in honey by LC-MS/MS

Chloramphenicol (CAP) is a broad spectrum antibiotic used in the treatment of human and animal diseases. However, CAP can exhibit toxic effects in certain susceptible individuals, causing bone marrow depression, including fatal aplastic anemia. As this condition is dose-independent, CAP has been banned for use in food-producing animals, including honeybees. In this study, a quick, simple and low-cost routine analytical method was developed for the screening and confirmation of chloramphenicol in honey by LC-MS/MS. Sample clean-up takes only two steps without SPE procedure and with recoveries >97%. Honey samples were selected from several producers in Brazil and diluted in a small amount of water. After fortification and addition of d (s)-chloramphenicol as internal standard, the samples were extracted with ethyl acetate. Complete validation of the method was performed on the basis of EU decision 2002/657. Within-laboratory CV reproducibility at the lowest concentration was <10%. An evaluation of two different methods to calculate the decision limit and detection capability gave 0.08 μg kg(-1) for CCα and 0.12 μg kg(-1) for CCβ.     

Method validation for the determination of total mercury in fish muscle by cold vapour atomic absorption spectrometry

A method was validated for the determination of total Hg in fish muscle using continuous flow cold vapour atomic absorption (CVAAS) after microwave digestion in closed vessels. The method was validated according to European Union Regulations 333/2007 and 657/2002, considering the maximum level for the metal in fish, established by European Union regulation 1881/2006. The procedure for determining linear range, selectivity, recovery, precision, trueness, decision limit (CCα), detection capability (CCβ), measurement uncertainty and robustness of the method is reported. The results of the validation process demonstrate the method fulfils the provisions of the Commission Regulation. The selectivity study indicated that there was no matrix effect on the calibration curve between the concentration range of 1.0 and 30.0 μg Hg l(-1). The mean recovery calculated at six levels of fortification was in the range of 94-104%. The limit of detection (LOD) and limit of quantification (LOQ) values were 4.90 and 15.7 μg kg(-1), while the CCα and CCβ values were 0.517 and 0.533 mg kg(-1), respectively, for the maximum contaminant level of 0.500 mg kg(-1). The relative expanded measurement uncertainty of the method was 0.055 mg kg(-1). The method was not affected by slight variations of some critical factors (ruggedness minor changes) as sample mass and volume of the HNO(3) and H(2)O(2) used in the digestion step. The method allowed accurate confirmation analyses of the CRM DORM 3. In fact, the Z-scores attained in a proficiency test round were well below the reference value of 2.0, proving the excellent performance of the laboratory.     

QuEChERS-液相色谱-串联质谱法测定葵花籽中30种农药残留

目的 建立QuECHERS-液相色谱-串联质谱法测定葵花籽中30种农药残留的分析方法.方法 葵花籽用1％乙酸-乙腈溶液提取,QuEChERS试剂净化,液相色谱-串联质谱法(liquid chromatography-tandem mass spectrometry,LC-MS/MS)测定,多反应监测模式(multipl...     

Determination of tadalafil and N-desmethylsibutramine in health and dietary supplements using ultra-performance liquid chromatography (UPLC) coupled with quadrupole-time-of-flight mass spectrometry (Q-TOF MS)

The adulteration of dietary supplements with drugs is potentially dangerous for human health. In this study, a method was used to test simultaneously for the presence of three synthetic PDE-5 inhibitors (sildenafil, vardenafil and tadalafil), and sibutramine and its two major metabolites (N-desmethylsibutramine and N-didesmethylsibutramine) using ultra-performance liquid chromatography (UPLC) coupled with quadrupole-time-of-flight mass spectrometry (Q-TOF MS) in dietary supplements. This approach with UPLC/Q-TOF MS uses the high accurate mass of six compounds for identification and has a short run time. The recovery was from 87% to 113%; precision was less than 12.8%. The limit of detection and limit of quantification were from 0.4 to 2.0 μg kg(-1) and from 1.3 to 6.0 μg kg(-1), respectively. This method allows easy and fast analysis and detection of diverse adulterants.     

液相色谱-串联质谱法测定猪肉中13种磺胺类药物残留

利用液相色谱-电喷雾串联质谱建立猪肉中13种磺胺类药物残留的同时、快速分析方法.猪肉样品中的残留药物用1%乙酸-乙腈均质提取,浓缩后用0.1%甲酸-乙腈定容,并用正己烷液-液分配除脂后,用Kinetex C18色谱柱(100mm×2.1mm,2.6μm)分离,以0.1%甲酸-乙腈作为流动相梯度洗脱,采用电喷雾正离子电离,多反应监测模式检测,基质匹配外标法定量.13种磺胺类药物在1~300μg/kg范围内线性关系良好,定量限均小于10μg/kg.在1、2μg/kg和10μg/kg这3个添加水平下,回收率在60.0%~78.1%之间,相对标准偏差(RSD)在0.24%~8.88%之间.此方法操作简便,灵敏度、准确度、精密度均满足残留分析的要求.     

Citrinin Determination in Red Fermented Rice Products by Optimized Extraction Method Coupled to Liquid Chromatography Tandem Mass Spectrometry (LC‐MS/MS)

A rapid and sensitive method was developed and validated for citrinin determination in red fermented rice products by liquid chromatography tandem mass spectrometry (LC‐MS/MS) under the selected reaction monitoring mode. Sample preparation was especially focused, and the quantitative methods of LC‐MS/MS and high‐performance liquid chromatography with fluorescence detection (HPLC‐FLD) were compared. In red fermented rice samples, the limit of detection was 1.0 μg/kg for LC‐MS/MS compared to 250 μg/kg for HPLC‐FLD, the limit of quantification was 3.0 μg/kg for LC‐MS/MS compared to 825 μg/kg for HPLC‐FLD. High correlation coefficient was obtained (R² = 0.999) within the linear range (0.1 to 100 μg/L) in the MS method. The recoveries ranging from 80.9% to 106.5% were obtained in different spiking concentrations. The average intra‐ and inter‐day accuracy ranged from 75.4% to 103.1%, and the intra‐ and inter‐day precisions were from 3.3% to 7.9%. The developed method was applied to 12 commercial red fermented rice products, and citrinin was found in 10 samples ranging from 0.14 to 44.24 mg/kg. Compared to traditional qualitative and quantitative methods, the newly developed LC‐MS/MS method for citrinin determination includes the merits of using a small amount of extraction solvent, simple preparation steps, and high sensitivity.     

基于QuEChERS-GC-MS/MS法同时测定猕猴桃中15种有机磷农药残留

建立改良的QuEChERS方法结合气相色谱-三重四极杆串联质谱（GC-MS/MS）法同时检测猕猴桃中15种有机磷农药残留的分析方法。样品前处理采用改进的QuEChERS方法,乙腈-乙酸乙酯（1∶1,V/V）作为提取溶剂,经100 mg乙二胺-N-丙基硅烷（PSA）、200 mg十八烷基硅烷键合硅胶（C18）、40 mg石墨化碳黑（GCB）、300 mg无水硫酸镁固相分散净化,用GC-MS/MS法在多反应监测（MRM）模式下分析检测,内标法定量。结果表明,15种有机磷农药在质量浓度0.05～1.00 μg/mL范围内线性关系良好,相关系数R2均＞0.998,方法检出限为0.001 2～0.326 5 μg/kg,定量限为0.004～1.088 μg/kg;其平均加标回收率为76.95%～101.2%,精密度试验结果的相对标准偏差（RSD）（n=6）为0.21%～10.05%。该方法具有净化效果好、重复性强、灵敏度高等优势,适用于猕猴桃样品中多种农药残留的同时检测。     

高效液相色谱-串联质谱法测定动物肌肉组织中氨基甲酸酯类杀虫剂及其代谢物残留

建立动物组织中氨基甲酸酯类杀虫剂及其代谢物(共16种)残留的高效液相色谱-串联质谱分析方法。样品经乙腈提取、浓缩、净化,液相色谱串联质谱测定,内标法定量。16种杀虫剂在1.0～100μg/L范围内线性关系良好(r＞0.9959);方法定量限为0.5～2.5μg/kg;样品添加5.0、10.0、20μg/kg时,加标回收率为71.4%～105.5%;相对标准偏差为3.2%～13.7%。该方法具有简便快捷、灵敏度高的特点,适用于动物肌肉中氨基甲酸酯类杀虫剂及其代谢物残留量的检测。     

液相色谱-串联质谱法测定糖果中2-(4-甲氧基苯氧基)-丙酸钠甜味抑制剂

建立了高效液相色谱-串联质谱法检测糖果中的2-(4-甲氧基苯氧基)-丙酸钠的方法。采用水溶液(pH8.0):乙腈=1.5:1 (V/V)作为提取溶剂,然后用正己烷去除油脂,取水层清液过膜,进行检测。采用电喷雾负离子模式(ESI-)和选择反应监测扫描(SRM)模式,外标法定量。结果表明,该方法在6.25~100 μg/L范围内线性关系良好(相关系数r=0.9998),检出限为8.0 μg/kg,定量限为20.0 μg/kg。回收率为82.7%~90.7%,相对标准偏差为2.5%~4.2%。该方法前处理简单、重现性好、灵敏度高,为糖果中的2-(4-甲氧基苯氧基)-丙酸钠的检测提供一种新的选择。     

液相色谱-串联质谱法测定畜禽表皮中松香酸和脱氢松香酸

采用液相色谱-串联质谱法对畜禽表皮组织中的松香酸和脱氢松香酸含量进行检测。样品中的松香酸和脱氢松香酸用乙腈提取,经HLB固相萃取柱净化,采用液相色谱-串联质谱进行检测。色谱柱：反相C18柱（100 mm×2.1 mm,2 μm）;流动相：0.1%甲酸溶液和0.1%甲酸-甲醇溶液（10∶90,V/V）;流速：0.3 mL/min;质谱分析采用电喷雾电离正离子模式检测,扫描方式为多反应监测。结果表明：松香酸线性范围为2.0～100 μg/L,检出限为1.1 μg/kg,定量限为3.6 μg/kg;脱氢松香酸线性范围为5.0～250 μg/L,检出限为3.9 μg/kg,定量限为13 μg/kg。在5.0～200 μg/kg添加量范围内松香酸和脱氢松香酸的回收率为81.7%～93.7%,相对标准偏差均在12%以内。方法用于实际样品分析,多个畜禽表皮样品中检出松香酸及脱氢松香酸,采用松香甘油酯进行脱毛的鸭皮中也检出了一定含量的松香酸及脱氢松香酸。本实验建立的分析方法简便快捷,具有较好的灵敏度和可靠性,可用于畜禽表皮组织中松香酸和脱氢松香酸的定量确证分析。