Development of a Liquid Chromatography Tandem Mass Spectrometric Method for Simultaneous Determination of 15 Aminoglycoside Residues in Porcine Tissues

A quantitative and confirmatory method has been developed for simultaneous determination of 15 aminoglycoside (AG) residues in porcine tissues (muscle, liver and kidney) by liquid chromatography tandem mass spectrometry (LC-MS/MS). The analytes were extracted from different matrices with aqueous trichloroacetic acid solution (5 %, w/v) followed by solid phase extraction (SPE) clean-up under optimised conditions. Due to the different pK _a values of the compounds, two consecutive SPE steps using Oasis HLB cartridges were used to purify all 15 AGs from sample extracts, with 9 AGs quantitatively retained on Oasis HLB cartridges at pH?<1 and the other 6 AGs retained at pH 8.5. The analytes were separated on a reversed-phase C18 column and eluted with water and acetonitrile containing the ion-pair reagent heptafluorobutyric acid (HFBA). The LC-MS/MS method was validated according to Decision 2002/657/EC. The optimised procedure was successfully applied to analyse 100 real porcine tissue samples (60 muscles, 20 livers and 20 kidneys) collected from local markets in southern China, demonstrating that the method is robust and useful for determination of residues of the 15 target AGs in porcine tissue samples.     

Simultaneous Determination of Nine Quinolones in Food by Liquid Chromatography with Fluorescence Detection

A confirmatory analytical method for simultaneous determination of nine regulated quinolones (Council Regulation 2377/90/ECC) in six matrices of animal origin is proposed. The sample pretreatment involves double step liquid extraction with acetonitrile and purification by solid-phase extraction on Oasis HLB cartridges. The quinolones were separated by liquid chromatography on C18 Zorbax column with gradient elution program. Aqueous formic acid, methanol, and acetonitrile were used as a mobile phase. A multi-wavelength excitation/emission program was used for sensitive fluorescence detection of quinolones. The proposed sample pretreatment protocol was applied to each of the six studied matrices without any modification. The method was validated according to Commission Decision 2002/657 EC. Residues were quantified down to 15 μg kg^?1 with limits of detection and quantification ranging from 3 to 50 μg kg^?1 and from 7.5 to 100 μg kg^?1, respectively. The recoveries at the maximum residual limits (MRLs) were between 77 and 120 % with RSD values lower than 30 %. For quinolones without established MRL or maximum required performance limit, the accuracy and precision of the method were estimated at concentration levels corresponding to the lowest linear calibration point and recoveries between 70 and 130 % were achieved. Decision limits, detection capability, and linear range in eggs, milk, fish, ovine muscle, chicken muscle, and porcine kidney are also reported.     

Confirmatory Method for the Determination of Amphenicols in Muscle and Kidney of Several Animal Species

A rapid and reliable method using liquid chromatography (UHPLC) tandem mass spectrometry (MS/MS) to identify and quantify chloramphenicol (CAP), thiamphenicol (TAP), florfenicol (FF) and its marker residue florfenicol amine (FFA) has been developed and validated. FF and TAP are both allowed substances and different MRLs have been established for different matrices and species. However, CAP is a well-known forbidden substance with a MRPL of 0.3 μg kg^?1 for all matrices. It was relevant to develop this confirmatory method to quantify simultaneously the four amphenicols in kidney and muscle of all species, to make more effective the official control programme for this group of substances. The variety of levels of interest to be studied for the allowed compounds and the different chemical behaviour of FFA have been the main difficulties in the development and validation of the method. The method has been validated in swine, bovine, caprine, equine, porcine, aquaculture species, rabbit and poultry muscle and kidney (except aquaculture species, rabbit and poultry). Performance criteria have been calculated in accordance with Commission Decision 2002/657/EC. The proposed procedure is being applied to the official control in the Valencian Region after being successfully evaluated by ENAC (Spanish national body for the accreditation).     

Analysis of trace residues of tetracyclines in animal tissues and fluids using metal chelate affinity chromatography/HPLC

A novel method of analysis for the trace residue determination of tetracyclines in animal tissues and fluids has been developed. Clean-up of sample extracts is based upon the specific ability of tetracyclines to chelate with divalent metal ions (metal chelate affinity chromatography, MCAC) and determination made by high-performance liquid chromatography. The method has been tested for the determination of oxytetracycline (OTC), tetracycline (TC) and chlortetracycline (CTC) in porcine kidney and muscle, ovine kidney, bovine kidney and milk, and trout muscle. Recoveries at the 0.05 mg/kg level for OTC, TC and CTC respectively were 75%, 63%, 73% in porcine kidney, 77%, 79%, 76% in porcine muscle, 85%, 54%, 53% in bovine kidney, 78%, 63%, 57% in ovine kidney, 75%, 58%, 56% in fish (trout) muscle, and 80%, 59%, 59% in bovine milk. At this level both within- and between-batch precision, as measured by the coefficient of variation (CV), was less than 10%. Determination to the 0.01 mg/kg level was carried out in all cases, although the method becomes less precise. The method has been used for several months and found to be both reliable and sufficiently rapid for use as a routine quantitative screening procedure. When coupled with liquid chromatography-mass spectrometry (LC-MS) it is suitable for use as a confirmatory method. Analysis of animals treated with tetracyclines has been carried out.     

Detection of macrocyclic lactones in porcine liver,meat and fish tissue using LC–APCI–MS–MS

A selective and sensitive method for the simultaneous determination of five avermectins (abamectin, ivermectin, doramectin, emamectin and eprinomectin) and one milbemycin (moxidectin) in porcine liver, bovine meat and fish tissue was developed. The method involved extraction with acetonitrile and purification by C₁₈ solid-phase extraction. Detection was carried out using liquid chromatography coupled to multiple mass spectrometry (LC–MS²) equipped with APCI in the negative mode. This method was validated according to the requirements of Commission Decision EC/2002/657 (Implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results. Off J Eur Commun. L221: 8–36.). In addition to the linear response (R ² between 59 and 97%), good repeatability (CV between 20 and 35%), reproducibility (CV between 20 and 35%) and detection (CCα) and quantification (CCβ) limits were obtained for all compounds in all matrices considered.     

Vitamin A and beta-carotene in bovine and porcine plasma, liver, corpora lutea, and follicular fluid

Bovine and porcine blood plasma, liver, corpora lutea, and follicular fluid were obtained from local abattoirs for study of distribution of vitamin A and beta-carotene. Retinol, retinyl esters, and beta-carotene were separated on alumina columns and subjected also to thin-layer chromatography. Retinol and retinyl esters were in corpora lutea and follicular fluid of both species. Concentrations of beta-carotene were high in bovine plasma, corpus luteum, and follicular fluid. In contrast, beta-carotene was lower in porcine tissues. Retinol, retinyl esters, and beta-carotene were closely correlated in bovine follicular fluid and blood plasma; however, correlations between bovine plasma and corpora lutea were not significant except for retinol. Only porcine retinol was closely correlated with plasma and follicular fluid, whereas correlations were nonsignificant between plasma and corpora lutea retinol, retinyl esters, and beta-carotene. Further studies, therefore, are needed to elucidate the physiological role of vitamin A and beta-carotene in regulating ovarian functions.     

Determination of the hormonal growth promoter 17α-methyltestosterone in food-producing animals: Bovine hair analysis by HPLC–MS/MS

This paper describes the development, validation and application of a confirmatory method to detect 17α-methyltestosterone (MT) in bovine hair, to aid in controlling the administration of this growth promoter in meat-producing animals. After cryogenic grinding, MT was removed from the hair matrix using a single step extraction procedure with acetonitrile. Hydroxylamine derivatisation was used to enhance analyte determination with an electrospray ionisation (ESI) source. Determination was carried out using a triple quadrupole liquid chromatography tandem mass spectrometer (LC–MS/MS) in multiple reaction monitoring mode (MRM). The method was validated in accordance with the criteria defined in Commission Decision 2002/657/EC and using deuterated testosterone (T-d₃) as the internal standard. The decision limit (CCα) was 0.07 ng g⁻¹ and the detection capability (CCβ) was 0.12 ng g⁻¹. Repeatability was CV% (7%), within-laboratory reproducibility was CV% (11.0%), and trueness was (87%). Applicability of the method was demonstrated in an animal study. Samples obtained from animal experiments were analyzed and the presence of MT was confirmed.     

基于超高效液相色谱－串联质谱法的纺织品中7种尼泊金酯类防腐剂测定

 利用超高压液相色谱-电喷雾串联四极杆质谱(UPLC-MS/MS)联用技术,建立了一种能在7min内快速分离和测定纺织品中7种尼泊金酯类防腐剂的方法。样品经超声提取、浓缩,甲醇+水(1+1)溶解,采用ACQUITY UPLC?BEH C18柱(50 mm × 2.1 mm i.d.,1.7μm),以甲醇-水溶液为流动相,梯度洗脱,目标分析物采用UPLC-MS/MS进行分析,以保留时间和离子对进行定性和定量测定,在ESI(-)和MRM监测模式下进行样品分析。该方法检出限(LOD)为4～12μg/kg;在0.1～1.0μg/mL范围内线性关系良好,添加浓度分别为0.5mg/kg,1.0mg/kg和3.0mg/kg时,添加回收率范围为76.09 ± 4.92～106.65 ± 4.90％,相对标准偏差为4.60～14.01％。     

A Simple and Fast Method for the Determination of 20 Veterinary Drug Residues in Bovine Kidney and Liver by Ultra-High-Performance Liquid Chromatography Tandem Mass Spectrometry

A simple and rapid sample preparation method was developed and validated for multi-class analysis of veterinary drug residues in bovine kidney and liver. Sample preparation procedure was performed using acetonitrile and trichloroacetic acid for protein precipitation followed by ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) analysis. The proposed method was validated according to the criteria of the Commission Decision 2002/657/EC evaluating linearity, selectivity, accuracy and precision, determination limit (CCα), and detection capacity (CCβ). Linearity presented r ² ≥ 0.99 for all the target compounds, and recoveries ranged from 80 to 110 % with RSD ≤17 % for intra- and inter-day assay. Values of CCα and CCβ ranged from 11 to 1096 and from 12 to 1191 μg kg^?1, respectively. The proposed sample preparation followed by UHPLC-MS/MS analysis was suitable for the determination of 20 veterinary drug residues in bovine kidney and liver in routine analysis. Method applicability was evaluated using commercial samples.     

Determination of sulbactam in raw bovine milk by isotope dilution–ultra-high-performance liquid chromatography–tandem mass spectrometry

We developed a sensitive and selective isotope dilution ultra-high-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) method for the determination of sulbactam residue in raw bovine milk. Sulbactam and internal standard, sulbactam-d₅, were extracted from raw bovine milk via liquid-liquid extraction and enriched with strong anion exchange solid-phase extraction cartridges and finally analyzed by using UPLC-MS/MS with multiple reaction monitoring mode. The method was validated according to European regulations. The calibration curve showed good linearity, with a correlation coefficient of 0.9998. Decision limit and detection capability of sulbactam were determined by matrix calibration curve and were 0.0445 and 0.0517 μg/L, respectively. The recoveries of sulbactam in fortified raw bovine milk ranged from 72.1 to 91.5%, with the intra- and interday relative standard deviations ranging from 3.0 to 18.9%. Furthermore, the developed method was applied to analyzing real raw bovine milk samples collected from dairy farms in Beijing, China. Sulbactam was not determined in all samples. The proposed method could ultimately serve as a methodological foundation for the determination of sulbactam in different types of raw milk and dairy products.     

气相色谱-质谱联用法测定葡萄酒中三种邻氨基苯甲酸酯类葡萄香精

建立了气相色谱-质谱联用法（GC-MS）测定葡萄酒中邻氨基苯甲酸酯类（邻氨基苯甲酸甲酯、N-甲基邻氨基苯甲酸甲酯、邻 氨基苯甲酸乙酯）葡萄香精的方法。 样品用正己烷作为提取溶剂,经过液-液萃取净化,气相色谱-质谱仪的选择离子监测模式进行测 定,外标法定量。 3种邻氨基苯甲酸酯类葡萄香精在0.05～1.00 mg/L范围内呈现良好的线性关系,相关系数R2均＞0.998,该方法定量 限为5 μg/L,样品平均加标回收率为86.2%～102.4%,精密度实验结果相对标准偏差（RSD）为1.02%～8.75%（n=6）。 该方法简便、快速、 准确、灵敏,具有良好的精密度及准确性,适用于葡萄酒中邻氨基苯甲酸酯类葡萄香精的测定。     

气相色谱-质谱联用法检测食品塑料包装材料中的16种邻苯二甲酸酯

目的建立气相色谱-质谱联用法检测食品塑料包装材料中的16种邻苯二甲酸酯。方法样品经粉碎后,加入10 mL正己烷超声提取30 min,经0.45μm滤膜过滤后,采用DB-5MS色谱柱分离,供GC-MS分析。质谱采用电子轰击离子源(electron impact ion source,EI)和选择离子监测(selected ion monitor,SIM),以外标法定量。结果 16种目标物在0.5~50 mg/L范围内线性关系良好,线性相关系数介于0.9995~1.0000,检出限为0.5 mg/kg,在0.5、1.0和2.5 mg/kg 3个加标水平的平均回收率为65.3%~111.3%,相对标准偏差为0.6%~9.9%(n=6)。结论该方法简便、快速、准确,可应用于食品塑料包装中邻苯二甲酸酯的检测。     

气相色谱-质谱法测定微波食品中17种邻苯二甲酸酯

建立了同时测定微波食品中17种邻苯二甲酸酯的气相色谱质谱(GC-MS)分析方法。本方法首次采用将微波食品按照不含油食品和含油食品进行分类前处理。不含油微波食品经正己烷提取GC-MS直接检测;含油微波食品经正己烷-乙腈提取,PSA/Silica复合玻璃固相萃取柱净化,浓缩定容后,GC-MS检测,外标法定量。17种邻苯二甲酸酯在0.02~1.00mg/L浓度范围内,线性相关良好,相关系数(R2)大于0.99。17种邻苯二甲酸酯的检出限为0.05mg/ kg。加标实验平均回收率为85.7%~105.1%,相对标准偏差(RSD)小于8.9%。在82份市售微波食品样品检测中,总体阳性检出率为17.1%,其中DBP的检出率为9.8%,污染水平在ND~3.51mg/kg之间;DEHP的检出率为11.0%,污染水平在ND~3.26mg/kg之间;有2份样品超过国家安全限量值要求,具有一定的潜在风险。该方法简便、准确、可靠,适用于微波食品中17种邻苯二甲酸酯类物质的测定。     

Determination of naturally occurring progestogens in bovine milk as their oxime derivatives using high performance liquid chromatography–electrospray ionization–tandem mass spectrometry

BACKGROUND: Hormones and hormone‐like substances which are present in the environment have been repeatedly accused of being the cause of most endocrine disruption. However, the possible role of endogenous hormones in food of animal origin deserves to be discussed as well. The relation between steroid hormones and several human health problems has been previously reported, such as prostate and breast cancer, perturbation of human reproduction and endocrine disruption on humans and wildlife. This research is particularly concerned with cow's milk, which contains a considerable amount of sex hormones. RESULTS: A liquid chromatography–tandem mass spectrometry method has been developed for the simultaneous detection and quantification of four naturally occurring steroid hormones in commercial bovine milk (pregnenolone (P₅), progesterone (P₄), 17‐hydroxypregnenolone (17‐OHP₅), 17‐hydroxyprogesterone (P₄)). Oxime derivatives of steroids were analyzed in positive ionization and multiple reaction monitoring mode. Methodology has been validated according to Decision 2002/657/EC criteria. CONCLUSION: This method has been successfully used in real samples. It is fast and easy‐handling and provides a useful tool for the assessment of progestogens in bovine milk. Copyright © 2010 Society of Chemical Industry     

改进QuEChERS方法结合气相色谱串联质谱检测黄瓜中的邻苯二甲酸酯类

目的建立Fe_3O_4磁性纳米材料QuEChERS结合气相色谱串联质谱联用仪检测黄瓜中15种邻苯二甲酸酯类残留的方法。方法样品采用乙腈超声提取,经无水MgSO_4和NaCl盐析离心后,通过Fe_3O_4磁性纳米材料结合C_(18)和GCB吸附剂净化,采用气相色谱串联质谱MRM模式测定。结果在20~2000μg/kg范围内线性关系良好(r~2≥0.9985),检出限(S/N=3)为0.37~1.58μg/kg。进行了20、50和200μg/kg3个添加浓度的15种邻苯二甲酸酯类的回收率试验,回收率在84.9%~111.6%;RSD为0.41%~6.84%。结论该方法准确、灵敏,符合多残留检测和痕量分析的技术要求,适用黄瓜等蔬菜中邻苯二甲酸酯类残留的分析。     

Analysis of enrofloxacin and its metabolite ciprofloxacin in bovine and porcine muscle by high-performance liquid chromatography following cation exchange clean-up.

A simple and rapid method of analysis for the trace residue determination of enrofloxacin and its metabolite ciprofloxacin has been developed. Clean-up of the samples is by cation exchange solid phase extraction (SPE) and determination made by high-performance liquid chromatography using a base-deactivated column and fluorescence detection. The method has been validated for the determination of residues in bovine and porcine muscle tissue and bacon. Recoveries at the 0.010 mg kg-1 level for enrofloaxacin and ciprofloxacin respectively were 90%, 75% in bovine muscle, 75%, 54% in porcine muscle and 81%, 63% in bacon. Determination to the 0.001 mg kg-1 level in bovine muscle and to the 0.002 mg kg-1 level in porcine muscle and bacon was also carried out. The method has been used as a quantitative screening procedure.     

Evidence of non-extractable florfenicol residues: development and validation of a confirmatory method for total florfenicol content in kidney by UPLC-MS/MS

The parent compound florfenicol (FF) is a broad-spectrum antibacterial compound licensed in the UK for use in cattle, pigs and the aquaculture industry. The analysis of porcine tissues in this study demonstrates that significant amounts of solvent non-extractable FF-related residues are present in incurred tissues (kidney and muscle) from treated animals. The results indicate that methods based on solvent extraction alone may carry a significant risk of reporting false-negative results. The use of a strong acid hydrolysis step prior to solvent extraction of tissue samples is necessary for an accurate estimate of the total tissue FF content. A robust and sensitive method for the determination of total FF residue content in kidney samples by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) has been developed and validated. This method covers the synthetic amphenicol drug FF and its metabolites, measured as the marker residue florfenicol amine (FFA) as per Commission Regulation (EU) No. 37/2010. Non-extractable and intermediate metabolites are converted to the hydrolysis product FFA, and then partitioned into ethyl acetate. Extracts are solvent exchanged prior to a dispersive solid-phase extraction step, then analysed using an alkaline reverse-phase gradient separation by UPLC-MS/MS. The method was validated around the maximum residue levels (MRLs) set out in Regulation (EU) No. 37/2010 for bovine kidney in accordance with Commission Decision No. 2002/657/EC. The following method performance characteristics were assessed during a single laboratory validation study: selectivity, specificity, sensitivity, linearity, matrix effects, accuracy and precision (decision limit (CCα) and detection capability (CCβ) were determined).     

A new,direct analytical method using LC-MS/MS for fatty acid esters of 3-chloro-1,2-propanediol (3-MCPD esters) in edible oils

A new, direct analytical method for the determination of 3-chloro-1,2-propanediol fatty acid esters (3-MCPD esters) was developed. The targeted 3-MCPD esters included five types of monoester and 20 types of diester. Samples (oils and fats) were dissolved in a mixture of tert-butyl methyl ether and ethyl acetate (4:1), purified using two solid-phase extraction (SPE) cartridges (C₁₈ and silica), then analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Five monoesters and five diesters with the same fatty acid group could be separated and quantified. Pairs of 3-MCPD diesters carrying the same two different fatty acid groups, but at reversed positions (sn-1 and sn-2), could not be separated and so were expressed as a sum of both compounds. The limits of quantification (LOQs) were estimated to be between 0.02 to 0.08?mg?kg^?1, depending on the types of 3-MCPD ester. Repeatability expressed as relative standard deviation (RSD_r%) varied from 5.5% to 25.5%. The new method was shown to be applicable to various commercial edible oils and showed levels of 3-MCPD esters varying from 0.58 to 25.35?mg?kg^?1. The levels of mono- and diesters ranged from 0.10 to 0.69?mg?kg^?1 and from 0.06 to 16?mg?kg^?1, respectively.     

Development of an HPLC–UV method for the simultaneous determination of tetracyclines in muscle and liver of porcine,chicken and bovine with accelerated solvent extraction

A simple and especially rapid method-using accelerated solvent extraction (ASE) and HPLC has been developed for the quantitative determination of oxytetracycline, tetracycline, chlortetracycline, minocycline, methacycline, demeclocycline and doxycycline in muscle and liver of porcine, chicken and bovine. Samples of muscle and liver were extracted with trichloracetic acid/acetonitrile using ASE instrument, parameters such as extraction temperature (40–80 °C) and pressure (45–85 bar) were investigated and the selected extraction (60 °C, 65 bar) was most effective. The limits of detection were lower than 10 μg/kg and limits of quantification no more than 15 μg/kg for all compounds in muscle and liver. The recoveries of tetracyclines spiked at levels of muscle 50–150 μg/kg, liver 150–450 μg/kg, averaged from 75.0% to 104.9% with the relative standard deviation values less than 10%. The method was applied to determine 30 real porcine livers. It is demonstrated that the new method is robust for detection and quantification of seven tetracycline residues in muscle and liver of porcine, chicken and bovine.     

气相色谱-质谱法测定食用植物油中氯丙醇酯的含量方法验证研究

目的建立一种同时测定食用植物油中3-氯-1,2-丙二醇脂肪酸酯(3-MCPD酯)、2-氯-1,3-丙二醇脂肪酸酯(2-MCPD酯)、1,3-二氯-2-丙醇脂肪酸酯(1,3-DCP酯)和2,3-二氯-1-丙醇脂肪酸酯(2,3-DCP酯)含量的气相色谱-质谱(GC-MS)方法。方法植物油样品经叔丁基甲基醚-乙酸乙酯溶液(8∶2,V/V)溶解,在甲醇钠-甲醇溶液中发生酯键断裂反应,用冰乙酸中和正己烷去脂,经硅藻土小柱净化,七氟丁酰基咪唑(HFBI)衍生,以GC-MS法测定,采用同位素内标进行定量。结果经5家检验机构验证,4种氯丙醇酯(以对应的氯丙醇计)在0.02~0.4 mg/L范围内线性良好,相关系数r~2均大于0.999,在0.2~2.0 mg/kg水平的加标回收率在78.5%~109.8%之间,RSD为2.2%~18.8%,检出限均为0.1 mg/kg。结论 2014年英国FAPAS分析实验室能力验证和5家机构协同验证的结果证实,该方法线性良好,准确度、精密度高,检测成本较低,适用于食用植物油中氯丙醇酯的检测。