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.     

Validation of a liquid chromatography-tandem mass spectrometry method for the simultaneous determination of sulfonamides,trimethoprim and dapsone in muscle,egg, milk and honey

A quantitative multi-residue method that includes 13 sulfonamides, trimethoprim and dapsone was developed and validated according to Commission Decision 2002/657/EC for muscle, milk egg and honey samples. For all matrices, the same extraction procedure was used. Samples were extracted with an acetone/dichloromethane mixture and cleaned up on aromatic sulfonic acid (SO₃H) SPE cartridges. After elution and concentration steps, analytes were identified and quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Data were acquired according to the multiple reaction-monitoring approach (MRM) and analytes were quantified both by the isotope dilution and the matrix-matched approaches calculating the response factors for the scanned product ions. The developed method shows good linearity, specificity, precision (repeatability and within-laboratory reproducibility), and trueness. Estimated CCβ for sulfonamides ranged between 5.6 and 8.2 µg kg^?1 for eggs, between 11.1 and 69.9 µg kg^?1 for milk, between 64.7 and 87.9 µg kg^?1 for muscle, and between 2.7 and 5.3 µg kg^?1 for honey. CCβ values for dapsone were 3.1, 0.6, 0.7 and 1.5 µg kg^?1 and for trimethoprim were 3.1, 6.7, 81.7 and 3.0 µg kg^?1 calculated for eggs, milk, muscle and honey, respectively. Recovery for all matrices was in the range from 89.1% and 109.7%. In matrix effect testing, no significant deviations were found between different samples of muscle and milk; however, a matrix effect was observed when testing different types of honey. The validation results demonstrate that the method is suitable for routine veterinary drug analysis and confirmation of suspect samples.     

Single-Laboratory Validation for the Determination of Aflatoxin B1, B2, G1, and G2 in Foods Based on Immunoaffinity Column and Liquid Chromatography with Postcolumn Derivatization and Fluorescence Detection

This study presents a method validation procedure for the determination of aflatoxin B₁, B₂, G₁, and G₂ in hazelnut, hazelnut paste, walnut, peanut, pistachio, corn, and wheat. The method consisting of clean-up with immunoaffinity column, high performance liquid chromatography with postcolumn derivatization and fluorescence detection was validated in accordance with Commission Regulation 2004/882/EC. The selectivity, linearity, decision limit, detection capability, detection and quantification limits, precision, recovery, ruggedness, and measurement uncertainty of the method were determined. The limit of detection and limit of quantification values (μg/kg) were: aflatoxin B₁, 0.02, 0.07; aflatoxin B₂, 0.01, 0.02; aflatoxin G₁, 0.02, 0.07; and aflatoxin G₂, 0.01, 0.03. The relative standard deviation values for the repeatability and within-laboratory reproducibility were below 4 and 5 %, respectively. The recovery values of the spiked samples ranged from 80 to 105 %. These results complied with minimum performance criteria established by regulation 2006/401/EC. Therefore, the procedure can be implemented for the routine analysis of aflatoxins in the studied matrices.     

Evaluation of a Selective Approach for the Determination of 5-Nitroimidazoles in Aquaculture Products by Capillary Liquid Chromatography Using Molecularly Imprinted Solid-Phase Extraction

Capillary liquid chromatography with UV detection is proposed for the determination of 5-nitroimidazole residues in aquaculture products. The separation was carried out in a C18 column at 20 °C, using a mobile phase consisting of water and acetonitrile, at 7 μL/min and 320 nm as detection wavelength. Furthermore, full loop injection mode (8 μL) was selected and water was considered as injection solvent. The optimized method was applied to the monitoring of nine 5-nitroimidazoles, including three metabolites, in crab, salmon, prawn, and velvet swimming crab. Molecularly imprinted solid-phase extraction has been evaluated for sample cleanup. The method was characterized in all the matrices in terms of linearity (R ² ≥ 0.9964), precision (repeatability, RSD ≤ 7.9%, and reproducibility, RSD ≤ 11.1%) and trueness (recoveries between 80.4 and 108.7%). Decision limits, CCα, ranging from 0.2 to 1.5 μg/kg and detection capabilities, CCβ, from 0.2 to 1.8 μg/kg, were obtained.     

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.     

Determinative and confirmatory method for residues of tetracyclines in honey by LC-MS/MS

A limited number of substances are authorised for the treatment of bees. Maximum residue limits (MRLs) are set for tetracyclines in several matrices, but not for honey. Nevertheless, tetracycline antibiotics may be used in order to prevent bacterial diseases and the loss of honey bee populations. In this study, a sensitive multi-residue LC-MS/MS method was developed and optimised for the quantitative and qualitative determination of tetracycline residues in honey. Homogenisation of samples under acidic conditions was performed and solid-phase extraction was carried out. The eluate was evaporated under nitrogen and dissolved in an aqueous methanol solution prior to filtration. A mobile phase composed of acetic acid–water and acetic acid–acetonitrile was used. Separation of tetracycline, oxytetracycline, chlortetracycline and doxytetracycline was achieved by using gradient elution on a C18 chromatography column. The analytical method was validated according to Commission Decision 2002/657/EC by the analysis of spiked samples around the recommended concentration of 20 μg kg^?1 by EURL Guidance Paper, December 2007. A matrix effect was observed, so quantification was based on an external matrix calibration curve. Calculated decision limits (CCα) were lower than 10 μg kg^–1 for all tetracyclines. Good linearity, repeatability and within-laboratory reproducibility were achieved.     

Simultaneous determination of nine acaricides and two metabolites in comb honey by LC/MS/MS

ABSTRACT

We developed a method for the simultaneous determination of acaricides in comb honey using LC/MS/MS. Because methods for honey analysis had not previously been applied to comb honey, we modified three techniques for sample preparation and LC/MS/MS conditions. First, we used a modified QuEChERS method that changed the extraction solution from ethyl acetate to acetonitrile. Second, we replaced the InertSep® MA-1 (30 mg, 1 ml) clean-up cartridge with an Oasis® HLB (60 mg, 3 ml). Third, we changed the ionisation mode from ESI to atmospheric pressure chemical ionisation (APCI). With these modifications, sample matrices had no effect on the identification and quantification of analytes, using an external solvent calibration curve. We verified this new method with nine acaricides and two metabolites on comb honey and honey samples from three different honey origins. The trueness ranged from 74.0 to 99.4%. The relative standard deviation of repeatability (RSD_r) ranged from 0.8 to 14.8% and that of within-laboratory reproducibility (RSD_WR) ranged from 1.3 to 14.8%. All criteria met Japanese validation guidelines. The LOQ was 1.0 μg kg^–1 for all analytes. We applied this method to 10 comb honey and 31 honey samples commercially available in Tokyo. From the results of the analysis of 41 samples, we observed that amitraz remained as N-(2,4-dimethylphenyl)-N-methylformamidine (DMPF) in 9 comb honey and 23 honey samples and that their residual concentrations were less than 20 μg kg^–1. Using this new method, we improved recovery and precision, which enabled precise quantitative determination. Furthermore, the residual amitraz value in honey determined by both this new and the previous method were in good agreement.     

Determination of Seven <Emphasis Type="Italic">N</Emphasis>-nitrosamines in Agricultural Food Matrices Using GC-PCI-MS/MS

The quantitative analytical methods for seven N-nitrosamines including N-nitrosodimethylamine (NDMA), N-nitrosomethylethylamine (NMEA), N-nitrosodiethylamine (NDEA), N-nitrosodibutylamine (NDBA), N-nitrosopiperidine (NPIP), N-nitrosopyrrolidine (NPYR), and N-nitrosomorpholine (NMOR) were established for agricultural food matrices. Four food matrices were used for the method development: rice soup as a fatless solid matrix, apple juice as a fatless liquid matrix, corn oil as a fat-rich liquid matrix, and 20 % alcohol as an alcohol matrix. A combination of solid-supported liquid-liquid extraction (SLLE) using Extrelut NT and a solid phase extraction (SPE) using Florisil was employed for fatless matrices. For an alcohol matrix, only SLLE was used without SPE, and liquid-liquid extraction (LLE) was established for a fat-rich matrix. The extract was analyzed by gas chromatography-positive chemical ionization-tandem mass spectrometry (GC-PCI-MS/MS) using ammonia gas as an ion source. Linearity, recovery, repeatability, inter-day precision, reproducibility, and uncertainty were evaluated for method validation using four matrices. Method detection limits for all of the investigated N-nitrosamines were ranged from 0.10 to 0.18 μg/kg for the rice soup, from 0.10 to 0.19 μg/kg for the apple juice, 0.10 μg/kg for the corn oil, and from 0.10 to 0.25 μg/kg for 20 % alcohol, depending on N-nitrosamines. Established methods were applied to determine seven N-nitrosamines in some agricultural food products.     

Analytical Procedures for the Determination of Aflatoxin B1 in Eggs of Laying Hens Using Immunoaffinity Columns and Liquid Chromatography with Post-Column Derivatisation and Fluorescence Detection

An analytical procedure for the determination of aflatoxin B₁ in eggs was introduced and validated in laboratory 1. The method consisted of the extraction of aflatoxin B₁ from a sample, purification of the extract with solvents, immunoaffinity column cleanup and the determination by liquid chromatography with post-column bromination and fluorescence detection at λ _ex ?=?362 nm and λ _em ?=?425 nm. The method was transferred to laboratory 2, where it was modified and validated. The limit of detection (LOD) and limit of quantification (LOQ) obtained in laboratory 1 were 2 and 6 ng/kg, respectively, and 2 and 5 ng/kg in laboratory 2, respectively. The repeatability of measurements in laboratory 1, represented by differences between results of duplicate measurements, was 10 ng/kg at the contamination level of 50 ng/kg. At the same concentration level, the standard deviation (s _R) and the relative standard deviation (RSD _R) for the within-laboratory reproducibility were 5.5 ng/kg and 11 %, respectively, and the measurement uncertainty was ±10 ng/kg. The mean recovery was 70 %. In laboratory 2, the repeatability of measurements at the contamination level of 20 ng/kg, represented by the standard deviation (s _R), repeatability (r) and relative standard deviation (RSD _R) was 4 ng/kg, 11 ng/kg and 20 %, respectively, and the recovery was 67 %. The results indicate that the procedures are suitable for the determination of aflatoxin B₁ in eggs and can be implemented for the routine analysis. Using the procedure validated in laboratory 1, 25 samples from farms in Slovenia were analysed. In none of the analysed samples, aflatoxin B₁ was detected.     

A New Simplified and Stability Indicating Liquid Chromatography Method for Routine Analysis of Isoflavones Aglycones in Different Complex Matrices

In this work, a stability indicating method for routine analysis of isoflavones in different matrices was developed and validated. In order to simplify the analytical method, the glycosides were previously hydrolyzed to the corresponding aglycone forms. The separation of all isoflavone aglycones was achieved in 23 min, with a total time of analysis of 30 min, using trifluoroacetic acid 0.1 % (v/v) and acetonitrile:trifluoroacetic acid (100:0.01, v/v) as mobile phase. The LC method specificity was evaluated by the analysis of isoflavone standards submitted to acidic, alkali, neutral, oxidative, and photolytic stress conditions. The isoflavones degraded in alkali at 60 °C or in alkali under ulraviolet C (UVC) radiation, forming, in both conditions, three degradation products D1, D2, and D3 which were analyzed by liquid chromatography mass spectroscopy (LC-MS/MS). The method showed linearity higher than 0.999 with the concentration ranged from 0.1 to 10 μg ml^-1 for all isoflavone aglycones. The limits of quantitation obtained using calibration curves were from 0.28 to 0.37 μg ml^-1 and the intermediary precision at three levels (2, 6, and 10 μg ml^-1) showed RSD values between 0.03 and 0.25 %. After the performed validation, the LC method was applied to compare the isoflavone aglycones content in three different matrices: Glycine max beans, Glycine max dry extract, and isoflavone aglycone loaded nanoemulsion. The repeatability data showed RSD values between 0.02 and 1.41 % and the intermediary precision at three levels showed RSD values between 0.05 and 1.99 %. The recovery data of the isoflavone aglycones standards in the matrices at three levels were between 90.74 and 106.43 %.     

Validation of an enzyme-linked immunosorbent assay for qualitative screening of neomycin in muscle, liver, kidney, eggs and milk

A rapid and sensitive enzyme-linked immunosorbent assay (ELISA) was used for the qualitative screening analysis of neomycin in food of animal origin (muscle, liver, kidney, eggs and milk) at levels corresponding to the European Union maximum residue limit (MRL) set for this substance. The method validation was performed according to the criteria of Commission Decision 2002/657/EC established for qualitative screening methods. In this regard, the following parameters were determined: detection capability (CCβ), specificity, detection limit (LOD), quantification limit (LOQ), recovery, precision, linearity and ruggedness. LODs ranged from 5.7 microg kg(-1) in kidney to 29.3 microg kg(-1) in milk; LOQs ranged from 11.4 microg kg(-1) in kidney to 59.7 microkg(-1) in eggs. The recoveries from spiked samples at the MRL, half the MRL and double the MRL levels ranged from 65.8% to 122.8%, with a coefficient of variation (CV) between 5.9% and 28.6%. The CCβ value was less than the MRL for all examined matrices. Moderate variations of some critical factors in the sample pretreatment for muscle, milk and eggs were deliberately introduced for ruggedness evaluation and had a slight but not statistically significant effect on method performance. The proposed method is suitable for qualitative screening analysis of neomycin in the above-mentioned food in conformity with current European Union performance requirements.     

Distribution and elimination of levamisole in eggs and tissues after oral administration to laying hens,determined by LC-MS/MS

Levamisole was administered to laying hens, and concentrations in eggs and tissues (thigh muscle, breast muscle, liver and kidney) were determined by a newly developed liquid chromatography tandem mass spectrometry method, which allowed trace level quantification of levamisole. The adopted analytical method showed good sensitivity, repeatability and percentage of recovery from spiked matrices. Maximum concentrations of levamisole were found on the first day after the administration (531.1 μg/kg in liver, 164.3 μg/kg in egg yolk, 130.7 μg/kg in kidney, 78.0 μg/kg in breast muscle, 70.7 μg/kg in thigh muscle and 64.0 μg/kg in egg white), after which there is a decline. The compound was rapidly eliminated from eggs, with a half-life of 1.3 days. Elimination appeared to be slower in thigh muscle (3.5 days), breast muscle (3.4 days) and liver (3.3 days). According to this experiment, the levamisole withdrawal periods calculated for eggs, liver, kidney, breast muscle and thigh muscle in laying hens were 14.1, 6.1, >4.0, 14.5 and 13.0 days, respectively. The longest time for levamisole residues to be completely released from tissues was seen in liver samples (37.4 days), followed by thigh muscle, breast muscle and kidney. Elimination from eggs was fastest (16.4 days for levamisole residues to drop below the method quantification limit).     

Comparison of four analytical techniques based on atomic spectrometry for the determination of total tin in canned foodstuffs

Different techniques for the determination of total tin in beverages and canned foods by atomic spectrometry were compared. The performance characteristics of inductively coupled plasma-mass spectrometry (ICP-MS), hydride generation-inductively coupled plasma-atomic emission spectrometry (HG-ICP-AES), electrothermal atomisation-atomic absorption spectrometry (ETA-AAS) and inductively coupled plasma-atomic emission spectrometry (ICP-AES) were determined in terms of linearity, precision, recovery, limit of detection, decision limit (CCα) and detection capability (CCβ) (Decision 2002/657/EC). Calibration ranges were covered from ng l?1 to mg l?1 level. Limits of detection that ranged from 0.01, 0.05, 2.0 to 200 μg l?1 were reached for ICP-MS; HG-ICP-AES; ETA-AAS and ICP-AES, respectively. Precision, calculated according to ISO 5725-2 for repeatability and within-laboratory reproducibility and expressed as relative standard deviation (RSD), ranged from 1.6% to 4.9%; and recovery, based on Decision 2002/657/EC, was found to be between 95% and 110%. Procedures for the mineralisation or extraction of total tin were compared. Wet digestion, sequentially, with nitric acid and hydrogen peroxide provided the best results. The influence of possible interferences present in canned food and beverage was studied, but no interference in the determination of tin was observed. Since maximum levels for tin established by European Union legislation vary from 50 mg kg?1 in canned baby foods and infant foods up to 200 mg kg?1 in canned food, ICP-AES was chosen as the preferred technique for routine analysis thanks to its good precision, reliability and ease of use. The accuracy of this routine method was confirmed by participation in six proficiency test schemes with z-scores ranging from -1.9 to 0.6. Several canned foodstuffs and beverage samples from a local market were analysed with this technique.     

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.     

Simultaneous Determination of Fluoxastrobin and Tebuconazole in Cucumber and Soil Based on Solid-Phase Extraction and LC-MS/MS Method

Based on aqueous acetonitrile extraction (AAE) and solid-phase extraction (SPE) pretreatments, an analytical method was first introduced for the simultaneous determination of E/Z-fluoxastrobins and tebuconazole in cucumber and soil. The method validation obtained satisfactory results in terms of linearity, matrix effect (? 17.7–2.9%), fortified recovery (78.4–108.0%), precision (1.1–11.9%), and sensitivity (the limits of quantification were 5 μg/kg). During application of the method, the recommended method was used to estimate the dissipation and residues of fluoxastrobin and tebuconazole in cucumber and soil in field tests. The results showed that the half-lives of fluoxastrobin and tebuconazole were 6.5–8.3 days in cucumber and 11.6–12.2 days in soil. In the terminal residue experiment, the residues of fluoxastrobin and tebuconazole were 33–389 and 35–522 μg/kg in cucumber, respectively. Referencing the maximum residue limits (MRLs) of fluoxastrobin (0.5 mg/kg, US Environmental Protection Agency) and tebuconazole (1.0 mg/kg, China), 1 day is recommended as the preharvest interval for the application of fluoxastrobin and tebuconazole in cucumber.     

Laboratory validation of an LC-MS/MS method for the detection of ractopamine,clenbuterol and salbutamol in bovine and swine muscle at sub-μg kg–1 regulatory limits

ABSTRACT

Ractopamine (RAC), is a β-adrenergic agonist increasingly used in the swine and cattle industry. This compound redirects nutrients to favour leanness rather than fat deposition, improves growth and carcass traits gaining higher economic benefit to producers. Countries around the world are split over whether to allow the use of RAC in meat production. Clenbuterol (CLB) and salbutamol (SLB) are anillinic and phenolic β-agonists, respectively, with the same capacity of producing economic benefits for the meat sector. However, they are prohibited because of the potentially adverse reactions they can cause in consumers. The three β-agonist compounds have been included in the Brazilian National Regulatory Survey and consequentially there is an eminent need for reliable methods capable of detecting those substances at the same time and reduce analytical costs. Therefore, an LC-MS/MS method for the simultaneous determination of residual RAC, CLB and SAL in swine and cattle muscle was developed and validated with quantification levels respecting the action levels established for Brazil which are 0.1, 0.2 and 5 µg kg^–1 for RAC, CLB and SAL, respectively. Samples were quantified using RAC-d₅, CLB-d₉ and SLB-d₆ as internal standards. The validation was performed according to European Union Decision 2002/657, which includes criteria (CCα, CCβ, recovery, repeatability, reproducibility and calibration curve). The method meets the Brazilian regulatory requirement that establishes criteria and procedures for the determination of parameters such as CCα, CCβ, precision and recovery. CCα values were 0.02, 0.21 and 5.42 µg kg^–1 for RAC, CLB and SAL, respectively, in bovine and swine muscle samples; CCβ values were 0.03, 0.22 and 5.8 µg kg^–1 for RAC, CLB and SAL, respectively, in bovine and swine muscle samples. Average recoveries fortified with 0.05–7.5 µg kg^–1 of the studied β-agonist leads around 95%. The method was demonstrated to be suitable for the determination of RAC, CLB and SLB in swine and cattle muscle samples.     

Determination of Melatonin in Cow’s Milk by Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS)

The aim of the present study is the development of a rapid and accurate analytical method to determine melatonin in milk. In this method, melatonin extracted from milk with ethyl acetate, after application of reversed phase liquid chromatographic separation melatonin, was detected and quantified with tandem mass spectrometry. Performance of method was tested by conducting method validation study wherein limit of detection, limit of quantification, linearity recovery, and precision parameters were determined. Limit of detection and limit of quantification were calculated as 0.016 and 0.054 μg/kg, respectively. Recovery of the method was checked at two different concentration namely 0.5 and 2.5 μg/kg and determined as 106 and 100%, respectively. Precision of the method was determined as interday and intraday precision. Intraday precision calculated as 3.92 and 2.15% for 0.5 and 2.5 μg/kg spike levels. In this study, we describe an analytical method for determination of melatonin in milk with LC-MS/MS. This method offers an efficient, rapid, and easy sample preparation procedure with high selectivity and good sensitivity for the determination of melatonin in milk.     

Development and validation of rapid multiresidue and multi-class analysis for antibiotics and anthelmintics in feed by ultra-high-performance liquid chromatography coupled to tandem mass spectrometry

A new multi-residue method for the analysis of veterinary drugs, namely amoxicillin, chlortetracycline, colistins A and B, doxycycline, fenbendazole, flubendazole, ivermectin, lincomycin, oxytetracycline, sulfadiazine, tiamulin, tilmicosin and trimethoprim, was developed and validated for feed. After acidic extraction, the samples were centrifuged, purified by SPE and analysed by ultra-high-performance liquid chromatography coupled to tandem mass spectrometry. Quantitative validation was done in accordance with the guidelines laid down in European Commission Decision 2002/657/CE. Matrix-matched calibration with internal standards was used to reduce matrix effects. The target level was set at the authorised carryover level (1%) and validation levels were set at 0.5%, 1% and 1.5%. Method performances were evaluated by the following parameters: linearity (0.986 < R² < 0.999), precision (repeatability < 12.4% and reproducibility < 14.0%), accuracy (89% < recovery < 107%), sensitivity, decision limit (CCα), detection capability (CCβ), selectivity and expanded measurement uncertainty (k = 2).This method has been used successfully for three years for routine monitoring of antibiotic residues in feeds during which period 20% of samples were found to exceed the 1% authorised carryover limit and were deemed non-compliant.     

Multi-residue and multi-class method for the determination of antibiotics in bovine muscle by ultra-high-performance liquid chromatography tandem mass spectrometry

A multi-residue quantitative screening method covering 41 antibiotics from 7 different families, by ultra-high-performance–liquid-chromatography tandem mass spectrometry (UHPLC–MS/MS), is described. Sulfonamides, trimethoprim, tetracyclines, macrolides, quinolones, penicillins and chloramphenicol are simultaneously detected after a simple sample preparation of bovine muscle optimized to achieve the best recovery for all compounds. A simple sample treatment was developed consisting in an extraction with a mixture of acetonitrile and ethylenediaminetetraacetic acid (EDTA), followed by a defatting step with n-hexane. The methodology was validated, in accordance with Decision 2002/657/EC by evaluating the required parameters: decision limit (CCα), detection capability (CCβ), specificity, repeatability and reproducibility. Precision in terms of relative standard deviation was under 20% for all compounds and the recoveries between 91% and 119%. CCα and CCβ were determined according the maximum residue limit (MRL) or the minimum required performance limit (MRPL), when required.     

Development of a Headspace-Solid Phase Microextraction-Gas Chromatography/Mass Spectrometry (HS-SPME-GC/MS) Method for the Determination of Benzene in Soft Drinks

A method based on headspace-solid phase microextraction and gas chromatography with mass spectrometry has been developed and validated for the determination of benzene in soft drinks. The extraction step was optimized using a rotatable central composite design including the following experimental variables: extraction temperature, extraction time, sample weight, and salt concentration. The optimized procedure, which was carried out at 30 °C during 30 min by using a 75 μm carboxen-polydimethylsiloxane fiber, showed good linearity within the concentration range 0–25 μg?kg^?1 (r ² ?>?0.999), mean recoveries from 97.5 to 103.1 %, and coefficients of variation from 1.5 to 13.4 % for repeatability and from 1.5 to 15.7 % for within-laboratory reproducibility. Limits of detection and quantification were calculated at 0.02 and 0.08 μg?kg^?1, respectively. The method was applied to determine the concentrations of benzene in 77 samples of beverages from the Brazilian market. Levels from <0.08 to 10.84 μg?kg^?1 were obtained, which are comparable to those verified in other countries. Most of the samples (72.2 %) contained benzene up to 1 μg?kg^?1.