Development of an analytical methodology to quantify melamine in milk using micellar liquid chromatography and validation according to EU Regulation 2002/654/EC

Melamine is a toxic triazine, illegally used as an additive in milk to apparently increase the amount of protein. A chromatographic procedure using a C18 column and a micellar mobile phase of sodium dodecyl sulphate (0.05 M) and propanol (7.5%), buffered at pH 3, and a detection set by absorbance at 210 nm, was reported for the resolution and quantification of melamine in liquid and powdered milk samples. In this work, samples were diluted with a SDS solution and were directly injected, thus avoiding long extraction and experimental procedures. Melamine was eluted in nearly 9.3 min without overlapping the protein band or other endogeneous compounds. The optimal mobile phase composition was taken using a chemometrical approach that considers the retention factor, efficiency and peak shape. Validation was performed following the European Commission's indications (European Decision 2002/657/EC), and the main analytical parameters studied were: linearity (0.02-100 ppm; r² = 0.999), limit of detection (5 ppb), intra- and inter-day precision (R.S.D. <7.6% and <9.7%, respectively) and robustness (R.S.D. <7.4% for retention time and <5.0% for area). Sensitivity was adequate to detect melamine under the safety limits proposed by the US FDA. Finally, recoveries for several milk samples were found in the 85-109% range.     

Validation according to European Commission Decision 2002/657/EC of a confirmatory method for aflatoxin M1 in milk based on immunoaffinity columns and high performance liquid chromatography with fluorescence detection

A high performance liquid chromatographic method with fluorimetric detection for the determination of aflatoxin M₁ (AFM₁) in milk has been optimized and validated according to Commission Decision 2002/657/EC by using the conventional validation approach. The procedure for determining selectivity, recovery, precision, decision limit (CC_α), detection capability (CC_β) and ruggedness of the method has been reported. The results of the validation process demonstrate the agreement of the method with the provisions of Commission Regulation 401/2006/EC. The mean recovery calculated at three levels of fortification (0.5, 1.0, and 1.5-fold the MRL) was 91% and the maximum relative standard deviation value for the within-laboratory reproducibility was 15%. Limit of detection (LOD) and limit of quantitation (LOQ) values were 0.006 μg kg⁻¹ and 0.015 μg kg⁻¹ while the CC_α and CC_β values were 0.058 μg kg⁻¹ and 0.065 μg kg⁻¹, respectively. The relative expanded measurement uncertainty of the method was 7%. The method was not affected by slight variations of some critical factors (ruggedness minor changes) as pre-treatment and clean-up of milk samples, thermal treatment and different storage conditions, as well as by major changes valued in terms of milk produced by different species (buffalo, goat and sheep). The method allowed accurate confirmation analyses of milk samples, resulted positive by the screening method. In fact, the Z-score values attained in a proficiency test round were well below the reference value of 1, proving the excellent laboratory performances.     

Tetracycline residues in royal jelly and honey by liquid chromatography tandem mass spectrometry: validation study according to Commission Decision 2002/657/EC

A specific, sensitive and robust liquid chromatography tandem mass spectrometry method for determining oxytetracycline, tetracycline, chlortetracycline and doxycycline in royal jelly and honey samples is presented. Extraction of drug residues was performed by ammonium acetate buffer as extractant followed by a clean-up with metal chelate affinity chromatography and solid-phase extraction. Tetracycline analysis was performed using liquid chromatography–electrospray ionisation–tandem mass spectrometry. The presented method is the first validated for royal jelly and in accordance with the requirements set by Commission Decision 2002/657/EC. Recoveries of the methods, calculated spiking the samples at 5.0, 10.0, 20.0 and 30.0 μg kg⁻¹, were 79% to 90% for honey and 77% to 90% for royal jelly. The intra-day precision (RSD) ranged between 8.1% and 15.0% for honey and from 9.1% to 16.3% for royal jelly, while inter-day precision values were from 10.2% to 17.6% and from 10.6% to 18.4% respectively for honey and royal jelly. Linearity for the four analytes was calculated from 5.0 to 50.0 μg kg⁻¹. The decision limits (CCα) ranged from 6.2 to 6.4 μg kg⁻¹ and from 6.1 to 6.5 μg kg⁻¹ for honey and royal jelly, respectively. Detection capabilities values (CCβ) ranged between 7.2 and 7.7 μg kg⁻¹ and from 7.3 to 7.9 μg kg⁻¹ respectively for honey and royal jelly. The developed method is currently in use for confirmation of the official control analysis of honey and royal jelly samples.     

Use of capillary electrophoresis with laser-induced fluorescence detection to screen and liquid chromatography–tandem mass spectrometry to confirm sulfonamide residues: Validation according to European Union 2002/657/EC

A multiresidue method is described for determining six sulfonamides (SAs) (sulfadiazine, sulfathiazole, sulfamethazine, sulfamethoxazole, sulfaquinoxaline and sulfadimethoxine) in liver by a capillary electrophoresis screening method and a liquid chromatography coupled to tandem mass spectrometry confirmatory assay. Samples were prepared by homogenizing the tissue, with sodium hydroxide and acetonitrile. After evaporation, extracts were injected in the capillary electrophoresis system or mass spectrometry system for confirmatory analysis. The detection of analytes was achieved by laser-induced fluorescence in capillary electrophoresis. Procedures were validated according to the European Union regulation 2002/657/EC determining specificity, selectivity and detection capability for screening method and decision limit, detection capability, specificity, selectivity, trueness and precision for confirmation method. The results of validation process demonstrate that the method is suitable for application in Brazilian statutory veterinary drug residue surveillance programs. Capillary electrophoresis was proved to be a fast, robust method with low time and reagents consumption.     

Multiresidue analysis of sulfonamides in meat by supramolecular solvent microextraction,liquid chromatography and fluorescence detection and method validation according to the 2002/657/EC decision

A multiresidue method was described for determining eight sulfonamides, SAs (sulfadiazine, sulfamerazine, sulfamethoxypyridazine, sulfachloropyridazine, sulfadoxine, sulfamethoxazole, sulfadimethoxine and sulfaquinoxaline) in animal muscle tissues (pork, chicken, turkey, lamb and beef) at concentrations below the maximum residue limit (100 μg kg⁻¹) set by the European Commission. The method was based on the microextraction of SAs in 300-mg muscle samples with 1 mL of a supramolecular solvent made up of reverse micelles of decanoic acid (DeA) and posterior determination of SAs in the extract by LC/fluorescence detection, after in situ derivatization with fluorescamine. Recoveries were quantitative (98–109%) and matrix-independent, no concentration of the extracts was required, the microextraction took about 30 min and several samples could be simultaneously treated. Formation of multiple hydrogen bonds between the carboxylic groups of the solvent and the target SAs (hydrogen donor and acceptor sum between 9 and 11) were considered as the major forces driving microextraction. The method was validated according to the European Union regulation 2002/657/EC. Analytical performance in terms of linearity, selectivity, trueness, precision, stability of SAs, decision limit and detection capability were determined. Quantitation limits for the different SAs ranged between 12 μg kg⁻¹ and 44 μg kg⁻¹, they being nearly independent of matrix composition. Repeatability and reproducibility, expressed as relative standard deviation, were in the ranges 1.8–3.6% and 3.3–6.1%. The results of the validation process proved that the method is suitable for determining sulfonamide residues in surveillance programs.     

In-house validation of a liquid chromatography/electrospray tandem mass spectrometry method for confirmation of chloramphenicol residues in muscle according to Decision 2002/657/EC

In this work we present an in-house validation study for the confirmatory analysis of chloramphenicol (CAP) in muscle according to the Commission Decision 2002/657/EC requirements. CAP is extracted in acetonitrile and after liquid-liquid partitioning with n-hexane is identified and quantitatively determined by ion trap liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI-MS/MS) analysis in the negative ion mode. CAP was identified using the precursor ion and at least two product ions, meeting the qualitative and quantitative criteria set by the European Commission in the Decision 2002/657/EC for confirmation of prohibited veterinary drug residues. We calculated mean drug recoveries, CCalpha and CCbeta of the method, and reported data on specificity, ruggedness and within-laboratory reproducibility. Finally, we point out and discuss some problems and questions arising from controversy about the application of Decision 2002/657/EC.     

Development and validation of an HPLC method for the determination of penicillin antibiotics residues in bovine muscle according to the European Union Decision 2002/657/EC

A high-performance liquid chromatographic method was developed for the determination of five penicillins: penicillin G (PENG), penicillin V (PENV), oxacillin (OX), cloxacillin (CLO), and dicloxacillin (DICLO), in bovine muscle. Samples were macerated with a mixture of H(2)O/CH(3)CN (1:1) and purified using RP-8 Adsorbex SPE cartridges after centrifugation, with mean recovery from spiked samples higher than 89%. The separation of the examined penicillins was achieved on an analytical column, an Inertsil C8 5 microm, 250x4 mm(2), at ambient temperature. The mobile phase consisted of 0.1% TFA/ACN 50:50 v/v delivered isocratically at a flow rate of 1.1 mL/min. Analytes were monitored at 240 nm. The procedure was validated according to the European Union Decision 2002/657/EC by means of selectivity, stability, decision limit, detection capability, accuracy, and precision. Method's LOQ values achieved were 54 microg/kg for PENG and DICLO, 46 microg/kg for PENV, 16 microg/kg for OX, and 43 microg/kg for DICLO. The detection capabilities (CC(beta)) were 73.6 microg/kg for PENG, 29.1 microg/kg for PENV, 350.6 microg/kg for OX, 379.9 microg/kg for CLO, and 355.8 microg/kg for DICLO. The method was applied to various samples from the local market. Two penicillins were identified by photodiode array (PDA) detection and quantified.     

Development and validation of an HPLC confirmatory method for the determination of seven tetracycline antibiotics residues in milk according to the European Union Decision 2002/657/EC

An HPLC method with diode-array detection, at 355 nm, was developed and validated for the determination of seven tetracyclines (TCs) in milk: minocycline (MNC), TC, oxytetracycline (OTC), methacycline (MTC), demeclocycline (DMC), chlortetracycline (CTC), and doxycycline (DC). Oxalate buffer (pH 4) was used with 20% TCA as a deproteinization agent for the extraction of analytes from milk followed by SPE. The separation was achieved on an Inertsil ODS-3, 5 microm, 250 x 4 mm(2 )analytical column at ambient temperature. The mobile phase, a mixture of A: 0.01 M oxalic acid and B: CH(3)CN, was delivered using a gradient program. The procedure was validated according to the European Union decision 2002/657/EC determining selectivity, stability, decision limit, detection capability, accuracy, and precision. Mean recoveries of TCs from spiked milk samples (50, 100, and 200 ng/g) were 93.8-100.9% for MNC, 96.8-103.7% for OTC, 96.3-101.8% for TC, 99.4-107.2% for DMC, 99.4-102.9% for CTC, 96.3-102.7% for MTC, and 94.6-102.1% for DC. All RSD values were lower than 8.5%. The decision limits CC(a) calculated by spiking 20 blank milk samples at MRL (100 microg/kg) ranged from 101.25 to 105.84 microg/kg, while detection capability CC(b )from 103.94 to 108.88 microg/kg.     

False‐positive liquid chromatography/tandem mass spectrometric confirmation of sebuthylazine residues using the identification points system according to EU directive 2002/657/EC due to a biogenic insecticide in tarragon

In pesticide residue analysis using liquid chromatography/tandem mass spectrometry (LC/MS/MS) the confirmation of a sebuthylazine finding in a tarragon (Artemisia dranunculus) sample was demonstrated to be false positive. A coeluting interfering matrix compound produced product ions in MS/MS analysis, perfectly corresponding to the multiple reaction monitoring (MRM) of two sebuthylazine transitions. Using the EU directive 2002/657/EC which regulates the confirmation of suspected positive findings would have resulted in a false‐positive finding. A third LC/MS/MS transition with a deviant ion ratio and a gas chromatography (GC)/MS/MS analysis revealed the false‐positive results. With optimized high resolving ultra‐performance liquid chromatography (UPLC) conditions it was possible to separate spiked sebuthylazine from the interfering matrix compound. Using its exact mass and isotope ratios from LC/time‐of‐flight (TOF) MS measurements, the compound was identified as nepellitorine, a – not surprising – endogenous alkamide in tarragon (Arthemisia dranunculus). False‐positive results, especially in heavy matrix samples such as herbs, can be dealt with by further confirmatory analysis, e.g. a third transition, GC analysis if possible or more advantageous by an orthogonal criterion like exact mass. Copyright © 2009 John Wiley & Sons, Ltd.     

Simple assay for monitoring seven quinolone antibacterials in eggs: Extraction with hot water and liquid chromatography coupled to tandem mass spectrometry: Laboratory validation in line with the European Union Commission Decision 657/2002/EC

A simple and rapid method able to determine residues of seven quinolone antibacterials in whole eggs is presented here. This method is based on the matrix solid-phase dispersion technique with hot water as extractant followed by liquid chromatography–tandem mass spectrometry. After depositing 1.5 g of an egg sample containing the analytes and the analyte surrogate (norfloxacin) on sand (crystobalite), this material was packed into an extraction cell. Quinolones were extracted by flowing 6 mL of water acidified with 50 mmol/L formic acid through the cell heated at 100 °C. After pH adjustment and filtration of the extract, 100 μL of it was injected into the LC column. MS data acquisition was performed in the multiple reaction monitoring mode, selecting two precursor ion to product ion transitions for each target compound. Hot water appeared an efficient extracting medium, since absolute recoveries of the analyte in egg at the level of 20 ng/g were 89–103%. Estimated limits of quantification (S/N = 10) were 0.2–0.6 ng/g. Based on the EU Commission Decision 2002/657/EC, the method was validated in terms of ruggedness, specificity, linearity, within-laboratory reproducibility, decision limit (CCα and detection capability (CCβ). Depending on the particular analyte, CCαs ranged between 0.41 and 2.6 ng/g, while CCβs were 0.64–3.7 ng/g. The method was linear in the 3–30 ng/g range, with typical R² values higher than 0.97. The within-laboratory reproducibility (n = 21) at 6 ng/g level was in the 9.0–12% range. After validation, a depletion study of enrofloxacin and one of its metabolites, i.e. ciprofloxacin, in eggs was conducted.     

Development of a methodology to quantify tamoxifen and endoxifen in breast cancer patients by micellar liquid chromatography and validation according to the ICH guidelines

A simple micellar liquid chromatographic procedure is described to determine tamoxifen and endoxifen in plasma. For the analysis, tamoxifen and endoxifen solutions were diluted in water and UV-irradiated for 20 min to form the photocycled derivative with a phenanthrene core which shows intense fluorescence. Samples were then directly injected, thus avoiding long extraction and experimental procedures. The resolution from the matrix was performed using a mobile phase containing 0.15 mol L⁻¹ SDS-7% n-butanol at pH 3, running at 1.5 mL min⁻¹ through a C18 column at 40 °C. Detection was carried out by fluorescence, and the excitation and emission wavelengths were 260 and 380 nm, respectively. The chromatographic analysis time was 20 min. The analytical methodology was validated following the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines. The response of the drugs in plasma was linear in the 0.5-15 μg mL⁻¹ range, with r² > 0.99. Accuracy and precision were <14% in both cases. Limits of detection and quantification (ng mL⁻¹) in plasma were 75 and 250 for endoxifen, and 50 and 150 in tamoxifen. The method developed herein does not show interferences by endogenous compounds. Finally the analytical method was used to determine the amount of tamoxifen and endoxifen in several plasma samples of breast cancer patients from a local hospital.     

Development and validation of an HPLC confirmatory method for residue analysis of ten quinolones in tissues of various food-producing animals, according to the European Union Decision 2002/657/EC

The aim of this work was to develop an HPLC method for the simultaneous determination of ten quinolones: enoxacin, ofloxacin, norfloxacin, ciprofloxacin, danofloxacin, enrofloxacin, sarafloxacin, oxolinic acid, nalidixic acid, and flumequine, in various tissues of food-producing animals. Separation was achieved on a PerfectSil Target column (250 mm x 4 mm, ODS-3, 5 microm), by MZ-Analysentechnik (Germany), at room temperature. The mobile phase consisted of 0.1% TFA-CH(3)OH-CH(3)CN and was delivered by a gradient program of 35 min. The detection and quantitation was performed on a photodiode array detector at 275 and 255 nm. Caffeine (7.5 ng/microL) was used as the internal standard (IS). Analytes were isolated from tissue samples by 0.1% methanolic TFA solution. SPE, using LiChrolut RP-18 cartridges, was applied for further purification. The extraction protocol was optimized and the final recoveries varied between 92.0 and 107.4%. The method was fully validated according to Commission Decision 2002/657/EC. Limits of quantitation for the examined quinolones extracted from each tissue were much lower than the respective Maximum Residue Levels, ranging between 30 and 50 microg/kg for bovine tissue, between 30 and 55 microg/kg for ovine tissue, and between 40 and 50 microg/kg for porcine tissue.     

Evaluation of the limit of performance of an analytical method based on a statistical calculation of its critical concentrations according to ISO standard 11843: Application to routine control of banned veterinary drug residues in food according to European Decision 657/2002/EC

Traceability of the measurement of analytical parameters capable of evaluating the performance of methods is an important concept for the assessment of quality for the routine control, especially for residue monitoring of non-authorized medicinal substances in food from animal origin. The European Decision no. 657/2002/EC recommends to calculate two statistical limits, CCα and CCβ, which allow to evaluate the critical concentrations above which the method reliably distinguish and quantify a substance taking into account the variability of the method and the statistical risk to take a wrong decision. The calculation, which can be derived from the ISO standard no. 11843 is applied on a routine basis. An example displays a very simple way for evaluating the performance of an LC-MSMS method which has been validated a few years ago and is qualified onto a Micromass Quattro LCZ tandem mass spectrometer to monitor and confirm the nitrofuran metabolite residues in food from animal origin. Community Reference Laboratory for Antimicrobial Veterinary Drug Residue Control in Food from Animal Origin     

Development and validation according to European Union Decision 2002/657/EC of an HPLC‐DAD method for milk multi‐residue analysis of penicillins and amphenicols based on dispersive extraction by QuEChERS in MSPD format

A precise and reliable method for milk residue analysis regarding five penicillins and three amphenicols by HPLC‐diode array detection has been developed herein. The chromatographic separation was performed using a mobile phase of CH₃COONH₄ (0.05 M) and ACN delivered by gradient program on a Kinetex^?‐C₁₈ core‐shell, 2.6 μm column, starting at a volume ratio of 95:5 and ending at 60:40 after 17 min, remaining stable for 3 more min. A modified matrix solid phase dispersion procedure was applied for the extraction and clean‐up procedure of antibiotics using a mixture of Strata by Phenomenex and QuEChERS as a sorbent. The method was validated at the respective 0.5× MRL, MRL and 1.5 ×MRL level for each compound. Results were quantitated against the internal standard paracetamol (2 ng/μL) according to the matrix‐matched approach. The method was validated in line with the EC guidelines as cited in the Decision 2002/657/EC. The within‐laboratory reproducibility, expressed as a RSD, never exceeded 16%. All decision limit (CCα) values lay in the range between 35.2 and 56.3 μg/kg and the corresponding results for detection capability (CCβ) were 39.9 and 61.9 μg/kg. Ruggedness was estimated according to the Youden approach.