Usefulness of parallel factor analysis to handle the matrix effect in the fluorescence determination of tetracycline in whey milk

The determination of tetracycline by fluorescence spectrophotometry in complex matrices has some difficulties, because the presence of other compounds in the matrix affects the analytical signal. In this work, the effect of some inorganic species that are present in whey milk on the fluorescence signal of tetracycline is studied using a D-optimal experimental design. Next, an experimental strategy is proposed in conjunction with Parallel Factor Analysis, PARAFAC, modeling that leads to suitably modeling the severe matrix effect in the determination of tetracycline in whey milk. A specific design is performed in such a way that the lack of trilinearity due to the effect of the presence of interferents on the signal is obviated. Then, ten test samples from three brands of milk, spiked with different quantities of tetracycline and measured in 2 days were analysed using this methodology (mean of the absolute value of the relative errors: 5.1%). The developed analytical method fulfils the property of trueness, the relative errors being, both in calibration and prediction, inside the interval set by Commission Decision 2002/657/EC at these concentration levels. Decision limits (CCα) at x₀ = 0 μg L⁻¹ and at x₀ = 100 μg L⁻¹ were 13.2 and 112.4 μg L⁻¹ respectively, for α = 0.05; whereas detection capabilities (CCβ) were 25.9 μg L⁻¹ and 124.4 μg L⁻¹ respectively for α = β = 0.05.     

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.     

A dual validation approach to detect anthelmintic residues in bovine liver over an extended concentration range

This paper describes a method for the detection and quantification of 38 residues of the most widely used anthelmintics (including 26 veterinary drugs belonging to the benzimidazole, macrocyclic lactone and flukicide classes) in bovine liver using two different protocols for MRL and non-MRL levels. A dual validation approach was adopted to reliably quantify anthelmintic residues over an extended concentration range (1-3000 μg kg⁻¹). Sample extraction and purification was carried out using a modified QuEChERS method. A concentration step was included when analysing in the low μg kg⁻¹ range. Rapid analysis was carried out by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), which was capable of detecting residues to <2 μg kg⁻¹. The method has been single-laboratory validated according to the 2002/657/EC guidelines and met acceptability criteria in all but a few cases. The inclusion of 19 internal standards, including 14 isotopically labelled internal standards, improved accuracy, precision, decision limit (CCα) and detection capability (CCβ).     

Three-way models and detection capability of a gas chromatography-mass spectrometry method for the determination of clenbuterol in several biological matrices: the 2002/657/EC European Decision

Clenbuterol has been extracted by mixed solid-phase extraction from two biological matrices (bovine hair and urine) and detected by GC/MS (selected ion monitoring (SIM) and full-SCAN modes). The analytical signal has been modelled with univariate and three-way models, namely DTLD, PARAFAC, PARAFAC2, Tucker3 and trilinear PLS. Since clenbuterol is a banned substance a comparative study of the capability of detection (CCβ, X₀=0) has been performed as a function of the sample (hair, 74 μg kg⁻¹ and urine, 0.36 μg l⁻¹), the mode in which the signals are monitored (SCAN, 283 μg kg⁻¹ and SIM, 74 μg kg⁻¹) and the statistical model (univariate, 283 μg kg⁻¹ and trilinear PLS, 20.91 μg kg⁻¹). The capability of detection has been calculated as stated in ISO 11843 and Decision 2002/657/EC setting in all cases the probabilities of false positive and of false negative at 0.05.The identification of the mass spectra must be done to confirm the presence of clenbuterol and has been carried out through PARAFAC. The correlation coefficient between the spectra estimated by PARAFAC and the library spectra is 0.96 (hair, SCAN mode) and 1.00 (hair and urine, SIM mode).The Decision 2002/657/EC advocates the use of independent mass fragments to identify banned compounds. These recommendations together with the effect of the number of ions registered on the capability of detection have lead us to select five uncorrelated fragments (86, 243, 262, 264 and 277) from the data set of 210 ions by hierarchical clustering of variables.     

Enzyme immunoassay for semicarbazide--the nitrofuran metabolite and food contaminant

Semicarbazide (SEM), the marker residue for the banned nitrofuran veterinary antibiotic nitrofurazone (NFZ), has been detected regularly in foods (47% of recent nitrofuran EU Rapid Alerts involve SEM). However, the validity of SEM as a definitive marker for NFZ has been undermined by SEM arising from other sources including azodicarbonamide, a plastics blowing agent and flour treatment additive. An inexpensive screening test for SEM in food matrices is needed—all SEM testing currently uses expensive LC-MS/MS instrumentation. We now report the first production of antibodies against derivatised SEM. A novel carboxyphenyl SEM derivative was used to raise a polyclonal antibody that has been incorporated into a semi-quantitative microtitre plate ELISA, validated according to the criteria set out in Commission Decision 2002/657/EC, for use with chicken muscle. The antibody is highly specific for derivatised SEM, cross-reactivity being 1.7% with NFZ and negligible with a wide range of other nitrofurans and poultry drugs. Samples are derivatised with o-nitrobenzaldehyde and simultaneously protease digested before extraction by cation exchange SPE. The ELISA has a SEM detection capability (CC_β) of 0.25 μg kg⁻¹ when a threshold of 0.21 μg kg⁻¹ is applied to the selection of samples for confirmation (lowest observed 0.25 μg kg⁻¹ fortified sample, n = 20), thus satisfying the EU nitrofurans’ minimum required performance limit of 1 μg kg⁻¹. NFZ-incurred muscles (12) containing SEM at 0.5-5.0 μg kg⁻¹ by LC-MS/MS, all screened positive by this ELISA protocol which is also applicable to egg and chicken liver.     

Determination and identification,according to European Union Decision 2002/657/EC,of malachite green and its metabolite in fish by liquid chromatography–tandem mass spectrometry using an optimized extraction procedure and three-way calibration

This paper reports a multiresponse optimization of an extraction procedure in the simultaneous determination of malachite green (MG) and its metabolite (leucomalachite green, LMG) in fish by liquid chromatography with triple quadrupole mass spectrometry (LC–MS/MS). Prior to optimization, the active factors of the extraction procedure were determined by a screening experimental design. Then, in the optimal experimental conditions of the extraction, MG and LMG have been determined by using a three-way calibration model based on parallel factor analysis (PARAFAC). The procedure fulfils the performance requirements for a confirmatory method established by the European Union Decision 2002/657/EC. This norm establishes maximum permitted tolerances for relative abundance of the precursor/product ion pairs. There is a reported contradiction in the literature related to the fact that there are standard samples whose concentration is greater than CCα but the maximum permitted tolerances are not fulfilled in the identification of the analytes. In this work, it is shown that with the information provided by PARAFAC this contradiction is avoided. The figures of merit for PARAFAC and univariate calibration procedures were evaluated under optimal conditions in the extraction step. The figures of merit obtained were in the range of 0.13–0.23 μg kg⁻¹ for the decision limit, CCα, (α = 0.01) and 0.22–0.39 μg kg⁻¹ for the detection capability, CCβ, (β = 0.05), whereas mean relative errors in absolute value were in the range of 2.8–4.6% for MG and LMG with PARAFAC calibration. The proposed optimized extraction procedure using a PARAFAC calibration was also applied in the determination of MG and LMG in gilthead bream samples: the decision limit was in the range of 0.45–0.55 μg kg⁻¹, the detection capability was in the range of 0.76–0.92 μg kg⁻¹ for MG and LMG. Trueness was likewise confirmed and the mean of the absolute values of relative errors were between 4.2% and 7.2%.     

In-house validation of liquid chromatography tandem mass spectrometry for determination of semicarbazide in eggs and stability of analyte in matrix

An improved LC-MS/MS method for the determination of semicarbazide in whole egg is described. Waters OASIS-MCX cation exchange purification cartridges increased the sensitivity for analysis by LC-MS/MS. The validation study was carried out according to criteria and requirements of Commission Decision 2002/657/EC for confirmatory analysis and provided the data as follows: The correlation coefficient for the matrix calibration curve, in the range of 0–5 μg kg⁻¹, was r = 0.9968. The detection capability and decision limit, measured according to ISO11843-2, were CCα = 0.20 μg kg⁻¹ and CCβ = 0.25 μg kg⁻¹. Repeatability (CVSr) and within-laboratory reproducibility (CVSwr) determined for the concentration levels of 0.2, 0.5 and 1.0 μg kg⁻¹ SEM ranged from 11.9 to 5.7% and 11.8 to 6.3%, respectively. The validated method was applied to investigate SEM stability in incurred materials (egg homogenates) during long-term storage at −20 °C and 4 °C. The study proved by a two-sampling test that SEM at levels of 17. 7, 1.2, 10.6 and 0.47 μg kg⁻¹ was stable for up to 12 months.     

A multivariate multianalyte screening method for sulfonamides in milk based on front-face fluorescence spectroscopy

Screening methods are used to detect the presence of a substance or class of substances at the level of interest and are specifically designed to avoid false compliant results. They should allow the running of a high number of samples per day at a low cost under routine conditions. In this work, a rapid and simple method for the screening of six sulfonamides (sulfadiazine, SD; sulfamerazine, SMR; sulfamethazine, SMT; sulfachloropyridazine, SCP; sulfathiazole, STZ and sulfamethoxazole, SMO) in milk samples is proposed and assessed according to the criteria required by the European Regulation, Decision 2002/657/EC. The method is based on modelling front-face fluorescence emission spectra by means of partial least squares class modelling (PLS-CM). The milk samples are pre-treated with a single easy step of derivatization with fluorescamine.After confirming that the method has equal analytical sensitivity for all the six sulfonamides, it is established that the multivariate analytical sensitivity at 100 μg L⁻¹ is 37.5 μg L⁻¹ when analysing a mixture of six sulfonamides added to different brands of milk and measured in different days. In addition, the method is applied to samples from 11 commercial brands of milk. For β = 0.05, threshold value established by the Decision 2002/657/EC for this method, the probability of false non-compliance, α, is equal to 0.17, allowing the suitable screening of these six sulfonamides.     

Development and validation of an ultra-high performance liquid chromatography tandem mass spectrometry method for quantifying thyreostats in urine without derivatisation

Thyreostatic drugs, illegally administrated to livestock for fattening purposes, are banned in the European Union since 1981 (Council Directive 81/602/EC). For monitoring their illegal use, sensitive and specific analytical methods are required. In this study an UHPLC-MS/MS method was described for quantitative analysis of eight thyreostatic drugs in urine, this without a derivatisation step. The sample pretreatment involved a reduction step with dithiothreitol under denaturating conditions at 65 °C, followed by liquid-liquid extraction with ethyl acetate. This analytical procedure was subsequently validated according to the EU criteria (2002/657/EC Decision), resulting in decision limits and detection capabilities ranging between 1.1 and 5.5 μg L⁻¹ and 1.7 and 7.5 μg L⁻¹, respectively. The method obtained for all, xenobiotic thyreostats, a precision (relative standard deviation) lower than 15.5%, and the linearity ranged between 0.982 and 0.999. The performance characteristics fulfill not only the requirements of the EU regarding the provisional minimum required performance limit (100 μg L⁻¹), but also the recommended concentration fixed at 10 μg L⁻¹ in urine set by the Community of Reference Laboratories. Future experiments applying this method should provide the answer to the alleged endogenous status of thiouracil.     

Supramolecular solvents in solid sample microextractions: Application to the determination of residues of oxolinic acid and flumequine in fish and shellfish

Supramolecular solvents are here proposed firstly as extractants in solid sample microextractions. The approach was evaluated by extracting flumequine (FLU) and oxolinic acid (OXO), two widely used veterinary medicines, from fish and shellfish muscle using a supramolecular solvent made up of decanoic acid (DeA) reverse micelles. The antibiotics were extracted in a single step (∼15 min), at room temperature, using 400 μL of solvent. After centrifugation, an aliquot of the extract was directly analyzed by liquid chromatography and fluorescence, without the need of clean-up or solvent evaporation. Contrary to the previously reported methods, both OXO and FLU were quantitatively extracted from fish and shellfish, independently of sample composition. The high extraction efficiencies observed for these antibiotics were a consequence of their amphiphilic character which resulted in the formation of DeA-OXO and DeA-FLU mixed aggregates. The quality parameters of this quantitative method including sensitivity, linearity, selectivity, repeatability, trueness, ruggedness, stability, decision limit and detection capability were evaluated according to the 2002/657/EC Commission Decision. Quantitation limits in the different samples analyzed (salmon, sea trout, sea bass, gilt-head bream, megrim and prawns) ranged between 6.5 and 22 μg kg⁻¹ for OXO and, 5 and 15 μg kg⁻¹ for FLU. These limits were far below the current maximum residue limits (MRLs) set by the European Union (EU) (i.e. 100 and 600 μg kg⁻¹, for OXO and FLU, respectively). The trueness of the method was determined by analyzing a Certified Reference Material (CMR, BCR^®-725) consisting of a lyophilised salmon tissue material. Recoveries for fortified samples (50–100 μg kg⁻¹ of OXO and 50–600 μg kg⁻¹ of FLU) and their relative standard deviations were in the intervals 99–102% and 0.2–5%, respectively. The repeatability, expressed as relative standard deviation, was 3.6% for OXO and 2.3% for FLU ([OXO] = [FLU] = 200 μg kg⁻¹ and n = 11).     

Rapid multi-residue method for the quantitative determination and confirmation of glucocorticosteroids in bovine milk using liquid chromatography-electrospray ionization-tandem mass spectrometry

Dexamethasone, betamethasone and prednisolone are synthetic glucocorticosteroids authorised for therapeutic use in bovine animals within the European Union. Dexamethasone and betamethasone are used mainly for the treatment of metabolic and inflammatory diseases. Prednisolone is used to treat bovine mastitis. Maximum residue limits (MRLs) of 0.3 μg kg⁻¹ for both dexamethasone and betamethasone and 6.0 μg kg⁻¹ for prednisolone in bovine milk have been established. 6α-Methylprednisolone and flumethasone are not authorised for use in bovine animals and are completely banned in bovine milk. The proposed method is based on deprotenisation of milk using 20% (w/v) trichloroacetic acid. Samples are filtered using glass microfibre filters and subject to clean-up using OASIS HLB solid phase extraction. Separation was achieved on a Hypercarb 100 mm × 2.1 mm × 5 μm column. Mobile phase was: 90/10 acetonitrile/0.1% formic acid in water; flow rate was 600 μL min⁻¹. The method allowed the rapid identification and confirmation of the five glucocorticosteroids according to the criteria laid down in Commission Decision 2002/657/EC. Matrix calibration curves for all compounds were linear in the interval 0.0 MRL to 2.0 MRL with a correlation coefficient (r²) higher than 0.96. Relative recoveries ranged from 97% for betamethasone to 111% for prednisolone. Precision at the MRL ranged from 3.8% for prednisolone to 13.8% for betamethasone. Decision limits, CCα, and detection capability, CCβ have been calculated for all compounds.     

Detection capability of tetracyclines analysed by a fluorescence technique: comparison between bilinear and trilinear partial least squares models

According to the committee decision of 12 August 2002 (2002/657/EC) the capability of detection, CCβ, must be set in all analytical methods not only at concentration levels close to zero but also at the maximum permitted limit (PL). In this work we describe a methodology which evaluates the capability of detection of a fluorescence technique with soft calibration models (bilinear and trilinear PLS) to determine tetracyclines (group B1 substances from annex 1 of Directive 96/23/EC). Its estimation is based on the generalisation of the procedure described in International Union of Pure and Applied Chemistry and in the ISO standard 11843 for univariate signals which evaluates the probabilities of false positive (α) and false negative (β). The capability of detection, CCβ, estimated from the second-order signal and the trilinear PLS model is 9.93 μg l⁻¹ of tetracycline, 17.75 μg l⁻¹ of oxytetracycline and 26.31 μg l⁻¹ of chlortetracycline, setting α and β at 0.05. The capability of detection, CCβ, determined around the PL (100 μg kg⁻¹ in milk and muscle) with the second-order signal is 109.4 μg l⁻¹ of tetracycline, 117.0 μg l⁻¹ of oxytetracycline and 124.9 μg l⁻¹ of chlortetracycline, setting α and β at 0.05. The results were compared with those obtained with zero and first-order signals. The effect of the interferences on the capability of detection was also analysed as well as the number of standards used to build the models and their calibration range.When a tetracycline is quantified in presence of uncalibrated ones by means of the trilinear PLS model the errors oscillate between 14.70% for TC and 9.57% for OTC.     

Trace analysis of endectocides in milk by high performance liquid chromatography with fluorescence detection

An analytical method has been developed for the simultaneous determination of the following endectocide drugs in milk: ivermectin, abamectin, doramectin, moxidectin, eprinomectin, emamectin and nemadectin. Samples were extracted with acetonitrile, purified with solid-phase extraction on a reversed phase C₈, derivatised with N-methylimidazole, trifluoroacetic anhydride and acetic acid to a stable fluorescent derivative, and were further analysed by gradient high performance liquid chromatography (HPLC) on an endcapped reversed phase Supelcosil LC-8-DB. The derivatisation step was mathematically optimised and the method was validated according to the requirements of Commission Decision 2002/657/EC, using fortified raw bovine milk. Mean recovery was between 78 and 98%. The repeatability (CV_r) and within-laboratory reproducibility (CV_W) ranged from 4.6 to 13.4% and from 6.6 to 14.5%, respectively. Decision limits (CCα) for analytes with MRL values, namely eprinomectin and moxidectin, were determined to be 24.8 and 50.6 μg kg⁻¹, respectively. CCα values for unauthorised endectocides ranged from 0.1 to 0.2 μg kg⁻¹. Due to high acceptability regarding the required criteria and applicability to ovine and caprine milk, giving similar results, this multi-analyte method has been successfully implemented in pharmacokinetic research studies as well as statutory residue monitoring in Slovenia.     

Simultaneous determination of 11 drugs belonging to four different groups in human urine samples by reversed-phase high-performance liquid chromatography method

A new, accurate, sensitive and fast reversed-phase high-performance liquid chromatography (RP-HPLC) as an analytical method for the quantitative determination of 11 drugs in human urine was worked out, optimized and validated. The objects of analysis were imipenem (IMP), paracetamol (PAR), dipyrone (DPR), vancomycin (VCM), amikacin (AMK), fluconazole (FZ), cefazolin (CFZ), prednisolone (PRE), dexamethasone (DEX), furosemide (FUR) and ketoprofen (KET) belonging to four different groups (antibiotics, analgesic, demulcent and diuretic). For HPLC analysis, diode array (DAD) and fluorescence (FL) detectors were used. The separation of analyzed compounds was conducted by means of a LiChroCART^® Purospher^® C₁₈e (125 mm × 3 mm, particle size 5 μm) analytical column with LiChroCART^® LiChrospher^® C₁₈ (4 mm × 4 mm, particle size 5 μm) pre-column with gradient elution. Analyzed drugs were determined within 20 min. The mobile phase was comprised of various proportions of methanol, acetonitrile and 0.05% trifluoroacetic acid in water. AMK was separated and determined from human urine using ortho-phthaldialdehyde-3-mercaptopropionic acid (OPA-3-MPA) as a fluorescent reagent by RP-HPLC-FL. The following retention times for drugs IMP, PAR, DPR, VCM, AMK, FZ, CFZ, PRE, DEX, FUR and KET in human urine were found: 4.01 min, 4.86 min, 6.71 min, 8.14 min, 9.46 min, 10.01 min, 10.90 min, 13.34 min, 14.06 min, 16.03 min and 18.98 min, respectively. Excellent linearity was obtained for compounds in the range of concentration: 0.35-42 μg ml⁻¹, 0.5-45 μg ml⁻¹, 4.5-38 μg ml⁻¹, 0.25-25 μg ml⁻¹, 0.5-35 μg ml⁻¹, 0.25-22 μg ml⁻¹, 0.03-52 μg ml⁻¹, 0.15-25 μg ml⁻¹, 0.25-28 μg ml⁻¹, 0.05-18 μg ml⁻¹ and 0.15-35 μg ml⁻¹ for IMP, PAR, DPR, VCM, AMK, FZ, CFZ, PRE, DEX, FUR and KET, respectively. The limits of detection (LOD) and limits of quantification (LOQ) for analyzed drugs were calculated in all cases and recovery studies were also performed. Ten human urine samples obtained from patients treated in hospital have been tested. In analyzed samples, one or more drugs from the 11 examined drugs were detected. The concentrations of examined drugs in urine samples ranged between: 1.5-12 μg ml⁻¹ of PAR, 5.2-11.5 μg ml⁻¹ of DPR, 0.13-9.5 μg ml⁻¹ of CFZ and 0.1-8 μg ml⁻¹ of FUR. This method can be successfully applied to routine determination of all these drugs in human urine samples.     

Rapid confirmatory method for the determination of 11 nitroimidazoles in egg using liquid chromatography tandem mass spectrometry

A rapid confirmatory method has been developed and validated for the simultaneous identification, confirmation and quantitation of 11 nitroimidazoles in eggs by liquid chromatography tandem mass spectrometry (LC–MS/MS). The method is validated in accordance with Commission Decision 2002/657/EC and is capable of analysing metronidazole (MNZ), dimetridazole (DMZ), ronidazole (RNZ), ipronidazole (IPZ) and their hydroxy metabolites MNZ-OH, HMMNI (hydroxymethyl, methyl nitroimidazole), IPZ-OH. The method is also capable of analysing carnidazole (CRZ), ornidazole (ORZ), tinidazole (TNZ) and ternidazole (TRZ). MNZ, DMZ and RNZ have been assigned a recommended level (RL) of 3 μg kg⁻¹ by the Community Reference Laboratory (CRL) in Berlin. The developed method described in this study is easily able to detect all the nitroimidazole compounds investigated at this level and below. Egg samples are extracted with acetonitrile, and NaCl is added to help remove matrix contaminants. The acetonitrile extract undergoes a liquid–liquid wash step with hexane; it is then evaporated and reconstituted in mobile phase. The reconstituted samples are analysed by liquid chromatography tandem mass spectrometry (LC–MS/MS). The decision limits (CCα) range from 0.33 to 1.26 μg kg⁻¹ and the detection capabilities (CCβ), range from 0.56 to 2.15 μg kg⁻¹. The results of the inter-assay study, which was performed by fortifying hen egg samples (n = 18) on three separate days, show the accuracy calculated for the various analytes to range between 87.2 and 106.2%. The precision of the method, expressed as %CV values for the inter-assay variation of each analyte at the three levels of fortification (3, 4.5 and 6.0 μg kg⁻¹), ranged between 3.7 and 11.3%. A Day 4 analysis was carried out to examine species variances in eggs from different birds such as duck and quail and investigating differences in various battery and free range hen eggs.     

Fluorescence quantification of tetracycline in the presence of quenching matrix effect by means of a four-way model

In this work, a four-way tensor is used to model the quenching effect in fluorescent measurements. By means of the analysis of excitation-emission matrices obtained in the determination of tetracycline in tea, which acts as quencher, it is shown as the impossibility to use a calibration, or an addition standard based on a three-way model. It is analysed the quencher multiplicative effect made on the tetracycline signal by means of an ANOVA. However, by arranging the experimental data in a four-way tensor, it is viable to perform a calibration based on the parallel factor analysis, PARAFAC, decomposition and a four-way partial least squares, 4-PLS, regression to quantify the tetracycline in the presence of the matrix quencher effect. 4-PLS calibration provides better results. In the range from 40 to 220 μg L⁻¹ gives an average of relative errors in absolute value equal to 8.02% in prediction (3.40% in calibration). The repeatability as standard deviation in this range is 5.08 μg L⁻¹ and the method is accurate, slope and intercept being statistically equal to 1 and 0, respectively when a regression calculated versus true concentration is performed. Moreover, it has a decision limit (CCα) of 13.87 μg L⁻¹ for a probability of false positive, α, equal to 0.05 and a capability of detection (CCβ) of 26.63 μg L⁻¹ (for probabilities of false positive, α, false negative, β, equals to 0.05).     

Validation of a high-performance liquid chromatographic method with fluorescence detection for the simultaneous determination of tetracyclines residues in bovine milk

A high-performance liquid chromatography-fluorescence detection method was optimized and validated to determine tetracyclines residues in bovine milk. Post-column derivatization using metal complexation in non-aqueous reagent increased the fluorescence of chelates by a factor up to 2.54 compared to water (signal-to-noise ratio enhancement). Overall recoveries ranged from 61 to 115%, with RSD_r from 5 to 15% (n = 54). Detection limits ranged from 5 to 35 μg kg⁻¹. Limits of quantification were established at 50 μg kg⁻¹. Decision limits (CCα) were 109, 108 and 124 μg kg⁻¹ and detection capabilities (CCβ) 119, 117 and 161 μg kg⁻¹ for oxytetracycline, tetracycline and chlortetracycline, respectively. The method was applied successfully in a national monitoring program.     

Transfer assessment of fipronil residues from feed to cow milk

Fipronil, a phenylpyrazole insecticide introduced for pest control on a broad range of crops, can also affect non-target insects such as honeybees. More widely, non-target environment such as milk produced by dairy cows fed with maize silage from treated seeds (=silage T) can be affected. To assess the potential transfer of fipronil residues (sulfone, sulfide, fipronil, desulfinyl and amide), a methodology including gas chromatography coupled with tandem mass spectrometry (GC-MS/MS) analysis was developed and validated according to the 2002/657/EC decision, in order to reach a level of quantification below 0.1 μg L⁻¹ in milk and 0.1 μg kg⁻¹ in plants. Twelve dairy cows were fed with silage T during 4 months. Concentration of fipronil in treated seeds was estimated at 1 g kg⁻¹, whereas silage from these seeds contained 0.30 ± 0.05 μg kg⁻¹ of dry material of fipronil, 0.13 ± 0.03 μg kg⁻¹ of dry material of sulfone. Sulfide residues were below the limit of quantification. Silage from untreated seeds (=silage U) presented traces of fipronil and sulfone, respectively at 0.04 ± 0.06 and 0.02 ± 0.03 μg kg⁻¹ of dry material. Contribution of fipronil residues from supplies was insignificant. During administration of silage T, only sulfone residues were quantified in milk. The average concentration was 0.14 ± 0.05 μg L⁻¹. Before and after administration, sulfone residues were detected but not quantifiable (<0.025 μg L⁻¹). Our results suggest a transfer of fipronil from feed to milk under its sulfone form. Moreover, traces of fipronil residues in maize U, soya, wheat and straw show a diffuse contamination of this pesticide in the environment.     

An amperometric method for the detection of amitrole, glyphosate and its aminomethyl-phosphonic acid metabolite in environmental waters using passive samplers

The herbicides amitrole and glyphosate, and its metabolite aminomethyl-phosphonic acid (AMPA), in water samples have been directly analysed by high-performance liquid chromatography using an electrochemical (EC) detector. Limits of detection of 0.3 μg mL⁻¹ for glyphosate, 0.05 μg mL⁻¹ for AMPA and 0.03 μg mL⁻¹ for amitrole were comparable to those obtained by other authors using EC and also by liquid chromatography coupled to mass spectrometry, but the latter method requires derivatisation and pre-concentration of the sample whereas EC methods show similar sensitivity without the need of any derivatisation. The method was specifically designed to analyse extracts from passive samplers used for monitoring of polar herbicide residues in waters. To this purpose, three types of Empore^® disks were tested for their ability to adsorb and desorb these ionic, polar analytes. A procedure for their extraction from the membranes and reducing the interferences from other substances present in natural waters (i.e. humic acids) is described. The method is simple, does not require sophisticated equipment and is valid for the analysis and monitoring of herbicides residues using passive samplers.     

Targeted analysis with benchtop quadrupole–orbitrap hybrid mass spectrometer: Application to determination of synthetic hormones in animal urine

Sensitive and unequivocal determination of analytes/contaminants in complex matrices is a challenge in the field of food safety control. In this study, various acquisition modes (Full MS/AIF, Full MS + tMS/MS, Full MS/dd MS/MS and tSIM/ddMS/MS) and parameters of a quadrupole–orbitrap hybrid mass spectrometer (Q Exactive) were studied in detail. One of the main conclusions has been that, reducing the scan range for Full MS (using the quadrupole) and targeted modes give higher signal-to-noise (S/N) ratios and thereby better detection limits for analytes in matrix. The use of Q Exactive in a complex case, for the confirmatory analysis of hormones in animal urine is presented. A targeted SIM data dependent MS/MS (tSIM/ddMS/MS) acquisition method for determination of eight synthetic hormones (trenbolone, 17α ethinylestradiol, zeranol, stanozolol, dienestrol, diethylstilbestrol, hexestrol, taleranol) and a naturally occurring hormone (zearalenone) in animal urine were optimized to have sensitive precursors from targeted SIM mode and trigger MS/MS scans over the entire chromatograph peak. The method was validated according to EC/657/2002. CCα (decision limit) for the analytes ranged between 0.11 μg L⁻¹ and 0.69 μg L⁻¹ and CCβ (detection capability) ranged between 0.29 μg L⁻¹ and 0.90 μg L⁻¹.