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 reliable high-performance liquid chromatography with ultraviolet detection for the determination of sulfonamides in honey

The sulfonamides are stable chemotherapeutics used against the bacterial disease affecting bees, known as American foulbrood (Bacillus larvae), so their residues could appear in the honey of treated bees. Their presence at a concentration above the limit value is a potential hazard to human health. Brazilian authorities have included in the National regulatory monitoring program, the control of the three most widely used sulfonamides in honey production, i.e., sulfathiazole, sulfamethazine and sulfadimethoxine. A method for the determination of residual sulfonamides in honey, using sulfapyridine as an internal standard has been developed, optimized and validated. Some changes were implemented on current available methodologies for the analysis of sulfonamides in honey in order to adopt such procedures to Brazilian honey samples. Sulfonamides were extracted from honey with dichloromethane after dissolution with 30% sodium chloride, and cleaned up with solid phase extraction on Florisil columns. The eluate was analyzed by high-performance liquid chromatography with ultraviolet detection. The limit of detection was determined at 3 μg kg⁻¹, 4 μg kg⁻¹ and 5 μg kg⁻¹ for sulfathiazole, sulfamethazine and sulfadimethoxine, respectively with average recoveries of 61.0% for sulfathiazole; 94.5% for sulfamethazine and 86.0% for sulfadimethoxine at the 100 μg kg⁻¹ level. As the final step of validation procedure, the analysts were submitted to a blind spiked sample prepared by the quality assurance officer which results were successfully obtained regarding recovery and deviations.     

Determination of sulfonamides in soil samples based on alumina-coated magnetite nanoparticles as adsorbents

In this study, alumina-coated magnetite nanoparticles (Fe₃O₄/Al₂O₃ NPs) were synthesized, and they were applied to the analysis of sulfonamides (SAs) including sulfadiazine (SDZ), sulfamerazine (SMR), sulfamethoxazole (SMX), sulfamonomethoxine (SMM), sulfamethoxydiazine (SMD), sulfadimethoxine (SDM) and sulfaquinoxaline (SQX) in different soil samples based on magnetic solid-phase extraction (MSPE). The extraction and concentration process was carried out in a single step by mixing the extraction solvent, magnetic adsorbents and soil sample under ultrasonic action. Then, the adsorbents were isolated from the complicated matrix easily with an external magnetic field. The SAs desorbed from the adsorbents were determined by liquid chromatography-tandem mass spectrometry. Compared with traditional methods, the MSPE method simplified the operation procedure and reduced the analysis time. Under the optimum conditions, the recoveries of SDZ, SMR, SMX, SMM, SMD and SDM by analyzing the five spiked soil samples were between 71% and 93% except for SQX (42-60%). This may be due to the stronger hydrophobic property of SQX. Detection limits of SAs were between 0.37 and 6.74 ng g⁻¹. It was also found that the “aging” effect of SAs contaminated soil could cause the recoveries to decrease.     

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.     

Simultaneous determination of tetracyclines in poultry muscle by capillary zone electrophoresis

A capillary zone electrophoresis method with UV detection was developed for the simultaneous detection and quantification of three tetracyclines in chicken meat samples: tetracycline (TC), oxytetracycline (OTC) and doxycycline (DOC). The separation conditions were: a running buffer containing 30 mM sodium phosphate, 2 mM EDTA disodium salt and 2.5% 2-propanol, pH 12.0, a 5 s hydrodynamic injection and a 14 kV separation voltage. Two different clean-up methodologies were employed: solid-phase extraction with C₁₈ cartridges and ion exchange with Amberlite XAD7 resin. Analytes were detected at 360 nm in less than 12 min. LODs ranged from 61 μg kg⁻¹ for OTC to 68 μg kg⁻¹ for DOC with C₁₈ cartridges, and 81 μg kg⁻¹ for DOC to 89 μg kg⁻¹ for TC with Amberlite XAD7 resin. The recoveries for TC, OTC and DOC obtained by both methods were between 85 and 95%, and the peak area repeatability for all of the samples was below 5% in all cases. Twenty-four samples of commercial chicken drumsticks were examined with both clean-up methodologies. In nine cases (37.5%) TC was detected, in a range from 197.8 to 2564.3 μg kg⁻¹, and in seven cases (29.2%) OTC was detected in a range from 83.0 to 2049.3 μg kg⁻¹. DOC was not detected in any of the tested samples. This method would be useful for the routine monitoring of TCs residues in poultry muscle.     

Application of a modified enzyme-linked immunosorbent assay for 3-amino-2-oxazolidinone residue in aquatic animals

Furazolidone has been banned from use in food animals because of its carcinogenicity and mutagenicity, but its continued misuse is widespread in aquacultures. Therefore, there is an urgent need for a simple, reliable, and rapid method for the detection of its marker residue, 3-amino-2-oxazolidinone (AOZ), in aquatic products. In this regard, we modified a simplified indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) to address this need. A good linearity was achieved over a concentration range of 0.05-12.15 μg L⁻¹, and the IC₅₀ value was 0.96 μg L⁻¹. The sample preparation was simple and effective included water bath treatments, acid hydrolysis combined with overnight derivatization of AOZ by benzaldehyde. The limit of detection and the limit of quantification were 0.15 and 0.3 μg kg⁻¹. The recoveries of AOZ in all tissues were between 78.0-95.3% at the levels of 0.3, 1.0, and 2.0 μg kg⁻¹. The inter-assay variability was less than 19.1%. The modified ic-ELISA was applied in quantification of AOZ elimination in carp. The results showed that AOZ was quite difficult to eliminate. Good correlations of the results obtained by ELISA and LC-MS/MS were observed in incurred carp muscle (r = 0.9923) and carp plasma (r = 0.9915) at the levels of 2.5-571.8 μg kg⁻¹ (μg L⁻¹). Better results were obtained by modified ic-ELISA when compared with commercial ELISA kit. Therefore, the present assay is considered a rapid, accurate, reliable, and inexpensive method for the detection of furazolidone-residues in the edible tissues of aquatic animals.     

Concentration levels of total and methylmercury in mussel samples collected along the coasts of Sardinia Island (Italy)

This paper reports the assessment of the total mercury (T-Hg) and methylmercury (MeHg) contamination of mussel samples collected by two sampling campaigns from along the coastline of Sardinia (Italy). T-Hg has been determined by a direct mercury analyser (DMA) whereas MeHg has been determined by gas chromatography-mass spectrometry (GC-MS) after acid extraction, and employs a novel NaBPh₄ derivatization method. The evaluation of the quality of measurements was carried out by analysing candidate certified reference material (CRM) BCR 710, for MeHg and T-Hg, and CRM IAEA-350 for T-Hg. In the analysed samples, the T-Hg concentrations range from 35 to 115 μg kg⁻¹ and from 40 to 830 μg kg⁻¹, for the two sampling campaigns, respectively, whereas the MeHg concentrations range from l5 to 51 μg kg⁻¹ and from 17 to 116 μg kg⁻¹. Consequently, the MeHg/T-Hg ratios range from 0.33 to 0.91 and from 0.14 to 0.98, respectively. Despite the increasing trend of Hg concentration from the first to the second sampling campaign, the T-Hg concentration of all the samples was much below the 0.5 μg g⁻¹ WHO limit, and the MeHg values ranged between 2.2 and 17.2 μg kg⁻¹, not exceeding the 43.5 μg kg⁻¹ tolerable daily residue level calculated for Italy.     

Enzyme-assisted extraction and liquid chromatography mass spectrometry for the determination of arsenic species in chicken meat

Chicken is the most consumed meat in North America. Concentrations of arsenic in chicken range from μg kg⁻¹ to mg kg⁻¹. However, little is known about the speciation of arsenic in chicken meat. The objective of this research was to develop a method enabling determination of arsenic species in chicken breast muscle. We report here enzyme-enhanced extraction of arsenic species from chicken meat, separation using anion exchange chromatography (HPLC), and simultaneous detection with both inductively coupled plasma mass spectrometry (ICPMS) and electrospray ionization tandem mass spectrometry (ESIMS). We compared the extraction of arsenic species using several proteolytic enzymes: bromelain, papain, pepsin, proteinase K, and trypsin. With the use of papain-assisted extraction, 10 arsenic species were extracted and detected, as compared to 8 detectable arsenic species in the water/methanol extract. The overall extraction efficiency was also improved using a combination of ultrasonication and papain digestion, as compared to the conventional water/methanol extraction. Detection limits were in the range of 1.0–1.8 μg arsenic per kg chicken breast meat (dry weight) for seven arsenic species: arsenobetaine (AsB), inorganic arsenite (As^III), dimethylarsinic acid (DMA), monomethylarsonic acid (MMA), inorganic arsenate (As^V), 3-nitro-4-hydroxyphenylarsonic acid (Roxarsone), and N-acetyl-4-hydroxy-m-arsanilic acid (NAHAA). Analysis of breast meat samples from six chickens receiving feed containing Roxarsone showed the presence of (mean ± standard deviation μg kg⁻¹) AsB (107 ± 4), As^III (113 ± 7), As^V (7 ± 2), MMA (51 ± 5), DMA (64 ± 6), Roxarsone (18 ± 1), and four unidentified arsenic species (approximate concentration 1–10 μg kg⁻¹).     

Determination of benzimidazolic fungicides in fruits and vegetables by supramolecular solvent-based microextraction/liquid chromatography/fluorescence detection

A supramolecular solvent consisting of vesicles, made up of equimolecular amounts of decanoic acid (DeA) and tetrabutylammonium decanoate (Bu₄NDe), dispersed in a continuous aqueous phase, is proposed for the extraction of benzimidazolic fungicides (BFs) from fruits and vegetables. Carbendazim (CB), thiabendazole (TB) and fuberidazole (FB) were extracted in a single step and no clean-up or concentration of extracts was needed. The high extraction efficiency obtained for BFs was a result of the different types of interactions provided by the supramolecular solvent (e.g. hydrophobic and hydrogen bonds) and the high number of solubilisation sites it contains. Besides simple and efficient, the proposed extraction approach was rapid, low-cost, environment friendly and it was implemented using conventional lab equipments. The target analytes were determined in the supramolecular extract by LC/fluorescence detection. They were separated in a Kromasil C₁₈ (5 μm, 150 mm × 4.6 mm) column using isocratic elution [mobile phase: 60:40 (v/v) 50 mM phosphate buffer (pH 4)/methanol] and quantified at 286/320 nm (CB) and 300/350 nm (TB and FB) excitation/emission wavelengths, respectively. Quantitation limits provided by the supramolecular solvent-based microextraction (SUSME)/LC/fluorescence detection proposed method for the determination of CB, TB and FB in fruits and vegetables were 14.0, 1.3 and 0.03 μg kg⁻¹, respectively, values far below the current maximum residue levels (MRLs) established by the European Union, i.e. 100-2000 μg kg⁻¹ for CB, 50-5000 μg kg⁻¹ for TB and 50 μg kg⁻¹ for FB. The precision of the method, expressed as relative standard deviation, for inter-day measurements (n = 13) was 3.3% for CB (50 μg kg⁻¹), 3.5% for TB (10 μg kg⁻¹) and 2.8% for FB (0.5 μg kg⁻¹) and recoveries for fruits (oranges, tangerines, lemons, limes, grapefruits, apples, pears and bananas) and vegetables (potatoes and lettuces) fortified at the μg kg⁻¹ level were in the interval 93-102%.     

Optimization of LC-MS/MS using triple quadrupole mass analyzer for the simultaneous analysis of carbosulfan and its main metabolites in oranges

This paper describes an analytical method involving a simple solvent extraction for the simultaneous liquid chromatography coupled to quadrupole tandem mass spectrometry (LC-MS/MS) determination of carbosulfan, its most toxic metabolite -carbofuran -, and its other main metabolites - 3-hydroxycarbofuran, 3-ketocarbofuran, 3-hydroxy-7-phenolcarbofuran, 3-keto-7-phenolcarbofuran, 7-phenolcarbofuran and dibutylamine - in oranges. Chromatography was performed on a Zorbax Bonus-RP (150 mm × 2.1 mm, 5 μm). The mobile phase was a ternary gradient water-methanol-acetonitrile with 1.0 mM ammonium acetate at flow rate of 0.2 ml min⁻¹. The LC separation and MS/MS optimization were studied to select the most appropriate operating conditions. The method developed has also been validated. The limits of quantification (LOQs) were from 1 μg kg⁻¹ for carbofuran to 10 μg kg⁻¹ for 3-keto-7-phenolcarbofuran. Extracts spiked with carbosulfan and its metabolites, at LOQ level, yielded average recoveries in the range 60-94%, with relative standard deviations (R.S.D.s) less than 15%. Calibration curves for carbosulfan and its metabolites (range LOQ-1000LOQ) were linear, with coefficients of correlations better than 0.990. The method was successfully applied to establish the primary degradation products in oranges treated with carbosulfan. The LC-MS/MS method developed is simple, rapid, and suitable for the quantification and confirmation of carbosulfan and seven of its main metabolites in orange at levels lower than 10 μg kg⁻¹.     

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.     

Multi-residue method for the determination of organochlorine pesticides in fish feed based on a cleanup approach followed by gas chromatography–triple quadrupole tandem mass spectrometry

A multi-residue method for the determination of organochlorine pesticides in fish feed samples was developed and optimized. The method is based on a cleanup step of the extracted fat, carried out by liquid–liquid extraction on diatomaceous earth cartridge with n-hexane/acetonitrile (80/20, v/v) followed by solid phase extraction (SPE) with silica gel–SCX cartridge, before the identification and quantification of the residues by gas chromatography–triple quadrupole tandem spectrometry (GC–MS/MS). Performance characteristics, such as accuracy, precision, linear range, limits of detection (LOD) and quantification (LOQ), for each pesticide were determined. Instrumental LODs ranged from 0.01 to 0.11 μg L⁻¹, LOQs were in the range of 0.02–0.35 μg L⁻¹, and calibration curves were linear (r² > 0.999) in the whole range of explored concentrations (5–100 μg L⁻¹). Repeatability values were in the range of 3–15%, evaluated from the relative standard deviation of six samples spiked at 100 μg kg⁻¹ of fat, and in compliance with that derived by the Horwitz's equation. No matrix effects or interfering substances were observed in fish feed analyses. The proposed method allowed high recoveries (92–116%) of spiked extracted fat samples at 100 μg kg⁻¹, and very low LODs (between 0.02 and 0.63 μg kg⁻¹) and LOQs (between 0.05 and 2.09 μg kg⁻¹) determined in fish feed samples.     

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.     

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).     

The status of pesticide pollution in Tanzania

The paper summarises the findings of recent studies carried out to assess the levels of pesticide residues in water, sediment, soil and some biota collected from different parts of Tanzania. Although the intention is to cover the whole country, so far the studies have focused on areas with known large-scale pesticide use (Southern Lake Victoria and its basin, TPC sugar Plantations in Kilimanjaro region, Dar es Salaam coast, Mahonda-Makoba basin in Zanzibar) and a former pesticide storage area at Vikuge Farm in Coast region). Analysis of the cleaned extracts in GC-ECD/NPD revealed the dominance of organochlorines in all samples. Generally, low levels of residues were found in areas associated with agricultural pesticide use but the levels in the former storage areas were substantially high. DDT and HCH were dominant in all the studied areas. In the former areas, levels of ∑DDT in water, sediments and soil were up to 2 μg L⁻¹, 700 μg kg⁻¹ and 500 μg kg⁻¹, respectively, while those of ∑HCH were up to 0.2 μg L⁻¹, 132 μg kg⁻¹ and 60 μg kg⁻¹, respectively. The levels in aquatic biota were much higher than those in the water most likely due to bioaccumulation. In the former storage area at Vikuge the levels of pesticides in the topsoil were alarmingly high. Their concentrations were up to 282,000 mg kg⁻¹ dry weight for ∑DDT and up to 63,000 mg kg⁻¹ for ∑HCH. A herbicide, pendimethalin [N-(1-ethylpropyl)-2,6-dinitro-3,4-xylidine], was also found at concentrations up to 41,000 mg kg⁻¹ dry weight. Thus the total pesticide content in the soil was almost 40%. Following these findings the area is now earmarked to be a demonstration site for a proposed GEF project ‘Bioremediation of POPs impacted soils in East Africa’.     

Determination of naphthalene-derived compounds in apples by ultra-high performance liquid chromatography-tandem mass spectrometry

Naphthylacetic acid, naphthyloxy acetic acid and naphthylacetamide belong to a group of synthetic substances known as “auxin-like” compounds which are used as growth regulators in vegetables and fruits due to their structure similarities with the indoleacetic acid, the most important plant auxin. This paper reports a selective, sensitive and fast ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC–MS/MS) method for the determination of naphthylacetamide (NAD) and the isomers (α and β) of naphthylacetic acid (NAA) and naphthyloxy acetic (NOA) acid in apple samples. A baseline separation between the respective isomers was achieved using an RP-Amide column with gradient elution. The UHPLC-MS/MS method developed, using electrospray and selected reaction monitoring (SRM) acquisition mode led to a reliable determination of these family of compounds in apple samples at low quantitation levels, down to 1.0 μg kg⁻¹ and 0.25 μg kg⁻¹ respectively. For confirmation of NAA accurate mass measurement is proposed giving at these conditions quantitation limits of 10 μg kg⁻¹ for this compound. The UHPLC-MS/MS method developed was used for the analysis of apple samples harvested in three different apple fields from Lleida (Spain) during the blooming period. NAD and NAA were found in samples collected during 4–5 weeks after application at concentrations between the quantification limits and 43 μg kg⁻¹ and 24 μg kg⁻¹, respectively.     

Application of a quick,easy, cheap,effective, rugged and safe-based method for the simultaneous extraction of chlorophenols,alkylphenols, nitrophenols and cresols in agricultural soils,analyzed by using gas chromatography–triple quadrupole-mass spectrometry/mass spectrometry

Due to the different physico-chemical properties of phenols, the development of a methodology for the simultaneous extraction and determination of phenolic compounds belonging to several families, such as chlorophenols (CPs), alkylphenols (APs), nitrophenols (NTPs) and cresols is difficult. This study shows the development and validation of a method for the analysis of 13 phenolic compounds (including CPs, APs, NTPs and cresols) in agricultural soils. For this purpose, a quick, easy, cheap, effective, rugged and safe (QuEChERS)-based procedure was developed, validated and applied to the analysis of real samples. A derivatization step prior to the final determination by gas chromatography (GC) coupled to a triple quadrupole analyzer operating in tandem mass spectrometry (QqQ-MS/MS) was performed by using acetic acid anhydride (AAA) and pyridine (Py). The optimized procedure was validated, obtaining average extraction recoveries in the range 69–103% (10 μg kg⁻¹), 65–98% (50 μg kg⁻¹), 76–112% (100 μg kg⁻¹) and 76–112% (300 μg kg⁻¹), with precision values (expressed as relative standard deviation, RSD) ≤ 22% (except for 4-chlorophenol) involving intra-day and inter-day studies. Furthermore, 15 real soil samples were analyzed by the proposed method in order to assess its applicability. Some phenolic compounds (e.g. 2,4,6-trichlorophenol or 4-tert-octylphenol) were found in the samples at trace levels (<10 μg kg⁻¹).     

Determination of ochratoxin A in wine grapes: comparison of extraction procedures and method validation

A method for determination of ochratoxin A (OTA) in wine grapes is described, using extraction with a hydrogen carbonate and polyethylene glycol (PEG) solution (5% NaHCO₃ and 1% PEG 8000), followed by immunoaffinity clean-up and liquid chromatography with fluorescence detection. Validation was made with spiked samples, in levels of 0.05 and 1 μg kg⁻¹, with average recovery rates of 76% and relative standard deviations in repeatability and intermediate precision conditions of 8 and 12%, respectively. The limit of detection and limit of quantification in grapes were established at 0.004 and 0.007 μg kg⁻¹, respectively. To evaluate further the accuracy and efficiency of this method, naturally contaminated grapes were also analysed by another method that involves extraction with acidified methanol, at levels ranging from 0.05 to 37 μg kg⁻¹, and the results compared. A good correlation (r=0.9996) was found, with better performances in terms of precision for the new method. A survey was conducted on wine grapes from 11 Portuguese vineyards, during the harvest of 2002, using the proposed method. OTA was detected in three out of the 11 samples, at levels ranging from 0.035 to 0.061 μg kg⁻¹.The new method meets all the criteria of the European Commission directive 2002/26/CE, that lays down the sampling and the analysis methods for the official control of OTA levels in foodstuffs. It is reliable for low levels of contamination (ng kg⁻¹), and avoids the use of organic solvents in the extraction step.     

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.     

Screening of salbutamol residues in swine meat and animal feed by an enzyme immunoassay in Taiwan

An ELISA was developed for routine examination for extensive monitoring and screening programs for the residues of salbutamol in swine serum, animal feed, meat, and meat-related products destined for human consumption in Taiwan. Objectives of the study were to investigate the use of a new immunoassay for the detection of salbutamol residues in swine meat and animal feed samples, and to compare with a commercial kit in field test screens. A fast, simple and reliable sample preparation method for the determination of salbutamol was established. Field trials with 222 swine meat and 120 animal feed samples that were taken from local meat markets, auction markets and feed mills. The application and the results of two ELISA kits (a homemade and a commercial kit) for the screening of salbutamol were presented. Adopting 2 μg kg⁻¹ salbutamol as a cut-off value for swine meat, the commercial β-agonist ELISA had a sensitivity of 85.3% and a specificity of 95.2% versus GC-MS at a cut-off of 2 μg kg⁻¹. The homemade salbutamol ELISA had a sensitivity of 100% and a specificity of 90.9% and gave no false-negative rate results. Furthermore, adopting 20 μg kg⁻¹ salbutamol as a cut-off value for animal feed, both the commercial and homemade ELISA showed 100% sensitivity and 100% specificity of the assays. In conclusion, a sensitive, specific salbutamol polyclonal antibody-based ELISA has been developed that could serve as a rapid screening assay, and the detection of positive samples at the place of sampling can result in more effective control of the illegal use of β-agonists.