Comprehensive screening and quantification of veterinary drugs in milk using UPLC–ToF-MS

Ultra-performance liquid chromatography combined with time-of-flight mass spectrometry (UPLC–ToF-MS) has been used for screening and quantification of more than 100 veterinary drugs in milk. The veterinary drugs represent different classes including benzimidazoles, macrolides, penicillins, quinolones, sulphonamides, pyrimidines, tetracylines, nitroimidazoles, tranquillizers, ionophores, amphenicols and non-steroidal anti-inflammatory agents (NSAIDs). After protein precipitation, centrifugation and solid-phase extraction (SPE), the extracts were analysed by UPLC–ToF-MS. From the acquired full scan data the drug-specific ions were extracted for construction of the chromatograms and evaluation of the results. The analytical method was validated according to the EU guidelines (2002/657/EC) for a quantitative screening method. At the concentration level of interest (MRL level) the results for repeatability (%RSD < 20% for 86% of the compounds), reproducibility (%RSD < 40% for 96% of the compounds) and the accuracy (80–120% for 88% of the compounds) were satisfactory. Evaluation of the CCβ values and the linearity results demonstrates that the developed method shows adequate sensitivity and linearity to provide quantitative results. Furthermore, the method is accurate enough to differentiate between suspected and negative samples or drug concentrations below or above the MRL. A set of 100 samples of raw milk were screened for residues. No suspected (positive) results were obtained except for the included blind reference sample containing sulphamethazine (88 μg/l) that tested positive for this compound. UPLC–ToF-MS combines high resolution for both LC and MS with high mass accuracy which is very powerful for the multi-compound analysis of veterinary drugs. The technique seems to be powerful enough for the analysis of not only veterinary drugs but also organic contaminants like pesticides, mycotoxins and plant toxins in one single method.     

Isotopic pattern and accurate mass determination in urine drug screening by liquid chromatography/time-of-flight mass spectrometry

An efficient method was developed for toxicological drug screening in urine by liquid chromatography coupled with electrospray ionization time-of-flight mass spectrometry. The method relies on a large target database of exact monoisotopic masses representing the elemental formulae of reference drugs and their metabolites. Mass spectral identification is based on matching measured accurate mass and isotopic pattern (SigmaFit) of a sample component with those in the database. Data post-processing software was developed for automated reporting of findings in an easily interpretable form. The mean and median of SigmaFit for true-positive findings were 0.0066 and 0.0051, respectively. The mean and median of mass error absolute values for true-positive findings were 2.51 and 2.17 ppm, respectively, corresponding to 0.65 and 0.60 mTh. For routine screening practice, a SigmaFit tolerance of 0.03 and a mass tolerance of 10 ppm were chosen. Ion abundance differences from urine extracts did not affect the accuracy of the automatically acquired SigmaFit or mass values. The results show that isotopic pattern matching by SigmaFit is a powerful means of identification in addition to accurate mass measurement.     

Multi-residue determination of 115 veterinary drugs and pharmaceutical residues in milk powder,butter, fish tissue and eggs using liquid chromatography–tandem mass spectrometry

A simple and sensitive multi-residue method for the determination of 115 veterinary drugs and pharmaceuticals, belonging in more than 20 different classes, in butter, milk powder, egg and fish tissue has been developed. The method involves a simple generic solid–liquid extraction step (solvent extraction, SE) with 0.1% formic acid in aqueous solution of EDTA 0.1% (w/v)–acetonitrile (ACN)–methanol (MeOH) (1:1:1, v/v) with additional ultrasonic-assisted extraction. Precipitation of lipids and proteins was promoted by subjecting the extracts at very low temperature (−23 °C) for 12 h. Further cleanup with hexane ensures fat removal from the matrix. Analysis was performed by liquid chromatography coupled with electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS). Two separate runs were performed for positive and negative ionization in multiple reaction monitoring mode (MRM). Particular attention was devoted to extraction optimization: different sample-to-extracting volume ratios, different concentrations of formic acid in the extraction solvent and different ultrasonic extraction temperatures were tested in butter, egg and milk powder samples. The method was also applied in fish tissue samples. It was validated, on the basis of international guidelines, for all four matrices. Quantitative analysis was performed by means of standard addition calibration. For over 80% of the analytes, the recoveries were between 50% and 120% in all matrices studied, with RSD values in the range of 1–18%. Limits of detection (LODs) and quantification (LOQs) ranged from 0.008 μg kg⁻¹ (oxfendazole in butter) to 3.15 μg kg⁻¹ (hydrochlorthiazide in egg). The evaluated method provides reliable screening, quantification, and identification of 115 veterinary drug and pharmaceutical residues in foods of animal origin and has been successfully applied in real samples.     

Atmospheric pressure gas chromatography–time-of-flight-mass spectrometry (APGC–ToF-MS) for the determination of regulated and emerging contaminants in aqueous samples after stir bar sorptive extraction (SBSE)

This work presents the development, optimization and validation of a multi-residue method for the simultaneous determination of 102 contaminants, including fragrances, UV filters, repellents, endocrine disruptors, biocides, polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and several types of pesticides in aqueous matrices. Water samples were processed using stir bar sorptive extraction (SBSE) after the optimization of several parameters: agitation time, ionic strength, presence of organic modifiers, pH, and volume of the derivatizing agent. Target compounds were extracted from the bars by liquid desorption (LD). Separation, identification and quantification of analytes were carried out by gas chromatography (GC) coupled to time-of-flight (ToF-MS) mass spectrometry. A new ionization source, atmospheric pressure gas chromatography (APGC), was tested. The optimized protocol showed acceptable recovery percentages (50–100%) and limits of detection below 1 ng L⁻¹ for most of the compounds. Occurrence of 21 out of 102 analytes was confirmed in several environmental aquatic matrices, including seawater, sewage effluent, river water and groundwater. Non-target compounds such as organophosphorus flame retardants were also identified in real samples by accurate mass measurement of their molecular ions using GC-APGC–ToF-MS. To the best of our knowledge, this is the first time that this technique has been applied for the analysis of contaminants in aquatic systems. By employing lower energy than the more widely used electron impact ionization (EI), AGPC provides significant advantages over EI for those substances very susceptible to high fragmentation (e.g., fragrances, pyrethroids).     

Coumarins as new matrices for matrix-assisted laser-desorption/ionization Fourier transform ion cyclotron resonance mass spectrometric analysis of hydrophobic compounds

Hydrophobic compounds with hydroxyl, aldehyde or ketone groups are generally difficult to detect using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), because these compounds have low proton affinity and are poorly ionized by MALDI. Herein, coumarins have been used as new matrices for MALDI-MS analysis of a variety of hydrophobic compounds with low ionization efficiency, including steroids, coenzyme Q10, a cyclic lipopeptide and cholesterol oleate. Five coumarins, including coumarin, umbelliferone, esculetin, 7-hydroxycoumarin-3-carboxylic acid (HCA) and 6,7-dihydroxycoumarin-3-carboxylic acid (DCA), were compared with the conventional matrices of 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid (CHCA). Coumarins with hydroxyl or carboxylic acid groups enabled detection. Taking DCA as an example, this matrix proved to be superior to DHB or CHCA in detection sensitivity, stability, spot-to-spot and sample-to-sample reproducibility, and accuracy. DCA increased the stability of the target compounds and decreased the loss of water. The [M + Na]⁺ peaks were observed for all target compounds by adding NaCl as an additive, and the [M − H₂O + H]⁺ and [M + H]⁺ peaks decreased. DCA was selected for the identification of sterols in yeast cells, and thirteen sterols were detected by Fourier transform ion cyclotron resonance (FT ICR) mass spectrometry. This work demonstrates the potential of DCA as a new matrix for detection of hydrophobic molecules by MALDI-MS and provides an alternative tool for screening sterols in antifungal research.     

Fully automated screening of veterinary drugs in milk by turbulent flow chromatography and tandem mass spectrometry

There is an increasing interest in screening methods for quick and sensitive analysis of various classes of veterinary drugs with limited sample pre-treatment. Turbulent flow chromatography in combination with tandem mass spectrometry has been applied for the first time as an efficient screening method in routine analysis of milk samples. Eight veterinary drugs, belonging to seven different classes were selected for this study. After developing and optimising the method, parameters such as linearity, repeatability, matrix effects and carry-over were studied. The screening method was then tested in the routine analysis of 12 raw milk samples. Even without internal standards, the linearity of the method was found to be good in the concentration range of 50 to 500 μg/L. Regarding repeatability, RSDs below 12% were obtained for all analytes, with only a few exceptions. The limits of detection were between 0.1 and 5.2 μg/L, far below the maximum residue levels for milk set by the EU regulations. While matrix effects—ion suppression or enhancement—are obtained for all the analytes the method has proved to be useful for screening purposes because of its sensitivity, linearity and repeatability. Furthermore, when performing the routine analysis of the raw milk samples, no false positive or negative results were obtained.     

A validated analytical method for the determination of perfluorinated compounds in surface-, sea- and sewagewater using liquid chromatography coupled to time-of-flight mass spectrometry

Perfluorinated compounds (PFCs), which are extensively used in a wide variety of applications because of their specific surfactant properties, have recently appeared as an important new class of global environmental pollutants. Quantitative analysis of PFCs in aqueous matrices remains, however, a challenging task. During this study, a new analytical method for the determination of 14 PFCs in surface-, sewage- and seawater was developed and validated. The target analytes were extracted using solid-phase extraction followed by liquid chromatography coupled to a time-of-flight mass spectrometer (LC–ToF-MS). The use of very narrow mass tolerance windows (<10 ppm) resulted in a highly selective MS-technique for the detection of PFCs in complex aqueous matrices. Validation of this analytical method in surface-, sewage- and seawater resulted in limits of quantification (LOQs) varying from 2 to 200 ng L⁻¹, satisfying recoveries (92–134%), and good linearity (R² = 0.99 for most analytes). Analysis of samples of the North Sea, the Scheldt estuary, and three harbours of the Belgian coastal region led to the detection of four different PFCs. Perfluorooctane sulfonate (PFOS) was found to be the most abundant PFC in levels up to 38.9 ng L⁻¹.     

Comparison of several extraction techniques for multiclass analysis of veterinary drugs in eggs using ultra-high pressure liquid chromatography-tandem mass spectrometry

This study compared four extraction methods for the simultaneous determination of tetracyclines, macrolides, quinolones, sulphonamides and anthelmintics (including benzimidazoles and avermectins) in eggs by ultra-high pressure liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). Solvent extraction, solid-phase extraction (SPE), matrix solid-phase dispersion (MSPD) and modified QuEChERS procedure were compared in terms of recovery and number of veterinary drugs extracted. The solvent extraction procedure with a clean-up step provided better results than the other tested procedures. The QuEChERS procedure was simpler and faster, but extracted fewer compounds than solvent extraction. MSPD did not extract tetracyclines and quinolones, whereas macrolides and tetracyclines were not extracted when SPE was applied. The solvent extraction procedure was validated, obtaining recoveries ranging from 60% (sulfaquinoxaline) to 119% (levamisole) with repeatability values (expressed as relative standard deviations, RSDs) lower than 20% at two concentration levels (10 and 100 μg kg⁻¹), except for erythromycin, emamectin and ivermectin that showed RSD values close to 25% at 10 μg kg⁻¹. Limits of quantification (LOQs) were always equal or lower than 5 μg kg⁻¹. Finally the method was applied to egg samples, and erythromycin, enrofloxacin, difloxacin, thiabendazole, emamectin and fenbendazole were detected in four samples.     

Development of fast screening methods for the analysis of veterinary drug residues in milk by liquid chromatography-triple quadrupole mass spectrometry

Two rapid multi-residue screening methods for the determination of 21 veterinary drugs in milk by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) have been developed and compared. For both methods, veterinary drugs were extracted from milk samples using a rapid extraction procedure based on the modification of the well-known QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) method, and no further clean-up steps were necessary. One screening method was based on the selection of a characteristic neutral loss or product ion of the various families of compounds, whereas another one was based on the choice of a selected reaction monitoring (SRM) for each compound. These methods were compared with regards to false negatives, cut-off values and the unreliability region. The total run time for both methods was 3 min, allowing quick selection of samples that contained veterinary drugs. Non-negative samples were re-analyzed by the UHPLC-MS/MS confirmation/quantification method, which consisted in the monitoring of two SRM for each compound. The methods were validated according to international guides. The proposed analytical methods allow for the identification and confirmation of the target veterinary drugs at trace levels employing quick analysis time.     

Evaluating polymer monolith in-tube solid-phase microextraction coupled to liquid chromatography/quadrupole time-of-flight mass spectrometry for reliable quantification and confirmation of quinolone antibacterials in edible animal food

A simple, rapid, and sensitive method is presented to determine seven trace quinolone antibacterials simultaneously in milk, egg, chicken and fish. This method is based on the combination of polymer monolith in-tube solid-phase microextraction with liquid chromatography and electrospray ionization quadrupole time-of-flight mass spectrometry (LC/ESI-QTOF-MS). LC/ESI-QTOF-MS offers the capability of unequivocal identification of target compounds from complex matrices, as well as the possibility of quantitation at low-level concentrations in real samples. The extraction was performed with a poly(methacrylic acid-ethylene glycol dimethacrylate) monolithic column. Under the optimized extraction conditions, good extraction efficiencies for the targets were obtained with no matrix interference in the subsequent LC/ESI-QTOF-MS. Good linearities were obtained for seven quinolones with the correlation coefficients (R) above 0.9951. The limits of detection (S/N = 3) for seven quinolones were found to be 0.3–1.2 ng/g in egg, 0.2–3.0 ng/mL in milk, 0.2–0.7 ng/g in chicken and 0.2–1.0 ng/g in fish. The recoveries of quinolones spiked in four different matrices ranged from 80.2 to 115.0%, with relative standard deviations less than 14.5%. The developed method was applied for the determination of quinolone residues in animal-producing food, and the positive samples were confirmed with high number of identification points (IPs) according to the IP system defined by the European Union (Commission Decision 2002/657/EC).     

High resolution orbitrap mass spectrometry in comparison with tandem mass spectrometry for confirmation of anabolic steroids in meat

A prominent trend which has been observed in recent years in the analysis of veterinary drugs and growth-promoting agents is the shift from target-oriented procedures, mainly based on liquid chromatography coupled to triple-quadrupole mass spectrometry (LC-QqQ-MS), towards accurate mass full scan MS (such as time of flight (ToF) and Fourier Transform (FT) Orbitrap MS). In this study the applicability of high resolution single-stage-Orbitrap-MS for confirmatory analysis of growth-promoting agents in meat was compared to that of a QqQ-MS. Validation according to CD 2002/657/EC demonstrated that steroid analysis based on Orbitrap MS, operating at a resolution of 50,000 FWHM, is indeed capable to compete with QqQ-MS in terms of selectivity/specificity, while providing excellent linearity (for most compounds >0.99) but somewhat inferior sensitivity. Indeed, CC_αs reached from 0.04–0.88 μg kg⁻¹ for the 34 anabolic steroids upon MS/MS detection, while upon Orbitrap MS detection a range of 0.07–2.50 μg kg⁻¹ was observed. Using QqQ-MS adequate precision was obtained since relative standard deviations, associated with the repeatability and intra-laboratory reproducibility, were below 20%. In the case of Orbitrap MS, for some compounds (i.e. some estrogens) this threshold was exceeded and thus poor precision was observed, which is possibly caused by the lack in sensitivity. Overall, it may be concluded that Orbitrap-MS offers an adequate performance in terms of linearity and precision but lacks in sensitivity for some of the compounds.     

Development and validation of a rapid assay based on liquid chromatography–tandem mass spectromtetry for determining macrolide antibiotic residues in eggs

A simple and rapid method able to determine residues of erythromycin A, tylosin and tilmicosin in whole eggs is presented here. The analytical protocol involves a one-step extraction followed by liquid chromatography (LC)–tandem mass spectrometry. Analytes were extracted from 1 g of egg spiked with an internal standard (josamycin) with acetonitrile. In terms of accuracy, matrix effect and ion signal stability, no extract cleanup was found to be necessary. After partial solvent removal, the final extract was injected into the LC column. Extraction was effective, since absolute recovery of the analyte in egg at their maximum residue limit (MRL) level was 85–102%. Estimated limits of quantification (S/N = 10) were 0.2–0.5 ng/g. Based on the EU Commission Decision 2002/657/EC, the method was in-house validated in terms of ruggedness, specificity, linearity, within-laboratory reproducibility, decision limit (CCα) and detection capability (CCβ). The within-laboratory reproducibility, expressed as RSD (n = 18 at the MRL levels), was not higher than 13%. After validation, a short study on EA depletion in eggs was conducted after administration of this drug to laying hens.     

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.     

Rapid automatic identification and quantification of compounds in complex matrices using comprehensive two-dimensional gas chromatography coupled to high resolution time-of-flight mass spectrometry with a peak sentinel tool

Comprehensive two-dimensional gas chromatography coupled to mass spectrometry (GC × GC–MS) is a powerful tool for comprehensive analysis of organic pollutants. In this study, we developed a powerful analytical method using GC × GC for rapid and accurate identification and quantification of compounds in environmental samples with complex matrices. Specifically, we have developed an automatic peak sentinel tool, T-SEN, with free programming software, R. The tool, which consists of a simple algorithm for on peak finding and peak shape identification, allows rapid screening of target compounds, even for large data sets from GC × GC coupled to high resolution time of flight mass spectrometry (HRTOFMS). The software tool automatically assigns and quantifies compounds that are listed in user databases. T-SEN works on a typical 64 bit workstation, and the reference calculation speed is 10–20 min for approximately 170 compounds for peak finding (five ion count setting) and integration from 1–2 GB of sample data acquired by GC × GC–HRTOFMS. We analyzed and quantified 17 PCDD/F congeners and 24 PCB congeners in a crude lake sediment extract by both GC × GC coupled to quadrupole mass spectrometry (qMS) and GC × GC–HRTOFMS with T-SEN. While GC × GC–qMS with T-SEN resulted in false identification and inaccurate quantification, GC × GC–HRTOFMS with T-SEN provided correct identification and accurate quantification of compounds without sample pre-treatment. The differences between the values measured by GC × GC–HRTOFMS with T-SEN and the certified values for the certified reference material ranged from 7.3 to 36.9% for compounds with concentrations above the limit of quantification. False positives/negatives were not observed, except for when co-elution occurred. The technique of GC × GC–HRTOFMS in combination with T-SEN provides rapid and accurate screening and represents a powerful new approach for comprehensive analysis.     

Development and validation of a method for the detection and confirmation of biomarkers of exposure in human urine by means of restricted access material-liquid chromatography–tandem mass spectrometry

The present article describes the development and validation of a LC–MS/MS method for the determination and confirmation of biomarkers of exposure to different types of xenobiotics in human urine. The method combines the use of a restricted access material (RAM) coupled on-line to a LC–IT-MS system; in this way, a rapid and efficient matrix cleanup was achieved, reducing manual sample preparation to freezing and sample filtration. The ion trap (IT) mass spectrometry detector provided the selectivity, sensitivity and ruggedness needed for confirmatory purposes. The on-line RAM-LC–MS/MS method developed here has been validated as a quantitative confirmatory method according to the European Union (EU) Decision 2002/657/EC. The validation steps included the verification of linearity, repeatability, specificity, trueness/recovery, reproducibility, stability and ruggedness in fortified urine samples. Repeatability and within-laboratory reproducibility, measured as intraday and interday precisions, were evaluated at two concentration levels, being 12.7% or below at the concentration corresponding to the quantification limits. Matrix effects and non-targeted qualitative analyses were also evaluated in fortified urine samples. Decision limits (CC_α) and detection capabilities (CC_β) were in the range of 3.6–16.5 and 6.0–28.1 ng mL⁻¹ respectively. The results of the validation process revealed that the proposed method is suitable for reliable quantification and confirmation of biomarkers of exposure to xenobiotics in human urine at low ng mL⁻¹ levels. In addition, working in Data-Dependent Scan mode the proposed method can be used for the screening of these compounds in urine samples.     

Rapid determination of sulfonamides in milk samples using fluorescence spectroscopy and class modeling with n-way partial least squares

In this paper, a methodology to evaluate the probability of false non-compliance and false compliance for screening methods, which give first or second-order multivariate signals is proposed. For this task 120 samples of 6 different kinds of milk have been measured by excitation-emission fluorescence. The samples have been spiked with different amounts of three sulfonamides (sulfadiazine, sulfamerazine and sulfamethazine). These substances have been classified in group B1 (veterinary medicines and contaminants) of annex I of Directive 96/23/EC. The European Union (Commission Regulation EC no. 281/96) has set the maximum residue level (MRL) of total sulfonamides at 100 μg kg⁻¹ in muscle, liver, kidney and milk.The work shows that excitation-emission fluorescence together with the partial least squares class modeling (PLS-CM) procedure may be a suitable and cheap screening method for the total amount of sulfonamides in milk. Three models, PLS-CM, have been built, for the emission and excitation spectra (first-order signals) and for the excitation-emission matrices (second-order signals). In all the cases it reaches probabilities of false compliance below 5% as required by Decision 2002/657/EC.With the same flourescence signals, the total quantity of sulfonamide was calibrated using 2-PLS, 3-PLS and PARAFAC regressions. Using this quantitative approach, the capability of detection, CCβ, around the MRL has been estimated between 114.3 and 115.1 μg kg⁻¹ for a probability of false non-compliance and false compliance equal to 5%.     

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 3-monochloropropane-1,2-diol and acrylamide in food by gas chromatography-triple quadrupole mass spectrometry with coupled column separation

Both 3-monochloropropane-1,2-diol (3-MCPD) and acrylamide are contaminants found in heat-processed foods and their related products. A quantitative method was developed for the simultaneous determination of both contaminants in food by gas chromatography-triple quadrupole mass spectrometry (GC–MS/MS). The analytes were purified and extracted by the matrix solid-phase dispersion extraction (MSPDE) technique with Extrelut NT. A coupled column (a 3 m Innowax combined with a 30 m DB-5 ms) was developed to separate both compounds efficiently without derivatization. Triple quadrupole mass spectrometry in multiple reaction monitoring mode (MRM) was applied to suppress matrix interference and obtain good sensitivity in the determination of both analytes. The limit of detection (LOD) in the sample matrix was 5 μg kg⁻¹ for 3-MCPD or acrylamide. The average recoveries for 3-MCPD and acrylamide in different food matrices were 90.5–107% and 81.9–95.7%, respectively, with the intraday relative standard deviations (RSDs) of 5.6–13.5% and 5.3–13.4%, respectively. The interday RSDs were 6.1–12.6% for 3-MCPD and were 5.0–12.8% for acrylamide. Both contaminants were found in samples of bread, fried chips, fried instant noodles, soy sauce, and instant noodle flavoring. Neither 3-MCPD nor acrylamide was detected in the samples of dairy products (solid or liquid samples) and non-fried instant noodles.     

Improved compatibility of liquid chromatography with electrospray tandem mass spectrometry for tracing occurrence of barbital homologous residues in animal tissues

This work describes a liquid chromatography–tandem mass spectrometry (LC–MS/MS) procedure for multiplex screening, ultratrace quantification and reliable confirmation of barbital series residues in animal-derived food matrices. The method is developed based on a distinct dependency of the electrospray ionization (ESI) response of nine structural homologues on LC eluent properties and gas-phase ion chemistry during the ESI process. The “wrong-way-round” negative ionization aspect has been explored to optimize the compatibility of the hyphenated LC–MS/MS technique, which facilitates detection limits at 30–100-fold lower than 0.01 ppm without derivatization or post-column basification step. A mobile phase using methanol modified with 0.01% acetic acid is adopted to achieve an approximately 2–9-fold increase in signal-to-noise ratio over the results under suboptimal conditions. There is no significant differential matrix effects or deuterium isotope effects on chromatographic retention and ESI responsiveness at all levels across the different analyte–matrix pairs. Mean recoveries ranged from 79.6% (barbital) to 108% (secobarbital) at fortified levels of 0.5–20 ng/g within relative standard deviations less than 11%. Between-run repeatability and within-laboratory reproducibility were 3–11% and 5–13%, respectively. An ion ratio criterion for valid detection limit data for simultaneous screening of homologous multiresidues in complex sample matrices is proposed. The satisfactory applicability of the newly described procedure to 43 real samples including pork, poultry meat, swine liver, fish tissue and shrimp muscle demonstrated the LC–MS/MS technique with facile sample handling can serve as an attractive alternative analytical method accepted for regulatory purpose.     

Determination of chiral pharmaceuticals and illicit drugs in wastewater and sludge using microwave assisted extraction,solid-phase extraction and chiral liquid chromatography coupled with tandem mass spectrometry

This is the first study presenting a multi-residue method allowing for comprehensive analysis of several chiral pharmacologically active compounds (cPACs) including beta-blockers, antidepressants and amphetamines in wastewater and digested sludge at the enantiomeric level. Analysis of both the liquid and solid matrices within wastewater treatment is crucial to being able to carry out mass balance within these systems. The method developed comprises filtration, microwave assisted extraction and solid phase extraction followed by chiral liquid chromatography coupled with tandem mass spectrometry to analyse the enantiomers of 18 compounds within all three matrices. The method was successfully validated for 10 compounds within all three matrices (amphetamine, methamphetamine, MDMA, MDA, venlafaxine, desmethylvenlafaxine, citalopram, metoprolol, propranolol and sotalol), 7 compounds validated for the liquid matrices only (mirtazapine, salbutamol, fluoxetine, desmethylcitalopram, atenolol, ephedrine and pseudoephedrine) and 1 compound (alprenolol) passing the criteria for solid samples only. The method was then applied to wastewater samples; cPACs were found at concentration ranges in liquid matrices of: 1.7 ng L⁻¹ (metoprolol) – 1321 ng L⁻¹ (tramadol) in influent, <LOD (desmethylcitalopram and metoprolol) – 506 ng L⁻¹ in effluent, and in solid matrix digested sludge: 0.4 ng g⁻¹ (metoprolol) – 275 ng g⁻¹ (citalopram). Enantiomeric profiling revealed that studied compounds were present in analysed samples in non-racemic composition. Furthermore, enantiomeric composition of studied analytes differed in liquid and solid matrices. This demonstrates that not analysing the solid fraction of wastewater may lead to over-estimation of the removal rates of cPACs as well as possible misrepresentation of the enantiomeric fraction of the compounds as they leave the wastewater treatment plant. Consequently risks from cPACs entering the environment might be higher than anticipated.