Alida A. M. Stolker

Multi-residue screening of veterinary drugs in egg, fish and meat using high-resolution liquid chromatography accurate mass time-of-flight mass spectrometry

The last 2 years multi-compound methods are gaining ground as screening methods. In this study a high-resolution liquid chromatography combined with time-of-flight mass spectrometry (HRLC-ToF-MS) is tested for the screening of about 100 veterinary drugs in three matrices, meat, fish and egg. While the results are satisfactory for 70-90% of the veterinary drugs, a more efficient sample preparation or extract purification is required for quantitative analysis of all analytes in more difficult matrices like egg. The average mass measurement error of the ToF-MS for the veterinary drugs spiked at concentrations ranging from 4 to 400 microg/kg, is 3.0 ppm (median 2.5 ppm) with little difference between the three matrices, but slightly decreases with increasing concentration. The SigmaFit value, a new feature for isotope pattern matching, also decreases with increasing concentration and, in addition, shows an increase with increasing matrix complexity. While the average SigmaFit value is 0.04, the median is 0.01 indicating some high individual deviations. As with the mass measurement error, the highest deviations are found in those regions of the chromatogram where most compounds elute from the column, be it analytes or matrix compounds. The median repeatability of the method ranges from 8% to 15%, decreasing with increasing concentration, while the median reproducibility ranges from 15% to 20% with little difference between matrices and concentrations. The median accuracy is in between 70% and 100% with a few compounds showing higher values due to matrix interference. The squared regression coefficient is >0.99 for 92% of the compounds showing a good overall linearity for most compounds. The detection capability, CCbeta, is within 2 times the associated validation level for >90% of the compounds studied. By changing a few conditions in the analyses protocol and analysing a number of blank samples, it was determined that the method is robust as well as specific. Finally, an alternative validation strategy is proposed and tested for screening methods. While the results calculated for repeatability, within-lab reproducibility and CCbeta show a good comparison for the matrices meat and fish, and a reasonable comparison for the matrix egg, only 27 analyses are required to obtain these results versus 63 analysis in the traditional, 2002/657/EC, approach. This alternative is suggested as a cost-effective validation procedure for screening methods.

Generic sample preparation combined with high-resolution liquid chromatography–time-of-flight mass spectrometry for unification of urine screening in doping-control laboratories

A unification of doping-control screening procedures of prohibited small molecule substances—including stimulants, narcotics, steroids, β2-agonists and diuretics—is highly urgent in order to free resources for new classes such as banned proteins. Conceptually this may be achieved by the use of a combination of one gas chromatography–time-of-flight mass spectrometry method and one liquid chromatography–time-of-flight mass spectrometry method. In this work a quantitative screening method using high-resolution liquid chromatography in combination with accurate-mass time-of-flight mass spectrometry was developed and validated for determination of glucocorticosteroids, β2-agonists, thiazide diuretics, and narcotics and stimulants in urine. To enable the simultaneous isolation of all the compounds of interest and the necessary purification of the resulting extracts, a generic extraction and hydrolysis procedure was combined with a solid-phase extraction modified for these groups of compounds. All 56 compounds are determined using positive electrospray ionisation with the exception of the thiazide diuretics for which the best sensitivity was obtained by using negative electrospray ionisation. The results show that, with the exception of clenhexyl, procaterol, and reproterol, all compounds can be detected below the respective minimum required performance level and the results for linearity, repeatability, within-lab reproducibility, and accuracy show that the method can be used for quantitative screening. If qualitative screening is sufficient the instrumental analysis may be limited to positive ionisation, because all analytes including the thiazides can be detected at the respective minimum required levels in the positive mode. The results show that the application of accurate-mass time-of-flight mass spectrometry in combination with generic extraction and purification procedures is suitable for unification and expansion of the window of screening methods of doping laboratories. Moreover, the full-scan accurate-mass data sets obtained still allow retrospective examination for emerging doping agents, without re-analyzing the samples.

High sensitive multiresidue analysis of pharmaceuticals and antifungals in surface water using U-HPLC-Q-Exactive Orbitrap HRMS. Application to the Danube river basin on the Romanian territory

The occurrence of 67 pharmaceutical and antifungal residues in the Danube river on the Romanian territory was studied by using solid-phase extraction (SPE) and LC-Q Exactive Orbitrap high resolution MS in both full scan (FS) MS and targeted MS/MS modes. A single-laboratory validation procedure was carried out for the determination of 67 compounds in FSMS mode evaluating selectivity, sensitivity, linearity, precision and accuracy. The method showed satisfactory analytical performance. The evaluation of the recovery concluded that 75% of the compounds show recoveries between 85 and 115% and 10% of the compounds show recoveries between 85% and 65%. The level of detection was lower than 5 ng l(-1) for 66% of the compounds, between 5 and 10 ng l(-1) for 22% and between 10 and 25 ng l(-1) for 14% of the compounds. The coefficients of determination R(2) were higher than 0.99 for 79% of the compounds, over a linearity range of 2.5-50 ng l(-1). Targeted MS/MS analysis, performed in addition to the full scan acquisition was used for confirmatory purpose. Twenty samples of Danube water and three of the main tributaries were collected in May, July, August and October 2014. Analysis of the selected water samples revealed the occurrence of 23 compounds such as diclofenac, carbamazepine, sulfamethoxazole, tylosin, indomethacin, ketoprofen, piroxicam, together with antifungals like thiabendazole, and carbendazim. Carbamazepine was detected in 17 samples, the maximum concentration being 40 ng l(-1). The highest concentration reached was 166 ng l(-1) for diclofenac.

Ultrasonic or accelerated solvent extraction followed by U-HPLC-high mass accuracy MS for screening of pharmaceuticals and fungicides in soil and plant samples

Different veterinary pharmaceuticals are used in agricultural livestock becoming a source of environment contamination. Furthermore, no regulation exists for the concentration limits of pharmaceuticals in soil or water. Monitoring programs for environment contamination with pharmaceuticals are needed, requiring new sensitive and selective screening methods. The present study focuses on developing a method for the simultaneous scanning of forty-two compounds (pharmaceuticals, azole biocides and fungicides) in soil and plant material samples. For extraction purposes the use of ultrasonic assisted and accelerated solvent extraction (ASE) were compared. The extract was purified and concentrated by applying a solid phase extraction step followed by ultra-high-performance-chromatographic separation and accurate-mass spectrometric detection using Exacte Orbitrap technology (FWHM 50,000). The effects of the different extraction solvents and conditions on the extraction efficiency were tested. Although both extraction approaches are applicable the optimal extraction efficiency was obtained by applying accelerated solvent extraction using solvent mixtures containing acetone for soil and methanol for plant samples. An ASE process has been validated for the determination of selected pharmaceuticals and fungicides in soil and in plant material. The recoveries from soil samples were >70% for more than 68% of the compounds. The levels of detection were ≤10 μg kg(-1) for 93% of the compounds tested. The recoveries from plant material were >70% for 64% of the compounds tested. The levels of detection were ≤10 μg kg(-1) for 66% of the compounds. The developed method was used to screen soil and plant material collected throughout the Netherlands and oxytetracycline residues were detected.

Uptake of oxytetracycline, sulfamethoxazole and ketoconazole from fertilised soils by plants

This study was performed to investigate the potential for a set of two antibiotics and one antifungal compound to be taken up from the soil by plants. Plants are used for animal or human consumption, and so the measured concentrations in the plant material will be used to model potential human exposure to these compounds. The uptake by two types of plants (grass and watercress) from two types of soil was studied. The compounds used for these experiments were sulfamethoxazole, oxytetracycline and ketoconazole at concentrations of 5 and 10 mg kg−1 in the soil. The compounds of interest were extracted out of the plant matrix by applying accelerated solvent extraction. Analyses were carried out by a LC–MS/MS. From the results, it was concluded that the plant materials used for this study were able to take up sulfamethoxazole and ketoconazole when the soil was contaminated with these compounds at a concentration ranging from 5 to 10 mg kg−1. Sulfamethoxazole was detected in all samples, at levels ranging from 7 to 21 µ kg−1 for grass and 4 to 7.5 µ kg−1 for watercress. For ketoconazole, the results showed low absorption. Oxytetracycline was not detected in any sample. A partition-limited model approach was applied for the comparison of experimental and estimated data, and the relationship between physicochemical properties of the compounds and plant uptake was highlighted.

Determination of the stability of antibiotics in matrix and reference solutions using a straightforward procedure applying mass spectrometric detection

The stability of an antibiotic is a very important characteristic, especially in the field of antibiotic residue analysis. During method development or validation, the stability of the antibiotic has to be demonstrated no matter if the method is used for screening, confirmation, qualitative or quantitative analysis. A procedure for testing the stability of antibiotics in solutions and food samples using LC-MS/MS is described. The procedure is based on the assumption that the antibiotics are stable when stored at −70°C. Representative solutions or spiked samples containing the antibiotic were stored at the temperature to be tested (−18 or 4°C) and at −70°C. After a selected storing time samples were moved from the chosen storage temperature to −70°C. At the end of the study, all samples – per class of antibiotic – were analysed in one batch. By applying statistical models, it was finally concluded in which circumstances the antibiotic is stable. The stability of 60 antibiotics belonging to the classes of tetracyclines, sulphonamides, quinolones, penicillins, macrolides and aminoglycosides were tested. The stability of solutions containing tetracyclines and penicillins is only guaranteed for 3 months while stored at −18°C. Solutions of all other antibiotics tested are stable for at least 6 or 12 months when stored at 4°C. In muscle tissue stored at −18°C no severe degradation of the tested antibiotics was observed, with the exception of the penicillins. The stability data reported here are useful as a reference for laboratories carrying out validation studies of analytical methods for antibiotic (residue) detection. The data should save the time needed for long-term stability testing of solutions and samples.

Carry-over of veterinary drugs from medicated to non-medicated feeds in commercial feed manufacturing plants

Different compound feeds have to be manufactured in the same production line. As a consequence, traces of the first produced feed may remain in the production and get mixed with the next feed batches. This “carry-over” is unavoidable, and so non-medicated feed can be contaminated with veterinary drugs like antibiotics added to the previous batch of medicated feed. To monitor the carry-over of antibiotics in the Netherlands, 21 feed mills were visited and 140 samples of flushing feeds were collected and analysed for containing residues of antibiotics. Results show that 87% of all samples contain concentrations of antibiotics in the range of 0.1–154 mg/kg. It is expected that these levels – which are in the same range as previously found for the nowadays banned antimicrobial growth promoters (AMGPs) – have an effect on the occurrence of microbial resistance. Analysis of a second set of samples collected at four different feed mills directly after the production of oxytetracycline-medicated feed demonstrated that the first part of a flushing feed has much higher contamination than the last part of the batch. Furthermore, it was demonstrated that the carry-over percentage shows no correlation with the carry-over determined by one of the standard GMP+ procedures. These observations, unavoidable carry-over, inhomogeneous batches of feed with antibiotics and difficulties to predict the carry-over levels, together with the awareness of the increasing problem of microbial resistance, motivated the NEVEDI, association of Dutch Feed Producers, to announce that they will voluntarily stop the production of medicated feed in 2011. The alternatives for medicated feed are for example water or milk medication or the use of top-dressings at the farm. The consequences and possible new risks of carry-over at the farm are not completely clear yet.

Response to letter regarding “Evidence of natural occurence of the banned antibiotic chloramphenicol in herbs and grass”

We were surprised to read that the natural occurrence of chloramphenicol (CAP) was reported as long ago as 2003 by Hanekamp et al. [1]. We missed that information, probably because their findings were not published in a peer-reviewed journal. In our paper [2], we did not claim to be the first to suggest the natural occurrence of CAP; on the contrary, we included several references from earlier studies. We stated that our paper was the first to describe the detection of CAP in plant materials, sample material which had not been addressed before. Furthermore, there is an important issue which is not raised by Hanekamp et al. and that is the issue of ‘criteria for confirmation of the identity of a compound’. In EU legislation, viz. Commission Decision 2002/657/EC, it is described how to confirm the identity of a banned compound, such as CAP. The confirmation of the identity of a compound should comply with the identification points approach. A suitable way is the use of tandem mass-spectrometric detection in which two (specific) product ions are monitored and the ion ratios have to be within a predefined tolerance interval. Although Hanekamp et al. suggested the natural occurrence of CAP in 2003, it is also important to mention that their findings were not supported by confirmatory analysis and validation data. Theoretically, it cannot be excluded that the results published at that time should be regarded as false positives. In summary, in our opinion we were the first to publish fully confirmed non-compliant findings (according to 2002/657/EC) of the natural occurrence of CAP in plant materials. Nevertheless, we do think Hanekamp et al. have an interesting opinion about the established ‘tolerance levels’ of banned antibiotics and we appreciate their contribution.