Linda A.M. Stolker

Newly identified degradation products of ceftiofur and cephapirin impact the analytical approach for quantitative analysis of kidney

This paper describes our research on the degradation of ceftiofur and cephapirin at physiological temperatures in kidney extract and in alkaline and acidic solution, conditions that regularly occur during sample preparation. Degradation products were identified using LC-ToF/MS, NMR and microbiological techniques. Additionally kinetics of the degradation processes were studied. A slight instability of cephapirin and desfuroylceftiofur was observed at elevated temperatures. Ceftiofur and cephapirin degraded immediately and completely in an alkaline environment, resulting in inactive degradation products. Ceftiofur and cephapirin also degraded immediately and completely in kidney extract resulting in both formerly reported metabolites as well as not previously reported products. Our research shows that conditions often occurring during the analysis of ceftiofur or cephapirin result in rapid degradation of both compounds. From this it is concluded that underestimation of the determined amounts of ceftiofur and cephapirin is likely to occur. Therefore, a new approach is needed for the analysis of both compounds newly identified degradation products.

The analysis of animal faeces as a tool to monitor antibiotic usage

The analysis of antibiotics in animal faeces is important to obtain more insight in the possible formation of bacterial resistance in the animals׳ gut, to learn about the dissemination of antibiotics to the environment, to monitor trends in antibiotic usage and to detect the illegal and off-label use of antibiotics. To facilitate these studies a comprehensive method for the analysis of trace levels of 44 antibiotic compounds including tetracyclines, quinolones, macrolides and sulfonamides in animal faeces by liquid chromatography in combination with tandem mass spectrometric (LC-MS/MS) detection is reported. The method is fully validated according to European regulation and showed satisfactory quantitative performance according to the stringent criteria adopted, with the exception of some of the macrolide compounds, which can be analysed with somewhat high measurement uncertainty. A large survey was carried out monitoring swine and cattle faeces and the outcomes were striking. In 55% of the swines, originating from 80% of the swine farms and in 75% of the calves, originating from 95% of the cattle farms, antibiotics were detected. Oxytetracycline, doxycycline and sulfadiazine were the most detected antibiotics, followed by tetracycline, flumequine, lincomycin and tylosin. Over 34% of the faeces samples contained two or more different antibiotics with a maximum of eight. Possible explanations for these findings are given and the effects are discussed.

Discrimination of eight chloramphenicol isomers by liquid chromatography tandem mass spectrometry in order to investigate the natural occurrence of chloramphenicol

This paper describes the discrimination of eight different isomers of chloramphenicol (CAP), an antibiotic banned for use in food producing animals, by reversed phase and chiral liquid chromatography in combination with tandem mass spectrometric detection. Previously, by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) the presence of CAP was confirmed in some grass and herb samples collected on Mongolian pastures up to concentrations of 450 μg kg(-1). It was not possible to establish the cause of CAP residues which has initiated research on the natural occurrence of this drug. CAP occurs in the para-configuration and in the meta-configuration and contains two chiral centers thus eight different isomeric configurations exist, namely four (RR, SS, RS, SR) meta-stereoisomers and four para-stereoisomers. It is known that only RR-p-CAP has antimicrobial properties. To find out if the CAP detected in the plant material samples is the active configuration, a high resolution reversed phase LC-MS/MS system was tested for its ability to separate the different isomers. This system proved to be able to discriminate between some isomers, but not between RR-p-CAP and SS-p-CAP, also called dextramycin. Despite a detailed elucidation of the product ions and the fragmentation patterns of all isomers, MS/MS did not add sufficient specificity for full discrimination of the isomers. Therefore a chiral liquid chromatographic separation with MS/MS detection that is able to distinguish all isomers was developed and finally the isomeric ratio of non-compliant plant material samples and some CAP formulations was determined using this system. This showed that Mongolian grass and herb samples only contain the biological active isomer of CAP as do the obtained formulations. Therefore the CAP present in the plant material might origin from the production by soil organisms or from a manufactured source.

Influence of natural organic matter on the screening of pharmaceuticals in water by using liquid chromatography with full scan mass spectrometry

The influence of natural organic matter on the screening of pharmaceuticals in water was determined by using high resolution liquid chromatography (HRLC) combined with full scan mass spectrometry (MS) techniques like time of flight (ToF) or Orbitrap MS. Water samples containing different amount of natural organic matter (NOM) and residues of a set of 11 pharmaceuticals were analyzed by using Exactive Orbitrap™ LC-MS. The samples were screened for residues of pharmaceuticals belonging to different classes like benzimidazoles, macrolides, penicillins, quinolones, sulfonamides, tetracyclines, tranquillizers, non-steroidal anti-inflammatory drugs (NSAIDs), anti-epileptics and lipid regulators. The method characteristics were established over a concentration range of 0.1-500 μg L(-1). The 11 pharmaceuticals were added to two effluent and two influent water samples. The NOM concentration within the samples ranged from 2 to 8 mg L(-1) of dissolved organic carbon. The HRLC-Exactive Orbitrap™ LC-MS system was set at a resolution of 50,000 (FWHM) and this selection was found sufficient for the detection of the list of pharmaceuticals. With this resolution setting, accurate mass measurements with errors below 2 ppm were found, despite of the NOM concentration of the different types of water samples. The linearities were acceptable with correlation coefficients greater than 0.95 for 30 of the 51 measured linearities. The limit of detection varies between 0.1 μg L(-1)and 100 μg L(-1). It was demonstrated that sensitivity could be affected by matrix constituents in both directions of signal reduction or enhancement. Finally it was concluded that with direct shoot method used (no sample pretreatment) all compounds, were detected but LODs depend on matrix-analyte-concentration combination. No direct relation was observed between NOM concentration and method characteristics. For accurate quantification the use of internal standards and/or sample clean-up is necessary. The direct shoot method is only applicable for qualitative screening purposes. The use of full scan MS makes it possible to search for unknown contaminants. With the use of adequate software and a database containing more than 50,000 entries a tool is available to search for unknowns.

Gynaecomastia linked to the intake of a herbal supplement fortified with diethylstilbestrol.

This study reports the findings of a supplement marketed on the Internet for prostate problems. The supplement was orally taken by a 60-year-old man with divergent hormonal levels and who was surgically treated for gynaecomastia: development of abnormally large mammary glands in males. The supplement showed a strong effect in a yeast oestrogen bioassay, expressing a yeast-enhanced green fluorescent protein (yEGFP) upon exposure to oestrogens. Using both nuclear magnetic resonance (NMR) and a gradient liquid chromatographic time-of-flight mass spectrometric (LC/TOF-MS) method, the response was shown to be caused by very high levels of diethylstilbestrol, known for causing gynaecomastia. The gynaecomastia was most probably caused by this orally taken ‘natural’ herbal supplement, as the patients hormonal levels also returned to normal again when stopping the use of it. This case demonstrates that physicians need to be aware of the use of supplements with illegal components that may be responsible for unwanted side-effects.

Quantitative trace analysis of eight chloramphenicol isomers in urine by chiral liquid chromatography coupled to tandem mass spectrometry.

Chloramphenicol is a broad-spectrum antibiotic with, apart from its human medicinal use, veterinary abuse in all major food-producing animals. Chloramphenicol occurs in four stereoisomers (all para-nitro substituted) and furthermore four meta-nitro analogs of chloramphenicol exist. In this paper these are referred to as eight chloramphenicol isomers. According to EU regulations an analytical method should be able to discriminate the analyte from interfering substances that might be present in the sample, including isomers. For the first time a quantitative method for the analysis of trace levels of eight chloramphenicol isomers in urine by chiral liquid chromatography in combination with tandem mass spectrometric detection is reported. The separation of the isomers on the analytical column, the clean-up of urine and the selectivity of the monitored product ions turned out to be critical parameters. To obtain reproducible retention isocratic elution on a chiral AGP column was applied. For urine samples matrix compounds present in the final extract caused decreased retention of the isomers on the chiral stationary phase and a lack of chromatographic resolution. Therefore an extended clean-up procedure that combines solid phase extraction and liquid-liquid extraction had to be developed. The final method was fully validated and showed satisfactory performance for all isomers with decision limits (CCα) ranging from 0.005 to 0.03 μg L(-1) and within-laboratory reproducibility of all isomers below 20% at the minimum required performance limit level of 0.3 μg L(-1).

Are antibiotic screening approaches sufficiently adequate? A proficiency test

A proficiency test including the screening analysis of antibiotics in beef using cryogenicly minced materials was organized by RIKILT in 2009. The test included blank beef samples and beef samples spiked with either flumequine or a combination of lincomycin and spectinomycin around the maximum residue limits [1]. The suitability of the materials was demonstrated with a homogeneity and a stability study. This study showed that cryogenically minced spiked muscle material is suited for proficiency tests aiming at the screening and the confirmatory analysis. Of the 26 participants, 23 carried out their in-house screening approach involving microbial, biochemical or instrumental methods, or a combination of these to cover the broad range of antibiotic groups. The false negative rate was 73% for microbial methods, 50% for biochemical and 22% for instrumental methods. These results indicate that substantial effort is needed to improve screening approaches and that more regular proficiency tests are needed to reveal the shortcomings in the currently applied screening methods.

Assessment of liquid chromatography–tandem mass spectrometry approaches for the analysis of ceftiofur metabolites in poultry muscle

The use of cephalosporin antibiotics in veterinary practice is likely to play an important role in the development of β-lactam-resistant bacteria. To detect off-label cephalosporin antibiotic usage, an analytical method is needed that, besides the native compound, also detects their active metabolites. In this paper, the applicability of three approaches for the quantitative analysis of ceftiofur using LC–MS/MS is assessed, viz. (A) analysis of ceftiofur, desfuroylceftiofur and/or desfuroylceftiofur cystein disulfide, (B) derivatisation of ceftiofur metabolites to desfuroylceftiofur acetamide and (C) chemical hydrolysis using ammonia, to produce a marker compound for ceftiofur. We found that approach A was not suited for quantitative analysis of total ceftiofur concentration or for effectively detecting off-label use of ceftiofur. Approach B resulted in adequate quantitative results, but was considered a single compound method because it depends on cleavage of a thioester group, which is present in only a limited number of cephalosporin antibiotics. Approach C showed adequate quantitative results but, in contrast to approach B, it is applicable to a range of cephalosporin antibiotics. Therefore, it is applicable as a broad quantitative screening of cephalosporin compounds in poultry tissue samples to indicate off-label use of cephalosporins in poultry breeding. Based on this study, it was concluded that approach C is the most suitable to detect off-label use of a range of cephalosporin antibiotics.