LC-MS residue analysis of antibiotics : what selectivity is adequate?

In residue analysis of antibiotics quantitative and qualitative aspects are involved in declaring a sample non-compliant. The quantitative aspect regards the determination of the amount of the compound present in the sample. Validation procedures are available to determine the uncertainty of this result, which is taken into account in the decision making process. The qualitative aspect regards the confirmation of the identity of the compound present. In this, selectivity is the main parameter which is defined as the ability of a method to discriminate the analyte being measured from other substances. A trend observed in residue analysis is towards more generic methods for the detection of a broad range of compounds in a single run. As a result, by definition, selectivity is compromised. Procedures to determine the uncertainty of the qualitative aspect are lacking and, as a result, whether or not a method is adequately selective is a matter of experts’ judgment. In this thesis a method is presented for grading selectivity of methods using liquid chromatography coupled to tandem mass spectrometry. Based on the outcome it can be stated if selectivity is adequate and thus if a confirmatory result stands strong when challenged in a court case. If selectivity is found inadequate, additional measures can be taken like the selection of another product ion or the use of a third product ion to obtain adequate selectivity. Furthermore, two examples of analyses are presented in which selectivity plays an important role. First, the analysis of the banned antibiotic chloramphenicol (CAP). CAP contains two chiral centers and the nitro-group can either be para- or meta-substituted. Therefore, eight different isomers of CAP occur of which only RR-p-CAP is antimicrobially active. In the analysis of CAP, extreme selectivity is needed to distinguish the antimicrobially active compound from its inactive isomers. A method applying chiral liquid chromatography with tandem mass spectrometry was developed to discriminate antimicrobially active CAP for its inactive isomers. Also the research for the possible natural occurrence of this drug is presented. It is shown that CAP can be produced in unamended soil by Streptomyces venezuelaein appreciable amounts and that crops can take up CAP from soils. Therefore, it is concluded that CAP can occur in crops and animal feed due to its natural production by soil bacteria. Second, the development of a multi-ß-lactam method is presented. In this method a derivatization is applied to be able to effectively detect off-label ceftiofur use. In this selectivity is intentionally compromised and no unequivocal confirmation can be carried out using this method. The developed method is applicable to a wide range of ß-lactam antibiotics including penicillins, cephalosporins and carbapenems and is the best method available today for effective monitoring of off-label ß-lactam usage in poultry breeding. </p

A study of the origin of chloramphenicol isomers in honey

Due to the unexpected detection of chloramphenicol isomer residues in honey, we have studied the hypothesis of unauthorized or unintended use of unregistered veterinary drug preparations. First, we have investigated honey samples in which a discrepancy was observed between the results of the immunological screening methods and the confirmatory liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. In all samples, previously identified to be contaminated with the banned antibiotic chloramphenicol according to LC-MS/MS only, the presence of dextramycin (SS-para isomer of chloramphenicol) was detected by chiral LC-MS/MS. The source of dextramycin in honey was investigated by studying the preparations utilized in apiaries from which the above non-compliant honey samples have been received. In all these preparations (beehive strips applied against the mite Varroa destructor) chloramphenicol was detected in the concentrations ranging from 33 to 34,400 μg kg-1. Chiral LC-MS/MS demonstrated the presence of chloramphenicol and dextramycin in different ratios, and it was concluded that these preparations can be the source of chloramphenicol and dextramycin residues in honey. These preparations were of foreign production and are not officially registered in accordance with current legislation

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

Застосування нового УЕРХ-МС/МС мультиметоду визначення залишкових кількостей антимікробних препаратів для контролю безпеки меду

The article presents the results of the developed UPLC-MS/MS multimethod application for quantitative determination of different groups of antimicrobials residues in honey. According to the European RASFF system, despite the ban on the use of antimicrobials in beekeeping, there are still many reports of antibiotics detection in honey. Today there are no harmonized global requirements for honey safety, which are to be mandatory for honey exports and imports worldwide, and thus the instrumental abilities of the control laboratory (e.g., CCα and CCβ) often are the main criteria of honey batches compliance. The development and use of multianalyte methods have obvious advantages, viz. shortened time of the analysis, reduced consumption of necessary consumables and reagents, the decrease of utilization time of laboratory equipment etc. Main advantages of our developed multimethod are the simplicity, rapidity and cost-effectiveness, since the analysis is performed in one sample preparation without the use of filtration or solid-phase extraction. This method provides selective determination of target analytes with LODs in the ranges from 0.05&nbsp;μg/kg for chloramphenicol to 2&nbsp;μg/kg for sulfanilamide and tetracycline; and it exceeds the requirements of the MRPLs or regulatory limits provided with a sufficient level of precision of the method and a high percentage of recoveries, which is in accordance with the requirements of Decision 2002/657/EC. The technique was tested in the analysis of natively contaminated and fortified honey samples, as well as in the interlaboratory studies. During the period of 2018-2019, 809 export lots of honey were analyzed for safety, as well as 946 samples were tested during honey incoming control using our developed multimethod. The most frequently investigated analytes in honey were metronidazole and chloramphenicol, viz. 68% and 52% of the analyzed samples, respectively. This may be related to the risks of unauthorized use by apiaries of human medicine drugs as well as the counterfeit veterinary preparations containing these antibiotics, but which are not declared. With regard to chloramphenicol, the main reason for detecting its residues in honey today is the illegal use of preparations containing a mixture of chloramphenicol and its SS-para-stereoisomer dextramycin. The isomer has no therapeutic effect, but has repeatedly been found in counterfeit preparations for bees and, as a consequence, in honey. It was found that among the samples for incoming honey control 297 contained target analytes at the level above the detection limit of the method. Most often, viz. in 28% of cases, the regulatory limit (3&nbsp;μg/kg) was exceeded for sulfathiazole, which may be used to treat and prevent American foulbrood, one of the most dangerous bee diseases. In few cases, the analyzed homogenized honey samples contained simultaneously five or six target analytes at concentration levels above their LOD. This clearly demonstrates the fact that Ukrainian beekeepers occasionally use unregistered antimicrobials for the treatment and/or prevention of bee diseases. The data obtained do not claim to be highly representative, since they are based on the analysis of honey samples directly from the customers and not obtained as a result of monitoring by random sampling.У статті представлено результати впровадження розробленого УЕРХ-МС/МС мультиметоду для кількісного визначення залишків різних груп антимікробних препаратів у меді. Основною перевагою цього методу є простота, експресність та економічна ефективність, оскільки аналіз проводиться за однієї пробопідготовки. Методику апробовано при аналізі нативно контамінованих та фортифікованих зразків меду, а також при проведенні міжлабораторних досліджень. За період 2018-2019 років з використанням розробленого нами мультиметоду було проаналізовано 809 експортних партій меду за показниками безпеки, а також 946 зразків – для вхідного контролю сировини. Найчастіше проводились дослідження меду на вміст залишків метронідазолу та хлорамфеніколу, а саме – 68&nbsp;% та 52&nbsp;% аналізованих зразків, відповідно. Виявлено, що серед зразків вхідного контролю меду 297 містили цільові аналіти на рівні, вищому за межу виявлення методу. Це чітко свідчить про непоодинокі факти використання українськими бджолярами незареєстрованих антимікробних препаратів для лікування та/або профілактики хвороб бджіл. Отримані дані не претендують на високу репрезентативність, оскільки базуються на аналізі зразків меду безпосередньо від замовників, а не отримані внаслідок моніторингу шляхом випадкового вибору проб