Development and validation of chromatographic methods for screening and subsequent quantification of suspected illegal antimicrobial drugs encountered on the Belgian market

Abstract

Estimations, made by the World Health Organization (WHO), state that 10% of the medical products in low-and middle-income countries are substandard or falsified (SF). Among them, antibiotics and antimalarials are the most commonly reported since 2013. Besides the fact that falsification is a crime, the worldwide use of poor quality antimicrobials could result in treatment failures, stronger antimicrobial resistance and even the promotion of the emergence of superbugs. Therefore, simple and accurate analytical methods are necessary, which are capable to detect and quantify a wide range of antimicrobials in suspected illegal products. In this work, a screening and a quantification method using ultra-high performance liquid chromatography with tandem mass spectrometry (UHPLC-MS 2) and diode array detection (UHPLC-DAD), respectively were developed and validated. These methods could be used for routine analysis and enable a more in-depth characterization of SF-antimicrobials. According to their popularity as SF-antimicrobials, 31 antibiotics, 3 antibacterial agents, 1 antifungal agent and 1 beta-lactamase inhibitor, covering eleven different antibacterial classes, were selected. The UHPLC-MS 2 screening method with gradient elution is able to selectively detect these 36 compounds within 18 min (including wash and equilibration step). It was validated for sensitivity, selectivity and matrix effects. Within an analysis time of 32 min, the UHPLC-DAD method could quantify 32 compounds (4 showed insufficient UV absorbance) and resulted in sufficient selectivity, necessary since some SF-antimicrobials may include more than one antimicrobial component. This quantification method was validated for the positive hits found during screening tests of suspected illegal samples. This resulted in a validation set of 11 antimicrobials and 1 betalactamase inhibitor. The ''total error'' approach in accordance with the validation requirements of ISO-17025 was employed for the validation. 57 real-life illegal samples, seized by inspectors from the Belgium Federal Agency for Medicinal and Health Products (FAMHP), were analyzed using the two described methods. About half of them were not compliant and some samples that contained clavulanic acid showed a serious reduction in the amount of this molecule (in one sample only 14% of the claimed dosage was found). These quality issues might be attributed to either poor manufacturing, storage or transportation conditions.

Evaluation of impurities and dissolution profiles of illegal antimicrobial drugs encountered in Belgium

“…Quantifications of co‐trimoxazole and amoxicillin‐containing samples were performed on an Aquity UPLC system (Waters, Milford, CT, USA) consisting of a binary solvent manager, a sample manager and a DAD detector following the method developed by Tie et al Data integration and analysis were performed with Empower 3 Citrix software.…”

“…Individual stock solutions (1 mg/mL) were prepared by dissolving the standard in the corresponding solvent (Table ) . Calibration standards were prepared by diluting the respective stock solutions as indicated in Table .…”

“…Besides the global efforts and directive regulations against SF medical products, local laboratories also need efficient analytical methods to survey the (illegal) drug market. The majority of papers related to SF antimicrobials deal with the determination of APIs using liquid chromatography‐ultra violet (LC−UV) and/or liquid chromatography−mass spectrometry (LC–MS) . However, other parameters may also have a tremendous influence on drug efficacy and toxicity.…”

Substandard and falsified antimicrobials: A potential biohazard in disguise?

“…2 The past decade has been marked with numerous actions that were taken to prevent SF medical products from infiltrating legal supply chains. [5][6][7][8][9][10] The majority of these analytical findings is focused on the chemical characterization, i.e. This directive provides a preventive system for falsified medicines, e.g.…”

“…4 In addition to the regulatory efforts, many efficient analytical methods were also developed for the analysis and risk evaluation of SF medical products. [5][6][7][8][9][10] The majority of these analytical findings is focused on the chemical characterization, i.e. presence of the active pharmaceutical ingredients (APIs) and impurities.…”