Validation of a single liquid chromatography‐tandem mass spectrometry approach for oxytetracycline determination in bull plasma, seminal plasma and urine

Abstract Oxytetracycline is a broad‐spectrum antibiotic, which inhibits protein synthesis and is generally used for the treatment of pneumonia, shipping fever, leptospirosis and wound infections in cattle and swine. The present work proposes a novel liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for oxytetracycline quantification in bull plasma, seminal plasma and urine, requiring limited sample treatment before analysis. Extraction with trichloroacetic acid followed by dilution of the supernatant in mobile phase proved to be effective in all three matrices, allowing to rapidly process large batches of samples. Sharp and symmetrical peak shape was obtained using a BEH C18 reversed‐phase column in a chromatographic run of just 3.5 min. The mass spectrometer operated in positive electrospray ionization mode and monitored specific transitions for oxytetracycline (461.1 → 425.8) and the internal standard demeclocycline (465.0 → 447.6). The method was validated over concentration ranges suitable for field concentrations of oxytetracycline found in each matrix, showing good linearity during each day of testing (R 2 always >0.99), as also confirmed by analysis of variance (ANOVA) and lack‐of‐fit tests. Excellent accuracy and precision were demonstrated by calculated bias always within ±15% and CV% below 10% at all quality control (QC) levels in the three matrices. Matrix effect and recovery were investigated for both analytes, which showed consistent and comparable behaviour in each matrix. To our knowledge, this is the first validated approach for mass spectrometric determination of oxytetracycline in seminal plasma and urine. The method was successfully applied to samples collected during a pharmacokinetic study in bulls, allowing to assess the oxytetracycline concentration–time profile in plasma, seminal plasma and urine.

the supernatant in mobile phase proved to be effective in all three matrices, allowing to rapidly process large batches of samples. Sharp and symmetrical peak shape was obtained using a BEH C18 reversed-phase column in a chromatographic run of just 3.5 min. The mass spectrometer operated in positive electrospray ionization mode and monitored specific transitions for oxytetracycline (461.1 ! 425.8) and the internal standard demeclocycline (465.0 ! 447.6). The method was validated over concentration ranges suitable for field concentrations of oxytetracycline found in each matrix, showing good linearity during each day of testing (R 2 always >0.99), as also confirmed by analysis of variance (ANOVA) and lack-of-fit tests. Excellent accuracy and precision were demonstrated by calculated bias always within ±15% and CV% below 10% at all quality control (QC) levels in the three matrices. Matrix effect and recovery were investigated for both analytes, which showed consistent and comparable behaviour in each matrix. To our knowledge, this is the first validated approach for mass spectrometric determination of oxytetracycline in seminal plasma and urine. The method was successfully applied to samples collected during a pharmacokinetic study in bulls, allowing to assess the oxytetracycline concentration-time profile in plasma, seminal plasma and urine.

| INTRODUCTION
Tetracyclines (TCs) were first described in the scientific literature and commercialized with clinical success starting from the late 1940s. Due to their broad spectrum of antibacterial activity, easy availability, good treatment efficacy and low costs, TCs are now widely used in both veterinary and human medicine. 1 To this class belongs oxytetracycline (OTC; molecular formula C 22 H 24 N 2 O 9 , MW 460.4 g/mol), an antibiotic produced by fermentation of certain strains of Streptomyces rimosus. This compound is an effective protein synthesis inhibitor in Gram-positive and Gram-negative bacteria that targets the 30S ribosomal subunit and specifically impairs the enzyme-binding of aminoacyl-t-RNA to the ribosomal acceptor site. 2 OTC is generally used for the treatment of pneumonia, transport fever, leptospirosis and wound infections in cattle and pigs. 1 To study TCs' pharmacokinetic properties, selective and sensitive analytical methods allowing their quantification in different biological matrices are needed. Immunoassay approaches have been described 3 but with limited specificity and reproducibility. Liquid chromatography-tandem mass spectrometry (LC-MS/ MS) is considered the gold standard for drug measurement in biological samples, and several methods have been developed to quantify TCs in human and swine plasma, 4,5 honey, 6 muscle, 7 milk, 8 shrimps, 9 medicated feed 10 and manure. 11 However, OTC was successfully determined in plasma and seminal plasma by liquid chromatography coupled with photodiode array, 12 PDA 13 or UV 14 detectors. OTC quantification by mass spectrometry has been performed only in plasma. 15,16 To our knowledge, analytical approaches for its quantification in seminal plasma and urine by LC-MS/MS have not yet been described. The aim of this study was therefore the development and validation of a single method for OTC determination in plasma, seminal plasma or urine by LC-MS/MS. The approach was then applied to samples of three matrices collected during a pharmacokinetic study in bulls.

| Sample preparation
The same extraction procedure was applied to plasma, seminal plasma (diluted 1:10 in water) and urine (diluted 1:20 in water), adapting a previously described approach. 5

| Method validation
The method was validated in plasma, seminal plasma and urine using as reference the current European Medicines Agency guideline on bioanalytical method validation during three separated days of testing. 17 The following parameters were assessed: selectivity, linearity, sensitivity, accuracy, precision, extraction recovery, matrix effect and carry-over.  This is not only a single approach that can be applied to three different matrices for OTC quantification but is also, to our knowledge, The results of the validation, however, suggest that this did not affect the overall performances of the method in either of the two matrices.

| CONCLUSIONS
This work describes for the first time a single LC-MS/MS validated method for the quantification of OTC in bull plasma, seminal plasma and urine. We believe that this approach could be employed not only for research purposes but also in routine clinical laboratory testing involving a still effective and widely used antimicrobial as OTC.