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Abstract INTRODUCTION: Antihypertensives (AHT) is a class of drugs that are used to treat hypertension. The compliance to antihypertensive therapy is critical to achieve adequate blood pressure and avoid complications such as stroke or myocardial infarction. Therapeutic drug monitoring (TDM) is a reliable approach to assess AHT compliance, moreover TDM helps to distinguish non-compliance from patients with true resistant hypertension.
OBJECTIVES: The aim of this work, based on the physician’s requests, was to develop, validate and implement method for quantification of 21 AHT in serum samples suitable for use in routine laboratory practice.
METHODS: Methods for quantification of AHT in serum are based on reversed phase liquid chromatography and ESI+ ionization. Sample preparation requires single step of protein precipitation. Methods development and validation were performed on Agilent 6460 Triple Quadrupole LC/MS System. Compounds were separated on an Agilent Poroshell 120 2.7 µm, 2.1 x 100 mm HPLC Column using gradient elution (0.1 % formic acid in water as mobile phase A and methanol or acetonitrile as mobile phase B). For determination of analytical parameters patient samples, commercially available materials (Chromsystems) and inhouse spiked calibrators and controls were used. For quantification of serum concentrations isotopically labeled internal standards were used.
RESULTS: Two LC-MS/MS methods for 14 AHT have been developed and validated so far. The first method is dedicated for 7 analytes – Irbesartan, Losartan, Telmisartan, Valsartan, Amlodipine, Nitrendipine, Indapamide. The second method is dedicated for 7 analytes – Acebutolol, Bisoprolol, Betaxolol, Carvedilol, Atenolol, Nebivolol, Metoprolol. For both methods the intra-assay and inter-assay coefficients of variation (CVs) were below 10 %. Recoveries, determined by standard addition, at normal and pathological concentration levels ranged between ±15 %. Methods provided linearities in the relevant concentration range (5 - 1000 µg/l, for LLOQs CVs were below 10 %. Stability of inhouse prepared calibrators is being tested at different storage conditions, -20 ˚C and -80 ˚C. As far, after 2 months, all analytes are stable and there is no significant difference observed between calibrators stored at -20 ˚C and calibrators stored at -80 ˚C (more data will be collected).
CONCLUSION: Two methods for determination of 14 AHT have been developed so far. The analytical performance of the developed methods is satisfactory. Both methods fulfilled requirements for validation parameters for all analytes. Presented approach with the difference only in mobile phase B and gradients allows for easy and fast changing between methods with minimal manual intervention and provides optimized workflows in daily laboratory routine practice. |
= Discovery stage.
= Translation stage.
= Clinically available.
