Determination of rifampicin in human plasma and blood spots by high performance liquid chromatography with UV detection: A potential method for therapeutic drug monitoring
Introduction
Tuberculosis (TB), an infection caused by Mycobacterium tuberculosis currently infects approximately one third of the world's population. There are approximately eight million new cases each year worldwide, almost all of them preventable or treatable [1]. Incomplete treatment of TB is common and is usually a consequence of non-compliance with the therapeutic regime or an interrupted supply of drugs. Therapeutic drug monitoring (TDM) may provide a means of determining compliance, particularly in remote areas of developing countries. However, outpatient treatment of TB patients raises logistical obstacles for TDM. The convenience of non-hospital based blood sampling could be of immense benefit to the quality of treatment for these patients. Sampling for TDM can be undertaken by collecting blood on specimen collection cards similar to the Guthrie cards used to test for phenylketonuria in neonates. These cards do not require specific storage or transportation conditions and blood dried on the cards is considered the lowest risk by the International Air Transportation Association [2].
Rifampicin (RIF, Fig. 1) is an important first line drug prescribed throughout TB therapy [3], often as part of fixed dose combination (FDC) tablets, which may also contain isoniazid and pyrazinamide. Although FDCs simplify the prescribing process and encourage compliance [4], the absorption of RIF from these formulations may vary, especially in tablets containing isoniazid [5], and contribute to treatment failure. Treatment failure and the development of drug resistance may be attributed to non-compliance with the treatment regime, poor bioavailability of RIF in some preparations, including some FDCs as described; counterfeit preparations, or malabsorption of RIF. While directly observed therapy (DOT) may address the first issue, it cannot address treatment failure due to the latter two causes. Currently, plasma levels of RIF are not monitored routinely in TB patients but it is clear that this would be advantageous if a simple and effective quantitative test were available. A number of methods for the determination of RIF in plasma have been reported [6], [7], [8], [9], [10], [11], [12], [13]. These methods are characterised by lengthy sample preparation procedures [10], [12], non-ideal chromatographic retention parameters (low analyte capacity factor) [6], [7], [12] and poor selectivity in the presence of the major metabolite (desacetlyrifampicin (DRIF)) [7], [8], [10] and the degradant (RIF-quinone) [6], [7], [8], [9], [10], [12], [13].
In the present study, an optimised sample preparation technique for quantitative and reproducible recovery of RIF from human plasma is described. A validated HPLC method for subsequent quantification is reported and its suitability in the monitoring of RIF in dried blood on specimen collection cards is demonstrated. These improved techniques should aid the TDM of RIF in TB patients.
Section snippets
Chemicals and materials
RIF (95%, w/w), papaverine hydrochloride (≥99.0%, w/w) and sulindac (99.0%, w/w) were all obtained from Sigma–Aldrich (Dorset, UK). Rifabutin (96.3%, w/w) was obtained from the European directorate for the quality of medicines (EDQM, France). Desacetylrifampicin (DRIF) was kindly provided by Professor Mitchison (St. George's Hospital, London, UK). All solvents were of HPLC grade and were obtained from VWR Ltd. (Lutterworth, UK). Ammonium acetate was obtained from BDH (Lutterworth, UK) and
Specificity of analysis
Fig. 2a and b shows the chromatograms obtained for the analysis of RIF (tR = 10.9 min) at the LOQs in plasma and blood spots, respectively. To determine the resolution of RIF from DRIF, three different batches of blank plasma and whole blood were spiked with both RIF and DRIF and extracted as described. The resulting solutions, along with aqueous solutions containing RIF and DRIF were analysed by HPLC. In all studies, RIF was well resolved from its major metabolite, DRIF, with resolution factor
Discussion
TDM of RIF is not routinely carried out in TB patients. However, due to the problems with malabsorption and erratic compliance, such testing would be advantageous. There are many examples in the literature of the extraction and analysis of RIF in plasma [6], [7], [8], [9], [10], [11], [12], [13]. SPE using silica based reverse phase (RP) cartridges has been used to extract RIF from plasma [7], [8], [15] with reasonable success: recoveries ranged from 88 to 98% and linearity has been
Conclusions
The assay procedures described are suitable for the quantification of RIF in both plasma and blood spots. Linearity, accuracy, precision and resolution from major degradants and metabolites have been demonstrated and both methods are sensitive for the detection of therapeutic concentrations of RIF in both plasma and blood spots. Both methods afford high, consistent recovery of RIF. These methods could be used for therapeutic drug monitoring of rifampicin in TB patients to identify cases of RIF
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