Abstract
A simple and reliable high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for the determination of colistin A and colistin B in human plasma was developed and validated. Clarithromycin was used as an internal standard (IS). Plasma extraction was performed using C-18 cartridges and methanol containing 0.1% formic acid. Analysis was performed using Atlantis dC18 (2.1 × 100 mm, 3 μm) column at room temperature and a mobile phase of 0.2% formic acid in acetonitrile and water (50:50, v:v), delivered at a flow rate of 0.2 ml/minute. Eluent was detected in the positive ion mode using electrospray ionization at the following transitions of mass to charge (m/z): colistin A, 585.6 → 101.4; colistin B, 578.7 → 101.3; and IS, 748.6 → 158.4. No interference by components of blank plasma or commonly used drugs was observed. The relationship between colistin A colistin B concentrations and their corresponding peak height ratios to the IS was linear over the range of 0.05 - 10 μg/ml. Inter-day coefficient of variation and bias were, respectively, ≤11.5% and −3.0 to 6.0 for colistin A and ≤9.9 and −4.7 to 3.0 for colistin B. Mean extraction recovery of colistin A, colistin B, and the IS were 97%, 94%, and 97%, respectively. The method was applied to assess the stability of colistin A and colistin B in processed samples (24 hr. at room temperature, 48 hours at −20°C) and unprocessed samples (24 hr. at room temperature, 8 weeks at −20°C) and after three cycles of freeze and thaw found to be ≥87%.
Highlights
Colistin A and colistin B are part of polmyxcin E, together with other polypeptides
We report a simple, precise, and rapid LCMS/MS assay for measurement of colistin A and colistin B in human plasma using clarithromycin as an internal standard in order to produce reliable measurement of both colistin A and colistin B
Five aliquots of each quality control (QC) sample were extracted and immediately analyzed, five aliquots were allowed to stand on the bench-top for 24 hours at room temperature before being processed and analyzed, five aliquots were stored at −20 ̊C for eight weeks before being processed and analyzed, and five aliquots were processed and stored at room temperature for 24 hours or 48 hours at −20 ̊C before analysis
Summary
Colistin A and colistin B are part of polmyxcin E, together with other polypeptides They constitute more than 85% of polymyxcin E by weight. Colistin is one of the oldest antibiotics but is used as last-line treatment for infections caused by multi-drug-resistant gram-negative bacteria [2] [3] It is commercially available as colistin methanesulfonate for intravenous administration and as colistin sulfate for oral use. Several analytical methods are reported for measurement of colistin in various biological matrices, including human plasma and urine [5]-[17] These include thin layer chromatography [6], isotachophoresis [7], capillary electrophoresis [8], and high performance liquid chromatography with ultraviolet [9] or fluorescence detection after dervatization with 9-fluorenylmethyl chloroformate [10] [11] [12] [13] [14]. We report a simple, precise, and rapid LCMS/MS assay for measurement of colistin A and colistin B in human plasma using clarithromycin as an internal standard in order to produce reliable measurement of both colistin A and colistin B
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