LC-MS/MS Method for Determination of Colistin in Human Plasma: Validation and Stability Studies

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%.


Introduction
Colistin A and colistin B are part of polmyxcin E, together with other polypeptides. Commonly, they constitute more than 85% of polymyxcin E by weight.
They have similar chemical structure with colistin A having one extra methylene moiety [1]. 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. Pharmacokinetic of colistin is very variable and it has narrow therapeutic window [4], necessitating careful drug monitoring.
In this paper, 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.

Instrument
Sep-Pak classic C-18 cartridges (Waters Corporation, Milford, MA, USA) was used for analyte extraction from plasma samples. Chromatography was performed on an LCMS/MS system consisting of Water Alliance 2695 separation module (Waters Associates, Inc Milford, MA, USA) for solvent delivery and sample introduction, and a Micromass Quattro micro API bench-top triple quadruple mass spectrometer (Micromass, Manchester, UK) interfaced with a Z-spray ESI source as detector. Analysis was performed using reversed phase Atlantis dC 18 column (2.1 × 100 mm, 3 µm) protected by guard column Symmetry C 18 (3.9 × 20 mm, 5 µm) at room temperature. Mass Lynx software (Ver 4.0) working under Microsoft Window XP professional environment was used to control the instrument, data acquisition, peak integration, peak smoothing, and signal-tonoise ratio measurement.

Chromatographic Conditions
The mobile phase consisted of 0.2% formic acid in acetonitrile and water (50:50, v:v). It was degassed before use and delivered at a flow rate of 0.2 ml/minute at ambient temperature with a run time of 4 minutes. The electrospray ionization source was operated in the positive-ion mode at a capillary voltage of 3.5 kV and a cone voltage of 35 V. Nitrogen was used as nebulizing and desolvation gas at a flow rate of 50 and 600 L/hr, respectively. Argon was used as the collision gas at a pressure of 1.28 × 10 −3 mbar. The optimum collision energy for colistin and the IS was 25 eV. The ion source and the desolvation temperatures were maintained at 120˚C and 350˚C, respectively. Colistin A, colistin B, and the IS were detected in the positive ion using multiple reactions monitoring mode at the following transitions of mass to charge (m/z): 585. 6 → 101.4, 578.7 → 101.3, and 748.6 → 158.4, respectively.

Sample Preparation
Aliquots of 1 ml blank plasma, calibration curve, or QC samples were allowed to equilibrate to room temperature. To each tube, 150 µl of the IS 0.1 µg/ml solution was added and vortexed for 20 seconds. Before loading samples, Sep-Pak C-18 cartridges were conditioned with 1 ml methanol followed by 2 ml water.
Then the sample (1 ml) was loaded followed by 1 ml water. Samples were eluted with 1 ml methanol containing 0.1% formic acid. The eluates were then evaporated to dryness under a gentle nitrogen stream in a heating block at 45˚C, the residues were reconstituted in 100 µl of 0.1% formic acid in water, transferred into auto-sampler vial, and 10 µl were injected into the LC-MS/MS system.

Stability Studies Int. J. Analytical Mass Spectrometry and Chromatography
for stability studies. Five aliquots of each QC sample were extracted and immediately analyzed (baseline), five aliquots were allowed to stand on the bench-top for 24 hours at room temperature before being processed and analyzed (counter stability, 24 hours at room temperature), five aliquots were stored at −20˚C for eight weeks before being processed and analyzed (long term freezer storage stability), and five aliquots were processed and stored at room temperature for 24 hours or 48 hours at −20˚C before analysis (autosampler stability). Finally, fifteen aliquots of each QC sample were stored at −20˚C for 24 hours, they were then left to thaw completely at room temperature unassisted. Five aliquots of each QC sample were extracted and analyzed and the rest returned to −20˚C for another 24 hours. The cycle was repeated three times (freeze-thaw stability).

Method Validation
The method was validated according to standard procedures described in the US Food and Drug Administration bioanalytical method validation guidance [18].
The validation parameter included specificity, linearity, accuracy, precision, recovery, and stability.

Specificity
We screened six blank plasma samples and seven commonly used drugs including aspirin, acetaminophen, ranitidine, nicotinic acid, caffeine, diclofenac and omeprazole for interferences with colistin A and colistin B, or IS. No drug or endogenous component co-eluted with colistin A, colistin B or the IS. Figure   2(A) depicts a representative chromatogram of drug free human plasma (blank) used in preparation of standards and quality control samples. Figure 2

Linearity and Limit of Detection and Quantification
Linearity of the assay was evaluated by analyzing a series of standard mixtures containing colistin A and colistin B in human plasma at nine concentrations over the range of 0.05 -10.0 μg/ml. Corresponding peak height ratios to the IS and concentrations were subjected to regression analysis. Mean equations obtained were y = 0.0979x − 0.0029, r 2 = 0.9901 (n = 10) and y = 0.0461x − 0.0012, r 2 = 0.9921, (n = 10) for colistin A and colistin B, respectively. The suitability of the calibration curves was confirmed by back calculating the concentration of colistin A and colistin B in human plasma from calibration curves ( Table 1). All calculated concentrations were well within the acceptable limits. Figure 3 represents

Recovery
Extraction recovery of colistin A and colistin B at four concentrations (0.05, 0.15, 5.0 and 9.0 µg/ml) and the IS at one concentration (0.1 µg/ml) were determined by comparing peak heights of spiked-before-extraction samples and spiked-after-extraction samples (5 sets). Mean measured extraction recovery of colistin A, and colistin B was 97%, and 94%, respectively. Recovery of IS was 97%.

Matrix Effect
Matrix effect was quantitatively evaluated by comparing peak heights of colistin

Stability
Colistin (  Stability (%) = mean measured concentration (n = 5) at the indicated time divided by mean measured concentration (n = 5) at baseline × 100. Spiked plasma samples were processed and analyzed immediately (baseline, data not shown), after 24 hours at room temperature (24 hrs RT), after freezing at −20˚C for 8 weeks (8 wks, −20˚C), or processed and then analyzed after storing for 24 hours at room temperature (24 hrs, RT) or 48 hours at −20˚C (48 hrs, −20˚C).

Conclusion
The described LCMS/MS assay is simple, precise, and accurate, making it suitable for therapeutic drug monitoring and pharmacokinetic analysis. It requires 1 ml plasma sample, and the analysis was completed within four minutes. Assay was applied successfully to monitor stability of colistin under various conditions generally encountered in the clinical laboratories.