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%.

Cardiovagal Tone: A Predictor of Heart Rate Adjusted Augmentation Index in Men but Not in Women

Purpose: Aortic augmentation index (AIx) and cardiovagal tone (CVT) are indicators of cardiovascular health. Associations between these variables provide information about their roles in cardiovascular disease. However, evaluating these associations from a gender perspective and gaining an understanding of the relationship between cardiorespiratory fitness and AIx based on gender can provide additional information. Therefore, we examined the relationships between CVT, measured as the log transformed high-frequency power of R-R intervals from electrocardiogram measurements (lnHFR-R);cardiorespiratory fitness, measured as maximum oxygen consumption (VO2max);and AIx at a heart rate of 75 beats·min-1(AIx@75). We hypothesized that the relationships between CVT, cardiorespiratory fitness, and AIx@75 would differ based on gender. Methods: We examined the associations between lnHFR-R, VO2max, and AIx@75 in 41 women and 39 men. Power spectral density analysis of heart rate variability determined lnHFR-R, radial tonometry determined AIx@75 and the Bruce Treadmill Protocol determined VO2max. Results: In men, lnHFR-R positively correlated with and significantly predicted AIx@75 (P = 0.005) but not in women (P = 0.49). For every unit increase in lnHFR-R, there was a 4.6 unit increase in AIx@75 in men versus a 1.0 unit increase in women. There was a significant inverse relationship between VO2max and AIx@75, with VO2max being a significant predictor of AIx@75 in men (P = 0.01). For every unit increase in VO2max, there was a 0.60 unit decrease in AIx@75 in men versus a 0.2 unit decrease in women. Conclusions: The associations between CVT and AIx@75 and between cardiorespiratory fitness and AIx@75 differ by gender. CVT and cardiorespiratory fitness are significant predictors of AIx@75 in men but not in women.