Effects of transport conditions and freeze–thaw cycles on the concentration of polymyxin E in human whole blood and plasma

Polymyxin E has a narrow therapeutic window and significant toxicity, necessitating precise blood concentration monitoring. Its prodrug, colistin methanesulfonate sodium (CMS), undergoes conversion to polymyxin E in blood, a process influenced by temperature and time. This study aimed to identify suitable whole-blood transport conditions and acceptable plasma freeze–thaw limits for reliable polymyxin E quantification in real-world centralized or referred therapeutic drug monitoring (TDM) workflows. Fresh venous blood samples ( n = 18) were collected and promptly delivered on ice to the laboratory. After gentle inversion, each sample was divided into 11 aliquots. One aliquot was centrifuged immediately, and the plasma was stored at − 80 °C as the baseline. The remaining ten aliquots were allocated into two groups (five aliquots per group) and stored at room temperature (RT) or 4 °C for 2, 4, 6, 8, or 12 h before centrifugation and plasma storage (− 80 °C). To assess the effect of freeze–thaw cycling, 4 plasma aliquots from each fresh blood sample underwent 1–4 complete cycles of freezing at − 20 °C and thawing at RT. All samples were analyzed by liquid chromatography–tandem mass spectrometry (LC–MS/MS). In peak samples, polymyxin E concentrations increased, with mean %Bias values of 6.43% within 12 h at 4 °C and 11.38% within 4 h at RT. Trough samples showed decreasing concentrations, with mean %Bias values of − 3.40% at RT and − 7.66% at 4 °C over 12 h. No significant changes occurred within three freeze–thaw cycles. However, the fourth cycle induced significant concentration increases in both peak (19.46%) and trough samples (13.40%), yet these changes showed no correlation with initial concentrations. For reliable quantification of polymyxin E, 4 °C is the optimal transport temperature for whole-blood samples. In cases of logistical constraints, peak samples could be stored for up to 4 h at RT or 12 h at 4 °C, whereas trough samples under a twice-daily dosing regimen could be stored for up to 12 h at both temperatures. Additionally, plasma samples should undergo no more than three freeze–thaw cycles.