www.klinickafarmakologie.cz / Klin Farmakol Farm 2023;37(3):89-92 / KLINICKÁ FARMAKOLOGIE A FARMACIE 91 PŮVODNÍ PRÁCE Assay of colistin A, B and colistin methanesulfonate in human plasma by LC-MS/MS and short-term plasma stability of R2 = 0,9995 for colistin A and R2 = 0,9982 for colistin B. The results of the inter-day and intraday measurements for method validation are accurate and precise, with an error not exceeding 15 % with the LOQ set at 0,15 mg/L. The validation confirmed the reliability of the LC-MS method for measuring concentrations of colistin in human plasma. Summary information on the analysis parameters is given in Table 1. The CMS concentration was measured indirectly by acid hydrolysis, for which we found it most useful to use 15 µl of 1 M sulfuric acid. After 30 minutes, the hydrolysis was stopped by adding 30µl of 1M sodium hydroxide. As this is an indirect method, it is necessary to back-calculate the CMS concentrations from the difference: CMS = COLtotal – COLbefore hydrolysis where COLtotal is the concentration after hydrolysis of CMS to colistin and COLbefore hydrolysis is the circulating concentration of colistin formed by endogenous transformation of the prodrug to its active form. Long-term stability studies of colistin showed no degradation in stock solutions and patient plasma samples stored at −70 °C for at least 90 days. Also, no degradation was observed in three freeze-thaw cycles (data not shown). The short-term stability of colistin was measured for three concentration points at room temperature (RT) and 37 °C (Table 2). After 24 hours at RT, the degradation of the samples reached almost 10 %. However, the degradation of colistin was more significant at 37 °C. Already after 30 minutes, degradation reaching up to 25 % was observed, the average degradation for all samples was 11 %. CMS stability in plasma samples at a selected concentration of 10mg/L was studied at room temperature and 37 °C. As expected, the conversion of CMS to colistin is more significant at 37 °C than at RT. At elevated temperatures, we observe the conversion of CMS into its colistin Tab. 1. Summary information on the analysis parameters Chromatographic conditions HPLC: Shimadzu, Prominence LC–20A Column: Arion® Polar C18 (250 × 4,6 mm; 5 mm) Mobile phase A: Mobile phase B: 0,1% formic acid in water 0,1% formic acid in methanol (40:60, v/v) Flow rate: 0,8 ml/minute Column temperature: 35 °C The volume of injection: 10 µl Analysis time: 3 minutes MS/MS detection Mass spectrometer: Shimadzu, LCMS-8045 Ionization mode: ESI positive Ion transition monitored: Colistin A 585,55 → 101,05 Colistin B 578,5 → 101,15 IS 602,4 → 101,1; 120,15; 86,15 Validation parameters Calibration curve range (mg/l) 0,15–30 Limit of quantification – LOQ (mg/l) 0,2 Limit of detection – LOD (µg/l) 4,7 Recovery (%) 81 Tab. 2. Stability of colistin A and B (%) at three concentration levels (2; 10; 20 mg/L) in human plasma Colistin A Colistin B Time 2 mg/L 10 mg/L 20 mg/L 2 mg/L 10 mg/L 20 mg/L RT 0,5 h 99,28 101,28 100,18 107,01 105,14 103,41 1 h 98,90 97,86 99,05 92,93 97,28 104,80 2 h 96,92 97,66 96,08 92,15 95,31 99,89 5 h 93,28 92,16 95,64 92,22 94,15 96,57 24 h 90,57 92,71 91,37 90,15 93,93 96,46 37 °C 30 min 91,65 92,76 91,21 74,96 92,76 92,17 RT – room temperature Tab. 3. Stability of CMS (%) at 10 mg/L concentration level in human plasma 37 °C RT °C Time COL A COL B COL A COL B 0,5 h 103,7 103,1 100,1 100,6 1 h 108,0 109,2 102,0 105,6 2 h 114,0 116,3 108,1 110,3 3 h 117,6 119,9 111,7 113,8 RT – room temperature Fig. 1. Representative chromatograms of calibration point 10 mg/L (1.a) and patient sample (1.b) 1.a 1.b Time (min) 5 000 000 4 500 000 4 000 000 3 500 000 3 000 000 2 500 000 2 000 000 1 500 000 1 000 000 500 000 0 5 000 000 4 500 000 4 000 000 3 500 000 3 000 000 2 500 000 2 000 000 1 500 000 1 000 000 500 000 0 Absolute intensity 0 0,5 1 1,5 2 2,5 3 0 0,5 1 1,5 2 2,5 3 IS colistin B colistin A IS colistin A colistin B Time (min) Absolute intensity
RkJQdWJsaXNoZXIy NDA4Mjc=