Simultaneous determination of colistin sulfate and tigecycline in human plasma by liquid chromatography-tandem mass spectrometry method.

OBJECTIVE: To establish a high-performance liquid chromatography-tandem mass spectrometry method (HPLC-MS/MS) to simultaneously determine colistin sulfate and tigecycline in human plasma. METHODS: Polymyxin B1 internal standard (20 µL) was added into 200 µL of plasma sample. The samples were treated with methanol-5% trichloroacetic acid (50:50, V/V) solution, and the protein precipitation method was adopted for post-injection analysis. The chromatographic column was a Dikma C18 (4.6 mm × 150 mm, 5 µm). For the mobile phase, 0.1% formic acid in aqueous solution was used for phase A, 0.1% formic acid in acetonitrile solution for phase B, and gradient elution was also applied. The flow rate was 0.8 mL/min, the column temperature was 40 °C, and the injection volume was 10 µL; Electrospray ionization and multiple reaction ion monitoring were adopted and scanned by the HPLC-MS/MS positive ion mode. RESULTS: The endogenous impurities in the plasma had no interference in the determination of the analytes. There existed a good linear relationship of colistin sulfate within the range of 0.1-10 µg/mL (R2 = 0.9986), with the lower limit of quantification (LLOQ) of 0.1 µg/mL. There existed a good linear relationship of tigecycline within the range of 0.05-5 µg/ mL (R2 = 0.9987), with the LLOQ of 0.05 µg/mL. The intra- and inter-day relative standard deviations of colistin sulfate and tigecycline were both less than 15%, and the accuracy was between 88.21% and 108.24%. The extraction had good stability, the extraction recovery rate was 87.75-91.22%, and the matrix effect was 99.40-105.26%. CONCLUSION: This study successfully established a method for simultaneously detecting colistin sulfate and tigecycline plasma concentrations. The method was simple, rapid, and highly sensitive and could be applied for therapeutic medication monitoring.

A simple and accurate HFCF-UF method for the analysis of homocysteine, cysteine, cysteinyl-glycine, and glutathione in human blood.

The presence of reduced aminothiols, including homocysteine (Hcy), cysteine (Cys), cysteinyl-glycine (CG), and glutathione (GSH), is significantly increased in the pathological state. However, there have been no reports on the relationship between reduced aminothiols (Hcy, Cys, CG, and GSH) and different genders, ages, and drug combinations in human blood. The accurate quantification of these reduced thiols in biological fluids is important for monitoring some special pathological conditions of humans. However, the published methods typically not only require cumbersome and technically challenging processing procedures to ensure reliable measurements, but are also laborious and time-consuming, which may disturb the initial physiological balance and lead to inaccurate results. We developed a hollow fiber centrifugal ultrafiltration (HFCF-UF) method for sample preparation coupled with a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method and used it to determine four reduced aminothiols (Hcy, Cys, CG, and GSH) in human blood for the first time. A total of 96 clinical patients were enrolled in our study. The influence of different genders, ages, and drug combinations on the levels of four reduced thiols in human blood was also discussed by SPSS 24.0. The sample preparation was simplified to a single 5 min centrifugation step in a sealed system that did not disturb the physiological environment. The validation parameters for the methodological results were excellent. The procedure was successfully applied to monitoring the concentrations of four reduced aminothiols (Hcy, Cys, CG, and GSH) in 96 clinical blood samples. There were no significant differences in Hcy, Cys, CG, or GSH for the different genders, ages, or combinations with methotrexate or vancomycin (P > 0.05). However, there was a significant increase in Hcy concentration in patients treated with valproic acid who were diagnosed with epilepsy (p=0.0007). It is advisable to measure reduced Hcy level in patients taking valproic acid. The developed HFCF-UF method was simple and accurate. It can be easily applied in clinical research to evaluate oxidative stress in further study.

Relationship Between the Free and Total Methotrexate Plasma Concentration in Children and Application to Predict the Toxicity of HD-MTX.

High-dose methotrexate (HD-MTX) can be highly effective as well as extremely toxic. Many drug molecules can bind to plasma proteins to different extents in vivo, whereas only the free drug can reach the site of action to exert a pharmacological effect and cause toxicity. However, free MTX concentrations in plasma have not been reported. Traditional analyses of free drugs are both cumbersome and inaccurate. We collected 92 plasma samples from 52 children diagnosed with ALL or NHL or other lymphomas that were treated with HD-MTX. The hollow fiber centrifugal ultrafiltration (HFCF-UF) was used to prepare plasma samples for analysis of the free MTX concentration. Protein precipitation was employed to measure the total MTX concentration. The HFCF-UF is a simple method involving a step of ordinary centrifugation; the validation parameters for the methodological results were satisfactory and fell within the acceptance criteria. A linearity coefficient r 2 of 0.910 was obtained for the correlation between the free and total MTX plasma concentrations in 92 plasma samples. However, the free and total MTX concentrations was only weakly correlated in 16 clinical plasma specimens with total MTX concentrations >2 µmol L-1 (r 2 = 0.760). Both the free and total MTX concentrations at 42 h were negatively correlated with the creatinine clearance (CCr) level (P = 0.023, r = -0.236 for total MTX and P = 0.020, r = -0.241for free MTX, respectively). The free MTX concentration could not be accurately estimated from the total MTX concentration for patients with high MTX levels which are conditions under which toxic reactions are more likely to occur. High plasma MTX levels could become a predictor of the occurrence of MTX nephrotoxicity to draw people's attention. The proposed HFCF-UF method is a simple and accurate way to evaluate efficacy and toxicity in clinical therapeutic drug monitoring.