Development and validation of a bioanalytical assay for the measurement of total and unbound teicoplanin in human serum

Background: The glycopeptide teicoplanin is considered first-line treatment for severe infections caused by Gram-positive bacteria. Individualized treatment of teicoplanin is gaining interest. As only protein-unbound drug is pharmacologically active, a sensitive assay measuring unbound and total teicoplanin is indispensable for pharmacological research and dose optimization. Objectives: To develop and validate a UPLC-MS/MS method to quantify unbound and total teicoplanin in human serum. Methods: The developed assay was validated according to the ICH guideline M10 on Bioanalytical Method Validation and study sample analysis. Unbound teicoplanin was obtained by ultrafiltration. The assay was cross-validated with a quantitative microsphere (QMS) immunoassay in a side-by-side comparison using 40 patient samples. Results: With the developed and validated method, all main teicoplanin components (A2-1, A2-2/A2-3, A2-4/A2-5 and A3-1) can be quantified. Total run time was 5.5 min. Concentration range was 2.5-150 mg/L for total and 0.1-25 mg/L for unbound teicoplanin. Precision (coefficient of variation) and accuracy (bias) of total teicoplanin were 5.97% and 107%, respectively, and 7.17% and 108%, respectively, for unbound teicoplanin. Bland-Altman analysis showed total concentrations measured with the UPLC-MS/MS method were equivalent to the results of the QMS immunoassay. A total of 188 samples from 30 patients admitted to the ICU and haematology department were measured; total concentrations ranged between 2.92 and 98.5 mg/L, and unbound concentrations ranged between 0.37 and 30.7 mg/L. Conclusions: The developed method provided rapid, precise and accurate measurement of unbound and total teicoplanin. The developed method is now routinely applied in pharmacological research and clinical practice.