Quantification of vancomycin in human serum by LC-MS/MS

Abstract

Background: The aim of our work was to develop and validate a reliable LC-MS/MS-based measurement procedure for the quantification of vancomycin in serum, to be applied in the context of efforts to standardize and harmonize therapeutic drug monitoring of this compound using routine assays. Methods: Sample preparation was based on protein precipitation followed by ultrafiltration. In order to minimize differential modulation of ionization by matrix constituents extended chromatographic separation was applied leading to a retention time of 9.8 min for the analyte. Measurement was done by HPLC-ESI-MS/MS. For internal standardization the derivative vancomycin-glycin (ISTD) prepared by chemical synthesis was used, HPLC conditions ensured coelution of ISTD with the analyte. Results: In a bi-center validation total CVs of < 4% were observed for quality control material ranging from 5.3 mg/L to 79.4 mg/L; accuracy was ± 4%. No relevant ion suppression was observed. Comparative measurement of aliquots from 70 samples at the two validation sites demonstrated close agreement. Conclusions: Employing a closely related homologue molecule for internal standardization and the use of MS/ MS following highly efficient sample pre-fractionation by HPLC, the method described here can be considered to offer the highest level of analytical reliability realized so far for the quantification of vancomycin in human serum. Thus, the method is suitable to be used in a comprehensive reference measurement system for vancomycin.

Factors Impacting Unbound Vancomycin Concentrations in Different Patient Populations

“…Also, it is well known that some immunoassay methods are prone to interference by paraproteins or rheumatic factor (18) or can cross-react with crystalline degradation products, which comprise inactive metabolites of vancomycin found in plasma or serum samples from renally impaired and dialysis patients (19)(20)(21). As LC-MS/MS methods are more specific, it is generally accepted that they are less likely to suffer from these issues (22). Differences in unbound vancomycin concentrations between immunoassays and HPLC were recently illustrated in a study by Crandon et al in which unbound vancomycin concentrations were measured by an FPIA and an in-house-developed HPLC method (9).…”

Determination of Vancomycin in Human Plasma, Bone and Fat by Liquid Chromatography/Tandem Mass Spectrometry

“…Various analytical methods have been developed for measuring vancomycin in liquid samples only, such as plasma/serum, urine, human drainage tissue fluid and bronchoalveolar lavage fluid, including HPLC methods [8][9][10] and LC-MS/MS methods [11][12][13][14][15]. A few methods have been reported for the determination of vancomycin in both liquid and solid samples, such as immunoassays for plasma/serum and bone [16,17], HPLC method for plasma, bone and atrial appendage tissue [18], and LC-MS/MS for serum, perivascular fat and atrial wall samples [19].…”

An Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry Method for the Quantification of Vancomycin Requiring Only 2 &#181;L of Rabbit Serum

“…The current UHPLC-MS/MS method is superior to the PETINIA technique as it requires only 2 µL of sample compared to the large volumes (50 µL to fill the sample cuvette) required by the immunoassay. Other LC-MS/MS assays determined VAN in human [8] or rat plasma [7], and human serum [6].…”

“…There are several methodologies available for the measurements of VAN in biological matrices that include radioimmunoassays, fluorescence immunoassays, fluorescence polarization immunoassays [3], particle enhanced turbidimetric inhibition immunoassays (PETINIA) [4], liquid chromatography (LC) [5], and LC-tandem mass spectrometry (LC-MS/MS) [6] [7] [8] [9]. These methods were successfully used to measure VAN in a variety of matrices, but they had their limitations.…”

“…These methods were successfully used to measure VAN in a variety of matrices, but they had their limitations. They either were not sufficiently precise [3], suffered from interference [4], had insufficient sensitivity [5] [6] [8], or required large sample volumes [6] [8] [7] [9]. Large sample volumes are sometimes difficult to obtain, especially in studies where VAN is determined in the samples of small laboratory animals, such as rabbits, where the availability of the biological matrix is limited.…”