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Development, Validation, and Application of an UHPLC-MS/MS Method for Simultaneous Determination of Vancomycin, Meropenem, and Sulbactam in Human Blood Plasma and Cerebrospinal Fluid
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- 10 May 2025
- 03 Jul 2025
- 08 Jan 2026
Abstract
Introduction
Postoperative intracranial infections present significant therapeutic challenges in neurosurgery, with inadequate cerebrospinal fluid drainage and antimicrobial resistance constituting primary risk factors. The escalating prevalence of extensively drug-resistant pathogens necessitates combination therapy, particularly vancomycin-meropenem-sulbactam (VCM-MER-SUL) regimens for multidrug-resistant infections. Marked interpatient pharmacokinetic variability complicates dosing, prompting the development of a UHPLC-MS/MS method for precise therapeutic drug monitoring (TDM) of these agents.
Methods
Chromatographic separation was achieved on a BEH C18 column (Waters, 2.1 × 50 mm, 1.7 µm particles) with 0.1% formic acid in water (A) and methanol (B) under gradient elution conditions: 95% A; 0.7 min, 70% A; 1.0 min, 5% A; 2.0 min, 5% A; 2.1 min, 95% A; 3.5 min, 95% A. The column oven and autosampler temperatures were maintained at 37°C and 15°C, respectively. The mobile phase flow rate was set at 0.4 mL/min.
The ion transition was m/z 725.5 > 144.0 for VCM, m/z 383.7 > 141.1 for MER, m/z 234.1 > 124.0 for SUL, and m/z 390.0 > 114.1 for MER-D6 (internal standard). The one-step precipitation by acetonitrile was used for sample pretreatment.
Results
The calibration range for VCM and SUL was established between 0.1 and 40 mg/L, whereas for MER, it was set from 0.02 to 8 mg/L. In all cases, the linearity of the calibration curves was deemed satisfactory; the method demonstrated acceptable accuracy and precision, with intra-day and inter-day bias ranging from -9.98% to 13.40%, while the corresponding imprecision remained below 12.85%. Additionally, the variability in internal standard-normalized recovery and matrix effects was controlled, showing coefficients below 12.76% and 8.77%, respectively. Stability assessments confirmed that all analytes remained within acceptable limits under the tested conditions.
Discussion
Initial trials with an acetonitrile-water mobile phase yielded poor peak symmetry and inadequate resolution for all analytes. Subsequent systematic evaluation of organic modifiers (acetonitrile vs methanol) and formic acid concentrations identified a 0.1% formic acid aqueous/methanol gradient as optimal. This condition, implemented on a BEH C18 column at 37°C, delivered symmetric peaks for vancomycin, meropenem, and sulbactam within 3.5 min—markedly faster than most reported methods. A single-step acetonitrile protein precipitation doubled vancomycin recovery relative to methanol while maintaining high efficiency for the other analytes. The method’s calibration ranges, matrix effects, recoveries, and long-term stability under clinically relevant storage and handling conditions were fully validated and concordant with literature benchmarks.
Conclusion
The method is fast, sensitive, accurate, and reliable, and has been verified in the study. The streamlined one-step protein precipitation method for sample preparation, coupled with rapid chromatographic separation (3.5 minutes), demonstrated suitability for clinical applications.
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