Determination of the free and total concentrations of vancomycin by two-dimensional liquid chromatography and its application in elderly patients

• 2D-LC to determine the free and total concentrations of vancomycin in elderly patients.
• 2D-LC with special RP1–IEX–RP2 column system.
• 2D-LC with unique “Assistant Flow” technology.

A robust two-dimensional liquid chromatography (2D-LC) method for determining the free and total concentrations of vancomycin in plasma was developed and validated. The 2D-LC system, which exhibited a strong capacity for inhibiting interference, comprised a unique RP1–IEX–RP2 column system and an “Assistant Flow” configuration. Ultrafiltration technology was employed to separate free vancomycin from the protein-bound fraction in human plasma. The influence of ultrafiltration conditions on the free vancomycin concentration was evaluated. The calibration curve was linear over the 0.195–49.92 μg/ml range for the free and total vancomycin concentrations. The within- and between-run precision ranges were 1.5–3.9% and 2.0–4.7% for the total concentration, 1.4–3.3% and 2.4–4.0% for the free concentration, respectively. Ultrafiltration was susceptible to variations in the experimental conditions, including the centrifugation time, the centrifugal force, and the nominal molecular weight limit of the ultrafiltration membrane. A total of 101 serum samples from 84 elderly patients were analyzed by this method. The free vancomycin concentration was 5.88 ± 3.75 μg/ml (range: 0.240–16.79 μg/ml), the total concentration was 12.36 ± 5.36 μg/ml (range: 2.16–27.14 μg/ml), and the unbound fraction was 45.6 ± 18.8% (range: 11.1–96.9%). There was a poor correlation between the free and total vancomycin concentrations (R2 = 0.596, p < 0.05). This method appears to be sensitive, precise, selective, and suitable for use in protein-binding studies of vancomycin.