Chromatography columns packed with thermoresponsive-cationic-polymer-modified beads for therapeutic drug monitoring

Abstract

Therapeutic drug monitoring, which is used to determine appropriate drug doses, is critical in pharmacological therapy. In this study, we developed thermoresponsive chromatography columns with various cationic properties for effective therapeutic drug monitoring. Thermoresponsive cationic copolymer poly(N-isopropylacrylamide-co-n-butyl methacrylate-co-N,N-dimethylaminopropyl acrylamide) (P(NIPAAm-co-BMA-co-DMAPAAm))-modified silica beads, which were used as the chromatographic stationary phase, were prepared by modifying the radical initiator of the silica beads, followed by radical polymerization. Characterization of the prepared silica beads demonstrated that thermoresponsive polymers with various cationic properties successfully modified the beads. The elution behavior of several steroids in the prepared bead-packed columns at various temperatures indicated that the optimal column operating temperature was 30 °C. Appropriate measurement conditions for 13 drugs were investigated by varying the cationic properties of the columns and the pH of the mobile phase. Drug concentrations in serum samples were determined using the developed columns and mobile phases with a suitable pH. Voriconazole concentrations in human serum samples were determined using the developed columns with all-aqueous mobile phases. We anticipate that the developed chromatography columns can be used for therapeutic drug monitoring because drug concentrations can be measured using all-aqueous mobile phases that are suitable in clinical settings.

Figure 1

Thermoresponsive anion-exchange chromatography for therapeutic drug monitoring. (A) Schematic diagram of the preparation of a thermoresponsive cationic hydrogel. (B) Temperature-responsive property change of the modified copolymer on silica beads. (C) Electrostatic and hydrophobic interactions between the thermoresponsive cationic hydrogel and drugs in a chromatography column.

Figure 2

(A) FT-IR spectra and (B) SEM images of the prepared beads. Scale bar: 2 μm.

Figure 3

Chromatograms of steroids obtained using the prepared thermoresponsive-cationic-polymer-hydrogel-modified silica beads. Mobile phase: pure water; mobile phase flow rate: 1.0 mL/min; detection wavelength: 254 nm. Peaks 1: hydrocortisone, 2: prednisolone, 3: dexamethasone, 4: hydrocortisone acetate, and 5: testosterone.

Figure 4

Chromatograms of drugs obtained using the suitable columns packed with thermoresponsive cationic-copolymer-modified silica beads. Mobile phase: 10 mmol/L CH₃COONH₄ (pH 4.7) or 10 mmol/L CH₃COONH₄ (pH 6.8); mobile phase flow rate: 1.0 mL/min. Detection wavelengths for each drug are summarized in Table S2.

Figure 5

Chromatograms of the drugs with serum proteins obtained using the suitable columns packed with thermoresponsive cationic-copolymer-modified silica beads. Mobile phase: 10 mmol/L CH₃COONH₄ (pH 4.7) or 10 mmol/L CH₃COONH₄ (pH 6.8); flow rate: 1.0 mL/min. Detection wavelengths for each drug are summarized in Table S2.

Figure 6

Calibration curves of the drugs with serum proteins (n = 5).

Figure 7

Chromatograms of clinical samples collected from patients who were administered voriconazole. Column: PN-0; mobile phase: 10 mmol/L CH₃COONH₄ (pH 4.7); mobile phase flow rate: 1.0 mL/min; detection wavelength: 250 nm.