Temperature responsive chromatography for therapeutic drug monitoring with an aqueous mobile phase

Abstract

Therapeutic drug monitoring is a key technology for effective pharmacological treatment. In the present study, a temperature-responsive chromatography column was developed for safe and simple therapeutic drug monitoring without the use of organic solvents. Poly(N-isopropylacrylamide) (PNIPAAm) hydrogel-modified silica beads were prepared via a condensation reaction and radical polymerization. The temperature-dependent elution behavior of the drugs was observed using a PNIPAAm-modified silica-bead packed column and an all-aqueous mobile phase. Sharp peaks with reproducible retention times were observed at temperatures of 30 °C or 40 °C because the PNIPAAm hydrogel on the silica beads shrinks at these temperatures, limiting drug diffusion into the PNIPAAm hydrogel layer. The elution behavior of the sample from the prepared column was examined using a mixture of serum and model drugs. The serum and drugs were separated on the column at 30 °C or 40 °C, and the concentration of the eluted drug was obtained using the calibration curve. The results show that the prepared chromatography column would be useful for therapeutic drug monitoring because the drug concentration in serum can be measured without using organic solvents in the mobile phase and without any need for sample preparation.

Figure 1

Schematic illustration of the preparation of thermoresponsive polymer hydrogel modified beads (A) and therapeutic drug monitoring using temperature-responsive chromatography with all-aqueous mobile phase (B).

Figure 2

Characterization of the prepared beads. (A) Fourier transfer infrared spectra of the prepared beads. The dashed lines (i) and (ii) indicate the peak attributed to C=O stretching vibrations and N–H bending vibrations, respectively. (B) SEM images of the prepared beads. (1) aminopropyl silica beads, (2) V-501 immobilized silica beads, (3) PNIPAAm modified silica beads prepared via polymerization for 5 h, and (4) PNIPAAm modified silica beads prepared via polymerization for 18 h. Scale bars denote 5 μm.

Figure 3

Chromatograms of hydrophobic steroids using crosslinked PNIPAAm modified-bead packed columns using beads prepared via polymerization for (A) 5 h, and (B) 18 h. The mobile phase was pure water. The flow rate of the mobile phase was 1.0 mL/min. Detection at 254 nm. Peak 1 = hydrocortisone, peak 2 = prednisolone, peak 3 = dexamethasone, peak 4 = hydrocortisone acetate, and peak 5 = testosterone.

Figure 4

Chromatograms of antiepileptic drugs (A–C) and anti-arrhythmic drugs (D–F) using crosslinked PNIPAAm modified-bead packed columns. (A) Lamotrigine, (B) Carbamazepine, (C) Phenytoin, (D) Quinidine, (E) Propafenone, and (F) Disopyramide. The mobile phase was 10 mM CH₃COONH₄ (pH 6.75) buffer solution with a flow rate of 1.0 mL/min.

Figure 5

Chromatograms of (A) digoxin (cardiac glycoside), (B) vancomycin (antibacterial drug), (C) mycophenolic acid (immune-suppressing drug), and (D) methotrexate (anti-cancer drug) using crosslinked PNIPAAm modified-bead packed columns. Mobile phases were 10 mM CH₃COONH₄ (pH 6.75) buffer solution and 10 mM CH₃COONH₄ (pH 4.8) buffer solution with a flow rate of 1.0 mL/min.

Figure 6

The retention time of drugs used in therapeutic drug monitoring using PNIPAAm hydrogel modified-bead columns. Retention times of mycophenolic acid and methotrexate were obtained using pH 4.80 CH₃COONH₄ buffer solution as mobile phase. All other drugs used pH 6.75 CH₃COONH₄ buffer solution mobile phase.

Figure 7

Chromatograms of antiepileptic drug and anti-arrhythmic drug with serum proteins using crosslinked PNIPAAm modified-bead packed columns. (A) Carbamazepine, (B) phenytoin, (C) lamotrigine, (D) quinidine, (E) propafenone, and (F) disopyramide. Mobile phase was 10 mM CH₃COONH₄ (pH 6.75) buffer solution for carbamazepine, phenytoin, lamotrigine, and disopyramide, and 10 mM CH₃COONH₄ buffer solution (pH 4.80) for quinidine and propafenone. The mobile phase flow rate was 1.0 mL/min.

Figure 8

Chromatograms of (A) digoxin, (B) vancomycin, (C) mycophenolic acid, and (D) methotrexate in serum using crosslinked PNIPAAm modified-bead packed columns. The mobile phase was 10 mM CH₃COONH₄ buffer solution (pH 6.75) for digoxin, and 10 mM CH₃COONH₄ buffer solution (pH 4.80) for vancomycin, mycophenolic acid, and methotrexate. The mobile phase flow rate was 1.0 mL/min.