Determination of isopropyl-β-D-thiogalactopyranoside in recombinant human growth hormone using reversed-phase high performance liquid chromatography and pulsed electrochemical detection

doi:10.3724/SP.J.1123.2016.10037

Abstract

Abstract:

A novel method for the determination of isopropyl-β-D-thiogalactopyranoside (IPTG), a sulfur-containing compound, a pharmaceutical additive in commercial recombinant human growth hormone (rhGH), has been developed using pulsed electrochemical detection (PED), following liquid chromatographic separation. A Dionex-500 ion chromatograph coupled with an electrochemical detector was employed, equipped with a gold working electrode and an Acclaim 300 analytical C18 column, with a mobile phase consisting of sodium acetate (NaOAc) buffer (pH 5.45, 0.01 mol/L) and acetonitrile (ACN) (90:10, v/v). The C18 phase has very high surface coverage, resulting in high capacity. Electrochemical characterization via cyclic voltammetry is the basis of optimization of the integrated pulsed amperometric detection (IPAD) waveform. PED at a gold electrode has proven to be selective for sulfur-containing compounds under mildly acidic conditions. Upon optimization, IPTG was found to have a limit of detection of 1 μg/L (0.1 pmol) with 25 uL injection volume. This method was successfully applied to the determination of IPTG in rhGH samples with the characteristics of simplicity, high sensitivity and good repeatability. The assay is also useful, efficient and low-cost in monitoring disulfide formation kinetics and assuring quality control for biopharmaceuticals.

Key words: isopropyl β-D-thiogalactopyranoside (IPTG), pulsed electrochemical detection (PED), recombinant human growth hormone (rhGH), reversed-phase high performance liquid chromatography (RP-HPLC), sulfur, thiol

CLC Number:

O658

WANG Li, LIN Birong. Determination of isopropyl-β-D-thiogalactopyranoside in recombinant human growth hormone using reversed-phase high performance liquid chromatography and pulsed electrochemical detection[J]. Chinese Journal of Chromatography, 2017, 35(3): 308-313.

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