反相高效液相色谱-脉冲电化学检测法测定重组人生长激素中异丙基-β-D-硫代半乳糖苷（英文）

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

采用反相高效液相色谱(RP-HPLC)-脉冲电化学检测(PED)法测定了重组人生长激素(rhGH)中的诱导剂异丙基-β-D-硫代半乳糖苷(IPTG)。rhGH样品经超滤离心后,用超纯水提取,经C18色谱柱分离,用脉冲电化学器检测。结果表明,样品中添加0.02~0.10 mg/L的IPTG,其回收率为100%~102%,相对标准偏差(n=3)小于10%。在优化的条件下,IPTG的检出限可达1μg/L(0.1 pmol,25μL)。该方法简便高效,具有良好的灵敏度、回收率和重复性。

A novel method for the determination of isopropyl-β-D-thiogalactopyranoside （IPTG）, a sulfurcontaining compound, a pharmaceutical additive in commercial recombinant human growth hormone （rhGH）, has been developed using pulsed electrochemical detection （PED）, following liquid chromatographic separa-tion. 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 elec-trode has proven to be selective for sulfur-containing compounds under mildly acidic conditions. Upon optimiza-tion, IPTG was found to have a limit of detection of 1μg/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 sim-plicity, 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.