Pichette A, Drouin N, Girard M
Bureau of Biologics and Radiopharmaceuticals, Therapeutic Products Programme, Health Canada, Sir F.G. Banting Research Center, Ottawa, Ontario.
J Chromatogr A. 2000 Aug 18;890(1):127-33. doi: 10.1016/s0021-9673(00)00594-x.
Human parathyroid hormone (1-84) is a naturally occurring polypeptide that acts as the major regulator of calcium ion homeostasis. It can be efficiently produced through both synthetic and biosynthetic routes and, as such, highly selective analytical methods are required for the detection of a wide range of impurities. Herein we report on the development of an ion-pairing reversed-phase HPLC method for the analysis of human parathyroid hormone and the separation of impurities including a major, unidentified impurity detected in synthetic preparations. This impurity could not be resolved using trifluoroacetic acid-based methods generally used for monitoring purity levels in commercial products. Separation conditions consisted of a gradient elution of 0.155 M sodium chloride containing 0.037 M sodium pentanesulfonate, pH 5.6, as mobile phase A and acetonitrile as mobile phase B. Separations were carried out on an octadecylsilyl silica column maintained at 50 degrees C. Both column temperature and pH of mobile phase A significantly affected the separation of the major impurity. The major impurity eluted after the main human parathyroid peak and was detected in the two commercial synthetic products analyzed. Several minor impurities eluting before and after the main peak were also detected. Purity levels measured by the developed HPLC method (method C) were similar to those previously measured by capillary electrophoresis. Analysis of purified recombinant human parathyroid hormone did not show the presence of this impurity. This method offers a significant advantage for the purity assessment of human parathyroid hormone.
人甲状旁腺激素（1 - 84）是一种天然存在的多肽，是钙离子稳态的主要调节因子。它可以通过合成和生物合成途径有效生产，因此，需要高选择性的分析方法来检测各种杂质。本文报道了一种离子对反相高效液相色谱法的开发，用于分析人甲状旁腺激素并分离杂质，包括在合成制剂中检测到的一种主要的未鉴定杂质。使用通常用于监测商业产品纯度水平的基于三氟乙酸的方法无法分离这种杂质。分离条件包括以含有0.037 M戊烷磺酸钠的0.155 M氯化钠（pH 5.6）作为流动相A进行梯度洗脱，以乙腈作为流动相B。在保持在50℃的十八烷基硅烷硅胶柱上进行分离。柱温和流动相A的pH值都对主要杂质的分离有显著影响。主要杂质在人甲状旁腺主峰之后洗脱，并在分析的两种商业合成产品中检测到。还检测到了在主峰之前和之后洗脱的几种次要杂质。通过开发的高效液相色谱法（方法C）测量的纯度水平与先前通过毛细管电泳测量的纯度水平相似。对纯化的重组人甲状旁腺激素的分析未显示该杂质的存在。该方法为人甲状旁腺激素的纯度评估提供了显著优势。
