Estradiol valerate (EST) and Norethisterone acetate (NOR) are co-formulated as a hormone replacement therapy. Naturally occurring steroidal hormones like estradiol, estrone and estriol are also considered as impurities in drug substance. Chromatographic separation of these five steroidal hormones was developed by HPLC and TLC. Reversed-phase chromatography was applied using either acetonitrile–methanol containing 0.05% β-cyclodextrin as a mobile phase for HPLC with gradient elution and chloroform–propanol–formic acid (5:3:2, by volume) as a developing system in TLC. The methods have been applied for determination of EST and NOR in presence of other natural congeners in biological samples as well as in drug products as official impurities. Solid-phase extraction was applied for saliva sample cleaning using testosterone as internal standard whereas ampoules and tablets formulations were used in pharmaceutical applications. The methods have been validated for bioanalytical and pharmaceutical analysis following the respective FDA and ICH guidelines. The obtained results were statistically compared with the official methods for both drugs.

中文翻译：

---

天然同源物存在下合成雌激素和孕酮的色谱分离：在唾液和药物样品中的应用

戊酸雌二醇 (EST) 和醋酸炔诺酮 (NOR) 共同配制为一种激素替代疗法。天然存在的甾体激素如雌二醇、雌酮和雌三醇也被认为是原料药中的杂质。这五种甾体激素的色谱分离是通过 HPLC 和 TLC 开发的。反相色谱使用含 0.05% β-环糊精的乙腈-甲醇作为流动相进行 HPLC 梯度洗脱，氯仿-丙醇-甲酸（5:3:2，体积比）作为 TLC 中的展开系统。该方法已用于测定生物样品中存在的其他天然同系物以及作为官方杂质的药物产品中的 EST 和 NOR。固相萃取用于唾液样品清洁，使用睾酮作为内标，而安瓿和片剂制剂用于药物应用。这些方法已经按照各自的 FDA 和 ICH 指南进行了生物分析和药物分析的验证。将所得结果与两种药物的官方方法进行统计学比较。