背景:外源性孕激素是激素避孕和计划生育的有效工具。孕激素可以作为口服药丸,肌肉注射或皮下注射,阴道环,或宫内节育器。药物浓度可基于递送途径和持续时间而变化。测量血浆中的合成类固醇可以帮助确定产品的依从性,评估药物-药物相互作用,和意外怀孕的调查。
方法:在无药K2EDTA血浆中加入合成类固醇依托孕烯(ETO),左炔诺孕酮(LNG),醋酸甲羟孕酮(MPA),和norethisterone(NET)。血浆与同位素标记的内标结合,和药物通过液-液萃取法提取。然后对样品进行液相色谱-串联质谱(LC-MS/MS)分析。根据监管建议对该方法进行了验证。使用上述孕激素中的一种在个体的残余血浆样品群中评估测定。
结果:ETO的分析测量范围,MPA,NET为20-10,000pg/mL;液化天然气的主要线性为20-20,000pg/mL。该方法对所有孕激素均显示出可接受的精密度和准确度。在室温储存下并通过3个冻融循环建立72小时的稳定性。所有分析物在室温孵育25小时的全血中是稳定的,并在40°C和100%湿度下2小时。对血浆中掺入的所有分析物观察到离子抑制;平均离子抑制为31.6%,66.6%,ETO的32.1%和41.2%,液化天然气,MPA,和NET,分别。然而,内标显示出可比的离子抑制,相对矩阵效应很小。ETO,液化天然气,MPA,NET也可以在K3EDTA血浆和血清中准确定量。使用激素避孕药在个体的残余样品中成功测量了孕激素。
结论:用于定量ETO的多重LC-MS/MS测定法,液化天然气,MPA,NET已经开发和验证。该测定符合可接受的性能特征,可用于下游研究以评估孕激素药理学。
BACKGROUND: Exogenous progestins are an effective tool for hormonal contraception and family planning. Progestins may be delivered as oral pills, intramuscular or subcutaneous injections, vaginal rings, or intrauterine devices. Drug concentrations may vary based on the route and duration of delivery. Measurement of synthetic steroids in blood plasma can aid in determination of product adherence, evaluation of drug-drug interactions, and investigation of unintended pregnancies.
METHODS: Drug-free K2EDTA plasma was spiked with the synthetic steroids etonogestrel (ETO), levonorgestrel (LNG), medroxyprogesterone acetate (MPA), and
norethisterone (NET). Plasma was combined with isotopically labeled internal standards, and drugs were extracted via liquid-liquid extraction. Samples were then subjected to liquid chromatographic-tandem mass spectrometric (LC-MS/MS) analysis. The method was validated in accordance with regulatory recommendations. The assay was evaluated in a cohort of remnant plasma samples in individuals using one of the aforementioned progestins.
RESULTS: The analytical measuring range for ETO, MPA, and NET was 20-10,000 pg/mL; the primary linearity for LNG was 20-20,000 pg/mL. The method showed acceptable precision and accuracy for all progestins. Stability was established for 72 h with room temperature storage and through 3 freeze-thaw cycles. All analytes were stable in whole blood incubated at room temperature for 25 h, and at 40°C and 100% humidity for 2 h. Ion suppression was observed for all analytes spiked in plasma; average ion suppression was 31.6%, 66.6%, 32.1% and 41.2% for ETO, LNG, MPA, and NET, respectively. However, internal standards showed comparable ion suppression, and relative matrix effects were minimal. ETO, LNG, MPA, and NET could also be quantified accurately in K3EDTA plasma and serum. Progestins were successfully measured in remnant samples from individuals using hormonal contraceptives.
CONCLUSIONS: A multiplexed LC-MS/MS assay for the quantification of ETO, LNG, MPA, and NET has been developed and validated. The assay met acceptable performance characteristics and may be used in downstream studies to evaluate progestin pharmacology.