开发并验证了一种通过液相色谱-串联质谱法测定血浆中长效激素避孕药的多重分析方法。
Development and validation of a multiplexed assay for the measurement of long-acting hormonal contraceptives in plasma via liquid chromatography-tandem mass spectrometry.
机构信息
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA; Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
出版信息
J Pharm Biomed Anal. 2023 May 10;228:115321. doi: 10.1016/j.jpba.2023.115321. Epub 2023 Mar 5.
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 KEDTA 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 KEDTA 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.
背景
外源性孕激素是激素避孕和计划生育的有效工具。孕激素可以通过口服药丸、肌肉或皮下注射、阴道环或宫内节育器来给药。药物浓度可能因给药途径和持续时间而有所不同。测量血浆中的合成类固醇可以帮助确定产品的依从性、评估药物相互作用，并调查意外怀孕。
方法
无药物 KEDTA 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）和去氧孕烯（NET）。将血浆与同位素标记的内标混合，然后通过液-液萃取提取药物。样品随后进行液相色谱-串联质谱（LC-MS/MS）分析。该方法按照监管建议进行了验证。该测定方法在使用上述孕激素之一的个体的残留血浆样本中进行了评估。
结果
ETO、MPA 和 NET 的分析测量范围为 20-10,000 pg/mL；LNG 的主要线性范围为 20-20,000 pg/mL。该方法对所有孕激素均表现出可接受的精密度和准确度。室温储存和 3 次冻融循环下，稳定性可维持 72 小时。所有分析物在室温孵育 25 小时、40°C 和 100%湿度下孵育 2 小时的全血中均稳定。所有分析物在血浆中添加时均观察到离子抑制，ETO、LNG、MPA 和 NET 的平均离子抑制率分别为 31.6%、66.6%、32.1%和 41.2%。然而，内标表现出可比的离子抑制，相对基质效应最小。ETO、LNG、MPA 和 NET 也可以在 KEDTA 血浆和血清中准确定量。成功地在使用激素避孕药的个体的残留样本中测量了孕激素。
结论
已经开发和验证了用于定量 ETO、LNG、MPA 和 NET 的多重 LC-MS/MS 测定法。该测定法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。
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