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Development and validation of a multiplexed assay for the measurement of long-acting hormonal contraceptives in plasma via liquid chromatography-tandem mass spectrometry.
J Pharm Biomed Anal. 2023 May 10; 228:115321.JP

Abstract

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.

Links

Publisher Full Text (DOI)

Authors+Show Affiliations

Knezevic CE
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA; Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Parsons TL
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Gollings R
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Pandey A
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Marzinke MA
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA; Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA. Electronic address: Mmarzin1@jhmi.edu.

MeSH

FemaleHumansContraceptive AgentsProgestinsChromatography, LiquidTandem Mass SpectrometryEdetic AcidLevonorgestrelSteroidsMedroxyprogesterone AcetatePlasma

Pub Type(s)

Journal Article

Language

eng

PubMed ID

36924631

Citation

Knezevic, Claire E., et al. "Development and Validation of a Multiplexed Assay for the Measurement of Long-acting Hormonal Contraceptives in Plasma Via Liquid Chromatography-tandem Mass Spectrometry." Journal of Pharmaceutical and Biomedical Analysis, vol. 228, 2023, p. 115321.
Knezevic CE, Parsons TL, Gollings R, et al. Development and validation of a multiplexed assay for the measurement of long-acting hormonal contraceptives in plasma via liquid chromatography-tandem mass spectrometry. J Pharm Biomed Anal. 2023;228:115321.
Knezevic, C. E., Parsons, T. L., Gollings, R., Pandey, A., & Marzinke, M. A. (2023). Development and validation of a multiplexed assay for the measurement of long-acting hormonal contraceptives in plasma via liquid chromatography-tandem mass spectrometry. Journal of Pharmaceutical and Biomedical Analysis, 228, 115321. https://doi.org/10.1016/j.jpba.2023.115321
Knezevic CE, et al. Development and Validation of a Multiplexed Assay for the Measurement of Long-acting Hormonal Contraceptives in Plasma Via Liquid Chromatography-tandem Mass Spectrometry. J Pharm Biomed Anal. 2023 May 10;228:115321. PubMed PMID: 36924631.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Development and validation of a multiplexed assay for the measurement of long-acting hormonal contraceptives in plasma via liquid chromatography-tandem mass spectrometry. AU - Knezevic,Claire E, AU - Parsons,Teresa L, AU - Gollings,Ryann, AU - Pandey,Aashish, AU - Marzinke,Mark A, Y1 - 2023/03/05/ PY - 2023/02/09/received PY - 2023/02/27/revised PY - 2023/02/28/accepted PY - 2024/05/10/pmc-release PY - 2023/3/31/medline PY - 2023/3/17/pubmed PY - 2023/3/16/entrez KW - Etonogestrel KW - Hormonal contraceptives KW - LC-MS KW - Levonorgestrel KW - Mass spectrometry KW - Medroxyprogesterone acetate KW - Norethisterone KW - Progestins SP - 115321 EP - 115321 JF - Journal of pharmaceutical and biomedical analysis JO - J Pharm Biomed Anal VL - 228 N2 - 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. SN - 1873-264X UR - https://www.unboundmedicine.com/medline/citation/36924631/Development_and_validation_of_a_multiplexed_assay_for_the_measurement_of_long_acting_hormonal_contraceptives_in_plasma_via_liquid_chromatography_tandem_mass_spectrometry_ DB - PRIME DP - Unbound Medicine ER -