title: Development and Validation of LC-MS/MS Assay for the Quantification of Progesterone in Rat Plasma and its Application to Pharmacokinetic Studies.
abstract: A sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the determination of progesterone levels in rat plasma. Progesterone-d9 was used as an internal standard (IS). Samples were prepared using salting-out assisted liquid/liquid extraction (SALLE), and the extracts were injected directly onto the LC-MS/MS system. The chromatographic separation was achieved on a CAPCELL PAK C18 MGIII column (100 mm × 2.0 mm, i.d. 5 µm) using methanol and aqueous 0.1% formic acid solution gradient as the mobile phase with a constant flow rate of 0.45 mL/min. Electrospray ionization in the positive-ion mode was employed. Multiple reaction monitoring of the precursor to product ion pairs, from m/z 315.20 to m/z 109.10 for progesterone and from m/z 324.26 to m/z 113.07 for the IS, was used for quantification. Good linearity was observed over the concentration range of 0.05-20.00 ng/mL with a weighted (1/x(2)) linear regression. The intra- and inter-day precision (% relative standard deviation [RSD]) across 3 validation days over the entire concentration range was lower than 6.7%. Accuracy (% nominal) determined at 5 quality control concentrations was between 94.0 and 103.7%. The validation method was applied in a pharmacokinetic study evaluating progesterone levels after intramuscular or vaginal administration to ovariectomized (OVX) rats. The area under the plasma concentration-time curve (AUC) calculated after intramuscular administration was more than 4 times higher than the AUC measured following vaginal administration of a comparable dose.
authors.affiliation: Pharmacokinetics Research Department, Developmental Research Center, ASKA Pharmaceutical Co., Ltd, Kawasaki, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Sasaki
authors.affiliation: Pharmacokinetics Research Department, Developmental Research Center, ASKA Pharmaceutical Co., Ltd, Kawasaki, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Ochiai
authors.affiliation: ASKA Pharma Medical Co., Ltd, Kawasaki, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Takahashi
authors.affiliation: Pharmacokinetics Research Department, Developmental Research Center, ASKA Pharmaceutical Co., Ltd, Kawasaki, Japan.
authors.forename: R
authors.initials: R
authors.lastname: Suzuki
authors.affiliation: Pharmacokinetics Research Department, Developmental Research Center, ASKA Pharmaceutical Co., Ltd, Kawasaki, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Minato
authors.affiliation: Pharmacokinetics Research Department, Developmental Research Center, ASKA Pharmaceutical Co., Ltd, Kawasaki, Japan.
authors.forename: A
authors.initials: A
authors.lastname: Fujikata
mesh_terms: D000282:Administration, Intravaginal; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D005260:Female; D007273:Injections, Intramuscular; D059625:Liquid-Liquid Extraction; D011374:Progesterone / Q000008:administration & dosage / Q000097:blood* / Q000493:pharmacokinetics*; D051381:Rats; D053719:Tandem Mass Spectrometry / Q000379:methods* / Q000592:standards*
chemical_list: D011374:Progesterone