Drug Res (Stuttg) 2015; 65(09): 484-489
DOI: 10.1055/s-0034-1389967
Original Article
© Georg Thieme Verlag KG Stuttgart · New York

Development and Validation of LC-MS/MS Assay for the Quantification of Progesterone in Rat Plasma and its Application to Pharmacokinetic Studies

M. Sasaki
1   Pharmacokinetics Research Department, Developmental Research Center, ASKA Pharmaceutical Co., Ltd, Kawasaki, Japan
,
H. Ochiai
1   Pharmacokinetics Research Department, Developmental Research Center, ASKA Pharmaceutical Co., Ltd, Kawasaki, Japan
,
K. Takahashi
2   ASKA Pharma Medical Co., Ltd, Kawasaki, Japan
,
R. Suzuki
1   Pharmacokinetics Research Department, Developmental Research Center, ASKA Pharmaceutical Co., Ltd, Kawasaki, Japan
,
K. Minato
1   Pharmacokinetics Research Department, Developmental Research Center, ASKA Pharmaceutical Co., Ltd, Kawasaki, Japan
,
A. Fujikata
1   Pharmacokinetics Research Department, Developmental Research Center, ASKA Pharmaceutical Co., Ltd, Kawasaki, Japan
› Author Affiliations
Further Information

Publication History

received 10 July 2014

accepted 01 September 2014

Publication Date:
29 September 2014 (online)

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/x2) 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.

 
  • References

  • 1 Fatemi HM, Popovic-Todorovic B, Papanikolaou E et al. An update of luteal phase support in stimulated IVF cycles. Hum Reprod Update 2007; 13: 581-590
  • 2 Tavaniotou A, Smitz J, Bourgain C et al. Comparison between different routes of progesterone administration as luteal phase support in infertility treatments. Hum Reprod Update 2000; 6: 139-148
  • 3 Tai SS, Xu B, Welch MJ. Development and evaluation of a candidate reference measurement procedure for the determination of progesterone in human serum using isotope-dilution liquid chromatography/tandem mass spectrometry. Anal Chem 2006; 78: 6628-6633
  • 4 Tang YQ, Weng N. Salting-out assisted liquid-liquid extraction for bioanalysis. Bioanalysis 2013; 5: 1583-1598
  • 5 Wu H, Zhang J, Norem K et al. Simultaneous determination of a hydrophobic drug candidate and its metabolite in human plasma with salting-out assisted liquid/liquid extraction using a mass spectrometry friendly salt. J Pharm Biomed Anal 2008; 48: 1243-1248
  • 6 Houghton R, Pita CH, Ward I et al. Generic approach to validation of small-molecule LC-MS/MS biomarker assays. Bioanalysis 2009; 1: 1365-1374
  • 7 Bonnamy PJ, Benhaim A, Leymarie P. Changes of progesterone content of rat uterine flushings in relation to serum concentrations of progesterone during the oestrous cycle. J Reprod Fertil 1992; 96: 233-239
  • 8 Pepe GJ, Rothchild I. Metabolic clearance rate of progesterone: Comparison between ovariectomized, pregnant, pseudopregnant and deciduoma-bearing pseudopregnant rats. Endocrinology 1973; 93: 1200-1205
  • 9 Gangrade NK, Boudinot FD, Price JC. Pharmacokinetics of progesterone in ovariectomized rats after single dose intravenous administration. Biopharm Drug Dispos 1992; 13: 703-709
  • 10 Bawarshi-Nassar RN, Hussain AA, Crooks PA. Nasal absorption and metabolism of progesterone and 17β-estradiol in the rat. Drug Metab Dispos 1989; 17: 248-254