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.

LC-MS/MS Method for Rapid Quantification of Progesterone in Rabbit Plasma and Its Application in a Pharmacokinetic Study of the Transdermal Formulation

“…The LOD and LOQ were at 0.3 and 1 ng/mL, respectively, which were low enough for determining progesterone concentration in rabbit plasma. The LOD of the method was not as low as that of Sasaki's study [ 13 ], but it is fit for the purpose of analyzing the level of progesterone in plasma, which is usually higher than 1 ng/mL. The method was linear from 1 to 200 ng/mL with the coefficient of determination ( R 2 ) being higher than 0.99 ( Figure 3 ).…”

“…The recovery when using MgSO 4 was low because some water might still be in the acetonitrile layer. Although the salting-out assisted liquid/liquid extraction (SALLE), described by Sasaki et al [ 13 ], used CH 3 COONH 4 as a salting-out agent, this study showed that using CH 3 COONH 4 will result in incomplete separation between aqueous and organic phase.…”

“…With ammonium acetate as the salting-out agent, this method was applied to analyze progesterone in rat plasma from 0.05 to 20 ng/mL. The heavy matrix effect was controlled using the calibration curve on the surrogate matrix (water) [ 13 ].…”

Assessing the precision of point-of-care immunological analyzer for swift progesterone measurement and guidance for determining the optimal breeding time in bitches

“…Within the domain of veterinary practice, diverse techniques exist for quantifying sP4, encompassing radioimmunoassay (RIA) [14,15] liquid chromatography-tandem mass spectrometry (LC-MS) [16][17][18], and chemiluminescence immunoassay (CLIA) [15,[19][20][21]. Furthermore, recent progress encompasses the introduction of point-of-care sP4 measurement analyzers, exemplified by the rapid fluorescence immunochromatography assay (RFICA) and surface plasmon field-enhanced fluorescence spectroscopy (SPFS) [22][23][24][25].…”

Accuracy and precision guidelines for optimal breeding time in bitches using in-house progesterone measurement compared with chemiluminescent microparticle immunoassay

“…Several qualitative, quantitative, and semi-quantitative methods for progesterone measurements are available for veterinarian practitioners. In general, quantitative assays are preferred, such as radioimmunoassay (RIA) [9,10], liquid chromatography-tandem mass spectrometry [11][12][13], the more recently introduced enzyme-linked fluorescence assay, and chemiluminescence immunoassay (CLIA) [10,[14][15][16].…”