LC-MS/MS Method for Progesterone Quantitation in Cattle Plasma

Title: Quantitation of Progesterone in Bovine Plasma Using LC-MS/MS: A Comparison with Radioimmunoassay

Journal: Theriogenology

Published: 2012

Background

Progesterone (P4) quantitation in cattle is essential for effective estrus synchronization protocols that enhance reproductive efficiency. Traditionally, radioimmunoassay (RIA) has been used for this purpose due to its simplicity and sensitivity. However, RIA suffers from specificity issues, including cross-reactivity and matrix interferences. Liquid chromatography-tandem mass spectrometry (LC-MS/MS), particularly with atmospheric pressure photoionization (APPI), offers high specificity and avoids the drawbacks of RIA. This study aimed to develop and validate a rapid and efficient LC-MS/MS method for P4 quantitation in bovine plasma without the need for derivatization, and to compare its performance against RIA using samples from cows treated with different commercial intravaginal P4-releasing devices.

Materials & Methods

An LC-MS/MS method using atmospheric pressure photoionization (APPI) was developed and validated for quantifying progesterone (P4) in bovine plasma. Medroxyprogesterone acetate (MPA) was used as an internal standard. Plasma samples were prepared by liquid-liquid extraction without derivatization. Specificity, linearity, accuracy, precision, recovery, matrix effect, limits of detection (LOD), and quantitation (LOQ) were evaluated according to international bioanalytical validation guidelines.

For comparison, P4 was also quantified by radioimmunoassay (RIA). Both methods were applied to plasma samples from ovariectomized (OVX) cows treated with three commercial intravaginal progesterone-releasing devices (IPRDs) over 192 hours.

Results

LC-MS/MS Validation:

P4 was detected as [M+H]+ (m/z 315), with two major fragments (m/z 109 and 123) used for quantitation.
No matrix interference was observed; specificity was confirmed.
Calibration curve was linear (R² = 0.999) from 0.25 to 10 ng/mL.
LOD and LOQ were 0.08 ng/mL and 0.25 ng/mL, respectively.
Intra- and interassay accuracy ranged from 90.0% to 103.2%; precision (RSD) ranged from 2.3% to 10.0%.
Recovery rates were 83%–89%; matrix effect was minimal (< 10%).

Application to IPRD Devices:

P4 kinetics for all three devices were similar (P > 0.05), with peak levels reached within 4–6 hours and a steady state between 96–192 hours.
Cmax ranged from 5.91 to 7.04 ng/mL.

RIA Performance:

High sensitivity (0.003 nmol/L), with intra- and interassay CVs of 2.03% and 4.72%, respectively.
RIA consistently showed higher P4 values than LC-MS/MS due to possible cross-reactivity and matrix effects.

Method Comparison:

P4 kinetics measured by both methods were similar, but LC-MS/MS offered greater specificity and avoided overestimation observed in RIA.

APPI mass spectrum of progesterone showing a peak at m/z 315 and its molecular structure; MS/MS spectrum displays fragment ions at m/z 109 and 123 for quantification. (A) Representative APPI mass spectrum of methanolic progesterone (P4), with the protonated molecular ion [P4 + H]+ at m/z 315. Inset shows the molecular structure of P4. (B) APPI-MS/MS spectrum of [P4 + H]+ (m/z 315), highlighting major fragment ions at m/z 109 and 123, used for P4 quantification via LC-MS/MS.

Chromatograms showing the selectivity of the liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method for progesterone (P4) quantitation. (A) A blank plasma extraction and (B) a plasma extract that contains P4 and medroxyprogesterone 17-acetate (MPA) at the limit of quantitation (LOQ) are represented.

Mean values of progesterone (P4) concentration provided by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in three pairs of cows; each pair had an intravaginal progesterone-releasing device (A, B, and C) in place for 192 h (P > 0.05).

Mean values from the comparison of radioimmunoassay (RIA) and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) progesterone (P4) quantitation in plasma samples from cows (N = 2) with an intravaginal progesterone releasing device A (a), B (b), or C (c) in place for 192 h.

Reference

Fernandes, R. M. T., et al. "LC-MS/MS quantitation of plasma progesterone in cattle." Theriogenology 76.7 (2011): 1266-1274. https://doi.org/10.1016/j.theriogenology.2011.05.033

* For Research Use Only. Not for use in diagnostic procedures.

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