This assay was originally developed for the World Health Organization for use in clinical trials and pharmacokinetic studies of contraceptive steroid and hormone replacement therapy (HRT) preparations. The method is an extraction RIA and not subject to the well-documented interference problems associated with direct serum/plasma immunoassays.

Medroxyprogesterone Acetate in the sample (200µL of serum or plasma) is extracted with diethyl ether. The extract is dried and redissolved in assay buffer. The redissolved extract is assayed for Medroxyprogesterone Acetate by 3H-RIA (tritium labelled radioimmunoassay with charcoal separation). All samples, standards and quality control specimens are assayed in duplicate.

The assay is controlled through the use of at least 3 Internal Quality Control samples in every run. In addition the performance of every run is closely monitored for various parameters including imprecision, antibody binding of tracer, non-specific binding and reproducibility of the standard curve.

• Standard Curve: The range of the standard curve is 0 to 6500 pmol/L (2519 pg/mL).
• Precision: The between-run precision of the assay is generally less than 10% CV.
• Sensitivity: The assay reliably measures Medroxyprogesterone Acetate concentrations as low as 150 pmol/L (58 pg/mL).

The cross-reactions of the assay (estimated as the relative potency causing a 50% displacement of zero binding) for each substance tested are shown below:

Substance % Cross Reaction Medroxyprogesterone Acetate 100 6b-hydroxyprovera 64.6 6-dehydroprovera (megestrol acetate) 9.5 17a-hydroxy 6a-methyl progesterone 0.02 20a-dihydroprogesterone <0.02 20b-dihydroprogesterone <0.02 17a-hydroxyprogesterone <0.02

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