MSACL 2023 Abstract

Introduction
Intrahepatic Cholestasis of Pregnancy (ICP) is a medical condition occurring during pregnancy which can lead to complications for both the mother and the baby. Patients with ICP often experience symptoms late in pregnancy and are diagnosed in part with measurement of serum bile acids. There is considerable interest in identifying biomarkers that would allow earlier detection of patients at risk of ICP. It has been previously reported that sulfated progesterone metabolites (“ProgS”) have promise as an early stage biomarker to predict ICP. The five metabolites validated were 5b-Pregnan-3a20a-diol Sulfate (PM3S), 5a-Pregnan-3a-ol-20-one Sulfate (PM4S), 5a-Pregnan-3b-ol-20-one- Sulfate (PM5S), 5a-Pregnan-3a20a-diol Disulfate (PM2DiS) and 5b-Pregnan-3a20a-diol Disulfate (PM3DiS).

Methods
An analytical method was developed using a TX-4 HPLC system (Thermo-Fisher) with Agilent® 1200 pumps (Agilent Technologies, Inc.) and a Sciex® 5500 (Danaher) triple quadrupole mass spectrometer.
Calibration curves based on purified naturally labeled compounds (ASI, Inc) were prepared in 6% albumin. Sample preparation consisted of an initial isotope dilution using deuterated heavy isotope internal standards (ASI, Inc), followed by protein precipitation, evaporation and reconstitution. Three independent reversed phase analytical methods were used with alkaline water/acetonitrile solvent gradients to achieve chromatographic separation of the five ProgS species. Negative mode electrospray ionization (ESI) was used for detection in Multiple Reaction Monitoring (MRM) mode.

Results
Analytical range was 1 – 500 ng/mL for each analyte (up to 2,500 ng/mL with dilution). Inter-assay precision ranged from 7.2–10.4% and inter-assay accuracy ranged from 93.6-106.7%. Reference intervals for adult females were developed using residual specimens. No interference was observed in the presence of hemolysis, icteric or lipemia. No difference in result was observed when using collection tubes containing EDTA, Heparin or gel barriers. Specimen stability was interrogated at ambient, refrigerated and frozen conditions.

Conclusion
A fully validated assay for five sulfated metabolites of progesterone has been validated and may be useful in the investigation and early risk detection for intrahepatic cholestasis of pregnancy.