Development and validation of a liquid chromatography-tandem mass spectrometry method for the quantification of twenty-five steroids in equine serum.

Overview

• This research developed and validated a precise liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to simultaneously measure 25 different steroids in horse blood serum.
• The method addresses limitations of current immunoassays and could improve monitoring of equine pregnancy and related conditions.

Background and Importance

• Steroids play crucial roles in equine physiology, especially during pregnancy, where they serve as important biomarkers.
• Existing steroid measurement techniques mainly use immunoassays, which often suffer from problems like cross-reactivity, leading to inaccurate results.
• Accurate and specific quantification methods are needed to better monitor equine pregnancy, diagnose placentitis, and determine fetal sex.

Method Development

• Researchers designed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) protocol capable of detecting and quantifying 25 steroids simultaneously, including progestogens, corticosteroids, androgens, and estrogens.
• The sample preparation involved:
  • Protein precipitation to extract steroids from serum.
  • Evaporation to concentrate the analytes.
  • Derivatization (chemical modification) to enhance detection sensitivity and specificity.
  • Reconstitution of samples before introduction into the LC-MS/MS system.
• A surrogate matrix (a replacement fluid free of endogenous steroids) was used in calibration and validation to prevent interference from naturally occurring steroids in samples.

Validation Process

• Validation followed Clinical and Laboratory Standards Institute (CLSI) guidelines, adapted to this context, evaluating:
  • Linearity: ability to produce results proportional to concentration.
  • Trueness: accuracy or closeness to true steroid levels.
  • Precision: reproducibility within and between days.
  • Limits of detection and quantification (LOD and LOQ): smallest amounts reliably measured.
  • Measurement uncertainty: margin of error estimation.
  • Recovery: efficiency of extracting steroids from serum.
  • Matrix effects: influence of serum components on measurement accuracy.
  • Carryover: contamination from previous samples.
  • Selectivity: ability to measure target steroids without interference.
  • Stability: steroid stability during sample handling and storage.
• Calibration curves employed the best weighted linear or quadratic regression models to optimize data fitting.

Validation Results

• Linearity was excellent for all steroids with R values greater than 0.990, indicating strong proportionality between concentration and signal.
• Trueness ranged from -9.0% (slight underestimation) to 2.3% (slight overestimation), demonstrating high accuracy.
• Precision was very good with intra- and inter-day variability under 6.3%, ensuring consistent results across runs.
• Lower limits of quantification varied from 2.07 to 2250 pg/mL, consistent with physiological concentrations in horses.
• Extraction recovery averaged between 24.3% and 114.9%, reflecting efficient and acceptable steroid extraction from serum.
• Matrix effects were within acceptable ranges, indicating minimal interference from serum components.
• Accuracy during recovery tests ranged from 94.4% to 98.9%, validating the method’s reliability.
• No carryover or interfering compounds were detected, confirming measurement specificity.
• Measurement uncertainty for all steroids remained below 15%, reflecting good confidence in reported values.

Significance and Potential Applications

• This is the first LC-MS/MS method partially validated according to CLSI standards for quantifying 24 steroids in horse serum.
• The method offers enhanced sensitivity and specificity over immunoassays, overcoming their known drawbacks.
• It provides a robust analytical tool that can be used for:
  • Monitoring pregnancy status in mares by tracking steroid profiles.
  • Diagnosing placentitis, a placental inflammation that can threaten pregnancy.
  • Determining fetal sex based on steroid hormones.
• The validated approach can support veterinary diagnostics, research into equine reproductive health, and improve outcomes in equine breeding management.