(T1030-06-36) Liquid Chromatography – Tandem Mass Spectrometry for Quantification of Zymostenol and Lathosterol in Human Serum with High Specificity and Low Background Noise

Purpose: Zymostenol and Lathosterol are precursors for cholesterol biosynthesis. They are often used as biomarkers for certain aging related diseases. Zymostenol and Lathosterol have the same molecular weight and similar structures, which makes them very difficult to separate  in biological matrix. In order to support clinic studies, we developed and validated a specific method for Zymostenol and Lathosterol in human serum. In this method, Zymostenol and Lathosterol were separated by a reverse phase column, and monitored in positive APCI MRM mode on a Sciex 5500 LC-MS/MS System. This method showed high specificity and low background noise for Zymostenol and Lathosterol in human serum.

Methods: Zymosternol-d₇ and Lathosterol-d₇ were used as the internal standards for Zymostenol and Lathosterol. Analytes and internal standards were extracted from human serum using liquid-liquid extraction with hexane.The LC-MS/MS analysis was carried out with a Sciex Triple Quad 5500 mass spectrometer coupled with a Waters Acquity UPLC system. The chromatographic separation was achieved on a Phenomenex Kinetex® F5, 2.6 µm, 150 × 2.1 mm column with run time (22 min). The mass spectrometer was operated in positive APCI mode. The MRM transitions were monitored at m/z 369.4 ® 161.2 for Zymostenol; 376.4 ® 95.0 for its internal standard, Zymostenol-d_7; .at m/z 369.3 ® 161.1 for Lathosterol, and 376.4 ® 95.1 for its internal standard, Lathosterol-d₇.  A linear regression (1/concentration²) was established from 0.1 µg/mL to 10 µg/mL for Zymostenol and from 0.2 µg/mL to 20 µg/mL for Lathosterol in human serum.

Results: Excellent linearity was obtained with a correlation coefficient ≥0.9966 for Zymostenol and ≥0.9932 for Lathosterol.  The high dynamic calibration range was reached due to eliminated background noise.  For Zymostenol, including LLOQ, the intra‑day CV ranged from 1.2% to 14.8% and the biases of the means ranged from -10.4% to 8.0%, the inter‑day CV ranged from 3.8% to 11.9% and the biases of the means ranged from -6.5% to -2.3%; For Lathosterol, including LLOQ, the intra‑day CV ranged from 1.5% to 7.7% and the biases of the means ranged from -10.1% to 18.4%, the inter‑day CV ranged from 3.1% to 12.5% and the biases of the means ranged from -9.2% to 2.0%,. The method has been successfully validated and applied to the analysis of the clinical samples.

Conclusion: A high specificity and low background noise validated method for the determination of Zymostenol and Lathosterol in human serum has been established with short run (22 min) and low sample volume (0.1mL).

References: Molecules 2020, 25, 4116; doi:10.3390/molecules25184116