(M1330-01-01) Quantitation of Total and Free Cortisol in Cynomolgus Monkey Serum Using LC-MS/MS

Purpose: Cortisol is a hormone that reflects an individual's stress level in response to various environmental stimuli. Chronic exposure to elevated cortisol has been strongly linked to long-term health conditions, including cardiovascular diseases and obesity. In many stress-related studies, cortisol levels in saliva, blood, or urine are commonly measured as biomarkers. However, 80%~90% of total cortisol binds to transcortin (corticosteroid-binding globulin (CBG)), about 10% of total cortisol binds to albumin, and merely 5% of total cortisol exists in its bioactive form as unbound free cortisol.  (Choi, 2022). Therefore, accurate measurement of free cortisol in biofluid samples is critical in supporting pharmacological study and clinical decisions. Traditionally, free cortisol has been extracted using dialysis or ultrafiltration and quantified by immunoassay (Vogeser, 2007). These methods are labor-intensive and require relatively large sample volumes (approximately 1 mL). Here, an innovative and rapid LC-MS/MS method has been developed for the quantification of total and free cortisol in cynomolgus monkey serum. The method requires only 100 µL of sample and allows for simultaneous detection of both cortisol forms, achieving a sensitivity of 1 ng/mL.<

Methods: A filter with 30 kDa molecular weight cutoff was utilized to separate the free cortisol via ultrafiltration. Unfiltered samples were used to measure total cortisol, and ultrafiltrate from the same sample was used to measure free cortisol. Cortisol was extracted by a protein precipitation method using cortisol-d4 as internal standard. Extracted samples were analyzed using a ScieX 5500+ platform paired Shimadzu nexera-2 HPLC system with a Waters Acquity BEH C18 column (50× <21 mm, 1.7µm). We monitored the following transitions: Cortisol (363.2-121.0) and Cortisol-d4 (367.2-121.0). A surrogate matrix (0.05% Tween-80) was used to prepare the calibration standards and quality control samples, as cortisol endogenously presents in cynomolgus monkey serum. Tween-80 was used to reduce non-specific binding in the surrogate matrix.

Results: Precision and Accuracy: To assess extraction recovery, cortisol-d4 was used as a surrogate in both the surrogate matrix and blank monkey serum. The results demonstrated comparable recovery across both matrices using the current extraction method. The quantitation method covers a calibration range of 1.00~1,000 ng/mL, encompassing both the total cortisol levels and free cortisol levels in 6 individual lots of monkey serums. Linearity and representative chromatograms are in Figure 1. Stability and Dilution integrity: Commercially available monkey serum was used to evaluate the stability of total and free cortisol across four freeze/thaw cycles over a 5-day storage period at −80 °C (Figure 2-A). The results demonstrated that neither freeze/thaw cycles nor short-term storage affected the free cortisol fraction, supporting the reliability of the data under typical sample handling conditions. However, a shift in the free cortisol fraction was observed when serum samples were diluted with the surrogate matrix, while total cortisol levels remained unchanged (Figure 2-B), This highlights the importance of avoiding dilution with surrogate matrix when measuring free cortisol along with total cortisol. Recovery test: Cortisol recovery during extraction was evaluated for both total and free cortisol (Figure 3). For total cortisol, spike-in recovery was assessed by adding 100 ng/mL of cortisol to 6 lots of monkey serum. For free cortisol, cortisol-d4 was spiked into ultrafiltered monkey serum. All samples were then subjected to ultrafiltration, and the peak areas of cortisol or cortisol-d4 were measured before and after filtration. Recovery was determined to be 107.7% for total cortisol and 88.6% for free cortisol, indicating acceptable extraction efficiency for both forms.

Conclusion: This method successfully quantifies both total and free cortisol simultaneously from cynomolgus monkey serum, demonstrating good precision and accuracy. When samples are analyzed without dilution, both total and free cortisol levels in monkey serum remain stable under typical laboratory handling and storage conditions.

References: Choi, M. H. (2022). Clinical and technical aspects in free cortisol measurement. . Endocrinology and Metabolism, 37(4), 599-607.
SUEDA, K. S. (1986). Comparative study of primates' transcortin: immunoreactivity and steroid-binding activity. Endocrinologia japonica, 33(2), 143-150.
Vogeser, M. M. (2007). Free serum cortisol: quantification applying equilibrium dialysis or ultrafiltration and an automated immunoassay system. Clin Chem Lab Med, 521-525.

Figure 1: A. Representative Linearity and B Representative chromatograms for matrix blank, IS only, and LLOQ from qualification run.

Figure 2: A. Total cortisol level before and after 5-day, 4 F/T cycles, and B. Free cortisol level before and after 5-day, 4 F/T cycles. No significant change observed. B. Total cortisol level before and after diluted 2X using surrogate matrix remains similar, Free cortisol level significantly increased after dilution 2X using surrogate matrix. No significant difference in overall comparison between total cortisol before and after dilution. *: P<0.05.

Figure 3: The spike in cortisol recovery was ~107. 7% in overall 6 lots of cynomolgus monkey serum, and the recovery of isotope-labeled cortisol from ultrafiltration was ~ 88.6% in Lot 7 of monkey serum.