Cortisol in Oral Fluids as a Biomarker for Stress Assessment

Authors: Mégane Moreau, Serge Auger, Pierre Picard and Jean Lacoursière
Themes: High-Throughput, Oral Fluids, Cortisol, LDTD-MS/MS
Download PDF


The rapid quantification of cortisol in oral fluids can be useful for the screening of adrenal pathologies like Cushing’s syndrome1,2. Saliva analysis of cortisol can be use as biomarker for stress assessment. It was shown that salivary cortisol can provide important information about hypothalamic-pituitary-adrenal (HPA) axis activity under normal and stress conditions3.

Our goal for this application note is to develop a diagnostic tool to rapidly quantify cortisol in saliva, which will allow the rapid diagnosis of certain adrenal pathologies or as a biomarker for stress assessment.

LDTD-MS/MS offers specificity combined with an ultra-fast analysis for an unrivaled quantification method. To develop this application, we focused on performing an efficient sample preparation, which will increase the speed of the analysis process.


Sample Preparation Method

Stock solutions of Cortisol were prepared in methanol at 1 mg/mL. Then, artificial saliva samples were spiked to generate a calibration curve and QCs.


Automated Sample Extraction

The Azeo Liquid Handler (Figure 3) is used to extract the samples using the following conditions:



Figure 3 – Automated extraction system



LDTD®-MS/MS Parameters


Model: Luxon S-960, Phytronix

Carrier gas: 6.0 L/min (air)

Laser pattern:



MS model: QTrap® System 5500, Sciex.

IonSpray Voltage: -4200

Temperature, GS1 and GS2 set to zero.

Scan Time: 50 msec

Analysis Method: Negative MRM mode


Table 1 – MRM transitions for LDTD-MS/MS

Transition CE (V)
Cortisol 361.2 → 331.1 -15
Cortisol-d6 367.1 → 335.0 -15


Results and Discussion

Validation Test

Calibration curves ranging from 1 to 20 ng/mL were prepared in synthetic saliva. Sets of QCs were prepared (LLOQ, QCL, QCM, QCH, and ULOQ) in synthetic saliva. Replicate extractions were deposited onto a LazWell™ plate and dried before analysis. The peak area against the internal standard (IS) ratio was used to normalize the signal.



The calibration curves were plotted using the peak area ratio and the nominal concentration of standards. For the linearity test, the following acceptance criteria was used:


Table 2 shows the inter-day correlation coefficients for Cortisol. Values greater than 0.995 are obtained. Figure 4 shows a typical calibration curve result for Cortisol.


automated extraction system
Figure 4 – Cortisol calibration curve



Table 2 – Inter-day calibration curve correlation coefficients

  Cortisol R
Curve 1 Y = 0.15169 X + 0.06757 0.99623
Curve 2 Y = 0.15002 X + 0.066340 0.99700
Curve 3 Y = 0.13005 X + 0.06599 0.99741
Curve 4 Y = 0.08740 X + 0.06370 0.99789
Curve 5 Y = 0.11970 X + 0.05502 0.99517


Precision and Accuracy

For the accuracy and precision evaluation, the following acceptance criteria were used:


For the intra and inter-run precision and accuracy experiment, each QC was analyzed in sextuplicate, on four different runs. Table 3 and 4 show the intra and inter-run precision and accuracy results for Cortisol. The obtained %CV was below 15% and the accuracy was within 15% of the nominal value.


Table 3 – Intra-Run Precision and Accuracy of Cortisol

Conc (ng/mL) 1 3 10 15 20
N 6 6 6 6 6
Mean (ng/mL) 1.0 2.7 9.6 14.9 21.4
%CV 5.0 3.5 4.3 3.0 2.6
% Nom 102.0 89.9 96.3 99.5 107.0



Table 4 – Inter-Run Precision and Accuracy of Cortisol

Conc (ng/mL) 1 3 10 15 20
N 12 30 30 30 12
Mean (ng/mL) 1.0 2.8 9.4 14.9 20.8
%CV 7.6 11.0 7.4 9.6 10.9
% Nom 104.2 93.3 94.2 99.3 104.0


Wet Stability of Sample Extracts

Following the extraction, sample extracts are kept at 4°C in closed containers protected from light. After five days, sample extracts are spotted on a LazWell™ plate, dried and analyzed. The precision and accuracy results of QCs in synthetic saliva samples are reported in Figure 5. All the results are within the acceptable criteria range for one week at 4°C.


Dry Stability of Samples Spotted in LazWell™

Extracted samples are spotted onto a LazWell™ plate, dried and kept at room temperature for 2 hours before analysis. The precision and accuracy results of QCs in synthetic saliva samples are reported in Figure 5. the results are within the acceptable criteria range for 2 hours at room temperature.


Precision data




accuracy data
Figure 5 – Wet and Dry Stability of Cortisol in Synthetic Saliva Sample. A) Precision data; B) Accuracy data


Cortisol study

The study of cortisol in saliva over a period of 24 hours was carried out. The results obtained agree with those obtained in Chan et al. (2020).  The level of cortisol rises rapidly when an individual wakes up and decreases throughout the day until bedtime.


Cortisol Study
Figure 6 – Cortisol study (one day)


The Luxon Ion Source combined with the Sciex QTrap® 5500 mass spectrometer system enables the rapid analysis of cortisol in oral fluids in less than 10 seconds.