High Performance Liquid Chromatography Mass Spectrometry Series Method For Salivary Free Cortisol Level In Diagnosis Of Cushing Syndrome

Objectives. Cushing syndrome is a series of symptoms and signs resulting from excess glucocorticoid due to the disorder of hypothalamic-pituitary-adrenal axis, which is caused by a variety of reasons. The diagnosis of Cushing syndrome mainly depends on the cortisol level in serum and urine, while there are several inevitable factors which effect the reliability of the current testing measures. Since it is necessary for serum cortisol to bind corticosteroid-binding globulin and albumin, only 3%~5% of total serum cortisol is free molecules, which means the test of serum cortisol level is affected by liver function and pregnancy(1 Guang Ning). Besides, the sample collection and volume calculation of 24-hour urine are not easy. Salivary cortisol is different from serum and urine cortisol, which exists mainly as free molecules. Also, it has been proved that salivary cortisol is parallel to serum cortisol level at the rate of 70%. Other advantage of salivary cortisol test is the easy access to sample collection. Several studies about the test of salivary cortisol have been under research abroad in order to reduce the suffering of patients(2 Brazil).Methods: We continuously collected the saliva samples and clinical records of patients who were diagnose of Cushing syndrome during October to November of 2015 in Peking Union Medical College Hospital. Healthy people were selected as the control group. After being dealt with by proteomics technology, the salivary cortisol level of the samples were tested through high performance liquid chromatography mass spectrometry series method (HPLC/MS/MS). Statistical analysis was performed with IBM SPSS Statistics 20.0.Results: We included 10 patients diagnosed of Cushing syndrome and 7 healthy people as control group.(1) We established the reliable and effective extraction method of saliva.(2) We also established both external and internal standard methods of mass spectrometer method for salivary cortisol.(3) The cortisol circadian in healthy people is high level during daytime and low level at night.(4) The cortisol circadian in Cushing syndrome patients are absent.(5) The results of cortisol circadian through the tests of serum total cortisol and salivary cortisol in Cushing syndrome patients match with each other.Conclusions: Salivary cortisol testing is a reliable and effective technology. HPLC-MS allows the testing of cortisol level through a scale of 1~100ng/ml, which has a better operability. Due to the reality of low incidence of Cushing syndrome in China, this research only contains a small sample size. A further repetitive research with larger sample size is needed in the future in order to make this new test method of salivary cortisol into clinical usage.