Bedtime Salivary Cortisol and Cortisone by LC-MS/MS in Healthy Adult Subjects: Evaluation of Sampling Time

Abstract

The measurement of late-night salivary cortisol is a mainstay in the diagnosis of Cushing syndrome. Furthermore, the measurement of salivary cortisol is useful in assessing the cortisol awakening response. Because the salivary glands express 11-β-hydroxysteroid dehydrogenase, the measurement of salivary cortisone may improve the performance of salivary corticosteroid measurements. We measured salivary cortisol by enzyme immunoassay (EIA) and salivary cortisol and cortisone by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in only 50 µL of saliva sampled from 54 healthy subjects (aged 20 to 64 years). We allowed patients to sample at their normal bedtime (2025 to 2400 hours) to answer a common question as to whether sampling at the normal bedtime is equivalent to the standard required sampling at 2300 to 2400 hours. We found that the salivary cortisol and cortisone results by LC-MS/MS correlated well with salivary cortisol measured with the US Food and Drug Administration-cleared EIA. Furthermore, the upper limit of normal of salivary cortisol by EIA for bedtime samples was lower than the previously published upper limit of normal with sampling required at 2300 to 2400 hours. There were no significant effects of age or sex on any of the salivary steroid measurements. We conclude that (i) salivary cortisol and cortisone can be reliably measured by LC-MS/MS in small volumes of saliva and (ii) that patients can be evaluated using saliva sampled at their normal bedtime, rather than being required to stay awake until 2300 to 2400 hours.

Prospective Evaluation of Late-Night Salivary Cortisol and Cortisone by EIA and LC-MS/MS in Suspected Cushing Syndrome

“…A total of 1453 salivary samples from 705 patients were collected during this time using a standard Salivette (Sarstedt, Newton, NC). In addition to the standard FDA-cleared EIA-F performed on these samples [ 27 ], each sample underwent additional testing to obtain LCMS-F and LCMS-E concentrations [ 26 ]. The 1453 samples were first subdivided into 2 groups ( Fig.…”

“…1 ): samples with all normal late-night salivary steroid data and samples with at least one abnormal value among the 3 salivary analyses (EIA-F, LCMS-F, and/or LCMS-E). Abnormal late-night salivary results were defined as EIA-F ≥3.3 nmol/L; LCMS-F ≥2.8 nmol/L, and LCMS-E ≥8.7 nmol/L based on our previously published contemporaneous normative data using the same analytic methods in samples collected in the same manner [ 26 ]. We identified 501 samples from a total of 283 patients with at least one abnormal salivary result and 952 samples in 422 patients without increases in any salivary result.…”

“…Salivary cortisol was measured by EIA using an FDA-cleared method (Salimetrics, State College, PA) as described in detail previously [ 26 , 37 ]. The lower detection limit is 0.3 nmol/L.…”

“…Salivary cortisol and cortisone were measured by LC-MS/MS as described by us in detail previously [ 26 ]. The functional sensitivity, set at a threshold CV of 10%, was 0.053 (SD, 0.004) nmol/L for cortisol and 0.053 (SD, 0.002) nmol/L for cortisone.…”

“…Late-night salivary cortisol (LNSC) measured by immunoassay is a useful and simple means to screen patients for suspected hypercortisolism and has been recommended by expert guidelines as an important first-line diagnostic test [ 24 , 25 ]. A commonly used cortisol enzyme immunoassay (EIA-F) is inexpensive, Food and Drug Administration (FDA)-cleared, available internationally, and readily performed in clinical laboratories [ 26 , 27 ]. Furthermore, high-throughput platform cortisol immunoassay systems are widely available at reference laboratories around the world, although there are significant differences in their performance and reference ranges [ 28 , 29 ].…”