Simultaneous Quantification of Dexamethasone and Cortisol for the Dexamethasone Suppression Test Using LC-MS/MS: Adaptation of a Commercial CE-IVD Steroid Assay.

BACKGROUND

The dexamethasone suppression test (DST) is a key diagnostic tool for evaluating disorders of the hypothalamic-pituitary-adrenal (HPA) axis. Interpretation of DST results can be confounded by inadequate dexamethasone exposure due to pharmacokinetic variability or patient nonadherence. Simultaneous measurement of dexamethasone and cortisol by LC-MS/MS improves diagnostic accuracy by distinguishing true biological nonsuppression from insufficient drug exposure and prevents analytical interferences observed with cortisol immunoassays.

METHODS

A commercially available CE-IVD LC-MS/MS steroid panel (Chromsystems) was adapted for the simultaneous quantification of dexamethasone and cortisol in human plasma. The method was validated for linearity, accuracy, precision, selectivity, carry-over, matrix effects, and stability. Method comparison was performed using 26 clinical DST samples analyzed using immunoassay and external LC-MS testing. The proposed dexamethasone cutoff of 1.3 ng/mL (3.3 nmol/L) was evaluated in 62 patient samples.

RESULTS

The method demonstrated excellent linearity, with intra- and interassay accuracy and precision within ±15% for both analytes. No relevant carry-over or matrix effects were observed. Agreement with reference methods was 91% ± 6% for cortisol and 95% ± 7% for dexamethasone. Among the 62 DST samples, 48 showed appropriate suppression [cortisol <18.12 ng/mL (<50 nmol/L)] with dexamethasone levels above the cutoff. In contrast, 3 samples with insufficient suppression [cortisol >48.6 ng/mL (134.1 nmol/L)] had subthreshold dexamethasone levels, whereas 11 samples showed elevated cortisol levels despite sufficient dexamethasone exposure, indicating possible HPA axis dysfunction.

CONCLUSIONS

The proposed rapid and robust LC-MS/MS method enables reliable, simultaneous quantification of dexamethasone and cortisol. The assay supports accurate DST interpretation by identifying cases of pharmacokinetic variance or nonadherence.