Dexamethasone suppression test: development of a method for simultaneous determination of cortisol and dexamethasone in human plasma by liquid chromatography/tandem mass spectrometry.

BACKGROUND: Dexamethasone is a synthetic glucocorticoid and is analogous to cortisol. It is used in the low-dose overnight dexamethasone suppression test (LDODST) to diagnose hypercortisolism in patients suspected to be suffering from Cushing's syndrome (CS). Measuring plasma dexamethasone in conjunction with measuring the amount of cortisol following the LDODST may allow clinicians to improve the diagnosis of CS. METHODS: Plasma samples were cleaned up by solid-phase extraction before analysis. Liquid chromatographic separation was carried out under reversed-phase conditions prior to detection by tandem mass spectrometry. The analytes were determined in the presence of deuterated internal standards cortisol-d4 and dexamethasone-d4. RESULTS: Limit of quantitation (LOQ) was 1.89 nmol/L for dexamethasone and <0.02 µmol/L for cortisol. Recoveries of both analytes ranged from 80.2% to 114.4%. Intra- and interassay coefficients of variation were <15%. The concentration of dexamethasone and cortisol was determined in 62 patients after performing LDODST. Dexamethasone concentrations ranged from 3.0 to 21.5 nmol/L (median 7.4 nmol/L) for 57 of these samples. For five patients the concentration was 0.22 µmol/L). CONCLUSIONS: A method for the simultaneous measurement of dexamethasone and cortisol in human plasma by liquid chromatography/tandem mass spectrometry has been developed and validated. The method is suitable for controlling the compliance to the LDODST and for determining the cortisol plasma concentration after the test. The interpretation of LDODSTs was improved by the simultaneous determination of both analytes.

Beta thalassemia IVS-I-5(G-->C) heterozygosity masked by the presence of HbJ-Meerut in a Dutch-Indian patient.

We describe the genotype/phenotype correlation in a 35 year old anemic female referred to our laboratory because a fast eluting minor fraction on HPLC, mild hemolysis and hematological parameters suggesting a Thalassemia trait, eventually in combination with iron depletion. Direct sequencing of the alpha globin genes revealed heterozygosity for HbJ-Meerut, a Glu-->Ala substitution at residue 120 not justifying the hematological parameters. No other point mutations were found on the alpha genes and Gap-PCR excluded the 6 common deletion defects. Direct sequencing of the beta-globin genes revealed the IVS-I-5 (G-->C) transversion in absence of the elevated HbA2 levels usually measured in carriers of this beta-Thalassemia mutation. The HbA2 tetramer in the presence of HbJ-Meerut divides in two parts. One alphaN2/delta2 migrating on the right spot on HPLC. The other alphaJ2/delta2 migrating under the HbA fraction. Classic alkaline electrophoresis and the modern capillary electrophoresis CE showed these two tetramers and the reduction of the elevated HbA2 level of the beta-Thalassemia trait by at least 20% due to HbA2 Meerut.