Steroid profiling by gas chromatography-mass spectrometry and high performance liquid chromatography-mass spectrometry for adrenal diseases

Abstract

The ability to measure steroid hormone concentrations in blood and urine specimens is central to the diagnosis and proper treatment of adrenal diseases. The traditional approach has been to assay each steroid hormone, precursor, or metabolite using individual aliquots of serum, each with a separate immunoassay. For complex diseases, such as congenital adrenal hyperplasia and adrenocortical cancer, in which the assay of several steroids is essential for management, this approach is time consuming and costly, in addition to using large amounts of serum. Gas chromatography/mass spectrometry profiling of steroid metabolites in urine has been employed for many years but only in a small number of specialized laboratories and suffers from slow throughput. The advent of commercial high-performance liquid chromatography instruments coupled to tandem mass spectrometers offers the potential for medium- to high-throughput profiling of serum steroids using small quantities of sample. Here, we review the physical principles of mass spectrometry, the instrumentation used for these techniques, the terminology used in this field and applications to steroid analysis.

Fig. 1

a Schematic diagram of a simple mass spectrometer with source, quadrupole, and detector, sorting ions by m/z. b Generation of a mass spectrum for a pure compound. The molecular ions fragment, and the charged fragments are separated by the quadrupole and quantitated by the detector, yielding a standard mass spectrum. c Mass spectrum of testosterone. d Schematic diagram of a collision cell. Following ionization, molecular ions are accelerated into the collision cell via electrostatic repulsion where they collide with gas molecules in the collision cell through a process called collision induced dissociation (CID). CID imparts internal energy to the molecular ion, which induces fragmentation

Fig. 2

Schematic diagram of triple-quadrupole LC-MS/MS process and instrumentation. In the upper right, pre-analytical sample cleanup with optional derivatization prepares the sample for injection. Analytes are resolved on the HPLC column, and the effluent is introduced into the mass spectrometer. Steroid molecules are ionized in the source, selected in Q1, fragmented in the collision cell (Q2), and sorted in Q3. The detector monitors the intensity of the chosen (quantifier) Q3 fragments derived from the ions selected in Q1 at the appropriate times in the chromatogram to perform the measurement

Fig. 3

Simultaneous quantitation of 11-deoxycorticosterone (11-DOC), testosterone, and 17-hydroxyprogesterone (17OH-Prog) by LC-MS/MS. a Chromatogram showing tracked MRMs for the three analytes. Note that 11-DOC and 17OH-Prog are isobaric and use identical MRMs; however, chromatography separation allows individual quantitation. In addition, T and 17OH-Prog overlap chromatographically, but the different MRMs allows individual quantitation. b Mass spectrum of 17OH-Prog, showing small molecular ion (331.0, [M + H]⁺) and fragments, with the 108.9 (109) amu fragment as base peak used for quantitation