Optimizing adrenal vein sampling in primary aldosteronism subtyping through LC–MS/MS and secretion ratios of aldosterone, 18-oxocortisol, and 18-hydroxycortisol

Abstract

Adrenal venous sampling (AVS) is the gold standard for identifying curable unilateral aldosterone excess in primary aldosteronism (PA). Studies have demonstrated the value of steroid profiling through liquid chromatography–tandem mass spectrometry (LC–MS/MS) in AVS interpretation. First, the performance of LC–MS/MS and immunoassay in assessing selectivity and lateralization was compared. Second, the utility of the proportion of individual steroids in adrenal veins in subtyping PA was analyzed. We enrolled 75 consecutive patients with PA who underwent AVS between 2020 and 2021. Fifteen adrenal steroids were analyzed in peripheral and adrenal veins through LC–MS/MS before and after adrenocorticotropic hormone (ACTH) stimulation. Through selectivity index that was based on cortisol and alternative steroids, LC–MS/MS rescued 45% and 66% of failed cases judged by immunoassay in unstimulated and stimulated AVS, respectively. LC–MS/MS identified more unilateral diseases than did immunoassay (76% vs. 45%, P < 0.05) and provided adrenalectomy opportunities to 69% of patients judged through immunoassay to have bilateral disease. The secretion ratios (individual steroid concentration/total steroid concentration) of aldosterone, 18-oxocortisol, and 18-hydroxycortisol were novel indicators for identifying unilateral PA. The 18-oxocortisol secretion ratio of ≥0.785‰ (sensitivity/specificity: 0.90/0.77) at pre-ACTH and aldosterone secretion ratio of ≤0.637‰ (sensitivity/specificity: 0.88/0.85) at post-ACTH enabled optimal accuracy for predicting ipsilateral and contralateral disease, respectively, in robust unilateral PA. LC–MS/MS improved the success rate of AVS and identified more unilateral diseases than immunoassay. The secretion ratios of steroids can be used to discriminate the broad PA spectrum.

Determination of three C18‐oxygenated steroids in adrenal lesion segments in primary aldosteronism by super‐selective adrenal venous sampling and LC/ESI–MS/MS

“…Taking 18-oxoF as an example, the LLOQ (2 ng/ml) of our method was unremarkable, which was partly due to the use of the Shimadzu LCMS-8030 + mass spectrometer, which was the low-end model of Shimadzu's LC/MS/MS instrument lineup (discontinued model) and had been used for over 10 years in our laboratories. An additional cause for this was the smaller sample volume (10 μl) used in this study compared with those (100-250 μl) in the other studies (Chang et al, 2023;Chen et al, 2022;Nakamura et al, 2011;Peitzsch et al, 2015;Satoh et al, 2015;Tezuka et al, 2021). The small sample volume impaired the concentration-based LLOQ but simplified the sample pretreatment owing to reduced matrix-derived interference; the unremarkable LLOQ of our method was an unavoidable trade-off for the effort-saving pretreatment procedure (only deproteinization) with a small volume sample.…”

“…The ssAVS serum sample (10 μl) was deproteinized in acetonitrile containing the IS, then the supernatant was used for the steroid quantification after the solvent was replaced by the mobile phase. As previously described, in the most reported LC/MS/MS assays for quantifying the serum/plasma 18-oxoF and the related steroids, SPE was performed in combination with deproteinization (Chang et al, 2023;Chen et al, 2022;Eisenhofer et al, 2016;Nakamura et al, 2011;Peitzsch et al, 2015;Satoh et al, 2015) (Table S1 in the Supporting Information), whereas our method required only deproteinization. This deproteinization-based pretreatment significantly reduced not only the processing time but also cost compared with the SPE-based pretreatment; we could treat 10 samples inside 40 min only with a centrifuge by the deproteinization-based method.…”

“…Because all the steroids examined in this study including IS had the 3-oxo-4-ene steroid structure (α,β-unsaturated ketone), they were efficiently ionized during the positive ESI-MS to produce the protonated molecules ([M + H] + ) with satisfactory intensities (Higashi et al, 2007). As discussed in other papers (Chang et al, 2023;Chen et al, 2022;Peitzsch et al, 2015;Turcu et al, 2020), by the collisional activation of their [M + H] + , the product ions were observed with sufficient intensities at m/z 315.2, 313.2, 267.2 and 323.2 for ALD, 18-oxoF, 18-OHF and IS, respectively (the product ion spectra are shown in Figure S1 in the Supporting Information). Based on these results, the SRM transitions described in Section 2.2 were used for quantifying ALD, 18-oxoF and 18-OHF in the ssAVS serum samples.…”

“…Several studies have noted that the serum/plasma concentration of 18-oxoF might be a promising index for distinguishing between APA and BAH compared with those of ALD and another C18-oxygenated steroid, 18-hydroxycortisol (18-OHF); the 18-oxoF concentrations in the peripheral blood samples primarily collected from antebrachial veins and adrenal central venous blood samples collected by cAVS were higher in the APA patients than in the BAH patients (Chang et al, 2023;Eisenhofer et al, 2016;Mulatero et al, 2012;Nakamura et al, 2011;Satoh et al, 2015;Tezuka et al, 2021). However, the peripheral and adrenal central venous blood is diluted with blood from other organs or unaffected segments of the adrenal glands; therefore, their steroid concentrations might not accurately reflect those in the ALD-hypersecreting segments, such as the ALD-producing tumor (APT) and hyperplastic (HP) tissues.…”

“…Although immunoassays have been used for quantifying the serum/plasma 18-oxoF (Morra di Cella et al, 2002;Mulatero et al, 2012), LC/MS/MS now has the central role for this purpose owing to its high specificity and capability of simultaneous quantification of the related steroids (Chang et al, 2023;Chen et al, 2022;Eisenhofer et al, 2016;Nakamura et al, 2011;Peitzsch et al, 2015;Satoh et al, 2015;Tezuka et al, 2021;Turcu et al, 2020). Most methods require solid-phase extraction (SPE) in addition to deproteinization prior to the LC/MS/MS analysis, although some of them enabled the simultaneous quantification of 15 or more steroids (Chang et al, 2023;Eisenhofer et al, 2016;Peitzsch et al, 2015) and acquired remarkable sensitivity (lower limit of quantification, LLOQ, on the order of pg/ml) (Chen et al, 2022). In certain cases, derivatization was employed to enhance the electrospray ionization (ESI)-MS/MS sensitivity of the 18-oxoF and other steroids (Nakamura et al, 2011;Satoh et al, 2015).…”