Nicola Gray (Presenter)
Authorship: Nicola GRAY (1), Mikaël LEVI (2)
(1) SHIMADZU UK Limited, Milton Keynes, UK, (2) SHIMADZU Corporation, MS Business Unit, Kyoto, Japan
The measurement of the aldosterone-renin-ratio (ARR) is a recommended screening tool for primary aldosteronism. Aldosterone is classically measured in plasma/serum, while renin is often quantified by its activity, measuring angiotensin-I generation in a defined time range. Several reports describe the measurement of aldosterone or plasma renin activity (PRA) by LC-MS/MS, but no method has yet proposed a combined assay. We present an assay to measure low aldosterone levels as well as low PRA in a single sample with a simplified workflow.
Hypertension or high blood pressure is a highly prevalent disease. Secondary hypertension is caused by kidney or endocrine disorders. To define the cause of the secondary hypertension the measurement of aldosterone-renin-ratio (ARR) is a recommended tool, especially for the screening of primary aldosteronism.
Aldosterone is classically measured in plasma/serum, while renin is quantified by means of its activity, measuring the amount of angiotensin-I produced in a defined time range referred to as plasma renin activity (PRA). Many reports on the measurement of aldosterone or PRA by LC-MS/MS have been published but no method has yet been proposed to combine both in a single assay.
Here we present a method that allows the measurement of low aldosterone levels as well as low PRA in a single sample with a simplified workflow.
Plasma (100 µL) from blood collected on EDTA-K3 is buffered and spiked in duplicate with internal standards. One sample is kept at +4°C to be used a reference (t0) while the other one is incubated at 37°C for 1 hour (t1). After incubation, both samples were precipitated and the supernatant submitted to online SPE-UHPLC-MS/MS. 50 µL of extract was injected on a short C18 guard column for clean-up then transferred to an UHPLC C18 column. The total cycle time was 7 minutes.
Multiple Reaction monitoring (MRM) acquisition was operated in positive and negative electrospray ionization for angiotensin-I and aldosterone, respectively, using polarity switching on the LCMS-8060 (Shimadzu Corporation). 13C11,15N2-angiotensin-I and 2H4-aldosterone were used as quantification internal standards (ISTD). 13C6,15N1-angiotensin-II was used as internal standard for angiotensin-II monitoring.
As angiotensin-I is cleaved into angiotensin-II by the angiotensin converting enzyme (ACE), it was necessary to ensure that ACE is effectively inhibited during sample incubation. For this purpose, angiotensin-II and its internal standard are monitored in samples. In addition, the MRM of angiotensin-I ISTD cleaved into angiotensin-II is also monitored. This is made possible by the different labelling of ISTD. Results show that the use of EDTA in the incubation buffer efficiently inhibited ACE. However, as this does not significantly increase the cost or workload, it is recommended to keep monitoring these additional MRMs for quality assessment.
Quantification of angiotensin-I was performed from 0.023 to 77.2 nmol/L. PRA is calculated as the difference in angiotensin-I concentration between t1 and t0 divided by the incubation time. Therefore, PRA as low as 0.023 nmol/L/hr was achievable. Quantification of aldosterone was possible from 111 to 4162 pmol/L. Measured concentrations were not significantly different at t0 and t1. ARR is then calculated as the ratio of the values obtained for aldosterone and plasma renin activity. All the calculations were performed automatically by the acquisition and processing software LabSolutions (Shimadzu Corporation).
Intra and inter-assay precision measurement were performed using Bio-Rad Lyphochek Hypertension control samples (3 levels). PRA and aldosterone intra and inter-assay concentration imprecision was less than 10 %. As no consensus value for PRA or aldosterone is given for these control samples, accuracy could not be estimated. Nonetheless, calculated values were in close accordance with those indicated on certificates.
Conclusions & Discussion
We present an assay to measure low aldosterone levels as well as low PRA in a single sample with a simplified workflow to measure the aldosterone-renin-ratio.
References & Acknowledgements:
IP Royalty: no
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