A widely applicable plasma renin activity assay by LC-MS/MS with offline solid phase extraction

Abstract

Background

The measurement of plasma renin activity using LC-MS/MS is an attractive alternative to radioimmunoassay techniques. A published method for renin activity by LC-MS/MS uses equipment that is not available in all LC-MS/MS laboratories. Here, we present an offline modification to allow users of LC-MS/MS to perform this analysis without specialist equipment.

Methods

Samples were prepared in duplicate and incubated for 6.5 h and 24 h. Solid phase extraction was performed offline using Waters Oasis MAX ion-exchange 96-well plate. A method comparison was performed between this assay and a previously published assay using on-line solid phase extraction.

Results

The offline method for plasma rennin activity gave similar results to the on-line method across the concentration range. Analytical performance was also found to be comparable and the use of a 24 h incubation for low samples was also deemed unnecessary.

Discussion

The method described is an acceptable alternative to a previously published assay which does not require the use of highly specialist equipment and will be applicable to the majority of LC-MS/MS users.

Introduction

The measurement of plasma renin activity (PRA) using LC-MS/MS as an alternative to radioimmunoassay techniques is often favourable due to decreased safety risks and a more rapid turnaround of samples. A previously published method demonstrates that the measurement of PRA can be achieved using LC-MS/MS; however, this on-line solid phase extraction (SPE) method uses equipment that is not available in all laboratories. ¹ We present an offline SPE alternative to allow users of LC-MS/MS to perform this analysis without specialist equipment.

Materials and methods

During routine use the PRA results from the 6.5 h and the 24 h plates were similar, questioning whether the 24 h plate was required. To determine if the 24 h plate is required to improve assay sensitivity with the on-line sample preparation, results (n = 51) by the on-line method were compared between the 6.5 h and 24 h plates using Analyse-it for Microsoft Excel.

Plasma samples (n = 40) for the method comparison were prepared and incubated as described previously by Carter et al. ¹ These samples had previously been run on multiple assays and were selected to cover the concentration range. After the reaction was stopped using a cold 2% (v/v) ammonia solution (Fisher, Loughborough, UK), the samples were ready for extraction using an Oasis MAX SPE 96-well plate (Waters, Manchester UK). The extraction was performed using a positive pressure manifold (Waters, Manchester, UK). The plate was conditioned with 2 × 1 mL of 2% (v/v) formic acid (BDH, Lutterworth, UK) in 60% (v/v) methanol (Fluka, Poole, UK) followed by 1 mL of methanol. The wells were then equilibrated using 1 mL of 1% (v/v) ammonia. The samples were then transferred from the wells of the incubation plate to the SPE plate using a multichannel pipette, left to stand for 5 min, and samples were forced into the plate. The wells were then washed with 2 × 1 mL of 1% (v/v) ammonia in 35% (v/v) methanol followed by 2 × 1 mL of distilled water. A collection plate was then placed under the SPE plate and Angiotensin I was eluted with 2% (v/v) formic acid in methanol. The eluate was evaporated to dryness using heated (60℃) air and the extract was subsequently reconstituted in 100 µL of 0.2% (v/v) formic acid in 10% (v/v) methanol. After vortex mixing for 1 min and centrifugation at 2600g for 5 min, analysis was performed using a Waters Acquity UPLC and Xevo TQ mass spectrometer with conditions described by Carter et al. ¹

Inter-assay imprecision was assessed using Bio-Rad Hypertension quality control material (Bio-Rad, Hemel Hempsted, UK).

Results

When comparing PRA results below 0.3 nmol/L/h between the 6.5 h and 24 h incubation plates, the Passing–Bablok analysis showed: 24 h plate = 0.94 × 6.5 h plate − 0.03 nmol/L/h. The Bland–Altman bias plot is shown in Figure 1. This confirmed that the results were comparable in this low activity range, deeming the 24 h incubation unnecessary. Subsequent comparisons were performed using the 6.5 h plates only and results below the lower limit of quantitation were not included in the analysis.

Figure 1.

Difference between low renin activities obtained after either a 6.5 h or 24 h incubation.

The results from the offline preparation described above were compared to the original method of Carter et al. For all samples (n = 40) covering the PRA range of 0.1 to 10 nmol/L/h, the Passing–Bablok comparison showed: offline = 1.10 × on-line–0.02 nmol/L/h. For samples < 3 nmol/L/h (n = 33), the Passing–Bablok comparison showed: offline = 1.07 × on-line–0.01 nmol/L/h. The Pearson correlation coefficient was r = 1.0 at these low concentrations.

The imprecision observed with the new sample preparation was noted to be superior to the on-line assay. The offline assay gave CVs of 7.5, 6.7 and 6.0% at plasma renin activities of 1.20, 2.98 and 9.86 nmol/L/h while the on-line assay gave CVs of 12.5, 6.6 and 7.4% at similar activities. This improved precision was also demonstrated by a superior lower limit of quantitation of 0.05 nmol/L/h.

Conclusions

We have described an alternative sample preparation for PRA analysis which does not require expensive on-line SPE equipment. The offline technique does require some amount of extra time for the preparation of the samples by using a positive pressure manifold for the SPE. However, the time required to prepare and prime the on-line SPE equipment has also been removed, thus almost neutralizing the time required for the offline extraction. The assay described here shows superior precision to the on-line method and also removes the need for a 24 h incubation, therefore reducing the turnaround times for this analysis. The lower limit of quantitation of the previous assay was 0.1 nmol/L/h using the 24 h incubation; this assay shows no decline in performance at these low concentrations without a 24 h incubation (Figure 1). The type of analysis described here can be applied to measure renin activity in most LC-MS/MS laboratories.