Study of the involvement of amlodipine in the mechanism of death in a sample of patients at the University Hospital of Dijon

Abstract

Aim

Amlodipine is a calcium channel blocker, used in cardiac pathologies. At toxic doses, amlodipine is responsible for death by cardiogenic shock and vasoplegia. Its pharmacokinetic properties suggest that post-mortem redistribution is possible, precluding interpretation based on living data (generally, between 6.5 and 20.9 μg/L). Our objective was to determine the toxic concentrations of amlodipine post-mortem.

Method

Plasma samples were analyzed using a Q-Exactive Focus high-resolution mass spectrometer coupled to the Transcend LX2 UHPLC system (Thermo Scientific™). The various compounds in the liquid sample were extracted using in-line extraction due to two different TurboFlow™ columns, assembled in series. The first is a Cyclone™ and the second is a Phenyl™ (50 mm × 0.5 mm) (Thermo Scientific™). After extraction, the compounds were separated by an analytical column, here, a phenylhyexyl™ column (100 mm × 2.1 mm, 2.6 μm) (Thermo Scientific™). The composition of mobile phase A was water with 0.1% formic acid and 2 mM ammonium formate. For phase B, acetonitrile:methanol:water (49.5: 49.5: 1 v/v) with 0.1% formic acid and 2 mM ammonium formate was used. Phase C was a mixture of acetone:acetonitrile:isopropanol (20: 40: 40 v/v). All solvents were of LCMS quality. The flow rate of the mixture of mobile phases A and B was set at 2 mL/min for the TurboFlow™ columns and 0.5 mL/min for the analytical column. The temperature of the analytical column was set at 40 °C. Once the sample had been extracted and separated, it was transferred to the mass spectrometry system. The sample is ionized by an H-ESI probe, switching positive and negative. Amlodipine ionizes positively. The ions are analyzed by Full Scan MS and a ddMS² analysis is applied to fragment the ions and obtain confirmation. The calibration curve ranged from 0.05 to 0.25 μg/L. With the autopsy findings, we classified the data into three groups, independent of the toxicological findings: unrelated death (group 1), possibly related death (group 2), and Amlodipine-related death (group 3). All samples were post-mortem femoral whole blood.

Results

Of the 31 cases, 1 case was classified in group 3 (Amlodipine concentration 275 μg/L); 13 in group 2 (median concentration 83.0 μg/L [42; 127.5]); and 17 cases in group 1 (median concentration 49 μg/L [35.75; 85.25]).

Conclusion

Our results show that in group 1 (death not related to Amlodipine), the median concentration of 49 μg/L is 2.33 times higher than the therapeutic concentration observed in living patients.

According to our results, post-mortem concentrations below 85.25 (3rd quartile) μg/L, can be considered non-toxic. The use of therapeutic concentrations in living patients is therefore not reliable in post-mortem for this drug with a strong cardiovascular tropism. A larger-scale study would allow the definition of reference tables for post-mortem toxic concentrations of Amlodipine.