Simultaneous Determination of Carvedilol, Enalaprilat, and Perindoprilat in Human Plasma Using LC–MS/MS and Its Application to a Pharmacokinetic Pilot Study

Abstract

A method for the extraction and quantification of carvedilol, enalaprilat, and perindoprilat in 50 µL human plasma, using high-performance liquid chromatography with tandem mass spectrometry (LC–MS/MS) detection was developed and validated. Samples were prepared via protein precipitation with chromatographic separation on a Restek Ultra II Biphenyl column using gradient elution at a corresponding flowrate of 300 µL/min. Electrospray ionisation with mass detection at unit resolution in the multiple reaction monitoring (MRM) mode on an AB Sciex API 5500 mass spectrometer was used. Accuracy, precision, selectivity, sensitivity, matrix effects, recovery, process efficiency, and stability were assessed over the validation period. The assay was validated over the calibration range 0.2–200 ng/mL for all three analytes. The inter- and intra-day precision expressed as the coefficient of variation (CV) and accuracy (%Nom) all fell within acceptable limits. The overall recovery was calculated as 72.9%, 77.1%, and 77.0% for carvedilol, enalaprilat, and perindoprilat respectively, with the recovery being shown to be reproducible at the low, medium and high end of the calibration range for all three analytes. The method proved to be specific for all three analytes with no significant matrix effects observed. The validated method facilitated the analysis of carvedilol, enalaprilat, and perindoprilat in human plasma collected from adults as part of a pilot pharmacokinetic study. This validated analytical method lays the foundation for determining adherence in heart failure patients prescribed with carvedilol, enalapril and perindopril.

Validation of a quantitative multiplex LC-MS/MS assay of carvedilol, enalaprilat, and perindoprilat in dried blood spots from heart failure patients and its cross validation with a plasma assay

“…LC-MS/MS and chromatographic conditions similar to our previously developed plasma assay was used [10] . Electrospray ionization (ESI) in the positive ionisation mode was employed for detection on an AB Sciex API 5500 Qtrap mass spectrometer (AB Sciex™, Germany).…”

“…The mobile phase was held at 90 % B for 2.5 min, after which it was reduced back to 5 % B in 0.1 min. A 3.4-minute equilibration period preceded the next injection [10] .…”

“…Stock solutions were prepared for testing via dilution with methanol. UV absorbances at 206 nm for enalaprilat and perindoprilat and 242 for carvedilol were assessed as an expression of the concentrations of the analytes [10] .…”

“…Enalapril and perindopril are both ACE-I medications, with their active metabolites being enalaprilat and perindoprilat, respectively. A direct and objective method to evaluate the adherence of patients suffering from HF is to quantify the concentration of carvedilol as well as the metabolites enalaprilat and perindoprilat [10] .…”

“…The method described here is, as far as we know, the first to allow for the simultaneous quantification of carvedilol, enalaprilat, and perindoprilat in DBS. Paired plasma and DBS samples were analysed from a pharmacokinetic study using the validated DBS method and our previously validated plasma method [10] . Plasma sample concentrations were predicted from DBS sample concentrations using Deming regression and the differences between the derived and observed plasma samples were evaluated using Bland-Altman plots.…”