A simple and rapid UPLC/MS/MS method has been developed and validated for the analysis of digoxin and metildigoxin in whole blood. Samples were prepared by SPE extraction with Oasis HLB columns. Separation was achieved with an ACQUITY UPLC HSS T3 column (
Digitalis glycosides are the drugs of choice for the treatment of congestive heart failure and certain disturbances in cardiac rhythm, producing a positive inotropic activity and increase, myocardial contractility [
Chemical structure of digoxin (C41H64O14).
Chemical structure of metildigoxin (C42H66O14).
These compounds have a narrow therapeutic range, so they can frequently lead to intoxication, involving suicide, homicide, and accidental poisoning cases [
In this study, we have developed a sensitive and rapid method for the identification and quantification of digoxin and metildigoxin in whole blood by UPLC-MS/MS, after a SPE extraction technique.
Chromatographic separation was carried out on an ACQUITY UPLC system (Waters) with an ACQUITY TQD Mass Detector (Waters). Solid-phase extractions were carried out on an automatic extractor Aspec XL (Gilson).
Pure digoxin, metildigoxin, and d3-digoxin (internal standard) were purchased from Chemos (Germany). Each standard compound was dissolved in methanol (1 mg/mL for digoxin and 0.5 mg/mL for metildigoxin and d3-digoxin) and stored at −20°C. Working solutions were also prepared in methanol. Ammonium formate was purchased from Sigma-Aldrich (USA). Ammonium acetate was purchased from Merck (Darmstadt, Germany). All the other solvents were analytical or HPLC grade and were purchased from E. Merck (Darmstadt, Germany). Water was purified by a Milli-Q system obtained from Millipore (Molsheim, France). The mobile phase was filtered with a 0.20
Chromatographic separation was performed with an ACQUITY UPLC HSS T3 column (
Compound | Transition | Cone voltage (V) | Collision energy |
---|---|---|---|
Digoxin | 30 | 16 | |
30 | 8 | ||
Metildigoxin | 30 | 15 | |
30 | 9 | ||
d3-digoxin | 30 | 15 |
Control and calibration samples were prepared by spiking drug-free
There are various studies in the literature reporting the development of methods for digoxin determination in human plasma, serum, and urine by liquid chromatography-mass spectrometry LC-MS [
In the present study, a sensitive and rapid UPLC-MS/MS method for the determination of digoxin and metildigoxin in
MRM chromatograms of whole blood samples spiked with 1 ng/mL of digoxin [(a) and (b)] and metildigoxin [(d) and (e)]; and with 5 ng/mL of d3-digoxin, internal standard (c).
Thus, validation studies were performed separately for each substance, in whole blood samples. The chromatographic method described had a run time of only 1.5 min, with digoxin detection at a retention time of 0.78 and 0.92 min for metildigoxin (Figure
Product ion mass spectra of digoxin.
Product ion mass spectra of metildigoxin.
The developed method was validated according to the guidelines of the International Conference on Harmonization (ICH) for the validation of the following parameters: selectivity/specificity, linearity, limits of detection and quantification, recovery, and intra- and interday precision [
To evaluate peak purity and selectivity, 10 different blank samples (no analyte or internal standard was added) were analyzed to check for peaks that might interfere with the detection of the analyte or internal standard (IS). Also, negative samples (blank blood samples + IS) were analyzed, to verify the absence of native analyte in the IS solution. In addition, ten blood samples spiked with the 2 analytes but without IS were analysed, in order to verify no interferences from the analytes in the IS retention time. To assess possible interferences, 10 different samples were spiked with a mixture of various benzodiazepines (bromazepam, nordiazepam, 7-aminoflunitrazepam, flunitrazepam, oxazepam, diazepam, clonazepam, temazepam and midazolam) at a concentration of 25 ng/mL and with digoxin and metildigoxin at 5 ng/mL. All 10 samples were free of coeluting peaks at the retention times of the corresponding studied substances and their respective deuterated IS. Analysis of negative samples also demonstrated that the IS did not contain relevant amounts of native cardiac glycosides. None of the 10 compounds tested showed any interference when added to a blood sample with 5 ng/mL digoxin and metildigoxin.
The limit of detection (LOD) was estimated from extracted samples spiked with decreasing concentrations of the studied compounds
Simple linear regression analyses were performed, with calibration curves constructed from peak area ratios, spiking whole blood samples with the studied substances at 10 different concentrations of the cardiac glycosides covering the range of (0.3–10) ng/mL. Ten calibrators were used to generate the standard curve, each calibrator injected in three aliquots. Calibration curves showed a linear relationship for digoxin with a correlation coefficient of 0.994 and for metildigoxin with a correlation coefficient of 0.996.
Intraday and interday coefficients of variation values were determined by replicate analyses (
Recovery and precision for digoxin in spiked whole blood samples.
Concentration (ng/mL) | Mean (ng/mL) | Extraction recovery (%) | CV (%) | |
Intraday | Interday | |||
1 | 4.56–10.00 | 7.80 | ||
3 | 1.95–7.35 | 4.83 | ||
8 | 0.78–9.91 | 5.82 |
Recovery and precision for metildigoxin in spiked whole blood samples.
Concentration (ng/mL) | Mean (ng/mL) | Extraction recovery (%) | CV (%) | |
Intraday | Interday | |||
1 | 4.56–9.90 | 7.45 | ||
3 | 4.82–9.62 | 7.51 | ||
8 | 2.32–9.80 | 6.65 |
The recovery of SPE was determined by repeated analysis of five samples spiked at three different levels of digoxin and metildigoxin concentration (1, 3, and 8 ng/mL). The extraction recovery was determined by comparing the representative peak areas of extracted drug-free samples spiked before extraction with the peak area of drug-free samples fortified after the extraction at the same concentration levels. Tables
In summary, this paper describes an UPLC-MS/MS procedure for quantitative analysis of digoxin and metildigoxin in
P. Melo and R. Machado equally contributed to the work.