Postmortem Femoral Blood Concentrations of Amlodipine

Journal of Analytical Toxicology, Vol. 35, May 2011

Postmortem Femoral Blood Concentrations of Amlodipine Kristian Linnet*, Lotte Markussen Lang, and Sys Stybe Johansen Section of Forensic Chemistry, Dept. of Forensic Medicine, Faculty of Health Sciences, University of Copenhagen, Denmark

Abstract Postmortem femoral blood concentrations of the calcium blocker amlodipine were determined by LC–MS–MS and compiled for the years 2003–2010. The cause of death was classified as unrelated to amlodipine in 38 cases in which the concentration ranged from 0.006 to 0.13 mg/kg (median 0.048 mg/kg), a range that exceeds published in vivo therapeutic serum levels by several-fold. In three cases, amlodipine was judged to be a contributing factor to death and concentrations ranged from 0.29 to 0.44 mg/kg. This concentration range is of the same order of magnitude as published serum levels for clinical toxicity cases. One fatality was ascribed to amlodipine poisoning with a concentration of 0.90 mg/kg, which is similar to values observed in previously published fatality cases. One suspected drug–drug interaction case in which the amlodipine level (0.17 mg/kg) was considered elevated due to inhibition of the cytochrome P450 3A4 enzyme by the azole antifungal drug, fluconazole, was detected. In conclusion, it is important to establish postmortem reference concentrations rather than relying on in vivo therapeutic serum levels, which may be too low and lead to false conclusions in postmortem cases.

whole blood levels (5,6). Druid and Holmgren (7) and Reis et al. (8) compiled postmortem drug levels and divided them into three categories; A: concentrations in cases ascribed to fatal intoxication with the drug as the sole cause of death, B: concentrations where the drug is considered a contributing cause of death, and C: concentrations observed when the drug is not considered to be related to the cause of death, (e.g., suicide by hanging or shooting). This categorization is primarily based on the circumstances and not on the observed drug concentrations. Other authors have also provided postmortem reference values for various drugs (e.g., for olanzapine and quetiapine) (9,10). The effects of postmortem redistribution and differences between serum and whole blood concentrations are taken into account in these values. In this study, the postmortem concentration ranges for amlodipine observed over recent years is presented, and the results are discussed relative to previous published findings. In addition, a small series of in vivo whole blood measurements is presented.

Experimental Introduction Amlodipine is a dihydropyridine calcium blocker widely used in the treatment of hypertension. The daily dose is 2.5–10 mg, and the drug has a relatively long half-life (on average 34 h) (1). The long half-life results in a stable steady-state level, which is appropriate for treatment of hypertension. However, in overdose cases, the long half-life results in prolonged intoxication periods with profound hypotension and reflex tachycardia, which requires intensive therapy (2). In addition, coma and death may occur at high doses (3,4). In postmortem cases, amlodipine blood levels may not be comparable to in vivo therapeutic levels in serum because of postmortem redistribution and differences between serum and

* Author to whom correspondence should be addressed. Section of Forensic Chemistry, Frederik Femtes Vej 11, DK-2100 Copenhagen Ø, Denmark. E-mail: [email protected].

Amlodipine from Pfizer was used as a reference material, and stock solutions of 1000 mg/L in methanol were prepared. Dibenzepin from Novartis Healthcare (Basel, Switzerland) was used as an internal standard (IS) and a working solution of 0.20 mg/L in acidic methanol (0.1% formic acid in methanol) was prepared. Acetonitrile and methanol (LC–MS grade) were obtained from Fisher Scientific UK (Leicestershire, U.K.) and formic acid (98–100% GR for analysis) from Merck (Darmstadt, Germany). Purified water was obtained from a Millipore Synergy UV water purification system (Millipore A/S, Copenhagen, Denmark). Acidic water (0.1% formic acid in water) and acidic methanol were prepared and stored at 4°C. Blank human whole blood (BB) was obtained from the Blood Bank at Copenhagen University Hospital (Copenhagen, Denmark) and preserved by adding 1% sodium fluoride. BB was used for the negative control, blank with IS, spiked calibrators, and quality control samples. Authentic forensic samples of whole blood were preserved with 1% sodium fluoride. The calibration stan-

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dards in whole blood (0, 0.002, 0.050, and 0.500 mg/kg) were prepared for each run using dilutions of the stock solution in acidic methanol. Quality control (QC) samples in whole blood were prepared at two concentrations (0.020 and 0.200 mg/kg) and stored at –80°C. QC samples were analyzed in each run.

under a stream of nitrogen at 40°C, and the remains were reconstituted in 200 µL solvent (1:4:5 acetonitrile/methanol/ acidic water). The reconstituted solution was mixed thoroughly, and after another centrifugation for 10 min, the solution was moved to an amber vial. Five microliters of each extract was injected onto the LC–MS–MS system.

Analytical method

For whole blood protein precipitation: 0.200 g blood was mixed with 1200 µL cold acidic methanol and spiked with 25 µL 0.20 mg/L IS. To improve the precipitation a disposable plastic stick was added to the solution before mixing for 3 min, the samples were frozen for 30 min at –20°C. The supernatant was removed to another glass after centrifugation at 5°C for 10 min at 1600 × g. The methanolic phase was evaporated

Apparatus

Chromatographic separation was performed using an Agilent 1100 series high-performance liquid chromatography (HPLC) system from Agilent Technologies (Waldbronn, Germany) comprised of a binary pump, an autosampler, and a thermostated column compartment fitted with a Zorbax SB C18 column (2.1 × 50-mm i.d. 3.5 µm, Agilent Technologies) and a Gemini C18 guard column (4 × 2.0 mm, Phenomenex, Alleroed, Denmark). The eluant was diverted to waste for the first 2.0 min after injection. An elution gradient consisting of acidic water (A) and acidic acetonitrile (B) (5% up to 90% B) separated the compounds within 11 min (Figure 1). The flow rate was 0.3 mL/min at a column temperature of 60°C. A tandem mass spectrometer, Quattro micro from Waters (Manchester, United Kingdom), was coupled to the HPLC system. Data were acquired in the positive ionization mode with an electrospray source using MassLynx 4.1 software (Waters). Multiple Reaction Monitoring (MRM) analysis was used for data collection—two traces per target compound (408 → 237(Q); 408 → 294) with a cone voltage of 15V and collision energy at 10eV. The trace for the IS was 296 → 251 with a cone voltage of 30 V and collision energy at 20eV. The source and desolvation temperatures were 120°C and 300°C, respectively. Argon was used as the collision gas at 0.004 mBar. The absolute recoveries of amlodipine and IS were 80% and 71%, respectively. There was a linear range from 0.002 to 1.0 mg/kg for amlodipine. The imprecision (CV) of the QC samples was less than 15% at the low level and 10% at the high level, and the bias was below 15%. The ion ratio was 1.46 ± 10%. Two cases, examined in 2003 and in the beginning of 2004, were analyzed using an HPLC–diode-array detection method described previously (11). Cases

Figure 1. Ion chromatogram (quantitative trace, qualitative trace and IS trace) of blank whole blood (A), standard at 0.002 mg/kg blood (B), and case with 0.016 mg/kg amlodipine in postmortem blood (C).

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Over the years 2003–2010, amlodipine was measured in postmortem femoral blood in 52 cases. In 38 cases, the cause of death was classified as unrelated to amlodipine (Category C cases). These cases included 25 cases of natural death (mostly


cardiovascular and/or pulmonary diseases), 7 cases of poisoning with psychoactive drugs and/or alcohol, 2 cases of drowning, 2 cases of suffocation, 1 traffic accident, and 1 homicide committed with an axe. For the remaining 14 cases, amlodipine was considered a possible contributor to death in 5 cases, whereas the cause of death for the remaining 9 cases was uncertain, and these cases were not included in the Categories (Table I). Of the five cases, one case was classified as a Category

A case. The deceased had been depressed for a long period, and further investigation revealed tablet residues in the stomach content indicating possible overdose. A high blood concentration of amlodipine was found (0.94 mg/kg), and the amounts of venlafaxine (1.3 mg/kg), citalopram (0.18 mg/kg), and oxazepam (0.23 mg/kg) corresponded to therapeutic use. Three cases were classified as Category B cases, where amlodipine was considered a contributing cause of death. In the first of these

Table I. Nine Cases with Undetermined Cause of Death Case No. (age)

Amlodipine Concentration (mg/kg)

Other Compounds (mg/kg)

Diseases and Autopsy Findings

1 (85 y)

0.11

Propanolol 0.13 Bromazepam 0.08 7-Aminonitrazepam 0.11

Probable pneumonia Cardiac disease

Undetermined Cardio-pulmonary disease might be a contributing factor

2 (64 y)

0.057

Dipyridamole 0.31 Metronidazole 13

Heart disease Diabetes

Undetermined Cardiac disease might be a contributing factor

3 (58 y)

0.051

Ketobemidone 0.15 Mianserine 0.055 Citalopram 0.40 Alprazolam 0.004 Diazepam 0.025 Flunitrazepam 0.002

Psychosis Depression Drug abuse Generalized arteriosclerosis

Undertermined Drug effects might be a factor

4 (45 y)

0.040

Ketobemidone 0.030 Quetiapine 0.069 Levomepromazine 0.078 Salicylic acid 204 Codeine 0.093

Alcoholic Psychosis

Undetermined

5 (51 y)

0.087

Fluoxetine 0.11 Norfluoxetine 0.14

Alcoholic Depression Diabetes Liver disease

Undetermined

6 (54 y)

0.040

Lithium 0.3 Metoprolol 0.058 Zuclopenthixol 0.02

Pulmonary disease Schizophrenic

Undetermined

7 (59 y)

0.041

Morphine 0.024 Codeine 0.074 7-Aminoclonazepam 0.26 Ibuprofen 18

Alcoholic Gastric ulcer Generalized arteriosclerosis

Undetermined

8 (49 y)

0.034

Quetiapine 0.004 Oxazepam 0.020 Zopiclone 0.002

Schizophrenic Cardiac disease

Undetermined Cardiac disease might be a contributing factor

9 (48 y)

0.023

Amitriptyline 0.050 Nortriptyline 0.017 Phenytoin 10 Levetiracetam 5.2

Psychosis Epilepsy Pneumonia

Undetermined Pneumonia might be a contributing factor

Cause of Death

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cases, a combined poisoning of amlodipine (0.29 mg/kg) and diltiazem (21 mg/kg) was detected. The second case was a combined poisoning of amlodipine (0.41 mg/kg) and mirtazapine (5.4 mg/kg). In the last Category B case, amlodipine (0.44 mg/kg) was the only compound detected; however, the subject’s health was compromised by leukemia. The day prior to death, the deceased had fainted and was subsequently evaluated by a medical doctor, who determined that the incident was due to heat exhaustion. The reason for the relatively high amlodipine concentration is unclear; however, it is possible that the high level is due to compromised metabolism. The last of the five cases (amlodipine, 0.17 mg/kg) involves a possible drug-drug interaction that may have contributed to the cause of death. This case is treated separately in the Results and Discussion section. Finally, for comparison, cases from living subjects who were prescribed amlodipine and involved in criminal or traffic cases for the period 2003–2010 were included (n = 7, Category D).

Results and Discussion Category A–D cases

centrations of Category C and D cases, but it has not been determined for amlodipine. Further, the serum levels may refer to trough values, whereas the Category D values originate from samples taken at random time points. A suspected drug–drug interaction case

A 77-year-old disabled woman was found dead in her wheelchair. She had slipped down the seat and the safety belt ended up under her arms pressing on her throat. On the basis of the autopsy, the pathologist concluded that the cause of death was suffocation. The woman had been treated for hypertension with amlodipine and for depression with venlafaxine. Analysis of femoral blood showed the presence of venlafaxine (0.16 mg/kg), O-desmethylvenlafaxine (0.52 mg/kg), amlodipine (0.17 mg/kg), and the antifungal compound fluconazole (only qualitatively identified). It was concluded that venlafaxine and O-desmethylvenlafaxine were present in typical therapeutic amounts (8), and the amlodipine concentration was higher than expected for therapeutic conditions. There were no signs suggesting an overdose (the deceased had been medicated by nurses). Fluconazole is a strong inhibitor of the cytochrome P450 enzymes 3A4, 2C9, and 2C19, as are other azole antifungal compounds (21). Amlodipine is metabolized by CYP3A4, and it is therefore likely that the relatively high amlodipine concentration was a result of inhibition of the metabolism of the compound (22). Experimental studies have shown that the strong cytochrome P450 3A4 inhibitor itraconazole, another azole antifungal compound, inhibits the metabolism of amlodipine and elevates the plasma concentration (23). It might be further speculated that the possible toxic effects led to dizziness (because of possible low blood pressure) and weakness, which could have contributed to the woman slipping down the seat.

Amlodipine concentrations recorded in the case scenarios, Categories A–D, are displayed in Table II and the amlodipine values reported in the literature are indicated in Table III (12– 17). The Category A case had an amlodipine concentration (0.90 mg/kg) comparable to those reported in the literature for fatalities (0.9–2.7 mg/kg). In a prior fatality case investigated by our laboratory, amlodipine was measured in muscle tissue and determined to be 2.3 mg/kg (11). The concentrations were somewhat lower in the Category B cases, 0.29–0.44 mg/kg, which partly overlaps with the serum concentration range found in clinical intoxicaTable II. Blood Concentrations for Categories A–D (mg/kg) tion cases (Table III). The concentrations of the Category C A B C D cases, which ranged from 0.006 to 0.13 (n = 1) (n = 3) (n = 38) (n = 7) mg/kg, were considerably higher than reported in vivo therapeutic serum levels, Single values or range 0.94 0.29, 0.41, 0.44 0.006–0.13 0.006–0.086 10–90-percentile – – 0.016–0.098 – 0.001–0.024 mg/L (Table III) (18–20). If Median – 0.41 0.048 0.018 this therapeutic range is used for postmortem cases, many cases are likely to be misinterpreted as displaying a toxic range rather than a usual therapeutic level, which has been the sitTable III. Reported Amlodipine Concentrations in the uation for a long period in our laboratory. It is observed that Literature the median (0.041 mg/kg) and range of the values in Table I Range largely overlap with the Category C values. The concentraMatrix (mg/kg or mg/L) References tions of the Category D cases ranged from 0.006 to 0.086 mg/kg and tended to be slightly lower than the Category C cases (meFatalities Blood 0.9–2.7 3,4 dian 0.018 vs. 0.048; 0.05 < P < 0.10, Mann-Whitney test), but (peripheral) also considerably higher than the cited in vivo therapeutic serum levels. Although this tendency towards a difference in Clinical serum 0.067–0.39 15–17 intoxication cases concentration between Categories C and D cases should not be over-interpreted, it may at least partly be a reflection of postTherapeutic/ serum 0.001–0.024 12–14 mortem redistribution of amlodipine. The serum/blood dispharmacokinetic tribution ratio may be a factor in the difference between the studies cited in vivo serum therapeutic levels and the full blood con-

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Conclusions In the present study, postmortem reference values for amlodipine concentrations in femoral blood were compiled. It was concluded that the reference values for cases presumed to be unrelated to amlodipine toxicity were several-fold higher than generally referenced in vivo therapeutic serum values. Levels related to toxicity were of the same order of magnitude as previously reported. In addition, a case involving a possible drug–drug interaction between amlodipine and fluconazole, related to inhibition of the cytochrome P450 3A4 enzyme, was reported.

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Manuscript received October 8, 2010; revision received November 16, 2010.

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