Journal of Analytical Toxicology 2014;38:231 –232 doi:10.1093/jat/bku015 Advance Access publication March 26, 2014
Case Report
Case Report of Ivabradine Intoxication Franc¸ois Mathiaux1, Sylvain Dulaurent1, Fre´de´ric Julia2 and Jean-Michel Gaulier1* 1
Biological and Forensic Toxicology Unit, Pharmacology and Toxicology Department, University Hospital of Limoges, 2 Avenue Martin-Luther King, 87024 Limoges cedex, France and 2Emergency Department, University Hospital of Limoges, 2 Avenue Martin-Luther King, 87024 Limoges cedex, France *Author to whom correspondence should be addressed. Email: [email protected]
Ivabradine is a drug used for the treatment of angina and chronic heart failure in cases of intolerance or insufficiency of response to betablocker treatment. A 47-year-old man was admitted to the emergency department of the hospital for a voluntary intoxication with 280 mg of ivabradine: he presented drowsiness and a mild sinusal bradycardia (50 bpm) associated with a well-tolerated low blood pressure at 100/ 50 mmHg. No complication was noted and he was discharged from the hospital on Day 3. A method for ivabradine assay in serum was obtained using liquid chromatography coupled with a mass spectrometry detection method. After a deproteinization step using QuECHERS salts and acetonitrile, a chromatographic separation was performed using a 5-mm 50 3 2.1 mm Xterraw column (Waters, France). Detection was performed using an LTQ linear ion-trap mass spectrometer equipped with an electrospray ionization source used in a positive ionization mode (ThermoFisher Scientific, San Jose CA, USA) and a detection in full MS2 scan. The limit of quantification of ivabradine was 10 mg/L, and the method was linear up to 1000 mg/L. The ivabradine concentration in the patient’s serum was 375 mg/L. This concentration value was >30 times those measured after therapeutic doses intakes. Nevertheless, the bradycardia was no more severe than the one observed with therapeutic dosage. In conclusion, this case tends to show an absence of correlation between blood concentration and severity of the troubles in cases of overdosage. Introduction Ivabradine is a drug commercialized in France under the brand name Procoralan. It is used for the treatment of angina and chronic heart failure in cases of intolerance or insufficiency of response to beta-blocker treatment (Figure 1). Ivabradine acts by direct inhibition of the spontaneous depolarization of the sinusal node, without any activity on the signal conduction in auricular or ventricular cavities. The clinical effect of this drug is a decrease of the heart rate which seems to be dose dependent, at least in therapeutic dosages (5–15 mg orally per day) (1). There is very little information about the consequences of ivabradine overdosage: that is, no cases are registered neither in the French poison control center nor in the MeSH database with research including the terms ‘ivabradine’ and ‘overdosage’, ‘ivabradine’ and ‘intoxication’. Nevertheless, owing to its pharmacodynamic properties and some adverse events observed before and after marketing authorization (atrioventricular block, syncope and cardiac arrest), intoxication of ivabradine is thought to induce severe bradycardia and acute heart failure (2). In addition, its large volume of distribution is in favor of prolonged effects. In the absence of documented issues, we propose to report an ivabradine intoxication case. Case story A 47-year-old man was admitted to the emergency department of the hospital for a voluntary intoxication: 280 mg of ivabradine
(56 5 mg tablets) associated (not proved) with an unknown dose of bromazepam. He presented drowsiness and a mild sinusal bradycardia (50 bpm) associated with a well-tolerated low blood pressure at 100/50 mmHg. No abnormality was found on the successive electrocardiograms, with the exception of a sinusal bradycardia. Initial treatment consisted of just one injection of atropine (1 mg) and a gastric wash. The patient was kept under medical supervision for 48 h before psychiatric care. No complication was noted, and the patient left the emergency department on Day 3. A blood sample was collected 1.5 h after the intoxication for ivabradine determination. It is of note that the hypothesis of a bromazepam intake by the patient was not investigated by the clinicians as from their clinical point of view and in the presented case, such benzodiazepine intoxication only required monitoring, without any specific dispositions. Materials and methods A method for ivabradine assay in serum was obtained using liquid chromatography coupled with a mass spectrometry detection method. Extraction was performed using the following procedure: after addition of 20 mL of 5 mg/L mebutamate solution (internal standard) to 100 mL patient’s serum, 5 mg QuEChERS (‘Quick, Easy, Cheap, Effective, Rugged and Safe’) salt mixture were added to the sample. This mixture is the combination of four salts: eight weight parts of MgSO4; four weight parts of NaCl; four weight parts of C6H5Na3O7.2H2O and one weight part of C6H6Na2O7.1.5H2O (3). After addition of 500 mL of acetonitrile, 1 min of vigorous shaking and centrifugation (10 000 rpm for 10 min), 1 mL of the organic phase was injected in the chromatographic system. The chromatographic system consisted in two Shimadzu LC-20 AD pumps, SIL-20 AC autosampler (Shimadzu, Marne-laValle´e, France). Chromatographic separation was performed on an Xterra MSC18 5-mm (50 2.1 mm, i.d.) column (Waters, France), using a gradient of (A) ammonium formate 2 mM pH 3.0 in water, and (B) a mixture of acetonitrile with 10% A (v/v) as mobile phase (constant flow rate of 200 mL/min), programmed as follows: 0 – 2.5 min, 20% (B); 2.5 – 5 min, increase from 20 to 90% (B); 5 – 6.5 min, 90% (B); 6.5 – 7 min, decrease from 90 to 20% (B); 7 – 11.5 min, column equilibration with 20% (B). The total chromatographic run time was 11.5 min. All chromatographic solvents were degassed with helium beforehand. Mass spectrometric analyses were conducted using a ThermoFisher Scientific (San Jose, CA, USA) LTQ linear ion-trap mass spectrometer equipped with an electrospray ionization source. The main parameter settings in the positive ionization mode were: ionspray voltage, 4.5 kV; sheath gas flow rate, 12;
# The Author [2014]. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
Figure 1. Ivabradine formula. Chemical name: 2H-3-Benzazepin-2-one, 3-(3-(((3,4dimethoxybicyclo(4.2.0)octa-1,3,5-trien-7-yl)methyl)methylamino)propyl)-1,3,4, 5-tetrahydro-7,8-dimethoxy-, (S)- Formula: C 27 – H 36 – N 2 – O 5 . MM: 468. CAS registry number: 0155974-00-8.
Aux gas flow rate, 10; sweep gas flow rate, 12; capillary voltage, 15 V; capillary temperature, 3008C and tube lens, 65 V. Acquisition was performed in full MS2 mode following a parent mass for ivabradine (m/z 469.30) and mebutamate (m/z 233.15). Ionic transitions were obtained by extraction of daughter ions [ivabradine quantification (m/z 262.00) and confirmation (m/z 177.00 and 206.00)/mebutamate (m/z 172.11)] from the full MS2 spectrum of the corresponding parent ion. In addition, detection of N-desmethylivabradine, the main metabolite of ivabradine, was performed in full MS2 using its main following transitions (m/z 455.3 ! 262.00, m/z 455.3 ! 177.00 and m/z 455.3 ! 206.00). By means of ad hoc ivabradine (Sigma Aldrich, France) additions to free human serum, a calibration curve (10, 50, 200, 500 and 1000 mg/L) was constructed using a quadratic function with weighting factor 1/x. Validation procedure was performed according to the French Society of Analytical Toxicology recommendations (4) and proposed experiments, evaluations and acceptance criteria for the validation of new analytical methods to be used in single-case studies of analysis of rare analytes (5). The intra-assay precision and accuracy were assessed at low and high concentrations relative to calibration range: analysis of five spiked serum samples at each concentration. The limit of detection (LOD) was defined as the lowest concentration giving a response of at least three times the average of the baseline noise. The limit of quantification (LOQ) was defined as the lowest concentration that could be measured with an intra-assay precision CV% (coefficient variation %) and a relative bias ,20%. Recoveries were determined in triplicate at three concentration levels (10, 100 and 1000 mg/L) by comparing the analyte/internal standard peak area ratios obtained after the extraction of spiked samples with those of serum free extracts further spiked with ivabradine. Carry over was evaluated, by analyzing a sample of mobile phase subsequently to the highest calibration level (n ¼ 5). The absence of any signal on the corresponding chromatogram was verified. Ion suppression phenomenon was studied following the experimental system previously proposed by Antignac et al. (6). Briefly, a standard solution of ivabradine (at 100 mg/L) was continuously and directly infused into the mass spectrometer interface. A simultaneous LC flow containing either a pure mobile phase or a blank biological extract (serum from 10 non-drug consumers) was introduced through a tee. Evolution of the signal of the compounds of interest was studied to evaluate the presence and intensity of ion suppression.
Results This method exhibits LOD and LOQ for ivabradine of 5 and 10 mg/L, respectively. Intra-assay precision CV and relative bias
232 Mathiaux et al.
(%) were 6.3 and 25.2 (10 mg/L), 3.7 and 8.0 (200 mg/L) and 8.3 and 29.0 (1000 mg/L). Inter-assay precision CV and relative bias (%) were 5.0 and 5.1 (10 mg/L), 5.5 and 4.1 (200 mg/L) and 3.6 and 21.1 (1000 mg/L). Mean observed recoveries were .95, 94 and 75% at 10, 200 and 1000 mg/L, respectively. Concerning matrix effects, no influence of interfering compounds on the signal was observed for all the analytes: no significant signal loss or increase was observed at the retention time windows of ivabradine and the IS. In the patient’s serum, ivabradine concentration was 375 mg/L, and its metabolite (N-desmethylivabradine) was also detected. This assay was made after gastric wash at the theoric peak concentration owing to a blood collection 1.5 h after ivabradine intake. This concentration value was .30 times those measured after therapeutic doses intakes: in a clinical study in 44 healthy male volunteers treated with 7.5 mg of ivabradine and 4 mg of reboxetine, the mean plasma concentration of ivabradine (blood sampling 13 h after ivabradine intake) was 9.7 + 0.8 mg/L, with 25 mg/L as the highest value (7). In this case study, the serum concentration of ivabradine was consistent with the high dose intake (280 mg). Nevertheless, the bradycardia was no more severe than the one observed with therapeutic dosage. In fact, hypotension is certainly the noteworthy clinical effect in this case. Indeed, this observed hypotension (100/50 mmHg) was severe in regard to the associated mild bradycardia, especially in this patient treated with bradycardics for a long time.
Conclusion To the best of our knowledge, this is the first reported case of ivabradine intoxication documented with blood concentration. This case tends to show an absence of correlation between blood concentration and severity of the effects in cases of overdosage.
References 1. Riccioni, G. (2010) Ivabradine: from molecular basis to clinical effectiveness. Advances in Therapy, 27, 160– 167. 2. Deedwania, P. (2013) Selective and specific inhibition of If with ivabradine for the treatment of coronary artery disease or heart failure. Drugs, 73, 1569– 1586. 3. Lehotay, S.J., Son, K.A., Kwon, H., Koesukkwiwat, U., Fu, W., Mastovska, K. et al. (2010) Comparison of QuEChERS sample preparation methods for the analysis of pesticide residues in fruits and vegetables. Journal of Chromatography A, 127, 2548– 2560. 4. Gruson, A., Dingeon, B., Dreyfuss, M.F., Gaillard, Y., Le Bouil, A., Lhermitte, M. et al. (2005) Help for validation of analytical procedures in toxicology and therapeutic drug monitoring. Annales Toxicologie Analytique, 17, 1–20. 5. Peters, F.T., Drummer, O.H., Musshoff, F. (2007) Validation of new methods. Forensic Sciences International, 165, 216–224. 6. Antignac, J.-P., de Wasch, K., Monteau, F., De Brabander, H., Andre, F., Le Bizec, B. (2005) The ion suppression phenomenon in liquid chromatography– mass spectrometry and its consequences in the field of residue analysis. Analytica Chimica Acta, 529, 129– 136. 7. Zoerner, A.A., Schroeder, C., Kayacelebi, A.A., Suchy, M.T., Gutzki, F.M., Stichtenoth, D.O. et al. (2013) A validated, rapid UPLC-MS/MS method for simultaneous ivabradine, reboxetine, and metoprolol analysis in human plasma and its application to clinical trial samples. Journal of Chromatography B, Analytical Technologies in the Biomedical and Life Sciences, 927, 105– 111.
Case Report
Case Report of Ivabradine Intoxication Franc¸ois Mathiaux1, Sylvain Dulaurent1, Fre´de´ric Julia2 and Jean-Michel Gaulier1* 1
Biological and Forensic Toxicology Unit, Pharmacology and Toxicology Department, University Hospital of Limoges, 2 Avenue Martin-Luther King, 87024 Limoges cedex, France and 2Emergency Department, University Hospital of Limoges, 2 Avenue Martin-Luther King, 87024 Limoges cedex, France *Author to whom correspondence should be addressed. Email: [email protected]
Ivabradine is a drug used for the treatment of angina and chronic heart failure in cases of intolerance or insufficiency of response to betablocker treatment. A 47-year-old man was admitted to the emergency department of the hospital for a voluntary intoxication with 280 mg of ivabradine: he presented drowsiness and a mild sinusal bradycardia (50 bpm) associated with a well-tolerated low blood pressure at 100/ 50 mmHg. No complication was noted and he was discharged from the hospital on Day 3. A method for ivabradine assay in serum was obtained using liquid chromatography coupled with a mass spectrometry detection method. After a deproteinization step using QuECHERS salts and acetonitrile, a chromatographic separation was performed using a 5-mm 50 3 2.1 mm Xterraw column (Waters, France). Detection was performed using an LTQ linear ion-trap mass spectrometer equipped with an electrospray ionization source used in a positive ionization mode (ThermoFisher Scientific, San Jose CA, USA) and a detection in full MS2 scan. The limit of quantification of ivabradine was 10 mg/L, and the method was linear up to 1000 mg/L. The ivabradine concentration in the patient’s serum was 375 mg/L. This concentration value was >30 times those measured after therapeutic doses intakes. Nevertheless, the bradycardia was no more severe than the one observed with therapeutic dosage. In conclusion, this case tends to show an absence of correlation between blood concentration and severity of the troubles in cases of overdosage. Introduction Ivabradine is a drug commercialized in France under the brand name Procoralan. It is used for the treatment of angina and chronic heart failure in cases of intolerance or insufficiency of response to beta-blocker treatment (Figure 1). Ivabradine acts by direct inhibition of the spontaneous depolarization of the sinusal node, without any activity on the signal conduction in auricular or ventricular cavities. The clinical effect of this drug is a decrease of the heart rate which seems to be dose dependent, at least in therapeutic dosages (5–15 mg orally per day) (1). There is very little information about the consequences of ivabradine overdosage: that is, no cases are registered neither in the French poison control center nor in the MeSH database with research including the terms ‘ivabradine’ and ‘overdosage’, ‘ivabradine’ and ‘intoxication’. Nevertheless, owing to its pharmacodynamic properties and some adverse events observed before and after marketing authorization (atrioventricular block, syncope and cardiac arrest), intoxication of ivabradine is thought to induce severe bradycardia and acute heart failure (2). In addition, its large volume of distribution is in favor of prolonged effects. In the absence of documented issues, we propose to report an ivabradine intoxication case. Case story A 47-year-old man was admitted to the emergency department of the hospital for a voluntary intoxication: 280 mg of ivabradine
(56 5 mg tablets) associated (not proved) with an unknown dose of bromazepam. He presented drowsiness and a mild sinusal bradycardia (50 bpm) associated with a well-tolerated low blood pressure at 100/50 mmHg. No abnormality was found on the successive electrocardiograms, with the exception of a sinusal bradycardia. Initial treatment consisted of just one injection of atropine (1 mg) and a gastric wash. The patient was kept under medical supervision for 48 h before psychiatric care. No complication was noted, and the patient left the emergency department on Day 3. A blood sample was collected 1.5 h after the intoxication for ivabradine determination. It is of note that the hypothesis of a bromazepam intake by the patient was not investigated by the clinicians as from their clinical point of view and in the presented case, such benzodiazepine intoxication only required monitoring, without any specific dispositions. Materials and methods A method for ivabradine assay in serum was obtained using liquid chromatography coupled with a mass spectrometry detection method. Extraction was performed using the following procedure: after addition of 20 mL of 5 mg/L mebutamate solution (internal standard) to 100 mL patient’s serum, 5 mg QuEChERS (‘Quick, Easy, Cheap, Effective, Rugged and Safe’) salt mixture were added to the sample. This mixture is the combination of four salts: eight weight parts of MgSO4; four weight parts of NaCl; four weight parts of C6H5Na3O7.2H2O and one weight part of C6H6Na2O7.1.5H2O (3). After addition of 500 mL of acetonitrile, 1 min of vigorous shaking and centrifugation (10 000 rpm for 10 min), 1 mL of the organic phase was injected in the chromatographic system. The chromatographic system consisted in two Shimadzu LC-20 AD pumps, SIL-20 AC autosampler (Shimadzu, Marne-laValle´e, France). Chromatographic separation was performed on an Xterra MSC18 5-mm (50 2.1 mm, i.d.) column (Waters, France), using a gradient of (A) ammonium formate 2 mM pH 3.0 in water, and (B) a mixture of acetonitrile with 10% A (v/v) as mobile phase (constant flow rate of 200 mL/min), programmed as follows: 0 – 2.5 min, 20% (B); 2.5 – 5 min, increase from 20 to 90% (B); 5 – 6.5 min, 90% (B); 6.5 – 7 min, decrease from 90 to 20% (B); 7 – 11.5 min, column equilibration with 20% (B). The total chromatographic run time was 11.5 min. All chromatographic solvents were degassed with helium beforehand. Mass spectrometric analyses were conducted using a ThermoFisher Scientific (San Jose, CA, USA) LTQ linear ion-trap mass spectrometer equipped with an electrospray ionization source. The main parameter settings in the positive ionization mode were: ionspray voltage, 4.5 kV; sheath gas flow rate, 12;
# The Author [2014]. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
Figure 1. Ivabradine formula. Chemical name: 2H-3-Benzazepin-2-one, 3-(3-(((3,4dimethoxybicyclo(4.2.0)octa-1,3,5-trien-7-yl)methyl)methylamino)propyl)-1,3,4, 5-tetrahydro-7,8-dimethoxy-, (S)- Formula: C 27 – H 36 – N 2 – O 5 . MM: 468. CAS registry number: 0155974-00-8.
Aux gas flow rate, 10; sweep gas flow rate, 12; capillary voltage, 15 V; capillary temperature, 3008C and tube lens, 65 V. Acquisition was performed in full MS2 mode following a parent mass for ivabradine (m/z 469.30) and mebutamate (m/z 233.15). Ionic transitions were obtained by extraction of daughter ions [ivabradine quantification (m/z 262.00) and confirmation (m/z 177.00 and 206.00)/mebutamate (m/z 172.11)] from the full MS2 spectrum of the corresponding parent ion. In addition, detection of N-desmethylivabradine, the main metabolite of ivabradine, was performed in full MS2 using its main following transitions (m/z 455.3 ! 262.00, m/z 455.3 ! 177.00 and m/z 455.3 ! 206.00). By means of ad hoc ivabradine (Sigma Aldrich, France) additions to free human serum, a calibration curve (10, 50, 200, 500 and 1000 mg/L) was constructed using a quadratic function with weighting factor 1/x. Validation procedure was performed according to the French Society of Analytical Toxicology recommendations (4) and proposed experiments, evaluations and acceptance criteria for the validation of new analytical methods to be used in single-case studies of analysis of rare analytes (5). The intra-assay precision and accuracy were assessed at low and high concentrations relative to calibration range: analysis of five spiked serum samples at each concentration. The limit of detection (LOD) was defined as the lowest concentration giving a response of at least three times the average of the baseline noise. The limit of quantification (LOQ) was defined as the lowest concentration that could be measured with an intra-assay precision CV% (coefficient variation %) and a relative bias ,20%. Recoveries were determined in triplicate at three concentration levels (10, 100 and 1000 mg/L) by comparing the analyte/internal standard peak area ratios obtained after the extraction of spiked samples with those of serum free extracts further spiked with ivabradine. Carry over was evaluated, by analyzing a sample of mobile phase subsequently to the highest calibration level (n ¼ 5). The absence of any signal on the corresponding chromatogram was verified. Ion suppression phenomenon was studied following the experimental system previously proposed by Antignac et al. (6). Briefly, a standard solution of ivabradine (at 100 mg/L) was continuously and directly infused into the mass spectrometer interface. A simultaneous LC flow containing either a pure mobile phase or a blank biological extract (serum from 10 non-drug consumers) was introduced through a tee. Evolution of the signal of the compounds of interest was studied to evaluate the presence and intensity of ion suppression.
Results This method exhibits LOD and LOQ for ivabradine of 5 and 10 mg/L, respectively. Intra-assay precision CV and relative bias
232 Mathiaux et al.
(%) were 6.3 and 25.2 (10 mg/L), 3.7 and 8.0 (200 mg/L) and 8.3 and 29.0 (1000 mg/L). Inter-assay precision CV and relative bias (%) were 5.0 and 5.1 (10 mg/L), 5.5 and 4.1 (200 mg/L) and 3.6 and 21.1 (1000 mg/L). Mean observed recoveries were .95, 94 and 75% at 10, 200 and 1000 mg/L, respectively. Concerning matrix effects, no influence of interfering compounds on the signal was observed for all the analytes: no significant signal loss or increase was observed at the retention time windows of ivabradine and the IS. In the patient’s serum, ivabradine concentration was 375 mg/L, and its metabolite (N-desmethylivabradine) was also detected. This assay was made after gastric wash at the theoric peak concentration owing to a blood collection 1.5 h after ivabradine intake. This concentration value was .30 times those measured after therapeutic doses intakes: in a clinical study in 44 healthy male volunteers treated with 7.5 mg of ivabradine and 4 mg of reboxetine, the mean plasma concentration of ivabradine (blood sampling 13 h after ivabradine intake) was 9.7 + 0.8 mg/L, with 25 mg/L as the highest value (7). In this case study, the serum concentration of ivabradine was consistent with the high dose intake (280 mg). Nevertheless, the bradycardia was no more severe than the one observed with therapeutic dosage. In fact, hypotension is certainly the noteworthy clinical effect in this case. Indeed, this observed hypotension (100/50 mmHg) was severe in regard to the associated mild bradycardia, especially in this patient treated with bradycardics for a long time.
Conclusion To the best of our knowledge, this is the first reported case of ivabradine intoxication documented with blood concentration. This case tends to show an absence of correlation between blood concentration and severity of the effects in cases of overdosage.
References 1. Riccioni, G. (2010) Ivabradine: from molecular basis to clinical effectiveness. Advances in Therapy, 27, 160– 167. 2. Deedwania, P. (2013) Selective and specific inhibition of If with ivabradine for the treatment of coronary artery disease or heart failure. Drugs, 73, 1569– 1586. 3. Lehotay, S.J., Son, K.A., Kwon, H., Koesukkwiwat, U., Fu, W., Mastovska, K. et al. (2010) Comparison of QuEChERS sample preparation methods for the analysis of pesticide residues in fruits and vegetables. Journal of Chromatography A, 127, 2548– 2560. 4. Gruson, A., Dingeon, B., Dreyfuss, M.F., Gaillard, Y., Le Bouil, A., Lhermitte, M. et al. (2005) Help for validation of analytical procedures in toxicology and therapeutic drug monitoring. Annales Toxicologie Analytique, 17, 1–20. 5. Peters, F.T., Drummer, O.H., Musshoff, F. (2007) Validation of new methods. Forensic Sciences International, 165, 216–224. 6. Antignac, J.-P., de Wasch, K., Monteau, F., De Brabander, H., Andre, F., Le Bizec, B. (2005) The ion suppression phenomenon in liquid chromatography– mass spectrometry and its consequences in the field of residue analysis. Analytica Chimica Acta, 529, 129– 136. 7. Zoerner, A.A., Schroeder, C., Kayacelebi, A.A., Suchy, M.T., Gutzki, F.M., Stichtenoth, D.O. et al. (2013) A validated, rapid UPLC-MS/MS method for simultaneous ivabradine, reboxetine, and metoprolol analysis in human plasma and its application to clinical trial samples. Journal of Chromatography B, Analytical Technologies in the Biomedical and Life Sciences, 927, 105– 111.
