Journal of Analytical Toxicology, Vol. 16, November/December 1992
I Case Report
Toxicological Findings in a Fatal Case of Acebutolol
Self-Poisoning A. T r a c q u i t, P. Kintz 1, P. W e n d l i n g 2, S. R i t t e r - L o h n e r 1, P. M a n g i n 1, and A. J a e g e r 2 tnstitut de M&decine Logs~e, 11 rue Humann, 67085 Strasbourg Cedex, France 2Service de Rdanimation M~dicale et Centre Anti-Poisons, Pavilion Pasteur, CHRU/HOpitaux Universitaires de Strasbourg, 67091 Strasbourg Codex, France
l Abst_ra_ct~ A fatal case of acebutolol self-poisoning is presented. After single-step liquid-liquid alkaline extraction, acebutolol was Identified by using an HPLC/DAD screening procedure. By means of a specific HPLC method, acebutolol was then quantified In a large range of postmortem samples. The blood acebutolol concentration was 34.7 p,g/mL. The tissue distribution of the drug Is discussed In the light of the existing literature.
Introduction
Acebutolol (3'-acetyl-4'-(2-hydroxy-3-isopropylaminopropoxybutyranilide)) (Figure l) is a B-adrenoceptor blocking agent (BB) with cardioselectivity, moderate membrane stabilizing activity, and little intrinsic sympathomimetic activity (1,2). The drag has been available in France since 1976 under the name Sectral | and is widely prescribed for hypertension, dysrhythmias, angina pectoris, and migraine at dosages of 0.2 to 1.2 g daily (1-3). Acebutolol poisonings have been very seldom described. We present here a fatal case of acebutolol self-poisoning in which the drug was identified by using a recently developed HPLC/DAD screening method and then quantified in blood, urine, and a large range of postmortem samples.
C a s e History
An 18-year-old-girl was admitted to the intensive care unit with cardiovascular shock due to a self-poisoning. One hour before admission, she had suddenly lost consciousness and developed hypotension (blood pressure 80160 mmHg, pulse rate 60/min) and hypoventilation. She was treated by tracheal intubation, artificial ventilation, dopamine (5/ag/kg,min), and molar sodium bicarbonate infusion (100 mL). On admission, she was in coma (Glasgow stade 4) and circulatory arrest with bradycardia at 50/rain and widening of the QRS complexes (0.12 s). After iv epinephrine (5 rag), molar sodium bicarbonate infusion (250 mL), and artificial ventilation with 100% O2, blood pressure in-
398
creased to 110/90 mmHg; however, the shock persisted and pulmonary oedema occurred. A hemodynamic study by Swan-Ganz catheter showed a severe cardiogenic shock: cardiac index 1.03 L/min-mz, pulmonary capillary wedge pressure 25 mmHg, systemic vascular resistances 44.6 I.U./m2, left ventricular systolic work index 8.2 g/m2. Treatment with epinephrine (5.5 I.tg/kg,min), isoprenaline (1.6 !t~/kg.min), norepinephrine, milrinone (200 mg iv), glucagon (10 mg iv) and oral activated charcoal (50 g) was ineffective and the patient died 10 h after admission. Further questioning of the parents revealed that she had ingested about 24 g of acebutolol. Autopsy findings were unremarkable. In order to perform the toxicological analysis, the following postmortem samples were collected: blood, urine, vitreous humor (1.6 mL), liver, kidney, myocardium, brain, lung, spleen, uterus, squelettic muscle (psoas), and adipose tissue.
Experimental
Materials. Acebutolol and oxprenolol were kindly donated by their respective manufacturers (Specia and Ciba-Geigy, France). Methanol, tetrahydrofuran, and acetonitrile were HPLC grade and purchased from Merck, ER.G. All other chemicals and reagents were analytical grade and obtained from Merck and Prolabo, France. Instrumentation System 1. Drug screening was performed using an HPLC/DAD apparatus consisting of a quaternary pump (Waters 600 E) with an autoinjector (Waters 715 Ultra Wisp); the detector was a Waters CH3--CH2--CH2--CO--NH
O -- C H 2 - - C H O H
Figure 1. Chemicalstructure of acebutolol.
Reproduction (photocopying) of editorial content of this journal is prohibited without publisherspermission.
-- C H 2 - N H-- C H ( C I ~ ) 2
Journal of Analytical Toxicology, Vol. 16, November/December 1992
991 photodiode array spectrophotometer (wavelength 190 to 800 nm) with resolution and spectra sampling interval set at 1.3 nm and 1 s, respectively. The column was a 4-~n Nova Pak CI 8 (Waters) (300 x 3.9 mm i.d.) set at 30~ The eluent consisted of methanol, tetrahydrofuran, and 10-2M KH2PO4, pH 2.6 buffer (65:5:30, v/v). Elution was performed isocratically (flow rate 0.8 mlJmin, average operating pressure 2850 lb/in.2). Under these operating conditions, the lower limit of detectability for acebutolol was about 110 ng/mL. System 2. Acebutolol was then quantified by using a previously described specific HPLC method (4,5). The system consisted of a pump (Waters 810) and an autoinjector (Waters WISP 710B) coupled to a multiwavelength spectrophotometer (Waters 490) set at 230 nm. An RSil CN (Alltech) 10-1am column (300 x 4.1 mm i.d.) was used at ambient temperature, The eluent was a mixture of water, acetonitrile, and 0.1M NaH2PO4, pH 3.0 buffer (60:30: i0, v/v). The flow rate was 1.0 mL/min, with an average operating pressure of 1000 lb/in 2. Under these operating conditions, the detection limit for acebutolol had been previously estimated (4) at 17 ng/mL. The main values of reproducibility for acebutolol were 5.3% within-run precision (8 plasma samples spiked at 100 ng/mL) and 7.0% day-to-day precision (plasma sample spiked at 100 ng/mL, daily dosage during 3 weeks). In addition, the method was linear for acebutolol over a range of 50 to 10,000 ng/mL.
cedure indicated the presence of acebutolol and diazepam (Figure 2). A fluorescence polarization immunoassay (Abbott ADx), which is systematically performed in parallel to HPLC/DAD, was positive only for benzodiazepines, which confirmed the previous findings. This th'st step of the analysis excluded the following substances: barbiturates, antidepressants, antipsychotics (phenothiazines, butyrophenones, and benzamides), antihistaminics, salicylates, acetaminophen, calcium channel blockers, opiates, cocaine, amphetamine derivatives, and cannabinoids. The concentrations of acebutolol in the postmortem samples are summarized in Table I. The blood concentration of diazepam was 0.84 pg/mL (dosage by GC). A small amount of ethanol (0.12 mg/mL) was also measured in blood (dosage by headspace GC).
Discussion
Acebutolol fatalities appear to be extremely rare. To the best of our knowledge, only nine cases had been previously reported (6-14) (Table II). Main symptoms in severe acebutolol overdose are delayed conduction and hypotension, as with other fiBs. Massive ingestions cause widening of QRS (due to the stabilizing membrane effect, which occurs only at supratherapeutic levels [2]), first- to high-degree atrioventricular block, and then asystole. Electromechanical dissociation seems frequent (15,16), but bradycardia may be lacking (16,17). Therapeutic steady-state plasma concentrations of acebutuolol usually range from 0.5 to 1.2 pg/mL (when 0.9 to 1.2 g given daily) (3). Although fiB are claimed to possess wide toxic-therapeutic margins (1), death was observed following ingestion of a dose as low as 4.0 g in a healthy young female (10). On the
Procedure
Drug screening. To 2.0 mL postmortem blood in a 15-mL centrifuge tube were added 5.0 mL of chloroform-2-propanol-nheptane (60:14:26, v/v) and 1.5 mL of a saturated NHaCI buffer solution (pH 9.5). After horizontal agitation (10 min) and centrifugation (10 min at 2800 g), the lower organic phase was removed and evaporated at 45~ in a rotary evaporator (Speed Vac Concentrator A290, Savant Instruments). The residue was dissolved in 100 IlL of the eluent and 50 p.L was injected into the System 1. Acebutolol quantification. 1-mL samples of biological fluids (pure and 1:5 diluted with distilled water) and tissue homogenates (one part tissue homogenized in four parts distilled water with an IKA UltraTurrax homogenizer) were extracted by adding 3 mL diethylether-dichloromethane (80:20, v/v), 200/aL _ i 1N NaOH, and 50 laL ofa 10-~g/mL methanolic solution of oxprenolol as an internal standard (IS). After agitation and centrifugation, the organic supernatant was removed and backextracted with 100 ILL 0.IN H2SO 4, from which 70 pL was injected into System 2. Quantification was achieved for each sample by comparing peak area ratios (acebutolol:IS) with the previously stored specific calibration curve. . . . .
I
.
.
.
I
.
I
/, ';
. . . .
Results
The HPLC/DAD screening pro-
~
/ 2
. .
",
,.,
/ i
2
...........,.... .....
. . . .
.
J
i 9
"
I
IH
4
HPLC-13AD chromatogram (displayed at 254 nm) of the blood extract. Peak 1 (3.84 rain): acebutolol; Peak 2 (6.01 rain): diazeparn. Inset: superposition of UV spectra (200 to 400 nm) of Peak 1 (full line) and acebutolol (dotted line); similarity 967/1000.
Figure 2.
399
Jourr~at ot Anatytica~ Toxicotogy, Vot, t6, NovembeflOecember 1992
other hand, survival has occurred with 7.6-, 8.8-, 9.6-, 11.2-, and 13.6-g overdoses ( 15-19), which confirms the absence of a close correlation between ingested dose and toxic effects (20). Tissue distribution of acebutolol had been investigated in few studies (6,11,13) (Table llI). These results show a tendency for the drug to accumulate in kidneys, where it undergoes primary elimination (1,2), and then in the liver. The lower levels were measured in brain and adipose tissue; in comparison to other 13Bs such as pmpranolol, acebutolol exhibits a marked hydrophilic character and consequently enters deep body compartments only with difficulty (1).
The recent introduction of reliable photodiode array detectors (DAD) has dramatically improved the selectivity of HPLC and afforded it a number of advantages previously attributed only to GC/MS, at about one-third to one-fourth the cost. Consequently, HPLC/DAD is achieving greater prominence in drug screening applications (21-24). The present work represents a preliminary application of such a technique, which is being developed in our laboratory. Further results will be subsequently published.
References Table I. Tissue Distribution of Acebutolol Concentration (IJg/mL or pg]g)
Tissue/fluid Blood Urine Vitreous humor Liver Kidney My0cardium Lung Brain Spleen Uterus Adipose tissue Squelettic muscle
34.7 614.0 17.9 55.8 93.2 57.8 58.4 6.5 91.8 54.2 7.3 62.9
(psoas)
Table II. Fatalities Involving Acebutoloh Summary of the Literature (1979-1991) Author
Subject
Amount Bloodlevel
(Ref.)
Year
(sex, age)
Ingested (g)
(pg/mL)
Klug (6) Harzer (7) Bayer (8) Eibs (9) Eibs (10) Chaumont (11) Jean (12) Weiser (13) Chennebault (14) Tracqui
1979 1979 1981 1982 1982 1983 1984 1985 1986 1991
M32 ? M15 F15 F30 M22 F M49 F20 F18
12.0 (?) 6.0 8.6 8,0 4.0 8,8 (?) 8.0 8.0 16.0 24.0 (?)
33.0 25.0 21.5 13.4 149.0 51.0 34.7
Table Iil. Tissue Distribution in Acebutolol Fatalities Present
Klug
Tissue/fluid
study
(6)
Blood Urine Bile Liver Kidney
34.7 614.0 55.8 93.2
33.0 1330.0 40.0 140.0
My0cardium Lung
57.8 58.4
-
Brain Spleen Squelettic muscle
6.5 91.8 62.9
18.0
400
Chaumont (11) 21.5 303.7 415.5 122.8 R: 142.0 L: 138.8 81.8 R: 34.5 L: 15,8 0.6 38.6 -
Welser (13) 149.0 51.9 127.7 25.7 22.1 48.0 24.4
1. MJ. Ellenhom and e.G. Barceloux. Medical Toxicology--Diagnosis and Treatment of Human Poisoning. Elsevier, Amsterdam, 1988, pp. 187-94. 2. U. Borchard, Pharmakologie der B-Rezeptorenblocker. Aesopus Vedag, Basel, 1983, pp. 9-38. 3. Clarke's Isolation and Identification of Drugs in Pharmaceuticals, Body Fluids, and Post-Mortem Material A.C, Moffat, Ed., The Pharmaceutical Press, London, 1986, p. 309. 4. A Tracqui, P. Kintz, J. Himber, A.A.J, Lugnier, and P. Mangin. A specific HPLC method for determination of B-blockers topically used in ophthalmological diseases. Forensic Sci. Int. 38:37-41 (1988). 5. A. Tracqui, P. KintZ, P. Mangin, and B. Lenoir. Self-poisoning with the f3blocker bisoprolol. Hum. Exp. ToxicoL 9:255-56 (1990). 6. E. Klug and V. Schneider. Tbd{iche Vergittung mit Acebutolol (Prant), Z. Rechtsmed. 83:325-30 (1979). 7. K. Harzer. TSdliche Vergiftung mit Acebutolol (Prent), Toxichem 7:(1979), cited in Reference 6. 8. H. Beyer, P. Blank, L. Kelch, D. Oltaamanns, and T. Shultek. Letale Vergiftung mit Acebutolol. Intensive Mad. 18:83-85 (1981). 9. H.G. Eibs, U. Oberdisse, and U. Brambach. Vergiftungen mit 8-Rezeptoranblockem bei Kindern und Jugendlichen, Monatsschr. Kinderheilkd. 130:292-95 (1982). 10. H.G. Eibs, U. Oberdisse, and U. Brambach. Intoxikation mit Beta-Rezeptorenblockern. Dtsche. med. Wschr. 107:1139-43 (1982). 11. A.J, Chaumont, P. Mangin, and A.A.J. Lugnier. Suicide par absorption massive d'un b~ta-bloquant: I'ac~butolol. J. Med. Leg.--Droit Mad. 26: 139-42 (1983). 12. P. Jean, J. Arditti, J, Jouglard, J. Bues-Charbit, J.P. Sommadossi, N. Fomaris, and J,P. Cane. intoxication aigu~ par I'ac~butolol. Apropos d'un cas avec dosages de I'ac~butolol inchangd et de ses m~tabolites. Thdrapie 39:49-50 (1984). 13. J,R. Weiser and I. Gerlin. LetaJe Vergiftung mit AcebutoloL Dtsche. mad, Wschr. 110:1793-95 (1985). 14. J.M. Chennebault, A. Turcant, P. Harry, P. Alquier, and P. Allain. Intoxication mortelle par ace~butolol. Th~Jrapie41:143 (1986). 15. M. Freysz, D. Honnart, A. Besan(;on, and M. Wilkening. Cardiac arrest after beta-blocker poisoning. Crit, Care Mad. 14:837-38 (1986). 16. B. Journe, R, Berlault, M. Buffet, M. Jaussaud, M e . Rambourg, and G.A. Says, Intoxications volontaires par acebutolol. Nouv. Presse Mad. 11: 8569-70 (1982). 17. B. Sangster, D. de Wildt, and A. van Dijk. A case of acebutolol intoxication. Clin. ToxicoL 20:69-77 (1983). 18. P. Massari, B. Courbon, J.M Droy, E. Moirot, G. Bonmarchand, and J. Leroy. Intoxication grave par l'acdbutotol avec ~tat de choc et bloc intraventriculaire. R(}an. Soins Intens. Mad. Urg. 1:25-26 (1985). 19. M Lewis, J. Kallenbach, and C. Germond. Survival following massive overdose of adrenergic blocking agents (acebutolol and labetalol). Eur. Heart J. 4:328-32 (1983). 20. P. Auz6py, N. Boukara, C. Richard, and J.E GiudJcelli. Intoxication aigu~ par les b6te-bloquants chez I'adulte. Apropos de sept cas. Ann. CardioL AngeioL 32:253-58 (1983). 21. B.K, Logan, D.T. Stafford, I,R Tebbett, and C.M. Moore. Rapid screening for 100 basic drugs and metabolites in urine using cation exchange solidphase extraction and high-performance liquid chromatography with diode array detection. J. Anal ToxicoL 14:154-59 (1990). 22. M. Bogusz and M. Wu. Standardized HPLC/DAD system, based on retention indices and spectral library, applicable for systematic toxicological screening. J. Anal ToxicoL 15:188-97 (1991). 23. A. Tracqui, P. Kintz, and P. Mangin. La chromatographie liquide haute performance avec d~tection par barrette de diodes: un nouvel outil au service du laboratoire de toxicologie m~dico-16gale. J. Med. Leg.--Droit Mad. 35: 37-40 (1992). 24. A, Tracqui, P. Kintz, and P. Mangin. Simple and rapid screening procedure for 27 neuroleptics using HPLC/PDA. J. Liq. Chromatogr. 15:1381-96 (1992). Manuscript received November 7, 1991 ; revision received May 5, 1992.
I Case Report
Toxicological Findings in a Fatal Case of Acebutolol
Self-Poisoning A. T r a c q u i t, P. Kintz 1, P. W e n d l i n g 2, S. R i t t e r - L o h n e r 1, P. M a n g i n 1, and A. J a e g e r 2 tnstitut de M&decine Logs~e, 11 rue Humann, 67085 Strasbourg Cedex, France 2Service de Rdanimation M~dicale et Centre Anti-Poisons, Pavilion Pasteur, CHRU/HOpitaux Universitaires de Strasbourg, 67091 Strasbourg Codex, France
l Abst_ra_ct~ A fatal case of acebutolol self-poisoning is presented. After single-step liquid-liquid alkaline extraction, acebutolol was Identified by using an HPLC/DAD screening procedure. By means of a specific HPLC method, acebutolol was then quantified In a large range of postmortem samples. The blood acebutolol concentration was 34.7 p,g/mL. The tissue distribution of the drug Is discussed In the light of the existing literature.
Introduction
Acebutolol (3'-acetyl-4'-(2-hydroxy-3-isopropylaminopropoxybutyranilide)) (Figure l) is a B-adrenoceptor blocking agent (BB) with cardioselectivity, moderate membrane stabilizing activity, and little intrinsic sympathomimetic activity (1,2). The drag has been available in France since 1976 under the name Sectral | and is widely prescribed for hypertension, dysrhythmias, angina pectoris, and migraine at dosages of 0.2 to 1.2 g daily (1-3). Acebutolol poisonings have been very seldom described. We present here a fatal case of acebutolol self-poisoning in which the drug was identified by using a recently developed HPLC/DAD screening method and then quantified in blood, urine, and a large range of postmortem samples.
C a s e History
An 18-year-old-girl was admitted to the intensive care unit with cardiovascular shock due to a self-poisoning. One hour before admission, she had suddenly lost consciousness and developed hypotension (blood pressure 80160 mmHg, pulse rate 60/min) and hypoventilation. She was treated by tracheal intubation, artificial ventilation, dopamine (5/ag/kg,min), and molar sodium bicarbonate infusion (100 mL). On admission, she was in coma (Glasgow stade 4) and circulatory arrest with bradycardia at 50/rain and widening of the QRS complexes (0.12 s). After iv epinephrine (5 rag), molar sodium bicarbonate infusion (250 mL), and artificial ventilation with 100% O2, blood pressure in-
398
creased to 110/90 mmHg; however, the shock persisted and pulmonary oedema occurred. A hemodynamic study by Swan-Ganz catheter showed a severe cardiogenic shock: cardiac index 1.03 L/min-mz, pulmonary capillary wedge pressure 25 mmHg, systemic vascular resistances 44.6 I.U./m2, left ventricular systolic work index 8.2 g/m2. Treatment with epinephrine (5.5 I.tg/kg,min), isoprenaline (1.6 !t~/kg.min), norepinephrine, milrinone (200 mg iv), glucagon (10 mg iv) and oral activated charcoal (50 g) was ineffective and the patient died 10 h after admission. Further questioning of the parents revealed that she had ingested about 24 g of acebutolol. Autopsy findings were unremarkable. In order to perform the toxicological analysis, the following postmortem samples were collected: blood, urine, vitreous humor (1.6 mL), liver, kidney, myocardium, brain, lung, spleen, uterus, squelettic muscle (psoas), and adipose tissue.
Experimental
Materials. Acebutolol and oxprenolol were kindly donated by their respective manufacturers (Specia and Ciba-Geigy, France). Methanol, tetrahydrofuran, and acetonitrile were HPLC grade and purchased from Merck, ER.G. All other chemicals and reagents were analytical grade and obtained from Merck and Prolabo, France. Instrumentation System 1. Drug screening was performed using an HPLC/DAD apparatus consisting of a quaternary pump (Waters 600 E) with an autoinjector (Waters 715 Ultra Wisp); the detector was a Waters CH3--CH2--CH2--CO--NH
O -- C H 2 - - C H O H
Figure 1. Chemicalstructure of acebutolol.
Reproduction (photocopying) of editorial content of this journal is prohibited without publisherspermission.
-- C H 2 - N H-- C H ( C I ~ ) 2
Journal of Analytical Toxicology, Vol. 16, November/December 1992
991 photodiode array spectrophotometer (wavelength 190 to 800 nm) with resolution and spectra sampling interval set at 1.3 nm and 1 s, respectively. The column was a 4-~n Nova Pak CI 8 (Waters) (300 x 3.9 mm i.d.) set at 30~ The eluent consisted of methanol, tetrahydrofuran, and 10-2M KH2PO4, pH 2.6 buffer (65:5:30, v/v). Elution was performed isocratically (flow rate 0.8 mlJmin, average operating pressure 2850 lb/in.2). Under these operating conditions, the lower limit of detectability for acebutolol was about 110 ng/mL. System 2. Acebutolol was then quantified by using a previously described specific HPLC method (4,5). The system consisted of a pump (Waters 810) and an autoinjector (Waters WISP 710B) coupled to a multiwavelength spectrophotometer (Waters 490) set at 230 nm. An RSil CN (Alltech) 10-1am column (300 x 4.1 mm i.d.) was used at ambient temperature, The eluent was a mixture of water, acetonitrile, and 0.1M NaH2PO4, pH 3.0 buffer (60:30: i0, v/v). The flow rate was 1.0 mL/min, with an average operating pressure of 1000 lb/in 2. Under these operating conditions, the detection limit for acebutolol had been previously estimated (4) at 17 ng/mL. The main values of reproducibility for acebutolol were 5.3% within-run precision (8 plasma samples spiked at 100 ng/mL) and 7.0% day-to-day precision (plasma sample spiked at 100 ng/mL, daily dosage during 3 weeks). In addition, the method was linear for acebutolol over a range of 50 to 10,000 ng/mL.
cedure indicated the presence of acebutolol and diazepam (Figure 2). A fluorescence polarization immunoassay (Abbott ADx), which is systematically performed in parallel to HPLC/DAD, was positive only for benzodiazepines, which confirmed the previous findings. This th'st step of the analysis excluded the following substances: barbiturates, antidepressants, antipsychotics (phenothiazines, butyrophenones, and benzamides), antihistaminics, salicylates, acetaminophen, calcium channel blockers, opiates, cocaine, amphetamine derivatives, and cannabinoids. The concentrations of acebutolol in the postmortem samples are summarized in Table I. The blood concentration of diazepam was 0.84 pg/mL (dosage by GC). A small amount of ethanol (0.12 mg/mL) was also measured in blood (dosage by headspace GC).
Discussion
Acebutolol fatalities appear to be extremely rare. To the best of our knowledge, only nine cases had been previously reported (6-14) (Table II). Main symptoms in severe acebutolol overdose are delayed conduction and hypotension, as with other fiBs. Massive ingestions cause widening of QRS (due to the stabilizing membrane effect, which occurs only at supratherapeutic levels [2]), first- to high-degree atrioventricular block, and then asystole. Electromechanical dissociation seems frequent (15,16), but bradycardia may be lacking (16,17). Therapeutic steady-state plasma concentrations of acebutuolol usually range from 0.5 to 1.2 pg/mL (when 0.9 to 1.2 g given daily) (3). Although fiB are claimed to possess wide toxic-therapeutic margins (1), death was observed following ingestion of a dose as low as 4.0 g in a healthy young female (10). On the
Procedure
Drug screening. To 2.0 mL postmortem blood in a 15-mL centrifuge tube were added 5.0 mL of chloroform-2-propanol-nheptane (60:14:26, v/v) and 1.5 mL of a saturated NHaCI buffer solution (pH 9.5). After horizontal agitation (10 min) and centrifugation (10 min at 2800 g), the lower organic phase was removed and evaporated at 45~ in a rotary evaporator (Speed Vac Concentrator A290, Savant Instruments). The residue was dissolved in 100 IlL of the eluent and 50 p.L was injected into the System 1. Acebutolol quantification. 1-mL samples of biological fluids (pure and 1:5 diluted with distilled water) and tissue homogenates (one part tissue homogenized in four parts distilled water with an IKA UltraTurrax homogenizer) were extracted by adding 3 mL diethylether-dichloromethane (80:20, v/v), 200/aL _ i 1N NaOH, and 50 laL ofa 10-~g/mL methanolic solution of oxprenolol as an internal standard (IS). After agitation and centrifugation, the organic supernatant was removed and backextracted with 100 ILL 0.IN H2SO 4, from which 70 pL was injected into System 2. Quantification was achieved for each sample by comparing peak area ratios (acebutolol:IS) with the previously stored specific calibration curve. . . . .
I
.
.
.
I
.
I
/, ';
. . . .
Results
The HPLC/DAD screening pro-
~
/ 2
. .
",
,.,
/ i
2
...........,.... .....
. . . .
.
J
i 9
"
I
IH
4
HPLC-13AD chromatogram (displayed at 254 nm) of the blood extract. Peak 1 (3.84 rain): acebutolol; Peak 2 (6.01 rain): diazeparn. Inset: superposition of UV spectra (200 to 400 nm) of Peak 1 (full line) and acebutolol (dotted line); similarity 967/1000.
Figure 2.
399
Jourr~at ot Anatytica~ Toxicotogy, Vot, t6, NovembeflOecember 1992
other hand, survival has occurred with 7.6-, 8.8-, 9.6-, 11.2-, and 13.6-g overdoses ( 15-19), which confirms the absence of a close correlation between ingested dose and toxic effects (20). Tissue distribution of acebutolol had been investigated in few studies (6,11,13) (Table llI). These results show a tendency for the drug to accumulate in kidneys, where it undergoes primary elimination (1,2), and then in the liver. The lower levels were measured in brain and adipose tissue; in comparison to other 13Bs such as pmpranolol, acebutolol exhibits a marked hydrophilic character and consequently enters deep body compartments only with difficulty (1).
The recent introduction of reliable photodiode array detectors (DAD) has dramatically improved the selectivity of HPLC and afforded it a number of advantages previously attributed only to GC/MS, at about one-third to one-fourth the cost. Consequently, HPLC/DAD is achieving greater prominence in drug screening applications (21-24). The present work represents a preliminary application of such a technique, which is being developed in our laboratory. Further results will be subsequently published.
References Table I. Tissue Distribution of Acebutolol Concentration (IJg/mL or pg]g)
Tissue/fluid Blood Urine Vitreous humor Liver Kidney My0cardium Lung Brain Spleen Uterus Adipose tissue Squelettic muscle
34.7 614.0 17.9 55.8 93.2 57.8 58.4 6.5 91.8 54.2 7.3 62.9
(psoas)
Table II. Fatalities Involving Acebutoloh Summary of the Literature (1979-1991) Author
Subject
Amount Bloodlevel
(Ref.)
Year
(sex, age)
Ingested (g)
(pg/mL)
Klug (6) Harzer (7) Bayer (8) Eibs (9) Eibs (10) Chaumont (11) Jean (12) Weiser (13) Chennebault (14) Tracqui
1979 1979 1981 1982 1982 1983 1984 1985 1986 1991
M32 ? M15 F15 F30 M22 F M49 F20 F18
12.0 (?) 6.0 8.6 8,0 4.0 8,8 (?) 8.0 8.0 16.0 24.0 (?)
33.0 25.0 21.5 13.4 149.0 51.0 34.7
Table Iil. Tissue Distribution in Acebutolol Fatalities Present
Klug
Tissue/fluid
study
(6)
Blood Urine Bile Liver Kidney
34.7 614.0 55.8 93.2
33.0 1330.0 40.0 140.0
My0cardium Lung
57.8 58.4
-
Brain Spleen Squelettic muscle
6.5 91.8 62.9
18.0
400
Chaumont (11) 21.5 303.7 415.5 122.8 R: 142.0 L: 138.8 81.8 R: 34.5 L: 15,8 0.6 38.6 -
Welser (13) 149.0 51.9 127.7 25.7 22.1 48.0 24.4
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