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Blood concentrations of tetracaine and its metabolite following spinal anesthesia
Forensic Science International 116 (2001) 9±14
Blood concentrations of tetracaine and its metabolite following spinal anesthesia Keiko Kudoa, Yukiko Hinoa, Noriaki Ikedaa,*, Hidefumi Inoueb, Shosuke Takahashib a
Department of Forensic Pathology and Sciences, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan b Department of Anesthesiology & Critical Care Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan Received 19 May 2000; received in revised form 22 June 2000; accepted 27 June 2000
Abstract Blood concentrations of tetracaine and its metabolite, p-butylaminobenzoic acid, were measured after spinal anesthesia with tetracaine which had been administered to patients under going orthopedic surgery. Tetracaine, an ester anesthetic, was given to 10 patients, the dose was 8±14 mg, and blood samples were collected 1, 2 and 6 h after the injection of tetracaine. We used gas chromatography/mass spectrometry for purposes of analysis. Tetracaine was not detected in any blood sample, but the metabolite was detected in each sample with the mean concentrations of 126.5, 97.9 and 43.3 ng/ml at 1, 2 and 6 h, respectively. This data will be useful in determination of the cause of death after spinal anesthesia with tetracaine. # 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Forensic toxicology; Spinal anesthesia; Tetracaine; p-Butylaminobenzoic acid; Mepivacaine
1. Introduction Tetracaine, also known as amethocaine, is an estertype local anesthetic. This drug is a very potent, longacting agent with a low therapeutic dose and is commonly used to induce spinal anesthesia [1]. It is known that the use of local anesthetic in spinal anesthesia occasionally results in sudden death of the patient [2], and thus it is important to clarify the cause of death. We have experienced a case where a patient died after being given tetracaine to induce spinal anesthe-
*
Corresponding author. Tel.: 81-92-642-6124; fax: 81-92-642-6126. E-mail address: [email protected] (N. Ikeda).
sia. Based on the autopsy ®ndings and microscopic analysis, high spinal anesthesia by tetracaine was suspected and further toxicological examinations were carried out. Since no data were available on the concentrations of tetracaine and its metabolite in human samples following spinal anesthesia, we devised a sensitive method to analyze tetracaine and its metabolite, p-butylaminobenzoic acid in human tissues, and successfully measured the blood and tissue concentrations of the drugs in the patient [3]. To estimate the concentrations of the drugs in our case, blood levels of tetracaine and its metabolite following normal spinal anesthesia were performed for comparison. We therefore measured the blood concentrations of tetracaine and its metabolite in 10 patients who were given intraspinal tetracaine.
0379-0738/01/$ ± see front matter # 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 9 - 0 7 3 8 ( 0 0 ) 0 0 3 4 2 - X
10
K. Kudo et al. / Forensic Science International 116 (2001) 9±14
analysis. Respiratory, cardiovascular or neurological side effects were nil in all patients during and after the surgery.
2. Experimental 2.1. Subjects Candidates for the study were 10 Japanese patients 24±71 years of age who were admitted to Kyushu University Hospital for orthopedic surgery. Every patient signed informed consent on blood sampling. Demographic data and the doses of tetracaine and mepivacaine for the 10 patients are summarized in Table 1. 2.2. Medication Twenty milligram of tetracaine hydrochloride was dissolved in 5 ml normal saline solution for injection to make up a 0.4% solution, and 2±3.5 ml of this solution was injected into each patient once at lumber 3/4 for spinal anesthesia. Surgery was begun 42± 64 min after the injection of tetracaine, and continued for 30±230 min. To maintain the state of anesthesia, 1±2% mepivacaine was given as epidural anesthesia 9±94 min after the initial anesthesia by tetracaine, and added as needed. The total amount of mepivacaine injected into each patient is shown in Table 1. Blood was obtained from a dorsalis pedes artery at 1, 2 and 6 h after the injection of tetracaine. Neostigmine solution, an esterase inhibitor, was added to the blood sample (0.15 mmol in 100 ml distilled water/0.5 ml blood) immediately after collection of blood samples. The samples were stored in a freezer at ÿ208C until
2.3. Reagents Mepivacaine hydrochloride was provided by Fujisawa Pharmaceutical Company (Osaka, Japan). Tetracaine hydrochloride, dibucaine hydrochloride, pbutylaminobenzoic acid and p-dimethylaminobenzoic acid were purchased from Wako Pure Chemical Industries (Osaka, Japan), Teikoku Chemical Industries (Osaka, Japan), Aldrich Chemical (Milwaukee, WI) and Ishizu Seiyaku (Osaka, Japan), respectively. N(tert-butyldimethylsilyl)-N-methyltri¯uoroacetamide (MTBSTFA) used for tert-butyldimethylsilyl (tBDS) derivatization was purchased from GL Sciences (Tokyo, Japan). Acetonitrile and tert-butyl methyl ether were of analytical grade and were puri®ed by distillation. Other chemicals used were of analytical grade. 2.4. Analysis of blood samples The concentrations of tetracaine, mepivacaine and p-butylaminobenzoic acid in whole blood samples were determined, using our method [3] but with some modi®cations. Tetracaine and mepivacaine determination: 0.5 ml of whole blood sample was mixed with 3.0 ml distilled water; 1.0 ml, 0.2 M sodium acetate±acetic acid buffer
Table 1 Demographic data and the dose of local anesthetics Patients
Diagnosis
Type of operation
Age
Sexb
Weight (kg)
Tetracaine Mepivacaine (mg) (mg)
1 2 3 4 5 6 7 8 9 10
Osteonecrosis of the knee joint Coxarthrosis of the hip joint (AOa) Coxarthrosis of the hip joint Coxarthrosis of the hip joint Coxarthrosis of the hip joint Coxarthrosis of the hip joint (AOa) Meniscus injury Coxarthrosis of the hip joint Necrosis of the femoral head Necrosis of the femoral head
Arthroscope, tibial osteotomy Nail extraction Arthroscope, valgus osteotomy (hip) Arthroscope Varus osteotomy (hip) Nail extraction Arthroscopic repair of the meniscus Total hip replacement Femoral neck prosthetic replacement Arthroscope, varus osteotomy (hip)
69 36 48 25 24 51 60 63 71 27
F F M F M F F F F F
46 62 65 43 63 45 53 60 49 54
8 12 8 12 14 12 10 12 10 12
a b
AO: after operation. F: female; M: male.
100 320 640 573 380 197 150 237 337 590
K. Kudo et al. / Forensic Science International 116 (2001) 9±14
(pH 3.8) and 2 ml of internal standard solution (IS1: 1000 ng dibucaine) in a 30 ml centrifuge tube. To the mixture, 10 ml of tert-butyl methyl ether was added and the preparation was shaken for 10 min and centrifuged at 850 g for 15 min. The aqueous layer was transferred to a 10 ml centrifuge tube, adjusted to ca. pH 9.5 with 100 ml of 20% sodium carbonate. To the mixture, 2 ml of tert-butyl methyl ether was added, the preparation was shaken for 10 min then centrifuged at 850 g for 15 min. The organic layer was evaporated to dryness under a stream of nitrogen. The residue was dissolved in 50 ml of tert-butyl methyl ether, and a 2 ml aliquot of the solution was injected into a GC/MS. p-Butylaminobenzoic acid determination: 0.5 ml of each sample was mixed with 3.0 ml distilled water; 1.0 ml, 0.2 M sodium acetate±acetic acid buffer (pH 3.8) and 2 ml of internal standard solution (IS2: 200 ng p-dimethylaminobenzoic acid) in a 30 ml centrifuge tube. To the mixture, 10 ml of methylene chloride was added and the preparation was shaken for 10 min and centrifuged at 850 g for 15 min. The organic layer was evaporated to dryness under a stream of nitrogen at 708C. The residue was dissolved in 50 ml of acetonitrile, and 10 ml of MTBSTFA was added to the solution for derivatization. The mixture was kept at 608C for 1 h in a heating block and a 2 ml aliquot of the solution was injected into the GC/MS. Two extracts each containing tetracaine and mepivacaine, or the metabolite were separately introduced to GC/MS, and same conditions of GC/MS were used for each extract. The apparatus used was a HewlettPackard 5989A GC/MS system. A fused-silica capillary column, HP-1 (12 m 0:2 mm i.d., 0.33 mm ®lm thickness), coated with 100% dimethylpolysiloxane stationary phase was used. Splitless injection mode was selected with a valve off time of 2 min. The GC/ MS conditions were as follows: the initial temperature 1008C was held for 2 min, then the temperature was programmed to 3008C at a rate of 208C/min and maintained for 1 min. Temperatures of the injection port and transfer line were each maintained at 2808C. Helium was used as the carrier gas with a ¯ow rate of 1 ml/min. Selected ion monitoring (SIM) mode was used. For determination of each compound, ions of m/z 176, 98, 116, 250 and 178 were selected for tetracaine, mepivacaine, dibucaine (IS1), p-butylaminobenzoic acid and p-dimethylaminobenzoic acid (IS2), respectively.
11
3. Results and discussion 3.1. Analysis of tetracaine, mepivacaine and pbutylaminobenzoic acid Our method developed for the analysis of tetracaine [3] was examined to determine tetracaine and mepivacaine in whole blood, simultaneously. These two drugs were well separated on the chromatogram, and could be determined without any interfering peaks. On the other hand, the extraction ef®ciency of p-butylaminobenzoic acid sometimes varied by a three-step solvent extraction procedure, probably due to protein in the whole blood. This problem was overcome by single extraction of the metabolite with methylene chloride. Protein in the whole blood denatured well with methylene chloride and clean extracts gave constant results in GC/MS, without further puri®cation. Typical chromatograms obtained from patient's whole blood are shown in Figs. 1 and 2. 3.2. Concentrations of tetracaine and its metabolite in blood Blood concentrations of p-butylaminobenzoic acid and mepivacaine in the patients are shown in Tables 2 and 3. Tetracaine was not detected in any sample, even when the samples were analyzed immediately after drawing the blood. Concentrations of tetracaine in plasma or serum have been measured by other investigators. Tetracaine was not detected in the sera from 10 children after application of 4% tetracaine cream (40 mg) to each hand [4] and in the plasma of children after application of a solution (3 ml) containing tetracaine (0.5%, 15 mg), epinephrine (0.05%) and cocaine (11.8%) for lacerations requiring suture repair [5]. To our knowledge, there is only one report of detection of tetracaine in plasma from 3 of the 10 patients after application of 5% tetracaine cream (100 mg) [6]. The levels of tetracaine in their paper were stated to be 20± 200 ng/ml. Since tetracaine is rapidly hydrolyzed to metabolites by pseudocholine esterase in blood [7], detection of tetracaine in blood was considered to be dif®cult in practical cases of spinal anesthesia. Therefore, we considered that tetracaine was not a proper compound to be analyzed for diagnosing the cause of death after spinal anesthesia.
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K. Kudo et al. / Forensic Science International 116 (2001) 9±14
Fig. 1. SIM chromatograms of the extract from whole blood of patient 1, collected 6 h after injection of tetracaine.
p-Butylaminobenzoic acid, was detected in every sample with the concentrations of 126:5 49:7, 97:9 33:7 and 43:3 16:6 ng/ml at 1, 2 and 6 h, respectively. These are the ®rst blood levels after injecting tetracaine to induce spinal anesthesia. When the blood levels were corrected by weight corresponding to 0.2 mg/kg injection of tetracaine, the coef®cient of variation was signi®cantly reduced from 39, 34, and 38% to 24, 18 and 21% at 1, 2 and 6 h after the tetracaine injection, respectively. Therefore, a rough
estimation of blood levels is feasible when the dose of tetracaine given and weight of patient are known. Concentrations of p-butylaminobenzoic acid were also determined by two groups of investigators after application of tetracaine cream [4,6]. They detected the metabolite in plasma from 2 of the 10 patients [6], and 7 of the 10 patients [4] with levels of 0.20±0.82 and 0.06±1.8 mg/ml, respectively. The reason why the metabolite was only detected in part of the samples in their study probably relates to the low sensitivity of the
Fig. 2. SIM chromatograms of the derivatized extract from whole blood of patient 1, collected 1 h after injection of tetracaine.
K. Kudo et al. / Forensic Science International 116 (2001) 9±14
13
Table 2 Concentrations of p-butylaminobenzoic acid in whole blood of patients (ng/ml)a Patients
p-Butylaminobenzoic acid 1h
2h
6h
1 2 3 4 5 6 7 8 9 10
93.8 (110.4) 93.8 (98.7) 46.5 (77.5) 157.4 (112.4) 135.0 (122.7) 241.0 (178.5) 95.5 (100.5) 156.8 (156.8) 131.2 (131.2) 113.5 (103.2)
73.4 (86.4) 81.5 (85.8) 40.5 (67.5) 133.5 (95.4) 82.8 (75.3) 169.3 (125.4) 78.8 (82.9) 111.8 (111.8) 102.2 (102.2) 105.4 (95.8)
39.4 29.3 24.1 56.1 41.4 87.0 38.5 34.8 40.6 41.6
Mean S:D: CV (%)
126:5 49:7 119:2 28:2 39 (24)
97:9 33:7 92:9 16:3 34 (18)
43:3 16:6 41:5 8:6 38 (21)
a
Values in parenthesis are values corrected by weight corresponding to the 0.2 mg/kg injection of tetracaine.
analytical method they used. p-Butylaminobenzoic acid was stable for several months in whole blood in storage at ÿ208C. Therefore, this metabolite was considered to be a proper target compound to determine when a forensic diagnosis of tetracaine poisoning is required. The obtained blood levels can serve as basic data when estimating blood levels in practical cases. 3.3. Concentration of mepivacaine in human blood Mepivacaine concentrations in blood varied depends on the dosage and the time of injection as shown in Table 3. The highest concentration was 4.6 mg/ml at 2 h in patient 10. Takasaki et al. reported data on blood levels of mepivacaine during continuous Table 3 Concentrations of mepivacaine in whole blood of patients (ng/ml) Patients
1 2 3 4 5 6 7 8 9 10
(46.4) (30.8) (40.2) (40.1) (37.6) (64.4) (42.7) (34.8) (40.6) (37.8)
Mepivacaine lh
2h
6h
91.2 943.7 953.2 741.0 496.8 125.4 418.8 142.7 2272.3 3465.6
677.5 2712.5 1022.5 1527.8 1627.0 896.5 858.9 899.7 2307.6 4609.4
427.6 1084.5 1164.4 996.9 1525.8 903.8 302.6 2123.2 2547.8 3626.9
epidural anesthesia [8]. In their study with doses of 200±300 mg given initially and 200 mg every hour thereafter, blood mepivacaine concentrations exceeded 5 mg/ml at about 2.5 h and approached 7.0 mg/ml at 4.5 h. The mean maximum concentration was reported to be 7:67 0:85 mg/ml at 5.5 h. The levels where mepivacaine was used as supplemental anesthesia in our study were much less than those in their study [8]. 4. Conclusion Blood concentrations of tetracaine and its metabolite, p-butylaminobenzoic acid, following spinal anesthesia induced by tetracaine were measured. Our ®ndings will be useful for diagnosing the cause of death when tetracaine is given to induce spinal anesthesia. Acknowledgements We thank M. Ohara for language assistance.
References [1] R.S. Altman, R. Smith-Coggins, L.L. Ampel, Local anesthetics, Ann. Emerg. Med. 14 (1985) 1209±1217.
14
K. Kudo et al. / Forensic Science International 116 (2001) 9±14
[2] M. Fukui, Medicolegal study of spinal anesthesia, Jpn. J. Legal Med. 24 (1970) 136±158. [3] Y. Hino, N. Ikeda, K. Kudo, A. Tsuji, Sensitive and selective determination of tetracaine and its metabolite in human samples by gas chromatography±mass spectrometry, J. Anal. Toxicol. 24 (2000) 165±169. [4] H.J.M. Van Kan, A.C.G. Egberts, W.P.M. Rijnvos, N.J. Ter Pelkwijk, A.W. Lenderink, Tetracaine versus lidocaineprilocaine for preventing venipuncture-induced pain in children, Am. J. Health-Syst. Pharm. 54 (1997) 388±392. [5] T.E. Terndrup, H.C. Walls, P.J. Mariani, D.P. Gavula, C.M. Madden, R.M. Cantor, Plasma cocaine and tetracaine levels
following application of topical anesthesia in children, Ann. Emerg. Med. 21 (1992) 162±166. [6] B. Mazumdar, A.A. Tomlinson, G.C. Faulder, Preliminary study to assay plasma amethocaine concentrations after topical application of a new local anaesthetic cream containing amethocaine, Br. J. Anaesth. 67 (1991) 432±436. [7] J.R. Kambam, B. Horton, W.C.V. Parris, S.A. Hyman, M.L. Berman, B.V.R. Sastry, Pseudocholinesterase activity in human cerebrospinal ¯uid, Anesth. Analg. 68 (1989) 486±488. [8] M. Takasaki, T. Oh-oka, K. Doi, Y. Kosaka, Blood levels of mepivacaine during continuous epidural anesthesia, Anesth. Analg. 66 (1987) 337±340.
Blood concentrations of tetracaine and its metabolite following spinal anesthesia Keiko Kudoa, Yukiko Hinoa, Noriaki Ikedaa,*, Hidefumi Inoueb, Shosuke Takahashib a
Department of Forensic Pathology and Sciences, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan b Department of Anesthesiology & Critical Care Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan Received 19 May 2000; received in revised form 22 June 2000; accepted 27 June 2000
Abstract Blood concentrations of tetracaine and its metabolite, p-butylaminobenzoic acid, were measured after spinal anesthesia with tetracaine which had been administered to patients under going orthopedic surgery. Tetracaine, an ester anesthetic, was given to 10 patients, the dose was 8±14 mg, and blood samples were collected 1, 2 and 6 h after the injection of tetracaine. We used gas chromatography/mass spectrometry for purposes of analysis. Tetracaine was not detected in any blood sample, but the metabolite was detected in each sample with the mean concentrations of 126.5, 97.9 and 43.3 ng/ml at 1, 2 and 6 h, respectively. This data will be useful in determination of the cause of death after spinal anesthesia with tetracaine. # 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Forensic toxicology; Spinal anesthesia; Tetracaine; p-Butylaminobenzoic acid; Mepivacaine
1. Introduction Tetracaine, also known as amethocaine, is an estertype local anesthetic. This drug is a very potent, longacting agent with a low therapeutic dose and is commonly used to induce spinal anesthesia [1]. It is known that the use of local anesthetic in spinal anesthesia occasionally results in sudden death of the patient [2], and thus it is important to clarify the cause of death. We have experienced a case where a patient died after being given tetracaine to induce spinal anesthe-
*
Corresponding author. Tel.: 81-92-642-6124; fax: 81-92-642-6126. E-mail address: [email protected] (N. Ikeda).
sia. Based on the autopsy ®ndings and microscopic analysis, high spinal anesthesia by tetracaine was suspected and further toxicological examinations were carried out. Since no data were available on the concentrations of tetracaine and its metabolite in human samples following spinal anesthesia, we devised a sensitive method to analyze tetracaine and its metabolite, p-butylaminobenzoic acid in human tissues, and successfully measured the blood and tissue concentrations of the drugs in the patient [3]. To estimate the concentrations of the drugs in our case, blood levels of tetracaine and its metabolite following normal spinal anesthesia were performed for comparison. We therefore measured the blood concentrations of tetracaine and its metabolite in 10 patients who were given intraspinal tetracaine.
0379-0738/01/$ ± see front matter # 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 9 - 0 7 3 8 ( 0 0 ) 0 0 3 4 2 - X
10
K. Kudo et al. / Forensic Science International 116 (2001) 9±14
analysis. Respiratory, cardiovascular or neurological side effects were nil in all patients during and after the surgery.
2. Experimental 2.1. Subjects Candidates for the study were 10 Japanese patients 24±71 years of age who were admitted to Kyushu University Hospital for orthopedic surgery. Every patient signed informed consent on blood sampling. Demographic data and the doses of tetracaine and mepivacaine for the 10 patients are summarized in Table 1. 2.2. Medication Twenty milligram of tetracaine hydrochloride was dissolved in 5 ml normal saline solution for injection to make up a 0.4% solution, and 2±3.5 ml of this solution was injected into each patient once at lumber 3/4 for spinal anesthesia. Surgery was begun 42± 64 min after the injection of tetracaine, and continued for 30±230 min. To maintain the state of anesthesia, 1±2% mepivacaine was given as epidural anesthesia 9±94 min after the initial anesthesia by tetracaine, and added as needed. The total amount of mepivacaine injected into each patient is shown in Table 1. Blood was obtained from a dorsalis pedes artery at 1, 2 and 6 h after the injection of tetracaine. Neostigmine solution, an esterase inhibitor, was added to the blood sample (0.15 mmol in 100 ml distilled water/0.5 ml blood) immediately after collection of blood samples. The samples were stored in a freezer at ÿ208C until
2.3. Reagents Mepivacaine hydrochloride was provided by Fujisawa Pharmaceutical Company (Osaka, Japan). Tetracaine hydrochloride, dibucaine hydrochloride, pbutylaminobenzoic acid and p-dimethylaminobenzoic acid were purchased from Wako Pure Chemical Industries (Osaka, Japan), Teikoku Chemical Industries (Osaka, Japan), Aldrich Chemical (Milwaukee, WI) and Ishizu Seiyaku (Osaka, Japan), respectively. N(tert-butyldimethylsilyl)-N-methyltri¯uoroacetamide (MTBSTFA) used for tert-butyldimethylsilyl (tBDS) derivatization was purchased from GL Sciences (Tokyo, Japan). Acetonitrile and tert-butyl methyl ether were of analytical grade and were puri®ed by distillation. Other chemicals used were of analytical grade. 2.4. Analysis of blood samples The concentrations of tetracaine, mepivacaine and p-butylaminobenzoic acid in whole blood samples were determined, using our method [3] but with some modi®cations. Tetracaine and mepivacaine determination: 0.5 ml of whole blood sample was mixed with 3.0 ml distilled water; 1.0 ml, 0.2 M sodium acetate±acetic acid buffer
Table 1 Demographic data and the dose of local anesthetics Patients
Diagnosis
Type of operation
Age
Sexb
Weight (kg)
Tetracaine Mepivacaine (mg) (mg)
1 2 3 4 5 6 7 8 9 10
Osteonecrosis of the knee joint Coxarthrosis of the hip joint (AOa) Coxarthrosis of the hip joint Coxarthrosis of the hip joint Coxarthrosis of the hip joint Coxarthrosis of the hip joint (AOa) Meniscus injury Coxarthrosis of the hip joint Necrosis of the femoral head Necrosis of the femoral head
Arthroscope, tibial osteotomy Nail extraction Arthroscope, valgus osteotomy (hip) Arthroscope Varus osteotomy (hip) Nail extraction Arthroscopic repair of the meniscus Total hip replacement Femoral neck prosthetic replacement Arthroscope, varus osteotomy (hip)
69 36 48 25 24 51 60 63 71 27
F F M F M F F F F F
46 62 65 43 63 45 53 60 49 54
8 12 8 12 14 12 10 12 10 12
a b
AO: after operation. F: female; M: male.
100 320 640 573 380 197 150 237 337 590
K. Kudo et al. / Forensic Science International 116 (2001) 9±14
(pH 3.8) and 2 ml of internal standard solution (IS1: 1000 ng dibucaine) in a 30 ml centrifuge tube. To the mixture, 10 ml of tert-butyl methyl ether was added and the preparation was shaken for 10 min and centrifuged at 850 g for 15 min. The aqueous layer was transferred to a 10 ml centrifuge tube, adjusted to ca. pH 9.5 with 100 ml of 20% sodium carbonate. To the mixture, 2 ml of tert-butyl methyl ether was added, the preparation was shaken for 10 min then centrifuged at 850 g for 15 min. The organic layer was evaporated to dryness under a stream of nitrogen. The residue was dissolved in 50 ml of tert-butyl methyl ether, and a 2 ml aliquot of the solution was injected into a GC/MS. p-Butylaminobenzoic acid determination: 0.5 ml of each sample was mixed with 3.0 ml distilled water; 1.0 ml, 0.2 M sodium acetate±acetic acid buffer (pH 3.8) and 2 ml of internal standard solution (IS2: 200 ng p-dimethylaminobenzoic acid) in a 30 ml centrifuge tube. To the mixture, 10 ml of methylene chloride was added and the preparation was shaken for 10 min and centrifuged at 850 g for 15 min. The organic layer was evaporated to dryness under a stream of nitrogen at 708C. The residue was dissolved in 50 ml of acetonitrile, and 10 ml of MTBSTFA was added to the solution for derivatization. The mixture was kept at 608C for 1 h in a heating block and a 2 ml aliquot of the solution was injected into the GC/MS. Two extracts each containing tetracaine and mepivacaine, or the metabolite were separately introduced to GC/MS, and same conditions of GC/MS were used for each extract. The apparatus used was a HewlettPackard 5989A GC/MS system. A fused-silica capillary column, HP-1 (12 m 0:2 mm i.d., 0.33 mm ®lm thickness), coated with 100% dimethylpolysiloxane stationary phase was used. Splitless injection mode was selected with a valve off time of 2 min. The GC/ MS conditions were as follows: the initial temperature 1008C was held for 2 min, then the temperature was programmed to 3008C at a rate of 208C/min and maintained for 1 min. Temperatures of the injection port and transfer line were each maintained at 2808C. Helium was used as the carrier gas with a ¯ow rate of 1 ml/min. Selected ion monitoring (SIM) mode was used. For determination of each compound, ions of m/z 176, 98, 116, 250 and 178 were selected for tetracaine, mepivacaine, dibucaine (IS1), p-butylaminobenzoic acid and p-dimethylaminobenzoic acid (IS2), respectively.
11
3. Results and discussion 3.1. Analysis of tetracaine, mepivacaine and pbutylaminobenzoic acid Our method developed for the analysis of tetracaine [3] was examined to determine tetracaine and mepivacaine in whole blood, simultaneously. These two drugs were well separated on the chromatogram, and could be determined without any interfering peaks. On the other hand, the extraction ef®ciency of p-butylaminobenzoic acid sometimes varied by a three-step solvent extraction procedure, probably due to protein in the whole blood. This problem was overcome by single extraction of the metabolite with methylene chloride. Protein in the whole blood denatured well with methylene chloride and clean extracts gave constant results in GC/MS, without further puri®cation. Typical chromatograms obtained from patient's whole blood are shown in Figs. 1 and 2. 3.2. Concentrations of tetracaine and its metabolite in blood Blood concentrations of p-butylaminobenzoic acid and mepivacaine in the patients are shown in Tables 2 and 3. Tetracaine was not detected in any sample, even when the samples were analyzed immediately after drawing the blood. Concentrations of tetracaine in plasma or serum have been measured by other investigators. Tetracaine was not detected in the sera from 10 children after application of 4% tetracaine cream (40 mg) to each hand [4] and in the plasma of children after application of a solution (3 ml) containing tetracaine (0.5%, 15 mg), epinephrine (0.05%) and cocaine (11.8%) for lacerations requiring suture repair [5]. To our knowledge, there is only one report of detection of tetracaine in plasma from 3 of the 10 patients after application of 5% tetracaine cream (100 mg) [6]. The levels of tetracaine in their paper were stated to be 20± 200 ng/ml. Since tetracaine is rapidly hydrolyzed to metabolites by pseudocholine esterase in blood [7], detection of tetracaine in blood was considered to be dif®cult in practical cases of spinal anesthesia. Therefore, we considered that tetracaine was not a proper compound to be analyzed for diagnosing the cause of death after spinal anesthesia.
12
K. Kudo et al. / Forensic Science International 116 (2001) 9±14
Fig. 1. SIM chromatograms of the extract from whole blood of patient 1, collected 6 h after injection of tetracaine.
p-Butylaminobenzoic acid, was detected in every sample with the concentrations of 126:5 49:7, 97:9 33:7 and 43:3 16:6 ng/ml at 1, 2 and 6 h, respectively. These are the ®rst blood levels after injecting tetracaine to induce spinal anesthesia. When the blood levels were corrected by weight corresponding to 0.2 mg/kg injection of tetracaine, the coef®cient of variation was signi®cantly reduced from 39, 34, and 38% to 24, 18 and 21% at 1, 2 and 6 h after the tetracaine injection, respectively. Therefore, a rough
estimation of blood levels is feasible when the dose of tetracaine given and weight of patient are known. Concentrations of p-butylaminobenzoic acid were also determined by two groups of investigators after application of tetracaine cream [4,6]. They detected the metabolite in plasma from 2 of the 10 patients [6], and 7 of the 10 patients [4] with levels of 0.20±0.82 and 0.06±1.8 mg/ml, respectively. The reason why the metabolite was only detected in part of the samples in their study probably relates to the low sensitivity of the
Fig. 2. SIM chromatograms of the derivatized extract from whole blood of patient 1, collected 1 h after injection of tetracaine.
K. Kudo et al. / Forensic Science International 116 (2001) 9±14
13
Table 2 Concentrations of p-butylaminobenzoic acid in whole blood of patients (ng/ml)a Patients
p-Butylaminobenzoic acid 1h
2h
6h
1 2 3 4 5 6 7 8 9 10
93.8 (110.4) 93.8 (98.7) 46.5 (77.5) 157.4 (112.4) 135.0 (122.7) 241.0 (178.5) 95.5 (100.5) 156.8 (156.8) 131.2 (131.2) 113.5 (103.2)
73.4 (86.4) 81.5 (85.8) 40.5 (67.5) 133.5 (95.4) 82.8 (75.3) 169.3 (125.4) 78.8 (82.9) 111.8 (111.8) 102.2 (102.2) 105.4 (95.8)
39.4 29.3 24.1 56.1 41.4 87.0 38.5 34.8 40.6 41.6
Mean S:D: CV (%)
126:5 49:7 119:2 28:2 39 (24)
97:9 33:7 92:9 16:3 34 (18)
43:3 16:6 41:5 8:6 38 (21)
a
Values in parenthesis are values corrected by weight corresponding to the 0.2 mg/kg injection of tetracaine.
analytical method they used. p-Butylaminobenzoic acid was stable for several months in whole blood in storage at ÿ208C. Therefore, this metabolite was considered to be a proper target compound to determine when a forensic diagnosis of tetracaine poisoning is required. The obtained blood levels can serve as basic data when estimating blood levels in practical cases. 3.3. Concentration of mepivacaine in human blood Mepivacaine concentrations in blood varied depends on the dosage and the time of injection as shown in Table 3. The highest concentration was 4.6 mg/ml at 2 h in patient 10. Takasaki et al. reported data on blood levels of mepivacaine during continuous Table 3 Concentrations of mepivacaine in whole blood of patients (ng/ml) Patients
1 2 3 4 5 6 7 8 9 10
(46.4) (30.8) (40.2) (40.1) (37.6) (64.4) (42.7) (34.8) (40.6) (37.8)
Mepivacaine lh
2h
6h
91.2 943.7 953.2 741.0 496.8 125.4 418.8 142.7 2272.3 3465.6
677.5 2712.5 1022.5 1527.8 1627.0 896.5 858.9 899.7 2307.6 4609.4
427.6 1084.5 1164.4 996.9 1525.8 903.8 302.6 2123.2 2547.8 3626.9
epidural anesthesia [8]. In their study with doses of 200±300 mg given initially and 200 mg every hour thereafter, blood mepivacaine concentrations exceeded 5 mg/ml at about 2.5 h and approached 7.0 mg/ml at 4.5 h. The mean maximum concentration was reported to be 7:67 0:85 mg/ml at 5.5 h. The levels where mepivacaine was used as supplemental anesthesia in our study were much less than those in their study [8]. 4. Conclusion Blood concentrations of tetracaine and its metabolite, p-butylaminobenzoic acid, following spinal anesthesia induced by tetracaine were measured. Our ®ndings will be useful for diagnosing the cause of death when tetracaine is given to induce spinal anesthesia. Acknowledgements We thank M. Ohara for language assistance.
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