Drugs and Poisons in Humans - A Handbook of Practical Analysis (Part 28)

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Drugs and Poisons in Humans - A Handbook of Practical Analysis (Part 28)

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Introduction: Butyrophenone drugs including haloperidol are being widely used in the field of psychiatry. The acute butyrophenone poisoning incidents sometimes take place; in such cases, the analysis of a butyrophenone becomes necessary in forensic toxicology or clinical toxicology. Their analysis is being made by GC [1–4], GC/MS [5–6], HPLC [7–15] and LC/MS [16,17]. Six butyrophenones are now available as ethical drugs in Japan ( Fig. 2.1); the most typical ones are haloperidol and bromperidol, which most frequently cause poisoning incidents among butyrophenones. These drugs are rapidly metabolized in human bodies into reduced haloperidol and reduced bromperidol, respectively. In this...

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  1. 3.2 II.3.2 Butyrophenones by Kazuo Igarashi Introduction Butyrophenone drugs including haloperidol are being widely used in the field of psychiatry. The acute butyrophenone poisoning incidents sometimes take place; in such cases, the analysis of a butyrophenone becomes necessary in forensic toxicology or clinical toxicology. Their anal- ysis is being made by GC [1–4], GC/MS [5–6], HPLC [7–15] and LC/MS [16,17]. Six butyro- phenones are now available as ethical drugs in Japan ( > Fig. 2.1); the most typical ones are haloperidol and bromperidol, which most frequently cause poisoning incidents among butyro- phenones. These drugs are rapidly metabolized in human bodies into reduced haloperidol and reduced bromperidol, respectively. In this chapter, the methods of GC/MS, HPLC and LC/MSa are presented for analysis of haloperidol, bromperidol and their reduced forms. ⊡ Figure 2.1 Structures of butyrophenones. © Springer-Verlag Berlin Heidelberg 2005
  2. 264 Butyrophenones GC/MS analysis Reagents and their preparation • Haloperidol can be purchased from Sigma (St. Louis, MO, USA) and other manufacturers; bromperidol, reduced haloperidol and reduced bromperidol from Research Biochemical International (Natick, MA, USA). • A 4-g aliquot of NaOH and 6 g NaCl are dissolved in distilled water to prepare 100 mL solution (1 M NaOH solution)b. • n-Hexane/isopropanol (95:5, v/v) mixture solution • 0.1 M Hydrochloric acid solution • As internal standard (IS)c, bromperidol (500 ng/mL in 0.1 M hydrochloric acid solution) is used for analysis of haloperidol, and vise versa. • Preparation of standard solutions: haloperidol or bromperidol solutions at 2–50 ng/mL in 0.01 M hydrochloric acid are prepared, and each 2-mL aliquot is placed in a 15-mL volume glass centrifuge tube with a ground-in stopper. GC/MS conditions Instrument: an Agilent 5890 GC instrument (Agilent Technologies, Palo Alto, CA, USA) con- nected with a JEOL Automass quadrupole mass spectrometer (JEOL, Tokyo, Japan). GC column: an HP-5 fused silica capillary column (30 m × 0.32 mm i. d., film thickness 0.25 µm, Agilent Technologies); column (oven) temperature: 100 °C (1 min) → 30 °C/min → 270 °C (30 s) → 5 °C/min → 290 °C (5 min); injection temperature: 260 °C; separator tempera- ture: 280 °C; carrier gas: He; its flow rate: 1.5 mL/min; MS ionization mode: EI; electron energy: 70 eV; detector voltage: 750 V; ion source temperature: 280 °C. Procedured i. A 2-mL volume of urine or blood, 0.05 mL IS and 0.5 mL of 1 M NaOH are placed in a 15-mL volume glass centrifuge tube with a ground-in stopper and mixed well, followed by addi- tion of 6 mL of the mixture of n-hexane/isopropanol and its shaking for 20 min. ii. After centrifugation at 600 g for 5 min, 5.5 mL of the upper organic layer is transferred to another 15-mL volume glass centrifuge tube, followed by the addition of 1.5 mL of 0.1 M hydrochloric acid solution and vigorous shaking for 20 min. iii. After centrifugation at 600 g for 5 min, the upper organic layer is discarded; the aqueous phase is again washed with 1 mL of the mixture of n-hexane/isopropanol by shaking it for 30 s. iv. After centrifugation at 600 g for 5 min, 1.2 mL of the lower aqueous phase is transferred to a 10-mL volume glass centrifuge tube with a ground-in stopper, followed by addition of 0.2 mL of 1 M NaOH and 1 mL of the n-hexane/isopropanol mixture, and vigorous shaking for 30 s. v. After centrifugation at 600 g for 5 min, the upper organic layer is transferred to a small glass test tube and evaporated to dryness. vi. The residue are dissolved in 20 µL ethanol.
  3. HPLC and LC/MS analysis 265 vii. For quantitation, the selected ion monitoring (SIM) mode of GC/MS is employed using ions at m/z 224 for haloperidol and m/z 268 for bromperidol; peak area ratios of haloperi- dol or bromperidol to IS are plotted against various concentrations of the test compound spiked to blank blood or urine to draw a calibration curve. A peak area ratio of a test specimen is applied to the calibration curve to calculate its concentration. Assessment of the method The butyrophenone drugs analyzable by GC or GC/MS in the underivatized forms are halo- peridol, bromperidol, moperone and floropipamide; but for timiperone and spiperone, satis- factory peaks cannot be obtained. TICs and SIM chromatograms of haloperidol, bromperidol and their reduced forms are shown in > Fig. 2.2. The detection limit was about 10 pg in an injected volume for both halo- peridol and bromperidol; the recoveries were also excellent. The separation ability of GC or GC/MS is much superior to that of HPLC or LC/MS. HPLC and LC/MS analysis Reagents and their preparation The sources for acquisition of haloperidol, bromperidol and their reduced forms is the same as described in the GC/MS section. ⊡ Figure 2.2 TICs and SIM chromatograms by GC/MS for the authentic standards of butyrophenone drugs (100 ng/mL each) (A) and for a serum extract from a poisoned patient (B). m/z 224: haloperidol (HP) and reduced haloperidol (RHP); m/z 268: bromperidol (BP, IS) and reduced bromperidol (RBP).
  4. 266 Butyrophenones HPLC analysis Instrument: an SPD-M10A photodiode array detector (DAD), a CTO-10A column oven, an SIL-10A autosampler and an LC-10AD pump system (all from Shimadzu Corp., Kyoto, Japan). HPLC conditions; column: Cosmosil 5CN-MS (150 × 4.6 mm i. d., particle diameter 5 µm, Nacalai Tesque, Kyoto, Japan; mobile phase: acetonitrile/methanol/20 mM ammonium acetate aqueous solution/triethylamine (20:25:55:0.1, v/v, to be adjusted to pH 4.7 with phosphoric acid); flow rate: 1.0 mL/min; column (oven) temperature: 40 °C; detection wavelength: 220 nm. LC/MS analysis Instrument: a 2690 Alliance HPLC pump system (Waters, Milford, MA, USA) connected with a Micromass Quattro Ultima desktop quadrupole MS-MS instrument (Micromass, Manchester, UK) LC/MS conditions; column: Cosmosil 5CN-MS (150 × 4.6 mm i. d., particle diameter 5 µm, Nacalai Tesque); mobile phase: methanol/20 mM ammonium formate aqueous solution (60:40, v/v); flow rate: 0.6 mL/min; column (oven) temperature: 40 °C; interface: electrospray ionization (ESI); ion source temperature: 120 °C; temperature for removing solvent: 350 °C; gas for removing solvent: 600 L/h; spray (cone) voltage: 35 eV. Procedure i. The procedure i–v described in the GC/MS analysis section is followed for a urine or blood specimen to obtain a residue containing butyrophenones. The residue is dissolved in 0.1 mL of the mobile phase of HPLC or LC/MS. ii. For HPLC-DAD and LC/MS, 50 and 20 µL of the above solution are injected, respec- tively. iii. For the SIM of LC/MS, the ions at m/z 376, 378 and 422 are used for detection of halo- peridol, reduced haloperidol and bromperidol, respectively. iv. For both HPLC and LC/MS, bromperidol is used as IS for quantitation of haloperidol, and vice versa. v. For both HPLC and LC/MS, the peak area ratio of a test compound to IS obtained from a test specimen is applied to a calibration curve constructed in advance to calculate the con- centration of the test compound. Assessment of the methods > Figure 2.3 shows chromatograms for haloperidol, reduced haloperidol and bromperidol (IS) obtained by HPLC-DAD. The optimum detection wavelength was 220 nm; when meas- ured at 250 nm, reduced haloperidol could not be detected. The detection limit obtained by HPLC-DAD was about 5 ng in an injected volume for all compounds. HPLC-DAD is advantageous over HPLC-UV in that the sensitivity can be enhanced by measuring a target compound at the wavelength of its absorbance maximum, or by shifting the
  5. Poisoning cases, and toxic and fatal concentrations 267 ⊡ Figure 2.3 HPLC-DAD chromatograms for the authentic haloperidol (HP) and reduced haloperidol (RHP) (100 ng/mL each) (A) and for a serum extract from a poisoned patient (B). wavelength from its maximum to avoid impurity peaks. Also by measuring an absorbance spectrum of a compound, it is possible to make tentative identification. By LC/MS analysis, the sensitivity and specificity are much higher. Distinct peaks of all compounds appear ( > Fig. 2.4); the detection limit by LC/MS was about 2 pg in an injected volume. For sensitive analysis of timiperone and spiperone with relatively high molecular weights, LC/MS may be most suitable. Poisoning cases, and toxic and fatal concentrations A 2-year plus 5 month-old female and an 11-month-old male [18] had ingested 265 mg haloperidol in total (combined amount for both children); both were brought to a hospital in the comatose state and showed bradycardia, hypotension and sinus arrhythmia. Mannitol was injected into the female child intravenously; though the consciousness was gradually recovered 24 h after admission, neurological symptoms, such as tremor, muscle stiffness and dyskinesia of the face, appeared. Thus, diphenhydramine was injected into her intravenously; she recov- ered 4 days after admission. For the male baby, similar treatments, such as intravenous injec- tion of mannitol and diphenhydramine, were carried out, but the neurological symptoms were not improved easily; it took as long as 7 days for his recovery. Therapeutic and toxic blood levels of haloperidol were reported to be 5–40 and 50–100 ng/ mL, respectively; therapeutic blood levels of bromperidol 2–20 ng/mL [19]. Therapeutic and toxic blood levels of floropipamide were reported to be 0.1–0.4 and 0.5–0.6 ng/mL, respec- tively [20].
  6. 268 Butyrophenones ⊡ Figure 2.4 SIM chromatograms by LC/MS for the authentic haloperidol (HP) and bromperidol (BP) (100 ng/mL each) (A) and for a serum extract from a poisoned patient (B). m/z 376.5: HP; m/z 378.5: HP and RHP; m/z 422.4: BP=IS.
  7. Poisoning cases, and toxic and fatal concentrations 269 Notes a) When GC/MS, HPLC and LC/MS are compared for analysis of haloperidol and bromperi- dol, the LC/MS seems of the best choice for their trace analysis. The HPLC method enables highly sensitive detection (ng/mL) of the compounds with an electrochemical detector (ECD). Since, in this chapter, poisoning cases with ingestion of large amounts of drugs are assumed, HPLC analysis with a photodiode array detector can be realized for several ten ng/mL of the drugs. GC/MS is unexpectedly not so highly sensitive; it requires a condensa- tion step. b) NaCl was added to the 1 M NaOH solution, because it increases extraction efficiency due to its salting-out effect. c) There is a report using a haloperidol analog as IS, in which chlorine is substituted for the fluorine; but this compound is usually difficult to be obtained. Therefore, bromperidol was used as IS for analysis of haloperidol and vice versa. This is because both drugs are not simultaneously administered in most cases. d) All glasswares, including glass centrifuge tubes with ground-in stoppers, are preferably treated for inactivation with dimethylsilyl coating, because trace amounts of drugs are easily adsorbed to their surfaces, causing variation of results. References 1) Seno H, Suzuki O, Kumazawa T et al. (1989) Rapid isolation with Sep-Pak C18 cartridges and wide-bore capillary gas chromatography of some butyrophenones. Z Rechtsmed 102:127–132 2) Tyndale RF, Inaba T (1990) Simultaneous determination of haloperidol and reduced haloperidol by gas chroma- tography using a megabore column with electron capture detection: application to microsomal oxidation of reduced haloperidol. J Chromatogr 529:182–188 3) Ulrich S, Meyer FP, Neuhof S et al. (1995) Megabore capillary gas-liquid chromatographic method with nitro- gen-phosphorus selective detection for the assay of haloperidol and reduced haloperidol in serum: results of therapeutic drug-monitoring during acute therapy of eight schizophrenics. J Chromatogr B 663:289–296 4) Tokunaga H, Kudo K, Imamura T (1996) Screening of antipsychotic drugs by wide-bore capillary gas chromato- graphy with nitrogen phosphorus detection. Jpn J Legal Med 50:196–202 5) Hattori H, Suzuki O, Brandenberger H (1986) Positive- and negative-ion mass spectrometry of butyrophenones. J Chromatogr 382:135–145 6) Couper FJ, McIntyre IM, Drummer OH (1995) Detection of antidepressant and antipsychotic drugs in postmor- tem human scalp hair. J Forensic Sci 40:87–90 7) Nilsson LB (1988) Reversed-phase ion pair liquid chromatographic method for the determination of low con- centrations of haloperidol in plasma. J Chromatogr 431:113–122 8) Hariharan M, Kindt EK, Van Noord T et al. (1989) An improved sensitive assay for simultaneous determination of plasma haloperidol and reduced haloperidol levels by liquid chromatography using a coulometric detector. Ther Drug Monit 11:701–707 9) Park KH, Lee MH, Lee MG (1991) Simultaneous determination of haloperidol and its metabolite, reduced halo- peridol, in plasma, blood, urine and tissue homogenates by high-performance liquid chromatography. J Chro- matogr 572:259–267 10) Eyles DE, Whiteford HA, Stedman TJ et al. (1992) Determination of haloperidol and reduced haloperidol in the plasma and blood of patients on depot haloperidol. Psychopharmacology 106:268–274 11) Fang J, Gorrod JW (1993) High-performance liquid chromatographic method for the detection and quantitation of haloperidol and seven of its metabolites in microsomal preparations. J Chromatogr B 614:267–273 12) Aravagiri M, Marder SR, Van Putten T et al. (1994) Simultaneous determination of plasma haloperidol and its metabolite reduced haloperidol by liquid chromatography with electrochemical detection: plasma levels in schizophrenic patients treated with oral or intramuscular depot haloperidol. J Chromatogr B 656:373–381
  8. 270 Butyrophenones 13) Igarashi K, Kasuya F, Abe T et al. (1995) Simultaneous determination of haloperidol and its neurotoxic metabo- lite in plasma and brain tissue from schizophrenic patients treated with haloperidol using HPLC and solid- phase extraction. Jpn J Forensic Toxicol 13:31–38 14) Pan L, Rosseel MT, Belpaire FM (1998) Comparison of two high-performance liquid chromatographic methods for monitoring plasma concentrations of haloperidol and reduced haloperidol. Ther Drug Monit 20:224–230 15) Walter S, Bauer S , Roots I et al. (1998) Quantification of the antipsychotics flupentixol and haloperidol in human serum by high-performance liquid chromatography with ultraviolet detection. J Chromatogr B 720:231–237 16) Igarashi K, Shigee Y, Kasuya F et al. (1997) Analysis of haloperidol and its neurotoxic pyridinium metabolite in biological samples by liquid chromatography/mass spectrometry. Jpn J Forensic Toxicol 15:44–54 17) Hoja H, Marquet P, Verneuil B et al. (1997) Determination of haloperidol and its reduced metabolite in human plasma by liquid chromatography-mass spectrometry with electrospray ionization. J Chromatogr B 688:275– 280 18) Yamazaki F, Mori H (eds) (2000) Guide to Acute Poisonings by Medical Drugs. Van Medical, Tokyo, p 56 (in Japanese) 19) Uges DRA (1997) Blood level data. In: Brandenberger H, Maes RAA (eds) Analytical Toxicology for Clinical, Forensic and Pharmaceutical Chemists. Walter de Gruyter, Berlin, pp 707–718 20) Uges DRA, Conemans JMH (2000) Antidepressants and antiphychotics. In: Bogusz MJ (ed) Handbook of Analytical Separations Vol.2, Forensic Science. Elsevier, Amsterdam, pp 229–257

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