3.2 © Springer-Verlag Berlin Heidelberg 2005 II.3.2 Butyrophenones by Kazuo Igarashi Introduction Butyrophenone drugs including haloperidol are being widely used in the  eld of psychiatry.  e acute butyrophenone poisoning incidents sometimes take place; in such cases, the analysis of a butyrophenone becomes necessary in forensic toxicology or clinical toxicology.  eir 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.  ese 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/MS a are presented for analysis of haloperidol, bromperidol and their reduced forms. Structures of butyrophenones. ⊡ Figure 2.1 264 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.,  lm 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  ow rate: 1.5 mL/min; MS ionization mode: EI; electron energy: 70 eV; detector voltage: 750 V; ion source temperature: 280 °C. Procedure d 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. A er 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. A er 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. A er 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. A er centrifugation at 600 g for 5 min, the upper organic layer is transferred to a small glass test tube and evaporated to dryness. vi.  e residue are dissolved in 20 µL ethanol. 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  e butyrophenone drugs analyzable by GC or GC/MS in the underivatized forms are halo- peridol, bromperidol, moperone and  oropipamide; 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.  e detection limit was about 10 pg in an injected volume for both halo- peridol and bromperidol; the recoveries were also excellent.  e 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  e sources for acquisition of haloperidol, bromperidol and their reduced forms is the same as described in the GC/MS section. 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). ⊡ Figure 2.2 HPLC and LC/MS analysis 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);  ow 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);  ow 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.  e procedure i–v described in the GC/MS analysis section is followed for a urine or blood specimen to obtain a residue containing butyrophenones.  e 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.  e optimum detection wavelength was 220 nm; when meas- ured at 250 nm, reduced haloperidol could not be detected.  e 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 shi ing the 267 wavelength from its maximum to avoid impurity peaks. Also by measuring an absorbance spectrum of a compound, it is possible to make tentative identi cation. By LC/MS analysis, the sensitivity and speci city 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 a er admission, neurological symptoms, such as tremor, muscle sti ness and dyskinesia of the face, appeared.  us, diphenhydramine was injected into her intravenously; she recov- ered 4 days a er 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.  erapeutic 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].  erapeutic and toxic blood levels of  oropipamide were reported to be 0.1–0.4 and 0.5–0.6 ng/mL, respec- tively [20]. 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). ⊡ Figure 2.3 Poisoning cases, and toxic and fatal concentrations 268 Butyrophenones 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. ⊡ Figure 2.4 269 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.  e 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 e ciency due to its salting-out e ect. c)  ere is a report using a haloperidol analog as IS, in which chlorine is substituted for the  uorine; but this compound is usually di cult to be obtained.  erefore, bromperidol was used as IS for analysis of haloperidol and vice versa.  is 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 C 18 cartridges and wide-bore capillary gas chromatography of some butyrophenones. 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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 . brought to a hospital in the comatose state and showed bradycardia, hypotension and sinus arrhythmia. Mannitol was injected into the female child intravenously;. 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