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

In this chapter, the methods of GC/MS, HPLC and LC/MSa are presented for analysis of haloperidol, bromperidol and their reduced forms.. HPLC and LC/MS analysis Reagents and their prepara

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Introduction

Butyrophenone drugs including haloperidol are being widely used in the fi eld of psychiatry

Th e acute butyrophenone poisoning incidents sometimes take place; in such cases, the analysis

of a butyrophenone becomes necessary in forensic toxicology or clinical toxicology Th 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 Th 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/MSa

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., fi 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 fl ow 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 Aft 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 Aft 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 Aft 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 Aft 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 Th e residue are dissolved in 20 µL ethanol

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 bromperihaloperi-dol 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

Th e butyrophenone drugs analyzable by GC or GC/MS in the underivatized forms are halo-peridol, bromhalo-peridol, moperone and fl 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 Th e detection limit was about 10 pg in an injected volume for both halo-peridol and bromhalo-peridol; the recoveries were also excellent Th 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

Th 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

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);

fl 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); fl 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 Th 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 Th 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 bromhalo-peridol, 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 Th e optimum detection wavelength was 220 nm; when meas-ured at 250 nm, reduced haloperidol could not be detected Th 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 shift ing the

wavelength from its maximum to avoid impurity peaks Also by measuring an absorbance spectrum of a compound, it is possible to make tentative identifi cation

By LC/MS analysis, the sensitivity and specifi 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 aft er admission, neurological symptoms, such as tremor, muscle stiff ness and dyskinesia

of the face, appeared Th us, diphenhydramine was injected into her intravenously; she recov-ered 4 days aft 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

Th 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] Th erapeutic and toxic blood levels of fl 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

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

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 Th 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 effi ciency due

to its salting-out eff ect

c) Th ere is a report using a haloperidol analog as IS, in which chlorine is substituted for the

fl uorine; but this compound is usually diffi cult to be obtained Th erefore, bromperidol was

used as IS for analysis of haloperidol and vice versa Th 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

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270 Butyrophenones

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