Tài liệu Drugs and Poisons in Humans - A Handbook of Practical Analysis (Part 27) ppt

3.13.1
© Springer-Verlag Berlin Heidelberg 2005
II.3.1 Phenothiazines
by Akira Ishii and Yoshinao Katsumata
Introduction
Phenothiazine drugs, including chlorpromazine and levomepromazine, have been being
widely used as neuroleptics ( major tranquilizers), antiparkinsonian drugs and antihistaminics
for a long time [1].
> Table 1.1 shows chemical structures of representative phenothiazines.
 ese drugs show blocking action on D
2
receptors of dopaminergic neurons; there is close
relationship between the receptor blocking and tranquilizing actions.  e dopamine D
2
recep-
tor-blocking actions provoke extrapyramidal symptoms, such as muscular sti ness, tremor
and ptyalism. Orthostatic hypotension, arrythmia and icterus are occasionally found a er ad-
ministration of phenothiazines as side e ects.
 e number of phenothiazine poisoning cases is relatively small, as compared with the
extensive use of this group of drugs [2]. However, until now, many phenothiazine poisoning
cases, including fatal ones [3, 4], were reported.
Phenothiazines were analyzed by various methods, such as GC, GC/MS, HPLC and LC/
MS.  e methods by GC and GC/MS are relatively simple, but most of the methods reported
used packed columns or wide-bore capillary columns [5, 6], which did not give high sensi-
tivity.
Hattori et al. [7] reported a highly sensitive method for detecting phenothiazine in body
 uids using GC- surface ionization detection ( SID)
a
. Although the SID detector has an advan-
tage in that each compound having a tertiary amino group can be analyzed with high sensitiv-
ity and speci city, it is not a detector commonly available. In this chapter, the methods

of qualitative and quantitative analysis of phenothiazines in human body  uids using useful
GC/MS and LC/MS are presented.
GC/MS analysis
Reagents and their preparation
i. Reagents
Chlorpromazine, tri upromazine, promethazine and thioridazine can be purchased from
Sigma (St. Louis, MO, USA). Pure powder of levomepromazine was donated by Mitsubishi
Welpharma, Osaka, Japan.
Other common chemicals were of the highest purity commercially available.
256 Phenothiazines
⊡ Table 1.1
Structures of phenothiazine drugs
TIC and mass chromatograms (MCs) for 5 phenothiazines. 1: triflupromazine; 2: promethazine;
3: chlorpromazine; 4: levomepromazine; 5: thioridazine. For the authentic sample, the mixture
of 4 ng each of the compounds was directly injected into GC/MS. A 200-ng aliquot each of 5
compounds had been spiked into 1 mL whole blood or urine and extracted with a Sep-Pak C
18
cartridge; the residue was dissolved in 100 µL methanol and a 2- µL of it was injected into GC/MS.
257
ii. Preparation
Each drug is dissolved in methanol to prepare 1 mg/mL solution
b
. Many of the phenothiazine
drugs are in the forms of hydrochloride salt. For example, to prepare 1 mg/mL solution of the
free form of chlorpromazine, the amount of chlorpromazine hydrochloride to be dissolved in
1 mL methanol is calculated as follows.
1 mg (MW of the free form 318.9 MW of the salt form 355.4) = 1.11 mg
 e 1 mg/mL solution of each phenothiazine drug is diluted with methanol appropriately,
according to need.
GC/MS conditions

GC column: an Rtx-1 fused silica capillary column (30 m × 0.32 mm i.d.,  lm thickness
0.25 µm, Restek, Bellefonte, PA, USA)
c
GC conditions; instrument: a Shimadzu GC-17A gas chromatograph connected to MS
(Shimadzu Corp., Kyoto, Japan)
d
; column (oven) temperature: 150 °C (1 min) →15 °C/min
e
→
290 °C (10 min); injection temperature: 270 °C; interface temperature; 270 °C; carrier gas: He;
its  ow rate: about 1.5 mL/min; injection: splitless mode for 1 min followed by the split mode.
MS conditions; instrument: a Shimadzu QP-5050A quadrupole mass spectrometer
f
; mea-
surement: scan mode; ionization: positive ion EI; ionization current: 60 µA; electron energy:
70 eV; scan range: m/z 50–400; scan speed: 1,000 amu/s.
Procedure
i. A 10-mL volume of methanol and 10 mL distilled water are passed through a Sep-Pak C
18

cartridge; this procedure is repeated twice (in total 3 times) to activate the cartridge
g
.
ii. To 1 mL of whole blood or urine, are added 8 mL distilled water
h
, internal standard (IS)
solution and 1 mL of 1 M sodium bicarbonate solution. For IS, a non-target phenothiazine
drug (200 ng)
i
is selected for use.

iii.  e mixture solution is poured into the cartridge, followed by washing with 10 mL distilled
water twice and by elution with 3 mL of chloroform/acetonitrile (8:2).
iv.  e upper aqueous layer of the eluate is removed with a Pasteur pipette; the organic phase
is evaporated to dryness under a stream of nitrogen.  e residue is dissolved in 100 µL of
methanol and a 2-µL of it is injected into GC/MS.
v. For each target compound, a suitable ion (m/z) is selected; a peak area ratio of the target
compound to IS is obtained.
vi. For quantitation with mass chromatograms (MCs), a  xed amount of IS and various con-
centrations of a target compound are added to 1 mL each of blank whole blood or urine,
and processed as above. At least 4 vials containing di erent concentrations of the com-
pound are prepared to construct a calibration curve, consisting of drug concentration on
the horizontal axis and of peak area ratio of a drug to IS on the vertical axis.  e above peak
area ratio obtained from a specimen is applied to the calibration curve to obtain its concen-
tration.
GC/MS analysis
258 Phenothiazines
Assessment of the method
> Figure 1.1 shows a TIC and MCs for 5 phenothiazine drugs obtained by GC/MS. Panels A
and B show a TIC and an MC for the authentic compounds, respectively; panels C and D those
for extracts of human whole blood and urine, respectively. In the TIC, small peaks of 5 com-
pounds could be discernible for the authentic sample, but could not for the extracts of whole
blood or urine spiked with 200 ng each/mL of phenothiazines due to the appearance of big
impurity peaks (data not shown). In MCs, every compound could be detected.  e detection
limits
j
obtained by mass chromatography were 25–200 ng/mL depending on the kinds of phe-
nothiazines. Since the toxic concentrations of phenothiazines are several hundred ng–1 µg/mL,
the sensitivity of the present method is su cient to detect the toxic levels.  e recovery rate
was not lower than 80 %; in some experiments, it apparently exceeded 100 %
k

.
 e GC/MS analysis for phenothiazines shows some disadvantages as follows. ① Pheno-
thiazines with long side chains are not suitable for GC/MS analysis; especially, those with long
piperazinyl or hydroxyl side chains cannot be detected by either GC or GC/MS. ② Prometha-
zine, isothipendyl and ethopropazine do not give molecular ions, but give big fragment peaks
at m/z 58 or 72 only, which are not useful for identi cation of the compounds. For analysis of
such compounds, the following LC/MS becomes very useful.
LC/MS analysis [8]
See [8].
Reagents and their preparation
i. Reagents
Prochlorperazine, tri uoperazine, perphenazine,  uphenazine, propericiazine and thiorida-
zine can be purchased from Sigma (St. Louis, MO, USA). Pure powder of perazine and clospi-
razine was donated by Mitsubishi Welpharma, Osaka, Japan; that of  upentixol by Takeda
Chem. Ind. Ltd., Osaka, Japan; that of thioproperazine by Shionogi & Co., Ltd., Osaka, Japan;
that of thiethylperazine by Sandoz, Basel, Switzerland.
ii. Preparation
 e above compounds are all in the salt forms; 1 mg free base/mL methanolic solution is pre-
pared for each compound.
LC/MS conditions
LC column: a Capcell Pak C
18
UG-80 capillary column (S-5 µm, 250 × 1.0 mm i.d., Shiseido,
Tokyo, Japan).
LC conditions; instrument
l
: an HP-1100 Series LC chromatograph (Agilent Technologies,
Palo Alto, CA, USA); mobile phase A: distilled water containing 0.1 % formic acid and 10 mM
259
ammonium acetate; mobile phase B: 100 % acetonitrile. A gradient elution with solutions A

and B was performed; the initial composition of 70 % A plus 30 % B was linearly changed to
the  nal composition of 10 % A plus 90 % B during 40 min; the  ow rate was 50 µL/min.
MS conditions; instrument: an MAT LCQ ion-trap MS instrument ( ermoFinnigan, San
Jose, CA, USA); interface: electrospray ionization (ESI) in the positive mode; capillary tem-
perature: 230 °C; spray needle voltage: + 5.5 kV; sheath gas pressure: 80 units; assisted gas pres-
sure: 15 units; detection: mass chromatography in the full scan mode
Procedure
i. To 1 mL whole blood, are added an appropriate amount of IS, 3 mL distilled water and
0.5 mL of 1 M sodium bicarbonate solution. A er well mixing, the mixture is centrifuged
at 3,000 rpm for 10 min. For IS, one of other phenothiazine drugs is selected.
ii. A 1-mL volume of methanol and 1 mL distilled water are passed through an Oasis HLB
3 cc cartridge (Waters, Milford, MA, USA) to activate it.
iii.  e above supernatant fraction obtained at the step i) is poured into the cartridge at a
 ow rate not greater than 2 mL/min.
iv.  e cartridge is washed with 2 mL distilled water, and the target compound and IS are
eluted with 2 mL acetonitrile.
v.  e eluate is evaporated to dryness under a stream of nitrogen.
vi.  e residue is dissolved in 50 µL methanol, followed by addition of 100 µL distilled water
with mixing. A 100-µL aliquot of it is injected into LC/MS.
vii. Using a speci c ion of each target compound, a peak area ratio of the compound to IS is
obtained.
viii.  e method for construction of a calibration curve for a target compound by LC/MS is
essentially similar to that for GC/MS. At least 4 vials containing various concentrations of
a target compound are prepared for a calibration curve.
Assessment of the method
All eleven kinds of phenothiazine drugs gave the base peaks of protonated molecular ions.
Under the present conditions, many phenothiazines could be extracted from whole blood with
high e ciencies; 2 ng (on-column) of many compounds could be detected as distinct peaks.
However, peaks of perazine, prochlorperazine, thiethylperazine and perphenazine were small
and also interfered with by impurity peaks; low concentrations of these compounds in human

whole blood and urine cannot be analyzed by LC/MS. To overcome this di culty, detection
using LC/tandem MS seems useful. For the details of the tandem MS for phenothiazines, the
readers can refer to the reference [8]. According to the reference, every phenothiazine with a
long side chain can be speci cally detected with a high S/N ratio. A report dealing with the
combination of LC/MS/MS with solid-phase microextraction ( SPME) for phenothiazines was
also published [9].
LC/MS analysis