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 260 Phenothiazines Toxic and fatal concentrations Serum concentrations of chlorpromazine, the most typical phenothiazine, were reported to be 0.05–0.5 µg/mL as therapeutic levels and 0.5–1 µg/mL as toxic ones [10]. Blood concentrations of chlorpromazine were reported to be 0.5–2 µg/mL as toxic levels and not lower than 2 µg/mL as fatal ones [11]. Blood concentrations of other phenothiazines; perazine: therapeutic levels 0.025–0.1 µg/mL and toxic level 0.5 µg/mL; perphenazine: therapeutic levels 0.0004–0.03 µg/ mL and toxic level 0.05 µg/mL; promazine: therapeutic levels 0.1–0.4 µg/mL and toxic levels 2–3 µg/mL; thioridazine: therapeutic levels 0.2–1 µg/mL and toxic level 2 µg/mL [10].  e therapeutic and toxic concentrations are markedly di erent according to di erent kinds of phenothiazines.  ioridazine was reported to show higher cardiotoxicity than other phenothi- azines [12]. Notes a)  e GC- SID is commercially available from Shimadzu Corp., Kyoto, Japan; the detector can be attached to a GC-14 type of instruments.  e detector speci cally responds to com- pounds with a tertiary amino group. For biomedical specimens, such as blood and urine, containing large amounts of amino compounds, the GC-SID can detect compounds with a tertiary amino group with much higher speci city and sensitivity than GC-NPD. b) The methanolic standard solution at 1 mg/mL is stable for at least 2–3 weeks at 4 °C. c)  is column corresponds to the DB-1 (J&W); any capillary column with 100 % dimethylpo- lysiloxane can be used, regardless of its manufacturer. However, when many impurity peaks appear in background upon analysis by GC/MS, so-called “low bleed MS column” can be tried. When satisfactory separation of compounds cannot be achieved with such non-polar columns, slightly and intermediately polar columns (DB-5 and DB-17) are worth trying. d) Any type of gas chromatographs, to which a capillary column can be attached, can be used. e)  e temperature program conditions also a ect the separation ability for compounds, together with the type of a column to be used. According to Hattori et al. [7], the tempera- ture-elevating program was 6 °C/min. f)  e quadrupole type mass spectrometer is generally cheaper and relatively easy to be han- dled. Any type of GC/MS, such as the sector and ion-trap types, can be used. GC/MS/MS can be also used to perform even more speci c detection; this was omitted, because it is beyond the policy of this book. g) According to the original method by Suzuki et al. [13] reported in 1989, the cartridge had been activated by passing 10 mL each of chloroform/methanol (9:1), methanol and water; the procedure had been repeated 2–3 times. However, for the recent lots of the same type of cartridges, the above procedures oppositely cause the appearance of many impurity peaks.  e activation should be mildly made with methanol and water only to get good results. h)  e  rst addition of distilled water is aimed to completely hemolyze the blood specimen. According to the original method, 16 mL distilled water had been added to 1 mL whole blood, followed by addition of 3 mL of 1 M sodium bicarbonate solution. In the experience of the authors, only with half the amount of distilled water (8 mL), almost the same results could be obtained without any problem. 261 i) In the report of Hattori et al. [7], 100 pmol each (about 30–40 ng) of phenothiazines had been added. j) If the selected ion monitoring mode is used, the sensitivity is increased 10–100 fold. k) As probable reasons of such a phenomenon, the slight thermal decomposition of the authentic standard during the step of GC, and/or protection of the target phenothiazine from its thermal decomposition or from adsorption to the column by certain compound(s) being contained in specimen extracts, can be mentioned. l) Any LC instrument, to which a capillary LC column can be attached and which enables a low  ow rate of 50–200 µL/min, can be used, regardless of its manufacturer. References 1) Baldessarini RJ, Tarazi FI (2001) Drugs and the treatment of psychiatric disorders. In: Goodman Gilman A, Hard- man JG, Limbard LE (eds) Goodman & Gilman’s The Pharmacological Basis of Therapeutics, 10th edn. McGraw- Hill, New York, pp 485–520 2) Meredith TJ, Vale JA (1985) Poisoning due to psychotropic agents. Adverse Drug React Acute Poisoning Rev 4:83–126 3) Geraut A, Kintz P, Traqui A et al. (1994) Toxicological findings in a fatality involving chlorpromazine. Bull Int Assoc Forensic Toxicol 24 (2):27–30 4) Kintz P, Berthault F, Traqui A et al. (1995) A fatal case of alimemazine poisoning. J Anal Toxicol 19:591–594 5) Ishikawa Y, Suzuki O, Hattori H (1990) Positive-and negative-ion mass spectrometry and rapid clean-up of 19 phenothiazines. Forensic Sci Int 44:93–105 6) The Pharmaceutical Society of Japan (ed) (1992) Standard Methods of Chemical Analysis in Poisoning – With Commentary, 4th edn. Nanzando, Tokyo, pp 150–158 (in Japanese) 7) Hattori H, Yamamoto S, Iwata M et al. (1992) Sensitive determination of phenothiazines in body fluids by gas chromatography with surface ionization detection. J Chromatogr 579:247–252 8) Seno H, Hattori H, Ishii A et al. (1999) High performance liquid chromatography/electrospray tandem mass spectrometry for phenothiazines with heavy side chains in whole blood. Rapid Commun Mass Spectrom 13:2394–2398 9) Kumazawa T, Seno H, Watanabe-Suzuki K et al. (2000) Determination of phenothiazines in human body fluids by solid-phase microextraction and liquid chromatography/tandem mass spectrometry. J Mass Spectrom 35:1091–1099 10) Uges DRA (1997) Blood level data. In: Brandenberger H, Maes RAA (eds) Analytical Toxicology for Clinical, Fo- rensic and Pharmaceutical Chemists. Walter de Gruyter, Berlin, pp 707–718 11) Moffat AC, Jackson JV, Moss MS et al. (eds) (1986) Clarke’s Isolation and Identification of Drugs. The Phamaceu- tical Press, London, pp 460–461 12) Buckley NA, Whyte IM, Dawson AH (1995) Cardio-toxicity more common in thioridazine overdose than with other neuroleptics. J Toxicol Clin Toxicol 33:199–204 13) Suzuki O, Kumazawa T, Seno H et al. (1989) Rapid isolation with Sep-Pak C 18 cartridges and wide-bore capillary gas chromatography of some barbiturates. Med Sci Law 29:242–248 Toxic and fatal concentrations . 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. GC/MS and LC/MS are presented. GC/MS analysis Reagents and their preparation i. Reagents Chlorpromazine, tri upromazine, promethazine and thioridazine can