Sensitive spectrophotometric method for determination of some phenothiazine drugs

Article abstract of DOI:10.1007/s11164-014-1838-8

A new simple and sensitive spectrophotometric method for some phenothiazine derivatives has been developed. The proposed method is based on the reaction of phenothiazine derivatives promethazine hydrochloride, chlorpromazine hydrochloride, triflupromazine hydrochloride, prochlorperazine, and trifluoperazine with potassium iodate followed by reaction of liberated iodine with leuco crystal violet (LCV) and measurement of the color of the oxidized LCV at 598 nm. The method showed a good linearity in the ranges 0.05-4.0, 0.02-2.0, 0.05-5.0, 0.1-8.0, and 0.05-2.0 μg mL^-1 respectively. The optimum conditions and other analytical parameters were evaluated. The proposed methods have been applied successfully to the analysis of phenothiazine derivatives in pure form and in their dosage forms, and no interference was observed from common excipients present in pharmaceutical formulations.

Full text of DOI:10.1007/s11164-014-1838-8

Res Chem Intermed
DOI 10.1007/s11164-014-1838-8
Sensitive spectrophotometric method for determination
of some phenothiazine drugs
•
Kanchan Upadhyay Anupama Asthana
Raunak Kumar Tamrakar
•
Received: 15 June 2014 / Accepted: 29 September 2014
Ó Springer Science+Business Media Dordrecht 2014
Abstract A new simple and sensitive spectrophotometric method for some pheno-
thiazine derivatives has been developed. The proposed method is based on the reaction of
phenothiazine derivatives promethazine hydrochloride, chlorpromazine hydrochloride,
triflupromazine hydrochloride, prochlorperazine, and trifluoperazine with potassium
iodate followed by reaction of liberated iodine with leuco crystal violet (LCV) and
measurement of the color of the oxidized LCV at 598 nm. The method showed a good
linearity in the ranges 0.05–4.0, 0.02–2.0, 0.05–5.0, 0.1–8.0, and 0.05–2.0 lg mL-1
respectively. The optimum conditions and other analytical parameters were evaluated.
The proposed methods have been applied successfully to the analysis of phenothiazine
derivativesin pure form and in their dosage forms, and no interferencewasobserved from
common excipients present in pharmaceutical formulations.
Keywords Spectrophotometric Á Phenothiazine Á Drug Á LCV
Introduction
Phenothiazines are a very significant class of organic compounds with potent
physiological activity (Fig. 1) [1]. Phenothiazines are neuroleptics used for the
K. Upadhyay (&)
Department of Chemistry, Shri Shankarahcarya Vidyalaya, Aamdi nagar, Hudco, Bhilai 490006,
India
e-mail: kanchanupadhyay83@gmail.com
A. Asthana
Department of Chemistry, Govt. V.Y.T.PG. Autonomous College, Durg, India
R. K. Tamrakar
Department of Applied Physics, Bhilai Institute of Technology (Seth Balkrishan Memorial), Near
Bhilai House, Durg 491001, C.G., India
123

K. Upadhyay et al.
treatment of moderate and severe mental and emotional conditions. They are also
used as antipsychotics, anticholinergics and antihistamines [2]. Many phenothi-
azine derivatives and their formulations are officially in British pharmacopoeia [3]
and Indian pharmacopoeia [4]. The increasing use of phenothiazine derivatives in
medicine has boosted the development of several methods for their determination
in pure form and in pharmaceutical formulations. The methods used for their
determination include spectrofluorometry [5, 6], conductometry [7], fluoroimmu-
noassay [8], HPLC [9, 10], HPTLC [11] polarography [12], GLC [13],
chemiluminescence [14], and capillary zone electrophoresis [15]. The official
methods presented in the British pharmacopoeia for phenothiazines consist of non-
aqueous potentiometric titrimetry or spectrophotometry in the ultraviolet region,
depending upon the derivative [5]. The first method is time consuming and care
must be taken with the second one since many organic compounds absorb in this
region of the spectrum.
Many spectrophotometric methods have already been reported for the determi-
nation of phenothiazines. They are generally based on redox reaction [16, 17], ion
pair complex formation [18, 19], binary complex formation [20], ternary complex
formation [21], diazo-coupling [22], and oxidative coupling [23, 24].
HPLC, GC, and HPTLC methods which are widely used for pharmaceutical
analysis are accurate and precise with good reproducibility, but the cost of analysis
is quite high owing to expensive instrumentation, reagents and expertise. Hence, it
is worthwhile to develop simpler and cost-effective methods for simultaneous
estimation of drugs for routine analysis of formulations. Spectrophotometric
methods fulfil such requirements [25–30].
Some of the spectrophotometric methods found in the literature are reasonably
rapid and selective, but suffer from a number of disadvantages such as low
sensitivity, narrow dynamic range, heating step, extraction step, poor stability of the
colored species, and instrumental or procedural complications.
The 2-substituted and 10-substituted phenothiazine derivatives exhibit many
chemical properties, apart from interesting medicinal qualities [31]. Phenothiazine
and its derivatives are characterized by low ionization potentials [32]. They are
easily oxidized by different chemicals, and by electrochemical, photochemical,
and enzymatic methods [33]. Oxidation is interesting for the determination of the
phenothiazines, because it improves the spectroscopic properties for UV–Visible
and fluorescence detection. General approaches for the oxidation of the
phenothiazines are based on either photochemical [34] or chemical oxidation
[35–38, 44].
The present investigation aims to develop a more sensitive and cost-effective
method for the determination of phenothiazine in pure form and pharmaceutical
formulations. The method employs potassium iodate as an oxidizing agent and
leuco crystal violet (LCV) as a chromogenic reagent. The proposed methods have
been demonstrated to be superior to the reported methods with respect to their
speed, simplicity, sensitivity, and cost-effectiveness.
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Sensitive spectrophotometric method
Fig. 1 Structure of the basic
phenothiazine molecule
Experimetal
Apparatus
A Varian Carry 50 Bio UV spectrophotometer with 1-cm matched quartz cell was
used for all absorbance measurements, and a Systronics type 331- pH meter was
employed for the pH measurements.
Reagents and solutions
All chemicals used were of analytical reagent grade and double-distilled water was
used throughout the experiment.
Standard drug solution
A standard solution of 1,000 lg mL-1 of phenothiazine derivatives was prepared by
dissolving accurately weighed 100 mg of pure drug in distilled water, diluted to
100 mL. The stock solution was diluted stepwise to get working concentrations.
Potassium iodate solution: A 5 9 10-2-M aqueous solution was prepared in
deionized water.
Leuco crystal violet (LCV) [Eastman Kodak Co]: An amount of 250 mg of LCV
was dissolved in 200 mL distilled water containing 3 mL 85 % phosphoric acid
(Merck) and the volume was made up to 1 L with distilled water and stored in an
amber-colored bottle away from sunlight.
Sodium hydroxide solution: A 0.1-M aqueous solution was prepared.
Hydrochloric acid solution: A 2-M aqueous solution was prepared.
Procedure
Preparation of calibration curve
Different aliquots of phenothiazine derivatives containing promethazine (0.05–4.0
lg mL-1), chlorpromazine (0.02–2.0 lg mL-1), triflupromazine (0.05–5.0 lg mL-1),
trifluperazine (0.05–2.0 lg mL-1), and prochlorperazine (0.1–8.0 lg mL-1) were
123

K. Upadhyay et al.
0.5
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
490
540
590
640
690
740
790
Wave length (nm)
Fig. 2 Absorption maximum for the colored product
accurately measured and transferred into a series of 25-mL standard flasks and the volume
was adjusted to 5.0 mL by adding distilled water. To each flask, 1 mL of 1 M HCl, 1 mL
ofLCV and1 mLof potassium iodatewasadded. Thecontentsweremixedwell andflasks
were allowed to stand for 10 min with occasional shaking, then the pH of each mixture
was adjusted with sodium hydroxide solution. The volume was diluted to the mark with
water, mixed well, and the absorbance was measured at 598 nm against reagent blank
(Fig. 2, 3).
Procedure for tablet
Twenty tablets were weighed and finely powdered. An accurately weighed portion
of the powder equivalent to 50 mg of the phenothiazine salt was transferred into a
100-mL calibarated flask and diluted to volume with water. Using a mechanical
stirrer, the powder was completely disintegrated and the solution was filtered. A
suitable aliquot of this solution in the individual phenothiazine working range was
treated as described in the recommended procedure.
Results and discussion
The proposed method is based on the oxidation of phenothiazine derivatives with
KIO3 in acidic medium. Phenothiazine derivatives are reported to be oxidized to
123

Sensitive spectrophotometric method
9
(a) PH
(b) CPH
(c) TPH
8
(d) PCPM
(e) TFPH
7
6
5
4
3
2
1
0
0
1
2
3
4
5
6
7
8
concentration (µg mL-1
)
Fig. 3 Calibration graph for a chlorpromazine, b promethazine, c proclorpromazine, d trifluoperazine,
e triflupromazine
Scheme 1 Mechanism of oxidation of phenothiazine
form sulfoxides. The process proceeds in two steps; the first is reversible electron
abstraction from phenothiazine to the colored semiquinol cationic radical. The
radical is stable for a certain period of time depending upon phenothiazine
substitution, pH and buffer used [37]. The second irreversible step leads to the
generation of the colorless sulfoxide [36, 38] (Scheme 1).
To optimize the detection system, the UV–Vis spectra of the phenothiazines were
recorded; similar spectroscopic properties were observed for all analytes. The
respective data are presented in Table 1. The phenothiazines and their
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K. Upadhyay et al.
123

Sensitive spectrophotometric method
Scheme 2 Color reaction
corresponding sulfoxide are characterized by similar UV–Vis spectroscopic
properties, with a slightly red shifted absorption maximum for the sulfoxide, e.g.,
at 340 nm for chlorpromazine. The radical cation exhibits an additional absorption
maximum at 529 nm with small molar absorptivity.
In the proposed method, when KIO3 and the phenothiazines are mixed in
solution, the formation of red color is observed, which is associated with the
additional absorption band as described above. This color quickly fades away. As a
result of the reaction between KIO3 and phenothiazines with sulfoxides, I2 is
liberated. The liberated iodine is treated with LCV to form CV dye which gives an
absorption maximum at 598 nm. The intensity of the color of the dye is directly
proportional to the concentration of phenothiazines (Scheme 2).
Optimization of reaction variables
Effect of reagent concentrations
For method A, the influence of iodate and LCV concentrations on the colored
reaction has been checked. Optimization revealed that the 4.5 9 10-2–6 9 10-2
M
of KIO3 gives the maximum and stable absorbance and that the intensity decreases
with the increase in iodate concentration. Hence, 1 mL of 5 9 10-2 M KIO3
solution was used for the studies (Fig. 4).
The concentration of LCV on its absorbance showed that maximum absorbance
was found after the addition of 0.8 mL of LCV and it remained constant at higher
concentrations; hence, 1 mL of LCV was used for further work (Fig. 5).
Effect of time, temperature and pH
The reasonable reaction time was 10 min and delay up to 30 min had no effect on
the absorbance, while the developed color remained stable for several days (Fig. 6).
The oxidation of phenothiazine derivatives with iodate was studied at the
123

K. Upadhyay et al.
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
0
1
2
3
4
5
6
7
8
[KIO3]x 10-2 M
Fig. 4 Effect of potassium iodate concentration on oxidation of phenothiazine
0.6
a
0.5
0.4
0.3
0.2
0.1
0
0.5
0.7
0.9
1.1
1.3
1.5
Amount of LCV (mL)
Fig. 5 Effect of the amount of LCV on the color reaction
temperature range between 20 and 40 °C. The rate of oxidation decreased slightly
with increasing temperature. Therefore, working at room temperature, i.e. 25 °C,
was adopted (Fig. 7).
The effect of various acids like HNO3, H2SO4, HCl, HClO4, etc. were checked
and the best results were found in HCl medium. A pH 2–3 was required for
liberation of I2 which gives a blue color with LCV at pH 4–4.5. After pH 5, the
solution becomes turbid. Initial pH was maintained by 1 mL of 1 M HCl; later on,
the pH was adjusted up to 4 by using 0.1 M NaOH solution (Fig. 8).
123

Sensitive spectrophotometric method
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
0
5
10
15
20
25
30
Time (min)
Fig. 6 Effect of time on the reaction
0.5
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
0
5
10
15
20
25
30
35
40
45
50
Temperature(°C)
Fig. 7 Effect of temperature on the oxidation of phenothiazine
Method validation
The absorbance versus concentration was plotted and a linear correlation was found
(Fig. 3). Beer’s law range, the molar absorptivity and Sandell’s sensitivity are given
in Table 2. The limits of detection, limits of quantification, regression equation, and
correlation coefficients values are also given in Table 2.
123

K. Upadhyay et al.
0.5
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
0
1
2
3
4
5
6
pH
Fig. 8 Effect of pH on the color reaction
The accuracy and precession of the analytical method have been evaluated. For
accuracy, the percentage relative error between the measured concentrations and the
chosen concentration was calculated. The precision of the method was calculated in
terms of intermediate precision (intra-day and inter-day). The SD and RSD values
of intra-day and inter-day studies showed that precision was good (Table 3).
Selectivity
To determine the selectivity of the proposed method, the analytical placebo was
analyzed by the proposed methods. It was confirmed that the change in the
absorbance with respect to the blank was caused only by the change in analyte
concentration. To identify the interference by common tablet excipients, a synthetic
mixture with the composition of phenothiazine derivatives (50 mg), talc (80 mg),
starch (160 mg), calcium gluconate (80 mg), lactose (80 mg), sodium alginate
(40 mg) and magnesium stearate (40 mg) was prepared and subjected to analysis by
the proposed methods after solution preparation using the procedure described for
tablets. The percentage recoveries suggesting no interference by the excipients in
the assay of phenothiazine under the described optimum conditions (Table 4).
Application to formulations
The proposed method was applied to the determination of phenothiazines in tablets
and injections. The results in Table 4 show that the method is applicable for the
determination of phenothiazines and that the excipients in the dosage forms do not
interfere. A statistical comparison of the results by the proposed methods and a
literature method [3] showed that the results agree well with the claim and are also
in agreement with the results obtained by the literature method. Statistical analysis
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Sensitive spectrophotometric method
123

K. Upadhyay et al.
Table 3 Inter-day and intra-day precision and accuracy studies for method A
Phenothiazine taken Intra-day (n = 7)
in lg mL-1
Found
Inter-day (n = 5)
Precisiona Accuracyb Found Precisiona Accuracyb
PH
1
1.07 0.077 7.19
2.04 0.079 3.87
3.08 0.14 4.49
7
1.02 0.022
2.19
3.59
7.21
2
2
2.07
2.76
2.09 0.019
3.10 0.25
4.5
3.33
3
CPH
0.5
0.51 0.022 4.26
1.02 0.019 1.82
1.714
2.14
1.14
0.51 0.019
1.05 0.045
1.52 0.027
3.66
4.27
1.77
2
1
5.2
1.6
1.5
1.51 0.25
1.69
TPH
1
1.02 0.051 5.04
2.03 0.051 2.53
1.71
1.57
1.67
1.07 0.084
2.04 0.12
4.15 0.22
7.87
5.73
3.52
6.8
2
2.0
4
4.06 0.14
3.52
3.75
PCPM
2
2.07 0.19
4.04 0.16
6.18 0.25
9.53
3.93
4.01
3.38
0.93
2.98
2.1 0.26
4.16 0.21
6.08 0.23
12.37
4.95
3.70
5.4
4
6
4.05
1.43
TFPH
0.5
1
0.51 0.021 4.26
1.02 0.019 1.82
1.52 0.026 1.69
1.71
2.14
1.14
0.52 0.052 10.12
1.08 0.130 12.00
3.2
8.4
1.5
1.56 0.099
6.40
3.73
a
Relative standard deviation (%)
b
Bias%: (found-taken/taken) 9 100
Table 4 Determination of CPHa in the presence of excipients
S. no.
Material
Amount (mg)
% recovery of CPH % RSDb
1
2
3
4
5
6
7
8
a
Lactose
40
40
30
40
30
20
10
0.1
98 1.41
98.6 1.10
99.7 1.46
100.0 0.76
99.6 0.49
100.6 0.84
101.2 0.90
99.8 1.60
Dextrose
Sucrose
Starch
Talc
Stearic acid
Sodium alginate
Ascorbic acid
1 lg mL-1 of CPH taken
b
Average of five determinations
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Sensitive spectrophotometric method
Table 5 Results of assay of tablets by the proposed methods and statistical evaluation
Tablet brand
name
Nominal amount
(mg/tab)
Literature
methoda
Proposed
methoda
t testb
(
)
F testb
PH
Phenargan
Phenargan
Avomine
CPH
10
25
25
10.05 0.32
24.88 0.38
25.02 0.33
10.2 0.20
24.92 0.27
25.14 0.34
0.886
0.189
0.575
2.56
1.98
1.06
Megatil
Megatil
Emetil
25
100
100
24.96 0.31
99.01 0.46
100.33 0.38
24.98 0.33
99.4 0.58
99.8 0.34
0.098
1.316
1.882
1.132
1.59
1.25
TPH
Siquil (tab)
Siquil (inj.)
PCPM
10
10
9.8 0.23
9.97 0.26
9.9 0.11
0.5854
1.464
1.28
2.11
10.03 0.16
Emidoxine
Stemetil
TFPH
5
5
4.82 0.23
5.36 0.35
4.98 0.34
4.84 0.52
0.8646
0.6926
2.18
2.21
Trazine
Trazine
Espazine
a
5
10
25
4.76 0.24
10.02 0.28
25.17 0.17
5.1 0.39
10.0 0.37
24.96 0.33
1.607
0.317
1.271
2.64
1.75
4.74
Mean value of five determinations
b
Tabulated t value at the 95 % confidence level is 2.78; tabulated F value at the 95 % confidence level is
6.39
of the results using Student’s t test for accuracy and F test for precision revealed no
significant difference between the proposed method and the literature method at
95 % confidence level with respect to accuracy and precision (Table 5).
Conclusion
In conclusion, a simple, rapid, and cost-effective method for the determination of
phenothiazine derivatives has been developed and validated. The proposed method
is more sensitive than many existing methods, and is free from such experimental
variables as heating or an extraction step (Table 6). The stability of the color system
is an advantage over the earlier methods. The method provides sensitivity
comparable to that achieved by sophisticated and expensive techniques like HPLC.
Thus, it can be used as an alternative for rapid and routine determination of bulk
samples and tablets.
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