1. science
2. mathematics
3. statistics

Short communication
Received: 3 November 2013,
Revised: 18 February 2014,
Accepted: 25 February 2014
Published online in Wiley Online Library: 10 April 2014
(wileyonlinelibrary.com) DOI 10.1002/bio.2673
Spectrofluorimetric determination of
amisulpride and bumidazone in raw
materials and tablets
M. I. Walash, F. Belal, M. M. Tolba and M. I. Halawa*
ABSTRACT: A highly sensitive, simple and rapid spectrofluorimetric method was developed for the determination of amisulpride (AMS) and bumidazone (BUM) in tablet form. The proposed method is based on measuring the native fluorescence of
the studied drugs in methanol at 360 and 344 nm after excitation at 276 and 232 nm for AMS and BUM, respectively. The
fluorescence–concentration plots were rectilinear over the ranges of 5.0–60.0 ng/mL for AMS and 0.5–5.0 μg/mL for BUM.
The lower detection limits were 0.70 ng/mL and 0.06 μg/mL, and the lower quantification limits were 2.0 ng/mL and
0.18 μg/mL for AMS and BUM, respectively. The method was successfully applied for the analysis of AMS and BUM in commercial
tablets. Statistical evaluation and comparison of the data obtained using the proposed and comparison methods revealed good
accuracy and precision for the proposed method. Copyright © 2014 John Wiley & Sons, Ltd.
Keywords: amisulpride; bumidazone; spectrofluorimetry; tablets
Introduction
Chemically, amisulpride (AMS) is 4-amino-N-{[(2RS)-1-ethylpyrrolidin-2-yl]methyl}-5(ethyl sulfonyl)-2-methoxybenzamide (Fig. 1a).
AMS is a selective D2–D3 antagonist that has been reported to be
effective in the treatment of schizophrenia and major depressive
disorder (1). AMS has been the subject of monographs in the British
Pharmacopoeia (2) and the European Pharmacopoeia (3). Reviewing the literature revealed that only a few analytical methods
have been reported for the determination of AMS including spectrophotometry (4–9), high-performance liquid chromatography
(HPLC) (10–13) and liquid chromatography–mass spectrometry
(LC-MS) (14–18). Bumadizone calcium semi-hydrate (BUM) is
butylmalonic acid mono-(1,2-diphenylhydrazide) calcium semihydrate; (Fig. 1b). It is used as a non-steroidal anti-inflammatory
drug and has a peripheral analgesic effect (1). A literature survey
revealed that there are only three chromatographic methods for
the determination of BUM (19–21). To the best of our knowledge,
nothing has been published concerning the specrofluorimetric
determination of AMS and BUM in tablet form. The current study
aimed to develop and validate a simple, rapid and sensitive
spectrofluorimetric method for the determination of AMS and
BUM utilizing their native fluorescence in methanol.
labeled as containing 50 mg of AMS, were from of Al-Andalus
Medical Company, Cairo, Egypt. Bumadizone calcium semihydrate was kindly provided by October Pharma S.A.E. Company
(6th October City, Egypt). Octomotol W tablets (batch #
B1830212), labeled as containing 110 mg of BUM, were from
October Pharma S.A.E. Company, 6th October City, Egypt.
Sodium dodecyl sulfate (SDS) solution and cetyl trimethyl ammonium bromide (CTAB; 99%) were purchased from Winlab Ltd
(Market Harborough, UK). Methanol, acetonitrile and n-propanol
were obtained from Sigma-Aldrich (Munich, Germany).
Dimethylsulfoxide (DMSO) was purchased from Riedel-de Häen
(Seelze, Germany), dimethyl formamide (DMF) was obtained from
El-Nasr Pharmaceutical Chemical Co. (ADWIC; Egypt), and
hydroxypropyl-β-cyclodextrin (HP-β-CD) was obtained from Merck
(Darmstadt, Germany).
Tween-80, methyl cellulose, ethanol, glacial acetic acid, sodium acetate trihydrate and boric acid were all obtained from
El-Nasr Pharmaceutical Chemical Co.
SDS, CTAB, methylcellulose, HP-β-CD and Tween-80 were prepared as 0.1% w/v aqueous solutions, acetate buffer (pH 3.0–5.5)
and borate buffer (pH 6.0–10.0) solutions were freshly prepared.
Standard solutions
Experimental
Apparatus
All fluorescence measurements were made using a RF-1501 Shimadzu
spectrofluorometer, equipped with a 150 W xenon arc lamp.
Materials and reagents
1202
Amisulpride, lot # 2AMS0361011 was kindly provided by Sigma
Pharmaceutical Industries, Egypt. Amipride tablets (batch # 11950),
Luminescence 2014; 29: 1202–1205
Stock solutions equivalent to 100.0 μg/mL of AMS and BUM were
prepared by dissolving 10 mg of each in 100 mL of methanol
with the aid of an ultrasonic bath. Working standard solutions
of 1.0 μg/mL for AMS and 10.0 μg/mL for BUM were prepared
by appropriate dilution of the stock solutions with methanol.
* Correspondence to: M. Halawa, Department of Analytical Chemistry,
Faculty of Pharmacy, University of Mansoura, 35516, Mansoura, Egypt.
E-mail: m_halawa88@hotmail.com
Department of Analytical Chemistry, University of Mansour, Mansour, Egypt
Copyright © 2014 John Wiley & Sons, Ltd.
Spectrofluorimetric determination of amisulpride and bumidazone
Table 1. Analytical performance data for the determination
of the studied drugs using the proposed method
Parameter
Figure 1. Structural formulae of the studied drugs. (a) Amisulpride, (b) bumidazone.
Solutions of AMS were protected from light with aluminum foil.
All solutions were stored in a refrigerator and found to be stable
for at least 10 days without alteration.
Construction the calibration graphs
Accurately measured aliquots of the suitable drug working standard solutions were transferred into a series of 10 mL volumetric
flasks so that the final concentration was in the range of
5.0–60.0 ng/mL for AMS or 0.5–5.0 μg/mL for BUM. The volume
was completed with methanol. The fluorescence intensity was
measured at 360 nm after excitation at 276 nm for AMS or at
344 nm after excitation at 232 nm for BUM. The relative fluorescence intensity (RFI) was plotted against the final concentration
of the drug to obtain the caliberation graph. Alternatively, the
corresponding regression equations were derived.
Procedures for tablets
An accurately weighed quantity of the mixed contents of 10
powdered tablets equivalent to 10.0 mg of either AMS or BUM
was transferred into a 100 mL volumetric flask and ~ 80 mL of
methanol was added. The contents of the flask were sonicated
for 30 min, made up to the mark with the same solvent and
filtered through a cellulose acetate syringe filter. Further dilution
with methanol was performed to obtain a working standard solution that was assayed by subjecting it to the general procedure
described above (Construction of calibration graphs). The nominal content was calculated from a previously plotted calibration
graph or using the corresponding regression equation.
Results and discussion
Both AMS and BUM were found to exhibit an intense native fluorescence in methanolic solution at 360 and 344 nm after excitation at 276 and 232 nm (Fig. 2), respectively. As a consequence,
we aimed to utilize these emission bands to explore a new
methodology for the analysis of AMS and BUM in tablet form.
5.0–60.0
109.00
1.24 × 104
0.9999
3.40
2.48
74.52
1.13
0.70
2.00
0.5–5.0
–16.80
1.74 × 102
0.9999
3.93
3.08
1.01
1.21
0.06
0.18
Optimization of experimental conditions
Effect of different organized media. Different surfactants including anionic (SDS), cationic (CTAB) and non-ionic (Tween-80),
and different macromolecules such as β-CD, HP-β-CD and methylcellulose (1 mL of a 0.1% w/v freshly prepared aqueous solution of
each) were investigated. It was found that Tween-80 caused a very
slight increase in the RFI of both drugs, whereas all the other media
caused a slight decrease when added to the methanolic solution
of the drug (final concentration 60 ng/mL for AMS and 4.0 μg/mL
for BUM). This may be attributed to the high background fluorescence intensity of the blank. Therefore, no surfactant was used in
the study.
Effect of pH. The influence of pH on the fluorescence of AMS
and BUM was studied using different types of buffers covering
the whole pH range. For both drugs, the use of buffer did not enhance the RFI over the pH range studied. It was found that the
maximum RFI was achieved in methanol without the addition
of any buffer.
Effect of diluting solvent. The effect of different diluting
solvents on the RFI of AMS and BUM was investigated using water, ethanol, methanol, n-butanol, DMF, DMSO, acetonitrile and
n-propanol. It was found that methanol was the best solvent
for dilution, as it gave the highest RFI and the lowest blank
reading with reproducible results. A distinct and sharp decrease
in the relative fluorescence intensities of both drugs was observed upon using water, acetonitrile and ethanol. n-Propanol
and n-butanol were not selected due to the high blank reading.
However, DMSO and DMF greatly quenched the fluorescence of
Fluorescence Intensity
Fluorescence Intensity
Linearity range
Intercept (a)
Slope (b)
Correlation coefficient (r)
SD of residuals (Sy/x)
SD of intercept (Sa)
SD of slope (Sb)
Percentage relative standard
deviation, % RSD
Limit of detection, LOD
Limit of quantitation, LOQ
1000.00
1000.00
-
0.00
Wavelength(nm)
-
C
C
C
220
AMS (ng/mL) BUM (μg/mL)
500
0.00
220
C
Wavelength (nm)
a
500
b
Luminescence 2014; 29: 1202–1205
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/luminescence
1203
Figure 2. Fluorescence spectra of the studied drugs in methanol: (a) 60.0 ng/mL of AMS, (b) 4.0 μg/mL of BUM. (A, B) Excitation spectra, (A′, B′) emission spectra, (C, C′)
excitation spectra and emission spectra of methanol.
M. I. Walash et al.
the studied drugs, because they exhibited an intersystem crossing process (behavior similar to the heavy atom effect) (22).
Validation of the method
Linearity. Calibration graphs for the determination of AMS
and BUM using the proposed methods were constructed by
plotting RFI against the cincentration of the drugs (μg/mL). The
graphs were found to be rectilinear over concentration ranges
of 5.0–60.0 ng/mL for AMS and 0.5–5.0 μg/mL for BUM. Linear
regression analysis of the data gave the following equations:
RFI = 109.00 + 1024 × 104 C (r = 0.9999) for AMS
RFI = –16.80 + 1.74 × 102 C (r = 0.9999) for BUM
where RFI is the relative fluorescence intensity, C is the
concentration of the drug in ng/mL for AMS and in μg/mL
for BUM and r is the correlation coefficient. Statistical analysis
(24) of the data gave a high value for the correlation coefficient (r) of the regression equation, small values for the
standard deviation of the residuals (Sy/x), intercept (Sa) and
slope (Sb), and a small value for the percentage relative
standard deviation (% RSD) and the percentage relative error
(Table 1). These data proved the linearity of the calibration
graphs.
Limits of quantification and limits of detection. Values for
limits of quantification (LOQ) and limits of detection (LOD) were
calculated according to the following equations (23):
LOQ = 10 Sa/b LOD = 3.3 Sa/b
where Sa is the standard deviation of the intercept of the regression line and b is the slope of the calibration graph.
Table 2. Assay results for the determination of the studied drugs in tablet form using the proposed method
Parameter
Proposed method
AMS
Amount taken
(ng/mL)
Amipride® 50 mg tablets
(AMS 50 mg/tablet)
20.0
40.0
60.0
Amount found
(ng/mL)
19.975
39.731
60.711
Mean
± SD
t
F
BUM
Octomotol® w tablets
(BUM 110 mg/tablet)
Amount taken
(μg/mL)
1.0
2.0
4.0
Amount found
(μg/mL)
0.998
1.028
4.004
Mean
± SD
t
F
Comparison method (9,21)
%
Found
99.88
99.35
101.19
100.14
0.95
0.62 (2.78)
1.48 (19.0)
%
Found
99.80
101.40
100.10
100.43
0.85
0.38 (2.78)
2.17 (19.0)
Amount taken
(μg/mL)
4.0
6.0
8.0
Amount taken
(μg/mL)
6.0
8.0
10.0
Amount found
(μg/mL)
4.0780
5.9814
8.0320
Amount found
(μg/mL)
5.928
8.016
10.130
%
Found
101.95
99.69
100.40
100.68
1.16
%
Found
98.80
100.20
101.30
100.10
1.25
Each result is the average of three separate determinations. Values in parentheses are the tabulated t and F values at P = 0.05 (24).
Table 3. Precision data for the determination of the studied drugs using the proposed method
Parameter
AMS (ng/mL)
Intraday
% Found
Interday
Mean
SD
% RSD
% Found
Mean
S.D.
% RSD
BUM (μg/mL)
10.0
20.0
60.0
0.5
2.0
5.0
98.75
100.45
101.15
100.12
1.23
1.23
100.82
99.90
101.30
100.67
0.71
0.71
101.23
100.29
98.95
100.16
1.15
1.14
98.48
100.30
98.80
99.19
0.97
0.98
99.80
100.40
101.88
100.69
1.07
1.06
99.60
101.12
100.20
100.31
0.77
0.76
99.30
98.22
101.00
99.51
1.40
1.41
100.40
100.10
99.20
99.90
0.62
0.63
98.25
100.29
101.76
100.10
1.77
1.77
99.25
100.50
100.42
100.06
0.70
0.70
101.98
100.40
99.20
100.52
1.40
1.39
99.60
100.80
101.20
100.53
0.83
0.83
1204
Each result is the average of three separate determinations.
wileyonlinelibrary.com/journal/luminescence
Copyright © 2014 John Wiley & Sons, Ltd.
Luminescence 2014; 29: 1202–1205
Spectrofluorimetric determination of amisulpride and bumidazone
Accuracy and precision. Statistical analysis (24) of the results
obtained with the proposed and comparison methods (9,21)
using the Student’s t-test and variance ratio F-test showed no
significant differences between the two methods in regarding
accuracy and precision (Table 2).
Intraday precision was evaluated by determining three concentrations of each drug in the pure form on three successive
occasions. Interday precision was also evaluated by replicate
analysis of three concentrations for a period of three successive
days. The results of the intraday and interday precision are summarized in Table 3. RSD values were found to be very small, indicating reasonable repeatability and intermediate precision for
the proposed method.
Pharmaceutical applications
The proposed method was successfully applied to the determination of AMS and BUM in commercial tablet form (Table 2) without
interference from common excipients. The average percentages
found for different concentrations were based on the average of
three replicate determinations. The results shown in Table 2 are
in good agreement with those obtained using comparison
methods (9,21). Statistical analysis of the results obtained using
the Student’s t-test and variance ratio F-test (24) showed no
significant difference between the performance of the two
methods regarding accuracy and precision, respectively.
Conclusion
The developed spectrofluorimetric method provided a reliable,
reproducible assay for AMS and BUM in bulk material and tablet
form. The proposed method is rapid, less time-consuming and
does not require the elaborate treatment associated with
chromatographic methods; moreover, it is sensitive, with no
need for derivatization reactions. By virtue of its simplicity and
rapidity, the proposed method could be applied in quality
control laboratories as an alternative to existing HPLC methods.
Acknowledgements
The authors extend their appreciation to Analytical Chemistry
Department Mansoura University for providing instruments and
chemicals. Also Sigma company and October Pharma S.A.E. for
kindly providing pure powders of amisulpride and bumadizone.
References
1. Sweetman SC. Martindale, the complete drug reference. 37th ed.
London: Pharmaceutical Press, 2009: 29–30; 1063–64.
2. The British pharmacopoeia. Vol. II. London: HMSO, 2010.
3. The European pharmacopoeia VII. Vol. II. Strasbourg: European Directorate for the Quality of Medicines and Heathcare, 2011:1376.
4. Devadasu C, Ravisankar P, Babu P, Devala RG. Spectrophotometric
methods for the analysis of amisulpride in pharmaceutical dosage
forms. Ind Pharm 2011;10(103):70–4.
5. Venumadhav E, Nishat A, Swetha A, Neeha T, Bhargavi P, Devala RG.
New spectrophotometric methods for the determination of
amisulpride in pharmaceutical dosage forms. J Chem Pharm Sci
2010;3:195–7.
6. Sangita S, Madhurjya N, Vipul P, Hiren P, Dipti D. Development and
validation of spectrophotometric methods for estimating amisulpride in pharmaceutical preparations. Anal Sci 2010;26:485–9.
7. Humaira S, Dey A, Raju A, Sanaullah S, Ali A, Pasha K. Spectrophotometric determination of amisulpride. Turk J Pharm Sci 2009;6:221–9.
8. Humaira S, Dey AK, Sanaullah S, Raju SA. Spectrophotometric determination of amisulpride. Int J Chem Sci 2009;7:912–18.
9. Humaira S, Dey AK, Raju SA, Sanaullah S. Development and
validation of spectrophotometric method for determination of
amisulpride in pharmaceutical dosage forms. Int J Chem Sci
2008;6:437–40.
10. Madhurjy N, Rikesh P, Sangita S. Isocratic liquid chromatographic
method for analysis of amisulpride in pharmaceutical preparations.
Int J PharmTech Res 2011;3:1909–15.
11. Chatterjee B, Das A, Chakraborty US, Bhaumik U, Sengupta P, Pal TK.
Development and validation of method for quantification of
amisulpride in human plasma. Res J BioTechnol 2008;3:235–8.
12. Weiqiao H, Jun Z, Caiyun C, Weizhong L. Determination of
amisulpride in plasma by HPLC with UV detection. Zhongguo Yiyuan
Yaoxue Zazhi 2007;27:1192–4.
13. Pehourcq F, Ouariki S, Begaud B. Rapid high-performance liquid
chromatographic measurement of amisulpride in human plasma:
application to manage acute intoxication. J Chromatogr B
2003;789:101–5.
14. Robert S Identification of photodegradation product of amisulpride
by ultra-high-pressure liquid chromatography–DAD/ESI–quadrupole
time-of-flight-mass spectrometry. J Pharm Biomed Anal
2011;56:904–10.
15. Ramakotaiah M, Kanchanamala K, Reddy KC, Rao BC, Kottapalli CB.
Development and validation of amisulpride in human plasma by
HPLC coupled with tandem mass spectrometry and its application
to a pharmacokinetic study. Sci Pharm 2011;79:583–99.
16. Couchman L, Morgan PE, Flanagan RJ. Basic drug analysis by
strong cation-exchange liquid chromatography–tandem mass
spectrometry: simultaneous analysis of amisulpride, metamfetamine
and amfetamine in serum/plasma. Biomed Chromatogr 2011;
25:867–72.
17. Eva S, Dimitri G, Jochen B. Identification and quantification of 30 antipsychotics in blood using LC-MS/MS. J Mass Spectrom
2010;45:915–25.
18. Shangrong Z, Yuguan W, Qiongying L. Determination of amisulpride
in plasma by LC-MS/MS. Zhongguo Xiandai Yingyong Yaoxue
2009;26:483–6.
19. Spahn H, Mutschler E. Quantitative determination of bumadizone,
phenylbutazone and oxyphenbutazone in human plasma by high
performance liquid chromatography. Arzn Forsch 1981; 31:495–9.
20. Hermansson I, Hermansson J. Direct resolution of nonsteroidal
antiinflammatory drugs on α1- acid glycoprotein column poster
presentaion. Poster presentation at the 13th International Symposium
on Column Liquid Chromatography, Stockholm, 1989.
21. Ali WN, ZaaZaa AH, Abdelkawy M, Magdy AM. Simultaneous HPTLC
and RP-HPLC methods for determination of bumadizone in the
presence of its alkaline-induced degradation product. Biomed
Chromatogr 2012;26:1143–9.
22. Skoog DA, Holler FJ, Crouch SR. Principles of instrumental analysis. 6th
ed. Stamford, CT: Cengage, 2007:399–429.
23. ICH harmonized tripartite guidelines, validation of analytical procedures: text and methodology, Q2(R1), 2005. www.ich.org/LOB/media/MEDIA417.pdf (accessed 15 February 2008).
24. Miller JC, Miller JN. Statistics and chemometrics for analytical chemistry.
5th ed. Harlow, UK: Pearson Education, 2005:39–73;107–49.
1205
Luminescence 2014; 29: 1202–1205
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/luminescence