Int J Pharm Pharm Sci, Vol 7, Issue 1, 178-184Original Article


SPECTROPHOTOMETRIC DETERMINATION OF ARIPIPRAZOLE, CLOZAPINE AND SULPIRIDE BY ION- PAIR EXTRACTIONIN IN TABLETS AND BIOLOGICAL FLUIDS

AKRAM M. ELDIDAMONY1, SAMEH M. HAFEEZ2, MOGEDA M. ABDEL HAFEZ3

1Chemistry Department, Faculty of Science, Zagazig University, Zagazig 44519, Egypt, 2Ismailia Chemical Laboratory, Forensic Medicine Authority, Justice Ministry, Egypt, 3Plant Protection Research Institute, Agriculture Research Center, Dokki, Egypt.
Email: ak_eldidamony@yahoo.com

Received: 24 Sep 2014 Revised and Accepted: 20 Oct 2014

ABSTRACT

Objective: Simple, sensitive and extraction-free spectrophotometric methods have been developed for the determination of three antipsychotics drugs, namely aripiprazole (ARP), clozapine (CLP) and sulpiride (SUP) both in tablets and in biological fluids.

Methods: Two spectrophotometric methods are based on the formation of yellow colored ion-pair complexes between the studied drugs and two sulphonphthalein acid dyes, bromophenol blue (BPB) and bromothymol blue (BTB) with absorption maxima at 408 and 406 nm, respectively.

Results: Molar ratio of formed ion-pair complex was found to be 1:1 as deduced by Jobʼs method. Several parameters such as pH and buffer type, reagent volume, sequence of addition and effect of extracting solvent were optimized to achieve high sensitivity, stability, low blank reading and reproducible results. Various analytical parameters have been evaluated and the results have been validated by statistical data. A proposal for the reaction pathway was postulated.

Conclusion: The proposed methods were successfully applied to the analysis of commercial tablets containing the drugs and the results were in good agreement with those obtained with reported methods. The proposed methods were further applied to the determination of the studied drugs in spiked human serum and urine.

Keywords: Aripiprazole, Clozapine, Sulpiride, Ion-pair complex, Bromophenol blue, Bromothymol blue.

INTRODUCTION

Aripiprazole (ARP), clozapine (CLP) and sulpiride (SUP) are structurally related atypical antipsychotics. They are used in the treatment of schizophrenia and other psychotic syndromes. It is reported that they are effective in the treatment of both positive and negative symptoms of schizophrenia, and that they are less likely to produce extra pyramidal side effects when compared with classical antipsychotics. The advantages of the therapeutic profile of the three drugs have led to increasing use of them in treatment of schizophrenic patients [1, 2].

However, high doses of these atypical antipsychotics are suspected to pose an increased risk for extra pyramidal side effects or other side effects [1, 3–7]. Aripiprazole (ARP) (Fig. 1a), chemically 7-[4-[4-(2, 3-dichlorophenyl) piperazine -1- yl] butoxy] - 3, 4-dihydro-1H-quinolin-2-one. A survey of pertinent literature revealed that few analytical methods reported for determination of aripiprazole in pharmaceutical dosage forms and biological samples include chromatographic [8–16] and spectrophotometric [17, 18] methods.

Clozapine (CLP) (Fig. 1b), (8-chloro-11-(4-methyl-piperazin-1-yl)- 5H-dibenzo[b,e] [1,4]-diazepine; is an atypical antipsychotic drug. Different methods for the analysis of clozapine have been reviewed. These methods include high performance liquid chromatography [19–22], liquid chromatography [23, 24], gas chromatography [25], capillary zone electrophoresis [26, 27] and linear scan voltammetry [28–30]. Few photometric [31, 32] and fluorimetric [33] methods have been reported for the analysis of clozapine. Sulpiride (SUP) (Fig. 1c), 5-(Aminosulfonyl)-N-[(1-ethyl-2-pyrrolidinyl)methyl]-2-methoxy-benzamide. A review of the literature revealed that several analytical methods have been described for the determination of sulpiride in pharmaceuticals or biological fluids, including spectrophotometric [34–37], fluorimetric [38], chromatographic [39–44], electrophoretic [45-47], voltammetric [48] and chemiluminometric [49]; however, the methods proposed for the analysis of biological fluids suffer the inconvenience of time-consuming procedures and expensive instrumentation.

Most of the reported methods (except spectrophotometric methods) are either not appropriately sensitive or tedious and utilized expensive instruments that are not available in most quality control laboratories and the procedures are not simple to perform. Therefore the aim of the present work is to develop a simple, accurate, sensitive and low-cost spectrophotometric method for the quantification of three antipsychotic drugs, namely aripiprazole, clozapine and sulpiride. The proposed methods are based on the ability of the studied drugs to form stable ion-pair complexes with BPB and BTB.

MATERIALS AND METHODS

Apparatus

All the absorbance spectral measurements were made using spectroscan 80 D double-beam UV/Visible spectrophotometer (Biotech Engineering Ltd. (UK), with wavelength range 190 nm ~ 1100 nm, spectral bandwidth 2.0 nm, with matched quartz cells. The pH values of buffer solutions were measured using Jenway instrument pH-meter (combined electrode).

Reagents and solutions

All of the chemicals used were of analytical or pharmaceutical grade and used without further purification. Double distilled de-ionized water was used to prepare all solutions.

  1. A 1×10- of bromophenol blue and bromothymol blue (Aldrich Co., Ltd., Gillingham-Dorst, Germany), were prepared by dissolving 66.998 mg and 54.022 mg from each dye in 2 ml methanol then, add 20 ml distilled water and diluted to 100 ml in a calibrated flask with distilled water to the mark.
  2. Series of buffer solutions of KCl-HCl (pH 1.0-2.2), NaOAc-HCl (1.99-4.92) and NaOAc-AcOH (3.4-5.6) pH were prepared by standard methods.
  3. Pharmaceutical grade of ARP, CLP and SUP certified to be 99.85% pure was obtained as gift was kindly supplied from Egyptian International Pharmaceutical Industries Company (EIPICo), Egypt. Stock solutions of pure ARP, CLP and SUP were prepared separately by dissolving accurately weighed 10 mg of each drug in 1.0 ml of concentrated sulphuric acid and finally the volume was made up to 100 ml with distilled water (100 μg/ml).

General recommended procedures

Procedures for calibration curves

Into a series of separated funnels, accurately measured aliquots of ARP, CLP and SUP, in the concentration range shown in (Table 1) were pitted out. A volume of 2.0 ml of 5×10- BPB or BTB was added. Then, 2.0 ml of the optimum pH value for each system as recorded in table 1was added and the volume was completed to 10 ml with distilled water. The ion-pairs were extracted with 10 ml of dichloromethane by shaking for 2.0 min and then, the combined dichloromethane extracts were dried over anhydrous sodium sulphate. The absorbance of colored ion-pair complexes were measured within 20 min of extraction against the reagent blank prepared in the same manner except an addition of drugs. In both the methods, a standard curve was prepared by plotting the increasing absorbance values versus concentrations of drug. A linear equation for the standard curve was calculated by linear regression.

Procedure for tablets

At least ten tablets of the drugs were weighted into a small dish, powdered and mixed well. A portion equivalent to 10 mg of ARP, CLP and SUP was weighted and dissolved in distilled water with 1.0 ml of concentrated sulphuric acid, filtered into a 100 ml calibrated flask and diluted to volume with water. Solutions of working range concentration were prepared by proper dilution of this stock solution with water and followed the above procedure for the calibration curve.

Procedures for human serum and urine

The proposed methods were applied to the determination of the studied drugs in spiked urine and serum provided from several healthy volunteers. Spiked urine was 50-fold diluted with distilled water. A 10 ml of serum sample was deproteinzed by adding 5 ml of acetonitrile in a centrifuge for 5 min at 1000 rpm. The supernatant was used to investigate recovery. Add an aliquot of standard aqueous solution of each drug to 1.0 ml of diluted urine or serum. Proceed as described above. A blank value was determined by treating drug-free urine and drug -free serum in the same way. The absolute recovery was determined for each drug by comparing the representative absorbance of the treated urine or serum samples with the absorbance of the standard drug at the same concentration.

(a) ARP


(b) CLP


(c) SUP

Fig. 1: Chemical structure of the studied drugs.


Fig. 2: Absorption spectra of CLP (6 µg/ml) with dyestuff: A-bromophenol blue, B-bromthymol blue


Fig. 3: Proposed reaction pathway for the formation of ion-pair complex between CLP and BTB measured spectrophotometrically at 406 nm

.

Fig. 4: Calibration curves for determination of a-ARP (2–20µg/ml), b-CLP (1–11µg/ml) and c-SUP (1–11µg/ml) using BPB.


Fig. 5: Calibration curves for determination of a-ARP (1–14µg/ml), b-CLP (1–7µg/ml) and c-SUP (1–11µg/ml) using BTB

RESULTS AND DISCUSSION

Spectral characteristics

Absorption spectra of the yellow color of CLP–BPB and BTB ion-pair complex with a maximum absorbance (λmax) at 408 and 406 nm, respectively is shown in (Fig. 2). The colorless blanks have practically negligible absorbance.

Optimization of the reaction conditions

A number of preliminary experiments established optimum conditions necessary for rapid and quantitative formation of colored ion-paired complexes to achieve the maximum stability and sensitivity. Optimum condition was fixed by varying one parameter at a time while keeping other parameter constant and observing its effect on the absorbance.

Effect of buffer type and pH

It was observed that the effective extraction of the complex depends on the type of buffer used and its pH. The effect of pH was studied by extracting the colored complexes in the presence of various buffers such as KCl–HCl (pH 1.0-2.2), NaOAc–HCl (pH 1.99-4.92) and NaOAc–AcOH (pH 3.6-5.6). It is evident that the maximum color intensity and maximum absorbance were found in NaOAc_HCl buffer. It is evident that the absorbance of ion pair complex with BPB was found to be maximal at pH 3, while with BTB the absorbance was maximal at pH 3 for ARP and pH 4.4 for both CLP and SUP. Buffer volume was determined by applying the same experiment and variation the volume regularly (0.5-4.0 ml). The higher absorbance value obtained at using 2.0 ml of buffer solutions.

Effect of reagent concentration

The ARP, CLP and SUP concentrations were kept constant, while the concentrations of BPB or BTB was varied from 0.5–4.0 ml of 5×10-3M. The results showed that the absorbance of the extracted ion-pairs increased by increasing the BPB or BTB concentrations till 2.0 ml. After this volume, the absorbance remains constant by increasing the volume of the reagents. So an excess of reagents has no effect on the determination of the drugs.

Table 1: Analytical parameters and optical characteristics of the studied drugs

Parameters

BPB

BTB

ARP

CLP

SUP

ARP

CLP

SUP

lmax (nm)

408

408

408

406

406

406

pH

3

3

3

3

4.4

4.4

Beer’s law limit, µg/ml

2–20

1–11

1–11

1–14

1–7

1–11

Molar absorptivity,
l mol-1 cm-1

2.4×104

3.9×104

4.01×104

4.10×104

6.17×104

4.30×105

Sandell’s sensitivity,
Ng/cm2

10.8

8.3

8.5

10.7

5.2

7.9

Correlation coefficient (r)

0.9996

0.9992

0.9999

0.9991

0.9993

0.9998

Linear regression equation*

 

 

 

 

 

 

Intercept (a)

0.0641

0.1680

0.1440

0.0410

0.2511

0.1371

Slope (b)

0.0506

0.1030

0.1058

0.0839

0.1587

0.1128

S y/x

0.0082

0.0159

0.0037

0.0179

0.0127

0.0071

S. D. of slope (Sb)

6.7×10-3

2×10-3

4.8×10-3

1.8×10-3

2.9×10-3

9.1×10-3

S. D. of intercept (Sa)

0.020

0.025

0.006

0.029

0.024

0.011

LOD, µg/ml

0.2503

0.1230

0.1212

0.1359

0.0813

0.1134

LOQ, µg/ml

0.8338

0.4098

0.4036

0.4528

0.2714

0.3778

*A= a+ b C, where A is the absorbance and C is the concentration of drug in µg/ml.

Table 2: Evaluation of day accuracy and precision of the proposed methods

Method Drugs

Drug

taken,

µg/ml

Drug

found,

µg/ml

 Recoverya, %

RSDb,

%

RE,

%
BPB ARP 1.6 1.59 100.002 0.420 -0.625
4.0 3.99 99.999 0.207 -0.250
8.0 7.9 99.998 0.172 -1.250
CLP 0.8 0.79 99.999 0.428 -1.250
1.6 1.59 99.991 0.264 -0.625
2.4 2.39 99.999 0.384 -0.416
SUP 0.8 0.79 98.999 0.264 -1.250
2.4 2.39 99.998 0.351 -0.416
3.2 3.19 99.989 0.493 -0.312
BTB ARP 0.8 0.799 99.996 0.351 -0.125
3.2 3.199 99.999 0.405 -0.031
1.0 0.999 99.991 0.258 -0.100
CLP 0.8 0.799 99.999 0.496 -0.125
1.6 1.599 99.999 0.237 -0.062
2.4 2.399 99.997 0.364 -0.041
SUP 0.8 0.799 99.999 0.799 -0.125
2.4 2.401 100.002 0.223 0.041
4.0 3.991 99.999 0.141 -0.225

aMean value of five determinations, bRE: Relative error.

Table 3: Recovery of the studied drugs in pharmaceutical formulations using the proposed methods

Method Drugs

Drug

taken,

µg/ml

Drug

formulation

Drug

found,

µg/ml

 Recoverya, %

RSD,

%

REb,

%
BPB ARP 1.6

Aripiprex

10mg/tablet

 1.599 99.999 1.028 -0.062
4.0 3.999 99.997 0.544 -0.025
8.0 7.999 100.009 0.326 -0.012
CLP 0.8

Clozapex,

100mg/tablet

 0.801 100.001 0.844 0.125
1.6 1.599 99.999 0.821 -0.062
2.4 2.399 97.999 0.781 -0.041
SUP 0.8

Dogmatil,

200mg/tablet

 0.799 99.992 0.524 -0.125
2.4 2.399 101.010 0.906 -0.041
3.2 3.199 99.999 0.993 -0.031
BTB ARP 0.8

Aripiprex

10mg/tablet

 0.799 99.997 0.975 -0.125
3.2 3.199 99.989 0.854 -0.031
4.0 3.999 101.074 0.749 -0.025
CLP 0.8

Clozapex,

100mg/tablet

 0.799 99.999 0.834 -0.125
1.6 1.599 100.256 0.473 -0.062
2.4 2.399 99.998 0.187 -0.041
SUP 0.8

Dogmatil,

200mg/tablet

 0.799 101.003 0.830 -0.125
2.4 2.399 99. 981 1.063 -0.041
3.2 3.201 100.002 0.993 0.031

aMean value of five determinations, bRE: Relative error.

Table 4: Recovery of the studied drugs from spiked human urine

Method Drugs

Drug added,

µg/ml

Drug

found,

µg/ml

Recovery a,

%

RSD,

%

REb,

%
BPB ARP 1.6 1.599 99.999 1.046 -0.062
2.0 1.999 100.010 0.857 -0.050
1.0 1.001 98.996 0.399 0.100
CLP 0.8 0.799 101.209 0.525 -0.125
1.6 1.599 99.994 1.097 -0.062
2.4 2.399 102.341 1.030 -0.041
SUP 0.8 0.799 99.999 1.056 -0.125
1.6 1.599 99.993 1.021 -0.062
4.0 3.990 101.008 0.615 -0.250
BTB ARP 0.8 0.799 99.999 0.726 -0.125
3.2 3.199 98.750 0.416 -0.031
4.0 3.999 100.896 1.052 -0.025
CLP 0.8 0.799 102.700 0.958 -0.125
1.6 1.601 100.001 0.733 0.062
2.4 2.399 99.999 0.992 -0.041
SUP 0.8 0.799 100.003 0.976 -0.125
2.4 2.399 99.996 0.726 -0.041
3.2 3.199 99.991 0.761 -0.031

aMean value of five determinations, bRE: Relative error.

Choice of organic solvents

Different organic solvents as dichloromethane, carbon tetrachloride, chloroform and ether were tested as extractive solvents for the proposed method. Dichloromethane was preferred to other solvents for its selective and obtained the highest absorbance with dichloromethane.

It was also observed that only one extraction was adequate to achieve a quantitative recovery of the complexes and the shortest time to reach the equilibrium between both phases.

Shaking time of 0.5-5 min provided constant absorbance and hence, 2.0 min was selected as the optimum shaking time.

Reaction mechanism

Clozapine forms ion-pair complex with BPB or BTB dye, since it contains the tertiary amino group which is protonated. In the ring of 1H-[1, 4] diazepine, protonation is very difficult due to resonance and steric effects.

Therefore, the only site in CLP vulnerable for protonation is the nitrogen bonded to electron donating methyl group in the piperazine ring [50] and finally the protonated CLP forms ion-pair with the BPB or BTB dye. The suggested reaction pathway for the reaction product of CLP - BTB ion–pair complex formation for example, is given in Fig. 3.

Table 5: Recovery of the studied drugs from spiked human serum

Method Drugs

Drug added,

µg/ml

Drug

found, µg/ml

Recovery a,

%

RSD,

%

REb,

%
BPB ARP 1.6 1.599 99.999 1.046 -0.062
4.0 4.001 100.003 0.857 0.025
8.0 7.999 101.023 0.399 -0.125
CLP 0.8 0.799 102.198 0.525 -0.125
1.6 1.599 99.999 1.097 -0.062
2.4 2.399 100.006 1.030 -0.041
SUP 0.8 0.799 99.991 1.056 -0.125
2.4 2.401 99.999 1.021 0.041
3.2 3.199 99.986 0.615 -0.031
BTB ARP 0.8 0.799 99.994 1.173 -0.125
3.2 3.199 100.488 1.098 -0.031
4.0 3.999 98.986 0.996 -0.025
CLP 0.8 0.799 101.023 1.002 -0.125
1.6 1.599 102.344 0.886 -0.062
2.4 2.399 100.010 0.344 -0.041
SUP 0.8 0.799 99.816 0.548 -0.125
2.4 2.399 99.999 0.829 -0.041
3.2 3.201 102.001 0.790 0.031

aMean value of five determinations, bRE: Relative error.

Effect of temperature on the colored complexes

The effect of temperature on colored complexes was studied over the range 20-35 °C. It was found that the absorbance of the ion pair complex was constant up to 30 °C. At higher temperatures, the drug concentration was found to increase due to the volatile nature of the dichloromethane. Therefore, the temperature chosen was 30 °C as the best temperature for micro-determination of the drugs under study in pure and pharmaceutical formulations.

Effect of shaking time for extraction

Shaking time ranging from 0.5-4.0 min was tested to ascertain the extraction of the complex. Maximum and constant absorbance value was obtained when extracted after 1.5 min shaking. Therefore, shaking time of 2.0 min was maintained throughout the experiment.

Composition of the ion-pair complexes

In order to establish the molar ratio between ARP, CLP and SUP on one side and BPB or BTB reagent used on the other, Job’s method of continuous variation was applied [51]. In this method, 5×10-3 M solutions of drugs and reagent were mixed in varying volume ratios in such a way that the total volume of each mixture was the same. The absorbance of each solution was measured and plotted against the mole fraction of the drug. This procedure showed that a (1: 1) complex was formed through the electrostatic attraction between the positively charged drug, D+ ions and negatively charged reagent, R-, ions. The extraction equilibrium can be represented as follows:

D+(aq) + R-(aq) ↔ D+R-(aq) ↔ D+R-(org)

Where D+ and R- represent the protonated drug and the anion of the reagent, respectively, and the subscripts “aq” and “org” refer to the aqueous and organic phases, respectively.

Quantification

Under the optimum conditions described above, the calibration graphs were constructed for the investigated drugs. The molar absorptivity, Sandellʼs sensitivity, concentration range, regression equation and correlation coefficient for each drug are tabulated in (Table 1). A linear relationship was found between the absorbance at λmax. Regression analysis of Beer’s law plotted at λmax reveals a good correlation (Figs. 4, 5). The graphs showed a negligible intercept, which was calculated by the least-squares method’s regression equation, A = a + bC (where A is the absorbance of 1.0 cm layer, b is the slope, a is the intercept and C is the concentration of the measured solution in µg/ml. The high molar absorptivities of the resulting colored complexes indicated high sensitivity of the method (2.40×104 – 4.30×105). The SUP - BTB method was found to be the most sensitive of all these methods with high ε value. The limit of detection (LOD) and limit of quantitation (LOQ) are calculated according to ICH guidelines [53]. The results are as shown in (Table 1).

Accuracy and precision

In order to determine the accuracy and precision of the recommended procedure five replicate determinations at three different concentrations of the studied drugs were carried out. Precision and accuracy were based on the calculated relative standard deviation (RSD, %) and relative error (RE, %) of the found concentration compared to the theoretical one, respectively and indicate that the proposed method is highly accurate and reproducible (Table 2).

Analysis of dosage forms

To evaluate the validity and reproducibility of the methods, known amounts of the

ARP, CLP and SUP drugs were added to the previously analyzed pharmaceutical preparations and the mixtures were analyzed by the proposed methods. The percent recoveries are given in table 3. Interference studies revealed that the common excipients and other additives such as lactose, starch, gelatin, talc and magnesium trisilicate, that are usually present in the tablet dosage forms did not interfere at their regularly added levels.

Analysis of biological fluids

The high sensitivity of the proposed method, also allowed the in vitro determination of ARP, CLP and SUP in spiked human serum and urine samples. Thus the proposed method is sufficient for routine estimation of the drugs in human serum and urine. A prior extraction step by the same organic solvent was adopted before application of the method. The results obtained are satisfactorily accurate and precise (Tables 4, 5).

CONCLUSION

A significant advantage of the extractive spectrophotometric methods is that it can be applied for the determination of individual compounds in a multicomponent mixture. The proposed methods make use of simple reagent which an ordinary analytical laboratory can afford, and the procedures do not involve any critical reaction conditions or tedious sample preparation. The methods are highly reliable owing to the stability of the ion-pair complex and acid / base forms of the dye, which are ultimately measured. Moreover, the methods are accurate, reproducible, adequately sensitive and free from interference caused by the excipients expected to be present in tablets. The methods were successfully applied to the spiked human serum and urine.

CONFLICT OF INTERESTS

Declared None

REFERENCES

  1. Li KY, Li H D. A typical antipsychotic of pharmacokinetics of quetiapine. Chin J Clin Pharmacol 2002;18:227-31.
  2. Ereshefsky L. Pharmacokinetics and drug interactions: update for new antipsychotics. J Clin Psychiatry1996;57:12-25.
  3. Davis PC, Wong J, Gefvert O. Analysis and pharmacokinetics of quetiapine and two metabolites in human plasma using reversed-phase HPLC with ultraviolet and electrochemical detection. J Pharm Biomed Anal 1999;20:271-82.
  4. Raggi MA. Therapeutic drug monitoring: chemical-clinical correlations of atypical antipsychotic drugs. Curr Med Chem 2002;14:1397-449.
  5. Yoshimura R, Ueda N, Nakamura J. Possible relationship between combined plasma concentrations of risperidone plus 9-hydroxyrisperidone and extra pyramidal symptoms. Preliminary study. Neuropsychobiol 2001;44:129-33.
  6. Gerlach J. Improving outcome in schizophrenia: the potential importance of EPS and neuroleptic dysphoria. Ann Clin Psychiatry 2002;14:47-57.
  7. Weigmann H, Hartter S, Maehrlein S. Simultaneous determination of olanzapine, clozapine and demethylated metabolites in serum by on-line column-switching high-performance liquid chromatography. J Chromatogr B Biomed Sci Appl 2001;759:63-71.
  8. Thakkar RS, Saravaia HT, Ambasana MA, Kaila O, Shah AK. A chromatographic determination of aripiprazole using HPLC and UPLC: A comparative validation study. Indian J Pharm Sci 2011;73:439-42.
  9. Babu GR, Rao JS, Kumar KS, Reddy PJ. Stability indicating liquid chromatographic method for aripiprazole. Asian J Pharm Anal 2011;1:3-7.
  10. Akamine Y, Yasui-Furukori N, Kojima M, Inoue Y, Uno T. A sensitive column-switching HPLC method for aripiprazole and dehydroaripiprazole and its application to human pharmacokinetic studies. J Sep Sci 2010;33:3292-8.
  11. Lancelin F, Djebrani K, Tabaouti K, Kraoul L, Brovedani S, Paubel P, et al. Development and validation of a high-performance liquid chromatography method using diode array detection for the simultaneous quantification of aripiprazole and dehydro-aripiprazole in human plasma. J Chromatogr B Anal Technol Biomed Life Sci 2008;867:15-9.
  12. Koduri SV, Buchireddy SR, Madhusudhan G, Mukkanti K, Srinivasulu P. Stress degradation studies on aripiprazole and development of a validated stability indicating LC method. Chromatographia 2008;68:635-40.
  13. Zuo X-c, Wang F, Xu P, Zhu R-h, Li H-de. LC-ESI-MS for rapid and sensitive determination of aripiprazole in human plasma. Chromatographia 2006;64:387-91.
  14. Shimokawa Y, Akiyama H, Kashiyama E, Koga T, Miyamoto G. High performance liquid chromatographic methods for the determination of aripiprazole with ultraviolet detection in rat plasma and brain: application to the pharmacokinetic study. J Chromatogr B Anal Technol Biomed Life Sci 2005;821:8-14.
  15. Ashour S, Kattan N. Sensitive method for the quantitative determination of risperidone in tablet dosage form by high-performance liquid chromatography using chlordiazepoxide as internal standard. Int J Biomed Sci 2013;9:91-7.
  16. Sandeep K, Induri M, Sudhakar M. Validated spectrophotometric quantification of aripiprazole in pharmaceutical formulations by using multivariate technique. Adv Pharm Bull 2013;3:469-72.
  17. Jain R, Kashaw SK, Jain R, Mishra P, Kohli DV. Visible spectrophotometric method for the determination of aripiprazole in tablets. Indian J Pharm Sci 2011;73:74-6.
  18. Kalaichelvi R, Thangabalan B, Rao DS, Jayachandran E. UV Spectrophotometric determination of aripiprazole in bulk and pharmaceutical formulation. E-J Chem 2009;6:S87-S90.
  19. Mosier KE, Song JX, McKay G, Hubbard JW, Fang J. Determination of clozapine, and its metabolites, N-desmethylclozapine and clozapine N-oxide in dog plasma using high-performance liquid chromatography. J Chromatogr B-Anal Technol Biomed Life Sci 2003;783:377-82.
  20. Liu YY, van Troostwijk LJAED, Guchelaar HJ. Simultaneous determination of clozapine, norclozapine and clozapine-N-oxide in human plasma by high-performance liquid chromatography with ultraviolet detection. Biomed Chromatogr 2001;15:280-6.
  21. Hariharan U, Hariharan M, Naickar JS, Tandon R. Determination of clozapine and its two major metabolites in human serum by liquid chromatography using ultraviolet detection. J Liq Chromatogr Related Technol 1996;19:2409-17.
  22. Aravagiri M, Marder SR. Simultaneous determination of clozapine and its N-desmethyl and N-oxide metabolites in plasma by liquid chromatography/electrospray tandem mass spectrometry and its application to plasma level monitoring in schizophrenic patients. J Pharm Biomed Anal 2001;26:301-11.
  23. Vardakou I, Dona A, Pistos C, Alevisopoulos G, Athanaselis S, Maravelias C, et al. Validated GC/MS method for the simultaneous determination of clozapine and norclozapine in human plasma. Application in psychiatric patients under clozapine treatment. J Chromatogr B 2010;878:2327-32.
  24. Zhou DW, Li FM. Determination of free clozapine concentration in serum and plasma by capillary electrophoresis-frontal analysis. Acta Chim Sinica 2004;62:1256-9.
  25. Jin WR, Xu Q, Li W. Determination of clozapine by capillary zone electrophoresis following end-column amperometric detection with simplified capillary/electrode alignment. Electrophor 2000;21:1415-20.
  26. Mashhadizadeh MH, Afshar E. Electrochemical investigation of clozapine at TiO2 nanoparticles modified carbon paste electrode and simultaneous adsorptive voltammetric determination of two antipsychotic drugs. Electrochim Acta 2013;87:816-23.
  27. Arvand M, Shiraz MG. Voltammetric determination of clozapine in pharmaceutical formulations and biological fluids using an in situ surfactant-modified carbon ionic liquid electrode. Electroanal 2012;24:683-90.
  28. Ozkan SA, Senturk Z. Study on the determination of clozapine by voltammetry. Analusis 1996;24:73-5.
  29. Mohamed AA, Al-Ghannam SM. Spectrophotometric determination of clozapine based on its oxidation with bromate in a micellar medium. Farmacol 2004;59:907-11.
  30. Sastry CSP, Rekha TV, Satyanarayana A. Spectrophotometric determination of clozapine in pharmaceuticals. Mikrochim Acta 1998;128:201-5.
  31. Gfeller GC, Frey G. Investigation and automation of the fluorometric ion pair method for the determination of several amines in low dosage pharmaceuticals. Fresenius J Anal Chem 1978;291:332-6.
  32. Liang F, Terry AV, Bartlett MG. Determination of aripiprazole in rat plasma and brain using ultra-performance liquid chromatography/electrospray ionization tandem mass spectrometry. Biomed Chromatogr 2012;26:1325-32.
  33. Bhanotu B, Srinath P, Kedarnath J. Development, Estimation and validation of aripiprazole in bulk and its pharmaceutical formulation by HPLC method. Int J Chem Tech Res 2012;4:124-8.
  34. El Walily AFM, El Gindy A, Bedair MF. Application of first-derivative UV spectrophotometry, TLC-densitometry and liquid chromatography for the simultaneous determination of mebeverine and sulpiride. J Pharm Biomed Anal 1999;21:535-48.
  35. Attia KA, Abou-Seada HH, Nassar MW. Colorimetric determination of certain dopamine antagonists in pharmaceutical preparation. Egypt J Biomed Sci 2003;12:199-208.
  36. Radwan MF. Spectrophotometric determination of three benzamide antipsychotic in pure andpharmaceutical preparation using charge transfer complex. Egypt J Biomed Sci 2003;11:1-12.
  37. Zayed S. Simultaneous determination of mebeverine hydrochloride and sulpiride using the firstderivativesof ratio spectra and chemometric methods. Anal Sci 2005;8:985-9.
  38. Buna M, Aaron JJ, Prognon P, Mahuzier G. Effects of pH and solvent on the fluorescence properties of biomedically important benzamides. Application to determination in drugs and inhuman urine. Analyst 1996;121:1551-6.
  39. Chiba R, Soukura M, Tatsuta S. Solid-phase extraction and determination of sulpiride in human serum by high performance liquid chromatography using electrogenerated chemiluminescence detection. Anal Lett 2011;44:1559-69.
  40. Naguib IA, Abdelkawy M. Development and validation of stability indicating HPLC and HPTLC methods for determination of sulpiride and mebeverine hydrochloride in combination. Eur J Med Chem 2010;45:3719-25.
  41. Tokunaga H, Kudo K, Jitsufuchi N, Ohtsuka Y, Imamura T. Sensitive determination of sulpiride in human plasma by high-performance liquid chromatography. J Chromatogr B 1997;691:203-7.
  42. Huang MC, Ho HO, Yeh GC, Ke WT, Lin LC, Hsu TM, et al. Development of a high-performance liquid chromatographic method for bioanalytical applications with sulpiride. J Chromatogr B 2001;763:157-63.
  43. Chiba R, Ogasawara A, Kubo T, Yamazaki H, Umino M, Ishizuka Y. Direct determination of benzamides in serum by column-switching high-performance liquid chromatography. Anal Sci 2003;19:785-9.
  44. Kirchherr H, Kuehn-Velten WN. Quantitative determination of forty-eight antidepresants and antipsychotics in human serum by HPLC tandem mass spectrometry: a multi-level, single-sample approach. J Chromatogr B 2006;843:100-13.
  45. Xu X, Stewart JT. Chiral analysis of selected dopamine receptor antagonists in serum using capillary electrophoresis with cyclodextrin additives. J Pharm Biomed Anal 2000;23:735-43.
  46. Liu JF, Cao WD, Qiu HB, Sun XH, Yang XR, Wang EK. Determination of sulpirideby capillary electrophoresis with end-column electrogenerated chemiluminescence detection. Clin Chem 2002;48:1049-58.
  47. Li J, Zhao F, Ju H. Simultaneous electroluminescence determination of sulpiride and tiapride by capillary electrophoresis with cyclodextrin additives. J Chromatogr B 2006;835:84-9.
  48. Farghaly O. Adsortive stripping voltammetric determination of the antidepressant drug sulpiride. J Pharm Biomed Anal 2000;23:783-91.
  49. Aly FA, Alarfaj NA, Alwarthan AA. Flow injection chemiluminometric analysis of some benzamides by their sensitizing effect on the cerium-sulphite reaction. Talanta 2001;54:714-25.
  50. Harikrishna K, Nagaralli BS, Seetharamappa J. Extractive spectrophotometric determination of sildenafil citrate (viagra) in pure and pharmaceutical formulations. J Food Drug Anal 2008;16:11-7.
  51. Job P. Formation and stability of inorganic complexes in solution. Ann Chim 1928;9:113–23.
  52. InczedyJ. Analytical application of complex equilibria. Ed., Budapest: Wiley; 1976.
  53. Miller JN, Miller JC. Statistics and chemometrics for analytical chemistry. 5th ed. Prentice Hall, England; 2005.