LIST OF PAPERS PUBLICATIONS OF THE AUTHOR

1. S. M. Mallegowda, H. D. Revanasiddappa, H. N. Deepakumari, Spectrophotometric

methods for the determination of nimodipine in pure and in pharmaceutical

Preparations, Jordan Journal of Chemistry 2011, 6 , 413.

2. S. M. Mallegowda, H. N. Deepakumari and H. D. Revanasiddappa,

Spectrophotometric determination of valsartan using p-Chloranilic acid as π-Acceptor

in Pure and in Dosage Forms, Journal of Applied Pharmaceutical Science 2013, 3

113.

3. S. M. Mallegowda, H. D. Revanasiddappa, development and validation of indirect

visible spectrophotometric method for doxepine and dothiepine in pure and the tablet

dosage forms, Jordon Journal of Pharmaceutical Sciences 2013, 6, 1.

PUBLICATIONS NOT FORMING THE PART OF THE THESIS

1. H. N. Deepakumari, S. M. Mallegowda, K. B. Vinay, H. D. Revanasiddappa,

Simple and extraction free spectrophotometric methods for risperidone in pure

form and in dosage forms, Chemical Industry & Chemical Engineering

Quarterly 2013, 19, 121.

2. H. D. Revanasiddappa, H. N. Deepakumari, S. M. Mallegowda, development

and validation of indirect spectrophotometric methods for lamotrigine in pure

and the tablet dosage forms, Analele Univ. Bucuresti. Chimie 2011, 20, 49.

3. H. D. Revanasiddappa, H. N. Deepakumari, S. M. Mallegowda, K. B. Vinay,

Facile spectrophotometric determination of nimodipine and nitrazapam in

pharmaceutical preparations, Analele Univ.Bucuresti.chimie. 2011, 20, 189.

4. H. D. Revanasiddappa, S. M. Mallegowda, H. N .Deeepakumari, K. B. Vinay,

Spectrophotometric determination of nitrazepam and nimodipine in pure and

the tablet dosage forms, Asian Journal of Biochemical and Pharmaceutical

Research, 2011, 1, 70.

5. H. D. Revanasiddappa, H. N. Deepakumari, S. M. Mallegowda, K. B. Vinay,

Development and validation of spectrophotometric methods for nitrazepam,

lamotrigene in pure and the tablet dosage forms, Вестник Московского

Университета 2012, 53, 358.

LIST OF CONFERENCE ATTENDED

1. National conference on “The emerging areas in chemistry NACEAC-2009”,

organized by Department of studies in chemistry, Manasagangotri, university

of Mysore, Mysore India on 31st July and 1

st August 2009.

2. National conference on “Recent trends in analytical techniques” (NCRTAT-

2011) organized by D.R.M .Science college Davanagere, Karnataka, India on

19th

February 2011.

3. National conference on “Recent trends in chemistry (RTC-2011)” organized

by P.E.S. college of Science, Arts and commerce, Mandya, India on 16th

and

17th

September 2011.

Journal of Applied Pharmaceutical Science Vol. 3 (01), pp. 113-116, January, 2013 Available online at http://www.japsonline.com DOI: 10.7324/JAPS.2013.30122 ISSN 2231-3354

Spectrophotometric Determination of Valsartan using p-Chloranilic Acid as π-Acceptor in Pure and in Dosage Forms

S. M. Mallegowda, H. N. Deepakumari and H. D. Revanasiddappa* Department of Chemistry, University of Mysore, Manasagangotri, Mysore-570 006, India.

ARTICLE INFO

ABSTRACT

Article history: Received on: 10/12/2012 Revised on: 29/12/2012 Accepted on: 10/01/2013 Available online: 28/01/2013

The aim of this work was to develop a simple, sensitive and extraction free spectrophotometric method for the quantitative estimation of valsartan in both pure and in pharmaceutical preparations. The developed method is based on the charge transfer complexation reaction between valsartan (VRT) as n- electron donor and p-chloranilic acid (p-CA) as π-acceptor. VRT reacts with p-CA in methanol to produce a bright pink colored complex with a maximum absorption at 530 nm. Beer's law was obeyed in the concentration range of 5-50 µg/mL. The linear regression equation of the calibration graph is A = 0.0081+0.0092C with a regression coefficient (r) of 0.9976 (n = 7). The molar absorptivity is calculated to be 2.06 × 103 L mol–1 cm–1 and the Sandell sensitivity is 0.1025 μg cm–2. The limits of detection (LOD) and quantitation (LOQ) values are calculated according to ICH guidelines. The method developed is successfully applied to the determination of VRT in dosage forms.

Key words: Charge-transfer, spectrophotometry, valsartan, dosage forms.

INTRODUCTION

Chemically valsartan (VRT) is (S)-3-methyl-2-(N-{[2'-(2H -1 ,2 ,3 ,4-tetrazol-5-yl) biphenyl -4-yl] methyl} pentanamido) butanoic acid (Fig. 1) has an empirical formula of C24H29N5O3 is a potent angiotensin receptor π antagonist with particularly high affinity for the type I (AT1) angiotensin receptor. By blocking the action of angiotensin, valsartan dilates blood vessels and reduces blood pressure (Marks, 2007).

Fig. 1: Structure of valsartan.

Several analytical methods have been reported for the determination of VRT in combination with other anti‐ hypertensive

agents in pharmaceutical formulations. Very few methods have been appeared in the literature for the determination of valsartan individually, those include high performance liquid chromate-graphy (Zarghi et al., 2008, Li et al., 2000, Gonzales et al., 2000, Francotte et al.,1996, Parambi et al., 2011), RP-HPLC (Vinzuda et al., 2010, Manoranjani et al., 2011, Patnaik et al., 2011), UV- and second derivative-spectrophotometric and LC method (Tatar et al., 2002), HPLC with UV Detection (Piao et al.,2008) and uv-spectrophotometry (Gupta et al., 2010) and spectrofluorimetric determination of losartan and valsartan in human urine (Gagigal et al., 2001) and simultaneous determination of valsartan and hydrochlorothiazide in tablets by first-derivative ultraviolet spectrophotometry and LC [Satana et al., 2001]. The VRT is not yet appeared in any pharmacopoeia either alone or in combinations with other drugs.

The purpose of the present work is the development of a simple spectrophotometric method for the determination of VRT in bulk and in pharmaceutical formulations through the formation of charge transfer complexation reaction between valsartan (VRT) as n- electron donor and p-chloranilic acid (p-CA) as π-acceptor.

* Corresponding Author Dr. H. D. Revanasiddappa, Email: hdrevanasiddappa@yahoo.com Tel: +0821 2419669

114 Mallegowda et al. / Journal of Applied Pharmaceutical Science 3 (01); 2013: 113-116

MATERIALS AND METHODS

Apparatus Absorbance measurements for spectrophotometric analysis were performed using a Systronics Model 166 digital spectrophotometer provided with 1-cm matched quartz cells. Reagents and standards Analytical reagent grade chemicals and reagents were used. p-chloranilic acid (0.05 %, w/v) It was freshly prepared by dissolving 0.05 g p-chloranilic acid (Rolex, Mumbai, India) in 100 mL acetone. Standard VRT solution The pure grade VRT, certified to be 99.99% was received from Cipla India Ltd., Mumbai, India, as a gift sample and was used as such. A stock standard solution equivalent to 100 µg/mL of VRT was prepared by dissolving 10 mg of the pure drug in 100 mL methanol. Pharmaceutical formulations of VRT such as VALZAAR [Torrent], DIOVAN [Novartis Pharma] were purchased from local markets. Procedure for calibration graph Accurately measured aliquots of standard solution of VRT (100µg/mL) corresponding to 0.5, 1.0, 1.5, 2.0, …………..5.0 mL were transferred into a series of 10 mL calibrated flasks using a micro burette. To this solution was added 3.5 mL 0.05 % p-CA, then shake well and the contents were diluted to the mark with methanol and mixed well. The absorbance of the bright pink colored complex was measured at 530 nm after 5 min against the reagent blank prepared similarly, but without drug content. Procedure for dosage forms In order to determine the contents of VRT in commercial dosage forms (label claim: 40 and 80 mg tablet), the contents of ten tablets were weighed accurately and ground into a fine powder. An amount of powder equivalent to 10 mg of VRT was accurately weighed and transferred into two separate 100mL calibrated flasks and 30 mL of methanol was added. The content was shaken for about 30 min; the volume was diluted to the mark with methanol and mixed well and filtered using a Whatman no.41 filter paper. The filtrate containing VRT at a concentration 100 µg/mL was subjected to analysis by the procedure described above. RESULTS AND DISCUSSION

Chemistry of the method The method developed involves charge-transfer complex formation between the basic nitrogenous VRT as n-donor and p-chloranilic acid (p-CA) in polar solvent as π-acceptor. The formed charge transfer (C-T) complex was subsequently dissociated into

radical anions, which are colored species. The absorbance of the colored complex was measured at 530 nm, and it was observed as shown in the following equation:

Thus, p-CA was used as reagent in the proposed method

for the estimation of VRT. The possible reaction pathway for VRT - p-CA complex is shown in scheme1.

Scheme. 1: Proposed reaction scheme. The influence of various factors on the formation of charge-transfer complex viz., reagent concentration, reaction time and stability of the colored complex were studied and maintained throughout the experiment to determine the quantity of VRT in bulk and in dosage forms. Highest absorbance values were obtained with 3.5 mL of 0.05 % p-CA, which remained unaffected by further addition of p-CA. Thus, 3.5 mL of the reagent was used for the determination in the developed method. It was observed that the formed complex was stabilized within 5 min in the

Mallegowda / Journal of Applied Pharmaceutical Science 3 (01); 2013: 113-116 115

developed method. The color of the C-T complex remained stable at room temperature (27 ± 3 ºC) for a period of 2 h. Method validation The developed method was validated in terms of linearity and sensitivity, limit of detection (LOD) and limit of quantitation (LOQ), precision, accuracy, selectivity and recovery following the ICH guidelines (International Conference On Harmonization, guidelines). Linearity, sensitivity, limits of detection and quantification A calibration graph was constructed using a standard solution of VRT at respective wavelength and concentration of VRT in the ranges is given in Table 1. Under the optimum experimental conditions, a linear relationship existed between the absorbance and concentration of the drugs. The regression analysis of the calibration curve using the method of least-squares was made to calculate the slope (b), intercept (a) and correlation co-efficient (r) values are presented in Table 1. The optical characteristics such as absorption maxima, Beer’s law limit, molar absorptivity and Sandell’s sensitivity value of the method are also given in Table 1. Table. 1: Analytical and regression parameters of the proposed methods.

*y=a+bx, where c is the concentration of VRT in µg/mL and y is the absorbance at the respective λmax., Sa is the standard deviation of the intercept, Sb is the standard deviation of the slope. Intra and inter-day precision and accuracy The accuracy of an analytical method expresses the closeness between the proposed method and reference method. Further, the accuracy and precision (intra-day) of the proposed method were evaluated by replicate analysis (n=5) of calibration standards at three different concentration levels in the same day. Precision and accuracy of inter-day were measured by performing the calibration standards at cited three concentrations on five consecutive days. Both precision and accuracy were based on the calculated percent relative standard deviation (RSD, %) and percent relative error (RE, %) values, respectively for

thedeveloped method were found to be satisfactory. The analytical results obtained from this investigation are summarized in Table 2. Application to analysis of pharmaceutical samples The validity of the proposed method was ascertained by the statistical comparison of the results obtained by a reference method (Patnaik et al., 2011) with the proposed method by applying Student’s t-test for accuracy and F- test for precision in some commercial formulations. The results were compared with those of the reported method. Statistical analysis of the results using the Student’s-t and F-tests revealed no significant difference between the reported method at the 95% confidence level with respect to accuracy and precision (Table 3). Table. 3: Results of determination of VRT in tablets and statistical comparison with the reference method.

Tablet brand Name

Nominal amount mg per

tablet

Found*(% of nominal amount ± SD)

Reference method Proposed method

VALZAARa 40 100.88±0.48 99.62±0.704

t = 1.68, F = 2.15

DIOVANb 80 100.80 ±0.46 100.38±0.854

t = 0.51, F = 3.45

Marketed by: a. [Torrent], b. [Novartis Pharma] *Mean value of five determinations Tabulated t and F-values at 95 % confidence level are 2.77 and 6.39, respectively. Recovery study To test the applicability of the proposed method, recovery experiments were carried out by standard addition method. In this study, pre-analyzed tablet powder was spiked with pure drug at three different concentrations and the total was found by the proposed method. Each determination was repeated three times. The recovery of the pure drug added was quantitative and revealed that co-formulated substances did not interfere in the determination. The results of recovery study are compiled in Table 4. Table. 4: Results of recovery experiments via the standard addition technique.

Tablet brand name

VRT mg per tablet

Pure VRT added, µg/mL

Total found µg/mL

Pure VRT recovered*

% ± SD

VALZAARa 40

20 20.02 100.22 ± 0.77 30 29.83 99.16 ± 0.88 40 39.85 99.49 ±0.46

DIOVANb 80

20 19.91 99.13±0.77 30 30.20 100.98±0.85 40 40.31 101.03±0.95

a. * Mean value of three determinations. b. Marketed by: a. [Torrent], b. [Novartis Pharma]

Parameter Method λmax nm 530 Beer’s law range (µg/mL ) 5.0 – 50 Molar absorptivity (ε), (L mol-1cm-1) 2.06 x 10 3 Sandell sensitivity ( µg cm-2) 0.1025 Intercept (a) 0.0081 Slope (b) 0.0092 Correlation coefficient (r) 0.9976 Sa 0.0170 Sb 0.0004 LOQ ( µg/mL ) 1.1169 LOD ( µg/mL ) 0.3686

Table. 2: Evaluation of intra-day and inter-day accuracy and precision results. VRT taken,

μg/mL intra-daya inter-dayb

VRT foundc, μg/mL Precisiond Accuracy e VRT foundc, μg/mL Precisiond Accuracy e

Method 10 9.93±0.10 1.02 0.66 9.97±0.25 2.50 0.21 20 19.97±0.06 0.30 0.17 19.84±0.13 0.63 0.81 40 40.07±0.33 0.83 0.18 39.79±0.29 0.74 0.52

Mean value of five determinations, b. Mean value of five determinations, c. Mean value of three determinations, d. Relative standard deviation (%), e. Bias%: (found-taken/taken)×100.

116 Mallegowda et al. / Journal of Applied Pharmaceutical Science 3 (01); 2013: 113-116

CONCLUSION

The proposed method is simple and sensitive and in addition, the method has wider linear dynamic range with good accuracy and precision which could be applied for the determination of valsartan in bulk drug and dosage forms. From the calculated t- and F-values at the 95 % confidence level, it is clear that, the results obtained by the proposed method are in good agreement with those obtained by the reference method. The small values of R.E and R.S.D. indicate the reliability, accuracy and precision of suggested procedure. The results obtained in Table 3 are considered to be of high accuracy and can be successfully applied to the routine assay of valsartan in pharmaceutical formulations. ACKNOWLEDGEMENT

The authors are grateful to Cipla, India Ltd, for the generous supply of pure drug sample. The authors SM and HND are thankful to the University of Mysore, Mysore for providing necessary facilities. REFERENCES

Francotte E., Davatz A., Richert P. Development and validation of chiral high performance liquid chromatographic methods for the quantitation of valsartan and of the tosylate of valinebenzyl ester. J. Chromatogr. 1996; 686: 77–83

Gupta KR., Wadodkar AR., Wadodkar SG. UV-Spectrophotometric methods for estimation of Valsartan in bulk and tablet dosage form. Int. J. Chem Tech Res. 2010; 2: 985-989

Gagigal E., Gonzalez L., Alonso RM., Jimenez RM. Experimental design methodologies to optimise the 1121–113

Gonzales L., Alonso RM., Jimenez RM. A high performance liquid chromatographic method for screeing angiotensin π receptor antagonists in human urine. Chromatographia. 2000; 52: 735–740

International Conference On Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use, ICH Harmonised Tripartite Guideline, Validation of Analytical Procedures: Text and Methodology Q2(R 1), Complementary Guideline on Methodology, dated 06 November 1996, incorporated in November 2005, London.

Li Y., Zhao Z., Chen X., Wang J., Guo J., Xiao F. HPLC determination of valsartan in human plasma. Yaowu Fenxi Zazhi. 2000; 20: 404–406

Marks JW. (2007-02-15). "Valsartan, Diovan". MedicineNet. Retrieved 2010-03-04

Manoranjani M., Bhagyakumar T. RP-HPLC method for the estimation of valsartan in pharmaceutical dosage forms. Int. J. Sci. Innov. Dis. 2011; 1: 101-108

Parambi DGT., Mathew M., Ganesan V. A validated stability indicating HPLC method for the determination of Valsartan in tablet dosage forms. J. Appl. Pharm. Sci. 2011; 1: 97-99

Patnaik A., Shetty M., Sahoo S., Nayak DK., Veliyath SK. A new RP-HPLC method for the determination of valsartan in bulk and its pharmaceutical formulations with it’s stability indicative studies. Pharma Sci. Monit. 2011; 2: 43-53

Piao ZZ., Lee ES., Tran HTT., Lee BJ. Improved Analytical Validation and Pharmacokinetics of Valsartan Using HPLC with UV Detection. Arch. Pharm. Res. 2008; 3: 1055-1059

Satana E., Altinay S., Goger NG., Ozkan SA., Senturk Z. Simultaneous determination of valsartan and hydrochlorothiazide in tablets by first-derivative ultraviolet spectrophotometry and LC. J. Pharm. Biomed. Anal. 2001; 25: 1009–1013

Tatar S., Saglık S. Comparison of UV- and second derivative-spectrophotometric and LC methods for the determination of valsartan in pharmaceutical formulation. J. Pharm. Biomed. Anal. 2002; 30: 371–375

Vinzuda DU., Sailor GU., Sheth NR. RP-HPLC Method for Determination of Valsartan in Tablet Dosage Form. Int. J. Chem Tech Res. 2010; 2: 1461-1467.

Zarghi A., Shafaati A., Foroutan SM., Khoddam A., Madadian B. Rapid quantification of valsartan in human plasma by HPLC using monolithic column. Scientia Pharmaceutica. 2008; 76: 439–450

How to cite this article:

S. M. Mallegowda, H. N. Deepakumari and H. D. Revanasiddappa., Spectrophotometric Determination of Valsartan using p-Chloranilic Acid as π-Acceptor in Pure and in Dosage Forms. J App Pharm Sci. 2013; 3 (01): 113-116.

Jordan Journal of Chemistry Vol. 6 No.4, 2011, pp. 413-422

413  

JJC

Spectrophotometric Methods for the Determination of Nimodipine in Pure and in Pharmaceutical Preparations

Hosakere D. Revanasiddappa*, Shiramahally M. Mallegowda, Hemavathi N. Deepakumari and Kanakapura B. Vinay

Department of Chemistry, University of Mysore, Manasagangothri, Mysore-570 006, India.

Received on March 8, 2011 Accepted on Aug. 11, 2011

Abstract Four simple, sensitive, accurate and rapid visible spectrophotometric methods (A, B, C

and D) have been developed for the estimation of nimodipine in both in pure and pharmaceutical

preparations. They are based on the diazotization of reduced nimodipine with nitrous acid

followed by coupling with acetylacetone (Method A), diphenylamine (Method B), citrazinic acid

(Method C) and chromotropic acid (Method D) to form respective colored azo-dyes and

exhibiting absorption maxima (λmax) at 420, 540, 440 and 520 nm, respectively. The colored azo-

dyes formed are stable for more than 2h. Beer's law was obeyed in the concentration range of 0-

35, 0–25, 0-35 and 0-35 µg/mL for methods A, B, C and D, respectively. The results of the four

analyses have been validated statistically and by recovery studies. The results obtained in the

proposed methods are in good agreements with labeled amounts, when marketed

pharmaceutical preparations are analyzed.

Keywords: Diazotization; Visible spectrophotometry; Nimodipine; Pharmaceutical

preparation.

Introduction Nimodipine (NMD) belongs to the class of pharmacological agents known as a

calcium channel blockers. It is chemically known as isopropyl 2-methoxy ethyl- 1,4-

dihydro-2,6-dimethyl-4-(m-nitrophenyl)-3,5-pyridine dicarboxylate (Figure 1).

Nimodipine is used to treat symptoms resulting from a ruptured blood vessel in the

brain (hemorrhage). It increases blood flow to injured brain tissues. It has been

officially determined by liquid chromatographic method with UV detection[1-2]. Many

chromatographic techniques have been used for the analysis of NMD in biological

fluids include HPLC[3], LC-MS-MS[4], LC-MS[5] and GC with electron capture

detection[6]. These techniques are highly expensive and not affordable for the routine

analysis of dosage forms. Various analytical techniques that have been reported for

this drug in therapeutics include atomic absorption spectrophotometry[7],

polarography[8], spectrofluorimetry[9] and spectrophotometry [10-14].

                                                            * Corresponding author: e-mail: hdrevanasiddappa@yahoo.com ; Tel: +0821 2419669.

414  

Figure 1: Structure of nimodipine

Many of the reported methods are time consuming, requires expensive

experimental set up [7], and polarographic methods are less sensitive [8]. Besides, the

reported spectrophotometric methods are less sensitive [10-14], requires heating

conditions [10] and costly chromogenic reagent [12], (Table 1). Considering these

demerits, there was a need to develop more advantageous spectrophotometric method

for the determination of NMD in bulk sample and in tablets.

Table 1: Comparision of the Performance characteristic of the existing visible

spectrophotometric methods with the proposed methods Sl No Reagent/s used Methodology Linear range,

µg mL-1 and molar

absorptivity, L moL-1 cm-1

Remarks Ref

1. 4-dimethyl amino

benzaldehyde in

methanol

Condensation product was

measured at 580 nm

-

requires

heating

condition

10

2. N-(1-naphthyl)ethylene

diamine dihydrochloride

Colored azo dye was

measured at 550 nm

0-40.0 Less

sensitive

11

3. Paradimethylaminocinn

amaldehyde (PDACA)

Folin Ciocalteu reagent

The absorbance of Schiff

base was measured at

510 nm

The colored complex was

measured at 640 nm

0.5-4.0

5.0-25.0

Uses

costly

reagent

less

sensitive

12

4. β-naphthol Orange-red colored azo

dye measured at 555 nm

0-10.0

(€=7.74×102)

Less

sensitive

13

5. Metol-dichromate

Charge transfer complex

measured at 520 nm

0-70.0

(€=3.15×103)

Requires

strict pH

control

14

415  

The present communication describes four sensitive, rapid, simple, accurate and

cost-effective spectrophotometric methods for the determination of NMD in

pharmaceutical preparation by utilizing the aromatic amino group present in reduced

nimodipine and its ability to diazotize with nitrous acid in acidic medium and the formed

diazonium ion couples with the coupling agents such as diphenylamine in acidic

condition (method B) while acetyl acetone (method A), citrazinic acid (method C) and

chromotropic acid (method D) in basic medium.

Materials and Methods Apparatus

All absorbance measurements were performed using a Systronics Model 166

digital spectrophotometer with 1cm matched quartz cells.

Material and reagents

All chemicals used were of analytical grade. Sodium nitrite, hydrochloric acid

and sodium hydroxide (E. Merck, India Ltd), sulfamic acid (Qualigens), citrazinic acid

(Fluka, Switzerland), chromotropic acid and acetyl acetone (BDH Chemicals, Poole,

England) were used as received. Double distilled water was used for dilution and

preparation of all reagents. Nimodipine pure drug was obtained from Cipla, Ltd.,

Mumbai, India as a gift sample and used as received. Nimodip [USV, Corvette] was

purchased from the market.

Working standard solution

Accurately 10 mg of pure nimodipine was weighed and dissolved in 5 mL

methanol and to that 5 mL 4 N HCl and 1 g zinc dust were added in a 100 mL

volumetric flask and after 20 min, the contents were filtered using Whatman No. 41

filter paper. Stock solution was prepared with the distilled water in a 100 mL calibrated

flask to get a standard solution of 100 µg mL-1.

Assay

Method A

An aliquot of standard solution containing 0-3.5 mL (100 µg mL-1) of NMD was

transferred into a series of 10 mL calibrated flasks. To this solution was added 1 mL

sodium nitrite (0.1%), and the acidity was adjusted with 1 mL of 1 M hydrochloric acid.

The solution was shaken thoroughly for 2 min to allow diazotization reaction to go to

completion. Then, 1 mL of 2% sulfamic acid was added to each flask. Then, 2 mL each

of the 5% acetylacetone and 4 M sodium hydroxide were added and the contents were

diluted to the mark with distilled water and mixed well. The absorbance of the yellow

colored azo-dye was measured at 420 nm against reagent blank prepared similarly,

but without drug content. The amount of nimodipine in the pure sample and also in the

drug formulations was computed from the concurrent calibration curve or the

regression equation.

416  

Method B

Into a series of 10 mL volumetric flasks were added different aliquots of

nimodipine in the concentration range of 0-25 µg mL-1, 1 mL sodium nitrite (0.1% w/v)

and 1 mL of 1 M hydrochloric acid. After 10 min, 1 mL of sulfamic acid (2% w/v) was

added to each flask. After 1 min, 2 mL diphenylamine (0.5% w/v) was added and the

volumes were made up to the mark with 6 M hydrochloric acid and mixed well. The

absorbance of the violet colored azo-dye was measured at 540 nm against reagent

blank, 10 min later. The amount of nimodipine present in the sample was computed

from calibration curve or the regression equation.

Method C

Different aliquots of nimodipine in the final concentration of 0-35 µg mL-1 were

transferred into a series of 10 mL volumetric flasks. To this, 1 mL each of sodium nitrite

(0.1% w/v) and 1 M hydrochloric acid were added. After 3 min, 1 mL of sulfamic acid

(2% w/v) was added to each flask. Volumes of 2 mL citrazinic acid (0.1% w/v) and 2

mL 4 M sodium hydroxide were added after 1 min. The contents were made up to the

mark with distilled water and mixed well. The absorbance of the orange -yellow colored

azo-dye was measured at 440 nm against reagent blank prepared similarly, but without

drug content. The amount of nimodipine present in the sample was computed from

calibration curve.

Method D

Various aliquots of nimodipine in the final concentration of 0-35 µg mL-1 were

transferred into a series of 10 mL volumetric flasks. To each flask, 1 mL sodium nitrite

(0.1% w/v), and 1 mL of 1 M hydrochloric acid were added at room temperature. After

10 min, 1 mL of sulfamic acid (2% w/v) was added to each flask. After 1 min, 2 mL

chromotropic acid (0.2% w/v) and 2 mL 4 M sodium hydroxide were added, and then

contents were made up to the mark with distilled water and mixed well. The

absorbance of the red colored azo-dye was measured at 520 nm against reagent

blank. The amount of nimodipine present in the sample was computed from calibration

curve.

Procedure for tablet dosage forms

Twenty tablets each containing 30 mg nimodipine were taken and crushed into a

fine powder using a pestle and mortar. Tablets powder equivalent to 10 mg of the drug

weighed accurately and dissolved in 20 mL of methanol. To this, 5 mL 4 N hydrochloric

acid and 1 g of zinc dust were added into a 100 mL volumetric flask and shaken

thoroughly for about 30 min. The volume was made up to the mark with water, mixed

well and filtered using Whatman No. 41 filter paper. Appropriate aliquots of the drug

solution were taken and the proposed standard procedures were followed for analysis

of the drug content.

417  

Results and Discussion The methods involve the diazo-coupling reaction of reduced NMD with acetyl

acetone, diphenylamine, citrazinic acid and chromotropic acid to produce colored azo-

dyes with a maximum absorption at 420 (acetyl acetone), 540 (diphenylamine), 440

(citrazinic acid) and at 520 nm (chromotropic acid). The absorption spectra of the

above dyes are presented in figure 2. Two steps are involved in the reaction that

produces the colored dye. In the first step, reduced NMD is treated with nitrite solution

in acidic condition, undergoes diazotization to give diazonium chloride ion. In the

second step, the diazonium ion is coupled with the coupling agents such as acetyl

acetone (Method A), diphenylamine (Method B), citrazinic acid (Method C) and

chromotropic acid (Method D), to form respective colored azo-dyes in an alkaline

medium for methods A, C and D, while in acidic medium for method B. The proposed

chemical reactions are shown in scheme 1.

Figure 2: Absorption spectra for 10 µg mL-1 NMD (Methods A-D).

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

400 420 440 460 480 500 520 540 560 580 600

Absorba

nce

(Wavelength) λ, nm

Blank

Method A (AA)

Method B (DPA)

Method C (CZA)

Method D (CTA)

418  

Scheme 1: Proposed reaction pathway for NMD with AA, DPA, CZA and CTA.

The factors affecting the color development, reproducibility, sensitivity and

adherence to Beer’s law were investigated and are reported below.

Effect of reagents

The effect of concentration of different reagents such as acetyl acetone,

diphenylamine, citrazinic acid and chromotropic acid was studied by measuring the

absorbance at specified wavelengths in the standard procedure for solutions

containing a fixed concentration of NMD (5 µg mL-1) and varying amounts of cited

reagents (1-5 mL). A volume of 2 mL each reagent solutions in a total volume of 10 mL

were found to be sufficient in methods A-D.

The constant absorbance readings were obtained in the range of 0.5 - 3 mL of

0.1 % sodium nitrite for all the methods. Thus, 1 mL of sodium nitrite solution was used

(methods A-D) in a total volume of 10 mL of reaction mixture. The excess of nitrite

could be removed by the addition of 1 mL of 2 % sulfamic acid.

419  

Effect of acid concentration

Diazotization was carried out at room temperature and cooling to 0-5˚C was not

necessary. The hydrochloric acid concentration was studied and 1 mL of 1 M acid

concentration was fixed for getting constant and maximum absorbance values.

Effects of alkali or acid

The optimum concentration of sodium hydroxide leading to a maximum intensity

of the azo-dye was found to be 2 mL of 4 M in the final solution (for methods A, C and

D). Higher concentrations of alkali may lead to partial decolorization of the dye. For

method B, the violet colored azo-dye was stable and gave maximum absorbance

values when reaction mixture was diluted with 6 M hydrochloric acid. Thus, 6 M

hydrochloric acid was used throughout as diluents in method B.

Effect of reaction time

The colored azo-dyes developed rapidly after addition of the reagents and

attained maximum intensity after about 10 min at room temperature in methods A-D.

The formed azo-dyes in each reagent system were stable for a period of more than 2 h

for NMD.

Method validation

According to the ICH guidelines [15], all the methods were validated for linearity

and sensitivity, limit of detection (LOD) and limit of quantitation (LOQ), precision,

accuracy, selectivity and recovery.

Linearity, sensitivity, limits of detection and quantitation

Under established experimental conditions for all the methods A-D, a linear

correlation was found between the absorbance at respective wavelengths and

concentration of NMD in the ranges are given in table 2. Regression analysis of the

calibration curve using the method of least-squares was made to calculate the slope

(b), intercept (a) and correlation co-efficient (r) for each method (methods A-D) and the

values are presented in table 2. The optical characteristics such as absorption

maxima, Beer’s law limit, molar absorptivity and Sandell’s sensitivity values of all four

methods are also given in table 2. The limit of detection (LOD) and limit of

quantification (LOQ) evaluated as per ICH guidelines and the values for each method

are also summarized (Table 2).

420  

Table 2: Optical characteristics and precision.

Parameter Method A Method B Method C Method D AA DPA CZA CTA

λmax nm 420 540 440 520 Beer’s law range ( µg mL-1)

0 – 35 0 – 25 0 – 35 0 – 35

Molar absorptivity (ε), (L mol-1 cm-1)

0.421x10 4 1.32x10 4 0.762x10 4 0.264x10 4

Sandell sensitivity ( µg cm-2)

0.0993 0.0325 0.0549 0.1583

Intercept (a) 0.0040 0.0101 0.0104 -0.0125 Slope (b) 0.0096 0.0299 0.0173 0.0074 Correlation coefficient (r) 0.9993 0.9998 0.9996 0.9995 Sa 0.0099 0.1029 0.0352 0.0125 Sb 0.0003 0.0043 0.0011 0.0004 LOQ( µg mL-1) 0.239 0.0602 0.451 1.568 LOD( µg mL-1) 0.079 0.0198 0.149 0.517

*y=a+bx, where c is the concentration of nimodipine in µg mL-1 and y is the absorbance at the respective λmax., Sa is the standard deviation of the intercept, Sb is the standard deviation of the slope.

Precision and accuracy. The precision and accuracy of the methods developed were evaluated by

replicate analysis of drug samples at three different concentrations (low, medium and

high) (Table 3). The RSD (%) values of within–day studies showed that the precision

was good (Table 3). The values percentage relative error between the concentrations

of NMD for taken and found showed the accuracy of the methods. The results obtained

are presented in table 3 and show that the accuracy is good.

Table 3: Evaluation of accuracy and precision

Method NMD taken µg

mL-1

NMD found* µg

mL-1

RE

%

SD

SEM

RSD

%

ROE**

%

10 10.09 -0.99 0.17 0.07 1.72 ±1.72

Method A 20 19.41 2.96 0.18 0.07 0.94 ±0.94

30 30.82 -2.72 0.30 0.11 0.97 ±0.96

Method B

10

15

10.07

14.94

-0.71

0.43

0.07

0.07

0.03

0.03

0.68

0.47

±0.68

±0.47

20 19.73 1.36 0.13 0.05 0.67 ±0.67

10 10.01 -0.06 0.09 0.03 0.88 ±0.88

Method C 20 20.20 -1.02 0.16 0.06 0.79 ±0.79

30 30.06 -0.21 0.26 0.10 0.86 ±0.86

Method D

10

20

30

9.98

19.87

30.89

0.16

0.65

-2.98

0.19

0.23

0.41

0.07

0.09

0.15

1.88

1.16

1.32

±1.88

±1.16

±1.32

RE. relative error; SD. Standard deviation; SEM. Standard error of mean; RSD. Relative standard deviation; ROE. Range of error; * Mean value of five determinations **At the 95% confidence level for 6 degrees of freedom

421  

Selectivity

The recommended procedures were applied to the analysis of synthetic mixtures

prepared in the laboratory. The usual diluents and excipients such as talc (20 mg),

dextrose (50 mg), starch (10 mg), sodium alginate (15 mg), gelatin (20 mg) and acacia

(10 mg) were found not to interfere with the analysis by the proposed methods and the

results were obtained in the range 99.5% to 101.5%. These results further showed

clearly the accuracy and precision of the developed methods.

Applications

The methods developed were applied to the quantitative determination of NMD

in pharmaceutical samples and the results are presented in table 4. The results of an

assay of Nimodip were statistically compared with the reference method [11] by applying

the Students-t test for accuracy and F-test for precision. At 95% confidence level, the

calculated t- and F- values do not exceed the theoretical values (Table 4). Therefore,

there is no significant difference between the proposed and the reference method,

indicating that the method is as accurate and precise as the reference method [11]. The

reliability of the method for analysis of pharmaceutical sample was checked by

recovery experiments, which gave quantitative results with appropriate reproducibility.

The accuracy and validity of the suggested methods were further demonstrated

by performing recovery studies through standard addition technique. The results are

given in table 5.

Table 4: Results of determination of NMD in tablets and statistical comparison with the

reference method. Tablet brand Name

Nominal amount mg per tablet

Found**(% of nominal amount±SD) Reference method[11]

Method A Method B Method C Method D

Nimodip* 30 mg 101.4±0.26 101.3±0.45 t=0.521, F=2.996

101.76±0.29 t=1.484, F=1.236

100.35±0.61 t=2.209, F=5.504

101.07±0.40 t=0.2404, F=2.367

*Marketed by: [USV, Corvette]; **Mean value of five determinations Tabulated t and F-values at 95% confidence level are 2.77 and 6.39, respectively.

Table 5: Results of recovery by standard-adddition method.

* Mean of three determinations

Method Tablet studied

Labeled Amount, mg/tab

NTZ in tablet µg

mL-1

NTZ Pure added, µg mL-1

Total found µg mL-1

% Recovery* ± S.D

A Nimodip 30 10.09 5 15.08 99.85±0.29 10 20.10 100.14±0.26 15 25.15 100.41±0.09

B Nimodip 30 5.13 2.5 07.61 99.22±1.04 5 10.13 99.99±0.52 7.5 12.70 100.95±0.35

C Nimodip 30 10.09 5 15.14 101.06±0.52 10 19.95 98.58±0.26

D

Nimodip

30

10.09

15 5

10 15

25.15 15.05 20.23 25.15

100.41±0.10 99.15±0.79

101.44±0.26 100.41±0.95

422  

Conclusions The developed visible spectrophotometric methods are simple, sensitive,

accurate, precise, reproducible and economical and can be successfully applied for the

routine estimation of nimodipine in bulk and in pharmaceutical dosage forms. The

value of standard deviation was satisfactorily low and recovery was close to 100%

which indicates the reproducibility and accuracy of the four methods. These suggested

methods are unaffected by slight variation in the experimental conditions. Added

advantages of the proposed methods are not required cooling (0-5˚ C) for

diazotization.

Acknowledgements The authors are grateful to Cipla Ltd, Mumbai, India, for the generous supply of

pure drug sample. HND and SMM thank the authorities of the University of Mysore,

Mysore for laboratory facilities.

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