Indian Journal of Chemical Technology Vol. 16, September 2009, pp. 431-436

New spectrophotometric methods for the estimation of Lercanidipine hydrochloride T Manikya Sastry1* & K Ramakrishna2 1

Department of Chemistry, GVP College of Engineering, Visakhapatnam 530 048, India 2 Department of Chemistry, GIS, GITAM University, Visakhapatnam 530 045, India Email: [email protected] Received 26 December 2008; revised 30 June 2009

Three simple and sensitive visible spectrophotometic methods (A-C) have been developed for the assay of Lercanidipine hydrochloride (LER) in bulk and formulations. Method A is based on the formation of colour species by condensation reaction of drug with vanillin (λmax=600). Method B is based on the formation of colour species between the drug and citric acid/acetic anhydride (λmax=555) by means of forming inner salt formation. Method C is based on the formation of colour species by charge transfer complex formation with chloranil-acetaldehyde (λmax=585). The results of analysis have been validated statistically. Recoveries range from 99.4 to 100.5% for the routine assay of LER formulations. Keywords: Spectrophotometry, Lercanidipine hydrochloride, CiA/Ac2O, Vanillin, TQ-CH3CHO.

Lercanidipine hydrochloride (LER) is chemically 2-{(3,3-diphenylpropyl) methylamine}-1,1-dimethylethyl-methyl 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridine carboxylic ester hydrochloride (Fig. 1). This drug is used as a calcium channel blocker in the treatment of hypertension1. The drug is official in Merck Index2 and Martindale3. In literature, a number of analytical methodshave been described for estimation of LER. These include HPLC4,5, electrophoresis6, LC-MS7,8-11, extractive spectrophotometric12, and few visible spectrophotometric13-22 methods. With an aim to develop relatively cheap and useful method for the laboratories with modest infra structure, three simple, sensitive, accurate, reproducible, reliable and economical analytical methods for the estimation of LER in bulk drug and formulations have been developed.

Experimental Procedure Instrumentation

All spectral and absorbance measurements were made on a Elico SL-177 model visible spectrophotometer with 1 cm matched glass cells and on UNICAM UV 500 spectrophotometer made by Thermo Electron Corporation. All pH measurements were made on a Elico LI 120 digital pH meter. Reagents

All the reagents were of analytical grade and all solutions were prepared in double distilled water. Citric acid/Ac2O (E.Merck; 12%, 6.24 × 10-1M), was prepared by dissolving it in methanol initially followed by dilution with acetic anhydride. Vanillin (VN) (BDH; 0.4%, 2.63 × 10-3M) was prepared by dissolving it in methanol. Chloranil (TQ), (BDH; 0.1%, 4.06×10-3M), was prepared by dissolving it in 1,4-dioxan. Acetaldehyde (Qualigens), 1,4-dioxan (Qualigens), DMF (Qualigens), H2SO4 (E.Merck; 36 N), and acetic anhydride (Qualigens) were used without any further purification. Standard drug solution

Fig. 1 Chemical structure of lercanidipine

About 100 mg of bulk drug was dissolved in 10.0 mL methanol and reduced using standard literature method23. The reduced drug solution was diluted to 100 mL with methanol to prepare stock solution (1.542 × 10-3M) and was diluted stepwise

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with the same solvent to obtain working standard solution of concentration 1.542 × 10-5M for method A. The stock solution (1.542 × 10-3M) in methanol was evaporated to dryness and mixed with 10.0 mL of 10% Na2CO3 solution. The resulting solution was transferred into 150 mL separating funnel and extracted with 3 × 25 mL portions of chloroform and combined chloroform layer was brought up to 100 mL with the same solvent to get free base stock solution (1.542 × 10-3M). 2.0 mL of the free base stock solution (1.542 ×10-3M) was diluted stepwise with the chloroform to obtain working standard solution of concentrations 3.086 × 10-3M for method B. 50.0 mL of the free base stock solution (1.542 × 10-3M) in chloroform was evaporated to dryness in boiling water bath. The residue was cooled and then dissolved in DMF to prepare stock solution (1.542 × 10-3M). The standard stock solution (1.542 × 10-3M) was used as it is as working standard solution for method C. Recommended procedures Method A

Aliquots of standard drug solution (LER: 0.5 – 2.5 mL, 10 µg/mL) were taken into a series of calibrated tubes. Then 2.0 mL of 2.63×10-3M vanillin and 3.0 mL of concentrated sulphuric acid (36 N) were added successively and the total volume in each flask was brought to 10.0 mL by the addition of methanol and placed in heating water bath for 15 min at 50°C. Then the flasks were cooled and the volume made up to 15.0 mL with methanol and the absorbances were measured after 10 min at 600 nm against a reagent blank prepared in a similar way. The stability of coloured species was found to be 30 min. The concentration of drug in a sample was computed from Beer-Lambert plot. Method B

Aliquots of standard drug solution (1.0–3.0 mL, 20 µg.mL-1) in free base form in chloroform were taken into a series of graduated tubes and gently evaporated on boiling water bath to dryness. To this 10.0 mL of 6.24×10-1M CiA/Ac2O reagent was added and the flasks were immersed in boiling water bath for 50 min. The tubes were cooled to room temperature and the volume was made up to 10.0 mL with acetic anhydride. The absorbances of the coloured solutions were measured at 555 nm against a reagent blank within the stability period of 30 min. The amount of drug (LER) was computed from its calibration curve.

Method C

Aliquots of standard drug solution (LER: 1.0 – 3.0 mL, mg/mL) in free base form were taken into a series of 10.0 mL calibrated tubes. The solution in each test tube was evaporated to dryness and diluted with DMF to 3.0 mL. Then 0.5 mL of acetaldehyde and 2 mL of 4.06 ×10-3M chloranil were added and allowed to stand for 20 min at room temperature. The solutions were made up to 10.0 mL with 1,4-dioxan and the absorbances were measured at 585 nm (for LER) against a reagent blank prepared simultaneously. The stability of coloured species were found to be 15 min. The amount of the each drug was calculated from the appropriate calibration graph. Pharmaceutical formulations

Since only two formulations are available for LER (Tablets), formulations of different batches were collected and analyzed as 4 sets to verify the validity of proposed methods. Accurately weighed quantity of tablet powder equivalent to 100 mg of LER was extracted with warm chloroform (3 × 25.0 mL portions) and filtered. The volume of combined extract evaporated to dryness, reduced as described in the preparation of standard drug solution (1.542 × 10-3M) and working standard solution of concentration 1.542 × 10-5M (Method A). Further, the released free base stock solution (1.542 × 10-3M) was made as described in the preparation of bulk drug and working standard solutions of concentration 3.086 × 10-5M (Method B). The free base stock solution (1.542 × 10-3M) was evaporated to dryness, cooled and then dissolved in DMF to prepare stock solution (1.542 × 10-3M) which was used as it is as working standard solution (for method C). Further, the UV spectrophotometric method which was suggested for the identification of LER has been moulded for its assay and chosen as the reference method28 for ascertaining the accuracy of the proposed methods. Results and Discussion The optimum conditions for each method were established by varying single parameter at one time24 while keeping the others fixed and observing the effect produced on the absorbance of the coloured species. Optimum conditions and chemistry of coloured species Method A

2.0 mL of vanillin (2.63×10-3 M), 3.0 mL of 36 N H2SO4, order of addition is drug, vanillin, con. H2SO4 and use of methanol solvent for final dilution were identified as

SASTRY & RAMAKRISHNA: SPECTROPHOTOMETRIC ESTIMATION OF LERCANIDIPINE HYDROCHLORIDE

the optimum conditions for this method. The absorbance of coloured species was measured after 10 min. The λmax (nm) and εmax (mol-1/cm) values were found to be 600 nm and 2.6447×105. Sastry and Sastry25 explained the formation of coloured species by the condensation of reduced nitro aromatic derivatives with vanillin. Among the different aldehydes p-dimethylamino benzaldehyde (PDAB), p-dimethylamino cinnamaldehyde (PDAC) and vanillin (p-hydroxy-m-methoxybenzaldehyde) were tried for developing the colour in acidic condition, vanillin was found to be superior for its sensitivity. In the present investigation, the imino (-NH-) group present in the drug (LER) developed colour to produce the condensed product with vanillin and the absorption maximum was found at 600 nm (Scheme 1). Method B

10.0 mL of citric acid/Ac2O reagent (6.245×10-1M), 50 min heating time on boiling water bath, use of Ac2O for final dilution were identified as optimum conditions. The absorbance of coloured species was measured at 555 nm after 5 min. In the present investigation the tertiary amino group present in the side chain of drug (LER) permits the development of new spectrophotometric method for its determination through the formation of red-violet colour species due to internal salt formation26 between aconitic anhydride and LER (Scheme 2). Method C

The optimum conditions of the method were identified as: 0.5 mL of acetaldehyde, 2.0 mL of

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chloranil (4.06×10-3M), 20 min waiting period before final dilution, 1,4-dioxan solvent for final dilution. The absorbance values were measured after 5 min. The λmax (nm) and εmax (mol-1/cm) values were found to be 585 nm and 1.283×104. In the present investigation, the drug (LER) contain secondary imino group which reacts initially with acetaldehyde giving enamine which then reacts with chloranil (TQ) by nucleophilic attack giving blue coloured substituted enamine derivative27 (Scheme 3). Analytical data

The optical characteristics such as Beer’s law limits, molar absorptivity, sandell’s sensitivity, and regression characteristics are given in Table 1. The accuracy of the method for the drug (LER) was ascertained by comparing the result by proposed methods and reference method (UV), statistically24 by t- and F- tests (Table 2). An additional check of accuracy of the proposed method, recovery experiments were performed by adding a fixed amount of the drug to the pre-analyzed formulation and results are given in Table 2. This comparison shows that there is no significant difference between the results obtained by the proposed methods and reference method28. The proposed methods are simple, rapid, economical and sensitive with a range of 0.33 -1.67 µg mL-1 (Method A), 2-16 µg mL-1 (Method B) and 100-300 µg mL-1(Method C) when compared to literature methods20 (Table 3).

Scheme 1 Formation of colour species by condensation reaction with vanillin

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Scheme 2 Formation of colour species by condensation reaction with citric acid/acetic anhydride

Scheme 3 Formation of colour species by charge transfer complex formation with chloranil-acetaldehyde

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Table 1 Optical characteristics, precision, accuracy of proposed methods for the determination of LER Vanillin CiA/Ac2O TQ-CH3CHO Optical characteristics (Method A) (Method B) (Method C) 600 555 585 λmax (nm) 0.33 – 1.67 2–6 100 – 300 Beer’s Law limits (µg/mL) 1.27 Detection limit (µg/mL) 8.5 × 10-3 3.44 × 10-2 Molar absorptivity (L mol-1cm-1) 2.64 × 105 5.54 × 104 1.28 × 104 Sandell’s sensitivity (µg/cm2/0.001 Absorbance unit) 2.5 × 10-3 1.17 × 10-2 5.05 × 10-2 Regression equation (y = a + bC)∗ Slope (b) Standard deviation on slope (Sb) Intercept (a) Standard deviation on intercept (Sa) Standard error of estimation (Se) Correlation coefficient (r) Optimum photometric range (µg/mL) Relative Standard Deviation** % range of error (confidence limit) 0.05 level 0.01 level % error in bulk sample***

4.04 × 10-1 1.03 ×10-3 1.52 × 10-3 1.14 × 10-3 1.094 × 10-3 0.9999 0.48-1.62 0.53

8.54 × 10-2 2.30 × 10-4 8.0 × 10-4 9.8 × 10-4 7.3 × 10-4 0.9999 2.3-5.8 0.80

2.0 × 10-3 6.0 × 10-6 -1.9 × 10-3 9.95 × 10-4 9.49 × 10-4 0.9999 102.6-297.5 0.4683

0.55 0.87 0.24

0.84 1.32 -0.08

0.49 0.77 -0.25

∗ y = a + bC where C is the concentration of analyte in µg/mL and y is the absorbance unit ∗∗Calculated from six determinations ∗∗∗Average error of three determinations Table 2 Assay of LER in pharmaceutical formulations Amount found by proposed methods Reference % Recovery by proposed methods*** (mg)** method28

Formulations* Labelled amount (mg) Vanillin CiA/Ac2O TQ-CH3CHO (Method A) (Method B) (Method C) Tablet I

10

II

10

III

10

IV

10

9.92±0.13 F = 1.81 t =1.52 9.83±0.11 F = 1.12 t =1.84 10.11±0.1 F = 1.52 t =1.64 9.96±0.05 F = 4.87 t =0.94

9.97±0.13 F = 1.65 t =0.35 9.88±0.06 F = 4.15 t =1.85 10.08±0.11 F = 1.17 t =1.34 10.13±0.11 F = 1.13 t =1.89

9.90±0.06 F = 2.82 t = 2.16 9.89±0.14 F = 1.28 t =1.26 10.13±0.1 F = 1.31 t =1.7 9.97±0.09 F = 1.59 t =2.17

Vanillin (Method A)

CiA/Ac2O (Method B)

TQ-CH3CHO (Method C)

99.16 ±1.34

99.71 ±1.27

99.05 ±0.59

10.07 ± 0.12 98.34 ±1.13

98.83 ±0.58

98.88 ±1.35

9.92 ± 0.12 101.1 ±1.0

100.8 ±1.1

101.3 ±1.1

9.92 ± 0.12 99.77 ±0.52

101.3 ±1.1

99.75 ±0.91

9.96± 0.10

*Four different samples of tablets **Average + standard deviation of six determinations, the t and F-test values refer to comparison of the proposed method with the reference method. Theoretical values of 95% confidence limit, F = 5.05, t = 2.57 ***After adding 3 different amounts of the pure labeled to the pharmaceutical formulations, each value is an average of 3 determinations Table 3 Comparison of spectrophotometic methods for Lercanidipine hydrochloride Reagents used

λmax (nm)

HNO2/NED Fe(NO3)3 VN CiA/Ac2O TQ-CH3CHO

530 390 600 555 585

Beer’s Law limits (µg/mL) 5-25 5-25 0.33-1.67 2-6 100-300

Correlation coefficient (r) 0.9989 0.9978 0.9999 0.9999 0.9999

Molar absorptivity (L mol-1cm-1) 0.463 x 104 1.468 x 104 2.64 x 105 5.54 x 104 1.28 x 104

Reference 20 20 Present paper Present paper Present paper

NED- N-(1-naphthyl) ethylene diamine dihydrochloride; VN- Vanillin; CiA- Citric acid; Ac2O- Acetic anhydride; TQ –Chloranil

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Interference studies

The ingredients usually present in the preparation of formulations such as yellow oxide of iron and titanium dioxide anticipated in pharmaceutical formulations did not interfere with the assay of LER by proposed methods. Commercial formulations (tablets) containing LER were successfully analyzed by the proposed methods. The values obtained by the proposed and reference methods for formulations were compared statistically with t- and F- tests and found not to differ significantly (Table 2). Conclusion Unlike the GLC and HPLC procedures, spectrophotometer involved in the present procedure is cheap and simple to operate. The high sensitivity, selectivity and simplicity of the methods proposed make them ideally suitable for the assay of LER in pharmaceutical formulations. The ingredients usually present in pharmaceutical formulations do not interfere in the colour development by proposed methods. All the proposed methods are simple, economical and do not require much of instrumentation. Acknowledgements One of the author (TMS) is thankful to the Management of Gayatri Vidya Parishad College of Engineering, Visakhapatnam for providing facilities. References 1 McClellan K J & Jarvis B, Drugs, 60 (2000) 1123. 2 Budavari S, Merck Index, 12th edn (Merck & Co. Inc., New York), 1996. 3 Reynolds F E J, Martindale the Extra Pharmacopoeia, 32nd edn (The Pharmaceutical Press, London), 1999. 4 Alvarez-Lueje A, Pujol S, Squella J A & Nunez-Vergara L J, J Pharm Biomed Anal, 31 (2003) 1. 5 Barchielli M, Dolfini E, Farina P, Leoni B, Targa G, Vinaccia V & Tajana A, J Cardiovasc Pharmacol, 29 (1997) S1.

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