Simultaneous UV Spectrophotometric Method for Estimation of Enalapril Maleate and Lercanidipine HCl in Synthetic Mixture

 

Pratik N. Shah*, Bhavini N. Patel and Chaganbhai N. Patel

Shree Sarvajanik Pharmacy College, Nr. Arvind Baug, Mehsana (Gujarat), India 384001.

*Corresponding Author E-mail: pratik_pharma_2005@yahoo.com

 

ABSTRACT:

Two simple, accurate, precise, reproducible, requiring no prior separation and economical procedures for simultaneous estimation of Enalapril Maleate and Lercanidipine HCl in Synthetic mixture form have been developed. First method employs formation and solving of simultaneous equation using 209 nm and 238 nm as two analytical wavelengths for both drugs in methanol. The second method is Q value analysis based on measurement of absorptivity at 216 nm (as an iso-absorptive point) and 209 nm. Enalapril Maleate and Lercanidipine HCL at their respective λ max 209 and 238 nm and at isoabsorptive point 216 nm shows linearity in a concentration range of 4-14 µg/mL. Recovery studies range from 98.15 to 101.14% for Enalapril Maleate and 98.77% to 101.1% for Lercanidipine HCl in case of simultaneous equation method and 98.29% to 100.23% for Enalapril Maleate and 99.85% to 101.16 for Lercanidipine HCL in case of Q - analysis method confirming the accuracy of the proposed method. The proposed method is recommended for routine analysis since it is rapid, simple, accurate and also sensitive and specific by no heating and no organic solvent extraction

 

KEYWORDS: Enalapril Maleate, Lercanidipine HCl, λmax, Simultaneous equation method, Q analysis

 


1. INTRODUCTION:

Enalapril maleate (EM) is an ACE inhibitor used in the treatment of hypertension and heart failure. Enalapril showes its activity to enalaprilat to which it is converted after oral administration1. The structure of EM is shown in Fig 1(a). EM is the subject of a monograph in the Indian Pharmacopoeia(IP)2, the United States Pharmacopoeia (USP)3 and the British Pharmacopoeia (BP)4. The USP recommends an HPLC method for the raw material and tablets with UV detection at 210nm while the BP recommends an aqueous titration for the raw material with sodium hydroxide and potentiometric end point detection and an HPLC method for the tablets. The therapeutic importance of EM initiated several reports for its determination, both in formulations and in biological fluids, viz: spectrophotometry5–10, several HPLC methods10, fluorimetry11, flow injection chemiluminescence method12, polarography9, stripping voltammetry13, and capillary electrophoresis.

 

Lercanidipine HCL (LH) is a dihydropyridine calcium-channel blocker used in the treatment of hypertension. The structure of LH is shown in Fig 1(b).  LH is given by mouth as the hydrochloride in a usual initial dose of 10 mg once daily before food intake, and increased if necessary1.

 

There are several reports on the determination of LH, viz: spectrophotometry14-16, voltammetr,17,18, HPLC methods19–21, and capillary electrophoresis22. LH is a good option to combine with angiotensin converting enzyme inhibitors to optimize control of blood pressure, even in patients with other cardiovascular risk factors. It has an agonistic effect, decreasing high blood pressure without increasing adverse events. LH and EM are established antihypertensive agents23. EM/LH 10mg/10mg or 20mg/10 mg, once daily, significantly reduced sitting diastolic blood pressure and sitting systolic blood pressure, relative to 10mg LH once daily. Fixed-dose LH/EM was generally well tolerated, with a tolerability profile similar to that of either of the individual drugs alone or placebo24.  To the best of our knowledge, no UV Spectrophotometric method has been yet described for the simultaneous determination of the binary mixture of EM and LH.

Figure 1(a):  Chemical structure of Enalapril Maleate

 

Figure 1(b): Chemical structure of Lercanidipine Hydrochloride

 

The aim of the present work is to develop a feasible, sensitive, and specific UV Spectrophotometric method for the analysis of the investigated drugs. Adaptation of the proposed procedures to the analysis of the synthetic mixtures is also an important task. Comparison of the suggested method is also investigated against reported methods.

 

2. MATERIALS AND METHODS:

2.1 Materials:

Spectral runs were made on a Shimadzu UV-Visible spectrophotometer, model- 1700 (Japan) was employed with spectral bandwidth of 0.5 nm and wavelength accuracy of ± 0.3 nm with automatic wavelength corrections with a pair of 10 mm quartz cells. Glasswares used in each procedure were soaked overnight in a mixture of chromic acid and sulphuric acid rinsed thoroughly with double distilled water and dried in hot air oven. Enalapril Maleate and Lercanidipine HCL standards were obtained from Torrent Pharma, Ahmedabad, India. Methanol of analytical reagent grade was purchased by Loba Chemie Pvt. Ltd.(India). All the solutions were protected for light and were analyzed on the day of preparations.

 

2.2 Selection of common solvent:

Methanol of analytical reagent grade was selected as common solvent for developing spectral characteristics of drug. The selection was made after assessing the solubility of both the drugs in different solvents.

 

2.3 Preparation of Standard Drug Solution:

Standard stock solutions containing Enalapril Maleate (EM) and Lercanidipine HCL (LH) were prepared individually by dissolving 10 mg of EM and 10 mg LH separately in 20 ml of methanol. It was then sonicated for 10 minutes and the final volume of both the solutions were made up to 100 ml with methanol to get stock solutions containing 100 µg/ml each of EM and LH in two different 100 ml volumetric flasks. For calibration curves of both drugs,series of solutions were prepared containing  EM 4,6,8,10,12,14 µg/ml and LH 4,6,8,10,12,14 µg/ml by diluting the standard stock solution with methanol in standard volumetric flasks(10ml)

 

2.4 Determination of Absorption Maxima:

By appropriate dilution of two standard drug solutions with methanol, solutions containing 10 µg/ ml of EM and 10 µg/ ml of LH were scanned separately in the range of 200- 400 nm to determine the wavelength of maximum absorption for both the drugs EM and LH showed absorbance maxima at 209 nm (λ1) and 238 nm (λ2) respectively. The overlain spectra showed λmax of both drugs and also isoabsorptive points at 216 nm which is shown in Fig. 2.

 

Figure 2: Overlain Spectra of Enalapril Maleate and Lercanidipine HCL

 

2.5 Method I (Simultaneous equation method):

Two wavelengths selected for the method are 209 nm and 238 nm that are absorption maximas of EM and LH respectively in methanol. The stock solutions of both the drugs were further diluted separately with methanol to get a series of standard solutions of 4-14 µg /ml concentrations. The absorbances were measured at the selected wavelengths and absorptivities (A 1%, 1 cm) for both the drugs at both wavelengths were determined as mean of five independent determinations. Concentrations in the sample were obtained by using following equations-

CX =   …………Eq(1)

Cy =   …………Eq(2)

 

Where, A1 and A2 are absorbances of mixture (test) at 209 nm and 238 nm respectively, ax1 and ax2 are absorptivities of  EM at λ1 and  λ2 respectively and ay1 and ay2 are absorptivities of  LH at λ1 and λ2 respectively. Cx and Cy are concentrations of EM and LH respectively.

 

2.6 Method II (Absorbance ratio or Q-analysis method):

From the overlain spectrum of EM and LH, two wavelengths were selected one at 216 nm which is the isoabsorptive point for both the drugs and the other at 209 nm which is λmax of EM . The absorbances of the sample solutions prepared in a similar manner as in the previous method, were measured and the absorptivity values for both drugs at the selected wavelengths were also calculated. The method employs Q values and the concentrations of drugs in sample solution were determined by using the following formula,

 

For EM

C1 ×

 

For LH

C2 ×

 

Where,

Q0 =

Q1 =

 

Q2 = 

 

A = Absorbance of sample at isoabsorptive point,

a1 and a2 = Absorptivities of  EM and LH respectively at isoabsorptive point.

 

2.7 Application of the proposed method for the determination of EM and LH in Synthetic Mixture:

Synthetic Mixture containing EM 10 mg and LH 10 mg in combination were prepared in laboratory by using suitable excipients (prepared by mixing 10.0 mg EM, 10.0mg LH, 5.0 mg talc powder, 20.0 mg maize starch and lactose (excipient) to 200 mg tablet). The ratio of EM and LH in the samples was  taken 1:1. For analysis, the drug mixture containing 10mg EM and 10mg LH shaken vigorously with  methanol for 15min.Then solution was filtered through Whatman filter paper no 41 and then final volume of the solution was made up to 100ml with methanol to get the stock solution containing 100 µg/ml of EM and LH. Appropriate aliquots of EM and LH within Beer’s Law limit were taken.

 

 

Table 1: Results of analysis of Synthetic Mixture

Synthetic Mixture

 

Method I

Method II

EM

LH

EM

LH

%Conc. Estimated* (Mean ± %R.S.D.)

102.7±

0.17

98.7±

0.53

101.33±

0.43

97.67±

0.27

 

 

In Method I, the concentration of both EM and LH were determined by measuring the absorbance of the sample at 209 nm and 238 nm. Values were substituted in the respective formula to obtain concentrations.

 

For Method II, the concentration of both EM and LH were determined by measuring absorbance of the sample at 216 nm and 209 nm and values were substituted in the respective formula to obtain concentrations. Results of synthetic mixture are shown in Table- 1.

 

3. VALIDATION:

The method was validated according to ICH Q2R1 guidelines for validation of analytical procedures in order to determine the LOD, LOQ, linearity, sensitivity, precision and accuracy for the analyte. [25]

 

3.1 Limit of Quantification (LOQ) And Limit of Detection (LOD):

LOD and LOQ were calculated statistically from formula:

 

LOD = 3.3 (SD / S),           LOQ = 10 (SD / S)

 

Where, SD: Standard Deviation of y- intercepts of regression lines,

S: Slope of the calibration curve.

The LOD and LOQ of EM and LH by statistical and visualization methods were mentioned  in Table- 2.

 

3.2 Linearity:

The linearity of measurement was evaluated by analyzing different concentration of the standard solution of EM and LH. For simultaneous equation method and Q analysis, the Beer- Lambert’s concentration range was found to be 4-14 µg/ml for EM and LH. Calibration curve of  EM at 209 nm and 216 nm are shown in Fig. 3 and 5 respectively and calibration curve of  LH at 238 nm and 216 nm are shown in Fig. 4 and 6 respectively.

 

Figure 3: Calibration Curve of Enalapril Maleate at 209nm

 

Figure 4: Calibration Curve of Lercanidipine HCL at 238 nm

 

Figure 5: Calibration Curve of Enalapril Maleate at 216nm

 

 

Figure 6: Calibration Curve of Lercanidipine HCL at 216nm

 

 

Table 2: Linearity data for EM and LH

Parameter

Method I

Method II

EM

LH

EM

LH

Beer’s law limit (µg/ml)

4-14

4-14

4-14

4-14

Correlation coefficient (R2)

0.9993

0.9995

0.9996

0.9996

Molar absorptivity (lit/mole/cm)

25782

21994

24376

31940

Sandell's sensitivity

(μgcm-3 AU-1)

0.008

0.02

0.0099

0.01

Slope

0.066

0.041

0.10

0.044

Intercept

0.007

0.011

-0.06

0.048

LOD(µg/ml)

0.18

0.28

0.18

0.28

LOQ(µg/ml)

0.54

0.76

0.54

0.76

 

3.3 Accuracy:

To ascertain the accuracy of the proposed methods, recovery studies were carried out by standard addition method at three different levels (80%, 100% and 120%). Percent recovery for EM and LH, by both methods was found in the range of 98% to 102% as per ICH which is shown in Table- 3.

 

Table 3: Result from Recovery studies

Met-hod

Drug

Label claim

%Recovery* (Mean±%R.S.D)

80%

100%

120%

I

EM

10

99.76±0.37

98.15±0.34

101.14±0.54

LH

10

98.77±0.56

99.87±0.24

101.1±0.64

II

EM

10

98.29±0.53

99.15±0.97

100.23±0.69

LH

10

101.16±0.31

99.85±0.38

100.8±0.4

*Average of six determinations; R.S.D.; Relative Standard Deviation

 

3.4 Precision:

Precision was studied to find out intra and inter-day variations in the test method of EM and LH. Calibration curves prepared in medium were run in triplicate in same day and for three days. %RSD were calculated which should be less than 2 %. The results are tabulated in    Table- 4.

 

 

Table 4: Results of Precisions

Day

Method I

Method II

%R.S.D

%R.S.D

EM

LH

EM

LH

Intraday

0.18-0.64

0.75-1.02

0.23-0.67

0.73-1.09

Interday

0.58-1.11

1.42-1.57

0.64-1.23

1.35-1.55

 

 

4. RESULTS AND DISCUSSION:

The overlain spectra of EM and LH exhibit λmax of 209 nm and 238 nm for EM and LH respectively which are quite separated from each other. Additionally one isoabsorptive point was observed at 216 nm. This wavelength was selected for simultaneous estimation of EM and LH for Q value analysis and it is assume to be sensitive wavelength. Standard calibration curves for EM and LH were linear with correlation coefficients (r2) values in the range of 0.9993-0.9996 at all the selected wavelengths and the values were average of six readings with standard deviation in the range of 0.08 – 0.56. The calibration curves were repeated six times in a day and the average % RSD was found to be 0.18 to 0.64 for method I and 0.23 to 0.67 for  method II for EM and 0.75 to 1.02 for method I and 0.73 to 1.09 for method II for LH, similarly the method was repeated for six different days and average % RSD was found to be 0.58 to 1.11 for method I and 0.64 to 1.23 for method II for EM and 1.42 to 1.57 for method I and 1.35 to 1.55 for method II for LH. The accuracy of the method was confirmed by recovery studies from synthetic mixture at three different levels of standard additions. Recovery in the range of 98 – 102% justifies the accuracy of method.

 

 

5. CONCLUSIONS:

The most striking feature of this method is its simplicity and rapidity, non- requiring- consuming sample preparations such as extraction of solvents, heating, degassing which are needed for HPLC procedure. These are new and novel methods and can be employed for routine analysis in quality control analysis. The described methods give accurate and precise results for determination of Enalapril Maleate and Lercanidipine HCL mixture in marketed formulation.

 

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Received on 02.04.2011       Modified on 14.04.2011

Accepted on 01.05.2011      © RJPT All right reserved

Research J. Pharm. and Tech. 4(7): July 2011; Page 1118-1122