Spectrofluorometric Determination of Lisinopril dihydrate and Methyl- Dopa in Bulk and Pharmaceutical Formulation by Using Dansyl Chloride

A novel spectrofluorometric method has been developed for quantitative determination of Lisinopril dihydrate and methyldopa either in pure form or in pharmaceutical preparation. The principle of the method based on the reaction of each drug with dansyl chloride [5-(Dimethyl Amino) Naphthalene-1-Sulfonyl chloride] by the aid of sodium hydroxide solution of pH 8.5 resulted in a product of a high fluorescent intensity which was then measured at 511 nm and 516 nm, for lisinopril and methyldopa respectively after excitation at 323 nm. Factors affecting reaction development were studied and optimized. The proposed method was found to be sensitive, selective and reproducible. Calibration curves were linear over the concentration ranges of 3–20 for lisinopril and 7–25 μg/mL for methyldopa. Results obtained from tablets analysis were compared statistically with reported reference methods.


Apparatus
 Fluorescence measurements were carried out on a FP-6300 spectro-fluorimeter (Jasco, Japan) equipped with a 150 W xenon lamp and 1 cm quartz cells.
 Consort P400 ® digital pH-meter for pH adjustment.

Materials and reagents
All solvents and reagents used throughout the work were of analytical grade and double distilled water was used.
S420171 (KAHIRA, Egypt).Dansyl chloride (DNS-Cl) was purchased from Cornell Lab Company, solution of DNS-Cl was freshly prepared at 3.0 mg/ml in acetone.(Stable for one week).Sodium hydroxide was purchased from Research lab fine chemical industry, solution of NaOH was freshly prepared at 20 mg/ml in distilled water and pH was adjusted by 1M of (34%) HCl to 8.5.

Preparation of stock standard drug solution:
A stock standard solution of lisinopril (1mg/ml) was prepared by weighing accurately 0.1 gm of pure drug and dissolving in 100 ml distilled water.stock standard solution of methyl dopa (1mg/ml) was prepared by weighing accurately 0.1 gm of pure drug and dissolving in 100 ml methanol.

Preparation of working standard solution and construction of calibration curve:
Aliqout containing from 1 to 25 μg/mL of (LIS) and (MD) was prepared by transferring certain increasing volume from each drug to a series of 10 ml volumetric flasks, followed by adding 0.3 ml of standard NaOH, mixed well, then 0.

Optimization of reaction conditions
Different experimental factors including pH, volume of buffer, amounts of reagent, temperature and reaction time were studied and optimized by changing each factor individually keeping the others constant.

Effect of pH:
The influence of pH on the fluorescence intensity of the reaction product was examined using sodium hydroxide solution over the pH range from 6 to 11.0 since DNS-Cl reacts under alkaline conditions.The maximum fluorescence intensity was obtained when the reaction was carried out with NaOH solution of pH 8.5.

Effect of sodium hydroxide volume:
The influence of volume of NaOH on the fluorescence intensity of the reaction product was examined using different volume over the range from 0.1 to 0.7 ml.Maximum fluorescence intensity was obtained when the reaction was carried out using 0.3 ml NaOH solution of pH 8.5 as shown in Figure 5.

Effect of dansyl chloride volume:
The influence of the volume of dansyl chloride solution was examined by addition of different volumes of 3.0 mg/ml reagent in the range of 0.2 to 1 ml.A maximum fluorescence intensity was obtained when 0.5 ml of dansyl chloride solution was utilized as depicted in Figure 6.

Effect of time and temperature:
In this study, the reaction between the two drugs and dansyl chloride was performed using pH 8.5 at different temperatures (25°C, 35°C, 40°C, and 50°C) for various time intervals (10, 20, 30, 35 and 40 min).As it is seen in Figure 7 and 8, the reaction was found to be completed after 30 and 35 min for (LIS) and (MD) respectively at 35°C.

Stoichiometry of the reaction
The molar ratio of the reagent and the two drugs in the reaction was studied by using the continuous variation method (Job ҆ s method) [28].The molar ratio was found to be 1: 2 (drug: reagent) and 1:1 (drug: reagent) for Lisinopril and methyl-dopa respectively as seen in Figure 9.

Method validation
The validity of the proposed method was tested regarding linearity, range, limits of detection, limits of quantification, accuracy, precision, robustness and specificity according to ICH recommendations [29].Where S is the standard deviation of the three replicate determination and K is the slope of calibration graph.The results are summarized in Table 2. L Mol -1 cm -1 *Mean of three different experiments; ** Calculated in the basis of molecular weight of the drug.

Table 2:
Statistical data for the reaction of lisinopril and methyl dopa with dansyl chloride.

Accuracy and precision:
Accuracy of the proposed methods was checked by performing recovery experiments through standard addition technique.The results are shown in Table 3 indicates good accuracy.The precision of the method was calculated in term of intermediate precision (intraday and inter-day).Three different concentrations five times of lisinopril and methyl dopa were analyzed during the same day (intra-day precision) and five consecutive days (inter-day precision).The standard analytical errors, relative standard deviations (RSD) and recoveries obtained by the proposed method were found to be acceptable.The results are summarized in Table 4.  3.4.5.Robustness: Robustness was achieved by making small incremental change in volume of sodium hydroxide and volume of reagent.The effect of changes was studied on the percent recovery of drugs proved that all changes had a negligible influence on the results as seen in Table 5.

Sinopril
Table 5: Results of the robustness for the determination of lisinopril and methyl dopa using DNS-Cl.

Analysis of pharmaceutical preparations:
The proposed methods were applied to the analysis of the drug in dosage forms and the results were statistically compared with reported reference methods by calculating Student's tand F-values.The evaluated t-and F-values were less than the tabulated values at the 95% confidence level.Results listed in Table 6 showing that there is no statistical significance difference between the proposed and reference methods.

Conclusion
The proposed method was simple, fast, selective, sensitive, specific, reproducible and not very expensive.Validation experiments proved that results were linear over the mentioned working range.No interference was observed from the described drugs and their common excipients.Also there was no need for extraction procedure.

Acknowledgment
Authors are grateful to all members of staff of Medicinal chemistry department (Zagazig University) also to all members of staff of analytical chemistry department and laboratories staff (Tanta University)

Figure 4
Figure 4 illustrate the effect of pH.

Figure 4 :
Figure 4: Effect of the pH on the development of the reaction product of (LIS) and (MD) (10 µg/mL) with DNS-Cl.

Figure 5 :
Figure 5: Effect of volume of NaOH solution on development of the reaction product of (LIS) and (MD) (10 µg/mL) with DNS-Cl.

Figure 6 :
Figure 6: Effect of volume of DNS-Cl solution on development of the reaction product of (LIS) and (MD) (10 µg/mL).

3. 4 . 1 .
Linearity: Under the optimum experimental conditions, standard calibration curves were constructed at five concentration levels by plotting the values of florescence intensity versus the final concentrations (μg/ml) as shown in Figure10.The correlation coefficient was 0.9999 and 0.9997 for (LIS) and (MD) respectively, indicating good concentration range of 3.0 to 20.0 and 7.0 to 25.0 μg/ml for (LIS) and (MD) respectively.The intercept, slope, Correlation coefficient for the calibration data are summarized in Table1.Calibration graph is described by the equation (Y= a+bX), (Where Y= florescence intensity, a= intercept, b= slope and X= concentration in μg.ml -1 ).

2 .
Limits of detection and limits of quantification: The limit of detection (LOD) was calculated according to the equation [LOD = 3.3 S /K].The limit of quantification (LOQ) was calculated by the equation [LOQ = 10 S/K]

Table 1 :
Statistical and analytical parameters of (LIS) and (MD) determination.

Table 3 :
Application of standard addition technique for the determination of Sinopril ® and Aldomet® tablets form through reaction with DNS-Cl.

Table 4 :
Results of the intraday and inter-day precision for the determination of lisinopril and methyl dopa using DNS-Cl.

Sinopril ® tablet Aldomet ® tablet Reported reference method number [133] Proposed method Reported reference method number [153] Proposed method Mean recovery* ± SD 100
Average of three experiments; a and b are Theoretical Student t-values and F-ratio at p=0.05. *

Table 6 :
Statistical data for the determination of pharmaceutical tablets Sinopril® and Aldomet® through the proposed methods using DNS-Cl compared with the reference methods.