200 mg of chemical (Sigma-Aldrich) were dissolved in water and brought to 100 mL with water. Freshly prepared ninhydrin solution was always used.

The solution was prepared by dissolving 319 mg of chemical (Honeywell Fluka) in water and diluting to 100 mL in a calibrated flask.

Pharmacopeial standard sample of lisinopril dihydrate was provided by Sigma-Aldrich (≥ 98%, HPLC).

The used dosage forms of lisinopril: Lisinopril – Astrapharm (Ukraine) (20 mg), Lisinopril-KRKA (Slovenia) (20 mg), Lisinopril-Teva (Germany) (20 mg).

Different aliquots of 100 µg/mL lisinopril methanol solution (40–60 µg/mL) were accurately measured and transferred in heating tubes. 1.1 mL of 0.2% solution of ninhydrin was added to each tube. The mixture was kept in a water bath at 95 ± 2 °C for 25 minutes, then cooled to room temperature and transferred into a 25 mL volumetric flask. The volume was made up to the mark by adding water. The absorbance was measured at 400 nm against the reagent blank, which was similarly prepared by omitting the drug. The calibration curve was performed by plotting the measured absorbance values versus concentration.

Twenty tablets were accurately weighed and powdered. A quantity of powder containing 25 mg of lisinopril was transferred into a 25 mL volumetric flask with 15 mL methanol. The mixture was shaken for 15 min, diluted to volume with methanol and then filtered using 0.2 µm Nylon filter membrane. The filtrate was subsequently subjected to analysis using the above described procedure.

Different aliquots of 10 mg/mL lisinopril aqueous solution (0.5–2.1 mg/mL) were accurately measured and transferred into a 25 mL volumetric flask. 10.0 mL of 0.02 M solution of copper (II) sulfate was added to each tube. The volume was made up to the mark by adding water. The absorbance was measured at 730 nm against the reagent blank, which was similarly prepared by omitting the drug. The calibration curve was performed by plotting the measured absorbance values versus concentration.

Thirty tablets were accurately weighed and powdered. A quantity of powder containing 0.37 g of lisinopril was transferred into a 50 mL volumetric flask with 35 mL water. The mixture was shaken for 15 min, diluted to volume with water and then filtered using 0.2 µm Nylon filter membrane. The filtrate was subsequently subjected to analysis using the above described procedure. Aliquots of 5 mL lisinopril aqueous solution was accurately measured and transferred into a 25 mL volumetric flask. 10.0 mL of 0.02 M solution of copper (II) sulfate was added to each tube. The volume was made up to the mark by adding water. The absorbance was measured at 730 nm against the reagent blank, which was similarly prepared by omitting the drug. The calibration curve was performed by plotting the measured absorbance values versus concentration.

The ninhydrin has been known as a reagent for the detection of amino acids and amines for many years and therefore, a number of theories have been put forward to explain the mechanism of its reaction. It was suggested that the reactions of ninhydrin with amine, amino acids and imino acids all proceed by the same mechanism (McCaldin 1960) to give diketohydrindylidene–diketohydrindamine. This compound would further react with amino group to give the product which absorbed maximally at 400 nm and 560 nm. The optimum conditions for determination of lisinopril were established via a number of preliminary experiments. Lisinopril interacts with ninhydrin in aqueous medium via oxidative deamination of the primary aliphatic amino group of the lisyne rest contained in the lisinopril molecule followed by the condensation of the reduced ninhydrin to form the colored reaction complex with λmax at 400 nm and 560 nm. Results of experiments suggested that a higher sensitivity could be achieved at λmax 400 nm, which was selected for the following studies (Fig. 2 and Scheme I).

Figure 2. 

Absorption spectrum of lisinopril for method I.

Scheme I. 

Suggested reaction pathway between lisinopril and ninhydrin (Sbârcea et al. 2014).

Different parameters such as the temperature, heating time, reagents concentration have been analyzed, in order to render the optimal conditions for reaction. It has been noted that the complete color development was attained at 95 ± 2 °C. Optimum reaction time has been determined by heating the reaction mixture on a water bath at 95 ± 2 °C. A heating time of 25 minutes was found as optimal for the development of the purple color product (Fig. 3).

Figure 3. 

Effect of heating time on the formation of coloured product.

In order to investigate the effect of ninhydrin concentration on the reaction product color, the change in absorbance generated by varying the concentration of ninhydrin on fixed concentration of lisinopril (40 µg/mL) has been measured against reagent blank. The optimum value was found to be 1.1 mL of 0.2% ninhydrin (Fig. 4).

Figure 4. 

Effect of ninhydrin concentration on the absorbance at λmax of the coloured product.

To establish the analytical sensitivity of valsartan with ninhydrin, the sensitivity of the reaction was calculated. The molar absorption index (ε) was 2.44 × 10³, the specific absorption (a) was 6.02 × 10^-1, and the Sendel coefficient (Ws) was 1.66 × 10^-1.

In neutral media lisinopril forms with Cu²⁺ ions a blue complex compound. Figure 5 shows the spectrum of this compound with an absorption maximum at λ = 730 nm. Schemes II, III present proposal of the reaction pathway between lisinopril and copper (II) sulfate and suggested reaction mechanism for reaction between lisinopril and copper (II) sulfate.

Figure 5. 

Absorption spectrum of lisinopril for method II.

Scheme II. 

Proposal of the reaction pathway between lisinopril and copper (II) sulfate.

The stoichiometry of the reaction was determined using Job’s method of continuous variation (Job 1936). Master equimolar solutions (1×10^-3 M) of copper (II) sulfate with lisinopril were prepared. The method revealed 1:2 ratio (copper (II) sulfate:lisinopril). The results obtained from molar ratio studies were in agreement with the suggested reaction mechanism (Scheme III) (Fig. 6).

Figure 6. 

The absorption spectra for the blue end product in terms of study Job’s method.

Scheme III. 

Suggested reaction mechanism for reaction between lisinopril and copper (II) sulfate.

To establish the analytical sensitivity of valsartan with copper (II) sulfate, the sensitivity of the reaction was calculated. The molar absorption index (ε) was 0.13 × 10³, the specific absorption (a) was 3.08 × 10^-3, and the Sendel coefficient (Ws) was 2.40 × 10^-3.

Beer’s law limit, molar absorptivity, detection limit, regression equation and correlation coefficient were obtained by least square treatment of results (ICH 2005). The linear relationship was found between absorbance at λmax and concentration of drug in the range 40–60 µg/mL (Method I) and 0.592–2.072 mg/mL (Method II). Regression analysis of Beer’s law plot at 400 nm yielded the regression equation, y = 7.4929x – 0.0545 (Method I) and at 730 nm y = 0.0443x – 0.0832 (Method II). High values of correlations coefficient (R² = 0.9917 (Method I) and R² = 0.999 (Method II)) and small values of intercept validated the linearity of calibration curve and obedience to Beer’s law. Calibration curves are presented in Figure 7A, B. The range of application for Method I was narrow but these results did affect other validation parameters.

Figure 7. 

(A) Calibration curve (Method I) (B) Calibration curve (Method II).

The ICH guidelines were followed in order to determine the LOD and LOQ. Accordingly, the method based on the standard deviation of the response and the slope has been applied, so that 3.3 and 10 times the standard deviation values of y-intercept of regresion line and the regression equation were used to calculate the LOD and LOQ. The LOD and LOQ values were calculated to be 6.91 µg/mL and 23.01 µg/mL respectively (Method I) and 0.11 mg/mL and 0.36 mg/mL respectively (Method II).

The proposed methods were tested in order to assess its selectivity using the artificial mixture for analysis. It has been confirmed that the measured absorbance was only produced by the analyte. A synthetic mixture was prepared, containing lisinopril (20 mg), calcium hydrogen phosphate, mannitol (E 421), corn starch, magnesium stearate, colloidal anhydrous silica. The extract was yielded according to the procedure that was described for tablets and subsequently analyzed using the procedure previously described. The replicate analysis (n = 5) for a concentration level of 52 µg/mL lisinopril has yielded the % lisinopril recovery at 100.42 ± 1.25 (Method I) and for a concentration level of 1.78 mg/mL has yielded the % lisinopril recovery at 100.85 ± 1.41 (Method II), and thus revealed that the inactive ingredients did not interfere with lisinopril determination.

Intra-day and inter-day precision values have been calculated by replicate analysis (n = 5) of calibration standard, at three different concentration levels, during the same day, and then during 5 consecutive days. The RSD (%) values of intra-day and inter-day measurements have indicated a good precision. (Table 1). Accuracy, defined as the closeness between the reference and the found values, has been evaluated, on the other hand, as percentage relative error between the measured and theoretical concentration of lisinopril. The results are presented in Table 1, and show good accuracy for developed methods.

The proposed methods were applied for the quantification of lisinopril in tablets pertaining to three commercial formulations. The results as presented in Table 2 reveal no significant differences between the proposed methods. The Student’s t- and the F-values at 95% confidence level are less than the theoretical one, but nevertheless confirming a good agreement between the results obtained by the proposed methods.

The evaluation of robustness was carried out at the stage of development of spectrophotometric methods for the determination of valsartan during the establishment of optimal conditions for the course of reactions and determination of factors that may affect the optical density (stability of solutions over time). It was found that the studied solutions were stable for at least 45 minutes (Figs 8, 9).

Figure 8. 

Graph of the dependence of the adsorption of the reaction product of valsartan with ninhydrin on time.

Figure 9. 

Graph of the dependence of the adsorption of the reaction product of valsartan with copper (II) sulfate on time.

Analytical eco-scale is a semi-quantitative assessment tool commonly used for examining the greenness of analytical methods in a comparative manner (Van Aken et al. 2006; Peleshok et al. 2021b, 2021c; Gałuszka et al. 2012). It is based on assigning a numerical score, penalty points, for every step in the whole analytical method of analysis that may affect the green system such as solvents, reagents, their amounts, energy consumption, occupational risk and waste generated hazards.

Table 3 summarizes the results of developed methods found to be an excellent green analysis with a score of 89 (Method I) and 93 (Method II).