Corresponding author: Liliya Logoyda (
Two simple, rapid and green spectrophotometric methods are described for the determination of lisinopril medicines. The determination is based on the reaction of the primary amino group of the lisinopril with ninhydrin in aqueous medium (Method I) and reaction on the carboxylic group of the lisinopril with copper (II) sulfate (Method II). For both methods, optimal spectrophotometric conditions were established. 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. 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). Intra-day and inter-day accuracy and precision were in acceptable limits. The proposed methods were applied for the quantification of lisinopril in tablets pertaining to three commercial formulations. Analytical eco-scale for greenness assessment of the proposed spectrophotometric methods showed that both methods correspond to excellent green analysis.
Nowadays, hypertension is becoming a worldwide problem. Several medicines used for treatment hypertension. Lisinopril is a competitive inhibitor of angiotensin-converting enzyme (
Chemical structure of lisinopril.
Analytical methods of analysis such as HPLC (
The present paper describes a rapid, simple and green visible spectrophotometric methods for the determination of lisinopril in medicines. The determination is based on the reaction of the primary amino group of the lisinopril with ninhydrin in aqueous medium (Method I) and reaction on the carboxylic group of the lisinopril with copper (II) sulfate (Method II).
We aimed to develop and validate rapid, simple and green visible spectrophotometric methods for the determination of lisinopril in medicines.
A double – beam Shimadzu UV-Visible spectrophotometer, with spectral bandwidth of 1 nm wavelength accuracy ±0.5 nm, Model –UV 1800 (Japan), Software UV-Probe 2.62, and a pair of 1 cm matched quartz cells, was used to measure absorbance of the resulting solution. Designed in accordance with the governing Japanese and European Pharmacopoeia, the new UV-1800 UV-VIS spectrophotometer achieves a resolution of 1 nm, the highest in its class, in a compact design.
All the chemicals were used of analytical reagent grade.
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 (
Absorption spectrum of lisinopril for method I.
Suggested reaction pathway between lisinopril and ninhydrin (
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.
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.
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 (
In neutral media lisinopril forms with Cu2+ ions a blue complex compound. Figure
Absorption spectrum of lisinopril for method 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 (
The absorption spectra for the blue end product in terms of study Job’s method.
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 (
Beer’s law limit, molar absorptivity, detection limit, regression equation and correlation coefficient were obtained by least square treatment of results (
(
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 (
Intra-day and inter-day precision values have been calculated by replicate analysis (
Intra-day and inter-day accuracy and precision.
Method | Lisinopril taken, µg/mL (Method I), mg/mL (Method II) | Intra-day accuracy and precision | Inter-day accuracy and precision | ||||
---|---|---|---|---|---|---|---|
Lisinopril found, µg/mL (Method I), mg/mL (Method II) | Lisinopril found, µg/mL (Method I), mg/mL (Method II) | ||||||
I | 40 | 39.97 | 0.65 | 1.09 | 40.11 | 0.74 | 1.06 |
50 | 50.07 | 0.60 | 1.18 | 49.86 | 0.85 | 1.14 | |
60 | 60.13 | 1.09 | 1.31 | 60.07 | 0.56 | 1.01 | |
II | 0.59 | 0.5901 | 0.74 | 1.06 | 0.5904 | 0.36 | 1.02 |
1.48 | 1.4795 | 0.49 | 1.53 | 1.4803 | 0.45 | 1.08 | |
2.07 | 2.0689 | 0.82 | 1.37 | 2.0692 | 0.51 | 1.04 |
The proposed methods were applied for the quantification of lisinopril in tablets pertaining to three commercial formulations. The results as presented in Table
Determination of lisinopril formulation by the proposed methods.
Tablet brand name | Label claim, mg/tablet | Found (label claim ± SD), % | |
---|---|---|---|
Method I | Method II | ||
Lisinopril – Astrapharm (Ukraine) | 20 | 101.075 ± 0.648 | 100.142 ± 0.311 |
t = 1.66 | t = 2.17 | ||
F = 3.46 | F = 3.76 | ||
Lisinopril-KRKA (Slovenia) | 20 | 100.346 ± 0.412 | 101.205 ± 0.695 |
t = 2.16 | t = 1.64 | ||
F = 2.87 | F = 3.74 | ||
Lisinopril-Teva (Germany) | 20 | 100.961 ± 0.569 | 100.975 ± 0.529 |
t = 2.03 | t = 1.98 | ||
F = 3.38 | F = 3.34 |
Tabulated t-value at 95% confidence level is 2.77; Tabulated F-value at 95% confidence level is 6.39.
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
Graph of the dependence of the adsorption of the reaction product of valsartan with ninhydrin on time.
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 (
Table
Analytical eco-scale for greenness assessment of the developed spectrophotometric methods.
Parameters | Penalty points (PP) | |
---|---|---|
Method I | Method II | |
|
||
Methanol | 4 | – |
Water | – | 0 |
Ninhydrin | 1 | – |
Copper (II) sulfate | – | 1 |
Energy consumption | 1 | 1 |
Occupational hazards | 0 | |
Waste | 5 | 5 |
Total penalty points (PP) | 11 | 7 |
Analytical Eco-scale score | 89 | 93 |
Comment | Excellent green analysis | Excellent green analysis |
Two simple, rapid and green spectrophotometric methods were developed for the determination of lisinopril medicines. The determination was based on the reaction of the primary amino group of the lisinopril with ninhydrin in aqueous medium (Method I) and reaction on the carboxylic group of the lisinopril with copper (II) sulfate (Method II). Optimal spectrophotometric conditions were established. As a result of calculations of analytical indicators of sensitivity of reactions it was established that reaction of valsartan with ninhydrin has higher sensitivity, than reaction of valsartan with copper (II) sulphate that was testified by high value of molar coefficient of absorption and low value of an opening minimum. The proposed methods were validated for selectivity, linearity, limits of detection and quantification, precision and accuracy. The results of the assay of medicines of the developed methods are highly reliable and reproducible and are in good agreement with the label claim of the drugs. The developed methods can help research studies, quality control and routine analysis with lesser resources available.
Authors are grateful to the Ministry of Health of Ukraine Fund for providing scholarship for studies related to solutions for development of original combinations of antihypertensive agents, their analysis and standardization (0120U104201 (№509 date 24.02.2020)).