Perindopril Erbumine its Molecular Formula is C₁₉H₃₂N₂O₅C₄H₁₁N {Tertbutylammonium (2S, 3a S, 7a S)-1-N-[(S)-1-ethoxycarbonyl butyl]-(-alanyl) perhydroindole-2-carboxylate} and its molecular weight is 441.61g/mol; it is a dipeptide monoester with perhydroindole group {Fig.(1, a)}.

Enalapril maleate is a prodrug that is hydrolyzed in the body to enalaprilat. The molecular formula of enalapril maleate is C₂₀H₂₈N₂O₅•C₄H₄O₄{(S)-1-[N-[1 (ethoxycarbonyl)-3-phenylpropyl]-L-alanyl]-L-proline, (Z)-2-butenedioate salt (1:1)} and its molecular weight is 492.53g/mol, it is a derivative of two amino acids L-alanine and L-proline{Figure (1,b)}. Both drugs are inhibitors of angiotensin converting enzyme (ACEI). They are widely used in the management of essential hypertension, stable chronic heart failure, myocardial infarction and diabetic nephropathy. They act mainly by suppressing the formation of angiotensin II by blocking its formation via rennin and angiotensin I^1-5. Concerning Enalapril maleate’s physical and chemical characteristics; it is white to off-white crystalline powder which is practically sparingly soluble in water, soluble in ethanol, and freely soluble in methanol^6-7. The official methods of analysis of ENL in Pharmaceuticals are high-performance liquid chromatography (HPLC) in USP⁶ and British Pharmacopeia (BP)⁷.

While, the perindopril erbumine’s is a white or almost white, crystalline powder, slightly hygroscopic; perindopril erbumine is freely soluble in water (60% w/w), alcohol and chloroform. Literature survey showed that, perindopril active pharmaceutical ingredient (API) is official in British Pharmacopoeia⁸.

An overview of PPE and ENL methods have been reported for the quantitative determination of PPE and ENL by one of the spectroscopic techniques (UV-Visible spectrophotometry^9-26, separation technique such as High-Performance Liquid chromatography (HPLC)^27-30, fluorescence spectroscopy^31-35. All these methods are costly with regard to instrumentation set up cost and complex with regard to technology.

Pharmaceutical’s dosage formulations are needed for effective analytical procedure and quality control of PPE and ENL in clinical and pharmaceutical practices.

Bromocresol green (BCG) (Fig. (1, c)) is an anionic dye and that can be protonated or deprotonated to form yellow or blue, respectively. BCG was used to make ion-pair complex, which was applied to determine many pharmaceutical compounds by extractive and direct spectrophotometric^36-44. However, the direct formulation of ion-pair complexes between BCG and PPE or ENL has not been studied. Method based on ion-pair complexes between analytes and BCG into a suitable organic solvent is also simple, fast, and cheap.

In this paper, for the first time, has been investigated direct spectrophotometric method based on the formation of ion-pair complex between Perindopril Erbumine and Enalapril Maleate with BCG into chloroform. Some effective conditions on the formation of complexes such as organic solvent, the concentration of dye, temperature, and time were systematically studied. The present methods were also applied to determine PPE and ENL in some pharmaceutical formulations including single dose tablets and combination dose tablets.

(a) (b)

(c)

Figure 1: Chemical structures of Perindopril Erbumine (a), Enalapril Maleate (b), and Bromocresol Green (c).

MATERIALS AND METHODS:

Apparatus:

A double beam UV-visible spectrophotometer (JASCO, model V650, Japan) with 1.00cm of path length quartz cells was used to measure all sample absorbance. A high-performance liquid chromatography system (Shimadzu) equipped with degasser (DGU 20 A3), a pump (LC-20AT), an auto-sampler (SIL-20A), an oven (CTO-20A) and Shimadzu photo diode array detector (SPD-M20A). Ultrasonic processor (Powersonic, model 405, Korea) was used to sonicate the sample solutions. Adjustable micro pipettes covering a volume range from 8 to 1000µL (ISO-LAB, Germany), were used for preparation of the experimental solutions. Analytical balance (Sartorius, model 2474, Germany).

Materials and Reagents:

All used chemicals and solvents were of analytical grade. BCG (BDH, ENGLAND), Chloroform (Merck), Methanol (Merck) Dichloromethane (Panereac), Dichloroethane (Panereac), Aceton (Scharlau) and Acetonitrile (Scharlau). Pharmaceutical grade: Peindopril Erbumine was received from Shanghai Longyu Biotechnology Co., Ltd, China.

Enalapril Maleate was received from Zhejiang Changming Pharmaceutical Co., Ltd, China.

Pharmaceutical Formulations:

Following dosage forms containing PPE and ENL were purchased from local pharmacy and employed in the study:

Revosyl tablets, Ibn-Alhaytham pharmaceutical industry, Aleppo- Syria, (2018/2021), labeled to contain 4/8mg of PPE. Revosyl plus tablets, Ibn-Alhaytham pharmaceutical industry, Aleppo- Syria, (2018/2021), labeled to contain 4/1.25mg of PPE and IPD. Copendil tablets, Asia pharmaceutical industry, Aleppo- Syria, (2019/2022), labeled to 4/8mg of PPE. Enapril tablets, Asia pharmaceutical industry, Aleppo- Syria, (2019/2022), labeled to contain 5/10/20mg of ENL. Enapril Plus tablets, Asia pharmaceutical industry, Aleppo- Syria, (2019/2022), labeled to contain 5/12.5 and 10/25 of ENL and HCTZ. Eanlapril ALFares tablets, Fares pharmaceutical co, Damascus- Syria, (2018/2021), labeled to contain 5/10/20mg of ENL.

Preparation of Standard Solutions and General Procedure:

A stock solution of PPE and ENL (1 mg/mL) in chloroform. working standard solution containing 100 µg/mL was prepared by appropriate dilution. stock solution of BCG (1. 10^-3M) was prepared in chloroform.

Construction of Calibration Curves:

A series of 5mL volumetric flask, the volumes of working solutions of the drug in different concentration ranges PPE (2-20µg/mL) and ENL (8-44µg/mL), were transferred. 1.5mL of BCG solution was added to each. After that, the volume was adjusted with chloroform. yellow-colored chloroform containing the ion-pair complexes were measured at 414, 415nm for PPE and ENL respectively, against the reagent blanks. At each concentration, the experiment was repeated five times. colored chromogen complexes are stable for 24 hours.

Samples Preparation:

Weigh and mix the contents of twenty tablets of PPE and ENL drugs, an accurately weighed amount of powder equivalent to labeled content (4mg and 8mg) of PPE and (5mg, 10 and 20mg) of ENL transferred into a 25-mL

beaker and sonicated for 30 minutes to completely disintegrate the powder in chloroform. Filter through a Whatman paper (N^o 4) and fill up to 25mL with chloroform in a volumetric flask. Further dilutions were made to obtain an appropriate concentration (among the range of linearity) and then the general procedures were conducted.

Validation with reference method:

Enalapril maleate was determined by UV spectrophotometric method using phosphate buffer pH =4 with a maximum absorbance at 208nm. The calibration curve linear was reported in the reference method in the range 1-20μg/mL²².

Perindopril erbumine was determined by RP-HPLC method using Chromatographic Conditions: mobile phase was a mixture of phosphate buffer (pH=4.5) and acetonitrile (60:40 v/v) with flow rate of 1.3mL/min, stationary phase was column C18 with photo diode array detector (λ =242nm), temperature was maintained at 45ºC, and the injection volume of samples was 10μL. The calibration curve linear was reported in the reference method in the range 8 - 80μg/mL³⁰.

RESULTS AND DISCUSION:

Optimum Reaction Conditions:

Effect of organic solvents:

Six organic solvents including chloroform (CH), acetone (AC), dichloroethane (DCE), dichloromethane (DCM), acetonitrile (ACN) and methanol (ME) were used to study the effect of solvents to ion-pair formation between PPE, ENL and BCG. Figure 2 shows that chloroform is the most suitable solvent for both PPE and ENL with low blank absorbance, and lowest standard deviation. It implies that chloroform is the best solvent to achieve good recoveries of the complexes with the shortest time to reach the equilibrium processes.

Effect of Dye Concentration:

Effect of dye concentrations was studied by adding different volumes of BCG from 0.1 to 2.0mL with a fixed concentration of PPE (10μg/mL) and ENL (20 μg/mL). Figure 4 shows that the maximum absorbance of the complex PPE: BCG was achieved with 0.5mL and ENL: BCG was with 1.0mL of BCG (1.10^-3 M) for PPE and ENL and excess dye did not aﬀect the absorbance of the complexes and it is kept as constant for further studies. (Fig. 3).

Effect of Time:

Effect of time on the formation and stability of the ion-pair complexes was investigated by measuring the absorbance of the ion associates with increasing time from 0 to 2.0 hours. The ion-pair complexes (PPE: BCG, ENL: BCG) were formed directly after the mixing.

Effect of Temperature:

Effect of temperature on the formation and stability of the ion-pair complexes (PPE: BCG, ENL: BCG) has a negligible effect on these procedures in the range of (15-45^oC).

Stability of the formed colored complexes:

Absorbance of formed complexes was measured by intervals in order to determine the period of stability. The results showed that the absorbance of complexes (PPE: BCG – ENL: BCG) were stable for a period up to 24 hours to start then decline obviously.

Stoichiometry of Ion-Pair Complexes:

Job’s method:

Job’s method of continuous variation of equimolar solutions were employed to evaluate stoichiometry of the complexes. Standard solution of drug and reagent stock solution was prepared at the same concentration 1.10⁻³ M.

A series of the solutions were prepared in which the total volume of drug and reagent was kept in concentration of 80µM of PPE, ENL and BCG, whereas the absorbance was measured at 414nm for PPE and 415nm for ENL. Absorbance was plotted against the mole fraction of the drug. The symmetrical bell of job’s plot indicates that drug-dye ion-pair complexes (PPE: BCG – ENL: BCG) were found to be 1 :1 (Figure 4).

Molar ratio method:

The stoichiometry of drug-dye ion-pair complex was studied by molar ratio method, where the concentration of reagent was constant 4*10^-5M and the concentrations of PPE were among the range of 0.5*10^-5 - 10*10^-5 M and the range of ENL were 1*10^-5 - 10*10^-5 M.

The plot of relation between absorbance and molar ratio [Drug]/[Reagent] confirmed that the stoichiometric ratio for drug-dye ion-pair complexes of (PPE: BCG, ENL: BCG) are 1:1 (Figure 4).

Validation of the Present Methods:

The proposed methods are validated according to ICH recommendations Q2(R1)⁴⁵. The parameters that have been investigated are indicated below.

Linearity, Sensitivity, and Limits of Detection and Quantification:

A linear relationship between the measured absorbance and the concentration range studied for PPE and ENL as shown in Figure 5 and the correlation coefficient (R²) of at least 0.9998 for PPE and 0.9994 for ENL were achieved. The limit of detection (LOD) and quantification (LOQ) of the methods are determined by 3.3(SD /b) and 10(SD /b), respectively, where SD is the standard deviation of blank absorbance values and b is the slope of the calibration curve equation. The LOD and LOQ values, slope, and intercept of linear graphs for PPE and ENL and analytical parameters presented in Table 1.

Accuracy and Precision:

The accuracy of the methods determined by preparing solutions of three different concentrations of PPE and ENL and analyzing them in three replicates. The proposed methods were evaluated as percentage relative standard deviation (RSD%). In order to confirm repeatability and intermediate precision, the measurement of three replicates of three concentrations (4-8-10µg/ml) and (16-20-24µg/ml) of PPE and ENL, respectively were repeated during a period of three days including three replicates for each concentration per day. RSD% was less than 2%. The accuracy and precision as showed in Table 2. The low values of the RSD confirm the high precision and the good accuracy of the present methods.

Figure 5: Calibration curves for PPE and ENL at 414 and 415 nm, respectively.

Table 1: Analytical characteristics of the proposed methods (n = 5).

Parameters	Proposed method for Enalapril	Proposed method for Perindopril
Wavelengths λ_max (nm)	415	414
Stability (h)	24	24
Stoichiometric ratio	1: 1	1: 1
Beer’s law range (μg/mL)	8-44	2-20
LOD (μg/mL)	0.230	0.125
LOQ (μg/mL)	0.697	0.381
Molar absorptivity (L/mol.cm)	19331	44045
Sandell’s sensitivity SS (μg/cm²)	2.01*10^-6	2.22*10^-6
Regression equation (Y = b x + c), where Y is the absorbance, b is the slope, c is the intercept, and x is the concentration in μg/mL
Slope (b)	0.0389	0.0979
Intercept (c)	0.0031	0.0183
Correlation coefficient (R²)	0.9994	0.9998

Robustness:

For the evaluation of the methods robustness, some parameters were interchanged: Scan speed, interval slop, wavelength range. The capacity remains unaffected by small deliberate variations. The results showed no statistical differences, suggesting that the developed methods were robust (Table 3).

Selectivity and Effect of Interferences:

The effect of commonly utilized excipients in drugs formulation was studied. The investigated PPE and ENL were studied with various excipients such as magnesium stearate, glucose, lactose, starch, sodium chloride and ENL with hydrochlorothiazide (HCTZ) and PPE with indapamide (IPD). The HCTZ and ENL were prepared in the proportion (8μg/mL ENL, 20ug/mL HCTZ) corresponding to their amounts in the pharmaceutical forms. The ENL was evaluated by recovery and the results in Table 4 show that recoveries are in the range of 99.95-100.46, demonstrating that there is no interference of HCTZ when ENL in pharmaceutical forms are qualified by spectrophotometric method using ion-pair formation with BCG. In other words, the presented method has a high selectivity for the determination of ENL in its single and mixture dosage forms with HCTZ.

The IPD and PPE were prepared in the proportion (16 μg/mL PPE, 8ug/mL IPD) corresponding to their amounts in the pharmaceutical forms. The results show that there is slight interference of IPD when PPE. Whereas, IPD showed an ion –pair complex with BCG and low absorbance of the formed complex, which affects the selectivity of the PPE in its pharmaceutical forms that contain IPD and PPE (Table 4).

Analysis of Pharmaceutical Formulations:

The presented methods were applied successfully for the determination of PPE and ENL in the pharmaceutical formulations and the results are shown in Table 5. Three replicated determinations were measured. Table 5 shows that satisfactory recoveries from 99.77% to 100.37% for PPE and 98.84% to 100.05% for ENL, that indicate high accuracy of the presented methods in determine PPE and ENL in their Tablets.

Moreover, the obtained results were compared to PPE and ENL reference methods^30-22 and t- test/ F- test were calculated; Table 5.

Table 2: Evaluation of accuracy and precision of the proposed methods (n =3).

Drug	Taken concentration (μg/mL)	Intra-day Precision and Accuracy			Inter-day Precision and Accuracy
Drug	Taken concentration (μg/mL)	Found concentration ± CL (μg/mL)	Recovery (%)	RSD%	Found concentration ± CL (μg/mL)	Recovery (%)	RSD%
ENL	16	16.030±0.201	100.18	0.51	16.053±0.087	100.33	0.218
	20	20.012±0.104	100.05	0.177	20.036±0.037	100.18	0.076
	24	24.044±0.228	100.22	0.46	24.033±0.100	100.13	0.168
PPE	4	4.045±0.091	100.12	0.936	4.015±0.032	100.37	0.329
	8	8.018±0.044	100.22	0.225	8.018±0.025	100.22	0.129
	10	10.013±0.034	100.13	0.148	10.036±0.247	100.36	0.995

Table 3: Robustness for the proposed methods (n=3).

Drug		Enalapril			Perindopril
Parameters	Changes	Found concentration (μg/mL)	RSD%	Recovery (%)	Found concentration (μg/mL)	RSD%	Recovery (%)
Wavelength λ _MAXnm	+2 nm	19.975	0.022	99.97	4.007	0.514	100.17
	+2 nm	27.998	0.0224	99.99	10.003	0.425	100.003
	-2 nm	19.974	0.03	99.97	4.001	0.531	100.02
	-2 nm	27.977	0.098	99.92	9.999	0.534	99.99
Interval width	1 nm	20.011	0.572	100.05	4.06	0.577	101.5
	1 nm	27.98	0.11	99.93	10.021	0.264	100.21
	2 nm	20.005	0.108	100.02	3.999	0.412	99.97
	2 nm	27.999	0.149	99.99	10.002	0.251	100.02
Scan speed	Fast	20.019	0.854	100.09	4.004	0.549	100.1
	Fast	28.031	0.23	100.1	9.95	0.49	99.5
	Slow	19.994	0.855	99.97	4.031	0.52	100.77
	Slow	28.006	0.28	100.02	10.016	0.636	100.16

Table 4: Selectivity of the proposed method in the combined pharmaceutical dosages containing ENL / HCTZ and PPE / IDP (n = 3).

Pharmaceutical preparation	Labeled amount (mg/form)	Found concentration ± CL (μg/mL) (ENL)	*SD	RSD%	Recovery (%)
Enapril Asia plus 5mg ENL/ 12.5 HCTZ	5/12.5	4.997 ± 0.063	0.025	0.321	99.95
Enapril Asia plus 10mg ENL/ 25 HCTZ	10/25	10.046 ± 0.036	0.014	0.184	100.46
Pharmaceutical preparation	Labeled amount (mg/form)	Found concentration ± CL (μg/mL) (PPE/IDP)	*SD	RSD%	Recovery (%)
Revosyl plus 4mg PPE/ 1.25 IPD	4/1.25	4.41 ± 0.118	0.047	0.271	110.25

Table 5: Determination of the studied drugs in its pharmaceutical preparations using proposed methods and comparing with reference methods (n = 3).

Pharmaceutical Preparation	Labeled amount (mg/form)	Recovery (%) ± SD (R %)		Statistic Tests
Pharmaceutical Preparation	Labeled amount (mg/form)	Proposed method	Reference method [22-30]	F-test	t-test
Enapril Asia	5	4.945 ± 0.040 (98.90 %)	4.941 ± 0.060 (98.83 %)	2.25	0.16
	10	9.884 ± 0.076 (98.84%)	9.856 ± 0.055 (98.56%)	1.90	0.62
	20	20.014 ± 0.032 (100.05%)	20.032 ± 0.041 (100.16 %)	1.64	0.94
Enalapril Alfares	5	4.946 ± 0.033 (98.93 %)	4.954 ± 0.041 (99.08 %)	1.54	0.40
	10	9.902 ± 0.132 (99.02%)	9.883 ± 0.144 (98.83 %)	1.19	0.22
	20	19.805 ± 0.141 (99.02%)	19.862 ± 0.096 (99.31 %)	2.15	0.67
Copendil	4	3.996 ± 0.067 (99.92%)	3.995 ± 0.102 (99.87%)	2.31	0.01
Copendil	8	8.030 ± 0.047 (100.37%)	8.003± 0.061 (100.03%)	1.68	0.60
Revosyl	4	3.990± 0.028 (99.77%)	3.994 ± 0.035 (99.85%)	1.56	0.15
Revosyl	8	8.023± 0.032 (100.29%)	8.001± 0.038 (100.02%)	1.40	0.76

*Average of three determinations. At 95% confidence limit the tabulated t- and F value at four degree of freedom are t=4.303 and F=16.00.

DISCUSSION:

In This study, extractive free methods were developed and validated to be carried out in short time at room temperature.

CONCLUSION:

These methods have reported new, free extractive spectrophotometric methods using BCG for the determination of perindopril erbumine and enalapril maleate in pure and pharmaceuticals drugs (tablets) in both single and companied dosage with hydrochlorothiazide and enalapril maleate and perindopril erbumine and Indapamide. The presented methods have advantages of other reported methods which characterized by simplicity, they do not need heating or wait time after ion-pair formation, not even adjusting pH compared with the methods of extraction. It is considered of high sensitivity and selectivity of enalapril alone or when companied with diuretic hydrochlorothiazide in pharmaceutical preparations and considered of high sensitivity and of perindopril in its pharmaceutical single dosage preparations. Also, there is no interference with excipients. The limit of detection (LOD) value is 0.125ug/mL of PPE and 0.230ug/mL of ENL and the limit of quantification (LOQ) is 0.381 ug/mL of PPE and 0.697ug/mL of ENL. The stoichiometry complex of PPE and ENL determined by Job’s method and Molar ratio method was found to be 1:1 for both drugs. The statistical parameters and the recovery data indicate good accuracy and precision of the methods and suitable for routine analysis of drugs in pharmaceutical formulations. Therefore, the validated methods could be useful for routine quality control assay of PPE and ENL in pure and dosage forms.

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Received on 25.05.2020 Modified on 11.08.2020

Research J. Pharm. and Tech. 2021; 14(6):3276-3282.

DOI: 10.52711/0974-360X.2021.00570


*Figure 2: effect of solvents on the ion-pair complexes formation (20 µg/mL of PPE and 20 µg/mL of ENL with BCG 310^-4 M).**	Figure 3: Effect of dye concentration on the absorbance of 10 μg/mL PPE and 20 μg/mL of ENL.