The ACE inhibitors block the angiotensin converting enzyme that cleaves the terminal two peptides from angiotensin I (decapeptide) to form the potent vasoconstrictor angiotensin II (octapeptide) [1,2] and lower the BP by reducing peripheral vascular resistance without increasing cardiac output rate and contractility. Comparatively, all ACE inhibitors (Figure 1) have similar antihypertensive efficacy they effectively block the conversion of angiotensin 1 to angiotensin II and all have similar therapeutic indications, adverse effect profile and contraindications.

Figure 1: Structure of ACE inhibitors.

Lisinopril, (S)-1-[N²-(1-carboxy-3-phenyl propyl) -L- lysyl] -Lproline dihydrate a lysine analogue of enalaprilate, is a white to off-white crystalline, odorless powder Enalapril(S)-1-[N-[1-(ethoxycarbonyl)-3- phenylpropyl]-L-alanyl]-L-proline,(Z)-2- butenedioate is the maleate salt of enalapril a derivative of 2-amino acid, L-alanine and L-proline. Enalapril is a pro-drug it is bioactivated by hydrolysis of the ethyl ester to enalaprilate active form.

Captopril 1-[(2S)-3-mercapto-2-methyl-1-oxo-proptonyl]-Lproline is the first orally active and specific inhibitor of angiotensinconverting enzyme. Fosinopril sodium chemically designated as L-proline, 4-cyclohexyl-l-[[[2-methyl-l-(l¬oxopropoxy) propoxy] (4-phenylbutyl) phosphinyl] acetyl]-, sodium salt, is an ester prodrug which is hydrolyzed to pharmacologically active fosinopril at a specific competitive inhibitor of angiotensin converting enzyme.

Several methods has been reported for determination of lisinopril [3-8] enalapril [9-15], captopril [16-21] and fosinopril [22] by HPLC alone or in combination with other drugs, but there is no single method reported for determination of these ACE inhibitors together.

The measurement of ACE-Inhibitors serum concentration is essential to optimize therapy and to avoid sub therapeutic or toxic concentrations particularly for patients with both impaired renal and hepatic functions. This paper describes an isocratic reversed-phase high performance liquid chromatographic method for the determination of four ACE-Inhibitors: lisinopril, enalapril, captopril and fosinopril. The isocratic HPLC method uses a simple mobile phase, UV-detection at 225 nm, and a simple sample-preparation step for serum samples. UV-detection is simple, rapid, selective, and reproducible, and sensitivity is adequate for routine use. Direct determination of the examined compounds in small volumes of human blood serum can be accomplished by protein precipitation using acetonitrile. The method was validated in terms of linearity, accuracy, precision, sensitivity, selectivity, and stability.

Wavelength selection

In addition, the UV spectra of individual drugs were recorded in the wavelength range from 200 to 400 nm and overlap to each other. It was found that all the drugs exhibited the high response at 225 nm.

Sample preparation

Standard stock solution was prepared by dissolving 10 mg each of enalapril, captopril, lisinopril and fosinopril in the mobile phase and the volume made up to 100 mL with the same solvent. For the calibration curve, six calibrators of drugs were prepared by making serial dilutions from stock solution over the range of 5-100 μgmL^-1.

Pharmaceutical grade enalapril was gifted by MSD Laboratories Limited, Karachi, Pakistan. Captopril, lisinopril and fosinopril were gifted from Bristol Meyers Pvt Ltd, Atco Laboratories Ltd respectively. Formulations of enalapril namely (renitec 10 mg), captopril (capoten 25 mg), lisinopril (lisinopril 10 mg) and fosinopril (fosinopril 10 mg) were obtained from retail pharmacies. All reagent used were of HPLC grade. Methanol and acetonitrile used were of HPLC grade from E. Merck, Germany. Orthophosphoric acid 85% used were of analytical grade (Merck, Germany). HPLC grade water was used to prepare mobile phase. Stock solutions and working solution were prepared daily. All solutions were filtered through 0.45 μm filter and degassed using sonicator.

Instrumentation

Shimadzu HPLC system equipped with a LC-10 AT VP pump, an SPD-10 AV VP UV–VIS dual wave length detector, integrated via Shimadzu model CBM-102 Communication Bus Module to P-IV computer. Shimadzu CLASS-GC software (Version 5.03) was used for data acquisition and mathematical calculations. Rheodyne manual injector fitted with a 20 μL loop. Chromatographic separation was carried out on a Purospher STAR RP-and Hypersil ODS column C-18 column (250×4.6 mm, with a particle size of 10 μ) and DGU- 14 AM on-line degasser. UV visible 1601 Shimadzu double beam spectrophotometer.

Assay in formulation

Twenty tablets of each lisinopril, enalapril, captopril and fosinopril were weighed to obtain the average tablet weight and then powdered. Equivalent to 10 mg of enalapril, 25 mg of captopril, 10 mg of lisinopril and 10 mg of fosinopril were weighed and the solution was made up to 100 mL with mobile phase. The resulting mixture was allowed to stand for 1 hour with intermittent sonication to ensure complete solubility of the drug (stock solution). This stock solution was filtered and the filtrate was diluted to the desired concentration. All sample and standard solutions were filtered through 0.45 μm filter paper before injection into the system. A placebo tablet was also subjected to the same process as discussed above. The possibility of excepients interference in the analysis was studied.

Assay in serum

Plasma samples, obtained from healthy volunteers, were collected and stored. To 1.0 mL of plasma, 9.0 mL of acetonitrile was added; the mixture was vortexed for 1 min and than centrifuged for 10 min at 10,000 rpm and the supernatant was filtered by 0.45-micron membrane filter. An aliquot of serum sample was fortified with lisinopril, enalapril, captopril and fosinopril to achieve final concentration.

Method validation

To optimize the operating conditions for isocratic RP-LC detection of all analytes, a number of parameters such as the mobile phase composition, pH and the flow rate were varied. Various ratios (50:50, 60:40, 70:30, v/v) of methanol: water was tested as starting solvent for system suitability study. The variation in the mobile phase led to considerable changes in the chromatographic parameters, like peak symmetry, capacity factor and retention time. The pH effect showed that optimized conditions are reached when the pH value was 3, producing well resolved and sharp peaks for all drugs assayed. Henceforth, in the present method pH adjusted to 3 using wavelength 225 nm (isobestic point) was used. However, the ratio of (60:40 v/v) ACN: water pH adjusted to 3 with phosphoric acid as mobile phase (filtered through a 0.45 micron filter), a flow rate of 1.0 mLmin^-1 was chosen as optimal condition. The International Conference on Harmonization [23] has defined these validation parameters which are summarized below.

System suitability

Before sample analysis, six consecutive replicate analysis of the drugs at a concentration of 100 μgmL^-1 for lisinopril, enalapril, captopril and fosinopril were assessed for repeatability, peak symmetry, theoretical plates and resolution. Results are depicted in Table 1, sharp and symmetrical peaks were obtained with good base line resolution (Figure 2).

Figure 2: Representative chromatogram of ACE inhibitors in API.

Table 1: System suitability parameters sensitivity of the proposed method.

Specificity

The specificity of the method was observed by analyzing the pharmaceutical formulation. The excepients did not show any interference with the peak of ACE inhibitors. No significant changes in AUC or retention time of the drug in presence of excepients.

Range and linearity

Linearity studies were performed on 6 different concentrations. The calibration curves were linear in the range of 5-100 μgmL^-1 for lisinopril, enalapril, captopril and fosinopril with correlation coefficient (r²) of ≥ 0.9997 (Table 2). Statistical soft ware was used to generate linear regression equations for all calibration curves.

Analytes	Goodness of fit (r2)	Standard error	Standard error of estimate	Intercept value	Slope	P
Bulk material
Lisinopril	0.999	10514	18750	31517	49133	0
Enalapril	0.999	4432	6217	47360	49953	0
Captopril	0.996	10515	1475	5399	60415	0
Fosinopril	0.999	7206	10108	32219	34600	0
In serum
Lisinopril	0.999	11058	18512	26548	49331	0
Enalapril	0.998	4352	6105	48474	49959	0
Captopril	0.999	7642	1020	5672	6381	0
Fosinopril	0.999	7378	10350	33125	34595	0

Table 2: Regression statistics of the proposed method.

Limit of detection and quantitation

LOD=3.3?/S and LLOQ= 0?/S; where ο is the standard deviation of the lowest standard concentration and S is the slope of the standard curve. The LOD for this assay were 0.9, 3.9, 2.0 and 0.5 ngmL^-1 and limit of quantitation were 2.8, 12, 8 and 0.7 ngmL^-1 for lisinopril, enalapril, captopril and fosinopril respectively.

Accuracy and precision

The precision was investigated with respect to repeatability (intraday precision) and intermediate precision (inter-day precision) and was expressed as relative standard deviation (RSD). These experiments were repeated over three days period to evaluate inter mediate precision. Accuracy of the method was determined by adding known amount of all analytes (lisinopril, enalapril, captopril and fosinopril) in the pharmaceutical formulations. All measurements were performed in triplicates (Tables 3 and 4).

	Lisinopril			Enalapril			Captopril			Fosinopril
Conc.	RSD	Rec	Rec. Conc.	RSD	Rec	Rec. Conc.	RSD	Rec.	Rec. Conc.	RSD	Rec.	Rec. Conc.
5	0.77	99.6	4.98	0.9	101.76	5.35	0.94	100.54	5.22	0.94	100.5	40.22
10	0.07	101.47	10.15	0.79	100.2	10.1	0.6	100.37	10.3	0.6	100.4	80.3
15	0.8	99.95	14.99	0.02	100.87	15.5	0.46	100.06	15.07	0.46	100.1	120.1
20	0.65	99.85	19.97	0.75	100.11	20.1	0.12	100.43	20.69	0.12	100.4	160.7
25	0.18	100.07	25.01	0.7	100.09	25.1	0.09	100.11	25.21	0.09	100.1	200.2
50	0.11	100.02	50.01	0.22	99.98	50	0.14	99.97	50.9	0.14	99.97	399.9
Spiking (Bulk material -- % level)
80	0.12	99.95	8	0.37	99.92	7.99	0.12	99.95	8	0.25	100.2	8.01
100	0.22	100.06	10.01	0.16	99.9	9.99	0.22	100.06	10	0.12	99.95	10
120	0.36	99.69	11.96	0.09	99.92	12	0.36	99.69	12	0.06	99.88	11.99
Spiking (Serum -- % level)
80	0.13	100.02	8	0.34	100.05	8	0.37	99.92	7.99	0.25	100.2	8.01
100	0.15	100.17	10.02	0.13	99.87	9.99	0.16	99.9	9.99	0.12	99.95	10
120	0.332	100.11	12.01	0.09	99.94	12	0.09	99.92	11.99	0.06	99.88	11.99

Recovery (Rec.) and RSD in %; Concentration (Conc.) and Recovered Concentration (Rec. Conc.) in μgmL^-1.

Table 3: Method accuracy from recovery assays for the studied analytes.

Conc.	Day 1 (%RSD)				Day 2 (%RSD)				Day 3 (%RSD)
µgmL-1	LSP	ENP	CAP	FSP	LSP	ENP	CAP	FSP	LSP	ENP	CAP	FSP
(Bulk material)
5	0.351	0.016	0.390	0.044	0.062	0.459	0.309	0.122	0.115	0.341	0.069	0.076
10	0.116	0.025	0.101	0.098	0.070	0.048	0.140	0.014	0.079	0.249	0.020	0.136
15	0.149	0.035	0.007	0.024	0.009	0.018	0.117	0.291	0.032	0.061	0.118	0.112
20	0.003	0.070	0.055	0.000	0.015	0.219	0.012	0.104	0.012	1.005	0.064	0.004
25	0.017	0.019	0.081	0.023	0.065	0.019	0.124	0.006	0.011	0.003	0.260	0.063
50	0.056	0.014	0.110	0.055	0.029	0.402	0.002	0.107	0.004	0.620	0.049	0.024
(Serum)
5	0.452	1.009	0.021	0.380	0.083	0.509	0.060	0.003	0.125	0.341	0.069	0.076
10	0.353	0.431	0.039	0.488	0.030	0.208	0.160	0.050	0.079	0.249	0.020	0.536
15	0.098	0.095	0.049	0.162	0.024	0.021	0.004	0.119	0.032	0.061	0.108	0.112
20	0.140	1.007	0.620	0.123	0.103	0.107	0.043	0.021	0.012	1.005	0.004	0.098
25	0.006	0.011	0.108	0.079	0.028	0.008	0.001	0.035	0.010	0.003	0.260	0.163
50	0.187	0.109	0.143	0.083	0.006	0.303	0.006	0.005	0.008	0.620	0.049	0.224

Table 4: Intra-and inter day, variation\intermediate precision of the method.

Ruggedness

The ruggedness was established by determining lisinopril, enalapril, captopril and fosinopril using the same chromatographic system and the same column by two analysts on different days. The results indicated that the method was capable with high precision. Additionally good separations were achieved which suggested that the method was selective for all components under the test (Table 4).

Robustness

Robustness of the methods was checked by making small deliberate changes in the mobile phase (± 5%), pH (± 0.01) and flow rate. These variations had a minor effect in operating conditions. One parameter was changed in the experiments at a time and assays were carried out in triplicate.

Applicability of method

The developed method was applied for the determination of drug content in marketed formulations (Figure 3). Similarly the % recoveries and r² of dugs in presence of serum shown in (Figure 4) that the applicability of method for therapeutic purposes also.

Figure 3: Representative chromatogram of ACE inhibitors in formulations.

Figure 4 Representative chromatogram of ACE inhibitors in human serum.

After studying all the results obtained by HPLC studies it was concluded that the present method was fast and easy to perform, having low LLOQ and LLOQ values, was found to be linear up to a wide range of analytes and showing high % of recoveries. So, this method is good for the simultaneous determination of ACE Inhibitors (lisinopril, enalapril, captopril and fosinopril) in dosage formulation. This method is used for therapeutic purpose also. Moreover, in quality control lab it can be effectively and efficiently used to save time and excessive use of chemicals by preparing single samples for all the drugs to be assayed.