1. INTRODUCTION:

Cephradine (6R,7R)-7-[(2R)-2-amino-2-(cyclohexa-1,4-dien-1-yl)acetamido]-3-methyl-8-oxo-5-thia-1-azabicyclo [4.2.0]oct-2-ene-2-carboxylic acid. Cephradine like all Cephalosporins,, inhibit bacterial growth by interfering with a specific step in bacterial cell wall synthesis[1]. The first generation cephalosporins are active mainly against gram-positive bacteria and to a relative extent against gram-negative organisms [2].

Several methods have been developed for their determination of cephradine, including spectrophotometric methods [3-7], spectroflurometry [8-12], thin layer chromatography [13], high-performance liquid chromatography (HPLC) [14-19], electro chemical methods [20].

Captopril, 1 - [(2S)- 3 - mercapto -2 -methylpropionyl]-L-proline, (CPT) is an angiotensin-converting enzyme inhibitor, which reduces peripheral resistance end lowers blood pressure. It is extensively used for the treatment of hypertension and congestive failure [21]. Several methods have been developed for its determination, including specrootomtric methds[22-31], spectroflurometry [32 - 35], high-performance liquid chromatography (HPLC) [36 - 40], Electro chemical methods [41-46].

O-Phthalaldehyde has been recently shown to be a useful reagent for chemical modification of compounds containing the SH and NH₂ groups. It reacts covalently with closely spaced sulfahydryl and amino group respectively, to yield fluorescent isoindole derivatives [47].

A quantitative spectrophotometric method determination of ACE inhibitor captopril (SH) group and first generation cephalosporine cephradine (NH₂) group. The developed method is based on reaction of amino and thiol with o-phthalaldehyde to form isoindole derivatives. This method could be used for their analysis in pure forms and in pharmaceutical formulations.

2. EXPERIMENTAL:

2.1. Apparatus:

Labomed^® Spectro UV-VIS Double Beam (UVD-2950) Spectrophotometer with matched 1 cm quartz cells connected to windows compatible computer using UV Win 5 Software v5.0.5 (U.S.A).

Thermostatically controlled Water bath (WISD laboratory instruments, Korea).

Consort P400^® digital pH-meter for pH adjustment.

2.2. Materials and reagents

All solvents and reagents were of analytical grade and double distilled water was used throughout the work.

O-Phthaldhyde (Sigma). It was prepared as 0.25mg ml^-1 in methanol (for spectrophotometric analysis).

Borate buffer pH 9.5 was prepared from 0.1M boric acid (El Nasr Chemical Co. Cairo Egypt) and the pH was adjusted to pH 9.5 with 2M NaOH (El Nasr Chemical Co. Cairo Egypt) aqueous solution.

Cefradine (EIPICO) 10th of Ramadan City, Egypt, purity 96.46 %. Certified to contain 100% and was prepared as 1 mg ml^-1 in water (for spectrophotometric analysis).

Captopril (EIPICO), 10th of Ramadan City, Egypt, purity 97.5 %. It was prepared as 0.25 mg ml^-1 in water.

2.3. Pharmaceutical preparations:

Capotril® tablets used labeled to contain 25 mg captopril per each tablet (Batch No. 1041).

Pharcocef® vial labeled to contain 500 mg cefradine per each vial (Batch No. 00202912).

2.4. General Procedure.

2.4.1. Procedure for captopril. 1 ml captopril solution was transferred to 10-ml calibrated flasks (for spectrophotometric analysis). 1 ml OPA was added followed by 1 ml cefradine solution and 0.4 ml borate buffer, the volume was shaked well and completed with distilled water, and contents were mixed, and left for 10 minutes, absorbance was measured at 334 nm against blank which was prepared by the same manner except addition of captopril.

2.4.2. Procedure for capotril tablets. An amount equivalent to 50 mg of captopril was taken from the thoroughly powdered capotril^® tablets and transferred to 50 ml volumetric flask, shaked well and completed to the volume with water, filtered and then 25 ml were transferred to 100 ml volumetric flask and volume was completed by water (for spectrophotometry) and The assay was continued as mentioned under procedure 2.4.1.

2.4.3. Procedure for cefradine. 1ml cefradine solution was transferred to 10 ml calibrated flasks. 1 ml OPA was added followed by 1 ml captopril solution and 0.4 ml borate buffer, the volumes were shaked well and completed with water, contents were mixed and absorbance was measured at lmax 334 nm after 10 minutes against blank which was prepared by the same manner except addition of cefradine.

2.4.4 Procedure for pharcocef vials. An amount equivalent to 100 mg of cefradine was taken from the thoroughly powdered pharcocef^® vial and dissolved in water in 100-ml volumetric flask and volume was completed with distilled water. First 10 ml was discarded and the assay was continued as mentioned under procedure 2.4.3.

3. RESULTS AND DISCUSSION

OPA reacts in alkaline medium with substances bearing primary amino group to form isoindole derivatives in the presence of mercaptans. This type of derivatization can possibly be applied to primary aliphatic amines and to wide range of mercaptans to determine them spectrophotometrically [48].

In this procedure the cefradine (primary amino compound) and captopril (mercapto compound) react with OPA in borate buffer at pH 9.5 to form isoindole derivative with new lmax at 334 nm (fig. 6). The method was utilized to determine captopril using OPA and cefradine as reagent, and to determine cefradine using OPA and captopril as reagent.Other cephalosporins which contain amino group attached to heterocyclic ring as cefepime, cefotaxime and cefotriaxone cannot be determined by this method. This may be due to low basicity of amino group attached to heterocyclic ring.

OPA Isoindole derivative

Figure 1. Proposed reaction between O-phthalaldhyde, thiol, and amine for formation of isoindole derivative at pH 9.5.

The effect of concentration of the reagents was studied and showed that the excess of the reagents has no effect on the intensities. Calibration graphs were constructed by plotting the absorbance as a function of the concentration.

3.1. Absorption spectra:

Absorption spectra for determination of cefradine and Captopril were studied over range of 200 - 500 nm. After reaction of cefradine (primary amino compound) and captopril (mercapto compound) with OPA in borate buffer at pH 9.5 to form isoindole derivative with maximum lmax at 334 nm while maximum lmax of OPA at 245 nm as shown at fig.1.

Figure 1. Absorption spectrum of reaction product of captopril (a), cefradine (b) with OPA in borat buffer at pH 9.5 and OPA (c).

3.2. Effect of pH:

Different PHs were tested for this reaction ranged from pH 5 to pH 10 the most precise and accurate results obtained at PH 9.5. Therefore, PH 9.5 was used throughout experiments (Fig.2).

Figure 2. Effect of pH on the absorbance of the reaction coloured product at 334 nm.

3.3. Effect of OPA volume:

OPA volume was studied by varying the reagent volume while other factors were held constant. It was found that 1 ml OPA gave the highest absorbance (Fig. 3).

Figure 3. Effect of OPA volume on the absorbance of the reaction coloured product at 334 nm.

3.4. Effect of time:

Time required for the reaction to proceed after reagent addition was studied. The reaction was found to be complete in 10 minutes while contact times up to 25 minutes had been examined and no higher absorbance was detected (Fig.4).

Figure 4. Effect of time on the absorbance of the reaction coloured product at 334 nm.

3.5. Effect of Borate Buffer volume:

Borete Buffer volume was studied by varying their volume while other factors were held constant. It was found that 0.4 ml Borate Buffer give the highest absorbance.(Fig.5).

Figure 5. Effect of Borete Buffer volume on the absorbance of the reaction coloured product at 334 nm.

3.6. Effect of temperature:

Temperature required for the reaction to proceed after reagent addition was studied. The reaction was found to be complete at room temperature (25±5 ºC) while temperature up to 100 ºC had been examined and no higher absorbance was detected (Fig.6).

Figure 6. Effect of temperature on the absorbance of the reaction coloured product at 334 nm.

3.7. Method validation:

The developed methods were validated according to international conference on harmonization guidelines [49].The linearity range of absorbance as a function of drug concentration (Table 1) provides good indication about sensitivity of reagents used. Calibration curves have correlation coefficients (r) higher than 0.999 indicating good linearity. The accuracy of the methods was determined by investigating the recovery of drugs at concentration levels covering the specified range (three replicates of each concentration). The results showed excellent recoveries (table 2). Also, the Limit of detection (L.D.), Limit of quantitation (L.Q.), and Molar absorbitivity were calculated. Intra - day precision was evaluated by calculating standard deviation (SD) of five replicate determinations using the same solution containing pure drug (table 5). The SD values revealed the high precision of the methods For inter - day reproducibility on a day - to - day basis, a series was run, in which the standard drug solutions were analyzed each for five days(table 5). The robustness of the methods was evaluated by making small changes in pH, the volume of buffer, the volume of OPA and time where the effect of the changes was studied on the percent recovery of drugs (table 6). The changes had negligible influence on the results as revealed by small SD values.

3.6. Applications:

Some Pharmaceutical formulations containing stated drugs have been successfully analyzed by the proposed methods. Results obtained were compared to those obtained by applying reported reference methods by the reaction with 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) in the presence of sodium tetraborate in absolute methanol in case of captopril [29] and the reaction of hydrolysate with 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) in the presence of HCl in case of cefradine[7]. Since Student’s t-test and F-test were performed for comparison. Results are shown in tables 2 and 3 where the calculated t and F values were less than tabulated values which in turn indicate that there is no significant difference between proposed methods and reference ones relative to precision and accuracy.

4. CONCLUSION:

Unlike GC and HPLC techniques, spectrophotometry is simple and inexpensive. The proposed methods require only OPA reagent and borate buffer which are cheaper and readily available, pH adjustment is required and the procedures do not involve any critical reaction conditions or tedious sample preparation. Moreover, methods are simple, fast, accurate, adequately sensitive and free from interference by common additives and excipients which make it as choice for routine quality control analysis. The proposed spectrophotometric methods overcome the problem of low absorptivity of cited ACE inhibitors in UV region. The recovery % obtained by the proposed methods is between 98.3% and 101.6%, within the acceptance level of 95% to 105%. The methods have been successfully applied for the analysis of marketed tablets and vials.

Table(1). Analytical parameters for the determination of cefradine and captopril.

OPA		PARAMETERS
Captopril	Cefradine
334	334	λmax, nm
1	1	Volume of OPA, ml
0.4	0.4	Volume of buffer, ml
25±5	25±5	Temperature, ºC
10	10	Time of reaction, min
9.5	9.5	pH
0.5-2.5	0.1-0.7	Beer's law limits, µg/ml
y=0.324x+0.045	y=1.066 x + 0.053	Regression equation
0.999	0.999	Correlation Coefficient

bx, where y is the absorbance, a is the intercept, b is the slope and x is the concentration in µg/ml.

Table(2). Results of the analysis for determination of cefradine and captopril using isoindole derivatives method.

Parameters	OPA
	Cefradine*			Captopril*
	Taken µg/ml	Found µg/ml	Recovery %	Taken µg/ml	Found µg/ml	Recovery %
	0.1	0.098	98.31	0.5	0.494	98.77
	0.2	0.196	98.31	1	1.006	100.6
	0.3	0.3043	101.44	1.5	1.5277	101.8
	0.4	0.405	101.36	2	2.0277	101.38
	0.5	0.508	101.69	2.5	2.472	98.895
	0.6	0.603	100.49
	0.7	0.691	98.72
Mean			100.047			100.30
±SD			1.544			1.409
±RSD			1.544			1.405
±SE			0.4657			0.446
Variance			2.4489			1.986
Slope			1.086			0.3246
L.D.			0.0295			0.148
L.Q.			0.09841			0.494

Apparent Molar absorbitivity L.Mol^-1.cm^-1			440630.2			79382.9

* Average of three independent procedures.

Table (3). Statistical analysis of results obtained by the proposed methods applied on captopril (capotril^®) tablets compared with reference method.

Parameters	Proposed method	Reported method[29].
N	5	5
Mean Recovery	99.83	98.53
±SD	1.346	1.21
±RSD	1.348	1.23
±SE	0.602	0.429
Variance	1.81	1.48
Student-t	1.61(2.57)a
F-test	1.23(6.256)b

a and b are the Theoretical Student t-values and F-ratios at p=0.05.

Table (4). Statistical analysis of results obtained by the proposed methods applied on cefradine (pharcocef^®) vials compared with reference method.

Parameters	Proposed method	Reported method [7].
N	5	5
Mean Recovery	100.158	98.468
±SD	0.952	1.29367
±RSD	0.950	1.3138
±SE	0.4256	0.4312
Variance	0.905	1.674
Student-t	2.34(2.57)a
F-test	1.85(6.256)b

a and b are the Theoretical Student t-values and F-ratios at p=0.05.

Table (5).Results of the intraday and interday precision for the determination of cefradine, and captopril using isoindole derivatives method.

intraday and interday precision
Intraday		Conc.ug/ml	Drug
RSD	mean ± SD	Conc.ug/ml	Drug
0.68	100.9 ± 0.675	0.5	cefradine
0.38	101.32 ± 0.38	2	captopril
interday		conc.ug/ml	drug
RSD	mean ±SD	conc.ug/ml	drug
0.45	99.8± 0.46	0.5	cefradine
0.75	101.28 ± 0.748	2	captopril

Table(6).Results of the robustness for the determination of cefradine, and captopril using isoindole derivatives method.

Mean of recovery ± SD		Parameters
Captopril	Cefradine
98.6 ±1.6	98.3 ± 1.47	pH 8.9
101.7 ± 0.17	101.8± 0.82	pH 9.1
99.08 ± 1.4	100.37 ± 0.75	Buffer0.38ml
100.8 ± 0.47	101.1± 0.43	Buffer0.42ml
98.5 ± 1.7	98.87± 1.44	OPA0.95 ml
101.5 ± 0.15	101.6 ± 0.81	OPA1.05ml
99.38±1.2	98.5 ±1.8	Time9 min.
101.5± 0.15	101.6± 0.81	Time11 min.

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Received on 09.12.2013 Accepted on 15.02.2014

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