1. INTRODUCTION:

Captopril (CAP) is an angiotensin converting enzyme inhibitor. It is chemically known as (2S)-1-[(2S)-2-Methyl-3-sulphanylpropanoyl] pyrrolidine-2-carboxylic acid¹ (Figure.1a). It has generally worked as orally moderating heart failure medicine² and antihypertensive³. Captopril characterizes from other ACE inhibitors by its short half-life and it is an example of the sulfhydryl-containing group^4,5.

Hydrochlorothiazide (HCTZ) chemical name is 6-Chloro-3,4-dihydro-2H-1,2,4 benzothiadiazine-7-sulphonamide 1,1-dioxide (Figure.1b). It belongs to a class of medicines named thiazide diuretics antihypertensive⁶.

Hydrochlorothiazide binds to the carbonic anhydrase enzyme and inhibits it. It is usually used alone or in combining with other drugs for prevention of kidney stones and management of hypertension, diabetes insipidus, symptomatic edema, congestive heart failure, renal tubular acidosis, edema, and hypoparathyroidism and used in treating of osteoporosis⁷.

Figure 1. Chemical structures of (a) Captopril and (b) Hydrochlorothiazide

There are many articles mentioned for simultaneous estimation of captopril and hydrochlorothiazide in pharmaceutical dosage form including spectrophotometry^8-11, electrochemical analysis¹², and High-performance liquid chromatography (HPLC)^13-17 , but there is no method available for their determination using FT-IR spectroscopy.Various studies reported methods for determining hydrochlorothiazide in combination with otherdrugs^18-24. FTIR is a spread spectroscopic method used by pharmacologist scientists for the qualitative analysis of drugs because of its easy accessibility and ingrained simplicity in most of the research laboratories. The diverse pharmaceutical preparations including gases, films, liquids, surfaces, powders, fibers and solutions, can all be examined with a suitable choice of this sampling technique^25,26.

In recent years, Fourier transform infrared spectroscopy (FTIR) has been widely used for the quantitative determination for drugs and pharmaceutical dosage forms including roxithromycin²⁷, amoxicillin²⁸, acyclovir²⁹, furosemide³⁰, rosuvastatin calcium³¹, ibuprofen and paracetamol³², ceftazidime³³, ampicillin sodium³⁴, gliclazid³⁵, and omperazol³⁶.

Based on the above, in this study the aim was to develop a new, simple, fast, and unpublished method for simultaneous determination of Captopril and Hydrochlorothiazid in raw material and pharmaceutical dosage forms. This technique can be used as an advanced and environmental favorable choice in quality control.

2. EXPERIMENTAL:

2.1 Chemicals and reagents:

Working standard of Hydrochlorthiazid (Purity 99.55%) and Captopril (Purity 100.3%) were provided as a gift from Medico Laboratories, Homs- Syria and used without further purification. The diverse solid pharmaceutical formulations were obtained from national drug stores. Potassium bromide (Merck KGaA, Germany) was also used as a diluent and was previously dried at 120 for 2 h. The adjuvants present in the pharmaceutical formulations (avicil, crospovidone, talc, and magnesium stearate) were obtained from the pharmaceutical laboratories.

2.2. Equipment and software:

The FT-IR analysis was carried out on ALPHA-T spectrophotometer (Bruker, Germany). The device was controlled by IR spectra analysis software system OPUS 6.5 version for data analysis. FT-IR spectrum was registered in the Mid-range between 4000 and 400cm^-1 using 16 scans for every sample at a resolution of 4cm^-1. Generated spectrums were automatically corrected by KBr background spectrum which were previously measured. The other equipment used are as follows: oven (Memmert, Germany), analytical balance ±0.1mg (Sartorius, Germany), pestle and agate mortar, hydraulic press (Specac, United Kingdom) and 13mm evacuable pellet die (Specac, United Kingdom) were used for the preparation of pellets.The development of calibration curves was performed using Microsoft Excel (2013).

2.3. Calibration curve:

A stock mixture powder of equal concentration of Hydrochlorothiazide and Captopril combination in oven-dried KBr was prepared to obtain a KBr pellet of 100 mg. The mixture was put into 13mm evacuable die and was compressed using a press device under pressure of 10 tons for two minutes. Five different concentrations within the range of 0.4-2% w/w were made and measured at wavenumbers 4000-400cm-1. The preparation was repeated six times.

2.4. Interference study:

The interaction effect of excipients which present in commercial preparations has been studied. Each substance of the excipients (Avicil - Crospovidone - Talc - Magnesium stearate) and (Standard mix) were taken and measured separately.

2.5. Sample preparation procedure:

Twenty tablets were each weighed, and the average was calculated. All tablets were crushed and powdered until homogeneous. Suitable amount of these powders, which it was equivalent to the labeled content of one tablet was diluted by potassium bromide for getting 1.2% w/w concentration and condensed in the 13-mm evacuable die at a pressure of 10 tons for 2 min.

2.6. Method validation:

Method validation was achieved under a variety of United States Pharmacopeia 30 recommended test conditions and ICH^37,38,39,40.

3. RESULTS AND DISCUSSION:

3.1. Development of HCTZ and CAP assay method:

The FT-IR spectrum for pure mixture samples of HCTZ and CAP exhibited many absorbance bands. The centerd regions of 3115.92-3222.1cm^-1 and 1689.91-1800.73 cm^-1 corresponding to (NH2) and ester (C=O) functional groups for HCTZ and CAP respectively were selected for quantitative determination. The absorbance spectra for the diluted samples of various concentrations and selected peaks are shown in (Figure 2). The peak height (intensity) for the selected peaks were plotted against the concentrations to preparation of calipration curve using Microsoft Excel 2013.

The FT-IR spectrum in (Figure 3) shows that there is no overlapping with excipients used in producing of tablet dosage form.

Figure 2: Group of FTIR transmission spectra of HCTZ and CAP standards with prominent bands for quantitative

Figure 3: FT-IR spectrum of a: avecil, b: magnesium stearate, c: crosspovidone and d: talc

Figure 4: Calibration curves of CAP and HCTZ

3.2. Methodvalidation:

3.2.1. Linearity and range:

Five standard mixtures of HCTZ and CAP were prepared within the range (0.4, 0.8, 1.2, 1.6, 2 w/w %) in KBr. Each concentration was repeated six times in FT-IR. A curve between standard concentrations and peak height (intensity) values of selected peaks was plotted. The equation of the calibration curve based on the peak response was y = 0.3068x + 0.1485 with R² of 0.9989 for Captopril , and y = 0.09x + 0.0433 with R² of 0.9982 for Hydrochlorothiazid. (Figure 4) shows the result of linearity for HCTZ and CAP. Linear regression data, and ranges were listed in Table.1. The response of the selected peaks to the quantitative analysis in various concentrations of the CAP and HCTZ standards by FT-IR transmission shows in (Figure 5).

Figure 5: The response of selected peaks of CAP (A) and HCTZ (B) standards by FT-IR transmission

3.2.2. Accuracy:

Concentrations of 0.4, 0.8, 1.2, 1.6, and 2 w/w % have been used to study the accuracy of CAP and HCTZ. Recovery was defined by comparing the nominal concentration with the obtained concentration. Recovery of Captopri ranged between (98.19% and 101.99%) with mean recovery of 100.195% and RSD% of 1.13% Table 1.

Whereas for Hydrochlorothiazid recovery was (98.16%–101.94%) with mean recovery of 100.042% and RSD% of 1.38% Table 2. The recovery of the Captopril and Hydrochlorothiazid by proposed method is satisfactory as RSD% is not more than +2.0% and mean recovery between 98.0 – 102.0%.

Table 1: Accuracy of Captopril assay.

Conc. w/w%	Peak intensity	Calc. Conc.	Recovery %	AVR ^a	±SD	RSD %
0.4	0.273	0.41	101.450	99.821	1.630	1.63
0.8	0.391	0.79	98.802	100.160	1.245	1.24
1.2	0.524	1.22	101.994	100.545	1.660	1.65
1.6	0.637	1.59	99.515	99.923	0.407	0.41
2	0.768	2.02	100.962	100.527	1.048	1.04
Total				100.195	1.127	1.13

^an= 3

Table 2. Accuracy of Hydrochlorothiazid assay.

Conc. w/w%	Peak intensity	Calc. Conc.	Recovery%	AVR ^a	±SD	RSD %
0.4	0.079	0.397	99.17	100.09	1.604	1.60
0.8	0.114	0.786	98.19	99.58	1.389	1.39
1.2	0.151	1.197	99.72	100.03	1.414	1.41
1.6	0.19	1.630	101.88	100.49	1.837	1.83
2	0.224	2.008	100.39	100.02	1.697	1.70
Total				100.04	1.382	1.38

^an=3

Table 3: Results of repeatability of the developed FTIR method

Drug	Peak intensity									AVR ^a	±SD	RSD%
	1	2	3	4	5	6	7	8	9
Captopril (1.2 % w/w)	0.521	0.532	0.521	0.522	0.52	0.53	0.527	0.525	0.529	0.525	0.004	0.76
Hydrochlorothiazid (1.2 % w/w)	0.158	0.156	0.153	0.152	0.15	0.155	0.153	0.15	0.152	0.153	0.003	1.96

^an= 9

Table 4: Intermediate precision of Captopril and Hydrochlorothiazide

No. of measurement	Conc. w/w%		Peak intensity
Day.1	Captopril	Hydrochlorothiazide	Captopril	Hydrochlorothiazide
1	1.2	1.2	0.521	0.158
2	1.2	1.2	0.532	0.156
3	1.2	1.2	0.521	0.153
4	1.2	1.2	0.522	0.152
5	1.2	1.2	0.52	0.15
6	1.2	1.2	0.53	0.155
Day.2
1	1.2	1.2	0.53	0.16
2	1.2	1.2	0.529	0.152
3	1.2	1.2	0.531	0.157
4	1.2	1.2	0.527	0.156
5	1.2	1.2	0.537	0.153
6	1.2	1.2	0.533	0.154

AVR ^a			0.527	0.154
SD			±0.006	±0.003
RSD %			1.14	1.95

^an = 12

3.2.3. Precision:

3.2.3.1.Repeatability:

The standard concentrations 1.2% of CAP and 1.2% of HCTZ have been measured nine times. SD and RSD% of the response (peak intensity) have been calculated and the results were illustrated in Table 3. The RSDs % for repeatability of standard preparation were 0.76% for CAP and 1.96% for HCTZ; this proves that the precision of the proposed method is satisfying as RSD% is less than +2.0%.

3.2.3.2. Intermediate Precision:

Intermediate precision was confirmed by the determination of the following concentrations: 1.2% and 1.2% for HCTZ and CAP; respectively in two different days. The RSD% for intermediate precision of HCTZ is 1.95, whereas it is 1.14 for CAP suggesting that the precision of the proposed method is satisfying as RSD% is less than +2.0% (Table 4).

3. 2. 5. Limit of quantification and detection (LOQ and LOD):

The LOQ (Limit of Quantification) and LOQ (Limit of detection) were calculated from the calibration curve equation, using the following formula:

LOD: 3.3 σ / S

LOQ: 10 σ / S

Where:

σ = the standard deviation of the response, (It was calculated based on residual standard deviation of a regression line)

S = the slope of the calibration curve

The LOD values of CAP and HCTZ were 0.08 and 0.103 respectively, whereas the LOQ values were 0.245 for CAP and 0.313 for HCTZ

3.3. Pharmaceuticals assay:

Two Pharmaceuticals were analyzed for the combination of HCTZ and CAP (A^®and B^®) using the validated method.Quantification was performed in triplicates and the experiment yielded data listed in Table 5.®

The acceptable range mentioned in USP Pharmacopeia for Captopril and Hydrochlorothiazide Tablets is 90%–110% of the labeled amounts³⁷.

The actual contents of CAP and HCTZ in A^® were 98.91% and 99.66%; respectively of the labeled claims with RSDs % (0.67 and 1.84). The actual contents of CAP and HCTZ in B^® were 97.83% and 96.92%; respectively of the labeled claims with RSDs % (0.34 and 1.89).

Table 5: Results of pharmaceuticals assay (A^®&B^®) using the developed FTIR method

pharmaceuticals	A^®		B^®
Active ingredient (mg)	CAP 50 mg	HCTZ 25 mg	CAP 25 mg	HCTZ 12.5 mg
Theoretical concentrations (w/w %) ^a	1.2	1.2	1.2	1.2
Peak intensity	0.515	0.153	0.509	0.148
	0.510	0.149	0.510	0.146
	0.513	0.151	0.507	0.150
Found values (w/w %)	1.195	1.219	1.175	1.163
	1.178	1.174	1.178	1.141
	1.188	1.197	1.169	1.186
AVR ^b	1.187	1.196	1.174	1.163
SD±	0.008	0.022	0.004	0.022
RSD	0.67	1.84	0.34	1.89
content %	98.91	99.66	97.83	96.92

^asuitable concentrations in the range of linearity, which they were prepared by diluting an amount equivalent to the labeled content of each pharmaceutical. (Mentioned in sample preparation paragraph).

^bn= 3

4. CONCLUSION:

This method for simultaneous determination of CAP and HCTZ in pharmaceutical tablets was suitable analytical method, which was, cheap, eco-friendly. This technique is appropriate for substances with solubility problems because it does not require any extraction step of sample. The proposed FTIR method opens a unique window into the possibility of applying this technique in quality control (QC) analysis routinely performed in pharmaceutical manufacture laboratories.

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Received on 22.11.2022 Modified on 31.12.2022

Research J. Pharm. and Tech 2023; 16(8):3869-3874.

DOI: 10.52711/0974-360X.2023.00638