INTRODUCTION:

Hydrochlorothiazide (HCTZ) (Figure 1A) is chemically 6-chloro-1, 1 dioxo-3, 4-dihydro-2H-1, 2, 4-benzo thiadiazine-7-sulfonamide¹. Hydrochlorothiazide (Figure 1A) belongs to the thiazide class of diuretics and acts on kidneys to reduce sodium reabsorption in the distal convoluted tubule. This increases the osmolarity in the lumen causing less water to be reabsorbed from the collecting ducts, finally increasing urinary output. It is often used in the treatment of hypertension, congestive heart failure, symptomatic edema and the prevention of kidney stones. Telmisartan (TM) is chemically 2-(4-{[4-methyl-6-(1methyl-1H-1, 3-benzodiazol-2-yl)-2-propyl-1H-1, 3-benzodiazol-1-yl] methyl} phenyl) benzoic acid.

Telmisartan (Figure 1B) also acts as a selective modulator of peroxisome proliferator activated receptor gamma (PPAR-γ), a central regulator of insulin and glucose metabolism.

Telmisartan displaces angiotensin II with very high affinity from its binding site at the AT1 receptor subtype, which is responsible for the known actions of angiotensin II. Telmisartan does not exhibit any partial agonist activity at the AT1 receptor. Telmisartan binds selectively with the AT1 receptor and does not reveal relevant affinity for other receptors nor does it inhibit human plasma renin or block ion channels. It is believed that the dual mode of action of Telmisartan may provide protective benefits against the vascular and renal damage caused by diabetes and cardiovascular disease². Telmisartan has binding affinity 3000 times with AT-2 receptor than AT-1 receptor.

Figure 1A: Hydrochlorothiazide (HCTZ)

Figure 1B: Telmisartan (TM)

Literature survey revealed that Lories et al developed first derivative, ratio derivative spectrophotometry, TLC densitometry, and spectrofluorimetry³ for the simultaneous determination of Telmisartan and Hydrochlorothiazide. Shah et al developed HPTLC⁴ method and Yan et al established LC-MS⁵ method in human plasma for the simultaneous quantitation of Telmisartan and Hydrochlorothiazide. Very few HPLC methods were developed^6-12for the simultaneous determination of Telmisartan and Hydrochlorothiazide in human plasma, pharmaceutical dosage forms and impurities. In the present study the authors proposed a new stability indicating RP-HPLC method for the simultaneous determination of Telmisartan and Hydrochlorothiazide on gradient mode and the method was validated as per ICH guidelines.

MATERIALS AND METHODS:

Chemicals and reagents:

Telmisartan standard (purity 99.0%) and Hydrochlorthiazide standard (Purity 99.0%) were obtained from SUN Pharma (India). Acetonitrile (ACN) (HPLC grade), formic acid, HCl, NaOH and hydrogen peroxide (30% w/w) solution were purchased from Merck (India) and other chemicals and solvents used were of analytical grade. Water (HPLC grade) was obtained from Milli-Q water system. The combination of Telmisartan and Hydrochlorthiazide is available as tablets commercially with brand names TEMAS-HT ® (Genesis Biotech Inc.), ASTEL-H (A S Pharmaceutical (India) Pvt. Ltd and ZITELMI-H ® (FDC Limited) (Telmisartan 40 mg and Hydrochlorthiazide 12.5mg).

Preparation of stock solution:

Telmisartan (1600μg/ml) and Hydrochlorothiazide (5000 μg/ml) stock solutions were prepared by accurately weighing 40mg of TM and 125mg of HCTZ in a 25mL volumetric flask with mobile phase. Standard solution prepared by further diluting 5mL of the stock solution to 50mL with mobile phase. Working standard solutions were prepared on daily basis from the stock solutions by dilution with mobile phase and the solutions were filtered through 0.45μm membrane filter prior to injection.

Preparation of Formic acid buffer (0.1% v/v) solution:

1mL of Formic acid (HPLC grade) was diluted with water in a 1000mL volumetric flask (0.1% v/v) and sonicated to dissolve completely and filtered through 0.45µm filter.

Instrumentation and chromatographic conditions:

Liquid chromatographic separation was achieved by using a X Bridge shield RP C-18 (150mm × 3.0mm, 3.5 µm) column of Waters HPLC with Empower2 software and photodiode array detector, maintained at 45ºC. Gradient mode elution was performed using Acetonitrile and 0.1% v/v formic acid. The overall run time was 20 min. and the flow rate of the mobile phase was 0.8 mL/min. The wavelength of the PDA detector was set at 235nm. 5µl of sample was injected into the HPLC system.

Method validation¹³:

The method was validated for the following parameters: linearity, limit of quantitation (LOQ), limit of detection (LOD), precision, accuracy, system suitability, selectivity and robustness.

Linearity:

A series of solutions were prepared by diluting the stock solutions of Hydrochlorothiazide (2-600µg/ml) and Telmisartan (0.64-200µg /ml) with mobile phase. 5µl of mixture of these solutions (n=3) was injected in to the HPLC system and the peak area of each of the drug was noted from the chromatogram.

Precision, Accuracy and Robustness:

The intra-day precision of the assay method was evaluated by carrying out 6 independent assays of Telmisartan and Hydrochlorothiazide (Telmisartan 160 μg/mland Hydrochlorothiazide 500μg/ml) and the % RSD was calculated. The accuracy of the assay method was evaluated in triplicate at three concentration levels (80, 100 and 120%), and the percentage recoveries were calculated. Standard addition and recovery experiments were conducted to determine the accuracy of the method for the quantification of Hydrochlorothiazide & Telmisartan respectively in the drug product and the % RSD was calculated. The robustness of the assay method was established by introducing small deliberate changes in the HPLC conditions which included flow rate (0.72 and 0.88 mL/min), percentage of acetonitrile in the mobile phase (absolute ±2% composition) and column oven temperature (± 5°C).

Analysis of commercial formulations:

Twenty tablets of two different brands containing Telmisartan and Hydrochlorothiazide were procured from the local medical store and powder equivalent to 12.5mg Hydrochlorothiazide and 40mg Telmisartan was accurately weighed, diluted, sonicated for 30 minutes and finally made up to volume with mobile phase in a 25 mL volumetric flask and diluted further as per the requirement. The contents were and 5µl of solution was injected into the HPLC system and the filtered through 0.45µm filter corresponding peak areas of the two drugs were noted. The percentage recovery was calculated from the linear regression equation using the mean peak area obtained from the respective chromatograms.

Forced degradation studies¹⁴:

Forced degradation studies are carried out to establish the degradation pathways of drug molecules and the sensitivity/resistance of the drug molecules towards the stressed conditions. Forced degradation studies were intended to ensure the effective separation of Telmisartan and Hydrochlorothiazide in presence of their degradants.

Acidic degradation:

The combined solution of Telmisartan and of Hydrochlorothiazide was treated with 1N HCl and refluxed for 2 hours in thermostat maintained at 80°C. The stressed sample was cooled, neutralized and diluted with mobile phase as per the requirement and filtered through 0.45µm filters. 5µL of this solution was injected into the HPLC system and the peak area of the chromatograms was noted.

Alkaline degradation:

The combined formulation of Telmisartan and of Hydrochlorothiazide was treated with 0.1N NaOH and refluxed for 2 hours in thermostat maintained at 80°C. The stressed sample was cooled, neutralized and diluted with mobile phase as per the requirement and filtered through 0.45µm filter. 5µL of this solution was injected into the HPLC system and the peak area of the chromatograms was noted.

Oxidative degradation:

The combined formulation of Telmisartan and of Hydrochlorothiazide was treated with 1% H₂O₂ and refluxed for 2 hours in thermostat maintained at 80°C. The stressed sample was cooled and diluted with mobile phase as per the requirement and filtered through 0.45 µm filter. 5µL of this solution was injected into the HPLC system and the peak area of the chromatograms was noted.

RESULTS AND DISCUSSION:

A new stability indicating RP-HPLC method has been developed for the simultaneous determination of Telmisartan and Hydrochlorothiazide on gradient mode and the method was validated as per ICH guidelines. The present proposed method was compared with the previously published methods in the literature in Table 1.

Table 1: Literature survey

Mobile phase (v/v)	λ (nm)	Linearity (mg/mL)	Comments	Ref.
Chloroform: Methanol: Toluene (2:5:5) (ng/spot)	272	250-500 (TM) 200-700 (HCTZ)	HPTLC	4
Acetonitrile-10 mM Ammonium acetate: Formic acid (Gradient mode) Probenecid (Internal standard)	-	0.001-0.6	LC-MS/MS Human plasma	5
Methanol: 10 mM Ammonium acetate solution (pH 6.0) (35:65) (Gradient mode)	270	1-10 (TM) 0.31-3.12 (HCTZ)	HPLC Human plasma	6
Methanol: Acetonitrile (70: 30); Rabeprazole (Internal standard)	270	2-10 (TM) 0.62-3.12 (HCTZ)	HPLC	7
Solvent A (Potassium dihydrogen phosphate and Sodium 1- Hexane sulphonic acid aq. sol) (pH 3.0 with o-phosphoric acid): Solvent B (Acetonitrile: Methanol) (80:20) (Gradient mode)	270	150-960 (TM) 150-960 (HCTZ)	HPLC (Impurities)	8
Buffer and mixture, acetonitrile and methanol (50:50) (pH adjusted to 3.0 with o-phosphoric acid)	298 (TM) 270 (HCTZ)	50-150	HPLC Rt 18.43 min (TM) Rt 8.11 min (HCTZ)	9
50 mM Ammonium acetate: Acetonitrile (60:40) (pH 5.5 adjusted with acetic acid) Methyl paraben (Internal standard)	260	10-150 (TM) 5-75 (HCTZ)	HPLC	10
Acetonitrile:0.05 M KH₂PO₄ (pH 3.0) (60:40)	271	4.1-20.48 (TM) 1.28-6.4 (HCTZ)	HPLC	11
Potassium dihydrogen ortho phosphate (pH 3.0 with ortho phosphoric acid): Acetonitrile (40: 60)	271	4-12 (TM) 1.3-3.9 (HCTZ)	HPLC	12
0.1% Formic acid: Acetonitrile (Gradient mode)	254	0.64-200 (TM) 2-600 (HCTZ)	HPLC Stability indicating	Present work

Table 2: Details of columns used during method optimization

S. No.	Column particulars	Observation
1	Luna C-18(2) (150 x 3.0mm, 3.5µm)	HCTZ peak eluting in the void
2	YMC pro pack C-18 (150 x 3.0mm, 3.5µm) 45°C	HCTZ peak eluting in the void
3	Kromasil C-18 (150 x 3.0mm, 3.5µm) Flow rate 1.1 mL/min	HCTZ peak eluting in the void. Telmisartan peak co-eluting with the blank peak.
4	X Bridge RP-18 (150 x 3.0mm, 3.5µm)	Telmisartan peak co-eluting with the blank peak
5	X Bridge Shield RP-18 (150 x 3.0mm, 3.5µm)	Telmisartan peak was separated from blank peak

Method development and optimization:

Selection of mobile phase and its pH

0.1% formic acid (volatile buffer) was chosen as aqueous phase and acetonitrile as organic phase.

Selection of column:

For selection of column, a spiked sample of Telmisartan was prepared with Hydrochlorothiazide and injected HPLC system with different columns. The required system suitability criterion was obtained using X Bridge Shield RP-18 (150 x 3.0mm, 3.5µm) column (Table 2). The chromatogram of the blank was shown in Figure 2.

Method validation:

Linearity:

Hydrochlorothiazide obeys Beer-Lambert’s law over the concentration range 2-600µg/ml and Telmisartan 0.64-200µg/ml respectively (Table 3). The representative chromatogram of Hydrochlorothiazide and Telmisartan was shown in Figure 3A. The linear regression equations were found to be y = 10664x - 4074.7 (R² = 0.9998) and y = 28449x+4219.9 (R² = 0.9999) for Hydrochlorthiazide (Figure 4A) and Telmisartan (Figure 4B) respectively.

Limit of quantification (LOQ) and limit of detection (LOD):

The LOQ and LOD were determined based on the 10 and 3.3 times the standard deviation of the response, respectively, divided by the slope of the calibration curve as described in International Conference on Harmonization guidelines Q2 (R1). The LOQ and LOD for Telmisartan were found to be 0.419μg/ml and 0. 1361μg/ml respectively whereas the LOQ and LOD for Hydrochlorothiazide were found to be 1.864μg/ml and 0.593μg/mlrespectively.

Table 3: Linearity of Telmisartan and Hydrochlorthiazide

Hydrochlorothiazide		Telmisartan
Conc. (μg /ml)	*Mean peak area	Conc. (μg /ml)	*Mean peak area
0	0	0	0
2	19842	0.64	18194
20	226974	1.28	37664
40	423748	6.4	181012
60	646779	12.8	359427
120	1280485	38.4	1102791
200	2134881	64	1815267
275	2908549	88	2506874
350	3709773	112	3221542
400	4199869	128	3701542
500	5304681	160	4521634
600	6469869	200	5671289

*Mean of three replicates

Table 4A: Precision studies of Hydrochlorothiazide (500 μg/ml)

S. No.	Intra Day		Inter Day
S. No.	*Mean peak area ± SD	% RSD	*Mean peak area ± SD	% RSD
1	5316987 ± 7975.481	0.15	5306988 ± 12206.072	0.23
2	5309984 ± 9026.973	0.17	5319846 ± 12767.630	0.24
3	5315266 ± 9567.479	0.18	5306984 ± 11144.666	0.21
4	5309456 ± 6371.347	0.12	5316981 ± 10102.264	0.19
5	5315648 ± 8505.037	0.16	5369796 ± 9665.633	0.18
6	5309564 ± 11150.084	0.21	5306984 ± 11144.666	0.21

*Mean of three replicates

Table 4B: Precision studies of Telmisartan (160 μg/ml)

S. No.	Intra Day		Inter Day
S. No.	*Mean peak area ± SD	% RSD	*Mean peak area ± SD	% RSD
1	4516986 ± 5420.383	0.12	4536987 ± 9981.371	0.22
2	4526987 ± 14033.66	0.31	4529984 ± 5435.981	0.12
3	4525564 ± 10408.797	0.23	4526932 ± 4979.625	0.11
4	4519875 ± 5875.838	0.13	4535486 ± 6349.680	0.14
5	4518956 ± 11749.29	0.26	4529631 ± 8606.299	0.19
6	4523654 ± 8497.943	0.19	4536796 ± 5444.155	0.12

*Mean of three replicates

Table 5A: Accuracy study of Hydrochlorothiazide

Drug added (mg)	Drug recovered (mg)	% Recovery	Statistical analysis
10.611	10.221	101.03	Mean = 100.74 SD = 0.61 % RSD = 0.61
10.214	10.615	100.04
10.191	10.321	101.05
12.629	10.307	101.14	Mean = 99.70 SD = 0.54 % RSD =0.54
12.698	12.655	100.21
12.820	12.668	99.76
15.166	12.709	99.13	Mean = 99.22 SD = 0.10 % RSD = 0.1
15.323	15.038	99.16
15.823	15.194	99.16

Table 5B: Accuracy study of Telmisartan

Drug added (mg)	Drug recovered (mg)	% Recovery	Statistical analysis
32.89	32.37	98.42	Mean = 99.08 SD = 0.91 % RSD = 0.92
32.78	32.82	100.12
32.09	31.67	98.69
40.04	39.98	99.85	Mean = 99.46 SD = 0.81 % RSD = 0.81
40.58	39.98	98.52
40.00	40.00	100.00
48.44	48.47	100.06	Mean = 99.40 SD = 1.04 % RSD = 1.05
48.89	48.01	98.20
47.86	47.83	99.94

Table 6: Robustness Study of Hydrochlorothiazide and Telmisartan

Conditions	Parameter	System suitability
		Hydrochlorothiazide			Telmisartan
		Tailing factor	Theoretical plates	% RSD	Tailing factor	Theoretical plates	% RSD
Flow rate (± 0.08, mL/min)	0.72	1.19	2690	0.12	1.02	56798	0.09
Flow rate (± 0.08, mL/min)	0.88	1.14	2754	0.51	1.04	56849	0.31
Mobile phase composition Acetonitrile: 0.1% formic acid (± 2%, v/v)	58:42	1.15	2789	0.09	1.11	57889	0.19
	62:38	1.12	2776	0.21	1.02	58794	0.26
Column oven temperature (± 5°C)	40°C	1.16	2681	0.08	1.14	55646	0.21
Column oven temperature (± 5°C)	50°C	1.12	2793	0.23	1.06	58649	0.34

*Mean of three replicates

Analysis of commercial formulations (Tablets):

The two different brands containing Telmisartan (40mg) and Hydrochlorothiazide (12.5mg) were analyzed by following the optimized method so developed and the results were tabulated (Table 7). The chromatogram obtained was shown in Figure 3B. The percentage recovery was calculated from the linear regression equation using the mean peak area obtained from the respective chromatograms and it was found to be 99.60-100.08. No interference of excipients was observed.

Table 7: Analysis of commercial formulation (Tablets)

Brand name	Labeled amount (mg)		*Amount found (mg)		*Recovery (%)
Brand name	TM	HCTZ	TM	HCTZ	TM	HCTZ
Brand I	40	12.5	39.86	12.45	99.65	99.60
Brand II	40	12.5	39.90	12.51	99.75	100.08

*Mean of three replicates

Forced degradation studies:

Acidic degradation:

The chromatogram shows peaks at 2.900 min and 8.692 min indicating the elution of Hydrochlorothiazide and Telmisartan respectively. On heating with 1 N HCl for 2 hours, about 0.35 % of TM has undergone degradation where as HCTZ is so stable as no decomposition was observed. The purity angle (0.061) was less than the purity threshold (1.018) indicating that TM peak was well separated from the degradants and there is no interference of degradants (Figure 5A). Similarly, the purity angle (0.348) was less than the purity threshold (1.008) for HCTZ. Therefore, it can be concluded that the method is specific and selective.

Alkaline degradation:

The chromatogram shows peaks at 2.891 min and 8.708 min indicating the elution of Hydrochlorothiazide and Telmisartan respectively. On heating with 0.1 N NaOH for 2 hours, no degradation was observed for both TM as well as HCTZ. The purity angle (0.064) was less than the purity threshold (1.020) indicating that TM peak was well separated from the degradants indicating that no interference of degradants (Figure 5B). Similarly, the purity angle (0.325) was less than the purity threshold (1.007) for HCTZ. Therefore, it can be concluded that the method is specific and selective.

Oxidative degradation:

The chromatogram shows peaks at 2.883 min and 8.723 min indicating the elution of Hydrochlorothiazide and Telmisartan respectively. On refluxation with hydrogen peroxide no degradation was observed for both TM as well as HCTZ (Figure 5C). The purity angle (0.052) was less than the purity threshold (1.010) indicating that TM peak was well separated from the degradants indicating that no interference of degradants. Similarly, the purity angle (0.311) was less than the purity threshold (1.006) for HCTZ. Therefore, it can be concluded that the method is specific and selective.

The system suitability tests were performed to ensure that the complete testing system was suitable for the intended application. The tailing factor was 1.16 (HCTZ) and 1.03 (TM) which is <1.5–2 or <2 and the theoretical plates were found to be 2866 (HCTZ) and 53864 (TM) which is >2000. Telmisartan is more sensitive towards alkaline conditions in comparison to other degradations whereas Hydrochlorthiazide is slight sensitive towards alkaline and oxidative conditions in comparison to acidic conditions. A brief summary was given in Table 8.

Figure 5: Typical chromatogram of Hydrochlorothiazide (500μg/ml) and Telmisartan (1600μg/ml) during forced degradation studies

Table 8: Forced degradation studies of Hydrochlorthiazide and Telmisartan

Stress conditions	*Mean peak area		*Drug recovered (%)		(%) Drug decomposed		Purity angle		Purity threshold
Stress conditions	HCTZ	TM	HCTZ	TM	HCTZ	TM	HCTZ	TM	HCTZ	TM
Untreated	5316925	4808877	100	100	-	-	0.351	0.057	1.007	1.016
Acidic Hydrolysis	5332227	4792236	100.29	99.65	-	0.35	0.348	0.061	1.008	1.018
Alkaline Hydrolysis	5342449	4806716	100.48	99.96	-	0.04	0.325	0.064	1.007	1.020
Oxidative degradation	5324728	4756904	100.15	98.92	-	0.08	0.311	0.052	1.006	1.010

*Mean of three replicates

CONCLUSION:

The present developed stability indicating RP-HPLC method was simple, specific, precise, accurate and robust and therefore it can be applied for the pharmacokinetic studies as well as for the determination of Hydrochlorthiazide and Telmisartan in pharmaceutical dosage forms.

ACKNOWLEDGEMENT:

The authors are grateful to SUN Pharma (India) for providing the gift samples of Hydrochlorthiazide and Telmisartan.

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Received on 19.12.2020 Modified on 14.01.2021

Research J. Pharm. and Tech. 2021; 14(4):2081-2088.

DOI: 10.52711/0974-360X.2021.00369