INTRODUCTION:

Trimetazidine Dihydrochloride [1-(2,3,4-trimethoxybenzyl)-piperazine dihydrochloride (Fig. 1) is a highly effective anti-ischemic agent. It controls ionic and extracellular exchanges, which reduces cellular oedema produced by anoxia. And also it corrects the aberrant flow of ions across the cell membrane caused by ischemia^1,2.

Trimetazidine Dihydrochloride (TMZ)can be used as a monotherapy in patients with angina pectoris. It also used in those patients whose symptoms are not well managed by nitrates, beta-blockers, or calcium antagonists³. Literature survey reveals that few methods havebeen reported like spectrophotometric^4-8, HPLC^9-12, HPTLC^13,14, GC/MS¹⁵for the determination of TMZ in bulk and pharmaceutical formulation.

Chemically Metoprolol Tartrate is (RS)-1-isopropylamino-3-p-(2-methoxyethyl) phenoxypropan-2-ol tartrate with Molecular formula (C₁₅H₂₅NO₃)_2,C₄H₆NO₆ and molecular weight 684.81^16,17. Acute myocardial infarction, heart failure, angina pectoris, and mild to moderate hypertension are all treated with Metoprolol Tartrate (METO), a cardio-specific β1-adrenergic blocking drug. It can also be used to treat supraventricular and tachyarrhythmia’s, as well as migraine headache prophylactic. Metoprolol preferentially inhibits cardiac β 1-adrenergic receptors at low dosages while having minimal effect on β2-adrenergic receptors in the lungs and vascular smooth muscle¹⁸. Literature survey reveals that few methods like HPLC^19-29, HPTLC^30-36, Spectrometric^37,38, UPLC-MS-MS³⁹, LC-MS⁴⁰ for the determination of Metoprolol tartrate in bulk and pharmaceutical formulation. On the contrary to the best of our knowledge, there is no official method for the stability-indicating simultaneous estimation of Trimetazidine Hydrochloride and Metoprolol Tartrate.

Fig 1 :- Structure of Trimetazidine Hydrochloride

Fig 2 :- Structure of Metoprolol Tartrate

EXPERIMENTAL:

Chemicals and Reagents:

Trimetazidine Dihydrochloride was kindly gifted by USV Pvt. Ltd., Mumbai. And Metoprolol Tartrate is provided by M/s Shreya Pharmaceuticals Ltd., Aurangabad. All chemicals and reagents like Methanol, n-Butanol, Chloroform, Sodium Hydroxide (NaOH), Hydrochloric Acid (HCl), Hydrogen Peroxide(H₂O₂) were procured from LOBA CHEME PVT. LTD., Mumbai_.

Instrumentation and Chromatographic conditions:

CAMAG HPTLC system equipped with Linomat 5 sample applicator (Muttenz, Switzerland) was operated under a gentle stream of nitrogen, coupled with a Hamilton microliter syringe (100μl) (Hamilton, Switzerland). CAMAG TLC SCANNER 3 controlled by WinCATS software version 1.4.3. was utilized for the application and detection of spots respectively. Chromatographic separation was carried on TLC aluminum plates pre-coated with silica gel 60 F₂₅₄ (10 × 10 cm) and development was carried out in twin trough glass chamber. Chamber saturation is done for 30 min under room temperature. The plates were developed and air-dried at room temperature. Densitometric scanning was performed at wavelength 225nm.

Preparation of Standard Stock Solutions:

An accurately weighed 7mg of TMZ and 10mg of METO were transferred into two separate 10ml volumetric flasks, and the volume was made up with methanol, to get separate standard stock solutions of TMZ (700μg/mL) and METO (1000μg/mL) respectively.

Selection of wavelength:

A solution of 20µg/ml was prepared from standard stock solution of TMZ (1000μg/mL) and METO (1000μg/mL) and scanned over 200- 400nm using UV – Spectrophotometer. The maximum absorbance was shown at 223nm for Trimetazidine Dihydrochloride and 227nm for Metoprolol Tartrate respectively, hence 225 nm was selected as analytical wavelength. UV overlay spectrum of Trimetazidine Dihydrochloride and Metoprolol Tartrate is given in Fig.3.

Fig. 3: Overlay spectrum of TMZ 20ppm and METO 20ppm.

Preparation of Standard and Sample Solutions:

Added 0.7ml from 1000μg/mL of TMZ and 1ml from 1000μg/mL of METO into 10ml volumetric flask and diluted up to mark. This final sample solution contains 70μg/mL TMZ and 100μg/mL METO and is used for spotting on TLC plate.

Preparation of Solution for Assay:

The spiked blend was prepared by thorough mixing such that it contains 175mg TMZ, 250mg METO and 500mg of common excipient blend. Amount equivalent to 70 mg of TMZ was taken and transferred to 10 ml volumetric flask and the volume was made up to 10 ml with methanol. Then sonicated for 15min on ultra-sonic bath and filtered through whatman filter paper to get standard stock solution of TMZ (700μg/mL) and METO (1000μg/mL). From the standard stock solution, working standard solution was prepared using methanol as final diluent to obtain 70μg/mL TMZ and 100μg/mL METO.

METHOD DEVELOPMENT AND OPTIMIZATION:

Initially different solvent combinations with different polarity were tried as mobile phase. The optimized mobile phase was n-butanol: methanol: ammonia (10:4:2 v/v/v) wherein acceptable peak shapes and resolution value was obtained. The detection was carried out at wavelength 225 nm for TMZ and METO. The Rf value of TMZ and METO were found to be 0.26 ± 0.02 and 0.70 ± 0.02 respectively and a typical densitogram shown in Fig. 4.

Fig. 4: Densitogram of TMZ (1400ng/band) and METO (2000ng/band).

FORCED DEGRADATION STUDIES:

Stress testing was carried out according to guidelines ICH Q1A (R2). The study was carried out by exposing both the drugs separately to different stress conditions for the different time interval. The reproducibility of the optimized condition is checked by verification of percent recovery. A stressed sample at high concentration was spotted and multi-wavelength scanning was done for detection of degradation products. We had referred to other chromatographic methods in literature to execute the work^42-50

Acid and Alkali Catalysed Hydrolysis:

1ml of stock solution of both the drugs (700μg/mL and 1000μg/mL of TMZ and METO respectively) was mixed with 1ml of 1N HCl/ 1N NaOH and methanol was added to make up the volume up to 10ml and sample was kept in dark for 4 hrsand application was carried out.

Oxidative Degradation:

1ml of stock solution of TMZ and METO separately (700μg/mL and 1000μg/mL) was mixed with 1ml of 30% w/v H₂O₂. The volume was made up to 10ml with methanol. The solution were kept in dark at room temperature for 4 hrs then applied on a TLC plate.

Thermal Degradation:

For the thermal degradation both the bulk drug in solid state were exposed to the temperature of 60⁰C for 18hrs, cooled to room temperature. Then sample was weighed and final dilution of strength 70μg/mL (TMZ) and 100μg/mL (METO) were prepared and applied on TLC plate.

Photolytic Degradation:

Both the APIs in solid state were exposed to UV energy not less than 200 Watt-hours/square meter separately and to fluorescence illumination not less than 1.2million lux hours. The final dilution of strength 70μg/mL (TMZ) and 100μg/mL (METO) were prepared and applied on TLC plate.

METHOD VALIDATION:

The method has been validated according to the guidelines ICH Q2(R1) for parameters such as specificity, linearity, range, precision, accuracy, limit of detection (LOD), limit of quantitation (LOQ), and robustness.

RESULTS:

The study was carried out by exposing both the drugs to different stress conditions such as acid, alkali, oxidative, thermal and photolytic at different time interval. We have optimized the stressed condition based on recovery and reproducibility testing. A higher concentration of the stressed sample was also applied to look for degradation product which is shown in Fig. 5 and Fig. 6 for TMZ and METO. The summary shown in Table 1.

Fig. 5: Multi-wavelength scanning of the stress degradation condition of TMZ from 205-295nm. (track 2: standard 70ppm, track 3: UV: track 4: fluorescence, track 5: thermal, track 7: acid, track 9: base, track 11: oxidation, track 1,6,8,10: blank).

Fig. 6: Multi-wavelength scanning of the stress degradation condition of METO from 205-295nm. (track 2: standard 70ppm, track 3: UV, track 4: fluorescence, track 5: thermal, track 7: acid, track 9: base, track 11: oxidation, track 1,6,8: blank).

Table. 1: Summary of forced degradation

Parameter	Condition	% Degradation
Parameter	Condition	TMZ	METO
Acid Hydrolysis	1N HCl, 4 hrs	14.01%	36.93%
Base Hydrolysis	1N NaOH, 4 hrs	16.86%	22.32%
Oxidation	30% w/v H₂O_2,4 hrs	15.40%	39.72%
Thermal	60⁰C 18 hrs	17.7%	37.17%
UV-Light	Not less than 200 Watt-hours/square meter	4.85%	9.60%
Cool White Fluorescent light	1.2 million lux Hours	7.28%	4.3%

Linearity:

The mixture was made up of 70μg/mL TMZ and 100 μg/mL METO. Different volumes (10-50μl) of the mixture were spotted on the plate to get the range of 700-3500ng/band for TMZ and 1000-5000ng/band for METO. The simple regression equation method was used to establish the relationship between amounts spotted and peak area. The detail of regression is shown in the table. 2, linearity densitogram shown in fig.7.

Table. 2: Regression details of TMZ and METO

Parameter	TMZ	METO
Range	700-3500 ng/band	1000-5000 ng/band
Correlation Coefficient (R²)	0.998	0.9945
Linearity Equation	y = 2.4851x + 1749.6	y = 2.0911x + 2703.3

Fig. 7: 3-D Densitogram of TMZ (Rf:0.26) and METO (Rf:0.70)

Assay:

A blend of commonly used excipients was spiked using TMZ and METO. The amount equal to an average weight of tablet was taken and dispersed in methanol and sonicated then filtered through whatman filter paper to obtain a concentration equivalent to 70μg/mL and 100 μg/mL for TMZ and METO respectively and it was analyzed by the developed HPTLC method. The assay results are summarized in table 3.

Table. 3: Assay of TMZ and METO

Drug	TMZ	METO
Concentration taken (ng/band)	1400	2000
% Drug content	100.41	98.71
% RSD	1.314	1.047

* Average of six determinations

Accuracy:

The standard addition method was used to carry out the recovery studies. Pure drug substance was spiked in blend prepared for assay at three different levels 50%, 100% and 150%. Three replicates of three concentrations were assessed. The results are summarized in table 4.

Table. 4: Recovery study of TMZ and METO

Parameter	TMZ			METO
% level	50	100	150	50	100	150
Initial amount	1400	1400	1400	2000	2000	2000
Amount added(ng/band)	700	1400	2100	1000	2000	3000
% Recovery	99.68	100.01	98.30	98.32	99.98	99.52

Precision:

The precision was checked for both intra-day and inter-day studies. Intra-day precision was evaluated by applying 6 replicates of 1400ng/band (TMZ) and 2000 ng/band (METO). Six replicates were also analysed on three consecutive days to evaluate the inter-day precision. The method was found to be precise as % RSD was less than 2% and results are summarized in table 5

Table. 5: Precision study of TMZ and METO

Parameter	TMZ		METO
Condition	Intra-day	Inter-day	Intra-day	Inter-day
Concentration(ng/band)	1400	1400	2000	2000
% Relative Standard Deviation(%RSD)	1.04	1.31	1.57	1.38

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

LOD and LOQ were calculated by formula 3.3 σ/S and 10 σ/S respectively. Where σ is the standard deviation of the response of lowest concentration and S is the slope of the calibration curve. The results are summarized in table 6.

Table. 6: LOD and LOQ study of TMZ and METO

Parameter	TMZ	METO
LOD (ng/band)	70.69	87.46
LOQ (ng/band)	214.23	265.03

Robustness:

Robustness was carried out by doing small and deliberate changes to the optimized method like change in mobile phase ratio, saturation time, change in wavelength. The results are summarized in table 7.

Table. 7: Robustness study of TMZ and METO

Parameter	Condition	% RSD of TMZ	% RSD of METO
Mobile phase ratio	n-butanol: methanol: ammonia (9.8:4.2:2 v/v/v).	1.62	1.11
Mobile phase ratio	n-butanol: methanol: ammonia (10.2:3.8:2 v/v/v).	1.44	1.04
Saturation time	27 min	1.72	1.49
Saturation time	33 min	1.03	1.12
Change in wavelength	223 nm	1.19	1.82
Change in wavelength	227 nm	1.60	1.32

Validation results proved that the developed method performs well with specificity, linearity, range, accuracy, precision. The summary of the validation parameter is summarized in table 8.

Table.8: Summary of validation parameter of TMZ and METO

Sr. No	Parameter		TMZ	METO
1	Linearity and Range		y=2.4994x + 1709.6 700-3500 ng/band	y=2.0911x + 2703.3 1000-5000 ng/band
2	Precision (%RSD)	Intra day	1.04	1.57
2	Precision (%RSD)	Interday	1.31	1.38
3	Assay (%)		1.31	1.04
4	Accuracy (%Recovery)	50%	99.68	98.32
		100%	100.01	99.98
		150%	98.30	99.52
5	LOD(ng/band)		70.69	87.46
6	LOQ(ng/band)		214.23	265.03
7	Robustness		Robust	Robust

DISCUSSION:

The prime objective of present work was to know the stability of TMZ and METO under various stress conditions, because number of research articles found during literature search did not show matching results. The strength of combination which studied here is 35mg of SOF and 50 mg of METO.

Few methods were found for both drugs separately, but we could not find any method reported for combined study of TMZ and METO. Sreelatha Nagula et. al.¹² has reported complete degradation in acidic condition and for alkaline degradation and stable for thermal condition. In our study, stress degradation percentage obtained is relatively lesser, though performed with similar stress conditions.

We found METO to be more sensitive to acid hydrolysis and oxidation similar tothe results reported by Mohammad Yunoos et. al²¹.

The HPTLC has advantage that is requires less mobile phase. We could optimize a tertiary mobile phase that gives well resolved peaks and peak shape is also acceptable with sufficient saturation time of mobile phase. The maximum absorption was observed at 223 nm and 227nm for TMZ and METO respectively. The forced degradation conditions were optimised by changing the strength of reagent and its exposure time. Here, during stress degradation study, it was observed that the TMZ and METO is relatively stable in photolytic condition, and TMZ is less stable thermal condition and in other hydrolytic conditions. METO has been found to be sensitive to oxidative condition followed by thermal, acid, base. No degradation product was found under any stress condition. In order to look for the degradation product peaks, higher concentrations of samples prepared for stress degradation study were spotted on TLC plate. In view of fact that degradation product can have different absorbance so multi wavelength scanning was also performed. By doing this the obtained results were confirmed.

The developed HPTLC method was validated as per ICH guideline and complies with the validation parameters like linearity and range, accuracy, precision, robustness LOD and LOQ. The R square value > 0.99 was obtained. The assay results are found to be 100.41 and 98.71 % for TMZ and METO respectively which indicates that method does not have interference from other excipients present in drug product. The samples can be prepared and analysed at room temperature without the special precaution so the method is simple and efficient which can be used for routine stability monitoring study of trimetazidine dihydrochloride and metoprolol tartrate. Now, this optimised method can be considered as good alternative for analysis of the studied anti-ischemic drugs with excellent recovery results. We had referred to other chromatographic methods in literature to execute the work^51-53

CONCLUSION:

A simple and rapid stability indicating HPTLC method was developed and validated for trimetazidine dihydrochloride and metoprolol tartrate in combination. The method covers a wide range like 700-3500 ng/band for TMZ and 1000-5000 ng/band for METO. From the observations, it is concluded that both drugs were stable under UV-light and cool white fluorescent light exposure. The multi-wavelength scanning was used to confirm non-interference by the degradation product. It was concluded that the validated method can be used for stability studies of trimetazidine dihydrochloride and metoprolol tartrate in combination.

ACKNOWLEDGEMENTS:

Authors are thankful to the principal and the management of the AISSMS College of Pharmacy, Pune, Maharashtra, India for providing required facilities for research work. The authors are also grateful to USV Pvt. Ltd., Mumbai, India for providing Trimetazidine dihydrochloride and M/s Shreya Pharmaceuticals Ltd., Aurangabad, for providing Metoprolol tartrate as gift samples.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 14.03.2024 Revised on 10.08.2024

Accepted on 04.11.2024 Published on 28.02.2025

Available online from March 04, 2025

Asian Journal of Pharmaceutical Analysis. 2025;15(1):13-19.

DOI: 10.52711/2231-5675.2025.00003

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