INTRODUCTION:

Trimetazidine dihydrochloride (TD), chemically called as 1-[(2,3,4-trimethoxyphenyl) methyl] piperazine dihydrochloride, is a pharmaceutical molecule with anti-ischemic, antineoplastic and immunomodulating features^1-6. TD is medically effective in the management and prophylaxis of angina pectoris. In ischemic cases, TD optimizes the energy metabolism of myocardial and cardiac activity.

The process of TD chemical synthesis includes piperazine and 2, 3, 4-trimethoxy benzaldehyde as raw materials and formic acid as catalyst⁷. The 2, 3, 4-trimethoxybenzaldehyde is a medicinal intermediate which is used in the manufacturing of several compounds.

The synthesis of 2, 3, 4-trimethoxybenzaldehyde includes gallic acid and dimethyl sulfate as raw material and alkylate reagent, respectively in the presence of sodium hydroxide⁸. Pyrogallol is generated as an intermediate while 2, 3, 4-trimethoxybenzaldehyde is formed from gallic acid.

Intake of pyrogallol is toxic to ruminants⁹. Ingestion or prolonged skin application of pyrogallol has been reported with extreme acute poisoning in humans¹⁰. A variety of clinical studies in people exposed to pyrogallol was a contact sensitizer^11-13. The manufacturing companies follow different purification strategies during the manufacturing of drug to remove process related impurities. Despite of several purification steps, there is a possibility for the existence of pyrogallol as process related residual impurity in the final product of TD. The presence of pyrogallol can affect the efficiency, safety, and quality of TD. In general, a standard of exposure of 1.5 μg per day for any impurity can be viewed as a specific appropriate qualification requirement for marketing application support¹⁴. Maximum daily dose of TD is 0.070 g. Thus, the acceptable exposure levels of pyrogallol based on Threshold of Toxicological Concern was considered as 21.42 ppm¹⁵.

Different techniques like HPLC^16-21, UPLC^22,23, Mass spectrometry²⁴ and NMR²⁴ were applied to assess and characterize related substances in active pharma ingredient. The quantification of different impurities of TD in active pharm ingredient and/or drug formulations was addressed in some reports. Quantification of N-formyl trimetazidine (by LC-MS)²⁵, 2,3,4-trimethoxybenzyl alcohol & 2,3,4-trimethoxybenzaldehyde (by GC/MS)²⁶, 2,3,4-trimethoxybenzaldehyde, 1,4-bis(2,3,4-trimethoxybenzyl) piperazine & 1-Ethyl-4-(2,3,4-trimethoxybenzyl) piperazine (by RP-HPLC)²⁷, piperazine carboxaldehyde, trimethoxybenzyl alcohol & trimethoxybenzaldehyde (by RP-HPTLC)²⁸ were reported.

The quantification of process-related impurity of TD, pyrogallol, in TD drug material was not recorded till now. This work describes a simple, reliable and selective gradient HPLC method for the separation of pyrogallol followed by its quantification in TD drug material. The method mentioned has been validated and has proven to be effective for determining the quality of TD drug material.

EXPERIMENTAL:

Chemicals and solvents:

All chemicals used in this project like potassium dihydrogen phosphate, disodium hydrogen phosphate, phosphoric acid and solvents like methanol were acquired from Merck, Mumbai, India. TD active pharm ingredient and pyrogallol were procured from GVK Biosciences Private Limited, Hyderabad, India. Milli Q ultra-pure water obtained from Milli Q purification system was used throughout the project.

Apparatus:

Waters Acquity ARC e2695 model HPLC system with quaternary pump, degasser, autoinjector and Waters model-2998 photodiode array detector was used. Develosil ODS MG-5 250 x 4.6 mm, 5 µ column was employed for separation of pyrogallol. Samples were weighed using Sartorius, model - CPA225D analytical balance. Fast clean ultrasonic cleaner model - f1756 sonicator was used for sonication.

Conditions to evaluate pyrogallol:

10 µl sample was injected in to Develosil ODS MG-5 (250 × 4.6 mm, 5 µ) column for analysis. The temperatures at the column and sample injector was adjusted at 40 °C and 5 °C, respectively. Gradient elution method was managed to analyse pyrogallol in TD utilizing a photodiode array absorbance detection system adjusted at 240 nm. The mobile phase consisted of two solvent systems with 1.2 ml/min flow rate. Solvent system A (SSA) consisted of 0.02M potassium dihydrogen phosphate and 0.03 M disodium hydrogen phosphate. Solvent system B (SSB) was methanol. Adjusted the mobile phase pH to 2.8 with phosphoric acid. The gradient elution scheme was tuned as follows: 90% SSA and 10% SSB at initial zero time; 85% SSA and 15% SSB at 10 min; 80% SSA and 20% SSB at 20 min; 30% SSA and 70% SSB at 28 min; 30% SSA and 70% SSB at 40 min; 90% SSA and 10% SSB at 42 min; 90% SSA and 10% SSB at 45 min. The solvent systems (SSA and SSB) were filtered via 0.22 μ nylon filter after preparation. The total time of chromatography for one sample was 45 min. Water and methanol at proportion 90:10 (vol by vol) was utilized as diluent.

Pyrogallol Stock and Standard Solutions:

Pyrogallol stock solution (1050 μg / ml) was made with a diluent. Appropriate pyrogallol stock solution aliquots were moved into 50 ml volumetric flasks using pipettes. The solutions were diluted with diluent up to 50 ml volume to obtain penultimate pyrogallol concentrations of 6.3 ppm, 10.5 ppm, 21 ppm, 25.2 ppm and 31.5 ppm.

Trimetazidine Dihydrochloride Sample Solution:

Accurately weighed 500 mg of TD drug material into a 10 ml flask, dissolved through sonication for 10 min and diluted to 10 ml volume with diluent.

Calibration Curve of Pyrogallol:

Pyrogallol working solutions with a concentration range of 6.3 ppm to 31.5 ppm were analyzed utilizing the condition presented in the "Conditions to evaluate pyrogallol" section. The pyrogallol peak areas were scaled opposed to the appropriate pyrogallol concentrations to generate the calibration graph for pyrogallol. Regression analysis was done to generate the regression equation for pyrogallol.

Evaluation of Pyrogallol Content in Trimetazidine Dihydrochloride:

For this, one injection of diluent, 3 injections of pyrogallol working solution (21 ppm) and one injection of TD sample solution were made into the column. The samples were analyzed utilizing the condition presented in the "Conditions to evaluate pyrogallol" section. The pyrogallol content in the TD sample was evaluated using formula:

Where,

A _Sample=Pyrogallol peak area in TD sample

A _Standard = Average area of pyrogallol peak

W _Standard= Weight of pyrogallol

W _Sample= Weight of TD

RESULTS AND DISCUSSION:

Method Development:

Different solvent systems (0.1% trifluoracetic acid in water as SSA and 0.1% trifluoracetic acid in acetonitrile as SSB; 0.1% perchloric acid in water as SSA and 0.1% perchloric acid in acetonitrile as SSB; 10 mM ammonium bicarbonate in Water as SSA and acetonitrile as SSB; 0.02M potassium dihydrogen phosphate and 0.03 M disodium hydrogen phosphate as SSA and methanol as SSB) with diverse pH units were checked. Three different columns (X-Bridge C18 column with measurements150 × 4.6 mm 3.5 µm; X-SELECT CSH C18 column with measurements 150 × 4.6 mm 3.5 µm; Develosil ODS MG-5 column with measurements 250 × 4.6 mm, 5 µm) with temperatures 25 ^oC and 40 ^oC were checked. Best peak shape, acceptable peak purity and good response were obtained using Develosil ODS MG-5 column with 40 ^oC and 0.02M potassium dihydrogen phosphate and 0.03 M disodium hydrogen phosphate as SSA in combination with methanol as SSB. The optimized flow rate and pH of mobile solvent system was 1.2 ml/min and 2.8 units, respectively. Various gradients have been inspected, and the one with the optimal results was: 90% SSA and 10% SSB at initial zero time; 85% SSA and 15% SSB at 10 min; 80% SSA and 20% SSB at 20 min; 30% SSA and 70% SSB at 28 min; 30% SSA and 70% SSB at 40 min; 90% SSA and 10% SSB at 42 min; 90% SSA and 10% SSB at 45 min. 240 nm was the preferred wavelength because pyrogallol showed good response at this nanometres. The chromatogram recorded in optimal conditions for the standard solution of pyrogallol is displayed in Figure 1.

Figure 1: Chromatogram of pyrogallol in optimal conditions

METHOD VALIDATION:

The HPLC method has been verified for pyrogallol assay in TD implementing the suggestions defined in the Industry Guidelines ICH Q2 (R1)²⁹.

Specificity:

The specificity was verified by injecting the diluent blank, standard pyrogallol solution (21 ppm), pyrogallol-spiked TD sample at concentration of 21 ppm. In Figure 2, Chromatogram A corresponds to standard pyrogallol solution with retention time of 6.246 min. Chromatogram B and C represents diluent blank and pyrogallol-spiked TD sample, respectively devoid of interfering peaks at pyrogallol’s retention time. The specificity was further verified by determining the peak purity of pyrogallol in standard pyrogallol solution and pyrogallol-spiked TD sample. Pyrogallol’s purity angle was 0.197 and 0.474 in standard pyrogallol solution and pyrogallol-spiked TD sample, respectively (Figure 3). Pyrogallol’s purity threshold was 0.640 in standard pyrogallol solution and 1.040 in pyrogallol-spiked TD sample (Figure 4). The peak purity angle value of pyrogallol was lower than that of purity threshold value both in the standard pyrogallol solution and also the pyrogallol-spiked TD sample. This is an evidence of method’s specificity.

Chromatogram A

Chromatogram B

Chromatogram C

Figure 2: Chromatogram A – standard pyrogallol solution: Chromatogram B – diluent blank: Chromatogram C - pyrogallol-spiked TD sample

Peak purity flag A

Peak purity flag B

Figure 3: Peak purity flag A – standard pyrogallol solution: Peak purity flag B –pyrogallol-spiked TD sample

Limits of Quantification (LOQ) and Detection (LOD):

Both the parameters were computed using the ratio of signal to noise. Ratio of signal to noise should be equal or greater than 3 and 10 for LOD and LOQ, respectively. The LOQ for pyrogallol parallel to a ratio of signal to noise of 18 was 6.3 ppm. The LOD for pyrogallol parallel to a ratio of signal to noise of 7 was 1.89 ppm. The LOQ of pyrogallol was further verified by determining the precision at LOQ pyrogallol concentration level. The resulting RSD values were 0.06% for these analyses. The low values of LOD and LOQ is an evidence of method’s sensitivity.

Linearity:

The linearity was validated by evaluating a set of distinct concentrations of pyrogallol (n = 5) over the 6.3 ppm (LOQ) – 31.5 ppm (150 % of the specification limit, 21 ppm) concentration range. The subsequent linear regression equation was developed by treating the calibration data with least squares regression analysis: AP = 289.543 c – 95.967 (r² = 0.9990), where ‘AP’ and ‘c’ are the area of pyrogallol and concentration of pyrogallol, respectively. The r² value, which is greater than 0.99, is an evidence of method’s linearity.

Precision:

The precision of the procedure was studied at 21 ppm concentration level for pyrogallol-spiked TD sample using six repeated determinations. The standard deviation and RSD values for pyrogallol peak area from six repeated determinations were calculated. The standard deviation and RSD values were 9.352 and 0.150%, respectively. The low RSD value, which is less than 10%, is an evidence of method’s precision.

Accuracy:

The accuracy of the procedure was studied at LOQ (6.3 ppm), 100% (21 ppm) specification limit and 150% (31.5 ppm) specification limit concentration levels for pyrogallol-spiked TD sample using three repeated determinations. The percentage recovery values for pyrogallol at three concentration levels from three repeated determinations were calculated. The mean pyrogallol recovery value was 118.54% at LOQ level, 108.45% at 100% level and 98.34% at 150% level. The better recovery values, which for each Level are within the range of 70-130%, are evidence for the method’s accuracy.

Robustness:

The robustness of the procedure was studied at 21 ppm concentration level for pyrogallol. Robustness was assessed by assessing the impact of slight differences in flow rate and temperature of the column. The flow rates studied during robustness were 1.5 ml/min and 1.0 ml/min. the temperatures of the column studied during robustness were 35 ^oC and 45 ^oC. The optimized column temperature and flow rate were 40 ^oC and 1.2 ml/min. The retention time of pyrogallol in both conditions (optimized and altered) were determined and calculated the percentage difference (Table 1). The percent difference values, which are less than 15%, are evidence for the method’s robustness.

Table 1: Robustness data for pyrogallol

Condition tested	Retention time of pyrogallol		Difference (%)
Condition tested	Optimized condition	Altered condition	Difference (%)
1.5 ml/min flow rate	6.6	7.1	-7.5
1.0 ml/min flow rate	6.6	6.0	9.09
35 ^oC column temperature	6.6	6.3	4.5
40 ^oC column temperature	6.6	6.7	-1.5

Analysis of Pyrogallol in TD Batch Samples:

Four TD batch samples were analysed by the proposed procedure and the data pertaining to the same was presented in Table 2. Pyrogallol residues were not detected in any of the TD batch samples analysed.

Table 2: Analysis of pyrogallol in TD batch samples

Sample No.	Batch No.	Concentration of pyrogallol detected (ppm)
1	TM219001	Not detected
2	TM219003	Not detected
3	TM201019 GTI	Not detected
4	TM201319 GTI	Not detected

CONCLUSION:

This is the very first HPLC procedure developed to selectively quantify pyrogallol in TD drug material. The method can be utilized for separating and identifying the pyrogallol in TD drug material, as well as for the quality control of TD drug material. The method demonstrated good analytical efficiency with satisfactory precision, robustness and accuracy. Based on the observations and results of this investigation, it was concluded that the procedure developed can be employed for testing TD drug material for pyrogallol content.

ACKNOWLEDGEMENTS:

The authors thank management of GVK Biosciences Private Ltd, Hyderabad, for facilities, co-operation and support during the study.

CONFLICTS OF INTEREST:

Declares that there is no conflict of interest.

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Received on 12.06.2020 Modified on 28.11.2020

Research J. Pharm. and Tech 2021; 14(11):5803-5807.

DOI: 10.52711/0974-360X.2021.01009