INTRODUCTION:

2,3,4-Trimetazidine Dihydrochloride (TMZ-Di HCl) is a drug for angina pectoris, Trimetazidine dihydrochloride is described as the first cytoprotective anti-ischemic agent developed. Which improves myocardial glucose utilization through inhibition of fatty acid metabolism, also known as fatty acid oxidation inhibitor.^[12][13] (Figure 1)

Figure 1: Trimetazidine Dihydrochloride

Figure 2: 2,3,4-trimethoxybenzaldehyde (Impurity A)

Figure 3 : 1,4-bis(2,3,4-trimethoxybenzyl)piperazine (Impurity B)

Figure 4 : 1-Ethyl-4-(2,3,4-trimethoxybenzyl)piperazine.

Experimental Data:

Trimetazidine Dihydrochloride and its three impurities were provided by Ultratech India limited (Mumbai). Methanol (HPLC grade) and Water (HPLC grade) were purchased from Merck.(Mumbai, India).

Instrumentation

The HPLC system (Thermo) consisted of a U.V. Visible detector, Column used was octadecylsilyl silica gel for chromatography R (5 μm) with a pore size of 10 nm, column size: l = 0.15 m, Ø = 4.6 mm of (Peerless, Chromatopak), at column temperature: 30 °C. pH meter of Labindia make.

Chromatographic conditions

The chromatographic analysis was performed on Chromatopak Peerless -C18 analytical column with a mobile phase composed of buffer: Methanol (50:50v/v) (buffer pH 3.0, adjusted with orthophosphoric acid) and was isocratically eluted at a flow rate of 1.2 mL min^-1. Column oven temperature was 30ºC. A small sample volume of 20 μL was used for each sample run, being injected into the HPLC system. The chromatogram was monitored with UV detection at a wavelength of 240 nm and the total run time was 50 min. Diluent used was HPLC grade water.

Preparation of Buffer Solution

Buffer solution was prepared by dissolving 3.2 g of sodium hepta sulphonic acid in 1L std volumetric flask, dissolved with HPLC grade water pH adjusted to 3.0, with orthophosphoric acid.

Method development

The objective of this study was to develop a method for estimation of TMZ- Di HCl and its related substance under isocratic conditions. The mobile phase used was the mixture of Methanol with buffer in different ratios. The mixture of methanol: buffer (pH 3.0) in the ratio of [50:50] (v/v) was proved to be most effective mixture than the other mixtures used for better elution. The flow rates tested were 0.8, 1.0, 1.2 and 1.4 mL. Among them, flow rate of 1.2 mL was selected for the assay because of better elution of the peak. The column oven temperature selected as 30ºC for better peak shape and elution of peak. The above mentioned chromatographic conditions proved to provide a better and symmetric elution of TMZ DiHCl and its related substance. The optimum wave length for detection was 240 nm.^[8]

Standard solution Preparation-

1. About 100 mg of Trimetazidine Dihydrochloride Reference standard was accurately weighed and transferred in 100 mL volumetric flask, dissolved in diluent upto the mark. (1000 ppm)

2. About 10 mg of Impurity A was dissolved with diluent upto the mark in 100 ml volumetric flask (100 ppm).

3. About 10 mg of Impurity B was dissolved with diluent upto the mark in 100 ml volumetric flask (100 ppm).

4. About 10 mg of Impurity C was dissolved with diluent upto the mark in 100 ml volumetric flask (100 ppm).

This solution was further diluted with diluent to obtain required ppm solutions.

Method Validation:

The proposed method for estimation of related substances of Trimetazidine di hydrochloride is validated as per the United States Pharmacopoeia and ICH guidelines ^.[9][10][11]

1) Specificity

Specificity is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present. Typically these might include impurities, matrix, etc. here study was done using Impurities.^[1][2]

(2) Linearity

The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample, was studied by analyzing five concentrations of the drug, and process was repeated for five times each. It was done over the range of 80 -120 µg ml for TMZ Di HCl and 8-12 µg ml for Impurities A, B, C

(3) Precision-

The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. The precision of the method was demonstrated by

1. Repeatability:

Repeatability expresses the precision under the same operating conditions over a short interval of time. Repeatability is also termed intra assay precision, was studied by injecting 100 µg /ml of the drug (TMZ DiHCl) process was repeated for three times each.

2. Intermediate:

Intermediate precision expresses within-laboratories variations:

i. Analysis on different days: was studied by injecting 100 µg/ ml of the drug, and process was repeated next day for three times each.

ii. Changing Chemist (3 chemists)

(4) Accuracy

The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found. The recovery studies were carried out at three levels of 80%, 100% and 120 % and the percentage recovery was calculated.

(5) Robustness-

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage and done by observing - influence variations of buffer pH in a mobile phase, changing wavelength and flow rate.

(6) Limit of detection-

Limit of Detection (LOD) The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample, which can be detected but not necessarily quantities as an exact value. Based on the Standard Deviation of the Response and the Slope, The detection limit (DL) may be expressed as:

3.3σ

DL = -------------.

Where, σ = the standard deviation of the response for the lowest conc. in the range.

S = the slope of the calibration curve.

Limit of Quantification (LOQ) The quantitation limit of an individual analytical

procedure is the lowest amount of analyte in a sample, which can be quantitatively determined with suitable precision and accuracy. Based on the Standard Deviation of the Response and the Slope, The quantitation limit (QL) may be expressed as:

10σ

QL = ------------.

Where, σ = the standard deviation of the response for the lowest conc. in the range

S = the slope of the calibration curve.

RESULTS AND DISCUSSION:

Specificity:

Impurities were added to the stock solution and the mixture was subjected to chromatographic analysis and it was observed that impurity peaks were well resolved from peak of Trimetazidine Dihydrochloride (fig 5); system suitability parameters are shown in (Table 1). The method was considered to be specific since there was no interfering peak at the retention time of Trimetazidine Dihydrochloride and also the peak was well resolved from the peaks of all impurities.^[3][4]

Linearity:

The data obtained in the linearity experiments was subjected to linear-regression analysis. A linear relationship between peak areas and concentrations was obtained in the range of 80- 120 µg ml-1 with r = 0.9520 (Table 2) for TMZ Di HCl and for process impurity range was 8-12 µg ml-1 with r =0.998 imp A r=0.9228 imp B r=0.9984 imp C.^[5] (Table 3)

Precision:

The developed method was found to be precise as the % RSD value for repeatability studies was less than 2.0%, where as the %RSD for inter-day precision was lower than that of repeatability study. (Refer Table 4, 5 and 6).

Accuracy

The results of recovery studies for accuracy was calculated. Recovery observed was (97.86 -99.17%) for TMZ Di-HCl process impurities (Refer table 8).

Limit of Detection (LOD) and Limit of Quantitation (LOQ)

The LOD and LOQ were found to be 2.89 ppm and 8.77 ppm for TMZ Di HCl and for corresponding process impurities LOD was 0.09 ppm, 0.39 ppm 0.14 ppm LOQ was 0.20,1.18, 0.43 respectively. (Refer Table 7 )

DISCUSSION:

Impurities determination is an integral part of pharmaceutical analysis. Here a specific, accurate, precise and cost effective method for estimation of Trimetazidine dihydrochloride in the presence of its impurities was developed which fulfill all parameters of validation as per given in the ICH guidelines.^[6][7]

RESULT:

Main objective of this analytical method development was to separate Trimetazidine Dihydrochloride from Imp A, Imp B and Imp C. Different Mobile phases and different stationary phases were tried but effective chromatographic separation was achieved with a stainless steel column 0.15 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel. Flow rate of mobile phase was adjusted to 1.2 ml/min. Mobile phase composed of buffer : Methanol (50:50v/v) Buffer was prepared by dissolving 3.2 g of sodium hepta sulphonic acid in 1L std volumetric flask, dissolved with HPLC grade water. Adjusted pH 3.0, with orthophosphoric acid. UV detector was set at 240 nm with column temperature 30°C. Peak shapes and separation of TMZ- Di HCl and impurities were as follows:

Table 1 Retention Time

Sr.No	Name of API & its Impurity	Retention Time
1	Blank	--------
2	Trimetazidine Dihydrochloride (100 ppm)	15.52
3	Impurity A (10 ppm)	6.71
4	Impurity B (10 ppm)	38.51
5	Impurity C (10 ppm)	4.91

Specificity and selectivity studies results

Selectivity of the method was performed by separately injecting individual impurities and none of these impurities were seen to interfere with the Trimetazidine Dihydrochloride peak with minimum resolution of 1.5 between any two peaks. No interference of blank was observed (fig 6)

Fig 6 Typical chromatogram of Blank

Table 2 Trimetazidine Dihydrochloride Precision

Volume Of Stock Solution (ml)	Final dilution (ml)	Final Conc. (mg/ml)		Area		Mean Area	Relative standard deviation (%)
0.8	10	80	3858.32	3875.44	3890.07	3874.61	0.41
0.9	10	90	4034.87	4065.72	4079.05	4059.88	0.55
1.0	10	100	4147.03	4161.49	4163.36	4157.29	0.21
1.1	10	110	4485.98	4499.12	4512.56	4499.22	0.29
1.2	10	120	4578.49	4554.45	4542.10	4558.35	0.40
						Average	0.37
						Slope	18.06
						*Co-rel	0.9520

Table 3: Process Impurities

Volume Of Stock Solution (ml)	Final dilution (ml)	Conc in ppm	Mean Area of Imp A	Mean Area of Imp B	Mean Area of Imp C
0.8	10	8	897.99	972.38	663.68
0.9	10	9	1005.41	1073.07	792.89
1.0	10	10	1323.05	1089.28	947.51
1.1	10	11	1424.80	1090.64	965.01
1.2	10	12	1595.97	1363.53	1023.70
*RSD			0.45	0.86	0.42
Slope			181.53	79.98	89.21
*Co-rel			0.9998	0.9228	0.9984

· Co-rel : Correlation Coefficient

· RSD : Relative standard Deviation

Linearity

Linear calibration plot for the method was obtained over the calibration ranges tested.

Stock solution : TMZ Di HCl (1000 ppm) and Imp A , Imp B ,Imp C (100 ppm)

Table 5- Intra day Precision

Injection Details	Standard Deviation	Relative standard Deviation
Trimetazidine Dihydrochloride	9.30	0.21
Impurity A	4.20	0.36
Impurity B	1.71	0.15
Impurity C	8.23	1.25

Precision

Five replicate injections of Trimetazidine Dihydrochloride (100 ppm) and process Impurity A, Impurity B and Impurity C each of 10 ppm was made. The results for each impurity are summarized in the following table:

Table 6 - Change of chemist

Injection Details	Standard Deviation	Relative standard Deviation
Trimetazidine Dihydrochloride	9.44	0.22
Impurity A	6.70	0.63
Impurity B	10.14	0.96
Impurity C	4.16	0.60

Limit of Detection and Limit of Quantifications

The results of each impurity are summarized in the following table:

Table 7 LOD/LOQ

	Average Standard Deviation	Slope of Calibration Curve	Detection Limit in ppm	Quantitation Limit in ppm
Trimetazidine Dihydrochloride	15.85	18.0681	2.89	8.77
Impurity A	5.113	181.53	0.09	0.20
Impurity B	9.513	79.98	0.392	1.189
Impurity C	3.870	89.21	0.143	0.43

Table 8 - Accuracy (Mean recovery of all impurities at each level )

Sr.No	Percentage	Impurity A	Impurity B	Impurity C
1	Level - 1 80%	99.39	95.78	99.25
2		99.10	95.25	99.68
3		99.27	97.42	99.72
4	Level 2 -100%	99.23	98.89	99.98
5		99.70	99.47	99.72
6		99.36	99.89	99.36
7	Level 3 -120%	99.40	99.55	94.69
8		98.68	99.60	97.83
9		98.40	96.97	95.70
	Mean	99.17	98.09	98.43
	SD	0.39	1.77	1.95
	RSD (%)	0.40	1.81	1.98

Recovery limit - 90% -110% RSD NMT 2.0%

Robustness:

The method was tested for capacity to remain unaffected by small variation in method parameters, such as

1. Change of Flow rate

2. Change of Wavelength

3. Change of pH

Sample of Trimetazidine Dihydrochloride and its process impurities were analyzed for the same. It was observed that the method is unaffected by small changes in the experimental conditions. Which confirms robustness of the method.

CONCLUSION:

Isocratic HPLC method has been developed for Trimetazidine Dihydrochloride in presence of its process related impurities. The proposed method is simple accurate, precise, specific and the method is suitable for use of routine quality control of drug, due to following reasons

A. Analytical method is found to be specific as proved by injecting known amount of component into the chromatogram.

B. Limit of quantification and limit of detection for Trimetazidine Dihydrochloride and process related. Impurities have been established and are found to be within the range.

C. Analytical method is found to be linear over a specific range.

D. Analytical method is found to be precise and accurate.

E. Analytical method is found to be robust.

Hence method is completely evaluated for its specificity linearity, precision, accuracy, robustness, ruggedness, limit of quantification and detection.

REFERENCES:

1. X. Zuo, F. Wang , P. Xu, R. Zhu, H. Li, Chromatogr., 2006, 64, 387.

2. K. Masanori, M. Yasuo, H. Yukihiro, O. Takahiko, J. Chromatogr. B., 2005, 822, 294.

3. K. Kirschbaum, M. Müller, G. Zernig, A. Saria, A. Mobascher, J. Malevani, C. Hiemke,

4. Clinical Chem., 2005, 51, 1718.

5. Nandini R. Pai* and Swapnali Patil Der Pharmacia Sinica, 2013, 4(2):76-84

6. Chromatogr. B., 2007, 856, 57.

7. V. Shah, K. Kamal, D. Shrikant, J. Iain, P. Jerome, Y. Avraham, L. Thomas, C. Viswanathan C. Edgar, R. McDowall, A. Kenneth, S. Sidney, J. Pharm. Sci., 1992, 81, 309.

8. Nandini R. Pai et al Der Pharmacia Sinica, 2012, 3 (5):526-535

9. International Conferences on Harmonization Q3A (R2). Draft Revised Guidance

10. Impurities in New Drug Substances,(2006).

11. International Conferences on Harmonization Q2 (R1). Validation of analytical procedures: Text and methodology, (2005).

12. www.wikipedia.org

13. www.drugs.com

Received on 08.06.2013 Modified on 30.06.2013

Research J. Pharm. and Tech. 6(9): September 2013; Page 1014-1018