Novel RP-HPLC method for quantification of 2-(dimethyl amino) thioacetamide impurity in Nizatidine drug substance

 

Naga Jhansi Tamanampudi^1,2*, Naga Raju Rajana^1,2, Pavan Kumar Dharmasanam^1,2,

Jaya Deep Kumar Lilakar¹, Nageswara Rao Gollapalli²

¹Department of Analytical Research and Development, Reddy’s Laboratories Ltd., Visakhapatnam, 530045, Andhra Pradesh, India.

²Department of Inorganic and Analytical chemistry, Andhra University, Visakhapatnam, 530003,

Andhra Pradesh, India.

 

ABSTRACT:

This study describes the development and quantification of reverse phase HPLC method for the genotoxic impurity i.e. 2- (dimethyl amino) thioacetamide commonly named as DIS. It is one of the key raw material for the synthesis of Nizatidine product. There is no method is available for the determination of DIS in any active pharmaceutical ingredients/drug substance. After several development trials, DIS quantification at extremely low level was accomplished on YMC PACK ODS A, 15cm x 4.6mm, 3.0µm column using the combination of sodium acetate and n-Heptane sulfonic acid sodium salt with additives of Acetic acid and Tri ethyl amine as buffer and acetonitrile as organic modifier. Diluent was selected as water. The flow was selected as 1.5mL/min and temperature of column was maintained at 35°C. The wavelength was selected at 254nm and injection volume was 10µL. The limit of detection and limit of quantification were 20ppm and 50ppm respectively. The method was validated and found to be precise, accurate and robust. The method can be used for determination of 2- (Dimethyl amino) thioacetamide, which is the key starting material for preparation of Nizatidine.

 

 

 

INTRODUCTION:

Nizatidine is a histamine H2-receptor antagonist that inhibits stomach acid production, and commonly used in the treatment of peptic ulcer disease (PUD) and gastroesophageal reflux disease (GERD). It was developed by Eli Lilly and is marketed under the trade names Tazac and Axid certain preparations of nizatidine are now available over the counter in various countries including the United States. Nizatidine has been used experimentally to control weight gain associated with some antipsychotic medication^1,2. 2- (dimethyl amino) thioacetamide hydrochloride is used in the preparation of thiazole derivatives. 2-(dimethyl amino) thioacetamide is an impurity of the histamine H2-receptor antagonist Nizatidine (N598500)^3,4. DIS impurity details were captured in Fig 1 and preparation of Nizatidine from DIS as starting material was shown in Fig 2.

 

Fig: 1 Details of DIS impurity

 

Name: 2-(dimethyl amino) thioacetamide hydrochloride; Molecular Formula: C₄H₁₁ClN₂S; Molecular weight: 154.66; CAS Number: 27366-72-9

 

Fig: 2 Synthetic schemes of Nizatidine from DIS starting material

 

MATERIALS AND METHODS:                                                                   

Samples and reagents:

The drug development samples of Nizatidine and 2- (Dimethyl amino) thioacetamide were received from process development laboratory of Reddy’s Laboratories Ltd., Ammonium acetate, n-Heptane sulfonic acid sodium salt, Ortho phosphoric acid (AR grade), Tri ethyl amine and acetonitrile (HPLC grade) were purchased from Merck (India) Limited. Milli-Q grade water was obtained from the Milli-Q Plus water purification system used.

 

Instrumentation:

High performance liquid Chromatography (RP-HPLC) instrument used with Agilent Infinity 1260 series. Weighing of standards and test samples were carried out using analytical balance with Sartorius make and MSA 225S-100-DA model. The obtained chromatographic data were integrated through empower-3 software.

 

Chromatographic Conditions:

Quantification was carried out on YMC PACK ODS A, 15cm x 4.6mm, 3.0µm. Mobile phase-A was selected as 50mM ammonium acetate and 10mM n-Heptane sulfonic acid sodium salt in to a 1000mL of water and added 0.3% (v/v) of Acetic acid and 0.1% (v/v) of tri ethyl amine. Mobile phase –B was selected as 100% Acetonitrile. Diluent was selected as water and injection volume was 10µL. Column flow maintained at 1.5 mL/min with gradient program. Gradient program (Time/%B): 0/8, 5/8, 5.1/85, 9/85, 9.1/8 with 10 min post injection delay. The temperature of column oven was maintained at 35°C. Based on UV spectra of DIS, wavelength for the detection was selected as 254nm. To avoid more pressure on the column injection volume was set as 10µL. Sample concentration was taken as 1.0 mg/mL to get symmetry in the peak shape.

 

Standard and Sample Preparation:

Related substance by HPLC was performed with 1.0 mg/mL test concentration. %RSD of 0.1% 2- (Dimethyl amino) thioacetamide standard areas from six replicate standard injections was monitored as system suitability criteria. System suitability results were tabulated in Table 1.

 

Table: 1 System Suitability results of 2- (Dimethyl amino) thioacetamide

Name of the injection	Area of DIS
0.1% DIS standard	18993
0.1% DIS standard	19144
0.1% DIS standard	18864
0.1% DIS standard	18487
0.1% DIS standard	18385
0.1% DIS standard	18291
Average	18694
SD	353
% RSD	1.9%

Acceptance criteria: % of RSD for area should not be more than 10%

 

RESULTS AND DISCUSSION:

Method development and optimization:

As per the literature search several methods such as spectroscopic methods^5,6,7 and chromatographic methods ^8,9,10 are available for the determination of Nizatidine and its potential impurities. To the best our knowledge there is no analytical method is available for the quantification of 2- (dimethyl amino) thioacetamide. Hence the development trials were started from the basic mobile phase such as 0.1% Formic acid in water and Methanol ^11,12. But the peak elution pattern was not satisfactory (Trial-1). The buffer strength was increased by selecting the acetate buffer, there was a slight separation of DIS peak from the sample matrix (Trial-2).

 

For the symmetrical peak elution introduced phosphoric acid as an additive and the mode elution was changed from isocratic to gradient. Due to the acidic buffer the retention time of the DIS was drastically changed to early retention rimes. But there was a slight tailing in the elution of DIS peak (Trial-3). To get symmetry in the peak shape added the tri ethyl amine to the buffer and column temperature was also increased to 35° C (Trial-4). Observed satisfactory peak shape and no interference from the sample matrix. Details of method development trials are tabulated in Table 2. Method development chromatograms are shown in Fig 3.

 

 

Table 2. Results method development trials

Trials	Column	Dimensions	Mobile phase	Column temp (°C)	Conclusion
1	X-Bridge C18	150X4.6mmX3.0µ	Formic acid in water/Methanol	25 °C	DIS peak was not separated from the sample peak
2	YMC PACK ODS A	150X4.6mmX3.0µ	NaOAc Buffer/Methanol	25 °C	No separation form DIS peak and sample matrix
3	YMC PACK ODS A	150X4.6mmX3.0µ	NaOAc Buffer / H3PO4 additive	25 °C	Peak shape is not good
4	YMC PACK ODS A	150X4.6mmX3.0µ	NaOAc and n-Heptane Sodium salt/ H3PO4 & TEA	35 °C	Good separation from sample Matrix

 

 

Fig: 3 Method development chromatograms

 

Validation:

This analytical method was validated to establish the suitability of the proposed method for the intended purpose. More specifically, it is a matter of establishing documented evidence providing a higher level of assurance with respect to the consistency of the method and results. It evaluates the product against defined specifications. The validation parameters viz., specificity, accuracy, precision, linearity, limit of detection, limit of quantitation, robustness, system suitability have to be evaluated as per the ICH guidelines ^13,14.

 

Specificity:

The specificity of an analytical method is the ability to assess unequivocally the analyte in the presence of components that may be expected to be present, such as impurities, degradation products, and matrix components. The basic criteria to show the specificity is no peak should be interfere at the retention time of peak of interest. 2-(dimethyl amino) thioacetamide is eluted at ~Retention time of ~3.0 min and there are no other peaks eluted at this particular RT.

 

Limit of detection (LOD) and Limit of Quantification (LOQ):

 Limit of detection and Limit of quantification values for 2- (dimethyl amino) thioacetamide was established by preparing the known concentration solutions from their stock solutions that would give a signal to noise ratio of 3:1 and 10:1 respectively. The LOQ Precision was carried out by preparing six individual solutions at LOQ level. Calculated % RSD for the areas of 2- (dimethyl amino) thioacetamide.

 

Accuracy at the Limit of quantification level was performed by preparing three accuracy solutions of Nizatidine by spiking 2- (Dimethyl amino) thioacetamide at LOQ level concentration. Calculated % recovery against the impurity spiked. LOD and LOQ results were tabulated in Table 3. Precision at LOQ and LOQ Recovery results were captured in Table 3 & 4. Blank, LOD and LOQ chromatograms are shown in Fig 4-6.

 

Table: 3 LOD and LOQ results

Parameter	Conc (ppm)	Area	S/N ratio
LOQ solution	50	1066	12
LOD solution	20	478	4.4

 

Table: 4 Precision at LOQ level results

Name of the injection	Area of DIS
Precision at LOQ level Prep-1	885
Precision at LOQ level Prep-2	821
Precision at LOQ level Prep-3	887
Precision at LOQ level Prep-4	823
Precision at LOQ level Prep-5	986
Precision at LOQ level Prep-6	911
Average	885
SD	62
% RSD	7.0

 

Fig: 4, 5, 6 Blank, LOD and LOQ chromatogram of 2-(dimethyl amino) thioacetamide.

 

Linearity:

The linearity is one of the essential parameters of validation, which gives the assurance on reported results by using the subject method at different concentration levels. Linearity was executed by preparing the solutions of 2-(dimethyl amino) thioacetamide at different concentration levels i.e., from LOQ to 0.15% against the target analyte concentration. Plotted the linearity graphs of peak areas versus concentration. These linearity curves give the information related to slope, intercept, correlation coefficients of regression and percent y-intercept. Linearity results are tabulated in Table 6 and calibration curves were shown in Figure 7.

 

Table: 5 Accuracy at LOQ results

S.No.	Name of the solution	Amount spiked (ppm)	Amount recovered (ppm)	% Recovery
1	Accuracy at LOQ preparation-1	51.125	52.82	103.3
2	Accuracy at LOQ preparation-2		53.56	104.8
3	Accuracy at LOQ preparation-3		50.19	98.2
Average recovery at LOQ (%)				102.1

 

Table: 6 Linearity Results

Name of the solution	Con. of solution (ppm)	Area
Linearity solution-1	50	880
Linearity solution-2	250	4452
Linearity solution-3	500	8952
Linearity solution-4	750	14206
Linearity solution-5	1000	19025
Linearity solution-6	1250	23958
Linearity solution-7	1500	28648
Slope		19.33
Y-Intercept		-342.08
% Y-Intercept		-1.80
Correlation coefficient		0.9998

 

Fig: 7 Linearity graph of 2- (Dimethyl amino) thioacetamide

 

Precision:

The precision was carried out by calculating the %RSD for the content of 2- (dimethyl amino) thioacetamide in each precision preparation. The precision solutions were prepared by spiking the 2- (dimethyl amino) thioacetamide impurity at specification level in to six different flasks. Each preparation was injected as per the developed method conditions. The same parameter was executed by changing the column, analyst and instrument in the same laboratory in order to establish the intermediate precision. Method precision and intermediate precision results were captured in Table 7.

 

Table: 7 Precision results of 2- (dimethyl amino) thioacetamide

Name of the injection	Area of DIS	Name of the injection	Area of DIS
Method Precision preparation-1	16582	Intermediate Precision preparation-1	18452
Method Precision preparation-2	17025	Intermediate Precision preparation-2	17582
Method Precision preparation-3	16982	Intermediate Precision preparation-3	18120
Method Precision preparation-4	17256	Intermediate Precision preparation-4	18563
Method Precision preparation-5	17631	Intermediate Precision preparation-5	17852
Method Precision preparation-6	16523	Intermediate Precision preparation-6	18423
Average	16999.8	Average	18165.3
SD	416.4	SD	386.6
% RSD	2.4	% RSD	2.1

 

Table: 8 Accuracy at 50%, 100% and 150% level

S. No.	Name of the solution	Spiked (ppm)	Recovered (ppm)	% Recovery
1	Accuracy at 50% preparation-1	499.8	505.8	101.2
2	Accuracy at 50% preparation-2		521.3	104.3
3	Accuracy at 50% preparation-3		495.3	99.1
Average recovery at 50 (%)				101.5
1	Accuracy at 100% preparation-1	1002.5	997.5	99.5
2	Accuracy at 100% preparation-2		1027.5	102.5
3	Accuracy at 100% preparation-3		1005.8	100.3
4	Accuracy at 100% preparation-4		983.1	98.1
5	Accuracy at 100% preparation-5		1023.1	102.1
6	Accuracy at 100% preparation-6		972.5	97.0
Average recovery at 100 (%)				99.9
1	Accuracy at 150% preparation-1	1505.9	1498.5	99.5
2	Accuracy at 150% preparation-2		1520.9	101.0
3	Accuracy at 150% preparation-3		1482.3	98.4
Average recovery at 150 (%)				99.6

 

Accuracy:

In order to carry out an accuracy parameter, solutions were prepared at three different concentrations i.e., 0.05, 0.10 and 0.15% w/w of analyte concentration. The % recovery for 2- (Dimethyl amino) thioacetamide impurity was calculated as an amount of impurity obtained against an amount of impurity spiked. Accuracy results were captured in Table 8.

 

Robustness:

The robustness is one of the validation parameters, which gives the measure method capacity to remain unaffected by intentional variations in method parameters and it is the indication of its consistency during normal usage. The robustness of the RP-HPLC method was executed by applying the changes in the method parameters from original experimental conditions such as flow rate was modified 1.2 mL/min. Wavelength was modified as 250 nm and 260 nm, instead of at 254 nm. Temperature of the column oven was changed to 30° C and 40 °C instead of 35 °C. The mobile phase composition was also modified as the ratio of organic modifier was studied with the variation of ±10.0% (As such). Results were recorded at all modified conditions, i.e. temperature, wavelength, flow rate and organic modifier and tabulated in Table 9.

 

Table: 9 System suitability results in Robustness parameter

Parameter	Results (% RSD of DIS)
System Suitability (As such cond.)	1.9
Robustness
Flow Variation (1.2 mL/min)	2.1
Column Temperature Variation (30º C)	3.2
Column Temperature Variation (40º C)	1.4
Wavelength at 250	2.6
Wavelength at 260	1.2
(-) In organic modifier- MP-A : MP-B:: 96:4 (v/v)	1.7
(+) In organic modifier - MP-A : MP-B:: 96:4 (v/v)	0.9

 

CONCLUSION:

A method for the quantification of 2- (Dimethyl amino) thioacetamide by RP-HPLC in nizatidine drug substance API has been successfully developed and validated as per the ICH guidelines. As per the literature search there is no analytical method was available for the determination of 2- (Dimethyl amino) thioacetamide. The validation results shown, that proposed RP-HPLC method was precise, accurate, linear and specific. Hence this method can be conveniently used to identify and quantify 2- (Dimethyl amino) thioacetamide in regular Nizatidine test samples.

 

ACKNOWLEDGEMENTS:

The first authors wish to thank the organization of Reddy’s Laboratories Ltd., for allowing this work to be published. Support extended by all the colleagues of Analytical Research and development department and Process Research and development department is thankfully acknowledged.

 

CONFLICTS OF INTERESTS:

The authors declare that they have no conflict of interest.

 

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Received on 15.04.2020                    Modified on 10.05.2020

Accepted on 24.05.2020                   ©AJRC All right reserved