Method Development and Validation for Simultaneous Determination of Isoniaizid and Atazanavir in Pure and Pharmaceutical Formulation by using HPLC

 

Prem Kumar Bichala^1*, R. Suthakaran², Ch. Shankar³

¹Associate Professor, Department of Pharmaceutical Analysis, Vijaya College of Pharmacy, Munaganoor – 501511, Hyderabad, Telangana, India

²Professor and Principal, Department of Pharmaceutical Chemistry, Vijaya College of Pharmacy, Munaganoor – 501511, Hyderabad, Telangana, India

³Associate Professor, Department of Pharmaceutical Analysis, Vijaya College of Pharmacy, Munaganoor – 501511, Hyderabad, Telangana, India

 

ABSTRACT:

A rapid and precise reverse phase high performance liquid chromatographic method has been developed for the validated of Isoniazid and Atazanavir, in its pure form as well as in tablet dosage form. Chromatography was carried out on a Phenomenex Gemini C18 (4.6 x 150mm, 5µm) column using a mixture of Methanol: TEA Buffer pH 4.5 (35:65) as the mobile phase at a flow rate of 1.0ml/min, the detection was carried out at 240 nm. The retention time of the Isoniazid and Atazanavir was 2.256, 5.427 ±0.02min respectively. The method produce linear responses in the concentration range of 5-25mg/ml of Isoniazid and 25-125mg/ml of Atazanavir. The method precision for the determination of assay was below 2.0%RSD. The method is useful in the quality control of bulk and pharmaceutical formulations.

 

 

 

INTRODUCTION:

Isoniazid (Figure 1) was chemically Pyridine-4-carbohydrazide an Anti-Bacterial Agent. Isoniazid is a bactericidal agent active against organisms of the genus Mycobacterium. Isoniazid is a prodrug and must be activated by bacterial catalase. Half life of Isoniazid is for Fast acetylators: 0.5 to 1.6 hours and for Slow acetylators: 2 to 5 hours.

 

 

Fig. 1 Chemical Structure of Isoniazid

 

Atazanavir (Figure 2) was chemically methyl N-[(1S)-1-{[(2S,3S)-3-hydroxy-4-[(2S)-2-[(methoxycarbonyl)amino]-3,3-dimethyl-N'-{[4-(pyridin-2-yl)phenyl]methyl}butanehydrazido]-1-phenylbutan-2-yl]carbamoyl}-2,2-dimethylpropyl]carbamate, an Anti-Retroviral Agent. Atazanavir selectively inhibits the virus-specific processing of viral Gag and Gag-Pol polyproteins in HIV-1 infected cells by binding to the active site of HIV-1 protease, thus preventing the formation of mature virions. Half life of Atazanavir is 6.5 hours

 

 

Fig. 1 Chemical Structure of Atazanavir

 

MATERIALS AND METHODS:

The various materials and equipments used for the present study are summarized as follows.

 

Table 1. List of various equipments used

S.No	Instruments	Model
1	HPLC	WATERS Alliance 2695 separation module, Software: Empower 2, 996 PDA detector.
2	UV-VISIBLE Spectophotmeter	Shimadzu
3	pH meter	LabIndia
4	Weighing machine	Sartorius
	Digital ultra sonicator	Labman

 

Table 2. List of various materials used

S. No	Name of the material	Company
1	Isoniazide	Sura labs Pvt.Ltd(Gift sample)
2	Atazanavir	Sura labs Pvt.Ltd(Gift sample)
3	Water and Methanol for HPLC	LICHROSOLV (MERCK)
4	Acetonitrile for HPLC	Merck
5	Triethylamine	Merck

 

Optimized Chromatographic Conditions

 

Mobile Phase: Methanol: TEA buffer	65:35 (v/v)
Flow rate	1ml/min
Column	Phenomenex Gemini C18 (4.6×150mm, 5µ)
Detector wavelength	240nm
Injection volume	10 ml
Run time	10 min
Temperature	40ºC

 

 

 

Preparation of mobile phase:

350 ml (35%) of Methanol, 650 ml of Triethylamine buffer (65%) were mixed well and sonicated for 10 minutes and then filtered through 0.45 µ filter under vacuum conditions. The prepared solution was used as mobile phase.

 

Preparation of standard and sample solutions of Isoniazid and Atazanavir:

Preparation of Standard Stock Solution:

An accurately weighed quantity of Isoniazide (10mg) and Atazanavir (10 mg) were transferred into a 10ml of clean dry volumetric flasks add about 7mL of Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent.

 

Standard solution:

The 100 percent mixed standard solution of Isoniazide and Atazanavir was prepared by transferring 0.15 ml of Isoniazide and 0.75 ml Atazanavir to the 10 ml volumetric flasks and made up to the mark with dilute up to the mark with diluents.

 

Preparation of Sample Stock Solution:

20 Tablet contents were weighed and triturate to fine powders. An accurately weighed 10 mg equivalent weight of Isoniazide and Atazanavir sample into a 10mL clean dry volumetric flask and add about 7mL of Diluent and sonicated to dissolve it completely and make volume up to the mark with the same solvent.

 

Sample solution:

From this stock solution pipette 0.15 ml of Isoniazide and 0.75 ml Atazanavir above stock solution into a 10ml volumetric flask and dilute up to the mark with diluent.

 

RESULTS AND DISCUSSION:

The developed method of analysis was validated as per the ICH for the parameters like system suitability, specificity, linearity, precision, accuracy, robustness and system suitability, limit of detection (LOD) and limit of quantitation (LOQ).

 

System suitability:

System suitability test was carried out on freshly prepared mixed standard solution of Isoniazid and Atazanavir. 20 µL of the standard solution was injected under optimized chromatographic conditions and retention time, theoretical plates, area and tailing factor parameters were studied to evaluate the suitability of system and results were presented in Table 3.

 

 

 

 

 

Table 3 Data of System suitability

Injection no	Isoniazid				Atazanavir
	Retention time	Peak area	Efficiency (Th. Plates)	Asymmetry	Retention time	Peak area	Efficiency (Th. Plates)	Asymmetry
1	2.247	86092	5506	1.36	5.452	376065	1.04	15.0
2	2.246	85626	5674	1.2	5.484	373325	1.5	15.5
3	2.248	85557	5298	1.2	5.491	373435	1.2	15.3
4	2.252	86141	5032	1.0	5.482	375113	1.1	15.1
5	2.248	86557	5812	1.33	5.491	373435	1.2	15.2
Mean		85994				37427
SD		410.662				1247.001
%RSD		0.4				0.3

 

Specificity:

Specificity of the HPLC method was demonstrated by the separation of the analytes from other potential components such as impurities, degradants or excipients. A volume of 20 µl of working placebo sample solution was injected and the chromatogram was recorded. No peaks were found at retention time of 2.2 and 5.4 min. Hence, the proposed method was specific for Isoniazid and Atazanavir. The chromatogram of placebo was shown in Fig. 3. The specificity results were presented in Table 4 and 5.

 

 

Figure 3: Chromatogram of Blank

 

 

Figure 4: Chromatogram of Sample

 

Table 5 a Specificity Data of Sample

Name	Retention time	Peak area	Efficiency (Th. Plates)	Asymmetry	Resolution
Isoniazid	2.247	86092	9506	1.36	16.42
Atazanavir	5.452	376779	9511	1.38

 

 

Figure 5: Chromatogram of Standard

 

Table 5 b Specificity Data of Standard

Name	Retention time	Peak area	Efficiency (Th. Plates)	Asymmetry	Resolution
Isoniazid	2.256	84994	3535	1.32	16.27
Atazanavir	5.427	377906	9101	1.03

 

Linearity:

Calibration curves for Isoniazid and Atazanavir were prepared individually. Aliquots of 0.05, 0.1, 0.15, 0.2, 0.25 ml of sample stock solutions were transferred individually to the 10 ml of volumetric flasks and made up to the mark with mobile phase to get concentration of 5, 10, 15, 20, 25 µg/ ml of Isoniazid. Aliquots of 0.25, 0.5, 0.75, 0.1, 1.25 ml of sample stock solutions were transferred individually to the 10 ml of volumetric flasks and made up to the mark with mobile phase to get concentration of 25, 50, 75, 100, 125 µg/ ml for Atazanavir respectively. An aliquot (20 µl) of each solution was injected under the operating chromatographic condition as described above and responses were recorded. Calibration curves were constructed by plotting the peak areas versus the concentration and the regression equations were calculated is shown in Fig.6 and 7 and results were presented in Table 6.

 

 

Figure.6. Calibration curve of Isoniazide

 

 

Figure.7. Calibration curve of Atazanavir

 

Table: 6. Data of linearity

S. No	Isoniazid		Atazanavir
	Working conc. (µg/ ml)	Peak area	Working conc. (µg/ ml)	Peak area
1	5	51080	25	224573
2	10	92208	50	441895
3	15	139140	75	635379
4	20	180998	100	842226
5	25	223920	125	1041381
Correlation Coefficient (r)		0.999	0.999
Slope (m)		8893	8289
Intercept (c)		3394	12813

 

Precision:

To check the intra-day and inter-day variation of the method, standard concentration was subjected to the proposed HPLC method of analysis. The precision of the proposed method i.e. the intra and inter-day variations in the peak area of the drug solutions was calculated in terms of percent RSD. A statistical evaluation revealed that the relative standard deviation of drugs at different concentration levels for 6 injections was less than 2.0. The results for intra-day and inter-day precision were presented in Table 6 and Table 7 respectively.

 

Accuracy:

The recovery studies were carried out for the accuracy parameter. Accuracy at different concentrations (50%, 100%, and 150%) were prepared and the % recovery was calculated. The percentage recovery was found to be within the limit i.e. 98-102%). The results obtained for recovery at 50%, 100%, 150% are within the limits. Hence method is accurate. The results were presented in Table 8

 

Table: 7. Precision data for Isoniazid and Atazanavir

Injection No.	Isoniazid		Atazanavir
	Retention time (min)	Peak area	Retention time (min)	Peak area
1	2.248	84029	5.284	366831
2	2.245	84202	5.293	368856
3	2.242	84745	5.306	370174
4	2.239	85442	5.319	370603
5	2.243	85535	5.346	372578
6	2.246	85699	5.352	376550
Mean		84942		370932
SD		720.3716		3349.09
%RSD		0.8		0.9

 

 

 

 

Table: 8. Accuracy data for Isoniazid and Atazanavir

%Concentration (at specification Level)	Isoniazid			Atazanavir
	Amount Added (ppm)	Amount Found (ppm)	% Recovery	Amount Added (ppm)	Amount Found (ppm)	% Recovery
50	7.5	7.47	99.6	37.5	37.4	99.7
100	15	14.8	98.6	75	74.7	99.6
150	22.5	22.1	98.2	112.5	112.5	100
Mean % Recovery			98.8%			99.7%

 

 

 

Table: 9. Robustness data for flow rate variation

Flow rate (ml/min)	Isoniazid			Atazanavir
	Retention time (min)	Efficiency (Th. Plates)	Asymmetry	Retention time (min)	Efficiency (Th. Plates)	Asymmetry
1.0	2.256	5535	1.32	5.427	9101	1.01
0.9	2.505	5891	1.27	5.599	9407	1.03
1.1	2.046	5085	1.20	4.576	9584	0.98

 

Limit of Detection and Limit of Quantification:

LOD and LOQ were determined by using the formula based on the standard deviation of the response and the slope. LOD and LOQ were calculated by using equations, LOD = 3.3 × σ / s and LOQ=10×σ/S., The results were presented in Table 10.

 

Where

σ = Standard deviation of the response

S = Slope of the calibration curve

 

Robustness:

The robustness was performed for the flow rate variations from 0.9 ml/min to 1.1ml/min and mobile phase ratio variation from more organic phase to less organic phase ratio for Isoniazid and Atazanavir. The method is robust only in less flow condition and the method is robust even by change in the Mobile phase ±5%. The standard and samples of Isoniazid and Atazanavir were injected by changing the conditions of chromatography. The results were presented in Table 9.

 

 

 

Table: 10. Data table of LOD and LOQ for Isoniazid and Atazanavir

Drug	LOD (µg/ml)	LOQ (µg/ml
Isoniazid	0.54µg/ml	1.6µg/ml
Atazanavir	2.4µg/ml	7.3µg/ml

 

 

CONCLUSION:

In the present investigation, a simple, sensitive, precise and accurate RP-HPLC method was developed for the quantitative estimation of Isoniazid and Atazanavir in bulk drug and pharmaceutical dosage forms. This method was simple, since diluted samples are directly used without any preliminary chemical derivatisation or purification steps. Isoniazid and Atazanavir was freely soluble in ethanol, methanol and sparingly soluble in water. Methanol: TEA Buffer pH 4.5 (35:65) was chosen as the mobile phase. The solvent system used in this method was economical. The %RSD values were within 2 and the method was found to be precise. The results expressed inTablesfor RP-HPLC method was promising. The RP-HPLC method is more sensitive, accurate and precise compared to the Spectrophotometric methods. This method can be used for the routine determination of Isoniazid and Atazanavirin bulk drug and in Pharmaceutical dosage forms.

 

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Received on 16.05.2018       Accepted on 11.10.2018     

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 2018; 8(4): 203-208.

DOI: 10.5958/2231-5675.2018.00037.6