INTRODUCTION:

Posaconazole is chemically, 4-[4-[4-[4-[[ (3R,5R)-5- (2,4-difluorophenyl)tetrahydro-5- (1H-1,2,4-triazol-1-ylmethyl)-3-furanyl]methoxy]phenyl]-1-piperazinyl]phenyl]-2-[ (1S,2S)-1 -ethyl-2-hydroxypropyl]-2,4-dihydro-3H-1,2,4-triazol-3-one which is used as an anti- fungal agent [1]. It is the first azole agent to demonstrate activity against the zygomycetes, a difficult-to-treat family that includes Mucor and Rhizopus species [2].

An extensive literature survey discloses some works related to posaconazole assay in biological fluids applying mainly chromatographic methods [3–9]. Kim et al. (2000) [3] validated a reversed-phase HPLC method, using 0.09 M ammonium phosphate monobasic acetonitrile- triethylamine (530:470:0.5 v/v/v) mobile phase for pharmacokinetics studies in dog serum with limit of quantification of 0.05 μg ml⁻¹.

In 2003 [4], the same group evaluated the presence of posaconazole active metabolites in human plasma by HPLC and microbiological assay, applying a mobile phase composed of 0.09 M ammonium phosphate buffer (pH 4.5) –acetonitrile- methylene chloride—triethylamine (1060: 940: 10:1 v/v), a C₁₈ column and 262 nm UV detection for the chromatographic method. Literature survey further revealed the availability of LC-MS/MS methods [5,7] to determine posaconazole in human plasma. Considering concomitant analysis of other azoles drugs and posaconazole in human plasma/serum, some works were published using liquid chromatography with UV and MS/MS detection, respectively [6, 8]. Ekiert et al. [9] made a review about the chromatographic and electrophoretic methods applied to azoles determination, including posaconazole.

Considering the analysis in bulk or pharmaceutical products, there is no work published and no monograph available in pharmacopoeias. So, the objective of this work is to develop and validate a stability-indicating high performance liquid chromatographic method for determination of posaconazole in bulk, according to official guidelines [10–11], with no necessity of buffers in the mobile phase. Since no HPLC method is reported for simultaneous estimation of posaconazole and its five impurities, therefore, in the present work, a successful attempt has been made to estimate the drug and its impurities in API [12-13].

EXPERIMENTAL:

Chemicals:

Samples of posaconazole and its related impurities were procured from Neuland Pharma Research, Hyderabad. HPLC grade methanol, were purchased from Merck, Darmstadt, Germany. High purity water was prepared by using Millipore Milli-Q plus water purification system. All samples and impurities used in this study were of greater than 99.0% purity.

Equipment:

The HPLC system, used for method development, forced degradation studies and method validation was Waters 2695 binary pump plus auto sampler and a 2996 photo diode array detector with empower 2 software (Waters Corporation, MA, USA). The method was carried out on Inertsil ODS-3V C₁₈ (150 x 4.6mm with 5µ particle size) column as a stationary phase. The output signal was monitored and processed using empower software on pentium computer (Digital equipment Co). Water bath equipped with temperature controller was used to carry out degradation studies for all solution. Photo stability studies were carried out in a photo stability chamber (Mack Pharmatech, Hyderabad, India). Thermal stability studies were performed in a dry air oven (Mack Pharmatech, Hyderabad, India). Posaconazole batch sample was obtained from Neuland Pharma Research, Hyderabad. All the chemicals and reagents used were of analytical grade. The synthetic scheme for the synthesis of posaconazloe is shown in Figure 17.

Chromatographic conditions:

The mobile phase used for the present investigation consisted of A and B. The mobile phase A employed was water and mobile phase B used is methanol. The flow rate of the mobile phase was 1.0 mL/ min. The HPLC gradient program was set as: time (min)/% solution B: 0.01/55, 15/70, 20/70, 30/90, 35/90, 37/55 and 45/55. The column temperature was maintained at 25˚C and the detection was monitored at a wavelength of 225and 260 nm. The injection volume was 10 μL. Mobile phase was used as diluent.

Preparation of solutions:

Weigh accurately 25mg of standard posaconazole and transfer into a 25 mL volumetric flask, dissolve and dilute up to the mark with diluent. By using this solution, prepare the following solutions.

(a) Reference solution 0.15% (1.5µg/mL)

Transfer 0.15 mL of standard solution into a 100 mL volumetric flask, dissolve and dilute up to the mark with diluent.

(b) Unspiked sample solution

Weigh accurately 50 mg of sample (it refers to the production batches of various posaconazole samples) and transfer into a 50 mL volumetric flask. Dissolve and dilute up to the mark with diluent. Prepare the sample solutions separately and label as preparation-1 & 2.

(c) Spiked sample solution

Weigh accurately 100 mg of sample, transfer into a 100 mL volumetric flask. Add 0.15 mL of intermediate-A, intermediate-B, formyl, diastereomer and benzyl posaconazole impurities from their respective stock solutions (prepared 1 mg/mL concentration of all five impurities separately) and make up to the volume with diluent and mixed well.

Procedure for recording chromatograms:

The optimized chromatographic conditions were set as mentioned earlier and a steady base line was recorded. After the stabilization of base line 0.15 % reference solution was injected and the chromatograms were recorded for six replicate injections. The unspiked and spiked sample solutions were injected separately and sample chromatograms were recorded in duplicate until the reproducibility of the peak areas were satisfactory.

METHOD VALIDATION:

After the method development, the method is validated in terms of parameters like specificity, system suitability, linearity, LOD, LOQ, precision, accuracy, and robustness parameters as per ICH guidelines.

System suitability studies

System-suitability tests are an integral part of method development and are used to ensure adequate performance of the chromatographic system. Retention time (RT), number of theoretical plates (N), tailing factor (T), and peak asymmetry (AS), resolution (RS) were evaluated for five replicate injections of the drug. The system suitability test was performed using six replicate injections of standards before analysis of samples.

Precision:

Precision is a measure of degree of reproducibility and repeatability of the analytical method and is usually expressed as the relative standard deviation.

System precision:

Weigh accurately 100 mg of standard, transfer into a 100 mL volumetric flask. Add 0.15 mL of intermediate-A, intermediate-B, formyl, diastereomer and benzyl posaconazole impurities from their respective impurity stock solutions and make up to the volume with diluent and mix well. Inject the above solutions into the chromatograph and measure the area for all six individual injections. Calculate the % RSD for area of all six replicate injections.

Method precision:

Weigh accurately 100 mg of sample, transfer into a 100 mL volumetric flask. Add 0.15 mL of Intermediate-A, Intermediate-B, Formyl, Diastereomer and Benzyl Posaconazole impurities from their respective impurity stock solutions and make up to the volume with diluent and mix well. Inject these solutions into the chromatograph and measure the area for all six individual injections. Calculate the % RSD for area of all six replicate injections.

Linearity:

It’s the ability of the method to elicit test results that is directly proportional to the analyte concentration within a given range.

Preparation of linearity solution-1 (LOQ Level):

Transfer 0.1 mL of sample solution in to 100 mL volumetric flask and add 0.15 mL of Intermediate-A, Intermediate-B, Formyl and Benzyl Posaconazole and Diastereomer impurities from their respective stock solutions and make up to the mark with diluent and mix well. Based on the signal-to-noise ratio obtained, LOQ solutions for impurities and posaconazole were prepared to obtain the signal-to-noise ratio about 10.

Preparation of linearity solution-2 (25%):

Transfer 0.025 mL of Posaconazole standard in to 100 mL volumetric flask and add 0.0375 mL of Intermediate-A, Intermediate-B, Formyl, BenzylPosaconazole and Diastereomer impurities from their respective stock solutions. Make up to the mark with diluent.

Preparation of linearity solution-3 (50%):

Transfer 0.05 mL of Posaconazole standard in to 100 mL volumetric flask and add 0.075 mL of Intermediate-A, Intermediate-B, Formyl, Benzyl posaconazole and Diastereomer impurities from their respective stock solutions. Make up to the mark with diluent.

Preparation of linearity solution-4 (100%):

Transfer 0.1 mL of Posaconazole standard in to 100 mL volumetric flask and add 0.15mL of Intermediate-A, Intermediate-B, Formyl, Benzyl posaconazole and Diastereomer impurities from their respective stock solutions. Make up to the mark with diluent.

Preparation of linearity solution-5 (125%):

Transfer 0.125 mL of Posaconazole standard in to 100 mL volumetric flask and add 0.1875 mL of Intermediate-A, Intermediate-B, Formyl, Benzyl posaconazole and Diastereomer impurities from their respective stock solutions. Make up to the mark with diluent.

Preparation of linearity solution-6 (150%):

Transfer 0.15 mL of Posaconazole standard in to 100 mL volumetric flask and add 0.225 mL of Intermediate-A, Intermediate-B, Formyl, Benzyl posaconazole and Diastereomer impurities from their respective stock solutions. Make up to the mark with diluent.

Procedure:

The above solutions were injected into HPLC and the areas were recorded. Plot the linearity graphs of Posaconazole form-1, Intermediate-A, Intermediate-B, Formyl impurity and Benzyl Posaconazole impurity separately by extrapolating concentration vs. area. Calculate the regression coefficient for Posaconazole form-1, Intermediate-A, Intermediate-B, Formyl, Diastereomer and Benzyl Posaconazole impurities.

Accuracy:

Preparation of LOQ level solution:

Weigh accurately 100 mg of sample into a 100 mL volumetric flask. Make up to the volume with LOQ solution and mix well.

Preparation of 100% level solution:

Weigh accurately 100 mg of sample into a 100 mL volumetric flask. Add 0.15 mL of Intermediate-A, Intermediate-B, Formyl, Diastereomer and Benzyl Posaconazole impurities from their respective stock solutions and make up the volume with diluent and mix well.

Preparation of 150% level solution:

Weigh accurately 100 mg of sample into a 100 mL volumetric flask. Add 0.225 mL of Intermediate-A, Intermediate-B, Formyl, Diastereomer and Benzyl Posaconazole impurities from their respective stock solutions and make up the volume with diluent and mix well.

Limit of detection:

Detection limit is determined based on signal-to-noise ratio.

Preparation of LOD Reference solution:

Preparation of LOD solution:

Based on the signal-to-noise ratio obtained from the LOD Reference solution, LOD solutions for impurities and Posaconazole were prepared to obtain the signal-to-noise ratio about 3.

Limit of quantitation:

Based on the signal-to-noise ratio obtained from the LOD Reference solution, LOQ solutions for impurities and Posaconazole form-1 were prepared to obtain the signal-to-noise ratio about 10.

Robustness:

(a) Effect of variation in Flow rate:

Analyse the sample at 1.0 mL/min ± 0.1 mL/min flow rate by keeping remaining conditions same. Analyse the samples and find out the impurities in Posaconazole form-1.

Condition-1: 0.1 mL increase in flow rate (1.1 mL/min)

Condition-2: 0.1 mL decrease in flow rate (0.9 mL/min)

(b) Effect of variation in oven temperature:

Analyse the sample at 250C ± 20C temperature by keeping remaining conditions same.

Condition-1: 20C increase in oven temperature (27⁰C)

Condition-2: 20C decrease in oven temperature (23⁰C)

Forced degradation studies:

Liquid state degradation:

Acid Hydrolysis:

Weigh accurately 20 mg of sample into a 20 mL volumetric flask. Add about 2 mL of 1N HCL dissolve and dilute up to the mark with diluent. Analyse the sample at room temperature at regular intervals i. e 0Hr, 24Hr and 24Hr reflux at 100⁰C.

Base Hydrolysis:

Weigh accurately 20 mg of sample into a 20 mL volumetric flask. Add about 2 mL of 1N NaoH dissolve and dilute up to the mark with diluent. Analyze the sample at room temperature at regular intervals i. e 0Hr, 24Hr and 24Hr reflux at 100⁰C.

Oxidation:

Weigh accurately 20 mg of sample into a 20 mL volumetric flask. Add about 2 mL of 3% H₂O₂ and dilute with the diluent up to the mark. Analyze the sample at room temperature at regular intervals i.e. 0Hr, 24Hr and 24Hr reflux at 100⁰C.

Solid state degradation UV Irradiation at 254 nm Spread nearly 500 mg of sample into a petridish and place into a UV cabinet at 254 nm for 24Hrs at room temperature. Remove the sample from UV light. Weigh accurately 20 mg of test sample and transfer into 20 mL volumetric flask dissolve and dilute up to the mark with diluent.

Thermal Degradation (about 105⁰C):

Place nearly 500 mg of sample in an oven at 105^OC for 24 Hrs and keep tightly closed, protected from moisture and light. Weigh accurately 20 mg of test sample and transfer into 20 mL volumetric flask dissolve and dilute up to the mark with diluent.

RESULTS AND DISCUSSION:

After the optimization of chromatographic conditions, estimation of process related impurities were carried out by the developed RP-HPLC method. Standard and sample solutions of Posaconazole were injected separately and chromatograms were recorded as shown in Fig (1 to 6) and in table1, respectively.

Linearity:

This was performed by preparing standard solutions of Posaconazole at different concentration levels of LOQ, 25, 50, 100, 125 and 150%. Twenty microlitres of each solution was injected in to the HPLC system. The peak responses were measured at about 225 and 260 nm and the corresponding chromatograms were recorded. The results are shown in table 2.

Precision:

Precision was performed by injecting six replicates of standard (system precision) and sample (method precision) solutions which were prepared and analyzed. The resulting chromatograms were recorded. The percent relative standard deviation (% RSD) was calculated and the results are incorporated in Table 3and 4, respectively

Accuracy:

Accuracy of the method was determined by standard addition method. The standard addition method was performed at LOQ, 100% and 150% level. The resulting solutions were analyzed in triplicate at each level as per the ICH guidelines. The percent recovery was calculated and results are presented in tables 5-9, respectively.

Limit of Detection (LOD):

LOD is defined as lowest concentration of analyte that can be detected, but not necessarily quantified, by the analytical method. Limit of detection is determined by the analysis of samples with known concentrations of analyte and by establishing the minimum level at which the analyte can be reliably detected.The results are shown in table 10.

Limit of quantification (LOQ):

LOQ is the concentration that can be quantitated reliably with a specified level of accuracy and precision. The results are incorporated in table 11, respectively.

Robustness:

Robustness of the developed method was demonstrated by purposely altering and evaluating the experimental conditions. Robustness of method was carried out with variation of flow rate ± 0.1 ml/min and variation of oven temperature ± 2°C.

Specificity:

Specificity is the ability of the analytical method to measure the analyte free from interference due to other components. Specificity was determined by comparing test results obtained from analyses of sample solution containing ingredients with that of test results those obtained from standard drug. Chromatograms for blank, standard & samples were recorded and they represent no interference.

Forced degradation studies :

They are performed as a part of specificity studies. Stability of a drug product or a drug substance is a critical parameter which may affect purity, potency and safety. Changes in drug stability can risk patient safety by formation of a toxic degradation product(s) or deliver a lower dose than expected. Therefore it is essential to know the purity profile and behavior of a drug substance under various environmental conditions. The forced degradation chromatograms under different conditions are shown from Figures 7-15and table 12, respectively.

CONCLUSIONS:

From the computer assisted literature survey, no method was established for the determination of process related impurities in Posaconazole API. It was concluded that there was no sensitive method reported for the estimation of the above selected drug, which promote to pursue the present work. The scope and objective of the present work is to develop and validate a new simple RP-HPLC method for the estimation of process related impurities in API. In the present investigation, Waters HPLC with PDA detector and Inertsil ODS-3V C₁₈column was selected. Injection volume of 10µL was used and the components were eluted with the mobile phase consisting of water and methanol by gradient programme. The flow rate was found to be optimized at1.0mL/min and the detection was carried out at225 and 260 nm respectively.

Precision of the developed method was studied under system precision and method precision. In System precision the %RSD for the area of known impurities obtained from six replicate injections of spiked solution were found within the acceptance criteria. Hence the system is precise. In Method precision the % RSD for the area of known impurities obtained from six different sample preparations were found within the acceptance criteria. Hence the method is precise. The precision for the known impurities at LOQ level were within acceptance criteria. The method is linear for Posaconaole and known impurities from LOQ to 150% level with respect to limit. The regression coefficients were found to be more than 0.99 of acceptance criteria. Hence the method is linear. The accuracy is studied for the known impurities from LOQ to150%level. The % Recovery of known impurities at LOQ and 100 % level were within 85 to115% and for 150% level were found within 80 to120%, which were all within acceptance criteria. Hence the method is accurate. In Robustness study, the deliberate changes from the actual method for flow rate (1.0 ± 0.1 mL) and oven temperature (25°C ± 2°C) were studied. The % variation for the impurities of Posaconazole obtained from the Robustness study and Method precision was found to be less than 10. Hence the method is Robust. Forced degradation studies revealed that the drug is stable in all stress conditions and the method is capable of resolving the degradation products from the Posaconazole peak. Finally the method is able to separate all process related impurities (Intermediate-A, Intermediate-B, Diastereomer, Formyl and Benzyl Posaconazole from the peak of Posaconazole. The method has been established to be specific, accurate, precise, linear, reproducible and robust one and is therefore suitable for routine analysis of Posaconazole in API. Method validation studies have been performed as per ICH guidelines and hence it may be implemented in research institutions, quality control department in industries, approved testing laboratories.

Figure 1: Chromatogram of Posaconazole standard at 225 nm (conc. 0.15 %)

Figure 2: Chromatogram of Posaconazole standard at 260nm (conc. 0.15 %)

_{Figure 3: C}_h_{romatogram
of Posaconazole
un-spiked sample
at 225nm (conc. 1mg/mL)}

Figure 4: Chromatogram of Posaconazole un-spiked sample at260nm

Figure 5: Chromatogram of Posaconazole sample spiked with 0.15%of known_{impurities at225 nm}

Figure 6: Chromatogram of Posaconazole sample spiked with 0.15%of known impurities at260 nm

Figure 7: Chromatogram of acid (1N HCl) stressed sample (24Hr reflux at 100°C) at 225 nm

Figure 8: Chromatogram of acid (1N HCl) stressed sample (24Hr reflux at 100°C) at260 nm

Figure 9: Chromatogram of alkali (1NNaoH) stressed sample (24Hr refluxat100°C) at 225 nm

Figure 10: Chromatogram of alkali (1NNaOH) stressed sample (24Hr refluxed at100°C) at 260 nm

Figure 11: Chromatogram of 3% H₂O₂stressed sample (24Hr refluxat100°C) at 225 nm

Figure 12: Chromatogram of 3%H₂O₂stressed sample (24Hr refluxat100°C) at 260 nm

Figure 13: Chromatogram of Thermal (105°C for24Hrs) stressed sample at 225 nm

Figure 14: Chromatogram of Thermal (105°C for24Hrs) stressed sample at 260 nm

Figure 15: Chromatogram of UV treated sample at 225nm

Figure 16: Chromatogram of UV treated sample at260nm

NaoH DMSO

Starting Material 1 (Intermediate A)

Starting Material 2 (Intermediate B)

Stage 1( Benzyl posaconazole)

PD/C Formic acid

Stage 2 Posaconazole

Diasteromer Impurity Formyl impurity

Figure 17: Synthetic scheme of Posaconazole

Table 1: Retention time and relative retention time for the determination of _{chromatographic
purity by
HPLC}

Name^(min)	^R_t^about	RRT about	RRF
Posaconazole	26.31	-	1.00
Intermediate-A impurity	15.9	0.60	0.66
Intermediate-Bimpurity	20.34	0.77	0.91
Diastereomer impurity	24.24	0.92	0.85
Formylimpurity	27.45	1.04	0.70
Benzyl Posaconazole Impurity	32.12	1.22	0.62

Table 2: Linearity Results

Component Name	Regressionco-efficient R²(NLT 0.99)	Y-intercept
Posaconazole	0.9997	-864.14
Intermediate-A impurity	0.9984	-353.79
Intermediate-Bimpurity	0.9991	-876.96
Diastereomer impurity	0.9992	-1485.4
Formylimpurity	0.9984	-426.37
Benzyl Posaconazole impurity	0.9995	-596.76

Table 3: System precision results

AREA
S. No	Intermediate-A (225nm)	Intermediate-B	Diastereomer	Formyl	Benzyl Posaconazole
1	25117	34283	38398	28835	27367
2	25352	34319	38847	27442	27318
3	25924	33699	38828	27312	28416
4	24927	34410	38143	28039	27290
5	25570	34170	38550	27511	27971
6	25497	34766	38548	26996	27583
Avg	25398	34275	38552	27689	27658
%RSD (NMT10%)	1.39	1.01	0.69	2.37	1.63

Table 4: Method precision results

AREA
S. No	Intermediate-A (225nm)	Intermediate-B	Diastereomer	Formyl	Benzyl Posaconazole
1	25466	34336	38535	27451	26115
2	25241	34619	38634	27925	26911
3	25659	34205	39240	28211	26733
4	25553	34593	39128	28266	26681
5	25595	34904	38882	27155	26247
6	25900	34393	38511	27831	25926
Avg	25569	34508	38822	27807	26436
%RSD 0.85 (NMT10%)		0.72	0.80	1.56	1.49

Table 5: Recovery data of Intermediate-A impurity

Conc. Level	Spiked Content (µg/mL)	Obtained Content (µg/mL)	%Recovery	Mean %Recovery	Acceptance criteria	%RSD (NMT10%)
	0.169	0.152	89.94
LOQ	0.169	0.141	83.43	88.16	100±20	3.58
	0.169	0.154	91.12
	1.515	1.554	102.60
100%	1.515	1.562	103.08	103.28	100±15	0.85
	1.515	1.578	104.16
	2.107	2.144	101.75
150%	2.107	2.120	100.60	100.98	100±15	0.92
	2.107	2.120	100.60

Table 6: Recovery data of Intermediate-B impurity

Conc.

Level

Spiked Content

(µg/mL)

Obtained content

(µg/mL)

%Recovery

Mean

%Recovery

Acceptance

criteria

%RSD

(NMT10%)

LOQ

0.151

0.132

0.137

0.131

87.42

90.73

86.75

88.3

100±20

1.56

100%

1.523

1.591

1.580

1.590

104.45

103.75

104.42

104.2

100±15

0.72

150%

2.265

2.425

2.463

2.471

107.08

108.72

109.09

108.3

100±15

1.38

Table 7: Recovery data of Formyl impurity

Conc.

Level

Spiked content

(µg/mL)

Obtained content

(µg/mL)

% Recovery

Mean

%Recovery

Acceptance

criteria

%RSD

(NMT10%)

LOQ

0.128

0.122

0.113

95.31

88.28

90.62

100±20

5.92

100%

1.508

1.572

1.611

1.591

104.25

106.82

105.49

105.52

100±15

1.56

150%

2.223

2.222

2.208

2.181

106.82

105.49

99.14

100±15

0.59

Table 8: Recovery data of Diastereomer impurity

Conc.

Level

Spiked content

(µg/mL)

Obtained content

(µg/mL)

Mean

%Recovery

Acceptance

criteria

%RSD

(NMT10%)

Mean

%Recovery

LOQ

0.179

0.163

0.168

0.161

91.06

93.85

89.94

91.61

100±20

0.66

100%

1.545

1.486

1.511

1.515

96.15

97.80

98.06

97.3

100±15

0.8

150%

2.232

2.517

2.521

2.519

112.75

112.95

112.88

112.86

100±15

0.49

Table 9: Recovery data of Benzyl Posaconazole impurity

Conc. Level	Spiked content (µg/mL)	Obtained content (µg/mL)	Mean %Recovery	Acceptance criteria	%RSD (NMT10%)	Mean %Recovery
	0.144	0.131	90.97
LOQ	0.144	0.135	93.75	92.6	100±20	1.29
	0.144	0.134	93.06
	1.523	1.553	101.98
100%	1.523	1.551	101.85	101.61	100±15	1.49
	1.523	1.523	101.01
	2.319	2.380	102.62
150%	2.319	2.373	102.34	102.63	100±15	0.71
	2.319	2.387	102.93

Table 10: LOD and LOQ results

S. No	Component Name	LOD (µg/mL)	LOQ (µg/mL)
1	Posaconazole	0.0455	0.138
2	Intermediate-A impurity	0.0558	0.169
3	Intermediate-Bimpurity	0.0498	0.151
4	Diastereomer impurity	0.0591	0.179
5	Formyl impurity	0.0422	0.128
6	Benzyl Posaconazole impurity	0.0475	0.144

Table 11: LOQ precision results

AREA
Intermediate - A		Intermediate-B	Diastereomer	Posaconazole	Formyl	Benzyl Posaconazole
Conc.(µg/mL)	0.169	0.151	0.179	0.138	0.128	0.144
1 2 3 4 5 6 Mean %RSD	2619 2605 2689 2533 2638 2646 2622 1.98	3050 2068 1828 2160 2055 2195 2970 1.54	2995 2911 2947 2976 2969 2964 3004 5.15	3283 2937 3122 2780 3226 2962 3159 6.16	2292 3111 3162 2900 3459 3039 2264 1.54	2202 2273 2223 2308 2263 2224 2085 6.74

Table 12: Forced degradation results

Peak purity

S.NO

Stressed Condition

Total impurities (%)

%Purity

Purity angle

Purity threshold

1N HCL

1N NaOH

^{3% H}₂^O₂

Thermal

0.8625

1.7756

0.1312

0.0506

99.14

100.0

98.22

99.87

99.95

0.56

1.01

0.78

1.11

1.46

1.03

11.17

4.07

19.07

2.29