devlopement and validation of stability indicating rp …
TRANSCRIPT
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DEVLOPEMENT AND VALIDATION OF STABILITY INDICATING
RP-HPLC METHOD FOR SECNIDAZOLE GRANULES
Digambar Ashok Nagare*, Dr. Vipul P. Patel1, Sima Gosavi
1 and
Tushar N. Deore
1
*(M- Pharmacy Department of Pharmaceutical Quality Assurance).
1Sanjivani College of Pharmaceutical Education and Research, Kopargaon. Tal. Kopargaon
Dist. Ahamadnagar (India).
ABSTRACT
Objective of the present work is to develop and validate a simple, cost
effective, sensitive and fast RP-HPLC method for the analysis of
Secnidazole Granules. A Waters HPLC system with Inertsil ODS-3V,
C18, 150 mm, 5μm column is employed for the analysis using 0.01 M
Potassium Dihydrogen Orthophosphate Buffer Solution, Acetonitrile
And Methanol in the ratio of (70:25:5 v/v) as mobile phase. Signal
from Secnidazole is detected at 228 nm by UV Spectrophotometer.
The proposed method is fully validated and found to be linear over a
workable drug concentration, accurate, precise and robust. This fast
and inexpensive method and also this method is HPLC Equivalent to UPLC. It is suitable for
research laboratories as well as for quality control analysis in pharmaceutical industries.
KEYWORDS: Secnidazole, RP-HPLC, Waters system , validation etc.
INTRODUCTION
Secnidazole is an antiprotozoal drug. It is derivatives of 5-nitroimidazole which is closely
related to metronidazole (Figure 1). It is category of6 Anti-infective drugs, Anti-protozoal
drugs, Antiamebic and Antigiradiasis drugs. It is of Synthetic Origin and belongs to
Nitroimidazole. Secnidazole Belonfs to Amebicides pharmacological group.
Secnidazole is used to treat intestinal ameobiasis, trichomonosis, fiardiasis and bacterial
viginosis. These disease symptoms can be treated with single onces only dose of secnidazole
granules. However, prolonged plasma half life of of secnidazole makes its first choice for
treatment of many disease (Gillis and Wiseman, 1996) Various method validations for
Article Received on
10 July 2018,
Revised on 30 July 2018,
Accepted on 20 August 2018
DOI: 10.20959/wjpps20189-12300
*Corresponding Author
Digambar Ashok Nagare
(M- Pharmacy Department of
Pharmaceutical Quality
Assurance).
WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES
SJIF Impact Factor 7.421
Volume 7, Issue 9, 1041-1063 Research Article ISSN 2278 – 4357
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secnidazole has been developed using different analytical method for the determination of
secnidazole inpharmaceutical dosagr form formulation (Misiuk, 2010, Farooqui t al., 2010,
Bansode et al., 2013, Jain et al., 2014, Baraka et al., 2014). These method used eitheralone or
combination with other drugs. HPLC method more suitable than other because operational
pressures are significantly higher and require very small sample amount to be separated
(Shabir, 2003).
Among the existing method for determination of secnidazole in pharmaceutical dosage form,
most are time consuming, expensive, complex in nature and damage susceptibility of the
column (Alhalabi et al., 2012, Yanamndra et al., 2011). The HPLC method development for
secnidazole which are superior to prior developed method with respect to cost of analysis and
simplification (Sharmin et al., 2016). The Aim of present work was to attempt the
development of new RP-HPLC method for secnidazole Granules which are Superior to prior
developed method and validation with respect to analysis Time and sensitivity. When the
method Developed HPLC Equivalent to UPLC it must be validated to check that it meet
performance charecteristics. Typical analytical characteristic used in method validation are
accuracy, precision, specificity, linearity, range, ruggedness, robustness and force
degradation study. Further validated as per ICH Guideline.
MATERIALS AND METHODS
Chemical and reagents
Pharmaceutical grade of secnidazole was supplied as a generous gift from Alkem
laboratories, saver, Mumbai, secnidazole 1000 mg. Methanol HPLC grade from Fischer
scientific Mumbai, HPLC water and Distill water were used.
Secnidazole Granule working standard,
Source: Alkem Laboratories Ltd, Mumbai
Potency: 95.2 % (on as basis as Secnidazole Granules)
Chemicals Grade Supplier/Manufacturer
Potassium Dihydrogen Ortho-Phosphate AR Merck
Acetonitrile HPLC Fischer Scientific
Water HPLC PureLab
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Instrument used and chromatographic condition
HPLC system Compromised to column oven (Model: CT-10 ASvp Made by Shimadzu
corporation Japan), Prominanace liquid chromatographs, (Model: Shimadzu, Chromolean
Software, Version 7.0, PDA Detector), Waters System Liquid chromatographs, ( Model:
Waters 2489, Empower Software, Version-3.0, UV Detector-SPD 20A, Japan), Analytical
Balance, (Make: Sartorious And Metlor Toledo), Photo stability Chamber (Make:
Newtronic), Oven (Make: Thermolab), The chromatographic separation were performed
Using Inertsil ODS -3V, C18, 150mm x 4.6mm, 5µm as column, Phosphate Buffer :
Acetonitrile : Methanol (70:25:5, V/V), as Mobile Phase, Flow rate 1.5 ml/min with isocratic
elution, Run time 4 min and retation time of 2.2 min etc. The wavelength 228nm was
detected by UV Spectrometer (Model: UV-1800, made by Shimadzu corporation (Figure 2).
Figure 1. Chemical Structure of Secnidazole Figure 2. UV Spectrum of Secnidazole
Preparation of 0.01 M Potassium Dihydrogen Orthophosphate Solution
Dissolved 1.36g of Potassium Dihydrogen Orthophosphate in 1000ml of water.
Preparation of mobile phase
Prepared mixture of 0.01M Potassium Dihydrogen Orthophosphate buffer solution,
Acetonitrile and Methanol in the ratio of 70:25:5. Filter through 0.45µ membrane filtered and
degassed.
Standard Preparation
An accurately Weighed about 25mg of Secnidazole was placed into a 50 ml volumetric flask
and added 40ml of mobile phase, Then Sonicate to dissolved and diluted to the mark with
mobile phase. Then, 5ml were Withdrawn in a 50 ml Volumetric flask. The Volume was
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made with mobile phase to made final concentration 50 µl/ml. The Solution were passed
through 0.45µ PVDF filter/ 0.45µ nylon filter before injection.
Sample Preparation
Weighed and pool the content of 5 Sachets. Weighed and transfer quantity of sample
equivalent to about 500mg Secnidazole into a 250ml volumetric flask and, Added about 160
ml of mobile phase, sonicate for 15 minutes with intermittent shaking and make up the
volume with mobile phase. Filtered through 0.45µ PVDF filter/ 0.45µ nylon filter used. Then,
5ml were Withdrawn in a 200 ml Volumetric flask. The Volume was made with mobile phase
to made final concentration 50 µl/ml.
Method development and optimization
For the HPLC method determination of secnidazole with an intention to develop a precise,
accurate, simple, rapid, sensitive, and specific way, isocratic RP-HPLC method was
optimized. Secnidazole was very soluble in methanol, ethanol, chloroform, acetic acid, water
and soluble in acetonitrile. But Methanol, Acetonitrile and water easily available and low
cost. So, different mobile phase were tried with different fraction of Phosphate buffer,
methanol and acetonirile. A satisfactory resolution was achieved using a mixture of 0.01M
Potassium Dihydrogen Orthophosphate buffer solution, Acetonitrile and Methanol in the ratio
of 70:25:5 (V/V). The optimum wavelength for detection of secnidazol was 228nm. The flow
rate of secnidazole 1.5 ml/min at ambient temperature for column oven 30°C and Sample
compartment temp 25°C. The injection Volume was 20µl and run time was 4 min. The
retention time of secnidazole was observed at 2.2 min (Figure 3).
Figure 3: Typical Chromatogram of secnidazole Drug.
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SYSTEM SUITABILITY AND METHOD VALIDATION
System Suitability Parameters
To ascertain resolution and reproducibility of proposed chromatographic system for
estimation of Secnidazole Granules, system suitability parameters like theoretical
plates(USP), area precision, retention time precision were studied. Standard stock solution
containing Secnidazole (50µg/ml) was used for analysis. The filtrate (20 µl) was injected into
the column and chromatographed using optimized chromatographic conditions. The system
suitability test was performed from six replicate injections of standard solution. The
corresponding chromatograms were recorded at 228 nm. System suitability parameters were
calculated (Table 1 and Table 2).
All the system suitability parameters assessed as per ICH guidelines
% Potency of the STD (on as is basis) 95.2
Table 1.
Injection Area of STD
(Secnidazol) Injection
Area of STD
(Secnidazol) Injection
Area of STD
(Secnidazol)
1 630042 1 630042 1 630042
2 630333 2 630333 2 630333
3 625791 3 625791 3 625791
4 629909 4 629909 4 629909
5 633253 5 633253 5 633253
Mean 629866 Bkt. Std.-1 631541 Bkt. Std.-2 628485
SD 2661.2
% RSD 0.42 Mean 630145 Mean 629636
SD 2476.6 SD 2446.1
% RSD 0.39 % RSD 0.39
Sample Dilution Wt. of Sample (mg)/ Wt. of secnidazole granules Spl. Dil.-2 5
250 200
Table 2.
Sample
No.
Wt. of Sample (mg)/
Secnidazole granules
Area %
Assay Injection 1 Injection 2 Average Area
1 1185.95 639789 639446 639618 98.08
2 1187.39 638062 639156 638609 97.81
3 1185.13 639255 639662 639459 98.13
4 1183.75 637671 634952 636312 97.76
5 1184.87 635971 640077 638024 97.93
6 1183.54 636903 637321 637112 97.90
Average 97.94
SD 0.146
% RSD 0.15
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Linearity
Suitable dilutions using mobile phase were made from the standard stock solutions containing
50 µg/ml of Secnidazole to prepare range of standard solutions containing six different
concentrations of analyses. Five replicates per concentration were injected. The linearity of
the relationship between peak area and concentration was determined by analyzing five
standard solutions over the concentration range 35-65µg/ml for Secnidazole. The calibration
curve data is presented in table 3 and 4 for Secnidazole and the calibration curve is presented
in (Table 3). (acceptance criteria: Not less than 0.999).
Secnidazole Standard Preparation For Linearity
STD weight 26.22 Std.Dil.2 5
50 50
Table 3: Calibration curve of Secnidazole.
Injection Area of STD Injection Area of Standard
1 656382 1 656382
2 655732 2 655732
3 656597 3 656597
4 656492 4 656492
5 656194 5 656194
Mean 656279 B.std-1 656301
SD 340.417 Mean 656283
% RSD 0.05 SD 304.606
% RSD 0.05
Fig 4 Linearity Plot for Secnidazole.
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Table 4: Linearity of Secnidazole.
Level
Spike
level in
%
Vol. Added
from stock
solution-1 (ml)
Diluted
to (ml)
Concentration
(mcg/ml) Inj-1 Inj-2
Average
Area
1 70 3.5 50 34.95 456370 456519 456445
2 80 4.0 50 39.94 521866 521496 521681
3 90 4.5 50 44.93 587640 587433 587537
4 100 5.0 50 49.92 655396 655893 655645
5 110 5.5 50 54.92 723150 723307 723229
6 120 6.0 50 59.91 789702 790505 790104
7 130 6.5 50 64.90 855761 856058 855910
Slope 13385
Intercept -12436
Corelation
Coefficient
0.99998
Analysis of Secnidazole granules Sample
Weighed accurately Granules and a quantity of granules equivalent to 500 mg of Secnidazole
was weighed in 250 ml of volumetric flask and dissolved in the 100 ml of methanol with the
aid of ultrasonication for 15 min and make up the volume with mobile phase upto the mark.
Further dilute 5 to 200 ml with mobile phase, filtered through PVDF 0.45 filter.From this
solution appropriate dilutions were made and injected in to the system to get the
chromatogram. The results obtained are shown in Table 5.
Table 5: Analysis of Secnidazole granules and Precision of Secnidazole.
Sample
No.
Wt. of Sample (mg)/ No.
of Tabs/Caps
Area of sample %
Assay Injection 1 Injection 2 Average Area
1 1183.77 658212 657949 658081 99.78
2 1186.68 651550 661189 656370 99.28
3 1187.40 658352 657770 658061 99.47
4 1187.29 658191 658357 658274 99.51
5 1184.97 658766 658676 658721 99.78
6 1183.01 654223 653894 654059 99.23
Average 99.51
SD 0.236
% RSD 0.24
Precision
The precision of the developed HPLC method was determined by preparing the Secnidazole.
The result for Secnidazole was shown in Table 6.
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Table 6: Precision.
Injection Area of STD
1 639040
2 638671
3 638343
4 637989
5 638213
6 638302
Mean 638426
SD 373.0
% RSD 0.06
Accuracy
To check the accuracy of the proposed method, recovery studies were carried out according
to ICH guidelines by applying the standard addition method to known amount of Secnidazole
corresponding to 70, 100 and 130%. From the secnidazole working standard weighed
accurately 25 mg of 50 ml volumetric flask add 40 ml of mobile phase, sonicate to dissolve
the drug and make up the volume of mobile phase. (Conc.50 mg/ml.) And prepare sample
solution, 70%, 100% and 130%. Analysis was performed as per the procedure given under
the Secnidazole Granules analysis. The recovery studies were performed three times at each
level. The results obtained are shown in Table 7.
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Table 7: Results of the recovery analysis of Secnidazole Granules.
Level No
Amount
Added in
mg
Actual
Amount
added in mg
Area-
Inj.1
Area-
Inj.2
Average
area
Amount
Found
in mg
%
Recovery Average SD %RSD
Sample-1
(70%) 351.63 334.75 437186 436449 436818 333.51 99.63
99.91 0.252 0.25 Sample-2
(70%) 351.79 334.90 439004 439292 439148 335.29 100.12
Sample-3
(70%) 350.44 333.62 436775 436942 436859 333.54 99.98
Sample-1
(100%) 501.64 477.56 620671 621299 620985 474.12 99.28
99.30 0.136 0.14 Sample-2
(100%) 502.10 478.00 621138 620545 620842 474.01 99.17
Sample-3
(100%) 501.37 477.30 621863 621503 621683 474.65 99.44
Sample-1
(130%) 650.89 619.65 806231 806295 806263 615.58 99.34
99.29 0.056 0.06 Sample-2
(130%) 651.25 619.99 806745 806038 806392 615.67 99.30
Sample-3
(130%) 651.31 620.05 805718 806041 805880 615.28 99.23
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RESULT AND DISCUSSION
Precision
1. System precision
Prepared standard solution as per test method and injected for 6 times in HPLC system.
%RSD for peak areas of Secnidazole was calculated and results are tabulated in Table 8.
Acceptance criteria
The % relative standard deviation (% RSD) for peak areas of Secnidazole should not be more
than 2.0.
Table 8: System Precision.
Sr. No. Area
1 639040
2 638671
3 638343
4 637989
5 638213
6 638302
Mean 638426
SD 373.0
% RSD 0.06
Conclusion
The %RSD value indicates an acceptable level of precision of the analytical system for the
assay of Secnidazole in Secnidazole Granules (2g Secnidazole per Sachet).
2. Method precision
Six samples of same batch were analysed as per test method. The % assay of Secnidazole in
Secnidazole Granules (2g Secnidazole per Sachet) in each six samples was calculated and the
results are tabulated in table 9.
Acceptance criteria
The % relative standard deviation (% RSD) for % Assay of Secnidazole in Secnidazole
Granules (2g Secnidazole per Sachet) for the six samples should not be more than 2.0.
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Table 9: Method Precision.
Sr. No. % Assay
1 99.78
2 99.28
3 99.47
4 99.51
5 99.78
6 99.23
Mean 99.51
SD 0.236
% RSD 0.24
Conclusion
The %RSD value indicates an acceptable level of precision of the analytical method for the
assay of Secnidazole in Secnidazole Granules (2g Secnidazole per Sachet).
3. Intermediate precision (Ruggedness)
Ruggedness of the method was verified by analysing the six samples of same batch which
was used for method precision as per test method by different analyst using different
instrument and different column on different day. The % assay of Secnidazole in Secnidazole
Granules (2g Secnidazole per Sachet) was determined. Calculated %RSD for % assay of
Secnidazole in six samples and overall %RSD for ruggedness results with the method
precision results are tabulated in table 10.
Acceptance criteria
The % relative standard deviation (%RSD) for % assay of the six samples should not be more
than 2.0 and the overall % RSD should not be more than 2.0.
Table 10: Intermediate Precision.
Sr. No. Analyst-I Analyst-II
1 99.78 98.08
2 99.28 97.81
3 99.47 98.13
4 99.51 97.76
5 99.78 97.93
6 99.23 97.90
Mean 99.51 97.94
SD 0.236 0.146
% RSD 0.24 0.15
Over all Mean 98.74
Over all SD 0.824
Over all % RSD 0.83
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Conclusion
The %RSD value indicates an acceptable level of intermediate precision of the analytical
method for the assay of Secnidazole in Secnidazole Granules (2g Secnidazole per Sachet).
4. Specificity
Blank (mobile phase), Placebo, standard and sample solution were injected into the HPLC
system. There was no interference from the blank and placebo at the retention time of
Secnidazole peak. Peak purity data reveals that Secnidazole peak was homogeneous and there
were no co-eluting peaks at the retention time of Secnidazole peak. The Retention time and
peak purity data for Secnidazole peak from control sample are summarized in table 11. Refer
fig. 6 for chromatogram of blank and fig. 7 for chromatogram of placebo. Refer fig. 8 for
chromatogram and fig 9 purity plot of control sample and refer fig. 24 for chromatogram of
standard.
Acceptance criteria
a. No peak shall be eluted at the retention time Secnidazole peak in blank and placebo.
b. Peak purity of Secnidazole peaks shall be passed for control sample.
[Note: for Empower software Purity angle shall be less than purity threshold. Peak shall not
be have any purity flag.]
Table 11: Specificity.
Sample type Retention
Time (min)
Purity
Angle
Purity
Threshold
Purity
Flag
Standard
(Secnidazole Drug) 2.247 - - -
Control sample
(Secnidazole
Granules)
2.253 0.049 0.241 No
Conclusion
The method is specific for the assay of Secnidazole in Secnidazole Granules (2g Secnidazole
per Sachet).
5 Stability in analytical solution
Stability of Secnidazole peak in analytical solution was verified by analyzing the standard
solutions and sample solutions, initially and also at different time intervals as mentioned
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below by storing in sample compartment of HPLC instrument at 25°C. Calculated the %
assay for standard solution and sample solutions. The results are tabulated in table 12 & 13.
Acceptance criteria
The solution should be considered as stable if the differences in the assay are not more than
2.0 at each time interval.
Table 12: Solution Stability For Standard Solution.
Time in hours % Assay Difference
Initial 101.08 -
3 101.00 0.08
7 101.13 0.05
10 101.46 0.38
12 101.77 0.69
18 101.67 0.59
20 102.06 0.98
22 102.61 1.53
25 101.96 0.88
Table 13: Solution Stability For Sample Solution.
Time in hours % Assay Difference
Initial 99.80 -
3 99.95 0.15
8 100.12 0.32
11 100.41 0.61
14 100.90 1.10
18 100.65 0.85
20 100.71 0.91
23 100.81 1.01
25 100.86 1.06
Conclusion
The difference in assay in both the standard and sample solutions were within the acceptance
criteria, hence it was concluded that the standard solution and sample solution is stable up to
25 hours at 25°C
6. Linearity
Linearity for Secnidazole performed in the range of 34.95mcg/ml to 64.90mcg/ml (about
70% to 130%) test concentration. A graph was plotted with concentration (in mcg/ml) on x-
axis and peak areas on y-axis. Slope, y-intercept, correlation coefficient (r-value) and residual
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sum of squares (RSS) were determined. The results are tabulated in table 14 for Secnidazole.
The results are represented graphically in figure 4.
Acceptance criteria
The correlation coefficient (r) value should not be less than 0.999.
Table 14: Linearity for Secnidazole.
Spike level in % Concentration mcg/ml Area
70 34.95 456445
80 39.94 521681
90 44.93 587537
100 49.92 655645
110 54.92 723229
120 59.91 790104
130 64.90 855910
Slope 13385
y-intercept -12436
r-value 0.99998
RSS 2372433263
Conclusion
The detector response of Secnidazole is directly proportional to concentration ranging from
70% to 130% test concentration i.e. 34.95mcg/ml to 64.90mcg/ml.
7. Accuracy
Placebo was spiked with the known amount of Secnidazole at 70%, 100% and 130% of test
concentration as Secnidazole in Secnidazole Granules (2g Secnidazole per sachet). The
amount of Secnidazole was quantified as per the test method. The percentage recovery was
calculated from the amount found and the actual amount added. The results are tabulated in
Table 15.
Acceptance criteria
The % recovery of Secnidazole at each spiking level should be in between 98.0 and 102.0 and
% RSD should not be more than 2.0. The overall average %recovery should be between 98.0
to 102.0 with %RSD not more than 2.0
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Table 15: Accuracy.
Level
no/Spike
level in %
Actual Amount of
Scnidazole added
in mg
Amount of
Scnidazole
found in mg
%Recovery Mean SD %
RSD
Level – 1
(70%)
334.75 333.51 99.63
99.91 0.252 0.25 334.90 335.29 100.12
333.62 333.54 99.98
Level – 2
(100%)
477.56 474.12 99.28
99.30 0.136 0.14 478.00 474.01 99.17
477.30 474.65 99.44
Level – 3
(130%)
619.65 615.58 99.34
99.29 0.056 0.06 619.99 615.67 99.30
620.05 615.28 99.23
Over all mean 99.50
Over all SD 0.341
Over all % RSD 0.34
Conclusion
The analytical method meets the pre-established acceptance criteria for accuracy study as per
protocol. Hence the method is accurate for the assay of Secnidazole in Secnidazole Granules
(2g Secnidazole per Sachet).
8. Range
Range inferred from the data of linearity, accuracy and precision experiments.
Conclusion
a) The method was found to be linear in the range of 70% to 130% of test concentration. (i.e.
Secnidazole: 50 mcg/ml as 100%).
b) The method was found to be accurate in the range of 70% to 130% of test concentration
for Secnidazole (i.e.Secnidazole: 50 mcg/ml as 100%).
9 Filter paper selection study
Selection of filter paper was evaluated by preparing the assay preparation in triplicate as per
test method. Filtered the test solution through 0.45µm PVDF filter and 0.45µm Nylon filter
analysed the samples against centrifuged a portion of sample solution. The % assay of
Secnidazole was calculated and compared the results with method precision results. Results
are tabulated in table 16.
Acceptance criteria
The difference in the results shall not be more than 2.
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Table 16: Filter Paper Selection Study.
Filter used Sample % Assay Difference
Centrifuged sample
1 98.14 -
2 98.20 -
3 97.97 -
0.45µm PVDF filter
1 98.08 0.06
2 97.81 0.39
3 98.13 0.16
0.45µm Nylon filter
1 98.03 0.11
2 97.66 0.54
3 97.83 0.14
Conclusion
0.45µm PVDF filter and 0.45µm Nylon filter are suitable for filtering the sample solution of
Secnidazole Granules (2g Secnidazole per sachet).
10. Robustness
Robustness of the method was verified by deliberately varying the following instrumental
conditions.
a. By changing the temperature by 5°C
b. By changing the flow rate by 10%
c. By changing the organic content in mobile phase by 2% absolute.
System suitability was evaluated in each condition and sample was analyzed in triplicate. The
system suitability parameters are tabulated in table-17.
Acceptance criteria
The overall %RSD for %assay of above results with method precision results should not be
more than 2.
Table 17: Robustness.
Sr. No. I II III IV V VI VII
1 99.78 98.55 98.62 98.81 98.77 98.01 97.93
2 99.28 99.82 98.65 98.80 98.63 97.98 98.01
3 99.47 98.46 98.68 98.81 98.80 97.88 98.26
4 99.51 - - - - - -
5 99.78 - - - - - -
6 99.23 - - - - - -
Over all Mean 99.32 99.22 99.27 99.25 98.99 99.03
Over all SD 0.509 0.468 0.397 0.432 0.799 0.750
Overall % RSD 0.51 0.47 0.40 0.44 0.81 0.76
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Conclusion
The method is robust for change in flow rate, change in column oven temperature and change
in organic content in mobile phase.
11 Summary of system suitability
System suitability was evaluated by injecting standard solution during various days of
validation. The tailing factor for the first standard injection and % relative standard deviation
for the peak areas of Secnidazole from five replicate injections of standard solution were
verified at every stage. The results are tabulated in table 18 Refer fig. 23 for the
representative chromatogram of standard solution.
Table 18: Summary of System Suitability.
Sr.
No. Name of Experiment
Tailing
Factor %RSD
1 System precision, Method precision and Solution Stability 1.3 0.06
2 Recovery 1.3 0.13
3 Linearity 1.3 0.05
4 Robustness : Minus Flow 1.3 0.04
5 Plus Flow 1.3 0.07
6 Minus Temperature 1.3 0.04
7 Plus Temperature 1.3 0.04
8 Minus Organic 1.3 0.05
9 Plus Organic 1.3 0.06
10 Ruggedness / Filter Paper Study 1.2 0.42
11 Force Degradation and Specificity 1.1 0.37
Acceptance Criteria
1. % RSD (Relative Standard Deviation) of five replicate injections of Standard preparation
for Secnidazole peak should not be more than 2.0.
2. The tailing factor for Secnidazole peak should not be more than 2.0.
12. Forced Degradation
Forced degradation study was carried out by treating the sample under the following
conditions.
a) Degradation by hydrochloric acid (Acid treated sample)
Sample and placebo were treated with 5 mL of 0.1N hydrochloric acid solution for 24 hours.
Treated samples solution was neutralized with 5mL of 0.1N sodium hydroxide solution.
Treated samples solutions were analyzed as per the test method.
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b) Degradation by sodium hydroxide (Base treated sample)
Sample and placebo were treated with 5mL of 0.1N sodium hydroxide solution for 24 hours.
Treated samples solution was neutralized with 5 mL of 0.1N hydrochloric acid solution.
Treated sample solutions were analysed as per the test method.
c) Degradation by hydrogen peroxide (Peroxide treated sample)
Sample and placebo were treated with 5mL of 3.0% hydrogen peroxide solution for 24 hours.
Treated sample solutions were analysed as per the test method.
d) Degradation by thermal (Heat treated sample)
Sample and placebo were kept in oven at 60°C for about 24 hours. Treated sample were
analysed as per the test method.
e) Degradation by hydrolysis (Water treated sample)
The sample and placebo were treated with 5ml water for 24 hours. The treated sample
solutions were analysed as per test method.
f) Degradation by UV –Visible light (UV-visible treated sample)
Sample and placebo were exposed to UV light of about 200 watt hours/square meter and to
visible light for about 1.2 million lux hours in Photo stability chamber. Treated sample were
analysed as per the test method.
g) Degradation by Humidity
Sample and placebo were exposed at 25ºC temperature and 90% relative humidity prepared
with supersaturated solution of potassium nitrate for 24hours. Treated samples solutions were
analyzed as per the test method.
The results of forced degradation studies are summarized in table 19. Refer fig.9.10,11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21 and 22 for chromatograms and purity plot of treated sample
solutions.
Acceptance criteria
The peak purity for Secnidazole peaks shall be passed.
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Table 19: Forced Degradation.
Condition % Assay % Degradation
w.r.t. Control
Purity
Angle
Purity
Threshold
Purity
Flag
Untreated Sample 98.20 - - - -
Acid Treated sample 95.18 3.08 0.051 0.243 No
Base Treated sample 90.15 8.20 0.047 0.242 No
Peroxide Treated sample 95.11 3.15 0.054 0.246 No
Water Treated sample 96.15 2.09 0.051 0.246 No
Heat Treated sample 95.74 2.51 0.049 0.241 No
UV Treated sample 97.54 0.67 0.048 0.241 No
Humidity Treated Sample 95.60 2.65 0.045 0.242 No
Conclusion
The method is stability indicating for assay of Secnidazole in Secnidazole Granules (2g
Secnidazole per Sachet).
Fig.5: HPLC Chromatogram of Blank Fig.6: HPLC Chromatogram of placebo
Fig.7: HPLC Chromatogram of
control sample
Fig.8: Purity plot of control sample.
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Fig.9: HPLC Chromatogram of Acid
treated sample
Fig.10 Purity plot of Acid treated sample
Fig.11: HPLC Chromatogram of Base
treated sample Fig.12 Purity plot of Base treated sample
Fig.13: HPLC Chromatogram of Peroxide
treated sample
Fig.14: Purity plot of Peroxide treated
sample
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Fig.15 HPLC Chromatogram of water
treated sample
Fig.16 Purity plot of water treated sample
Fig.17: HPLC Chromatogram of heat
treated sample Fig.18 Purity plot of heat treated sample
Fig.19: HPLC Chromatogram of humidity
treated sample
Fig.20 Purity plot of humidity treated
sample
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Fig.21 HPLC Chromatogram of UV
treated sample Fig.22 Purity plot of UV treated sample
Fig. 23: HPLC Chromatogram of standard (Secnidazole Drug).
CONCLUSION
The proposed HPLC methods are simple, accurate, and precise for Secnidazole Six replicate
samples of Secnidazole Granules were Analyzed HPLC methods and the results were
correlated.
A simple, specific, linear, precise and accurate HPLC method has been developed and
validated for quantitative determination of Secnidazole Granules formulation. The method is
very simple and specific as peak of Secnidazole is well separated and there is no interference
by excipient with total run time of 4 min and regular simple mobile phase which makes it
especially suitable for routine quality control analysis work.
The HPLC method for identification of Secnidazole Granules formulation is simple, accurate,
and precise and requires a very small amount of mobile phase, compared to UPLC method.
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