DEVLOPEMENT AND VALIDATION OF STABILITY INDICATING RP …

www.wjpps.com Vol 7, Issue 9, 2018.

1041

Nagare et al. World Journal of Pharmacy and Pharmaceutical Sciences

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


1042


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


1043


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


1044


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.


1045


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


1046


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.


1047


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.


1048


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.


1049


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


1050


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.


1051


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


1052


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


1053


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


1054


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


1055


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.


1056


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


1057


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.


1058


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.


1059


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.


1060


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


1061


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


1062


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.


1063


REFERENCES

1. Sharmin T. Analytical method development and validation of Secnidazole in the tablet

dosage form by RP-HPLC method. International Current Pharmaceutical Journal, 2016;

5(4): 41-44.

2. Alhalabi, Z. Separation and assay of antiprotozoal imidazole derivatives (metronidazole,

tinidazole secnidazole) By RP-HPLC. International Journal of Pharmaceutical Science

Review and Research, 2012; 13(1): 1-18.

3. Bansode, A.S and Shelke, V. Stability indicating UV-spectrophotometric method for

Secnidazole in bulk and pharmaceutical formulation. International Journal of Universal

Pharmacy and Bio Sciences, 2013; 2(4): 19-26.

4. Elsadek, M.E. and Ibrahim, A.M. HPLC method for the simultaneous determination of

Secnidazole, Omeprazole and Amoxicillin mixture in pure forms and pharmaceutical

formulations. Asian Journal of Pharmaceutical Analysis And Medicinal Chemistry, 2014;

2(4): 197-207.

5. Farooqui, N.A. and Smith, A.Analytical Method Development and Validation of

Secnidazole Tablets by RP-HPLC. Journal of Pharmaceutical Sciences and Research,

2010; 2(7): 412-416.

6. Gillis, J.C. and Wiseman, L.R. Secnidazole. A review of its antimicrobial activity,

pharmacokinetic properties and therapeutic use in the management of protozoal infections

and bacterial vaginosis. Drugs, 1996; 51: 621-638.

7. ICH. (2005) International conference on harmonization of technical requiremnts for

registration of pharmaceuticals for human use. ICH Harmonised Triparatite Guideline,

Validation and Analytical Procedures: Text and Methodology, Q2(R1).

8. Jain P.S. and Lohar, T.R. Development and validation of stability-indicating HPTLC

method for estimation of secnidazole in bulk drug and pharmaceutical dosage form.

Journal of Advanced Drug Delivery, 2014; 1(4): 144-156.

9. Misiuk, W. The role of assay methods in characterizing the quality of bulk

pharmaceuticals. Journal of Pharmacy and Bioallied Science, 2010; 2(2): 88-92.

10. Yanamandra, R., Chaudhary, A. Development of a RP-UPLC method for the

simultaneous analysis of Secnidazole, Fluconazole, and Azithromycin: Application in

pharmaceuticals and human serum. International Journal of PharmTech Research, 2011;

3(2): 1198-1207.

Documents

DEVLOPEMENT AND VALIDATION OF STABILITY INDICATING RP …