Dissolution Test Of Deprim Forte Tablets Biology Essay

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Abstract:

Deprim Forte tablets have as main actives sulphamethoxazole and trimethoprim. The combination of Sulphomethoxazole and Trimethoprim (SMX-TMP) is well known as co-trimoxazole. At the first place the product was approved for chronic urinary tract infections, shigellosis, and proven pneumocystis carini pneumonia and otitis media by the US Food and Drug Administration (FDA). The following report is referred on the procedure taken for the validation of HPLC dissolution test of deprim forte tablets. The method validation of HPLC dissolution test, consists a series of tests to confirm its ability to act for its purpose. This series of tests are specificity, linearity, accuracy, precision, intermediate precision, standard and samples stability in solution and robustness. Each of these tests has its acceptance criteria that should meet. If not then the test is not accepted. For every test the solutions are injected in HPLC chromatograph and analysed there. Standards solutions are prepared and injected in HPLC to get the calibration curve that gives the amount of anything that is injected after. The specific dissolution test method is followed for the preparation of the samples. Before each test a system suitability test is made to check if the system is adequate for the test to be performed. After checking that the criteria are met the next step take place. When all of the steps are done and the acceptance criteria are met then the method can be used for routine testing of Deprim Forte tablets.

Acknowledgements:

I am truly indebted and thankful to Dr Peter Clemenson for encourage me to go for a placement and also for the contact that we had during the time that I have been in Cyprus.

Of course the dissertation would not be possible without the help and guidance from staff of Remedica Ltd Cyprus and in particular Remedica's QC Manager who accepted me in the company for the practical section of my dissertation.

I owe sincere and earnest thankfulness to Remedica's Head of Analytical Development, R&D department who gave me the title of the dissertation and helped all this time with the practical and for the solutions that gave me in any questions or issues that I had.

I would like to thank also Remedica's QC Analyst who was my trainer for the HPLC and for the software.

I also would like to thank my parents and my friends who supported me all of this time.

Table of contents:

Introduction:

Deprim Forte tablets have as many active ingredients sulphomethoxazole and trimethoprim. Individually, sulphomethoxazole included in sulphonamides and so like them it is a structural analogue of p-aminobenzoic acid which is the main building block used by bacteria to synthesize dihydrofolic acid that is the first step to folic acid. Bacterial dihydropteroate synthetase is inhibited by sulphomethoxazole as a consequence of blocking incorporation of p-aminobenzoic acid into dihydrofolic acid. The conversion of hydrofolic acid to tetrahydrofolic acid which is the metabolically active cofactor for the synthesis of purines, thymidine and DNA is inhibited by trimethoprim through its binding with dihydrofolate reductase.

Sulphomethoxazole -Trimethoprim (SMX-TMP) is well known as co-trimoxazole. At the first place it was approved for chronic urinary tract infections, shigellosis, and proven pneumocystis carini pneumonia and otitis media by the US Food and Drug Administration (FDA). Also, in 1973 it was approved for the treatment of initial episodes of uncomplicated UTI, acute exacerbations of chronic bronchitis, traveller's diarrhea due to enterotoxigenic Escherichia coli and prevention of Pc Pin immunosuppressed patients. Due to the failure of SMX-TMP to eliminate the organism and prevent post streptococcal sequelae must not be used for the treatment of tonsidlopharyngitis.

In order to validate a method certain steps should take place. The steps were referred before in the explanation of the method validation procedure.

Specificity: The specificity test is the capability to measure accurately and specifically of the analyte of interest. It is in presence of other components that could be expected to be present in the sample matrix. Components could be other active ingredients, excipients, impurities and degradation products. Resolution, plate count and tailing (symmetry) factor are the parameters that measured and documented specificity.

Before each test the system suitability check was taking place by injecting six times the standard solution at 100% level. The acceptance criteria for this test were that the system suitability criteria should be met. This means that the test was valid only if the resolution between the peaks of the two actives was at least 4.0 and the RSD [%] of both main peak areas for the six replicate injections was no more that 2.0%. In addition to that any interference of the peaks due to mobile phase, solvents and placebo between the active peaks has as a result the failure of the test.

Linearity and range: The purpose of the linearity is to obtain test results which were directly proportional to the concentration of the analyte in the sample in a given range. The range is the breach between the lower concentration which is 50% and the high concentration which is 120%. The variance of the slope of the regression line is what linearity is noted. Then a graph was plotted between the mean peak area of each level and the concentration in g/ml for each level. If R2 > 0.99 then the linearity is expressed.

Accuracy: In the accuracy test the closeness of agreement between the true value and the value measured is studied. Accuracy is determined by analyzing samples added with known amounts of impurities for the quantitation of impurities. For the document of the accuracy according to ICH guideline nine determinations of minimum tree concentration levels covering the specified range should be studied. The results are expressed in percentage recovery.

Precision: Precision test is the scale of repeatability of an analytical method under normal operation and is most of the times expressed as the percent relative standard deviation. Precision is measured at three different levels which are repeatability, intermediate precision and reproducibility. In repeatability test the results should be operated under the same conditions over a short time. The range it should be covered by six determinations at 100% of the test or target concentration. The concentration of the compound of interest is the target concentration. The intermediate precision is the different variations that take place in the conditions, in the analyst, in equipment and it should happen in different day. Reproducibility associates to the results of collaborative studies between laboratories. In documentation included standard deviation, RSD, coefficient of variation and the confidence interval.

Stability in standards and samples: In this test the stability of the standards and samples are expressed. If the standards and samples showed to be stable then they can be used in more than one day for analysis. The range of standards covers from 80%, 100% and 120% concentration and three samples are tested.

Robustness: Robustness is the ability of a method to stay unaffected by small, intentional variations in method parameters. In order to achieve some method parameters for the HPLC method and dissolution method are varied. For the HPLC method the parameters that varied are the organic composition of the mobile phase, the pH of the buffer, the temperature of the column and the flow of the system. In addition, if the results of a method or other measurements are suggestible to variations in method parameters that should be sufficiently controlled and a precautionary statement should be included in the method documentation. For dissolution method the parameters that changed are the rotation of the paddles, the temperature of the medium and the medium in a non-deareated phase.

Experimental:

The instruments that were used for the achievement of the test were High Performance Liquid Chromatography (HPLC) and the Dissolution method using an Electrolab instrument.

High Performance Liquid Chromatography:

The system should be cleaned before each test. Therefore for the cleaning of the system a cleaning procedure was followed. There are four lines that can be used in this instrument. Line A is the line for the acetonitrile, Line B is the line for the buffer, Line C in the line for the methanol and Line D is the line for the water. Not necessarily all the lines are used each time, only the ones that are needed for the analysis. First the seal wash took place with an organic component for the cleaning of the system, and then the needle wash occurs for the cleaning of the syringe. After that a dry prime to the lines that used is carried out in order to remove any liquid or air exists in the lines to have a normal pressure in the system. Finally the wet prime in the lines that used occurs to fill the line with components that are used to check that the reservoirs are full and they can pass through the outlet. The wet prime for each line is done with 100% of the solvent that respects to each line. In order for a sample to be analysed it has to pass several steps and then to the detector and finally appeared in the computer. The solvent that was used was 20% acetonitrile and 80% buffer. The buffer was water with 1 ml of triethylamine for each 1000 ml of water and the adjustment of the pH at 5.9 was made by 0.1% glacial acetic acid. The solvent is first taken and then passed through the pump and through the injector the sample goes through and mixed with the solvent. Hence, the mixture passes through the column which can be normal or reversed. In the column the mobile phase interferes with the stationary phase. In this case the stationary phase is the silica which is a polar component; therefore components which are polar have less retention time than non-polar components. After the components pass through the column then the detector in a certain wavelength measures the UV absorption of the elluent. Based on the chromatographs trimethorpim has less retention time than sulphamethoxazole. This concludes that trimethoprim is more polar than sulphamethoxazole so the interference with the stationary phase is less.

Dissolution method:

"Dissolution testing is required for all solid oral Pharmacopeial dosage forms in which absorption of the drug is necessary for the product to exert the desired therapeutic effect. Exceptions are for tablets meeting a requirement for completeness of solution or for rapid (10 to 15 minutes) disintegration for soluble or radiolabeled drugs." (ref: world health organization-power point)

Apparatus:

The apparatus that is used are paddles or baskets. Baskets are used for tablets that are very small and they cannot dissolve very well by paddles. On the other hand paddles are used for large tablets and capsules. In the case of Deprim Forte paddles were used as the tablet are large.

In dissolution method the instrument that was used was the (Electrolab). In this instrument there are 8 vessels in which 6 are used for the samples. In each vessel medium is added according to the protocol of each drug. The maximum volumes of medium that can be used in these vessels are 1000 ml. The medium is also referred on the protocol depending on where will the drug dissolved in humans. The pH also should have in-vivo relevance. In addition, water may be used as medium, on the other side the effect of the formulation on the pH of water must be investigated and if it changes, the use of buffers or HCl should be considered.

For the Deprim Forte dissolution method 0.1N HCl was used in a volume of 900 ml in a temperature of 37oC. In almost all of the cases the 6 vessels were filled by 900 ml 0.1 N HCl. Then the paddles had a rotation of 75 rpm and they were rotated for 60 min. When the 60 min were passed the sample was withdrawn with a PE filter 10 μm in a syringe of 10 ml.

The procedure taken for the preparation of the samples was the same for every sample. With a pipette 5 ml of the sample was placed in a 200 ml volumetric flask and it was filled up with mobile phase and then it was injected in HPLC chromatograph for analysis.

RESULTS:

SYSTEM SUITABILITY TESTING:

This section will be carried out in accordance with the guidelines of the US Pharmacopoeia. This procedure will be carried out prior to all other chromatographic determinations. This test shows if the system is suitable for a further analysis. If the parameters are not meeting the criteria then the test is not acceptable.

Suitability parameters

Sulphamethoxazole

Trimethoprim

Limits

Theoretical plates

5783

6682

≥2000

Symmetry factor

0.7

1.1

≤2.0

Resolution

7.98

≥ 4.0

RSD of Replicate injections

0.0

0.0

≤2.0%

All the suitability parameters meet the criteria.

SPECIFICITY

Test of the actives

In order to be known the retention times of each active two solution of each active was prepared separate at 100% level. For the 100% Trimethoprim around 32 mg were used and for the 100% Sulphamethoxazole around 160 mg were used. Each amount of the active was placed in a different volumetric flask of 100 ml and filled up with mobile phase and finally 100 μl of each solution was injected to the HPLC chromatograph.

3.2 Interference with Mobile phase

To check if there is any interference with mobile phase, 100 μl of the mobile phase was injected to the HPLC chromatograph.

There were no interfering peaks from the mobile phase at the retention time of the principal peaks.

Interference with medium

To check if there is any interference with the medium - 0.1 N Hydrochloric acid-that was used in dissolution procedure, 100 μl of the medium was injected to the HPLC chromatograph.

There were no interfering peaks from the medium at the retention time of the principal peaks.

3.4 Interference with excipients

280 mg of the placebo tablet mixture (the amount present in one DEPRIM 960 TABLET- worst case scenario) was added in dissolution vessel containing 900 ml of medium at 37oC and stirred at 75 rpm for 60 minutes. From each vessel 10 ml was withdrawn and filtered through PE filter and injected to the HPLC chromatograph.

There were no interfering peaks from the placebo at the retention time of the principal peaks.

Interference with solvents mixtures

Two solvent mixtures were prepared in order to check if there is any interference with methanol and the medium. Solvent mixture 1 included 3 ml of methanol diluted up to 200 ml volumetric flask with the mobile phase. Solvent mixture 2 included 5 ml of medium diluted up to 200 ml volumetric flask with the mobile phase. In both cases 100 μl of each were injected in HPLC chromatograph.

There were no interfering peaks from medium or methanol at the retention time of the principal peaks.

Test solutions and standard solution

In consideration of checking if the retention time of the standard solution at 100% was the same as the test solution both solutions were prepared and 100 μl was injected from each in HPLC chromatograph.

The retention times were the same.

All the criteria were met.

LINEARITY AND RANGE

Solution preparation

The solutions in the concentration levels showed in the table below were prepared by a stock solution. The stock solution was prepared by around 160 mg of sulphamethoxazole and around 32 mg of trimethoprim into a 100 ml volumetric flask filled up with methanol. Then 15 ml were pipette into a 50 ml volumetric flask and filled up with mobile phase. Then based on the table below each solution was prepared and injected twice in HPLC chromatograph starting from solution with the lowest concentration.

Concentration level (%)

Concentration of Sulphamethoxazole [%w/v]

Concentration of Trimethoprim [%w/v]

Volume of the stock solution taken (ml)

Volume of the volumetric flask placed (ml)

50%

0.0012

0.00024

5.0

200

60%

0.00144

0.000288

3.0

100

70%

0.00168

0.000336

7.0

200

80%

0.00192

0.000384

4.0

100

100%

0.0024

0.00048

5.0

100

120%

0.00288

0.000576

6.0

100

The mean value of the peak area was taken for each concentration level for both injections and a graph for each active was plotted between the actual concentration and the mean peak area in order to show the linearity.

(missing graphs)

Concentration level (%)

Mean peak area for both injections

Concentration of Sulphamethoxazole [%w/v]

Actual amount of Sulphamethoxazole (g)

50%

2682172

0.0012

0.00121

60%

3224570

0.00144

0.00145

70%

3778054

0.00168

0.00169

80%

4285161

0.00192

0.00194

100%

5337603

0.0024

0.00242

120%

6401157

0.00288

0.00290

Concentration level (%)

Mean peak area for both injections

Concentration of Trimethoprim [%w/v]

Actual amount of Trimethoprim (g)

50%

167531

0.00024

0.0002478

60%

200639

0.000288

0.0002973

70%

235963

0.000336

0.0003469

80%

267253

0.000384

0.0003964

100%

332999

0.00048

0.0004955

120%

399549

0.000576

0.0005946

Suitability parameters

Sulphamethoxazole

Trimethoprim

Limits

Theoretical plates

5024

5041

≥2000

Symmetry factor

0.7

1.1

≤2.0

Resolution

7.50

≥ 4.0

RSD of Replicate injections

0.1

0.2

≤2.0%

All the suitability parameters meet the criteria.

ACCURACY

Solutions preparation

Three artificial mixtures were prepared in the way referred before in triplicate at three different levels 50%, 100% and 120% substance. Each of these mixtures was added to a dissolution vessel containing 900 ml of the medium at 37oC and stirred at 150 rpm for 60 minutes. 10 ml of each vessel was withdrawn and filtered through PE filter and injected into the HPLC chromatograph. Accuracy % recovery was calculated using this equation:

The range of the individual recoveries should be between 95%-105% in order this test to be accurate.

The preparation of the separate weightings of synthetic mixtures of the product components was according to the table below:

LEVEL (%)

Weight of Sulphamethoxazole (mg)

Weight of Trimethoprim(mg)

Weight of placebo (mg)

50%

400

80

280

50%

50%

100%

800

160

280

100%

100%

120%

960

192

280

120%

120%

For Trimethoprim: C%=99.5% - C = 0.995

LEVEL (%)

AMOUNT ADDED (mg)

AMOUNT

AMOUNT FOUND

% RECOVERY

50%

80.0

0.2211

0.2302

104.1%

50%

80.0

0.2211

0.2267

102.5%

50%

79.0

0.2184

0.2216

101.5%

100%

161.0

0.4450

0.4322

97.1%

100%

159.0

0.4395

0.4336

98.7%

100%

159.0

0.4395

0.4466

101.6%

120%

192.0

0.5307

0.5268

99.3%

120%

193.0

0.5334

0.5139

96.3%

120%

192.0

0.5307

0.5214

98.2%

Equation to find the amount:

For Sulphamethoxazole: C%=99.9% - C = 0.999

LEVEL (%)

AMOUNT ADDED (mg)

AMOUNT

AMOUNT FOUND

% RECOVERY

50%

400.0

1.1100

1.1012

99.2%

50%

400.0

1.1100

1.0927

98.4%

50%

401.0

1.1128

1.0745

96.6%

100%

800.0

2.2200

2.1706

97.8%

100%

798.0

2.2145

2.1809

98.5%

100%

802.0

2.2256

2.2082

98.2%

120%

962.0

2.6696

2.6168

98.0%

120%

961.0

2.6668

2.5961

97.3%

120%

962.0

2.6696

2.6577

99.6%

Equation to find the amount:

Suitability parameters

Sulphamethoxazole

Trimethoprim

Limits

Theoretical plates

3863

3504

≥2000

Symmetry factor

0.8

1.3

≤2.0

Resolution

5.7

≥ 4.0

RSD of Replicate injections

0.1

0.4

≤2.0%

Acceptance criteria

Sulphamethoxazole

Trimethoprim

Limits

RSD -50%

1.2%

2.2%

≤5%

RSD-100%

0.9%

1.9%

≤5%

RSD -120%

1.2%

1.3%

≤5%

PRECISION

Repeatability

Repeatability expresses the precision under the same operating conditions over a short interval of time.

For the quantitative determination precision under repeatability study six test solutions as per the dissolution method were prepared to be evaluated and then analyzed using the HPLC chromatograph. 5 ml of each test solution were taken and dissolved in 200 ml flask filled up with mobile phase. Each solution was injected once in the HPLC chromatograph.

6 injections from the same vessel:

Sample

Sulphamethoxazole

Trimethoprim

1

97.9%

102.4%

2

97.7%

102.2%

3

90.5%

96.0%

4

97.7%

102.6%

5

97.7%

102.8%

6

90.4%

95.2%

Mean Value

95.3%

100.2%

RSD of peak area

3.7%

3.8%

1 injection from 6 different vessels:

Sample

Sulphamethoxazole

Trimethoprim

1

90.4%

95.3%

2

95.8%

101.8%

3

96.6%

102.1%

4

94.0%

97.8%

5

97.5%

102.7%

6

94.0%

98.6%

Mean Value

94.7%

99.7%

RSD of peak area

2.5%

2.8%

Intermediate precision:

Intermediate precision expresses within-laboratories variation: different days, different analysts and different equipment.

6 injections from the same vessel:

Sample

Sulphamethoxazole

Trimethoprim

1

99.6%

97.6%

2

98.3%

99.2%

3

99.2%

100.3%

4

100.0%

98.4%

5

97.4%

98.1%

6

98.8%

99.3%

Mean Value

98.9%

98.8%

RSD of peak area

0.9%

1.0%

1 injection from 6 different vessels:

Sample

Sulphamethoxazole

Trimethoprim

1

100.7%

99.1%

2

99.5%

98.7%

3

98.8%

98.3%

4

99.8%

98.6%

5

99.7%

97.5%

6

91.2%

91.4%

Mean Value

98.3%

97.3%

RSD of peak area

3.5%

3.0%

ROBUSTNESS:

The robustness of an analytical procedure is a measure of its capacity to remain affected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage.

7.2 Filtration effect:

To check if there is any filtration effect was made by changing the filters for standards and test solutions. The filters that were used for standards were PTFE, PVDF and NYLON 0.45 μm and for test solutions PE 10 μm, PTFE, PVDF and NYLON 0.45 μm.

7.2.1 Solutions preparation:

Standards of 80%, 100% and 120% level were prepared and three test solutions.

Each solution was injected once.

The compare was between the standard unfiltered and filtered with the filters referred above and for the test solutions between the centrifuged and the filtered using the filters referred above.

7.2.2 Data analysis

Standard 1

Sulphamethoxazole

Trimethoprim

% Difference

Sulphomethoxaloze

% Difference

Trimethoprim

Area Unfiltered

4559914

272900

-

-

PTFE

4490399

268795

1.5%

1.5%

PVDF

4452494

269628

2.4%

1.2%

NYLON

4503696

267693

1.2%

1.9%

Standard 2

Sulphamethoxazole

Trimethoprim

% Difference

Sulphomethoxaloze

% Difference

Trimethoprim

Area Unfiltered

5534871

332786

-

-

PTFE

5562973

330375

0.5%

0.7%

PVDF

5497368

331806

0.7%

0.3%

NYLON

5562662

330811

0.5%

0.6%

Standard 3

Sulphamethoxazole

Trimethoprim

% Difference

Sulphomethoxaloze

% Difference

Trimethoprim

Area Unfiltered

6516416

395009

-

-

PTFE

6516698

389225

0.0%

1.5%

PVDF

6440980

391260

1.2%

0.9%

NYLON

6488734

390727

0.4%

1.1%

The area percentage difference for each active peak should be not more than 2.0%. The results met the criteria.

Test solution 1

Sulphamethoxazole

Trimethoprim

% Difference

Sulphomethoxaloze

% Difference

Trimethoprim

Area Centrifuged

4974162

292907

-

-

PE

4853418

286119

2%

2%

PTFE

4949042

292063

0.3%

0.5%

PVDF

4986771

294405

0.5%

0.3%

NYLON

4964595

293247

0.2%

0.1%

Test solution 2

Sulphamethoxazole

Trimethoprim

% Difference

Sulphomethoxaloze

% Difference

Trimethoprim

Area

Centrifuged

5002070

297522

-

-

PE

4897552

291555

2%

2%

PTFE

4981945

296040

0.4%

0.5%

PVDF

4932722

293844

1.4%

1.2%

NYLON

4989886

296966

0.2%

0.2%

Test solution 3

Sulphamethoxazole

Trimethoprim

% Difference

Sulphomethoxaloze

% Difference

Trimethoprim

Area Centrifuged

4799292

308052

-

-

PE

4803805

308574

0.1%

0.2%

PTFE

4824532

307714

0.5%

0.1%

PVDF

4797997

307318

0.0%

0.2%

NYLON

4828958

308802

0.6%

0.2%

Suitability parameters

Sulphamethoxazole

Trimethoprim

Limits

Theoretical plates

4683

5283

≥2000

Symmetry factor

0.7

1.2

≤2.0

Resolution

6.71

≥ 4.0

RSD of Replicate injections

0.1

0.1

≤2.0%

All the suitability parameters meet the criteria.

No significant difference was noticed between the chromatograms under the filtration effect study.

7.3 DISSOLUTION METHOD

The conditions of dissolution test should be varied to assess their impact on the dissolution results obtained. The dissolution test should be done under nominal and altered conditions. The following changes were applied comparing to the nominal conditions.

Non-deareated medium in place of a deareated medium

Rotation of the paddle : 75 rpm ± 5 rpm

Medium temperature: 37.0 oC ± 5oC

7.3.1 Solutions preparation

All the solutions were prepared as it was mentioned above.

7.3.2 Data analysis

Comparison and assessment had done of the dissolution results that were obtained for each set of conditions.

Test solution

LOT: 49608

Sulphamethoxazole

(mean of 6 test solutions)

Trimethoprim

(mean of 6 test solutions)

% DIFFERENCE FROM TARGET FOR TRIMETHORPIM

% DIFFERENCE FROM TARGET FOR SULPHAMETHOXAZOLE

Target solution-(75 rpm, 37oC, deareated medium)

97.8%

97.1%

-

-

Non-deareated medium

95.8%

97.5%

2.0%

0.4%

Temperature of medium at 36.5 oC

95.9%

98.3%

1.9%

1.2%

Test solution

LOT: 49606

Sulphamethoxazole

(mean of 6 test solutions)

Trimethoprim

(mean of 6 test solutions)

% DIFFERENCE FROM TARGET FOR TRIMETHORPIM

% DIFFERENCE FROM TARGET FOR SULPHAMETHOXAZOLE

Target solution-(75 rpm, 37oC, deareated medium)

94.7%

99.7%

-

-

Rotation at 70 rpm

99.3%

98.5%

4.6%

1.2%

Rotation at 80 rpm

98.2%

96.2%

3.5%

3.5%

Temperature of medium at 37.5 oC

97.9%

97.1%

3.2%

2.6%

The % difference of each change should be not more than 5% absolute. The criteria were met.

Suitability parameters for Target (lot:49608)

Sulphamethoxazole

Trimethoprim

Limits

Theoretical plates

4549

3308

≥2000

Symmetry factor

0.8

1.3

≤2.0

Resolution

5.91

≥ 4.0

RSD of Replicate injections

0.1%

0.2%

≤2.0%

Suitability parameters for medium temp: 36.5oC

Sulphamethoxazole

Trimethoprim

Limits

Theoretical plates

5454

5350

≥2000

Symmetry factor

0.7

1.1

≤2.0

Resolution

7.89

≥ 4.0

RSD of Replicate injections

0.1%

0.2%

≤2.0%

Suitability parameters for non-deareated medium

Sulphamethoxazole

Trimethoprim

Limits

Theoretical plates

5404

5072

≥2000

Symmetry factor

0.7

1.2

≤2.0

Resolution

7.79

≥ 4.0

RSD of Replicate injections

0.7%

0.6%

≤2.0%

Suitability parameters for rotation with 70 rpm

Sulphamethoxazole

Trimethoprim

Limits

Theoretical plates

4599

3296

≥2000

Symmetry factor

0.8

1.3

≤2.0

Resolution

5.94

≥ 4.0

RSD of Replicate injections

0.0%

0.1%

≤2.0%

Suitability parameters for rotation with 80 rpm

Sulphamethoxazole

Trimethoprim

Limits

Theoretical plates

4549

3308

≥2000

Symmetry factor

0.8

1.3

≤2.0

Resolution

5.91

≥ 4.0

RSD of Replicate injections

0.0%

0.1%

≤2.0%

Suitability parameters for medium with temp: 37.5oC

Sulphamethoxazole

Trimethoprim

Limits

Theoretical plates

5081

4950

≥2000

Symmetry factor

0.7

1.1

≤2.0

Resolution

5.28

RSD of Replicate injections

0.1%

0.0%

≤2.0%

7.4 In house HPLC house

Same as the dissolution method some changes applied in HPLC analytical procedure. The nominal conditions would be compared to the altered conditions. The changes that applied are showed below:

Flow rate: 1.4 ml/min ± 0.2 ml/min (1.2 ml/min-1.6 ml/min)

Organic component in the mobile phase: A: 20% B: 80% ± 10% relative (A: 10-25% B: 90-75%)

pH of the aqueous component of the mobile phase: 5.9 ± 0.2 (5.7-6.0)

column temperature: 30oC ± 5 oC (25-32 oC)

7.4.1 Solutions preparation

Three samples were prepared by the dissolution procedure and analyzed using the HPLC analytical procedure being validated under nominal and altered conditions.

7.4.2 Data analysis

A comparison between the nominal conditions and the altered conditions will be obtained.

Nominal conditions

Sulphamethoxazole

Trimethoprim

Sample 1

99.5%

97.8%

Sample 2

99.2%

98.3%

Sample 3

99.7%

99.7%

Change in flow (1.2 ml/min)

Sulphamethoxazole

% Difference from nominal conditions

Trimethoprim

% Difference from nominal conditions

Sample 1

100.1%

0.6%

101.6%

3.7%

Sample 2

99.8%

0.6%

102.4%

4.1%

Sample 3

100.4%

0.7%

101.8%

2.1%

Change in flow (1.6 ml/min)

Sulphamethoxazole

% Difference from nominal conditions

Trimethoprim

% Difference from nominal conditions

Sample 1

99.4%

0.1%

99.1%

1.2%

Sample 2

99.3%

0.1%

99.3%

1.0%

Sample 3

99.7%

0.0%

99.1%

0.6%

Change organic component (A: 10% - B: 90%)

Sulphamethoxazole

% Difference from nominal conditions

Trimethoprim

% Difference from nominal conditions

Sample 1

99.8%

0.3%

101.2%

3.3%

Sample 2

99.9%

0.7%

101.2%

2.9%

Sample 3

100.5%

0.8%

101.1%

1.4%

Change organic component (A: 25% - B: 75%)

Sulphamethoxazole

% Difference from nominal conditions

Trimethoprim

% Difference from nominal conditions

Sample 1

98.5%

1.0%

100.1%

2.2%

Sample 2

99.4%

0.2%

99.7%

1.4%

Sample 3

100.0%

0.3%

101.4%

1.7%

Change pH (pH=5.7)

Sulphamethoxazole

% Difference from nominal conditions

Trimethoprim

% Difference from nominal conditions

Sample 1

100.3%

0.8%

101.6%

3.7%

Sample 2

100.1%

0.9%

102.3%

4.0%

Sample 3

100.4%

0.7%

101.6%

1.9%

Change of pH (pH=6.0)

Sulphamethoxazole

% Difference from nominal conditions

Trimethoprim

% Difference from nominal conditions

Sample 1

98.1%

1.4%

96.4%

1.5%

Sample 2

98.6%

0.6%

96.1%

2.2%

Sample 3

99.0%

0.7%

98.4%

1.3%

Change of temperature (25oC)

Sulphamethoxazole

% Difference from nominal conditions

Trimethoprim

% Difference from nominal conditions

Sample 1

99.6%

0.1%

100.9%

3.0%

Sample 2

99.6%

0.4%

100.2%

1.9%

Sample 3

102.4%

2.7%

100.5%

0.8%

Change of temperature (32oC)

Sulphamethoxazole

% Difference from nominal conditions

Trimethoprim

% Difference from nominal conditions

Sample 1

98.6%

0.9%

99.1%

1.2%

Sample 2

99.2%

0.0%

98.2%

0.1%

Sample 3

99.7%

0.0%

100.9%

1.2%

Suitability parameters:

Suitability parameters for flow 1.2 ml/min

Sulphamethoxazole

Trimethoprim

Limits

Theoretical plates

3027

7163

≥2000

Symmetry factor

0.6

1.1

≤2.0

Resolution

5.16

≥ 4.0

RSD of Replicate injections

0.1

0.2

≤2.0%

Suitability parameters for flow 1.6 ml/min

Sulphamethoxazole

Trimethoprim

Limits

Theoretical plates

5299

4980

≥2000

Symmetry factor

0.7

1.2

≤2.0

Resolution

8.1

≥ 4.0

RSD of Replicate injections

0.1

0.2

≤2.0%

Suitability parameters for composition A: 10% B: 90%

Sulphamethoxazole

Trimethoprim

Limits

Theoretical plates

5782

3298

≥2000

Symmetry factor

0.8

1.4

≤2.0

Resolution

6.59

≥ 4.0

RSD of Replicate injections

0.2

0.1

≤2.0%

Suitability parameters for composition A: 25% B: 75%

Sulphamethoxazole

Trimethoprim

Limits

Theoretical plates

4315

3403

≥2000

Symmetry factor

0.8

1.4

≤2.0

Resolution

5.99

≥ 4.0

RSD of Replicate injections

0.1

0.2

≤2.0%

Suitability parameters for pH:5.7

Sulphamethoxazole

Trimethoprim

Limits

Theoretical plates

5884

4824

≥2000

Symmetry factor

0.7

1.1

≤2.0

Resolution

7.82

≥ 4.0

RSD of Replicate injections

0.2

0.1

≤2.0%

Suitability parameters for pH: 6.0

Sulphamethoxazole

Trimethoprim

Limits

Theoretical plates

5933

4961

≥2000

Symmetry factor

0.7

1.1

≤2.0

Resolution

6.35

≥ 4.0

RSD of Replicate injections

0.0

0.5

≤ 2.0%

Suitability parameters for temperature 25oC

Sulphamethoxazole

Trimethoprim

Limits

Theoretical plates

3850

5391

≥2000

Symmetry factor

0.7

1.1

≤2.0

Resolution

7.23

≥ 4.0

RSD of Replicate injections

0.1

0.3

≤ 2.0%

Suitability parameters for temperature 32oC

Sulphamethoxazole

Trimethoprim

Limits

Theoretical plates

4874

4044

≥2000

Symmetry factor

0.7

1.1

≤2.0

Resolution

5.35

≥ 4.0

RSD of Replicate injections

0.1

1.1

≤2.0%

Acceptance criteria:

% Difference should not be more than ± 5% absolute for both actives.

RSD % of the both main peak area for six replicate injections should not be more than 2.0%

The resolution between the peaks due to Trimethoprim and Sulphamethoxazole should be at least 4.0

All the criteria were met.

STABILITY OF STANDARDS AND SAMPLE SOLUTIONS

The stability of standards and sample solution test were done in order to verify that samples and standards can be used in period of 24 hour. Injections were happened between the periods of 24 hours.

Solution preparation

Standards of three different levels-80%, 100%, 120% level- and three test solutions were prepared. Each solution was injected once.

For Sulphamethoxazole:

% Difference of the area after 6 hrs

% Difference of the area after 8 hrs

%Difference of the area after 24 hrs

Standard 1

0.8%

0.9%

1.4%

Standard 2

0.5%

0.3%

1.2%

Standard 3

0.3%

0.3%

1.0%

For Trimethoprim:

% Difference of the area after 6 hrs

% Difference of the area after 8 hrs

% Difference of the area after 24 hrs

Standard 1

1.0%

0.7%

1.1%

Standard 2

0.2%

0.0%

0.6%

Standard 3

1.6%

1.5%

2.2%

Acceptance criteria:

The area percentage difference for Trimethoprim and Sulphamethoxazole peaks should be not more than 3%

Chromatographic picture of the solutions tested should not show any significant changes during the stability study.

All criteria were met.

SAMPLES:

For Sulphamethoxazole:

% Difference of the area after 2 hrs

% Difference of the area after 4 hrs

% Difference of the area after 6 hrs

% Difference of the area after 22 hrs

Sample 1

2.0%

1.3%

0.5%

2.9%

Sample 2

1.2

0.3

0.0

2.9

Sample 3

1.7

1.1

0.9

1.3

For Trimethoprim:

% Difference of the area after 2 hrs

% Difference of the area after 4 hrs

% Difference of the area after 6 hrs

% Difference of the area after 22 hrs

Sample 1

2.2

2.0

1.2

2.7

Sample 2

2.4

2.3

1.9

2.3

Sample 3

2.9

2.9

2.8

1.6

Discussion:

In a method validation a series of test should followed in order to approve a drug to be sold in the market. If all of the results meet the acceptance criteria of each test then the drug is free to the market. In this test Deprim Forte met the acceptance criteria of each test.

In more details, linearity of an analytical procedure is its ability to obtain test results which are directly proportional to the concentration of analyte in the sample in a range of 50%-120% level. The regression was >0.99 therefore, the amount of each active in the different concentrations was increasing in a same way.

The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found. Accuracy was determined by composing a "placebo-mixture" of the excipients of the product and adding known amounts of the drug substance. The criteria were met in this test as well.

The precision of an analytical procedure expresses the closeness of agreement between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed condition. Precision may be considered at three levels which are repeatability, intermediate precision and reproducibility. Each level studies a different element. Repeatability expresses the results after a short time of period under the same conditions. In this test results were taken after 6 injections from the same vessel and 6 injections from 6 different vessels. The results were between the range of 95%-105% which was the limit. Intermediate precision test was made by a second analyst; the injections that were made were the same as in the repeatability test. But in order to complete the reproducibility test the injections of the samples were made in different equipment to check if there is any significant difference between the equipment of the second analyst. From the results taken the only difference that was noticeable was the small change in the time of the actives. Based on these observations the change in retention time was caused by the change in the HPLC machine as there was no other changes applied.

In robustness different conditions were tried than the nominal in HPLC conditions and dissolution conditions. First the filtration effect was studied. In this test standards and samples were filtered by using different kinds of filters to compare with the unfiltered for the standards and the centrifuged for the samples. The purpose of this test was to check if there was any significant difference between them. The filters used for the standards were PTFE 0.45 μm, PVDF 0.45 μm and NYLON 0.45 μm. After processing the data all of the filters were able for the filtration of the standards but the use of the NYLON 0.45 μm according to the results. For the study of the samples the filters that were used were PE 10 μm, PTFE 0.45 μm, PVDF 0.45 μm and NYLON 0.45 μm. Subsequently, the data were managed and the conclusion taken was that in samples the best filter was NYLON 0.45 μm as in the standards.

The conditions in HPLC that were altered were the flow of the mobile phase, the composition of the organic component, the pH of the buffer and the temperature of the column. The nominal conditions were 1.4 ml/min for the flow, A: 20% acetonitrile B: 80% buffer, 30oC the temperature of the column and 5.9 the pH of the buffer. The % difference between the nominal and the altered conditions was between the 3% and the chromatographs had no significant different from the nominal conditions. Exceptions were the results for pH=6.1 and the temperature of the column at 35oC where the chromatograph had resolution less than 4.0 and the composition of the organic component at A: 30% and B: 70% where the peak of trimethoprim had an interference with the mobile phase. Another conditions were tried in place of them such as pH=6.0, the temperature of the column 32oC and the composition A: 25% and B: 75%; the results taken from these changes were met the criteria. Therefore, conditions with pH=6.1, temperature of the column at 32oC and the composition of the organic component at A: 30% and B: 70% were not suitable for used in the analysis of Deprim Forte.

The changes applied in the dissolution method were the rotation, the temperature of the medium and the use of non-deareated medium in the place of deareated medium. The nominal conditions were 37.0 oC the temperature of the medium, deareated medium and the rotation at 75 rpm. After the changes were applied and processing the results of them they were no significant difference at the chromatographs. Hence, all of the alterations done in this test can be used as the results met the criteria.

Finally the stability of standards and samples were studies whether can be used in a period of 24 hours. For the needs of this test 3 test solutions were prepared and 3 standards. The standards were injected at 6, 8 and 24 hours and the samples at 2, 4, 6 and 22 hours. The standards had no large different between this period of time. On the other hand samples at the time of 22 hours had larger different. Through the pass of time the percentage difference from the 0 hrs was getting larger but still in the limits for the samples therefore is still suitable for injection after 24 hours.

Conclusion:

Deprim Forte is a new version of deprim tablets. Deprim Forte has as actives Sulphamethoxazole and Trimethoprim. This medicine was approved for chronic urinary tract infections, shigellosis, and proven pneumocystis carini pneumonia and otitis media by the US Food and Drug Administration (FDA). Also, in 1973 it was approved for the treatment of initial episodes of uncomplicated UTI, acute exacerbations of chronic bronchitis, traveller's diarrhea due to enterotoxigenic Escherichia coli. In order for this medicine to be sold in the market first it should by somehow be approved that it is suitable for use. The tests that should be taken will cover the specificity, linearity, accuracy, precision, robustness and the stability of standards and samples. Each test studies a different element. Specificity test is the capability to measure accurately and specifically of the analyte of interest. The purpose of the linearity is to obtain test results which were directly proportional to the concentration of the analyte in the sample in a given range. In the accuracy test the closeness of agreement between the true value and the value measured is studied. Precision test is the scale of repeatability of an analytical method under normal operation and is most of the times expressed as the percent relative standard deviation. In stability test the stability of the standards and samples are expressed. Robustness is the ability of a method to stay unaffected by small, intentional variations in method parameters.

Concluding, when all of these tests took place as it was referred above the results has met all of the acceptance criteria. Once these acceptance criteria were met the method of the procedure will be validated and it can be used from now on for the analysis of new portions of these medicines. The conditions those are suitable for further analysis for the dissolution method 75 rpm for the rotation of the paddles, 37oC the temperature of the medium that is used for the vessels and the medium should be deareated. The conditions of HPLC are 1.4 ml/min flow of the mobile phase, 30oC the temperature of the column, 20% acetonitrile and 80% buffer the composition of the components and the pH of the buffer at 5.9. In addition to this the samples and the standards as it was proven from the results they can be used for the analysis in a period of 24 hours. Deprim forte is now available for distribution in many countries.

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