Experimental Plan For Accelerated Stability Studies Biology Essay

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Distilled water used was obtained from an all glass electrically heated still and kept stored in a 5 liters well leached and stopper bottle,pH 5.8±as determined by corning pH meter and surface tension 72± 0.2m Nm-1 at 20oC.

Friction and shock are the forces that induce tablets to chip, cap or break. As a result equipment such as Curio Friabilator was developed. The unit causes self abrasion of the tablets as the cylinder section rotates. The tablets also undergo shock as they fall 6 inches on each turn. After 4 minutes of this process or 100 revolutions, the tablets are weighed and compared to their original weight.

The value is expressed as percent w/w.Acceptable limits of weight loss are less than 1 percent. Generally, when capping occurs or tablets break during the test, friability values are not valid and are not calculated. When tablets lose moisture on aging, they may become more friable. (Javaid, 1993)

The apparatus was manufactured by Curio, model FB-098.It was supplied by Technologies link, Islamabad Pakistan.

3-Disintegration Test Apparatus:

The apparatus was manufactured by Curio, model DS-0702.It was supplied by Technologies link, Islamabad Pakistan. It was manufactured under specification

4-Double Beam UV Spectrophotometer (Hitachi, U-2001 Spectrophotometer, Japan)

Determination of spectrophotometer measurements are based on the absorption of radiant energy by organic molecules at a particulate wavelength in the ultraviolet(UV,180-380nm)or visible region(380-800nm).The absorption of light at specific wavelengths due to electronic excitation is generally characteristic of the chemical nature of the absorbing species and can be used for the determination of substances.

According to Beer Lambert's law, the absorbance at a specific wavelength is directly proportional to the concentration of solute and path length of the sample.

Mathematically, it is expressed as,

A=acl

Where:

A=Absorbance,

a= Absorptivity,

c=Concentration and

l=Thickness of the cell or path length (Javaid, 1993)

3.3-METHODS:

1. Length and Thickness:

The length and thickness of tablets was measured with micrometer (vernier) calipers. Mean of six tablets were taken for accurate result.

2. Hardness:

The hardness of tablets were determined by placing a tablet between right and left plungers and then start checking hardness by turning on hardness tester, until the tablet fractured and noted the reading. The same process with 4 tablets and the average force in kg was calculated. Mean of six tablets were taken for accurate reading.

The apparatus consist of control panel, the detail is given below:

Controls :

1. Start

It is used to start the Apparatus.

2. Stop

It is used to stop the Apparatus.

3. Print

It is used to print the results.

4. Mode

It is used to change the units i.e. (kg & Ibs).

Operation

Power on the apparatus by the Button on the Back side of the apparatus.

The Screen will show the zeroing position then ready for test thickness.

Then press start push Button.

Open the pan and place the tablet in the edges between two sliding bars.

The apparatus will automatically give the thickness and then shift to hardness. Result will be in kgs.

Record the reading and repeat on at least two more tablets.

After operation clean the apparatus and close the pan.

The apparatus was supplied by Technologies Link, Islamabad Pakistan.

3. Weight Variation test:

20 tablets were randomly taken then the individual and total weight were determined by analytical balance and average weight was calculated .The result of tablets was compared with B.P specification.

4. Disintegration Test:

The U.S.P disintegration procedure was used in which six tablets from each brand was kept in basket assembly suspended in a beaker containing distilled water maintaining at 37±0.5°C with up and down frequency of 27 to 33 cycles per minute, the time required for complete disintegration of six tablets with in U.S.P specification.

Operation:

It is established to provide guidelines for the use DISINTEGRATION TEST APPARATUS

ON the instrument form the button present at the right lateral side of the instrument.

Press the "Power" button and then "HEAT (1)" & "STIRR (2)" buttons present on the front panel.

Heating is start till the temperature of the liquid is 360 to 380C. The heating process is indicated by blinking of red light under "HEAT (1)" button. When the temperature reaches 370C, the red light stops blinking and the green light begins to blink.

For time setting there are two modes i.e. "Stopwatch" and "Lab counter".

Press "M" button to toggle between "Stopwatch" & "Lab counter" mode. The "M" button has two i.e. â-º (above) and â-„ (below) indicating towards lab counter and stopwatch mode respectively. The lamination of "â-„" or "â-º" with red light indicates that the respective mode is activated.

When using "Stopwatch" mode, simply press the "ST/STOP (3)" button followed by pressing "OSCI (8)", the instrument starts operation.

To stop the stopwatch, press "ST/STOP (3)". Press "RESET (4)" to get zero time on the display.

When using the lab counter mode, press Shift button, "00.00" is displayed on the screen. The two zeroes at the right side to the dot shows time in seconds and those at the left side shows time in minutes. Press Shift button repeatedly to shift to the desired position. The activated zero digit starts blinking.

Press set button to set the specified time.

Press "ON/OFF (7)" button, the time starts in the countdown manner. When the time reaches zero, the instrument beeps.

5. Friability Test:

For friability test, 10 tablets were randomly taken, then the individual and total weights were determined by analytical balance and average weight was calculated. The tablets also undergo shock and abrasion as they fall 6 inches on each turn. After 100 revolutions, the tablets were weight and compared to their original weight. The loss due to abrasion or friction or shock is the measure of tablet friability.

Operation:

It is established to provide guidelines for the use of FRIABILITY TEST APPARATUS.

Accurately weigh required number of tablets and place in the drum. Fix the drum on the Friabilator.

Press "2(shift)" button, "00.00" is displayed on the LCD. The two zeroes at the right side of the dot show seconds while those at the left side are for minutes. Press the shift button repeatedly to shift to the desired position. The activated zero digit starts blinking.

Press "1(set)" button to set the desired time.

Press "3(start/stop)" button, the time starts in a countdown manner.

When the time is over the apparatus starts beep.

6.Double beam UV Spectrophotometer:

The term used to designate instrument which have a radiant energy-dispersing device, such as a radiant power is called Spectrophotometer.

In Double beam spectrophotometer a source of UV (deuterium) or visible (tungsten) energy is required to emit intense and uniform radiations. A monochromator is used to isolate the desired wavelength which enters the sample compartment through a narrow slit and passes through the sample contained in a quartz or glass cell. Appropriate photo cell give information about the relative absorption by producing an electrical signal proportional to the transmitted light. Upon amplification of this signal the absorbance along with wavelength of the sample or its spectrum may be recorded.

Operation:

It is established to provide guidelines for the use of UV-VISIBLE SPECTROPHOTOMETER.

Start the computer.

Switch on the UV-Visible spectrophotometer. A starting process begins.

When the following is displayed on the screen;

(1) Set method. (2) Run Method. (3) Set Curve. (4) List Methods

Press "ESC" button and then "YES/REPEAT" button. Now the screen shows the following;

(1) Print Parameters. (2) Serial Control. (3) Mechanical Test. (4) Low Power.

Press "2" button, "Serial Control (Y/N)?" is shown on the screen.

Press "YES/REPEAT" button. Now the spectrophotometer is connected to computer.

Double click on the "SF 1700" icon on the desktop of computer. A "Metro lab" dialogue box appears.

"SPECTRA" MODE:

Click on "Lamp" and then click on "Spectra"; the "Spectra" dialogue box appears.

Go to "file" menu and click "new spectra"; the "New Spectra" dialogue box appears.

Enter the desired wavelength range and click "OK".

"Insert Cuvette" box appears. Insert the cuvette, filled with blank in to the cuvette holder and click "OK".

The blank scanning process starts. When the blank is scanned, an "Input a Name" box appears.

Enter the name of solution and click "OK". The solution scanning process starts.

When the scanning is completed, the spectrum is appeared on the screen. Find out the wavelength of maximum absorbance.

To save the spectrum, go to file menu and click on "save". Save the spectrum in the desired destination.

In similar way scan both sample and standard solutions.

"PHOTOMETRIC READING" MODE:

Click on "Lamp".

Click on "Photometric Reading"; the "Photometric Reading" dialogue box appears.

Enter the desired wavelength in "wavelength".

Insert cuvette, filled with blank, in to the cuvette holder and click "Read Blank".

Remove the cuvette from the holder and rinse several times with test solution. Now insert the cuvette filled with test solution into the cuvette holder and click "Read Sample".

Note the absorbance of test solution under "Absorbance".

Measure the absorbance of both standard and sample solutions in similar way and deduce the result by comparison.

Determination of pH:

pH is defined as, For practical purposes, its definition is enough for experiment.

pH should not be checked in oily phase preparation.

8. Clarity:

A liquid is considered clear if its clarity is the same as that of water R or of the solvent used when examined under the conditions given above, or if its opalescence is not more pronounced than that of reference suspension I.

Particulate Contamination: Visible Particles :

Apparatus:

The apparatus consists of following procedure:

_a matt-glass white panel of appropriate size held in a perpendicular position,

_a non-glass white panel of appropriate size held in a perpendicular position next to the black panel,

_an adjustable lamp holder fitted with a appropriate, shaded, white-light source and with a suitable light diffuser(a viewing illuminator containing two 13 W fluorescent tubes, each 525mm in length, is suitable).The intensity of illuminatination at the viewing point is maintained between 2000 lux and 3750 lux, however higher values are preferable for coloured glass and plastic containers.

Method:

Remove any supportive labels from the container and wash and dry the outside. Gently swirl or invert the container, confirm that air bubbles are not introduced, and observe for about 5 seconds in front of the black panel. At last, record the presence of any particles.

13. Climatic Chamber

Operation:

It is established for Climatic Chamber

Connect the unit power cord to a ground 3-prong.

Ensure the correct assembling by moving the wheel manually to check free movement of all loading parts and then switch ON.

In case of any abnormal sound from machine during its operation stop the machine and report to maintenance department.

14- Drug Assay Procedure for Tablets and Injections:

Drug assay:

The assay for the active ingredient(s) in the formulations will be performed using U.V spectroscopic method (newly developed or non-pharmacopoeia method). The assays will be repeated three times and the results will be presented as the mean of 3 determinations (± standard deviation) at lambda (ʎ) max 265nm.

Testing procedure for injections:

Preparation of standard solution: Take 100mg of VitaminD3 in 50ml of volumetric flask. Add 10ml formic acid in same volumetric flask and sonicate it for some time. Add methanol to make up the volume up to the mark (50ml).

Then, take 2ml from the stock solution into 100ml volumetric flask.Then,add 1ml of 2,4 dinitrophenylhydrazin compound in 100ml volumetric flask and make up the volume up to the mark up of the flask.Then,take absorbance at lambda max 265nm.

Blank=Methanol.

Preparation of sample: Take 100mg equivalent of VitaminD3 in 50ml of volumetric flask. Add 10ml formic acid in same volumetric flask and sonicate it for some time. Add methanol to make up the volume up to the mark (50ml).

Then, take 2ml of filtrate solution, through filtration(0.45 micron filter) process, into 100ml volumetric flask.Then,add 1ml of 2,4 dinitrophenylhydrazin compound in 100ml volumetric flask and make up the volume up to the mark up of the flask.Then,take absorbance at lambda max 265nm.

Blank=Methanol.

U.V spectroscopic method is used to determine the drug assay by using following formula

%Assay = Absorbance of sample x 100

Absorbance of standard

Testing procedure for Tablets:

Preparation of standard solution: Take 100mg of VitaminD3 in 50ml of volumetric flask. Add 10ml formic acid in same volumetric flask and sonicate it for some time. Add methanol to make up the volume up to the mark (50ml).

Then, take 2ml from the stock solution into 100ml volumetric flask.Then,add 1ml of 2,4 dinitrophenylhydrazin compound in 100ml volumetric flask and make up the volume up to the mark up of the flask.Then,take absorbance at lambda max 265nm.

Blank=Methanol.

Preparation of sample: Take 100mg equivalent of VitaminD3 in 50ml of volumetric flask. Add 10ml formic acid in same volumetric flask and sonicate it for some time. Add methanol to make up the volume up to the mark (50ml).

Then, take 2ml of filtrate solution, through filtration(0.45 micron filter) process, into 100ml volumetric flask.Then,add 1ml of 2,4 dinitrophenylhydrazin compound in 100ml volumetric flask and make up the volume up to the mark up of the flask.Then,take absorbance at lambda max 265nm.

Blank=Methanol.

U.V spectroscopic method is used to determine the drug assay by using following formula

%Assay = Absorbance of sample x 100

Absorbance of standard

CHAPTER NO 4

ACCELERATED STABILITY STUDY DATA

ACCELERATED STABILITY DATA OF VITAMIN D3 (CHOLECALCIFEROL) TABLETS:

Stability tests & Acceptance criteria:

The test parameters mentioned in the stability specification given below will be monitored during stability study of the drug substance. The batches of the product kept on stability at different storage conditions, should comply with these specifications. In case of any aberrant data or an OOS situation, a thorough investigation shall be performed and based on the findings a decision will be made with regards to further testing or disposition of the batch.

A-Stability Specification

(Qalsan D Tablets)

Note: The requirement for disintegration does not apply to Chewable Calcium Carbonate Tablets (B.P, 2008, Volume 3).

STABILITY DATA

(ACCELERATED STUDY)

Figure 6: Graph highlights relationship between Absorbance and Wavelength (nm) of Qalsan D in three different intervals (Batch 001, 002 and 003).

(a)At 0 month (Batch No:001).

(b)At 3 month (Batch No:001) .

(c)At 6 month (Batch No:001).

(d)At 0 month (Batch No:002).

(e)At 3 month (Batch No:002).

(f)At 6 month (Batch No:002).

(g)At 0 month (Batch No:003).

(h)At 3 month (Batch No:003).

(i)At 6 month (Batch No:003).

B-Stability Specification

(Calgo Tablets)

Note: The requirement for disintegration does not apply to Chewable Calcium Carbonate Tablets (B.P, 2008, Volume 3).

STABILITY DATA

(ACCELERATED STUDY)

Figure 7: Graph highlights relationship between Absorbance and Wavelength (nm) of Calgo Tablets in three different intervals (Batch 001, 002 and 003).

(a)At 0 month (Batch No:001).

(b)At 3 month (Batch No:001).

(c)At 6 month (Batch No:001).

(d)At 0 month (Batch No:002).

(e)At 3 month (Batch No:002).

(f)At 6 month (Batch No:002).

(g)At 0 month (Batch No:003).

(h)At 3 month (Batch No:003).

(i)At 6 month (Batch No:003).

C-Stability Specification

(Osam-D Tablets)

STABILITY DATA

(ACCELERATED STUDY)

Figure 8: Graph highlights relationship between Absorbance and Wavelength (nm) of Osam-D in three different intervals (Batch 001, 002 and 003).

(a)At 0 month (Batch No:001).

(b)At 3 month (Batch No:001).

(c)At 6 month (Batch No:001).

(d)At 0 month (Batch No:002).

(e)At 3 month (Batch No:002).

(f)At 6 month (Batch No:002).

(g)At 0 month (Batch No:003).

(h)At 3 month (Batch No:003).

(i)At 6 month (Batch No:003).

ACCELERATED STABILITY DATA OF VITAMIN D3 (CHOLECALCIFEROL) INJECTIONS:

Stability tests & Acceptance criteria:

The test parameters mentioned in the stability specification given below will be monitored during stability study of the drug substance. The batches of the product kept on stability at different storage conditions, should comply with these specifications. In case of any aberrant data or an OOS situation, a thorough investigation shall be performed and based on the findings a decision will be made with regards to further testing or disposition of the batch.

A-Stability Specification

(Indrop D Injection)

Figure 9: Graph highlights relationship between Absorbance and Wavelength (nm) of Indrop D Injection in three different intervals (Batch 001, 002 and 003).

(a)At 0 month (Batch No:001).

(b)At 3 month (Batch No:001).

(c)At 6 month (Batch No:001).

(d)At 0 month (Batch No:002).

(e)At 3 month (Batch No:002).

(f)At 6 month (Batch No:002).

(g)At 0 month (Batch No:003).

(h)At 3 month (Batch No:003).

(i)At 6 month (Batch No:003).

B-Stability Specification

(ED-3 Injection)

Figure 10: Graph highlights relationship between Absorbance and Wavelength (nm) of ED-3 Injection in three different intervals (Batch 001, 002 and 003).

(a)At 0 month (Batch No:001).

(b)At 3 month (Batch No:001).

(c)At 6 month (Batch No:001).

(d)At 0 month (Batch No:002).

(e)At 3 month (Batch No:002).

(f)At 6 month (Batch No:002).

(g)At 0 month (Batch No:003).

(h)At 3 month (Batch No:003).

(i)At 6 month (Batch No:003).

C-Stability Specification

(Cara-In-D Injection)

Figure 11: Graph highlights relationship between Absorbance and Wavelength (nm) of Cara-In-D Injection in three different intervals (Batch 001, 002 and 003).

(a)At 0 month (Batch No:001).

(b)At 3 month (Batch No:001).

(c)At 6 month (Batch No:001).

(d)At 0 month (Batch No:002).

(e)At 3 month (Batch No:002).

(f)At 6 month (Batch No:002).

(g)At 0 month (Batch No:003).

(h)At 3 month (Batch No:003).

(i)At 6 month (Batch No:003).

Table 10: Comparative Accelerated Stability Studies of Different Brands of Cholecalciferol (Vitamin D3) Tablets.

CHAPTER NO 5

STATISTICAL GLOSSARY:

OR

"It is the least/minimum most value, for the study concerned " that differentiate between the critical and acceptance region.

Significance level:

The criterion used for rejecting the null hypothesis is called significance level. The 0.05 or 5% value is significance level.

Standard deviation:

The descriptive statistic, in which there is a measure of dispersion, or spread, of sample data around the mean and all the data in a sample is used is called as Standard deviation.

Variable:

It is the thing measured, counted, or identified, the thing of interest. It is the fundamental element of a statistical analysis. e.g., Birth weight of a newborn infant, the time a clock fails or practically any definable quantity.

Note:

I used Minitab 15 Statistical software for my Research data analysis by applying two way ANOVA test.

STATISTICAL DATA OF ACCELERATED STABILITY STUDY OF VITAMIN D3 (CHOLECALCIFEROL) TABLETS AND INJECTIONS:

Statistical Data of Accelerated Stability Studies of Qalsan D Tablets in Two-way ANOVA: Responses versus Months, batches (Based on Assay result)

Source DF SS MS F P

Months 2 3.7727 1.88634 14.75 0.014

Batches 2 9.6977 4.84884 37.90 0.003

Error 4 0.5117 0.12793

Total 8 13.9821

S = 0.3577 R-Sq = 96.34% R-Sq(adj) = 92.68%

Individual 95% CIs For Mean Based on Pooled StDev

Months Mean -+---------+---------+---------+--------

0 101.493 (-------*-------)

3 100.497 (--------*-------)

6 99.927 (--------*-------)

-+---------+---------+---------+--------

99.40 100.10 100.80 101.50

Individual 95% CIs For Mean Based on Pooled StDev

Batches Mean -+---------+---------+---------+--------

1 100.443 (----*-----)

2 101.997 (-----*-----)

3 99.477 (-----*-----)

-+---------+---------+---------+--------

99.0 100.0 101.0 102.0

Results: Here, I have observed a p value of 0.014 for the average analysis of the affects of different methods for months. In this conditional probability, if my hypothesis is true, reveals that three methods significantly differ from each other in giving the response for the understudy assay result. My was set at 0.05 and the p value "the observed " is much less than 0.05.

In this research, I have also observed a p value of 0.003 for the average analysis of the affects of different methods for batches. In this conditional probability, if my hypothesis is true, reveals that three methods significantly differ from each other in giving the response for the understudy assay result. My was set at 0.05 and the p value "the observed " is much less than 0.05.

Statistical Data of Accelerated Stability Studies of Indrop D Injection in Two-way ANOVA: Responses versus Months, Batches (Based on Assay result)

Source DF SS MS F P

Months 2 2.77416 1.38708 11.47 0.022

Batches 2 2.96176 1.48088 12.24 0.020

Error 4 0.48378 0.12094

Total 8 6.21969

S = 0.3478 R-Sq = 92.22% R-Sq(adj) = 84.44%

Individual 95% CIs For Mean Based on

Pooled StDev

Months Mean -----+---------+---------+---------+----

0 100.943 (-------*-------)

3 100.000 (-------*-------)

6 99.623 (-------*-------)

-----+---------+---------+---------+----

99.40 100.10 100.80 101.50

Individual 95% CIs For Mean Based on

Pooled StDev

Batches Mean ------+---------+---------+---------+---

1 100.070 (-------*-------)

2 100.943 (-------*-------)

3 99.553 (-------*-------)

------+---------+---------+---------+---

99.40 100.10 100.80 101.50

Results: Here, I have observed a p value of 0.022 for the average analysis of the affects of different methods for months. In this conditional probability, if my hypothesis is true, reveals that three methods significantly differ from each other in giving the response for the understudy assay result. My was set at 0.05 and the p value "the observed " is much less than 0.05.

In this research study, I have also observed a p value of 0.020 for the average analysis of the affects of different methods for batches. In this conditional probability, if my hypothesis is true, reveals that three methods significantly differ from each other in giving the response for the understudy assay result. My was set at 0.05 and the p value "the observed " is much less than 0.05.

Statistical Data of Accelerated Stability Studies of Osam-D Tablets in Two-way ANOVA: Responses versus Months, Batches(Based on Assay result)

Source DF SS MS F P

Months 2 75.0064 37.5032 16.61 0.012

Batches 2 5.8756 2.9378 1.30 0.367

Error 4 9.0290 2.2572

Total 8 89.9110

S = 1.502 R-Sq = 89.96% R-Sq(adj) = 79.92%

Individual 95% CIs For Mean Based on

Pooled StDev

Months Mean ------+---------+---------+---------+---

0 103.613 (-------*-------)

3 99.857 (-------*-------)

6 96.547 (-------*-------)

------+---------+---------+---------+---

96.0 99.0 102.0 105.0

Individual 95% CIs For Mean Based on

Pooled StDev

Batches Mean -----+---------+---------+---------+----

1 101.147 (-----------*-----------)

2 99.383 (-----------*-----------)

3 99.487 (-----------*-----------)

-----+---------+---------+---------+----

98.0 100.0 102.0 104.0

Results: Here, I have observed a p value of 0.012 for the average analysis of the affects of different methods for months. In this conditional probability, if my hypothesis is true, reveals that three methods significantly differ from each other in giving the response for the understudy assay result. My was set at 0.05 and the p value "the observed " is much less than 0.05.

In this stability study, I have also observed a p value of 0.367 for the average analysis of the affects of different methods for batches. In this conditional probability, if my hypothesis is false, reveals that three methods significantly not differ from each other in giving the response for the understudy assay result. My was set at 0.05 and the p value "the observed " is much greater than 0.05.

Statistical Data of Accelerated Stability Studies of ED-3 Injection in Two-way ANOVA: Responses versus Months, Batches(Based on Assay result)

Source DF SS MS F P

Months 2 75.0064 37.5032 16.61 0.012

Batches 2 5.8756 2.9378 1.30 0.367

Error 4 9.0290 2.2572

Total 8 89.9110

S = 1.502 R-Sq = 89.96% R-Sq(adj) = 79.92%

Individual 95% CIs For Mean Based on

Pooled StDev

Months Mean ------+---------+---------+---------+---

0 103.613 (-------*-------)

3 99.857 (-------*-------)

6 96.547 (-------*-------)

------+---------+---------+---------+---

96.0 99.0 102.0 105.0

Individual 95% CIs For Mean Based on

Pooled StDev

Batches Mean -----+---------+---------+---------+----

1 101.147 (-----------*-----------)

2 99.383 (-----------*-----------)

3 99.487 (-----------*-----------)

-----+---------+---------+---------+----

98.0 100.0 102.0 104.0

Results: Here, I have observed a p value of 0.012 for the average analysis of the affects of different methods for months. In this conditional probability, if my hypothesis is true, reveals that three methods significantly differ from each other in giving the response for the understudy assay result. My was set at 0.05 and the p value "the observed " is much less than 0.05.

In this research case, I have also observed a p value of 0.367 for the average analysis of the affects of different methods for batches. In this conditional probability, if my hypothesis is false, reveals that three methods significantly not differ from each other in giving the response for the understudy assay result. My was set at 0.05 and the p value "the observed " is much higher than 0.05.

Statistical Data of Accelerated Stability Studies of Calgo Tablets in Two-way ANOVA: Responses versus Months, Batches (Based on Assay results)

Source DF SS MS F P

Months 2 92.917 46.4586 12.42 0.019

Batches 2 33.250 16.6252 4.45 0.096

Error 4 14.961 3.7402

Total 8 141.128

S = 1.934 R-Sq = 89.40% R-Sq(adj) = 78.80%

Individual 95% CIs For Mean Based on

Pooled StDev

Months Mean -------+---------+---------+---------+--

0 107.970 (-------*-------)

3 104.537 (------*-------)

6 100.120 (------*-------)

-------+---------+---------+---------+--

100.0 104.0 108.0 112.0

Individual 95% CIs For Mean Based on Pooled StDev

Batches Mean +---------+---------+---------+---------

1 102.180 (----------*---------)

2 103.657 (----------*---------)

3 106.790 (---------*---------)

+---------+---------+---------+---------

99.0 102.0 105.0 108.0

Results: Here, I have observed a p value of 0.019 for the average analysis of the affects of different methods for months. In this conditional probability, if my hypothesis is true, reveals that three methods significantly differ from each other in giving the response for the understudy assay result. My was set at 0.05 and the p value "the observed " is much less than 0.05.

In this case of research study, I have also observed a p value of 0.096 for the average analysis of the affects of different methods for batches. In this conditional probability, if my hypothesis is wrong, reveals that three methods significantly not differ from each other in giving the response for the understudy assay result. My was set at 0.05 and the p value "the observed " is greater than 0.05.

Statistical Data of Accelerated Stability Studies of Cara-In- D Injection in Two-way ANOVA: Responses versus Months, Batches(Based on Assay result)

Source DF SS MS F P

Months 2 92.917 46.4586 12.42 0.019

Batches 2 33.250 16.6252 4.45 0.096

Error 4 14.961 3.7402

Total 8 141.128

S = 1.934 R-Sq = 89.40% R-Sq(adj) = 78.80%

Individual 95% CIs For Mean Based on

Pooled StDev

Months Mean -------+---------+---------+---------+--

0 107.970 (-------*-------)

3 104.537 (------*-------)

6 100.120 (------*-------)

-------+---------+---------+---------+--

100.0 104.0 108.0 112.0

Individual 95% CIs For Mean Based on Pooled StDev

Batches Mean +---------+---------+---------+---------

1 102.180 (----------*---------)

2 103.657 (----------*---------)

3 106.790 (---------*---------)

+---------+---------+---------+---------

99.0 102.0 105.0 108.0

Results: Here, I have observed a p value of 0.019 for the average analysis of the affects of different methods for months. In this conditional probability, if my hypothesis is true, reveals that three methods significantly differ from each other in giving the response for the understudy assay result. My was set at 0.05 and the p value "the observed " is much less than 0.05.

In this study, I have also observed a p value of 0.096 for the average analysis of the affects of different methods for batches. In this conditional probability, if my hypothesis is false, reveals that three methods significantly not differ from each other in giving the response for the understudy assay result. My was set at 0.05 and the p value "the observed " is much higher than 0.05.

Table 12: Parameters of the Statistical analysis of results in determination of assay content of Cholecalciferol (Vitamin D3) Tablets during Accelerated Stability Study conditions.

STATISTICAL HYPOTHESIS TESTING DATA (MONTHS AND BATCHES)

a. Statistical hypothesis testing Data of Qalsan D Tablets (Months).

b. Statistical hypothesis testing Data of Qalsan D Tablets (Batches).

c. Statistical hypothesis testing Data of Calgo Tablets (Months).

d. Statistical hypothesis testing Data of Calgo Tablets (Batches).

f. Statistical hypothesis testing Data of Indrop D Injection (Months).

f. Statistical hypothesis testing data of Osam-D Tablets (Batches).

Statistical hypothesis testing data of Indrop D injection (Batches).

Statistical hypothesis testing data of Osam-D Tablets (Months).

Statistical hypothesis testing data of ED-3 Injection (Months).

Statistical hypothesis testing data of ED-3 Injection (Batches).

Statistical hypothesis testing data of Cara-In-D Injection (Months).

Statistical hypothesis testing data of Cara-In-D Injection (Batches).

CHAPTER NO 6

RESULTS AND DISCUSSIONS:

Three brands of Cholecalciferol (Vitamin D3) tablets i.e., (a) Qalsan D, (b) Calgo and (c) Osam-D containing 125, 125 and 400 IU respectively of Cholecalciferol were used in this study.

Qalsan D tablet in record of assay content had no degradation observed and was stable at temperature 40±2 oC and Relative humidity75±5% while Osam-D and Calgo in record of assay content show little bit degradation at given accelerated stability study parameters.

The brands of Cholecalciferol tablets were found to comply with the Pharmacopoeial requirements as shown in table (10) as regards to their variation in assay contents, hardness, friability, disintegration and moisture content.

The assay content tests of tablet were carried out by using methanol as blank, formic acid and dinitrophenylhydrazin supply by reliable sources at ÊŽmax 265nm, using Double beam UV Spectrophotometer.

The percent of Qalsan D tablet samples of Batch 001, 002, 003 were analyzed and checked at month (0, 3, 6) at specific temperature and relative humidity. Graph was plotted between wavelength and absorbance as shown in figure (6).

From assay content behavior of these Cholecalciferol tablets, it is evident from table (10), ASS data and figure (6) that in first 6 months of Batch 001, results decreased from 100.89% to 100 %. In Batch 002, having 6 months study, results illustrated downward condition from 102.92% to 101.35%, while in Batch 003 having 6 months ASS, result move from 100.67% to 98.43%.

Similarly both "p" values of Qalsan D tablets were placed within the limit i.e., NMT 0.05, as shown in statistical data.

The percent of Calgo tablet samples of Batch 001, 002, 003 were analyzed and checked at month (0, 3, 6) at specific temperature and relative humidity. Graph was plotted between wavelength and absorbance as shown in figure (7).

From assay content behavior of these Cholecalciferol tablets, it is evident from table (10), ASS data and figure (7) that in first 6 months of Batch 001, results decreased from 105.63% to 98.19 %. In Batch 002, having 6 months study, results illustrated downward condition from 107.79% to 97.50%, while in Batch 003 having 6 months ASS, result move from 110.49% to 104.67%.

Similarly out of two "p" values, one "p" value of Calgo tablets was out of the limit while other meets the requirement, i.e., NMT 0.05, as shown in statistical data.

The percent of Osam-D tablet samples of Batch 001, 002, 003 were analyzed and checked at month (0, 3, 6) at specific temperature and relative humidity. Graph was plotted between wavelength and absorbance as shown in figure (7).

From assay content behavior of these Cholecalciferol tablets, it is evident from table (10), ASS data and figure (8) that in first 6 months of Batch 001, results decreased from 104.54% to 96.18 %. In Batch 002, having 6 months study, results illustrated downward condition from 102.69% to 97.27%, while in Batch 003 having 6 months ASS, result move from 103.61% to 96.19%.

Similarly out of two "p" values, one "p" value of Osam-D tablets was out of the limit while other meets the requirement, i.e., NMT 0.05, as shown in statistical data.

In second study, three brands of Cholecalciferol (Vitamin D3) injections i.e., (a) Indrop D, (b) ED-3 and (c) Cara-In-D containing 200,000 IU/1ml (5mg/1ml) each of Cholecalciferol were used.

Indrop D in record of assay content show no degradation and was stable at temperature 40±2 oC and Relative humidity75±5% while ED-3 and ED-3 and Cara-In-D injection in record of assay content show degradation and were unstable at temperature 40±2 oC and Relative humidity75±5%.

The brands of Cholecalciferol injections were found to comply with the Pharmacopoeial requirements as shown in table (11) as regards to their variation in assay contents.

The assay content tests of injections were carried out by using methanol as blank, formic acid and dinitrophenylhydrazin supply by reliable sources at ÊŽmax 265nm, using Double beam UV Spectrophotometer.

The percent of Indrop D tablet samples of Batch 001, 002, 003 were analyzed and checked at month (0, 3, 6) at specific temperature and relative humidity. Graph was plotted between wavelength and absorbance as shown in figure (9).

From assay content behavior of these Cholecalciferol injections, it is evident from table (11), ASS data and figure (9) that in first 6 months of Batch 001, results decreased from 100.44% to 99.77 %. In Batch 002, having 6 months study, results illustrated downward condition from 100.44% to 101.35%, while in Batch 003 having 6 months ASS, result move from 100.67% to 98.66%.

Similarly two "p" values of Indrop D were placed within the limit, i.e., NMT 0.05, as shown in statistical data.

The percent of ED-3 injection samples of Batch 001, 002, 003 were analyzed and checked at month (0, 3, 6) at specific temperature and relative humidity. Graph was plotted between wavelength and absorbance as shown in figure (10).

From assay content behavior of these Cholecalciferol injections, it is evident from table (11), ASS data and figure (10) that in first 6 months of Batch 001, results decreased from 104.54% to 96.18 %. In Batch 002, having 6 months study, results illustrated downward condition from 102.69% to 97.27%, while in Batch 003 having 6 months ASS, result move from 103.61% to 96.19%.

Similarly out of two "p" values, one "p" value of ED-3 injection was out of the limit while other meets the requirement, i.e., NMT 0.05, as shown in statistical data.

The percent of Cara-In-D injection samples of Batch 001, 002, 003 were analyzed and checked at month (0, 3, 6) at specific temperature and relative humidity. Graph was plotted between wavelength and absorbance as shown in figure (11).

From assay content behavior of these Cholecalciferol injections, it is evident from table (11), ASS data and figure (11) that in first 6 months of Batch 001, results decreased from 105.63% to 98.19 %. In Batch 002, having 6 months study, results illustrated downward condition from 107.79% to 97.50%, while in Batch 003 having 6 months ASS, result move from 110.49% to 104.67%.

Similarly out of two "p" values, one "p" value of Cara-In-D injections was out of the limit while other meets the requirement, i.e., NMT 0.05, as shown in statistical data.

Referring to different assay results in table (10) and (11) of Cholecalciferol tablets and injections, the difference in the rates of assay content can be approximately be explained on the basis of oxidative degradation of Cholecalciferol, since nature of Cholecalciferol degradation is unknown (Hoffmann-La-Roche, 1989).

Therefore, the stability is only explained by carrying out the assay for Cholecalciferol after storage under the chosen exaggerated conditions.

This behavior of degradation from four brands of Cholecalciferol suggests that in these particular environmental conditions i.e., temperature 40±2 oC and Relative humidity75±5% affect the quality of Cholecalciferol molecule.

In first case, Qalsan D tablet has stable Cholecalciferol molecule while, Calgo and Osam D tablet required special attention from the start of manufacturing up to final packaging.

In second case of study, Indrop D has stable Cholecalciferol molecule while ED-3 and Cara-In-D injection require special care during manufacturing from first step to last one.

In our studies, we confirmed the previous findings that Cholecalciferol (Vitamin D3) degrades rapidly at high temperatures and relative humidity. The fastest degradation occurs at the combination of elevated temperature and high relative humidity, when all samples degraded within several days.

CHAPTER NO 7

CONCLUSIONS AND SUGGESTIONS:

CONCLUSIONS:

From results and their discussion, the main conclusions of the present investigation on the accelerated stability study of different brands of Cholecalciferol tablets and injections may be summarized as follows:

The stability of the Cholecalciferol was investigated under various conditions. Cholecalciferol tablet and injection samples were found to be sensitive to elevated temperatures as well as to high relative humidity. The most stable form is obtained in Qalsan D tablet and Indrop D injection formulations.

The Qalsan D tablet and Indrop D injection have stable Cholecalciferol molecule for 2 years while degradation of Cholecalciferol in Calgo tablet, Osam-D tablet, ED-3 injection and Cara-In-D injection highlight unstability of molecule under stress conditions and should be degraded completely before 2 years.

The difference in assay contents with the progress of month and batch can be explained on the basis of oxidative degradation phenomenon.

Lowering of assay result and change in structure of molecule are basic research accelerated stability results for antioxidant like Cholecalciferol.

Three brands of tablets, accordingly of the degree of oxidative degradation of Cholecalciferol are arranged in following sequence:

Qalsan D › Osam-D › Calgo

Three brands of injections, accordingly of the degree of oxidative degradation of Cholecalciferol are arranged in following sequence:

Indrop D › ED-3 › Cara-In-D

SUGGESTIONS:

Some of the most complex formulations include tablet and parenteral vitamins/multivitamins preparations. These may be exposed to temperature and relative humidity during manufacturing, storage, and administration causing vitamin interaction and loss of the individual vitamins. Since Cholecalciferol is particularly sensitive to temperature and humidity and is normally present in micro quantities, its loss even in small amounts may deprive the patient of the required dose of the vitamin.

In view of the sensitivity of Cholecalciferol and the influence of factors such as temperature, relative humidity, light, oxygen level and wavelength, presence of other vitamins, in a multivitamin preparation, or active/excipients of drug product, an evaluation of stability characteristics of Cholecalciferol extremely difficult. Therefore, the approach adopted in this study work to simplify the system initially to binary mixtures can serve as a model for further studies.

This may be used to conduct further systematic studies on the evaluation of Cholecalciferol stability in tablets and injections mixtures.

Identification of the unknown oxidation products of Cholecalciferol in tablet and injection.

Study of the relationship of degradation rates with temperature and relative humidity in a particular medium and prediction of normal shelf-life of Vitamin D3 tablet and injection.

Development of Stability-determining methods for the assay of a Cholecalciferol in the presence of its degradation product.

Study of the relationship of degradation rates with viscosity of parenteral medium.

Study of the relationship of degradation rates of vitamin D3 with dielectric constant of the medium.

Study of the degradation rates of Cholecalciferol using different parameters suggested above under anaerobic conditions.

Study of the relationship of degradation rates with Non-Pharmacopoeial methods applied during analysis of Cholecalciferol tablet and injection.

CHAPTER NO 8

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