Estimating Concentration of Calcium Pantothenate in Vitamin C Tablets

This lab report discusses the microbial approach used to evaluate the concentration of calcium pantothenate in vitamin C tablets. It explains the procedure of the turbidimetric assay with Lactobacillus plantarum and provides the results and discussion.

Running Head:
Name :Arshpreet kaur
Student no: 300960214
Course code:
Section :109
Date:
Partner’s name: Parimala vidya
TITLE
To Estimate the Concentration of Calcium Pantothenate in Commercial Sample of Vitamin C
Tablets by Using Turbidimetric Assay with Lactobacillus Plantrum.

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Abstract
Microbial approach which is employed to evaluate the concentration of the calcium
Pantothenate in particular amino acids and nutrients is known as Calcium Pantothenate test.
Calcium Pantothenate is a type of salt of the pantothenic corrosive that is vital for human
health. It is correlated with various capacities such as, digestion of sugars and fat, cholesterol,
combination of steroid hormones, and different absence and mixes of the nutrient, which can
causes extreme complication such as, kidney impairment, degeneration of spinal string and
mind, and so on.
In the laboratory, we evaluated the quantity of Pantothenate with the nutrient C cases
(quantities in the possibility of 10 ng/ml) in B complex. At that time the arrangement of
Pantothenate standard arrangement was organized in the laboratory. For the inoculation of the
sans Pantothenate development medium, Lactobacillus plantarum is used. Single drop of
Lactobacillus plantarum is supplemented in each of the standard containers, blank, analyte,
inoculated before hatching, which is done at 35° Celsius for about 24 hours. Several
processes (known fixations) were prepared and various questions were organized before
determining the transmittance, after the time frame of hatching by operating
spectrophotometer (wavelength 590nm) along with cuvettes. After that, the figure of
transmittance is evaluated, which helps in providing the combination of calcium Pantothenate
(due to bacterial infection, the media changes to turbid). To estimate the exact concentration
of (C18H32CaN2O10) calcium pantothenate in complex B cases, the curve is applied.

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Table of Contents
Introduction................................................................................................................................3
Materials and methods:..............................................................................................................4
Culture used...........................................................................................................................4
Pantothenate standard solution...............................................................................................4
Sample used...........................................................................................................................4
Sterile water...........................................................................................................................5
Sterile saline...........................................................................................................................5
Equipments:............................................................................................................................5
Method...................................................................................................................................5
Results:.......................................................................................................................................6
Figure 1..................................................................................................................................8
Figure 2..................................................................................................................................9
Discussion :................................................................................................................................9
References................................................................................................................................11

3Pharmaceutical Lab Report
Introduction
Pantothenic corrosive is also called B nutrient (nutrient B5) or Pantothenate. It is a
found by Roger J. Williams in the year 1919 and is soluble in water. It is used as basic
supplement for some living beings. Some living beings require the pantothenic corrosive to
incorporate CoA (coenzyme- A), and in addition it is also used for the assimilation of sugar,
protein and fats. It is the amide among the β-alanine and the β-alanine. Pantothenic acid is
found in nearly every nutrient, in some nutrient it is present in low quantity ad in some foods
such as, vegetables, meat, avocado, regal jam, yogurt, eggs and grain oats. It is commonly
found in the liquid sample, calcium Pantothenate and Provitamin Pantothenate. Pantothenic
corrosive is present as a fixing in healthy skin substances and hair. The isomer of the
pantothenic corrosive, D isomer (dextrorotatory) pose biologic movement. The effect of the
D isomer (dextrorotatory) is hindered by the (levorotatory) L. For this the free hydrated
medium is used, which is Pantothenate medium. In the medium all the essential nutrients are
present which are vital for the survival of Lactobacillus development. In addition,
development of Lactobacillus plantarum provides unusual reaction in present of the calcium
Pantothenate fixation and can be implemented by Turbidimetrically. Lactobacillus has
numerous different strains. One of the strain is Lactobacillus plantarum, which is generally
found in the ripened items and in different vital plant material.
It was first isolated from the saliva and is a gram positive bacteria. This gram positive
bacteria is beneficial for the human health such as, in skin.
Several procedures have been evaluated in performing the pantothenic corrosive test.
Measure of Calcium Pantothenate is applied in the research center in order to check the
combination of calcium Pantothenate. Calcium Pantothenate (C18H32CaN2O10 ) in the nutrient

4Pharmaceutical Lab Report
sample, which is reliant on the Turbidimetric examine. In the media, the Calcium
Pantothenate is added.
Lactobacillus plantarum strain was used and is inoculated in each of the test tubes.
Arrangement were established. 2 arrangement of the standard with 5 test tubes each and 2
arrangement of the analytes with 4 of each test tubes were set up. Appropriate amount of
analyte or standard, medium and water is supplied in each of the test tubes (Table 1: obscure
and clear example test tubes and planning of the standard. 2 of the test tubes were considered
as spaces, one of them is clear and uninoculated ( that is B-U) and clear and vaccinated. A
single drop of Lactobacillus plantarum culture is supplied to each of the test tubes, with the
exclusion of B-U.it is then brooded afterwards at 35° C for about 24 hours.
After the addition of 1 drop of CH2O (formaldehyde) with the spectrophotometer at
590 nm of wavelength, the transmittance of both the standard and the analyte is measured.
The normal is then used in plotting the standard curve against the two (in X pivot, % T is
plotted and Y axis, Pantothenate fixation is plotted). It is done to estimate the concentration
of calcium Pantothenate.
Materials and methods:
Culture used: Lactobacillus plantarum is used in the experiment. It is incubated at in the
Lactobacillus MRS (De Man, Rogosa and Sharpe agar) stock at 35° Celsius for about 24
hours. It is then centrifuged at for about 10 minutes at 2000 rpm for 10 minutes and after that
pellet was collected. It is then resuspended in 10 ml of saline and later vortexed in order to
blend. It is then again centrifuged for some time and then the saline was discarded.
Pantothenate standard solution: By using the Difco pantothenate medium (which is in the
powder form) the procedure is being set up at the concentration of 20ng/ml.

5Pharmaceutical Lab Report
Sample used: vitamin C capsules (life brand ; expiry date is July, 2021and Lot no :
LOT808156) with 10 pills of B complex was used as the tests. It is then mixed with distilled
water and then diluted. It is diluted in order to coordinate the standard arrangement’s (20
ng/ml) dilution standard.
Preparation of medium: At first, 5 tubes in the 2 sets are utilized to prepare standards for
the standard and set aside. It is labelled as S-1-1, S-2-2… … and so on. 4 tubes in the 2 sets
are kept for analyte and set aside by labelling as A-1-1, A-2-2…. After that, clear
uninoculated and clear vaccinated. From the table 1, appropriate amount of water, medium
and standard/analyte is further added to each of the cylinder.
Sterile water: The sterile water is utilize to formulate capsules. Sample dilution is also
prepare by using the sterile water in a volumetric flasks.
Sterile saline: Lactobacillus plantarum is washed by using the sterile saline.
Equipments: cuvettes, spectrophotometer, vortex mixtures and formaldehyde.
Method: To check the concentration of C18H32CaN2O10 (Calcium Pantothenate) in the test
media, which us the B complex pills, 2 out of the 5 tubes were analysed and the 2 set of the 4
tubes consisting of analyte were also arranged. They are then labelled accordingly. To each of
the tube 5 ml of the medium is supplied in order the manage the volume up to 10 ml followed
by 1 drop of Lactobacillus plantarum
After that, the tubes were incubated for about 24 hours at 35° Celsius and after that, it
is supplied with formaldehyde. The tubes are then observed under the spectrophotometer.
To set the spectrophotometer, it is warmed for about 15 minutes and then the
wavelength is set at 590nm. Each of the tubes were mixed properly and after that, the
cuvettes are cleaned sensibly before to get the accurate at precise outcomes before using each

6Pharmaceutical Lab Report
time. At first, the Uninoculated clear is used. It is done to set transmittance at 100% and then
the immunized clear was operated to reset the transmittance at100% once more.
The transmittance of all the significant amount of tubes, were measured and based on that, a
standard curve was then plotted in order to discern the potency.
Results:
% transmittance of blank inoculated and uninoculated is set at 100%
Table 1 : % transmittance of the standard arrangement determined utilizing
spectrophotometer at 590 nm of standard tubes incubated at 35 degree Celsius for 16 to
24 hours pursued by the expansion of formaldehyde.
Number of tubes % transmittance
(set 1)
% transmittance
(set 2)
Average Calcium pantothenate
concentration
1 77.4 70.2 73.8 2
2 60.6 58.8 59.7 4
3 52.6 51.8 52.2 6
4 45.2 40.3 42.7 8
5 42.3 35.6 38.9 10

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Above table shows that the percentage transmittance decreased but concentration of
calcium pantothenate increased.
Table 2 : % transmittance of the analyte determined utilizing spectrophotometer at
wavelength 590nm of tubes with analyte, incubated at 35degree Celsius for 24 hours
pursued by the addition of 1 drop of formaldehyde.%
Number of
tubes
% T (set 1) % T ( set 2 ) Average Estimated
potency from
curve
Actual
potency / ml
1 16.8 15.6 16.2 0.4 0.4
2 11.4 10.4 10.9 3.2 1.6
3 8.3 7.7 8.0 5.0 1.6
4 6.5 6.3 6.4 6.6 1.6
The above table showing that, the commercial sample concentration increased and
transmittance percentage decreased.
Calculated Average actual potency
(0.4 +1.6 +1.6 +1.6 ) = 5.3= 1.328 ng/ml
4 4
= 0.00132ug/ml

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Therefore, mg/capsule = 11.95 mg/capsule
% potency was 65. 3% rather than 18.3%.
GRAPH
1 2 3 4 5 6 7 8 9 10 11
0
10
20
30
40
50
60
70
80
f(x) = − 4.34 x + 79.5
% Transmittance vs concentration of calcium pantothenate
X-axis: concentration of calcium pantothenate
Y-axis: % transmittance
Figure 1: The line graph depicts the bend of % transmittance and convergence of
pantothenate (ng/ml) of standard calcium pantothenate at 590nm wavelength.

9Pharmaceutical Lab Report
1 2 3 4 5 6 7 8 9
0
10
20
30
40
50
60
70
80
90
f(x) = − 7.41 x + 92.1
% transmittance vs concentration of calcium pantothenate
Figure 2: The line graph depicts the bend of % transmittance and convergence of
pantothenate (ng/ml) of analyte calcium pantothenate at 590nm wavelength.
Discussion :
Calcium pantohenate is a nutrient B5 and water dissolvable which is fundamental for
water solvent nutrient B5 which is extremely basic for unsaturated fats, proteins and starches
digestion and go about as a cofactor for compound CoA. In the test, business nutrient B test
was evaluated by Turbidimetric examine. In addition, The development rate of a
microorganism which requires a nutrient is seen in the media that contain different known
amounts of foodstuff arrangement containing obscure measure of nutrients
( Russel,1997).From the outcomes, part of transmittance on spectrophotometer was taken at
wavelength 590 nm.. The pantothenic corrosive realized focus was exist in given standard
example tubes, by which information on diagram was intended for Estimate power. Besides,

10Pharmaceutical Lab Report
real intensity of cases was determined. The outcomes clears up that there was an expansion in
the media focus which contains supplements for Lactobacillus plantarum development and
that lead to decrease the level of transmittance because of more ingestion of light
( Transmittance and absorbance are contrarily relative). Also, there was no turbidity in the
vaccinated clear cylinder in light of the fact that there was just water and media in the
cylinder rather than Calcium pantothenate. As per the down to earth, discovering fixation was
not exactly the genuine. Subsequently, the range was not adequate scope of given nutrient
that was water solvent. This was because of any slip-up in performing weakening, getting
ready media and measure of culture drop that was included the cylinders. Besides, it was
influenced because of instrumental blunder, spectrophotometer instrument was utilized in the
trial to note or check the level of transmittance in standard arrangement. Here and there
instrument shows variety in the perusing after moment which contrarily aggravates the
centralization of the example in the cylinders. Along these lines, that was the reasons which
was in charge of the low fixation ( mg/case). At last, the microbiological examine is the
refined strategy which is suitable for B nutrients and for their edifices to direct their focus
that depends on turbidimetric strategy.

11Pharmaceutical Lab Report
References
Appendix
Calculation of calcium pantothenate in the commercial vitamin sample:
Estimated potency for tubes was 0.4, 3.2, 5.0, 6.6
Actual Potency =
0.4 /1= 0.4 ng/ml
3.2 /2= 1.5 ng/ml
5.0 /3 = 1.6 ng/ml
6.6 /4= 1.6 ng/ml
Average actual potency = (0.4+1.6+1.66+1.65)
4
=5.3/4
= 0.00132ug/ml
mg per capsule = Average potency in ug /ml × 10 × volume of blender (ml)
Dilution from jar ×Number of tablets× 1000
= 0.00132 × 10 × 460
5×10 -5 × 10 × 1000
= 11.95 mg /capsule

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Calculation of % potency or percent label claim
=Actual mg /per capsule
Amount of claimed table × 100
= 11.95
18.3
= 65. 3 %