Calcium Pantothenate Assay

The Assay of Calcium Pantothenate Standard with L. plantarum and a Vitamin B sample using the

Turbidimetric Method

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Calcium Pantothenate Assay

ABSTRACT

In this experiment, a calcium pantothenate assay was performed utilizing the turbidimetric measurement

method. A commercial Vitamin B complex product was experiment to determine the actual

concentration and potency comparing it to the claimed potency on the product label. The microorganism

that was utilized was Lactobacillus plantarum, which was further inoculated in the pantothenate medium

specifically made for the proliferation of L. plantarum. The turbidity growth was analyzed using the

Spectrophotometer to calculate the amount of light that passes through each standard solution and for

each unknown solution based on the concentrations aliquoted as transmittance %. The reading values of

the standard solutions obtained were then plotted on a graph created on the excel software. This was to

determine the unknown’s concentration based on the standard curve created. The estimated potency and

the actual potency in % were determined, along with the dilution ratio occurred for the stock solution, to

calculate the actual concentration mass in each tablet. Based on the mass in mg, the % label claim was

calculated allowing for the comparison of the acceptable range for water soluble vitamin C of 95% to

130%.

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Calcium Pantothenate Assay

TABLE OF CONTENTS

Introduction ------------------------------------------------------------------------------------------------------------ 3

Materials and Methods ----------------------------------------------------------------------------------------------- 4

Results ------------------------------------------------------------------------------------------------------------------ 5

Table 1.0 – Standard Solution Results -------------------------------------------------------------------- 6

Table 2.0 – Analyte Solution Reults ---------------------------------------------------------------------- 7

Table 3.0 – Blank Solution Results ----------------------------------------------------------------------- 7

Figure 1.0 – Calcium Pantothenate Graph --------------------------------------------------------------- 8

Table 4.0 – Actual Potency Results ---------------------------------------------------------------------- 9

Vitamin Assay Calculations ------------------------------------------------------------------------------- 9

Discussion -------------------------------------------------------------------------------------------------------- 10,11

Reference ------------------------------------------------------------------------------------------------------------ 12

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Calcium Pantothenate Assay

INTRODUCTION

Calcium pantothenate, also known as a Vitamin B5 water soluble salt, is an essential component

for human health. The component of the vitamin is a necessity for the metabolism of macromolecules,

such as lipids and carbohydrates. The deficiency of this vitamin can cause illnesses such as, dermatitis,

kidney damage, necrosis, and more (Khan, N & Jameel, N., 2019). There are a variety of bacterial

strains that can grow in the presence of calcium pantothenate and other strains that cannot grow.

Lactobacillus plantarum is a microbial culture that can grow in the presence of calcium pantothenate.

The methodology of the calcium pantothenate assay will determine the concentration of the

calcium pantothenate standard solution using the turbidimetric measurements. This will allow the

determination of the unknown analyte solution in a standard curve graph (Bazyleu, n.d). The unknown

product tested was: Life Brand Vitamin B Complex with Vitamin C; no gluten, lactose, or soy. The

manufacture of this product was Shoppers Drug Mart with a lot number of 808156. The expiration date

was February 2021.

Lactobacillus plantarum was the specific culture experimented in the calcium pantothenate assay.

Calcium pantothenate is a great source for the growth of L. plantarum. The pantothenate assay medium

contains all the necessary nutrients needed for growth when vitamins when not added (Zimbro, 2009).

The addition of pantothenate allows for the utilization of the turbidimetric method to measure the

concentration of L. plantarum growth in the broth medium (Zimbro, 2009).

The turbidity is measured by analyzing the transmittance in percent using the Spectrophotometer.

The Spectrophotometer measures the amount of light that passes through the pantothenate medium when

L. plantarum proliferates. The calcium pantothenate standard solution was used as a known

concentration in order to determine the unknown commercial vitamin solution from the standard curve

(Bazyleu, n.d)

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Calcium Pantothenate Assay

MATERIALS AND METHODS

The preparation of the experimental set-up

To prepare a series of 2 sets of test tubes for the turbidimetric testing, a series of 5 test tubes/set (10 in

total) were set up in a test tube rack that will be aliquoted by the pantothenate standard solution. A series

of 4 test tubes/set (8 in total) were set up that contained the unknown analyte solution. A series of 2 test

tube/set (4 in total) were prepared for the blank solutions: one blank test tube was uninoculated and the

other was inoculated with Lactobacillus plantarum. All the test tubes were properly labeled according to

the table provided: S-1-1 to S-5-1 for the standard solution; A-1-1 to A-4-1 for the unknown solution; B-

U-1; and, B-I-1 (another set was labelled). A series of material were obtain and provided: a commercial

bottle of vitamin-pantothenate in a form of tablets was given in a stock solution blended with dH 2O; a

sterile 120 mL of Difco pantothenate medium was provided; a 35 mL of a pantothenate solution at a

concentration of 20ng/mL; A bottle of sterile water to prepare the vitamin-analyte solutions with the

blended sample in three 100 mL volumetric flasks; A series of 5 mL and 10 mL sterile pipettes; and, a

Spectrophotometer for the analysis of the test tube samples.

The preparation of the standards, analyte, and the blank solutions

The pantothenate standard solutions were prepared by the lab technologist. The 184 mg (10 tablets) of

sample for the unknown was already prepared by blending with 460 mL of dH2O. The unknown analyte

was further diluted with three 100 mL volumetric flasks. A 5 mL aliquot of unknown analyte was

transferred using a 10 mL sterile pipette into the first volumetric flask with 100 mL of dH 2O filled. A 1

mL aliquot of the first diluted solution was transferred into a second volumetric flask with 100 mL of

dH2O filled. A 10 mL aliquot of the second flask sample was transferred into a third volumetric flask

with 100 mL dH2O filled. The blank solutions did not contain any pantothenate samples.

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Calcium Pantothenate Assay

The preparation of the culture, standard, unknown, and blank solutions.

A 1 mL aliquot L. plantarum culture was diluted into a 10 mL sterile pantothenate medium. The two

blank solution set were aliquoted with 5 mL of sterile water. A 5 mL aliquot of sterile medium provided

in a jar was transferred into the both blank test tubes. The blank solutions did have any vitamin samples

added. A 1 drop aliquot of L. plantarum culture was transferred into the inoculated blank test tube

(labelled B-I-1 and 2) only. The uninoculated blank tube (B-U-1 and 2) did not contain L. plantarum

culture. The series of standard test tubes were diluted: a 1 mL aliquot of pantothenate standard solution

was transferred into the S-1-1/2 test tubes using a 10 mL pipette; a 2 mL aliquot of standard into the S-2-

1/2 tubes; a 3 mL aliquot standard into the S-3-1/2 tubes; a 4 mL aliquot standard into S-4-1/2; and, a 5

mL aliquot standard into the S-5-1/2 tubes. A 5 mL aliquot of sterile pantothenate medium was

transferred into all the standard test tubes. A 1 drop aliquot of L. plantarum was transfer into all standard

test tubes. The four unknown analyte test tube solutions from A-1-1/2 to A-4-1/2 were aliquoted the

same way as the standards. All the test tube solutions were incubated at 35oC for 24 hours.

Spectrophotometer set up and the turbidimetric method

The spectrophotometer was set-up and adjusted to a 590 nm in wavelength. A 2mL aliquot was

transferred into a micro test tube for each standard, and unknown analyte readings from least turbid to

most turbid. The spectrophotometer was first calibrated with the uninoculated blank solution to a

transmittance of 100%, then with the inoculated blank solution calibrated to 100% again. The readings

of each transmittance were recorded for all sample test tube sets, then averaged. The excess culture was

disposed of in the empty container for each micro tube sample.

RESULTS

The turbidity was measured with a Spectrophotometer at the wavelength of 590nm to evaluate the

transmittance (%) of 4 sets of samples. Two sets of samples contained the pantothenate standard

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Calcium Pantothenate Assay

solution inoculated with Lactobacillus plantarum and two sets of unknown analyte solutions inoculated

with the Vitamin B complex. The uninoculated blank solution were used to standardize the

Spectrophotometer, then calibrated using the inoculated blank solution with L. plantarum to 100% for

the series of inoculated sample test tubes.

Table 1.0 – The averaged results of the turbidimetric measurements for the 2 sets of the pantothenate

standard solutions with a concentration of 2ng/mL, 4ng/mL, 6ng/mL, 8ng/mL, and 10ng/mL measured

by percent transmittance inoculated with L. plantarum incubated at 35oC for 24 hours.

Concentration
Standard Sample(s) Transmittance % Average %
(ng/mL)

S-1-1 16.6
2 10.7
S-1-2 4.8

S-2-1 9.0
4 8.1
S-2-1 7.2

S-3-1 8.1
6 7.0
S-3-2 5.8

S-4-1 5.6
8 5.9
S-4-2 6.2

S-5-1 3.0
10 3.9
S-5-2 4.7

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Calcium Pantothenate Assay

Table 2.0 - The averaged results of the turbidimetric measurements of the 2 sets of the unknown analyte

solutions inoculated with the Vitamin B complex incubated at 35oC for 25 hours. The concentration was

calculated based on the graph of the standard curve.

Concentration
Analyte Sample(s) (ng/mL) Transmittance (%) Average (%)
from graph

A-1-1 13.2
0.1 25.3
A-1-2 37.4

A-2-1 6.3
3.7 8.5
A-2-2 10.6

A-3-1 2.6
9.8 4.7
A-3-2 6.7

A-4-1 2.2
12.8 3.7
A-4-2 5.2

Table 3.0 – The transmittance results obtained for the uninoculated blank solution and the inoculated

blank solution with L. plantarum incubated at 35oC for 24 hours.

Blank Sample(s) Transmittance (%)

Uninoculated 100

Inoculated with L. plantarum 93.3

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Calcium Pantothenate Assay

The calcium pantothenate standard solutions were plotted on excel to create a standard curve with a

proper trend line. Based on the standard curve, the concentration of the unknown vitamin B complex

samples was determined (concentration values on Table 2.0).

Figure 1.0 – The graph for the calcium pantothenate standard solution with a series concentration of

2ng/mL, 4ng/mL, 6ng/mL, 8ng/mL, and 10ng/mL. The unknown analyte concentrations were

determined from the standard curve.

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Calcium Pantothenate Assay

The % label claim or the % potency of the unknown vitamin B complex solution were determined

through various calculations: the estimated potency and the actual potency were determined by the

concentration obtained by the graph; the mass the unknown analyte in ug for each concentration; the

stock solution dilution ratio; and, the amount of tablets experimented. The actual mass per capsule for

the unknown analyte would be compared to the expected mass of 18.3mg in the bottled sample provided

to determine if the actual mass is not overage and is in the acceptable range of 95% - 130%.

Table 4.0 – The results obtained of the estimated potency and the average of the actual potency in

ug/mL from the volume of the unknown analyte aliquoted.

Analyte aliquoted (mL) Transmittance (%) EST Potency (ug/mL) Actual Potency (ug/mL)

1 25.3 0.0001 0.0001

2 8.5 0.0037 0.0019

3 4.7 0.0098 0.0033

4 3.7 0.0128 0.0032

AVERAGE 0.0021

Mass of the analyte (ug): 0.0021 x 10 = 0.021ug

Dilution ratio of the blended sample:

Flask #3 (20ng = 0.02ug); blended jar (400ug)

DR = Cfinal / Cinitial = 0.02ug/400ug = 0.00005  1:20,000 dilution

0.0021 x 10 x 460
actual potency x 10 x mL of blender sample
Mg/capsule:
dilutionratio x 10 tablets x 1000
=
( 1
20,000) x 10 x 1000
= 19.32mg/capsule

% label claim:

(actual mg/capsule x 100) / (claimed mg/capsule)

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Calcium Pantothenate Assay

(19.32mg x 100) / (18.40mg) = 105%

DISCUSSION

In this experiment, a sample of a calcium pantothenate standard solution was experimented to

compare with a commercial Vitamin B complex from a drug store. An assay was prepared through

series of the calcium pantothenate standard solutions that were inoculated with Lactobacillus plantarum

and for an unknown analyte sample that was inoculated with the commercial vitamin product in tablets

that were blended with dH2O. To determine its actual potency or the actual concentration of the calcium

pantothenate in the commercial vitamin B product, the turbidimetric method was utilized using a

Spectrophotometer to determine the percent transmittance of the samples. The commercial vitamin

sample was compared to the calcium pantothenate standard solution prepared, to determine the true

concentration of the unknown vitamin sample. Based on the data collected, a standard curve was created

for the standard solution to determine the estimated concentrations for the unknown vitamin sample and

determining the estimated and the actual potency percentage depending on the amount of volume

aliquoted for each unknown sample. The claimed concentration of commercial pantothenic acid

(calcium pantothenate) was 18.4 mg. By utilizing the vitamin assay calculations, the actual

concentration of calcium pantothenate per capsule was 19.32mg. This value concludes that the actual

mass of the calcium pantothenate is slightly higher than the claimed mass of 18.5 mg.

The potency of every vitamin is important to determine in order to know the right concentration

needed for a human to remain healthy. Too much potency can create an “overdose” to your body

causing even more illnesses or symptoms. Too little potency can create vitamin deficiency in humans, so

by determining the true potency of vitamins, such as vitamin B complexes, through assays and

turbidimetric techniques, one can obtain a healthy amount of vitamin concentration based on human

studies.

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Calcium Pantothenate Assay

The results of the experiment determined an increase of potency by 0.92 mg. This value can be a

result in sources of errors while performing the experiment. The result of the higher potency value can

be due to aliquoting a higher volume of standard or analyte solution in the sample tubes. It can also be

an inaccuracy of the other components of aliquoted; such as, the water, the pantothenate medium, and/or

the amount of L. plantarum culture added). The calibration of the Spectrophotometer is very crucial step

as well. The lack of calibration using the blank solutions created will disrupt the transmittance value and

give an inaccurate reading. Not only that, but calibrating can ensure that the Spectrophotometer is

functioning properly even though a blank is added. The transmittance reading could have been

inaccurate due to the improper aliquot of the sample in the cuvette; such as, inserting the cuvette with

bubbles in the sample in the Spectrophotometer and/or fingerprints were all over the cuvette disrupting

the light passage.

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 Calcium Pantothenate Assay

References

Bazyleu, A. (2017). BI 209

from eCentennial:https://e.centennialcollege.ca/d2l/le/content/641833/viewContent/7417516/View

Khan, N., & Jameel, N. (2020, May). PANTOTHENIC ACID –A DIETARY ABUNDANT VITAMIN.

Retrieved March 20, 2021, from https://ijbaf.com/abspdf/MS_IJBAF_2020_2338.pdf

Zimbro, M. J., & Power, D. A. (2009). Difco & BBL manual: Manual of microbiological culture media

(2nd ed.). Becton Dickinson and Co.

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