In this slide contains introduction, principle, precautions, solution and assay method for vitamin B series. Presented by: P. VENKATESH (Department of pharmaceutical analysis), RIPER, anantapur

1. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 1
A Seminar as a part of curricular requirement
for I year M. Pharm I semester.
Presented by
Mr P.Venkatesh.
(Reg. No. 20L81S0714)
Under the guidance of
Dr. K.Vinod kumar ,M.pharm,PhD.
Professor Pharmaceutical Analysis,
Head of department -Pharmaceutical analysis.
MICROBIAL ASSAY Of
VITAMIN OF B SERIES

2. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 2
• Introduction.
• Principle.
• Quick view of B series vitamins.
• Microorganisms used.
• Precautions.
• Solutions and inoculum preparations.
• Assay methods.

3. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 3
• Microbial assay
Also Known as microbiological assay.
• Microbial assay could be a sort of biological assay
designed to analyse the compounds that have impact
on microorganisms.
• Hepls to estimate
-Concentration.
-Effency of antibiotics.
•
Introduction

4. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 4
PRNCIPLE
• Vitamins are important growth factors required for
growth and multiplication of certain microorganisms.
• The microorganisms require growth factors in micro or
nanograms.
• The basis of this assay is to measure the ability of test
organism to synthetise the factor by utilising the
substance being assayed under a proper nutrition
conditions.
• Factor-growth ,nutrition condition-cyanocobalamine.

5. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 5
PRNCIPLE
• Microbial assay of vitamin is based on
Test concentration of vitamin is estimated with
known concentration of standard.
Comparison of growth
of microorganisms

6. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 6
• Thiamine (B1 ) :
Organism :lactobacillus varidescens.
Defiency : memory loss, confusion hallucination.
Daily intake :
Quick view of vitamin B series
Years Male Female
9-13 0.9 mg 0.9mg
14-18 1.2mg 1.0mg
19-50 1.2mg 1.1mg
51 above 1.2mg 1.1mg

7. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 7
• Riboflavin(B2) :
Organism: lactobacillus casei.
Defiency : skin disorders ,chelosis,hair loss,reproductive
problems.
Daily intake :
Quick view of vitamin B series
2-5 years 2.1mg
6-11 years 2.2mg
12-19 years 2.3mg

8. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 8
• Niacin(B3) :
Organism : lactobacillus plantarum.
Defiency: pellegra ,dermatitis, diarrhea and can result in
death Occurs due to genetic disorders.
Daily intake :
Quick view of vitamin B series
Adult men 19
above
16 mg
Adult women 19
above
14 mg
Pregnant women 18 mg
Brest feeding
women
17 mg

9. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 9
• Pantothenic acid (B5) :
• Organism: lactobacillus plantarum.
• Defiency: inherited disorder called pantothenate kinase
associated neuro degeneration - can't use pathothenic acid
.
• Daily intake:
Quick view of vitamin B series
9- 13 years 4 mg
14-18 years 5 mg
19 years above 5 mg
Pregnant women 6 mg

10. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 10
• Pyrodoxin(B6) :
Organism : saccharomyces uvarum.
Defency : skin rashes, cracked lips, impaired immune
function, nerve pain.
Daily intake :
Quick view of vitamin B series
9-13 years 1.0mg 1.0mg
14-18 years 1.3mg 1.2mg
19-50 years 1.3mg 1.3 mg
50 above 1.7mg 1.5mg

11. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 11
• Folic acid(B9) :
Organism : lactobacillus casei.
Defiency : megaloblastic anemia .
Daily intake:
Quick view of vitamin B series
Adult 400mcg
Pregnant women 1000mcg

12. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 12
• Biotin(H) :
Organism : lactobacillus plantarum.
Defiency: rare inherited disorder where body is not able to
use biotin, defiency caused by mutation.
Daily intake :
Quick view of vitamin B series
Years Male Female
4-8 12mcg 12mcg
9-13 20mcg 20mcg
14-18 25mcg 25mcg
19
above
30mcg 30mcg

13. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 13
Quick view of vitamin B series
Stick model structure. Skeletal model structure

14. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 14
• Also known as vitamin B12.
• It is a water soluble vitamin.
• Structure is similar to that of heme where iron is replaced
with cobalt as center of molecule.
Quick view of cyanocobalamine

15. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 15
Quick view of cyanocobalamine
9-13 years 1.8mcg/day
14 above years 2.4mcg/day
Pregnant women 2.6mcg/day
Brest feeding women 2.8mcg/day

16. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 16
• Defiency causes pernicious anemia.
• Helps in maintaining healthy nervous system.
• Aids in production of DNA and RNA.
• Help to keep red blood cells healthy.
Quick view of cyanocobalamine

17. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 17
Quick view of vitamin B series

18. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 18
MICROORGANISM USED
• microorganism used here are
Lactobacillus leichmanni
• Test organism selected must be capable of utilizing free
cyanocobalamine.
• Gram negative bacteria.
• Non- pathogenic.
• Easy to culture and easily available.
• Isolated from - Milk
- Cheese and dairy products.

19. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 19
• Great care must be taken to avoid contamination of
preparations.
• All glasswares must be free from chemicals and
microbes.
• Glassware must be heated at 250°C for at least 1hour
before use.
• The whole expirement must be carried under asceptic
conditions.
• Asceptic conditions - laminor air flow conditions
PRECAUTIONS

20. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 20
• Standard B12 stock solution.
• Standard B12 solution.
• Test solution to be assayed.
• Basal medium stock solution.
• Suspension medium.
• Preparation of inoculum.
SOLUTIONS, INOCULUM PREPERATION

21. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 21
Continuation ..........

22. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 22
Continuation ..........

23. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 23
• Basal medium stock solution :
Continuation ..........

24. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 24
• Suspension medium :
• Prepare 100 ml solution by mixing equal volume of basal
medium stock solution and distilled water.
• Preparation of inoculum:
• Transfer a loop full of lactobacillus liechmanni from recent
subculture into two tubes each containing 10 ml of sterile
culture medium.
• Incubate the tube for 18 to 24 hours at 37 degrees
centigrade.
Continuation ..........

25. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 25
Continuation ..........

26. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 26
• Assay is performed by two methods
Titrimetric method.
Turbidimetric method.
• Titrimetric method :
• Clean the test tube and add
0,0.5,1.0,2.0,2.5,3.0,3.5,4.0,4.5 and 5.0 ml of standard
cyanocobalamine solution.
• To each tube add 5 ml of basal medium solution.
ASSAY METHODS

27. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 27
• Volume of each is adjusted with 10 ml distilled water .
• In another 4 test tubes add 1,2,3,4 ml of test solution
which is to be assayed.
• Volume of each is adjusted with 10 ml of distilled water.
• Sterilize all the test tubes in autoclave at 121°C for 15
minutes.
• Cool the test tube to room temperature.
• Inoculate a drop inoculum prepared.
Continuation........

28. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 28
• Incubate the test tube for 64 to 72 hours at a temperature
range of 30 to 37 degrees centigrade.
• After incubation content of each test tube is titrated with
0.05 N NaOH using bromothymal blue as an indicator until
green colour appears.
• Record all tritant reading clearly.
Continuation........

29. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 29
• Turbidimetric method :
• Also performed by Turbidimetric method.
• By using un inoculated media as blank and adjusting
transmittance at 640 nm.
• Transmittance values are noted.
• Plot a graph by considering transmittance verses
standard cyanocobalamine solution.
Continuation........

30. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 30
Continuation........
Transmittance
Value
Standard cyanocobalamine
Solution

31. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 31
• Principle :
Vitamin B12 is extracted form sample using
Sodium acetate buffer in presence of sodium cyanide.
at100 degrees centigrade
Extract are purified and concentrated with
Immunoaffinity column.
HPLC method of analysis

32. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 32
• Sample preparation :
• If the sample is in powder form weigh 25 g of sample and
taken in a beaker.
• Add 200 g of sample and suspension is homogenized with
polytron.
• Extraction
• weigh 60 grams of above solution and taken in 250 ml flat
bottom amber glass flask.
• Add 1 ml sodium cyanide solution.
• If sample contain starch add 0.05g of alpha amylase .
HPLC method of analysis

33. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 33
• Mix thoroughly and incubate for 15 minutes .
• Add 25 ml of sodium acetate solution and place the flask in
boiling water bath.
• Quantitatively transfer the content of the flask into 100 ml
ambered volumetric flask.
• Filter the content and pass through immunoaffinity
column
• Analysis:
• Make an injection of standard and test solution.
• Measure Chromatographic peak responce.
HPLC method of analysis

34. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 34
• Chromatographic conditions:
Flow rate 0.4 ml/min
Injection volume 50 micro liters
Detector UV
Detection wave length 361 nm
Mobile phase A-1000ml water+TFA 250ml
B-1000mlacetonytril+TFA 250ml
Elution Gradient
HPLC method of analysis

35. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 35
• Identification:
Identify B12 peak in chromatogram of test solution
comparing with that
retention time and UV spectrum of corresponding peak of
obtained from standard.
Quantification :
HPLC method of analysis

36. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 36
Where A=Response of peak obtained for the sample.
I=Intercept of calibration curve.
S= slope of calibration curve.
V0= volume of test solution in ml.
V2=Volume in which aliquite of sample
Solution from immunoaffinity cleanup.
m= weight of test portion in grams.
V1=Volume of aliquot of sample solution
loaded into affinity column.
HPLC method of analysis

37. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 37
• Bishoni kapil,kataria Mahesh, Microbial assay of vitamin B,
international resurch jonoural of pharmacy, 2012,3(2),
p.no-74-81.
• Garrat,D.C.,The quantitative analysis of drugs 2nd
edition.India,vallabh prakashan, 2001,p.no-813-825.
• Jain,N.K..Pharmaceutical microbiology, 2nd edition. India
Macmillan ltd,New Delhi.p.no-37-107.
• Esther Campos Gimenez., Improved AOAO first action
2011.08 for the analysis of vitamin B12, Journal of AOAC
international, 2014,vol.37 no -5 ,p.no-3,4,5.
References

38. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 38