Vitamin assay by Microbiology

1.0 PURPOSE

This procedure ensures that the Microbiological assay of Vitamins is conducted as per Pharmacopoeial Specification.

2.0 SCOPE

This procedure covers assay of Vitamins by Microbiological method.

3.0 RESPONSIBILITY

Head – Quality control

Microbiologist

Assistants

4.0 PROCEDURE (Cyanocobalamin, Folic Acid, Calcium Pantothenate and Biotin assays)

The following procedure is provided for the determination of vitamins as an ingredient in multi-vitamin preparations. In this method assays were conducted in duplicate series of six level assay.

4.1 Cleaning of Glassware

All glass wares including test tubes, volumetric flasks, buretes. pipettes. conical flasks. glass rods should be soaked in chromic acid over night.

4.2 Chromic Acid Preparation

Take 200gm of Sodium dichromate (or) Potassium dichromate in a 2000ml conical flask, dissolve with 100ml of water and add carefully 1500ml of concentrated sulphuric acid through the sides of the flask. Wear safety gloves during this preparation. when the mixture acquires a green colour , it should be discarded under continuously following water. The glassware should be soaked in chromic acid mixture overnight and then ,rinsed with tap water, then with demineralized water, followed by distilled water.

Note:

Under any circumstances, the glass wares of Vitamins should not be mixed with that of antibiotics.

4.3 Transferring of cultures

The following cultures are used for analysis.

S.No---Name of the organisms---NCIM No.----Analysis of

01. Lactobacillus leichmannii---2071---Cyanocobalamin

02. Streptococcus faecalis---2080---Folic acid

03. Lactobacillus plantarum---2083--Calcium Pantothenate and Biotin

These cultures are maintained in stabs. Dissolve the Specified amount of culture medium (for 100ml) in a conical flask, add the required amount of water and heat the mixture on a water bath to dissolve. Pour approximately 5ml portions of this media in test tubes, plug these tubes with cotton and sterilise at 121C for 15 minutes. After sterilisation cool the tubes immediately in an upright position. Label these tubes neatly and store in the refrigerator. Each culture should be transferred once in two days under aseptic condition. Before culture transfer, the laminar flow bench should be wiped with 70% v/vIPA, and the person transferring the cultures should wash hands with antiseptic solutions. During inoculation the inoculation needle should be properly sterilised by flame. For that hold the needle in a vertical position, to immerse the needle in the flame wait for a few seconds, so that the full needle becomes red hot and then turn the needle to horizontal position and heat again. This should be repeated twice or thrice to sterilise the needle perfectly. To inoculate the tubes, hold the charged sterilised needle in right hand and the culture media on the left hand. Hold the culture in a nearly horizontal position close to the f1ame.

Remove the cotton plug of the tube by grasping it with the little finger of the hand holding the needle. Flame the mouth of the culture tube and rotate it to kill any organism that may fall in the culture. After flaming, insert the hot needle along with the side of the tube until the needle is cool and then insert the needle into the culture stab. Remove the needle from the tube and again flame the mouth of the tube, with the needle being close to the flame. Take the stab in which the culture to be transferred and after flaming the mouth of the tube, allow the inoculating needle to stab the solid media. A maximum of 3 stabs can be made in a tube. Flaming the mouth of the tube after inoculation is essential and important. Sterilise the needle properly before transferring other cultures. Incubate the transferred new culture at 37C for 16 to 24 hours. This is known as stock culture. Fresh stab cultures should be prepared for assay and do not use them for preparing lnoculum if more than 4 days old.

4.4 Preparation of Inoculum Broth

Inoculum broth should be prepared on the previous day of the assay. For this take the required amount of dehydrated inoculum broth media in a conical flask and add 20ml of hot water to dissolve it. Pour 10ml portions of this media in two test tubes and sterilise at 121C for 15 minutes. After cooling it transfer the culture from the stab to the broth by using the sterile needle.

4.5 Previous day of the assay

All the Glass apparatus should be clean and neat. Prepare the inoculum broth, sterilise it transfer the culture and incubate it. Prepare for each assay, normal saline (0.9% w/v Sodium Chloride) of about 150ml in a 250ml conical flask, from this take 50ml, and add 10ml each in 5 test tubes pack properly with cellophane paper and sterilise it. Pack properly with cellophane paper one burette for each assay and sterilise. Pack centrifuge tubes two numbers for each assay and sterilise. Make arrangement for the sufficient sterile test tubes. (For each standard preparation 16 test tubes and for one batch for each assay 4 test tubes are required).

Bring the distilled water from water plant. During dilution care should be taken to use the water properly without contaminating it. While making up water should be transferred from a clean conical flask, so that the mouth of the conical flask should not touch the rim of the standard flask. After each dilution wash the hands with antiseptic solution.

4.6 Medias and Reagents Required

4.6.1 Media

(i)For Cyanocobalamin -- B12 Culture Agar, broth and Assay media.

(ii)For Folic Acid -- Folic acid Culture Agar , Inoculum broth and Assay media.

(iii)For Calcium Pantothenate -- Calcium Pantothenate Agar, Inoculum broth and Assay media.

(iv) For Biotin -- Calcium Pantothenate culture Agar, Inoculum broth and Biotin assay media.

4.6.2 Reagents

4.6.2.1 Aqueous Extracting Solution for Cyanocobalamin

Disodium Phosphate - 1.29gm

Citric Acid anhydrous - 1.1gm

Sodium metabisulphite - 1.0gm

Water - 100ml

4.7 for Folic acid

Dilute Ammonia solution

4.8 For Calcium pantothenate

(i)0.2 N Acetic acid

(ii) 1.66% w/v Sodium acetate solution

4.9 Preparation of Assay Media

Weigh required amount of Dehydrated assay media in a clean butter paper and transfer to a clean conical flask. During weighing the balance and pan should be cleaned and two or three butter paper should be kept over the pan in order to avoid chemical contamination. The conical flask that is going to be used should be rinsed two or three times with water. Hot boiling water should be added to the media to dissolve it completely. Arrange clean, Sterile test tubes in a stand separately for each assay as follows: B, 0, 1.0, 1.5, 2.0, 3,0, 4.0, and 5.0 in duplicates where 'B' is the uninoculated blank, and ‘0’ is the inoculated blank. For sample take 4 test tubes per batch marked as 1.0, 2.0, 3.0. and 4.0 without any duplicate for each assay. Transfer the standard and sample solution of required amount to the respective tubes and make up all the test tubes to 5 ml with water. To the tubes 'B' and ‘0’ add 5ml of water. To all these tubes add 5ml of assay media including the blanks. Plug the tubes with cotton and then cover with cellophane paper and sterilize at 121C for 5 minutes. Care should be taken to maintain the pressure and temperature for exactly 5 minutes. After sterilisation, cool the tubes immediately, to avoid the colour formation, in ice water taken in a plastic trays. The tubes are now ready for inoculation.

4.10 Preparation of Inoculum

Transfer the broth culture, which was incubated overnight, to centrifuge tubes, near the flame and centrifuge for 15 minutes. Discard the supernatant liquid and slowly tap the centrifuge tube with the thumb, so that it comes in to solution . Wash these cells five times with saline. After the final washing is over, transfer the cells in to the conical flasks containing sterile saline. These diluted broth culture is referred to as the inoculum. Take these inoculum in a sterile 10ml burette under aseptic conditions. Keep the burette stopper open, so that the inoculum flows in drops and inoculate the tubes by adding uniformly one drops of inoculum and flame the mouth of tube before and after Inoculation. Do not Inoculate the first blank 'B'. After the inoculation shake the tubes individually and keep in the incubator at 37 + 0.5degC for 16 to 24 hours. After incubation, measure the transmittance at a wave length of 530nm. With the transmittance set 100 for the uninoculated blank (B) take the transmittance of the inoculated blank (0), with the transmittance set at 100 for the inoculated blank (o), take the transmittance for the remaining tubes.

4.11 ASSAY FOR CYANOCOBALAMIN

4.11.1Cyanocobalamin Standard Solution

Dissolve 25mg equivalent of Cyanocobalamin in 500ml of water. From this dilute 5ml to 200ml (1.25mcg per ml) with 25% v/v alcohol. This is stock solution, this should be prepared once in 30 days and store in a refrigerator. On the day of analysis when the stock solution comes to room temperature dilute 2ml to 100ml with water. From this dilute 10ml to 250ml with water. From this dilute 2ml to 100ml with water (0.02ng per ml).

SAMPLE DILUTIONS

4.11.1.1 Liquid (Claim 2.5mcg per 15ml)

Pipette 25 ml of sample in a 200ml volumetric flask, add 25ml of aqueous extracting solution and make up to volume with water. Dilute 10ml of the resulting solution to 250ml with water. From this dilute 2ml to 100ml with water.

4.11.1.2 Liquid (Claim 2.5mcg per 10ml)

Pipette 20ml sample in a 250ml volumetric flask, add 25ml of B12 extractions buffer, and make up to volume with water. From this dilute 10ml to 250ml with water. From this dilute 2ml to 100 ml with water.

4.11.1.3 Liquid (Claim 3mcg per 15ml)

Pipette 20ml sample in a 200ml volumetric flask, add 25ml of B12 extraction buffer, and make up to volume with water. From this dilute 10ml to 250ml with water. From this dilute 2ml to 100ml with water.

4.11.1.4 Syrup (Claim 5mcg per 10ml)

Pipette 5ml sample in a 200ml volumetric flask, add 25ml of B12 extractions buffer, and make up to volume with water. From this dilute 5ml to 100ml with water. From this dilute 1ml to 50 ml with water.

4.11.1.5 Syrup (Claim 5mcg per 10ml)

4.11.1.6 Syrup(Claim 1.0mcg per 10ml)

Pipette 15ml sample in a 100ml volumetric flask, add 25ml of B12 extractions buffer, and make up to volume with water. From this dilute 10ml to 250ml with water. From this dilute 2ml to 100 ml with water.

4.11.1.7 Capsules (Claim 2.5mcg per capsule)

Weigh accurately about 615mg of powdered capsules (equivalent to 4 mcg of B12) in a 200ml volumetric flask add 25ml of B12 extraction buffer, and make up to volume with water. From this dilute 10 ml to 250 ml with water. From this dilute 2 ml to 100 ml with water.

4.11.1.8 Capsules (Claim 2.5mcg per capsule)

Weigh accurately about 800mg of powdered capsules (equivalent to 4 mcg of B12) in a 200ml volumetric flask add 25ml of B12 extraction buffer, and make up to volume with water. From this dilute 10ml to 250ml with water. From this dilute 2ml to 100ml with water.

4.11.1.9 Capsule (Claim 5mcg per capsule)

Weigh accurately about 800mg of powdered capsules (equivalent to 4mcg of B12) in a 200ml volumetric flask, add 25ml of B12 extraction buffer, and make up to volume with water. From this dilute 10ml to 250ml with water. From this dilute 1ml to 100ml with water.

4.11.1.10 Capsules: (Claim 2.5 mcg per Capsule)

Weigh accurately about 960mg of powdered capsules (equivalent to 4mcg of B12) in a 200ml volumetric flask add 25ml of B12 extraction buffer, and make up to volume with water. From this dilute 10ml to 250ml with water. From this dilute 2ml to 100ml with water.

4.11.1.11 (Claim 5mcg per Capsule)

Weigh accurately about 300mg of powdered capsules in a 200ml volumetric flask, add 25ml of B12 extractions buffer, and make up to volume with water. From this dilute 10ml to 250ml with water. From this dilute 2ml to 100 ml with water.

4.11.1.12 Capsule (Claim 5mcg per 10ml)

4.11.1.13 Tablets (Claim 3 mcg per tablet)

Weigh accurately about 855mg (UCT) (or) 1.3gm (SCT) of powdered tablet (equivalent to 3.5mcg of B12) in a 200ml volumetric flask, add 25ml of B12 extraction buffer, and make up to volume with water. From this dilute 10ml to 250ml with water. From this dilute 2ml to 100ml with water.

4.11.1.14 Injection(Claim 5 mcg per ml)

Pipette 2ml of injection in a 250ml volumetric flask, add 25ml of B12 extraction buffer, and make up to volume with water. From this dilute 5ml to 250ml with water. From this dilute 2ml to 100ml with water.

4.11.1.15 Vitamin B Complex with B12 Injection (Claim 4mcg per ml)

Pipette 2ml of Injection in a 200ml volumetric flask, add 25ml of B12 extraction buffer, and make upto volume with water. Dilute 5ml to 250ml with water. From this dilute 2ml to 100ml with water.

4.12 ASSAY FOR FOLIC ACID

4.12.1 Folic acid Standard Solution

Take 25 mg equivalent of Folic Acid in a 200ml volumetric flask, add 5ml of dilute ammonia solution to dissolve, and make up to 200ml with water (125mcg per ml). This is stock solution keep this in the refrigerator. On the day of analysis, when the stock solution comes to room temperature, dilute 2ml to 200ml with water. From this dilute 2ml to 100 ml with water. From this dilute 2ml to 100ml with water (0.5ng per ml).

SAMPLE DILUTIONS

4.12.1.1 Liquid (Claim 0.75mg per 15ml)

Pipette 25ml of sample in a 200ml volumetric flask add 3 ml dilute ammonia solution and make upto volume with water. From this dilute 10ml to 250ml with water. From this dilute 2ml to 100ml with water. From this dilute 20ml to 250ml with water.

4.12.1.2 Liquid (Claim 0.5 mg per 10 ml)

Pipette 20ml of sample, in a 100ml volumetric flask, add 3ml of dilute ammonia solution and make up to volume with water. From this dilute 5ml to 50ml with water. From this dilute 2ml to 100ml with water. From this dilute 2ml to 100ml with water.

4.12.1.3 Liquid(Claim 0.5 mg per 15ml)

Pipette 20 ml of sample in a 100 ml volumetric flask, add 3ml of dilute ammonia solution and make up to volume with water. From this dilute 10ml to 250ml with water. From this dilute 2ml to 100ml with water. From this dilute 20ml to 250ml with water.

4.12.1.4 Syrup (Claim 0.5 mg per 10 ml)

Pipette 15ml of sample, in a 100ml volumetric flask, add 3ml of dilute ammonia solution and make up to volume with water. From this dilute 5ml to 50ml with water. From this dilute 2ml to 100ml with water. From this dilute 2ml to 100ml with water.

4.12.1.5 Capsules (Claim 0.75 mg per capsule) (or) Emvit Capsules (0.5mg per capsule)

Weigh accurately about 615 mg of powdered capsule (equivalent to 1.23mg of Folic Acid) in a 200ml volumetric flask, add 3ml of dilute ammonia solution and make up to volume with water. From this dilute 10ml to 250ml with water. From this dilute 2ml to 100ml with water. From this dilute 20 ml to 250ml with water.

4.12.1.6 Capsules (Claim 0.5mg per Capsule)

Weigh accurately about 1.95gm of powdered capsule (equivalent to 1.5mg of Folic Acid) in a 100ml volumetric flask, add 3ml of dilute ammonia solution, and make up to volume with water. From this dilute 5ml to 100ml with water. From this dilute 2 ml to 100 ml with water. From this dilute 2ml to 100ml with water.

4.12.1.7 Capsules (Claim 0.75mg per capsule)

Weigh accurately about 825mg of powdered capsule (equivalent to 1.3mg of Folic acid) in a 200ml volumetric flask, add 3ml of dilute ammonia solution, and make up to volume with water. From this dilute 10ml to 250ml with water. From this dilute 2ml to 100ml with water. From the dilute 20ml to 250ml with water.

4.12.1.8 Capsules (Claim 0.5 mg per capsules)

Weigh accurately about 2.0gm of powdered capsule (equivalent to 1.5mg of Folic Acid) in a 100ml volumetric flask, add 3ml of dilute ammonia solution, and make up to volume with water. From this dilute 5ml to 100ml with water. From this dilute 2ml to 100ml with water. From this dilute 2ml to 100ml with water.

4.12.1.9 Tablet (Claim 0.5mg per Tablet)

Weigh accurately about 920mg (UCT) or 1.4 gm (SCT) of powdered tablet (equivalent to 0.65 mg of Folic Acid) in a 100ml volumetric flask, add 3ml of dilute ammonia solution, and make up to volume with water. From this dilute 10ml to 250ml with water. From this dilute 2ml to 100ml with water. From this dilute 20ml to 250ml with water.

4.13 ASSAY FOR CALCIUM PANTOTHENATE

4.13.1Calcium Pantothenate Standard Solution

Take 25mg equivalent of Calcium Pantothenate in a 250ml volumetric flask and make up to volume with water (100mcg per ml). This is stock solution. Keep this in the Refrigerator. On the day of analysis, when the stock solution comes to room temperature, dilute 5ml to 200ml with water. From this dilute 2ml to 200ml with water (0.025mcg per ml).

4.13.1.1 Liquid (Claim 5mg per 15ml)

Pipette 15ml of sample in a 200ml volumetric flask, and make up to volume with water. From this dilute 5ml to 200ml with water. From this dilute 2 ml to 100ml with water.

4.13.1.2 Liquid (Claim 5mg per 15ml)

Pipette 10ml of sample in a 250ml volumetric flask, and make up to volume with water. From this dilute 10ml to 250ml with water. From this dilute 5 ml to 200ml with water.

4.13.1.3 Capsules (Claim 5 mg per capsule)

weigh accurately about 450 mg of powdered capsule (equivalent to 8.2mg of Calcium Pantothenate) in a 200ml volumetric flask, and make up to volume with water. From this dilute 5ml to 200ml with water. From this dilute 2ml to 100ml with water.

4.13.1.4 Capsule (Claim 5mg per capsule)

Weigh accurately about 615mg of powdered capsule (equivalent to 8.2mg of Calcium Pantothenate) in a 200ml volumetric flask, and make up to volume with water. From this dilute 5ml to 200ml with water. From this dilute 2ml to 100ml water.

4.13.1.5 Capsule (Claim 5mg per capsule)

Weigh accurately about 245mg of powdered capsule (equivalent to 8.2mg of Calcium Pantothenate) in a 200ml volumetric flask, and make up to volume with water. From this dilute 10ml to 250ml with water. From this dilute 5ml to 200ml water.

4.13.1.6 Capsule (Claim 5mg per capsule)

4.13.1.7 Tablet (Claim 1mg per tablet)

Weigh accurately about 780mg of powdered tablet (equivalent to 1mg of Calcium Pantothenate) in a 100ml volumetric flask and make up to volume with water. From this dilute 5ml to 200ml with water. From this dilute 2ml to 100ml with water.

4.13.1.8 Vitamin B-Complex Tablet (Prophylactic) (Claim 1 mg per tablet)

Weigh accurately about 260mg of powdered tablet (equivalent to 2mg of Calcium Pantothenate) in a 200ml volumetric flask and make upto volume with water. From this dilute 5ml to 200ml with water. From this dilute 10ml to 100ml with water

4.14 ASSAY FOR BIOTIN

4.14.1 Biotin Standard Solution

Take 25mg equivalent of Biotin in a 250ml volumetric flask add 125 ml of water and keep the flask on the direct flame. Allow the solution to boil ,then cool and allow the temperature to come down to room temperature then add 125 ml of alcohol .Now cool to room temperature and make up to volume with water (100mcg per ml). This is stock solution. Keep this in the Refrigerator. On the day of analysis, when the stock solution comes to room temperature dilute 2ml to 200ml with water. From this dilute 2ml to 200ml with water. From this dilute 10ml to 250ml with water. (0.4ng per ml).

SAMPLE DILUTIONS

4.14.1.1 Liquid (Claim 0.1 mg per 15ml)

Pipette 20ml of sample in a 100ml volumetric flask and make up to volume with water. From this dilute 5ml to 250ml with water. From this dilute 2ml to 250ml with water.

4.14.1.2 Capsules (Claim 30mcg per capsule)

Weigh accurately about 780mg of powdered capsule in a 200ml volumetric flask, and make up to volume with water. From this dilute 10ml to 100ml with water. From this dilute 1ml to 100ml with water.

4.14.1.3 Tablet (Claim 0.1mg per Tablet)

Weigh accurately about 1.4gm (SCT) or 900mg (UCT) of powdered tablet(equivalent to 0.125mg of Biotin) in a 100ml volumetric flask and make up to volume with water. From this dilute 5ml to 250ml with water. From this dilute 2ml to 250ml with water.

5.0 ABBREVATIONS: -Nil-

6.0 REVISION HISTORY :