Method of Analysis for Vitamin E or Tocopheryl Acetate

1. Description

Almost white fine granular powder

2. Identification

Record I. R. absorption spectrum of the sample as KBr pellet, by taking 2-3 mg of sample in 150-200 mg of KBr, and compare it with similarly recorded the spectrum of vitamin E acetate working reference standard.

3. Loss on drying

Crush the powder quickly if the test substance is in form of large crystal, reduce the particle size to about 2 mm. Take accurately weighed 1 g substance in a clean and dried (for 30 minutes under the same conditions to be employed in the determination) preweighed glass stoppered shallow weighing bottle. Cover the stopper and gently shake to distribute test substance as evenly as practicable to a depth of about 5 mm and more than 10 mm height. Place the bottle in the oven and remove the cover and leave it in the oven. Dry the sample at 105 ± 2° for 4 hours. On opening the chamber, immediately close the bottle, transfer it to a desiccator and bring it to room temperature. Weigh the bottle and calculate the % of loss on drying as

% Loss on drying = ( Lx100 )/W

Where,

L: Loss of weight (g)

W: Weight of substance (g)

4. Acid Value

Dissolve about 2.0 g of the substance being examined, accurately weighed, in 50 ml of a mixture of equal volumes of ethanol (95 %) and ether, previously neutralized with 0.1 M potassium hydroxide to phenolphthalein solution. If the sample does not dissolve in the cold solvent, connect the flask with a reflux condenser and warm slowly, with frequent shaking, until the sample dissolves. Add 1ml of phenolphthalein solution and titrate with 0.1 M potassium hydroxide until the solution remains faintly pink after shaking for 30 seconds. Calculate the acid value from the expression.

Acid value = 5.61 n/w

Where n = the number of ml of 0.1M potassium hydroxide required;

w = the weight, in g, of the substance.

5. Free Tocopherol 

Weigh accurately about 0.5 g, dissolve in 100 ml of 0.25M ethanolic sulphuric acid, add 20 ml of water and 0.1 ml of a 0.25% w/v solution of diphenylamine in sulphuric acid and titrate with 0.01M ceric ammonium nitrate until a blue color is produced that persists for at least 5 seconds. Repeat the operation without the substance being examined. The difference between the titrations represents the amount of ceric ammonium nitrate required. Each ml of 0.01M ceric ammonium nitrate is equivalent to 0.002154 g of tocopherol

6. Heavy metals

Standard solution

Into a 50-ml Nessler cylinder pipette 1.0 ml of lead standard solution (1 ppm Pb) and dilute with water to 25 ml. Adjust with dilute acetic acid or dilute ammonia solution to a pH between 3.0 and 4.0, dilute with water to about 35 ml and mix.

Test solution

Weigh in a suitable crucible 1.0 g of the substance, add sufficient sulphuric acid to wet the sample, ignite carefully at a low temperature until thoroughly charred. Add to the charred mass 2 ml of nitric acid and 5 drops of sulphuric acid and heat cautiously until white fumes are no longer evolved. Ignite, preferably in a muffle furnace, at 500° to 600° , until the carbon is completely burnt off. Cool, add 4 ml of hydrochloric acid, cover, digest on a water-bath for 15 minutes, uncover and slowly evaporate to dryness on a water-bath. Moisten the residue with 1 drop of hydrochloric acid, add 10 ml of hot water and digest for 2 minutes. Add ammonia solution dropwise until the solution is just alkaline to litmus paper, dilute to 25 ml with water and adjust with dilute acetic acid to a pH between 3.0 and 4.0. Filter, if necessary, rinse the crucible and the filter with 10 ml of water, combine the filtrate and washings in a 50-ml Nessler cylinder, dilute with water to about 35 ml and mix.

Procedure

To each of the cylinders containing the standard solution and test solution respectively add 10 ml of freshly prepared hydrogen sulphide solution, mix, dilute to 50 ml with water, allow to stand for 5 minutes and view downwards over a white surface; the colour produced with the test solution is not more intense than that produced with the standard solution.

7. Microbial Limits

i) Total aerobic viable count

Media and buffer
I. Fluid Casein Digest-Soy Lecithin-Polysorbate 20 Medium

Pancreatic Digest of Casein: 20 g

Soy Lecithin: 5 g

Polysorbate 20: 40 mL

Water: 960 mL

Dissolve the pancreatic digest of casein and soy lecithin in 960 ml of water. heating in a water bath at 48° to 50° for about 30 minutes to effect solution. Add 40 ml of polysorbate 20. Mix and dispense as desired.

II. Sabouraud Chloramphenicol Agar Medium

Casein enzymic hydrolysate: 5.0g

Peptic Digest of animal: 5.0 g

Tissue: 5.0 g

Dextrose: 0.05 g

Agar: 15.00g

Water: 1000 ml

pH after sterilisation : 5.6 ± 0.2

III. Soybean-Casein Digest Agar Medium

Pancreatic Digest of Casein: 15.0g

Papaic Digest of Soybean Meal: 5.0 g

Sodium Chloride: 5.0 g

Agar: 15.0 g

Water: 1000 ml

pH after sterilisation : 7.3 ± 0.2

Procedure 

Dissolve 10 gm of the sample in Fluid Casein Digest-Soy Lecithin-Polysorbate 20 medium to make 100 ml. Dilute further if necessary, the fluid so that 1 ml will be expected to yield between 30 and 300 colonies. Pipet 1 ml of the final dilution onto each of two sterile petri dishes, Promptly add each dish 15 to 20 ml of Soybean-Casein Digest Agar Medium that previously has been melted and cooled to approximately 45°. Cover the petri dishes, mix the sample with the agar by tilting or rotating the dishes, and allow the contents to solidify at room temperature. Invert the petri dishes, and incubate for 48 to 72 hours. Following incubation, examine the plates for growth, count the number of colonies and express the average for the two plates in terms of the number of microorganisms per g or per ml of specimen. If no microbial colonies are recovered from the dishes representing the initial 1:10 dilution of the specimen, express the results as "less than 10 microorganisms per ml of specimen.

ii) Total Combined Molds and Yeasts Count

Procedure
Dissolve 10 g of the sample in Fluid Casein Digest-Soy Lecithin-Polysorbate 20 Medium to make 100 ml. Dilute further if necessary, the fluid so that 1 ml will be expected to yield between 20 and 80 colonies. Pipet 1 ml of the final dilution onto each of two sterile petri dishes, Promptly add each dish 15 to 20 ml of Sabouraud Chloramphenicol Agar Medium that previously has been melted and cooled to approximately 45°. Cover the petri dishes, mix the sample with the agar by tilting or rotating the dishes, and allow the contents to solidify at room temperature. Invert the petri dishes, and incubate for 5 to 7 days at 20° to 25°. Following incubation, examine the plates for growth, count the number of colonies and express the average for the two plates in terms of the number of microorganisms per g or per ml of specimen. If no microbial colonies are recovered from the dishes representing the initial 1:10 dilution of the specimen, express the results as "less than 10 microorganisms per g/ml of specimen.

iii) Test for Staphylococcus aureus and Pseudomonas aeruginosa

To 10 g of the sample add Fluid Soybean-Casein Digest Medium to make 100 mL, mix, and incubate. Examine the medium for growth, and if growth is present, use an inoculating loop to streak a portion of the medium on the surface of Vogel-Johnson Agar Medium (or Baird-Parker Agar Medium, or Mannitol-Salt Agar Medium) and of Cetrimide Agar Medium, each plated on petri dishes. Cover and invert the dishes, and, incubate. If upon examination, none of the plates contains colonies having the characteristics listed in Tables 1 and 2 for the media used, the test specimen meets the requirements freedom for Staphylococcus aureus and Pseudomonas aeruginosa.

Confirm any suspect colonial growth on one or more of the media as Pseudomonas aeruginosa by means of the oxidase test. Upon the colonial growth place or transfer colonies to strips or disks of filter paper that previously has been impregnated with N,N-dimethyl-p-phenylenediamine dihydrochloride: if there is no development of a pink color, changing to purple, the specimen meets the requirements of the test for the absence of Pseudomonas aeruginosa. The presence of Pseudomonas aeruginosa may be confirmed by other suitable cultural and biochemical tests, if necessary.

Table – 1: Morphologic characteristics of Staphylococcus aureus

on selective agar media

Selective Medium	Characteristic colonial morphology	Gram stain
Vogel-Johnson Agar Medium	Black surrounded by yellow zone	Positive cocci (in clusters)
Mannitol-Salt Agar Medium	Yellow colonies with yellow zones	Positive cocci (in clusters)
Baird-Parker Agar Medium.	Black, shiny, surrounded by clear zones 2 to 5 mm	Positive cocci (in clusters)

Table – 2: Morphologic characteristics of pseudomonas aeruginosa

on selective and diagnostic agar media

Selective Medium	Characteristic colonial morphology	Fluorescence in ultraviolet light	Oxidase test	Gram stain
Cetrimide Agar Medium	Generally greenish	Greenish	Positive	Negative rods
Pseudomonas Agar Medium for Detection of   Fluorescin	Generally colorless to yellowish	Yellowish	Positive	Negative rods
Pseudomonas Agar Medium for Detection of Pyocyanin	Generally greenish	Blue	Positive	Negative rods

Coagulase Test (For Staphylococcus Aureus)

With the aid of inoculating loop, transfer representative suspect colonies from the surfaces of the Vogel-Johnson Agar Medium (or Baird-Parker Medium. or Mannitol-Salt Agar Medium) to individual tubes, and containing 0.5 ml of mammalian, preferably rabbit or horse, plasma with or without suitable additives. Incubate in a water bath at 37°, examining the tubes at 3 hours and subsequently at suitable intervals up to 24 hours. Test positive and negative controls simultaneously with the unknown specimens. If no coagulation in any degree eg. the specimen meets the requirements of the test for the absorbance of Staphylococcus aureus.

Oxidase And Pigment Tests (For Pseudomonas Aeruginosa)

With the aid of an inoculating loop, streak representative suspect combines from the agar surface of Cetrimide Agar Medium on the agar infaces of Pseudomonas Agar Medium for Detection of Fluorescin and Pseudomonas Agar Medium for Detection of Pyocyanin contained in petri dishes. If numerous colonies are to be transferred. the surface of each plate into quadrants. each of which may be inoculated from a separate colony. Cover and invert the inoculated media and incubate at 35 ± 2° for not less than three days. Examine the streaked surfaces under ultraviolet light. Examine the plates to determine whether colonies having the characteristics listed in Table 2 are present.

8. Assay of Tocopheryl Acetate

Reagents
(1) Isopropyl alcohol: HPLC grade

(2) Acetonitrile: HPLC grade

(3) Water: HPLC grade

(4) Orthophosphoric acid: AR grade

Mobile phase 

Prepare a homogenous and degassed mixture of isopropyl alcohol, acetonitrile and 0.1M orthophosphoric acid in the ratio of 270 : 730 : 5.

Standard preparation 

Transfer about 50 mg of accurately weighed, vitamin E acetate WRS into a 50 ml- volumetric flask, dissolve with sonication and dilute to volume with methanol. Centrifuge to get a clear solution.

Sample preparation 

Transfer about 50 mg of accurately weighed sample into a 50 ml- volumetric flask, dissolve with sonication and dilute to volume with methanol. Centrifuge to get a clear solution.

Instrumental conditions 

Column : Lichrosphere RP-18, 250 x 4.0 mm, 5 µ (Merck)

Flow rate : 1.0 ml/min

Detector: UV at 284 nm

Injection volume: 20 µl

Runtime: 20 min

Procedure 

(1) Set up the chromatographic system as described under instrumental conditions.

(2) Determine the system precision by six replicate injections of vitamin E acetate standard preparation. The relative standard deviation should be not more than 2.0 %. The retention time of the vitamin E acetate peak is about 10 minutes. The column efficiency determined by the number of theoretical plates is not less than 3000 and the tailing factor is not more than 1.5 for the vitamin E acetate peak.

(3) Prepare samples, make injections and calculate mean area counts for each sample.

Calculation

Content of vitamin E acetate (% LC) =      AT x D x WS x P x 100    

                                                                 AS x WT x (100-L)  

                                                       

Where :

                        AT       =          Average area count of vitamin E acetate peak in test preparation

                        AS       =          Average area count  of vitamin E acetate peak in standard preparation

                       WS       =          Weight of vitamin E acetate WRS in mg

                       WT       =          Weight of sample in mg

                        D         =          Dilution factor

                        P          =          Percentage purity of vitamin E acetate WRS  on as is basis.

                        L          =          % Loss on drying

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