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Antioxidants

Antioxidants are development, manufacture and sold for use in feed, food and industrial users.

Oxidation is the major cause of deterioration of food and feed products which contain fats and oils, because it is one of the main causes of rancidity in these products. Oxidation is a "weak point" of almost all fats and vegetable oils, even those containing natural antioxidants.

The factors to consider, when discussing deterioration of fats and oils and methods of prevention, include biochemical processes (enzyme caused deterioration), chemical processes (oxygen caused deterioration or auto-oxidation), nature and condition of substrates, required storage time, light, temperature, etc.

Three types of additives are commonly used to prevent the deterioration of fats and oils: antioxidants, their synergists, and preservatives (antimicrobial agents). It should be noted that antioxidants and preservatives act independently of each other. 

Antioxidants are added to fats and oils in minute quantities, because of cost factors and regulatory requirements. Another reason is inversion; when an excess of antioxidants acts in the opposite way and actually promotes oxidation. Antioxidants provide an alternate path for oxidation which does not involve the substrate, e.g. fats and oils. The antioxidant does not function indefinitely; it is destroyed in the process.

Peroxide value is one of the most important criteria used to monitor the oxidation process. In the course of oxidation, the peroxide value first increases very slowly (the induction period). When this period ends, the peroxide value starts growing very rapidly, and signs of deterioration become evident. The efficacy of antioxidants can be expressed as a protective factor (PF):

         Induction period with antioxidant
PF  =  ________________________________
        Induction period without antioxidant

This formula shows that the role of antioxidants is to extend the induction period and thus prolong the product's shelf life.

The induction period varies for different fats and oils depending on their chemical composition. Even for the same fats and oils, it can vary, depending on such pro-oxidative factors as light, temperature, metal traces, size of contact surface between air and fats, etc.

Very often, when added in allowable concentration levels, antioxidants fail to protect fats and oils from oxidation. Fortunately their action can be improved by the addition of synergists.

Synergists have little or no activity alone, but enhance the activity of true antioxidants. The most common synergists are phosphoric acid, citric acid, ascorbic acid and ascaorbyl palmitate. The main function of synergists is to form complexes with the pro-oxidative metal traces that are found in the most fats and oils. Some synergists also may participate in regeneration of exhausted antioxidants. The antioxidative action is not reduced until both antioxidant and synergist are completely consumed.

Obviously, there is no magical antioxidant formulation to solve the oxidation problem for all fats and oils. Some of the antioxidants manufactured and used in formulations are:

BHA / BHT / Ethoxyquin / Propyl Gallate / TBHQ / Tocopherols (See sections below)

THE USE OF ANTIOXIDANTS AND ANTIMICROBIALS IN FOOD PRESERVATION

The are two basic ways in which food products can spoil, or undergo an unwanted chemical change:
     1. spontaneous oxidation by atmospheric oxygen
     2. chemical reactions catalyzed by microbes
These can include all of biochemistry, hydrolysis, reduction, and not surprisingly oxidation as well (Hius in't Veld, Leninger).

Food preservation strategies follows the same logic. Some methods prevent oxidation by adding antioxidants to be product, others act by eliminating the microbes by heating, irradiation or antimicrobials. The best methods are those which address both sides of the problem: hermetic sealing of sterilized food and freezing. The last two strategies provide a way to prevent chemical changes for a long time, but tend to be expensive. Since ancient times humanity learned many ways to address the two sides of food preservation issue inexpensively: if milk is going to sour anyway - control the process and find uses for sour milk. Fruit will both oxidize and rot -  preserve it by increasing the concentration of acid or sugar (or both) until neither reaction occur anymore. However in the process, instead of preserving the original substance, a new food was created. This is not always an option. A more balanced way of preserving food can be found using modern knowledge of chemistry.

Antioxidant use alone provides no antibacterial action. Moreover, antioxidants improve the environment for bacterial action, because reactive oxygen species are harmful to microorganisms (Kim et al, Hayashi et al, Stephens et al). Addressing both sides of the food spoilage problem, addition of a natural antimicrobial substance may not only help prevent bacterial growth but increase the effectiveness of the antioxidant. Coca Cola company is adding Phosphoric acid to its trademark beverage for that reason.

When an abundant source of food is encountered, bacteria may form communities within  a matrix secreted by the bacteria. More than one species may be included. This biofilm provides a relatively protected environment for the bacteria (March et al). One of the goals of food preservation is to prevent the formation of dense bacterial populations. Isolated pockets of bacterial growth may degrade food even if most of the food is clean and adequately treated with antioxidants. This is another reason why initial antimicrobial treatment of food and antioxidation alone may not be sufficient. A combination of antioxidant and bacteriostatic additives provides an optimal solution to the problem of preservation of sensitive substances such as fat and oils.

References 

Coel, M., "Ethoxyquin: Science vs. Marketing", Petfood Industry, Sept.-Oct. 1995:8.

Sherwin, E. R., "Antioxidants for Vegetable Oils", J. Am. Oil Chem. Soc., 53:430 (1976).

Cort, W. M., "Antioxidant Activity of Tocopherols, Ascorbyl Palimate and Their Mode of Action", J. Am. Oil Chem. Soc. 51:322 (1974).

Allen, J. C. and Hamilton R. J., "Rancidity in Foods", Applied Science, Elsevier (1989).

Hudson B. J. F., "Food Antioxidants", Applied Science, Elsevier (1990).

Leninger textbook of biochemistry

Iowa State University food safety consortium web site: www.foodsafery.iastate.edu 


BHA

BHA can easily by applied to food items due to its outstanding solubility in fats and oils. BHA adds excellent stability to food products, fats, oils, vitamins, and pet foods.

Butylated Hydroxyanisole

C11H16O2
Formula wt 180.25
INS: 320
CAS: [25013-16-5]

Description
BHA is predominately 3-tert-buryl-4-hydroxyanisole (3-BHA), with varying amounts of 2-tert-butyl-4-hydroxyanisole (2-BHA). It occurs as a white or slightly yellow, waxy solid having a faint characteristic odor. It is insoluble in water, but is freely soluble in alcohol and in propylene glycol. It melts between 48o and 63o.

Functional Use in Foods: Antioxidant.

Requirements
Identification: To 5 mL of a 1 in 10,000 solution of the sample in 72% alcohol add 2 mL of sodium borate TS and 1 mL of a 1 in 10,000 solution of 2,6-dichloroquinonechlorimide in absolute alcohol, and mix. A blue color develops.
Assay: Not less than 98.5% of C11H16O2.
Heavy Metals (as Pb): Not more than 10 mg/kg.
Residue on Ignition: Not more than 0.05%.

Tests
Assay: 
Internal Standard Solution:    Dissolve about 500 mg of 4-tert-butylphenol, accurately weighed, in acetone in a 100-mL volumetric flask, add acetone to volume, and mix.
Standard Preparation:    Dissolve together, accurately weighed quantities of USP reference standards 3-tert-butyl-4-hydroxyanisole and 2-tert-butyl-4-hydroxyanisole to final concentrations of 9 mg/mL and 1 mg/mL, respectively, in Internal Standard Solution.
Assay Preparation:    Dissolve about 100 mg of Butylated Hydroxyanisole, accurately weighed, in the Internal Standard Solution in a 10-mL volumetric flask, dilute with the Internal Standard Solution to volume, and mix.
Chromatographic System:   The gas chromatograph is equipped with a flame-ionization detector and contains a 1.8-m x 2-mm (id) stainless-steel column packed with 10% silicone GE XE-60; the column is maintained isothermally at a temperature between 175o and 185o, and helium is used as the carrier gas, at a flow rate of 30 mL/min. Chromatograph a sufficient number of injections of the Standard Preparation, and record the areas as directed under Procedure, to ensure that the relative standard deviation does not exceed 2.0% for the 3-tert-butyl-4-hydroxyanisole isomer and 6.0% for the 2-tert-butyl-4-hydroxyanisole isomer; the resolution between the isomers is not less than 1.3, and the tailing factor doesn't exceed 2.0.
Procedure:    Separately inject suitable portions (about 5 mL) of the Standard Preparation and the Assay Preparation into the gas chromatograph, and record the chromatograms. Measure the areas under the peaks for each isomer and the internal standard in each chromatogram, and calculate the quantity, (I), in mg, of each isomer in the Butylated Hydroxyanisole by the formula

I = 10 Cs ( Ru / Rs )

in which Cs is the concentration, in mg/mL, of the isomer in the Standard Preparation; Rs is the ratio of the area of the isomer to that of the internal standard in the chromatogram from the Standard Preparation; and Ru is the ratio of the area of the isomer to that of the internal standard in the chromatogram from the Assay Preparation. Calculate the weight, in mg, C11H16O2 in the Butylated Hydroxyanisole by adding the quantities of the two isomers.
Heavy Metals:    Prepare and test a 2-g sample as directed in Method II under the Heavy Metals Test, using 20 mg of lead ion (Pb) in the control (Solution A).
Residue on Ignition:    Ignite 10 g as directed in the general method, Appendix IIC.

Packaging and storage    Store in well-closed containers.

Reference: FCC IV


BHT

BHT offers excellent solubility in fats and oils and provides excellent stability in cooked foods. Used alone or in combination with other antioxidants. It improves stability to fats, oils, and cereals.

Butylated Hydroxytoluene
2,6-Di-tert-butyl-p-cresol

C15H24O
Formula wt 220.35
INS: 321
CAS: [128-37-0]

Description
A white crystalline solid having a faint characteristic odor. It is insoluble in water and in propylene glycol, but is freely soluble in alcohol

Functional Use in Foods: Antioxidant.

Requirements
Identification: To 10 mL of a 1 in 100,000 solution of the sample in methanol add 10 mL of water, 2mL of sodium nitrite solution (3 in 1000), and 5 mL of dianisidine solution (200 mg of 3,3'-dimethoxybenzidine dihydrochloride dissolved in a mixture of 40 mL of methanol and 60 mL of 1 N hydrochloric acid). An orange-red color develops within 3 min. Add 5 mL of chloroform, and shake. The chloroform layer exhibits a purple or magenta color that fades when exposed to light.
Assay: Not less than 99.0% of C15H24O.
Heavy Metals (as Pb): Not more than 10 mg/kg.
Residue on Ignition: Not more than 0.002%.

Tests
Assay:    Its solidification point is not lower than 69.2o, indicating a purity of not less than 99.0% of C15H24O.
Heavy Metals:    Prepare and test a 2-g sample as directed in Method II under the Heavy Metals Test, (Appendix IIIB) using 20 g of lead ion (Pb) in the control (Solution A).
Residue on Ignition:    Transfer a 50-g sample into a tared crucible, ignite until thoroughly charred, and cool. Moisten the ash with 1 mL of sulfuric acid, and complete the ignition by heating for 15-min periods at 800o 25o to constant weight.

Packaging and storage    Store in well-closed containers.

Reference: FCC IV


Ethoxyquin

The best known antioxidant for animal fats, fish oil and feedstuff. It is available in both liquid and dry form. 

Ethoxyquin
6-Ethoxy-1,2-dihydro-2,2,4-trimethylquinoline

C14H19NO
Formula wt, monomer 217.31
INS: 324
CAS: [91-53-2]

Description
Ethoxyquin is a mixture consisting predominantly of the monomer (C14H19NO). It occurs as a clear liquid that may darken with age without affecting its antioxidant activity. Its specific gravity is about 1.02, and its refractive index is about 1.57.

Functional Use in Foods: Antioxidant.

Requirements
Identification: A solution of 1 mg of the sample in 10 mL of acetonitrile exhibits a strong fluorescence when viewed under short-wavelength ultraviolet light.
Assay: Not less than 92.0% of C14H19NO.
Heavy Metals (as Pb): Not more than 10 mg/kg.

Tests
Assay:    Transfer about 200 mg of the sample, accurately weighed, into a 150-mL breaker containing 50 mL of glacial acetic acid, and immediately titrate with 0.1 N perchloric acid in glacial acetic acid, determining the endpoint potentiometrically. Perform a blank determination and make any necessary correction. Each mL of 0.1 N perchloric acid is equivalent to 21.73 mg of C14H19NO(monomer).
Heavy Metals:    Prepare and test a 2-g sample as directed in Method II under the Heavy Metals Test, (Appendix IIIB) using 20 g of lead ion (Pb) in the control (Solution A).

Packaging and storage    Store in tightly closed carbon steel or black iron (not rubber, neoprene, or nylon) containers in a cool, dark place. Prolonged exposure to sunlight causes polymerization.

Reference: FCC IV


Propyl Gallate

The most effective naturally derived antioxidant for the food industry. Propyl Gallate is made from natural Gallic Acid, which is obtained by the hydrolysis of tannins from Tara pods. It exhibits excellent antioxidant activity in food and vegetable oils, especially in combination with Ascorbyl Palmitate.

Propyl Gallate
Gallic Acid, Propyl Ester

C10H12O5
Formula wt 180.20
INS: 310
CAS: [121-79-9]

Description
A fine, white to nearly white, odorless powder having a slightly bitter taste. It is slightly soluble in water and freely soluble in alcohol and in either.

Functional Use in Foods: Antioxidant.

Requirements
Identification: Place about 5 g of the sample and several boiling chips in a 500-mL round-bottom flask, connect a water-cooled condenser to the flask, and introduce a steady stream of nitrogen into the flask, maintaining the flow of nitrogen at all times during the remainder of the procedure. Pour 100 mL of 1 N sodium hydroxide through the top of the condenser, heat the solution to boiling, boil for 30 min, and cool. Place the reaction flask in an ice bath, and slowly, with occasional swirling, add dilute sulfuric acid (10%) until a pH of 2 to 3 is obtained (using pH paper). Filter the precipitate through a sintered-glass crucible, wash with a minimum amount of water, and dry at 110o for 2 h. The gallic acid so obtained melts at about 240o with decomposition.
Assay: Not less than 98.0% and not more than 102.0% of C10H12O5 after drying.
Heavy Metals (as Pb): Not more than 10 mg/kg.
Loss on Drying: Not more than 0.5%.
Melting Range: Between 146o and 150o.
Residue on Ignition: Not more than 0.1%.

Tests
Assay:    Transfer about 200 mg, previously dried at 110o for 4 h and accurately weighed, to a 400-mL beaker, dissolve it in 150 mL of water, and heat to boiling. With constant and vigorous stirring, add 50 mL of bismuth nitrate TS, continue stirring and heating until precipitation is complete, and cool. Filter the yellow precipitate on a tared sintered-glass crucible, wash it with cold dilute nitric acid (1 in 300), and dry at 110o to constant weight. The weight of the precipitate so obtained, multiplied by 0.4866, represents its equivalent of C10H12O5.
Heavy Metals:    Prepare and test a 2-g sample as directed in Method II under the Heavy Metals Test, (Appendix IIIB) using 20 g of lead ion (Pb) in the control (Solution A).
Loss on Drying:    Appendix IIC - Dry at 110o for 4 h.
Melting Range:    Determine as directed for Melting Range or Temperature, Appendix IIB, after drying at 110o for 4 h.
Residue on Ignition:    Appendix IIC - Ignite 2 g as directed in the general method.

Packaging and storage    Store in well-closed containers.

Reference: FCC IV


TBHQ

TBHQ is an antioxidant which offers excellent solubility in fats and oils. Used alone or in combination with other antioxidants it adds stability to fats, oils, cereals and packaging materials.

TBHQ
tert-Butylhydroquinone; Mono-tert-butylhydroquinone

C10H14O2
Formula wt 166.22
INS: 319
CAS: [1948-33-0]

Description
A white, crystalline solid having a characteristic odor. It is soluble in alcohol and in ether, but is practically insoluble in water.
Functional Use in Foods: Antioxidant.

Requirements
Identification: Dissolve a few mg of the sample in 1 mL of methanol, and add a few drops of a 25% solution of dimethylamine in water. A pink to red color is produced.
Assay: Not less than 99.0% of C10H14O2.
t-Butyl-p-benzoquinone: Not more than 0.2%
2,5-Di-t-butylhydroquinone: Not more than 0.2%
Heavy Metals (as Pb): Not more than 10 mg/kg.
Hydroquinone: Not more than 0.1%.
Melting Range: Between 126.5o and 128.5o.
Toluene: Not more than 0.0025%.
Ultraviolet Absorbance (polynuclear hydrocarbons): Passes test.

Tests
Assay:    Transfer about 170 mg of the sample. previously ground to a fine powder and accurately weighed, into a 250-mL, wide-mouth Erlenmeyer flask, and dissolve in 10 mL of methanol. Add 150 mL of water, 1mL of 1 N sulfuric acid, and 4 drops of diphenylamine indicator (3 mg of p-diphenylaminesulfonic acid sodium salt per mL of 0.1 N sulfuric acid), and titrate with 0.1 N ceric sulfate to the first complete color change from yellow to red violet. Record the volume, in mL, of 0.1 N ceric sulfate required as V. Calculate the percentage of C10H14O2 in the sample, uncorrected for hydroquinone (HQ) and 2,5-di-tert-butylhydroquinone (DTBHQ), by the formula

8.311N ( V - 0.1 mL ) / W

in which 0.1 mL represents the volume of ceric sulfate solution consumed by the primary oxidation products of tert-Butylhydroquinone ordinarily present in the sample; N is the exact normality of the ceric sulfate solution; and W is the weight of the sample taken, in g. Record the uncorrected percentage thus calculated as A. If HQ and DTBHQ are present in the sample, they will be included in the titration. Calculate the corrected percentage of C10H14O2 in the sample by the formula

A - ( % HQ x 1.51 ) - ( % DTBHQ x 0.75)

using the respective values for percentage of HQ and percentage of DTBHQ as determined under 2,5-Di-t-butylhydroquinone and Hydroquinone.
t-Butyl-p-benzoquinone:
Standard Preparation    Transfer about 10 mg of FCC Mono-tertiary-butyl-p-benzoquinone Reference Standard, accurately weighed, into a 10-mL volumetric flask, dissolve in chloroform, dilute to volume with the same solvent, and mix.
Sample Preparation    Transfer about 1 g of the sample, previously reduced to a fine powder in a high-speed blender and accurately weighed, into a 10-mL volumetric flask, dilute to volume with chloroform, and shake for 5 min to extract the t-butyl-p-benzoquinone. Filter through a Millipore filter or equivalent, before use in the Procedure below.
Procedure    Fill the reference cell with chloroform and the sample cell with the Standard Preparation, place the cell in the respective reference and sample beams of the spectrophotometer, and record the infrared spectrum from 1600 to 1775 cm-1. On the Spectrum draw a background line from 1612 to 1750 cm-1, and determine the net absorbance (AS) of the standard Preparation at 1659cm-1. Similarly, obtain the spectrum of the Sample Preparation, and determine its net absorbance (AU) at 1659 cm-1. Calculate the percentage of t-butyl-p-benzoquinone in the sample by the formula

100 x ( AU / AS ) x ( WS / WU )

in which WS is the exact weight, in mg, of the Reference Standard taken, and WU is the exact weight, in mg, of the sample taken.
2,5-Di-t-butylhydroquinone and Hydroquinone:
Stock Solutions    Weigh accurately about 50 mg each of hydroquinone (HQ), 2,5-di-t-butylhydroquinone (DTBHQ), and methyl benzoate (internal standard), transfer into separate 50-mL volumetric flasks, dilute to volume with pyridine, and mix.
Calibration Standards    Into separate 10-mL volumetric flasks add 0.50, 1.00, 2.00, and 3.00 mL of the HQ stock solution, then to each flask add 2.00 mL of the methyl benzoate (internal standard) stock solution, dilute each to volume with pyridine, and mix. In the same manner prepare four DTBHQ calibrating solutions. Prepare the trimethylsilyl derivative of each solution as follows: Add 9 drops of calibration solution to a 2-mL serum vial, cap the vial, evacuate with a 50-ml gas syringe, add 250 L of N,O-bis-trimethylsilylacetamide, and heat at about 80o for 10 min. Chromatograph 10-L portions of each standard in duplicate, and plot the concentration ratio of HQ to internal standard (X-axis) against the response ratio of HQ to internal standard (Y-axis). Plot the same relationships between DTBHQ and the internal standard.
Sample Preparation and Procedure    Transfer about 1 g of the sample, accurately weighed, into a 10-mL volumetric flask, add 2.00 mL of the methyl benzoate internal standard stock solution, dilute to volume with pyridine, and mix. Prepare the trimethylsilyl derivative as described above under Calibration Standards, and then chromatograph duplicate 10-L portions to obtain the chromatogram. The approximate peak times, in min, are methyl benzoate, 2.5; TMS derivative of HQ, 5.5; TMS derivative of tert-Butylhydroquinone, 7.3; TMS derivative of DTBHQ, 8.4.
Calculation    Determine the peak areas (response) of interest by automatic integration or manual triangulation. Calculate the response ratio of HQ and DTBHQ to internal standard. From the calibration curves determine the concentration ratio of HQ and DTBHQ to internal standard, and calculate the percentage of HQ and the percentage of DTBHQ in the sample by the formula

A = Y x I x 10 / S

in which A is the percentage of HQ or the percentage of DTBHQ in the sample; Y is the concentration ratio (X-axis on calibration curve); I is the percentage (w/v) of internal standard in the Sample Preparation; and S is the weight of sample taken, in g.
Packaging and storage    Store in well-closed containers.
Reference: FCC IV


Tocopherols

The most commonly used natural antioxidant for foodstuffs, pharmaceuticals and cosmetics. Natural tocopherols are obtained as an ancillary product in the production of vegetable oils. They are relatively mild compounds, suitable for use in food products, cosmetics, and those vegetable oils which have low tocopherol content.

Tocopherol

C29H50O2
Formula wt 430.71
INS: 307
CAS: [2074-53-5]

Natural Mixture of alpha, beta, gamma and delta tocopherols

Description
A form of vitamin E. It occurs as a yellow to amber, nearly odorless, clear, viscous oil that oxidizes and darkens in air and on exposure to light. It is insoluble in water, is freely soluble in alcohol, and is miscible with acetone, chloroform, ether, fats, and vegetable oils.

Functional Use in Foods: Nutrient; dietary supplement; antioxidant.

Requirements
Labeling: Label claims in terms of former International Units (IU) should be based on the following 1 mg DL-a-tocopherol = 1.1 IU
Identification: 
   A.     Dissolve about 10 mg of the sample in 10 mL of absolute alcohol, add with swirling 2 mL of nitric acid, and heat at about 75o for 15 min. A bright red to orange color develops.
   B.    The retention time of the major peak (excluding the solvent peak) in the chromatogram of the Assay Preparation is the same as that of the Standard Preparation, both relative to the internal standard, as obtained in the Assay.
   C.    If the isomeric form is not otherwise known, determine the optical rotation on a 1 in 10 solution of the sample in chloroform. The specific rotation is not appreciable (approximately +/- 0.05o).
Assay: Not less than 96.0% and not more than 102.0% of C29H50O2.
Heavy Metals (as Pb): Not more than 10 mg/kg.
Acidity: Passes test.

Tests
Note: In the following Assay, use low-actinic glassware for all solutions containing tocopherols.
Assay: 
Internal Standard Solution:    Prepare a solution in n-hexane containing 3 mg of hexadecyl hexadecanoate, accurately weighed, in each mL
Standard Preparation:    Dissolve about 30 mg of USP Alpha Tocopherol Reference Standard, accurately weighed, in 10.0 mL of the Internal Standard Solution.
Assay Preparation:    Dissolve about 30 mg of the sample, accurately weighed, in 10.0 mL of the Internal Standard Solution.
Chromatographic System:   Use a gas chromatograph equipped with a flame-ionization detector and a glass-lined sample-introduction system or on-column injection. Under typical conditions, the instrument contains a 2-m x 4-mm borosilicate glass column packed with 2% to 5% methylpolysiloxane gum on 80- to 100-mesh acid-base washed silanized chromatographic diatomaceous earth. The column is maintained isothermally between 240o and 260o, the injection port at about 290o, and the detector block at about 300o. The flow rate of dry carrier gas is adjusted to obtain a hexadecyl hexadecanoate peak approximately 10 to 20 min after sample introduction when a 2% column is used, or 30 to 32 min when a 5% column is used.
Note: Cure and condition the column as necessary.
System Suitability:    Chromatograph a sufficient number of injections of a mixture in n-hexane of 1 mg/mL each of USP Alpha Tocopherol Reference Standard and USP Alpha Tocopheryl Acetate Reference Standard, as directed under Calibration, to ensure that the resolution factor, R, is not less than 1.0.
Calibration:    Chromatograph successive 2- to 5-mL portions of the Standard Preparation until the relative response factor, F. is constant (i.e., within a range of approximately 2%) for three consecutive injections. If graphic integration is used, adjust the instrument to obtain at least 70% maximum recorder response for the hexadecyl hexadecanoate peak. Measure the areas under the major peaks occurring at relative retention times of approximately 0.51 (a-tocopherol) and 1.00 (hexadecyl hexadecanoate), and record the values as AS and AI, respectively. Calculate the relative response factor, F, by the formula

 ( AS / AI ) x ( CI / CS )

in which CI and CS are the exact concentrations, in mg/mL, of hexadecyl hexadecanoate and of USP Alpha Tocopherol Reference Standard in the Standard Preparation, respectively.
Procedure:    Inject a suitable portion (2 to 5 mL) of the Assay Preparation into the chromatograph, and record the chromatogram. Measure the areas under the major peaks occurring at relative retention times of approximately 0.51 (a-tocopherol) and 1.00 (hexadecyl hexadecanoate), and record the values as aU and aI, respectively. Calculate the weight, in mg, of DL-a-tocopherol in the sample by the formula

( 10CI / F ) x ( aU / aI )

Heavy Metals:    Place a 2-g sample in a silica crucible, and proceed as directed in Method II under the Heavy Metals Test, Appendix IIIB, using 20 mg of lead ion (Pb) in the control (Solution A).
Acidity:    Dissolve 1.0 g of the sample in 25 mL of a mixture of equal volumes of alcohol and ether that has been neutralized to phenolphthalein TS with 0.1 N sodium hydroxide, add 0.5 mL of phenolphthalein TS, and titrate with 0.1 N sodium hydroxide until the solution remains faintly pink after shaking for 30 s. Not more that 10 mL of 0.1 N sodium hydroxide is required.

Packaging and storage    Store in tight containers blanked by inert gas and protected from heat and light.

Reference: FCC IV


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