USP 37 Chemical Tests / 〈401〉 Fats and Fixed Oils 1

2 mL, a more dilute titrant may be used, or the sample size

may be adjusted accordingly. The results may be expressed
〈401〉 FATS AND FIXED OILS in terms of the volume of titrant used or in terms of the
equivalent volume of 0.1 N potassium hydroxide or 0.1 N
sodium hydroxide.
The following definitions and general procedures apply to If the oil has been saturated with carbon dioxide for the
fats, fixed oils, waxes, resins, balsams, and similar purpose of preservation, gently reflux the alcohol–ether so-
substances. lution for 10 min before titration. The oil may be freed from
carbon dioxide also by exposing it in a shallow dish in a
vacuum desiccator for 24 h before weighing the test
PREPARATION OF SPECIMEN specimens.

If a specimen of oil shows turbidity owing to separated Method II

stearin, warm the container in a water bath at 50° until the
oil is clear, or if the oil does not become clear on warming,
pass it through dry filter paper in a funnel contained in a Procedure—Prepare 125 mL of a solvent mixture consist-
hot-water jacket. Mix thoroughly, and weigh at one time as ing of equal volumes of isopropyl alcohol and toluene.
many portions as are needed for the various determinations, Before use, add 2 mL of a 1% solution of phenolphthalein in
preferably using a bottle that has a pipet dropper or a isopropyl alcohol to the 125-mL mixture, and neutralize
weighing buret. If the specimen is solid at room tempera- with alkali to a faint but permanent pink color. Accurately
ture, keep it melted until the desired portions of specimen weigh the appropriate amount of well-mixed liquid sample
are withdrawn. indicated in Table 1, and dissolve it in the neutralized sol-
vent mixture. If the test specimen does not dissolve in the
cold solvent, connect the flask with a suitable condenser
SPECIFIC GRAVITY and warm slowly, with frequent shaking, until the specimen
dissolves. Shake vigorously while titrating with 0.1 N potas-
Determine the specific gravity of a fat or oil as directed in sium hydroxide VS or 0.1 N sodium hydroxide VS to the
Specific Gravity 〈841〉. first permanent pink of the same intensity as that of the
neutralized solvent before mixing with the sample. Calculate
the Acid Value as indicated in Method I.
MELTING TEMPERATURE
Table 1
Determine the melting temperature as directed for Class II
substances (see Melting Range or Temperature 〈741〉). Sample Weight
Acid Value (g)
0–1 20
ACID VALUE (FREE FATTY ACIDS) 1–4 10
4–15 2.5
The acidity of fats and fixed oils in this pharmacopeia may
be expressed as the number of mL of 0.1 N alkali required 15–74.9 0.5
to neutralize the free acids in 10.0 g of substance. Acidity ≥75.0 0.1
frequently is expressed as the Acid Value, which is the num-
ber of mg of potassium hydroxide required to neutralize the
free acids in 1.0 g of the substance. Unless directed other-
wise in the individual monograph, use Method I. ESTER VALUE
The Ester Value is the number of mg of potassium hydrox-
Method I ide required to saponify the esters in 1.0 g of the substance.
If the Saponification Value and the Acid Value have been
Procedure—Unless directed otherwise, dissolve about determined, the difference between these two represents
10.0 g of the substance, accurately weighed, in 50 mL of a the Ester Value, i.e., Ester Value = Saponification Value – Acid
mixture of equal volumes of alcohol and ether (which has Value.
been neutralized to phenolphthalein with 0.1 N potassium Procedure—Place 1.5–2 g of the substance, accurately
hydroxide or 0.1 N sodium hydroxide, unless otherwise weighed, in a tared, 250-mL flask, add 20–30 mL of neu-
specified) contained in a flask. If the test specimen does not tralized alcohol, and shake. Add 1 mL of phenolphthalein
dissolve in the cold solvent, connect the flask with a suitable TS, and titrate with 0.5 N alcoholic potassium hydroxide VS
condenser and warm slowly, with frequent shaking, until until the free acid is neutralized. Add 25.0 mL of 0.5 N alco-
the specimen dissolves. Add 1 mL of phenolphthalein TS, holic potassium hydroxide VS, and proceed as directed in
and titrate with 0.1 N potassium hydroxide VS or 0.1 N Saponification Value, beginning with “Heat the flask” and
sodium hydroxide VS until the solution remains faintly pink omitting the further addition of phenolphthalein TS. Calcu-
after shaking for 30 s. Calculate either the Acid Value or the late the Ester Value:
volume of 0.1 N alkali required to neutralize 10.0 g of speci-
men (free fatty acids), whichever is appropriate. Calculate Result = [Mr × (VB − VT) × N]/W
the Acid Value:
Mr = molecular weight of potassium hydroxide, 56.11
Result = (Mr × V) × (N/W) VB = volume of 0.5 N hydrochloric acid consumed in the
blank test (mL)
Mr = molecular weight of potassium hydroxide, 56.11 VT = volume of 0.5 N hydrochloric acid consumed in the
V = volume (mL) actual test (mL)
N = normality of the potassium hydroxide solution or the N = exact normality of the hydrochloric acid
sodium hydroxide solution W = weight of the substance taken for the test (g)
W = weight of the sample taken (g)
If the volume of 0.1 N potassium hydroxide VS or 0.1 N
sodium hydroxide VS required for the titration is less than
2 〈401〉 Fats and Fixed Oils / Chemical Tests USP 37

HYDROXYL VALUE IODINE VALUE

The Hydroxyl Value is the number of mg of potassium The Iodine Value represents the number of g of iodine
hydroxide equivalent to the hydroxyl content of 1.0 g of the absorbed, under the prescribed conditions, by 100 g of the
substance. substance. Unless otherwise specified in the individual mon-
Pyridine–Acetic Anhydride Reagent—Just before use, ograph, determine the Iodine Value by Method I.
mix 3 volumes of freshly opened or freshly distilled pyridine
with 1 volume of freshly opened or freshly distilled acetic Method I (Hanus Method)
anhydride.
Procedure—Transfer a quantity of the substance, deter- Procedure—Transfer an accurately weighed quantity of
mined by reference to Table 2 and accurately weighed, to a sample, as determined from Table 3, into a 250-mL iodine
glass-stoppered, 250-mL conical flask, and add 5.0 mL of flask, dissolve it in 10 mL of chloroform, add 25.0 mL of
Pyridine–Acetic Anhydride Reagent. Transfer 5.0 mL of iodobromide TS, insert the stopper in the vessel securely,
Pyridine–Acetic Anhydride Reagent to a second glass-stop- and allow it to stand for 30 min protected from light, with
pered, 250-mL conical flask to provide the reagent blank. Fit occasional shaking. Then add, in the order named, 30 mL of
both flasks with suitable glass-jointed reflux condensers, potassium iodide TS and 100 mL of water, and titrate the
heat on a steam bath for 1 h, add 10 mL of water through liberated iodine with 0.1 N sodium thiosulfate VS, shaking
each condenser, and heat on the steam bath for 10 min thoroughly after each addition of thiosulfate. When the io-
more. Cool, and to each add 25 mL of butyl alcohol, previ- dine color becomes quite pale, add 3 mL of starch TS, and
ously neutralized to phenolphthalein TS with 0.5 N alcoholic continue the titration with 0.1 N sodium thiosulfate VS until
potassium hydroxide, by pouring 15 mL through each con- the blue color is discharged. Perform a blank test at the
denser and, after removing the condensers, washing the same time with the same quantities of the same reagents
sides of both flasks with the remaining 10-mL portions. To and in the same manner (see Titrimetry 〈541〉, Residual Titra-
each flask add 1 mL of phenolphthalein TS, and titrate with tions). Calculate the Iodine Value:
0.5 N alcoholic potassium hydroxide VS, recording the vol-
ume, in mL, consumed by the residual acid in the test solu- Result = [Ar × (VB − VS) × N]/(10 × W)
tion as T and that consumed by the blank as B. In a 125-mL
conical flask, mix about 10 g of the substance, accurately Ar = atomic weight of iodine, 126.90
weighed, with 10 mL of freshly distilled pyridine, previously VB = volume of 0.1 N sodium thiosulfate VS consumed
neutralized to phenolphthalein TS, add 1 mL of phenol- by the blank test (mL)
phthalein TS, and titrate with 0.5 N alcoholic potassium hy- VS = volume of 0.1 N sodium thiosulfate VS consumed
droxide VS, recording the volume, in mL, consumed by the by the actual test (mL)
free acid in the test specimen as A. Calculate the Hydroxyl N = exact normality of the sodium thiosulfate VS
Value: W = weight of the substance taken for the test (g)
NOTE—If more than half of the iodobromide TS is absorbed
Result = [(Mr × N)/W] × {B + [(W × A)/C] − T } by the portion of the substance taken, repeat the determi-
nation, using a smaller portion of the substance under
Mr = molecular weight of potassium hydroxide, 56.11 examination.
N = exact normality of the alcoholic potassium
hydroxide
W = weight of the substances taken for the acetylation Table 3. Sample Weights
(g) Iodine Value Weight (g)
C = weight of the substances taken for the free acid de- Expected ±0.1
termination (g) <5 3.0
If the Acid Value for the test substance is known, calculate 5–20 1.0
the Hydroxyl Value:
21–50 0.4
Result = [(Mr × N)/W] × (B − T) + Acid Value 51–100 0.2
101–150 0.13
Mr = molecular weight of potassium hydroxide, 56.11 151–200 0.1
N = exact normality of the alcoholic potassium
hydroxide
W = weight of the substance taken for the acetylation
(g) Method II

Table 2
Potassium Iodide Solution—Dissolve 10.0 g of potas-
sium iodide in water to make 100 mL. Store in light-resistant
Weight of containers.
Hydroxyl Value Test Specimen Starch Indicator Solution—Mix 1 g of soluble starch
Range (g) with sufficient cold water to make a thin paste. Add, while
0–20 10 stirring, to 100 mL of boiling water. Mix, and cool. Use only
20–50 5 the clear solution.
50–100 3 Procedure—Melt the sample, if it is not already liquid.
100–150 2 [NOTE—The temperature during melting should exceed the
150–200 1.5 melting point of the sample by NMT 10°.] Pass through two
200–250 1.25
pieces of filter paper to remove any solid impurities and the
last traces of moisture. The filtration may be performed in
250–300 1.0 an air oven at 100° but should be completed within 5 min
300–350 0.75 ± 30 s. The sample must be absolutely dry. All glassware
must be absolutely clean and completely dry. After filtration,
allow the filtered sample to achieve a temperature of
68°–71 ± 1° before weighing the sample. Once the sample
has achieved a temperature of 68°–71 ± 1°, immediately
weigh the sample into a 500-mL iodine flask, using the
USP 37 Chemical Tests / 〈401〉 Fats and Fixed Oils 3

weights and weighing accuracy noted in the accompanying trimetry 〈541〉, Residual Titrations). The titration also can be
table. [NOTE—The weight of the substance must be such carried out potentiometrically. Calculate the Saponification
that there will be an excess of iodochloride TS of 50%–60% Value:
of the amount added, that is, 100%–150% of the amount
absorbed.] Add 15 mL of a fresh mixture of cyclohexane Result = [Mr × (VB – VT) × N]/W
and glacial acetic acid (1:1), and swirl to dissolve the sam-
ple. Add 25.0 mL of iodochloride TS, insert the stopper se- Mr = molecular weight of potassium hydroxide, 56.11
curely in the flask, and swirl to mix. Allow it to stand at VB = volume of 0.5 N hydrochloric acid consumed in the
25 ± 5°, protected from light, with occasional shaking, for blank test (mL)
1.0 or 2.0 h, depending on the Iodine Value (IV) of the VT = volume of 0.5 N hydrochloric acid consumed in the
sample: IV < 150, 1.0 h; IV ≥ 150, 2.0 h. Then, within 3 min actual test (mL)
after the indicated reaction time, add, in the order named, N = exact normality of the hydrochloric acid
20 mL of Potassium Iodide Solution and 150 mL of recently W = weight of the substance taken for the test (g)
boiled and cooled water, and mix. Within 30 min, titrate If the oil has been saturated with carbon dioxide for the
the liberated iodine with 0.1 N sodium thiosulfate VS, while purpose of preservation, expose it in a shallow dish in a
stirring by mechanical means after each addition of thiosul- vacuum desiccator for 24 h before weighing the test
fate. When the yellow iodine color has almost disappeared, specimens.
add 1–2 mL of Starch Indicator Solution, and continue the
titration with 0.1 N sodium thiosulfate VS until the blue
color is discharged. Perform a blank test at the same time UNSAPONIFIABLE MATTER
with the same quantities of the same reagents and in the
same manner (see Titrimetry 〈541〉, Residual Titrations). Cal- The term “unsaponifiable matter” in oils or fats refers to
culate the Iodine Value as indicated in Method I. those substances that are not saponifiable by alkali hydrox-
ides but are soluble in the ordinary fat solvents, and to
products of saponification that are soluble in such solvents.
PEROXIDE VALUE Procedure—Transfer about 5.0 g of the oil or fat, accu-
rately weighed, to a 250-mL conical flask, add 50 mL of an
The Peroxide Value is the number that expresses, in millie- alcoholic potassium hydroxide solution prepared by dissolv-
quivalents of active oxygen, the quantity of peroxide con- ing 12 g of potassium hydroxide in 10 mL of water and di-
tained in 1000 g of the substance. [NOTE—This test must be luting this solution with alcohol to 100 mL, and heat the
performed promptly after sampling to avoid oxidation of flask on a steam bath under a suitable condenser to main-
the test specimen.] tain reflux for 1 h, swirling frequently. Cool to a tempera-
Procedure—Unless otherwise directed, place about 5 g ture below 25°, and transfer the contents of the flask to a
of the substance, accurately weighed, in a 250-mL conical separator with a polytetrafluoroethylene stopcock, rinsing
flask fitted with a ground-glass stopper. Add 30 mL of a the flask with two 50-mL portions of water that are added
mixture of glacial acetic acid and chloroform (3:2), shake to to the separator (do not use grease on stopcock). Extract
dissolve, and add 0.5 mL of saturated potassium iodide solu- with three 100-mL portions of ether, combining the ether
tion. Shake for exactly 1 min, and add 30 mL of water. Ti- extracts in another separator containing 40 mL of water.
trate with 0.01 N sodium thiosulfate VS, adding the titrant Gently rotate or shake the separator for a few minutes.
slowly with continuous shaking, until the yellow color is al- [NOTE—Violent agitation may result in the formation of a
most discharged. Add 5 mL of starch TS, and continue the difficult-to-separate emulsion.] Allow the mixture to sepa-
titration, shaking vigorously, until the blue color is dis- rate, and discard the lower aqueous phase. Wash the ether
charged. Perform a blank determination under the same extract with two additional 40-mL portions of water, and
conditions. [NOTE—The volume of titrant used in the blank discard the lower aqueous phase. Wash the ether extract
determination must not exceed 0.1 mL.] Calculate the Per- successively with a 40-mL portion of potassium hydroxide
oxide Value: solution (3 in 100) and a 40-mL portion of water. Repeat
this potassium hydroxide solution–water wash sequence
Result = [1000 (VT − VB) × N]/W three times. Wash the ether extract with 40-mL portions of
water until the last washing is not reddened by the addition
VT = volume of 0.01 N sodium thiosulfate consumed in of 2 drops of phenolphthalein TS. Transfer the ether extract
the actual test (mL) to a tared flask, and rinse the separator with 10 mL of ether,
VB = volume of 0.01 N sodium thiosulfate consumed in adding the rinsings to the flask. Evaporate the ether on a
the blank test (mL) steam bath, and add 6 mL of acetone to the residue. Re-
N = exact normality of the sodium thiosulfate solution move the acetone in a current of air, and dry the residue at
W = weight of the substance taken for the test (g) 105° until successive weighings differ by NMT 1 mg. Calcu-
late the percentage of unsaponifiable matter in the portion
of oil or fat taken:
SAPONIFICATION VALUE
Result = 100 × (WR/WS)
The Saponification Value is the number of mg of potas-
sium hydroxide required to neutralize the free acids and sa- WR = weight of the residue (g)
ponify the esters contained in 1.0 g of the substance. WS = weight of the oil or fat taken for the test (g)
Procedure—Place 1.5–2 g of the substance in a tared, Dissolve the residue in 20 mL of alcohol, previously neu-
250-mL flask, weigh accurately, and add to it 25.0 mL of tralized to the phenolphthalein endpoint, add phenolphthal-
0.5 N alcoholic potassium hydroxide. Heat the flask on a ein TS, and titrate with 0.1 N alcoholic sodium hydroxide
steam bath, under a suitable condenser to maintain reflux VS to the first appearance of a faint pink color that persists
for 30 min, frequently rotating the contents. [NOTE—Reflux for NLT 30 s. If the volume of 0.1 N alcoholic sodium hy-
time can be up to 90 min to ensure complete saponifica- droxide required is greater than 0.2 mL, the separation of
tion, depending on the type of ester to be tested.] Then the layers was incomplete; the residue weighed cannot be
add 1 mL of phenolphthalein TS, and titrate the excess po- considered “unsaponifiable matter,” and the test must be
tassium hydroxide with 0.5 N hydrochloric acid VS. Perform repeated.
a blank determination under the same conditions (see Ti-
4 〈401〉 Fats and Fixed Oils / Chemical Tests USP 37

SOLIDIFICATION TEMPERATURE OF FATTY Table 5 (Continued)

ACIDS Carbon- Number of
Chain Double
Preparation of the Fatty Acids—Heat 75 mL of glycer- Percentage Fatty Acid Ester Length Bonds
in–potassium hydroxide solution (made by dissolving 25 g 48.0 Methyl laurate 12 0
of potassium hydroxide in 100 mL of glycerin) in an 800-mL 15.0 Methyl myristate 14 0
beaker to 150°, and add 50 mL of the clarified fat, melted if 7.0 Methyl palmitate 16 0
necessary. Heat the mixture for 15 min with frequent stir- 3.0 Methyl stearate 18 0
ring, but do not allow the temperature to rise above 150°.
Saponification is complete when the mixture is homogene- 12.0 Methyl oleate 18 1
ous and no particles cling to the beaker at the meniscus. 3.0 Methyl linoleate 18 2
Pour the contents of the beaker into 500 mL of nearly boil-
ing water in an 800-mL beaker or casserole, add slowly 0.5 N Methanolic Sodium Hydroxide Solution—Dis-
50 mL of dilute sulfuric acid [made by adding water and solve 2 g of sodium hydroxide in 100 mL of methanol.
sulfuric acid (3:1)], and heat the solution, with frequent stir- Test Solution—[NOTE—If fatty acids containing more
ring, until the fatty acids separate cleanly as a transparent than 2 double bonds are present in the test specimen, re-
layer. Wash the acids with boiling water until free from sul- move air from the flask by purging it with nitrogen for a
furic acid, collect them in a small beaker, place on a steam few minutes.] Transfer about 100 mg of the test specimen
bath until the water has settled and the fatty acids are clear, to a 50-mL conical flask fitted with a suitable water-cooled
filter into a dry beaker while hot, and dry at 105° for 20 reflux condenser and a magnetic stir bar. Add 4 mL of 0.5 N
min. Place the warm fatty acids in a suitable container, and Methanolic Sodium Hydroxide Solution, and reflux until fat
cool in an ice bath until they congeal. globules disappear (usually 5–10 min). Add 5 mL of a solu-
Test for Complete Saponification—Place 3 mL of the tion prepared by dissolving 14 g of boron trifluoride in
dry acids in a test tube, and add 15 mL of alcohol. Heat the methanol to make 100 mL, swirl to mix, and reflux for 2
solution to boiling, and add an equal volume of 6 N ammo- min. Add 4 mL of chromatographic n-heptane through the
nium hydroxide. A clear solution results. condenser, and reflux for 1 min. Cool, remove the con-
Procedure—Using an apparatus similar to the Congealing denser, add about 15 mL of saturated sodium chloride solu-
Temperature Apparatus specified therein, proceed as directed tion, shake, and allow the layers to separate. Pass the n-
for Procedure in Congealing Temperature 〈651〉, reading “so- heptane layer through 0.1 g of anhydrous sodium sulfate
lidification temperature” for “congealing point” (the terms (previously washed with chromatographic n-heptane) into a
are synonymous). The average of NLT four consecutive read- suitable flask. Transfer 1.0 mL of this solution to a 10-mL
ings of the highest point to which the temperature rises is volumetric flask, dilute with chromatographic n-heptane to
the solidification temperature of the fatty acids. volume, and mix.
System Suitability Solution—Transfer about 20 mg each
of stearic acid, palmitic acid, and oleic acid to a 25-mL coni-
FATTY ACID COMPOSITION cal flask fitted with a suitable water-cooled reflux condenser
and a magnetic stir bar, and proceed as directed for Test
Standard Solution—Prepare an ester mixture of known Solution beginning with “Add 5.0 mL of a solution prepared
composition containing the esters required in the individual by dissolving”.
monograph. This Standard Solution may contain other com- Chromatographic System (see Chromatography 〈621〉)—
ponents. [NOTE—Ester mixtures are available commercially The gas chromatograph is equipped with a flame-ionization
from Nu-Chek-Prep, Inc., PO Box 295, Elysian, MN 56028. detector, maintained at a temperature of about 260°, a
Typical Nu-Chek-Prep ester mixtures useful in this test in- splitless injection system, and a 0.53-mm × 30-m fused-silica
clude Nu-Chek 17A and Nu-Chek 19A.] Nu-Chek mixture capillary column bonded with a 1.0-µm layer of phase G16.
17A has the following composition: The chromatograph is programmed to maintain the column
temperature at 70° for about 2 min after injection, then to
Table 4 increase the temperature at the rate of 5°/min to 240°, and
finally to maintain this temperature for 5 min. The injection
Carbon- Number of port temperature is maintained at about 220°. The carrier
Percent- Chain Double gas is helium with a linear velocity of about 50 cm/s.
age Fatty Acid Ester Length Bonds Chromatograph the System Suitability Solution, and record
1.0 Methyl myristate 14 0 the peak responses as directed for Procedure: the relative
4.0 Methyl palmitate 16 0 retention times are about 0.87 for methyl palmitate, 0.99
3.0 Methyl stearate 18 0 for methyl stearate, and 1.0 for methyl oleate; the resolu-
3.0 Methyl arachidate 20 0 tion, R, between methyl stearate and methyl oleate is NLT
1.5; and the relative standard deviation of the peak area
3.0 Methyl behenate 22 0
responses for the palmitate and stearate peaks for replicate
3.0 Methyl lignocerate 24 0 injections is NMT 6.0%. The relative standard deviation of
45.0 Methyl oleate 18 1 the peak area response ratio of the palmitate to stearate
15.0 Methyl linoleate 18 2 peaks from these replicate injections is NMT 1.0%.
3.0 Methyl linolenate 18 3 Procedure—Separately inject equal volumes (about 1 µL)
20.0 Methyl erucate 22 1 of the Standard Solution and the Test Solution into the chro-
matograph, record the chromatograms, identify the fatty
Nu-Chek mixture 19A has the following composition: acid ester peaks in the chromatogram of the Test Solution by
comparing the retention times of these peaks with those in
the chromatogram of the Standard Solution, and measure
Table 5 the peak areas for all of the fatty acid ester peaks in the
Carbon- Number of
Chain Double
Percentage Fatty Acid Ester Length Bonds
7.0 Methyl caprylate 8 0
5.0 Methyl caprate 10 0
USP 37 Chemical Tests / 〈401〉 Fats and Fixed Oils 5

chromatogram from the Test Solution. Calculate the percent- 15 s. Allow the upper layer to become clear, and transfer to
age of each fatty acid component in the test specimen: a separate tube. Shake the methanol layer once more with
1.0 mL of 2,2,4-trimethylpentane, and combine the 2,2,
Result = 100 × (A/B) 4-trimethylpentane extracts. Wash the combined extracts
with two quantities, 1 mL each, of water, and dry over an-
A = area of the peak response obtained for each individ- hydrous sodium sulfate.
ual fatty acid ester component Test Solution 2 (for triglycerides)—Transfer the equiva-
B = sum of the peak areas of all of the peaks, excluding lent amount of sample used to prepare Test Solution 1 to a
the solvent peak, in the chromatogram from the Test 10-mL volumetric flask, and dissolve in and dilute with Inter-
Solution nal Standard Solution to volume. Gentle heating (up to 60°)
may be applied to obtain a clear solution. Then proceed as
OMEGA-3 FATTY ACIDS DETERMINATION directed in Test Solution 1 starting with “Transfer 2.0 mL”.
AND PROFILE Test Solution 3 (for ethyl esters)—In a 10-mL volumetric
flask, dissolve the mass of sample to be examined, accord-
The following procedure may be used for the determina- ing to Table 6, in the Internal Standard Solution, and dilute
tion of eicosapentaenoic acid (EPA) (C20:5 n-3), docosahex- with the same solution to volume. Gentle heating (up to
aenoic acid (DHA) (C22:6 n-3), and total omega-3 acids ob- 60°) may be applied to obtain a clear solution.
tained from fish, plant, or microbial sources in bulk oils and Test Solution 4 (for ethyl esters)—Transfer the equivalent
encapsulated oil, either as triglycerides or as ethyl esters. amount of sample used to prepare Test Solution 3 to a
The term “triglyceride” is applicable to algal oils, fish oils, 10-mL volumetric flask, and dissolve in and dilute with Anti-
fish liver oils, and products containing omega-3-acids in tri- oxidant Solution to volume.
glycerides form. The results are expressed as free fatty acids Standard Solution 1a—Transfer 60 mg of USP Docosa-
or ethyl esters. Perform the tests as rapidly as possible. Pro- hexaenoic Acid Ethyl Esters RS , accurately weighed, to a
tect the solutions from actinic light, oxidizing agents, oxida- 10-mL volumetric flask, and dissolve in and dilute with Inter-
tion catalysts, and air. nal Standard Solution to volume. Gentle heating (up to 60°)
may be applied to obtain a clear solution.
Content of EPA and DHA Standard Solution 1b—Transfer 90 mg of USP Eicosa-
pentaenoic Acid Ethyl Esters RS, accurately weighed, to a
10-mL volumetric flask, and dissolve in and dilute with Inter-
USP Reference Standards 〈11〉 nal Standard Solution to volume. Gentle heating (up to 60°)
USP Docosahexaenoic Acid Ethyl Ester RS may be applied to obtain a clear solution.
USP Eicosapentaenoic Acid Ethyl Ester RS Standard Solution 1a and Standard Solution 1b are ready
USP Methyl Tricosanoate RS for ethyl esters analysis. For triglycerides analysis, continue
Antioxidant Solution—Dissolve an accurately weighed with Standard Solution 2a and Standard Solution 2b.
quantity of butylated hydroxytoluene in 2,2,4-trimethylpen- Standard Solution 2a—Transfer 2.0 mL of Standard Solu-
tane to obtain a solution with a concentration of 0.05 mg tion 1a to a glass tube, and evaporate the solvent with a
per mL. gentle stream of nitrogen. Then proceed as directed for Test
Internal Standard Solution—Transfer an accurately Solution 1 starting with, “Add 1.5 mL”.
weighed quantity of USP Methyl Tricosanoate RS to a volu- Standard Solution 2b—Transfer 2.0 mL of Standard Solu-
metric flask. Dissolve in Antioxidant Solution, and dilute with tion 1b to a glass tube, and evaporate the solvent with a
the same solvent to obtain a solution with a concentration gentle stream of nitrogen. Then proceed as directed for Test
of about 7.0 mg/mL. [NOTE—Guard the solution against Solution 1 starting with, “Add 1.5 mL”.
evaporation during use.]
System Suitability Solution 1—Transfer 0.300 g of
methyl palmitate, 0.300 g of methyl stearate, 0.300 g of
Table 6 methyl arachidate, and 0.300 g of methyl behenate, accu-
Amount of Sample rately weighed, to a 10-mL volumetric flask, and dissolve in
Approx. Sum to Be Weighed and dilute with Antioxidant Solution to volume.
EPA + DHA (g) System Suitability Solution 2—[NOTE—This solution
30%–50% 0.4–0.5 must be prepared only for triglycerides and only if tetracos-
50%–70% 0.3 15-enoic acid methyl ester is not clearly observed in the
>70% 0.25 chromatogram obtained with Test solution 2.] Transfer
55.0 mg of docosahexaenoic acid methyl ester and about
5.0 mg of tetracos-15-enoic acid (nervonic acid) methyl es-
Test Solution 1 (for triglycerides)—In a 10-mL volumetric ter, accurately weighed, to a 10-mL volumetric flask, and
flask, dissolve the mass of sample to be examined, accord- dissolve in and dilute with Antioxidant Solution to volume.
ing to Table 6, in Antioxidant Solution, and dilute with the Chromatographic system
same solution to volume. Transfer 2.0 mL of this solution to (See Chromatography 〈621〉, System Suitability.)
a glass tube, and evaporate the solvent with a gentle stream
of nitrogen. Add 1.5 mL of a 2% (w/v) solution of sodium Mode: GC
hydroxide in methanol, cap tightly with a polytetrafluoro- Detector: Flame ionization
ethylene-lined cap, mix, and heat in a boiling water bath for Column: 0.25-mm × 25-m fused–silica capillary column
7 min. Cool, add 2 mL of boron trichloride–methanol solu- bonded with a 0.20-µm film of phase G16
tion (120 g in 1000 mL of methanol), cover with nitrogen, Temperatures
cap tightly, mix, and heat in a boiling water bath for 30 Injection port
min. Cool to 40°–50°, add 1.0 mL of 2,2,4-trimethylpen-
tane, cap, and mix on a vortex mixer or shake vigorously for Split injection: 250°
at least 30 s. Immediately add 5 mL of saturated sodium Splitless injection: 90°–250°
chloride solution containing 1 volume of sodium chloride Detector: 270°
and 2 volumes of water. [NOTE—Shake from time to time. Column: See Table 7a or Table 7b.
Before use, decant the solution from any undissolved sub-
stance, and filter if necessary.] Cover with nitrogen, cap,
and mix on a vortex mixer or shake thoroughly for at least
6 〈401〉 Fats and Fixed Oils / Chemical Tests USP 37

Table 7a (Split Injection) Analysis (for triglycerides)

Hold Samples: Standard Solution 2a, Standard Solution 2b,
Time Test Solution 1, and Test Solution 2
Initial Hold Tempera- Final at Final Inject each sample in duplicate and measure the peak re-
Tempera- Time ture Tempera- Tempera- sponses. Compare the chromatographic profile of Test Solu-
ture at 170° Ramp ture ture tion 2 with that of Test Solution 1 and identify the internal
(°) (min) (°/min) (°) (min) standard peak present in Test Solution 2. Calculate the per-
170 2 3 240 2.5 centage of EPA or DHA in the triglycerides sample taken:
Result = (RU/RS) × (WS/WU) × F × 100
Table 7b (Splitless Injection) RU = ratio of peak response of either EPA or DHA to the
Temp- Temp- Hold corrected peak response of the internal standard in the
era- era- Time chromatogram of Test Solution 2, calculated:
Ini- ture ture To at
tial Hold Ramp To Ramp Final Final Result = 1/[(rU2/rT2) − (rU1/rT1)]
Tem- Time Num- Temp- Num- Temp- Tem-
per- at ber 1 era- ber 2 era- pera- [NOTE—If rU1 = 0 due to no peak observed at the locus of
ature 90° (°/ ture (°/ ture ture the internal standard in the chromatogram of Test solution
(°) (min) min) (°) min) (°) (min) 1, then RU = rT2/rU2.]
90 2 30 170 3 240 2 rU2 = peak response of the internal standard in the chro-
matogram of Test Solution 2
rT2 = peak response of EPA or DHA in the chromatogram
Carrier gas: Helium of Test Solution 2
Flow rate: 1 mL/min rU1 = peak response of any peak at the locus of the
Split flow ratio: 200:1. [NOTE—If necessary, adjust the internal standard in the chromatogram of Test Solution 1
split ratio and/or sample dilution to obtain a tailing factor of rT1 = peak response of EPA or DHA in the chromatogram
0.8–1.5 for the fatty acid methyl ester peaks in System Suita- of Test Solution 1
bility Solution 1, the methyl or ethyl ester peaks of eicosa- RS = ratio of peak response of either DHA or EPA to the
pentaenoic acid and docosahexaenoic acid in Test Solution 1 peak response of the internal standard in the chromato-
and Test Solution 4, at the same time observing that for Test gram of Standard Solution 2a or Standard Solution 2b
Solution 1 or Test Solution 4 any peaks due to the corre- WS = weight of DHA taken for preparing Standard Solu-
sponding esters of C18:3 n-3, C18:4 n-3, C20:4 n-3, C21:5 tion 1a or weight of EPA taken for preparing Standard
n-3, and C22:5 n-3 are clearly detectable. If splitless injec- Solution 1b (mg)
tion mode is used, solutions should be further diluted 1 in WU = weight of sample taken for preparing Test Solution
200 with Antioxidant Solution.] 2 (mg)
F = factor to express content of DHA as free fatty acids,
Injection size: 1 µL 0.921; and factor to express content of EPA as free fatty
System suitability (for triglycerides) acids, 0.915
Samples: System Suitability Solution 1 and Test Solution Analysis (for ethyl esters)
2 or System Suitability Solution 2 (if applicable) Samples: Standard Solution 1a, Standard Solution 1b,
System suitability (for ethyl esters) Test Solution 3, and Test Solution 4
Sample: System Suitability Solution 1 Inject each sample in duplicate and measure the peak re-
System suitability requirements sponses. Compare the chromatographic profile of Test Solu-
Theoretical area percentages (System Suitability Solution 1): tion 3 with that of Test Solution 4 and identify the internal
The area percentages, after adjusting for the actual weights, standard peak present in Test Solution 3. Calculate the per-
of the peaks of methyl palmitate, methyl stearate, methyl centage of EPA or DHA in the ethyl esters sample taken:
arachidate, and methyl behenate, each should be within Result = (RU/RS) × (WS/WU) × 100
±1.0% (absolute) of theoretical values provided in Table 8.
RU = ratio of peak response of either EPA or DHA to the
Table 8 corrected peak response of the internal standard in the
Theoretical Area (%) in a chromatogram of Test Solution 3, calculated as follows:
Solution of Equal Weights of
Methyl Palmitate, Methyl
Result = 1/[(rU3/rT3) − (rU4/rT4)]
Stearate, Methyl Arachidate,
Fatty Acid Methyl Ester and Methyl Behenate
[NOTE—If rU4 = 0 due to no peak observed at the locus of
the internal standard in the chromatogram of Test solution
Methyl palmitate 24.37 4, then RU = rT3/rU3.]
Methyl stearate 24.84 rU3 = peak response of the internal standard in the chro-
Methyl arachidate 25.23 matogram of Test Solution 3
Methyl behenate 25.56 rT3 = peak response of EPA or DHA in the chromatogram
of Test Solution 3
[NOTE—The theoretical area (%) in a solution of equal rU4 = peak response of any peak at the locus of the
weights (Table 8) is derived from the calculated theoretical internal standard in the chromatogram of Test Solution 4
response factors, which are 1.049, 1.029, 1.013, and 1.000 rT4 = peak response of EPA or DHA in the chromatogram
for methyl palmitate, methyl stearate, methyl arachidate, of Test Solution 4
and methyl behenate, respectively.] RS = ratio of peak response of either DHA or EPA to the
Resolution: For triglycerides (Test Solution 2 or System peak response of the internal standard in the chromato-
Suitability Solution 2): NLT 1.2 between the docosahexaenoic gram of Standard Solution 1a or Standard Solution 1b
acid methyl ester and tetracos-15-enoic acid methyl ester WS = weight of DHA taken for preparing Standard Solu-
peaks tion 1a or weight of EPA taken for preparing Standard
Solution 1b (mg)
USP 37 Chemical Tests / 〈401〉 Fats and Fixed Oils 7

WU = weight of sample taken for preparing Test Solution Table 9 (Continued)

3 (mg) Volume Scale Division
(mL) (mL)
Content of Total Omega-3 Acids (for 25–50 25.0
triglycerides) 50–100 50.0

Calculate the percentage of the total omega-3 acids in Procedure—Place 50.0 mL of benzene in each of two
the portion of triglycerides sample taken: centrifuge tubes, and to each tube add 50.0 mL of the oil,
warmed if necessary to re-incorporate separated stearin, and
Result = EPA + DHA + [(An− 3 × (EPA + DHA)/(AEPA + ADHA)] mixed thoroughly at 25°. Insert the stopper tightly into the
tubes, shake them vigorously until the contents are mixed
EPA = content of EPA, from the test for Content of EPA thoroughly, and then immerse the tubes in a water bath at
and DHA (%) 50° for 10 min. Centrifuge for 10 min. Read the combined
DHA = content of DHA, from the test for Content of EPA volume of water and sediment at the bottom of each tube.
and DHA (%) Centrifuge repeatedly for 10-min periods until the combined
An− 3 = sum of the areas of the peaks corresponding to volume of water and sediment remains constant for three
C18:3 n-3, C18:4 n-3, C20:4 n-3, C21:5 n-3, and C22:5 consecutive readings. The sum of the volumes of combined
n-3 methyl esters in the chromatogram obtained with water and sediment in the two tubes represents the per-
Test Solution 1 centage, by volume, of water and sediment in the oil.
AEPA = area of the peak corresponding to the EPA methyl
ester in the chromatogram obtained with Test Solution 1
ADHA = area of the peak corresponding to the DHA ANISIDINE VALUE
methyl ester in the chromatogram obtained with Test So-
lution 1 The Anisidine Value is defined as 100 times the optical
density measured in a 1-cm cell of a solution containing 1 g
of the substance to be examined in 100 mL of a mixture of
Content of Total Omega-3 Acids (for ethyl solvents and reagents according to the following method
esters) described. [NOTE—Carry out the operations as rapidly as
possible, avoiding exposure to actinic light.]
Calculate the percentage of the total omega-3 acids in Test Solution A—Dissolve 0.500 g of the substance to be
the portion of ethyl esters sample taken: examined in isooctane, and dilute with the same solvent to
Result = EPA + DHA + [(An − 3 × (EPA + DHA)/(AEPA + ADHA)] 25.0 mL.
Test Solution B—To 5.0 mL of Test Solution A add 1.0 mL
EPA = content of EPA, from the test for Content of EPA of a 2.5-g/L solution of p-anisidine in glacial acetic acid,
and DHA (%) shake, and store protected from light.
DHA = content of DHA, from the test for Content of EPA Standard Solution—To 5.0 mL of isooctane add 1.0 mL
and DHA (%) of a 2.5-g/L solution of p-anisidine in glacial acetic acid,
An − 3 = sum of the areas of the peaks corresponding to shake, and store protected from light.
C18:3 n-3, C18:4 n-3, C20:4 n-3, C21:5 n-3, and C22:5 Procedure—Measure the absorbance of Test Solution A at
n-3 ethyl esters in the chromatogram obtained with Test 350 nm using isooctane as the blank. Measure the absorb-
Solution 4 ance of Test Solution B at 350 nm exactly 10 min after its
AEPA = area of the peak corresponding to the EPA ethyl preparation, using the Standard Solution as the compensa-
ester in the chromatogram obtained with Test Solution 4 tion liquid. Calculate the Anisidine Value from the
ADHA = area of the peak corresponding to the DHA ethyl expression:
ester in the chromatogram obtained with Test Solution 4
Result = [25 × (1.2AS − AB)]/m
WATER AND SEDIMENT IN FIXED OILS AS = absorbance of Test Solution B at 350 nm
AB = absorbance of Test Solution A at 350 nm
Apparatus—The preferred centrifuge has a diameter of m = weight of the substance to be examined in Test
swing (d = distance from tip to tip of whirling tubes) of Solution A (g)
38–43 cm and is operated at a speed of about 1500 rpm. If
a centrifuge of different dimensions is used, calculate the
desired rate of revolution: TOTAL OXIDATION VALUE (TOTOX)
Total Oxidation Value is defined:
Result = 2PV + AV
The centrifuge tubes are pear-shaped and are shaped to
accept closures. The total capacity of each tube is about PV = Peroxide Value
125 mL. The graduations are clear and distinct, reading up- AV = Anisidine Value
ward from the bottom of the tube according to the scale
shown in Table 9.
TRACE METALS
Table 9
Volume Scale Division
(mL) (mL) Apparatus
0–3 0.1
The apparatus typically consists of the following:
3–5 0.5
Digestion Flasks—Use a polytetrafluoroethylene flask
5–10 1.0
with a volume of about 120 mL, fitted with an airtight clo-
10–25 5.0 sure, a valve to adjust the pressure inside the container, and
a polytetrafluoroethylene tube to allow the release of gas.
8 〈401〉 Fats and Fixed Oils / Chemical Tests USP 37

System—Make the flask airtight, using the same torsional substance.] For routine measurements, 3 standard solutions,
force for each of them. Blank Solution 1, and Test Solution 1 are prepared and ex-
Microwave Oven—It has a magnetron frequency of amined.
2450 MHz with a selectable output from 0 to 630 ± 70 W in Use Test Solution 1 and Blank Solution 1 as prepared previ-
1% increments, a programmable digital computer, a poly- ously or as indicated in the monograph. Prepare NLT 3 stan-
tetrafluoroethylene-coated microwave cavity with a variable dard solutions containing all the metal elements to be
speed exhaust fan, a rotating turntable drive system, and tested. The expected absorbance value in Test Solution 1 for
exhaust tubing to vent fumes. each metal element should be within its corresponding cali-
Atomic Absorption Spectrometer—It is equipped with a brated absorbance range, preferably in the middle of the
hollow-cathode lamp as the source of radiation and a deute- calibrated absorbance range. Any reagents used in the prep-
rium lamp as a background corrector. The system is fitted aration of Test Solution 1 are added at the same concentra-
with the following: tion to the standard solutions.
1. A graphite furnace as the atomization device for cad- Introduce each of the solutions into the instrument using
mium, copper, iron, lead, nickel, and zinc. the same number of replicates for each of the solutions to
2. An automated continuous-flow hydride vapor genera- obtain a steady reading.
tion system for arsenic and mercury. Prepare a calibration curve from the mean of the readings
obtained with the standard solutions by plotting the means
as a function of concentration. Determine the concentration
General Procedure of the element in Test Solution 1 from the curve obtained.
Standard Additions—Add to at least four identical volu-
Caution—When using closed high-pressure digestion vessels metric flasks equal volumes of Test Solution 1 as prepared
and microwave laboratory equipment, the safety precautions previously or as indicated in the monograph. Add to all but
and operating instructions given by the manufacturer must be one of the flasks progressively larger volumes of a standard
followed. solution containing a known concentration of the test ele-
[NOTE—If an alternative apparatus is used, adjustment of ment to produce a series of solutions containing steadily
the instrument parameters may be necessary.] increasing concentrations of that element known to give re-
Cleaning—Clean all the glassware and laboratory equip- sponses in the linear part of the curve. Dilute the contents
ment with a 10-mg/mL solution of nitric acid before use. of each flask with the solvent specified in the monograph to
Trace Metal-Free Nitric Acid—Nitric acid meets the re- volume, and mix. The flask without an addition of standard
quirements with the maximum values for arsenic (As), cad- solution is labeled as the test solution.
mium (Cd), copper (Cu), iron (Fe), mercury (Hg), lead (Pb), Introduce each of the solutions into the instrument, using
nickel (Ni), and zinc (Zn) equal to 0.005, 0.005, 0.001, the same number of replicates for each of the solutions, to
0.02, 0.002, 0.001, 0.005, and 0.01 ppm, respectively. obtain a steady reading.
Trace Metal-Free Hydrochloric Acid—Hydrochloric acid Plot the absorbances of the standard solutions and the
meets the requirements with the maximum values for As, test solution versus the added quantity of test element.
Cd, Cu, Fe, Hg, Pb, Ni, and Zn equal to 0.005, 0.003, [NOTE—The test solution should be plotted as if it had a
0.003, 0.05, 0.005, 0.001, 0.004, and 0.005 ppm, respec- content of added test element equivalent to 0 mg or µg.]
tively. Extrapolate the line joining the points on the graph until it
meets the concentration axis. The distance between this
Trace Metal-Free Sulfuric Acid—Sulfuric acid meets the point and the intersection of the axes represents the con-
requirements with the maximum values for As, Cd, Cu, Fe, centration of test element in the test solution.
Hg, Pb, Ni, and Zn equal to 0.005, 0.002, 0.001, 0.05,
0.005, 0.001, 0.002, and 0.005 ppm, respectively.
Test Stock Solution—In a digestion flask place about Specific Tests
0.5 g of fatty oil, accurately weighed, as indicated in each
individual monograph. Add 6 mL of Trace Metal–Free Nitric
Acid and 4 mL of Trace Metal–Free Hydrochloric Acid. Close CADMIUM (CD), COPPER (CU), IRON (FE), LEAD (PB),
the flask.
NICKEL (NI), AND ZINC (ZN)
Blank Stock Solution—Mix 6 mL of Trace Metal-Free Ni-
tric Acid and 4 mL of Trace Metal–Free Hydrochloric Acid in a
digestion flask. Standard Stock Solution—Prepare a solution containing
known concentrations of 5 µg/mL for each test element.
Test Solution 1—Place the digestion flask containing the
Test Stock Solution in the microwave oven. Carry out the Standard Solutions—In three identical 10-mL volumetric
digestion in three steps according to the following program: flasks introduce 10, 20, and 40 µL of Standard Stock Solu-
80% power for 15 min, 100% power for 5 min, and 80% tion, respectively. To each flask add 5.0 mL of Test Solution
power for 20 min. 1, dilute with water to volume, and mix.
At the end of the cycle allow the flask to cool. Add 4 mL Test Solution 2—In a 10-mL volumetric flask add
of Trace Metal-Free Sulfuric Acid to the flask. Repeat the di- 5.0 mL of Test Solution 1, dilute with water to volume, and
gestion program. After completing the digestion, allow the mix.
flask to cool to room temperature. Open the digestion flask, Blank Solution 2—In a 10-mL volumetric flask add
and transfer the clear, colorless solution obtained into a 5.0 mL of Blank Solution 1, dilute with water to volume, and
50-mL volumetric flask. Rinse the digestion flask with 2 mix.
quantities, 15 mL each, of water, and collect the rinsings in Procedure—Measure the content of Cd, Cu, Fe, Pb, Ni,
the volumetric flask. Add 1.0 mL of a 10-mg/mL solution of and Zn using a suitable graphite furnace atomic absorption
magnesium nitrate and 1.0 mL of a 100-mg/mL solution of spectrophotometer. Concomitantly determine the ab-
ammonium dihydrogen phosphate to the volumetric flask. sorbances of Blank Solution 2, the Standard Solutions, and
Dilute with water to volume, and mix. This solution is Test Test Solution 2 at least three times each. The absorbance
Solution 1. value of Blank Solution 2 is subtracted from the value ob-
Blank Solution 1—Place the digestion flask containing tained using the Standard Solutions and Test Solution 2. Pro-
Blank Stock Solution in the microwave oven. Proceed as di- ceed as directed in the Standard Additions method in Gen-
rected in Test Solution 1 beginning with “Carry out the di- eral Procedure. See Table 10 for instrumental parameters that
gestion in three steps according to the following program”. may be used.
Direct Calibration—[NOTE—Concentrations of the stan-
dard solutions will depend on the metal contents of the test
USP 37 Chemical Tests / 〈401〉 Fats and Fixed Oils 9

ARSENIC AND MERCURY STEROL COMPOSITION

Measure the content of arsenic and mercury against their
standard solutions at a known concentration using the Di-
rect Calibration method in General Procedure, with an auto- Separation of the Sterol Fraction
mated continuous-flow hydride vapor generation system.
For the 1 ppm arsenic specification limit and 1 ppm mer- Reference Solution A—Dissolve an accurately weighed
cury specification limit, prepare three working calibration so- quantity of cholesterol in chloroform to obtain a solution of
lutions with known concentrations of 5, 10, and 20 ng/mL 5% (w/v).
for each test element, respectively. Developing Solvent System—A mixture of toluene and
The absorbance value of the blank solution is automati- acetone (95:5) or a mixture of hexane and ether (65:35)
cally subtracted from the value obtained using the test Test Solution A—Accurately weigh 5 g of the test sub-
solution. stance into a 250-mL flask. Add 50 mL of alcoholic potas-
Arsenic— sium hydroxide TS 2 (2 N alcoholic potassium hydroxide),
Blank Solution 3—Add 1.0 mL of a 200-mg/mL solution and heat to gentle boiling with continuous vigorous stirring
of potassium iodide to 19.0 mL of Blank Solution 1 prepared until saponification takes place (the solution becomes clear).
previously. Allow this solution to stand at room temperature Continue heating for a further 20 min, and add 50 mL of
for about 50 min or at 70° for about 4 min. water from the top of the condenser. Cool the flask to ap-
Test Solution 3—Add 1.0 mL of a 200-mg/mL solution of proximately 30°. Transfer the contents of the flask to a
potassium iodide to 19.0 mL of Test Solution 1 prepared pre- 500-mL separating funnel with several rinses of water,
viously. Allow this solution to stand at room temperature for amounting in all to about 50 mL. Add approximately 80 mL
about 50 min or at 70° for about 4 min. of ether, shake vigorously for approximately 30 s, and allow
to settle. [NOTE—Any emulsion can be destroyed by adding
Acid Reagent 1: Trace Metal-Free Hydrochloric Acid small quantities of ethyl or methyl alcohol by means of a
Reducing Reagent 1: A 6-mg/mL solution of sodium te- spray.] Separate the lower aqueous phase, and collect it into
trahydroborate in a 5-mg/mL solution of sodium hydroxide a second separating funnel. Perform two further extractions
The instrumental parameters in Table 11 may be used. on the water–alcohol phase in the same way, using
Mercury— 60–70 mL of ether on each occasion. Pool the ether extracts
Blank Solution 4—Proceed as directed in Blank Solution 3. into a single separating funnel, and wash with water, 50 mL
at a time, until the wash water is no longer alkaline to phe-
Test Solution 4—Proceed as directed in Test Solution 3. nolphthalein. Dry the ether phase with anhydrous sodium
Acid Reagent 2: A 515-mg/mL solution of Trace Metal- sulfate, and filter on anhydrous sodium sulfate into a previ-
Free Hydrochloric Acid ously weighed 250-mL flask, washing the funnel and filter
Reducing Reagent 2: A 10-mg/mL solution of stannous with small quantities of ether. Distill the ether down to a
chloride in a 200-mg/mL solution of Trace Metal-Free Hydro- few mL, and bring to dryness under a slight vacuum or in a
chloric Acid stream of nitrogen. Complete the drying at 100° for ap-
The instrumental parameters in Table 11 may be used. proximately 15 min, and then weigh after cooling in a des-
iccator. Dissolve the unsaponifiables so obtained in chloro-
Table 11 form to obtain a solution with a concentration of
approximately 5%.
As Hg
Test Solution B—Treat 5 g of canola oil in the same way
Wavelength (nm) 193.7 253.7 as prescribed for the test substance in Test Solution A, begin-
Slit width (nm) 0.2 0.5 ning with “Add 50 mL of alcoholic potassium hydroxide TS
Lamp current (mA) 10 4 2 (2 N alcoholic potassium hydroxide)”.
Acid reagent flow rate (mL/min) 1.0 1.0 Test Solution C—Treat 5 g of sunflower oil in the same
Reducing reagent flow rate way as prescribed for the test substance in Test Solution A,
(mL/min) 1.0 1.0 beginning with “Add 50 mL of alcoholic potassium hydrox-
Flow rate for the blank, stan- ide TS 2 (2 N alcoholic potassium hydroxide)”.
dard, test solutions (mL/min) 7.0 7.0 Procedure—Immerse the thin-layer chromatographic
Quartz Quartz plate (see 〈621〉), 20-cm × 20-cm silica gel on polyester with
a layer thickness of 200 µm and particle size of 5–17 µm,
1
Absorption cell (heated) (unheated)
Background corrector Off Off
completely in the 0.2 N alcoholic potassium hydroxide for
10 s, allow to dry in a fume cupboard for 2 h, and finally
Nitrogen flow rate (L/min) 0.1 0.1 place at 100° for 1 h. [NOTE—Remove from the validated
heating device, and keep the plate in a desiccator until re-

Table 10
Cd Cu Fe Pb Ni Zn
Wavelength (nm) 228.8 324.8 248.3 283.5 232 213.9
Slit (nm) 0.5 0.5 0.2 0.5 0.2 0.5
Lamp current (mA) 6 7 5 5 10 7
Ignition temperature (°) 800 800 800 800 800 800
Atomization 2500
temperature (°) 1800 2300 2300 2200 2000
Background corrector On Off Off Off Off Off
Nitrogen flow (L/min) 3 3 3A commercial TLC plate
1 3 may be obtained from
3 Sigma-Aldrich, catalog
3
#z122785.
10 〈401〉 Fats and Fixed Oils / Chemical Tests USP 37

quired for use. The plates must be used within 15 days. Reference Solution E—To 9 parts of the sterols separated
Thin-layer chromatographic plates that do not require the from canola oil by thin-layer chromatography, add 1 part of
preconditioning also are commercially available.] Use a sepa- cholesterol. Treat the mixture in the same way as directed in
rate plate for each test solution. Test Solution D.
Place a mixture of toluene and acetone (95:5) or a mix- Reference Solution F—Treat the sterols separated from
ture of hexane and ether (65:35) in the chamber to a depth sunflower oil by thin-layer chromatography in the same way
of approximately 1 cm. Close the chamber with the appro- as directed in Test Solution D.
priate cover, and leave for at least 30 min. Strips of filter Chromatographic System (see 〈621〉)—The gas chro-
paper dipping into the eluant may be placed on the internal matograph is equipped with a flame-ionization detector and
surfaces of the chamber. [NOTE—The developing mixture a glass or fused-silica capillary column 20–30 m in length,
should be replaced for every test to ensure reproducible elu- internal diameter 0.25–0.32 mm, entirely coated with a
tion conditions.] Apply 0.3 mL of Test Solution A approxi- 0.10- to 0.30-µm layer of stationary phase G27 or G36. The
mately 2 cm from the lower edge in a streak that is as thin injection port temperature is maintained at 280°, the detec-
and as uniform as possible. In line with the streak, place tor temperature is maintained at 290°, and the column tem-
2–3 µL of Reference Solution A at one end of the plate. De- perature is maintained at 260 ± 5°. The carrier gas is either
velop the chromatograms in an equilibrated chamber with helium with a linear velocity of 20–35 cm/s or hydrogen
the Developing Solvent System until the solvent front reaches with a linear velocity of 30–50 cm/s. A split ratio of 1:50 to
approximately 1 cm from the upper edge of the plate. Re- 1:100 is used. Chromatograph Reference Solution E and Ref-
move the plate from the developing chamber, and evapo- erence Solution F, and record the peak responses as directed
rate the solvent under a current of hot air [NOTE—avoid in Procedure: the retention time should be 20 ± 5 min for β-
excessive heat] or by leaving the plate for a short while sitosterol, and all the sterols present must be separated. The
under a hood. Spray the plate with a 0.2% alcoholic solu- chromatogram obtained with Reference Solution E shows four
tion of 2,7-dichlorofluorescein, and examine in UV light at principal peaks corresponding to cholesterol, brassicasterol,
254 nm. [NOTE—The plates pretreated with UV indicator campesterol, and β-sitosterol; and the chromatogram ob-
also are commercially available and are used equivalently.] tained with Reference Solution F shows four principal peaks
In each of the plates, mark the limits of the sterol band corresponding to campesterol, stigmasterol, β-sitosterol, and
identified by being aligned with the stain obtained from Ref- ∆7-stigmastenol. The retention times of the sterols with ref-
erence Solution A along the edges of the fluorescence, and erence to β-sitosterol are given in Table 12.
additionally include the area of the zones 2–3 mm above
and below the visible zones corresponding to Reference Solu-
tion A. Remove the silica gel in the marked area into a filter Table 12. Relative Retention Times of Sterols for Two Different
2
funnel with a G3 porous septum. Add 10 mL of hot chloro- Columns
form, mix carefully with the metal spatula, filter under vac- Identification G36 Column G27 Column
uum, and collect the filtrate in the conical flask attached to Cholesterol 0.67 0.63
the filter funnel. Wash the residue in the funnel three times
Brassicasterol 0.73 0.71
with ether, about 10 mL each time, and collect the filtrate
in the same flask attached to the funnel. Evaporate the fil- 24-Methylene-cholesterol 0.82 0.80
trate to a volume of 4–5 mL, transfer the residual solution to Campesterol 0.83 0.81
a previously weighed 10-mL test tube with a tapering bot- Campestanol 0.85 0.82
tom and a sealing stopper, and evaporate to dryness by Stigmasterol 0.88 0.87
mild heating in a gentle stream of nitrogen. Dissolve the ∆7-Campesterol 0.93 0.92
residue in a few drops of acetone, and evaporate again to
∆5,23-Stigmastadienol 0.95 0.95
dryness. Place at 105° for approximately 10 min, allow to
cool in a desiccator, and weigh. Clerosterol 0.96 0.96
Treat Test Solution B and Test Solution C the same way as β-Sitosterol 1.00 1.00
directed for Test Solution A. Sitostanol 1.02 1.02
∆5-Avenasterol 1.03 1.03
Determination of the Sterols ∆5,24-Stigmastadienol 1.08 1.08
∆7-Stigmastenol 1.12 1.12
Test Solution D—To the test tube containing the sterol ∆7-Avenasterol 1.16 1.16
fraction separated from the test substance by thin-layer
chromatography add a freshly prepared mixture of anhy- Procedure—Separately inject equal volumes (about 1 µL)
drous pyridine, hexamethyldisilazane, and chlorotrimethylsi- of Test Solution D, Reference Solution E, and Reference Solution
lane (9:3:1) [NOTE—This reagent also is commercially availa- F into the chromatograph, record the chromatograms, and
ble and is used equivalently.] in the ratio of 50 µL for every measure the peak areas for the sterols. Calculate the per-
mg of sterols, avoiding any uptake of moisture. Insert the centage of each individual sterol in the sterol fraction of the
stopper into the test tube, and shake carefully until the ste- test substance taken:
rols are completely dissolved. Allow it to stand for at least
15 min at ambient temperature, and centrifuge for a few Result = 100 × (A/S)
minutes if necessary. Use the supernatant. [NOTE—The slight
opalescence that may form is normal and does not cause an A = area of the peak due to the sterol component to be
anomaly. However, the formation of a white floc or the ap- determined
pearance of a pink color is indicative of the presence of S = sum of the areas of the peaks due to the compo-
moisture or deterioration of the reagent. If these occur, the nents indicated in Table 12
test must be repeated.]
2
A commercial product may be obtained from Kimble/Kontes as a filter,
buchner with fritted disc, Kimax 28400-152.