SALATRIM
Prepared at the 59th JECFA (2002) and published in FNP 52 Add 10
(2002), superseding specifications prepared at the 49th JECFA (1997),
published in FNP 52 Add 5 (1997). The safety evaluation of salatrim could
not be completed at the 59th JECFA.
DEFINITION Salatrim is the acronym for short- and long-chain acyl triglyceride
molecules. Salatrim is prepared by non-enzymatic inter-esterification of
triacetin, tripropionin, tributyrin, or their mixtures with hydrogenated
canola, soybean, cottonseed, or sunflower oil. Triglycerides with three
short-chain fatty acids (SCFA) are removed in the process. Stearic acid is
the predominant long-chain fatty acid (LCFA).
Structural formula
where 14 _ (x + y + z) _ 42 for those triglycerides with either one LCFA
and two SCFA or two LCFA and one SCFA.
Assay Not less than 87% triglycerides. Not less than 90% of the triglycerides with
a SCFA-to-LCFA mole ratio in the range 0.5-2.0. Not more than 70% by
weight of saturated LCFA.
DESCRIPTION Clear, slightly amber liquid to a light-coloured waxy solid at room
temperature. Free of particulate matter and of foreign or rancid odour.
FUNCTIONAL USES Reduced-energy fat and oil replacement.
CHARACTERISTICS
IDENTIFICATION
Solubility (Vol. 4) Soluble in hexane, cyclohexane, acetone, ether, tetrahydrofuran, and
liquid triglyceride oils. Insoluble in water
Melting range (Vol. 4) 16-71°, depending on triglyceride composition.
Infrared spectrum (Vol. 4) The infrared absorption spectrum of the sample, contained in a sodium
chloride cell or between salt plates, corresponds to the infrared spectrum
in the Appendix to this monograph.
PURITY
Water (Vol. 4) Not more than 0.3% (Karl Fischer method).
Sulfated ash (Vol. 4) Not more than 0.1%
Test 2 g of the sample (Method I for solid samples; Method II for liquid
samples).
Free fatty acids (Vol. 4) Not more than 0.5% (as oleic acid). Use an equivalence factor (e) of 28.2.
Monoglycerides Not more than 2%
See description under METHOD OF ASSAY.
Peroxide value Not more than 2.0
See description under TESTS.
Unsaponifiable matter Not more than 1.0%
See description under TESTS.
Lead (Vol. 4) Not more than 0.1 mg/kg
Determine using an atomic absorption technique appropriate to the
specified level. The selection of sample size and method of sample
preparation may be based on the principles of the method described in
Volume 4, “Instrumental Methods.”
.
TESTS
PURITY TESTS
Peroxide value Reagents
Acetic acid-chloroform solution: Mix 3 volumes of acetic acid with 2
volumes of chloroform.
Potassium iodide solution, saturated: Dissolve excess potassium iodide in
freshly boiled water. Excess solid must remain. Store in the dark. Test
daily by adding 0.5 ml to 30 ml of the acetic acid-chloroform solution, then
add 2 drops of starch TS. If the solution turns blue, requiring more than 1
drop of 0.1 N sodium thiosulfate to discharge the colour, prepare a fresh
solution.
Procedure
Weigh accurately about 5 g of the sample into a 250-ml Erlenmeyer flask.
Add 30 ml of the acetic acid-chloroform solution and swirl to dissolve. Add
0.5 ml of the saturated potassium iodide solution, allow to stand with
occasional shaking for 1 min, and add 30 ml of water. Slowly titrate with
0.01 N sodium thiosulfate, swirling the flask vigorously until the yellow
colour is almost gone. Add about 0.5 ml of starch TS and continue the
titration while swirling the flask vigorously to release all the iodine from the
chloroform layer until the blue colour disappears.
Perform a blank determination and make any necessary correction.
Peroxide value = (S x N x 1000)/W
where
S = ml of N sodium thiosulfate
N = normality of sodium thiosulfate
W = weight of the sample (g)
Unsaponifiable matter Maxwell, R.J., Reimann, K.A., and Percell, K. (1981) Determination of the
Unsaponifiable Matter in Fatty Acids by a Rapid Column Method, JAOCS
58:1002-1004.
Reagents
Calcium Chloride-Celite Mixture: Using a mortar and pestle, grind 1 part
anhydrous calcium chloride with 1 part water; add 3 parts Celite 545
(grade: not acid-washed, C-212). Grind to a uniform consistency. The
mixture may be stored in a covered amber jar for up to one month.
Potassium Hydroxide-Celite Mixture: For multiple analyses, prepare in lots
of 75 g or more. Using a mortar and pestle, grind 2 parts potassium
hydroxide pellets with 1 part water (CAUTION: considerable heat is
generated; wear eye protection and gloves); add 4 parts Celite 545. Grind
to a uniform consistency. The mixture may be stored in a covered amber
jar for up to 10 days.
Procedure
Saponification: Place 10 g Potassium Hydroxide-Celite mixture in a 400-ml
mortar; accurately weigh 5 g of sample (WS) and transfer to the mortar.
Grind the mixture until the sample is uniformly distributed. Add another 10
g of Potassium Hydroxide-Celite mixture; grind to uniform consistency.
Transfer the mixture to a jar. Using a pestle, transfer any residual sample
by sweeping 5 g Celite 545 along the sides of the mortar and into the jar.
Shake until the mixture is uniform. Heat for 20-30 min in a 130o oven.
Gravimetric Extraction: Transfer the cooled mixture to the mortar; regrind
(approximately 30 sec) to a uniform granular consistency. Loosely fit a
plug of glass wool into the tip of a glass chromatography column (30 mm
i.d.; 30 cm long, overall, with a drip tip 5 cm x 8 mm o.d.). Pack the column
with 5 g of Calcium Chloride-Celite mixture. Transfer the contents of the
mortar to the column. Pack to a total bed height of 50-60 mm. Place a
150-ml tared flask under the column. Qualitatively transfer residue from
the mortar to the column using about 25 ml of dichloromethane. Once this
solution has percolated into the column bed, add sufficient
dichloromethane so that the column bed is wet and a few drops of eluate
have been collected in the flask. Charge the column with 150 ml of
dichloromethane, and collect the entire volume in the flask (approximately
25 min). Remove the solvent under a stream of nitrogen with gentle
heating while the eluate is being collected. Take the contents of the flask
to constant weight under vacuum. Determine the weight of the residue
(WR).
To check for completeness of extraction, add 20 ml of dichloromethane to
the column and collect in a second tared flask. Evaporate the contents of
the second flask to dryness and examine for residue. Determine the
weight of the residue (WR1), if present. If residue is observed, repeat the
procedure using an additional 20 ml of dichloromethane.
The total residue weight and the weight of the original sample are used to
calculate the percent unsaponifiable matter:
[(WR + WR1 + ...)/WS] x 100
Lead (Vol. 4) Determination by graphite furnace atomic absorption spectrophotometry.
(Food Chemicals Codex, 4th Ed. (1996), National Academy of Sciences,
Washington, DC, pp. 765-766).
Apparatus
An atomic absorption spectrophotometer (Perkin-Elmer Model 3100 or
equivalent) fitted with a graphite furnace (Perkin-Elmer HGA 600 or
equivalent), a lead hollow cathode lamp (Perkin-Elmer or equivalent) with
argon as the carrier gas.
Follow the manufacturer's directions for setting the appropriate instrument
parameters for lead determination. (NOTE: Use reagent-grade chemicals
with as low a lead content as practicable; use high-purity water and
gases.) Prior to this analysis, rinse all glassware and plasticware twice
with 10% nitric acid and twice with 10% hydrochloric acid. Then rinse
thoroughly with high-purity water, preferably obtained from a mixed-bed
strong acid/strong base ion-exchange cartridge capable of producing
water that has an electrical resistivity of 12 to 15 megohms.
Solutions
Hydrogen Peroxide-Nitric Acid Solution: Mix together equal volumes of
10% hydrogen peroxide and 10% nitric acid (NOTE: use extreme caution;
wear gloves and protective eyeware).
Butanol-Nitric Acid Solution: Introduce approximately 500 ml of n-butanol
in a 1000-ml volumetric flask. Slowly add 50 ml nitric acid. Dilute to
volume with n-butanol and mix.
Lead Nitrate Stock Solution: Using glassware that is free of lead salts,
dissolve 159.8 mg of reagent-grade lead nitrate in 100 ml of Hydrogen
Peroxide-Nitric Acid Solution. Dilute to 1000.0 ml with Hydrogen Peroxide-
Nitric Acid Solution and mix. Each ml of this solution contains the
equivalent of 100 µg of lead ion.
Standard Lead Solution: On the day of use, pipet 10.0 ml of Lead Nitrate
Stock Solution into a 100.0-ml volumetric flask, add Hydrogen Peroxide-
Nitric Acid Solution to the mark and mix. Each ml of Standard Lead
Solution contains the equivalent of 10 µg of lead ion.
Standard Solutions: Into separate 100-ml volumetric flasks, pipet 0.2, 0.5,
1, and 2 ml, respectively, of Standard Lead Solution; dilute to volume with
Butanol-Nitric Acid Solution. These solutions contain 0.02, 0.05, 0.1, and
0.2 µg lead per ml, respectively.
Sample Solution: (NOTE: Perform this procedure in a fume hood).
Accurately weigh 1 g of the sample, and transfer to a large test tube. Add
1 ml of nitric acid. Place the test tube in a rack in a boiling water bath. As
soon as the rusty tint (reddish-brown colour) is gone, add 1 ml of 30%
hydrogen peroxide dropwise (in order to avoid a vigorous reaction) and
wait for bubbles to form. Stir with an acid-washed plastic spatula if
necessary. Remove the test tube from the water bath and let it cool.
Transfer the solution to a 10-ml volumetric flask and dilute to volume with
Butanol-Nitric Acid Solution. Use this solution for analysis.
Tungsten Solution: Transfer 0.1 g of tungstic acid (H2WO4) and 5 g of
sodium hydroxide pellets into a 50-ml plastic bottle. Add 5.0 ml of high-
purity water (distilled water may be redistilled from an all-glass apparatus
or may be passed down a column of cation exchange resin, e.g.,
Amberlite IR 120(H)), and mix. Heat the mixture in a hot water bath until
completely dissolved. Cool and store at room temperature.
Procedure
Place the graphite tube in the furnace. Inject a 20-µl aliquot of Tungsten
Solution into the graphite tube, using a 300-ml/min argon flow and the
following sequence of conditions: dry at 110o for 20 sec, char at 700o to
900o for 20 sec and, with the argon flow stopped, atomize at 2700o for 10
sec; repeat using a second 20-µl aliquot. Clean the quartz windows.
(NOTE: The sample injection technique is the most crucial step in
controlling the precision of the analysis; the volume of the sample must
remain constant. Rinse the µl pipet tip (Eppendorf or equivalent) three
times with either the Standard Solutions or Sample Solution before
injection. Use a fresh pipet tip for each injection and start the atomization
process immediately after injecting the sample. Between injections, flush
the graphite tube to eliminate any residual lead by purging at a high
temperature, as recommended by the manufacturer.)
With the hollow cathode lamp aligned for maximum absorbance, and the
wavelength set at 283.3 nm, atomize 20-µl aliquots of the four Standard
Solutions, using a 300-ml/min argon flow and the following sequence of
conditions: dry at 110o for 30 sec (20 sec ramp period and 10 sec hold
time), char at 700o for 42 sec (20 sec ramp period and 22 sec hold time)
and, with the argon flow stopped, atomize at 2300o for 7 sec.
Plot a standard curve of concentration, in µg/ml, of each Standard Solution
versus its maximum absorbance value compensated for background
correction, as directed for the particular instrument. Atomize 20 µl of the
Sample Solution under conditions identical to those for the Standard
Solutions and measure its background-corrected maximum absorbance.
From the standard curve, determine the concentration of lead in the
Sample Solution, C, in µg/ml, Calculate the concentration of lead in the
Salatrim sample, in mg/kg, using:
10C/W
where W is the mass, in g, of the sample.
METHOD OF
ASSAY
[Characterization of Triacylglycerols in Saturated Lipid Mixtures with
Application to Salatrim 23CA (Huang et al, J. Agric. Food Chem., (1994)
42, 453).]
I. Determination of
monoacylglyceride and
triacylglyceride content
Principle
This method permits the quantitation of monoglycerides (MAG) with one
LCFA, and triglycerides (TAG) with the same acyl carbon number (ACN)
in Salatrim by high-temperature capillary gas chromatography (HTCGC).
The ACN is the sum of the number of carbons of each carboxylic acid
sidechain of each TAG. (E.g., the ACN for tristearin is 54 (i.e., 3 x 18); the
ACNs for both dipropionylstearoylglycerol and diacetylarachidoylglycerol
are 24 (i.e., [(2 x 3) + 18] and [(2 x 2) + 20], respectively). MAG and TAG
are identified by comparison with standards. The weight percent of each
MAG and TAG in Salatrim is determined from the peak areas and
calibration curves constructed from data from analyses of standard
solutions.
Materials
MAG Standard: Monopentadecanoin (mono-C15) and monostearin
(mono-C18); purity: 99% minimum (available from Nu Check Prep., Inc.,
Elysian, MN, USA).
TAG Standards: Tricaproin (tri-C6), triheptanoin (tri-C7), tricaprylin (tri-C8),
trinonanoin (tri-C9), tricaprin (tri-C10), triundecanoin (tri-C11), trilaurin (tri-
C12), tritridecanoin (tri-C13), trimyristin (tri-C14), tripentadecanoin (tri-
C15), tripalmitin (tri-C16), triheptadecanoin (tri-C17), and tristearin (tri-
C18); purity: 99% minimum (available from Nu Check Prep., Inc., Elysian,
MN, USA).
Sample Preparation
Internal Standard Stock Solution: Dissolve 200 mg of tri-C11 in 2 litres of
undecane/toluene (95/5, v/v; spectroscopic grade).
Standard Solutions: Standard Solution (Group 1) - To each of twenty-two
10-ml volumetric flasks, add the thirteen TAG Standards so that each
flask, respectively, will contain 1600, 800, 400, 200, 100, 50, 32, 25, 20,
18, 16, 14, 12, 10, 9, 8, 7, 6, 5, 4, 3, and 2 mg/l of each TAG when filled to
the mark with the Internal Standard Stock Solution (tri-Cll; 100 mg/l).
Standard Solution (Group 2) - To each of seven 10-ml volumetric flasks,
add the two MAG Standards so that each flask, respectively, will contain
500, 250, 125, 62.5, 31, 15.6, and 7.8 mg/l of each MAG when filled to the
mark with the Internal Standard Stock Solution (tri-C11; 100 mg/l).
Salatrim Solution: Accurately weigh 2 g Salatrim into a 1-L volumetric
flask. Dilute to volume with Internal Standard Stock Solution.
Procedure
1. Instrumentation and Analysis Parameters
Instrument: Hewlett-Packard 5890 Series II GC equipped with flame-
ionization detector (FID), pressure programmable on-column injector, HF
7673 auto-sampler, and HP Series II integrator; or equivalent.
GC Column: Chrompack SIM-DIST CB fused-silica column (Chrompack
Inc., Raritan, NJ, USA) 5 m x 0.32 mm i.d.; 0.1 µm film thickness, or
equivalent. A deactivated fused-silica pre-column (0.5 m x 0.53 mm i.d.)
coupled to the analytical column via a butt connector (Quadrex Corp., New
Haven, CT, USA) or equivalent.
Oven Temperature: 140o to 350o at 10o/min; total run time 21.0 min.
Injector temperature: Track mode "ON" (injector temperature follows the
oven temperature conditions).
Injection Mode: On-column injection.
Sample Size: Inject 0.5 µl.
FID Temperature: 375o.
Flow Rate: Hydrogen gas constant flow mode "ON"; pressure: 5.5 psi
(140o).
2. Quantitation of MAG
Calibration Curve and Response Factor (RF) Determination for Mono-C15
and Mono-C18
Analyze each of the Standard Solutions of Group 2 using a sample
injection volume of 0.5 µl. From each chromatogram, establish the
response factors (RFi) for the two MAG using the formula:
RFi = (CIS/Ci) x (Ai/AIS)
where AIS is the peak area of the Internal Standard (tri-C11); Ai is the peak
area of a MAG; CIS is the concentration of the Internal Standard (100
mg/l); and Ci is the concentration (mg/l) of a MAG.
For each MAG, construct a calibration curve by plotting the peak area
ratios of (Ai/AIS) (x-axis) versus the RFi (y-axis) for each solution.
Weight Percent of MAG
Analyze the Salatrim sample using a sample injection of 0.5 µl. From the
chromatograms, obtain the peak area of each MAG (Ai) and the Internal
Standard (AIS). Example chromatograms of Salatrim are provided in the
Appendix to this monograph. Calculate the peak area ratio (Ai/AIS) and
determine the response factor for each MAG (RFi) from the calibration
curve. The concentration (µg/ml) of each MAG (Ci) in the Salatrim sample
is:
Ci = (CIS/RFi) x (Ai/AIS)
where CIS is the concentration of the Internal Standard (tri-C11; 100 mg/l).
The weight percent of each MAG in the Salatrim sample ((W%)i) is:
(W%)i = (Ci/CT) x 100
where CT is the concentration of Salatrim Solution (2000 mg/l). The total
weight percent of MAG in the Salatrim sample is:
Ó i (W%)i.
3. Quantitation of TAG with the Same ACN
Determination of the Response Factors for TAG Standards
Analyze each of the Standard Solutions of Group 1 using a sample
injection volume of 0.5 µl. The response factor for each TAG standard, j,
is:
RFn,j = (CIS/Cn,j) x (An,j/AIS)
where AIS is the peak area of the Internal Standard; An,j is the peak area of
the TAG standard with ACN = n (where n = 18-54 and the ACNs of the
TAG standards are as follows: tri-C6, 18; tri-C7, 21; tri-C8, 24; tri-C9, 27;
tri-C10, 30; tri-C11, 33; tri-C12, 36; tri-C13, 39; tri-C14, 42; tri-C15, 45; tri-
C16, 48; tri-C-17, 51; and tri-C18, 54)and concentration Cn,j (mg/l); CIS is
the concentration of the Internal Standard (100 mg/l).
The relative peak area of each TAG standard with ACN = n and
concentration Cn,j to that of the Internal Standard is given by:
RAn,j = An,j/AIS
The response factors and relative peak areas for each TAG with ACN = (n
+ 1) and with ACN = (n + 2) are calculated from the measured response
factors and relative peak areas for TAG standards with ACN = n and (n +
3) at the same concentration, Cn,j, according to the following:
RFn+1,j = RFn,j + (RFn+3,j - RFn,j) x 1/3
RAn+1,j = RAn,j + (RAn+3,j - RAn,j) x 1/3
RFn+2,j = RFn,j + (RFn+3,j - RFn,j) x 2/3
RAn+2,j = RAn,j + (RAn+3,j - RAn,j) x 2/3
Determination of the Response Factors for Salatrim
For the Salatrim Sample, the response factor for a TAG with a relative
peak area RAn,j is calculated from:
RFn,j = RFn,j-1 + (RFn,j+1 - RFn,j-1) x [(RAn,j - RAn,j-1)/(RAn,j+1 - RAn,j-1)]
where RFn,j+1 and RFn,j-1 are the response factors and RAn,j+1 and RAn,j-1
are the relative peak areas, respectively, of the TAG standards with the
same ACN value. RAn,j+1 and RAn,j-1 must meet the following condition:
RAn,j-1 < RAn,j < RAn,j+1
where RAn,j is the ratio of the peak area of the TAG in Salatrim with ACN =
n, (An,j), to the peak area for the Internal Standard (AIS). (NOTE: The
notation "j+1", “j”, and "j-1" denotes consecutive concentration values in
the series of standard solutions. For example, for a TAG standard with
ACN = n and concentrations of 100, 200, and 400 mg/L, the relative peak
areas RAn,j+1 and RAn,j-1 correspond to data for standards with
concentrations of 400 mg/l and 100 mg/l, respectively.)
Weight Percent Determination of Salatrim TAG Components
The concentrations (mg/l) of TAG in the Salatrim Sample with ACN = n are
given by
Cn,j = (CIS/RFn,j) x (An,j/AIS)
where CIS is the concentration of the Internal Standard (tri-C11; 100 mg/l).
The weight percent of a TAG with ACN = n in Salatrim is:
(W%)n = (Cn,j/CT) x 100
where CT is the concentration of the Salatrim sample (2000 mg/l).
The total weight percent of TAG in the Salatrim sample is:
Ó n (W%)n
II. Determination of the
SCFA/LCFA mole ratio
Principle
Triglycerides are converted to fatty acid butyl esters and