National Institute of Standards & Technology
Report of Investigation
Reference Material 8037
Krill Oil
Reference Material (RM) 8037 is intended primarily for use in evaluation of analytical methods for determining fatty
acids in fish oils, dietary supplements, and similar materials. This RM provides a common matrix to those in the
natural products community who wish to conduct research studies. A unit of RM 8037 consists of three screw-top
tins, each containing approximately 4.5 mL of krill oil.
The development of RM 8037 was a collaboration between the National Institute of Standards and Technology (NIST)
and the National Institutes of Health Office of Dietary Supplements (NIH-ODS).
Reference Mass Fraction Values of Fatty Acids: Reference mass fraction values for fatty acids in RM 8037 are
provided in Table 1. A NIST reference value is a noncertified value that is the best estimate of the true value based
on available data; however, the value does not meet the NIST criteria for certification [1] and is provided with an
associated uncertainty that may reflect only measurement reproducibility, may not include all sources of uncertainty,
or may reflect a lack of sufficient statistical agreement among multiple analytical methods. The reference mass
fraction values were derived from results reported by NIST and/or collaborating laboratories. Values are reported on
an as-received basis in mass fraction units of free fatty acids in RM 8037 [2].
Expiration of Value Assignments: RM 8037 is valid, within the measurement uncertainty specified, until
31 December 2029, provided the RM is handled and stored in accordance with instructions given in this Report of
Investigation (see “Instructions for Storage and Use”). This report is nullified if the RM is damaged, contaminated,
or otherwise modified.
Maintenance of RM Value Assignment: NIST will monitor this RM over the period of its value assignment. If
substantive technical changes occur that affect the value assignments before the expiration of this report, NIST will
notify the purchaser. Registration (see attached sheet or register online) will facilitate notification.
Coordination of the technical measurements leading to the value assignment of this RM was performed by
C.A. Rimmer and B.J. Place of the NIST Chemical Sciences Division.
Technical consultation was provided by J.M. Betz (NIH-ODS). Acquisition and preparation of the material was
coordinated by M.M. Schantz formerly of NIST.
Coordination of the distribution of materials and reporting of measurement results for an interlaboratory comparison
exercise were performed by M.M. Phillips and L.J. Wood of the NIST Chemical Sciences Division and by W. Koshute
and B. St. Amant of the Grocery Manufacturers Association (GMA).
Analytical measurements at NIST were performed by M.M. Schantz.
Statistical analysis was provided by J.H. Yen of the NIST Statistical Engineering Division.
Support aspects involved in the issuance of this RM were coordinated through the NIST Office of Reference Materials.
Carlos A. Gonzalez, Chief
Chemical Sciences Division
Gaithersburg, MD 20899 Steven J. Choquette, Director
Report Issue Date: 20 April 2020 Office of Reference Materials
RM 8037 Page 1 of 4
Analysts at the following laboratories performed measurements that contributed to the value assignment of fatty acids
in RM 8037 Krill Oil as part of a GMA Food Industry Analytical Chemists Share Group (FIACSG) interlaboratory
comparison exercise: ConAgra Foods (Omaha, NE); Covance Inc.(Madison, WI); Covance Pte. Ltd. (Asia); The
Synergy (Singapore); Covance Inc. (Harrogate, UK); Del Monte Foods (Walnut Creek, CA); Eurofins Central
Analytical Laboratories (Metairie, LA); Eurofins Scientific (Des Moines, IA); General Mills Inc. (Golden
Valley, MN); Hormel Foods (Austin, MN); Krueger Food Labs (Chelmsford, MA); Land O’ Lakes (Arden
Hills, MN); Mereiux NutriSciences Brasil (Sao Paolo, Brazil); Nestlé Brasil Ltda. (Sao Paolo, Brazil); Nestle Quality
Assurance Center (Dublin, OH); Schwan Food Company (Salina, KS); The J.M. Smucker Co. (Orrville, OH); and
The National Food Laboratory (Livermore, CA).
NOTICE TO USERS: RM 8037 IS INTENDED FOR RESEARCH USE; NOT FOR HUMAN OR ANIMAL
CONSUMPTION.
INSTRUCTIONS FOR STORAGE AND USE
Storage: The RM should be stored frozen (–20 °C or colder), in original unopened tins, until required for use. Value
assignment does not apply to contents of previously opened and stored tins as the stability of fatty acids has not been
investigated in previously opened tins.
Use: Before use, the contents of the tin should be allowed to warm to room temperature and mixed thoroughly. To
relate analytical determinations to the values in this Report of Investigation, a minimum test portion size of 0.2 g
should be used to ensure valid results. Results obtained in analyses should include their own estimates of uncertainty
and can be compared to the non-certified or reference values using procedures described in reference 3.
SOURCE, PREPARATION, AND ANALYSIS(1)
Source and Preparation: The RM is a krill oil prepared by a commercial manufacturer. A 5 kg container of krill
oil was received at NIST from a commercial manufacturer of krill oil. The oil was warmed to room temperature and
gently rotated to mix the contents. Approximately 4.5 mL of krill oil was transferred into screw-top tins, then stored
at –20 °C.
Analytical Approach for Determination of Fatty Acids: Value assignment of the mass fractions of fatty acids in
RM 8037 was based on the combination of measurements made using gas chromatography with flame ionization
detection (GC-FID) at NIST and collaborating laboratories, where appropriate.
GC-FID measurements at NIST: Mass fractions of free fatty acids were determined from duplicate, nominal 0.2 g
test portions from 10 screw-top tins of RM 8037. The test portions of krill oil were weighed into 15 mL centrifuge
tubes with Teflon caps. One milliliter of chloroform was added to each tube volumetrically and 1 mL of internal
standard solution containing myristic acid-d27 and palmitic acid-d31 was added gravimetrically. Additions of 1.5 mL
of 1.0 mol/L NaOH (in methanol); 2.0 mL of 14 % (volume fraction) boron trifluoride (BF3 in methanol); 3 mL of
40 mg/L BHT (in hexane); and 5 mL of saturated NaCl (aq.) were added and, after each addition, the samples were
blanketed with N2, capped, and heated in a dry bath. A portion of the hexane/BHT layer was then transferred to an
autosampler vial for GC-FID analysis. Analysis was by GC-FID using a 0.32 mm × 30 m FAMEWAX column with
a 0.25 µm film. Calibrants were prepared gravimetrically from SRM 2377 Fatty Acid Methyl Esters in 2,2,4-
Trimethylpentane, at levels intended to approximate the levels of fatty acids in RM 8037. A single internal standard
solution was used for the calibrants and samples. Calculations are based on average response factors for the calibrants.
Collaborating Laboratories’ Analyses: The GMA FIACSG laboratories were asked to use their usual methods to
make single measurements on test portions taken from each of two tins of RM 8037 for measurements of fatty acids.
Because of the variability among data provided by laboratories participating in an interlaboratory comparison exercise,
the median of laboratory means is used, with the uncertainty estimated using the median absolute
deviation (MADe) [4]. The median of the collaborating laboratories’ means was combined with NIST data for
calculation of reference values of fatty acids, where appropriate, with the uncertainty estimated using the standard
error of the mean of laboratory means. The methods used by collaborating laboratories were all reported as utilizing
a sample hydrolysis and derivatization step followed by analysis using GC-FID.
(1) Certain commercial equipment, instruments, or materials are identified in this report of investigation in order
to specify adequately the experimental procedure. Such identification does not imply recommendation or endorsement by the
National Institute of Standards and Technology, nor does it imply that the materials or equipment identified are necessarily the best
available for the purpose.
RM 8037 Page 2 of 4
Homogeneity Assessment: The homogeneity of fatty acids was assessed at NIST using the methods and test portion
sizes described above. Analysis of variance showed possible statistically significant heterogeneity for
(Z)-9-Tetradecenoic Acid (C14:1), Octadecanoic Acid (C18:0), (Z)-9-Octadecenoic Acid (C18:1 n-9),
(Z)-11-Octadecenoic Acid (C18:1 n-7), and (Z,Z)-9,12-Octadecadienoic Acid (C18:2 n-6), and the uncertainties for
these values incorporate an additional component for possible heterogeneity.
Value Assignment: For calculation of assigned values for analytes that were measured only at NIST, the mean values
from NIST results were used. The collaborating laboratories reported the individual results for each of their analyses
for a given analyte. The mean of each laboratory’s results was then determined. For calculation of assigned values
for analytes that were measured only by the GMA FIACSG laboratories, the median of the laboratory means was used.
For analytes that were also measured by NIST, the mean of the individual sets of NIST data were averaged with the
median of the individual GMA FIACSG laboratory means.
Reference Mass Fraction Values for Fatty Acids as Free Fatty Acids: Each reference mass fraction value is the
mean result of a NIST analysis using a single method or the mean from the combination of NIST results with the
median of the mean results provided by collaborating laboratories where appropriate. Values are expressed as
x ± U95%(x), where x is the estimated value and U95%(x) is the expanded uncertainty of the value. The method-specific
value of the analyte lies within the interval x ± U95%(x) with about a 95 % confidence [5-7]. The measurands are the
mass fractions of fatty acids in krill oil listed in Table 1, on an as-received basis, as determined by the method
indicated. Metrological traceability is to the measurement processes and standards used by NIST and collaborating
laboratories.
Table 1. Reference Mass Fraction Values for Fatty Acids (as Free Fatty Acids) in RM 8037
Analyte Common Name Mass Fraction
(mg/g)
Octanoic Acid (C8:0)(a) Caprylic Acid 0.061 ± 0.008
Decanoic Acid (C10:0) (a)
Capric Acid 3.15 ± 0.23
Dodecanoic Acid (C12:0) (a,b)
Lauric Acid 1.35 ± 0.13
Tetradecanoic Acid (C14:0) (a,b)
Myristic Acid 60 ± 11
(Z)-9-Tetradecenoic Acid (C14:1)(a,b) Myristoleic Acid 1.05 ± 0.10
Hexadecanoic Acid (C16:0)(a,b) Palmitic Acid 128.5 ± 7.8
(Z)-9-Hexadecenoic Acid (C16:1 n-7) (a,b)
Palmitoleic Acid 33.8 ± 2.9
Octadecanoic Acid (C18:0) (a,b)
Stearic Acid 6.9 ± 1.3
(Z)-9-Octadecenoic Acid (C18:1 n-9) (a,b)
Oleic Acid 60.8 ± 4.9
(Z)-11-Octadecenoic Acid (C18:1 n-7)(a,b) Vaccenic Acid 39.7 ± 2.4
(Z,Z)-9,12-Octadecadienoic Acid (C18:2 n-6) (a,b)
Linoleic Acid 16.3 ± 2.5
(Z,Z,Z)-9,12,15-Octadecatrienoic Acid (C18:3 n-3) (a,b)
α-Linolenic Acid 8.50 ± 0.98
(Z,Z,Z)-6,9,12-Octadecatrienoic Acid (C18:3 n-6) (a,b)
γ-Linolenic Acid 1.17 ± 0.29
Docosanoic Acid (C22:0)(a,b) Behenic Acid 0.31 ± 0.09
(Z)-11-Eicosenoic Acid (C20:1 n-9)(a) Gondoic Acid 3.88 ± 0.16
(Z,Z,Z,Z)-5,8,11,14-Eicosatetraenoic Acid (C20:4 n-6) (a,b)
Arachidonic Acid 2.49 ± 0.10
(Z)-13-Docosenoic Acid (C22:1 n-9) (a,b)
Erucic Acid 3.50 ± 0.51
(Z,Z,Z,Z,Z)-5,8,11,14,17-Eicosapentaenoic Acid (a,b)
EPA 118.8 ± 3.8
(Z,Z,Z,Z,Z)-7,10,13,16,19-Docosapentaenoic Acid (C22:5 n-3)(a,b) DPA 3.28 ± 0.14
(Z,Z,Z,Z,Z,Z)-4,7,10,13,16,19-Docosahexaenoic Acid (C22:6 n-3)(a,b) DHA 74 ± 11
Tetracosanoic Acid (C24:0)(a) Lignoceric Acid 0.430 ± 0.042
(Z)-15-Tetracosenoic Acid (C24:1 n-9)(a) Nervonic Acid 1.08 ± 0.14
(a) NIST GC-FID.
(b) Collaborating Laboratories. Reported methods included hydrolysis and derivatization followed by GC-FID.
RM 8037 Page 3 of 4
REFERENCES
[1] May, W.; Parris, R.; Beck, C.; Fassett, J.; Greenberg, R.; Guenther, F.; Kramer, G.; Wise, S.; Gills, T.;
Colbert, J.; Gettings, R.; MacDonald, B.; Definitions of Terms and Modes Used at NIST for Value-Assignment
of Reference Materials for Chemical Measurements; NIST Special Publication 260-136; U.S. Government
Printing Office: Washington DC (2000); available at https://www.nist.gov/system/files/documents/srm/SP260-
136.PDF (accessed Apr 2020).
[2] Thompson A.; Taylor, B.N.; Guide for the Use of the International System of Units (SI); NIST Special
Publication 811; National Institute of Standards and Technology; U.S. Government Printing Office:
Washington, DC (2008); available at https://www.nist.gov/pml/special-publication-811 (accessed Apr 2020).
[3] Sharpless, K.E.; Duewer, D.L.; Standard Reference Materials for Analysis of Dietary Supplements; J. AOAC
Int., Vol. 91, pp. 1298–1302 (2008).
[4] Huber, P.J; Robust Statistics; John Wiley: New York (1981).
[5] JCGM 100:2008; Evaluation of Measurement Data — Guide to the Expression of Uncertainty in Measurement;
(GUM 1995 with Minor Corrections); Joint Committee for Guides in Metrology (JCGM) (2008); available at
https://www.bipm.org/utils/common/documents/jcgm/JCGM_100_2008_E.pdf (accessed Apr 2020); see also
Taylor, B.N.; Kuyatt, C.E.; Guidelines for Evaluating and Expressing the Uncertainty of NIST Measurement
Results; NIST Technical Note 1297: U.S. Government Printing Office: Washington, DC (1994); available at
https://www.nist.gov/pml/nist-technical-note-1297 (accessed Apr 2020).
[6] JCGM 101:2008; Evaluation of Measurement Data – Supplement 1 to the Guide to the Expression of
Uncertainty in Measurement — Propagation of Distributions using a Monte Carlo Method;
JCGM (2008); available at https://www.bipm.org/utils/common/documents/jcgm/JCGM_101_2008_E.pdf
(accessed Apr 2020).
[7] Efron, B.; Ribshirani, R.J.; An Introduction to the Bootstrap; Chapman & Hall, London, UK (1993).
Users of this RM should ensure that the Report of Investigation in their possession is current. This can be
accomplished by contacting the SRM Program at: telephone (301) 975-2200; fax (301) 948-3730; e-
mail [email protected]; or via the Internet at https://www.nist.gov/srm.
RM 8037 Page 4 of 4
