A Reference Material Suite for Evaluating Seafood Authenticity and Safety

Abstract

Materials

Debra L. Ellisor1, Melissa Phillips

2

, Benjamin Place

2

, Catherine Rimmer

2

, Laura Wood

2

1 National Institute of Standards and Technology Materials Measurement Laboratory Charleston, South Carolina USA

2 National Institute of Standards and Technology Materials Measurement Laboratory Gaithersburg, Maryland USA

All materials used to generate the seafood suite were obtained

from sources verified authentic by the National Oceanic and

Atmospheric Administration Marine Forensics Unit:

o Wild-caught Coho salmon (NIST RM 8256)

- O. kisutch hook and line caught ~15 mi off the coast of Alaska

o Aquacultured Coho salmon (NIST RM 8257)

- O. kisutch procured from a land-based aquaculture facility in

Washington

o Wild-caught shrimp (NIST RM 8258)

- F. aztecus trawl caught off the coast of South Carolina

o Aqaucultured shrimp (NIST RM 8259)

- L. vannamei procured from an aquaculture facility in Texas

The authors would like to thank the National Oceanic and Atmospheric Administration Marine Forensics Unit for help in procuring and verifying sources of authentic material and the 

Reference Material Production team at the National Institute of Standards and Technology, Charleston for their assistance in processing and cryomilling the materials

Processing Methods Future Work and References

Analytical Methods

Analytical Results

o Salmon was scaled, fileted, chopped (including skin) and frozen

o Shrimp was headed, peeled, chopped and frozen

o Materials were cryohomogenized three times using a Palla VMKT Vibrating cryomill, generating a fine fresh-frozen powder

Seafood is one of the most highly-traded international

commodities. Pricing at import is determined by weight, species

and provenance, including whether the food is from aquacultured

or wild-caught sources. Each of these characteristics can be

falsified, resulting in an inflated payout, encouraging unsustainable

practices and affecting the US domestic seafood economy. The

inclusion of additives such as gels, dyes and antibiotics commonly

found in fish feed can also endanger the health of consumers. The

US Food and Drug Administration (FDA) and US Customs and

Border Protection (CBP) would benefit from seafood authenticity

reference materials to aid in identifying cases of import fraud. To

address this need, the National Institute of Standards and

Technology (NIST) is using shrimp and salmon, two of the three

most consumed seafood products in the US, to develop

authenticity reference materials. Wild-caught and aquacultured

Coho salmon and wild-caught and aquacultured shrimp were

procured from reliable sources and processed to include only the

edible portions. Each of the four materials was cryomilled and

bottled at the NIST Reference Material Production Facility in

Charleston, SC. Preliminary analysis reveals differences in

metabolomic profiles between materials of differing origin. The

anticipated differences in DNA profiles, elemental ratios and total

polar/semi-polar constituents will allow regulatory agencies to

better identify fraudulent trade items at import and provide a

standardization tool for typical analyses conducted at inspection

laboratories.

Processing Results

o Fresh-frozen powder was subsampled during each of the three

rounds of cryohomogenization for a preliminary homogeneity

assessment using a Malvern Mastersizer 3000 for laserdiffraction particle size analysis

o A small amount of powder was added to ethanol dispersant in

the sampling reservoir and 10 measurements were made per

subsample to obtain an average particle size for each round of

milling

o Three rounds of cryomilling proved sufficient to effectively

homogenize the materials according to ISO standard 13320 for

particle size distribution (1)

o 2 g aliquots of each wild-caught and aquacultured salmon

material were extracted using hydroxylamine and acetonitrile,

dried with magnesium sulfate and vortexed

o Supernatant was transferred to a clean tube, taken to dryness and

reconstituted in ascorbic acid solution in acetonitrile according

to AOAC published methods (2)

o Extracts were analyzed by LC-MS/MS using high resolution

accurate mass (HRAM) with a Thermo Vanquish UPLC, Agilent

XDB-C18 column, and coupled to a Thermo Fusion Lumos to

generate full MS (120,000 resolution) and MS/MS (15,000

resolution) with high collisional dissociation (HCD)

o The data were analyzed using Compound Discoverer 2.1 to

identify feature differences between the wild-caught and

aquacultured materials

o Associated p-values were corrected using a BenjaminiHochberg (BH) procedure to control the false discovery rate

1. ISO 13320 (2009). Particle size analysis – Laser diffraction

methods

2. Schneider and Andersen (2015). Journal of AOAC International.

98(3), 658-670

0

1

2

3

4

5

6

0.1 1 10 100 1000 10000

Volume Density (%)

Size classes (μm)

Mill 1

Mill 2

Mill 3

Mill 1 Mill 2 Mill 3

Avg Dv(50) (μm) 111.00 58 39

1x SD 3.75 0.955 0.238

RSD (%) 3.39 1.65 0.61

o A volcano plot was used to visualize feature differences between

replicate aliquots of wild-caught and aquacultured salmon

materials

o Statistical differences were evident between the two materials

using a log fold Δ of 2 and a BH-adjusted p-value of 0.05

o Preliminary analysis of the two salmon materials suggests they

are fit-for-purpose in providing the authenticity community a

tool to help verify seafood source at import

o In the future, HRAM MS and MS/MS scans of the wild-caught

and aquacultured shrimp materials will be compared to ensure

differences can be identified

o All materials will be analyzed by targeted and non-targeted LCHRMS, ICP-MS and GC-MS using optimal sample preparation

methods to identify polar and non-polar compounds useful in

differentiating wild-caught and aquacultured materials

o Materials will be assessed by genetic methods for species

determination and to evaluate models for use in species and

provenance confirmation

o All RMs will be made available to the seafood authenticity

community to aid in identification and source of imported goods

o Specific compounds differentially present in the two salmon

materials were explored

o Ethoxyquin, a fat stabilizer used in fish feed in the US, was

found in the aquacultured, but not the wild-caught salmon

o Docosahexaenoic acid, an omega-3 fatty acid was found at

much higher levels in the wild-caught salmon compared to the

aquacultured salmon

Ethoxyquin Docosahexaenoic acid Peak Area (10

6

)

4

3

1

2

Peak Area (10

6

)

40

30

10

0

20

5

Environmental 

Health

Human

Health

Economic

Success

SUSTAINABILITY

0

5

10

15

20

25

30

35

40

-15 -10 -5 0 5 10 15 20

-log10 BH adjusted p

-value

log2 fold-change

1718 1190