A CONTRACT LABS APPROACH FOR PROTEIN ID AND CONTENT DETERMINATION - - PowerPoint PPT Presentation

A CONTRACT LAB’S APPROACH FOR PROTEIN ID AND CONTENT DETERMINATION

Spencer Carter, PhD August 28, 2018 AOAC Annual Meeting

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• Estimated that 4 billion metric tons of food

protein is produced globally

• Estimated that $94M was lost by changing the

nitrogen-to-protein factor for dairy products from 6.38 to 6.25 in Europe in 2006

• Proteins make up $4.7B dollars in the Sports

Nutrition industry, which represents 70% of the total revenue in that category

SIGNIFICANCE OF PROTEINS

PROTEIN ADULTERATION

• Non-selective protein methods have fueled the potential to adulterate

samples with non-proteins and give inaccurate results

• Melamine, urea, free amino acids cannot be differentiated using Kjeldahl,

Dumas methods and have been adulterants

• Public health is still at risk; Economics still push adulteration

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• Historically, no direct analysis of proteins for

identification

• Instead, an indirect approach is taken. For

example, FCC monograph for whey protein identification requires:

• Ash
• Fat
• Lactose
• Loss on drying
• Nitrogen (and apply conversion factor)

PROTEIN IDENTIFICATION

NEW APPROACH FOR PROTEIN IDENTIFICATION

• Dyad Labs (previously Genysis Labs) developed a LC/MS/MS method for

identification of proteins in raw materials and finished goods

• Method is part of Dyad Labs ISO 17025 accreditation scope
• Method received First Action status for AOAC Official Method of Analysis in

December 2017

• AOAC 2017.11 Pea, Rice and Soy Proteins
• AOAC 2017.12 Milk Protein

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AOAC 2017.11 AND 2017.12 METHOD SUMMARY

• Enzymatic digestion using trypsin
• Separate peptides using Phenomenex Luna C18, 150x2 mm, 3 µm
• Shimadzu Nexera for 7.5 minute gradient with A: 0.1% formic acid in water

and B) acetonitrile

• Sciex API 5500 (triple quadrupole) to monitor three peptides from each

protein source

• Three peptide peak transitions each have peak area > ~2000 ppm = positive

confirmation of protein

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• Specific regions of the

protein’s amino acid sequence were selected for analysis

• Region is unique to the

protein being identified

• Tryptic peptides from

the sequence are monitored

PEPTIDE SELECTION

PEPTIDE SELECTION EXAMPLE:

PEA VS. SOY

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Pea Vicilin : PFNLRSRGPIYSNEFGKFFEITPEKNPQLQDLDIFVNSVEIKEGSLLLPHYNSRAIVIVTVNEGKGDFELVGQRNENQQ Soy ß-conglycinin : PFNLRSRNPIYSNNFGKFFEITPEKNPQPRDLDIFLSSVDINEGALLLPHFNSKAIVILVINEGDANIELVGIKE—QQQ GPIYSNEFGK NPIYSNNFGK FFEITPEK FFEITPEK GDFELVGQR AIVILVINEGDANIELVGIK EGSLLLPHYNSR DLDIFLSSVDINEGALLLPHFNSK

Same cleavage sites Different sequences Unique peptides Same cleavage sites Same sequences Identical peptides Different cleavage sites Unique peptides

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EXAMPLE: PEA RAW MATERIAL SAMPLE

Rice Pea Milk

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EXAMPLE: MILK FINISHED PRODUCT SAMPLE

Rice Pea Milk

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• Kjeldahl used most for

contract lab testing in nutraceutical/food industry

• Dumas being used

more

• Other techniques:

amino acids, dye- binding, etc.

PROTEIN CONTENT TESTING

• 1. Acid digestion converts nitrogen to ammonium sulfate
• 2. Neutralize to convert to free ammonia
• 3. Distill ammonia into boric acid
• 4. Back titrate with alkali
• 5. Convert nitrogen concentration to protein using conversion ratio

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KJELDAHL METHOD SUMMARY

• 1. Combust samples at high temp with oxygen to form water,

carbon dioxide, nitrogen

• 2. Remove water and carbon dioxide using column
• 3. Nitrogen is measured using a thermal conductivity detector
• 4. Convert nitrogen concentration to protein using conversion ratio

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DUMAS METHOD SUMMARY

• Dumas determines total nitrogen including inorganic fractions like

nitrite and nitrate

• Kjeldahl determines only organic nitrogen and ammonia
• Neither method is selective, since protein is not directly analyzed
• Neither method can detect adulteration; separate analyses by other

methods must be used to do so

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KJELDAHL/DUMAS SELECTIVITY

KJELDAHL/DUMAS ACCURACY AND PRECISION

• Dumas typically provides slightly higher recovery than Kjeldahl
• Dyad Labs analyzed 25 different samples; Mean recoveries:
• Lysine, nicotinic acid, and other compounds have difficulty digesting and are not

fully recovered using Kjeldahl

• AOAC 2001.11 reports recovery of lysine at only 87%
• Kjeldahl and Dumas provide similar, tight precision

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KJELDAHL DUMAS

80.9% 81.3%

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MASTER TITLE STYLE

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KJELDAHL/DUMAS ANALYSIS TIME

KJELDAHL (HRS) DUMAS (HRS) Sample Collection 0.5 0.5 Sample Prep 1.5 0.5 Instrument Time 2.5 1.5* Total Time 4.5 2.5 Total Labor 4.5 1 For a batch of ~15 samples, average time for Dyad technician:

*Dyad instrument can run unattended

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• Chemicals/standards/reagents

used in Kjeldahl:

• Concentrated Sulfuric Acid
• Sodium Hydroxide
• Bromothymol Blue
• 95% Alcohol
• Hydrochloric Acid
• Boric Acid
• Catalyst: copper sulfate +

potassium sulfate

• Gases used in Dumas:
• helium
• oxygen

KJELDAHL/DUMAS SAFETY

KJELDAHL/DUMAS COST

• Estimated* cost per sample for a batch of ~ 15 samples:

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KJELDAHL DUMAS

$7.50 $4.00

*based on typical batch size; includes direct costs only, such as labor, consumables, and instrument maintenance

• Dyad charges same price for Kjeldahl or Dumas test

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MASTER TITLE STYLE

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COMPARISON

KJELDAHL DUMAS

Analysis Time Ease of Operation Cost Safety Accuracy Precision Selectivity

X X

• LC/MS/MS provides more selective identification of proteins than
• ther methods
• Dumas is the preferred approach for quantitative testing
• For total protein testing, the approach Dyad takes is LC/MS/MS for

identification, Dumas for quantitative testing, and targeted tests for positive confirmation of potential adulterants

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CONCLUSIONS

Thank You.