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Direct determination of MCPD esters and glycidyl esters by LC-TOFMS

May 16-19, Phoenix, AZ

AOCS Meeting

Mark W. Collison, Ph. D., Director, Analytical Chemistry,

Troy Haines, Analytical Laboratory Manager, ADM Research

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What are MCPD and glycidol?

• Monochloropropanediol

- Byproduct found in in soy sauce and hydrolyzed vegetable protein produced by HCl digestion

- Also reported previously in HCl hydrolyzed oils

- Reported in the last several years to be widespread in vegetable oils using chemical tests.

• Glycidol

- Also reported to be present in vegetable oils by the same chemical tests.

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MCPD Esters and Glycidyl Esters

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Chemical Methods of Analysis• Fat + Sodium Methoxide → FAME + glycerol etc.

• Acidify and extract using concentrated sodium chloride to partition (converts glycidol to MCPD)

• React with phenylboronate or other derivatizing agents

• Analyze MCPD-phenylboronate by GCMS.

• The DGF chemical method will be the one we compared with: DGF c-iii_18 _09_e14

• Alternative procedure utilized acid hydrolysis

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Why Develop an LCMS method?•The DGF method does not work adequately.

-ISTD recovery is acceptable ONLY if NaCl is used in the wash-Loss of virtually all ISTD if Na2SO4 is used in the wash-Conversion to the brominated form if NaBr is used in the wash.

•The DGF method claims the method analyzes the total of MCPD plusglycidol, but MCPD and glycidol have not been shown to be the only precursors that can produce MCPD under the reaction conditions.

•The difference between the two treatments in the DGF method has been implied as a measure of glycidol, but there is no evidence glycidol is the only substance which contributes to this difference in results.

•LCMS provides direct analysis of MCPD mono-esters and di-esters and glycidyl esters without chemical modifications that can giveincorrect results.•LCMS provides very specific correct identification of analytes.•LCMS allows use of isotopic internal standards for very accurateanalysis.

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Effect of Salts on the DGF method –Loss of MCPD in the absence of chloride

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Effect of Salts on the DGF method –Conversion of MCPD to MBPD in the presence of bromide

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LC-TOFMS Method for MCPD esters and glycidyl esters

•MCPD diesters do not ionize well to MH+ ions by either ESI or APCI•Current approach was used previously for diglycerides [1]•Low concentration of sodium acetate (~26 µM) in the mobile phase allows ESI ionization and detection of sodiated ions [MNa+]•High resolution TOF-MS detection allowed elimination of background interferences for certain MCPD esters.

-Eliminated by setting m/z resolution to 50 ppm (±0.016 da)

[1] Hannah L. Callender, Jeffrey S. Forrester, Pavlina Ivanova, Anita Preininger, Stephen Milne, and H. Alex Brown, “Quantification of Diacylglycerol Species from Cellular Extracts by Electrospray Ionization Mass Spectrometry Using a Linear Regression Algorithm” Anal. Chem. 2007, 79, 263-272

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LC-TOFMS

•Detects both mono- and di-esters of MCPD•Detection limit for diester standards is 10-25 µg/kg•Limit of quantitation for total MCPD esters in vegetable oil samples is about ~500 µg/kg

(~100 µg/kg MCPD equivalent concentration)

•Method also optimized for detection of glycidyl esters•Detection Limit of individual GS standards is ~5 µg/kg•Limit of quantitation for total glycidyl esters in vegetable oils ~200 µg/kg

(~40 µg/kg glycidol equivalent concentration)

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Standards• 3-MCPD monopalmitate, 3-MCPD monostearate, 3-MCPD

dipalmitate, were purchased from Toronto Research Chemicals, Inc (North York, ON, Canada).

• Glycidyl stearate was purchased from TCI America (Portland, OR, USA).

• Glycidyl linolenate, glycidyl linoleate, glycidyl oleate and glycidyl palmitate were purchased from Wako Pure Chemical Industries (Tokyo, Japan).

• Glycidyl-31d-palmitate synthesized enzymatically as internal standard for glycidyl esters

• 3-MCPD mono- and diesters of oleic, linoleic and mixed fatty acids, and deuterated internal standard (dioleyl-d5-MCPD), and glycidyl esters of palmitic, oleic and mixed fatty acids were synthesized enzymatically.

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LC and TOF-MS conditions• Methanol-Sodium Acetate - Methanol containing 0.26 mM sodium acetate

was prepared by adding 21.3 mg sodium acetate to 1 liter methanol.

• HPLC Mobile Phase A - 90% methanol 10% acetonitrile with 0.026 mM sodium acetate. Mix 100 mL Methanol-Sodium Acetate, 800 mL methanol and 100 mL acetonitrile.

• HPLC Mobile Phase B - 80% methylene chloride 10% methanol 10% acetonitrile with 0.026 mM sodium acetate. Mix 100 mL Methanol-Sodium Acetate, 800 mL methylene chloride and 100 mL acetonitrile.

• ISTD solution: 2030 µg/mL dioleyl-d5-MCPD and 180 µg/mL glycidyl 31d-palmitate in HPLC Mobile B

• Sample Preparation:-In a 12 x 32 HPLC vial, accurately weigh 1 drop (~20-25 mg) oil-Add 975 µL ISTD Solution.

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LC and TOF-MS conditions

• HPLC Column: Phenomenex Luna 3 µm C18 ,100Å, 50 x 3.0 mm.

• Flow Rate - 0.25 mL / min.

• TOF-MS –• Agilent 6210 TOF-MS upgraded to 4 GHz

- ESI at 3.5 kV- Gas Temp. 300°C

- Drying Gas 5 L/min.

- Nebulizer Pressure 50 psi

- Mass Range 300 to 700 m/z

• Polarity Positive, Instrument Mode 4 GHz, Data Storage - Centroid

• Monoheptadecanoin and Dinonadecanoin as Mass Reference Ions

Gradient ProgrammingRun Time % B0 min. 05 min. 015 min. 6516 min. 10020 min. 100

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571 ppb Glycidyl Palmitate485 ppb Glycidyl Stearate568 ppb Glycidyl Oleate478 ppb Glycidyl Linoleate444 ppb Glycidyl Linolenate

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5693 ppb 3-MCPD Monopalmitate2338 ppb 3-MCPD Monostearate12730 ppb 3-MCPD Monooleate10396 ppb 3-MCPD Monolinoleate1516 ppb 3-MCPD Monolinolenate

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7180 ppb 3-MCPD Palmitic-Palmitic Diester4347 ppb 3-MCPD Oleic-Oleic Diester10981 ppb 3-MCPD Oleic-Linoleic Diester6935 ppb 3-MCPD Linoleic-Linoleic Diester2022 ppb 3-MCPD Linoleic-Linolenic Diester2030 ppb d5-3-MCPD Oleic-Oleic Diester (ISTD)

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732 ppb 3-MCPD Palmitic-Oleic Diester924 ppb 3-MCPD Palmitic-Linoleic Diester251 ppb 3-MCPD Oleic-Stearic Diester147 ppb 3-MCPD Linolenic-Linolenic Diester135 ppb 3-MCPD Palmitic-Linolenic Diester

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GL, ng/mL 4.79 11.96 23.93 47.85 119.63 239.25 478.5

Typical Standard Curve

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MCPD Ester and Glycidyl Ester Content of Commercial Vegetable OilsSamples are from a variety of producers and were obtained from grocery stores and commercial suppliers

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Results for Commercial Refined Oils

•MCPD monoesters are never found in deodorized vegetable oils. •Glycidol is not likely to be the precursor of MCPD esters in commercial vegetable oils.

•MCPD diesters have not been found in commercial vegetable oils other than some samples of palm oil.

•Glycidyl esters were found in significant quantities in some commercial vegetable oils.

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DGF Method compared to LCMS method

DGF method predicts higher total MCPD + glycidol concentrations than LCMS when MCPD esters and / or glycidyl esters are present.

DGF Method still gives positive results even when MCPD and glycidyl esters are not detected by LC-TOFMS.

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MCPD Esters can form from contact with hydrochloric acid under mild conditions.

Conditions of acid treatment: 0.1 v/wt% acid, 2 min shear mix, 60 min stir mix, 70 C, water wash to neutral

Note that glycidyl stearate was not destroyed during this mild acid treatment and was not the precursor for MCPD formation.

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MCPD Esters can form from contact with HCl treated adsorbents during vegetable oil processing

* manufacturers confirmed that HCl was used in the acid washingSamples deodorized after treatments at 260 C, 30 min, 3 mm Hg, 3% steam sparge.Original experiment was an attempt to remove precursors of glycidyl esters and MCPD esters. RB Palm Oil was free of detectable MCPDE and GE prior to the experiment

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Glycidyl esters

•Diglyceride content seems to correlate somewhat with glycidyl ester content. High diglyceride content in oils predisposes them to formation of glycidyl esters

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Challenges in getting correct results from the LC-TOFMS method

• The instrument gets dirty quickly from the sodium in the mobile phase and requires a quick cleaning every day prior to use. More extensive cleaning is required weekly in heavy use.

• Certain parts in the ESI source corrode quickly and require frequent replacement. ESI Nebulizer needles are replaced weekly in heavy use.

• Close attention to internal standard areas and frequent check standards are needed to be certain that loss of intensity has not occurred.

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Conclusions• The harsh chemistry of the DGF method creates incorrect results in

the analysis of MCPD and glycidyl esters.

• Chemistry capable of transesterifying oils needs to be avoided in analysis of MCPD and glycidyl esters.

• Direct analysis by LCMS looks extremely promising.

• MCPD esters can form from contact with hydrochloric acid and from acid washed absorbents during vegetable oil processing.

• Glycidyl esters are widely present in vegetable oils. Glycidyl esters seem to be a much more widespread issue than MCPD esters.

• Glycidyl esters are not the precursor of MCPD esters.

• High diglyceride content predisposes oils to glycidyl ester formation

• Glycidyl esters and MCPD esters can be greatly reduced by processing.

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Laboratory treatments of soy, corn and palm oilMCPD diesters found by LC-TOFMS

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Recent Research• GCMS Confirmation of the MCPD esters found by LC-TOFMS.

- Samples were injected on a DB5 column, analyzed by MS on a Shimadzu QP-2010 Plus

- Samples that were negative by LC-TOFMS for MCPD esters had no detectable MCPD esters by GCMS

- Samples which were positive for MCPD esters by LC-TOFMS showed the same detectable MCPD esters by GCMS

- Difficult to maintain sensitivity due to detector fouling

- Complete chromatographic resolution of MCPD esters from corresponding glycerides is essential

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Atmospheric Pressure Photoionization (APPI)

• Utilizes UV light to ionize nebulized samples.

• Syagen is the patent holder and makes all APPI sources currently sold.

M + hν → M+ + e-

• MH+ ions are normally seen, though M+ ions are sometimes seen.

M+ + SH → MH+ + S

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APPI – MCPD monoesters and glycidyl esters

Addition of acetone as a dopant increases response of MCPD monoesters and glycidyl esters and decreases response of monoglycerides

All of which have identical LCMS spectra

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APPI – MCPD diesters

Favored reaction for saturated diesters

Favored reaction for unsaturated diesters

Addition of acetone as a dopant increases response of MCPD monoesters and glycidyl esters and decreases response of monoglycerides

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