OverviewPurpose. Develop a method for the rapid analysis of 4-methylimidazole in beer.Methods. Reverse phase HPLC-MS/MSResults. A simple dilute-and-shoot procedure using a labeled internal standard, coupled with the high selectivity a mass spectrometer, can provide a rugged method for the analysis for 4-methylimidazole in beer.

IntroductionMolecular Weight: 82.11

4-Methylimidazole (4-MeI) is highly water soluble and relatively ubiquitous.

Sources of 4-MeI Class III and IV caramel coloring (ammoniated caramel colors) Fermentation byproduct Beverages, Soy Sauce, Worcestershire Sauce, Ammoniated Livestock Feed

California Prop 65 Listing January 2011, 4-MeI was added to the Prop 65 List.

Decision to list the chemical based on toxicology report issued by the NTP (National Toxicology Program).

4-MeI considered to be a known carcinogen.

NSRL (No Significant Risk Level) set at 16 µg/day - Any 12 oz beverage containing a 4-MeI concentration of 45 ng/mL or more

would put you over this limit.

By January 7, 2012, companies selling products containing 4-MeI must provide a “clear and reasonable” warning before knowingly and intentionally exposing anyone to the chemical.

All finished beer products sold in California will require the manufacturer to know their 4-MeI content. Therefore, an efficient, rugged method for the rapid determination of 4-MeI content is needed. This presentation focuses on a simple approach to achieving this.

Keys to AnalysisUtilization of Mass Spectrometer Detector High selectivity of detector Provides mass confirmation of multiple daughter ions in addition to retention time

confirmation in the chromatography

Use of a Stable Labeled Internal Standard Makes a dilute-and-shoot procedure possible due to the ability of the internal

standard to compensate for matrix effects

Verification That the Internal Standard Does Not Contribute to 4-MeI Prior to use, the internal standard

must be tested for contribution to the analyte channel.

A pure solution of 4-methyl-d3-imidazole at 10.0 ng/mL was prepared and analyzed.

Internal standard tested clean, as documented at right.

Method for the Rapid Determination of 4-Methylimidazole in Beer Using LC-MS/MS

Matthew Eckert, Covance Laboratories Inc., Madison, Wisconsin

Materials/Methods

Pure Low Standard 4-MeI 83→56

Pure Low Standard 4-Me-d3-I 86→59

Pure High Standard 4-MeI 83→56

Pure High Standard 4-Me-d3-I 86→59

Blank “Amber” Beer Extract 4-MeI 83→56

Blank “Amber” Beer Extract 4-Me-d3-I 86→59

Summary of Results

Reagents Concentrated Ammonium

Hydroxide (37%), J.T. Baker Purified Water, Milli-Q Acetonitrile, HPLC grade,

Sigma-Aldrich

Matrix “Amber” Beer

Standards 4-Methylimidazole, Sigma-Aldrich 4-Methyl-d3-imidazole, C/D/N Isotoptes

Equipment Column: Varian Polaris 3 C-18A, 150 x 4.6 mm, 3 µm HPLC: Shimadzu CBM 20 series Detector: AB Sciex (API 4000) mass spectrometer Analytical Balance, Sartorius Volumetric flasks and pipettes Positive displacement pipettes Sonicator bath Millex PTFE 0.2 µm syringe filter 2-mL glass injection vials

Stock/Standard Solutions 4-MeI stock solution, ~100 µg/mL prepared in purified water 4-Me-d3-I internal standard stock solution, ~100 µg/mL prepared in purified water 4-MeI intermediate solution, 100 ng/mL, prepared in purified water 4-Me-d3-I intermediate internal standard solution, 100 ng/mL prepared in

purified water Pure Calibrator solutions (n=6) with 4-MeI concentrations of 0.200, 0.400, 1.00,

2.00, 10.0, and 50.0 ng/mL, each with a 4-Me-d3-I concentration of 2.00 ng/mL All solutions stored refrigerated, protected from light - Stock stability for at least 60 days has been established

Extraction Procedure Remove carbonation from the beer sample by placing in a sonicator bath for

~20 minutes Accurately measure a 1.00 mL aliquot of degassed beer into a 10-mL volumetric

flask Spike the sample with 0.200 mL of 100 ng/mL 4-Me-d3-I intermediate internal

standard solution Bring to final volume with purified water and mix thoroughly Filter diluted sample with a PTFE 0.2 µm syringe filter into an injection vial for

analysis

HPLC Conditions Mobile Phase A: 5 mM

ammonium hydroxide in water Mobile Phase B: Acetonitrile Flow rate: 0.400 mL/min Column temperature: 40ºC Initial conditions: 2% Mobile

Phase B Gradient Time (min) % B 0.25 2 10.0 40 15.0 10 20.0 2 22.0 Controller Stop

Mass Spectrometer Parameters ESI+ (Electrospray ionization, positive

mode) CAD Gas: Nitrogen, setting 11 Curtain Gas: Nitrogen, setting 25 Ion Source Gas 1: Nitrogen, setting 70 Ion Source Gas 2: Nitrogen, setting 80 Source Temperature 550ºC Ionspray Voltage: 2500 V

Mass Dwell Time Collision Compound Transition (msec) Energy (eV)4-MeI Quantification ion 83→56 100 264-MeI Qualification ion 1 83→42 100 384-MeI Qualification ion 2 83→68 100 394-Me-d3-I 86→59 100 28

Assay LOD 2.00 ng/mL (low standard

concentration 0.200 ng/mL x dilution factor of 10)

Mean Concentration (n=18) 5.17 ng/mL, RSD 2.94%

Spiked Recoveries (n=27) Mean accuracies 87.9% - 95.8% RSD range 4.51% - 5.43%

Linearity Linear regression (1/x weighting) r = 0.9996 to 0.9999 over three batches

Conclusions Utilization of HPLC-MS/MS for analysis of 4-MeI is desirable Availability of labeled internal standard for 4-MeI makes dilute-and-shoot analysis

possible with its ability to account for matrix effects A quality, rugged method can be still be obtained while having a simple

extraction procedure

References“Solid-phase extraction of 4(5)-methylimidazole (4-MeI) and 2-acetyl-4(5)-(1,2,3,4-tetrahydroxybutyl)-imidazole (THI) from foods and beverages with subsequent liquid chromatographic-electrospray mass spectrometric quantification”, by Klejdus, B., Moravcova J., Lojkova L., Vacek J., Kuban V., Journal of Separation Science, 2006, 29, 378-384.Reproductive and Cancer Hazard Assessment Branch Office of Evironmental Health Hazard Assessment California Environmental Protection Agency, Package 32b, March 2009.NTP Technical Report on the Toxicity Studies of 2- and 4-Methylimidazole. National Toxicology Program, Toxicity Report Series Number 67, April 2004, NIH Publication No. 04-4409.www.caramel.com, “Technical Overview of Caramel Colors”www.ChemSpider.com

Presented at the74th ASBC Annual MeetingFort Myers, Florida11-15 June 2011

Method for the Rapid Determination of 4-Methylimidazole in Beer Using LC-MS/MS

Matthew EckertCovance Laboratories Inc.Madison, Wisconsin

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