Melatonin Analysis in Food by Chromatography

Jack OwensTaylor PatekAlyssa Rosas

Victoria PeckhamChris Omiwade

DJ Riddle

Chuck RosalesHaris Shafi

Rohit RaghuJustin TeperaBen Simpson

4/24/14

(Tandem Mass Spectrometry)

Cell 8

Anatomy of Analysis

Poor Bob…Hey There Bob!Anatomy of Analysis: Tomato Edition

Melatonin on the Circadian Sleep Cycle

• What does it do?• How it affects Circadian rhythm• Naturally produced by body– Amount depends on environment

• Found everywhere– Fun Fact!

Why is the Analysis Important?

• Melatonin as an antioxidant–Can terminate NO, OH, O2

-

–Amphiphilic–Cascade reaction

Objective of the Analysis

• To determine the amount of melatonin present in different foods

• Find a reliable method by which to do this

Fundamental Principles

Reversed Phase Chromatography

• Solute (in mobile phase) + hydrophobic immobilized = hydrophobic interaction

• Molecules must possess some degree of hydrophobic character

• Excellent recovery and resolution• Binding interaction results in increased

entropy

The System Used

• Stationary phase is hydrophobic and nonpolar.– diisobutyl n-octadecylsilane

• Mobile phase: 0.1% FA in water and 0.1% FA in MeCN (65:35 v/v)

• Solute binds to the stationary phase• Water around stationary phase is

displacedincrease in entropy

Strategy Used

• Sample Preparation– Low Moisture samples (green tea, walnut, cacao,

bread)– High Moisture samples (sour cherry, tomato, olive,

yogurt)– Liquid samples (red wine, beer)

• Ethanol Extraction– Supernatants obtained, chilled, filtered

Strategy (cont’d)

• Sample Prep (cont’d)– Evaporated– Re-dissolved– Put in autosampler

• Time for some chromatographic action!

Chromatography

• HPLC system• Liquid Solvent- formic acid/acetonitrile mixture• Autoinjector• Rapid Resolution column• Electrospray allows for detection• Multiple Reaction Monitoring by triple

quadrupole mass spectrometer

Triple Quadrupole Mass Spectrometer

Formic Acid, Acetonitrile, Water mixture

Gaseous/Liquid Waste

Overall Setup

Electrospray Overview

Triple Quadrupole Mass Spectrometer Overview

Measurable Quantity

• Isolated via liquid chromatography• Quantifiable species: a specific molecular

fragment to each species– Melatonin: 174.2/233.2– Melatonin Isomer: 216.2/233.2

• Recovery Percentage

Calculating the Result

• Standard solutions for ion fragment ratios to concentration plotted

• Experimental samples placed on the plot to find concentrations

Melatonin Standard Walnut

Red WineBeer

Not a bad melatonin content, Bob!

Accuracy and Precision

• Sample Preparation• Solvent Used• Reverse Phase Chromatography• Triple Quadrupole Spectrometer

Discussion of Results• High melatonin isomer content

– red wine 170.7 ng/ml– Beer 14.3 ng/ml

• Low melatonin isomer content– probiotic yogurt 0.9 ng/g– Black olives 0.1 ng/g

• Deduced- yeast fermented>bacteria fermented• Thermal processing of food affects melatonin levels• Concentration of the melatonin isomer∝ activity of yeast• Melatonin Isomer concentration>Melatonin concentration

QUIZ TIME

Don’t get too excited, Bob… there’s candy at stake!

Q: Can you name 2 things that melatonin does in your body?

A:1)Regulates Circadian

Rhythm/sleep cycle2)Helps eliminate free radicals3)Antioxidant

Q: How does the triple quadrupole spectrometer increase the specificity of the experiment?

A: It is able to filter out ions based on their mass to charge ratio

Q: What type of chromatography is evident in this process, and what is the exact name of this process?

A:• Reverse phase chromatography• HPLC (High Pressure Liquid

Chromatography)

Q: Why does the sample need to hydrophobic?

A: Because the stationary phase is hydrophobic, and you need to promote adsorption to achieve results

References"ZORBAX Rapid Resolution | Agilent." ZORBAX Rapid Resolution | Agilent. N.p., 2014. Web. 23 Apr. 2014.

"Fundamentals of a First-Choice Mobile Phase : SHIMADZU (Shimadzu Corporation)." Shimadzu Corporation.N.p., n.d. Web. 23 Apr. 2014.

Kocadagli, Tolgahan, Cemile Yilmaz, and Vural Gokmen. "Determination of Melatonin and Its Isomer in Foodsby Liquid Chromatography Tandem Mass Spectrometry." Diss. Hacettepe U, 2013. Journal of

Agricultural and Food Chemistry 62.13 (2013): 2900-905. ScienceDirect. Elsevier. Web. 23 Apr. 2014.

Yokoyama, et al. Chemical Geology. 1999. 157.(3-4)

Reversed Phase Chromatography: Principles and Methods. Amersham Biosciences. Edition AA

http://www.webmd.com/brain/pineal-glandhttp://www.psychiatrictimes.com/sleep-disorders/role-melatonin-circadian-rhythm-sleep-wake-cyclew.webmd.com/sleep-disorders/tc/melatonin-overviewhttp://www.chemspider.com/Chemical-Structure.872.htmlhttp://www.immunehealthscience.com/foods-with-melatonin.html