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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
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