Biological MS by Isotope

DilutionBy: Cryst Bambo

What is Isotope Dilution?

Isotopes

versions of an atom or an element that have the same number of protons, but

different numbers of neutrons.

i.e. 12C, 13C and 14C

Dilution

A technique that allows the process of reducing the concentration of a sample in a

solution.

Isotope Dilution

An analytical technique used for quantity determination by the addition of known

amounts of isotopically-enriched substance to the analyzed sample.

History – A Cup of Tea

Springtime of 1913

George Hevesy (1885) drinking a cup of tea at the Manchester Physics

Laboratory with his colleague Henry Moseley.

Hevesy wanted to determine the fate of the individual water molecules

contained in the cup of tea consumed, Moseley did not. 1915, Moseley

was shot by a Turkish sniper.

1933

E. Hofer, his new drinking buddy, consumed 2000 ml aliquots of

deuterated tea. Tested their own urine samples.

half of the body’s water turned over every 9 days and the body’s

water content is at 43 liters.

Isotope Dilution Mass Spectrometry(IDMS)

Isotope dilution is an analytical technique used in combination with mass

spectrometry to determine the concentration of wanted element in

unknown samples.

A known amount of “spike” with known elemental concentration

and isotopic abundances is added to

sample with unknown

elemental concentration but

known isotopic abundances.

Requirements:

1) The sample has natural (or known) isotopic abundance.

2) The spike and sample isotopic ratios are different.

Isotope Dilution Mass Spectrometry(IDMS)

An Extension of the Isotope Dilution Method. Science, 107(2774), pp.228-228.

Advantages:

Most accurate and precise method for quantitative elemental concentrations

Partial loss of analyte during preparation is compensated for since physical and chemical interferences are not an issue -- will cancel out as they will affect each isotope identically

Ideal form of internal standardization since another isotope of the same element is used in this capacity

Disadvantages:

– Generally only applicable to multiple-isotopic elements

– Need an enriched isotope spike for the analyte of interest - not always available or sometimes at very high cost

– Need two interference free isotopes

– VERY time consuming

Isotope Dilution Mass Spectrometry(IDMS)

Figure. 4-Methylimidazole

4-Methylimidazole (4-MEI)

A heterocyclic organic compound formed in many ways; fermentation

through alcohol production and Maillard Reaction.

Browning reaction that occurs between amino acids and

carbohydrates when they are heated together.

4-MEI can be found in roasted foods, grilled meats, coffee in types of

caramel colouring.

Caramel colour most used food-beverage colouring; i.e. beers, sodas

Implies that commercially available beverages contain 4-MEI

1975 WHO

Caramel colouring showed disturbed immune functions in rodents.

National Toxicology Program (NTP, USA, 2007)

4-MEI showed carcinogenic activity in male and female mice; labeled

as carcinogen,

established No Significant Risk Level (NSRL) of 29 ug/day.

European Food Safety Authority (EFSA) has stated that 4-MEI is safe and is

not a health concern for Europeans exposed in caramel coloured foods.

ADI of 100mg/kg bw/day.

4-Methylimidazole (4-MEI)

25mL of sample (beverage) was decarbonated via a mechanical shaker.

100uL of the aliquot was diluted and fortified with d6-4-MEI to 50pg/uL.

A 5uL of volume was injected for LC-MS/MS analysis with water as a blank

control.

LC-MS/MS analysis was carried via C8-reverse phase using 0.05% Formic

acid in both water (MP A) and methanol (MP B).

Case Study: 4-Methylimidazole (4-MEI)

Results: 4-MEI

4-MEI in Beverages

Pepsi and butter beer exceed the NSRL of 29ug/L.

According to EFSA, assuming an average of BW is 80kg.

Using the highest MEI-detected, pepsi at 211.5ug, the daily intake per

can would equal to 0.003mg/kg bw/day. Which is significantly smaller

than the recommended 100mg/kg bw/day.

Health canada has stated that 4-MeI levels found in foods do not represent

a risk.

Conclusion

Isotope dilution with d6-4-MEI and analysis via LC-MS/MS allowed the

accurate quantification of 4-MEI present in a variety of alcoholic and non

alcoholic beverages.

The amount of 4-MEI in alcoholic beverages exceed the NSRL level of

29ug/L while the three of the non alcoholic beverages (pepsi, diet pepsi,

butter beer) exceed the threshold value.

None of the samples exceed the EFSA ADI guideline.

Reference

Abdallah, M., Harrad, S. and Covaci, A. (2009). Isotope Dilution Method for Determination of Polybrominated Diphenyl Ethers Using Liquid Chromatography Coupled to Negative Ionization Atmospheric Pressure Photoionization Tandem Mass Spectrometry: Validation and Application to House Dust. Analytical Chemistry, 81(17), pp.7460-7467.

BLOCH, K. and ANKER, H. (1948). An Extension of the Isotope Dilution Method. Science, 107(2774), pp.228-228.

Blom, K. (1987). Average mass approach to stable isotope dilution mass spectrometry. Organic Mass Spectrometry, 22(8), pp.530-533.

Ratnayake, G., Halldorson, T., Bestvater, L. and Tomy, G. (2015). Determination of 4(5)-methylimidazole in carbonated beverages by isotope-dilution liquid chromatography-tandem mass spectrometry. Food Additives & Contaminants: Part A, 32(7), pp.1075-1082.

Present Regulation

In 2012, coca-cola and Pepsi announced they had their caramel supplier

alter their manufacturing processes to meet the 29.ug/L standard. Changes

have already been made in California.

Levels of 4-MEI in Europe on the other hand remains the same since it

passes the EFSA ADI