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Dr. Jens Dahlmann, Applied Biosystems, Germany

Advantages in Food Analysis UsingLC/MS/MS Techniques – Applications for the Determination of Valuable and

Harmful Compounds

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Outline of talk

Analysis of flavonoides

Analysis of vitamins by LC/MS/MS

Some examples of phospholipid analysis

Determination of cyanobacterial toxins in healthsupplements and in drinking water

Multi-Component analysis of pesticides in fruits

Contamination of foodstuff by antibiotics

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Analysis of flavonoides

Quercetin

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Information about flavonoidsA ubiquitous group of polyphenolic substances which arepresent in most plants, concentrating in seeds, fruit skinor peel, bark, and flowers

The structure includes two benzene rings on either sideof a 3-carbon ring. Multiple combinations of hydroxylgroups, sugars, oxygens, and methyl groups attached tothese structures create the various classes of flavonoids

Flavonoids have been shown in a number of studies tobe potent antioxidants, capable of scavenging hydroxylradicals, superoxide anions, and lipid peroxy radicals

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LC/MS/MS chromatogram of selected flavonoid standards

Phenomenex, SynergieTM, 50 x 2 mm, 3 µ, eluent A: water with 0,1 % acetic acid, B: acetonitril, start with 95 % A 80 % B in 8 min, flow 400 µL/min, API 4000TM LC/MS/MS system

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Quercetin, rutin and epigallocatechin gallate in red wine

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Quercetin, rutin and epigallocatechin gallate in green tea

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Quercetin, rutin and epigallocatechin gallate in black tea

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Different sources for the flavonoides quercetin, rutinand epigallocatechin gallate

1

2

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2

3

3

1

2

red wine

green tea

black tea

quercetin EGCG rutin

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Analysis of vitamins by LC/MS/MS

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Vitamin B 2 - riboflavin

An essential function of riboflavin is to metabolizecarbohydrates, proteins (amino acids), and fatsfor use by the body

Riboflavin is also necessary for red blood cellformation, antibody production, cell respiration,normal fetus growth, the development of bodytissues (like hair, skin, and nails)

Vitamin B 2 maintenance mucous membranesin the digestive tract

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Vitamin B2, Positive TurboIonSpray (TIS)TM

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Vitamin B 6 - pyridoxine

Vitamin B 6 plays a role in the synthesis of antibodiesby the immune system, which are needed to fightmany diseases

It helps maintain normal nerve function and also actsin the formation of red blood cells

Vitamin B 6 is also required for the chemical reactionsneeded to digest proteins

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Vitamin B 6, Positive TurboIonSpray (TIS)TM

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Biotin

Biotin is used in cell growth, the production offatty acids, metabolism of fats, and proteins

Biotin not only assists in various metabolicchemical conversions, but also helps with the transferof carbon dioxide

Biotin is also helpful in maintaining a steadyblood sugar level

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Biotin, Negative TurboIonSpray (TIS)TM

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Vitamin D promotes the body's absorption of calcium,which is essential for the normal development andmaintenance of healthy teeth and bones - calcium isalso important to nerve cells

Vitamin D also helps maintain adequate blood levelsof calcium and phosphorus

Vitamin D

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HPLC Column: 50x2.1 mm, 3 µ, C-8 (ThermoHypersilKeystone)

Mobile Phase A: 0.1% aqueous Formic Acid

Mobile Phase B: Methanol

Flow rate: 400 µl/min, gradient method

Selected Ion Monitoring (SIM) for D vitamins in human plasma

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Extracted patient plasma sample showing full metabolite profile

25 (OH) Vit D 3

25 (OH) Vit D 2

1,25 (OH) Vit D 2

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XIC of +MRM (1 pair): 381,3/363,3 amu from Sample 1 (AcnEx+100ppb) of AcnEx+100pp... Max. 1,7e4 cps.

1 2 3 4 5 6 7 8 9 10 11 12Time, min

0,0

1000,0

2000,0

3000,0

4000,0

5000,0

6000,0

7000,0

8000,0

9000,0

1,0e4

1,1e4

1,2e4

1,3e4

1,4e4

1,5e4

1,6e4

1,7e4 9,94

7,87

7,347,229,69 10,61

7,026,401,96

1,25 di-hydroxy vitamin D3, 100 ng/L

API 2000TM LC/MS/MS system, PhotoSprayTM Ionization, 2 dim. LC

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Vitamin E - alpha-Tocopherol

Vitamin E is an antioxidant that protects body tissuefrom damage caused by free radicals. Free radicals canharm cells, tissues, and organs

Vitamin E is also important in the formation of red bloodcells and it helps the body to use vitamin K

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Vitamin E, Positive TurboIonSpray (TIS)TM

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Strategy for vitamin analyses by LC/MS/MS

Vitamin B 2 & B 6 and rutin (water soluble), mobilephase: MeOH:water, possibly with 0.1% formic acid ona C-18 column with TISTM ionization

Vitamin D2 & D3 and E with PhotoSprayTM (or APCI),mobile phase: MeOH:acetonitrile 25:75 (isocratic)

25 (OH) Vit. D 2 and D 3 can be best ionized with TISTM

D vitamins will lose water and should be optimized onthis ion [M+H-H2O]+

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Some examples of phospholipid analysis

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Phospholipids are diglycerides that are covalently bonded to aphosphate group by an ester linkage. Amino groups can be additionally attached to the phosphate

The hydrocarbon chains are hydrophobic. The phosphate andamino groups make that portion of the molecule hydrophilic.The result is an amphiphilic molecule

Amphiphilic lipids are major constituents of cell membranes –polar heads are facing aqueous surroundings (e.g. cytosol) andhydrophobic tails facing each other

This ideal combination of physical and chemical functionalitymakes phospholipids an indispensable component in cosmeticproducts

Facts about phospholipids

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Basic structure of a phospholipid molecule

fatty acidfatty acid phosphate

glycerol

e.g. choline

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Chromatographic separation of:

1) sphingomyelin,2) phosphatidylethanolamin and3) phosphatidylserin

1)

2)

3)

TIC

Phenomenex, LunaTM, 150 x 2 mm, 3 µ, eluent A: chloroform, B: methanolcontaining 10 mM NH4Ac, start with80 % A 80 % B in 12 min, flow 400 µL/min, API 4000TM LC/MS/MS system

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Product-Ion-spectrum of phosphatidylcholin, ESI

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Q1 Scan of phosphatidylethanolamin

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Determination of cyanobacterial toxins inhealth supplements and in drinking water

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Cyanobacteria as a dietary supplement

cyanobacteria are advertise as:

having a high content of omega-3 fatty acids

are an excellent source of amino acids

supports the immune system by stimulating theproduction and movement of “natural killer cells”

stimulate macrophage activity

what most people don´t know:

some cyanobacteria produce toxins – thesesubstances are tumor promotors as well as hepatotoxic

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O

H H O

NH

NHO CH2

NNH

XNH

Y

H

H

H

COOH

H3C

COOH

CH3

O

H3C

H

H

OCH3

H3C CH3 H

O

H

H

CH3

1

2

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5

6 7

ss

s

ss

R

R

R

Adda

D-Glu Mdha

D-Ala

D-Me-Asp

General structure of microcystins – one class of cyanotoxins

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0 5 10 15 20 25 30

inte

nsity

[cps

]

0

10000

20000

30000

time [min]

1

2

3

4

5

6

7

8

9

10

1112

1 = Saxitoxin2 = Anatoxin-A3 = Domoic acid4 = Microcystin-RR5 = Nodularin6 = Microcystin-YR7 = Microcystin-LR8 = Microcystin-LA9 = Microcystin-LW10 = Microcystin-LF11 = Okadaic acid12 = DTX-1

MID-chromatogram of various cyanobacterial toxins

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m/z [dalton]100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000

inte

nsity

[cps

]

0

500

1000

1500

2000

* 84.2

116.2

135.3

157.3

155.2 213.2

228.3

272.3

311.2

343.2

375.3

426.4

440.4

513.2 [M-2H]2+

* 72.2

m/z [Da] Fragmente72 Ala +H84 Dhb +H116 Asp +H135 Charakterist. Adda-Fragment155 Dhb Ala +H157 Arg +H157 Arg +H213 Glu Dhb +H228 Ala Arg +H272 Arg Asp +H272 Asp Arg +H311 Dhb Ala Arg +H343 Ala Arg Asp +H375 C11 H14 O + Glu Dhb +H426 Dhb Ala Arg Asp +H428 Arg Asp Arg +H440 Glu Dhb Ala Arg +H

NH

O

NH

NHO

NH

NH

H

COOH

H3CO

H3C

H O

H

H HH

H

OCH3

H3C CH3 NH

COOHO

NH

H

O

NH2

NH

NH

O

NH

NH

NH2

CH3

Structure elucidation on an API 2000TM LC/MS/MS system

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Bloom of PlanktothrixRubescense in „LakeBehlendorf“, Germany during spring 2003Photo by LANU, SWH

cyanobacteria are often found in drinking water reservoirs

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Multi-Component analysis of pesticides in fruits

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Substance or mixture of substances intended forpreventing, destroying, repelling, or mitigating anypest (insects, animals, unwanted plants (weeds),fungi, or microorganisms)

Pesticides can cause health problems, suchas birth defects, nerve damage, and cancer

World Health Organization (WHO) estimatesone million pesticides poisoning cases and20,000 deaths every year globally

What are pesticides?

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XIC of +MRM (97 pairs): 246.9/169.0 amu from Tomate03_MSCE_25_A.wiff, Smoothed, Smooth... Max. 1.9e4 cps.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21Time, min

0.0

5000.0

1.0e4

1.5e4

2.0e4

2.5e4

3.0e4

3.5e4

4.0e4

4.5e4

5.0e4

5.5e4

6.0e4

6.5e4

7.0e4

7.5e4

8.0e4

8.5e4

9.0e4

9.5e49.8e4

3.60

Calibration 0.001 – 0.1mg/kg(max. tolerance level 0.01mg/kg)

Standard containing 100 pesticides

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Column: Phenomenex AquaTM, 50x2 mm, 5 µmFlow: 200 µL/minGradient: H2O/CH3OH + 5 mM NH4form

API 2000™ LC/MS/MS systemPositive TurboIonSprayTM sourceQuantitation in MRMDwell time per MRM = 25msec

Extraction with ChemElut and Filtration

Analytical conditions

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Positive real sampleXIC of +MRM (99 pairs): 246.9/169.0 amu from 07S_02_030_Salat_SCE_blank_A.wiff, Smoothe... Max. 4.2e5 cps.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21Time, min

0.0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

1.8e5

2.0e5

2.2e5

2.4e5

2.6e5

2.8e5

3.0e5

3.2e5

3.4e5

3.6e5

3.8e5

4.0e5

4.2e53.38

residue of pesticides in strawberries

oxydemeton-methyl

metalaxyl

demeton-S-methyl-sulfone

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Contamination of foodstuff by antibiotics

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Antibiotics are used in the treatment of infectionscaused by bacteria. It works by killing bacteria orpreventing their growth

Antibiotics should only be used for seriousinfections in which other medicines do not work

Antibiotics may cause some serious side effects

Antibiotics were found in e.g milk, honey, and shrimps

What are antibiotics?

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Selected standards of antibioticsXIC of +MRM (9 pairs): Period 1, 445.1/341.0 amu from Sample 43 (100ng/ml 1ml/min) of Data... Max. 1350.0 cps.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0Time, min

0.00

5.00e4

1.00e5

1.50e5

2.00e5

2.50e5

3.00e5

3.50e5

4.00e5

4.50e5

5.00e5

5.50e5

6.00e5

6.50e5

7.00e5

7.50e5

8.00e5

8.50e5

9.00e5

9.50e5

1.00e6

1.05e61.09e6

SulfaguanidineSulfathiazol

Sulfamethazine

Sulfamerazine

Chloramphenicol

Sulfamethoxazol

Oxytetracycline Chlortetracycline

Positive ESI Negative ESI

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Simple sample preparation for theanalysis in honey

Dissolving of 1 g honey in 1 mL H2O

Adding 200 uL ISTD (CAP-D5, ca 0.1 µg/ml in methanol)

Adding 4 mL of ethyl acetate

Extraction in ultra sound bath

Centrifugation

Taking 1 mL of the upper phase

Evaporating the solvent and dissolving the residuein 1 mL of water

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Column: monolythic Chromolith SpeedROD (Merck), RP18 (50x4.6mm)

Flow rate: 1mL/min - 5mL/min (no split API 4000TM)Gradient: H2O/CH3OH + 0.1% formic acid

API 4000™ LC/MS/MS systemNegative Turbo V™ sourceQuantitation in MRM

Mix of antibiotics:Positive and negative ion mode by different periods

Analytical conditions

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Contaminated real sample (honey)TIC: from Sample 44 (Honig 1ml/min) of Data Quantitation_pos neg switching.wiff (Turbo Spray), Smoothed Max. 4.0e4 cps.

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0Time, min

0.0

5000.0

1.0e4

1.5e4

2.0e4

2.5e4

3.0e4

3.5e4

4.0e41.1

3.2

0.9 2.3

TIC: from Sample 30 (Honig 5ml/min) of Data Quantitation_pos neg switching.wiff (Turbo Spray), Smoothed, Smoothed Max. 9822.9 cps.

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0Time, min

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

9823 0.2

0.7

0.50.2

1mL/min

5mL/min

Water diluted honeyFound: sulfaguanidine

oxytetracyclinechloramphenicol

Low ppb level

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Acknowledgements• André Schreiber (Applied Biosystems, Germany)• Bruno Casetta (Applied Biosystems, Italy)• Kristin von Czapiewski (Applied Biosystems, Germany)• Stephan Lock (Applied Biosystems, UK)• Fans Schoutsen (Applied Biosystems, The Netherlands)• Ales Merta (Applied Biosystems, Czech Republic)

Presented applications are available as hard copies or electronically

Please contact us:

jens.dahlmann@eur.appliedbiosystems.comales.merta@eur.appliedbiosystems.com

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Legal acknowledgements

• For Research Use Only. Not for use in diagnostic procedures.

• ABI PRISM, Analyst, Applied Biosystems, and TurboIonSpray are registered trademarks and AB (Design), API 2000, API 3000, API 4000, Q TRAP, 4000 Q TRAP, Applera, Curtain Gas, and IonSprayare trademarks of Applera Corporation or its subsidiaries in the US and/or certain other countries.

• PhotoSpray is a trademark of Applied Biosystems/MDS Sciex, a joint venture between Applera Corporation and MDS, Inc.

• LINAC is a trademark of MDS Inc.

• All other trademarks are the sole property of their respective owners.

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Metabolomics using an electrochemical cell