Advantages in Food Analysis Using LC/MS/MS Techniques ...schulzov/HPLC/LCMS food anal...
Transcript of Advantages in Food Analysis Using LC/MS/MS Techniques ...schulzov/HPLC/LCMS food anal...
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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
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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:
[email protected]@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