rapid determination of antioxidant polyphenols in ... determination of antioxidant polyphenols in...
TRANSCRIPT
Rapid Determination of Antioxidant Polyphenols in Beverages and Herbal Supplements
Pranathi R. Perati, Brian De Borba, Deepali Mohindra, and Jeffrey RohrerOct 6th
2011
●
Antioxidants protect cells from damage caused by free radicals
●
Excess production of free radicals or decrease in antioxidants lead to oxidative stress, which is implicated in cancer, Alzheimer's disease, and aging
●
The body's ability to produce antioxidants is controlled by genetic makeup and is influenced by exposure to environmental factors such as diet and smoking
●
Increasing dietary intake of antioxidants helps mitigate damage due to oxidative stress
Antioxidants
Flavonoids
●
Flavonoids are polyphenolic compounds that are ubiquitous in plants
●
The three classes of flavonoids that have attracted most attention in nutraceuticals and functional foods are:
Flavanols or catechins Good sources are tea, chocolate, red wine, apples and berries
Anthocyanins Responsible for the red, orange, and blue colors of most fruits and flowers Pomegranates, bilberries, grapes, blueberries are all good sources Pomegranates and bilberries have higher total polyphenol concentrations than most common fruit juices and berries
Isoflavonoids Soy is a good source
Tea
•
An agricultural product derived from the Camellia sinensis plant that is prepared and cured by various methods
•
Four major varieties include:•
White tea•
Green tea•
Oolong tea•
Black tea
•
Processing of teas•
White/green tea: not oxidized•
Oolong tea: partially oxidized•
Black tea: fully oxidized
http://www.teazonline.com/tea-production.htm
Processing of Camellia sinensis (Tea)
Pan Frying Green Tea
Health Benefits of Catechins
•
Strong antioxidant activity: superior to ascorbic acid (vitamin C) and tocopherol
(vitamin E)•
Evaluated for its effectiveness in a number diseases:•
Cardiovascular•
Cancer (skin, breast, lungs, colon, and bladder)•
Neurodegenerative•
Some studies have characterized green tea as:•
Antiangiogenic
(prevents tumor blood vessel growth)•
Antimutagenic•
Helping to prevent the development of atherosclerotic plaques•
Helping to protect against Parkinson’s and Alzheimer’s disease•
Providing antidiabetic
effects (in animals)
8
Global Tea Market
●
Import/export of tea●
Largest exporters:
●
Consumption of green tea●
Largest consumers: China (largest producer/consumer), Hong Kong, Taiwan, Middle East, and Japan
Chemistry of Catechins
•
Characteristics of catechins•
Two benzene rings (A & B)•
A dihydropyran
heterocycle
(C) with –OH on C3•
The (A) ring is similar to resorcinol
•
The (B) ring is similar to catechol
•
Two chiral
centers at C2 and C3•
Four diastereoisomers: two in trans config
(catechin), two in cis
config
(epicatechin)
28471
Catechins
In Green Tea
Epicatechin Gallate
Epigallocatechin
Epigallocatechin Gallate
Gallocatechin
Gallocatechin GallateCatechin Epicatechin
EGCG: most abundant catechin
(up to 65%, 100–200 mg)
Catechin
+ GC: trace amounts
28472
Analytical Methods To Determine Catechins
INA Method 111.002 USP Method HPLC-ED Our Method
Column MetaChem Polaris™
Amide C18, 5 μm, 4.6 × 250 mm
C18 RP, 5 μm, 4.6 × 250 mm
C18, 5 μm, 4.6 × 150 mm Acclaim®
120 C18, 2.2 μm, (2.1 × 150 mm)
Mobile Phases A: 0.1% Phosphoric acidB: Acetonitrile
A: 0.35% Phosphoric acidB: 95:5 Acetonitrile: CH3
OH
A: 50 mM Sodium phosphate with 1% CH3
OH (pH 3.0)B: 50 mM Sodium phosphate with 10% CH3
OH and 60% CH3
OH (pH 3.45)
A: 0.1%TFA, 5% ac
et
on
itri
le
B: 0.1%TFA, in ac
et
on
itri
le
Total Run Time 28 min 90 min 50 min 25 min
Flow Rate 1.0 mL/min 0.8 mL/min 1.0 mL/min 0.45 mL/min
Column Temperature
35 ºC 25 ºC 35 ºC 25 ºC
Detection UV at 280 nm UV at 278 nm CoulArray® UV at 280 nm
Injection Volume
10 μL 15 μL 50 μL 1 μL
Column:
Thermo Scientific Acclaim™ 120, C18, 2.2 μm, analytical
(2.1 × 150 mm)Eluent:
A: 0.1% TFA, 5% AcetonitrileB: 0.1% TFA in Acetonitrile
Gradient:
0.0–1.2 min, 100% A1.2–15.5 min, 28.5% BHold for 1.5 min at 28.5% B
Temp.:
25 °CFlow: 0.45 mL/minDetection: Absorbance, UV, 280 nmInj. Vol.: 1.0 µL
Peaks: 1. Gallic Acid
50.0 µg/mL2. Gallocatechin
1003. Epigallocatechin
1004. Caffeine
35.05. Catechin
100 6. Epicatechin
1007. EGC Gallate
1008. Gallocatechin Gallate
1009. Epicatechin Gallate
1000 2 4 6 8 10 12 15
-10
100
1
2 3
4
5 6
7 8
9
Minutes
Separation of a Mixed Catechin Standards
AU
EGC, Catechin, EGCG, EC, GCG, and ECG at concentrations of 15, 1, 30, 1, 2, and 3 μg/mL were used for precision studies.
Linearity, LOD, and LOQ of Catechins
Analyte Range(μg/mL)
Correlation Coefficient (r2)
LOD (μg/mL)
LOQ (μg/mL)
RSD
Ret. Time*(n=30)
Peak Area*(n=30)
Gallocatechin 3.56–75 0.9993 1.17 3.56 0.11 1.17
Epigallocatechin 1.8–50 0.9993 0.59 1.8 0.18 1.45
Catechin 0.78–50 0.9992 0.20 0.78 0.13 1.19
Epicatechin 0.59–50 0.9999 0.20 0.59 0.06 1.51
Epigallocatechin Gallate 1.17–150 0.9994 0.39 1.17 0.04 1.00
Gallocatechin Gallate 1.2–10 0.9998 0.39 1.20 0.02 1.37
Epicatechin gallate 1.56–50 0.9995 0.39 1.56 0.02 1.30
NIST Collaborative Study: Catechins in Green Tea
•
Samples Received from NIST:•
Controls: Spray-dried green tea extract•
Samples: Ground and homogenized green tea tablets•
Analytes of interest (7 catechins): C, EC, ECG, EGC, EGCG, GC, GCG
•
NIST Sample and control Prep Procedure:•
Extraction solution: 70/30 H2
O/CH3
OH containing 0.05% formic acid•
Control: 20 mg/14 mL solvent + 5 min sonication•
Sample: 60 mg extracted with two portions of 5 mL of solvent + 90 min sonication
•
All samples filtered with 0.45 mm cellulose acetate filter prior
to analysis
Analyte NIST Controls (mg/g)
Certified values
NIST Control (mg/g)
NIST Sample (mg/g)
Certified values
NIST Sample (mg/g)
Gallocatechin 22.8 24 ± 1 7.84 7.6
Epigallocatechin 84.7 88 ± 3 29.6 30.7
Catechin 9.7 9.8 ± 0.4 2.46 2.6
Epicatechin 47.3 46 ± 2 11.9 12
Epigallocatechin Gallate 427 417 ± 16 80.8 71.1
Gallocatechin Gallate 40.9 38 ± 3 4.46 4.6
Epicatechin Gallate 76.8 94 ± 5 17.4 17.1
Total Catechins 710 716.8 ± 27.4 154 145.7
Summary of Catechin Evaluation of NIST Control Material (SRM 3255) and Reference Samples (SRM 3256) Compared to Certified Values
Column: Thermo Scientific Acclaim™ 120, C18, 2.2 μm (2.1 × 150 mm)
Eluent:
A: 0.1% TFA, 5% Acetonitrile B: 0.1% TFA in Acetonitrile
Gradient: 0.0–1.2 min, 100% A1.2–15.5 min, 28.5% BHold for 1.5 min at 28.5% B
Temp.: 25 °CFlow: 0.45 mL/minDetection: Absorbance, UV 280 nmInj. Vol.: 1.0 µLSample: 1:20 Dilute white tea
Peaks: 1. Gallic Acid
—mg/g*2. Gallocatechin
15.83. Epigallocatechin
16.5
4. Caffeine
—5. Catechin 3.126. Epicatechin 2.737. EGC Gallate 42.68. Gallocatechin Gallate 8.839. Epicatechin Gallate 8.96
* Calculated concentrationMinutes0 2 4 6 8 10 12 14 15
-1
1
2 3
4
6
7
8
9
5
10
mAU
Separation of Catechins in White Tea
Column:
Thermo Scientific Acclaim™
120, C18, 2.2 μm (2.1 × 150 mm)
Eluent:
A: 0.1% TFA, 5% Acetonitrile
B: 0.1% TFA in Acetonitrile Gradient: 0.0–1.2 min, 100% A
1.2–15.5 min, 28.5% BHold for 1.5 min at 28.5% B
Temp.: 25 °CFlow: 0.45 mL/minDetection: Absorbance, UV 280 nmInj. Vol.: 1.0 µLSample: 1:20 Dilute green tea
Peaks: Brand A mg/g* Brand B mg/g*1. Gallic Acid —
—2. Epigallocatechin 45.1
43.53. Caffeine
——4. Catechin
3.45 3.575. Epicatechin 6.14 6.086. EGC Gallate
64.0
60.67. Gallocatechin Gallate 6.74 6.658. Epicatechin Gallate 9.81
12.8
* Calculated concentrations0 5 10 15Minutes
-2.3
8.7
12
3
5
6
7
8
4
Brand A
Brand B
mAU
Separation of Catechins in Green Tea
Column:
Thermo Scientific Acclaim™
120, C18, 2.2 μm (2.1 × 150 mm)
Eluent:
A: 0.1% TFA, 5% Acetonitrile B: 0.1% TFA in Acetonitrile
Gradient:
0.0–1.2 min, 100% A
1.2–15.5 min, 28.5% BHold for 1.5 min at 28.5% B
Temp.: 25 °CFlow: 0.45 mL/minDetection:
Absorbance, UV 280 nmInj. Vol.:
1.0 µLSample:
1:20 Dilute black tea
Peaks: 1. Gallic Acid
—
mg/g*2. Epigallocatechin
27.83. Caffeine
—4. Catechin 4.355. Epicatechin 2.256. EGC Gallate
12.37. GC Gallate 9.208. Epicatechin Gallate
7.47
* Calculated concentrations
0 5 10 15-1
15
12
3
5
6
7
8
4
mAU
Minutes
Separation of Catechins in Black Tea
28265
●
Five different samples were studied (n=3)●
White tea, white and green tea blend, two commercial brands of green tea, and black tea
Retention time RSDs ranged from 0.01–0.36
Peak Area RSDs ranged from 0.33–1.63
Between day Peak Area RSDs ranged from 1.04–2.15
Sample Analysis Precision
Recovery of Catechins in TeasSample Analytes Amount Spiked
μg/mL % Recovery
Green Tea Brand A
Gallocatechin 1.0 85.4
EpiGallocatechin 15.0 100
Catechin 1.0 101
Epicatechin 1.5 90.9
Epigallocatechin Gallate 15.0 100
Galocatechin Gallate 2.0 95.5
Epicatechin Gallate 3.0 96.5
White Tea
Gallocatechin 4.0 104
EpiGallocatechin 4.0 92.1
Catechin 1.0 99.8
Epicatechin 1.0 112
Epigallocatechin Gallate 10.0 93.3
Galocatechin Gallate 3.0 105
Epicatechin Gallate 3.0 84.2
White Tea and Green Tea
Gallocatechin 1.0 93.4
EpiGallocatechin 6.5 91.3
Catechin 1.0 94.3
Epicatechin 1.0 96.4
Epigallocatechin Gallate 10.0 100
Galocatechin Gallate 1.0 97.7
Epicatechin Gallate 2.0 93.6
Basic Structure of Anthocyanins
R3′ R5′ Anthocyanidin
H H Pelargonidin
OH H Cyanidin
OH OH Delphinidin
OCH3 OH Petunidin
OCH3 H Peonidin
OCH3 OCH3 Malvidin
28447
HO
OH
O+
R3′
R5′
OH
OH
•
Punica granatum, (pomegranates) belong to the family Punicaceae
•
Pomegranates first originated in central Asia, and spread to the
rest of the world
•
Today pomegranates are cultivated in Asia, Europe, North Africa,
and North America in subtropical and tropical areas and in varied climatic conditions
•
Pomegranates are still a minor horticultural crop
Anthocyanins in Pomegranates
Health Benefits of Consuming PJ
•
Fights breast cancer
•
Helps prevent lung cancer
•
Slows prostate cancer
•
Keeps prostate-specific antigen (PSA) levels stable
•
Helps protect the neonatal brain
•
Helps prevent osteoarthritis
•
Helps protect the arteries
•
Helps prevent Alzheimer's disease
•
Lowers cholesterol
•
Lowers blood pressure
•
Helps provide dental protection
http://www.healthdiaries.com
Incentives to Adulterate Pomegranate Juice
•
Global supply shortage encourages manufacturers to stretch the finite quantity further
•
Sellers have higher profit margin with lower production cost
•
Inexpensive fillers like apple juice and grape juice are more readily available
•
Bottlers are able to enter the market at competitive prices
•
Polyphenols: anthocyanins
•
Sugar profiles•
Glucose:mannitol ratios of 4:15•
Glucose:fructose ratios of 0.8:1.0•
Sucrose, sorbitol, and maltose not found in pomegranate juice
•
Organic acids•
Predominantly citric and malic acid present•
Oxalic and tartaric acid present in low amount
•
Potassium content
Chemical Profiling Methods to Determine the Authenticity of Pomegranate Juice (PJ)
Analytical Methods to Determine Anthocyanins
•
Thin-layer chromatography methods: to qualify the anthocyanidin content after anthocyanin hydrolysis
•
AOAC Official Method 2005.02: pH Differential Method
•
Liquid chromatography (LC): the best technique for standardization of anthocyanin extracts
•
Other methods do not allow complete separation of all the constituents•
Samples used in other methodologies require acid hydrolysis
Bilberry: low-growing deciduous shrub belonging to the family Ericaceae
Found in woods and on heaths, chiefly in hilly districts of Great Britain, northern Europe, and Asia
Closely related to the native North American wild blueberry, but one characteristic difference is that they produce single or paired berries on the bush, unlike blueberries that grow in clusters.
Bilberry –
Vaccinium myrtillus
Bilberry –
Vaccinium myrtillus
Bilberries are also smaller, darker, hard, less juicy, easier to transport, and have a different flesh color than blueberries.
The two berries also have different phytochemical profiles with the anthocyanin content of fresh bilberry fruits being almost four times higher than blueberries.
The high price of the extract makes it more susceptible to adulteration.
Chronic venous insufficiency - veins in the legs that carry blood to the heart are damaged
Promotes urinary tract health
Diabetes - Bilberry leaves have traditionally been used to control blood sugar levels in people with diabetes
Atherosclerosis - strengthen blood vessels, improve circulation, and prevent the oxidation of LDL ("bad") cholesterol, a major risk factor for atherosclerosis (plaque that blocks blood vessels, leading to heart attack and stroke)
Diarrhea and wounds - Used in European medicine for nearly a thousand years to treat diarrhea Anti-inflamatory tannins act as astringent (constricting and tightening)
Vision - British fighter pilots in World War II reported improved night-time vision after eating bilberry jamTreatment for retinopathy Prevent cataracts
Health Benefits of Bilberries
Method A Determination of Anthocyanins in
PJ
Method B Determination of Anthocyanins in
BilberriesColumn Thermo Scientific Acclaim RSLC 120,
C18, 2.2 μm Analytical, 2.1 × 150 mm
Thermo Scientific Acclaim RSLC 120 C18, 2.2 μm Analytical, 2.1 × 150 mm
Mobile phases A: 9% Acetonitrile, 10% formic acidB: 36% Acetonitrile, 10% formic acid
A: 10% Formic AcidB: 10% Formic Acid, 22.5% Methanol, 22.5% Acetonitrile
Total run time 10 min 35 min
Flow Rate 0.475 mL/min 0.475 mL/min
Column temperature
30 °C 35 °C
Detection Vis at 540 nm Vis at 520 nm
Injection Volume 0.5 μL 2 μL
System Backpressure
~6025–6200 psi during the gradient ~6700–7400 psi during the gradient
Analytical Conditions
Column:
Thermo Scientific Acclaim™
120, C18, 5.0 μm (4.6 × 250 mm)
Eluent:
A: 10% MeCN, 10% Formic acid B: 40% MeCN, 10% Formic acid
Gradient: 0.0–2.5 min –
100% A2.5–30.00 min –
28.5% BHold for 15 min at 28.5% B
Temp.:
30 °CFlow: 1.0 mL/minDetection: Absorbance, Vis 540 nmInjection Volume:
2 µL
Peaks:
Conc. (μg/mL)1. Delphinidin 3,5-diglucoside
20.02. Cyanidin 3,5-diglucoside
20.03. Delphinidin 3-glucoside
20.0
4. Pelargonidin 3,5-diglucoside
20.05. Cyanidin 3-glucoside 20.0 6. Pelargonidin 3-glucoside 20.0
0 2 4 6 8 10 12 14 16 18 20-0.5
10
mA
U
Minutes
12
3
4
5
6
Separation of a Mixed Anthocyanin Standards Specific to PJ
Column:
Thermo Scientific Acclaim™
120, C18, 2.2 μm (2.1 × 150 mm)
Eluent:
A: 10% Formic acid B: 10% Formic acid, 22.5% Methanol 22.5% MeCN
Gradient:
0.0–12.0 min –
9% B12.0–25.0 min –
35% B25.0–30.0 min –
50% B30.0–35.0 min –
9% BTemp.:
35 °CFlow:
0.475 mL/minDetection: Absorbance, Vis 520 nmInjection Volume: 2.0 µLSample: 125 µg/mL15 Anthocyanin Standard
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30-0.5
0
8
Minutes
mAU
Separation of a 15 Anthocyanin Standard Specific to Bilberries
Peaks: Conc. in μg/mL1. Dp3Gal
—2. Dp3Glu
20.13. Cy3Gal
11.0 4. Dp3Ara
—5. Cy3Glu
12.36. Pet3Gal —7. Cy3Ara
—8. Delphinidin
—9. Pet3Glu
9.9110. Peo3Gal
3.91 11. Pet3Ara
—12. Peo3Glu
—13. Mal3Gal
8.7614. Peo3Ara
12.715. Cyanidin
—16. Mal3Glu —17. Mal3Ara
—18. Petunidin
—19. Peonidin
—20. Malvidin
—
12 3
4
5
67
8
9
1011
12
1314
15
1617
18
19
20
Column:
Thermo Scientific Acclaim™ 120, C18, 2.2 µm (2.1 × 150 mm)
Eluent:
A: 10% CH3
CN, 10% formic acidB: 40% CH3
CN, 10% formic acid Gradient: 0.0–0.9 min: 100%
A 0.9–8.0 min: 28.5% B
Hold for 2 min at 28.5% BTemp: 30 °CFlow: 0.475 mL/minDetection:
Absorbance, vis 540 nmInjection Volume:
0.5 μLSample: 1:5 Dilute 100% Pomegranate juice
Peaks:
Conc. (μg/mL)*1. Dp3,5 65.5 2. Cy3,5
123 3. Dp3 27.5 4. Pg3,5 8.00 5. Cy3
1216. Pg3 4.45
* Calculated Concentrations
mA
U
Minutes0 1.3 2.5 3.8 5
-0.2
6
1
2
3
4
5
6
0
27831
Separation of Anthocyanins in Pomegranate Juice
Column:
Thermo Scientific Acclaim™ 120, C18, 2.2 µm (2.1×150 mm)
Eluent:
A: 10% CH3
CN, 10% formic acidB: 40% CH3
CN, 10% formic acidGradient: 0.0–0.9 min: 100% A
0.9–8.0 min: 28.5% B
Hold for 2 min at 28.5% BTemp.: 30 °CFlow: 0.475 mL/minDetection:
Absorbance, vis 540 nmInjection Volume:
0.5 µLSample: 1:5 Dilute Pomegranate/wildberry juice
Peaks:
μg/mL*1. Dp3,5
39.02. Cy3,5 54.03. Dp3
10.04. Unknown ––5. Unknown ––6. Cy3 50.57. Unknown
––
* Calculated Concentrations
Minutes0 1.3 2.5 3.8 5.0
-0.1
3
mA
U
1 2
3
6
4
5
7
0
27833
Anthocyanins in Pomegranate/Wildberry Juice
Column:
Thermo Scientific Acclaim™ 120, C18, 2.2 µm (2.1 × 150 mm)
Eluent:
A: 10% CH3
CN, 10% formic acidB: 40% CH3
CN, 10% formic acid Gradient:
0.0–0.9 min: 100% A0.9–8.0 min: 28.5% B
Hold for 2 min at 28.5% BTemp: 30 °CFlow: 0.475 mL/minDetection:
Absorbance, vis 540 nmInjection Volume: 0.5 µLSample: Pomegranate/Cherry
Peaks: μg/mL1. Dp3,5
1.502. Unknown —3. Unknown
—4. Unknown
—
Minutes
0 1.3 2.5 3.8 5-0.25
7
mA
U
1
2
34
0
27832
* Calculated Concentrations
Anthocyanins in Pomegranate/Cherry Juice
Column:
Thermo Scientific Acclaim™ 120, C18, 2.2 µm (2.1 × 150 mm)
Eluent:
A: 10% CH3
CN, 10% formic acid
B: 40% CH3
CN, 10% formic acid Gradient:
0.0–0.9 min: 100% A
0.9–8.0 min: 28.5% B
Hold for 2 min at 28.5% BTemp:
30 °CFlow:
0.475 mL/minDetection:
Absorbance, vis 540 nmInjection Volume:
0.5 µLSample: A: 1:5 dilute Simulated adulterated
pomegranate juice (PJ)B: 1:5 dilute Grape juice
Peaks:
A
B
(μg/mL)*
(μg/mL)*1. Dp3,5
33.5 ––
2. Cy3,5 61.0
4.953. Dp3 26.0
34.04. Cy3
73.0
37.15. Pg3,5 3.10
7.036. Pg3 2.10
––
* Calculated Concentrations
0 2.5 5 7.5 10-0.1
3.5
mA
U
Minutes
B
A0
1
2
3
4
5 6
2
3
4
5
27834
Comparison of Anthocyanins in Simulated Adulterated PJ with Grape Juice
Analyte Rangeμg/mL
Coefficient of Determination
r2
LOD(μg/mL)
LOQ(μg/mL)
RSD
Ret. Time(n=30)
Peak Area(n=30)
Method A
Dp3,5 0.31–160 0.9992 0.21 0.66 0.06 1.82Cy3,5 0.31–160 0.9995 0.19 1.25 0.12 1.60Dp3 0.31–160 0.9996 0.12 0.63 0.06 1.45
Pg3,5 0.31–160 0.9984 0.37 1.25 0.07 1.80Cy3 0.31–160 0.9994 0.15 1.25 0.06 1.46Pg3 0.31–160 0.9996 0.20 0.63 0.09 1.70
Method B
Dp3Glu 0.39–50 0.9997 1.56 6.25 0.15 0.60Cy3Gal 0.39–25 0.9996 0.78 3.13 0.12 0.97Cy3Glu 0.39–25 0.9999 0.78 3.13 0.14 1.29Pet3Glu 0.39–25 0.9999 0.20 0.78 0.08 1.51 Peo3Gal 0.39–10 0.9988 0.56 2.34 0.03 1.05 Peo3Ara 0.39–25 0.9997 0.78 3.13 0.03 0.50Mal3Gal 0.39–25 0.9993 0.20 0.78 0.01 0.72
Data for Linearity, LOD and LOQ of Anthocyanins for Methods A and B
Column:
Thermo Scientific Acclaim™ 120, C18, 2.2 µm (2.1 × 150 mm)
Eluent:
A: 10% formic acid B: 10% formic acid, 22.5% methanol, 22.5% acetonitrile
Gradient:
0.0–12.0 min –
9% B12.0–25.0 min –
35% B25.0–30.0 min –
50% B30.0–35.0 min –
9% BTemp.:
35 °CFlow:
0.475 mL/minDetection:
Absorbance, Vis 520 nmInjection Volume:
2.0 µLSample: Eye antioxidant
Peaks: Conc. in μg/mL1. Dp3Gal
—2. Dp3Glu
5.153. Cy3Gal
2.23 4. Dp3Ara
—5. Cy3Glu
2.026. Pet3Gal —7. Cy3Ara
—8. Pet3Glu
1.899. Peo3Gal
0.2810. Pet3Ara
—11. Mal3Gal
4.2712. Peo3Ara
1.4913. Cyanidin
—14. Mal3Glu
—0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
-0.1
0
1.1
Minutes
mAU
Anthocyanins in Bilberry-Based Eye Antioxidants
1
2
34
5 67
8
9
10
1112
13
14
Anthocyanins in Bilberry Nutritional Supplement Brand A Column:
T hermo Scientific Acclaim™ 120, C18, 2.2 µm (2.1 × 150 mm)
Eluent:
A: 10% formic acid B: 10% formic acid, 22.5% methanol, 22.5% acetonitrile
Gradient:
0.0–12.0 min –
9% B12.0–25.0 min –
35% B25.0-30.0 min –
50% B30.0–35.0 min –
9% BTemp.:
35 °CFlow: 0.
475 mL/minDetection:
Absorbance, Vis 520 nmInjection Volume:
2.0 µLSample:
Nutritional Supplement A
Peaks:
Conc. in μg/mL1. Dp3Gal
—2. Dp3Glu
21.73. Cy3Gal
11.9 4. Dp3Ara
—5. Cy3Glu 10.66. Pet3Gal —7. Cy3Ara
—8. Delphinidin
—9. Pet3Glu 8.7610. Peo3Gal 3.31 11. Pet3Ara
—12. Peo3Glu
—13. Mal3Gal 6.2614. Peo3Ara 8.8815. Cyanidin
—16. Mal3Glu —17. Mal3Ara
—28. Petunidin
—19. Peonidin
—20. Malvidin
—0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
-1
0
12
Minutes
mAU
1
2 3
4
5 6
7
8
9
10
11
12
13
14
15
16
17 181920
Column:
Thermo Scientific Acclaim™
120, C18, 2.2 µm (2.1 × 150 mm)
Eluent:
A: 10% Formic Acid B: 10% Formic Acid, 22.5% methanol, 22.5% acetonitrile
Gradient:
0.0–12.0 min –
9% B12.0–25.0 min –
35% B25.0–30.0 min –
50% BHold for 5 min at 9% B
Temp.:
35 °CFlow:
0.475 mL/minDetection:
Absorbance, Vis 520 nmInjection Volume:
2.0 µLSample:
Nutritional Supplement B
Peaks:
Conc. in μg/mL1. Dp3Gal
—2. Dp3Glu
20.83. Cy3Gal
10.24. Dp3Ara
—5. Cy3Glu
9.996. Pet3Gal —7. Cy3Ara —8. Delphinidin
—9. Pet3Glu
8.4010. Peo3Gal
1.12 11. Pet3Ara
—12. Peo3Glu
—13. Mal3Gal
3.2714. Peo3Ara
6.9615. Cyanidin
—16. Mal3Glu —17. Mal3Ara
—28. Petunidin
—19. Peonidin
—20. Malvidin
—
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30-0.5
0
7
Minutes
mAU
Anthocyanins in Bilberry-Based Nutritional Supplement Brand B
1
2
3
45
6
7
8
9
10
11
12
13
14
15
16
1718
1920
Sample Analytes Amount μg/mL Amount Spiked μg/mL % Recovery
1:5 dilute 100% pomegranate juice
Dp3,5 13.2 15.0 101.8
Cy3,5 23.8 25.0 98.0
Dp3 6.50 5.0 106.9
Pg3,5 1.04 1.0 104.1
Cy3 23.6 25.0 102.2
Pg3 0.88 1.0 108.3
1:5 dilute 100% grape juice
Dp3,5 < LOD 2.5 104.6
Cy3,5 1.05 1 105.1
Dp3 5.12 5 89.5
Pg3,5 < LOD 1 87.3
Cy3 4.24 5 85.5
Pg3 0.86 1 102.2
1:5 dilute simulated adulterated pomegranate juice
Dp3,5 6.5 10.0 102.6
Cy3,5 12.8 10.0 80.1
Dp3 5.48 5.0 103.4
Pg3,5 0.56 0.75 102.0
Cy3 14.9 10.0 97.2
Pg3 0.43 0.75 87.6
Recovery of Anthocyanins in Various Juice Matrices
Sample Analytes Amountμg/mL
Amount Spiked μg/mL % Recovery
Eye Antioxidant
Dp3Glu 6.24 10 89.3Cy3Gal 2.53 3.0 94.8Cy3Glu 2.45 3.0 81.1Peo3Gal 0.28 0.5 96.2Pet3Glu 2.25 3.0 92.6Mal3Gal 0.53 0.5 94.2Peo3Ara 1.78 3.0 91.2
Brand A Bilberry-Based Nutritional Supplement
Dp3Glu 24.3 25 90.2Cy3Gal 13.5 10 82.8Cy3Glu 12.01 10 78.7Peo3Gal 3.26 4.0 98.1Pet3Glu 9.94 10 78.4Mal3Gal 6.47 10 107Peo3Ara 10.3 10 104
Brand B Bilberry-Based Nutritional Supplement
Dp3Glu 20.1 20 97.6Cy3Gal 9.86 10 84.0Cy3Glu 9.47 10 93.6Peo3Gal 1.64 2.0 105Pet3Glu 8.01 10 83.7Mal3Gal 2.76 3.0 101Peo3Ara 6.07 5.0 108
Recovery of Anthocyanins in Herbal Supplement Matrices
Sample Analysis Precision
•
Method A: Five different samples were studied (n=3)•
Pomegranate juice, grape juice, pomegranate/wildberry, pomegranate/cherry, and simulated adulterated pomegranate juice
•
Retention time RSDs ranged from 0.004–0.178•
Peak area RSDs ranged from 0.85–1.71•
Between-day peak area RSDs ranged from 1.18–2.68
•
Method B: Three different samples were studied (n=3)•
Eye Antioxidant, Brand A and B Bilberry-Based Nutritional supplements
•
Retention time RSDs ranged from 0.01–0.17•
Peak area RSDs ranged from 0.65–6.00•
Between-day peak area RSDs ranged from 1.5–6.93
Conclusions
●
The three methods discussed are simple, sensitive, and accurate methods to separate and quantify catechins and anthocyanins with minimal
sample preparation
●
Commercially available teas with varying concentrations of catechins ranging from 2.25 mg/g to 427 mg/g were determined
●
The method described in the study was used to determine and quantify catechins in NIST control material (SRM 3255) and sample (SRM 3256)
●
The values determined using the method were in agreement with the NIST certified values
●
Several herbal supplements and juices with varying anthocyanin concentrations ranging from 0.2 μg/mL to 123 μg/mL were determined
Thank You