Reference standards of all six anthocyanins i.e. Petunidin chloride, Pelargonidin chloride, Malvidin chloride, Delphinidin chloride, Peonidin chloride and.
O P T I M I Z AT I O N A N D VA L I DAT I O N O F A FA S T U P LC M E T H O D FO R T H E Q UA N T I F I C AT I O N O F A N T H O C YA N I N S
BISWATRISH SARKAR ASSISTANT PROFESSOR DEPARTMENT OF PHARMACEUTICAL SCIENCES
Plants of interest
Syzygium cumini Clitoria ternatea (Labiateae)
Hibiscus rosa-sinensis (Malvaceae)
(Myrtaceae)
Hibiscus sabdariffa (Malvaceae)
Ocimum tenuiflorum (Lamiaceae) © Biswatrish Sarkar, B.I.T, Mesra. Ranchi. India
TYPES OF ANTHOCYANIDINS? 400 naturally occurring anthocyanidins or aglycones are found till date (Kong et al. 2003, Phytochemistry, 64,923–933) Only six anthocyanidins are common in higher plants OH
Pelargonidin (Pg) O
HO
+
Orange-red OH
OH
Mol. Wt.= 271 OCH 3
Peonidin (Pn)
OH
O
HO
Petunia species
Orange-red
+
Paeonia officinalis
OH
Mol. Wt.= 301
OH
OH
Cyanidin (Cy)
Orange-red
+
HO
O
OH OH
OH
Mol. Wt.= 287
Petunia species
© Biswatrish Sarkar, B.I.T, Mesra. Ranchi. India
TYPES OF ANTHOCYANIDINS? OCH3
Malvidin (Mv)
OH
+
HO
O
Bluish-red
OCH3 OH
Mol. Wt.= 331
OH
Primula farinosa
Petunidin (Pt) O
HO
H3CO OH
+
Bluish-red
OH OH
Mol. Wt.= 317
OH
Pisum sativum
OH
Delphinidin (Dp)
OH
Bluish-red
+
HO
O
OH OH
OH
Mol. Wt.= 303 Petunia species
© Biswatrish Sarkar, B.I.T, Mesra. Ranchi. India
Identification and quantification techniques for anthocyanins
• Spectroscopy UV-Vis, MS, NMR etc.
• Chromatography – TLC, HPTLC, HPLC, CCC, UPLC etc.
• Capillary Electrophoresis Method of choice: LC-MS or LC-MS/MS or LC NMR © Biswatrish Sarkar, B.I.T, Mesra. Ranchi. India
WHY UPLC?? • Smaller particle size: 1.7 µm
•Narrow Band- Sharp Peak • Increased sensitivity
• Increased resolution-
More information from complex samples like flavonoids
• Increased speed-
Ultra Fast Analysis
•Increased throughput
Traditional HPLC is time consuming Sample: Wines (Pinot noir and Cabernet Sauvignon)*** Sample Preparation: Pinot: Filter detection of anthocyanins followed by Hydrolysis Cabernet Sauvignon: Hydrolysis/ Saponification followed by Hydrolysis Guard Column: PLRP-S (30×5 mm) Column/ Stationary Phase: PLRP-S (250×4.6 mm, 5 µm) Mobile Phase:
A: 4% H3PO4 /H2O; B: CH3CN
Gradients
Pinot Noir
Cabernet Sauvignon
Time (min.)
%A
%B
Time (min.)
%A
%B
0
90
10
10
90
10
12
65
35
17
65
35
27
90
10
0 10 35 55 62 67
90 90 30 70 0 90
10 20 70 30 100 10
***Wightman et al. (1997); Am. J. Enol. Vitic. 48:1:39-48
© Biswatrish Sarkar, B.I.T, Mesra. Ranchi. India
Preparation of reference standards and samples for Liquid Chromatography of Anthocyanin Reference standards of all six anthocyanins i.e. Petunidin chloride, Pelargonidin chloride, Malvidin chloride, Delphinidin chloride, Peonidin chloride and Cyanidin chloride were purchased from Extrasynthese SAS (Genay, France) Solutions were prepared by dissolving 0.1 mg respective anthocyanin in 1 ml of methanol. For anthocyanin sample preparation 1 g of powdered plant parts were extracted with 2 ml solvent (80/20 acetonitrile/0.3%phosphoric acid), vortexed for 5-10 sec and then centrifuged at 5000g for 10 mins and supernatant was collected. The cleared lysates were passed through Sep-Pak plus short tC18 cartridges (Waters Corporation, Milford, MA, USA) and then passed through a 0.20 µm membrane (Ministart Ny25 Sartorius AG Goettingen, Germany). A portion of the sample (1 ml) was acid hydrolysed by the addition of 2M HCl at 150°C for 30 min in a sealed ampoule to get the anthocyanins. © Biswatrish Sarkar, B.I.T, Mesra. Ranchi. India
Separation of anthocyanins using Ultra Performance Liquid Chromatography (UPLC)
Column : Acquity UPLC BEH C18 RP (2.1 mm × 50 mm; 1.7 μm particle size).
Flow rate: 0.5 ml min-1
Injection volumes: 2 μl
Eluents:
Temperature of column: 40°C
Temperature of sample manager: 5°C
TUV detector set at: 525 nm
Gradient: A linear gradient (curve no. 6)
Pressure limits: 0 psi low; 15000 psi high, highest pressure observed 9346 psi
Instrument operations, data acquisition and processing: EmPower2 Chromatographic Data Software (Waters Corporation, MA, USA).
Samples were identified by the comparison of retention times with the corresponding retention times of standards.
A : 0.3% Phosphoric Acid in Water B : 100% Acetonitrile.
Time (min.) Initial 4.0 4.2
Flow (ml/min) 0.50 0.50 0.50
%A
%B
89.0 80.0 90.0
11.0 20.0 10.0
© Biswatrish Sarkar, B.I.T, Mesra. Ranchi. India
Mixture of 6 Major Anthocyanidin Standards
© Biswatrish Sarkar, B.I.T, Mesra. Ranchi. India
Calibration Curves Cyanidin Y = 8.75e + 003x – 4.11e + 004 LOD = 0.694105 LOQ = 2.082314 R2 = 0.995174
Delphinidin
Y = 8.09e + 003x – 1.41e + 004 LOD = 0.415148 LOQ = 1.245445 R2 = 0.996500
© Biswatrish Sarkar, B.I.T, Mesra. Ranchi. India
Calibration Curves Malvidin Y = 6.35e + 003x – 1.08e + 005 LOD = 0.044128 LOQ = 0.132384 R2 = 0.995170
Pelargonidin
Y = 7.78e + 003x – 8.17e + 004 LOD = 1.03891 LOQ = 3.116731 R2 = 0.993178
© Biswatrish Sarkar, B.I.T, Mesra. Ranchi. India
Calibration Curves Peonidin Y = 8.99e + 003x – 3.87e + 004 LOD = 0.411004 LOQ = 1.233011 R2 = 0.998079
Petunidin
Y = 1.67e + 004x + 1.14e + 005 LOD = 0.056584 LOQ = 0.169752 R2 = 0.998700
© Biswatrish Sarkar, B.I.T, Mesra. Ranchi. India
Hibiscus rosa-sinensis
© Biswatrish Sarkar, B.I.T, Mesra. Ranchi. India
Hibiscus sabdariffa © Biswatrish Sarkar, B.I.T, Mesra. Ranchi. India
Clitoria ternatea
© Biswatrish Sarkar, B.I.T, Mesra. Ranchi. India
Ocimum tenuiflorum
© Biswatrish Sarkar, B.I.T, Mesra. Ranchi. India
Syzygium cumini
© Biswatrish Sarkar, B.I.T, Mesra. Ranchi. India
Recovery Studies for Syzygium cumini Spiked Amounts
Anthocyanidins
500 ng
1000 ng
1500 ng
Percentage Recovered Cyanidin
73.42
67.59
56.99
Delphinidin
92.58
83.57
89.87
Malvidin
99.79
96.26
93.84
Petunidin
70.38
62.83
56.05
© Biswatrish Sarkar, B.I.T, Mesra. Ranchi. India
Identification of anthocyanin using Mass Spectrometry Unhydorlyzed plant extract from all the samples were analysed by UPLC coupled with ESI-MS/MS (UPLC and Q-TOF-Micro). The Quadrapole instrument was operated in ESI positive mode with following settings: Capillary Voltage: 3.0 Kv Cone voltage: 30 V Desolvation temp. : 250°C. Cone gas and desolvation gas flow: 150 and 1200 L h-1 Collision gas: High purity argon gas Collision energy: 30 eV. Mass spectra was recorded in continuum mode across the range 100 to1000 m/z.
Anthocyanins were identified based on the mass fragmentation obtained. © Biswatrish Sarkar, B.I.T, Mesra. Ranchi. India
Mass Spectrometric Profile of Anthocyanin Standards
Delphinidin (301.6863)
Malvidin (329.6037)
© Biswatrish Sarkar, B.I.T, Mesra. Ranchi. India
Mass Spectrometric Profile of Anthocyanin Standards
Pelargonidin (269.8420)
Peonidin (299.7162)
© Biswatrish Sarkar, B.I.T, Mesra. Ranchi. India
Mass Spectrometric Profile of Anthocyanin Standards
Petunidin (315.6581)
Cyanidin (285.7382)
© Biswatrish Sarkar, B.I.T, Mesra. Ranchi. India
Mass Spectrometric Profile of Anthocyanins from H. rosa-sinensis Fresh Petals
Cyanidin (285.8118)
Delphinidin (301.7619)
© Biswatrish Sarkar, B.I.T, Mesra. Ranchi. India
Mass Spectrometric Profile of Anthocyanins from H. rosa-sinensis Fresh Petals Petunidin (315.7354)
Malvidin(329.6915)
Pelargonidin(269.9373)
© Biswatrish Sarkar, B.I.T, Mesra. Ranchi. India
Mass Spectrometric Profile of Anthocyanins from H. rosa-sinensis Fresh Petals
Peonidin (299.7915)
© Biswatrish Sarkar, B.I.T, Mesra. Ranchi. India
Mass Spectrometric Profile of Anthocyanins from H. rosa-sinensis Dry Petals
Malvidin
Petunidin
Pelargonidin
Delphinidin
© Biswatrish Sarkar, B.I.T, Mesra. Ranchi. India
Mass Spectrometric Profile of Anthocyanins from H. sabdariffa Dry Calyces
Malvidin
Petunidin
Delphinidin
© Biswatrish Sarkar, B.I.T, Mesra. Ranchi. India
Mass Spectrometric Profile of Anthocyanins from S. cumini
Malvidin
Mass Spectrometric Profile of Anthocyanins from O. tenuiflorum
Cyanidin
© Biswatrish Sarkar, B.I.T, Mesra. Ranchi. India
Mass Spectrometric Profile of Anthocyanins from O. tenuiflorum
Peonidin
© Biswatrish Sarkar, B.I.T, Mesra. Ranchi. India
Summary of Mass Spectroscopy Results Standards
Fragment m/z
Cyanidin
285.7382
Hibiscus rosasinensis Fresh(HRF) 285.8118
Hibiscus rosasinensis Dried(HRD) Absent
Hibiscus sabdariffa Fresh(HS) Absent
Delphinidin
301.6863
301.7619
301.7955
301.7955
Malvidin
329.6037
329.6915
329.7002
329.6915
Pelargonidin
269.8420
269.9373
269.9453
Absent
Petunidin
315.6581
315.7354
315.7439
315.7525
Peonidin
299.7162
299.7915
Absent
Absent
© Biswatrish Sarkar, B.I.T, Mesra. Ranchi. India
MS/MS Spectra of S. cumini
493.00
331.33
Malvidin
Malvidin 3-glucoside
MS/MS
655.17 Malvidin 3,5-diglucoside
MS
© Biswatrish Sarkar, B.I.T, Mesra. Ranchi. India
Hypothetical Fragmentation Pattern of Malvidin 3,5-diglucoside +MS2
+MS OCH 3
Malvidin 3- glucoside
OH O
HO
+
O
HO
OCH 3
HO HO H H
O O
HO H
HO
HO HO H H
OH
HO
OCH 3
OCH 3
O H
HO H H
OH
O O
HO H
H
OH
HO HO H H HO
O H
C29H35O17
m/z 655.577 Da
C6H11O5 163.0606 Da
+
HO H H
O O
HO H
H
OH
H
C23H24O12 m/z 492.1268 Da
+
OCH 3 O
HO
OH
H
H
O
HO
+
OCH 3
OH
Malvidin
OH
O
O
C17H13O7
m/z 329.0661 Da
C6H11O5 163.0606 Da
HO HO H H
O H
HO
OH
H
C6H11O5 163.0606 Da
Malvidin 3,5-diglucoside
© Biswatrish Sarkar, B.I.T, Mesra. Ranchi. India
ACKNOWLEDGEMENT Supervisors: Dr. Kunal Mukhopadhyay Dr. D. Sasmal Analytical & Technical: Dr. Manish Kumar Dr. Inamul Haque Pritesh Vyas Bratati Chaudhary
Day to Day trouble shooting: Dr. Animesh Ghosh Shailesh Kumar Singh
© Biswatrish Sarkar, B.I.T, Mesra. Ranchi. India
Sincerely Acknowledges: BIRLA INSTITUTE OF TECHNOLOGY, MESRA, RANCHI. INDIA UNIVERSITY GRANTS COMMISSION, GOVT. OF INDIA MINISTRY OF FOOD PROCESSING INDUSTRIES, GOVT. OF INDIA
&
IWA 2011 © Biswatrish Sarkar, B.I.T, Mesra. Ranchi. India
© Biswatrish Sarkar, B.I.T, Mesra. Ranchi. India
