Identification of Aqueous Extract of Red Amaranth …
171
Molar extinction coefficient of cyanidine-3-glucoside was 26,900 molar in
L mol
−1 cm
−1 . Results were expressed as milligrams of cyanidine-3-glucoside
equivalent (CGE) 100 g_1 DW.
3.7 Identification of Red Amaranth Aqueous Extract
by HPLC Methods
For removing the sugar and acid from the aqueous extract, 1% trifluoroacetic acid
(TFA) in methanol was added to it and then it was pass through a supelclean LC18 cartridge (María de Lourdes Vargas y Vargas et al 2013). A high-performance
liquid chromatography (HPLC) (Alliance 2695 HPLC system; Waters Corporation,
Massachusetts, MA) is equipped with a binary pump, a dual λ absorbance UV
detector 2487, an inline degasser, and the Empower 2 software. The separations
were performed using Symmetry C-18 reversed-phase column (250 mm × 4.6 mm
length, 5 μm particle size) at 30 °C. Acidified water with 0.1% TFA (v/v) was used
as mobile phase A and HPLC grade acetonitrile with 0.035% formic acid (v/v) was
used as phase B. The injection volume was 20 μl. The following gradients were used:
10%–11% of B in 12 min, 11%–12% in 8 min, 12%–13% in 5 min, 13%–18% in
10 min, and 18% of B maintained for 25 min. The anthocyanins were analyzed with
a visible detector at 520 nm. Before injecting it, the sample was filtered through a
0.45 μm acrodisc (Millipore, Bedford, MA). The flow rate was kept at 1 ml/min. For
the identification of anthocyanin cyaniding-3-glucoside was used as a standard. The
amount of phenolic compound was expressed as milligram per gram of the extract.
3.8 Qualitative and Quantitative Analysis of Aqueous Red
Amaranth Leaves by LC-MS
Analyses were performed using a Waters Xevo–G2 XS Q-TOF mass C-18 column
(100 × 2.1 mm i.d.; particle size 5 μm) and as mobile phase a gradient of 0.1%
formic acid as eluent A and acetonitrile (LC-MS grade, J. T Becker) as eluent B. The
flow rate was 0.250 ml/min and the amount injected was 10 μl. Here, the column
back pressure was used as 1490 bar and the temperature was set at 25 °C. An electrospray ionization source (source block temperature 130 °C, desolvation temperature
300 °C, capillary voltage 3 kV, cone voltage 35 V) was used when the desolvation
and cone gas flow rate were 650 and 50 L/h, respectively. The flow generated by
chromatographic separation was directly injected into the electro-spray ion source
for mass detection. Positive ion as well as negative ion mode was used for mass
detection.
171
Molar extinction coefficient of cyanidine-3-glucoside was 26,900 molar in
L mol
−1 cm
−1 . Results were expressed as milligrams of cyanidine-3-glucoside
equivalent (CGE) 100 g_1 DW.
3.7 Identification of Red Amaranth Aqueous Extract
by HPLC Methods
For removing the sugar and acid from the aqueous extract, 1% trifluoroacetic acid
(TFA) in methanol was added to it and then it was pass through a supelclean LC18 cartridge (María de Lourdes Vargas y Vargas et al 2013). A high-performance
liquid chromatography (HPLC) (Alliance 2695 HPLC system; Waters Corporation,
Massachusetts, MA) is equipped with a binary pump, a dual λ absorbance UV
detector 2487, an inline degasser, and the Empower 2 software. The separations
were performed using Symmetry C-18 reversed-phase column (250 mm × 4.6 mm
length, 5 μm particle size) at 30 °C. Acidified water with 0.1% TFA (v/v) was used
as mobile phase A and HPLC grade acetonitrile with 0.035% formic acid (v/v) was
used as phase B. The injection volume was 20 μl. The following gradients were used:
10%–11% of B in 12 min, 11%–12% in 8 min, 12%–13% in 5 min, 13%–18% in
10 min, and 18% of B maintained for 25 min. The anthocyanins were analyzed with
a visible detector at 520 nm. Before injecting it, the sample was filtered through a
0.45 μm acrodisc (Millipore, Bedford, MA). The flow rate was kept at 1 ml/min. For
the identification of anthocyanin cyaniding-3-glucoside was used as a standard. The
amount of phenolic compound was expressed as milligram per gram of the extract.
3.8 Qualitative and Quantitative Analysis of Aqueous Red
Amaranth Leaves by LC-MS
Analyses were performed using a Waters Xevo–G2 XS Q-TOF mass C-18 column
(100 × 2.1 mm i.d.; particle size 5 μm) and as mobile phase a gradient of 0.1%
formic acid as eluent A and acetonitrile (LC-MS grade, J. T Becker) as eluent B. The
flow rate was 0.250 ml/min and the amount injected was 10 μl. Here, the column
back pressure was used as 1490 bar and the temperature was set at 25 °C. An electrospray ionization source (source block temperature 130 °C, desolvation temperature
300 °C, capillary voltage 3 kV, cone voltage 35 V) was used when the desolvation
and cone gas flow rate were 650 and 50 L/h, respectively. The flow generated by
chromatographic separation was directly injected into the electro-spray ion source
for mass detection. Positive ion as well as negative ion mode was used for mass
detection.