Técnica Analitos
HPLC- eupatin, casticin,
API ESI chrysoplenetin, cirsilineol
HPLC- casticin, eupatin,
APCI artemetin,
chrysosplenetin,
chrysosplenol-D, cirsilineol
HPLC- artemisinin
ESI- Otros flavonoides
MS/MS (identificación)
HPLC- (+)-catechin, (-)-
DAD epicatechin, quercetin,
myricetin, kaempferol, p-
coumaric conjugates,
caffeic or ferulic
conjugates, chlorogenic,
sinapic, cinnamic, gallic
acid, bezoic acid, gentisic
acid, p-anisic acid
Condiciones extracción óptimas
Hojas secas, molidas (10 g) + 100
ml disolvente [éter petróleo, n-
hexano, DCM, acetona, MeOH,
EtOH (96, 80, 60%), agua 85-90ºC]
for 72 h; maceración 72 h
Concentración
Hojas (100 g) +1 l DCM; maceración
72 h oscuridad
Sequedad 40ºC, P reducida
Redisolución ACN
Hojas secas (500 mg) + 13 ml agua
hirviendo, 1, 24, 48 h
Liofilización
Redisolución MeOH:agua (9:1)
Hojas secas (0,3 g) + 3 ml MeOH,
agitador rotatorio, 24 h
Centrifugación 1600 g, 10 min
Observaciones Separación/Identificación Ref.
↑ flavonoids totales: Columna: Purospher®Star RP-18, 250 × 4,6 mm, 5 μm 1
EtOH, acetone A: water pH 3.2 FA, B: ACN, C: MeOH.
↑ eupatin: EtOH; ↑ Oven T: 26°C
casticin: acetona Gas T: 350°C, flow rate: 10 L/min, nebuliser P: 30 psi, Q T
30°C, Capillary V: 3500 V, FS: m/z 100-800, mode: positive
ion; Scan time: 1 s
Casticin: [M + H]+ 375 Columna: AscentisExpress C18, 150 × 4,6 mm, 2,7 μm 2
Eupatin: [M + H]+ 361
Vol iny: 2 μL,
A: water 0,1% FA, B: A) and ACN 0,1% FA; isocrático 45%B
Flow-rate:1.0 mL/min
Mode: positive
APCI conditions: m/z 100–700, interval 0.5 s, scanspeed
1500 amu/s, N2 flow 4.0 L/min, Interface T 350ºC, heatblock
T 300 ºC, DL T 300 ºC, DL V -34 V, probe V +4.5 kV, Q-array
V 1.0 V, detection gain 1.05 kV
Artemisinin (MW 282): Columna: Gemini C18, 250 mm x 4.60 mm, 5 μm 3
MS1 [M+Na]+ 305 A: H2O FA 0.1%, pH 4 NaOH, B, ACN FA 0.1%; 12% B in A to
MS3 [M+NaCO]+ 277,0 25% B at 60 min, 60% B at 80 min and 100% B at 85 min
(30%), [M+Na-CH2CO]+ Flow rate 10 l/min
263,0 (100%), [M+Na- Modo: positive and negative
CO-H20]+ 259,1 (27%) Capillary V 4000 V, nebulizer gas (N2) 15 psi, drying gas (N2)
350ºC 5 l/min. FS: 100–2200 scan time 13,000 m/z/s
Columna: 100 RP-18, 25cm x 4mm, 5m 4
Fase móvil: MeOH 5% FA; gradiente lineal 15% a 35% en 15
min; e isocrático hasta 20 min
Fllow rate: 1mL/min
Volumen inyección: 20L
HPLC- Compuestos fenólicos Hojas secas (50 g) +500 ml - Phenomenex Gemini C18 column, 250 mm x 3.0 mm, 5 m 5
DAD- (identificación) disolvente (acetone, MeOH) Volumen inyección: 10 L
ESI/MSn maceración 24 h A: ACN 0,1% FA, B: agua 0,1/ FA; 20% A (0 min), 25% A (10
Sequedad 40ºC, P reducida min), 25% A (20 min), 50% A (40 min), 100% A (42–47 min),
Redisolución ACN:agua (20:80), 5 20% A (49–55 min); flow rate 0.4 mL/min
mg residuo/ml T columna: 30ºC
Ion trap MS-ESI, modo negativo, m/z 100–1000, scan speed
13,000 Da/s
Drying gas (N2) flow 10.0 mL/min, nebulizing gas (N2) P 50
psi; nebulizer T 365ºC, capillary potential +400 V, collision
gas (He) P 1 · 10-5 mbar, collision energy 40 V
MSn analysis: auto MSn mode, isolation width 4.0 m/z, 10-
1000 m/z, fragmentation amplitude 1.0 V (MSn up to MS4)
and two precursor ions.
LC-DAD- Compuestos fenólicos Hojas (1 g) + 100 ml MeOH, US 40 Columna Zorbax XDB-C18 5 m, 4.6mmx250 mm 6
ESI-MSn (identificación) min Volumen inyección: 10 l
A: ACN, B: water–0,3% acetic acid; 12–20% A at 0–10 min,
hold for 5 min, linearly gradient to 46% A in 30 min, then
linearly gradient to 80% A at 60 min; flow rate 0.8 mL/min
T columna: 25ºC
Ion trap MS-ESI, modo negativo, FS m/z 100–1000
He collision gas; N2 nebulizing gas
Ion spray V 4.5 kV; sheath gas 55 arbitrary units; auxiliary
gas 5U; capillary T, 350ºC; capillary V-4V; tuve lens offset
voltage -40V
Isolation width precursor ions 3.0 U
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