Study of the extraction procedure by experimental design and validation of a LC method for determination of flavonoids in Citrus bergamia juice.
A reversed-phase high-performance liquid chromatographic (HPLC) separation with photo-diode array detection was developed for the simultaneous determination of flavonoids extracted from Citrus bergamia juice. It employs a C18 reversed-phase column and a linear gradient elution system with methanol/water with 5% acetic acid (v/v), as mobile phase. The method was validated in terms of detection limits (LOD), quantitation limits (LOQ), linearity, precision and accuracy. Limits of detection ranged from a low of 0.007 mg ml(-1) (narirutin) to a high of 0.018 mg ml(-1) (didymin). The limits of quantitation were between a low of 0.011 mg ml(-1) (7-OH flavanone) and a high of 0.024 mg ml(-1) (didymin). An excellent linear response was observed over the range specified for all analytes, as confirmed by the correlation coefficient with ranged from 0.9982 and 0.9999. The intra-day R.S.D.% ranged from 0.11 to 3.64%. The intermediate precision R.S.D.% were not higher than 7.62%. The accuracy of the method was confirmed with an average recovery ranging, except for neoeriocitrin, between 88.07% and 102.45%. Since the extraction conditions can affect analyte recovery, a suitable optimization strategy of the procedure was needed. The experimental parameters optimized were extraction time, temperature, and solvents. A multivariate approach was used to provide direct evaluation of the selected variables and related interactions. The D-optimal design was constructed by applying the exchange algorithm. All experimental results were computed by NEMROD-W software. This methodology led us to obtain the best recovery for all the flavonoids in the least number of experiments.
Calabrò
M L
ML
Dipartimento Farmaco-Chimico, Facoltà di Farmacia, Università di Messina, Viale Annunziata, 98168 Messina, Italy.
Galtieri
V
V
Cutroneo
P
P
Tommasini
S
S
Ficarra
P
P
Ficarra
R
R
D002853:Chromatography, Liquid / Q000379:methods* / Q000592:standards; D002957:Citrus; D005419:Flavonoids / Q000737:chemistry / Q000302:isolation & purification*; D005638:Fruit; D010936:Plant Extracts / Q000737:chemistry / Q000302:isolation & purification*; D012107:Research Design / Q000592:standards
D005419:Flavonoids; D010936:Plant Extracts