A sensitive chemiluminescence (CL) method for the determination of hesperetin at nanogram levels was first presented. It was based on the inhibitory effect of hesperetin on luminol-dissolved oxygen CL reaction in a flow injection system. The decrements of CL intensity were logarithm over the concentrations of hesperetin in a range of 0.5 to 1000 ng mL−1, with a detection limit of 0.2 ng mL−1 (
Hesperetin (5, 7, 3′-trihydroxy-4′-methoxy-flavanone, Figure
Structure of hesperetin.
Methods previously employed for the determination of hesperetin involve high performance liquid chromatography (HPLC) with UV or MS detector [
In this work, it was found that hesperetin could inhibit the CL intensity from luminol-dissolved oxygen reaction, and the CL intensity decrements were logarithm over hesperetin concentrations ranging from 0.5 to 1000 ng mL−1, with a detection limit (LOD) of 0.2 ng mL−1 (
All chemicals were of analytical reagent grade. Deionized water purified in a Milli-Q system (Millipore, Bedford, MA, USA) was used throughout. Luminol (Fluka, Biochemika, Switzerland) was purchased from Xi’an Medicine Purchasing and Supply Station, China. Luminol (2.5 × 10−2 mol L−1) stock solution was prepared in 1.0 × 10−1 mol L−1 NaOH solution. Hesperetin was supplied by Shaanxi Entry-Exit Inspection and Quarantine Bureau (Shaanxi, China). Hesperetin stock solution (10
The FI-CL system (Xi’an Remax Analysis Instrument Co. Ltd., Xi’an, China) was depicted in Figure
Schematic diagram of the FI-CL system for the determination of hesperetin.
As shown in Figure
Hesperidin in PCR (the dried ripe pericarp of
Human serum samples from healthy volunteers were provided by the Hospital of Northwest University. The spiked samples were prepared by adding known quantities of hesperetin into 0.1 mL of each serum samples. After homogenization, the spiked serum samples with appropriate dilution were taken for hesperetin determination.
The relative CL intensity-time profile of luminol-dissolved oxygen-hesperetin was given in Figure
Relative CL intensity: time profile of different reactions. Luminol: 25
The effect of luminol concentration on the CL intensity was investigated over a range of 1.0 × 10−7 to 1.0 × 10−4 mol L−1, and 2.5 × 10−5 mol L−1 luminol was selected as the optimum concentration giving a stable and strong CL intensity. Considering the nature of luminol CL reaction favoring in alkaline medium, 2.5 × 10−2 mol L−1 NaOH was added into the solution to enhance the CL intensity.
The effect of flow rate and mixing tube lengths was also examined. 2.0 mL min−1 flow rate and 5.0 cm mixing tube length were chosen in the subsequent experiments as a good compromise of sensitivity, reagent consumption, and reproducibility.
Under the optimum experimental conditions given above, the standard solutions of hesperetin were determined. It was found that the CL intensity decrements were linear with the logarithm of hesperetin concentrations ranging from 0.5 to 1000 ng mL−1, giving the calibration equation of
The interference of potentially interfering species were tested by adding increasing amounts of interfering substance to the standard solution of hesperetin (10 ng mL−1) and the error was controlled at 5% level. The tolerable concentrations of interfering species were 100
The possible mechanism of luminol-dissolved oxygen-hesperetin reaction was discussed in detail. Firstly, the CL emission spectrum showed that the maximum CL emission wavelength was at 425 nm. This result suggested that luminol could react with dissolved oxygen to yield
2.5 g PCR were pretreated by the modified method which was described in the experimental section and then suitable aliquots from above prepared samples were taken for the indirect determination of hesperidin. In order to evaluate the validity of the proposed method, recovery studies were carried out and the results were listed in Table
Results of hesperidin in PCR (
Samples number | Added ng mL−1 | Found ng mL−1 | RSD % | Recovery % | Content in PCR % | |
---|---|---|---|---|---|---|
1 | 0 | 199 | 1.9 | 95.0 | 8.0 | |
100 | 294 | 1.4 | ||||
2 | 0 | 201 | 1.5 | 103.9 | 8.1 | |
100 | 305 | 1.2 | ||||
3 | 0 | 205 | 1.5 | 107.0 | 8.3 | 8.1 ± 0.2 |
200 | 419 | 0.9 | ||||
4 | 0 | 197 | 2.0 | 99.7 | 7.9 | |
200 | 396 | 1.0 | ||||
5 | 0 | 203 | 1.9 | 96.9 | 8.2 | |
300 | 494 | 0.7 | ||||
6 | 0 | 200 | 1.9 | 104.0 | 8.0 | |
300 | 512 | 0.6 |
Hesperetin in the spiked serum samples prepared in the experimental section were measured, and the results were listed in Table
Hesperetin determination in spiked human serum samples (
Samples number | Added ng mL−1 | Found ng mL−1 | RSD % | Recovery % | Proposed method/spiked |
---|---|---|---|---|---|
1 | 0 | 19 | 1.5 | 97.0 | 9.7/10.0 |
20 | 39 | 1.0 | |||
2 | 0 | 21 | 1.4 | 106.6 | 10.6/10.0 |
20 | 43 | 0.9 | |||
3 | 0 | 20 | 1.6 | 98.2 | 9.8/10.0 |
20 | 39 | 1.0 | |||
4 | 0 | 21 | 1.3 | 102.4 | 10.5/10.0 |
30 | 52 | 0.8 | |||
5 | 0 | 20 | 1.5 | 97.0 | 9.7/10.0 |
30 | 49 | 0.9 | |||
6 | 0 | 21 | 1.6 | 103.7 | 10.3/10.0 |
30 | 52 | 0.8 |
A simple and rapid CL method for the determination of hesperetin at nanogram levels was proposed for the first time. A comparison of the presented FI-CL method and the previously reported methods for the determination of hesperetin was given in Table
Comparison of different methods for hesperetin determination.
Methods | Linear range ng mL−1 | LOD ng mL−1 | Samples | References |
---|---|---|---|---|
MEKC-UV |
|
6000 | Propolis | [ |
HPLC-UV |
|
500 | Human urine |
[ |
Rat urine | ||||
|
100 | Human urine | [ | |
GC-MS |
|
2.0 | Human plasma |
[ |
Human urine | ||||
HPLC-MS/MS |
|
0.5 | Rat plasma | [ |
FI-CL |
|
0.2 | PCR | This study |
Human serum |
The authors gratefully acknowledge the financial support from the NWU Graduate Innovation and Creativity Funds (no. 09YZZ45 and no. 10YZZ29) and the open fund from the Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, China.