Successive cartridge clean-up method for the simultaneous determination of matrine and oxyma- trine in biopesticides containing Sophora flavescens extract was developed and validated by UPLC. The clean-up method was established with ENVI-Carb (0.5 g) and C18 SPE (0.5 g) cartridges for the bioactive alkaloid in biopesticides from S. flavescens, and the eluate was analyzed to quantify the matrine and oxymatrine by UPLC. The developed method was validated, and the recovery and LOQ of both materials were 105.0% and 103.6%, and 0.050 and 0.684 mg·kg-1, respectively. Of the twenty one samples, the total content of matrines were analyzed by using the developed method and the result showed the developed successive clean-up method could contribute to the manufacture and control of biopesticides including matrines, and can be ap- plied to the method development for the analysis of alkaloid materials in biopesticides.
Recently, biopesticide with broad-spectrum to pest control has been actively investigated for its sustainability to avoid disrupting the ecosystem and environmental contamination originated from synthetic alternatives [
Sophora flavescens is a traditional Chinese medicinal herb and has been used for anti-tumor, viral hepatitis and anti-arthritis [
Many researchers reported different extractions and clean-up methods to achieve the quantification of bioac- tive materials with tedious and/or repeated chromatography based on the polarity of chemicals [
Matrine (purity 99.9%) and oxymatrine (purity 94.3%) were purchased from ChromaDex (Irvine, California, USA). High performance liquid chromatography (HPLC) grade methyl alcohol (MeOH), acetonitrile, and ace- tone were purchased from Tedia (Ohio, USA). Hydrophilic lipophilic balanced (HLB) SPE (60 mg, 3 mL) from Waters (Milford, USA) were used, and six carbonaceous SPE cartridges were purchased; ENVI-Carb SPE car- tridge (0.25 g, 3 mL and 0.5 g, 6 mL) from Supelco (Philadelphia, USA), activated carbon (0.5 g, 6 mL) from Silycycle (Quebec, Canada), Carbograph SPE tubes (0.5 g, 8 mL and 1 g, 15 mL) from Chrom Tech (Minnesota, USA), and Bond Elut Carbon (0.5 g, 6 mL) from Agilent (California, USA). C18 SPE (0.5 g, 6 mL) was from Phenomenex (California, USA). Twenty one commercial biopesticides containing S. flavescens extract were purchased from 14 Korean local companies.
The HLB clean-up procedure as reported by Lee et al. was followed [
In order to compare the recovery rate of matrine and oxymatrine, the six carbonaceous SPE were tested. Each cartridge was placed onto female-luer port of the SPE vacuum manifold and conditioned with 3 mL of MeOH and 3 mL of DW. A portion of 1 mL aqueous solution of matrine (10 mg·L−1), oxymatrine (10 mg·L−1), and the biopesticides diluted 10 times with DW, were loaded each into conditioned cartridges, respectively, and were eluted with MeOH (3 Í 3 mL) after washing/non-washing with 12 mL of DW. Eluate evaporated slowly under reduced pressure, and the dried residues were re-dissolved into 1 mL of MeOH for analysis. UPLC system (Wa- ters, MA, USA) with UV detector was used for matrine and oxymatrine analysis.
ENVI-Carb SPE cartridge (0.5 g) was selected according to the result of Section 2.2. The second clean-up procedure was conducted with C18 SPE cartridge (0.5 g) to remove unknown impurities for reduce the burden on UPLC. C18 SPE cartridge was placed to the bottom of the ENVI-Carb cartridge after DW washing as men- tioned in Section 2.2. The eluate of ENVI-Carb was fully loaded to the C18 cartridge and the eluate was col- lected. Then the C18 cartridge was additionally eluted with 9 mL of MeOH. The second clean-up efficiency was confirmed with absorbance of eluate from 210 to 800 nm using UV-Vis spectrophotometer (Lambda 650, Perkin-Elmer, London, UK).
Matrine and oxymatrine were analyzed on a Waters UPLC system. One microliter of a sample was introduced to a Waters Acquity BEH Phenyl column (1.7 mm particle size, 3.0 × 100 mm, Waters, Boston, USA). The column was maintained at a temperature of 40˚C and a constant flow rate of 0.5 mL·min−1. The UPLC was operated us- ing a gradient mobile phase of solvent A (0.05% formic acid in DW) and B (acetonitrile). The gradient was 0% - 10% B (0 - 10 min), 10% - 50% B (10 - 15 min), and 50% - 100% B (15 - 17 min). The absorbance of matrine and oxymatrine was measured at 217 nm.
Sample preparation and analytical methods were validated in terms of linearity, limit of quantitation (LOQ) and recovery. The linearities of the calibration curve for matrine and oxymatrine were obtained from the concentra- tions 1, 5, 10, 25 and 50 mg·L−1, respectively. LOQs for matrine and oxymatrine were calculated from the con- centration that produced a signal-to-noise ratio of 10. Recoveries of matrine and oxymatrine were tested by spiking the standard material at a concentration of 10 mg·L−1 in the extract of neem and its biopesticide that were not containing the analytes. The precision of the method was expressed in percentage (%) of relative stan- dard deviation (RSD).
Recently, Lee et al. reported the analytical method using HLB SPE and UPLC for the quantitative analysis of bioactive limonoids in the biopesticides containing neem extract [
Furthermore, the LC condition had to be optimized to get the best result particularly on good sensitivity for matrine and oxymatrine. Acquity UPLC BEH Phenyl column has been known to provide complementary selec- tivity to C18 stationary phase, especially for aromatic compounds. However, C18 column was used for HPLC analysis of matrine and oxymatrine in previous studies [
UPLC chromatogram of matrine and oxymatrine in biopesticide
This study tried to find out the suitable SPE for the clean-up of biopesticides containing S. flavescens extract and various supplementary materials, and selected carbonaceous SPE. Carbonaceous SPE can be applied to the aqueous sample unlikely to silica gel or Florisil cartridge. Also, many reports have described the extraction of polar analytes and/or multi-residue extractions such as chloroaniline, chlorophenols, and polar pesticides with graphitized carbons [
Six carbonaceous cartridges were tested to compare the recovery rate of matrine and oxymatrine with or without washing step. The recoveries of matrine and oxymatrine both with and without DW washing were 78.6% - 131.0% in ENVI-Carb (0.25 and 0.5 g) and Bond Elut Carbon tube (0.5 g), whereas below 48% in the other SPE cartridge (
To improve data reproducibility, the second purification step was taken into consideration, and C18 SPE was adopted to remove non-polar impurities. The C18 SPE and ENVI-Carb SPE were connected sequentially after washing step of the method 2.3, and MeOH and acetonitrile were tested as elution solvents to find the suitable one because the two solvents were shown to have the good recoveries above 95%. The ENVI-Carb and C18 SPE were eluted with 3 × 3 mL MeOH or acetonitrile, then 9 mL MeOH or acetonitrile was additionally eluted to C18 SPE cartridge. The spectra for the eluates of two solvents were measured with UV-visible spectrophotometer, and MeOH showed better impurity removing efficiency than acetonitrile on the comparison of Abs217 (
. Linear corre
. Method validation result for matrine and oxymatrine
Materials | Linearity (r2) | Recovery (%) | LOQ (mg·L−1) | RSD (%) | |
---|---|---|---|---|---|
Inter-day | Intra-day | ||||
Matrine | 0.9986 | 105.0 ± 1.39† | 0.266 | 5.1 | 2.9 |
Oxymatrine | 0.9996 | 103.6 ± 2.05 | 0.684 | 5.9 | 4.2 |
†The data represent the mean values ± SD of three replicates.
lations from 1 to 50 mg·L−1 for both matrine and oxymatrine were detected with coefficient correlations of 0.9986 and 0.9996, respectively. The method LOQs of matrine and oxymatrine were 0.226 and 0.684 mg·L−1, respectively. Validation of the analytical method was conducted in terms of recovery of the spiked sample that did not contain S. flavescens. Recoveries of 10 mg·L−1 matrine and oxymatrine were found to be 105.0 and 103.6% using the established method. The inter- and intra-day precision of the method were determined from recovery of both materials on three separate days. The method was reliable since the RSD percentage (2.9% - 5.9%) below 15, which was the normal percent value. These results suggest that the experimental procedure including clean-up and instrumental analysis was suitable for use in the analysis of targeted materials in biopesticide samples.
The developed method for matrine and oxymatrine analysis was applied to the extract of S. flavescens and its biopesticides. Matrine and oxymatrine in all samples were detected at 0.00 - 0.66 and
UV-Vis spectra of MeOH (a) and acetonitrile (b) eluate of biopesticide sample with and without C18 SPE cartridge clean-up
per insect, respectively. If we only consider the concentration of alkaloidal matrines as bioactive materials, the contents of matrine and oxymatrine in some products among the tested biopesticides were assumed to be sufficient as crop protection agents. However, if we consider the practical use guideline of biopesticide, the concentration of the active ingredient would be expected to be lower than EC50 and LD50 level, because the general guideline for field application was suggested be 100 - 1000 folds diluted with water. In a survey of matrines present in biopesticides from S. flavescens, the concentration of bioactive materials showed wide ranges and these results would be related to the crop protection efficiency. Thus, the quality control of the commercial biopesticide should be performed.
This study successfully developed and validated the clean-up method by using carbonaceous and C18 SPE car- tridge for the simultaneous determination of matrine and oxymatrine in biopesticides containing S. flavescens extract. This is the first report of the simultaneous analysis of matrine and oxymatrine in biopesticides which are constituted with various interferences of instrumental analysis such as pigments, lipids, and surfactants. This method could be contributed to the manufacture for quality control and to the method development for the quantitative analysis or effective purification of alkaloidal indicator in other biopesticides.
Flow chart for matrine and oxymatrine analyses in biopesticides
. Matrine and oxymatrine content in S. flavescens extract and its biopesticides
Samples | Matrine (%) | Oxymatrine (%) | Total (%) |
---|---|---|---|
S. flavescens extract | 0.31 | 0.31 | |
1 | 0.37 | 0.10 | 0.47 |
2 | 0.15 | 0.15 | |
3 | 0.00 | 0.00 | 0.00 |
4 | 0.01 | 0.01 | |
5 | 0.00 | 0.00 | |
6 | 0.27 | 0.05 | 0.32 |
7 | 0.23 | 0.23 | |
8 | 0.59 | 0.59 | |
9 | 0.18 | 0.18 | |
10 | 0.12 | 0.16 | 0.28 |
11 | 0.16 | 0.16 | |
12 | 0.24 | 0.24 | |
13 | 0.05 | 0.14 | 0.19 |
14 | 0.35 | 0.35 | |
15 | 0.45 | 0.45 | |
16 | 0.66 | 0.05 | 0.71 |
17 | 0.35 | 0.12 | 0.47 |
18 | 0.01 | 0.01 | |
19 | 0.14 | 0.14 | |
20 | 0.32 | 0.32 | |
21 | 0.23 | 0.12 | 0.35 |
This study was carried out with the support of “Cooperative Research Program for Agricultural Science & Technology Development (PJ009219)”, Rural Development Administration and “Research Program for Agri- cultural Science & Technology Development (PJ008468)” and “Postdoctoral Fellowship Program of Chemical Safety Division”, National Academy of Agricultural Science, Rural Development Administration, Republic of Korea.