Background
Methods
Reagents
Plant materials
Batch no. | Species | Origin | Collection year | Content of KAa (mg/g) | Content of KBa (mg/g) |
---|---|---|---|---|---|
LC 1 |
L. chinense
| Jiangxi | 2011 | 9.65 ± 0.26 | 5.03 ± 0.13 |
LC 2 |
L. chinense
| Jiangxi | 2011 | 4.38 ± 0.27 | 13.27 ± 0.04 |
LC 3 |
L. chinense
| Jiangxi | 2011 | 3.33 ± 0.18 | 13.50 ± 0.18 |
LC 4 |
L. chinense
| Jiangxi | 2011 | 1.82 ± 0.13 | 3.39 ± 0.22 |
LC 5 |
L. chinense
| Gansu | 2011 | 3.83 ± 0.03 | 13.29 ± 0.14 |
LC 6 |
L. chinense
| Shangxi | 2011 | 6.34 ± 0.11 | 22.08 ± 0.11 |
LC 7 |
L. chinense
| Neimenggu | 2011 | 1.13 ± 0.02 | 3.49 ± 0.02 |
LC 8 |
L. chinense
| Neimenggu | 2011 | 4.87 ± 0.04 | 14.27 ± 0.27 |
LC 9 |
L. barbarum
| Ningxia | 2011 | 1.86 ± 0.09 | 6.80 ± 0.18 |
LC 10 |
L. barbarum
| Ningxia | 2011 | 1.93 ± 0.02 | 6.65 ± 0.01 |
LC 11 |
L. barbarum
| Ningxia | 2011 | 4.06 ± 0.23 | 16.88 ± 0.38 |
LC 12 |
L. barbarum
| Ningxia | 2011 | 1.47 ± 0.01 | 4.76 ± 0.23 |
LC 13 |
L. barbarum
| Ningxia | 2011 | 3.96 ± 0.29 | 19.02 ± 0.11 |
LC 14 |
L. chinense
| Hong Kong | 2011 | 1.29 ± 0.03 | 3.47 ± 0.15 |
LC 15 |
L. chinense
| Hong Kong | 2012 | 4.25 ± 0.04 | 18.45 ± 0.60 |
LC 16 |
L. chinense
| Hong Kong | 2012 | 1.75 ± 0.02 | 7.60 ± 0.02 |
Chromatographic conditions
Sample preparation
Method validation
Statistical analysis
Results and discussion
Establishment of HPLC conditions for determination of kukoamines
Selection of column, mobile phase, and detection wavelength
Aqueous phasea (v/v) | Analytes | Retention time (min) | Platesb
| Rc
| Td
|
---|---|---|---|---|---|
Water | KA | 15.3 | 126 | 0.8 | 2.30 |
KB | 20.9 | 52 | 4.40 | ||
0.2% Formic acid (v:v) pH 2.5 | KA | 34.6 | 4557 | 1.0 | 3.02 |
KB | 37.8 | 4632 | 3.06 | ||
0.1% Phosphate acid (v:v) pH 2.0 | KA | 35.1 | 3954 | 0.7 | 2.01 |
KB | 37.9 | 3249 | 2.52 | ||
0.1% Trifluoroacetic acid (v:v) pH 2.0 | KA | 47.3 | 10,091 | 2.6 | 1.10 |
KB | 37.1 | 10,887 | 1.12 |
Influence of pH on the stability of the kukoamines
Optimization of kukoamine extraction procedures
Method validation
Parameter | Kukoamine A | Kukoamine B |
---|---|---|
Linearity (12 points) | 3.91–250.00 mg/L; r2 = 0.9999a
| 4.12–263.50 mg/L; r2 = 0.9999a
|
Precision (RSD)b, % | 1.29 | 0.57 |
Repeatability (RSD)c, % | 1.81 | 0.92 |
Mean recovery (n = 5)d
| 95.89% 90.03–102.30% | 100.32% 98.49–101.67% |
MDL (µg/mL) | 1.76 | 1.86 |
LOQ (µg/mL) | 8.78 | 9.29 |
Quantification results and proposed contents
Parameters | KA | KB |
---|---|---|
Caa
| 3.49 | 10.75 |
SDb
| 2.51 | 6.31 |
RSDc
| 71.91 | 58.73 |
nd
| 16 | |
t
critical
e
| 2.131 | |
uc (C)f
| 0.46 | 2.67 |
Calculated limitg
| 1.45 | 4.72 |
Outlierh
| 0 | 0 |
Failing rate (%)i
| 12.50 | 18.75 |