Quality control of Lycium chinense and Lycium barbarum cortex (Digupi) by HPLC using kukoamines as markers
© The Author(s) 2017
Received: 2 November 2016
Accepted: 6 December 2016
Published: 9 January 2017
Lycii Cortex (LyC), composed of Lycium chinense and Lycium barbarum cortex and having the Chinese name Digupi, is used to treat chronic diseases like cough, hypertension, and diabetes in Eastern Asia. However, chromatographic methods, such as TLC and HPLC, to determine the phytochemical composition of LyC have not been included in any official compendiums. This study aims to establish a validated HPLC method for quality control of LyC.
Kukoamines A and B (KA and KB, respectively) were selected as markers for the HPLC method. An acetic acid solution was adopted for sample extraction because it facilitated the release of kukoamines and effectively prevented their degradation. Optimal separation of the kukoamine isomers was achieved on hydrophilic ligand-coated C18 columns with a gradient elution of acetonitrile and 0.1% (v/v) trifluoroacetic acid. The average contents and proposed contents for LyC were calculated with a t test and an uncertainty test based on 16 batches of authentic samples.
The method was validated with linearity (r2 = 0.9999 for both KA and KB), precision (RSD = 1.29% for KA and 0.57% for KB), repeatability (RSD = 1.81% for KA and 0.92% for KB), and accuracy (recovery of 90.03–102.30% for KA, and 98.49–101.67% for KB), indicating that the method could offer reliable results for quality control analysis of LyC. At the 95% confidence level, the calculated content limits were 1.45 mg/g for KA and 4.72 mg/g for KB.
Compared with conventional morphological identification, the HPLC method involving KA and KB contents offers precise, objective, and quantitative results for quality control of LyC.
Lycii Cortex (LyC) is used to treat chronic diseases like cough, hypertension, and diabetes in Eastern Asia [1–3]. Although pharmacopeias from China, Japan, and Korea have officially stipulated the source of LyC as the root bark of Lycium chinense and Lycium barbarum [4–6], the herb is often misused or adulterated with other cortexes having similar appearances. Contemporary authentication of LyC relies on morphological identification [4–6]. In contrast, inspections based on characteristic constituents are practical and objective, because monitoring of phytochemical markers is normally not only genus-specific, but also herbal function-related [7–9].
Chromatographic techniques are commonly used for both qualitative and quantitative purposes with reliable results . However, a genuine chromatographic method for LyC involving unique markers is still missing in official compendiums. Although some phytochemicals such as phenolic acids and flavonoids have been recommended as markers for quality assessment for LyC [11–13], they are neither unique nor abundant in this herb. Hence, they should not be adopted.
Recently, kukoamines A and B (KA and KB, respectively) were recommended by us as unique markers for LyC , because they are truly bioactivity-related, genus-specific, and content-abundant in this herb. Although the LC–MS/MS method is considered to provide simultaneous and precise determination of numerous phytochemical constituents [14, 15], the high cost of the equipment and its operation has made this method less practical for daily quality control inspections.
This study aims to provide a validated method for quality control of LyC with reference to KA and KB. Based on the developed method, multiple batches of LyC were investigated and the content limits were statistically calculated.
Production areas and species of Lycii Cortex samples and the contents of kukoamines A and B
Content of KAa (mg/g)
Content of KBa (mg/g)
9.65 ± 0.26
5.03 ± 0.13
4.38 ± 0.27
13.27 ± 0.04
3.33 ± 0.18
13.50 ± 0.18
1.82 ± 0.13
3.39 ± 0.22
3.83 ± 0.03
13.29 ± 0.14
6.34 ± 0.11
22.08 ± 0.11
1.13 ± 0.02
3.49 ± 0.02
4.87 ± 0.04
14.27 ± 0.27
1.86 ± 0.09
6.80 ± 0.18
1.93 ± 0.02
6.65 ± 0.01
4.06 ± 0.23
16.88 ± 0.38
1.47 ± 0.01
4.76 ± 0.23
3.96 ± 0.29
19.02 ± 0.11
1.29 ± 0.03
3.47 ± 0.15
4.25 ± 0.04
18.45 ± 0.60
1.75 ± 0.02
7.60 ± 0.02
LyC sliced samples, with the commercial names Hong Digupi (RLyC) and Bai Digupi (WLyC), were provided by herb stores from the Hehuachi herbal market in Chengdu (Sichuan Province, China). These samples had unclear sources, and were classified based on traditional experience. LyC extracts (Nos. 01–03) were bought from herbal extract companies in Sichuan, Shanxi, and Hubei. These extracts were claimed to be extracted from LyC.
The HPLC analysis was performed on an Agilent 1260 system (Agilent, USA) equipped with an on-line degasser (G1322A; Agilent, USA), a binary bump (G1312C; Agilent, USA), an autosampler (G1329B; Agilent, USA), a column oven (G1316A; Agilent, USA), and a diode array detector (G1315D; Agilent, USA). A Zorbax C18 SB-AQ column (250 mm × 4.6 mm i.d., 5 μm; Agilent, USA) was used for the separation. A mobile phase consisting of 0.1% TFA (A) and acetonitrile (B) was applied for the separation with the following gradient program: 0–15 min, 12–16% B; 15–35 min, 16–22% B. The flow rate was 1.0 mL/min and the column oven was set at 40 °C. The detection wavelength was 280 nm.
Each LyC sample was cut into small pieces and pulverized into a powder (passed through a 200-mesh sieve). The herbal powder (0.5 g) was accurately weighed. The extraction solvent was a 50% methanol aqueous solution containing 0.5% acetic acid (v/v). After addition of 20 mL of the extraction solvent, the mixture was sonicated in an ultrasonic bath (100 W, AC-120H; MRC, Germany) for 30 min and then centrifuged at 2880×g for 10 min (Allegra X-15R; Beckman Coulter, USA). The supernatant was transferred into a 50-mL volumetric flask and the pellet was re-extracted with another 20 mL of extraction solvent for 30 min. After centrifugation, the extracted solution was transferred to the same volumetric flask. Next, 10 mL of extraction solvent was used to wash the tube, and the washing solution was also added to the volumetric flask and filled to the mark. The prepared sample solution was filtered through a 0.22-μm pore-size filter before injection.
The method detection limit (MDL) and limit of quantitation (LOQ) were determined according to the guidelines in Validation of Analytical Procedures: Methodology (Q2B) .
The contents of samples were presented as the mean ± standard deviation. The proposed content limit for each analyte was calculated according to the contents from multiple batches with consideration of the standard uncertainty of the precision, bias, purity of reference substance, and water content (the calculation equation is shown in Additional file 1). The calculation was based on the ISO “Guide to the Expression of Uncertainty in Measurement” , EURACHEM/CITAC document “Quantifying Uncertainty in Analytical Measurement” , and LGC document “Development and Harmonization of Measurement Uncertainty Principles” . The mean differences for KA and KB in the two species of L. chinense and L. barbarum were evaluated by analysis of variance (ANOVA). If the P value was less than 0.05, a follow-up post hoc test was conducted.
Results and discussion
Establishment of HPLC conditions for determination of kukoamines
Selection of column, mobile phase, and detection wavelength
KA and KB are water-soluble spermine alkaloids with multiple amino and amide groups [22, 23] that cause peak tailing on the C18 packing material. Three kinds of C18 columns with special coatings on the packing surface, i.e., Zorbax C18 BDS, Zorbax C18 Extended, and Zorbax C18 SB-AQ (all from Agilent, USA), were compared for the analyte retention behaviors. The kukoamines showed better retention on the Zorbax C18 SB-AQ column, owing to the better affinity of the extremely water-soluble analytes for the hydrophilic packing surface.
Influence of acids on chromatographic behaviors of kukoamines
Aqueous phasea (v/v)
Retention time (min)
0.2% Formic acid (v:v) pH 2.5
0.1% Phosphate acid (v:v) pH 2.0
0.1% Trifluoroacetic acid (v:v) pH 2.0
Influence of pH on the stability of the kukoamines
Optimization of kukoamine extraction procedures
Linearity, precision, repeatability, recovery, MDL, and LOQ of the established method
Linearity (12 points)
3.91–250.00 mg/L; r2 = 0.9999a
4.12–263.50 mg/L; r2 = 0.9999a
Precision (RSD)b, %
Repeatability (RSD)c, %
Mean recovery (n = 5)d
Quantification results and proposed contents
The LyC samples from L. chinense and L. barbarum did not differ significantly from each other in their kukoamine contents when compared by one-way ANOVA (Additional file 1). These findings demonstrated that both plants can produce LyC with equivalent quality.
Mean contents and proposed content limits for kukoamines A and B in Lycii Cortex
t critical e
Failing rate (%)i
Application of the HPLC method for quality control of LyC
Compared with the conventional morphological identification, the proposed HPLC method for monitoring the characteristic constituents KA and KB provides a simple and universal way to measure the quality of LyC samples. The method and calculated content limits presented in this article could support amendments to the quality standards for LyC in official compendiums.
The developed method could be applied for quality inspection of LyC in the food and pharmaceutical manufacturing industries.
high performance liquid chromatography
limit of quantification
method detection limit
relative standard deviation
LYY and DR conceived and designed the study. CHY secured financial support and oversaw the whole study. DR, LYY, and HWL performed the experiments. HYQ performed the statistical analysis. LYY and DR drafted the manuscript and CHY revised it. All authors read and approved the final manuscript.
The authors also extend their gratitude to Mr. William T. Mahan for language editing.
The authors declare that they have no competing interests.
Hong Kong Chinese Materia Medica Standards, Department of Health, Hong Kong Government, SAR, China, for the CityU project (No. 9210029).
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