Chinese Journal of Natural Medicines 2014, 12(10): 07860793
Chinese Journal of Natural Medicines
Simultaneous quantitative determination of five alkaloids in Catharanthus roseus by HPLC-ESI-MS/MS ZHANG Lin1, 2, 3†, GAI Qing-Hui1, 2, 3†, ZU Yuan-Gang1, 2, 3*, YANG Lei1, 2, 3, MA Yu-Liang1, 2, 3, LIU Yang1, 2, 3 1
State Engineering Laboratory of Bio-Resource Eco-Utilization, Northeast Forestry University, Harbin 150040, China;
2
Engineering Research Center of Forestry Bio-preparation, Ministry of Education, Northeast Forestry University, Harbin 150040, China;
3
Key Laboratory of Forest Plant Ecology, Ministry of Education; Northeast Forestry University, Harbin, 150040, China Available online October 2014
[ABSTRACT] AIM: To establish a method to simultaneously determine the main five alkaloids of Catharanthus roseus for trace samples, a high-performance liquid chromatography–electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) analysis method was developed. METHOD: The five Catharanthus alkaloids, vinblastine, vincristine, vinleurosine, vindoline, and catharanthine were chromatographically separated on a C18 HPLC column. The mobile phase was methanol−15 nmolL–1 ammonium acetate containing 0.02% formic acid (65 : 35, V/V). The quantification of these alkaloids was based on the Multiple Reaction Monitoring (MRM) mode. RESULTS: This method was validated, and the results achieved the aims of the study. The intra- and inter-day precision and accuracy of the five alkaloids were within 1.2%−11.5% (RSD%) and −10.9%−10.5% (RE%). The recovery rates of the five alkaloids of samples were from 79.9% to 91.5%. The five analytes were stable at room temperature for 2 h, at 4 °C for 12 h, and at −20 °C for two weeks. The developed method was applied successfully to determine the content of the five alkaloids in three plant parts of three batches of C. roseus with a minute amount collected from three regions of China. CONCLUSION: The HPLC-ESI-MS/MS method can be used for the simultaneous determination of five important alkaloids in trace C. roseus samples. [KEY WORDS] Catharanthus roseus; HPLC-ESI-MS/MS; Monomeric and bisindole alkaloids
[CLC Number] R917
[Document code] A
[Article ID] 2095-6975(2014)10-0786-08
Introduction Vinblastine (VBL), vincristine (VCR), vinleurosine (VLS), vindoline (VDL), and catharanthine (CTR) are important monoterpenoid indole alkaloids derived from the periwinkle plant, Catharanthus roseus (L.) G. Don (C. roseus), a [Received on] 23-Mar.-2013 [Research Funding] This project was supported by the Fundamental Research Funds for the Central Universities (No. DL09BA21) and Special Fund for Forestry Scientific Research in the Public Interest (No. 201204601). [*Corresponding author] ZU Yuan-Gang: Prof., Tel: 86-45182191387, Fax: 86-451-82192223, E-Mail: [email protected] † Co-first author These authors have no conflict of interest to declare. Published by Elsevier B.V. All rights reserved
tropical perennial subshrub known to produce about 130 alkaloids [1]. VBL and VCR are currently used widely to treat Hodgkin’s disease and the neoplasms including breast, bladder, lung cancer, lymphomas, and leukemia [2-3]. They inhibit the division and growth of cancer cells, and are considered as anti-mitotic or anti-microtubule agents, or mitotic inhibitors [4-7]. VLS is used in the treatment of Hodgkin's disease, acute lymphoblastic leukemia and lymphoblastosarcoma [8]. VDL and CTR are precursors for the synthesis of VBL [9]. The structures of VBL, VCR, VLS, VDL and CTR are shown in Fig. 1. Because of the trace amounts (0.01−0.1 mg/g DW) of VBL and VCR in the plant [10], their quantitative analysis in the plant has been difficult. In attempts to improve the determination of these alkaloids in C. roseus, several studies have been reported [11-19]. The current method for the determination of these alkaloids mainly uses high-performance liquid
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Fig. 1
Structures of VBL, VCR, VLS, VDL, CTR, and IS
chromatography (HPLC) with ultra-violate absorbance, fluorescence, or electrochemical detection [11-13]. High- performance thin-layer chromatography [14], gas chromatography [15], supercritical fluid chromatography [16], radioimmunoassay [17] and capillary zone electrophoresis [18-19] are also used in the analysis and the quantification of the Catharanthus alkaloids. However, the threshold of sensitivities of these methods for detecting the alkaloids is high. Therefore, a more sensitive method with high resolution for analyzing the important monoterpene alkaloids in C. roseus would be highly desirable. A better sensitivity of the Catharanthus alkaloids could be achieved using ESI-MS or ESI-MS/MS compared to other detectors. Choi et al. analyzed the VBL content of supercritical fluid extracts by HPLC-ESI-MS [20], and He et al. analyzed VDL, CTR, serpentine, and ajmalicine in the C. roseus cell line C20hi by UPLC-MS [21]. The results showed that the limits of quantification were 0.4 g·mL−1 for VBL [20], 1.52 ng·mL−1 for VDL, and 2.32 ng·mL−1 for CTR [21]. In recent years, high performance liquid chromatography combined with tandem mass spectrometry (HPLC-MS/MS) has been successfully applied to determine the contents of trace bioactive components in plant medicines [22-26]. Although mass spectrometry is a more expensive and complex option than other detectors, LC-MS/MS is a simple and rapid analytical method with no need for a clean-up step for the detection of trace compounds from a complex plant matrix due to the high selectivity of multiple reaction monitoring (MRM) quantitative analysis mode of MS/MS detection, thus it can reduce the analysis time. To date, no report has been published on the use of LC-MS/MS for the simultaneous quantitative determination of VBL, VCR, VLS, VDL, and CTR in C. roseus plant material. In the present work, LC-MS/MS with ESI was used to quantitatively determine VBL, VCR, VLS, VDL, and CTR in C. roseus simultaneously, and application of the established method was investigated. This work may provide a valuable method for the quantification of trace Catharanthus alkaloids and their precursors.
Experimental Chemicals and reagents Reference standards of VBL, vinorebine [VNB, as an internal standard (IS), Fig. 1] were purchased from Sigma (St. Louis, USA) (purity > 98%). VCR, VLS, VDL, and CTR were purchased from Shanghai Tauto Biotech Co., Ltd. (Shanghai, China) (purity > 98%); All standards were dissolved in methanol and stored in the dark at 4 °C. HPLC grade methanol was purchased from J&K Chemical Ltd. (Beijing, China). Formic acid and ammonium acetate were purchased from Dikma Technology Inc. (Richmond Hill, USA). Deionized water for HPLC analysis was purified with a Milli-Q Ultrapure water system from Millipore Corp. (Billerica, MA, USA). Other reagents were all analytical grade chemicals. Plant material Three batches of C. roseus crude samples were collected from different regions of China. The samples of north, middle, and south regions of China were from Heilongjiang (cultivation park of Northeast Forestry University), Zhejiang (cultivation park of Zhejiang Hisun Pharmaceutical Co., Ltd.) and Hainan province (cultivation park of Hainan Catharanthus Research and Development Center), respectively, and were identified by Prof. NIE Shao-Quan from the Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry University, Harbin, China. The material was dried in the shade and was kept at −20 °C prior to use. Preparation of stock solutions, calibration samples, and quality control (QC) samples Each reference standard and IS solution at a concentration of 1.2 mg·mL−1 in methanol for optimization of MS conditions was prepared, respectively. A mixed stock solution containing 0.2 mg·mL−1 of the five reference standards and IS in methanol for chromatographic separation was prepared. A series of working standard solutions were prepared by successive dilution of the mixed stock solution with methanol as the
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calibration standards to yield final concentrations of 1.5, 3.1, 24.8, 97.6, 390.6, 1 562.5, and 6 250.0 ng·mL−1. The quality control samples were prepared at concentrations of 12.4, 97.6 and 781.3 ng·mL−1 for VBL, VCR, and VLS, and 24.8, 390.6, and 1 562.5 ng·mL−1 for VDL and CTR for the low, medium and high concentration QC samples, respectively. Sample preparation The leaves, stems, and roots of C. roseus were ground from lumps into powders. Dried powder (0.2 g) was extracted with IS-water−0.1% H2SO4 methanol (1 : 1, V/V, 10 mL × 3, the IS concentrations were kept constant 500 ng·mL−1 in all sample solutions.) in an ultra-sonic bath for 30 min and were evaporated in vacuum at 45 °C until no methanol was distilled. Then the acidic extracts were extracted with ethyl acetate (10 mL × 3) after they were adjusted pH value to 10 (using 25% NH3·H2O). The ethyl acetate portions were evaporated in vacuum at 45 °C and reconstituted in mobile phase (2 mL) to obtain the test solutions for analysis. The samples were filtered through a 0.45 μm syringe filter prior to injection. Calibration curve, lower limits of quantification (LLOQ) and lower limits of detection (LLOD) The calibration curves were constructed by plotting the peak area ratio of the analytes to IS versus analyte concentration. The calibration curves were fitted through a 1/x2 weighted linear least squares regression model. The LLOQ and LLOD were determined based on a signal-to-noise ratio of at least 10 : 1 and 3 : 1 and LLOQ was defined as the lowest analytical concentration of the calibration curve. Precision and accuracy The intra- and inter-day precision and accuracy of the method were determined by analyzing six replicates of the QC samples at three concentration levels of the five analytes on the same day, and on three consecutive days, respectively. The accuracy and precision are expressed in terms of relative error (RE) and relative standard deviation (RSD), respectively. Recovery test Recoveries were calculated for the three levels of QC samples (n = 6 for each concentration). The recovery was evaluated by comparing the peak areas of the five analytes from the QC samples with the corresponding average peak area of each analyte spiked into the post-extraction supernatant. Stability test The stability of the five analytes in samples was examined by analyzing replicates (n = 6) of the three levels of QC samples under different conditions. The room temp stability was assessed by analyzing samples kept at room temp for 2 h. The long-term stability was assessed by analyzing samples kept at −20 °C for 2 weeks. The stability of the post-preparation stability was assessed by analyzing samples left at 4 °C for 12 h after sample preparation. All stability testing QC samples were determined by using the freshly prepared standard samples. The samples were considered
stable if the assay values were within an acceptable deviation from the nominal concentration (± 15%). HPLC-ESI-MS/MS analysis An Agilent 1100 series HPLC system equipped with a binary pump and a degasser (Agilent, Waldbronn, Germany). HPLC analysis was carried out on a C18 column (Agilent Eclipse C18, 4.6 μm × 150 mm, 5 μm). The mobile phase was methanol−15 mmol·L−1 ammonium acetate containing 0.02% formic acid (65 : 35, V/V). The flow rate was 1 mL·min−1, and the injection volume was 5 μL. The detection was performed using Applied Biosystems Sciex API 3000 triple-quadruple mass spectrometers (Sciex, Toronto, Canada) equipped with an electron spray ionization interface and AnalystTM 1.4 controlling software. Nitrogen was used as a nebulizing gas and curtain gas at a pressure of 10 psi and 12 psi, and temperature at 300 °C. HPLC-ESI-MS/MS analyses were carried out in the positive ion mode with the scan range m/z 100−1,000, and the quantitative determination of the five alkaloids was performed using the MRM mode. The MS parameters for each alkaloid were optimized by direct infusion of the standards of each alkaloid into the source using the syringe pump.
Results and Discussion Optimization of LC-ESI-MS/MS conditions Full scan positive ion ESI mass spectra were obtained for each of the alkaloids by direct infusion of the standards of VBL, VCR, VLS, VDL, CTR, and IS, respectively. In these experiments, it was discovered that the response of alkaloids observed in positive ion mode was higher than that in negative ion mode. Thus, positive ion mode was finally employed. The deprotonated molecular ions [M + H]+ for each of the above mentioned alkaloids and the IS were observed at m/z 811.5, 825.3, 809.7, 457.4, 337.4, and 779.4, respectively. In addition to full scan mass spectra, collision induced dissociation was undertaken in their MS/MS fragmentation mode to yield product ion mass spectra. While infusing each of the standards, the collision energy (CE) was varied from 10 to 80 V to find the optimal CE. The most abundant fragment ions for each alkaloid were chosen for MRM quantification. The optimal MS, MS/MS conditions for fragment ions and the main fragment ions of each alkaloid and the ion pairs for MRM experiments are listed in Table 1. Fig. 2 shows the product ions scan spectra of the analytes and IS. The composition and ratio of the mobile phase is important for the HPLC-ESI-MS/MS determination of the five Catharanthus alkaloids. Several mobile phases with different composition and ratio were used to investigate the chromatographic behavior, and it was found that methanol-15 mmol·L−1 ammonium acetate containing 0.02% formic acid (65 : 35, V/V) was optimal for the separation of the five alkaloids and IS. All the peaks of the analytes and IS were detected with excellent resolution as well as peak shapes,
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Table 1 MS, MS/MS conditions and ESI-MS/MS data for five Catharanthus alkaloids Compounds
DP (V)
EP (V)
FP (V)
CE (V)
CXP (V)
NEB (psi)
CUR (psi)
CAD (psi)
Precursor ion [M + H]+ (m/z)
Major product ions (m/z)
Ion pairs (m/z)
VBL
120
47
9
811.5
793.1, 355.3, 337.5, 224.4
811.5→355.3
VCR
120
53
12
825.3
765.1, 556.4, 341.2, 144.1
825.3→765.1
VLS
100
50
8
809.7
748.8, 353.0
809.7→353.0
VDL
130
45
14
457.4
397.3, 188.3
457.4→188.3
CTR
100
30
13
337.4
173.3, 144.2, 93.2
337.4→144.2
IS
65
41
9
779.4
658.6, 457.4, 323.4
779.4→323.4
10
400
10
12
6
Notes: DP, Delustering potential; EP, Entrance potential; FP, Focusing potential; CE, Collision energy; CXP, Collision cell exit potential; NEB, Nebulizing gas; CUR, Curtain gas; CAD, Collision gas
Fig. 2
Product ion mass spectra of A) VBL; B) VCR; C) VLS; D) VDL; E) CTR, and F) IS
and no interference from endogenous substances was observed at the retention time of the analytes and IS. A typical MRM chromatogram of the standards and the sample is shown in Fig. 3. The retention times of VCR, VBL, VLS, VDL, CTR, and IS were 2.51, 4.42, 4.57, 6.10, 7.31, and 2.97 min, respectively. Method validation Linearity, lower limits of quantification (LLOQ) and lower limits of detection (LLOD) The typical equations of calibration curves and the linearity ranges for the five analytes are shown in Table 2. All correlation coefficients were higher than 0.9950. Calibration data for each alkaloid were obtained using the optimized HPLC-MS/ MS conditions. The response profile was determined and observed to be linear for each of the alkaloids within the linear ranges. Each concentration level was analyzed in triplicate. The results showed that there was excellent correlation between the ratio of peak area of each alkaloid versus IS and concentration for each alkaloid within the linearity. The LLOQs of VBL, VCR, and VLS were 1.5 ng·mL−1, while the LLOQs of VDL and CTR were 3.1 ng·mL−1. The LLODs of VBL, VCR, and VLS were 0.75 ng·mL−1, while the LLODs of VDL and CTR were 1.5 ng·mL−1. These LLOQs are sufficient for content detection of the five
Catharanthus alkaloids. Precision and accuracy The intra- and inter-day precision and accuracy values for the QC samples are summarized in Table 3. The intra- and inter-day precision and accuracy were evaluated by determination of standard QC samples at three concentration levels of the five analytes. The intra- and inter-day precisions (RSD) of these analytes were all less than 11.5% and 9.0%, while the accuracy was within ± 10.9% for all the analytes. The values for accuracy and precision demonstrated that the method is reliable and reproducible. Recovery The recoveries for the five analytes are summarized in Table 4. Mean absolute recoveries of VBL, VCR, VLS, VDL, and CTR were 82.6%−91.2%, 82.8%−91.5%, 87.0%−89.5%, 79.9%−90.7%, and 83.0%−85.2% at three QC levels. The recoveries were consistent and reproducible. Stability The stability of all the analytes was assessed under various conditions. The results presented in Table 4 indicate that the five analytes were stable in the extract sample at room temp for 2 h and at −20 °C for two weeks. No significant degradation of five analytes was observed during the 2 h storage at room temp and −20°C for two weeks. Post-preparative
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Fig. 3 Representative MRM chromatograms of A) five Catharanthus alkaloids and IS standard; B) C. roseus leaf sample (A1, B1 VCR; A2, B2 VBL; A3, B3 VLS; A4, B4 VDL; A5, B5 CTR; and A6, B6 IS
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Table 2 Regression equations, linear ranges, correlation coefficients, LLOQ, and LLOD of the Catharanthus alkaloids Alkaloid
Regression equation
Linear range (ng·mL−1)
LLOQ (ng·mL−1)
r2 (n = 6)
LLOD (ng·mL−1)
VBL
Y = 0.373 1x + 0.076 8
1.5−1 562.5
0.999 5
1.5
0.75
VCR
Y = 0.580 1x + 0.147 3
1.5−1 562.5
0.998 0
1.5
0.75
VLS
Y = 0.282 1x + 0.049 6
1.5−1 562.5
0.997 9
1.5
0.75
VDL
Y = 0.385 3x + 0.802 6
3.1−6 250.0
0.995 3
3.1
1.5
CTR
Y = 0.552 2x + 0.158 0
3.1−6 250.0
0.998 9
3.1
1.5
Table 3 Precision and accuracy for the Catharanthus alkaloids (n=3 days, six replicates per day) Alkaloid
Nominal concentration (ng·mL−1)
VBL
12.4
VCR
VLS
VDL
CTR
Intra-day
Inter-day
Precision (RSD%)
Accuracy (RE%)
c/(ng·mL−1)
Precision (RSD%)
Accuracy (RE%)
13.7 ± 0.9
6.2
10.5
12.8 ± 1.1
8.5
3.5
97.6
94.6 ± 4.3
4.5
–3.1
94.6 ± 4.3
4.5
–3.1
781.3
762.3 ± 17.6
2.3
–2.4
752.3 ± 38.1
5.1
–3.7
c/(ng·mL−1)
12.4
13.2 ± 1.2
8.8
6.7
11.3 ± 0.6
4.9
–8.9
97.6
94.1 ± 3.1
3.3
–3.6
94.1 ± 4.8
5.1
–3.6
781.3
756.3 ± 20.8
2.7
–3.2
726.3 ± 37.3
5.1
–7.0
12.4
13.1 ± 1.5
11.5
5.6
11.4 ± 1.0
9.0
–8.1
97.6
93.9 ± 2.0
1.5
–3.8
93.8 ± 1.3
1.4
–1.4
781.3
763.1 ± 13.9
1.8
–2.3
752.6 ± 11.9
1.6
–3.7
24.8
25.6 ± 2.9
11.3
4.8
25.9 ± 2.3
9.0
6.1
390.6
368.2 ± 30.9
8.4
–5.7
374.8 ± 19.4
5.2
–4.0
1562.5
1528.4 ± 30.9
2.0
–2.2
1 518.4 ± 47.5
3.1
–2.8
24.8
22.0 ± 2.1
9.4
–9.9
21.7 ± 1.7
7.7
–10.9
390.6
407.0 ± 22.3
5.5
4.2
410.4 ± 25.4
6.2
5.1
1 562.5
1523.5 ± 17.9
1.2
–2.5
1492.9 ± 46.8
3.1
–4.5
Table 4 Recovery and stability for the analytes (n = 6) Extraction recovery
Alkaloid
Spiked concentration (ng·mL−1)
Mean (%)
VBL
12.4
91.2
VCR
VLS
VDL
CTR
Stability (RE%)
RSD (%)
Short-term (2 h at room temp)
Post-preparative (12 h at 4 °C)
Long-term (2 weeks at −20 °C)
12.5
–1.2
–2.9
–10.8
97.6
82.6
5.3
–0.6
–3.1
–6.3
781.3
86.5
7.9
1.2
–7.5
–5.2
12.4
82.8
13.7
2.7
1.3
3.4
97.6
86.2
4.9
–1.5
–5.9
–2.7
781.3
91.5
6.0
–3.4
–11.8
–5.5
12.4
88.9
9.2
1.6
0.3
0.8
97.6
89.5
4.2
–0.9
–5.6
–4.7
781.3
87.0
6.1
–2.1
–9.9
–6.1
24.8
79.9
11.0
1.9
7.8
2.4
390.6
90.7
8.3
3.5
10.7
6.2
1562.5
80.1
7.8
2.6
9.6
7.3
24.8
85.2
13.0
3.8
4.2
–3.1
390.6
83.6
6.2
5.4
8.1
2.9
1 562.5
83.0
7.9
1.6
6.6
6.7
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stability of the alkaloids also showed that no significant degradation occurred when the extracted samples were kept at 4 °C for 12 h. The responses varied from −11.8% to 10.7% at all tested concentrations. Method application - quantitative analysis of C. roseus samples The validated method was successfully applied for the quantitative analysis studies of five Catharanthus alkaloids in C. roseus samples. Three batches of C. roseus samples were collected from the southern, middle, and northern regions of China (Hainan, Zhejiang, and Heilongjiang Provinces), and the leaves, stems, and roots were quantitatively analyzed using the developed LC-ESI-MS/MS method. The results (Table 1) showed that the contents of the five alkaloids varied between the different regions of China, and the different crude plant parts. For the C. roseus leaves and stems, the contents of VBL, VCR and VLS in samples from Heilongjiang are lower than those of Zhejiang and Hainan provinces,
but the contents of VDL and CTR of the samples from Heilongjiang are higher than the other two provinces. The difference between the contents in the samples from the three regions showed that the five alkaloids may vary based on the variety of temperature and sunlight. It was reported previously that the biosynthesis of bis-indole alkaloids depended on light, and that UV can induce VDL and CTR to synthesize VBL [27]. So the sufficient sunlight and moderate temperatures of Zhejiang and Hainan provinces may be benefit VBL synthesis. Therefore, the VBL content of the samples from these two provinces are much higher than Heilongjiang’s. The VCR and VLS contents in the Zhejiang and Hainan samples are also higher than in Heilongjiang samples because of the its bis-indole alkaloid structure as VBL. From the results, it was found that the VBL, VCR, and VLS contents are very low in three parts (leaves, stems and root). So the developed LC-ESI-MS/MS method is a new, more sensitive method for VBL, VCR, and VLS determination.
Table 5 Content of five Catharanthus alkaloids in C. roseus samples from three different regions of China Source Heilongjiang, China
Zhejiang, China
Hainan, China
Analysis part
VBL (µg·g−1, DW)
VCR (µg·g−1, DW)
CTR (µg·g−1, DW)
38.5
3.3
6.1
288.3
206.8
stems
45.7
2.4
4.5
220.6
128.4
roots
9.5
1.9
2.4
16.8
21.2
leaves
102.9
11.0
15.3
232.9
134.4
stems
55.9
6.1
7.8
80.7
49.6
roots
21.9
3.4
4.1
13.2
92.8
leaves
113.2
9.6
17.6
215.3
113.5
stems
70.5
5.0
12.3
39.2
24.7
roots
31.3
5.3
7.2
8.9
116.0
[3]
A sensitive LC-ESI-MS/MS method for the determination of VBL, VCR, VLS, VDL, and CTR was developed, validated, and applied for their quantitative analysis in C. roseus. The described LC-ESI-MS/MS method was sensitive, with high accuracy and a short run time of 10 min, and met all the requirements for effective plant extract analysis. With no reports on HPLC-MS or HPLC-MS/MS methods for the quantitative determination of the five Catharanthus alkaloids, this is the first report of the simultaneous quantitative study of VBL, VCR, VLS, VDL, and CTR in C. roseus extracts. The method presented is valuable for providing a procedure for the future analysis of trace components in C. roseus.
References
[2]
VDL (µg·g−1, DW)
leaves
Conclusions
[1]
VLS (µg·g−1, DW)
Noble RL. The discovery of the Vinca alkaloids - chemotherapeutic agents against cancer [J]. Biochem Cell Biol, 1990, 68 (12): 1344-1351. Armstrong JG, Dyke RW, Fouts PJ. Vinblastine sulfate treatment of Hodgkin's disease during a pregnancy [J]. Science, 1964, 143 (3607): 703.
Sertel S, Fu YJ, Zu YG, et al. Molecular docking and pharmacogenomics of Vinca alkaloids and their monomeric precursors, vindoline and catharanthine [J]. Biochem Pharmacol, 2011, 81 (6): 723-735. [4] Himes RH. Interactions of the Catharanthus (Vinca) alkaloids with tubulin and microtubules [J]. Pharmacol Ther, 1991, 51 (2): 257-267. [5] Jordan MA, Thrower D, Wilson L. Mechanism of inhibition of cell proliferation by Vinca alkaloids [J]. Cancer Res, 1991, 51 (4): 2212-2222. [6] Zhou XJ, Rahmani R. Preclinical and clinical pharmacology of Vinca alkaloids [J]. Drugs, 1992, 44 (Suppl 4): 1-16, 66-69. [7] Okouneva T, Hill BT, Wilson L, et al. The effects of vinflunine, vinorelbine, and vinblastine on centromere dynamics [J]. Mol Cancer Ther, 2003, 2 (5): 427-436. [8] Schneider M, Band P, Amiel JL, et al. Leurosine, the 3rd alkaloid from Vinca rosea, in the treatment of Hodgkin's disease, acute lymphoblastic leukemia and lymphoblastosarcoma [J]. Sem Hop, 1966, 42 (49): 2952-2954. [9] Pennanen S, Huhtikangas A. Photochemical one-pot synthesis of vinblastine and vincristine [J]. Photochem Photobiol, 1990, 51 (5): 515-518. [10] Mu FS, Yang L, Wang W. et al. Negative-pressure cavitation extraction of four main vinca alkaloids from Catharanthus
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roseus leaves [J]. Molecules, 2012, 17 (8): 8742-8752. [11] Naaranlahti T, Ranta VP, Jarho P, et al. Electrochemical detection of indole alkaloids of Catharanthus roseus in highperformance liquid chromatography [J]. Analyst, 1989, 114 (10): 1229-1231. [12] Volkov SK, Grodnitskaya EI. Application of high-performance liquid chromatography to the determination of vinblastine in Catharanthus roseus [J]. J Chromatogr B Biomed Appl, 1994, 660 (2): 405-408. [13] Tikhomiroff C, Jolicoeur M. Screening of Catharanthus roseus secondary metabolites by high-performance liquid chromatography [J]. J Chromatogr A, 2002, 955 (1): 87-93. [14] Horvath P, Ivanyi G. Quantitative analysis of natural drugs III. Densitometric determination of vinblastine and other alkaloids of Catharantus roseus [J]. Acta Pharm Hung, 1982, 52 (4): 150-157. [15] Ylinen M, Suhonen P, Naaranlahti T, et al. Gas chromatographic-mass spectrometric analysis of major indole alkaloids of Catharanthus roseus [J]. J Chromatogr, 1990, 505 (2): 429-434. [16] Balsevich J, Hogge LR, Berry AJ, et al. Analysis of indole alkaloids from leaves of Catharanthus roseus by means of supercritical fluid chromatography/mass spectrometry [J]. J Nat Prod, 1988, 51 (6): 1173-1177. [17] Deus-Neumann B, Stöckigt J, Zenk MH. Radioimmunoassay for the quantitative determination of catharanthine [J]. Planta Med, 1987, 53 (2): 184-188. [18] Inhou C, Joy AB, Earl LW, et al. Quantification of vincristine and vinblastine in Catharanthus roseus plants by capillary zone electrophoresis [J]. J Chromatogr A, 1996, 755 (2): 281-288. [19] Barthe L, Ribet JP, Pelissou M, et al. Optimization of the separation of Vinca alkaloids by nonaqueous capillary electro-
phoresis [J]. J Chromatogr A, 2002, 968 (1-2): 241-250. [20] Choi YH, Yoo KP, Kim J. Supercritical fluid extraction and liquid chromatography-electrospray mass analysis of vinblastine from Catharanthus roseus [J]. Chem Pharm Bull, 2002, 50 (9): 1294-1296. [21] He LH, Yang L, Xiong AZ, et al. Simultaneous quantification of four indole alkaloids in Catharanthus roseus cell line C20hi by UPLC-MS [J]. Anal Sci, 2011, 27 (4): 433-438. [22] Liu W, Fu YJ, Zu YG, et al. Negative-pressure cavitation extraction for the determination of flavonoids in pigeon pea leaves by liquid chromatography-tandem mass spectrometry [J]. J Chromatogr A, 2009, 1216 (18): 3841-3850. [23] Zu YG, Yan MM, Fu YJ, et al. Determination and quantification of astragalosides in Radix Astragali and its medicinal products using LC-MS [J]. J Sep Sci, 2009, 32 (4): 517-525. [24] Liu W, Kong Y, Zu YG, et al. Determination and quantification of active phenolic compounds in pigeon pea leaves and its medicinal product using liquid chromatography-tandem mass spectrometry [J]. J Chromatogr A, 2010, 1217 (28): 4723-4731. [25] Li QY, Su L, Zu YG, et al. Quantification of CPT13 in rat plasma using LC-MS/MS for a pharmacokinetic study [J]. J Chromatogr B Analyt Technol Biomed Life Sci, 2011, 879 (7-8): 461-466. [26] Hua X, Fu YJ, Zu YG, et al. Determination of pinostrobin in rat plasma by LC-MS/MS: Application to pharmacokinetics [J]. J Pharm Biomed Anal, 2011, 56 (4): 841-845. [27] Wang H, Sun M, Wu CL, et al. Advances in the study on critical steps in the biosynthesis pathway of Catharanthus alkaloids and the regulation of their metabolism [J]. China J Chin Mat Med, 2001, 26 (10): 656-659.
Cite this article as: ZHANG Lin, GAI Qing-Hui, ZU Yuan-Gang, YANG Lei, MA Yu-Liang, LIU Yang. Simultaneous quantitative determination of five alkaloids in Catharanthus roseus by HPLC-ESI-MS/MS [J]. Chinese Journal of Natural Medicines, 2014, 12 (10): 786-793.
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Chinese Journal of Natural Medicines
Simultaneous quantitative determination of five alkaloids in Catharanthus roseus by HPLC-ESI-MS/MS ZHANG Lin1, 2, 3†, GAI Qing-Hui1, 2, 3†, ZU Yuan-Gang1, 2, 3*, YANG Lei1, 2, 3, MA Yu-Liang1, 2, 3, LIU Yang1, 2, 3 1
State Engineering Laboratory of Bio-Resource Eco-Utilization, Northeast Forestry University, Harbin 150040, China;
2
Engineering Research Center of Forestry Bio-preparation, Ministry of Education, Northeast Forestry University, Harbin 150040, China;
3
Key Laboratory of Forest Plant Ecology, Ministry of Education; Northeast Forestry University, Harbin, 150040, China Available online October 2014
[ABSTRACT] AIM: To establish a method to simultaneously determine the main five alkaloids of Catharanthus roseus for trace samples, a high-performance liquid chromatography–electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) analysis method was developed. METHOD: The five Catharanthus alkaloids, vinblastine, vincristine, vinleurosine, vindoline, and catharanthine were chromatographically separated on a C18 HPLC column. The mobile phase was methanol−15 nmolL–1 ammonium acetate containing 0.02% formic acid (65 : 35, V/V). The quantification of these alkaloids was based on the Multiple Reaction Monitoring (MRM) mode. RESULTS: This method was validated, and the results achieved the aims of the study. The intra- and inter-day precision and accuracy of the five alkaloids were within 1.2%−11.5% (RSD%) and −10.9%−10.5% (RE%). The recovery rates of the five alkaloids of samples were from 79.9% to 91.5%. The five analytes were stable at room temperature for 2 h, at 4 °C for 12 h, and at −20 °C for two weeks. The developed method was applied successfully to determine the content of the five alkaloids in three plant parts of three batches of C. roseus with a minute amount collected from three regions of China. CONCLUSION: The HPLC-ESI-MS/MS method can be used for the simultaneous determination of five important alkaloids in trace C. roseus samples. [KEY WORDS] Catharanthus roseus; HPLC-ESI-MS/MS; Monomeric and bisindole alkaloids
[CLC Number] R917
[Document code] A
[Article ID] 2095-6975(2014)10-0786-08
Introduction Vinblastine (VBL), vincristine (VCR), vinleurosine (VLS), vindoline (VDL), and catharanthine (CTR) are important monoterpenoid indole alkaloids derived from the periwinkle plant, Catharanthus roseus (L.) G. Don (C. roseus), a [Received on] 23-Mar.-2013 [Research Funding] This project was supported by the Fundamental Research Funds for the Central Universities (No. DL09BA21) and Special Fund for Forestry Scientific Research in the Public Interest (No. 201204601). [*Corresponding author] ZU Yuan-Gang: Prof., Tel: 86-45182191387, Fax: 86-451-82192223, E-Mail: [email protected] † Co-first author These authors have no conflict of interest to declare. Published by Elsevier B.V. All rights reserved
tropical perennial subshrub known to produce about 130 alkaloids [1]. VBL and VCR are currently used widely to treat Hodgkin’s disease and the neoplasms including breast, bladder, lung cancer, lymphomas, and leukemia [2-3]. They inhibit the division and growth of cancer cells, and are considered as anti-mitotic or anti-microtubule agents, or mitotic inhibitors [4-7]. VLS is used in the treatment of Hodgkin's disease, acute lymphoblastic leukemia and lymphoblastosarcoma [8]. VDL and CTR are precursors for the synthesis of VBL [9]. The structures of VBL, VCR, VLS, VDL and CTR are shown in Fig. 1. Because of the trace amounts (0.01−0.1 mg/g DW) of VBL and VCR in the plant [10], their quantitative analysis in the plant has been difficult. In attempts to improve the determination of these alkaloids in C. roseus, several studies have been reported [11-19]. The current method for the determination of these alkaloids mainly uses high-performance liquid
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Fig. 1
Structures of VBL, VCR, VLS, VDL, CTR, and IS
chromatography (HPLC) with ultra-violate absorbance, fluorescence, or electrochemical detection [11-13]. High- performance thin-layer chromatography [14], gas chromatography [15], supercritical fluid chromatography [16], radioimmunoassay [17] and capillary zone electrophoresis [18-19] are also used in the analysis and the quantification of the Catharanthus alkaloids. However, the threshold of sensitivities of these methods for detecting the alkaloids is high. Therefore, a more sensitive method with high resolution for analyzing the important monoterpene alkaloids in C. roseus would be highly desirable. A better sensitivity of the Catharanthus alkaloids could be achieved using ESI-MS or ESI-MS/MS compared to other detectors. Choi et al. analyzed the VBL content of supercritical fluid extracts by HPLC-ESI-MS [20], and He et al. analyzed VDL, CTR, serpentine, and ajmalicine in the C. roseus cell line C20hi by UPLC-MS [21]. The results showed that the limits of quantification were 0.4 g·mL−1 for VBL [20], 1.52 ng·mL−1 for VDL, and 2.32 ng·mL−1 for CTR [21]. In recent years, high performance liquid chromatography combined with tandem mass spectrometry (HPLC-MS/MS) has been successfully applied to determine the contents of trace bioactive components in plant medicines [22-26]. Although mass spectrometry is a more expensive and complex option than other detectors, LC-MS/MS is a simple and rapid analytical method with no need for a clean-up step for the detection of trace compounds from a complex plant matrix due to the high selectivity of multiple reaction monitoring (MRM) quantitative analysis mode of MS/MS detection, thus it can reduce the analysis time. To date, no report has been published on the use of LC-MS/MS for the simultaneous quantitative determination of VBL, VCR, VLS, VDL, and CTR in C. roseus plant material. In the present work, LC-MS/MS with ESI was used to quantitatively determine VBL, VCR, VLS, VDL, and CTR in C. roseus simultaneously, and application of the established method was investigated. This work may provide a valuable method for the quantification of trace Catharanthus alkaloids and their precursors.
Experimental Chemicals and reagents Reference standards of VBL, vinorebine [VNB, as an internal standard (IS), Fig. 1] were purchased from Sigma (St. Louis, USA) (purity > 98%). VCR, VLS, VDL, and CTR were purchased from Shanghai Tauto Biotech Co., Ltd. (Shanghai, China) (purity > 98%); All standards were dissolved in methanol and stored in the dark at 4 °C. HPLC grade methanol was purchased from J&K Chemical Ltd. (Beijing, China). Formic acid and ammonium acetate were purchased from Dikma Technology Inc. (Richmond Hill, USA). Deionized water for HPLC analysis was purified with a Milli-Q Ultrapure water system from Millipore Corp. (Billerica, MA, USA). Other reagents were all analytical grade chemicals. Plant material Three batches of C. roseus crude samples were collected from different regions of China. The samples of north, middle, and south regions of China were from Heilongjiang (cultivation park of Northeast Forestry University), Zhejiang (cultivation park of Zhejiang Hisun Pharmaceutical Co., Ltd.) and Hainan province (cultivation park of Hainan Catharanthus Research and Development Center), respectively, and were identified by Prof. NIE Shao-Quan from the Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry University, Harbin, China. The material was dried in the shade and was kept at −20 °C prior to use. Preparation of stock solutions, calibration samples, and quality control (QC) samples Each reference standard and IS solution at a concentration of 1.2 mg·mL−1 in methanol for optimization of MS conditions was prepared, respectively. A mixed stock solution containing 0.2 mg·mL−1 of the five reference standards and IS in methanol for chromatographic separation was prepared. A series of working standard solutions were prepared by successive dilution of the mixed stock solution with methanol as the
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calibration standards to yield final concentrations of 1.5, 3.1, 24.8, 97.6, 390.6, 1 562.5, and 6 250.0 ng·mL−1. The quality control samples were prepared at concentrations of 12.4, 97.6 and 781.3 ng·mL−1 for VBL, VCR, and VLS, and 24.8, 390.6, and 1 562.5 ng·mL−1 for VDL and CTR for the low, medium and high concentration QC samples, respectively. Sample preparation The leaves, stems, and roots of C. roseus were ground from lumps into powders. Dried powder (0.2 g) was extracted with IS-water−0.1% H2SO4 methanol (1 : 1, V/V, 10 mL × 3, the IS concentrations were kept constant 500 ng·mL−1 in all sample solutions.) in an ultra-sonic bath for 30 min and were evaporated in vacuum at 45 °C until no methanol was distilled. Then the acidic extracts were extracted with ethyl acetate (10 mL × 3) after they were adjusted pH value to 10 (using 25% NH3·H2O). The ethyl acetate portions were evaporated in vacuum at 45 °C and reconstituted in mobile phase (2 mL) to obtain the test solutions for analysis. The samples were filtered through a 0.45 μm syringe filter prior to injection. Calibration curve, lower limits of quantification (LLOQ) and lower limits of detection (LLOD) The calibration curves were constructed by plotting the peak area ratio of the analytes to IS versus analyte concentration. The calibration curves were fitted through a 1/x2 weighted linear least squares regression model. The LLOQ and LLOD were determined based on a signal-to-noise ratio of at least 10 : 1 and 3 : 1 and LLOQ was defined as the lowest analytical concentration of the calibration curve. Precision and accuracy The intra- and inter-day precision and accuracy of the method were determined by analyzing six replicates of the QC samples at three concentration levels of the five analytes on the same day, and on three consecutive days, respectively. The accuracy and precision are expressed in terms of relative error (RE) and relative standard deviation (RSD), respectively. Recovery test Recoveries were calculated for the three levels of QC samples (n = 6 for each concentration). The recovery was evaluated by comparing the peak areas of the five analytes from the QC samples with the corresponding average peak area of each analyte spiked into the post-extraction supernatant. Stability test The stability of the five analytes in samples was examined by analyzing replicates (n = 6) of the three levels of QC samples under different conditions. The room temp stability was assessed by analyzing samples kept at room temp for 2 h. The long-term stability was assessed by analyzing samples kept at −20 °C for 2 weeks. The stability of the post-preparation stability was assessed by analyzing samples left at 4 °C for 12 h after sample preparation. All stability testing QC samples were determined by using the freshly prepared standard samples. The samples were considered
stable if the assay values were within an acceptable deviation from the nominal concentration (± 15%). HPLC-ESI-MS/MS analysis An Agilent 1100 series HPLC system equipped with a binary pump and a degasser (Agilent, Waldbronn, Germany). HPLC analysis was carried out on a C18 column (Agilent Eclipse C18, 4.6 μm × 150 mm, 5 μm). The mobile phase was methanol−15 mmol·L−1 ammonium acetate containing 0.02% formic acid (65 : 35, V/V). The flow rate was 1 mL·min−1, and the injection volume was 5 μL. The detection was performed using Applied Biosystems Sciex API 3000 triple-quadruple mass spectrometers (Sciex, Toronto, Canada) equipped with an electron spray ionization interface and AnalystTM 1.4 controlling software. Nitrogen was used as a nebulizing gas and curtain gas at a pressure of 10 psi and 12 psi, and temperature at 300 °C. HPLC-ESI-MS/MS analyses were carried out in the positive ion mode with the scan range m/z 100−1,000, and the quantitative determination of the five alkaloids was performed using the MRM mode. The MS parameters for each alkaloid were optimized by direct infusion of the standards of each alkaloid into the source using the syringe pump.
Results and Discussion Optimization of LC-ESI-MS/MS conditions Full scan positive ion ESI mass spectra were obtained for each of the alkaloids by direct infusion of the standards of VBL, VCR, VLS, VDL, CTR, and IS, respectively. In these experiments, it was discovered that the response of alkaloids observed in positive ion mode was higher than that in negative ion mode. Thus, positive ion mode was finally employed. The deprotonated molecular ions [M + H]+ for each of the above mentioned alkaloids and the IS were observed at m/z 811.5, 825.3, 809.7, 457.4, 337.4, and 779.4, respectively. In addition to full scan mass spectra, collision induced dissociation was undertaken in their MS/MS fragmentation mode to yield product ion mass spectra. While infusing each of the standards, the collision energy (CE) was varied from 10 to 80 V to find the optimal CE. The most abundant fragment ions for each alkaloid were chosen for MRM quantification. The optimal MS, MS/MS conditions for fragment ions and the main fragment ions of each alkaloid and the ion pairs for MRM experiments are listed in Table 1. Fig. 2 shows the product ions scan spectra of the analytes and IS. The composition and ratio of the mobile phase is important for the HPLC-ESI-MS/MS determination of the five Catharanthus alkaloids. Several mobile phases with different composition and ratio were used to investigate the chromatographic behavior, and it was found that methanol-15 mmol·L−1 ammonium acetate containing 0.02% formic acid (65 : 35, V/V) was optimal for the separation of the five alkaloids and IS. All the peaks of the analytes and IS were detected with excellent resolution as well as peak shapes,
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Table 1 MS, MS/MS conditions and ESI-MS/MS data for five Catharanthus alkaloids Compounds
DP (V)
EP (V)
FP (V)
CE (V)
CXP (V)
NEB (psi)
CUR (psi)
CAD (psi)
Precursor ion [M + H]+ (m/z)
Major product ions (m/z)
Ion pairs (m/z)
VBL
120
47
9
811.5
793.1, 355.3, 337.5, 224.4
811.5→355.3
VCR
120
53
12
825.3
765.1, 556.4, 341.2, 144.1
825.3→765.1
VLS
100
50
8
809.7
748.8, 353.0
809.7→353.0
VDL
130
45
14
457.4
397.3, 188.3
457.4→188.3
CTR
100
30
13
337.4
173.3, 144.2, 93.2
337.4→144.2
IS
65
41
9
779.4
658.6, 457.4, 323.4
779.4→323.4
10
400
10
12
6
Notes: DP, Delustering potential; EP, Entrance potential; FP, Focusing potential; CE, Collision energy; CXP, Collision cell exit potential; NEB, Nebulizing gas; CUR, Curtain gas; CAD, Collision gas
Fig. 2
Product ion mass spectra of A) VBL; B) VCR; C) VLS; D) VDL; E) CTR, and F) IS
and no interference from endogenous substances was observed at the retention time of the analytes and IS. A typical MRM chromatogram of the standards and the sample is shown in Fig. 3. The retention times of VCR, VBL, VLS, VDL, CTR, and IS were 2.51, 4.42, 4.57, 6.10, 7.31, and 2.97 min, respectively. Method validation Linearity, lower limits of quantification (LLOQ) and lower limits of detection (LLOD) The typical equations of calibration curves and the linearity ranges for the five analytes are shown in Table 2. All correlation coefficients were higher than 0.9950. Calibration data for each alkaloid were obtained using the optimized HPLC-MS/ MS conditions. The response profile was determined and observed to be linear for each of the alkaloids within the linear ranges. Each concentration level was analyzed in triplicate. The results showed that there was excellent correlation between the ratio of peak area of each alkaloid versus IS and concentration for each alkaloid within the linearity. The LLOQs of VBL, VCR, and VLS were 1.5 ng·mL−1, while the LLOQs of VDL and CTR were 3.1 ng·mL−1. The LLODs of VBL, VCR, and VLS were 0.75 ng·mL−1, while the LLODs of VDL and CTR were 1.5 ng·mL−1. These LLOQs are sufficient for content detection of the five
Catharanthus alkaloids. Precision and accuracy The intra- and inter-day precision and accuracy values for the QC samples are summarized in Table 3. The intra- and inter-day precision and accuracy were evaluated by determination of standard QC samples at three concentration levels of the five analytes. The intra- and inter-day precisions (RSD) of these analytes were all less than 11.5% and 9.0%, while the accuracy was within ± 10.9% for all the analytes. The values for accuracy and precision demonstrated that the method is reliable and reproducible. Recovery The recoveries for the five analytes are summarized in Table 4. Mean absolute recoveries of VBL, VCR, VLS, VDL, and CTR were 82.6%−91.2%, 82.8%−91.5%, 87.0%−89.5%, 79.9%−90.7%, and 83.0%−85.2% at three QC levels. The recoveries were consistent and reproducible. Stability The stability of all the analytes was assessed under various conditions. The results presented in Table 4 indicate that the five analytes were stable in the extract sample at room temp for 2 h and at −20 °C for two weeks. No significant degradation of five analytes was observed during the 2 h storage at room temp and −20°C for two weeks. Post-preparative
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Fig. 3 Representative MRM chromatograms of A) five Catharanthus alkaloids and IS standard; B) C. roseus leaf sample (A1, B1 VCR; A2, B2 VBL; A3, B3 VLS; A4, B4 VDL; A5, B5 CTR; and A6, B6 IS
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Table 2 Regression equations, linear ranges, correlation coefficients, LLOQ, and LLOD of the Catharanthus alkaloids Alkaloid
Regression equation
Linear range (ng·mL−1)
LLOQ (ng·mL−1)
r2 (n = 6)
LLOD (ng·mL−1)
VBL
Y = 0.373 1x + 0.076 8
1.5−1 562.5
0.999 5
1.5
0.75
VCR
Y = 0.580 1x + 0.147 3
1.5−1 562.5
0.998 0
1.5
0.75
VLS
Y = 0.282 1x + 0.049 6
1.5−1 562.5
0.997 9
1.5
0.75
VDL
Y = 0.385 3x + 0.802 6
3.1−6 250.0
0.995 3
3.1
1.5
CTR
Y = 0.552 2x + 0.158 0
3.1−6 250.0
0.998 9
3.1
1.5
Table 3 Precision and accuracy for the Catharanthus alkaloids (n=3 days, six replicates per day) Alkaloid
Nominal concentration (ng·mL−1)
VBL
12.4
VCR
VLS
VDL
CTR
Intra-day
Inter-day
Precision (RSD%)
Accuracy (RE%)
c/(ng·mL−1)
Precision (RSD%)
Accuracy (RE%)
13.7 ± 0.9
6.2
10.5
12.8 ± 1.1
8.5
3.5
97.6
94.6 ± 4.3
4.5
–3.1
94.6 ± 4.3
4.5
–3.1
781.3
762.3 ± 17.6
2.3
–2.4
752.3 ± 38.1
5.1
–3.7
c/(ng·mL−1)
12.4
13.2 ± 1.2
8.8
6.7
11.3 ± 0.6
4.9
–8.9
97.6
94.1 ± 3.1
3.3
–3.6
94.1 ± 4.8
5.1
–3.6
781.3
756.3 ± 20.8
2.7
–3.2
726.3 ± 37.3
5.1
–7.0
12.4
13.1 ± 1.5
11.5
5.6
11.4 ± 1.0
9.0
–8.1
97.6
93.9 ± 2.0
1.5
–3.8
93.8 ± 1.3
1.4
–1.4
781.3
763.1 ± 13.9
1.8
–2.3
752.6 ± 11.9
1.6
–3.7
24.8
25.6 ± 2.9
11.3
4.8
25.9 ± 2.3
9.0
6.1
390.6
368.2 ± 30.9
8.4
–5.7
374.8 ± 19.4
5.2
–4.0
1562.5
1528.4 ± 30.9
2.0
–2.2
1 518.4 ± 47.5
3.1
–2.8
24.8
22.0 ± 2.1
9.4
–9.9
21.7 ± 1.7
7.7
–10.9
390.6
407.0 ± 22.3
5.5
4.2
410.4 ± 25.4
6.2
5.1
1 562.5
1523.5 ± 17.9
1.2
–2.5
1492.9 ± 46.8
3.1
–4.5
Table 4 Recovery and stability for the analytes (n = 6) Extraction recovery
Alkaloid
Spiked concentration (ng·mL−1)
Mean (%)
VBL
12.4
91.2
VCR
VLS
VDL
CTR
Stability (RE%)
RSD (%)
Short-term (2 h at room temp)
Post-preparative (12 h at 4 °C)
Long-term (2 weeks at −20 °C)
12.5
–1.2
–2.9
–10.8
97.6
82.6
5.3
–0.6
–3.1
–6.3
781.3
86.5
7.9
1.2
–7.5
–5.2
12.4
82.8
13.7
2.7
1.3
3.4
97.6
86.2
4.9
–1.5
–5.9
–2.7
781.3
91.5
6.0
–3.4
–11.8
–5.5
12.4
88.9
9.2
1.6
0.3
0.8
97.6
89.5
4.2
–0.9
–5.6
–4.7
781.3
87.0
6.1
–2.1
–9.9
–6.1
24.8
79.9
11.0
1.9
7.8
2.4
390.6
90.7
8.3
3.5
10.7
6.2
1562.5
80.1
7.8
2.6
9.6
7.3
24.8
85.2
13.0
3.8
4.2
–3.1
390.6
83.6
6.2
5.4
8.1
2.9
1 562.5
83.0
7.9
1.6
6.6
6.7
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stability of the alkaloids also showed that no significant degradation occurred when the extracted samples were kept at 4 °C for 12 h. The responses varied from −11.8% to 10.7% at all tested concentrations. Method application - quantitative analysis of C. roseus samples The validated method was successfully applied for the quantitative analysis studies of five Catharanthus alkaloids in C. roseus samples. Three batches of C. roseus samples were collected from the southern, middle, and northern regions of China (Hainan, Zhejiang, and Heilongjiang Provinces), and the leaves, stems, and roots were quantitatively analyzed using the developed LC-ESI-MS/MS method. The results (Table 1) showed that the contents of the five alkaloids varied between the different regions of China, and the different crude plant parts. For the C. roseus leaves and stems, the contents of VBL, VCR and VLS in samples from Heilongjiang are lower than those of Zhejiang and Hainan provinces,
but the contents of VDL and CTR of the samples from Heilongjiang are higher than the other two provinces. The difference between the contents in the samples from the three regions showed that the five alkaloids may vary based on the variety of temperature and sunlight. It was reported previously that the biosynthesis of bis-indole alkaloids depended on light, and that UV can induce VDL and CTR to synthesize VBL [27]. So the sufficient sunlight and moderate temperatures of Zhejiang and Hainan provinces may be benefit VBL synthesis. Therefore, the VBL content of the samples from these two provinces are much higher than Heilongjiang’s. The VCR and VLS contents in the Zhejiang and Hainan samples are also higher than in Heilongjiang samples because of the its bis-indole alkaloid structure as VBL. From the results, it was found that the VBL, VCR, and VLS contents are very low in three parts (leaves, stems and root). So the developed LC-ESI-MS/MS method is a new, more sensitive method for VBL, VCR, and VLS determination.
Table 5 Content of five Catharanthus alkaloids in C. roseus samples from three different regions of China Source Heilongjiang, China
Zhejiang, China
Hainan, China
Analysis part
VBL (µg·g−1, DW)
VCR (µg·g−1, DW)
CTR (µg·g−1, DW)
38.5
3.3
6.1
288.3
206.8
stems
45.7
2.4
4.5
220.6
128.4
roots
9.5
1.9
2.4
16.8
21.2
leaves
102.9
11.0
15.3
232.9
134.4
stems
55.9
6.1
7.8
80.7
49.6
roots
21.9
3.4
4.1
13.2
92.8
leaves
113.2
9.6
17.6
215.3
113.5
stems
70.5
5.0
12.3
39.2
24.7
roots
31.3
5.3
7.2
8.9
116.0
[3]
A sensitive LC-ESI-MS/MS method for the determination of VBL, VCR, VLS, VDL, and CTR was developed, validated, and applied for their quantitative analysis in C. roseus. The described LC-ESI-MS/MS method was sensitive, with high accuracy and a short run time of 10 min, and met all the requirements for effective plant extract analysis. With no reports on HPLC-MS or HPLC-MS/MS methods for the quantitative determination of the five Catharanthus alkaloids, this is the first report of the simultaneous quantitative study of VBL, VCR, VLS, VDL, and CTR in C. roseus extracts. The method presented is valuable for providing a procedure for the future analysis of trace components in C. roseus.
References
[2]
VDL (µg·g−1, DW)
leaves
Conclusions
[1]
VLS (µg·g−1, DW)
Noble RL. The discovery of the Vinca alkaloids - chemotherapeutic agents against cancer [J]. Biochem Cell Biol, 1990, 68 (12): 1344-1351. Armstrong JG, Dyke RW, Fouts PJ. Vinblastine sulfate treatment of Hodgkin's disease during a pregnancy [J]. Science, 1964, 143 (3607): 703.
Sertel S, Fu YJ, Zu YG, et al. Molecular docking and pharmacogenomics of Vinca alkaloids and their monomeric precursors, vindoline and catharanthine [J]. Biochem Pharmacol, 2011, 81 (6): 723-735. [4] Himes RH. Interactions of the Catharanthus (Vinca) alkaloids with tubulin and microtubules [J]. Pharmacol Ther, 1991, 51 (2): 257-267. [5] Jordan MA, Thrower D, Wilson L. Mechanism of inhibition of cell proliferation by Vinca alkaloids [J]. Cancer Res, 1991, 51 (4): 2212-2222. [6] Zhou XJ, Rahmani R. Preclinical and clinical pharmacology of Vinca alkaloids [J]. Drugs, 1992, 44 (Suppl 4): 1-16, 66-69. [7] Okouneva T, Hill BT, Wilson L, et al. The effects of vinflunine, vinorelbine, and vinblastine on centromere dynamics [J]. Mol Cancer Ther, 2003, 2 (5): 427-436. [8] Schneider M, Band P, Amiel JL, et al. Leurosine, the 3rd alkaloid from Vinca rosea, in the treatment of Hodgkin's disease, acute lymphoblastic leukemia and lymphoblastosarcoma [J]. Sem Hop, 1966, 42 (49): 2952-2954. [9] Pennanen S, Huhtikangas A. Photochemical one-pot synthesis of vinblastine and vincristine [J]. Photochem Photobiol, 1990, 51 (5): 515-518. [10] Mu FS, Yang L, Wang W. et al. Negative-pressure cavitation extraction of four main vinca alkaloids from Catharanthus
– 792 –
ZHANG Lin, et al. / Chin J Nat Med, 2014, 12(10): 786793
roseus leaves [J]. Molecules, 2012, 17 (8): 8742-8752. [11] Naaranlahti T, Ranta VP, Jarho P, et al. Electrochemical detection of indole alkaloids of Catharanthus roseus in highperformance liquid chromatography [J]. Analyst, 1989, 114 (10): 1229-1231. [12] Volkov SK, Grodnitskaya EI. Application of high-performance liquid chromatography to the determination of vinblastine in Catharanthus roseus [J]. J Chromatogr B Biomed Appl, 1994, 660 (2): 405-408. [13] Tikhomiroff C, Jolicoeur M. Screening of Catharanthus roseus secondary metabolites by high-performance liquid chromatography [J]. J Chromatogr A, 2002, 955 (1): 87-93. [14] Horvath P, Ivanyi G. Quantitative analysis of natural drugs III. Densitometric determination of vinblastine and other alkaloids of Catharantus roseus [J]. Acta Pharm Hung, 1982, 52 (4): 150-157. [15] Ylinen M, Suhonen P, Naaranlahti T, et al. Gas chromatographic-mass spectrometric analysis of major indole alkaloids of Catharanthus roseus [J]. J Chromatogr, 1990, 505 (2): 429-434. [16] Balsevich J, Hogge LR, Berry AJ, et al. Analysis of indole alkaloids from leaves of Catharanthus roseus by means of supercritical fluid chromatography/mass spectrometry [J]. J Nat Prod, 1988, 51 (6): 1173-1177. [17] Deus-Neumann B, Stöckigt J, Zenk MH. Radioimmunoassay for the quantitative determination of catharanthine [J]. Planta Med, 1987, 53 (2): 184-188. [18] Inhou C, Joy AB, Earl LW, et al. Quantification of vincristine and vinblastine in Catharanthus roseus plants by capillary zone electrophoresis [J]. J Chromatogr A, 1996, 755 (2): 281-288. [19] Barthe L, Ribet JP, Pelissou M, et al. Optimization of the separation of Vinca alkaloids by nonaqueous capillary electro-
phoresis [J]. J Chromatogr A, 2002, 968 (1-2): 241-250. [20] Choi YH, Yoo KP, Kim J. Supercritical fluid extraction and liquid chromatography-electrospray mass analysis of vinblastine from Catharanthus roseus [J]. Chem Pharm Bull, 2002, 50 (9): 1294-1296. [21] He LH, Yang L, Xiong AZ, et al. Simultaneous quantification of four indole alkaloids in Catharanthus roseus cell line C20hi by UPLC-MS [J]. Anal Sci, 2011, 27 (4): 433-438. [22] Liu W, Fu YJ, Zu YG, et al. Negative-pressure cavitation extraction for the determination of flavonoids in pigeon pea leaves by liquid chromatography-tandem mass spectrometry [J]. J Chromatogr A, 2009, 1216 (18): 3841-3850. [23] Zu YG, Yan MM, Fu YJ, et al. Determination and quantification of astragalosides in Radix Astragali and its medicinal products using LC-MS [J]. J Sep Sci, 2009, 32 (4): 517-525. [24] Liu W, Kong Y, Zu YG, et al. Determination and quantification of active phenolic compounds in pigeon pea leaves and its medicinal product using liquid chromatography-tandem mass spectrometry [J]. J Chromatogr A, 2010, 1217 (28): 4723-4731. [25] Li QY, Su L, Zu YG, et al. Quantification of CPT13 in rat plasma using LC-MS/MS for a pharmacokinetic study [J]. J Chromatogr B Analyt Technol Biomed Life Sci, 2011, 879 (7-8): 461-466. [26] Hua X, Fu YJ, Zu YG, et al. Determination of pinostrobin in rat plasma by LC-MS/MS: Application to pharmacokinetics [J]. J Pharm Biomed Anal, 2011, 56 (4): 841-845. [27] Wang H, Sun M, Wu CL, et al. Advances in the study on critical steps in the biosynthesis pathway of Catharanthus alkaloids and the regulation of their metabolism [J]. China J Chin Mat Med, 2001, 26 (10): 656-659.
Cite this article as: ZHANG Lin, GAI Qing-Hui, ZU Yuan-Gang, YANG Lei, MA Yu-Liang, LIU Yang. Simultaneous quantitative determination of five alkaloids in Catharanthus roseus by HPLC-ESI-MS/MS [J]. Chinese Journal of Natural Medicines, 2014, 12 (10): 786-793.
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