J S S
ISSN 1615-9306 · JSSCCJ 38 (12) 2007–2192 (2015) · Vol. 38 · No. 12 · June 2015 · D 10609
JOURNAL OF
SEPARATION SCIENCE
Methods Chromatography · Electroseparation Applications Biomedicine · Foods · Environment
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J. Sep. Sci. 2015, 38, 2007–2013
Awantika Singh1,2 Vikas Bajpai1,2 Sunil Kumar1 Kamal Ram Arya2,3 Kulwant Rai Sharma4 Brijesh Kumar1,2 1 Sophisticated
Analytical Instrument Facility Division, CSIR-Central Drug Research Institute, Lucknow, India 2 Academy of Scientific and Innovative Research, New Delhi, India 3 Botany Division, CSIR-Central Drug Research Institute, Lucknow, India 4 Department of Forest Products, Dr YS Parmar University of Horticulture and Forestry, Nauni, Solan, Himachal Pradesh, India Received January 18, 2015 Revised March 25, 2015 Accepted March 25, 2015
Research Article
Quantitative determination of isoquinoline alkaloids and chlorogenic acid in Berberis species using ultra high performance liquid chromatography with hybrid triple quadrupole linear ion trap mass spectrometry Berberis species are well known and used extensively as medicinal plants in traditional medicine. They have many medicinal values attributable to the presence of alkaloids having different pharmacological activities. In this study, a method was developed and validated as per international conference on harmonization guidelines using ultra high performance liquid chromatography with hybrid triple quadrupole-linear ion trap mass spectrometry operated in the multiple reaction monitoring mode for nine bioactive compounds, including protoberberine alkaloids, aporphine alkaloids and chlorogenic acid. This method was applied in different plant parts of eight Berberis species to determine variations in content of nine bioactive compounds. The separation was achieved on an ACQUITY UPLC CSHTM C18 column using a gradient mobile phase at flow rate 0.3 mL/min. Calibration curves for all the nine analytes provided optimum linear detector response (with R2 ࣙ0.9989) over the concentration range of 0.5–1000 ng/mL. The precision and accuracy were within RSDs ࣘ2.4 and ࣘ2.3%, respectively. The results indicated significant variation in the total contents of the nine compounds in Berberis species. Keywords: Berberis / Isoquinoline alkaloids / Multiple reaction monitoring / Quantification DOI 10.1002/jssc.201500063
Additional supporting information may be found in the online version of this article at the publisher’s web-site
1 Introduction Plants of the genus Berberis belong to family Berberidaceae and they are extensively used medicinal plants in traditional medicine [1]. It has about 400–450 species found in Asia, Mediterranean region and America [2]. In India, most Berberis species are available in Himalayas at an altitude of 1000– 3000 m and in Nilgiri hills at an altitude of 1000–2400 m [3]. They are spiny, deciduous, evergreen or semi-evergreen shrubs or small trees with characteristic yellow wood and flowers [4]. Their medicinal values may be attributed to the presence of alkaloids, mainly ‘berberine’, which is an isoquinoline alkaloid [1]. Berberine is responsible for many Correspondence: Dr. Brijesh Kumar, Sophisticated Analytical Instrument Facility, CSIR-Central Drug Research Institute, Lucknow226031, India E-mail: [email protected], [email protected] Fax: +91 0522 2623405
Abbreviations: MRM, multiple reaction monitoring; PBA, Protoberberine alkaloid
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pharmacological activities like anti-diabetic, anti-diarrhea, anti-hepatitis, anti-inflammatory, anti-microbial, anti-tumor, anti-oxidant, hepato-protective, cardiotonic, skin related problems, laxative, anti-depressant, immuno-modulatory, and neuro-protective [1, 2, 5–9]. Local people use decoctions of the root and stem bark of Berberis species viz B. aristata, B. lycium, and B. chitria for the treatment of conjunctivitis, ophthalmic problems, bleeding piles, ulcers, skin diseases, jaundice, enlarged liver, and enlarged spleen by local people [1, 2]. Berberis aristata is one of the most important species, used as a raw drug in different ayurvedic and homeopathic formulations [10,11]. Overconsumption of B. aristata by many herbal industries has led to the possibility of swapping this species with B. asiatica, B. chitria, B. lycium, and so on [7, 12, 13]. Several analytical methods are reported for the quantification of some alkaloids in Berberis species [2]. Although it has been previously reported that the maximum content of berberine is present in the root part of Berberis species [1, 2, 14], but no reliability could be established in the results with respect to species. HPLC–UV, high-performance TLC,
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and TLC were used to obtain the content of berberine [2]. Using high-performance TLC methods, methanolic extracts of B. asiatica yielded higher berberine content (4.3%) in comparison to B. lycium (4.0%) and B. aristata (3.8%), whereas methanolic extracts of B. aristata yielded higher berberine content (2.8%) in comparison to B. asiatica (2.4%) [14, 15]. Similarly, Srivastava et al. (2006) have also reported that the methanolic extract of B. chitria yielded 3.16% of berberine content by HPTLC method [13]. Andola et al. (2010c) have reported that B. pseudumbellata (roots and stem bark) yielded higher berberine content in the summer season while contrary to this Rashmi et al. (2009) have reported that B. aristata (roots) yielded higher berberine content (1.86%) in the winter samples using HPLC method [16, 17]. These variations may be due to the difference in the species of the plants, location or the analytical techniques used for the analysis. However, the analytes in these literatures were identified only by their retention time (tR), UV spectra, and CAMAG TLC system. Compared with these analytical methods, the UHPLC– ESI-MS/MS analytical method in multiple reaction monitoring (MRM) mode is a more powerful approach [18]. This method rapidly quantifies multiple components in a complex matrix due to its rapid separation power, greatest sensitivity and high specificity [19–21]. Although, we have previously done quantification of berberine, jatrorrhizine, palmatine, magnoflorine, and chlorogenic acid in different part of B. petiolaris but quantification of these bioactive compounds have not yet been carried out in other Berberis species [1]. In this paper, a previously developed UHPLC–ESI-MS/MS method is modified for the quantification of nine bioactive compounds, the characteristic compounds of the genus Berberis [2, 7], by UHPLC coupled with triple quadrupole-linear ion trap mass spectrometry (QqQLIT -MS/MS) within MRM mode [1]. This method is applied in different plant parts such as leaf, root, stem, fruit and flower of eight Berberis species (BAR: B. aristata; BAS: B. asiatica; BC: B. chitria; BJ: B. jaeschkeana; BK: B. koehneana; BL: B. lycium; BP: B. petiolaris; BU: B. pseudoumbellata) to determine variations in content of nine bioactive compounds.
2 Materials and methods 2.1 Materials LC–MS-grade methanol, acetonitrile and formic acid, purchased from Sigma–Aldrich (St. Louis, MO, USA), were used in mobile phase and sample preparation throughout the LC–MS studies. AR grade ethanol, purchased from Merck Millipore (Darmstadt, Germany), was used in the preparation of ethanolic extract. Ultra-pure water, obtained from Direct-Q system (Millipore, Billerica, MA, USA), was used throughout the analysis. Standard compounds of berberine, palmatine, jatrorrhizine, magnoflorine, tetrahydroberberine, tetrahydropalmatine, glaucine, and isocorydine were purchased from Shanghai Tauto Biotech (Shanghai, China). C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chlorogenic acid was purchased from Extrasynthese (Genay Cedex, France). The structures of these standard compounds are shown in Fig. 1. Plant materials were collected from the different region of India. Voucher specimen numbers of Berberis chitria (BC), B. jaeschkeana (BJ), B. koehneana (BK), B. lycium (BL) and B. pseudoumbellata (BU) are 13503, 13504, 13513, 13502 and 13501, respectively, collected from Churdhar, Himachal Pradesh, India and deposited in the Department of Forest Products, Dr. Y. S. Parmar University of Horticulture and Forestry, Nauni, Solan, Himachal Pradesh, India. B. aristata (BAR) and B. asiatica (BAS) were collected from Chaura forest, Loharkhet, district Bageshwar of Kumaon, Uttarakhand, India, whereas B. petiolaris (BP) was collected from Pandukholi Forest, Almora, Uttarakhand, India. Voucher specimen numbers of BAR, BAS and BP are KRA-24415, KRA-24456 and KRA-24410, respectively, and deposited in the departmental Herbarium of Central Drug Research Institute, Lucknow, Uttar Pradesh, India.
2.2 Extraction and sample preparation 50 g powder of each dried plant parts of selected Berberis species were extracted with 500 mL ethanol (100%) in an ultrasonic water bath for 15 min and then kept under room temperature. After 24 h, the extracts were filtered through filter paper (Whatman No. 1) and residues were re-extracted three times with fresh solvent following the same procedure. The combined filtrates of each sample were evaporated to dryness under reduced pressure at 20–50 kPa and temperature at 40⬚C using a Buchi rotary evaporator (Flawil, Switzerland). Stock solutions (1 mg/mL) of each sample were prepared in methanol and filtered through a 0.22 m PVDF membrane (Merck Millipore, Darmstadt, Germany).
2.3 Preparation of standard solutions Stock solutions of berberine, palmatine, jatrorrhizine, tetrahydroberberine, tetrahydropalmatine, magnoflorine, isocorydine, glaucine and chlorogenic acid were prepared separately in methanol (1000 g/mL). Then, methanol stock solutions containing the nine analytes were prepared and diluted in appropriate concentration to yield a series of concentrations, within the ranges from 0.5 to 1000 ng/mL. The calibration curves were constructed by plotting the value of peak areas versus the value of concentrations of each analyte. All stock solutions were stored at –20⬚C until use.
2.4 Instrumentation Quantitative analysis was performed on a 4000 QTRAPTM MS/MS system, hybrid triple quadrupole-linear ion trap mass spectrometer (Applied Biosystem; Concord, ON, Canada), hyphenated with a Waters ACQUITY UPLCTM system (Waters; Milford, MA, USA) via an pneumatically-assisted electrospray www.jss-journal.com
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Figure 1. Structures and MS/MS spectrum of (A) Chlorogenic acid; (B) Magnoflorine; (C) Isocorydine; (D) Glaucine; (E) Jatrorrhizine; (F) Tetrahydropalmatine; (G) Tetrahydroberberine; (H) Palmatine; and (I) Berberine.
(Turbo VTM source with TurboIonSprayTM probe and APCI probe) interface. The Waters ACQUITY UPLCTM system was equipped with a binary solvent manager, sample manager, column compartment and photodiode array detector (PAD).
parameters are shown in Table 1. Quadrupole 1 and quadrupole 2 were maintained at unit resolution.
2.5 Data processing 2.4.1 UHPLC conditions Chromatographic separation of compounds was obtained with an ACQUITY UPLC CSHTM C18 column (1.7 m, 2.1 × 100 mm) operated at 25⬚C.The mobile phase, which consisted of a 0.1% formic acid aqueous solution (A) and acetonitrile (B), was delivered at a flow rate of 0.3 mL/min under a gradient program: 5% (B) initial to 1.0 min, 5–20% (B) from 1.0 to 2.0 min, 20–30% (B) from 2.0 to 3.0 min, 30–90% (B) from 3.0 to 4.0 min, maintained at 90% (B) from 4.0 to 5.0 min and back to initial conditions from 5.0 to 5.5 min. The sample injection volume used was 1 L. 2.4.2 MS conditions Positive pneumatically assisted ESI was used for sample introduction, ionization process and operated in the MRM R probe was vertically positioned mode. A Turboionspray 11 mm from the orifice and charged with 5500 V. Source dependent parameters such as temperature, GS1, GS2 and curtain gas were set at 550⬚C, 50 psi, 50 psi, and 20 psi, respectively. The collision-activated dissociation gas was set as medium and the interface heater was on. High-purity nitrogen was used for all the processes. The compound-dependent C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
AB Sciex Analyst software version 1.5.1 was used to control the LC–MS/MS system and for data acquisition and processing. All the statistical calculations related to quantitative analysis were performed on Graph Pad Prism software version 5.
3 Results and discussion 3.1 Optimization of UHPLC–ESI-MS/MS condition For quantification of chlorogenic acid and isoquinoline alkaloids in the genus Berberis, standards chlorogenic acid (1), magnoflorine (2), isocorydine (3), glaucine (4), jatrorrhizine (5), tetrahydropalmatine (6), tetrahydroberberine (7), palmatine (8), and berberine (9) were studied [2, 7]. Preliminary, compounds dependent MRM parameters (DP: declustering potential; EP: entrance potential; CE: collision energy; and CXP: cell exit potential) were optimized to achieve the most specific and stable MRM transitions (precursor-toproduct ions). To accomplish this target, each selected analyte (10 ng/mL) was directly injected into the ESI source of mass spectrometer by continuous infusion. The optimized mass spectrometric conditions for the detection of analytes www.jss-journal.com
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Table 1. The optimized compound dependent MRM parameters and transitions for each analyte in the UPLC-ESI-MS/MS analysis
Peak No.
RT (min)
Analyte
Precursor ion (Da)
DP (V)
EP (V)
CE (eV)
Quantifiera)
Qualifiera)
1 2 3 4 5 6 7 8 9
1.24 1.39 1.61 1.81 1.94 1.96 2.09 2.21 2.38
Chlorogenic acid Magnoflorine Isocorydine Glaucine Jatrorrhizine Tetrahydropalmatine Tetrahydroberberine Palmatine Berberine
355 [M+H]+ 342 [M]+ 342.1 [M+H]+ 356.3 [M+H]+ 338 [M]+ 356.2 [M+H]+ 340 [M+H]+ 352.2 [M]+ 336 [M]+
50 50 73 101 50 86 55 32 40
12 10 4.5 8 10 7 10 10 10
15 27 27 20 55 35 35 40 45
355→163 (12) 342→296.7 (20) 342.1→279.2 (12) 356.3→325.3 (16) 338→307.3 (15) 356.2→192.1 (7) 340→176 (9) 352.2→336 (15) 336→320 (5)
355→145 (15) 342→282 (12) 342.1→311.1 (8) 356.3→294.1 (15) 338→322.1 (11) 356.2→165.1 (5) 340→149.1 (5) 352.2→308.1 (16) 336→292.2 (8)
RT: Retention Time; DP: Declustering Potential; EP: Entrance Potential; CE: Collision Energy; a) Cell Exit Potential (CXP in V) is given in brackets
were achieved in positive-ion mode. Analytes 1, 3, 4, 6, and 7 showed [M+H]+ ion while analytes 2, 5, 8, and 9 showed [M]+ ion in Q1 MS scan and, therefore, selected as the precursor ions for MS/MS fragmentation analysis of analytes. DP and EP were optimized to obtain the maximum sensitivity of [M+H]+ and [M]+ ions in Q1 multiple ion scan (Q1 MI). Identification of the fragment ions and selection of CE for each analyte were carried out in the product ion scan. Their MS/MS spectra are shown in Fig. 1. Furthermore, CE and CXP were optimized for two transitions to acquire the maximum sensitivity in the MRM scan but only one transition was monitored in quantitative analysis of samples due to the lack of sensitivity of the other observed product ions. The most prominent MRM transition was selected as a quantifier and other one as a qualifier in specificity test only. Table 1 shows the optimized parameters for all the analytes. The source dependent parameters (curtain gas, GS1, GS2, and ion source temperature) were optimized for the highest abundance of precursor-to-product ions in the FIA by operating UHPLC. With respect to UHPLC separation, different parameters were examined and compared. Acetonitrile displayed stronger elution ability, which shortened the elution time with good resolving power than methanol in the test. Therefore, aqueous acetonitrile was chosen as the mobile phase other than aqueous methanol. Formic acid was used as a modifier to obtain desirable peak shapes and degree of separation than acetic acid (Supporting information; Fig. S1). Different flow rates (0.2, 0.3, 0.4, 0.5 mL/min) and column temperatures (20, 25, 30, or 35⬚C) were examined in the test. The results showed that the desirable resolution in shortest analysis time were achieved when the aqueous formic acid (0.1%) and acetonitrile system was used at a flow rate of 0.3 mL/min and column temperature at 25⬚C. MRM extracted ion chromatogram of analytes are shown in Fig. 2.
3.2 Analytical method validation The proposed UHPLC–MRM method for quantitative analysis was validated according to the guidelines of international
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conference on harmonization (ICH, Q2R1) by determining specificity, linearity, lower limit of detection (LOD), lower limit of quantification (LOQ), precision, solution stability, and recovery [22]. 3.2.1 Specificity Exact identification of each analyte in the samples is a prerequisite for successful quantification. UHPLC–ESI-MS/MS method was employed to analyze the two MRM signals (quantifier and qualifier) in the sample matrix. All of the peaks of the target analytes in B. asiatica fruit and B. jaeschkeana root were unambiguously identified by comparison of retention time, quantifier and qualifier transitions in MRM chromatogram of standards (Table 1). 3.2.2 Linearity, LODs, and LOQs Linearity, LODs, and LOQs of nine analytes were determined by serial dilution of sample solutions using the described UHPLC–ESI-MS/MS method. The linearity of calibration was performed by the analyte peak area (y) versus concentration (x) and constructed with a weighting (1/x2 ) factor by least-squares linear regression. Good linear relationship between the peak area and concentration was obtained for each of the nine analytes over the tested concentration range with a correlation coefficient (R2 ) of ࣙ0.9989. LODs and LOQs were the concentrations of the compound at which S/Ns were detected as 3:1 and 10:1, respectively (Table 2). The LOD for each analyte varied from 0.12–2.92 ng/mL and LOQ from 0.26–8.79 ng/mL. 3.2.3 Precision, stability, and accuracy Precision of the developed method was determined at three different levels i.e. intraday variation, interday variation, and reproducibility, according to ICH guideline [22]. Intra- and inter-day variations of the method were determine by analyzing known concentrations of the nine analytes in the six replicates during a single day and by triplicating the
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Figure 2. Extracted ion chromatograms of (A) Chlorogenic acid; (B) Magnoflorine; (C) Isocorydine; (D) Glaucine; (E) Jatrorrhizine; (F) Tetrahydropalmatine; (G) Tetrahydroberberine; (H) Palmatine and; (I) Berberine, in MRM mode.
Table 2. Linearity, LOD, LOQ, precisions, stability and recovery results of investigated components
Linearity
Precision RSD (%)
Recovery
Analytes
Regression equation
R2
Linear range (ng/ml)
LOD (ng/ml)
LOQ (ng/ml)
Intraday (n = 6)
Interday (n = 5)
Reproducibility (n = 5)
Stability
Mean (n = 6)
RSD (%)
1 2 3 4 5 6 7 8 9
y = 610*x -19.7 y = 743*x - 43.7 y = 6060*x - 266 y = 3690*x - 49.8 y = 1100*x - 7.66 y = 14600*x - 448 y = 15200*x + 2350 y = 989*x + 870 y = 1030*x - 487
1.0000 0.9999 0.9991 0.9990 0.9988 0.9998 0.9998 0.9992 0.9989
0.5-1000 0.5-250 0.5-1000 0.5-1000 0.5-1000 0.5-200 0.5-200 0.5-50 1-100
0.12 0.22 0.36 0.19 0.87 0.08 0.48 2.92 1.53
0.35 0.67 1.08 0.56 2.65 0.26 1.46 8.79 4.64
2.40 1.42 1.48 0.65 1.53 2.10 1.13 0.40 1.52
2.39 2.25 1.65 0.74 0.96 2.37 2.05 0.89 1.68
1.07 0.41 1.11 0.69 1.02 0.75 0.38 3.1 0.75
2.82 2.64 1.98 2.21 2.02 3.27 3.80 1.38 1.72
91.2 97.49 99.6 101.5 99.01 102.1 102.7 101.3 98.6
1.2 2.3 0.92 1.49 1.32 1.6 1.03 0.74 1.11
y: peak area; x: concentration of compound (ng/ml); LOD: limit of detection: S/N = 3; LOQ: limit of quantification: S/N = 10
experiments on five successive days, respectively. RSD values for precision were in the range of 0.40–2.40% for intraday assays, 0.74–2.39% for interday assays (Table 2). Six different sample solutions of B. asiatica flower in parallel were extracted and analyzed with the proposed method to evaluate the reproducibility. The RSD values of the nine compounds were ࣘ 3.1%, which showed high reproducibility of the method. Stability of sample solutions stored at room temperature was investigated by replicate injections of the sample solution at 0, 2, 4, 8, 12, and 24 h. The RSD values of stability of the nine analytes were ࣘ 3.80% (Table 2). To evaluate the accuracy of this method, a recovery test was applied by the standard addition method. The mixed standard solutions with three
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different spike levels (low, middle and high) were added into a sample and analyzed using the above mentioned method in triplicate experiments. The recovery was calculated by the formula: recovery = (a–b)/c×100%, where a is the detected amount, b is the original amount and c is the spiked amount. The results showed that the developed analytical method was simple, reliable and reproducible with good recovery in the range of 91.2–102.7% (RSD ࣘ 2.3%) for all analytes (Table 2). In the term of LOD, LOQ, precision, stability and accuracy, this method is more sensitive compared to earlier reported methods for similar type of compounds using UHPLC–ESIMS/MS, HPLC–ESI-MS/MS, and HPLC–DAD methods [20, 21, 23, 24]. Therefore, this method was applied to quantify selected analytes in crude extract of B. species.
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Table 3. Contents (in mg/g) of nine bioactive compounds in the different parts of eight Berberis species
Analyte content (mg/g) Sample Name BAR
BAS
BC
BJ
BK
BL
BP
BU
Flower Leaf Root Stem Fruit Leaf Root Stem Leaf Root Stem Leaf Root Stem Leaf Root Stem Leaf Root Stem Shoot Fruit Leaf Root Stem Leaf Root Stem
1
2
3
4
5
6
7
8
9
Total (mg/g)
Total (% w/w)
191.3 38 nd nd 86.3 66.1 nd nd 21.8 nd nd 48.5 nd nd 104.1 nd nd 35.2 nd nd nd 38.4 189.4 nd 4.9 31.4 nd 3.5
0.5 0.4 6.8 26.3 1.8 0.4 17.8 33.8 0.5 16.4 65 0.8 42 66.4 0.8 20.6 34.2 0.3 7.8 64.3 7.28 2.9 1.1 79 285 0.7 53.95 184.1
1.3 nd 0.25 0.34 1.8 0.7 nd 0.5 0.16 0.3 0.9 nd 0.8 0.6 nd nd 0.45 nd nd 0.72 0.2 0.6 nd nd nd nd 0.53 0.31
0.4 nd nd nd 0.2 nd nd nd nd 0.06 0.05 nd 0.06 0.05 nd nd nd nd nd nd nd nd nd 0.06 0.06 nd 0.08 0.06
0.07 0.2 8.7 6.7 0.3 0.08 12.1 4.2 0.1 14.4 0.5 0.09 2.8 1.9 0.3 10.5 3.5 0.17 27.9 1.8 32.4 0.23 0.3 9.2 3.4 0.13 3.1 2
nd nd nd 0.28 0.37 0.1 nd 0.1 nd nd nd 0.1 0.31 0.2 nd nd nd nd nd nd nd 0.1 nd nd nd nd nd nd
0.05 nd 0.003 0.5 0.1 nd 0.05 0.04 nd nd nd nd 0.18 nd nd nd nd 0.4 nd 0.15 0.03 0.002 nd nd nd nd nd nd
0.004 bdl 78.9 7.1 0.01 0.002 89.2 11.4 0.02 38.2 0.13 0.03 5.1 1.6 0.3 87.4 4.2 2.4 53.8 1.8 55.43 1.3 0.6 5.7 1.14 bdl 8.1 5.7
1.7 1.9 121.8 102.3 3 1.6 78.1 29.9 2 106 2 1.9 11.2 6.4 7.1 144.9 29.5 3.6 172.8 18.2 158.5 3.4 3.6 24.1 5.2 0.91 43.8 14.6
195.3 40.5 216.5 143.5 94.2 69 197.3 79.9 24.6 175 68.6 51.4 62.5 77.2 112.6 263.4 71.9 42.1 262.3 87 253.8 46.9 195 118.1 299.7 33.1 109.6 210.3
19.53 4.05 21.65 14.35 9.42 6.9 19.73 7.99 2.46 17.5 6.86 5.14 6.25 7.72 11.26 26.34 7.19 4.21 26.23 8.7 25.38 4.69 19.5 11.81 29.97 3.31 10.96 21.03
nd: not detected; bdl: below detection level
3.3 Method application The newly developed UHPLC–ESI-MS/MS method was applied successfully to determine all the nine bioactive compounds in the ethanolic extracts of eight Berberis species. The content of each analyte was calculated from the corresponding calibration curve and summarized in Table 3. The analytical results indicated significant variations of their contents among the samples. 3.3.1 Protoberberine alkaloids (PBAs) Five PBAs were quantified in eight Berberis species including different plant parts. In PBAs, the contents of quaternary PBAs (Berberine, Jatrorrhizine, and Palmatine) were detected in almost all the samples. They were identified as the major constituent in root part of all the Berberis species and the least amount was detected in leaf part (Table 3). Berberine was found the most abundant PBAs in Berberis species and it was present in the highest amount in BL root (172.8 mg/g) followed by BL shoot (158.5 mg/g), BK root (144.9 mg/g), BAR root (121.8 mg/g), and BC root (106 mg/g). It was also
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showed that BL root, BL shoot, BK root, and BC root may be used for swapping of B. aristata (BAR) in herbal industries [2]. Jatrorrhizine was detected in higher quantity in BL shoot (32.4 mg/g), BL root (27.9 mg/g), and BC root (14.4 mg/g) while palmatine was detected in higher quantity in BAS root (89.2 mg/g) and BK root (87.4 mg/g). Tetrahydroprotoberberine alkaloids were detected in lesser quantity in BAR, BAS, BJ, BL, and BP and it was not detected in BC, BK, and BU. Tetrahydroberberine and tetrahydropalmatine were detected maximum in BAR stem (0.5 mg/g) and BAS fruit (0.37 mg/g), respectively.
3.3.2 Aporphine alkaloids Magnoflorine, isocorydine, and glaucine were detected higher in the stem part of Berberis species. Magnoflorine was detected as major constituent in BP stem (285 mg/g) and BU stem (184.1 mg/g) while isocorydine and glaucine were maximum in BAS fruit (1.8 mg/g) and BAR flower (0.4 mg/g), respectively.
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3.3.3 Acid Chlorogenic acid was a major constituent in flower and leaf part of Berberis species. It was detected in significant amounts in BAR flower (191.3 mg/g), BP leaf (189.4 mg/g), and BK leaf (104.1 mg/g) and was absent in root part of all the Berberis species.
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[4] Anonymous, The Wealth of India, A Dictionary of Indian Raw Materials, Vol 2 (B). Publications and Information Directorate CSIR, New Delhi, 1988, pp 114–118. [5] Tiwari, B. K., Khosa, R. L., Int. J. Pharm. Pharm. Sci. 2010, 2, 92–97. [6] Grycova, L., Dostal, J., Marek, R., Phytochemistry 2007, 68, 150–175. [7] Karimov, A., Chem. Nat. Compd. 1993, 29, 415–438.
4 Concluding remarks The present study involved quantification of nine bioactive compounds (protoberberine alkaloids, aporphine alkaloids, and chlorogenic acid) in different plant parts of eight Berberis species. An UHPLC–ESI-MS/MS method under MRM mode was successfully developed and validated as per ICH guidelines and it was applied in eight Berberis species. Results indicate that all the nine bioactive compounds are the most abundant in BP stem. Chlorogenic acid, magnoflorine, and berberine are found abundant in leaf, stem and root part, respectively, of all the Berberis species. Results also indicated that the roots of B. lycium, B. koehneana, and B. chitria may be used for swapping of B. aristata. This information was lacking in the literature and it could be helpful for better swapping of Berberis species. Awantika Singh is thankful to UGC, New Delhi, for a fellowship. A grateful acknowledgement is made to the Department of Science and Technology for Grant SB/EMEQ-095 and SAIFCDRI, Lucknow, India, where all the mass spectral analysis were carried out. This is CDRI communication no 8975. The authors have declared no conflict of interest.
5 References
[8] Singh, J., Kakkar, P., J. Ethnopharmacol. 2009, 123, 22– 26. [9] Semwal, B. C., Gupta, J., Singh, S., Kumar, Y., Giri, M., Int. J. Green Pharm. 2009, 3, 259–262. [10] Shenoy, P. K. R., Yoganarasimhan, S. N., Indian J. Tradit. Know. 2009, 8, 272–274. [11] Srivastava, S. K., Khatoon, S., Rawat, A. K. S., Mehrotra, S., Pushpangadan, P., Nat. Prod. Sci. 2001, 7, 102–106. [12] Andola, H. C., Gaira, K. S., Rawal, R. S., Muniyari, M. S., Bhatt, I. D., Chem. Biodivers. 2010a, 7, 415–420. [13] Srivastava, S. K., Rawat, A. K. S., Srivastava, M., Mehrotra, S., Nat. Prod. Sci. 2006, 12, 19–23. [14] Andola, H. C., Rawal, R. S., Rawat, M. S. M., Bhatt, I. D., Purohit, V. K., Asian J. Biotechnol. 2010b, 2, 239–245. [15] Srivastava, S. K., Rawat, A. K. S., Mehrotra, S., Pharm. Biol. 2004, 42, 467–473. [16] Andola, H. C., Rawal, R. S., Rawat, M. S. M., Bhatt, I. D., Purohit, V. K., India Med. Plants 2010c, 2, 111–115. [17] Rashmi, P. J., Rajasekaran, A., Rekha, P., Singh, Y. P., Pharmacog. Mag. 2009, 5, 355–358. [18] Grace, P. B., Mistry, N. S., Carter, M. H., Leathem, A. J. C., Teale, P., J. Chromatogr. B, 2007, 853, 138–146. [19] Wei, Z., Pan, Y., Li, L., Huang, Y., Qi, X., Luo, M., Zu, Y., Fu, Y., J. Sep. Sci. 2014, 37, 3045–3051. [20] Liu, J., Wu, H., Zheng, F., Liu, W., Feng, F., Xie, N., J. Sep. Sci. 2014, 37, 2513–2522. [21] Qiana, X. C., Zhang, L., Tao, Y., Huanga, P., Li, J. S., Chai, C., Li, W., Dia, L. Q., Cai, B. C., J. Pharm. Biomed. Anal 2015, 105, 64–73.
[1] Singh, A., Bajpai, V., Srivastava, M., Arya, K. R., Kumar, B., Rapid Commun. Mass Spectrom. 2014, 28, 2089– 2100.
[22] ICH Guideline. Q2 (R1), Validation of analytical procedures: Text and Methodology. International Conference on Harmonization, Geneva, 2005. p. 1.
[2] Bhardwaj, D., Kaushik, N., Phytochem. Rev. 2012, 11, 523–542.
[23] Ren, L., Xue, X., Liang, X., J. Sep. Sci. 2013, 36, 1389– 1396.
[3] Rajasekaran, A., Kumar, N., Indian J. Tradit. Know. 2009, 8, 562–563.
[24] Chen, Y., Li, M., Liu, J., Yan, Q., Zhong, M., Liu, J., Di, D., Liu, J., J. Sep. Sci. 2015, 38, 9–17.
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ISSN 1615-9306 · JSSCCJ 38 (12) 2007–2192 (2015) · Vol. 38 · No. 12 · June 2015 · D 10609
JOURNAL OF
SEPARATION SCIENCE
Methods Chromatography · Electroseparation Applications Biomedicine · Foods · Environment
12 15
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J. Sep. Sci. 2015, 38, 2007–2013
Awantika Singh1,2 Vikas Bajpai1,2 Sunil Kumar1 Kamal Ram Arya2,3 Kulwant Rai Sharma4 Brijesh Kumar1,2 1 Sophisticated
Analytical Instrument Facility Division, CSIR-Central Drug Research Institute, Lucknow, India 2 Academy of Scientific and Innovative Research, New Delhi, India 3 Botany Division, CSIR-Central Drug Research Institute, Lucknow, India 4 Department of Forest Products, Dr YS Parmar University of Horticulture and Forestry, Nauni, Solan, Himachal Pradesh, India Received January 18, 2015 Revised March 25, 2015 Accepted March 25, 2015
Research Article
Quantitative determination of isoquinoline alkaloids and chlorogenic acid in Berberis species using ultra high performance liquid chromatography with hybrid triple quadrupole linear ion trap mass spectrometry Berberis species are well known and used extensively as medicinal plants in traditional medicine. They have many medicinal values attributable to the presence of alkaloids having different pharmacological activities. In this study, a method was developed and validated as per international conference on harmonization guidelines using ultra high performance liquid chromatography with hybrid triple quadrupole-linear ion trap mass spectrometry operated in the multiple reaction monitoring mode for nine bioactive compounds, including protoberberine alkaloids, aporphine alkaloids and chlorogenic acid. This method was applied in different plant parts of eight Berberis species to determine variations in content of nine bioactive compounds. The separation was achieved on an ACQUITY UPLC CSHTM C18 column using a gradient mobile phase at flow rate 0.3 mL/min. Calibration curves for all the nine analytes provided optimum linear detector response (with R2 ࣙ0.9989) over the concentration range of 0.5–1000 ng/mL. The precision and accuracy were within RSDs ࣘ2.4 and ࣘ2.3%, respectively. The results indicated significant variation in the total contents of the nine compounds in Berberis species. Keywords: Berberis / Isoquinoline alkaloids / Multiple reaction monitoring / Quantification DOI 10.1002/jssc.201500063
Additional supporting information may be found in the online version of this article at the publisher’s web-site
1 Introduction Plants of the genus Berberis belong to family Berberidaceae and they are extensively used medicinal plants in traditional medicine [1]. It has about 400–450 species found in Asia, Mediterranean region and America [2]. In India, most Berberis species are available in Himalayas at an altitude of 1000– 3000 m and in Nilgiri hills at an altitude of 1000–2400 m [3]. They are spiny, deciduous, evergreen or semi-evergreen shrubs or small trees with characteristic yellow wood and flowers [4]. Their medicinal values may be attributed to the presence of alkaloids, mainly ‘berberine’, which is an isoquinoline alkaloid [1]. Berberine is responsible for many Correspondence: Dr. Brijesh Kumar, Sophisticated Analytical Instrument Facility, CSIR-Central Drug Research Institute, Lucknow226031, India E-mail: [email protected], [email protected] Fax: +91 0522 2623405
Abbreviations: MRM, multiple reaction monitoring; PBA, Protoberberine alkaloid
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pharmacological activities like anti-diabetic, anti-diarrhea, anti-hepatitis, anti-inflammatory, anti-microbial, anti-tumor, anti-oxidant, hepato-protective, cardiotonic, skin related problems, laxative, anti-depressant, immuno-modulatory, and neuro-protective [1, 2, 5–9]. Local people use decoctions of the root and stem bark of Berberis species viz B. aristata, B. lycium, and B. chitria for the treatment of conjunctivitis, ophthalmic problems, bleeding piles, ulcers, skin diseases, jaundice, enlarged liver, and enlarged spleen by local people [1, 2]. Berberis aristata is one of the most important species, used as a raw drug in different ayurvedic and homeopathic formulations [10,11]. Overconsumption of B. aristata by many herbal industries has led to the possibility of swapping this species with B. asiatica, B. chitria, B. lycium, and so on [7, 12, 13]. Several analytical methods are reported for the quantification of some alkaloids in Berberis species [2]. Although it has been previously reported that the maximum content of berberine is present in the root part of Berberis species [1, 2, 14], but no reliability could be established in the results with respect to species. HPLC–UV, high-performance TLC,
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and TLC were used to obtain the content of berberine [2]. Using high-performance TLC methods, methanolic extracts of B. asiatica yielded higher berberine content (4.3%) in comparison to B. lycium (4.0%) and B. aristata (3.8%), whereas methanolic extracts of B. aristata yielded higher berberine content (2.8%) in comparison to B. asiatica (2.4%) [14, 15]. Similarly, Srivastava et al. (2006) have also reported that the methanolic extract of B. chitria yielded 3.16% of berberine content by HPTLC method [13]. Andola et al. (2010c) have reported that B. pseudumbellata (roots and stem bark) yielded higher berberine content in the summer season while contrary to this Rashmi et al. (2009) have reported that B. aristata (roots) yielded higher berberine content (1.86%) in the winter samples using HPLC method [16, 17]. These variations may be due to the difference in the species of the plants, location or the analytical techniques used for the analysis. However, the analytes in these literatures were identified only by their retention time (tR), UV spectra, and CAMAG TLC system. Compared with these analytical methods, the UHPLC– ESI-MS/MS analytical method in multiple reaction monitoring (MRM) mode is a more powerful approach [18]. This method rapidly quantifies multiple components in a complex matrix due to its rapid separation power, greatest sensitivity and high specificity [19–21]. Although, we have previously done quantification of berberine, jatrorrhizine, palmatine, magnoflorine, and chlorogenic acid in different part of B. petiolaris but quantification of these bioactive compounds have not yet been carried out in other Berberis species [1]. In this paper, a previously developed UHPLC–ESI-MS/MS method is modified for the quantification of nine bioactive compounds, the characteristic compounds of the genus Berberis [2, 7], by UHPLC coupled with triple quadrupole-linear ion trap mass spectrometry (QqQLIT -MS/MS) within MRM mode [1]. This method is applied in different plant parts such as leaf, root, stem, fruit and flower of eight Berberis species (BAR: B. aristata; BAS: B. asiatica; BC: B. chitria; BJ: B. jaeschkeana; BK: B. koehneana; BL: B. lycium; BP: B. petiolaris; BU: B. pseudoumbellata) to determine variations in content of nine bioactive compounds.
2 Materials and methods 2.1 Materials LC–MS-grade methanol, acetonitrile and formic acid, purchased from Sigma–Aldrich (St. Louis, MO, USA), were used in mobile phase and sample preparation throughout the LC–MS studies. AR grade ethanol, purchased from Merck Millipore (Darmstadt, Germany), was used in the preparation of ethanolic extract. Ultra-pure water, obtained from Direct-Q system (Millipore, Billerica, MA, USA), was used throughout the analysis. Standard compounds of berberine, palmatine, jatrorrhizine, magnoflorine, tetrahydroberberine, tetrahydropalmatine, glaucine, and isocorydine were purchased from Shanghai Tauto Biotech (Shanghai, China). C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chlorogenic acid was purchased from Extrasynthese (Genay Cedex, France). The structures of these standard compounds are shown in Fig. 1. Plant materials were collected from the different region of India. Voucher specimen numbers of Berberis chitria (BC), B. jaeschkeana (BJ), B. koehneana (BK), B. lycium (BL) and B. pseudoumbellata (BU) are 13503, 13504, 13513, 13502 and 13501, respectively, collected from Churdhar, Himachal Pradesh, India and deposited in the Department of Forest Products, Dr. Y. S. Parmar University of Horticulture and Forestry, Nauni, Solan, Himachal Pradesh, India. B. aristata (BAR) and B. asiatica (BAS) were collected from Chaura forest, Loharkhet, district Bageshwar of Kumaon, Uttarakhand, India, whereas B. petiolaris (BP) was collected from Pandukholi Forest, Almora, Uttarakhand, India. Voucher specimen numbers of BAR, BAS and BP are KRA-24415, KRA-24456 and KRA-24410, respectively, and deposited in the departmental Herbarium of Central Drug Research Institute, Lucknow, Uttar Pradesh, India.
2.2 Extraction and sample preparation 50 g powder of each dried plant parts of selected Berberis species were extracted with 500 mL ethanol (100%) in an ultrasonic water bath for 15 min and then kept under room temperature. After 24 h, the extracts were filtered through filter paper (Whatman No. 1) and residues were re-extracted three times with fresh solvent following the same procedure. The combined filtrates of each sample were evaporated to dryness under reduced pressure at 20–50 kPa and temperature at 40⬚C using a Buchi rotary evaporator (Flawil, Switzerland). Stock solutions (1 mg/mL) of each sample were prepared in methanol and filtered through a 0.22 m PVDF membrane (Merck Millipore, Darmstadt, Germany).
2.3 Preparation of standard solutions Stock solutions of berberine, palmatine, jatrorrhizine, tetrahydroberberine, tetrahydropalmatine, magnoflorine, isocorydine, glaucine and chlorogenic acid were prepared separately in methanol (1000 g/mL). Then, methanol stock solutions containing the nine analytes were prepared and diluted in appropriate concentration to yield a series of concentrations, within the ranges from 0.5 to 1000 ng/mL. The calibration curves were constructed by plotting the value of peak areas versus the value of concentrations of each analyte. All stock solutions were stored at –20⬚C until use.
2.4 Instrumentation Quantitative analysis was performed on a 4000 QTRAPTM MS/MS system, hybrid triple quadrupole-linear ion trap mass spectrometer (Applied Biosystem; Concord, ON, Canada), hyphenated with a Waters ACQUITY UPLCTM system (Waters; Milford, MA, USA) via an pneumatically-assisted electrospray www.jss-journal.com
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Figure 1. Structures and MS/MS spectrum of (A) Chlorogenic acid; (B) Magnoflorine; (C) Isocorydine; (D) Glaucine; (E) Jatrorrhizine; (F) Tetrahydropalmatine; (G) Tetrahydroberberine; (H) Palmatine; and (I) Berberine.
(Turbo VTM source with TurboIonSprayTM probe and APCI probe) interface. The Waters ACQUITY UPLCTM system was equipped with a binary solvent manager, sample manager, column compartment and photodiode array detector (PAD).
parameters are shown in Table 1. Quadrupole 1 and quadrupole 2 were maintained at unit resolution.
2.5 Data processing 2.4.1 UHPLC conditions Chromatographic separation of compounds was obtained with an ACQUITY UPLC CSHTM C18 column (1.7 m, 2.1 × 100 mm) operated at 25⬚C.The mobile phase, which consisted of a 0.1% formic acid aqueous solution (A) and acetonitrile (B), was delivered at a flow rate of 0.3 mL/min under a gradient program: 5% (B) initial to 1.0 min, 5–20% (B) from 1.0 to 2.0 min, 20–30% (B) from 2.0 to 3.0 min, 30–90% (B) from 3.0 to 4.0 min, maintained at 90% (B) from 4.0 to 5.0 min and back to initial conditions from 5.0 to 5.5 min. The sample injection volume used was 1 L. 2.4.2 MS conditions Positive pneumatically assisted ESI was used for sample introduction, ionization process and operated in the MRM R probe was vertically positioned mode. A Turboionspray 11 mm from the orifice and charged with 5500 V. Source dependent parameters such as temperature, GS1, GS2 and curtain gas were set at 550⬚C, 50 psi, 50 psi, and 20 psi, respectively. The collision-activated dissociation gas was set as medium and the interface heater was on. High-purity nitrogen was used for all the processes. The compound-dependent C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
AB Sciex Analyst software version 1.5.1 was used to control the LC–MS/MS system and for data acquisition and processing. All the statistical calculations related to quantitative analysis were performed on Graph Pad Prism software version 5.
3 Results and discussion 3.1 Optimization of UHPLC–ESI-MS/MS condition For quantification of chlorogenic acid and isoquinoline alkaloids in the genus Berberis, standards chlorogenic acid (1), magnoflorine (2), isocorydine (3), glaucine (4), jatrorrhizine (5), tetrahydropalmatine (6), tetrahydroberberine (7), palmatine (8), and berberine (9) were studied [2, 7]. Preliminary, compounds dependent MRM parameters (DP: declustering potential; EP: entrance potential; CE: collision energy; and CXP: cell exit potential) were optimized to achieve the most specific and stable MRM transitions (precursor-toproduct ions). To accomplish this target, each selected analyte (10 ng/mL) was directly injected into the ESI source of mass spectrometer by continuous infusion. The optimized mass spectrometric conditions for the detection of analytes www.jss-journal.com
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Table 1. The optimized compound dependent MRM parameters and transitions for each analyte in the UPLC-ESI-MS/MS analysis
Peak No.
RT (min)
Analyte
Precursor ion (Da)
DP (V)
EP (V)
CE (eV)
Quantifiera)
Qualifiera)
1 2 3 4 5 6 7 8 9
1.24 1.39 1.61 1.81 1.94 1.96 2.09 2.21 2.38
Chlorogenic acid Magnoflorine Isocorydine Glaucine Jatrorrhizine Tetrahydropalmatine Tetrahydroberberine Palmatine Berberine
355 [M+H]+ 342 [M]+ 342.1 [M+H]+ 356.3 [M+H]+ 338 [M]+ 356.2 [M+H]+ 340 [M+H]+ 352.2 [M]+ 336 [M]+
50 50 73 101 50 86 55 32 40
12 10 4.5 8 10 7 10 10 10
15 27 27 20 55 35 35 40 45
355→163 (12) 342→296.7 (20) 342.1→279.2 (12) 356.3→325.3 (16) 338→307.3 (15) 356.2→192.1 (7) 340→176 (9) 352.2→336 (15) 336→320 (5)
355→145 (15) 342→282 (12) 342.1→311.1 (8) 356.3→294.1 (15) 338→322.1 (11) 356.2→165.1 (5) 340→149.1 (5) 352.2→308.1 (16) 336→292.2 (8)
RT: Retention Time; DP: Declustering Potential; EP: Entrance Potential; CE: Collision Energy; a) Cell Exit Potential (CXP in V) is given in brackets
were achieved in positive-ion mode. Analytes 1, 3, 4, 6, and 7 showed [M+H]+ ion while analytes 2, 5, 8, and 9 showed [M]+ ion in Q1 MS scan and, therefore, selected as the precursor ions for MS/MS fragmentation analysis of analytes. DP and EP were optimized to obtain the maximum sensitivity of [M+H]+ and [M]+ ions in Q1 multiple ion scan (Q1 MI). Identification of the fragment ions and selection of CE for each analyte were carried out in the product ion scan. Their MS/MS spectra are shown in Fig. 1. Furthermore, CE and CXP were optimized for two transitions to acquire the maximum sensitivity in the MRM scan but only one transition was monitored in quantitative analysis of samples due to the lack of sensitivity of the other observed product ions. The most prominent MRM transition was selected as a quantifier and other one as a qualifier in specificity test only. Table 1 shows the optimized parameters for all the analytes. The source dependent parameters (curtain gas, GS1, GS2, and ion source temperature) were optimized for the highest abundance of precursor-to-product ions in the FIA by operating UHPLC. With respect to UHPLC separation, different parameters were examined and compared. Acetonitrile displayed stronger elution ability, which shortened the elution time with good resolving power than methanol in the test. Therefore, aqueous acetonitrile was chosen as the mobile phase other than aqueous methanol. Formic acid was used as a modifier to obtain desirable peak shapes and degree of separation than acetic acid (Supporting information; Fig. S1). Different flow rates (0.2, 0.3, 0.4, 0.5 mL/min) and column temperatures (20, 25, 30, or 35⬚C) were examined in the test. The results showed that the desirable resolution in shortest analysis time were achieved when the aqueous formic acid (0.1%) and acetonitrile system was used at a flow rate of 0.3 mL/min and column temperature at 25⬚C. MRM extracted ion chromatogram of analytes are shown in Fig. 2.
3.2 Analytical method validation The proposed UHPLC–MRM method for quantitative analysis was validated according to the guidelines of international
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conference on harmonization (ICH, Q2R1) by determining specificity, linearity, lower limit of detection (LOD), lower limit of quantification (LOQ), precision, solution stability, and recovery [22]. 3.2.1 Specificity Exact identification of each analyte in the samples is a prerequisite for successful quantification. UHPLC–ESI-MS/MS method was employed to analyze the two MRM signals (quantifier and qualifier) in the sample matrix. All of the peaks of the target analytes in B. asiatica fruit and B. jaeschkeana root were unambiguously identified by comparison of retention time, quantifier and qualifier transitions in MRM chromatogram of standards (Table 1). 3.2.2 Linearity, LODs, and LOQs Linearity, LODs, and LOQs of nine analytes were determined by serial dilution of sample solutions using the described UHPLC–ESI-MS/MS method. The linearity of calibration was performed by the analyte peak area (y) versus concentration (x) and constructed with a weighting (1/x2 ) factor by least-squares linear regression. Good linear relationship between the peak area and concentration was obtained for each of the nine analytes over the tested concentration range with a correlation coefficient (R2 ) of ࣙ0.9989. LODs and LOQs were the concentrations of the compound at which S/Ns were detected as 3:1 and 10:1, respectively (Table 2). The LOD for each analyte varied from 0.12–2.92 ng/mL and LOQ from 0.26–8.79 ng/mL. 3.2.3 Precision, stability, and accuracy Precision of the developed method was determined at three different levels i.e. intraday variation, interday variation, and reproducibility, according to ICH guideline [22]. Intra- and inter-day variations of the method were determine by analyzing known concentrations of the nine analytes in the six replicates during a single day and by triplicating the
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Figure 2. Extracted ion chromatograms of (A) Chlorogenic acid; (B) Magnoflorine; (C) Isocorydine; (D) Glaucine; (E) Jatrorrhizine; (F) Tetrahydropalmatine; (G) Tetrahydroberberine; (H) Palmatine and; (I) Berberine, in MRM mode.
Table 2. Linearity, LOD, LOQ, precisions, stability and recovery results of investigated components
Linearity
Precision RSD (%)
Recovery
Analytes
Regression equation
R2
Linear range (ng/ml)
LOD (ng/ml)
LOQ (ng/ml)
Intraday (n = 6)
Interday (n = 5)
Reproducibility (n = 5)
Stability
Mean (n = 6)
RSD (%)
1 2 3 4 5 6 7 8 9
y = 610*x -19.7 y = 743*x - 43.7 y = 6060*x - 266 y = 3690*x - 49.8 y = 1100*x - 7.66 y = 14600*x - 448 y = 15200*x + 2350 y = 989*x + 870 y = 1030*x - 487
1.0000 0.9999 0.9991 0.9990 0.9988 0.9998 0.9998 0.9992 0.9989
0.5-1000 0.5-250 0.5-1000 0.5-1000 0.5-1000 0.5-200 0.5-200 0.5-50 1-100
0.12 0.22 0.36 0.19 0.87 0.08 0.48 2.92 1.53
0.35 0.67 1.08 0.56 2.65 0.26 1.46 8.79 4.64
2.40 1.42 1.48 0.65 1.53 2.10 1.13 0.40 1.52
2.39 2.25 1.65 0.74 0.96 2.37 2.05 0.89 1.68
1.07 0.41 1.11 0.69 1.02 0.75 0.38 3.1 0.75
2.82 2.64 1.98 2.21 2.02 3.27 3.80 1.38 1.72
91.2 97.49 99.6 101.5 99.01 102.1 102.7 101.3 98.6
1.2 2.3 0.92 1.49 1.32 1.6 1.03 0.74 1.11
y: peak area; x: concentration of compound (ng/ml); LOD: limit of detection: S/N = 3; LOQ: limit of quantification: S/N = 10
experiments on five successive days, respectively. RSD values for precision were in the range of 0.40–2.40% for intraday assays, 0.74–2.39% for interday assays (Table 2). Six different sample solutions of B. asiatica flower in parallel were extracted and analyzed with the proposed method to evaluate the reproducibility. The RSD values of the nine compounds were ࣘ 3.1%, which showed high reproducibility of the method. Stability of sample solutions stored at room temperature was investigated by replicate injections of the sample solution at 0, 2, 4, 8, 12, and 24 h. The RSD values of stability of the nine analytes were ࣘ 3.80% (Table 2). To evaluate the accuracy of this method, a recovery test was applied by the standard addition method. The mixed standard solutions with three
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different spike levels (low, middle and high) were added into a sample and analyzed using the above mentioned method in triplicate experiments. The recovery was calculated by the formula: recovery = (a–b)/c×100%, where a is the detected amount, b is the original amount and c is the spiked amount. The results showed that the developed analytical method was simple, reliable and reproducible with good recovery in the range of 91.2–102.7% (RSD ࣘ 2.3%) for all analytes (Table 2). In the term of LOD, LOQ, precision, stability and accuracy, this method is more sensitive compared to earlier reported methods for similar type of compounds using UHPLC–ESIMS/MS, HPLC–ESI-MS/MS, and HPLC–DAD methods [20, 21, 23, 24]. Therefore, this method was applied to quantify selected analytes in crude extract of B. species.
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Table 3. Contents (in mg/g) of nine bioactive compounds in the different parts of eight Berberis species
Analyte content (mg/g) Sample Name BAR
BAS
BC
BJ
BK
BL
BP
BU
Flower Leaf Root Stem Fruit Leaf Root Stem Leaf Root Stem Leaf Root Stem Leaf Root Stem Leaf Root Stem Shoot Fruit Leaf Root Stem Leaf Root Stem
1
2
3
4
5
6
7
8
9
Total (mg/g)
Total (% w/w)
191.3 38 nd nd 86.3 66.1 nd nd 21.8 nd nd 48.5 nd nd 104.1 nd nd 35.2 nd nd nd 38.4 189.4 nd 4.9 31.4 nd 3.5
0.5 0.4 6.8 26.3 1.8 0.4 17.8 33.8 0.5 16.4 65 0.8 42 66.4 0.8 20.6 34.2 0.3 7.8 64.3 7.28 2.9 1.1 79 285 0.7 53.95 184.1
1.3 nd 0.25 0.34 1.8 0.7 nd 0.5 0.16 0.3 0.9 nd 0.8 0.6 nd nd 0.45 nd nd 0.72 0.2 0.6 nd nd nd nd 0.53 0.31
0.4 nd nd nd 0.2 nd nd nd nd 0.06 0.05 nd 0.06 0.05 nd nd nd nd nd nd nd nd nd 0.06 0.06 nd 0.08 0.06
0.07 0.2 8.7 6.7 0.3 0.08 12.1 4.2 0.1 14.4 0.5 0.09 2.8 1.9 0.3 10.5 3.5 0.17 27.9 1.8 32.4 0.23 0.3 9.2 3.4 0.13 3.1 2
nd nd nd 0.28 0.37 0.1 nd 0.1 nd nd nd 0.1 0.31 0.2 nd nd nd nd nd nd nd 0.1 nd nd nd nd nd nd
0.05 nd 0.003 0.5 0.1 nd 0.05 0.04 nd nd nd nd 0.18 nd nd nd nd 0.4 nd 0.15 0.03 0.002 nd nd nd nd nd nd
0.004 bdl 78.9 7.1 0.01 0.002 89.2 11.4 0.02 38.2 0.13 0.03 5.1 1.6 0.3 87.4 4.2 2.4 53.8 1.8 55.43 1.3 0.6 5.7 1.14 bdl 8.1 5.7
1.7 1.9 121.8 102.3 3 1.6 78.1 29.9 2 106 2 1.9 11.2 6.4 7.1 144.9 29.5 3.6 172.8 18.2 158.5 3.4 3.6 24.1 5.2 0.91 43.8 14.6
195.3 40.5 216.5 143.5 94.2 69 197.3 79.9 24.6 175 68.6 51.4 62.5 77.2 112.6 263.4 71.9 42.1 262.3 87 253.8 46.9 195 118.1 299.7 33.1 109.6 210.3
19.53 4.05 21.65 14.35 9.42 6.9 19.73 7.99 2.46 17.5 6.86 5.14 6.25 7.72 11.26 26.34 7.19 4.21 26.23 8.7 25.38 4.69 19.5 11.81 29.97 3.31 10.96 21.03
nd: not detected; bdl: below detection level
3.3 Method application The newly developed UHPLC–ESI-MS/MS method was applied successfully to determine all the nine bioactive compounds in the ethanolic extracts of eight Berberis species. The content of each analyte was calculated from the corresponding calibration curve and summarized in Table 3. The analytical results indicated significant variations of their contents among the samples. 3.3.1 Protoberberine alkaloids (PBAs) Five PBAs were quantified in eight Berberis species including different plant parts. In PBAs, the contents of quaternary PBAs (Berberine, Jatrorrhizine, and Palmatine) were detected in almost all the samples. They were identified as the major constituent in root part of all the Berberis species and the least amount was detected in leaf part (Table 3). Berberine was found the most abundant PBAs in Berberis species and it was present in the highest amount in BL root (172.8 mg/g) followed by BL shoot (158.5 mg/g), BK root (144.9 mg/g), BAR root (121.8 mg/g), and BC root (106 mg/g). It was also
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showed that BL root, BL shoot, BK root, and BC root may be used for swapping of B. aristata (BAR) in herbal industries [2]. Jatrorrhizine was detected in higher quantity in BL shoot (32.4 mg/g), BL root (27.9 mg/g), and BC root (14.4 mg/g) while palmatine was detected in higher quantity in BAS root (89.2 mg/g) and BK root (87.4 mg/g). Tetrahydroprotoberberine alkaloids were detected in lesser quantity in BAR, BAS, BJ, BL, and BP and it was not detected in BC, BK, and BU. Tetrahydroberberine and tetrahydropalmatine were detected maximum in BAR stem (0.5 mg/g) and BAS fruit (0.37 mg/g), respectively.
3.3.2 Aporphine alkaloids Magnoflorine, isocorydine, and glaucine were detected higher in the stem part of Berberis species. Magnoflorine was detected as major constituent in BP stem (285 mg/g) and BU stem (184.1 mg/g) while isocorydine and glaucine were maximum in BAS fruit (1.8 mg/g) and BAR flower (0.4 mg/g), respectively.
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J. Sep. Sci. 2015, 38, 2007–2013
3.3.3 Acid Chlorogenic acid was a major constituent in flower and leaf part of Berberis species. It was detected in significant amounts in BAR flower (191.3 mg/g), BP leaf (189.4 mg/g), and BK leaf (104.1 mg/g) and was absent in root part of all the Berberis species.
Liquid Chromatography
2013
[4] Anonymous, The Wealth of India, A Dictionary of Indian Raw Materials, Vol 2 (B). Publications and Information Directorate CSIR, New Delhi, 1988, pp 114–118. [5] Tiwari, B. K., Khosa, R. L., Int. J. Pharm. Pharm. Sci. 2010, 2, 92–97. [6] Grycova, L., Dostal, J., Marek, R., Phytochemistry 2007, 68, 150–175. [7] Karimov, A., Chem. Nat. Compd. 1993, 29, 415–438.
4 Concluding remarks The present study involved quantification of nine bioactive compounds (protoberberine alkaloids, aporphine alkaloids, and chlorogenic acid) in different plant parts of eight Berberis species. An UHPLC–ESI-MS/MS method under MRM mode was successfully developed and validated as per ICH guidelines and it was applied in eight Berberis species. Results indicate that all the nine bioactive compounds are the most abundant in BP stem. Chlorogenic acid, magnoflorine, and berberine are found abundant in leaf, stem and root part, respectively, of all the Berberis species. Results also indicated that the roots of B. lycium, B. koehneana, and B. chitria may be used for swapping of B. aristata. This information was lacking in the literature and it could be helpful for better swapping of Berberis species. Awantika Singh is thankful to UGC, New Delhi, for a fellowship. A grateful acknowledgement is made to the Department of Science and Technology for Grant SB/EMEQ-095 and SAIFCDRI, Lucknow, India, where all the mass spectral analysis were carried out. This is CDRI communication no 8975. The authors have declared no conflict of interest.
5 References
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