Accepted Manuscript

Title: Simultaneous quantitative determination of bioactive

terpene indole alkaloids in ethanolic extracts of Catharanthus
roseus (L.) G. Don by ultra high performance liquid
chromatography-tandem mass spectrometry

Authors: Sunil Kumar, Awantika Singh, Brijesh Kumar,

Bikarma Singh, Lal Bahadur, Mohan Lal

PII: S0731-7085(17)32037-X
DOI: https://doi.org/10.1016/j.jpba.2017.12.040
Reference: PBA 11693

To appear in: Journal of Pharmaceutical and Biomedical Analysis

Received date: 9-8-2017

Revised date: 18-12-2017
Accepted date: 19-12-2017

Please cite this article as: Sunil Kumar, Awantika Singh, Brijesh Kumar, Bikarma
Singh, Lal Bahadur, Mohan Lal, Simultaneous quantitative determination of bioactive
terpene indole alkaloids in ethanolic extracts of Catharanthus roseus (L.) G.Don by
ultra high performance liquid chromatography-tandem mass spectrometry, Journal of
Pharmaceutical and Biomedical Analysis https://doi.org/10.1016/j.jpba.2017.12.040

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Simultaneous quantitative determination of bioactive terpene indole alkaloids in ethanolic

extracts of Catharanthus roseus (L.) G. Don by ultra high performance liquid

chromatography-tandem mass spectrometry

Sunil Kumara, Awantika Singha,b, Brijesh Kumara,b,*, Bikarma Singhc, Lal Bahadurd, Mohan Lale

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a
Sophisticated Analytical Instrument Facility, CSIR-Central Drug Research Institute, Lucknow-

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226031, Uttar Pradesh, India

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Academy of Scientific and Innovative Research (AcSIR), New Delhi-110025, India

c
Biodiversity and Applied Botany Division, CSIR-Indian Institute of Integrative Medicine, Canal

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Road Jammu-180001, India

d
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Plant Diversity, Systematics and Herbarium Division, CSIR-National Botanical Research
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Institute, Rana Pratap Marg, Lucknow-226001, Uttar Pradesh, India
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e
Division of Medicinal Aromatic & Economic Plants, CSIR-North-East Institute of Science &
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Technology (CSIR-NEIST) Jorhat 785 006, Assam, India

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*Corresponding author.
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E-mail: brijesh_kumar@cdri.res.in, gbrikum@yahoo.com (B. Kumar)

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Graphical Abstract
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Highlights
 A UHPLC-ESI-MS/MS method in MRM mode was developed and validated.
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 Validation was based on calibration curve, linearity, LOD, LOQ and precision.
 UHPLC-ESI-MS/MS method was successfully applied in ethanolic extracts of leaf, stem and root of 39
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samples of C. roseus.
 Serpentine was detected as one of the most abundant alkaloid amongst 10 alkaloids.
 PCA was discriminated all C. roseus samples collected from five states of India.
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Abstract
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A rapid, sensitive and reproducible method using ultra-high-performance liquid chromatography

coupled with electrospray ionization hybrid triple quadrupole-linear ion trap mass spectrometry
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(UHPLC-ESI-QqQLIT-MS/MS) in multiple reaction monitoring (MRM) mode was developed

and validated for simultaneous quantitation of anticancer (vincristine, vinblastine, vindesine),

antihypertensive (ajmaline, ajmalicine, reserpine), aphrodisiac (yohimbine), sedative (serpentine)

agents, dietary supplement (vinpocetine, yohimbine) and precursor of vinblastine (vindoline)

from crude extracts of Catharanthus roseus. The precursor to product ion transitions for these

compounds were observed at m/z 327→144, 355→144, 754→355, 353→144, 349→317,

825→225, 811→224, 458→188, 351→280 and 609→195, respectively in positive ionization

mode. Chromatographic separation of all targeted TIAs was performed on ACQUITY UPLC

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BEH™ C18 column (1.7 μm, 2.1 mm × 50 mm). The calibration curves were linear within the

concentration range 0.5-1000 ng/mL and correlation coefficients (R2) were closer to 1. Limit of

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detection (LOD) and limit of quantitation (LOQ) ranged from 0.039–0.583 ng/mL and 0.118–

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1.767 ng/mL, respectively. The intra-day (0.23-2.71% RSD) and inter-day (0.40-2.90% RSD)

precision, stability (0.69-3.45% RSD) and recovery (99.63–104.30% ±%RSD  3.03%) were

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acceptable indicating good accuracy of the developed method. The method was successfully
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applied in ethanolic extracts of 39 samples of C. roseus parts (leaf, stem and root) collected from
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five different locations in India. Serpentine was detected as one of the most abundant TIA.
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Principal component analysis (PCA) was able to successfully discriminate among C. roseus

samples on the basis of content of targeted TIAs.

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Keywords: Catharanthus roseus; Terpene Indole alkaloids; Multiple reaction monitoring

1. Introduction
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The ajmaline, yohimbine vindesine, ajmalicine, serpentine, vincristine, vinblastine,

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vindoline, vinpocetine and reserpine are naturally occurring bioactive terpene indole alkaloids

(TIAs). They are found in family Apocynaceae (Catharanthus and Rauwolfia spp. (Fig. 1) [1-2].
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Vincristine, vinblastine and vindesine have been reported as potent chemotherapeutic or

anticancer agent. Vincristine and vinblastine used as drug for the treatment of various

carcinomas, including pediatric acute leukemia, malignant lymphoma, and breast cancer. They

are also useful to treat adult Hodgkin’s lymphomas, non-Hodgkin’s lymphomas,

rhabdomyosarcoma, neuroblastoma, Wilms’ tumor, and multiple myeloma [3-5]. Vindoline is

the precursor of vinblastine [5], similarly serpentine as a source of ajmalicine and also exhibited

antiarrhythmics activity [6]. Vinpocetine is a synthetic derivative of vincamine. It has been

reported to have cerebral blood-flow enhancing and neuroprotective effects, and used as drug for

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the treatment of cerebrovascular disorders and age-related memory impairment [7-8]. It is

marketed as dietary supplements in United States (US) [9]. Ajmaline, ajmalicine, reserpine are

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antihypertensive agents and used extensively for the treatment of cardiovascular diseases such as

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high blood pressure [11-14]. Yohimbine is a potent aphrodisiac agent marketed as a dietary

supplement for sexual enhancement [15-16].

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C. roseus (L.) G. Don formerly known as Vinca rosea is well-known medicinal and
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ornamental plant belonging to Apocynaceae family. Eight Catharanthus spp. are reported out of
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these, seven are endemic to Madagascar, while C. roseus is widely distributed throughout the
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world. According to Indian Traditional Medicine (Ayurveda) and Traditional Chinese Medicine
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(TCM), the extract of C. roseus is used for the treatment of several diseases, such as diabetes,
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malaria and Hodgkin's lymphoma [1-5]. Due to commercialization of bioactive TIAs especially

vincristine and vinblastine from C. roseus several analytical methods have been applied for
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identification and quantitation during last two decades. Vindoline, vindolidine, vincristine and

vinblastine reported from crude extracts of C. roseus using high-performance liquid

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chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) [17-

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24]. Gas chromatography mass spectrometry (GC-MS) has been utilized for volatile compounds

[25-28]. Large numbers of papers on quantitative analysis of TIAs from crude extracts of leaf

and root of C. roseus and biological samples have been published using high-performance thin

layer chromatography (HPTLC) and high-performance liquid chromatography (HPLC) [29-32].

Semi-quantitative analysis of vincristine and vinblastine from somatic embryogenesis of C.

roseus has been reported using flow-injection electrospray ionization mass spectrometry (ESI-

MS) [19]. Similarly, quantitative analysis of vinblastine, vindoline and catharanthine has been

reported by capillary electrophoresis (CE) and capillary electrophoresis–mass spectrometry (CE-

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MS) [33-36]. Liu et al. [37] conducted quantitative analysis of tabersonine, serpentine, vindoline,

catharanthine, tryptamine, and vincamine in C. roseus and V. minor using HPLC-MS/MS. The

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quantification of vinpocetine in dietary supplements has been reported by Avula et al [9] using

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ultra high-performance liquid chromatography with diode-array detection (UHPLC-PDA).

Similarly, vinblastine, vindoline, ajmalicine, catharanthine, and vinleurosine have been

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quantified in C. roseus using liquid chromatography-ion trap mass spectrometry (LC-IT-MS)

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[21]. High-performance liquid chromatography with diode-array detection (HPLC–DAD) has
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been used for the identification and quantification of 19-S-vindolinine, vindolinine, ajmalicine
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and ajmalicine isomer, tabersonine, catharanthine, serpentine and a serpentine isomer in C.

roseus roots. [22]. Supercritical fluid extraction and liquid chromatography-electrospray mass
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analysis have been used for the analysis of vinblastine from C. roseus [38]. Yang et al [39]
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described simultaneous estimation of vinpocetine, its metabolites and apovincaminic acid, in

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beagle plasma by liquid chromatography- tandem mass spectrometry (LC-MS/MS). A solid-

liquid extraction method was used for quantitative determination of vinpocetine and its
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metabolite in rat plasma by LC-MS/MS [40]. Liquid chromatography-fourier transform ion

cyclotron resonance tandem mass spectrometry (LC–FTICR–MS/MS) was applied for analysis
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of monoterpene indole alkaloids in C. roseus [41]. Hydrophilic interaction liquid

chromatography separation mode (HILIC) coupled to electrospray tandem mass spectrometry

(ESI-MS/MS) have been used for quantitative analysis of vindesine in human plasma [4].
Quantification of catharanthine, taberosinine, vindoline, vinblastine and vincristine was achieved

by LC-MS/MS analysis using selected reaction monitoring (SRM) [42]. Recently identification,

characterization and distribution of 72 terpene indole alkaloids were reported in ethanolic

extracts of Catharanthus roseus using HPLC/ESI-QTOF-MS/MS and the study of their

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geographical variation [43]. Reported HPLC and HPTLC methods are comparatively less

sensitive and less accurate than LC-MS/MS. Therefore, HPLC and HPTLC are not very suitable

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for quantitation of vincristine and vinblastine in crude extracts due to lack of sensitivity and

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accuracy [44-45]. It is very important to establish a more accurate and sensitive UHPLC-ESI-

QqQLIT-MS/MS method in MRM mode for the rapid simultaneous quantitative determination of

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bioactive TIAs in C. roseus.

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The aim of this work was to develop and validate a method in MRM mode using ultra-
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high-performance liquid chromatography coupled with electrospray ionization-hybrid triple
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quadrupole-linear ion trap mass spectrometry (UHPLC-ESI-QqQLIT-MS/MS) for rapid

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simultaneous quantitative determination of ten TIAs in ethanolic extracts of 39 samples of C.

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roseus collected from five states namely Jammu and Kashmir, Uttar Pradesh, Assam, Arunachal

Pradesh and Bihar in India. PCA was used to study the variation in all samples.
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2. Material and Methods

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2.1. Chemicals

AR grade ethanol was purchased from Merck Millipore (Darmstadt, Germany) and used
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for phytochemical extraction. LC–MS grade acetonitrile, methanol and formic acid were

purchased from Sigma-Aldrich (St. Louis, MO, USA) and used throughout the LC-MS study.

Ajmaline, vindesine, ajmalicine, serpentine, vincristine, vinblastine, vindoline, vinpocetine and

reserpine were purchased from Sigma-Aldrich. Yohimbine was purchased from ChemFaces

(Wuhan, Hubei, China). Ultra-pure water obtained from Direct-Q system (Millipore, Billerica,

MA, USA) was used throughout the analysis. All standards have purity  95%

2.2. Plant materials

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Leaves, stems and roots of C. roseus were collected from Jammu (Jammu and Kashmir),

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Arrah (Bihar), Itanagar and Pasighat (Arunachal Pradesh), Jorhat (Assam) and Lucknow (Uttar

Pradesh) as given in Table 1. C. roseus was identified by Dr. Bikarma Singh (Scientist, CSIR-

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IIIM, Jammu). The plant materials were shade dried and grinded using a grinding machine

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(Decibel, Lab Willey Griender, New Delhi, India)

2.3. Extraction N
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The powdered plant material (~10 g dry weight of each) was placed in conical flask with
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(500 mL) ethanol (100 mL) followed by sonication for 30 min at 30˚C using ultrasonicator (53
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KHz, Bandelin SONOREX, Berlin) and then kept at room temperature for 24 h. The extracts
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were filtered through filter paper (Whatman No. 1) and filtrates were collected. The residues

were re-extracted with fresh solvent thrice. The combined filtrates were evaporated to dryness
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under reduced pressure at 20-50 kPa at 40˚C using a rotary evaporator (Buchi Rotavapor-R2,

Flawil, Switzerland).
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2.4. Sample preparation for standards and extracts

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All standards and extracts were weighed accurately (approximate 1 mg) and separately.

Each sample was dissolved in methanol according to weight for preparation of 1 mg/mL stock

solutions. After that each solution was sonicated for 30 min and filtered through a 0.22-µm

PVDF membrane (Millex-GV, PVDF, Merck Millipore). Therefore, obtained stock solutions
were 1 mg/mL. A series of separate and mixed standards (concentrations: 0.1-1000 μg/mL) were

prepared from 1 mg/mL stock solutions for plotting the calibration curve. Each standard stock

solution (10 μg/mL) was diluted with methanol to final working solutions during parameters

optimization. All stock solutions and extracts were stored at -4˚C.

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2.5. Instrumentation and analytical conditions

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The UHPLC–ESI-MS/MS analysis was performed on a Waters Acquity UPLCTM system

(Waters, Milford, MA, USA) interfaced with a hybrid linear ion trap with triple-quadrupole mass

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spectrometer (API 4000 QTRAP™ MS/MS system from AB Sciex, Concord, ON, Canada)

equipped with an electrospray (Turbo V) ion source. The Waters Acquity UPLCTM system was

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equipped with a binary solvent manager, sample manager, column oven and photodiode array
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detector (PDA). Analyst software version 1.5.1 AB (Sciex) was used for data acquisition and
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processing.

2.5.1. Chromatographic conditions

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ACQUITY UPLC BEH™ C18 column (1.7 μm, 2.1 mm × 50 mm) was used for the
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chromatographic separation of TIAs at 35°C column temperatures. Formic acid (0.1%) (A) in
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water and acetonitrile (B) were used as a mobile phases with the flow rate of 0.3 mL/min under a

gradient program: 15-15% (B) initial to 0.5 min, 15-24% (B) from 0.5-1.5 min, 24–27% (B)
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from 1.5-2.5 min, 27–40% (B) from 2.5-5.0 min, 40–45% (B) from 5.0-6.50 min, 45-50% (B)

from 6.50-7.50 min, 50-98% (B) from 7.50-8.0 and held 98% for 1.5 min., then returned back to
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initial condition. The sample injection volume was 4.0 μL.

2.5.2. Mass spectrometric conditions

 Precursor ions scan was performed in positive ionization ESI mode from m/z 100-1000

for screening or detection of targeted TIAs. Source dependent parameters, ion spray voltage,

source temperature (TEM), nebulizer gas (GS 1), heater gas (GS 2) and curtain gas were

optimized at 5500 V, 550°C, 50 psi, 50 psi and 20 psi, respectively. Nitrogen used as nebulizer,

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heater, curtain, collision-activated dissociation gas was set as medium and the interface heater

was on. Each standard solution (10 μg/mL) was taken separately in methanol with the rate at 10

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μL/min for optimization of mass spectrometric conditions by direct infusion using a Harvard ‘22’

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syringe pump (Harvard Apparatus, South Natick, MA, USA). The most abundant fragment ions

were selected for MRM transition. The compound-dependent parameters are shown in Table 2.

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3. Results
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3.1. Optimization of MS/MS conditions for standards
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The compound-dependent MRM parameters such declustering potential (DP), entrance potential

(EP), collision energy (CE) and cell exit potential (CXP) for ajmaline yohimbine, ajmalicine,
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serpentine and reserpine were used in present study as previously optimized for simultaneous
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quantitative determination of TIAs from ethanolic extracts of seven Rauwolfia species [46].
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Similarly, in the present study, DP, EP, CE and CXP for vindesine, vincristine, vinblastine,

vindoline and vinpocetine were optimized. To achieve highest abundance of precursor-to-product

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ions the source parameters such as curtain gas, GS1, GS2, and ion source temperature were

optimized. The MS spectra of vindesine, vincristine, vinblastine, vindoline and vinpocetine

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showed protonated precursor ion as [M+H]+ at m/z 754, 825, 811, 458 and 351, respectively in

Q1 scan. The [M+H]+ ion of vindesine, vincristine, vinblastine, vindoline and vinpocetine

yielded abundant product ions at m/z 355, 224, 225, 188 and 280 in Q2 scan and these were

selected for MRM transitions for quantitation (Fig. 2).

3.2. Optimization of UHPLC conditions

Vinblastine and vincristine have approximately the same polarity due to their structural

similarities (Fig. 1). Hence, column and mobile phase selection were critical to separate these

analytes properly. Two type of reverse phase columns, Acquity UPLC CSHTM C18-column (2.1

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mm ×100 mm, 1.7 μm) and Acquity UPLC BEH™ C18-column (2.1 mm × 50 mm,1.7 μm),

were tried but satisfactory separation was achieved only on Acquity UPLC BEH C18 column (2.1

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mm × 50 mm, 1.7 μm). Similarly, different concentrations (0.05%, 0.1% and 0.2%) of formic

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acid were checked to optimize ionization. A solvent system of 0.1% formic acid and acetonitrile

was found optimum for the separation of these analytes. Curtain gas, GS 1, GS 2 and ion source

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temperature were also optimized to obtain highest abundance of precursor and product ions.
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Different column temperatures (20, 25, 30 and 35°C) and flow rates (0.20, 0.25, 0.30, 0.35, 0.40,
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0.45 and 0.50 mL/min) were also examined. Satisfactory resolution in minimum analysis time
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was achieved at a flow rate of 0.3 mL/min at 35°C column temperature. Hence, a mobile phase
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consisting of 0.1% formic acid and acetonitrile at the flow rate 0.3 mL/min at 35°C on Acquity
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UPLC BEH™ C18 column (2.1 mm × 50 mm, 1.7 μm) was finally selected for analysis. MRM

extracted ion chromatograms (EICs) and total ion chromatogram (TIC) of analytes are shown in
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Fig. 3 and Fig. 4.

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3.3. Analytical method validation

Calibration curves, lower limit of detection (LOD), lower limit of quantification (LOQ),
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precision, solution stability and recovery were determined according to international conference

on harmonization (ICH, Q2R1) [47] to validate UHPLC-ESI-QqQLIT-MS/MS method.

3.3.1. Linearity, LOD and LOQ

 The calibration curves were constructed between peak areas ratio versus the

concentration of analytes solution. The linear regression equations and their correlation

coefficients are listed in Table 3. The estimated correlation coefficients (R2) ranged from 1-

0.9931. Finally, linear regression equations and their correlation coefficients showed good

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linearity for all analytes. The LOD and LOQ were in agreement with the baseline as a signal-to-

noise ratio (S/N=3/10) of 3 and 10, respectively. The LODs and LOQs for all the analytes ranged

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from 0.039–2.121 ng/mL and 0.118–7.375 ng/mL, respectively.

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3.3.2. Precision, stability and recoveries

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The intra-day and inter-day precisions were examined by six replicates during a single

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day and in triplicate for three consecutive days for each analytes. The %RSD for intra-day and
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inter-day precision ranged from 0.23-2.71% and 0.40-2.90%, respectively. Replicate injections
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of the sample solution were examined during 0, 2, 4, 8, 12 and 24 h at room temperature to

investigate the stability analytes in solution. The %RSD values for repeats were found between
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0.69-3.45%. The average recoveries were determined by spiking known amounts of all the
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analytes (high, middle and low) into the sample. The recovery was calculated using formula:

recovery = (a − b)/c×100%, where ‘a’ is the detected amount, ‘b’ is the original amount and ‘c’
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is the spiked amount. The recoveries of the targeted analytes ranged from 99.63–104.30% with
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RSD  3.03% which promising the accuracy of the present method (Table 3).

3.4. Application: Quantitation of TIAs in C. roseus

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To check the utility and performance of the developed and validated method, it was

applied for quantitative analysis of 10 TIAs in ethanolic extracts of 39 samples of C. roseus leaf,

stem and root. The content of ajmaline, yohimbine, vindesine, ajmalicine, serpentine, vincristine,
vinblastine, vindoline, vinpocetine and reserpine are presented in Table 4. Serpentine was

detected as the most abundant among all the targeted analytes. The highest content of serpentine

was obtained in Uttar Pradesh stem sample C-5S (50.078 mg/g), followed by Bihar stem sample

CR-3R (49.851 mg/g) and Arunachal Pradesh root sample C-44R (49.337 mg/g). Present results

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showed that serpentine content was found highest amongst quantified compounds in the earlier

reports in aqueous extract of C. roseus by HPLC–DAD–ESI-MS/MS analysis [22], Ajmalicine

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was detected in high abundance in root samples C-11R (17.675 mg/g), C-44R (16.766 mg/g) and

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CR-3R (15.630 mg/g. Likewise high content of vincristine was observed in CR-4R (0.659 mg/g)

followed by C-41L (0.523 mg/g) and CR-2L (0.510 mg/g), whereas approximately same content

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of vinblastine was detected in C-44R (1.537) and CR-4R (1. 638 mg/g). Similarly, CR-41L

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(1.293) and CR-2R (1.284) gave approximately same content of vinblastine. High content of
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vindoline was detected in CR-2L (19.463 mg/g) followed by CR-3L (18.540 mg/g) while
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samples C-2R (2.433 mg/g), C-5R (2.263 mg/g) and C-44R (2.153 mg/g) have shown

approximately same content of yohimbine. High content of vindesine was detected in sample C-
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2R (3.247 mg/g) followed by C-44L (2.978 mg/g). Content of vindesine was ranged from 1.552-
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3.247 mg/g. Very low amounts of ajmaline (<0.140 mg/g) was detected in all the samples.
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Similarly, vinpocetine and reserpine were detected (< 0.056 mg/g) and (0.055 mg/g),

respectively.
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3.3. Principal component analysis

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PCA is one of the most widely used multivariate techniques useful in dealing with large data sets

for discrimination among the samples [50-52]. PCA was applied on the basis of content of 10

targeted TIAs (ajmaline, vindesine, ajmalicine, serpentine, vincristine, vinblastine, vindoline,

vinpocetine and reserpine). The content of TIAs having low contributions were dropped. The
PCA score plots of leaf (A), stem (B) and root (C) are shown in Fig. 5. The correlation of PC1

and PC2 showed 71.27 %, 62.66 % and 66.63 % in leaf, stem and root on the basis of content of

content of 10, 8 and 7 TIAs, respectively.

PCA model of leaf (A) showed all samples closer to each other except samples from

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Jammu and Kashmir (Canal Road Jammu) and Arunachal Pradesh (Itanagar). Similarly, samples of

stem and root from all locations have not shown any correlation. Result obtained by UHPLC-

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ESI-QqQLIT-MS/MS combined with PCA was effective to study the variations and discriminated

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amongst the C. roseus samples.

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4. Discussion

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The serpentine was detected high amongst quantified compounds (Table 4), as reported
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in aqueous extract of C. roseus by HPLC–DAD–ESI-MS/MS analysis [21]. This may be due to
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in vivo conversion of ajmalicine into serpentine [22]. Chen et al. [34] have simultaneously

determined vinblastine (0.00619 mg/g) and its monomeric precursors vindoline (0.0718 mg/g)
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and catharanthine (0.134 mg/g) by CE-MS using extraction and fractionation procedure.
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Similarly, 0.1469 and 0.3169 mg/g of serpentine was reported in methanol extracts of V. minor
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and C. roseus, respectively by Liu et al. [37] using LC-MS/MS. Favretto et al. [19] conducted

semi-quantitative method by flow-injection electrospray ionization mass spectrometry and

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reported vinblastine and vincristine ranged from 0.001971-0.0145 mg/g and 0.000349-0.00222

mg/g respectively. Choi, et al. [38] quantified vinblastine and vincristine by LC-MS using
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supercritical fluid extraction with carbon dioxide-methanol-triethytamin extraction procedure in

aerial parts and root which showed high amount of vinblastine (0.4660 mg/g) than root (0.00630

mg/g) whereas vincristine was reported in trace. Digvijay et al. [48] have reported simultaneous

quantitation of TIAs and their precursors in both leaves and roots of different genotypes using
LC. The amount of serpentine was ranged from 0.0034% to 0.6490%, ajmalicine 0.0085% to

0.2910% (dry weight) in methanolic extracts. All these results were reported as percentage or

using different extractions and fractionations procedures with different type of solvent

combinations which make it difficult to compare with the present results [21-22, 31, 34, 38, 44,

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48-49]. In present work we have simultaneously determined targeted TIAs by UHPLC-ESI-

QqQLIT-MS/MS method in MRM mode from ethanolic extracts.

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The vincristine and vinblastine were fairly distributed in the Jammu-Kashmir and

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Arunachal Pradesh samples. Vincristine was detected high in the leaf and root of all the samples.

Jammu - Kashmir and Arunachal Pradesh samples showed high content of vincristine.

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Vinblastine was comparatively more abundant in the leaf and root samples of Jammu-Kashmir,

UP and Arunachal Pradesh samples.

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4. Conclusion

An UHPLC-ESI-QqQLIT-MS/MS method in MRM mode was developed and validated

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for the simultaneous determination of targeted TIAs. The method showed acceptable accuracy,
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repeatability and precision for both inter- and intra-day validation, with LODs, LOQs according
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to ICH guideline. Method was successfully applied in ethanolic extracts of 39 samples of leaf,

stem and root of C. roseus. The present results showed high content of vincristine and
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vinblastine in Jammu and Kashmir. Vinblastine was also found in Arunachal Pradesh. Variations

were observed among the samples collected from different geographical locations. To the best of
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our knowledge this is the first report of the simultaneous estimation of TIAs from C. roseus

extracts. PCA was able to successfully discriminate among all the samples collected from five

states of India.
 Conflict of interest Authors declare no conflict of interest.

References

Acknowledgement

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Grateful acknowledgement is made to SAIF, CSIR-CDRI, Lucknow, India where all

mass spectral studies were done. SK thanks to his Principal MK Govt. Degree College Ninowa,

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Farrukhabad (CSJM University Kanpur, India) for his support.Q. Pan, N.R. Mustafa, K. Tang,

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Y.H. Choi, R. Verpoorte, Monoterpenoid indole alkaloids biosynthesis and its regulation in

Catharanthus roseus: a literature review from genes to metabolites, Phytochem. Rev.15 (2016)

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Captions

SC
Fig.1. Structures of 10 targeted terpene indole alkaloids.

U
Fig. 2. MS/MS spectra of vindesine, vincristine, vinblastine, vindoline and vinpocetine.

N
Fig.3. Extracted ion chromatograms of ajmaline, yohimbine, vindesine, ajmalicine, serpentine,
A
vincristine, vinblastine, vindoline, vinpocetine and reserpine.
M

Fig.4. Total ion chromatogram (TIC) of ajmaline (1), yohimbine (4), vindesine (3), ajmalicine
D

(4), serpentine (5), vincristine (6), vinblastine (7), vindoline (8), vinpocetine (9) and reserpine
TE

(10).

Fig.5. PCA score plots of leaf (A), stem (B) and root (C) on the basis of content of 10 (ajmaline,
EP

vindesine, ajmalicine, serpentine, vincristine, vinblastine, vindoline, vinpocetine and reserpine),

CC

8 (ajmaline, yohimbine, vindesine, ajmalicine, serpentine, vincristine, vinblastine and reserpine)

and 7 (ajmaline, yohimbine, vindesine, serpentine, vincristine, vinblastine and reserpine) TIAs,
A

respectively.
A Figr-1

CC
EP
TE
D
M
A
N
U
SC
RI
PT
A Figr-2

CC
EP
TE
D
M
A
N
U
SC
RI
PT
A Figr-3

CC
EP
TE
D
M
A
N
U
SC
RI
PT
 Figr-4
A
CC
EP
TE
D
M
A
N
U
SC
RI
PT
A Figr-5

CC
EP
TE
D
M
A
N
U
SC
RI
PT
Table 1

Plant materials and its collection details.

S. State Code Voucher Place of Part Deposition of

No. number collection Voucher

PT
number

RI
1. Jammu and CSIR-IIIM),

Kashmir Jammu, India.

SC
CR-1L RRLH- Deichek, Leaf

U
52986 Akhnoor, , J&K

CR-1S
N Stem
A
M

CR-1R Root

CR-2L RRLH- IIIM Campus, Stem

D

9932 Jammu, J&K

TE

CR-2S Leaf
EP

CR-2R Root
CC

CR-4L RRLH- Canal Road Leaf

52952 Jammu, J&K

A

CR-4L Root

CR-4L Stem
2. Uttar Pradesh CSIR-NBRI,

Lucknow,
C-1L 102857 Ashiyana, Leaf
Uttar Pradesh,
Lucknow
India
C-1S Stem

PT
C-1R Root

RI
C-2L 102858 Leaf

SC
C-2S Stem

U
C-2R Root

C-3L 102859 N Leaf

A
C-3S Stem
M

C-3R Root
D

C-4L 102860 Leaf

TE

C-4S Stem
EP

C-4R Root
CC

C-5L 102861 Leaf

C-5S Stem
A

C-5R Root

C-6L 102862 Leaf

 C-6S Stem

C-6R Root

3. Assam C-11L RRLJM Mariani Leaf CSIR-NEIST)

1578 Jorhat, Assam,

PT
India.
C-11S Stem

RI
C-11R Root

SC
4. Arunachal CSIR- CSIR-

Pradesh NEIST, Jorhat,

U
Assam, India.
C-41L RRLJM N
Itanagar Leaf
A
1579
M

C-41S Stem
D

C-41R Root
TE
EP

C-44L RRLJM Pasighat Leaf

1581
CC

C-44S Stem
A

C-44R Root

5. Bihar
 CR-3L RRLH- Arrah, Bhojpur, Leaf CSIR-IIIM),

52987 Bihar Jammu, India.

CR-3S Root

CR-3R Stem

PT
RI
SC
U
N
A
M
D
TE
EP
CC
A
Table 2

Compound-dependent MRM parameters including declustering potential (DP), entrance potential

(EP), collision energy (CE) and cell exit potential (CXP) 10 analytes.

RT Precursor ion DP EP Product ion

PT
S. No. Analyte CE (eV) CXP
(min) (m/z) (V) (V) (m/z)

RI
1. 2.09 Ajmaline 327 [M+H]+ 141 9 50 40 144

SC
Yohimbin
2. 2.25 355 [M+H]+ 144 9 34 13 144
e

U
2.37 Vindesine 754 [M+H]+ 40 3 59 16 355
3.
N
A
Ajmalicin
4. 3.10 353 [M+H]+ 120 6 33 22 144
M

Serpentin
D

5. 3.26 349 [M]+ 128 5.7 34 15 317

e
TE

Vincristin
825 [M+H]+
EP

6. 3.35 15 10 69 9 225
e
CC

Vinblasti
7. 4.00 811 [M+H]+ 50 10 62 9 224
ne
A

8. 4.07 Vindoline 458 [M+H]+ 40 7 46 7.6 188

32
 Vinpoceti
9. 4.98 351 [M+H]+ 40 10 41 12 280
ne

10. 5.92 Reserpine 609 [M+H]+ 140 7.80 40 32 195

PT
RI
Table 3

SC
Regression equation, correlation coefficients, linearity ranges, limits of detection (LOD), limit of

quantitation (LOQ), intra-day and inter-day precisions, stability and recovery for 10 analytes.

U
Ser Precision

ial
Regressi
Linea
LOD N
LOQ
(%RSD) Stabili
Mean
A
r
Analyt on recoveries
no. R 2 ty
M
range Intra- Inter-
e Equatio ± RSD
day day %RSD
n (%)
D

(ng/mL)
(n=6) (n=3)
TE

1. Ajmali 4190x- 0.99 0.1- 0.03 100.64±0.

0.118 1.45 2.87 2.77
EP

ne 49.7 98 500 9 38

2. Yohim 1000x- 0.99 0.4- 0.29 101.26±0.

CC

0.892 2.58 2.72 2.45

bine 89.2 99 250 4 46
A

3. Vindes 3690x - 0.99 0.5- 0.04 101.42±1.

0.134 2.37 1.83 3.45
ine 49.8 982 1000 4 53

33
 4. Ajmali 4770x+ 0.99 0.1- 0.55 102.02±3.
1.666 2.59 2.52 2.94
cine 795 98 250 0 09

5. Serpen 1410x+ 0.99 0.5- 0.43 101.71±0.

1.326 0.23 0.40 0.69
tine 187 31 500 7 80

PT
6. Vincri 2921x- 0.99 0.04 99.70±1.8
1-75 0.147 0.74 0.58 0.90

RI
stine 43.2 88 88 6

SC
7. Vinbla 622x- 0.99 0.5- 0.38 101.24±0.
1.178 1.73 1.39 2.66
stine 73.3 93 100 8 68

U
8. Vindol 17100x 0.99 0.5- 0.25 99.63±0.2

ine +1320 98 75 4
N
0.771 0.51 0.41 1.22
2
A
9. Vinpo 1550x+ 1- 0.58 101.69±2.
M

1 1.767 1.44 2.44 2.66

cetine 274 500 3 07
D

10. Reserp 2480x+ 0.99 0.5- 0.34 104.30±2.

TE

1.032 2.71 2.90 1.97

ine 256 97 1000 0 04
EP

Table 4
CC

Alkaloid content (mg/g dry extract) of ethanol extracts from C. roseus samples collected at
A

different locations of India

34
 Samp
Stat
le mg/g (dry extract) ±SD
e
code

Ajmal Yohim Vindes Ajmali Serpent Vincri Vinbla Vindoli Vinpo Reserp

ine bine ine cine ine stine stine ne cetine ine

PT
Jam 0.021

RI
mu CR1 ±0.00 0.237± 1.679± 0.559± 3.441±0 0.189± 0.266± 13.317 0.001± 0.004±

& L 1 0.019 0.029 0.167 .122 0.094 0.048 ±0.075 0.074 0.002

SC
Kash
0.039
mir

U
±0.00 0.281± 1.872± 0.642± 11.946± 0.148± 0.289± 3.260± 0.007± 0.017±

CR1S 6 0.012 0.023 0.240 0.050 0.054 0.019 0.030 0.003 0.007

0.047
N
A
CR1 ±0.01 0.611± 2.181± 2.554± 36.684± 0.237± 0.969± 0.658± 0.005± 0.003±
M

R 0 0.008 0.060 0.299 0.096 0.117 0.038 0.091 0.003 0.002

D

0.018
TE

CR2 ±0.00 0.256± 2.158± 0.174± 10.090± 0.510± 0.726± 19.463 0.006±

L 6 0.041 0.030 0.074 0.057 0.161 0.244 ±0.025 bdl 0.001

EP

0.059

±0.02 0.243± 1.957± 0.438± 20.775± 0.183± 0.541± 1.675± 0.004± 0.006±
CC

CR2S 5 0.042 0.040 0.071 0.293 0.069 0.102 0.012 0.004 0.004

0.060
A

CR2 ±0.02 0.667± 1.830± 2.666± 39.078± 0.162± 1.284± 0.754± 0.020± 0.001±

R 0 0.010 0.053 0.200 0.070 0.036 0.022 0.037 0.008 0.005

35
 0.067

CR4 ±0.03 0.176± 1.748± 0.306± 2.868±0 0.287± 0.732± 7.173± 0.006± 0.012±

L 8 0.011 0.037 0.071 .039 0.019 0.049 0.240 0.002 0.002

0.054

±0.02 0.233± 2.244± 0.768± 19.775± 0.214± 0.665± 0.566± 0.002± 0.003±

PT
CR4S 9 0.011 0.018 0.038 0.160 0.038 0.044 0.114 0.001 0.002

RI
0.054

CR4 ±0.04 0.316± 2.328± 0.124± 4.927±0 0.659± 1.638± 9.690± 0.002± 0.001±

SC
R 2 0.014 0.023 0.026 .028 0.027 0.302 0.161 0.001 0.001

Uttar 0.037

U
Prad ±0.01 0.260± 1.819± 0.263± 4.279±0 0.358± 0.765± 12.398 0.001±

esh C-1L 7 0.062 0.027 0.055 N

.011 0.034 0.030 ±0.390 bdl 0.004
A
0.030
M

±0.01 0.359± 1.861± 1.463± 16.764± 0.265± 1.056± 3.344± 0.017±

C-1S 2 0.018 0.037 0.033 0.095 0.034 0.027 0.438 bdl 0.007
D

0.036
TE

±0.02 0.613± 1.866± 2.172± 35.239± 0.177± 0.585± 0.636± 0.023± 0.001±

C-1R 4 0.040 0.042 0.081 0.050 0.026 0.120 0.033 0.005 0.001
EP

0.027
CC

±0.00 0.317± 1.814± 0.362± 5.139±0 0.246± 0.534± 12.437 0.002± 0.004±

C-2L 3 0.010 0.013 0.046 .051 0.046 0.048 ±0.038 0.001 0.004
A

0.031

±0.00 0.590± 1.843± 2.143± 33.057± 0.143± 0.434± 1.853± 0.001± 0.020±

C-2S 1 0.010 0.011 0.094 0.084 0.056 0.046 0.027 0.054 0.032

36
 0.083

±0.01 2.433± 3.247± 6.939± 40.330± 0.091± 0.553± 0.423± 0.002± 0.004±

C-2R 1 0.015 0.044 0.042 0.555 0.012 0.037 0.066 0.04 0.001

0.022

±0.00 0.189± 1.657± 0.277± 2.932±0 0.258± 0.739± 7.803± 0.007±

PT
C-3L 5 0.041 0.025 0.028 .048 0.077 0.044 0.059 bdl 0.004

RI
0.023

±0.00 0.475± 1.762± 2.554± 25.727± 0.157± 0.413± 1.444± 0.003±

SC
C-3S 6 0.008 0.034 0.281 0.212 0.084 0.071 0.038 bdl 0.005

0.059

U
±0.02 0.627± 1.839± 2.361± 34.644± 0.145± 0.756± 0.566± 0.056± 0.007±

C-3R 0 0.07 0.042 0.461 0.285N 0.041 0.029 0.035 0.039 0.004
A
0.058
M

±0.01 0.277± 1.724± 0.263± 5.803±0 0.341± 0.648± 11.313 0.001±

C-4L 8 0.010 0.051 0.048 .117 0.050 0.035 ±0.212 bdl 0.000
D

0.085
TE

±0.01 1.572± 1.754± 3.653± 31.255± 0.082± 0.335± 0.144± 0.009±

C-4S 2 0.012 0.050 0.381 0.136 0.015 0.043 0.040 bdl 0.010
EP

0.088
CC

±0.01 1.389± 1.804± 5.438± 42.469± 0.078± 0.840± 0.151± 0.006±

C-4R 0 0.047 0.011 0.477 0.216 0.017 0.042 0.020 bdl 0.004
A

0.016

±0.00 0.139± 1.627± 0.165± 17.187± 0.194± 0.458± 5.301± 0.036±

C-5L 4 0.019 0.011 0.038 0.062 0.098 0.091 0.382 bdl 0.011

37
 0.065

±0.00 1.034± 2.113± 2.205± 50.078± 0.201± 0.420± 2.413± 0.006±

C-5S 5 0.014 0.089 0.049 0.011 0.012 0.030 0.378 bdl 0.003

0.104

±0.00 2.263± 1.552± 10.374 47.109± 0.177± 0.540± 0.224± 0.006±

PT
C-5R 9 0.015 0.033 ±0.517 0.073 0.026 0.050 0.077 bdl 0.001

RI
0.051

±0.00 0.539± 1.768± 0.384± 8.196±0 0.423± 0.859± 14.437 0.005±

SC
C-6L 5 0.041 0.039 0.327 .095 0.055 0.028 ±0.065 bdl 0.003

0.025

U
±0.00 0.309± 1.757± 0.520± 11.265± 0.388± 0.765± 3.090± 0.001± 0.043±

C-6S 5 0.008 0.049 0.424 N

0.092 0.141 0.022 0.121 0.87 0.030
A
0.051
M

±0.00 1.316± 1.738± 6.544± 29.660± 0.173± 0.652± 0.224± 0.005±

C-6R 3 0.031 0.052 0.078 0.346 0.027 0.036 0.018 bdl 0.004
D

Assa 0.017
TE

m C- ±0.00 0.141± 1.873± 0.377± 22.079± 0.273± 0.347± 6.803± 0.002± 0.004±

11L 4 0.040 0.026 0.035 0.060 0.108 0.032 0.215 0.001 0.003
EP

0.043
CC

C- ±0.00 0.258± 2.302± 0.679± 16.365± 0.226± 0.557± 2.670± 0.005±

11S 3 0.018 0.247 0.020 0.099 0.062 0.033 0.192 bdl 0.005
A

0.064

C- ±0.00 1.525± 2.062± 17.675 42.254± 0.104± 0.463± 0.166± 0.001± 0.004±

11R 8 0.041 0.024 ±0.069 0.100 0.021 0.035 0.013 0.001 0.005

38
Arun 0.016

acha C- ±0.00 0.255± 2.978± 0.381± 5.198±0 0.488± 0.963± 14.405 0.005± 0.006±

l 44L 1 0.018 0.020 0.021 .086 0.088 0.033 ±0.382 0.003 0.004

Prad
0.053
esh
C- ±0.01 0.356± 2.175± 1.441± 28.454± 0.175± 0.285± 0.430± 0.003± 0.004±

PT
44S 5 0.043 0.031 0.040 0.095 0.030 0.001 0.160 0.002 0.004

RI
0.140

C- ±0.01 2.153± 2.949± 16.766 49.337± 0.253± 1.537± 0.229± 0.001± 0.036±

SC
44R 0 0.044 0.042 ±0.213 0.510 0.015 0.040 0.061 0.001 0.042

0.028

U
C- ±0.01 0.242± 2.137± 0.970± 7.496±0 0.523± 1.293± 9.791± 0.001±

41L 7 0.040 0.048 0.040 N

.413 0.028 0.474 0.216 bdl 0.007
A
0.050
M

C- ±0.00 0.522± 2.194± 5.487± 33.761± 0.152± 0.426± 1.663± 0.003± 0.044±

41S 5 0.012 0.018 0.335 0.193 0.044 0.013 0.041 0.003 0.035
D

0.105
TE

C- ±0.00 1.559± 2.170± 11.475 33.576± 0.161± 0.850± 0.021± 0.004±

41R 5 0.020 0.058 ±0.100 0.183 0.037 0.115 0.004 bdl 0.003
EP

Biha 0.016
CC

r CR3 ±0.00 0.242± 2.197± 0.186± 9.657±0 0.409± 0.750± 18.540 0.005±

L 2 0.010 0.080 0.014 .035 0.013 0.012 ±0.265 bdl 0.001

A

0.025

±0.01 0.185± 1.839± 0.162± 12.911± 0.171± 0.450± 1.084± 0.006± 0.055±

CR3S 0 0.011 0.030 0.034 0.061 0.033 0.045 0.009 0.003 0.002

39
 0.049

CR3 ±0.00 1.802± 1.975± 15.630 49.851± 0.144± 0.613± 0.053± 0.004± 0.015±

R 1 0.009 0.018 ±0.420 0.141 0.078 0.064 0.027 0.005 0.003

L: Leaf; S: Stem; R: Root; nd: not detected, bdl: Below Detection limit

PT
RI
SC
U
N
A
M
D
TE
EP
CC
A

40