Studies on structural elucidation of delphinium alkaloids by using LC-ESI-MS technique

3399 | P a g e F e b r u a r y 1 9 , 2 0 1 5 Studies on structural elucidation of delphinium alkaloids by using LCESI-MS technique. Bilal Ahmad Dar, Mushtaq Ahmad Qurishi, Aijaz Hussain Kanth University of Kashmir, Hazratbal, Srinagar, Kashmir, India. darbilal29@yahoo.in University of Kashmir, Hazratbal, Srinagar, Kashmir, India. qurishi@gmail.com Department of chemistry GDC Bemina, Srinagar, Kashmir. Aijazkanth@rediffmail.com ABSTRACT 1. A rapid,sensitive and specific liquid chromatography-electron spray-mass spectrometry (LC-ESI-MS) method to identify the different diester-diterpenoid and monoester type alkaloids from delphinium cashmerianum Collected from Sopi Kargil. Chromatographic separation were achieved on c-18 coloumn and peaks determined by mass spectrometry in positive and negative modes.The validated method led to tentative identification of eight alkaloids on the basis of their retention times and fragmentation patterns. Results showed that the positive mode response was much higher than the negative ion mode .chromatographic conditions were optimized to obtain high resolution and short run time. Indexing terms/


INTRODUCTION
It is the genus belonging to family renunculaceae comprising of more than 300 species of perennial flowering plants distributed in Asia, North America, Europe and tropical Africa 1 .The members of this genus are toxic to humans and livestock 2 . The perennial species of Delphinium along with the annual species of consolidate are commonly called as Larkspur 3,4 . In the flowering season of June -July the plant has raceme of colored flowers varying from purple to blue and red and white. In most species the flower has five petal like sepals joined together with a spur(hence common name) enclosing four similarly colored petals. They possess small black and shiny seeds.
Delphinium cashmerianum Royle (Kashmir Larkspur )is a perennial herb found in the Himalayas, from Pakistan to Uttarakhand, at altitudes of 2700-4500 m. It is characterized by woolly-haired, conspicuously veined bluish-purple flowers, 2-3 cm across clustered in a dense flat-topped head. Flowers have a stout spur 1.5 cm long. Leaves are rounded in outline, deeply lobed, 3-5 cm across. It is quite similar to Musk Larkspur which is found only above altitudes of 4500 m, and has larger, more inflated flowers.
The majority of phytochemical investigation on Delphinium species (Renunculaceae) have been carried out for diterpenoidalkaloid.AlkaloidslikeNeoline,Chasmanine,Homochasmanineand Delphisine were reported by pelletier 5

Extraction
The air dried, finely powdered root material (5Kg) was extracted for 72 hours with methanol to afford the respective extract, which was concentrated under reduced pressure and was coded as DEL.

Reagents and chemicals
HPLC grade acetonitrile, methanol and formic acid were purchased from Sigma Aldrich. HPLC grade water (18.2MὨ) was procured from Milli-Q water system(USA). other chemicals were of analytical grade and are available commercially.

LC System
LC analysis was carried out by an Agilent1260 infinity series consisting of a pump, detector, an auto sampler and a column component. The samples were separated on chromo lith RP-18e column (4.6 mm ID, 50mm length) Merck at room temperature. The mobile phase consisted of (A) aqueous formic acid(0.1 %) and (B) methanol and the elution gradient was set as follows : ; 0-8 min, linear gradient from 12% to 25% of B; 8-12 min, isocratic conditions at 25% of B; 12-16 min, linear gradient from 25% to 40% of B; 16-40 min, linear gradient from 40 to 50% of B, 40-50 min, linear gradient from 50 to 100% of B. Flow rate: 1 ml/min.

LIQUID-CHROMATOGRAPHY TANDEM-MASS-SPECTROMETRY (LC-ESI-MSMS)
For LC-ESI-MS n e xperiments, LC-MS QqQ-6410B equipment (Agilent Technologies) comprising a chromatographic system 1260 Infinity (Agilent Technologies) coupled with an Agilent Triple Quad mass spectrometer fitted with an ESI source was used. The conditions of ESI source were set as follows: sheath and auxillary gases flow, 25 and 3 arbitrary units, respectively; spray voltage, 4KV; capillary temperature 325 o C and tube length voltage: 120 V. The samples were analysed in both positive and negative modes and full scan mass range was set between m/z 100 -1200 with the aquisition of centroided-type mass spectra. Accurate mass analyses were calibrated according to manufacturers guidelines. In the MS n experiments, data dependent MS n scanning was performed to minimize total analysis time as it can trigger fragmentation spectra of target ions and prevent repitition by d ynamic exclusion settings. The software of the Agilent technolgies was used for data analysis. After the automated removal of noise and baseline signals, the theoritical extract chromatogram of main alkaloids was shown in the plot (fig 3)  F e b r u a r y 1 9 , 2 0 1 5   fig-3 Total chromatogram of Delphinium cashmerianum.

RESULTS AND DISCUSSION
For LC-MS e xperiments different fragmentation measurements were conducted in independent LC -MS n e xperiments. In the first measurement the survey scan was performed in analyser at (R=30,000) followed by MS 2 scan at (R=15,000). The accurate tandem mass spectra can provide strong evidences for identification of fragments and neutral loss. In the separate measurement in which ESI MS 2 and MS 3 e xperimens using dynode detection provided more fragment ions and order of fragmentation could be determined to show evidence of structural identification. Fifteen alkaloids were picked from the chromatogram at the retention times from 2 -20 min. Among them nine compounds were identified as 14 -2methylbutylnudicaudiline, 14-cis cinnamoyl nudicaudiline, Davisinol, uncinitine, condelphine, Peregrine, Karakoline and 10-hydroxy methyllyoaconitine. by comparing their retention times and on the basis of their fragmentation patterns in MS-MS data. The other compounds were tentatively identified by comparing their mass data with those reported in literature. The Retention time values and mass data of deduced compounds from pe aks are summarised in table 3. The fragmentation patterns of the identified compounds is shown in (fig 4). F e b r u a r y 1 9 , 2 0 1 5