trans 5-o - caffeoylquinic acid ( 1 ) , quercetin-3-o - rutinoside ( 2 ) , luteolin-5-o - rutinoside ( 3 ) and luteolin-7-o - rutinoside ( 4 ) were separated from the plant material , its purity was checked by hplc and structure elucidated by ms spectral data .
water was purified by a milli - q system from millipore ( milford , usa ) .
analyses were performed on agilent 1200 chromatograph equipped with a diode array detector and mass detector in series ( agilent technologies , waldbronn , germany ) .
a phenomenex luna -c18 , 4.6150 mm , particle size 5 m with suitable guard column was employed for the separation . the binary mobile phase consisted of solvents a as 0.1% formic acid in water and solvents b as acetonitrile .
the gradient elution started with 10% b and changed to 75% b in 20 min , then reached 95% b in 27 min .
after each run the chromatographic system was set to 10% b in 4 min and equilibrated for 4 min .
the flow rate was 1.0 ml / min and injection volume was 5 l . separation of trans and cis isomer of 5-o - caffeoylquinic acid was attempted by using a zorbax sb phenyl , 4.6100 mm , particle size 1.8 m with suitable guard column .
the binary mobile phase consisted of solvents a as 0.1% formic acid in water and solvents b as acetonitrile .
the isocratic elution started with 20% b and 80% a. the flow rate was 0.8 ml / min and injection volume was 5 l .
spectral data for all peaks were recorded in the range of 200 - 400 nm .
the mass detector was an ion trap spectrometer ( agilent lc / msd trap xct ) equipped with an electrospray ionization interface and controlled by lcmsd software .
the ionization conditions were adjusted at 300 and 3.5 kv for capillary temperature and voltage , respectively .
the nebulizer pressure was 40 psi and the nitrogen flow rate was 8 l / min .
collision - induced fragmentation experiments were performed in the ion trap using helium as a collision gas , with voltage cycles from 0.3 up to 2 v. all mass spectrometry data were recorded in negative ion mode .
the screening was performed in full scan covering the range from m / z 100 up to 1000 ; multiple reaction monitoring ( mrm ) mode .
the flowers of nerium indicum were collected from the surroundings of vellore , in november 2013 , and identified at the department of chemistry , thiruvallur university . the plant material was air dried , smashed into powder and stored in a desiccator .
the flowers of nerium indicum ( 50 g ) were extracted 3 times with meoh under reflux for 3 h. evaporation of the solvent under reduced pressure provided the meoh extract ( 5.1 g ) .
to separate the compound , preparative liquid chromatography was done using acetonitrile - formic acid - water as mobile phase and the octadecylsilane as a stationary phase .
the flowers of nerium indicum were collected from the surroundings of vellore , in november 2013 , and identified at the department of chemistry , thiruvallur university . the plant material was air dried , smashed into powder and stored in a desiccator .
the flowers of nerium indicum ( 50 g ) were extracted 3 times with meoh under reflux for 3 h. evaporation of the solvent under reduced pressure provided the meoh extract ( 5.1 g ) . to separate the compound ,
preparative liquid chromatography was done using acetonitrile - formic acid - water as mobile phase and the octadecylsilane as a stationary phase .
a method coupling hplc with diode - array detector ( dad ) and electrospray ionization mass spectrometry with an ion trap analyzer was optimized for the separation and identification of phenolic acid in the extract of nerium indicum flowers .
different mobile phase compositions were screened to obtain chromatograms with good resolution within an acceptable time of analysis .
formic acid , as solvent a , and acetonitrile , as solvent b , were chosen for the gradient elution .
especially the formic acid and acetonitrile are volatile and thus compatible with lc / ms system .
formic acid ( lower ph values ) ensures better sample separation but shortens the hplc column lifetime and affects esi ionization .
compound 1 ( trans 5-o - caffeoylquinic acid ) with tr of 5.5 min , compound 2 ( quercetin-3-o- rutinoside ) with tr of 7.4 min , compound 3 ( luteolin-5-o - rutinoside ) with tr of 7.9 min and compound 4 ( luteolin-7-o - rutinoside ) with tr of 8.1 min .
* dad1 , sig=325.00 , 4.00 ref = off , ext of 2210nif.d compound 1 observed as a [ m - h ] - ion peak at m / z 353 in the esi mass spectrum ( fig .
2 ) and based on literature data compound 1 was confirmed as a 5-o - caffeoyl quinic acid . by using zorbax sb phenyl column
able to separate cis and trans isomer of 5-o - caffeoyl quinic acid ( fig .
3 ) gave an [ m - h]-ion peak at m / z 353 in the esi mass spectrum ( fig .
uv spectra also suggest that trans isomer is more absorption value compared to cis isomer ( fig .
mass spectral value of compound 2 showed a quasi - molecular ion [ m - h ] - at m / z 609 , compared with literature data compound 2 was identified as a quercetin-3-o - rutinoside ( fig .
tic / ms chromatogram of crude methanolic extract of nerium indicum flowers from hplc-(- ) esi - ms .
m / z value of 353.0 with tr of 5.5 min , m / z value of 609.3 with tr of 7.4 min , m / z value of 593.3 with tr of 7.9 min and m / z value of 593.3 with tr of 8.1 min .
hplc - dad chromatogram of cis and trans isomer of 5-o - caffeoyl quinic acid .
cis isomer with tr of 1.4 min and trans isomer with tr of 2.2 min .
tic / ms chromatogram of cis and trans isomer of 5-o - caffeoyl quinic acid from hplc-(- ) esi - ms .
cis isomer with m / z value of 362.9 and trans isomer with m / z value of 363.0 .
cis isomer with absorption maximum at 216 and 328 nm similarly trans isomer with absorption maximum at 220 , 242 and 326 nm .
esi - ms of compound 3 , 4 showed a quasi - molecular ion [ m - h ] - at m / z 593 , both are same mass value only position is different and based on literature data compounds 3 and 4 identified as luteolin-5-o - rutinoside and luteolin-7-o - rutinoside ( fig .
2 ) . based on the above data two phenolic acids , trans 5-o - caffeoylquinic acid and cis 5-o - caffeoylquinic acid ( fig . 6 ) .
three flavonoid glycosides , quercetin-3-o - rutinoside , luteolin-5-o - rutinoside and luteolin-7-o - rutinoside ( fig .
a. quercetin-3-o - rutinoside , b. luteolin-5-o - rutinoside , c. luteolin- 7-o - rutinoside .
 four major compounds were separated and identified from the methanol extracts of nerium indicum flowers ( arali ) using hplc and mass spectral data . through mass data , 
 the chemical structures were elucidated as : trans5-o - caffeoylquinic acid ( 1 ) , quercetin-3-o- rutinoside ( 2 ) , luteolin-5-o - rutinoside ( 3 ) and luteolin-7-o - rutinoside ( 4 ) . 
 in addition , the cis isomers of 5-o - caffeoylquinic acid in nerium indicum flowers were confirmed by mass , hplc and uv . 
 the structures of these compounds confirmed with the help of liquid chromatography mass spectrometry . 
MATERIALS AND METHODS
Plant material:
Extraction and isolation:
RESULTS AND DISCUSSION