hplc method development and validation of cytotoxic agent phenyl-heptatriyne in bidens pilosa with...

HPLC method development and validation ofcytotoxic agent phenyl-heptatriyne in Bidenspilosa with ultrasonic-assisted cloud pointextraction and preconcentrationPriyanka Trivedi,a Jonnala Kotesh Kumarb Arvind Singh Negia andKaruna Shankera*

ABSTRACT: Extraction and pre-concentration of a bioactive marker compound, phenyl-1,3,5-heptatriyne from Bidens pilosa,prior to HPLC has been demonstrated using both organic and ecofriendly solvents. Non-ionic surfactants, viz. Triton X-100,Triton X-114 and Genapol X-80, were used for extraction. No back-extraction or liquid chromatographic steps were requiredto remove the target phytochemical from the surfactant-rich extractant phase. The optimized cloud point extraction proce-dure has been shown to be a potentially useful methodology for the preconcentration of the target analyte, with a precon-centration factor of 4–99. Moreover, the method is simple, sensitive, rapid and consumes lesser solvent than traditionalmethods. An isocratic chromatographic separation and quantitation was accomplished on a C18 column with acetonitrile–acidified aqueous as mobile phase at a flow rate of 1.0 mL/min, UV detection at 254 nm and specificity with photo diode-arraydetector (PDA) and MS. Under the optimum experimental conditions recovery was satisfactory (99.18–100.33%) withoutinterference from the surfactant. The method seems to be reliable with intraday precision and interday precision below 2.0%.Good linearity was obtained in the working range from 7.5 to 30 mg/mL with correlation coefficient >0.99. The limits ofdetection and quantitation were 1.84 and 6.13 mg/mL, respectively. The method was validated following international guide-lines and successfully applied for quantitative assays of cytotoxic compound phenyl-1,3,5-heptatriyne in Bidens pilosa.Copyright © 2010 John Wiley & Sons, Ltd.

Keywords: Bidens pilosa; cloud-point preconcentration; micelle-mediated extraction; RP-HPLC; Triton X-100

IntroductionBidens pilosa (Asteraceae), commonly known as Spanish needles,is an annual weed distributed widely in the tropical and subtropi-cal regions of the world. Although it is native weed of SouthAmerica, it is widely used in folk medicine in several countries ofAsia and Africa (Ashafa and Afolayan, 2009; Kviecinski et al., 2008;Lee et al., 2008, Lin et al., 2008) and also southern India(Sundararajan et al., 2006) to treat glandular sclerosis, wounds,colds and flu, acute or chronic hepatitis and urinary tract infec-tions. It is reported to have the pharmaceutical effect on malaria,skin, stomach and liver disorders (Brandao et al., 1997), as ananti-inflammatory, antiseptic, blood pressure lowering agent andhypoglycemic (Chien et al., 2009; Dimo et al., 2002; Hsu et al.,2009; Marles and Farnsworth, 1995; Pereira et al., 1999; Ubillaset al., 2000). Several phytochemicals such as acetylacetone(Kumar and Sinha, 2003), polyacetylenes (Alvarez et al., 1996),flavonoids (Brandao et al., 1998; Wang et al., 1997), a diterpenoid(Zulueta et al., 1995), phenylheptatriyne (Kumari et al., 2009),phenylpropanoids (Sashida et al., 1991; Kumar and Sinha, 2003),polyacetylenes (Wu et al., 2004) and polyphenols (Hoffmann andHolzl, 1989) have been reported from this weed. The presence ofdiverse secondary metabolites has motivated researchers toexplore the possibility of new lead molecules with anti-malarialand anti-cancer potential (Kumari et al., 2009) as well as to ratio-nalize to its use in folk and traditional medicine.

Currently, ultrasonic- and microwave-assisted extraction inaddition to cold and hot percolation are employed as a rapidextraction method for natural products (Shanker et al., 2008; Wuand Zhang, 2010; Yadav, 2009), but these lack pre-concentration.Pre-treatment methods such as liquid–liquid extraction (LLE)usually require a large volume of toxic organic solvent, even assolid-phase extraction (SPE) needs organic solvent for the elutionstep (Huie, 2002). Unfortunately, both of these pre-treatmentprocesses suffer with the drawbacks of large sample volume,time-consuming procedure and sometimes compromised recov-ery (Jiang et al., 2010). Undoubtedly, the most popular extraction

* Correspondence to: K. Shanker, Analytical Chemistry Department, CentralInstitute of Medicinal and Aromatic Plants, Council of Scientific and Indus-trial Research (CSIR), PO-CIMAP, Lucknow-226015, India. E-mail: [email protected]

a Analytical Chemistry Department, Central Institute of Medicinal and Aro-matic Plants, Council of Scientific and Industrial Research (CSIR), PO-CIMAP,Lucknow 226015, India

b CIMAP Research Centre, Boduppal, PO-Uppal, Hyderabad 500039, India

Abbreviations used: CPE, cloud point extraction; PHT, phenyl-1,3,5-heptatriyne; UAMME, ultrasonic assisted micelle-mediated extraction.

Research Article

Received 1 May 2010, Accepted 30 June 2010 Published online in Wiley Online Library: 01 September 2010

(wileyonlinelibrary.com) DOI 10.1002/bmc.1505

697

Biomed. Chromatogr. 2011; 25: 697–706 Copyright © 2010 John Wiley & Sons, Ltd.

method is solvent extraction. The choice of solvent dependsgreatly on its properties and is obviously the most critical factorto consider (Kim and Verpoorte, 2010). Therefore, development ofa simple and environment friendly method is of growing interestin natural product research (Kiathevest et al., 2009; Zhou et al.,2008).

The micelle-mediated extraction and cloud-point preconcen-tration (CPE) method offers a convenient alternative to the con-ventional extraction systems (Martıınez et al., 1999; Wang, 2007).Nonionic surfactant micelle solutions exist as a single homoge-neous isotropic phase at temperatures below their cloud point.However, if the solution temperature is raised above the cloudpoint, such solutions become turbid and phase separate to yielda surfactant-lean phase (aqueous phase) and small volumesurfactant-rich (coacervate) phase. Target analyte species oftendifferentially partition between these two phases (Delgado et al.,2004; Hung et al., 2007; Merino et al., 2003). Extractions based onsuch phenomenon are referred to as cloud point extractions(CPEs). A plethora of reports have concerned the utilization ofsuch a CPE approach as a means by which to extract and enrichinorganic, organic and biological analytes prior to their analysisby spectroscopy, capillary electrophoresis or liquid chromatogra-phy (Bosch Ojeda et al., 2009; Carabias-Martínez et al., 2000), butvery limited work on natural products (Choi et al., 2003; Fanget al., 2000; He et al., 2005; Sun et al., 2008) has been reported inthe literature.

To the best of our knowledge no validated method for extrac-tion and pre-concentration prior to HPLC analysis of B. pilosa-based phenyl-heptatriynes has been reported so far. However, aneffort (Chien et al., 2009) has been made to define the chemot-axonomic correlation of the antidiabetic potential of B. pilosavarieties on the basis of polyacetylene glycosides content usinggradient elution in reverse-phase chromatography, which suffersseveral drawbacks of non-validation of HPLC method, time-consuming sample preparation and longer analysis time. Thepresent paper describes a method that is fast and simple, and thetargeted marker compound (phenyl-1,3,5-heptatriyne, PHT) canbe extracted selectively. The comparison of recovery of PHT withconventional extraction processes with common organic sol-vents as well as non-ionic surfactants has been evaluated for firsttime. The feasibility and effectiveness of employing non-organicsolvents containing three common non-ionic surfactants (TritonX-100, Triton X-114 and Genapol X-80) as the extracting mediumusing both ultrasonic and microwave techniques were evaluatedby comparison with conventional extraction solvents (hexane,ethylacetate and methanol). Extraction efficiency was optimizedon various experimental parameters, such as temperature, time,concentration of the surfactant and salt concentration. Addition-ally, the advantages and limitations of solvents and techniquesfor quantitative estimation of biologically active PHT in B. pilosausing both organic as well as eco-friendly solvents for the pre-concentration of the active ingredients without disturbing high-performance liquid chromatography (HPLC) analysis wasdemonstrated.

Experimental

Chemicals and Bioactive Marker Compound

Triton X-100 (polyethylene glycol mono[4-(1,1,3,3-tetramethylbutyl)-phenyl] ether with an average ethylene oxide (EO) chain length of n = 9.5;TX-100), Triton X-114 (polyethylene glycol mono[4-(1,1,3,3-

tetramethylbutyl)-phenyl] ether with an average ethylene oxide (EO)chain length of n = 7.5; TX-114) and Genapol X-080 (isotridecyl poly eth-ylene glycol ether) were purchased from Sigma-Aldrich (St Louis, MO,USA) and used without further purification. The bioactive marker, PHT,was isolated (Kumari et al., 2009) in our laboratory. The physicochemicalproperties of PHT and surfactant are summarized in Table 1. The solventsused for extraction and chromatographic analysis were either analyticalor HPLC-grade, purchased form E. Merck Ltd, Mumbai, India. Before use,the solvents were filtered through a 0.45 mm Millipore membrane (Milli-pore, Billerica, MA) after sonication for 15 min.

Plant Material and Sample Preparation

The leaves of B. pilosa were collected from Garhwal, Uttaranchal, India andidentified by a taxonomist; a voucher specimen (collection no. 7702) wasdeposited in our Biodiversity Department. The shade-dried plant materialwas stored in a dry place at room temperature following good storagepractices until use. Dried and finally milled aerial parts (100 mg) of B.pilosa were extracted using different techniques and solvents such ashexane, ethyl acetate and methanol to determine the total PHT content inthe particular extract. Extraction was optimized as to solvents, time andtechniques (Table 2). The representative chromatogram of reference PHTand B. pilosa ultrasonic extraction with common organic solvents aredepicted in Fig. 1.

Instrumentation

LC-PDA-MS (Shimadzu, Japan) consisting of an analytical column (Waterssymmetry®, 250 ¥ 4.6 mm, 5mm), pumps (LC-20AD), autoinjector (SIL-10AF) and PDA (SPD-M20A), was used for analysis. A sonicator (Micro-clean 109, Oscar Ultrasonic, Mumbai, India) was used for samplepreparation. Hyphenated LC-PDA-MS (Prominence LC and massMS-2010EV, Shimadzu, Japan) was used for mass spectra.

Ultrasonic Assisted micelle-mediated Extraction

Powders of B. pilosa (100 mg) were accurately weighed and placed in a15 mL centrifuge tube; 10 mL non-ionic surfactant (Genapol X-080, TritonX-100 and Triton-X114) solution (2–10%) (v/v) was added. The tube wascapped and blended adequately, then placed in the ultrasonic cleaningbath for ultrasonic extraction. During extraction, mixing was provided byan ultrasonic bath (Microclean-109, Oscar Ultrasonics, Mumbai, India,30.0 ¥ 25.0 ¥ 12.5 cm, 34 � 3 kHz, PZT Sandwich type six transducer,250 W). For ultrasonic-assisted micelle-mediated extraction (UAMME),the extraction time was 45 h. Two sets of UAMME experiment werecarried out: at a controlled temperature at 50 � 2°C, and at the risingtemperature from 50 to 63°C, which was caused by the 45 min ultrasonicexposure. To control the temperature of water in the ultrasonic bath,water in the bath was circulated and regulated at the constant desiredtemperature. The experimental conditions for UAMME were optimized.After ultrasonic-assisted extraction, the B. pilosa extracts were centri-fuged at 4000 rpm for 20 min, then the supernatant was collected andoptimized for pre-concentration of PHT.

Cloud-point Pre-concentration

To study the bi-phasic behavior of PHT in respective non-ionic surfac-tants, i.e. formation of a layer of organic-rich coacervate and largeaqueous extract, the cloud point was achieved by adding salts. Sodiumchloride and ammonium sulfate (40–90 g/L) were added to the samplesolution. The sample solution was then kept in a thermostatic water bathat 50°C for 20–30 min until the solution completely separated into twodistinct phases. The upper phase was the small volume of surfactant-richphase and the lower phase was the large volume of the aqueous phase.After centrifugation for 20 min at 4000 rpm, the aqueous phase waspipetted out, and the sticky surfactant-rich phase was left in the tube. A1.0 mL aliquot of methanol was added to lower the viscosity of thesurfactant-rich phase, and 10 mL of the solution was injected into theHPLC system for PHT analysis after filtration with a 0.45mm syringe nylon

698

P. Trivedi et al.

Biomed. Chromatogr. 2011; 25: 697–706wileyonlinelibrary.com/journal/bmc Copyright © 2010 John Wiley & Sons, Ltd.

Tab

le1.

Che

mic

alan

dp

hysi

calp

rop

ertie

sof

the

mar

ker

chem

ical

and

surf

acta

nts

Nam

eA

bb

revi

atio

nSt

ruct

ure

Form

ula

l max

(nm

)C

MC

(mM

)M

olec

ular

wei

ght

(g/m

ol)

Phen

yl-1

,3,5

-hep

tatr

iyne

PHT

C13

H8

238,

250,

291,

310

—16

4

Trito

nX-

100

[pol

yeth

ylen

egl

ycol

p-(1

,1,3

,3-

tetr

amet

hylb

utyl

)-p

heny

leth

er]

TX-1

00O

OH n=

10

C34

H62

O11

275

0.13

–0.3

062

5

Trito

nX-

114

[1,1

,3,3

-tet

ram

ethy

lbut

yl)p

heny

l-p

olye

thyl

ene

glyc

ol]

TX-1

14O

OH n=

8

C30

H54

O9

223

0.20

–0.3

553

7

Gen

apol

X-80

[olig

oeth

ylen

egl

ycol

mon

oalk

ylet

her]

GP-

80

n=8

OO

HC

H3

C30

H62

O10

210

0.6–

0.15

583

699

HPLC method development and validation of cytotoxic agent phenyl-heptatriyne

Biomed. Chromatogr. 2011; 25: 697–706 wileyonlinelibrary.com/journal/bmcCopyright © 2010 John Wiley & Sons, Ltd.

filter. The representative chromatograms of B. pilosa ultrasonic assistedextraction with non-ionic surfactant are depicted in Fig. 2.

Preconcentration Factor of the CPE Method

The preconcentration factor was calculated as the ratio between thevolume of the original sample solution and the volume of the obtained

surfactant-rich phase (Wu and Huang, 1998). For the convenient mea-surement of the volume of the surfactant-rich phase, larger volumes of5% surfactant solutions were used and the following experiment wascarried out: powders of B. pilosa (100 mg) were accurately weighed andplaced into a 15 mL centrifuge tube, 10 mL 5% Triton X-100, Triton X-114and Genapol X-080 solution and 80 g/L sodium chloride were addedunder the same experimental conditions used for phase separation (ther-

Table 2. Extractability of different solvents and techniques for marker PHT from the aerial part of B. Pilosa

Solvent Amount of compound quantifieda (mg/g)Cold percolationb Hot extractionc Ultrasonicationd Microwavee

Hexane 0.0040 0.0056 0.0061 NSEthylacetate 0.0158 0.0238 0.0255 0.0231Methanol 0.0023 0.0036 0.0038 0.0032CPE (Triton X-100) NS NS 0.0235 0.0058CPE (Triton X-114) NS NS 0.0020 0.0019CPE (Genapol X-80) NS NS 0.0110 0.0056a Dry weight basis; NS, not studied.b Cold percolation (3 ¥ 15 mL, 10 h extraction time at room temperature).c Hot extraction (3 ¥ 15 mL, 30 min extraction time at 50°C).d Ultrasonication (15 min extraction time).e Microwave (3 ¥ 15 mL, at 90 W, 50°C for 3 min extraction time).

0.0 5.0 10.0 15.0 20.0 25.0 min

0.00

0.25

0.50

0.75

1.00

mAU(x100)Ch1-254nm,4nm (1.00)

100.0 125.0 150.0 175.0 m/z0.00

0.25

0.50

0.75

1.00

Inten.(x1,000,000)

17

3

13

3

19

8

17

9

12

8

14

5

13

7

18

71

91

11

7

16

5

14

9

10

5

15

9 163

[M+H2O2]-

[M-H]-

0.0 5.0 10.0 15.0 20.0 25.0 min

0.0

1.0

2.0

3.0

4.0

5.0

mAU(x100)Ch1-254nm,4nm (1.00)

200 250 300 350 nm

0

500

1000

1500

2000

2500

mAU

250

202

238

310

291

A

B

PHT

PHT

Figure 1. Representative HPLC chromatograms: (A) reference phenyl-1,3,5-heptatriyne (PHT-0.33 mg/mL); (B) ethylacetate extract of aerial part of Bidens pilosa (100 mg/mL). Characteristic UVand ESI-APCI(-ve) mass spectra of PHT are shown in the inset of the chromatogram.

700

P. Trivedi et al.


mostat at 50°C and centrifuging at 4000 rpm for 20 min). The reportedvalues were the averages of triplicate determinations (Table 3).

Chromatographic Conditions

Various mobile phase compositions, viz. methanol–water, acetonitrile–water with and without acid additives, were tried on diverse stationaryphases. Finally, a better separation was achieved with a mobile phasecomposition of acetonitrile–water containing 1.0% acetic acid (80:20 v/v).A flow rate of 1.0 mL/min and column temperature of 30°C was main-tained throughout the run. Data acquisition was performed in the rangeof 200–400 nm by injecting 10 mL volume of each reference PHT andsample solution. The column eluent after PDA detection was divided in a1:4 ratio and used as an inlet for MS (ESI-APCI dual probe) detection to

0.0 5.0 10.0 15.0 20.0 25.0 min

0.00

0.25

0.50

0.75

1.00

1.25mAU(x10)Ch1-254nm,4nm (1.00)

0.0 5.0 10.0 15.0 20.0 25.0 min

0.00

0.25

0.50

0.75

1.00mAU(x100)Ch1-254nm,4nm (1.00)

0.0 5.0 10.0 15.0 20.0 25.0 min

0.0

1.0

2.0

3.0

4.0

5.0

mAU(x10)Ch1-254nm,4nm (1.00)

A

B

C

PHT

PHT

PHT

Figure 2. Representative HPLC chromatograms B. pilosa (100 mg/mL) extracted with (A)Triton X-100, (B) Triton X-114 and (C) Genapol X-80 surfactant in 5% concentration usingultrasonic at 50°C for an incubation time of 45 min and cloud point pre-concentration withNaCl (80 g/L).

Table 3. Influence of the different concentration of surfac-tants on pre-concentration factor

Surfactant (%) Ratio of volume of aqueous and surfactantlayers (Va/Vs)

Triton X-100 Triton X-114 Genapol X-80

2 99.00 99.00 99.005 9.00 19.00 9.007 6.14 15.67 5.6710 4.00 15.67 4.71

701



monitor the total ion chromatogram in the mass range 100–200. Thecomponents were identified by comparison of their retention times withthose of authentic standards under identical analysis conditions and theUV and mass spectra with our in-house PDA and MS library. Representa-tive chromatograms of standard and B. pilosa extracts prepared with dif-ferent solvents extract are shown in Fig. 1.

Validation of the Developed Method

The linearity of the method was established by triplicate injections in therange of 7.5 mg/mL to 30 mg/mL. Intraday precision was calculated usingfive injections of the concentration in the higher range (30 mg/mL) on thesame day. These studies were repeated in different weightings on differ-ent days to determine the inter-day precision. The specificity of themethod was established in three ways, first by studying peak purity plotsusing a PDA detector, second by UV–vis spectra matching and finally bymass spectral matching. Limits of detection (LOD) and limits of quantifi-cation (LOQ) were determined using the linear regression equation. Thefollowing equations were applied: LOD = 3Sy,x/b and LOQ = 10Sy,x/b, whereSy,x is the standard deviation of the y-value distribution around the regres-sion line and b is the slope of the calibration curve. The calibration curveswere determined using the least-squares method, for independent vari-able (x) the concentration (mg/mL-1) and for dependent variable (y) thedetector response in terms of peak area of PHT.

Statistical Analysis

Data were processed and recorded as means � standard deviation oftriplicate measurements. Analyses of variance (ANOVA) and significancedifferences between the means, least square regression and residualanalysis (Shanker et al., 2007; Yadav et al., 2009) were performed usingGraph PAD Prism version 4.0 for Windows (GraphPad Software, San Diego,CA, USA).

Results and Discussion

Extraction Efficiency of Common Organic Solvents

The technique of extraction from plant matrix was optimized bycomparing results of the PHT content using cold and hot extrac-tion, ultra-sonication and microwave extraction. Cold extractionwas performed by allowing the plant sample contact with therespective solvent for 10 h, while hot extraction was performedfor 15, 30, 45 and 60 min at a constant temperature of 50°C.Ultrasonic extraction was performed at 15, 30, 45 and 60 min at afixed temperature of 50°C. The microwave-assisted extractionwas performed at 90 W, 50°C for 3 min extraction time usingkitchen microwave (Whirlpool, Noida, India). The variation in PHTcontent in samples of different extraction techniques was foundto vary dynamically by 0.0023–0.0255 mg/g (Table 2). It is evidentthat among common organic solvent ultrasonic extraction(30 min at 50°C) with ethylacetate solvent is a better choice forsample preparation due to comparative high extraction effi-ciency (0.571%). From the results of PHT content, the ultrasoni-cation technique was found to be the most suitable due to theshorter sample preparation time, better extraction efficiency andcost effectiveness. The extraction efficiency with ecofriendly non-ionic surfactant was also assessed and it was found that TritonX-100 is a relatively better choice. Based on the extraction effi-ciency experiments, ultrasonic extraction with ethylacetate for30 min was selected for PHT determination in B. pilosa and recov-ery studies.

Optimization of the Extraction andPreconcentration Process

Nonionic surfactants are used for cloud-point extraction of PHTbecause their cloud-point temperatures are lower than those of

cationic or anionic surfactants. Therefore, the need for over-heating can be avoided. Conditions were optimized that couldpotentially affect extraction efficiency, for example concentra-tion of surfactant, salt concentration and extraction and incuba-tion times, were studied by comparing the peak areas of PHTobtained from plant material extraction

Three surfactants, viz. Triton X-100, Triton X-114 and GenapolX-080, were tested as extraction solvents. Unlike Genapol X-80,Triton X series showed high UV absorbance and gave very broadpeaks in the HPLC chromatogram, but did not interfere with thedetermination of PHT as it eluted before it (Fig. 2a–c). GenapolX-080 has eight oxyethylene units and tridecyl alkyl moieties anddoes not absorb above 210 nm, having the advantage of non-interference due to the absence of any aromatic moiety butshowed lower extractability of PHT. Therefore, Genapol X-080 wasnot found to be a suitable CPE surfactant in this study (Fig. 2c).However several reports of successful extraction procedures (Heet al., 2005; Sanz et al., 2005; Sirimanne et al., 1998) based onusing Genapol X-080 have been reported. The amount of surfac-tant required to achieve quantitative extraction of the analytewas studied. Figure 3(c) shows the maximum signals (area underpeak) obtained for PHT as a function of Triton X series with NaClsalt addition; this may be due to lower viscosity of Triton X seriesthan Genapol X-80. UAMME with Triton X-100 solution was foundto give slightly higher PHT recovery than Genapol X-080 andTriton X-114. Triton X-100 has lower viscosity, thus the ultrasonicwave may occur more easily because the intensity applied couldmore easily exceed the molecular forces of the liquid. In additionto this, the lower viscosity of Triton X-100 resulted in higher dif-fusivity, causing the solution to easily diffuse into the pores of theplant materials (Paleologos et al., 2005).

The effect of surfactant concentration on extract solution wasinvestigated and the results are shown in Fig. 3(a). The incubationtemperature was maintained at 50°C. The effect of incubationtime was also studied and 45 min time was found to be optimumfor both phase separation as well as recovery (Fig. 3b). As theconcentration of the surfactant increases, the PHT content inorganic rich layer increases, but at 7 and 10% the volumedecreases. However, the recovery of PHT remained constant withfurther increase in concentration to maintain the materialbalance as the concentration of surfactant in aqueous phaseremained almost constant. The maximum recovery was obtainedwith Triton X-100 with 5% concentration.

The nature of the salt and its concentration to achieve thecloud point of the respective surfactant affects not only the pre-concentration factor but also the recovery of PHT (Fig. 3c, d).Sodium chloride at 80 g/L was found to recover the maximumPHT from plant material when extracted with Triton X-100.While the addition of ammonium sulfate at lower amounts (40and 50 g/L) recovered the maximum PHT irrespective of surfac-tant, at higher concentrations the signal intensity of PHTremained unaffected. Based on this observation, Triton X-100(5%) was used for extraction at 50°C with an incubation time of45 min. The phase separation was obtained by adding sodiumchloride (80 g/L).

LC Method Validation

Validation was performed in compliance with International Con-ference on Harmonization (ICH, 2005) and IUPAC (IUPAC, 2002)using adequate statistical estimates (%RSD, Student’s t-test, leastsquare regression and residual analysis; Steele and Torrie, 1980).

702

P. Trivedi et al.


Linearity and sensitivities (LOD and LOQ). Five test solutionsranging from 7.5 to 30 mg/mL of PHT were prepared by dilutionfrom stock solution of 0.3 mg/mL and tested individually for lin-earity. The linear regression curve was obtained by plotting theUV detector response in terms of peak area of PHT at each level(y-axis) against the concentration (x-axis) of each injection(Table 4).

Linearity was checked for three consecutive days for the sameconcentration range from different stock solutions. A good cor-relation (r2 = 0.9989) with slope significant deviation from zerowas found on computation. Moreover, the intercept was not sta-tistically significant (p = 0.0524). A statistical residual analysis wasalso performed for each point of concentration range and corre-sponding to the difference between estimated and mean con-

Figure 3. (A) Effect of surfactant concentration on the extraction efficiency of PHT (experimental conditions: 50°C, minimum 80 g/L NaCl, 45 min, UAE).(B) Effect of incubation time on the extraction efficiency of PHT (experimental conditions: 5% surfactant, 50°C, minimum 80 g/L NaCl, 45 min, UAE). (C)Effect of NaCl concentration on the extraction efficiency of PHT (experimental conditions: 5% surfactant, 50°C, 45 min, UAE). (D) Effect of (NH4)2SO4

concentration on the extraction efficiency of PHT (experimental conditions: 5% surfactant, 50°C, 45 min UAE).

703



centrations. Residual plot analysis demonstrated that residualswere randomly distributed around the zero value. This confirmsthe choice of the linear model for PHT analysis. The sensitivity ofthe method was calculated as the limits of detection and quan-titation (LOD and LOQ). The LOD within a linearity of 0.9988 at95% confidence was 1.84 mg/mL and the LOQ was 6.13 mg/mL(Table 4).

Specificity. For any method to be specific, the measured peakmust be only related to the substance intended to be analyzed inthe presence of potential impurities and/or co-eluting compo-nents. The specificity with regard to other co-eluting compo-nents was investigated. It was found that under optimizedchromatographic conditions closely eluting peaks did not inter-fere with PHT (Figs 1 and 2). In both standard and sample tracksthe peak purity of PHT was greater than 0.999. The UV–vis spec-trum of standard PHT was matched with the UV–vis spectra ofPHT in the B. pilosa sample track. The UV–vis spectral matchingwas found to be 0.9998, which was further ensured by massspectra matching (-ve mode). The MS[ESI-APCI] spectra of the markercompound (Fig. 1b) in standard solution and B. pilosa extractwere obtained using a hyphenated instrument (LC-PDA-MS). Themass spectra were processed using a standard method of inte-gration and stored in the library of Lab Solution 3.21 software.The mass spectrum of the corresponding peak of PHT in sampletrack was matching with the standard track. A 64% matching ofPHT in ESI negative ionization mode was observed.

Precision. The precision of the method was measured by meansof repeatability and intermediate precision. Repeatability wasestimated for five determinations of PHT on the same day. Inter-mediate precision was evaluated by triplicate qunantitation ofPHT in B. pilosa extract on three different days. The intra-day andinter-day precision values of measured concentration of PHTwere calculated from linearity plots. In both situations, the rela-

tive standard deviation (RSD) values are approximately 1.0%.which is considered to be acceptable (Table 4).

Accuracy. Accuracy of the method was tested in terms of per-centage recovery. Recovery of PHT was evaluated at three sampleconcentrations of B. pilosa (50, 100 and 150 mg/mL; equivalentPHT content 12.1, 24.2 and 48.4 mg). A known amount (0.03 mg) ofPHT from the stock solution of of 30 mg/mL was added to the B.pilosa sample and extracted following the procedures describedabove. A percentage RSD value was determined as the differencebetween measured and expected values. The average recoverywith %RSD values is presented in Table 5.

Robustness. The robustness of the method was determined bymeasuring the effect of small and deliberate changes in the ana-lytical parameters on retention time and area under the peak. Theparameters that were taken into consideration were mobilephase composition with acid additive, flow rate and temperature.Each time only one parameter was changed while the otherswere kept constant. The concentration of acetic acid has a rela-tively higher influence on peak area. The standard deviations(%RSD) of retention time and peak area counts were calculatedfor each parameter and standard deviation (%RSD) valuesshowed the robustness of the method (Table 6).

ConclusionThe present work demonstrated the qualitative and quantitativeanalysis with common organic solvents and also an alternativemethod for effective extraction and concentration of phenyl-1,3,5-heptatriyne from the aerial part B. pilosa. Ultrasonic-assistedextraction with ethylacetate solvent was found to be suitablewith a run time of 10 min because of its simplicity, precision,accuracy and sensitivity. Alternatively, Triton X-100 gave compa-rable extraction efficiency of hydrophobic phenyl-1,3,5-

Table 4. Overview of method development for the quantitation of PHT in B. Pilosa

Parameters PHT

Retention time (min) (mean � SD) 8.522 � 0.1LinearityWorking concentration range 7.5–30 mg/mLRegression equation y = (20580 � 214)x - 121.6Correlation coefficient (r2) 0.9989Goodness of fit (Sy.x) 126,100Level of significant (p-value) <0.01Sensitivity

Limit of detection (LOD; mg/mL) 1.84Limit of quantitation (LOQ; mg/mL) 6.13

Precision and accuracyInstrumental (peak %RSD; n = 5; 30 mg/mL) 1.41Intra-day

Repeatability, PHT content (mean � SD; n = 9; %RSD) 0.0242 � 0.0007(1.03)

Inter-dayReproducibility, PHT content (mean � SD; day 1/

day 2/day 3; n = 3; %RSD)0.0241 � 0.0007/0.0251 � 0.0009/0.0235 � 0.0005

(1.03/1.03/1.02)One-way ANOVA (Bonferroni’s multiple comparison test)

t-value (day 1 vs day 2/day 1 vs day 3/ day 2 vs day 3)0.7044/0.6084/1.313 (p > 0.05)

704

P. Trivedi et al.


heptatriyne without a concentration step. The method isenvironment friendly and uses low extraction temperature,system energy and non-toxic solvent. It can be concluded that,because of the simple extraction procedure, high precision andaccuracy, the method may be useful for screening cytotoxicphenyl-1,3,5-heptatriyne of hydrophobic nature in raw materialsof B. pilosa as well as in the derived product.

AcknowledgementsThis work was financially supported as a Young Research Project(YSP-16) under Major Laboratory Programme 9.16 of the Insti-tute. The authors are grateful to Professor Ram Rajsekharan,Director, CIMAP, and Dr MM Gupta, Head, Analytical Chemistry forhelpful discussions and kind support during the progress of thework.

ReferencesAlvarez L, Marquina S, Villarreal ML, Alonso D, Aranda E, Delgado G. Bio-

active polyacetylenes from Bidens pilosa. Planta Medica 1996; 62: 355.Ashafa AOT and Afolayan AJ. Screening the root extracts from Biden pilosa

L. var. radiata (Asteraceae) for antimicrobial potentials. Journal ofMedicinal Plants Research 2009; 3: 568–572.

Bosch Ojeda C and Sánchez Rojas F. Separation and preconcentration bya cloud point extraction procedure for determination of metals: anoverview. Analytical and Bioanalytical Chemistry 2009; 394: 759–782.

Brandao MGL, Krettli AU, Soares LSR, Nery CGC and Marinuzzi HC. Anti-malarial activity of extracts and fractions from Bidens pilosa and Bidenspecies Asteraceae correlated with the presence of acetylenes and fla-vonoid compounds. Journal of Ethnopharmacology 1997; 57: 131–138.

Brandao MGL, Nery CGC, Mamao MAS and Krettli AU. Two methoxylatedflavone glycosides from Bidens pilosa. Phytochemistry 1998; 48: 397.

Carabias-Martínez R, Rodríguez-Gonzalo E, Moreno-Cordero B, Pérez-Pavón JL, García-Pinto C and Fernández Laespada E. Surfactant cloudpoint extraction and preconcentration of organic compounds prior tochromatography and capillary electrophoresis. Journal of Chromatog-raphy A 2000; 902: 251–265.

Chien SC, Young PH, Hsu YJ, Chen CH, Tien YJ, Shiu SY, Li TH, Yang CW,Marimuthu P, Tsai LFL and Yang WC. Anti-diabetic properties of threecommon Bidens pilosa variants in Taiwan. Phytochemistry 2009; 70:1246–1254.

Choi MPK, Chan KKC, Leung HW, Huie CW. Pressurized liquid extraction ofactive ingredients (ginsenosides) from medicinal plants using non-ionic surfactant solutions. Journal of Chromatography A 2003; 983:153–162.

Delgado B, Pino V, Ayala JH, Gonžalez V, Afonso AM. Nonionic surfactantmixtures: a new cloud-point extraction approach for the determina-tion of PAHs in seawater using HPLC with fluorimetric detection. Ana-lytica Chimica Acta 2004; 518: 165–172.

Dimo T, Rakotonirina SV, Tan PV, Azay J, Dongo E and Cros G. Leaf metha-nol extract of Bidens pilosa prevents and attenuates the hypertensioninduced by high-fructose diet in Wistar rats. Journal of Ethnopharma-cology 2002; 83: 183–191.

Fang Q, Yeung HW, Leung HW and Huie CW. Micellemediated extractionand preconcentration of ginsenosides from Chinese herbal medicine.Journal of Chromatography A 2000; 904: 47–55.

He J, Zhao Z, Shi Z, Zhao M, Li Y and Chang W. Analysis of isoflavonedaidzein in Puerariae radix with micelle-mediated extraction and pre-concentration. Journal of Agriculture and Food Chemistry 2005; 53:518–523.

Hoffmann B and Holzl J. Chalcone glucosides from Bidens pilosa. Phy-tochemistry 1989; 28: 247–249.

Hsu YJ, Lee TH, Chang LTC, Huang YT and Yanga WC. Anti-hyperglycemiceffects and mechanism of Bidens pilosa water extract. Journal of Eth-nopharmacology 2009; 122: 379–383.

Huie CW. A review of modern sample-preparation techniques for theextraction and analysis of medicinal plants. Analytical and Bioanalyti-cal Chemistry 2002; 373: 23–30.

Hung KC, Chen BH and Yu LE. Cloud-point extraction of selected polycy-clic aromatic hydrocarbons by nonionic surfactants. Separation andPurification Methods 2007; 57: 1–10.

ICH. (Q2) Proceedings of the International Conference on Harmonization.IFPMA, Geneva, November, Step 4 Version, 2005.

IUPAC. Harmonized guidelines for single-laboratory validation ofmethods of analysis (IUPAC Technical Report). Pure and Applied Chem-istry 2002; 74: 835–855.

Jiang Y, David B, Tu P and Barbin Y. Recent analytical approaches inquality control of traditional Chinese medicines—a review. AnalyticaChimica Acta 2010; 657: 9–18.

Kiathevest K, Goto M, Sasaki M, Pavasant P and Shotipruk A. Extractionand concentration of anthraquinones from roots of Morinda citrifoliaby non-ionic surfactant solution. Separation and Purification Technol-ogy 2009; 66: 111–117.

Kim HK and Verpoorte R. Sample preparation for plant metabolomics.Phytochemical Analytical 2010; 21: 4–13.

Kumar JK and Sinha AK. A new disubstituted acetyl acetone from theleaves of Bidens pilosa Linn. Natural Product Research 2003; 17: 71–74.

Table 5. Results of recovery test

PHT in B. pilosasample (mg)

Spiked amount(mg)

Theoreticalvalue (mg)

Observedvalue (mg)

Recovery(%)

Average recovery(% RSD)

12.1 0.03 12.13 12.07 99.51 99.64 (0.55)12.16 100.2512.03 99.18

24.2 0.03 24.23 24.17 99.75 99.94 (0.19)24.22 99.9624.26 100.12

48.4 0.03 48.43 48.47 100.08 100.20 (0.12)48.59 100.3348.52 100.19

Table 6. Results of robustness data

Parameters Variations (%RSD)Retention

timeArea under

peak

Mobile phase compositionAcetonitrile (�1.0%) 0.32 1.86AcOH (�1.0%) 0.18 2.14

Flow rate (�10%) 0.42 1.45Column temperature (�2°C) 0.48 2.02

705



Kumari P, Misra K, Sisodia BS, Faridi U, Srivastava S, Luqman S, Darokar MP,Negi AS, Gupta MM, Singh SC and Kumar JK. A promising anticancerand antimalarial component from the leaves of Bidens pilosa. PlantaMedica 2009; 75: 59–61.

Kviecinski MR, Felipe KB, Schoenfelder T, Wiese LPL, Rossi MH, Goncalez E,Felicio JD, Filho DW and Pedrosa RC. Study of the antitumor potentialof Bidens pilosa (Asteraceae) used in Brazilian folk medicine. Journal ofEthnopharmacology 2008; 117: 69–75.

Lee TH, Lu CK, Kuo YH, Lo JM and Lee CK. Unexpected novel pheophytinperoxides from the leaves of Biden pilosa. Helvetica Chimica Acta 2008;91: 79–84.

Lin YL, Chang CC and Lee IJ. Review on phytochemical study of asteraceaein Taiwan (1996–2005). Journal of Chinese Medicine 2008; 19: 135–149.

Marles RJ and Farnsworth NR. Antidiabetic plants and their active con-stituents. Phytomedicine 1995; 2: 137–189.

Martıınez RC, Gonzalo ER, Alvarez JD and Meındez JH. Cloud point extrac-tion as a preconcentration step prior to capillary electrophoresis. Ana-lytical Chemistry 1999; 71: 2468–2474.

Merino F, Rubio S and Bendito DP. Mixed aggregate-based acid-inducedcloud-point extraction and ion-trap liquid chromatography–massspectrometry for the determination of cationic surfactants in sewagesludge. Journal of Chromatography A 2003; 998: 143–154.

Paleologos EK, Giokas DL and Karayannis MI. Micelle-mediated separationand cloud-point extraction. Analytical Chemistry 2005; 24(5): 426–436.

Pereira RL, Ibrahim T, Lucchetti L, da Silva AJ and Goncalves de Moraes VL.Immunosuppressive and anti-inflammatory effects of methanolicextract and the polyacetylene isolated from Bidens pilosa L. Immunop-harmacology 1999; 43: 31–37.

Sanz CP, Halko R, Ferrera ZS and Rodriguez JJS. Combination of micro-wave assisted micellar extraction and liquid chromatography for thedetermination of organophosphorous pesticides in soil samples.Journal of Chromatography A 2005; 1078: 13–21.

Sashida Y, Ogawa K, Kitada M, Karikome H, Mimaki Y and Shimomura H.New aurone glucosides from Bidens pilosa. Chemical and Pharmaceu-tical Bulletin 1991; 39: 709–11.

Shanker K, Fatima A, Negi AS, Gupta VK, Darokar MP, Gupta MM andKhanuja SPS. RP-HPLC method for the quantitation of glabridin inYashti-madhu (Glycyrrhiza glabra). Chromatographia 2007; 65: 771–774.

Shanker K, Singh SC, Pant S, Srivastava P, Pandey R, Verma RK and GuptaMM. Quantitative TLC analysis of sterol (24b-ethylcholesta-5,22E,25-triene-3b-ol) in Agnimantha (Clerodendrum phlomidis Linn). Chro-matographia 2008; 67: 269–274.

Sirimanne SR, Patterson DG, Ma L and Justice JB. Application of cloud-point extraction-reversed-phase high-performance liquid chroma-tography: a preliminary study of the extraction and quantification ofvitamins A and E in human serum and whole blood. Journal of Chro-matography B 1998; 716: 129–137.

Steele RGD and Torrie JH. Principles and Procedures of Statistics. McGraw–Hill: New York, 1980.

Sun C, Xie Y, Tian Q and Liu H. Analysis of glycyrrhizic acid and liquiritin inliquorice root with microwave-assisted micellar extraction and pre-concentration. Phytochemical Analysis 2008; 19: 160–163.

Sundararajan P, Dey A, Smith A, Doss AG, Rajappan M and Natarajan S.Studies of anticancer and antipyretic activity of Bidens pilosa wholeplant. African Health Science 2006; 6: 27–30.

Ubillas RP, Mendez CD, Jolad SD, Luo J, King SR, Carlson TJ and Fort DM.Antihyperglycemic acetylenic glucosides from Bidens pilosa. PlantaMedica 2000; 66: 82–83.

Wang J, Yang H, Lin ZW and Sun HD. Flavonoids from. Bidens pilosa var.radiata. Phytochemistry 1997; 46: 1275–1278.

Wang Z. The potential of cloud point system as a novel two-phase parti-tioning system for biotransformation. Applied Microbiology and Bio-technology 2007; 75: 1–10.

Wu LW, Chiang YM, Chuang HC, Wang SY, Yang GW and Chen YH. Poly-acetylenes function as anti-angiogenic agents. PharmaceuticalResearch 2004; 21: 2112–2119.

Wu R and Zhang ZQ. Microwave-assisted extraction and high-performance liquid chromatography for simultaneous determinationof osthole and isoimperatorin in Libanotis buethorimensis. AnalyticalLetters 2010; 43: 34–44.

Wu YC and Huang SD. Cloud point preconcentration and liquid chro-matographic determination of aromatic amines in dyestuffs. AnalyticaChimica Acta 1998; 373: 197–206.

Yadav AK, Yadav D, Shanker K, Verma RK, Saxena AK and Gupta MM.Flavone glycoside based validated RP-LC method for quality evalua-tion of Prishniparni (Uraria picta). Chromatographia 2009; 69: 653–658.

Zhou J, Suna XL and Wang SW. Micelle-mediated extraction and cloud-point preconcentration of osthole and imperatorin from Cnidiummonnieri with analysis by high performance liquid chromatography.Journal of Chromatography A 2008; 1200: 93–99.

Zulueta MCA, Tada M and Ragasa CY. A diterpene from Bidens pilosa.Phytochemistry 1995; 38: 1449–1450.

706

P. Trivedi et al.


hplc method development and validation of cytotoxic agent phenyl-heptatriyne in bidens pilosa with...

Documents