LJLC #725692, VOL 36, ISS 19

DETERMINATION OF PACLITAXEL BY 3D VIEW LC-DIODE ARRAYUV: ITS APPLICATION TO AN IN SITU CLOSED LOOPRE-CIRCULATING INTESTINE ABSORPTION STUDY IN RATS

Sekar Vasanthakumar, Viswanathan Karthikeyan, Ali Rashid, andRanendra N. Saha

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DETERMINATION OF PACLITAXEL BY 3D VIEW LC-DIODE ARRAY UV: ITS APPLICATION TO AN

IN SITU CLOSED LOOP RE-CIRCULATING INTESTINE ABSORPTION STUDY IN

RATS & Sekar Vasanthakumar, Viswanathan Karthikeyan, Ali Rashid, and Ranendra N. Saha

DETERMINATION OF PACLITAXEL BY 3D VIEW LC-DIODE ARRAYUV: ITS APPLICATION TO AN IN SITU CLOSED LOOPRE-CIRCULATING INTESTINE ABSORPTION STUDY IN RATS

Sekar Vasanthakumar, Viswanathan Karthikeyan, Ali Rashid, and5Ranendra N. Saha

Department of Pharmacy, Birla Institute of Technology and Science (BITS-Pilani),Formulation Development and Pharmacokinetic Laboratory, Rajasthan, India

& Paclitaxel (TAX) amount remaining to be absorbed from the intestine in the in situ closed loop,re-circulating absorption samples were estimated by the 3D view LC-diode array method. The Log

10ARA versus time graph (r2) of TAX loaded nanoparticles (NP)s explains that there is exponentialdisappearance of TAX from the intestine, which indicates TAX NPs follows first order absorptionkinetic. The calculated Ka and t1=2 of TAX and TAX loaded NPs were 0.9, 1.02, 1.38, and 1.74=min and 46.20, 40.66, 30.09, and 23.88min, respectively, shows the rapid absorption of TAXNPs from the intestine than the pure drug TAX. The 3D view chromatogram, counter view graph,

15UV, and IR spectrum of the degradation samples confirms the selectivity and specificity of thedeveloped method. The peak purity curve of all the samples shows that the similarity curve didnot intersect the threshold curve and the rectangular ratiograms, in addition, demonstrates the sel-ectivity and specificity nature of the 3D view LC-method. The accuracy of the developed methodswas tested by placebo spiking and standard addition method. The LOD and LLOQ of the methods

200.39lgmL,�1 1.18 lgmL�1 and 2.96 ngmL,�1 8.96 ngmL�1 respectively.

Keywords 3D chromatogram, closed loop re-circulating absorption, counter view plot,paclitaxel, purity curve, ratiogram

INTRODUCTION

Paclitaxel (TAX) is the most unique natural diterpene pseudoalkaloid25isolated from the bark of Taxus brevifolia (pacific or western yew tree).

TAX is the preferred anticancer drug in the past three decades for manytumors including gastric, breast, ovarian, head, neck, and lung tumors

Address correspondence to Ranendra N. Saha, FD-III, Department of Pharmacy FormulationDevelopment and Pharmacokinetic Laboratory, Birla Institute of Technology and Science (BITS),Pilani, Rajasthan, India. E-mail: rnsvks@yahoo.co.in

Journal of Liquid Chromatography & Related Technologies, 36:1–33, 2013Copyright # Taylor & Francis Group, LLCISSN: 1082-6076 print/1520-572X onlineDOI: 10.1080/10826076.2012.725692

3b2 Version Number : 7.51c/W (Jun 11 2001)File path : P:/Santype/Journals/TandF_Production/LJLC/v36n19/LJLC725692/LJLC725692.3dDate and Time : 03/06/13 and 13:17

Journal of Liquid Chromatography & Related Technologies, 36:1–33, 2013Copyright # Taylor & Francis Group, LLCISSN: 1082-6076 print/1520-572X onlineDOI: 10.1080/10826076.2012.725692

because of its unique mechanism of action[1] and clinically effectivetherapeutic benefit.[2]

30TAX has wide limited physiochemical properties[3] which makes itdifficult to formulate TAX as the delivery system. TAX is currently marketedas an intravenous solution using Cremophore EL which produces fatalhypersensitivity reaction at the dose (175 mg=m2) used in the cancerpatients. This results in plenty of particulate drug delivery system (PDDS)

35for TAX in literature in the last five years such as microparticles (MPs), lipo-some, nanoparticles (NPs), and microemulsion.[4,5] Estimation of drugcontent in drug loaded particles and their respective drug release in thedissolution medium is a routine work in the development of PDDS. Inliterature, researchers used complicated procedures and methods[6–9] to

40estimate TAX from PDDS. In formulation development, the optimizeddelivery systems are subjected to an in situ rat intestine absorption studyto determine the rate of absorption and absorption half-life. In most ofthe formulation research and development labs, those who work withTAX use a lesser amount of drug in the trial batch because TAX is costly.

45All this issue clearly says that there is always need for sensitive (with low,limit of detection [LOD], and lower limit of quantification [LLOQ]), selec-tive, accurate, and specific method for the estimation of TAX in PDDSwhich can be easily adapted for the routine analysis in any laboratories.Hence, development of simple, rapid, and routine LC method which can

50be applied to estimate TAX from PDDS, selectively and specifically isrequired. If the developed method can be applicable to estimate the in situsample, it is more convenient to develop a successful formulation.

Fonseca et al.[8]Q1 used HPLC method to determine TAX content in NPs,

where specific amount of lyophilized NPs was dissolved in 1 mL dichloro-55methane (DCM) and mixed with 5 mL of acetonitrile:2 mM phosphoric

acid solution (50:50, v=v). The mixture was then vortexed vigorously for5 min and DCM was evaporated under a nitrogen stream until a clear sol-ution was obtained. The final solution was diluted with acetonitrile:2 mMphosphoric acid solution (50:50, v=v) and used for TAX analysis. This com-

60plicated and sophisticated procedure may not be suitable for routine analy-sis. Hence, there is a need for more simple and routine analytical methodand procedure to determine TAX from the Nanoparticulate Drug DeliverySystem (NPDDS). Many bioanalytical methods have been developed todetermine TAX in biomatrix (serum, plasma and tissue) in the past one

65decade like HPLC-UV, immunoassays, capillary electrophoresis, andLCMS-MS.

Recently, we published a 3D view LC-UV method for the estimation ofTAX from micro volume rat plasma.[1] All these methods cannot beadopted directly for the quantification of TAX in PDDS. The 3D view LC

70diode array analytical method is the ideal choice for the development of

2 S. Vasanthakumar et al.

simple, rapid, selective, sensitive, and accurate method because it allow theanalyst to record chromatogram in 3D view which clearly shows the interfer-ences of other co-eluting peak with the drug peak and the counter view plotexplains better the lack of possible interaction with other co-eluting or

75degradation product peaks.Our laboratory is working on formulating novel drug delivery systems

for many challenging drugs.[5] Our group has published many analyticaland bioanalytical chromatography methods and spectrophotometric=spec-trofluorimetric methods for different classes of drugs.[10–12]

80To the best of our knowledge and a thorough literature search revealedthat there is no simple, selective, and specific method for TAX determi-nation in bulk and NPDDS by 3D view LC-diode array UV with peak puritydetermination by counter view plot, ratiograms, and purity curve. There isno reported method for the determination of absolute recovery of TAX

85from the NPDDS by placebo spiking and standard addition method.In the present study, an attempt has been made to develop and validate a

simple, rapid, sensitive, accurate, reproducible, selective, and specific 3D viewLC- diode array UV method for quantification of TAX in bulk and NPDDS.The developed 3D view LC method was applied to estimate TAX content in

90NPDDS, TAX release in dissolution medium from NPs and in addition, itwas used for the estimation of TAX in the in situ closed loop re-circulatingabsorption study samples. With the 3D view LC-diode array UV method, a sim-ple, selective, accurate, and precise UV-spectrophotometric method was alsodeveloped and validated. The advantages of the present method over the

95previously published methods are given in Table 1.

EXPERIMENTAL

Materials and Methods

ChemicalsA generous gift sample of TAX (assay 99.95%) was provided by GetWell

100Pharmaceuticals, India. Solvents acetonitrile, ethanol, and dimethylsulfox-ide (DMSO) used were of UV and HPLC grade from Spectrochem, India.All other chemicals used were analytical grade procured from S.D. FineChemicals Ltd. India. Fresh triple distilled water (TDW) prepared in quartzdistillation assembly was used throughout the experiment after filtering

105through, 0.22 mm membrane using Millipore ultra-filtration system(Millipore, France).

PolyE-caprolactone (PCL, molecular weight 40,000), Poly (DL-lactic-co-glycolic acid) (PLGA, 50:50, inherent viscosity 0.15–0.25dL=g (lit), averagemolecular weight 5,000–15,000), Poly (DL-lactic acid) (PLA, inherent

3D View LC-Diode Array UV Meth 3

TABLE

1C

om

par

iso

no

fP

ub

lish

edM

eth

od

toth

eP

rese

nt

3DV

iew

LC

-Dio

de

Arr

ayM

eth

od

Fo

rmu

lati

on

Sam

ple

An

alys

isP

roce

du

re

Dis

adva

nta

geC

om

par

edto

Pre

sen

t3D

Vie

wL

C-D

iod

eA

rray

Met

ho

dR

efer

ence

PL

GA

and

PL

AN

Ps

Dru

gco

nte

nt

and

In-v

itro

dis

solu

tio

n

TA

Xre

leas

edin

the

dis

solu

tio

nm

ediu

mw

asex

trac

ted

wit

h1

mL

of

DC

M,

afte

rth

atD

CM

was

evap

ora

ted

.T

oth

ed

ryre

sid

ue,

acet

on

itri

le:w

ater

(50:

50v=

v)w

asad

ded

and

anal

yzed

by

HP

LC

.

Ext

ract

ion

effi

cien

cyo

fD

CM

was

no

tgi

ven

.L

LE

isn

ot

ago

od

met

ho

do

fex

trac

tio

nfo

rro

uti

ne

anal

ysis

.

Xie

and

Wan

g[9]

PC

L=P

F-6

8N

Ps

Dru

gco

nte

nt

and

In-v

itro

dis

solu

tio

n

NP

sw

ere

dis

solv

edin

DC

Mu

nd

ervi

goro

us

vort

exin

gan

dth

isso

luti

on

was

tran

sfer

red

to5

mL

of

acet

on

itri

le:

wat

er(5

0:50

,v=v)

mix

ture

.D

CM

was

evap

ora

ted

ina

nit

roge

nat

mo

sph

ere

and

itis

filt

ered

thro

ugh

0.22

mmsy

rin

gefi

lter

toge

tth

ecl

ear

solu

tio

nan

dit

isu

sed

for

dru

gco

nte

nt

anal

ysis

by

HP

LC

.T

AX

rele

ased

inth

ed

isso

luti

on

med

ium

was

extr

acte

dw

ith

2m

Lo

fD

CM

and

then

DC

Mw

asev

apo

rate

du

nd

ern

itro

gen

stre

am.

To

the

dry

resi

du

e5

mL

of

mo

bil

ew

asad

ded

and

anal

yzed

by

HP

LC

.

Co

mp

lica

ted

extr

acti

on

pro

ced

ure

,so

ph

isti

cate

dsa

mp

lep

roce

ssin

g,ti

me

con

sum

ing

extr

acti

on

step

,an

dex

trac

tio

nef

fici

ency

of

DC

Mw

asn

ot

give

n.

Hen

ce,

this

met

ho

dm

ayn

ot

be

suit

able

for

rou

tin

ean

alys

is.

Inth

ep

rese

nt

met

ho

dN

Ps

are

dig

este

dw

ith

acet

on

itri

lew

ith

30m

inso

nic

atio

n.

Zh

ang

etal

.[23

]

PL

A-P

EG

NP

sD

rug

con

ten

tan

dIn

-vit

rod

isso

luti

on

Nan

osu

spen

sio

n=d

isso

luti

on

sam

ple

was

dri

edu

nd

era

nit

roge

nst

ream

,an

dth

end

isso

lved

in1

mL

of

DC

Man

dth

en5

mL

of

acet

on

itri

le:

wat

er(5

0:50

v=v)

was

add

edan

dvo

rtex

edvi

goro

usl

yfo

r2

min

.N

itro

gen

stre

amw

asin

tro

du

ced

toth

esa

mp

leu

nti

lth

ep

oly

mer

pre

cip

itat

esan

da

clea

rso

luti

on

was

ob

tain

ed.

Th

ep

reci

pit

ated

po

lym

erso

luti

on

was

cen

trif

uge

dfo

r10

min

at50

0rp

man

dth

esu

per

nat

ant

was

coll

ecte

d.

No

wth

esu

per

nat

ant

was

was

hed

wit

h5

mL

of

acet

on

itri

le:

wat

er(5

0:50

,v=v)

and

cen

trif

uge

dat

5000

rpm

for

10m

in.

Th

esu

per

nat

ant

was

coll

ecte

dan

dvo

lum

em

ade

wit

hac

eto

nit

rile

:wat

er(5

0:50

,v=v)

and

the

dru

gco

nte

nt

was

det

erm

ined

usi

ng

HP

LC

.

Th

eex

trac

tio

nef

fici

ency

of

this

met

ho

dw

as95

.6�

2.6%

.T

he

lin

eari

tyo

fth

ism

eth

od

was

0.05

–50mg

=m

L,

inth

ep

rese

nt

met

ho

dth

eli

nea

rity

was

10–1

500

ng=

mL

.T

he

pre

sen

tm

eth

od

ism

ore

sen

siti

ve,

LO

Q8.

96an

dL

OD

2.96

ng=

mL

.T

he%

reco

very

of

the

pre

sen

tm

eth

od

sw

asin

the

ran

geo

f99

.18

to10

1.92

and

99.8

1to

100.

37[n

¼9,

RSD

(%)¼

0.09

to2.

30an

d0.

04to

0.31

,re

spec

tive

ly].

Pu

lkki

nen

etal

.[24

]

PL

GA

NP

sD

rug

con

ten

tN

Ps

wer

ed

isso

lved

in1

mL

of

DC

Man

d0.

5m

Lo

fac

eto

nit

rile

,vo

rtex

edan

dth

enag

itat

edat

37� C

for

Co

mp

lica

ted

extr

acti

on

pro

ced

ure

,ti

me

con

sum

ing

extr

acti

on

step

and

extr

acti

on

effi

cien

cyo

fD

CM

Ch

akra

vart

hi

etal

.[25

]

4

30m

in.

Nit

roge

nst

ream

was

intr

od

uce

dto

evap

ora

teal

lso

lven

tan

dth

en5

mL

of

acet

on

itri

lew

asad

ded

toth

eex

trac

tan

dag

itat

edo

vern

igh

tin

are

cip

roca

lsh

akin

gb

ath

at37

� Can

d50

rpm

tod

isso

lve

TA

X.

Th

isso

luti

on

was

anal

yzed

tod

eter

min

eth

ed

rug

con

ten

t.

was

no

tgi

ven

.H

ence

,th

ism

eth

od

may

no

tb

esu

itab

lefo

rro

uti

ne

anal

ysis

.

PE

G-p

oly

cyan

oac

r-yl

ate

NP

sD

rug

con

ten

tN

Ps

wer

ed

isso

lved

in2

mL

of

DC

Man

dsh

aken

,th

en4

mL

of

am

ixtu

reo

fac

eto

nit

rile

:w

ater

(1:1

,v=v)

was

add

ed.

Nit

roge

nst

ream

was

intr

od

uce

dto

evap

ora

ted

DC

Man

dth

eso

luti

on

was

anal

yzed

for

dru

gco

nte

nt

by

HP

LC

.

Co

mp

lica

ted

extr

acti

on

pro

ced

ure

may

no

tb

esu

itab

lefo

rro

uti

ne

anal

ysis

.X

uet

al.[2

6]

PL

GA

NP

sD

rug

con

ten

tN

Ps

wer

ed

isso

lved

in1

mL

of

DC

Man

dm

ixed

wit

h5

mL

of

anac

eto

nit

rile

:2

mM

ph

osp

ho

ric

acid

solu

tio

n(5

0:50

,v=v)

.T

he

mix

ture

was

then

vort

exed

vigo

rou

sly

for

5m

inan

dD

CM

was

evap

ora

ted

un

der

an

itro

gen

stre

amu

nti

la

clea

rso

luti

on

was

ob

tain

ed.

Th

efi

nal

solu

tio

nw

asd

ilu

ted

wit

hac

eto

nit

rile

:2

mM

ph

osp

ho

ric

acid

solu

tio

nan

du

sed

for

TA

Xan

alys

isu

sin

gH

PL

C.

Use

of

bu

ffer

inth

eex

trac

tio

nst

epis

no

ta

sim

ple

pro

ced

ure

,h

ence

may

no

tb

esu

itab

lefo

rro

uti

ne

anal

ysis

.L

LE

isn

ot

ago

od

met

ho

do

fex

trac

tio

nfo

rro

uti

ne

anal

ysis

.

Fo

nse

caet

al.[2

7]

PL

GA

NP

sD

rug

con

ten

tan

dIn

-vit

rod

isso

luti

on

Per

cen

td

rug

rele

ase

was

esti

mat

edb

yd

isso

lvin

gth

ely

op

hil

ized

po

wd

erin

1m

Lo

fac

eto

nit

rile

for

16h

rat

37� C

.

Pro

cess

ing

tim

eis

very

hig

h,i

nth

ep

rese

nt

met

ho

dit

was

on

ly30

min

,w

ith

son

icat

ion

.N

ot

mo

resu

itab

lefo

rro

uti

ne

anal

ysis

.

De

etal

.[18

]

MP

EG

-PL

AD

rug

con

ten

tan

dIn

-vit

rod

isso

luti

on

NP

sw

ere

dis

solv

edco

mp

lete

din

1m

Lo

fD

CM

and

the

solv

ent

was

evap

ora

ted

.T

he

resi

du

ew

asre

con

stit

ute

din

5m

Lm

ob

ile

ph

ase,

acet

on

itri

le:w

ater

(50:

50,v=v)

and

anal

yzed

for

TA

Xco

nte

nt

by

HP

LC

Th

eT

AX

rele

ased

inth

ed

isso

luti

on

med

ium

was

extr

acte

dw

ith

1m

LD

CM

and

anal

yzed

by

HP

LC

.

Ext

ract

ion

effi

cien

cyo

fD

CM

was

no

tgi

ven

.L

LE

isn

ot

ago

od

met

ho

do

fex

trac

tio

nfo

rro

uti

ne

anal

ysis

.E

xtra

ctio

no

fT

AX

fro

mth

ed

isso

luti

on

med

ium

usi

ng

DC

Mis

no

tsi

mp

lem

eth

od

for

rou

tin

ean

alys

is.

Do

ng

and

Fo

ng[2

8]

PL

GA

NP

sD

rug

con

ten

tT

AX

fro

mth

eN

Ps

was

extr

acte

dw

ith

1m

lo

fm

eth

ano

lo

na

shak

erat

100

rpm

for

48h

r.T

AX

con

ten

tin

the

sup

ern

atan

tw

asan

alyz

edb

yH

PL

C.

Mo

bil

ep

has

eu

sed

was

amm

on

ium

acet

ate

(10

mM

,p

H4.

0)an

dac

eto

nit

rile

inth

era

tio

of

45:5

5(v=v)

,b

ut

inth

ep

rese

nt

met

ho

d,

the

mo

bil

ep

has

eu

sed

was

very

sim

ple

,u

seo

fb

uff

erca

nb

eav

oid

edb

ecau

seit

crea

tes

add

itio

nal

pre

ssu

reo

nth

eco

lum

nw

hic

hm

ayh

ind

erco

nti

nu

esan

alys

is.

Ext

ract

ion

of

TA

Xfr

om

the

NP

sta

kes

48h

rw

hic

h

Ch

avan

pat

ilet

al.[2

9]

(Continued

)

5

TABLE1

Co

nti

nu

ed

Fo

rmu

lati

on

Sam

ple

An

alys

isP

roce

du

re

Dis

adva

nta

geC

om

par

edto

Pre

sen

t3D

Vie

wL

C-D

iod

eA

rray

Met

ho

dR

efer

ence

clea

rly

says

that

itis

no

tsu

itab

lefo

rro

uti

ne

anal

ysis

.P

LA

MP

sD

rug

con

ten

tan

dIn

-vit

rod

isso

luti

on

MP

sw

ere

dis

solv

edin

1m

Lo

fD

CM

,th

en20

mL

of

60:4

0ac

eto

nit

rile

:w

ater

add

ed.

Th

etw

ocl

ear

ph

ase

wer

eal

low

edto

sep

arat

ew

her

eP

LA

pre

cip

itat

ein

the

inte

rfac

e.T

he

pac

lita

xel

con

ten

tin

the

org

anic

and

aqu

eou

sp

has

ew

ere

det

erm

ined

by

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110viscosity 0.55–0.75dL=g (lit), average molecular weight 75,000–120,000)and Pluronic F 68 (F68, average molecular weight˜ 8,400) were purchasedfrom Sigma-Aldrich chemicals, United States of America. Surgical instru-ments scissors, forceps, suture thread, glass hypodermic syringes, threeway stopcocks, and so forth, required for the surgery, were procured from

115the Niraj Industries Pvt. Ltd. India.

Instruments

UV-SpectrophotometerA double-beam Jasco (Tokyo Japan) UV-spectrophotometer model

120V630 connected to computer (DELL), which is loaded with spectramanager software. This software was used for spectral and fixed wavelengthanalysis and for the data acquisition and processing. For the inter-instrument repeatability study, a double-beam Jasco (Tokyo Japan) UV–VIS–NIR (near IR) spectrophotometer model V570 connected to

125computer (LG Studioworks 440 si) was used.

Liquid ChromatographyThe UFLC (Ultra-Fast Liquid Chromatography) system (Shimadzu pro-

minence Japan) consisting of a pump (LC-20AD) with integrated systemcontroller auto sampler (SIL-20AC) and variable wavelength UV detector

130diode array detector (SPD-M20A). Data acquisition and analysis was per-formed in postrun analysis using LC solution software (Shimadzu Japan).The column and UFLC system were kept in ambient condition throughoutthe development and validation experiment.

Peristaltic Pump135The peristaltic pump (Heidolph Mehrkanal Pumpenkopf C4

524-80420-00 AC 230 V 5060 HZ 125 W Germany) is assembled with a pumpdrive (Heidolph Pumdrive PD 5101) and a cassette (Heidolph cassettesmall). The peristaltic pump controls the circulation of drug solutionthroughout the intestine in re-circulating manner through the silicone

140tubing (116’ i.d. x 316’ o.d).

Chromatographic SeparationThe chromatographic separation of TAX was carried out in reverse

phase Luna C8(2) end capped 250� 4.60 mm column with the followingspecification 5mm sphere shape particles 100 A pore size 400 m2g surface

3D View LC-Diode Array UV Meth 7

145area 13.5% carbon load and calculated bonded phase coverage of5.50 mmole m2 (PhenomenexUSA).

UV-Spectrophotometric ConditionsThe spectrum analysis was carried out between 200–400 nm and the

fixed wavelength measurement was recorded at 229 nm using 10 mm150matched quartz cells at a speed of 400 nm=min using medium response

and 1.0 nm bandwidth.

Method Development and Validation

Preparation of Stock, Calibration Curve and Quality Control (QC) Standardsfor UV-Spectrophotometric Method

155A master stock solution of TAX (1 mg mL�1) was prepared in ethanol.To get desired calibration curve standards, 6–34mg mL�1 different aliquots(30–170 mL) were taken from master stock solution into a series of standard5 mL volumetric flask and volume made up through diluent acetonitrileand water, 85:15%, vv.

160On three consecutive days of validation, three separate series of eight cali-bration curve standards were prepared fresh and their absorbance wasrecorded in fixed wavelength mode at 229 nm keeping diluent as blank. QCsamples were prepared at three concentrations lower (LQC 6mg mL�1),medium (MQC 18mg mL�1), and higher (HQC 30mg mL�1) from the master

165stock solution like the calibrator sample.

Preparation of Stock Calibration Curve and QC Standards for LC MethodTax master stock solution of 1 mg mL�1 in DMSO was prepared and

stored in glass ampoules at �20�C. This stock was further serially dilutedto get working standard in the range of 30–0.2mg mL�1 from this nine

170calibration curve standards (10–1500 ng mL�1) were prepared.On each day of validation QC samples were prepared at 10, 25, 500, and

1500 ng mL�1 from independent stock and working standard like thecalibrator sample.

Selectivity=Specificity175In case of UV-spectrophotometry method, selectivity is performed by

spiking 50 and 100% of TAX as per the label claim (6 mg mL�1) into thePCL placebo NPs and the spectrum was compared with the freshly pre-pared pure drug calibrator sample 6 and 18 mg mL�1 (n¼ 5).

8 S. Vasanthakumar et al.

The overlaid spectrum and accuracy in terms of % recovery confirm the180selectivity of the method. Paired t-test was performed at 95% level of signif-

icances to the spiked sample and to the pure drug sample.The selectivity of the developed 3D view LC method at LLOQ

(10 ng mL�1) was determined by placebo and spiked-placebo analysis tech-nique. In this method, on three consecutive days the placebo PCL NPs for-

185mulation chromatograms was compared with the fresh chromatogram ofdrug spiked at LLOQ level in placebo PCL NPs formulation standardsand QC standard LLOQ. In addition, the % recovery of TAX was comparedbetween QC standard LLOQ and drug spiked in placebo PCL NPs. On theother hand, the placebo chromatogram was compared with the diluent

190chromatogram. The PCL NPs formulation standard samples were pro-cessed for analysis by the procedure given in the sample preparation andall the samples were prepared in triplicate on each day of analysis.

Forced Degradation Study

Oxidative Stress Treatment (OST)195TAX was intentionally subjected to oxidation by treating with 6% v=v

hydrogen peroxide (HPO), HPO can be used in the range of 3–30% foroxidizing any drug molecules. To the 2 mg of drug taken in 5 mL of volu-metric flask, 2 mL of HPO (6% vv) was added and vortexed in multi holdervortex mixer (SPINIX Multilab, India) for 2 min. This one milligram per

200milliliter drug solution was kept at 25�C for 12 hr, in a temperature con-trolled orbital shaking incubator chamber (MAC Macro Scientific Works(R) Delhi India). The oxidized drug solution was analyzed by thedeveloped 3D view LC method.

Forced Hydrolytic Degradation Study (FDS)205TAX was subjected to hydrolysis in acidic and alkaline condition by a

microwave based fast and effective technique.[11] The drug suspension1 mg mL�1 was prepared by adding 5 mL of 0.1 N HCl or 0.1 N NaOH toa 5-mL volumetric flask which contain 5 mg of TAX for acidic=alkalinehydrolysis. The prepared drug solution was hydrolyzed by subjecting it to

210microwave radiation (LG microwave appliance LG Grill intellowaveMG-555 F frequency 2450 MHz 2650 W 230v-50 Hz LG Electronics IndiaPvt. Ltd.) for 20 min by 10 cycle with 2.45 GHz 300 W and 80% intensity.During the radiation treatment, care was taken to avoid bubbling of thesolution. Once the treatment was over, the samples were allowed to cool

215and then neutralized to stop further reaction and then analyzed by thedeveloped 3D view LC method.

3D View LC-Diode Array UV Meth 9

Thermal Stress Treatment (TST)TAX was subjected to thermal stress treatment[13] in liquid state by

exposing the drug solution to 90�C in a temperature controlled oven220(MAC Macro scientific works (R) Delhi India). Drug solution of

1 mg mL�1 was prepared in ethanol and exposed to the above temperaturefor 12 hr and then the sample was suitable diluted and analyzed by thedeveloped method.

The selectivity and specificity of the developed method was tested by225observing the retention time of TAX in the degraded sample through 3D

view mode and by calculating the percent TAX degraded. The 3D viewchromatogram was constructed using absorbance range from 225 to230 nm; this range was selected because TAX was having good absorbencyin this nanometer.

230The 3D view LC diode array method further confirms the selectivity andspecificity by constructing a counter view graph, which will clearly showswhether TAX was separated from the degradation products. In addition,the 3D view LC diode array UV method is used to record UV spectra corre-sponding to a specific time period. In the present method, UV spectrum of

235the degraded samples which was recorded simultaneously during chroma-tographic analysis, was obtained.

Specificity will be further confirmed by constructing the peak puritycurve by total peak and three point view method at the retention time ofTAX. The peak purity curve result was supported by the ratiogram which

240is constructed by plotting ratio of absorbency=response of TAX at three dif-ferent wavelengths in the range of 227–232 over the retention time of TAXat 6.66� 0.18 min. The rectangular ratiograms show that the peak was pureand, hence, the developed method was selective and specific.

Precision

245Precision of the developed methods were tested in different headsrepeatability with intra-day precision and reproducibility by intermediate(inter-day) precision. Intra-day precision experiment was performed byanalyzing six=nine separate QC samples within a day on three differentoccasions; it gives the precision with in the same concentration (n¼ 6 or

250n¼ 9) in a day and between the same set of concentrations (n¼ 18=27)in a day. Inter-day precision was performed with the QC samples betweendays for three days (n¼ 18=27). In addition, a UV-spectrophotometricmethod was validated for inter-instrument repeatability where six sets ofQC samples were analyzed using a different instrument with the same speci-

255fication. The precision of the developed methods were represented byactual mean concentration and % RSD.

10 S. Vasanthakumar et al.

Accuracy

In addition to the intra-day and inter-day accuracy, developed methodswere supported by studying the TAX recovery by two unique techniques,

260placebo spiking and standard addition using NPDDS. In the case ofUV-spectrophotometric and LC method placebo spiking was performedby adding a known amount of drug from standard solution at five differentconcentrations 6, 9, 12, 15, and 18mg mL�1 and 50, 75, 100, 125, and 150%of label claim respectively to placebo PCL NPs. In the LC method, standard

265addition was performed by adding known amounts of drug at five differentconcentrations of 50, 75, 100, 125, and 150% of label claim from standardsolution to the pre-analyzed (150–450 ng mL-1) TAX loaded NPDDS. InUV-spectrophotometric method, standard addition was accomplished withthree concentrations (6, 12, and 18 mg mL-1) of pure drug solutions to

270pre-analyzed (10 and 12.2 mg mL-1) drug loaded PCL NPs. The accuracyresults were represented by mean absolute recovery, RSD (%) and Bias (%).

Sensitivity

The sensitivity of the developed UV-Spectrophotometric method wasrepresented by the limit of detection (LOD) and the limit of quantitation

275(LOQ). The LOD and LOQ were determined using the standard formula3.3 rs�1 and 10.00 rs�1, respectively, where s- is the slope of the calibrationcurve and r- is the SD of y-intercept of regression equation or SD of intercept.

The LOD and LOQ of the developed 3D view LC method wereobtained by the signal-to-noise ratio and by the slope calculation. The

280LOQ determination was performed on five different days (n¼ 5) by spikingan aliquot from working standard to diluent to obtain the desired concen-tration of the lowest calibrator. The results with precision less than 20%,accuracy of 80–120%, and signal-to-noise ratio of 10:1 confirms the LOQof the developed method.

285Robustness and Stability Study

Robustness of the developed UV-Spectrophotometric method wasdetermined by making a small deliberate change to the optimized internalparameter. In this study, the diluent composition acetonitrile:water waschanged from 85:15% v=v to 80:20% v=v (Diluent-1) and 90:10% v=v

290(Diluent-2), respectively. The robustness result was represented by meanrecovery (%) and RSD (%).

The stability of TAX in the diluent medium (acetonitrile:water, 85:15)at 4�C was monitored using the validated UV-Spectrophotometric method.

3D View LC-Diode Array UV Meth 11

The QC samples were prepared fresh and their respective spectra were295recorded using the developed method. Now all the QC samples were stored

at 4�C at a predetermined time point of 8 and 72 hr, once again the spectrawere recorded. The overlay of the 8 and 72 hr spectra, over the fresh QCsample spectrum demonstrates the stability of TAX in the selected diluent.The stock solution stability of TAX in ethanol at 4�C was determined for

300one month at 18 mg mL�1 and it was represented by recovery (%).

System Suitability

The repeatability or routine analysis of the developed 3D view LCmethod within the validated limits was checked by system suitability para-meters. The parameters used in this study are Capacity factor (k), Tailing

305factor (T), No of theoretical plates, Height equivalent to theoretical plates(HETP), Effective plate number (Neff), Height equivalent to one effectiveplate (HEFF), and Reduced plate height (h). All the aforementioned para-meters were calculated using the standard formula.[1]

Application of the Developed Methods

310Preparation and Characterization of Polymeric NanoparticlesTAX loaded and placebo polymeric nanoparticles (NPs) were prepared

by a nanoprecipitation method. In brief, 50 mg of polymer (PLGA, PLA,and PCL) was dissolved in 5 mL of acetone and this organic phase waspoured into the aqueous phase (water) which contains 0.5% w=v of stabili-

315zer PF-68. During this process the aqueous phase was sonicated usingmicrotip probe sonicator (Microson, Misonix, USA) under controlled tem-perature (cooling bath maintained at 5�C� 1.00) with intensity 15 W for15 min. Once the organic phase was added to the aqueous phase, the milkynanodispersion was formed instantaneously. The formed nanodispersion

320was kept for stirring under magnetic stirrer to evaporate the organicsolvent for 3 hr and then the dispersion was subjected to Rotavapor (Buchi,Switzerland) under reduced pressure at 40�C for �2 hr to reduce the vol-ume �10 mL. The nanodispersion was filtered through syringe filter holder(Axiva, India) with a 0.44-mm filter membrane (Millipore Co., USA) to

325remove the free TAX and aggregates. The entire nanodispersion was cen-trifuged at 14,000 rpm at 15�C for 10 min (Cooling Compufuge, Remi,Mumbai, India) in three cycles. The nanoparticles get settled and super-natant was analyzed for free drug content and the sediment nanoparticlewas freeze-dried. The prepared polymeric NPs were used for characteriza-

330tion (size, polydispersity index, zeta potential, and drug content) and forin vitro dissolution study.

12 S. Vasanthakumar et al.

The prepared PLGA, PCL, and PLA NP formulations shows size rangefrom 173.8 to 198.3 nm with good polydispersity index range from 0.144to 0.348 and the zeta potential value, �28.2 to �36.7 mV, respectively. The

335PCL NPs was used in the accuracy and selectivity=sensitivity study. Theprepared PLGA, PCL, and PLA NP formulation was used for the in vitrodissolution and in situ closed loop re-circulating absorption study. Thedeveloped method was applied to estimate drug content in the PLGA,PCL, and PLA NP formulations and their respective in vitro dissolution

340samples.

Closed Loop Re-Circulating Absorption StudyThe in situ absorption study of TAX in pure form and TAX loaded NPs

were performed in young healthy male Wistar rats (160–220 g, 12–15weeks) obtained from the Central Animal Facilities BITS (Pilani, India).

345The study design and protocol used in this experiment was described else-where with slight modifications.[4] The study protocol was approved by theInstitute Animal Ethical Committee (protocol no: IEACRES51 REV 01)before starting the experiment.

Animals used in this experiment were housed in polypropylene cages350filled with sterile paddy husk, had access to water ad libitum and main-

tained at 22� 2�C, 50–60% relative humidity, under 12:12 hr light–darkcycle. The rats were housed three per cage; they were marked by head,tail, and body method with picric acid. All the animals were kept underthese conditions for acclimatization, one week before the experiment

355was initiated. The rats were fasted overnight before the commencementof surgery for absorption study. Before the beginning of the experiment,the peristaltic pump designed to deliver the drug solution to theintestine through the silicone cannula was calibrated to deliver solutionat 0.9 mL=min.

360Drug Solutions for Absorption StudyFive milligrams of TAX were weighed in a 5-mL volumetric flask and it

was dissolved in DMSO. The volume was made up to five milliliters toobtain a concentration of 1 mg=mL. One milliliter from the stock solutionwas spiked into a Sorensen buffer of pH 7.4 which contains 5% v=v of etha-

365nol to get 100 mg=mL concentration. This solution was vortexed at 37�C for15 min in a temperature controlled orbital shaking incubator chamber(MAC, Macro scientific works (R), Delhi, India) to get clear solution, whichis used for the in situ absorption study. The selected TAX loaded NPs for-mulations (PLGA, PCL, and PLA) were weighed equivalent to 1 mg of TAX

370and suspended in Sorensen buffer (pH 7.4) to obtain a concentration of100 mg=mL.

3D View LC-Diode Array UV Meth 13

Preparation of Intestinal Loop and Surgery ProcedureThe fasted rates were anaesthetized fifteen minutes before surgery by

intraperitoneal (i.p) administration of urethane, 1% w=v solution at 1 g=375kg dose. The rat was placed on a heating chamber (Tarsons, SPINOT digital

magnetic stirrer hot plate) whose temperature was maintained at 37� 3�Cthroughout the experiment. The abdominal region of the rat was depilatedand a midline incision was made to expose the small intestine. The exposedintestine was treated frequently with perfusion solution until the surgery

380was completed. The intestinal loop of 10 cm length was measured leavingthe initial 3 cm from the pyloric end of the stomach with help of silk suturethread of 10 cm. The silicone cannula was inserted isoperistaltically at theproximal end of the duodenum and the tube (drug receiving) was heldin place by tying with silk suture thread. At the other end of the 10 cm loop,

385a small slit was made and the cannula was inserted antiperistaltically at thedistal end of the ileum and held in place (drug collecting). While tying thecannula to the intestine, care was taken not to disturb the blood vessels sup-plying the gut. The entire intestine was flushed and cleaned with the per-fusion solution (37� 3�C) and then air was pumped through the

390cannula to remove the trapped perfusion solution throughout there-circulating path. The prepared intestinal loop was kept in place at theabdominal cavity without disturbing the other organs and packed withsuture. The entire surgery was performed as quickly as possible to makesure that the animal would be alive throughout the experiment. Then,

395the drug reservoir was connected to the other end of the isoperistalticallyinserted cannula (drug receiving), which takes drug solution to the intes-tine at 0.9 mL=min flow rate. The drug reservoir was continuously stirredat 300 RPM using the magnetic stirrer (Tarsons, SPINOT digital magneticstirrer). The other end of the antiperistaltically inserted tube, which col-

400lects the drug solution after absorption, was placed inside the drug reser-voir in this way the method becomes the closed loop re-circulatingabsorption method. At predetermined time intervals 0, 5, 10, 15, 20, 25,30, 45, 60, 75, and 90 min, 100 mL of sample was taken from the reservoirand stored in a 1.5-mL centrifuge tube at �20�C until analysis.

405Estimation of TAX in NPs and in in-Vitro Release Medium. The developedmethod was applied to determine the drug content of TAX in the preparedPLGA, PCL, and PLA NP formulations. In addition it is also used to findthe amount of TAX released from the respective NP formulation in the dis-solution medium. The dissolution study was performed in phosphate buf-

410fer pH 7.4 with 1% Tween 80 at 37� 0.5�C. The samples were processedin triplicate as per the sample processing procedure given in the followingsection.

14 S. Vasanthakumar et al.

Estimation of TAX in In-Situ Absorption Samples. The re-circulating loop insitu absorption model samples were estimated by the developed 3D view

415LC-method to determine absorption rate constant (Ka) and absorptionhalf-life (t½) of pure drug and TAX loaded NPs (PLGA, PCL, and PLA).The samples were processed in triplicate as per the procedure given inthe next section.

Sample Preparation and Procedure

420An accurately weighed amount of freeze dried placebo=TAX loadedNPs equivalent to 1 mg of TAX was taken in a 50-mL of volumetric flask.The particles were digested with 25 mL of acetonitrile by ultrasonicationusing the ultra-sonic bath (6.5 L Toshibha Laboratory testing instrumentsDelhi India) for 30 min and then the volume was made up to 50 mL with

425acetonitrile. The processed samples were centrifuged in cooling Compu-fuge CPR 24 (REMI India) at 12,000 rpm for 20 min at 4�C. The super-natant was suitably diluted with respective diluent and then analyzed bythe developed methods. The dissolution sample was digested with requiredvolume of acetonitrile by sonication for 20 min and the clear solution was

430used for analysis by the developed method.The in situ samples collected at predetermined time points were stored

at �20�C until analysis. During analysis, the in situ samples were thawedunassisted at room temperature and centrifuged in cooling compufugeCPR 24 (REMI, India) at 12,000 rpm for 15 min at 4�C. The supernatant

435was suitably diluted and then sonicated using ultra-sonic bath before analy-sis (6.5 L, Toshibha, Laboratory testing instruments, Delhi, India) for20 min. The TAX amount remaining to be absorbed was estimated by thedeveloped 3D view LC method.

RESULT AND DISCUSSION

440Chromatographic Separation

From our previous study results,[1] we have chosen Millipore water asthe aqueous phase and acetonitrile as the organic phase for the separationof TAX in the C8 column. Even though ammonium acetate buffer showedgood separation, Millipore water was selected because the buffer creates

445additional pressure on the column and takes more time to wash the col-umn.[14] The Luna C8 column provides significantly improved perfor-mance over the traditional C8 phases due to high surface coverage andslightly low carbon load than original Luna C8 (14.75%).

3D View LC-Diode Array UV Meth 15

TAX was estimated selectively, specifically, and sensitively at 229 nm by450isocratic reversed-phase 3D view LC-diode array UV method at a 1-mL

min�1 flow rate. The isocratic method is very simple to optimize and toobtain more appropriate and acceptable separation of the analyte in theselected column than the gradient elution.[15] The main aim of any analyti-cal method is to use the method routinely in any laboratory; the routine

455analysis is difficult with gradient elution. In addition there are reports stat-ing that gradient elution is a slower technique and provides ghost peak andbase line noise during routine analysis.[14]. This results in inaccurate deter-minations which result in poor accuracy and precision. In the literature,there are methods where the flow rate is more than 1 mL min�1.[16,17]

460Hence, the method used more organic phase and, in most cases, it is acet-onitrile that is very costly. The optimized mobile phase of the developed 3Dview LC method was acetonitrile and Millipore water (60:40%, v=v) whichprovided moderate and quick retention time (6.66� 0.18 min) with betterpeak properties and selectivity=specificity for TAX and degradation

465products.In the literature, there are reports using complicated and time consum-

ing extraction procedures to estimate TAX in the NPs and in the in vitrodissolution sample (Table 1). Li and Feng[6] used DCM to extract thereleased TAX in the dissolution sample from the polymeric NPs. To the

470extracted TAX, a mixture of acetonitrile and water (50:50, v=v) was addedafter the DCM had evaporated. In this, the reported extraction efficiencywas 37% only and there was inefficient extraction. The extraction pro-cedure adopted was not able to extract TA� 100%, which resulted in inap-propriate release results. The extraction procedure described in the

475present 3D view LC method is a simple, single step extraction with acetoni-trile, and is economical when compared to the reported procedure usingnitrogen stream and membrane filter (Table 1). The developed 3D viewLC method does not use any complicated extraction procedure to estimateTAX even in the in situ closed loop re-circulating absorption samples. De

480et al.[18] used a simple extraction procedure to estimate TAX from theNPs, where TAX was extracted using a small amount of acetonitrile(1 mL). The time for extraction of TAX was a lengthy 16 hr; this makesthe method not more suitable for routine analysis. In the developed 3Dview LC method, TAX was extracted from NPs by simple extraction using

485acetonitrile within 30 min with extraction efficiency, 99.65 to 102.62%.Liggins et al.[19] used a lengthy, tedious, and complicated procedure toestimate TAX from microsphere, which makes this procedure not as suit-able for routine analysis. The developed UV-spectrophotometer methodestimated TAX from the drug loaded NPs with low % relative error, 0.39

490to 1.08. There was no significant difference in the TAX determination inNPs by the UV-spectrophotometer method (99.62–102.60%) when

16 S. Vasanthakumar et al.

compared to the developed 3D view LC-diode array method (98.63–102.62%). By using liquid–liquid extraction (LLE) method, it has beenreported that the liquid solvents cause insufficient efficiency of extrac-

495tion to permit routine analysis. It is well-known that the LLE methodhas more inconsistent variation in extraction between samples. Hence,using DCM to extract TAX from the NPs and from the dissolutionmedium is not an ideal routine method to estimate TAX. The sampleprocessing procedure of the present method takes less time, 30 min

500(with sonication) and there is no time consuming step, such as evapor-ation of solvent and shaking of the sample for more than 10 hr as inLLE (Table 1). The highlight of the 3D view LC method is that it is usedto estimate TAX in the in situ closed loop recirculating absorption sam-ples with a simple, rapid, and sensitive (LLOQ 10 ng=mL) extraction

505procedure.Badea et al.[20] developed a rapid, (total run time is 35 min) gradient

HPLC method for the determination of TAX in pharmaceutical formu-lation which contains cremophore as pharmaceutical excipients. In this,TAX was eluted at 12.5 min and the excipient interferences peaks were

510observed between 15 to 35 min. It is a dedicated method for in-processdeterminations of TAX in cremophore contained pharmaceutical formu-lation; this method cannot be used routinely for the estimation of TAXin other pharmaceutical dosage forms such as polymeric NPs, because itis a time consuming method; the single analysis takes 35 min. In the present

515method, TAX was eluted at 6.66� 0.02 min by isocratic reverse phase 3DView LC-diode array UV with total run time of 10 min and there were nointerferences from the excipients observed up to 30 min, which is con-firmed from the 3D view chromatogram (Figure 1) and counterview graph(Figure 2).

520To demonstrate the specificity and selectivity, Badea et al.[20] inten-tionally added nine different related=degradation product of TAX andexcipient cremophore to the pure drug TAX. In this separation, excipi-ent interferes with the separation of related=degradation product thusthis method was not used to control the purity of finished product. This

525method may not be suitable for routine analysis of TAX in other dosageforms, in vitro and in situ samples. In the present method, it was wellestablished with 3D chromatogram, counter view plot, peak purity deter-mination, and ratiogram, even in the presence of TAX degradation pro-ducts, TAX was selectively and specifically estimated and separated from

530the degradation peaks. In the present 3D view LC method, there wereno interferences of excipients (polymers and stabilizers) in the esti-mation of TAX were clearly shown by the standard addition and pla-cebo spiking method and their respective chromatograms (Figures 3and 4).

3D View LC-Diode Array UV Meth 17

535Linearity

The typical best-fit linear regression equation for the methods wasobtained after mathematical treatment (least square method) of the resultsby plotting the absorbance=peak area versus the concentration in the concen-tration range of 6–34mg mL�1 and 10–1500 ng mL�1 (Figure 1), respectively.

540The typical representative three dimensional view chromatogram of TAX cali-bration curve is illustrated in Figure 1. The obtained regression equations areabsorbance¼ 0.0363 x concentration of TAX (mg mL�1)þ 0.0233 and peak

FIGURE 1 Representative 3D chromatograms of calibration curve of TAX A) Blank or Diluent; B)Standard at 10 ng=mL; C) Standard at 25 ng=mL; D) Standard at 500 ng=mL; E) Standard at 750 ng=mL;and F) Standard at 1500 ng=mL. (Color figure available online.)

18 S. Vasanthakumar et al.

FIGURE 2 Representative counter view plot and UV spectrum of degradation sample. A) Counter viewplot of Standard 750 ng=mL; B) Counter view plot of FDS-HCL; C) Counter view plot of OST; D) Coun-ter view plot of TST; E) UV spectrum of 750 ng=mL; F) UV spectrum of FDS-HCL; and G) UV spectrumof OST H) UV spectrum of TST. (Color figure available online.)

FIGURE 3 Base shift overlay representative chromatogram demonstrating the selectivity of TAX deter-mination in presences of excipients: (a) Calibration standard LLOQ 10 ng=mL; (b) Drug (10 ng=mL)spiked in blank placebo NPs formulation standards; (c) Blank placebo NPs formulation standard; and(d) Diluent.

3D View LC-Diode Array UV Meth 19

area (mV s)¼ 211.59� concentration of TAX (ng mL�1)� 792.26, respect-ively. The goodness of fit of regression equation in the selected concentration

545range for TAX in both the method was linear with significant correlation coef-ficient r2¼ 0.9941 and 0.9999, respectively. The % bias range from 0.62 to�6.58 and 0.70 to �2.51, respectively, for the entire calibration curve concen-trations of this low % bias supports the selected linear model with univariantregression and it also shows the goodness of fit. The % error calculated for

550the entire range of calibration points were between 0.09 to 0.37 and 0.02 to0.53, respectively. The goodness of fit was further supported with the low stan-dard error of estimate 2.96� 10�2 and 5.72 and low standard error of mean0.0015 and 126.48, respectively. The other statistical analysis on slope andintercept of the calibration curve confidence interval standard deviation and

555standard error further confirms the excellent linearity of TAX in the concen-tration range of 6–34mg mL�1 and 10–1500 ng mL�1 for respective methods.The obtained slope values without intercept for the developed methods were0.0373 and 210.78, respectively. The LC method slope value fell within the 95%confidence interval of slope limits (Table 2); hence, the linearity of the

560developed LC method could practically be in the range of 0–1500 ng mL�1,but it is not in case of developed UV method. The present developed method

FIGURE 4 A) Representative overlaid chromatogram of placebo spiking and standard addition withzoom view. B) Representative chromatogram of acid hydrolytic (100 mM HCl) degradation of TAX withtheir peak purity curve and ratiogram. C) Representative chromatogram of forced oxidative (6% v=vH2O2) degradation of TAX with their peak purity curve and ratiogram. D) Representative chromato-gram of thermal stress degradation of TAX with their peak purity curve and ratiogram.

20 S. Vasanthakumar et al.

linearity range (10–1500 ng mL�1) was more sensitive than recently publishedmethods.[14,15] The linearity range of the method of Anupama et al.[14] fordetermination of TAX in amphiphilic polymer matrix was 2–50mg mL�, which

565is not as sensitive as the present developed method.A one-way analysis of variance test (ANOVA) was performed on each

replicate response (absorbance=peak area) of the eight=nine calibrationconcentration of TAX the determination of best fit linear equation. TheANOVA results of the developed UV and LC methods showed that the cal-

570culated F-value 1.95� 10�4 and 3.11� 10�5 was found to be lower than thecritical F- value 2.09 and 2.41, respectively, at a 95% level of significancewhich supports the best-fit linear equation. The test of the intercept wasperformed using the t-statistic at a 95% confidence interval; the tdf 0.929and 6.11� 10�6, respectively, shows that the intercept was not significantly

575(0.05 level) different from zero, and thus, once again, supports the best fitlinear equation (Table 2).

TABLE 2 Summary of Statistical Data of Optical Characteristics and Validation Parameters of theDeveloped Methods

Parameter Observed Values

UV- spectroscopic methodConfidence interval of slope (S.E.a) 0.03613 to 0.03650 (8.92� 10�5)Confidence interval of intercept (S.E.) 0.02030 to 0.02632 (1.43� 10�3)Standard deviation of intercept (Sc) 2.68� 10�4

Standard deviation of slope 4.28� 10�3

t-value for interceptb (tab¼ 2.11) 0.929 (P-value 0.39)Calculated F-value (critical F-value)c 1.95� 10�4 (2.09)

System suitability (Optical characteristics)

Molar Absorptivity - 3.15� 104 L M�1 cm�1

Specific Absorptivity �3.70� 10�2 mL mg�1 cm�1

Sandell’s sensitivity �2.70� 10�2mg cm�2

Specificity and Selectivity- tCal (tCrit)d 1.88 (4.30)

3D view LC-diode UV Method95% Confidence interval of slope (S.E.e) 210.47 to 212.71 (0.437)95% Confidence interval of intercept (S.E) 593.33 to 991.19 (77.42)Standard deviation of intercept (Sc) 189.64Standard deviation of slope 1.07t-value for interceptf (tab¼ 2.57) 6.11� 10�6 (P-value 0.999)Calculated F-value (critical F-value)g 3.11� 10�5 (2.41)Specificity and Selectivity- tCal (tCrit)

h 0.83 (2.78)

aStandard error of mean.bCalculated at p¼ 0.05 level of significance, using the test of the intercept (tdf¼ j C – a j = Sc).cCalculated based on one-way ANOVA test at P¼ 0.05 level of significance.dCalculated at P¼ 0.05 level of significance (d.f. 2) based on paired t-test.eStandard error of mean.fCalculated at p¼ 0.05 level of significance, using the test of the intercept (tdf¼ j C – a j = Sc).gCalculated based on one-way ANOVA test at P¼ 0.05 level of significance.hCalculated at P¼ 0.05 level of significance (d.f. 4) based on paired t-test, A-Based on the formula 3.3

rs�1, B-Based on the formula 10 rs�1, where r- standard deviation of intercept and s- slope of the cali-bration curve.

3D View LC-Diode Array UV Meth 21

Selectivity/Specificity

The developed methods were found to be selective and specific; it dif-ferentiates TAX from the other unexpected substances in the NP formu-

580lation samples (Figures 3 and 4), and the method produces responseonly to TAX at 229 nm (Figures 1 and 5A) where it is monitored and quan-tified in the studied formulation samples (Figure 4).

The experimental result of selectivity and specificity shows that spectraof TAX spiked to PCL placebo NPs at two different concentrations 6 and

58518 mg mL�1 overlaid with the freshly prepared calibrator sample spectrumof the same concentrations, respectively (Figure 5A). The recovery (%)of the analyzed samples was 99.88� 1.02 and 100.23� 0.45, respectively;this shows that the excipients in the NPs formulation do not influence inthe estimation of TAX. The paired t-test result of the developed methods

590showed that the calculated t-value (1.88 and 0.83) was found to be less thanthe critical t–value (4.30 and 2.78), which indicated that there were no stat-istical significant (P¼ 0.05) differences between mean absorbance=peakarea of TAX spiked PCL NPs and pure drug calibrator samples. The baseshift overlay chromatogram (Figure 3) clearly explains that there was no

595interference from the excipients used in the PCL NPs preparation overthe diluent chromatogram (Figure 3d) at the RT of TAX, which confirmedthe selectivity of the developed 3D view LC method. The chromatogramFigure 3a and b shows that the response of TAX at LLOQ in the calibratorsample and the drug spiked (10 ng mL�1) in blank placebo NPs formu-

600lation standard were overlaid; hence, this, once again, shows that themethod can selectively and specifically determine TAX at 229 nm. Finally,the recovery (%) of TAX at two different samples at the same concentrationof the QC standard LLOQ (99. 96� 0.21) and drug spiked placebo NPs for-mulation samples (100.01� 0.21) confirmed that the method was selective

605and specific in determining TAX in the presence of expected excipients atLLOQ levels.

Forced Degradation Study: Peak Purity Curve and Ratiogram

Forced degradation studies were conducted to generate degradationproducts of TAX to check for the selectivity and specificity of the developed

6103D view LC method. All the degradation samples were analyzed by the 3Dview LC method and the chromatogram was recorded up to 60 min. TAX isa complex molecule that has numerous hydrolysable ester groups and witha strained oxetane ring that undergoes cleavage in acid catalyzed con-ditions.[21] The presence of ester amide acetyl acetoxy ester free hydroxyl

615functional groups makes TAX prone to hydrolysis and oxidation under dif-ferent applied stress conditions. As per guidelines, the degradation study

22 S. Vasanthakumar et al.

FIGURE 5 A) Overlaid selectivity indicating UV-visible absorption spectra of TAX (j pure drug at twoconcentrations 6 and 18mg=mL; & 50 and 150% drug spiked placebo sample; zoom view of the selec-tivity absorption spectra is showed inside the spectrum); and B) Overlaid stability indicating UV-visibleabsorption spectra of quality control samples (6, 18, and 30mg=mL) of TAX in diluent medium (acet-onitrile and water 85:15) at 0 hr, 8 hr, and 78 hr. (j TAX at 0 hr; — TAX at 8 hr; and ~ TAX at 78 hr zoomview of the spectra is showed inside the spectrum). (Color figure available online.)

3D View LC-Diode Array UV Meth 23

protocols were optimized to make sure that 100% of TAX would not bedegraded in the given set of stress conditions.[13] The developed 3D viewLC-method not only helps in separating the TAX from the degradation pro-

620duct but also helps to determine the drug accurately with high sensitivity(LLOQ, 10 ng=mL) and specificity (Table 2).

The novel microwave FDS by acidic and alkaline condition generatedhydrophilic and hydrophobic degradation products, which elutes at 2.35,3.55, 3.86, 4.08, 4.95, 5.68, 7.39, 9.61, and 15.06 and 2.34, 3.87, 6.33, and

6256.73 min, respectively. The TST of TAX generated hydrophilic and hydro-phobic degradation products eluting at 2.68, 7.64, and 9.11 min, respect-ively. The OST of TAX generated only hydrophilic degradation productsthat elutes at 4.10 min. The developed 3D view LC method separatesTAX from all the degradation products completely, which was clearly shown

630by the counter view graph (Figure 2) and ratiogram (Figure 4).There was less degradation of TAX, 19.62%, during OST among the

FDS-HCL (91.04%), FDS-NaOH (92.97%), and TST (67.51%). The peakpurity curve of all the degradation samples clearly shows that the similaritycurve did not intersect the threshold curve; hence, there are no impurities

635or degradants coeluting with TAX at the estimated wavelength. The peakpurity index (PPI) and single point threshold (SPT) of the degradationsamples, FDS-HCL (0.995600 and 0.880975), FDS-NaOH (0.999577 and0.723763), TST (0.999986 and 0.946592), and OST (1.0000 and0.992731) were always close to one and the PPI was always greater than

640the SPT which shows the absences of degradation product co-eluting withTAX at 229 nm in the developed 3D view method. The positive MPPI ofall the degradation samples, FDS-HCL, FDS-NaOH TST, and OST were114035, 275813, 53393, and 7268, respectively, which shows the specificquantification of TAX in the degraded sample by the developed 3D view

645LC-method. In addition, the contour view plot (Figure 2) shows that thedegradation peaks does not overlap with TAX peak and the optimum wave-length (229 nm) where TAX was well-separated from the degradation peakscan be observed only by this 3D view LC diode array method, which was notpossible with a single-wavelength monitor. The 3D view LC-diode array UV

650method allowed the analyst to record UV spectra (Figure 2) correspondingto a specific time period when the sample was analyzed chromatographi-cally. The recorded UV spectrum of the entire degraded sample(Figure 2D, G, and H) was similar to the pure TAX (Figure 2E) at the detec-tion wavelength 229 nm.

655The three point view results of peak purity supports the total peakmode results in determining TAX specifically by the developed 3D viewLC-method. In case of three point view, the upslope purity (UP) value ofall the degradation samples, 0.635258, 0.749008, 0.992718, and 0.118460was always greater than down slope purity (DSP) value 0.116591,

24 S. Vasanthakumar et al.

6600.563228, 0.323086, and 0.084852, respectively. This results in positive 3point purity (PP) value 0.375925, 0.656118, 0.657902, and 0.101656 of allthe degradation samples, FDS-HCL, FDS-NaOH TST, and OST, respectively.The rectangular ratiograms (Figure 4) obtained for the entire degradationsample in selected wavelength 229 nm at the TAX RT shows that only TAX

665was determined specifically by the developed 3d view LC method. Thedegradation of TAX was performed only to show the selectivity and speci-ficity of the 3D view LC method and, hence, the degradation products werenot separated in the present study. The structural elucidation of the degra-dation product is out of scope of the paper, but infrared (FTIR) spectrum

670for the degraded samples was taken (Figure 6). In the IR spectrum,Figure 6C represents the pure drug, where the following characteristic bandswere observed, O-H stretching vibration band at 3600 cm�1, C-H stretchingband at 3200 cm�1, aromatic ring and C-O-C stretching peaks at1500–1200 cm�1. The peak around 1700 cm�1 corresponded to saturated

675ketone groups and the band around 1680 cm�1 represented the amidegroup. Figure 6A, B, and D represent OST, FDS-HCL, and TST degradationsample IR spectrum, where there is reduction in the O-H stretching vibrationband in all the degraded samples when compared to pure TAX. The charac-teristic band C=O at about 1700 cm�1 was reduced in all the degradation

680sample and the peak for amide completely disappeared in the OST sampleand the band was reduced in the other degradation samples, FDS-HCLand TST. The aromatic ring and C-O-C stretching peaks at 1500–1200 cm�1

was reduced in the entire degraded sample. This shows that TAX wasdegraded partially in all the stress conditions. The pure TAX was selectively

685and specifically separated and estimated by the 3D view LC method.

FIGURE 6 FTIR spectra of A) OST, B) FDS-HCL, C) TAX, and D) TST.

3D View LC-Diode Array UV Meth 25

Accuracy

The accuracy results of the developed UV and 3D view LC method showsRSD (%) less than 2.29 and 0.64 with mean recovery (%) range from 99.62to 102.60 and 98.63 to 102.62 and the relative (%) error range from 0.39 to

6901.08 and 0.07 to �1.37, respectively. The developed method has shown highmean absolute recovery at all five concentration levels tested and the resultswere consistent with low SD in placebo spiking and standard addition method.The mean absolute recovery (%) of TAX in case of placebo spiking ranges from99.65 to 101.56 and 99.81 to 100.37 with low RSD (%) of 0.21 and 0.04 for the

695highest concentration (18mg mL�1 and 450 ng mL�1) and the highest RSD(%) 2.30 and 0.21 (second highest) for the lowest concentration (6mg mL�1

and 150 ng mL�1), respectively. The mean recovery (%) of standard additionmethod was in the range of 99.83 to 102.62 and 99.94 to 100.13, respectively,with RSD (%) less than 2.23 and 0.199, respectively. Hence, we can conclude

700from the statistical evaluation [RSD (%)] that the recoveries were normally dis-tributed around the mean; thus, the data obtained were homogenous in nat-ure. This shows that the TAX recovery was high at each time of analysis andthe developed extraction procedure was efficient in extracting TAX and thedeveloped methods were accurate in determining TAX. One more important

705inference from this accuracy study was that there was no significant inter-ference of the excipients (PF-68, PCL, PLGA, and PVA) at 229 nm where thedrug was monitored and determined. This was supported by the bias (%) rangeobtained for each technique of the developed methods 0.21 to �0.89 and 0.01to �0.19, respectively. The overlay chromatogram (Figure 4) represents the

710blank placebo NP formulation standards and the blank diluent chromatogramwhere there was no interference from the excipients at the retention time(6.64� 0.31) of TAX at 229 nm. In Figure 4A the overlay chromatogram of300 ng mL�1 and 100% addition of LC (label claim) once again confirmed thatthere was no interference from the formulation in determining TAX by the

715developed LC method. The super imposable spectrum (Figure 5) obtainedfor pure drug TAX and drug spiked placebo samples were also in agreementwith the LC chromatogram results. As per our knowledge, this is the first reportthat describes the accuracy of the developed LC and UV-spectrophotometermethod in terms of standard addition and placebo spiking for TAX determi-

720nation. In a previously published method,[20] the accuracy in terms of recoverywas found to be in the range of 98.2 to 100.5 with RSD (%) of 0.83, and in thepresent method it was found to be in the range of 98.63 to 102.62 with RSD (%)range from 0.04 to 0.64, respectively.

Precision

725The repeatability study results [RSD (%)] of the developed UV and 3DLC method ranged from 0.22 to 2.63 and 0.06 to 2.35, respectively. The

26 S. Vasanthakumar et al.

intra-day precision of the QC samples on three different occasions within aday was always less than 2.53 and 1.14 and with least RSD (%) of 0.27 and0.03. In case of inter-day precision the RSD (%) was in the range of 1.02 to

7301.93 and 0.13 to 1.74, respectively. The study results on QC samples showsthat there was no significant variation in absorbance and peak area in therespective method, which demonstrates that the entire calibration range ofthe methods had good repeatability and reproducibility. This shows thatthe method had good agreement among the individual homogenous sam-

735ples tested. The repeatability of the QC samples set on each day of vali-dation was less than 2.63 and 2.35, respectively. In the case of LOQ, itwas always less than 1.95 and 1.18 with least RSD (%) of 0.98 and 0.63,respectively. This once again shows the sensitivity of the method at the levelof LOQ with an acceptable limit of RSD (%). The concentration range of

740QC samples during inter-day precision of the developed UV method was5.79 to 6.20, 18.21 to 18.22, and 30.34 to 30.61mg mL�1, respectively withRSD (%) range from 1.02 to 1.93, respectively. In the case of the developed3D view LC method, the mean concentration and SD was satisfying and theRSD (%) was in the range of 0.13 to 1.74, respectively. The inter-instrument

745repeatability [RSD (%)] of the developed UV method was in the range of0.21 to 2.30, respectively. The intra-day and inter-day precision results[RSD (%)] are low compared to Badea et al.[20] of 1.33 to 3.53 and Anu-pama et al.[14] of 1.06 to 1.77 and 0.77 to 1.53, respectively. The studyresults clearly show that the developed method can be used routinely in

750any analytical lab.

Sensitivity

The LOD and LOQ of the developed UV method were found to be 0.39and 1.18 mg mL�1, respectively. The developed calibration curve of themethod has shown high magnitude of slope (0.0363� 0.00027) less SD

755of intercept (0.0043) and low standard error of mean slope and intercept0.0015, 0.0014, and 0.00009, respectively. The developed UV method wasfound to be sensitive; even a small change in concentration can bedetected. The LOD and LOQ of the developed 3D view LC method byslope calculation were found to be 2.96 and 8.96 ng mL�1, respectively.

760The LOD, 4 ng=mL of the developed 3D view method, satisfies the signalto noise ratio 2:1. The calibration point starts from LOQ (10 ng mL�1) asit satisfies the signal to noise ratio condition, 10:1, with precision less than0.12% RSD and accuracy 98.63� 0.12%. In addition, the peak propertiessuch as retention time 6.64� 0.31; peak area 1278.87� 3.89, and T� 2 of

765TAX at LOQ were not affected during the accuracy and precision study.Badea et al.[20] method LOQ and LOD were found to be 240 and

3D View LC-Diode Array UV Meth 27

72 ng mL�1, which is high when compared to the present method. Inaddition there are methods where the LOQ and LOD were notreported.[22] The sensitivity of the present 3D view LC method was high

770when compared to the recently published method[14] whose LOD andLOQ was 0.17 and 0.5mg mL�1, respectively.

Robustness and Stability Study

A deliberate change in composition of diluent in determination of TAXat 229 nm in the UV-method shows that there was no significant change in

775absorbance at the selected diluent composition in the tested concentra-tions. The mean recovery (%) ranged from 99.93 to 102.66 and 99.64 to102.84 with SD less than 1.25 and 1.75 and RSD (%) less than 1.26 and1.70, respectively. The stability of TAX in the selected diluent was moni-tored at 4�C using the developed UV method. The superimposed spectra

780of the 8th and 72nd hr samples over the fresh sample spectra (Figure 5B)of the selected QC samples shows that TAX is stable over the evaluated timeof 72 hr and temperature 4�C in the selected diluent acetonitrile:water85:15%, v=v. The stock solution stability result shows that TAX was stablewith recovery (%) of 101.21� 0.45 in ethanol for one month at 4�C.

785System Suitability and TAX Stability

The developed LC method was found to be suitable in terms of systemperformances as per the system suitability evaluation parameter results.The capacity factor (k) of TAX in the selected column and mobile phasewas 3.36 which is greater than 2 which shows that the TAX spends enough

790time to obtain optimum resolution with the selected stationary phase versusthe mobile phase. The k value above one shows that the TAX iswell-separated from the solvent peak. The highest number of theoreticalplates (11016.6) shows the sharpness of the peak and therefore theefficiency of the column. The higher the number of plate the column

795efficiency is more. The column efficiency was supported by the smallerHETP 0.023. The tailing factor (T) of 1.25, which is less than 2, shows thatthe TAX peak is symmetrical. The other system suitability parameters likeNeff (6518.7), HEFF (0.038), and h (2203.32) demonstrate the suitabilityand repeatability of the developed method in the respective conditions.

800Estimation of TAX in Different Samples

The drug content of TAX loaded PCL and PLGA NP formulationsdetermined by the developed UV-spectroscopy and 3D view LC methods

28 S. Vasanthakumar et al.

were found to be 100.89� 0.56 and 101.06� 0.75 and 100.02� 0.04 and100.03� 0.05, with low % bias, 0.25 to 0.33 and 0.018 to 0.023, respectively.

805The recovery of TAX from the NP formulations by the developed methodswas close to 100%, which shows the accuracy of the method in determiningTAX in polymeric NPs. The estimated mean concentration (mg mL�1) ofthe two formulations by the developed methods has low standard deviation0.10 and 0.14 and 0.04 and 0.04, respectively, which shows the precision of

810the developed methods. The one-way ANOVA test result shows that there isno significant different in concentration determination between the NPformulations and pure drug, this is confirmed by less calculated F-valuethan the Fcrit. The Fcalc for UV and LC method was 0.35 and 0.69, respect-ively, which is less than the Fcrit 3.88 and the mean SS for the developed

815methods were 5.41� 10�3 and 1.55� 10�2 and 3.21� 10�2 and4.61� 10�2, respectively, between and within group. This result shows thatthe developed methods were suitable for estimation of TAX in NP formula-tions with high accuracy and precision. In literature there are complicated,time consuming, and sophisticated procedures to estimate TAX content in

820the polymeric NPs (Table 1). An extensive literature search (Table 1)shows, many researchers used LLE method to extract TAX from NPs. Ithas been reported that the LLE method shows poor efficiency of extractionand it exhibits concentration dependent recovery.

There many complicated procedures used in literature to extract TAX825from NPs, such as evaporating the extracted solvent system by using nitro-

gen stream, using a mixture of extracting solvent with buffer and using aspecial solvent system such as tetrahydrofuran. There is a method that useda membrane filter to filter the extracted TAX (Table 1) before LC analysis.The present 3D view LC method procedure is simple, rapid, and economi-

830cal when compare to the reported method using LLE.Q2

The developed 3D view LC method was used to find the TAX releasefrom the PLGA and PCL NPs. The prepared NPs show a biphasic drugrelease pattern with an initial burst release and then slow and controlledrelease up to 48 hr (Figure 7). The initial burst release in the profile is

835due to release of poorly entrapped drug from the surface of the NPs.The slow and controlled release of TAX from NPs is due to the slow dissol-ution and diffusion of the entrapped drug from the polymeric matrix NPs.In a recent procedure,[18] 1 mL of acetonitrile was added to PLGA NPs andextract TAX from NPs for 16 hr; the extraction procedure is simple but the

840time to extract TAX is high, hence this method may not be suitable for rou-tine analysis. Chavanpatil et al.[29] used 1 mL of methanol to extract TAXfrom PLGA NPs by shaking at 100 rpm for 48 hr; this method was not rapidin determining TAX and hence it may not be suitable for routine analysis.In the present 3D view LC method, TAX from the polymeric NPs was

845extracted by digesting NPs with acetonitrile by sonication for 30 min; this

3D View LC-Diode Array UV Meth 29

simple and rapid method can be used for routine analysis of TAX in pureform, NPs and in in vitro dissolution samples. This result shows that thedeveloped 3D view LC method was selective=specific in determining TAXin the in vitro dissolution medium in the presence of formulation excipients.

850The developed 3D view LC method was used to estimate TAX amountremaining to be absorbed from the intestine at different time intervals ofthe in situ absorption samples. The Log ARA versus time graph of puredrug and TAX loaded PLGA, PCL, and PLA NP formulations shows lin-earity with significant correlation coefficient 0.9894, 0.9733, 0.9713, and

8550.9751, respectively. The Log ARA versus time graph (Figure 8) was almosta straight line (r2) for all the tested samples with exponential disappear-ance of TAX from the intestine, which indicates TAX followed first orderabsorption kinetic at the intestine. The calculated Ka and t1=2 of pure drugand TAX loaded NPs were 0.9 1.02, 1.38, and 1.74=min and 46.20, 40.66,

86030.09, and 23.88 min, respectively. The increased Ka and decreased t1=2 ofthe prepared NPs than the pure drug TAX clearly show the rapid absorp-tion of TAX NPs from the intestine. The increase in absorption of TAXNPs was attributed to their nanometer size and, in addition, the polymerof NPs altered the physiochemical properties of the drug to cross the mem-

865brane barrier for absorption.Apart from the benefit of estimating TAX content in the polymeric NPs

with the simple and efficient extraction procedure, this is the first reportthat shows the application of 3D view LC method in estimating the TAX

FIGURE 7 Linearity graph of in-situ rat intestine closed loop recirculating absorption study of puredrug paclitaxel and drug loaded NPs (mean �S.D, n¼ 3).

30 S. Vasanthakumar et al.

amount remaining to be absorbed from the rat intestine by the closed loop870recirculation absorption study.

CONCLUSION

Successfully, a rapid, sensitive, accurate, precise, and specific-stabilityindicating LC-diode array UV and simple UV-spectrophotometric methodwas developed and validated for determination of TAX. A stringent novel

875microwave assisted hydrolysis protocol was used for the first time in TAXdegradation for representing the selectivity and specific-stability indicatingnature of the developed LC-method. The validated methods were appliedfor the estimation of TAX in bulk, NPDDS, and in-situ absorption samples.TAX was monitored at 229 nm with retention time of 6.66� 0.18 min with

880total run time of 10 min. The typical best-fit linear regression equations ofthe developed methods were absorbance (AU)¼ 0.0363�Conc. (mg mL�1)þ 0.0233 and peak area (mV s)¼ 211.59� conc. (ng mL�1)� 792.26 withr2¼ 0.9941 and 0.9999, respectively. The linearity range of the methodswas 6–34 mg mL�1 and 10–1500 ng mL�1, respectively. The system suitability

885parameters showed that the method has good reproducibility in terms ofsystem performance in the respective conditions. The peak purity curveof all the samples showed that the similarity curve did not intersect thethreshold curve, and the rectangular ratiograms, in addition, demonstratedthe selectivity and specific-stability indicating nature of the developed

FIGURE 8 In-vitro drug release of paclitaxel from the NPs formulation (mean �S.D, n¼ 3).

3D View LC-Diode Array UV Meth 31

890LC-method. The accuracy of the developed methods was tested by placebospiking and standard addition method. The selectivity of the developedmethods was well-established at the limit of quantification. The LOD andLLOQ of the methods 0.39 mg mL�1, 1.18 mg mL�1, and 2.96 ng mL�1,8.96 ng mL�1, respectively.

895ACKNOWLEDGMENTS

Author Sekar Vasanthakumar acknowledges the Council of Scientificand Industrial Research (CSIR- India) for providing the Senior ResearchFellowship (SRF). The authors are also thankful to Getwell Pharmaceuti-cals, India, for the generous gift samples of Paclitaxel.

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