review of hplc methods & hplc-ms detection for direct determination of aspirin with its metabolites...

ds and HPLC methodsic detection for directn with its metabolite(s)aa

asintis necessary to quantitate aspirin along with its metabolite(s) inteethalatoigh

eth

of 2.54.0 h. SA further undergoes direct conjugation with glycine Blood or its derived products (serum and plasma) are typically

Special issue: review

1 Published online in Wiley Online Library: 2 February 2012

906to form salicyluric acid (SUA, Fig. 1) and with glucuronic acid toform acyl and phenyl glucuronide conjugates (Mason and Winer,1981). Earlier work also reported that salicyluric acid forms adouble conjugate with glucuronic acid (Hutt et al., 1982). To aminor extent, salicylic acid is metabolized by hydroxylation togentisic acid (GA; 2,5-dihydroxybenzoic acid, Fig. 1), which itselfis either conjugated with glycine to give gentisuric acid (Fig. 1,GUA) or is glucuronidated (Wilson et al., 1978).

ScopeThe purpose of this review is: (a) to enlist the various HPLC andLC-MS/MS methods in biological matrices for direct quantitation

the choice of sampling matrix for detecting and quantifyingdrugs in in vivo models. ASA is rapidly hydrolyzed by esterasesin the gut wall, liver, plasma and red blood cells to form SA, witha half-life of only 20min (Reilly and FitzGerald, 1988). Measure-ment of ASA in studying the metabolism of ASA is meaningful

* Correspondence to: RameshMullangi, DrugMetabolism and Pharmacokinetics,Jubilant Biosys Ltd, Industrial Suburb, Yeshwanthpur, Bangalore-560 022,India. E-mail: [email protected]

a Drug Metabolism and Pharmacokinetics, Jubilant Biosys Ltd, IndustrialSuburb, Yeshwanthpur, Bangalore-560 022, India

b Vanthys Pharmaceutical Development (Pvt) Ltd, Phoenix Pinnacle, Ulsooranalgesic, anti-inammatory and antipyretic drug and the mostwidely used anionic drug in the world. In addition, low-doseaspirin is employed as an antithrombotic agent to inhibitcyclooxygenase-dependent platelet aggregation (Hennekenset al., 1989). After administration of aspirin, it is rapidly hydrolyzedin the body (by ubiquitous esterases in the gut wall, liver and othertissues) to produce salicylic acid (SA, Fig. 1, CAS no. 69-72-7), whichis the principle metabolite and primarily responsible for thepharmacological activity of aspirin. The half-life of aspirin inplasma is about 20min, suggesting that it is a short-livedentity (Reilly and FitzGerald, 1988; Kwong, 1987). However, theresidence of SA is relatively longer in the circulation, with a half-life

for the quantication of aspirin along with its metabolites ormetabolites alone. Hence the focus of our article is to providea comprehensive review of both HPLC and LC-MS/MS methodsand as well as discussing the advantages and limitations of thesemethods. In order to make it more benecial to the readership,we have provided a concise compilation of the validation detailsand applicability of the HPLC and LC-MS/MS methods in Table 1and 2, respectively.

Sample matrixReview of HPLC methowith mass spectrometrdetermination of aspiriin various biological mRamesh Mullangia*, Kuldeep Sharma

ABSTRACT: Aspirin, the most widely used drug in the world, hpharmacologically active entity, but is also biotransformedsimilar pharmacologic/pharmacodynamic properties. Hence itvarious biological matrices accurately and precisely to correlaprovides a comprehensive overview of various bioanalytical mquantitation of aspirin along with its metabolite(s). The reviewprocessing, internal standard selection, conditions for chromconclusions for reported assays in a structured manner. Copyr

Keywords: aspirin; metabolites; HPLC; LC-MS/MS; bioanalytical m

IntroductionAspirin (acetyl salicylic acid, ASA, Fig. 1, CAS no. 50-78-2) is an

Received: 22 November 2011, Accepted: 5 December 201

(wileyonlinelibrary.com) DOI 10.1002/bmc.2694of aspirin alone or along with its metabolites, viz. salicylic acid,salicyluric acid and gentisic acid, with other relevant informationsuch as sample processing details, chromatographic conditions,validation parameters in tabular format; and (b) discuss relevant

Biomed. Chromatogr. 2012; 26: 906941 Copyright 2012 Johnwith pharmacological/pharmacodynamic activity. This paperods (HPLC and LC-MS/MS) that have been reported for directso provides general information on sample collection, samplegraphic separation, succinct validation data and applicablet 2012 John Wiley & Sons, Ltd.

ods; review

bioanalytical strategies and considerations for method develop-ment to aid the LC-MS/MS analysis of aspirin and its metabolites.

Kwong (1987) reviewed most of the reported HPLC methodstricesand Nuggehally R. Srinivasb

been known to mankind for over a century. It is not only theo a major metabolite, i.e. salicylic acid, which also exhibitsRoad, Bangalore-560 001, India

Abbreviations used: ASA, acetyl salicylic acid; GA, gentisic acid; GUA,gentisuric acid; LLE, liquidliquid extraction; PPT, protein precipitation;SA, salicylic acid; SPE, solid-phase extraction; SUA, salicyluric acid.

Wiley & Sons, Ltd.

O CH3

OH

O

c ac

Hydrolysis

id (

lic p

Review of aspirin bioanalytical methodsonly if in vitro hydrolysis of ASA to SA is inhibited/arrested.Therefore blood specimens should be collected in chilled tubescontaining esterase inhibitor. Nieder and Jaeger (1983)published the results of extensive studies on ASA hydrolysis in

OHO

OH

OH

NHO

OH

Aspirin or acetyl salicylic acid (ASA) Salicyli

Gentisic acid (GA)

by esterases

Hydroxylation

Gentisuric ac

conjugationwith glycine

conjugationwith glucuronic acid

Gentisic acid glucuronide

Figure 1. MetaboOHO Obiological matrices and concluded that enzymatic hydrolysis ofASA is drastically reduced by potassium uoride; with additionalcooling, the loss of ASA was less than 1% in 15min in plasma.Further, no detectable loss was seen in 5 days for frozen plasmasamples (at 1 C) while only a 510% loss was observed after4weeks of storage. Interestingly, McMahon and Kelly (1998)published their observations, in which the acidication ofplasma samples appeared to be a more important driver thantemperature in terms of the stability of ASA. Numerous methodshave been reported for quantitation of ASA and its metabolites(mostly deacetylated product, i.e. salicylic acid) in plasma, whileonly one has been reported for serum (Wahlin-Boll et al., 1981).Several methods have been reported for urine as the choice ofmatrix for analysis (Amick and Mason, 1979; Harrison et al.,1980; Bakar and Niazi, 1983; Mays et al., 1984; Krivoskov et al.,1996). However as reported by Krivoskov et al. (1996), ASA iseliminated from systemic circulation quickly but not excretedin urine, which unequivocally points to its fast deacetylation inkidney, as the drug is ltered in glomeruli into primary urine.SA is a dominant plasma metabolite and also excreted in urine(40% of administered dose). In addition, some authors have alsoreported methods in tissue homogenates (Reidl, 1983) and skin(Pirola et al., 1998).

Sample preparation

Efcient sample preparation is key requirement for a goodanalytical method that leads to improved selectivity, sensitivityand ruggedness. Removal of interfering matrix compounds/

Biomed. Chromatogr. 2012; 26: 906941 Copyright 2012 Johncomponents is required to lower or eliminate the risk of matrixeffects in LC-MS procedures. During the pre-treatment, endoge-nous components such as proteins, salts and lipids are removedfrom biological matrix. These components can inuence the

OH

OH

NHO

OH

O

OH

OH

id (SA) Salicyluric acid (SUA)

conjugationwith glycine

Acyl and phenylglucuronides ofsalicylic acid

conjugationwith glucuronic acid

GUA)

athway of aspirin.ionization efciency of MS and therefore canmodify the sensitivityof method. The most widely used techniques for sample prepara-tion are liquidliquid extraction (LLE), solid-phase extraction (SPE)and protein precipitation (PPT).

Protein precipitation. Almost all methods reported for quanti-cation of ASA, SA and other metabolites include the addition ofacid to sample matrix to bring down the sample matrix pH closeto the pKa of ASA (i.e. 3.5). The earliest method reported by Chamet al. (1980) included addition of an equal volume of methylcyanide (acetonitrile) and analysis of supernatant layer forestimation of ASA and SA in human plasma. In 1981, Rumble etal. reported a precipitation method with methanol, which wasdeveloped with the advantages of lower injection volume andaddition of an internal standard (IS) in the methanolic solution.Further, several other methods with acetonitrile (Bakar and Niazi,1983; Kees et al., 1996) and perchloric acid (OKruk et al., 1984;Krivoskov et al., 1996) were reported. All of these methods weredeveloped for UV detection (235280nm). Recently in 2009, Xuet al. reported their method utilizing acidied acetonitrile asprecipitation solution and mass spectrometric detection. Themethodwas validated for the intended usage, although thematrixeffect and ion suppression were evaluated for ASA and SA andslight ion suppression was seen from the results reported (i.e.recoveries for ASA concentrations of 8 and 400ng/mL were 51and 84%, while those for SA concentrations of 80 and 4000ng/mLrecoveries were 102 and 60%). From the literature data it is appar-ent that the precipitation method should not be the primarychoice for processing such complex analytes.

Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/bmc

907

Table

1.Su

mmaryvalid

ationof

vario

uspu

blishe

dHPL

Cmetho

ds

Ana

lyte(s)

Autho

rsSampleproc

essing

details

Chrom

atog

raph

icco

ndition

sVa

lidationpa

rameters

App

licab

leco

nclusion

s

Aspirin,

salicylic

acid,2,3-

DHBA

and

gentisicacid

Yamam

otoet

al.,

2007

Matrix

:rat

plasma

(100

mL).

System

:HPL

Cwith

UVde

tector.

Line

arity

:60

20,00

0ng

/mLfor

aspirin

andsalicylic

acid

and

200

20,000

ng/m

Lfor

2,3-DHBA

andge

ntisicacid

(r2>

0.99

9foralla

nalytes).

Autho

rsdiscussedtherestric

ted

access

med

ia(RAM)co

lumns

[mad

eup

ofmethy

lcellulose

(MC)

immob

ilizedsilicamaterials

bond

edto

octade

cylsilanized

(ODS)

silica]

advantag

esfor

sepa

ratio

nof

broa

dspectrum

ofdrug

s(cationic,an

ionic,an

dne

utral).

MC-stron

gan

ion-

exch

ange

(MC-SAX)

hastrim

ethy

lam

mon

ium

grou

pson

pore

surface

ofMCsilica,used

foran

ionic

analytes

on-line

extractio

nfrom

biosam

ples.V

arious

mob

ileph

ase

combina

tions

andinue

nceof

orga

nicsolven

ton

compo

und

reco

very

over

MC-SAX-SP

Eco

lumn

wereevalua

ted.

Thismetho

dwas

successfully

appliedto

quan

titate

aspirin

,salicylicacid,2,3-DHBA

and

gentisicacid

follo

wingi.v.

administrationof

aspirin

torats

at2.5mg/kg

dose.Intheplasma

samples

2,3-DHBA

andge

ntisic

acid

couldno

tbe

determ

ined

,he

nceph

armacok

inetic(PK)

pro

lesof

aspirin

andsalicylicacid

wereestablishe

dan

dtheseresults

werein

agreem

entwith

earlier

repo

rted

values.

Extractio

n:to

analiquo

tof

plasmaeq

ualv

olum

eof

0.6%

phosph

oricacid

aque

oussolutio

nwas

adde

dan

dcentrifug

edfor10

min

at10

00rpm

andtheco

nten

tswere

load

edon

SPEco

lumn

foron

-line

sample

extractio

nan

den

richm

ent.Fo

llowing

thistheen

riche

dsamples

werean

alyzed

onan

analyticalco

lumn.

Colum

n:forLC

-UVan

alysis:M

C-

SAX(10

4mm,5

0mm

,12

nmpo

resize)maintaine

dat

35 C;w

hereas

forSP

E-LC

-UVan

alysis:

MC-SAX-SP

Ewas

used

asextractio

nco

lumnan

dYM

CHyd

rosphe

reC18(150

4.6mm,5

mm,1

2nm

pore

size)was

used

asan

analytical

column.

Selectivity

:noen

doge

nous

interferen

cewas

observed

from

lasm

asamples

atthe

retentiontim

esof

the

analytes.

Internal

stan

dard:n

oIS

was

used

.Mob

ileph

ase:

grad

ient

elution

with

mob

ileph

aseAan

dB.

Mob

ileph

aseAisamixture

ofwater

ACN(100

0:10

,v/v)

containing

0.2%

TFAan

dmob

ileph

aseBisamixture

ofwater

ACN(100

:900

,v/v)

containing

0.1%

TFA.Initia

llymob

ileph

aseBwas

0%,

which

was

increasedto

30%

by18

min,after

which

the

conc

entrationwas

held

at30

%for2min.A

fter

each

run

both

SPEan

dan

alytical

columnwereushe

dwith

mob

ileph

aseB.

Flow

-ratefor

SPEan

dan

alytical

column

was

2.8an

d1mL/min,

respectiv

ely.

Accuracyan

dprecision:

intra-

andinter-da

yprecisionwere

0.1

5.8%

and0.2

11.4%,

respectiv

ely,foralla

nalytes.

For2,3-DHBA

andge

ntisic

acid,p

recision

andaccu

racy

couldno

tbe

determ

ined

below

60ng

/mLbe

causeof

interferen

cefrom

plasma

samples.T

heintra-

andinter-

dayaccu

racy

were92

.61

12%

and98

.21

03.5%,

respectiv

ely.

Detectio

n:l m

axsetat

235nm

.Vo

lumeof

injection:

10mL

.

R. Mullangi et al.

Biomed. Chromatogr. 2012; 26: 906941Copyright 2012 John Wiley & Sons, Ltd.wileyonlinelibrary.com/journal/bmc

908

Table

1.(Con

tinue

d)

Ana

lyte(s)

Autho

rsSampleproc

essing

details

Chrom

atog

raph

icco

ndition

sVa

lidationpa

rameters

App

licab

leco

nclusion

s

Retentiontim

e:~13

.5,1

5,18

.5an

d21

min

forge

ntisicacid,

2,3-DHBA

,aspirinan

dsalicylic

acid,respe

ctively.

Totalrun

time:

23min.

Aspirin

Abu

-Qarean

dAbo

u-Don

ia,

2001

Matrix

:rat

plasmaan

durine(200

mL).

System

:HPL

Cwith

UVde

tector.

Line

arity

:100

100

0ng

/mL.

Thismetho

dwas

used

toqu

antitate

aspirin

individu

ally

orin

combina

tionwith

pyrid

ostig

mine

brom

ide,

caffeine

and

acetam

inop

henin

ratplasma,

urinean

dtissues

inaPK

stud

y.

Extractio

n:an

aliquo

tof

plasma/urinewas

acidied

with

1Macetic

acid

(pH5),vortex

mixed

andcentrifug

edfor5min

at10

00gan

dthesupe

rnatan

twas

load

edon

C18Se

pPa

kVa

c3cc,5

00mg(pre-

cond

ition

edwith

3mL

ACNan

d3mLwater),

washe

dwith

2mLwater

andna

llyeluted

twice

with

2mLMeO

H.The

combine

delua

tewas

conc

entrated

to50

0mL

unde

rge

ntle

stream

ofnitrog

enan

dtran

sferredinto

HPL

Cvialsforan

alysis.

Colum

n:mB

onda

pakC18(300

3.9mm,1

0mm

)co

upledto

agu

ardco

lumn(Sup

elco

,20

0

4mm,5

mm)

maintaine

dat

ambien

troom

tempe

rature.

Limitof

detection:

200ng

/mL.

Internal

stan

dard:n

oIS

was

used

.Mob

ileph

ase:

grad

ient

elution

ataow

-rateof

1mL/min.

Selectivity

:noen

doge

nous

interferen

cefrom

plasmaan

durineat

theretentiontim

esof

thean

alytes.

Detectio

n:l m

axsetat

280nm

.Absolutereco

very:8

8.6

9.3

and85

.9

9.8%

inplasma

andurine,

respectiv

ely.

Volumeof

injection:

10mL

.Accuracyan

dprecision:

precisionan

daccu

racy

was

foun

dto

beacceptab

le.

Retentiontim

e:8.8,

9.9,

10.4

and11

.5foracetam

inop

hen,

pyrid

ostig

minebrom

ide,

caffeine

andaspirin

,respectiv

ely.

Totalrun

time:

15min.

(Con

tinue

s)

Review of aspirin bioanalytical methods

Biomed. Chromatogr. 2012; 26: 906941 Copyright 2012 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/bm

9c

09

Table

1.(Con

tinue

d)

Ana

lyte(s)

Autho

rsSampleproc

essing

details

Chrom

atog

raph

icco

ndition

sVa

lidationpa

rameters

App

licab

leco

nclusion

s

Aspirinan

dsalicylicacid

McM

ahon

and

Kelly,1

998

Matrix

:hum

anplasma(200

mL).

System

:HPL

Cwith

UVde

tector.

Line

arity

:0.1

5mg

/mLfor

aspirin

andan

d0.25

15mg

/mLforsalicylic

acid

(r>

0.99

9forbo

than

alytes).

Autho

rsevalua

tedvario

usan

ticoa

gulants(lithium

hepa

rin,

EDTA

,uo

ride/ED

TAan

dcitrate)

tobe

used

durin

gbloo

dco

llection

andfoun

dthat

minim

aldifferen

cein

plasma

pro

les.

Fluo

ride/ED

TAwas

thech

oice

ofan

ti-co

agulan

tas

itga

vecleane

rba

selin

ech

romatog

ramswith

blan

kplasma

andinhibits

theactio

nof

plasma

esterases,which

catalyzesin

vitro

hydrolysisof

aspirin

.Byco

nduc

ting

aserie

sof

expe

rimen

tstheau

thors

reco

mmen

dedthat

acidicatio

nof

plasmasamples

ismoreim

portan

tthan

maintaining

them

inch

illed

cond

ition

tominim

izethe

hydrolysisof

aspirin

.Thisco

lumn-

switc

hing

metho

dwas

used

forthe

quan

ticatio

nof

aspirin

and

salicylicacid

follo

wingoral

administrationof

600mgof

aspirin

tohe

althyhu

man

voluntee

run

derfedcond

ition

s.

Extractio

n:to

analiquo

tof

chilled

plasmaeq

ual

volumeof

0.2

Mortho-

phosph

oricacid

was

adde

d,vo

rtex

mixed

andcentrifug

edat

5800

gfor3min

(toremov

etheclou

dine

ss,ifan

y)an

d20

0mL

was

load

edov

eron

-line

SPEco

lumn

(PEEKcartrid

geha

ving

Hyp

ersilC

18,30mm

)and

eluted

with

water

ortho-ph

osph

oricacid

(100

0:1,

v/v,pH

2.5).

Colum

n:Nuc

leosilC8(250

4.6mm,5

mm)co

upledto

aHyp

ersilC

8gu

ardco

lumn

(10

4mm,1

0mm

)maintaine

dat

ambien

troom

tempe

rature.

Limitof

detection:

0.04

mg/m

Lforbo

ththean

alytes.

Internal

stan

dard:n

oIS

was

used

.Mob

ileph

ase:

isoc

ratic

mob

ileph

aseco

mprising

water

MeO

HA

CN

ortho

-ph

osph

oricacid

(650

:20

0:15

0:1,

v/v/v/v,na

lpH

2.6)

ataow

-rateof

1mL/min.

Selectivity

:noen

doge

nous

interferen

ceat

theretention

times

ofthean

alytes

evalua

tedfrom

sixdifferen

tsources.Anu

mbe

rof

possible

common

lyco

administered

drug

s(19drug

s)were

evalua

tedan

dfoun

dthat

only

xylazine

andprazosin

interferewith

the

chromatog

raph

icresolutio

nof

aspirin

andsalicylicacid.

Detectio

n:l m

axsetat

225nm

.Re

covery:absolutereco

very

at0.5an

d5mg

/mLwas

foun

dto

be99

and10

0%foraspirin

;10

4an

d10

1%forsalicylic

acid.T

herelativ

ereco

very

at0.5an

d5mg

/mLwas

101an

d94

%foraspirin

;88an

d90

%forsalicylicacid.

R. Mullangi et al.


910

Table

1.(Con

tinue

d)

Ana

lyte(s)

Autho

rsSampleproc

essing

details

Chrom

atog

raph

icco

ndition

sVa

lidationpa

rameters

App

licab

leco

nclusion

s

Volumeof

injection:

Not

applicab

leAccuracyan

dprecision:

intra-

andinter-assayprecisionan

daccu

racy

foun

dto

beacceptab

leforbo

thaspirin

andsalicylicacid.

Retentiontim

e:11

.5an

d15

.6min

foraspirin

andsalicylic

acid,respe

ctively.

Stab

ility:aspirinwas

foun

dto

bestab

lefor24

hat

room

tempe

rature,at4 C

forup

to48

han

dat3

0 C

and

throug

h2F/Tcycles.A

fter

thesetim

epe

riods

thelevels

ofen

doge

nous

interferen

ceincreased,

which

adversely

affected

thequ

anti

catio

nof

both

analytes.

Totalrun

time:

22min.

Aspirinan

dsalicylicacid

Pirola

etal.,19

98Matrix

:hum

anskin

and

plasma.

System

:HPL

Cwith

UVde

tector.

Line

arity

:0.1

100

and

0.1

5mg

/cm

2foraspirin

and

salicylicacid

intape

strip

ping

s;0.1

2an

d0.1

50mg

/mLforaspirin

andsalicylicacid

inplasma

(r2>

0.99

97forbo

than

alytes).

Noaspirin

orsalicylicacid

was

detected

inplasmafollo

wing

topicala

pplicationof

750mg

aspirin

.Followingoral

administrationof

500mgof

aspirin

both

aspirin

andsalicylicacid

were

quan

tied

usingthismetho

dan

dthevalues

werecloseto

theearlier

repo

rted

values.

Extractio

n:to

thetape

strip

ping

s3mLof

ACN

was

adde

d,sonicated

for15

min

and

centrifug

edfor10

min

at15

000g.

Thealiquo

twas

used

forHPL

Can

alysis.

Colum

n:LiChrosph

er10

0RP

18

(250

4mm,5

mm)co

upled

toa2cm

pre-co

lumn(lled

with

analytical

column

material)maintaine

dat

ambien

troom

tempe

rature.

Selectivity

:noen

doge

nous

interferen

ceat

theretention

times

ofthean

alytes

and

correspo

ndinginternal

stan

dards.

Toan

aliquo

tof

plasma

(1mL),ISsolutio

n(200

mg)and

1mLof

2M

HClw

eread

ded,

vortex

mixed

for1min

and

centrifug

edfor10

min

at15

00gan

dthe

solutio

nwas

extracted

overtSP

Eco

lumn

(IsoluteC8,p

re-w

ashe

dwith

2vo

lsof

MeO

Han

d1vo

l.of

0.1

MHCl)

andwashe

dwith

5vo

ls

Mob

ileph

ase:

isoc

ratic

mob

ileph

aseco

mprising

water

pho

spha

tebu

ffer

(pH2.5)

ACN,35:40

:25(v/v/v)

ataow

-rateof

1mL/min.

Absolutereco

very:>

98%

for

aspirin

,salicylicacid

inbo

ththematric

es.

(Con

tinue

s)


Biomed. Chromatogr. 2012; 26: 906941 Copyright 2012 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/bmc

911

Table

1.(Con

tinue

d)

Ana

lyte(s)

Autho

rsSampleproc

essing

details

Chrom

atog

raph

icco

ndition

sVa

lidationpa

rameters

App

licab

leco

nclusion

s

of2

MHCla

ndtheSP

Ewas

kept

indriedstate

for15

min.A

fter

15min,

SPEwas

na

llyeluted

with

2

500mL

ofMeO

H1

%NH4OH

ACN,5

0:30

:20(v/v/v).

Com

bine

delua

teswere

used

forHPL

Can

alysis.

Internal

stan

dard:

piroxicam

(for

tape

strip

ping

s)an

dph

enob

arbital(for

plasma).

Detectio

n:l m

axsetat

234nm

Accuracyan

dprecision:

intape

strip

ping

s,meanintra-da

yaccu

racy

was

98.8

and99

.3%

with

aprecisionof

2.3an

d1.8%

foraspirin

andsalicylic

acid,respe

ctively.Similarly

theinter-da

yaccu

racy

was

100.2an

d98

.8%

with

aprecisionof

2.8an

d1.5%

for

aspirin

andsalicylicacid,

respectiv

ely.

Volumeof

injection:

501

00mL

.In

plasma,

meanintra-da

yaccu

racy

was

101.3an

d99

.7%

with

aprecisionof

4.3

and3.3%

foraspirin

and

salicylicacid,respe

ctively.

Similarly

theinter-da

yaccu

racy

was

98.1

and99

.5%

with

aprecisionof

3.8an

d1.7%

foraspirin

andsalicylic

acid,respe

ctively.

Retentiontim

e:6.8,

8.7,

16.0

and21

.6min

foraspirin

,salicylicacid,p

heno

barbita

lan

dpiroxicam,respe

ctively.

Stab

ility:aspirinan

dsalicylic

acid

werefoun

dto

bestab

lein

both

matric

esforon

emon

that2

5 C.

Totalrun

time:

~25

and30

min

forplasmaan

dtape

strip

ping

s,respectiv

ely.

Aspirinan

dsalicylicacid

Kees

etal.,19

96Matrix

:hum

anplasma(200

mL).

System

:HPL

Cwith

UVde

tector.

Line

arity

:0.2

20mg

/mLfor

aspirin

andan

d0.5

50mg

/mL

forsalicylicacid

(r2>

0.99

97forbo

than

alytes).

Thismetho

dwas

used

forthe

analysisof

human

plasmasamples

follo

wed

byad

ministrationof

100

500mgof

aspirin

tohe

althy

R. Mullangi et al.


912

Table

1.(Con

tinue

d)

Ana

lyte(s)

Autho

rsSampleproc

essing

details

Chrom

atog

raph

icco

ndition

sVa

lidationpa

rameters

App

licab

leco

nclusion

s

human

voluntee

rsin

abioe

quivalen

cestud

y.Extractio

n:to

analiquo

tof

plasmaeq

ualv

olum

eof

ISsolutio

n,which

brings

thepH

ofmixture

to2.7.

Tothis,4

00mL

ofACN

was

adde

dmixed

and

after15

min,the

conten

tswere

centrifug

edat

10,500

gfor1min

andthe

supe

rnatan

twas

tran

sferredinto

1.5mL

tube

sco

ntaining

100

200mgsodium

chlorid

e.Th

esuspen

sion

was

vortex

mixed

andincu

batedat

4 C

for10

min.

Follo

wingincu

batio

ntheco

nten

tswere

centrifug

edfor1min

at10

,500

gan

d20

0mL

oforga

niclayerwas

tran

sferredinto

HPL

Cvialsforan

alysis.

Colum

n:Nov

apak

C18(10

4mm,4

mm)maintaine

dat

35 C.

Limitof

detection:

75an

d10

0pg

onco

lumnforaspirin

and

salicylicacid,respe

ctively.

Internal

stan

dard:2

-methy

lben

zoicacid

(5mg

/mLin

1:1mixture

of0.2

MHCla

nd0.2

M

ortho-ph

osph

oricacid).

Mob

ileph

ase:

isoc

ratic

mob

ileph

aseco

mprising74

0mL

water,9

00mL

ortho-ph

osph

oricacid

and18

0mLACN(na

lpH2.5)

ataow

-rateof

1mL/min.

Selectivity

:noen

doge

nous

interferen

ceat

theretention

times

ofthean

alytes.

Detectio

n:l m

axsetat

237nm

.Absolutereco

very:1

06.8

8.4,

121.7

4.8an

d12

9.0

2.1%

foraspirin

,salicylicacid

and

IS,respe

ctively.

Volumeof

injection:

10mL

.Accuracyan

dprecision:

precisionan

daccu

racy

determ

ined

at10

0ng

/mLfor

aspirin

andsalicylicacid

and

foun

dto

beacceptab

le.

Retentiontim

e:2.1,

2.9,

4.2,

6.8

and8.9min

forge

ntisicacid,

salicyluricacid,aspirin,

Stab

ility:aspirinwas

foun

dto

bestab

lein

human

plasma

for3mon

thsat7

0 C,b

utat3

0 C

itde

compo

sedto

(Con

tinue

s)



913

Table

1.(Con

tinue

d)

Ana

lyte(s)

Autho

rsSampleproc

essing

details

Chrom

atog

raph

icco

ndition

sVa

lidationpa

rameters

App

licab

leco

nclusion

s

salicylicacid

and

IS,respe

ctively.

salicylicacid

with

in3wee

ksto

5%an

d7wee

ksto

13%.A

t2

4 C

aspirin

degrad

ation

was

5%with

in1han

dad

ditio

nof

potassium

uo

rideha

sno

inue

nce.

Totalrun

time:

10min.

Aspirin,

salicylic

acid

and

salicyluricacid

Krivoskov

et

al.,

1996

Matrix

:hum

anplasmaan

durine(200

mL).

System

:HPL

Cwith

UVde

tector.

Line

arity

:0.2

10mm

ol/L

forall

analytes

(r2>

0.99

9for

both

analytes).

Bloo

dsamples

(1mL)

wereco

llected

into

tube

sco

ntaining

hepa

rin(50IU)an

dsodium

uo

ride(4

mg

per1.5mLbloo

d)an

dim

med

iately

centrifug

edto

harvestplasma.

Plasmawas

stored

at5

6 C

until

analysis(w

ithin

onewee

k).The

metho

dwas

used

toqu

antitate

aspirin

,salicylicacid

andsalicyluric

acid

follo

wingoral

administration

of30

mgaspirin

andalso

toqu

antitatesalicylicacid

and

salicyluricacid

follo

wingoral

administrationof

304

00mg

ofaspirin

.

Extractio

n:an

aliquo

tof

plasma

was

deproteinizedwith

50mL

of35

%pe

rchloric

acid,vortexmixed

for

10san

dcentrifug

edat

12,000

gfor10

min

and

50mL

supe

rnatan

twas

tran

sferredinto

HPL

Cvialsforan

alysis.

Colum

n:Se

paronSG

XC18(150

3.3mm)maintaine

dat

45 C.

Absolutereco

very:w

ere

901

05%

foralla

nalytes.

Internal

stan

dard:n

oIS

was

used

.Mob

ileph

ase:

isoc

ratic

mob

ileph

aseco

mprisingwater

85%

phosph

oricacid

butan

ol

tetrab

utylam

mon

ium

hydrox

ide

MeO

H(134

:1:1:63,

v/v/v/v)

ataow

-rate

of0.9mL/min.

Detectio

n:l m

axsetat

237an

d30

5nm

forplasmaan

durine

samples,respe

ctively.

Retentiontim

e:2.86

,3.78an

d7.12

min

foraspirin

,salicylic

acid

andsalicyluric

acid,respe

ctively.

Totalrun

time:

10min.

Aspirin,

salicylic

acid

and

gentisicacid

Klim

eset

al.,19

92Matrix

:rab

bitwho

lebloo

d,plasmaan

disolated

erythroc

ytes.

System

:HPL

Cwith

UVde

tector.

Line

arity

:20

400,

100

500an

d2

40mg

/mLforaspirin

,salicylicacid

andge

ntisic

acid,respe

ctively(r>

0.98

for

alla

nalytesin

threematric

esexcept

for

gentisic

acid

in

Tothehe

parin

ized

rabb

itbloo

dpo

tassium

uo

ride(50mL

/5mL)

was

adde

dto

preven

thy

drolysisof

aspirin

bych

olinesterases.Plasma

anderythroc

ytes

(byspinning

plasmaat

1500

gfor10

min)were

harvestedfrom

bloo

d.Who

le

R. Mullangi et al.


914

Table

1.(Con

tinue

d)

Ana

lyte(s)

Autho

rsSampleproc

essing

details

Chrom

atog

raph

icco

ndition

sVa

lidationpa

rameters

App

licab

leco

nclusion

s

plasma,whe

rein

itwas

r>

0.89

).bloo

d,erythroc

ytes

andplasma

werefrozen

until

analysis.This

metho

dwas

used

toqu

antitate(up

to2h)

aspirin

,salicylicacid

and

gentisicacid

inrabb

itwho

lebloo

d,plasmaan

derythroc

ytes

follo

wing

i.v.adm

inistrationof

aspirin

(Aspeg

icinjection)

torabb

itsat

100mg/kg

dose.Followingi.v.

administrationat

3min,aspirin

even

lydistrib

uted

betw

een

erythroc

ytes

andplasmaan

dits

levelsde

creasedrapidlyin

both

matric

es.G

entisicacid

was

not

detected

inerythroc

tyes

inthe

course

of2hPK

stud

ytim

e.

Extractio

n:bloo

dsamples:

toan

aliquo

tof

bloo

d(500

mL),10

mLIS

solutio

nwas

adde

dan

dthebloo

dwas

hemolyzed

byad

ding

900mL

ofwater.T

heco

nten

tswereshaken

for5min,son

icated

for

5min

andleftasidefor

5min

atroom

tempe

rature.T

henthe

samplewas

acidied

with

300mL

ofHCl(3

mol/L),shaken

for5min

and6mLof

dich

lorometha

newas

adde

d,centrifug

edfor5

min

at19

30g.

The

orga

niclayer(5

mL)

was

evap

orated

todryn

ess

unde

rnitrog

enstream

andresidu

ewas

reco

nstituted

in50

mLof

mob

ileph

asean

dused

forHPL

Can

alysis.

Colum

n:Sepa

ronSG

XC18

(150

3.2mm,5

mm)

maintaine

dat

ambien

troom

tempe

rature.

Limitof

detection:

20ng

/mLfor

aspirin

andsalicylicacid

inbloo

dan

dplasma;60

ng/m

Lin

erythroc

ytes.For

gentisic

acid

itwas

50ng

/mLin

bloo

dan

dplasma.

Erythroc

ytes:5

00mL

was

proc

essedin

thesame

man

neras

describ

edfor

bloo

dsamples

except

using40

0mL

ofHCl

insteadof

300mL

for

acidicatio

nan

dcentrifug

ationwas

7min.

Mob

ileph

ase:

isoc

ratic

mob

ileph

aseco

mprisingMeO

H

water,80:10

0,v/v(na

lpH2.5

with

5%pe

rchloricacid)at

aow

-rateof

0.5mL/min.

Selectivity

:noen

doge

nous

interferen

ceat

theretention

times

ofthean

alytes

inallthe

matric

es.

Plasma:50

0mL

was

proc

essedin

thesame

man

neras

describ

edfor

bloo

dsamples

except

Detectio

n:l m

axsetat

236nm

.Absolutereco

very:m

ean

reco

very

was

98.9

1.8,

90.7

4.2an

d10

0

1.6%

foraspirin

from

who

lebloo

d,

(Con

tinue

s)



915

Table

1.(Con

tinue

d)

Ana

lyte(s)

Autho

rsSampleproc

essing

details

Chrom

atog

raph

icco

ndition

sVa

lidationpa

rameters

App

licab

leco

nclusion

s

using20

0mL

ofHCl

insteadof

300mL

foracidicatio

n.

erythroc

ytes

andplasma,

respectiv

ely;70

.9

1.5an

d61

.7

3.8,

80.1

1.4%

for

salicylicacid

from

who

lebloo

d,erythroc

ytes

and

plasma,respectiv

ely;

50.3

3.7an

d67

.2

3.3%

forge

ntisicacid

from

who

lebloo

dan

dplasma,

respectiv

ely.

Internal

stan

dard:

benzan

ilide

.Re

tentiontim

e:3.0,

5.3,

8.4an

d14

.2min

forge

ntisicacid,

aspirin

,salicylicacid

andIS,

respectiv

ely.Salicyluricacid

eluted

at4min

had

endo

geno

usinterferen

ce.

Precisionan

daccu

racy:

intra-

andinter-da

yprecision

andaccu

racy

werefoun

dto

bewith

inacceptab

lelim

its(w

ithCV%

0.99

9forbo

than

alytes).

Owingto

post-colum

nhy

drolysisan

duo

rescen

cede

tection,

aspirin

can

bede

tectab

leas

low

as2ng

/mLin

plasma.Autho

rsop

timized

thepH

,tempe

rature

oftheco

ilan

dtim

edu

ratio

nto

controlthe

rate

ofaspirin

hydrolysis.T

hismetho

dis

used

toqu

antitateaspirin

and

salicylicacid

follo

wingoral

administrationof

50mgof

aspirin

tohe

althyhu

man

voluntee

rs.

Extractio

n:to

analiquo

tof

plasma,3mLof

diethy

lethe

r,50

mLof

21.25%

phosph

oricacid

(85%

ortho-ph

osph

oric

acid

water,1

:3,v/v)

weread

dedvo

rtex

mixed

for10

min

and

centrifug

edfor5min

at15

00g.

Totheethe

rph

ase1

50mL

of10

mM

phosph

atebu

ffer

(pH

7.4)

was

adde

d,vo

rtex

mixed

andcentrifug

ed.

Theethe

rlayerwas

evap

orated

unde

rnitrog

enstream

andleft

over

aque

ouslayerwas

kept

oniceun

tilHPL

Can

alysis.

Colum

n:Sp

herisorbODS2

(50

4.6mm,5

mm)

maintaine

dat

ambien

troom

tempe

rature.

Selectivity

:noen

doge

nous

interferen

ceat

theretention

times

ofthean

alytes.N

ointerferingpe

akswerefoun

din

extracts

ofplasmafrom

patie

ntstaking

med

ications

(alm

ost30

drug

s).

Mob

ileph

ase:

isoc

ratic

mob

ileph

aseco

mprising40

%MeO

Hwith

0.08

5%ph

osph

oricacid

inwater

ataow

-rateof

1mL/min.S

odium

hydrox

ide

(0.5

M)at

0.15

mL/min

was

merge

dviaT-piecewith

the

elue

ntfrom

theco

lumn,

which

isow

edthroug

hreactio

nco

il(16m

0.25

mm,i.d.w

ithco

ildiam

eter

of6cm

),which

isim

mersedin

anoilb

atch

maintaine

dat

Absolutereco

very:7

4.6

4.4

and70

.2

3.8%

foraspirin

in0.5an

d1mLplasma,

respectiv

ely;77

.0

2.4an

d66

.4

1.6%

forsalicylicacid

in0.5an

d1mLplasma,

respectiv

ely.

(Con

tinue

s)



917

Table

1.(Con

tinue

d)

Ana

lyte(s)

Autho

rsSampleproc

essing

details

Chrom

atog

raph

icco

ndition

sVa

lidationpa

rameters

App

licab

leco

nclusion

s

Internal

stan

dard:n

oIS

was

used

.60

C.T

hesepa

ratedan

dhy

drolyzed

aspirin

was

measuredas

salicylicacid

byuo

rescen

cede

tector

with

anexcitatio

nwavelen

gthof

310nm

anda38

9nm

emission

.Re

tentiontim

e:~5.0,

7.0an

d12

.0min

forsalicyluricacid,

aspirin

andsalicylicacid,

respectiv

ely.

Volumeof

injection:

50mL

.

Accuracyan

dprecision:

foun

dto

bewith

inthe

acceptab

lelim

its.

Totalrun

time:

12min.

Aspirinan

dsalicylicacid

Gaspa

rian

dLo

catelli,1

987

Matrix

:hum

anplasma

(200

mL).

System

:HPL

Cwith

UVde

tector.

Line

arity

:0.1

20mg

/mLfor

aspirin

andsalicylicacid

(r2>

0.99

9forbo

than

alytes).

Bloo

dsamples

(1mL)

wereco

llected

into

chilled

tube

sco

ntaining

10mL

hepa

rin(100

0U/m

L)an

dpo

tassium

uo

ride(10mL

50%

w/v

inwater).Plasmawas

harvested

immed

iately

byspinning

at0 C

andstored

at8

0 C

until

analysis

(with

inon

ewee

k).A

lthou

ghdich

lorometha

nega

vereco

very

>90

%foraspirin

inhe

althy

voluntee

rs,ratsan

drabb

its,the

reco

very

was

poor

from

urem

icpa

tientsplasma.Onlyhe

xane

elim

inated

interferen

cean

dga

vego

odreco

very

ofaspirin

from

urem

icpa

tientsplasma.Noloss

ofsalicylicacid

was

observed

durin

gevap

orationstep

.The

valid

ityof

themetho

dwas

determ

ined

byassessingtheplasma

conc

entrations

ofaspirin

and

salicylicacid

follo

wingi.v.

administrationof

aspirin

atado

seof

100mg/m

2to

aurem

icpa

tient,

who

ison

regu

larhe

mod

ialysis.

Extractio

n:to

analiquo

tof

plasma,15

mLof

7M

H3PO

4,80mgNaC

land

40mL

water

MeO

H(1:1,

v/v)

and50

mLIS

solutio

nweread

ded,

vortex

mixed

for15

s,then

8mLof

hexane

was

adde

dan

dshaken

for10

min

at0 C.T

heorga

niclayerwas

dried

unde

rnitrog

enstream

andtheresidu

ewas

reco

nstituted

in20

0mL

mob

ileph

asean

dwas

used

forHPL

Can

alysis.

Colum

n:Lich

rosorb

RP8(250

4mm,7

mm)maintaine

dat

ambien

troom

tempe

rature.

Selectivity

:noen

doge

nous

interferen

ceat

theretention

times

ofthean

alytes.

Internal

stan

dard:p

-toluic

acid

(20mg

/mLin

ACN

water,7

0:30

,v/v).

Mob

ileph

ase:

isoc

ratic

mob

ileph

aseco

mprisingACN

water

(pH2.5with

H3PO

4),30

:70,

v/vat

aow

-rateof

1mL/min.

Absolutereco

very:2

7

3an

d54

2%foraspirin

and

salicylicacid,respe

ctively.

Detectio

n:l m

axsetat

229nm

.Accuracyan

dprecision:

foun

dto

bewith

intheacceptab

lelim

its.

Retentiontim

e:5.9,

8.0an

d9.6

min

foraspirin

,salicylicacid

andIS,respe

ctively.

Volumeof

injection:

100mL

.To

talrun

time:

12min.

R. Mullangi et al.


918

Table

1.(Con

tinue

d)

Ana

lyte(s)

Autho

rsSampleproc

essing

details

Chrom

atog

raph

icco

ndition

sVa

lidationpa

rameters

App

licab

leco

nclusion

s

Aspirinan

dsalicylicacid

Tebb

ettet

al.,

1985

Matrix

:hum

anserum

(500

mL).

System

:HPL

Cwith

UVde

tector.

Line

arity

:10

120mg

/mLfor

both

thean

alytes

(r2>

0.99

8forbo

than

alytes).

Autho

rssugg

estedthat

thismetho

dcanbe

routinelyused

inho

spita

lsto

quan

titateaspirin

andsalicylic

acid

alon

gwith

paracetamol.

Extractio

n:an

aliquo

tof

serum

was

acidied

with

100mL

ofHCla

ndextractedthric

ewith

2mLof

chloroform

ACN(60:40

,v/v).Th

eco

mbine

dorga

nic

extracts

were

evap

orated

todryn

ess

andreco

nstituted

in10

0mL

ofmob

ileph

ase

andwas

used

for

HPL

Can

alysis.

Colum

n:Sp

herisorbODS(250

4.5mm,5

mm)maintaine

dat

ambien

troom

tempe

rature.

Internal

stan

dard:n

oIS

was

used

.Mob

ileph

ase:

isoc

ratic

mob

ileph

aseco

mprisingACN

MeO

Hw

ater,2

5:10

:65

(v/v/v)

na

lpH3.0with

ortho-ph

osph

oricacid

ata

ow

-rateof

1mL/min.

Limitof

detection:

2ng

onco

lumnforbo

than

alytes.

Detectio

n:l m

axsetat

234nm

.Se

lectivity

:noen

doge

nous

interferen

cefrom

theblan

kserum

attheretentiontim

esof

thean

alytes.

Retentiontim

e:~3.5an

d5.0

min

foraspirin

,and

salicylic

acid,respe

ctively.

Absolutereco

very:9

710

0%forbo

than

alytes.

Volumeof

injection:

20mL

.To

talrun

time:

9min.

Aspirinan

dsalicylicacid

Bran

donet

al.,

1985

Matrix

:hum

anplasma

(1mL).

System

:HPL

Cwith

UVde

tector.

Line

arity

:0.025

0.5

and

0.5

5mg/mLas

low

conc

entrationcu

rvean

d0.5

10an

d5

70mg/mLas

high

conc

entrationcu

rvefor

aspirin

andsalicyluricacid,

respectiv

ely(r2>

0.99

8forbo

than

alytes).

Peroxide

-freediethy

lether

oran

hydrou

sdiethy

lether

(usedafter

72h)

show

edch

romatog

raph

icinterferen

cewith

aspirin

.To

preven

ttheen

zymatichy

drolysis

ofaspirin

into

salicylicacid,

physostig

minesulfa

tewas

adde

dto

bloo

dsamples.Thismetho

dwas

used

tode

term

inethePK

parametersof

aspirin

andsalicylic

Extractio

n:to

analiquo

tof

plasma,20

0mL

of1

M

Colum

n:mBo

ndap

akC18(300

3.9mm,1

0mm

)co

upledto

aSe

lectivity

:noen

doge

nous

interferen

ceat

theretention

(Con

tinue

s)



919

Table

1.(Con

tinue

d)

Ana

lyte(s)

Autho

rsSampleproc

essing

details

Chrom

atog

raph

icco

ndition

sVa

lidationpa

rameters

App

licab

leco

nclusion

s

acid

follo

wingoral

administration

ofeither

100or

600mgof

glycinated

aspirin

toov

ernigh

tfasted

healthyhu

man

voluntee

rs.

Bloo

dwas

collected

into

tube

having

lithium

hepa

rinan

dph

ysostig

minesulfa

te(100

mLof

2104M).Bloo

dsamples

were

centrifug

edwith

in5min

and

stored

at2

0 C

until

analysis.The

C maxan

dT m

axob

served

foraspirin

were1.7

0.47

mg/mLan

d26

.7

10.8

min,respe

ctivelywith

at

of14

.7min;for

salicylicacid

theC m

ax

andT m

axwere6.63

1.42

mg/mL,

respectiv

elyan

d1.67

0.88

hwith

at

of1.61

h.Alltheob

tained

values

werein

agreem

entwith

the

earlier

repo

rted

values.

HCla

nd10

mLof

anhy

drou

sdiethy

lether

weread

dedan

dthe

tube

swereclosed

,vo

rtex

mixed

for5min

andcentrifug

edat

1500

gfor4min.The

orga

nic

layerwas

driedun

der

gentle

stream

ofnitrog

enwhile

keep

ing

thetube

sin

anice

water

bath

(topreven

tsublim

ationof

salicylic

acid).Re

sidu

ewas

dissolvedin

200mL

ofmob

ileph

asean

dused

forHPL

Can

alysis.IS

solutio

nwas

adde

dinto

tube

san

ddriedbe

fore

adding

plasma.

guardco

lumn(23

3.9mm,

mBo

ndap

akC18

material)maintaine

dat

47 C.

times

ofthean

alytes.

Interferen

cefrom

othe

r46

drug

san

dfrom

plasmaof

patie

ntsreceivingothe

rmed

ications

was

tested

and

foun

dthat

therewas

nointerferen

ceexcept

with

methy

clothiazide.

Internalstan

dard:m

-anisic

acid

(25mL

of50

mg/L

inMeO

H).

Mob

ileph

ase:

isoc

ratic

mob

ileph

aseco

mprising0.13

mL

ortho-ph

osph

oricacid,1

0mL

1-bu

tano

l,27

0mLMeO

Han

d72

0mLwater

delivered

ata

ow

-rateof

1.8mL/min.

Absolutereco

very:9

6.4

100.3

and76

.79

1.2%

,respe

ctively,

foraspirin

andsalicylic

acid,respe

ctively.

Detectio

n:l m

axsetat

234nm

.Re

tentiontim

e:5.6,

8.0an

d9.6min

foraspirin

,salicylic

acid

andIS,respe

ctively.

Volumeof

injection:

510

0mL

.To

talrun

time:

10min.

Aspirin,

salicylic

acid,g

entisic

acid

and

salicyluricacid

OKruket

al.,19

84Matrix

:hum

anan

drat

plasmaor

serum

(200

mL)an

draturine.

System

:HPL

Cwith

UVde

tector.

Line

arity

:15

00mg

/mLfor

aspirin

andsalicylicacid;

160

mg/m

Lforge

ntisicacid

andsalicyluricacid

inplasma

(r>

0.99

3foralla

nalytes).In

raturinethelin

earityrang

eforsalicylicacid

was

110

0mg

/mL(r>

0.99

6).

Bloo

dsamples

wereco

llected

into

chilled

tube

sco

ntaining

5mg/mL

potassium

uo

ride(25%

)to

preven

taspirin

hydrolysis.Sim

ilarly

follo

wingco

llectionof

urine

samples,the

yweremixed

with

equa

lvolum

eof

10Mhy

droc

hloric

acid

andstored

at8

0 C

until

R. Mullangi et al.


920

Table

1.(Con

tinue

d)

Ana

lyte(s)

Autho

rsSampleproc

essing

details

Chrom

atog

raph

icco

ndition

sVa

lidationpa

rameters

App

licab

leco

nclusion

s

furthe

ran

alysis.A

nticoa

gulant

EDTA

was

preferredov

erhe

parin

asbe

nzyl

alco

hol,which

isused

asapreservativ

ean

dinterferes

with

theretentiontim

eof

salicyluric

acid.A

utho

rsevalua

tedtheeffect

ofMeO

Hco

ncen

trationin

mob

ileph

asean

ddifferen

tpH

cond

ition

son

thech

romatog

raph

icresolutio

nof

thean

alytes.Thismetho

dwas

successfully

used

tostud

ythePK

ofaspirin

andits

metab

olite

sin

rats

andhu

man

.Ratsweregiven

i.v.infusionat

ado

seof

200mg/kg

andserum

was

analyzed

foraspirin

andits

metab

olite

s.In

ratserum

aspirin

,salicylicacid

andge

ntisic

acid

werede

tectab

lebu

tsalicyluric

acid

was

notde

tectab

le.The

serum

half-lifeof

aspirin

was

78min,

which

isless

than

plasmaha

lf-life

(15min)in

man

.Followingoral

administrationof

aspirin

tablets(4

32

5mg),p

lasm

awas

analyzed

andtim

evs

conc

entrationpro

lesof

aspirin

,salicylicacid

andsalicyluricacid

werede

term

ined

,whe

reas

gentisic

acid

was

quan

tiab

leon

lyat

0.5an

d4hon

ly.

Extractio

n:plasma/serum

samples,toan

aliquo

tof

plasmaor

serum

20mL

of30

%pe

rchloricacid

containing

ISsolutio

n(0.02%

)an

d20

0mL

ofMeO

Hweread

ded

vortex

mixed

and

centrifug

edat

9000

gfor4min.Following

centrifug

ationthe

supe

rnatan

twas

tran

sferredinto

HPL

Cvialsforan

alysis.

Colum

n:C8(10

4mm,4

mm)

coup

ledto

agu

ardco

lumn

(40

2mm,3

038

mm)

maintaine

dat

ambien

troom

tempe

rature.

Selectivity

:noen

doge

nous

interferen

ceat

theretention

times

ofthean

alytes

inplasma/serum.N

early

19be

nzoicacid

deriv

atives

were

tested

fortheirinterferen

ceon

thech

romatog

raph

icresolutio

n(in

serum/plasm

a)of

thesean

alytes

andfoun

dthat

allo

fthem

eluted

before

therstan

alyte(i.e.

gentisic

acid)un

derthis

chromatog

raph

icco

ndition

s.In

raturinealso

therewas

interferen

ceat

theretention

timeof

salicylicacid

andIS.

Urin

esamples,after

thaw

ingurinesamples,

400mL

ofurinesample

was

tran

sferredinto

2mLglassam

poules

ushe

dwith

nitrog

enan

dsealed

immed

iately.

Theam

poules

were

heated

for3hat

120 C

tohy

drolyzethe

conjug

ates.Following

cooling,

tothe20

mLof

hydrolyzed

mixture,

20mL

ofIS

solutio

n(0.02%

),18

0mL

ofdistilled

water

and

200mL

MeO

Hwere

adde

d;theco

nten

tswerecentrifug

edat

9000

gfor2min

and

20mL

ofsupe

rnatan

twas

used

forHPL

Can

alysis.

Mob

ileph

ase:

isoc

ratic

mob

ileph

aseco

mprisingMeO

H

0.1%

KH2PO

4bu

ffer

(pH3.9),

35:6

5(v/v)at

aow

-rateof

2mL/min.

Recovery:fou

ndto

be80

86%

for

the

analytes

inserum/plasm

a.Accuracyandprecision

:precision

andaccuracy

determ

ined

at100ng

/mL(fo

raspirin

and

salicylic

acid)and

foun

dto

beacceptable.

Internal

stan

dard:

3,4,5-trim

etho

xy-

benzalde

hyde

.

Detectio

n:l m

axsetat

235nm

forplasma/serum

analysis,

whe

reas

forurinesamples

thel m

axwas

setat

313nm

.

(Con

tinue

s)



921

Table

1.(Con

tinue

d)

Ana

lyte(s)

Autho

rsSampleproc

essing

details

Chrom

atog

raph

icco

ndition

sVa

lidationpa

rameters

App

licab

leco

nclusion

s

Volumeof

injection:

20mL

.Re

tentiontim

e:3.3,

4.1,

5.5,

6.7

and8.7min

forge

ntisicacid,


salicylicacid

andIS,

respectiv

ely,in

plasma/

serum.Inraturinethe

retentiontim

eforsalicylic

acid

andIS

was

6.0an

d7.5min,respe

ctively.

Totalrun

time:

10min.

Aspirin,

salicylic

acid,g

entisic

acid

and

salicyluricacid

Mayset

al.,19

84Matrix

:plasm

aan

durine

ofrabb

itsan

dhu

man

s.System

:HPL

Cwith

UVde

tector.

Line

arity

:0.2

100

mg/m

Lfor

aspirin

andsalicylicacid;

0.2

4mg

/mLforsalicyluric

acid

inplasma.In

urine:2

20,

203

00an

d20

020

00mg

/mL

forge

ntisicacid,salicylicacid

andsalicyluricacid,

respectiv

ely(r2>

0.99

9fora

llan

alytes

inbo

thmatric

es).

Duringtheextractio

nproc

essthe

loss

ofaspirin

was

minim

al(~2.2%

)andtheacidicpH

cond

ition

ofmob

ileph

asealso

helped

toredu

cethe

aspirin

hydrolysis.Autho

rsrepo

rted

that

loss

ofsalicylicacid

owingto

sublim

ationwas

also

minim

al,w

hich

was

dueto

thecompo

nentsof

plasmaandurine.App

licationof

this

metho

dwas

show

nin

ahu

man

PKstud

yfollowing

oral

administration

of975mg

aspirin

tablet.Th

eC m

ax

and

T max

observed

foraspirin

acid

were11.7

1.7mg

/mLand20

min,

respectiv

elywith

at

of30

.6min;

forsalicylic

acid

theC m

axan

dT m

ax

werefoun

dto

be56

.7

4.8mg

/mL

and2.37

0.37

h,respectiv

elywith

at

of2.70

h.All

the

obtained

values

werein

agreem

entwith

the

earlier

repo

rted

values.

Therecoverie

sof

free

salicyluric

acid,con

juga

tedsalicyluricacid,

free

salicylicacid,con

juga

ted

salicylicacid

andge

ntisicacid

from

human

urinewere61

.3,7.4,8.5,9.6

and1.1%

,respe

ctively.Th

emetho

dwas

also

used

tode

term

inethePK

ofaspirin

andits

metab

olite

sfollowingi.v.

Extractio

n:plasma

samples,toan

aliquo

tof

human

plasma(1

mL),

150mL

of3

Mortho-

phosph

oricacid,

400mgof

sodium

chlorid

ean

d12

mL

DCM

containing

IS(3mg

/mL)wereadded.Th

econten

tsin

tube

were

shaken

for10

min

at300rpm

andcentrifug

ed.

Theup

perlayerwas

discarde

dan

dthe

orga

niclayer(8

mL)

was

driedun

derredu

ced

pressure

andtheresidu

ewas

dissolvedin

200mL

ofmob

ileph

aseand

used

forH

PLCan

alysis.In

case

ofrabb

itplasma

Colum

n:Nuc

leosil

C18(250

4.6mm,5

mm)maintaine

dat

ambien

troom

tempe

rature.

Selectivity

:noen

doge

nous

interferen

ceat

theretention

times

ofthean

alytes

inplasma,whe

reas

inblan

kurinesalicyluricacid

was

observed

infew

human

sas

itisano

rmal

constitue

nt.

Similarly

infew

urineblan

ksinterferen

ceat

thege

ntisic

acid

retentiontim

eas

this

interferen

ceisag

lyco

neof

endo

geno

usgluc

uron

ide.

Inrabb

itplasmathereisno

interferen

ceforaspirin

and

salicyluricacid,b

utshow

edsalicylicacid

(upto

0.2mg

/mL).Rab

bitb

lank

urine

show

ed6

25an

d6

19mg

/mLof

salicylicacid

andsalicyluricacid,

respectiv

ely,an

dinterferen

ce

R. Mullangi et al.


922

Table

1.(Con

tinue

d)

Ana

lyte(s)

Autho

rsSampleproc

essing

details

Chrom

atog

raph

icco

ndition

sVa

lidationpa

rameters

App

licab

leco

nclusion

s

administrationof

aspirin

atado

seof

25mg/kg

torabb

its.

only500mL

was

analyzed

inthesimilar

lines,and

thequ

antity

ofsaltandph

oshp

horic

acid

redu

ceby

half.

ofge

ntisicacid

was

high

inblan

k,which

didno

tpe

rmit

toqu

antitatege

ntisicacid.

Urin

esamples,inurine

aspirin

metab

olite

swerean

alyzed

bymixing1mLof

urine

with

equa

lvolum

eof

0.2

Macetatebu

ffer

(pH5),a

drop

ofch

loroform

and20

mLof

b-gluc

uron

idasean

dincu

batedfor20

hat

37 C

onashaking

water

bath.Following

incu

batio

nthesolutio

nwas

tran

sferredinto

a10

mLvo

lumetric

ask

andthevo

lumewas

mad

eup

with

buffer

andfrom

this1mLwas

adde

dinto

acentrifug

etube

containing

12mL

ofDCM

(9mg

/mL)

and

thesameam

ountsof

saltan

dph

oshp

horic

acid

asab

ovean

dproc

essedin

similar

lines

ofplasmasamples.

Fortheestim

ationof

salicylicacid

and

salicyluricacid

the

residu

ewas

reco

nstituted

in1mLof

mob

ileph

ase,

whe

reas

forestim

ationof

entisic

acid

thereco

nsti-tutio

nvo

lumewas

500mL

.

Mob

ileph

ase:

isoc

ratic

mob

ileph

aseco

mprising5m

M

phosph

atebu

ffer

(pH2.5)

MeO

HA

CN,6

8:16

:16,

v/v/v,

ataow

-rateof

1.3mL/min.

Absolutereco

very:from

plasma

thereco

very

foraspirin

,salicylicacid

andsalicyluric

acid

was

92

1,85

4an

d52

8%,respe

ctively;

whe

reas

from

urinethe

reco

very

forsalicylicacid,

salicyluricacid

andge

ntisic

acid

was

98

4,93

7an

d55

3%,respe

ctively.

Internal

stan

dard:

mep

heny

toin.

Detectio

n:l m

axsetat

237nm

except

forge

ntisicacid

Stab

ility:e

xtracted

samples

inmob

ileph

ased

show

ed4%

(Con

tinue

s)



923

Table

1.(Con

tinue

d)

Ana

lyte(s)

Autho

rsSampleproc

essing

details

Chrom

atog

raph

icco

ndition

sVa

lidationpa

rameters

App

licab

leco

nclusion

s

estim

ationin

urineforwhich

thel m

axsetat

330nm

.loss

ofaspirin

atroom

tempe

rature.

Retentiontim

e:4.5,5.7,7.4,10

.0an

d11

.6min

forge

ntisicacid,


salicylicacid

andIS,

respectiv

ely.

Volumeof

injection:

10mL

except

forge

ntisicacid

estim

ationin

urine,whe

rethe

injectionvo

lumewas

50mL

.To

talrun

time:

12min.

Aspirinan

dsalicylicacid

Niede

ran

dJaeg

er,

1983

Matrix

:hum

anbloo

dan

dplasma.

Extractio

n:plasmawas

extractedwith

ethe

r:he

xane

(80:20

,v/v),

vortex

mixed

and

centrifug

ed.T

heorga

nic

layerwas

evap

orated

todrynessandreconstituted

inmob

ileph

aseandused

forH

PLCanalysis.Entire

samples

processin

gwas

done

inan

ice-bath

inordertoavoidtheloss

ofsalicylicacidby

sublimation.

Internalstan

dard:n

oIS

was

used

.

System

:HPL

Cwith

UVde

tector.

Line

arity

:0.1

10an

d0.1

40mg

/mLforaspirin

and

salicylicacid,respe

ctively

(r2>

0.99

8forbo

than

alytes).

Enzymatichy

drolysisof

aspirin

was

redu

cedby

proc

essing

thebloo

dun

derch

illed

cond

ition

san

dby

additio

nof

potassium

uo

ride.

Furthe

rtheplasmasamples

were

stab

leby

storingthem

inde

epfree

zerfor5da

ys.Evenafter

storag

efor4wee

ksthe

degrad

ationof

aspirin

was

510

%.

Thismetho

dwas

used

toqu

antitatebo

thaspirin

and

salicylicacid

inhu

man

plasma

follo

wingoral

administrationof

500mgof

aspirin

tablet.

Colum

n:Hyp

ersilO

DS(250

4mm,5

mm)at

ambien

troom

tempe

rature.

Absolutereco

very:~

93an

d88

%foraspirin

andsalicylic

acid,respe

ctively.

Mob

ileph

ase:

isoc

ratic

mob

ileph

aseco

mprisingACN

5%

aceticacid,2

0:80

(v/v)at

aow

-rateof

2.5mL/min.

Accuracyan

dprecision:

foun

dto

bewith

inthe

acceptab

lelim

its.

Detectio

n:l m

axsetat

237nm

.Re

tentiontim

e:3.54

and

4.85

min

foraspirin

and

salicylicacid,respe

ctively.

Volumeof

injection:

20mL

.

Aspirin,

salicylic

acid,salicyluric

acid

and

gentisicacid

Bakaran

dNiazi,

1983

Matrix

:rat

plasmaan

durine.

System

:HPL

Cwith

UVde

tector.

Line

arity

:0.5

200

mg/m

L(in

plasma)

and6

200mg

/mL

(inurine)

foralla

nalytesin

both

matric

es(r>

0.99

9for

alla

nalytesin

both

matric

es).

Asau

thorsutilizeddirect

precipita

tion,

thevaria

tionin

the

data

was

considerab

lyde

creased.

Extractio

n:plasma

samples,analiquo

tof

plasma(50mL

)was

precipita

tedwith

100mL

ofACN

containing

IS,

vortex

mixed

for1min

andcentrifug

edfor5

Colum

n:mBo

ndap

akC18

maintaine

dat

ambien

troom

tempe

rature.

Absolutereco

very:the

mean

reco

very

was

61.6,9

4.1,

99.5

and90

.7%

forge

ntisicacid,

salicyluricacid,aspirinan

dsalicylicacid,respe

ctively,in

plasma;whe

reas

thereco

very

ofge

ntisicacid,salicyluric

R. Mullangi et al.


924

Table

1.(Con

tinue

d)

Ana

lyte(s)

Autho

rsSampleproc

essing

details

Chrom

atog

raph

icco

ndition

sVa

lidationpa

rameters

App

licab

leco

nclusion

s

min

at15

,000

rpm

and

thesupe

rnatan

twas

used

forHPL

Can

alysis.

acid,O

-anisicacid

and

salicylicacid

was

97,9

5,92

and91

%,respe

ctively,

from

urine.

Urin

esamples,1

mLof

urinewas

acidied

with

0.5mLof

6MHCl,then

6mLof

anhy

drou

sethe

rwas

adde

dan

dmixed

for15

min

and

centrifug

edfor10

min

at20

00rpm

and5mLof

ethe

rlayerwas

sepa

rated.

Totheethe

rlayer,1mLof

0.1

M

(pH7)

phosph

atebu

ffer

was

adde

d,mixed

for1

5min

andcentrifug

edfor

10min

at20

00rpm

and

theethe

rlayerwas

aspiratedan

dbu

ffer

solutio

nwas

used

for

analysis.

Mob

ileph

ase:

isoc

ratic

mob

ileph

aseco

mprisingwater

MeO

Hg

lacial

aceticacid,

64:35:1(v/v/v)at

aow

-rate

of2mL/min.

Accuracyan

dprecision:

foun

dto

bewith

intheacceptab

lelim

its.

Internal

stan

dard:o

-toluic

acid

(dissolved

inwater

ACN,2

:1,v/v;

100mg

/mLIS

solutio

nforplasmalin

earity

having

conc

entration

>20

and10

mg/m

LIS

solutio

nforplasma

linearityha

ving

conc

entration

40

and

20mg

/mLIS

solutio

nfor

plasmalin

earityha

ving

Detectio

n:l m

axsetat

238an

d30

5nm

forplasmaan

durine

samples

analysis,respe

ctively.

Retentiontim

e:3.0,

3.7,

4.8,

5.0,

7.1an

d10

.2min

forge

ntisic

acid,salicyluricacid,aspirin,

o-an

isicacid,salicylicacid

and

o-toluicacid,respe

ctively.

Volumeof

injection:

20mL

.To

talrun

time:

~12

min.

(Con

tinue

s)



925

Table

1.(Con

tinue

d)

Ana

lyte(s)

Autho

rsSampleproc

essing

details

Chrom

atog

raph

icco

ndition

sVa

lidationpa

rameters

App

licab

leco

nclusion

s

conc

entration

0.99

3foralla

nalytes).

Enzymatichy

drolysisof

aspirin

was

performed

byco

llectingbloo

dinto

tube

sco

ntaining

sodium

uo

ride

andhe

parin

(4mgof

sodium

uo

ridean

d50

IUof

hepa

rinfor

1.5mLbloo

d).The

vialswerekept

oniceno

long

erthan

30min

before

proc

essing

.Plasm

awas

harvestedfrom

bloo

dby

centrifug

ingat

1500

gfor1

0min

atroom

tempe

rature.T

hismetho

dwas

used

toqu

antitateaspirin

,salicylicacid

andsalicyluricacid

inrabb

itplasmaan

dtissues

follo

wing

i.v.adm

inistrationof

aspirin

at50

mg/kg

dose.G

entisicacid

could

notbe

detected

inplasma

follo

wingaspirin

administration.

Thismetho

dwas

also

used

toqu

antitateaspirin

,salicylicacid

and

salicyluricacid

inplasmasamples

collected

from

healthyhu

man

voluntee

rsfollo

wingoral

administrationof

a65

0mgoral

dose

ofaspirin

effervescent

tablet.

Gen

tisicacid

was

notde

tected

inhu

man

plasma.

Extractio

n:plasma

samples,toan

aliquo

tof

plasma(200

mL),50

mLco

ncen

trated

phosph

oricacid

and

600mL

ethy

lacetate

weread

ded,

vortex

mixed

andcentrifug

edat

600gfor10

min

at10

C.The

supe

rnatan

t(400

mL)was

stored

at2

6 C

until

analysis.

Before

HPL

Can

alysis

thesupe

rnatan

twas

driedin

anice-ba

thun

derage

ntle

stream

ofairan

dtheresidu

ewas

dissolvedin

200mL

ofmob

ileph

asean

d10

0mL

was

used

for

HPL

Can

alysis.

Colum

n:LiChrosorbRP

18(150

4mm,5

mm)maintaine

dat

45 C.

Limitof

detection:

0.5an

d2.5mg

/mLforaspirin

and

gentisicacid;0

.2mg

/mLfor

salicylicacid

andsalicyluric

acid.

Tissue

homog

enate,50

0mgof

tissuewas

homog

enized

with

2mL

ofdistilled

water

and

proc

essedas

describ

edforplasmasamples.

Mob

ileph

ase:

isoc

ratic

mob

ileph

aseco

mprisingMeO

H

water,4

0:60

(v/v),na

lpH

3.0(adjustedwith

0.00

5M

phosph

oricacid

andsodium

hydrox

ide)

ataow

-rate

of1.5mL/min.

Absolutereco

very:93

4,95

4,10

2

3an

d10

1

3%for

aspirin

,salicylicacid,

salicyluricacid

andge

ntisic

acid,respe

ctively,from

rabb

itplasma.From

human

plasma

thereco

very

was

98

3,89

5,

94

3an

d10

1

2%foraspirin

,salicylicacid,

salicyluricacid

andge

ntisic

acid,respe

ctively.

Urin

esamples,u

rinewas

10-folddilutedwith

water

andproc

essedas

Detectio

n:l m

axsetat

280nm

.Se

lectivity

:nointerferen

cefrom

asco

rbicacid,cod

eine

,caffeine

,inu

lin,

R. Mullangi et al.


926

Table

1.(Con

tinue

d)

Ana

lyte(s)

Autho

rsSampleproc

essing

details

Chrom

atog

raph

icco

ndition

sVa

lidationpa

rameters

App

licab

leco

nclusion

s

describ

edforplasma

samples.

dextroprop

oxyp

hene

andTEA

attheretentiontim

eof

the

analytes.P

aracetam

olinterfered

with

salicylic

acid

retentiontim

e.Internal

stan

dard:n

oIS

was

used

.Re

tentiontim

e:2,

2.7,

3.7an

d5.5min

forge

ntisicacid,

salicyluricacid,aspirinan

dsalicylicacid,respe

ctively.

Stab

ility:aspirinstab

ility

was

assessed

for27

days

at5,2

6an

d8

0 C

anditwas

foun

dthat

only

at8

0 C

was

little

hydrolysisob

served

.Vo

lumeof

injection:

100mL

.

Aspirin,

salicylic

acid

and

salicyluricacid

Buskin

etal.,19

82Matrix

:hum

anplasmaan

durine.

System

:HPL

Cwith

UVde

tector.

Line

arity

:0.05

1an

d1

10mg/L

aslow

andhigh

calib

ratio

ncu

rves

foraspirin

and

salicyluricacid

inplasma;

110

and10

100

mg/Las

low

andhigh

calib

ratio

ncu

rves

salicylicacid

inplasma.Lo

wan

dhigh

calib

ratio

ncu

rves

with

conc

entrationrang

eof

102

00an

d20

020

00mg/L;

510

0an

d10

075

0mg/L;

140

and40

40mg/Lfor

salicylicacid,salicyluricacid

andge

ntisicacid,

respectiv

ely.Fo

rtotal

salicylates

andge

ntisates

the

low

andhigh

calib

ratio

ncu

rverang

eswere20

400

and40

040

00mg/Lan

d2

40an

d20

400

mg/L,

respectiv

ely.

Bloo

dsamples

wereco

llected

into

chilled

tube

sco

ntaining

lithium

hepa

rinan

dpo

tassium

uo

ride(to

preven

taspirin

hydrolysis).Plasma

samples

werestored

at2

0 C

until

analysis.The

metho

dwas

appliedto

analysisof

plasma

samples

collected

from

healthy

human

voluntee

rsfollo

wingoral

administrationof

a65

0mgoral

dose

ofaspirin

.

Extractio

n:plasma

samples,toan

aliquo

tof

1mLplasma,1mLof

aque

ousox

alicacid

(1mol/L)an

d1mLof

aque

ousIS

solutio

n(0.7mg

)an

d10

mLof

ethe

rhe

xane

(1:1,v/v)

weread

dedvo

rtex

Colum

n:Sp

herisorbODS(250

4.6mm,5

mm)maintaine

dat

ambien

troom

tempe

rature

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