-
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.
Biomed. Chromatogr. 2012; 26: 906941Copyright 2012 John Wiley & Sons, Ltd.wileyonlinelibrary.com/journal/bmc
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)
Review of aspirin bioanalytical methods
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.
Biomed. Chromatogr. 2012; 26: 906941Copyright 2012 John Wiley & Sons, Ltd.wileyonlinelibrary.com/journal/bmc
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)
Review of aspirin bioanalytical methods
Biomed. Chromatogr. 2012; 26: 906941 Copyright 2012 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/bmc
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.
Biomed. Chromatogr. 2012; 26: 906941Copyright 2012 John Wiley & Sons, Ltd.wileyonlinelibrary.com/journal/bmc
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)
Review of aspirin bioanalytical methods
Biomed. Chromatogr. 2012; 26: 906941 Copyright 2012 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/bmc
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)
Review of aspirin bioanalytical methods
Biomed. Chromatogr. 2012; 26: 906941 Copyright 2012 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/bmc
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.
Biomed. Chromatogr. 2012; 26: 906941Copyright 2012 John Wiley & Sons, Ltd.wileyonlinelibrary.com/journal/bmc
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)
Review of aspirin bioanalytical methods
Biomed. Chromatogr. 2012; 26: 906941 Copyright 2012 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/bmc
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.
Biomed. Chromatogr. 2012; 26: 906941Copyright 2012 John Wiley & Sons, Ltd.wileyonlinelibrary.com/journal/bmc
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)
Review of aspirin bioanalytical methods
Biomed. Chromatogr. 2012; 26: 906941 Copyright 2012 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/bmc
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,
salicyluricacid,aspirin,
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.
Biomed. Chromatogr. 2012; 26: 906941Copyright 2012 John Wiley & Sons, Ltd.wileyonlinelibrary.com/journal/bmc
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)
Review of aspirin bioanalytical methods
Biomed. Chromatogr. 2012; 26: 906941 Copyright 2012 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/bmc
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,
salicyluricacid,aspirin,
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.
Biomed. Chromatogr. 2012; 26: 906941Copyright 2012 John Wiley & Sons, Ltd.wileyonlinelibrary.com/journal/bmc
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)
Review of aspirin bioanalytical methods
Biomed. Chromatogr. 2012; 26: 906941 Copyright 2012 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/bmc
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.
Biomed. Chromatogr. 2012; 26: 906941Copyright 2012 John Wiley & Sons, Ltd.wileyonlinelibrary.com/journal/bmc
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