Iran

nian Jo
ournall of Bassic Med
dical Science
S es
ijbms.mum
ms.ac.ir

Determ
mination n of valproic
v c acid in human p plasma using
disperssive liq
quid‐liq
quid mmicroexttractionn follow
wed byy gas
chromaatograpphy‐flam me ionizzation detection
n
Rana Fazeli‐Bakhtiyari 1, 2, Vahid
d Panahi‐Azzar 3, Moham
mmad Hosssein Sorourraddin 2, Abolghasem
Jouyban 4*
1 Liver and Gastrointestinal Disseases Research Center, Tabriz U University of Medical Sciences, Tabriz,
T Iran
2 Department oof Analytical Chemistry, Faculty of
o Chemistry, Un niversity of Tabrriz, Tabriz, Iran
3 Drug Applied Research Center r, Tabriz University of Medical SSciences, Tabriz, Iran
4 Pharmaceuticcal Analysis Reseearch Center andd Faculty of Pharrmacy, Tabriz Un niversity of Medical Sciences, Taabriz, Iran

ARTICLE INFO ABSTRACT

Article type: Objecttive(s): Dispersiive liquid‐liquid
d microextractio on coupled withh gas chromato ography (GC)‐
Original article flame ionization deteector was develo oped for the dettermination of vvalproic acid (VPA) in human
ma.
plasm
Article history:
Materrials and Methodds: Using a syring ge, a mixture of suitable extractiion solvent (40 µl
µ chloroform)
Received: Nov 8
8, 2014
and disperser (1 ml a cetone) was quiickly added to 10 0 ml of diluted pplasma sample containing VPA
Accepted: Sep 5
5, 2015
(pH, 1.0;
1 concentratioon of NaCl, 4% (w/v)), resultin ng in a cloudy solution. After centrifugation
(60000 rpm for 6 min)), an aliquot (1 µl)
µ of the sedimented organic phhase was remove ed using a 1‐µl
Keywords: GC miicrosyringe and injected into the GC system for analysis. One va variable at a timee optimization
Dispersive liqu
uid‐liquid‐ methood was used to sstudy various pa arameters affecting the extractioon efficiency of target
t analyte.
microextractionn Then, the developed m method was fully validated for its accuracy, preecision, recovery y, stability, and
Gas chromato ography‐flame‐ robustness.
ionization detector Resultts: Under the ooptimum extracttion conditions, good linearity range was obttained for the
Human plasmaa calibration graph, witth correlation cooefficient higher than 0.998. Limmit of detection and lower limit
Valproic acid of quaantitation were 3.2 and 6 μg/m ml, respectively. The
T relative stanndard deviation ns of intra and
inter‐day analysis of examined compound were lesss than 11.5%. T The relative reccoveries were
foundd in the range off 97 to 107.5%. Finally,
F the valid
dated method w was successfully applied to the
analyssis of VPA in pattient sample.
Concllusion: The preesented method has acceptable e levels of preccision, accuracy y and relative
recovery and could bee used for therap peutic drug monitoring of VPA inn human plasma a.

►Please citee this article ass:

Fazeli‐Bakhtiyaari R, Panahi‐Azaar V, Sorouraddin MH, Jouyban A. Determination of valproic acid in human pllasma using disp
persive liquid‐
liquid microexttraction followed
d by gas chromatography‐flame ionization detecction. Iran J Basic Med Sci 2015; 18:979‐988.

Introducttion with contactless co onductivity deetection (CD) (18,

( 19), are
Valproic acid (2‐prop pylpentanoic acid, VPA) iis a the methods tha at were usedd for determ mination of
simple eightt carbon braanched‐chain fatty acid w with VPA.. Additionally y, due to volatility of VPA, gas
unique anticconvulsant prroperties and d is used in the chromatography (GC)( (20, 21) is often used.. In order to
treatment off epilepsy, bippolar disorderr and prophylaaxis prevvent severe taiiling of the fat
atty acid peak,, on‐column
of migraine headaches (1 1–5). Hence, monitoring d drug and pre‐column derivatization
d have also bee
en used (22,
levels in various matricees is particullarly valuablee in 23). Analysis
A PA in biologicaal samples is difficult due
of VP
epilepsy forr effective th herapeutic drrug managem ment to th
he presence of o proteins, saalts and vario ous organic
(6–9). Theraapeutic serum/plasma conccentration of V VPA comp pounds in sa amples. Hencee, sample pre eparation is
is between 220–100 µg/mll during controlled therapy but cruciial in drug ana
alysis, which inncludes both analyte
a pre‐
its toxic seru
um/plasma co oncentration may reach 120– conccentration and d sample cleaanup (24). So ome sample
150 µg/ml ((10). Physicocchemical and pharmacokin netic prepparation techn niques based on protein precipitation
p
properties off VPA are listeed in Table1. High
H performaance (PPTT) (18), liquid d‐liquid extraaction (LLE) (22), solid‐
liquid chrommatography (HPLC)
( with ultraviolet ((UV) phasse extraction (SPE) (16), solid‐phase microextr‐
detection (1 11, 12), fluo orescence dettection (13, 14) actio
on (SPME) (2 23, 25–27), hhollow fiber‐lliquid‐phase
or coupled with masss spectrome etry (MS) ((15– micrroextraction (H HF‐LPME) (288) and disperrsive liquid–
17) and ccapillary eleectrophoresis (CE) coup pled liquid
d microextra action (DLLM ME) (19, 29) have been
*Correspondiing author: Abolghaasem Jouyban. Facu
ulty of Pharmacy, T
Tabriz University of
o Medical Sciences, Tabriz, Iran. Tell: +98‐41‐3337932
23; Fax: +98‐41‐
33363231; emaill: [email protected]
 Fazeli‐Bakhtiyyari et al Gas ch
hromatographic determination of valproic acid

developed fo or this purposse. LLE is time e‐consuming and Germ

many). Distilleed water wass used for pre
eparation of
uses large aamounts of potentially
p tox
xic or hazard dous aqueeous solutionss.
solvents (30). In addition n, the resulting g extract mayy be
transferred, evaporated to t dryness and reconstitu uted Instrrumentation:: GC‐FID
with a suitabble solvent priior to analysiss. Compared w with An
A Agilent 7890A gas chromatog graph with
LLE, SPE is a selective sam mple preparation method tthat split//splitless inle
et and FID wass used for sep paration and
uses a packed solid sorben nt (silica or po
olymer) to isoolate determination of VPA. Optimuum flow rates of carrier
the desired aanalyte. Neverrtheless, poten ntial variabilitty of (N2) and detecttor gases, ssuch as hyd drogen and
SPE packings, irreversiblee adsorption of o some analyytes comp pressed air were 1, 440 and 300 ml/min,
on SPE ccartridges and a more‐co omplex metthod respectively. He ettich centri rifuge, mode el D‐7200
developmentt are some of the draw wbacks that are (Germany) was used u for cenntrifuging. Injection port
presented byy this techniqu ue (31). SPME E was introdu uced temp perature of 27 70 °C in the spplitless mode and
a a purge
in the early 1
1990s as a solv vent‐free proccess for extractcting timee of 30 secc were seleccted as opttimal state.
the analytes from aqueous samples or headspace off the Sepaaration was ca arried out on aan HP‐5 capilllary column
samples (32 2). Despite itts obvious ad dvantages, SP PME (30 m × 0.32 mm m i.d., 0.25 μm m film thicknnesses). The
suffers fromm some drawb backs, for exa ample: expenssive ovenn temperature e was prograammed as folllows: initial
SPME fibers are fragile an nd quite senssitive to comp plex temp perature of 80 0 °C (held 1 mmin), from 80 °C to 140 °C
matrices succh as plasm ma (33). LPM ME is a solve vent‐ at a rate
r of 15 C/m ° min, and held at 140 °C for 2 min. Then
minimized ssample pretreeatment proccedure, in wh hich raiseed at 40 C/m
° min to 250 °C and held for 5 min. The
only a few microliters of solvents are used (34, 35). FID temperature
t was
w maintaineed at 280 °C.
Several differrent modes of o LPME have been develop ped,
such as staticc LPME, dynaamic LPME an nd HF‐LPME ((36).
It should be nnoted that in most
m cases, LP PME methodss are Sammple preparattion
time‐consum ming and equiilibrium could d not be attaiined Plassma treatmen nt
even after a llong extraction n time (31). Recently,
R Assad di et A standard stocks solutionn of sodium m valproate
al developed d a simple an nd novel LPME E method, wh hich (10000 mg/l) was prepared in methanol an nd stored at
was named aas DLLME (37 7, 38). In this method, a waater‐ 4°C. Working solu utions were pprepared by dilution with
miscible dispperser solventt containing a water‐immisc
w cible deion nized water. Free drug plasma sam mples were
extraction soolvent is injectted into the aqqueous solutioon of obtained from Irranian Bloodd Transfusion n Research
analytes. A clloudy solutionn (a mixture off water, disperrser Centter (Tabriz, Iran) and keptt at –20 °C un ntil analysis.
solvent, and d extraction n solvent) is i formed and For preparation of desiredd concentratiion (6‐140
consequentlyy the equilib brium state achieved
a quicckly. µg/mml) of VPA in plasma, 1 ml of drug‐free plasma was
After phasee separation,, enriched analyte a can be spikeed with kno own amountss of the VPA A standard
determined by analytical systems. To our knowled dge, soluttion and kep pt at room teemperature for f 20 min.
there is no D DLLME couplled with gas chromatograp
c phy‐ Thenn for precipitation of plasm ma proteins, acetonitrile
flame ionizzation detecctor (GC‐FID D) method for was added to pla asma sample in the ratio of 1:1 and
determinatio on of VPA in human
h plasmaa in the literatture. vorteexed for 1 min.
m Then it w was centrifug
ged at 6000
The present w work is the firrst report of coombination off the rpm for 5 min. 1 ml of thee clear superrnatant was
DLLME meth hod with GC‐F FID, without an ny derivatizattion, transsferred in a 10.0 ml volumeetric flask andd 0.4 g NaCl
for the deterrmination of VPAV in human n plasma. Sevveral was added. Follow wing this, it w
was diluted to
t the mark
factors that in
nfluence the microextractio
m on efficiency w were withh deionized water
w and the pH of obtain ned solution
comprehensiively examineed in detail an nd the optimiized was adjusted to 1.00 by 1 M HCCl. In order to o reduce the
microextracttion conditions were establiished. Finally,, the matrrix effect of the plasma sam mple, the superrnatant was
developed m method was vaalidated accorrding to the FFood dilutted 10‐fold with
w deionizeed water and d then was
and Drug Ad dministration (FDA) guidance and applied d to subjeected to the microextractio
m on procedure.
a real samplee analysis.
Prepparation of re
eal plasma saample
Materialss and Meth
hods Blood
B sample was obtainedd from a male patient (35
Chemicals aand reagents years old) who had signed thee consent forrm that was
Sodium valproate waas kindly do onated by R Rouz apprroved by the Ethics
E Commit ittee, Tabriz University
U of
Darou Pharrmaceutical Co. C (Tehran, Iran). Dichlooro‐ Medical Sciences. This patientt had been ad dministered
methane, tettrachloroethy ylene, chlorofo
orm, and carb bon VPA (125 mg), fluurazepam, bippyridine, rispe eridone, and
tetrachloridee as extraction solven nts and otther proppranolol. 5 ml of bloodd was colle ected in a
chemicals ssuch as metthanol, aceto one, acetonittrile, hepaarinized tube at
a 2 hr after ddrug intake. Bllood sample
tetrahydrofuuran (THF), sodium chloridec (NaaCl), was centrifuged immediatelyy and the plasma p was
hydrochloricc acid (HCl), and
a sodium hy ydroxide (NaOOH) sepaarated and subjected
s to the propossed DLLME
were purch hased from Merck
M Compa any (Darmsttadt, methhod.

980 Iran
I J Basic Medd Sci, Vol. 18, No.10, Oct 2015
Gas chromatog
graphic determin
nation of valproic acid Fazeli‐Bak
khtiyari et al

Table 1. Ph
hysicochemical an
nd pharmacokine
etic properties of vvalproic acid

Physicochemical propertties
Molecullar structure Molecularr weight (g/mole)) Meltting point (˚C) Log P pKa
CH
H3
HO
144.21 120‐121 2.8 4.7
O CH
H3
Pharmaacokinetic propertties

Plasma protein
Therapeeutic range (µg/m
ml) Toxicc range (µg/ml) Halff‐life [ t0.5 (h)] Bioavailabiility
binding (%
%)
80‐955
20‐100 120‐1
150 9‐18
8 70‐100

Physicochem
mical properties calculated
c using ACD/Labs
A softwar
are version 11.0

DLLME proccedure samples

s unde
er different sstorage cond ditions, and
In the next step, 10 mll of diluted pllasma samplee method
m robusstness were also evaluated. Further
(described aabove) was transferred
t into
i a 12‐mll details
d on the
e validation reesults were described
d in
glass test tub
be with conic bottom, and 1 ml acetonee the
t following section.
s
(as disperseer) containin ng 40 µl chlloroform (ass
extraction ssolvent) wass rapidly ad dded to thee Results
R
solution usinng a 5‐ml syyringe, which immediatelyy Optimization
O of the didispersive liq quid–liquid
resulted in a cloudy solutiion. After centrifugation att microextracti
m ion
6000 rpm fo or 6 min, orgganic phase was
w settled too Extraction efficiency oof DLLME depends
d on
the bottom of the tube.. An aliquot (1µl) of thee several
s parameters. Onee variable at a a time
sedimented organic phase was removed using a 1‐‐ optimization
o method wass used to stu udy factors
µl GC micro osyringe (Hamilton, Switzzerland) and d affecting
a the extraction
e effificiency. Some
e important
injected into the GC system
m for analysiss. parameters
p such
s as typpes of extra action and
dispersive
d solvents and ttheir volume es, pH, salt
Assay validaation effect,
e sample volume, centtrifugation ratte, and time
Method vvalidation waas done acco ording to thee were
w investiga ated.
FDA recommendationss. For quantification,,
calibration curves weree constructe ed on threee Selection
S of th
he extractionn solvent
different dayys. Linear ran
nge, correlatio
on coefficient,, Chlorinated solvents are re denser than n water and
and limit off detection (LLOD) was calculated from m are
a the most widely
w used soolvents in DL LLME due to
calibration curve. Thee lowest and a highestt being
b easily removed froom the botttom of the
concentratioons of calibrattion curve werre selected ass conical
c vial after cenntrifugation. Therefore,
the lower lim
mit of quantiffication (LLOQ Q) and upperr dichlorometha
d ane, tetrachlooroethylene, chloroform,
limit of quaantification (UULOQ), while the relativee and
a carbon te etrachloride w were used ass extraction
standard devviations (%R RSDs) of threee replicationss solvents.
s For this
t purpose, 500 µl of spiiked plasma
were less thhan 20% and d 15%, respecctively. Intra‐‐ (140
( µg/ml) was
w transferreed into 1.5 ml Eppendorff
and inter‐d day precisio on and acccuracy weree tubes.
t In the next
n step, acettonitrile was added with
determined by measurin ng plasma qu uality controll 1:1
1 ratio. The mixture wass vortexed forr 1 min and
samples (Q QCs) at low w, medium and high h centrifuged
c for
f 5 min at 6000 rpm. r After
concentratioon levels of VPPA. Relative recovery (RR) precipitation
p of
o proteins, 0.55 ml of clear supernatant
s
of the samp ple preparatioon method was computed d solution
s was transferred in a 5.0 ml volumetric flask
using the folllowing equatiion: and
a deionized d water and 0.2 g NaCl was added
before
b pH adjustment.
a DDLLME proccedure was
CFouund  CReal performed
p by
b various volumes of o selected
RR  100 extraction
e solvents mixed with 1 ml methanol
m to
CAdded give
g equal volume of the seedimented ph hase (40 µl).
where CFoundd is the analytte concentration measured d The
T obtained results reveaaled (Figure 1) 1 that VPA
from the sam mple after annalyte addition, CReal is thee was
w extracted into chlorofoorm better tha an the other
native analyyte concentrration and CAdded is thee solvents.
s Therefore, chlorroform was selected as
concentratioon of addeed analyte. Specificity,, extraction
e solv
vent for furtheer studies.
selectivity o
of method, sttability of VPPA in plasmaa

Iran J Basic Meed Sci, Vol. 18, No.

N 10, Oct 2015 9881
 Fazeli‐Bakhtiyyari et al Gas ch
hromatographic determination of valproic acid

Selection of the disperserr Effect

E of salt addition
a
Selection
n of dispersionn solvent is ve
ery importantt With incre easing the ioonic strength
h, solubility
in DLLME. T The disperser is a miscible solvent with h of
o the analytes in the aquueous phase e decreases
both aqueou us and organicc phases. A clooudy solution n and
a extractio on efficiencyy can be enh hanced. To
containing fiine droplets of
o the extractiion solvent iss evaluate
e thiis parameteer, 1 ml of o acetone
formed when n a mixture of
o extraction and
a disperserr containing
c 677 µl of chlorooform was used for the
solvents is injected into an aqueous sample.. extraction
e of
o VPA froom aqueouss solution
Therefore, a large surfacee area for mass transfer iss containing
c vaarious conceentrations off NaCl from
obtained. Exxtraction efficciency can be e significantlyy 0 to 10% (w/ /v).
increased byy effective diispersion of an a extraction n According g to the obtaiined results (Figure 3)
solvent into aqueous phaase. So 1 ml of methanol,, peak
p area was slighhtly increa ased with
acetonitrile, acetone or THHF was mixed d with 67 μl off increasing th he concentraation of NaCll up to 4%
chloroform aand rapidly injected
i into the aqueouss due
d to saltin ng out effectct and decreeased after
sample. Due to the resultts, acetone wa as selected ass that
t due to t increasiing volume e of the
the disperserr because of formation
f of a cloudy statee sedimented
s phase andd dilution. Therefore,
with very fin
ne droplets annd consequenttly increasingg further
f studies were perfformed in the presence
the extractio
on capability of
o the VPA (Fig gure 2). of
o 4% (w/v) NaCl.

Optimization
O of extractionn solvent volu
ume
In micro oextraction methods, typically
microliter
m vo
olumes of aan organic solvent are
used.
u Therrefore, prreconcentrattion and
extraction
e efficiency
e ccan be significantly
improved. Ex xtraction solvvent volume e is usually
selected
s as low as posssible to obtain higher
extraction
e effficiencies annd lower tox xic effects.
Extraction
E solvent volum me was eva aluated by
injecting 1 ml
m of acetonne containing g different
volumes
v of chloroform (440, 50, 67, 75, and 100
Figure 1. Effectt of extraction sollvent type on the microextraction
n μl).
μ The ressults (Figurre 4) show w that the
efficiency. Exxtraction condditions: extraction solvent,, analytical
a signal decreeases gradu ually with
dichloromethan ne (150 µl) , tetrachloroethy ylene (100 µl),, increasing thet extracttion solventt volume.
chloroform (67 7 μl), carbon tetrachloride (60 0 μl); disperserr
solvent, methaanol (1 ml); saample volume, 5 ml; analytee Therefore,
T 40 µl was chhosen as the e optimum
concentration, 7 µg/ml of sodium valpro oate; pH, 2.0; volume
v of thee extraction solvent.
concentration o of NaCl, 4% (w w/v); extraction time, ~0 min;
centrifugation ttime, 5 min and centrifugation sp peed, 6000 rpm..
The bars indicatte the standard deeviations (n=3)

Figure 2. Effeect of disperserr kind on the microextraction n Figure

F 3. Effect of salt addittion on the microextraction
efficiency. Extraaction conditionss: extraction solv
vent, chloroform
m efficiency.
e Extrraction condittions: extraction solvent,
(67 μl); dispersser solvent volumme, 1 ml; samplle volume, 5 ml; chloroform
c (67 μl);
μ disperser soolvent, acetone (1 ml); sample
analyte concenttration, 7 µg/ml of sodium valproate; pH, 2.0; volume,
v 5 ml; analyte concenttration, 7 µg/m ml of sodium
concentration o of NaCl, 4% (w w/v); extraction time, ~0 min; valproate;
v pH, 2.0;
2 extraction ttime, ~0 min; centrifugation
c
centrifugation ttime, 5 min and centrifugation sp peed, 6000 rpm.. time, 5 min and d centrifugation speed, 6000 rpm. The bars
The bars indicatte the standard deeviations (n=3) in
ndicate the standdard deviations (n=3)

982 Iran
I J Basic Medd Sci, Vol. 18, No.10, Oct 2015
Gas chromatog
graphic determin
nation of valproic acid Fazeli‐Bak
khtiyari et al

4000
4000 3500
3500 3000
3000 2500

Peak area
2500 2000
Peak area

2000 1500
1500 1000
1000 500
500 0
0 0,25
0 0,5 00,75 1 1,5
1 2
40 50 67 75
5 100
Dispeerser volume (ml)
(
Extraction
n solvent volume (µl)
Figure
F 5. Effect of disperser voolume on the microextraction
m
efficiency.
e Extracttion conditions: extraction solvent, chloroform
Figure 4. Efffect of extracttion solvent volume on the
(40 µl); disperser solvent, acettone; sample volume,
v 5 ml;
microextraction n efficiency. Exxtraction conditiions: extraction
n
analyte
a concentra
ation, 7 µg/ml oof sodium valprroate; pH, 2.0;
solvent, chlorofform; disperser solvent, acetone
e (1 ml); sample
concentration
c of NaCl, 4% (w/v /v); extraction time,
t ~0 min;
volume, 5 ml;; analyte conceentration, 7 µg/ /ml of sodium
centrifugation
c tim
me, 5 min and ceentrifugation speeed, 6000 rpm
valproate; pH H, 2.0; concenttration of NaC Cl, 4% (w/v);
The
T bars indicate the standard devviations (n=3)
extraction timme, ~0 min; centrifugation tim me, 5 min and d
centrifugation speed, 6000 rpm. The bars indicate the
standard deviations (n=3) 5000

Optimizatio on of disperseer volume 4000

Disperserr volume haas a key role in DLLME 3000
Peak area

procedure. In low disp perser volum mes, DLLME

performancee is disrupted whereas in highh volumes,, 2000
the solubilityy of the analy
yte in the aque
eous phase iss
1000
increased. T To study th his parameter, differentt
volumes of aacetone (0.25‐2 ml) contaiining 40 µl off 0
chloroform w were investiggated. The obttained resultss 2,5 5 7,5 10
were illustraated in Figuree 5. From the ese results itt
Sampple volume (m
ml)
was concluded that analyttical signal increases up too
1 ml due to the cloudy state
s being we ell formed. A
Figure
F 6. Effectt of sample vollume on the microextraction
further incrrease in disp perser volum me results in n efficiency.
e Extrraction condittions: extraction solvent,
decreased peak areas; th his may be be ecause largerr chloroform
c (40 µl);
µ disperser soolvent, acetone (1 ml); analyte
disperser vo olume increasses the aqueo ous solubilityy concentration,
c 7 µg/ml of ssodium valproa ate; pH, 2.0;
of VPA. For this reason, 1 ml acetone was selected d concentration
c off NaCl, 4% (w//v); extraction time,
t ~0 min;
centrifugation
c time, 5 min andd centrifugation speed, 6000
as optimum m volume of disperser in n subsequentt rpm.
r The bars inddicate the standaard deviations (n=3)
experimentss.
Optimization
O of centrifugaation rate and time
Optimizatio on of plasma sample
s volum
me The extraction equilibbrium can be b attained
Diluted plasma sam mple volume effect wass quickly
q after adding
a mixtuure of the exttraction and
studied in four levels fromm 2.5 to 10 mll containing 7 disperser
d solvvents. In DLLLME processs, the most
µg/ml of VPA A. For this purrpose, 0.25, 0.5, 0.75, and 1 time
t consumin ng step is centtrifugation. Th
he effects of
ml of spikedd plasma (140 0 µg/ml) were e mixed with h centrifugation
c n rate and timme were exam mined in the
the acetonitrrile in 1:1 (v/v
v) ratio. After precipitation
n range
r of 3000–6000 rrpm and 2–20 2 min,
with acetoniitrile, 0.25, 0.5, 0.75, and 1 ml of clearr respectively.
r According too the obtain ned results
supernatant solution weree used for pre eparing 2.5, 5,, (Figures
( 7A and
a 7B) 60000 rpm and 6 min were
7.5, and 10 ml of samplee solutions. Ba asically, peakk selected
s as cen
ntrifuge rate aand time, resppectively.
areas shouldd be increased d when the sample volumee
is increased.. This is due to
t the additio onal amountss Effect
E of pH
of VPA in thhe aqueous so olution. Howe ever, ratio off The effect of pH was sttudied ranging from 1 to
organic phase/aqueous phase, red duces when n 10,
1 and 1 M HCl or NaOH H was used for the pH
sample volu ume increasees. Therefore e, extractionn adjustment.
a VPA
V is a weakk acid with a pKa of 4.7
recovery deccreases in higgher volumes. As shown in n and
a is completely ionized aat high pH. Th he results in
Figure 6, p peak area in ncreases with h increasingg Figure
F 7C indiicate that peaak area decrea
ases in high
sample size. Therefore, 10 ml was used as thee pH.
p Therefore e, pH of 1 w was chosen for further
optimum ssample volu ume in the followingg experiments.
e
experimentss.

Iran J Basic Meed Sci, Vol. 18, No.

N 10, Oct 2015 9883
 Fazeli‐Bakhtiyyari et al Gas ch
hromatographic determination of valproic acid

Table
T 2. Quantita
ative features of proposed metho
od for valproic
5000
A 4500 acid
a determination in plasma sampples
4000
3500 Parameter Valpro
oic acid
3000
Peak area

2500
2000 Linear range (µg/ml)
( 6–1
140
1500 Slope 724.2
1000 Slope standardd errors 13.65
500 Intercept 65.59
0 Intercept standard errors 5.3
32
3000 3600 40
000 4200 4800
0 5200 6000 Correlation co
oefficient (r2) 0.9
998
Number of datta points 10
LOD (µg/ml) 3.2
Centrifu
ugation speed
d (rpm) LLOQ (µg/ml) 6.0
ULOQ (µg/ml)) 140.0

5500
B 5000 highest
h conce entrations of calibration curve were
4500 selected
s as LLOQ and ULOQ Q. LOD was caalculated for
4000
3500 an
a S/N ratio of o 3, baselinee noise was measured
m at
Peak area

3000 different
d placees of the basseline void off VPA peak.
2500
2000 Signal
S height was converrted into concentration
1500 through
t the height
h of the peak of the VPA at the
1000
500 LLOQ.
L LOD, LLOQ and ULO OQ were 3.2, 6 and 140
0 (µg/ml),
( resspectively. O Obtained re esults are
2 4 6 8 10 15 20 presented
p in Table
T 2.
Centriifugation time
e (min)
Precision
P and
d accuracy
The mean intra‐ and innter‐day assay
y precisions
5500 for
f all QC sa amples were determined at low (8
C 5000 µg/ml),
µ mediu
um (40 µg/m ml) and high (120 µg/ml)
4500
4000 concentration
c levels of VPPA and were e expressed
3500 as
a RSD%. By B comparinng the calcculated QC
Peak area

3000 concentration
c s with nom minal values, accuracies
2500 were
w obtained by comput uting the rela
ative errors
2000
1500 (REs).
( RSD% and RE% were less than 11.5%
1000 and
a 7.5%, resspectively. Thhe results we ere given in
500 Table
T 3.
0
1 2 4 6 8 10 Specificity
S and selectivity
pH The specifficity of the method wass evaluated
by
b analyzing batches of blank plasm ma and the
Figure 7. Op ptimization of (A) centrifugatiion speed, (B)
centrifugation ttime and (C) pH. Extraction condiitions: extraction
n results
r demonstrated thatt there is no significant
solvent, chlorofform (40 µl); dissperser solvent, acetone (1 ml); interference at
a the retentioon time of VP
PA. Some of
sample volume,, 10 ml; analyte concentration,
c 7 µg/ml
µ of sodium
m the
t most fre equently useed antiepile eptic drugs
valproate; conceentration of NaCCl, 4% (w/v); extraction time, ~00 (AEDs)
( such as gabbapentin, la amotrigine;
min for A‐C; (A)) pH, 2.0; centriffugation time, 5 min;
m (B) pH, 2.0;
centrifugation sspeed, 6000 rpm m; (C) centrifugattion speed, 60000 phenobarbita
p al, primidonee, carbamazepine, and
rpm; centrifugaation time, 6 minn. The bars indica ate the standard
d phenytoin
p are
e used in VPA A combinationn therapy.
deviations (n=3)

Table
T 3. Intra‐ an
nd inter‐day analyytical precision and
a accuracy of
proposed
p method d for determinattion of valproic acid
a in plasma
Validation rreport samples
Linearity annd calibrationn curves
Three callibration curv
ves of VPA we ere preparedd Nominal
N Inter‐assay
Intra‐assay
in 3 differen
nt days at 10 increasing co oncentrationss concentration
c precision
precision
Accuracy
ranging fromm 6–140 (µg/m ml) in plasmaa samples andd (µg/ml)
( (n=5) (RSD %) (RE %)
(RSD %) (n=55)
(n=15)
the analysis was carried out in triplicates for eachh
concentratioon. During method vallidation, thee 8 8.7 11.5 7.5
calibration ccurves were liinear over the
e therapeuticc 40
4 2.8 5.7 5.4
concentratioon range (r2 > 0.998). The e lowest and 120
1 0.8 1.2 1.8

984 Iran
I J Basic Medd Sci, Vol. 18, No.10, Oct 2015
Gas chromatog
graphic determin
nation of valproic acid Fazeli‐Bak
khtiyari et al

Presence of non‐volatile and basic dru ugs poses no

problems du ue to the very
y different chharacteristics
of the AEDs (boiling po oint, pKa an nd volatility)
(39). These drugs show no interferen nce with the
present anaalysis becausee basic drugss get ionized
in acidic m medium, thu us this form m is poorlyy
extracted annd in most cases,
c chromaatography off
above meentioned d
drugs with
hout priorr
derivatizatio
on is not posssible.

Recovery
RRs of VPA spikeed in plasm ma at threee
concentratio
on levels weree calculated byb comparingg
real values w
with those meeasured using g the presentt
extraction pprocedure. Thhe RRs% for VPA weree
Figure
F 8. Typical chromatogram obtained from spiked plasma
between 97 and 107.5% %. From recovery data in n extracted
e by prooposed method. (a) blank (b) plasma
p sample
Table 4, it ccan be foundd that the me ethod allowss spiked with sodiuum valproate (200 µg/ml). In both
h cases DLLME
determinatioon of VPA in a complex ma atrix (plasma) method
m was performed and 1 µµl of the collectted phase was
without a siggnificant mattrix effect. Fig
gure 8 showss in
njected into GC C. Extraction coonditions: extra
action solvent,
chloroform
c (40 µl);
µ disperser soolvent, acetone (1 ml); sample
typical chroomatograms of a blank plasma and d volume,10
v ml; pH,
p 1.0; concenntration of NaC Cl, 4% (w/v);
plasma spikeed with 20 µg//ml VPA afterr DLLME. extraction
e time, ~0 min; centrrifugation speed d, 6000 rpm;
centrifugation
c tim
me, 6 min
Stability stu
udy
Stability was also inv vestigated in three levels Analysis
A of a patient's
p sam
mple
of VPA and d after differrent storage e conditions; In order to evaluate metthod performance for the
short‐term (12 hr) room m temperaturre and three monitoring
m off VPA in real samples, plassma sample
freeze‐thaww (‐20 to 25 ˚C) cycles. According
A to of
o an epileptic patient wass extracted according
a to
the obtained results no o significant degradation n the
t proposed method. The patient had plasma p level
was observved for VPA under differrent storage of
o 17 µg/ml. Figure
F 9 showws typical chrromatogram
conditions. TThe results arre given in Ta
able 5. of
o a real samp ple. Note thatt no interferinng peaks in
the
t retention time of VPA A are observ ved and the
Robustness of the method appearance
a off the chromattogram is verry similar to
Robustness o of the method d was checke ed by varyingg those
t of spike
ed plasma in FFigure 8. It caan be found
method paraameters such as pH of sam mple solution,, that
t this methhod is applicabble for the dettermination
ionic strengtth, centrifugattion rate, and
d time. Effects of
o VPA levelss in patient plasma for therapeutic
of the followwing changess in extractio on conditionss purposes.
p
were determ mined: NaCl content
c in sam
mple solution n
adjusted byy (±1% w/v v), sample solution pH Discussion
D
adjusted byy (up to +0.5 and +1 pH units),, This work
k explains a w well‐known microextrac‐
m
centrifugatioon rate and time
t adjusted
d by (± 1000 tion
t procedurre (DLLME) fo for quantificattion of VPA
rpm and ±1 min), respecttively. Results presented in n in
n plasma sa amples. Plasm ma samples are more
Table 6 sho ow RRs% at these
t conditions were alll challenging
c n this respecct because plasma
in p can
below 96.5 % and thesee changes arre within thee emulsify
e organnic solvents tto some extennt. Thus, the
limits that prroduce accepttable results. problems
p asso
ociated with thhe matrix effe
ects should

Table 4. Relativve recoveries off valproic acid ob

btained by propoosed method in plasma
p samples spiked at 8, 40 aand 120 µg/ml

Nominal concenttration Relative reecovery

Found concentraation Accurracy (RE %)
(µg/ml) (n=5) (RR%) ± SD
(µg/ml) ± SD (n=
=5)

8 8.6±0.02 7.5 107.5±±0.02

40 38.8±0.04 ‐3 97±0..03
120 118.4±0.05 ‐1.3 98.7±00.05

SD: Standard dev

viation

Iran J Basic Meed Sci, Vol. 18, No.

N 10, Oct 2015 9885
 Fazeli‐Bakhtiyyari et al Gas ch
hromatographic determination of valproic acid

Table 5. Stabiliity data for valprroic acid in plasm

ma samples obtaained by the pro
oposed method

Room tem
mperature stabi lity Freeze–thhaw stability
Found concentration
c
Nominal und
Fou ve recovery
Relativ
(µg//ml) ± SD Accuracy Relative recovery Acccuracy
concentration concen
ntration (%
%) ± SD
(RE %) (%) ± SD (REE %)
=3)
(µg/ml) (n= (µg/mll) ± SD
8 8.8±0.04 10 110±0.09 8.6±0.05 77.5 107
7.5± 0.12
40 8.8±0.06
38 ‐3 97±0.05 38.0±
± 0.11 ‐5 95
5± 0.09
120 113.8±0.05 ‐5.2 94.8±0.07 116.4± 0.06 ‐3 97
7± 0.08

Figure 9. Typical chromatograam obtained froom real plasma ssample extracteed by proposed method. (a) bellongs to the dru
ug‐free plasma
sample and (b)) belongs to thee plasma sample
e from patient w
with epilepsy. Exxperimental con
nditions were thhe same as those
e described in
Figure 8

be reduced d in quantiitative bioan nalysis. Thee should

s be no oted that MSS is not available in all
optimized mmethod preseents an improvement in n laaboratories anda is not rroutine like FID. CE‐CD
work‐flow ccompared to common app plied samplee methods
m witth DLLME aand/or PPT T (18, 19)
preparation and analyssis technique es, i.e., SPE
E employed
e conntactless condductivity detecction, which
followed byy liquid chrommatography withw tandemm is not a stan ndard detectioon system available
a on
mass spectroometry. This carefully connducted workk commercial
c C instrumennts. Four GC‐FID based
CE
is presented in a very con
ncise and clear way so thatt methods
m (266–28, 40) w were also re eported for
it could bee used as a guideline for method d determination
d n of VPA in pllasma sample es. As it can
development and validattion in similarr fields. Withh be
b seen, all of these m methods requ uire a long
comparison of the propossed method with
w others, itt extraction
e tim
me to reach equuilibrium of analyte
a from
was found thhat for a num
mber of GC me ethods (5, 22,, the
t sample ma atrix.
23) cited inn Table 7, column
c deacctivation andd In additionn, a few of thee reported wo orks carried
chemical d derivatization of VPA could c be a out
o full valid dation concerrning FDA and/ora ICH
restriction factor when n compared d with thee guidelines.
g Heence, the impoortance of this validated
proposed m method, especcially for routtine analysis.. method
m is the
e rapidity of sample prepa aration and
Methods rep ported in refferences (11, 15, 16, 23) t simplicity and versatiliity of instrum
the mental setup
have used mmore sophisticaated instrumeentations andd that
t make feassible the deteermination of this analyte
time‐consumming sample preparation
p procedures.
p Itt inn real samples.

Table 6. Evaluaation of the prop

posed method ro
obustness for exttraction and ana
alysis of valproic
c acid in spiked pplasma samples

Level Nom
minal concentration (µg/ml) Found conccentration Accuracy (RE
E %) Relaative recovery (%
%) ± SD
(n=3) (µg/ml) ± SSD (n=3)
1 8 7.5±00.08 ‐6 94.0± 0.06
2 8 7.7± 0
0.07 ‐3.5 96.5± 0.09
3 8 7.2± 0
0.06 ‐9.8 90.2± 0.07
1: pH=1.5, 3% (w/v) NaCl, speed
s and time of
o centrifugationn: 5000 rpm for 5 min
% (w/v) NaCl, speeed and time of centrifugation:
2: pH=1, 4% c 6
6000 rpm for 6 min
m
3: pH=2, 5%
% (w/v) NaCl, speeed and time of centrifugation:
c 7
7000 rpm for 7 min
m

986 Iran
I J Basic Medd Sci, Vol. 18, No.10, Oct 2015
 Gas chromatog
graphic determin
nation of valproic acid Fazeli‐Bak
khtiyari et al

Table 7. Comparison of the pro

oposed method with
w other methhods

Method
M Samp
ple Validation Lin
near range / (µg/
/ml) Extracttion LODD/ Ref
time / m
min (µg//ml)
LLE‐GC‐FID
L a Human serum
s No
N method valid dation 10‐160 5 5 (5
5)
LLE‐HPLC‐UV
L b Human plasma
p No stability teest 1.25‐320 4 0.1
10 (11)
SPE‐LC‐MS‐MS
S c Human plasma
p Yes 2‐200 NRjj NR (15)
SPE‐LC‐MS‐MS
S Human plasma
p Yes 2.03‐152.25 NRR NR (16)
PPT‐CE‐CD
P d Human plasma
p No selectivity, stabiility test 2‐150 ~0 0.024 (18)
DLLME‐CE‐CD
D e Human plasma
p No
N method valid dation 0.40‐300 ~0 08
0.0 (19)
LLE‐GC‐FID
L Human plasma
p No selectivity ttest 0.45‐100 5 0.1
15 (22)
HS‐SPME‐GC‐MS
H f Human plasma
p No selectivity, stabiility test 2‐100 20 NR (23)
HS‐SPME‐GC‐FID
H Dg Human serum
s No
N method valid dation 0.20‐100 15 07
0.0 (26)
HS‐SPME‐GC‐FID
H D Human serum
s No
N method valid dation 0.25‐100 10 1.7 (27)
HF‐LPME‐GC‐FID
H Dh Rat plaasma No
N method valid dation 0.05‐10 30 0.017 (28)
HS‐LPME‐GC‐FID
H Di Human serum
s No
N method valid dation 2‐20 20 80
0.8 (4
40)
DLLME‐GC‐FID
D Human plasma
p Yes 6‐140 ~0 3.2 This work
w

aLLE‐GC‐FID:
L Liq
quid liquid extracction‐ gas chrom
matography‐ flamme ionization dettector
bLLE‐HPLC‐UV:
L L
Liquid liquid extrraction‐high perrformance liquid
d chromatograph hy‐ultraviolet de etection
cSPE‐LC‐MS‐MS:
S Solid‐phase extrraction‐ liquid ch
hromatography‐‐tandem mass sp pectrometry
dPPT‐CE‐CD: Prottein precipitation‐capillary electtrophoresis‐conttactless conducttivity detection
eDLLME‐CE‐CD:
D D
Dispersive liquidd‐liquid microextraction‐capillarry electrophoressis‐contactless co onductivity deteection
fHS‐SPME‐GC‐MS
H S: Headspace ‐so olid‐phase microextraction‐gas cchromatography y‐mass spectrometry
gHS‐SPME‐GC‐FID
H D: Headspace ‐so olid‐phase microoextraction‐gas cchromatography y‐ flame ionization detector
hHF‐LPME‐GC‐FI D: Hollow fiber‐liquid‐phase microextraction‐ga as chromatograp phy‐flame ioniza ation detector
iHS‐LPME‐GC‐FID
H D: Headspace ‐liqquid‐phase micrroextraction‐gass chromatograph hy‐flame ionizatiion detector
jNR:
N Not reported d

Conclusio
on 7. Krasowsk ki MD. Therappeutic drug monitoring
m of
In this sttudy, DLLME method follo owed by GC‐‐FID the newer anti‐epilepsy
a medications. Pharmaceu‐
ticals 2010; 3:1909–1935.
analysis wass established for determin nation of VPA A in 8. Musenga A, Saracino MA, Sani G, Raggi MA.
human plasm ma. Compared d with the oth
her methods, this Antipsychoticc and antieppileptic drugs in bipolar
technique p provided several advan ntages includ ding disorder: th he importancce of therap peutic drug
simplicity o of operation n, less solv vent and tim me‐ monitoring. Curr
C Med Chem m 2009; 16:146 63–1481.
consumption n, low cost an nd excellent sample clean n‐up 9. Kang J, Paark YS, Kim SH H, Kim SH, Jun MY. Modern
for the deteection of VPA in plasma a and in thee its methods for analysis of aantiepileptic drugs
d in the
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I J Basic Medd Sci, Vol. 18, No.10, Oct 2015