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 m microexttraction n follow wed by y gas chromaatograp phy‐flam me ionizzation detection n



Rana Fazeli‐Bakhtiyari 1, 2, Vahid d Panahi‐Azzar 3, Moham mmad Hosssein Sorourraddin 2, Abolghasem Jouyban 4**

Liver and Gastrointestinal Disseases Research Center, Tabriz U University of Medical Sciences, T Tabriz, Iran Department o of Analytical Chemistry, Faculty o of Chemistry, Un niversity of Tabrriz, Tabriz, Iran 3 Drug Applied Research Center r, Tabriz University of Medical SSciences, Tabriz, Iran 4 Pharmaceutic cal Analysis Reseearch Center and d Faculty of Pharrmacy, Tabriz Un niversity of Medical Sciences, Taabriz, Iran 1 2



A R T I C L E I N N F O

T R A C T A B S T

Article type:

Original article

Article history:

Received: Nov 8 8, 2014 Accepted: Sep 5 5, 2015

Keywords:

Dispersive liqu uid‐liquid‐ n microextraction Gas chromato ography‐flame‐ ionization detector Human plasmaa Valproic acid



Objecttive(s): Dispersiive liquid‐liquid d microextractio on coupled withh gas chromato ography (GC)‐ flame ionization deteector was develo oped for the dettermination of vvalproic acid (VPA) in human ma. plasm Materrials and Methodds: Using a syring ge, a mixture of suitable extractiion solvent (40 µ µl chloroform) and disperser (1 ml a cetone) was quiickly added to 10 0 ml of diluted pplasma sample containing VPA 1 concentratioon of NaCl, 4% (w/v)), resultin ng in a cloudy solution. After centrifugation (pH, 1.0; (6000 0 rpm for 6 min)), an aliquot (1 µ µl) of the sedimented organic phhase was remove ed using a 1‐µl GC miicrosyringe and injected into the GC system for analysis. One va variable at a time e optimization metho od was used to sstudy various pa arameters affecting the extractioon efficiency of ttarget analyte. Then, the developed m method was fully validated for its accuracy, preecision, recovery y, stability, and robustness. Resultts: Under the ooptimum extracttion conditions, good linearity range was obttained for the calibration graph, witth correlation co oefficient higher than 0.998. Lim mit of detection and lower limit of qua antitation were 3.2 and 6 μg/m ml, respectively. T The relative stanndard deviation ns of intra and inter‐day analysis of examined compound were lesss than 11.5%. T The relative reccoveries were d in the range of f 97 to 107.5%. F Finally, the valid dated method w was successfully applied to the found 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 cite e 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 the methods tha at were usedd for determ mination of Valproic acid (2‐prop pylpentanoic acid, VPA) iis a VPA. VPA, gas . Additionally y, due to volatility of with simple eightt carbon braanched‐chain fatty acid w is often used. chro matography ( (GC) (20, 21) . In order to unique anticconvulsant prroperties and d is used in the prev vent severe taiiling of the fat atty acid peak,, on‐column treatment off epilepsy, bip polar disorderr and prophylaaxis and pre‐column d derivatization have also bee en used (22, of migraine headaches (1 1–5). Hence, monitoring d drug 23). A Analysis of VP PA in biologicaal samples is difficult due levels in various matricees is particullarly valuablee in he presence of o proteins, saalts and vario ous organic epilepsy forr effective th herapeutic drrug managem ment to th comp pounds in sa amples. Hencee, sample pre eparation is (6–9). Theraapeutic serum/plasma conccentration of V VPA cruciial in drug ana alysis, which inncludes both a analyte pre‐ is between 2 20–100 µg/mll during controlled therapy but conc centration and d sample clea anup (24). So ome sample its toxic seru um/plasma co oncentration may reach 120– prep paration techn niques based on protein precipitation p 150 µg/ml ((10). Physicocchemical and pharmacokin netic (PPT T) (18), liquid d‐liquid extraaction (LLE) (22), solid‐ properties off VPA are listeed in Table1. H High performaance phasse extraction (SPE) (16), solid‐phase microextr‐ liquid chrom matography (HPLC) ( with ultraviolet ((UV) actio on (SPME) (2 23, 25–27), hhollow fiber‐lliquid‐phase detection (1 11, 12), fluo orescence dettection (13, 14) micrroextraction (H HF‐LPME) (288) and disperrsive liquid– or coupled with masss spectrome etry (MS) ((15– liquid d microextra action (DLLM ME) (19, 29) have been 17) and ccapillary eleectrophoresis (CE) coup pled





ulty of Pharmacy, T Tabriz University o of Medical Sciences, Tabriz, Iran. Tell: +98‐41‐3337932 23; Fax: +98‐41‐ *Correspondiing author: Abolghaasem Jouyban. Facu 33363231; emaill: [email protected]

 Fazeli‐Bakhtiyyari et al    









developed fo or this purposse. LLE is time e‐consuming and uses large aamounts of potentially p tox xic or hazard dous solvents (30). In addition n, the resulting g extract mayy be transferred, evaporated to t dryness and reconstitu uted with a suitab ble solvent priior to analysiss. Compared w with LLE, SPE is aa selective sam mple preparation method tthat uses a packed solid sorben nt (silica or po olymer) to isoolate the desired aanalyte. Neverrtheless, poten ntial variabilitty of SPE packings, irreversiblee adsorption of o some analyytes on SPE ccartridges and a more‐co omplex metthod developmentt are some of the draw wbacks that are presented byy this techniqu ue (31). SPME E was introdu uced in the early 1 1990s as a solv vent‐free proccess for extract cting the analytes from aqueous samples or headspace of f the samples (32 2). Despite itts obvious ad dvantages, SP PME suffers from m some drawb backs, for exa ample: expenssive SPME fibers are fragile an nd quite senssitive to comp plex matrices succh as plasm ma (33). LPM ME is a solve vent‐ minimized ssample pretreeatment proccedure, in wh hich only a few microliters of solvents are used (34, 35). Several differrent modes of o LPME have been develop ped, such as staticc LPME, dynaamic LPME an nd HF‐LPME ((36). It should be n noted that in m most cases, LP PME methodss are time‐consum ming and equiilibrium could d not be attaiined even after a llong extraction n time (31). R Recently, Assad di et al developed d a simple an nd novel LPME E method, wh hich was named aas DLLME (37 7, 38). In this method, a waater‐ miscible disp perser solvent t containing a w water‐immisccible extraction so olvent is injectted into the aq queous solutioon of analytes. A clloudy solution n (a mixture off water, disperrser solvent, and d extraction n solvent) is i formed and consequentlyy the equilib brium state achieved a quicckly. After phasee separation,, enriched analyte a can be determined by analytical systems. To our knowled dge, there is no D DLLME couplled with gas chromatograp c phy‐ flame ionizzation detecctor (GC‐FID D) method for determinatio on of VPA in h human plasma a in the literatture. The present w work is the firrst report of co ombination off the DLLME meth hod with GC‐F FID, without an ny derivatizattion, for the deterrmination of V VPA in human n plasma. Sevveral factors that in nfluence the m microextractio on efficiency w were comprehensiively examineed in detail an nd the optimiized microextracttion conditions were establiished. Finally, , the developed m method was vaalidated accorrding to the FFood d to and Drug Ad dministration (FDA) guidance and applied a real samplee analysis.



Materialss and Meth hods

Chemicals a and reagents Sodium valproate waas kindly do onated by R Rouz Darou Pharrmaceutical Co. C (Tehran, Iran). Dichlooro‐ methane, tettrachloroethy ylene, chlorofo orm, and carb bon tetrachloridee as extraction solven nts and otther chemicals ssuch as metthanol, aceto one, acetonittrile, tetrahydrofu uran (THF), sodium chloride c (NaaCl), hydrochloricc acid (HCl), aand sodium hy ydroxide (NaO OH) were purch hased from Merck M Compa any (Darmsttadt,

980

Gas ch hromatographic determination of of valproic acid

Germ many). Distille ed water wass used for pre eparation of aqueeous solutionss. Instrrumentation:: GC‐FID An A Agilent 7890A gas chromatog graph with split/ /splitless inle et and FID wass used for sep paration and determination of VPA. Optimuum flow rates of carrier (N2) and detecttor gases, ssuch as hyd drogen and comp pressed air were 1, 440 and 300 ml/min, respectively. He ettich centri rifuge, mode el D‐7200 (Germany) was used u for cenntrifuging. Injection port temp perature of 27 70 °C in the spplitless mode a and a purge timee of 30 secc were seleccted as opttimal state. Sepaaration was ca arried out on aan HP‐5 capilllary column (30 m × 0.32 mm m i.d., 0.25 μm m film thickn nesses). The oven n temperature e was prograammed as folllows: initial temp perature of 80 0 °C (held 1 m min), from 80 °C to 140 °C ° at a rrate of 15 C/m min, and held at 140 °C for 2 min. Then ° raiseed at 40 C/m min to 250 °C and held for 5 min. The FID ttemperature w was maintaineed at 280 °C.



Sam mple preparattion Plassma treatmen nt A A standard stock s solutionn of sodium m valproate (100 00 mg/l) was prepared in methanol an nd stored at 4°C. Working solu utions were pprepared by dilution with deion nized water. Free drug plasma sam mples were obtained from Irranian Bloodd Transfusion n Research Centter (Tabriz, Iran) and kept t at –20 °C un ntil analysis. For preparation of desired d concentratiion (6‐140 µg/m ml) of VPA in plasma, 1 ml of drug‐free plasma was spikeed with kno own amountss of the VPA A standard soluttion and kep pt at room teemperature for f 20 min. Then n for precipitation of plasm ma proteins, acetonitrile was added to pla asma sample in the ratio of 1:1 and vorteexed for 1 min. m Then it w was centrifug ged at 6000 rpm for 5 min. 1 ml of thee clear superrnatant was transsferred in a 10.0 ml volumeetric flask and d 0.4 g NaCl was added. Follow wing this, it w was diluted to t the mark with h deionized water w and the pH of obtain ned solution was adjusted to 1.00 by 1 M HCCl. In order to o reduce the matrrix effect of the plasma sam mple, the superrnatant was dilutted 10‐fold with w deionizeed water and d then was subjeected to the m microextractioon procedure.



Prep paration of re eal plasma saample Blood sample B was obtainedd from a male patient (35 years old) who had signed thee consent forrm that was apprroved by the E Ethics Commit ittee, Tabriz U University of Medical Sciences. This patientt had been ad dministered VPA (125 mg), flu urazepam, bippyridine, rispe eridone, and prop pranolol. 5 ml of bloodd was colle ected in a hepaarinized tube a at 2 hr after ddrug intake. Bllood sample was centrifuged immediatelyy and the plasma p was sepaarated and subjected s to the propossed DLLME meth hod.

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

HO O

CH H3 CH H3

Molecularr weight (g/mole)) 144.21

Meltting point (˚C)

Log P

pKa

120‐121

2.8

4.7

Pharmaacokinetic propertties Therapeeutic range (µg/m ml)

Toxicc range (µg/ml)

Halff‐life [ t0.5 (h)]

Bioavailabiility

20‐100

120‐1 150

9‐18 8

70‐100

Plasma protein binding (% %) 80‐95 5

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



DLLME proccedure In the next step, 10 mll of diluted pllasma sample e (described aabove) was transferred t into i a 12‐mll glass test tub be with conic bottom, and 1 ml acetone e (as disperseer) containin ng 40 µl chlloroform (as s extraction ssolvent) wass rapidly ad dded to the e solution usin ng a 5‐ml sy yringe, which immediately y resulted in aa cloudy solutiion. After centrifugation at t 6000 rpm fo or 6 min, orgganic phase was w settled to o the bottom of the tube.. An aliquot (1µl) of the e sedimented organic phase was removed using a 1‐‐ µl GC micro osyringe (Hamilton, Switzzerland) and d injected into the GC system m for analysiss.

Assay valida ation Method vvalidation waas done acco ording to the e FDA recommendationss. For quantification, , calibration curves weree constructe ed on three e different dayys. Linear ran nge, correlatio on coefficient, , and limit of f detection (L LOD) was calculated from m calibration curve. Thee lowest and a highest t concentratio ons of calibrattion curve werre selected as s the lower lim mit of quantiffication (LLOQ Q) and upper r limit of quaantification (U ULOQ), while the relative e standard devviations (%R RSDs) of three e replications s were less th han 20% and d 15%, respecctively. Intra‐‐ and inter‐d day precisio on and acccuracy were e determined by measurin ng plasma qu uality control l samples (Q QCs) at low w, medium and high h concentratio on levels of VP PA. Relative recovery (RR) of the samp ple preparatio on method was computed d using the folllowing equatiion:

RR 

CFouund  CReal 100 CAdded

where CFoundd is the analytte concentration measured d from the sam mple after an nalyte addition, CReal is the e native analyyte concentrration and CAdded is the e concentratio on of addeed analyte. Specificity, , selectivity o of method, sttability of VP PA in plasma a

Iran J Basic Meed Sci, Vol. 18, No. N 10, Oct 2015

samples s unde er different sstorage cond ditions, and method m robusstness were also evaluated. Further details d on the e validation reesults were described d in the following s t section.

Results R

Optimization O of the di dispersive liq quid–liquid microextracti m ion Extraction efficiency oof DLLME depends d on several s parameters. Onee variable at a a time optimization o method wass used to stu udy factors affecting a the extraction e effificiency. Some e important parameters p such s as typpes of extra action and dispersive d solvents and ttheir volume es, pH, salt effect, sample e volume, centtrifugation ratte, and time were investiga w ated. Selection of th S he extraction n solvent Chlorinated solvents are re denser than n water and are the most w a widely used soolvents in DL LLME due to being b easily removed froom the botttom of the conical c vial after cenntrifugation. Therefore, dichlorometha d ane, tetrachlooroethylene, chloroform, and a carbon te etrachloride w were used ass extraction solvents. For t s this purpose, 500 µl of spiiked plasma (140 µg/ml) w ( was transferreed into 1.5 ml Eppendorff tubes. In the n t next step, acettonitrile was added with 1:1 ratio. The 1 mixture wass vortexed forr 1 min and centrifuged c for f 5 min at 6000 rpm. r After precipitation o p of proteins, 0.55 ml of clear ssupernatant solution was t s ransferred in a 5.0 ml volumetric flask and a deionized d water and 0.2 g NaCl was added before b pH adjustment. a D DLLME proccedure was performed p by b various volumes of o selected extraction e solvents mixed with 1 ml methanol m to give equal vol g ume of the seedimented ph hase (40 µl). The T obtained results reveaaled (Figure 1) 1 that VPA was extracted w into chlorofoorm better tha an the other solvents. s Therefore, chlorroform was selected as extraction solv e vent for furtheer studies.

9881

 Fazeli‐Bakhtiyyari et al    







Selection of the disperserr Selection n of dispersion n solvent is ve ery important t in DLLME. T The disperser is a miscible solvent with h both aqueou us and organicc phases. A clo oudy solution n containing fiine droplets o of the extractiion solvent is s formed when n a mixture o of extraction a and disperser r solvents is injected into an aqueous sample. . Therefore, a large surfacee area for mass transfer is s obtained. Exxtraction efficciency can be e significantly y increased byy effective diispersion of an a extraction n solvent into aqueous phaase. So 1 ml of methanol, , acetonitrile, acetone or TH HF was mixed d with 67 μl of f chloroform aand rapidly injected i into the aqueous s sample. Due to the resultts, acetone wa as selected as s the disperserr because of fformation of a a cloudy state e with very fin ne droplets an nd consequenttly increasing g the extractio on capability o of the VPA (Fig gure 2). Figure 1. Effectt of extraction sollvent type on the microextraction n efficiency. Exxtraction cond ditions: extraction solvent, , dichloromethan ne (150 µl) , tetrachloroethy ylene (100 µl), , chloroform (67 7 μl), carbon tetrachloride (60 0 μl); disperser r solvent, methaanol (1 ml); saample volume, 5 ml; analyte e concentration, 7 µg/ml of sodium valpro oate; pH, 2.0; 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)



Gas ch hromatographic determination of of valproic acid

Effect of salt a E addition With incre easing the ioonic strength h, solubility of the analyt o es in the aquueous phase e decreases and a extractio on efficiencyy can be enh hanced.  To evaluate e thiis parameteer, 1 ml of o acetone containing 67 c 7 µl of chlorooform was used for the extraction e of o VPA froom aqueouss solution containing va c arious conceentrations off NaCl from 0 to 10% (w/ 0 /v). According g to the obtaiined results (Figure 3) peak p area was slighhtly increa ased with increasing th he concentraation of NaCll up to 4% due d to saltin ng out effect ct and decre eased after that t due to t increasiing volume e of the sedimented s phase andd dilution. Therefore, further studi f es were perfformed in the presence of 4% (w/v) o NaCl. Optimization O of extraction n 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 of c v hloroform (440, 50, 67, 75, and 100 μl). μ The ressults (Figurre 4) show w that the analytical a signal decreeases gradu ually with increasing the t extracttion solventt volume. Therefore, T 40 µl was chhosen as the e optimum volume of the v e extraction solvent.



Figure 2. Effeect of disperserr kind on the microextraction n efficiency. Extraaction conditionss: extraction solv vent, chloroform m (67 μl); dispersser solvent volum me, 1 ml; samplle volume, 5 ml; analyte concenttration, 7 µg/ml of sodium valproate; pH, 2.0; 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)



982

Figure F 3. Effect of salt addittion on the microextraction efficiency. e Extrraction condittions: extraction solvent, chloroform (67 μ c μl); disperser soolvent, acetone (1 ml); sample volume, v 5 ml; analyte concenttration, 7 µg/m ml of sodium valproate; v pH, 2.0; 2 extraction ttime, ~0 min; centrifugation c time, 5 min and d centrifugation speed, 6000 rpm. The bars in ndicate the stand dard deviations (n=3)



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  

Peak area  

4000 3500 3000 2500 2000 1500 1000 500 0

4000 3500 3000 2500 2000 1500 1000 500 0 0,25 0

40

50

67

75 5

100

Extraction n solvent volume (µl)  Figure 4. Efffect of extracttion solvent volume on the microextraction n efficiency. Exxtraction conditiions: extraction n solvent, chlorofform; disperser solvent, acetone e (1 ml); sample volume, 5 ml;; analyte conceentration, 7 µg/ /ml of sodium valproate; pH H, 2.0; concenttration of NaC Cl, 4% (w/v); extraction tim me, ~0 min; centrifugation tim me, 5 min and d centrifugation speed, 6000 rpm. The bars indicate the standard deviations (n=3)

Optimizatio on of disperseer volume Disperserr volume haas a key role in DLLME procedure. In low disp perser volum mes, DLLME performancee is disrupted whereas in h high volumes, , the solubilityy of the analy yte in the aque eous phase is s increased. T To study th his parameter, different t volumes of aacetone (0.25‐2 ml) contaiining 40 µl of f chloroform w were investiggated. The obttained results s were illustraated in Figuree 5. From the ese results it t was concluded that analyttical signal increases up to o 1 ml due to the cloudy state s being we ell formed. A further incrrease in disp perser volum me results in n decreased peak areas; th his may be be ecause larger r disperser vo olume increasses the aqueo ous solubility y of VPA. For this reason, 1 1 ml acetone was selected d as optimum m volume of disperser in n subsequent t experimentss. Optimizatio on of plasma ssample volum me Diluted plasma sam mple volume effect was s studied in four levels from m 2.5 to 10 mll containing 7 µg/ml of VPA A. For this purrpose, 0.25, 0.5, 0.75, and 1 ml of spiked d plasma (140 0 µg/ml) were e mixed with h the acetonitrrile in 1:1 (v/v v) ratio. After precipitation n with acetoniitrile, 0.25, 0.5, 0.75, and 1 ml of clear r supernatant solution weree used for pre eparing 2.5, 5, , 7.5, and 10 ml of samplee solutions. Ba asically, peak k areas should d be increased d when the sample volume e is increased.. This is due tto the additio onal amounts s of VPA in th he aqueous so olution. Howe ever, ratio of f organic phase/aqueous phase, red duces when n sample volu ume increasees. Therefore e, extraction n recovery deccreases in higgher volumes. As shown in n Figure 6, p peak area in ncreases with h increasing g sample size. Therefore, 10 ml was used as the e optimum ssample volu ume in the following g experimentss.

Iran J Basic Meed Sci, Vol. 18, No. N 10, Oct 2015

0,5

00,75

1

1,5 1

2

Dispeerser volume ((ml)  Figure F 5. Effect of disperser voolume on the microextraction m efficiency. e Extracttion conditions: extraction solvent, chloroform (40 µl); disperser solvent, acettone; sample volume, v 5 ml; analyte a concentra ation, 7 µg/ml oof sodium valprroate; pH, 2.0; concentration c of NaCl, 4% (w/v /v); extraction time, t ~0 min; centrifugation c tim me, 5 min and ceentrifugation spe eed, 6000 rpm The bars indicate T the standard devviations (n=3)

5000 4000 Peak area  

Peak area  



3000 2000 1000 0 2,5

5 7,5 10 Sampple volume (m ml) 

Figure F 6. Effectt of sample vollume on the microextraction efficiency. e Extrraction condittions: extraction solvent, chloroform (40 µ c µl); disperser soolvent, acetone (1 ml); analyte concentration, c 7 7 µg/ml of ssodium valproa ate; pH, 2.0; concentration c off NaCl, 4% (w//v); extraction time, t ~0 min; centrifugation c time, 5 min andd centrifugation speed, 6000 rpm. The bars ind r dicate the standaard deviations (n=3)

Optimization O of centrifugaation rate and time The extraction equilibbrium can be b attained quickly q after adding a mixtuure of the exttraction and disperser d solv vents. In DLLLME processs, the most time consumin t ng step is centtrifugation. Th he effects of centrifugation c n rate and tim me were exam mined in the range r of 3000–6000 rrpm and 2–20 2 min, respectively. r According too the obtain ned results (Figures ( 7A and a 7B) 60000 rpm and 6 6 min were selected as cen s ntrifuge rate aand time, resp pectively. Effect of pH E The effect of pH was sttudied ranging from 1 to 10, 1 and 1 M HCl or NaOH H was used for the pH adjustment. a VPA V is a weakk acid with a a pKa of 4.7 and is comple a tely ionized aat high pH. Th he results in Figure 7C indi F icate that peaak area decrea ases in high pH. p Therefore e, pH of 1 w was chosen for further experiments. e

9883

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Table T 2. Quantita ative features of proposed metho od for valproic acid determinatio a n in plasma sampples

5000

A  4500 Peak area  

4000 3500 3000 2500 2000 1500 1000 500 0 3000 3600 40 000 4200 4800 0 5200 6000 Centrifu ugation speed d (rpm) 

Parameter Linear range ((µg/ml) Slope Slope standard d errors Intercept Intercept standard errors Correlation co oefficient (r2) Number of datta points LOD (µg/ml) LLOQ (µg/ml) ULOQ (µg/ml))





4

6

8

10

15

20

Peak area  

Centriifugation time e (min) 

Peak area  

5500 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 1

2

4

6

8

10

pH  Figure 7. Op ptimization of (A) centrifugatiion speed, (B) centrifugation ttime and (C) pH. Extraction condiitions: extraction n solvent, chlorofform (40 µl); dissperser solvent, acetone (1 ml); sample volume,, 10 ml; analyte cconcentration, 7 µ µg/ml of sodium m valproate; conceentration of NaC Cl, 4% (w/v); extraction time, ~0 0 min for A‐C; (A)) pH, 2.0; centriffugation time, 5 m min; (B) pH, 2.0; centrifugation sspeed, 6000 rpm m; (C) centrifugattion speed, 6000 0 rpm; centrifugaation time, 6 min n. The bars indica ate the standard d deviations (n=3)

Validation rreport Linearity an nd calibration n curves Three callibration curv ves of VPA we ere prepared d in 3 differen nt days at 10 increasing co oncentrations s ranging from m 6–140 (µg/m ml) in plasma a samples and d the analysis was carried out in triplicates for each h concentratio on. During method vallidation, the e calibration ccurves were liinear over the e therapeutic c concentratio on range (r2 > 0.998). The e lowest and







Precision and P d accuracy The mean intra‐ and innter‐day assay y precisions for f all QC sa amples were determined at low (8 µg/ml), mediu µ um (40 µg/m ml) and high (120 µg/ml) concentration c levels of VPPA and were e expressed as a RSD%. By B comparinng the calcculated QC concentration c s with nom minal values, accuracies were w obtained by comput uting the rela ative errors (REs). ( RSD% and RE% were less than 11.5% and a 7.5%, resspectively. Thhe results we ere given in Table 3. T



C 



highest h conce entrations of calibration curve were selected as LL s OQ and ULOQ Q. LOD was ca alculated for an a S/N ratio of o 3, baselinee noise was measured m at different d place es of the basseline void off VPA peak. Signal S height was converrted into concentration through t the height h of the peak of the VPA at the LLOQ. L LOD, LLOQ and ULO OQ were 3.2, 6 and 140 (µg/ml), ( resspectively. O Obtained re esults are presented in T p Table 2.

4500 4000 3500 3000 2500 2000 1500 1000 500 0 2

Valpro oic acid 6–1 140 724.2 13.65 65.59 5.3 32 0.9 998 10 3.2 6.0 140.0





5500

B  5000

984

Gas ch hromatographic determination of of valproic acid



Specificity an S d selectivity The specifficity of the method wass evaluated by b analyzing batches of blank plasm ma and the results r demonstrated thatt there is no significant interference a at the retentioon time of VP PA. Some of the t most fre equently useed antiepile eptic drugs (AEDs) ( such as gabbapentin, la amotrigine; phenobarbita p al, primidonee, carbamazepine, and phenytoin are p e used in VPA A combination n therapy. Table 3. Intra‐ an T nd inter‐day analyytical precision a and accuracy of proposed p method d for determinattion of valproic acid a in plasma samples Nominal N concentration c (µg/ml) (n=5) (

Inter‐assay precision (RSD %) (n=15)

Intra‐assay precision (RSD %) (n=5 5)

Accuracy (RE %)

8 40 4 120 1

8.7 2.8 0.8

11.5 5.7 1.2

7.5 5.4 1.8

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 ch haracteristics 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 poorly y extracted an nd in most cases, c chroma atography of f above meentioned drugs d with hout prior r derivatizatio on is not posssible. Recovery RRs of VPA spikeed in plasm ma at three e concentratio on levels weree calculated b by comparing g real values w with those meeasured using g the present t extraction p procedure. Th he RRs% for VPA were e between 97 and 107.5% %. From recovery data in n Table 4, it ccan be found d that the me ethod allows s determinatio on of VPA in aa complex ma atrix (plasma) without a siggnificant mattrix effect. Fig gure 8 shows s typical chro omatograms of a blank plasma and d plasma spikeed with 20 µg//ml VPA afterr DLLME. Stability stu udy Stability was also inv vestigated in three levels of VPA and d after differrent storage e conditions; short‐term (12 hr) room m temperaturre and three freeze‐thaw w (‐20 to 25 ˚C) cycles. According A to the obtained results no o significant degradation n was observved for VPA under differrent storage conditions. T The results arre given in Ta able 5. Robustness of the method Robustness o of the method d was checke ed by varying g method paraameters such as pH of sam mple solution, , ionic strengtth, centrifugattion rate, and d time. Effects of the follow wing changess in extractio on conditions s were determ mined: NaCl content c in sam mple solution n adjusted byy (±1% w/v v), sample solution pH adjusted byy (up to +0.5 and +1 pH units), , centrifugatio on rate and time t adjusted d by (± 1000 rpm and ±1 min), respecttively. Results presented in n Table 6 sho ow RRs% at these t conditions were alll below 96.5 % and thesee changes arre within the e limits that prroduce accepttable results.



Figure F 8. Typical chromatogram obtained from spiked plasma extracted e by pro oposed method. (a) blank (b) plasma p sample spiked with sodiu um valproate (200 µg/ml). In both h cases DLLME method m was performed and 1 µµl of the collectted phase was in njected into GC C. Extraction coonditions: extra action solvent, chloroform c (40 µl); µ disperser soolvent, acetone (1 ml); sample volume,10 v ml; pH, p 1.0; concenntration of NaC Cl, 4% (w/v); extraction e time, ~0 min; centrrifugation speed d, 6000 rpm; centrifugation tim c me, 6 min

Analysis of a p A patient's sam mple In order to evaluate metthod performance for the monitoring of m f VPA in real samples, plassma sample of o an epileptic patient wass extracted according a to the proposed t method. The patient had p plasma level of 17 µg/ml. F o Figure 9 show ws typical chrromatogram of o a real samp ple. Note thatt no interferin ng peaks in the t retention time of VPA A are observ ved and the appearance of a f the chromattogram is verry similar to those of spike t ed plasma in FFigure 8. It ca an be found that this meth t hod is applicabble for the dettermination of o VPA levelss in patient plasma for therapeutic purposes. p

Discussion D

This work k explains a w well‐known microextrac‐ m tion t procedurre (DLLME) fo for quantificattion of VPA in n plasma sa amples. Plasm ma samples are more challenging c in n this respecct because plasma p can emulsify organ e nic solvents tto some exten nt. Thus, the problems asso p ociated with thhe matrix effe ects should



Table 4. Relativve recoveries off valproic acid ob btained by propoosed method in p plasma 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

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Gas ch hromatographic determination of 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

8 40

Found cconcentration (µg//ml) ± SD 8.8±0.04 8.8±0.06 38

120

113.8±0.05

Nominal concentration =3) (µg/ml) (n=

Freeze–thhaw stability

Accuracy (RE %)

Relative recovery (%) ± SD

und Fou concen ntration (µg/mll) ± SD

Acccuracy (REE %)

ve recovery Relativ (% %) ± SD

10 ‐3

110±0.09 97±0.05

8.6±0.05 38.0± ± 0.11

77.5 ‐5

107 7.5± 0.12 95 5± 0.09

‐5.2

94.8±0.07

116.4± 0.06

‐3

97 7± 0.08







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

be reduced d in quantiitative bioan nalysis. The e optimized m method preseents an improvement in n work‐flow ccompared to common app plied sample e preparation and analyssis technique es, i.e., SPE E followed by y liquid chrom matography with w tandem m mass spectro ometry. This carefully con nducted work k is presented in a very con ncise and clear way so that t a guideline for method d it could bee used as a development and validattion in similarr fields. With h comparison of the propossed method w with others, it t was found th hat for a num mber of GC me ethods (5, 22,, 23) cited in n Table 7, column c deacctivation and d chemical d derivatization of VPA could c be a restriction factor when n compared d with the e proposed m method, especcially for routtine analysis. . Methods rep ported in refferences (11, 15, 16, 23) have used m more sophisticaated instrume entations and d time‐consum ming sample p preparation p procedures. It t



should s be no oted that MSS is not available in all laaboratories and a is not rroutine like FID. CE‐CD methods m witth DLLME aand/or PPT T (18, 19) employed con e ntactless condductivity detecction, which is not a stan ndard detectioon system available a on commercial c CE C instrumennts. Four GC‐FID based methods m (26 6–28, 40) w were also re eported for determination d n of VPA in pllasma sample es. As it can be b seen, all of these m methods requ uire a long extraction tim e me to reach equuilibrium of a analyte from the sample ma t atrix. In addition n, a few of thee reported wo orks carried out o full valid dation concerrning FDA and/or a ICH guidelines. g He ence, the impoortance of this validated method m is the e rapidity of sample prepa aration and and versatiliity of instrum the simplicity t mental setup that make feas t sible the deteermination of this analyte in n real samples.

posed method ro obustness for exttraction and ana alysis of valproic c acid in spiked pplasma samples Table 6. Evaluaation of the prop 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±0 0.08 ‐6 94.0± 0.06 2 8 7.7± 0 0.07 ‐3.5 96.5± 0.09 7.2± 0 0.06 3 8 ‐9.8 90.2± 0.07

1: pH=1.5, 3% (w/v) NaCl, sspeed and time o of centrifugation n: 5000 rpm for 5 5 min % (w/v) NaCl, speeed and time of ccentrifugation: 6 6000 rpm for 6 m min 2: pH=1, 4% 3: pH=2, 5% % (w/v) NaCl, speeed and time of ccentrifugation: 7 7000 rpm for 7 m min

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Gas chromatog graphic determin nation of valproic acid Fazeli‐Bak khtiyari et al  

Table 7. Comparison of the pro oposed method w with other methhods Method M Samp ple Validation a LLE‐GC‐FID L b LLE‐HPLC‐UV L c SPE‐LC‐MS‐MS S SPE‐LC‐MS‐MS S d PPT‐CE‐CD P e DLLME‐CE‐CD D LLE‐GC‐FID L f HS‐SPME‐GC‐MS H HS‐SPME‐GC‐FID H Dg HS‐SPME‐GC‐FID H D HF‐LPME‐GC‐FID H D h HS‐LPME‐GC‐FID H Di DLLME‐GC‐FID D

Human sserum Human p plasma Human p plasma Human p plasma Human p plasma Human p plasma Human p plasma Human p plasma Human sserum Human sserum Rat plaasma Human sserum Human p plasma

No method valid N dation No stability teest Yes Yes No selectivity, stabiility test No method valid N dation No selectivity ttest No selectivity, stabiility test No method valid N dation No method valid N dation No method valid N dation No method valid N dation Yes

Lin near range / (µg/ /ml) 10‐160 1.25‐320 2‐200 2.03‐152.25 2‐150 0.40‐300 0.45‐100 2‐100 0.20‐100 0.25‐100 0.05‐10 2‐20 6‐140

Extracttion time / m min 5 4 NRjj NR R ~0 ~0 5 20 15 10 30 20 ~0

LOD D / (µg/ /ml) 5 5 0.1 10 NR NR 0.024 08 0.0 0.1 15 NR 07 0.0 1.7 0.017 80 0.8 3.2

Ref (5 5) (11) (15) (16) (18) (19) (22) (23) (26) (27) (28) (4 40) This w work



LLE‐GC‐FID: Liq L quid liquid extracction‐ gas chrom matography‐ flam me ionization dettector LLE‐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: Prot tein precipitation‐capillary electtrophoresis‐conttactless conducttivity detection D eDLLME‐CE‐CD: D Dispersive liquid d‐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 micro oextraction‐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 ‐liq quid‐phase micrroextraction‐gass chromatograph hy‐flame ionizatiion detector jNR: Not reported N d a

b

Conclusio on

In this sttudy, DLLME method follo owed by GC‐‐FID analysis wass established for determin nation of VPA A in human plasm ma. Compared d with the oth her methods, this technique p provided several advan ntages includ ding simplicity o of operation n, less solv vent and tim me‐ consumption n, low cost an nd excellent sample clean n‐up for the deteection of VPA in plasma a and in the e its therapeutic rrange. Thereffore, the validated method can be utilized ass a routine an nalytical metho od in therapeeutic drug monitoring studies.

Acknowle edgment

The auth hors would lik ke to thank th he Iranian Blood Transfusion Research Cen nter, Tabriz, IIran for donatting drug‐free plaasma sampless.

Reference es

1. Maccdonald RL, Kelly KM. Antiepileptic d drug mechan nisms of action. Epilepsia 199 95; 36:S2–S12. 2. Johaannessen CU, JJohannessen SI. Valproate: p past, present,, and future. CN NS Drug Rev 2003; 9:199–21 16. 3. Warrner A, Privittera M, Bates D. Standardss of laborato ory practice: antiepileptic drug monitorring. Clin Cheem 1998; 44:10 085–1095. 4. Lösccher W. Valp proate: a re eappraisal of its pharmaacodynamic prroperties and d mechanismss of action. P Prog Neurobio ol 1999; 58:31– –59. 5. Arraanz Peña MI. Rapid gas chromatograp phic determiination of valproic v acid d in serum m. J Chromaatogr 1981; 225:459–462. 6. Kum mps AH. Theerapeutic drug monitoringg: a comprehensive and critical revie ew of analyttical nvulsive drugss. J Neurol 19 982; methods for anticon 16. 228:1–1

Iran J Basic Meed Sci, Vol. 18, No. N 10, Oct 2015

7. Krasowsk ki MD. Therappeutic drug monitoring m of the newer anti‐epilepsy a medications. Pharmaceu‐ ticals 2010; 3:1909–1935. 8. Musenga A, Saracino MA, Sani G, Raggi MA. Antipsychoticc and antieppileptic drugs in bipolar disorder: th he importancce of therap peutic drug monitoring. C Curr Med Chem m 2009; 16:146 63–1481. 9. Kang J, Pa ark YS, Kim SH H, Kim SH, Jun MY. Modern methods for analysis of aantiepileptic drugs d in the uids for pharrmacokinetics, bioequival‐ biological flu ence and th herapeutic druug monitoring g. Korean J Physiol Pharm macol 2011; 155:67–81. 10. Regenthal R, Krueger M M, Koeppel C, Prreiss R. Drug Levels: therapeutic andd toxic serum/plasma ns of commoon drugs. J Clin Monit concentration Comput 1999 9; 15:529–544.. 11. Amini H, JJavan M, Ahmaadiani A. Devellopment and validation off a sensitive aassay of valproic acid in human plasma by hhigh‐performance liquid chromatograp phy without prior deriv vatization. J Chromatogr B B 2006; 830:3668–371. 12. Kushida K, K Ishizaki T. CConcurrent de etermination of valproic acid a with otheer antiepilepttic drugs by high‐perform mance liquidd chromato ography. J Chromatogr 1 1985; 338:131 –139. 13. Lin MC, K Kou HS, Chen CCC, Wu SM, Wu u HL. Simple and sensitive fluorimetricc liquid chro omatography method for the determinaation of valprroic acid in omatogr B 20004; 810:169–172. plasma. J Chro 14. Wolf JH, Veenma‐vann der Duin L, Korf J. Automated analysis a proceddure for valp proic acid in blood, serum and braain dialysate by high‐ performance liquid chromatograp phy with bromomethyllmethoxycoum marin as fluorescent label. J Chromatogr 1 1989; 487:4966–502. 15. Jain D, Subbaiah G, Sanyyal M, Shrivasttav P. A high throughput and a selective m method for the e estimation of valproic acid an antieepileptic drug g in human

9887

 Fazeli‐Bakhtiyyari et al    









plasma by tandem LC C–MS/MS. Tala anta 2007; 72::80– 88. 16. Gao S, Miao H, Tao X, Jiang B, Xiao o Y, Cai F, et al. LC– n of MS/MS method for simultaneous determination or metabolites iin human plasm ma. J valproicc acid and majo Chromattogr B 2011; 87 79:1939–1944. 17. Chen ng H, Liu Z, Blum W, Byrrd JC, Klisovicc R, Grever MR, et al. Quaantification of valproic acid and pyl‐4‐pentenoiic acid in hum man its metaabolite 2‐prop plasma using HPLC‐M MS/MS. J Chromatogr B 20 007; 6–212. 850:206 18. Belin GK, Kräheenbühl S, Ha auser PC. Diirect determiination of valp proic acid in biological fluidss by capillaryy electroph horesis wiith contacttless conducttivity detection. J Chrom matogr B 20 007; 847:205 5–209. 19. Pham H Morand R, Krähenbüh hl S, m TTT, See HH, Hauser PC. Determination of free and a total valp proic ma by capillarry electrophorresis acid in human plasm ntactless conductivity detecttion. J Chromattogr with con B 2012; 907:74–78. h R, Schmid RD, Weidemann n G. 20. Degel F, Heidrich Quantitaative determin nation of valprroic acid by meeans of gas chromatograp phic headspace analysis. Clin 29–36. Chim Accta 1984; 139:2 21. Cooreman S, Cuyp pers E, De Donccker M, Van Heee P, broeck W, Neeels H. Comp parison of th hree Uyttenb immuno oassays and one GC‐MS method for the determiination of valproic acid. Imm muno‐Anal Biol Spe 2008; 23:240–244. d F, 22. Ahmadkhaniha R, Rastkari N, Kobarfard man H, Ahmadk khaniha O, Keb briaeezadeh A A. An Pakdam improveed GC method d for rapid an nalysis of valp proic acid in human plasma without derrivatization. Iraan J Pharm SSci 2007; 9:37‐‐42. 23. Den ng C, Li N, Ji J, Yang B, Duan D G, Zhangg X. Develop pment of water‐phase derivatizaation followed d by solid‐ph hase microexttraction and gas chromattography/masss spectrom metry for fast determiination of vallproic acid in n human plassma. Rapid Commun Mass SSpectrom 2006; 20:1281–12 287. n‐Bjergaard S,, Rasmussen KE. 24. Ho TS, Pedersen phase microexxtraction of pro otein‐bound drrugs Liquid‐p under non‐equilibriu um conditionss. Analyst 20 002; 8–613. 127:608 25. Kroggh M, Johansen n K, Tønnesen F, Rasmussen n KE. Solid‐ph hase microextrraction for the e determinatio n of the freee concentratio on of valproic acid in hum man plasma by capillaary gas chrromatographyy. J 673:299–305. Chromaatogr B 1995; 6 26. Mattin AA, Biparv va P, Amanzad deh H, Farhad di K. uminum layerred double hy ydroxide–titan nium Zinc/Alu dioxide composite nanosheet n film m as novel ssolid phase microextracttion fiber for the gas chromattographic dettermination of o valproic aacid. Talanta 2013; 103:207–213. mi P, 27. Faraajzadeh MA, Faarhadi K, Matiin AA, Hashem



988

Gas ch hromatographic determination of of valproic acid

Jouyban A. Headspace H soliid‐phase micrroextraction‐ gas chromato ography methood for the determination of valproic acid d in human sserum, and formulations f using hollow w‐fiber coatedd wire. Anall Sci 2009; 25:875–879. M, Farhadi K, Bahram M. 28. Jahangiri S, Hatami M Hollow‐fiber‐‐based LPME as a reliablle sampling method for gas g chromatoographic deterrmination of pharmacokinetic parameteers of valproicc acid in rat matographia 22013; 76:663–6 669. plasma. Chrom 29. Sobhi HR,, Kashtiaray A,, Farahani H, A Abrahimpour F, Esrafili A. A Quantitatioon of valpro oic acid in pharmaceuticcal preparationns using dispe ersive liquid‐ liquid miccroextraction followed by gas chromatograp phy‐flame ionnization detecttion without prior derivattization. Drug Test Anal 20 010; 2:362– 366. 30. Namera A, A Saito T. Reecent advance es in unique sample prep paration techhniques for bioanalysis. Bioanalysis 2013; 5:915–9332. Y, Abdel‐Rehim m M. Sample e treatment 31. Ashri NY based on extrraction techniqques in biological matrices. Bioanalysis 2011; 3:2003–22018. 32. Lord H, Pawliszyn J. Evolution of solid‐phase ogr A 2000; microextractiion technologyy. J Chromato 885:153–193. 33. Arthur CL, Pawlisszyn J. So olid phase microextractiion with therm mal desorption n using fused silica optical ffibers. Anal Chhem 1990; 62:2 2145–2148. 34. Jeannot M MA, Cantwell FFF. Solvent microextraction into a single d drop. Anal Chem m 1996; 68:22 236–2240. 35. He Y, Lee e HK. Liquid‐pphase microexttraction in a single drop off organic solveent by using a cconventional microsyringe. Anal Chem 19997; 69:4634– –4640. z ‐Yazdi A, Amiri A. Liquid‐phase L 36. Sarafraz microextractiion. Trends Annal Chem 2010; 29:1‐14. 37. Rezaee M, M Assadi Y, Miilani Hosseini MR, Aghaee E, Ahmadi F, Berijani S. D n of organic Determination compounds in i water usingg dispersive liquid–liquid l microextractiion. J Chromatoogr A 2006; 11 116:1–9. 38. Berijani S S, Assadi Y, Anbbia M, Milani H Hosseini MR, Aghaee E. Dispersive liquuid‐liquid microextraction combined with gas chromatog graphy‐flame photometric detection. V Very simple, rapid and m for the determ mination of sensitive method organophosphorus pesticiddes in water. J Chromatogr A 2006; 1123:1–9. 39. Volmut J, Matisová E, Phham Thi Ha. Simultaneous determination n of six antieppileptic drugs by capillary gas chromato ography. J Chroomatogr B 199 90; 527:428– 435. mmadi A, Alizadeh A N. 40. Shahdoussti P, Moham Determination of valproic aacid in human n serum and pharmaceuticcal preparatioons by headsp pace liquid‐ phase micro oextraction gaas chromatog graphy‐flame ionization de etection withoout prior derivatization. J Chromatogr B B 2007; 850:1228–133.

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