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Journal of Analytical Methods in Chemistry
Volume 2016, Article ID 5976324, 12 pages

Research Article
Determination of Levetiracetam in Human
Plasma by Dispersive Liquid-Liquid Microextraction Followed
by Gas Chromatography-Mass Spectrometry

Greyce Kelly Steinhorst Alcantara,1 Leandro Augusto Calixto,2
Luiz Alberto Beraldo de Moraes,3 Regina Helena Costa Queiroz,4
Anderson Rodrigo Moraes de Oliveira,3 and Cristiane Masetto de Gaitani1
Department of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo,
14040-903 Ribeirão Preto, SP, Brazil
Department of Exact and Earth Sciences, Institute of Environmental, Chemical and Pharmaceutical Sciences,
Federal University of São Paulo, 09972-270 Diadema, SP, Brazil
Departament of Chemistry, Faculty of Philosophy, Sciences and Letters of Ribeirão Preto, University of São Paulo,
14040-901 Ribeirão Preto, SP, Brazil
Department of Clinical Analysis, Toxicology and Food Science, Faculty of Pharmaceutical Sciences of Ribeirão Preto,
University of São Paulo, 14040-903 Ribeirão Preto, SP, Brazil

Correspondence should be addressed to Leandro Augusto Calixto; lcalixto@unifesp.br

Received 26 June 2016; Accepted 1 September 2016

Academic Editor: Antonio V. Herrera-Herrera

Copyright © 2016 Greyce Kelly Steinhorst Alcantara et al. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.

Levetiracetam (LEV) is an antiepileptic drug that is clinically effective in generalized and partial epilepsy syndromes. The use of this
drug has been increasing in clinical practice and intra- or -interindividual variability has been exhibited for special population. For
this reason, bioanalytical methods are required for drug monitoring in biological matrices. So this work presents a dispersive liquid-
liquid microextraction method followed by gas chromatography-mass spectrometry (DLLME-GC-MS) for LEV quantification
in human plasma. However, due to the matrix complexity a previous purification step is required. Unlike other pretreatment
techniques presented in the literature, for the first time, a procedure employing ultrafiltration tubes Amicon (10 kDa porous
size) without organic solvent consumption was developed. GC-MS analyses were carried out using a linear temperature program,
capillary fused silica column, and helium as the carrier gas. DLLME optimized parameters were type and volume of extraction
and dispersing solvents, salt addition, and vortex agitation time. Under chosen parameters (extraction solvent: chloroform, 130 ?L;
dispersing solvent: isopropyl alcohol, 400 ?L; no salt addition and no vortex agitation time), the method was completely validated
and all parameters were in agreement with the literature recommendations. LEV was quantified in patient’s plasma sample using
less than 550 ?L of organic solvent.

1. Introduction disorders, such as autism, anxiety, and bipolar disorders
[1]. A unique pharmacokinetic profile [4–6] and multiple
Levetiracetam (LEV; Figure 1(a)) belongs to a generation mechanisms of action have differentiated LEV from other
of antiepileptic drugs that have been recommended for the antiepileptic drugs [3, 7]. According to the Subcommittee
treatment of epilepsy as either monotherapy in the case of the International League Against Epilepsy, the thera-
of partial seizures or as an adjunctive therapy for partial, peutic plasmatic concentration of LEV was set from 12 to
myoclonic, and tonic-clonic seizures [1–3]. In addition, 46 mg L−1 [5, 8]. However, concentrations of LEV outside
LEV exhibits benefits for other neurologic and psychiatric this range can be measured in special groups, such as elderly

0 (MQC). The reagents (analytical grade) were dichloromethane high preenrichment. Methanol. solid-phase extraction 2. expected plasma concentration. tem used during the analyses was composed of a GC-2010 tion solvent and the aqueous phase. The GC-MS sys- surface area is formed between the fine droplets of the extrac. The 2. there are not many works in biological matrices have been reported. and hazardous solvents. USA). USA) was prepared at the concentration of result in signal suppression when liquid chromatography 1 mg mL−1 in methanol. this procedure can Louis. Baker (Philipsburg. extraction from Tedia (Fairfield. USA). [9–24]. For this purpose. carryover or cross-contamination ?g mL−1 in human plasma. GC System and Analytical Conditions.2.0 (LLOQ). analytical chemists have focused on the devel. Germany). and a lack of selectivity. Water used and dispersing solvent is quickly injected by microsyringe to prepare the solutions was purified using a Milli-Q Plus into the sample. Acetonitrile and chloroethylene were pur- has received special attention in the analytical chemical field. and dispersing solvent. Additionally. all of which were obtained from . a large 2. Since 90s. hand. Chloroform was acquired vent system composed of sample (aqueous phase). the analyte transference plus Gas Chromatograph coupled to a mass spectrome- toward the extraction phase occurring almost instantly [27– try model QP2010 Series Plus system equipped with an 30]. St. oped to extract organic compounds from simple aque- Chromatographic methods for the quantification of LEV ous samples [30]. this include high-performance liquid chromatography (HPLC) work will employ for the first time the DLLME to quantify an and gas chromatography (GC) with various detection systems antiepileptic drug from human plasma by GC-MS. (CBZ. Brazil). These situations could modify the analyzed by an appropriate analytical technique [31]. the cartridges used for the extraction are relatively 10. Experimental (SPE). Louis. USA).0. pregnant patients. with electron impact (70 eV) microdroplets are sedimented at the bottom of the extraction as the ionization source. USA) and sodium chloride was solvent. 60. SPE is a more selective technique. and involves multiple steps high (HQC) samples and the lowest limit (LLOQ) and upper [26. DLLME burg. remove the plasmatic protein. Reagents. MO. SP. organic solvents. previously reported methods are mainly based on the conventional sample preparation procedure such as liquid-liquid extraction (LLE). highlighting the microextraction procedures. 4. pediatric populations. selected as internal standard (IS) (Sigma Aldrich. the coprecipitation of concentrations range of 20-800 ?g mL−1 . containing the target analyte. the extraction solvent autosampler model AOC20i. After the centrifugation step. is tedious.0. In addition. These methods employing highly complex matrix [31].0 (HQC). The calibration curve was the ionization suppression is seldom observed. so limit (ULOQ) of quantification. USA). All these solutions were stored at −20∘ C in glass microextractions require low consumption of sample and tubes.0 (LQC). Nevertheless. and plasmatic protein precipitation (PPP) [9–23]. Standard PPP procedure is considered a fast and easy method to stock solution of LEV purchased from Sigma Aldrich (St. At this moment. 27]. The mixture of the extraction purchased from Merck (Darmstadt. however. both acquired from Macron Chemicals (Philips- Introduced in 2006 by Rezaee and collaborators. NJ. Chemicals. cloudy state due to the microdroplets of the extraction solvent dispersed into the aqueous phase. Bedford. and suitable cleanup procedure. carbon It is a miniaturized kind of LLE which requires microliters of tetrachloride. Moreover. and patients tube and further collected by microsyringe and afterwards with renal impairment.0 (ULOQ) expensive. Conversely. and acetone. NJ. the MO. and 80. which may lead to a higher This microextraction procedure has been widely devel- and toxic level of LEV [5]. and isopropyl alcohol were acquired from JT extraction solvent [28].2 Journal of Analytical Methods in Chemistry CH3 O NH2 N N O O NH2 (a) (b) Figure 1: Chemical structures: (a) levetiracetam and (b) carbamazepine (IS). the free human plasma with the working solutions we obtained target analyte(s) may be occluded in the protein pellets [25]. besides they present adequate selectivity. seven calibration standards (CS) and five levels of quality The LLE procedure usually requires more consumption of control (QC) defined by low (LQC). CT. A solution of carbamazepine can occur [27]. people. Figure 1(b)) at the concentration of 100 ?g mL−1 was opment of the new novel sample preparation techniques. creating a System (Millipore. In general. 20.1. On the other prepared at the concentrations of 2. medium (MQC). 40. chased from Synth (Diadema. and Standard Solutions. Appropriate dilutions were then coupled to mass spectrometry (LC/MS) is used due to the made with methanol to obtain working stock solutions at presence of a precipitation agent. By spiking drug- interfering species. The principle consists of a ternary sol.

50 ?L of the IS solution between LEV and the IS versus the LEV concentrations.0 statistical program (State College.53 software is summarized in Figure 2. NV. The procedure 123 for LEV and m/z 193 for the IS. the calibration curve range (LLOQ. Sample Preparation mitted to ultrafiltration procedure (described in Section 2. Validation method was carried out participate in the research. In order to assess the carryover effect.4. and the results were weighted by 1/?.4. sources of the blank matrix (drug-free human plasma). LabSolution 2. Linear (? = 4) 500 ?L of human plasma sample.4. fine droplets of the extraction solvent were pure helium (99. A patient treated with LEV received all information about the study protocol and gave written informed consent to 2. 400 ?L of film thickness) fused silica capillary columns from SGE isopropyl alcohol (dispersing solvent) containing 130 ?L of Analytical Science (Ringwood. was obtained over a mass range from m/z 40 to 400 to and the solution was transferred to a clean tube for solvent confirm the identification of the analytes. These ultrafiltration tubes were centrifuged was also evaluated and it was defined as the lowest concen- at 1800 ×g for 20 minutes using a CF-15 centrifuge (Hitachi tration that can be quantified reliably with an accuracy and Koki. and the vortex agita- tion time (assisted DLLME). The ton. Simultaneously. Quantification of evaporation under a gentle stream of compressed air. The method linearity was evaluated by tube (Becton Dickinson. The results of each parameter were analyzed using for this study. So the conical tube was for 2 min before finally being increased by 20∘ C min−1 to subjected to centrifugation at 1800 ×g for 5 minutes.3. which . The chromatographic 2. The LEV and the IS was carried out in selected ion monitoring residue was solubilized in 200 ?L of methanol. the full scan mode The volume of the sedimented phase (?L) was determined. Germany).25 ?m was added in a 10 mL conical bottom glass tube. DLLME. in which 500 ?L of plasma samples was procedures. an aliquot of 1.4. the ionic strength. Japan).5. The 2. %) and as permeated phase was subjected to DLLME procedure. The expressed as relative standard deviation (RSD. (aqueous solution containing LEV and IS) was transferred to Within-day accuracy and precision were evaluated using a conical bottom glass tube for further DLLME optimization five replicates (? = 5). 50 ?L of regression was performed by plotting the peak area ratios the LEV solution (400 ?g mL−1 ). Australia).Journal of Analytical Methods in Chemistry 3 Shimadzu Technologies (Kyoto. PA. analytical time was 9 min. Plasma Samples. a cloudy solution column temperature was then programmed to increase to (aqueous phase/extraction solvent/dispersing solvent) was 230∘ C at a rate of 20∘ C min−1 . Validation Method. The Hemotherapy Center of Ribeirão Preto the Minitab 14.0 mL separations were made using Rtx-5ms (5% phenyl/95% of pretreated plasma (described in Section 2.0 mL of ultrapure water were added. After 270∘ C (and again held for 1 min). membrane of 10 kDa porous size from Millipore (Darm. 0. In conical bottom glass tubes calibration curve range was from 2 to 80 ?g mL−1 . The lower limit of quantification (LLOQ) of the method stadt. and 1. The aqueous phase with a 1.318).0 mL microsyringe (Gastight. LQC.25 mm i.1) and then DLLME procedure (described in Section 2. washing procedure of Amicon ultrafiltration tubes consisted The selectivity was assessed by analyzing six individual of the use of ultrapure water at least four times. from Shimadzu was used to control the GC-MS system and All optimization parameters. and 1. MQC. plasma with 50 ?L of the standard solution of LEV. This solution was sub- 2. (100 ?g mL−1 ). 50 ?L of the IS. 46]. USA).999%) was set at 1 mL min−1 . such as the type of extrac- for data acquisition. Kyoto. and centrifuged of operation. 30 m length × 0.0 mL of ultrapure water.4. Reno.1. and 1 ?L of the (SIM) mode at the following mass/charge (m/z) ratios: m/z sample was injected into the GC-MS system. France) and centrifuged at employing seven different concentrations of LEV in quadru- 1800 ×g for 5 min. at which point it was held quickly formed in the conical tube.4. it plicate (? = 4).0 mL of the permeated phase precision below 20% of a nominal value [45].1) samples dimethyl polysiloxane. For DLLME procedure.2). Hamil- initial temperature of the column oven was 150∘ C. were applied in quadruplicate 2. Then. At the same moment. %). 1. tion and dispersing solvents. The Ethics Committee of the College of experiments (? = 4). donated drug-free plasma samples from healthy volunteers. The percentage of relative error (RE. After separation of the plasma sample. The precision and accuracy parameters were at 1800 ×g for 20 minutes (blank plasma samples). USA). The extraction efficiency was deter- Pharmaceutical Sciences from Ribeirão Preto. Ultrafiltration Procedure. Between-day precision and ultrapure water were added in Amicon ultrafiltration tubes.2. Japan). respectively [45]. Blood samples of patients treated according to the EMA guidelines on bioanalytical method with LEV (±5 mL) were collected in a Vacutainer heparinized validation [45]. the volume of extraction and dispersing solvents.d. and HQC) an aliquot of 500 ?L of human plasma sample and 1100 ?L of of the standard solution of LEV. So the Amicon ultrafiltration tubes were reused spiked with a minimum of four concentration levels covering among the analysis. approved the protocol parameter. University of mined by plotting the peak area of LEV versus the evaluated São Paulo (protocol number 898. the statistical parameters of the calibration curve The tubes were vortex for 1 minute and this solution was were calculated by analysis of variance (ANOVA) and linear transferred to a 15 mL Amicon ultrafiltration tube with a regression [32. and the total concentrated in the bottom of the tube (sedimented phase). A was stored in a polypropylene tube and kept frozen at −20∘ C calibration curve was obtained by spiking 500 ?L of human until the analysis. Meylan. The column flow rate using centrifugation. accuracy were assessed for at least three consecutive days after DLLME extraction of ULOQ solution. After that. The injection and chloroform (extraction solvent) was injected rapidly into the ionization source temperatures were 250∘ C and 220∘ C.

a GC application in biological matrices were showed in Table 1. nominal concentration value [45]. and no peaks autoinjector conditions (24 hours.0 2. included four normal plasma sources. at a LQC (4. To conduct the from endogenous compounds were observed at the retention stability tests. several ramping speeds and temperatures were quantification (80 ?g mL−1 ). 22 ± 2∘ C). The carryover parameter was assessed by injecting blank Under these chromatographic conditions.0 7. The LEV concentrations nique widely employed to extract analytes from environmen- obtained from the stability tests were compared with the tal samples but it is seldom applied for the extraction of LEV concentrations obtained from freshly prepared samples analytes from biological samples. The IS source. The initial oven temperature was 150∘ C. dilution was done ten times for the DLLME from human plasma by GC-MS. the following stability tests were performed: (i) sample were eluted at the same time of LEV (4. this work . no method has to reduce the matrix effect and then centrifuged was done. a blank sample was injected and the storage conditions to which the analyte was subjected.0 ?g mL−1 ).1.0 Centrifugation Centrifugation Analysis 1.2.5 1.2 min and 7. and accurate the determination of letrozole in human plasma. Mashayekhi et 3. Ultrafiltration Procedure. Some strategies have been adopted to remove the influence of this matrix during the DLLME procedure.0 Plasma samples + Permeated phase Cloudy Extraction analytes solution phase collection Figure 2: Steps of DLLME procedure. and reached a final temperature of 270∘ C (held for 1 min). and (iii) m/z 123 was selected for further analysis of LEV. respectively.4 Journal of Analytical Methods in Chemistry Rapid injection of dispersing and extraction solvent 3. and and the IS signals under the GC-MS conditions previously then it was increased by 20∘ C/min to 230∘ C (held for 2 min) established [45]. another single ion temperature conditions (8 h on the bench-top). During that the concentration of LEV had not been affected by the selectivity evaluation. The samples were considered stable if the mean from a biological matrix could interact with organic solvents. [32] reported the DLLME procedure for the quantification of CBZ where a suitable amount of acetonitrile was employed 3. injecting the calibration LEV standard at the upper limit of However. system with electron impact as the ionization source and an Different from the previous published papers.5 2. 500 ?L of human plasma was spiked with LEV time of LEV or the IS. DLLME procedure is a tech- procedures with the replicates. Based on that goal.0 6. than 20% of the LOQ and 5% for the IS [45]. a hemolyzed plasma Rtx-5ms fused silica capillary column was employed. Initially.0 ?g mL−1 ) and at a HQC (60. Based on this result. the peaks from endogenous compounds of human plasma Therefore. After submitting samples at storage and preparation conditions. According to the EMA (2011). the retention times plasma samples (without the standard LEV spike) after were 4.2 min) using freeze (−20∘ C)/thaw (22 ± 2∘ C) cycles. the SIM-mode chosen for the quantification of The stability parameter was determined in order to ensure single ions was m/z 126 for LEV and m/z 193 for the IS. Adopted methodology for plasma LEV analysis employing the DLLME different approaches of pretreatment as well as DLLME as sample preparation technique. Chromatographic Separation. been described in the literature for the quantification of LEV After filtering. Results and Discussion al. Endogenous compounds (? = 4).5 0 4. evaluated in order to obtain a GC run-time that was as short the carryover effect on the blank sample should not be greater as possible. In the same way. (ii) short-term room SIM-mode m/z 126. the IS was added and developed ultrafiltration and DLLME 3.0 5. One of the aims of this procedure. and a hyperlipidemic plasma source in order to chosen for analysis was CBZ solution prepared at 100 ?g mL−1 verify that no endogenous peak would interfere in the LEV in methanol. concentrations at each concentration were within ±15% of the and a suitable sedimented phase could not be formed [28]. fast. [25] carried out work was to develop a sensitive.2 min for LEV and the IS. selective. To date.0 0. Rezaee et al.

and dilution Chloroform (88 ?L) Acetone (1000 ?L) HPLC-UV/Vis [41] Five antiarrhythmic drugs Plasma Precipitation with acetonitrile Dichloromethane (100 ?L) Acetonitrile (1340 ?L) HPLC-UV/Vis [42] Benzodiazepines Plasma Precipitation with acetonitrile Chloroform (220 ?L) Acetonitrile (3200 ?L) HPLC-UV/Vis [43] Efavirenz Plasma Sulfosalicylic acid (4%) Chloroform (100 ?L) Acetonitrile (1300 ?L) HPLC-UV/Vis [44] 5 . and dilution Carbamazepine Chloroform (78 ?L) Ethanol (1000 ?L) HPLC-UV/Vis [32] (1 : 10) Urine Urine: dilution (1 : 5) Amitriptyline. and Urine Carbon tetrachloride (20 ?L) Acetonitrile (500 ?L) HPLC-UV/Vis [33] of upper phase thioridazine Clozapine and Urine Centrifugation at 4000 rpm/15 min Carbon tetrachloride (40 ?L) Ethanol (200 ?L) HPLC-UV/Vis [34] chlorpromazine Plasma: precipitation with acetone (1 : 1) and Plasma Plasma: chloroform (100 ?L) Plasma: acetone (500 ?L) Losartan and carvedilol dilution (1 : 5) HPLC-UV/Vis [35] Urine Urine: pH adjustment (acid solution) Urine: chloroform (160 ?L) Urine: acetone (400 ?L) 7-Aminoflunitrazepam Urine Ammonia (0.45 ?m) and dilution (1 : 2) Piroxicam Urine Enzymatic conjugation Chloroform (70 ?L) Methanol (700 ?L) Espectrofotometer [38] Plasma Plasma and urine centrifugation. and dilution (1 : 20) Methadone Saliva Saliva: centrifugation and dilution (1 : 100) Chloroform (250 ?L) Methanol (2500 ?L) HPLC-UV/Vis [39] Sweat: successive baths of ethanol (30 min each Sweat bath) Precipitation with trichloroacetic acid (10%). decantation. and Fentanyl. filtration (0.45 ?m) clomipramine. filtration. Plasma centrifugation. Table 1: DLLME application in biological matrices and pretreatment approaches.45 ?m). and dilution Opium alkaloids Urine Centrifugation. Warfarin Plasma 1-Octanol (150 ?L) Methanol (150 ?L) HPLC-UV/Vis [40] refrigeration (4∘ C/20 min).45 ?m).2 M) Dichloromethane (250 ?L) Isopropyl alcohol (500 ?L) LC-ESI-MS/MS [36] Plasma: filtration (0. alfentanil. Extracting solvent volume Dispersing solvent volume Analytes Sample Pretreatment Analysis method References (?L) (?L) Plasma: precipitation with acetonitrile. dilution (1 : 4). Journal of Analytical Methods in Chemistry Centrifugation for 15 min and filtration (0. Urine filtration. and Plasma precipitation with methanol (1 : 2) Chloroform (142 ?L) Methanol (2000 ?L) HPLC-UV/Vis [37] sufentanil Urine Urine: filtration (0.

efficiency of LEV decreased. There was no carryover effect among the analysis. The selection of the demonstrated that the extraction efficiency of LEV slightly extraction solvent type is a fundamental step in the DLLME increased after the addition of isopropyl alcohol in amounts procedure [32].6 Journal of Analytical Methods in Chemistry presents for the first time the use of ultrafiltration procedure extraction was observed by using a fixed volume of 210 ?L of as previous treatment of human plasma of DLLME. soluble in ethanol. methanol.2. LEV is an antiepileptic drug novel with polar of chloroform into the aqueous phase. such as dichloromethane. trifuged at 1800 ×g for 20 minutes. the reproducibility was poor. a very low amount nature and it is fairly challenging for DLLME technique. 400. in ultrafiltration tubes previously used with ULOQ of LEV. All of these DLLME parameters evaluated were carried out in quadruplicate (? = 4).05. for the subsequent DLLME optimization. the area peaks associated with aqueous phase.1. range while keeping the volume of isopropyl alcohol constant (210 ?L): 50. The maximum value was reached when choose solvents with a higher density than water and a adding 400 ?L of isopropyl alcohol (Figure 3(d)). the behavior and extraction capability for the analyte of interest cloudy state did not form very well.3. and chloroform. However. The most important a conical bottom glass tube for further DLLME optimization. was evaluated mixing 90 ?L of size of molecules. Due to the low volume of presents a significant tendency to remain in the aqueous the sedimented phase. [48]. Excluding isopropyl alcohol.3. LEV of sedimented phase was formed. The influence of precision and accuracy values were in agreement with the the volume of chloroform was evaluated in the following specification of the regulatory agency. and the vortex agitation time (assisted chosen to perform the DLLME procedure (Figure 3(c)). a new analyte of interest and the IS. volume of the sedimented phase was observed when carbon without adding organic solvent. confidence level of 0. the extraction [34. respectively. This mixture was injected directly by effect of the plasma samples. The 3. 170 and 236 daltons. it seems that by tetrachloroethylene. After the addition of 50 ?L 3. Further. Therefore. and 1. During this step. The effect of these solvents on LEV 400 ?L). in quadruplicate. Therefore. syringe into 1. In order to reach the best extraction efficiency of LEV. and the IS solution (100 ?g mL−1 ). Effect of Dispersing Solvent Volume. Effect of Extraction Solvent Volume. tetrachloride was used as the extraction solvent. So 500 ?L aliquot of the plasma sample. and practically insoluble in n-hexane López-Nogueroles et al. chloroform was chosen as the extraction solvent water were placed into Amicon ultrafiltration tubes and cen.0 mL of the permeated phase containing the nominated permeated phase. once 3. 130. The difficulty in collecting low amounts in chloroform and methanol. Selection of Dispersing Solvent. So 49]. There was no statistically significant difference considering a type and volume of the extraction and dispersing sol. Isopropyl alcohol assisted of the use of ultrapure water at least four times to remove the in the dispersion of the extraction solvent inside aqueous plasma proteins. 300. resulting in less waste and. there is possibility to extract the drug from and 170 ?L of chloroform. it was selected for further experiments once it a blank plasma sample (without LEV and IS) was processed showed the best efficiency of extraction of LEV. DLLME) were optimized. 210 ?L with 10 kDa size which it enables it to block the plasmatic of each selected dispersing solvent. by adding 130 ?L [4]. was transferred to 3. To verify the applicability of the developed method was confirmed by effect of dispersing solvent volumes was investigated by checking a plasma sample from a patient who had daily taken mixing 130 ?L of chloroform with 200. ionic strength. and LEV. 1. leading a formation of an adequate cloudy state [47– effect of the membranes of Amicon ultrafiltration tubes. it is highly important to from 200 to 350 ?L. Selection of Extraction Solvent. Thereby. making phase once it has low octanol-water partition coefficient it impossible to use this amount of extraction solvent for [33]. Afterwards. further increasing the isopropyl alcohol volume (more than show these characteristics. was obtained. sparingly of the sedimented phase has been previously reported by soluble in acetonitrile. Furthermore. Chlorinated solvents. LEV were compared using analysis of variance (ANOVA). which pre- consequently. Probably low solubility in water that shows good chromatographic at lower volumes of isopropyl alcohol (less than 400 ?L). During these experiments. and 170 ?L. the drug shows that it is freely soluble quantitative analysis. vents this sample from being further collected and analyzed. containing LEV and IS. and thus. Chloroform showed higher extraction efficiency probably ously spiked with the LEV standard solution (400 ?g mL−1 ) because LEV is highly soluble in chloroform (Figure 3(a)).0 mL of ultrapure Therefore. A limpid aqueous phase. The washing procedure consisted in human plasma (Figure 3(b)). The results 3. So the washing procedure was suitable. 90. DLLME. Nevertheless. proteins and permit the passage of the LEV and IS due to low acetone. 350. the method assessed the carryover phase. diminishing the environmental concern. which was previ. all other dispersing The Amicon ultrafiltration tubes were reused during solvents demonstrated similar extraction efficiency for LEV developing of the method. no statistically significant difference was observed by . 130 ?L of chloroform was vents. chloroform. point for the choice of a dispersing solvent is its miscibility in Membrane of the Amicon ultrafiltration tubes was porous both aqueous phase and organic phase [25]. 35]. and isopropyl alcohol. 500 ?L of the isopropyl alcohol and then rapidly injecting the samples into 1. carbon tetrachloride.0 mL of the aqueous phase. acetonitrile. methanol (dispersing solvent) combined with 90 ?L of each Ultrafilters were successfully applied to reduce the matrix chlorinated solvent. However. Nevertheless.0 mL of the permeated phase. a low pretreatment for complex matrix by DLLME was introduced.

DLLME has the 47]. (e) vortex agitation time. 28. 400 ?L of isopropyl alcohol large surface area between the aqueous and extraction phases. Effect of Vortex Agitation Time. In addition to the from the aqueous phase to the extraction phase [25. vortex- advantage of saving time [29].3. (b) dispersing solvent type. Therefore. therefore. (c) extraction solvent (chloroform) volume. and (f) salt addition. To increase the extraction efficiency of LEV.Journal of Analytical Methods in Chemistry 7 40000 40000 30000 30000 Peak area Peak area 20000 20000 10000 10000 0 0 Chloroform Dichloromethane Tetrachloroethylene Methanol Isopropyl alcohol Acetonitrile Acetone Extraction solvent type Dispersing solvent type (a) (b) 100000 120000 100000 80000 80000 60000 Peak area Peak area 60000 40000 40000 20000 20000 0 0 50 90 130 170 200 300 350 400 500 Chloroform volume (?L) Isopropyl alcohol volume (?L) (c) (d) 120000 120000 100000 100000 80000 80000 Peak area Peak area 60000 60000 40000 40000 20000 20000 0 0 0 10 20 30 0 3 5 8 10 Vortex agitation time (seconds) Salt addition (%) (e) (f) Figure 3: Optimization of DLLME procedure: (a) extraction solvent type. and the analyte transfer is almost immediate 3. features of other microextraction techniques. 30. the equilibrium state can be achieved quickly (a few seconds).05). (d) dispersing solvent (isopropyl alcohol) volume.5. This is due to the extremely mixing after the formation of the cloudy mixture has been . ANOVA (? > 0. was selected for further optimization.

10.95% = 2.7 50. The linear equation calculated by the efficiency of LEV from human plasma (Figure 3(f)). the outcome of the analysis centrifugation times greater than 5. Therefore. and LLOQ (?g mL−1 ) 2 400 volumes (?L) 500 Precision (RSD.8 Extraction solvent Accuracy (RE. Salt Addition.15 and tetrachloroethylene ? value 0. 20.84.15) was smaller transfer to the organic solvent droplets [36. Defined Optimized parameters conditions Parameters LEV Methanol. Therefore. 350. acetonitrile.0011 acetone Regression coefficient (?) 0.7%. the calculated ? value (4931.0040 Isopropyl Dispersing solvent types alcohol. . Extraction solvent types carbon tetrachloride. 3.0 mL of ultrapure water was added 0. an aliquot of 500 ?L of human plasma and thaw cycles. Linear range (?g mL−1 ) 2–80 dichloromethane.0 mL of obtained permeated phase.05 (95% significance level). 400. solvent into the aqueous phase until centrifugation without Afterwards. So. Validation Method. and 80 ?g mL−1 . Linearity of the analytical method Stability tests assured the LEV stability in the biological was investigated by employing seven concentrations of LEV matrix. According to previous studies. such as the freeze in four replicates.84) and the ? value was higher the subsequent experiments were carried out without the than 0. 60 (HQC). The results 40 (MQC). polar molecules such as LEV can participate for the lack of fit should be performed [52]. and 170 130 volumes (?L) a Calibration curves were prepared in quadruplicate (? = 4) for concentra- NaCl concentration (%) 0–10 0 tions of 2. %) 5. The final conditions for the DLLME in quintuplicate. where ? is the ratio Type of shaking between the analyte peak area and the IS peak area. a test this process. 20. Table 3: Linearity and limit of quantification of the LEV method for analyses of human plasma. Water least squares method was ? = 0. RSD = 5. samples spiked with 50 ?L of LEV and 50 ?L of IS in order to and autoinjector conditions (Table 6) showed no significant obtain the final concentrations of 2 (LLOQ).9988.0040? + 0. and the extraction systems. (seconds) b experimental ?value < ?crit. The out effect was assessed by adding NaCl ranging from 3 to 10% curve calibration was linear over the concentration range (w/v) to the aqueous phase in order to increase the extraction from 2 to 80 ?g mL−1 . The results are showed in Table 3. 1. the centrifugation step than tabulated ? value (2. 90. Thus.and between-days precision and accuracy the separation of the sedimented phase and promoting good evaluated for at least four concentrations of the LEV standard. and ? is the concentration of the measured solution in ?g mL−1 . The agitation time varied from 10 to 30 seconds after the cloudy state formation. 130. However.05 (95% significance level). 50]. short-term room temperature conditions. all tests performed. the analytical curve showed heteroscedasticity behavior. Chloroform Experimental ? valueb 1.0 minutes do not promote by the regression method is linear and showed no lack of fit higher efficiencies of the extraction [47]. as determined by ANOVA.8 Journal of Analytical Methods in Chemistry Table 2: DLLME parameters. Thus.8%). %) 0. ? > 10 ?g mL−1 of CBZ. The results were weighted (1/?) because the residual analysis of 3. The solution was transferred to Amicon ultrafiltra- the time after injecting the mixture of extraction/dispersing tion tubes and it was centrifuged at 1800 ×g for 20 minutes. 10. and alcohol Interceptb 0.37 Dispersing solvent 200. extraction efficiency. 4. reducing the water quantity available to dissolve the target According to the AMC (Analytical Methods Committee) [51]. For lack of fit. and 80 (ULOQ) ?g mL−1 of LEV and showed no statistically significant difference (ANOVA. the measured procedure are summarized in Table 2. Then.71) was higher (formation of a large surface area). concentration at each concentration level was within ±15% of the nominal concentration and for the LLOQ within ±20%. and 1800 ×g were set as suitable conditions for achieving The within.85) and its ? value was smaller was a fundamental procedure to separate both the aqueous than 0. proposed (assisted DLLME). ? = ?? + ?. thus RE = 0. 5 minutes (Table 4). 300. As a result. According to the statistical tests. This test evaluates in electrostatic interactions with the salt ions in solution the variance of the residual values for each concentration and can consequently reduce the ability of the analyte to [53].3. in competition with necessarily the outcome of a linear relationship. isopropyl Slopea 0. 50].2) procedure vortex-mixing.4. ? is the 0–30 0 intercept. than the tabulated ? value (2. analyte and thereby leading the analyte molecules to the a value of the regression coefficient (?) close to unity is not extraction solvent [36. the salting.6. 4 (LQC). Therefore.0011 (? = 0.05) when compared with the tubes without vortex agitation and the conical glass tubes were submitted to vortex for 1 (Figure 3(e)). the extraction time was defined as minute. the calculated ? value (1. 60.9988 Chloroform. Once the mixture reached a cloudy state regression model. instability. DLLME (described in Section 2. ? is the slope. whereas for the linear addition of salt. In the DLLME procedure. are given in Table 5.4. was developed in 1. molecules form hydrated spheres around the salt ions. 40.

000 2.85 Residual error 0.1 sample using a miniaturized sample preparation technique. [9] showed a total run-time (?g mL−1 ) (?g mL−1 ) for the LEV of 14.6%).5 to 45 ?g mL−1 .1 has no described method to quantify LEV from biological LEV 4. Df: degrees of freedom. Our developed method presented a wider linear range Nominal Measured compared to this previous method. 2. for DLLME.0 57. 40.15 0.0 4.4 accomplishment of the DLLME procedure.50 (II) 0. demonstrating it is within the therapeutic .0 38.6 suitable cleanup procedure of the biological samples and to extract the LEV despite its polar nature from aqueous phase Between-day employing only 130 ?L of chloroform. even in complex 60.05.0 6.00005 Linear Lack of fit 0. Until this moment. method where the linear range was from 2.0 2. and their The analytical parameters of the proposed method have been chromatograms are shown in Figure 4.00005 No lack of fit a ? value: significance level of 0.2 −8. relative error expressed as percentage (%). LEV 1.00103 19 0.8 8. ?tab : tabulated ? test).1 5. [21] described a GC-NPD and RE = 5.9 −3. The ultrafilters were reused among analysis after checking carryover effect.6 4. the literature 2.9 10. Previously to DLLME.0 8.0 6. The GC-MS method Precision Accuracy Analyte concentration concentration RSDa (%) REa (%) developed by Isoherranen et al. DLLME procedure was able to promote a 60. relative error deviation expressed as percentage (%).0 7.0 7. SIM mode is (I) m/z 123 and (II) m/z 193.26854 1 0.6 −3.and between-days accuracy and precision. solvents.4 3.1 to remove endogenous compounds from the plasma allowing 40.6 the ultrafiltration procedure was employed for the first time LEV 4.00030 5 0.0 min). The plasmatic concen- compared to earlier reported methods for the quantitation tration of LEV in the patient was 25 ?g mL−1 (RSD = 3. Patient plasma samples and drug-free plasma were extracted under optimized conditions (? = 3).0 39.0 7.0 5.2% of LEV.Journal of Analytical Methods in Chemistry 9 Table 4: ANOVA results for the linearity of LEV (SS: sum of squares. which is longer time than our pro- Within-day posed run-time (9. ANOVA SS Df MS ?calc ? valuea ?tab Regression model 0.00 0.0 1.26854 4931.00006 1. this one does not require the use of any organic b RE. The proposed analytical method was used to analyze a human plasma sample from a patient who had taken LEV 4. MS: mean squares.0 4. Method Application and (1000 mg once per day) in order to evaluate its applica- Comparison to Other Methods bility. Table 5: Within.0 8.1 11.0 2.84 Pure error 0.1 3.75 (?V) IS 0.0 In this regard.6 1.0 (Minutes) (Minutes) (a) (b) Figure 4: GC-MS chromatograms obtained from the analysis of an extracted patient plasma sample: (a) patient sample after the levetiracetam intake (plasma concentration of levetiracetam was 25 ?g mL−1 ) and (b) a drug-free plasma sample. Unlike other pretreatments described in the literature a RSD.0 61.25 (I) (I) (II) 4.6 matrix. Greiner-Sosanko et al.0 4.71 0. ?calc : calculated ? test.00073 14 0.7 min.0 4.0 −2.6 −5.379 2.0 5.

Oláh. antiepileptic drugs in human plasma by liquid chromatogra- HPLC method for estimation of levetiracetam in pharmaceuti. K. an analytical method has been described Biomedical Research. pp. Jain. Soback. G. 5. S. 2. by liquid chromatography–tandem mass spectrometry: applica- tion to therapeutic drug monitoring. Chromatographic Science. Seo. monitoring and toxicology: a procedure for the monitoring 1909–1935. vol. Kim. P.-B. A. Isoherranen.. S. F. vol. 2014. accurate. no. “Determination of levetiracetam in human plasma by liquid chromatography/electrospray tandem mass spectrometry and Acknowledgments its application to bioequivalence studies. relative error deviation expressed as percentage (%). Soldin.3 5. Jayashankar. N. the process have been reduced. 17. L. 49. no. E. 50. M. pp. and precise. French and F. 2000. 3. N. and V. pp. M. vol. 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