determination of naproxen in human plasma by gc-ms

J. Sep. Sci. 2014, 37, 997–1003 997

Bilal Yilmaz1

Huseyin Sahin2

Ali Fuat Erdem3

1Department of AnalyticalChemistry, Faculty of Pharmacy,Ataturk University, Erzurum,Turkey

2Department of EmergencyMedicine, Faculty of Medicine,Ataturk University, Erzurum,Turkey

3Department of Anesthesiologyand Reanimation, Faculty ofMedicine, Sakarya University,Sakarya, Turkey

Received October 8, 2013Revised December 18, 2013Accepted January 20, 2014

Research Article

Determination of naproxen in human plasmaby GC–MS

This paper describes a GC–MS method for the determination of naproxen in humanplasma. Naproxen and internal standard ibuprofen were extracted from plasma usinga liquid–liquid extraction method. Derivatization was carried out using N-methyl-N-(trimethylsilyl)trifluoroacetamide. The calibration curve was linear between the concentra-tion range of 0.10–5.0 �g/mL. Intra- and interday precision values for naproxen in plasmawere <5.14, and accuracy (relative error) was better than 4.67%. The extraction recoveriesof naproxen from human plasma were between 93.0 and 98.9%. The LOD and LOQ ofnaproxen were 0.03 and 0.10 �g/mL, respectively. Also, this assay was applied to determinethe pharmacokinetic parameters of naproxen in six healthy Turkish volunteers who hadbeen given 220 mg naproxen.

Keywords: Ibuprofen / Liquid–liquid extraction / Naproxen / Pharmacokinetics /ValidationDOI 10.1002/jssc.201301105

1 Introduction

Naproxen (6-methoxy-�-methyl-2-naphthalene acetic acid), anonsteroidal anti-inflammatory drug (NSAID) derived frompropionic acid, is widely used to moderate pain relief in thetreatment of many diseases [1]. NSAIDs, including naproxen,are commonly employed to reduce ongoing inflammation,pain, and fever, since they are able to block [2] the cyclooxy-genase (Cox) enzymes (Cox-1 and Cox-2) that both produceprostaglandins; these classes of compounds have several im-portant functions, such as the promotion of inflammation,pain, and fever [3]. However, prostaglandins produced by theCox-1 enzyme are also able to protect the stomach, supportplatelets, and blood clotting. Thus, NSAIDs can cause ul-cers in the stomach and promote bleeding after an injury orsurgery. Moreover, they are associated with other serious sideeffects, i.e. kidney failure and with a number of minor sideeffects, such as nausea, vomiting, diarrhea, constipation, de-creased appetite, rash, dizziness, headache, and drowsiness.When anti-inflammatory treatments become chronic, as inrheumatoid arthritis, the patients are exposed to the drugsfor prolonged periods. The potential misuse and involuntaryintake of naproxen as residues in food could pose a health risk

Correspondence: Assoc. Prof. Dr. Bilal Yilmaz, Department of Ana-lytical Chemistry, Faculty of Pharmacy, Ataturk University, 25240,Erzurum, Turkey Erzurum, TurkeyE-mail: [email protected]: +90-0442-2360962

Abbreviations: AUC, area under the curve; Cox, cy-clooxygenase; ICH, International Conference on Har-monization; IS, internal standard; MSTFA, N-methyl-N-(trimethylsilyl)trifluoroacetamide; NSAID, nonsteroidalantiinflammatory drug; QC, quality control

in people, e.g. allergy, severe gastrointestinal lesions, changesin renal function, and nephrotoxicity [4].

Several methods have been reported for the determina-tion of naproxen including HPLC with UV [5–12] or fluores-cence detection [13–17], LC–MS/MS [18,19], GC–MS [20,21],and GC with flame ionization detection [22, 23] in plasma,serum, and urine.

The present work describes the O-silylation ofthe hydroxyl group of naproxen using N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) as a silylatingreagent. This is followed by determination of naproxen aftera derivatization procedure in human plasma using internalstandard (IS) methodology by GC–MS. The developedmethod was validated by using linearity, stability, precision,accuracy, and sensitivity parameters according to Interna-tional Conference on Harmonization (ICH) guidelines [24].

The advantages of the present method include a simplesingle step extraction procedure using inexpensive chemicalsand a short run-time. Besides, this method was used to assaythe naproxen in plasma samples obtained from six healthymale volunteers. At the same time, this method was used fora pharmacokinetic study after therapeutic doses of naproxen.

2 Materials and methods

2.1 Chemicals and reagents

Naproxen sodium and IS ibuprofen were obtained fromSigma–Aldrich (St. Louis, MO, USA). MSTFA, ethyl acetate,hexane, dichloromethane, acetonitrile, butanol, and chlo-roform were purchased from Sigma–Aldrich. Aleve tablet

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998 B. Yilmaz et al. J. Sep. Sci. 2014, 37, 997–1003

(220 mg naproxen sodium) was obtained from a pharmacy(Erzurum, Turkey).

2.2 Apparatus and analytical conditions

Chromatographic analysis was carried out on an Agilent6890N gas chromatography system equipped with 5973 se-ries mass selective detector, 7673 series autosampler, andchemstation (Agilent Technologies, Palo Alto, CA, USA). AHP-5 MS column with 0.25 �m film thickness (30 m ×0.25 mm id) was used for separation. Splitless injectionwas used and the carrier gas was helium at a flow rate of1 mL/min. The MS detector parameters were transfer linetemperature 280�C, solvent delay 3 min, and electron energy70 eV.

2.3 Preparation of stock solutions

A stock solution (1 mg/mL) of naproxen was prepared inacetonitrile. The initial stock solution was further diluted inacetonitrile to produce solutions of naproxen (10 �g/mL).Calibration standards of naproxen at concentrations of 0.10–5.0 �g/mL was prepared by spiking appropriate amount ofthe standard solutions in blank plasma. Standard solutionswere stored at +4�C. IS stock solution was made at an initialconcentration of 1 mg/mL. The working solution of IS wasprepared by dissolving in acetonitrile to obtain a concentra-tion of 1.0 �g/mL.

2.4 Preparation of quality control samples

For quality control (QC) study, the concentrations of 0.3,1.5, and 4.5 �g/mL naproxen were added to human plasma,respectively. Volumes (0.1 mL) from standard solution ofnaproxen were added to normal human plasma to get low,middle, and high QC samples, respectively, and stored at−20�C. The QC samples were taken out from storage foranalysis to determine intra- and interday precision and accu-racy.

2.5 Extraction procedure

The samples were extracted by SPE cartridge (C18, 3 mL, 500mg, Bond-elut, Agilent). The cartridge was conditioned with3 mL methanol and 3 mL water. The frozen plasma sam-ples (0.5 mL) were thawed at 25�C (room temperature), and0.75 mL water and IS (0.1 mL at 1.0 �g/mL concentration)were added to the plasma. The mixture was vortexed andtransferred to SPE cartridge. Then, the cartridge was washedwith a mixture of acetonitrile/water (2 mL, 15/85) and 3 mLhexane. Eluate was collected from 3 mL acetonitrile. The elu-ate was evaporated at 50�C under nitrogen. The residue wasdissolved in acetonitrile and MSTFA (50:50, v/v), and 1 �Lvolume was injected into the GC–MS system. The extraction

recoveries of naproxen from human plasma were between53.6 and 68.6%.

Therefore, the liquid–liquid extraction method wasused in this study. Several solvents (ethyl acetate, hexane,dichloromethane, acetonitrile, butanol, and chloroform) weretested for the extraction. Finally, ethyl acetate/hexane (2:3,v/v) proved to be the most efficient in extracting naproxenfrom human plasma. Blood samples were collected intothe tubes containing disodium EDTA and centrifuged at4500 × g for 10 min. A 0.5 mL volume of the resultantplasma samples was spiked with 0.1 mL of naproxen (0.10,0.25, 0.50, 1.0, 2.0, 3.0, 4.0, and 5.0 �g/mL), 0.1 mL of IS(1.0 �g/mL) and 0.5 mL H3PO4 solution. After vortex mix-ing for 5 s, 4 mL of ethyl acetate and hexane was added(2:3, v/v), the mixture was vortexed for 30 s and then cen-trifuged at 3000 × g for 3 min. Plasma samples were extractedonly once. The organic layer was transferred into another tubeand evaporated to dryness at room temperature under nitro-gen gas. The dry residue was dissolved in 100 �L of a mixtureof acetonitrile and MSTFA (50:50, v/v). The mixture was vig-orously shaken and then delayed at room temperature for10 min. One microliter of sample was injected into theGC–MS system.

2.6 Collection of samples

Prior to the study, the clinical protocol was approved by theEthics Committee of Faculty of Medicine, Ataturk University.All volunteers were given written informed consent to partici-pate in the study according to the principles of the Declarationof Helsinki. The volunteers who submitted the agreementsto attend this project were medically examined and threevolunteers were selected (39.2 ± 2.65 years; 74.2 ± 1.87 kg;172 ± 5.67 cm) for pharmacokinetics study for naproxen. Thesubjects were required to abstain from taking any other drugfor three days prior the start of test. They were also forbiddento smoke or drink alcohol or xanthine-containing beveragesfor 24 h before the beginning of the study until its end. Sixvolunteers received an oral tablet (Aleve) containing 220 mgof naproxen. Then they were allowed to drink water. The totalamount of water drunk during the day was 1500 mL. Thevolunteers sitting during lunch had normal activity (stand-ing or sitting) during the study, but were never in a supineposition during the 16 h after administration. Blood sampleswere taken into EDTA tubes at 0, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 6.0,8.0, 10, 12, and 16 h after oral administration. Blood sampleswere centrifuged at 4500 × g for 10 min and the plasma wasseparated and kept frozen at −20�C until analysis.

3 Results and discussion

3.1 Method development and optimization

Naproxen is a polar molecule, so a capillary column coatedwith 5% phenyl and 95% dimethylpolysiloxane was used for


J. Sep. Sci. 2014, 37, 997–1003 Gas Chromatography 999

Figure 1. MS spectra afterderivatization of naproxen (A)and ibuprofen (IS) (B) withMSTFA.

separation. The injection port and detector temperature wasset to 250�C. Different temperature programs were investi-gated to give an optimum temperature program as follows:initial temperature was 150�C, held for 1 min, increased to220�C at 20�C/min held for 1 min, and finally to 300�C at10�C/min with a final hold of 1 min. The injector volume was1 �L in splitless mode.

MSTFA reacts to replace labile hydrogen atoms on a widerange of polar compounds with a TMS group and is usedto prepare volatile and thermally stable derivatives for GC–MS [25, 26]. To increase the performance of the gas chro-matographic separation, naproxen and IS were derivatizedusing MSTFA (Fig. 1). The hydroxy (–OH) groups were con-verted to the corresponding silyl (–O–TMS) groups. To con-firm the complete derivatization of naproxen and IS, eachcompound was derivatized and analyzed separately. After es-tablishing the optimum reaction conditions, the compoundswere mixed together and then derivatized in order to performa simultaneous analysis.

The effects of time and temperature on the reaction wereinvestigated. Therefore, naproxen and IS were dissolved inacetonitrile. One hundred microliters of MSTFA solution wasadded to 100 �L of 2.0 �g/mL naproxen solution and reactedat room temperature—50 and 75�C for 5, 10, and 20 min. Theresulting samples were quantitated by GC–MS system. Afterstanding for 10 min at room temperature, maximum peakareas were quantitated.

3.2 Validation of the method

To evaluate the validation of the present method, parameterssuch as specificity, linearity, precision, accuracy, LOD andLOQ, recovery, and stability were investigated according toICH validation guidelines [24].

3.2.1 Specificity

The fragment ions (m/z 185 and 73) were used for the quan-titation of naproxen and IS in the selected-ion monitoringmode. The retention times of naproxen-TMS and IS-TMSderivatives were 7.6 and 4.5 min, respectively, and the to-tal run-time of analysis was 8 min. Representative chro-matograms of (a) drug-free plasma, (b) the plasma spiked withnaproxen (5.0. �g/mL) and IS (1.0 �g/mL), and (c) plasmaobtained at 1.0 h after a single dose of 220 mg naproxen aregiven in Fig. 2. There is no interference in the chromatogramof drug-free plasma.

Drug-free plasma was spiked with therapeutic concentra-tions of such drugs that included some drugs (carvedilol,atenolol, mexiletine, flurbiprofen, medazepam, diazepam,disulfiram, estradiol valerate, and medroxyprogesterone ac-etate). The retention times for these drugs under the chro-matographic conditions for the naproxen assay were deter-mined and found not to interfere with the retention time ofnaproxen and IS.

3.2.2 Linearity

The linearity of an analytical method is its ability within adefinite range to obtain results directly proportional to theconcentrations (quantities) of an analyte in the sample. Theconcentrations of the spiked naproxen in human plasma were0.10, 0.25, 0.50, 1.0, 2.0, 3.0, 4.0, and 5.0 �g/mL with constantconcentration of IS (1.0 �g/mL). The calibration curves wereestablished by plotting the ratio of the peak areas of naproxenand IS obtained after extraction of the spiked plasma sample.The equation of the mean calibration curve obtained fromeight points was y = 114.3x + 13.24 with a correlation co-efficient (r = 0.999). The linear regression equation was cal-culated by the least squares method using Microsoft Excel R©



Figure 2. Representative chro-matograms of (A) drug-freeplasma, (B) the plasma spikedwith naproxen (5.0 �g/mL) andIS (1.0 �g/mL), (C) plasma ob-tained at 1.0 h after a single oraldose of 220 mg naproxen.

program. SDs of the slope, intercept, and correlation coeffi-cient for the calibration curves were 0.128, 1.386, and 3.58 ×10−3, respectively.

3.2.3 Precision and accuracy

Assay precision was determined by repeatability (intraday)and intermediate precision (interday). Repeatability duringthe same day and intermediate precision on different days(three days) were evaluated with six replicates of QC sam-ples. The accuracy of analytic method was assessed as thepercentage relative error. The accuracy and precision of themethod were evaluated with QC samples at concentrationsof 0.3, 1.5, and 4.5 �g/mL. The intra- and interday accuracyand precision results are shown in Table 1. The intra- and

interday precision of the QC samples were satisfactory withRSD <5.14% and accuracy with relative error within ±4.67%(should be <15 according to ICH guidance).

3.2.4 Recovery

The recovery of an analyte is the extraction efficiency of ananalytical process, reported as a percentage of the knownamount of an analyte carried through the sample extractionand processing steps of the method. Liquid–liquid extractionwas used for the sample preparation in this work. Finally,ethyl acetate/hexane (2:3, v/v) proved to be the most efficientin extracting naproxen from human plasma. Spiked plasmasamples were prepared three times at all levels (0.10, 0.25,0.5, 1.0, 2.0, 3.0, 4.0, and 5.0 �g/mL) of the calibration graph

Table 1. Precision and accuracy of naproxen in human plasma

Intraday Interday

Added Found Precision Accuracyc) Found Precision Accuracyc)

(�g/mL) (Mean ± SDa)) RSD (%)b) (Mean ± SDa)) RSD (%)b)

Plasma poolsd)

0.3 0.286 ± 0.012 4.19 −4.67 0.292 ± 0.015 5.14 −2.671.5 1.43 ± 0.038 2.66 −1.38 1.44 ± 0.048 3.33 −4.004.5 4.43 ± 0.189 4.27 −1.56 4.58 ± 0.208 4.54 1.78

a) SD of six replicate determinations.b) Average of six replicate determinations.c) % Relative error = found − added)/added × 100.d) Plasma volume (0.5 mL).



Table 2. Recovery of naproxen in human plasma

Added Found(�g/mL) (Mean ± SDa)) Recovery (%) RSD (%)b)

0.10 0.093 ± 0.006 93.0 6.450.25 0.235 ± 0.011 94.0 4.680.5 0.476 ± 0.027 95.2 5.671.0 0.954 ± 0.052 95.4 5.452.0 1.978 ± 0.115 98.9 5.813.0 2.931 ± 0.139 97.7 4.744.0 3.918 ± 0.259 97.9 6.615.0 4.906 ± 0.288 98.1 5.87

a) SD of three replicate determinations. b) Average of three repli-cate determinations.

of naproxen. The recovery of naproxen was determined bycomparing the peak areas measured after analysis of spikedplasma samples with those found after direct injection ofstandard solutions at the same concentration levels. The ex-traction recoveries of naproxen from human plasma werebetween 93.0 and 98.9% as shown in Table 2.

3.2.5 LOD and LOQ

The LOD is a parameter that provides the lowest concentra-tion in a sample that can be detected from background noisebut not quantitated. The LOQ is defined as the lowest con-centration of analyte that can be determined with acceptableprecision and accuracy. The LOD and LOQ were studied totest the sensitivity of the method. The LOD defined as S/N =3 (RSD < 20% for LOD) was found to be 0.03 �g/mL. TheLOQ defined as S/N = 10 (RSD < 10% for LOQ) was found tobe 0.10 �g/mL. Both accuracy and precision of these valueswere well within the proposed criteria (RSD < 20%).

3.2.6 Stability

The stability of naproxen and IS solutions was tested at severalstorage conditions (room temperature for 24 h and −20�C fortwo weeks). The freeze–thaw stability of naproxen in plasmasamples was evaluated over three freeze–thaw cycles. Stabilitycontrol plasma samples in triplicate at the levels of 0.10, 1.5,and 4.5 �g/mL for plasma were immediately frozen at −20�C,and thawed at room temperature for three consecutive times.After that, the samples were processed and assayed. The sta-bility of naproxen in spiked plasma samples stored at roomtemperature for 24 h and −20�C for two weeks was evalu-ated as well. Long-term stability was assessed using samplesstored at −20�C over a period of four weeks. The results ofthese stability studies are given in Table 3 and percent ratiosare within the acceptance range of 90–110%.

3.2.7 Matrix effect

The matrix effect was defined as the direct or indirect al-teration or interference in response due to the presence of

Table 3. Stability of naproxen in human plasma (n = 3)

Treatment Recovery (Mean ± SD)Plasma concentration(�g/mL)

0.10 1.5 4.5

Three freeze–thaw cycles 92.18 ± 3.132 91.12 ± 2.736 94.21 ± 2.356Stored at RT for 24 ha) 90.16 ± 3.244 92.26 ± 5.263 93.34 ± 2.567Stored at −20�C for 2 wk 91.46 ± 2.432 92.25 ± 3.726 91.16 ± 4.621Stored at −20�C for 4 wk 92.19 ± 2.862 91.03 ± 2.086 93.15 ± 4.342

a) RT, room temperature.

unintended analytes or other interfering substances in thesample [27]. The matrix effect of naproxen was investigatedby comparing amount of naproxen solutions with processedblank samples reconstituted with naproxen solutions. Theblank plasmas used in this study were from six differentbatches of human blank plasma. If the ratio was <85% or>115%, a matrix effect was implied. The relative matrix ef-fect of naproxen at three different concentrations (0.5, 2.0,and 4.0 �g/mL) was <±2.30% (Table 4). The results showedthat there was no matrix effect of the analytes observed fromthe matrix of plasma in this study.

3.2.8 Data analysis

The maximum plasma concentration (Cmax) and time toreach maximum concentration (Tmax) were directly deter-mined from the plasma concentration versus time curves.The area under the curve from 0 to t (AUC0–t) was calculatedby the linear trapezoidal rule. The AUC from 0 h to infinity(AUC0–∞) was estimated by summing the area from 0 to t(AUC0–t) and t to infinity (AUCt–∞), where AUCt–∞ = Ct/Kel,with Ct is defined as the last measured plasma concentra-tion at time t and Kel, the slope of the terminal portion ofthe ln(plasma concentration) versus time curve. The elimina-tion half-life (t1/2) was calculated using the pharmacokineticrelationship t1/2 = ln(2)/Kel [28].

3.2.9 Pharmacokinetic study

To check the clinical applicability of the method, the phar-macokinetic parameters of naproxen was investigated in sixhealthy male volunteers after a single oral administration of220 mg of the drug. The amount of naproxen was deter-mined between 0 and 16 h in human plasma. The meanplasma concentration–time curve is shown in Fig. 3. It showsthe mean plasma concentration profile (including the SDs) ofnaproxen after oral administration of a 220 mg formulationtablet. The mean peak concentration (Cmax) of 43.1 �g/mLfor naproxen was attained at 1.50 h after administration ofthe drug. The mean half-life was 14.7 h. The mean valuesof pharmacokinetic parameters estimated by the computerprogram WinNonlin with noncompartmental method wereshown in Table 5. The pharmacokinetics results obtained



Table 4. Matrix effect evaluation of naproxen and IS in human plasma (n = 3)

Samples Concentration level (�g/mL) A B Matrix effect (%)(Mean ± SD) (Mean ± SD)

Naproxen 0.5 0.487 ± 0.052 0.492 ± 0.063 98.92.0 1.972 ± 0.182 2.018 ± 0.012 97.74.0 3.962 ± 0.047 4.036 ± 0.038 98.2

IS 1.0 0.983 ± 0.087 1.032 ± 0.097 95.3

(A) The amount of naproxen and IS derivatized in blank plasma sample’s reconstituted solution (the final solution of blank plasma afterextraction and reconstitution).(B) The amount of naproxen and IS derivatized with MSTFA.

using this method are in agreement with studies reportedpreviously [8, 29, 30].

3.2.10 Comparison of the methods

Today, GC–MS is a good alternative for LC–MS/MS in casethe latest expensive technique is not available in the labora-tory. As compared to LC–MS/MS, high-resolution capillaryGC–MS has been less frequently used [25, 31, 32].

Sultan et al. [18] and Miksa et al. [19] described two meth-ods to simultaneously quantify and identify naproxen andother NSAIDs in human plasma, employing LC–MS in afull-scan MS mode. Both methods were robust and reliable,however, their run-times were approximately 15 min withrather high LOQ values of 2 and 20 �g/mL, respectively.

During method development, it became evident thatnaproxen and IS were very sensitive to matrix effects dur-ing the derivatization process in plasma. Sample preparationtechniques, such as liquid–liquid extraction were used in or-der to minimize matrix suppression effects.

GC–MS method sensitivity is not enough for the deter-mination of naproxen in plasma. For this reason, MSTFAis used to increase sensitivity. In this study, the purpose of

Figure 3. Mean plasma naproxen concentration–time profile forsix volunteers after a single oral dose of naproxen, 220 mg.

Table 5. Mean pharmacokinetic parameters of naproxen for sixvolunteers after oral administration of Aleve tablet(220 mg)

Parameters (Mean ± SD) RSD (%)

Maximum plasma concentration 43.1 ± 5.104 11.84Cmax (�g/mL)Time required for maximum plasma

concentration (Tmax)1.50 ± 0.186 12.40

AUC 526.3 ± 47.12 8.95AUC(0–16 h) (�g/mL h)AUC at infinite timeAUC(0–∞) (�g/mL h) 653.2 ± 47.42 7.26Plasma half-life (t1/2, h) 14.7 ± 1.252 8.52

the derivatization reaction is to increase sensitivity thus it ispossible to work at low concentrations.

When this method is applied to plasma samples, its sensi-tivity was found to be adequate for pharmacokinetic studies.The present method has the following advantages over thereported method [18, 19]. The LOQ of the reported methodswere 2.0 and 20 �g/mL, whereas the present method LOQwas 0.10 �g/mL.

The calibration curve of naproxen was linear over theconcentration range of 0.10–5.0 �g/mL for plasma, whichis as good as or superior to that reported in other papers[5–8, 11, 13–16, 18, 21–23].

Naproxen was extracted from human plasma with an SPEprocedure reported in other papers [11, 12, 17]. This methodis also the most comprehensive method, which can extractnaproxen in a single extraction procedure.

In this study, the recovery percentage of naproxen is high[13, 14, 19], extraction processes do not take much time [11,12, 17], additionally, the retention time is short, which is anadvantage [5, 9, 13, 15, 18, 19, 21].

4 Concluding remarks

In the present work, a simple and sensitive GC–MS methodhas been developed for the determination of naproxen in hu-man plasma. The method was completely validated using sen-sitivity, stability, specificity, linearity, accuracy, and precision



parameters for determination of naproxen in human plasma.Additional advantages of this method include small samplevolume (0.5 mL), good extraction recovery from plasma, anda readily available IS. Also, the extraction and derivatizationprocedures in this study were simple. Therefore, the methodcan be very useful and an alternate to performing pharmacoki-netic studies in determination of naproxen for clinical use.

We are thankful to Vedat Akba (Criminal Police Laboratory,25060, Erzurum, Turkey) for their help in GC–MS analyses. Also,the authors would like to thank all the adults who participated inthis study.

The authors have declared no conflict of interest.

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determination of naproxen in human plasma by gc-ms

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