Simultaneous determination of fluoxetine and its major active metabolite norfluoxetine in human plasma by LC-MS/MS using supported liquid extraction

Research article

Received 6 September 2010, Revised 18 November 2010, Accepted 19 November 2010 Published online in Wiley Online Library: 10 February 2011

(wileyonlinelibrary.com) DOI 10.1002/bmc.1597

Simultaneous determination of fluoxetine andits major active metabolite norfluoxetine inhuman plasma by LC‐MS/MS using supportedliquid extractionYu Li*, Thomas Emm and Swamy Yeleswaram

ABSTRACT: A rapid, sensitive and selective bioanalytical method was developed for the simultaneous determination offluoxetine and its primary metabolite norfluoxetine in human plasma. Sample preparation was based on supported liquidextraction (SLE) usingmethyl tert‐butyl ether to extract the analytes fromhumanplasma. Chromatographywas performed on aSynergi 4 μ polar‐RP column using a fast gradient. The ionization was optimized using ESI (+) and selectivity was achieved bytandem mass spectrometric analysis using MRM functions, m/z 310→44 for fluoxetine, m/z 296→134 for norfluoxetine andm/z 315→44 for fluoxetine‐d5 (internal standard). The method is linear over the range of 0.05 –20 ng/mL (using a humanplasma sample volume of 0.1 mL) with a coefficient determination of greater than 0.999. The method is accurate and precisewith intra‐batch and inter‐batch accuracy (%bias) of <±15%andprecision (%CV) of < 15% for both analytes. A run time of 4minmeans a high throughput of samples can be achieved. To our knowledge, this method appears to be the most sensitive onereported so far for the quantitation of fluoxetine and norfluoxetine and can be used for routine therapeutic drugmonitoring orpharmacokinetic studies. Copyright © 2011 John Wiley & Sons, Ltd.

Keywords: LC‐MS/MS determination; fluoxetine; norfluoxetine; human plasma; supported liquid extraction

* Correspondence to: Yu Li, Drug Metabolism and Biopharmaceutics, IncyteCorporation, Experimental Station, Route 141 & Henry Clay Road, Wilmington,DE 19880, USA. E-mail: [email protected]

Drug Metabolism and Biopharmaceutics, Incyte Corporation, ExperimentalStation, Route 141 & Henry Clay Road, Wilmington, DE 19880, USA

Abbreviations used: ACN, acetonitrile; FA, formic acid; LLE, liquid–liquidextraction; SLE, supported liquid extraction; SSRIs, selective serotoninreuptake inhibitors.

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IntroductionFluoxetine (Prozac®) is currently one of the widely prescribedselective serotonin reuptake inhibitors (SSRIs) for the treatmentof depression, bulimia nervosa and obsessive–compulsivedisorder (Shen et al., 2002). It is extensively metabolized in theliver to its primary active metabolite, norfluoxetine (Green et al.,2002). Because of its increasing popularity and important role inmodern therapy, reliable analytical methods for monitoringfluoxetine and its primary active metabolite in biological fluidsare highly desirable (Souverain et al., 2003).

Although several bioanalytical methods have been reportedfor the determination of fluoxetine and norfluoxetine inbiological fluids, these methods have a lower limit ofquantification (LLOQ) that may be inadequate for a pharmaco-kinetic study or therapeutic drug monitoring (Sutherland et al.,2001; Green et al., 2002; Li et al., 2002; Shen et al., 2002;Souverain et al., 2003; Chu and Metcalfe, 2007; Franceschi et al.,2009). In addition, the majority of the methods used conven-tional liquid–liquid extraction (LLE) which is labor intensive,time‐consuming and very difficult to automate since it oftenrequires off‐line processing such as mixing or centrifuging, andis therefore not well suited to high throughput bioanalyticalsample preparation. The supported liquid extraction (SLE) is analternative approach and can be easily automated. The SLEprovides high analyte recoveries while eliminating emulsionformation and other liquid handling issues associated with theconventional LLE (Williams et al., 2007).

The aim of this study was to establish a simple, selective andhighly sensitive bioanalytical method combining the supported

Biomed. Chromatogr. 2011; 25: 1245–1251 Copyright © 2011 John

liquid extraction, reversed‐phase HPLC and MS/MS detection toperform simultaneous determination of trace levels of fluoxetineand its major active metabolite norfluoxetine in human plasma.This is the first report of using SLE sample preparation methodfor the quantitation of fluoxetine and norfluoxetine in humanplasma. The performance of the method was evaluatedaccording to the current US Food and Drug Administrationbioanalytical method validation guidelines (US Department ofHealth and Human Services et al., 2001). The structures offluoxetine, norfluoxetine and fluoxetine‐d5, the internal stan-dard used for the analysis of both compounds, are presented inFig. 1.

Experimental

Chemicals, reagents and materials

Fluoxetine hydrochloride, norfluoxetine hydrochloride and fluoxetine‐d5hydrochloridewere purchased from Sigma (St Louis, MO, USA). HPLC‐grade

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5

O

F3C

O

H2NNH

O

NH

DD

D

DD

Fluoxetine Norfluoxetine Fluoxetine-d5 (IS)

F3C F3C

Figure 1. Structures of fluoxetine, norfluoxetine and fluoxetine‐d5 (internal standard).

Y. Li et al.

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acetonitrile (ACN), methanol (MeOH), ACS‐grade formic acid (FA),methyl tert‐butyl ether (MTBE) and sodium hydroxide (NaOH) weresupplied by EM Science (Gibbstown, NJ, USA). De‐ionized water(H2O) was prepared in‐house using a Milli‐Q water system providedby Millipore (Bedford, MA, USA). Blank human plasma with EDTA asanticoagulant was purchased from Biological Specialty (Colmar, PA,USA). The Isolute 200 mg SLE + supported liquid extraction platewas obtained from Biotage (Charlottesville, VA, USA).

Instrumentation

Chromatographic separations were performed with Shimadzu LC‐10ADVP binary pumps and SCL‐10AVP controller (Columbia, MD, USA)and LEAP CTC HTS PAL autosampler with stack cooling option(Carrboro, NC, USA). Mass spectrometric analyses were performedusing Applied Biosystems Sciex API 3000 triple quadruple massspectrometer equipped with Analyst 1.3.1 software (Toronto, Canada).The supported liquid extraction was conducted with TomTec Quadra96®model 320 liquid handling system equipped with vacuum manifold(Hamden, CT, USA).

Preparation of standard and QC samples

Stock solutions of fluoxetine, norfluoxetine and fluoxetine‐d5 wereprepared separately in methanol at a concentration of 1 mg/mL. Aworking solution of 1000 ng/mL containing fluoxetine and norfluoxe-tine was prepared by diluting both stock solutions with methanol andused for the preparation of standard curves and quality control (QC)samples.

Duplicate calibration standards were prepared freshly for each batchby serially diluting the working solution with blank human plasma togive eight appropriate final concentrations: 0.05, 0.1, 0.4, 1, 2.5, 5, 10 and20 ng/mL. The QC samples, at concentrations of 0.05, 0.2, 8 and16 ng/mL in human plasma, were prepared similarly with the differentstock solutions.

Fluoxetine‐d5 was used as the internal standard (IS). The internalstandard working solution was prepared by diluting fluoxetine‐d5 stocksolution (1 mg/mL) with 25% acetonitrile to a final concentration of100 ng/mL. All stock and working solutions were stored at −20°C.

Table 1. The mass spectrometer parameters for fluoxetine, norfl

Compounds Retention time (min) MRM transition (m/z

Fluoxetine 2.2 310→ 44Norfluoxetine 2.1 296→ 134Fluoxetine‐d5 2.2 315→ 44

DP, declustering potential; FP, focusing potential; EP, entrance po

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Sample preparation

An aliquot of 0.1 mL human plasma sample was mixed with 0.09 mL of0.5 M NaOH and 0.01 mL of internal standard working solution(100 ng/mL), then loaded onto the Isolute SLE + supported liquidextraction plate. A pulse of vacuum was applied to initiate flow onto theplate and the samples left to absorb for 5 min. Elution was broughtabout by the addition of 1 mL of MTBE. The extract was evaporated todryness and the residue was reconstituted into 0.1 mL of initial mobilephase. An aliquot of 0.01 mL reconstituted sample was injected onto theLC‐MS/MS system.

LC‐MS/MS conditions

A Synergi 4 μ polar‐RP 50× 2.0 mm column (Phenomenex, Torrance, CA,USA) was used at ambient temperature. Mobile phase solvents A and Bwere 0.1% formic acid in water and acetonitrile, respectively. The mobilephases were delivered at a flow rate of 0.3 mL/min, using a gradientperformed as follows: mobile phase B increased from 25 to 85% within3 min, then decreased to 25% over 0.1 min and held for 0.9 min toequilibrate the column prior to the next injection. Under theseconditions, the respective retention times for norfluoxetine, fluoxetineand fluoxetine‐d5 (IS) were approximately 2.1, 2.2 and 2.2 min,respectively. The total run time was 4 min for each injection.

The mass spectrometric conditions were optimized by infusing a neatsolution of the analytes and IS with a syringe pump. An electrospray ionsource was operated in positive ion mode at 400°C. Data were acquiredin multiple‐reaction monitoring (MRM) mode. The dwell time for eachprecursor‐product ion transition was set to 300 ms. The optimizedMS/MS parameters for this method are presented in Table 1.

Method validation

Method validation was performed to evaluate the specificity, linearity,sensitivity, accuracy, precision, recovery, matrix effect and stabilityaccording to current FDA bioanalytical method validation guidelines.

The specificity of the method is documented by the absence ofinterferences from endogenous substances from drug‐free humanplasma. To evaluate assay specificity, six independent lots of human

uoxetine and fluoxetine‐d5 (internal standard)

) DP (V) FP (V) EP (V) CE (eV) CXP (V)

30 160 7 29 1625 150 6 10 1230 160 7 29 16

tential; CE, collision energy; CXP, collision cell exit potential.

Biomed. Chromatogr. 2011; 25: 1245–1251Wiley & Sons, Ltd.

LC‐MS/MS determination of fluoxetine in human plasma

control plasma were extracted and analyzed for endogenous co‐elutinginterference.

To demonstrate the linearity and sensitivity of the method, doublecalibration curves for each batch were determined using calibrationstandards ranging from 0.05 to 20 ng/mL at eight concentration levels(0.05, 0.1, 0.4, 1, 2.5, 5, 10 and 20 ng/mL). The lower limit of quantitation(LLOQ) and the upper limit of quantitation (ULOQ) of this method were0.05 and 20 ng/mL, respectively. The linearity was evaluated by linearregression analysis, which was calculated by least square regressionmethod based on the peak area ratio (analyte/internal standard) vsconcentration.

To evaluate the accuracy and precision of the method, fourconcentration levels of QC samples (0.05, 0.2, 8 and 16 ng/mL) wereprepared and interpolated against the respective calibration curves. Theintra‐day assay variations were determined by quintuplicate analyses ofthe four levels of QC samples in each batch. The inter‐day assayvariations were determined by quintuplicate analyses of the four levelsof QC samples on three different batches. The acceptance criteria foreach calibration standard and quality control sample were that theaccuracy (%bias) and precision (%CV) must not exceed 15% (< 20% atthe LLOQ).

The percentage recovery (or extraction efficiency) was determined bymeasuring an extracted QC sample against a post‐extraction spiked QCsample:

%Recovery ¼ Response of extracted QCResponse of post‐extraction spiked QC

100%

The matrix effect was measured by referring the post‐extraction spikedQC sample to the mobile phase spiked QC sample:

Matrix effect ¼ Response of post‐extraction spiked QCResponse of mobile phase spiked QC

The absence of a matrix effect was indicated by a ratio of 1. No responsedue to total matrix suppression would give a value of 0 (Shi et al., 2003).

To determine the recovery and matrix effect, quintuplicate post‐extraction spiked QC samples and mobile phase spiked QC sampleswere analyzed along with the extracted QC samples and calibrationstandards. The stability for each analyte was evaluated in human plasmaat ambient temperature, for extracted samples placed in an autosamplerat 4°C, and following three separate freeze–thaw cycles. All stabilityexperiments were performed with two different concentration levels (0.2and 16 ng/mL) in quintuplicate.

×

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Results

MS/MS optimization

Quantitation was conducted by operating the mass spectrom-eter in the positive ion mode. The mass spectrometer wasprogrammed to transmit the protonated molecules (precursor)through the first quadrupole (Q1). Following collision‐induceddissociation (fragmentation) in the collision cell with nitrogengas, product ions were transmitted through the third quadru-pole (Q3). The positive ion full scan indicated the presence ofthe molecular ion [M+] as the predominant ion for eachcompound with m/z values of 310, 296 and 315 for fluoxetine,norfluoxetine and fluoxetine‐d5 (internal standard), respectively.The most abundant product ion obtained from fluoxetine andfluoxetine‐d5 at m/z 44 can be attributed to the dimethyliminium ion (C2H6N). The major fragment ion of m/z 134 fornorfluoxetine implied that the precursor ion could be broken bythe loss of trifluoromethyl phenol group (CF3–C6H4–O–) fromthe branch chain. The transitions of m/z 310→ 44 for fluoxetine,


m/z 296→ 134 for norfluoxetine and m/z 315→ 44 forfluoxetine‐d5 (IS) were selected for multiple reaction monitoring(MRM), permitted sensitive and selective detection of theanalytes and internal standard.

Extraction optimization

Supported liquid extraction is an alternative sample preparationtechnique which is analogous to traditional LLE, but can be easilyautomated without the problems presented by LLE. The IsoluteSLE + plate is packed with an optimized grade of diatomaceousearth, providing reproducible flow characteristics from well towell. When the aqueous biological fluid sample is applied, itspreads over the surface of the support, and is absorbed. Analytesof interest remain on the surface of the packing material, formingthe interface for extraction. When thewater immiscible extractionsolvent is applied, analytes are efficiently desorbed and thesolvent is collected. Several SLE bioanalytical LC‐MS/MS assayshave been developed and validated using SLE plates packed withdiatomaceous earth material (Wang et al., 2002; O’Maille et al.,2008; Jiang et al., 2009; Pan et al., 2010). The SLE proceduredeveloped for the isolation of fluoxetine, norfluoxetine andfluoxetine‐d5 (IS) from human plasmawas simple and automated.Topromote even flow, humanplasma sampleswere diluted 1:1 v/vwith an aqueous buffer prior to loading. To enhance extractionefficiency, pH adjustment was used to suppress ionization. Fourcommon water immiscible extraction solvents were investigated:dichloromethane (DCM), MTBE/DCM=2:1 (v/v), MTBE, and ethylacetate. The MTBE was selected as the extraction solvent forfluoxetine and norfluoxetine, since minimal ion suppression wasobserved and excellent analyte recovery was obtained by using it.

Specificity

Six different donors of blank human plasma were screened andno endogenous human plasma components were observed atthe retention times corresponding to the analytes. Therepresentative LC‐MS/MS chromatograms of the analytes inextracted blank human plasma and LLOQ (0.05 ng/mL) areillustrated in Figs 2 and 3.

Linearity

A regression analysis of the peak area ratio vs concentrationshowed linearity over the range of 0.05–20 ng/mL for bothfluoxetine and norfluoxetine in human plasma. The calibrationcurves were calculated using weighted linear regression, withweight = 1/x2 in the equation, y= ax+ b, where y is the peak arearatio of analyte to internal standard, a is the slope of calibrationcurve, b is the intercept and x is the analyte concentration(ng/mL). Coefficients of determination (r 2) of 0.9990 or betterwere obtained in the validation experiments. Tables 2 and 3summarize the accuracy, precision and parameter data of thecalibration curves.

Lower limit of quantitation

The lower limit of quantitation (LLOQ) is defined as the lowestconcentration on the calibration graph for which an acceptableaccuracy (nominal ± 20%) and precision (<20% CV) wereobtained. The current assay has a LLOQ of 0.05 ng/mL for eachanalyte based on 0.1 mL of human plasma sample volume and a

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7

XIC of +MRM (3 pairs): 310.1/44.0 Da from Sample 16 (B+IS) of SLE_FLU_040909.wiff (Turbo Spray) Max. 40.0 cps.

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8Time, min

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8Time, min

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8Time, min

0

10

20

30

40

Inte

nsity

, cps Fluoxetine

XIC of +MRM (3 pairs): 296.1/134.1 Da from Sample 16 (B+IS) of SLE_FLU_040909.wiff (Turbo Spray) Max. 40.0 cps.

0

10

20

30

40

Inte

nsity

, cps Norfluoxetine

XIC of +MRM (3 pairs): 315.3/44.0 Da from Sample 16 (B+IS) of SLE_FLU_040909.wiff (Turbo Spray) Max. 4.1e4 cps.

0.0

1.0e4

2.0e4

3.0e4

4.0e4

Inte

nsity

, cps

2.19

Fluoxetine-d5 (IS)

Figure 2. Representative chromatogram of extracted blank human plasma.

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signal–noise ratio of more than 10. Fifteen replicates (fivereplicates in each batch × three batches) of the LLOQ QC sampleswere used to evaluate the inter‐day accuracy and precision at thelow end of the assay range in three separate runs. For fluoxetine,

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8Ti

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8Ti

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8Ti

XIC of +MRM (3 pairs): 310.1/44.0 Da from Sample 17 (Std 0.05) of SLE_FLU_040909.wiff

0

50

100

150

200

250

294

Inte

nsity, cps

XIC of +MRM (3 pairs): 296.1/134.1 Da from Sample 17 (Std 0.05) of SLE_FLU_040909.wif

0

100

200

300

Inte

nsity, cps

XIC of +MRM (3 pairs): 315.3/44.0 Da from Sample 17 (Std 0.05) of SLE_FLU_040909.wif

0.0

1.0e4

2.0e4

3.0e4

4.0e4

Inte

nsity, cps

Figure 3. Representative chromatogram of fluoxetine and norfluoxetine fr


the inter‐day bias and CV values for LLOQ samples at 0.05 ng/mL(n=15) were 3.4 and 3.3%, respectively. For norfluoxetine, theinter‐day bias and CV values for LLOQ samples at 0.05 ng/mL(n=15) were 5.2 and 2.8%, respectively (Table 4).

2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8me, min

2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8me, min

2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8me, min

(Turbo Spray), Smoothed Max. 294.2 cps.

Max. 331.7 cps.

Max. 4.4e4 cps.

2.20

Fluoxetine

f (Turbo Spray), Smoothed

2.06

Norfluoxetine

f (Turbo Spray), Smoothed

2.19

Fluoxetine-d5 (IS)

om an extracted human plasma LLOQ (0.05 ng/mL) sample.


Table 2. Analytical performance of fluoxetine and norfluoxetine in human plasma assay: back‐calculated concentrationsa ofcalibration standards

Compound Nominal concentration (ng/mL)0.05 0.1 0.4 1 2.5 5 10 20

Fluoxetine Calculated 0.0502 0.101 0.391 0.987 2.50 5.04 10.0 20.4% Bias −0.3 0.5 −2.3 −1.3 0.2 0.8 0.2 1.8% CV 10.0 3.1 4.9 3.6 2.1 1.8 4.0 1.9

Norfluoxetine Calculated 0.0511 0.104 0.371 0.962 2.46 5.15 9.98 20.9% Bias 2.0 3.5 −7.4 −3.9 −1.7 3.1 −0.2 4.3% CV 3.9 12.7 12.1 5.8 6.4 2.8 5.4 2.8

aA linear regression method was used with 1/concentration2 as the weighting factor.

Table 3. Analytical performance of fluoxetine and norfluoxetine in human plasma assay: calibration curve parametersa

Compound Run number Slope Intercept R‐squared

Fluoxetine 1 0.121 0.00103 0.99782 0.111 0.00112 0.99643 0.116 0.00111 0.9980

Mean 0.116 0.00109 0.9974SD 0.00500 0.0000493 0.000872

Norfluoxetine 1 0.144 0.000491 0.99822 0.128 0.000431 0.99683 0.130 0.000419 0.9928

Mean 0.134 0.000447 0.9959SD 0.00872 0.0000386 0.00280

aA linear regression method was used with 1/concentration2 as the weighting factor.


Accuracy and precision

The intra‐day and inter‐day accuracy and precision weredetermined by calculating daily (intra‐day assay) and overall(inter‐day assay) bias and CV values for QC samples that wereassayed in three analytical runs. Table 4 summarizes the intra‐day and inter‐day accuracy and precision for fluoxetine andnorfluoxetine in human plasma. For fluoxetine, the intra‐daybias and CV values ranged from −4.7 to 3.8% and from 1.4 to8.0%, respectively. The overall inter‐day bias and CV valuesranged from −3.0 to 6.0% and from 3.3 to 4.7%, respectively. Fornorfluoxetine, the intra‐day bias and CV values ranged from−10.0 to 5.1% and from 2.8 to 9.2%, respectively. The overallinter‐day bias and CV values ranged from −10.6 to 5.6% andfrom 2.8 to 10.6%, respectively.

Recovery

The overall extraction recoveries from human plasma were70.8% for fluoxetine and 82.3% for norfluoxetine. These valueswere determined at three concentration levels (0.2, 8 and16 ng/mL). The recovery of fluoxetine‐d5 (IS) was 80.0%. Theobserved recoveries for the analytes and internal standardillustrated the suitability of the extraction procedure.

12

Matrix effect and carryover

There was no significant signal suppression due to matrix effectduring the ionization process. The average matrix effects


measured from human plasma at three different levels (0.2,8 and 16 ng/mL) were 0.95 for fluoxetine and 0.97 fornorfluoxetine, respectively. These values indicated an insignifi-cant ion suppression effect present from human plasma matrix.The matrix did not adversely affect the precision, accuracy,sensitivity and selectivity of the assay as all validation acceptancecriteria were met. Carryover was determined by injecting anextracted control plasma sample immediately following thehighest calibrator. No such carryover was observed in this assay.

Stability

The bench‐top (6 h at ambient temperature), freeze–thaw (threecycles) and autosampler (48 h at 4°C) stability were evaluatedand the results are presented in Table 5. All stability experimentswere performed at two different concentration levels (0.2 and16 ng/mL) in five replicates. Fluoxetine and norfluoxetine werefound to be stable in human plasma at ambient temperature forat least 6 h and as a reconstituted extract at 4°C (thetemperature of cooled autosampler tray stacks) for at least48 h. After 6 h at ambient temperature, the concentration offluoxetine in human plasma decreased by 5.4 and 5.0% at thelow and high concentration levels, respectively, from theircorresponding nominal concentrations. Similarly, the concen-tration of norfluoxetine decreased by 2.1 and 1.0% at the lowand high concentration levels, respectively. The stability offluoxetine and norfluoxetine in human plasma under repeatedfreeze–thaw cycles was also examined. After three freeze–thaw


49

Table 5. Analytical performance of fluoxetine and norfluoxetine in human plasma assay: bench‐top, freeze–thaw and autosamplerstability

Compound Nominalconcentration

(ng/mL)

Bench‐top stability(6 h at ambient temp)

Freeze–thaw stability(three cycles)

Autosampler stability(48 h at 4°C)

Calculatedconcentration

(ng/mL)

Differencefrom nominal

(%)


(ng/mL)


(%)


(ng/mL)


(%)

Fluoxetine 0.2 0.189 −5.4 0.190 −5.0 0.192 −4.216 15.2 −5.0 15.9 −0.4 15.5 −2.9

Norfluoxetine 0.2 0.196 −2.1 0.191 −4.6 0.202 0.916 15.8 −1.0 15.4 −3.8 16.1 0.6

Table 4. Analytical performance of fluoxetine and norfluoxetine in human plasma assay: intra‐ and inter‐day accuracy andprecision of QC samples

Compound Nominalconcentration

(ng/mL)

Intra‐day variation (n=5) Inter‐day variation (n=15)Calculated (ng/mL) Bias (%) CV (%) Calculated (ng/mL) Bias (%) CV (%)

Fluoxetine 0.05 0.0505 0.9 1.4 0.0517 3.4 3.30.2 0.191 −4.7 7.2 0.194 −3.0 4.78 8.30 3.8 5.5 8.48 6.0 3.4

16 16.5 3.2 8.0 16.8 4.8 4.7Norfluoxetine 0.05 0.0525 5.1 3.0 0.0526 5.2 2.8

0.2 0.180 −10.0 2.8 0.179 −10.6 10.68 8.07 0.9 7.7 8.45 5.6 6.6

16 16.2 1.0 9.2 16.5 2.8 7.6

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cycles, the proportion of fluoxetine remaining, relative to thenominal values, was 95.0 and 99.6% at the low and highconcentrations, respectively. The proportion of norfluoxetineremaining was 95.4 and 96.2% at the low and high concentra-tions, respectively. Therefore, fluoxetine and norfluoxetine inhuman plasma can tolerate at least three freeze–thaw cycleswithout significant degradation.

ConclusionA new LC‐MS/MS method for the simultaneous quantitation offluoxetine and its major metabolite norfluoxetine in humanplasma using an SLE approach was developed and validated.The method combines a variety of convenient features in termsof simplicity, selectivity, sensitivity, precision, accuracy, rapidityand very satisfactory reproducibility.

Several LC‐MS/MS methods have been reported for thedetermination of fluoxetine and norfluoxetine in biologicalmatrices. Of those methods, none was capable of achieving aLLOQ as low as 0.05 ng/mL, which may be required for tracelevel drug monitoring. Recently, one LC‐MS/MS method fordetermination of fluoxetine and norfluoxetine in human serumwith a LLOQ of 0.3 ng/mL was published (Franceschi et al.,2009). However, this method needs a large sample volume (upto 1 mL). The new method with SLE approach described here istherefore the most sensitive one so far reported for thequantitation of fluoxetine and norfluoxetine in biological


matrices after taking into consideration both sample size(0.1 mL) and lower LLOQ (0.05 ng/mL). Also, the new methodinvolves the first use of the SLE plate for the quantitation offluoxetine and norfluoxetine in human plasma, which allows asignificant reduction in the overall sample preparation time andprovides at least 2‐fold increased extraction throughputcompared with conventional LLE. By using the simple SLEprocedure with the Isolute SLE + plate, the sample preparationcan be completed within 10 min. In addition, SLE extractsexhibit reduced matrix interferences and increased recovery incomparison to the recovery achieved with conventional LLE(Li et al., 2002).

In conclusion, the high sensitivity, high recovery, rapidity inchromatographic separation (4 min) and simplicity in samplepreparation (10 min) of this method allow the determination oftrace levels of fluoxetine and norfluoxetine in biologicalmatrices in high throughput fashion. Therefore, this novelmethod can be used for routine therapeutic drug monitoring orfor supporting pharmacokinetic studies.

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Simultaneous determination of fluoxetine and its major active metabolite norfluoxetine in human plasma by LC-MS/MS using supported liquid extraction