Development and validation of dried blood spots technique for quantitative determination of topiramate using liquid chromatography-tandem mass spectrometry

Research article

Received: 22 January 2013, Revised: 17 February 2013, Accepted: 18 February 2013 Published online in Wiley Online Library: 26 March 2013

(wileyonlinelibrary.com) DOI 10.1002/bmc.2905

1054

Development and validation of dried bloodspots technique for quantitativedetermination of topiramate using liquidchromatography–tandem mass spectrometryTanja Vnu�cec Popova*, Lea Cvitkovi�c Mari�ci�ca, Helena Prosenb andDarinka Brodnjak Von�cinac

ABSTRACT: An LC-MS/MS method for determination of the anti-epileptic drug topiramate (TPM) in dried blood spot (DBS)samples was developed and validated. DBS samples were prepared by spotting 30 mL of spiked whole blood onto FTATM

DMPK-C cards and drying for at least 3 h. Six-millimetre punched spots were then extracted by using a mixture of methanoland water (90:10, v/v) with deuterated internal standard (topiramate-d12). The extracted samples were injected into a liquidchromatograph equipped with a tandem mass spectrometric detector. Negative ions were monitored in the selected reactionmonitoring mode and transitions m/z 338.2!78.1 and m/z 350.3!78.1 were used for the quantitative evaluation of TPMand internal standard, respectively. The results obtained from validation were statistically evaluated according to therequirements of the European Medicines Agency and US Food and Drug Administration regulatory guidelines. The linearityof the method was checked within a concentration range from 10 to 2000 ng/mL. The validation results indicate that themethod is accurate, precise, sensitive, selective and reproducible. Copyright © 2013 John Wiley & Sons, Ltd.

Keywords: topiramate; liquid chromatography tandem mass spectrometry; dried blood spots; pharmacokinetic study

* Correspondence to: Tanja Vnu�cec Popov, Krka, d.d., Novo mesto, Šmarje�skacesta 6, SI-8501 Novo mesto, Slovenia. Email: [email protected]

a Krka, d.d., Novo mesto, Šmarje�ska cesta 6, SI-8501 Novo mesto, Slovenia

b University of Ljubljana, Faculty of Chemistry and Chemical Technology,A�sker�ceva 5, 1000 Ljubljana, Slovenia

c University of Maribor, Faculty of Chemistry and Chemical Engineering,Smetanova 17, SI-2000 Maribor, Slovenia

Abbreviations used: DBS, dried blood spot; EBF, European BioanalysisForum; SRM, selected reaction monitoring mode; TPM, topiramate.

IntroductionTopiramate [TPM, 2,3:4,5-bis-O-(1-methylethylidene)-b-D-fructopyranose sulfamate] is an anti-epileptic drug that is usedfor the treatment of various forms of epilepsy in adults andchildren and for the prevention of frequently recurring migrainesin adults (Shank et al., 2000). The collection of whole bloodsamples on paper, known as the dried blood spots technique(DBS), dates back to the early 1960s, when it was used fordetecting metabolic problems in newborns (Guthrie and Suzi,1963; Li and Tse, 2010).

Over the last few years, bioanalysts, pharmacokineticists andclinical investigators have rediscovered the technique of driedblood spots as an alternative to the traditional plasma collectionin pharmacokinetic studies. For that reason the EuropeanBioanalysis Forum (EBF) organized a symposium in June 2010,with the objective of bringing together experts from differentfields in order to discuss various perspectives on dried bloodspots technique, in order to try and provide answers to issuesthat still remain and to present the current status of thistechnique (Abbott et al., 2010).

DBS offers a number of advantages over conventionalwhole blood or serum sample collection. These advantages aremanifold and relate to ethical, practical, scientific and costconsiderations. DBS provides less invasive sampling (finger orheel prick rather than conventional venous cannula), easiertransfer and simpler storage (no need for dry ice or freezers).Furthermore, DBS provides the ethical benefits of reducedblood volume (<100 mL compared with the >1.0 mL blood that

Biomed. Chromatogr. 2013; 27: 1054–1061 Copyright © 2013 John

is usually obtained during the conventional blood samplecollection), as well as a reduced risk of HIV/AIDS or any otherkind of infection (Abbott et al., 2010; Li and Tse, 2010).

The increased use of DBS in regulated bioanalysis hasstimulated the bioanalytical community to look more closelyinto the validation requirements of DBS methods. The EBFsummarized their current thinking in recommendations on thevalidation of bioanalytical methods for dried blood spots. Therecommendations outlined are intended to provide guidancefor method validation and analysis of DBS samples. Theecommendations reflect on (potential) differences and specialconsiderations needed for method validation and sampleanalysis for DBS compared with traditional liquid assays(Timmerman et al., 2011).

Several previous reports have described the determinationof topiramate in conventional plasma and serum samples

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DBS method for determination of topiramate using LC-MS/MS

using analytical methods including HPLC with UV detection(Bahrami et al., 2005), gas chromatography (Wolf et al., 2000),fluorescence polarization immunoassay (Christensen et al.,2002), liquid chromatographic–mass spectrometry (Chen andCarvey, 1999) and liquid chromatography–tandem mass spec-trometry (LC-MS/MS; Chen and Carvey, 2001; Park et al., 2008;Matar, 2010; Tai et al., 2011). These analytical methods haveusually adopted liquid–liquid extraction procedures that involvetedious drying and reconstitution processes.

In 2008, la Marca et al. reported a rapid method for TPM assayusing DB. However, at that time there were no recommenda-tions for the validation of bioanalytical methods for DBS;therefore the method was not tested for intra- and inter-dayprecision of quality control samples, hematocrit effect, carry-over, extraction recovery, sample dilution and matrix effect.

As described in the European Medicines Agency (EMEA)guide, the measurement of drug concentrations in biologicalmatrices (such as serum, plasma, blood, urine and saliva) is animportant aspect of medicinal product development. The resultsof clinical trials, including bioequivalence studies, are used tomake critical decisions supporting the safety and efficacy of amedicinal drug substance or product. It is therefore paramountthat the applied bioanalytical methods used are well character-ized, fully validated and documented to a satisfactory standard,in order to yield reliable results (EMEA, 2011).

This paper presents a method for the quantification oftopiramate in human blood from DBS samples. The methodwas validated according to the EBF recommendations. Theresults of validation tests were evaluated according to theinternationally used acceptance criteria described by the USFood and Drug Administration (FDA, 2001) and EMEA (2011).

A fully validated DBS method in the concentration range10–2000 ng/mL followed by LC-MS/MS performed with onlya few drops of blood is therefore highly suitable for applicationin bioequivalence studies and pharmacokinetic studies involvinginfants and very young children.

Table 1. Tandem mass spectrometer main workingparameters

Ionization ESI�

Ion transition m/zTPM 338.2! 78.1

Experimental

Chemicals and reagents

Topiramate and its structurally analogous internal standard weresupplied as certified reference compounds. Topiramate was obtainedfrom the US Pharmacopeia (USA) and the internal standard (IS)topiramate-d12 was purchased from TLC PharmaChem (Canada).

All of the chemicals and reagents were of analytical grade, thesolvents being of HPLC grade. The methanol and water were obtainedfrom Merck (Germany), the acetonitrile from J.T.Baker (Netherlands),and the ammonia solution 25% from Fluka (Germany). Human wholeblood with K3-EDTA anticoagulant was supplied by Trina BioreactivesAG (Switzerland).

IS 350.3! 78.1Collision energy eVTPM 28IS 29Desolvation temperature 220�CSource temperature 120�CCapillary voltage 2.95 kVCone voltage 43.00 VDesolvation gas flow 750 L/hCone gas flow 96 L/h

TPM, Topiramate. 10

Instrumental and operating conditions

Instrumentation. A tandem triple quadrupole mass spectrometerQuattro Premier Micromass (Waters Corporation, Milford, MA, USA)equipped with a liquid chromatographic system (Waters 2795 AllianceHT system, Waters Corporation, Milford, MA, USA) was used for theLC-MS/MS analysis. The peak area ratios for the analyte vs internalstandard were generated by the MassLynx software version 4.0.

Equipment. FTATM DMPK-C DBS cards were purchased from the GEHealthcare division of Whatman (USA). All spots were punched on


cutting mats purchased from the GE Healthcare division of Whatman(USA) using a Harris Uni-Core puncher (6 mm).

HPLC-MS/MS. In order to achieve quantitative determination ofTPM in dried blood spot samples, the electrospray ionization interfaceparameters were optimized for the maximum abundance of themolecular ions. The acquisition parameters were determined by thedirect infusion of a 200 ng/mL solution of TPM and IS into the massspectrometer at a flow rate of 50 mL/min. The variable mass spectro-metric conditions were optimized when the method for determinationof topiramate in human plasma was developed (the article ‘Determina-tion of topiramate in human plasma using liquid chromatographytandem mass spectrometry’ has been accepted for publication inActa Chimica Slovenica). The main working parameters are summarizedin Table 1.

Topiramate and internal standard from human K3-EDTA whole bloodwere extracted from DBS cards. The extracted samples were injected intoa liquid chromatograph equipped with a tandem mass spectrometricdetector. Negative ions were monitored in the selected reaction moni-toring (SRM) mode. The SRM transitions at m/z 338.2! 78.1 and m/z350.3! 78.1 were selected for the quantification of TPM and IS,respectively. The mass spectra of TPM and IS are presented in Fig. 1.Quantitation was performed using weighted linear regression analysis(1/c) of the peak area ratios between the analyte and internal standard.

Chromatographic conditions. The autosampler temperature was keptat 23�C. A Gemini C18 110 Å column from Phenomenex (50� 2 mm i.d.,5 mm) preceded by a Zorbax Eclipse XDB C8 guard column from AgilentTechnologies (12.5� 2.1 mm i.d., 5 mm, Agilent) was used for theseparation. The columns were operated at ambient temperature.

The mobile phase consisted of acetonitrile and 0.1% ammoniasolution (pH 11.12) at a ratio of 75:25 (v/v). The flow rate was set to0.4 mL/min. The sample loop and injection volume were 50 mL. Theretention time was 0.93 min for analyte and IS, which yielded a totalrun time of 2 min. Figure 2 shows the representative LC-MS/MSchromatograms for TPM and IS in DBS samples.

Standard solutions, calibration curve and quality controlsamples. Stock solutions of TPM and IS (topiramate-d12) wereprepared by dissolving an appropriate amount of analyte in metha-nol. Working standard solutions for TPM were prepared from a stocksolution using acetonitrile–water (80:20, v/v) as a diluent. All thesolutions were stored at +5 (�3�C).

Calibration standards were freshly prepared on a daily basis by spikingdrug-free human K3-EDTA whole blood with appropriate TPM workingsolutions, in order to produce eight TPM concentrations (10, 20, 50,100, 500, 1000, 1500 and 2000 ng/mL). Quality control (QC) samples were

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A

B

m/z60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500

%

0

100

m/z60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500

%

0

100

O

O

O

O

SO

O

NH2

Me

MeO

O

MeMe

O

O

O

O

SO

O

NH2

CD3

CD3O

O

CD3

D3C

Figure 1. Product ion mass spectra and chemical structure of (A) topiramate (TPM) and (B) topiramate-d12.

A B

C D

min0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00

%

0

100

F2:MRM of 1 channel,ES-350.20 > 78.10

4.224e+001mrm-2912-2-4 Smooth(Mn,2x2) Slepa kri

1.220.480.02 0.91 1.801.56

min

%

0

100

F1:MRM of 1 channel,ES-338.1 > 78.14.108e+001

mrm-2912-2-4 Smooth(Mn,2x2) Slepa kri

1.880.450.230.01 1.230.83 1.44

min0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00

%

0

100


5.058e+003mrm-2912-2-5 Smooth(Mn,2x2) Slepa kri+IS IS;0.93;968.74;5005

min

%

0

100


mrm-2912-2-5 Smooth(Mn,2x2) Slepa kri+IS

1.831.070.190.53 0.71 1.46

min0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00

%

0

100


4.911e+003

mrm-2912-2-6 Smooth(Mn,2x2) S1

IS;0.93;944.83;4832

min

%

0

100



TPM;0.92;26.99;139

0.45 1.85

min0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00

%

0

100


5.258e+003


IS;0.93;986.82;5180

min

%

0

100



TPM;0.92;5865.84;30909

Figure 2. Representative LC-MS/MS chromatograms for topiramate (upper trace) and the internal standard (lower trace) in the human blood spotsamples. (A) Blank human dried blood spot (DBS) sample; (B) blank human DBS sample spiked with IS; (C) lower limit of quantitation DBS sample at10 ng/mL of TPM; (D) upper limit of quantitation DBS sample at 2000 ng/mL of TPM.

T. Vnu�cec Popov et al.

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prepared in a similar manner at three TPM concentration levels: low,medium and high (30, 250 and 1750 ng/mL). At least two replicates ofeach QC sample were analyzed together with the calibration curve andunknown samples within one sequence.

Sample preparation. Aliquots (30 mL) of calibration standards andQC samples were spotted onto FTATM DMPK-C DBS cards and allowedto dry at room temperature for at least 3 h before the circles werepunched out (Fig. 3). Before the analysis, the spotted cards were storedat room temperature in a sealed plastic bag containing dry desiccantuntil the analysis was performed.

A disk 6 mm in diameter was punched out of the centre of a DBScard and put into a clean tube. Then it was extracted by the additionof 200 mL aliquots of methanol–water (90:10, v/v) containing internalstandard (15 ng/mL), followed by vortex-mixing for approximately10 min. The tube was centrifuged for 5 min at 3000g and the superna-tant transferred to a clean tube in order to be analyzed by LC-MS/MS.

Table 2. Inter- and intra-day accuracy and precision of TPMdetermination in dried blood spot (DBS) samples

Nominalconcentration

Results and discussionMethod validation was performed according to EBF recommenda-tions (Timmerman et al., 2011) and the guidelines for bioanalyticalassays in biomatrices (FDA, 2001; EMEA, 2011). The validatedparameters were selectivity, accuracy and precision, lower limitof quantification, linearity, matrix factor, extraction recovery,dilution integrity, stability, carry-over, effect of hematocrit andinfluence of blood volume.

(ng/mL) Intra-day (N= 8) Inter-day (N= 6)

CV(%)

Accuracy(%)

CV(%)

Accuracy(%)

LLOQ 10 8.5 102.0 8.2 100.9QC1 30 9.6 104.6 4.5 95.0QC2 250 5.2 99.9 3.6 96.2QC3 1750 3.1 101.0 2.8 96.7

Specificity

The specificity of the developed method was determined byanalyzing the blank DBS samples from six different sources ofhuman blood and by comparing them with chromatograms ofDBS samples spiked with TPM and IS. No peak was found atthe retention time of TPM and IS in six of the blank DBS samples.

Figure 3. Images of (A) freshly spotted card with 30 mL of whole humanpunched out of FTATM DMPK-C cards.


Accuracy and precision

Intra-day and inter-day precision and accuracy of the method wereobtained by the analysis of the DBS QC samples’ replicates at threeconcentration levels. The precision of the method was expressed asthe coefficient of variation (CV) of the repeatable assays, andaccuracy was calculated as the ratio between the mean found andnominal TPM concentrations. Table 2 shows a summary of the indi-vidual QC data obtained during the six validation runs. As can beseen, the assay for TPM was both accurate and precise betweenthe days and within a day for each level. The inter-day precision(CV) of the assay was no more than 4.5% at the three concentrationlevels of the QC samples, and the CV for the intra-day precision wasno more than 9.6%. The intra- and inter-day accuracies of the assayranged from 95.0 to 96.7% and from 99.9 to 104.6%, respectively.

Lower limit of quantification

The sensitivity (lower limit of quantification; LLOQ) was deter-mined by whole blood spiked with TPM at the lowest calibration

blood; (B) drying of spotted cards at room temperature; and (C) spots


1057

y = 0.0028x - 0.0371R² = 0.99385.000

6.000

M/IS


1058

level (10.0 ng/mL), spotted on FTATM DMPK-C cards and analyzedindependently from the calibration curve. Satisfactory intra-dayand inter-day precision and accuracy were found at this concentra-tion. The signal-to-noise ratio at LLOQ was >10. The detailedresults are presented in Table 2.

0.000

1.000

2.000

3.000

4.000

0 500 1000 1500 2000

Pea

k ar

ea r

atio

of

TP

Nominal conc. TPM (ng/mL)

Figure 4. The response of the instrument with regard to theconcentrations of TPM in the DBS samples as an average of sixcalibration curves.

Linearity

For the validation of linearity, 30 mL of calibration standards inwhole blood ranging from 10 to 2000 ng/mL were spotted ontoFTATM DMPK-C cards, over different days. The calibration curveswere constructed from the linear regression analysis of thepeak–area ratio vs the concentration of TPM, respectively,weighted with 1/concentration. A weighted linear regressioncurve was determined to best represent the concentration/detector response relationship for TPM in dried blood spots. Thecorrelation coefficient (r) was 0.9971 or better. The precision andaccuracy of the calibration samples’ and calibration curves’parameters of TPM in the DBS samples are presented in Table 3.The average curve of the six calibration curves data is shown inFig. 4. The CV at each level was ≤6.6%, with accuracy ranging from92.8 to 105.3%.

Matrix factor

The matrix factor (MF) was calculated by comparing theresponses for TPM in the extracted blank DBS samples fromeight different sources (spiked with low and high QC standardsand IS) with the responses for TPM in the standard solution sam-ples (low and high QC standard solutions and IS solution). Themean MF of TPM was 0.124 and the mean MF for IS was 0.113.The IS normalized MF was also calculated by dividing the MFof the analyte by the MF of the IS. The mean IS-normalized MFwas 1.085 and the CV of the IS-normalized MF was 12.1%.

In the DBS samples the influence of matrix was observed.However, the internal standard compensated for this effect – aMF of the same order of magnitude was present for the internalstandard. The mean value of IS-normalized MF shows thatmatrix effect is irrelevant for the accuracy and precision of thedeveloped method.

Table 3. Precision and accuracy of calibration samples and calibr

Nominal concentration (ng

10.0 20.0 50.0 100 500 100

Calculated concentration of TPM (ng/mRun 1 10.44 19.99 49.57 96.42 511.22 95Run 2 10.20 22.90 47.69 92.58 488.20 92Run 3 10.70 21.24 51.93 95.07 453.73 88Run 4 10.63 21.55 49.41 98.74 451.49 88Run 5 10.32 20.19 48.23 106.80 456.03 95Run 6 10.91 19.03 48.58 106.78 434.19 94Mean 10.53 20.82 49.40 99.40 465.81 92SD 0.262 1.366 1.467 6.063 28.319 3CV (%) 2.5 6.6 3.0 6.1 6.1Accuracy (%) 105.3 104.1 98.8 99.4 93.2 9N 6 6 6 6 6

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Effect of hematocrit

It is necessarily to test the effect of hematocrit on the determina-tion of active analytes in the blood. Usually, if blood istaken from healthy subjects, the hematocrit level will notinterfere with the determination process. This was also con-firmed by our research when we analyzed eight DBS samplesat a low QC level (30 ng/mL) in one single run and evaluatedthem on a calibration curve prepared from another source.The results indicate that human whole blood samples withhematocrit values ranging from 34 to 45% gave acceptablequantitation results. The accuracy of each hematocrit wascalculated. Table 4 presents the accuracy and precision data ofthe hematocrit effect.

However, if blood is taken from a subject suffering fromphysiological conditions or undergoing medical treatmentthat affects the hematocrit level, it is imperative to adequatelycorrect the concentration of active analytes (Vu et al., 2011).

ation curve parameters of TPM in DBS samples

/mL) Slope Intercept Correlation, r

0 1500 2000

L)9.39 1516.35 2016.62 0.00266 0.00742 0.99987.97 1536.38 2054.07 0.00267 0.00162 0.99916.80 1525.61 2134.92 0.00286 0.00033 0.99727.37 1580.30 2080.50 0.00286 �0.00181 0.99748.58 1592.15 1987.71 0.00280 0.00115 0.99889.26 1651.30 1958.95 0.00283 0.00284 0.99718.23 1567.02 2038.803.824 51.248 64.3263.6 3.3 3.22.80 104.5 101.96 6 6

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Table 4. Hematocrit effect

Nominal concentration of TPM, 30 ng/mL

Hematocrit value

0.34 0.36 0.37 0.38 0.41 0.42 0.44 0.45 Accuracy, mean (%)

Found concentration of TPM (ng/mL)23.40 28.90 28.58 27.53 26.29 32.29 25.38 25.7226.38 29.95 29.42 29.28 25.84 28.65 30.58 30.6028.31 28.42 27.33 29.75 32.08 30.02 30.09 30.14

Mean 26.03 28.95 28.44 28.85 28.07 30.32 28.68 28.82SD 2.474 0.562 1.052 1.170 3.480 1.838 2.871 2.695CV (%) 9.5 1.9 3.7 4.1 12.4 6.1 10.0 9.3Accuracy (%) 86.8 96.5 94.80 96.2 93.6 101.1 95.6 96.1 95.1


Extraction recovery

Aliquots of 10 mL of blank whole blood samples (N=8) and 10 mLof pooled whole blood samples (N= 8) at three QC concentra-tions were spotted on FTATM DMPK-C cards and the wholespot was punched out. Each punch of the blank DBS samplewas then extracted by adding 200 mL aliquots of QC solutionsand the QC DBS samples were extracted according to their usualprocedure.

The recovery of TPM was evaluated by comparing theextracted DBS samples (mean analyte responses of eightprocessed samples at 30, 250 and 1750 ng/mL; pre-extractionspiked samples) with the reference DBS samples (mean analyteresponses of eight replicates of QC solutions at three concentra-tions that were added to the blank DBS samples; post-extractionspiked samples).

The mean recoveries of TPM at 30, 250 and 1750 ng/mL were89.0, 90.2 and 98.6%, with CV <6.6%. The results of extractionrecovery are presented in Table 5.

Dilution integrity

The dilution of DBS samples was checked at a twofold value – atthe highest concentration level of the QC samples (3500 ng/mL).

Table 5. Extraction recovery data of TPM in DBS samples

QC1

Area response Reference Extract Refe

76 72 685 78 980 78 675 75 572 81 680 84 679 82 681 69 6

Mean 79 77 6SD 4.0 5.1CV (%) 5.1 6.6N 8 8Recovery (%) 98.6


A 30 mL aliquot of whole blood was taken from a previouslyprepared pool at a concentration of 3500 ng/mL and spottedonto FTATM DMPK-C card. After drying, each 6 mm punch wasindividually extracted with 200 mL methanol–water (90:10, v/v)containing internal standard (15 ng/mL). A 100 mL aliquot ofeach DBS extract was diluted with 100 mL of the blank DBSextract. Eight replicates with dilution factor 2 were analyzed.No significant deviation in the accuracy of TPM concentrationby sample dilution was observed. The dilution accuracy was105.4%, which is evident from Table 6.

Stability

The stability of TPM in solutions, in whole blood, in dried bloodspots on FTATM DMPK-C cards, and the extracted samplesprepared for LC-MS/MS analysis was tested at two differentconcentration levels (30 and 1750 ng/mL). The mean concentra-tion at each level (Found) was compared with the nominal con-centrations (Nom). The results are listed in Table 7 and indicatethat the analyte remained considerably stable under each ofthe above-mentioned conditions.

Solution stability. The stability of TPM stock and workingsolutions as well as IS stock and working solutions was checked

QC2 QC3

rence Extract Reference Extract

49 608 4904 454782 591 4923 441268 596 5095 437789 598 4834 420632 611 4864 438785 600 5026 425986 540 4890 447091 618 5004 451860 595 4943 439735.4 24.0 89.9 118.85.4 4.0 1.8 2.78 8 8 8

90.2 89.0


1059


1060

by analyzing stability samples stored at +5� 3�C and comparingthem with the samples of freshly prepared solutions. No signifi-cant changes for stock and working solutions stabilities of TPMand IS after 208 and 212 days at +5� 3�C) were observed.

Stability in whole blood. Eight replicates of 30 mL wholeblood samples were spotted onto FTATM DMPK-C cards afterstorage at +5� 3�C for 48 h.

Stability in DBS on FTATM DMPK-C card. Eight replicates of30 mL whole blood samples were spotted on FTATM DMPK-C cardand stored desiccated at room temperature for 194 days.

Stability of sample extracts. Extracts from DBS validationwere re-injected after storage at room temperature for 48 h,and evaluated on the freshly prepared calibration standardextract.

Post-preparative and instrumentation carry-over

In order to determine the physical carry-over from one card toanother when stored together, and spot-to-spot carry-overoriginating from the punching device, the blank whole bloodwas spotted onto a place between two spots of the calibrationcurve’s highest concentration level of the 2000 ng/mL. After3 h of drying, the spotted cards were stored for 48 h at room

Table 6. Dilution integrity of DBS samples

Nominal concentration (ng/mL), 1750

Sample no. Dilution factor, 2

1 1927.692 1871.653 1739.744 1820.125 1902.586 1877.707 1841.908 1749.37Mean 1843.84SD 64.752CV (%) 3.5Dilution accuracy (%) 105.4N 8

Table 7. Stability of TPM in DBS samples

Nominal concentration (ng/mL), N= 8 Calculated conce

Stability of sample extracts at +23�C after 48 h301750Stability in whole blood 48 h at +5�C301750Stability in DBS on DMPK-C card after 194 days301750

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temperature in a sealed plastic bag containing dry desiccant.Six replicates of the blank DBS were punched out withoutpreviously cleaning the punching device that had been usedfor punching spots of the highest concentration level. For allthe carry-over blank samples, the peak area at the retention timeof TPM was <20% relative to the peak area of the lowestcalibration sample at 10 ng/mL TPM.

In order to verify that there was no carry-over in the LC-MS/MSinstrument, the solution of the mobile phase was injectedimmediately after the DBS sample extracts of the highestconcentration of the calibration curve had been injected withsix consecutive injections. There were no peaks observed atthe retention times of the TPM and IS.

Influence of blood volume

The spotting on the FTATM DMPK-C cards was done withaccurate and calibrated pipettes. Nevertheless, the spotting ofdifferent blood volumes on the cards was researched to seewhether the results obtained during the research process wouldbe comparable to the results medical staff would obtain inclinics or with patients at home.

Replicates of 15, 25 and 35 mL whole blood were spotted ontoFTATM DMPK-C cards at a concentration of 1000 ng/mL. Afterdrying, the 6 mm punches were extracted according to thedescribed procedure. The replicates were evaluated on thecalibration curve prepared with 30 mL of whole blood.

ntration (ng/mL), N= 8 CV (%) F/Nom (%)

27.36 9.2 �8.81638.42 4.3 �6.4

27.02 9.0 �9.91990.14 3.2 13.7

27.02 9.0 �9.91990.14 3.2 13.7

Table 8. Influence of blood volume

Nominal concentration of TPM, 1000 ng/mL

Spotted volume (mL)

15 25 35Found concentration of TPM (ng/mL)982.0 958.88 950.90958.94 977.30 1130.96973.45 1098.67 1079.16

Mean 971.46 1011.62 1053.67SD 11.658 75.951 92.696CV (%) 1.2 7.5 8.8F/Nom (%) �2.9 1.2 5.4

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The obtained results were all within 15% of nominal valueand are presented in Table 8. This means that blood volumehad no influence on the accuracy of TPM determination fromDBS samples.

ConclusionA simple and rapid bioanalytical assay was developed andvalidated using the DBS and LC-MS/MS techniques. Thevalidated DBS method, which complies with the regulatoryguidelines, demonstrated acceptable linearity, precision, accu-racy, selectivity, sensitivity and recovery. No significant degrada-tion of topiramate was observed in the stability DBS samplesafter the storage in a desiccated plastic bag at room tempera-ture for 194 days. This demonstrates that samples in this formcan be stored for long periods of time prior to analysis, withoutthe need for refrigeration. Successful validation confirmed thatthis method is suitable for the determination of topiramate inDBS samples in pharmacokinetic and bioequivalence studies.

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Development and validation of dried blood spots technique for quantitative determination of topiramate using liquid chromatography-tandem mass spectrometry