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Journal of Chromatography B
j ourna l ho me page: www.elsev ier .com/ locate /chromb
evelopment and validation of a simple and sensitive method foruantification of levodopa and carbidopa in rat and monkey plasmasing derivatization and UPLC–MS/MS
enkatraman Junnotula ∗, Hermes Licea-Perezioanalytical Science and Toxicokinetics, Platform Science and Technology, GlaxoSmithkline Pharmaceuticals, 709 Swedeland Road, King of Prussia,A 19406, USA
r t i c l e i n f o
rticle history:eceived 3 December 2012ccepted 4 March 2013vailable online 14 March 2013
eywords:evodopaarbidopa
a b s t r a c t
A simple, selective, and sensitive quantitative method has been developed for the simultaneous determi-nation of levodopa and carbidopa in rat and monkey plasma by protein precipitation using acetonitrilecontaining the derivatizing reagent, flourescamine. Derivatized products of levodopa and carbidopa wereseparated on a BEH C18 column (2.1 mm × 50 mm; 1.7 �m particle size) using ultra high performanceliquid chromatography (UHPLC) without any further purification. Tandem mass spectrometry (MS/MS)was used for detection. The method was validated over the concentration range of 5–5000 ng/mL and3–3000 ng/mL for levodopa and carbidopa, respectively in rat and monkey plasma. Due to the poor sta-
bility of the investigated analytes in biological matrices, a mixture of sodium metabisulfite and hydrazinedihydrochloride was used as a stabilizer. This method was successfully utilized to support pharmacoki-netic studies in both species. The results from assay validations and incurred samples re-analysis showthat the method is selective, sensitive and robust. To our knowledge, this is the first UHPLC–MS/MSbased method that utilizes derivatization with fluorescamine and provides adequate sensitivity for bothlevodopa and carbidopa with 50 �L of sample and a run time of 3.5 min.
at plasma and monkey plasma
. Introduction
Parkinson’s disease is a neurodegenerative disorder of the cen-ral nervous system and symptoms of this disease result from loss ofeurons in the substantia nigra, a region of the midbrain [1]. Loss ofhese neurons leads to a deficiency of dopamine neurotransmittern the brain that may lead to Parkinson’s disease. Symptoms of thisisease can be alleviated by oral administration of levodopa, theiological precursor to dopamine [1]. Following oral administra-ion, levodopa exhibits a significant first pass effect with substantialnzymatic decarboxylation, giving rise to dopamine. After high-ose administration, elevated levels of systemic dopamine mayause side effects such as nausea, vomiting, and cardiac arrhyth-ias [2]. To prevent these side effects and for better therapeutic
fficacy, levodopa is often administered along with carbidopa,hich is an inhibitor of the levodopa decarboxylase enzyme [3].
dministration of levodopa in combination with carbidopa helps
o control dopamine levels in appropriate manner and reduceside effects. Because of the therapeutic importance of the levodopa
and carbidopa combination, there has been a continuing interestin improving existing bioanalytical methods for the quantificationof these drugs in biological matrices. However, quantification ofthese drugs is challenging due to their poor stability, low molec-ular mass and high polarity [4]. In the literature, a few analyticalmethods based on liquid chromatographic separations with differ-ent types of detection have been reported [4–12]. One of the mostwidely used approaches is liquid chromatography with electro-chemical detection [4–9]. Although the electrochemical detectionapproach provides good sensitivity, it lacks on selectivity, simplic-ity and ease of use. The other approach used is a combination ofsolid phase extraction (SPE) and liquid chromatography with tan-dem mass spectrometry (LC–MS/MS) [11–13]. In our initial work,protein precipitation and solid phase extractions were evaluatedas sample extraction procedures. However, poor and inconsistentrecoveries were observed. This is likely related to the poor stabilityof levodopa and carbidopa during sample handling. Also, chromato-graphic retention and peak shapes on reverse phase columns werevery sensitive to the high content of organic solvents in the sam-ple extracts. To improve chromatography either sample dilution
with water or solvent evaporation was required. Sample dilutionwas limited by the sensitivity while the evaporation step resultedin considerable loss of both levodopa and carbidopa. Therefore, aderivatization procedure with fluorescamine for the determination
f these drug molecules in rat and monkey plasma was developed.he use of this simple derivatization technique allowed improve-ents in stability, retention on column, and sensitivity.
. Experimental
.1. Chemicals and reagents
Acetonitrile, methanol, isopropanol, acetone, phosphoric acid,odium metabisulfite, hydrazine dihydrochloride, fluorescamine,evodopa, and carbidopa were purchased from Sigma–Aldrich (St.ouis, MO, USA). Levodopa-[2H3] and carbidopa-[2H3] were pur-hased from TLC PharmaChem, Inc. (Vaughan, Ontario, Canada).mmonium acetate, and formic acid were purchased from EMD
Gibbstown, NJ, USA). Water from Millipore purification systemas used. Rat and monkey plasma were obtained from Biorecla-ation Inc. (East Meadow, NY, USA).
.2. Equipment
An Eppendorf 5810R centrifuge with a rotor capacity forour 96-well plates (Brinkmann Instrument, Westbury, NY, USA),nd a Mettler UMX2 balance (Hightown, NJ, USA) were used. AomTec Quadra 3 (Hamden, CT, USA) was used for liquid trans-er. Polypropylene tubes (1.4 mL), 96-well matrix tube platesnd yellow caps from Micronic B.V. (Platinastraat 51, 8211 ARelystad, Netherlands) were employed during protein precipita-ion extraction, while polypropylene tubes along with 96-welllate clear silicone w/PTFE coating CapMat from Arctic White LLCBethlehem, PA, USA) were used for sample introduction to theHPLC–MS/MS. Separations were performed on an ACQUITYTM
PLC integrated system from Waters (Milford, MA, USA), consistingf a sample manager combined with a sample organizer capablef holding ten 96-deep well plates, and a binary solvent man-ger, combined with a triple quadrupole mass spectrometer API000 (Applied Biosystems/MDS-Sciex, Concord, Ontario, Canada)or analyte detection.
.3. Preparation of calibration standards and quality control (QC)amples
Stock solutions of levodopa, carbidopa, and their correspondingnternal standards were individually prepared in water contain-ng H3PO4 (0.1% v/v) at final concentration of 1 mg/mL. All stockolutions and working solutions were stored at 4 ◦C until use. Theorking stock solution of levodopa/carbidopa at 200/120 �g/mLas prepared fresh in water containing H3PO4 (0.1% v/v). One work-
ng stock solution (from individual weigh out) was used to preparealibration standards of levodopa/carbidopa on wet ice in rat oronkey plasma in the presence of stabilizers, sodium metabisulfite
0.244% w/v) and hydrazine dihydrochloride (0.244% w/v) at 5/3,0/6, 25/15, 100/60, 300/180, 750/450, 1500/900, 2500/1500 and000/3000 ng/mL using serial dilution. The working stock solutionfrom second weigh out) was used to prepare QC samples on wetce in rat and monkey plasma in the presence of stabilizers, sodium
etabisulfite (0.244% w/v) and hydrazine dihydrochloride (0.244%/v) at 5/3, 15/9, 250/150, 4000/2400 and 5000/3000 ng/mL using
erial dilution. QC samples were divided into 0.4 mL aliquots on wetce and frozen at −80 ◦C or extracted.
.4. Sample preparation for the analysis of levodopa and
arbidopa
Rat or monkey plasma samples (50 �L) were transferred toorresponding 1.4 mL polypropylene tubes on wet ice. Then
omatogr. B 926 (2013) 47– 53
25 �L of internal standard solution of levodopa/carbidopa in ace-tonitrile at 500/250 ng/mL was spiked into tubes followed bybriefly vortex-mixing. Subsequently, samples were precipitatedwith 150 �L of acetonitrile containing derivatization reagent flu-orescamine (5 mg/mL, prepared fresh). Sample tubes were cappedand vortex-mixed thoroughly for approximately 5 min, followedby centrifuging at least for 5 min at approximately 3220 × g. Then,150 �L of supernatant was transferred into clean tubes, capped, andincubated at 37 ◦C for 60 min. Following centrifugation, the sampleswere analyzed without further purification using UHPLC–MS/MS.
2.5. Chromatographic conditions for levodopa and carbidopa
The analytical column used was a BEH C18, 2.1 mm × 50 mmwith 1.7 �m particle size, from Waters Co. The column temperaturewas maintained at 65 ◦C and the sample compartment was kept at4 ◦C. Mobile phase A consisted of 5 mM ammonium formate/formicacid at 1000/1 (v/v) and mobile phase B was acetone. The UHPLCsystem was held at 25% B for 0.2 min followed by a linear gradientfrom 25% B to 50% B in 1.8 min. The conditions were then was heldat 90% B for 0.9 min to remove late eluting substances from thecolumn, after which the system was returned to initial conditions.The total run time including the sample load was approximately3.5 min and the flow rate was maintained constant at 0.5 mL/minthroughout the run. Eluent from the column was diverted from themass spectrometer during 0–0.6 min and 2–3.5 min to maintain theinstrument as clean as possible. A typical injection volume of 5 �Lin a 10 �L loop (partial loop injection mode) was used. Injectioncarry-over was assessed with each analytical run by inclusion of ahigher limit of quantification (HLQ) sample followed by an injectionof a blank. No significant carry-over was noted in any analyticalrun.
2.6. Mass spectrometric conditions
An API-5000 with a TurboIonspray interface (TIS) was operatedin the positive ionization mode. The instrument was optimized forfluorescamine derivatized products of levodopa, [2H3]-levodopa,carbidopa, and [2H3]-carbidopa, by infusing corresponding deriva-tized solutions at 100 ng/mL in acetonitrile/water (50/50, v/v) usingflow rate at 300 �L/min through an Agilent pump 1100 series (PaloAlto, CA, USA) directly connected to the mass spectrometer. TheMRM transitions of m/z 458 → 440, m/z 461 → 442, m/z 487 → 293,and m/z 490 → 293 were chosen for levodopa, [2H3]-levodopa,carbidopa, and [2H3]-carbidopa, respectively. The optimized massspectrometric conditions for levodopa and carbidopa included thefollowing conditions: TIS source temperature, 650 ◦C; TIS voltage,5500 V; curtain gas, 40 psi (nitrogen); nebulizing gas (GS1), 60 psi(zero air); TIS gas (GS2), 80 psi (zero air); collision energy for lev-odopa and carbidopa, were 24 and 10 eV respectively; declusteringpotential for levodopa and carbidopa, were 60 and 90 eV respec-tively. However the collision energy was deoptimized for carbidopato achieve linearity across the calibration range.
2.7. Data analysis
MS data were acquired and processed (integrated) usingthe proprietary software application AnalystTM (Version 1.4.2,Applied Biosystems/MDS-Sciex, Canada). Calibration plots of ana-lyte/internal standard peak area ratio versus levodopa andcarbidopa concentrations were constructed and a weighted 1/x2
quadratic regression was used for both analytes. Concentrations of
levodopa and carbidopa in QC samples were determined from theappropriate calibration line, and used to calculate the bias and pre-cision of the method with an in-house LIMS (Study ManagementSystem, SMS2000, version 2.1, GlaxoSmithKline).
V. Junnotula, H. Licea-Perez / J. Chromatogr. B 926 (2013) 47– 53 49
ivatiz
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Fig. 1. Structures of levodopa, carbidopa and their der
. Results and discussion
.1. Challenges during method development
The objective was to develop simple and reliable assay for deter-ination of levodopa and carbidopa in rat or monkey plasma using
ow sample volume (as low as 50 �L) and to achieve at least 5 ng/mLs a lower limit of quantification (LLQ) for both of these compounds.n initial attempts, protein precipitation extraction and solid phasextraction (SPE) with hydrophobic, anionic and cationic sorbents asample clean up procedures were examined. However, the follow-ng challenges were encountered: (1) both levodopa and carbidopare not stable in rat or monkey plasma requiring the use of thetabilizers, sodium metabisulfite and hydrazine dihydrochloride,nd handling on wet ice [13] during sample preparation; (2) poornd inconsistent recoveries; (3) peak shape and retention wereery sensitive to high content of organic solvents on reverse phasehromatographic conditions and requiring either dilution of sam-les with water or solvent evaporation; (4) sensitivity was notnough after dilution; (5) both compounds were not stable duringhe evaporation step. To overcome these challenges we explorederivatization of levodopa and carbidopa with fluorescamine.
.2. Derivatization of levodopa and carbidopa with fluorescamine
Fluorescamine (see Fig. 1) is known to readily react withrimary amines in aqueous media [14–16]. In addition, reac-ions of fluorescamine with levodopa and its biologically relevant
etabolite, dopamine were well documented [17–19]. Since bothevodopa and carbidopa (see Fig. 1) have a primary amine it
as decided to explore the derivatization with fluorescamine toddress some of the challenges encountered when developing an
ssay for determination of levodopa and carbidopa in plasma. Asart of investigation, plasma samples containing levodopa andarbidopa were precipitated and derivatized in acetonitrile con-aining fluorescamine. Reaction conditions including concentration
ed products following incubation with fluorescamine.
of fluorescamine, reaction time and temperature were optimizedfor better recovery of derivatized products from plasma samples.Concentrations of fluorescamine in acetonitrile ranging from 1to 10 mg/mL were evaluated. The recovery of the fluorescaminederivatives of levodopa and carbidopa was similar at both 5 mg/mLand 10 mg/mL and therefore, 5 mg/mL was selected to maintainsamples as clean as possible. It is important to note that bothlevodopa and carbidopa readily react with fluorescamine and gen-erate intermediate products with characteristic precursor [M+H]+
ions 476 for levodopa and 505 for carbidopa. These intermediateproducts undergo dehydration to generate relatively more stablefluorescamine derivatives (see Fig. 1) with characteristic precur-sor [M+H]+ ions 458 for levodopa and 487 for carbidopa. Therefore,as part of accelerating dehydration process, incubation times ran-ging from 0 min to 60 min and temperatures of 37 ◦C and 60 ◦Cwere investigated. Although an incubation temperature of 60 ◦Cdecrease the dehydration time for derivatized levodopa it wasnoticed that derivatized carbidopa was not stable at this tem-perature. Therefore, the dehydration was monitored at 37 ◦C atdifferent incubation times. An increase in sensitivity was observedfor derivatized levodopa with increasing incubation time up to60 min at 37 ◦C. However, there was no significant change in sensi-tivity of derivatized carbidopa with incubation. Therefore, for thisassay, the incubation time and temperature were kept at 60 minand 37 ◦C, respectively.
In order to clean up the investigated derivatives solid phaseand liquid–liquid extraction procedures were evaluated. How-ever, these derivatives were poorly recovered when using theseclean up steps. Therefore, it was decided to inject the samples onUHPLC–MS/MS without further purification. A diverting valve wasused to divert part of the eluents away from the mass spectrometerto keep instrument as clean as possible.
Derivatization of levodopa and carbidopa with fluorescaminewas shown to improve the sensitivity, chromatographic peak shapeand retention. In addition, derivatization facilitated injection ofsamples after protein precipitation containing high organic content
50 V. Junnotula, H. Licea-Perez / J. Chromatogr. B 926 (2013) 47– 53In
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ig. 2. Representative UHPLC–MS/MS chromatograms of (A) double blank, (B) lowerimit of quantification sample, (C) higher limit of quantification sample, and (D)nternal standard for levodopa.
f precipitation solvent, acetonitrile (∼78% v/v) without affectinghe peak shape. Therefore, sample dilution with water or dry downtep was not required for acceptable peak shape.
.3. Selectivity and linearity
The selectivity of the method was assessed by monitoring char-cteristic precursor [M+H]+ to product ion transitions, 458–440,87–293, 461–442, and 490–293, which are consistent with thetructures of fluorescamine derivatives of levodopa, carbidopa,2H3]-levodopa, and [2H3]-carbidopa, respectively (see Fig. 1).hese transitions were used as multiple reaction monitoring tran-itions to ensure high selectivity. The selectivity of the method wasstablished by the analysis of double blank (with no internal stan-ards) samples of control rat plasma in the presence of stabilizers,odium metabisulfite (0.244% w/v) and hydrazine dihydrochloride0.244% w/v) from 6 individual lots from bioreclamation (West-ury, NY, USA). The selectivity of the method was also assessed byhe inclusion of blank (with internal standard) and double blankwith no internal standard) samples prepared from pooled controlat plasma in the presence of stabilizers in this validation. Repre-entative chromatograms of a double blank sample, quality controlample at the LLQ, quality control sample at the HLQ, and inter-al standards for derivatized levodopa and carbidopa are shown
n Figs. 2 and 3 respectively. No unacceptable interferences at theetention times of levodopa or carbidopa or their internal standardsere observed.
The linearity of the method was assessed using linear regressionith both 1/x2 and 1/x. However, it was observed that responses for
evodopa and carbidopa derivatives were not linear for the 1000-old assay range. Therefore, quadratic regression (y = ax2 + bx + c
Fig. 3. Representative UHPLC–MS/MS chromatograms of (A) double blank, (B) lowerlimit of quantification sample, (C) limit of quantification sample, and (D) internalstandard for carbidopa.
with 1/x2 weighing, where “x” is the analyte concentration and “y” isthe peak area ratio of the analyte to the internal standard) was usedfor both levodopa and carbidopa in the assay validation. The meanconcentrations and inter-assay variation for calibration standardsof levodopa and carbidopa are presented in Table 1. The calibra-tion curve parameters for levodopa and carbidopa are presented inTable 2. The correlation coefficients obtained using 1/x2 weightedquadratic regressions were better than 0.9965 for levodopa and0.9990 for carbidopa.
3.4. Bias and precision
The bias and precision for levodopa and carbidopa were assessedin three core validation runs (see Table 3). The maximum bias(%) observed for levodopa and carbidopa were −14.2% and 13.4%,respectively. The maximum within- and between-run precision(%) values observed for levodopa were 13.6% and 4.5%, respec-tively. The maximum within- and between-run precision (%) valuesobserved for carbidopa were 7.2% and 6.5%, respectively. As definedby the lower and upper validation sample concentrations possess-ing acceptable accuracy and precision, the validated range of thismethod for levodopa and carbidopa from 50 �L of rat plasma was5–5000 ng/mL and 3–3000 ng/mL respectively.
3.5. Stability of levodopa and carbidopa in stock solutions
The stability of analytical stock solutions of levodopa and car-
bidopa was assessed in water with H3PO4 (0.1% v/v) at 1 mg/mLstored at 4 ◦C for 31 and 20 days respectively and at ambienttemperature for 6 h by diluting stock solutions to an appropriateconcentration and analysing by UHPLC–MS/MS. The mean analyte
V. Junnotula, H. Licea-Perez / J. Chromatogr. B 926 (2013) 47– 53 51
Table 1Inter-assay variation for calibration standards of levodopa and carbidopa.
o internal standard peak area ratio of a stock solution after storageas compared to that of a freshly prepared solution. The differencesere less than 5%, and indicate that levodopa and carbidopa are sta-
le in analytical solutions of water with H3PO4 (0.1% v/v) stored at◦C for 31 and 20 days, respectively, and at ambient temperature
or at least 6 h.
.6. Stability of levodopa and carbidopa in blood
The stability of levodopa and carbidopa in rat whole blood sam-les in the absence of stabilizers stored on wet ice was assessed by
able 3ean, bias and precision for quality control samples of levodopa and carbidopa.
Levodopa
Concentration (ng/mL) 5 15 250 4000
Run1, n = 6Mean 4.81 15.20 266.24 4169.87
Precision (%CV) 8.0 5.0 2.6 1.8
Bias (%) −3.9 1.4 6.5 4.2
Run2, n = 6Mean 4.56 15.85 253.21 4036.45
Precision (%CV) 13.6 8.1 1.8 2.7
Bias (%) −8.8 5.7 1.3 0.9
Run3, n = 6Mean 4.29 15.16 254.67 4074.87
Precision (%CV) 12.0 4.5 1.3 1.9
Bias (%) −14.2 1.0 1.9 1.9
Overall totals, n = 18Mean 4.55 15.40 258.04 4093.73
Precision (%CV) 11.6 6.2 3.0 2.5
Bias (%) −9.0 2.7 3.2 2.3
Between-run precision (%) 3.3 0.2 2.6 1.4
3.14E−04 0.99901.43E−03 0.99946.51E−04 0.9992
comparing the mean analyte to internal standard peak area ratio ofsamples extracted after storage for 60 min for both levodopa andcarbidopa against those of the samples extracted immediately uponspiking. The maximum bias (%) observed for levodopa was less than3.5%, and indicates that levodopa is stable in rat whole blood storedon wet ice at least for 60 min. However, carbidopa was not stablefor 60 min and the maximum bias (%) observed was greater than
−36.8%. Therefore, stability of carbidopa in rat blood was assessedfor 20 min on wet ice. Under these conditions the maximum bias(%) observed for carbidopa was less than −5.8%, and indicates thatcarbidopa is stable in rat whole blood stored on wet ice at least for
52 V. Junnotula, H. Licea-Perez / J. Chromatogr. B 926 (2013) 47– 53
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ig. 4. Individual plasma concentration time profiles of levodopa following a singleral combination administration of carbidopa/levodopa at 15/60 mg/kg to male andemale rats.
0 min. Similar results were observed for the stability of levodopand carbidopa in monkey whole blood. This indicates that bloodamples must be processed to plasma samples within 20 min afterample collection followed by treatment with the stabilizer.
.7. Stability of levodopa and carbidopa in plasma
The stability of levodopa and carbidopa in fortified rat plasmaamples in the presence of stabilizers and stored on wet ice wasssessed at 15 and 4000 ng/mL for levodopa and 9 and 2400 ng/mL
or carbidopa (in replicates of 6) by comparing the mean concentra-ions of samples extracted after storage for 24 h on wet ice againsthe nominal concentrations. The maximum bias (%) observed forevodopa and carbidopa was less than 4.1% and −10.0% respectively.
Mean
0 100 0 200 0
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Fig. 6. Representative incurred graph for lev
Fig. 5. Individual plasma concentration time profiles of carbidopa following a singleoral combination administration of carbidopa/levodopa at 15/60 mg/kg to male andfemale rats.
Data indicates that both levodopa and carbidopa are stable in ratplasma in the presence of stabilizers and stored on wet ice at leastfor 24 h.
3.8. Stability of levodopa and carbidopa in plasma during freezethaw cycles
The stability of levodopa and carbidopa in spiked rat plasmasamples in the presence of stabilizers after 3 freeze–thaw cyclesfrom −80 ◦C to storage on wet ice conditions was assessed at 15
and 4000 for levodopa, and 9 and 2400 ng/mL for carbidopa (inreplicates of 6) by comparing the mean concentrations against thenominal concentrations. The maximum bias (%) observed for lev-odopa and carbidopa was less than 14.5% and 3.1% respectively.
Result
3000 400 0 500 0
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Carbidopa
odopa and carbidopa from rat plasma.
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V. Junnotula, H. Licea-Perez /
he results indicate that levodopa and carbidopa are stable in ratlasma after at least 3 freeze–thaw cycles from −80 ◦C to wet iceondition.
.9. Matrix dilution
The ability to dilute samples containing levodopa and carbidopat concentrations above the HLQ was demonstrated by performing
replicate 10-fold dilutions of rat plasma samples in the presence oftabilizers at 25,000 ng/mL for levodopa and 15,000 ng/mL for car-idopa. Concentrations of levodopa and carbidopa in these matrixilution samples were determined and corrected for the dilutionactor. The bias (%) and precision (%) values observed for levodopaere −0.8 and 3.3, respectively. The bias (%) and precision (%) val-es observed for carbidopa were −0.4 and 1.8, respectively. Data
ndicating that a 10-fold dilution of rat plasma samples containingevodopa and carbidopa above the HLQ is valid.
.10. Stability in processed samples
The stability of derivatized levodopa and carbidopa in processedamples derived from 50 �L of rat plasma in the presence of stabi-izers was assessed by re-injecting a validation run after storage at◦C temperature for 72 h. The maximum bias (%) observed for lev-dopa and carbidopa was less than 8.9% and 1.7%, respectively. Theccuracy, precision and sensitivity of these samples were foundo be acceptable on re-injection, indicating that the processedamples were stable when stored at 4 ◦C temperature for at least2 h.
.11. Application of validated method to a pharmacokinetic study
After successful validation, this method was used to supportharmacokinetic study where the levels of levodopa and carbidopa
n rat plasma were monitored. As part of pharmacokinetic study,evodopa and carbidopa were administered orally to both malend female rats. Data reveal that levodopa and carbidopa con-entrations were quantifiable up to at least 6 h after dosing (seeigs. 4 and 5). The maximum plasma concentrations of levodopaere observed between 0.25 and 1 h post dose and the maxi-um plasma concentrations of carbidopa were observed at 1 h
ost dose. There were no marked (>2-fold) differences in systemicxposure between male and female rats for both carbidopa andevodopa.
.12. Incurred sample reproducibility
Reproducibility and ruggedness of the method was evaluatedy performing incurred sample reproducibility (ISR) analysis. Asart of this analysis, 21 samples from pharmacokinetic study wereeanalyzed after approval of the original assay results. All of thencurred sample results were within the limits of ±20% of the
ean of the reanalysis result and its corresponding original resultor levodopa (see Fig. 6). Only two of the incurred sample resultsere outside the limits of ±20% of the mean of the reanaly-
is result and its corresponding original result for carbidopa (see
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omatogr. B 926 (2013) 47– 53 53
Fig. 6). The results confirmed reproducibility and ruggedness of themethod.
3.13. Transfer of method to monkey plasma
This method was successfully transferred and validated inmonkey plasma. Validation data for monkey plasma (data notshown) demonstrates that method is reliable and reproducible. Thevalidated method for monkey plasma was also used for pharma-cokinetic analysis. Incurred data from the pharmacokinetic studyconfirm the ruggedness of the method.
4. Conclusion
A simple and sensitive method was developed for the quantifi-cation of levodopa and carbidopa in rat and monkey plasma usinga derivatization with fluorescamine. This method facilitates lowsample volumes (50 �L), good mass spectral ionization, and col-umn retention. The method was validated in both rat and monkeyplasma. Validation data shows that method is selective, sensitiveand robust. Further this method provides wider dynamic range(1000-fold) for both levodopa and carbidopa with quadratic regres-sion (1/x2).
Acknowledgements
The authors would like to acknowledge Christopher Evans, Yan-wen Qian, Gordon Dear and Eric Yang from the Department of DrugMetabolism and Pharmacokinetics at GlaxoSmithKline for theirassistance with this project.
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