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228 ZAHRA SAFAEI et al.the determination of fluconazole with good accuracyat low drug concentrations in plasma using single-stepextraction procedure. We also demonstratethe applicability of this method for pharmacokineticstudies in humans.MATERIALS AND METHODSChemicalsFluconazole and methocarbamol (internal standard)were supplied by Pars-Daru Pharmaceuticals(Tehran, Iran). Fluconazole is available as oral capsulescontaining 150 mg of fluconazole and otherinactive ingredients. HPLC-grade acetonitrile andall other chemicals were obtained from Merck(Darmstadt, Germany). Water was obtained by doubledistillation process and purified additionallywith a Milli-Q system (Millipore, Bedford, MA,USA).Instruments and chromatographic conditionsThe chromatographic apparatus consists of amodel Wellchrom K-1001 pump, a model Rheodyne7125 injector and a model K 2501 UV detector connectedto a model Eurochrom 2000 integrator, allfrom Knauer (Berlin, Germany). The separation wasperformed on MZ C8 (125 ◊ 4.0 mm, 5 µm) columnfrom Merck (Darmstadt, Germany). The wavelengthwas set at 261 nm. The mobile phase consisted ofacetonitrile ñ potassium dihydrogen phosphatebuffer (15 : 85, v/v), pH 3.0, at a flow rate of 1.5mL/min. The mobile phase was prepared daily anddegassed by ultrasonication before use.Standard solutionsStock solution (5 mg/mL) of fluconazole andmethocarbamol (10 mg/mL) were prepared inmethanol. The working standard solutions wereobtained by dilution with methanol.Sample preparationTo 450 mL of plasma in a glass-stoppered 15mL centrifuge tube were added 50 mL of methocarbamolas internal standard (10 mg/mL), 500 mL ofacetonitrile and 100 mg NaCl. After mixing (30 s),the mixture was centrifuged for 15 min at 8000 rpm.The organic phase was separated and dried underargon. Then, 50 mL of mobile phase was added and40 mL of supernatant was injected into liquid chromatograph.Biological samplesTwelve male healthy volunteers were includedin this study. The study protocol was approved bythe Ethics Committee of Shahid Beheshti Universityof Medical Sciences and written informed consentwas obtained from the volunteers. Fluconazole wasadministered in a single dose of 150 mg to the volunteersafter overnight fasting. Plasma samples werecollected at several intervals after dosing and thenfrozen immediately at ñ20 O C until assayed.StabilityThe stability of fluconazole was assessed forspiked plasma samples stored at ñ20 O C for up to twomonths and at ambient temperature for at least 24 h.The stability of stock solutions stored at ñ20 O C wasdetermined for up to one month by injecting appropriatedilutions of stock solution in distilled water onday 1, 15, and 30 and comparing their peak areaswith fresh stock solution prepared on the day ofanalysis. Samples were considered to be stable, ifthe assay values were within the acceptable limits ofaccuracy and precision.Plasma calibration curves and quantitationBlank plasma was prepared from heparinizedwhole blood samples collected from healthy volunteersand stored at ñ20 O C. After thawing, 50 µL offluconazole working standards were added to yieldfinal concentrations of 0.1, 0.25, 0.5, 1, 2, 3, and 4µg/mL. Internal standard solution was added to eachof these samples and the samples were then preparedfor analysis as described above. Calibration curvewas constructed by plotting peak area ratio (y) offluconazole to the internal standard versus fluconazoleconcentrations (x). A linear regression was usedfor quantitation.PrecisionThe precision of an analytical methoddescribes the closeness of individual measures offluconazole when the analytical procedure is appliedrepeatedly to multiple aliquots of a single homogeneousvolume of the biological matrix. The precisionis reflected by the standard deviation, while relativeprecision is calculated as the coefficient ofvariation (CV), i.e., relative standard deviation (%RSD). Calculations of precision are largely independentof the number of replicates, except thatmore replicates may give a better estimate of thepopulation variance. The measure RSD can beexplaining by: repeatability (intra-day precision)and reproducibility (between laboratories precision).Intra-day precision was determined by repeatabilityof the method with obtained data on the same dayunder similar conditions from the precision experimentsfor five multiple injections at three different
Determination of fluconazole in human plasma by reverse phase... 229dilutions (0.8, 1.6, and 3.2 µg/mL). The concentrationof the sample is then obtained by measuring thepeak area and the coefficients of variation were calculated,being within the acceptable criteria of lessthan 0.1 (Table 1).Inter-day precision was obtained from reproducibilityof the method with obtained data byrepeating the assay five times (n = 5) on five differentdays, by injecting standard fluconazole at threedifferent dilutions (0.8, 1.6, and 3.2 µg/mL). Thecoefficients of variation value were found to be lessthan 0.1 (Table 2).AccuracyAccuracy is the degree of agreement of a measuredvalue with an accepted reference value. Theaccuracy of the method is determined by replicateanalysis of samples containing known amounts ofthe fluconazole at three different concentrationsequivalent to (0.8, 1.6, and 3.2 µg/mL) of the activepharmaceutical ingredient, by adding a knownamount of fluconazole standard, and calculating therecovery of fluconazole with recovery and coefficientsof variation for each concentration (Table 3).Limit of quantification (LOQ) and recoveryThe limit of detection (LOD) expresses thelowest concentration of analyte that can be detectedfor a given type of sample, instrument, and method,but not necessarily quantified under the statedexperimental conditions. The limit of quantitation(LOQ) is the lowest concentration of analyte in asample that can be determined with acceptable accuracy(70 ñ 120%) and precision (< 20%). LOD andLOQ were estimated using the following formulas:LOD = 3.3σ/S and LOQ = 10σ/Swhere σ represents the standard deviation of theresponse (estimated based on the calibration curve),and S is the slope of the calibration curve. The LODand LOQ values were 20 ng/mL and 61 ng/mL,Table 1. Results of precision study (intra-day) for the fluconazole analysis in plasma (n = 5).SampleConcentrationInjection no.(µg/mL) 1 2 3 4 5Mean SD CV0.8 0.60 0.69 0.66 0.63 0.67 0.65 0.03 0.05Fluconazole 1.6 1.40 1.44 1.41 1.37 1.19 1.36 0.10 0.073.2 3.22 2.44 2.9043 2.83 2.77 2.83 0.28 0.10Table 2. Results of precision study (inter-day) for the fluconazole analysis in plasma (n = 5).SampleConcentrationInjection no.(µg/mL) 1 2 3 4 5Mean SD CV0.8 0.60 0.65 0.63 0.61 0.63 0.63 0.02 0.03Fluconazole 1.6 1.40 1.20 1.42 1.17 1.37 1.31 0.11 0.093.2 3.22 2.58 2.81 2.51 2.83 2.79 0.28 0.10Table 3. Results of recovery study for the fluconazole analysis in plasma (n = 3).SampleConcentrationInjection no.(µg/mL) 1 2 3Mean STD0.8 106.75 107.92 108.36 107.68 0.83Fluconazole 1.6 110.06 109.46 111.62 110.38 1.113.2 112.21 109.73 110.56 110.83 1.26
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290 TOMASZ KOSMALSKI et al.the lite
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Synthesis and in vitro antiprolifer
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308 ASHRAF M. MOHAMED et al.Cevipab
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312 ASHRAF M. MOHAMED et al.Table 2
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314 ASHRAF M. MOHAMED et al.Table 3
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328 MANSURAH A. ABDULAZEEZ et al.34
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330 SAMINA AFZAL et al.is used as a
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332 SAMINA AFZAL et al.Detection of
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334 SAMINA AFZAL et al.CONCLUSIONAm
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336 BEATA PASKER et al.inhibitory o
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338 BEATA PASKER et al.Figure 2. Ef
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340 BEATA PASKER et al.In conclusio
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342 NASER VAHED DEHKORDI et al.EXPE
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344 NASER VAHED DEHKORDI et al.Anti
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358 OLADAPO A. ADETUNJI et al.In th
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378 SHENG LI et al.the developed an
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380 SHENG LI et al.Fisherís exact
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406 ANNA CWYNAR et al.Consequently,
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