Received: 9 June 2014, Revised: 7 August 2014, Accepted: 11 August 2014 Published online in Wiley Online Library: 10 September 2014
(wileyonlinelibrary.com) DOI 10.1002/bio.2764
Simultaneous determination of desloratadineand montelukast sodium using second-derivative synchronous fluorescencespectrometry enhanced by an organizedmedium with applications to tablets andhuman plasmaF. A. Ibrahim,a N. El-Enany,a R. N. El-Shahenya,b* and I. E. Mikhaila
* Correspondence to: Rania Nabih El-Shaheny, Department of AnalyticalChemistry, Faculty of Pharmacy, Mansoura University, 35516 Mansoura,Egypt. E-mail: [email protected]
a Department of Analytical Chemistry, Mansoura University, 35516Mansoura, Egypt
b Department of Pharmaceutical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan
48
IntroductionDesloratadine (DSL), 8-chloro-6,11-dihydro-11-(4-piperidylidene)-5H-benzo[5,6]cyclohepta[1,2-b]pyridine (Fig. 1a), is a non-sedatingantihistamine used in the symptomatic relief of allergic conditionsincluding rhinitis and urticaria (1). Montelukast sodium (MKT),sodium 1-[({(R)-m-[(E)-2-(7-chloro-2-quinolyl)-vinyl]-α-[o-(1-hydroxy-1methylethyl)phenethyl]benzyl}thio)methyl] cyclopropane ace-tate (Fig. 1b), is a selective leukotriene receptor antagonist. It isused in the management of chronic asthma, allergic rhinitis andas prophylaxis for exercise-induced asthma (1). DSL/MKT combina-tion preventive therapy has been demonstrated to be superior toeither drug administered alone in reducing both early and lateasthmatic responses (2,3). Hence, the two drugs have beenrecently co-formulated as combined tablets in a 1: 2 DSL: MKTmedicinal ratio (Telekast D tablets, product of Lupin LaboratoriesLtd, Mumbai, India andMondeslor tablets, product of Sun Pharma-ceutical Industries, Ltd, Mumbai, India) (4–6).
Some analytical methods have been reported for the simulta-neous determination of the two drugs in their co-formulatedtablets, for example, HPLC (4–6), HPTLC (7) and spectrophotom-etry (8–13). However, these methods (4–13) exhibit poor sensi-tivity, in addition to the elaborate treatments and expensivesolvents needed for HPLC procedures (4–6). Moreover, the use
of organic solvents has a harmful impact on the environmentand the analyst. This encouraged us to develop a new simple,sensitive, selective, rapid and green spectrofluorimetric methodfor the simultaneous determination of the two drugs.In fluorimetric methods, high sensitivity and selectivity are
generally expected. However, problems of selectivity can occurin multi-component analysis because of the overlap of thebroadband spectra. Synchronous fluorescence spectroscopy(SFS) has been found to have several advantages, such as simplespectra, high selectivity and low interference (14). Because of itssharp, narrow spectrum, SFS serves as a simple and effectivemethodology for obtaining data for quantitative determinationin a single measurement. SFS techniques are classified according
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Figure 1. Structural formula of (a) desloratadine and (b) montelukast sodium.
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to different scanning modes of monochromators into constantwavelength, variety angle and constant energy (14). At present,the constant wavelength method, in which a constant differencebetween the emission and excitation wavelengths (Δλ) is main-tained, is used most extensively in pharmaceutical analysis. It hasattracted the attention of many researchers and has beenapplied for the determination of several combined drugs (15,16).
When conventional spectra are strongly overlapped, the SFStechnique reduces the extent of overlapping but does not allowthe mixture to be adequately resolved. However, complete reso-lution is still possible if the derivative synchronous spectrum isrecorded. The main advantages of derivative SFS are selectivity,low cost, simplicity and rapidness (17). The combination of SFSand derivatives is more advantageous than the use of theconventional emission spectrum in terms of sensitivity, becausethe amplitude of the derivative signal is inversely proportional tothe bandwidth of the original spectrum (18). Recently, derivativesynchronous fluorimetry has been utilized for the determinationof different drugs in their dosage forms and biological fluids(19–23).
Both DSL and MKT were reported to exhibit native fluores-cence that was remarkably enhanced in the presence of orga-nized media (24,25). Because of the strong overlapping of theiremission spectra, it was difficult to determine both drugs simul-taneously by conventional spectrofluorimetry. The aim of thepresent study is to develop and validate a sensitive, selective,rapid, economic and simple method for the simultaneous deter-mination of DSL and MKT based on the second-derivative oftheir synchronous spectra. The synchronous fluorescencecharacteristics of DSL and MKT were investigated in different or-ganized media to achieve the best conditions for highest sensi-tivities. Based on the enhancement effect of carboxy methylcellulose sodium (CMC) on synchronous fluorescence intensity(SFI) of both drugs, a sensitive and selective second-derivativesynchronous fluorescence spectroscopy method was developedfor the simultaneous determination of DSL and MKT withoutprior separation steps. To the best of our knowledge, to dateno fluorescence-based method has been reported for theanalysis of DSL and MKT in their binary mixture evidencingnovelty of the developed method.
Experimental
Instruments
An LS 45 luminescence spectrometer (Perkin-Elmer, Beaconsfield,UK) equipped with a 150W xenon arc lamp and grating excitationand emission monochromators was used. Slit widths for bothmonochromators were set at 10nm with a scan rate of600 nm/min. A 1 cm quartz cell was used. Derivative spectra wereevaluated using Fluorescence Data Manager (FLDM) software(Perkin-Elmer). An NV P-901 digital pH meter (Consort, Turnhout,
Belgium) was used to adjust the pH of the buffer solutions. ASigma Centrifuge (Sigma, laborzentrifugen GmbH, Ostrode,Germany) and a VM-300P vortex mixer (Gemmy Industrial Corp.,Taiwan) were used.
Materials
Pure samples of DSL and MKT were kindly provided by Schering-Plough (Kenilworth, NJ, USA) and Merck research laboratory(Boston, MA, USA), respectively. Laboratory-prepared tablets con-taining DSL and MKT in the pharmaceutical ratio of 1: 2, respec-tively, were prepared by thoroughly mixing 5mg DSL+10mgMKT+20mg talc powder + 15mg maize starch+15mg lactose+10mg magnesium stearate/tablet. Singulair® tablets (Batch #A23001/S) and Aerius® tablets (Batch # 2STBABQAO1) were gener-ously provided as gifts fromMerck Co. (Cairo, Egypt) and Schering-Plough Co. (Brussels, Belgium), respectively. Plasma samples wereobtained from Mansoura University Hospital blood bank and keptfrozen at –20 °C until use after gentle thawing.
Chemicals and reagents
All chemicals were of analytical reagent grade and solvents wereof spectroscopic grade. Distilled water was used throughout thestudy. CMC (prepared as a 0.1% w/v solution), disodium hydro-gen phosphate, citric acid, boric acid, sodium hydroxide andmethanol were purchased from ADWIC Co. (Cairo, Egypt). Inac-tive ingredients used in the preparation of tablets (talc powder,maize starch, lactose and magnesium stearate) were also ob-tained from ADWIC Co. McIlvaine’s buffer solutions coveringthe pH range of 2.2–6.5 were prepared by mixing appropriatevolumes of 0.1M citric acid with 0.2M disodium hydrogenphosphate (26). Borate buffer solutions covering the pH rangeof 8.5–10.0 were prepared by mixing appropriate volumes of0.2M boric acid and 0.2M sodium hydroxide.
Standard solutions
Standard solutions of DSL (100μg/mL) and MKT (100μg/mL)were prepared in methanol. MKT stock solution, calibration stan-dards and quality control samples were protected from light bywrapping in aluminum foil.
General procedures
Construction of the calibration graphs. Accurately measured aliquotsof DSL and MKT standard solutions covering working concentra-tions of 0.10–2.00 and 0.20–2.00μg/mL for DSL and MKT, respec-tively, were transferred into a series of 10mL volumetric flasks. A0.3mL aliquot of McIlvaine’s buffer (pH2.3) was added along with0.4mL of 0.1% CMC. The solutions were made up to the volumewith distilled water and mixed well. The synchronous fluorescencespectra of the solutions were recorded by scanning both mono-chromators at Δλ =160nm and a scan rate of 600nm/min using10nm excitation and emission windows. A blank experiment wasperformed simultaneously. The second-derivative synchronousfluorescence spectra (SDSFS) of DSL and MKT were derived fromthe normal synchronous fluorescence spectra using FLDM soft-ware. For best sensitivity and smoothing, 40 points were used forderiving the SDSFS. The peak amplitudes of the SDSFS were esti-mated at 288 and 385nm for DSL and MKT, respectively. The peakamplitudes of the SDSFS were plotted vs. final concentration of the
Luminescence 2015; 30: 485–494Wiley & Sons, Ltd.
Flu
ores
cenc
e in
tens
ity
Wavelength (nm)
Figure 2. Normal fluorescence spectra of DSL and MKT. (A, A′) Excitation andemission spectra of DSL (1.00 μg/mL). (B, B′) Excitation and emission spectra ofMKT (2.00 μg/mL).
Sync
hron
ous
fluo
resc
ence
inte
nsit
y
Wavelength (nm)
Figure 3. Synchronous fluorescence spectra of DSL at 285 nm in the presence ofMKT: (A1–5) 2.00, 1.00, 0.80, 0.30 and 0.10μg/mL DSL; and (B) 2.00 μg/mL MKT.
Determination of desloratadine/montelukast by synchronous fluorimetry
48
drug (μg/mL) to obtain the calibration graphs. Alternatively, thecorresponding regression equations were derived.
Analysis of Laboratory-preparedMixtures of DSL andMKT. Aliquotsof DSL and MKT standard solutions in the pharmaceutical ratio of1: 2 (as in co-formulated tablets) were transferred together into aseries of 10mL volumetric flasks. A 0.3mL aliquot of McIlvaine’sbuffer (pH2.3) and 0.4mL of CMC were added. The solutions weremade up to the volume with distilled water and mixed well. Then,the procedure described under ‘Construction of the calibrationgraphs’ was followed. Percentage recoveries of each drug werecalculated using the corresponding regression equation.
Analysis of tablets. Laboratory-prepared tablets containing DSLand MKT in a pharmaceutical ratio of 1: 2 were prepared. A quan-tity of the powdered tablets equivalent to 5.0mg DSL and 10.0mgMKT was transferred into a 100mL volumetric flask and the vol-ume was made up to the mark with methanol. For commerciallyavailable single-ingredient tablet formulations (Singulair® andAerius® tablets), 10 tablets were accurately weighed and finely pul-verized. A quantity of the powdered tablets equivalent to 10.0mgMKT and 5.0mg DSL was transferred into a 100mL volumetricflask and the volume was made up to the mark with methanol.The contents of the flasks were sonicated for 30min then filtered.Different volumes of the tablet extracts were accurately trans-ferred into a series of 10mL volumetric flasks. The proceduredescribed under ‘Construction of the calibration graphs’ wasfollowed. The nominal contents of tablets were calculated usingthe corresponding regression equations.
Determination of MKT in spiked human plasma. One milliliter ofhuman plasma was transferred into a series of small centrifugationtubes and spiked with increasing concentrations of standard MKTsolution (final concentration from 0.30 to 0.60μg/mL), then 5.0mLof acetonitrile was added to denature the plasma proteins. Thesamples were vortexed for 1min, then centrifuged for 15min at3000 rpm. The supernatants were carefully aspirated into smallbeakers and evaporated to dryness at room temperature. Theresidueswere reconstitutedwith 1.0mL ofmethanol and transferredinto a series of 10mL volumetric flasks. McIlvaine’s buffer (0.3mL)and 0.1% CMC (0.4mL) were added, the solutions were diluted tothe volume with distilled water and mixed well. A blank plasma ex-periment was performed simultaneously. The procedure describedunder ‘Construction of the calibration graphs’was applied. The peakamplitudes of the SDSFS of MKT were plotted vs. final concentra-tions of the drug (μg/mL) to obtain the calibration graph. Alterna-tively, the corresponding regression equation was derived.
Results and discussion
Fluorescence characteristics of desloratadine andmontelukast sodium
Both DSL and MKT were reported to exhibit native fluorescencethat is intensified significantly in the presence of organized media(24,25). DSL was determined by conventional spectrofluorimetryin acetate buffer of pH 4.5 in the presence of SDS at 290/438nm(24). Meanwhile, MKT was determined via inclusion complexformation with heptakis-(2,6-di-O-methyl)-β-cyclodextrin at281/395nm (25). Studying the influence of various organizedmedia on the fluorescence spectral properties of DSL and MKTrevealed that CMC exhibits best enhancement effect for both com-pounds. The excitation and emission spectra of DSL and MKT in aCMC–McIlvaine’s buffer (pH2.3) system are shown in Fig. 2. The
optimum excitation/emission wavelengths were 285/450 nm forDSL and 350/505 nm for MKT. It was clear that the spectra of DSLand MKT overlapped greatly. Therefore, the simultaneous analysisof DSL and MKT in mixtures by conventional spectrofluorimetry isnot feasible.
Synchronous fluorescence spectroscopy of desloratadineand montelukast sodium
The SFS technique allows the stronger peaks to be increasedselectively by using a suitable Δλ. Consequently, compared withconventional fluorescence, SFS can provide higher selectivity foridentification and estimation, which is essential for the simulta-neous determination of multi-component systems without pre-vious physical separation (17). First, it was necessary to recordthe normal synchronous fluorescence spectra for both DSL andMKT. Figure 3 illustrates the SFS of different concentrations ofDSL at 285 nm in the presence of MKT and Fig. 4 the SFS of dif-ferent concentrations of MKT at 340 nm in the presence of DSL.Overlapping of the synchronous fluorescence spectra of bothdrugs led us to derivation of them for simultaneous determina-tion of both compounds in the presence of each other withoutprior extraction or physical separation steps. The fluorescencespectra of the two compounds were resolved entirely byadopting SDSFS. Figure 5 shows that DSL can be determinedat 288 nm in the presence of MKT and Fig. 6 illustrates the pos-sibility of MKT determination at 385 nm in the presence of DSL.
Optimization of experimental conditions
Different experimental parameters affecting the spectral charac-teristics, SFI and resolution of synchronous fluorescence spectra
Figure 4. Synchronous fluorescence spectra of MKT at 340 nm in the presence ofDSL: (A1–5) 2.00, 1.40, 0.80, 0.50, and 0.20μg/mL MKT; and (B) 2.00μg/mL DSL.
Pea
k am
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ude
Wavelength (nm)
Figure 5. Second-derivative synchronous fluorescence spectra of different con-centrations of DSL at 288 nm in the presence of MKT: (A1–5) 2.00, 1.00, 0.80, 0.30and 0.10μg/mL DSL; and (B) 2.00μg/mL MKT.
Pea
k am
plit
ude
Wavelength (nm)
Figure 6. Second-derivative synchronous fluorescence spectra of different con-centrations of MKT at 385 nm in the presence of DSL: (A1–5) 2.00, 1.40, 0.80,0.50, and 0.20μg/mL MKT; and (B) 2.00μg/mL DSL.
Table 1. Effect of Δλ on the SFI of DSL (2.00 μg/mL) andMKT (2.00 μg/mL)
of DSL and MKT were carefully studied and optimized. Thesefactors were studied in turn while keeping all others constant.
Selection of optimum instrumental parameters forderivative-synchronous fluorescence scanning. Differentinstrumental parameters affecting the SFI, spectral shape andresolution of the spectra of DSL and MKT including Δλ, no ofpoints of derivative spectra and scan speed, were carefully
studied and optimized. The Δλ value is one of the most impor-tant elements for performing the SFS technique, because itdirectly influences spectral shape, bandwidth, peak locationand signal intensity (17). With the purpose of selecting theoptimum Δλ, a wide range of Δλ (20–180 nm) was examinedby obtaining the synchronous fluorescence spectra of the twocompounds. Δλ of 160 nm was chosen as the optimum, becauseit resulted in two distinct peaks with good shape, highestsensitivities and minimum spectral interference caused by eachcompound in the mixture. At other Δλ values, weaker SFI forboth drugs with poor separation was obtained. Table 1 illus-trates the influence of Δλ on SFI and λmax of both drugs.
The number of points of derivative spectra was also studiedover the range of 30–99. Forty points was selected as theoptimum for recording the SDSFS in this study. It was found thatincreasing the number of points resulted in decrease in thesensitivity, meanwhile with number of points fewer than 40;irregular peaks were obtained specially for DSL.
Another instrumental parameter that may affect the SDSFSshape is the scan speed. Experimental trials revealed that thescan rate has no significant effect on the shape of the SDSFSof DSL and MKT; hence 600 nm/min was selected for this study.
Effect of pH and volume of buffer. The influence of pH onthe SFI of the studied drugs was investigated over the pH rangeof 2.2–6.5 using McIlvaine’s buffer, and 8.5–10.0 using boratebuffer (Fig. 7a). It can be noted that maximum SFI of DSL was ob-tained over pH range of 2.2–2.4 after which a gradual decreasein SFI was observed with increasing pH. For MKT, maximum SFIwas achieved over a pH range of 2.2–5.0 after which furtherincrease of pH resulted in gradual decrease in SFI. Hence,McIlvaine’s buffer of pH 2.3 was selected as the optimum forthis study yielding maximum SFI for both drugs. Studying theinfluence of the volume of McIlvaine’s buffer revealed that0.3mL was sufficient for maximum SFI for both DSL and MKT(Fig. 7b).
Effect of different organized media. In order to obtain bestanalytical characteristics for the simultaneous determination ofDSL and MKT, the influence of different organized media onSFI of both drugs was investigated using 0.5mL of 0.1% (w/v)solutions of CMC, gelatin (macromolecules), sodium dodecylsulfate (SDS, anionic surfactant) and cetrimide (cationic surfactant).It was found that both SDS and CMC enhanced the SFI of DSL,while, gelatin and cetrimide had no significant effect (Fig. 8a). ForMKT, only CMC enhanced the SFI, whereas gelatin and cetrimide
Luminescence 2015; 30: 485–494Wiley & Sons, Ltd.
Figure 9. Effect of different solvents on the SFI of DSL (2.00μg/mL) and MKT(2.00μg/mL).
(a)
(b)
Figure 7. (a) Effect of pH on the SFI of DSL (2.00μg/mL) and MKT (2.00 μg/mL). (b)Effect of volume of McIlvaine’s buffer on the SFI of DSL (2.00μg/mL) and MKT(2.00μg/mL).
(a)
(b)
Figure 8. (a) Effect of different organized media (0.5mL of 0.1% solution of each)on the SFI of DSL (2.00μg/mL) and MKT (2.00μg/mL). (b) Effect of volume of CMC(0.1% w/v) on the SFI of DSL (2.00μg/mL) and MKT (2.00μg/mL).
Determination of desloratadine/montelukast by synchronous fluorimetry
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decreased it slightly and SDS had no effect (Fig. 8a). So, to achievethe best sensitivities for both compounds, CMC was selected as theoptimum fluorescence enhancer in this study, which increases theSFI of DSL by 216% and that of MKT by 28%. The percentenhancement of SFI was calculated as:
% Enhancement of SFI
¼ SFI in CMC=buffer system – SFI in aqueous=buffer systemð ÞSFI in aqueous=buffer system
� 100%
The enhancement effect of CMC on the SFI of DSL and MKT isprobably due to a very rigid microenvironment, which is capableof restricting the freedom of fluorophores and consequentlydiminishes the probabilities of non-radiative processes and pro-vides a relatively high viscous microenvironment that can inhibitquenching by molecular oxygen. These factors might increasethe fluorescence quantum yield and enhance the fluorescencesignals (27).
The use of CMC as a non-toxic and biodegradable fluores-cence enhancer (28), together with the exclusion of hazardousreagents or harsh conditions, softens the experimental condi-tions required for simultaneous assay of DSL and MKT, makingthe proposed method an example of green analytical chemistry.
Effect of volume of CMC. The effect of CMC volume on theSFI of the two studied drugs was also considered. Increasingthe volume of CMC produced a proportional increase in the SFI
of DSL and MKT up to 0.3–0.5mL, after which any further in-crease resulted in a decrease in the SFI (Fig. 8b). Eventually,0.4mL was chosen as the optimum volume of CMC in this study.
Effect of diluting solvent. Dilution with different solventssuch as, water, methanol, ethanol, dimethyl formamide (DMF),acetone and acetonitrile was attempted. As can be seen in Fig. 9,the SFI of DSL increased in water compared with other solvents,whereas that of MKT was maximum in ethanol compared withother solvents. Because of the presence of DSL at a lowerconcentration than MKT in the co-formulated tablets, we hadto predominate the effect of diluting solvent on SFI of DSL.Therefore, water was selected as the optimum diluting solvent.
Using water as a diluent in the present method provides extrabenefits with regard to safety for the analyst and the environ-ment, cost-effectiveness, simplicity and convenience. Theseadditional advantages together with the high sensitivityachieved make the method superior to previously reportedmethods for the simultaneous analysis of DSL and MKT.
Stability of standard solutions. Because of the light sensitiv-ity of MKT (29), its stock solution, calibration standards and qual-ity control samples were protected from light by wrapping inaluminum foil. By contrast, DSL standard solution was found tobe stable for 7 days when kept in the refrigerator at 4 °C.
Influence of time on SFI of desloratadine and montelukastsodium. The effect of time on the development and stabilityof the SFI of both drugs was also studied. It was found that thefluorescence emission developed instantaneously and remainedstable for at least 2 h.
Method validation
The validity of the proposed method was tested regarding line-arity, range, limit of quantitation (LOQ), limit of detection (LOD),accuracy, precision, robustness, selectivity and specificity accord-ing to ICH Q2 (R1) recommendations (30).
Linearity and range. Calibration graphs were obtained byplotting the values of the peak amplitude of the SDSFS vs. thefinal drug concentrations (μg/mL). The calibration graphs werefound to be rectilinear over the concentration ranges cited inTable 2. The validity of the proposed methods was proven bystatistical regression analysis of the data (31). The small valuesof the standard deviation of the residuals (Sy/x), standard devia-tion of the intercept (Sa) and standard deviation of the slope(Sb) indicate low scattering of the points around the calibrationlines proving linearity of the proposed method.
The results of the proposed method are 4–200 times moresensitive than the reported chromatographic methods (4–7) forthe simultaneous determination of DSL and MKT. Moreover,the proposed method is 20–90 and 10–90 times more sensitivethan the reported spectrophotometric methods (8–13) for DSLand MKT, respectively. This high sensitivity makes the proposedmethod superior to all methods reported in the literature.
Limits of quantitation and limits of detection. LOQ was de-termined according to ICH Q2 (R1) recommendation (30) by es-tablishing the lowest concentrations that can be measured
Table 2. Analytical performance data for the propos
Parameter
Concentration range (μg/mL)LOD (μg/mL)LOQ (μg/mL)Regression equationa (D2 = a+ bC) DCorrelation coefficient (r)Standard deviation of the residuals (Sy/x)Standard deviation of the intercept (Sa)Standard deviation of the slope (Sb)% RSD% ErroraD2, the peak amplitudes of the SDSFS; a, intercept; C
below which the calibration graphs are nonlinear. LOD was alsodetermined by evaluating the lowest concentrations of theanalytes that can be readily detected. LOQ and LOD were calcu-lated as (30):
LOQ ¼ 10Sa=b
LOD ¼ 3:3Sa=b
Where, Sa is the standard deviation of the intercept of the re-gression line, and b is the slope of the regression line. The resultsare summarized in Table 2.
Accuracy. To test the accuracy of the proposed method, it wasapplied for the determination of pure samples of DSL and MKTover the concentration ranges cited in Table 3. The results werein good agreement with those obtained using the comparisonspectrophotometric method (10). Statistical evaluation of theproposed method using Student’s t-test and the variance ratioF-test (31) revealed no significant differences between the per-formance of the proposed and comparison methods regardingaccuracy and precision (Table 3).
Precision
The repeatability (intraday precision) of the proposed methodwas tested by applying the method for the determination ofthree concentrations of DSL and MKT in the pure form at threesuccessive times within the same day (30). The results are pre-sented in Table 4.
Intermediate precision (interday precision) was tested by rep-licate analysis of three concentrations of DSL and MKT in thepure form over a period of three successive days (30). The resultsare also summarized in Table 4.
The data obtained from the precision study indicate the highprecision of the developed method as revealed by small valuesof % RSD and % error (Table 4).
Robustness. The robustness of the proposed method wasdemonstrated by the constancy of the SFI of both drugs with de-liberate minor changes in the experimental parameters. Minorintentional variation in pH (2.3 ± 0.1), changes in the buffervolume (0.3 ± 0.1mL) and in CMC volume (0.4 ± 0.1mL) did notsignificantly affect the analytical response.
Determination of desloratadine/montelukast by synchronous fluorimetry
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Selectivity. The selectivity of the proposed method for thesimultaneous determination of the cited drugs in binary mix-tures was evaluated through the preparation and analysis oflaboratory-prepared mixtures of DSL/MKT at a ratio of 1: 2. Thesecond-derivative signal of DSL was measured at 288 nm, whichis considered to be a zero-crossing point for MKT, and that ofMKT was measured at 385 nm, which is the zero-crossing pointfor DSL (Fig. 10). As obvious from Table 5, the adequate recov-ered concentrations in conjunction with the small values of %RSD (<1.0) and % error (<0.6) confirm the ability of the pro-posed method to resolve and quantify each drug in this binarymixture with acceptable analytical performance and withoutany interference from the co-existing drug.
Specificity. The specificity of the proposed method wasinvestigated by observing any interference encountered fromcommon tablets excipients such as lactose, maize starch, talcpowder, magnesium stearate, calcium hydrogen phosphateand microcrystalline cellulose. These additives did not interferewith the proposed method.
Determination of desloratadine/montelukast by synchronous fluorimetry
MKT in laboratory-prepared co-formulated tablets. Moreover,the method was extended to the determination of the twodrugs in commercially available single-ingredient tablets. Theaverage percent recoveries of different concentrations werebased on the average of three replicate determinations. Theobtained results were compared with those of the comparisonmethod (10) using Student’s t-test and variance ratio F-test(31). The obtained t- and F-values indicated no significant differ-ences between the performance of the two methods regardingaccuracy and precision (Tables 6 and 7).
Biological application. The high sensitivity of the proposedSDSFS method allowed the determination of MKT in spikedhuman plasma (Fig. 11). Following the oral administration of10mg MKT tablet formulation, a mean peak plasma concentra-tion (Cmax) of 0.5357μg/mL was achieved in ~ 3.6 h (32). Thisvalue lies within the working concentration range of the pro-posed method. The Cmax of DSL is reported to be 3.3 ng/mL(33). So, the current method was applied for the quantitative de-termination of MKT only in spiked human plasma. A simple pro-tein precipitation procedure described by Sripalakit et al. (32)was employed, excluding multiple extraction steps with hazard-ous organic solvents.
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Validation of the proposed method for the determination ofMKT in spiked human plasma
Linearity, range, LOD and LOQ. The second derivative signalof MKT in spiked plasma was plotted against drug concentration(μg/mL) to obtain the calibration graph. Linear regression analy-sis (31) of MKT in spiked human plasma gave the followingregression equation:
2D ¼ –5:96 þ 44:11C r ¼ 0:9982ð Þ:
Where, 2D is the peak amplitude of the SDSFS of MKT, C is theconcentration of the drug (μg/mL) and r is the correlation coef-ficient. Excellent linearity was achieved over a concentrationrange of 0.30–0.60μg/mL of MKT.
LOD and LOQ for the analysis of MKT in human plasma werealso calculated according to ICH recommendations (30) andfound to be 0.09 and 0.27μg/mL, respectively.
Accuracy. The accuracy of the proposed method was evalu-ated by analyzing plasma samples spiked with different concen-trations of MKT. The mean recoveries of MKT in spiked human
plasma samples based on the average of three independent de-terminations were 100.11 ± 2.44% at 385 nm (Table 8).
Precision. Intra- and interday precision were evaluatedthrough replicate analyses of plasma samples spiked with MKTat three times within the same day and on three consecutivedays. The small values of % RSD and % error proved the highprecision of the developed method for the determination ofMKT in plasma (Table 9).
Specificity. The specificity of the proposed method for thedetermination of MKT in human plasma was verified by theabsence of any potential interference from plasma matrix andendogenous components at the wavelength selected for SDSFSanalysis of MTK (385 nm), as illustrated in Fig. 11.
ConclusionA new, simple, rapid and sensitive method was developed for thesimultaneous determination of DSL and MKT in co-formulateddosage forms in which second-derivative synchronous fluores-cence spectroscopy enables the concurrent determination ofDSL and MKT by applying the zero-crossing technique withoutprior separation steps, thereby saving time. The developedmethod could be applied to the analysis of both drugs in theirco-formulated dosage forms and, because of to its high sensitiv-ity, could be applied to the determination of MKT in spiked
human plasma with good accuracy and precision. The proposedmethod combines many analytical merits such as simplicity, sen-sitivity, selectivity and rapidness. In addition, the methodexcludes the need for sophisticated instrumentation or lengthysample pretreatment. Moreover, the simplicity of the methodwas illustrated by the minimum requirement for chemicals andorganic solvents, which suggests that the proposed method iscost-effective and environmentally friendly.
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