Rapid and Sensitive Determination Of Montelukast in Human Plasma .pdf

8/12/2019 Rapid and Sensitive Determination Of Montelukast in Human Plasma .pdf

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Research Article Open Access

Bioequivalence &

Bioavailability

Shafaati et al. J Bioequiv Availab 2010, 2:6http://dx.doi.org/10.4172/jbb.1000046

Volume 2(6): 135-138 (2010) - 135J Bioequiv AvailabISSN:0975-0851 JBB, an open access journal

Keywords: Montelukast; Determination; Human Plasma; HPLC;Monolithic Column

Introduction

Montelukast sodium [1] is described chemically as [R-(E)]-1-[[[1-[3-[2-(7-chloro-2 quinolinyl) ethenyl]phenyl]-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio] methyl] cyclopropane acetic acid,

monosodium salt (Figure 1). Montelukast is a selective and orally activeleukotriene receptor antagonist that inhibits the cysteinyl leukotrieneCysL

1 receptor [2,3]. Te drug is indicated or the prophylaxisand

chronic treatment o asthma in adults and pediatric patients 12months o age and older. It is also indicated or prevention o exercise-induced bronchoconstriction in patients 15 years o age and older andor the relie o symptoms o seasonal allergic rhinitis in adults andpediatric patients 2 years o age and older [2-4]. Montelukast is rapidlyabsorbed ollowing oral administration and is extensively metabolized.Montelukast and its metabolites are excreted almost exclusively via thebile[5]. In several studies, the mean plasma hal-lie o montelukastranged rom 2.7 to 5.5 hours in healthy young adults [5,6]. Tepharmacokinetics o montelukast is nearly linear or oral doses up to50 mg [6].

In order to study the pharmacokinetics o montelukast in humans,a sensitive and reliable assay o the drug in biological fluids is necessary.Various analytical methods have been reported or the determinationo montelukast in bulk or pharmaceutical dosage orms [7-10] andin biological fluids [1,10-13]. Recently, [14] published an excellentpaper on development and validation o a bioanalytical method ordetermination o montelukast in human plasma using HPLC. In theirpaper, they also reviewed and compared the published HPLC methodsor determination o montelukast in human plasma. Compared to otherreports, the method reported by Sripalakit and colleagues was simpleand the total run time (i.e. 9 minutes) was less than the time previouslyreported or similar methods. Furthermore, they used meenamic acidas internal standard, which is available commercially. In our study, we

have developed and validated a very simple method based on using amonolithic column, by which the consumption o acetonitrile in themobile phase has been reduced rom 70% to 56%. Moreover, although

we have achieved a base line resolution between the internal standardand the major peak, the lag time between the two peaks (which is morethan 4 minutes in Sripalakits paper) has been reduced to less than 2minutes. Tis in turn, led to a shortened analysis time (


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Volume 2(6): 135-138 (2010) - 136 J Bioequiv AvailabISSN:0975-0851 JBB, an open access journal

Citation: Shafaati A, Zarghi A, Foroutan SM, Khoddam A, Madadian B (2010) Rapid and Sensitive Determination Of Montelukast in Human Plasma

by High Performance Liquid Chromatographic Method Using Monolithic Column: Application to Pharmacokinetic Studies. J Bioequiv Availab

2: 135-138. doi:10.4172/jbb.1000046

method is less hazardous to human health and to the environment aswell as being aster and more economic.

Materials and MethodsChemicals and reagents

Montelukast sodium and ethoxiquine standard powders werekindly gifed by Sobhan Daru Pharmaceutical Company (ehran,Iran). HPLC grade acetonitrile, analytical grade disodium hydrogenphosphate, phosphoric acid, and all other chemicals were purchasedrom Merck (Darmstadt, Germany). High purity water was supplied byMilli-Q Water Purification System (Millipore, Billerica, MA).Humanplasma was supplied by Noor Educational and Research Institute(ehran, Iran).

Chromatographic conditions

Te HPLC system (Knauer, Germany) consisted o a K1001 pump

and a RX-10AXL spectrofluorometric detector and controlled byEurochrom 2000 (Integration Package) sofware.Te analytical columnemployed was Chromolith RP (100mm4.6mm i.d.) purchased romMerck (Germany). Te mobile phase was comprised o acetonitrle50mM phosphate buffer pH 7.0 (56% - 46% v/v, respectively). Te mobilephase was prepared reshly and was filtered beore use. All separationswere perormed isocratically at a flow rate o 2.0ml/min and columncondition was maintained at ambient temperature. Te detector wasoperated at an excitation wavelength o 350 nm and an emissionwavelength o 450 nm.

Standard solutions

Blank plasma was prepared rom heparinized whole-blood samplescollected rom healthy volunteers and stored at -20C. Stock solution omontelukast sodium was prepared with concentration o 0.01 mg/mlin water and diluted with drug ree human plasma or the mobile phaseto the desired concentration. Due to the sensitivity o montelukast tothe light [8,9], exposure o the drug solution to the light was careullyavoided. Ethoxyquine was dissolved in acetonitrile at a concentration o0.4 mg/ml. A series o plasma standard solutions o montelukast sodiumwere prepared with concentrations o 20, 200, 350, 500, 650 and 800 ng/ml, and each one containing the internal standard at a concentrationo 40ng/ml. Also, quality control standard solutions were preparedcontaining low (100 ng/ml), median (300 ng/ml) and high (600 ng/ml)concentrations o the drug in plasma to assess accuracy o the methodand intra- and inter-day variations. All solutions o montelukast werekept at 4C and all plasma solutions o the drug were stored at -20Cuntil analysis.

Biological samplesMontelukast was administered in a single dose o 10 mg to twelve

healthy volunteers afer overnight asting. Blood samples (3-4 ml) werecollected at several intervals afer dosing and transered into heparinizedtubes. Afer centriugation at 5000 rpm or 10 min, resultant plasmawas separated and rozen immediately at -20C until assayed.

Sample preparation

All plasma samples were thawed at room temperature immediatelybeore assayed. o a 500l o plasma sample in a glass-stoppered 15ml centriuge tube 50l o the internal satndard solution (600 ng/ml)and 200l saturated solution o NaCl were added [15]. Te mixture wasmixed (30 s) and was centriuged or 10 min at 8000 rpm. Ten, 20l othe supernatant was injected into liquid chromatography.

Stability

Te stability o montelukast was assessed during all steps o the

method including storage and analysis. No changes in stability overthe period o one month were observed (provided that all samples andsolutions are kept in amber glass containers or protected rom light by

wrapping containers and tubes in aluminum oil).

Selectivity and specificity

Control human plasma, obtained rom 12 healthy volunteers,

was assessed by the procedure as described above and compared withrespective plasma samples to evaluate selectivity o the method.

Precision and accuracy

Te precision and accuracy o the method were examined by addingknown amounts o montelukast (20, 300 and 600 ng/ ml to pool plasma(quality control samples). For intra-day precision and accuracy fivereplicate quality control samples at each concentration were assayed onthe same day. Te inter-day precision and accuracy were evaluated on

three different days.Limit of quantification (LOQ) and recovery

For the concentration to be accepted as LOQ, the percent deviation

rom the nominal concentration (accuracy) and the relative standarddeviation must be 10% and less than 10%, respectively, considering atleast five times the response compared to the blank response [15]. Terelative analytical recovery or plasma at three different concentrationso montelukast (100, 300 and 600 ng/ ml) was determined. Averagerecovery o montelukast was determined by comparing AUC obtainedafer injection o the processed QC samples with those achieved bydirect injection o the same amount drug in distilled water at differentconcentrations (six samples or each concentration level).

Plasma calibration curves and quantitationAfer thawing, a series o plasma standard solutions o montelukast

sodium were prepared with concentrations o 20, 200, 350, 500, 650 and800 ng/ml, each one containing the internal standard at a concentrationo 40 ng/ml.Te samples were then prepared or analysis as describedabove. Calibration curves were constructed by plotting peak area ratio(y) o montelukst to the internal standard versus the drug concentration(x). A linear regression was used or quantitation.

Pharmacokinetic design and analysis

Te proposed method was applied to evaluate the pharmacokinetico montelukast in 12 healthy male volunteers with a mean age o 25.9 4.2 years and body mean weight o 72/7 2.5 kg . Each volunteer was

orally administered 10mg montelukast (Sobhan Daru PharmaceuticalCompany, ehran, Iran) under asting conditions. Blood sampleswere collected in heparinized tubes beore and at 0.5, 1, 1.5, 2, 2.5,3, 3.5, 4, 5, 7, 10 and 24 h afer dosing and centriuged to obtain theplasma raction. Te plasma samples were kept in cryogenic vial storedat 20C until analysis. Montelukast pharmacokinetic parameterswere determined by non-compartmental methods. Elimination rateconstant (K) were estimated by the least-square regression o plasmaconcentrationtime data points lying in the terminal log-linear region

o the curves. Hal-lie (1/2

) was calculated as 0.693 divided by K. Tearea under the plasma concentrationtime curve rom time zero to thelast measurable concentration at time t (AUC

0t) was calculated using

the trapezoidal rule (Foroutan et al, 2003). Te area was extrapolatedto infinity (AUC

0) by addition o C

t/Kto AUC

0twhere C

tis the last

detectable drug concentration. Peak plasma concentration (Cmax) andtime to peak concentration (

max) were derived rom the individual

subject concentration time curves.
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Results

Chromatography separation

Under the chromatographic conditions described, montelukastand the internal standard peaks were well resolved. No intereringpeaks rom endogenous plasma components were observed. ypicalchromatograms o blank plasma in comparison to spiked samplesanalyzed or pharmacokinetics study are shown in Figure 2. Temonolithic column has lower flow impedance comparing to theparticulate packings, and thereore allows applying higher flowrate and easy optimization o chromatographic conditions [16] toobtain desirable resolution in a short time (


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significant difference was observed between our pharmacokinetic dataand results reported in the literature [5,6].

Discussion

Te aim o our study was to develop a rapid and sensitive methodor the routine analysis o biological samples in pharmacokineticmontelukast research. Tis method is well suited or routine applicationin the clinical laboratory because o the speed o analysis and simpleextraction procedure. Over 300 plasma samples were analyzed by thismethod without any significant loss o resolution. No change in thecolumn efficiency or increase in backpressure was observed over theentire study time, thus proving its suitability.

In the present method we used a protein precipitation extraction,HPLC on a monolithic column and fluorescence detection to givehigh sensitivity [16]. Te published HPLC methods or determinationo montelukast in human plasma were summarized in an excellentpaper published by [14]. Tey presented in their paper, a simplebioanalytical assay or determination o montelukast in human plasmaand its application to a pharmacokinetic study and compared to thepreviously reported methods. Ten, they concluded that their methodis advantageous over most o the reported methods because o theshorter analysis time and availability o the internal standard. In ourmethod, in this work, total analysis time is even shorter and separationachieved in less than 5 minutes. Moreover, separation was perormedon a monolithic column which resulted in lower consumption othe acetonitrile as an organic modifier in the mobile phase. Further

reduction in the acetonitrile consumption was resulted due the shortertotal analysis time. Tereore, the present method is less hazardous tohuman health and to the environment as well as being aster and moreeconomic. Tese results were obtained along with the same sensitivityand selectivity as described by [14].

Te present method proved to be reproducible and reliable basedon the results o validation assessment. Also, sensitivity and selectivityo the method allowed us to apply it successully or the routine analysiso biological samples in pharmacokinetic research on montelukast.

Conclusions

We developed a rapid, simple, accurate and reproducible methodor determination o montelukast in plasma. Te proposed method is

Figure 3: Mean plasma concentrationtime prole of montelukast in healthy

volunteers (n = 12) after a single dose of 10 mg montelukast.

0

200

400

600

800

0 5 10 15 20 25

Time (h)

ng/ml

less hazardous to human health and to the environment as well as beingaster and more economic. Tis method will permit pharmacokineticand pharmacodynamic studies o the drug in humans.

Acknowledgements

This work was supported by Noor Research and Educational Institute.

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Rapid and Sensitive Determination Of Montelukast in Human Plasma .pdf

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