Original Article 617
Development of an LC-Tandem Mass Spectrometry Method for the Separation of Montelukast and its Application to a Pharmacokinetic Study in Humans
Affiliations
Key words ▶ montelukast ● ▶ gliclazide ● ▶ LC-MS/MS ● ▶ pharmacokinetic ●
E. Ezzeldin1, 2, M. H. Tammam2, N. F. AboTalib2 1 2
Drug Bioavailability Laboratory, College of Pharmacy, King Saud University, Saudi Arabia Drug Bioavailability Center, National Organization for Drug Control and Research, (NODCAR), Cairo, Egypt
Abstract
▼
Accurate, precise, and sensitive LC-MS/MS assay method for the determination of montelukast (MO) in human plasma samples using gliclazide (GL) as internal standard was developed and applied in pharmacokinetics study. MO extracted by protein precipitation using acetonitrile. Chromatographic separation was carried out using a Agilant Triple quadrupoles mass spectrometer with API source with an Agilant SB-
Introduction
▼
received 05.11.2013 accepted 10.01.2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1366986 Published online: February 5, 2014 Drug Res 2014; 64: 617–622 © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence Dr. M. Tammam Drug Bioavailability Center National Organization for Drug Control and Research P.O. Box 29 Cairo Egypt Tel.: + 20/233/367 954 Fax: + 20/233/79 445 [email protected]
Montelukast sodium is 1-[({(R)-m-[(E)-2-(7chloro-2-quinolyl) vinyl]-α-[o-(1 – hydroxyl – 1 – methylethyl)phenethyl] -benzyl}thio)methyl] ▶ Fig. 1). MO is a selective cyclopropaneacetate (● leukotriene receptor antagonist with actions and use similar to those of zafirlukast [1]. The drug is indicated for the prophylaxis and chronic treatment of asthma in adults and pediatric. It is also indicated for prevention of exercise induced bronchoconstriction and for the relief of symptoms of seasonal allergic rhinitis [2–4]. Montelukast is rapidly absorbed following oral administration and is extensively metabolized. Montelukast and its metabolites are excreted almost exclusively via the bile [5]. In several studies, the mean plasma half-life of montelukast ranged from 2.7 to 5.5 h in healthy young adults [5, 6]. The pharmacokinetics of montelukast is nearly linear for oral doses up to 50 mg [6]. In order to study the pharmacokinetics of montelukast in humans, a sensitive and reliable assay of the drug in biological fluids is necessary. The methods for determination of montelukast involved several analytical techniques such as voltametric [7], capillary electrophoresis [8], spectroflurometry [9] spectrophotometry [10] and liquid chromatography [11–25]. Some of
C18 (50 × 4.6 mm), 1.8 μm particle size column. A mobile phase consisting of acetonitrile: 0.1 % formic acid (84:16) was delivered. Calibration curves were linear in the concentration range of (10.00–800.00) ng/ml. The bioanalytical method for determination of MO was successfully applied to assess pharmacokinetics of montelukast. The LLOQ was sensitive enough for detecting terminal phase concentrations of the drug. This study showed that developed method is suitable for MO pharmacokinetic study.
these methods were developed in bulk or pharmaceutical dosage forms [19–23] and in biological fluid [9–18, 24, 25]. Some of these methods involve tedious procedure and too many steps which do not satisfy the determination of the samples [7–10]. Quantification of MO in human plasma using HPLC was developed by few authors [12–17], which involves longer run time and are more expensive. Quantification of MO in human plasma using LC-MS/MS was developed [11, 24]. In some of the previous methods the samples were pretreated with liquid-liquid extraction (LLE) [11, 17] or the preparation of sample was performed by plasma protein precipitation [12– 16, 18, 24, 25]. Some methods are expensive as they need either chiral, switching column [13, 14] or need 96-well sample preparation microtiter plate [15, 16]. In the present study, a sensitive, simple (one step) and accurate LC-MS/MS method for the determination of MO in human plasma based on the commercially available internal standard GL, the use of low amount of plasma sample (100 μl), low amount of solvent for precipitation and use of 0.1 % formic acid instead of buffers which affects the life span of the separation column.
Ezzeldin E et al. Montleukast Analysis & Pharmacokinetics … Drug Res 2014; 64: 617–622
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Authors
618 Original Article Extraction procedure MO extracted by protein precipitation using acetonitrile. 10.0 μl of internal standard solution was added to 100.0 μl plasma. The mixture was then vortex-mixed for 30 s and centrifuged with 300.0 μl acetonitrile for 10 min at 4 000 rpm. 20.0 μl of supernatant was injected to LC-MS/MS instrument.
Method validation
The analytical method established was to be applied to samples obtained from a pharmacokinetic study in healthy volunteers who received oral dose of MO.
Material and Methods
▼
Standards and chemicals Singulair 10 mg tablets (Merk &Co. Inc-USA). MO standard material (Merk), 99.5 % potency, GL standard material, 99.22 % potency (Sigma-Aldrich) and formic acid (Romil chemicals, England). Acetonitrile and methanol (HPLC grade, Alpha Chemicals, Egypt).
Methods
▼
Bioanalytical phase Preparation of standards and quality control samples All reagents used were of HPLC grade. A stock solution of MO (100.0 μ/ml) was prepared in methanol. Working standards of the drug (10.0–800.0 ng/ml) were prepared by serial dilution of the stock solution in methanol. A working standard solution of the GL (2.5 μg/ml) was prepared in methanol. All solutions were stored at 4 °C.
Liquid chromatography and mass spectrometric condition Chromatographic separation was carried out using Agilant Triple quadrupoles mass spectrometer with API source with an Agilant SB-C18 (50 × 4.6 mm), 1.8 μm particle size column. A mobile phase consisting of acetonitrile: 0.1 % formic acid (84:16) was delivered with a flow rate of 0.6 mL/min. Mass spectra were obtained using an electrospray ionization source operated in the multiple reaction monitoring (MRM) modes. Samples introduction and ionization were in the positive ion mode. The cone voltage was set as 135 V for both MO and GL. The capillary voltages was optimized at 4 000 V. Argon was used as collision gas. The collision energy was set at 20 and 15 for both MO and GL. The gas flow was optimized during tuning as 8 L/min and nebulizer pressure was 30 psi. The source temperature was 325 °C for both MO and GL. The mass transitions ion-pair selected were m/z 586.2→568.3 for MO and m/z 324.4→127.2 for GL. The data acquisition software used was Agilant MassHunter software. For quantification, the peak area ratios of the target ions of the drugs to those of the internal standard were compared with weighted (1/concentration2) least squares calibration curves in which the peak area ratios of the calibration standards were plotted vs. their concentrations.
Analytical method for determination of MO in human plasma was developed and partially validated according to FDA Bioanalytical method validation guidelines [26].
Linearity: The linearity of the method was determined by analysis of 6 standard plots associated with 7-point standard calibration curve. The ratio of area response for analyte to IS was used for regression analysis. Each calibration curve was analyzed individually by using least square weighted (1/x2) linear regression. The concentration of volunteers samples were calculated from the calibration curve (y = bx + a). Recovery: The analytical absolute recovery of MO was determined at concentrations of 10.0, 20.0, 40.0, 80.0, 120.0, 400.0 and 800.0 ng/ml (n = 3). Drug-free plasma was spiked with known amounts of the drug to achieve the concentration previously specified. These samples were processed by the analytical method applied in this study, and peak area was compared with the peak area obtained by direct injection of the same concentrations of the drugs dissolved in the mobile phase. Limit of detection (LOD) and limit of quantification (LOQ): The LOD was determined by using the signal-to-noise ratio and comparing test results from samples with known concentrations of analyte against blank samples. The analyte concentration that produced a signal-to-noise ratio of 3:1 was accepted as the LOD. The LOQ was estimated by analyzing samples with known amounts of MO at progressively lower concentrations, starting at the lower end of the calibration curves. The LOQ was considered as the concentration level in which accuracy and precision were still better than 20 %. Precision and accuracy: Inter- and intra-assay precision and accuracy were determined by analyzing 6 replicates at 7 different plasma concentrations (10.0, 20.0, 40.0, 80.0, 120.0, 400.0 and 800.0 ng/ml) on 3 different days. The criteria for acceptability of the data included accuracy within ± 15 % standard deviation (SD) from the nominal values and a precision of within ± 15 % relative standard deviation (RSD), except for lower limit of quantitation (LOQ), where it should not exceed ± 20 %. Stability: Low, medium and high quality control samples (n = 6) corresponding to 40.0, 120.0 and 800.0 ng/ml, respectively were retrieved from deep freezer after 3 freeze–thaw cycles according to the clinical protocols. Samples were frozen at − 80 °C in 3 cycles of 24.0, 36.0, and 48.0 h. In addition, the long-term stability of MO in quality control samples was also evaluated by analysis after 6 weeks of storage at − 80 °C. Autosampler stability was studied following 24 h-storage period in the autosampler tray. Bench top stability was studied for 12-h period. Stability samples were processed and extracted along with the freshly spiked calibration curve standards. The precision and accuracy for the stability samples must be within ≤ 15 and ± 15 %, respectively, of their nominal concentrations.
Ezzeldin E et al. Montleukast Analysis & Pharmacokinetics … Drug Res 2014; 64: 617–622
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Fig. 1 Chemical structure of montelukast.
Original Article 619
Pharmacokinetic study 6 healthy adult male volunteers were included in this study. Male healthy volunteers, 22–37 years of age, with not more than ± 10 % from ideal weight (54–100 kg, the describe weights for Men – 1983 Metropolitan Height and Weight table) for their age were considered eligible. On the basis of medical history, clinical examination and laboratory investigations (haematology, blood biochemistry, urine analysis and viruses scanning), none of the participants has revealed any medical abnormality. In addition, the included subjects have no history of hospitalization or involvement in any clinical trials within 12 weeks, and none of them had received any regular course of drug therapy within 4 weeks prior to this study. Informed written consent was obtained from the volunteers and the protocol of the study was approved by the Ethical Committee of National Organization for Drug Control and Research. Each subject received Singulair 10 mg tablets. The order of product administration was done after an overnight fast of 10.0 h. Food is only allowed after 4 h of montelukast administration. Similar meals were standardized in amount and composition (protein, carbohydrate and vegetable) and served after 4 and 8 h post dosing. Blood samples were collected in heparinized tube, at 0.0 (pre-dose), 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 7.0, 10.0, 12.0 and 24.0 h after drug administration. The samples were centrifuged and the obtained plasma was frozen at − 80 °C until analyzed.
Results and Discussion
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The goal of this work was to develop and validate a simple, rapid and sensitive assay method for determination of MO in human plasma and its application in pharmacokinetics. To achieve the goal, during method development, different options were evaluated to optimize sample extraction, detection parameters and chromatography.
Validation of method of analysis Electrospray ionization (ESI) was evaluated to get a better response from analytes. It was found that the best signal was achieved with ESI positive ion mode. The product ion spectrum for MO and GL yielded high abundance fragment ions of m/z
▶ Fig. 2). Further optimiza568.3 and m/z 127.2, respectively (● tion in chromatographic conditions increased the signal of analytes. Several trials were done to select the best ratio of the mobile phase components to achieve the best resolution between montelukast and the internal standard. It was found that the ratio 86 % acetonitrile and 0.1 % formic acid was excellent in separation. This mobile phase was delivered at a flow rate of 0.6 ml/min. The average retention times of the internal standard and montelukast were 1.4 and 2.3 min, respectively. Glaclizide, easily available as a pharmaceutical substance, so it was selected among many compounds we tested as internal standard. Under these chromatographic conditions described, montelukast and the internal standard peaks were well resolved. No interfering peaks from endogenous plasma components were observed. The representative typical MRM chromatograms of blank plasma in comparison to plasma spiked with gliclazide, spiked with montelukast and representative plasma sample from healthy volunteer analyzed for pharmacokinetic study ▶ Fig. 3). The standard calibration curve for were shown in (● montelukast relating concentration to peak area ratio was found to be linear within the range of 10.0–800.0 ng/ml (r2 = 0.997 with ▶ Table 1, ● ▶ Fig. 4). low limit of quantification of 10.0 ng/ml) (● The intraday precision and the accuracy value of the method were < 15 %. Inter-day precision values were found to be < 15 % ▶ Table 1). The recovery ranged from 72.77 to 104.83 % for MO (● ▶ Table 2 summarizes the concentrations of 10.0–800.0 ng/ml. ● freeze and thaw stability, short term stability, long-term stability and auto sampler stability data of MO. All the results showed the reliable stability behavior during these tests and there were no stability-related problems during the routine analysis of samples for the bioavailability study. The present method is characterized from the other previous methods in the using small volume of plasma and acetonitrile. Extraction procedure involved one step of protein precipitation by acetonitrile which is simpler than liquid-liquid extraction [11, 17]. MO has been separated using reversed C18 column rather than dual column [14, 15], switching column [14] system or chiral column [13].
Ezzeldin E et al. Montleukast Analysis & Pharmacokinetics … Drug Res 2014; 64: 617–622
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Fig. 2 Mass spectra of the positive ion of montelukast (MO) and gliclazide (GL).
620 Original Article
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Fig. 3 Representation MRM chromatograms of a blank plasma,b plasma spiked with 25 ng/ml of gliclazide (GL), c Plasma spiked with 400 ng /ml of montelukast, d plasma spiked with 25 ng/ml of gliclazide and 400 ng/ml montelukast and e plasma sample from healthy volunteer at 3.5 h of after an oral administration of 10 mg of montelukast.
Table 1 Intra-day and inter-day reproducibility of standard plasma calibration curves of montelukast. Theoretical concentration (ng/ml) Parameters
Inter-days repeatability 10
Mean Precision Accuracy ( %)
10.18 9.37 101.75
20 18.78 9.48 93.92
40 39.25 11.53 98.14
80 80.46 11.88 100.57
120
Intra-day repeatability 400
800
10
Concentration found (ng/ml) 107.34 433.97 796.40 10.18 10.41 12.02 14.96 8.88 89.45 108.49 99.55 101.78
20
40
80
120
400
17.89 2.37 89.45
35.97 12.41 89.92
72.92 9.56 91.15
96.19 9.31 80.15
374.47 9.87 93.62
800 705.73 14.35 88.22
Pharmacokinetic study
Safety evaluation
Successful completion of the clinical phase was done by 6 subjects in the single-dose fasting pharmacokinetic study. Following oral administration, mean plasma concentration-time profile of MO best fit a non-compartment in all subjects. Plasma con▶ Fig. 5. The pharmacokicentration-time profile was shown in ● ▶ Table 3. The median t netic parameters were shown in ● max, Cmax and AUC values were similar to those reported in the literature [12, 25].
Having completed the study, subject underwent a through physical assessment and vital signs evaluation on follow-up examination to assure their safety. Clinical assessment for all subjects was carried out to evaluate their tolerability to the study medications. Subjects demonstrated good tolerance to the study drug.
Ezzeldin E et al. Montleukast Analysis & Pharmacokinetics … Drug Res 2014; 64: 617–622
Original Article 621
procedure. The lag time between the main peak of drug and the internal standard is about 1 min. This in turn, led to a shortened analysis time ( < 5 min). This method characterized also by use of small volume of plasma and acetonitrile as precipitating agent in drug extraction.
Competing Interests
▼ Fig. 4 Standard calibration curve for montelukast (mean peak area ratio vs. concentration of montoleukast).
The authors declare that they have no conflict of interests.
Reference
Accuracy (mean ± CV %) 40.0 ng/ml
120.0 ng/ml
Short-term stability 104.45 ± 10.38 105.32 ± 11.60 (6 h, room temperature) Long-term stability 111.31 ± 6.99 104.03 ± 5.15 (42 days, − 80 °C) Freeze and thaw stability 106.48 ± 9.24 106.69 ± 5.66 (3 cycles, − 80 °C–room temperature)
800.0 ng/ml 93.60 ± 9.75 96.74 ± 10.33 103.29 ± 12.28
Fig. 5 Mean plasma concentration-time profiles after single oral dose (10 mg montelukast).
Table 3 Pharmacokinetic parameters of montelukast. Parameters Cmax (ng/ml) Tmax (Median) (h) AUC0–24 (ng.h/ml) AUC0–inf (ng.h/ml) t1/2 (h) MRT (h) Kel (h)
Mean ± SD 503.6 ± 204.1 3.5 3 628.3 ± 1 582.5 4 038.5 ± 1 651.7 6.2 ± 1.1 9.3 ± 1.4 0.11 ± 0.01
Conclusion
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The aim of our study was to develop a rapid and sensitive method for the analysis of plasma samples in pharmacokinetic montelukast. This method is well suited for pharmacokinetics studies because of the speed of analysis and simple extraction
▼
The title of this article has been corrected.
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Ezzeldin E et al. Montleukast Analysis & Pharmacokinetics … Drug Res 2014; 64: 617–622
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Ezzeldin E et al. Montleukast Analysis & Pharmacokinetics … Drug Res 2014; 64: 617–622
Development of an LC-Tandem Mass Spectrometry Method for the Separation of Montelukast and its Application to a Pharmacokinetic Study in Humans
Affiliations
Key words ▶ montelukast ● ▶ gliclazide ● ▶ LC-MS/MS ● ▶ pharmacokinetic ●
E. Ezzeldin1, 2, M. H. Tammam2, N. F. AboTalib2 1 2
Drug Bioavailability Laboratory, College of Pharmacy, King Saud University, Saudi Arabia Drug Bioavailability Center, National Organization for Drug Control and Research, (NODCAR), Cairo, Egypt
Abstract
▼
Accurate, precise, and sensitive LC-MS/MS assay method for the determination of montelukast (MO) in human plasma samples using gliclazide (GL) as internal standard was developed and applied in pharmacokinetics study. MO extracted by protein precipitation using acetonitrile. Chromatographic separation was carried out using a Agilant Triple quadrupoles mass spectrometer with API source with an Agilant SB-
Introduction
▼
received 05.11.2013 accepted 10.01.2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1366986 Published online: February 5, 2014 Drug Res 2014; 64: 617–622 © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence Dr. M. Tammam Drug Bioavailability Center National Organization for Drug Control and Research P.O. Box 29 Cairo Egypt Tel.: + 20/233/367 954 Fax: + 20/233/79 445 [email protected]
Montelukast sodium is 1-[({(R)-m-[(E)-2-(7chloro-2-quinolyl) vinyl]-α-[o-(1 – hydroxyl – 1 – methylethyl)phenethyl] -benzyl}thio)methyl] ▶ Fig. 1). MO is a selective cyclopropaneacetate (● leukotriene receptor antagonist with actions and use similar to those of zafirlukast [1]. The drug is indicated for the prophylaxis and chronic treatment of asthma in adults and pediatric. It is also indicated for prevention of exercise induced bronchoconstriction and for the relief of symptoms of seasonal allergic rhinitis [2–4]. Montelukast is rapidly absorbed following oral administration and is extensively metabolized. Montelukast and its metabolites are excreted almost exclusively via the bile [5]. In several studies, the mean plasma half-life of montelukast ranged from 2.7 to 5.5 h in healthy young adults [5, 6]. The pharmacokinetics of montelukast is nearly linear for oral doses up to 50 mg [6]. In order to study the pharmacokinetics of montelukast in humans, a sensitive and reliable assay of the drug in biological fluids is necessary. The methods for determination of montelukast involved several analytical techniques such as voltametric [7], capillary electrophoresis [8], spectroflurometry [9] spectrophotometry [10] and liquid chromatography [11–25]. Some of
C18 (50 × 4.6 mm), 1.8 μm particle size column. A mobile phase consisting of acetonitrile: 0.1 % formic acid (84:16) was delivered. Calibration curves were linear in the concentration range of (10.00–800.00) ng/ml. The bioanalytical method for determination of MO was successfully applied to assess pharmacokinetics of montelukast. The LLOQ was sensitive enough for detecting terminal phase concentrations of the drug. This study showed that developed method is suitable for MO pharmacokinetic study.
these methods were developed in bulk or pharmaceutical dosage forms [19–23] and in biological fluid [9–18, 24, 25]. Some of these methods involve tedious procedure and too many steps which do not satisfy the determination of the samples [7–10]. Quantification of MO in human plasma using HPLC was developed by few authors [12–17], which involves longer run time and are more expensive. Quantification of MO in human plasma using LC-MS/MS was developed [11, 24]. In some of the previous methods the samples were pretreated with liquid-liquid extraction (LLE) [11, 17] or the preparation of sample was performed by plasma protein precipitation [12– 16, 18, 24, 25]. Some methods are expensive as they need either chiral, switching column [13, 14] or need 96-well sample preparation microtiter plate [15, 16]. In the present study, a sensitive, simple (one step) and accurate LC-MS/MS method for the determination of MO in human plasma based on the commercially available internal standard GL, the use of low amount of plasma sample (100 μl), low amount of solvent for precipitation and use of 0.1 % formic acid instead of buffers which affects the life span of the separation column.
Ezzeldin E et al. Montleukast Analysis & Pharmacokinetics … Drug Res 2014; 64: 617–622
Downloaded by: NYU. Copyrighted material.
Authors
618 Original Article Extraction procedure MO extracted by protein precipitation using acetonitrile. 10.0 μl of internal standard solution was added to 100.0 μl plasma. The mixture was then vortex-mixed for 30 s and centrifuged with 300.0 μl acetonitrile for 10 min at 4 000 rpm. 20.0 μl of supernatant was injected to LC-MS/MS instrument.
Method validation
The analytical method established was to be applied to samples obtained from a pharmacokinetic study in healthy volunteers who received oral dose of MO.
Material and Methods
▼
Standards and chemicals Singulair 10 mg tablets (Merk &Co. Inc-USA). MO standard material (Merk), 99.5 % potency, GL standard material, 99.22 % potency (Sigma-Aldrich) and formic acid (Romil chemicals, England). Acetonitrile and methanol (HPLC grade, Alpha Chemicals, Egypt).
Methods
▼
Bioanalytical phase Preparation of standards and quality control samples All reagents used were of HPLC grade. A stock solution of MO (100.0 μ/ml) was prepared in methanol. Working standards of the drug (10.0–800.0 ng/ml) were prepared by serial dilution of the stock solution in methanol. A working standard solution of the GL (2.5 μg/ml) was prepared in methanol. All solutions were stored at 4 °C.
Liquid chromatography and mass spectrometric condition Chromatographic separation was carried out using Agilant Triple quadrupoles mass spectrometer with API source with an Agilant SB-C18 (50 × 4.6 mm), 1.8 μm particle size column. A mobile phase consisting of acetonitrile: 0.1 % formic acid (84:16) was delivered with a flow rate of 0.6 mL/min. Mass spectra were obtained using an electrospray ionization source operated in the multiple reaction monitoring (MRM) modes. Samples introduction and ionization were in the positive ion mode. The cone voltage was set as 135 V for both MO and GL. The capillary voltages was optimized at 4 000 V. Argon was used as collision gas. The collision energy was set at 20 and 15 for both MO and GL. The gas flow was optimized during tuning as 8 L/min and nebulizer pressure was 30 psi. The source temperature was 325 °C for both MO and GL. The mass transitions ion-pair selected were m/z 586.2→568.3 for MO and m/z 324.4→127.2 for GL. The data acquisition software used was Agilant MassHunter software. For quantification, the peak area ratios of the target ions of the drugs to those of the internal standard were compared with weighted (1/concentration2) least squares calibration curves in which the peak area ratios of the calibration standards were plotted vs. their concentrations.
Analytical method for determination of MO in human plasma was developed and partially validated according to FDA Bioanalytical method validation guidelines [26].
Linearity: The linearity of the method was determined by analysis of 6 standard plots associated with 7-point standard calibration curve. The ratio of area response for analyte to IS was used for regression analysis. Each calibration curve was analyzed individually by using least square weighted (1/x2) linear regression. The concentration of volunteers samples were calculated from the calibration curve (y = bx + a). Recovery: The analytical absolute recovery of MO was determined at concentrations of 10.0, 20.0, 40.0, 80.0, 120.0, 400.0 and 800.0 ng/ml (n = 3). Drug-free plasma was spiked with known amounts of the drug to achieve the concentration previously specified. These samples were processed by the analytical method applied in this study, and peak area was compared with the peak area obtained by direct injection of the same concentrations of the drugs dissolved in the mobile phase. Limit of detection (LOD) and limit of quantification (LOQ): The LOD was determined by using the signal-to-noise ratio and comparing test results from samples with known concentrations of analyte against blank samples. The analyte concentration that produced a signal-to-noise ratio of 3:1 was accepted as the LOD. The LOQ was estimated by analyzing samples with known amounts of MO at progressively lower concentrations, starting at the lower end of the calibration curves. The LOQ was considered as the concentration level in which accuracy and precision were still better than 20 %. Precision and accuracy: Inter- and intra-assay precision and accuracy were determined by analyzing 6 replicates at 7 different plasma concentrations (10.0, 20.0, 40.0, 80.0, 120.0, 400.0 and 800.0 ng/ml) on 3 different days. The criteria for acceptability of the data included accuracy within ± 15 % standard deviation (SD) from the nominal values and a precision of within ± 15 % relative standard deviation (RSD), except for lower limit of quantitation (LOQ), where it should not exceed ± 20 %. Stability: Low, medium and high quality control samples (n = 6) corresponding to 40.0, 120.0 and 800.0 ng/ml, respectively were retrieved from deep freezer after 3 freeze–thaw cycles according to the clinical protocols. Samples were frozen at − 80 °C in 3 cycles of 24.0, 36.0, and 48.0 h. In addition, the long-term stability of MO in quality control samples was also evaluated by analysis after 6 weeks of storage at − 80 °C. Autosampler stability was studied following 24 h-storage period in the autosampler tray. Bench top stability was studied for 12-h period. Stability samples were processed and extracted along with the freshly spiked calibration curve standards. The precision and accuracy for the stability samples must be within ≤ 15 and ± 15 %, respectively, of their nominal concentrations.
Ezzeldin E et al. Montleukast Analysis & Pharmacokinetics … Drug Res 2014; 64: 617–622
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Fig. 1 Chemical structure of montelukast.
Original Article 619
Pharmacokinetic study 6 healthy adult male volunteers were included in this study. Male healthy volunteers, 22–37 years of age, with not more than ± 10 % from ideal weight (54–100 kg, the describe weights for Men – 1983 Metropolitan Height and Weight table) for their age were considered eligible. On the basis of medical history, clinical examination and laboratory investigations (haematology, blood biochemistry, urine analysis and viruses scanning), none of the participants has revealed any medical abnormality. In addition, the included subjects have no history of hospitalization or involvement in any clinical trials within 12 weeks, and none of them had received any regular course of drug therapy within 4 weeks prior to this study. Informed written consent was obtained from the volunteers and the protocol of the study was approved by the Ethical Committee of National Organization for Drug Control and Research. Each subject received Singulair 10 mg tablets. The order of product administration was done after an overnight fast of 10.0 h. Food is only allowed after 4 h of montelukast administration. Similar meals were standardized in amount and composition (protein, carbohydrate and vegetable) and served after 4 and 8 h post dosing. Blood samples were collected in heparinized tube, at 0.0 (pre-dose), 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 7.0, 10.0, 12.0 and 24.0 h after drug administration. The samples were centrifuged and the obtained plasma was frozen at − 80 °C until analyzed.
Results and Discussion
▼
The goal of this work was to develop and validate a simple, rapid and sensitive assay method for determination of MO in human plasma and its application in pharmacokinetics. To achieve the goal, during method development, different options were evaluated to optimize sample extraction, detection parameters and chromatography.
Validation of method of analysis Electrospray ionization (ESI) was evaluated to get a better response from analytes. It was found that the best signal was achieved with ESI positive ion mode. The product ion spectrum for MO and GL yielded high abundance fragment ions of m/z
▶ Fig. 2). Further optimiza568.3 and m/z 127.2, respectively (● tion in chromatographic conditions increased the signal of analytes. Several trials were done to select the best ratio of the mobile phase components to achieve the best resolution between montelukast and the internal standard. It was found that the ratio 86 % acetonitrile and 0.1 % formic acid was excellent in separation. This mobile phase was delivered at a flow rate of 0.6 ml/min. The average retention times of the internal standard and montelukast were 1.4 and 2.3 min, respectively. Glaclizide, easily available as a pharmaceutical substance, so it was selected among many compounds we tested as internal standard. Under these chromatographic conditions described, montelukast and the internal standard peaks were well resolved. No interfering peaks from endogenous plasma components were observed. The representative typical MRM chromatograms of blank plasma in comparison to plasma spiked with gliclazide, spiked with montelukast and representative plasma sample from healthy volunteer analyzed for pharmacokinetic study ▶ Fig. 3). The standard calibration curve for were shown in (● montelukast relating concentration to peak area ratio was found to be linear within the range of 10.0–800.0 ng/ml (r2 = 0.997 with ▶ Table 1, ● ▶ Fig. 4). low limit of quantification of 10.0 ng/ml) (● The intraday precision and the accuracy value of the method were < 15 %. Inter-day precision values were found to be < 15 % ▶ Table 1). The recovery ranged from 72.77 to 104.83 % for MO (● ▶ Table 2 summarizes the concentrations of 10.0–800.0 ng/ml. ● freeze and thaw stability, short term stability, long-term stability and auto sampler stability data of MO. All the results showed the reliable stability behavior during these tests and there were no stability-related problems during the routine analysis of samples for the bioavailability study. The present method is characterized from the other previous methods in the using small volume of plasma and acetonitrile. Extraction procedure involved one step of protein precipitation by acetonitrile which is simpler than liquid-liquid extraction [11, 17]. MO has been separated using reversed C18 column rather than dual column [14, 15], switching column [14] system or chiral column [13].
Ezzeldin E et al. Montleukast Analysis & Pharmacokinetics … Drug Res 2014; 64: 617–622
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Fig. 2 Mass spectra of the positive ion of montelukast (MO) and gliclazide (GL).
620 Original Article
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Fig. 3 Representation MRM chromatograms of a blank plasma,b plasma spiked with 25 ng/ml of gliclazide (GL), c Plasma spiked with 400 ng /ml of montelukast, d plasma spiked with 25 ng/ml of gliclazide and 400 ng/ml montelukast and e plasma sample from healthy volunteer at 3.5 h of after an oral administration of 10 mg of montelukast.
Table 1 Intra-day and inter-day reproducibility of standard plasma calibration curves of montelukast. Theoretical concentration (ng/ml) Parameters
Inter-days repeatability 10
Mean Precision Accuracy ( %)
10.18 9.37 101.75
20 18.78 9.48 93.92
40 39.25 11.53 98.14
80 80.46 11.88 100.57
120
Intra-day repeatability 400
800
10
Concentration found (ng/ml) 107.34 433.97 796.40 10.18 10.41 12.02 14.96 8.88 89.45 108.49 99.55 101.78
20
40
80
120
400
17.89 2.37 89.45
35.97 12.41 89.92
72.92 9.56 91.15
96.19 9.31 80.15
374.47 9.87 93.62
800 705.73 14.35 88.22
Pharmacokinetic study
Safety evaluation
Successful completion of the clinical phase was done by 6 subjects in the single-dose fasting pharmacokinetic study. Following oral administration, mean plasma concentration-time profile of MO best fit a non-compartment in all subjects. Plasma con▶ Fig. 5. The pharmacokicentration-time profile was shown in ● ▶ Table 3. The median t netic parameters were shown in ● max, Cmax and AUC values were similar to those reported in the literature [12, 25].
Having completed the study, subject underwent a through physical assessment and vital signs evaluation on follow-up examination to assure their safety. Clinical assessment for all subjects was carried out to evaluate their tolerability to the study medications. Subjects demonstrated good tolerance to the study drug.
Ezzeldin E et al. Montleukast Analysis & Pharmacokinetics … Drug Res 2014; 64: 617–622
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procedure. The lag time between the main peak of drug and the internal standard is about 1 min. This in turn, led to a shortened analysis time ( < 5 min). This method characterized also by use of small volume of plasma and acetonitrile as precipitating agent in drug extraction.
Competing Interests
▼ Fig. 4 Standard calibration curve for montelukast (mean peak area ratio vs. concentration of montoleukast).
The authors declare that they have no conflict of interests.
Reference
Accuracy (mean ± CV %) 40.0 ng/ml
120.0 ng/ml
Short-term stability 104.45 ± 10.38 105.32 ± 11.60 (6 h, room temperature) Long-term stability 111.31 ± 6.99 104.03 ± 5.15 (42 days, − 80 °C) Freeze and thaw stability 106.48 ± 9.24 106.69 ± 5.66 (3 cycles, − 80 °C–room temperature)
800.0 ng/ml 93.60 ± 9.75 96.74 ± 10.33 103.29 ± 12.28
Fig. 5 Mean plasma concentration-time profiles after single oral dose (10 mg montelukast).
Table 3 Pharmacokinetic parameters of montelukast. Parameters Cmax (ng/ml) Tmax (Median) (h) AUC0–24 (ng.h/ml) AUC0–inf (ng.h/ml) t1/2 (h) MRT (h) Kel (h)
Mean ± SD 503.6 ± 204.1 3.5 3 628.3 ± 1 582.5 4 038.5 ± 1 651.7 6.2 ± 1.1 9.3 ± 1.4 0.11 ± 0.01
Conclusion
▼
The aim of our study was to develop a rapid and sensitive method for the analysis of plasma samples in pharmacokinetic montelukast. This method is well suited for pharmacokinetics studies because of the speed of analysis and simple extraction
▼
The title of this article has been corrected.
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