Highly Sensitive Micellar Enhanced Spectrofluorimetric Method for Determination of Mirtazapine in Tablets and Human Urine : Application to In Vitro Drug Release and Content Uniformity Test

1Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia 2Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr El-Aini Street, Cairo 11562, Egypt 3Department of Chemistry, Faculty of Science and Technology, El-Neelain University, P.O. Box 12702, 12702 Khartoum, Sudan 4Department of Aviation Medicine, King Abdulaziz Medical City, Ministry of National Guard, P.O. Box 22490, Riyadh 11426, Saudi Arabia


Introduction
Mirtazapine (MRZ; [1,2,3,4,10,14b-hexahydro-2-methylpyrazino- [2,1a] pyrido [2,3-c] benzazepine]) (Figure 1) belongs to chemical class of piperazinoazepines.MRZ is an antidepressant of the second generation and a member of noradrenergic and specific serotonergic class.MRZ acts by increasing serotonin and norepinephrine discharge, maybe due to its antagonistic action on  2 -adrenergic heteroreceptors and autoreceptors.High efficiency of MRZ as an antidepressant drug is due to its unique mechanism which differs from the other drugs belonging to the same class [1].Currently, MRZ is utilized for treatment of number of disorders including obsessive-compulsive disorder [2], posttraumatic stress disorder [3], generalized anxiety disorder [4], and postchemotherapy nausea and vomiting [5] (due to its antiemetic properties).Regarding MRZ dose, it initiates with 15-30 mg in the first day of administration and then increases according to the patients' needs [6].Upon extensive survey in the literature, it was found that several techniques were adopted for assaying MRZ in REMERON tablets including spectrophotometry [7], spectrofluorimetry [8], high performance liquid chromatography (HPLC) [8], and capillary zone electrophoresis (CZE) [8].MRZ was determined in biological fluids utilizing spectrofluorimetry [9], HPLC [10][11][12], gas chromatography (GC) [13], capillary electrophoresis (CE) [14], and high performance thin layer chromatography (HPTLC) [15].For analysis of MRZ in urine, there are only few techniques reported for that aim.These techniques include GC coupled with tandem mass spectrometry [16,17], liquid chromatography coupled with mass spectrometry [18], and CE [19].These techniques suffered from several disadvantages such as being laborious, time consuming, and in need for costly apparatuses that lessened their availability in developing communities.Therefore, the aim of the current study is to develop simple and sensitive spectrofluorimetric method for assaying MRZ in dosage form and urine samples.Moreover, the adopted method was applied for testing REMERON tablets content uniformity and in vitro drug release from REMERON tablets following USP guidelines.

Apparatus
(i) All fluorescence measurements were performed on Jasco FP-8200 Fluorescence Spectrometer (Jasco Corporation, Japan) equipped with 150 W xenon lamp and 1 cm quartz cells with bandwidth of 5 nm for excitation and emission monochromators.All data acquisition was handled utilizing SpectraManager5 software.(ii) pH was measured and adjusted using Hanna pH-Meter (Romania).(iii) In vitro drug release was tested using automatic dissolution tester (8-cup system) (Abbota Corporation, New Jersey, United States).The volumetric flask was sonicated for 30 min and then diluted to the mark with the same solvent.This solution (0.3 mgmL −1 ) was filtered with chromafil5 Xtra 0.2 m filter paper and diluted with ACN to get appropriate concentrations of MRZ for subsequent analysis.Finally, the procedures designated under "Construction of Calibration Graph" were followed.

Reagents and
2.6.Determination of Human Urine Samples.One mL of free drug human urine was spiked with 20 L of various MRZ standard solutions and mixed for 30 min.A volume of one milliliter of NaOH 100 mM/glycine buffer (pH∼11) was added and then mixing took place for 10 s.Liquid-liquid extraction was accomplished using five milliliters of diethyl ether and the solution was placed in the vortex mixer for 30 seconds followed by centrifugation for 15 minutes at 10,000 rpm (for complete phases separation).Then three milliliters of the upper organic layer was transferred into glass vials and dried utilizing gentle stream of nitrogen.Finally, the residue was reconstituted in methanol and appropriate dilutions were performed with ACN to yield final MRZ concentrations of 100 ng/mL, 200 ng/mL, 300 ng/mL, and 400 ng/mL.The procedures termed under "Construction of Calibration Graph" were performed.Treatment of a blank urine sample took place in a similar way.FI was measured at 403 nm after excitation at 320 nm.Linearity was checked by computing regression equation and MRZ concentrations were calculated.

Procedure for Content Uniformity Testing for MRZ.
Analysis of number of REMERON tablets was carried out by applying the procedure adopted under "Determination of Tablet Samples" for assaying MRZ.USP guidelines [20] (Chapter 905: Uniformity of Dosage Units) were followed for studying the uniformity of REMERON tablets.

Procedure for In Vitro Drug Release Test (Dissolution Test)
for MRZ.For testing in vitro release of REMERON tablets, dissolution procedure of USP [20] was followed utilizing USP apparatus II.A volume of 900 mL of 0.1 N HCl solution, kept at ambient temperature, was utilized for dissolution of the cited tablets and stirred at a speed of 50 rpm for 25 min.A volume of five mL of REMERON tablet sample was taken and filtered using 0.45 m syringe filter.The volume was kept constant by substitution of the withdrawn volume by dissolution medium prepared freshly at the selected time intervals.Afterwards, the same procedures mentioned under "Construction of the Calibration Graph" were followed for assaying of MRZ in these filtered samples.

Results and Discussion
Spectrofluorimetry, as an analytical technique, was characterized by number of advantages including sensitivity and simplicity which offered multiapplication for this technique.Sensitivity allowed the determination of MRZ in urine while simplicity was beneficial for analysis of its dosage form in addition to content uniformity and dissolution rate testing.In this study, sensitivity of spectrofluorimetry was enhanced by addition of surface active agents which resulted in micelle formation.Micellar enhanced spectrofluorimetry was adopted and applied in assaying several active ingredients in many reports [21][22][23][24][25][26][27] due to the ability of the micelle to enhance RFI of these drugs in addition to the exclusion of organic solvents and thus increase the greenness of the adopted procedure.RFI of the cited drug A concentration of 1% w/v for different surfactants was chosen as it is higher than the reported critical micelle concertation (cmc) of these surfactants.Maximum RFI was attained utilizing SLS (Figure 3).For optimization of the concentration of organized media, various volumes of 1% w/v SLS were added to MRZ solution.It was clear from Figure 4 that optimum RFI for MRZ was reached by utilizing a final concentration of 1.5 × 10 −3 Molar SLS (equivalent to 0.2 mL 1% SLS) and any extra addition of SLS had no influence on the MRZ response.Therefore, 1.75 × 10 −3 Molar SLS (equivalent to 0.3 mL 1% w/v) was added in all subsequent experiments.

Effect of pH.
Different buffers and acids (covering pH range 1-12) were examined to optimize the influence of pH on RFI of MRZ and MRZ-SLS and SLS, as shown in Figure 5.
It was clear from that figure that RFI of MRZ alone was optimum at highly acidic solutions and decreased remarkably as pH increased.This may be attributed to alteration of MRZ ionization.Regarding SLS surfactant, its RFI was almost nil in all pH range which considered an advantage for utilizing this anionic surfactant for MRZ determination in this work.Ultimately, maximum RFI for MRZ-SLS was attained at acidic solutions (specifically 0.1 N H 2 SO 4 ) and RFI decreased noticeably on increasing pH values.These findings assure that MRZ at acidic pH attained positive charge and hence it is logic to interact well with the negatively charged sulphonyl group of SLS.This thought was supported by Chemicalize [28] calculations.The interaction of MRZ-SLS was proposed in Scheme 1.

Effect of Diluting Solvent.
Various solvents were examined as diluting solvents in the current study.Among the tested solvents, the highest response was attained using water as displayed in Figure 6.This offered an advantage for the proposed procedure due to the absence of the organic solvent.This behavior may be attributed to the increment of medium polarity by water which in turn increased the interaction between water molecules and MRZ ones (Figure 6).

Effect of
(3) (4) micellar medium was developed immediately and was stable for at least 30 minutes.

Validation of the Method
4.1.Linearity and Range.The calibration curve was linear through the concentration range of 1-500 ng/mL as displayed in Table 1.
From Table 1, it can be seen that the linearity was proven from the high values of the determination coefficient () and low values of standard deviation of residuals ( / ), intercept (S a ), slope (S b ), % RSD, and % error [29].4.2.Sensitivity.Calculation of limit of quantitation (LOQ) and limit of detection (LOD) was carried out in accordance with the ICH Q2 (R1) recommendation [30].Their values were abridged in Table 1.These values were computed by applying the next equation: where ℎ is the SD of the intercept of regression line of calibration curve while  is the slope of regression line of the calibration curve (Table 1).

Accuracy.
Testing the accuracy of the suggested procedure took place by analyzing different samples of MRZ standard solution equivalent to different concentrations.Mean % recovery was 99.05 ± 1.83 as displayed in Table 2 which in turn reflects the high accuracy of the current spectrofluorimetric method.Additionally, the accuracy of the current procedure was assessed by investigating the results of analyzing REMERON tablets and spiked urine samples as seen in Table 2.

Repeatability and Reproducibility.
Intra-and interassay precision were evaluated by analyzing various MRZ concentrations (20, 100, 200, and 400 ng/mL) in triplicate in one day and in three successive days, respectively.Mean % recoveries were almost equal to 100% and the relative standard deviation values (%RSD) were small.These values are evidence for the precision and accuracy of the suggested procedure (Table 3).changes of the experimental conditions.It was found that small changes that possibly occur during the experimental runs will have no significant effect on the fluorescence intensity of the cited drug.

Robustness.
4.6.Specificity.For investigation of the specificity of the proposed procedure, interferences that may result from inactive ingredients of tablets were observed.It was clear from the results of analysis of REMERON tablets that these inactive ingredients did not exhibit any interference as can be seen in Table 2.

Conclusion
Micellar enhanced spectrofluorimetric methodology was conducted for assaying MRZ in different matrices.The proposed method is characterized by its simplicity, sensitivity, and rapidity when compared to the reported chromatographic methodology.The current procedure was applied with a great success for assaying MRZ in REMERON tablets and spiked human urine.There were no interferences raised from the common inactive ingredients of REMERON tablets or the endogenous compounds in urine samples.The current study was extended for application of content uniformity testing.Additionally, it has been utilized for dissolution testing of MRZ tablets.Economically, the method offered cheap analytical procedure for assaying MRZ as it depended on measuring intrinsic fluorescence response of MRZ, rather than utilizing expensive derivatizing analytical reagents.Additionally, this technique belongs to green analytical methodology, as there is no utilization of organic solvent.

Competing Interests
The authors declare that there is no conflict of interests regarding the publication of this paper.[20] 3.81 Max.allowed AV (L1) [20] 15
Materials.MRZ was kindly gifted by Organon, OSS, and utilized without additional purification.All the chemicals used were of Analytical Reagents grade, and the solvents were of HPLC grade.
Fluorescence Spectra and Characteristics of MRZ.Molecular emission behavior of a certain compound is represented by two spectra.The first one represents the process of electronic excitation and the second one represents the process of emission.Thus molecular emission spectra are characterized by two wavelengths: excitation and emission wavelengths ( ex / em ).Figures2(a) and 2(b) showed that MRZ have  ex / em of 320/403 nm.This labeled the native fluorescence property of MRZ.Upon addition of SLS to MRZ standard solution, the RFI was improved remarkably as shown in peak number (2) in Figure2(a).
(MRZ) was affected by number of factors including pH, type, and concentration of surfactant, diluting solvent, and others.Optimization of these factors was done by adjusting each factor individually while the other factors kept constant or what we call "one factor at a time optimization strategy."3.1.

Table 1 :
Table 4summarizes the method robustness by evaluating the liability of determinations to deliberate Analytical performance data for the spectrofluorimetric determination of MRZ.
a Percentage relative standard deviation for three replicate samples.b Percentage relative error for three replicate samples.c Limit of detection.d Limit of quantitation.

Table 2 .
5.1.Application of Procedure to Analysis of MRZ in REMERONTablets.REMERON tablets were assayed efficiently by the suggested spectrofluorimetric procedure.Mean percent recoveries of various concentrations depended on the mean of triplicate determinations.The % recovery ranged from 98.37 ± 1.96 as displayed in Table2.These results indicated the high accuracy of the method.

Table 2 :
Results of the determination of MRZ in pure form, REMERON tablets (Lot number 783147), and urine samples.

Table 3 :
Accuracy and precision data for the determination of MRZ by the proposed method.

Table 4 :
Robustness of the proposed spectrofluorimetric method.Mean of three determinations, b following the general calibration procedures. a

Table 5 :
Results of weight variation testing of MRZ tablets using the proposed method.