Development and validation of a stability indicating HPLC-diode array-fluorescence method for the determination of meclofenoxate hydrochloride and p-chlorophenoxyacetic acid

Article history: Received on: 30/04/2016 Revised on: 14/05/2016 Accepted on: 21/06/2016 Available online: 28/07/2016 The present work deals with the development and validation of a stability indicating HPLC-diode arrayfluorescence method (HPLC-DAD-FL) for the determination of meclofenoxate hydrochloride (MFX) and its hydrolytic degradation product p-chlorophenoxyacetic acid (pCPA). The chromatographic separation was achieved using a reversed phase Zorbax Eclipse SB-C18 column (250 x 4.6 mm i.d., 5 μm particle size), a mobile phase composed of 20 mM phosphate buffer, adjusted to pH 3, and acetonitrile (65: 35, v/v); and isocratic flow at a rate of 1 mL min -1 .The detection was carried out using a diode array detector (DAD) at 225 nm and a fluorescence detector (FL) at λex/em, 225/310 nm. The linear dynamic ranges were found to be 0.5-100 and 0.25-100 μgmL -1 (DAD); and 0.05-20 and 0.01-8 μg mL -1 (FL) for MFX and pCPA, respectively. Detection limits were 0.05 and 0.04μgmL -1 (DAD); and 0.005 and 0.001 μg mL -1 (FL) for MFX and pCPA, respectively. The investigated method was applied to the assay of MFX in tablets and vials dosage forms and also to the kinetics stability studies of MFX in different media. Further, the dissolution rate of the tablets was examined using the proposed method.

To our knowledge, no HPLC-fluorescence detection method has been reported for the determination of MFX or pCPA.The first target of this work was to establish stability indicating assay method, so the method was applied to study the forced hydrolytic, oxidative and photolytic degradation of MFX.MFX showed good resolution from all degradation products.It should be noted that the other reported methods have considered only the hydrolytic degradation.
Another target was to apply the developed method to the kinetic stability study of MFX, to investigate its half-life time in aqueous and non aqueous solutions to give enough information about the most appropriate solvent for the stability of MFX standard solutions and the best pH for analytical method optimization.Also, the method was applied to the determination of MFX and its degradation product, pCPA in pharmaceutical formulations and for MFX tablets dissolution testing.

Instrumentation
The HPLC equipment was Agilent 1200 series (Agilent Technologies, Santa Clara, CA, USA) (quaternary pump, vacuum degasser) with auto-sampler (G13298 1260 series ALS) and two detectors: diode array and multiple wavelength detector (G1315 C/D and G1365 C/D); and a fluorescence detector (G1321C 1260 series FLD) connected to a computer loaded with Agilent ChemStation Software.The column used was Zorbax Eclipse SB-C18 (250 x 4.6 mm i.d., 5 µm particle size) Agilent.The flow rate was maintained at 1 mL min -1 and the injection volume was 20 μL.The mobile phase was prepared daily, filtered through a 0.45 μm membrane filter.
The photo-degradation was carried out using an ultra violet lamp of the Horizontal Laminar Flow Clean Work Bench modelNU-201 and NU-201(E).The dissolution testing of MFX Lucidril ® tablets was performed on a six station dissolution apparatus, Hanson Research Corp., Northridge California,USA.

Materials and reagents
Authentic sample of MFX,98% was purchased from Alfa Aesar, a Johnson Matthey Company, Karlsruhe, Germany.Lucidril ® tablets labeled to contain 250 mg MFX per tablet and Luciforte ® vials labeled to contain 500 mg MFX per vial, both are manufactured by Minapharm, Egypt and purchased from local market.HPLC grade acetonitrile and methanol were purchased from LAB-SCAN Analytical Sciences, Poland.HPLC grade ophosphoric acid (LOBA chemie PVT-LTD.Mumbai, India) was used.Disodium hydrogen phosphate, sodium hydroxide, hydrochloric acid, 30% hydrogen peroxide were of analytical grade.High purity distilled water was used.

Chromatographic conditions
The chromatographic separation was performed using a reversed phaseZorbax Eclipse SB-C18 column (250 x 4.6 mm i.d., 5 µm particle size) Agilent maintained at 25°C, eluted with mobile phase at a flow rate of 1 mL min -1 .The mobile phase consisted of 20mM phosphate buffer adjusted to pH 3 (using o-phosphoric acid) and acetonitrile (65: 35, v/v), filtered prior to use through 0.45 µm nylon filter and degassed prior to use.The injection volume was20 µL.The eluents were monitored using DAD at 225 nm and FL at λ ex/em , 225/310 nm.

Preparation of pCPA
pCPA was prepared by alkaline hydrolysis of MFX using 2 M NaOH following the reported method (El-Bardicy et al., 2007 a).

Preparation of solutions Preparation of stock and working standard solutions
Stock solution of 1000 μgmL -1 of MFX in 0.1 M HCl was prepared and kept in the refrigerator.Working standard solutions were prepared by suitable dilution with the mobile phase to cover the concentration range of 0.5-100 μg mL -1 and 0.05-20 μg mL -1 for DAD and FL, respectively.The stock solution should be freshly prepared daily.
Stock solution of 1000 μg mL -1 of pCPA was prepared by dissolving pCPA powder in 2 mL of methanol followed by dilution with distilled water.Working standard solutions were prepared by suitable dilution with the mobile phase to cover the concentration range of 0.25-100 μg mL -1 and 0.01-8 μg mL -1 for DAD and FL, respectively.

Preparation of dosage form solutions Preparation of MFX tablets solution
A total of 20 Lucidril ® tablets were weighed and finely powdered.An accurate weight of the finely powdered sample equivalent to 50 mg of MFX was extracted into about 2 mL methanol with the aid of sonication for 1 min, using ultrasonic bath, then 20 mL of 0.01 M HCl were added and the sonication was continued for further 10 min.The mixture was filtered into a 50-ml volumetric flask.The residue was washed with two 10-ml portions of 0.01 M HCl and washings were added to the filtrate and diluted to volume with 0.01 M HCl.The concentration of this prepared assay solution is 1000 µg mL -1 .Further suitable dilutions were made with the mobile phase before chromatographic analysis.

Preparation of MFX vials sample solution
Fifty mg from Lucifort ® vial was transferred into 50-mL volumetric flask containing about 2 mL of methanol.The powder was dissolved and the volume was completed with 0.01 M HCl to prepare 1000 µg mL -1 assay vials solution.Further suitable dilutions were made with the mobile phase before chromategraphic analysis.

Procedure for forced degradation study
Forced degradation of MFX was carried out under neutral/ acid/ base hydrolytic, oxidative and photolytic stress conditions.MFX powder was dissolved in distilled water (for neutral hydrolysis, using 6h study time), 0.1 M HCl (for acid hydrolysis, using 6h study time), 0.1 M NaOH (for base hydrolysis, using 5 min study time), all kept at ambient temperature.After the degradation time specified was elapsed, a neutralization step of acidic and alkaline degraded solution was done.Chromatographic analysis of the degraded samples was carried out, taking into consideration a suitable dilution with the mobile phase, to get concentrations of 100 and 10 µgmL -1 MFX for the DAD and FL, respectively, as initial concentrations.
For the oxidative degradation, MFX drug powder was dissolved in 8% H 2 O 2 (prepared by dilution of hydrogen peroxide 30 % with distilled water), and allowed for stress degradation for 24h at ambient temperature.Another experiment was performed using 10% H 2 O 2 for 6 h at 80°C.Chromatographic analysis of the degraded samples was carried out, taking into consideration a suitable dilution with the mobile phase, to get concentration of 500 µgmL -1 MFX for the DAD and FL, as initial concentration.
Photo-degradation was carried out on solid drug substance and on solutions of MFX prepared in 0.01 M HCl and in distilled water.The solid and the solutions were irradiated with UV radiation having peak intensity at 254 nm for 3 h.After the specified time, the mobile phase was used to reconstitute the powder and to suitably dilute the solutions in order to get concentrations of 100 and 10 µg mL -1 MFX for the DAD and FL, respectively as initial concentrations.

Kinetic and stability study of MFX
Into a set of volumetric flasks, separate accurate volumes of a freshly prepared stock standard solution of MFX, 1000 µg mL -1 in distilled water, were diluted with a suitable volume of the medium to be investigated, to give final concentrations of 100 and 20 µgmL -1 MFX for the DAD and FL, respectively.The kinetic studies were performed at ambient temperature.The concentration of the remaining MFX was monitored at suitable time intervals, taking into consideration that the analysed solution was double diluted with the mobile phase before chromatographic analysis.

In vitro dissolution study of MFX tablets
In vitro dissolution testing of Lucidril ® tablets was performed with USP apparatus 2 (paddle method) using dissolution medium, 900 mL of HCl of pH 1.2 as simulated gastric medium.A paddle stirrer was used for 60 min at a stirring rate of 100 rpm.The temperature of the cell was maintained at 37 ± 0.5°C.
Five mL sample aliquots were withdrawn at time intervals of 5 min, and immediately replaced with equal volume of the fresh dissolution medium to maintain constant total volume.These aliquots were filtered with 0.45 µm millipore filter and diluted 20 folds and 200 folds for DAD and FL, respectively, with the mobile phase before chromatographic analysis.The drug dissolved was calculated from chromatographic analysis data with reference to a standard MFX solution analysed in parallel.Then, the cumulative percentages of the dissolved drug were calculated.
The dissolution data were obtained by averaging three parallel studies.

Method development and optimization of chromatographic conditions
In developing and optimization of the HPLC method, 3 objectives were considered; first, establishing a stability indicating method for the analysis of MFX and pCPA, with best resolution from oxidative degradation products, second, setting conditions which favor the stability of MFX as it is highly susceptible to hydrolysis, third, maximizing the sensitivity, especially speaking, fluorescence detection.
The first study in the method development was concerned with the chromatographic separation of MFX and pCPA, using two tested columns; Zorbax SB-C8 (250 x 4.6 mm) and Zorbax Eclipse SB-C18 (250 x 4.6 mm);and a mobile phase consisting of 20mM acetate buffer adjusted to pH 3 (using acetic acid) : methanol (65: 35, v/v).The results referred to un-significant difference, regarding retention, resolution and peak symmetry.The C18 column was selected for the following studies.The second study aimed to compare between 20mM phosphate buffer of pH 3 and 20mM acetate buffer of pH 3, both at a ratio of buffer: methanol (65: 35, v/v), The results referred to un-significant difference, regarding retention, resolution, peak symmetry and UV detection sensitivity.However, phosphate buffer showed fluorescence intensity (peak area) nearly double that of acetate buffer, so phosphate buffer was chosen as optimum for the following studies.The next study dealt with the comparison between methanol and acetonitrile as an organic modifier, both at a ratio of 20mM phosphate buffer of pH 3: organic modifier (65: 35, v/v).About 35 % enhancement in FL detection was observed with acetonitrile with no noticeable difference in chromatographic performance and in UV detection sensitivity.Accordingly, acetonitrile was selected as the optimum organic modifier.Other study was performed using 20mM phosphate buffer of pH 3, with variation of acetonitrile ratio from 25% to 60%.The results showed that at acetonitrile % > 40%, shorten analysis time with poor resolution was obtained while acetonitrile % ≤ 30% resulted in marked retardation in chromatographic separation.Accordingly, acetonitrile % between 35% and 40% seems to be the best.Although 40% acetonitrile showed slight enhancement in FL detection, relative to 35%acetonitrile, the latter was chosen as optimum as it allows better resolution of MFX and pCPA from oxidative degradation products.
The final study, in optimizing chromatographic conditions, targeted pH.Two factors were considered, the pHdependent stability of MFX and the pK a of pCPA (3.56).The pH limit for best stability of MFX could be deduced from reported first order rate constant, k (min -1 ) at pH 4 (1.19 x 10 -4 ) and pH 5 (2.39 x 10 -4 ), both correspond to t 90% 15h and 6.6h, respectively (Cooke et al., 1979;Ohta et al., 1986).So, working in pH ranged from 2 to 5 is practically possible, however, at pH 2, pCPA showed significant retarded elution, also at pH ≥ 4, it directed towards too early elution (ionized species predominate), resulting in poor resolution from MFX peak.Accordingly, pH 3 was chosen as a working pH.It was advantageous that the isocratic elution achieved good chromatographic performance and analysis time less than 20 min for separation of MFX from all possible degradation products.DAD was set at 225 nm to allow for best method sensitivity.Both MFX and pCPA show strictly overlapping UV spectra showing maximum absorption at 225 nm.
To our knowledge, no reported article has studied the native fluorescence properties of MFX to establish an analytical method.pCPA is the hydrolytic product, also the active metabolite, of MFX(Cooke et al., 1979).Its structure is closely related to chlorophenoxyacetic acid herbicides.Photodegraedinduced fluorimetric methods for the determination of chlorophenoxyacetic acid herbicides have been developed (Eremin et al., 1996;Garcia et al., 1996;Garcia-Campana et al., 2001;Almansa Lopez et al., 2003).The authors have claimed that all chlorophenoxyacetic acid herbicides have no native fluorescence.However, Schussler (1990) has developed an HPLC-fluorescence detection method based on measurement of native fluorescence . of chlorophenoxy derivatives, without any derivatization, in water down to ng levels.He showed that4-chloro-2methylphenoxyacetic acid and 4-chloro-2-methylphenoxybutanoic acid are natively fluorescent species, while 2,4dichlorophenoxyacetic acid and 2,4-dichlorophenoxybutanoic acid are not fluorescent.The present work targeted the native fluorescence of MFX and pCPA to establish a new analytical HPLC-FL method.The FL detector was set at λ ex/ em , 225/310 nm, to achieve optimum sensitivity.
Figure 1 shows the typical chromatograms for the separation of MFX and pCPA mixture at retention times 3.81 ± 0.06 and 9.82 ± 0.08 min, respectively using the proposed HPLC-DAD-FL method.
System Suitability Parameters were assessed to ensure adequate performance of the chromatographic system.Retention time (R t ), capacity factor (k), number of theoretical plates (N), peak resolution (R s ), selectivity (α) and tailing factor (T) were evaluated for six replicate injections of MFX and pCPA standard solutions at concentrations of 20 and 8 μg mL -1 , respectively.The results presented in Table1 are within the acceptable limits.
Fig. 1: HPLC chromatogram of 20-µL injection of (A) a mixture containing 40µg mL -1 MFX and 15µg mL -1 pCPA using DAD at 225 nm, (B) a mixture containing 5µg mL -1 MFX and 1µg mL -1 pCPA using FL at λex/em, 225/310nm.16.61 a system suitability parameters were evaluated for six replicate injections of MFX and pCPA standard solutions at concentrations of 20 and 8 μg mL -1 , respectively, calculated as average of DAD and FL data.

Results from forced degradation studies
The study aimed to evaluate the specificity of the method towards the separation of MFX from the potential degradation products.The hydrolytic degradation of MFX (in neutral, acidic and alkaline solutions) is well known and is a matter of many researches (Cooke et al., 1979;Tatsuhara and Tabuchi, 1980;Yoshioka et al., 1982;Yoshioka et al., 1983;Ohta et al., 1986;Araman et al., 1992;Otsuka et al., 1994;El-Bardicy et al., 2007 a;El-Bardicy et al., 2007b;El-Nashar et al., 2012).Table2 demonstrates the % remaining of MFX, it is clear that the reaction is base-catalysed.The chromatograms (Figures 2-4) show good resolution of MFX from the hydrolytic product, pCPA.It should be noted that, N,N-dimethylamino ethanol is not detected as it is a non UV absorbing species.
Forced oxidative degradation was performed with 8% H 2 O 2 for 24 h at ambient temperature and with 10% H 2 O 2 for 6h at 80°C.The DAD and FL chromatograms of the stressed samples showed peaks for MFX, pCPA and three oxidation products I, II, and III at 12.2, 14.60 and 15.80 min, respectively, with good resolution of MFX and pCPA from the oxidation products (Figure 5).Analogus experiment was done on pCPA, HPLC analysis showed the same oxidation products I, II, and III at the same retention times (Figure 6).The stress oxidation reaction of MFX and pCPA can be explained in the view of chromatographic results.MFX is highly liable to hydrolysis, even in H 2 O 2 aqueous solution, which most probably occurs in preference to oxidation.The readiness of MFX to get oxidized is slow.Identical oxidation products in the chromatograms of MFX and pCPA (as proved by retention times and UV spectra obtained by DAD) agreed with our assumption that these are the products of oxidation of pCPA.This means that hydrolysis of MFX occurs first then followed by oxidation of the hydrolytic product.Only small fraction of pCPA gets oxidized, even with 10% H 2 O 2 and heating for 6h at 80°C.This is in line with the reported oxidative degradation reaction of pCPA by H 2 O 2 which has been catalized photochemically to force the reaction to completion (Lang and Lunák, 2002).The oxidation products are natively fluorescent species, detected in the FL chromatogram.
HPLC chromatograms of photo-degraded MFX (UV irradiated solid or solutions) showed only pCPA peak, with no photo-degradation products.The hydrolysis of MFX was monitored in the % remaining of MFX (Table 2).
In all stress degradation studies, the peak purity data for MFX and pCPA were assessed (Table 2).

Linearity and concentration ranges
Standard solutions of six concentration levels, for MFX and pCPA, were prepared and injected twice for each level onto the HPLC system.Table 3 presents the linearity data and statistical parameters including concentration ranges, slope (b) and intercept (a) of the linear regression equations, correlation coefficients and standard deviations of the intercept (S a ), slope (S b ) and residuals (S y/x ).Regression analysis for the calibration curves showed good linear relationship over the concentration ranges stated in the table as judged by the correlation coefficient values (r > 0.9999)and RSD% of the slope (S b %) which did not exceed 0.76%.

Limits of detection and quantitation
The limit of detection (LOD) and limit of quantitation (LOQ) were calculated in accordance to the USP and ICH guidelines based on signal-to-noise ratio values (ICH, 2005).Data given in Table 3 reveals that the proposed method is sensitive enough to detect concentrations down to 0.05 and 0.04 μg mL -1 (DAD); and 0.005 and 0.001μg mL -1 (FL) for MFX and pCPA, respectively.

Accuracy
Accuracy of the developed methods was determined by performing recovery experiments.Known amounts of standards MFX and pCPA at three concentration levels were fortified to preanalysed degraded samples (MFX sample at 20 μg mL -1 concentration level was exposed to oxidative degradation using 10% H 2 O 2 at 80°C for 6 h, pre-analysed results were 8.05μg mL -1 MFX and 5.06 μg mL -1 pCPA).Three replicate samples of each concentration level were prepared and the percentage recovery at each level was determined (Table4), taking into consideration that the peak area of the standard added was calculated by the difference in peak area between fortified and unfortified samples.The mean percentage recoveries obtained ranged from 98.2 to 101.6%.The percentage relative standard deviation (RSD%) did not exceed 1.5 %. .50 a standards fortified to pre-analysed degraded samples containing 8.05 μg mL -1 MFX and 5.06 μg mL -1 pCPA.b recovery obtained by difference in peak area between fortified and unfortified pre-analysed degraded samples.c standard deviation (n=3).

Precision
Intra-day and inter-day precision were evaluated by analyzing three replicates over three concentration levels of standards MFX and pCPA, within the same day (intra-day repeatability) and on three consecutive days (inter-day precision).The intra-day and inter-day precision (% RSD) was found to be less than 2% (Table5), indicating that the method is precise.

Specificity
The specificity of the developed HPLC-DAD-FL method was demonstrated by its ability to measure accurately and specifically MFX and pCPA in the presence of other potential degradants or excipients (ICH, 2005).
Photodiode array detector was used to evaluate the homogeneity and purity of the chromatographic peaks.Chromatographic peak purity data was obtained from the Agilent Chemstation Software (Stahl, 2003).A similarity factor of 0 indicates no match and that of 1000 indicates identical spectra.Generally, values very close to the ideal similarity factor (greater than 995) indicate that the spectra are very similar (Stahl, 2003).The similarity factors for, MFX and pCPA peaks in the chromatograms of stressed samples, assayed tablets and vials were found to be in the range of 999.983-1000.All these factors lie within the corresponding automatically computed noise threshold limits indicating pure peaks.The threshold limits were found to be in the range of 999.936-999.974.
The specificity of the HPLC methods is also illustrated from chromatograms obtained with the mixture solution of standard MFX/pCPA, stressed samples, tablets solution and vials solution.Well-resolved peaks were observed for MFX and pCPA in all the studied chromatograms(Rs =16.61), and for pCPA and oxidation product I in the stressed samples chromatograms (Rs =3.85).Additionally, either of MFX or pCPA showed identical retention times in all the studied chromatograms.No interfering peaks from any of the excipients appear in tablets chromatogram.
Method specificity was further confirmed by the recovery experiments carried out on standards MFX and pCPA added to degraded samples (Table 4).This indicates that the .developed method was specific and can be applied to simultaneous estimation of MFX and pCPA in kinetic stability study of MFX and in analysis of pharmaceutical formulations, to monitor the % degraded.

Stability of Solutions
Standard solution of MFX, prepared in 0.1 M HCl, is stable at ambient temperature for up to8h only (regarding the t 98% , time required for the drug concentration to fall to 98% of the original value), so it should be freshly prepared daily.Standard solution of pCPA prepared in methanol/water is stable and can be kept in refrigerator for about one week.The stability was verified by chromatographic analysis and monitoring the respective retention time and peak area.

Robustness
To evaluate the robustness of the developed HPLC-DAD-FL method, small deliberate variations in optimized methods parameters were made once at a time (ICH, 2005).The parameters included variation of flow rate (± 0.05 mL min -1 ),detection wavelengths (± 1 nm), pH of the phosphate buffer (± 0.05), and % of acetonitrile in the mobile phase (± 0.5%).Replicate injections (n=3) of standard solutions (40 µg mL -1 MFX and 15µg mL -1 pCPA for DAD) (5µg mL -1 MFX and 1 µg mL -1 pCPA for FL) were used.These variations have no significant effect on the peak area, retention time, tailing factor and resolution of the analytes.

Assay of MFX Tablet and Vial dosage forms
The developed HPLC-DAD-FL method was applied to the assay of MFXcommercial pharmaceutical formulations (Lucidril ® tablets and Lucifort ® vials).Both the intact drug and its hydrolytic degradation product, pCPA were determined in 3 different batches.It should be noted that the samples were extracted with methanol prior to 0.01 M HCL to dissolve any present pCPA.The stability of MFX in methanol/ HCl was proved in the current kinetic study.In all batches, the mean % of MFX found was in the range of 95.9 -96.8% and for pCPA, the mean amount found was in the range of 5.53 -6.66 mg per tablet and 10.40 -12.03 mg per vial (Table 6).The hydrolysis of MFX in .commercial dosage forms is attributed to heat and humidity of storage conditions.The results agreed with the reported study on the hydrolysis of MFX in commercial tablets (Yoshioka et al., 1983).

Kinetic stability studies of MFX
Hydrolysis of MFX, in aqueous solution, is known to be first order reaction and pH dependent (Ohta et al., 1986).Some kinetic studies on MFX stability have been reported.These include MFX stability in solid dosage form to investigate effect of temperature and humidity (Yoshioka et al., 1982;Yoshioka et al., 1983), hydrolysis of MFX in buffer solutions at pH 4 to 11 (Cooke et al., 1979)and degradation reaction of MFX in 1M, 1.5M and 2M NaOH (El-Bardicy et al., 2007 a).
In the present work, the kinetic studies on MFX stability aimed to serve drug analysis, in view of selecting the most appropriate solvents to prepare stable MFX solutions or even optimization of the analytical method conditions (like pH of the solution, % organic solvent) to compromise drug stability and method sensitivity.So, the current kinetic study included stability of MFX in aqueous solutions of different pH values and in aqueous/organic solvent mixtures, as summarized in Table 7.The developed HPLC-DAD-FL method was applied to monitor the percentage remaining of MFX in the investigated media.The kinetic parameters were calculated using equations ( 1) and (2) (Sinko, 2006): where, k is the first order reaction rate constant, C 0 is the concentration of MFX at time t = 0 and C t is its concentration at time t.The degradation reaction rate constant was calculated from the slope of regression line representing the correlation of log % remaining of MFX and time.The kinetic parameters, presented in Table 7, include: k (rate constant), t 1/2 (half-life time) and t 90% (time required for the drug concentration to fall to 90% of the original value).
From the data given in Table 7, our findings could be reviewed in the following remarks: first, MFX is not stable enough As a conclusion, some points, seem to be valuable and should be high-lighten.First, MFX exhibits best stability in aqueous acidic solution of pH 1-3 (HCl, H 2 SO 4 and acetate buffer).Second, methanol fastens the degradation reaction, as seen in mixtures of methanol with water, HCl, and buffers of pH 3 and 7.4.However, acetonitrile and methanol added to acidic aqueous solutions, still keep good stability required for performing analytical experiments.

In vitro dissolution study of tablet dosage form
The developed HPLC-DAD-FL method was applied to calculate the amount of MFX released from Lucidril ® tablets (Table 8).The results showed that more than 75% of the drug dissolved within 20 min and about 93% of the labeled MFX per tablet eventually dissolved in 60 min.The dissolution profile for .tablets was considered satisfactory and agreed with the results reported for lucidril ® tablets, manufactured by Minapharm, Egypt (El-Nashar et al., 2012).

CONCLUSION
The present work described a simple, sensitive and reliable HPLC-DAD-FL method for the assay of MFX and pCPA.The paper is the first to exploit the native fluorescence properties of MFX and its hydrolytic product, pCPA, to establish a sensitive HPLC method.The limits of quantitation were found to be 18 and 5 ngmL -1 for MFX and pCPA, respectively with fluorescence detection without any derivatization.
The developed method is superior to the other previously reported analytical methods, as the present work considered hydrolytic, oxidative and photolytic degradations.To our knowledge, the previous attempts have concerned only with hydrolytic degradation.The developed HPLC-DAD-FL method was successfully applied to the analysis of MFX tablets and vials, to determine the intact drug as well as the hydrolytic product, pCPA, also applied to the dissolution testing of MFX tablets.Further, the method was applied to kinetic studies on MFX hydrolytic degradation reaction.Kinetic parameters including degradation rate constant, t 1/2 and t 90% were estimated.The kinetic data are especially valuable to give attention about the most appropriate media, which favor MFX stability, to be a guide in establishing analytical method conditions.

Table 1 :
System suitability parameters a of the developed HPLC-DAD-FL method.

Table 3 :
Analytical parameters for the determination of MFX and pCPA using the proposed HPLC-DAD-FL method.
a Sa,Standard deviation of the intercept.b Sb, Standard deviation of the slope.c Sy/x, Standard deviation of residuals ( standard error of estimates) .d Limit of detection.e Limit of quantitation.

Table 4 :
Accuracy of the proposed HPLC-DAD-FL method.

Table 5 :
Intra-day and inter-day precision of the proposed HPLC-DAD-FL method.

Table 6 :
Assay results of MFX and pCPA in commercial pharmaceutical preparations using the proposed HPLC-DAD-FL method.

Table 7 :
Kinetic parameters of MFX in different media using the proposed HPLC-DAD-FL method.t, time required for MFX to fall to 50% (t1/2) and 90% (t90%) of the original value.composed of 18.06 mg disodium phosphate and 17.61 mg potassium dihydrogen o-phosphate ,pH 6.82.
b c

Table 8 :
Dissolution study of Lucidril ® tablets using the proposed HPLC-DAD-FL method.The dissolution data were obtained by averaging three parallel studies.