Development and Validation of Densitometry-TLC Stability Indicating Methods for Quantitative Determination of Azelastine Hydrochloride and Emedastine Difumarate in their Drug Products

Stability indicating densitometry-TLC assay was established and validated for determination of azelastine hydrochloride and emedastine difumarate in the presence of their acid and oxidative degradants. Forced degradation was performed using 30% H2O2 and 5 M HCl. The method was based on thin-layer chromatographic separation of the two drugs from their degradants, using methanolammonia (9.5:0.5, v/v) as developing system, followed by densitometric measurements of the intact drug spots at 292 and 283 nm, respectively. The linear range was 0.5 10.0 μg/spot, with mean recoveries of 100.09 ± 0.53% and 100.36 ± 0.40%. The proposed method was successfully applied for the routine quality control analysis of the two drugs in synthetic mixtures and commercially available preparations. The degradation products were identified by IR and MS and the pathways were illustrated. The method was validated according to ICH.

In the literature, no method is available so far for separation and structure elucidation of the hydrolytic and oxidative degradants of AZT and ETD.Therefore, the aim of the present work is to establish densitometry-TLC method for the selective determination of both drugs.
 A Shimadzu GCMS-QP1000 EX quadrupole spectrometer.EI ionization was performed with an electron energy of 70 eV.The ion source temperature was 200 o C, scan mode was ACQ, and scan speed was 769 U s -1 .

Materials and Reagents
Azelastine-HCl was kindly supplied from European Egyptian Pharm Co., Egypt, with certified purity of 99.00%.Zalastine® Nasal Spray labeled to contain 1 mg azelastine-HCl per mL (BN 7579001, European Pharm Co., Egypt) and Azelast® Eye Drops, labeled to contain 0.5 mg azelastine-HCl per mL (BN 86872, product of El-Kahira Pharm and Chem Ind Co., EPCI, Egypt) were purchased from the local market.

Standard Solutions
Standard stock solutions of AZE and ETD (1 mg mL) were prepared in methanol and diluted with methanol to obtain working solutions of 5 -100 μg mL -1 for each drug.
The stock solutions were stable for one week at 4 o C.

Acid degradants
About 50 mg of azelastine-HCl or emedastine difumarate were refluxed with 50 mL 5 M HCl at 100 o C for 36 hours or 7 hours respectively, then neutralized with 5 M NaOH and evaporated to dryness under vacuum.The residue of each drug was extracted with 3x10 mL methanol, filtered into 50 mL volumetric flasks and the volume was completed with methanol.The obtained solutions were labeled to contain the acid degradants derived from 1 mg mL -1 of each drug.

Oxidative degradants
About 50 mg of each drug were weighed in 50 mL volumetric flask, completed to the mark with 30% H 2 O 2 , and left in the dark for 24 hours for azelastine-HCl and 6 hours for emedastine difumarate.Both solutions were evaporated to dryness under vacuum.
The residues were dissolved in 40 mL methanol and transferred separately to 50 mL volumetric flasks.The volume was completed with methanol to obtain a solution labeled to contain the oxidative degradants derived from 1 mg mL -1 of each drug.

Densitometry-TLC Method
TLC was performed on 20 x 20 cm aluminum plates precoated with silica gel F254, 10 µL of each azelastine-HCl or emedastine difumarate were applied to the plates with 25 µL Hamilton microsyringe.Ascending development of the plates, with methanol-10% ammonia(9.5:0.5, v/v) as mobile phase, was performed.After development, the plates were air-dried and scanned at 292 nm and 283 nm for AZT and ETD respectively in reflection photo mode and zigzag scan, with swing width=10.

UNDER PEER REVIEW Laboratory Prepared Mixtures
Aliquots of each standard drug solution (1 mg mL -1 ) equivalent to 90 -10 µg were transferred into a series of 10 mL volumetric flasks.Then mixed with volumes of the corresponding drug -acid or oxidative degradants (prepared as mentioned under 2.4 ) derived from 10 -90 µg azelastine-HCl or emedastine difumarate.Volumes were completed with methanol and the detailed under, "2.5 Densitometry-TLC Method" were followed.Intact drug concentrations were calculated from the corresponding regression equation.

Application to Pharmaceutical Formulations
The content of five bottles of Zalastine nasal spray or twelve bottles of Azelast eye drops were mixed and a volume equivalent to 25 mg azelastine-HCl was evaporated under vacuum.The residue was extracted with 2 x 10 mL methanol and filtered into 25 mL volumetric flask, and completed with methanol.
The contents of eighteen Emedastine 0.05% ophthalmic bottles were mixed and volume equivalent to 25 mg emedastine base was evaporated under vacuum and above details were followed.The obtained methanolic solutions labelled to contain 1 mg mL -1 of the each drug were analyzed by the proposed densitometric-TLC method as described under " Densitometry-TLC Method ".The concentration of each drug was calculated from the corresponding regression equation.

Results and Discussion
Forced degradation of both azelastine-HCl and emedastine difumarate has been studied through acid and oxidative stress conditions.Partial hydrolysis (about 50% as measured by the proposed densitometry TLC method) of azelastine-HCl was achieved after reflux with 5 M HCl for 36 hours or about 65% of emedastine difumarate after reflux with 5 M HCl for 7 hours.Also partial oxidative degradation (about 50%) of azelastine-HCl was obtained by keeping 1 mg mL -1 solution of it in 30% H 2 O 2 for 24 hours.While complete oxidative degradation of emedastine difumarate was obtained by keeping 1 mg mL -1 solution in 30% H 2 O 2 for 6 hours.

Separation and identification of degradants
The methanolic extracts of acid-hydrolysis and oxidative degradation products of each drug was tested by TLC on silica gel 60 F254 plates.Different developing systems were tried as mixture of toluene -methanol -chloroform -10% ammonia, and mixture of dichloromethane -methanol -triethylamine in different ratios no separation was achieved.Complete resolution of each drug from its degradants was UNDER PEER REVIEW achieved upon using a mixture of methanol -10% ammonia (9.5: 0.5 v/v) with detection under UV lamp at 254 nm.
For acid hydrolyzed azelastine-HCl, two spots with R f 0.66 and R f 0.84 were appeared and two spots at R f 0.73 and R f 0.86 for its oxidative degradation products.
Whereas the intact drug spot was at R f 0.59, Figure (1).However, for emedastine difumarate one spot at R f 0.73 for acid hydrolyzed solution and one spot at R f 0.4 for its oxidative degradant.Intact emedastine base spot appeared at R f 0.6 and difumarate spot appeared at R f 0.79 as shown in Figure (1).
The degradants of each drug was subsequently separated on preparative TLC plates using the same developing solvents and extracted with methanol.The methanolic solutions were evaporated under vacuum, the residues were confirmed by IR on KBr discs and mass spectroscopy, and the results are given in Table 1.The suggested pathway of azelastine HCl and emedastine difumarate degradation are shown in Scheme (1&2).

Linearity range
Good correlation was found to exist between the peak areas of the separated spots and drug concentration over the range of 0.5 -10 g/spot for azelastine-HCl and emedastine difumarate as indicated by correlation coefficient ( r= 0.9993 -0.9997),Table 2.

The limit of detection (LOD)
The LOD was calculated to be 0.031 and 0.042g/spot for azelastine-HCl and emedastine base, respectively, Table 2.

Accuracy
The previously mentioned procedure under linearity was repeated three times for five different concentrations within the linearity range.The mean percentage recoveries were ranged between 100.09% and 100.36% for the two drugs respectively, Table 2.

Precision
The precision of the assay (within assay and between assays) was determined for both drugs in triplicate at five concentration levels for each drug using the previous mentioned procedure under linearity in the same day Table 2.The intra day, RSD were 0.641-1.27%and 0.367-1.09%while intermediate RSD were 0.891-1.42%and 0.438 -1.31% for AZT and EDT respectively, Table 2.

UNDER PEER REVIEW Selectivity
The selectivity of the proposed method was revealed by analyzing laboratory prepared mixtures of intact drugs with each of its degradants.The method was applicable for the selective determination of intact AZT in presence of, 10 -45 % of its acid or oxidative degradants(Table 3) with mean recoveries of 99.42 ±1.59 % and 100.2 ± 1.44 % respectively.Table (4) shows that EDT could be determined in presence of up to 59 % of its acid degradant or up to 80 % of oxidative degradant with mean recoveries of 99.34 ± 1.55 and 99.58 ± 1.16%, respectively.

Robustness
Robustness was assessed by evaluating the influence of small variation of experimental variables as developing system composition, saturation time and temperature on reliability of the method.For mobile phase, methanol: ammonia 10%, 9.5:0.4 or 9.5:0.3gave RSD% not exceeding 1.52% for azelastine-HCl and 1.44% for emedastine.The small change in temperature 23, 25, 27 o C or saturation time (15±2 min) did not significantly affect the results.

Application of the proposed densitometric-TLC method
The proposed method was applied for the determination of the two drugs in their pharmaceutical preparations.The results revealed good recoveries ± RSD% of 99.33 ±1.210 %, 99.25 ± 1.176% for azelastine-HCl in Zalastine nasal spray or Azelast eye drops and 99.37 ±1.410 % for emedastine base in Emedastine ophthalmic solution.
Statistical analysis of the results obtained by the suggested method compared with the manufacturer [18] or official method for azelastine-HCl [4] and emedastine difumarate [1] revealed no significant difference within a probability of 95%; Table (5).However, the proposed densitometric-TLC method is more sensitive and more selective than the manufacturer or reported methods in being stability indicating one.
The validity of the proposed method was further assured by applying the standard addition technique.The mean percentage recoveries ± RSD% were 99.74 ± 1.381 and 99.56 ± 1.121 for azelastine-HCl and 99.95 ±1.412 for emedastine base; Table (6).

Conclusion
The suggested densitometric-TLC method proved to be pure green analytical chemistry, as the solvent used have minimal hazards.The method can be used for routine analysis of azelastine hydrochloride and emedastine in their drug substances, drug products and in presence of their acid and oxidative degradants.The method

Table 3 . Determination of azelastine-HCl in mixtures with its acid or oxidative degradation products by the proposed densitometric-TLC method Intact* (µg/spot) Acid degradants (µg/spot ) Recovery % of intact* Oxidative degradants (µg/spot ) Recovery% of intact a
a Added + remained in degraded solution.

Table 4 . Determination of emedastine base in mixtures with its acid or oxidative degradation products by the proposed densitometric- TLC method Intact* (µg/spot) Acid degradants (µg/spot ) Recovery% of intact a Intact (µg/spot)
a Added + remained in degraded solution.

Table 5 . Statistical analysis of the results obtained by the proposed densitometric-TLC and manufacturer or official methods for the determination of azelastine-HCl and emedastine base Paramet ers
The values between paranthesis are the theoretical values of t and F at (p=0.05).

Table 6 . Application of standard addition technique for the determination of azelastine HCl and emedastine base by the proposed densitometric-TLC method Conc.(µg/spot ) Zalastine nasal spray Azalast eye drop Emedastine ophthalmic soln.
a Average of three determinations.