Journal of Chromatographic Science 2015;53:503– 510

doi:10.1093/chromsci/bmu075 Advance Access publication July 10, 2014 Article

Development and Validation of a Stability-Indicating Micellar Liquid Chromatographic Method

for the Determination of Timolol Maleate in the Presence of Its Degradation Products
Mohamed S. Rizk1, Hanan A. Merey2, Shereen M. Tawakkol1 and Mona N. Sweilam1*
1
Department of Analytical Chemistry, Faculty of Pharmacy, Helwan University, Ain Helwan, Cairo 11790, Egypt, and 2Department of
Analytical Chemistry, Faculty of Pharmacy, Cairo University, Kasr El Aini Street, Cairo 11562, Egypt

*Author to whom correspondence should be addressed. Email: monanabil@helwan.edu.eg, dr.monanabil@gmail.com

Received 18 September 2013; revised 10 May 2014

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A stability-indicating micellar liquid chromatographic (MLC) method in pharmaceutical dosage form under simulating Egyptian sum-
was developed and validated for the quantitative determination of ti- mer climate.
molol maleate (TM) in the presence of its degradation products result-
ing from accelerated degradation in a run time not more than 8 min.
TM was subjected to stress conditions of hydrolysis (including alka- Experimental
line, acidic and thermal hydrolysis) and oxidation. An isocratic, rapid Reagents and chemicals
and mobile phase saving the micellar LC method was developed with (i) TM pure sample was kindly supplied by Sigma Pharmaceu-
a BioBasic phenyl column (150 3 1.0 mm, 5 mm particle size) and a ticals Industries, Egypt, and its purity was found to be
micellar mobile phase composed of 0.1 M sodium dodecyl sulfate, 100.0 + 0.697 according to a comparison method (7).
10% of 1-propanol and 0.1% of triethylamine in 0.035 M ortho- (ii) Timololw 0.5% eye drops, labeled to contain 6.8 mg/mL TM
phosphoric acid. The flow rate of the mobile phase was 0.1 mL/min. equivalent to 5.0 mg/mL Timolol, manufactured by Egyptian
UV detection was adjusted at 298 nm and performed at room temper- International Pharmaceuticals Industries Co. (EIPICO),
ature. The method has been validated according to the International Egypt, were purchased from a local market.
Conference on Harmonisation guidelines. The method is successfully (iii) Ortho-phosphoric acid (85%, w/w) (HPLC grade), triethyl-
applied for the determination of TM in bulk powder and pharmaceu- amine (TEA) and sodium dodecyl sulfate (SDS, 99%) were
tical dosage form. purchased from Riedel-de Häen (Seelze, Germany).
1-Propanol (HPLC grade) and sodium hydroxide were pur-
chased from Sigma-Aldrich (Steinheim, Germany).
Hydrochloric acid (30%, w/v) and hydrogen peroxide
Introduction (30%, w/v) were purchased from Prolabo (West Chester,
PA, USA). Deionized water was obtained from Purelab flex
Timolol maleate (TM) is a nonselective b-adrenergic receptor
(Marlow, UK).
antagonist indicated orally for treating heart attacks and hyper-
tension, and topically for treating glaucoma by reducing aque-
ous humor production through blockage of the b-receptors on Instrumentation and chromatographic conditions
the ciliary epithelium (1). Several methods have been reported A Thermo Fisher Scientific HPLC Accela autosampler system
for the determination of TM including titrimetric official meth- equipped with an HPLC Accela photodiode array (PDA) detector
ods of USP (2) and BP (3), spectrophotometric determination (80 Hz version) (CA, USA) and an Accela 600 pump (CA, USA)
for TM alone (4 – 6) or in mix with other drugs (7 – 10), densi- was used. The injection volume was 20 mL. Analytical data were
tometric determination (11 – 13), high-performance liquid stored in a computer equipped with Chromoquest version 5 soft-
chromatography (HPLC) determination with other drugs ware. The pH was measured with Jenway pH meter 3510 (Essex,
(14 – 20), stability-indicating HPLC (21), chiral liquid chroma- UK). The mobile phase was filtered through Charles Austen
tography for enantiomer separation (22, 23), capillary electro- Pumps Ltd filter (model-B100 SE; UK), using a nylon membrane
phoresis (24, 25), chemiluminescence determination (26) and of 0.2 mm (Waters Corporation; Cork, Ireland). Ultrasonic used
voltammetric determination (27, 28). Micellar liquid chromato- was Power Sonic 410 (Treviglio, Italy). The analyses were carried
graphic (MLC) is an alternative to conventional reversed-phase out on a BioBasic phenyl column (150  1.0 mm, 5 mm particle
liquid chromatography with aqueous organic mobile phase in size; Thermo Corporation, CA, USA), as a stationary phase. The
which the mobile phase is aqueous solution of a surfactant at a mobile phase was prepared by transferring 28.84 g of SDS
concentration above the critical micelle concentration. Micellar (0.1 M), 100 mL of 1-propanol (10%) and 1 mL of TEA (0.1%)
LC has many advantages over RPLC, as it enhances the separation into a 1-L volumetric flask and then completed to volume with
selectivity; it has the ability of separation of hydrophilic and hy- 0.035 M ortho-phosphoric acid. The apparent pH was adjusted
drophobic analytes in the same run without gradient elution to 2.8 if necessary with ortho-phosphoric acid. The mobile
and enables the direct injection of untreated physiological fluids. phase was filtered and sonicated for 30 min before use. The
The aim of this work is to develop a novel stability study for TM flow rate of the mobile phase was 0.1 mL/min. The wavelength
under stress condition of hydrolysis (including alkaline, acidic of the PDA detector was set at 298 nm to detect TM and its deg-
and thermal hydrolysis) and oxidation of TM. There are no re- radation products. All analyses were carried out at room
ported HPLC methods for stability study of TM in pure form or temperature.
# The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com
Preparation of solutions Thermal degradation
Accurately weighed amounts of (20.0 mg) TM powder were
Preparation of standard solutions transferred into five 250-mL conical flasks, and 50 mL of double
Standard stock solutions. Standard stock solution of TM distilled water was added and refluxed for time intervals 5, 30, 60,
(1.0 mg/mL): An accurately weighed 100.0 mg of TM was 90 and 120 min at 1008C, respectively. After cooling, 1 mL from
transferred into a 100-mL volumetric flask, dissolved and each solution was transferred separately into a 10-mL volumetric
completed to volume with mobile phase. flask and then the volume was completed with mobile phase. The
solution was filtered through a disposable syringe filter (0.2 mm),
and triplicate 20 mL were injected into the column.
Standard working solutions. Standard working solution of
TM (0.1 mg/mL): A portion of 10 mL of the previously Degradation of eye drops under conditions simulating the
prepared standard stock solution was transferred into a 100-mL Egyptian summer ambient conditions
volumetric flask and then completed to volume with mobile
The dosage form was exposed to 408C and 65% relative humidity
phase.
in an incubator for 2 months. Timololw 0.5% dosage form was

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kept in a jar containing saturated solution of sodium nitrite,
Preparation of eye drop solutions which gives 65% relative humidity (29), then the jar was kept
Eye drop stock solution. Stock solution equivalent to 136 mg/ in an incubator that was adjusted to 408C, taking samples at
mL of TM was prepared by transferring 1 mL of Timololw 0.5% time intervals of 10, 30 and 60 days. At each time interval, 1 mL
eye drops into a 50-mL volumetric flask and then completed to of the dosage form was taken into a 50-mL volumetric flask and
volume with mobile phase. completed to volume with mobile phase and then 2.94 mL was
transferred from it into a 10-mL volumetric flask and the volume
was completed with mobile phase. The solution was filtered
Eye drop working solutions. Portions of 2.5, 3.75 and 5 mL of through a disposable syringe filter (0.2 mm), and triplicate
the previously prepared eye drops stock solution were accurately 20 mL were injected into the column.
transferred separately into three 10-mL volumetric flasks and
then completed to volume with mobile phase.
Procedure

Forced degradation conditions Linearity

The linearity of the method was evaluated at six concentration lev-
Oxidative degradation els. Aliquots equivalent to 50–2,500 mg of TM from its respective
An accurately weighed 20.0 mg of TM powder was transferred standard working solutions (0.1 mg/mL for TM) were transferred
into a 250-mL conical flask and 2 mL of 0.3% H2O2 was added separately into a series of 25-mL volumetric flasks and completed
and then evaporated until dryness (2 min). The residue was dis- to volume with mobile phase. The solutions were filtered through
solved and quantitatively transferred into a 50-mL volumetric a disposable syringe filter (0.2 mm) before column injection. Then,
flask using deionized water and then the volume was completed 20 mL aliquots of each solution were injected and eluted with the
with it. Triplicate 20 mL from a 40 mg/mL solution were injected mobile phase under the previously described chromatographic
into the column after filtration. conditions. The peak areas were recorded and then the relative
peak areas were calculated for each concentration relative to
the external standard (40 mg/mL) of TM. The average of the rela-
Alkaline degradation tive peak areas of TM was plotted versus the corresponding con-
An accurately weighed 20.0 mg of TM powder was transferred centrations in mg/mL to obtain the calibration graph. Alternatively,
into a 250-mL conical flask, and 50 mL of 0.1 N NaOH was the corresponding regression equation was derived.
added and refluxed for 5 min at 1008C. After cooling, 1 mL of
the solution was neutralized with HCl and then the volume Application to pharmaceutical dosage form and
was completed with mobile phase in a 10-mL volumetric flask. degradable solutions of TM
The solution was filtered through a disposable syringe filter The selectivity of the proposed MLC method was evaluated by
(0.2 mm), and triplicate 20 mL were injected into the column. injecting the previously prepared solutions that exposed to dif-
ferent forced degradation conditions. Furthermore, the prepared
Acidic degradation working solutions of Timololw 0.5% eye drops were analyzed as
Accurately weighed amounts of (20.0 mg) TM powder were mentioned under “chromatographic conditions”. The concentra-
transferred into five 250-mL conical flasks, and 50 mL of 0.1 N tions of TM were calculated from their corresponding regression
HCl was added in each flask and refluxed for time intervals of equation, and system suitability parameters were calculated.
5, 30, 60, 90 and 120 min at 1008C, respectively. After cooling,
1 mL from each solution was transferred separately into a Sample solution and mobile phase stability
10-mL volumetric flask and neutralized with NaOH, and then Evaluation of the stability of TM solutions was achieved by quan-
the volume was completed with mobile phase. The solution tification of TM on seven successive days and comparison with
was filtered through a disposable syringe filter (0.2 mm), and trip- freshly prepared solutions. Similarly, the stability of the mobile
licate 20 mL were injected into the column. phase was checked.

504 Rizk et al.

Results 5 mm particle size), Biobasic C18 (100  1.0 mm, 5 mm particle
The current International Conference on Harmonisation (ICH) size), Biobasic C8 (100  1.0 mm, 5 mm particle size), Phenyl
guidelines require that stability analysis should be done by Hypersil (250  4.6 mm, 5 mm particle size) and Biobasic phenyl
using stability-indicating assay methods, developed and validated (150  1.0 mm, 5 mm particle size) columns. Experimental trials
after stress testing on the drug under a variety of conditions, in- revealed that all tried columns showed bad separation and bad
cluding hydrolysis (at various pH values), oxidation and thermal resolution of peaks except Phenyl Hypersil that showed good
degradation (30). separation but within long analysis time (15 min) and peak
broadening. The BioBasic phenyl column (150  1.0 mm i.d.,
5 mm particle size, Thermo Corporation) was the most suitable
Optimization of chromatographic conditions one giving narrower symmetric peaks and highest number of the-
To investigate the chromatographic performance, different col- oretical plates within a reasonable analytical time (8 min)
umns had been tried that include Hypersil Gold amino (100  (Figure 1). The mobile phase composition was optimized to pro-
1.0 mm, 5 mm particle size), Biobasic cyano (100  2.1 mm, vide sufficient selectivity and sensitivity in a short separation

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Figure 1. ML Chromatograms of TM and its alkaline degradation products separated on (A) BioBasic C8 column (100  1.0 mm, 5 mm particle size), (B) BioBasic C18 (100 
1.0 mm, 5 mm particle size), (C) BioBasic CN column (100  2.1 mm, 5 mm particle size), (D) Hypersil Gold amino (100  1.0 mm, 5 mm particle size), (E) Phenyl Hypersil
(250  4.6 mm, 5 mm particle size) and (F) BioBasic phenyl column (150  1.0 mm, 5 mm particle size).

MLC Method for the Determination of TM 505

time. To select the optimum pH value for the analysis of TM in and longer time of analysis was observed. The best compromise
the presence of its degradation products, the pH of the mobile in terms of run time, efficiency and peak symmetry was achieved
phase was studied over the range of 2.5 – 7. The mobile phase upon using a mobile phase containing 0.1 M SDS. To study the
with pH values 2.8 + 0.2 provided suitable peak symmetry and influence of the concentration of 1-propanol on the peak of
better peak shape. Increasing the pH of the mobile phase offered TM, it was varied over the range of 5 – 15%. As expected, the
bad separation of the degradates and broadening of the TM retention of TM decreases as percentage of organic modifier
peaks. SDS concentration was varied over the range of 0.08 – increases. In addition, peak broadening was observed at low con-
0.15 M. Using mobile phase containing 0.15 M SDS, high efficien- centrations of 1-propanol. A concentration of 10% of 1-propanol
cy and decreasing the retention time was obtained but overlap- was chosen as the optimal concentration, where it offers a good
ping between peaks of TM and one of its degradate was occurred, separation between TM and its degradates, good peak symmetry
while lower concentration of SDS below 0.1 M, peak broadening and reasonable analysis time. TEA concentration was varied over

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Figure 2. ML Chromatograms under optimum chromatographic conditions represent 40 mg/mL of TM under various stress conditions: (A) TM standard, (B) TM and its oxidative
degradation product, (C) TM and its alkaline degradation products, (D) TM after acidic degradation, (E) TM after thermal degradation and (F) 40 mg/mL of Timololw 0.5% eye drops
after 2 months under 408C and 65% humidity.

506 Rizk et al.

the range of 0.05 –0.3%. It was found that the peak tailing, peak The accuracy of bulk powder was found to be 100.4 + 1.196
asymmetry and number of theoretical plates were better with for TM, using the proposed MLC method. Statistical comparison
0.1% TEA. The effect of flow rate of the mobile phase on the re- of the results obtained by the proposed MLC method with those
tention of TM was investigated over the range of 0.05 –0.1 mL/ obtained by the comparison method revealed no significant dif-
min. A flow rate of 0.1 mL/min was chosen because it provides ferences between the performances of the two methods (33)
better peak shape within a reasonable time. For the flow rate (Table II).
higher than 0.1 mL/min, the column pressure became a problem. Precision of the assay was determined in relation to repeatabil-
After optimization of these variables, the best peak shape and ity (intraassay) and intermediate precision (interassay). The rela-
the lowest peak tailing were achieved with well-defined peaks tive standard deviation (RSD) values were ,2%, demonstrating
and good sensitivity within a reasonable analytical run time. It that the method was precise. Good recoveries were obtained
was observed that satisfactory resolution of TM (retention time for each concentration, confirming that the method was accurate
5.8 min) and its degradation products (retention time of 1.8 (Table III).
and 4.3 in addition to 2.8 min for alkaline and oxidative degra- The selectivity was examined for nondegraded and degraded
dates) formed under various stress conditions was achieved samples [the solutions of TM after stress conditions of alkaline hy-

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when analysis of stressed samples was performed on an HPLC drolysis (0.1 M NaOH refluxed at 1008C), acid hydrolysis (0.1 M
system using a BioBasic phenyl column (150  1.0 mm i.d., HCl refluxed at 1008C), oxidation (0.3% H2O2) and thermal deg-
5 mm particle size) and a mobile phase, composed of 0.1 M radation (1008C)]. The MLC method for the determination of TM
SDS, 10% of 1-propanol and 0.1% of TEA in 0.035 M ortho- was found to be selective in the presence of degradation products
phosphoric acid. The detection was carried out at 298 nm. The as shown in Figure 2. Peaks were symmetrical, clearly separated
mobile phase flow rate was 0.1 mL/min. Typical retention time of from each other (Figure 2). Furthermore, the selectivity of the
TM was 5.8 min (Figure 2), and peak asymmetry was 1.25. proposed MLC method was established by its ability to determine
TM in eye drops without interference from common eye drop
additives, indicating selectivity of the method.
Method validation The LOD and LOQ for TM were calculated practically from the
The MLC method was validated according to the ICH Q2 (R1) signal-to-noise ratio and it was found that LOD ¼ 0.56 mg/mL
recommendations (31). The method was validated for parame- and LOQ ¼ 1.86 mg/mL.
ters such as system suitability, linearity, limit of detection
(LOD), limit of quantitation (LOQ), accuracy, precision and selec- Table II
tivity. Stability of sample solution and mobile phase were also Statistical Analysis of the Results Obtained by the Proposed MLC Method and the Comparison
determined. Method (7) for TM Pure Bulk Powder

As the system suitability test is an integral part of chromato- Value MLC proposed method Comparison methodb (7)
graphic method development and it is used to verify that the sys- Mean 100.4 100.0
tem is adequate for the analysis to be performed, the parameters +SD 1.196 0.697
for TM were evaluated. The suitability of the chromatographic % RSD 1.191 0.697
n 6 7
system was determined according to USP guidelines (2) and Variance 1.430 0.486
with acceptance of the obtained parameter values (32) (Table I). Student’s t- test 0.752 (2.201)a
Under the optimum chromatographic conditions, the concen- F-test 2.94 (4.39) a
trations of TM were proportional to the relative peak areas, in the a
The values between parenthesis are the corresponding theoretical values of t and F at P ¼ 0.05
concentration range of 2– 100 mg/mL, by adopting the external (33).
b
standard method for calibration. The comparison method involving spectrophotometric determination by the ratio difference method
The regression equation was computed by measuring the peak amplitudes at DP ¼ 260 –290 nm for BT and at DP ¼ 295– 330 nm for TM.

RPA ¼ 0:025C þ 0:0044 r ¼ 0:9999;

Table III
where RPA is the relative peak area, C is the concentration of TM Assay Parameters and Method Validation for TM by the Proposed MLC Method
in mg/mL and r is the correlation coefficient.
Parameter TM

Table I Validation of regression equation

Parameters Required for System Suitability of MLC Method of TM Slopea 0.025
Intercepta 0.0044
Parameter Obtained value Reference value (32) Correlation coefficient (r) 0.9999
for TM Validation of response
Concentration range (mg/mL) 2 –100
Number of theoretical plates, N 626 Increases with efficiency of the separation LOD (mg/mL) 0.56
Capacity factor, K0 2.75 1 –10 acceptable LOQ (mg/mL) 1.86
a (relative retention time) a1,2 ¼ 1.35 a.1 Average accuracy (%) 100.4
a1,3 ¼ 2.02 +SD (precision) 1.196
Resolution factor, Rs R1,0 ¼ 6.45 R . 1.5 Percentage RSD (SD  100/X) p 1.191
R1,2 ¼ 1.91 Percentage Error (% RSD/ n) 0.486
b
R1,3 ¼ 4.01 Repeatability + %RSD 100.8 + 0.892
Tailing factor, T 1.25 T ¼ 1 for a typical symmetric peak Intermediate precisionb + %RSD 99.5 + 1.406

a
0, 1, 2 and 3 are the mobile phase peak, TM peak, TM alkaline degradate at 4.34 min (its Rs from the Average of three determinations.
b
first alkaline degradate ¼ 4.39) and oxidative degradate, respectively. 3  3 (concentrations 20, 40 and 60 mg/mL of TM were measured three times).

MLC Method for the Determination of TM 507

 No significant changes were observed in standard solution or The validation sheet of the proposed MLC method, according
mobile phase responses, relative to freshly prepared ones. The re- to the ICH Q2 (R1) recommendations (31) of linearity and
sults obtained in both cases proved that the sample solution and range, accuracy, precision, LOD and LOQ, is presented in
mobile phase used during the assay were stable up to 7 days. Table III.

Table IV Application to pharmaceutical formulation

Determination of TM in Timololw 0.5% Eye Drops by the Proposed MLC and Application of Standard
Addition Technique Satisfactory results were obtained for the determination of TM in
Timololw 0.5% eye drops by the proposed MLC method, which
Product Proposed MLC Standard addition proved to be valid and applicable for the analysis without any in-
method
Claimed Added Found Recoverya terference of the additives or preservatives. The accuracy of the
(mg/mL) (mg/mL) (mg/mL) (%)
TM in Timololw 10.00 9.82 98.2
proposed procedure was assessed by applying the standard addi-
0.5% eye dropsb 20.00 20.00 19.89 99.5 tion technique, and the results are shown in Table IV.
B. no. 1202133 40.00 39.59 99.0

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Mean 100.9 98.9
+SD 0.513 0.656 Investigation of stability of TM by the proposed
Percentage RSD 0.508 0.663
chromatographic method
a
Each result is the average of three separate determinations. During stability studies, 20 –80% degradation of the substance to
b
Timololw 0.5% labeled to contain 5.0 mg timolol (6.8 mg TM) per milliliter. be examined is to be achieved to qualify the assay method as able

Figure 3. Spectra of separated TM degradates measured by PDA: (A) TM alkaline degradate at 1.88 min. Maximum wavelength at 298 nm. (B) TM alkaline degradate at 4.34 min.
Maximum wavelength at 295 nm. (C) TM oxidative degradate at 2.88 min. Maximum wavelength at 302 nm.

508 Rizk et al.

Table V
Recovery and Degradation % of TM after Stress Testing by the Proposed MLC Method

Stress condition Time Degradationa (%) Recoverya

(%)
Oxidativeb (2 mL, 0.3% H2O2 at 1008C) Evaporation until dryness 72.4 27.6
Alkalineb (50 mL, 0.1 N NaOH refluxed at 1008C) 5 min 30.1 69.9
Acidicb (50 mL, 0.1 N HCl refluxed at 1008C) 5 –120 min No degradation 99.7
Thermalb (50 mL H2O refluxed at 1008C) 5 –120 min No degradation 100.0
Simulating Egyptian summer ambientc (408C and 65% humidity) 10 –60 days No degradation 99.8

a
Each result is the average of three separate determinations, and the percentage degradation was calculated using the peak area of TM.
b
Stress conditions performed on bulk powder.
c
Stress condition performed on dosage form.

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to indicate stability (34). TM was observed to be significantly li- routine analysis studies of TM in bulk powder and dosage form in
able to oxidative degradation more than alkaline one. It was quality control laboratories, which is our aim.
found to be resistant to acidic hydrolysis and thermal hydrolysis From the chromatographic point of view, the proposed meth-
even in high temperatures (1008C). Chromatograms of solutions od is considered to be an economical method, due to the very
obtained after degradation under various conditions are shown in low flow rate (0.1 mL/min), and a cheap HPLC method, due to
Figure 2. The main degradation products had retention times of low mobile phase consumption (.10 times), in comparison
1.8, 2.8 and 4.3 min. Figure 3 shows the spectra of the separated with other HPLC methods.
degradates scanned by PDA. The results of degraded dosage form,
under conditions simulating the Egyptian summer ambient con-
ditions, show stability of TM for 2 months. The results of forced
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MLC Method for the Determination of TM 509

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