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A validated and highly selective high-performance thin-layer chromatography (HPTLC) method was developed for the determination of ketorolac tromethamine (KTC) with phenylephrine hydrochloride (PHE) (Mixture 1) and with febuxostat (FBX) (Mixture 2) in bulk drug and in combined dosage forms. The proposed method was based on HPTLC separation of the drugs followed by densitometric measurements of their spots at 273 and 320 nm for Mixtures 1 and 2, respectively. The separation was carried out on Merck HPTLC aluminum sheets of silica gel 60 F254 using chloroform–methanol–ammonia (7:3:0.1, v/v) and (7.5:2.5:0.1, v/v) as mobile phase for KTC/PHE and KTC/FBX mixtures, respectively. Linear regression lines were obtained over the concentration ranges 0.20–0.60 and 0.60–1.95 µg band−1 for KTC and PHE (Mixture 1), respectively, and 0.10–1.00 and 0.25–2.50 µg band−1 for KTC and FBX (Mixture 2), respectively, with correlation coefficients higher than 0.999. The method was successfully applied to the analysis of the two drugs in their synthetic mixtures and in their dosage forms. The mean percentage recoveries were in the range of 98–102%, and the RSD did not exceed 2%. The method was validated according to ICH guidelines and showed good performances in terms of linearity, sensitivity, precision, accuracy and stability.
Ketorolac tromethamine (KTC) (Figure 1), chemically known as (±)-5-benzoyl-2,3-dihydro-1H-pyrrolizine-1-carboxylic acid, compound with 2-amino-2-(hydroxymethyl)-1,3-propanediol (1:1) (1), is a pyrrolizine carboxylic acid derivative structurally related to indometacin. KTC is a nonsteroidal anti-inflammatory drug (NSAID), used principally as an analgesic. KTC is used intramuscularly, intravenously or orally in the short-term management of moderate to severe postoperative pain. KTC eye drops are used to relieve ocular itching associated with seasonal allergic conjunctivitis. Also, it is used for the topical treatment of cystoids macular edema and for the prevention and reduction of inflammation associated with ocular surgery (2). The United States Pharmacopeia (USP) describes a high-performance liquid chromatography (HPLC) procedure with ultraviolet (UV) detection for the assay of KTC both in bulk form and in tablets and injections (1). Alternatively, several methods have been described in the literature for the determination of KTC in its pharmaceutical dosage forms or in biological samples. Examples of these methods in pharmaceutical dosage form are various spectrophotometric and spectrofluorometric methods (3), HPLC with UV detection (4), high-performance thin-layer chromatography (HPTLC) (5), capillary chromatography (6) and micellar electrokinetic chromatography (7). Few articles reported the determination of pharmaceutical mixtures containing KTC with sparfloxacin using an HPLC method (8) and gatifloxacin using an HPTLC method (9).
Phenylephrine hydrochloride (PHE) (Figure 1), chemically known as (1R)-1-(3-hydroxyphenyl)-2-(methylamino)ethanol hydrochloride (10), is a sympathomimetic with mainly direct effects on adrenergic receptors. It has mainly alpha-adrenergic activity without significant stimulating effects on the central nervous system at usual doses. It is used either topically or orally, for the symptomatic relief of nasal congestion and is frequently included in preparations intended for the relief of cough and cold symptoms. In ophthalmology, PHE is used as a mydriatic and conjunctival decongestant (2). It is official in both USP and British Pharmacopoeia (BP), where the USP describes an iodometric titration (1) and the BP performs a potentiometric titration (10) for its assay in bulk form. An HPLC method with UV detection is described by the USP for analysis of PHE in different dosage forms (injection, nasal jelly, nasal solutions and ophthalmic solutions) (1) and by the BP for the assay of PHE in eye drops (10). PHE injection is assayed spectrophotometrically in the BP (10). The scientific literature comprises a wide variety of PHE assays in its pharmaceutical dosage forms or in biological samples. Anodic stripping voltammetry (11), UV spectrophotometric methods (12), color reactions with spectrophotometric determinations (13) and spectrofluorometry (14) were used in the assay of PHE in different pharmaceutical dosage forms. Many articles reported the determination of PHE in pharmaceutical mixtures using HPLC (15–17), capillary electrophoresis (18, 19), HPTLC (20), spectrophotometry (21, 22), spectrofluorometry (23) and UPLC (24).
Febuxostat (FBX) (Figure 1), chemically known as 2-[3-cyano-4-(2-methylpropoxy)phenyl]-4-methylthiazole-5-carboxylic acid (2), is a nonpurine, selective inhibitor of xanthine oxidase and used in treatment of chronic gout (2). FBX was assayed by HPLC in many pharmaceutical dosage forms (25, 26). Few articles reported the determination of pharmaceutical mixtures containing FBX with KTC using HPLC (27) and spectrophotometric (28) methods and with Naproxen using an HPLC method (29).
KTC (0.3%) and PHE (1%) are co-formulated in a fixed-dose combination vial, which is added to ophthalmic irrigation solution used during cataract surgery or intraocular lens replacement and is indicated for maintaining pupil size by preventing intraoperative miosis and reducing postoperative ocular pain. To the best of our knowledge, no attempts have yet been made to assay this drug combination by any analytical method including HPTLC.
KTC (20 mg) and FBX (80 mg) are co-formulated in a fixed-dose combination tablet for the treatment of chronic gout and management of its associated pain. Few reports in the scientific literature can be found for the simultaneous determination of KTC and FBX including HPLC (27) and spectrophotometric (28) methods. To the best of our knowledge, no attempts have yet been made to analyze this drug combination by any HPTLC method.
The aim of this work is the development of a simple, rapid and reliable HPTLC method for the simultaneous determination of KTC in its two binary mixtures with PHE and with FBX. HPTLC is becoming a routine analytical technique due to its advantages of low operating cost, high sample throughput and minimum sample clean up. The major advantage of HPTLC is that several samples can be run simultaneously using a small quantity of mobile phase unlike HPLC, thus lowering analysis time and cost per analysis.
HPTLC plates (20 × 10 cm, aluminum plates with 250 µm thickness precoated with silica gel 60 F254) were purchased from E. Merck (Darmstadt, Germany). The samples were applied to the plates using a 100 µL CAMAG microsyringe (Hamilton, Bonaduz, Switzerland) in the form of bands using a Linomat IV applicator (CAMAG, Muttenz, Switzerland). The slit dimension was kept at 5.00 × 0.45 mm, and 20 mm s−1 scanning speed was employed. Ascending development of the mobile phase was carried out in a CAMAG 20 cm × 10 cm twin trough glass chamber. The optimized chamber saturation time for mobile phase was 30 min at room temperature (25 ± 2°C). Densitometric scanning was performed at 273 and 320 nm on a CAMAG TLC Scanner 3 operated in the reflectance–absorbance mode and controlled by CAMAG CATS software (V 3.15). The source of radiation utilized was a deuterium lamp emitting a continuous UV spectrum between 190 and 400 nm.
Pharmaceutical grades of KTC, PHE and FBX were kindly supplied by Pharonia Pharmaceuticals (New Borg El-Arab City, Alexandria, Egypt) and were certified to contain 99.98, 99.99 and 99.80%, respectively. Methanol (S.D. Fine-Chem Limited [SDFCL], India) was of analytical grade. HPLC-grade chloroform (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland) was used. Laboratory-made pharmaceutical preparations containing mixture 1 (KTC and PHE) in the same concentrations present in the 5 mL eye vials OMIDRIA™ (1% phenylephrine and 0.3% ketorolac injection) were prepared containing citric acid and sodium citrate as buffer system. As for Mixture 2, Ketolac™ labeled to contain 10 mg KTC manufactured by Amriya Pharmaceuticals and Feburic™ labeled to contain 80 mg FBX manufactured by Hikma-Pharm were used to analyze KTC and FBX in their laboratory-made pharmaceutical preparations.
Standard solutions containing 0.2, 0.6 and 0.6 mg mL−1 of KTC, PHE and FBX were prepared separately by dissolving the reference materials in methanol. Regarding the stability of solutions of the drugs, stock solutions were stored at 4°C in amber glass vessels and were found to be stable for at least 10 days. The working solutions were prepared by dilution of the standard solution with methanol. For Mixture 1, different volumes corresponding to concentrations in the range of 0.02–0.06 and 0.06–0.195 mg mL−1 for KTC and PHE, respectively, were diluted with methanol in 10 mL volumetric flasks. For Mixture 2, different volumes corresponding to concentrations in the range of 0.01–0.1 and 0.025–0.25 mg mL−1 for KTC and FBX, respectively, were diluted with methanol in 10 mL volumetric flasks.
From each working standard solution, 10 µL portions were spotted as bands on a HPTLC plate to obtain final concentrations of KTC, PHE and FBX as cited in Table I. The bands were separated by a distance of 10 mm apart and 15 mm from the bottom of the plate. Triplicate applications were made for each solution. The plate was then developed using chloroform–methanol–ammonia (33%) (7:3:0.1, v/v) as a mobile phase for Mixture 1 (KTC and PHE) and using chloroform–methanol–ammonia (33%) (7.5:2.5:0.1, v/v) as a mobile phase for Mixture 2 (KTC and FBX). The approximate time of plate development was 10 min. Densitometric scanning was performed at 273 and 320 nm for Mixtures 1 and 2, respectively. The peak areas were plotted against the corresponding concentrations to obtain the calibration graph for each compound. The concentrations of KTC, PHE and FBX either in synthetic mixtures or in final dilutions of laboratory-made pharmaceutical preparations were computed from the corresponding calibration graphs.
For Mixture 1, a volume of 5 mL solution containing 1% PHE and 0.3% KTC was prepared by dissolving 50 and 15 mg PHE and KTC, respectively, in water for injection containing citric acid and sodium citrate (previously prepared by dissolving 100 mg citric acid and 100 mg sodium citrate in 1,000 mL water for injection) in a 5-mL volumetric flask. The solution was sonicated for 30 min. The 5 mL solution was transferred into a 100-mL volumetric flask. For Mixture 2, ten tablets of Ketolac™ and Feburic™ each were separately weighed and ground. A portion of the separate tablet powders equivalent to ~80 mg FBX and 20 mg KTC was weighed and mixed, then was accurately transferred into a 100-mL volumetric flask using ~30 mL methanol. The sample solution of the flask was sonicated for 30 min. Dilution was made to volume in both flasks with methanol followed by filtration through Whatman No. 1 filter paper. Three final dilutions containing 0.06, 0.12 and 0.18 mg mL−1 PHE and 0.02, 0.04 and 0.06 mg mL−1 KTC for Mixture 1 and 0.08, 0.12 and 0.20 mg mL−1 FBX and 0.02, 0.03 and 0.05 mg mL−1 KTC for Mixture 2 were prepared in methanol. The general procedure described under construction of calibration graphs was followed.
The experimental conditions for the HPTLC method such as mobile phase composition and wavelength of detection were optimized to provide accurate, precise and reproducible, compact, flat bands for the simultaneous determination of KTC and PHE/FBX.
Figure 2 shows that the two compounds in both mixtures could be separated with good resolution as sharp and symmetrical peaks upon the use of a mobile phase consisting of chloroform–methanol–ammonia in the ratio of (7:3:0.1, v/v) and (7.5:2.5:0.1, v/v) for the determination of KTC/PHE (Mixture 1) and KTC/FBX (Mixture 2), respectively. Well-defined bands for the two drugs in both mixtures were obtained when the chamber was saturated with the mobile phase at room temperature for at least 30 min. The length of the chromatogram run was ~90 mm. After development, the plates were dried in air for 5 min. Slit dimensions was 5.00 × 0.45 mm, and the scanning speed was 20 mm s−1. Densitometric measurements were performed with a CAMAG TLC Scanner 3 in the reflectance—absorbance mode operated by CAMAG TLC software. The optimized chromatographic conditions gave compact spots for the cited drugs at the specific Rf values mentioned in Table I. Different scanning wavelengths were tried, and 273 and 320 nm were chosen for Mixtures 1 and 2, respectively. The optimum bandwidth chosen was 6 mm, taking into consideration the range of concentrations applied and number of tracks. All tracks were scanned efficiently at the same wavelength (273 and 320 nm for Mixtures 1 and 2, respectively).
ICH guidelines (30) for method validation were followed for the developed HPTLC method.
The linearity of the proposed method was evaluated by analyzing series of different concentrations of each of KTC, PHE and FBX. According to ICH, at least five concentrations must be used. Under the experimental conditions described, the graphs obtained by plotting peak areas of the two drugs versus concentrations (in the ranges stated in Table I) showed linear relationships. The slopes, intercepts and correlation coefficients obtained by the linear least squares regression treatment of the results are also given. The smaller the standard error of the estimate (Sy/x) obtained, the closer the points are to the straight line. High values of correlation coefficients together with the F-values indicate the good linearity of the calibration graphs (31, 32).
The limit of detection (LOD) is considered to be the concentration which has a signal-to-noise ratio of 3:1. For the limit of quantification (LOQ), the ratio considered is 10:1 with a % RSD value <2% (1). Using the proposed method, the LOD and LOQ for each compound were calculated and are presented in Table I. Their values indicate high sensitivity of the proposed method.
For each drug, method repeatability was tested through the analysis of standard solutions prepared in triplicates at three concentration levels within the same day. On the other hand, the intermediate precision was examined by analyzing standard solutions prepared at the same concentration levels repeated over 3 days. Tables II and III comply the obtained percentage relative standard deviation (RSD%) values, which in all cases were equal or less than 2%. Accordingly such low values of RSD% can indicate the satisfactory level of precision of the proposed method. In addition, the method can be deemed accurate as shown by the recovered concentrations and the values of percentage relative error (Er %) that did not exceed ±2% (Tables II and III).
Robustness of the proposed method was evaluated by analyzing KTC, PHE and FBX at three concentration levels as cited in Table IV for precision. The parameters studied were mobile phase composition, mobile phase volume, duration of saturation and time from chromatography to scan. It was found that small deliberate variation in the above parameters had no significant influence on the determination of any of the two drugs using the proposed method. The low values of RSD along with nearly unchanged Rf values obtained after introducing small deliberate changes in the method parameters indicated the robustness of the developed method (Table IV).
The selectivity of the proposed method for the simultaneous determination of the cited drugs was assessed through the analysis of laboratory-prepared synthetic mixtures and laboratory-prepared dosage forms. The synthetic mixtures were prepared in order to contain a combination of KTC and PHE (Mixture 1) and KTC and FBX (Mixture 2) at different ratios within their linearity ranges mentioned in Table I. The recovered concentration for each drug was further calculated from the corresponding regression equation. As seen in Tables V and andVI,VI, the acceptable values of the found concentration, RSD % (<2%) and percentage relative error Er % (within ±2%) confirm the accuracy, precision and selectivity of the developed method. The same good results were obtained upon analyzing the laboratory-prepared dosage forms in the presence of different inactive ingredients.
The specificity of the method was also ascertained through the peak purity profiling performed by the Wincats software. The peak purity profiling was an adequate tool for peak identity and purity confirmation. This was done by comparing the Rf values of the drug peaks in samples to that of the standard. Also, upon comparing the recorded spectra at different points of the chromatographic peak, the calculated correlation was not less than 0.999 indicating the homogeneity of the peaks (33). Moreover, achieving a perfect overlap of the recorded spectra of each drug in standard and combined dosage form confirms the peak purity and identity (Figure 3).
The stability of KTC, PHE and FBX in their solutions during analysis was investigated. Solutions of the three drugs were prepared and stored at room temperature for 1, 3 and 5 h, and then they were analyzed using the proposed method. The results showed that the Rf value and peak area of all drugs remained almost unchanged (% RSD < 2%), and no additional peaks were found in the chromatograms throughout the analysis time indicating that the drug solutions were stable for at least 5 h, which was sufficient for the whole analytical process. Stock solutions were stored at 4°C in amber glass vessels and were found to be stable for at least 10 days.
The time for which the sample is left to stand prior to chromatographic development can influence the stability of separated spots and is required to be investigated for validation (34). Two-dimensional chromatography, using the same solvent system, was used to find out any decomposition occurring during spotting and development. Since no decomposition was observed during spotting and development using the proposed conditions, this indicated the stability of the three drugs in their solutions.
Due to the unavailability of the commercial dosage form in the local market, laboratory-prepared solutions for Mixture 1 and the mixture of Ketolac™ and Feburic 80™ tablets for Mixture 2 were prepared and analyzed by the proposed HPTLC method. The active ingredients KTC, PHE and FBX eluted at their specific Rf values as shown in Figure 2. No interfering peaks were observed from any of the inactive ingredients. Peak purity was verified through the peak purity profiling (Figure 3). Furthermore, a methanol extract of the inactive ingredients used in the preparation of laboratory-made solutions (citric acid and sodium citrate) for Mixture 1 and of inactive ingredients used in the preparation of tablets (maize starch, microcrystalline cellulose, magnesium stearate, HPMC and colloidal silica) for Mixture 2 was chromatographed using the developed procedure then scanned at the working wavelengths. The chromatograms of the inactive ingredients extract illustrated in Figure 4 reveal the absence of any interfering peaks at the retention time of each drug.
Recovered concentrations were calculated using an external standard method. The contents of both drugs in solutions of each mixture were determined by analysis of three independently prepared solutions each repeated three times, and percentage recovery of concentrations were calculated using similarly treated standard solutions. The assay results revealed that satisfactory accuracy and precision as values of the mean percentage recovery of concentrations were between 100.03 and 100.90 with RSD % values between 0.85 and 1.74 (Table VII). Furthermore, comparison reference methods (22, 27) were applied for drugs estimation in their mixtures. Recovery data obtained from the proposed method were statistically compared with those of the reference methods using the Student t-test and the variance ratio F-test. In both tests, the calculated values did not exceed the theoretical ones at the 95% confidence level, which indicated that there were no significant differences between the recoveries obtained from the developed method and those of the reference methods (Table VII). It is evident from these results that the proposed method is applicable to the assay of both mixtures with satisfactory level of accuracy and precision.
Optimization of method parameters is important for the simultaneous determination of the three drugs in their mixtures. On the basis of minimum tailing and maximum separation of the drugs, different solvent systems were tried. Initially, a system using methanol and distilled water in different ratios was tried. KTC, PHE and FBX bands were not separated enough and were very close to baseline. When chloroform replaced distilled water, the distance travelled by developed KTC, PHE and FBX bands increased, and the required separation between the two bands in each mixture was optimized, but PHE and FBX bands suffered tailing which was corrected using very small volumes of ammonia. Different ratios of chloroform to methanol and ammonia were tried to obtain the optimum separation of bands. The greatest differences between the Rf values of the KTC and PHE in Mixture 1 (0.08 ± 0.01 for PHE and 0.47 ± 0.02 for KTC) and KTC and FBX in Mixture 2 (0.61 ± 0.02 for KTC and 0.84 ± 0.02 for FBX) with minimum tailing were obtained by using a mobile phase consisting of chloroform–methanol–ammonia (7:3:0.1, v/v) and (7.5:2.5:0.1, v/v), respectively. The optimum time for chamber saturation with mobile phase is not less than 30 min. It was required to eliminate the edge effect and to avoid unequal solvent evaporation losses from the developing plate that could lead to various types of random behavior resulting in a lack of Rf values reproducibility. For the selection of optimum scanning wavelength, different wavelengths were tried for the simultaneous determination of drugs in their dosage forms considering their ratios. For Mixture 1, wavelength 273 nm gave reasonable response with KTC and PHE. The ratio of KTC–FBX in Mixture 2 was 1:4, so a wavelength of 320 nm was chosen to give the maximum response of minor component KTC in its combined dosage form with FBX.
The method was fully validated with respect to ICH guidelines. The method was tested for its adequate capability of resolving and simultaneously quantifying the investigated drugs over a wide range of ratios within solutions (Tables V and andVI).VI). In the same time, in the presence of different inactive ingredients in the combined dosage form, the method was capable of selectively detect and quantify the active ingredient with good accuracy and precision. The method was validated with respect to specificity by the aid of peak purity profiling. This was done by recording the UV absorption spectrum at several points across each chromatographic peak by the TLC scanner, the software is capable of evaluating the purity of the peaks through two main steps. At first, the correlation coefficient (rs,m) between the spectra extracted at peak start and peak maximum and the correlation coefficient (re,m) between the spectra extracted at peak end and peak maximum are calculated. Second, the software interprets mathematically the significance of the correlation values and gives a decision about the purity of the designated peak (33). The spots were declared pure as the spectra extracted at different points across the absorption spectra were superimposed on one another and the calculated correlation coefficients values were not less than 0.999. Moreover, the recorded spectra of each drug in standard and combined dosage form shows a perfect overlap confirming the peak purity and identity (Figure 3).
Reviewing the literature exposed that there were no reports for the application of a TLC-based method for the assay of Ketorolac mixture with phenylephrine or FBX mixture. HPTLC exhibits several advantages. Unlike HPLC, HPTLC is fast and consumes by far less amount of solvents and therefore can be regarded as more economic and environment friendly. The proposed methods met the ICH validation acceptance criteria. The selectivity of the proposed methods was evaluated through the analysis of several laboratory-prepared synthetic mixtures at different ratios within the linearity ranges of the drugs. In addition, the applicability of the proposed methods to real life situations was assessed through the analysis of laboratory-made solutions containing Mixture 1 in the same concentrations present in the 5 mL eye vials OMIDRIA™ (1% phenylephrine and 0.3% ketorolac injection) and mixture of Ketolac™ and Feburic 80™ tablets for Mixture 2 and satisfactory results were obtained. As a result, the present HPTLC method could be advantageous in content uniformity testing and quality control laboratories.