Journal of Chromatographic Science, 2016, Vol. 54, No. 5, 819–828 doi: 10.1093/chromsci/bmv250 Advance Access Publication Date: 3 February 2016 Article
Article
Development and Validation of a HighPerformance Thin-Layer Chromatographic Method for the Simultaneous Determination of Two Binary Mixtures Containing Ketorolac Tromethamine with Phenylephrine Hydrochloride and with Febuxostat Fawzy A. El Yazbi1,*, Ekram M. Hassan1, Essam F. Khamis1, Marwa A.A. Ragab1, and Mohamed M.A. Hamdy2 1
Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, University of Alexandria, Elmessalah, Alexandria 21521, Egypt, and 2Analytical and Pharmaceutical Chemistry Department, Faculty of Pharmacy and Drug Manufacturing, Pharos University in Alexandria, Canal El-Mahmoudia Street, Smouha, Alexandria, Egypt *Author to whom correspondence should be addressed. Email:
[email protected] Received 25 September 2015; Revised 18 November 2015
Abstract 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.
Introduction 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)
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Figure 1. Chemical structures of (A) PHE, (B) FBX and (C) KTC.
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-cyano4-(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
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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.
Experimental Instrumentation 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
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Simultaneous Determination of Two Binary Mixtures Containing KTC with PHE and with FBX utilized was a deuterium lamp emitting a continuous UV spectrum between 190 and 400 nm.
Materials and reagents 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 and calibration graphs Preparation of standard and working solutions 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. Chromatographic conditions and construction of a calibration graph 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.
Analysis of laboratory-prepared pharmaceutical preparations 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.
Results Method development and optimization 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
Table I. Regression and Statistical Parameters (n = 6) Parameters
−1
Linearity range (µg band ) LOD (µg band−1) LOQ (µg band−1) Intercept (a) Slope (b) Correlation coefficient (r) Sa Sb Sy/x F Significance F
Mixture 1 (both scanned at 273 nm)
Mixture 2 (both scanned at 320 nm)
KTC
PHE
KTC
FBX
0.20–0.60 0.03 0.10 328.89 5,036.07 0.9991 55.78 125.04 42.03 1,622.25 3.37 × 10−5
0.60–1.95 0.15 0.50 531.54 3,512.52 0.9993 78.10 67.31 71.98 2,723.41 8.07 × 10−7
0.10–1.00 0.03 0.10 4023.67 16,515.52 0.9997 143.54 213.19 159.54 6,001.47 1.66 × 10−7
0.25–2.50 0.06 0.20 −176.52 11,731.55 0.9993 368.72 221.51 474.22 2,804.94 7.61 × 10−7
Sa is standard deviation of intercept, Sb is standard deviation of slope and Sy/x is standard deviation of residuals.
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Figure 2. A typical HPTLC chromatogram of (A) 0.30 and 0.90 μg band−1 of KTC and PHE, respectively, in their mixture (Mixture 1) and (B) 0.50 and 2.00 μg band−1 of KTC and FBX, respectively, in their mixture (Mixture 2) using 10 µL band volume.
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).
Method validation ICH guidelines (30) for method validation were followed for the developed HPTLC method.
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Linearity and range 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). Limit of detection and limit of quantification 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
