untitled European Journal of Chemistry 4 (1) (2013) 29‐34 European Journal of Chemistry ISSN 2153‐2249 (Print) / ISSN 2153‐2257 (Online)  2013 EURJCHEM DOI:10.5155/eurjchem.4.1.29‐34.721 European Journal of Chemistry Journal homepage: www.eurjchem.com Comparative determination of miconazole, nystatin, hydrocortisone and neomycin by HPTLC/HPLC‐DAD Ismail Salama * and Mohamed Sayed Gomaa Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Suez Canal University, Ismailia 41552, Egypt *Corresponding author at: Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Suez Canal University, Ismailia 41552, Egypt. Tel.: +20.012.80327695; fax: +20.064.3230741. E‐mail address: ismail_mohamed@daad‐alumni.de (I. Salama). ARTICLE INFORMATION ABSTRACT Received: 28 December 2012 Received in revised form: 20 January 2013 Accepted: 20 January 2013 Online: 31 March 2013 KEYWORDS Two new validated methods for the quantification of miconazole (MIC), nystatin (NYS), hydrocortisone acetate (HCA) and neomycin (NS) by high performance thin layer chromatography (HPTLC)‐densitometry and reverse‐phase high‐performance liquid chromatography procedure coupled with photodiode array detector (RP‐HPLC‐DAD) were developed and compared. HPTLC separations were performed using a mobile phase, ethylacetate:methanol:glacial acetic acid (60:40:0.4, v:v:v) for MIC, NYS, HCA and ethylacetate for NS. For HPLC‐DAD analysis, using an isocratic elution system, separation of all compounds was achieved. Good resolution and quantization were achieved, accuracy and precision, as well as detection and quantitation limits of the two methods, were evaluated and compared. Excellent linearity was observed for all of the standard calibration curves, and the correlation coefficients were above 0.9997. HPTLC limits of quantitation were 15.37×10‐2, 9.8×10‐2, 13.32×10‐2 and 15.19×10‐2 µg/mL for MIC, NYS, HCA and NS respectively, whereas HPLC limits were 6.80×10‐2, 6.56×10‐2, 1×10‐1 and 10.16×10‐2 µg/band for MIC, NYS, HCA and NS respectively. In comparison with HPLC, HPTLC is less expensive and faster procedure, requiring 2‐3 h to analyse 10‐12 samples on a single plate. The developed methods were proved to be specific, robust and accurate for the determination of cited drugs in pharmaceutical preparations. HPTLC Nystatin Neomycin HPLC‐DAD Miconazole nitrate Hydrocortisone acetate 1. Introduction Miconazole nitrate (MIC) [l‐(2,4‐dichloro‐β‐((2,4‐ chlorobenzyl)oxy)phenethyl)imidazole], a synthetic imidazole derivative, is applied widely as the nitrate salt with a broad‐ spectrum antifungal activity [1,2]. It is established as a useful drug for the treatment of various systemic mycoses. It is also active against Gram‐positive bacteria. Nystatin (NYS) is a polyene antifungal antibiotic that is of particular interest because it exhibits remarkable action against a wide range of pathogenic and non‐pathogenic yeast and fungi [3,4]. Hydrocortisone acetate (HCA) is a human glucocortico‐ steroid which is often combined with nystatin and oxytetracycline [5] in different pharmaceutical preparations. Neomycin (NS) is a water‐soluble complex of amino‐ glycosides produced from the fermentation of the actinomycete Streptomyces fradiae [6‐9]. Neomycin is used to treat bacterial infections in animals because of their good spectrum of activity against gram‐negative bacteria. Several analytical methods in the literature describing the determination of miconazole nitrate [10‐14] or nystatin [15‐ 19] alone or in combination with other drugs have been reported. We have previously developed in our laboratory HPLC and chemometric methods for the simultaneous determination of MIC and NYS in their pharmaceutical formulations [20]. In the literature, various spectrophotometric methods for the determination of hydrocortisone [21,22] alone and with lidocaine [23], were reported. Other methods include determination of hydrocortisone in pharmaceuticals by TLC [24] and HPLC [25‐27] and in biological fluids [28‐30] using HPLC with different methods of detection. For neomycin a high‐performance liquid chromatography (HPLC)‐fluorescence after post column derivatization of serum [31], TLC after derivatization with fluram of plasma [32] or HPLC‐tandem‐MS [33] and a HILIC‐tandem‐MS method [34] was described. Neomycin lacks an usable chromophore however, derivatization of its primary amino groups is possible [35]. From the above‐cited literature, no analytical method has been reported for the simultaneous analysis of NS with primary amine containing drugs other than aminoglycosides using UV detection. The main advantage of the developed method is to separate and analyze NS without interference from other drugs, in the mixture, with its derivatization procedure. Furthermore, the main objectives of this work were to develop, validate and compare two new HPLC‐DAD and HPTLC methods for the simultaneous determination of NYS, MIC, HCA and NS in pharmaceutical preparations. 2. Experimental 2.1. Materials and reagents Miconazole nitrate, Nystatin and Neomycin sulphate were kindly supplied by medical union pharmaceuticals (MUP), Ismailia City, Egypt and certified to contain 99.8, 99.9 and 99.9%, respectively. Hydrocortisone acetate (98.75%) was from Egyptian International pharmaceutical industries company (EIPICO). 30 Salama and Gomaa / European Journal of Chemistry 4 (1) (2013) 29‐34 Commercial Monicure NH® vaginal suppositories (Batch no. 7361109) (Pharaonia Pharmaceuticals, Alexandria City, Egypt) were used. Each vaginal suppository was labeled to contain 100 mg (MIC), 100.000 IU = 20.5 mg (NYS), 50 mg (NS) and 5 mg (HCA). Phenylisocyanate (PIC) and triethylamine (TEA) were purchased from Sigma. HPLC grade methanol (TEDIA, USA).Distilled water for HPLC was obtained following distillation in glass and passage through a Milli‐Q® system (Millipore, Milford, MA, USA), filtered through 0.45 µm membrane filter (Merck, Germany), degassed for 30 minutes in an ultrasonic bath. All other chemicals and reagents used were of analytical grade unless indicated otherwise. 2.2. HPLC instrumentation and conditions HPLC analysis was carried out using a Hitachi LaChrom Elite liquid chromatograph L‐2000 series equipped with photodiode array detector model L‐2455, Hitachi La Chrom Elite (Tokyo, Japan), an Autosampler model L‐2200, Hitachi LaChrom Elite (Tokyo, Japan), column oven model L‐2300 Hitachi LaChrom Elite (Tokyo, Japan) and degasser built in model L‐2130 pump.The column (250 mm × 4.6 mm i.d.) was made of stainless steel and packed with Inertsil ODS‐3v (5 μm particle diameter, GL Sciences, Tokyo, Japan). Data acquisition was performed on EZChrom Elite software (Agilent Technologies). For MIC, NYS and HCA; the RP‐HPLC‐ DAD assay was carried out using an isocratic elution system with a flow rate of 1 mL min‐1. The mobile phase consisted of acetonitrile: 25 mM KH2PO4 buffer (pH = 2.8) in a ratio of 50:50 (v:v). For NS‐PIC; The mobile phase consisted of acetonitrile: 25 mM KH2PO4 buffer (pH = 2.8) (70:30, v:v). The DAD acquisition wavelength was set to scan from 200 to 400 nm, and all analyses were performed at ambient temperature (25 oC). Before use, the mobile phase was filtered through 0.45 µm membrane filters (Millipore, Milford, MA, USA) and degassed under vacuum. 2.3. High‐performance thin‐layer chromatography and instrumentation HPTLC analysis were performed by using the computerized Camag HPTLC system (Camag, Muttenz, Switzerland) consisting in an automatic delivery system (TLC Linomat IV) and in a UV densitometer (TLC Scanner II). Data were stored andprocessed by appropriate software (Cats 3 via RS232 interface). Separation was achieved on HPTLC precoated silica gel 60 F plates, 10×10 cm (Merck) using ethylacetate: methanol:glacial acetic acid (60:40:0.4, v:v:v) as mobile phase for MIC, NYS and HCA while ethylacetate was used for NS‐PIC. Samples were band applied (3 mm length) with a space of 4 mm under the nitrogen stream. Standard solutions of MIC, NYS, HCA and NS‐PIC were applied to the same plates in incremental concentrations to obtain the calibration curves for each compound. Chromatograms, developed in a saturated horizontal chamber, 10×10 cm (Camag Muttenz, Switzer land), were evaluated via peak height after scanning in absorbance‐ reflectance mode at 230 nm (MIC, NYS and HCA) and 240 (NS‐ PIC). Keeping slit width at 3 mm, slit length at 4 mm and scanning speed at 4 mm/s. 2.4. Preparation of standard solutions and calibrations 2.4.1. For HPLC method Stock standard solutions of MIC, NYS and HCA 1 mg/mL were prepared in methanol. While for NS 1 mg/mL was prepared in water. The prepared solutions were stored at 4 oC. The standard solutions were prepared by further dilutions of the stock standard solutions with mobile phase to reach the concentration ranges of 1‐50 µg/mL for MIC, 2‐100 µg/mL for NYS, 1‐35 µg/mL for HCA and 5‐50 µg/mL for NS. Triplicate 20 µL injections were made for each concentration and chromatographed under the specified conditions. The peak area values versus corresponding concentrations were plotted. Linear relationships were obtained. 2.4.2. For HPTLC method Stock standard solutions of the four drugs were prepared as for HPLC method. The standard solutions were prepared by further dilutions of the stock standard solutions with mobile phase to reach the concentration ranges of 3‐60 µg/band for MIC, 10‐70 µg/band for NYS, 7‐45 µg/band for HCA and 5‐100 µg/band for NS. 5 µL of each standard solution were applied to the HPTLC plates. Triplicate applications were made for each solution. The plates were developed using previously described mobile phases. The calibration curves were constructed relating the integrated area under the peak to the corresponding concentrations as µg/band. 2.5. Preparation of Neomycin‐PIC derivative For each concentration of the standard NS solutions in the linearity range, a 250 μL aliquot of each standard solution was added to 250 μL of phenylisocyanate solution (5 mg/mL in acetonitrile) and 250 μL of triethylamine solution (5 mg/mL in acetonitrile). The mixture was reacted at room temperature and the resulting solution was shaken for several times. 2.6. Sample preparation Five Monicure NH vaginal suppositories were accurately weighed and finely powdered in a mortar. An amount of the suppository mass equivalent to one suppository content (100 mg of MIC, 20.5 mg of NYS. 50 mg of NS and 5 mg of HCA) was dissolved in 60 mL of water. After 30 min of warming and mechanical shaking, the solution was filtered in a 100 mL volumetric flask. The residue was washed twice, each with 10 mL of water. The volume was then completed to 100 mL with water. The residue was dissolved in methanol and the same procedure was followed. Further dilutions of the filtrates were conducted with mobile phase to reach the calibration range. 3. Results and discussion We report here the separation and quantification of MIC, NYS, HCA, NS‐PIC by both HPLC‐DAD and HPTLC, and the analytical performance of the two methods was compared for their sensitivity, precision and linearity. NS is a weakly absorbing drug as it lacks a suitable pharmacophore and its quantification depends on the derivatisation of the drug with a suitable reagent to form a highly absorbing component that could be detected by HPLC/UV and HPTLC. However, one of the main problems of this concept is the interference from other amine containing drugs when present in mixture. This method depends on the separation of NS from MIC, NYS, HCA through differential solubility as the drug is water soluble while other components in the mixture are not. This is followed by derivatising the drug using phenyisocyanate as previously described in the literature [35]. Other components in the mixture are then easily determined as they all absorb well in the UV region. Other amine derivatising reagents as aldehydes and benzene sulphonyl chlorides were tried and phenylisocyanate gave the most accurate and reproducible results. 3.1. Optimization of the HPLC‐DAD method For separation of MIC, NYS and HCA; the mobile phase composition and pH of 25 mM KH2PO4 were studied and optimized. A successful separation was obtained with a mobile Salama and Gomaa / European Journal of Chemistry 4 (1) (2013) 29‐34 31 phase consisting of acetonitrile and 25 mM KH2PO4 buffer (pH = 2.8) in a ratio of 50:50 (v:v) at a flow rate of 1 mL/min. First by trying 60% MeOH : 30% Acetonitrile : 10 % KH2PO4, pH = 6, we found that the NYS peak was splitted and MIC eluted too late. Using 65% MeOH : 35% KH2PO4, pH = 3.1, NYS eluted at 24 min. An eluting system consisting of 60% MeOH: 30% acetonitrile: 10 % NH4 acetate, pH = 6, led to inadequate separation of HCA and NYS i.e eluted at the same retention time (3.6 min.). By decreasing acetonitrile:methanol ratio or using only methanol as organic modifier, we failed to separate NYS from HCA peaks with excessive tailing for the MIC peak. However, using only acetonitrile and KH2PO4 at apparent pH = 2.8, optimum resolution with reasonable retention time was observed. For NS‐PIC; a mobile phase, consisting of acetonitrile and 25 mM KH2PO4 buffer (pH = 2.8) in a ratio of 70:30 (v:v), was used at a flow rate of 1.5 mL/min. These conditions gave optimum resolution, clear baseline separation with reasonable retention time and no tailing of peaks of the studied compounds (Figure 1). Figure 1. (a) HPLC chromatogram (20 mL injection volume) of laboratory‐ prepared mixture of (1) NYS, (2) HCA and (3) MIC; (b) reaction mixture (4) NSC‐PIC and (5) NSB‐PIC. The three‐dimensional UV absorption spectra of the studied compounds (Figure 2) show that 230 nm is the wavelength of maximum absorbance for MIC, NYS and HCA (Panel a). For NS‐PIC, the wavelength of maximum absorbance was 240 nm (Panel b). The specificity of the HPLC‐DAD method is illustrated in Figure 1, in which complete separation of the three drugs (Figure 1a) and complete separation of Neomycin B‐PIC(NSB‐ PIC) (major) and Neomycin C‐PIC (NSC‐PIC) (minor) (Figure 1b) was observed. 3.2. Optimization of the HPTLC Method Experimental conditions such as mobile phase and wavelength of scanning were optimized to provide accurate, precise and reproducible results for the simultaneous determination of the four analytes. The wavelength of scanning was chosen to be 230 nm for NYS, MIC and HCA while for NS‐ PIC, it was 240 nm. The greatest difference between the Rf values of NYS, MIC and HCA with minimum tailing of NYS were obtained by using ethyl acetate:methanol:glacial acetic acid (60:40:0.4, v:v:v) while for NS‐PIC; using glacial acetic acid cause disappearance of its spot under UV light which may be attributed to the hydrolysis of the derivatised drug. NS‐PIC was eluted using only ethyl acetate. HPTLC densitograms for MIC, NYS and HCA are shown in the upper panel of Figure 3 and that for NSB‐PIC and NSC‐PIC are shown in the lower panel. Figure 2. HPLC‐DAD three‐dimensional spectra of (a) laboratory‐prepared mixture of (1) NYS, (2) HCA and (3) MIC; [b] reaction mixture (4) NSC‐PIC and (5) NSB‐PIC. 3.3. Validation of the methods 3.3.1. Linearity The linearity of the HPLC and HPTLC methods for determination of MIC, NYS, HCA and NS were evaluated by analyzing a series of different concentrations of each drug. In this study, six concentrations were chosen, ranging between 1‐ 50, 2‐100, 1‐35, 5‐50 µg/mL for MIC, NYS, HCA and NS respectively using HPLC method and 3‐60, 5‐70, 7‐45 and 5‐ 100 µg/band for MIC, NYS, HCA and NS respectively using HPTLC method. Each concentration was repeated three times, to provide information on the variation in peak area values between samples of the same concentration. The linearity of the calibration graphs was validated by the high value of the correlation coefficient and the intercept value, which was not statistically (p = 0.05) different from zero. Characteristic parameters for regression equations of the HPLC and HPTLC methods obtained by least‐squares treatment of the results are given in Table 1 and 2. 32 Salama and Gomaa / European Journal of Chemistry 4 (1) (2013) 29‐34 Table 1. Calibration curve data for MIC, NYS, HCA and NS using HPLC method. Regression parameters MIC NYS HCA NS Regression coefficient (r) a 0.9999 0.9999 0.9998 0.9998 Calibration range (µg/mL) 1‐50 2‐100 1‐35 5‐50 Detection limit (LOD) (µg/mL) 2.00×10‐2 19.68×10‐3 3×10‐2 30.47×10‐3 Quantitation limit (LOQ) (µg/mL) 6.80×10‐2 6.56×10‐2 1×10‐1 10.16×10‐2 Slope 37.39×103 2.13×103 3.42×105 1.94×103 Standard deviation of slope 3.27×102 0.17×102 4.38×103 0.25×102 Confidence limit of the slope b 37.07×103‐37.71×103 2.11×103 ‐2.15×103 3.37×105‐3.46×105 1.92×103‐1.97×103 Intercept ‐5.54×103 ‐0.19×102 0.09×105 ‐0.87×102 Standard deviation of the intercept 9.19×103 7.85×102 1.23×105 6.41×102 Confidence limit of the intercept b ‐14.46×103‐3.39×103 ‐7.82×102‐7.44×102 ‐1.09×105‐1.29×105 ‐7.10×102‐5.36×102 a The degree of freedom = 5. b Confidence limit = 95%. Table 2. Calibration curve data for MIC, NYS, HCA and NS using HPTLC method. Regression parameters MIC NYS HCA NS Regression coefficient (r) a 0.9997 0.9999 0.9997 0.9997 Calibration range (µg/band) 3‐60 5‐70 7‐45 5‐100 Detection limit (LOD) (µg/mL) 46.12×10‐3 3×10‐2 39.98×10‐3 45.59×10‐3 Quantitation limit (LOQ) (µg/mL) 15.37×10‐2 9.8×10‐2 13.32×10‐2 15.19×10‐2 Slope 1.01×103 23.29×103 1.59×103 1.00×103 Standard deviation of slope 0.19×102 2.93×102 0.27×102 0.19×102 Confidence limit of the slope b 0.98×103‐1.03×103 23.00×103 ‐23.57×103 1.56×103‐1.61×103 0.98×103‐1.02×103 Intercept ‐1.09×102 ‐12.59×103 3.39×102 ‐1.83×102 Standard deviation of the intercept 7.60×102 14.31×103 4.74×102 7.48×102 Confidence limit of the intercept b ‐8.48×102‐6.28×102 ‐26.49×103‐1.31×103 ‐1.21×102‐8.00×102 ‐9.09×102‐5.44×102 a The degree of freedom = 5. b Confidence limit = 95%. Figure 3. (a) HPTLC chromatogram of laboratory‐prepared mixture of (1) NYS, (2) MIC and (3) HCA; (b) reaction mixture (4) NSB‐PIC, (5) NSC‐PIC and (6) PIC. 3.3.2. Limits of detection (LOD) and quantification (LOQ) The limit of detection (LOD) and limit of quantification (LOQ) were calculated according to the current ICH guidelines as the ratio of 3.3 and 10 standard deviations of the blank (n = 7) respectively, against the slope of the calibration line [36]. The LOD and LOQ are given in Table 1 and 2. 3.3.3. Precision The intra‐day and inter‐day variations of the two methods were determined using three replicate injections of three different concentrations, which were prepared and analysed on the same day and on three different days over a period of two weeks, respectively (Table 3 and 4). These data indicate a considerable degree of precision and reproducibility for the methods both during one analytical run and between different runs. By comparison of coefficient of variation (CV) for the two methods, HPLC appeared somewhat more precise than HPTLC, particularly at low analyte concentrations. 3.3.4. Accuracy The interference of excipients in the pharmaceutical formulations was studied in detail by the HPLC and HPTLC methods. For this reason, the standard addition method was applied to the pharmaceutical formulation containing these compounds. In application of standard addition method to the pharmaceutical formulation, the mean percentage recoveries and their standard deviations for the proposed methods were calculated (Table 5). According to the obtained results, satisfactory precision and accuracy were observed for these methods. Consequently, the excipients in pharmaceutical formulation do not interfere in the analysis of these compounds in the pharmaceutical formulation. 3.3.5. System suitability Resolution (Rs) is a measure of the degree of separation between adjacent peaks. A value of 1.5 for resolution implies a complete separation of the two compounds [37]. Additionally, British Pharmacopoeia specifies that the symmetry factor of a principal peak must be between 0.8 and 1.5 [37]. Resolutions and other system suitability parameters were calculated for MIC, NYS, HCA and NS. Their values were found to be acceptable (Table 6). 3.3.6. Ruggedness and robustness tests As recommended in the ICH guidelines and the Dutch Pharmacists guidelines, a robustness assessment was performed during the development of the analytical procedure [38]. Salama and Gomaa / European Journal of Chemistry 4 (1) (2013) 29‐34 33 Table 3. Intra‐day and inter‐day precision of MIC and NYS standard solutions by HPLC method. Compound Theoretical concentrations (µg/mL) Intra‐day Precision Inter‐day Precision Recovery%±S.D CV% Recovery%±S.D CV% 4 99.02±0.63 0.64 98.88±0.90 0.91 MIC 12 98.02±0.73 0.74 98.86±0.89 0.90 20 99.72±0.56 0.56 99.26±0.67 0.68 20 99.04±0.96 0.97 100.04±1.17 1.17 NYS 60 98.96±0.83 0.84 99.06±0.93 0.94 100 99.80±0.69 0.69 99.50±1.03 1.04 HCA 4 99.87±0.69 0.69 99.73±1.98 1.98 12 99.82±0.49 0.49 100.12±0.84 0.84 20 100.04±0.39 0.39 100.09±0.73 0.73 NS 20 99.14±0.68 0.69 99.92±2.01 2.01 60 99.93±0.56 0.56 99.85±0.89 0.89 100 100.02±0.41 0.41 100.07±0.75 0.75 Table 4. Intra‐day and inter‐day precision of MIC and NYS standard solutions by HPTLC method. Compound Theoretical concentrations (µg/band) Intra‐day Precision Inter‐day Precision Recovery%±S.D CV% Recovery%±S.D CV% 4 97.40±0.73 0.75 97.89±0.94 0.96 MIC 12 97.02±0.83 0.86 98.06±0.92 0.94 20 98.62±0.66 0.67 98.26±0.77 0.78 20 98.10±0.96 0.98 99.34±1.32 1.33 NYS 60 98.06±0.93 0.95 98.30±0.99 1.01 100 98.08±0.89 0.91 98.70±1.13 1.15 HCA 4 98.07±0.79 0.81 98.93±2.01 2.05 12 98.04±0.69 0.70 99.02±0.99 1.00 20 99.04±0.49 0.50 99.39±0.85 0.86 NS 20 98.14±0.78 0.80 98.72±2.01 2.04 60 97.93±0.66 0.67 98.95±0.99 1.00 100 99.02±0.61 0.62 99.47±0.88 0.89 Table 5. Determination of MIC, NYS, HCA and NS in Monicure NH® vaginal suppositoriesa using the proposed HPLC and HPTLC methods. MIC NYS HCA NS HPLC Mean recovery (%)±SD 100.61±1.03 99.57±1.01 99.91±0.66 100.26±0.82 t (2.18)b (4.28)b f HPTLC Mean recovery (%)±SD 100.14±1.04 99.96±0.78 99.89±0.74 99.89±1.05 t 0.56 0.32 0.31 0.34 f 1.11 1.01 1.69 1.05 Standard addition technique c HPLC [Mean recovery (%)±SD] 101.15±1.09 99.95±0.85 100.17±0.33 99.55±0.93 HPTLC[Mean recovery (%)±SD] 101.10±0.88 100.10±1.04 100.18±0.94 99.92±0.91 a Monicure NH® vaginal suppositories labeled to contain 100 mg MIC, 20.6 mg NYS, 5 mg HCA and 50 mg NS per suppository. b Theoretical values for t and F at p = 0.05. c For standard addition of 50% of the nominal content. Table 6. Parameters required for system suitability testing of the proposed HPLC method. Parameters NYS HCA MIC NSC‐PIC NSB‐PIC Resolution (RS) 3.83 ‐ 14.20 1.63 ‐ Selectivity (α) 1.63 ‐ 2.21 1.18 ‐ Symmetry factor (T) 1.01 1.04 1.10 1.09 1.00 Capacity factor (k') 0.90 1.47 3.25 1.20 1.42 Number of theoretical plates (N) 757 4432 2501 1716 5791 HETP (cm/plate) 0.03 0.005 0.01 0.014 0.004 The ruggedness [39] of the two methods was assessed by comparison of the intra‐day and inter‐day assay results for MIC, NYS, HCA and NS that were performed by two analysts. The CV% values for intra‐day and inter‐day assays of the four analytes in the Monicure NH vaginal suppositories performed in the same laboratory by two analysts did not exceed 3.8%, indicating the ruggedness of the two methods. In addition, the robustness of the method was investigated under a variety of conditions, including changes of the flow rate, PH and mobile phase composition [40]. 3.4. Analysis of MIC and NYS in Monicure NH vaginal suppositories The two methods were applied to the determination of MIC, NYS, HCA and NS in commercial Monicure NH vaginal suppositories. Recoveries were calculated using external regression equations. No interfering peaks were observed from any of the excipients. The assay results revealed satisfactory accuracy and precision, as indicated by the recovery and SD values (Table 5). Recovery data resulting from the proposed HPLC and HPTLC methods were statistically compared with those of the reported HPLC methods: [14,17,30,39] for MIC NYS, HCA and NS, respectively, using one way ANOVA. It was found that the calculated F values did not exceed the critical value of the F‐ ratio at α = 0.05, indicating no significant differences between the proposed and reported methods (Table 7) 3.5. Methods comparison The two proposed analytical methods were compared in term of sensitivity, precision and accuracy to further assess their applicability in the analysis of real samples. 34 Salama and Gomaa / European Journal of Chemistry 4 (1) (2013) 29‐34 Table 7. ANOVA test for statistical comparison of the recovery data results obtained by the proposed HPLC and HPTLC methods and reported methodsa. Compound Source of variation Sum of squares (SS) Degree of freedom (df) Mean sum of squares (MS) F‐ratio b P‐value MIC Between 1.86 2 0.93 0.83 0.46 Within 13.48 12 1.12 Total 15.34 14 ‐ NYS Between 3.60 2 1.80 1.67 0.23 Within 12.91 12 1.08 Total 16.51 14 ‐ HCA Between 0.06 2 0.03 0.24 0.79 Within 1.42 12 0.12 Total 1.48 14 ‐ NS Between 0.06 2 0.03 0.19 0.83 Within 1.71 12 0.14 Total 1.76 14 ‐ a Reported methods are [14,17,30,39] for MIC NYS, HCA and NS, respectively. b The critical value of F‐ratio is 3.89 at α = 0.05. As shown in Table 1 and 2, compared to HPTLC method, The HPLC‐DAD method was found to be more sensitive (linearity range, LOD) in quantitating the cited drugs. The quantitation limits of the cited drugs using the HPLC are lower than those obtained using the HPTLC method. The overall precision (CV%) (Table 3 and 4) and accuracy (recovery %) (Table 5) of the HPLC‐DAD method are superior to that of the HPTLC method particularly at low analyte concentrations. However the HPTLC method is advantageous with respect to simplicity and duration. 4. Conclusion In conclusion, our study suggests that HPTLC is an acceptable technique only for samples with high concentrations of MIC, NYS, HCA and NS. However, we should consider that HPTLC is cheaper and faster than HPLC, since on a single plate at least 10‐15 samples can be analysed in 2‐3 h. Acknowledgement The authors would like to acknowledge financial assistance from Faculty of Pharmacy, Suez Canal University, Ismailia, Egypt. References [1]. Reynolds, J. E. F. (ed). Martindale: The Extra Pharmacopoeia, 29th ed., Pharmaceutical Press, London, 1989, pp. 430, 666. [2]. Hardman, J. G.; Limbird, L. E. (eds). Goodman & Gilman’s: The Pharmacological Basis of Therapeutics, 9th ed., McGraw‐ Hill, New York, 1996. [3]. USP DI, 24th edition, Drug Information for the Health Care Professional, 2004. [4]. Sweetman, S. C. (ed). Martindale: The complete drug reference, 35th ed., Pharmaceutical Press, London, 2002, p. 392. [5]. Lemus, G. J. M.; Perez, A. J. Chromatographia 2002, 55, 749‐753. [6]. Waksman, S. A. Neomycin. Nature, Formation, Isolation, and Practical Application, Rutgers University Press, New Brunswick, NJ, 1953. [7]. Waksman, S. A.; Lechevalier, H. A. Guide to the Classification and Identification of the Actinomycetes and their Antibiotics, The Williams & Wilkins Co., Baltimore, MD, 1953. [8]. Waksman, S. A. Neomycin. Its Nature and Practical Application, The Williams & Wilkins Co., Baltimore, MD, 1958. [9]. Rinehart, K. L. The neomycins and related antibiotics, in: Chemistry of Microbial Products E. R. Squibb Lectures on Chemistry of Microbial Products, Institute of Microbiology, John Wiley & Sons, Inc. , New York, NY, 1964. [10]. Kobylinska, M.; Kobylinska, K.; Sobik, B. J. Chromatogr B: Biomed. Appl. 1996, 685, 191‐195. [11]. Goger, N. G.; Gokcen, L. Anal. Lett. 1999, 32, 2595‐2602. [12]. Wrobel, K.; Wrobel, K.; de la Garza Rodriguez, I. M.; Lopezde‐Alba, P. L.; Lopez Martinez, L. J. Pharm. Biomed. Anal. 1999, 20, 99‐105. [13]. Khashaba, P. Y.; El‐Shabouri, S. R.; Emara, K. M.; Mohamed, A. M. J. Pharm. Biomed. Anal. , 2003, 22, 363‐376. [14]. Akay, C.; Ozkan, S. A.; Senturk, Z.; Cevheroglu, S. Il Farmaco 2003, 57, 953‐957. [15]. Lupan, L.; Bandula, R.; Vasilescu, M.; Bercu, C. Fresenius J. Anal. Chem. 1996, 355, 409‐411. [16]. Botsoglou, N. A.; Fletouris, D. J. J. Agr. Food Chem. 1996, 44, 1271‐ 1274. [17]. Groll, A. H.; Mickiene, D.; Werner, K.; Pistelli, S. C.; Walsh, T. J. J. Chrom. B 1999, 735, 51‐57. [18]. Yeo, S. K.; Lee, H. K.; Li, S. F. Y. J. Chrom. A 1991, 585, 133‐137. [19]. Raith, K.; Althoff, E.; Banse, J.; Neidhardt, H.; Neubert, R. H. H. Electrophoresis 1998, 19, 2907‐2911. [20]. Heneedak, H.; Salama, I.; Mostafa, S.; El‐Sadek, M. J. Chrom. Sci. 2012, 50, 855‐861. [21]. Bonazzi, D.; Andrisano, V.; Gatt, R.; Cavrini, V. J. Pharm. Biomed. Anal. 1995, 13, 1321‐1329. [22]. Amin, A. S. Anal. Lett. 1996, 29, 1527‐1537. [23]. Medenica, M.; Ivanovic, D.; Markovic, S.; Malenovic, A.; Jancic, B. Pharmind. 2004, 66, 330‐333. [24]. Gagliardi, L.; De Orsi, D.; Giudice, M. R. D.; Gatta, F.; Porra, R.; Chimenti, P.; Tonelli, D. Anal. Chim. Acta. 2002, 457, 187‐198. [25]. Chauhan, V.; Conway, B. Chromatographia 2005, 61, 555‐559. [26]. Hajkova, R.; Solich, P.; Dvocak, J.; Cicha, J. J. Pharm. Biomed. Anal. 2003, 32, 921‐927. [27]. Jancic‐stojanovic, B.; Malenovic, A. J. AOAC Int. 2010, 93, 102‐107. [28]. Hay, M.; Mormed, P. J. Chrom. B. 1997, 702, 33‐39. [29]. Grippa, E.; Santini, L.; Castellano, G.; Gatto, M. T.; Leone, M. G.; Saso, L. J. Chrom. B 2000, 738, 17‐25. [30]. Majid, O.; Akhlaghi, F.; Lee, T.; Holt, D. W.; Trull, A. Ther. Drug Monit. 2001, 23, 163‐168. [31]. Shaikh, B.; Jackson, J.; Guyer, G.; Ravis, W. R. J. Chrom. B: Biomed. Appl. 1991, 571, 189‐198. [32]. Medina, M. B.; Unruh, J. J. J. Chrom. B 1995, 663, 127‐135. [33]. Oertel, R.; Renner, U.; Kirch, W. J. Pharm. Biomed. Anal. 2004, 35, 633‐ 638. [34]. Oertel, R.; Neumeister, V.; Kirch, W. J. Chrom. A 2004, 1058, 197‐201. [35]. Byoung‐Hyoun, K.; Suk Chin, L.; Hye Jin, L.; Jong Hoa, O. Biomed. Chrom. 2003, 17, 396‐ 403. [36]. International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, ICH Harmonised Tripartite Guideline‐Validation of Analytical Procedures: Text and Methodology Q2(R1), Current Step 4 version, London, 2005. [37]. The British Pharmacopoeia, Her Majesty’s Stationery Office. London, UK, 2008, pp 404‐405, 485‐486, 1080‐1081, 1382, 2519‐2520, 2559‐ 2560, 2592‐2593, 2766‐2767, 2861. [38]. Zeaiter, M.; Roger, J. M.; Maurel, V. B.; Rutledge, D. N. Trends. Anal. Chem. 2004, 23, 157‐170. [39]. Mulholland, M. Trends Anal. Chem. 1988, 7, 383‐389. [40]. Heyden, Y. V.; Nijhuis, A.; Verbeke, J. S.; Vandeginste, B. G. M.; Massaret, D. L. J. Pharm. Biomed. Anal. 2001, 24, 723‐753.