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Comparative Study of RP-HPLC Versus TLC-Spectrodensitometric Methods Applied for Binary Mixtures of Fluoroquinolones and Corticosteroids
Abstract and Figures
Reversed phase high-performance liquid chromatography (RP-HPLC) and thin-layer chromatography (TLC)-spectrodensitometric methods have been developed and validated for the separation and quantitation of two binary mixtures: Ofloxacin (OFX) and dexamethasone (DXM) in eye preparation; ciprofloxacin hydrochloride (CIP) and hydrocortisone (HYD) in ear preparation. The linearity ranges of RP-HPLC methods were found to be (2.5-45 mu g mL(-1)) for OFX, (2.5-50 mu g mL(-1)) for DXM and (1-8 mu g mL(-1)) for both CIP and HYD. The percentage recoveries/relative standard deviation (RSD) were found to be 100.36/1.38, 100.13/1.49, 99.98/0.61 and 100.28/1.27, respectively. The linearity ranges of TLC-spectrodensitometric methods were found to be (0.5-2 mu g band-1), (0.5-3.5 mu g band-1), (0.2-1.6 mu g band-1), and (0.6-2 mu g band-1) for OFX, DXM, CIP, and HYD, respectively. The percentage recoveries/RSD were found to be 99.98/1.06, 99.93/1.18, 99.74/1.27, and 99.94/1.54, respectively. A comparative study was conducted to show the advantages of the proposed methods which showed that the TLC-spectrodensitometric methods were simpler, more sensitive, and economic, while RP-HPLC methods were more precise and robust. The methods were validated in compliance with the ICH guidelines and were successfully applied for determination of the selected drugs in their laboratory-prepared mixtures and commercial dosage forms.
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... They then expanded their exploration into instrumentation, utilizing reversed-phase high-performance liquid chromatography-UV detection (RP-HPLC-UV) and TLC-spectrodensitometric detection (TLC-spectrodensitometric) methods to separate and quantify the same binary mixture of CIP and HYD in their ear preparation. Their work showcased innovation in pharmaceutical analysis and expanded analytical capabilities [24]. However, the utilization of multiple instruments under different conditions for the separation of the same mixture raises a logical question regarding the comparison of their operation and the determination of preferences. ...
... Subsequently, the plates were air-dried. More information about the chromatographic conditions and separation trials using RP-HPLC-UV and TLC-spectrodensitometric methods can be found in Table S3 and the study of Elgizawy et al. [24]. The mixture solutions were then formed by thoroughly mixing the liquids in the same order. ...
... Among all methods, RP-HPLC-UV had the lowest SDs due to its high automation level, reducing errors. The net assay results are found in Table S11 for UV-spectrophotometric methods and Table S12 for chromatographic methods, which are extracted from the developed methods [20,24]. ...
This study aims to prefer the suitability of an analytical approach developed for simultaneously determining ciprofloxacin hydrochloride (CIP) and hydrocortisone (HYD) in their ear drop dosage form. The preference between the three known instruments was utilized, namely UV-spectrophotometry, reversed phase-high-performance liquid chromatography (RP-HPLC)-UV, and thin-layer chromatography (TLC)-densitometry. The instrumentational studies determined that the mathematical UV methods (utilized various manipulation designs such as the isoabsorptive point, absorbance ratio, extended ratio subtraction, ratio difference, and mean centering of ratio spectra) had linearities in the range of 2.0–14.0 and 1.0–14.0 µg·mL⁻¹, the RP-HPLC-UV method showed a linearity range of 1.0–8.0 µg·mL⁻¹, and the TLC-spectrodensitometric method had linearity ranges of 0.2–1.6 µg/band and 0.6–2.0 µg/band for both CIP and HYD, respectively. The analytical performance, validity, and greenness of the approaches were evaluated through the benchmarking of key performance indicator (KPI)-based standards and a SWOT (strengths, weaknesses, opportunities, and threats) analysis. The KPIs and SWOT study focused on several aspects, including (1) the selectivity and robustness of the methods, (2) sensitivity, (3) accuracy and precision, (4) applicability, (5) whiteness, (6) greenness, and (7) blueness. However, the assessment of whiteness, greenness, and blueness was conducted using well-known ecological assessment tools such as the RGB12 Algorithm, Analytical Eco-Scale, AGREE, GAPI, and Blue Applicability Grade Index. In conclusion, based on the findings, UV-spectrophotometry emerged as the most practically convenient approach. It demonstrated advantages based on the predetermined KPI-based standards. Furthermore, UV-spectrophotometry was deemed to be the most environmentally friendly option.
... It is used for topical application in the treatment of various skin disorders. A survey of the literature revealed the reported methods for the determination of HCA such as ultraviolet (UV) spectrophotometry [1,2], high-performance liquid chromatography (HPLC) [3][4][5], thin-layer chromatography (TLC) [6,7], micellar electrokinetic capillary chromatography [8,9], and capillary electrophoresis [10]. Fusidic acid (FSA) is an antimicrobial substance produced by the growth of certain strains of Fusidium coccineum. ...
... This method offers a simple way for quantification directly on TLC plate by measuring the optical density of the separated bands. The amounts of compounds are determined by comparing to a standard curve from reference materials chromatographed simultaneously under the same condition [5,[24][25][26][27]. ...
This work presents a comparative study on the development and validation of two analytical techniques applied for the simultaneous determination of hydrocortisone acetate (HCA), fusidic acid (FSA), methyl paraben (MPB), and propyl paraben (PPB) formulated as a topical cream. The first technique was thin-layer chromatography (TLC)-densitometric method, which was developed by separating the four components on silica gel 60 F254 using methylene chloride-methanol-benzene in the ratio of 10:2:5, v/v, as the developing system, followed by densitometric measurement of the bands at 240 nm. The second technique was the chemometric method using two models: principle component regression model (PCR) and partial least squares (PLS). The suggested techniques were validated in compliance with the International Conference on Harmonization (ICH) guidelines and were successfully applied for the determination of the quaternary mixtures as prepared synthetically in laboratory and in the commercial topical pharmaceutical formulation.
... It is used for topical application in the treatment of various skin disorders. A survey of the literature revealed the reported methods for the determination of HCA such as ultraviolet (UV) spectrophotometry [1,2], high-performance liquid chromatography (HPLC) [3][4][5], thin-layer chromatography (TLC) [6,7], micellar electrokinetic capillary chromatography [8,9], and capillary electrophoresis [10]. Fusidic acid (FSA) is an antimicrobial substance produced by the growth of certain strains of Fusidium coccineum. ...
... This method offers a simple way for quantification directly on TLC plate by measuring the optical density of the separated bands. The amounts of compounds are determined by comparing to a standard curve from reference materials chromatographed simultaneously under the same condition [5,[24][25][26][27]. ...
Sodium cromoglicate (SCG), antihistaminic agent, and tetryzoline hydrochloride (TZH), a sympathomimetic agent, are formulated together as an ophthalmic preparation. An ultra-performance liquid chromatographic method with UV detection (UPLC-UV) was developed and validated for the quantitative determination of SCG and TZH in rabbit aqueous humor. Due to the instability of both SCG and TZH under alkaline conditions, the UPLC method was applied for their determination in the presence of their possible degradation impurities. The separation was performed using C18 column (1.7μm particle size) and isocratic elution system with methanol: 1% o-phosphoric acid (65: 35, v/v).The optimum flow rate was 0.5ml/min and the detection was done at 230nm. The suggested method was validated in compliance with the ICH guidelines and was successfully applied for determination of sodium cromoglicate (SCG) and tetryzoline HCl (TZH) as prepared synthetically in laboratory mixtures, and in the presence of their alkali-induced degradation impurities. The suggested method was effectively applied the determination of spiked rabbit aqueous humor samples as well as commercial pharmaceutical formulation.
... Although spectroscopic methods and TLC are simple and rapid, they have low sensitivity, accuracy, and specificity and might require sample pretreatment. Thus, they are incapable of accurate quantification on their own or require a densitometer for the quantitative aspect (Elgizawy et al., 2014). Specific methods, such as NMR spectroscopy, have also been used (McEwen et al., 2012); however, this technique requires sophisticated instruments and a special solvent. ...
Topical steroids are usually used for the treatment of inflammation and irritation of the skin; however, their inappropriate use results in serious side effects. Thus, the Thai Food and Drug Administration regulation forbade the use of corticosteroids in cosmetics. In this study, a method of high-performance liquid chromatography with a photodiode array detector was developed and validated for the simultaneous determination of three corticosteroids in cream formulation: triamcinolone acetonide (TA), betamethasone valerate (BV), and clobetasol propionate (CP). Chromatographic separation was performed on ACE® C18 (4.6 mm × 150 mm, 5 μm particle size) using a mobile phase consisting of water and acetonitrile in gradient elution. The detection wavelength was set at 254 and 240 nm. The validation results revealed the specificity and linearity of the proposed method with r≥0.999 within the concentration range of 2.5–50 μg/mL. The relative standard deviation (%) of the repeatability and intermediate precision of all corticosteroid samples at three different concentration levels were in the range of 0.04%–2.04% and 0.41%–4.93%, respectively, and the recovery (%) was 98.72 %–105.41%. The detection limits of TA, BV, and CP were 0.69, 0.46, and 0.48 μg/mL, respectively, and their quantitative limits were 2.08, 1.39, and 1.47 μg/mL, respectively. The validated method can be applied to quantify the amount of active compounds in commercial formulations and to determine the corticosteroid adulterants present in cream samples from online shops.
... Chromatography is the benchmark among other analytical techniques for several reasons including: providing reliable analytical data with good accuracy and precision [15,16]; the possibility of separation and identification of countless mixtures with complexity such as enantiomers [17][18][19][20] or natural extracts [21][22][23] and the availability of different detection approaches that serves the diversity of samples' nature such as ultraviolet detection [24,25], fluorescence [26] or mass spectrometry [27]. Ultra-Performance liquid chromatography (UPLC) is an effective technique defined by smaller particle size, extended capacity with decreased time and solvent consumption. ...
Facing the pandemic COVID-19 is of highest priority for all researchers nowadays. Recent statistics indicate that the majority of the cases are home-treated. Two drugs of interest, Guaifenesin and Bromohexine HCl, are among the add-on therapy for treatment of COVID-19 mild cases, which has raised the need for their simultaneous determination. The analysis of the two drugs of interest was described using ultra-performance liquid chromatography–tandem mass spectrometric (UPLC–MS/MS) in plasma of healthy human volunteers using tetryzoline HCl as an internal standard (IS) after liquid-liquid extraction. The applied chromatographic conditions were Kinetex C18 (100Å, 2.6 µm X 50 mm X 4.6 mm) column and a mixture of methanol: water (95: 5, v/v) as a mobile phase at flow rate 1 mL/min. The positive ionization mode was used for detecting the ions, by observing the pairs of transition m/z 199< 125 for GUF, m/z 377< 114 for BRM and m/z 201 <131 for IS. The linearity range was from 50-1500 ng/mL for GUF and 0.5-50 µg/mL for BRM. Limit of detection (LOD) was found to be 35.16 and 0.43 ng/ml for GUF and BRM, respectively. The method was validated according to FDA guidance. The proposed method was assessed to be more eco-friendly versus the reported method using the greenness assessment tools: National Environmental Methods Index (NEMI), Assessment of Green Profile (AGP), Green Analytical Procedure Index (GAPI) and Eco-Scale. The proposed method was applied for the application of a pilot pharmacokinetic study.
... It is used for topical application in the treatment of various skin disorders. A survey of the literature revealed the reported methods for the determination of HCA such as UV spectrophotometry [1,2], HPLC [3][4][5], TLC [6,7], micellar electrokinetic capillary chromatography [8,9] and capillary electrophoresis [10]. Fusidic acid (FSA) is an antimicrobial substance produced by the growth of certain strains of Fusidium coccineum. ...
The persistence of antimicrobial drugs in the aquatic environment has raised critical concerns about their possible impact on drinkable water quality and human health. The Nile River is experiencing water...
Corticosteroids are anti-inflammatory and immunosuppressant drugs. Topical corticosteroids formulations (ointments, creams, gels) are used in the treatment of different types of dermatitis and urticaria. Considering their therapeutic and whitening effects, they are frequently used for counterfeiting of cosmetic products. Corticosteroids can cause different local and systemic side effects. HPLC method is often chosen for their analysis, because it is selective, sensitive, precise, simple and fast.
The aim of this study was optimization and validation of RP-HPLC method with UV detection for determination of trace levels of corticosteroids in ambiphilic creams. This method is used for qualitative and quantitative analysis of evaluated corticosteroids.
Mometasone furoate, hydrocortisone acetate, fluocinonide, fluocinolone acetonide, betamethasone, betamethasone dipropionate and triamcinolone acetonide were evaluated. Separation was performed on Inertsil® ODS-3V 250 × 4.6 mm, 5 μm chromatographic column. Mobile phase was mixture of acetonitrile and water 50:50 (v/v) with gradient elution and flow rate 1 mL min ⁻¹ . Column temperature was held on 40 °C and UV detection was performed at 240 nm.
Selectivity, linearity, accuracy, precision and limit of quantification (LOQ) were evaluated. Method is selective because ambiphilic cream base peaks and corticosteroids peaks were not overlapping. Linearity was confirmed since correlation coefficient was 1 for all compounds. Accuracy and precision were evaluated for hydrocortisone acetate and betamethasone dipropionate. Determined Recovery values were in range of 70–130%. Both RSD values (21.46% and 9.59%) were lower than 30%. Method is highly sensitive since LOQ concentrations were in ng mL ⁻¹ range.
All evaluated parameters of validation were in accordance with regulatory requirements. Validated RP-HPLC method can be used for qualitative and quantitative analysis of selected corticosteroids in ambiphilic creams.
This work represents a comparative study of different approaches of manipulating ratio spectra, applied on a binary mixture of ciprofloxacin HCl and dexamethasone sodium phosphate co-formulated as ear drops. The proposed new spectrophotometric methods are: ratio difference spectrophotometric method (RDSM), amplitude center method (ACM), first derivative of the ratio spectra (¹DD) and mean centering of ratio spectra (MCR). The proposed methods were checked using laboratory-prepared mixtures and were successfully applied for the analysis of pharmaceutical formulation containing the cited drugs. The proposed methods were validated according to the ICH guidelines. A comparative study was conducted between those methods regarding simplicity, limitations and sensitivity. The obtained results were statistically compared with those obtained from the reported HPLC method, showing no significant difference with respect to accuracy and precision.
High-performance liquid chromatography (HPLC), ultra-performance liquid chromatography (UPLC) and rapid resolution liquid
chromatographic (RRLC) methods have been developed and validated for the separation and quantitation of both or either of
two ternary mixtures present in ophthalmic solutions. The first mixture contains chloramphenicol, dexamethasone sodium phosphate
and tetryzoline HCl (TZH); while the second one contains ofloxacin, prednisolone acetate and TZH. Both preparations contain
benzalkonium chloride as a preservative. The columns used were a HPLC column (C18 5 µm particle size), a RRLC column (C18 2.6 µm particle size) and a UPLC column (C18 1.7 µm particle size). A comparative study was conducted to illustrate the effect of the change in column particle size and
dimensions on the other chromatographic conditions, backpressure and the separation of both ternary mixtures. The methods
were validated as per ICH guidelines where accuracy, repeatability, interday precision and robustness were found to be within
the acceptable limits. The RRLC column provided shorter run time and better resolution than HPLC, while the UPLC column gave
the shortest run time for all columns. The RRLC column resulted in minimum backpressure, so it could be used with any HPLC
instrument, which makes the method more practical and economic. The results obtained from the proposed methods were statistically
compared with official ones where no significant difference was observed.
Three different accurate, sensitive and reproducible stability-indicating methods for the determination of pipazethate HCl in the presence of its alkaline degradation product are presented. The first method is based on ratio-spectra 1st derivative (RSD1) spectrophotometry of the drug at 305nm, over a concentration range of 10–70μgmL−1 with mean percentage recovery of 99.69±1.10. The second method utilises quantitative densitometric evaluation of thin-layer chromatography of pipazethate HCl in the presence of its alkaline degradation product, using methanol: ethyl acetate: ammonia (8:2:0.2, v/v/v) as a mobile phase. Chromatograms are scanned at 251nm. This method analyses pipazethate HCl in a concentration range of 4–14μg/spot with mean percentage recovery of 100.19±0.77. The third method is an HPLC method for the simultaneous determination of pipazethate HCl in the presence of its alkaline degradation product. The mobile phase consists of methanol: ammonium sulphate (1%), pH=5.7, (80:20, v/v). The standard curve of pipazethate HCl shows a good linearity over a concentration range of 5–200μgmL−1 with mean percentage recovery of 100.67±0.91. These methods were successfully applied to the determination of pipazethate HCl in bulk powder, laboratory-prepared mixtures containing different percentages of the degradation product and pharmaceutical dosage forms. The validity of results was assessed by applying standard addition technique. The results obtained were found to agree statistically with those obtained by a reported method, showing no significant difference with respect to accuracy and precision.
Two simple and sensitive high performance liquid chromatographic (HPLC) methods have been developed for the simultaneous determination of three different quinolones: enrofloxacin, lomefloxacin and ofloxacin in their pure and dosage forms, one with reversed phase HPLC and the other with ion-pair HPLC. In reversed phase HPLC, method (A), the mobile phase consists of 2.18% aqueous solution of KH2PO4 with pH adjusted to 2.4±0.2 with acetonitrile (80:20; v/v), the mobile phase pumped at flow rate of 1.2mlmin−1. A Neucleosil C18 column (10μm, 100Å), 250mm length×4.6mm diameter was utilized as stationary phase. Detection was affected spectrophotometrically at 294nm. While in ion-pair HPLC, method (B), the mobile phase was aqueous solution of 0.65% sodium perchlorate and 0.31% ammonium acetate adjusted to pH 2.2±0.2 with orthophosphoric acid: acetonitrile (81:19; v/v), the mobile phase pumped at flow rate of 1.5mlmin−1. A μ bondapack C18 column (10μm, 100Å), 250mm length×4.6mm diameter was utilized as stationary phase. Detection was affected spectrophotometrically at 294nm. Linearity ranges for enrofloxacin, lomefloxacin and ofloxacin were 4.0–108, 7.0–112 and 8.0–113μgml−1, respectively using method A and 8.0–112, 7.0–112 and 5.0–105μgml−1, respectively applying method B. Minimum detection limits obtained were 0.013, 0.023 and 0.035μgml−1 for enrofloxacin, lomefloxacin and ofloxacin, respectively using method A, and 0.028, 0.023 and 0.011μgml−1 using method B. The proposed methods were further applied to the analysis of enrofloxacin in injection and tablets containing the ofloxacin and lomefloxacin drugs, and the results were satisfied.
A liquid chromatographic method was developed and validated for the simultaneous determination of ofloxacin and dexamethasone sodium phosphate in bulk and pharmaceutical formulations. Optimum separation was achieved in less than 5 min using a C18 column (250 mmx4.6 mm i.d, 5µ particle size) by isocratic elution. The mobile phase consisting of a mixture of mixed phosphate buffer (pH 4) and acetonitrile (50:50, v/v) was used. Column effluents were monitored at 236 nm at a flow rate of 1ml/min. Retention times of ofloxacin and dexamethasone sodium phosphate were 2.4 and 4 min respectively. The linearity of ofloxacin and dexamethasone sodium phosphate was in the range of 3-18 µg/ml and 1-6 µg/ml respectively. Developed method was economical in terms of the time taken and amount of solvent consumed for each analysis. The method was validated and successfully applied to the simultaneous determination of ofloxacin and dexamethasone sodium phosphate in bulk and pharmaceutical formulations.
A simple, accurate, sensitive and validated RP-HPLC method for simultaneous determination of Cefixime and Ofloxacin in combined tablet dosage form has been developed. Separation was carried out on Jasco HPLC system equipped with HiQ-SiL C 8 Neosphere column (150 × 4.6 mm i.d.) and UV/VIS detector using Methanol: 0.025 mM potassium dihydrogen phosphate buffer in ratio of (70:30, v/v) as mobile phase and detection was carried out at 290 nm. Ambient temperature conditions were maintained. Results were linear in the range of 1-10 μg/mL for both the drugs. The method has been successfully applied for the analysis of drugs in pharmaceutical formulation. Results of analysis were validated statistically and by recovery studies.
A spectrofluorimetric study of the acid–base properties and of the complexation behavior of the fluoroquinolone antibiotics ofloxacin (OF), norfloxacin (NOR), ciprofloxacin (CIP) and pefloxacin (PEF) has been performed. Their dissociation constants were determined by a combined potentiometric/fluorimetric technique. All studied fluoroquinolones form fluorescent complexes with Sc3+ in slightly acidic solutions (pH=4.2, λex∼280 nm, λem∼430 nm (480 nm for OF). A simple, rapid and sensitive spectrofluorimetric method based on the formation of scandium complexes has been developed for the determination of OF, NOR, CIP and PEF in aqueous solutions. Calibration graphs for all four fluoroquinolones were linear up to 1.0 μM, with results having a mean relative error of 3.2%. The 3σ detection limits are 1.1, 0.6, 0.5 and 1.0 nM for OF, NOR, CIP and PEF, respectively. The method has been successfully applied to the determination of NOR in synthetic serum samples (5.0–50.0 μM) after deproteinization with acetonitrile (serum–acetonitrile, 1 : 2 v/v) with a mean recovery of 93.4±1.1% (n=3).
Two spectrophotometric methods are described and applied to resolve ternary mixtures of the corticosteroid hydrocortisone (HYD) and the antibiotics nystatin (NYS) and oxytetracycline (OXY). The simultaneous determination of these three compounds was firstly accomplished by a derivative method using the “ratio spectrum-zero crossing derivative” and then by multivariate methods partial least squares (PLS)-1, -2 and principal component regression (PCR). Multivariate calibration methods provide, specially PLS-2 in this case, a clear example of the high resolving powder of these techniques. The two described procedures do not require any separation step. Repeatability and reproducibility studies were achieved over two series of 10 standards for each compound showing no significant differences at 95% confidence level in the four spectrophotometric methods. A comparison of the derivative and multivariate calibration results obtained in pharmaceutical formulations was performed resulting in agreement of the values obtained and the results was confirm by a high-pressure liquid chromatography (HPLC) method.
Betamethasone (BM) is an active pharmaceutical ingredient (API) or an intermediate which is used to manufacture various finished pharmaceutical products. Betamethasone is also used as a starting material to manufacture other APIs that are related to this steroid family. It is quite a challenging task to separate dexamethasone (DM) peak (the alpha epimer) and other structurally related compounds from BM. A stability-indicating reversed-phase high performance liquid chromatography (RP-HPLC) method has been developed which can separate and accurately quantitate low levels of DM and other related compounds from BM and also from each other. This method was successfully validated for the purpose of conducting stability studies of betamethsone in quality control (QC) laboratories. The stability-indicating capability of this method was demonstrated by adequate separation of DM and all the degradation product peaks from BM peak and also from each other in aged stability samples of betamethasone. A gradient mobile phase system consisting of (A) water:acetonitrile (90:10, v/v) and (B) acetonitrile:isopropanol (80:20, v/v) was used with an ACE Phenyl column (10 cm × 4.6 mm, 3 μm particles, 100 Å pore size) and an ultraviolet (UV) detection at 240 nm.
Simple, accurate, sensitive and validated UV spectrophotometric, chemometric and HPTLC-densitometric methods were developed for determination of oxybutynin hydrochloride (OX) in presence of its degradation product and additives in its pharmaceutical formulations. Method A is the first derivative of ratio spectra (DD(1)) which allows the determination of OX in presence of its degradate in pure form and tablets by measuring the peaks amplitude at 216 nm. Method B and C are principal component regression (PCR) and partial least-squares (PLS) for determination of OX in presence of its degradate in pure form, tablets and syrup. While, the developed high performance thin layer chromatography HPTLC-densitometric method was based on the separation of OX from its degradation product, methylparaben and propylparaben followed by densitometric measurement at 220 nm which allows the determination of OX in pure form, tablets and syrup. The separation was achieved using HPTLC silica gel F(254) plates and chloroform:methanol:ammonia solution:triethylamine (100:3:0.5:0.2, v/v/v/v) as the developing system. The accuracy, precision and linearity ranges of the developed methods were determined. The results obtained were statistically compared with each other and to that of a reported HPLC method, and there was no significant difference between the proposed methods and the reported method regarding both accuracy and precision.