Stability indicating high-performance thin-layer chromatographic determination of gatifloxacin as bulk drug and from polymeric nanoparticles

A simple, sensitive, selective, precise and stability indicating high-performance thin-layer chromatographic method for determination of gatifloxacin both as a bulk drug and from polymeric nanoparticles was developed and validated as per the International Conference on Harmonization (ICH) guidelines. The method employed thin-layer chromatography (TLC) aluminium plates precoated with silica gel 60F-254 as the stationary phase and the mobile phase consisted of n-propanol-methanol-concentrated ammonia solution (25%) (5:1:0.9, v/v/v). This solvent system was found to give compact spots for gatifloxacin (R_f value of 0.60 ± 0.02). Densitometric analysis of gatifloxacin was carried out in the absorbance mode at 292 nm. The linear regression analysis data for the calibration plots showed good linear relationship with r = 0.9953 with respect to peak area in the concentration range of 400-1200 ng spot⁻¹. The mean value (±S.D.) of slope and intercept were 9.66 ± 0.05 and 956.33 ± 27.67, respectively. The method was validated for precision, accuracy, ruggedness and recovery. The limits of detection and quantitation were 2.73 and 8.27 ng spot⁻¹, respectively. Gatifloxacin was subjected to acid and alkali hydrolysis, oxidation, photodegradation and dry heat treatment. The drug undergoes degradation under acidic and basic conditions and upon wet and dry heat treatment. The degraded products were well separated from the pure drug. The statistical analysis proves that the developed method for quantification of gatifloxacin as bulk drug and from polymeric nanoparticles is reproducible and selective. As the method could effectively separate the drug from its degradation products, it can be employed as stability-indicating one.     

Stability-indicating high-performance thin-layer chromatographic determination of levonorgestrel and ethinyloestradiol in bulk drug and in low-dosage oral contraceptives

A stability-indicating high-performance thin-layer chromatography (HPTLC) method was developed and validated for simultaneous determination of steroidal hormones levonorgestrel and ethinyloestradiol both in bulk drug and in low-dosage oral contraceptives. Optimization of conditions for the spectrodensitometric procedure was reached by eluting HPTLC silica gel plates in a 10 cm × 10 cm horizontal chamber. The solvent system consisted of hexane-chloroform-methanol (1.0:3.0:0.25, v/v/v). This system was found to give compact, dense and typical peaks for both levonorgestrel (R_f = 0.65 ± 0.03) and ethinyloestradiol (R_f = 0.43 ± 0.02). Densitometric analysis of the drugs was carried out in the reflectance mode at 225 nm by using a computer controlled densitometric scanner. The calibration curves of levonorgestrel and ethinyloestradiol were linear in the range of 200-800 and 40-160 ng per spot, respectively. The method was validated for precision, robustness and recovery. As the proposed method can effectively separate the drugs from their degradation products, it can be employed as a stability-indicating method.     

Development and validation of a high-performance thin-layer chromatographic method for determination of ofloxacin residues on pharmaceutical equipment surfaces

An HPTLC method with densitometric quantification using fluorescence at 313 nm was developed and validated for the determination of ofloxacin residue in controlling pharmaceutical equipment cleanliness. Simulated samples at a residue level of 1 mg/m2 were prepared by spreading the calculated amount of ofloxacin solution on 1, 5, and 10 dm2 stainless steel surfaces. After evaporation of the solvent, the residue was removed by two ethanol wetted cotton swabs, which were thereafter extracted with the mixture of ethanol and Na2EDTA-water solution at pH 11 for 15 min with sonication. The extract and standards were applied on HPTLC silica gel 60 plates and then developed in a horizontal developing chamber from both sides using ethanol-conc. ammonia (4+1, v/v) as the mobile phase. The mean recovery (n=6) at 1 mg/m2 from 1, 5, and 10 dm2 was 95.3, 88.6, and 89.7% with the CV values 3.78, 4.41, and 4.97%, respectively. The absolute detection limit was 0.6 ng and the quantitation limit was 2 ng, but it was shown that these can be improved by immersion of the developed plate into a solution of liquid paraffin-n-hexane (1+2, v/v) to approximately 0.25 and 0.9 ng, respectively. The LOD of the method using detection without paraffin-n-hexane was 3, 0.6, and 0.3 microg/m2 by swabbing 1, 5, and 10 dm2, respectively. The method can be applied to routine control of pharmaceutical equipment cleanliness by sampling from stainless steel surface areas of 1 to 10 dm2 with acceptable residue limit/surface of 1 mg/m2.     

Determination of chenodeoxycholic acid on 3-cyanopropyltrichlorosilane-treated high-performance thin-layer chromatographic plates

Summary Chenodeoxycholic acid in commerical drugs was analyzed by high-performance, thin-layer chromatography (HPTLC) using a cyanoalkyl chemically-bonded stationary phase, prepared by treating pre-coated silica with 3-cyanopropyltrichlorosilane (3CPTS). The 3CPTS-treated plates were used to evaluate commerical chenodeoxycholic acid in drug capsules. After development with methanol on the 3CPTS-treated plate, the chenodeoxycholic acid spot in the chromatogram was measured with a TLC densitometer equipped with a dual-wave length TLC scanner at λ=370nm. By this method, the fluorescence intensity of chenodeoxycholic acid was measured within 2.6% error over the range 30–240ng and the limit of detection was 30ng.     

Preparation and evaluation of mono-, di- and triphenyl-treated plates for the high-performance thin-layer chromatographic analysis of bufadienolides inBufonis venenum

Summary The retention and selectivity behaviour of bufadienolides (cinobufagin, resibufogenin, bufalin) inBufonis venenum have been studied by high-performance thin-layer chromatography on phenyl-modified silica plates, prepared from solutions of phenyldimethylethoxysilane, diphenylmethylethoxysilane or triphenylethoxysilane in toluene. Using various acetonitrile-distilled or methanol-distilled water mixtures as eluents, the bufadienolides have been separated on all the plates studied, but with different degrees of resolution. The selectivity for the separation of cinobufagin, resibufogenin, and bufalin between solutes and stationary phases is discussed. It is shown that monophenyl and diphenyltreated plates are more suitable for the selective separation of cinobufagin, resibufogenin, and bufalin inBufonis venenum than triphenyl-treated plates.     

Validated high-performance thin-layer chromatography method for determination of trigonelline in herbal extract and pharmaceutical dosage form

A new, simple, sensitive, selective, precise and robust high-performance thin-layer chromatographic (HPTLC) method for analysis of trigonelline was developed and validated for the determination of trigonelline in herbal extracts and in pharmaceutical dosage forms. Analysis of trigonelline was performed on TLC aluminium plates pre-coated with silica gel 60F-254 as the stationary phase. Linear ascending development was carried out in twin trough glass chamber saturated with mobile phase consisting of n-propanol-methanol-water (4:1:4, v/v/v) at room temperature (25 ± 2 °C). Camag TLC scanner III was used for spectrodensitometric scanning and analysis in absorbance mode at 269 nm. The system was found to give compact spots for trigonelline (R_f value of 0.46 ± 0.02). The linear regression analysis data for the calibration plots showed good linear relationship with r² = 0.9991 ± 0.0002 in the concentration range 100-1200 ng spot⁻¹ with respect to peak area. According to the International Conference on Harmonization (ICH) guidelines the method was validated for precision, recovery, robustness and ruggedness. The limits of detection and quantification were determined. The trigonelline content of herbal extracts quantified and estimated from the formulation was found to be well within limits (±5% of the labeled content of the formulations). Statistical analysis of the data showed that the method is reproducible and selective for the estimation of trigonelline.     

Development and validation of RP-HPLC method for the determination of stigmasterol in the botanical extract of Ficus deltoidea

A simple, rapid, accurate and precise RP-HPLC method was developed for the determination of stigmasterol in botanical extract of Ficus deltoidea. Separation was achieved with acetonitrile and acetic acid in water (75:25% v/v) in isocratic mode at 210?nm. Single sharp peak of standard stigmasterol was detected at retention time 3.17?min which overlay with the peak of plant extract at 3.14?min. The calibration curve was found to be linear in a concentration range of 2–10?μg/ml with correlation coefficient of 0.998. The LOD and LOQ were found to be 1.50?μg/ml and 4.55?μg/ml respectively. Accuracy and precision was determined with overall recovery of 99.6–100.1% for stigmasterol and RSD values in both intra-day and inter-day repeatability assay lesser than 0.340%, respectively. The robustness study also indicated that there is no influence of minor changes in detecting wavelength and flow rate of mobile phase on the response.     

Development and validation of a reversed-phase ion-pair high-performance liquid chromatographic method for the determination of risedronate in pharmaceutical preparations

A stability indicating, reversed-phase ion-pair high-performance liquid chromatographic method was developed and validated for the determination of risedronate in pharmaceutical dosage forms. The determination was performed on a BDS C₁₈ analytical column (250 mm × 4.6 mm i.d., 5 μm particle size); the mobile phase consisted of 0.005 M tetrabutylammonium hydroxide and 0.005 M pyrophosphate sodium (pH 7.0) mixed with acetonitrile in a ratio (78:22, v/v) and pumped at a flow rate 1.00 mL min⁻¹. The ultraviolet (UV) detector was operated at 262 nm. The retention times of magnesium ascorbyl phosphate, which was used as internal standard and risedronate were 4.94 and 5.95 min, respectively. The calibration graph was ranged from 2.50 to 20.00 μg mL⁻¹, while detection and quantitation limits were found to be 0.48 and 1.61 μg mL⁻¹, respectively. The intra- and inter-day percentage relative standard deviations, %R.S.D., were less than 5.9%, while the relative percentage error, %E_r, was less than 0.4%. The method was applied to the quality control of commercial tablets and content uniformity test and proved to be suitable for rapid and reliable quality control.     

Application of a validated stability-indicating densitometric thin-layer chromatographic method to stress degradation studies on moxifloxacin

A simple, sensitive, selective, precise and stability-indicating high-performance thin-layer chromatographic (HPTLC) method for densitometric determination of moxifloxacin both as a bulk drug and from pharmaceutical formulation was developed and validated as per the International Conference on Harmonization (ICH) guidelines. The method employed TLC aluminium plates pre-coated with silica gel 60F-254 as the stationary phase and the mobile phase consisted of n-propanol-ethanol-6 M ammonia solution (4:1:2, v/v/v). Densitometric analysis of moxifloxacin was carried out in the absorbance mode at 298 nm. Compact spots for moxifloxacin were found at R_f value of 0.58 ± 0.02. The linear regression analysis data for the calibration plots showed good linear relationship with r = 0.9925 in the working concentration range of 100-800 ng spot⁻¹. The method was validated for precision, accuracy, ruggedness, robustness, specificity, recovery, limit of detection (LOD) and limit of quantitation (LOQ). The LOD and LOQ were 3.90 and 11.83 ng spot⁻¹, respectively. Drug was subjected to acid and alkali hydrolysis, oxidation, dry heat, wet heat treatment and photodegradation. All the peaks of degradation products were well resolved from the standard drug with significantly different R_f values. Statistical analysis proves that the developed HPTLC method is reproducible and selective. As the method could effectively separate the drug from its degradation products, it can be employed as stability-indicating one. Moreover, the proposed HPTLC method was utilized to investigate the kinetics of the acidic and alkaline degradation processes at different temperatures. Arrhenius plot was constructed and apparent pseudo-first-order rate constant, half-life and activation energy were calculated. In addition the pH-rate profile for degradation of moxifloxacin in constant ionic strength buffer solutions within the pH range 1.2-10.8 was studied.     

Development and validation of a hydrophilic interaction liquid chromatographic method for determination of aromatic amines in environmental water

A simple, precise, and accurate hydrophilic interaction liquid chromatographic (HILIC) method has been developed for the determination of five aromatic amines in environmental water samples. Chromatography was carried out on a bare silica column, using a mixture of acetonitrile and a buffer of NaH₂PO₄–H₃PO₄ (pH 1.5, containing 10 mM NaH₂PO₄) (85:15, v/v) as a mobile phase at a flow rate of 1 mL min⁻¹. Aromatic amines were detected by UV absorbance at 254 nm. The linear range of amines was good (r² > 0.998) and limit of detection (LOD) within 0.02–0.2 mg L⁻¹ (S/N = 3). The retention mechanism for the analytes under the optimum conditions was determined to be a combination of adsorption, partition and ionic interactions. The proposed method was applied to the environmental water samples. Aromatic amines were isolated from aqueous samples using solid-phase extraction (SPE) with Oasis HLB cartridges. Recoveries of greater than 75% with precision (RSD) less than 12% were obtained at amine concentrations of 5–50 μg L⁻¹ from 100 mL river water and influents from a wastewater treatment plant (WWTP). The present HILIC technique proved to be a viable method for the analysis of aromatic amines in the environmental water samples.     

Development and validation of a normal-phase high-performance thin layer chromatographic method for the analysis of sulfamethoxazole and trimethoprim in co-trimoxazole tablets

Pneumocystis carinii pneumonia (PCP) is often the ultimate mortal cause for immunocompromised individuals, such as HIV/AIDS patients. Currently, the most effective medicine for treatment and prophylaxis is co-trimoxazole, a synergistic combination of sulfamethoxazole (SMX) and trimethoprim (TMP). In order to ensure a continued availability of high quality co-trimoxazole tablets within resource-limited countries, Medicines Regulatory Authorities must perform quality control of these products. However, most pharmacopoeial methods are based on high-performance liquid chromatographic (HPLC) methods. Because of the lack of equipment, the Tanzania Food and Drugs Authority (TFDA) laboratory decided to develop and validate an alternative method of analysis based on the TLC technique with densitometric detection, for the routine quality control of co-trimoxazole tablets. SMX and TMP were separated on glass-backed silica gel 60 F₂₅₄ plates in a high-performance thin layer chromatograph (HPTLC). The mobile phase was comprised of toluene, ethylacetate and methanol (50:28.5:21.5, v:v:v). Detection wavelength was 254 nm. The R_f values were 0.30 and 0.61 for TMP and SMX, respectively. This method was validated for linearity, precision, trueness, specificity and robustness. Cochran's criterion test indicated homoscedasticity of variances for the calibration data. The F-tests for lack-of-fit indicated that straight lines were adequate to describe the relationship between spot areas and concentrations for each compound. The percentage relative standard deviations for repeatability and time-different precisions were 0.98 and 1.32, and 0.83 and 1.64 for SMX and TMP, respectively. Percentage recovery values were 99.00% ± 1.83 and 99.66% ± 1.21 for SMX and TMP, respectively. The method was found to be robust and was then successfully applied to analyze co-trimoxazole tablet samples.     

Development and validation of a thin-layer chromatographic method for determination of chloramphenicol residues on pharmaceutical equipment surfaces

A thin-layer chromatographic (TLC) method with densitometric quantitation using the absorption reflectance mode at 280 nm was developed and validated for the determination of chloramphenicol residues in controlling pharmaceutical equipment cleanliness. Simulated samples at residue levels 0.5, 1, and 1.2 mg/m2 were prepared by spreading the calculated amount of chloramphenicol solution on a 10 dm2 stainless steel surface. After evaporation of the solvent, the residue was removed by 2 methanol-wetted cotton swabs, which were then extracted with methanol. The extract was applied on a high-performance TLC (HPTLC) silica gel F254 plate together with standards ranging from 10 to 60 ng. Plates were developed in a horizontal developing chamber from both sides (36 applications per plate) by using n-hexane-ethyl acetate (35 + 65, v/v) as developing solvent. The mean recovery (n=6) at 1 mg/m2 was 95.8%, and the coefficient of variation was 5.8%. The absolute detection limit was 3 ng, and the quantitation limit 10 ng. The method detection limit was 0.3 mg/m2 by swabbing 2.5 dm2 and 0.075 mg/m2 by swabbing 10 dm2. Chloramphenicol was stable on the plate 2 h before and 24 h after development. Additionally, it was stable during 7 days storage on the cotton swabs in the solvent at room temperature and in diluted standard solution stored in darkness at 4 degrees C. The method can be applied to routine control of pharmaceutical equipment cleanliness by sampling from the stainless steel surface areas of 2.5 to 10 dm2, and an acceptable residue limit of 1 mg/m2.     

A new validated high-performance liquid chromatographic method for the determination of EGIS-9933 in rat plasma

Summary A new highly sensitive high-performance liquid chromatographic (HPLC) procedure for determination of EGIS-9933 (a newly developed anxiolytic compound) in rat plasma is described. A gradient, elution method with UV detection at 270 nm has been developed using a mobile phase of a mixture of A: methanol:acetonitrile 1:9 and B:0.5% triethilamine in water, the pH of B was adjusted to 3 with phosphoric acid. Solid phase extraction (SPE) was used for the sample preparation. The calibration was linear in the 10–10000 ng mL⁻¹ concentration range. The limit of quantification was 10 ng mL⁻¹. The bioanalytical method was validated according to internationally accepted criteria for biological samples. Presented at: Balaton Symposium on High-Performance Separation Methods, Siófok, Hungary, September 3–5, 1997.     

A versatile high-performance thin-layer chromatographic method for the simultaneous determination of five antihypertensive drugs: method validation and application to different pharmaceutical formulations

JPC – Journal of Planar Chromatography – Modern TLC - A selective high-performance thin-layer chromatography (HPTLC) densitometric method has been developed for the simultaneous...     

Expert systems for method development and validation in HPLC

ESCA, Expert Systems Applied to Chemical Analysis, started its research in March 1987, with the aim of building prototype expert systems for HPLC method development. Results of this research have been published as the work has progressed. The project is now completed and this paper summarises some of the overall project conclusions. Seven different expert systems have been built which tackle problems throughout the process of method development, four stand-alone systems and three integrated systems. The object of ESCA was to evaluate the applicability of expert system technology to analytical chemistry and not all the systems were built for commercial uses. Many of the systems tackle problems specific to one or more of the partners and thus may not be useful outside this environment. However, the results of the work are still pertinent to analysts wishing to build their own systems. These results are described, however, the emphasis of the paper is on those systems developed for method validation.Method validation for HPLC is a complex task which requires many characteristics of the method to be tested, e.g. accuracy, precision, etc. The expert systems built within ESCA concern the validation of precision. Two systems were developed for repeatability testing and ruggedness testing. The method validation process can be divided into several discrete stages, these include: (1) The selection of the method feature to test, for instance which factors can influence the ruggedness of a method. (2) The definition of a test procedure, for instance an efficient statistical design. (3) The execution of experiments and the interpretation of results. (4) A diagnosis of any observed problem. This paper describes these two systems in some detail and summarises some of the results obtained from their evaluation. It concludes that expert systems can be useful in solving analytical problems and the integration of several expert systems can provide extremely powerful tools for the analyst.     

Development and validation of a new high-performance liquid chromatographic method for the loperamid hydrochloride determination in drugs

A selective, precise and new high-performance liquid chromatographic method for the analysis of loperamid hydrochloride in pharmaceutical formulations was developed and validated. The mobile phase consisting buffer (sodium-octansulphonate, triethylamine and ammonium hydroxide) in water: acetonitriie (45: 55, v/v) (pH 3.2). The absorbance was monitored with a DAD detector at 226 nm. The flow rate was 1.5 cm³ min⁻¹. The linearity (r = 0.9947) and the recovery (98.58–100.42%) were found to be satisfactory. The detection and quantitation limits were found to be 0.95 and 3.12 μg cm⁻³. The results demonstrated that the procedure was accurate, precise and reproducible. It can be suitably applied for the estimation of lopera-mid hydrochloride in pharmaceutical formulations. The article is published in the original.     

Development and validation of a pre-column reversed phase liquid chromatographic method with fluorescence detection for the determination of primary phenethylamines in dietary supplements and phytoextracts

A sensitive and selective reversed-phase liquid chromatographic (RP-LC) method was developed and validated to determine octopamine, tyramine and Tyrosine (Tyr) in complex matrices as formulations and phytoextracts (Citrus aurantium), after pre-column derivatization with o-phthaldialdehyde (OPA) reagent. The chromatographic separations were performed at room temperature on a Phenomenex Luna C18 column using methanol and sodium acetate buffer (pH 5.5) by varying composition gradient elution as mobile phase and detected flurometrically at λ(em)=455 nm with λ(ex)=340 nm. The results obtained by the proposed method were compared with those achieved by a validated direct RP-LC method with fluorescence detection at λ(em)=310 nm with λ(ex)=275 nm, as reference method, using a Phenomenex Gemini C18 column under isocratic elution conditions with acetonitrile and sodium 1-heptanesulphonate (pH 3), as mobile phase. The higher sensitivity of the derivatization method (detection limit about 0.06 pmol) allowed the sure determination of octopamine present in traces in the examined samples. The repeatability of method (RSD) was ≤1.90% and there was no significant difference between repeatability and intermediate precision data. Recovery studies showed good results 99.5-101.3% with RSD ranging from 0.8 to 1.2%. All analyses were performed by mild conditions in absence of preliminary difficult extraction methodologies or laborious step of sample pre-treatment.     

A validation protocol for the HPTLC standardization of herbal products: application to the determination of acteoside in leaves of Plantago palmata Hook. f.s

Formal validation, that is the study of the analytical performances of a method, is recognized as the best safeguard against the generation and publication of data with low reliability.Although the topic of HPTLC validations has been largely investigated, there is still a need for a general validation method applicable whenever a blank matrix cannot be reconstituted, notably herbs and their extracts.This work proposes two validation schemes aiming at generate linearity, accuracy and precision data in a minimal number of HPTLC plates, taking the standardization of Plantago palmata as an example with both UV and visible (post-chromatographic derivatization with a sulphuric acid-vanillin reagent) detections. A major problem associated with HPTLC determinations is underlined, namely the low range of linearity which makes spiking studies quite difficult as care must be taken to avoid overloading, whereas keeping the analyte detectable in blank extracts and avoiding spikes too close to endogenous levels. A second problem is the use of general post-chromatographic derivatization reagents that compromise the selectivity of the method by reacting with compounds that may not be resolved from the compound of interest. The use of such reagents is clearly not without danger, especially given the relatively low resolution of planar chromatography.In conclusion, the retained validation protocol effectively yields the main validation data whereas allowing to pinpoint major analytical drawbacks. It was not possible to simultaneously validate aucubin and acteoside assays as both analytes are present at too different levels/detectabilities.     

Development and validation of a high-performance liquid chromatographic method for determination of eprosartan in bulk drug and tablets

A simple, precise, and accurate isocratic RP-HPLC method was developed and validated for determination of eprosartan in bulk drug and tablets. Isocratic RP-HPLC separation was achieved on a Phenomenex C18 column (250 x 4.6 mm id, 5 microm particle size) using the mobile phase 0.5% formic acid-methanol-acetonitrile (80 + 25 + 20, v/v/v, pH 2.80) at a flow rate of 1.0 mL/min. The retention time of eprosartan was 7.64 +/- 0.05 min. The detection was performed at 232 nm. The method was validated for linearity, precision, accuracy, robustness, solution stability, and specificity. The method was linear in the concentration range of 10-400 microg/mL with a correlation coefficient of 0.9999. The repeatability for six samples was 0.253% RSD; the intraday and interday precision were 0.21-0.57 and 0.33-0.71% RSD, respectively. The accuracy (recovery) was found to be in the range of 99.86-100.92%. The drug was subjected to the stress conditions hydrolysis, oxidation, photolysis, and heat. Degradation products produced as a result of the stress conditions did not interfere with detection of eprosartan; therefore, the proposed method can be considered stability-indicating.     

Thin-layer chromatographic and high-performance thin-layer chromatographic ready-for-use layers with lipophilic modifications

Stable silica gel sorbents with aliphatic or aromatic groups are formed by chemical modifications of the silanol groups with special reactive silanes. Various lipophilic surface modifications on silica gels with varying pore structures are tested with regard to their chemical and physico-chemical characteristics, their wettability and their chromatographic retention data. The main problem in TLC is the preparation of abrasion-resistant layers on glass or on foils which meet the usual high standard of quality and are also suited for quantitative determinations. Thin-layer chromatography on reversed-phase layers can only be performed if the complete wettability of the lipophilic stationary phase in dry form is guaranteed by the mobile phase. Adsorption-chromatographic separations with lipophilic eluents and reversed-phase partition-chromatographic separations with hydrophilic eluents are performed, for example, with dyes, with polycyclic aromatic hydrocarbons and with lipids. The great differences in selectivity caused by the various modifications of the sorbent and the varying eluent composition are remarkable. Ready-for-use layers with lipophilic surface modifications complement the existing range of pre-coated layers and thus widen the application of TLC and HPTLC considerably.