Development and validation of a simple thin layer chromatographic method for the analysis of artemisinin in Artemisia annua L. plant extracts

Owing to the development of parasite resistance to standard antimalarial treatments like chloroquine and sulfadoxine-pyrimethamine, the demand for Artemisia annua, a key ingredient for new and highly effective antimalarial drugs, is huge. Therefore selective and precise methods to determine the content of artemisinin in dry plant material and in raw impure extracts are needed. In this work a method is described for the clear separation and extraction of artemisinin from other plant components in the Artemisia annua L. plant by thin-layer chromatography (TLC). To obtain optimal extraction and recovery efficiency, several parameters were evaluated, including choice of extraction solvent, TLC plate type and sensitivity between UV and visible light. Method validation was performed on both the dry plant material and non-purified plant extracts. Toluene presented the highest extraction efficiency compared with petroleum ether, hexane and methanol. Reversed-phase plates showed more concentrated spots than normal-phase plates, while the sensitivity of the analysis in UV was comparable to that in visible light but less precise. The impure plant extracts were analyzed by both TLC and HPLC-UV at 215 nm and both methods met the requirements for linearity, selectivity, precision and accuracy. Hence, the proposed TLC method can easily be used for both qualitative and quantitative control of the raw plant extract in areas where advanced methods are scarce.     

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.     

Highly sensitive high-performance thin-layer chromatography method for the simultaneous determination of molnupiravir,favipiravir, and ritonavir in pure forms and pharmaceutical formulations

Favipiravir, molnupiravir, and ritonavir have been recently approved as the first oral antivirals for treatment of SARS-CoV-2 viral infections. Their combination was reported in several clinical studies, alternatively, to enhance the viral eradication and improve patient's recovery times and rates. Being all orally administered, therefore, the development of new sensitive and validated methodologies for their simultaneous determination is a necessitate. In the proposed research, a sensitive, selective, and simple high-performance thin layer chromatography method was developed and validated for determination of favipiravir, molnupiravir, and ritonavir. Silica gel 60F254 thin layer chromatography plates were used as stationary phase for this separation using mobile phase composed of methylene chloride:ethyl acetate:methanol:25% ammonia (6:3:4:1, v/v/v/v). Densitometric detection was performed at wavelength 289 nm. Peaks of favipiravir, molnupiravir, and ritonavir were resolved at retention factors 0.22, 0.42, and 0.63, respectively. The proposed method was found linear within the specified ranges of 3.75–100.00 μg/mL for molnupiravir and favipiravir, and 2.75–100.00 μg/mL for ritonavir. Limits of detection were found to be 1.12, 1.21, and 0.89 μg/mL for favipiravir, molnupiravir, and ritonavir, respectively. This is the first method to be reported for the simultaneous determination of the cited three antiviral drugs. The method was assessed on novel greenness metrics.     

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.     

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 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.     

Validated high-performance thin-layer chromatographic method for quantification of gallic acid and ellagic acid in fruits of Terminalia chebula,Phyllanthus emblica,and Quercus infectoria

A simple and rapid high-performance thin-layer chromatographic method for quantification of gallic acid and ellagic acid in dried fruits of Terminalia chebula, Phyllanthus emblica, and Quercus infectoria has been developed. The chromatographic development was carried out on precoated silica gel 60 F₂₅₄ plates in a mixture of toluene:ethyl acetate:chloroform:formic acid (4:8:1:3 v/v/v/v). The plate was scanned densitometrically at a wavelength of 280 nm. The retention factor value of gallic acid and ellagic acid was found to be 0.63 ± 0.2 and 0.53 ± 0.1, respectively. The developed method was validated in terms of linearity, precision, accuracy, sensitivity, robustness, specificity and stability as per the international conference of harmonization guidelines. The method showed good linear relationship over a range of 100–600 ng/band (gallic acid) and 100–500 ng/band (ellagic acid) with a regression coefficient (r²) of 0.997 (gallic acid) and 0.996 (ellagic acid). The method showed high accuracy (99.65%–100.85%). The percentage relative standard deviation of intra-day and inter-day precision studies was not more than 2%. The method is highly robust and has displayed high specificity. The developed method is new, simple, and accurate and can be successfully employed in routine analysis of raw materials and formulations containing gallic acid and ellagic acid.     

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.     

Development and validation of stability indicating chromatographic methods for simultaneous determination of citicoline and piracetam

Citicoline and piracetam were subjected separately to different stress conditions as recommended by the international conference on harmonization. In addition, new stability indicating thin layer chromatographic and ultra high performance liquid chromatographic methods have been developed and validated for simultaneous determination of citicoline and piracetam in presence of their degradation products. Separation on the proposed thin layer chromatographic method was carried out using a developing system containing methanol:chloroform:ammonium chloride buffer (9:1:2, v/v/v) on silica gel plates at 230 nm. On the other hand, the mobile phase in the ultra high performance liquid chromatographic method was composed of water (containing 0.1% triethylamine):ethanol (92:8, v/v). The flow rate was 1 mL/min and ultraviolet detection was at 230 nm. Moreover, results of the developed methods were statistically compared to those obtained by the reported high‐performance liquid chromatography method and no significant difference between them was found. The greenness profile of ultra high performance liquid chromatographic method was assessed and compared with those of the previously published high‐performance liquid chromatography methods, it was noticed that the proposed ultra high performance liquid chromatographic method more environmentally friendly and greener than other methods.     

Development and validation of a multiresidue method for determination of 37 pesticides in soil using GC-NPD

In this study, a multiresidue analytical method for the detection of 37 pesticides in a soil matrix was developed and validated. The soil sample was fortified with a known quantity of pesticides at two different concentration levels (0.1 and 0.01 µg/g) and the analytes were extracted via a liquid–solid extraction method. The pesticides were separated on an HP5 capillary column and were analyzed with a gas chromatograph coupled to a nitrogen–phosphorous detector (GC‐NPD). Method validation was accomplished with good linearity (r² = 0.994–0.999) within a considerable range of concentrations. Satisfactory recoveries (70.5–110.4%) were obtained with 32 pesticides at both spiking concentration levels, whereas five pesticides—dimepiperate, buprofezin, prometryn, pirimicarb, and fludioxonil—were recovered at relatively low levels (43.6–61.8%). The applicability of the method was demonstrated by analyzing field samples collected from 24 different sites around Yeongsan and Sumjin rivers in the Republic of Korea. No residues of the selected pesticides were detected in any of the samples. The developed method could be employed as a simple and cost‐effective method for the routine detection and analysis of 37 pesticides in soil samples. Copyright © 2010 John Wiley & Sons, Ltd.     

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.     

Advances in the thin-layer chromatographic forensic analysis of inks

Advances in the use of thin layer and high-performance thin-layer chromatography for analysis of inks and ink writings in forensic investigations are reviewed. Experimental materials and techniques such as sample preparation, layers, sample application, development, detection, documentation, and interpretation of results are described and selected applications for several ink types are given. Some additional analytical techniques that complement thin-layer chromatography are also mentioned. Citations to important literature, from 2008 to 2016, are included.     

Development and validation of high-performance liquid chromatography method for determination of clarithromycin in pharmaceutical tablets

Clarithromycin is a very important macrolide antibiotic used to treat bacterial infections in human and veterinary medicine. This study reports the development and validation of cost-effective, simple, precise, accurate, and robust high-performance liquid chromatography (HPLC) for the determination of clarithromycin (CLA) in tablets. Reversed-phase chromatography was conducted using a standard column at 55°C with ultraviolet detection at 215 nm. A mobile phase consisting of acetonitrile –2-methyl-2-propanol –potassium phosphate buffer was used at a flow rate of 1.0 mL/min. The proposed method displayed good linearity, precision, accuracy, robustness, and specificity. The present HPLC was compared with capillary electrophoresis and bioassay methods and the results indicated that there was no significant difference between these methods. Moreover, the obtained results demonstrated the validity of the isocratic HPLC, which allows reliable quantitation of CLA in pharmaceutical samples. Thus, it can be used as a substitute alternative methodology for the routine quality control of this medicine, in situations where other methods are less accessible in the laboratory.