The presented study describes the development, optimization, and validation of a method for the determination of four structurally different anthelmintics (benzimidazole, diphenyl sulfide, imidazothiazole, hexahydropyrazine, and tetrahydropyrimidine) in water samples. The studied compounds were albendazole, febantel, levamisole, praziquantel, and pyrantel pamoate. The method involves solid-phase extraction and preconcentration of the anthelmintics, separation by thin-layer chromatography (TLC), and quantification by videodensitometry. TLC and preparative TLC separation was performed on silica gel 60 F254 plates with chloroform-methanol-formic acid as the mobile phase. Spots were detected and quantified at λ = 254 nm by videodensitometry. The method provides a linear response over a concentration range of 200–1500 µg L−1, depending on the anthelmintic with a correlation coefficient of 0.9888 in most cases, except for albendazole (0.9634) and pamoic acid (0.9654) as a secondary component of pyrantel pamoate. Also, the method has revealed limits of detection (100–300 µg L−1), good precision (intra- and inter-day) with a relative standard deviation below 13%, and recoveries above 95% for all investigated pharmaceuticals. The developed method has been successfully applied to the analysis of anthelmintics in production wastewater samples obtained from the local pharmaceutical industry. In order to determine the structures of two compounds detected by TLC of pyrantel pamoate, the spots were separated by preparative TLC, and the structures of the isolated samples were analyzed using 1H and 13C nuclear magnetic resonance (NMR) spectroscopy.
Background: The fruits of Benincasa hispida (Thunb.) Cogniaux, a ‘rasayana’ in ‘Ayurveda’, are enriched with several secondary metabolites, and rutin is one of them. Fruits are used for their anabolic, brain tonic, carminative, diuretic, memory enhancer, refrigerant, and vitalizer properties. Objective: In view of the fact that herbal medicines and/or products are facing challenges towards global acceptance due to the lack of universally accepted standardization method (s), the aim of the current investigation was to develop and validate a high-performance thin-layer chromatography (HPTLC)-densitometry method for the quantification of rutin in the hydroalcoholic extracts of the fruit pulp of B. hispida (HABH). Materials and methods: The separation was achieved in a solvent system consisting of ethyl acetate-formic acid-acetic acid-water at a ratio of 7.2:0.7:0.7:1.4 by volume on a TLC aluminum plate pre-coated with silica gel 60 F254. Quantifications were performed by densitometric scanning under a deuterium lamp at a wavelength of 268 nm in the absorbance mode. The precision, accuracy, and reproducibility of the HPTLC method were validated by the International Conference on Harmonization (ICH) guidelines. Results: The mobile phase employed for HPTLC/TLC resulted in good separation for rutin (RF = 0.357). The limit of detection and limit of quantification of the analysis were found to be 0.1 and 0.3 µg per band, respectively. The rutin concentration in the HABH was found to be 178.28 ± 3.62 µg in 10 mg of the extract. Conclusion: The method developed here is simple, fast, reliable, and sensitive and can be implemented in the analysis and routine quality control of B. hispida formulations containing rutin.
Three simple, sensitive, and validated methods were developed for the quantitative determination of fosinopril sodium (FOS) and hydrochlorothiazide (HCZ) in the presence of an HCZ impurity, chlorothiazide (CZ). The first method (I) was the ratio difference spectrophotometric method (RD), in which a standard spectrum of 8 µg mL−1 HCZ was used as a divisor, and the difference in amplitude values at 204.6 and 231.2 nm and 290 and 302.6 nm was used for the determination of FOS and CZ, respectively. Meanwhile, for the determination of HCZ, a standard spectrum of 6 µg mL−1 CZ was the chosen divisor, and the amplitude difference at 275 nm and 293.6 nm was selected for the calculation of its concentrations. The second method (II) was mean centering of ratio spectra spectro-photometric method (MCR), which depended on the implementation of the mean-centered ratio spectra in two successive steps and the measurement of the amplitudes of the mean-centered second ratio spectra at 243.4 nm for CZ and peak-to-peak amplitudes at 215.6 and 215.8 nm for FOS and at 223.8 and 224 nm for HCZ. On the other hand, the third method (III) was thin-layer chromatography (TLC)-densitometry at which the chromatographic separation of this ternary mixture was performed using pre-activated silica gel 60 F254 TLC plates and a developing system mixture consisting of ethyl acetate-chloroform-methanol-formic acid (60:40:5:0.5, by volume) with ultraviolet (UV) scanning at 215 nm. The developed methods were validated according to the International Conference of Harmonization (ICH) guidelines and were successfully used for the determination of FOS and HCZ in their pharmaceutical formulations. Also, a statistical comparison between the developed methods and the reported HPLC method was attained. Using Student's t-test and F-test, the results confirmed that there was not any significant difference between them regarding accuracy and precision.
A novel chromogenic reagent was developed for the identification and detection of organophosphorus herbicide. The organophosphorus herbicide glyphosate is abundantly used, despite being highly toxic to human beings. In the presented communication, we have developed a simple identification and detection technique for the identification of organophosphorus herbicides with the help of thin-layer chromatography. By using this technique, we can easily identify the presence of organophosphorus poisoning by the use of cobalt thiocyanate. By using the cobalt thiocyanate reagent, we can easily identify color change by high-performance thin-layer chromatography (HPTLC). We developed a simple HPTLC method for testing forensic samples. The results obtained are highly useful in the field of organic qualitative analysis, chemistry, forensic science, and toxicology.
We have recently reported on the effect of the environmental conditions on the quantity of diosgenin. Attempts for the simultaneous quantification of trigonelline and diosgenin using normal-phase silica gel plates were not successful. A high-performance thin-layer chromatography (HPTLC) method was developed using glass-backed plates coated with RP-18 silica gel 60 F254S and acetonitrile-water (7.5:2.5, V/V) as the mobile phase. Trigonelline and diosgenin peaks were well separated with RF values 0.29 ± 0.02 and 0.17 ± 0.01, respectively. The TLC plates were directly scanned at 267 nm for trigonelline and at 430 nm after derivatization with vanillin-sulfuric acid for diosgenin. Linear regression analysis revealed a good linear relationship between the peak area and the amounts of trigonelline and diosgenin in the range of 200–1400 and 50–300 ng per band, respectively. The method was validated in accordance with the International Conference on Harmonization (ICH) guidelines for precision, accuracy, and robustness.
Losartan potassium (LOS), used in the treatment of hypertension, is metabolized primarily by cytochrome P450. This study investigates the effect of quercetin (QU), a CYP3A4 inducer, on the pharmacokinetics of LOS in rats. A rapid, sensitive high-performance thin-layer chromatography (HPTLC) method was developed and validated for the bioanalysis of losartan using olmesartan as internal standard (IS). The salting-out assisted liquid-liquid extraction (SALLE) employing acetonitrile and MgCl2 gave high recovery of LOS (>90%). HPTLC separation, achieved on silica gel 60 F254 plates employing toluene-ethyl acetate-acetone-formic acid (4:4:1:0.5 V/V) as the mobile phase, with densitometric analysis at 240 nm, gave good linearity (50–1200 ng mL−1) with high intra-day and inter-day precision. LOS in plasma samples was stable when stored under different stability conditions. The pharmacokinetics of LOS was found to be significantly altered when co-administered with QU: Cmax = 809.8 ± 4.1 at 40 min (tmax) to Cmax = 1124.8 ± 86.6 ng mL−1 at 120 min (tmax). This study indicates the chances of herb-drug interaction when LOS is co-administered with QU, leading to its increased bioavailability, potentiating its side effect/toxic manifestation. As QU is abundantly present in herbs and dietary food, patients of LOS therapy need to be cautious while concurrently consuming herbal preparations containing QU. This study also demonstrates the utility of HPTLC as an effective tool for pharmacokinetics study for the estimation of herb-drug interactions.
Thin-layer chromatography is one of the most efficient analysis methods that have remained very popular for many decades, despite the high rates of modern science development and the relative simplicity of the method discussed. One should note that the frequency of published works is virtually an estimate of the value of the given method by chemists-analysts. The result of the conducted scientometric study permits to make the conclusion on the fact that the TLC method is most often used in its initial kind as a classical ascending elution of plates with a silica gel layer in a saturated chamber in spite of diversity of chambers, ways of elution, and stationary phases . In the present review article, the main directions of the method development, based on the comparison of published works on TLC during 2 time periods with an interval of 10 years (2008 and 2018), are presented.
2,4-Dichlorophenoxyacetic acid (2,4-D) is a phenoxy group of herbicide used worldwide. As it is extensively used, it has consequential problems on living beings. 2,4-D is degraded into the chlorinated phenols and catechols, and these phenol compounds are more hazardous than the parent 2,4-D herbicide. In this paper, an attempt is made to detect 2,4-dichlorophenol in 2,4-D poisoning cases from human viscera. Sensitive and selective detection of 2,4-dichlorophenol using high-performance thin-layer chromatography (HPTLC) is possible by coupling it with 4-amminoantipyrene in the presence of potassium ferricyanide. Standard 2,4-dichlorophenol and human visceral extract are allowed to run on an HPTLC plate with hexane, acetone, and ethyl acetate as the mobile phase. Mechanistically, 4-amminoantipyrene reacts with 2,4-dichlorophenol in the presence of potassium ferricyanide to form p-quinoneimide which is brick red in color. This known reaction is, for the first time, applied to detect 2,4-dichlorophenol in 2,4-D poisoning cases from human viscera. The formation of brick red color spot on the HPTLC plate allows the easy and confirmed detection of 2,4-dichlorophenol in 2,4-D poisoning case. This HPTLC method is simple and easy to work in laboratory. The reagents do not react with the parent 2,4-dichorophenoxyacetic acid and other organophosphorus, organochlorine, carbamate, and pyrethroid insecticides, i.e., these reagents are specific. The constituents of the viscera (amino acids, peptides, proteins, etc.) and plant material do not interfere with the reagents. The presence of 2,4-dichlorophenol in the same visceral sample is confirmed by gas chromatography-mass spectrometry (GC-MS). The detection limit of reagents for 2,4-dichlorophenol is approximately 0.5 µg.
The aim of this work was to establish qualitative and quantitative methods for studying Guyinye residue extracts and Turkish gall (TG) cream. This study involved qualitative and quantitative analyses of gallic acid and methyl gallate and determined their preliminary antioxidant activity by high-performance thin-layer chromatography (HPTLC) and thin-layer chromatography-1,1-diphenyl-2-trinitrobenzene hydrazine (TLC-DPPH) in Guyinye residue extracts and TG cream. The thin-layer plate was a polyamide film and glacial acetic acid, methanol, ethyl acetate, and formic acid (10:6:2:1, volume ratio) were used as the developing agent. The scanning wavelength was 280 nm. Results showed that the RF values of gallic acid and methyl gallate were 0.57 ± 0.05 and 0.72 ± 0.05, respectively, and their linearity ranges were 0.001–0.005 and 0.00025–0.00125 mg with correlation coefficients of 0.9990 and 0.9994, respectively, which indicated a good linear relationship. The detection limits of gallic acid and methyl gallate were 3 and 75 ng, respectively, and their quantification limits were 10 and 250 ng, respectively. The average recovery was 98.59% and 98.33%, and the relative standard deviation (RSD) was 2.49% and 3.55%, respectively. Gallic acid was more remarkable than methyl gallate in antioxidant activity. Thus, HPTLC combined with TLC-DPPH, which can rapidly and accurately determine gallic acid and methyl gallate in Guyinye residue extracts and TG cream, is a simple, accurate, and rapid qualitative and quantitative method.