High-performance thin-layer chromatography method for the quantification of quetiapine fumarate and its related genotoxic impurities using green solvents

A novel high-performance thin-layer chromatographic (HPTLC) analytical method has been developed and optimized for the quantification of quetiapine fumarate (QF) and its two genotoxic impurities in drug substance and drug product. The desired separation was achieved on 60F₂₅₄ pre-coated HPTLC plates using combination of green solvents, ethyl acetate‒ethanol‒n-heptane (5:1:4, V/V) as developing solvents. The detection wavelength used for quantification was 229 nm. QF and its two related genotoxic impurities, namely, 2-chloroaniline and 2-aminodiphenylsulfide, were well resolved from one another with retention factor values of 0.13 ± 0.02, 0.57 ± 0.02 and 0.76 ± 0.02, respectively. The optimized method was validated according to the guidelines laid down by the International Council for Harmonisation. The linearity was determined in the range of 100–600 ng/spot for QF and 10‒60 ng/spot for its two related genotoxic impurities; R² ≥ 0.993. The method exhibited precision along with good accuracy, where 0.51, 0.86 and 1.86. The percentage recoveries obtained for 2-chloroaniline and 2-aminodiphenylsulfide were 99.04‒101.04%. The developed method can be successfully used for the analysis of drug samples.

     

HPTLC–densitometric and HPTLC–MS methods for analysis of flavonoids

HPTLC silica gel plates without and with fluorescence indicator F₂₅₄ in combination with n-hexane–ethyl acetate–formic acid (20:19:1, v/v/v) as a developing solvent were explored for the HPTLC–densitometric and HPTLC–MS/(MSⁿ) analyses of flavonoids. Pre-development of the plates with chloroform–methanol (1:1, v/v) was needed for reliable HPTLC–densitometric analyses of flavonoid aglycones in the whole R_F range, while 2-step pre-development (1^st methanol–formic acid (10:1, v/v), 2^nd methanol), that decreased background signals of formic acid adducts, was required for HPTLC–MS analyses. Optimization with conditioning of the adsorbent layer with water before development and saturation of the twin trough chamber resulted in required decrease of the R_F values of studied flavonoids (flavone, apigenin, luteolin, chrysin, quercetin dihydrate, myricetin, kaempferide, kaempferol, naringenin, pinocembrin).

Detection was performed based on fluorescence quenching (on the plates with F₂₅₄), natural fluorescence and after post-chromatographic derivatization with natural product reagent without or with further enhancement and stabilization of fluorescent zones with polyethylene glycol (PEG 400 or PEG 4000) or paraffin–n-hexane reagents. For all three reagents, drying temperature and time passed after drying influenced the intensity, which was increasing the first 20?min, and the stability (less than 2?h for PEGs and at least 24?h for paraffin–n-hexane) of the standards’ zones.

Optimal wavelengths for densitometric evaluation were selected based on in-situ absorption spectra scanned before and after derivatization and after stabilization. The developed method was tested via analyses of propolis, roasted coffee, rose hip, hibiscus, rosemary and sage crude extracts. To further increase the reliability of the obtained densitometric results HPTLC–MS/(MSⁿ) analyses of all crude extracts were performed. Several phenolic and non-phenolic compounds were tentatively identified.

Some possible interferences with phenolic acids (chlorogenic acid, rosmarinic acid, protocatechuic acid, gallic acid, syringic acid, ellagic acid, trans-cinnamic acid, o-coumaric acid, m-coumaric acid, p-coumaric acid, caffeic acid, ferulic acid, sinapic acid) that are often present in the extracts together with flavonoids were also examined.     

Densitometric high-performance thin-layer chromatography methods for the quantification of oleuropein in Olea europaea leaves and pharmaceutical preparation utilizing normal- and reversed-phase silica gel plates

Two validated, simple and precise densitometric high-performance thin-layer chromatography (HPTLC) quantification methods were proposed for both qualitative and quantitative estimation of oleuropein in Olea europaea leaves and a pharmaceutical product utilizing normal-phase and reversed-phase silica gel TLC plates. In method I, 10 × 20 cm glass plates coated with 0.2 mm thin layers of normal-phase silica gel 60 containing F₂₅₄ (E-Merck, Germany) and a mixture of ethyl acetate‒methanol‒water (8:1:0.5, V/V) were used as the stationary and the mobile phase, respectively. Method II utilized 10 × 20 cm glass-backed plates supporting 0.2 mm layers of RP-18 silica gel 60 containing F₂₅₄ (E-Merck, Germany) as the stationary phase and green solvents mixture composed of ethanol‒water (5.5:4.5, V/V) as the mobile phase. The two methods resulted in sharp, symmetrical, well-resolved peaks at R_F values of 0.47 ± 0.02 and 0.78 ± 0.03 with linearity ranges 200‒1400 ng/spot (r² = 0.9994) and 200‒1400 ng/spot (r² = 0.9996) for method I and method II, respectively. Spots corresponding to oleuropein were scanned at 200 nm. The two methods complied with the ICH guidelines for validation. Due to simplicity, low cost and short analysis time, the methods can be good alternatives for the quality control of different products containing olive leaves extract or pure oleuropein.

     

Development and validation of a high-performance thin-layer chromatography method for the estimation of bromfenac in ophthalmic solution

A novel, simple, precise, specific, accurate high-performance thin-layer chromatography (HPTLC) method was developed and validated for the estimation of bromfenac in ophthalmic solution. Diclofenac sodium was used as an internal standard (IS) because of its structural resemblance with bromfenac to develop a more accurate and precise method. Silica gel 60 F₂₅₄ HPTLC plates were used to separate bromfenac from the formulation with a mobile phase consisting of toluene-ethyl acetate-glacial acetic acid (65:35:0.2, V/V). Densitometric scanning was performed at 274 nm after the HPTLC plates were air-dried. Well-resolved bands and good peak shapes were obtained for both bromfenac and diclofenac sodium, with retention factor (R_F) values of 0.28 and 0.44, respectively. The proposed method was validated as per International Council for Harmonisation Q2 (R1) guidelines for specificity, precision, robustness, accuracy, and recovery. The drug shows linearity in the concentration range of 60‒270 ng/band and the correlation coefficient was found to be 0.999. The mean percent recovery of bromfenac was found to be 100.7%. The limit of detection and limit of quantification values for bromfenac were found to be 7.4 ng/band and 22.5 ng/band, respectively. The method was found to be novel since no HPTLC methods have yet been reported for the estimation of bromfenac. The developed method was successfully applied for the quantitative analysis of the drug in the ophthalmic formulation.

     

A Validated Quantification of Benzocaine in Lozenges Using TLC and a Flatbed Scanner

We present a video-densitometric quantification method for benzocaine in lozenges. The quantification is based on a derivatisation reaction with 4-dimethylaminobenzaldehyde. Measurements were carried out using a 16-bit flatbed scanner. Benzocaine was separated to a distance of 50 mm in a vertical developing chamber without vapour saturation. We present an RP-18 phase separation on a cyanopropyl plate (Merck, Darmstadt, Germany) using water, CH₃CN, dioxane, ethanol, and NH₃ (25 %) (8 + 2 + 1 + 1 + 0.05, v/v) as the mobile phase. We also separated benzocaine in a normal phase system on silica gel 60 LiChrospere^® plates (Merck, Darmstadt, Germany) with the mobile phase MTBE/cyclohexane (1 + 1, v/v). The calibration functions for benzocaine in both separations were linear in the range from 1 to 1,000 ng per spot. The range of linearity covers two magnitudes of power because the Kubelka–Munk expression was used for data transformation. In the cyanopropyl-system, the benzocaine amount was quantified as 242.5 ± 18.2 ng in a spot or 6.86 ± 0.52 mg in a single lozenge. The amount of 7.0 mg benzocaine per lozenge was labelled. The combined uncertainty of sample and calibration measurements was statistically calculated using a significance level of α = 0.05 to a total relative uncertainty of 7.49 %. The separation method is inexpensive, fast and reliable.     

Simultaneous quantification of brexpiprazole and sertraline HCl in synthetic mixture by thin-layer chromatography method

According to the International Council for Harmonisation (ICH) Q2 (R1) guideline, a sensitive, precise, accurate and robust high-performance thin-layer chromatographic (HPTLC) method was developed and validated for the simultaneous quantification of a newer combination of brexpiprazole (BREX) and sertraline HCl (SERT) in bulk and synthetic mixture. Stationary phase selected was pre-coated silica gel aluminum plate 60 F_254, and n-propanol‒hexane‒toluene‒triethylamine (7:2:1:0.1, V/V) was used as developing mobile phase. An appreciable absorbance shows at 254 nm, therefore the common detection wavelength was selected for the simultaneous quantification of BREX and SERT. The method was validated for different parameters: linearity, precision, accuracy, robustness, limit of detection and limit of quantification as per ICH guideline. The correlation coefficients (r²) for BREX and SERT were found to be 0.9940 and 0.9911, respectively. The mean of percentage recoveries for BREX and SERT were found to be 99.40–102.10% and 99.52–101.05%, respectively. The proposed HPTLC method has potential application for the quantification of BREX and SERT simultaneously in bulk and synthetic mixture both qualitatively and quantitatively.

     

Stimulation quantification of four natural lipase inhibitors from Alismatis Rhizoma by high-performance thin-layer chromatography method

The lipase inhibitory activities of four main components from the rhizomes of Alisma orientale (Sam.) Juz. were evaluated by an in situ high-performance thin-layer chromatography (HPTLC)‒bioautographic assay taking orlistat as control standard. The order of relative activity was alisol B 23-acetate > alisol B > alisol A > alisol C 23-acetate. With that, an accurate, efficient and sustainable HPTLC method was developed to simultaneously determine the four lipase inhibitors from the methanolic extracts of Alismatis Rhizoma (AR). The method was carried out on HPTLC glassed plates (20 × 10 cm) coated with silica gel 60 F₂₅₄ (0.2 mm thickness) using a mixture of cyclohexane and ethyl acetate (1:1, V/V) as the mobile phase. The R_F values found for alisol B 23-acetate, alisol C 23-acetate, alisol B and alisol A were 0.62, 0.42, 0.28 and 0.09, respectively. The method was validated for specificity, linear range, precision, stability, and recovery. The results determined by scanning densitometry showed no significant difference to the results obtained by HPLC. The developed method was verified to be trustworthy for the evaluation of quality markers in AR.

     

Chromatographic methods development,validation and degradation characterization of the antithyroid drug Carbimazole

The current paper reports the development and validation of stability‐indicating HPLC and HPTLC methods for the separation and quantification of main impurity and degradation product of Carbimazole. The structures of the degradation products formed under stress degradation conditions, including hydrolytic and oxidative, photolytic and thermal conditions, were characterized and confirmed by MS and IR analyses. Based on the characterization data, the obtained degradation product from hydrolytic conditions was found to be methimazole—impurity A of Carbimazole as reported by the British Pharmacopeia and the European Pharmacopeia. A stability‐indicating HPLC method was carried out using a Zorbax Eclipse Plus CN column (150 × 4.6 mm i.d, 5 μm particle size) and a mobile phase composed of acetonitrile–0.05 m KH₂PO₄ (20: 80, v/v) in isocratic elution, at a flow rate of 1 mL/min. The method was proved to be sensitive for the determination down to 0.5% of Carbimazole impurity A. Additionally, a stability‐indicating chromatographic HPTLC method was achieved using cyclohexane–ethanol (9:1, v/v) as a developing system on HPTLC plates F₂₅₄ with UV detection at 225 nm. The proposed HPLC and HPTLC methods were successfully applied to Carbimazole^® tablets with mean percentage recoveries of 100.12 and 99.73%, respectively.     

Critical evaluation of experimental conditions influencing the surface-enhanced Raman spectroscopic (SERS) detection of substances separated by layer liquid chromatographic techniques

Summary Surface-enhanced Raman spectra (SERS) ofp-dimethylaminobenzylidenerhodanine have been recorded on silica gel 60 F₂₅₄ and Si60 F₂₅₄ Raman TLC plates. Spectra were enhanced by use of a silver sol prepared according to the modified Lee-Meisel procedure. The standard deviations of the intensities and the band ratios for the seven most intense peaks were calculated for 30 parallel measurements. Although the Raman plate gives more reproducible results, several experimental difficulties are encountered in the development of chromatograms. SERS detection of ascorbigen and 1′-methylascorbigen was performed after chromatography on silica gel 60 F₂₅₄ TLC and HPTLC plates and on Si60 F₂₅₄ Raman TLC plates. Traditional development was used for the silica gel 60 F₂₅₄ TLC plates and Si60 F₂₅₄ Raman plates, and the personal OPLC technique for the silica gel 60 F₂₅₄ HPTLC plates. It was found that the SERS spectrum gave information about the indole ring only. Because bonding of the analyte to the stationary phase results in a change in molecular conformation-in contrast with the behaviour of rhodanine-the type of the plateused and the development procedure employed can significantly influence the quality of the SERS spectrum. Presented at Balaton Symposium on High-Performance Separation Methods, Siófok, Hungary September 1–3, 1999     

Development of quantitative HPTLC-densitometry methods following a model approach for transfer of TLC screening methods for pharmaceutical products of atenolol,chloramphenicol, furosemide,glibenclamide, penicillin V potassium,and praziquantel

Transfer of six thin-layer chromatography (TLC) Global Pharma Health Fund E.V. Minilab manual protocols for detecting fake drugs in pharmaceutical products to quantitative high-performance TLC (HPTLC)-densitometry methods was performed following a previously published model process. The developed and validated methods for tablets or capsules containing atenolol, chloramphenicol, furosemide, glibenclamide, penicillin V potassium, and praziquantel involved use of a limited list of inexpensive, relatively nontoxic, readily available solvents and other reagents; silica gel 60?F₂₅₄ plates; automated bandwise sample and standard solution application; ascending mobile phase development of plates in a chamber; and automated slit scanning densitometry for detection, identification, and quantification. Validation data for methods developed in an early version of the transfer model process that did not include standard addition validation are reported for pharmaceutical products containing amitriptyline HCl, amodiaquine, diphenhydramine HCl, and mebendazole.     

Study of the degradation behavior of dapagliflozin propanediol monohydrate and metformin hydrochloride by a stability-indicating high-performance thin-layer chromatographic method

A simple, selective, precise, rapid and accurate stability-indicating high-performance thin-layer chromatography (HPTLC) method was developed and validated for the estimation of dapagliflozin and metformin in tablet dosage form. In this work, methanol–ethyl acetate–ammonium acetate (6:4:0.1, V/V) as the mobile phase and aluminum-backed TLC plates pre-coated with 250 µm layer of silica gel 60F₂₅₄ as the stationary phase were used for the estimation of dapagliflozin and metformin. The wavelength selected for detection was 220 nm. The linearity range was found to be 20–100 ng/spot (r² = 0.9985) for dapagliflozin and 500–2500 ng/spot (r² = 0.9984) for metformin. Validation of the developed method was performed as per the International Council for Harmonisation (ICH) guidelines. Stress testing of dapagliflozin and metformin was performed under acidic, alkaline, oxidative, photolytic and dry-heat degradation conditions. The chromatographic conditions successfully resolved dapagliflozin and metformin from their degradation products, formed under various stress conditions. From stress testing, dapagliflozin was found to be significantly degrading under acidic, alkaline, oxidative, photolytic and dry-heat degradation conditions, while metformin was found to be significantly degrading in acidic and alkaline degradation conditions and stable under oxidative, photolytic and dry-heat degradation conditions. Tablet dosage form of dapagliflozin and metformin was analyzed by the developed method.

     

Simultaneous Separation and Determination of Lamivudine and Zidovudine in Pharmaceutical Formulations Using the HPTLC Method

Abstract

A high-performance thin-layer chromatographic method (HPTLC) for the simultaneous determination of lamivudine and zidovudine in a binary mixture has been developed. The method developed was based on HPTLC separation of the two drugs followed by densitometric measurements of spots at 276 and 271 nm for lamivudine and zidovudine, respectively. Separation was carried out on Merck HPTLC silica-gel 60 F₂₅₄ plates, using toluene/chloroform/methanol (1:6:3 v:v) as the mobile phase. Validation of the method was performed based on The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines in terms of linearity, accuracy, precision, limit of detection, limit of quantification, and robustness. Second-order polynomial equations were obtained for the regression line in the ranges of 250–1400 and 250–1700 ng/spot for lamivudine and zidovudine respectively. Correlation coefficient (r) values were 0.9998 for both analytes. The method provides sufficient accuracy as indicated by recovery percentages given for lamivudine and zidovudine. For system precision study, the low coefficient of variation values (<2%) for both lamivudine and zidovudine ensured reproducible performance of the instrument. In the method precision study, coefficients of variation <2% were obtained, which showed that the proposed method provides acceptable intraday and interday variation. The detection and quantification limits and were 3.06 and 9.28 ng/spot for lamivudine and 3.34 and 10.13 ng/spot for zidovudine, respectively. Parameters such as mobile-phase composition, volume of mobile phase, time from spotting to development, and time from development to scanning were employed while testing for robustness of the method, and the standard deviation of peak areas was calculated for each parameter. The low coefficient of variation values indicated the robustness of the method. Statistical manipulation did not show any significant effect of one parameter over the others on the robustness of the method.     

Simultaneous determination of epimers (+)-catechin and (‒)-epicatechin in Onosma bracteatum Wall. using high-performance thin-layer chromatography‒mass spectrometry method

High-performance thin-layer chromatography‒mass spectrometry (HPTLC‒MS) method was developed for the estimation of epimers (+)-catechin (CA) and (‒)-epicatechin (ECA) in Onosma bracteatum Wall. Resolving these epimers is challenging and so method optimization was done for the selection of the stationary phase and the mobile phase to achieve their coherent separation. To further increase the reliability of the obtained densitometric results, HPTLC–MS analysis was performed. The genus Onosma L. is a species-rich genus that exhibits complex patterns of morphological and karyological diversity, and highly debatable taxonomic approaches. Thus, many similar species are described based on morphological differences and often quite ambiguous. To facilitate the identification of O. bracteatum, separation was achieved using pre-coated silica gel 60 F₂₅₄ HPTLC plate as the stationary phase and a mixture of diisopropyl ether–ethyl acetate–formic acid (9.0:0.2:0.7, V/V) as the mobile phase for the separation of epimers CA and ECA. Sample preparation, mobile phase selection and optimization were given importance to manage good resolution (R_F) of these markers. Flavan-3-ols CA and ECA were identified and confirmed on the basis of R_F and in situ UV and MS overlaid spectra with respective standards. The method was validated for linearity, inter-day precision, intra-day precision, repeatability, accuracy, specificity, limit of detection, and limit of quantification. The average recoveries for epimers CA and ECA from ethyl acetate extract fraction (MEF) were found 98.86 and 99.03% indicating the good reproducibility for each marker. The proposed validated HPTLC method is simple, accurate and reproducible and is the first report on the separation and quantification of the epimers CA and ECA in O. bracteatum using HPTLC–MS.

     

Development and validation of a simple HPTLC method for the determination of new hepatitis C subtype 4 antiviral agents in their tablet dosage form

A new, simple, precise, accurate and selective high-performance thin-layer chromatographic (HPTLC) method has been developed and validated for the simultaneous determination of ledipasvir and sofosbuvir in their tablet dosage form. Chromatographic separation was carried out on Merck TLC aluminum sheets of silica gel 60 F₂₅₄ using ethyl acetate:hexane:methanol in the ratio of 8:1.25:0.75 (% v/v) as the mobile phase followed by densitometric measurement at 256 nm. The method was validated in terms of linearity, accuracy, precision, limit of detection, limit of quantification and specificity in accordance with the International Conference on Harmonization (ICH) guidelines. The calibration curve was found to be linear between 60 to 1980 and 45 to 3600 ng/band for ledipasvir and sofosbuvir, respectively, with significantly high value of regression coefficient (r² > 0.9999) with linear and homoscedastic residuals. The limits of detection and quantification were found to be 16.5 and 50 ng/band, respectively, for ledipasvir and 13 and 39.5 ng/band, respectively, for sofosbuvir. Comparative study was performed between the developed HPTLC method and the reported high-performance liquid chromatography (HPLC) method. The quantitative results of the two analytical methods did not show statistically significant difference, whereas the developed HPTLC method is both time- and cost-effective.

     

Determination of Exemestane in bulk and pharmaceutical dosage form by HPTLC

An HPTLC method for analysis of Exemestane in bulk and pharmaceutical formulation has been established and validated. The analyte was separated on aluminium plates precoated with silica gel 60 F₂₅₄. The mobile phase was chloroform:methanol 9.2:0.8 (v/v). Quantification was done by densitometric scanning at 247 nm. Response was a linear function of Exemestane concentration in the range of 100–500 μg mL⁻¹. The limit of detection and quantification for Exemestane were 5.8 and 17.58 μg mL⁻¹, respectively. Average recovery of Exemestane was 100.1, which shows that the method was free from interference from excipients present in the formulation. The established method enabled accurate, precise, and rapid analysis of Exemestane in bulk as well as pharmaceutical formulation.     

Thermodynamic study of triclosan adsorption from aqueous solutions on activated carbon

The change in the thermodynamic properties of triclosan adsorption on three activated carbons with the different surface chemistry was studied through immersion calorimetry and equilibrium data; the amount adsorbed of triclosan (Q) during calorimetry was determined and correlated with the energy associated with adsorbate–adsorbent interactions in the adsorption process. It was noted that triclosan adsorption capacity decreases with an increase in oxygenated surface groups. For an activated carbon oxidized with HNO₃ (OxAC), the amount adsorbed was 8.50?×?10^?3 mmol g^?1, for a activated carbon without modification (GAC) Q?=?10.3?×?10^?3 mmol g^?1 and for a activated carbon heated at 1073 K (RAC1073) Q?=?11.4?×?10^?3 mmol g^?1. The adsorbed amounts were determined by adjusting the isotherms to the Sips model. For the activated carbon RAC1073, the immersion enthalpy (ΔH_imm) was greater than those of the other two activated carbons due to the formation of interactions with the solvent (ΔH_immOxAC?=?? 27.3 J g^?1?<?ΔH_immGAC?=?? 40.0 J g^?1?<?ΔH_imm RAC1073?=???60.7 J g^?1). The changes in the interaction enthalpy and Gibbs energy are associated with adsorbate–adsorbent interactions and side interactions such as the adsorbate–adsorbate and adsorbate–solvent interactions.

     

Quantification of <Emphasis Type="Italic">Lactobacillus</Emphasis> in fermented milk by multivariate image analysis with least-squares support-vector machines

This paper reports an approach for quantification of Lactobacillus in fermented milk, grown in a selective medium (MRS agar), by use of digital colour images of Petri plates easily obtained by use of a flatbed scanner. A one-dimensional data vector was formed to characterize each digital image on the basis of the frequency-distribution curves of the red (R), green (G), and blue (B) colour values, and quantities derived from them, for example lightness (L), relative red (RR), relative green (RG), and relative blue (RB). The frequency distributions of hue, saturation, and intensity (HSI) were also calculated and included in the data vector used to describe each image. Multivariate non-linear modelling using the least-squares support vector machine (LS-SVM) and a linear model based on PLS regression were developed to relate the microbiological count and the frequency vector. Feasibly models were developed using the LS-SVM and errors were below than 10% for Lactobacillus quantification, indicating the proposed approach can be used for automatic counting of colonies.     

Greenness assessment of a stability indicating simple inexpensive high-performance thin-layer chromatography–dual wavelength method for simultaneous determination of mometasone furoate and salicylic acid in complex matrix using analytical eco-scale

A stability-indicating validated high-performance thin-layer chromatography method was performed for the determination of mometasone furoate (MM) and salicylic acid (SLY), simultaneously within the concentration range of 0.1–1.6 μg/band for MM and 0.4–5 μg/band for SLY. This method was developed to assay the investigated drugs in the presence of their degradation products by alkaline, acidic, neutral, photolytic, and oxidative degradation. Separation was achieved using dual wavelength system, 250 nm for MM and 300 nm for SLY, with mobile phase composed of chloroform–ethanol (9:1, %v/v) and stationary phase of aluminum plates pre-coated with silica gel 60 F₂₄₅. The proposed method is well used for the assay and separation of MM and SLY in pure form and Elicasal^® ointment. The developed method has many advantages such as being rapid, selective and inexpensive. Such advantages promote the suggested method for the high throughput assay of MM and SLY mixture, in pure form and topical preparation. The developed method was validated according to the International Council for Harmonisation guidelines, in terms of linearity, limits of detection and quantification, precision, accuracy, robustness, and specificity. Assessment of greenness has been performed depending on analytical eco-scale approach.

     

Rapid separation of lactucin and lactucopicrin from Cichorium glandulosum by medium-pressure preparative liquid chromatography and quantitative analyses by high-performance thin-layer chromatography

Medium-pressure preparative liquid chromatography (MPPLC) was used to isolate and prepare lactucin and lactucopicrin from the whole herb of Cichorium glandulosum. After extracting the methanolic extract of the whole plant with petroleum ether and ethyl acetate several times to obtain ethyl acetate extract, the crude products, namely, lactucin and lactucopicrin were separated using MPPLC and thin-layer chromatography (TLC) tracking, and their purity rates reached more than 80%. The qualitative and quantitative analyses of lactucin and lactucopicrin were carried out by high-performance thin-layer chromatography (HPTLC) on the whole herb of C. glandulosum. The contents of lactucin and lactucopicrin were determined by scanning at 256 nm in the whole herb of C. glandulosum. The R_F values of lactucin and lactucopicrin were 0.42 ± 0.05 and 0.65 ± 0.05 with linear ranges of 0.498–2.988 and 0.499–2.994 μg/band, respectively. The correlation coefficients were 0.9938 and 0.9946, respectively, thereby showing a good linear relationship. The average recoveries were 99.96% and 99.52%, and the relative standard deviations (RSDs) were 2.49% and 2.45%, respectively. The crude products, namely, lactucin and lactucopicrin, can be isolated with high purity from the whole herbs of C. glandulosum by MPPLC. The lactucin and lactucopicrin contents of C. glandulosum can be determined rapidly and accurately by HPTLC.

     

Notiz zur densitometrischen Bestimmung von aromatischen Verbindungen durch Nitrierung auf HPTLC-Fertigplatten Kieselgel 60 F254

Zusammenfassung Verbindungen, die einen aromatischen Ring enthalten und unter kurzwelligem UV-Licht auf einer HPTLC-Fertigplatte Kieselgel 60 F₂₅₄ keine Absorption aufweisen, lassen sich durch Einwirkung nitroser Gase in Nitroverbindungen überführen, die unter kurzwelligem UV-Licht visuell nachweisbar sind. Nach der Chromatographie wird die HPTLC-Fertigplatte Kieselgel 60 F₂₅₄ im Trockenschrank erhitzt (15 min bei 160° C) und anschließend in heißem Zustand der Einwirkung von nitrosen Gasen (aus Salpetersäure 100%) ausgesetzt. Einwirkungszeit zur qualitativen Beurteilung der nitrierten Flecken: 3 min, zur quantitativen densitometrischen Messung: 10 min. Im Falle von Ephedrinhydrochlorid betrug die relative Standardabweichung 1,75%.

---

Note on the densitometric determination of aromatic compounds by nitration on pre-coated HPTLC plates Silica gel 60 F₂₅₄

Summary Compounds containing an aromatic ring and not exhibiting any absorption on a pre-coated HPTLC plate Silica gel 60 F₂₅₄ under short-wave UV light may be converted by nitrous gases into nitro compounds which are visually detectable under short-wave UV light. After chromatography the HPTLC plate Silica gel 60 Fe₂₅₄ is heated in a drying chamber (15 min at 160° C), and then exposed while still hot to nitrous gases (from 100% nitric acid). Exposure time for qualitative assessment of the nitrated spots: 3 min, and for quantitative densitometric measurement: 10 min. The relative standard deviation was 1.75% in the case of ephedrine hydrochloride.