Experimental and computational study of the pKa of coumaric acid derivatives

The dissociation constant (pK_a) of a drug is a key parameter in drug discovery and pharmaceutical formulation. The hydroxy substituent has a significant effect on the acidity of hydroxycinnamic acid. In this work, the acidic constants of coumaric acids are obtained experimentally by spectrophotometry using the chemometric method and calculated theoretically using ab initio quantum mechanical method at the HF/6‐31G* level of theory in combination with the SMD continuum solvation method. Rank annihilation factor analysis (RAFA) is an efficient chemometric technique based on the elimination of the contribution of one of the chemical components from the data matrix. RAFA cannot be performed because the pure spectrum of HA^? is not available. So, two‐rank annihilation factor analysis (TRAFA) is proposed for the determination of the pK_a OF H₂A. A comparison between the pK_a values obtained previously by TRAFA for the molecules o‐coumaric acid (4.13, 9.58), m‐coumaric acid (4.48, 10.35), and p‐coumaric acid (4.65, 9.92) makes it clear that there is good agreement between the results obtained by TRAFA and ab initio quantum mechanical method.     

Optimization of microwave‐assisted extraction followed by RP‐HPLC for the simultaneous determination of oleanolic acid and ursolic acid in the fruits of Chaenomeles sinensis

An optimized microwave‐assisted extraction (MAE) method and an efficient HPLC analysis method were developed for fast extraction and simultaneous determination of oleanolic acid and ursolic acid in the fruit of Chaenomeles sinensis. The open vessel MAE process was optimized by using a central composite experimental design. The optimal conditions identified were microwave power 600 W, temperature 52°C, solvent to material ratio 32 mL/g and extraction time 7 min. The results showed that MAE is a more rapid extraction method with higher yield and lower solvent consumption. The HPLC–photodiode array detection analysis method was validated to have good linearity, precision, reproduction and accuracy. Compared with conventional extraction and analysis methods, MAE–HPLC–photodiode array detection is a faster, convenient and appropriate method for determination of oleanolic acid and ursolic acid in the fruits of C. sinensis.     

Trace Determination of Domoic Acid in Sea Water and Phytoplankton by High-Performance Liquid Chromatography of the Fluorenylmethoxycarbonyl (FMOC) Derivative

Abstract

A method is presented for the trace determination of domoic acid, a neurotoxic amino acid responsible for cases of Amnesic Shellfish Poisoning resulting from the consumption of contaminated shellfish. The method involves pre-column derivatization with 9-fluorenylmethylchloroformate to form the FMOC derivative followed by reversed-phase HPLC with fluorescence detection. The detection limit for domoic acid in seawater and aqueous extracts is 15pg/mL (50 pM) using gradient elution, a 20μL injection volume, and a 2.1mm I.D. microbore column. Use of dihydrokainic acid as an internal standard improved quantitation. The method was applied to the detection of domoic acid in seawater, in phytoplankton cultures (Nitzschia pungens forma multiseries), and in natural mixed phytoplankton assemblages in estuarine waters.     

Estimation of hydrogen peroxide formed and residual ascorbate in the copper catalysed oxidation reaction of ascorbate at pH 7

A fluorescent method has been developed for determination of ascorbic acid concentrations. The method involves treatment of the ascorbic acid with Cu(II) and treating the hydrogen peroxide formed with horseradish peroxidase in the presence ofp-hydroxyphenylacetic acid, to form a fluorescentp-hydroxyphenylacetic acid dimer. The reaction is suitable for analysis of concentrations in the range from 50 M to 4 mM ascorbic acid solutions and can be used for analysis of pharmaceutical preparations but is unsuitable for analysis of ascorbate in preparations derived from natural sources. By using a slight modification of the analytical technique it is possible to measure the amount of hydrogen peroxide formed and the residual concentrations of ascorbic acid in solutions treated with varying amounts of copper ion.Abbreviations CySH cysteine - EDTA ethylenediamine tetraacetic acid - GSH reduce glutathione - HPAA hydroxyphenylacetic - HPLC high performance liquid chromatography - HRP horseradish peroxidase - TCA trichloracetic acid     

Separation and determination of the enantiomers of lactic acid and 2‐hydroxyglutaric acid by chiral derivatization combined with gas chromatography and mass spectrometry

Lactic acid and 2‐hydroxyglutaric acid are chiral metabolites that have two distinct d‐ and l ‐enantiomers with distinct biochemical properties. Perturbations of a single enantiomeric form have been found to be closely related to certain diseases. Therefore, the ability to differentiate the d and l enantiomers is important for these disease studies. Herein, we describe a method for the separation and determination of lactic acid and 2‐hydroxyglutaric acid enantiomers by chiral derivatization (with l‐ menthol and acetyl chloride) combined with gas chromatography and mass spectrometry. The two pairs of above‐mentioned enantiomers exhibited linear calibration curves with a correlation coefficient (R²) exceeding 0.99. The measured data were accurate in the acceptable recovery range of 88.17–102.30% with inter‐ and intraday precisions (relative standard deviations) in the range of 4.23–17.26%. The limits of detection for d‐ lactic acid, l‐ lactic acid, d‐ 2‐hydroxyglutaric acid, and l‐ 2‐hydroxyglutaric acid were 0.13, 0.11, 1.12, and 1.16 μM, respectively. This method was successfully applied to analyze mouse plasma. The d‐ lactic acid levels in type 2 diabetes mellitus mouse plasma were observed to be significantly higher (P < 0.05, t‐test) than those of normal mice, suggesting that d‐ lactic acid may serve as an indicator for type 2 diabetes mellitus.     

Internal rotation in the molecular ions of benzoic acid and salicylic acid. An INDO molecular orbital approach

An aspect of the mechanism of the hydroxyl H/ortho H scrambling in the molecular ion of benzoic acid was studied by the open-shell INDO molecular orbital method. Energies of te unrearranged structure, the planar structure in which H is attached to carbonyl oxygen and rotational conformers of the latter were studied to determine a pathway of minimum energy for rotation. The same calculations were made for salicylic acid, m-fluorobenzoic acid and p-fluorobenzoic acid. The results of these calculations agree quite well with experimental results.     

Use of the Synthesized Titania Monolith to Determine Benzoic Acid and Vanillin in Foodstuffs by HPLC

A simple and reliable HPLC method for the determination of benzoic acid and vanillin in food samples has been developed, in which a pure titania monolithic column synthesized through a template-free sol-gel synthesis route was used as chromatography column. To fully understand the retention mechanism of benzoic acid and vanillin on titania, acetonitrile (ACN) percentage, buffer concentration, and buffer pH of the mobile phase were investigated. The retention mechanism of benzoic acid and vanillin on the titania monolith column belongs to hydrophilic interaction and ligand exchange. When the high %ACN and appropriate acetate existed in eluent, the hydrophilic interaction was the dominant retention mode. Benzoic acid and vanillin in preserved fruit and jelly samples were successfully determined and quantitative analysis was carried out by external standard method with correlation coefficient (R ² ) of 0.9994 for benzoic acid and 0.9989 for vanillin. The relative standard deviations (RSDs) of benzoic acid and vanillin were 0.94% and 1.50%, respectively. The developed titania-based HPLC method is simple, rapid, accurate, and competent for the separation of polar and hydrophilic compounds, and this work has also promoted the application of titania monolith in chromatographic separation.     

TLC Densitometric Method for the Quantification of Eugenol and Gallic Acid in Clove

Eugenol and gallic acid are reported from the flower buds of Syzygium aromaticum (L.) Merr. & Perry (clove). Both the compounds have been shown to give interesting biological activities and hence serve as biomarkers. We report a simple TLC densitometric method for the quantification of eugenol and gallic acid in clove. The method was validated for precision, repeatability and accuracy. The method was found to be precise with RSD of 0.61 and 1.3 (intraday) and 0.96 and 0.24 (interday) for different concentrations of eugenol and gallic acid respectively. Instrumental precision was 0.24 and 0.21 (% CV) for eugenol and gallic acid respectively. Accuracy of the method was checked by conducting recovery study at two different levels for eugenol and gallic acid and the average percentage recoveries were found to be 99.79% and 97.90% respectively. The contents of eugenol and gallic acid in different samples of clove, as estimated by the proposed method, were found to be in the range of 12.9–14.6% and 0.31–0.61% respectively. The proposed HPTLC method for the estimation gallic acid and eugenol was found to be simple, precise, specific, sensitive and accurate and can be used for routine quality control of clove.     

Determination and tissue distribution comparisons of five xanthones after orally administering crude and processed gamboge

Caged polyprenylated xanthones are the main active ingredients isolated from the resin of Garcinia hanburyi, which has been reported to exhibit potential anticancer and anti-inflammatory activities. This study aimed to develop sensitive and specific ultra-performance liquid chromatography coupled with the triple quadrupole mass spectrometry method for investigating the tissue distribution of five xanthones in rats: β-morellic acid, isogambogenic acid, gambogenic acid, R-gambogic acid and S-gambogic acid. All tissue samples were prepared using the liquid–liquid extraction method and separated on a C₈ column with a gradient system. Detection was performed on a triple quadrupole mass spectrometer in multiple-reaction monitoring using positive ionization. The method established in this assay was successfully applied to the tissue distribution study of the five selected xanthones after orally administering crude and processed gamboge in rat tissues. The results indicated that these five xanthones were distributed to rat tissues rapidly and could be detected in all of the selected tissues after oral administration. After processing, the contents of R-gambogic acid and S-gambogic acid in the gastrointestinal tract were significantly reduced. The findings of this study might be helpful in further understanding the processing mechanism of gamboge and providing references for its reasonable clinical application.     

Ultra‐performance convergence chromatography for the quantitative determination of bioactive compounds in Aralia continentalis Kitagawa as quality control markers

A rapid ultra‐performance convergence chromatography method was developed for the quantitative determination of bioactive compounds in Aralia continentalis as quality control markers. Quantitative analysis indicated the presence of two major bioactive compounds: diterpenoid acids continentalic acid and kaurenoic acid. Using a Torus 1‐aminoanthracene column, continentalic acid and kaurenoic acid were separated in less than 8 min. The method was validated with respect to precision, accuracy, and linearity according to the International Conference on Harmonization guidelines. The optimized method exhibited a good linear correlation (r ² > 0.996), excellent precision (RSD < 1.0%), and acceptable recoveries (99.97–100.26%). Limits of detection for continentalic acid and kaurenoic acid were 0.068 and 0.097 μg/mL, respectively, while their corresponding limits of quantitation were 0.207 and 0.295 μg/mL. The system performance of ultra‐performance convergence chromatography was compared with that of conventional high‐performance liquid chromatography with respect to analysis time and efficiency. The proposed method was found to be reliable and convenient for the quantitative analysis of continentalic acid and kaurenoic acid in A. continentalis from South Korea and A. pubescens from China. This study is expected to serve as a guideline for the quality control of Aralia continentalis .     

Development of a validated high-performance thin-layer chromatography method for the standardization of an Ayurvedic formulation using berberine and ursolic acid

Giloy Tulsi tablet is an Ayurvedic preparation containing Tinospora cordifolia (Giloy) and Ocimum sanctum (Tulsi) and it is recommended for boosting the body’s immune response. This research work is about the marker-based standardization of this Ayurvedic preparation using high-performance thin-layer chromatography method. Standardization is based on the determination and quantification of the phytoconstituents berberine and ursolic acid present in Giloy Tulsi tablets. Separation was performed on pre-coated silica gel 60 F₂₅₄ plate as the stationary phase, with chloroform‒acetone‒formic acid (6:3.5:0.5, V/V) as the mobile phase. Identification and quantification were conducted densitometrically at 330 nm. The developed method resulted in good quality peak shape and enabled high-quality resolution of biomarkers. The R_F value for berberine (0.46 ± 0.02) and for ursolic acid (0.68 ± 0.02) in both reference standard and formulation were found to be comparable. The method was validated for specificity, linearity, limit of detection, limit of quantification, intra-day and inter-day precisions, accuracy, and robustness. The limit of detection values were 91 and 153 ng/band for berberine and ursolic acid, respectively. The limit of quantification values were 175 and 465 ng/band for berberine and ursolic acid, respectively. Regression analysis of the calibration data revealed a good linear relationship between peak area response and concentration in the range 200‒1000 ng/band for berberine (r² = 0.995) and 500‒2500 ng/band for ursolic acid (r² = 0.9968). The accuracy of the method, determined by measurement of recovery at three different levels, was in the range 98‒102% for both markers. These results are indication of reliability, reproducibility, accuracy, and precision of the method.

     

Determination of five nucleosides by LC‐MS/MS and the application of the method to quantify N6‐methyladenosine level in liver messenger ribonucleic acid of an acetaminophen‐induced hepatotoxicity mouse model

Ribonucleic acid N⁶‐methyladenosine methylation plays an important role in a variety of biological processes and diseases. Acetaminophen‐induced hepatotoxicity is one of the major challenges faced by clinicians. To date, the link between N⁶‐methyladenosine and acetaminophen‐induced hepatotoxicity has not been studied. In this study, a simple ultra high performance liquid chromatography with tandem mass spectrometry method was developed for the simultaneous determination of five nucleosides (adenosine, uridine, cytidine, guanosine, and N⁶‐methyladenosine) in messenger ribonucleic acid. After enzymatic digestion of messenger ribonucleic acid, the nucleosides sample was separated on an Acquity UPLC column with gradient elution using methanol and 0.02% formic acid water, and detected by a Qtrap 4500 mass spectrometer with an electrospray ionization mode. The method was validated over the concentration ranges of 4–800 ng/mL for adenosine, uridine, cytidine, and guanosine and 0.1–20 ng/mL for N⁶‐methyladenosine. It was successfully applied to the determination of N⁶‐methyladenosine levels in liver messenger ribonucleic acid in an acetaminophen‐induced hepatotoxicity mouse model and a control group. This study offers a method for the determination of nucleoside contents in epigenetic studies and constitutes the first step toward the investigation of ribonucleic acid methylation in acetaminophen‐induced hepatotoxicity, which will facilitate the elucidation of its mechanism.     

Quality assessment of Fructus Ligustri Lucidi by the simultaneous determination of six compounds and chemometric analysis

A comprehensive strategy was designed for the quality assessment of Fructus Ligustri Lucidi, a well‐known and commonly used herbal medicine in clinical practice in China. First, a simple and stable method of high‐performance liquid chromatography was developed for the simultaneous quantitative analysis of six compounds, namely, salidroside, nuzhenide, specnuezhenide, oleanic acid, ursolic acid, and acetyl oleanic acid in Fructus Ligustri Lucidi. The separation of analytes was conducted on a C₁₈ column (200 mm × 4.6 mm, 5 μm) at 30°C, and the wavelength of UV detector was set at 210 nm. In quantitative analysis, all of the calibration curves showed good linear regression (R² > 0.9994) within the tested ranges, and the mean recoveries of three different concentrations ranged from 95.21–102.34%. The described method was applied to determine 11 batches of samples collected from different stores in China. Then multiple chemometrics analysis including hierarchical cluster analysis and principal component analysis were performed to classify samples and search significant compounds. Three notable compounds, specnuezhenide, oleanic acid, and acetyl oleanic acid, were discovered for better quality control compared with those stated in the China pharmacopeia. The results demonstrated that this strategy could be readily utilized for the comprehensive quality control of Fructus Ligustri Lucidi.     

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 high‐performance liquid chromatography method for the simultaneous determination of aspirin and folic acid from nano‐particulate systems

Attention has shifted from the treatment of colorectal cancer (CRC) to chemoprevention using aspirin and folic acid as agents capable of preventing the onset of colon cancer. However, no sensitive analytical method exists to simultaneously quantify the two drugs when released from polymer‐based nanoparticles. Thus, a rapid, highly sensitive method of high‐performance liquid chromatography analysis to simultaneously detect low quantities of aspirin (hydrolyzed to salicylic acid, the active moiety) and folic acid released from biodegradable polylactide‐co‐glycolide (PLGA) copolymer nanoparticles was developed. Analysis was done on a reversed‐phase C₁₈ column using a photodiode array detector at wavelengths of 233 nm (salicylic acid) and 277 nm (folic acid). The mobile phase consisted of acetonitrile–0.1% trifluoroacetic acid mixture programmed for a 30 min gradient elution analysis. In the range of 0.1–100 μg/mL, the assay showed good linearity for salicylic acid (R² = 0.9996) and folic acid (R² = 0.9998). The method demonstrated good reproducibility, intra‐ and inter‐day precision and accuracy (99.67, 100.1%) and low values of detection (0.03, 0.01 μg/mL) and quantitation (0.1 and 0.05 μg/mL) for salicylic acid and folic acid, respectively. The suitability of the method was demonstrated by simultaneously determining salicylic acid and folic acid released from PLGA nanoparticles. Copyright © 2009 John Wiley & Sons, Ltd.     

Qualitative and quantitative analysis of phenolic acid glycosides in Ginkgo biloba L. leaf,G. biloba leaf extract and its injection

Ginkgo biloba L. leaf (GBL) is one of the most commonly used medicinal plants in the world. Phenolic acids with biological activities have a relatively high content in G. biloba leaf extracts (GBE); therefore they are of great significance for the quality control of GBL, GBE and its preparations. However, there have been few studies focused on their analysis. In this work, 12 phenolic acids, including 11 phenolic acid glycosides, were identified by liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC–Q-TOF/MS). Then, a method combining enzymolysis with HPLC was established for quantification of phenolic acid glycosides. It was found that the aglycones of phenolic acid glycosides mainly comprised five phenolic acids: 2,4,6-trihydroxybenzoic acid, protocatechuic acid, p-hydroxybenzoic acid, vanillic acid and p-coumaric acid. The quantitative method was validated, and the correlation coefficient (0.9993–0.9999), recovery (≥88.4%), repeatability (≤0.8%), and inter-day precision (≤5.5%) were satisfactory. Finally, the contents of glycosides of five phenolic acids in GBL, GBE and GBE injection from different sources were determined by the developed method. The method was accurate, repeatable and practicable, which could be helpful for the quantification of phenolic acid glycosides in other products containing GBL or GBE.     

The decarboxylation of methylmalonic acid and n-octadecylmalonic acid in normal alkanols

Rate constants and activation parameters are reported for the decarboxylation of methylmalonic acid and n-octadecylmalonic acid in three normal alkanols (hexanol-1, octanol-1, and decanol-1). Enthalpies of activation for both substrates in the various solvents are found to be a linear function of the number of carbon atoms or methylene groups in the hydrocarbon chain of the solvent. For both reaction series the isokinetic temperature is found to be equal to the melting point of the substrate. The free energy of activation at the isokinetic temperature in kcal/mole is 29.0 for n-octadecylmalonic acid and 29.4 for methylmalonic acid. Based on the results of the present investigation as well as on previously reported data in the case of malonic acid and n-butylmalonic acid, an empirical method of calculating the rate of reaction for the decarboxylation of malonic acid and its n-alkyl derivatives in normal alkanols is proposed. As a further test of the method of calculation the decarboxylation of n-dodecylmalonic acid in heptanol-1 at 110.30°C was studied. The calculated value of the pseudo-first-order specific reaction velocity constant of the reaction agreed with the experimental value to within about 0.1 percent.     

Quantification and antioxidant and anti-HCV activities of the constituents from the inflorescences of Scabiosa comosa and S. tschilliensis

To investigate the bioactive constituents of the inflorescences of Scabiosa comosa and S. tschilliensis, which are used traditionally for liver diseases, we tested the antioxidant activity using 2,2′-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), ferric reducing antioxidant potential (FRAP), and DPPH-ultra high performance liquid chromatography-mass spectrometer (UPLC-MS) assay. In addition, cell-based anti-HCV activity of the major compounds were evaluated. The plant extracts showed strong antioxidant activity. For the first time, 3,4-dicaffeoylquinic acid (DCQA), 3,5-DCQA and 4,5-DCQA were identified from genus Scabiosa. A UPLC-MS method in multiple reaction monitoring (MRM) mode was established to quantify 18 constituents in the inflorescences of Scabiosa. The 3,5-DCQA, chlorogenic acid and some glycosides of luteolin or apigenin were found to be the most abundant constituents. Chlorogenic acid and 3,5-DCQA showed excellent radical scavenging activity and demonstrated anti-HCV activity. These findings provided scientific evidences for the clinic use of this herbal medicine for liver diseases.     

UHPLC–MS/MS quantification combined with chemometrics for the comparative analysis of different batches of raw and wine‐processed Dipsacus asper

A rapid and sensitive ultra‐high performance liquid chromatography with tandem mass spectrometry approach was established for the simultaneous determination of 4‐caffeoylquinic acid, loganic acid, chlorogenic acid, loganin, 3,5‐dicaffeoylquinic acid, dipsacoside B, asperosaponin VI, and sweroside in raw and wine‐processed Dipsacus asper . Chloramphenicol and glycyrrhetinic acid were employed as internal standards. The proposed approach was fully validated in terms of linearity, sensitivity, precision, repeatability as well as recovery. Intra‐ and interassay variability for all analytes were 2.8–4.9 and 1.7–4.8%, respectively. The standard addition method determined recovery rates for each analytes (96.8–104.6%). In addition, the developed approach was applied to 20 batches of raw and wine‐processed samples of Dipsacus asper . Principle component analysis and partial least squares‐discriminate analysis revealed a clear separation between the raw group and wine‐processed group. After wine‐processing, the contents of loganic acid, chlorogenic acid, dipsacoside B, and asperosaponin VI were upregulated, while the contents of 3,5‐dicaffeoylquinic acid, 4‐caffeoylquinic acid, loganin, and sweroside were downregulated. Our results demonstrated that ultra‐high performance liquid chromatography with tandem mass spectrometry quantification combined with chemometrics is a viable method for quality evaluation of the raw Dipsacus asper and its wine‐processed products.     

Capillary zone electrophorsis for the analysis of naturally occurring 2‐hydroxycitric acids and their lactones

A simple capillary zone electrophoresis method with direct ultraviolet detection has been developed for the analysis of naturally occurring diastereomeric 2‐hydroxycitric acid lactones. Using 50 mM sodium phosphate buffer of pH 7, a baseline resolution R _s > 3.0 was observed for all organic acids selected for the present study. This method was employed for the quantitative determination of title acids present in the plant sources namely Garcinia cambogia fruit rinds and Hibiscus sabdariffa calyx. Conversion of 2‐hydroxycitric acids to their lactones on heating the above plant sources is deliberated. The Hydrolysis of hydroxycitric acid lactones in aqueous solution is reported for the first time.