High-performance Liquid Chromatography-diode Array Detection Method Research Articles

Eco-friendly solvent bar microextraction based on a natural deep eutectic solvent and multivariate optimization for simultaneous determination of spironolactone and canrenone in urine and plasma samples.

Simultaneous measurement of spironolactone and canrenone in urine and plasma provides valuable insight into renal function, and therapeutic efficacy and can be utilized to identify potential health risks and ensure patient safety throughout treatment. By adopting greener methods to analyze these compounds, significant reductions in the environmental impact of such studies can be achieved. For this purpose, a sensitive and eco-friendly solvent bar microextraction method using natural deep eutectic solvent (NDE) followed by high-performance liquid chromatography-diode array detection (HPLC-DAD) was developed to determine spironolactone and canrenone in urine and plasma samples. The extraction solvents were synthesized using NDE-based terpenoids containing menthol and camphor in various ratios. The extraction efficiency percentage (EE%) of both drugs was measured using response surface methodology (RSM) based on central composite design (CCD), and 29 extraction tests were conducted to determine the optimum conditions. Although all parameters were found to be significant, the extraction and elution times were critical for isolating the target analytes. Under optimized conditions, the linear dynamic ranges for spironolactone (SPI)/canrenone (CAN) were 11.7-104/13.1-104 μg L-1 and 21.7-104/24.6-104 μg L-1 in urine and plasma samples, respectively with R2 ≥ 0.993. The ranges of intra-/interprecision (relative standard deviation (RSD) %, n = 5) were 1.31-9.17%/ 2.4-11% with extraction recovery ≥ 88.6% for both drugs. The comparison findings with previously published methods confirmed that the developed NDE-solvent bar microextraction (SBME)-HPLC-DAD method for spironolactone and canrenone analysis displayed confident sensitivity, feasible operation, and simple analysis. Furthermore, the method's applicability and effectiveness were proven by successfully analyzing spironolactone and its metabolite canrenone in patients' urine and plasma samples.

HPLC method for quantifying verbascoside in Stizophyllum perforatum and assessment of verbascoside acute toxicity and antileishmanial activity.

We report the chemical composition of the crude leaf extracts obtained from Stizophyllum perforatum (Cham.) Miers (Bignoniaceae), a simple high-performance liquid chromatography-diode array detection (HPLC-DAD) method based on mangiferin as an internal standard to quantify verbascoside, and the verbascoside acute oral toxicity and antileishmanial activity. HPLC-high-resolution mass spectrometry-DAD (HPLC-HRMS-DAD) analyses of the crude ethanol S. perforatum leaf extracts (CE-1 and CE-2) revealed that verbascoside was the major constituent in both extracts. CE-1 was purified, and verbascoside and casticin, among other compounds, were isolated. The developed HPLC-DAD method was validated and met the required standards. Investigation of the CE-2 acute toxicity indicated a lethal dose (LD50) greater than 2,000 mg/kg of body weight. Both CE-1 and CE-2 exhibited antileishmanial activity. The isolated compounds, verbascoside and casticin, also displayed antileishmanial activity with effective concentrations (IC50) of 6.23 and 24.20 µM against promastigote forms and 3.71 and 18.97 µM against amastigote forms of Leishmania amazonensis, respectively, but they were not cytotoxic to J774A.1 macrophages. Scanning electron microscopy of the L. amazonensis promastigotes showed that the parasites became more rounded and that their plasma membrane was altered in the presence of verbascoside. Additionally, transmission electron microscopy demonstrated that vacuoles emerged, lipids accumulated, kinetoplast size increased, and interstitial extravasation occurred in L. amazonensis promastigotes exposed to verbascoside. These findings suggest that S. perforatum is a promising candidate for further in vivo investigations against L. amazonensis.

Development of a simple, eco-friendly HPLC-DAD method for tulathromycin analysis: Ensuring stability and assessing greenness

The core objective of this analytical study was to develop a simple, green, and efficient high-performance liquid chromatography-diode array detector (HPLC-DAD) method for analyzing Tulathromycin (TUL) in pure powder and injection solution without the need for complex derivatization procedures. A simple liquid mobile solution of 15 mL of phosphate buffer (pH = 7.5) and 85 mL of methyl alcohol was passed through a Hypersil BDS C18 (4.6 mm × 25 cm, 5µ) stationary phase in an isocratic mode. The flow speed was controlled at 1.0 mL/min, and the DAD detector was adjusted at 210 nm because of the extremely weak UV absorption characteristic of TUL. TUL was well separated and quantified in 10 min. The HPLC-DAD forced degradation assay was carried out in dissimilar stressful environments concerning international conference for harmonization (ICH) protocols. TUL was linearly quantified from 1000.0 to 3000.0 µg·mL−1. The moderate sensitivity of the HPLC-DAD is attributed to the aliphatic, less conjugated chemical structure of TUL. The maximum degradation ratios occurred in acidic and oxidative environments with proportions of 20.11 % and 18.87 %, respectively. Furthermore, the eco-friendly merits of the HPLC-DAD method were confirmed via the application of two greenness approaches, specifically the analytical greenness tool (AGREE) and the green analytical procedure index (GAPI). The seven green subdivisions in the GAPI pictogram and the total score of 0.72 in the AGREE pictogram illustrate the greenness appraisal of the RP-HPLC-DAD stability method. The innovative RP-HPLC–DAD method is the first sustainable stability-demonstrating assay for TUL in the co-existence of its degradation products in variable stressful environments.

Qualitative and quantitative evaluations of Chuanxiong dispensing granules by using chemical fingerprint in combination with chemometrics methods

To better elucidate the chemical constituents and evaluate the quality consistency of Chuanxiong dispensing granules (CDG), qualitative and quantitative analyses were performed in this study. Firstly, a high-performance liquid chromatography-diode array detector (HPLC-DAD) based fingerprint was constructed by 12 batches of CDGs from different manufacturers, in which 16 common peaks were assigned. Then, two of them were directionally isolated for structural elucidation. According to the nuclear magnetic resonance (NMR) and mass spectrometry (MS) spectra, 5,6-dihydrophthalic acid was identified as novel compound, and 8-O-4/8-O-4-dehydrotriferulic acid was firstly discovered in plant belonging to the genus Ligusticum. Secondly, a total of 46 components were detected in CDG using high performance liquid chromatography coupled with quadrupole-time of flight mass spectrometry (HPLC-Q-TOF-MS), and 14 of them were unambiguously identified by comparing with reference standards. Additionally, a HPLC-DAD method was firstly established to quantify 10 characteristic peaks specified in the China National Standard of CDG, and the results revealed that ferulic acid (1.71 mg/g), chlorogenic acid (1.14 mg/g), 5,6-dihydrophthalic acid (1.13 mg/g), and senkyunolide I (1.13 mg/g) are the major components in CDGs. Chemometrics analyses suggested that phenolic acids are more important than phthalides in discrimination of CDGs from different manufacturers.

Vortex-assisted dispersive liquid-liquid microextraction based on the hydrophobic deep eutectic solvent-based ferrofluid for extraction and detection of myclobutanil.

A vortex-assisted dispersive liquid-liquid microextraction (VA-DLLME) procedure using hydrophobic deep eutectic solvent-based ferrofluid (HDES-FF) as an extractant was established. The developed sample preparation method coupled with high-performance liquid chromatography-diode array detector (HPLC-DAD) was applied to the pretreatment and determination of myclobutanil (MYC) in fruit juice. Hydrophobic deep eutectic solvent, synthesized by n-decanoic acid and DL-menthol, was as a carrier and combined with magnetic nanoparticles (Fe3O4@OA) to form HDES-FF as an extractant with high extraction capacity. The synthesized materials were characterized by Fourier transform infrared (FT-IR) spectroscopy, X-ray diffraction (XRD), and vibrating sample magnetometer (VSM). Parameters affecting extraction efficiency were optimized using single-factor experiments and Box-Behnken design via response surface methodology (BBD-RSM). Parallel tests were performed three times under the optimal conditions predicted by the model, yielding an actual mean recovery of 94.77% with RSD of 2.7% (n = 3) and an enrichment factor of 41.8 ± 0.98 (mean value ± SD, n = 3). Under the optimal conditions, the linear range was 1.0-100.0µg·mL-1; the limit of detection (LOD) and limit of quantification (LOQ) were 0.25 and 0.80µg·mL-1, respectively. The average spiked recoveries in the samples ranged from 98.2 to 100.5% with intra-day relative standard deviations (RSDs) of 1.2-3.5% (n = 3) and inter-day RSDs of 1.1-3.8% (n = 3). Finally, the method was successfully applied to the determination of MYC antimicrobial agent in different fruit juice samples. The proposed HDES-FF-VA-DLLME/HPLC-DAD method was verified to widely apply to the extraction of triazole fungicides.

A Validated HPLC-Diode Array Detection Method for Therapeutic Drug Monitoring of Thiopurines in Pediatric Patients: From Bench to Bedside.

Thiopurine drugs are part of the therapeutic armamentarium for pediatric patients suffering from inflammatory bowel disease (IBD) and acute lymphoblastic leukemia (ALL). The therapeutic drug monitoring of these drugs, consisting of measurements of the thiopurine metabolites thioguanine nucleotides (TGN) and methylmercaptopurine nucleotides (MMPN) are used to optimize the effectiveness of treatment and prevent adverse effects. In this context, we developed and validated a high-performance liquid chromatography-diode array detection (HPLC-DAD) method for the simultaneous quantification of thiopurine metabolites according to the most recent International Council for Harmonisation (ICH) guidelines. The calibration curves were built in the clinically relevant range of concentrations for TGN of 300-12,000 nM and for MMPN of 3000-60,000 nM. The limit of detection and the lower limit of quantification were 100 and 300 nM for TGN and 900 and 3000 nM for MMPN, respectively. The percentage of inter-day accuracy and precision (CV%) varied between 85 and 104% and 1.6 and 13.8%. Stability was demonstrated for both of the metabolites for at least 50 days at -20 °C. The proposed HPLC-DAD method showed an appropriate selectivity, specificity, linearity, accuracy, precision and good applicability to samples from patients with IBD and ALL undergoing thiopurine treatment.

Hashish seizures in Algeria over the 2019-2020 period: The rise of potent hashish hybrids.

In Algeria, hashish is by far the most common illicit drug. This study explores Algerian hashish over a two-year period (2019-2020). A total of 2583 hashish samples from 1707 seizures for a total quantity of 108 tons were analyzed using a validated high-performance liquid chromatography-diode array detection method (HPLC-DAD). The yearly arithmetic mean tetrahydrocannabinol (THC) concentration shows relative stability-18.67% in 2019 and 19.03% in 2020 with an overall mean THC content of 18.87% and standard deviation of 10.99%. High-potency hashish (THC content>20%) is by far the most predominant type, representing almost 50% of the total Algerian hashish seizures (mean and median around 29%). The overall mean of cannabidiol (CBD) was 2.45%, and 12% of the total studied seizures were of very low CBD concentration (CBD content<1%). Three distinct hashish chemotypes were identified: Chemotype I described the traditional Moroccan hashish with THC content ranging from 0% to 16%, Chemotype II hashish included most of the seizures and characterized by THC content ranging from 16% to 30%, and Chemotype III was characterized by hashish potency higher than 30% and very low CBD content. The identified chemotypes I, II, and III were characterized in a ternary plot, and the relative contents (THC:CBD:CBN) were about 67%:29%:4%, 88%:9%:3%, and 96%:2%:2%, respectively.

Analysis of the Mycosporine-Like Amino Acid (MAA) Pattern of the Salt Marsh Red Alga Bostrychia scorpioides.

This study presents the validation of a high-performance liquid chromatography diode array detector (HPLC-DAD) method for the determination of different mycosporine-like amino acids (MAAs) in the red alga Bostrychia scorpioides. The investigated MAAs, named bostrychines, have only been found in this specific species so far. The developed HPLC-DAD method was successfully applied for the quantification of the major MAAs in Bostrychia scorpioides extracts, collected from four different countries in Europe showing only minor differences between the investigated samples. In the past, several Bostrychia spp. have been reported to include cryptic species, and in some cases such as B. calliptera, B. simpliciuscula, and B. moritziana, the polyphyly was supported by differences in their MAA composition. The uniformity in the MAA composition of the investigated B. scorpioides samples is in agreement with the reported monophyly of this Bostrychia sp.

Simultaneous quantifications of four purine derivatives biomarkers in cow milk by SPE HPLC-DAD

In this study, simultaneous quantification of allantoin, uric acid, xanthine, and hypoxanthine in cow milk by solid phase extraction (SPE) and high performance liquid chromatography-diode array detection (HPLC-DAD) method was perform. Five different SPE cartridges were tested in order to evaluate the isolation of purine derivatives (PD) from cow milk. Chromatography was carried out on ODS-2 Hypersil column and 0.05 M (NH&lt;sub&gt;4&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; buffer solution (pH = 7.76) as mobile phase. The HPLC-DAD validated method showed a linearity with regression coefficients higher than 0.999 and the limits of detection and quantification with values in the range 0.09–0.74 µg mL&lt;sup&gt;–1&lt;/sup&gt; and 0.27–2.24 µg mL&lt;sup&gt;–1&lt;/sup&gt;, respectively. The method showed good precision with a relative standard deviation (RSD) below 4.48%, while the accuracy ranged from 95.34 to 104.47% for all analytes. The best recovery degree of PD by SPE were obtained on Strata SCX cartridge for xanthine (87.79%) and hypoxanthine (89.02%); on Strata NH&lt;sub&gt;2&lt;/sub&gt; for allantoin (35.09%) and on Strata C8 for uric acid (101.08%). Finally, the HPLC-DAD method with SPE on SCX cartridges was applied to quantify the PD in a batch of thirty cow milk samples.

Development of an HPLC-DAD Method Combined with Chemometrics for Differentiating Geographical Origins of Chinese Red Wines on the Basis of Phenolic Compounds

In this work, a high-performance liquid chromatography-diode array detection (HPLC-DAD) method combined with chemometrics was developed for differentiating geographical origins of Chinese red wines. Phenolic compounds were firstly detected and quantified by direct injection HPLC-DAD method in thirty red wines originated from two famous wine-growing regions in China. Given the high complexity of the wine samples and the co-elution problems during chromatographic separation, multivariate curve resolution-alternating least squares (MCR-ALS) was utilized to extract pure chromatographic and spectral profiles of each co-eluted compound. Quantification of phenolic compounds was thus achieved even in the presence of overlapping peaks and uncalibrated interferents. The quantitative data of the phenolic compounds in wines provided by the MCR-ALS algorithm were afterwards analyzed by unsupervised principal component analysis (PCA) and hierarchical cluster analysis (HCA) methods and supervised orthogonal partial least squares-discrimination analysis (OPLS-DA) method. Both PCA and HCA results showed that the thirty red wines from different geographical origins could be obviously distinguished, and the OPLS-DA could successfully predict the geographical origins of red wines. This study demonstrated that the HPLC-DAD method combined with chemometrics can be used as an effective tool for differentiating geographical origins of Chinese red wines on the basis of phenolic compounds.

An HPLC-DAD Method to Quantify Flavonoids in Sonchus arvensis and Able to Classify the Plant Parts and Their Geographical Area through Principal Component Analysis

A simple and efficient method has been developed for the simultaneous determination of eight flavonoids (orientin, hyperoside, rutin, myricetin, luteolin, quercetin, kaempferol, and apigenin) in Sonchus arvensis by high-performance liquid chromatography diode array detector (HPLC-DAD). This method was utilized to differentiate S. arvensis samples based on the plant parts (leaves, stems, and roots) and the plant’s geographical origin. The chromatographic separation was carried out on a reverse-phase C18 column by eluting at a flow rate of 1 mL/min using a gradient with methanol and 0.2% aqueous formic acid. In the optimum conditions, the developed method’s system suitability has met the criteria of good separation. The calibration curve shows a linear relationship between the peak area and analyte concentration with a correlation coefficient (r2) &gt; 0.9990. The ranges for the analytes’ limits of detection and quantitation were 0.006–0.015 and 0.020–0.052 µg/mL, respectively. Intra-day and inter-day precision expressed in terms of RSD values were &lt;2%, and the accuracy range based on recovery was 97–105%. The stability of all analytes within 48 h was about 2%. By combining HPLC-DAD fingerprint analysis with chemometrics, the developed method can classify S. arvensis samples based on the plant parts and geographical origin.

Evaluation of Two Parts of Lithocarpus polystachyus Rehd. from Different Chinese Areas by Multicomponent Content Determination and Pattern Recognition.

The purpose of this work is to establish a new method using high-performance liquid chromatography-diode array detection (HPLC-DAD) with chemometrics analysis to determine the content of catechin, isoquercetin, astragalin, phloridzin, trilobatin, and phloretin for one flavanol and five flavonoids, filter out the key compounds, and evaluate the quality of 26 batches of tender leaves and flower spikes of Lithocarpus polystachyus Rehd. (LP) from ten areas in China. The result showed that the HPLC-DAD method had excellent performance for accurate quantification analysis. S3 (tender leaf from Lushan, Sichuan) had the highest contents for six measured chemicals with trilobatin content of up to 27.82% in dry weight. S22 (flower spike from Liangping, Chongqing) had the highest content of phloridzin (up to 7.28%). All samples were divided into three types based on spatial distribution using principal component analysis. The result showed that the tender leaves and flower spikes from the same areas had many similar properties, and there were significant differences between the samples from different regions. Furthermore, phloridzin and trilobatin were identified as chemical markers for quality evaluation of two parts with different tender leaves and flower spikes of LP from geographical areas by orthogonal partial least squares discrimination analysis. These results will be helpful to establish an effective and comprehensive evaluation system of the development and utilization of LP resources.

Razvoj SPE-HPLC-DAD metode za određivanje florfenikola i florfenikol amina u cerebrospinalnoj tekućini svinja

A study of florfenicol (FF) and its metabo- lite florfenicol amine (FFA) in pig cerebrospinal fluid was conducted following repeated intramuscular administration of the original (reference) and a generic veterinary medicinal product (VMP) under the same experimental conditions (20 mg FF/kg body weight, 48-hour interval). Both VMPs are solutions for injection containing FF as an active substance in the concentration of 300 mg/mL and have been authorized in Croatia for use in cattle and pigs. In this study, clinically healthy pigs were randomly divided into three groups. The first group was treated with the reference VMP, the second with the generic VMP, while the third served as the control group. Animals were sacrificed at 216, 288 and 384 hours after the first drug administration. Cerebrospinal fluid samples were analysed by the optimized and validated high-performance liquid chromatography-diode array detector method (HPLC-DAD). The solid-phase extraction (SPE) technique was chosen for sample preparation. The HPLC-DAD method provides good linearity over the concentration range of 0.05 to 5.00 μg/mL for FF and FFA. Limits of detection were 0.0023 μg/mL for FF and 0.0100 μg/mL for FFA. Extraction recoveries of FF were from 86.6% to 111.8%, and of FFA from 91.7% to 98.8%. The SPE-HPLC-DAD method has been demonstrated to be a selective, sensitive and suitable analytical method for the determination of FF and FFA in cerebrospinal fluid. The present study was based on a preliminary study that quantified FF in pig plasma at 216 hours after the first application of reference or generic VMP. However, FF and FFA were not detected in any of the cerebrospinal fluid samples during the experimental period. According to the nature of biological fluids, the SPE-HPLC-DAD method can be suitable for further pharmacokinetic studies of FF in pig plasma and serum after intramuscular administration of VMPs.

A novel HPLC-DAD method with dilute-and-shoot sample preparation technique for the determination of buprofezin, dinobuton and chlorothalonil in food, environmental and biological samples

ABSTRACT The development of agriculture is closely related to the use and application of pesticides in order to improve products. Excessive use of pesticides is indeed very dangerous to essential pests and it has a potential negative effect over human health, pollution of soil and waters. For these reasons, in this study, a dilute-and-shoot sample preparation procedure for simultaneous determination of three pesticides (buprofezin, dinobuton and chlorothalonil) was developed and validated for the first time combined with high-performance liquid chromatography–diode-array detector method in urine, serum, tomato, soil, commercial dosage form and water samples. Also, the degradation and behaviour of these pesticides were investigated under different conditions in the environment. HPLC analysis was performed with Kinetex C18 (150 mm × 4.6 mm; 5 μm) column with 1 mL min−1 flow rate of mobile phase which consisted of acetonitrile/buffer in the ratio of 75:25 (v/v) at 25.0°C. Calibration graphs showed a good linearity with a coefficient of determination (R 2) of at least 0.999 for all analytes. Intraday and interday precisions (expressed as RSD%) were lower than 1.66% for all analytes. The developed method was demonstrated to be sensitive, simple, robust and rapid for the simultaneously determination of analytes in different samples providing recoveries in the range between 92.3% and 109.74%.

Bioactive Constituents of F. esculentum Bee Pollen and Quantitative Analysis of Samples Collected from Seven Areas by HPLC.

Bee pollen contains all the essential amino acids needed by humans. China is the largest producer of bee pollen in the world. In the present study, we identified 11 fatty acids in F. esculentum bee pollen oil by GC-MS analysis, and 16 compounds were isolated from F. esculentum bee pollen by column chromatography and identified. A high-performance liquid chromatography-diode array detector (HPLC-DAD) method was established for the quality control of F. esculentum bee pollen. A validated HPLC-DAD method was successfully applied to the simultaneous characterization and quantification of nine main constituents in seven samples collected from seven different areas in China. The results showed that all standard calibration curves exhibited good linearity (R2 > 0.999) in HPLC-DAD analysis with excellent precision, repeatability and stability. The total amount in the samples from the seven regions ranged from 23.50 to 46.05 mg/g. In addition, seven compounds were studied for their bioactivity using enzymic methods, whereby kaempferol (3) showed high α-glucosidase inhibitory activity (IC50: 80.35 μg/mL), ergosterol peroxide (8) showed high tyrosinase inhibitory activity (IC50: 202.37 μg/mL), and luteolin (1) had strong acetylcholinesterase inhibitory activity (IC50: 476.25 μg/mL). All results indicated that F. esculentum bee pollen could be a nutritious health food.

Determination of Ibuprofen and Phenylephrine in Tablets by High-Performance Thin Layer Chromatography and in Plasma by High-Performance Liquid Chromatography with Diode Array Detection.

Two chromatographic methods (high performance thin layer chromatography (HPTLC) and high performance liquid chromatography-diode array detector (HPLC-DAD)), were addressed for the analysis of a mixture consisted of phenylephrine hydrochloride and ibuprofen in two forms bulk and their combined dosage form. This binary mixture is considered to be a challenging one as the two drugs differ greatly in their chemical and physical properties. Not only this affects their simultaneous analysis, but also hinders their simultaneous extraction from biological fluids as plasma. That is the reason the literature lacks any report for the simultaneous extraction and analysis of these drugs from biological fluids. The concentration ranges of both drugs were 0.1-2.5 μg/spot and 0.1-100 μg/mL by HPTLC and HPLC, respectively. Not only was the HPLC-DAD method applied to the investigated drugs determination in pharmaceutical preparations, but also in spiked human plasma. Extensive study was conducted to optimize their simultaneous extraction from plasma as it was a crucial step for the in vivo analysis. The results obtained by proposed methods and a reference one were statistically comparable by analysis of variance test. No significant difference was recorded between the mean percent levels determined by the proposed methods and the reference one.

Comprehensive determination of nine polyphenols in Polygoni Avicularis Herba with a new HPLC–DAD method and their correlation with the antioxidant activities

A simple high performance liquid chromatography (HPLC) method was established for identification and quantification of nine polyphenols (gallic acid, chlorogenic acid, caffeic acid, myricetrin, hyperoside, luteoloside, quercitrin-3-rhamnoside, quercetin and kaempferol) in Polygoni Avicularis Herba. The analysis was performed on a Pinnacle II C18 column (250 × 4.6 mm, 5.0 µm) with a gradient elution of an aqueous mobile phase (containing 0.3% acetic acid) modified by acetonitrile. Diode-array detector (DAD) was used to detect the analytes at four wavelengths (270, 326, 355, and 370 nm). The established method was validated with good linearity, precision, accuracy and reproducibility to determine the polyphenols in Polygoni Avicularis Herba. The applicability of this analytical method was confirmed by successful analysis of the herb samples. The results indicated that the contents of these polyphenols varied with different locations of the samples. The anti-oxidation activities of Polygoni Avicularis Herba were further evaluated based on DPPH, ABTS and superoxide anion free radical scavenging assay. The herb exhibited obvious radical scavenging capacity, and gallic acid, chlorogenic acid, myricetrin and hyperoside played important roles in this activity.

Quality Evaluation of the Traditional Medicine Majun Mupakhi ELA via Chromatographic Fingerprinting Coupled with UHPLC-DAD-Quadrupole-Orbitrap-MS and the Antioxidant Activity In Vitro.

By merging a high-performance liquid chromatography diode array detector (HPLC-DAD) method with high-performance thin-layer chromatography (HPTLC), an assay was developed for chemical fingerprinting and quantitative analysis of traditional medicine Majun Mupakhi ELA (MME), and constituent compounds were identified using HPLC coupled with UHPLC-DAD-Quadrupole-Orbitrap-MS method. In addition, the antioxidant capacity of MME was assessed based on the ability of components to scavenge radicals using in vitro method. Using a HPLC-DAD method with HPTLC easily validated the chemical fingerprinting results and quantified three characteristic components, namely, gallic acid (1), daidzein (2), and icariin (3), in commercial MMEs. The three compounds presented excellent regression values (R2 = 0.9999) in the ranges of the test and the method recovery was in the range from 100.49% to 100.68%. The fingerprints had 27 common characteristic peaks, of which 13 were verified by rapid UHPLC-DAD-Q-Orbitrap-MS analysis. In vitro antioxidant assays rapidly assessed and contrasted antioxidant activity or the free radical scavenging activity of the main polyphenolic classes in MMEs, and the antioxidant capacity was mostly affected by the presence of gallic acid. Thus, this study establishes a powerful and meaningful approach for MME quality control and for assessing in vitro antioxidant activity.

Separation of a mixture of eleven alkaloids by 2D-TLC on Multi-K CS5 plates and identification of analytes in Thalictrum foetidum root extract by TLC and HPLC—DAD

In this study, authors propose a simple and cost-effective two-dimensional thin-layer chromatography (2D-TLC) method for the simultaneous determination of eleven standards alkaloids: allocryptopine (A), berberine (Be), boldine (Bo), chelidonine (Ch), glaucine (G), papaverine (Pa), emetine (E), columbamine (Col), magnoflorine (M), palmatine (Pal), and coptisine (Cop). Separation of the alkaloid mixture was achieved by 2D-TLC using an aqueous mobile phase (RP) in the first dimension (80% methanol + water + 0.05 mL−1 diethylamine) and a normal phase (NP) in the second dimension (18% methanol, 18% acetone in 63% diisopropyl ether containing 1% ammonia, v/v) on bilayer Multi-K CS5 plates. The composition of the mobile phases was optimized in terms of retention, separation selectivity, spots symmetry, and system efficiency. The procedure was evaluated in terms of natural samples analysis. Magnoflorine and berberine were identified in Thalictrum foetidum root extract. Additionally, the alkaloids in the extract sample were confirmed by high-performance liquid chromatography—diode-array detection method.

Simultaneous determination of eleven alkaloids in Corydalis decumbens by HPLC with diode-array detection

A high-performance liquid chromatography—diode-array detection method was developed and validated to determine simultaneously eleven major alkaloids in Corydalis decumbens (Thunb.) Pers. The alkaloids detected were corlumidine, protopine, coptisine, tetrahydrojatrorrhizine, palmatine, berberine, sanguinarine, papaverine hydrochloride, tetrahydropalmatine, bicuculline, and corydaline. Chromatographic separation was achieved using a C-18 column with a mobile phase composed of A (0.2% acetic acid solution, adjusted with triethylamine to pH 5.0) and B (acetonitrile), with stepwise gradient elution. Ultraviolet diode-array detection was used; chromatograms were examined at the wavelength of 280 nm. The regression equations showed a good linear relationship between the peak area of each marker and concentration (r = 0.9994–0.9999). The recovery values ranged between 93.66% and 100.54%. The method was fully validated with respect to detection and quantification limits, precision, reproducibility, and accuracy. The described high-performance liquid chromatography (HPLC) method was successfully used for the differentiation and quantification of the eleven major alkaloids in C. decumbens (Thunb.) Pers. and can be considered an effective procedure for the analyses of this important class of natural compounds.