Validation of an analytical method by HPLC for the quantification of yangambin from the Ocotea duckei species / Validación de un método analítico por HPLC para la cuantificación de yangambin de la especie Ocotea duckei

Ocotea duckei , known as Louro - de - cheiro, belongs to the Lauraceae family and presents lignoid yangambine (YAN) as the main plant marker. This work aimed to develop and validate an analytical method by high performance liquid chromatography for the quantification of YAN. The sample used was the crude eth anolic extract (CEE) obtained from aerial parts. In the developed method, a C18 column was used. The mobile phase was composed of acetonitrile and water (45:55), whereas the method parameters included mobile phase flow rate at 0.8 mL/min, oven temperature at 40°C, and monitoring at 205 nm. In the validation, the parameters of selectivity, linearity, precision, accuracy, robustness, limits of detection and quantification were evaluated. As a result, the developed method is in accordance with the guidelines f or validation of analytical methods and presented satisfactory chromatographic parameters for YAN determination. Thus, the present analytical methodology can be applied in the quality control of O. duckei raw materials.

Ocotea duckei , conocida como Louro - de - cheiro, pertenece a la familia Lauraceae y presenta la yangambina lignoide (YAN) como principal marcador vegetal. Este trabajo tuvo como objetivo desarrollar y val idar un método analítico por cromatografía líquida de alta resolución para la cuantificación de YAN. La muestra utilizada fue el extracto etanólico crudo (EEC) obtenido de partes aéreas. En el método desarrollado se utilizó una columna C18. La fase móvil c onsistió en acetonitrilo y agua (45:55), mientras los parámetros del método incluyeron el caudal de la fase móvil a 0,8 m L /min, la temperatura del horno a 40°C y la monitorización a 205 nm. En la validación se evaluaron los parámetros de selectividad, line alidad, precisión, exactitud, robustez, límites de detección y cuantificación. Como resultado, el método desarrollado está de acuerdo con las pautas para la validación de métodos analíticos y presentó parámetros cromatográficos satisfactorios para la deter minación de YAN. Por lo tanto, la presente metodología analítica se puede aplicar en el control de calidad de las materias primas de O. duckei.

Poly-ε-caprolactone based nanoparticles for delivery of genistein in melanoma treatment

We developed poly-ε-caprolactone (PCL)-based nanoparticles containing D-α-tocopherol polyethylene glycol-1000 succinate (TPGS) or Poloxamer 407 as stabilizers to efficiently encapsulate genistein (GN). Two formulations, referred to as PNTPGS and PNPol, were prepared using nanoprecipitation. They were characterized by size and PDI distribution, zeta potential, nanoparticle tracking analysis (NTA), GN association (AE%), infrared spectroscopy (FT-IR), and differential scanning calorimetry (DSC). PNTPGS-GN exhibited a particle size of 141.2 nm, a PDI of 0.189, a zeta potential of -32.9 mV, and an AE% of 77.95%. PNPol-GN had a size of 146.3 nm, a better PDI than PNTPGS-GN (0.150), a less negative zeta potential (-21.0 mV), and an AE% of 68.73%. Thermal and spectrometric analyses indicated that no new compounds were formed, and there was no incompatibility detected in the formulations. Cellular studies revealed that Poloxamer 407 conferred less toxicity to PCL nanoparticles. However, the percentage of uptake decreased compared to the use of TPGS, which exhibited almost 80% cellular uptake. This study contributes to the investigation of stabilizers capable of conferring stability to PCL nanoparticles efficiently encapsulating GN. Thus, the PCL nanoparticle proposed here is an innovative nanomedicine for melanoma therapy and represents a strong candidate for specific pre-clinical and in vivo studie

A rapid and sensitive High-Performance Liquid Chromatography method with fluorescence detection for quantification of melatonin in small volume rat plasma samples: application to a preclinical study to determine the oral pharmacokinetics of melatonin under gestational conditions

Abstract A novel, simple and sensitive high-performance liquid chromatography with fluorescence detection method was developed and validated for the characterization of the preclinical pharmacokinetics of melatonin under pregnant conditions. Plasma samples (25 µL) were treated with 30 µL of ethanol absolute (containing the internal standard, IS). After a centrifugation process, aliquots of supernant (5 µL) were injected into the chromatographic system. Compounds were eluted on a Xbridge C18 (150 mm x 4.6 mm i.d., 5 µm particle size) maintained at 30°C. The mobile phase consisted in a mixture of aqueous solution of 0.4% phosphoric acid and acetonitrile (70:30 v/v). The wavelengths were set at 305 nm (excitation) and 408 nm (emission) and the total analysis time was 8 min/sample. All validation tests were obtained with accuracy and precision, according to FDA guidelines, over the concentration range of 0.005-20 µg/mL. Pharmacokinetic study showed that melatonin systemic exposure increased from day 14, with a significant difference at 19 days of gestation compared to the control group. Our findings suggest a decreased metabolism of melatonin as result of temporary physiological changes that occur throughout pregnancy. However, other maternal physiological changes cannot be ruled out.

Existence of advanced glycation / lipoxidation end-products in parenteral nutrition solutions and effects of infusion conditions on their levels

Abstract Infusion solutions must be stable from the production stage until the infusion stage. Some infusion fluids contain degradation products, known as advanced glycation end products (AGEs); however, it is unknown whether AGEs exist in parenteral nutrition solutions. We aimed to investigate this question and test the effect of infusion conditions on AGE formation in parenteral nutrition solution. Nine parenteral nutrition solutions were supplied by the pharmacy with which we collaborated. To simulate the infusion conditions, the solutions were held in a patient room with standard lighting and temperature for 24 hours. Samples were taken at the beginning (group A) and the end (24th hour, group B) of the infusion period. The degradation products were 3-deoxyglucosone, pentosidine, N-carboxymethyl lysine, and 4-hydroxynonenal, which we investigated by high-performance liquid chromatography-mass spectrometry (LC-MS) and Q-TOF LC/MS methods. Two of four degradation products, 4-hydroxynonenal and N-carboxymethyl lysine, were detected in all samples, and Group B had higher levels of both compounds compared to Group A, who showed that the quantities of these compounds increased in room conditions over time. The increase was significant for 4-hydroxynonenal (p=0.03), but not for N-carboxymethyl lysine (p=0.23). Moreover, we detected in the parenteral nutrition solutions a compound that could have been 4-hydroxy-2-butynal or furanone

Development, physicochemical evaluation, and in vivo permeation studies of topical formulations containing 0.1% tacrolimus

Abstract The objective of this paper was to develop and evaluate two semi-solid pharmaceutical forms containing 0.1% tacrolimus: cream (CRT01) and gel (GLT01). For the evaluation of physicochemical stability, at times 0, 30, 60 and 90 days, at 23°C and at 40°C, High Performance Liquid Chromatography coupled with a Diode Array Detector (HPLC-DAD) was employed. This method was developed and validated for tacrolimus quantification. The occlusivity test and skin permeation assay were also performed, using an animal model (Wistar rats), and the CRT01 and GLT01 were compared to the 0.1% tacrolimus ointment (PFU01) obtained from the University Pharmacy, Federal University of Rio de Janeiro, Brazil. CRT01 and GLT01 presented a homogeneous aspect and consistency adequate for topical products, along with sensory characteristics above PFU01. They also presented adequate physicochemical stability for 90 days and a lower occlusive effect than PFU01 (p<0.05). CRT01 showed greater affinity for the skin when compared to PFU01 and GLT01, with low systemic absorption. The CRT01 semi-solid formulation was considered the most adequate one to treat patients with atopic dermatitis or other dermatologic inflammatory diseases, promoting rational use of tacrolimus

Development and validation of an analytical method by HPLC-DAD for determination of caffeine in products based on guarana extracts (Paullinia cupana)

Abstract Guarana (Paullinia cupana) is a native plant from the Amazon whose seeds contain a high concentration of caffeine. Aqueous extract of guarana is widely used in the world. In this study, the objective was to develop and validate a High-Performance Liquid Chromatography method for the determination of caffeine in extracts and commercial beverages based on guarana. A sensitive, simple, and viable high performance liquid chromatographic method without the need of an analyte extraction procedure was developed and validated according to Brazilian and international requirements. The method presented high performance, fulfilling Brazilian and international requirements, in addition to allowing product compliance tests. Results confirmed high selectivity and linearity (>0.999) between 5 to 135 ug/mL, with no significant matrix effect. Detection and quantification limits were 0.02 µg/mL and 2 µg/mL, respectively. Precision was less than 4 %, and accuracy varied from 99.9-120 %. Applicability of the method was demonstrated by conducting a limited evaluation in products containing caffeine. Commercial extracts showed quite different caffeine levels, while carbonated drinks follow Brazilian and American recommendations. Our results indicate that the developed method can be used to evaluate the quality of the guarana extract and of products containing caffeine

Applicability of an HPLC method for analysis of alcoholic and glycolic Brazilian green-propolis extracts

Abstract Brazilian green propolis has been widely used in food and pharmaceutical products due to its valuable source of phenolic compounds and versatile biological activities. The development and validation of analytical methods are extremely useful for the characterization and quality control of products containing propolis. Therefore, the aim of this study was to optimize, validate and investigate the applicability of a reversed-phase HPLC method for analysis of different types of Brazilian green propolis extracts (glycolic and ethanolic). The method showed to be selective for the propolis phenolic markers. The analysis of variance and residues demonstrated that the method had significant linear regression, without lack of fit. It was also a precise, accurate and robust method, which was of utmost importance to analyze both glycolic and ethanolic extracts and at different concentrations. Moreover, as these products can display most complex matrices to analyze, a valid HPLC method can also prove to be specific and versatile.

Metabolic study of trimetazidine using ultra-high performance liquid chromatography-tandem mass spectrometry

Abstract In the present study, the application of ultra-high performance liquid chromatography-tandem mass spectrometry allowed us to study of known-as well as hitherto unknown-trimetazidine (TMZ) metabolites in human urine and to propose their renal excretion profiles. Urine samples from a healthy volunteer were analyzed at baseline and at 0-4 h, 4-8 h, 8-12 h, and 12-24 h after a single dose of TMZ. A dilute-and-shoot procedure was used as sample treatment before separation. Full-scan spectra of possible metabolites were acquired. Additionally, product ion scan spectra of precursor ions of interest were also acquired at two collision energies. Intact TMZ was a major excretion product, with a maximum concentration at 4-8 h after administration. Moreover, five minor metabolites were observed, namely trimetazidine-N-oxide (M1), N-formyl trimetazidine (M2), desmethyl-trimetazidine O-sulfate (M3), desmethyl-trimetazidine O-glucuronide (M4), and desmethyl-trimetazidine-N-oxide-O-glucuronide (M5). Metabolite M5 has not previously been reported. Excretion curves were constructed based on the chromatographic peak areas of specific mass transitions (precursor ion > product ion) related to each of the detected metabolites

Gastroprotective potential and mechanisms of action of Hedera nepalensis

Abstract Hedera nepalensis (H. nepalensis) , belonging to the family Araliaceae, is a medicinal plant traditionally used to treat stomach problems. The current study investigated the gastroprotective potential and the mechanism of action of H. nepalensis in diclofenac-and ethanol-induced ulcer models. Anti-oxidant and lipid peroxidation inhibitory prospects of H. nepalensis were checked out by free radical scavenging assay and UV spectrophotometer respectively. Effect of H. nepalensis on the pH, gastric total acidity of gastric juice and protective effects of H. nepalensis against ulcer models have been examined. Histopathological studies have been carried out. The aqueous methanol extract of H. nepalensis (100 µg/mL) showed anti-oxidant (83.55%) and lipid peroxidation inhibitory (70.88%) potential at 1000 µg/mL; the extract had no buffer potential. The extract (400 mg/kg) significantly (81.12% and 63.46%) showed gastroprotective effect in diclofenac and ethanol-induced rat ulcer models respectively. Histopathological studies confirmed the biochemical findings. FTIR analysis showed the presence of carboxylic acid, alkanes, conjugated alkanes, aldehydes and alkyl-aryl ethers. Gallic acid, M-coumaric acid and quercetin were found by HPLC analysis. H. nepalensis exhibited significant protection against diclofenac and ethanol induced gastric damage by anti-oxidant and lipid peroxidation suppression effects suggesting potential broad utility in treatment of diseases characterized with gastric damage.

Locust bean gum hydrogels are bioadhesive and improve indole-3-carbinol cutaneous permeation: influence of the polysaccharide concentration

Abstract The locust bean gum (LBG) is a polysaccharide with thickening, stabilizing and gelling properties and it has been used in the preparation of pharmaceutical formulations. Hydrogels (HGs) are obtained from natural or synthetic materials that present interesting properties for skin application. This study aimed to develop HGs from LBG using indole-3-carbinol (I3C) as an asset model for cutaneous application. HGs were prepared by dispersing LBG (2%, 3% and 4% w/v) directly in cold water. The formulations showed content close to 0.5 mg/g (HPLC) and pH ranging from 7.25 to 7.41 (potentiometry). The spreadability factor (parallel plate method) was inversely proportional to LBG concentration. The rheological evaluation (rotational viscometer) demonstrated a non-Newtonian pseudoplastic flow behavior (Ostwald De Weale model), which is interesting for cutaneous application. The HET-CAM evaluation showed the non-irritating characteristic of the formulations. The bioadhesive potential demonstrated bioadhesion in a concentration-dependent manner. Permeation in human skin using Franz cells showed that the highest LBG concentration improved the skin distribution profile with greater I3C amounts in the viable skin layers. The present study demonstrated the feasibility of preparing HGs with LBG and the formulation with the highest polymer concentration was the most promising to transport active ingredients through the skin.

Chromatographic study of sitagliptin and ertugliflozin under quality-by-design paradigm

Abstract The present study entails the systematic development and validation of a stability-indicating RP-HPLC method for the analysis of sitagliptin and ertugliflozin in a fixed-dose combination. Analytical quality by design (AQbD) concepts were used to define critical method variables, employing Pareto risk assessment and a Placket-Burman screening design, preceded by a Box-Behnken design with response surface analysis to optimise critical method parameters such as % acetonitrile (X1), buffer pH (X2) and column oven temperature (X3). Multiple response optimisation (Derringer's desirability) of variables was accomplished by studying critical analytical attributes, such as resolution, retention time and theoretical plates. The title analytes were separated effectively on a PRONTOSIL C18 column at 37 °C using a mobile phase of acetonitrile:acetate buffer, pH 4.4 (36:64 percent v/v), pumped at a flow rate of 1 mL/min, and UV detection at 225 nm. Linearity was observed over a concentration range of 25-150 µg/mL and 3.75-22.5 µg/mL at retention times of 2.82 and 3.92 min for sitagliptin and ertugliflozin, respectively. The method obeyed all validation parameters of the ICH Q2(R1) guidelines. The proposed robust method allows the study of the selected drugs in pharmaceutical dosage forms as well as in drug stability studies under various stress conditions.

HPLC-MS/MS method for determination of betamethasone in human plasma with application to a dichorionic twin pregnancy pharmacokinetic and placental transfers studies

Abstract Betamethasone (BET) is a synthetic glucocorticoid recommended for pregnant women at imminent risk of preterm birth before 34 weeks to reduce neonatal complications. There are different techniques to describe BET plasma quantification. However, none quantified the plasmatic concentration of BET in dichorionic (DC) twin pregnancies using LC-MS. Our objectives were to develop and validate a method for quantifying BET by LC-MS for pharmacokinetic (PK) and placental transfer studies in DC twin pregnancies. Blood samples were collected after intramuscular administration of a single BET dose containing 6 mg disodium phosphate + 6 mg acetate. BET was determined in plasma by liquid-liquid extraction. The method showed linearity in the range of 2-250 ng/mL, as well as precision and accuracy with a coefficient of variation and relative standard errors ≤ 15%. Additionally, the method presented selectivity and did not present matrix or carry-over effect. Stability tests also presented coefficient of variation and relative standard errors ≤ 15%. This is the first study which describe maternal and fetal plasma concentrations of BET in a DC twin pregnancy. The BET PK parameters were AUC0-∞, CL/F, Vd/F, Cmax, Tmax of 292.20 h*ng/mL, 39.08 L/h, 278.72 L, 25.55 ng/mL and 0.58 h, respectively. The placental transfer ratios of umbilical vein/maternal vein and intervillous space/maternal vein were 0.14 and 0.19 and 0.40 and 0.27 for both twins, respectively. However, a clinical study with more subjects is imperative to confirm this higher concentration of BET in the intervillous space

Development of a validated stability-indicating HPLC- DAD method for dasabuvir and the characterization of its degradation products using LC-QToF-MS/MS

Abstract A stability-indicating HPLC-DAD method was developed and validated for the simultaneous determination of dasabuvir and its degradation products in the pharmaceutical formulation. The proposed method utilized a Symmetry® C18 (4.6 x 75 mm, 3.5 µm) column, and the mobile phase consisted of an isocratic elution of formic acid (0.1%) and acetonitrile (55:45, v/v), at a flow of 1 mL min-1; analytes were detected at 244 nm. Dasabuvir was submitted to different stress degradation conditions, such as acidic, alkaline, neutral, thermal, oxidative and photolytic, and the structural elucidation of degradation products was performed using LC-QToF-MS/MS. The HPLC-DAD stability-indicating method was validated for selectivity, linearity, limit of detection and quantification, accuracy, precision and robustness, according to ICH guidelines. Dasabuvir produced two degradation products (DP1 and DP2) from the alkaline stress conditions, which were characterized in negative ion mode. Dasabuvir was linear in the range 9.78 to 136.92 µg mL-1, and DP and DP were linear in the range 2.9 to 20.2 µg mL-1 and 1.3 to 14.9 µg mL-1, respectively. The 1 2 recovery ranged between 99.16 and 100.86%, while precision ranged from 1.02 to 2.89%. As the method can effectively separate the dasabuvir from its degradation products and quantitate them, it may be employed as a stability-indicating method for the pharmaceutical formulation.

Clinical relevance of oxidative stress biomarkers in cardiovascular prevention / Relevância clínica de biomarcadores de estresse oxidativo na prevenção cardiovascular

Cardiovascular diseases involve hyperlipidemia, inflammation and oxidative stress. Although this relationship is well established, only biomarkers associated with hyperlipidemia and inflammation are currently in clinical practice for diagnosis and evaluation of patient treatment. Our hypothesis is that oxidative stress biomarkers may be an independent risk factor and may assist in cardiovascular risk stratification and contribute to improving current scores. Thus, the objective of this study was to investigate which are the biomarkers and methodologies were used in clinical studies in humans with different health conditions. With the results obtained in the first part, we selected studies conducted in healthy individuals and in individuals under primary and secondary cardiovascular prevention in order to evaluate the most frequent biomarkers, the results obtained according to the individual's profile and the methodology used, and correlate with different health conditions. We observed that malondialdehyde (MDA) was the most frequent lipid biomarker of oxidative stress applied in the studies, but it presented significant variability in the results and a weak correlation with clinical outcomes. The result of this study demonstrates the importance of carrying out a multicentric study to validate the MDA values in individuals with different health conditions and the standardization of the methodology based on high performance liquid chromatographyy (HPLC)

As doenças cardiovasculares envolvem hiperlipidemia, inflamação e estresse oxidativo. Embora essa relação esteja bem estabelecida, apenas biomarcadores associados à hiperlipidemia e inflamação são atuais na prática clínica para diagnóstico e avaliação do tratamento do paciente. Nossa hipótese é que biomarcadores de estresse oxidativo podem ser um fator de risco independente e podem auxiliar na estratificação de risco cardiovascular e contribuir para melhorar os escores atuais. Assim, o objetivo deste estudo foi investigar primeiramente quais são os biomarcadores e metodologias utilizados nos estudos clínicos em humanos em diferentes condições de saúde. Com os resultados obtidos na primeira etapa, selecionamos os estudos conduzidos em indivíduos saudáveis e em prevenção cardiovascular primária e secundária a fim de avaliar os biomarcadores mais utilizados, os resultados obtidos conforme o perfil do indivíduo e a metodologia utilizada e finalmente correlacionar com as diferentes condições de saúde. Observamos que o malondialdeído (MDA) foi o biomarcador lipídico de estresse oxidativo mais frequente nos estudos, porém apresentou importante variabilidade nos resultados e fraca correlação com desfechos clínicos. O resultado desse estudo demonstra a importância da realização de um estudo multicentrico para validação dos valores de MDA nos diferentes perfis de indivíduos e a padronização metodológica baseada na cromatografia líquida de alta eficiência (HPLC)

The Human Host Defense Peptide LL-37 Overexpressed in Lung Cell Lines by Methanolic Extract of Valeriana officinalis

Abstract The present study investigated the effects of valerian methanolic extract and valerenic acid on the expression of LL-37 gene and protein in A549 and MRC5 line cells. After preparing Valerian seeds, sowing them in March 2020, and harvesting the rhizome in October 2020, the extract was prepared from the valerian rhizome by maceration method. Valerian acid content was determined using high performance liquid chromatography (HPLC). Two cell lines (A549 and MRC-5) were used to study the effects of valerian extract, and the MTT test was used to evaluate cell viability. The expression of LL-37 mRNA and protein was assessed by Real-Time PCR and western blot, respectively. In vivo safety assessments and histopathological analysis were also conducted. Data was analyzed by Graphpad Prism 8 software. Valerian methanolic extract and valerenic acid upregulated the LL-37 mRNA and protein expression in both treated cell lines. Valerenic acid showed a greater effect on upregulating LL-37 expression than valerian methanolic extract. A549 cells were more sensitive to valerian methanolic extract compared to MRC5 cells, and its cell viability was reduced. Furthermore, liver and kidney-related safety assessments showed that valerian methanolic extract had no toxic effects. In general, it was concluded that the methanolic extract of valerian as well as the resulting valerenic acid as the most important component of the extract has the ability to upregulate LL-37expression. Therefore, methanolic extract of valerian and valerenic acid can be considered for improving the immune system.

Preservation of phenolic compounds on dried leaf infusion of Bauhinia forficata Link

Abstract Bauhinia forficata Link aqueous extract is usually recommended as a phytomedicine to reduce blood glucose levels and its biological activity has been linked to the presence of phenolic compounds from B. forficata preparations. Several drying processes are used in the production of dry herbal extracts, which may influence the chemical composition and efficacy of final herbal medicines. Due to significant chemical changes, defining appropriate drying processes is essential for phytopharmaceutical drug development. In view of this, we analyzed dried B. forficata leaf infusion (BFLI) extracts by HPLC-UV-MSn, followed by molecular networking analysis to evaluate the chemical profiles from dried extracts yielded by freeze-and spray-drying processes. The main metabolites detected included 11 ferulic/isoferulic acid derivatives and 13 glycosylated flavonoids. The qualitative chemical profiles were alike for both drying processes, whereas the relative abundance of some flavonoids was higher using spray-drying. Taken together, our results showed that freeze-and spray-drying preserved the phenolic profile of BFLI and suggested that spray-drying may be the most suitable to obtain its dried products. Along with studying the chemical profiles of dried herbal extracts, evaluating the influence of drying processes on the quality and chemical profiles of final products is pivotal and may benefit future research.

In vitro antibacterial-antibiofilm effect of Hypericum atomarium Boiss and chemical composition

Abstract Treatment with plant is considered an effective option against increased antibiotic resistance. In this study antibiofilm activity of methanol (CH3OH), chloroform (CHCl3), ethyl acetate (EtOAc) and water (H2O) extracts of Hypericum atomarium Boiss. which is member of Hypericum genus was evaluated in Pseudomonas aeruginosa PAO1 and antibacterial performance against Gram (+) and Gram (-) strains and also bioactive compounds of extract were analysed using by HPLC and GC-MS. According to antibacterial activity test results the extracts were effective all Gram (+) bacteria and Gram (-) Chromobacterium violaceum (MICs ranging from 0.42 µg/ml to 4.3 mg). Inhibition effect of biofilm formation was found to be different rate in extracts (methanol-63%, chloroform-52%). The major flavonoids were detected (−)-epicatechin (2388.93 µg/ml) and (+)-catechin (788.94 µg/ml). The main phenolic acids were appeared as caffeic acid 277.34 µg/ml and chlorogenic acid 261.79 µg/ml. And according to GC results α-pinene was found main compound for three solvent extracts methanol, chloroform and ethyl acetate 67.05, 62.69, 49.28% rate respectively

Structural Analysis and Characterization of 4-F-α-PVP Analog 4-F-3-Methyl-α- PVP Hydrochloride / 法医学杂志

OBJECTIVES@#To identify 1-(4-fluorophenyl)-2-(1-pyrrolidinyl) pentan-1-one (4-F-α-PVP) analog 1-(4-fluoro-3-methyl phenyl)-2-(1-pyrrolidinyl) pentan-1-one (4-F-3-Methyl-α-PVP) hydrochloride without reference substance.@*METHODS@#The direct-injection electron ionization-mass spectrometry (EI-MS), GC-MS, electrospray ionization-high resolution mass spectrometry (ESI-HRMS), ultra-high performance liquid chromatography-high resolution tandem mass spectrometry (UPLC-HRMS/MS), nuclear magnetic resonance (NMR), ion chromatography and Fourier transform infrared spectroscopy (FTIR) were integrated utilized to achieve the structural analysis and characterization of the unknown compound in the sample, and the cleavage mechanism of the fragment ions was deduced by EI-MS and UPLC-HRMS/MS.@*RESULTS@#By analyzing the direct-injection EI-MS, GC-MS, ESI-HRMS and UPLC-HRMS/MS of the compound in the samples, it was concluded that the unknown compound was a structural analog of 4-F-α-PVP, possibly with one more methyl group in the benzene ring. According to the analysis results of 1H-NMR and 13C-NMR, it was further proved that the methyl group is located at the 3-position of the benzene ring. Since the actual number of hydrogen in 1H-NMR analysis was one more than 4-F-3-Methyl-α-PVP neutral molecule, it was inferred that the compound existed in the form of salt. Ion chromatography analysis results showed that the compound contained chlorine anion (content 11.14%-11.16%), with the structural analysis of main functional group information by FTIR, the unknown compound was finally determined to be 4-F-3-Methyl-α-PVP hydrochloride.@*CONCLUSIONS@#A comprehensive method using EI-MS, GC-MS, ESI-HRMS, UPLC-HRMS/MS, NMR, ion chromatography and FTIR to identify 4-F-3-Methyl-α-PVP hydrochloride in samples is established, which will be helpful for the forensic science laboratory to identify this compound or other analog compounds.

Deciphering chemical and metabolite profiling of Chang-Kang-Fang by UPLC-Q-TOF-MS/MS and its potential active components identification / 中国天然药物

Chang-Kang-Fang (CKF) formula, a Traditional Chinese Medicine (TCM) prescription, has been widely used for the treatment of irritable bowel syndrome (IBS). However, its potential material basis and underlying mechanism remain elusive. Therefore, this study employed an integrated approach that combined ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) with network pharmacology to systematically characterize the phytochemical components and metabolites of CKF, as well as elucidating its underlying mechanism. Through this comprehensive analysis, a total of 150 components were identified or tentatively characterized within the CKF formula. Notably, six N-acetyldopamine oligomers from CicadaePeriostracum and eight resin glycosides from Cuscutae Semen were characterized in this formula for the first time. Meanwhile, 149 xenobiotics (58 prototypes and 91 metabolites) were detected in plasma, urine, feces, brain, and intestinal contents, and the in vivo metabolic pathways of resin glycosides were elaborated for the first time. Furthermore, network pharmacology and molecular docking analyses revealed that alkaloids, flavonoids, chromones, monoterpenes, N-acetyldopamine dimers, p-hydroxycinnamic acid, and Cus-3/isomer might be responsible for the beneficial effects of CKF in treating IBS, and CASP8, MARK14, PIK3C, PIK3R1, TLR4, and TNF may be its potential targets. These discoveries offer a comprehensive understanding of the potential material basis and clarify the underlying mechanism of the CKF formula in treating IBS, facilitating the broader application of CKF in the field of medicine.

Comprehensive quality evaluation of Tangwei capsule based on HPLC-QAMS multi-index quantitative determination combined with chemometrics and EW-TOPSIS method / 浙江大学学报·医学版

OBJECTIVES@#To detect the contents of Tangwei capsule main components with high performance liquid chromatography-quantitative analysis of multicomponents by single marker (HPLC-QAMS) method and to evaluate the quality with chemometrics and entropy weight-technique for order preference by similarity to an ideal solution (EW-TOPSIS).@*METHODS@#A symmetry C18 column and 0.1% formic acid-acetonitrile as mobile phase were used for HPLC of Tangwei capsule. The contents of 3'-hydroxypuerarin, puerarin, 3'-methoxypuerarin, methylnissolin-3-O-glucoside, calycosin, formononetin, rosmarinic acid, salvianolic acid B, dihydrotanshinone Ⅰ, cryptotanshinone, tanshinone Ⅰ, tanshinone ⅡA and cucurbitacin B in 15 batches of Tangwei capsule were determined simultaneously. The quality differences of 15 batches of samples were analyzed by chemometrics and EW-TOPSIS.@*RESULTS@#The HPLC-UV showed that 13 components had good linear relationships in corresponding concentration ranges (r≥0.9991). The relative standard deviations (RSD) of precision, repeatability and stability were all less than 2.00%. The average recovery rates were between 96.86% and 100.13%, and RSD were all less than 2.00%. Cluster analysis showed that 15 batches of samples were clustered into 3 groups. Partial least squares-discriminant analysis showed that salvianolic acid B, formononetin, puerarin, 3'-methoxypuerarin and rosmarinic acid were the main potential markers affecting the quality of Tangwei capsule. EW-TOPSIS analysis showed that the quality of S12-S15 was superior.@*CONCLUSIONS@#The analytical method established in this study can be used for the comprehensive evaluation of the quality of Tangwei capsule to provide laboratory support for its quality control and overall evaluation.