Anti-hyperglycemic fraction from Alternanthera sessilis L. leaves gets elucidated following bioassay-guided isolation and mass spectrometry

Abstract The anecdotal use of Alternanthera sessilis L. as a relief for diabetes has been known in the Philippines for generations, and antidiabetic activity of similar varieties in other countries is likewise documented. However, the compounds responsible for this activity remain unclear. This study aims to isolate the anti-hyperglycemic fraction of local A. sessilis leaves and identify the compounds in this fraction. Methanol extract of A. sessilis leaves and its hexane, ethyl acetate (ASE), and water fractions were administered to alloxan-induced diabetic mice. ASE (250mg/kg) had the highest anti-hyperglycemic activity at 6-h post-treatment (25.81%±12.72%), with almost similar blood glucose reduction rate as metformin (30.13±3.75%, p=0.767). Repeated fractionation employing chromatographic separation techniques followed by in vivo anti-hyperglycemic assay yielded partially purified subfractions. A. sessilis ethyl acetate subfraction 4-2 (100mg/kg) displayed remarkable suppression of blood glucose rise in diabetic mice at 6-h post-treatment (26.45±3.75%, p<0.0001), with comparable activity with metformin (100mg/kg, 27.87±5.65%, p=0.652). Liquid chromatography/mass spectrometry showed eight distinct peaks, with four peaks annotated via the Traditional Chinese Medicine library and custom library for A. sessilis. Among these, luteolin, apigenin, ononin, and sophorabioside were identified as putative compounds responsible for the anti-hyperglycemic activity. This result provided basis for the reported anecdotal claims and potential utility of the local variety of A. sessilis leaves as sources of anti-hyperglycemic agents

Quantitation of certolizumab pegol by validated liquid chromatography methods

Abstract Certolizumab pegol (CZP) is a Fab' fragment of the humanized antibody with anti-TNF-α activity that is indicated as therapy for Crohn's disease and rheumatoid arthritis. Using a BioSep-SEC-S3000 column (300 x 4.6 mm i.d., 5 µm particle size), a size exclusion liquid chromatography (SEC) method was developed. Mobile phase A consisted of 100 mM monobasic sodium phosphate and 200 mM sodium chloride (pH 7.0), while mobile phase B was ethanol (95:5, v/v), and the analysis was performed using a diode array detector (DAD) set to 214 nm and a flow rate of 0.5 ml min-1. In addition, a reversed-phase liquid chromatography (RP-LC) method based on gradient elution was developed on a Zorbax 300 SB C18 column (150 mm x 4.6 mm i.d., 3.5 µm particle size) kept at 80 °C. Mobile phase A was 0.1% (v/v) TFA in ultrapure water, and mobile phase B was a mixture of propanol, acetonitrile, ultrapure water and TFA (70 + 20 + 9.9 + 0.1, v/v) operated at a flow rate of 1.0 ml min-1, and DAD was applied at 214 nm. CZP elution was achieved with retention times of 5.6 min and 9.0 min for SEC and RP-LC, respectively.

Liquid chromatography-mass spectrometry for simultaneous determination of spironolactone and canrenone in plasma samples

Abstract n our study, we aimed to validate a method based on liquid chromatography-mass spectrometry (LC-MS) to quantify spironolactone (SPI) and its active metabolite canrenone (CAN) simultaneously in plasma samples to support in vivo experiments. Compounds were separated by using a C18 column with the isocratic elution of a mobile phase composed of 0.1% (v/v) formic acid in methanol-water (60:40 v/v) at a flow rate of 0.4 mL min−1. SPI and CAN were detected in na electrospray interface operating in a positive ionization mode and quantified using the selective ion mode monitoring of mass-charge ratios (m/z) of 439.0 for SPI and 363.1 for CAN. After calculating the matrix effect using theoretical equations, we observed the strong interference of plasma in the equipment-generated signal, which required creating analytical curves using the matrix as a solvent. The method was nevertheless linear (r 2 > 0.999) in a concentration range of 0.4-5.0 µg mL−1, as well as precise, with a coefficient of variation less than 5%. SPI's and CAN's recovery rates from the plasma ranged from 87.4% to 112.1%, while their limits of detection (i.e., 0.07 µg mL−1 and 0.03 µg mL−1, respectively) and quantification (i.e., 0.20 µg mL−1 and 0.08 µg mL−1, respectively) in the presence of plasma contaminants were low. Therefore, the bioanalytical method seems to be feasible for quantifying SPI and CAN in plasma

Development and validation of liquid chromatography-tandem mass spectrometry method to quantify dasatinib in plasma and its application to a pharmacokinetic study

Abstract Dasatinib, a potent oral multi-targeted kinase inhibitor against Src and Bcr-Abl, can decrease inflammatory response in sepsis. A simple and cost-effective method for determination of an effective dose dasatinib was established. This method was validated in human plasma, with the aim of reducing the number of animals used, thus, avoiding ethical problems. Dasatinib and internal standard lopinavir were extracted from 180 uL of plasma using liquid-liquid extraction with methyl tert-butil ether, followed by liquid chromatography coupled to triple quadrupole mass spectrometry in multiple reaction monitoring mode. For the pharmacokinetic study, 1 mg/kg of dasatinib was administered to mice with and without sepsis. The method was linear over the concentration range of 1-98 ng/mL for DAS in mice and human plasma, with r2>0.99 and presented intra- and interday precision within the range of 2.3 - 6.2 and 4.3 - 7.0%, respectively. Further intra- and interday accuracy was within the range of 88.2 - 105.8 and 90.6 - 101.7%, respectively. The mice with sepsis showed AUC0-t = 2076.06 h*ng/mL and Cmax =102.73 ng/mL and mice without sepsis presented AUC0-t = 2128.46 h*ng/mL. Cmax = 164.5 ng/mL. The described analytical method was successfully employed in pharmacokinetic study of DAS in mice.

Biotransformation differences of ginsenoside compound K mediated by the gut microbiota from diabetic patients and healthy subjects / 中国天然药物

Many natural products can be bio-converted by the gut microbiota to influence pertinent efficiency. Ginsenoside compound K (GCK) is a potential anti-type 2 diabetes (T2D) saponin, which is mainly bio-transformed into protopanaxadiol (PPD) by the gut microbiota. Studies have shown that the gut microbiota between diabetic patients and healthy subjects are significantly different. Herein, we aimed to characterize the biotransformation of GCK mediated by the gut microbiota from diabetic patients and healthy subjects. Based on 16S rRNA gene sequencing, the results indicated the bacterial profiles were considerably different between the two groups, especially Alistipes and Parabacteroides that increased in healthy subjects. The quantitative analysis of GCK and PPD showed that gut microbiota from the diabetic patients metabolized GCK slower than healthy subjects through liquid chromatography tandem mass spectrometry (LC-MS/MS). The selected strain A. finegoldii and P. merdae exhibited a different metabolic capability of GCK. In conclusion, the different biotransformation capacity for GCK may impact its anti-diabetic potency.

Proteomic Difference Analysis of Whole Blood and Bloodstains / 法医学杂志

OBJECTIVES@#To study the changes of protein levels in peripheral blood after it dried.@*METHODS@#The proteins from whole blood and bloodstains were detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and normalized by the label-free quantification (LFQ) method. The differential proteins were analyzed by using R 4.2.1 software, limma and edgeR package. The analysis of biological function, signaling pathway and subcellular localization for the differential proteins was then performed.@*RESULTS@#A total of 623 and 596 proteins were detected in whole blood and bloodstains, respectively, of which 31 were statistically significant in the quantitative results, including 10 up-regulated and 21 down-regulated proteins in bloodstains.@*CONCLUSIONS@#The protein abundances in whole blood and bloodstains are highly correlated, and the variation of protein abundances may be related to the changes of endogenous and structural proteins in cells. The application of proteomics technology can assist the screening and identification of protein biomarkers, thereby introducing new biomarkers for forensic research.

Detection and Analysis of Blood Dexmedetomidine in Drug-Facilitated Cases / 法医学杂志

OBJECTIVES@#To establish a simple and rapid qualitative and quantitative detection method of dexmedetomidine in blood.@*METHODS@#Blood was separated on the Allure PFP Propyl liquid chromatography column with isocratic elution after it was precipitated by acetonitrile and filtered. Qualitative and quantitative analysis of dexmedetomidine was performed using positive ion scan mode and multi-reaction monitoring mode.@*RESULTS@#The limit of detection of dexmedetomidine in blood was 0.2 ng/mL and the limit of quantification was 0.5 ng/mL. The linearity of the method was good in the range of 0.5-1 000 ng/mL, and the correlation coefficient was greater than 0.99. The accuracy of the method was 90.34%-112.67% and the extraction recovery was 50.05%-91.08%, with no significant matrix effect.@*CONCLUSIONS@#This method is simple, selective and suitable for the qualitative and quantitative analysis of dexmedetomidine in blood, which can provide a reference for drug-facilitated cases involving dexmedetomidine.

Excretion of three alkaloids from Simiao Pills in urine, feces, and bile between normal and type 2 diabetic rats / 中国中药杂志

This study investigated the differences in excretion kinetics of three alkaloids and their four metabolites from Simiao Pills in normal and type 2 diabetic rats. The diabetes model was established in rats by injection of streptozotocin, and the alkaloids in urine, feces, and bile of normal and diabetic rats were detected by LC-MS/MS to explore the effect of diabetes on alkaloid excretion of Simiao Pills. The results showed that 72 h after intragastric administration of the extract of Simiao Pills, feces were the main excretion route of alkaloids from Simiao Pills. The total excretion rates of magnoflorine and berberine in normal rats were 4.87% and 56.54%, which decreased to 2.35% and 35.53% in diabetic rats, which had statistical significance(P<0.05). The total excretion rates of phellodendrine, magnoflorine, and berberine in the urine of diabetic rats decreased significantly, which were 53.57%, 60.84%, and 52.78% of those in normal rats, respectively. After 12 h of intragastric administration, the excretion rate of berberine in the bile of diabetic rats increased significantly, which was 253.33% of that of normal rats. In the condition of diabetes, the excretion rate of berberine metabolite, thalifendine significantly decreased in urine and feces, but significantly increased in bile. The total excretion rates of jateorrhizine and palmatine in the urine increased significantly, and t_(1/2) and K_e changed significantly. The results showed that diabetes affected the in vivo process of alkaloids from Simiao Pills, reducing their excretion in the form of prototype drug, affecting the biotransformation of berberine, and ultimately increasing the exposure of alkaloids in vivo, which would be conducive to the hypoglycemic effect of alkaloids. This study provides references for the clinical application and drug development of Simiao Pills in diabetes.

Determination of plasma protein binding rate of Shuganning Injection using equilibrium dialysis and UPLC-MS/MS / 中国中药杂志

Traditional Chinese medicine(TCM) compound preparations have complex compositions. As a widely used TCM injection, Shuganning Injection, its in vivo processes are not yet fully understood. Determining the plasma protein binding rate is of great significance for pharmacokinetic and pharmacodynamic studies. In this experiment, the equilibrium dialysis method combined with UPLC-MS/MS technology was used to determine the plasma protein binding rates of 10 components, including p-hydroxyacetophenone, caffeic acid, baicalein, oroxylin A, geniposide, baicalin, cynaroside, oroxylin A-7-O-β-D-glucuronide, scutellarin, and hyperoside, in Shuganning Injection in rat and human plasma to provide a theoretical basis for further elucidating the in vivo processes of Shuganning Injection and guiding clinical medication. The results showed that, except for baicalein and geniposide, the plasma protein binding rates of the other eight components were higher in human plasma than in rat plasma, and there were interspecies differences. In human plasma, except for geniposide, caffeic acid, and baicalin, the plasma protein binding rates of the remaining seven components were above 80%, with baicalein and oroxylin A exceeding 90%. All components exhibit a high level of binding to plasma proteins, with the exception of geniposide.

Rapid determination of acetaminophen in plasma by LC-MS/MS / 中华劳动卫生职业病杂志

Objective: To establish a method for the rapid determination of acetaminophen (APAP) in human plasma by LC-MS/MS. Methods: The plasma samples were extracted by methanol and acetonitrile (1: 1) and purified directly. C(18) column was used for sample separation. The mobile phase were methanol (5 mmol/L ammonium acetate) and water (5 mmol/L ammonium acetate). Samples were analyzed by LC MS/MS with the electrospray ionization multi reaction monitoring (MRM) mode. Results: The calibration curves of APAP was linear in the concentration range of 0~10 mg/L, the correlation coefficient (r) was greater than 0.999 0. The relative standard deviation within and between batches was less than 10%. The recovery rate were 96.81%~101.7%. The detection limit of the method was 0.1 μg/L and the lower limit of quantification was 0.3 μg/L. Conclusion: This method has strong specificity, high sensitivity and reliable determination results. It is suitable for the rapid analysis of clinical plasma samples.

Analysis of Forty-Two Psychoactive Substances in a Single Hair by Micro-Segmental Technique / 法医学杂志

OBJECTIVES@#To establish an LC-MS/MS method based on single hair micro-segmental technique, and verify the detection of 42 psychoactive substances in 0.4 mm hair segments.@*METHODS@#Each piece of single hair was cut into 0.4 mm segments and extracted by sonication and the segments were immersed in dithiothreitol-containing extraction medium. Mobile phase A was the aqueous solution containing 20 mmol/L ammonium acetate, 0.1% formic acid, and 5% acetonitrile. Mobile phase B was acetonitrile. An electrospray ionization source in positive ion mode was used for data acquisition in multiple reaction monitoring (MRM) mode.@*RESULTS@#The 42 psychoactive substances in hair had a good linear relationship within their respective linear ranges (r>0.99), the limits of detection were 0.2-10 pg/mm, the limits of quantification were 0.5-20 pg/mm, the intra-day and inter-day precisions were 1.5%-12.7%, the intra-day and inter-day accuracies were 86.5%-109.2%, the recovery rates were 68.1%-98.2%, and the matrix effects were 71.3%-111.7%. The method was applied to hair samples collected from one volunteer at 28 d after a single dose of zolpidem, with zolpidem detected in 5 hairs was 1.08-1.60 cm near the root tip, and the concentration range was 0.62-20.5 pg/mm.@*CONCLUSIONS@#The micro-segmental technique of single hair analysis can be applied to the investigation of drug-facilitated sexual assault cases.

Contamination status and exposure risk of mycotoxins in Coicis Semen / 中国中药杂志

By investigating the contamination status and predicting the exposure risk of mycotoxin in Coicis Semen, we aim to provide guidance for the safety supervision of Chinese medicinal materials and the formulation(revision) of mycotoxin limit standards. The content of 14 mycotoxins in the 100 Coicis Semen samples collected from five major markets of Chinese medicinal materials in China was determined by UPLC-MS/MS. The probability evaluation model based on Monte Carlo simulation method was established after Chi-square test and One-way ANOVA of the sample contamination data. Health risk assessment was performed on the basis of margin of exposure(MOE) and margin of safety(MOS). The results showed that zearalenone(ZEN), aflatoxin B_1(AFB_1), deoxynivalenol(DON), sterigmatocystin(ST), and aflatoxin B_2(AFB_2) in the Coicis Semen samples had the detection rates of 84%, 75%, 36%, 19%, and 18%, and the mean contamination levels of 117.42, 4.78, 61.16, 6.61, and 2.13 μg·kg~(-1), respectively. According to the limit standards in the Chinese Pharmacopoeia(2020 edition), AFB_1, AFs and ZEN exceeded the standards to certain extents, with the over-standard rates of 12.0%, 9.0%, and 6.0%, respectively. The exposure risks of Coicis Semen to AFB_1, AFB2, ST, DON, and ZEN were low, while 86% of the samples were contaminated with two or more toxins, which needs more attention. It is suggested that the research on the combined toxicity of different mycotoxins should be strengthened to accelerate the cumulative exposure assessment of mixed contaminations and the formulation(revision) of toxin limit standards.

Determination of 10 mycotoxins in Hippophae Fructus medicinal and edible products by ultra-performance liquid chromatography- tandem mass spectrometry / 中国中药杂志

An analytical method for 10 mycotoxins in Hippophae Fructus medicinal and edible products was established in this study, and the contamination of their mycotoxins was analyzed. First of all, the mixed reference solution of ten mycotoxins such as aflatoxin, ochratoxin, zearalenone, and dexoynivalenol was selected as the control, and the Hippophae Fructus medicinal and edible products were prepared. Secondly, based on the ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS) technology, 10 mycotoxins in Hippophae Fructus medicinal and edible products were quantitatively investigated and their content was determined. Finally, the contamination of mycotoxins was analyzed and evaluated. The optimal analysis conditions were determined, and the methodological inspection results showed that the 10 mycotoxins established a good linear relationship(r>0.99). The method had good repeatability, test sample specificity, stability, and instrument precision. The average recovery rates of 10 mycotoxins in Hippophae Fructus medicinal products, edible solids, and edible liquids were 90.31%-109.4%, 87.86%-107.8%, and 85.61%-109.1%, respectively. Relative standard deviation(RSD) values were 0.22%-10%, 0.75%-13%, and 0.84%-8.5%, repsectively. Based on UPLC-MS/MS technology, the simultaneous determination method for the limits of 10 mycotoxins established in this study has fast detection speed, less matrix interference, high sensitivity, and accurate results, which is suitable for the limit examination of 10 mycoto-xins in Hippophae Fructus medicinal and edible products.

Detection of Carbamazepine and Its Metabolites in Blood Samples by LC-MS/MS / 法医学杂志

OBJECTIVES@#To establish a method for the detection of carbamazepine and its metabolites 10,11-dihydro-10,11-epoxycarbamazepine and 10,11-dihydro-10-hydroxycarbamazepine in blood samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS).@*METHODS@#The blood samples were treated with 1-butyl-3-methylimidazolium hexafluorophosphate as an extraction solvent. The samples were extracted by ultrasound-assisted extraction and separated by ZORBAX Eclipse Plus C18, 95Å column. The mobile phase A aqueous solution containing 0.1% formic acid and 10 mmol/L ammonium acetate, and mobile phase B mixed organic solvent containing acetonitrile/methanol (Vacetonitrile∶Vmethanol=2∶3) were used for gradient elution at the flow rate of 1.00 mL/min. An electrospray ion source in positive mode was used for detection in the multiple reaction monitoring.@*RESULTS@#The linearities of carbamazepine and its metabolites 10,11-dihydro-10,11-epoxycarbamazepine and 10,11-dihydro-10-hydroxycarbamazepine in blood samples were good within the corresponding range, with correlation coefficients (r) greater than 0.995 6. The limits of detection were 3.00, 0.40 and 1.30 ng/mL, respectively. The limit of quantitation were 8.00, 1.00 and 5.00 ng/mL, respectively. The extraction recoveries ranged from 76.00% to 106.44%. The relative standard deviations of the intra-day and inter-day precisions were less than 16%. Carbamazepine and its main metabolite 10,11-dihydro-10,11-epoxycarbamazepine were detected in blood samples of death cases with a mass concentration of 2.71 μg/mL and 252.14 ng/mL, respectively.@*CONCLUSIONS@#This method has high sensitivity and good selectivity, which is suitable for the detection of carbamazepine and its metabolites in blood samples, and can be used for carbamazepine-related forensic identifications.

Study on Clinical Application and Supervision of LC-MS in in Vitro Diagnosis Test / 中国医疗器械杂志

From the perspective of technical evaluation, this study reviewed the current situation of application and clinical application of medical device products were detected by liquid chromatography-tandem mass spectrometry in the market in recent years. The regulatory requirements of these products in China, USA, EU and Japan were compared and analyzed, and the monitoring situation of adverse events after listing, the standards for reference and the domestic and foreign regulatory documents were combined, the clinical application and regulatory risks of the product were analyzed. The problems such as pre-treatment, system matching, adequacy of performance index requirements, inter-room consistency, reference interval and registration unit were discussed and suggestions for supervision were given, with a view to the field of product R&D and production, review and approval of supervision to provide technical reference.

Therapeutic drug monitoring of gabapentin: the applicability in patients with neuropathic pain

Abstract Gabapentin is an antiepileptic drug prescribed for several neuropathic pain conditions. This study aimed to evaluate gabapentin (GAB) trough plasma concentration range and the applicability of therapeutic drug monitoring in patients with neuropathic pain. Fifty-three patients with neuropathic pain, aged 20 to 75, received gabapentin as treatment for at least 7 days. Gabapentin plasma concentration was sampled before GAB administration and quantified by liquid chromatography with a UV detector. GAB trough plasma concentration ranged between 0.40 and 11.94 µg/mL in patients with chronic neuropathic pain. No differences were observed in terms of GAB plasma concentrations between responsive and non-responsive patients. Our data suggest that the reference ranges suggested in the literature for patients with epilepsy should not be used for patients with neuropathic pain. Therapeutic drug monitoring of GAB was shown to be an important tool to assess treatment adherence.

Ferramenta in silico para desenvolvimento de métodos cromatográficos - aplicação em misturas de filtros solares / In silico tool for development of chromatographic methods - application in mixtures of sunscreensPalavras-chave em inglês

Planejamento de Experimentos (DoE) permite obter e explorar conhecimentos sobre inúmeros sistemas, facilitando a coleta de informações com reduzido número de experimentos. No entanto, DoE é restrito ao delineamento do desenho experimental. Para superar essa limitação e permitir uma previsão precisa dos tempos de retenção para uma seleção de filtros UV orgânicos sob diversas condições, usamos a Relação Quantitativa entre Estrutura e Retenção combinada com o método de Monte Carlo para desenvolver uma plataforma in silico capaz de prever o perfil cromatográfico de filtros UV orgânicos. Sete analitos foram usados para estabelecer o modelo de predição: benzofenona-3, avobenzona, ethilhexil triazona, octil dimetil PABA, metoxicinamato de octila, tinosorb® S e octocrileno. Os valores residuais obtidos no modelo de análise de regressão múltipla mostraram distribuição normal, homocedasticidade e independência. Os coeficientes de determinação (R2) e predição (R2 pred) foram de 99,82% e 99,71%, respectivamente. A plataforma in silico apresentou grande potencial para predição do perfil cromatográfico de filtros UV orgânicos, da coeluição de analitos, de seus parâmetros cromatográficos, além de permitir, sem experimentação, uma visão geral do comportamento de retenção de compostos sob diversas condições cromatográficas

Design of Experiments (DoE) allows obtaining and explorer knowledge about innumerous systems, facilitating the information collection with reduced number of experiments. However, DoE is restricted to the limited range which experimental design was delineated. In order to overcome this limitation and enable accurate prediction of retention times for a selection of organic UV filters under various conditions, we used the Quantitative Structure-Retention Relationships tool combined with Monte Carlo method to develop an in silico platform capable of predicting chromatographic profile of organic UV filters. Seven analytes were used to established to prediction model: benzophenone-3, butyl methoxydibenzoilmethane, ethylhexyl triazone, ethylhexyl dimetyl PABA, ethylhexyl methoxycinnamate, bisethylhexyloxyphenol methoxyphenyl triazine and octocrylene. Residual values obtained from multiple regression analysis model showed normal distribution, homoscedasticity, and independence. Determination (R2) and prediction (R2 pred) coefficients were found to be 99,82% and 99,71%, respectively. In silico platform presented great potential for predicting chromatographic profile of organic UV filters, analytes coelution, chromatographic parameters and allowing, without experimentation, an overview of retention behavior of compounds under various chromatographic conditions

Chromatographic profile of xanthones and flavonoids in the anti-dengue extracts of Fridericia samydoides (Cham. ) L. G. Lohmann (Bignoniaceae)

Abstract The flavonoids and xanthones present in the ethanol extracts of leaves and stems of Fridericia samydoides showed that anti-dengue activities in vitro were investigated qualitatively by liquid chromatography-ultraviolet-mass spectrometry in series. Nineteen flavones and fifteen xanthones were detected and characterized on the basis of their fragmentation pattern in the positive and negative ion mode tandem mass spectrometry spectra and ultraviolet bands. Acacetin, chrysin, vitexin, isovitexin, orientin, isoorientin, mangiferin, 2'-O-trans-caffeoylmangiferin, 2'-O-trans-coumaroylmangiferin and 2'-O-trans-cinnamoylmangiferin were identified by comparison with authentic samples. The other compounds detected were tentatively assigned by analysis of the spectral data and by comparison with literature reports. In addition, it performed the fractionation of the leaves extract leading to the isolation of mangiferin, isovitexin and isoorientin. All extracts and isolated compounds inhibited the Dengue virus replication cycle with EC50 less than 25.0 µg/mL for extracts and 272.5, 85.6 and 79.3 µg/mL for mangiferin, isovitexin and isoorientin, respectively.

Desenvolvimento de organogel injetável subcutâneo de Pluronic®-F127 e lecitina de soja (OPL) contendo meloxicam para uso veterinário / Development of a veterinary organogel formulation containing soy lecithin, Pluronic® F-127 and meloxicam for the treatment of acute and chronic pain in small animals

O organogel é formado por uma matriz tridimensional composta de filamentos que se auto-organizam em uma rede entrelaçada e que, por seu tipo de estrutura, pode ser utilizado com o objetivo de atuar como um implante que se forma in situ, sendo capaz de se comportar como uma forma farmacêutica de liberação prolongada. Esse trabalho tem, por tanto, o objetivo desse trabalho foi desenvolver, caracterizar, quantificar e traçar perfis de dissolução para formulações de organogel contendo meloxicam como principio ativo. O material está dividido em quatro capítulos, sendo apresentada inicialmente (I) revisão da literatura a respeito da lecitina de origem vegetal, com suas principais fontes de obtenção, como soja, girassol e colza, e também seu uso farmacêutico na obtenção de formulações como organogéis, microemulsões e lipossomas. Os demais capítulos abordam (II) desenvolvimento e otimização de uma formulação de organogel contendo lecitina de soja e Pluronic® F-127 como formadores da matriz tridimensional e meloxicam como principio ativo. (III) Desenvolvimento e validação de um método de quantificação do teor de meloxicam por cromatografia líquida de alta eficiência (CLAE). (IV) Desenvolvimento de um método de dissolução para formulações de organogel, que fosse capaz de ser utilizado na caracterização do perfil de dissolução de diferentes formulações. Com os resultados obtidos, foi possível desenvolver formulações de organogel contendo lecitina de soja, Pluronic® F-127 e meloxicam, assim como um método analítico validado para as analises de teor. Por fim, foram obtidos também os perfis de dissolução de duas formulações mais promissoras

Organogels are formed by a three-dimensional matrix composed of filaments that selforganize in an interlaced network and that, due to its type of structure, can be used with the objective of acting as an implant that forms in situ, being able to behave as an extendedrelease dosage form. This work has, therefore, the objective of this work was to develop, characterize, quantify and trace dissolution profiles for organogel formulations containing meloxicam as active ingredient. The material is divided into four chapters, initially presented (I) review of the literature on lecithin of plant origin, with its main sources of production, such as soybean, sunflower and rapeseed, and also its pharmaceutical use in obtaining formulations such as organogels , microemulsions and liposomes. The remaining chapters address (II) development and optimization of an organogel formulation containing soy lecithin and Pluronic® F-127 as three-dimensional matrix formers and meloxicam as an active ingredient. (III) Development and validation of a method for quantification of meloxicam content by high performance liquid chromatography (HPLC). (IV) Development of a dissolution method for organogel formulations, capable of being used to characterize the dissolution profile of different formulations. With the results obtained, it was possible to develop organogel formulations containing soy lecithin, Pluronic® F-127 and meloxicam, as well as a validated analytical method for content analysis. Finally, the dissolution profiles of two more promising formulations were also obtained

Development and validation of a HILIC-HPLC-ELSD method for simultaneous determination of glucosamine hydrochloride and chondroitin sulfate in dietary supplements

Abstract The objective of the present study is to develop and validate a simple, selective and accurate hydrophilic interaction liquid chromatography - a high performance liquid chromatography incorporating an evaporative light scattering detector (HILIC-HPLC-ELSD) method for simultaneously determining glucosamine hydrochloride and chondroitin sulfate in dietary supplements. The chromatographic separation was carried out on a ZIC-HILIC column (150 mm x 4.6 mm x 5µm) in isocratic system mode with a mobile phase of acetonitrile, 30 mM ammonium formate and water (77:20:3, v/v/v) at pH 4.5, a column temperature of 35°C, a flow rate of 1 mL.min-1, and an injection volume of 5 µL. An evaporative light scattering (ELS) detector was used. Effective separation was achieved by means of analyte resolution of more than 1.5 with an analysis run time of approximately 20 minutes. The linearity of glucosamine hydrochloride and chondroitin sulfate ranged from 0.4 to 2.5 mg.mL-1. The limits of the detection and quantification of glucosamine hydrochloride were 20 and 80 mg.mL-1 respectively, while for chondroitin sulfate they were 80 and 400 mg.mL-1. All validation parameters satisfied the acceptance criteria in accordance with International Conference on Harmonisation (ICH) guidelines. The method was successfully applied to the assay of commercial dietary supplement samples