Implementation of Two Chromatographic Methods for Simultaneous Quantitation of Thioctic Acid, Benfotiamine and Cyanocobalamin.

Two accurate and sensitive chromatographic methods have been introduced and validated for the simultaneous determination of thioctic acid, benfotiamine and cyanocobalamin in bulk powders and in their pharmaceutical formulation. Method A is reversed-phase ultra performance liquid chromatographic method with an isocratic elution, where a rapid separation was accomplished on a Zorbax C8 column using a mobile phase of acetonitrile:0.05 M phosphate buffer (pH 6 adjusted by o-phosphoric acid) (23:77, v/v). The retention times (tR) were 0.578, 0.852 and 1.376 for cyanocobalamin, benfotiamine and thioctic acid, respectively. The separated peaks were revealed at 210.0 nm. Method B is a thin-layer densitometric method where the separation of the studied drugs was carried out on silica gel plates using methanol-chloroform-heptane-1-sulphonic acid sodium salt (0.4%) (7:3:0.1, by volume) as a mobile phase, and scanning of the separated bands was done at 240.0 nm. The retardation factor (Rf) values were 0.17, 0.48 and 0.75 for cyanocobalamin, benfotiamine and thioctic acid, respectively. Validation of the methods was achieved following ICH guidelines and the applied methods succeeded to determine the cited drugs in their pure forms and capsules. Results were statistically compared to the manufacturer's method where no significant difference was observed.

Estimation of Lipoyllysine Content in Meat and Its Antioxidative Capacity.

During this research a simple, accurate, and environmentally friendly method to determine lipoyllysine and lipoic acid in meat was developed and validated. The presented approach was based on the hydrolysis of the proteins containing lipoic acid, reduction of disulfide bonds with tris(hydroxymethyl)phosphine, and precolumn derivatization of free thiol groups with 1-benzyl-2-chloropyridinium bromide long-term followed by HPLC separation with a diode-array detector. The method has been validated in accordance with the U.S. FDA guidelines and was linear in the range of 0.1-10 µmol/L in concentration with R2 values ≥0.9997 for both analytes. For lipoyllysine and lipoic acid, intra- and interday precision values were lower than 10%. The intraday accuracy values ranged from 91.0% to 99.4% for lipoyllysine and from 99.1% to 107.3% for lipoic acid, whereas the interday accuracy values for lipoyllysine and lipoic acid were 92.0-95.6% and 93.5-98.8%, respectively. Additionally, in this research the antioxidant activity of lipoyllysine and reduced lipoyllysine compound using spectrophotometric method with 1,1-diphenyl-2-picrylhydrazyl was examined for the first time. The data showed that dihydrolipoyllysine exhibits stronger antioxidant capacity than lipoyllysine based on a lower value of concentration required to achieve a 50% antioxidant effect in 1,1-diphenyl-2-picrylhydrazyl radical scavenging test.

Colorimetric sensor array for accurate detection and identification of antioxidants based on metal ions as sensor receptors.

There is an ongoing need to develop high-performance sensing strategy for detecting and discriminating antioxidants, primarily because of their role in medical diagnosis and food. In this regard, visual sensor arrays have been a subject of intensive research for such applications. To this end, we propose a colorimetric sensor array for accurate detection and identification of antioxidants, which is based on the reactions between 3,3',5,5'-tetramethylbenzidine (TMB) and metal ions as sensing receptors and the interactions between antioxidants and oxidized TMB (oxTMB). Different target antioxidants displayed diverse reduction abilities toward the oxTMB, creating distinct colorimetric response patterns. The combination of colorimetric response variation at color and absorbance at 652 nm enables the sensor array to provide a unique fingerprint pattern to each antioxidant. Linear discriminant analysis (LDA) and centroid diagrams show that the sensor array can well detect and discriminate the eight tested antioxidants, including lipoic acid (LIA), cysteine (Cys), tannin (TA), ascorbic acid (AA), glutathione (GSH), Uric Acid (UA), glycine (Gly), and dopamine (DA), with a high sensitivity in the range of nanomolar concentrations.

Prospective randomized multicentre comparison on sibling oocytes comparing G-Series media system with antioxidants versus standard G-Series media system.

RESEARCH QUESTION: Does the inclusion of three antioxidants (A3), acetyl-l-carnitine (ALC), N-acetyl-l-cysteine (NAC) and alpha-lipoic acid (ALA) improve human embryo development and pregnancy potential? DESIGN: Prospective randomized multicentre comparison of sibling oocytes. A total of 1563 metaphase II oocytes from 133 patients in two IVF centres. Day 3 embryo and day 5/6 blastocyst quality were assessed. Good embryo quality on day 3 was defined as 8 to 10 cells with even cells and low fragmentation; good quality blastocysts as 3BB or greater. Clinical outcome was assessed on transfers of fresh or vitrified-warmed blastocyst on day 5. RESULTS: Of the two-pronuclei, 40.7% (G-Series) and 50.2% (G-Series with A3 group) resulted in good quality embryos on day 3 (P < 0.05). The implantation rate by fetal sac was 39.2% and 50.6%, and by fetal heartbeat was 37.8% and 47.1% for the G-Series and G-Series with A3 group, respectively. When stratified by female patient age, patients 35-40 years had an implantation rate by fetal sac and heart of 23.5% in the G-Series compared with 57.5% (P < 0.05) and 50.0% (P < 0.05) in the A3 group. The ongoing pregnancies in patients 35-40 years were significantly higher in the A3 group (50%) compared with the control (25.8%) (P < 0.05). CONCLUSIONS: The presence of antioxidants during IVF and embryo culture for patients 35-40 years resulted in a significant increase in implantation and pregnancy rate. Supplementation of antioxidants to IVF and culture media may therefore improve the viability of human embryos in assisted reproductive technologies, plausibly through the reduction of oxidative stress.

Development and validation of a reversed-phase high-performance liquid chromatographic method for the quantitation and stability of α-lipoic acid in cosmetic creams.

OBJECTIVE: To develop and validate a simple reversed-phase HPLC method for the quantitation and evaluation of stability of α-lipoic acid in cosmetics, according to International Conference on Harmonization (ICH) Guidelines. METHODS: The chromatography was performed on a reversed-phase Luna C18, analytical column (150 × 4.6 mm id, 5 µm particle size) with a mobile phase of potassium dihydrogen phosphate (pΗ 4.5; 0.05 M) and acetonitrile (60:40, v/v) and a flow rate of 1.0 mL min-1 with UV detection at 340 nm. Accelerated and long-term stability studies of α-lipoic acid in cosmetic cream were conducted under various degradation conditions including acid, basis, oxidation, and thermal and photolytic degradation, according to European Medicines Agency Guidelines CPMP/ICH/2736/99. RESULTS: The limit of detection (LOD) for the cosmetic cream was 0.9 µg mL-1 and the limit of quantitation (LOQ) was 2.8 µg mL-1 , while the retention time was 7.2 min. The method proved to be linear, precise and accurate. The stability results demonstrated the selectivity of the proposed method to the analysis of α-LA, and the degradation products were determined and evaluated in specific stress conditions in cosmetic creams. The applicability of the method was tested in two different developed cosmetic products (cream with 1.5 % w/w and emulsion with 1.0 % w/w of LA) and proved to be reliable. CONCLUSION: A reversed-phase HPLC-UV method was developed and fully validated for the analysis of α-lipoic acid in cosmetics. It is the first reported application on the quantitation of lipoic acid in cosmetic creams, while at the same time evaluates the stability in forced degradation conditions, in new cosmetic formulations. It proved to be suitable for the reliable quality control of cosmetic products, with a run time of <8 min that allows for the analysis of large number of samples per day.

OBJECTIF: Développer et valider une méthode HPLC (chromatographie en phase liquide à haute performance) simple en phase inversée pour la quantification et l'évaluation de la stabilité de l'acide α-lipoïque dans les cosmétiques, conformément aux Directives de la Conférence internationale sur l'harmonisation (ICH). MÉTHODE: La chromatographie a été réalisée sur une colonne analytique Luna C18 en phase inversée (150 × 4,6 mm id, taille des particules 5 µm) avec une phase mobile de dihydrogénophosphate de potassium (pH 4,5 ; 0,05 M) et d'acétonitrile (60:40, v/v) et un débit de 1,0 ml min−1 avec détection UV à 340 nm. Des études de stabilité accélérée et à longterme de l'acide α-lipoïque dans les crèmes cosmétiques ont été menées dans diverses conditions de dégradation, notamment en milieu acide, basique, par oxydation et dégradation thermique et photolytique, conformément aux lignes directrices de l'Agence européenne des médicaments CPMP/ICH/2736/99. RÉSULTAT: La limite de détection (LD) pour la crème cosmétique était de 0,9 µg ml et la limite de quantification (LQ) était de 2,8 µml−1 , tandis que le temps de rétention était de 7,2 min. La méthode s'est avérée linéaire, précise et exacte. Les résultats de stabilité ont démontré la sélectivité de la méthode proposée pour l'analyse de l'acide α-lipoïque et les produits de dégradation ont été déterminés et évalués dans des conditions de stress spécifiques dans les crèmes cosmétiques. L'applicabilité de la méthode a été testée dans deux produits cosmétiques différents développés (crème avec 1,5 % p/p et émulsion avec 1,0 % p/p d'acide lipoïque) et s'est avérée fiable. CONCLUSION: une méthode HPLC en phase inversée avec détection UV a été développée et entièrement validée pour l'analyse de l'acide α-lipoïque dans les cosmétiques. Il s'agit de la première application signalée concernant la quantification de l'acide lipoïque dans les crèmes cosmétiques et permettant en même temps d'évaluer la stabilité des conditions de dégradation forcée dans les nouvelles formulations cosmétiques. Cette méthode s'est avérée adaptée au contrôle de qualité fiable des produits cosmétiques, avec une durée d'exécution < 8 min qui permet l'analyse d'un grand nombre d'échantillons par jour.

Integrative application of licorice root extract or lipoic acid with fulvic acid improves wheat production and defenses under salt stress conditions.

Although different plant extracts and plant growth regulators are used as biostimulants to support plants grown under salt stress conditions, little information is available regarding the use of licorice root extract (LRE) or lipoic acid (LA) as biostimulants. Studies on the application of LRE or LA in combination with fulvic acid (FA) as natural biostimulants have not been performed. Therefore, in this study, two pot experiments were conducted to evaluate the potential effects of LRE (5 g L-1) or LA (0.1 mM) supplemented as a foliar spray in combination with FA (0.2 mg kg-1 soil) on osmoprotectants and antioxidants, growth characteristics, photosynthetic pigments, nutrient uptake, and yield as well as on the anatomical features of the stems and leaves of wheat plants irrigated with three levels of saline water (0.70, 7.8, and 14.6 dSm-1). Moderate (7.8 dSm-1) and high (14.6 dSm-1) levels of salinity caused a significant (p ≤ 0.05) increase in the activities of SOD, APX CAT, POX, and GR as well as in electrolyte leakage, malondialdehyde level, and reactive oxygen species (O2‒ and H2O2) levels compared to those in controls (plants irrigated with tap water). However, the leaf relative water content, membrane stability index, NPK uptake, leaf area, plant height, spike length, straw yield, grain yield, and protein content of wheat grains significantly (p ≤ 0.05) decreased. Addition of LRE or LA and/or HA to wheat plants under saline stress significantly (p ≤ 0.05) enhanced their morphological and physio-biochemical characteristics in parallel with increases in the activities of enzymatic antioxidants. Salinity stress altered (p ≤ 0.05) wheat stem and leaf structures; however, treatment with LRE + FA significantly improved these negative effects. These findings indicate that FA + LRE treatment significantly improved the antioxidant defense system of the plants, thereby reducing ROS levels and increasing wheat growth and production under saline conditions.

P,N Codoped carbon dots as an efficient "off-on" fluorescent probe for lipoic acid detection and its cellular dual-color imaging.

A facile single hydrothermal method was developed to synthetize P,N codoped carbon dots (P,N/CDs), which show strong and stable fluorescence, good water solubility, low toxicity and good biocompatibility. Hence, a novel and efficient "off-on" P,N/CDs fluorescent probe was developed for the highly sensitive detection of lipoic acid (LA) for the first time. The fluorescence of the P,N/CDs was quenched by Cu2+ forming a P,N/CDs-Cu2+ complex, which acted as the "off" process, but Cu2+ could be removed by LA, due to stronger chelating between LA and Cu2+, forming a more stable complex, which recovered the fluorescence of the P,N/CDs, in order to achieve the "on" process. Under optimal conditions, the concentration of LA and the increased fluorescence intensity of the P,N/CDs-Cu2+ complex displayed a good linear relationship within the range of 0.05-28 µM, with a detection limit (S/N = 3) of 0.02 µM. The established "off-on" fluorescent probe was successfully applied to the analysis of LA in urine samples. The average recoveries were in the range of 98.3-101.5%, with a relative standard deviations of less than 3.1%. In addition, the P,N/CDs were also successfully applied to cellular dual-color imaging of live T24 cells. The results show that the P,N/CDs have great application potential in clinical diagnosis, bioassay and bioimaging. Graphical abstract.

A Rapid and Precise Liquid Chromatographic Method for Simultaneous Determination of Alpha Lipoic Acid and Docetaxel in Lipid-Based Nanoformulations.

Combinational drug delivery successfully merges the benefits of nanotechnology and combination therapy by providing diversity to improve the carrier properties and better control over tailoring them as per the need of cancer treatment. A combination of conventional chemotherapeutic agent; docetaxel (DTX) and antioxidant agent; alpha lipoic acid (ALA) which acts by preventing metastasis may fulfill idealness of control and targeted drug delivery against breast cancer. The objective of the current study is to develop a reverse-phase HPLC-UV method for simultaneous determination of DTX and ALA in lipid-based nanoformulations. DTX and ALA were separated on Intersil® ODS (C18) column (250 × 4.6 mm, 5 µm) with a mobile phase consisting of acetonitrile: sodium acetate buffer (pH 3.5; 10 mM) (65:35% v/v) run in isocratic mode at a flow rate of 1 mL/min. The developed method was validated as per ICH guidelines. The method showed linearity in the concentration range of 1-15 µg/mL for DTX and 2-30 µg/mL for ALA. It can detect minimum 200 ng/mL of DTX and 500 ng/mL of ALA. The method was further successfully applied in lipid-based formulation characterization. In conclusion, a simple, accurate and precise reverse-phase HPLC-UV method was established for simultaneous determination of DTX and ALA in nanoformulations.

Facile derivatization of ultratrace carboxylic acids in saliva for quantification by HPLC-MS/MS.

It remains an issue to directly quantify trace biologically important carboxyl compounds in body fluids. Herein we propose an innovative method to determine α-lipoic acid, 2-(ß-carboxyethyl)-6-hydroxy-2,7,8-trimethylchroman, prostaglandin E2, cholic acid, and chenodeoxycholic acid in saliva. The method consists of two successive steps: fast and direct labeling of the target analytes with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide followed by ultrahigh-performance liquid chromatography-tandem mass spectrometry analysis. The method exhibited a wide linear range from 2.5 to 2500 pg/mL, with linear coefficients greater than 0.9963 and limits of detection and quantification as low as 0.10 and 0.33 pg/mL, respectively. The method precision was evaluated, with relative standard deviations ranging from 2.12% to 10.63% for intraday assays and from 2.98% to 12.88% for interday assays. The recoveries were measured by our spiking saliva samples with standards at three different levels, and ranged from 72.5% to 98.0%. Real applicability was validated by direct quantification of trace target analytes in human saliva, with simple pretreatment, use of a small sample volume, and a short analysis time. Graphical abstract Sequential steps to extract, label, and determine the ultratrace carboxylic acids in saliva. CDCA chenodeoxycholic acid, γ-CEHC 2-(ß-carboxyethyl)-6-hydroxy-2,7,8-trimethylchroman, α-LA α-lipoic acid, PGE2 prostaglandin E2, UHPLC-MS/MS ultrahigh-performance liquid chromatography-tandem mass spectrometry.

Least median of squares and iteratively re-weighted least squares as robust linear regression methods for fluorimetric determination of α-lipoic acid in capsules in ideal and non-ideal cases of linearity.

This study outlines two robust regression approaches, namely least median of squares (LMS) and iteratively re-weighted least squares (IRLS) to investigate their application in instrument analysis of nutraceuticals (that is, fluorescence quenching of merbromin reagent upon lipoic acid addition). These robust regression methods were used to calculate calibration data from the fluorescence quenching reaction (∆F and F-ratio) under ideal or non-ideal linearity conditions. For each condition, data were treated using three regression fittings: Ordinary Least Squares (OLS), LMS and IRLS. Assessment of linearity, limits of detection (LOD) and quantitation (LOQ), accuracy and precision were carefully studied for each condition. LMS and IRLS regression line fittings showed significant improvement in correlation coefficients and all regression parameters for both methods and both conditions. In the ideal linearity condition, the intercept and slope changed insignificantly, but a dramatic change was observed for the non-ideal condition and linearity intercept. Under both linearity conditions, LOD and LOQ values after the robust regression line fitting of data were lower than those obtained before data treatment. The results obtained after statistical treatment indicated that the linearity ranges for drug determination could be expanded to lower limits of quantitation by enhancing the regression equation parameters after data treatment. Analysis results for lipoic acid in capsules, using both fluorimetric methods, treated by parametric OLS and after treatment by robust LMS and IRLS were compared for both linearity conditions.

Low-cost and disposable sensors for the simultaneous determination of coenzyme Q10 and α-lipoic acid using manganese (IV) oxide-modified screen-printed graphene electrodes.

In this work, for the first time, manganese (IV) oxide-modified screen-printed graphene electrodes (MnO2/SPGEs) were developed for the simultaneous electrochemical detection of coenzyme Q10 (CoQ10) and α-lipoic acid (ALA). This sensor exhibits attractive benefits such as simplicity, low production costs, and disposability. Cyclic voltammetry (CV) was used to characterize the electrochemical behavior of the analyte and investigate the capacitance and electroactive surface area of the unmodified and modified electrode surfaces. The electrochemical behavior of CoQ10 and ALA on MnO2/SPGEs was also discussed. Additionally, square wave anodic stripping voltammetry (SWASV) was used for the quantitative determination of CoQ10 and ALA. Under optimal conditions, the obtained signals are linear in the concentration range from 2.0 to 75.0 µg mL-1 for CoQ10 and 0.3-25.0 µg mL-1 for ALA. The low limits of detection (LODs) were found to be 0.56 µg mL-1 and 0.088 µg mL-1 for CoQ10 and ALA, respectively. Moreover, we demonstrated the utility and applicability of the MnO2/SPGE sensor through simultaneous measurements of CoQ10 and ALA in dietary supplements. The sensor provides high accuracy measurements, exhibiting its high potential for practical applications.

Effects of the supplementation with alpha-lipoic acid on muscular antioxidant biomarkers of trained mice / Efeitos da suplementação com ácido alfa-lipóico sobre biomarcadores antioxidantes musculares de camundongos treinados

BACKGROUND: Performing high intensity or exhaustive exercise can lead to muscle damage such as injuries, chronic fatigue and overtraining, partly due to the high synthesis of reactive oxygen species. The α-lipoic acid (ALA) and its reduced form, dihydrolipoic acid, act as potent antioxidant and eliminate free radicals. Although this response depends on the type of exercise and supplementation, animal and human studies have shown the benefits of antioxidant supplementation on the recovery of damages caused by exhaustive exercise, either by restoring antioxidant levels or by decreasing the damage. OBJECTIVE: We evaluated the effect of ALA supplementation on muscular biomarkers of oxidative stress following exhaustive exercise of trained mice. METHODS: Sixty mice were trained to swim for 6 weeks. On the last week, half of the animals were supplemented daily with 100 mg/kg of oral gavage of ALA in soy oil as a vehicle. The other half received just the vehicle. On the last day 20 animals from each group were submitted to an exhaustion protocol with 10% overweight attached to tail. Animals were euthanized on 3 moments: basal, just after the exhaustive protocol (0 h) and, 4 h after the exhaustive protocol. The gastrocnemius muscle was promptly excised and homogenized. The homogenates were used to estimate oxidative stress biomarkers. RESULTS: There was a simultaneous decrease of non-protein thiols and vitamin E after 4 h of exhaustive exercise in the ALA group (p<0.05) compared to the control group, suggesting the consumption of these compounds in the process of lipid peroxidation. Interestingly, there was an increase of nitrate and nitrite in ALA group (p<0.05) and a decrease in the control (p<0.05) compared to basal moment, possibly by activation of endothelial nitric oxide synthase. The total antioxidant capacity remained unchanged in the ALA group. CONCLUSION: The supplementation with ALA resulted in a protection against oxidative stress caused by exhaustive exercise.

CONTEXTO: A realização de exercício de alta intensidade ou exaustivo pode levar a danos musculares, como lesões, fadiga crônica e overtraining, em parte devido à alta síntese de espécies reativas de oxigênio. O ácido α-lipóico e sua forma reduzida, o ácido dihidrolipóico, atuam como potentes antioxidantes e eliminam os radicais livres. Apesar de depender do tipo de exercício e suplementação, estudos com animais e humanos mostram benefícios da suplementação com antioxidante na recuperação de danos causados pelo exercício exaustivo, seja restaurando os níveis de antioxidantes ou diminuindo os danos. OBJETIVO: Avaliar o efeito da suplementação com ácido α-lipóico sobre biomarcadores musculares de estresse oxidativo após o exercício exaustivo de camundongos treinados. METODOLOGIA: Os camundongos (n = 60) foram treinados em natação por 6 semanas. Na última semana, metade dos animais foram suplementados diariamente com gavagem oral de 100 mg / kg de ácido α-lipóico em óleo de soja como veículo. A outra metade recebeu apenas o veículo. No último dia 20 animais de cada grupo foram submetidos ao protocolo de exaustão com 10% de sobrepeso atado à cauda. Os animais foram eutanasiados em 3 momentos: basal, logo após o protocolo de exaustão (0 h) e 4 h após o protocolo de exaustão. O músculo gastrocnêmio foi imediatamente coletado e homogeneizado. Os homogeneizados foram usados para acessar os biomarcadores de estresse oxidativo. RESULTADOS: Houve diminuição simultânea de tióis não protéicos e vitamina E após 4 h de exercício exaustivo no grupo ácido α-lipóico (p <0,05) em relação ao grupo controle, sugerindo o consumo destes compostos no processo de peroxidação lipídica. Interessantemente, houve aumento de nitrato e nitrito no grupo ácido α-lipóico (p <0,05) e diminuição no controle (p <0,05) em relação ao momento basal, possivelmente pela ativação da óxido nítrico sintase endotelial. A capacidade antioxidante total permaneceu inalterada no grupo ácido α-lipóico. CONCLUSÃO: A suplementação com ácido α-lipóico resultou em proteção contra o estresse oxidativo causado pelo exercício exaustivo.

Development of Sensitive Analytical Approach for the Quantification of α-Lipoic Acid Using Boron Doped Diamond Electrode.

A boron doped diamond (BDD) electrode was investigated for use as an electrochemical sensor for α-lipoic acid (LA) using amperometric and differential pulse voltammetric detection. LA displays a well expressed oxidation peak at +0.9 V vs. Ag/AgCl in solutions with a pH value of 3. It was found that signals obtained are linearly related to the concentration range from 0.3 to 105 µM with detection limit of 0.088 µM. Interferences by common compounds such as ascorbic acid, uric acid and dopamine were tested and the method was successfully applied to the determination of LA in human body fluids where it gave recoveries in the range from 95 to 97%.

The Protective Effects of 5-Methoxytryptamine-α-lipoic Acid on Ionizing Radiation-Induced Hematopoietic Injury.

Antioxidants are prospective radioprotectors because of their ability to scavenge radiation-induced reactive oxygen species (ROS). The hematopoietic system is widely studied in radiation research because of its high radiosensitivity. In the present study, we describe the beneficial effects of 5-methoxytryptamine-α-lipoic acid (MLA), which was synthesized from melatonin and α-lipoic acid, against radiation-induced hematopoietic injury. MLA administration significantly enhanced the survival rate of mice after 7.2 Gy total body irradiation. The results showed that MLA not only markedly increased the numbers and clonogenic potential of hematopoietic cells but also decreased DNA damage, as determined by flow cytometric analysis of histone H2AX phosphorylation. In addition, MLA decreased the levels of ROS in hematopoietic cells by inhibiting NOX4 expression. These data demonstrate that MLA prevents radiation-induced hematopoietic syndrome by increasing the number and function of and by inhibiting DNA damage and ROS production in hematopoietic cells. These data suggest MLA is beneficial for the protection of radiation injuries.

Quantification of lipoic acid from skin samples by HPLC using ultraviolet, electrochemical and evaporative light scattering detectors.

Lipoic acid (LA) is an endogenous organosulfur compound with potent antioxidant property. LA is often used as a drug for the treatment of skin disorders. For the accomplishment of topical applications of LA appropriate drug quantification methods are essential. Thus far, no HPLC methods have been reported for the measurement of LA extracted from skin. In this article we report on the development and validation of three sensitive and specific HPLC methods for LA and dihydrolipoic acid (DHLA) using ultraviolet (UV), electrochemical (EC) or evaporative light scattering (ELS) detection. These methods demonstrate different linearity ranges. The chromatographic separations were performed by RP-HPLC (250 × 4 mm, 5 µm) with isocratic elution using an acidic mobile phase for the three detection techniques. The lower limits of detection and quantification were 0.04 and 0.08 ng LA, respectively, for HPLC coupled to ELS, an innovative detector for LA with high sensitivity. The extraction of LA from skin samples showed recoveries greater than 71%. The recovered LA concentrations from stratum corneum and epidermis+dermis layers were: 5.41 ± 0.56 and 4.92 ± 0.33 µg/mL, respectively for HPLC/UV and 6.52 ± 0.49 and 5.01 ± 0.41 µg/mL, respectively, for HPLC/EC for the added LA concentration (6.67 µg/mL), and 8.88 ± 0.46 and 8.95 ± 0.08 µg/mL, respectively, for HPLC/ELS for the added LA concentration (10 µg/mL). These three optimized HPLC methods allowed for a simple, rapid and reliable determination of LA in human skin. They should be useful for the development of drug delivery systems for topical applications of LA.

Gas-saturated solution process to obtain microcomposite particles of alpha lipoic acid/hydrogenated colza oil in supercritical carbon dioxide.

Alpha lipoic acid (ALA), an active substance in anti-aging products and dietary supplements, need to be masked with an edible polymer to obscure its unpleasant taste. However, the high viscosity of the ALA molecules prevents them from forming microcomposites with masking materials even in supercritical carbon dioxide (scCO2). Therefore, the purpose of this study was to investigate and develop a novel production method for microcomposite particles for ALA in hydrogenated colza oil (HCO). Microcomposite particles of ALA/HCO were prepared by using a novel gas-saturated solution (PGSS) process in which the solid-dispersion method is used along with stepwise temperature control (PGSS-STC). Its high viscosity prevents the formation of microcomposites in the conventional PGSS process even under strong agitation. Here, we disperse the solid particles of ALA and HCO in scCO2 at low temperatures and change the temperature stepwise in order to mix the melted ALA and HCO in scCO2. As a result, a homogeneous dispersion of the droplets of ALA in melted HCO saturated with CO2 is obtained at high temperatures. After the rapid expansion of the saturated solution through a nozzle, microcomposite particles of ALA/HCO several micrometers in diameter are obtained.

Estudo do efeito do Mito-TEMPO e do ácido lipoico sobre a cardiotoxicidade da dexorrubicina / Study of the effect of weather and Mito-lipoic acid on cardiotoxicity dexorrubicina

Introdução: A doxorrubicina (DOX) é um quimioterápico antracíclico amplamente usado para o tratamento de diversos tumores humanos, entretanto, o desenvolvimento de reações adversas à droga, em particular, cardiotoxicidade, tem limitado seu uso. Embora a toxicidade cardíaca induzida pela DOX pareça ser multifatorial, a hipótese mais investigada tem sido a formação de espécies reativas de oxigênio (ROS) e há evidências apontando para as mitocôndrias cardíacas como alvos primários da toxicidade da DOX. Esse dano oxidativo pode iniciar peroxidação lipídica e pode ser potencialmente limitado pelo uso de antioxidantes. Objetivo: O objetivo do presente estudo foi avaliar a possível eficácia do ácido lipoico (AL) e do Mito-TEMPO (Mito-T) como agentes protetores contra a cardiotoxicidade induzida pela DOX in vitro e in vivo e investigar se essa proteção pode afetar a atividade antitumoral da DOX. Método e Resultados: A capacidade do AL e Mito-T eliminar radicais livres foi avaliada usando o teste do 2,2-difenil-1-picril-hidrazila (DPPH). Menor atividade antioxidante do AL (29%) comparada ao Mito-T (63%) foi observada. DOX reduziu a viabilidade de células H9c2 (CI50 = 40,83 M, IC 95% = 28,64 – 58,21 M) e aumentou a concentração de malondialdeído (MLDA), um marcador de peroxidação lipídica, confirmando a citotoxicidade induzida pela DOX in vitro. O pré-tratamento com AL ou Mito-T não promoveu proteção contra o dano induzido pela DOX in vitro. Uma única injeção intraperitoneal (i.p.) de DOX (24 mg/kg de peso corpóreo) induziu redução significante no peso corpóreo (p<0,001), elevação da atividade sérica total de creatina quinase (p<0,05) e creatina quinase-MB (p<0,05), aumento na concentração de malondialdeído em mitocôndrias (p<0,05) e tecido cardíaco (p<0,01) em camundongos da linhagem C57BL/6 após 48 horas. O pré-tratamento dos animais com Mito-T (5 mg/kg de peso corpóreo, i.p., por dois dias, 48 e 24 horas antes da DOX) reduziu significativamente a peroxidação lipídica de mitocôndrias cardíacas (p<0,01) indicando o direcionamento do antioxidante para a mitocôndria. O tratamento com Mito-T ou AL, duas vezes, 24 e uma hora antes do tratamento com DOX, inibiu a atividade sérica de creatina quinase total (p<0,05). Além disso, o tratamento de camundongos apresentando tumor B16F10 com AL não interferiu na eficácia antitumoral da DOX. Conclusão: Os dados sugerem que a combinação de AL com DOX pode ser benéfica para o tratamento de câncer, entretanto, são necessárias novas investigações para confirmar essa suposição.

Introduction: Doxorubicin (DOX) is an anthracycline chemotherapeutic that is widely used for the treatment of many human tumours, however, the development of adverse drug reactions in particular cardiotoxicity has limited its use. Although doxorubicin-induced cardiac toxicity appears to be multifactorial, the most thoroughly investigated hypothesis has been the formation of reactive oxygen species (ROS) and there is evidence pointing to cardiac mitochondria as primary targets of the toxicity of DOX. This oxidative injury can initiate lipidic peroxidation and may be potentially limited by the use of antioxidants. Aim: The aim of the present study was to evaluate the possible efficacy of lipoic acid (LA) and Mito-TEMPO (Mito-T) as a protective agent against DOX-induced cardiotoxicity in vitro and in vivo and to investigate whether this protection may affect the antitumor activity of DOX. Method and Results: Free radical scavenging capacity of LA and Mito-T was assayed using 1,1-diphenyl-2-picrylhydrazy (DPPH) assay. Lower antioxidant activity for LA (29%) compared to Mito-T (63%) were observed. DOX reduced H9c2 viability (IC50 = 40.83 M, 95% CI = 28.64 – 58.21 M) and increased the levels of malondialdehyde (MLDA), a marker of lipid peroxidation, confirming DOX-induced cytotoxicity in vitro. Pretreatment with LA or Mito-T did not provide protection against DOX-induced damage in vitro. A single intraperitoneal (i.p.) injection of DOX (24 mg/kg body weight) induced a significant reduction in body weight (p<0.001), elevation of serum activity of total creatine kinase (p<0.05) and creatine kinase-MB (p<0.05), increase in malondialdehyde levels in cardiac mitochondria (p<0.05) and cardiac tissue (p<0.01) in C57BL/6 mice after 48 hours...

Chemical derivatization combined with capillary LC or MALDI-TOF MS for trace determination of lipoic acid in cosmetics and integrated protein expression profiling in human keratinocytes.

Lipoic acid (LA) is an essential cofactor in mitochondrial enzymes and an ideal antioxidant in prokaryotic and eukaryotic cells. Capillary liquid chromatography coupled with ultraviolet detection (CapLC-UV) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) are two environmentally friendly methods for determining LA. In this study, a pre-column microwave-assisted derivatization with 4-bromomethyl-6,7-dimethoxycoumarin enhanced the UV absorbance of LA and was monitored at 345 nm by CapLC-UV. Gradient separation was performed using a reversed-phase C18 column with a mobile phase consisting of acetonitrile-0.1% formic acid solution. The ionization of LA was increased, and the LA derivative was detected by MALDI-TOF MS at m/z 683 with an α-cyano-4-hydroxycinnamic acid matrix. The linear response ranged from 0.1 to 40 µM with a correlation coefficient of 0.999. The CapLC-UV and MALDI-TOF MS had detection limits of 5 and 4 fmol, respectively. These methods effectively detected LA in dietary supplements and cosmetics. Cellular proteomes of a human keratinocyte cell line (HaCaT) irradiated with UV radiation were also compared with and without LA treatment. The cellular proteomes were identified by nanoultra performance LC with LTQ Orbitrap system after trypsin digestion. Protein identification was performed by simultaneous peptide sequencing and MASCOT search. The analysis revealed changes in several proteins, including CDC42, TPI1, HNRPA2B1, PRDX1, PTGES3 and MYL6.

Chemistry of conjugation to gold nanoparticles affects G-protein activity differently.

BACKGROUND: Gold nanoparticles (AuNP) are extensively used as biophysical tools in the area of medicine and technology due to their distinct properties. However, vivid understanding of the consequences of biomolecule-nanomaterial interactions is still lacking. In this context, we explore the affect of conjugation of Gαi1 subunit (of heterotrimeric G-proteins) to AuNP and examine its consequences. We consider two bio-conjugation strategies covalent and non-covalent binding. RESULTS: Affinity of the AuNP to the Gαi1 is 7.58 × 10 12 M-1. AuNP conjugated Gαi1 exhibits altered kinetics of activation, non-covalent bio-conjugates displays retarded kinetics, up to 0.88 fold when GTPγS was used as ligand, of protein activation contrary to covalent conjugates which accelerates it to ~ 5 fold. Conjugation influence intrinsic Gαi1 GTPase function in conflicting modes. Non-covalent conjugation inhibits GTPase function (decrease in activity upto 0.8 fold) whilst covalent conjugation drastically accelerates it (12 fold increase in activity). Altered basal nucleotide uptake in both types of conjugates and GTPase function in non-covalent conjugate are almost comparable except for GTPase property of covalent conjugate. The effect is despite the fact that conjugation does not change global conformation of the protein. CONCLUSION: These findings provide clear evidence that nanoparticles, in addition to 'passive interaction' with protein (biomolecule), can interact "actively" with biomolecule and modify its function. This concept should be considered while engineering nanoparticle based delivery systems in medicine.

Determination of lipoic acid by flow-injection and high-performance liquid chromatography with chemiluminescence detection.

A new flow-injection (FI) and high performance liquid chromatography (HPLC) with chemiluminescence detection method has been proposed for the determination of α-lipoic acid (LA). The assay is based on the measurement of chemiluminescence (CL) produced during the reaction of α-lipoic acid with potassium permanganate in a sodium hexametaphosphate medium (pH 3). This reaction is accompanied by a weak CL, which is greatly increased in the presence of a formaldehyde solution. The proposed FI method allows the determination of LA over the range: 0.5-20µgmL(-1) with LOD 4×10(-3)µgmL(-1). An introduction of HPLC into the flow manifold improves selectivity of the method and allows the determination of LA in a complex sample. The chromatographic linear range is 2.5-30µgmL(-1) with LOD 1.774µgmL(-1). Chromatographic separation was achieved by isocratic elution (acetonitrile/potassium dihydrogen phosphate, pH 3, adjusted with phosphoric acid): 30/70 using a Cosmosil 5C(18)-MS-II (4.6mm×150mm I.D.) column at a flow rate of 1.0mLmin(-1). The presented methods were utilized to determine the α-lipoic acid content in "Alfa-lipoic acid" capsules and in food products.