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.