High-performance Liquid Chromatography System Research Articles

Characterization and antimicrobial potential of bioactive components of sonicated extract from garlic ( Allium sativum ) against foodborne pathogens

Garlic (Allium sativum) belongs to Liliaceae family. Plants belonging to this family are well known for their sulfur compounds besides phenolic substances. The current study was planned to explore the potential of sonication treatment combined with various solvents for extracting bioactive components (phenolics and allicin) of two local garlic varieties that is garlic white and garlic pink and to elucidate the antimicrobial potential of extracts against selected pathogens. Total phenolic contents in various extracts ranged 57.35 ± 4.77–86.18 ± 1.53mg GAE/100 g. Allicin as characterized through high-performance liquid chromatographic system was found in the range of 62.67 ± 13.49–425.6 ± 3.04 ppm. The minimum inhibitory concentration of garlic extracts as calculated by the broth dilution method ranged 25–133 mg/ml and biofilm formation was 45–82%. On an overall basis, garlic pink indicated higher bioactive components, antimicrobial potential against foodborne pathogens, and biofilm formation inhibition. Regarding solvents extracts with 50% methanol, as extraction solvent, indicated better results trailed by 50% ethanol and water. Practical applications Garlic extract is rich in bioactive moieties especially Allicin, other organosulfur and phenolics, having antioxidant and antimicrobial potential which can exert beneficial effects on human health. Antimicrobial potential of garlic extract can be utilized for killing foodborne pathogens and thus achieving food safety. Its use in meat, meat products, and fish can significantly reduce the pathogens associated with these food items. Moreover, both antioxidant and antimicrobial properties offer bio-preservation effect to these products and extend the shelf life. Biofilm formation inhibition properties of garlic extract can be utilized in food processing and preservation as well in food processing equipment. Further, garlic extracts can be used for preparation of nanoparticles with enhanced efficiency.

Neurochemical effects of motor cortex stimulation in the periaqueductal gray during neuropathic pain.

Motor cortex stimulation (MCS) is a neurosurgical technique used to treat patients with refractory neuropathic pain syndromes. MCS activates the periaqueductal gray (PAG) matter, which is one of the major centers of the descending pain inhibitory system. However, the neurochemical mechanisms in the PAG that underlie the analgesic effect of MCS have not yet been described. The main goal of this study was to investigate the neurochemical mechanisms involved in the analgesic effect induced by MCS in neuropathic pain. Specifically, we investigated the release of γ-aminobutyric acid (GABA), glycine, and glutamate in the PAG and performed pharmacological antagonism experiments to validate of our findings. Male Wistar rats with surgically induced chronic constriction of the sciatic nerve, along with sham-operated rats and naive rats, were implanted with both unilateral transdural electrodes in the motor cortex and a microdialysis guide cannula in the PAG and subjected to MCS. The MCS was delivered in single 15-minute sessions. Neurotransmitter release was evaluated in the PAG before, during, and after MCS. Quantification of the neurotransmitters GABA, glycine, and glutamate was performed using a high-performance liquid chromatography system. The mechanical nociceptive threshold was evaluated initially, on the 14th day following the surgery, and during the MCS. In another group of neuropathic rats, once the analgesic effect after MCS was confirmed by the mechanical nociceptive test, rats were microinjected with saline or a glycine antagonist (strychnine), a GABA antagonist (bicuculline), or a combination of glycine and GABA antagonists (strychnine+bicuculline) and reevaluated for the mechanical nociceptive threshold during MCS. MCS reversed the hyperalgesia induced by peripheral neuropathy in the rats with chronic sciatic nerve constriction and induced a significant increase in the glycine and GABA levels in the PAG in comparison with the naive and sham-treated rats. The glutamate levels remained stable under all conditions. The antagonism of glycine, GABA, and the combination of glycine and GABA reversed the MCS-induced analgesia. These results suggest that the neurotransmitters glycine and GABA released in the PAG may be involved in the analgesia induced by cortical stimulation in animals with neuropathic pain. Further investigation of the mechanisms involved in MCS-induced analgesia may contribute to clinical improvements for the treatment of persistent neuropathic pain syndromes.

청국장 중 바이오제닉 아민 및 무기원소함량 분석

The quantitative analyses of biogenic amines (BAs) in fermented food is one of the critical issues in food safety. Thirty-seven samples were analyzed in order to determine the BA content in Cheonggukjang. Additionally inorganic elements in 6 samples were analyzed using inductively coupled plasma atomic emission spectroscopy and inductively coupled plasma mass spectrometry. The BA content was analyzed using high-performance liquid-chromatography (HPLC) after derivatization of the samples by densyl chloride. Biogenic amines, namely, tryptamine (TRP), 2-phenylethylamine (PHE), putrescine (PUT), cadaverine (CAD), histamine (HIS), tyramine (TYR) spermidine (SPD), and spermine (SPM) were detected by monitoring the absorbance from the samples at 254 nm using the UV-vis detector attached to the HPLC system. The chromatograms were also verified for linearity and accuracy. The results showed that all the correlation coefficients (R2) were over 0.99, while the recovery was 89.7% -103.4%. The average BA contents in Cheonggukjang were as follows: TRP 43.8 mg/kg, PHE 11.8 mg/kg, PUT 10.6 mg/kg, CAD 10.3 mg/kg, HIS 3.8 mg/kg, TYR 113.7 mg/kg, SPD 37.6 mg/kg, SPM 11.5 mg/kg. The average contents of inorganic elements in Cheonggukjang were as follows Na 0.93%, K 0.55%, Ca 0.14%, P 0.30%, Mg 0.11%, S 0.17%, Fe 29.6 mg/kg, Mn 13.4 mg/kg, Zn 17.6 mg/kg, Cu 3.44 mg/kg, Sr 3.21 mg/kg, Mo 1.40 mg/kg, Ba 2.12 mg/kg, Se 0.071 mg/kg, Cd 0.014 mg/kg, As 0.011 mg/kg.

Classification of barberry genotypes by multivariate analysis of biochemical constituents and HPLC profiles.

Recently, there has been a growing interest in the use of edible barberry and their extracts as a source of natural antioxidants in food and pharmaceutical industries. The aim of this study was to evaluate the biochemical constituents of 18 samples of barberry fruits and classification of barberry genotypes by multivariate analysis. Total phenolic, total flavonoid, total anthocyanin, total tannin, total carbohydrate contents and antioxidant activity were determined using Folin-Ciocalteu, aluminum chloride, colorimetric, vanillin, anthron and DPPH (2,2'-diphenyl-1-picrylhydrazyl) assays, respectively. High-performance liquid chromatography (HPLC) system is used for quantitative determination of phytochemical constituents. The multivariate data analysis (principal component analysis and hierarchical cluster analysis) and heat map data visualisation techniques were performed to classify barberry genotypes using Minitab and GraphPad Prism software, respectively. The highest amounts of total phenolics and flavonoids were obtained in fruit extracts of G3 (Berberis vulgaris). The highest total anthocyanin content and antioxidant activity were observed in G8 (B. vulgaris) and G16 (B. vulgaris), respectively. HPLC analysis of phytochemicals (gallic acid, caffeic acid, chlorogenic acid, p-coumaric acid, cinnamic acid, rutin, apigenin, and quercetin) revealed that gallic acid and p-coumaric acid were found as the most abundant phytochemical compounds. Based on multivariate analysis and heat map visualisation techniques, Berberis genotypes were classified into three main clusters. These results showed that barberry species (especially B. vulgaris and B. carataegina) are promising sources of natural antioxidants and biochemical compounds beneficial to be used in the food industry and that the multivariate analysis was a suitable approach to classify the barberry samples.

Enhanced Production and Quantitative Evaluation of Nigericin from the Algerian Soil-Living Streptomyces youssoufiensis SF10 Strain

Nigericin, one of the main ionophoric polyethers produced by various Streptomyces strains, presents relevant biological activities including antibacterial and recently studied antitumor properties. This work describes the influence of different culture conditions on the production of this metabolite by Streptomyces sp. SF10, isolated from a semi-arid soil sample collected at Chélia Mountain, in Khenchela (Northeastern Algeria) and identified as Streptomyces youssoufiensis. The extracts from the strain, cultured in a solid state or submerged fermentation conditions, using several carbon sources at different pH values, in the presence or absence of iron (II) sulfate and in co-culture with other Streptomyces species, were analyzed using a high-performance liquid chromatography (HPLC) system equipped with an evaporative light scattering detector (ELSD). The best culture conditions provided a concentration of nigericin of 0.490 ± 0.001 mg/mL in the extract. The HPLC-ELSD method, optimized here for the quantitative detection of nigericin, can find wider applications in the analysis of several other metabolites characterized by a similar polycyclic polyether structure or, more generally, by the lack of significant chromophores in their molecular structure.

Reverse phase high-performance liquid chromatography (RP-HPLC) system for analysis of vitamin D3 soft capsules with soybean oil matrix

ObjectiveTo develop a method for the determination and impurity analysis of vitamin D3 soft capsules with soybean oil matrix in reverse phase high performance liquid chromatography (RP-HPLC) system. MethodsRP-HPLC system had Agilent Zorbax Eclipse XDB-C18 column (4.6 mm × 250 mm, 5 μm) with detection wavelength of 265 nm, column temperature of 25 ℃ and flow rate of 1 mL/min. Retention behaviors of vitamin D3 and its 3 isomers were studied by altering the mobile phase. Firstly, acetonitrile was mixed with different proportions of methanol, water and ethanol as the mobile phase to investigate the effects of these 4 mobile phase components on the retention behavior of vitamin D3 and its 3 main related substances (isomers) on a C18 column. Then, a suitable mobile phase was selected for content determination and impurity analysis according to the retention behavior study. ResultsThe recovery was only 80.55%-84.37% with 100% acetonitrile as the mobile phase.The addition of ethanol in acetonitrile was found to make remarkably significant improvement. Recovery rate was achieved between 98.07% and 103.23% with V (acetonitrile):V (ethanol)=90:10 as the mobile phase, while improving peak shape. The method showed good linearity [(0.52-5.2)×10-4 μmol/L, R2>0.999] and fine density (RSD < 2.32%) which can be used for determination. For impurities profile, it could be achieved using V (acetonitrile):V (water)=95:5 as the mobile phase which can obviate interference from soybean oil matrix. ConclusionsThe method established in this experiment can easily and accurately determine the content and impurity analysis of vitamin D3 soft capsules with soybean oil matrix in a RP-HPLC system.

Characterization of the Specific Mode of Action of a Chitin Deacetylase and Separation of the Partially Acetylated Chitosan Oligosaccharides.

Partially acetylated chitosan oligosaccharides (COS), which consists of N-acetylglucosamine (GlcNAc) and glucosamine (GlcN) residues, is a structurally complex biopolymer with a variety of biological activities. Therefore, it is challenging to elucidate acetylation patterns and the molecular structure-function relationship of COS. Herein, the detailed deacetylation pattern of chitin deacetylase from Saccharomyces cerevisiae, ScCDA2, was studied. Which solves the randomization of acetylation patterns during COS produced by chemical. ScCDA2 also exhibits about 8% and 20% deacetylation activity on crystalline chitin and colloid chitin, respectively. Besides, a method for separating and detecting partially acetylated chitosan oligosaccharides by high performance liquid chromatography and electrospray ionization mass spectrometry (HPLC-ESI-MS) system has been developed, which is fast and convenient, and can be monitored online. Mass spectrometry sequencing revealed that ScCDA2 produced COS with specific acetylation patterns of DAAA, ADAA, AADA, DDAA, DADA, ADDA and DDDA, respectively. ScCDA2 does not deacetylate the GlcNAc unit that is closest to the reducing end of the oligomer furthermore ScCDA2 has a multiple-attack deacetylation mechanism on chitin oligosaccharides. This specific mode of action significantly enriches the existing limited library of chitin deacetylase deacetylation patterns. This fully defined COS may be used in the study of COS structure and function.

Simultaneous Separation of Triacylglycerol Enantiomers and Positional Isomers by Chiral High Performance Liquid Chromatography Coupled with Mass Spectrometry.

The rapid and simultaneous separation of triacylglycerol (TAG) enantiomers and positional isomers was achieved using chiral high performance liquid chromatography (HPLC). TAGs composed of two fatty acids, which were both saturated (P: palmitic acid or S: stearic acid) and unsaturated (O: oleic acid or L: linoleic acid; e.g., sn-PPO/sn-OPP/sn-POP: 1,2-dipalmitoyl-3-oleoyl-sn-glycerol/1-oleoyl-2,3-dipalmitoyl-sn-glycerol/1,3-dilpalmitoyl-2-oleoylglycerol), were resolved into three peaks using CHIRALPAK IF-3 without recycling on the HPLC system. For example, the mixture of sn-PPO/sn-OPP/sn-POP was resolved in 30 min, although it took 150 min to resolve sn-PPO/sn-OPP using CHIRALCEL OD-RH in a previous study using a recycling HPLC system. This novel chiral HPLC method was applicable for the separation of other TAG isomers, including sn-OOP/sn-POO/sn-OPO, sn-PPL/sn-LPP/sn-PLP, sn-LLP/sn-PLL/sn-LPL, sn-SSO/sn-OSS/sn-SOS, sn-OOS/sn-SOO/sn-OSO, sn-SSL/sn-LSS/sn-SLS, and sn-LLS/sn-SLL/sn-LSL. For TAGs composed of three fatty acids containing both saturated and unsaturated fatty acids, the POL isomers were not sufficiently separated but the PSO and SOL isomers were partially separated into several peaks. Their elution order could be estimated by the fragment ions generated in the ion source of the mass spectrometer. However, TAGs consisting of only saturated or unsaturated fatty acids (e.g., sn-PSP/sn-PPS/sn-SPP and sn-OLO/sn-OOL/sn-LOO) were not separated. This novel chiral HPLC method is especially applicable for the analysis of TAG composition of semi-solid fats such as palm oil.

Determination of free nucleotides in infant formula by solid phase extraction-liquid chromatography

A method for the determination of five free nucleotides in infant formula was established. The analytes were extracted by ethylene diamine tetraacetic acid and sodium chloride solution, and purified by strong anion exchange (SAX) solid-phase extraction. An Atlantis T3 reverse-phase column was applied to separate the five free nucleotides. A high performance liquid chromatography (HPLC) system equipped with a diode array detector was applied to detect the target compound, and a series of external standards were used for quantification. Good linearities (r≥0.999) were observed, with the range of 0.5-10 mg/L for the five free nucleotides. When the addition levels were 0.05-0.50 g/kg, the recoveries were between 91.1% and 112%, and the precisions were between 2.3%-4.7%. The limits of detection (LODs) and the limits of quantification (LOQs) for the five free nucleotides ranged from 0.0010 to 0.0015 g/kg and from 0.0030 to 0.0045 g/kg. In conclusion, the proposed method has several merits. First, the high accuracy and precision indicated that the quality control sample had a relative deviation of less than 10% to the median. Second, perfect anti-disturbance was observed for the complex goat milk powder. Thus, this method can provide technical support for supervision departments and guarantees healthy development of the dairy industry.

Impact of solvent and supercritical fluid extracts of green tea on physicochemical and sensorial aspects of chicken soup

Designer foods carrying nutraceuticals or polyphenols are in fame to address varied free radical mediated disorders. In this context, green tea extract incorporation in designer products is considered as an effective approach to improve antioxidant capacity owing to epigallocatechin gallate (EGCG). In the current project, EGCG was extracted using solvent (acetone) and supercritical fluid (CO₂) extraction methods. Then, EGCG was quantified in the resultant extracts via high performance liquid chromatography (HPLC) system that showed an upper hand of EGCG in supercritical fluid extract (77.23 mg/g) as compared to acetonic extract (65.88 mg/g) at constant extraction temperature and time. In product development phase, different chicken soup prototypes were prepared such as S₀ (Control soup without green tea extract), S₁ (soup carrying 3% acetonic green tea extract) and S₂ (soup carrying 2.5% supercritical CO₂ green tea extract). The resultant products were then analyzed for physiochemical and sensory aspects during storage intervals; 0, 24, 48, 72 and 96 hr. Storage impacted significantly on some color values like a*, b* and chroma. The statistical analysis demonstrated obvious impact of treatments and storage on total soluble solids (TSS). Further, storage affected significantly on pH and acidity of the resultant samples. Green tea extract carrying products significantly improved TPC; S₁ (46.66 ± 2.39 mg GAE/100mL) and S₂ (49.19 ± 2.36 mg GAE/100mL) in contrast to control treatment (18.19 ± 0.89 mg GAE/100 mL). During storage, significant decline in TPC was noted from 39.43 ± 1.98 to 36.02 ± 1.82 mg GAE/100 mL. Moreover, considerable response of treatments was viewed on taste scores whilst, storage impacted remarkably on flavor, taste, texture and overall-acceptability, excluding organoleptic response regarding color. Conclusively, supercritical fluid green tea extract based designer chicken soup (S₂) has proven its relatively better antioxidants retention, storage stability and sensory profile in comparison to conventional solvent green tea extract based soup (S₁).

Simultaneous Determination of Kynurenine and Kynurenic Acid by High-Performance Liquid Chromatography Photoirradiation System Using a Mobile Phase Containing 18-crown-6.

A high-performance liquid chromatography (HPLC) system has been developed for the fluorometric determination of kynurenine (KYN) and kynurenic acid (KYNA) in human serum using a mobile phase containing 18-crown-6. A retention time of KYNA was adjusted with pH of phosphate buffer in 18-crown-6. KYN and KYNA were separated on a CAPCELLPAK C18 (250 × 4.6 mm i.d.). The mobile phase consisted of 35 mmol/L phosphate buffer (pH 8.0)/methanol (85/15, v/v) containing 35 mmol/L hydrogen peroxide and 10 mmol/L 18-crown-6. The retention times of KYN and KYNA were 18and 24 minutes, respectively. The calibration graphs of KYN and KYNA were linear over the range 180 to 2900 and 1 to 84 nmol/L by injecting a 50-μL volume of KYN and KYNA, respectively. Pretreatment of serum was achieved by deproteinization only. The mean recoveries of KYN and KYNA from serum were more than 97%.

Identification and Quantification of Carotenoids and Tocochromanols in Sorghum Grain by High-Performance Liquid Chromatography.

Carotenoids and tocochromanols are lipid-soluble secondary plant metabolites that are essential for the normal functioning of plants and, in some cases function as a source of vitamin A and E in humans. Enhancement of the provitamin A carotenoid and tocochromanols levels in sorghum and other cereal grains through traditional breeding and transgenic methods has increased in recent years with interest in biofortification of grains to combat micronutrient deficiencies in developing countries. With this increase in research, reliable methodology for the extraction, identification, and quantification of individual carotenoids and tocochromanols species from sorghum and other cereal grains is essential. Here, we describe a basic method for extraction of carotenoid and tocochromanols adapted to sorghum grain and chromatographic condition for separation, identification, and quantification of individual carotenoid using High-Performance Liquid Chromatography (HPLC) system.

Application of HPLC method in determination of miconazole nitrate in environmental samples from el-gharbia governorate in Egypt

This paper describes an enhanced High-performance liquid chromatography (HPLC) method for the analysis of miconazole in water samples. In this study, determination of miconazole has been carried out according to standard method for water and wastewater analysis. Samples of collected water were agriculture stream water, River Nile (Surface water samples) water and Hospital wastewater samples from El-gharbia governorate in Egypt. Miconazole was extracted by liquid-liquid extraction and analyzed by HPLC. The chromatographic separation was performed using a Phenomenex C8 column, flow rate of 0.8mL/min, and UV detection at 220nm. The optimized HPLC system was achieved using mobile phase composition containing methanol: water (85:15v/v). The intra-day and inter-day precisions were lower than 0.58 while the accuracy ranged from 99.06% to 101.53%. Finally, liquid-liquid phase extraction in combination with HPLC is a sensitive and effective method for the determination of Miconazole Nitrate in water samples. Miconazole was observed in some agricultural streams and waste water samples of El-gharbia governorate hospitals.

Zero electrical power pump for portable high-performance liquid chromatography.

A major trend in analytical chemistry is the miniaturization of laboratory instrumentation. We report a pump requiring no power to operate based on the controlled expansion of a pre-pressurised gas for use in portable applications of high-performance liquid chromatography. The performance of the gas pump is characterised and integrated into a compact liquid chromatography system capable of isocratic separations integrating an LED-based UV-absorption detector. The system weighed 6.7 kg when the mobile phase reservoir was fully charged with 150 mL solvent and included an on-board computer to control the system and analyse data. We characterise the flow-rate through chromatography columns with a variety of geometries and packing materials for a range of pressures up to 150 bar. The maximum variation in flow rate was measured to be 6.5 nL min-1, limited by the resolution of the flow detector. All tests were made on battery power and results are a mixture of those made in the laboratory and in the field. Additionally, we performed a series of 1 m drop tests on the device and show the system's high tolerance to mechanical shocks during operation in the field.

Low-cost customizable microscale toolkit for rapid screening and purification of therapeutic proteins.

Biopharmaceutical separations require tremendous amounts of optimization to achieve acceptable product purity. Typically, large volumes of reagents and biological materials are needed for testing different parameters, thus adding to the expense of biopharmaceutical process development. This study demonstrates a versatile and customizable microscale column (µCol) for biopharmaceutical separations using immobilized metal affinity chromatography (IMAC) as an example application to identify key parameters. µCols have excellent precision, efficiency, and reproducibility, can accommodate any affinity, ion-exchange or size-exclusion-based resin and are compatible with any high-performance liquid chromatography (HPLC) system. µCols reduce reagent amounts, provide comparable purification performance and high-throughput, and are easy to automate compared with current conventional resin columns. We provide a detailed description of the fabrication methods, resin packing methods, and µCol validation experiments using a conventional HPLC system. Finite element modeling using COMSOL Multiphysics was used to validate the experimental performance of the µCols. In this study, µCols were used for improving the purification achieved for granulocyte colony stimulating factor (G-CSF) expressed using a cell-free CHO in vitro translation (IVT) system and were compared to a conventional 1 ml IMAC column. Experimental data revealed comparable purity with a 10-fold reduction in the amount of buffer, resin, and purification time for the μCols compared with conventional columns for similar protein yields.

Detection and Quantification of Preservatives in Green Cosmetics

Abstract Preservatives are used in cosmetic formulations to provide protection against microbial growth to ensure the safety and the quality during their shelf life. Benzyl alcohol, sodium benzoate and sorbic acid are the preservatives that can be used in green cosmetic formulations. According to the cosmetic legislation usage of above indicated preservatives are restricted and thus determination and quantification of them from the cosmetic products have become important. In this study analytical methods are given to enable the determination of benzyl alcohol, sodium benzoate and sorbic acid. The methods are based on extracting the preservative agents from four different pharmaceutical form (solution, emulsion, gel and cream) of the various cosmetic matrices and later on detection in the scope of the basic chromatographic data and test parameters by High Performance Liquid Chromatography (HPLC) system with UV Detector.

Comparative study of aromatic and polyphenolic profiles of Croatian white wines produced by cold maceration

Aroma profiles and phenolic components of white wines made from ’Pošip’ and ’Škrlet’ (Vitis vinifera L.) both native Croatian, non-aromatic grape varieties enhances the knowledge about these varieties, prepared without maceration (control) and subjected to the pre-fermentative cold maceration (CM). Individual phenolic compounds in wines were determined by high-performance liquid chromatography (HPLC) system, while total phenolics and flavan-3-ols in wines were determined by spectrophotometric methods. CM had the significant impact on the total phenolic and flavan-3-ols composition of ’Škrlet’ wine. The significant increase of some individual phenolic compounds was observed in ’Pošip’ wine. Volatile aroma compounds in wines were determined by gas chromatography (GC) with the previous extraction on the solid phase micro extraction (SPME). The primary aroma compounds, i.e. terpenes were under the significant influence of CM process in ’Škrlet’ wine. Aroma profiles, based on the content of 24 specific compounds, grouped in eight aroma series that contribute to wine odor were developed. CM significantly increased five aroma series in ’Škrlet’ wine and only three in ’Pošip’ wine.

RP-HPLC-UV Method for Simultaneous Quantification of Second Generation Non-Steroidal Antiandrogens Along with their Active Metabolites in Mice Plasma: Application to a Pharmacokinetic Study.

A simple, specific and reproducible high-performance liquid chromatography (HPLC) assay method has been developed and validated for the quantitation of second generation antiandrogens and their active metabolites namely apalutamide, enzalutamide, N-desmethylenzalutamide (active metabolite of enzalutamide), darolutamide and ORM-15341 (active metabolite of darolutamide) in mice plasma. The method involves extraction of apalutamide, enzalutamide, N-desmethylenzalutamide, darolutamide and ORM-15341 along with internal standard (IS) from 100 µL mice plasma through a simple protein precipitation process. The chromatographic analysis was performed on a Waters Alliance HPLC system using a gradient mobile phase (comprising 10 mM ammonium acetate and acetonitrile in a flow-gradient) and X-Terra Phenyl column. The UV detection wave length was set at λmax 250 nm. Apalutamide, enzalutamide, N-desmethylenzalutamide, darolutamide and ORM-15341 and the IS eluted at 13.6, 11.4, 9.68, 6.11, 6.93 and 4.69 min, respectively with a total run time of 15 min. Method validation was performed as per regulatory guidelines and the results met the acceptance criteria. The calibration curve was linear over a concentration range of 209 - 5215 ng/mL (r 2=0.998). The intra- and inter-day precisions were in the range of 0.56-13.5 and 1.04-13.9%, respectively. The validated HPLC method was successfully applied to a pharmacokinetic study in mice.

Optimization of L-arginine and thiotriazoline compound analysis by high-performance liquid chromatography method

Cardiovascular diseases are the leading cause of death and disability in most countries of the world. Therefore, the crucial task of pharmacy and medicine is the creation of drugs for the treatment of these pathologies. As a result of pharmacological studies, the optimal ratio of L-arginine with thiotriazoline – 4:1 was found. It was proposed to use modern, high-precision method of high-performance liquid chromatography (HPLC) to determine the active substances in the dosage form. Objective. The purpose of our study is to develop a method for the simultaneous determination of L-arginine and thiotriazoline standardization in model mixture by high-performance liquid chromatography, in particular the selection of the mobile and stationary phase. Materials and methods . L-arginine (manufactured by Sigma-Aldrich, China), thiotriazoline (manufactured by the State Enterprise Chemical Reagents Plant of the Scientific and Technological Complex Institute of Single Crystals of the National Academy of Sciences of Ukraine) was used for the study. The studies were carried out using a modular BISCHOFF HPLC system (BISCHOFF Analysentechnik GmbH, Germany) with a Lambda 1010 spectrophotometric detector. Results: At first, the determination was carried out on the reverse phase, with L-arginine practically not retained (coming out at the “dead volume”), and the use of cyanated phase did not significantly affect the retention of L-arginine. Further, the retention time of L-arginine increased slightly under ion-pair chromatography on the reversed phase, in determining and using the eluent with tetrabutylammonium. The most acceptable result was obtained by using an ion buffer reagent acid buffer – 0.05 % solution of trifluoroacetic acid. Conclusion. Thus, according to the obtained results, we believe that the use of an acid buffer – 0.05 % solution of trifluoroacetic acid is one of the promising ways for the simultaneous determination of L-arginine with thiotriazoline in the model mixture along with the ion-pair reagent. In the future, we are going to use this technique for the standardization of a combined drug which includes L-arginine and thiotriazoline, in solid and liquid dosage forms.

The occurrence of acetaminophen/codeine as an adulterant in herbal analgesic supplements in Hamadan, Iran: A pilot study

Analgesics, such as acetaminophen (APAP) and codeine (COD), are used to adulterate medicinal herbs and/or herbal supplements. This study evaluated the APAP and COD levels in 60 herbal supplement and/or herb-based medicine samples collected from apothecaries in Hamadan, Iran. The samples were analysed using a high-performance liquid chromatography (HPLC) system. The results showed that 15% of the samples contained 38900–165200 ng/g and 31.1–603.3 ng/g of APAP and COD, respectively. Due to the side-effects of analgesic drugs in human, control of these drugs is recommended in herbal supplements.