High-Performance Liquid Chromatography Methods for Determining the Purity of Drugs with Weak UV Chromophores - A Review.

High-performance liquid chromatography (HPLC) is one of the most useful techniques for the separation and determination of new drugs with a complex nature. The selection of an HPLC detector depends on the chemical nature of molecules, potential impurities, matrix of the sample, sensitivity, availability, and/or cost of the detector. HPLC methods with UV/Vis detectors are the most used and simple analytical procedures in pharmaceutical applications, but it is limited to compounds that possess a chromophore. Hence, this review provides an overview on the development of analytical methods for compounds with weak chromophores. The review described selected papers about HPLC based methods in the PubMed, Scopus, Semantic Scholar and ScienceDirect databases, basically between 2006 and 2023. Of the analytical studies, the HPLC methods with UV-Vis, FLD, CAD, ELSD, RID, ECD, CLND and MS detection were found. This study is a comparison of different types of detection that are described in scientific literature and are routinely used for compounds with weak chromophores. It is expected that this review will be helpful for scientists in the analytical development fields to improve research related to the drug candidates and to ensure its quality according to regulatory levels.

Apigenin Modulates AnxA6- and TNAP-Mediated Osteoblast Mineralization.

Mineralization-competent cells like osteoblasts and chondrocytes release matrix vesicles (MVs) which accumulate Ca2+ and Pi, creating an optimal environment for apatite formation. The mineralization process requires the involvement of proteins, such as annexins (Anx) and tissue-nonspecific alkaline phosphatase (TNAP), as well as low molecular-weight compounds. Apigenin, a flavonoid compound, has been reported to affect bone metabolism, but there are doubts about its mechanism of action under physiological and pathological conditions. In this report, apigenin potency to modulate annexin A6 (AnxA6)- and TNAP-mediated osteoblast mineralization was explored using three cell lines: human fetal osteoblastic hFOB 1.19, human osteosarcoma Saos-2, and human coronary artery smooth muscle cells HCASMC. We compared the mineralization competence, the morphology and composition of minerals, and the protein distribution in control and apigenin-treated cells and vesicles. The mineralization ability was monitored by AR-S/CPC analysis, and TNAP activity was determined by ELISA assay. Apigenin affected the mineral structure and modulated TNAP activity depending on the concentration. We also observed increased mineralization in Saos-2 cells. Based on TEM-EDX, we found that apigenin influenced the mineral composition. This flavonoid also disturbed the intracellular distribution of AnxA6 and TNAP, especially blocking AnxA6 aggregation and TNAP attachment to the membrane, as examined by FM analysis of cells and TEM-gold analysis of vesicles. In summary, apigenin modulates the mineralization process by regulating AnxA6 and TNAP, as well as through various effects on normal and cancer bone tissues or atherosclerotic soft tissue.

Antixenosis in Glycine max (L.) Merr against Acyrthosiphon pisum (Harris).

To reveal the antixenosis potential against the pea aphid Acyrthosiphon pisum (Harris) (Hemiptera: Aphididae) we analyzed the pea aphid survival and probing behavior, and the quantitative and qualitative variation of flavonoids in the leaves of selected soybean Glycine max (L.) Merr (Fabaceae) cultivars 'Aldana', 'Annushka', 'Augusta', 'Madlen', 'Mavka', 'Simona', 'Violetta', and 'Viorica'. Aphid survival was drastically impeded on all cultivars. The electronic monitoring of aphid probing using the Electrical Penetration Graph (EPG) technique revealed that on all soybean cultivars, A. pisum readily probed into leaf tissues but the probes were usually terminated before reaching vascular tissues, which demonstrates the activity of antixenosis mechanisms in peripheral tissues epidermis and/or mesophyll in soybean leaves. The potency of antixenosis factors differed among soybean cultivars, which was reflected in differences in aphid survival and frequency and duration of phloem sap ingestion. Seven flavonoids were found: apigenin, daidzein, genistein, glycitein, isorhamnetin, kaempferol, and rutin, which occurred in different amount and proportion in individual cultivars. The content of apigenin and genistein in all soybean cultivars studied probably made them relatively unacceptable to A. pisum. Kaempferol in 'Aldana' might be responsible for the observed strong antixenosis resistance of this cultivar to A. pisum. The results of our survey provide the first detailed data that can be used for future studies.

Determining Potassium Bromate in the Inhalable Aerosol Fraction in Workplace Air with Ion Chromatography.

BACKGROUND: The article presents the results of studies performed in order to develop a new method of airborne potassium bromate(V) determination at workplaces. METHODS: The method is based on a collection of the inhalable fraction of potassium bromate(V) using the IOM Sampler, then extraction of bromates with deionized water and chromatographic analysis of the obtained solution. The analysis was performed using ion chromatography with conductometric detection. The tests were performed on a Dionex IonPac®AS22 analytic column (250 × 4 mm, 6 µm) with AG22 precolumn (50 × 4 mm 11 µm). RESULTS: The method provides for potassium bromate(V) determination within the concentration range of 0.043 ÷ 0.88 mg/m3 for an air sample of 0.72 m3 in volume, i.e., 0.1-2 times the exposure limit value as proposed in Poland. The method was validated in accordance with PN-EN 482. The obtained validation data are as follows: measuring range: 3.1-63.4 µg/mL, limit of detection (LOD) = 0.018 µg/mL and limit of quantification (LOQ) = 0.053 µg/mL. The developed method has been tested in the work environment, on laboratory employees having contact with potassium bromate(V). CONCLUSION: The analytical method allowed the determination of the inhalable fraction of airborne potassium bromate(V) at workplaces and can be used to assess occupational exposure.

Solid phase-extraction procedure for the determination of amitraz degradation products in honey.

Solid-phase extraction (SPE) was carefully optimised for preconcentration of 2-4-dimethylaniline (2,4-DMA) and 2,4-dimethylformanilide (DMF) from honey samples. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) was used for the separation and quantification of these analytes. By applying the SPE procedure with 200 mg of OASIS HLB sorbent, the limits of detection achieved in honey samples can be lowered to 0.41 µg·kg-1 for 2,4-DMA and 0.69 µg L-1 for DMF. The proposed method achieves good recoveries (81.1-114%) and precision (RSD 1.07-4.05%, n = 3) for analysed honey sample spiked at two concentration levels 1 µg L-1 and 2 µg L-1. The results demonstrated our method can be applied as a simple way for sample preparation of honey samples for determination of amitraz degradation products. In 1 out of 5 of the analysed Polish honey samples, the DMF residues exceed the maximum residual limits (0.2 mg·kg-1 for total amitraz residues).

Kairomone-like activity of bile and bile components: A step towards revealing the chemical nature of fish kairomone.

Despite the efforts of a number of research groups worldwide, we still have a poor understanding of the chemical nature of the fish kairomones which induce defensive morphology, life history and behavior in their planktonic prey. Bile excreted by foraging fish play a crucial role in their signaling systems. Using high-performance liquid chromatography (HPLC), we revealed the presence of primary and secondary bile acids and bile salts in fish-conditioned water, similar as in carp bile. Upon exposure to either fish bile or commercially acquired bile salts, Daphnia demonstrated similar changes in life history and behavior as when exposed to fish kairomones. The synergic effect of the injured Daphnia alarm substance with fish bile on Daphnia life history is similar to the adaptive effect of the same alarm substance combined with fish kairomones. This strongly supports the view that fish bile or selected bile acids/salts may be responsible for the biological activity of kairomones.

Application of Molecularly Imprinted Polymers for Bisphenols Extraction from Food Samples - A Review.

Bisphenols are the most-known endocrine disrupting compounds. Due to the possible migration of these compounds from plastics used as food packaging, the food may become contaminated. During past years, molecularly imprinted polymers (MIPs) were excessively being used as sorbents for bisphenols preconcentration from food samples. This review intended to comprehensively discuss and evaluate the recent applications of MIPs in determination of bisphenols in food samples. The fundamentals of MIPs devoted to preconcentration of bisphenols are outlined. Recent studies were discussed in terms of food sample preparation, determination methods, MIP synthesis, and extraction procedure aspects. The significant aspects of applications of the molecular imprinting polymers in food chemistry are evaluated focusing majorly on literature appeared in the last years.

Identification of proteins, drying oils, waxes and resins in the works of art micro-samples by chromatographic and mass spectrometric techniques.

Simplified method for simultaneous identification of proteins, drying oils, waxes, and resins in the works-of-art samples was developed. Liquid chromatography with mass spectrometry and gas chromatography with mass spectrometry were used to identify natural materials most frequently encountered in historical paintings. Protein binders were extracted with ammonia and purified using miniaturized solid-phase microextraction (Omix tips) to efficiently suppress matrix interferences. Zwitterionic stationary phase was used for separation of 16 underivatized amino acids analysis with hydrophilic interaction liquid chromatography that was subsequently quantified with liquid chromatography with mass spectrometry. Gas chromatography with mass spectrometry was used to analyze drying oils, waxes, and resins after one-step saponification/transmethylation with (m-trifluoromethylphenyl)trimethylammonium hydroxide (Meth-Prep II). While the drawback of this reagent is low reactivity towards hydroxyl groups, sample pretreatment was much simpler as compared to the other methods. Fatty acids derivatization with the Meth-Prep II reagent was compared with their silylation using N,O-bis(trimethylsilyl) trifluoroacetamide/trimethylchlorosilane mixture. It was concluded that fatty acids analysis as their methyl esters instead of trimethylsilyl esters had a minor impact on the method sensitivity. The developed method was used to analyze samples from 16th and 17th century historical paintings.

Application of Hydrophilic Interaction Liquid Chromatography for the Quantification of Flavonoids in Genista tinctoria Extract.

Hydrophilic interaction chromatography (HILIC) was employed to investigate chromatographic behavior of selected flavonoids from their different subgroups differing in polarity. Chromatographic measurements were performed on two different HILIC columns: unmodified silica (Atlantis-HILIC) and zwitterionic sulfoalkylbetaine (SeQuant ZIC-HILIC). Separation parameters such as content and type of organic modifier were studied. On ZIC column retention factors were observed to be inversely proportional to the buffer content in the mobile phase, which is the typical partitioning mechanism. In the case of bare silica column more or less apparent dual retention mechanism was observed, depending on the water component content in the mobile phase. ZIC-HILIC showed better selectivity (in comparison to silica column) with the detection limit of 0.01 mg/L (only for rutin was 0.05 mg/L). Finally, this chromatographic procedure was validated and applied for the determination of some flavonoids in Genista tinctoria L. extract.

Retention Study of Flavonoids Under Different Chromatographic Modes.

The goal of this study was to investigate the chromatographic behavior of selected flavonoids from their different subgroups (flavonols, flavanones, flavones and isoflavones) in hydrophilic interaction liquid chromatography (HILIC). Chromatographic measurements were made on two different HILIC columns: cross-linked DIOL (Luna HILIC) and zwitterionic sulfoalkylbetaine (SeQuant ZIC-HILIC). Separation parameters such as the content of acetonitrile and pH of an eluent were studied. On the ZIC column, the retention factors of flavonoids increased with decreasing water content in the mobile phase. The increase in pH of the aqueous component mainly affects the polarity of the analytes. DIOL stationary phase shows more or less apparent dual retention mechanism, HILIC at the acetonitrile (ACN) content ≥75% and reversed phase (RP) with lower content of organic modifier. In the presence of ammonium acetate in the mobile phase, the retention of flavonoids onto the DIOL column increases without change in the selectivity of the separations. The similar effect, but considerably smaller was observed for aglycones on the ZIC column. The retention of studied glycosides (hesperidin, rutin) decreases in the presence of salt in the mobile phase. The significantly higher mass spectrometry sensitivity was observed under HILIC conditions in comparison with the most often used RP LC due to much higher content of ACN in the mobile phase. Finally, under optimal chromatographic conditions, the method was validated and applied for the determination of flavonoids in chamomile (Matricaria chamomilla L.) infusion.

Effects of brewing process on phenolic compounds and antioxidant activity of herbs.

Traditionally used medicinal plants contain a wide range of polyphenolic compounds that act as powerful antioxidants. The content of phenolic compounds in the infusions and decoctions of chamomile (Matricaria chamomilla L.) and St. John's wort (Hypericum perforatum), which are traditionally used medicinal herbs, was evaluated via liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. To obtain relevant antioxidant/reducing capacity of the prepared extracts, cupric reducing antioxidant capacity and Folin-Ciocalteu assay were applied. Rutin and apigenin were the major flavonoids in the aqueous extract of chamomile, whereas the predominant phenolic compounds of St. John's wort water extracts were rutin and catechin followed by chlorogenic acid. A longer time of infusion and decoction of St. John's wort herb significantly affected the rutin content. The increase of extraction time had very little impact on the antioxidant activities for chamomile but considerably higher impact on those for St. John's wort.

Experimental Comparison of Efficiency of First Aid Dressings in Burning White Phosphorus on Bacon Model.

BACKGROUND: The aim of this study was to determine effectiveness of first aid dressings in extinguishing burning white phosphorous (WP), eliminating WP pieces from the surface, inhibiting re-ignition on the model (fresh bacon covered with military uniform), and preventing from late re-ignition caused by persistent WP pieces. MATERIAL AND METHODS: Burning WP was extinguished with several dressings: tactical Military Dressing (WJ10), wet gauze, 2 hydrocolloids, and 3 prototypes of hydrocolloids developed by the authors. RESULTS: All examined dressings were effective in extinguishing WP provided that the entire area of the burning substance was completely covered. Moist gauze was especially effective in extinguishing WP, and also removed and absorbed the majority of the WP mass, preventing deeper penetration of WP particles. The immediate re-ignition was observed when all the remaining examined dressings were removed from the bacon. A stream of water was dangerous, as it splashed and transferred pieces of WP around. CONCLUSIONS: Moist gauze placed on burning WP for approximately 3 min was most effective in extinguishing WP and removing most of the WP pieces. We recommend moist gauze, used once or twice, as the best primary means for WP elimination and preventing tissue penetration. As a dressing used for medical evacuation (MEDEVAC), or as a second step after complete removal of visible WP, innovative hydrocolloid or hydrogel dressings should be used.

Suitability of three-dimensional synchronous fluorescence spectroscopy for fingerprint analysis of honey samples with reference to their phenolic profiles.

The suitability of a right-angle geometry three-dimensional synchronous fluorescence spectroscopy (3D-SFS) for the differentiation and classification of different honey types, including acacia, buckwheat, heather, linden, multiflower and rape honeys, was evaluated with reference to their phenolic profiles. The fraction of phenolic compounds was initially isolated and pre-concentrated from analyzed samples by means of polymeric reversed phase Strata X solid phase extraction (SPE) cartridges. A thoughtful analysis of 3D-SF spectra of resulting methanolic extracts of honey samples containing the separated fraction of phenolics and standards of phenolic acids and flavonoids revealed that the proposed methodology based on SPE and 3D-SFS is capable of discriminating honey samples due to different floral sources in a fast and straightforward way.

Characterisation of honeys according to their content of phenolic compounds using high performance liquid chromatography/tandem mass spectrometry.

A simple, fast and specific high performance liquid chromatography separation with an electro-spray ionisation tandem mass spectrometry detection in a negative single reaction ion monitoring scan mode was developed and used for the characterization of Polish honeys according to the content of phenolic acids, including caffeic, chlorogenic, p-coumaric, ferulic, homogentisic, p-hydroxybenzoic and vanillic acids, and flavonoids, i.e., apigenin, genistein, hesperetin, kaempferol, luteolin, rhamnetin, rutin, tricetin and quercetin. Target compounds were isolated and pre-concentrated from the honey matrix by means of the solid phase extraction using Strata X (500mg) cartridges. Analysed honeys did not contain tricetin and genistein. Hesperetin was determined for the first time in heather and linden honeys while rutin in rape honey.

Effects of the operation parameters on HILIC separation of flavonoids on zwitterionic column.

The hydrophilic interaction chromatography (HILIC) coupled to mass spectrometry was employed to study retention behavior of several flavonoids from their different groups using the polymeric zwitterionic stationary phase (ZIC-pHILIC). It contains sulfobetaine-bonded ligand with an inner positively charged quaternary ammonium and an outer negatively charged sulfonate functional groups. Two organic solvents - acetonitrile (ACN) and methanol (MeOH) - were compared as a component of mobile phase. Separation parameters such as a content of organic modifier, pH of an eluent and a column temperature were studied. Retention of flavonoids is controlled primarily by a partition between the mobile phase eluent and a water-enriched layer on the hydrophilic stationary phase with some contribution from hydrogen bonding formation. Using MeOH, in contrast to ACN, strongly retained compounds (myricetin, morin, rutin and quercetrin) could be eluted under isocratic conditions. A better sensitivity was achieved with MeOH as mobile phase component, particularly for quercetin, naringenin and kaempferol. The method was applied to the determination of flavonoids in fruit juices.

Stability of bioactive polyphenols from honey during different extraction methods.

The LC-MS/MS technique was applied to the stability study of several flavonoids and phenolic acids in honey samples during the ultrasonic extraction (USE) and microwave-assisted extraction (MAE). Phenolic compounds from the standard mixture were stable under ultrasounds action with the mean recovery of (90.4%±7.1%), but during microwave-assisted extraction the benzoic acid derivatives and aglycones of flavonoids showed lower recovery (70-80%). In honey matrix, the phenolic acids and the glycosides exhibited the high stability for MAE and USE treatments. However, the recoveries of tested aglycones were below 10%. In the presence of an artificial sugar matrix, flavonols were almost completely degraded after successive treatment under MAE and USE conditions. The obtained results indicated that standard addition method for flavonoids quantification in honey samples should not be recommended. Application of the USE conditions provided higher and/or similar extraction yields for phenolic acids than usually applied shaking with solvent. It also allowed shortening the time required for the whole sample preparation procedure. Phenolic acids and glycosides such as quercetrin, rutin and hesperidin appeared to be stable under such conditions.

Screening of the antioxidant properties and polyphenol composition of aromatised green tea infusions.

BACKGROUND: Aromatised green teas are widely sold and popular owing to their fragrance. In this study the antioxidant activity of six commercial green tea infusions was assessed by three complementary assays. RESULTS: In order to evaluate the tea infusions as antioxidant sources, their phenolic content (Folin-Ciocalteu assay), cupric ion-reducing antioxidant capacity (CUPRAC) and 2,2-diphenyl-1-picryhydrazyl (DPPH) radical-scavenging ability were determined. Their content of polyphenols was also determined by high-performance liquid chromatography/mass spectrometry (HPLC/MS) in negative electrospray ionisation mode. Some teas with citrus aroma, besides having a relatively high content of catechins, also contain other phenolic compounds such as naringin and hesperidin. The three assays used to evaluate the antioxidant capacity of the tea infusions gave different rank orders. Therefore an antioxidant index was calculated for better correlation of the results, and its highest value was obtained for Clear Green Mint tea infusion. CONCLUSION: The studied teas, besides having a relatively high content of catechins, also contain naringin and hesperidin, which are not present in 'pure' green tea. It is important to run multiple assays to get a better estimate of the antioxidant capacity of a given sample.

Polyphenolic content and comparative antioxidant capacity of flavoured black teas.

The content of selected flavonoids and phenolic acids of 12 commercial flavoured black tea infusions was determined by high-performance liquid chromatography with tandem mass spectrometry in the negative electrospray ionization mode. Assays for the evaluation of their antioxidant capacity were carried out by cupric ion reducing antioxidant capacity, Folin-Ciocalteu assay, cyclic voltammetry and potentiometry. Flavonoids were predominantly present as glycosides in the studied infusions. Only quercetin as a free aglycone was found at trace levels. Notable amount of naringin and hesperidin was determined in teas with citrus aromas or fruits. These compounds were not found in premium black tea. Two principal groups of the studied teas were found using cluster analysis in relation to the values of their antioxidant activities.

Historical and archaeological textiles: an insight on degradation products of wool and silk yarns.

The characterisation of micro-samples from works of art and archaeological residues is a particularly complex task, due to the fact that only a relatively low amount of material is available for sampling, and compounds both derived from the target analytes and the matrix can be simultaneously present. Thus, sensitive, selective and reliable analytical procedures need to be developed. This paper presents the optimisation of an instrumental procedure based on liquid chromatography with mass spectrometric detection, which allows for determining selected analytes (anthraquinones, tannins, flavonoids), along with their known degradation products: phenolic acids. The instrumental parameters were optimised in terms of selecting the best ionisation source (APCI and ESI were compared), choosing the compound-dependant MS parameters and enhancing selectivity and sensitivity (SIM and MRM analyses were compared). The proposed procedure proved to be sensitive and selective, with limits of detection (0.4-20 ng/mL). The analytical procedure was validated by characterising reference materials, i.e. dyed and undyed woollen and silk yarns, both freshly prepared and artificially aged. Particularly, the study focused on the determination of 4-hydroxybenzoic acid, and on the correlation between its relative amounts with respect to ageing time. The optimised procedure was also applied to historical samples and proved fundamental in unravelling the complex composition of black dyed yarns collected from a medieval tapestry. The much degraded yarns were dyed with a tannin based dye, extracted from gallnuts, alder bark or sumac; the less degraded ones were coloured by superimposing colours with cochineal, madder, weld and indigo dye baths, and eventually by adding gallo-tannins as well.

Evaluation of the antioxidant properties of fruit and flavoured black teas.

OBJECTIVE: Antioxidant properties of the water extracts of the commercial bagged fruit and flavoured black teas were evaluated and compared with typical black teas of C. sinensis. METHODS: Folin-Ciocalteu (FC) assay, cupric ion reducing antioxidant capacity (CUPRAC) and DPPH radical method were used for these purposes. The content of selected flavonoids and phenolic acids was also determined by high-performance liquid chromatography with tandem mass spectrometry in the negative electrospray ionization mode. RESULTS: Flavoured black teas contain significantly higher level of catechins, quercetin, and rutin, while the content of chlorogenic and caffeic acids as well as naringin and hesperidin was higher in fruit teas. Supplementation with these flavonoids could reduce blood glucose. In FC and DPPH assays, the antioxidant properties of studied tea infusion increases in the order: fruit tea < flavoured black tea > premium black tea, while in CUPRAC method, some aromatized teas exhibit the highest antioxidant properties. Tea infusions with nice smell of fruits would also support the human diet with some source of antioxidants.