Simultaneous determination of free biliverdin and free bilirubin in serum: A comprehensive LC-MS approach.

BACKGROUND: Prognosis, diagnosis, and treatment of several diseases strongly rely on the sensitive, selective, and accurate determination of specific biomarkers in relevant biological samples. Free biliverdin and free bilirubin represent important new biomarkers of oxidative stress, however, the lack of suitable analytical methods for their determination has hindered progress in biomedical and clinical research. RESULTS: Here, we introduce a first comprehensive approach for robust and simultaneous determination of these bilins in serum using liquid chromatography - mass spectrometry (LC-MS). The developed analytical method exhibits linearity for both analytes within the concentration range of 0.5-100 nM, with limits of detection and quantitation determined at 0.1 nM and 0.5 nM, respectively. Moreover, several analytical pitfalls related to the intrinsic molecular structures of free bilirubin and free biliverdin and their trace concentration levels in biological samples are discussed here in detail for the first time. We have shown that the solubility, chemical stability, and affinity of these bilins to various materials strongly depend on the solvent, pH, and addition of stabilizing and chelating agents. Finally, the validated LC-MS method was successfully applied to the analysis of both bilins in fetus bovine serums, yielding higher free bilirubin/biliverdin ratios compared with previously reported values for human serum. SIGNIFICANCE: Failure to recognize and address the challenges presented here often leads to substantial analytical errors and consequently biased interpretation of the obtained results. This pertains not only to LC-MS, but also to many other analytical platforms due to the compound-derived sources of error.

Correction to "Esterification of Lutein from Japanese Knotweed Waste Gives a Range of Lutein Diester Products with Unique Chemical Stability".

[This corrects the article DOI: 10.1021/acssuschemeng.2c01241.].

Antioxidant-rich foods and nutritional value in daily kindergarten menu: A randomized controlled evaluation executed in Slovenia.

Little is known about Total Antioxidant Capacity (TAC) of daily kindergarten menus. The objective of the present study was to determine whether, antioxidant-enriched kindergarten menu had, compared to a standard one, more optimal proximate composition, energy value and higher antioxidant capacity of free and bound dietary antioxidants. Antioxidant-enriched kindergarten meals, on average, contained significantly more vegetables, nuts, and whole grain foods (p < 0.05) and the average proximate composition and mineral content were more consistent with Dietary Reference Intake (DRIs). Additionally, in antioxidant-enriched vs standard daily meals, average TAC was 1369 mg vs 586 mg vitamin C equivalent (determined by 2'-azinobis-[3-ethylbenzthiazoline-6-sulfonic acid] radical scavenging capacity (ABTS) assay) and 1734 mg vs 810 mg vitamin C equivalent determined by ferric reducing antioxidant power (FRAP) assay. Our study shed light on free and bound antioxidants in daily kindergarten meals and highlighted that supplementing kindergarten meals with foods rich in antioxidants can significantly improve dietary intakes.

Extraction of Polyphenols and Valorization of Fibers from Istrian-Grown Pomegranate (Punica granatum L.).

Pomegranate fruit is an ancient fruit that is used not only because of its deep-red color and tasty arils but also due to the health benefits of its extracts. Pomegranate is a valuable source of bioactive compounds, including colorful anthocyanins and other polyphenols. The main objective of the present study was to gain comprehensive knowledge of the phenolic composition and antioxidative activity of a new pomegranate cultivar, grown in Northwest Istria, a part of the North Adriatic coastal area. Various parts of the pomegranate fruit parts were extracted in 70% ethanol or water. Total phenolic content and antioxidative capacity were respectively determined with Folin-Ciocalteu reagent and ABTS radical. Phenolics were examined and analyzed with TLC, LC-MS, and HPLC. Pomegranate juice was prepared from red arils and after thermal treatment, the stability of anthocyanins was monitored for several months to understand the effect of storage. The highest total phenolics were determined in ethanol pomegranate peel extracts (30.5 ± 0.6 mg GAE/g DM), and water peel extracts exhibited the highest antioxidative activity (128 ± 2 µg TE/g DM). After five months of storage of thermally treated pomegranate juice, 50-60 percentage points increase in anthocyanin degradation was observed. Pomegranate peel was further tested as a sustainable inedible food source for papermaking. Due to the low content of cellulose and the high percentage of extractives, as well as a distinguished texture and appearance, the paper made from pomegranate peel is best suited for the production of specialty papers, making it particularly interesting for bioactives recovery, followed by material restructuring.

Esterification of Lutein from Japanese Knotweed Waste Gives a Range of Lutein Diester Products with Unique Chemical Stability.

A valorization strategy for an aggravating type of plant waste is put to the test herein. It envisions the use of Japanese knotweed green leaves as a sustainable source of free lutein, from which bioactive diesters could be prepared as potential value-added products with improved properties. To this end, 13 structurally distinct model lutein diesters were synthesized and the relationships between their structure and stability were systematically determined. The forced degradation data show that the stability of a particular lutein diester may depend to a large extent on the type of exposure (elevated temperature, light, oxidant, or acidic environment) and, more importantly, not every esterification attempt necessarily leads to an enhancement of lutein's chemical stability. However, three branched and bulky products-lutein di(2,2-dimethylpropanoate), lutein di(2-methylpropanoate), and lutein di(3-methylbutanoate)-proved to be particularly relevant, as they consistently exhibited 1.5-21-fold higher stability compared to free lutein, regardless of the stress conditions used. Finally, we show that the Japanese knotweed plant matrix had a significant negative or positive effect on pigment degradation kinetics that could not be easily predicted. Thus, the proposed valorization strategy is quite feasible, but the esterification approach should be tailored to the intended use of a lutein diester.

Structure Elucidation and Mitigation of Endogenous Interferences in LC-MS-Based Metabolic Profiling of Urine.

Liquid chromatography-mass spectrometry (LC-MS) is the main workhorse of metabolomics owing to its high degree of analytical sensitivity and specificity when measuring diverse chemistry in complex biological samples. LC-MS-based metabolic profiling of human urine, a biofluid of primary interest for clinical and biobank studies, is not widely considered to be compromised by the presence of endogenous interferences and is often accomplished using a simple "dilute-and-shoot" approach. Yet, it is our experience that broad obscuring signals are routinely observed in LC-MS metabolic profiles and represent interferences that lack consideration in the relevant metabolomics literature. In this work, we chromatographically isolated the interfering metabolites from human urine and unambiguously identified them via de novo structure elucidation as two separate proline-containing dipeptides: N,N,N-trimethyl-l-alanine-l-proline betaine (l,l-TMAP) and N,N-dimethyl-l-proline-l-proline betaine (l,l-DMPP), the latter reported here for the first time. Offline LC-MS/MS, magnetic resonance mass spectrometry (MRMS), and nuclear magnetic resonance (NMR) spectroscopy were essential components of this workflow for the full chemical and spectroscopic characterization of these metabolites and for establishing the coexistence of cis and trans isomers of both dipeptides in solution. Analysis of these definitive structures highlighted intramolecular ionic interactions as responsible for slow interconversion between these isomeric forms resulting in their unusually broad elution profiles. Proposed mitigation strategies, aimed at increasing the quality of LC-MS-based urine metabolomics data, include modification of column temperature and mobile-phase pH to reduce the chromatographic footprint of these dipeptides, thereby reducing their interfering effect on the underlying metabolic profiles. Alternatively, sample dilution and internal standardization methods may be employed to reduce or account for the observed effects of ionization suppression on the metabolic profile.

The influence of selected indigenous yeasts on Pinot Noir wine colour properties.

BACKGROUND: The use of indigenous selected starters in winemaking is gaining interest due to certain advantages for the sensory quality of the wine. The present work shows the results of a laboratory experiment in which the influence of selected indigenous yeasts on the colour characteristics of Pinot Noir was studied with the use of high hydroxycinnamate decarboxylase activity yeasts. Pichia guilliermondii ZIM624 and Wickerhamomyces anomalus S138 yeasts were used in sequential fermentation with two strains of Saccharomyces cerevisiae, the native ZIM2180 strain and commercial Fermol Premier Cru (FPC). RESULTS: In co-inoculation fermentations, non-Saccharomyces yeasts decreased colour intensity (on average by 25.5%). In wines fermented with ZIM624, the concentration of vinylphenolic pyranoanthocyanins increased (average concentration 1.5 mg L-1 ). However, vitisin concentration was significantly higher in S138 + FPC fermentation (1.3 mg L-1 and an average of 0.9 mg L-1 , respectively). Pinot Noir wines fermented with only ZIM2180 and sequential inoculation of ZIM624 + ZIM2180 resulted in significantly higher colour intensity (6.1 ± 0.0 AU and 4.4 ± 0.0 AU, respectively) and lower wine hue parameters compared to other wines. Sensory evaluation also showed that both wines had the highest perceived colour intensity and purple colour suggesting improvement in wine quality parameters. CONCLUSIONS: The results confirmed that selected indigenous starters made out of Saccharomyces and non-Saccharomyces yeasts can alter Pinot Noir wine colour parameters and improve wine colour properties. Those yeasts properties should be investigated prior to the development of new commercial starters but also be considered in large scale spontaneous fermentations of low colour intensity red wines like Pinot Noir. © 2021 Society of Chemical Industry.

Parameters for Irreversible Inactivation of Monoamine Oxidase.

The irreversible inhibitors of monoamine oxidases (MAO) slow neurotransmitter metabolism in depression and neurodegenerative diseases. After oxidation by MAO, hydrazines, cyclopropylamines and propargylamines form a covalent adduct with the flavin cofactor. To assist the design of new compounds to combat neurodegeneration, we have updated the kinetic parameters defining the interaction of these established drugs with human MAO-A and MAO-B and analyzed the required features. The Ki values for binding to MAO-A and molecular models show that selectivity is determined by the initial reversible binding. Common to all the irreversible inhibitor classes, the non-covalent 3D-chemical interactions depend on a H-bond donor and hydrophobic-aromatic features within 5.7 angstroms apart and an ionizable amine. Increasing hydrophobic interactions with the aromatic cage through aryl halogenation is important for stabilizing ligands in the binding site for transformation. Good and poor inactivators were investigated using visible spectroscopy and molecular dynamics. The initial binding, close and correctly oriented to the FAD, is important for the oxidation, specifically at the carbon adjacent to the propargyl group. The molecular dynamics study also provides evidence that retention of the allenyl imine product oriented towards FADH- influences the formation of the covalent adduct essential for effective inactivation of MAO.

Japanese and Bohemian Knotweeds as Sustainable Sources of Carotenoids.

Japanese knotweed (Fallopia japonica Houtt.) and Bohemian knotweed (Fallopia x bohemica) are invasive alien plant species, causing great global ecological and economic damage. Mechanical excavation of plant material represents an effective containment method, but it is not economically and environmentally sustainable as it produces an excessive amount of waste. Thus, practical uses of these plants are actively being sought. In this study, we explored the carotenoid profiles and carotenoid content of mature (green) and senescing leaves of both knotweeds. Both plants showed similar pigment profiles. By means of high performance thin-layer chromatography with densitometry and high performance liquid chromatography coupled to photodiode array and mass spectrometric detector, 11 carotenoids (and their derivatives) and 4 chlorophylls were identified in green leaves, whereas 16 distinct carotenoids (free carotenoids and xanthophyll esters) were found in senescing leaves. Total carotenoid content in green leaves of Japanese knotweed and Bohemian knotweed (378 and 260 mg of lutein equivalent (LE)/100 g dry weight (DW), respectively) was comparable to that of spinach (384 mg LE/100 g DW), a well-known rich source of carotenoids. A much lower total carotenoid content was found for senescing leaves of Japanese and Bohemian knotweed (67 and 70 mg LE/100 g DW, respectively). Thus, green leaves of both studied knotweeds represent a rich and sustainable natural source of bioactive carotenoids. Exploitation of these invaders for the production of high value-added products should consequently promote their mechanical control.

Evidence for a Cyanine Link Between Propargylamine Drugs and Monoamine Oxidase Clarifies the Inactivation Mechanism.

Successful propargylamine drugs such as deprenyl inactivate monoamine oxidase (MAO), a target in multi-faceted approaches to prevent neurodegeneration in the aging population, but the chemical structure and mechanism of the irreversible inhibition are still debated. We characterized the covalent cyanine structure linking the multi-target propargylamine inhibitor ASS234 and the flavin adenine dinucleotide in MAO-A using a combination of ultra-high performance liquid chromatography, spectroscopy, mass spectrometry, and computational methods. The partial double bond character of the cyanine chain gives rise to 4 interconverting geometric isomers of the adduct which were chromatographically separated at low temperatures. The configuration of the cyanine linker governs adduct stability with segments of much higher flexibility and rigidity than previously hypothesized. The findings indicate the importance of intramolecular electrostatic interactions in the MAO binding site and provide key information relevant to incorporation of the propargyl moiety into novel multi-target drugs. Based on the structure, we propose a mechanism of MAO inactivation applicable to all propargylamine inhibitors.

Kinetics, mechanism, and inhibition of monoamine oxidase.

Monoamine oxidases (MAOs) catalyse the oxidation of neurotransmitter amines and a wide variety of primary, secondary and tertiary amine xenobiotics, including therapeutic drugs. While inhibition of MAO activity in the periphery removes protection from biogenic amines and so is undesirable, inhibition in the brain gives vital antidepressant and behavioural advantages that make MAO a major pharmaceutical target for inhibitor design. In neurodegenerative diseases, MAO inhibitors can help to maintain neurotransmitter levels, making it a common feature in novel multi-target combinations designed to combat Alzheimer's disease, albeit not yet proven clinically. Vital information for inhibitor design comes from an understanding of the structure, mechanism, and kinetics of the catalyst. This review will summarize the kinetic behaviour of MAO A and B and the kinetic evaluation of reversible inhibitors that transiently decrease catalysis. Kinetic parameters and crystal structures have enabled computational approaches to ligand discovery and validation of hits by docking. Kinetics and a wide variety of substrates and inhibitors along with theoretical modelling have also contributed to proposed schemes for the still debated chemical mechanism of amine oxidation. However, most of the marketed MAO drugs are long-lasting irreversible inactivators. The mechanism of irreversible inhibition by hydrazine, cyclopropylamine, and propargylamine drugs will be discussed. The article finishes with some examples of the propargylamine moiety in multi-target ligand design to combat neurodegeneration.

High performance thin-layer chromatography-mass spectrometry enables reliable analysis of physalins in different plant parts of Physalis alkekengi L.

We developed first HPTLC and HPTLC-MS/MS methods which enable characterization of structurally similar and complex biologically active compounds - physalins - from crude extracts of Chinese lantern (Physalis alkekengi L.). Separation on HPTLC silica gel plates developed with ethyl acetate-toluene-formic acid (7:3:0.2, v/v) enabled densitometric screening of physalins in absorption and, after post-chromatographic derivatization with sulfuric acid reagent, also in fluorescence mode. Compared to existing (U)HPLC methods, in this case TLC provides an alternative selectivity, better sensitivity and higher resolution, which was exemplified by the separation of physalin L standard and its impurity, identified as 2,3,25,27-tetrahydrophysalin A. Strong ion suppression caused by the developing solvent additive - formic acid - was efficiently solved by two successive plate pre-developments with methanol-formic acid (9:1, v/v) and methanol. This significantly improved the sensitivity of HPTLC-MS/MS method, but also required a slightly modified developing solvent ethyl acetate-toluene-formic acid (6:4:0.2, v/v). Simultaneous hyphenation of HPTLC with a triple quadrupole and an ion trap mass analyzer enabled a reliable and straightforward non-targeted characterization of physalins from the same chromatographic zone (band) and determination of physalin types. The performance of developed HPTLC-densitometric and HPTLC-MS/MS methods was demonstrated by the analysis of physalins from the aqueous extracts, prepared by an optimized fast and simple extraction method under reflux. Variations in physalin profiles and abundances in different parts of P. alkekengi L. harvested at different stages of maturity were observed. This indicates that not all parts of the plant, or plant as a whole, are appropriate for specific medicinal applications. Husks are proposed as the most suitable plant part for P. alkekengi L. quality control, because they exhibited the most obvious MS2 fingerprints of physalins with minimal interferences.

High performance thin-layer chromatography-mass spectrometry of Japanese knotweed flavan-3-ols and proanthocyanidins on silica gel plates.

On-line elution based TLC-MS is now a well-established technique, but the quality of the data obtained can sometimes be hampered by a severe spectral background or by strong ion suppression, especially when silica gel plates are used in combination with an acidic modifier in the developing solvent. We solved this issue simply and efficiently using two pre-developments of the plates, firstly with methanol-formic acid (10:1, v/v) and secondly with acetonitrile-methanol (2:1, v/v). This solution resulted in significant improvement in the sensitivity of HPTLC-MS methods. The applicability of this approach was proven on analysis of flavan-3-ols and proanthocyanidins in crude extracts of Japanese knotweed (Fallopia japonica Houtt.) rhizomes. Separations on HPTLC silica gel and HPTLC silica gel MS grade plates using developing solvents toluene-acetone-formic acid (3:3:1, 6:6:1, 3:6:1, v/v) and dichloromethane-acetone-formic acid (1:1:0.1, v/v) were followed by post-chromatographic derivatization with 4-dimethylaminocinnamaldehyde (DMACA) detection reagent. Examination of the stability of the analytes on the start confirmed that the plates should be developed immediately after the application of standards and sample test solutions. In a five hours stability testing after development we discovered an unexpected phenomenon of enhanced absorption at 280nm. However, based on an experiment with post-chromatographic derivatization with DMACA detection reagent, the analytes were proven to be sufficiently stable in the time frame of an HPTLC-MS analysis. This was important for development of the first HPTLC-MS and HPTLC-MSn methods for identification of flavan-3-ols and B-type proanthocyanidins from monomers up to decamers. For the first time, based on this research methodology, trimers, trimer gallates, tetramer gallates, pentamers, pentamer gallates, hexamers, hexamer gallates, heptamers, octamers, nonamers and decamers were tentatively identified in Japanese knotweed rhizomes. Additionally, all developed HPTLC-MS methods have enabled simultaneous identification of stilbenes (resveratrol, piceatannol hexoside, piceid) and anthraquinones (emodin, emodin-O-hexoside, emodin-O-(acetyl)-hexoside and emodin-O-(6'-O-malonyl)-hexoside).

Non-targeted chromatographic analyses of cuticular wax flavonoids from Physalis alkekengi L.

Since Chinese lantern (Physalis alkekengi L.) represents a rich source of various bioactive secondary metabolites, there is an urge for its detailed characterization. Non-polar flavonoid aglycones represent one of the few bioactive species found in plant's cuticular waxes. The separation of flavonoids is already extensively covered in the literature, but methods dedicated to separation and identification of methylated flavonoids are rather scarce. In the present study a non-targeted approach for the separation, isolation and identification of methylated flavonoids present in P. alkekengi L. var. franchetii cuticular waxes was established. A rapid and simple separation on HPTLC silica gel was developed for preliminary screening of flavonoids. Fast HPLC-UV-MS(n) and HPLC-UV methods using a C6-Phenyl and a C18 stationary phase were also developed, respectively. In both cases, the right combination of temperature and tetrahydrofuran, as a mobile phase modifier, were shown to be crucial for a baseline separation of all studied compounds. By employing a semi-preparative analog of the C18 column, a simultaneous isolation of pure unknown analytes was achieved. Using these developed methods in combination with NMR, four 3-O-methylated flavonols were detected and identified in P. alkekengi L. var. franchetii cuticular waxes: myricetin 3,7,3'-trimethyl ether, quercetin 3,7-dimethyl ether, myricetin 3,7,3',5'-tetramethyl ether and quercetin 3,7,3'-trimethyl ether. Moreover, the simple and fast isocratic HPLC-UV-MS(n) method (under 8min) should prove useful in quality control of P. alkekengi L. var. franchetii by enabling chromatographic fingerprinting of external methylated flavonols. Finally, a rationale for the mechanism of separation of these metabolites by HPLC is also given, which establishes a foundation for future development of chromatographic methods for methylated flavonols and related compounds.

Anthocyanins in purple and blue wheat grains and in resulting bread: quantity, composition, and thermal stability.

The anthocyanin composition of blue (Triticum aestivum L., cv. Skorpion) and purple wheat (Triticum aethiopicum JAKUBZ cv. Abyssinskaja arrasajta cv. Abyssinskaja arrasajta), cultivated in the Czech Republic, and of the prepared whole blue and purple wheat bread was determined. In blue and purple wheat, 19 and 26 anthocyanins, respectively, were tentatively identified by liquid chromatography and mass spectrometry. The total content of anthocyanins determined in blue and purple wheat was 9.26 and 13.23 mgkg(-1), respectively. The breads were baked at 240 and 180 °C. Some significant differences in anthocyanins content were observed between breads prepared at different baking temperatures. The content of cyanidin-3-glucoside, delphinidin-3-glucoside and pelargonidin-3-glucoside was determinated in starting material, whole meal flours and baked breads. These kinds of wheat are suitable for baking bread, since intake of anthocyanins may play an important role in the prevention of human diseases.

A novel derivatization procedure and chiral gas chromatographic method for enantiomeric purity screening of L-carnitine.

L-Carnitine is used extensively in functional foods and food supplements; consequently, the control of its enantiomeric purity is of paramount importance. A new derivatization procedure and chiral gas chromatographic method with flame ionization detection, using a cyclodextrin based stationary phase, enables prompt, simple, and inexpensive screening of the enantiomeric ratio of L- and D-carnitine in samples with different matrices. Conversion of carnitine to beta-acetoxy-gama-butyrolactone was optimized for maximum conversion (98% of the desired product lactone was formed and 2% of the side product gama-crotonolactone) and minimum racemization (no changes at the chiral center were detected) and time consumption. As it is shown in this study, a fast gas chromatographic method, with total run time of 7 min, together with the new derivatization procedure enables an effective enantiomeric purity screening of L-carnitine in real samples such as food supplements and L-carnitine raw ingredient.

Addition of ß-lactoglobulin produces water-soluble shikonin.

Shikonin and its ester derivatives belong to a group of secondary metabolites with a wide array of beneficial effects on human health. However, shikonin is principally used in oil-based preparations due to the low solubility of the pigment in aqueous media, and the positive properties of shikonin are not exploited to their full potential. Such low aqueous solubility often results in poor bioavailability, makes shikonin undesirable for oral administration, and restricts its broadened use in the food and pharmaceutical industries. The purpose of this study was to enhance the aqueous solubility of shikonin by the addition of ß-lactoglobulin and to characterize the macromolecule-ligand binding interaction by means of spectrophotometry, spectrofluorometry, high-performance liquid chromatography, and mass spectrometry. In the presence of ß-lactoglobulin the solubility of shikonin is increased up to 181-fold. One shikonin molecule binds covalently to ß-lactoglobulin via Cys(121), whereas the remaining pigment molecules most probably bind to the protein via noncovalent interactions.

Applicability of analytical and preparative monolithic columns to the separation and isolation of major whey proteins.

The separation and isolation of major whey proteins is already extensively covered in the literature although no study has been published in which monolithic columns were used. In our research we present, for the first time, the use of short convective interaction media (CIM) monolithic columns for the separation of all major whey proteins and isolation of ß-lactoglobulin variant A and B (ß-LgA and ß-LgB) from a commercial product whey isolate (WI). Although our primary interest was directed towards finding a proper monolithic column and chromatographic conditions for the purification and isolation of ß-LgA and ß-LgB, three additional analytical LC methods, each having its own potential application target, were also developed in the course of our research. On the monolithic diethylaminoethyl convective interaction media analytical column (CIMac DEAE), the separation of major whey proteins was achieved by gradually lowering the pH of the mobile phase. The ever-so-hard obtainable linear external pH gradient was very linear in the range of pH 5.5-3 and the developed ion-exchange (IE) high-performance liquid chromatographic (HPLC) method was amenable to mass spectrometry (MS). A very fast baseline separation, with UV detection, of all major whey proteins was achieved on a prototype CIMac reversed-phase styrene-divinylbenzene (RP-SDVB) monolithic column in only 4 min and the performance of this column proved superior in comparison with the packed particle POROS perfusion column. The developed RP-HPLC-MS method is fast and, due to the MS detector, can offer low limits of detection and quantitation. Finally, in order to fulfill our primary interest, a scale-up method was developed, using a prototype 8 mL analogue of the CIMac RP-SDVB column, for the isolation of native and chemically unmodified ß-LgA and ß-LgB from WI with purities higher than 90% and 81%, respectively. The proteins were to be used in further protein-ligand binding studies. The developed methods excel in speed of the analysis, sensitivity, resolution, and simplicity. Thus, it is shown for the first time that short monolithic columns are applicable to the separation and isolation of major whey proteins and that their use has some obvious benefits.

Ultra-thin-layer chromatography mass spectrometry and thin-layer chromatography mass spectrometry of single peptides of angiotensin-converting enzyme inhibitors.

The separation of structurally related angiotensin-converting enzyme (ACE) inhibitors lisinopril, cilazapril, ramipril and quinapril and their corresponding active diacid forms (prilates) by conventional TLC silica gel 60 plates was contrasted with that afforded by monolithic ultra-thin-layer chromatographic (UTLC) plates. For the use of UTLC plates technical modifications of the commercially available equipments for the sample application, development and detection were made. Plates were developed in modified horizontal developing chamber using ethyl acetate-acetone-acetic acid-water (4:1:0.25:0.5, v/v). Detection of the separated compounds was performed densitometrically in absorption/reflectance mode at 220 nm and after exposure to iodine also by image analysis. The obtained results showed that monolithic layer is more efficient for the separation of structurally similar polar compounds, such as prilates than conventional silica layers. Identification of the compounds was confirmed by ESI-MS after their on-line extraction from the UTLC and TLC plates by means of Camag TLC-MS interface.

Identification of shikonin and its ester derivatives from the roots of Echium italicum L.

Nine shikonin pigments: shikonin (S), acetylshikonin (AS), propionylshikonin (PS), isobutyrylshikonin (IBS), tiglylshikonin (TS), 3,3-dimethylacrylshikonin (DAS), angelylshikonin (ANS), 2-methyl-n-butyrylshikonin (MBS) and isovalerylshikonin (IVS) were identified in the root epidermis of Echium italicum L. for the first time. A new thin-layer chromatographic (TLC) method for the separation of enantiomers alkannin and shikonin proved only shikonin after saponification of the root extract, and was afterwards esterified with the corresponding acyl chloride to acquire seven standard compounds (all except ANS). The developed isocratic high-peformance liquid chromatographic (HPLC) methods with VIS and mass spectrometry (MS) detection, allowed for the first time simultaneous separation of all nine compounds with similar structures including positional and geometric isomers in a short time. Structures of the main five compounds (AS, IBS, ANS, MBS, IVS) isolated from the extract by a new semi-preparative HPLC on C18 have additionally been confirmed by (1)H and (13)C nuclear magnetic resonance spectra, which were reported for AS and MBS for the first time.