Conventional High-performance Liquid Chromatography Methods Research Articles

Validation of An Efficient 2D-HPLC Method for The Determination of Pentazocine

Pentazocine is an opioid analgesic used to treat moderate to severe pains. The real analysis for the pharmaceutical products containing pentazocine is basically to ensure the correct dose and patient safety. This study developed and validated an improved high-performance liquid chromatography (HPLC) method in the selective and accurate quantification of pentazocine. Two-dimensional (2D) HPLC technique is employed to enhance the resolution and selectivity compared to conventional HPLC methods. The 2D-HPLC instrumentation consists of two C18 columns coupling with a switching valve to capture fractions from the first column, which is analyzed in the second column. The mobile phase was optimized to 45% acetonitrile and 55% water with 0.1% phosphoric acid. The method was validated in the International Conference on Harmonization guidelines and shows excellent linearity (R2=0.998), limit of detection of 1.58 μg/L, accuracy of 97.70–102.50%, and precision by relative standard deviation (RSD) of 1.02–4.20%. Selectivity was verified in resolving pentazocine from paracetamol, caffeine, ibuprofen, and oxycodone in laboratory mixtures. The utility of the 2D-HPLC method was demonstrated by accurate quantification of pentazocine in pharmaceutical injections and tablets unaffected in excipients. This research provides a rich validating technique to enhance the quality control testing of pharmaceuticals containing pentazocine.

Assessing the Comparative Efficacy of Multiple Whole-blood Count-based Testing Strategies for Thalassaemia Diagnosis

Context: Complete blood count (CBC) with confirmatory high-performance liquid chromatography (HPLC) is the gold standard for thalassemia screening. Because of the constraints of a high-pressure liquid chromatography facility, they develop alternative approaches to screening when the population to be screened is large. Aims: The objective of this meta-analysis is to evaluate the diagnostic accuracy of alternative approaches to thalassemia screening. This study is important, as accurate and accessible thalassemia screening can improve the quality of life for patients and reduce the burden on health-care systems. Settings and Design: The systematic review and meta-analysis followed the Preferred Reporting Items for Systematic Reviews and Meta-Analysis guidelines. Subjects and Methods: The present systematic review comprises 74,284 thalassemia-positive patients, identified using CBC and conventional HPLC methods was published between 2012 and 2022. After further screening, 23 studies were included in the present study. Statistical Analysis Used: The Moses-Littenberg Summary Receiver Operating Characteristic (SROC) model, the bivariate model, and the SROC reporting model are the examples of models that also consider sensitivity and specificity. Results: The total diagnostic odds ratio was 88.981 (95% confidence interval 37.645–210.321). Subgroup analysis was determined by a mixed-effects model, with I 2 (residual heterogeneity/unaccounted variability) at 90% and Partial Area under ROC curve (restricted to observed False Positive Rates and normalized) was 0.951. Conclusions: This systematic review investigated the efficacy of using CBC parameters to distinguish between iron deficiency anemia and the thalassemia trait. This can help reduce health-care costs and improve the accuracy of the results.

Enzymatic determination of carnosine in meat and fish using β-Ala-Xaa dipeptidase and histidine ammonia-lyase derived from Pseudomonas putida NBRC100650.

Carnosine is a naturally occurring dipeptide and a functional component in foods, while also showing health-promoting effects. Generally, food-derived carnosine is quantified via high-performance liquid chromatography (HPLC). We have developed a method for quantifying carnosine in foods using microbial enzymes, β-Ala-Xaa dipeptidase (BapA) and histidine ammonia-lyase (HAL). The carnosine concentrations in extracts of chicken, pork, beef, bonito, and tuna were determined via both HPLC and enzymatic determination. The carnosine contents measured via enzymatic determination were in agreement with those determined via conventional HPLC analysis. Relative standard-deviation values of the conventional HPLC method and the enzymatic determination of carnosine in foods were 0.728-5.76% and 0.504-4.58%, respectively. The recovery of carnosine in food extracts via enzymatic determination was 97-103%. Therefore, the developed enzymatic determination technique using BapA and HAL can be used for the determination of carnosine in meats and fishes with comparable accuracy to that of conventional HPLC analysis.

Evaluation of a new measurement method of indoxyl sulfate in hemodialysis patients.

Indoxyl sulfate (IS) is related to the development of cardiovascular disease and total mortality in dialysis patients. High-performance liquid chromatography (HPLC) is the conventional measurement approach. However, the HPLC method is difficult to perform in real time. Recently, the IS Assay Kit "NIPRO", which enables the measuring of total IS by the enzyme method, was developed. This new reagent allows the easy and quick measurement of many samples using the automatic biochemical analyzer. Moreover, it was reported that it demonstrated satisfactory analytical performance. If this enzyme method is useful for measuring IS in hemodialysis, we can expect that the mechanism in which the IS effects adversely on a body as uremic toxins will be clarified. However, the enzyme method is more easily influenced by other coexisting substances. In this study, we have assessed on how the uremic toxins and anticoagulation effect on this new reagent and evaluate whether it can be put into practice effectively for hemodialysis patients. For the enzyme method, accuracy, simultaneous repeatability, linearity, limit of detection, influence of coexisting materials, and correlation with the HPLC method were examined. Accuracy and simultaneous repeatability were satisfactory, and linearity was good. The limit of detection was acceptable, and there was no influence of coexisting materials. With regard to the correlation, the regression equation was y = 0.947X + 7.987 and the correlation coefficient (r) was 0.972. This new reagent showed sufficient fundamental performance and had a good correlation with the conventional HPLC method for assessing the plasma of dialysis patients.

New colorimetric method with bromocresol purple for estimating the redox state of human serum albumin.

Oxidative damage results in protein modification and is observed in many diseases, such as heart failure and renal insufficiency. Human serum albumin is an index of oxidative change and is conventionally measured using high-performance liquid chromatography (HPLC). Although this method is more sensitive than the colorimetric method, it is time-consuming for clinical practice and the sera must be stored at –80°C before analysis. To overcome these limitations, in the present study we developed a new reagent for a more rapid and convenient quantification of oxidative stress, involving determination of the ratio of human nonmercaptalbumin to total albumin using a colorimetric method with bromocresol purple. The clinical utility of the developed reagent was confirmed by demonstrating the consistently higher oxidative stress levels in dialysis patients than in healthy control subjects, matching the results of the conventional HPLC method. This novel approach could be a valuable tool for immediate estimation of the state of oxidative stress during the course of disease and treatment, and could aid clinical treatment decisions.

A review on the recent advances in HPLC, UHPLC and UPLC analyses of naturally occurring cannabinoids (2010-2019).

Organic molecules that bind to cannabinoid receptors are called cannabinoids, and they have similar pharmacological properties like the plant, Cannabis sativa L. Hyphenated liquid chromatography (LC), incorporating high-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography (UPLC, also known as ultrahigh-performance liquid chromatography, UHPLC), usually coupled to an ultraviolet (UV), UV-photodiode array (PDA) or mass spectrometry (MS) detector, has become a popular analytical tool for the analysis of naturally occurring cannabinoids in various matrices. To review literature on the use of various LC-based analytical methods for the analysis of naturally occurring cannabinoids published since 2010. A comprehensive literature search was performed utilising several databases, like Web of Knowledge, PubMed and Google Scholar, and other relevant published materials including published books. The keywords used, in various combinations, with cannabinoids being present in all combinations, in the search were Cannabis, hemp, cannabinoids, Cannabis sativa, marijuana, analysis, HPLC, UHPLC, UPLC, quantitative, qualitative and quality control. Since 2010, several LC methods for the analysis of naturally occurring cannabinoids have been reported. While simple HPLC-UV or HPLC-UV-PDA-based methods were common in cannabinoids analysis, HPLC-MS, HPLC-MS/MS, UPLC (or UHPLC)-UV-PDA, UPLC (or UHPLC)-MS and UPLC (or UHPLC)-MS/MS, were also used frequently. Applications of mathematical and computational models for optimisation of different protocols were observed, and pre-analyses included various environmentally friendly extraction protocols. LC-based analysis of naturally occurring cannabinoids has dominated the cannabinoids analysis during the last 10 years, and UPLC and UHPLC methods have been shown to be superior to conventional HPLC methods.

Response surface design as a powerful tool for the development of environmentally benign HPLC methods for the determination of two antihypertensive combinations: Greenness assessment by two green analytical chemistry evaluation tools.

Interest in implementing green chemistry principles in analytical chemistry has grown dramatically in the past few years. The solvents used have the major influence on the greenness of the method. Most conventional high-performance liquid chromatography methods employed utilize solvents that are "hazardous for the environment". In the present study, two-factor three-level response surface design was exploited to develop eco-friendly chromatographic methods for two different mixtures. The first one was atorvastatin and amlodipine and the second one was amlodipine, perindopril, and indapamide. As it is nontoxic to the environment, ethanol was used as the organic modifier in the mobile phase. The separation of the first mixture was attained using phosphate buffer (pH 7)/ethanol (42:58 v/v), and the second mixture was fully resolved using phosphate buffer (pH 5)/ethanol (40:60 v/v). The use of high-performance liquid chromatography allows excellent resolution in a short run time, hence, less waste was produced. The greenness of the developed methods was assessed by two evaluation tools, namely, National Environmental Methods Index and analytical eco-scale, and found to be excellent green analytical methods. Moreover, the developed methods were compared with other reported methods regarding accuracy and greenness and were found to be perfect alternatives to reported methods for separation and quantification of the mixtures.

A hybrid molecularly imprinted polymer coated quantum dot nanocomposite optosensor for highly sensitive and selective determination of salbutamol in animal feeds and meat samples.

A hybrid molecularly imprinted polymer (MIP)-coated quantum dot (QD) nanocomposite was synthesized and applied as a fluorescence probe for the highly sensitive and selective determination of salbutamol. The hybrid MIP-coated QD nanocomposite was synthesized via a copolymerization process in the presence of thioglycolic acid capped CdTe QDs with salbutamol as a template, 3-aminopropyltriethoxysilane as the functional monomer, and tetraethyl orthosilicate as a cross-linker. The optimum molar ratio of template, monomer, and cross-linker was 1:6:20. The fluorescence intensity of the hybrid MIP-coated QDs was efficiently quenched after salbutamol rebound to the recognition sites, as a result of charge transfer from QDs to salbutamol. The synthesized hybrid MIP-coated QD nanocomposite showed high sensitivity and good selectivity toward salbutamol. Under the optimal recognition conditions, the fluorescence intensity was quenched linearly with increasing concentration of salbutamol in the range from 0.10 to 25.0μgL-1, with a detection limit of 0.034μgL-1. The hybrid optosensor developed was successfully applied in the determination of salbutamol in animal feeds and meat samples. Satisfactory recoveries were obtained in the range from 85% to 98%, with a standard deviation of less than 8%. Furthermore, the accuracy of the hybrid MIP-coated QD nanocomposite was investigated by comparison with a conventional high-performance liquid chromatography method, with the results obtained with two methods agreeing well with each other. The advantages of this sensing method are simplicity, rapidity, cost-effectiveness, high sensitivity, and good selectivity. Graphical Abstract The synthesis of hybrid MIP-coated QDs nanocomposite.

Development and validation of an ultra-performance convergence chromatography method for the quality control of Angelica gigas Nakai.

Ultra-performance convergence chromatography, which integrates the advantages of supercritical fluid chromatography and ultra high performance liquid chromatography technologies, is an environmentally friendly analytical method that uses dramatically reduced amounts of organic solvents. An ultra-performance convergence chromatography method was developed and validated for the quantification of decursinol angelate and decursin in Angelica gigas using a CSH Fluoro-Phenyl column (2.1mm×150mm, 1.7μm) with a run time of 4min. The method had an improved resolution and a shorter analysis time in comparison to the conventional high-performance liquid chromatography method. This method was validated in terms of linearity, precision, and accuracy. The limits of detection were 0.005 and 0.004μg/mL for decursinol angelate and decursin, respectively, while the limits of quantitation were 0.014 and 0.012μg/mL, respectively. The two components showed good regression (correlation coefficient (r2 ) > 0.999), excellent precision (RSD < 2.28%), and acceptable recoveries (99.75-102.62%). The proposed method can be used to efficiently separate, characterize, and quantify decursinol angelate and decursin in Angelica gigas and its related medicinal materials or preparations, with the advantages of a shorter analysis time, greater sensitivity, and better environmental compatibility.

A Fast and Sensitive Method for the Detection of Leuprolide Acetate: A High-Throughput Approach for the In Vitro Evaluation of Liquid Crystal Formulations

The suitability of using fluorescence spectroscopy to rapidly assay drug release by quantifying the time-dependent increase in total intrinsic protein fluorescence was assessed. Leuprolide acetate, a synthetic nonapeptide analogue of gonadotropin-releasing hormone (GnRH or LHRH), is the active pharmaceutical ingredient used to treat a wide range of sex hormone-related disorders, including advanced prostatic cancer, endometriosis, and precocious puberty. During the in vitro evaluation of drug delivery technologies for leuprolide acetate, one of the most time-consuming steps is the detection and accurate quantification of leuprolide release from formulation candidates. Thus far, the dominant means for leuprolide detection involves conventional multistep high-performance liquid chromatography (HPLC) methods, requiring sampling, dilutions, sample filtration, and chromatography, which can take up to 40 min for each sample. With the increasing demand for assay adaptation to high-throughput format, here we sought to exploit fluorescence spectroscopy as a tool to develop a novel method to rapidly assay the in vitro release of leuprolide acetate. By utilizing the intrinsic fluorescence of the tryptophan (Trp) and tyrosine (Tyr) amino acid residues present in the leuprolide nonapeptide, the in vitro release from liquid crystal formulations was accurately quantified as a function of fluorescence intensity. Here, we demonstrate that assaying leuprolide release using intrinsic protein fluorescence in a 96-well format requiring volumes as low as 100 μL is a cost-effective, rapid, and highly sensitive alternative to conventional HPLC methods. Furthermore, the high signal-to-noise ratios and robust Z'-factors of >0.8 indicate high sensitivity, precision, and feasibility for miniaturization, high-throughput format adaptation, and automation.

Rapid Screening of Antioxidant Anthocyanins in Autochthonous Nero d’Avola Grape Clones by Pre-column DPPH Reaction Coupled to UHPLC-UV/Vis-IT-TOF: a Strategy to Combine Chemical data and Genetic Diversity

Grape is a rich source of bioactive compounds; among them anthocyanins are associated to many healthy properties, possessing a good antioxidant activity. In this work, we developed a fast and simple screening of antioxidant anthocyanins in six Sicilian Nero d’Avola autochthonous grape clones. The method was based on the pre-column reaction with 2,2′-diphenyl-1-picrylhydrazyl radical followed by the rapid separation by ultra high-pressure liquid chromatography coupled with ultraviolet and tandem mass spectrometry detection. Peak areas of antioxidant anthocyanins significantly reduced or even disappeared. The entire method took only 45 min per sample, showing good retention time and peak area repeatability with maximum CV% values ≤0.86 and 6.84, respectively. Samples rich in delphinidin derivatives showed lowest IC50 values, since those compounds possess the highest scavenging ability. The developed setup was less complex than online approaches and faster with respect to conventional high-performance liquid chromatography methods, taking advantage of ultra high-performance conditions, coupled for the first time with pre-column 2,2′-diphenyl-1-picrylhydrazyl assay. The proposed strategy is a valid tool for a rapid screening of antioxidant anthocyanins in grape samples, useful to correlate the genetic diversity with the production of secondary metabolites as well to assess their activity in nutraceutical products rich in anthocyanins.

Rapid and highly sensitive analysis of chlorophylls and carotenoids from marine phytoplankton using ultra-high performance liquid chromatography (UHPLC) with the first derivative spectrum chromatogram (FDSC) technique

We developed a rapid and highly sensitive analytical method for chlorophylls and carotenoids derived from marine phytoplankton using ultra-high performance liquid chromatography (UHPLC). High-performance liquid chromatography (HPLC) has been widely used in phytoplankton pigment analysis since the 1980s for estimating the abundance, composition, and photosynthetic physiology of natural algal assemblages or laboratory cultures. However, the run-time of the HPLC analyses is generally ca. 30min or more, which is time-consuming for analysts. Our UHPLC technique enabled us to complete the separations of chlorophylls and carotenoids from marine phytoplankton within 7min with similar resolution as conventional HPLC methods. The analytical method was tested on authentic pigment standards, marine phytoplankton cultures, and field samples that were collected from the central tropical and subarctic Pacific plus the neritic Bering Sea. Critical pigment pairs that generally co-eluted as a single peak were successively resolved by obtaining the first derivative spectrum chromatograms (FDSCs) with a photodiode array (PDA) detector based on differences in pigment absorption spectra, e.g., chlorophyll (Chl) c2 and Mg 2,4 divinyl (DV) pheoporphyrin a5 monomethyl ester (MgDVP), as well as DVChl b and Chl b. Because the maximum injection volume of UHPLC is generally lower than that of HPLC to minimize the unwanted broadening of chromatographic peaks, the detection sensitivity needed to be increased, especially for oligotrophic seawater samples with low pigment concentration. To overcome this sensitivity issue, a PDA detector equipped with an 85mm path length capillary cell was used with a fluorescence detector. As a result, the limit of quantitation (LOQ) as determined by absorbance was of the order of 0.1ng for chlorophylls and carotenoids. Furthermore, a bead-beating technique using N,N-dimethylformamide (DMF) and zirconia beads was utilized to minimize the volume of the organic solvent used for pigment extraction. Our UHPLC method can replace the conventional HPLC techniques, and allows us to yield high-throughput data of the chlorophylls and carotenoids derived from marine phytoplankton.

Enzyme-Linked Immunosorbent Assay for Hyodeoxycholic Acid in Pharmaceutical Products Using a Monoclonal Antibody

Herein is reported an immunochemical approach to determine hyodeoxycholic acid using hybridoma-secreting monoclonal antibodies. The hyodeoxycholic acid-specific antibody was produced by fusing splenocytes immunized with a hyodeoxycholic acid-bovine serum albumin conjugate with a hypoxanthine–aminopterin–thymidine-sensitive mouse myeloma cell line (SP2/0). The antibody was highly specific for hyodeoxycholic acid, with less than 0.05 percent cross-reactivity to over fifty structurally related compounds. The antihyodeoxycholic acid monoclonal antibody was then used to develop a rapid, specific, and sensitive indirect competitive enzyme-linked immunosorbent assay (icELISA) for the determination of hyodeoxycholic acid in pharmaceutical compounds. The linear dynamic range was from 0.48 to 62.5 nanograms per milliliter with an IC50 value of 8 nanograms per milliliter. The icELISA results correlated well with a conventional high-performance liquid chromatography method for the determination of hyodeoxycholic acid (R2 = 0.9982). This study shows that the icELISA method was successfully applied to the quantification of hyodeoxycholic acid in pharmaceutical products.

A Sequential Injection Analysis Method for the Determination of Glyphosate and Aminomethylphosphonic Acid in Water Samples

A conventional laboratory method for the determination of glyphosate and its degradation product aminomethylphosphonic acid (AMPA) is high performance liquid chromatography (HPLC) followed by post column derivatization with o-phthaldialdehyde (OPA). However, AMPA is partly decomposed in the process causing a deviation in the AMPA detection or even making a simultaneous detection of glyphosate and AMPA with the described postcolumn procedure impossible. We used a compact sequential injection analysis system and optimized the process conditions for both analytes independently from each other. The process conditions were adjusted to the different chemical characteristics of AMPA (primary amine) and glyphosate (secondary amine), which needs to be oxidized to a primary amine prior to the derivatization. An ion exchange column was included in the system to eliminate amines interfering with the method. Limits of detection of 16 and 9 µg L -1 for glyphosate and AMPA, respectively are similar to those achieved with HPLC methods reported in literature, but compared to conventional HPLC methods only small amounts of reagents are consumed.

Quality evaluation of Semen Cassiae (Cassia obtusifoliaL.) by using ultra‐high performance liquid chromatography coupled with mass spectrometry

A sensitive and reliable ultra-high performance liquid chromatography-electrospray ionization-tandem mass spectrometry has been developed and partially validated to evaluate the quality of Semen Cassiae (Cassia obtusifolia L.) through simultaneous determination of 11 anthraquinones and two naphtha-γ-pyrone compounds. The analysis was achieved on a Poroshell 120 EC-C(18) column (100 mm × 2.1 mm, 2.7 μm; Agilent, Palo Alto, CA, USA) with gradient elution using a mobile phase that consisted of acetonitrile-water (30 mM ammonium acetate) at a flow rate of 0.4 mL/min. For quantitative analysis, all calibration curves showed perfect linear regression (r(2) > 0.99) within the testing range. This method was also validated with respect to precision and accuracy, and was successfully applied to quantify the 13 components in nine batches of Semen Cassiae samples from different areas. The performance of developed method was compared with that of conventional high-performance liquid chromatography method. The significant advantages of the former include high-speed chromatographic separation, four times faster than high-performance liquid chromatography with conventional columns, and great enhancement in sensitivity. This developed method provided a new basis for overall assessment on quality of Semen Cassiae.

Characterization and relative response factor determination of process related impurity in Naproxen by nuclear magnetic resonance spectroscopy

In the impurity profile of naproxen around 0.6% unknown polar impurity was detected by high performance liquid chromatography (HPLC). The product ion spectrum of the impurity and Naproxen was recorded in LCMS/MS and the fragmentation pattern of the impurity was observed to be similar to the fragmentation pattern of Naproxen with only a difference of two atomic mass units. The high resolution mass spectrum (HRMS) of the impurity displayed a protonated molecular ion at m/z 229.0863, which corresponds to the pseudomolecular formula C14H13O3+. Based on LC/MS/MS, HRMS, 1D and 2D NMR data, the structure of the impurity was characterized as 2-(6-methoxynaphthalen-2-yl)acrylic acid. The acrylic acid impurity was synthesized in the laboratory and co injected in HPLC to confirm the retention time. RRF of the impurity was determined by 1H NMR method and also by conventional HPLC slope method and the RRF values are found to be 6.11 and 5.64, respectively. The values are comparable and 1H NMR method of RRF determination is complimentary and can be effectively used as an alternative method to conventional HPLC method especially in early stages of development when availability of impurity standards is not possible.

High-efficiency high performance liquid chromatographic analysis of red wine anthocyanins

The analysis of anthocyanins in natural products is of significant relevance in recent times due to the recognised health benefits associated with their consumption. In red grapes and wines in particular, anthocyanins are known to contribute important properties to the sensory (colour and taste), anti-oxidant- and ageing characteristics. However, the detailed investigation of the alteration of these compounds during wine ageing is hampered by the challenges associated with the separation of grape-derived anthocyanins and their derived products. High performance liquid chromatography (HPLC) is primarily used for this purpose, often in combination with mass spectrometric (MS) detection, although conventional HPLC methods provide incomplete resolution. We have previously demonstrated how on-column inter-conversion reactions are responsible for poor chromatographic efficiency in the HPLC analysis of anthocyanins, and how an increase in temperature and decrease in particle size may improve the chromatographic performance. In the current contribution an experimental configuration for the high efficiency analysis of anthocyanins is derived using the kinetic plot method (KPM). Further, it is shown how analysis under optimal conditions, in combination with MS detection, delivers much improved separation and identification of red wine anthocyanins and their derived products. This improved analytical performance holds promise for the in-depth investigation of these influential compounds in wine during ageing.

Clinical implications of the early detection of urinary microalbumin in diabetic patients using a new high performance liquid chromatography (HPLC) method

We assessed urine albumin in 180 diabetics by conventional immunonephelometry (IN) and HPLC methods. Those with HPLC but not IN detectable albuminuria had higher BP, worse glycaemic control and more vascular events (p<0.05 for all) but received fewer vascular risk reduction treatments.

Simultaneous determination of d-amino acids by the coupling method of d-amino acid oxidase with high-performance liquid chromatography

An enzymatic assay system of d-amino acids was established using the d-amino acid oxidase of Schizosaccharomyces pombe. In this method, the enzyme converts the d-amino acids to the corresponding α-keto acids, which are then reacted with 1,2-diamino-4,5-methylenedioxybenzene (DMB) in an organic solvent. The resultant fluorescent compounds are separated and quantified by high-performance liquid chromatography (HPLC). Use of an organic solvent following the α-keto acid modification with DMB prevents the non-enzymatic deamination of l-amino acids, which are generally present at much higher concentrations than d-amino acids in biological samples. With this method, d-Glu, d-Asn, d-Gln, d-Ala, d-Val, d-Leu, d-Phe, and d-Ile can be quantified in the order of micromolar, and other d-amino acids except d-Asp can be assayed within a sensitivity range of 50–100 μM. The established enzymatic method was used to analyze the d-amino acid contents in human urine. The concentration of d-Ser obtained using this enzymatic method (223 μM) was in good agreement with that obtained using the conventional HPLC method (198 μM). The enzymatic method also demonstrated that the human urine contained 5.45 μM of d-Ala and 0.91 μM of d-Asn. Both d-amino acids were difficult to be identified using the conventional method, because the large signals from l-amino acids masked those from d-amino acids. The enzymatic method that we have developed can circumvent this problem.

Evaluation of Polyphenol Composition in Red Leaves from Different Varieties of Vitis vinifera

In order to evaluate sustainable sources of high-quality pharmaceutical starting material, a comparative study of 135 samples of red vine leaves of different origin was conducted to determine flavonol, anthocyanin and polyphenol contents. Total flavonol content was found to be between 0.6 % and 3.5 %, anthocyanin content between 0.2 % and 1.45 % and polyphenol content between 4.6 % and 18.9 %. The results are discussed in view of published data on red vine leaf components. In order to define the optimum timeframe for collecting leaves without damaging their grape-bearing vines the ontogenetic development of red vine leaves constituents was studied. High-performance liquid chromatography (HPLC) methods used for determining anthocyanins and flavonols in red vine leaves were validated and findings were compared to results produced by assays described in the French Pharmacopoeia. Whereas the correlation between conventional photometric and HPLC methods was satisfactory for anthocyanins, the correlation between the pharmacopoeia assay for total polyphenols and the HPLC analysis for flavonols was poor. As flavonol compounds are considered relevant for the vasoprotective effect of red vine leaves, their content in starting material used in the production of herbal medicines needs to be quantified.