Comparison of Different Methods for the Determination of Phenylalanine Hydroxylase Activity in Rat Liver and Euglena gracilis

1984 ◽  
Vol 39 (7-8) ◽  
pp. 728-733 ◽  
Author(s):  
Rita M. Fink ◽  
Erich F. Elstner
Keyword(s):  
Rat Liver ◽  
Euglena Gracilis ◽  
Phenylalanine Hydroxylase ◽  
Hplc Method ◽  
Enzymic Activity ◽  
Hydroxylase Activity ◽  
Product Separation ◽  
Tyrosine Concentration ◽  
Layer Chromatography

Abstract Three different methods for the determination of phenylalanine hydroxylase activity have been compared: a) Differential photometric assay of the increase in tyrosine concentration in the presence of phenylalanine; b) Product separation by thin layer chromatography and scintillation counting of the [14C]tyrosine formed;c) HPLC separation and spectrofluorometric quantification of derivatized amino acids. A comparison of the activities of phenylalanine hydroxylase in rat liver and Euglena gracilis clearly showed that only rat liver contains this enzymic activity as shown by methods b) and c) although pseudo-activity of Euglena gracilis preparations was found during the spectrophotometric test a). The HPLC method proved to be the fastest, most reliable and convenient method for direct tyrosine determination and thus for measuring phenylalanine hydroxylase activity.

scholarly journals Analysis of FlavoneC-Glycosides in the Leaves ofClinacanthus nutans(Burm. f.) Lindau by HPTLC and HPLC-UV/DAD

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
June Lee Chelyn ◽  
Maizatul Hasyima Omar ◽  
Nor Syaidatul Akmal Mohd Yousof ◽  
Ramesh Ranggasamy ◽  
Mohd Isa Wasiman ◽  
...  
Keyword(s):  
Rapid Identification ◽  
Hplc Method ◽  
Chemical Markers ◽  
Step Method ◽  
Qualitative And Quantitative ◽  
Clinacanthus Nutans ◽  
Acetic Acid Water ◽  
Layer Chromatography ◽  
Tlc Separation

Clinacanthus nutans(family Acanthaceae) has been used for the treatment of inflammation and herpes viral infection. Currently, there has not been any report on the qualitative and quantitative determination of the chemical markers in the leaves ofC. nutans. TheC-glycosidic flavones such as shaftoside, isoorientin, orientin, isovitexin, and vitexin have been found to be major flavonoids in the leaves of this plant. Therefore, we had developed a two-step method using thin-layer chromatography (TLC) and high pressure liquid chromatography (HPLC) for the rapid identification and quantification of the flavonesC-glycosides inC. nutansleaves. The TLC separation of the chemical markers was achieved on silica gel 60 plate using ethyl acetate : formic acid : acetic acid : water (100 : 11 : 11 : 27 v/v/v/v) as the mobile phase. HPLC method was optimized and validated for the quantification of shaftoside, orientin, isovitexin, and vitexin and was shown to be linear in concentration range tested (0.4–200 μg/mL,r2≥ 0.996), precise (RSD ≤ 4.54%), and accurate (95–105%). The concentration of shaftoside, orientin, vitexin, and isovitexin inC. nutansleave samples was 2.55–17.43, 0.00–0.86, 0.00–2.01, and 0.00–0.91 mmol/g, respectively.

scholarly journals Determination of flavones in species of Thymus L. (Lamiaceae) from Macedonian flora

2001 ◽  
Vol 47 ◽  
pp. 9-14
Author(s):  
Svetlana Kulevanova ◽  
Marina Stefova ◽  
Tatjana Kadifkova Panovska ◽  
Jasmina Tonic ◽  
Trajce Stafilov
Keyword(s):  
Ethyl Acetate ◽  
High Performance ◽  
Gradient Elution ◽  
Hplc Method ◽  
Total Flavonoids ◽  
Column Temperature ◽  
Plant Samples ◽  
Layer Chromatography ◽  
Hydrolysis Of

Assay of flavonoids in extracts of seven Thymus L. (Lamiaceae) species from Macedonia including identification and quantification was performed. Extracts obtained after hydrolysis of air dried samples (A1) were analyzed by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). Luteolin and apigenin were identified in comparison to authentic standard substances. The content of total flavonoids in plant samples determined by UV-Vis spectrometry (with AlCl3) ranged from 0.05-0.13 %. Two other extracts were prepared by extraction with a mixture of ethanol:water (7:3, V/V), evaporation until only water remained and extraction first with diethylether (A2) and secondly with ethyl acetate (A3). The content of flavonoids in diethyl-ether and ethyl acetate extracts ranged from 52.5-244.4 mg·ml-1 and 48.7 -117.5 mg·ml-1, respectively. For quantification of luteolin and total flavonoids the HPLC method was applied, using reverse phase column C18, mobile phase consisting of 5% acetic acid and methanol in gradient elution mode and column temperature set to 40 o C. The content of luteolin in the plant samples ranged from 0.23-0.48 % (m/m), while the content of total flavonoids was found to be 0.26-0.52 %.

scholarly journals Development of Chromatographic Methods for Determination of Agrimoniin and Related Polyphenols in Pharmaceutical Products

2009 ◽  
Vol 92 (2) ◽  
pp. 410-418 ◽  
Author(s):  
Izabela Fecka
Keyword(s):  
Silica Gel ◽  
Extraction Procedure ◽  
Pharmaceutical Products ◽  
Hplc Method ◽  
Mobile Phases ◽  
Sample Extraction ◽  
Unmodified Silica ◽  
Short Time ◽  
Layer Chromatography

Abstract Thin-layer chromatography (TLC) and liquid chromatography (LC) methods were developed for the qualitative and quantitative determination of agrimoniin, pedunculagin, ellagic acid, gallic acid, and catechin in selected herbal medicinal products from Rosaceae: Anserinae herba, Tormentillae rhizoma, Alchemillae herba, Agrimoniae herba, and Fragariae folium. Unmodified silica gel (TLC Si60, HPTLC LiChrospher Si60) and silica gel chemically modified with octadecyl or aminopropyl groups (HPTLC RP18W and HPTLC NH2) were used for TLC. The best resolution and selectivity were achieved with the following mobile phases: diisopropyl etheracetoneformic acidwater (40 30 20 10, v/v/v/v), tetrahydrofuranacetonitrilewater (30 10 60, v/v/v), and acetoneformic acid (60 40, v/v). Concentrations of the studied herbal drugs were determined by using a Chromolith Performance RP-18e column with acetonitrilewaterformic acid as the mobile phase. Determinations of linearity, range, detection and quantitation limits, accuracy, precision, and robustness showed that the HPLC method was sufficiently precise for estimation of the tannins and related polyphenols mentioned above. Investigations of suitable solvent selection, sample extraction procedure, and short-time stability of analytes at storage temperatures of 4 and 20C were also performed. The percentage of agrimoniin in pharmaceutical products was between 0.57 and 3.23.

scholarly journals Simultaneous Determination of Alprazolam and Fluoxetine Hydrochloride in Tablet Formulations by High-Performance Column Liquid Chromatography and High-Performance Thin-Layer Chromatography

2009 ◽  
Vol 92 (4) ◽  
pp. 1082-1088 ◽  
Author(s):  
Rashmin B Patel ◽  
Mrunali R Patel ◽  
Madhira B Shankar ◽  
Kashyap K Bhatt
Keyword(s):  
Simultaneous Determination ◽  
Mobile Phase ◽  
High Performance ◽  
Hplc Method ◽  
Uv Detection ◽  
Fluoxetine Hydrochloride ◽  
Phase Quantification ◽  
Tablet Formulations ◽  
Layer Chromatography

Abstract This paper describes validated HPLC and HPTLC methods for simultaneous determination of alprazolam (ALP) and fluoxetine hydrochloride (FXT) in pure powder and formulation. The HPLC separation was achieved on a Nucleosil C8 column (150 mm length, 4.6 mm id, 5 m particle size) using acetonitrilephosphate buffer pH 5.5 (45 + 55, v/v) as the mobile phase at a flow rate of 1.0 mL/min at ambient temperature. The HPTLC separation was achieved on an aluminum-backed layer of silica gel 60F254 using acetonetolueneammonia (6.0 + 3.5 + 0.5, v/v/v) as the mobile phase. Quantification in the HPLC method was achieved with UV detection at 230 nm over the concentration range 414 g/mL for both drugs, with mean recovery of 99.95 0.38 and 99.85 0.56 for ALP and FXT, respectively. Quantification in the HPTLC method was achieved with UV detection at 230 nm over the concentration range of 4001400 ng/spot for both drugs, with mean recovery of 99.32 0.45 and 99.78 0.81 for ALP and FXT, respectively. These methods are simple, precise, and sensitive, and they are applicable for the simultaneous determination of ALP and FXT in pure powder and formulations.

Influence of amino acids on rat liver phenylalanine hydroxylase activity

1964 ◽  
Vol 206 (2) ◽  
pp. 341-344 ◽  
Author(s):  
R. A. Freedland ◽  
M. C. Krakowski ◽  
Harry A. Waisman
Keyword(s):  
Amino Acids ◽  
Enzyme Activity ◽  
Rat Liver ◽  
Phenylalanine Hydroxylase ◽  
Marked Decrease ◽  
Hydroxylase Activity ◽  
Short Term ◽  
Ad Libitum

The influence of oral and of injected amino acids on rat liver phenylalanine hydroxylase activity has been studied in both short-term and extended experiments. Phenylalanine added to the diet in moderate amounts ordinarily causes an increase in enzyme activity, but when excess phenylalanine was included in the diet, it caused a marked decrease in the enzyme activity in rats fed ad libitum. Tryptophan or tyrosine added to the diet in the absence of added phenylalanine increased the enzyme activity in these rats. Excess tryptophan, but not tyrosine, could counteract the effect of phenylalanine on this enzyme after an 18-hr fast. There were dramatic differences in phenylalanine hydroxylase activity when various amino acids were injected. Injection of tryptophan or glycine decreased the phenylalanine hydroxylase activity in rat liver. Injections of other amino acids, notably phenylalanine and tyrosine, had no effect on the phenylalanine hydroxylase activity 5 hr after injection.

scholarly journals DEVELOPMENT OF METHODS FOR DETERMINATION OF SPECIFIC IMPURITIES IN THE GLUTATIONION RESTORED SUBSTANCE

2019 ◽  
Vol 6 (6) ◽  
pp. 535-547
Author(s):  
K. A. Alexeeva ◽  
D. I. Pisarev ◽  
A. Yu. Malyutina ◽  
N. N. Boyko
Keyword(s):  
Amino Acids ◽  
High Performance ◽  
Hplc Method ◽  
Low Molecular Weight ◽  
Chromatography Method ◽  
Rp Hplc ◽  
Liquid Chromatography Method ◽  
Layer Chromatography ◽  
Better Than

Glutathione (γ-L-glutamyl-L-cysteinylglycine) is the most important low molecular weight intracellular thiol tripeptide consisting of three amino acids – glycine, cysteine and glutamic acid. In Russian pharmacopoeia there is no regulatory documentation for glutathione, therefore, the development of a pharmacopoeial item for the specified substance is a relevant problem.The aim of the article is the development of methods for determining foreign specific impurities in glutathione.Materials and methods. The substance of glutathione reduced (CAS 70-18-8, EC 2007254, Applichem, Germany) containing impurities, and a standard sample of reduced glutathione (Sigma Aldrich, Japan) were used as the objects of the study. The analysis was carried out by using a high-performance liquid chromatography method in the reverse phase version and a thin layer chromatography method. The chromatography using RP HPLC was performed after preliminary derivatization of glutathione and its specific impurities with dancil chloride. Specific impurities in glutathione are dipeptides and amino acids. Therefore, they, like glutathione, can react with dancil chloride. Dancil derivatives are formed, and they can be determined by chromatographic separation.Results. As a result of chromatography by the method of RP HPLC of derivatized dancil chloride glutathione it has been established that this reaction makes it possible to detect impurities in it. Glutathione derivatives are well separated by chromatography by implementing the method of RP HPLC and have different absorption maxima. The glutathione derivative had an absorption maximum at λmax=284 nm. The derivatives belonging to specific glutathione impurities absorb at λmax=288 nm and λmax=296 nm. The data obtained using RP HPLC were confirmed by TLC in the isopropanol-water (2:1) system. Three components were found out, one of which corresponds to glutathione, while two others are impurities.Conclusion. Methods for determining impurities in the glutathione substance using RP HPLC methods with preliminary derivatization with dancil chloride and TLC with ninhydrin detection have been worked out. A comparative analysis of the data obtained makes it possible to state that the OF-HPLC method with pre-column derivatization is more reliable, since it is more sensitive to impurities, and also makes it possible to study the UV profiles of impurity components better than the TLC method. Therefore, for the detection of impurities in the substance of glutathione, it is more preferable to use RP-HPLC with pre-column derivatization. The results of this study can be recommended for inclusion in the regulatory documentation on the substance of glutathione in the section “Impurities”.

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