scholarly journals Year-Long Validation Study and Reference Values for Urinary Amino Acids Using a Reversed-Phase HPLC Method

2001 ◽  
Vol 47 (3) ◽  
pp. 575-583 ◽  
Author(s):  
Rafael Venta
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Reference Values ◽  
Amino Acid Analysis ◽  
Reference Intervals ◽  
Reversed Phase ◽  
Hplc Method ◽  
Rp Hplc ◽  
Age Related

Abstract Background: Reversed-phase HPLC (RP-HPLC) has become an alternative to ion-exchange chromatography for amino acid analysis in biological fluids. However, validation studies for its urine application are limited, and the corresponding reference values have not been reported extensively. We studied the long-term performance of a commercial HPLC method for urine amino acid analysis and established specific age-related reference values for urine amino acid excretion. Methods: Method performance was continuously assessed by recovery and precision studies with urine samples and controls, respectively. Healthy individuals were prospectively analyzed throughout a 5-year period. Excretion of individual amino acids, expressed as mmol/mol of creatinine, was included in six age-related groups for random urine samples (0–1 month, 1–12 months, 1–3 years, 3–8 years, 8–16 years, and >16 years) and in two groups for 24-h urine collections (8–16 years and >16 years). Results: Over a 1-year period, CVs for retention times were <0.5% and 3.3% for within- and between-run imprecision, respectively. For amino acid concentrations, within-run CVs were 2.9–17% and between-run CVs were 7.1–46% for the same period. Amino acid recoveries were 78–122%. Reference intervals for 35 amino acids were calculated and compared with the concentrations observed in patients diagnosed with specific pathologies. A few statistically significant differences were found between the reference intervals derived using random and 24-h urine collections. Conclusions: Long-term reliability of the RP-HPLC method for urine amino acid analysis has been demonstrated. Representative age-related reference intervals for the RP-HPLC method in both random urine and 24-h urine collections have been established, and their feasibility for diagnosis of aminoaciduria has been shown. These intervals could serve as a guide for laboratories changing to HPLC methods.

Within-subject and between-subject biological variation of first morning void urine amino acids in 12 healthy subjects

2020 ◽  
Vol 58 (11) ◽  
pp. 1901-1909
Author(s):  
Hamit Hakan Alp ◽  
Halil İbrahim Akbay ◽  
Erdem Çokluk ◽  
Zubeyir Huyut ◽  
Sıddık Keskin ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Analysis ◽  
Healthy Subjects ◽  
Total Error ◽  
Reference Intervals ◽  
Biological Variation ◽  
Reference Change Value ◽  
Index Of Individuality ◽  
Variance Homogeneity

AbstractBackgroundUrine amino acid analysis is used for the assessment of various diseases. The aim of this study was to estimate the valid biological variation (BV) components (within- and between-subjects) required for the safe clinical application of free urine amino acids.MethodsFirst morning void urine samples were taken from 12 healthy subjects (five females, seven males) once a week for 10 consecutive weeks, and amino acid analysis was performed using an Agilent 6470 triple quadrupole tandem mass spectrometer instrument. The obtained data were subjected to normality, outlier and variance homogeneity analyses prior to coefficient of variation (CV) analysis. Within- and between-subject BV values (CVI and CVG) of 39 amino acids were determined for all subjects. In addition, the index of individuality (II), reference change value (RCV), imprecision, bias and total error were estimated using BV data obtained from our study.ResultsThe CVI values ranged from 8.9 (histidine) to 36.8% (trans-4-hydroxyprolin), while the CVG values ranged from 25.0 (1-methyl-L-histidine) to 63.3% (phenylalanine). The II value of most amino acids was less than 0.6 and ranged between 0.21 and 0.88. The imprecision, bias and total error ranged between 4.45 and 16.6, between 7.69 and 16.6, and between 18.4 and 43.2, respectively.ConclusionsThis study, designed according to a rigorous protocol, has the feature of being the first to give information about BV data of urine amino acids. We believe that the reference intervals have a limitation in the evaluation of consecutive results from an individual, so the use of RCV would be more appropriate.

scholarly journals Amino acid analysis at the picomole level. Application to the C-terminal sequence analysis of polypeptides

1981 ◽  
Vol 199 (3) ◽  
pp. 547-555 ◽  
Author(s):  
J Y Chang ◽  
R Knecht ◽  
D G Braun
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Analysis ◽  
Protein Hydrolysate ◽  
Visible Region ◽  
Reversed Phase ◽  
Terminal Sequence ◽  
Naturally Occurring ◽  
Ion Exchange Separation ◽  
Buffer Mixture

Amino acids labelled with dimethylaminoazobenzenesulphonyl chloride can be separated by reversed-phase high-pressure liquid chromatography and detected in the visible region (436 nm). All 19 naturally occurring amino acids can be separated on a Zorbax ODS column by employing two different gradient systems consisting of an acetonitrile/aqueous buffer mixture. As little as 2--5 pmol of an individual dimethylaminoazobenzenesulphonyl-amino acid can be quantitatively analysed with reliability, and only 10--30 ng of the dimethylaminoazobenzenesulphonylated protein hydrolysate is needed for each complete amino acid analysis. This new technique is as sensitive as any of the current amino acid analysis methods involving ion-exchange separation plus fluorescence detection, and is technically much simpler. By the combination of this sensitive amino acid-analysing technique with carboxypeptidase, we have been able to determine the C-terminal sequence of polypeptides at the picomole level.

Age-related reference values for urinary excretion of sialic acid and deoxysialic acid: application to diagnosis of storage disorders of free sialic acid

1995 ◽  
Vol 41 (4) ◽  
pp. 544-547 ◽  
Author(s):  
J Romppanen ◽  
I Mononen
Keyword(s):  
Sialic Acid ◽  
Urinary Excretion ◽  
Reference Values ◽  
Reference Interval ◽  
Reference Intervals ◽  
Mean Value ◽  
Hplc Method ◽  
Age Related ◽  
Free Sialic Acid ◽  
Storage Disorders

Abstract We have established by HPLC age-related reference intervals for sialic acid urinary excretion in 364 control individuals to assist in evaluating the clinical significance of the free sialic acid concentration in urine. In addition, an HPLC method for quantitative analysis of free deoxysialic acid was developed, and age-related reference intervals for excretion of this compound in urine were established. In patients with storage disorders of free sialic acid (n = 11) the sialic acid excretion was increased 2- to 35-fold, compared with the mean value of the control subjects in the corresponding age group, and exceeded the interval in each case. The excretion of deoxysialic acid was within the reference interval in all of the patients, indicating that its metabolism was not affected in the disorders. The age-related reference values assist in evaluating the excretion of free sialic acid in the diagnosis of storage disorders of free sialic acid, especially in young children.

Diet-related reference values for plasma amino acids in newborns measured by reversed-phase HPLC

1990 ◽  
Vol 36 (11) ◽  
pp. 1922-1927 ◽  
Author(s):  
P H Scott ◽  
S Sandham ◽  
S E Balmer ◽  
B A Wharton
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Reference Values ◽  
Detailed Examination ◽  
Reversed Phase ◽  
Reversed Phase Hplc ◽  
Highly Sensitive ◽  
Formula Group ◽  
Amino Acid Concentrations ◽  
Breast Fed

Abstract We have measured by reversed-phase HPLC concentrations of amino acids in plasma in groups of 80 normal appropriate-weight term babies fed from birth either a casein formula (WhiteCap SMA, n = 26), a whey formula (Gold Cap SMA, n = 26), or breast milk (n = 28). They were studied from day 11 to week 15 postpartum. The trend was towards an increase in amino acid concentrations in plasma with age, more marked in formula-fed than in breast-fed infants. Reference values were derived for each group. Both formula-fed groups showed several differences from the breast-fed group. Detailed examination indicated that tyrosine, phenylalanine, and methionine concentrations were increased in the casein-fed group greater than 20% of the time, but only threonine was similarly increased in the whey-fed group. Other amino acids, different ones for each formula group, were increased less frequently. There were no consistent correlations with any aspect of infant growth. Appropriate reference values are important for interpreting amino acid concentrations in plasma from newborns and for evaluating the effects of any future dietary modifications to infant formulas. HPLC analysis provides a suitable highly sensitive method for undertaking such studies.

scholarly journals Complexation of Calix[4]arene bis-Hydroxymethylenediphosphonic Acid with Amino acids. Binding Constants Determination by RP HPLC Method

2015 ◽  
Vol 3 (2) ◽  
pp. 93-100 ◽  
Author(s):  
Vitaly Kalchenko ◽  
Olga Kalchenko ◽  
Sergiy Cherenok
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Electrostatic Interactions ◽  
Water Solution ◽  
Binding Constants ◽  
Hplc Method ◽  
Log P ◽  
Rp Hplc ◽  
Solvophobic Interactions ◽  
Positively Charged

Host-Guest complexation of calixarene-bis-hydroxymethylenediphosphonic acid with 17 amino acids in water solution had been studied by the RP HPLC and molecular modelling methods. It had been shown the binding constants of the complexes are depended on the nature of the amino acid residue, log P and pKa of the acids. The complexation is mainly determined by the electrostatic interactions between the positively charged nitrogen atom of the amino acid and the negatively charged oxygen atom of phosphonic acid residue of the calixarene, the Host-Guest p-p, СН-p and solvophobic interactions.

scholarly journals Putative imbalanced amino acid metabolism in rainbow trout long term fed a plant-based diet as revealed by 1H-NMR metabolomics

2021 ◽  
Vol 10 ◽  
Author(s):  
Catherine Deborde ◽  
Blandine Madji Hounoum ◽  
Annick Moing ◽  
Mickaël Maucourt ◽  
Daniel Jacob ◽  
...  
Keyword(s):  
Amino Acids ◽  
Rainbow Trout ◽  
Amino Acid ◽  
Amino Acid Content ◽  
Proton Nuclear Magnetic Resonance ◽  
Free Form ◽  
Metabolomic Analysis ◽  
Nmr Metabolomics ◽  
H Nmr

Abstract The long-term effect of a plant (P)-based diet was assessed by proton nuclear magnetic resonance (1H-NMR) metabolomics in rainbow trout fed a marine fish meal (FM)–fish oil (FO) diet (M), a P-based diet and a control commercial-like diet (C) starting with the first feeding. Growth performances were not heavily altered by long-term feeding on the P-based diet. An 1H-NMR metabolomic analysis of the feed revealed significantly different soluble chemical compound profiles between the diets. A set of soluble chemical compounds was found to be specific either to the P-based diet or to the M diet. Pterin, a biomarker of plant feedstuffs, was identified both in the P-based diet and in the plasma of fish fed the P-based diet. 1H-NMR metabolomic analysis on fish plasma and liver and muscle tissues at 6 and 48 h post feeding revealed significantly different profiles between the P-based diet and the M diet, while the C diet showed intermediate results. A higher amino acid content was found in the plasma of fish fed the P-based diet compared with the M diet after 48 h, suggesting either a delayed delivery of the amino acids or a lower amino acid utilisation in the P-based diet. This was associated with an accumulation of essential amino acids and the depletion of glutamine in the muscle, together with an accumulation of choline in the liver. Combined with an anticipated absorption of methionine and lysine supplemented in free form, the present results suggest an imbalanced essential amino acid supply for protein metabolism in the muscle and for specific functions of the liver.

scholarly journals Development and validation of RP-HPLC method for estimation of brexpiprazole in its bulk and tablet dosage form using Quality by Design approach

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Amol S. Jagdale ◽  
Nilesh S. Pendbhaje ◽  
Rupali V. Nirmal ◽  
Poonam M. Bachhav ◽  
Dayandeo B. Sumbre
Keyword(s):  
Flow Rate ◽  
High Performance ◽  
Reversed Phase ◽  
Hplc Method ◽  
Control Analysis ◽  
Uv Detector ◽  
Mobile Phase Flow Rate ◽  
Rp Hplc ◽  
Quality By Design Approach ◽  
Bulk Drug

Abstract Background A new, sensitive, suitable, clear, accurate, and robust reversed-phase high-performance liquid chromatography (RP-HPLC) method for the determination of brexpiprazole in bulk drug and tablet formulation was developed and validated in this research. Surface methodology was used to optimize the data, with a three-level Box-Behnken design. Methanol concentration in the mobile phase, flow rate, and pH were chosen as the three variables. The separation was performed using an HPLC method with a UV detector and Openlab EZchrom program, as well as a Water spherisorb C18 column (100 mm × 4.6; 5m). Acetonitrile was pumped at a flow rate of 1.0 mL/min with a 10 mM phosphate buffer balanced to a pH of 2.50.05 by diluted OPA (65:35% v/v) and detected at 216 nm. Result The developed RP-HPLC method yielded a suitable retention time for brexpiprazole of 4.22 min, which was optimized using the Design Expert-12 software. The linearity of the established method was verified with a correlation coefficient (r2) of 0.999 over the concentration range of 5.05–75.75 g/mL. For API and formulation, the percent assay was 99.46% and 100.91%, respectively. The percentage RSD for the method’s precision was found to be less than 2.0%. The percentage recoveries were discovered to be between 99.38 and 101.07%. 0.64 μg/mL and 1.95 μg/mL were found to be the LOD and LOQ, respectively. Conclusion The developed and validated RP-HPLC system takes less time and can be used in the industry for routine quality control/analysis of bulk drug and marketed brexpiprazole products. Graphical abstract

SIMULTANEOUS ESTIMATION OF CURCUMIN AND GEFITINIB IN BULK AND TISSUE SAMPLES (PLASMA AND BRAIN HOMOGENATE) BY RP-HPLC: APPLICATION TO A DISTRIBUTION STUDY

INDIAN DRUGS ◽  
2019 ◽  
Vol 56 (07) ◽  
pp. 59-68
Author(s):  
H Mahajan ◽  
S Savale ◽  
P Nerkar ◽  
Keyword(s):  
Flow Rate ◽  
Mobile Phase ◽  
Internal Standard ◽  
Brain Homogenate ◽  
Reversed Phase ◽  
Hplc Method ◽  
Simultaneous Estimation ◽  
Bulk Analysis ◽  
Experimental Conditions ◽  
Rp Hplc

The present study was aimed at developing a Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) method for simultaneous determination of curcumin (CRM) and gefitinib (GFT) in bulk, plasma and brain homogenate. hydrochlorothiazide was used as an internal standard (IS). A new simple, rapid, selective, precise and accurate RP-HPLC method has been developed. The separation was achieved by using C-18 column (Qualisil BDS C18, 250 mm x 4.6 mm I.D.) coupled with a guard column of silica, mobile phase consisted of acetonitrile: water with 0.1% formic acid (30:70 v/v). The flow rate was 0.2 ml/min and the drug was detected using PDA detector at the wavelength of 242 nm. The experimental conditions, including the diluting solvent, mobile phase composition, column saturation and flow rate, were optimised to provide high-resolution and reproducible peaks. The method was developed and tested for linearity range of 10-60 μg/mL for bulk analysis and 200-800 ng/mL for plasma and brain homogenate. The developed method was validated as per ICH guidelines, in terms of linearity, application of the proposed method to bulk sample, recovery, precision, repeatability, ruggedness, sensitivity (LOD and LOQ) and robustness and stability study (short and long-term stabilities, freeze/thaw stability, post-preparative). The low value of % RSD showed that the method was precise within the acceptance limit of 2%. The developed method was successfully applied for the analysis of the drug in bulk as well as various marketed formulation and drug in plasma and brain distribution studies.

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