Amino Acids In Plasma Samples Research Articles

Altered metabolic pathways elucidated via untargeted in vivo toxicometabolomics in rat urine and plasma samples collected after controlled application of a human equivalent amphetamine dose

Amphetamine is widely consumed as drug of abuse due to its stimulating and cognitive enhancing effects. Since amphetamine has been on the market for quite a long time and it is one of the most commonly used stimulants worldwide, to date there is still limited information on its effects on the metabolome. In recent years, untargeted toxicometabolomics have been increasingly used to study toxicity-related pathways of such drugs of abuse to find and identify important endogenous and exogenous biomarkers. In this study, the acute effects of amphetamine intake on plasma and urinary metabolome in rats were investigated. For this purpose, samples of male Wistar rats after a single dose of amphetamine (5 mg/kg) were compared to a control group using an untargeted metabolomics approach. Analysis was performed using normal and reversed phase liquid chromatography coupled to high-resolution mass spectrometry using positive and negative ionization mode. Statistical evaluation was performed using Welch’s two-sample t test, hierarchical clustering, as well as principal component analysis. The results of this study demonstrate a downregulation of amino acids in plasma samples after amphetamine exposure. Furthermore, four new potential biomarkers N-acetylamphetamine, N-acetyl-4-hydroxyamphetamine, N-acetyl-4-hydroxyamphetamine glucuronide, and amphetamine succinate were identified in urine. The present study complements previous data and shows that several studies are necessary to elucidate altered metabolic pathways associated with acute amphetamine exposure.

A Dual Ligand Sol⁻Gel Organic-Silica Hybrid Monolithic Capillary for In-Tube SPME-MS/MS to Determine Amino Acids in Plasma Samples.

This work describes the direct coupling of the in-tube solid-phase microextraction (in-tube SPME) technique to a tandem mass spectrometry system (MS/MS) to determine amino acids (AA) and neurotransmitters (NT) (alanine, serine, isoleucine, leucine, aspartic acid, glutamic acid, lysine, methionine, tyrosine, and tryptophan) in plasma samples from schizophrenic patients. An innovative organic-silica hybrid monolithic capillary with bifunctional groups (amino and cyano) was developed and evaluated as an extraction device for in-tube SPME. The morphological and structural aspects of the monolithic phase were evaluated by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), nitrogen sorption experiments, X-ray diffraction (XRD) analyses, and adsorption experiments. In-tube SPME-MS/MS conditions were established to remove matrix, enrich analytes (monolithic capillary) and improve the sensitivity of the MS/MS system. The proposed method was linear from 45 to 360 ng mL−1 for alanine, from 15 to 300 ng mL−1 for leucine and isoleucine, from 12 to 102 ng mL−1 for methionine, from 10 to 102 ng mL−1 for tyrosine, from 9 to 96 ng mL−1 for tryptophan, from 12 to 210 ng mL−1 for serine, from 12 to 90 ng mL−1 for glutamic acid, from 12 to 102 ng mL−1 for lysine, and from 6 to 36 ng mL−1 for aspartic acid. The precision of intra-assays and inter-assays presented CV values ranged from 1.6% to 14.0%. The accuracy of intra-assays and inter-assays presented RSE values from −11.0% to 13.8%, with the exception of the lower limit of quantification (LLOQ) values. The in-tube SPME-MS/MS method was successfully applied to determine the target AA and NT in plasma samples from schizophrenic patients.

Determination of Amino Acids in Plasma Samples of Preeclampsia Patients by Liquid Chromatography Coupled to High-Resolution Mass Spectrometry

This work presents the application of a simple and sensitive method developed for the simultaneous analysis of the levels of twenty two underivatizated free L-amino acids in plasma samples of preeclampsia patients by high performance liquid chromatography coupled to high-resolution mass spectrometer (LC-HRMS) using hydrophilic interaction liquid chromatography (HILIC) silica column. The linearity was evaluated in the range of 0.009 to 8.018 µmol L-1 showing coefficients of determination (R2) in the range of 0.999 to 0.911 (p < 0.05). The intra- and inter-day precisions were above of 9.84 and 15.31, respectively. The limits of detection (LOD) and quantification (LOQ) were in the range of 0.003 to 0.683 µmol L-1 and 0.009 to 2.276 µmol L-1, respectively. The results of the investigation of free L-amino acids showed that the levels of GABA, Arg, Ser, Leu, Ile, Met, Val, Ala, Gly, Bet, His and Lys in plasma of patients with preeclampsia were significantly different from healthy patients.

Dispersive liquid‐liquid microextraction based on the solidification of floating organic droplets for preconcentration of amino acids in human plasma samples

AbstractIn this research, dispersive liquid‐liquid microextraction was established based on the solidification of floating organic droplets. This simple, fast, and eco‐friendly sample extraction method allows preconcentration and identification of amino acids in plasma samples through the use of high‐performance liquid chromatography with fluorescence detection. By application of central composite design, the main factors in the microextraction procedure were optimized. The optimal experimental parameters were: 330 μL of 1‐dodecanol as an extraction solvent, 340 μL of acetone as disperser solvent, 140 μL of methanol as diluent at 6.8 min of ultrasonic time, and finally cooling in an ice bath for 3 min. Under these optimal conditions, good responses for 19 amino acids were obtained in the concentration ranges of 0.5–400 μmol L−1, with coefficients of determination higher than 0.9993. Limits of detection were based on a signal‐to‐noise ratio of 3, and ranged from 0.09 to 0.85 μmol L−1. In conclusion, the results demonstrate that this method was successfully applied to determine the amino acids in plasma samples. The relative recoveries of the 19 amino acids ranged from 81 to 103%, with relative standard deviations ranging from 1 to 4%.

Label-free intrinsic imaging capillary zone electrophoresis analysis to detect homocysteine from blood serum for the detection of genetic metabolic disorders in new-born babies in Qatar

Abstract Over 14,000 babies are born in Qatar each year, and it is the State's intention to provide each with a health screen at birth for the timely diagnosis of inborn errors of metabolism. And since the population is characterized by a high consanguinity (estimates vary between 25–70%) from first-cousin marriages, congenital and genetic disorders are responsible for a major proportion of infant mortality, morbidity, and handicap birth defects and are relatively common among the population. Accurate and reliable quantification of amino acid (AA), generally in a plasma sample, allows early diagnosis of disorders such as phenylketonuria, tyrosinemia, maple syrup urinary disease, hyperornithinemia and citrullinemia. A deficiency of cystathionine B-synthase (CBS) can cause an autosomal recessive disorder of methionine and homocysteine (Hcy) metabolism known as homocystinuria which results in elevated concentrations of Hcy in plasma and urine. Clinical symptoms in untreated patients include progressive myopia and lens dislocation, thromboembolism, epilepsy, and mental retardation. Hcy is implicated as a wide variety of natal and other disorders – including Alzheimer's. The aim of this study is to develop novel screens for AA levels – and in particular Hcy - in the blood using novel approaches in capillary zone electrophoresis (CZE). In order to quantify relevant amino acids it is necessary to deplete proteins from a complex biological sample such as plasma. In this investigation two protein depletion protocols were investigated on human plasma containing Hcy by label-free intrinsic imaging in the UV using CZE. As with a majority of amino acids, Hcy has very little or no UV absorption and for this reason analysis of samples were performed on an advanced high performance capillary electrophoresis (HPCE) platform, by multiple-pixel multiple-imaging indirect UV measurements. Two complementary analysis workflows were deployed to take advantage of the time-development of the AAs in the analysis allowing accurate quantification and minimum inherent bias. Studies have been made with pure AAs and also with samples spiked in known quantities in blood which offer real clinical advantages. We have also developed methodologies for the simultaneous detection of several amino acids in plasma samples and limits of detection (LOD) and limits of quantification (LOQ) will be presented. These have been shown to be greatly improved with a pre-concentration technique known as ‘stacking’. Work has been undertaken in our labs in Qatar Science & Technology Park and in collaboration with Hamad Medical Center.

Classification of Diabetes Volunteers Based on Plasma Amino Acids Metabolic Profiling

To study the relation of plasma amino acids metabolic profiling and diabetes blood sugar level by using metabolomics, an on-line pre-column o-phthalic dicarboxaldehyde ( OPA) derivatization HPLC method for the relative quantitative determination of 21 amino acids in plasma samples was developed. The derivatization procedure was accomplished by an online auto-injector and the signals were detected with a UV detector. Mobile phase A was 10 mmol/L Na(2)HPO(4)-Na(2)B(4)O(7) buffer at pH 7.95, and B was acetonitrile-methanol-water (45:45:10 by volume). The elution program was 5% B at the start and 46% B at the end in 30 min. The 21 amino acids were satisfactorily separated in 27 min. Fifty-one plasma samples from diabetes volunteers were analyzed and were pre-divided into blood sugar ( B. S.) 9. 5 group. By SPSS and Matlab software platform, relation analysis was performed by using linear discriminant analysis, stepwise discriminant analysis, and scores plot models. By correlating the levels of blood sugar( B. S.) to 20 amino acids peak areas except glutamine, the results showed that the B. S. 9.5 group with a total hit ratio of 94.1%, stepwise discriminant analysis ( SDA) model indicated that 7 out of 20 amino acids,i.e., Arg, Cit, Leu, Asp, Asn, Thr and Trp essentially contributed to the above two groups division with the total hit ratio of 90.2%, which maybe have more means as potential marker groups for the early diagnosis of diabetes and in-depth research.

Diagnosis of maple syrup urine disease by determination of l-valine, l-isoleucine, l-leucine and l-phenylalanine in neonatal blood spots by gas chromatography–mass spectrometry

A novel method was developed for the diagnosis of maple syrup urine disease (MSUD) by the determination of l-valine, l-leucine, l-isoleucine and l-phenylalanine in dried blood spots of newborns by gas chromatography–mass spectrometry (GC–MS). The four amino acids were extracted from blood samples by methanol and derivatized by n-butanol and trifluroacetic anhydride under optimum reaction conditions. The corresponding single derivatives of the four amino acids were obtained under the optimum conditions. Their contents in blood samples were analyzed quantitatively by measurement of their derivatives by GC–MS in selected ion monitoring mode. MSUD can be diagnosed on the basis of the ratio of the total content of l-leucine and l-isoleucine to that of l-phenylalanine. The present method only took a short time to perform and required minimal sample preparation, which provided low detection limits and a relative standard deviation of less than 5.0%. The derivatization reactions of the four amino acids, l-valine, l-isoleucine, l-leucine and l-phenylalanine, with n-butanol and trifluroacetic anhydride were investigated and the optimum reaction conditions, including reaction time and temperature, were obtained and used for the determination of the amino acids in plasma samples.

Quantification of physiological amino acids by gradient ion-exchange high-performance liquid chromatography

A single-column gradient lithium ion-exchange chromatographic method with post-column derivatization and fluorimetric detection for the quantification of physiological amino acids is described. The method runs automatically, requires a minimum of sample preparation, separates all amino acids in plasma and cerebrospinal fluid, and most compounds in brain extract, in addition to some amino acids used therapeutically and in pharmacological studies. About 40 compounds can be quantitated within a run time of 3 h. The within-assay and between-assay coefficients of variations for principal amino acids in plasma samples are satisfactory. The system has performed conveniently and with high stability in the daily routine work and is cost-saving based on laboratory-prepared buffers.

Reversed-phase ion-pair chromatography of amino acids : Application to the determination of amino acids in plasma samples and dried blood on filter papers

Reversed-phase ion-pair chromatography of amino acids : Application to the determination of amino acids in plasma samples and dried blood on filter papers