Acids In Plasma Samples Research Articles

Analysis of 15 bile acids in human plasma based on C18 functionalized magnetic organic polymer nanocomposite coupled with liquid chromatography-tandem mass spectrometry

Because of the “enterohepatic circulation” of bile acid, liver damage can be reflected by monitoring the content of bile acid in the serum of the organism. To monitor the concentration of 15 bile acids in plasma samples, a new technique of PRiME (process, ruggedness, improvement, matrix effect, ease of use) pass-through cleanup procedure combined with high performance liquid chromatography-tandem quadrupole mass spectrometry (HPLC-MS/MS) was developed. The sorbent used in the PRiME pass-through cleanup procedure is a new type of magnetic organic resin composite nano-material modified by C18 (C18-PS-DVB-GMA-Fe3O4), which has high cleanup efficiency of plasma samples. It also shows good performance in the separation and analysis of 15 kinds of bile acids. Under the optimal conditions, the results show higher cleanup efficiency of C18-PS-DVB-GMA-Fe3O4 with recoveries in the range of 82.1–115 %. The limit of quantitative (LOQs) of 15 bile acids were in the range of 0.033 µg/L-0.19 µg/L, and the RSD values of 15 bile acids were in the range of 3.00–11.9 %. Validation results on linearity, specificity, accuracy and precision, as well as on the application to analysis of 15 bile acids in 100 human plasma samples demonstrate the applicability to clinical studies.

HBM4EU chromates study – PFAS exposure in electroplaters and bystanders

The study aims to reveal the exposure to perfluoroalkyl substances (PFAS) in workers in different industry sectors with exposures to hexavalent chromium (Cr(VI)). The PFAS exposure of in total 172 individuals from 4 countries was assessed by the determination of 8 perfluoroalkyl carboxylic acids and 4 perfluoroalkyl sulfonic acids in plasma samples. The participants were 52 chrome plating workers, 43 welders, 3 surface treating workers and 74 workers without any occupational Cr exposure as controls. Significant differences between workers with Cr exposure and controls were found for the perfluoroalkyl sulfonic acids, particularly for perfluorooctane sulfonic acid (PFOS). The median and maximum levels were, respectively, 4.83 and 789 μg/l for chrome plating workers, 4.97 and 1513 μg/l for welders, and 3.65 and 13.9 μg/l for controls. The considerably high PFOS exposure in Cr platers and welders can be explained by the former application of PFOS as mist suppressants in electroplating baths, which resulted in an exposure of the directly involved operators, but also of welders performing maintenance and repair service at these workplaces.

171 Supplementing Ca Salts of Soybean Oil Via Low-Moisture Molasses-Based Blocks to Improve Reproductive Performance and Overall Productivity of Beef Cows

Abstract This experiment evaluated reproductive and productive responses of beef cows receiving self-fed low-moisture blocks (LMB) enriched or not with Ca salts of soybean oil (CSSO) throughout the breeding season. Non-pregnant, suckling multiparous Angus-influenced cows (n = 554) were assigned to a fixed-time artificial insemination (AI) protocol (day -10 to 0) followed by natural service (day 15 to 70). Cows were managed in 12 groups (46 ± 4 cows/group) maintained in individual pastures, and groups received LMB enriched with 25% (as-fed basis) of CSSO or ground corn (CON) from day -10 to 100. Both treatments were designed to yield a daily LMB intake of 0.454 kg/cow (as-fed basis). Groups were enrolled in an estrus-synchronization + fixed-time AI protocol from day -10 to 0 (AI occurred on day 0) of the experiment and beginning 15 days after AI cows were exposed to mature Angus bulls for 55 d. Pregnancy status was verified on day 55 and 100 of the experiment, and cows that gave birth during the initial two weeks were considered pregnant to fixed-time AI. Calf birthdate and weight was recorded during the subsequent breeding season. All calves were weaned at the same time, and a calf BW and age were recorded. Blood samples were also collected from a subsample of cows on days -10, 0 (fixed-time AI), and 55 (pregnancy diagnosis). All data were analyzed using group as the experimental unit and Satterthwaite approximation to determine the denominator df for the tests of fixed effects. Quantitative data were analyzed with the MIXED procedure of SAS, and binary data analyzed with the GLIMMIX procedure of SAS with a binomial distribution and logit link function. Data were analyzed with group (treatment × location), cow(group), and location as random variables. Significance was set at P ≤ 0.05 and tendencies were determined if P > 0.05 and ≤ 0.10. Cows receiving CSSO had greater (P < 0.01) mean concentrations of ω-6 fatty acids in plasma samples collected on days 0 and 55. Cows receiving CSSO had greater (P = 0.05) pregnancy rate to fixed-time AI (67.2 vs. 59.3%), whereas final pregnancy rate did not differ (P = 0.92) between treatments. Pregnancy loss was less (P = 0.03) in CSSO cows (4.50 vs. 9.04%), which also calved earlier during the calving season (treatment × week; P = 0.04). Weaning rate tended to be greater (P = 0.09) in CSSO (84.8 vs. 79.4%), although calf weaning age and weight did not differ (P ≥ 0.72) between treatments. Kilos of calf weaned/cow exposed was greater (P = 0.04) in CSSO cows (234 vs. 215 kg). Therefore, supplementing CSSO to beef cows via LMB during the breeding season improved their reproductive and overall productivity during a cow-calf cycle.

Supplementing Ca salts of soybean oil via low-moisture molasses-based blocks to improve reproductive performance and overall productivity of beef cows

This experiment evaluated reproductive and productive responses of beef cows receiving self-fed low-moisture blocks (LMB) enriched or not with Ca salts of soybean oil (CSSO) throughout the breeding season. Non-pregnant, suckled multiparous Angus-influenced cows were assigned to a fixed-time artificial insemination (AI) protocol (day −10 to 0) followed by natural service (day 15–70). Cows were managed in 12 groups (46 ± 4 cows/group) maintained in individual pastures, and groups received LMB enriched with 25 % (as-fed basis) of CSSO or ground corn (CON) from day − 10 to 100. Both treatments were designed to yield a daily LMB intake of 0.454 kg/cow (as-fed basis). Cows receiving CSSO had greater (P < 0.01) mean concentrations of ω-6 fatty acids in plasma samples collected on days 0 and 55. Cows receiving CSSO had greater (P = 0.05) pregnancy rate to fixed-time AI (67.2 vs. 59.3 %), whereas final pregnancy rate did not differ (P = 0.92) between treatments. Pregnancy loss was less (P = 0.03) in CSSO cows (4.50 vs. 9.04 %), which also calved earlier during the calving season (treatment × week; P = 0.04). Weaning rate tended to be greater (P = 0.09) in CSSO (84.8 vs. 79.4 %), although calf weaning age and weight did not differ (P ≥ 0.72) between treatments. Kilos of calf weaned/cow exposed was greater (P = 0.04) in CSSO cows (234 vs. 215 kg). Therefore, supplementing CSSO to beef cows via LMB during the breeding season improved their reproductive and overall productivity during a cow-calf cycle.

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.

Tagging Fatty Acids Via Choline Coupling for the Detection of Carboxylic Acid Metabolites in Biological Samples

Background: Fatty acids and other metabolites containing a carboxyl group are of high interest in biomedicine because of their major role in many metabolic pathways and, particularly in the case of oxidised fatty acids, their high biological activity. Tagging carboxylic acid compounds with a permanent positive charge such as a quaternary ammonium compound could increase the LC-MS detection sensitivity and selectivity. This paper describes a new and novel strategy for analysing carboxylcontaining compounds in biological samples by ESI-MS through coupling to choline. Methods: Coupling of carboxylic acid derivatives in biological samples was performed by coupling to 2-Fluoro-1, 3 dimethyl –pyridinium (FDMP). The variation in the fatty acid profile of five different plasma samples was studied and was illustrated by using principal components analysis (PCA) to group the samples. Orthogonal partial least squares discriminant analysis (OPLS-DA) modelling was then applied to identify the fatty acids that were responsible for the variation. Results: The test results showed that choline coupling reactions were successful in detecting fatty acids, oxidised fatty acids and other compounds containing carboxylic acid groups in biological samples. The PCA results showed loadings of different fatty acids according to the plasma sample allowing identification of the fatty acids responsible for the observed variation. Conclusion: A new and easy tagging method was developed to detect carboxylic acids in plasma samples. The method proved to be precise and reproducible and can quantify fatty acid compounds to 50 ng/ml.

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.

LC-MS/MS Analysis of Bile Acids in In Vitro Samples.

Over the last decade, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become the method of choice for the quantification of bile acids (BA) and their conjugates in different matrices, such as plasma, blood, urine, and cell lysates. Numerous reports have indeed been published describing methods for quantitative determination of bile acids in plasma samples obtained during in vivo studies. However, information on bioanalytical methods suitable for determination of bile acids in in vitro samples remained scarce. Therefore, we presently report a simple and accurate LC-MS/MS method for the quantification of BA in cells (e.g., cultured human hepatocytes) and corresponding cell culture medium, obtained during in vitro experiments.

A UFLC-MS/MS method for the simultaneous determination of eight bioactive constituents from red wine and dealcoholized red wine in rat plasma: Application to a comparative pharmacokinetic study.

To explore whether alcohol has an effect on the pharmacokinetic behavior of phenolic acids, the main bioactive constituents in red wine, a highly sensitive and simple ultra-fast liquid chromatography coupled with triple quadrupole mass spectrometry (UFLC-MS/MS) method was developed for simultaneous quantitation of eight phenolic acids in plasma samples. Plasma samples were extracted by liquid-liquid extraction and the chromatographic separation was achieved on a Zorbax SB-C18 column within 7.0 min. Results of the validated method revealed that all of the calibration curves displayed good linear regression (r > 0.99). The intra- and inter-day precisions of the analytes were <14.0% and accuracies ranged from -8.5 to 7.3%. The extraction recoveries of the analytes were from 71.2 to 110.2% and the matrix effects ranged from 86.2 to 105.5%. The stability of these compounds under various conditions satisfied the requirements of biological sample measurement. The method was successfully applied to a comparative pharmacokinetic study of phenolic acids in rat plasma. For gallic acid and gentisic acid, the parameters AUC0-t and AUC0-∞ increased remarkably (p < 0.05) after oral administration of red wine, which suggested that alcohol might enhance their absorption. This is the first report to compare the pharmacokinetic behavior of phenolic acids in red wine and dealcoholized red wine.

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%.

Targeted metabolomics reveals altered oxylipin profiles in plasma of mild cognitive impairment patients

Mild cognitive impairment (MCI) is considered to be a prodromal stage of Alzheimer’s disease (AD), which is the most common type of dementia. Although MCI is a common clinical manifestation in the elderly, the pathology and molecular mechanisms are not fully understood. Oxylipins are a major class of lipid-derived signaling mediators, which have been implicated in the pathology of MCI and AD. In this study, we investigated the changes of oxylipin profiles in plasma of MCI patients. We performed a targeted liquid chromatography—mass spectrometry analysis to quantify 49 oxylipins and 4 polyunsaturated fatty acids in plasma samples of 60 clinically diagnosed MCI patients and 56 age- and gender-matched cognitively normal individuals. We found that the levels of linoleic acid (LA) and 7 oxylipins were significantly altered in MCI patients when compared to the controls. Notably, oxylipins synthesized through 5-lipoxygenase (5-LOX) and cytochrome P450 (CYP450) pathways of arachidonic acid (AA) or LA were elevated in MCI patients, which is in accordance with previously reports that oxylipins from the same pathways were increased in the brain tissues of AD and MCI patients, suggesting the potential correlations of oxylipin changes in 5-LOX and CYP450 pathways between the peripheral blood and the brain tissues in MCI and AD patients. This study is the first report on plasma oxylipin profiles in MCI patients, and disease-relevant changes of oxylipins and oxylipin pathways were identified. The results represent potentially an efficient method to monitor certain oxylipin changes in the brain tissues of MCI or AD patients.

Temporal trends of perfluoroalkyl acids in plasma samples of pregnant women in Hokkaido, Japan, 2003–2011

Perfluoroalkyl acids (PFAAs) are persistent organic pollutants that are used in a wide range of consumer products. Recent epidemiological studies have shown that prenatal exposure to toxic levels of PFAAs in the environment may adversely affect fetal growth and humoral immune response in infants and children. Here we have characterized levels of prenatal exposure to PFAA between 2003 and 2011 in Hokkaido, Japan, by measuring PFAA concentrations in plasma samples from pregnant women. The study population comprised 150 women who enrolled in a prospective birth cohort study conducted in Hokkaido. Eleven PFAAs were measured in maternal plasma samples using simultaneous analysis by ultra-performance liquid chromatography coupled to triple quadrupole tandem mass spectrometry. At the end of the study, in 2011, age- and parity-adjusted mean concentrations of perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluoroundecanoic acid (PFUnDA), perfluorododecanoic acid (PFDoDA), perfluorotridecanoic acid (PFTrDA), perfluorohexane sulfonate (PFHxS), and perfluorooctane sulfonate (PFOS) were 1.35ng/mL, 1.26ng/mL, 0.66ng/mL, 1.29ng/mL, 0.25ng/mL, 0.33ng/mL, 0.28ng/mL, and 3.86ng/mL, respectively. Whereas PFOS and PFOA concentrations declined 8.4%/y and 3.1%/y, respectively, PFNA and PFDA levels increased 4.7%/y and 2.4%/y, respectively, between 2003 and 2011. PFUnDA, PFDoDA, and PFTrDA were detected in the vast majority of maternal samples, but no significant temporal trend was apparent. Future studies must involve a larger population of pregnant women and their children to determine the effects of prenatal exposure to PFAA on health outcomes in infants and children.

An automated method for the analysis of phenolic acids in plasma based on ion-pairing micro-extraction coupled on-line to gas chromatography/mass spectrometry with in-liner derivatisation

A new method is presented for the analysis of phenolic acids in plasma based on ion-pairing ‘Micro-extraction in packed sorbent’ (MEPS) coupled on-line to in-liner derivatisation-gas chromatography–mass spectrometry (GC–MS). The ion-pairing reagent served a dual purpose. It was used both to improve extraction yields of the more polar analytes and as the methyl donor in the automated in-liner derivatisation method. In this way, a fully automated procedure for the extraction, derivatisation and injection of a wide range of phenolic acids in plasma samples has been obtained. An extensive optimisation of the extraction and derivatisation procedure has been performed. The entire method showed excellent repeatabilities of under 10% and linearities of 0.99 or better for all phenolic acids. The limits of detection of the optimised method for the majority of phenolic acids were 10ng/mL or lower with three phenolic acids having less-favourable detection limits of around 100ng/mL. Finally, the newly developed method has been applied in a human intervention trial in which the bioavailability of polyphenols from wine and tea was studied. Forty plasma samples could be analysed within 24h in a fully automated method including sample extraction, derivatisation and gas chromatographic analysis.

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.

Metabolism of primed, constant infusions of [1,2- 13C 2] glycine and [1- 13C 1] phenylalanine to urinary oxalate

Experiments in humans and rodents using oral doses of glycine and phenylalanine have suggested that the metabolism of these amino acids contributes to urinary oxalate excretion. To better define this contribution, we have examined the primed, constant infusion of [1- 13C 1] phenylalanine and [1,2- 13C 2] glycine in the postabsorptive state in healthy adults. Subjects were infused for 5 hours, hourly urines were collected, and blood was drawn every 30 minutes. Ion chromatography/mass spectrometry was used to measure [ 13C] enrichment in urinary oxalate, glycolate, and hippurate; and the enrichment of 13C-amino acids in plasma samples was measured by gas chromatography/mass spectrometry. Following infusion with either 6 μmol/(kg h) [1- 13C 1] phenylalanine or 6 μmol/(kg h) [1,2- 13C 2] glycine, no isotopic glycolate or oxalate was detected in urine. Based on the limits of detection of our ion chromatography/mass spectroscopy method, these data indicate that less than 0.7% of the urinary oxalate could be derived from phenylalanine catabolism and less than 5% from glycine catabolism. Infusions with high levels of [1,2- 13C 2] glycine, 60 μmol/(kg h), increased mean plasma glycine by 29% and the whole-body flux of glycine by 72%. Under these conditions, glycine contributed 16.0% ± 1.6% and 16.6% ± 3.2% to urinary oxalate and glycolate excretion, respectively. Experiments using cultured hepatoma cells demonstrated that only at supraphysiological levels (>1 mmol/L) did glycine and phenylalanine metabolism increase oxalate synthesis. These data suggest that glycine and phenylalanine metabolism make only minor contributions to oxalate synthesis and urinary oxalate excretion.

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.

Synthesis of the isotope‐labeled derivatization reagent for carboxylic acids, 7‐(N,N‐dimethylaminosulfonyl)‐4‐(aminoethyl)piperazino‐2,1,3‐benzoxadiazole (d6) [DBD‐PZ‐NH2 (D)], and its application to the quantification and the determination of relative amount of fatty acids in rat plasma samples by high‐performance liquid chromatography/mass spectrometry

The isotope-labeled benzofurazan derivatization reagent for carboxylic acids, 7-(N,N-dimethylaminosulfonyl)-4-(aminoethyl)piperazino-2,1,3-benzoxadiazole (d6) [DBD-PZ-NH2 (D)] was synthesized. DBD-PZ-NH2 (D) was used for the accurate quantification of fatty acids by liquid chromatography/mass spectrometry (LC/MS). The standard fatty acids were derivatized with DBD-PZ-NH2 (D) to the stable isotope-labeled compounds for the fatty acids derivatives of DBD-PZ-NH2 and used for the internal standards. The obtained calibration curves for fatty acids were linear over the range 0.1-200 microM (r2 > 0.999). Fatty acids in plasma samples were determined after derivatization with DBD-PZ-NH2 and analyzed by LC/MS using standard fatty acid DBD-PZ-NH2 (D) derivatives as internal standards. Furthermore, the relative amounts of fatty acids in two plasma samples were determined after derivatization with DBD-PZ-NH2 and DBD-PZ-NH2) (D). The isotope-labeled derivatization reagent was useful for accurate quantification and the determination of relative amounts of the metabolites in biological samples having the target functional group.

Stability of HCV, HIV-1 and HBV nucleic acids in plasma samples under long-term storage

The implementation of nucleic acid amplification technology (NAT) for detection of HCV, HIV-1 and HBV has undoubtedly contributed to the viral safety of blood, reducing the window period. One important matter related to the stability of RNA/DNA is the effect of the storage conditions on samples. In a previous work, we studied the stability of HCV RNA in plasma samples after storage at different temperatures. This work is an update on the follow-up of a sample containing 100 IU/ml HCV RNA for 5 years at −20 °C, showing no decrease in the initial titre. The nucleic acid stability of other viruses, such as HIV-1 and HBV, has also been studied. At −20 °C, samples containing HIV-1 were followed up for approximately 3 years and the results obtained show no decay in HIV-1 RNA detectability. Regardless of the HIV-1 RNA concentration, samples stored at 5 °C maintain their titre for at least 14 days. At 25 °C, the HIV-1 RNA half-life was determined at nearly 7 days. The HBV DNA, at 5 °C and 25 °C, is stable for at least 28 days, regardless of the initial titre.

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.