Statistical validation of sulfate quantification methods used for analysis of acid mine drainage

Turbidimetric method (TM), ion chromatography (IC) and inductively coupled plasma atomic emission spectrometry (ICP-AES) with and without acid digestion have been compared and validated for the determination of sulfate in mining wastewater. Analytical methods were chosen to compare the performance of a portable field turbidimetric instrument and to validate the underlying assumption utilized in conversion of total sulfur to sulfate during ICP-AES analysis. Accuracy and precision of analytical techniques were compared to one another using control and field samples collected from a mine site using the Bonferroni multiple comparison test. Effects of sample dilution, filter pore size and acidification on sulfate quantification were also studied. The results showed that IC and ICP-AES with and without acid digestion provided excellent recoveries in the case of control samples (within 90-110%). These analytical methods also showed lower relative standard deviation for both control and field samples. On the other hand, performance of the turbidimetric method was severely affected by sample dilution and acidification, and also revealed poor sulfate recoveries for control samples ranging from 0 to 83.5%. Analysis of variance (ANOVA) was used to evaluate the response (sulfate concentration) obtained from factorial design. Analytical method had significant effect (P < 0.0001) on the sulfate quantification. The interaction between determination method and sample dilution was more significant than other two-way interactions.     

Determination of creatinine-related molecules in saliva by reversed-phase liquid chromatography with tandem mass spectrometry and the evaluation of hemodialysis in chronic kidney disease patients

The serum concentrations of creatinine (Cre) and urea are used for the determination of the renal function. However, the use of blood is not always suitable due to the invasive, hygienic and infection problems during its sample collection and handling. In contrast, saliva is relatively clean and the samples can be quickly and noninvasively collected and easily stored. Therefore, the simultaneous determination of Arginine (Arg), creatine (Cr) and Cre in the saliva of chronic kidney disease (CKD) patients was performed by UPLC-ESI-MS/MS together with the saliva of healthy volunteers. The evaluation of hemodialysis of CKD patients was also carried out by the determinations before and after the dialysis. An HS-F5 column was used for the simultaneous determination of Arg, Cr and Cre in the saliva. These molecules were rapidly separated within 4 min and sensitively determined by the multiple reaction monitoring (MRM) of the precursor ion [M+H]⁺ → product ions (m/z 175.1 → 70.1 for Arg; m/z 132.0 → 44.1 for Cr; m/z 114.0 → 44.1 for Cre). The concentration of Cre in the CKD patients was higher than that in the healthy persons. The concentrations of Cre in the saliva of the patients before hemodialysis were moderately correlated with the serum Cre concentrations (R² = 0.661). Furthermore, the concentration in the saliva obviously decreased after hemodialysis (before 0.73 mg/dL, after 0.25 mg/dL; p < 0.02). Thus, the proposed detection method using saliva by UPLC-MS/MS is useful for the evaluation of the renal function in CKD patients. The present method offers a new option for monitoring the hemodialysis of CKD patients.     

LC-QTOF/MS metabolomic profiles in human plasma after a 5-week high dietary fiber intake

The objective was to investigate the alterations of plasma metabolome profiles to identify exposure and effect markers of dietary fiber intake. Subjects (n?=?25) aged 58.6 (1.1)?years (mean and SD) with a body mass index of 26.6 (0.5)?kg/m² were given a high fiber (HF) and a low fiber (LF) diet, in a 5-week randomized controlled crossover intervention. The HF diet consisted of oat bran, rye bran, and sugar beet fiber incorporated into test food products, whereas the LF diet was made of equivalent food products to the HF diet, but without adding fibers. Blood plasma samples were collected at the start and end of each intervention period and analyzed by LC-QTOF/MS. In total, 6 features in positive mode and 14 features in negative mode were significantly different between the HF and the LF diet (p?<?0.01, q?<?0.05). Two markers, 2,6-dihydroxybenzoic acid and 2-aminophenol sulfate, were increased after HF diet, along with a tentatively identified saponin derived from oat avenacosides. The untargeted metabolomics approach enabled the identification of two new markers of dietary fiber intake in human plasma. Further studies will be needed to verify if these markers could serve as compliance markers of fiber intake.     

Methadone concentrations in blood, plasma, and oral fluid determined by isotope-dilution gas chromatography–mass spectrometry

Methadone (MTD) is widely used for detoxification of heroin addicts and also in pain management programs. Information about the distribution of methadone between blood, plasma, and alternative specimens, such as oral fluid (OF), is needed in clinical, forensic, and traffic medicine when analytical results are interpreted. We determined MTD and its metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in blood, plasma, blood cells, and OF by gas chromatography–mass spectrometry (GC-MS) after adding deuterium-labeled internal standards. The analytical limits of quantitation for MTD and EDDP by this method were 20 and 3 ng/mL, respectively. The amounts of MTD and EDDP were higher in plasma (80.4 % and 76.5 %) compared with blood cells (19.6 % and 23.5 %) and we found that repeated washing of blood cells with phosphate–buffered saline increased the amounts in plasma (93.6 % and 88.6 %). Mean plasma/blood concentration ratios of MTD and EDDP in spiked samples (N?=?5) were 1.27 and 1.21, respectively. In clinical samples from patients (N?=?46), the concentrations of MTD in plasma and whole blood were highly correlated (r?=?0.92, p?<?0.001) and mean (median) plasma/blood distribution ratios were 1.43 (1.41). The correlations between MTD in OF and plasma (r?=?0.46) and OF and blood (r?=?0.52) were also statistically significant (p?<?0.001) and the mean OF/plasma and OF/blood distribution ratios were 0.55 and 0.77, respectively. The MTD concentration in OF decreased as salivary pH increased (more basic). These results will prove useful in clinical and forensic medicine when MTD concentrations in alternative specimens are compared and contrasted.     

New approach for measurement of non-SHBG-bound testosterone in human plasma

Testosterone (T) circulates in the blood tightly bound to sex hormone-binding globulin (SHBG) and weakly to albumin. Measuring protein unbound T (free) or non-SHBG-bound T rather than total T has been recommended for the evaluation of androgen disorders in humans. Ammonium sulfate precipitation has been widely used to separate [SHBG-T] complex from free and albumin-bound T. To achieve more specificity in this separation, we used monoclonal anti-SHBG antibody and developed a suitable and convenient immunoassay for measuring non-SHBG-bound T. Magnetic beads were covalently coupled to a monoclonal anti-SHBG antibody to capture [SHBG-T] complex from plasma samples. Magnetic separation was then performed to allow measurement of non-SHBG-bound T in the supernatant by direct radioimmunoassay. When 300 μL of plasma samples were incubated at room temperature with 10 μL of anti-SHBG beads, residual SHBG concentration was undetectable in the supernatant. The specificity of proteins retained on anti-SHBG beads was further demonstrated by peptide mass fingerprint on a MALDI-TOF analyzer. The non-specific adsorption of T on beads was low (5%), and dissociation of T from SHBG-T complex was less than 5% after 180 min of incubation. The plasma concentrations of non-SHBG-bound T using anti-SHBG beads were highly correlated to those obtained using ammonium sulfate precipitation. We conclude that SHBG immunocapture is a highly specific and useful tool for an experimental direct measurement of plasma non-SHBG-bound T. This methodology is also convenient and appropriate for routine and automated assay.     

Headspace Liquid-Phase Microextraction of Short-Chain Fatty Acids in Plasma, and Gas Chromatography with Flame Ionization Detection

A novel method for analysis of short-chain fatty acids (SCFA) in blood plasma has been developed by combining headspace liquid-phase microextraction (HS-LPME) and gas chromatography with flame ionization detection. HS-LPME conditions, for example working solution volume, salt concentration, and extraction temperature and time, were optimized by means of an L₉ (3⁴) orthogonal array design. The effects on extraction efficiency of the type of solvent and the volume of the microdrop, the pH of the sample matrix, and the stirring rate were examined. Under optimum conditions the linear calibration ranges for SCFA were from 0.08 to 80 µg mL^?1 (r ²>0.9921) with detection limits of 0.02 to 0.08 µg mL ^?1 and relative standard deviation less than 9.81%. Recovery from spiked blood plasma samples was more than 70.5%. The method was satisfactorily applied to analysis of SCFA in rat blood plasma samples from the blind-loop model.     

Bioappearance and pharmacokinetics of bioactives upon coffee consumption

Habitual consumption of medium amounts of coffee over the whole life-span is hypothesized to reduce the risk to develop diabetes type 2 (DM2) and Alzheimer’s disease (AD). To identify putative bioactive coffee-derived metabolites, first, pooled urine from coffee drinkers and non-coffee drinkers were screened by UPLC-HDMS. After statistical data analysis, trigonelline, dimethylxanthines and monomethylxanthines, and ferulic acid conjugates were identified as the major metabolites found after coffee consumption. For quantitative analysis of these markers in body fluids, targeted methods based on stable-isotope dilution and UPLC-MS/MS were developed and applied to plasma samples from a coffee intervention study (n?=?13 volunteers) who consumed a single cup of caffeinated coffee brew after a 10-day washout period. Chlorogenic acid-derived metabolites were found to be separated into two groups showing different pharmacokinetic properties. The first group comprised, e.g., ferulic acid and feruloyl sulfate and showed early appearance in the plasma (～1 h). The second group contained particularly chlorogenic acid metabolites formed by the intestinal microflora, appearing late and persisting in the plasma (>6 h). Trigonelline appeared early but persisted with calculated half-life times ～5 h. The plasma levels of caffeine metabolites significantly and progressively increased 2–4 h after coffee consumption and did not reach c _max within the time frame of the study. The pharmacokinetic profiles suggest that particularly trigonelline, caffeine, its metabolites, as well as late appearing dihydroferulic acid, feruloylglycine and dihydroferulic acid sulfate formed from chlorogenic acid by the intestinal microflora accumulate in the plasma due to their long half-life times during habitual consumption of several cups of coffee distributed over the day. Since some of these metabolites have been reported to show antioxidant effects in vivo, antioxidant-response-element activating potential, and neuroprotective properties, respectively, some of these key metabolites might account for the inflammation- and DM2/AD risk reducing effects reported for habitual life time consumption of coffee.

Suitability of methylmalonic acid and total homocysteine analysis in dried bloodspots

Methylmalonic acid (MMA) and total homocysteine (tHCYS) concentrations are used to detect acquired and inborn errors of cobalamin (vitamin B12, Cbl) metabolism and to evaluate the effect of therapeutic interventions. Dried blood spot sampling offers a patient-friendly and easy alternative to plasma sampling. However, dried blood spot concentrations are not necessarily equal to plasma concentrations. Therefore, the objective of this work was to establish the relationship between MMA and tHYS dried blood spot and plasma concentrations to facilitate clinical implementation of dried blood spot sampling. MMA and tHCYS in both plasma and DBS were validated on ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS). While position of the punch (in DBS) did affect tHCYS concentration, no influence of hematocrit (Ht) and blood volume on both MMA and tHCYS concentrations was observed. The plasma assay performed better than the DBS assay by most criteria. However, the DBS matrix was superior for tHCYS stability. Paired plasma and DBS samples were obtained from patients suspected for Cbl deficiency and from patients with a known inborn error of metabolism affecting MMA or tHCYS concentration. Based on the strong correlation of tHCYS in both matrices (y = 0.46 ± 1.12 (r² = 0.91)), determination of tHCYS in plasma can be replaced by tHCYS in DBS. However, for MMA, a correlation in the higher (pathological) range of MMA exist, but no correlation was observed in the lower ranges. Therefore the added value of MMA concentrations in DBS is currently unknown and should be further investigated.     

Glycation sites of human plasma proteins are affected to different extents by hyperglycemic conditions in type 2 diabetes mellitus

Glucose can modify proteins in human blood, forming early glycation products (e.g., Amadori compounds), which can slowly degrade to advanced glycation endproducts (AGEs). AGEs contribute significantly to complications of diabetes mellitus and, thus, represent markers of advanced disease stages. They are, however, currently unsuitable for early diagnosis and therapeutic monitoring. Here, we report sensitive strategies to identify and relatively quantify protein glycation sites in human plasma samples obtained from type 2 diabetes mellitus (T2DM) patients and age-matched nondiabetic individuals using a bottom-up approach. Specifically, Amadori peptides were enriched from tryptic digests by boronic acid affinity chromatography, separated by reversed-phase chromatography, and analyzed on-line by high-resolution mass spectrometry. Among the 52 Amadori peptides studied here were 20 peptides resembling 19 glycation sites in six human proteins detected at statistically significantly higher levels in T2DM than in the normoglycemic controls. Four positions appeared to be unique for T2DM within the detection limit. All 19 glycation sites represent promising new biomarker candidates for early diagnosis of T2DM and adequate therapeutic control, as they may indicate early metabolic changes preceding T2DM. Graphical Abstract

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LC–MS/MS method for simultaneous determination of serum p‐cresyl sulfate and indoxyl sulfate in patients undergoing peritoneal dialysis

p‐Cresol sulfate (pCS) and indoxyl sulfate (IS) are protein‐bound uremic toxins that accumulate in patients with chronic kidney disease (CKD). They are closely associated with the mortality rate of CKD and morbidity of cardiovascular disease. In the present study, we established a rapid method for determination of pCS and IS by HPLC‐MS/MS in serum samples from 205 CKD patients undergoing peritoneal dialysis. In brief, serum was extracted by acetonitrile and spiked with hydrochlorothiazide. The prepared sample was eluted through HPLC column (Agilent Zorbax SB‐C₁₈, 3.5 μm, 2.1 × 100 mm) with a mobile phase of acetonitrile and 10 mm ammonium acetate solution (10:90, v/v) for subsequent detection of pCS and IS by MS/MS. The linearity ranged from 50 to 10,000 ng/mL for pCS (r > 0.99), and from 500 to 10,000 ng/mL for IS (r > 0.99). The lower limit of quantification was 50 ng/mL for pCS, and 500 ng/mL for IS. Relative standard deviation (RSD) of intra‐ and inter‐day precision was within ±15%. The results showed that pCS and IS levels were partially correlated with renal function in CKD patients, and IS was directly related to serum creatinine and estimated glomerular filtration rate.     

Comparison of protein precipitation methods for sample preparation prior to proteomic analysis

Protein samples should be free of salt and other disturbing agents and have an appropriate concentration to be suitable for two-dimensional (2D) electrophoresis, the principal step of proteomics. To find the most efficient method for sample preparation, we used human plasma and compared four widely applied precipitation methods, using trichloroacetic acid (TCA), acetone, chloroform/methanol and ammonium sulfate, as well as ultrafiltration. Precipitation with TCA and acetone and ultrafiltration resulted in an efficient sample concentration and desalting. We also found that ammonium sulfate fractionation can efficiently remove albumin, which represents more than 50% of plasma proteins.     

Electrochemical detection of glucose from whole blood using paper-based microfluidic devices

Electrochemical paper-based analytical devices (ePADs) with integrated plasma isolation for determination of glucose from whole blood samples have been developed. A dumbbell shaped ePAD containing two blood separation zones (VF2 membranes) with a middle detection zone was fabricated using the wax dipping method. The dumbbell shaped device was designed to separate plasma while generating homogeneous flow to the middle detection zone of the ePAD. The proposed ePADs work with whole blood samples with 24–60% hematocrit without dilution, and the plasma was completely separated within 4 min. Glucose in isolated plasma separated was detected using glucose oxidase immobilized on the middle of the paper device. The hydrogen peroxide generated from the reaction between glucose and the enzyme pass through to a Prussian blue modified screen printed electrode (PB-SPEs). The currents measured using chronoamperometry at the optimal detection potential for H₂O₂ (−0.1 V versus Ag/AgCl reference electrode) were proportional to glucose concentrations in the whole blood. The linear range for glucose assay was in the range 0–33.1 mM (r² = 0.987). The coefficients of variation (CVs) of currents were 6.5%, 9.0% and 8.0% when assay whole blood sample containing glucose concentration at 3.4, 6.3, and 15.6 mM, respectively. Because each sample displayed intra-individual variation of electrochemical signal, glucose assay in whole blood samples were measured using the standard addition method. Results demonstrate that the ePAD glucose assay was not significantly different from the spectrophotometric method (p = 0.376, paired sample t-test, n = 10).     

Metabolic profiles of the Flos Abelmoschus manihot extract by intestinal bacteria from the normal and CKD model rats based on UPLC‐Q‐TOF/MS

Flos Abelmoschus manihot is a traditional herbal medicine widely used in clinical practice to tackle chronic kidney disease (CKD) for thousands of years. Nowadays, many studies indicate that gut bacteria are closely related to the progression of CKD and CKD‐related complications. In this study, a UPLC‐Q‐TOF/MS method coupled with the MetaboLynx™ software was established and successfully applied to investigate the metabolites and metabolic profile of Flos A. manihot extract by intestinal bacteria from normal and CKD rats. Eight parent components and eight metabolites were characterized by their protonated ions. Among these compounds, 15 were detected in the two group samples while M16 was only determined in the CKD model samples. Compared with the quercetin‐type glycosides, fewer myricetin‐type and gossypetin‐type metabolites were obtained in the two group samples. These metabolites suggested that deglycosylation and methylation are the major metabolic pathways of Flos A. manihot extract. Few differences of metabolite classes were observed in the two group samples. However, the concentrations of aglycones such as quercetin, myricetin and gossypetin in the normal samples were notably higher than those in the CKD model samples. The results are important in unravelling the pharmacological effects of A. manihot and clarifying its mechanism of action in vivo .     

Influence of sulfation conditions on the acidic and catalytic properties of sulfated alumina in isobutane alkylation with butylenes and <Emphasis Type="Italic">n</Emphasis>-pentane isomerization

The influence of the sulfation parameters (the source and concentration of sulfate ions) and the calcination temperature on the acidic and catalytic properties of sulfated alumina in the alkylation of isobutane with butylenes and n-pentane isomerization was studied. IR spectroscopy of adsorbed probe molecules and temperature-programmed desorption of ammonia were used to characterize the acidic properties of the catalysts. An increase in the content of sulfate groups to the value corresponding to a formal value of the monolayer capacity increases the activity of alkylation and the concentration of strong Brönsted sites. The dependence of the stability of activity in alkylation on the sulfate group concentration is extreme with a maximum at the concentration close to the monolayer capacity. It was concluded from the IR spectroscopic data that the decrease in the stability of activity with the further increase in the content of sulfate groups is due to an increase in the concentration of strong Lewis sites and/or an increase in the surface density of strong Brönsted sites. The absence of the correlation between the catalytic behavior of sulfated alumina samples in n-pentane isomerization and acidity indicates that paraffin activation on these samples occurs via the non-acidic mechanism.     

Sorption-catalytic determination of histamine and lysine after planar chromatography using surfactants

The effect of ionic surfactants on the separation characteristics of histamine and lysine in model mixtures has been studied by planar chromatography. It has been demonstrated that the impregnation of paper in paper chromatography and the modification of the mobile phase with solutions of sodium n-dodecyl sulfate in thin-layer chromatography improve the selectivity of separation. Use of n-dodecyl sulfate made possible an increase in selectivity and, in some cases, sensitivity of sorption-catalytic determination. A combination of planar chromatography with the subsequent sorption-catalytic determination directly on the support made us possible to develop methods for determining histamine and lysine in the concentration range 1 × 10^?12?5 × 10^?11 and 1 × 10^?7?1 × 10^?5 M, respectively. The developed methods have been used to determine histamine in human saliva and lysine in blood plasma and pharmaceutical products.     

Determination of 5-fluorouracil in plasma and whole blood by ion-pair high-performance liquid chromatography

5-Fluorouracil in blood or plasma was determined by extraction and column liquid chromatography. Acetonitrile was added to blood or plasma and the mixture was stirred and centrifuged. Zinc sulfate addition was followed by stirring and centrifuging. The acetonitrile was salted out with ammonium sulfate, and an aliquot was evaporated with nitrogen. The residue was dissolved in mobile phase and chromatographed. The stationary phase was a styrene-divinylbenzene copolymer, and the mobile phase was 10 mM tetrabutylammonium hydroxide-methanol (74:26). 5-Fluorouracil was detected by UV absorption at 266 nm. Time of the assay was less than 30 min. The detection limit was 10 ng/ml and the relative standard deviation was 4 to 10% depending on the concentration.     

肝素抗凝血浆与血清中微量元素测定的对比分析

目的研究肝素抗凝血浆代替血清进行微量元素测定的可行性。方法选择2014年6月在汉川市人民医院接受生化检验的272例患者,采用Beckman DXC800型全自动生化分析仪,同时检测患者的肝素抗血浆和血清中锌(Zn)、铁(Fe)、铜(Cu)3种微量元素的浓度。结果肝素抗凝血浆锌、铁与血清中的锌、铁离子浓度比较无显著差异,差异无统计学意义(P0.05);血浆中铜离子的浓度明显低于血清中铜离子的检测值,差异有统计学意义(P0.05),但通过回归方程可以校正。结论肝素抗凝检测方法能够有效避开血液的凝固过程,在短时间内分离标本并检测,从而减少放置误差。但肝素抗凝血浆铜的检验还应进行回归校正。     

Analysis of selected pesticides and alkylphenols in human cord blood by gas chromatograph-mass spectrometer

A total of seven pesticides and eight alkylphenols were monitored using this method for the determination of their trace levels in human cord blood. The pesticides are lindane, diazinon, α-endosulfan, β-endosulfan, endosulfan sulfate, chlorpyrifos and endrin; while the alkylphenols are 4-n-butylphenol, 4-n-pentylphenol, 4-n-hexylphenol, 4-t-octylphenol, 4-n-heptylphenol, nonylphenol, 4-n-octylphenol and bisphenol A. The pesticides and alkylphenols in the cord blood samples were extracted with solid phase extraction IST C18 cartridges and analyzed by selected ion monitoring mode using quadrapole detector in Shimadzu QP-5000 gas chromatograph-mass spectrometer. Trace levels of pesticide and alkylphenols in the range of non-detectable to 15.17 ng ml⁻¹, were detected in the human cord blood samples. This technique of monitoring the levels of endocrine-disruptors in blood samples is consistent, reliable and cost effective while reducing wastage of time and solvents.     

High-performance liquid chromatographic method for the direct determination of 4-methylumbelliferone and its glucuronide and sulfate conjugates. Application to studies in the single-pass in situ perfused rat intestine-liver preparation

A direct high-performance liquid chromatographic (HPLC) assay was developed for the separation and determination of 4-methylumbelliferone (4MU) and its glucuronide (MUG) and sulfate (MUS) conjugates in the cell-free perfusate ("plasma") from in situ perfused rat intestine-liver preparation. In addition, a procedure was developed to extract and determine 4MU in the whole blood perfusate. Perfusate plasma containing an internal standard (umbelliferone) was precipitated with methanol (1:4, v/v), and injected into a reversed-phase HPLC system with gradient elution. 4MU and the same internal standard were also extracted directly from the whole blood perfusate with ethyl acetate and injected into a reversed-phase HPLC system with isocratic elution. Inter- and intra-day precision studies (n = 5 for each) for both the plasma and whole blood procedures demonstrated relative standard deviation of less than 10% at all concentrations studied. The compounds were stable in either the plasma or blood extracts at room temperature for up to 72 h. The procedures were successfully used to analyze perfusate samples obtained from the single-pass in situ perfusion of rat intestine-liver system with either trace (0.95 nM) or 32.3 microM concentrations of 4MU. The intestine was responsible for the formation of most of the MUG formed by the intestine-liver preparation during steady-state perfusion with either input concentration of 4MU.     

Differential scanning calorimetry (DSC) analysis of human plasma in different psoriasis stages

Psoriasis vulgaris is a chronic autoimmune, inflammatory and proliferative skin disease. Recently, there is a need for new methods to detect and to monitor this dermatological syndrome at any stage. The application of differential scanning calorimetry (DSC) should be as a new diagnostic method for psoriasis detection and monitoring using human plasma. We aimed to detect blood plasma components with DSC in psoriasis patients. The study included 18 white adults (eight men and ten women; median age 55.7 years) who had underwent a full skin examination for psoriasis. According to the psoriasis area severity index (PASI) we selected them into three groups: PASI: 0 (symptomless), PASI: 1–6 (minimal symptoms), PASI: >7 (symptoms). According to medical treatment human blood plasma samples were collected from healthy controls, patients without or with therapy, and were analyzed by DSC technique. In this preliminary study we observed that thermal changes (T _m, calorimetric enthalpy) in blood plasma showed closed correlation with psoriasis severity and medical treatment. Further studies are needed to elucidate these relationships, but our application of the DSC method has provided a potential new tool for the early diagnosis and monitoring of psoriasis patients.