纳升液相色谱-高分辨串联质谱研究冻存条件对唾液多肽组的影响

唾液多肽组学为疾病相关的生物标记物研究提供了新方法,但冻存条件对分析结果的影响并不清楚。本研究采用氧化石墨烯-磷酸镧纳米复合材料分离富集唾液多肽,利用纳升液相色谱-高分辨串联质谱技术,考察唾液样品分别置于-80℃与-20℃冻存6个月后对唾液多肽组的影响。结果表明,在-80℃冻存条件下的唾液样品中,共鉴定出归属于33种蛋白的429条肽段;在-20℃冻存条件下的唾液样品中,鉴定出595条肽段,对应31种蛋白。实验结果表明,相比于-80℃,唾液置于-20℃条件下的新增加肽段主要来源于已有肽段的降解,并且唾液中的蛋白质也发生了一定降解。本研究在肽段序列水平上考察了冻存条件对多肽的影响,结果表明,-20℃冻存条件不适合长期保存用于多肽组分析的唾液样品。本研究结果可为相关医学研究提供借鉴。     

Pressurized liquid extraction of isoflavones from soybeans

Isoflavone derivatives from freeze-dried soybeans were extracted by pressurized liquid extraction (PLE) and determined by reverse-phase high performance liquid chromatography (HPLC) with both photo diode array and mass spectrometry (MS) detection. Both real and spiked samples were used in the development of the method.Several extraction solvents (methanol (MeOH) and ethanol (EtOH), 30-80% in water and water), temperatures (60-200 °C), pressures (100-200 atm), as well as the sample size (0.5-0.05 g) and cycle length (5-10 min) were studied for the optimization of the extraction protocol. The optimized extraction conditions for quantitative recoveries were: 0.1 g of sample, 100 °C, three (7 min) static extraction cycles and ethanol 70% as extracting solvent. The stability of the isoflavones during the PLE was also determined. Under PLE conditions, degradation of malonyl glucoside forms of the isoflavones takes place using temperatures higher than 100 °C whereas degradation of glucosides takes place above 150 °C. Using the optimized protocol, isoflavones can be extracted from freeze-dried soybeans without degradation.     

Inhibition of bacterial degradation of EtG by collection as dried urine spots (DUS)

Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are direct alcohol consumption markers widely used nowadays for clinical and forensic applications. They are detectable in blood and urine even after consumption of trace amounts of ethanol and for a longer time frame, being detectable even when no more ethanol is present. The instability of EtG against bacterial degradation in contaminated urine samples and/or the possible postcollection synthesis of this metabolite in samples containing, e.g., Escherichia coli and ethanol, may cause false identification of alcohol uptake. Therefore, it is of paramount importance to constrict these error sources by inhibition of any bacterial growth causing hydrolization or synthesis of EtG. This study evaluates a new method of collecting urine samples on filter paper, dried urine spots (DUS), for simultaneous detection of EtG, EtS and creatinine, having the great advantage of inhibiting bacterial activity. In addition, a method validation for the determination of EtG and EtS in DUS was performed according to the FDA guidelines. Sterile-filtered urine was spiked with EtG and EtS, inoculated with E. coli and incubated. Liquid and dried urine samples were collected after various time intervals up to 96 h. Liquid samples were frozen immediately after collection, whereas aliquots for DUS were pipetted onto filter paper, allowed to dry and stored at RT until analysis 1 week after. The specimens were analyzed by LC–ESI–MS/MS. As expected, degradation of EtG, but not of EtS, was observed in contaminated liquid urine samples. However, the specimens collected on filter paper and stored at RT showed no degradation during storage. Therefore, collecting urine samples on filter paper for EtG and EtS analysis turns out to be a reliable method to avoid bacterial degradation of EtG and EtS, and consequently, stabilization of these ethanol metabolites is achieved. In addition, simultaneous measurement of creatinine content as an indicator of urine dilution helps to interpret the results. Method validation for EtG and EtS in DUS was satisfactory, showing the linearity of the calibration curves in the studied concentration range, good precision, accuracy and selectivity.     

Stabilization of poly(lactic acid) by polycarbodiimide

Polycarbodiimide (CDI) was used to improve the thermal stability of poly(l-lactic acid) (PLA) during processing. The properties of PLA containing various amounts of CDI were characterized by GPC, DSC, rheology, and tensile tests. The results showed that an addition of CDI in an amount of 0.1-0.7 wt% with respect to PLA led to stabilization of PLA at even 210 °C for up to 30 min, as evidenced by much smaller changes in molecular weight, melt viscosity, and tensile strength and elongation compared to the blank PLA samples. In order to examine the possible stabilization mechanism, CDI was reacted with water, acetic acid, l-lactic acid, ethanol and low molecular weight PLA. The molecular structures of the reaction products were measured with FTIR. The results showed that CDI could react with the residual or newly formed moisture and lactic acid, or carboxyl and hydroxyl end groups in the PLA samples, and thus hamper the thermal degradation and hydrolysis of PLA.     

Sensitive determination of anions in saliva using capillary electrophoresis after transient isotachophoretic preconcentration

A transient isotachophoresis-capillary electrophoresis (tITP-CE) system for the determination of minor inorganic anions in saliva is described. The complete separation and quantification of bromide, iodide, nitrate, nitrite, and thiocyanate has been achieved with only centrifugation and dilution of the saliva sample. In-line tITP preconcentration conditions, created by introduction of the plugs of 5 mM dithionic acid (leading electrolyte) and 10 mM formic acid (terminating electrolyte) before and after the sample zone, respectively, allowed the limits of direct UV absorption detection (at 200 nm) to be up to 50-fold improved as compared with CE without tITP. As a result, nitrate and thiocyanate were still detectable at 4.6 and 3.8 μg l⁻¹, respectively, in 1000 times diluted saliva. The daily variations of anionic concentrations in saliva samples taken from a smoking health volunteer were discussed based on the results of tITP-CE analysis. It was confirmed that the thiocyanate concentration in saliva noticeably increased after smoking. This is apparently the first report on simultaneous quantification of more than four anionic salivary constituents using CE.     

Development of a non-competitive immunoassay for cortisol and its application to the analysis of saliva

A general method of performing non-competitive immunoassays for a low-molecular-mass analyte was developed and applied to cortisol determination in saliva samples. The method is based on the use of a “blocking reagent”, which is able to bind to antibody sites not occupied by the analyte, and in a stronger way than the analyte itself. When an enzyme-labelled analyte is added it substitutes the analyte in the antibody complex, but not the blocking reagent. The measured signal is linearly correlated to the concentration of the complex and, consequently, to the analyte concentration. The 3σ limit of detection (LOD, 0.2 nmol l⁻¹) obtained by the above method was 10 times lower than that obtained by the corresponding ELISA. As non-competitive immunoassays reported for small molecules up to now have been no more than just approaches, the suitability of the proposed assay for cortisol quantification in a real matrix was investigated. Human saliva was chosen as a matrix because of the need for very sensitive techniques to determine salivary cortisol content. The matrix effect was offset by performing the calibration experiments in acidic conditions (pH=5.6) and adding 0.1% of bovine serum albumin (BSA) to the buffer. In these conditions, the LOD was 1.4 nmol l⁻¹, which was adequate to measure normal levels of cortisol. Spiked samples were analysed and gave recoveries ranging from about 80 to 120%. Therefore, five subject samples, collected over 18 h showed salivary cortisol concentrations compatible with the circadian variation of reported normal values.     

Ageing properties of polyamide-12 pipes exposed to fuels with and without ethanol

The chemical and physical properties of polyamide-12 (PA12) fuel pipes/lines, aged for ≤2400 h at 110 °C, have been investigated. The pipes, containing fuel with or without ethanol, were either of a single PA12 layer, or of two PA12 layers surrounding a poly(vinylidene fluoride) barrier layer. The molar mass of the inner surface region obtained by size-exclusion chromatography was reduced during ageing, and optical microscopy revealed a surface that was partly dissolved in the presence of ethanol. Infrared spectroscopy revealed a rapid loss of plasticiser, especially in the presence of ethanol, and the fuel contained plasticiser and other polymer related components. Immersion tests at 60 °C showed that the swelling of the pipe and the amount of dissolved material were greatest for the fuels with intermediate ethanol content (50 vol.%). Aged samples experienced an increase in melting point, presumably, to a large extent, due to the loss of plasticiser and/or PA12-related components. In addition, for several samples, the crystallinity seemed to increase with ageing.     

Determination of drugs of abuse in water by solid-phase extraction,derivatisation and gas chromatography–ion trap-tandem mass spectrometry

An alternative method for the sensitive determination of several drugs of abuse and some of their metabolites in surface and sewage water samples is proposed. Analytes are concentrated using a solid-phase extraction (SPE) sorbent, converted into the corresponding trimethylsilyl derivatives and selectively determined by gas chromatography (GC) with tandem mass spectrometry (MS/MS) detection. Parameters affecting the performance of extraction, derivatisation and determination steps are systematically investigated. Moreover, the stability of target analytes in sewage water samples is discussed. Under final working conditions, water samples were adjusted at pH 8.5 and concentrated using a 200 mg OASIS HLB SPE cartridge. Analytes were sequentially eluted with ethyl acetate followed by acetone and silylated using N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA). The reaction was completed in 60 min at 80 °C and the mixture injected directly in the GC–MS/MS system without further purification. In most cases, analytes presented a poor stability in sewage water samples; however, once they are submitted to the SPE process, cartridges can be stored at −20 °C for at least 3 months without significant degradation and/or inter-conversion reactions of illicit drugs. The proposed method provided recoveries over 74% and LODs between 0.8 and 15 ng/L for river and treated wastewater samples. In the case of raw wastewater slightly worse recoveries, between 63 and 137%, and similar LODs were attained. Analysis of a limited number of waste and surface water samples confirmed the presence of several illicit drugs in the aquatic environment, with the highest levels and frequency corresponding to benzoylecgonine, the main metabolite of cocaine.     

Quantitative analysis of differentially expressed saliva proteins in human immunodeficiency virus type 1 (HIV-1) infected individuals

In the present study, we have established a new methodology to analyze saliva proteins from HIV-1-seropositive patients before highly active antiretroviral therapy (HAART) and seronegative controls. A total of 593 and 601 proteins were identified in the pooled saliva samples from 5 HIV-1 subjects and 5 controls, respectively. Forty-one proteins were found to be differentially expressed. Bioinformatic analysis of differentially expressed salivary proteins showed an increase of antimicrobial proteins and decrease of protease inhibitors upon HIV-1 infection. To validate some of these differentially expressed proteins, a high-throughput quantitation method was established to determine concentrations of 10 salivary proteins in 40 individual saliva samples from 20 seropositive patients before HAART and 20 seronegative subjects. This method was based on limited protein separation within the zone of the stacking gel of the 1D SDS PAGE and using isotope-coded synthetic peptides as internal standards. The results demonstrated that a combination of protein profiling and targeted quantitation is an efficient method to identify and validate differentially expressed salivary proteins. Expression levels of members of the calcium-binding S100 protein family and deleted in malignant brain tumors 1 protein (DMBT1) were up-regulated while that of Mucin 5B was down-regulated in HIV-1 seropositive saliva samples, which may provide new perspectives for monitoring HIV-infection and understanding the mechanism of HIV-1 infectivity.     

Determination of acetaldehyde in saliva by gas-diffusion flow injection analysis

The consumption of ethanol is known to increase the likelihood of oral cancer. In addition, there has been a growing concern about possible association between long term use of ethanol-containing mouthwashes and oral cancer. Acetaldehyde, known to be a carcinogen, is the first metabolite of ethanol and it can be produced in the oral cavity after consumption or exposure to ethanol. This paper reports on the development of a gas-diffusion flow injection method for the online determination of salivary acetaldehyde by its colour reaction with 3-methyl-2-benzothiazolinone hydrazone (MBTH) and ferric chloride. Acetaldehyde samples and standards (80 μL) were injected into the donor stream containing NaCl from which acetaldehyde diffused through the hydrophobic Teflon membrane of the gas-diffusion cell into the acceptor stream containing the two reagents mentioned above. The resultant intense green coloured dye was monitored spectrophotometrically at 600 nm. Under the optimum working conditions the method is characterized by a sampling rate of 9 h⁻¹, a linear calibration range of 0.5–15 mg L⁻¹ (absorbance = 5.40 × 10⁻² [acetaldehyde, mg L⁻¹], R² = 0.998), a relative standard deviation (RSD) of 1.90% (n = 10, acetaldehyde concentration of 2.5 mg L⁻¹), and a limit of detection (LOD) of 12.3 μg L⁻¹. The LOD and sampling rate of the proposed method are superior to those of the conventional gas chromatographic (GC) method (LOD = 93.0 μg L⁻¹ and sampling rate = 4 h⁻¹). The reliability of the proposed method was illustrated by the fact that spiked with acetaldehyde saliva samples yielded excellent recoveries (96.6–101.9%), comparable to those obtained by GC (96.4–102.3%) and there was no statistically significant difference at the 95% confidence level between the two methods when non-spiked saliva samples were analysed.     

Ultrasound-assisted extraction of isoflavones from soy beverages blended with fruit juices

A new method for the fast determination of isoflavones from soy beverages blended with fruit juices without the need of freeze-drying the sample was developed. During the method development, several parameters were studied: solvent (methanol and ethanol), sample:solvent ratio (5:1 to 0.2:1), temperature (10-60 °C) and extraction time (5-30 min). The most important parameter for the extraction of isoflavones from soy drinks was the sample:solvent ratio. The optimized method consists of extracting the sample with ethanol with a sample:solvent ratio of 0.2:1 on an ultrasound bath at 45 °C during 20 min. Also, samples were freeze-dried, extracted using conventional method and compared with the optimized method and no significant difference was observed on total and individual isoflavone concentration. The most representative samples from the Spanish market, with a wide variation of isoflavone concentration were analyzed using the optimized method. Differences between manufacturers reached an almost 10 times fold variation. Overall isoflavone concentration ranged from 6.7 to 58.2 mg L⁻¹.     

The effect of sample salt additives on capillary electrophoresis analysis of intact proteins using surface modified capillaries

The effect of adding alkali salts to protein samples for capillary electrophoretic (CE) analysis of intact proteins was studied. A high degree of peak stacking, even for large proteins, was found to occur when alkali salts were added to the sample. The addition of salt to the protein sample promotes a strong improvement in the peak efficiency of individual proteins giving up to 2.1 × 10⁶ apparent plates/m. The concentration of salt required in the sample to reach optimal peak efficiency show dependency on both the molecular weight and molar concentration of the protein. However, adding salt will, at a sufficiently high concentration, cause a mixture of proteins to co-migrate to one very sharp peak. The observed sample stacking effect was obtained with a number of different surface modified silica capillaries indicating a general phenomenon and not surface coating specific.     

Microstructure and electrical-optical properties of cesium tungsten oxides synthesized by solvothermal reaction followed by ammonia annealing

Cesium tungsten oxides (Cs_xWO₃) were synthesized by solvothermal reactions using ethanol and 57.1 vol% ethanol aqueous solution at 200 °C for 12 h, and the effects of post annealing in ammonia atmosphere on the microstructure and electrical-optical properties were investigated. Agglomerated particles consisting of disk-like nanoparticles and nanorods of Cs_xWO₃ were formed in the pure ethanol and ethanol aqueous solutions, respectively. The samples retained the original morphology and crystallinity after annealing in ammonia atmosphere up to 500 °C, while a small amount of nitrogen ion were incorporated in the lattice. The as-prepared Cs_xWO₃ sample showed excellent near infrared (NIR) light shielding ability as well as high transparency in the visible light region. The electrical resistivity of the pressed pellets of the powders prepared in pure ethanol and 57.1 vol% ethanol aqueous solution greatly decreased after ammonia annealing at 500 °C, i.e., from 734 to 31.5 and 231 to 3.58 Ω cm, respectively.     

Determination of some phthalate acid esters in artificial saliva by gas chromatography-mass spectrometry after activated carbon enrichment

The determination of six phthalate acid esters was achieved in artificial saliva using gas chromatography-mass spectrometry following activated carbon enrichment of samples. Central composite experimental design was applied to optimize method parameters, such as pH, adsorption time and amount of activated carbon. The best compromise of analytical conditions for the simultaneous determination of analytes from spiked artificial saliva were found to be: pH (3), adsorption time (30 min), activated carbon amount (1.8 g L⁻¹) and elution solvent (chloroform). These conditions were applied to study the migration of phthalate acid esters from different children's toys into saliva. A horizontal agitation method was applied to extract the analytes from plastic toys into saliva for 2 h at 37 °C. The detection limits of the method were in the range of 1.3-5.1 μg L⁻¹, while the relative standard deviation (%) values for the analysis of 100 μg L⁻¹ of the analytes were below 3.0% (n = 5). Di-2-ethylhexyl phthalate was the main analyte found in these samples.     

Thermo-oxidative stability of polypropylene/layered silicate nanocomposites

Several series of experiments were carried out to check the effect of components on the stability of PP/layered silicate nanocomposites. The amount of organophilic montmorillonite (OMMT) changed between 0 and 6, while that of maleated polypropylene (MAPP) between 0 and 50 vol%. The composites were prepared in an internal mixer at 190 °C. Mixing speed and time were changed to study the effect of processing conditions on stability. The structure of the samples was characterised by various methods, while stability by the induction time of oxidation (OIT), the onset temperature of degradation (OOT) and by colour. Contrary to numerous claims published in the literature, which indicate the positive effect of layered silicates on the stability of polymer nanocomposites, our results clearly proved that both OMMT and MAPP accelerate degradation during processing and deteriorate the properties of PP composites. Residual stability decreases drastically with increasing amounts of both components, chain scission leads to the decrease of viscosity and to inferior strength and deformability. In spite of expectations, the effect of the components is independent of each other. Discoloration is caused mainly by the inherent colour of the filler and it decreases with increasing exfoliation. The most probable reason for decreased stability is the reaction of the components with the stabilisers, but this explanation needs further verification. Processing conditions influence degradation considerably, increasing shear rate and longer residence times lead to more pronounced degradation. The basic stabilization of commercial grade polypropylenes is insufficient to protect the polymer against degradation and without additional stabilization processing under normal conditions results in products with inferior quality.     

Thermal degradation of keratin waste

Thermal degradation of sheep wool, human hair and chicken feathers was studied by TG-MSD/FTIR and by pyrolysis followed by GC-MSD analysis in order to identify the degradation compounds and the temperature range in which they are formed. Only small differences were found between the studied keratin samples. They consist mainly in shift of characteristic temperatures of degradation and in relative amounts of compounds in degradation products, especially in aqueous phase. Degradation started with formation of ammonia and CO₂ (from 167 and 197 °C respectively and with maximum evolution at 273 and 287 °C respectively), continues with formation of sulphur-containing inorganic compounds (SCS, SCO, H₂S and SO₂ at 240, 248, 255 and 253-260 °C respectively) and of water (255 °C). Thiols are formed in two stages (257 and 320 °C) while the evolution of nitriles is maximum around 340 °C and continues up to about 480 °C. Phenol and 4-methylphenol are the most important degradation compounds, formed at 370 and 400 °C respectively. Nitrogen was present mainly in aliphatic/aromatic nitriles, pyrroles, pyridines and amides while sulphur was found mainly as sulphides, thiols, thiazoles and thiophenes.     

Comparative study on hydrolytic degradation and monomer recovery of poly(l-lactic acid) in the solid and in the melt

The hydrolytic degradation of poly(l-lactide) (PLLA) and the formation of its monomer in the solid and in the melt were investigated at 120-150 °C (in the solid), at 160 °C (in the solid up to 40 min and in the melt exceeding 40 min), and at 170-190 °C (in the melt). Such state difference caused the difference in the degradation behavior of PLLA and the behavior of lactic acid formation, although the degradation of PLLA proceeds via a bulk erosion mechanism, regardless of its state. The crystalline residues were formed at the degradation temperatures below 140 °C, but not at the degradation temperatures above 160 °C. The lactic acid yield exceeding 95% can be successfully attained for all the temperatures of 120-190 °C. The activation energy for hydrolytic degradation values of PLLA were 69.6 and 49.6 kJ mol⁻¹ for the temperature ranges of 120-160 °C (in the solid) and 170-250 °C (in the melt), respectively, and are compared with the reported values.     

Biological reference materials for assaying human albumin in urine

Summary To assess the interlaboratory variation in the results of albumin measurements, we prepared albumin solutions in human urine at various concentrations within the normal range. Since some investigators have reported that albumin is unstable in some human urine samples stored at –20°C, we screened urine samples from 21 persons to identify samples that were stable under these conditions and that had low native albumin content. The urine of two donors met these criteria, and they provided urine, which we prefiltered, sterile-filtered, and spiked with commercially available human serum albumin. The albumin was characterized as pure by a Lowry assay of protein content with National Institute of Standards and Technology bovine serum albumin (standard reference material 926) as the standard and by the appearance of one band on agarose gel electrophoresis. To evaluate the necessity for additional stabilization when urine samples are stored at –20°C, a surfactant was included in one set of materials and not included in another. The materials with surfactant have been evaluated for 10.5 months and those without surfactant for 5 months. The preserved materials showed no significant loss of activity during this period. The unpreserved materials remained stable for 2 months, and then the two higher level materials appeared to loose activity. The negative slope of the highest level of unpreserved material was statistically significant (p=0.01) during this period. In our laboratory, the albumin recovered by enzyme immunoassay was 106.7% and 115.9% in two preserved normal-range materials and 102.2% and 106.3% in similar unpreserved materials.     

Optimization of a portable microanalytical system to reduce electrode fouling from proteins associated with biomonitoring of lead (Pb) in saliva

There is a need to develop reliable portable analytical systems for on-site and real-time biomonitoring of lead (Pb) from both occupational and environmental exposures. Saliva is an appealing matrix since it is easily obtainable, and therefore a potential substitute for blood due to existing reasonably good correlation between Pb levels in blood and saliva. The microanalytical system is based on flow-injection/stripping voltammetry with a wall-jet (flow-onto) microelectrochemical cell. Samples that contain as little as 1% saliva can cause electrode fouling, resulting in significantly reduced responsiveness and irreproducible quantitations. In addition, incomplete Pb release from salivary protein can also yield a lower Pb response than expected. This paper evaluates the extent of in vitro Pb-protein binding and the optimal pretreatment for releasing Pb from the saliva samples. Even in 50% by volume of rat saliva, the electrode fouling was not observed, due to the appropriate sample pretreatment and the constant flow of the sample and acidic carrier that prevented passivation by the protein. The system offered a linear response over a low Pb range of 1-10 ppb, low detection limit of 1 ppb, excellent reproducibility, and reliability. It also yielded the same Pb concentrations in unknown samples as did the ICP-MS. These encouraging results suggest that the microanalytical system represents an important analytical advancement for real-time non-invasive biomonitoring of Pb.     

Loss of selenium in water samples at natural levels during storage

Selenium losses in river, ground, snow-melt and tap water samples, and the recovery of selenite, selenate and selenomethionine added to purified water have been studied. In 1-litre high-density polyethylene bottles, tap, river and snow-melt water samples (at Se concentrations of 44.5–138 ng/l) could be stored at 4 °C for up to 15 days without Se losses. In similar samples stored at room temperature Se losses of 13–25% after 15 days were found, except for groundwater, which showed no Se losses during storage for 13 months at room temperature or at 4 °C. Selenite and selenate added to purified water were recovered without losses after 15 days at 4 °C, while 7.5% of selenomethionine was lost. The stability of different chemical forms of Se during storage followed the order: selenate > selenomethionine > selenite. It is recommended that unacidified water samples should not be kept in polyethylene bottles at room temperature for more than 1 week, nor stored at 4 °C for more than 2 weeks, before analysis for Se.