Determination and distribution of N-acetyl- and N-glycolylneuraminic acids in culture media and cell-associated glycoconjugates from human malignant mesothelioma and adenocarcinoma cells

Sialic acids containing glycoconjugates are very common in human neoplasias and their expression frequently correlates with malignant phenotype and the tumor grade. The majority of tumor markers containing sialic acids in man involve changes in the amount of total sialic acids and in the presence of the two main sialic acid types, Neu5Ac and Neu5Gc, and their derivatives. The aim of the present study was to examine whether malignant mesothelioma cell lines synthesize sialic acid containing glycoconjugates at both the extracellular and cell membrane levels and particularly whether the type and the content of Neu5Ac and Neu5Gc are of biological importance for mesothelioma cell differentiation and evaluation of its prognosis. The study was performed in three human malignant mesothelioma cell lines, two with a fibroblast like phenotype (STAV-FCS and Vester) and one of epithelial differentiation (STAV-AB), which developed from the pleural effusions of patients with malignant mesothelioma and in one human adenocarcinoma cell line (Wart). Neu5Ac and Neu5Gc were determined following a mild hydrolysis step and a sample clean-up procedure. The determination was performed by reversed-phase HPLC after the NeuAc and NeuGc had been converted to per-O-benzoylated derivatives. It was found that Neu5Gc is the major sialic acid in the culture media of all cell lines examined. Molar ratios of Neu5Ac to Neu5Gc showed that Neu5Gc is the predominant sialic acid in the culture medium of the fibroblast-like mesothelioma cells. Neu5Ac is almost undetectable in the cell membrane, whereas Neu5Gc is present in considerable amounts. The obtained results suggest that the type and the content of Neu5Ac and Neu5Gc in culture media are of biological importance for mesothelioma cell differentiation and may be of value in the evaluation of prognosis.     

Determination of sialic acids in infant formula by liquid chromatography tandem mass spectrometry

A liquid chromatographic/tandem mass spectrometric method using pneumatically assisted electrospray ionization (LC-ESI-MS/MS) was developed for determination of N-acetylneuramic acid and N-glycolylneuramic acid in infant formula. Reconstituted samples were hydrolysed in dilute sulfuric acid and deproteinized with acetonitrile. The extract was analysed directly without further clean-up by hydrophilic interaction chromatography. The substances were detected in negative ion mode and matrix matched standards were used for calibration. The relative intra-laboratory reproducibility standard deviation was better than 6% for both substances. An R2 of 0.985 was obtained by comparison with a classical colorimetric assay based on reaction with resorcinol. The developed method is expected to be applied for accurate routine analysis of infant formulas.     

Mass spectrometric analysis of the glycosphingolipid‐derived glycans from miniature pig endothelial cells and islets: identification of NeuGc epitope in pig islets

Glycosphingolipid (GSL) is a major component of the plasma membrane in eukaryotic cells that is involved directly in a variety of immunological events via cell‐to‐cell or cell‐to‐protein interactions. In this study, qualitative and quantitative analyses of GSL‐derived glycans on endothelial cells and islets from a miniature pig were performed and their glycosylation patterns were compared. A total of 60 and 47 sialylated and neutral GSL‐derived glycans from the endothelial cells and islets, respectively, were characterized by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) and collision‐induced fragmentation using positive‐ion electrospray ionization (ESI) ion‐trap tandem mass spectrometry (MS/MS). In accordance with previous immunohistochemistry studies, the α‐Gal‐terminated GSL was not detected but NeuGc‐terminated GSLs were newly detected from miniature pig islets. In addition, the neutral GSL‐derived glycans were relatively quantified by derivatization with carboxymethyl trimethylammonium hydrazide (so called Girard's T reagent) and MALDI‐TOF MS. The structural information of the GSL‐derived glycans from pig endothelial cells and islets suggests that special attention should be paid to all types of glycoconjugates expressed on pig tissues or cells for successful clinical xenotransplantation. Copyright © 2009 John Wiley & Sons, Ltd.     

AAPBA@KCC-1的合成及其对血清中唾液酸和神经节苷脂的分析应用

将3-丙烯酰胺基苯硼酸(AAPBA)修饰在介孔二氧化硅KCC-1的表面,合成AAPBA@KCC-1材料,并通过扫描电子显微镜、透射电子显微镜、N_2吸附解吸附测试和X射线光电子能谱对其进行表征;将AAPBA@KCC-1材料作为分散固相萃取(dSPE)的吸附剂,对血清样品中的唾液酸和神经节苷脂(GLS)进行提取和富集;采用傅里叶变换离子回旋共振质谱法和超高效液相色谱-离子淌度-四极杆飞行时间质谱法分别对AAPBA@KCC-1材料吸附得到的唾液酸和神经节苷脂进行分析。结果表明,AAPBA@KCC-1材料能够有效吸附唾液酸和神经节苷脂,简化了样品前处理的操作,可以进一步应用于生物样品中唾液酸和神经节苷脂的研究。     

A validated HPLC method with electrochemical detection for simultaneous assay of 5-aminosalicylic acid and its metabolite in human plasma

A high-performance liquid chromatographic method was developed, validated and applied to the simultaneous determination of 5-aminosalicylic acid (5-ASA) and its acetylated metabolite (acetyl-5-ASA) in human plasma. The method involves liquid-liquid extraction with methanol followed by isocratic reversed-phase chromatography on a Kromasil KR100 C(18) column with electrochemical detection. The recovery, selectivity, linearity, precision and accuracy of the method were evaluated from spiked human plasma samples. The effects of mobile phase composition, buffer concentration, mobile phase pH and concentration of organic modifiers on retention of 5-ASA, acetyl 5-ASA and internal standard were investigated. Limits' of detection were 5 ng/mL for 5-ASA and 10 ng/mL for acetyl-5-ASA, respectively. The method can be used for supporting therapeutical drug monitoring and pharmacokinetic studies.     

Simultaneous determination of niacin and its metabolites—nicotinamide,nicotinuric acid and N‐methyl‐2‐pyridone‐5‐carboxamide—in human plasma by LC‐MS/MS and its application to a human pharmacokinetic study

An LC‐MS/MS method for the simultaneous quantitation of niacin (NA) and its metabolites, i.e. nicotinamide (NAM), nicotinuric acid (NUA) and N‐methyl‐2‐pyridone‐5‐carboxamide (2‐Pyr), in human plasma (1 mL) was developed and validated using nevirapine as an internal standard (IS). Extraction of the NA and its metabolites along with the IS from human plasma was accomplished using a simple liquid–liquid extraction. The chromatographic separation of NA, NAM, NUA, 2‐Pyr and IS was achieved on a Hypersil‐BDS column (150 ¥ 4.6 mm, 5 mm) column using a mobile phase consisting of 0.1% formic acid : acetonitrile (20:80 v/v) at a flow rate of 1 mL/min. The total run time of analysis was 2 min and elution of NA, NAM, NUA, 2‐Pyr and IS occurred at 1.37, 1.46, 1.40, 1.06 and 1.27 min, respectively. A detailed validation of the method was performed as per the FDA guidelines and the standard curves were found to be linear in the range of 100–20000 ng/mL for NA; 10–1600 ng/mL for NUA and NAM and 50–5000 ng/mL for 2‐Pyr with mean correlation coefficient of ≥0.99 for each analyte. The method was sensitive, specific, precise, accurate and suitable for bioequivalence and pharmacokinetic studies. The developed assay method was successfully applied to a pharmacokinetic study in humans. Copyright © 2010 John Wiley & Sons, Ltd.     

Analysis of amino acids in individual human erythrocytes by capillary electrophoresis with electroporation for intracellular derivatization and laser-induced fluorescence detection

A method for monitoring amino acids in single erythrocytes is described. For intracellular derivatization, reagent fluorescein isothiocyanate (FITC) was introduced into living cells by electroporation. For an 8 microm erythrocyte, the analytes were diluted by a factor of only 1.6. After completion of the derivatization reaction, a single cell was injected into the separation capillary tip and lysed there. The derivatized amino acids were separated by capillary electrophoresis, followed by laser-induced fluorescence detection. Nine amino acids were quantitatively determined, with amounts of amino acids ranging from 3.8-32 amol/single cell.     

Simultaneous determination of low-molecular-weight organic acids and chlorinated acid herbicides in environmental water by a portable CE system with contactless conductivity detection

This report describes a method to simultaneously determine 11 low-molecular-weight (LMW) organic acids and 16 chlorinated acid herbicides within a single run by a portable CE system with contactless conductivity detection (CCD) in a poly(vinyl alcohol) (PVA)-coated capillary. Under the optimized condition, the LODs of CE-CCD ranged from 0.056 to 0.270 ppm, which were better than for indirect UV (IUV) detection of the 11 LMW organic acids or UV detection of the 16 chlorinated acid herbicides. Combined with an on-line field-amplified sample stacking (FASS) procedure, sensitivity enhancement of 632- to 1078-fold was achieved, with satisfactory reproducibility (RSDs of migration times less than 2.2%, and RSDs of peak areas less than 5.1%). The FASS-CE-CCD method was successfully applied to determine the two groups of acidic pollutants in two kinds of environmental water samples. The portable CE-CCD system shows advantages such as simplicity, cost effectiveness, and miniaturization. Therefore, the method presented in this report has great potential for onsite analysis of various pollutants at the trace level.     

Simultaneous determination of sunitinib and its two metabolites in plasma of Chinese patients with metastatic renal cell carcinoma by liquid chromatography–tandem mass spectrometry

A simple and rapid liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method was developed and validated for the simultaneous determination of sunitinib and its two metabolites in plasma of Chinese patients with metastatic renal cell carcinoma (mRCC). After simple one‐step protein precipitation with methanol–acetonitrile (1:1, v/v), all three analytes were separated on an Agilent Zorbax SB‐C₁₈ column using a gradient mobile phase consisting of water (0.1% formic acid)–acetonitrile (0.1% formic acid) at a flow rate of 0.50 mL/min. The detection was performed in multiple reaction monitoring mode, using the transitions of m/z 399.0 → 326.2, m/z 371.0 → 283.1, m/z 343.0 → 283.1 and m/z 386.3 → 122.2 for sunitinib, M1, M2 and buspirone, respectively. The method was linear over the range of 0.10–100 ng/mL for all three analytes using only 50 μL of plasma and the lower limit of quantifications for the three analytes were all 0.10 ng/mL. The intra‐day and inter‐day precisions were all less than 15% and the accuracies were within the range of ±15%; recoveries were between 85.0 and 115%. The validated method was successfully applied to an explorative pharmacokinetic study of sunitinib in Chinese patients with mRCC following multi‐dose oral administration. Copyright © 2012 John Wiley & Sons, Ltd.     

On-capillary derivatization and analysis of amino acids in human plasma by capillary electrophoresis with laser-induced fluorescence detection: application to diagnosis of aminoacidopathies

A capillary electrophoresis with laser-induced fluorescence detection method for the analysis of free amino acids (AA) in human plasma was developed. A mixture of 16 AA was on-capillary derivatized with 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ) and separated inside the capillary in less than 30 min using 70 mM borax-3.5 mM SDS pH 9.3 as running buffer. Four plasma samples from a healthy donor and patients suffering from phenylketonuria, propionic acidemia, and tyrosinemia type II were studied. Repeatabilities calculated as intra-day RSD (n = 3) values for the AA involved in these aminoacidopathies (glycine, phenylalanine, and tyrosine) were in the range of 0.3 to 1.2% for migration time and 3.7 to 8.2% for peak height. Reproducibilities calculated as inter-day RSD (n = 4) values for the same AA were between 0.7 and 1.4% for migration time and 4.7 and 9.1% for peak height. A fast qualitative analysis allowed the identification of the corresponding disease by comparing the electrophoretic profiles from the patient and the healthy donor and noting the increased level of the specific AA accompanying each individual disease. The results of the quantitative analysis for glycine, phenylalanine, and tyrosine in the plasma samples studied using the developed method showed a good agreement with those provided by the Center of Diagnosis of Molecular Diseases using a standard method for AA analysis.     

Phenylboronic acid modified solid‐phase extraction column: Preparation,characterization, and application to the analysis of amino acids in sepia capsule by removing the maltose

Maltose, a common auxiliary material of pharmaceutical preparation, may disturb the analysis of total amino acids in sepia capsule by aldolization. Therefore, it is necessary to remove the maltose through a convenient method. In this work, a phenylboronic acid modified solid‐phase extraction column has been synthesized and used to remove the maltose. The materials were synthesized by one step “thiol‐ene” reaction and the parameters of the column such as absorption capacity, recovery, and absorption specificity have been investigated. The results showed the column (0.5 cm of length × 0.5 cm of inner diameter) can absorb 4.6 mg maltose with a linear absorption and absorption specificity. Then this technique was applied in the quantification of amino acids in sepia capsule. After the optimization of the method, four kinds of amino acids, which were the most abundant, were quantified by high‐performance liquid chromatography with diode array detection. The amounts of the four kinds of amino acids are 1.5～2 times more than that without the treatment of solid‐phase extraction column, which almost overcomes the influence of the maltose. All the results indicate that the phenylboronic acid modified solid‐phase extraction column can successfully help to accurately quantify the total amino acids in sepia capsule.     

Determination of cell type‐specific proteome signatures of primary human leukocytes,endothelial cells,keratinocytes, hepatocytes,fibroblasts and melanocytes by comparative proteome profiling

Cells gain their functional specialization by different protein synthesis. A lot of knowledge with respect to cell type‐specific proteins has been collected during the last thirty years. This knowledge was built mainly by using antibodies. Nowadays, modern MS, which supports comprehensive proteome analyses of biological samples, may render possible the search for cell type‐specific proteins as well. However, a therefore necessary systematic MS study comprising many different cell types has not been performed until now. Here we present a proteome analysis strategy supporting the automated and meaningful comparison of any biological samples. We have presently applied this strategy to six different primary human cell types, namely leukocytes, endothelial cells, keratinocytes, hepatocytes, fibroblasts, and melanocytes. Comparative analysis of the resulting proteome profiles allowed us to select proteins specifically identified in one of the six cell types and not in any of the five others. Based on these results, we designated cell type‐specific proteome signatures consisting each of six such characteristic proteins. These signatures independently reproduced well‐known marker proteins already established for FACS analyses in addition to novel candidate marker proteins. We applied these signatures for the interpretation of proteome profiles obtained from the analyses of hepatocellular carcinoma‐associated tissue homogenates and normal liver tissue homogenates. The identification of members of the above described signatures gave us an indication of the presence of characteristic cells in the diseased tissues and thus supported the interpretation of the proteomics data of these complex biological samples.     

Simultaneous determination of short‐chain fatty acids in human feces by HPLC with ultraviolet detection following chemical derivatization and solid‐phase extraction segmental elution

Short‐chain fatty acids are currently the most studied metabolites of gut microbiota, but the analysis of them, simultaneously, is still challenging due to their unique property and wide concentration range. Here, we developed a sensitive and versatile high‐performance liquid chromatography with ultraviolet detection method, using pre‐column derivatization and solid‐phase extraction segmental elution, for the quantification of both major and trace amounts of short‐chain fatty acids in human feces. Short‐chain fatty acids were converted to 3‐nitrophenylhydrazine‐derived analytes, and then solid‐phase extraction segmental elution was used for extraction of major analytes and enrichment of trace analytes. The method validation showed limits of quantitation ?0.04 mM, and coefficient of determination > 0.998 at a wide range of 0.04–8.0 mM. The intra‐ and interday precision of analytes were all within accepted criteria, and the recoveries were 96.12 to 100.75% for targeted analytes in fecal samples. This method was successfully applied in quantification of eight analytes in human feces, which therefore could provide a sensitive and versatile high‐performance liquid chromatography with ultraviolet detection method for precise and accurate quantitation of short‐chain fatty acids in human feces.     

Synthesis and characterization of tributyltin derivatives from 4‐oxo‐4‐(arylamino)butanoic acids and their in vitro biological activity against cervical cancer cell lines

Uterine (cervix and corpus) cancer is one of the major causes of mortality in women in Mexico. Organotin carboxylated derivatives have shown high cytotoxic activity against various cell lines of human origin. We describe the synthesis of three new tri‐n‐butyltin derivatives from 4‐oxo‐4‐(arylamino)butanoic acids; their structures were confirmed using spectral data (¹H NMR, ¹³C NMR, ¹¹⁹Sn NMR and infrared), elemental analyses, mass spectrometry and X‐ray diffraction. All the tri‐n‐butyltin carboxylates exhibit ¹ J (^119/117Sn–¹³C) coupling satellites in solution and lie in the range 357 to 339 Hz, suggesting a tetrahedral geometry around the tin atom. The polymeric structures of two of the derivatives and the monomeric structure of another were confirmed using X‐ray crystallography. Using succinic anhydride as raw material, five N‐substituted succinamic acid compounds were synthesized by the acylation reaction with aniline, 4‐nitroaniline, 4‐nitro‐3‐(trifluoromethyl)aniline, 2‐amino‐5‐nitrothiazole and 4‐aminoantipyrine. From these compounds, five tin derivatives were prepared and their in vitro anti‐proliferative effect on HeLa, CaSki and ViBo cell lines was screened. All of the compounds showed potency against all three strains and null or low cytotoxic activity (necrotic) as well. The most potent of our derivatives as an anti‐proliferative agent against the three cell lines was tributylstannyl 4‐oxo‐4‐[(3‐trifluoromethyl‐4‐nitrophen‐1‐yl)amino]butanoate, exhibiting an IC₅₀ value of 0.43 μM against the HeLa cell line. Copyright © 2014 John Wiley & Sons, Ltd.     

Detection of Delta9-tetrahydrocannabinolic acid A in human urine and blood serum by LC-MS/MS

Delta9-tetrahydrocannabinolic acid A (Delta9-THCA-A) is the precursor of Delta9-tetrahydrocannabinol (Delta9-THC) in hemp plants. During smoking, the non-psychoactive Delta9-THCA-A is converted to Delta9-THC, the main psychoactive component of marihuana and hashish. Although the decarboxylation of Delta9-THCA-A to Delta9-THC was assumed to be complete--which means that no Delta9-THCA-A should be detectable in urine and blood serum of cannabis consumers--we found Delta9-THCA-A in the urine and blood serum samples collected from police controls of drivers suspected for driving under the influence of drugs (DUID). For LC-MS/MS analysis, urine and blood serum samples were prepared by solid-phase extraction. Analysis was performed with a phenylhexyl column using gradient elution with acetonitrile. For detection of Delta9-THCA-A, the mass spectrometer (MS) (SCIEX API 365 triple-quadrupole MS with TurboIonSpray source) was operated in the multiple reaction monitoring (MRM) mode using the following transitions: m/z357 --> 313, m/z357 --> 245 and m/z357 --> 191. Delta9-THCA-A could be detected in the urine and blood serum samples of several cannabis consumers in concentrations of up to 10.8 ng/ml in urine and 14.8 ng/ml in serum. The concentration of Delta9-THCA-A was below the Delta9-THC concentration in most serum samples, resulting in molar ratios of Delta9-THCA-A/Delta9-THC of approximately 5.0-18.6%. Only in one case, where a short elapsed time between the last intake and blood sampling is assumed, the molar ratio was 18.6% in the serum. This indicates differences in elimination kinetics, which need to be investigated in detail.     

Determination of 18 nucleobases, nucleosides and nucleotides in human peripheral blood mononuclear cells by isocratic solvent-generated ion-pair chromatography

The paper describes a method for the separation of 18 nucleotides, nucleosides and nucleobases by isocratic solvent-generated ion-pair chromatography in 55 min. It has been developed for pharmacodynamic monitoring of mycophenolic acid, the active metabolite of the immunosuppressant mycophenolate mofetil. The method was applied for the detection of mycophenolic acid-induced changes in inosine 5′-monophosphate dehydrogenase (IMPDH) activity in the lysate of human peripheral blood mononuclear cells.     

Accurate and sensitive determination of sildenafil,tadalafil, vardenafil,and avanafil in illicit erectile dysfunction medications and human urine by LC with quadrupole‐TOF‐MS/MS and their behaviors in simulated gastric conditions

The widespread use of phosphodiesterase‐5 inhibitors has attracted broad attention of counterfeiters to develop illicit erectile products with inaccurate amounts, unknown toxicity, and purity of active ingredients. Correspondingly, intake of these products endangers consumer health and needs to be screened for precautionary actions to reduce this risk. Therefore, in this study, a sensitive and rapid analytical method has been developed for simultaneous determination of selected phosphodiesterase‐5 inhibitors present in illicit erectile medications and human urine. Quantification of the analytes was performed by liquid chromatography coupled with quadrupole‐time‐of‐flight tandem mass spectrometry system. The chromatographic separation was successfully achieved with a run period of 8 min. Low detection limits were obtained in the range of 1.63–9.81 ng/g with relative standard deviations below 7.72% obtained using the replicate measurements of lowest concentration in calibration plots. The analytical performance of the proposed method proved good linearity, low detection limits, good accuracy and precision with high percent recoveries for human urine samples. Developed method was successfully applied to real samples including four different brands of illicit erectile medications. The results obtained revealed the presence of high levels of sildenafil in analyzed samples. The behaviors of selected phosphodiesterase‐5 inhibitors were also studied in simulated gastric conditions.     

Direct and simultaneous quantitation of 5‐aminolaevulinic acid and porphobilinogen in human serum or plasma by hydrophilic interaction liquid chromatography–atmospheric pressure chemical ionization/tandem mass spectrometry

Serum/plasma concentrations of 5‐aminolaevulinic acid (ALA) and porphobilinogen (PBG) are elevated in patients with acute hepatic porphyrias, especially during acute attacks. Current assays require lengthy sample pre‐treatment and derivatization steps. We report here a rapid, sensitive and specific hydrophilic interaction liquid chromatography–tandem mass spectrometry method for the direct and simultaneous quantitation of ALA and PBG in serum or plasma following simple protein precipitation with acetonitrile and centrifugation prior to injection. ALA and PBG were detected using selected reaction monitoring mode, following positive atmospheric pressure chemical ionization. Calibration was linear from 0.05 to 50 µmol/L for ALA and PBG. For both analytes, imprecision (relative standard deviation) was <13% and accuracy (percentage nominal concentrations) was between 92 and 107%. The method was successfully applied to the measurement of ALA and PBG in serum or plasma samples for the screening, biochemical diagnosis and treatment monitoring of patients with acute hepatic porphyrias. Copyright © 2012 John Wiley & Sons, Ltd.     

Targeted metabolomic analysis of 33 amino acids and biogenic amines in human urine by ion‐pairing HPLC‐MS/MS: Biomarkers for tacrolimus nephrotoxicity after renal transplantation

Calcineurin inhibitor nephrotoxicity, especially for the widely used tacrolimus, has become a major concern in post‐transplant immunosuppression. Multiparametric amino acid metabolomics is useful for biomarker identification of tacrolimus nephrotoxicity, for which specific quantitative methods are highlighted as a premise. This article presents a targeted metabolomic assay to quantify 33 amino acids and biogenic amines in human urine by high‐performance liquid chromatography coupled with tandem mass spectrometry. Chromatographic separation was carried out on an Agilent Zorbax SB‐C₁₈ column (3.0 × 150 mm, 5 μm) with addition of an ion‐pairing agent in the mobile phase, and MS/MS detection was achieved in both the positive and negative multiple reaction monitoring modes. Good correlation coefficients (r² > 0.98) were obtained for most analytes. Intra‐ and inter‐day precision, stability, carryover and incurred sample reanalysis met with the acceptance criteria of the guidance of the US Food and Drug Administration. Analysis on urine from healthy volunteers and renal transplantation patients with tacrolimus nephrotoxicity confirmed symmetric dimethylarginine and serine as biomarkers for kidney injury, with AUC values of 0.95 and 0.81 in receiver operating characteristic analysis, respectively. Additionally, symmetric dimethylarginine exhibited a tight correlation with serum creatinine, and was therefore indicative of renal function. The targeted metabolomic assay was time and cost prohibitive for amino acid analysis in human urine, facilitating the biomarker identification of tacrolimus nephrotoxicity.