Liquid Chromatography-tandem Mass Spectrometry Technique Research Articles

The serum proteomics tracking of hepatocellular carcinoma early recurrence following radical resection.

Purpose: There is still lacking of highly sensitive and specific biomarkers for the prediction of hepatocellular carcinoma (HCC) early recurrence, which has hindered further improvement of the clinical outcomes. We aim to find highly sensitive and specific biomarkers for the prediction of HCC recurrence.Patients and methods: By using isobaric tags for relative and absolute quantitation (iTRAQ)-based multidimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS) technique, we have quantitatively investigated and monitored the proteome alterations of a series of serum after radical resection during the follow-up of 4 HCC patients.Results: A total of 27 differentially abundant proteins (DAPs) in serum were identified to be closely associated with the early recurrence of HCC, and these DAPs were particularly concentrated within ERK1/2 and nuclear factor-κ beta signaling pathways, suggesting the dysregulation of these two pathways played an important role in the pathological process of HCC early recurrence. Further investigation of a cohort of patients confirmed that the high serum level of PGK1 was closely associated with HCC early recurrence and poor prognosis. In addition, the serum level of PGK1 could be complementary with AFP to further improve the sensitivity and specificity for predicting the relapse of HCC.Conclusion: PGK1 might be an independent factor for the recurrence of HCC. And the PGK1 could be complementary with AFP to further improve the sensitivity and specificity in prognostic prediction of HCC relapse.

Implementation of an objective test of medication adherence in a hypertensive population

To develop, implement and audit the performance of an objective liquid chromatography-tandem mass spectrometry (LC-MS/MS) technique for the detection of antihypertensive medications in urine. We have developed a novel LC-MS/MS method for the detection of 23 commonly prescribed antihypertensive medications in urine in a single assay. The method was first applied to the analysis of urine from a validation cohort of 49 individuals who were taking at least one antihypertensive agent in the screening …

Proteomic analysis of mouse ovaries during the prepubertal stages

Postnatal folliculogenesis, primordial follicle activation and follicular development at early stage are important for normal ovarian function and fertility, and a comprehensive understanding of this process under physiological condition is necessary. To observe the regulation and mechanism of ovarian follicle development during the prepubertal stages, we collected the mouse ovaries from three time points, including 1 day, 7 days, and 4 weeks after birth. We then performed a proteomic analysis using tandem mass tags (TMT) labeling combined with a two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS) technique. A total of 706 proteins were determined to be significant differential abundance (P-SDA). Sixty upregulated proteins and 12 downregulated proteins that were P-SDA and 3 significant KEGG pathways (P < 0.05) were found at 7 days vs. 1 day after birth, while 237 upregulated proteins, 271 downregulated proteins and 42 significant KEGG pathways were found for 4 weeks vs. 7 days after birth. Some vital genes (Figla, Ooep, Padi6, Zp3, Hsd3b1, cyp11a1), key pathways (ECM-receptor interaction, focal adhesion, ovarian steroidogenesis, complement and coagulation cascades, PI3K/Akt/mTOR), and metabolic regulation (energy metabolism, lipid metabolism, metal ion metabolism) were found to be related to the postnatal folliculogenesis, primordial follicle activation and follicular development. Finally, qRT-PCR and western blotting verified some vital genes and further elucidated the developmental process of follicles, and the results may contribute to the understanding of the formation and activation of primordial follicle and follicular development.Significance: This study offers the first proteomic insights into mechanisms of follicle development under physiological condition during the prepubertal stages. By comparing P-SDA of mouse ovaries during various period of age, our data reveals that the regulation of primordial follicle formation and activation is significantly different from that of follicular development. These findings demonstrate that many unique molecular mechanisms underlie ovarian development could be used for ovarian disease research.

A "Green" Homogenate Extraction Coupled with UHPLC-MS for the Rapid Determination of Diterpenoids in Croton Crassifolius.

Clerodane diterpenoids are the main bioactive constituents of Croton crassifolius and are proved to have multiple biological activities. However, quality control (QC) research on the constituents are rare. Thus, the major research purpose of the current study was to establish an efficient homogenate extraction (HGE) process combined with a sensitive and specific ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC–MS) technique together for the rapid extraction and determination of clerodane diterpenoids in C. crassifolius. All calibration curves showed good linearity (r > 0.9943) within the test ranges and the intra- and inter-day precisions and repeatability were all within required limits. This modified HGE–UHPLC–MS method only took 5 min to extract nine clerodane diterpenoids in C. crassifolius and another 12 min to quantify these components. The results indicated that the quantitative analysis based on UHPLC–MS was a feasible method for QC of clerodane diterpenoids in C. crassifolius, and the findings outlined in the current study also inferred the potential of the method in the QC of clerodane diterpenoids in other complex species of plants.

Compositional and structural analysis of glycosaminoglycans in cell-derived extracellular matrices.

The extracellular matrix (ECM) is a highly dynamic and complex meshwork of proteins and glycosaminoglycans (GAGs) with a crucial role in tissue homeostasis and organization not only by defining tissue architecture and mechanical properties, but also by providing chemical cues that regulate major biological processes. GAGs are associated with important physiological functions, acting as modulators of signaling pathways regulating several cellular processes such as cell growth and differentiation. Recently, in vitro fabricated cell-derived ECM have emerged as promising materials for regenerative medicine due to their ability of better recapitulate the native ECM-like composition and structure, without the limitations of availability and pathogen transfer risks of tissue-derived ECM scaffolds. However, little is known about the molecular and more specifically, GAG composition of these cell-derived ECM. In this study, three different cell-derived ECM were produced in vitro and characterized in terms of their GAG content, composition and sulfation patterns using a highly sensitive liquid chromatography-tandem mass spectrometry technique. Distinct GAG compositions and disaccharide sulfation patterns were verified for the different cell-derived ECM. Additionally, the effect of decellularization method on the GAG and disaccharide relative composition was also assessed. In summary, the method presented here offers a novel approach to determine the GAG composition of cell-derived ECM, which we believe is critical for a better understanding of ECM role in directing cellular responses and has the potential for generating important knowledge to use in the development of novel ECM-like biomaterials for tissue engineering applications.

Organic tracers from biomass burning in snow from the coast to the ice sheet summit of East Antarctica

Biomass burning is a significant process in the Earth system, driving ecosystem dynamics and changes in global vegetation, and affecting the carbon cycle and climate. Projections of future fire activities require an understanding of the connection between fire history and climate in the past. Polar snow/ice contain long-term records of past climates and fire activity and hold great promise to improve our understanding of wildfire patterns. Here, using ultra-high-performance liquid chromatography-tandem mass spectrometry techniques, we quantified three organic compounds (levoglucosan, vanillic, and syringic acids) released by biomass burning in snow samples collected along a 1250-km transect from the coast to the ice sheet summit Dome A in East Antarctica. Results indicate that these tracers are ubiquitous and have reached the ice sheet summit from the continental emissions in the Southern Hemisphere. These compounds showed high levels close to the coastal areas and decreased to a low level on the Antarctic plateau. The snow samples had similar levoglucosan/vanillic acid (∼45) and levoglucosan/syringic acid ratios (∼243) as aerosols from biomass burning. Multivariate analysis indicates that these compounds were likely derived from the burning of grasses and evergreen broadleaf trees that are widespread in Southern Hemisphere than from evergreen conifers that dominate northern hemisphere fire-prone ecosystems. Snow accumulation rate influenced the levels of these compounds, while coexisting ions had little effect on compound contents in the snow. The low concentrations of levoglucosan at inland sites (mean of 2.7 pg mL−1; versus 3.5 and 3.7 pg mL−1 in coastal and transition zones, respectively) could be associated with the oxidation by OH radicals under sunlight. Our analysis demonstrated that the ubiquity of multiple biomarkers from biomass burning in East Antarctic surface snow can provide baseline concentrations for future studies in Antarctica.

Serum Metabolomics Analysis in Wasp Sting Patients.

To analyze the dynamic changes of serum metabolomics in wasp sting victims, we collected serum from 10 healthy volunteers and 10 patients who had been stung 3 hours, 24 hours, and 72 hours before sample collection. We analyzed the metabolomics by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) techniques and then performed enrichment analysis. A total of 838 metabolites were identified. Serum metabolomics analysis using MetaboAnalyst revealed 289 metabolites that were significantly different among patients in the 3-hour group versus healthy controls (P<0.001). Pathway analysis of those metabolites indicated that those metabolic sets were associated with sphingolipid metabolism. Based on the differences among the control, 3-hour, 24-hour, and 72-hour groups, we classified serum metabolites into different categories. The first and second categories included 297 and 280 metabolites that were significantly different in terms of concentration among healthy controls versus the participants whose sera were analyzed 3 hours, 24 hours, and 72 hours after wasp stings. Pathway analysis of those metabolites indicated that those metabolic sets were associated with thiamine metabolism. The third category included 269 significant metabolites. The fourth category included 28 significant metabolites. Pathway analysis of the metabolites in third and fourth categories indicated that those metabolic sets were associated with phenylalanine, tyrosine, and tryptophan biosynthesis. The fifth category included 31 metabolites, which were not significantly different between the control and 3-hour groups but were higher in concentration in the 24-hour and 72-hour groups. Pathway analysis of the fifth category of significant metabolites identified linoleic acid metabolism. In conclusion, multiple metabolic pathways are associated with wasp stings, and these might provide a basis for exploring mechanisms of wasp sting injury and potential targets for therapy.

Bioanalytical technique for determination of tasimelteon in human plasma by LC-MS/MS and its application to pharmacokinetic study in humans.

A highly sensitive, specific and rapid liquid chromatography-tandem mass spectrometry technique for the quantification of tasimelteon in human plasma has been developed and validated using tasimelteon-d5 as internal standard. Liquid-liquid extraction technique with ethyl acetate was used for extraction of tasimelteon from the plasma. The chromatographic separation was achieved on an Agilent Zorbax, Eclipse, C18 (4.6 × 50 mm, 5 μm) column using a mobile phase of acetonitrile and 0.02% formic acid buffer (85:15, v/v) with a flow rate of 0.5 mL/min. A detailed method validation was performed as per the United States Food and Drug Administration guidelines. The linear calibration curve was obtained over the concentration range 0.30-299 ng/mL. The API-4000 liquid chromatography-tandem mass spectrometry was operated under multiple reaction monitoring mode during analysis. The validated method was successfully applied to estimate plasma concentration of tasimelteon after oral administration of a single dose of a 20 mg capsule in healthy volunteers under fasting conditions. The maximum concentration of the drug achieved in the plasma was 314 ± 147 ng/mL and the time at which this concentration was attained was 0.54 ±0.22 h.

α-Glucosidase immobilization on functionalized Fe3O4 magnetic nanoparticles for screening of enzyme inhibitors

Magnetic nanoparticles (MNPs) are widely used for the immobilization of enzyme owing to the unique properties such as good biocompatibility and rapid separation. Herein, we used Fe3O4 magnetic nanoparticles (Fe3O4 MNPs) as the carrier core with (3-aminopropyl)triethoxysilane (APTES) modification by our approach, in which α-glucosidase was stereoscopically immobilized on the surface of Fe3O4 MNPs via covalent binding. The result of immobilization was characterized by scanning electron microscope (SEM) and fourier transform-infrared spectroscopy (FT-IR). Then we optimized some key parameters of the immobilization reaction, including the ratio of MNPs to enzyme, GA concentration, crosslinking time and immobilization time. Moreover, under the optimal conditions, pH tolerance, thermo stability and reusability of the immobilized enzyme were investigated and compared with the free one. In order to evaluate the change of the affinity of the enzyme to its specific substrate after immobilization, the Michaelis-Menten constant (Km) was also studied. Finally, the immobilized α-glucosidase combining with high performance liquid chromatography-tandem mass spectrometry technique (HPLC-MS/MS) was applied to screen and identify eight inhibitors from Polygonum cuspidatum extract. These results indicated that the established method had the broad prospects for biotechnological applications.

Metabolism and elimination of the catechol-o-methyltransferase inhibitor tolcapone in the horse.

The metabolism of the masking agent tolcapone in the horse has been investigated. This substance was found to have undergone various chemical transformations that produced a large variety of phase I metabolites, as well as glucuronide and sulfate conjugation. Confirmation of the presence of tolcapone and the 3-O-methylated metabolite in the blood samples collected up to 240minutes and in urine obtained up to 24hours, was successfully conducted using both gas chromatography- and liquid chromatography-tandem mass spectrometry techniques. The 3-O-methyl tolcapone is the better marker to use in a screening method because, in comparison to tolcapone, we have found that this substance offers superior chromatographic performance that should potentially give a lower limit of detection.

Quick Multi-Class Determination of Residues of Antimicrobial Veterinary Drugs in Animal Muscle by LC-MS/MS.

On the basis of the highly sensitive and selective liquid chromatography-tandem mass spectrometry technique, a generic extraction solvent and a sample dilution method was developed for the residue analysis of different polar veterinary drugs known as fluoroquinolones, sulfonamides, macrolides, and tiamulin in chicken muscle. The results showed that the matrix-matched calibration curves of all 10 compounds were in an effective linear relationship (r2 ≥ 0.997) in the range of 0.2–100 μg L−1. At three spiking levels of 2 (5), 50, and 100 μg kg−1, average recoveries of analytes were between 67.1% and 96.6% with relative standard deviations of intra-day and inter-day below 20%. The limits of detection and limits of quantification of the method were in the range of 0.3–2.0 μg kg−1 and 2.0–5.0 μg kg−1, respectively, which were significantly lower than their maximum residue limits. In addition, the intensity of the target analytes and its corresponding matrix effects were obviously related to the sample dilution times (matrix concentration). There were no significant differences (p > 0.05) in the average content of almost any of the analytes in medicated chickens between this method and the method in the literature for determining analytes. Lastly, the proposed method was successfully applied for the simultaneous analysis of 10 common veterinary drugs in food animal muscle tissues.

Determination of Thyreostats in Urine Using Supported Liquid Extraction and Mixed-Mode Cation-Exchange Solid-Phase Extraction: Screening and Confirmatory Methods.

The application of thyreostats in livestock has been banned in the European Union since 1981, but these drugs are currently in the focus due to the natural occurrence of thiouracil (TU). Studies have been published on TU contamination in urine samples of animal and human origins without any drug administration of it. This paper presents new analytical methods to analyze thyreostats to support the legislation on the recommended concentration (RC) levels of these drugs. Both screening and confirmatory methods are developed for analyzing thyreostats in porcine and bovine urines using a liquid chromatography-tandem mass spectrometry technique. The new methods include a chemical derivatization with 3-iodobenzyl bromide, followed by novel purification approaches using supported liquid extraction and mixed-mode cation-exchange solid-phase extraction (SPE) for screening and confirmatory purposes, respectively. The optimized derivatization in combination with the cation-exchange SPE gives high sensitivity and reducing matrix effect of the analysis. The methods are validated in accordance with the guidelines for the validation of screening methods and European Commission Decision 2002/657/EC. The confirmatory method is used in the national monitoring plan. The detected levels of TU in urine samples are below the currently applicable RC level (10 μg L-1).

Development and validation of a LC-MS/MS assay for pharmacokinetic studies of complement C5a receptor antagonists PMX53 and PMX205 in mice

PMX53 and PMX205 are cyclic hexapeptide inhibitors of complement C5a receptors (C5aR1), that are widely used to study C5aR1 pathobiology in mouse models of disease. Despite their widespread use, limited information regarding their pharmacokinetics have been reported. Here, a bioanalytical method for the quantitative determination of PMX53 and PMX205 in plasma, brain and spinal cord of mice was developed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) techniques. The LC-MS/MS method was validated in all three matrices according to regulatory guidelines and successfully applied to pharmacokinetic studies of PMX53 and PMX205 in C57BL/6 J mice following intravenous administration. The developed method was highly sensitive and sufficiently accurate with a lower limit of quantification within the range of 3–6 ng/ml in extracted plasma samples and 3–6 ng/g in processed tissue samples, which outperforms previously published LC-MS/MS methods. The results thus support the suitability, reliability, reproducibility and sensitivity of this validated technique. This method can therefore be applied to perform a complete pre-clinical investigation of PMX53 and PMX205 pharmacokinetics in mice.

Dissipation behaviour and dietary risk assessment of boscalid, triflumizole and its metabolite (FM-6-1) in open-field cucumber based on QuEChERS using HPLC-MS/MS technique.

To resist plant diseases, boscalid and triflumizole have been applied to cucumbers frequently. However, the residue and dietary risk assessment of these fungicides in cucumber should be given attention for food safety. An effective and highly sensitive method based on the liquid chromatography-tandem mass spectrometry technique for simultaneous multidetermination of boscalid, triflumizole and its metabolite (FM-6-1) in a cucumber ecosystem was established and validated. Field experiments were conducted in three different locations, where boscalid and triflumizole (35% suspension concentration) were applied at 253 g of active ingredient (a.i.) per hectare (the recommended high dosage) and 379.5 g a.i. ha-1 (1.5 times the recommended high dosage) in each location. The limits of quantification and the limits of detection of the proposed method ranged from 0.01 to 0.05 mg kg-1 and 3.9×10-5 to 7.5×10-4 mg L-1 respectively. The mean recoveries and relative standard deviations of these compounds were 80-105% and 1.0-6.1% respectively. The dissipation dynamics of compounds followed pseudo-first-order kinetic models remarkably, with a half-value period of 2.3-40.8 days. The residues of boscalid and triflumizole in cucumber at harvest were below 0.66 mg kg-1 and 0.07 mg kg-1 respectively. The results of the dietary risk assessments have shown a low dietary risk of compounds in cucumber with hazard ratios <1 and hazard index <1. These results from the experiments are the most important for putting a guide on reasonable usage of these fungicides under the open-field conditions in China. © 2018 Society of Chemical Industry.

The Chinese herbal formula Zhibai Dihuang Granule treat Yin-deficiency-heat syndrome rats by regulating the immune responses

Ethnopharmacological relevanceZhibai Dihuang Granule (ZDG), a traditional Chinese medicine (TCM) made from eight Chinese herbs, has been classically used to treat Yin-deficiency-heat (YDH) syndrome. ZDG is well known with the therapeutic efficacy of nourishing Yin and decreasing internal heat in clinic, but the mechanism of ZDG's therapeutic effect is still not clear. Materials and methodsHigh doses of triiodothyronine (T3) were given intraperitoneally to induce Hyperthyroid YDH syndrome in SD rats. The animals were then treated with ZDG for one week. The iTRAQ-coupled with two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS) technique was used to screen the differentially expressed serum proteins between ZDG treated rats and YDH syndrome rats. The differentially expressed proteins were analyzed by bioinformatics method and were verified by enzyme-linked immunosorbent assay (ELISA). ResultsA total of 55 differentially expressed proteins were identified, including 23 up-regulated proteins (>1.25 fold, p < 0.05) and 32 down-regulated proteins (<0.80 fold, p < 0.05). Among the differentially expressed proteins, 26 proteins returned to normal after ZDG treatment. Bioinformatics analysis showed that these proteins were mainly involved in immune response, including regulation of immune system process, complement activation, and humoral immune response mediated by circulating immunoglobulin. ELISA revealed significantly increased levels of Zinc-alpha-2-glycoprotein (Azgp1), L-selectin, C-reactive protein (Crp), Plasminogen (Plg), Kininogen 1 (Kng1), and significantly decreased levels of Mannose binding lectin 2 (Mbl2) and Complement C1qb chain (C1qb) in ZDG treated rats compared with YDH syndrome rats. Bioinformatics analyses indicated that Azgp1 participated in antigen processing and presentation, Crp, C1qb, and Mbl2 were involved in complement activation, while L-selectin, Plg, and Kng1 were involved in regulating the inflammatory response. ConclusionsOur study provides experimental evidence to understand the therapeutic mechanism of ZDG in YDH syndrome. The results suggested that ZDG may regulate the complement activation and inflammatory response, and promote the ability to recognize antigens to alleviate YDH syndrome.

High Fundamental Frequency Quartz Crystal Microbalance (HFF-QCM) immunosensor for pesticide detection in honey

Quantification of chemical residues in honey is a market requirement to ensure consumer safety. The most common method used to analyze these compounds is the LC/MS/MS methodology, which requires highly qualified technicians and a tedious pre-treatment of the sample. The honey-packaging industry needs cheaper and faster alternatives for routine control. HFF-QCM (High Fundamental Frequency Quartz Crystal Microbalance) sensors are becoming a good option due to their high sensitivity, fast detection and low cost, while avoiding complex sample pre-treatment. The HFF-QCM technology is based on piezoelectric sensors with frequencies in the range from several tenths of MHz to hundreds of MHz. In this work a 100 MHz HFF-QCM sensor was used in a monoclonal antibody-based competitive immunoassay for specific bio-recognition of carbaryl pesticide as testing contaminant. The work intends to validate the use of HFF-QCM technology, in comparison with liquid chromatography-tandem mass spectrometry (LC-MS/MS) technique, for the detection of contaminants in honey. For this purpose, the validation criteria required by SANCO 12571/2013 guidance document were considered. The precision and accuracy (recovery) of both methods were determined by comparison of 5 replicates at 4 different concentrations (from 0 to 100 μg/kg) using the same honey matrix. HFF-QCM technology showed good accuracy, with recovery percentages always between 110 and 120%. As regards to precision, HFF-QCM coefficients of variation (CV) were around 10% higher than those recommended by GC SANCO 12571/2013. HFF-QCM limits of detection (LOD) and quantification (LOQ) were in the same order of magnitude as those for LC-MS/MS, which allows the analysis of carbaryl residues in honey under the established maximum residue limits (MRL), without sample pre-treatment. These results show that biosensors based on HFF-QCM technology has become a serious alternative to the traditional analytical techniques for food quality and safety applications.

Discriminating gastric cancer and gastric ulcer using human plasma amino acid metabolic profile.

Patients with gastric ulcer (GU) have a significantly higher risk of developing gastric cancer (GC), especially within 2 years after diagnosis. The main way to improve the prognosis of GC is to predict the tumorigenesis and metastasis in the early stage. The objective of this study was to demonstrate the ability of human plasma amino acid metabolic profile for discriminating GC and GU. In this study, we first used liquid chromatography-tandem mass spectrometry technique to characterize the plasma amino acid metabolism in GC and GU patients. Plasma samples were collected from 84 GC patients and 82 GU patients, and 22 amino acids were detected in each patient. Partial least squares-discriminant analysis model was performed to analyze the data of these amino acids. We observed seven differential amino acids between GC and GU. A regression analysis model was established using these seven amino acids. Finally, a panel of five differential amino acids, including glutamine, ornithine, histidine, arginine and tryptophan, was identified for discriminating GC and GU with good specificity and sensitivity. The receiver operating characteristic curve was used to evaluate diagnostic ability of the regression model and area under the curve was 0.922. In conclusion, this study demonstrated the potential values of plasma amino acid metabolic profile and metabolomic analysis technique in assisting diagnosis of GC. More studies are needed to highlight the theoretical strengths of metabolomics to understand the potential metabolic mechanisms in GC. © 2018 IUBMB Life, 70(6):553-562, 2018.

Phase I metabolism of the recently emerged synthetic cannabinoid CUMYL-PEGACLONE and detection in human urine samples.

Indole-, indazole-, or azaindole-based synthetic cannabinoids (SCs), bearing a cumyl substituent are a widespread, recreationally used subgroup of new psychoactive substances (NPS). The latest cumyl-derivative, CUMYL-PEGACLONE, emerged in December 2016 on the German drug market. The substance features a novel γ-carboline core structure, which is most likely synthesized to bypass generic legislative approaches to control SCs by prohibiting distinct core structures. Using liquid chromatography-tandem mass spectrometry and liquid chromatography-high resolution mass spectrometry techniques, the main in vivo phase I metabolites of this new substance were detected. A pooled human liver microsome assay was applied to generate in vitro reference spectra of CUMYL-PEGACLONE phase I metabolites. Additionally, 30 urine samples were investigated leading to 22 in vivo metabolites. A metabolite mono-hydroxylated at the γ-carbolinone core system and a metabolite with an additional carbonyl group at the pentyl side chain were evaluated as highly specific and sensitive markers to proof CUMYL-PEGACLONE uptake. Moreover, 3 immunochemical assays commonly used for SC screening in urine were tested for their capability of detecting the new drug but failed due to insufficient cross-reactivity.

Inhibitors of advanced glycation end products from coffee bean roasting by-product

The present study aimed to obtain novel information regarding the pathways of a polyphenol-rich coffee silverskin extract (CSE) for inhibiting the formation of advanced glycation products (AGE). Chlorogenic acid (CGA) is the main phenol present in coffee beans. The contribution of CGA to the antiglycoxidative properties of CSE was also evaluated by analysis of CGA standard. The CGA (90%) and CSE (49%) significantly inhibited (p < 0.001) the formation of fluorescent AGE in a protein–methylglyoxal (MGO) model system (37 °C for 96 h). The lack of MGO-derivative AGE in the presence of CGA and CSE was verified using liquid chromatography–tandem mass spectrometry techniques, which evidenced the almost complete absence of arginine adducts after 24 h of reaction. The inhibitory effect of polyphenols present in CSE may be associated to its carbonyl trapping capacity as well as to its ability to react with side-chains of protein amino residues blocking the reaction sites. In conclusion, aqueous CSE can be considered a natural source of various inhibitors of in vitro formation of AGE acting by different pathways.

Quality assessment of cellular and tissue-based products using liquid chromatography-tandem mass spectrometry

We are currently conducting clinical research on cell sheets for cartilage regeneration. One issue with the future use of chondrocyte sheets as cellular and tissue-based products is quality assessment. Currently, chondrocyte sheets are evaluated using invasive methods that cannot be performed on every sheet produced. We report here on our liquid chromatography-tandem mass spectrometry (LC-MS/MS) technique that allows the noninvasive assessment of every sheet using only 50 μl of culture medium. We found that LC-MS/MS could be used to confirm cell sheet viability through the measurement of glucose and glutamine uptake, to estimate extracellular matrix production by measuring serine consumption, to estimate cell kinetics by measuring cytidine and uracil concentrations, and to estimate melanoma inhibitory activity level by measuring pyridoxal concentration. LC-MS/MS may be useful for the noninvasive assessment of products to be used in regenerative medicine.