Use Of Liquid Chromatography Research Articles

Carryover analysis by liquid chromatography mass spectrometry in a multiproduct resin reuse context

The reuse of the same chromatography resin to purify multiple products is of high economic and ecologic interest in the pharmaceutical industry. An effective cleaning program to ensure no product carryover from one product to the subsequent is key to enable a multi-product use strategy. The methods utilised to assess product carryover during resin lifetime studies for a given product are not specific enough to detect carryover to support multi-product use. Here the proposed use of liquid chromatography coupled to mass spectrometry (LC-MS) is the analytical method of choice due to its high sensitivity and selectivity. A proof-of-concept study using protein A resin and two distinct IgG product streams produced by CHO cell lines was conducted. The experimental design was developed to determine any carryover from previous products after a multiple product Protein A resin reuse process, using LC-MS and capillary electrophoresis (CE). The capability of LC-MS coupled with bioinformatic analysis to detect carryover where CE with UV detection was not able to detect any carryover was demonstrated. The findings from this approach suggest that the acceptance of chromatographic resin reuse for multiple products will be dependent on the development of analytical/bioinformatics tools such as those proposed in this work.

B-155 Analysis of Monoclonal Antibodies Using a Quantification LC-MS/MS Kit - Spotlight on Infliximab

Abstract Background The use of immunoassays for measuring mAbs is well established but has some limitations, including poor performance, lack of standardization, a high cost when processing a limited number of samples, limited dynamic range, and the potential for cross-reactivity. Using LC-MS/MS associated with the ready-to-use commercial mAbXmise Kit is a simple way to implement mAbs measurement for infliximab, providing high analytical performance, ease of use, and flexibility. Methods The Promise Proteomics Inflammation mAbXmise Kit contains all the calibrators, QCs, internal standard and sample preparation consumables to run the LC-MS/MS method for infliximab. Briefly, 20µL samples are dispensed into the mAbXmise plates containing lyophilized stable labelled infliximab. Samples are transferred to the PuriXmise plate to perform the immunocapture of infliximab and allow washing of the samples. Samples are eluted into a collection plate, evaporated to dryness, resuspended, and the protease (CutXmise) is added to the sample to digest infliximab overnight. The digestion is quenched, with the samples immediately ready for analysis. Using an ACQUITY™ UPLC™ I-Class PLUS FL System, samples were injected onto a Waters™ XSelect™ Premier HSS T3 Column, eluted using a water/acetonitrile/formic acid gradient profile and quantified with both a Waters Xevo™ TQ-XS and Xevo TQ-S micro Mass Spectrometer. Results The method was shown to be linear from 2 - 100 µg/mL. Analytical sensitivity at lowest calibrator of 2µg/mL provided S/N (PtP) ≥15:1 for all tryptic peptides on both mass spectrometers. Coefficients of variation (CV) at 4 and 25µg/mL QC concentrations were all ≤ 10.0% (n = 5). The relative concentrations of the tryptic peptides were compared across 29 anonymized samples and good agreement was observed for GLE, SAV, DIL and ASA peptides. Conclusions We have demonstrated a clinical research method for quantifying Infliximab in plasma using a commercially available kit for sample preparation followed by LC-MS/MS analysis. The Promise Proteomics mAbXmise Kit aids in the day-to-day reproducibility of the immunocapture and tryptic digestion of IFX for targeted LC-MS/MS analysis with the correct peptide selection. Compared to a direct digest surrogate peptide workflow, the process used in the kit reduces matrix interference, improves analytical sensitivity, and enables the use of lower sample volumes. The method can be run on both the Xevo TQ-XS and Xevo TQ-S micro Mass Spectrometers which provide the dynamic range to quantify IFX across the expected range, and the selectivity and analytical sensitivity to obtain low-level quantification of the IFX surrogate peptide in plasma samples. The data presented combines the use of a kit dedicated to the preparation of samples and the use of liquid chromatography and mass spectrometry instrumentation to perform the quantitative analyses. The mAbXmise Kit described has not been cleared by any regulatory entity for diagnostic purposes outside of Europe. Analytical performance depends on the instrument characteristics and its settings. The end user is responsible for completion of the method development and validation. Proteomics mAbXmise Kits are not available for sale in all countries. For Research Use Only. Not for use in diagnostic procedures.

Secondary metabolites of perilla (<i>Perilla frutescens</i> (L.) Britton) of Far Eastern origin identified by tandem mass spectrometry

Perilla frutescens is a monotypic genus of annual herbaceous plants of dead nettles. The article presents a simultaneous evaluation of the presence of both phenolic compounds in P. frutescens seed extracts and compounds of other chemical classes. It has been confirmed that the seeds of P. frutescens varieties k-130, k-263, k-326 contain a large number of polyphenolic complexes, which are biologically active compounds. The complete metabolomic composition of perilla seed extracts is presented for the first time. The study utilized a tandem mass spectrometry method involving the use of liquid chromatography in parallel with an ion trap. Using tandem mass spectrometry, the presence of 60 compounds was identified, of which 41 compounds represent the group of polyphenolic compounds, as well as 19 compounds of other chemical groups in perilla extracts. The chemical components were identified by comparing their retention index, mass spectra and MS fragmentation with the home library database based on the data from other spectroscopic techniques such as nuclear magnetic resonance, UV-visible spectroscopy and mass spectrometry. Also, 29 chemical compounds were found for the first time, including 16 compounds of polyphenolic group and 13 compounds of other chemical groups. Lignans: syringaresinol and medioresinol; coumarins: umbelliferone and 4-hydroxycoumarin; flavones: luteolin 7-O-glucoside, diosmetin C-hexoside, chrysoeriol C-hexoside C-pentoside; flavanols: ampelopsin, astragalin, etc. were identified in perilla seeds for the first time. The data obtained will help intensify future research on the development and production of new drugs, dietary supplements and various functional, specialized and dietary products containing targeted perilla extracts.

LC and LC-MS/MS Studies for the Separation and Identification of Impurities and Degradation Products of Betaxolol.

Betaxolol (Bx) is a selective β1 receptor blocker used in the treatment of hypertension and glaucoma. The aim of the present work was to demonstrate an approach involving use of liquid chromatography (LC) and liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS) for the simultaneous separation, identification and characterization of impurities and of degradation products of betaxolol without their isolation from the reaction mixtures. At optimum condition, and according to ICH guidelines, the limit of detection (LOD) and limit of quantification (LOQ) for Bx are found to be 5.46 and 16.54μgmL-1, respectively. However, the LOD and LOQ for the major degradation product P6 were 2.15 and 6.53μgmL-1. Betaxolol was subjected to hydrolytic (acidic and basic) and oxidative, stress conditions according to International Conference on Harmonization (ICH) guideline Q1A (R2), and as results, the drug was found to be labile in acidic, basic and oxidative stress conditions. Based on LC-ESI/MS analysis, the found results revealed that Bx decomposes in acidic, basic and oxidizing environments. All degradation products were identified with the help of their fragmentation pattern and the masses obtained upon the MS analysis.

The Effect of Particle Monodispersity in HPLC Column Performance

Many developments in silica particles used in liquid chromatography have been well documented over the years. The move from irregular silica to spherical silica, the decrease in particle size from >5µm particle in HPLC to sub-2-µm particle size in UHPLC, the improved silica purity of type B silica over that of type A silica, and more recently the adoption of superficially porous particles versus traditional fully porous particles. One area of development that has been discussed less and is still open to debate is the particle size distribution (PSD) of these chromatographic materials. In this article, we discuss the move towards improved monodispersity of silica particles for use in high performance liquid chromatography (HPLC) and how the use of monodisperse particles can impact the resulting chromatographic parameters such as reduced plate height and column impedance. On a practical level we review how the reduction of PSD impacts efficiency, backpressure and sensitivity.

Separation and analysis of triazole antifungal in biological matrices by liquid chromatography: a review

Invasive fungal infections cause serious illness and death worldwide. Long-term therapeutic and preventative use of antifungal drugs in high-risk patients has caused resistance. Triazole antifungals are widely used to prevent and treat fungal infections, and therapeutic drug monitoring has been suggested to improve outcomes, reduce toxicity, and prevent drug resistance. Common methods used for monitoring triazole antifungal drugs in biological matrices such as blood, serum, and plasma include bioassay and instrumentation methods, especially liquid chromatography. Sample preparation is needed to remove interference from liquid chromatography for reliable results. This paper evaluates the use of liquid chromatography to analyze triazole antifungal agents. We provided various chromatographic techniques combined with different detector types to analyze triazole antifungal drugs in biological matrices. We also compared chromatography systems with different sample preparation methods in order to select the most suitable analytical method for bioanalysis.

Mechanochemical Degradation of Caffeine and Diclofenac Using Biochar of Fique Bagasse in the Presence of Al: Monitoring by Mass Spectrometry.

Much research has been carried out to remove emerging contaminants using diverse materials. Furthermore, studies related to pollutant degradation have increased over the past decade. Mechanochemical degradation can successfully decompose molecules that are persistent in the environment. In this study, the biochar of fique bagasse with mixtures SiO2, Al, Al2O3, and Al-Al2O3 was treated with a mechanochemical technique using a planetary ball mill to investigate the degradation of caffeine and diclofenac. These tests resulted in the transformation of caffeine and diclofenac due to the use of Al employing mechanochemistry. In fact, through the use of liquid chromatography coupled with mass spectrometry, eight and six subproducts were identified for caffeine and diclofenac, respectively. Additionally, analysis of the molecules proposed for caffeine and diclofenac transformation suggested hydroxylation, demethylation, decarboxylation, oxidation reactions, and cleavage of the C-C and C-N bonds in the pollutants studied. The formation of these transformation products could be possible by reductant oxygen species generated from the molecular oxygen in the presence of aluminum and the energy delivered for ball milling. The results obtained show the potential application in the environmental management of mechanochemical treatment in the elimination of emerging contaminants caffeine and diclofenac.

Kwas benzoesowy Metoda oznaczania w powietrzu na stanowiskach pracy

Benzoic acid is an organic compound that belongs to the group of aromatic carboxylic acids. It is mainly used in the production of phenol, caprolactam and benzoic salts, as a food and pharmaceutical preservative, and in the production of herbicides, insecticides and bactericides. According to the Regulation of the European Parliament and of the Council (WE 1272/2008), benzoic acid is classified as a substance that is harmful to the lungs, irritates the skin and causes eye damage. The aim of the study was to develop a method for the determination of benzoic acid for the assessment of occupational exposure within 1/10–2 of the proposed MAC value. The method involves taking the inhalable fraction of airborne benzoic acid onto a glass fiber filter coated with sodium carbonate(IV), desorption with a solution of methanol in water and then determining the benzoic acid content of the sample by the use of liquid chromatography with diode array detector (UHPLC-DAD). Validation requirements presented in European standard PN-EN 482 were fulfilled during the tests. The method enables determination of benzoic acid in air at concentrations of 0.05 to 1 mg/m3 . The method for determining benzoic acid has been recorded in the form of an analytical procedure (see Appendix). This article discusses the problems of occupational safety and health, which are covered by health sciences and environmental engineering.

Sustainable chromatographic quantitation of multi-antihypertensive medications: application on diverse combinations containing hydrochlorothiazide along with LC–MS/MS profiling of potential impurities: greenness and whiteness evaluation

Cardiovascular disorders are among the leading causes of death worldwide, especially hypertension, a silent killer syndrome requiring multiple drug therapy for appropriate management. Hydrochlorothiazide is an extensively utilized thiazide diuretic that combines with several antihypertensive drugs for effective treatment of hypertension. In this study, sustainable, innovative and accurate high performance liquid chromatographic methods with diode array and tandem mass detectors (HPLC–DAD and LC–MS/MS) were developed, optimized and validated for the concurrent determination of Hydrochlorothiazide (HCT) along with five antihypertensive drugs, namely; Valsartan (VAL), Amlodipine besylate (AML), Atenolol (ATN), Amiloride hydrochloride (AMI), and Candesartan cilextil (CAN) in their diverse pharmaceutical dosage forms and in the presence of Chlorothiazide (CT) and Salamide (DSA) as HCT officially identified impurities. The HPLC–DAD separation was achieved utilizing Inertsil ODS-3 C18 column (250 × 4.6 mm, 5 μm) attached with photodiode array detection at 225.0 nm. Gradient elution was performed utilizing a mixture of solvent A (20.0 mM potassium dihydrogen phosphate, pH 3.0 ± 0.2, adjusted with phosphoric acid) and solvent B (acetonitrile) at ambient temperature. Linearity ranges were 0.1–100.0 µg/mL for HCT, VAL, AML and CAN, 0.05 –100.0 µg/mL for both ATN and AMI and 0.05–8.0 µg/mL for both CT and DSA. Additionally, this work describes the use of liquid chromatography–electrospray–tandem mass spectrometry for the accurate detection and quantification of the impurities; CT and DSA in the negative mode utilizing triple quadrupole mass spectrometry. The linearity ranges for those impurities were 1.0–200.0 ng/mL and 5.0–200.0 ng/mL for CT and DSA, respectively. Developed methods’ validation was achieved in accordance with International Conference on Harmonization (ICH) guidelines. Upon applying liquid chromatographic techniques for the drug analysis, a green and sustainable assessment have to be handled due to the consumption of energy and many solvents. Through the use of the HEXAGON, Analytical Greenness (AGREE) and White Analytical Chemistry (WAC) tools, greenness and sustainability have been statistically assessed. The optimized HPLC–DAD and LC–MS/MS methods were fast, accurate, precise, and sensitive, and consequently could be applied for conventional analysis and quality control of the proposed drugs in their miscellaneous dosage forms for the purpose of reducing laboratory wastes, time of the analysis time, effort, and cost.Graphical

Targeted quantitative metabolomics with a linear mixed-effect model for analysis of urinary nucleosides and deoxynucleosides from bladder cancer patients before and after tumor resection.

In the present study, we developed and validated a fast, simple, and sensitive quantitative method for the simultaneous determination of eleven nucleosides and deoxynucleosides from urine samples. The analyses were performed with the use of liquid chromatography coupled with triple quadrupole mass spectrometry. The sample pretreatment procedure was limited to centrifugation, vortex mixing of urine samples with a methanol/water solution (1:1, v/v), evaporation and dissolution steps. The analysis lasted 20min and was performed in dynamic multiple reaction monitoring mode (dMRM) in positive polarity. Process validation was conducted to determine the linearity, precision, accuracy, limit of quantification, stability, recovery and matrix effect. All validation procedures were carried out in accordance with current FDA and EMA regulations. The validated method was applied for the analysis of 133 urine samples derived from bladder cancer patients before tumor resection and 24h, 2weeks, and 3, 6, 9, and 12months after the surgery. The obtained data sets were analyzed using a linear mixed-effect model. The analysis revealed that concentration level of 2-methylthioadenosine was decreased, while for inosine, it was increased 24h after tumor resection in comparison to the preoperative state. The presented quantitative longitudinal study of urine nucleosides and deoxynucleosides before and up to 12months after bladder tumor resection brings additional prospective insight into the metabolite excretion pattern in bladder cancer disease. Moreover, incurred sample reanalysis was performed proving the robustness and repeatability of the developed targeted method.

Evaluation of Bacillus subtilis Czk1 Metabolites by LC–MS/MS and Their Antifungal Potential against Pyrrhoderma noxium Causing Brow Rot Disease

Brown rot disease caused by Pyrrhoderma noxium is a widespread disease that severely affects the roots of rubber trees (Hevea brasiliensis Muell. Arg.). The economic losses, along with environmental and health problems arising from the use of disease control chemicals, have raised the interest of scholars to explore the use of biological control agents for the effective control of fungal pathogen P. noxium. Here, the inhibition effect of the culture filtrate of B. subtilis Czk1 on P. noxium was demonstrated. The findings indicate that the antifungal activity of this strain is mediated wholly or partly by compounds produced in the culture filtrate. The combined use of liquid chromatography–tandem mass spectrometry and antifungal activity assays rapidly identified compounds produced by B. subtilis Czk1. Metabolic profiles were assessed and used to identify major metabolites based on the scores of variable importance in the projection and the plot scores of principal component analysis. A total of 296 differential metabolites were screened, including 208 in positive ion mode and 88 in negative ion mode. Two key metabolites, diacetyl and trans-2-octenoic acid, were screened from 29 metabolites by antifungal activity assays. The median effective concentration (EC50) of trans-2-octenoic acid and diacetyl were 0.9075 mg/mL and 4.8213 mg/mL, respectively. The antifungal metabolites can disrupt the internal structure of the pathogenic fungal mycelium, thereby impeding its growth. This study is expected to contribute to the existing knowledge of Czk1-produced metabolites and their future antifungal applications. This study is also expected to provide a new biopreservative perspective on unexplored antifungal metabolites produced by Czk1 as a biocontrol agent.

Searching for pharmaceutically active products and metabolites in environmental waters of Peru by HRMS-based screening: Proposal for future monitoring and environmental risk assessment

The presence of pharmaceutical active products (PhACs) in the aquatic environment is a matter of current concern, and there is an increasing trend to include these compounds in water quality monitoring programs and environmental risk assessments. Several studies have reported the presence of PhACs in environmental waters worldwide, but only a few studies have focused on Latin American countries. Thus, available information on the occurrence of parent pharmaceuticals, especially their metabolites, is very scarce. Peru is one of the less monitored countries in terms of contaminants of emerging concern (CECs) in waters, and only one study has been found, which was focused on the quantification of selected PhACs in urban wastewater and surface water. The aim of this work is to complement the previous data reported on PhACs in the aquatic environment by application of a wide-scope high-resolution (HRMS)-based screening, making use of target and suspect approaches. In the present work, 30 pharmaceuticals, drugs or other compounds (sweeteners, UV filters, etc.) and 21 metabolites have been identified, with antibiotics (and metabolites) being the most prevalent compounds. The use of liquid chromatography (LC) coupled to ion mobility-HRMS allowed the tentative identification of parent compounds and metabolites, for which the analytical reference standard was not available, with a high level of confidence in their identification. Based on the results obtained, a strategy for the monitoring of PhACs and relevant metabolites in environmental waters from Peru and for subsequent risk assessment is proposed. Our data will also help to focus future studies to evaluate the removal efficiency of wastewater treatment plants and the impact of treated water in receiving water bodies.

Carpobrotus edulis leaf extract as green corrosion bio-inhibitor for copper in 3.5% NaCl solution

In this study, Carpobrotus edulis leaf extract (CELE) has been introduced as a new, eco-friendly, and green inhibitor of copper decomposition in a 3.5 wt.% sodium chloride solution. The water/ethanol extract of the leaves was used to investigate the chemical composition of CELE by the use of liquid chromatography combined with a mass spectrometry detector. Using polarization and electrochemical impedance spectroscopy, the dissolution rate of the copper samples in the presence and absence of various concentrations of CELE (2.5–20 wt.%) were studied, which the best being 20% with an inhibition efficiency of 87%. To evaluate the copper surface morphology and perform surface elemental analysis, scanning electron microscopy and energy dispersive spectroscopy were used. Also, the effect of temperature increment on IE% at the optimum concentration of CELE has been studied, which at 338 K was 57%. Isotherm studies showed that the CELE adsorption mechanism follows the Langmuir isotherm.

Insights into the Shape-Selective Retention Properties of Porous Graphitic Carbon Stationary Phases and Applicability for Polar Compounds

In this article, the ability of porous graphitic carbon (PGC) to discriminate between structurally similar compounds is described. After a brief overview of the particle technology and its use in liquid chromatography (LC), two case studies are presented using small-molecule probes to illustrate the importance on how molecular shape and the ability to interact with the graphitic surface lead to the resolution of structural isomers. Finally, an application of this technology in the area of glycan analysis is showcased where released N-glycans from human immunoglobulin G (IgG) were resolved without derivatization, enabling the glycan profile of a biotherapeutic to be determined.

Use of Liquid Chromatography--Tandem Mass Spectrometry to Quantify and Confirm the Fentanyl Metabolite N-[1-(2-Phenethy-4-Piperidinyl)] Maloanilinic Acid in Equine Urine for Doping Control.

Fentanyl, a powerful synthetic mu opioid receptor agonist, is banned in equine sports by the Association of Racing Commissioners International and the Fédération Équestre Internationale. The presence of fentanyl in equine blood has been confirmed during routine post-race screening for doping substances in the authors' laboratory. While fentanyl can be detected and confirmed in blood, it is rapidly metabolized, and screening for the metabolite N-[1-(2-phenethy-4-piperidinyl)] maloanilinic acid (PMA) in equine urine is expected to allow for a longer detection time. In this study, a quantitative and confirmatory liquid chromatography--tandem mass spectrometry (LC-MS-MS) method was developed for PMA analysis in equine urine. PMA was extracted by solid phase extraction, separated on a C18 column and detected using a triple quadrupole mass spectrometer. The mass spectrometer was operated in positive-ion mode, and multiple reaction monitoring was used to monitor product ions m/z 188, m/z 281 and m/z 323. The method was validated for extraction recovery, matrix effect, specificity, sensitivity, precision and accuracy, carryover and processed sample stability according to the guidelines of the US Food and Drug Administration for bioanalysis. The limits of detection and quantification were 5 and 10 pg/mL, respectively. Linearity was obtained over the concentration range of 10-10,000 pg/mL. To confirm PMA in equine urine, LC retention time, diagnostic product ions (m/z 188, m/z 281 and m/z 323) and product ion ratio were used as the criteria. The lowest concentration for confirmatory analysis was validated at 50 pg/mL. The method was applied to measure the PMA concentrations in equine urine following intravenous administration of fentanyl to a research horse and has confirmed the presence of PMA in post-race urine samples. This method is a valuable addition to the arsenal of equine doping control methods to combat illegal doping and protect racehorse health.

Optimization of tannin extraction from coconut coir through response surface methodology

Coconut coir is produced by extracting tiny grains of coir from coconut shell, which is an agricultural product that is abundant in Thailand. Coir is typically discarded, but it is a useful material because it contains tannins. Tannin is a polyphenolic compound that is prevalent in the plant kingdom, including in wood, bark, rhizomes, roots, and fruits. In this study, we extracted tannins from coconut coir using two types of solvents: water and ethanol. Optimization of the tannin content was achieved via RSM (response surface methodology) with the assistance of Design-Expert software. Meanwhile, the temperature (30–70 °C), ratio of solid to solvent (1:20–1:40), and solvent type (water or ethanol) were optimized via central composite design (CCD). The optimum model using analysis of variance revealed R2 = 0.9825, and adj.R2 = 0.9687. Temperature is affected by tannin content, and high temperatures result in a higher tannin content when using water than when using ethanol. Optimum conditions for coconut coir extraction using water with ethanol include a ratio for solid to solvent of 1:30 and the temperature set to 70 °C. The validated model exhibits errors of 8.24 and 11.08% for water and ethanol, respectively. Confirmation of the presence of tannins in the crude extract was carried out via Fourier-transform infrared spectroscopy and through the use of liquid chromatography with tandem mass spectrometry.

Optimization of extraction conditions for LC-ToF-MS analysis of mevalonate pathway metabolites in engineered E. coli strain via statistical experimental designs

Isoprenoids give rise to many functional products used today such as flavours, fragrances and even pharmaceutical compounds. Mevalonate pathway metabolites are the key intermediates that affect the production yield of isoprenoids. With increasing demand and benefit of isoprenoids, the present study adopts Analytical Quality-by-Design (AQbD) approach to establish an efficacious extraction protocol prior to the determination of mevalonate pathway metabolites in an engineered Escherichia coli model. The statistical experimental design approach, described in this work, has successfully validated an optimised sample preparation method i.e., using acetonitrile: 50 mM ammonium formate (pH 9.5) (7:3) (ACN73) at -20 °C for 10 min without solvent evaporation to retain the targeted mevalonate metabolites in engineered E. coli strain. The study also demonstrates the use of liquid chromatography paired with a Time-of-Flight Mass Spectrometer (LC-ToF-MS) for the quantitative analysis of the mevalonate pathway metabolites in E. coli. The analytical method was validated in accordance with guidelines in Metabolomics Standards Initiative and ICH Q2 (R1) with analyte spike recoveries at 80% and above. In short, the present study overcomes the one-variable-at-a-time (OVAT) limitations in analytical development, minimises metabolite losses and gives better cost and time efficiencies by eliminating the solvent evaporation and swapping process. This work highlights the importance of analytical methods development in microbial metabolomics studies.

Relative Quantification of Proteins in Formalin-Fixed Paraffin-Embedded Breast Cancer Tissue Using Multiplexed Mass Spectrometry Assays

The identification of clinically relevant biomarkers represents an important challenge in oncology. This problem can be addressed with biomarker discovery and verification studies performed directly in tumor samples using formalin-fixed paraffin-embedded (FFPE) tissues. However, reliably measuring proteins in FFPE samples remains challenging. Here, we demonstrate the use of liquid chromatography coupled to multiple reaction monitoring mass spectrometry (LC-MRM/MS) as an effective technique for such applications. An LC-MRM/MS method was developed to simultaneously quantify hundreds of peptides extracted from FFPE samples and was applied to the targeted measurement of 200 proteins in 48 triple-negative, 19 HER2-overexpressing, and 20 luminal A breast tumors. Quantitative information was obtained for 185 proteins, including known markers of breast cancer such as HER2, hormone receptors, Ki-67, or inflammation-related proteins. LC-MRM/MS results for these proteins matched immunohistochemistry or chromogenic in situ hybridization data. In addition, comparison of our results with data from the literature showed that several proteins representing potential biomarkers were identified as differentially expressed in triple-negative breast cancer samples. These results indicate that LC-MRM/MS assays can reliably measure large sets of proteins using the analysis of surrogate peptides extracted from FFPE samples. This approach allows to simultaneously quantify the expression of target proteins from various pathways in tumor samples. LC-MRM/MS is thus a powerful tool for the relative quantification of proteins in FFPE tissues and for biomarker discovery.

Proteomic Characterization of Virulence Factors and Related Proteins in Enterococcus Strains from Dairy and Fermented Food Products.

Enterococcus species are Gram-positive bacteria that are normal gastrointestinal tract inhabitants that play a beneficial role in the dairy and meat industry. However, Enterococcus species are also the causative agents of health care-associated infections that can be found in dairy and fermented food products. Enterococcal infections are led by strains of Enterococcus faecalis and Enterococcus faecium, which are often resistant to antibiotics and biofilm formation. Enterococci virulence factors attach to host cells and are also involved in immune evasion. LC-MS/MS-based methods offer several advantages compared with other approaches because one can directly identify microbial peptides without the necessity of inferring conclusions based on other approaches such as genomics tools. The present study describes the use of liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI–MS/MS) to perform a global shotgun proteomics characterization for opportunistic pathogenic Enterococcus from different dairy and fermented food products. This method allowed the identification of a total of 1403 nonredundant peptides, representing 1327 proteins. Furthermore, 310 of those peptides corresponded to proteins playing a direct role as virulence factors for Enterococcus pathogenicity. Virulence factors, antibiotic sensitivity, and proper identification of the enterococcal strain are required to propose an effective therapy. Data are available via ProteomeXchange with identifier PXD036435. Label-free quantification (LFQ) demonstrated that the majority of the high-abundance proteins corresponded to E. faecalis species. Therefore, the global proteomic repository obtained here can be the basis for further research into pathogenic Enterococcus species, thus facilitating the development of novel therapeutics.

Strategy for successful urine sample preparation for LC-MS/MS device at drug verification laboratory

Objectives
 The use of liquid chromatography with tandem mass spectrometry (LC-MS/MS) device has increased significantly in toxicology validation laboratories in recent years. The maintenance cost of this expensive equipment is high as well as the cost of consumables. Reduction of the matrix effect and preparation more clear samples are very important for the validation of methods in clinical laboratories. 
 The primary goal is to create a cleaner urine sample preparation technique to reduce the cost of maintenance of the LC-MS/MS device without affecting test results.
 Methods
 We prepared the patients’ urine in two different ways; routine urine preparation method and used our centrifuged method (14000 rpm, 10 minutes) for routine illicit substance use. The standard material used to determine whether there was a statistical difference in the urine sample with both different methods was added to both urine samples.
 Results
 Our findings showed that there was no statistical difference between the results of both methods for detection of illicit substance use. There was no difference between the high and low quantities of the 14 illicit substances measured and the centrifuged method and routine urine preparation methods (p