Capillary Chromatography Research Articles

Chiral separation by capillary electrokinetic chromatography with hydrophobic deep eutectic solvents as pseudo-stationary phases

This study demonstrated for the first time that hydrophobic deep eutectic solvents (HDESs) can be used in capillary electrophoresis (CE) for chiral separations. We found that the an HDES methyltrioctylammonium chloride:octanoic acid (N8881Cl:OctA) can exist in the form of nano-sized microdroplets in CE background electrolyte solutions, and show hydrophobic effects as a new type of pseudo-stationary phase (PSP) during CE separation. When used in combination with various cyclodextrin (CD)-type chiral selectors, the presence of N8881Cl:OctA significantly improved the enantioresolutions of several model drugs. Moreover, the migration time of the enantiomers can also be reduced when an anionic CD (e.g., carboxymethyl-β-cyclodextrin (CM-β-CD)) was used. Critical factors influencing the chiral separations were systematically investigated including the HDES concentration, hydrogen-bond acceptor (HBA)/hydrogen-bond donor (HBD) ratio, CD concentration, buffer pH, and applied voltage, etc. An insight into chiral recognition mechanism with HDES is provided for reference. A comparison of the chiral CE performance of HDESs with traditional surfactants was also performed to demonstrate their superiority as a new type of PSP.

Determination of gas-polydimethylsiloxane distribution constants of major Cannabis terpenes and terpenoids by capillary gas-liquid chromatography

Terpenes and terpenoids are the principal responsible for the aroma of Cannabis, playing an important role in the interaction with the environment. Analytical determination of these compounds can be done by headspace coupled to solid phase micro-extraction (HS-SPME) and then injected in a gas chromatograph. In the present study, we determined distribution constants between gas and polydimetylsiloxane (PDMS), a conventional SPME liquid phase, at three temperatures between 303.15 and 343.15 K for major Cannabis terpenes and terpenoids employing a method based in gas chromatography using four capillary columns for monoterpenes and five columns for sesquiterpenes. In addition, van't Hoff regressions (logKfg vs T−1) were obtained in order to estimate logKfg at 298.15 K aiming to compare with bibliographic values (experimental or estimated ones). An excellent agreement was found between them. The method, based on chromatographic theory is robust and relatively simple. It is expected that the herein obtained data could be useful for selecting SPME fiber type and dimensions, estimating extraction efficiencies, as well as to develop prediction models and validate them.

Determination of carbamazepine and its main metabolite in human hair by capillary electrophoresis and liquid chromatography techniques, both coupled with mass spectrometry

The aim of the research was to determine carbamazepine (CBZ) and its main metabolite 10,11-epoxy-10,11-dihydro-carbamazepine (CBZ-E) in human hair using the capillary electrophoresis (CE) system coupled with mass spectrometry detection (MS) and to compare the obtained results with the liquid chromatography (LC) technique, also coupled with the MS detector. Hair samples were prepared using microwave-assisted extraction (MAE) at 60 °C for 10 min in an alkaline solution (pH = 10) with ethyl acetate as the extraction solvent. In the frame of this study, the procedure for the separation of CBZ and CBZ-E using the CE-MS technique was developed. The best results were achieved using 10 mM ammonium acetate (pH=6.8) as the background electrolyte (BGE), after filling the capillary with 1% highly sulfonated β-cyclodextrin (HSβCD) in 10 mM ammonium acetate. Then, the validation parameters of the MAE/CE-MS and MAE/LC-MS methods such as: limit of detection (for CBZ are: 0.36 and 0.22 ng/mg, respectively; for CBZ-E 0.38 and 0.17 ng/mg, respectively), limit of quantification (for CBZ are: 0.86 and 0.72 ng/mg, respectively; for CBZ-E 0.94 and 0.56 ng/mg, respectively), precision (6.91–14.5% and 2.16–15.6%, respectively), recovery (87.7–102.7% and 88.9–105.5%, respectively), and matrix effect (99.5–111.0% and 98.9–115.1%, respectively) were defined and compared. Finally, the validated methods were applied to identify and quantify carbamazepine and its metabolite in hair in patients who received CBZ for medical reasons.

Characterization of the Ciguatoxin Profile in Fish Samples from the Eastern Atlantic Ocean using Capillary Liquid Chromatography-High Resolution Mass Spectrometry

Ciguatera Poisoning is an emerging risk in the east Atlantic Ocean. Despite characterization efforts, the complete profile of ciguatoxin chemical species in these waters is still unknown. These efforts have been complicated by a lack of reference materials and scarcity of fish contaminated with high levels of ciguatoxins. Development and application of analytical methods with enhanced selectivity and sensitivity is essential for ciguatoxin characterization. Here, we developed an analytical characterization approach using capillary liquid chromatography coupled to high resolution mass spectrometry applied to reference materials obtained from ciguatoxin contaminated fish. Capillary LC coupled mass spectrometry resulted in increased sensitivity leading to the confirmation of C-CTX1 as the principal ciguatoxin present in these samples. We also detected and structurally characterized minor C-CTXs analogues consisting of C-CTX3/4, hydroxy-, didehydro-, and methoxy- metabolites.

Recent developments and applications of capillary and microchip electrophoresis in proteomics and peptidomics (mid-2018-2022).

This review gives a wide overview of recent advances and applications of capillary electrophoresis and microchip capillary electrophoresis methods in the fields of proteomics and peptidomics in the period from mid-2018 up to the end of 2022. The methodological topics covering sample preparation and concentration techniques, hyphenation of capillary electrophoresis methods with mass spectrometry, and multidimensional separations by on-line or off-line coupled different capillary electrophoresis and liquid chromatography techniques are described and new developments in both bottom-up and top-down approaches in proteomics are presented. In addition, various applications of capillary electrophoresis methods in proteomic and peptidomic studies are demonstrated. They include monitoring of protein posttranslational modifications and applications in biological and biochemical research, clinical peptidomics and proteomics, and food analysis.

Compact capillary high performance liquid chromatography system for pharmaceutical on-line reaction monitoring.

Due to their size, conventional high performance liquid chromatographs (HPLCs) are difficult to place close to a reaction vessel within a pharmaceutical manufacturing or development site. Typically, long transfer lines are required to move sample from the reactor to the HPLC for analysis and high solvent usage is required. However, herein a compact and modular separation system has been developed to enable co-location of an HPLC with a small-scale reactor for reaction monitoring in the synthesis of active pharmaceutical ingredients. Using a framework based on capillary HPLC, a compact gradient separation system with a fully modular architecture is described. A custom miniature diode-array detector with a linear dynamic range (up to 1500 mAU at 210nm) was integrated and evaluated for on-line reaction monitoring. In evaluating system suitability, average peak area %RSD of <3%, and an average retention time %RSD of <0.7%, were achieved. To demonstrate practical utility, the compact system was coupled directly to an on-line lab-scale flow through reactor for continuous reaction monitoring in the laboratory fume hood, where a study of the 3rd Bourne reaction was used to compare the performance of the compact system with a commercially available process HPLC instrument (Waters PATROL UPLC). Further, 33 off-line samples from a continuous crystallization reactor were analysed and it was found that the developed compact HPLC system showed equivalent quantitative performance to an Agilent 1290 Infinity II HPLC system.

Simultaneous Determination of Glyphosate, AMPA and Inorganic Anions in Water Samples by Gradient Capillary Ion Chromatography

Simultaneous Determination of Glyphosate, AMPA and Inorganic Anions in Water Samples by Gradient Capillary Ion Chromatography

Capillary electrophoresis: Recent advancements and applications of micellar electrokinetic capillary chromatography

The article discusses capillary electrophoresis (CE) as an advanced technique used for the separation and detection of various pharmaceutical drugs. CE involves the application of high voltages across buffer-filled capillaries to produce separation based on various separation theories, including capillary zone electrophoresis (CZE), micellar electrokinetic capillary chromatography (MEKC), capillary gel electrophoresis, and capillary isoelectric focusing. While traditional CZE is not suitable for the separation of neutral substances, MEKC was developed by Shigeru Terabe in the early 1990s to expand the use of CE to neutral analytes that cannot be separated using straightforward free solution CE. MEKC employs an ionic micellar solution that interacts with the analytes through partitioning processes like a chromatographic technique. To create a pseudostationary phase, a surfactant such as sodium dodecyl sulfate (SDS) is added to the buffer solution at a concentration higher than its critical micellar concentration. The anionic SDS micelles are electrostatically drawn towards the anode, while the electro-osmotic flow carries the bulk solution toward the negative electrode due to the negative charge on the inside surface of the silica capillaries. When a neutral analyte is introduced into the micellar solution, a portion is integrated into the micelle, while the remaining fraction of the analyte migrates with the electroosmotic velocity. Separation depends on the individual partitioning equilibrium of the various analytes between the micellar and the aqueous phase. The bigger percentage of analyte dispersed inside the micelle, the slower it will travel. This article provides an in-depth understanding of the separation principle and the mechanism involved in MEKC, highlighting its usefulness in separating neutral analytes that cannot be separated using traditional CZE.

Application of an in-house packed octadecylsilica-functionalized graphene oxide column for capillary liquid chromatography analysis of hormones in urine samples

Graphene oxide-based LC stationary phases were developed and applied for separating hormones from urine using capillaryLC-MS/MS. Using two analytical approaches – direct injection and column-switching arrangement – it was possible to evaluate the chromatographic parameters and perform tests on the raw biological fluid. Two stationary phases (SPs) were produced, varying the amino silica support particle diameter (Si, 5, and 10 μm). Graphene oxide was covalently bonded to the surface of Si particles, and this material was functionalized by the insertion of octadecylsilica groups, generating the SiGO-C18. Infra-red spectroscopy assays revealed that both steps were successful – supporting GO onto Si and further C18 customization. Scanning electron microscopy showed spherical geometries with minor irregularities and narrow particle size distribution for the produced SPs. The GO-coating rate was higher on the Si particles of 10 μm. As a result, the 10 μm produced column reported better resolution, efficiency, and peak capacity. Therefore, this SiGO-C18 capillary column (100 mm × 0.32 mm i.d., 10 μm dp) was applied successfully in a column-switching method to separate hormones in urine. Linearity (R2 above 0.99), quantification limits (between 1.0 and 5 μg/L), and other figures of merit of the method were determined. It is worth mentioning that the SiGO-C18 capillaryLC column performed adequately, separating the target compounds in less than 6 min. We hope this work could significantly contribute to shedding some light on graphene-based materials as a promising class of stationary phase for miniaturized liquid chromatography.

Capillary liquid chromatography coupled with mass spectrometry for analysis of nanogram protein quantities on a wide-pore superficially porous particle column in top-down proteomics

In top-down proteomics experiments, intact protein ions are subjected to gas-phase fragmentation for MS analysis without prior digestion. This approach is used to characterize post-translational modifications and clipped forms of proteins, avoids several “inference” problems associated with bottom-up proteomics, and is well suited to the study of proteoforms. In the past decade, top-down proteomics has progressed rapidly, taking advantage of MS instrumentation improvements and the efforts of pioneering groups working to improve sample handling and data processing. The potential of this technology has been established through its successful use in a number of important biological studies. However, many challenges remain to be addressed like improving protein separation capabilities such that it might become possible to expand the dynamic range of whole proteome analysis, address co-elution and convoluted mass spectral data, and aid final data processing from peak identification to quantification. In this study, we investigated the use of a wide-pore silica-based superficially porous media with a high coverage phenyl bonding, commercially packed into customized capillary columns for the purpose of top-down proteomics. Protein samples of increasing complexity were tested, namely subunit digests of a monoclonal antibody, components of purified histones and proteins extracted from eukaryotic ribosomes. High quality mass spectra were obtained from only 100 ng of protein sample while using difluoroacetic acid as an ion pairing agent to improve peak shape and chromatographic resolution. A peak width at half height of about 15 s for a 45 min gradient time was observed on a complex mixture giving an estimated peak capacity close to 100. Most importantly, efficient separations were obtained for highly diverse proteins and there was no need to make method specific adjustments, suggesting this is a highly versatile and easy-to-use setup for top-down proteomics.

Towards the development of a reference measurement procedure for the quantification of neurofilament light chain

AbstractBackgroundNeurofilament‐light chain (Nf‐L) is an early marker monitoring axonal damage which occur in many forms of dementia. During neurodegeneration, Nf‐L is released into biological fluid and is quantifiable in cerebrospinal fluid (CSF) and blood. Currently, its quantification is performed by immunoassays. While those ones are sensitive and precise, the development of a reference measurement procedure (RMP) using mass spectrometry (MS) approaches traceable to the International System of Units (SI) is essential to achieve standardisation.Following the characterisation of three commercially available proteins as potential primary calibrator, one has been chosen and used for the method development. Here, we present the purity assessment and quantification traceable to the SI of the primary calibrator and preliminary data on the development of Nf‐L quantification in CSF by LC‐MS.MethodA recombinant Nf‐L protein from Promise was characterized, SI traceably quantified via amino acid analysis and used as primary calibrator for the development of a LC‐MS method for Nf‐L. A triple quadrupole (QQQ) mass spectrometer coupled to a capillary liquid chromatography (LC) was used to set up a multiple reaction monitoring (MRM) method to monitor Nf‐L tryptic peptides and assess the potential of developing a RMP for Nf‐L.ResultThe homogeneity of Nf‐L from Promise was assessed on 3 aliquots prepared at 1.7 nmol/g. The mass fraction content in each vial was estimated at 1nmol/g ± 0.02 nmol/g.A LC‐MRM method was developed to monitor Nf‐L tryptic peptides and assess their suitability for the development of a RMP. Fifteen peptides were selected. The limit of detection in solution is 1 ng/g for 6 peptides. The sample clean‐up was developed using Nf‐L spiked in artificial and real CSF. Several solid‐phase extraction cartridges were assessed in term of recovery and volume load. The use of protein precipitation was also assessed to increase the sensitivity. Preliminary experiments show a sensitivity of 100ng/g. Immunocapture will be used to achieve the required sensitivity.ConclusionA LC‐MRM based method and a sample clean‐up procedure were developed for the quantification of Nf‐L in CSF. A first assessment of sensitivity was carried out and the utilisation of immunocapture is on‐going to reach the required sensitivity.

Quantitation and speciation of inorganic arsenic in a biological sample by capillary ion chromatography combined with inductively coupled plasma mass spectrometry

The toxicity and biological activity of arsenic depend on its chemical form. In particular, inorganic arsenics are more toxic than organic ones. Apart from the determination of total arsenics, their accurate speciation is important for toxicity assessment. To separate arsenic species using a cation or an anion separation column, at least 0.5–1.0 mL of sample is required because conventional ion chromatography columns use a sample loop of 100–200 μL. It is thus difficult to analyze samples with small volumes, such as clinical and biological samples. In this study, a method for separating arsenic species using a 5-μL sample loop combined with a capillary ion exchange column has been developed for analyzing small volume of samples. The separated arsenics were determined by inductively coupled plasma mass spectrometry. By oxidizing As(III) to As(V) prior to analysis, the total inorganic arsenics, As(III) and As(V), could be well separated from the organic ones. Linear calibration curves (0.5–50 μg/kg) were obtained for total inorganic arsenics dissolved in water. Sub-picogram-level detection limit was obtained. The analytical capability of this method was successfully validated for certified reference materials, namely water and human urine, with total inorganic arsenic recovery efficiencies of 100% and 121%, respectively. Our method requires less than ~ 10 μL of sample and will be very useful to analyze valuable samples available in limited amounts.

In situ organic biosignature detection techniques for space applications

The search for life in Solar System bodies such as Mars and Ocean Worlds (e.g., Europa and Enceladus) is an ongoing and high-priority endeavor in space science, even ∼ five decades after the first life detection mission at Mars performed by the twin Viking landers. However, thein situdetection of biosignatures remains highly challenging, both scientifically and technically. New instruments are being developed for detecting extinct or extant life on Mars and Ocean Worlds due to new technology and fabrication techniques. These instruments are becoming increasingly capable of both detecting and identifyingin situorganic biosignatures that are indicative of life and will play a pivotal role in the search for evidence of life through robotic lander missions. This review article gives an overview of techniques used for space missions (gas chromatography, mass spectrometry, and spectroscopy), the further ongoing developments of these techniques, and ion mobility spectrometry. In addition, current developments of techniques used in the next-generation instruments for organic biosignature detection are reviewed; these include capillary electrophoresis, liquid chromatography, biosensors (primarily immunoassays), and nanopore sensing; whereas microscopy, biological assays, and isotope analysis are beyond the scope of this paper and are not covered.

Practical aspects of preparation, optimization, and evaluation of poly(styrene-divinylbenzene)porous-layer open tubular (PLOT) columns for capillary liquid chromatography

Using open tubular (OT) capillary columns in the nano and micro liquid chromatography scales has several advantages over conventional large-bore packed columns. Among the open tubular columns, those of the PLOT (porous layer open tubular) type are more suitable for liquid chromatography due to the porous character of the stationary phase that provides more significant interaction with the analytes. In addition, recent instrumental advances in capillaryLC coupled with mass spectrometry (MS) have opened up new avenues for developing and applying OT-LC-MS. This work discusses the possibility of applying PLOT columns in the capillary LC flow range (0.5–2 μL min−1) and describes the preparation, optimization, and evaluation of those columns. Poly(styrene-divinylbenzene) (PS-DVB) was used as the stationary phase, and the internal diameter of the columns discussed in this work was 10 and 25 μm. A comparison between these diameters is presented regarding fabrication and practical aspects of the operation. Additionally, for the 25 μm PLOT column, the gradient window and the flow rate effects on the system's performance were investigated. Flow rates of 1 μL min−1 and a gradient window of 60% (from 20 to 80% of acetonitrile) were the best conditions to work on this column in capillary LC conditions.

An efficient isoelectric focusing of microcolumn array chip for screening of adult Beta-Thalassemia

Traditional capillary isoelectric focusing (cIEF), liquid chromatography (LC) and capillary zone electrophoresis (CZE) still suffered from low resolution for hemoglobinopathy screening. Herein, a 30-mm pH 5.2–7.8 microcolumn IEF (mIEF) array chip was developed for hemoglobinopathy screening. As a proof of concept, adult beta-thalassemia was chosen as a model disease. In the method, blood samples were hemolyzed via hemolysin solution and loaded into the microcolumn. The experiments showed that (i) the species of Hb A, F, A2 and variants were clearly separated in the chip, and the resolution was greatly higher than the ones of LC/CZE/cIEF; (ii) up to 24 samples could be simultaneously analyzed in 12-min run; (iii) the intraday and interday RSDs were respectively 3.32–4.91 % and 4.07–5.33 %. The assays of mIEF to total 634 samples were compared with the ones of LC (n = 327) and PCR (n = 307). The cutoff of 3.5 % HbA2 led to the sensitivity of 100 % and specificity of 89.1 % for the mIEF-based screening; and there was 96.7 % coincidence between the methods of mIEF and PCR if refer Hb A2 and F. The method had the merits of facility, efficiency, specificity and sensitivity in contrast to the currently-used methods, implying its potential to screening of beta-thalassemia and hemoglobinopathies.

Investigation of the applicability of silica-graphene hybrid materials as stationary phases for capillary liquid chromatography

Graphene and graphene-derived substances are cutting-edge materials receiving increasing attention in the analytical chemistry field. Graphene oxide sheets bonded to amino silica particles functionalized with octadecyl (C18) groups and endcapped, also known as SiGO-C18ec, have been successfully employed as extraction phases and in analytical columns associated with conventional liquid chromatography (LC). In this work, SiGO-C18ec particles of 3, 5, and 10 µm nominal id were employed to pack capillary LC columns (100 mm long x 0.3 mm id), and their performance in the gradient mode was evaluated and compared. A 3 µm C18 capillary LC column (50 x 0.3 mm) was used as a reference column. Eight analytes having different polarities and topological surface areas were selected as a probe in this study: carbofuran clomazone, hexazinone, carbamazepine, citalopram, clomipramine, desipramine, and ochratoxin A. Studies about orthogonality were performed to investigate the orthogonality between the SiGO-C18ec and C18 phases. Among the SiGO-C18ec phases investigated, the column packed with 5 µm SiGO-C18ec particles presented the best peak capacity (29) in 15 min.Additionally, the performance of the columns packed with 5 µm SiGO-C18ec particles overcame the performance of the C18 columns used. Significant orthogonality was found between C18 and SiGO-C18ec packed columns; however, no significant differences were found between columns packed with SiGO-C18ec particles of different diameters.

On-line solid-phase extraction to enhance sensitivity in peptide biomarker analysis by microseparation techniques coupled to mass spectrometry: Capillary liquid chromatography versus capillary electrophoresis

In this study, on-line solid-phase extraction capillary liquid chromatography-mass spectrometry (SPE-CapLC-MS) and on-line solid-phase extraction capillary electrophoresis-mass spectrometry (SPE-CE-MS) were compared for the analysis of the opioid peptide biomarkers dynorphin A (1–7) (DynA), endomorphin 1 (End 1), and methionine-enkephalin (Met). First, a capillary liquid chromatography-mass spectrometry (CapLC-MS) method was established, which allowed limits of detection (LODs) of 0.5 μg/mL for Dyn A and Met, and 0.1 μg/mL for End 1. Then, a column switching setup operated by a 2-position/6-port micro-valve with a C18 enrichment column was assembled for SPE-CapLC-MS. Under optimized conditions, the LODs for the three peptides were lowered up to 1000-fold compared to CapLC-MS, until detecting 0.5 ng/mL concentrations. Repeatability (<0.2 % and <11 % RSD for retention times and peak areas, respectively), linearity (0.5–100 ng/mL), and durability (20 runs) of the enrichment column were appropriate, and the method was applied to analyze human plasma samples. Finally, the established SPE-CapLC-MS method was compared with a valve-free C18-SPE-CE-MS method previously described by our group for the analysis of these opioid peptides, using the same mass spectrometer. Both methods presented an evident difference regarding the need of a valve for the operation and allowed high preconcentration factors and quite similar LODs (until 0.5 and 0.1 ng/mL by SPE-CaLC-MS and SPE-CE-MS, respectively). Some other distinctions related to the instrumental set-up, procedure and method performance were also disclosed and discussed in detail.

Development of the Microemulsion Electrokinetic Capillary Chromatography Method for the Analysis of Disperse Dyes Extracted from Polyester Fibers.

The study aimed to develop a method for the separation of dispersed dyes extracted from polyester fibers. Nine commercially available disperse dyes, which were used to dye three polyester fabrics, were tested. Extraction of dyes from 1 cm long threads was carried out in chlorobenzene at 100 °C for 6 h. The separation was performed using microemulsion electrokinetic capillary chromatography (MEEKC) with photodiode array detection. Microemulsion based on a borate buffer with an organic phase of n-octane and butanol and a mixture of surfactants, sodium dodecyl sulphate and sodium cholate, were used. The addition of isopropanol and cyclodextrins to microemulsion resulted in a notable improvement in resolution and selectivity. The content of additives was optimized by using the Doehlert experimental design. Values of the coefficient of variance obtained in the validation process, illustrating the repeatability and intermediate precision of the migration times fit in the range of 0.11–1.24% and 0.58–3.21%, respectively. The developed method was also successfully applied to the differentiation of 28 real samples—polyester threads collected from clothing. The obtained results confirmed that proposed method may be used in the discriminant analysis of polyesters dying by disperse dyes and is promisingly employable in forensic practice.

Screening the extraction process of phenolic compounds from pressed grape seed residue: Towards an integrated and sustainable management of viticultural waste

The integrated valorisation of waste from the food chain to obtain value-added compounds with biological functionality will facilitate the transition to the era of a sustainable bioeconomy. To this end, an efficient matrix solid-phase dispersion (MSPD) extraction method was developed and optimized, using experimental factorial design and response surface methodology, for polyphenols recovery from pressed grape seeds obtained after the extraction of essential oils by cold pressing. Gallic, dihydroxybenzoic, p-coumaric and trans-ferulic acid, naringin, resveratrol, quercetin and kaempferol were quantified at 2.1–295 μg g−1 by capillary liquid chromatography coupled to a diode array detector and a mass analyser (cLC-DAD-MS). Furthermore, total antioxidant activity, free radical scavenging and lipid peroxidation suppression, together with the inhibition of beta-amyloid (Αβ42) protein aggregation, considered one of the main pathological effects of Alzheimer's disease, were evaluated. Potent lipid peroxidation inhibition (IC50 0.238 ± 0.003 ng g−1) was observed, along with the reduction of Αβ42 fibril width (9.4–54.8%) and aggregation. The results presented proved that the MSPD extraction method could be considered as an efficient and sustainable methodology to produce phenolic-rich extracts that may serve as an alternative antioxidant and neuroprotective ingredient for the food or pharmaceutical formulations, leading to the cascade valorisation of winery by-products.

Chemical inhibition of ER aminopeptidase 1 as a tool for regulating the immunopeptidome of cancer cells

The efficacy of cancer immunotherapy, including treatment with immune-checkpoint inhibitors, is often limited by ineffective presentation of antigenic peptides that can elicit T-cell mediated anti-tumor cytotoxic responses. Therefore, manipulating antigen presentation is an emerging approach for enhancing the immunogenicity of tumors in immunotherapy settings. ER aminopeptidase 1 (ERAP1) is an intracellular enzyme that trims peptides that can bind onto MHC class I molecules (MHC-I). We hypothesized that pharmacologically inhibiting ERAP1 in cells can regulate the global cellular immunopeptidome. we treated the A375 melanoma cell line with a recently developed potent ERAP1 inhibitor and analyzed the presented MHC-I peptide repertoire by isolating MHC-I, eluting the bound peptides and identifying them using capillary chromatography and tandem mass spectrometry. Although the inhibitor did not negatively affect overall MHC-I presence on the cell surface, it induced significant changes on the presented peptidomes, both at the qualitative and quantitative levels. Specifically, inhibitor treatment altered about half of the total 3204 identified peptides and about one third of the peptides predicted to be good ligands for MHC-I, affected length and sequence without however interfering with basic binding motifs. Surprisingly, the inhibitor enhanced predicted overall MHC-I binding affinity by reducing presentation of sub-optimal long peptides and generating many high-affinity 9-12mers suggesting that baseline ERAP1 activity in this cell line is destructive for many potential epitopes. Our results suggest that chemical inhibition of ERAP1 is a valid approach for manipulating the immunopeptidome of cancer with potential applications in standalone or combination immunotherapy.