Conventional Liquid Chromatography Research Articles

Eyes on the Prize: Overcoming Uncertainty to Realize the Power of 2D-LC Separations

Two-dimensional liquid chromatography (2D-LC) is a technique that extends the separation capabilities of conventional liquid chromatography by adding a second separation step to resolve compounds that are coeluted from a first column. This approach holds tremendous potential to solve difficult separation challenges in fields ranging from pharmaceutical analysis to biofuel characterization. Currently, method development is a significant bottleneck impeding more widespread implementation of 2D-LC methods, particularly for new users who are uncertain about how to proceed. In this month’s column, I highlight some of the primary considerations we face in method development and point to resources that can help users overcome uncertainty and develop highly effective 2D-LC methods.

Online Two-Dimensional Liquid Chromatography for Determination of Selected Flavonoids in Wine Samples

The development of two-dimensional chromatography systems is crucial in many scientific fields as it provides improved separation efficiency and selectivity of complex mixtures. By separating compounds based on two independent properties, these systems can offer a more detailed understanding of sample composition. This increased level of detail is particularly useful in industries such as pharmaceuticals, environmental analysis, and proteomics, where complex mixtures with similar properties need to be differentiated. A new chromatography system with two dimensions was developed to separate and quantify specific flavonoids in German wine samples. The first dimension used a homemade hydrophilic interaction liquid chromatographic column, while the second dimension used a reversed-phase liquid chromatographic column. This 2D ZIC-HILIC x RP technique was able to address the problem of peak overlapping that commonly occurs in one-dimensional liquid chromatography, especially for natural products. The study presents analytical and statistical data for both techniques, and suggests that the use of two-dimensional chromatography can improve the resolution of conventional one-dimensional liquid chromatography. In a conventional ZIC-HILIC-HPLC system, apigenin and naringenin were detected together but were successfully separated and measured using a 2DLC system. The limits of detection (LOD) for apigenin and naringenin were found to be 0.0705 µg/mL and 0.1300 µg/mL, respectively, while the limits of quantification (LOQ) were 0.2136 µg/mL and 0.3939 µg/mL, respectively. The recovery rate for both compounds was over 99%, with a relative standard deviation (RSD) of less than 1.5%. The linearity range for both compounds was between 0.005 µg/mL to 10 µg/mL, and the correlation coefficient (r2) was determined to be 0.9998. For hesperidin and rutin, the LOD and LOQ were found to be 0.0336 µg/mL and 0.1018 µg/mL, and 0.0120 µg/mL and 0.0363 µg/mL, respectively. The linearity range for both compounds was between 0.005 µg/mL to 12 µg/mL, and the correlation coefficient (r2) was determined to be 0.9999.

Quantitative Analysis of NDMA in Drug Products: A Proposed High-Throughput Approach Using Headspace–SIFT-MS

Since the initial 2018 recall of angiotensin receptor blockers due to unacceptable levels of mutagenic N-nitrosodimethylamine (NDMA) impurity, numerous drug products delivering diverse active pharmaceutical ingredients (APIs) have been recalled. Regulators and the industry are working together to understand and address this widescale problem. Conventional analysis of NDMA utilizes liquid or gas chromatography-based procedures that can involve complicated sample preparation and slow sample analysis. Selected ion flow tube mass spectrometry (SIFT-MS) analyses NDMA directly in the gas phase using soft chemical ionization, with an LOQ of 2 ng g−1. Through the novel application of the multiple headspace extraction (MHE) technique, NDMA was quantified directly and rapidly from the drug product without dissolution, at levels well below the regulatory acceptable intake of 96 ng day−1. A comparative analysis of recalled metformin using MHE-SIFT-MS and a conventional liquid chromatography–mass spectrometry/mass spectrometry (LC-MS/MS) method showed good agreement. Use of the novel MHE-SIFT-MS approach may enable a wider screening of drug products to be conducted, since it provides around a three-fold increase in daily sample throughput.

Accelerating Pharmaceutical Process Development with an Acoustic Droplet Ejection-Multiple Reaction Monitoring-Mass Spectrometry Workflow.

Fast-paced pharmaceutical process developments (e.g., high-throughput experimentation, directed evolution, and machine learning) involve the introduction of fast, sensitive, and accurate analytical assays using limited sample volumes. In recent years, acoustic droplet ejection (ADE) coupled with an open port interface has been invented as a sampling technology for mass spectrometry, providing high-throughput nanoliter analytical measurements directly from the standard microplates. Herein, we introduce an ADE-multiple reaction monitoring-mass spectrometry (ADE-MRM-MS) workflow to accelerate pharmaceutical process research and development (PR&D). This systematic workflow outlines the selection of MRM transitions and optimization of assay parameters in a data-driven manner using rapid measurements (1 sample/s). The synergy between ADE sampling and MRM analysis enables analytical assays with excellent sensitivity, selectivity, and speed for PR&D reaction screenings. This workflow was utilized to develop new ADE-MRM-MS assays guiding a variety of industrial processes, including (1) screening of Ni-based catalysts for C-N cross-coupling reaction at 1 Hz and (2) high-throughput regioisomer analysis-enabled enzyme library screening for peptide ligation reaction. ADE-MRM-MS assays were demonstrated to deliver accurate results that are comparable to conventional liquid chromatography (LC) experiments while providing >100-fold throughput enhancement.

Compressed fluid-based technology for downstream isolation of bluish anthocyanin-derived pigments obtained from blueberry surplus.

A dynamic compressed fluid-based separation process combining carbon dioxide and ethanol was explored to isolate portisins previously hemi-synthesized from blueberry surplus anthocyanins. The influence of process parameters such as pressure (100-500 bar), temperature (40-60 °C), and ethanol content in the compressed fluid mixture (20-50 wt%) on extraction yield, portisins yield, and portisins content in the extract was investigated. The two-step isolation process includes (1) a first step at 100 bar, 60 °C, and 20 wt% ethanol content in the compressed fluid mixture to remove the low polarity compounds; and (2) a second step at 500 bar, 40 °C, and 100 wt% ethanol to recover portisins, resulting in a 1.5-fold increase in portisins content. The performance of the two-step separation process was compared to centrifugal partitional chromatography and conventional reverse phase liquid chromatography already reported in terms of portisins content in the extract, process throughput, process efficiency, and total solvent used. The two-step separation process decreased the total solvent used, although with a decrease in the throughput and efficiency. Nevertheless, the choice of the best separation technology depends on the application, as these techniques result in different portisins purities. Overall, this study contributed to a scalable and more sustainable process for natural colorant production, specifically focusing on blue pigments, with several industrial applications.

A new single-reference quantitative method using liquid chromatography with relative molar sensitivity based on 1H-qNMR for khellactone esters from Peucedanum japonicum root extract.

Khellactone ester (KLE) quantification using the absolute calibration method is difficult owing to the unavailability of standard reagents that can guarantee purity. Herein, a new method was developed to quantify KLEs from Peucedanum japonicum root extracts using liquid chromatography (LC) without utilizing standards. This method used relative molar sensitivity (RMS) and 7-ethoxy-4-methylcoumarin as a single-reference (SR) compound instead of KLE standards. RMS is the sensitivity ratio of SR to analytes, determined using an offline combination of quantitative NMR and LC. LC was performed using a triacontylsilyl silica gel column of superficially porous particles with a ternary mobile phase. The range of the method was 2.60-509 µmol/L. The accuracy and precision were reasonable. This is the first study to apply the RMS method to both conventional LC and ultra-high-performance liquid chromatography using the same mobile phase and column. This method may aid the quality assurance of foods containing KLEs.

Assessment of ammonium fluoride as a mobile phase additive for sensitivity gains in electrospray ionization.

Ammonium fluoride has been shown to improve sensitivity when using electrospray ionization (ESI) coupled with mass spectrometry (MS). Recent internal investigation furthered that claim, through the observation of improved sensitivity when analyzing steroid molecules. This work focuses on extending those observations to other small molecules to understand the impact ammonium fluoride has on detection sensitivity with optimized instrument conditions. Using conventional liquid chromatography ESI-MS we investigated sensitivity differences between ammonium fluoride, formic acid, or ammonium hydroxide as mobile phase additives. Full source optimization was performed for nine compounds at three different organic concentrations (30%, 60%, or 90%) with formic acid, ammonium fluoride, and ammonium hydroxide adjustment. Optimization results were compiled to generate individual methods by compound, polarity, mobile phase, and organic concentration. Flow injection analysis was performed with fully optimized methods to compare compounds across different solvent systems under optimal conditions. Negative ESI data showed 2-22-fold sensitivity improvements for all compounds with ammonium fluoride. Positive ESI data showed>1-11-fold improvement in sensitivity for four of seven compounds and no change for three of seven compounds with ammonium fluoride. Ammonium fluoride improved ESI- sensitivity for all compounds studied when using optimized source conditions. Investigation with ESI+ analyses showed mixed results, with four of seven compounds showing improvement and others showing equivalency or slight loss in sensitivity, suggesting potential sensitivity gains for some analogs with ESI+.

Development of a liquid chromatography-based versatile bioanalysis for bevacizumab based on pretreatment combining aptamer affinity purification and centrifugal ultrafiltration concentration.

We report on the development of a versatile and accurate bioanalytical method for bevacizumab using a pretreatment method combining affinity purification with anti-idiotypic DNA aptamers and centrifugal ultrafiltration concentration, followed by liquid chromatography (LC)-fluorescence analysis. An affinity purification method using Sepharose beads as an affinity support removed immunoglobulin G and a large amount of coexisting substances in the serum sample. Purified bevacizumab was separated as a single peak by conventional LC and detected fluorometrically, showing good linearity (R2 = 0.999) in the range of 5-200μg/mL, sufficient to analyze bevacizumab concentrations in the blood of bevacizumab-treated patients. By combining this purification method with a concentration method using a centrifugal filtration device that inhibits non-specific adsorption of bevacizumab, the quantitative range was reduced by a factor of 10 while showing good linearity (R2 = 0.999) in the 0.5-20μg/mL range. The developed analytical method is expected to be used not only for general bioanalysis of therapeutic mAbs in clinical settings, but also for next-generation antibody drugs that show drug efficacy at low concentrations and for analysis of trace samples.

Hydrophilic interaction liquid chromatography with methanol-water eluent on a zeolite

BackgroundHydrophilic interaction chromatography (HILIC) works with organic solvent-water mixtures as eluent and is based on the formation of a water enriched liquid phase on the surface of a hydrophilic stationary phase. Hydrophilic solutes are retained on that stagnant water-rich film depending on the difference of solvation compared to the mobile phase composition. However, the enhancement of selectivity by increasing the fraction of organic cosolvent is coupled with a limitation the analyte solubility, and the improvement of the HILIC principle by new hydrophilic stationary phases is the remaining option. ResultsY-zeolite (faujasite, FAU type) in the Na+-form with an average particle diameter of 5 μm was used as packing material in a 125 mm long HPLC column. The chromatographic response of the column was tested in methanol-water mixtures as eluent after injection of several aliphatic alcohols, polyols and monosaccharides with eluent conditions where no separation occurs on diol functionalized silica. On the zeolite the retention time increases according to ethylene glycol < glycerol < erythritol < sorbitol < inositol. The separation principle is explained to be superposed by two effects: firstly, a partition equilibrium between the water-rich phase in the zeolite micropores exists, and secondly, selective interactions with the inner crystalline pore surface and fixed-position Na+ ions, both serving to enhance the selectivity. Furthermore, arabinose and fructose monosaccharides could be separated into their tautomeric forms. Only upon increasing the temperature from 20 to 60 °C the tautomeric pattern merges into a single peak. Significance and noveltyInstead of the stagnant water rich surface layer, zeolite micropores now take over that function. As a result, the selectivity among polyols and between α/β-arabinopyranose and β-fructopyranose/β-fructofuranose tautomers is extraordinary superior towards conventional hydrophilic interaction liquid chromatography (HILIC).

Comprehensive and heart-cutting multidimensional liquid chromatography-mass spectrometry and its applications in food analysis.

In food analysis, conventional one-dimensional liquid chromatography methods sometimes lack sufficient separation power due to the complexity and heterogeneity of the analyzed matrices. Therefore, the use of two-dimensional liquid chromatography (2D-LC) turns out to be a powerful tool to consider, especially when coupled to mass spectrometry (MS). This review presents the most remarkable 2D-LC-MS food applications reported in the last 10 years, including a critical discussion of the multiple approaches, modulation strategies as well as the importance of the optimization of the different analytical aspects that will condition the 2D-LC-MS performance. The presence of contaminants in food (food safety), the food quality, and authenticity or the relationship between the beneficial effects of food and human health are some of the fields in which most of the 2D-LC-MS applications are mainly focused. Both heart-cutting and comprehensive applications are described and discussed in this review, highlighting the potential of 2D-LC-MS for the analysis of such complex samples.

Comprehensive and heart-cutting multidimensional liquid chromatography-mass spectrometry and its applications in food analysis.

In food analysis, conventional one-dimensional liquid chromatography methods sometimes lack sufficient separation power due to the complexity and heterogeneity of the analysed matrices. Therefore, the use of two-dimensional liquid chromatography (2D-LC) turns out to be a powerful tool to consider, especially when coupled to mass spectrometry (MS). This review presents the most remarkable 2D-LC-MS food applications reported in the last 10 years, including a critical discussion of the multiple approaches, modulation strategies as well as the importance of the optimisation of the different analytical aspects that will condition the 2D-LC-MS performance. The presence of contaminants in food (food safety), the food quality and authenticity or the relationship between the beneficial effects of food and human health are some of the fields in which most of the 2D-LC-MS applications are mainly focused. Both heart-cutting and comprehensive applications are described and discussed in this review, highlighting the potential of 2D-LC-MS for the analysis of such complex samples.

Diluting modulation-based two dimensional-liquid chromatography coupled with mass spectrometry for simultaneously determining multiclass prohibited substances in cosmetics

Developing efficient and comprehensive screening methods for prohibited substances in cosmetics is critical for ensuring the quality and safety of cosmetics used in everyday life. This study proposed a heart-cutting two-dimensional liquid chromatography-mass spectrometry (2D-LC-MS) method based on online diluting modulation for detecting multiclass prohibited substances in cosmetics. The 2D-LC-MS method combines HILIC and RPLC techniques. Compounds near the dead time that the first dimensional HILIC could not separate were transferred to the second dimensional RPLC by valve switch, achieving good separation with a wide range of polarities. Moreover, the online diluting modulation solved the problem of mobile phase incompatibility, realizing an excellent column-head focusing effect and reducing the loss of sensitivity. Besides, the first dimensional analysis did not restrict the flow rate of the second dimensional analysis owing to the diluting modulation. We demonstrated the 2D-LC-MS system by determining 126 prohibited substances in cosmetic products, including hormones, local anesthetics, anti-infectives, adrenergic agents, antihistamines, pesticides, and other chemicals. All correlation coefficients of the compounds were above 0.9950. The LODs and the LOQs ranged from 0.000259 ng/mL to 16.6 ng/mL and 0.000864 ng/mL to 55.3 ng/mL, respectively. The RSDs% for intra-day and inter-day precision were within 6% and 14%, respectively. Compared with conventional one-dimensional liquid chromatography methods, the established method expanded the analytical coverage of cosmetics-prohibited substances with reduced matrix effects for most compounds and improved sensitivity for polar analytes. The results indicated that the 2D-LC-MS method was a powerful tool for screening multiclass prohibited substances in cosmetics.

Principles and potential of solvent gradient size-exclusion chromatography for polymer analysis

The properties of a polymeric material are influenced by its underlying molecular distributions, including the molecular-weight (MWD), chemical-composition (CCD), and/or block-length (BLD) distributions. Gradient-elution liquid chromatography (LC) is commonly used to determine the CCD. Due to the limited solubility of polymers, samples are often dissolved in strong solvents. Upon injection of the sample, such solvents may lead to broadened or poorly shaped peaks and, in unfavourable cases, to “breakthrough” phenomena, where a part of the sample travels through the column unretained. To remedy this, a technique called size-exclusion-chromatography gradients or gradient size-exclusion chromatography (gSEC) was developed in 2011. In this work, we aim to further explore the potential of gSEC for the analysis of the CCD, also in comparison with conventional gradient-elution reversed-phase LC, which in this work corresponded to gradient-elution reversed-phase liquid chromatography (RPLC). The influence of the mobile-phase composition, the pore size of the stationary-phase particles, and the column temperature were investigated. The separation of five styrene/ethyl acrylate copolymers was studied with one-dimensional RPLC and gSEC. RPLC was shown to lead to a more-accurate CCD in shorter analysis time. The separation of five styrene/methyl methacrylate copolymers was also explored using comprehensive two-dimensional (2D) LC involving gSEC, i.e. SEC × gSEC and SEC × RPLC. In 2D-LC, the use of gSEC was especially advantageous as no breakthrough could occur.

First proof-of-concept of UC/HILIC for extending the versatility of the current art of supercritical fluid separation

Supercritical Fluid Chromatography (SFC), a high-throughput separation technique, has been widely applied as a promising routine method in pharmaceutical, pesticides, and metabolome analysis in the same way as conventional liquid chromatography and gas chromatography. Unified chromatography (UC), an advanced version of SFC, which applied gradient elution with mobile phase changing continuously from supercritical to subcritical and to liquid states, can further extend the SFC applications. UC mostly applying the popular mobile phase of 95%:5%/Methanol:Water with additives allows to analyze many hydrophilic compounds. However, many of phosphorylated metabolites or multi carboxylic acids show very poor peak shapes or even can't be eluted under UC conditions, thus hampering the UC's metabolome coverage. In this study, we proposed the first proof-of-concept of UC/HILIC, a novel strategy to extend the current UC metabolome coverage by employing an aqueous gradient right after the UC gradient on a single packed column in a single measurement. The proposed method showed significant improvement regarding the chromatographic performance and metabolome coverage, while still maintaining the precision and high throughput in comparison with conventional UC methods.

Aqueous liquid chromatography with mobile phases of sodium dodecyl sulphate and ionic liquid

In conventional reversed-phase liquid chromatography (RPLC) with hydro-organic solvents, basic cationic solutes yield retained, broad, asymmetric peaks, owing to their interaction with free anionic silanols in the stationary phase. RPLC mobile phases to which the anionic surfactant sodium dodecyl sulphate (SDS), or an ionic liquid (IL) are added, have been proposed as solutions, since these additives are able to block the silanol effect thus improving the chromatographic performance. With these additives, it is however necessary to increase the elution strength by adding an organic solvent, such as an alcohol or acetonitrile. A novel aqueous liquid chromatographic mode (in the absence of organic solvent) is here proposed, where the mobile phases contain only a mixture of aqueous solutions of SDS and an IL derived from 1-alkyl-3-methylimidazolium associated to chloride, both environmentally friendly. When these reagents are added, the anionic surfactant adsorbed on the stationary phase is able to attract the cationic solutes, whereas the adsorbed IL cation repels them. The combination of both effects (attraction and repulsion) allows the modulation of retention, by varying the IL/SDS ratio. Given the character of the additives, a type of green liquid chromatography is achieved. In this work, the chromatographic behavior of six basic compounds of pharmaceutical interest, the β-adrenoceptor antagonists acebutolol, atenolol, carteolol, metroprolol, oxprenolol and propranolol, is examined. In order to assess the chromatographic behavior of the mixed mobile phases containing SDS and IL, changes in retention, peak profile and resolution of mixtures of the analytes were explored at varying concentration of the additives.

Driving under the Influence of Amphetamine: Analytical Evaluation of Illegal or Prescription Drug Intake Using Chiral UHPLC-MS-MS.

Differentiation between consumption of illegal and prescription drugs remains an important aspect in forensic toxicology. While illicit amphetamine is most often racemic, the medicinal drugs marketed in Denmark for the treatment of attention-deficit hyperactivity disorder contain the pure (S)-enantiomer or a prodrug thereof. In this study, we present a simple and efficient analytical workflow to provide information about the origin of amphetamine consumed in forensic cases concerning driving under the influence of drugs (DUID). Following quantification of amphetamine and methamphetamine using our conventional multi-target ultra-high performance liquid chromatography-tandem mass spectrometry method, determination of (R)- and (S)-amphetamine was performed by reinjecting the sample extract on a Phenomenex LUX® AMP chiral column using the same analytical instrument and mobile phases. Chiral separation was performed isocratic within a run time of 6 min. The analytical workflow was applied to blood samples from 5,248 suspected DUID cases within a 2-year period. Amphetamine was detected in 18.7% of the samples, of which both enantiomers were detected in 89.5% of the cases, indicating the consumption of illegal racemic amphetamine. In 6.1% of the positive cases, both amphetamine and methamphetamine were detected, indicating either co-consumption of both amphetamines or consumption of methamphetamine. In the remaining 4.4%, only (S)-amphetamine was detected indicating the consumption of prescription drugs containing (S)-amphetamine or a prodrug thereof. Implementation of a simple and rapid chiral method in the conventional analytical workflow for routine forensic casework proved to be an efficient way to elucidate whether a positive amphetamine result originates from illegal or prescription drug consumption, without increasing turnaround time nor costs to any significant extent, as no additional sample preparation was required.

High-throughput kinetic turbidity analysis for determination of amorphous solubility and excipient screening for amorphous solid dispersions

Many poorly water-soluble active pharmaceutical ingredients (APIs) rely on supersaturating formulations, such as amorphous solid dispersions (ASDs), to enhance oral bioavailability. ASDs kinetically trap amorphous solid drugs within polymer excipient matrices to maintain the amorphous drug states. The maximum solution concentration of the API in these formulations is known as the amorphous solubility. In early drug development with scarce material and time, high-throughput approaches to measuring amorphous solubility and screening excipient effects on crystallization risk offer significant benefits to preclinical formulation scientists. Here, we developed a high-throughput screening (HTS) workflow to quantify amorphous solubility and screen ASD excipients by automated kinetic turbidity analysis. Testing 20 model APIs with a wide range of biorelevant solubility, we demonstrated their apparent amorphous solubility determined by the HTS approach strongly correlated with quantification results using conventional liquid chromatography; while the real-time analysis significantly saved analytical time and experimental efforts. Furthermore, kinetic turbidity profiles elucidated distinct excipient effects on the precipitation process of APIs. These results were successfully translated to dissolution and precipitation behaviors of ASD formulations composed of the tested polymers. The high-throughput kinetic turbidity workflow presents a facile and information-rich approach for amorphous solubility screenings against excipients, and helps guide enabling formulation development.

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.

MetaProClust-MS1: an MS1 Profiling Approach for Large-Scale Microbiome Screening.

ABSTRACTMetaproteomics is used to explore the functional dynamics of microbial communities. However, acquiring metaproteomic data by tandem mass spectrometry (MS/MS) is time-consuming and resource-intensive, and there is a demand for computational methods that can be used to reduce these resource requirements. We present MetaProClust-MS1, a computational framework for microbiome feature screening developed to prioritize samples for follow-up MS/MS. In this proof-of-concept study, we tested and compared MetaProClust-MS1 results on gut microbiome data, from fecal samples, acquired using short 15-min MS1-only chromatographic gradients and MS1 spectra from longer 60-min gradients to MS/MS-acquired data. We found that MetaProClust-MS1 identified robust gut microbiome responses caused by xenobiotics with significantly correlated cluster topologies of comparable data sets. We also used MetaProClust-MS1 to reanalyze data from both a clinical MS/MS diagnostic study of pediatric patients with inflammatory bowel disease and an experiment evaluating the therapeutic effects of a small molecule on the brain tissue of Alzheimer’s disease mouse models. MetaProClust-MS1 clusters could distinguish between inflammatory bowel disease diagnoses (ulcerative colitis and Crohn’s disease) using samples from mucosal luminal interface samples and identified hippocampal proteome shifts of Alzheimer’s disease mouse models after small-molecule treatment. Therefore, we demonstrate that MetaProClust-MS1 can screen both microbiomes and single-species proteomes using only MS1 profiles, and our results suggest that this approach may be generalizable to any proteomics experiment. MetaProClust-MS1 may be especially useful for large-scale metaproteomic screening for the prioritization of samples for further metaproteomic characterization, using MS/MS, for instance, in addition to being a promising novel approach for clinical diagnostic screening.IMPORTANCE Growing evidence suggests that human gut microbiome composition and function are highly associated with health and disease. As such, high-throughput metaproteomic studies are becoming more common in gut microbiome research. However, using a conventional long liquid chromatography (LC)-MS/MS gradient metaproteomics approach as an initial screen in large-scale microbiome experiments can be slow and expensive. To combat this challenge, we introduce MetaProClust-MS1, a computational framework for microbiome screening using MS1-only profiles. In this proof-of-concept study, we show that MetaProClust-MS1 identifies clusters of gut microbiome treatments using MS1-only profiles similar to those identified using MS/MS. Our approach allows researchers to prioritize samples and treatments of interest for further metaproteomic analyses and may be generally applicable to any proteomic analysis. In particular, this approach may be especially useful for large-scale metaproteomic screening or in clinical settings where rapid diagnostic evidence is required.

The Future of Method Development for Two-Dimensional Liquid Chromatography – Work Smarter, Not Just Harder?

The potential for wider use of two-dimensional liquid chromatography (2D-LC) is becoming more evident as the complexity of samples that must be handled continues to increase in application areas ranging from biopharmaceuticals to biosourced consumer products. Although the sophistication and ease of use have improved in recent years for commercial 2D-LC instruments, many analysts are still intimidated by the method development process for 2D methods because of the larger number of variables involved compared to conventional liquid chromatography. In this article, I share my perspective on the trends in this area, and the developments we are likely to see in the field in the near future.