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A metal tube-in-manifold packed bed capillary column device, designed to overcome common limitations associated with capillary LC separations, is described. Experimental results of initial packing tests with sub-3 μm core-shell particles demonstrated efficiencies greater than 47,000 plates/m for a separation performed using the column device. Computational fluid dynamics (CFD) modeling of the multicomponent separation used for this work was validated against experimental LC results and the optimized model was able to effectively predict component peak retention times. However, the accuracy of predicted efficiencies requires further refinement. The tube-in-manifold design demonstrates that packed capillary columns with cylindrical cross-sectional channel geometry and ultrahigh pressure, low dead volume fluidic connections are achievable., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: M.K., D. J. C., T. A., and G.S. are employed by IDEX Health & Science, which is the assignee for patents related to the technology described in this work. Partial funding from IDEX Health & Science in support of this project was provided to C.P. and J.P.G. (Rowan University)., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

A unique strategy for developing porous membrane protected micro-solid phase extraction has been provided. An electrospun composite was fabricated on the sheet of membrane. To this end, NiFe-layered double hydroxide/Nylon 6 composite nanofibers were coated on a polypropylene membrane sheet followed by folding into a pocket shape, which were then utilized as a novel extractive device to extract of organophosphorus pesticides from fresh fruit juice samples prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The fabricated hybrid composites were successfully characterized. The effective parameters on extraction performance were investigated. LODs were 0.020-0.065 ng mL -1 . Excellent linearity (R 2 ≥0.996) was observed between 0.05 and 100.0 ng mL -1 . RSDs% were in the range of 3.1-5.8% (intra-day, n = 3) and 2.6-5.5% (inter-day, n = 3×3). Satisfactory related recovery values within the acceptable range of 90.7-111.2% with RSDs% below 6.7% were achieved for the analysis of real samples., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

In recent years, miniaturized analytical instruments have been developing to meet the needs of portable and rapid analysis. The key of miniaturized analytical equipment is the miniaturization and integration of functional modules. This paper aims to develop a miniaturized photometric detector and separation microfluidic chip for a liquid chromatography (LC) system. The detector uses a light-emitting diode to emit ultraviolet light, which is collimated by an internal double lens. A Z-shaped flow cell with a long optical path is designed and fabricated in the separation microfluidic chip with a three-layer structure, which provides a tubing-free connection between the separation and detection unit. Detector performance is evaluated using hemoglobin (Hb) samples, with an upper limit of detection linearity (95 %) of 0.345 AU and stray light level as low as 0.08 %. Additionally, the microchip channel can be filled with cation exchange resin and C18 particles. Finally, an ion LC system and a reversed-phase LC system were constructed based on the miniaturized photometric detector and two microchips with different packed columns, respectively, and were successfully used in the separation and detection of two metabolic markers (glycated hemoglobin or bilirubin). The results of this study are expected to facilitate the development of a portable LC system and their application in community health services and family health management of chronic diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Humic acid was the main compound in soil and reduced the availability of some organic compounds in soils. In this work, humic acid was immobilized for the first time on a homemade neutravidin poly(glycidyl methacrylate-co-ethylene dimethacrylate) capillary column with a 20 μm i.d. for the screening of potential ligands to humic acid and the evaluation of their molecular recognition mechanism. This homemade humic acid column enabling it to work at very low backpressure (0.60 MPa at 20 nl/min flow rate), had a long lifetime, excellent repeatability, and negligible non-specific binding sites. The performance of this affinity humic acid column was demonstrated by the evaluation of recognition assay for a series of known ligands of humic acid (a series of rodenticide molecules) which is the heart of the fragment-based drug design. In addition, this column was used successfully for highlighting the binding mechanism to humic acid of the severe acute respiratory syndrome coronavirus-2-spike protein. As well this new humic acid miniaturized liquid chromatography column developed in this work could be used in the feature for another solute molecule-humic acid binding studies or for a separative mode., (© 2023 Wiley-VCH GmbH.)

In metabolomics, retention prediction methods have been developed based on the structural and physicochemical characteristics of analytes. Such methods employ regression models, harnessing machine learning algorithms mapping experimentally derived retention time (t R ) analytes with various structural and physicochemical descriptors, known as Quantitative Structure Retention Relationships (QSRR) models. In the present study, QSRR models have been developed by applying four Machine Learning regression algorithms, i.e. Bayesian Ridge Regression (BRidgeR), Extreme Gradient Boosting Regression (XGBR) and Support Vector Regression (SVR) using both linear and non-linear kernels, all tested and compared for their retention prediction ability on experimentally derived and on publicly available chromatographic data, using Molecular Descriptors to describe the physical, chemical or structural properties of molecules. Various configurations of the available datasets, in terms of the highly-correlated features levels (defined as the maximum absolute value of the Pearson's correlation coefficient calculated between any pair of features) they contained, were analyzed in parallel. This is the first study, to the best of our knowledge, of the effect of collinearity on the performance of QSRR predictive models. In the vast majority of cases studied there was no statistically significant difference in the performance of the generated QSRR predictive models among the specified dataset configurations, indicative of the ability of the selected regression algorithms to effectively handle collinearity. In terms of the individual performance of the selected regression algorithms, no pattern was found where one algorithm (or class of algorithms) stood out significantly relative to the others among the study datasets., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Recently, numerous diagnostic approaches from different disciplines have been developed for SARS-CoV-2 diagnosis to monitor and control the COVID-19 pandemic. These include MS-based assays, which provide analytical information on viral proteins. However, their sensitivity is limited, estimated to be 5 × 10 4 PFU/ml in clinical samples. Here, we present a reliable, specific, and rapid method for the identification of SARS-CoV-2 from nasopharyngeal (NP) specimens, which combines virus capture followed by LC-MS/MS(MRM) analysis of unique peptide markers. The capture of SARS-CoV-2 from the challenging matrix, prior to its tryptic digestion, was accomplished by magnetic beads coated with polyclonal IgG-α-SARS-CoV-2 antibodies, enabling sample concentration while significantly reducing background noise interrupting with LC-MS analysis. A sensitive and specific LC-MS/MS(MRM) analysis method was developed for the identification of selected tryptic peptide markers. The combined assay, which resulted in S/N ratio enhancement, achieved an improved sensitivity of more than 10-fold compared with previously described MS methods. The assay was validated in 29 naive NP specimens, 19 samples were spiked with SARS-CoV-2 and 10 were used as negative controls. Finally, the assay was successfully applied to clinical NP samples (n = 26) pre-determined as either positive or negative by RT-qPCR. This work describes for the first time a combined approach for immuno-magnetic viral isolation coupled with MS analysis. This method is highly reliable, specific, and sensitive; thus, it may potentially serve as a complementary assay to RT-qPCR, the gold standard test. This methodology can be applied to other viruses as well., (© 2021. Springer-Verlag GmbH Germany, part of Springer Nature.)

The use of micro, capillary, and nano liquid chromatography systems for forensic analysis has excellent potential. In a field where sample size is often limited, several studies have presented the viability of capillary columns with microflow and nanoflow, and when using mass spectrometric analysis limits of detection can be improved. Reduction in flow rates result in significant reduction in operating costs. Recent advances in miniaturized liquid chromatography systems also aim at in-laboratory and on-site detection, which have already been applied to forensic drug cases. This critical review will discuss the advantages, disadvantages, and applicability of microflow and nano liquid chromatography. In this regard, included in this article is a discussion of some promising areas not yet applied to forensic research., (© 2021 Wiley-VCH GmbH.)

Sulfur mustard reacts with blood proteins, such as hemoglobin, to form stable adducts that can be used as long-lived biomarkers of exposure. These adducts can be analyzed by liquid chromatography coupled to tandem mass-spectrometry (LC-MS/MS) after an enzymatic digestion step. The objective of this study was to develop trypsin-based immobilized enzyme reactors (IMERs) in order to obtain a faster digestion of hemoglobin than the conventional in-solution digestion. Trypsin IMERs were synthetized by grafting the enzyme on a CNBr-Sepharose gel and the influence of several parameters on the digestion yields, such as the transfer volume between the injection loop and the IMER, the temperature and the digestion time was studied. The repeatability of the digestion on three laboratory-made IMERs was demonstrated for pure hemoglobin and hemoglobin previously exposed to different concentrations of sulfur mustard (RSD inferior to 13% and 21% respectively) and was better than that obtained for in-solution digestions (RSD inferior to 28% and up to 53% respectively). A preferential adduction of sulfur mustard on the histidine residues of hemoglobin was confirmed, for both in-solution and IMER digestion results. On a quantitative point of view, the performances of in-solution and IMER digestions were similar, with the theoretical possibility to detect peptides resulting from the in vitro incubation of hemoglobin in pure water with sulfur mustard at 7.5 ng⋅mL -1 . However, digestion on IMER proved to be more repeatable and 32 times faster than in-solution digestion, and a given IMER could be reused at least 60 times., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The gut microbiota is critical to the maintenance of physiological homeostasis and as such is implicated in a range of diseases such as colon cancer, ulcerative colitis, diabetes, cardiovascular diseases, and neurodegenerative diseases. Short chain fatty acids (SCFAs) are key metabolites produced by the gut microbiota from the fermentation of dietary fibre. Here we present a novel, sensitive, and direct LC-MS/MS technique using isotopically labelled internal standards without derivatisation for the analysis of SCFAs in different biological matrices. The technique has significant advantages over the current widely used techniques based on sample derivatization and GC-MS analysis, including fast and simple sample preparation and short LC runtime (10 min). The technique is specific and sensitive for the quantification of acetate, butyrate, isobutyrate, isovalerate, lactate, propionate and valerate. The limits of detection were all 0.001 mM except for acetate which was 0.003 mM. The calibration curves for all the analytes were linear with correlation coefficients r 2 > 0.998. The intra- and inter-day precisions in three levels of known concentrations were <12% and <20%, respectively. The quantification accuracy ranged from 92% to 120%. The technique reported here offers a valuable analytical tool for use in studies of SCFA production in the gut and their distribution to host tissues.

We report on a numerical simulation study of a number of potential column technology solutions to minimize the plate height contribution (H vh ) originating from the use of ultra-high pressures and their concomitant viscous heating effect. Looking as far as possible into the future of UHPLC, all main results are obtained for the case of a 2500 bar pressure gradient. However, to generalize the result, a correlation is given that can be used to interpolate the results to lower pressures with some 10% accuracy. For the considered case of a 2.1mm column, a liquid flow rate of 0.45 ml/min, an analyte with retention factor k(25°C)=3 and a retention enthalpy chosen such that ΔH R /R= -1000 K, it is found that, in order to keep the global plate height as measured at the column outlet (H vh,glob,out ) below 1 μm, the bed conductivity would need to be raised to λ bed =2.4 W/m•K, i.e., 4 times higher than a typical packed bed of fully-porous or core-shell silica particles. An equivalent effect on the band broadening could be obtained if it would be possible to replace the steel column wall with a low conductivity material. In this case, a wall conductivity of 0.25 W/m•K, i.e., 64 times smaller than the conductivity of steel, would be needed to keep H vh,glob,out below 1 μm. Results are also interpreted based on contour plots of the axial and radial velocity variation of a retained analyte., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier B.V.)

The mixing of two or more solvent streams to deliver a stable and accurate solvent composition is crucial to the performance, repeatability and reproducibility of a liquid chromatographic separation. We provide a theoretical treatment of axial mixing of a sequence of solvent packets with the framework of continuous stirred tank reactors (CSTRs) in series and investigate the tradeoffs presented between the primary goal of mixers (noise reduction) and it's necessary side-effects of gradient deformation and asymmetry. An experimental setup to mimic CSTR conditions was created using a stop-flow setup where the fluid flow was periodically paused and sonicated within pods of a certain volume. The effects of mixer volume relative to the volume of pump strokes and gradient volumes were investigated and discussed. A total mixer volume that is six-fold the pump stroke volume was found to be necessary to achieve sufficient (95%) noise reduction necessary for certain applications. A series of two or more CSTR elements was found to outperform a single CSTR element for larger mixer-to-pump stroke volume ratio in dampening baseline noise. For linear gradients, a gradient volume that is ten times larger than the mixer volume was found to sufficiently maintain gradient fidelity. For very small gradient volumes relative to the mixer volume, deformation of linear gradients was found to be significantly greater than predicted by the analytical solution. Furthermore, the nature of the solvent gradient deformation was asymmetric, with the latter half of the solvent gradient deforming significantly more than the first half. Combining analytically and numerically derived solutions for multiple CSTRs connected in series with experimental data, several suggestions can be made on mixer dimensions and design for a certain pump system and method transfer, given a pump stroke volume and gradient time., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Waters and Empower are trademarks of Waters Technologies Corporation. Autodesk Inventor is a trademark of Autodesk, Inc. Milli-Q is a trademark of Merck KGaA. Arduino is a trademark of Arduino AG. Wolfram Mathematica is a trademark of Wolfram Group, LLC., (Copyright © 2021. Published by Elsevier B.V.)

Modern analytical applications of liquid chromatography require more and more efficient columns. In this work, the possibility of utilizing particle size gradient in the chromatographic column was studied by a theoretical approach. In the course of our work three different scenarios of particle size gradients were considered with different shapes (linear, convex and concave). The evolution of bandwidth inside the column was plotted for each scenario. As a reference point, the bandwidth of the uniform column was used, which had the same pressure drop as the non-uniform column. According to our calculations, in isocratic elution mode, the non-uniform column does not offer any advantage compared to the uniform column, regardless the type of the particle size gradient. In gradient elution mode, however, extra band compression occurs was found. For negative particle size gradients, the final physical bandwidth was found to be approximately 1-4 % smaller than for uniform columns. This slight gain in efficiency in terms of bandwidth compression can be expanded to 5-8 % by the optimization of the limiting particle sizes. These optimized results are obtained when the final particle size is approximately 40% of the initial particle diameter., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier B.V.)

An on-surface multi-purpose autosampler was built for liquid chromatography-mass spectrometry (LC-MS) based on the autoTLC-MS interface, taking advantage of open-source hard- and software developments as well as 3D printing. Termed autoTLC-LC-MS system, it is introduced for orthogonal hyphenation of normal phase high-performance thin-layer chromatography with reversed phase high-performance LC (HPLC) and high-resolution MS (HRMS). For verification of its functionality, a multi-class antibiotic mixture was applied as a calibration band pattern on an adsorbent layer and detected by the Bacillus subtilis bioassay. This effect-image was uploaded as a template in the updated TLC-MS&#95;manager software. The clicked-on antibiotic zones were sequentially eluted without intervention from the planar counterpart (without bioassay) via a monolithic HPLC column into the HRMS system. For elution of antibiotics of 7 structural classes at 5 different calibration levels, the new on-surface autosampler achieved intra-day precisions of 2.1-14.1%, while inter-day precisions ranged 2.5-16.1% (all n = 3). The new hyphenation offers potential for planar sample clean-up prior to HPLC, concentration of liquid samples, increase of peak capacity and proof of peak purity or isomers. The integrated autoTLC-LC-MS system enabled high sample throughput, efficiency and reproducibility for the first time through fully automated TLC-LC-MS sequence operation. Its contact-closure signal functionality, versatile 3D printed planar sample holder and open-source software made it readily adjustable for new analytical tasks. Undoubtedly, any planar material can be investigated for leachables, such as textiles, foils, papers and other packagings, as well as planar biological samples for ingredients., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

This article describes the use of a new prototype column hardware made with 1.5 mm internal diameter (i.d.) and demonstrates some benefits over the 1.0 mm i.d. micro-bore column. The performance of 2.1, 1.5 and 1.0 mm i.d. columns were systematically compared. With the 1.5 mm i.d. column, the loss of apparent column efficiency can be significantly reduced compared to 1.0 mm i.d. columns in both isocratic and gradient elution modes. In the end, the 1.5 mm i.d. column is almost comparable to 2.1 mm i.d. column from a peak broadening point of view. The advantages of the 1.5 mm i.d. hardware vs 2.1 mm i.d. narrow-bore columns are the lower sample and solvent consumption, and reduced frictional heating effects due to decreased operating flow rates., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier B.V.)

The accurate determination of the pharmacokinetics (PK) of a candidate drug molecule is critical in both drug discovery and development. Over the last 30 years, the sensitivity and selectivity of LC/MS has resulted in it being established as the technology of choice for these studies. However, unwanted chemical interactions between analyte(s) and the metal components in a chromatography system can result in poor peak shape and reduction in signal response, which can adversely affect the analysis of low concentrations of drugs and their metabolites in biological samples. This study evaluated the benefits of employing an inert hybrid surface technology (HST) applied to the metallic components in the LC flow path, column frits and column wall to mitigate these interactions. The results obtained were compared with that of an identical conventional LC for the bioanalysis of two steroid phosphate drugs (dexamethasone phosphate and hydrocortisone phosphate) and an epidermal growth factor receptor (EGFR) inhibitor (gefitinib) in human plasma. The results showed that for the two steroid phosphates, the peak width was reduced by 20%, peak tailing factors reduced by up to 30% and the assay sensitivity improved by factors of 7.5 and 10. This resulted in a significant improvement in the limit of detection. The new LC system also improved the reproducibility of peak integration for gefitinib, thereby reducing assay coefficients of variation (%CV) from greater than 10% to less than 5% at the lower limit of quantification., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Severe acute malnutrition (SAM) may alter the pharmacokinetics (PK), efficacy, and safety of antiretroviral therapy. The phase IV study, IMPAACT P1092, compared PK, safety, and tolerability of zidovudine (ZDV), lamivudine (3TC), and lopinavir/ritonavir (LPV/r) in children with and without SAM., Materials and Methods: Children living with HIV 6 to <36 months of age with or without World Health Organization (WHO)-defined SAM received ZDV, 3TC, and LPV/r syrup for 48 weeks according to WHO weight band dosing. Intensive PK sampling was performed at weeks 1, 12, and 24. Plasma drug concentrations were measured using liquid chromatography tandem mass spectrometry. Steady-state mean area under the curve (AUC0-12h) and clearance (CL/F) for each drug were compared. Grade ≥3 adverse events were compared between cohorts., Results: Fifty-two children were enrolled across 5 sites in Africa with 44% (23/52) female, median age 19 months (Q1, Q3: 13, 25). Twenty-five children had SAM with entry median weight-for-height Z-score (WHZ) -3.4 (IQR -4.0, -3.0) and 27 non-SAM had median WHZ -1.0 (IQR -1.8, -0.1). No significant differences in mean AUC0-12h or CL/F were observed (P ≥ 0.09) except for lower 3TC AUC0-12h (GMR, 0.60; 95% CI, 0.4-1.0; P = 0.047) at week 12, higher ZDV AUC0-12h (GMR, 1.52; 1.2-2.0; P = 0.003) at week 24 in the SAM cohort compared with non-SAM cohort. Treatment-related grade ≥3 events did not differ significantly between cohorts (24.0% vs. 25.9%)., Conclusion: PK and safety findings for ZDV, 3TC, and LPV/r support current WHO weight band dosing of syrup formulations in children with SAM., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc.)

In this study, polyamide and MCI GEL® CHP20P were employed as stationary phases in medium pressure chromatography (MPC) for the efficient preparative separation of bergenin from Saxifraga atrata. Ethanol-water, methanol-water, and acetonitrile-water mobile phases all showed good enrichment capacity for bergenin fraction when polyamide was used as a stationary phase. After 5 cycles of polyamide MPC using acetonitrile/water, 1.2 g of bergenin fraction was isolated from 180 g Saxifraga atrata herb. Further purification of this fraction was conducted using MCI GEL® CHP20P styrene-divinylbenzene beads. The bergenin fraction was separated into two fractions, and after three runs of MPC, 714.2 mg of bergenin with purity above 99% was obtained. The results demonstrate that the combination of polyamide and styrene-divinylbenzene MPC can be utilized for preparative isolation of compounds from natural products with high yield and purity., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Introduction: In 2016 the American Academy of Ophthalmology(2016-AAO) recommended a maximum daily HCQ use of 5.0 mg/kg real body weight(RBW) taking into consideration minimizing eye toxicity. Retinopathy in systemic lupus erythematosus(SLE) patients was recently associated with obesity and this condition is progressively more common in these patients. However, the impact of obesity in HCQ blood levels remains controversial., Objective: To determine if the 2016-AAO recommendation based on RBW with and without maximum daily dose restriction results in adequate and safe blood levels in obese lupus nephritis(LN) patients., Methods: A cross-sectional study was performed with 108 LN patients under the prescribed 2016-AAO dose for at least 3 months. LN patients were assessed for demographic characteristics, body mass index(BMI), disease parameters, HCQ dose, concomitant treatment and HCQ blood levels measured by liquid chromatography-tandem mass spectrometry. Obesity was defined as BMI ≥30kg/m 2 ., Results: Obesity was identified in 35/108(32%) LN patients. The calculation of HCQ daily dosage revealed that obese patients were under a lower prescribed daily dose according to the real body weight (RBW) [4.4(2.9-5.4) vs. 4.9(4-5.5)mg/Kg/day, p < 0.001] due to the maximum limit used. Regardless of that the median of HCQ blood levels was significantly higher in obese compared to non-obese patients (1562 ± 548.6 vs. 1208 ± 448.9 ng/mL, p = 0.002). Further analysis of patients under the 20016-AAO recommendation by RBW without the restriction of maximum daily dose confirmed that in spite of comparable daily dose in 14 obese patients and 61 non-obese patients [4.8 (4.5-5.4) vs. 5.0(4.5-5.5) mg/kg, p = 0.312], the median of HCQ blood levels was significantly higher in obese patients than in non-obese (1734 ± 457.3 vs. 1189 ± 449.4 ng/mL, p < 0.001)., Conclusion: Obese patients under the 2016-AAO prescribed dose of HCQ based on RBW with and without maximum daily dose restriction have a very high HCQ blood levels compared to non-obese patients, with a potential increased risk of ocular toxicity. The use of 2016-AAO dose of HCQ according to the ideal body weight for this group of patients should be considered.Clinicaltrials.gov #NCT0312243.

Phytosterols and tocopherols are commonly used in food and pharmaceutical industries for their health benefits. Current analysis methods rely on conventional liquid chromatography, using an analytical column, which can be tedious and time consuming. However, simple, and fast analytical methods can facilitate their qualitative and quantitative analysis. In this study, a fast chromatography-tandem mass spectrometric (FC-MS/MS) method was developed and validated for the quantitative analysis of phytosterols and tocopherols. Omitting chromatography by employing flow injection analysis-mass spectrometry (FIA-MS) failed in the quantification of target analytes due to analyte-to-analyte interferences from phytosterols. These interferences arise from their ambiguous MS fingerprints that would lead to false identification and inaccurate quantification. Therefore, a C18 guard column with a 1.9 µm particle size was employed for FC-MS/MS under isocratic elution using acetonitrile/methanol (99:1 v / v ) at a flow rate of 600 µL/min. Analyte-to-analyte interferences were identified and eliminated. The false peaks could then be easily identified due to chromatographic separation. In addition, two internal standards were evaluated, namely cholestanol and deuterated cholesterol. Both internal standards contributed to the observed analyte-to-analyte interferences; however, adequate shift in the retention time for deuterated cholesterol eliminated its interferences and allowed for an accurate quantification. The method is fast (1.3 min) compared to published methods and can distinguish false peaks observed in FIA-MS. Seven analytes were quantified simultaneously, namely brassicasterol, campesterol, stigmasterol, β-sitosterol, α-tocopherol, δ-tocopherol, and γ-tocopherol. The method was successfully applied in the quantitative analysis of phytosterols and tocopherols present in the unsaponifiable matter of canola oil deodorizer distillate (CODD). β-sitosterol and γ-tocopherol were the most abundant phytosterols and tocopherols, respectively.

We report on the design and performance of in-house built column cartridges that can be directly screwed into the ports of a commercial rotor-stator valve to minimize extra-column band broadening and pressure-drop losses when pursuing ultra-fast separations such as those needed in 2D and 3D-LC separations. Two basic designs were evaluated and were compared with the results obtained with a commercial screw-in column cartridge. The system produces an extra-column band broadening as low as 0.05 to 0.1 μL 2 for the employed UV-detector set-up. Despite these very low values, the obtained separation efficiency of the in-house fabricated cartridge columns was very low, corresponding to a reduced minimal plate height around h=7 at the very best, which, for the 1.7 μm particle and 26.4 mm long columns corresponds to a number of theoretical plates of N=2200 under isocratic conditions. A similar poor performance was obtained with a commercial column cartridge with similar dimensions using the same set-up. One possible explanation of the observed performance could be found in the inner diameter of the column cartridges (i.d. =0.75 mm and 1 mm) which, for the employed sub 2-μm particles, falls into a region of column diameters that, according to literature models, is most likely to suffer from inherent packing problems., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

Rapid optimization of gradient liquid chromatographic (LC) separations often utilizes analyte retention modelling to predict retention times as function of eluent composition. However, due to the dwell volume and technical imperfections, the actual gradient may deviate from the set gradient in a fashion unique to the employed instrument. This makes accurate retention modelling for gradient LC challenging, in particular when very fast separations are pursued. Although gradient deformation has been addressed in method-transfer situations, it is rarely taken into account when reporting analyte retention parameters obtained from gradient LC data, hampering the comparison of data from various sources. In this study, a response-function-based algorithm was developed to determine analyte retention parameters corrected for geometry-induced deformations by specific LC instruments. Out of a number of mathematical distributions investigated as response-functions, the so-called "stable function" was found to describe the formed gradient most accurately. The four parameters describing the model resemble the statistical moments of the distribution and are related to chromatographic parameters, such as dwell volume and flow rate. The instrument-specific response function can then be used to predict the actual shape of any other gradient programmed on that instrument. To incorporate the predicted gradient in the retention modelling of the analytes, the model was extended to facilitate an unlimited number of linear gradient steps to solve the equations numerically. The significance and impact of distinct gradient deformation for fast gradients was demonstrated using three different LC instruments. As a proof of principle, the algorithm and retention parameters obtained on a specific instrument were used to predict the retention times on different instruments. The relative error in the predicted retention times went down from an average of 9.8% and 12.2% on the two other instruments when using only a dwell-volume correction to 2.1% and 6.5%, respectively, when using the proposed algorithm. The corrected retention parameters are less dependent on geometry-induced instrument effects., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Virotherapy, enabled by recent advances in the transdisciplinary field of biotechnology, has emerged as a powerful tool for use in anticancer treatment, gene therapy, immunotherapy, etc. Examining the effects of viruses and virus-derived immune-modulating therapeutics is of great fundamental and clinical interest. Here we describe a sample preparation protocol for metabolite extraction from virus-infected tissue, in addition to liquid chromatography-mass spectrometry conditions essential for subsequent analysis. This metabolomics approach delivers highly sensitive and specific metabolite information on various biospecimens. Such an approach may be adopted to monitor biological changes in over 30 relevant metabolic pathways in response to viral infection and also viral therapeutics.

The present study proposes a ready-to-use analytical expression to calculate the mobile zone mass transfer contribution (h Cm ) in packed bed columns. For this purpose, first high-accuracy computations of the band broadening in a perfectly ordered sphere array (fcc-arrangement, external porosity ε=0.40) were made using computational fluid dynamics (CFD), covering a broad range of zone retention factors (2≤k''≤18) and reduced velocities (0≤ν i ≤48). Subsequently, these data were used to determine the value of the geometrical constants in a number of possible analytical expressions for the h Cm -contribution. This fitting exercise showed the traditional literature approach, using the Wilson-Geankoplis correlation to calculate the dimensionless Sherwood (Sh) number for the mass transfer, leads to fitting errors on the h Cm -term as large as 150%. Instead, a new correlation for Sh is established. In addition, we also explored the difference in fitting accuracy between h Cm -expressions based on either a plug-flow or a laminar flow profile assumption. Surprisingly, no significant difference in fitting accuracy between both assumptions was observed. Finally, a best-fit analytical expression is proposed that can represent the CFD-computed band broadening data with an average absolute fitting error of Δh=0.005, corresponding to a relative error of 2.5% on the h Cm -term and of only 0.3% on the total plate height in a perfectly ordered sphere packing. Defining the presently investigated fcc-ordered sphere array with external porosity=40% as the reference geometry for a perfect sphere packing, the established expression can be used as a new yardstick expression against which the degree of eddy-dispersion can be measured., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Dual-channel chromatography was evaluated for pesticide residue analysis in fruits and vegetables and for unknown compounds detection. A dual-channel system was tested coupled to triple quadrupole and high-resolution mass spectrometry. The first part of the investigation was related to the improvement of the sample throughput with a 100 mm column. The dual-channel system provided the same analytical results as the single-channel system, however, with the throughput higher of about 70% (80 injections vs 137 injections in 24 h). Two types of calibration (in-channel and cross-channel) were checked. In the article, also solvent consumption is discussed. Six proficiency test samples were analysed to assess the quality of the results. Nor false positives neither false negatives were found. Calculated z-scores were typically <1. In the second part, a different approach was investigated. The 100 mm column was replaced by a 150 mm column keeping shorter run times than single channel system and 100 mm. The longer column improved the sensitivity and selectivity what was demonstrated in the target pesticide residue analysis. Additionally, the 150 mm column was compared with the 100 mm column in the analysis of unknown natural matrix compounds by high resolution mass spectrometry. The longer column allowed to detect up to 26% unknown compounds more than the shorter column., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

Rationale: Electrospray ionization mass spectrometry (ESI-MS) in conjunction with liquid chromatography (LC) can provide accurate quantitative data, but it is not well-suited for the rapid screening (RS) of analytes incurred into complex matrices. This study was designed to determine the usefulness of ESI for rapid detection and quantitation of veterinary drugs from complex biological matrices under near real-time conditions., Methods: Nine veterinary drugs or metabolites, clenbuterol, erythromycin, flunixin, 5-hydroxyflunixin, meloxicam, ractopamine, salbutamol, tylosin and zilpaterol, present in cow urine, sheep urine, sheep tissues (kidney, muscle, liver and lung) or pig kidney, were simultaneously analyzed. A simple sample clean-up procedure, which included dilution with 10% sodium carbonate followed by extraction with ethyl acetate, was used. For tissues, an additional pre-extraction with hexane was performed to remove fat prior to MS analysis. Samples were introduced into the mass spectrometer through the LC autosampler, but no chromatographic separation was employed. A Sciex 5600+ triple time-of-flight mass spectrometer with a dual-spray source interfaced with a Shimadzu Nexera LC system was used. Samples were analyzed in positive ion mode., Results: Sample extraction times were typically 10-30 min or less and instrumental analysis time was 1 min/sample. Regression coefficients of matrix-matched standard curves across all compounds ranged from 0.9701-0.9999 in urine (cow and sheep) and tissues (sheep kidney, liver, lung, muscle and pig kidney). Limits of detection ranged from 0.11 to 2.03 ng/mL across analytes in urine and 0.11 to 8.86 ng/g across tissues. Correlations between RS-ESI-MS and LC/MS/MS results were 0.956 to 0.998 for incurred residues of flunixin in cow urine, ractopamine in pig kidney and zilpaterol in sheep urine., Conclusions: RS-ESI-MS provided rapid, sensitive, and accurate analyses of nine veterinary drugs from complex matrices with very little sample preparation and produced quantitative data akin to LC/MS/MS., (Published 2020. This article is a U.S. Government work and is in the public domain in the USA.)

Protein glycosylation analysis is challenging due to the structural variety of complex conjugates. However, chromatographically separating glycans attached to tryptic peptides enables their site-specific characterization. For this purpose, we have shown the importance of selecting a suitable hydrophilic interaction liquid chromatography (HILIC) stationary phase in the separation of glycopeptides and their isomers. Three different HILIC stationary phases, i.e., HALO ® penta-HILIC, Glycan ethylene bridged hybrid (BEH) Amide, and ZIC-HILIC, were compared in the separation of complex N -glycopeptides of hemopexin and Immunoglobulin G glycoproteins. The retention time increased with the polarity of the glycans attached to the same peptide backbone in all HILIC columns tested in this study, except for the ZIC-HILIC column when adding sialic acid to the glycan moiety, which caused electrostatic repulsion with the negatively charged sulfobetaine functional group, thereby decreasing retention. The HALO ® penta-HILIC column provided the best separation results, and the ZIC-HILIC column the worst. Moreover, we showed the potential of these HILIC columns for the isomeric separation of fucosylated and sialylated glycoforms. Therefore, HILIC is a useful tool for the comprehensive characterization of glycoproteins and their isomers.

Columns of different structures have different potential kinetic performance - the trade-off between separation, time, and pressure. However, the full potential of a structure cannot always be realized in practically existing columns. Each combination of column efficiency, time, and pressure requires certain cross-sectional dimensions of the column flow-through channels. However, there are limits to the narrowest flow-through channels that can be manufactured with current technology. As a result, columns of some structures cannot be optimized for providing the required efficiency in the shortest time. Additionally, the full potential of its structure can be realized only if a column can operate at the highest pressure available from liquid chromatography (LC) equipment, has sufficient loadability, and satisfies other practical requirements. Equations tailored for a systematic approach to evaluation of factors affecting performance of optimized LC columns (effects of column structure, column dimensions, operational conditions, etc.) were developed. Parameters quantifying the performance of a specific column at and below its largest acceptable pressure were identified. New objective performance parameters of columns and their structures were introduced. Among them are the apparent structural quality factor accounting for the effect of insufficiently high pressure acceptable for the column, the dimensionless plate duration - the parameter of a column structure affecting its performance when the pressure is not limited, - and others. Applying the theory developed herein to published data, the performance of several differently structured columns is evaluated, and the factors affecting their comparative performance are discussed. In the final count, not the quality of a column structure, but practical factors such as the narrowest manufacturable flow-through channels can dominate the choice of the kinetically most suitable column for a practical LC analysis., Competing Interests: Declaration of Competing Interest I declare that I have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this manuscript., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Over the past several years, hyphenation of native (nondenaturing) liquid chromatography (nLC) methods, such as size exclusion chromatography (SEC), ion exchange chromatography (IEX), and hydrophobic interaction chromatography (HIC) with mass spectrometry (MS) have become increasingly popular to study the size, charge, and structural heterogeneity of protein drug products. Despite the availability of a wide variety of nLC-MS methods, an integrated platform that can accommodate different applications is still lacking. In this study, we described the development of a versatile, sensitive, and robust nLC-MS platform that can support various nLC-MS applications. In particular, the developed platform can tolerate a wide range of LC flow rates and high salt concentrations, which are critical for accommodating different nLC methods. In addition, a dopant-modified desolvation gas can be readily applied on this platform to achieve online charge-reduction native MS, which improves the characterization of both heterogeneous and labile biomolecules. Finally, we demonstrated that this nLC-MS platform is highly sensitive and robust and can be routinely applied in protein drug characterization.

This study ascertained the viability of a portable liquid chromatograph, operating at low microliter per minute flow, for the analysis of seized drugs at remote sites as well as in laboratory settings. Synthetic cathinones were screened using dual capillary columns in series, C8 and biphenyl, with on-column ultraviolet detection at 255 and 275 nm. The relative retention times of the two columns in series and their peak area absorbance ratio were used to determine if the 16 synthetic cathinones investigated could be uniquely identified in these conditions. Based on these parameters all of the analytes could be distinguished. Representative mixtures of synthetic cathinones were used to determine the repeatability, linearity, and limits of detection of the method. This cost effective and green instrumentation has the potential to satisfy minimum international guidelines for the analysis of seized drugs., (© 2020 Wiley-VCH GmbH.)

Miniaturized LC has evolved at an exponential rate over the last 50 years. In the past decade, it has received considerable attention in the field of bioanalytical separation science and technology due to the need to measure different classes of biomolecules present in a variety of matrixes on a global scale to gain a deeper understanding of complex biological processes. This field has become a dominant area underpinning the molecular omics research (e.g., proteomics, metabolomics, lipidomics, and foodomics), allowing key insights into the function and mechanism of small to very large biomolecules on a molecular level. This Feature highlights the recent advances in molecular omics focusing on miniaturized LC technology combined with mass spectrometry-based platforms, with a particular emphasis on the strategies adopted and applications using new and sensitive nanoscale analytical methodologies.

We report on a systematic and comprehensive (0.7 ≤ k'' ≤ 122) experimental study of the effect of the zone retention factor k'' on eddy dispersion (h eddy ) in packed bed columns for liquid chromatography. The values for h eddy are obtained by subtracting rigorously estimated contributions to the total plate height from longitudinal diffusion (h B ) and the mobile (h Cm ) and stationary zone (h Cs ) mass transfer resistances. For the first time, h Cm -values are calculated using an expression for the Sherwood number (Sh) that has been established and validated in the relevant velocity range. Experiments were carried out on both a fully-porous and a core-shell particle column. In both cases, the eddy dispersion systematically decreased with increasing retention factor k'', dropping 0.5 to 0.8 reduced plate height units when going from the lowest to the highest k''. To establish a simple empirical fitting equation that can represent the observed effects, the widely used power law-based Knox model has been extended to express the dependence of its A- and n-parameters on the retention factor., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

Two-dimensional liquid chromatography is increasingly being used to address challenging separations in fields ranging from pharmaceutical analysis to the food industry. A significant impediment to development of more methods is the lack of a complete theoretical foundation upon which sound development decisions can be made. One parameter that is currently not fully understood is the extent of filling of sampling loops in the case where effluent from the first dimension separation is transferred to the second dimension separation through this type of open loop interface. This is a highly important parameter because it is connected to several other variables in a 2D-LC system, including the first dimension flow rate, the sampling (modulation) time, and the loop volume. In this study we have used both numerical simulation methods and experimental measurements to understand the extent to which sampling loops can be filled before a significant fraction of the analyte is lost from the end of the loop. Variables included in the study are the analyte diffusion coefficient (D mol ), loop filling rate (F fill ), loop inner diameter or radius (R loop ) and loop volume (V loop ). For a straight loop capillary we find that analyte breakthrough curves (as measured at the loop outlet) depend only on a single the dimensionless parameter t * =V loop F fill ·D mol R loop 2 . As a function of this parameter, the fraction of analyte lost from the loop outlet for different extents of loop filling could be calculated, allowing to develop guidelines for the maximum permissible extent of filling before a specified level of analyte loss is reached. Breakthrough measurements using a coiled loop capillary show that less breakthrough is observed compared to the straight capillary at high filling flow rates, presumably due to secondary flows that increase radial dispersion. These measurements enabled the calculation of apparent radial diffusion coefficients for use with coiled capillaries such that the same relation for t* can be used to predict analyte loss due to breakthrough. These results should be very useful to practitioners of 2D-LC, enabling them to make rational decisions about the extent of loop filling on the basis of experimental conditions and analyte type., (Copyright © 2020. Published by Elsevier B.V.)

The fabrication of effective microchip liquid chromatography (LC) systems tends to be limited by the availability of suitable chromatographic columns. Herein, we developed a glass microchip LC system in which porous single-particle silica was adopted as frits and a freeze-thaw valve was utilized to achieve sample injection without interfering with sampling. The fabrication of single-particle-frit-based packed columns did not require an additional packing channel, thus avoiding dead volumes within the channel interface that can influence chromatographic separation. Further, the length of the packed column could be adjusted using the location of single-particle frits within the column channel. The fabricated frits exhibited high mechanical strength, good permeability, and tolerance for high pressures during chromatographic separation. In particular, the developed microchip LC system was able to withstand high separation pressures of more than 5000 psi. The microchip LC system was applied to the separation of neurotransmitters. Three different monoamine neurotransmitters were completely separated within 5 min with theoretical plate numbers on the order of 100,000 plates m -1 . The microchip LC system has a potential for application in a variety of fields including environmental analysis, food safety, drug analysis, and biomedicine., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A comprehensive linear gradient solvent system for centrifugal partition chromatography (CPC) was developed for the bioassay-guided isolation of natural compounds. The gradient solvent system consisted of three different ternary biphasic solvents types: n -hexane-acetonitrile-water (10:2:8, v / v ), ethyl acetate-acetonitrile-water (10:2:8, v / v ), and water-saturated n -butanol-acetonitrile-water (10:2:8, v / v ). The lower phase of the n -hexane-acetonitrile-water (10:2:8, v / v ) was used as the stationary phase, while its upper phase, as well as ethyl acetate-acetonitrile-water (10:2:8), and water-saturated n -butanol-acetonitrile-water (10:2:8, v / v ) were pumped to generate a linear gradient elution, increasing the mobile phase polarity. We used the gradient CPC to identify antioxidant response elements (AREs), inducing compounds from Centipeda minima , using an ARE-luciferase assay in HepG2 cells, which led to the purification of the active molecules 3-methoxyquercetin and brevilin A. The developed CPC solvent systems allow the separation and isolation of compounds with a wide polarity range, allowing active molecule identification in the complex crude extract of natural products.

The added value of dried blood spot (DBS) samples complementing the information obtained from commonly routine doping control matrices is continuously increasing in sports drug testing. In this project, a robotic-assisted non-destructive hematocrit measurement from dried blood spots by near-infrared spectroscopy followed by a fully automated sample preparation including strong cation exchange solid-phase extraction and evaporation enabled the detection of 46 lower molecular mass (< 2 kDa) peptide and non-peptide drugs and drug candidates by means of LC-HRMS. The target analytes included, amongst others, agonists of the gonadotropin-releasing hormone receptor, the ghrelin receptor, the human growth hormone receptor, and the antidiuretic hormone receptor. Furthermore, several glycine derivatives of growth hormone-releasing peptides (GHRPs), arguably designed to undermine current anti-doping testing approaches, were implemented to the presented detection method. The initial testing assay was validated according to the World Anti-Doping Agency guidelines with estimated LODs between 0.5 and 20 ng/mL. As a proof of concept, authentic post-administration specimens containing GHRP-2 and GHRP-6 were successfully analyzed. Furthermore, DBS obtained from a sampling device operating with microneedles for blood collection from the upper arm were analyzed and the matrix was cross-validated for selected parameters. The introduction of the hematocrit measurement method can be of great value for doping analysis as it allows for quantitative DBS applications by managing the well-recognized "hematocrit effect." Graphical abstract.

Over the past years, a great effort has been devoted to the development of new sorbents that can be used to pack or to coat extractive capillaries for in-tube solid-phase microextraction (IT-SPME). Many of those efforts have been focused on the preparation of capillaries for miniaturized liquid chromatography (LC) due to the reduced availability of capillary columns with appropriate dimensions for this kind of system. Moreover, many of the extractive capillaries that have been used for IT-SPME so far are segments of open columns from the gas chromatography (GC) field, but the phase nature and dimensions are very limited. In particular, polar compounds barely interact with stationary GC phases. Capillary GC columns may also be unsuitable when highly selective extractions are needed. In this work, we provide an overview of the extractive capillaries that have been specifically developed for capillary LC (capLC) and nano LC (nanoLC) to enhance the overall performance of the IT-SPME, the chromatographic separation, and the detection. Different monolithic polymers, such as silica C 18 and C 8 polymers, molecularly imprinted polymers (MIPs), polymers functionalized with antibodies, and polymers reinforced with different types of carbon nanotubes, metal, and metal oxide nanoparticles (including magnetic nanoparticles), and restricted access materials (RAMs) will be presented and critically discussed., Competing Interests: The authors declare no conflict of interest.

We report on an alternative experimental method to determine the transversal or radial dispersion coefficient (D rad ) in packed bed columns for liquid chromatography. The method uses a recently developed type of column end-fitting with an impermeable segmentation ring that splits the incoming flow in a central and peripheral part. Using this device and continuously sending a tracer-laden flow through the central inlet and a tracer-less flow through the peripheral inlet, a steady-state radial dispersion pattern is established which can be used to determine the degree of radial dispersion. The present part of the study lays the theoretical foundation for the method and shows the parameter sensitivity of the different possible data analysis variants. In addition, computational fluid dynamics simulations have been used to validate the established procedure (agreement between true and determined D rad -value better than 0.4%) as well as to study the effect of the most important error sources: the presence of the annular flow segmentation ring and the almost inevitable occurrence of mismatches between the central and the peripheral in- and outlet flows. In the latter case, a combined read-out method can be proposed that nearly perfectly compensates for the flow mismatch error (remaining error on the D rad -value smaller than 3%)., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

Enantiomeric separation of six β-adrenergic blockers was systematically studied for the first time on a polysaccharide-based chiral stationary phase, i.e. Chiralpak IB, under the normal-phase mode. The effect of alcohol modifiers, alcohol content and basic additive on enantiomeric separation was evaluated and optimized. Under the optimal conditions, the enantiomers of atenolol, bevantolol, cartelol, esmolol, metoprolol and propranolol were all baseline resolved with resolutions of 1.50, 8.56, 2.05, 2.11, 3.56 and 4.02, respectively. Additionally, molecular docking was tested to explain chiral recognition mechanisms of this set of the drug enantiomers on Chiralpak IB. The details of the various interactions affecting enantiomeric separation were confirmed from the molecular level and the modeling data were in agreement with the chromatographic results concerning the enantioselectivity., (© 2020 John Wiley & Sons, Ltd.)

Flow injection chromatography (FIC) or sequential injection chromatography (SIC) is a low-pressure liquid chromatography technique that uses flow injection or sequential injection hardware. Due to the constraints of this hardware, the separation resolution is low; often no more than 3-5 components are resolved. We have recently demonstrated the excellent resolving power of narrow open tubular (OT) columns for various biomolecules, and only moderate elution pressures are needed to carry out these separations. In this paper, we incorporate a narrow OT column with FIC and construct an FIC system using a pressure chamber and two injection valves to implement gradient elution. The resultant system not only improves the resolution but also reduces the system cost. When we use the system to separate peptides from trypsin-digested cytochrome C, we can resolve dozens of peptides (with resolutions of 0.5 or greater) at a speed of 12 samples per hour. When we use this system to separate a mixture containing 3 amino acids, we can base-line resolve these compounds at a speed of 1800 sample per hour., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Aim: A novel automated serial dried blood spot (DBS) sampler, 'Fluispotter', was tested for its sampling performance. Materials & methods: An LC-MS/MS method was developed for the analysis of cortisol in DBS samples serially spotted by Fluispotter. The cortisol concentrations in 148 paired DBS and plasma samples were compared across a hematocrit (HCT) range of 22-55%. Results: The interassay accuracy and precision were <10%. Overall assay bias was negligible across the HCTs tested when analyzing the whole-spot DBS samples. The accuracy and precision of the blood volume in 10 μl DBS samples spotted by Fluispotters and micropipettes were within 3%. Deming regression and Bland-Altman analysis showed a good agreement of DBS-predicted and measured plasma cortisol. Conclusion: The Fluispotter performed serial sampling with high accuracy and precision of the sample blood volume.

A newly developed portable capillary liquid chromatograph was investigated for the separation of various pharmaceutical and illicit drug compounds. The system consists of two high-pressure syringe pumps capable of delivering capillary-scale flow rates at pressures up to 10 000 psi. Capillary liquid chromatography columns packed with sub-2 μm particles are housed in cartridges that can be inserted into the system and easily connected through high-pressure fluidic contact points by simply applying a specific, predetermined torque rather than using standard fittings and less precise sealing protocols. Several over-the-counter analgesic drug separations are demonstrated, along with a simple online measurement of tablet dissolution. Twenty illicit drug compounds were also separated across six targeted drug panels. The results described in this study demonstrate the capability of this compact liquid chromatography instrument to address several important drug-related applications while simplifying system operation, and greatly reducing solvent usage and waste generation essential for onsite analysis., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

This protocol describes a simple single-step column purification (SSCP) of rAAV2 by gravity flow based on its affinity to heparin, without ultracentrifugation., (© 2020 Cold Spring Harbor Laboratory Press.)

Tartary buckwheat shell is an important by-product of Tartary buckwheat production. Previous studies shown that Tartary buckwheat shells are rich in flavonoids, which are responsible for their antioxidant properties. Due to lack of advanced separation technologies, the purification for Tartary buckwheat shell is still in the laboratory scale, and could not realize the industrialization production. According to the results of static adsorption experiment, AB-8 resin was selected for Tartary buckwheat shell flavonoids (TBSF) adsorption. The adsorption isotherm, resin adsorption thermodynamic and dynamic adsorption parameters were studied. And the adsorption of AB-8 resin for TBSF was determined as an endothermic process. Results of preparative chromatography experiment showed that TBSF could be efficiently purified by AB-8 resin. And the optimal parameters were: feed concentration 25 mg/mL, desorption flow rate 2.5 mL/min. Under these conditions, the TBSF were separated effectively. Results of liquid chromatography-mass spectrometer (LC-MS) indicated that there were seven kinds of flavonoids in Tartary buckwheat shell, which were mainly from the 40 and 60% of ethanol elution. Simulated moving bed (SMB) was applied for TBSF purification the first time in this study. The optimal conditions of SMB were as following: adsorption zone flow rate 7.0 mL/min, contaminant removal zone flow rate 17.9 mL/min, product elution zone flow rate 22.3 mL/min, regeneration zone flow rate 21.5 mL/min, water washing zone flow rate 27.5 mL/min, switching time 1260 S, and the purity and yield of TBSF was 90 ± 0.22% and 85 ± 0.28%, respectively. The IC50 values of α-glucosidase inhibition activities and DPPH scavenging activity of the purified TBSF were 57.09 ± 0.15 and 7.92 ± 0.23 μg/mL, respectively. The constituents of TBSF showed higher α-glucosidase inhibition activities and antioxidant than raw TBSF and rutin. The results suggest that SMB is a proper method for industrial production of TBSF, and SMB could be applied for other natural products purification., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

A facile strategy for highly efficient fabrication of chiral monolithic column was carried out by the simple "one-pot" hydroxymethyl polycondensation of native β-cyclodextrin (β-CD) and urea-formaldehyde (UF). In-situ rapid substitution of native β-CD and efficient polycondensation of β-CD products and UF oligomers was proposed and fulfilled in one pot. The feasibility, mechanism and key parameters of polycondensation reaction were discussed. The data on crosslinking polycondensation and reaction kinetics were further evaluated. Characteristics including the morphologies, permeability and structure stability of the resultant monolithic columns were also studied. Under the optimal conditions, the "one-pot" hydroxymethyl polycondensation was accomplished in an aqueous solution within only 10 mins. Satisfactory stability and repeatability were gained, and good enantioseparation of eight model enantiomers was successfully achieved with the resultant β-CD-based monolith. It was simple and highly efficient, and the organic solvents, special CD derivatives and tediously long-time polymerization reaction were successfully avoided, which might light a new access to rapid preparation of CD-chiral monolith for high-performance enantioseparation., Competing Interests: Declaration of Competing Interest The authors declare no Conflict of Interest., (Copyright © 2019. Published by Elsevier B.V.)

The analysis of complex mixtures of closely related species is quickly becoming a bottleneck in the development of new drug substances, reflecting the ever-increasing complexity of both fundamental biology and the therapeutics used to treat disease. Two-dimensional liquid chromatography (2D-LC) is emerging as a powerful tool to achieve substantial improvements in peak capacity and selectivity. However, 2D-LC suffers from several limitations, including the lack of automated multicolumn setups capable of combining multiple columns in both dimensions. Herein, we report an investigation into the development and implementation of a customized online comprehensive multicolumn 2D-LC-DAD-MS setup for screening and method development purposes, as well as analysis of multicomponent biopharmaceutical mixtures. In this study, excellent chromatographic performance in terms of selectivity, peak shape, and reproducibility were achieved by combining reversed-phase (RP), strong cation exchange (SCX), strong anion exchange (SAX), and size exclusion chromatography (SEC) using sub-2-μm columns in the first dimension in conjunction with several 3.0 mm × 50 mm RP columns packed with sub-3-μm fully porous particles in the second dimension. Multiple combinations of separation modes coupled to UV and MS detection are applied to the LC × LC analysis of a protein standard mixture, intended to be representative of protein drug substances. The results reported in this study demonstrate that our automated online multicolumn 2D-LC-DAD-MS workflow can be a powerful tool for comprehensive chromatographic column screening that enables the semi-automated development of 2D-LC methods, offering the ability to streamline full visualization of sample composition for an unknown complex mixture while maximizing chromatographic orthogonality. Graphical Abstract.

The preparation of a highly efficient chiral liquid chromatography (LC) column is explored by dynamically coating a reversed-phase porous silicon pillar array column with hydroxypropyl-β-cyclodextrin (Hp-β-CD) as the chiral selector. Analyte mixtures composed of non-steroidal anti-inflammatory drugs were tested to reveal the enantioseparation potential of the column. The mechanism of chiral discrimination was investigated. The adsorbed Hp-β-CDs on the column surface experience different interaction with enantiomers. The chiral stationary phase showed satisfying stability and could be easily restored by recovering the selector with sufficient flushing and repeating the loading procedure. The peak capacity of the column was evaluated, and it was found high enough to separate three enantiomer couples using a one-dimensional LC technique., Competing Interests: Declaration of Competing Interest Wim De Malsche is co-founder of PharmaFluidics and has shares of the company., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Differences in the characteristics of seventeen commercial C-30 liquid chromatographic columns were studied for the separation of carotenoid isomers. A mixture consisting of nine xanthophyll and hydrocarbon carotenoids were separated under conditions carefully chosen to reveal changes in selectivity. The influence of the mobile phase composition, column temperature, and mobile phase flow rate were evaluated. Shape selectivity was characterized with Standard Reference Material (SRM) 869b Column Selectivity Test Mixture, for correlation with carotenoid retention behavior. Regular changes were observed across a broad spectrum of shape selectivity characteristics as indicated by SRM 869b. Better separations of carotenoid isomers were achieved with C-30 columns than were possible with C-18 columns, even after optimization of separation conditions., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

A robust, portable and miniature battery powered gradient capillary liquid chromatograph (total weight ∼2.7 kg, without battery ∼2.0 kg), with integrated microfluidic injection, column heating and high sensitivity low-UV absorbance detection is presented. The portable capillary chromatograph, was applied with a packed reversed-phase capillary column (100 mm × 300 μm I.D., 5 μm ODS), housed within an integrated capillary column heater controlled by a proportional-integral-derivative (PID) chip module. The system delivered retention time and peak area relative standard deviation in isocratic mode of <0.7% (n = 10) and <3.3% (n = 10), respectively, and <0.1% (n = 10) and <2.3% (n = 10) respectively, for gradient elution mode. Detection was based upon a 255 nm light-emitting diode (LED) using one of two commercial capillary flow-cell options, namely a high sensitivity 12 nL Agilent capillary z-cell (HSDC) and a 45 nL Thermo Fisher Scientific UZ-View™ flow cell (UZFC). The HSDC, housed within a 3D printed detector arrangement, gave an effective pathlength of 1.01 mm (84% of nominal pathlength) and stray light of only 0.2%. Limits of detection for four test small molecule pharmaceuticals ranged from 65 to 101 μg L -1 based upon a 316 nL injection volume, with separation efficiencies of between 18,000 and 29,700 N m -1 , with sub-4 min run times. The portable capillary LC system was successfully coupled to a small footprint portable mass spectrometer (Microsaic 4500 MiD) to demonstrate compatibility and 'point-of-need' miniaturised LC-MS capability., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Proteomics is a large-scale study of proteins, aiming at the description and characterization of all expressed proteins in biological systems. The expressed proteins are typically highly complex and large in abundance range. To fulfill high accuracy and sensitivity of proteome analysis, the hybrid platforms of multidimensional (MD) separations and mass spectrometry have provided the most powerful solution. Multidimensional separations provide enhanced peak capacity and reduce sample complexity, which enables mass spectrometry to analyze more proteins with high sensitivity. Although two-dimensional (2D) separations have been widely used since the early period of proteomics, three-dimensional (3D) separation was barely used by low reproducibility of separation, increased analysis time in mass spectrometry. With developments of novel microscale techniques such as nano-UPLC and improvements of mass spectrometry, the 3D separation becomes a reliable and practical selection. This review summarizes existing offline and online 3D-LC platforms developed for proteomics and their applications. In detail, setups and implementation of those systems as well as their advances are outlined. The performance of those platforms is also discussed and compared with the state-of-the-art 2D-LC. In addition, we provide some perspectives on the future developments and applications of 3D-LC in proteomics.

Liquid chromatography (LC) based techniques in combination with mass spectrometry (MS) detection have had a large impact on the development of new pharmaceuticals in the past decades. Continuous improvements in mass spectrometry and interface technologies, combined with advanced liquid chromatographic techniques for high-throughput qualitative and quantitative analysis, have resulted in a wider scope of applications in the pharmaceutical field. LC-MS tools are increasingly used to analyze pharmaceuticals across a variety of stages in their discovery and development. These stages include drug discovery, product characterization, metabolism studies (in vitro and in vivo) and the identification of impurities and degradation products. The increase in LC-MS applications has been enormous, with retention times and molecular weights (and related fragmentation patterns) emerging as crucial analytical features in the drug development process. The goal of this review is to give an overview of the main developments in LC-MS based techniques for the analysis of small pharmaceutical molecules in the last decade and give a perspective on future trends in LC-MS in the pharmaceutical field.