Simultaneous LC-MS determination of glucose regulatory peptides secreted by stem cell-derived islet organoids.

For studying stem cell-derived islet organoids (SC-islets) in an organ-on-chip (OoC) platform, we have developed a reversed-phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS) method allowing for simultaneous determination of insulin, somatostatin-14, and glucagon, with improved matrix robustness compared to earlier methodology. Combining phenyl/hexyl-C18 separations using 2.1 mm inner diameter LC columns and triple quadrupole mass spectrometry, identification and quantification were secured with negligible variance in retention time and quantifier/qualifier ratios, negligible levels of carryover (<2%), and sufficient precision (±10% RSD) and accuracy (±15% relative error) with and without use of an internal standard. The obtained lower limits of quantification were 0.2 µg/L for human insulin, 0.1 µg/L for somatostatin-14, and 0.05 µg/L for glucagon. The here-developed RPLC-MS/MS method showed that the SC-islets have an insulin response dependent on glucose concentration, and the SC-islets produce and release somatostatin-14 and glucagon. The RPLC-MS/MS method for these peptide hormones was compatible with an unfiltered offline sample collection from SC-islets cultivated on a pumpless, recirculating OoC (rOoC) platform. The SC-islets background secretion of insulin was not significantly different on the rOoC device compared to a standard cell culture well-plate. Taken together, RPLC-MS/MS method is well suited for multi-hormone measurements of SC-islets on an OoC platform.

"Organ-in-a-Column" Coupled On-line with Liquid Chromatography-Mass Spectrometry.

Organoids, i.e., laboratory-grown organ models developed from stem cells, are emerging tools for studying organ physiology, disease modeling, and drug development. On-line analysis of organoids with mass spectrometry would provide analytical versatility and automation. To achieve these features with robust hardware, we have loaded liquid chromatography column housings with induced pluripotent stem cell (iPSC) derived liver organoids and coupled the "organ-in-a-column" units on-line with liquid chromatography-mass spectrometry (LC-MS). Liver organoids were coloaded with glass beads to achieve an even distribution of organoids throughout the column while preventing clogging. The liver organoids were interrogated "on column" with heroin, followed by on-line monitoring of the drug's phase 1 metabolism. Enzymatic metabolism of heroin produced in the "organ-in-a-column" units was detected and monitored using a triple quadrupole MS instrument, serving as a proof-of-concept for on-line coupling of liver organoids and mass spectrometry. Taken together, the technology allows direct integration of liver organoids with LC-MS, allowing selective and automated tracking of drug metabolism over time.

Electromembrane Extraction and Mass Spectrometry for Liver Organoid Drug Metabolism Studies.

Liver organoids are emerging tools for precision drug development and toxicity screening. We demonstrate that electromembrane extraction (EME) based on electrophoresis across an oil membrane is suited for segregating selected organoid-derived drug metabolites prior to mass spectrometry (MS)-based measurements. EME allowed drugs and drug metabolites to be separated from cell medium components (albumin, etc.) that could interfere with subsequent measurements. Multiwell EME (parallel-EME) holding 100 µL solutions allowed for simple and repeatable monitoring of heroin phase I metabolism kinetics. Organoid parallel-EME extracts were compatible with ultrahigh-performance liquid chromatography (UHPLC) used to separate the analytes prior to detection. Taken together, liver organoids are well-matched with EME followed by MS-based measurements.

Nuclear Magnetic Resonance Spectroscopy to Identify Metabolite Biomarkers of Nonresponsiveness to Targeted Therapy in Glioblastoma Tumor Stem Cells.

Glioblastoma is the most common and malignant brain tumor, and current therapies confer only modest survival benefits. A major obstacle is our ability to monitor treatment effect on tumors. Current imaging modalities are ambiguous, and repeated biopsies are not encouraged. To scout for markers of treatment response, we used NMR spectroscopy to study the effects of a survivin inhibitor on the metabolome of primary glioblastoma cancer stem cells. Applying high resolution NMR spectroscopy (1H resonance frequency: 800.03 MHz) to just 3 million cells per sample, we achieved sensitive and high resolving determinations of, e.g., amino acids, nucleosides, and constituents of the citric acid cycle. For control samples that were cultured, prepared, and measured at varying dates, peak area relative standard deviations were 15-20%. Analyses of unfractionated lysates were performed for straightforward compound identification with COLMAR and HMDB databases. Principal component analysis revealed that citrate levels were clearly upregulated in nonresponsive cells, while lactate levels substantially decreased following treatment for both responsive and nonresponsive cells. Hence, lactate and citrate may be potential markers of successful drug uptake and poor response to survivin inhibitors, respectively. Our metabolomics approach provided alternative biomarker candidates compared to spectrometry-based proteomics, underlining benefits of complementary methodologies. These initial findings make a foundation for exploring in vivo MR spectroscopy (MRS) of brain tumors, as citrate and lactate are MRS-visible. In sum, NMR metabolomics is a tool for addressing glioblastoma.

Nano liquid chromatography columns.

Nano liquid chromatography (nanoLC), with columns having an inner diameter (ID) of ≤100 µm, can provide enhanced sensitivity and enable analysis of limited samples. NanoLC has become an established tool in omics research, and is gaining ground in other applications as well. There are several variants and formats of nanoLC columns, including packed columns, monoliths, open tubular columns, and the pillar array format. Most applications are done with packed columns, while e.g. the monolith and open tubular columns are still less established as routine tools. The pillar array format is a new variant with excellent resolution and low backpressure, and has recently been commercialized and used for bio-applications. In this minireview, we summarize and discuss recent research on nanoLC column development and uses, focusing on literature between 2016 and medio 2019.

Selective Fishing for Peptides with Antibody-Immobilized Acrylate Monoliths, Coupled Online with NanoLC-MS.

An online microfluidics-mass spectrometry platform was developed for determining proteotypic peptides from in-solution digested samples. Accelerated and selective sample cleanup was achieved by integrating proteotypic epitope peptide immunoextraction with nano liquid chromatography-tandem mass spectrometry (online IE-nanoLC-MS/MS). Ten individually prepared 180 µm inner diameter capillaries with ethylene glycol dimethacrylate- co-vinyl azlactone (EDMA- co-VDM) monoliths were immobilized with anti-protein antibodies that are used in routine immunoassays of the intact small cell lung cancer biomarker ProGRP. The resulting AB columns provided linearity correlation coefficients of 0.96-0.99 for protein amounts and concentrations of 10 pg to 5 ng and 0.5-250 ng/mL, respectively. The columns/platform gave relative peak area RSDs below 15%. The IE-nanoLC-MS/MS platform provided a limit of detection (LOD) of 520 pg/mL of ProGRP in human serum. The approach was applicable for other matrixes and proteins, i.e., primary glioblastoma cells and endogenous αV integrin chain. Thus, EDMA- co-VDM monoliths immobilized with antibodies are suited for automated peptide capture in microfluidic formats.

Multichannel Open Tubular Enzyme Reactor Online Coupled with Mass Spectrometry for Detecting Ricin.

For counterterrorism purposes, a selective nano liquid chromatography-mass spectrometry (nanoLC-MS) platform was developed for detecting the highly lethal protein ricin from castor bean extract. Manual sample preparation steps were omitted by implementing a trypsin/Lys-C enzyme-immobilized multichannel reactor (MCR) consisting of 126 channels (8 µm inner diameter in all channels) that performed online digestion of proteins (5 min reaction time, instead of 4-16 h in previous in-solution methods). Reduction and alkylation steps were not required. The MCR allowed identification of ricin by signature peptides in all targeted mode injections performed, with a complete absence of carry-over in blank injections. The MCRs (interior volume ≈ 1 µL) have very low backpressure, allowing for trivial online coupling with commercial nanoLC-MS systems. The open tubular nature of the MCRs allowed for repeatable within/between-reactor preparation and performance.

Highly automated nano-LC/MS-based approach for thousand cell-scale quantification of side chain-hydroxylated oxysterols.

Iso-octyl chain-hydroxylated oxysterols were determined in attomoles per 10,000 cells concentrations in 10,000-80,000 cultured pancreatic adenocarcinoma cells, using a sensitive, highly automated nano-LC-ESI-MS-based method. Identified oxysterols included 24S hydroxycholesterol (24S-OHC), 25 hydroxycholesterol (25-OHC), and 27 hydroxycholesterol (27-OHC), while 20S hydroxycholesterol and 22S hydroxycholesterol were not detected. Lower mass limit of quantification was 23 fg (65 amol) for 25-OHC and 27-OHC (100 times lower than our previous method) and 54 fg (135 amol) for 24S-OHC, after derivatization into Girard T hydrazones and online sample cleanup using simplified and robust automatic filtration and filter back flushing solid phase extraction LC/MS/MS. The instrument configuration was easily installed using a commercial nano-LC/MS system. Recoveries in spiked sample were 96, 97, and 77% for 24S-OHC, 25-OHC, and 27-OHC, with within- and between-day repeatabilities of 1-21% and 2-20% relative SD, respectively. The study demonstrates the potential of nano-LC in lipidomics/sterolomics.

Quantification of nerve agent biomarkers in human serum and urine.

A novel method for rapid and sensitive quantification of the nerve agent metabolites ethyl, isopropyl, isobutyl, cyclohexyl, and pinacolyl methylphosphonic acid has been established by combining salting-out assisted liquid-liquid extraction (SALLE) and online solid phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS). The procedure allows confirmation of nerve agent exposure within 30 min from receiving a sample, with very low detection limits for the biomarkers of 0.04-0.12 ng/mL. Sample preparation by SALLE was performed in less than 10 min, with a common procedure for both serum and urine. Analyte recoveries of 70-100% were obtained using tetrahydrofuran as extraction solvent and Na2SO4 to achieve phase separation. After SALLE, selective analyte retention was obtained on a ZrO2 column by Lewis acid-base and hydrophilic interactions with acetonitrile/1% CH3COOH (82/18) as the loading mobile phase. The phosphonic acids were backflush-desorbed onto a polymeric zwitterionic column at pH 9.8 and separated by hydrophilic interaction liquid chromatography. The method was linear (R(2) ≥ 0.995) from the limits of quantification to 50 ng/mL, and the within- and between-assay repeatability at 20 ng/mL were below 5% and 10% relative standard deviation, respectively.

Separation optimization of long porous-layer open-tubular columns for nano-LC-MS of limited proteomic samples.

The single-run resolving power of current 10 µm id porous-layer open-tubular (PLOT) columns has been optimized. The columns studied had a poly(styrene-co-divinylbenzene) porous layer (~0.75 µm thickness). In contrast to many previous studies that have employed complex plumbing or compromising set-ups, SPE-PLOT-LC-MS was assembled without the use of additional hardware/noncommercial parts, additional valves or sample splitting. A comprehensive study of various flow rates, gradient times, and column length combinations was undertaken. Maximum resolution for <400 bar was achieved using a 40 nL/min flow rate, a 400 min gradient and an 8 m long column. We obtained a 2.3-fold increase in peak capacity compared to previous PLOT studies (950 versus previously obtained 400, when using peak width = 2σ definition). Our system also meets or surpasses peak capacities obtained in recent reports using nano-ultra-performance LC conditions or long silica monolith nanocolumns. Nearly 500 proteins (1958 peptides) could be identified in just one single injection of an extract corresponding to 1000 BxPC3 beta catenin (-/-) cells, and ~1200 and 2500 proteins in extracts of 10,000 and 100,000 cells, respectively, allowing detection of central members and regulators of the Wnt signaling pathway.

Determination of insulin secretion from stem cell-derived islet organoids with liquid chromatography-tandem mass spectrometry.

Organoids are laboratory-grown 3D organ models, mimicking human organs for e.g. drug development and personalized therapy. Islet organoids (typically 100-200 µm), which can be grown from the patient́s own cells, are emerging as prototypes for transplantation-based therapy of diabetes. Selective methods for quantifying insulin production from islet organoids are needed, but sensitivity and carry-over have been major bottlenecks in previous efforts. We have developed a reverse phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS) method for studying the insulin secretion of islet organoids. In contrast to our previous attempts using nano-scale LC columns, conventional 2.1 mm inner diameter LC column (combined with triple quadrupole mass spectrometry) was well suited for sensitive and selective measurements of insulin secreted from islet organoids with low microliter-scale samples. Insulin is highly prone to carry-over, so standard tubings and injector parts were replaced with shielded fused silica connectors. As samples were expected to be very limited, an extended Box-Behnken experimental design for the MS settings was conducted to maximize performance. The finale method has excellent sensitivity, accuracy and precision (limit of detection: ≤0.2 pg/µL, relative error: ≤±10%, relative standard deviation: <10%), and was well suited for measuring 20 µL amounts of Krebs buffer containing insulin secreted from islet organoids.

Automatic filtration and filter flush for robust online solid-phase extraction liquid chromatography.

Online solid-phase extraction-liquid chromatography (SPE-LC) with microbore or capillary columns was significantly improved regarding robustness, as an easily installed automatic filtration and filter flushing (AFFL) procedure was added to avoid system clogging. Specifically, an injected sample is passed through a union containing a stainless steel filter prior to SPE trapping. The filter stops any particulate matter from reaching the SPE. When the SPE is subsequently connected to the LC column by column switching, a separate pump backflushes the filter-union, removing the particulate matter off the filter after each injection. This feature greatly reduced backpressure buildup over the entire system.

Organophosphates in aircraft cabin and cockpit air--method development and measurements of contaminants.

Methods for measurements and the potential for occupational exposure to organophosphates (OPs) originating from turbine and hydraulic oils among flying personnel in the aviation industry are described. Different sampling methods were applied, including active within-day methods for OPs and VOCs, newly developed passive long-term sample methods (deposition of OPs to wipe surface areas and to activated charcoal cloths), and measurements of OPs in high-efficiency particulate air (HEPA) recirculation filters (n = 6). In total, 95 and 72 within-day OP and VOC samples, respectively, have been collected during 47 flights in six different models of turbine jet engine, propeller and helicopter aircrafts (n = 40). In general, the OP air levels from the within-day samples were low. The most relevant OP in this regard originating from turbine and engine oils, tricresyl phosphate (TCP), was detected in only 4% of the samples (min-max

Liquid chromatography, a key tool for the advancement of single-cell omics analysis.

Single-cell analysis can allow for an in-depth understanding of diseases, diagnostics, and aid the development of therapeutics. However, single-cell analysis is challenging, as samples are both extremely limited in size and complex. But the concept is gaining promise, much due to novel sample preparation approaches and the ever-improving field of mass spectrometry. The mass spectrometer's output is often linked to the preceding compound separation step, typically being liquid chromatography (LC). In this review, we focus on LC's role in single-cell omics. Particle-packed nano LC columns (typically 50-100 µm inner diameter) have traditionally been the tool of choice for limited samples, and are also used for single cells. Several commercial products and systems are emerging with single cells in mind, featuring particle-packed columns or miniaturized pillar array systems. In addition, columns with inner diameters as narrow as 2 µm are being explored to maximize sensitivity. Hence, LC column down-scaling is a key focus in single-cell analysis. But narrow columns are associated with considerable technical challenges, while single cell analysis may be expected to become a "routine" service, requiring higher degrees of robustness and throughput. These challenges and expectations will increase the need and attention for the development (and even the reinvention) of alternative nano LC column formats. Therefore, monolith columns and even open tubular columns may finally find their "killer-application" in single cell analysis.

Searching for a UV-filter in the eyes of high-flying birds.

The eye lens is a unique organ as no cells can be replaced throughout life. This makes it decisive that the lens is protected against damaging UV-radiation. An ultraviolet (UV)-absorbing compound of unknown identity is present in the aqueous humor of geese (wild and domestic) and other birds flying at high altitudes. A goose aqueous humor extract, that was believed to contain the UV protective compound which was designated as "compound X", was fractionated and examined using a variety of spectroscopic techniques including LC-MS and high field one- and two dimensional-NMR methods. A series of compounds were identified but none of them appeared to be the UV protective "compound X". It may be that the level of the UV protective compound in goose aqueous humor is much less than the compounds identified in our investigation, or it may have been degraded by the isolation and chromatographic purification protocols used in our investigations.

Tailored Polymer-Based Selective Extraction of Lipid Mediators from Biological Samples.

Lipid mediators, small molecules involved in regulating inflammation and its resolution, are a class of lipids of wide interest as their levels in blood and tissues may be used to monitor health and disease states or the effect of new treatments. These molecules are present at low levels in biological samples, and an enrichment step is often needed for their detection. We describe a rapid and selective method that uses new low-cost molecularly imprinted (MIP) and non-imprinted (NIP) polymeric sorbents for the extraction of lipid mediators from plasma and tissue samples. The extraction process was carried out in solid-phase extraction (SPE) cartridges, manually packed with the sorbents. After extraction, lipid mediators were quantified by liquid chromatography-tandem mass spectrometry (LC-MSMS). Various parameters affecting the extraction efficiency were evaluated to achieve optimal recovery and to reduce non-specific interactions. Preliminary tests showed that MIPs, designed using the prostaglandin biosynthetic precursor arachidonic acid, could effectively enrich prostaglandins and structurally related molecules. However, for other lipid mediators, MIP and NIP displayed comparable recoveries. Under optimized conditions, the recoveries of synthetic standards ranged from 62% to 100%. This new extraction method was applied to the determination of the lipid mediators concentration in human plasma and mouse tissues and compared to other methods based on commercially available cartridges. In general, the methods showed comparable performances. In terms of structural specificity, our newly synthesized materials accomplished better retention of prostaglandins (PGs), hydroxydocosahexaenoic acid (HDoHE), HEPE, hydroxyeicosatetraenoic acids (HETE), hydroxyeicosatrienoic acid (HETrE), and polyunsaturated fatty acid (PUFA) compounds, while the commercially available Strata-X showed a higher recovery for dihydroxyeicosatetraenoic acid (diHETrEs). In summary, our results suggest that this new material can be successfully implemented for the extraction of lipid mediators from biological samples.

2-D hydrophilic interaction liquid chromatography-RP separation in urinary proteomics--minimizing variability through improved downstream workflow compatibility.

Optimization of every step in a bottom-up urinary proteomics approach was studied with respect to maximize the protein recovery and making the downstream steps in the workflow fully compatible without compromising on the amount of information obtained. Sample enrichment and desalting using centrifugal filtration (5 kDa cut-off) yielded protein recoveries up to 97% when 8 M urea was used. Although yielding lower recoveries (88%), addition of Tris-HCl/NaCl was considered a better choice due to good down-stream compatibility. The consecutive depletion of HSA, using an immunoaffinity column was successfully adapted for use in urine. Separation of the trypsin generated peptides in an off-line 2-D chromatographic system consisting of a hydrophilic interaction liquid chromatography column, followed by a RP chromatography column showed a high peak capacity and good repeatability in addition to a high degree of orthogonality. All operations were modified in order to keep sample handling between every step to a minimum, reducing the variability of each process. In order to test the suitability of the full method in an extensive proteomic experiment, a urine sample from a kidney-transplanted patient was analyzed (n=6). The total variability of the method was identified with RSD values ranging from 11 to 30%. Eventually, we identified a total of 1668 peptides and 438 proteins from a single urine sample despite the use of low-resolution MS/MS equipment. The optimized and "streamlined" complex method has shown potential for use in future urinary proteomic studies.

Exposure to airborne organophosphates originating from hydraulic and turbine oils among aviation technicians and loaders.

This study describes the potential for occupational exposure to organophosphates (OPs) originating from turbine and hydraulic oils, among ground personnel within the aviation industry. The OPs tri-n-butyl phosphate (TnBP), dibutyl phenyl phosphate (DBPP), triphenyl phosphate (TPP) and tricresyl phosphate (TCP) have been emphasized due to their use in such oils. Oil aerosol/vapor and total volatile organic compounds (tVOCs) in air were also determined. In total, 228 and 182 OPs and oil aerosol/vapor samples from technician and loader work tasks during work on 42 and 21 aircrafts, respectively, were collected in pairs. In general, the measured exposure levels were below the limit of quantification (LOQ) for 84%/98% (oil aerosol) and 82%/90% (TCP) of the samples collected during technician/loader work tasks. The air concentration ranges for all samples related to technician work were

Compact semi-automatic incident sampler for personal monitoring of volatile organic compounds in occupational air.

Suddenly occurring and time limited chemical exposures caused by unintended incidents might pose a threat to many workers at various work sites. Monitoring of exposure during such occasional incidents is challenging. In this study a compact, low-weight and personal semi-automatic pumped unit for sampling of organic vapor phase compounds from occupational air during sporadic and suddenly occurring incidents has been developed, providing simple activation by the worker potentially subjected to the sudden occurring exposures when a trained occupational hygienist is not available. The sampler encompasses a tube (glass or stainless steel) containing an adsorbent material in combination with a small membrane pump, where the adsorbent is capped at both ends by gas tight solenoid valves. The sampler is operated by a conventional 9 V battery which tolerates long storage time (at least one year), and is activated by pulling a pin followed by automatic operation and subsequent closing of valves, prior to shipping to a laboratory. The adjustable sampling air flow rate and the sampling time are pre-programmed with a standard setting of 200 mL min(-1) and 30 min, respectively. The average airflow in the time interval 25-30 min compared to average airflow in the interval 2-7 min was 92-95% (n = 6), while the flow rate between-assay precisions (RSD) for six different samplers on three days each were in the range 0.5-3.7%. Incident sampler recoveries of VOCs from a generated VOC atmosphere relative to a validated standard method were between 95 and 102% (+/-4-5%). The valves that seal the sampler adsorbent during storage have been shown to prevent an external VOC atmosphere (500 mg m(-3)) to enter the adsorbent tube, in addition to that the sampler adsorbent is storable for at least one month due to absence of ingress of contaminants from internal parts. The sampler was also suitable for trapping of semi-volatile organophosphates.

Mass spectrometry-based measurements of cyclic adenosine monophosphate in cells, simplified using reversed phase liquid chromatography with a polar characterized stationary phase.

3', 5' - Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger that is involved in many cellular functions and biological processes. In several cell types, cholera toxin will increase the level of cAMP, which mediates toxic effects on cells. In this context, we have developed a fast and simple method based on extraction with 5% trichloroacetic acid (TCA) and quantitation with liquid chromatography-mass tandem spectrometry (LC-MS/MS) for measuring cAMP in cells. A main feature of the LC-MS method was employing a reversed phase C18 column (2.1 mm × 50 mm, 1.6 µm particles) compatible with a 100% aqueous mobile phase, providing retention of the highly polar analyte. Isocratic separations allowed for fast subsequent injections. Negative mode electrospray ionization detection was performed with a triple quadrupole (QqQ)MS. cAMP was extracted from cell samples (~106 cells per well) and spiked with a labelled internal standard, using 200 µL of 5% TCA. The extraction solvent was fully compatible for direct injection onto the reversed phase column. After 10 min incubation, the supernatant was removed, and 10 µL of the supernatant was directly analysed by LC-MS. The method was characterized by the simplicity of the extraction, and the speed (3 min retention time of cAMP), sensitivity (250 pg/mL detection limit), and selectivity (separation from interferences e.g. isomeric compounds) of the LC-MS method, and could be used for quantitation of cAMP in the range 1-500 ng/mL cell extract.