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.publishedVersio

Determination of perfluorooctane sulfonate and perfluorooctanoic acid in human plasma by packed capillary column switching coupled to micro-ESI-TOF-MS

ABSTRACT The present work displays capillary liquid chromatographic column switching methodology tailored for fast, sensitive and selective determination of perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) in human plasma using µ-electrospray ionization time-of-flight mass spectrometric detection. 50 µL plasma samples were diluted five times and underwent protein precipitation with TCA. The supernatants were loaded onto a 320 µm I.D. x 30 mm 10 µm Kromasil C18 pre-column, providing on-line sample clean-up and analyte enrichment, prior to back-flushed elution onto a 320 µm I.D. x 150 mm 3.5 µm Kromasil C18 analytical column. Loading flow rates up to 150 µL/min in addition to the use of an acetonitrile/water gradient containing 10 mM ammonium acetate provided a total analysis time of 12.5 minutes. Ionization was performed in the negative mode and PFOA was observed as [M-H] - at m/z 413.3 and [M-CO2-H]- at m/z 369.4. PFOS was observed as [M-H] - at m/z 499.2. The method was validated over the concentration range of 5-1250 ng analyte/mL plasma, yielding linearity correlation coefficients of 0.9964 and 0.9910 for PFOA and PFOS, respectively. The within-assay (n = 6) and between-assay (n = 6) precisions were in the range 1.9 - 20 % RSD and 9.6 -11.7 % RSD, respectively, and the recoveries were measured as 26.5% and 10.2 % for PFOA and PFOS, respectively. The mass limit of detection was 125 pg for both analytes, corresponding to a PFOA and PFOS concentration limit of detection of 0.1 ng/mL diluted plasma corresponding to 0.5 ng/mL plasma. The method was applied for the determination of PFOA and PFOS in a plasma sample obtained from a blood bank, but the compounds were not found above the detection limit

Liquid chromatography-mass spectrometry platform for both small neurotransmitters and neuropeptides in blood, With Automatic and robust solid phase extraction

Neurons communicate via chemical signals called neurotransmitters (NTs). The numerous identified NTs can have very different physiochemical properties (solubility, charge, size etc.), so quantification of the various NT classes traditionally requires several analytical platforms/methodologies. We here report that a diverse range of NTs, e.g. peptides oxytocin and vasopressin, monoamines adrenaline and serotonin, and amino acid GABA, can be simultaneously identified/measured in small samples, using an analytical platform based on liquid chromatography and high-resolution mass spectrometry (LC-MS). The automated platform is cost-efficient as manual sample preparation steps and one-time-use equipment are kept to a minimum. Zwitter-ionic HILIC stationary phases were used for both on-line solid phase extraction (SPE) and liquid chromatography (capillary format, cLC). This approach enabled compounds from all NT classes to elute in small volumes producing sharp and symmetric signals, and allowing precise quantifications of small samples, demonstrated with whole blood (100 microliters per sample). An additional robustness-enhancing feature is automatic filtration/filter back-flushing (AFFL), allowing hundreds of samples to be analyzed without any parts needing replacement. The platform can be installed by simple modification of a conventional LC-MS system

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

Combining HIC, SEC, and IEX with Fluorescence Polarization for Drug Target Discovery

Fluorescence polarization (FP) is a highly regarded technique for studying drug–protein interactions, but has limited value regarding protein mixtures. As a novel approach to drug target discovery, the possibility of combining FP with liquid chromatography (LC) was explored. Nondenaturing protein LC principles such as size-exclusion chromatography (SEC), hydrophobic interaction chromatography (HIC), and ion exchange chromatography (IEX) were found to be orthogonal and compatible with FP because the mobile phases used do not negatively affect detection. For simple protein mixtures, the SEC/HIC/IEX–FP approach was able to identify tankyrase as the target of a triazole-based inhibitor of the Wnt signaling pathway, which is heavily associated with colon cancer. However, the total peak capacity of the three LC dimensions was not sufficient to resolve at cell-proteome level, calling for higher resolution of intact proteins to enable stand-alone drug target discovery with LC and FP

Open tubular lab-on-column/mass spectrometry for targeted proteomics of nanogram sample amounts

A novel open tubular nanoproteomic platform featuring accelerated on-line protein digestion and high-resolution nano liquid chromatography mass spectrometry (LC-MS) has been developed. The platform features very narrow open tubular columns, and is hence particularly suited for limited sample amounts. For enzymatic digestion of proteins, samples are passed through a 20 µm inner diameter (ID) trypsin + endoproteinase Lys-C immobilized open tubular enzyme reactor (OTER). Resulting peptides are subsequently trapped on a monolithic pre-column and transferred on-line to a 10 µm ID porous layer open tubular (PLOT) liquid chromatography LC separation column. Wnt/ß-catenein signaling pathway (Wnt-pathway) proteins of potentially diagnostic value were digested+detected in targeted-MS/MS mode in small cell samples and tumor tissues within 120 minutes. For example, a potential biomarker Axin1 was identifiable in just 10 ng of sample (protein extract of ~1,000 HCT15 colon cancer cells). In comprehensive mode, the current OTER-PLOT set-up could be used to identify approximately 1500 proteins in HCT15 cells using a relatively short digestion+detection cycle (240 minutes), outperforming previously reported on-line digestion/separation systems. The platform is fully automated utilizing common commercial instrumentation and parts, while the reactor and columns are simple to produce and have low carry-over. These initial results point to automated solutions for fast and very sensitive MS based proteomics, especially for samples of limited size

Pancreas-on-a-Chip Technology for Transplantation Applications

Abstract Purpose of Review Human pancreas-on-a-chip (PoC) technology is quickly advancing as a platform for complex in vitro modeling of islet physiology. This review summarizes the current progress and evaluates the possibility of using this technology for clinical islet transplantation. Recent Findings PoC microfluidic platforms have mainly shown proof of principle for long-term culturing of islets to study islet function in a standardized format. Advancement in microfluidic design by using imaging-compatible biomaterials and biosensor technology might provide a novel future tool for predicting islet transplantation outcome. Progress in combining islets with other tissue types gives a possibility to study diabetic interventions in a minimal equivalent in vitro environment. Summary Although the field of PoC is still in its infancy, considerable progress in the development of functional systems has brought the technology on the verge of a general applicable tool that may be used to study islet quality and to replace animal testing in the development of diabetes interventions

Versatile, sensitive liquid chromatography mass spectrometry–Implementation of 10 μm OT columns suitable for small molecules, peptides and proteins

We have designed a versatile and sensitive liquid chromatographic (LC) system, featuring a monolithic trap column and a very narrow (10 μm ID) fused silica open tubular liquid chromatography (OTLC) separation column functionalized with C(18)-groups, for separating a wide range of molecules (from small metabolites to intact proteins). Compared to today’s capillary/nanoLC approaches, our system provides significantly enhanced sensitivity (up to several orders) with matching or improved separation efficiency, and highly repeatable chromatographic performance. The chemical properties of the trap column and the analytical column were fine-tuned to obtain practical sample loading capacities (above 2 μg), an earlier bottleneck of OTLC. Using the OTLC system (combined with Orbitrap mass spectrometry), we could perform targeted metabolomics of sub-μg amounts of exosomes with 25 attogram detection limit of a breast cancer-related hydroxylated cholesterol. With the same set-up, sensitive bottom-up proteomics (targeted and untargeted) was possible, and high-resolving intact protein analysis. In contrast to state-of-the-art packed columns, our platform performs chromatography with very little dilution and is “fit-for-all”, well suited for comprehensive analysis of limited samples, and has potential as a tool for challenges in diagnostics

Distinguishing Between Cyclopropylfentanyl and Crotonylfentanyl by Methods Commonly Available in the Forensic Laboratory

Background: The opioid analgesic fentanyl and its analogues pose a major health concern due to its high potency and the increasing number of overdose deaths worldwide. The analogues of fentanyl may differ in potency, toxicity, and legal status, and it is therefore important to develop analytical methods for their correct identification. This can be challenging since many fentanyl analogues are structural isomers. Two fentanyl isomers that have been in the spotlight lately due to difficulties regarding separation and identification are cyclopropylfentanyl and crotonylfentanyl, which have been reported to display nearly identical fragmentation patterns and chromatographic behavior. Methods: Chromatographic separation of cyclopropylfentanyl and crotonylfentanyl by ultra-high-performance liquid chromatography was investigated using 3 different stationary phases (high strength silica T3, ethylsiloxane/silica hybrid C18, and Kinetex biphenyl) using gradient elution with a mobile phase consisting of 10 mM ammonium formate pH 3.1 and MeOH. Detection was performed by tandem mass spectrometry. In addition, the major metabolites of the 2 compounds formed on incubation with human liver microsomes were identified by ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry analysis. Results: Baseline separation of cyclopropylfentanyl and crotonylfentanyl was achieved on the ethylsiloxane/silica hybrid C18 column with retention times of 6.79 and 7.35 minutes, respectively. The major metabolites of the 2 analogues formed by human liver microsomes differed, with the main biotransformation being N-dealkylation and carboxylation for cyclopropylfentanyl and crotonylfentanyl, respectively. We demonstrated the usefulness of the 2 approaches by unambiguously identifying cyclopropylfentanyl, as well as its metabolites, in 2 authentic postmortem blood samples. Conclusions: In this study, we successfully demonstrated that cyclopropylfentanyl and crotonylfentanyl can be distinguished by methods commonly available in forensic laboratories