On-line Sample Preparation Research Articles

Cannabidiol Determination on Peripheral Capillary Blood Using a Microsampling Method and Ultra-High-Performance Liquid Chromatography Tandem Mass Spectrometry with On-Line Sample Preparation.

The aim of this work is to evaluate volumetric absorptive microsampling (VAMS) from capillary blood as an alternative strategy for therapeutic drug monitoring (TDM) in patients treated with the newly available GW-purified form of cannabidiol (Epidiolex®). A fast ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) coupled to an online sample preparation system analysis was carried out on a Thermo Scientific Ultimate 3000 LC system coupled to a TSQ Quantiva triple quadrupole for the quantification of cannabidiol (CBD) and, in addition, delta-9-tetrahydrocannabinol (Δ9-THC). After validation using European Medicine Agency (EMA) guidelines the method was applied to samples obtained by finger prick of five pediatric patients treated with Epidiolex® and the results were compared to those obtained from venous blood and plasma. The method is linear in the range of 1–800 µg/L for both CBD and THC with intra- and inter-day precisions ranging from 5% to 14% and accuracies from −13% to +14% starting from 30 µL of sample. Stability in VAMS is ensured for up to 4 weeks at 25 °C thus allowing simple delivery. There was no difference (p = 0.69) between concentrations of CBD measured from VAMS sampled from capillary or venous blood (range: 52.19–330.14 or 72.15–383.45 µg/L) and those obtained from plasma (range: 64.3–374.09 µg/L) The VAMS-LC-MS/MS method represents a valid alternative strategy for therapeutic drug monitoring of patients treated with Epidiolex®.

On-line sample preparation for multiclass vitamin, hormone, and mycotoxin determination in chicken egg yolk using LC-MS/MS

Hen egg yolk from chicken eggs were examined for their mycotoxin, hormone, and fat-soluble vitamin content. A method was developed for multi-class analysis of egg yolk using a dilute/precipitate, centrifuge, and shoot process coupled with on-line sample clean-up using restricted access media with LC-MS/MS. Matrix effects were evaluated and a standard addition protocol with internal standards was chosen for analyte quantitation. With some minor exceptions, the method displayed good linearity (R2 > 0.99), with appropriate limits of detection (0.05–10 ng/g) and limits of quantitation (0.15–30 ng/g) for the analytes tested. Overall, it was discovered there was some variation between the different types of eggs tested in these experiments, especially with regard to their fat-soluble vitamin contents. Low-level mycotoxins were detected in most of the eggs tested. The challenges associated with developing a multi-class compound determination from a single analytical run were elucidated.

Consolidation of vitamin A and E methods onto a multiplexing liquid chromatography tandem mass spectrometry platform simplifies laboratory workflow

BackgroundVitamin A and E are routinely monitored to assess nutritional status. The most commonly used approach for their measurement involves laborious liquid-liquid extraction followed by high-performance liquid chromatography (HPLC) analysis on dedicated instrumentation. We describe a simple, rapid protocol for measurement of vitamin A and E and their integration into an existing online sample preparation liquid chromatography tandem mass spectrometry (SPLC-MS/MS) workflow. MethodsWe performed a method comparison between the SPLC-MS/MS and HPLC methods for vitamin A and E by measuring patient specimens across the concentration range 11–81 µg/dL for vitamin A and 1–18 mg/L for vitamin E. The analysis times on each platform were also compared. ResultsSPLC-MS/MS and HPLC methods were comparable with regards to analytical performance; mean bias across the measured range was 2.54% (95% CL: −11.56–16.64%) for vitamin A and −2.04% (95% CL: −18.20–14.12%) for vitamin E. Total analysis times were 7 min and 15 min for SPLC-MS/MS and HPLC respectively. ConclusionsThe development of a simplified sample preparation protocol and the use of multiplexing SPLC-MS/MS have reduced sample analysis times for vitamin A and E. This method has also optimized clinical workflow through consolidation of previously independent benches.

Improving Sensitivity and Throughput for Bioanalytical Measurements with Mass Spectrometry using Microfluidics and Online Sample Preparation

Improving Sensitivity and Throughput for Bioanalytical Measurements with Mass Spectrometry using Microfluidics and Online Sample Preparation

Investigating Monoliths (Vinyl Azlactone-co-Ethylene Dimethacrylate) as a Support for Enzymes and Drugs, for Proteomics and Drug-Target Studies.

Prior to mass spectrometry, on-line sample preparation can be beneficial to reduce manual steps, increase speed, and enable analysis of limited sample amounts. For example, bottom-up proteomics sample preparation and analysis can be accelerated by digesting proteins to peptides in an on-line enzyme reactor. We here focus on low-backpressure 100 μm inner diameter (ID) × 160 mm, 180 μm ID × 110 mm or 250 μm ID × 140 mm vinyl azlactone-co-ethylene dimethacrylate [poly(VDM-co-EDMA)] monoliths as supports for immobilizing of additional molecules (i.e., proteases or drugs), as the monolith was expected to have few unspecific interactions. For on-line protein digestion, monolith supports immobilized with trypsin enzyme were found to be suited, featuring the expected characteristics of the material, i.e., low backpressure and low carry-over. Serving as a functionalized sample loop, the monolith units were very simple to connect on-line with liquid chromatography. However, for on-line target deconvolution, the monolithic support immobilized with a Wnt pathway inhibitor was associated with numerous secondary interactions when exploring the possibility of selectively trapping target proteins by drug-target interactions. Our initial observations suggest that (poly(VDM-co-EDMA)) monoliths are promising for e.g., on-line bottom-up proteomics, but not a “fit-for-all” material. We also discuss issues related to the repeatability of monolith-preparations.

Impact of nitrate contamination on the analysis of carbon and oxygen isotopes in carbonate and a low-temperature phosphoric acid digestion approach for online sample preparation.

High-temperature phosphoric acid digestion combined with continuous-flow isotope ratio mass spectrometry (CF-IRMS) is one of the standard methods for the determination of carbon and oxygen isotopes in carbonate. However, the routine purification protocol has not been tested by samples with nitrate contamination. Here we show that the standard protocol is not capable of measuring such samples with satisfactory accuracy and precision. We tested the low-temperature (25°C) phosphoric acid digestion method combined with CF-IRMS for the measurement of natural carbonate samples with ~1% nitrate concentration and nitrate-doped carbonate standards. We also recorded the Raman spectra of the gases extracted from the nitrate-doped carbonate standards to identify the gas species that affect the results. The accuracy and precision of δ18 O values for nitrate-doped carbonate standards digested at 72°C are much poorer than routine measurements. For the measurements of doped standards reacted at 25°C, the accuracy and precision of the δ13 C and δ18 O values are indistinguishable from normal results. Raman spectra demonstrate that NO2 generated from high-temperature digestion is the possible interference species for oxygen isotope measurement. Low-temperature digestion is a reliable method for the measurement of carbon and oxygen isotopes in nitrate-contaminated carbonate samples because the formation of NO2 can be largely reduced. This approach can be applied to some nitrate-rich speleothems, evaporites and other samples with nitrate contamination.

Development of an online SPE-UHPLC-MS/MS method for the multiresidue analysis of the 17 compounds from the EU "Watch list".

During the last decades, the quality of aquatic ecosystems has been threatened by increasing levels of pollutions, caused by the discharge of man-made chemicals, both via accidental release of pollutants as well as a consequence of the constant outflow of inadequately treated wastewater effluents. For this reason, the European Union is updating its legislations with the aim of limiting the release of emerging contaminants. The Commission Implementing Decision (EU) 2015/495 published in March 2015 drafts a "Watch list" of compounds to be monitored Europe-wide. In this study, a methodology based on online solid-phase extraction (SPE) ultra-high-performance liquid chromatography coupled to a triple-quadrupole mass spectrometer (UHPLC-MS/MS) was developed for the simultaneous determination of the 17 compounds listed therein. The proposed method offers advantages over already available methods, such as versatility (all 17 compounds can be analyzed simultaneously), shorter time required for analysis, robustness, and sensitivity. The employment of online sample preparation minimized sample manipulation and reduced dramatically the sample volume needed and time required, dramatically the sample volume needed and time required, thus making the analysis fast and reliable. The method was successfully validated in surface water and influent and effluent wastewater. Limits of detection ranged from sub- to low-nanogram per liter levels, in compliance with the EU limits, with the only exception of EE2. Graphical abstract Schematic of the workflow for the analysis of the Watch list compounds.

Unattended reaction monitoring using an automated microfluidic sampler and on-line liquid chromatography

In-process sampling and analysis is an important aspect of monitoring kinetic profiles and impurity formation or rejection, both in development and during commercial manufacturing. In pharmaceutical process development, the technology of choice for a substantial portion of this analysis is high-performance liquid chromatography (HPLC). Traditionally, the sample extraction and preparation for reaction characterization have been performed manually. This can be time consuming, laborious, and impractical for long processes. Depending on the complexity of the sample preparation, there can be variability introduced by different analysts, and in some cases, the integrity of the sample can be compromised during handling. While there are commercial instruments available for on-line monitoring with HPLC, they lack capabilities in many key areas. Some do not provide integration of the sampling and analysis, while others afford limited flexibility in sample preparation. The current offerings provide a limited number of unit operations available for sample processing and no option for workflow customizability. This work describes development of a microfluidic automated program (MAP) which fully automates the sample extraction, manipulation, and on-line LC analysis. The flexible system is controlled using an intuitive Microsoft Excel based user interface. The autonomous system is capable of unattended reaction monitoring that allows flexible unit operations and workflow customization to enable complex operations and on-line sample preparation. The automated system is shown to offer advantages over manual approaches in key areas while providing consistent and reproducible in-process data.

Simultaneous determination of 12 vitamin D compounds in human serum using online sample preparation and liquid chromatography-tandem mass spectrometry

The development and validation of a method to simultaneously quantify 12 vitamin D compounds in human serum by LC–MS/MS is described. The main challenge was that of extracting and chromatographing vitamin D compounds with a range of polarities, both lipophilic and hydrophilic, in a single analytical procedure. The extractions of all 12 vitamin D compounds were achieved by an optimised protein precipitation method using acetonitrile as the precipitant, and the separation was accomplished by using a pentafluorophenyl (PFP) column. The sensitivity was increased by minimising matrix effects in MS detector rather than using a lengthy derivatisation procedure; an online solid phase extraction (SPE) using a PFP guard column was used for cleanup. Detection limits for all compounds were in the picomole range when using a 500μL sample volume. Recovery percentages ranged from 92% to 99%. LC–MS/MS resolution of all 12 vitamin D compounds, including the chromatographic separation of 25(OH)D3 from the isomer 3-epi-25(OH)D3 was achieved. Stable isotope labelled vitamin D compounds were used as internal standards for the quantification of all 12 vitamin D compounds. This is a simple yet accurate, selective, and sensitive method for the quantification of 12 major vitamin D compounds, including the sulfated forms, in human serum. The method is sufficiently robust to offer potential for use in routine analysis in a pathology laboratory setting.

The role of graphene-based sorbents in modern sample preparation techniques.

The application of graphene-based sorbents in sample preparation techniques has increased significantly since 2011. These materials have good physicochemical properties to be used as sorbent and have shown excellent results in different sample preparation techniques. Graphene and its precursor graphene oxide have been considered to be good candidates to improve the extraction and concentration of different classes of target compounds (e.g., parabens, polycyclic aromatic hydrocarbon, pyrethroids, triazines, and so on) present in complex matrices. Its applications have been employed during the analysis of different matrices (e.g., environmental, biological and food). In this review, we highlight the most important characteristics of graphene-based material, their properties, synthesis routes, and the most important applications in both off-line and on-line sample preparation techniques. The discussion of the off-line approaches includes methods derived from conventional solid-phase extraction focusing on the miniaturized magnetic and dispersive modes. The modes of microextraction techniques called stir bar sorptive extraction, solid phase microextraction, and microextraction by packed sorbent are discussed. The on-line approaches focus on the use of graphene-based material mainly in on-line solid phase extraction, its variation called in-tube solid-phase microextraction, and on-line microdialysis systems.

Two-Dimensional Capillary Electrophoresis with On-Line Sample Preparation and Cyclodextrin Separation Environment for Direct Determination of Serotonin in Human Urine.

An advanced two-dimensional capillary electrophoresis method, based on on-line combination of capillary isotachophoresis and capillary zone electrophoresis with cyclodextrin additive in background electrolyte, was developed for effective determination of serotonin in human urine. Hydrodynamically closed separation system and large bore capillaries (300–800 µm) were chosen for the possibility to enhance the sample load capacity, and, by that, to decrease limit of detection. Isotachophoresis served for the sample preseparation, defined elimination of sample matrix constituents (sample clean up), and preconcentration of the analyte. Cyclodextrin separation environment enhanced separation selectivity of capillary zone electrophoresis. In this way, serotonin could be successfully separated from the rest of the sample matrix constituents migrating in capillary zone electrophoresis step so that human urine could be directly (i.e., without any external sample preparation) injected into the analyzer. The proposed method was successfully validated, showing favorable parameters of sensitivity (limit of detection for serotonin was 2.32 ng·mL−1), linearity (regression coefficient higher than 0.99), precision (repeatability of the migration time and peak area were in the range of 0.02–1.17% and 5.25–7.88%, respectively), and recovery (ranging in the interval of 90.0–93.6%). The developed method was applied for the assay of the human urine samples obtained from healthy volunteers. The determined concentrations of serotonin in such samples were in the range of 12.4–491.2 ng·mL−1 that was in good agreement with literature data. This advanced method represents a highly effective, reliable, and low-cost alternative for the routine determination of serotonin as a biomarker in human urine.

Rugged Large Volume Injection for Sensitive Capillary LC-MS Environmental Monitoring.

A rugged and high throughput capillary column (cLC) LC-MS switching platform using large volume injection and on-line automatic filtration and filter back-flush (AFFL) solid phase extraction (SPE) for analysis of environmental water samples with minimal sample preparation is presented. Although narrow columns and on-line sample preparation are used in the platform, high ruggedness is achieved e.g., injection of 100 non-filtrated water samples did not result in a pressure rise/clogging of the SPE/capillary columns (inner diameter 300 μm). In addition, satisfactory retention time stability and chromatographic resolution were also features of the system. The potential of the platform for environmental water samples was demonstrated with various pharmaceutical products, which had detection limits (LOD) in the 0.05–12.5 ng/L range. Between-day and within-day repeatability of selected analytes were <20% RSD.

Nanofluidic device for continuous multiparameter quality assurance of biologics.

Process analytical technology (PAT) is critical for the manufacture of high-quality biologics as it enables continuous, real-time and on-line/at-line monitoring during biomanufacturing processes. The conventional analytical tools currently used have many restrictions to realizing the PAT of current and future biomanufacturing. Here we describe a nanofluidic device for the continuous monitoring of biologics' purity and bioactivity with high sensitivity, resolution and speed. Periodic and angled nanofilter arrays served as the molecular sieve structures to conduct a continuous size-based analysis of biologics. A multiparameter quality monitoring of three separate commercial biologic samples within 50 minutes has been demonstrated, with 20 µl of sample consumption, inclusive of dead volume in the reservoirs. Additionally, a proof-of-concept prototype system, which integrates an on-line sample-preparation system and the nanofluidic device, was demonstrated for at-line monitoring. Thus, the system is ideal for on-site monitoring, and the real-time quality assurance of biologics throughout the biomanufacturing processes.

Rhamnolipid biosurfactant analysis using online turbulent flow chromatography-liquid chromatography-tandem mass spectrometry

Rhamnolipids are biosurfactants produced by a variety of bacterial species that present a promising alternative to surfactants from petrochemical or oleochemical origin. The success of the fermentation is evaluated by subsequent qualitative and quantitative analysis. However, the sample preparation for high numbers of samples is often laborious and inefficient. In this study an online sample preparation is developed for the qualitative and quantitative analysis of rhamnolipids by LC–MS/MS. Online sample preparation is carried out on a TurboFlow Cyclone MAX column using turbulent flow chromatography. Sample preparation prior the analysis is minimized to a dilution and syringe filtration step leading to an instrumental analysis time of 33min. The limit of detection and the limit of quantification were 0.4ng and 0.6ng on column, respectively. Recovery of the main mono- and di-rhamnolipids from a fermentation sample was 102–104%. Additionally, the rhamnolipid biosynthetic precursors 3-hydroxy(alkanoyloxy)alkanoic acids (HAAs) are covered, albeit extraction is not quantitative (85–90%). The analysis of rhamnolipids from four different microbial species was in good agreement with previous reports. The presented method allows rapid and comprehensive analysis of rhamnolipids with minimal sample preparation directly from the fermentation broth. The application of complementary data-dependent MS/MS acquisition enables non-target screening of rhamnolipids.

Determination of Drugs by Online Column-Switching Liquid Chromatography

A fully automated and validated 2D online solid-phase extraction (SPE) high-performance liquid chromatographic method was described for the direct simultaneous determination of clenbuterol (CLEN) and doxazosin (DOX) in spiked human plasma samples. Chromatographic separation of the analytes was achieved by column-switching. SPE column packed with restricted access material for depleting matrix and a reversed phase HPLC column for eluting analytes were used with UV detection on 250 nm. A 10 mM acetate buffer (pH: 4) and acetonitrile mixture was used in gradient elution with 1 mL/min flow rate, as mass spectrometry friendly mobile phase. Calibration graphs were prepared for spiked plasma, and good linearity was achieved over a dynamic range of 75.0 ng/mL-200.0 µg/mL for CLEN and 37.5 ng/mL-100.0 µg/mL for DOX. Detection and quantification limits were 8.47 and 11.94 ng/mL, 25.66 and 36.18 ng/mL for CLEN and DOX, respectively. Online column-switching liquid chromatography technique combines the sample preparation by online SPE and separation steps in one allowing direct determination of drugs in biological fluids. This article could be assessed as a good example of combination of online sample preparation and separation, thus simultaneous determination of drugs in plasma samples.

Determination of Linezolid in Human Plasma Using Turbulent Flow Online Extractionand Tandem Mass Spectrometry

A rapid and sensitive HTLC-ESI-MS/MS method was developed for the determination of Linezolid in human plasma using an internal standard (cetirizine hydrochloride) with an advanced online sample preparation. This HTLC technique reduces the time required for sample cleanup since sample extraction and analysis are performed. A 10μl of prepared sample is directly injected into the HTLC-MS/MS system where analyte was retained on the extraction column (Cyclone P 50 × 0.5mm, 50μm) and washed away the waste with the help of extraction solvent. Then the analyte was eluted from the extraction column and transferred to the analytical column (Zorbex XDB C18 50 × 2.1mm, 5μm) using mobile phase of the mixture of 0.5% formic acid, 10mM ammonium formate and acetonitrile. The eluted analyte was then detected on mass spectrometer with ESI ion source and a positive selective reaction monitoring mode (SRM). The SRM transitions were m/z 383.20 → 337.20 for Linezolid and m/z 389.10 → 201.01 for internal standard. The developed method was validated as per USFDA guidelines. The method was linear over the concentration range of 0.409 – 20.310 ng/ml. The within batch and between batch accuracy for the three concentrations (LQC, MQC and HQC) were ranged from 98 -110.6% and 98 .6 – 108.3% respectively. The % RSD for all the QC samples was ranged from 3.0 – 8.1%. The percentage recovery of linezolid in HQC (16ng/ml), MQC (13ng/ml) and LQC (1,1ng/ml) was 60.3, 73 and 86.45% respectively. Stability studies were also performed and the results were within the acceptance range. This method was applied to the measurement of linezolid in human plasma and pharmacokinetic study.

Nearly 1000 Protein Identifications from 50 ng of Xenopus laevis Zygote Homogenate Using Online Sample Preparation on a Strong Cation Exchange Monolith Based Microreactor Coupled with Capillary Zone Electrophoresis.

A sulfonate-silica hybrid strong cation exchange monolith microreactor was synthesized and coupled to a linear polyacrylamide coated capillary for online sample preparation and capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) bottom-up proteomic analysis. The protein sample was loaded onto the microreactor in an acidic buffer. After online reduction, alkylation, and digestion with trypsin, the digests were eluted with 200 mM ammonium bicarbonate at pH 8.2 for CZE-MS/MS analysis using 1 M acetic acid as the background electrolyte. This combination of basic elution and acidic background electrolytes results in both sample stacking and formation of a dynamic pH junction. 369 protein groups and 1274 peptides were identified from 50 ng of Xenopus laevis zygote homogenate, which is comparable with an offline sample preparation method, but the time required for sample preparation was decreased from over 24 h to less than 40 min. Dramatically improved performance was produced by coupling the reactor to a longer separation capillary (∼100 cm) and a Q Exactive HF mass spectrometer. 975 protein groups and 3749 peptides were identified from 50 ng of Xenopus protein using the online sample preparation method.

Quantitative determination of opioids in whole blood using fully automated dried blood spot desorption coupled to on‐line SPE‐LC‐MS/MS

Opioids are well known, widely used painkillers. Increased stability of opioids in the dried blood spot (DBS) matrix compared to blood/plasma has been described. Other benefits provided by DBS techniques include point-of-care collection, less invasive micro sampling, more economical shipment, and convenient storage. Current methodology for analysis of micro whole blood samples for opioids is limited to the classical DBS workflow, including tedious manual punching of the DBS cards followed by extraction and liquid chromatography-tandem mass spectrometry (LC-MS/MS) bioanalysis. The goal of this study was to develop and validate a fully automated on-line sample preparation procedure for the analysis of DBS micro samples relevant to the detection of opioids in finger prick blood. To this end, automated flow-through elution of DBS cards was followed by on-line solid-phase extraction (SPE) and analysis by LC-MS/MS. Selective, sensitive, accurate, and reproducible quantitation of five representative opioids in human blood at sub-therapeutic, therapeutic, and toxic levels was achieved. The range of reliable response (R(2) ≥0.997) was 1 to 500 ng/mL whole blood for morphine, codeine, oxycodone, hydrocodone; and 0.1 to 50 ng/mL for fentanyl. Inter-day, intra-day, and matrix inter-lot accuracy and precision was less than 15% (even at lower limits of quantitation (LLOQ) level). The method was successfully used to measure hydrocodone and its major metabolite norhydrocodone in incurred human samples. Our data support the enormous potential of DBS sampling and automated analysis for monitoring opioids as well as other pharmaceuticals in both anti-doping and pain management regimens.

Online sample preparation integrated with LC/MS–MS for routine use in a clinical laboratory

Online sample preparation integrated with LC/MS–MS for routine use in a clinical laboratory

Generic on-line solid phase extraction sample preparation strategies for the analysis of drugs in biological matrices by LC–MS/MS

In the present work we investigate the integration of a single hardware platform (Prospekt-2) allowing on-line SPE with pre-/post-trapping dilution and direct injection of plasma extracts, and also compare the benefits and challenges of the different approaches for pharmaceutical drugs with heterogeneous physicochemical properties. In the first part, the generic use of on-line SPE with direct plasma injection or after protein precipitation was investigated for the quantitative analysis of talinolol. In the second part, pre-trapping and post-trapping dilution for on-line SPE is discussed for generic method development on an oxadiazole and its major metabolite. Finally, the difference of performance between direct plasma injection vs. off-line liquid–liquid extraction is also described for the quantification of buprenorphine and naltrexone down to 50 and 100pg/ml using a 0.25ml plasma aliquot. All assays were in human plasma and detection was performed by mass spectrometry detection either on simple or triple stage quadrupoles. Regardless of the tested strategy, assays were found linear, with precision and accuracy with <15% for all quality controls samples and <20% for lower limit of quantitation.