High-throughput Sample Analysis Research Articles

Comprehensive Targeted Metabolomic Assay for Urine Analysis.

Among all the human biological fluids used for disease biomarker discovery or clinical chemistry, urine stands out. It can be collected easily and noninvasively, it is readily available in large volumes, it is typically free from protein contamination, and it is chemically complex-reflecting a wide range of physiological states and functions. However, the comprehensive metabolomic analysis of urine has been somewhat less studied compared to blood. Indeed, most published metabolomic assays are specifically optimized for serum or plasma. In an effort to improve this situation, we have developed a comprehensive, quantitative MS-based assay for urine analysis. The assay robustly detects and quantifies 142 urinary metabolites including 28 amino acids and derivatives, 17 organic acids, 22 biogenic amines and derivatives, 40 acylcarnitines, 34 lipids, and glucose/hexose, among which 67 metabolites are absolutely quantified and 75 metabolites are semiquantified. All the analysis methods in this assay are based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) using both positive and negative-mode multiple reaction monitoring (MRM). The recovery rates of spiked urine samples at three different concentration levels, that is, low, medium and high, are in the range of 80% to 120% with satisfactory precision values of less than 20%. This targeted metabolomic assay has been successfully applied to the analysis of large numbers of human urine samples, with results closely matching those reported in the literature as well as those obtained from orthogonal analysis via NMR spectroscopy. Moreover, the assay was specifically developed in a 96-well plate format, which enables automated, high-throughput sample analysis. The assay has already been used to analyze more than 1800 urine samples in our laboratory since early 2019.

A Sectioning, Coring, and Image Processing Guide for High-Throughput Cortical Bone Sample Procurement and Analysis for Synchrotron Micro-CT

A Sectioning, Coring, and Image Processing Guide for High-Throughput Cortical Bone Sample Procurement and Analysis for Synchrotron Micro-CT

A Sectioning, Coring, and Image Processing Guide for High-Throughput Cortical Bone Sample Procurement and Analysis for Synchrotron Micro-CT.

Bone is a dynamic and mechanically active tissue that changes in structure over the human lifespan. The products of the bone remodeling process have been studied substantially using traditional two-dimensional techniques. Recent advancements in X-ray imaging technology via desktop micro-computed tomography (µCT) and synchrotron radiation micro-computed tomography (SRµCT) have allowed for the acquisition of high-resolution three-dimensional (3D) scans of a larger field of view (FOV) than other 3D imaging techniques (e.g., SEM) providing a more complete picture of microscopic structures within human cortical bone. The specimen should be accurately centered within the FOV, however, to limit the appearance of streak artifacts known to impact data analysis. Previous studies have reported procurement of irregularly shaped rectilinear bone blocks that result in imaging artifacts due to uneven edges or image truncation. We have applied a geological sampling protocol (coring) to procure consistently sized cortical bone core specimens for SRµCT experiments from the anterior aspect of human femora. This coring method is efficient and minimally destructive to tissue. It creates uniform cylindrical samples that decrease imaging artifacts by nature of being isometric during rotation and providing a uniform path length for X-ray beams throughout scanning. Image processing of X-ray tomographic data of cored and irregularly shaped samples confirms the potential of the technique to improve visualization and analysis of cortical bone microarchitecture. A goal of this protocol is to deliver a reliable and repeatable method for the extraction of cortical bone cores that is adaptable for various types of high-resolution bone imaging experiments. An overarching goal of the work is to create a standardized cortical bone procurement for SRµCT that is affordable, consistent, and straightforward. This procedure may further be adapted by researchers in related fields who commonly evaluate hard composite materials such as in biological anthropology, geosciences, or material sciences.

HPLC method development for fampridine using Analytical Quality by Design approach.

Offering a systematic and multivariate analysis of the analytical procedure, development and validation of HPLC methods using Quality by Design approach are in the limelight of current research trends. A new, experimental design-aided HPLC method for fampridine was developed and preliminarily validated according to current in-force international guidelines for linearity, accuracy, robustness and precision. The method offers a high throughput sample analysis, with an elution time of 2.9 minutes, and signal detection without excipient interference performed at 262 nm. The method proved to be linear between 1-15 µg mL-1 (R2= 0.9996). The mean recovery was found to be 98.7 ± 1.9 % in the tested range of 2.5-7.5 µg mL-1. Low RSD values (< 1 %) were obtained for both model, intra- and inter-day precision. The limit of detection and limit of quantification were 0.24 and 0.78 µg mL-1, resp. The method proved to be applicable for active substance assay in a pharmaceutical dosage form.

Precise determination of the molybdenum isotopic composition of urine by multiple collector inductively coupled plasma mass spectrometry.

Molybdenum (Mo) is predominantly expelled from the human body in urine. Consequently, urinary variability in the concentration and isotopic composition of Mo may encode valuable clinical information. To access this information, however, it is first necessary to develop and demonstrate a rapid, accurate and precise methodology capable of concentrating Mo from urine for isotope analysis. The utility of N-benzoyl-N-phenylhydroxylamine (BPHA) to effectively separate and purify Mo from urine samples without the need for acid digestion was tested. Following this approach, applying a double-spike mass bias correction, we determined the Mo isotopic compositions of a set of urine samples by multiple collector inductively coupled plasma mass spectrometry (MC-ICP-MS). Based on replicate analyses of an in-house urine standard, this approach demonstrates an external precision on δ98/95 Mo values of better than 0.08‰ (2SD, n = 15). Application to a sample set collected from healthy individuals in Guangzhou, China, provides the first suite of δ98/95 Mo measurements from urine samples. Samples from the female participants show δ98/95 Mo (‰) values (1.31 ± 0.19‰, Ave ± 2SD, n = 14) that are consistently lower than those from the male participants (1.55 ± 0.16‰, Ave ± 2SD, n = 17). The employed methodology is suitable for rapid, low-blank and high-throughput Mo isotope analysis of urine samples. Although resolvable δ98/95 Mo variability is seen in this preliminary dataset, the mechanism driving this variability is unknown. High-precision Mo isotopic analysis might be added to the urinalysis tool-kit, with the potential to provide valuable clinical information in the future.

A rapid method for simultaneous quantification of berberine, berbamine, magnoflorine and berberrubine in mouse serum using UPLC-MS/MS

Berberidis cortex, the dry bark of Berberis L., is used to treat diabetes and contains at least three bioactive components: berberine (BBR), berbamine (BBM) and magnoflorine (MGF). BBR in turn is metabolized into berberrubine (BRB). Although it is possible to quantify each of these components individually in serum, there are currently no methods for simultaneously quantifying all four. Here, we developed a specific and rapid method for simultaneously quantifying BBR, BBM, MGF and BRB in mouse serum using ultra high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Samples were pretreated by protein precipitation, separated using an ACQUITY UPLC® BEH C18 column and detected by a triple quadrupole mass spectrometer with electrospray ionization. The compound [9,10-(OC2H3)2]-BBR (d6-BBR) was used as internal standard for BBR and BRB, boldine (BOL) for MGF and tetrandrine (TET) for BBM. The m/z transitions for precursor/product ion pairs were 336.1/320.2 for BBR, 305.2/566.3 for BBM, 342.0/297.1 for MGF, 322.1/307.2 for BRB, 342.2/294.3 for d6-BBR, 312.2/580.3 for TET and 328.1/265.2 for BOL. We validated our method in terms of selectivity, linearity and lower limit of quantification, accuracy, precision, matrix effect and recovery, dilution integrity and stability. This method showed good linearity from 0.1 to 40 ng/mL for BBR, 8 to 3200 ng/mL for BBM, 5 to 2000 ng/mL for MGF and 0.2 to 80 ng/mL for BRB. The chromatographic run time was 3.9 min, and sample preparation took approximately 15 min per batch. Finally, we used our method to measure BBR, BBM, MGF and BRB in serum from diabetic mice after gavage administration of BBR hydrochloride, BBM hydrochloride, and MGF. This method is precise, accurate and suitable for high-throughput sample analysis.

Aspects of matrix and analyte effects in clinical pharmacokinetic sample analyses using LC-ESI/MS/MS – Two case examples

The increasing focus on high throughput sample analysis has led to the common practice of using simplest sample preparation method possible (i.e. protein precipitation) and shortest sample run-time possible. This means that there will be two aspects of compromise: the first compromise is made between sample cleanliness and sample preparation speed since protein precipitation does not provide very clean final extract; the second compromise is made between peak separation and run-time, meaning that sometimes overlap or co-elution of some peaks has to be accepted. The first compromise may lead to matrix effect, which is caused by co-eluting endogenous substances such as phospholipids. The second compromise can result in analyte effect, which is caused by co-eluting analyte(s). We have encountered the issue of matrix/analyte-mediated ion suppression in multiple preclinical and clinical pharmacokinetic projects during bioanalytical method development/validation or biological sample analysis of many small molecule drugs. As these matrix/analyte effects could occur in different situations with different “syndromes”, sometimes it can be easily overlooked, leading to unreliable result, poor sensitivity, and prolonged assay development process. To increase the awareness of this important issue, in this paper we presented two real case examples on signal suppression caused by either endogenous phospholipids or co-eluting analyte.

Microfluidic device for rapid investigation of the deformability of leukocytes in whole blood samples

The mechanical properties of cells, such as leukocytes, in a diseased state differ from those of healthy cells, typically due to their microstructure. The deformability of the cells through a constrictive area is analyzed by the applied stress to the cell. This study investigates the relationship between the sample flow speed and distribution of captured leukocytes based on the cell deformability using a microfluidic device. The device comprises of microfilters that serve as the filtration mechanism. The microfilter gap size gradually decreases from 15 to 3 µm to facilitate the deformability-based separation. Leukocytes have various sizes; hence, they can be separated by microfilters directly from whole blood samples without any cell clogging, and they do not require sample pre-processing such as centrifugation or red blood cell lysis. The distribution of leukocytes captured by the microfilters with respect to the sample flow speed can be analyzed; at higher sample flow speeds of 6 µL/min, small leukocytes with a size of 7 µm could not be captured and they passed through the smallest microfilter gap size of 3 µm. For smaller leukocytes, such as lymphocytes, the distributions are mainly at gap sizes of 4 µm to 8 µm, with most of the lymphocytes captured at the 6 µm microfilter gap size. We conclude that the distribution of the cells captured during the filtration varies depending on the microfilter gap sizes, applied sample flow speed, cell sizes, and the ability of the cells to deform. The deformability imaging profiles of the sample could be developed from the images of the cell distribution, which might be useful for preliminary screening in the clinical applications. This work presents the development of a simple device for the study of cell deformability as the results provide a biophysical marker in high throughput and bulk sample analyses.

Advancing Liquid Atmospheric Pressure Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Toward Ultrahigh-Throughput Analysis.

Label-free high-throughput screening using mass spectrometry has the potential to provide rapid large-scale sample analysis at a speed of more than one sample per second. Such speed is important for compound library, assay and future clinical screening of millions of samples within a reasonable time frame. Herein, we present a liquid atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) setup for high-throughput large-scale sample analysis (>5 samples per second) for three substance classes (peptides, antibiotics, and lipids). Liquid support matrices (LSM) were used for the analysis of standard substances as well as complex biological fluids (milk). Throughput and analytical robustness were mainly dependent on the complexity of the sample composition and the current limitations of the commercial hardware. However, the ultimate limits of liquid AP-MALDI in sample throughput can be conservatively estimated to be beyond 10–20 samples per second. This level of analytical speed is highly competitive compared with other label-free MS methods, including electrospray ionization and solid state MALDI, as well as MS methods using multiplexing by labeling, which in principle can also be used in combination with liquid AP-MALDI MS.

Design of a degenerate primer pair to target a bacterial functional community: The hppd bacterial gene coding for the enzyme targeted by herbicides, a study case

The present work aimed to design a degenerate primer pair to target a large part of the hppd soil bacterial community, possibly affected by herbicides. We validated these primers by qPCR and high-throughput sequencing analysis of soil samples.

Abnormal Liquid Chasing Effect in Paper Capillary Enables Versatile Gradient Generation on Microfluidic Paper Analytical Devices.

Microfluidic paper analytical devices (μPADs) are smart and accessible substituents to traditional counterparts in point-of-care tests (POCT), which exploited delicate control over passive fluid in microscale for rich functions. In this work, we are extending such control by introducing novel ways to generate 1D and 2D gradients on μPADs. It is achieved by using paper-capillary-based serial sampling. The paper capillary is composed of a concaved paper channel sealed with tape, with test pads properly distributed aside. In such a structure, the liquid can not only quickly and automatically flow along the capillary but also be continuously consumed by the peripheral test pads. Thus, when we do serial sampling, an abnormal liquid chasing effect can be observed, where the later liquid sample chases and surpasses the earlier parts in the paper capillary, leading to reversely ordered sample distribution compared with that in a typical glass capillary. This specialty allows for fast, ordered, and tunable sequential sampling and enables efficient generation of 1D and 2D concentration gradient with one, two, and even three components on μPADs. Besides, we verified the applicability of this technique for arrayed assays, including 1D serial dilution-based metal ion colorimetry and a 2D bacterial antibiotic susceptibility test for synergic effect evaluations, which paves the way for high-throughput sample analysis and information-rich condition screening on μPADs.

A 3D-printed modularized purification system for rapid, high-throughput MALDI-MS analysis of small-volume biological samples.

We report a modularized sample purification system (MSPS) fabricated by 3D printing for rapid and high-throughput MALDI-MS analysis of various small-volume biological samples. The MSPS presents distinct advantages such as customizability, portability, expandability, simple operation and high throughput and also keeps the flexibility of being customized with more functional modules in future applications.

Detection and Titration of Influenza A Virus Neuraminidase Inhibiting (NAI) Antibodies Using an Enzyme-Linked Lectin Assay (ELLA).

The neuraminidase (NA) of influenza A viruses (IAV) is a structurally and antigenically important envelope glycoprotein. There are eleven known subtypes of NA of which two, N1 and N2, circulate in swine. The sialidase activity of NA is required for the release of nascent virus particles from infected cell membranes and inhibition of NA enzymatic activity can significantly reduce virus titers and duration of infection. Efforts to improve IAV vaccine technology in humans have focused on the generation of neuraminidase inhibiting (NAI) antibodies and should be considered in swine as well. The enzyme-linked lectin assay (ELLA) conducted in 96-well plates has enabled high-throughput analysis of serum samples for NAI antibody titers. Through the use of reverse genetics, custom antigen panels and antisera can be generated to encompass the antigenically diverse population of NA that circulate in swine. The ELLA is a robust method to assess NAI antibody titers and characterize the antigenic difference between NA antigens.

Spray-Capillary: An Electrospray-Assisted Device for Quantitative Ultralow-Volume Sample Handling.

The analysis of low-volume samples provides valuable insight into complex biological systems. However, the proteomic and metabolomic analysis of low-volume samples remains challenging due to the lack of simple, efficient, and reproducible microsampling techniques. We have developed an electrospray-assisted device for quantitative low-volume sample extraction, referred to here as "Spray-Capillary". Stable electrospray was achieved through a chemically etched tip from a long (e.g., 50 cm) capillary with a conductive sheath flow. This electrospray provided the driving force to quantitatively draw low-volume samples into the capillary. We evaluated the precision and accuracy of sample injection volumes using our spray-capillary as the electrospray voltage, capillary ID, and column length were varied. Our results demonstrate that spray-capillary allows for reproducible and quantitative microsampling with low injection flow rates (as low as 15 pL/s). Furthermore, spray-capillary can be directly coupled with capillary zone electrophoresis (CZE) for separation. Overall, spray-capillary is a simple microsampling device that holds great potential for high-throughput quantitative omics analysis of ultralow-volume samples.

Inline cartridge extraction for rapid brain tumor tissue identification by molecular profiling

The development of perspective diagnostic techniques in medicine requires efficient high-throughput biological sample analysis methods. Here, we present an inline cartridge extraction that facilitates the screening rate of mass spectrometry shotgun lipidomic analysis of tissue samples. We illustrate the method by its application to tumor tissue identification in neurosurgery. In perspective, this high-performance method provides new possibilities for the investigation of cancer pathogenesis and metabolic disorders.

Rapid-melt DNP for multidimensional and heteronuclear high-field NMR experiments

Low sensitivity is the main limitation of NMR for efficient chemical analysis of mass-limited samples. Hyperpolarization techniques such as Dynamic Nuclear Polarization (DNP) have greatly improved the efficiency of NMR experiments. In this manuscript, we demonstrate a 400 MHz rapid-melt DNP setup. With this setup it is possible to perform liquid-state NMR experiments with solid-state DNP enhancement at high magnetic field. Sample volumes of 100 nL in fused-silica capillaries are detected using a stripline microcoil. Due to the small heat capacity of these samples it is possible to melt them with relatively low relaxation losses. With this 400 MHz setup, proton enhancements of up to −175 have been obtained in the liquid-state. The probe is double tuned, so it can be used for heteronuclear DNP-NMR and since the sample composition does not change during the experiment, it is possible to perform signal averaging and multidimensional experiments. This type of rapid-melt DNP setup thus allows for most types of liquid-state NMR experiments to be combined with efficient solid-state DNP. This makes rapid-melt DNP an interesting method for high-throughput chemical analysis of mass-limited samples.

High-Throughput and Direct Sample Screening Using a Laser Spray Ionization Miniature Mass Spectrometer

Over the past two decades, great efforts have been made toward mass spectrometer instrument miniaturization. With increasing analytical performances, miniature mass spectrometers are on the edge of being applied to more application scenarios. Besides sensitivity, mass resolution, and instrument portability, high-throughput and little or no sample preparation are also critical features in practical applications. In this study, we report the development of a miniature mass spectrometry (MS) system equipped with a 2D moving platform and a laser spray ionization (LSI) source. The method to make a patterned sample holder was also introduced and optimized for automatic high-throughput sample analyses. With the LSI source, analytes in complex matrix could be directly mass analyzed; in addition to the 2D moving platform, different samples could be analyzed in a high-throughput fashion. Results show that good linearity of quantitation could be achieved for multiple samples. Tens of nanograms of drugs, peptides, and vitamin B could be identified in diluted whole blood samples, and it takes 10 s on average to scan one sample.

Automatic Analyte-Ion Recognition and Background Removal for Ambient Mass-Spectrometric Data Based on Cross-Correlation.

Ambient mass spectrometry is a powerful approach for rapid, high-throughput, and direct sample analysis. Due to the open-air desorption and ionization processes, random fluctuations of ambient conditions can lead to large variances in mass-spectral signals over time. The mass-spectral data also can be further complicated due to multiple analytes present in the sample, background-ion signals stemming from the desorption/ionization source itself, and other laboratory-specific conditions (e.g., ambient laboratory air, nearby hardware). Thus, background removal and analyte-ion recognition can be quite difficult, particularly in non-targeted analyses. Here, we demonstrate the use of a cross-correlation-based approach to exploit chemical information encoded in the time domain to group/categorize mass-spectral peaks from a single analysis dataset. Ions that originate from ambient (or other) background species were readily flagged and removed from spectra; the result was a decrease in mass-spectral complexity by over 70% due to the removal of these background ions. Meanwhile, analyte ions were differentiated and categorized based on their time-domain profiles. With sufficient mass resolving-power and mass-spectral acquisition rate, isolated mass spectra containing ions from the same species in a sample could be extracted, leading to a reduction in mass-spectral complexity by more than 98% in some cases. The cross-correlation approach was tested with different ionization sources as well as reproducible and irreproducible sample introduction. Software built in-house enabled fully automated data processing, which can be performed within a few seconds. Ultimately, this approach provides an additional dimension of analyte separation in ambient mass-spectrometric analyses with information that is already recorded throughout the analysis.

Development of a rapid viability polymerase chain reaction method for detection of Yersinia pestis

Due to the occurrence of natural plague outbreaks and its historical usage as a biological weapon, Yersinia pestis is considered one of the high-priority biological threat agents. It can remain viable in certain environments including water for >100 days. Because of its slow-growth characteristic, it usually takes three or more days to detect and confirm the identity of viable Y. pestis cells by PCR, serological, or biochemical assays when using the traditional microbiological plate-culture-based analysis, and that too, assuming faster growing microbes present in a water sample do not mask the Y. pestis colonies and interfere with analysis. Therefore, a rapid-viability Polymerase Chain Reaction (RV-PCR) method was developed for detection of Y. pestis. The RV-PCR method combines 24 h-incubation broth culture in a 48-well plate, and pre- and post-incubation differential PCR analyses, thereby allowing for rapid and high-throughput sample analysis compared with the current plate culture method. One chromosomal and two plasmid gene target-based real-time PCR assays were down-selected, showing ca. 10 genome equivalent detection; the chromosomal assay was then used for RV-PCR method development. A 101-cell level (10–99 cells) sensitivity of detection was demonstrated even with complex sample backgrounds including known PCR inhibitors (ferrous sulfate and humic acid), as well as metal oxides and microbes present in Arizona Test Dust (ATD). The method sensitivity was maintained in the presence of dead Y. pestis cells up to 104 cells per sample. While affording high-throughput and rapid sample analysis, the 48-well plate format used in this method for sample enrichment significantly reduced labor requirements and generation of BioSafety Level-3 (BSL-3) laboratory waste as compared to the usual microbiological plate-culture-based methods. This method may serve as a model for other vegetative bacterial pathogens.

High sensitivity methods to quantify chloroquine and its metabolite in human blood samples using LC-MS/MS.

Aim:Chloroquine is an antimalarial drug used in the treatment of Plasmodium vivax malaria. Three methods to quantify chloroquine and its metabolite in blood matrices were developed and validated.Methodology & results:Different high-throughput extraction techniques were used to recover the drugs from whole blood (50 μl), plasma (100 μl) and dried blood spots (15 μl as punched discs) followed by quantification with LC–MS/MS. The intra- and inter-batch precisions were below 15%, and thus meet regulatory acceptance criteria.Conclusion:The developed methods demonstrated satisfactory validation performance with high sensitivity and selectivity. The assays used simple and easy to automate extraction techniques. All methods were reliable with robust performance and demonstrated to be suitable to implement into high-throughput routine analysis of clinical pharmacokinetic samples.