Capillary Electrophoresis Instrument Research Articles

Development of a capillary temperature control system for capillary electrophoresis instruments designed for spaceflight applications.

Capillary temperature control during capillary electrophoresis (CE) separations is key for achieving accurate and reproducible results with a broad array of potential methods. However, the difficulty of enabling typical fluid temperature control loops on portable instruments has meant that active capillary temperature control of in situ CE systems has frequently been overlooked. This work describes construction and test of a solid-state device for capillary temperature control that is suitable for inclusion with in situ instruments, including those designed for space missions. Two test articles were built, a thermal mass model (TMM) and a functional model (FM). The TMM demonstrated that temperature gradients could be limited using the proposed control scheme, and that our thermal modeling of the system can be relied on for future adaptations of physical geometries of the system. The FM demonstrated CE analytical performance while under active temperature control and that the device was compatible with the harsh thermal-vacuum environments that might be encountered during space flight.

Abstract 6423: KRAS mutation detection by Sanger sequencing using the SeqStudio Flex capillary electrophoresis instrument

Abstract The detection of mutations in the KRAS gene highly associated with cancer is one of the most widely used Sanger sequencing capillary electrophoresis (CE) applications in molecular diagnostics. In addition to their traditional role in cancer diagnostic, KRAS mutations have recently gained additional relevance as pharmacogenomics (PGx) markers for a significant number of new oncology therapeutics. KRAS mutation detection assays are used as companion diagnostics (CDx) for these cancer treatments. Analysis by Sanger Sequencing of KRAS mutations is cost effective and lends itself for use in resource limited healthcare settings. While there are currently no commercial kits for targeted sequencing of the KRAS mutation hotspots available, the online Applied Biosystems Primer Designer™ Tool (thermofisher.com) allows standardization of the PCR primer design between labs and repeat orders of the KRAS assays by using these commercially available assay ID numbers. Here we demonstrate the use of Primer Designer KRAS mutation detections assays for mutation in codon 12, 13 and 61 totaling to 8 allele variants. The data was generated on the SeqStudio Flex CE instrument using reference standard DNA as well as DNA extracted from FFPE solid tumor samples. We show accuracy of variant call (OPA 100% (64/64)) and concordance (OPA 98.3% (59/60)) in a side-by-side comparison of the SeqStudio Flex with the legacy Model 3500 Genetic Analyzer instrument using a set of commercially available (Horizon Discovery) KRAS reference DNA samples. Primary solid tumor samples are processed to Fresh Frozen Paraffin Embedded samples (FFPE) for further disease marker evaluation. These samples are stable for long term at ambient temperatures and provide the opportunity to evaluate prospective biomarkers at large scale to understand their potential utility. Discovery Life Sciences has generated a large-scale biorepository of human biospecimen, including FFPE, plasma/sera, buffy coat, and PBMCs. Archival FFPE samples from oncology indications associated with KRAS mutations, such as non-small cell lung cancer and colorectal cancer, were analyzed and identified samples with KRAS mutations, demonstrating the potential diagnostic utility of these assays. Finally, cell-free (cf) DNA from double spun plasma from relevant indications was also successfully evaluated for KRAS mutations, highlighting the potential for these assays in liquid biopsy studies. The study demonstrates the robustness of the Primer Designer KRAS mutation detection sequencing assays using reference standard DNA as well as DNA derived from FFPE DNA and cfDNA from patient derived tumor samples. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Achim Karger, Ami Patel, Kaylee Tseng, Camila Ornelas, Mary Napier, Rod Beck, Kerri Collwell, Shawn Fahl, Carole Bornarth. KRAS mutation detection by Sanger sequencing using the SeqStudio Flex capillary electrophoresis instrument [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6423.

Comparative study of the vibrating capillary nebulizer (VCN) and commercially available interfaces for on-line coupling of capillary electrophoresis with ICP-MS.

Capillary electrophoresis (CE) is a powerful and sensitive tool for speciation analysis when combined with inductively coupled plasmamass spectrometry (ICP-MS); however, the performance of this technique can be limited by the nature of pneumatic nebulizers. This study compares two commercially available pneumatic nebulizers to a newly introduced vibrating capillary nebulizer (VCN) for on-line coupling of CE with ICP-MS. The VCN is a low-cost, non-pneumatic nebulizer that is based on the design of capillary vibrating sharp-edge spray ionization. As a piezoelectrically driven nebulization source, the VCN creates an aerosol independent of gas flows and does not produce a low-pressure region at the nebulizer orifice. To compare the systems, we performed replicate analyses of sulfate in river water with each nebulizer and the same CE and ICP-MS instruments and determined the figures of merit of each setup. With the CE-VCN-ICP-MS setup, we achieved around 2-4 times lower sensitivity compared to the commercial setups. However, the VCN-based setup provided lower noise levels and better linear correlation from the analysis of calibration standards, which resulted in indistinguishable LOD and LOQ values from the in-house-built VCN-based and commercial setups for CE-ICP-MS analysis. The VCN is found to have the highest baseline stability with a standard deviation of 3500 cts s-1, corresponding to an RSD of 2.7%. High reproducibility is found with the VCN with a peak area RSD of 4.1% between 3 replicate measurements.

Multisite Verification of a Targeted CFTR Polymerase Chain Reaction/Capillary Electrophoresis Assay That Evaluates Pathogenic Variants Across Diverse Ethnic and Ancestral Groups.

Existing targeted cystic fibrosis screening assays miss important pathogenic CFTR variants in the ethnically diverse US population. To evaluate the analytic performance of a multiplex polymerase chain reaction (PCR)/capillary electrophoresis (CE) CFTR assay panel that simultaneously interrogates primary pathogenic variants of different ethnic/ancestral groups. Performance characteristic assessment and variant coverage comparison of the panel with a focus on ethnicity-specific CFTR variants were performed. Sample DNA was primarily from whole blood or cell lines. Detection of CFTR carriers was compared across several commercially available CFTR kits and recommended variant sets based on panel content. The panel interrogated 65 pathogenic CFTR variants representing 92% coverage from a recent genomic sequencing survey of the US population, including 4 variants with top 5 frequency in African or Asian populations not reflected in other targeted panels. In simulation studies, the panel represented 95% of carriers across the global population, resulting in 6.9% to 19.0% higher carrier detection rate compared with 10 targeted panels or variant sets. Precision and sensitivity/specificity were 100% concordant. Multisite sample-level genotyping accuracy was 99.2%. Across PCR and CE instruments, sample-level genotyping accuracy was 97.1%, with greater than 99% agreement for all variant-level metrics. The CFTR assay achieves 92% or higher coverage of CFTR variants in diverse populations and provides improved pan-ethnic coverage of minority subgroups of the US populace. The assay can be completed within 5 hours from DNA sample to genotype, and performance data exceed acceptance criteria for analytic metrics. This assay panel content may help address gaps in ancestry-specific CFTR genotypes while providing a streamlined procedure with rapidly generated results.

Interaction studies between human papillomavirus virus-like particles and laminin 332 by affinity capillary electrophoresis assisted by bio-layer interferometry

Human papillomavirus (HPV) interacts, in vitro, with laminin 332 (LN332), a key component of the extracellular matrix. In this study, we performed bio-layer interferometry (BLI) and affinity capillary electrophoresis (ACE) to investigate the binding properties of this interaction. Virus-like particles (VLPs), composed of the HPV16 L1 major capsid protein, were used as HPV model and LN332 as the VLPs binding partner. Using BLI, we quantitatively determined the kinetics of the interaction, via the measurement of VLP binding and release from LN332 immobilized onto the surface of aminopropylsilane biosensors. We found an averaged kon of 1.74 x 104 M−1s−1 and an averaged koff of 1.50 x 10−4 s−1. Furthermore, an ACE method was developed to study the interaction under physiological conditions, where the interactants are moving freely in solution, without any fluorescence labeling. Specifically, a constant amount of HPV16-VLPs was preincubated with increasing LN332 concentrations and then the samples were injected in the capillary electrophoresis instrument. A shift in the migration time of the HPV16-VLP/LN332 complexes, carrying an increasing number of LN332 molecules bound per VLP, was observed. The mobility of the complexes was found to decrease with increasing LN332 concentrations in the sample. It was used to quantify stability constant. From BLI and ACE approaches, we reported an apparent equilibrium dissociation constant in the nanomolar range (8.89 nM and 17.7 nM, respectively) for the complex between HPV16-VLPs and LN332.

Internal validation of the GA118-24B Genetic Analyzer, a stable capillary electrophoresis system for forensic DNA identification.

The GA118-24B Genetic Analyzer (hereafter, "GA118-24B") is an independently developed capillary electrophoresis instrument. In the present research, we designed a series of validation experiments to test its performance at detecting DNA fragments compared to the Applied Biosystems 3500 Genetic Analyzer (hereafter, "3500"). Three commercially available autosomal short tandem repeat multiplex kits were used in this validation. The results showed that GA118-24B had acceptable spectral calibration for three kits. The results of accuracy and concordance studies were also satisfactory. GA118-24B showed excellent precision, with a standard deviation of less than 0.1 bp. Sensitivity and mixture studies indicated that GA118-24B could detect low-template DNA and complex mixtures as well as the results generated by 3500 in parallel experiments. Based on the experimental results, we set specific analytical and stochastic thresholds. Besides, GA118-24B showed superiority than 3500 within certain size ranges in the resolution study. Instead of conventional commercial multiplex kits, GA118-24B performed stably on a self-developed eight-dye multiplex system, which were not performed on 3500 Genetic Analyzer. We compared our validation results with those of previous research and found our results to be convincing. Overall, we conclude that GA118-24B is a stable and reliable genetic analyzer for forensic DNA identification.

An open source 3D printed autosampler for capillary electrophoresis

BackgroundIn-house built capillary electrophoresis (CE) systems represent a significant share of laboratory instrumentation. In most of these instruments, sample injection is effected manually with low to moderate precision and requires skilled operators. Although few automated samplers have been previously developed, typically only one sample at a time can be injected. If a series of samples is to be analyzed, manual intervention is required. In the present work, we developed and constructed a fully automated, open source, CE autosampler, able to handle up to 14 different samples that can be used as a modular component of any in-house built CE instrument. ResultsAn inexpensive, 3D printed, open source, autosampler for CE was developed. The autosampler consists of two parts: an injection unit with carousel containing sample and electrolyte vials and a flushing unit, containing a miniature pressure/vacuum pump. The autosampler is operated by an Arduino Mega microcontroller and an Arduino code written in the laboratory. The injection sequence is entered through a keypad and LCD display by the user. The instrument can operate autonomously for extended periods of time. It was used for fully automated analysis and/or calibration of up to 14 samples with excellent injection repeatability reaching less than 2.7% RSD for peak areas. The sampler performance was tested with two independently built CE instruments, a CE system with contactless conductivity detection (C4D) and a CE system with laser induced fluorescence (LIF) detector. Significance and noveltyA novel, 3D printed, Arduino-based autosampler for CE was developed. The autosampler allows autonomous hydrodynamic injection of up to 14 different samples with fully programmable injection sequence, including capillary flushing and high voltage and data acquisition control. It provides the missing instrumental sampling setup for laboratory made CE instruments. It can be simply constructed based on the open-source blueprints in any laboratory and be a useful and time-saving add-on to any modular CE instrument.

Capillary electrophoresis instrumentation for determination of viscosity of deep eutectic solvents

Capillary electrophoresis instrumentation for determination of viscosity of deep eutectic solvents

Detection techniques for carbohydrates in capillary electrophoresis – a comparative study

Capillary electrophoresis methods for the separation of carbohydrates with four different detection techniques, namely direct UV, indirect UV, capacitively coupled conductivity, and laser-induced fluorescence detection, were tested and their performance was evaluated and compared in terms of linearity, limits of detection and quantitation, repeatability, recovery, analysis time, and sample treatment. The test set of analytes comprised sucrose, glucose, and fructose. The effect of using lactose as an internal standard on the individual methods was investigated, too. The results showed that laser-induced fluorescence detection is a technique of choice for applications requiring the detection of very low amounts of reducing carbohydrates. Contactless conductivity detection is favorable when detection sensitivity is not a crucial parameter but fast and reliable analysis is required. When only a UV detector is available as a standard part of capillary electrophoresis instruments, direct UV detection can be used when analysis time is not a critical parameter. For fast analysis with UV detectors, indirect UV detection is the technique of choice. Finally, to verify the applicability of the tested methods, samples of cola beverage, honey, and orange juice were analyzed and the results obtained by all four methods were compared.Graphical abstract

Fast, simple and calibration-free size characterization and quality control of extracellular vesicles using capillary Taylor dispersion analysis

This study reports the development of a Taylor Dispersion Analysis (TDA) method for the size characterization of Extracellular Vesicles (EVs), which are highly heterogeneous nanoscale cell-derived vesicles (30–1000 nm). Here, we showed that TDA, conducted in uncoated fused silica capillaries (50 µm i.d.) using a conventional Capillary Electrophoresis instrument, is able to provide absolute sizing (requiring no calibration) of bovine milk-derived EVs in a small sample volume (∼ 7 nL) and over their entire size range, even the smallest ones (< 70 nm) not accessible via other techniques that provide nanoparticle sizing in suspension. TDA size measurements were repeatable (RSD < 10%) and the average EV sizes were found in the range of 120–210 nm, in very good agreement with those measured with Nanoparticle Tracking Analysis, commonly used for EV characterization. TDA allowed quantitative estimation of EVs for concentrations ≥ 2 × 1011 EVs/mL. Furthermore, TDA was able to detect minor changes in EV size (i.e. by ∼25 nm upon interaction with specific anti-CD9 antibodies of ∼150 kDa), and to highlight the impact of extraction methods (i.e. milk pretreatment: freezing, acid precipitation or centrifugation; the type of size-exclusion chromatography column) and of fluorescent labeling (i.e. intravesicular or surface labeling) on the isolated EV population size. In parallel to EV sizing, TDA allowed to detect molecular contaminants (average sizes ∼1–13 nm) present within the sample, rendering this method a valuable tool to assess the quality and quantity of EV isolates.

Autonomous capillary electrophoresis processing and analysis of dried blood spots for high-throughput determination of uric acid

A new set-up for fully autonomous and high-throughput capillary electrophoresis (CE) analyses of dried blood spot (DBS) samples is presented. The DBS samples were prepared by collecting exactly 5 μL of capillary blood from a finger-prick onto a pre-punched DBS disc in a disposable plastic CE vial and by in-vial blood drying. The vials with the DBS samples were then loaded into a commercial CE instrument for a fully unmanned sample processing and analysis. A fused-silica capillary of the CE instrument was first used for the transfer of 100 μL of elution solvent to each vial, in-vial DBS elution, and in-vial eluate homogenization. The same capillary was also used for at-line injection, separation, and selective analysis of the resulting eluates. Novel CE sequences were tailor-programmed for consecutive processing and analyses of multiple DBSs, which facilitated a fully autonomous determination of uric acid with a throughput of 240 DBS samples per day (24 h). The presented analytical protocol (using 100 μm i. d./30 cm capillary; 30 mM 2-(N-morpholino)-ethanesulfonic acid, 30 mM l-histidine, and 30 μM cetyltrimethylammonium bromide background electrolyte solution; and UV detection at 292 nm) provided excellent precision at endogenous and spiked uric acid concentrations with RSD values of peak areas below 3.2%. Calibration curves were linear over the 33.3 − 1200 μM range (R2 better than 0.998), limits of detection and quantification in the original capillary blood were 10 and 33.3 μM, respectively, and were well below the uric acid clinical range (140–420 μM). The stability of uric acid in DBS samples stored at laboratory temperature for up to 2 months was also excellent demonstrating less than a 4.2% decrease in uric acid concentrations. The actual set-up might thus be highly attractive for clinical subjects and laboratories because it is minimally invasive and requires minimum intervention from laboratory staff.

On-line dual-stage enrichment via magneto-extraction and electrokinetic preconcentration: A new concept and instrumentation for capillary electrophoresis

This study reports on the development of a new concept of on-line dual preconcentration stages for capillary electrophoresis (CE), in which two completely different preconcentration approaches can be realized in the same capillary. In the first stage, a dynamic magneto-extraction of target analytes on circulating magnetic beads is implemented within the capillary. In the second one, electrokinetic preconcentration of eluted analytes via large volume sample stacking is carried out to focus them into a nano band, prior to CE separation of enriched analytes. To implement the dual-stage preconcentration operation, a purpose-made instrument was designed, combining electrophoretic and microfluidic modules to allow precise control of the movement of magnetic beads and analyte's flow. The potential of this new enrichment principle and its associated instrument was demonstrated for CE separation with light-emitting-diode-induced fluorescent (LEDIF) detection of target double-stranded DNA (ds-DNA). The workflow consists of purification and preconcentration of a target DNA fragment (300 bp) on negatively charged magnetic beads, followed by in-capillary elution and fluorescent labelling of the enriched DNA. Large volume sample stacking of the DNA eluent was then triggered to further preconcentrate the labelled DNA before its analysis by CE-LEDIF. An enrichment factor of 125 was achieved for the target DNA fragment. With our new approach, dual-stage sample pretreatment and CE separation can now be performed in-capillary without any mismatch of working volumes, nor any waste of pretreated samples.

A hydrodynamic injection approach for capillary electrophoresis using rotor-stator valves.

Sample injection is a critical step in a capillary electrophoresis (CE) analysis. Electrokinetic injection is the simplest approach and is often selected for implementation in portable CE instruments. However, in order to minimize the effect of sample matrix upon the results of a CE analysis, hydrodynamic injection is preferred. Although portable CE instruments with hydrodynamic injection have been reported, injection has always been performed at the grounded end of the capillary. This simplifies fluidic handling but limits coupling with electrochemical detectors and electrospray ionization-mass spectrometry (ESI-MS). We demonstrated previously that injection at the high-voltage (HV) end of the capillary could be performed using an HV-compatible rotary injection valve (fixed-volume injection). However, the mismatch between the bore sizes of the channels on the rotor-stator valve and the separation capillary caused peak tailing and undesired mixing, impairing analytical performance. In this work, we present an HV-compatible hydrodynamic injection approach that overcomes the issues associated with the fixed-volume injection approach reported previously. The performance of the CE instrument was demonstrated by analyzing a mixture of 13 amino acids by CE coupled to laser-induced fluorescence, which showed relative standard deviations for peak area and migration time below 5% and 1%, respectively, for triplicate analysis. Additionally, replicate measurements of a mixture of amino acids, peptides, nucleobases, and nucleosides by CE coupled to electrospray ionization-mass spectrometry (CE-ESI-MS) were performed to evaluate peak tailing, and results were similar to those obtained with a commercial CE-ESI-MS setup.

Portable capillary electrophoresis coupled with swab-based extraction device for cleaning validation in pharmaceutical facilities

All pharmaceutical manufacturers are required to verify that their production equipment is free from contaminants. Here, we report the capability of a fully automated portable capillary electrophoresis instrument with an integrated sample swab extraction – the Grey Scan ETD-100 – for the detection of pharmaceutical residues on surfaces of manufacturing equipment. Lidocaine was used as a model compound and could be recovered from a surface by swabbing, extracted from the swab, and analysed within 1 min. The recovery of lidocaine from a stainless-steel coupon was 81.3 %, with a LOD of 0.13 µg/swab. This fast, sensitive, and simple method implemented on a user-friendly portable CE instrument without the need for manual sample pre-treatment provides the possibility for on-site rapid determination of equipment cleanliness in the pharmaceutical industry.

Microscale Quantification of the Inhibition of Neuraminidase Using Capillary Nanogel Electrophoresis.

Neuraminidase inhibitors modulate infections that involve sialic acids, making quantitative analyses of this inhibitory effect important for selecting and designing potential therapeutics. An automated nanogel capillary electrophoresis system is developed that integrates a 5 nL enzyme inhibition reaction in line with a 5 min separation-based assay of the enzymatic product to quantify inhibition as the half maximal inhibitory concentration (IC50) and inhibitor constant (Ki). A neuraminidase enzyme from Clostridium perfringens is non-covalently immobilized in a thermally tunable nanogel positioned in the thermally controlled region of the capillary by increasing the capillary temperature to 37 °C. Aqueous inhibitor solutions are loaded into the capillary during the nanogel patterning step to surround the enzyme zone. The capillary electrophoresis separation provides a means to distinguish the de-sialylated product, enabling the use of sialyllactose which contains the trisaccharide motif observed on serine/threonine-linked (O-linked) glycans. A universal nanogel patterning scheme is developed that does not require pre-mixing of enzymes with inhibitors when an automated capillary electrophoresis instrument is used, thus reducing the consumption of enzymes and enabling adaption of the method to different inhibitors. The universal approach is successfully applied to two classical neuraminidase inhibitors with different electrophoretic mobilities. The IC50 and Ki values obtained for N-acetyl-2,3-dehydro-2-deoxyneuraminic acid (DANA) are 13 ± 3 and 5.0 ± 0.9 μM, respectively, and 28 ± 3 and 11 ± 1 μM, respectively, for Siastatin B. These values agree with literature reports and reflect the weaker inhibition anticipated for Siastatin B in comparison to DANA.

A voltage trade study for the design of capillary electrophoresis instruments for spaceflight.

Capillary electrophoresis (CE) systems have undergone extensive development for spaceflight applications. A flight-compatible high voltage power supply and the necessary voltage isolation for other energized components can be large contributors to both the volume and mass of a CE system, especially if typical high voltage levels of 25-30kV are used. Here, we took advantage of our custom CE hardware to perform a trade study for simultaneous optimization of capillary length, high voltage level, and separation time, without sacrificing method performance. A capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C4 D) method recently developed by our group to target inorganic cations and amino acids relevant to astrobiology was used as a test case. The results indicate that a 50cm long capillary with 15kV applied voltage (half of that used in the original method) can be used to achieve measurement goals while minimizing instrument size.

Selection of Optimal Conditions for the Separation of Curcuminoids by High-performance Capillary Electrophoresis (HPCE) Using Micellar Electrokinetic Chromatography (MEKC)

Introduction. Curcuminoids are natural polyphenolic colorants with a wide spectrum of pharmacological activity. Modern hybrid and classical methods are used for the determination of curcuminoids. However, the problem of the analysis of curcuminoids in the composition of plant and medicinal objects remains relevant. In this work, capillary electrophoresis (HPCE) in the variant of micellar electrokinetic chromatography with diode array detection (MEKC/DAD) was used as such a method. Aim. The purpose of this analysis is the choice of optimal conditions for the separation of curcuminoids using capillary electrophoresis in the form of micellar electrokinetic chromatography. Materials and methods. Separation of curcuminoids was carried out on an Agilent 7100 CE capillary electrophoresis instrument with a diode array detector and an Agilent Chem Station monitoring and data acquisition system. Borate buffer (20 mM, pH 9.3) with the addition of sodium dodecyl sulfate – SDS (30 mM) in ratios of 1 : 1 was used like the electrolyte. The sample was injected hydrodynamically – 50 mbar/3 sec, electrode voltage is +25 kV, quartz capillary – total capillary length/effective capillary length = 30/40 cm, ID = 50 µm, capillary temperature +20 ° С. The output of curcuminoids was monitored at the wavelength of the diode-matrix detector – λ max = 425 nm/4 nm, the refereed wavelength is 360 nm/100 nm. Results and discussion. Curcuminoids in a borate buffer solution with the addition of SDS are practically not separated, which is associated with a high activity of the electroosmotic flow, to suppress which 95 % ethyl alcohol was added. In the course of research, it was found that the addition of ethyl alcohol in an amount of 20 % relative to the buffer solution makes it possible to separate the mixture of curcuminoids. Conclusion. Thus, the conditions for the separation of the total curcuminoids by the HPCE method in the variant of micellar electrokinetic chromatography (MEKC) were selected. It has been established that for the separation of curcuminoids by this method, it is necessary to use an electrolyte consisting of a mixture of equal volumes of borate buffer (20 mM) and sodium dodecisulfate (30 mM) and ethyl alcohol in an amount of 20 % of the electrolyte volume. A further increase in the concentration of ethyl alcohol in the electrolyte is unreasonable, since it can adversely affect the stability of micelles.

Automated Sequential Injection-Capillary Electrophoresis for Dried Blood Spot Analysis: A Proof-of-Concept Study.

A hyphenated analytical platform that enables fully automated analyses of dried blood spots (DBSs) is proposed by the at-line coupling of sequential injection (SI) to capillary electrophoresis (CE). The SI system, exploited herein for the first time for unattended DBS handling, serves as the "front end" mesofluidic platform for facilitating exhaustive elution of the entire DBS by flow programming. The DBS eluates are thus free from hematocrit and nonhomogeneity biases. The SI pump transfers the resulting DBS eluates into CE sample vials through an internal port of the CE instrument and homogenizes the eluates, whereupon the eluted blood compounds are automatically injected, separated, and quantified by the CE instrument. The SI and CE are commercially available off-the-shelf instruments and are interconnected through standard nuts, ferrules, and tubing without additional instrumental adjustments. They are controlled by dedicated software and are synchronized for a fully autonomous operation. The direct determination of endogenous (potassium and sodium) and exogenous (lithium as a model drug) inorganic cations in DBS samples has been used for the proof-of-concept demonstration. The hyphenated SI-CE platform provides excellent precision of the analytical method with relative standard deviation (RSD) values of peak areas below 1.5 and 3.5% for intraday and interday analyses, respectively, of the endogenous concentrations of the two inorganic cations. For the determination of lithium, calibration is linear in a typical clinical range of the drug (R2 better than 0.9993 for 2-20 mg/L), RSD values of peak areas are below 4.5% (in the entire calibration range), the limit of detection (0.4 mg/L) and the limit of quantification (1.3 mg/L) are well below the drug's minimum therapeutic concentration (4 mg/L), and total analysis time is shorter than 5 min. The SI-CE platform reflects the actual trends in the automation of analytical methods, offers rapid and highly flexible DBS elution/analysis processes, and might thus provide a general solution to modern clinical analysis as it can be applied to a broad range of analytes and dried biological materials.

Portable fully automated oral fluid extraction device for illegal drugs

Currently used methods for in-field determination of illegal drugs involve various test kits based mainly on the immunoassay technique, where the presence of a compound of interest is assessed by antibody-antigen reaction and manifested by observable color change. Despite being accepted and widely used by police forces to test the presence of illegal drugs in a suspect person, these tests often suffer from unreliable results (high level of false-positive and/or false-negative) due to the cross-reactivity and difficulties with quantification. Therefore, we have developed a portable capillary electrophoresis instrument to determine illegal drugs in oral fluid collected from a suspected person. However, this drug analyzer has still required manual sample preparation. Therefore, this research aimed to develop, test, and validate a fully automated sample pretreatment (purification, extraction, pre-concentration) prototype compatible with the capillary electrophoresis drug of abuse analyzer and suitable for confirmatory analysis by mass spectrometry. The cotton swab from Salivette® oral fluid collector was examined and integrated into the fully automated extractor prototype. The recoveries for the automated extractor were between 18 and 20%, with repeatabilities within 5–11% for 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxy-N-ethylamphetamine (MDEA), cocaine (COC), and cocaethylene (COET). The developed extraction device was easy to use even for unskilled persons, required minimal liquid handling, and was applicable to use in field conditions.

EP377: Enabling single-platform testing and carrier screening of the FMR1, SMN1/2, and CFTR gene trio

Carrier screening can provide valuable information for couples to help guide their reproductive decision-making. Screening studies have shown that as many as 1 in 20 individuals has pathogenic variants associated with at least 1 of 3 conditions: spinal muscular atrophy (SMA), fragile X syndrome (FXS), and cystic fibrosis (CF). Many of these conditions cause severe symptoms, and SMA and CF can lead to premature death. Early detection can both inform reproductive choices and offer early therapeutic intervention to help mitigate the most severe symptoms if implemented early enough in the course of disease progression. The three genes associated with SMA, FXS and CF (SMN1, FMR1 and CFTR, respectively) each present unique technical challenges and currently require distinct molecular diagnostic methods and analysis platforms. As a result, a simple, cost-effective, and unified screening system is not yet available for this trio. Here we demonstrate the feasibility of a common, scalable workflow for analyzing combinations of PCR products from all 3 genes on a single capillary electrophoresis (CE) instrument. DNA samples were PCR amplified using AmplideX PCR/CE SMN1/2 Plus, FMR1, and CFTR kits (Asuragen) and electrophoresed on the Applied Biosystems 3500 and 3730 Series Genetic Analyzers (Thermo Fisher Scientific). PCR products from each of the 3 assays were generated according to kit instructions, and combined onto a single CE plate for injection using harmonized conditions. A collection of 48 unique genomic DNA samples isolated from human cell lines or whole blood were tested in singleton with each assay. Samples were designed to cover the breadth of possible genotypes detected by each kit, representing 0 to ≥4 copies of SMN1 or SMN2, FMR1 categorical genotypes of normal, premutation, intermediate, or full mutation [GJL1], and at least 23 unique CFTR variants representing the minimum recommended CFTR carrier screening variant panel from ACMG. Data were processed using AmplideX Reporter Software. Samples were analyzed using a single CE plate for all 3 assays injected with a single set of injection conditions, then compared to the same amplicons injected on separate CE plates for each assay using the conditions recommended for each assay. The overall percent agreement between the genotype calls of the co-injected samples and their individually injected counterparts was >98% for SMN1/2, FMR1, and CFTR on both the 3500 and 3730 CE instruments. Concordance was similar when compared to known reference results for each sample. The co-injection of SMN1/2, FMR1, and CFTR products did not alter the genotype category or copy number calls when compared to their respective, individually assessed results. Using this approach, comprehensive trio genotypes were generated within a single day; further, the system has the potential to process up to 1000 samples per day using a single 3730 CE instrument. The co-injection of PCR products of SMN1/2, FMR1, and CFTR genes can be achieved using commonly available high throughput CE instruments. This approach consolidates existing workflows and improves run and cost efficiencies, with the flexibility to accommodate a few samples at a time or up to 1000 per day. Our study demonstrates the potential for a unified single-instrument, single-software analysis of SMN1/2, FMR1, and CFTR using an accessible, easy-to-run system that runs on equipment that labs already have and can adapt to meet the increasing needs of carrier screening. This versatility can translate to significant cost savings for screening labs that typically rely on 2, 3, or even 4 unique methodologies (with reflexes) spanning different platform requirements, workflows, and analysis tools. The workflow similarities among the 3 PCR/CE assays also simplify their implementation in labs, as the protocols are largely identical.