Simple Sample Preparation Research Articles

Conveniently monitoring aldehyde changes in heated edible oils using miniaturized kapok fiber-supported liquid-phase extraction/in-situ derivatization coupled with liquid chromatography-tandem mass spectrometry

Heating edible oils generates aldehydes, potentially leading to adverse health effects, making their analysis essential for quality control. This study presents a convenient miniaturized kapok fiber-supported liquid-phase extraction/in-situ derivatization method for the simultaneous extraction and derivatization of aldehydes in oils. The method involves placing 150 mg oil into a 1 mL pipette tip packed with 25 mg kapok fiber, adding 150 μL ACN with 1.5 mg mL−1 DNPH, and post 30-minute static extraction, retrieving the extractant with a pipettor for liquid chromatography-tandem mass spectrometry analysis. By optimizing critical parameters through a Box-Behnken design, the method exhibits good linearity (1–500 ng g−1, R2 ≥ 0.991), low detection limits (0.2–1.0 ng g−1), excellent accuracy (95.3–107.1%) and high precisions (relative standard deviation < 7.9%). This method simplifies sample preparation processes, cuts solvent use, and facilitates automation. It effectively identifies ten aldehyde variations in six heated oils, displaying distinct profiles consistent with prior research.

Simultaneous quantification of 55 psychotropic drugs and metabolites in human plasma with a fast UPLC-MS/MS method

ObjectivesTherapeutic drug monitoring is strongly recommended for psychotropic drugs, which present a strong inter- and intra-individual variability due to multiple factors like inflammatory state, smoking, diet, drug interactions due to polypharmacy, and genetic profile. The aim of this study was to develop and validate a fast, simple, and sensitive method allowing the simultaneous quantification of a large number of psychotropic drugs. MethodsAfter a simple sample preparation with a one-step protein precipitation, a total of 55 compounds, including 22 antidepressants, 18 antipsychotics, 2 other psychotropic drugs (bupropion and nefopam), and their metabolites, was separated on a Waters Acquity HSS T3 ultra-performance liquid chromatography column, and subsequently detected and quantified by a triple quadrupole Quantis mass spectrometer with electrospray ionization operated in positive mode. ResultsTotal run time was only 5.7 min. Limits of detection ranged from 0.01 to 0.18 µg/L depending on compound. Measuring ranges were from 0.195 to 1000 µg/L depending on compound, and were defined according to therapeutic ranges. Inter- and intra-assay precisions values were less than 15 %. After validation, this method was successfully applied in daily practice for therapeutic drug monitoring of polymedicated psychiatric patients. ConclusionWe developed and validated one of the most sensitive and complete UPLC-MS/MS methods in psychopharmacology, allowing the simultaneous determination of 55 psychotropic drugs in only 5.7 min after a simple sample preparation. This method has been successfully used in daily practice for therapeutic drug monitoring of psychiatric patients and is especially useful in polymedicated patients.

Homogeneous liquid–liquid microextraction coupled with HPLC/DAD for determination of nirmatrelvir and ritonavir as COVID-19 combination therapy in human plasma

The study reports the development of a high-performance liquid chromatography/diode array detection method to measure the levels of nirmatrelvir and ritonavir in human plasma. These two antiviral medications are used for the treatment of COVID-19 and are marketed as Paxlovid®. The method employed sugaring-out induced homogeneous liquid–liquid microextraction to improve sensitivity. Optimization of the method was performed using the one variable at a time approach by adjusting several factors such as type of sugar, extractant, amount of sugar, volume of extractant, and pH of the aqueous sample to achieve the highest efficiency. The developed method was validated according to the Food and Drug Administration guidelines and demonstrated good linearity, accuracy, and precision. The range of linearity was from 1000 to 20,000 ng/mL for nirmatrelvir and 200 to 20,000 ng/mL for ritonavir with correlation coefficient values of 0.998 and 0.996, respectively. Selectivity studies revealed that no others peaks appeared in the retention times of the studied drugs. The stability of nirmatrelvir and ritonavir were also investigated through short term and three cycles of freeze–thaw, and both drugs were found stable. This analytical method could be useful for monitoring drug concentrations in patients undergoing treatment with these medications for COVID-19. In this work, for the first time, SULLME was used for the sensitive determination of nirmatrelvir and ritonavir in biological fluids. The developed method was able to determine both drugs in therapeutic levels with no need to sophisticated techniques like LC–MS. In addition to that, SULLME is considered a simple and green sample preparation in comparison with conventional sample preparation methods.

Monitoring Exposure to Five Chemical Warfare Agents Using the Dried Urine Spot Technique and Liquid Chromatography-Mass Spectrometry/Mass Spectrometry—In Vivo Determination of Sarin Metabolite in Mice

Dried urine spot (DUS) is a micro-sample collection technique, known for its advantages in handling, storage and shipping. It also uses only a small volume of urine, an essential consideration in working with small animals, or in acute medical situations. Alkyl-phosphonic acids are the direct and indicative metabolites of organophosphorus chemical warfare agents (OP-CWAs) and are present in blood and urine shortly after exposure. They are therefore crucially important for monitoring casualties in war and terror scenarios. We report here a new approach for the determination of the metabolites of five CWAs in urine using DUS. The method is based on a simple and rapid sample preparation, using only 50 µL of urine, spotted and dried on DBS paper, extracted using 300 µL methanol/water and analyzed via targeted LC-MS/MS. The detection limits for the five CWAs, sarin (GB), soman (GD), cyclosarin (GF), VX and RVX in human urine were from 0.5 to 5 ng/mL. Recoveries of (40–80%) were obtained in the range of 10–300 ng/mL, with a linear response (R2 &gt; 0.964, R &gt; 0.982). The method is highly stable, even with DUS samples stored up to 5 months at room temperature before analysis. It was implemented in a sarin in vivo exposure experiment on mice, applied for the time course determination of isopropyl methylphosphonic acid (IMPA, sarin hydrolysis product) in mice urine. IMPA was detectable even with samples drawn 60 h after the mice’s (IN) exposure to 1 LD50 sarin. This method was also evaluated in a non-targeted screening for multiple potential CWA analogs (LC-Orbitrap HRMS analysis followed by automatic peak detection and library searches). The method developed here is applicable for rapid CWA casualty monitoring.

SERS-based pH-Dependent detection of sulfites in wine by hydrogel nanocomposites

Sulfur dioxide (SO2) and sulfites are well-known additives in winemaking due to their preservative properties. Although they can prevent oxidation and inhibit microbial growth, they pose health risks and require limitations on their use. Consequently, the total level of SO2 is regulated and several quantification strategies have been proposed. The approved detection methods require the extraction of SO2 by heating and/or acid treatment. Then, iodine or acid/base titrations are conducted for the detection of liberated SO2. Although these methods can provide sensitive detection of SO2, they are complex, time-consuming, and require sample preparation steps and skilled operators. Thus, to overcome these disadvantages, an easy-to-use method, involving simple sample preparation steps, and offering high sensitivity and selectivity, is desirable. Herein, we introduce a SERS-based strategy for SO2 detection in liquids using hydrogel nanocomposites. The hydrogels are prepared by poly(ethylene glycol) diacrylate (PEGDA) in the presence of gold nanoparticles (AuNPs), acting as the SERS substrate. The use of hydrogels ensures a homogenous signal distribution and an efficient collection of SO2, and drying the hydrogels enhances and stabilizes the obtained SO2 signal. The detection strategy is based on the pH-dependent dissociation of SO2. By adjusting the pH value of wine to 10 through simple dilutions, SO2 can be directly detected in wine, down to 0.4 ppm, well below the regulatory limits. The proposed method allows for sensitive, direct, cost-effective detection of SO2 by eliminating the loss of the gaseous form of the sample and avoids titration-based detection methods.

Fatty acids analysis by capillary electrophoresis: Fundamentals, advantages and applications.

This review covers the know-how of the Grupo de Química Analítica e Quimiometria regarding the analysis of fatty acids by capillary electrophoresis acquired over its 20 years of existence. Therefore, the fundamentals, advantages, and applications of this technique for analyzing different fatty acids in samples such as food, oils, cosmetics, and biological matrices are presented and discussed. Capillary electrophoresis is, thus, shown as an interesting and valuable separation technique for the target analysis of these analytes as an alternative to the gas chromatography coupled to flame ionization detection, as it offers advantages over the latter such as low analysis times, low sample and reagent consumption, the use of a nondedicated column, and simpler sample preparation. In addition, the methods shown in this literature review can be useful for quality control, adulteration, and health-related research by regulatory agencies.

Interconnectable 3D-printed sample processing modules for portable mycotoxin screening of intact wheat

BackgroundThe increasing demand for food and feed products is stretching the capacity of the food value chain to its limits. A key step for ensuring food safety is checking for mycotoxin contamination of wheat. However, this analysis is typically performed by rather complex and expensive chromatographic methods, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS). These costly methods require extensive sample preparation that is not easily carried out at different points along the food supply chain. To overcome such challenges in sample processing, an inexpensive and portable sample preparation device was needed, that required low skill, for rapid sample-to-result mycotoxin screening. ResultsWe describe 3D-printed and interconnectable modules for simple, integrated and on-site sample preparation, including grinding of wheat kernels, and solvent-based extraction. We characterized these 3D-printed modules for mycotoxin screening and benchmarked them against a laboratory mill using commercial lateral flow device(s) (LFD) and in-house validated LC-MS/MS analysis. Different integrated sieve configurations were compared based on grinding efficiency, and we selected a sieve size of 2 mm allowing grinding of 10 g of wheat within 5 min. Moreover, 10 first time-users were able to operate the grinder module with minimal instructions. Screening for deoxynivalenol (DON) in naturally contaminated samples at the regulatory/legal limit (1.25 mg kg−1) was demonstrated using the developed 3D-printed prototype. The whole process only takes 15 min, from sample preparation to screening result. The results showed a clear correlation (R2 = 0.96) between the LFD and LC-MS/MS. SignificanceOur findings demonstrate the potential of 3D-printed sample handling equipment as a valuable extension of existing analytical procedures, facilitating the on-site implementation of rapid methods for the determination of mycotoxins in grains. The presented prototype is inexpensive with material costs of 2.5€, relies on biodegradable 3D printing filament and can be produced with consumer-grade printers, making the prototype readily available. As a future perspective, the modular character of our developed tool kit will allow for adaptation to other hard food commodities beyond the determination of DON in wheat.

Simultaneous determination of ergothioneine, selenoneine, and their methylated metabolites in human blood using ID-LC-MS/MS.

Ergothioneine and selenoneine are structurally related dietary antioxidants and cytoprotectants that may help prevent several chronic diseases associated with inflammation and aging. Both compounds share pharmacokinetic characteristics such as cellular uptake through the ergothioneine transporter, accumulation in red blood cells, and biotransformation to methylated metabolites. A rapid, sensitive, specific, precise, and cost-effective analytical method is required to further investigate the potential health benefits of these compounds, individually or combined, in large epidemiological studies. We developed and validated an isotope-dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) method for the simultaneous specific quantification of these analytes in human blood following a simple sample preparation consisting of dilution in aqueous dithiothreitol followed by centrifugal filtration. Chromatographic separation of the analytes is achieved using a reversed-phase chromatography within an 8-min run. Analyte detection is performed using triple quadrupole mass spectrometry in multiple reaction monitoring mode. Each analyte is quantified against its corresponding isotopically labeled internal standard either commercially available or synthesized in-house (77Se-labeled selenoneine compounds). The validated method demonstrates excellent linearity and very good precision (all CV < 10%). Matrix effects are minimal, suggesting that this method could easily be adapted to other matrices. Freeze/thaw cycles have little effect on methylated metabolites but significantly reduced concentrations of the parent compounds. The method was successfully applied to a small set of volunteer blood samples containing low levels of the analytes. The developed ID-LC-MS/MS method opens new avenues for exploring the roles of these bioactive compounds and their metabolites in human health and disease.

X-ray Fluorescence Analysis of Waste Sm-Co Magnets: A Rational Approach

Determination of the chemical composition of waste Sm-Co magnets is required for their efficient recycling. The non-stereotypical composition of said magnets makes an analysis extremely challenging. X-ray fluorescence spectrometry is a promising analytical tool for this task. It offers high accuracy and simplicity of sample preparation as it does not require sample dissolution. However, a serious limitation of X-ray fluorescence analysis is the spectral interference of matrix elements and impurities. In this work, a two-stage technique has been developed for the determination of the main components (Sm, Co) and impurities (Fe, Cu, Zr, Hf, Ti, Ni, Mn, Cr) in samples of spent samarium–cobalt magnets using wavelength dispersive X-ray fluorescence spectrometry. In order to overcome the main limitation of the chosen method and to maximize its capabilities of qualitative and quantitative analysis, we propose an approach to the selection of analytical lines and experimental conditions, as well as a preparation method for the calibration standards. The obtained results have been shown to have a good correlation with ICP-OES. The limits of detection are in the range of 0.001–0.02 wt%, and the limits of quantification are 0.003–0.08 wt%.

Simple andrapid detection ofthree amatoxins andthree phallotoxins inhuman body fluids by UPLC-MS-MS andits application in15 poisoning cases.

Amatoxins and phallotoxins are toxic cyclopeptides found in the genus Amanita and are among the predominant causes of foodborne sickness and poisoning-related fatalities in China. This study introduces and validates a simple, rapid and cost-effective ultra-performance liquid chromatography-mass spectrometry method for the simultaneous determination and quantification of α-amanitin, β-amanitin, γ-amanitin, phallisacin, phallacidin and phalloidin in human blood and urine. Quick therapeutic decision-making is supported by a 9 min chromatographic separation performed on a Waters Acquity UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 µm) using a gradient of high-performance liquid chromatography (HPLC)-grade water and methanol:0.005% formic acid. The analyte limit of quantification was 1-3 ng/mL in blood and 0.5-2 ng/mL in urine. Calibrations curves, prepared by spiking drug-free blood and urine, demonstrated acceptable linearity with mean correlation coefficients (r) greater than 0.99 for all phallotoxins and amatoxins. Acceptable intraday and interday precision (relative standard deviation <15%) and accuracy (bias, -4.8% to 13.0% for blood and-9.0% to 14.7% for urine) were achieved. The validated method was successfully applied to analyze 9 blood samples and 2 urine samples testing positive for amatoxins and/or phallotoxins. Amatoxins and/or phallotoxins were identified in each whole blood sample at a range of 1.12-5.63 ng/mL and in two urine samples from 1.01-9.27 ng/mL. The method has the benefits of simple sample preparation (protein precipitation) and wide analyte coverage, making it suitable for emergency quantitative surveillance toxicological analysis in clinics and forensic poisoning practice.

Mobile Affinity Selection Chromatography Analysis of Therapeutic Monoclonal Antibodies.

Federal regulatory agencies require continuous verification of recombinant therapeutic monoclonal antibody (mAb) quality that is commonly achieved in a two-step process. First, the host-cell proteome and metabolome are removed from the production medium by protein A affinity chromatography. Second, following recovery from the affinity column with an acidic wash, mAb quality is assessed in multiple ways by liquid chromatography-mass spectrometry (LC-MS). However, lengthy sample preparation and the lack of higher-order structure analyses are limitations of this approach. To address these issues, this report presents an integrated approach for the analysis of two critical quality attributes of mAbs, namely titer and relative aggregate content. Integration of sample preparation and molecular-recognition-based analyses were achieved in a single step utilizing an isocratically eluted mobile affinity selection chromatography (MASC) column. MASC circumvents the protein A step, simplifying sample preparation. Within 10 min, (i) mAbs are fluorescently coded for specific detection, (ii) monomers and aggregates are resolved, (iii) the mAb titer is quantified, (iv) relative aggregate content is determined, (v) analytes are detected, and (vi) the column is ready for the next sample. It is suggested herein that this mode of rapid quality assessment will be of value at all stages of discovery (screening, clone selection, characterization), process R&D, and manufacturing. Rapid monitoring of variant formation is a critical element of quality evaluation.

Natural thermometers in concrete: Stability and reliability verification of inclusion decrepitation thermoacoustic thermometry

The fire-exposure temperature of concrete directly determines the degree of damage to its microstructure and the performance of concrete. Therefore, an accurate assessment of the fire-exposure temperature of concrete is crucial. But to date, there is not yet an accurate, practical, and effective method for measuring the internal temperature of concrete after fire. Based on the discovery that fluid inclusions in concrete act as natural thermometers, this paper investigates the scientific principles and experimental evidence of the decrepitation thermometry of inclusions. By extracting quartz sand samples from the interiors of concrete members after a fire and performing an inclusion decrepitation test on these samples, the maximum fire-exposure temperature of concrete can be determined with a high degree of accuracy. The test results reveal that the natural sand used in construction typically contains fluid inclusions, indicating that the necessary conditions for inclusion decrepitation thermometry are satisfied for concrete structures. In this study, standard quartz sand samples were configured, and inclusion decrepitation temperature measurements were conducted after subjecting the samples to different heating rates, cooling methods, thermostatic durations, and standing times after fire. The test results indicate that the impact of the aforementioned factors on the inclusion decrepitation temperature measurement is not evident. The maximum relative error between all temperature measurement results and the actual heating temperature is <10 %, and the average relative error is <2.5 %. The experimental results show that fluid inclusions are natural thermometers in concrete. After a fire, the maximum temperature experienced in the concrete can be quantitatively assessed using the initial decrepitation temperature of the inclusion. This method possesses the advantages of being a scientific test method and providing accurate temperature measurements without any indirect analysis or calculation using simple test equipment, sample preparation, and operation.

Synthesis and Modification of Magnetic Nanoparticles for Biosensing and Bioassay Applications: A Review

Biosensors are analytical devices that use biological interactions to detect and quantify single molecules, clinical biomarkers, contaminants, allergens, and microorganisms. By coupling bioreceptors with transducers, such as nucleic acids or proteins, biosensors convert biological interactions into electrical signals. Electrochemical and optical transductions are the most widely used methods due to their high detection capability and compatibility with miniaturization. Biosensors are valuable in analytical chemistry, especially for health diagnostics, as they offer simplicity and sensitivity. Despite their usefulness, challenges persist in immobilizing biorecognition elements on the transducer surface, leading to issues such as loss of sensitivity and selectivity. To address these problems, the introduction of nanomaterials, in particular magnetic nanoparticles (MNPs) and magnetic beads, has been implemented. MNPs combine their magnetic properties with other interesting characteristics, such as their small size, high surface-to-volume ratio, easy handling, and excellent biocompatibility, resulting in improved specificity and sensitivity and reduced matrix effects. They can be tailored to specific applications and have been extensively used in various fields, including biosensing and clinical diagnosis. In addition, MNPs simplify sample preparation by isolating the target analytes via magnetic separation, thus reducing the analysis time and interference phenomena and improving the analytical performance of detection. The synthesis and modification of MNPs play a crucial role in adjusting their properties for different applications. This review presents an overview of the synthesis and surface modifications of magnetic nanoparticles and their contributions to the development of biosensors and bioassays for their applications across different areas. The future challenges of MNP synthesis and integration in assays are focused on their stability, multiplex detection, simplification and portability of test platforms, and in vivo applications, among other areas of development.

Molecular detection of per- and polyfluoroalkyl substances in water using time-of-flight secondary ion mass spectrometry.

Detection of per- and polyfluoroalkyl substances (PFASs) is crucial in environmental mitigation and remediation of these persistent pollutants. We demonstrate that time-of-flight secondary ion mass spectrometry (ToF-SIMS) is a viable technique to analyze and identify these substances at parts per trillion (ppt) level in real field samples without complicated sample preparation due to its superior surface sensitivity. Several representative PFAS compounds, such as perfluorooctanesulfonic acid (PFOS), perfluorobutanoic acid (PFBA), perfluoropentanoic acid (PFPeA), perfluoheptanoic acid (PFHpA), and perfluorononanoic acid (PFNA), and real-world groundwater samples collected from monitoring wells installed around at a municipal wastewater treatment plant located in Southern California were analyzed in this work. ToF-SIMS spectral comparison depicts sensitive identification of pseudo-molecular ions, characteristic of reference PFASs. Additionally, principal component analysis (PCA) shows clear discrimination among real samples and reference compounds. Our results show that characteristic molecular ion and fragments peaks can be used to identify PFASs. Furthermore, SIMS two-dimensional (2D) images directly exhibit the distribution of perfluorocarboxylic acid (PFCA) and PFOS in simulated mixtures and real wastewater samples. Such findings indicate that ToF-SIMS is useable to determine PFAS compounds in complex environmental water samples. In conclusion, ToF-SIMS provides simple sample preparation and high sensitivity in mass spectral imaging, offering an alternative solution for environmental forensic analysis of PFASs in wastewater in the future.

Elimination of matrix effects in urine for determination of ethyl glucuronide by liquid chromatography–tandem mass spectrometry

RationaleAlcohol use disorder affects 4% to 5% of the world's population. Analysis methods are available for various biological fluids to detect this disorder. Determination of ethyl glucuronide in urine by the liquid chromatography–tandem mass spectrometry (LC/MS/MS) method is frequently used in forensic toxicology. These analyses are known to cause matrix effects.MethodsThe presented study describes the elimination of matrix effects for ethyl glucuronide. This study used two different LC/MS/MS systems containing orthogonal and z‐spray ion sources. Ethyl glucuronide was analyzed in negative polarity in electrospray ionization. A different dilution method was chosen for each study. The methods were developed and validated according to the European Medicines Agency bioanalytical method validation parameters.ResultsThe lower limit of quantitation of the developed methods was 0.025 μg/mL for ethyl glucuronide. The calibration curve of ethyl glucuronide was between 0.025 and 100 μg/mL with a correlation coefficient of &gt;0.99 for the two methods.ConclusionsIt was determined that the analyses using the z‐spray ion source were more affected by the matrix effect. The two validated methods involve rapid analysis time and simple sample preparation. Also, the methods were applied to real patients' urine.

Cookie composition analysis by Fourier transform near infrared spectroscopy coupled to chemometric analysis

The consumption ofcookies is ever growing and during the COVID-19 pandemic reached record consumption values and it is imperative to guarantee the quality and safety of the products.Fourier transform near infrared (FT-NIR) spectroscopy, combined with chemometric techniques, provides a promising solution in that regard, due to its speed and simple sample preparation. The objective of this study was to investigate the possibilities of using FT-NIR to predict lipids, carbohydrates, fibers, proteins, salt and energy contents, as well as to identify cookies type and main cereals present in a batch of 120 commercially acquired samples. The prediction models were performed using ordinary least squares (OLS), partial least squares (PLS), and PLS based classification models including discriminant analysis (PLS-DA), k-nearest neighbors (PLS-kNN) and naïve Bayes (PLS-NB). The best prediction models allowed for good accuracies, with correlation coefficients higher than 0.9 for all studied nutritional parameters. PLS-kNN methodology was able to identify all 5 main cereals (wheat, integral wheat, oat, corn and rice) as well as the 14 types of cookies based on the nutritional contents. The developed methods were able to accurately identify the cookies type and composition, confirming the proposed methodology as a fast, reliable, environmentally friendly and non-destructive alternative to standard analytical methods.

B-299 Liquid Chromatography-mass Spectrometry Measurement of Drugs of Abuse and Alcohol Biomarker Phosphatidylethanol (peth 16:0/18:1) Using Volumetric Dried Blood Spot Device

Abstract Background Reliable biomarkers have great significance in the investigation of alcohol consumption patterns and in the treatment of alcohol-induced diseases. Direct biomarkers to detect alcohol intake are non-oxidative products of ethanol metabolism (1, 2). Phosphatidylethanol (PEth), blood-based direct alcohol biomarker, represents a group of unnatural phospholipids formed on leukocytes and predominantly erythrocyte membranes. The presence of PEth in blood samples is an indisputable indicator of ethanol consumption (3, 4). Moreover, detecting drugs of abuse (DOA) including opiates, amphetamines, cocaine metabolites, benzodiazepines, THC-metabolites, MDMA, methadone, fentanyl etc. is important to establish patterns of illicit drug usage. In recent years, DBS has been gaining interest in plenty of research areas owing to ease of specimen collection and storage (5, 6).The main objective of this study was to determine the concentration of PEth-16:0 18:1 in DBS samples collected using a volumetric dried blood spot (DBS) device (HemaxisTM DB) and to identify/quantify drugs of abuse (including 119 parameters-Jasem Clinical Toxicology mixture) using another blood spot punch of the same DBS sample. Methods According to Jasem method, DBS samples were prepared by sampling from spiked 20 µL of blood drop located onto paraffin film. PEth-16:0 18:1 and DOA were extracted from entire blood spot of DBS specimens implementing two different extraction reagents at room temperature for 10 min then subjected to HPLC system equipped with Agilent 6470 TQ. The total run times from injection to injection for PEth and DOA were 4.0 min and 12.0 min respectively. Results The linearity and accuracy of the methods were evaluated using 5 DBS calibrators levels for PEth and 6 DBS calibrators levels for DOA. Linearity was confirmed in the range 10 to 1000 ng/mL (r² &amp;gt; 0.995) for PEth 16:0/18:1 and in the range 2 to 100 ng/mL for DOA (r² &amp;gt; 0.99). For two methods linearity, RSD% (inter-intraday) and accuracy were within the analytical acceptable ranges. Conclusion The measurement of blood PEth levels provides a precious tool to determine the chronological profile based on retrospective alcohol intake in a reliable way. Besides, simultaneous analysis of DOA containing 119 parameters ensures comprehensive analytical solution and clinically relevant information. Both of Jasem LC-MS/MS approaches are centred on simple sample preparation following non-invasive sample collection method. Prior to rapid extraction, applying volumetric DBS device assures to collect fixed volume of blood regardless of blood haematocrit level making the measurements more consistent.

A-096 Novel Gastrointestinal Testing: Comparative Performance of the ALPCO Five-Plex Gastrointestinal Marker Assay compared to Conventional ELISA/CLIA testing for Human Stool Samples

Abstract Background All modern gastrointestinal (GI) testing laboratories face a similar challenge: How to test a panel of analytes from the same stool or plasma/serum sample and deliver clinically accurate results in a timely fashion. GI analytes are tested primarily on ELISAs that while accurate, manual ELISA testing is time-consuming and work intensive. In this study, we utilize the automated ALPCO five-plex assay (AutoPlex™) to measure five key GI biomarkers: Calprotectin (CP), Pancreatic Elastase (PE), Lactoferrin (LF), Eosinophil derived neurotoxin (EDN), and Hemoglobin (Hgb). These biomarkers are widely recognized indicators of gut inflammation and disease, including inflammatory bowel disease, pancreatic insufficiency, and colorectal cancer. The assay employs a simple and efficient sample preparation protocol and utilizes multiplexed bead-based technology to quantify each biomarker at the same dilution and time point in an automated fashion. In this study, we determine how this new assay performs against gold standard ELISA/CLIAs that measure the same analytes. Methods Capture and detector antibodies were optimized for each of the analytes to perform on beads, and buffers were optimized to allow for all the analytes to be measured simultaneously. A stool extraction device was utilized to extract 386 human stool samples from both healthy controls and populations with suspected GI impairment. Samples were analyzed using the ALPCO 5-Plex (AutoPlex) assay run on the Multiplier® - a fully-automated chemiluminescence analyzer (DYNEX® Technologies, Chantilly, VA), and conventional Chemiluminescence ELISA/CLIA methods. Results Comparative analysis of 386 human stool samples run on both the 5-Plex and ELISAs for each respective analyte, showed strong clinical correlation between the two platforms. With a clinical cut-off of 50 ug/g calprotectin had a 91% clinical correlation between the two platforms. Similarly, with a cut-off of 200 µg/g PE was at 91%, LF with a cut-off of 7.24 µg/g at 83%, EDN with a cut-off of 4.6 was at 97%, and Hgb with a cut-off of 20 µg/g was at 98% clinical correlation between the two platforms. Conclusions The results generated by ALPCO five-plex assay demonstrated on average &amp;gt;90% clinical matching for all 5 GI analytes tested when compared to their gold standard ELISA/CLIA counterparts. Since this assay allows for the simultaneous measurement of five common GI analytes that occur from a single stool extract in one concurrent and automated workflow, this saves time as it eliminates the need for multiple ELISA/CLIA tests and reduces processing time for stool samples.

A-250 Rapid molecular Identification of Herpes Simplex, Varicella-zoster, and Enteroviruses from Cerebrospinal Fluid

Abstract Background Accurate and efficient molecular testing of viral meningitis/encephalitis is crucial for proper patient care. Here we describe the clinical evaluation of the GenomEra® HSV-1/2, VZV + EV Assay Kit (Uniogen, Finland) allowing simultaneous detection of four key viruses responsible for causing meningitis/encephalitis, namely HSV-1 and HSV-2, VZV, and enteroviruses. Methods The study was carried out at three European institutions in 2022, with a total of 193 frozen, anonymized leftover cerebrospinal fluid (CSF) specimens analyzed retrospectively. For the GenomEra HSV-1/2, VZV + EV Assay Kit, 50 µL of CSF was mixed with lysis buffer in a 1:1 ratio, allowed to incubate for 1 min at room temperature, and then filtered by a 1-min centrifugation through an Extraction Column utilizing size-exclusion chromatography. A 35-µL sample of the flow-through was applied into the Test Chip and analyzed using the GenomEra CDX System. The cotemporally performed comparison tests included the FilmArray Meningitis/Encephalitis (ME) panel (bioMérieux), Xpert® EV Assay (Cepheid), artus VZV assay (Qiagen), and a well-established, validated non-commercial real-time RT-PCR assay for the detection of HSV-1/2, VZV, and EV. Results The GenomEra HSV-1/2, VZV + EV Assay Kit produced results in 70 min with a sensitivity and specificity of 92.2% (95% CI: 82.7–97.4%) and 99.9% (95% CI: 99.2–100%), respectively, as compared to the reference tests. Of the discordant results, five tested negative with the GenomEra assay, whereas the reference tests produced low positive results (Ct values ≥ 35). In addition, one specimen yielded a positive signal for VZV while remaining negative with the reference tests. Conclusion The GenomEra HSV-1/2, VZV + EV Assay Kit showed a high overall specificity and a high level of sensitivity particularly for herpes simplex and varicella-zoster viruses. For enteroviruses, while the specificity reached 100%, the sensitivity was not as high as with the reference methods. The high clinical performance, together with the simple sample preparation protocol and short turnaround time make the GenomEra assay a competitive molecular test especially for small and medium-sized laboratories that may not have a specialist in molecular biology. In addition, the GenomEra assay only requires a small amount of sample, conserving the valuable specimens for additional testing.

Development of Simplified and Efficient Sample Preparation Methods for the Analysis of Problem Material within the Diesel Fuel Delivery System.

A new approach for the analysis of diesel engine fuel filters has been developed. This method involves minimal to no sample preparation, allowing rapid and unbiased analysis of diesel fuel filters. In recent years, diesel fuel filter plugging incidences have increased in parallel with changing emissions legislation. Fuel filter blockages can result in increased emissions, reduced efficiency, and engine failure. It is not fully understood why fuel filter blockages occur; as a result, there has been an international increase in research into the cause of fuel filter plugging. The method discussed in this paper utilizes a thermal desorption (TD) style sample introduction technique that can be used in conjunction with gas chromatography-mass spectrometry (GC-MS) and presents a fast, simple, and more sustainable approach to the analysis of fuel filters. When required, an efficient and straightforward sample cleanup process was developed and was used to simplify and improve confidence in the data identification and assignment; this method is up to three orders of magnitude faster than some procedures adopted in the literature. Further complementary analytical techniques, such as ultrahigh-performance supercritical fluid chromatography-mass spectrometry (UHPSFC-MS) and high-resolution GC-MS, were used to access additional sample-specific information. This new approach has been successful in the identification of problematic materials deposited on blocked fuel filters, concurrent with recent research. This information can aid in the development of mitigation strategies to combat fuel filter plugging.