Linear Range Research Articles

Coffee Biomass-Based Carbon Material for the Electrochemical Determination of Antidepressant in Synthetic Urine

Escitalopram (ESC) is commonly prescribed as an antidepressant to enhance serotonin levels in the brain, effectively addressing conditions such as depression and anxiety. The COVID-19 pandemic, along with ongoing mental health crises, has exacerbated the prevalence of these disorders, largely due to factors such as social isolation, fear of the virus, and financial difficulties. This study presents the enhancement of a glassy carbon electrode (GC) through the incorporation of hydrochar (HDC) derived from spent coffee grounds and copper nanoparticles (CuNPs) for the detection of ESC in synthetic urine. Characterization of the nanocomposite was conducted using scanning electron microscopy (SEM), energy-dispersive spectroscopy (EDS), and cyclic voltammetry (CV). The analytical parameters were systematically optimized, and a sensing platform was utilized for the quantification of ESC via square-wave voltammetry (SWV). The established linear range was found to be between 1.0 µmol L−1 and 50.0 µmol L−1, with a limit of detection (LOD) of 0.23 µmol L−1. Finally, an electrochemical sensor was employed to measure ESC levels in synthetic urine, yielding recovery rates ranging from 91.7% to 94.3%. Consequently, the HDC-CuNPs composite emerged as a promising, sustainable, and cost-effective alternative for electroanalytical applications.

Design and synthesis of surface-decorated zinc-doped carbon quantum dots as fluorescent probes for tartrazine detection in real food samples exploiting the inner filter effect mechanism

We developed a rapid and sensitive method for detecting tartrazine (Tar), a common food colorant, using zinc-doped carbon quantum dots (Zn-doped CQDs). Synthesized from biowaste pigeon pod shells and zinc acetate, the Zn-doped CQDs exhibited a high quantum yield (47.63%) and were characterized by photo-stability, non-toxicity, and water solubility. The inner filter effect (IFE) allows selective attenuation of Zn-doped CQD fluorescence by Tar, enabling precise detection. The approach obtains a low detection limit of 20.35 ng/mL and a quantification limit of 61.69 ng/mL within a linear range of 0–100 ng/mL under optimal conditions when employing the Box-Behnken statistical design. The approach demonstrates excellent reproducibility, successfully detecting Tar in real food samples with recovery rates ranging from 89% to 102.2%. This high accuracy confirms the sensor's reliability and effectiveness in practical applications. This cost-effective and rapid technique presents a promising solution for monitoring Tar levels in food products.

LAMP-CRISPR/Cas12a-based impedimetric biosensor powered by Fe3O4@Au-(S-polyA-S)-Au for detection of SARS-CoV-2.

Alow-cost, lab-made polytetrafluoroethylene micro-cell, equipped with three electrodes, wasd evelopedfor the impedimetric detection of SARS-CoV-2. The gold working electrode was modified with a double-ended thiolated poly-adenine probe, which was conjugated with magnetic Fe₃O₄@Au nanoparticles (Fe3O4@Au-(S-polyA-S)-Au). After the loop-mediated isothermal amplification (LAMP) of viral RNA, the single-guide RNA (sgRNA), specifically bound to the SARS-CoV-2 target sequence, activates Cas12a. Cas12a then cleaved the immobilized probe. As a result, the magnetic Fe3O4@Au nanoparticles were released and adsorbed onto the gold electrode surface, using an external magnet. This process increased the physical surface area of the gold electrode, facilitating redox ion ([FeIII/II(CN)6]3-/4-) electron transfer. The decrease in the charge transfer resistance was utilized for SARS-CoV-2 detection. Our LAMP-CRISPR/Cas12a-based impedimetric biosensor, powered by Fe3O4@Au-(S-polyA-S)-Au, demonstrated impressive capabilities, including a remarkable detection limit of 0.8 aM (0.48 copies/µL) and a linear range of 0.01 to 36.06 fM.

B-015 Analytical Verification of MR-proADM Biomarker in Human EDTA Plasma on Immunoassay Analyzer LIAISON® XL of Diasorin

Abstract Background Adrenomedullin (ADM) is a potent vasodilator peptide of 52-aminoacid belonging to the calcitonin family of peptides. Midregional pro-ADM (MR-ProADM) is a precursor for ADM that circulates is in a 1:1 ratio with it, and proportionally represents its levels and activity. Increased ADM circulating concentrations have been described for several disease states, including dysfunction of the cardiovascular system and sepsis. However, reliable ADM quantification has been hampered by the short half-life, the existence of a binding protein, and its physical properties. Hence, quantificacion of MR-proADM represents a more suitable option. The biomarker MR-proADM has been shown to be an accurate prognostic marker for outcome prediction in patients with lower respiratory infections, sepsis, urinary tract infections, and kidney disease. The objective of this study was the verification of the metrological characteristics and the verification of the linearity of quantitative measurement range, as declared by the manufacturer, of the LIAISON® Brahms MR-proADMTM on Liaison® XL (DiaSorin). Methods The verification was performed with two levels of quality controls (QC) from the manufacturer (x̄L=1,19 nmol/L and x̄H=5,48 nmol/L), and residual EDTA plasma samples from patients with an active sepsis code. The evaluation included the assessment of: • Detection capability (including limit of quantification (LoQ) and limit of detection (LoD), measuring three samples at the manufacturer's stated concentration (0.21 nmol/L and 0.09 nmol/L, respectively) in duplicate within 4 runs). • Precision (CV, %) and accuracy (bias, %) within, measuring 10 replicates of each QC level; and between run, measuring 5 replicates for each QC level within 5 runs. • Linearity of the quantitative range of measurement (six intermediate samples prepared by mixing a high concentration sample- XH=9.64 nmol/L- and a low concentration sample, XL=0.59 nmol/L; measured in triplicate). All studies were performed in accordance with the recommendations of the Clinical Laboratory Standards Institute guidelines. Statistical analysis was performed using Microsoft Excel®, 2016. Results All results with diluted samples presented a signal (RLU) higher than that detected in samples without analyte, so the LoD was verified. The precision at the LoQ concentration was 26.7% (acceptance criteria was set as <20%). CV for within-run precision were 4.3% and 2.0% and for between-run precision 6.8% and 7.9% for low (L1) and high (L2) control level, respectively. The average bias was 3.60% for L1 and 4.23% for L2, meeting acceptance criteria (<8%). Linearity was confirmed between 0.59 and 9.64 nmol/L (R2 = 0.995). The biases between the expected and the obtained value were ≤7% through the quantitative range of measurement, except for the lowest concentration sample (close to the quantitation limit). Conclusions Our study showed good compliance with the metrological characteristics declared by the manufacturer for LoD, precision, bias and linearity range. Although the LoQ was not verified, it is outside the clinical decision value in septic patients (< 2.25 nmol/L) and it is not deemed a limitation for clinical practice. Additionally, the Liaison XL® platform permits us to configure an emergency protocol circuit (24/7) with a TAT of 35 min.

Advanced Electrochemical Biosensing toward Staphylococcus aureus Based on the RPA-CRISPR/Cas12a System and Conductive Nanocomposite.

Staphylococcus aureus (S. aureus) is a prevalent foodborne pathogen that poses significant challenges to food safety. Herein, a sensitive and specific electrochemical biosensor based on RPA-CRISPR/Cas12a is developed for evaluating S. aureus. In the presence of S. aureus, the extracted target DNA fragments are efficiently amplified by recombinase polymerase amplification (RPA). The designed crRNA, binding to Cas12a, effectively recognizes the target fragment cleaving hpDNA. The signal molecule of hpDNA is cleaved from the sensing interface, resulting in a reduction of current response. Under optimal experimental conditions, the developed electrochemical biosensor exhibits remarkable sensitivity in detecting S. aureus. The linear range for quantifying S. aureus in pure culture is 1.04 × 101-1.04 × 108 CFU/mL, with a detection limit as low as 3 CFU/mL. In addition, the biosensor enables the accurate and sensitive detection of S. aureus in milk within a linear range of 1.07 × 101-1.07 × 107 CFU/mL. The electrochemical biosensor enhances anti-interference capability owing to the specific amplification of RPA primers and the single-base recognition ability of crRNA. The RPA-CRISPR/Cas12a biosensor exhibits exceptional anti-interference capability, precision, and sensitivity, thereby establishing a robust foundation for real-time monitoring of microbial contamination.

B-093 Analytical performances of a novel fluorescent immunoassay of anti-Müllerian hormone and establishment of the reference intervals in Chinese children

Abstract Background Anti-Müllerian Hormone (AMH) plays a important role in children growth and the concentrations change with age as well as gender. This study aimed to evaluate the analytical performances of a novel AMH assay based on the cycle enhanced fluorescence immunoassay (CEFIA) and establish the pediatric reference intervals of AMH Methods The experiments on imprecision, sensitivity, linearity, linear range and comparison were carried out to evaluate the analytical performances. Furthermore, the concentrations of serum AMH in 238 females and 346 males aged 0-18 years were measured by the Pylon AMH assays and the reference ranges for ages group were calculated using robust methods. Results The repeatability and the within-laboratory imprecision CVs of the assay were 3.7% and 6.4% at at 2.25 ng/mL, and 4.6% and 6.4% at 15.49 ng/mL, respectively. The sensitivity of the assay (LoB =0.05 ng/mL, LoD= 0.1 ng/mL and LoQ= 0.3 ng/mL) was verified. The AMH Pylon assays showed good linearity within the detection range (between 0.1 and 24 ng/ml) and can report up to 480 ng/mL with 20x pre-dilution of samples. The AMH concentrations of serum samples measured by the Pylon assays correlated to those measured by the Elecsys assays. In the males under 18 years old, the AMH levels were high at birth (0-1 month: median 95.10 ng/mL) and continued to increase to a peak between 7 months to one year old (median 158.80 ng/mL) before they started to decrease with age to the lowest level between 15 and 18 years old (median 6.31 ng/mL). On the other side, the serum AMH concentrations in females were low at birth (0-1 month: median 0.20 ng/mL) and increased with age (15-18 years old: median 3.03 ng/mL). The reference intervals were provided for each gender and age groups,respectively. Conclusions The Pylon AMH assays showed good analytical performances, and the AMH reference intervals in chinese children determined may provide a basis for the use in clinical diagnosis and treatment of related diseases.

B-021 Impact of Assay Conditions on FT4 Measurement Accuracy of Equilibrium Dialysis LC/MS/MS Clinical Assay

Abstract Background Reliable FT4 measurements are vital for accurate diagnosis and effective management of thyroid disorders. However, currently used FT4 immunoassays (IAs) lack standardization, leading to significant variations between platforms. Equilibrium dialysis (ED) coupled with LC-MS/MS analysis, considered the gold standard for FT4 measurement, has been adopted as a reference measurement procedure (RMP) for IA standardization. This study aimed to establish a high-throughput version of the routine clinical FT4 assay based on ED-LC/MS/MS technology and assess the impact of altered assay conditions on measurement accuracy. Methods A commercially available micro-ED plate was employed for ED-LC/MS/MS analysis. FT4 extraction from the dialysate was performed by utilizing a 96-well C18 SPE plate, followed by FT4 quantitation via LC-MS/MS in positive ion mode. Certified primary reference material was used to calibrate the assay. To evaluate assay robustness, the effects of minor method modifications were studied, including incubation time of ED, serum/buffer ratios in ED inserts, ED incubation temperature, shaker speed during ED, serum sample dilution ratio, and dialysate sample dilution ratio. Additionally, short-term and long-term stability of serum FT4 were assessed. Results The developed ED-LC/MS/MS assay successfully resolved T4, T3, reverse triiodothyronine (rT3), and other interferences within 4 minutes using C18 chromatography. The assay's linear range (0.5-100 pg/mL) encompassed clinically relevant FT4 concentrations across hypo- and hyperthyroid patient samples. Excellent agreement was observed between the routine assay based on ED-LC/MS/MS and CDC RMP with a Deming regression slope near 1 (95%CI: 0.87-1.06). Furthermore, serum/buffer ratios from 1:1 to 1:2.5 in ED inserts, shaker speeds from 75 to 150 rpm, and ED incubation times from 16 to 20 hours did not significantly affect FT4 measurement. However, elevated ED temperature (38°C) led to a more than 10% increase in FT4 values. Serum FT4 remained stable when exposed to room temperature for 12 hours, five freeze-thaw cycles, and -70°C storage for 1.5 years. Additionally, dilution of sera by up to 3-fold before ED did not alter FT4 measurement results. Diluting dialysate samples at least 3 times prior to SPE extraction proved accurate for samples exceeding the calibration curve range. Conclusions In conclusion, we successfully developed a routine clinical FT4 assay based on ED-LC/MS/MS that generates data comparable to the RMP. The assay exhibits robustness, with several altered conditions not significantly impacting measurement accuracy. This high-throughput and accurate assay demonstrates suitability for both clinical laboratories and large-scale epidemiological studies. Disclaimer: The findings and conclusions in this report are those of the author(s) and do not necessarily represent the official position of the Centers for Disease Control and Prevention/the Agency for Toxic Substances and Disease Registry. Use of trade names is for identification only and does not imply endorsement by the Centers for Disease Control and Prevention, the Public Health Service, and the US Department of Health and Human Services.

B-177 Development of a Contrived Fecal Matrix for Clinical Validation of a LC-MS/MS Assay to Quantify Microbiota-derived Metabolites in Stool Specimens

Abstract Background The gut microbiota plays a critical role in health and its disruption can impact clinical outcomes in a variety of circumstances, including graft-versus-host disease following hematopoietic stem cell transplantation, Clostridioides difficile infections, and liver disease. Metagenomic and metabolomic analyses of fecal material is frequently performed in the research setting; however, evaluation of the gut microbiota is not yet incorporated into routine patient care. One barrier to its implementation is the lack of matrix-appropriate materials for validation of clinical assays. While surrogate fecal matrices are being developed for metagenomic sequencing, matrices specific for targeted metabolomics are currently unavailable. In this study, we designed a contrived fecal matrix to validate a new LC-MS/MS method for measurement of microbiota-derived metabolites from stool specimens. Methods Common food products and mucin were individually analyzed by LC-MS/MS for quantities of three microbiota-derived metabolites - butyrate, deoxycholic acid (DCA) and taurocholic acid (TCA). Food items with the lowest endogenous metabolite concentrations were selected to create a contrived fecal matrix with a consistency similar to human stool specimens. Calibrators and quality controls (QC) were then created by spiking butyrate, DCA, and TCA into either the contrived fecal matrix or 50% methanol (MeOH). The internal standards (ISTD) used were d7-butyrate, d4-DCA, and d4-TCA, respectively. Three human stool specimens were selected and spiked at a range of metabolite concentrations to assess matrix effects. All samples, calibrators and controls were homogenized in 80% MeOH extraction solvent containing the ISTDs using beadruptor tubes. The suspension was centrifuged and 25 µL of supernatant was derivatized using 12.5 µL 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 12.5 µL 3-nitrophenylhydrazine at 40°C for 30 minutes. For optimal separation of isomers, 2 µL of sample was injected and separated on a CORTECS T3 column (1.6 µm, 2.1 x 100 mm, Waters) at 45°C with a flow rate of 0.350 mL/min and run-time of 20 min using the Exion LC AD (Sciex). Analytes were detected using a Sciex QTRAP 6500 mass spectrometer with an electrospray ionization source. Results A contrived fecal matrix containing a mix of avocado, black beans, gluten-free flour, and cornstarch blended in water and mucin was created. The contrived matrix was confirmed to contain minimal to no endogenous levels of the 3 metabolites of interest: butyrate, DCA, and TCA. Validation of the LC-MS/MS assay using the contrived fecal matrix showed a linear analytical measurement range from 25-2000 µM for butyrate, 1-60 µM for DCA, and 0.5-640 µM for TCA. Intra-assay precision at 7 QC levels for all analytes was <10%. The matrix effects from use of the contrived fecal matrix compared to patient fecal samples were 140-148% for d7-butyrate and 101-112% for d4-DCA across multiple spiked analyte concentrations. Conclusions Our LC-MS/MS method quantifies butyrate, deoxycholic acid, and taurocholic acid, metabolites which serve as indicators of microbiota health in liver disease patients. The contrived fecal matrix provides an additional tool for the development and validation of clinical assays to assess the gut microbiota.

Software Integrated Personalized Biosensing Device for Serum Creatinine Detection Based on Boron doped MXene Nanohybrid.

In recent years, an increase in the number of chronic kidney disease (CKD) cases has led to a global health burden majorly affecting underdeveloped and developing nations. A key biomarker for assessing the kidneys' normal functioning is creatinine, which is filtered out from the blood by the kidney. Thus, timely and specific detection of creatinine becomes necessary for diagnosis and subsequent treatment of CKD. Hence, in this study, we have tried to develop a field-deployable, software-integrated immunosensor for the detection of creatinine in a serum sample. The immunosensor was developed by incorporating gold nanoparticles, boron doped MXene, polyaniline, and anticreatinine antibody using an appropriate bioconjugation reaction. The developed sensor was able to detect creatinine in a linear dynamic range of 10 nM to 0.1 M with a limit of detection of 1.72 (±0.07) nM. The sensor was integrated with an indigenously developed software named "CretCheck" which simplifies the process of data analysis. The software integrated personalized biosensing device was used to find the creatinine concentrations directly from the obtained analytical signals. The developed immunosensor with the integrated software can also be implemented directly in primary health care facilities for creatinine detection in the future.

Highly Specific Non-Enzymatic Electrochemical Sensor for the Detection of Uric Acid Using Carboxylated Multiwalled Carbon Nanotubes Intertwined with GdS-Gd2O3 Nanoplates in Human Urine and Serum.

Herein, the electrochemical sensing efficacy of carboxylic acid functionalized multiwalled carbon nanotubes (C-MWCNT) intertwined with coexisting phases of gadolinium monosulfide (GdS) and gadolinium oxide (Gd2O3) nanosheets is explored for the first time. The nanocomposite demonstrated splendid specificity for nonenzymatic electrochemical detection of uric acid (UA) in biological samples. It was synthesized using the coprecipitation method and thoroughly characterized. The presence of functional groups and disorder in the as-synthesized nanocomposite are confirmed using Fourier transform infrared spectroscopy and Raman spectroscopy. Furthermore, field emission scanning electron microscopy, high-resolution transmission electron microscope, X-ray powder diffraction, and X-ray photoelectron spectroscopy provides a clear understanding of the morphology, coexisting phases, and elemental composition of the as-synthesized nanocomposites. The differential pulse voltammetry technique was utilized to elaborate the electrochemical sensing of UA using a GdS-Gd2O3/C-MWCNT modified glassy carbon electrode (GCE), The sensor showed an enhanced current response by more than 2-fold compared to bare GCE. Also, the sensor's performance was further improved by dispersing the nanocomposite in an ionic liquid with the exceptional reproducibility (SD = 0.0025, n = 3). The fabricated UA sensor GdS-Gd2O3/C-MWCNT/IL/GCE demonstrated a wide linear detection range from 0.5-30 μM and 30-2000 μM, effectively covering the entire physiological range of UA in biological fluids with a limit of detection (LOD) of 0.380 μM (+3SD of blank) and a sensitivity of 356.125 μA mM-1 cm-2. Moreover, the electrodes exhibited storage stability for 2 weeks with decrease in zero-day current by only 4.5%. The sensor was validated by quantifying UA in 12 unprocessed clinical human urine and serum samples, and its comparison with the gold standard test yielded remarkable results (p < 0.05). Hence, the proposed nonenzymatic electrochemical UA sensor is selective, sensitive, reproducible, and stable, making it reliable for point-of-care diagnostics.

A-347 A Comprehensive Exdia TRF-LFIA for Simultaneous Quantification of GFAP and NT-proBNP in Distinguishing Ischemic and Hemorrhagic Stroke

Abstract Background The goal of this study is to create a highly sensitive time-resolved fluorescence lateral flow immunoassay (TRF-LFIA) capable of concurrently measuring glial fibrillary acidic protein (GFAP) and the N-terminal fragment of B-type natriuretic peptide precursor (NT-proBNP). This assay is designed as a diagnostic tool and aims to provide an algorithm for stroke management, specifically for distinguishing between Ischemic stroke (IS) and Hemorrhagic stroke (HS). However, LFIA to quantify simultaneous serum NT-proBNP and GFAP are not yet available. Methods We have developed and validated a novel TRF-LFIA for the simultaneous quantitative detection of NT-proBNP and GFAP. The sensitivity and reproducibility of the immunoassay were significantly improved by employing specific monoclonal antibodies linked to europium nanoparticles (EuNPs) that specifically target NT-proBNP and GFAP. The detection area on the nitrocellulose membrane featured sandwich-style complexes containing three test lines for NT-proBNP, GFAP, and Control. The fluorescence intensity of these test lines on the nitrocellulose membrane was measured using an in-house developed Exdia TRF-Plus analyzer. As proof-of-concept, we enrolled patients suspected of having a stroke who were admitted within a specific time frame (6 hours). A small amount of clinical specimen (serum) was used with a well-assembled cartridge for the measurement of GFAP and NT-proBNP. Results To optimize the LFIA, an EuNPs conjugated antibodies were investigated to improve the detection sensitivity and decrease the background signal as well shorten the detection time. The Exdia TRF-LFIA cartridge offers a wide linear dynamic detection range, rapid detection, high sensitivity, and specificity. The limit of detection was determined to be 98 pg/mL for NT-proBNP and 68 pg/mL for GFAP, with minimal cross-reactivity. There were 200 clinical human serum samples that were used to evaluate this platform with high correlation. By combining the results of NT-proBNP and GFAP, we formulated an algorithm for the clinical assessment of Ischemic Stroke (IS) and Hemorrhagic Stroke (HS). According to our proposed algorithm, the combination of GFAP and NT-proBNP emerged as the most effective biomarker combination for distinguishing between IS and HS. Conclusions Exdia TRF-LFIA shows great potential as a supplemental method for in vitro diagnostics in the laboratory or in other point-of-care testing (POCT) applications. Its development substantially decreases the diagnosis time for IS and HS. The proposed algorithm not only minimizes treatment delays but also lowers medical costs for patients.

Electrochemical Detection of H2O2 Using Bi2O3/Bi2O2Se Nanocomposites

The development of high-performance hydrogen peroxide (H2O2) sensors is critical for various applications, including environmental monitoring, industrial processes, and biomedical diagnostics. This study explores the development of efficient and selective H2O2 sensors based on bismuth oxide/bismuth oxyselenide (Bi2O3/Bi2O2Se) nanocomposites. The Bi2O3/Bi2O2Se nanocomposites were synthesized using a simple solution-processing method at room temperature, resulting in a unique heterostructure with remarkable electrochemical characteristics for H2O2 detection. Characterization techniques, including powder X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and scanning electron microscopy (SEM), confirmed the successful formation of the nanocomposites and their structural integrity. The synthesis time was varied to obtain the composites with different Se contents. The end goal was to obtain phase pure Bi2O2Se. Electrochemical measurements revealed that the Bi2O3/Bi2O2Se composite formed under optimal synthesis conditions displayed high sensitivity (75.7 µA µM−1 cm−2) and excellent selectivity towards H2O2 detection, along with a wide linear detection range (0–15 µM). The superior performance is attributed to the synergistic effect between Bi2O3 and Bi2O2Se, enhancing electron transfer and creating more active sites for H2O2 oxidation. These findings suggest that Bi2O3/Bi2O2Se nanocomposites hold great potential as advanced H2O2 sensors for practical applications.

A novel label-free electrochemical immunosensor based on Ce-MOF@AgAuNPs for highly sensitive detection of monensin

A novel electrochemical immunosensor for the sensitive detection of monensin (MON) was developed. The sensor platform utilized anti-MON monoclonal antibodies and a nanocomposite (Ce-MOF@AgAuNPs), composed of cerium-based metal-organic frameworks decorated with silver-gold nanoparticles. The physicochemical properties of the Ce-MOF@AgAuNPs nanocomposite were investigated using transmission electron microscopy, energy dispersive X-ray, UV–Vis spectrophotometry, and fourier transform infrared techniques. The stepwise fabrication process and electrochemical performance of the immunosensor were investigated through electrochemical impedance spectroscopy and cyclic voltammetry. The sensor exhibited excellent analytical performance for MON detection, with a wide linear response range from 0.05 to 250 ng mL−1 and a low LOD of 0.008 ng mL−1. Additionally, the immunosensor exhibited high accuracy, with recovery rates ranging from 97.4% to 103.2% in chicken liver and 96.0%–104.7% in milk. This developed immunosensor shows great potential for food safety applications, demonstrating high specificity, stability, and reproducibility in the detection of MON.

High-Throughput RPLC-MS/MS Quantification of Short- and Medium-Chain Fatty Acids.

Short- and medium-chain fatty acids (SMCFA) are monocarboxylic acids with a carbon chain length of 1-12 carbon atoms. They are mainly produced in humans by the gut microbiota, play crucial metabolic roles, are vital for intestinal health, and have multifaceted impact on immune and neurological functions. Accurate detection and quantification of SMCFA in different human biofluids is achieved using 3-nitro phenylhydrazine (3-NPH) derivatization of the free fatty acids followed by reverse phase liquid chromatography (RPLC) separation and detection by tandem mass spectrometry (MS/MS). Here, we describe the simultaneous measurement of 14 SMCFA and lactate in detail. All 3-NPH-SMCFA-hydrazones are separated in less than 5min with an 8-min total run time (injection-to-injection). Linear dynamic range over 0.1-500μM is achieved for most SCFAs, while it is 0.05-100μM for MCFAs. Validation of the procedure depicts good linearity (R2>0.98) and repeatability (CV≤20%). The lower limit of detection (LLOD) is 10-30nM. The lower limit of quantification (LLOQ) is 50-100nM for most analytes, while it is 0.5μM for acetate. In conclusion, the method offers several benefits compared to alternative methods regarding throughput, selectivity, sensitivity, and robustness.

A-114 Protective effect of leukocytes in preventing Cold Induced Hyperkalemia

Abstract Background Cellular components, especially PRBC units stored in blood banks show progressively increasing extracellular potassium because ATP dependent Na+/ K+ATPase that pumps potassium into cells is slowed at cold but K+ leak continues. The extracellular potassium levels in these units may go well above the range of linearity of some electrolyte analyzers and may need dilution to measure accurately. We wanted to see effect of Leukocytes on this progressive cold-storage hyperkalemia. Buffy-Coat, the leukocyte rich layer of spun whole blood, a discard product of blood banks using buffy-coat-method for components, was used as the source of leukocytes. Methods Buffy-Coat-bags (from Terumo Quadruple-bag-set, in CPD) were separated from 32 donations from male donors (with IRB, consent). Eight ABO,Rh compatible pools of four buffy each, were prepared and divided into 2 groups of four pools each:Leukoreduced pools (LR group): Each pool leukoreduced by a clinical-grade commercial Leukofilter (Mitra).Non-leukoreduced pools (NLR group) rich in leukocytes. Potassium/VBG on Cobas B221(Roche), and CBC were done on days 0, 3, 7 on each pool stored at 4oC. Results Potassium was significantly lower in NLR than LR pools on days 3 &amp; 7, and potassium had significant negative correlation with WBC count. Multiple-linear-regression and analysis of additional control experiments consistently showed that independent of days of storage, pH, and Hct, WBC-Count had significant negative correlation with Potassium. Conclusions Preanalytic artefacts like “seasonal-pseudohypokalemia” and pseudohypokalemia in a subset of AMLs (opposite of pseudohyperkalemia in fragile hyperleukocytosis) suggest that leukocytes may be capable of reducing extracellular potassium. Our in-vitro closed-system controlled experiments show that leukocyte-rich/Non-Leuko-Reduced (NLR) pools were relatively protected from hyperkaleimia than filtered leukocyte-poor/Leuko-Reduced (LR) buffy pools, and potassium has significant negative correlation with leukocyte count independent of storage-time, pH &amp; Hct. Though considered otherwise bad/aggravator for “PRBC-storage-lesion”, leukocytes may help abate progressive cold-storage-hyperkalemia.

B-268 Detection of xylazine in fentanyl-positive urine drug screen specimens using a quantitative LCMS/MS method

Abstract Background Fentanyl, a synthetic opioid used clinically for severe pain management, sedation, and as a perioperative analgesic, has been at the forefront of fatal drug-involved overdoses for almost a decade. Xylazine, an α-2 adrenergic receptor agonist, is an increasingly common adulterant in illicitly manufactured fentanyl products and analogs. Given its class, xylazine is non-responsive to naloxone, an opioid receptor antagonist used to reverse opioid overdose. Importantly, the U.S. Drug Enforcement Administration reported xylazine in 23% of seized fentanyl powders and 7% of fentanyl pills; additionally, a recent study by Kariisa et. al. identified an increase of xylazine presence in fatal fentanyl-involved overdoses (2.9% in January 2019 to 10.9% in June 2022) across twenty states.1 Collectively, these trends indicate a need for increased surveillance of xylazine in overdose-presenting patients to inform appropriate clinical responses. In this study, we developed a sensitive LC-MS/MS method for xylazine quantitation and used it to determine xylazine presence in our fentanyl positive patients. Methods An LC-MS/MS method for quantitation of xylazine from urine samples was developed using a Waters Xevo TQ-XS and reversed-phase CORTECS C8 2.7 µm column. MRMs and optimal MS tuning parameters were generated for xylazine and xylazine-d6 via IntelliStart, with transition ions of 221.18→90.04 (Quan) and 221.18→136.31 (Qual) selected for xylazine quantitation against a 227.23→89.98 xylazine-d6 transition as an internal standard. An LC method was developed using water (2 mM Ammonium Acetate; 0.1% Formic Acid v/v) and methanol (2 mM Ammonium Acetate; 0.1% Formic Acid v/v) gradient for a 4-minute runtime. Calibrators were prepared by addition of the pure drug to water, while QCs were prepared using healthy volunteer urine that was confirmed negative in a routine urine drug screen (UDS). Calibrators were value-assigned using a reference laboratory. A simple processing method consisting of the addition of 200 µL methanol containing 25 ng/mL xylazine-d6 to 50 µL of standards, QCs, or urine was used for sample preparation. Remnant fentanyl-positive patient samples (determined by ARK Fentanyl II Immunoassay - Abbott Architect C16000 analyzer) submitted for routine UDS evaluation were collected for xylazine analysis. Results Using custom xylazine standards, the method yielded a linear range from 2 ng/mL to 500 ng/mL with an average R2 of 0.9995 across runs (n = 12). The LLoQ for this assay was 2 ng/mL with a CV of 18.1%. The intra-assay CVs were &amp;lt; 5.0% and inter-assay CVs were &amp;lt; 15.0%. Of preliminary patient samples tested (n = 25), xylazine was detected in 32% of fentanyl-positive samples, ranging in concentration from 2.8 ng/mL to 192.8 ng/mL with an average of 64.8 ng/mL. Conclusions Here we report a simple LC-MS/MS method for quantitation of xylazine in remnant fentanyl-positive UDS specimens by LC-MS/MS. Preliminary testing revealed a xylazine positivity rate of 32% in fentanyl-positive patients. Uncomplicated sample work-up makes this method applicable to xylazine surveillance in all UDS-positive patient samples in high-throughput clinical laboratories. Reference: 1. Kariisa M, et. al. “Illicitly Manufactured Fentanyl-Involved Overdose Deaths with Detected Xylazine.” MMWR Morb Mortal Wkly Rep 2023;72:721-727.

A-051 Validation and Implementation of the Stanbio Beta-Hydroxybutyrate Assay on Roche c502 Analyzer

Abstract Background β-hydroxybutyrate (βOHB) serves as a crucial biomarker in assessing, managing, and monitoring ketosis in various clinical scenarios, such as diabetic ketoacidosis and instances of alcohol abuse combined with starvation. This study aimed to validate a βOHB assay (Stanbio Laboratory) conducted on a Roche Cobas c502 analyzer and establish performance specifications. Additionally, we conducted a method comparison for three assays: Randox D-3-Hydroxybutyrate (Ranbut) assay on Roche c501 analyzer, Abbott Freestyle Precision B-Ketone strip point-of-care test, and the Stanbio Laboratory β-Hydroxybutyrate LiquiColor assay on Roche c502 analyzer. Methods Precision, linearity, limit of quantification (LOQ), method comparison, and sample stability were determined according to Clinical and Laboratory Standards Institute (CLSI) guidelines, using commercial quality control materials (Randox Multisera) and residual patient samples (Lithium heparin anti-coagulated plasma). The reference interval was verified by residual samples from 20 healthy individuals (normal blood glucose, HbA1c, liver enzymes, and lipid panel results). Statistical analysis was performed by EP-Evaluator (v12). Results Intra-assay and total coefficient of variation (CV) were determined as 0% and 2.4% at 0.3 and 1.21 mmol/L, respectively. The linearity range of the assay was verified from 0.0 to 9.0 mmol/L. Using CV &amp;lt; 20% as criteria, the functional sensitivity (LOQ) was 0.10 mmol/L at least. Lithium heparin anti-coagulated plasma specimens were stable for 24 hours at room temperature or seven days at 4 - 8 ̊C. The manufacturer’s reference interval was verified as 0.00 - 0.40 mmol/L. The Stanbio βOHB assay on the Roche c502 analyzer demonstrated a strong agreement with the Randox D-3-Hydroxybutyrate assay on the Roche c501 analyzer with a slope of 1.053 (95% CI: 1.037, 1.070) and intercept of -0.005 (95% CI: -0.068, 0.059) (coefficient correlation R = 0.9992). A positive bias was observed compared to the Abbott Freestyle Precision B-Ketone strip POC test (mean bias = + 0.50 mmol/L; slope = 1.630, 95% CI: 1.404, 1.857; R = 0.9184), and this discrepancy occurred particularly at βOHB &amp;gt; 5.5 mmol/L measured by Abbott method. Conclusions The Standbio βOHB assay exhibited excellent precision and linearity across a brand range of concentrations. This assay strongly agrees with the Randox assay run on a similar instrument but discordance with Abbott’s POCT method at high βOHB concentration. Overall, the validated method provides a reliable and efficient means for measuring βOHB levels on a Roche chemistry analyzer, facilitating accurate diagnosis and monitoring of ketosis-related conditions in clinical settings.

A high-efficiency and selective fluorescent assay for the detection of tetracyclines

Tetracyclines (TCs) rank second globally in the use of animal infection therapy and animal husbandry as growth promoters among all antibiotics. However, large amounts of TCs residue in food products and more than 75% of TCs are excreted into the environment, causing adverse effects on the ecological system and human health. It has been challenging to simultaneously realize low-cost, rapid, and highly selective detection of TCs. Here, inspired by the fluorogenic reactions between resorcinol and catecholamines, we find the fluorescence quenching ability of tetracycline (TC) and firstly propose a fluorescent “turn-off” detection of TC using dopamine and 4-fluororesorcinol. The optimal reaction condition for the fluorescent assay is investigated and the optimized probe showed a good limit of detection (LOD of 1.7 µM) and a wide linear range (10 µM to 350 µM). Moreover, this fluorescent assay proved to be an effective tool for detecting TC in river, Sprite, and beer samples, which represent the aquatic environments and food and may contain tetracyclines residues. Finally, the high selectivity of the method for TC has been confirmed by eliminating the interference from common substances. The proposed strategy provides a high-efficiency and selective solution for the detection of TCs in environment and food and the application fields of this fluorescent assay could be further expanded in the future.

Advanced Electrochemical Monitoring of Carbendazim Fungicide in Foods Using Interfacial Superassembly of NRPC/NiMn Frameworks

A simple, sensitive and reliable sensing system based on nitrogen-rich porous carbon (NRPC) and transition metals, NRPC/Ni, NRPC/Mn and NRPC/NiMn was developed and successfully applied as electrode materials for the quantitative determination of carbendazim (CBZ). The synergistic effect of NRPC and bimetals with acceptable pore structure together with flower-like morphology resulted in producing a highly conductive and interconnected network in NRPC/NiMn@GCE, which significantly enhanced the detection performance of CBZ. The electrochemical behavior investigated by cyclic voltammetry (CV) showed improved CBZ detection for NRPC/NiMn, due to the controlled adsorption/diffusion process of CBZ by the NRPC/NiMn@GCE electrode. The influences of various factors such as pH, NRPC/NiMn concentration, CBZ concentration and scan rate were studied. Under optimal conditions, 0.1 M phosphate-buffered saline (PBS) with a pH of 7.0 containing 30 µg/mL NRPC/NiMn, a favourable linear range detection of CBZ from 5 to 50 µM was obtained. Moreover, a chronoamperometric analysis showed excellent repeatability, reproducibility and anti-interfering ability of the fabricated NRPC/NiMn@GCE sensor. Furthermore, the sensor showed satisfactory results for CBZ detection in real samples with acceptable recoveries of 96.40–104.98% and low RSD values of 0.25–3.45%.

B-069 Characterization of a panel of antibodies as diagnostics for NASH and NAFL

Abstract Background Nonalcoholic fatty liver disease (NAFLD) is on the rise in Western industrialized countries and associated comorbidities like e.g. cirrhosis are a major cause of liver transplantations in the USA. In NAFLD, excess fat is accumulated in the liver whereas this build-up is not caused by alcohol. NASH (nonalcoholic steatohepatitis) and NAFL (nonalcoholic fatty liver) are two forms of NAFLD. NAFL represents a stage of the disease in which the liver does not exhibit major signs of inflammation or damage, while NASH does. NAFL does not necessarily progress to cause liver damage. NASH on the other hand may cause fibrosis and lead to cirrhosis with a risk of progression into liver cancer. Since NAFLD shows a prevalence of over 24-25% in both the USA and Europe, diagnosis moves more and more into focus. The current gold standard for NASH diagnosis is liver biopsy, and NAFLD as such can also be diagnosed by ultrasound and MRI, among others. A quest for biomarkers is ongoing to develop blood tests that allow NAFLD diagnosis by immunoassays for example. We have developed high affinity and specificity antibodies against four highly discussed marker proteins for NAFLD: RBP4 (Retinol Binding Protein 4), FGF21 (Fibroblast Growth factor 21), CHI3L1 (Chitinase-3-like protein 1) and PAI-1 (Plasminogen activator inhibitor-1). All four markers are discussed to be elevated in NAFLD patient serum. Methods We characterized the developed anti-RBP4 and anti-FGF21 antibodies via an in-house developed Chemiluminescence Immunoassay (CLIA). Results We have determined the limit of quantification (LoQ), limit of detection (LoD) and linear range of measurable concentrations for our antibody pairs. We further conducted spike and recovery experiments showing the precision of the developed antibodies against RBP4 and FGF21. Conclusions Together, we present antibody pairs for RBP4 and FGF21 suitable as a part of a diagnostic panel that has high potential to be applied in the diagnosis of NAFLD.