Sample Recovery Research Articles

Metal organic framework derived composite as a new sorbent for micro-solid phase extraction of parabens from breast milk samples

In an attempt to enhance the adsorptive properties while addressing the limitations associated with powdered nature, zeolitic imidazolate framework (ZIF-67)-derived cobalt-doped nanoporous carbon (Co-NPC) was incorporated into chitosan and then shaped like hollow fiber by a simple casting method. Further modification with polyaniline (PANI) was also performed to improve extraction efficacy. The applicability of the modified hollow fibers was then investigated by packing them into a cartridge and utilizing them for conducting hollow fibers-packed in-cartridge micro solid-phase extraction (HF-IC µ-SPE) of parabens including methylparaben (MP), ethylparaben (EP), and propylparaben (PP) from human breast milk samples. Factors affecting extraction performance were studied using central composite design (CCD). Under the optimal conditions, good linearity was achieved within the range of 0.5–500 μg L−1 with the determination coefficient (R2) higher than 0.9901. All analytical parameters were obtained by liquid chromatography-tandem mass spectrometry (LC–MS/MS) analysis. In this regard, the limits of detection values (LODs) were 0.5 to 1.0 μg L−1. Intra- and inter-assay precision RSDs % were lower than 7.9 % and 8.4 %, respectively. Relative recoveries of breast milk samples were found in the range of 88.0–109.5 %. Accordingly, the novel nanocomposite sorbent based on PANI@Co-NPC/Chitosan hollow fiber was found to be an efficient, simple, and cost-effective packing material for HF-IC µ-SPE. It can also be offered as a promising alternative adsorbent to coated conventional hollow fiber.

Magnetic biochar prepared from a spent mushroom substrate as an adsorbent for the analysis of pyrethroids in environmental water samples

In this study, a spent mushroom substrate (SMS) from Lentinus edodes cultivation was used to prepare biochar (BC) and magnetic biochar (MBC) at high temperatures. The magnetic field strength of MBC was proved via VSM analysis. The results of the nitrogen adsorption‒desorption isotherms of BC and MBC showed that MBC exhibited stronger adsorption, and SEM was performed to compare the microstructures of BC and MBC. TEM was performed to compare the distributions of C, O, and Fe. The FT-IR and XRD results revealed changes in the structure and the formation of new substances after magnetization; that is, MBC was more conducive to the adsorption and recovery of pesticides. Moreover, a method for the detection of tetramethrin, beta-cypermethrin, and fenvalerate in water samples (tap water, lake water, and river water) was established, and this method combined the use of MBC as adsorbent and high-performance liquid chromatography. Moreover, great linearity of the method was obtained in the range of 0.5–2.5 μg/mL; the recoveries in real samples were in the range of 70.20–73.32%, and the RSDs were in the range of 0.22–1.09%. The method is simple and effective, is suitable for the detection of pyrethroids in water, and has certain application potential.

Structural optimization, characterization, and evaluation ofbinding mechanism of aptamers against bovine pregnancy-associated glycoproteins and their application in establishment of a colorimetric aptasensor using Fe-based metal-organic framework as peroxidase mimic tags.

Atruncated aptamer (designated A24-3) was identifiedthat specifically binds to bovine pregnancy-associated glycoproteins (bPAG9) with a low dissociation constant (2.04nM) through two truncation approaches. Circular dichroism spectroscopy indicated that A24-3 formed parallel G-quadruplexes, which was subsequently confirmed using nuclear magnetic resonance (NMR) spectroscopy. Furthermore, a molecular dynamics simulation was employed to investigate the recognition mechanism of A24-3 and bPAG9. Interaction analysis showed that A24-3 folded into a parallel G-quadruplex structure with three G-tetrads, primarily through numerous hydrogen bonds and hydrophobic and π-π interactions. Finally, a novel colorimetric aptasensor was developed for detecting bPAG9 using A24-3 and an Fe-based metal-organic framework as target recognition elements and enzyme mimics, respectively. The method demonstrated a broad detection range from 0.5 to 50ng/mL, with a low detection limit of 0.03ng mL-1, and exhibited a good recovery (91.0-102%) for bPAG9-spiked serum samples. Additionally, the aptasensor was successfully applied todetecting the pregnancy-specific biomarker bPAGs in serum samples.

Producing Activated Biochar from the Reuse of Discarded Coffee Grounds with Enhanced Adsorptive Features Explored Towards the Stripping Voltammetric Sensing of Copper (II)

AbstractThis work aims to allocate coffee grounds waste to the production of biochar from pyrolysis under different temperatures (350 °C and 500 °C), to add value to it. The resulting materials were characterized by immediate, elemental, thermogravimetric analysis, SEM, FT‐IR, XRD and Raman spectroscopy. Additionally, the need to activate the biochar produced for the desired use was evaluated. The obtained biochars were applied in electroanalysis in the manufacture of modified carbon paste electrodes. This choice was made due to the possibility of reaching a high sensitivity towards the determination of Cu2+ ions. The differential pulse adsorptive anodic stripping voltammetry technique was applied to perform the Cu2+ determination and, in this case, several parameters were optimized, including biochar’ percentage, pH of pre‐accumulation step and reduction/stripping step, pre‐accumulation time, potential and time of reduction of adsorbed‐Cu2+. From this, the proposed modified electrode showed a linear voltammetric response towards Cu2+ in the range of 66.1 to 3997.3 ppb, with a limit of detection of 31.8 ppb, in addition to having been satisfactorily applied in the analysis of water samples (recoveries close to 100 %). Thus, demonstrating the potential for reuse of discarded coffee grounds for the preparation of activated biochar with outstanding electrochemical performance.

Developing a New Method for the Estimation of Valsartan (VST) in Pharmaceutical Dosage Samples by Using Diazotization and Coupling Reactions Spectrophotometrically

Introduction: A new method for valsartan (VST) estimation in pharmaceutical dosage samples by spectrophotometry was developed. Method: The method was based on the formation of coloured dye by the diazotization reaction of 3-amino-2-naphthol with sodium nitrite in an acidic medium to form diazonium compounds, which were then coupled with VST in a basic medium. Results: The drug sample showed linearity in the range 4.6 - 24.2 μgmL-1, and the λmax was found at 432nm. Sandell’s sensitivity (9.950×10-3), Molar absorbtivity (Ɛ= 4.377×104), regression equa-tion (y= 0.0775x + 0.0041), correlation coefficient (r2= 0.968), detection limit (DL= 0.668) and quantitation limit (QL= 2.024) were evaluated. Conclusion: The percentage recovery of the drug samples was found to be 100%. This method successfully determined VST in Pharmaceutical dosage samples.

Controllable fabrication of high-quantum-yield bimetallic gold/silver nanoclusters as multivariate sensing probe for Hg2+, H2O2, and glutathione based on AIE and peroxidase mimicking activity

The grave threat posed by heavy metals and food hazards has increased the urgency of rapid and precise detection for food security and human health. Efficient multivariate sensing probes are imperatively required for sensing heavy metals and tumor markers, which are still facing great challenge in terms of multi-functional integration. Here, bimetallic gold-silver nanoclusters (NG-AuAgNCs) were developed with unique aggregation-induced-emission (AIE) property and peroxidase (POD) mimicking activity towards the efficient multivariate sensing via optimization of the precursors. The NG-AuAgNCs emitted at 614 nm and enable AIE feature with lifetime of 12.61 μs and high quantum yield of 40.5%. Possessing AIE and POD activity, the NG-AuAgNCs show great potential as fluorimetric and colorimetric dual-mode probe for multivariate sensing Hg2+, H2O2 and GSH, with good recoveries in real samples. The NG-AuAgNCs paper sensors further integrating with smartphone, achieved portable detection of Hg2+ with limit of detection (LOD) of 19 nM, while the colorimetric-mode presented consecutive response to H2O2 and GSH via a reversible oxidase tetramethylbenzidine process with LODs of 7.02 and 0.45 μM, respectively. This work not only demonstrates a multivariate probe for environment and human health, but also provides valuable insights for the function integration of the nanocluster via synthetic manipulation.

Metal-organic framework-derived hierarchical flower-like DyCo-layered double hydroxide amalgamated nitrogen-doped graphene for diphenylamine detection in fruit samples: Theoretical density functional theory interpretation

Diphenylamine (DPA) is an environmental pollutant that can be potentially toxic. As a result, it is crucial to use basic and affordable analytical techniques to detect DPA. Electrochemical detection of DPA is a cost-effective and simple method. Modifying the electrodes with nanomaterials can enhance the electrochemical characteristics and sensitivity of the sensor. Herein, metal-organic framework (MOF) derived dysprosium cobalt-layered double hydroxide integrated nitrogen-doped graphene (DyCo-LDH/NG) is reported for the fabrication of the DPA sensing platform. The electrochemical oxidation of DPA is enhanced by the exceptional electrocatalytic activity and electron transfer properties of the DyCo-LDH/NG nanocomposite. Interestingly, the glassy carbon electrode (GCE) modified with DyCo-LDH/NG nanocomposite demonstrates a large linear detection range (0.05–470 μM) and a low limit of detection (0.012 µM). The density functional theory (DFT) study is employed to examine the energy levels and electron transfer sites of DPA during the electro-oxidation process. Furthermore, the practical efficiency test of the developed DPA sensor demonstrates a substantial recovery in fruit samples.

NanoMIP beacons with a co-operative binding mechanism for the all-in-one detection of methamphetamine aptamer complexes

Methamphetamine is a highly addictive stimulant with significant public health implications, necessitating the development of rapid, sensitive, and reliable detection methods. Traditional analytical techniques, though accurate, often involve complex sample preparation, expensive equipment, and lengthy analysis times. This study presents the design, synthesis, and application of nanoMIP beacons with a unique co-operative binding mechanism for the detection of methamphetamine. NanoMIP beacons selective for methamphetamine/aptamer complexes were synthesized using a solid-phase synthesis method that involved the bio-conjugation of an aptamer on the stationary phase and the incubation of methamphetamine to form a methamphetamine-aptamer complex. The resultant nanoMIP beacons were eluted off the affinity column and characterised using transmission electron microscopy. The co-operative binding mechanism, which relies on the structure-switching capability of the aptamer upon analyte binding was demonstrated through comparison of the fluorescence quenching signal of a scrambled sequence and nanoNIP controls. The fluorescence quenching assay was established using a fixed optimal concentration of aptamer and varying amounts of methamphetamine. The nanoMIP beacons showed enhanced the sensitivity (LOD = 23 ± 3.5 nM) and excellent selectivity, with a 40-fold increase in quenching for methamphetamine compared to other illicit drugs. The nanoMIP beacons demonstrated acceptable sample recoveries in both urine diluent and 50% human serum. This work provides a new strategy for the development of hybrid nanoMIP/aptamer-based sensors and provides a robust analytical tool for combating methamphetamine abuse.

Study on the Properties of rhodamine B fluorescent probe Y2ST

Rhodamine B, hydrazine hydrate, and HCl generated the intermediate LBXJ. Next, LBXJ, chromone 3-formaldehyde, and 100 % ethanol prepared the probe Y2ST. NMR, IR, and MS. described the intermediate and probe structures. Following the determination of the structure, tests were conducted on the response time, pH value, linearity, detection limit, accuracy, coordination ratio, and recovery of samples. Additionally, the fluorescence selectivity of ions was compared. The rate of Zn2+ detection in zinc gluconate was 96.1 % at 5 × 10−6 mol/L--5 × 10−5 mol/L, while the detection limit of Y2ST was 0.7 × 10−6 mol/L. It can be applied to Zn2+ content determination.

Mo-doped carbon-dots nanozyme with peroxide-like activity for sensitive and selective smartphone-assisted colorimetric S2− ion detection and antibacterial application

Sulfur ion (S2−) plays a significant and considerable role in many living organisms and ecosystems, while its abnormal content can pose a serious hazard to human health and ecological environment. Hence, it is extremely meaningful to construct a highly sensitive and selective analytical platform for S2− detection in complex microenvironment, particularly in biological systems. In this study, phosphomolybdic acid and L-Arg were utilized to prepare a new molybdenum doped carbon-dots nanozyme (Mo-CDs) with great peroxidase-like activity by one-step hydrothermal approach. In the presence of H2O2, Mo-CDs converted 3,3′,5,5′-tetramethyl benzidine (TMB) into blue oxTMB, but S2− strongly reduced the blue solution to colorless and then brown, which established significant selectivity toward S2−. Mo-CDs illustrated a wide linear range (2.5 μM–900 μM) and low detection limit (LOD = 76 nM) by ultraviolet and smartphone-assisted visualized colorimetric analysis. Especially, the smartphone-assisted analysis platform successfully realized quick, portable, sensitive and visible identification of S2− with high recovery (95.7–106.7 %) and excellent specificity in water samples. More importantly, Mo-CDs was developed to antibacterial applications based on good peroxidase-like activity. This research not only constructed a new and efficient carbon-dots nanozyme and a low-cost, portable, visual analysis platform for real-time detection of S2−, but also proposed a novel design strategy and methodology for exploiting multifunctional nanozyme detection tool with great practical application.

Highly selective guanidine-linked covalent organic framework for efficient removal of perfluoroalkyl carboxylic acids from water samples

In this study, guanidine-linked covalent organic framework (TPDGCl) was synthesized via a solvothermal method. The presence of guanidine groups in its structure facilitates electrostatic interactions and provides hydrogen bonding sites, enabling the selective enrichment of perfluoroalkyl carboxylic acids (PFCAs) from environmental water samples. The material exhibits rapid kinetic mass transfer, which facilitates the efficient removal of samples containing PFCAs from aqueous solutions within 5 min. Furthermore, TPDGCl exhibits several advantageous properties, including high selectivity for PFCAs, a high adsorption capacity (2005 mg/g), excellent chemical stability, and remarkable recyclability (five cycles). Additionally, density functional theory (DFT) studies have demonstrated that the adsorption of PFCAs on TPDGCl is enhanced by ion exchange, hydrophobic interactions, hydrogen bonding interactions and ordered channel structures. Finally, TPDGCl was used as an adsorbent for dispersive solid-phase extraction (d-SPE), in combination with ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), to develop an analytical method for the efficient extraction and sensitive detection of PFCAs. The developed method exhibits highly sensitivity, with detection and quantification limits of 0.64–0.85 ng/L and 2.6–3.4 ng/L, respectively. The recoveries of actual water samples ranged from 75.4% to 113.7%. Moreover, TPDGCl effectively reduces the levels of PFCAs in real water samples to less than 70 ng/L within only 1 h. These results indicate that TPDGCl is an effective adsorbent for removing PFCAs from environmental water samples.

Liquid chromatography-mass spectrometric method for the simultaneous analysis of branched-chain amino acids and their ketoacids from dried blood spot as secondary analytes for the detection of maple syrup urine disease

BackgroundMaple syrup urine disease (MSUD) is an aminoacidopathy caused by a defective branched-chain alpha-ketoacid dehydrogenase complex, leading to the accumulation of branched-chain amino acids (BCAAs) and their respective keto acids (BCKAs). A comprehensive test was developed to measure BCAAs and BCKAs using LC-MS from dried blood spot (DBS) samples for the diagnosis and prevention of MSUD in newborns and infants. MethodsAnalytes were extracted from DBS using a methanol:0.1 % v/v formic acid solution (75:25) containing internal standards and analyzed on a Luna PFP column (150 mm × 4.6 mm, 3 µm) at a flow rate of 0.3 mL/min. The method was validated for linearity, accuracy, precision, recovery, carry-over, matrix effect, hematocrit, blood volume, and punch position effects. Biomarker stability in the matrix and stock solution was assessed. Correlation with the plasma method was determined using Pearson’s correlation coefficient and Bland-Altman analysis. The method established reference ranges for the Udupi district population in South India. ResultsThe method demonstrated linearity (r2 > 0.99), with a lower limit of detection at 2 µM (BCAA) and 1 µM (BCKA), and acceptable recovery of QC samples. Hematocrit, blood volume, punch position, and storage condition effects were within acceptable limits. Correlation and Bland-Altman analysis showed strong interconvertibility between plasma and DBS assays. Reference ranges for leucine, isoleucine, valine, KIC, KIV, and KMV were established. ConclusionThe developed DBS method, requiring no derivatization and involving simple sample preparation with short run times, is a cost-effective and reliable approach for the confirmatory diagnosis of MSUD.

Consolidated Curium Reprocessing Procedure Inspires Molecular Design for Sensitized Curium Circularly Polarized Luminescence.

Curium's stable redox chemistry and ability to emit strong metal-based luminescence make it uniquely suitable for spectroscopic studies among the actinide series. Targeted ligand and coordination compound design can support both fundamental electronic structure studies and industrial safeguards with the identification of unique spectroscopic signatures. However, limited availability, inherent radioactive hazards, and arduous purifications have long inhibited such investigations of this element. A consolidated reprocessing procedure for curium has been developed for the milligram scale. The recovery of not only standard legacy curium samples but also hazardous legacy perchlorate containing curium samples was achieved, culminating in column chromatography utilizing the extraction resin DGA (N,N,N',N'-tetra-2-ethylhexyldiglycolamide, branched). Surprisingly, controlled elution of the Cm band from the extraction resin was followed through bright pink luminescence triggered by an inexpensive hand-held UV-vis lamp (380-400 nm). This observation inspired the design of an enantiopure, C2-symmetrical ligand bearing a chiral (trans-1,2-diaminocyclohexane) backbone with achiral DGA moieties (N,N,N',N'-tetra-n-octylacetamide), that enabled rarely observed curium circularly polarized luminescence upon metal chelation. These combined achievements should unlock more luminescence and circularly polarized luminescence studies of curium, and enable the recovery of many curium and other trivalent actinide samples.

B-291 Validation of a Simple Qualitative Immunoassay for 21 Drugs of Abuse in Meconium on a Single Biochip Array

Abstract Background Drugs use during pregnancy is a major social and medical issue, which may lead to significant pre- and post-natal complications, such as preterm birth and newborn mortality. Exposed newborns may have long-term behavioral, cognitive, and developmental problems. Accurate detection of in utero drug exposure is crucial for timely medical intervention for the newborn and the mother. Meconium is the ‘gold standard' matrix for detection of drugs in newborn which reflects drug exposure during pregnancy. This study aims to develop and optimize a clinical assay for simultaneous screening of 21 drugs in meconium using a custom biochip array. Methods Custom biochip array (Randox Laboratories Ltd.) included 21 discrete test regions (DTRs) for amphetamine, methamphetamine, opioids/opiates (targeting morphine, oxymorphone, oxycodone, hydrocodone, 6-monoacetyl morphine, tramadol, buprenorphine, fentanyl, and methadone), phencyclidine (PCP), cocaine (targeting benzoylecgonine), benzodiazepines (targeting oxazepam, lorazepam, and clonazepam), zolpidem, barbiturates (targeting phenobarbital), ethyl glucuronide, methylphenidate, and marijuana metabolite (targeting Delta-9-Carboxy-Tetrahydrocannabinol [THC-COOH]). Patient samples were obtained from the residual meconium samples that had been previously used for other tests. Blank meconium samples were spiked at in-house cutoff, negative and positive assay control levels. All samples were extracted in specimen diluent using Biotage® Lysera and centrifuged. The supernatant was analyzed on a fully automated Randox Evidence+® Analyzer. A robotic arm pipetted 60 µL sample on to a biochip and then the analyzer performed a competitive chemiluminescent immunoassay. The results were generated as relative light units (RLUs). Results The sample results were determined to be “positive” or “negative” based on average RLU of cutoff controls for each analyte on an assay. The intra-assay (CV ≤ 15%) and inter-assay (CV ≤ 18%) precision was 100% agreement for positive and negative controls and there was no observed carryover for any of the analytes. The sample recovery was excellent with minimal to no matrix suppression for all analytes. Accuracy was performed by comparing spiked analyte results from Randox Evidence+® Analyzer with the expected results and all the spiked samples matched (positive or negative). When authentic patient or spiked results were compared with external laboratory test results, some discordant results were obtained. However, the discrepancies could be explained based on the different cutoffs (e.g., the Randox assay had lower cutoffs) or the known assay cross reactivities within the analytes of same class. These deviations did not affect clinical utility of the assay. Conclusions We validated a novel and simple qualitative chemiluminescent immunoassay in meconium using a custom biochip array. Unlike other routine screening/ELISA methods, which require separate assays for detection of similar drug targets, the custom biochip array offers a convenient, rapid and efficient method that utilizes only a single sample preparation for the screening of all 21 drug targets. Therefore, this technology has potential to be utilized as high-throughput screening assay. Like all qualitative assays, the limitation of this test is that all positive results will need to be assessed quantitatively for a definitive interpretation.

Analysis of tetrahydroisoquinolines formed after simultaneous consumption of ethanol and amphetamine or its derivatives by LC-MS/MS in human blood, brain, and liver tissue.

The effects of the simultaneous consumption of amphetamine or amphetamine derivatives and alcohol have not yet been adequately clarified, particularly concerning potential condensation products resulting from the endogenous reaction between these substances and their metabolites (e.g., acetaldehyde, a metabolite of ethanol). In this study, we developed an LC-MS/MS method employing liquid-liquid extraction for the qualitative detection of some relevant condensation products belonging to the class of tetrahydroisoquinolines and their derivatives in human blood, brain, and liver samples. This includes the analysis of the substrates amphetamine, methamphetamine, methylenedioxymethamphetamine, methylenedioxyamphetamine, as well as the condensation products 1,3-dimethyl-1,2,3,4-tetrahydroisoquinoline, N-methyl-1,3-dimethyl-1,2,3,4-tetrahydroisoquinoline, 1,3-dimethyl-7,8-methylenedioxy-1,2,3,4-tetrahydroisoquinoline, and N-methyl-1,3-dimethyl-7,8-methylenedioxy-1,2,3,4-tetrahydroisoquinoline. Therefore, the reference standards of the mentioned tetrahydroisoquinolines were synthesized in advance and the method was validated with regard to the question of the qualitative detection of these compounds. The validation parameters included selectivity, specificity, limit of detection, lower limit of quantification, recovery, matrix effects, and stability for blood, brain, and liver samples. Following the analysis of human blood and post-mortem tissue samples, evidence of the condensation product 1,3-dimethyl-1,2,3,4-tetrahydroisoquinoline originating from the interaction between amphetamine and acetaldehyde was identified in two liver samples. On the contrary, no evidence of this or other tetrahydroisoquinolines was found in the remaining tissue and serum samples.

A-318 Validation of umbilical cord blood as an alternate sample type for quantitative in vitro detection of glucose-6-phosphate dehydrogenase in neonates

Abstract Background Detection of glucose-6-phosphate dehydrogenase (G6PD) deficiency provides important information for neonatal clinical management. G6PD newborn screening is recommended using umbilical cord blood specimens in vulnerable patient populations.1 Also, cord blood is used by many clinicians instead of peripheral blood for neonatal screening of G6PD.2 Use of cord blood is a modification to the FDA-approved Pointe Scientific G6PD kinetic-340 nm assay. The aim of this study was to validate cord blood as an alternate specimen type for the Pointe Scientific G6PD assay. Methods Hemoglobin (Hgb) results are from Sysmex XN 9100™ analyzers that use SLS methodology with photometric detection. G6PD results are from Roche cobas® c502 analyzers with Pointe Scientific kinetic method and spectrophotometric detection of activity, expressed as U/g Hgb. Neonatal blood specimens were obtained as paired samples from heel sticks and umbilical cord collected in EDTA lavender top tubes. Accuracy acceptance criteria was defined as Deming regression slope between 0.90 and 1.10, r-value of >0.90 and bias ±15% and mixing study recovery of ±10%. Precision (intra-assay and inter-assay), reportable range and linearity, carryover, and stability were established using cord blood samples or control materials sourced from Thermo Fisher Scientific or Sysmex. Reference intervals3, analytical sensitivity, specificity, and clinical validity were transferred to cord blood due to comparability with whole blood FDA-approved method. EP Evaluator software by Data Innovations® was used for calculations. Results Direct comparison of G6PD results (U/g Hgb) from 125 paired samples yielded a 0.9025 correlation coefficient, 1.024 Deming slope, and -0.25% bias. Mixing and recovery of admixed samples gave % recoveries of 102, 103, 101, 102, 100, and 100 for G6PD (U/g Hgb), and 102, 102, 101, 104, 99, and 105 for Hgb (g/dL). Intra-assay precision results were 2.4, 0.6, 0.8, 0.5, 0.4 % CV for G6PD and Hgb, respectively. Inter-assay precision results were 3.1, 3.3, 0.7, 0.7, 0.5 % CV for G6PD and Hgb, respectively. Reportable range and linearity evaluation resulted in a maximum deviation from target recovery of 4%, and a slope of 0.979 for a range of 50 - 2740 U/L, and reportable range for Hgb was confirmed as 0.1 - 26.0 g/dL using controls (1% maximum deviation and 0.991 slope). Carryover was -5.8 (mean concentration) for G6PD and 0% for Hgb after testing 21 replicates of pooled high and low samples in a defined order. Testing 3 samples in 3 replicates at each defined time interval defined stability as 1 week refrigerated or 72 hours room temperature for G6PD, and 3 days refrigerated or 1 day room temperature for Hgb. Conclusions Regression statistics and mixing-recovery studies demonstrated accuracy for detecting G6PD (U/g Hgb) and Hgb (g/dL) from EDTA cord blood. Additional performance characteristics were transferrable for this specimen type from the FDA-approved method for whole blood. Umbilical cord blood is an acceptable alternate specimen type for quantitative detection of G6PD (U/g Hgb) using Pointe Scientific with Sysmex XN-9100™ and Roche cobas® c502 instrumentation.

B-115 Software Data Processing and Review LC-MS/MS Workflow Improvements for Clinical Research

Abstract Background In the field of quantitative LC-MS/MS, the need for efficient, accurate and user-friendly data review software is paramount, with the data generated in the clinical laboratory meeting local guidelines, as well as rigorous method performance characteristics defined by the FDA Bioanalytical Method Validation and CLSI C62-Ed2 guidelines. The quantitative LC-MS/MS workflow is dependent on interfacing to LMS/LIMS for sample traceability, batch analysis using matrix-matched external calibrators and quality controls for quantification and acceptance, and stable-isotope labeled internal standards to compensate for sample extraction, recovery, matrix effects and analytical variability. The demands on the laboratory to run multiplexed analyte panels and analyze a greater number of samples produces large amounts of analytical data which must still go through batch and quality review before release from the laboratory to the clinicians. The Waters Quan Review Software significantly reduces the burdensome aspects of batch quality control by introducing a series of innovative features. These include improvements in workflow, a modernized interface, and the ability to flag exceptions based on acceptance criteria. Methods Samples for three multiplexed LC-MS/MS panels with previously defined analytical method performance characteristics were acquired using Waters IVD Systems controlled by MassLynx software. The data was processed, quantified, and reviewed using both the existing TargetLynx XS IVD Application Manager and the new Quan Review Software. The three quantitative LC-MS/MS methods were the analysis of four immunosuppressants drugs in whole blood, a twelve steroid panel in plasma and a urine dilute and shoot method for a drugs of abuse panel containing greater than 80 analytes. Each method used matrix matched calibrators and QCs for quantification and batch control with performance characteristics including calibration linearity, carryover, precision and accuracy. Results The analytical performance data generated using the new MS Quan data review software met the same performance criteria as the TargetLynx XS data. For immunosuppressants, the % difference of the mean repeatability and total precision (%CV) for the individual analyte QCs processed using both software was within ±1.3% and ±4.4% respectively. The mean % bias of External Quality Assurance Samples (n=40) for each immunosuppressant processed using both software was within ±7.9%. The modernized, task-oriented accelerated workflows and focused review by exception reduced review time by up to 50%. Conclusions The new Quan Review Software generated results equivalent to the existing TargetLynx XS Application Manager. The software’s enhanced workflow capabilities streamlined the review process in a new user-friendly interface which reduced the training burden enabling scientists to generate high-quality data quickly and easily. The exception focused review functionality allowed tailored rule sets which reduced the time spent reviewing data by up to 50%. Whilst the batch-level review and task-oriented workflow improved batch acceptance and simplified the review of complicated datasets into easy tasks. By integrating these features, Data Review software offers a comprehensive solution that not only improves the efficiency and accuracy of batch quality control but also contributes to the overall reliability and integrity of quantitative LC-MS/MS analysis. For Research Use Only. Not for Use in Diagnostic Procedures.

B-258 Rapid clozapine blood levels on the Beckman Coulter DxC 500 AU

Abstract Background Clozapine is the most effective drug for treatment-resistant schizophrenia. Multiple guidelines strongly recommend clozapine therapeutic drug monitoring (TDM). Measuring clozapine blood levels provides essential information when there are questions of medication adherence, efficacy, toxicity, medication interactions, or concern over other factors that may influence clozapine levels. Turnaround time is essential for effective clinical decisions. However, with liquid-chromatography/mass spectrometry, clozapine levels can take days to report results. To address the medical need for a faster turnaround time, a rapid clozapine immunoassay was developed for automated clinical chemistry analyzers. The immunoassay measuring range is based on established APA Practice Guidelines and ASCP/AGNP Consensus Statement. We developed an analyzer application for the DxC 500 AU (time to first result < 9 minutes) to expand the availability of rapid clozapine blood level testing. Methods The performance of the FDA-cleared clozapine assay was evaluated on the DxC 500 AU according to CLSI guidelines. Precision testing was performed with three clozapine spiked pools over five days with five replicates per sample. Precision sample recovery was assessed to their theoretical spike concentration. Linearity was verified with five spiked serum samples covering the measuring range. Method comparison was performed with 40 remnant patient samples vs. the Beckman Coulter AU480. Results Repeatability CV was ≤5.5% and within-laboratory precision CV was ≤6.1%. Individual deviation was ±20% and mean deviation was ±2% for all samples. Deviation from linearity was <3%. Deming regression statistics for the method comparison were a slope of 0.987, an intercept of 5.7, and an R of 0.9948. The methods are statistically equivalent: the 99% confidence limits contained 1 and 0 for the slope and intercept respectively. Conclusions The precision, recovery, linearity, and method comparison results for clozapine on the DxC 500 AU demonstrate that the analyzer can be used to generate robust results for clozapine.

Porous mesh poly-L-Cysteine and lamellar polynicotinamide-based electrochemical sensor for melatonin detection in milk and tablets

An poly L-Cys/NADH/GCE electrochemical sensor for melatonin (MT) detection was developed by electropolymerising reticulated poly L-cysteine (poly L-Cys) and lamellar poly-nicotinamide adenine dinucleotide (NADH) on a glassy carbon electrode (GCE). The morphology, elemental composition, and elemental states of the fabricated electrochemical sensor were characterized through scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS), and X-ray photoelectron spectroscopy (XPS). Additionally, the electrochemical behavior of melatonin on poly L-Cys/NADH/GCE was investigated through electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), and differential pulse voltammetry (DPV). The results showed a linear relationship for MT across the concentration range of 0.1 to 1000.0 µmol/L, with linear equations Ip(µA) = 0.0978c(µmol/L) + 0.0981, R2 = 0.9937 (concentration range: 0.1–40.0 µmol/L), and Ip(µA) = 0.0137c(µmol/L) + 5.2741, R2 = 0.9636 (concentration range: 40.0–1000.0 µmol/L). The recoveries in milk samples ranged from 98.71 % to 107.98 %, with relative standard deviations (RSD) for actual tablet samples below 8.60 %. Furthermore, the electro-oxidation sites of MT and its reaction process were deduced by calculating electron orbitals and electron cloud diagrams.

Template-synthesizing hollow cubic CeO2/Co3O4 heterostructure as electrocatalysts for sensing hydrogen peroxide

H2O2 as an important intermediary is becoming more widely acknowledged, and interest in its detection has increased significantly. Herein, we developed a hollow cubic CeO2/Co3O4 heterostructure and further employed as non-enzymatic H2O2 detection sensor through a series of facile synthesis strategies using cobalt-based zeolitic imidazolate framework as a template. The synthesized CeO2/Co3O4 with huge cavity structure offers numerous electrocatalytic active sites for the target analyte, thus endowing its high sensitivity (60.5 μA. mM−1cm−2), excellent selectivity, broad detection range for H2O2 (10 μM -1 mM), and favorable working potential (-0.45 V). Not only that the CeO2/Co3O4 sensor achieved satisfactory recovery and accuracy in real lake-water samples.