Analytical Recovery Research Articles

Implementing Differential Recovery Corrections Enhances Accuracy of Mass Balances on Microplastics in Wastewater Treatment

This study examined the impact of using different analytical recovery practices to correct MP concentrations on the performance of count and mass balances of primary wastewater settling. Spiking tests using standard MPs were conducted to evaluate the influence of MP size and sample type on recovery. The derived recovery values were then used to correct MP concentrations and loads in a full-scale primary treatment facility. The results reveal substantial differences in recovery that were dependent upon MP size and sample type, underlining the necessity of incorporating these discrepancies when estimating MP concentrations. Omitting recovery considerations during MP concentration reporting led to underestimations of approximately 40%. In contrast, incorporating MP size and sample type during recovery calculations fostered a more accurate representation of MP concentrations, thereby enhancing the closure of balance models. While a count balance model was not apparently improved through the use differential recovery, the mass balance model showed a significant improvement in closure, reducing the lack of balance from 30.2% to 17.2%. This discrepancy was primarily attributed to the differing proportions of the primary sludge stream load in each model. These findings highlight the importance of incorporating differential recovery into mass balance models and demonstrate the complementary nature of count and mass balance approaches in understanding the fate and transport of MPs in WWTPs. The results of this study have significant implications for improving environmental assessments, policies and engineering design of wastewater treatment processes.

Direct colorimetric detection of triacetone triperoxide explosive by hydroxylamine-mediated fenton chemistry catalyzed by goethite nanoparticles

Due to the absence of nitro groups or aromatic structures in its structure, spectrophotometric methods that can directly determine triacetone triperoxide (TATP) are absent. In the proposed study, a molecular spectroscopic sensor was developed based on the decomposition of TATP by hydrolysis without the addition of external acid, allowing the direct determination of TATP. The detection principle of the developed sensor is based on the decomposition of TATP by the hydrochloric acid formed as a result of the reaction of TATP with hydroxylamine hydrochloride, taking advantage of the solubility of TATP in acetone, and the hydrogen peroxide formed as a degradation product to form hydroxyl radicals catalyzed by goethite nanoparticles. The formed hydroxyl radicals, thanks to their strong oxidation properties, oxidized N,N-dimethyl-p-phenylenediamine (DMPD) to the pink-colored DMPD•+ radical cation, and the direct determination of TATP was performed by measuring the absorbance of the colored product formed. Thus, a chemo-cybernetic sequence of reactions is proposed for intact TATP assay in a circular self-sustained system, where the reactants at a given step of reactions are supplied by the products of a previous step. By applying the developed method to TATP samples prepared in acetone, absorbances due to DMPD•+ were read at 554 nm to evaluate the results. A calibration curve was created between absorbance versus TATP concentration within a final range of 10 – 33 mg L-1. The limit of detection (LOD) and limit of quantification (LOQ) of the developed TATP method were 3.3 mg L-1 and 10.8 mg L-1, respectively. Five replicate measurements were performed to determine the intra-assay and inter-assay precision of the spectrophotometric determination of TATP, and the relative standard deviations (RSD %) for TATP were found as 2.70 % and 3.76 %, respectively. Interference analysis was carried out to see the effects of ions commonly present in soil/groundwater, and of the substances used as camouflage materials due to their color and appearance similarity to TATP. The selectivity of the method toward TATP was investigated by examining the analyte recovery from explosive mixtures. The developed method was validated against a reference GC–MS method using TATP-contaminated soil samples, and the results were compared statistically.

From Exhaust to Extraction: Evaluating Car Catalysts Waste for a Resilient Economy

Spent Automotive Catalytic Converters (SACC) are comprised of a support (a honeycomb ceramic structure) coated with a catalytic layer, where Platinum Group Elements (PGE), especially Pt and Pd, facilitate oxidation and reduction reactions to reduce hazardous emissions from car engines. This study provided information about various measurement procedures and principles for characterising SACC, revealing that SACC can release Potentially Toxic Elements (PTE) under environmental conditions. The SACC samples used primarily contain cordierite and moissanite, likely distinguishable upon visual inspection of waste piles. Unlike geological samples, the SACCs samples, considered as a homogeneous matrix, exhibit major elements such as Al, Si, Mg, and Ba, with minor elements including P, Na, Ca, Fe, Ti, Ce, and Zr, posing challenges for geoanalysts and environmental managers. Sequential extraction demonstrated high concentrations of PGE in the residual phase, especially Pt, Pd, and Rh. All other fractions, oxidisable, reducible and exchangeable, showed significant analytical recoveries of PTE such as Zn, Cu, and other trace elements. Watering bulk samples resulted in exceeded reference thresholds, with high Cd, Ni, and Zn, identifying SACC as a potentially hazardous materials. Toxicity tests on three aquatic species (A. fischeri, R. subcapitata, and D. magna) indicated both acute and chronic effects, further highlighting the need for proper waste management. The characterisation approach suggested here can help define the most appropriate SACC treatment demonstrating economic profit and ecological benefits.

Implementation of Sensitive Method for Determination of Benzophenone and Camphor UV Filters in Human Urine

The level of the human body’s burden of benzophenone and camphor ultraviolet (UV) filters can be estimated from their urinary levels. The present study describes the implementations and validation of the sensitive analytical method for the analysis of seven benzophenone and two camphor UV filters in urine. Sample preparation includes overnight enzymatic hydrolysis and ethyl acetate extraction followed by purification by dispersive solid-phase extraction using a sorbent Z-Sep. For the analysis, ultra-high performance liquid chromatography coupled with tandem mass spectrometry was used. Validation was performed using a Standard Reference Material® 3673 and an artificially contaminated urine sample. Target analyte recoveries ranged from 79–113% with repeatability expressed as a relative standard deviation of 2–15%. The limits of quantification were between 0.001 and 0.100 ng/mL in urine. This method was subsequently applied to examine the urine samples collected from Czech women. The analytes benzophenone-1 and 4-hydroxy-benzophenone were the most common analytes present in 100% of the samples, whereas benzophenone-3 was quantified in only 90% of the urine samples. The other four determined benzophenone derivatives were quantified in ≤33% of the samples. The derivatives of camphor were not detected in any samples. This method could be applied in biomonitoring studies.

Chromatographic determination of oxytetracycline in milk product samples using liquid-liquid microextraction procedure

Miniaturized procedures for chemical analysis give the possibility of a significant reduction in quantities of organic solvents used during isolation and determination processes. Liquid-liquid microextraction (LLME) using butanol as an extractant was investigated to selectively isolate oxytetracycline (OTC) antibiotic residues from dairy products. The optimal conditions of LLME were established after deproteinization of the samples. The isolation procedure for OTC was performed using 700 µL of butanol. The extracts were analysed by HPLC-UV and LC-MS/MS. A satisfactory analyte recovery was achieved in the range 97.6%–98.9%. The concentration of the linearity range of the studied antibiotic should allow its determination at the level of MRL values. The limit of detection during HPLC-UV and LC-MS/MS determination of OTC in milk samples was 9.9·10−8 mol L−1 (47.85 µg kg−1) and 5.7·10−9 mol L−1 (2.75 µg kg−1), respectively. The precision (1.7%–8.6%) of the methods using LLME isolation of OTC varied depending on the kind of sample and the chromatographic technique used. The developed methods were applied to the analysis of milk and cottage cheese to estimate the presence and detection of OTC content.

Optimization of the Combined Use of Z-Sep Plus and EMR-Lipid in QuEChERS Procedure for the Analysis of Eight Pesticides in Real Milk Samples

AbstractOne of the most applied procedures for the determination of trace analytes in complex matrices is QuEChERS (an acronym for Quick, Easy, Cheap, Effective, Rugged, and Safe). QuEChERS procedures include an extraction step followed by a dispersive solid-phase extraction (dSPE) for analytes cleaning-up from the matrix components. A challenging task in QuEChERS procedures is extracting and determining pesticides from samples of high fat such as milk samples. This challenge induced the innovation of new adsorbents for the clean-up step such as Z-Sep Plus® and EMR-Lipid® to enable removal of fatty matrix components without affecting the recovery of hydrophobic analytes. This work aims to apply experimental design to optimize the combined application of both QuEChERS clean-up adsorbents; Z-Sep Plus® and EMR-Lipid® in addition to other QuEChERS parameters in the determination of eight pesticides: hexachlorocyclohexane, dichlorodiphenyldichloroethane, dichlorodiphenyltrichloroethane, primiphos ethyl, diazinon, malathion, endrin, and dimethoate in milk matrix. This was augmented by optimization of GC–MS/MS and UPLC-MS/MS to detect and determine analytes in extracts. The experimental design of QuEChERS procedure enabled the optimization of Z-Sep Plus®- and EMR-Lipid®-added adsorbent amounts with other method parameters to enable the maximum recovery of analytes. Furthermore, the optimized methods enabled low detection limits of the studied pesticides within a short analysis time (28 min for GC and 12 min for LC methods, respectively). The procedure was validated according to European SANTE/11312/2021 Guideline. Quantitation limit ranged from 1.7 to 3.2 ng/mL for GC–MS/MS method and from 1.7 to 3 ng/mL for UPLC-MS/MS method. Greenness assessment of the methods followed four approaches indicating an excellent value of greenness for the proposed methods. Furthermore, 45 real milk samples collected from the Egyptian market were tested with the developed procedure for the presence of pesticides.

Simultaneous Electrochemical Determination of Sildenafil Citrate and Tadalafil Using a Multi Walled Carbon Nanotubes Modified Glassy Carbon Electrode by Adsorptive Stripping Linear Sweep Voltammetry

AbstractAn adsorptive stripping linear sweep voltammetric method was developed for the simultaneous determination of sildenafil citrate and tadalafil. The voltammetric working electrode was a glassy carbon electrode modified with carboxylic multi‐walled carbon nanotubes. The sensitivity of the developed voltammetric method was significantly increased by this modification. Both analytes spontaneously adsorbed on the modified electrode surface due to their adsorption affinities. The irreversible oxidation signals of both analytes were used as the analytical signals. The electrochemical behaviour of sildenafil citrate and tadalafil was assessed using cyclic voltammetry. The developed voltammetric method was linear in the range of 0.01–0.50 μM with a correlation coefficient of 0.9997 and 0.9985 for sildenafil citrate and tadalafil, respectively. The LOQs of the method for both analytes were 0.010 μM. The intra‐ and inter‐day reproducibility of the method was less than 10% RSD in all cases. The extraction recoveries for both analytes ranged between 83 and 94%. The applicability of the developed voltammetric method were investigated by determining the analyte in two different spiked samples (an energy drink and a traditional herbal product). Furthermore, the described method would represent the first electrochemical approach for the simultaneous detection of sildenafil citrate and tadalafil, providing an available alternative to current chromatographic techniques.

Determination of strobilurin fungicides residual in vegetables based on amino modified magnetic graphene oxide solid phase extraction coupled with GC–MS/MS

This research investigated the creation and utilization of an amino-functionalized magnetic graphene oxide (Fe₃O₄-NH₂@GO) nanocomposite as a selective sorbent for extracting and identifying strobilurin fungicides (SFs) from vegetable samples. The investigation utilized a method where magnetic solid phase extraction (MSPE) was integrated with Gas chromatography-triple quadrupole mass spectrometer (GC–MS/MS) for analysis. The physicochemical properties of synthesized sorbent were characterized using Scanning electron microscope (SEM) , Fourier infrared transform spectrum (FT-IR) , X Ray Diffraction (XRD), and vibrating sample magnetometry (VSM) techniques. The adsorption performance of Fe₃O₄-NH₂@GO aligned with the pseudo-second-order kinetics, Elovich, and Freundlich isothermal adsorption models, suggesting various interactions with SFs, including π-π interactions, hydrogen bonding, and electrostatic forces. The maximum adsorption capacity of two selected representative SFs (Picoxystrobin and E-metominostrobin) for the adsorbent were 55.85 mg/g and 47.35 mg/g, respectively. Additionally, the amino group exhibited notable potential for SFs adsorption. Parameters for MSPE, such as the quantity of sorbent, adsorption duration, eluent types and volumes, and sample pH, were optimized. Under optimal conditions, the developed method showed satisfactory performance within a range of 10 to 2000 µg L⁻¹ (R² ≥ 0.9969), low detection limits (0.01–0.080 µg/kg), high analyte recovery (80.5–104.3 %), and good precision (RSD ≤ 4.96 %, n = 6). This method is rapid, straightforward, environmentally friendly, demonstrates good sensitivity and providing a new perspectives on developing sorbents for SFs in complex food matrices. Also Fe₃O₄-NH₂@GO as a sorbent firstly used for extraction and enrichment of phenyl fungicides residues in food samples exhibits remarkable promise, thereby contributing to the advancement of pesticides residue determination methodologies.

Determination of furmonertinib in human plasma and cerebrospinal fluid by UPLC-MS/MS: Application in lung cancer patients with and without brain metastasis

Furmonertinib (AST2818) is a selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) being developed for the treatment of patients with EGFR mutation-positive non-small cell lung cancer. Quantification of furmonertinib in plasma and cerebrospinal fluid (CSF) can be used to assess penetration of furmonertinib into the central nervous system (CNS). This paper described ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) methods for quantification of furmonertinib in human plasma and CSF. Sample separation was achieved on a Kinetex C18 column (100 mm × 2.1 mm, 2.6 μm) after simple protein precipitation with acetonitrile. The mobile phase was composed of acetonitrile and 5 mM ammonium acetate with 0.2 % formic acid in water. Quantitative ion pairs were m/z 569.3 → 72.2 for furmonertinib and m/z 526.5 → 72.2 for aumolertinib, which was used as the internal standard (IS). The calibration curves showed good linearity (r2 > 0.99) over concentration range of 0.5–200 ng/mL(plasma sample) and 0.05–30 ng/mL(CSF sample). The precision (RSD) was ≤7.86 %, and the accuracy fell within the range of 96.2 % to 109.3 %, all meeting acceptance criteria. The matrix effect was from 94.3 % to 102.1 %. The recovery of analytes fell within the range of 93.3 % to 98.9 %. The established analytical methods showed great sensitivity, simplicity, accuracy and reliability for the analysis of furmonertinib in human plasma and CSF. This assay would be helpful to predict the effectiveness and toxicities of furmonertinib in the pursuit of precision medicine for lung cancer patients.

In-Syringe Vortex-Assisted Liquid-Liquid Microextraction Based on Natural Deep Eutectic Solvent for Simultaneous Determination of the Two Anticancer Polyphenols Chrysin and Resveratrol.

The simultaneous determination of multiple anticancer drugs in combination therapy poses a significant analytical challenge due to their complex nature and low concentrations. In this study, we propose an in-syringe vortex-assisted liquid-liquid microextraction (IS-VA-LLME), based on a green natural deep eutectic solvent (NaDES) for the simultaneous determination of two coadministered anticancer drugs (resveratrol and chrysin) prior to the HPLC-UV analysis, for the first time. The key parameters affecting the extraction efficiency, such as extraction solvent, vortex time, pH, and ionic strength were optimized. Under optimal conditions, the method demonstrates good linearity over the range of 0.05-15.0 μg/mL for RVT and 0.50-15.0 μg/mL for CHR with low limits of detection (LODs) of 16.78 and 161.60 ng/mL for RVT and CHR, respectively, confirming the high sensitivity of the method. The interday and intraday precision values, expressed as %RSDs, are below 2.0%, indicating good repeatability and reproducibility. Furthermore, the proposed method could be efficiently applied for the determination of the two drugs in human plasma and river water. The obtained results show satisfactory % recoveries (97.80%-102.04%), highlighting the accuracy and reliability of the developed method. The sustainability of the method was comprehensively evaluated using seven different tools. In conclusion, the developed IS-VA-LLME-NaDES allows for enhanced extraction efficiency, reduced extraction time, and improved recovery of the target analytes. This method holds great promise for applications in clinical and environmental research, enabling the precise quantification of these anticancer drugs in complex matrices.

Development of an ultrasensitive electrochemical aptasensor based on aptamer/rGO/SPGE for the detection of Botulinum neurotoxin A (BoTN/A) in food samples

Consumption of Botulinum neurotoxin A (BoTN/A) contaminated foods has increased the public health issues which necessitates the development of rapid and simple diagnostic tools for their sensitive detection. Electrochemical aptasensors can replace existing biosensors and traditional methods of BoTN/A detection in foods. These aptasensors are simple, quick response, economic, precise, sensitive, accurate and reproducible. In the present study, an electrochemical aptasensor was constructed by immobilizing BoTN/A specific aptamer and reduced graphene oxide (rGO) onto screen printed gold electrode (SPGE) for the detection of BoTN/A in spiked food samples. The specificity of this aptasensor was achieved by conjugating BoTN/A specific aptamer on rGO/SPGE. Test sample containing BoTN/A form aptamer-BoTN/A complex onto SPGE which was directly proportional to BoTN/A concentration. Formation of aptamer-BoTN/A complex reduced the surface of working SPGE for redox reactions. Thus decreased the current peaks that was taken as measuring signal of BoTN/A in test food sample. BoTN/A aptasensor showed optimum response at pH 6.5 and 30 °C temperature. The responsetime of BoTN/A/aptamer/rGO/SPGE was 6 s. In optimum conditions aptamer/rGO/SPGE BoTN/A aptasensor exhibited wide linearity ranges from 1-100 pM and 1 pM limit of detection (LOD). The analytical recoveries of aptamer/rGO/SPGE BoTN/A aptasensor at 5 pM and 10 pM BoTN/A were 98.5 % and 99.8 % respectively. The proposed aptasensor showed unique sensitivity, reproducibility and accuracy. Moreover, aptamer/rGO/SPGE aptasensor was used for the detection of BoTN/A in five different types of food samples.

Lipid and Corticosteroid Biomarkers Under the Influence of Bisphosphonates.

Detecting the use of bisphosphonates (BPs) in equine athletes is of interest to regulators and laboratories due to the threat to welfare issues for the potential to provide analgesic effects and manipulating bone structure. The detection of BPs in biological matrices is challenging due to erratic biological elimination and inconsistent analytical recoveries. Therefore, complementary approaches are needed to provide evidence of their misuse in racehorses. BPs have two sub-classes: nitrogenous and non-nitrogenous. This study investigated plasma elimination following administration of one example from each sub-class, together with changes in endogenous eicosanoid and corticosteroids. Zoledronic acid (ZA) and tiludronic acid (TA) were administered by IV infusion to 8 thoroughbred horses with an 11-month washout period between each administration. Sample preparation for quantification of BPs by liquid chromatography-tandem mass spectrometry (LC-MS/MS) utilised a two-step solid phase extraction (SPE) consisting of polymeric reversed-phase followed by weak anion exchange prior to derivatisation using trimethyl orthoacetate. Endogenous biomarkers were analysed after protein precipitation and SPE with polymeric reversed-phase prior to liquid chromatography-high resolution mass spectrometry (LC-HRMS) using data independent acquisition. The LC-MS/MS analysis showed ZA was undetectable after 8 h post-administration while TA was detected up to the final collection point of 28 days post-administration. The LC-HRMS analysis utilised targeted (i.e., prior inclusion list of compounds) approaches to monitor level changes of eicosanoid and corticosteroid biomarkers. Putative biomarkers were identified and now subject to validation for translation into routine sample analysis for improved retrospectivity to detecting BP misuse in equine plasma.

12260 Assessing The Impact Of Saliva Collection Devices On Analyte Recovery

Abstract Disclosure: D. Marshall: None. M. Dolan: None. M. Mctaggart: None. B.G. Keevil: None. Introduction: Analysis of saliva is gathering momentum, often conveying advantages over regular venepuncture, offering a convenient way of collecting multiple samples throughout the day with relative ease from the comfort of a patient’s home or workplace. The advent of more sensitive mass spectrometry equipment has opened the possibility of measuring analytes previously unsuitable, offering a multitude of steroid hormones for diagnosis and monitoring of a range of endocrine disorders. Traditionally, samples are collected onto a Sarstedt Salivette device comprising of a synthetic swab enclosed within a polypropylene tube. For certain analytes, the Salivette device is known to impede analyte recovery, resulting in the requirement for a passive drool sample which can be difficult and unpleasant to collect for some patients. Methods Previous work has been undertaken showing the SalivaBio Oral Swab (SOS) to have less of an impact on analyte recovery. Here, through addition of low, medium and high spikes and subsequent LC-MS/MS analysis of 19 different steroid hormones (11-dehydrocorticosterone, 11-deoxycorticosterone, 11-deoxycortisol, 11-hydroxyandrostenedione, 11-hydroxytestosterone, 11-ketoandrostenedione, 11-ketotestosterone, 17-hydroxyprogesterone, 18-hydroxycortisol, 18-oxocortisol, 21-deoxycortisol, aldosterone, androstenedione, corticosterone, cortisol, cortisone, dexamethasone, testosterone and tetrahydro11-deoxycortisol) we aimed to assess a broader repertoire of analytes and whether saliva collection devices impact on analyte recovery. Results With an acceptability criterion of 10% difference from baseline passive drool samples, Salivette devices were acceptable for 12 out of 18 analytes, SOS were acceptable for 16 analytes. Both devices were unacceptable for measurement of androstenedione, testosterone and 11-deoxycorticosterone. When 10% is deemed as an acceptable difference, the SOS performs better than the Salivette device for the steroid hormones investigated in this study. This may have implications for research studies, patient diagnosis and monitoring of endocrine disorders where saliva collection devices were previously deemed unacceptable due to issues with analyte recovery. Presentation: 6/2/2024

Liquid-Liquid Extraction Solvent Selection for Comparing Illegal Drugs in Whole Blood and Dried Blood Spot with LC-MS/MS.

This study focused on the simultaneous detection of amphetamine, 3,4-methyl enedioxy methamphetamine, morphine, benzoylecgonine, and 11-nor-9-carboxy- tetrahydrocannabinol (Δ9-THC-COOH) in whole blood and DBS. It is aimed to select a solvent mixture for liquid-liquid extraction (LLE) technique employing LC-MS/MS. The obtained DBS results were compared with the whole blood samples results. A simple, rapid, and reliable LC-MS/MS method was developed and validated for all analytes in whole blood and DBS. LC was performed on a Hypersil Gold C18 column an initial step with a gradient of 0.01 % formic acid, 5 mM ammonium format buffer in water, and acetonitrile at 0.3 ml/min with 7.5-min runtime. A methanol:acetonitrile (40:60 v/v) mixture was selected for both matrices. LOQ values were 10-25 ng/mL; linear ranges were LOQ-500 ng/ml for all analytes; correlation coefficients were greater than 0.99, and all calibrator concentrations were within 20%. Analytical recovery in blood and DBS ranged from 84.9-113.2% of the expected concentration for both intra and-inter day. Analytes were stable for 1, 10, and 30 days after three freeze/thaw cycles. It was determined that the variances of the results obtained with the two matrices in the comparison study were equal for each analyte, and the results were highly correlated (r=0.9625). A sensitive, accurate, and reliable chromatographic method was developed to determine amphetamine, MDMA, morphine, benzoylecgonine, and cannabis, by performing the same preliminary steps with whole blood and dried blood spots. It was observed that the results obtained in these two matrices were compatible and interchangeable when statistically compared.

Rapid bromate determination using short-column ion chromatography-mass spectrometry: Application to bromate quantification during ozonation

A streamlined, precise, and dependable method for the concurrent quantification of bromate has been established, employing ion chromatography in conjunction with electrospray ionization tandem mass spectrometry (IC-ESI-MS/MS). In an effort to expedite sample analysis duration, the AG18 short column was utilized for rapid separation of the specified analytes. Utilizing a blend of water and acetonitrile as the mobile phase within an optimized gradient elution setting, it was possible to achieve satisfactory resolution in under 3 minutes. Specifically, the mixing of acetonitrile in water samples improves the sensitivity and immunity of the method. This phenomenon may be attributed to the similar proportions of acetonitrile in the sample and the mobile phase, which effectively reduces the viscosity and surface tension of the droplets during the ESI process. Detection limits and quantification thresholds were 0.14 µg/L and 0.41 µg/L, respectively. Analyte recoveries, when spiked into tap water between 2.5 µg/L to 50 µg/L, ranged from 69.5 % to 116.7 %; precision metrics, measured as intra- and inter-day relative standard deviations, spanned 5.1–7.9 %. The devised approach encountered minimal interference from prevalent water matrix constituents, including chloride (50–200 mg/L), sulfate (25–200 mg/L), bicarbonate/carbonate (1–6 mM), and natural organic matter (1–8 mg/L). In environmental contexts, this methodology effectively tracked the production of bromate in controlled ozonation experiments conducted in the laboratory. Segmented ozone dosing reduced bromate production when the solution contained low concentrations of DOC, which provides an alternative strategy for controlling bromate production.

B-027 CLSI-Based Result Precision on the cobas ISE neo and cobas c 703 Analytical Units Across Two Sites in Europe

Abstract Background Novel cobas® pro integrated solutions analytical units, the cobas® ISE neo and cobas c 703 analytical units (all Roche Diagnostics International Ltd, Rotkreuz, Switzerland), were assessed at two sites in Europe (Aalst, Belgium and Heidelberg, Germany). During the system design validation, precision was evaluated under intended use conditions in a routine simulation setting. Methods Quality control (QC) materials at two analyte concentrations were measured per site for selected assays (ion selective electrodes [ISEs], general chemistries and specific proteins, and immunochemistry assays); analyte recovery per QC was closely monitored. Precision was evaluated at both study sites based on Clinical and Laboratory Standards Institute EP05-A3 guidelines over 21 days for 53 applications representing the entire assay menu: six ISE serum/plasma and urine applications; 40 clinical chemistry serum/plasma and urine applications (four were measured using cobas c 503 at one site only); and seven immunochemistry serum/plasma applications. Reproducibility under various stressed routine-like conditions, including provocations, was tested using routine simulation imprecision experiments. To test the interaction of new and existing analytical units under routine conditions, two test configurations were used: the first configuration comprised one cobas ISE neo, two cobas c 703, and one cobas e 801 analytical units; and the second configuration comprised one cobas ISE neo, one cobas c 703, one cobas c 503, and one cobas e 801 analytical units. The precision of batch type measurements was compared with the precision under routine simulated random access conditions. Random access coefficients of variance (CVs) that exceeded 1.5× reference batch CVs, or single measurements deviating by >10% from the batch mean, triggered in-depth analysis of the system components that contributed to the result. CVs for intermediate precision comprised measurements from ≤4 cobas c 703 reagent disks, dependent on the system set-up and application assignment. CVs for reproducibility were calculated across four ISE measuring units, ≤3 cobas c 703 reagent disks, or ≤4 cobas e 801 measuring cells. Results Using four analytical units, n=511 CVs for repeatability, intermediate precision, and reproducibility were calculated. For repeatability, the median CV for ISE (n=24) was 0.8%, for clinical chemistry (cobas c 703: n=146; cobas c 503: n=8) was 0.9%, and for immunochemistry (n=28) was 1.0%. For intermediate precision, the median CV for ISE (n=24) was 1.1%, for clinical chemistry (cobas c 703: n=146; cobas c 503: n=8) was 1.2%, and for immunochemistry (n=28) was 1.5%. For reproducibility, the median CV for ISE (n=12) was 0.9%, for clinical chemistry (cobas c 703: n=73) was 1.6%, and for immunochemistry (n=14) was 1.7%. Conclusions The novel cobas ISE neo and cobas c 703 analytical units demonstrated robust precision, comparable to the existing cobas pro integrated solutions analytical units, over a prolonged period.

B-295 Sensitive and Rapid Homogeneous Immunoassay for the Detection of Zolpidem and its Major Metabolite in Urine

Abstract Background Zolpidem, a schedule IV controlled substance under the Controlled Substances Act, is commonly known as Ambien and Ambien CR, a prescription only treatment for insomnia. Zolpidem is one of the most common prescribed sleep aids in the U.S. due to its rapid sedative effects. The recommended duration of treatment is between two days and four weeks, as long-term use of zolpidem increases the risk of habit formation. Addiction to zolpidem can cause severe side effects, including memory loss, delusion, and in extreme cases, fatal overdose. From 2005-2010, Zolpidem-related emergency room visits doubled, underscoring the need for accurate testing as zolpidem testing plays an important role in cases of misuse, diversion, and forensics. The sole commercially available homogeneous immunoassay for the detection of zolpidem in urine has a relatively high cutoff at 20 ng/mL and poor cross-reactivity (0.02%) to major metabolite zolpidem 4-carboxylic acid. ARK Diagnostics has developed the ARK™ Zolpidem Assay to detect zolpidem at a cutoff concentration of 10 ng/mL with high cross-reactivity to its metabolite, Zolpidem phenyl 4-carboxylic acid and no cross-reactivity to zaleplon and zopiclone at concentrations below 100,000 ng/mL. Methods The ARK™ Zolpidem Assay is a liquid stable homogeneous enzyme immunoassay, consisting of two reagents, with a cutoff concentration of 10 ng/mL and semi-quantitative range up to 40 ng/mL. Preliminary performance characterization for this assay was evaluated on the Beckman Coulter AU680 Automated Clinical Chemistry Analyzer. Precision, analytical recovery, specificity, Histogram Overlap Analysis of ±50% controls and the cutoff, and method comparison with LC-MS/MS were evaluated. Results In semi-quantitative mode, total precision ranged from 2.6 to 3.1% CV. Spiked recovery of zolpidem ranged from 88.6% (2.5 ng/mL) to 96.8% (10 ng/mL). The major metabolite, zolpidem phenyl-4-carboxylic acid, showed an approximate equivalence to the 10 ng/mL zolpidem cutoff at 30 ng/mL (33.3% cross-reactivity). Histogram overlap analysis showed no overlap between cutoff and control levels. Method correlation with LC-MS/MS using authentic urine samples showed an excellent agreement with a specificity of 100% and sensitivity of 100% (15 positives and 100 negatives). Conclusions The ARK™ Zolpidem Assay measures zolpidem and its major metabolite zolpidem phenyl-4-carboxylic acid in human urine with acceptable performance. The assay is sensitive, rapid, and applicable to a wide range of clinical chemistry analyzers.

B-182 Development and validation of an LC-MS/MS assay for simultaneous measurement of serum androstenedione and 17-hydroxyprogesterone in serum

Abstract Background Androstenedione and 17-hydroxyprogesterone are measured via immunoassays or high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) in clinical laboratories. Immunoassays have limited sensitivity and specificity for certain steroid hormones testing. We aimed to develop and validate a sensitive, accurate, and specific in-house LC-MS/MS assay to simultaneously measure both 17-hydroxyprogesterone and androstenedione in serum. Methods 13C-labeled androstenedione and 17-hydroxyprogesterone internal standards were added to calibrator, control, and patient samples. A hexane/ethyl acetate solution was used in a liquid-liquid extraction procedure. The organic layer was then removed and dried followed by reconstituting the extracts in 2.5 mM ammonium formate in 50% methanol. The HPLC mobile phase gradient began with 47%, 2.5 mM ammonium formate in water and 53%, 2.5 mM ammonium formate in methanol. This progressed to 56% of the methanol phase over three minutes. The mixtures were separated though an Agilent 1260 Infinity HPLC system with a Poroshell 120 EC-C18 analytical column (2.1 × 50 mm × 2.7 µm). The separated HPLC eluents then entered an Agilent 6460C QQQ mass spectrometer in positive ion mode through an electrospray ionization source where protonated androstenedione (m/z = 287.2) with the protonated androstenedione-[2, 3, 4-13C3] internal standard (m/z = 290.2) and protonated 17-hydroxyprogesterone (m/z = 331.2) with the protonated 17-hydroxyprogesterone-[2, 3, 4-13C3] (m/z = 334.2) were mass-selected for collision-induced dissociation. For androstenedione, respective quantifier and qualifier m/z values of 287.2 > 97.1 and 287.2 > 109.1 were used while respective quantifier and qualifier m/z values of 331.2 > 97.1 and 331.2 > 109.1 were used for 17-hydroxyprogesterone. Validation studies were performed including imprecision, lower limit of quantitation (LLOQ), analytical measurement range (AMR), interference, specificity, carryover, extraction recovery, matrix effect, and comparison with a reference laboratory LC-MS/MS method. The total allowable error limits used in this study were 23.5% for androstenedione and 30.2% for 17-hydroxyprogesterone. Results Total imprecision results across three concentrations yielded a maximum CV of 4.6 % for androstenedione and 6.5% for 17-hydroxyprogesteron. The AMRs of androstenedione and 17-hydroxyprogesterone were 0.2 to 13.4 and 0.1 to 22.0 ng/mL without significant carryovers. No significant interferences were observed from 11-deoxycorticosterone (700 ng/dL), testosterone (2500 ng/dL), DHEA (1000 ng/mL), bilirubin (30.1 mg/dL), hemoglobin (1032 mg/dL), or triglycerides (1858 mg/dL). The extraction recoveries of androstenedione and 17-hydroxyprogesterone were 98% and 81%, respectively. No significant matrix effects were identified. In comparison to the reference lab LC-MS/MS method, the in-house LC-MS/MS assay demonstrated a 0.523% bias for androstenedione and a -3.77% bias for 17-hydroxyprogesterone with both exhibiting excellent correlations with the reference lab measurements. More specifically, our results (as a function of values obtained from the reference lab) followed the relationship of y = 1.039x - 0.092 (R = 0.9951) for androstenedione and y = 0.957x + 0.01 (R = 0.9974) for 17-hydroxyprogesterone. Conclusions An LC-MS/MS assay is developed for simultaneous detection of serum androstenedione and 17-hydroxyprogesterone. The assay demonstrates acceptable imprecision, AMR, and accuracy. No significant carryover and interference from other structurally similar steroid hormones and other common interferents are identified.

Systematic Comparison of Extract Clean-Up with Currently Used Sorbents for Dispersive Solid-Phase Extraction

Dispersive solid-phase extraction (dSPE) is a crucial step for multiresidue analysis used to remove matrix components from extracts. This purification prevents contamination of instrumental equipment and improves method selectivity, sensitivity, and reproducibility. Therefore, a clean-up step is recommended, but an over-purified extract can lead to analyte loss due to adsorption to the sorbent. This study provides a systematic comparison of the advantages and disadvantages of the well-established dSPE sorbents PSA, GCB, and C18 and the novel dSPE sorbents chitin, chitosan, multi-walled carbon nanotube (MWCNT), and Z-Sep® (zirconium-based sorbent). They were tested regarding their clean-up capacity by visual inspection, UV, and GC-MS measurements. The recovery rates of 98 analytes, including pesticides, active pharmaceutical ingredients, and emerging environmental pollutants with a broad range of physicochemical properties, were determined by GC-MS/MS. Experiments were performed with five different matrices, commonly used in food analysis (spinach, orange, avocado, salmon, and bovine liver). Overall, Z-Sep® was the best sorbent regarding clean-up capacity, reducing matrix components to the greatest extent with a median of 50% in UV and GC-MS measurements, while MWCNTs had the largest impact on analyte recovery, with 14 analytes showing recoveries below 70%. PSA showed the best performance overall.

Reshaping Capillary Electrophoresis With State-of-the-Art Sample Preparation Materials: Exploring New Horizons.

Capillary electrophoresis (CE) is a powerful analysis technique with advantages such as high separation efficiency with resolution factors above 1.5, low sample consumption of less than 10µL, cost-effectiveness, and eco-friendliness such as reduced solvent use and lower operational costs. However, CE also faces limitations, including limited detection sensitivity for low-concentration samples and interference from complex biological matrices. Prior to performing CE, it is common to utilize sample preparation procedures such as solid-phase microextraction (SPME) and liquid-phase microextraction (LPME) in order to improve the sensitivity and selectivity of the analysis. Recently, there have been advancements in the development of novel materials that have the potential to greatly enhance the performance of SPME and LPME. This review examines various materials and their uses in microextraction when combined with CE. These materials include carbon nanotubes, covalent organic frameworks, metal-organic frameworks, graphene and its derivatives, molecularly imprinted polymers, layered double hydroxides, ionic liquids, and deep eutectic solvents. The utilization of these innovative materials in extraction methods is being examined. Analyte recoveries and detection limits attained for a range of sample matrices are used to assess their effects on extraction selectivity, sensitivity, and efficiency. Exploring new materials for use in sample preparation techniques is important as it enables researchers to address current limitations of CE. The development of novel materials has the potential to greatly enhance extraction selectivity, sensitivity, and efficiency, thereby improving CE performance for complex biological analysis.