Spike Recovery Studies Research Articles

Simultaneous Determination of Sulfamethoxazole and Trimethoprim from Clinical Urine and Blood Serum Samples by the Application of Poly(Cu2P4BCL4)/GCE.

Synthetic antibiotics known as sulfonamides suppress the synthesis of tetrahydrofolic acid, which cures respiratory tract infections and protozoal infections by preventing the creation of dihydrofolic acid. Electrochemical sensors based on tetrakis(1,10-phenanthroline)-μ-(4,4'-bipyridine) dicopper(II) chloride monohydrate ([P2Cu-Bip-CuP2]Cl4·H2O or simply Cu2P4BCl4) have been successfully applied for the determination of sulfamethoxazole (SMX) and trimethoprim (TMP) from samples. The experimental conditions and parameters were optimized to achieve the best electrode performances for simultaneous quantification of SMX and TMP. Based on the analysis of the effect of scan rate on the peak parameters, the R 2 for the peak current vs square root of the scan rate was greater than that of the peak current vs scan rate, indicating diffusion-controlled behavior of both species. The current intensities of both SMX and TMP were highly improved due to surface activation of the electrodes by electropolymerization. For SMX, the limit of detection was determined to be 27.94 nM, while for TMP, it was 21.56 nM, and the limit of quantifications was 71.88 nM, and the corresponding relative standard deviation for each was 0.74% and 0.11%. The constructed electrode was stored for varying durations ranging from two h to 2 days, and it was found to be above 97% stable after storing for 15 days. The real applicability of the suggested sensor for the simultaneous determination of SMX and TMP was verified by sensing clinical serum and urine samples and their spike recovery studies.

B-168 Development and Validation of a Quantitative Ultra-Performance Liquid Chromatography Quadrupole Time-of-Flight (UPLC-QToF) Method for Urine Organic Acid Analysis

Abstract Background Urine organic acid (UOA) analysis is essential for the diagnosis of inborn errors of metabolism (IEMs). Traditionally, UOA analysis is performed with gas chromatography-mass spectrometry (GC-MS) and requires time-consuming sample preparation steps including liquid-liquid extraction and derivatization. The rapid development of Ultra-Performance Liquid Chromatography Tandem Mass Spectrometry (UPLC-MS/MS) in the past few years provides the opportunity to perform UOA analysis with a dilute-and-shoot methodology. We describe the development and validation of a quantitative Ultra-Performance Liquid Chromatography Quadrupole Time-of-Flight (UPLC-QToF) method for UOA analysis. Methods Urine specimens were diluted to normalize creatinine concentrations to 1 mmol/L. 20 µL of urine specimen (diluted), calibrator, or quality control (QC) material was mixed with 400 µL of mobile phase A (0.05% formic acid in water) and a mixture of isotope-labeled internal standards. After centrifugation, 10 µL of the supernatant was analyzed using a Xevo G3 QTOF mass spectrometer (Waters) with a ACQUITYTM Premier HSS T3 1.8 µm VanGuardTM FIT 2.1 x 150 mm column (Waters). Data collection was performed with negative electrospray ionization (ESI) mode using the MSE method to produce fragment ions when applicable. Repeatability, reproducibility, and carryover were assessed using the QC materials. The analytical measuring range (AMR) was assessed using synthetic urine spiked with increasing concentrations of each organic acid. Accuracy was assessed by method comparison with the UOA test performed at Mayo Clinic Laboratory and by spike-recovery study using a pooled urine specimen. Matrix effect was also evaluated with matrix dilution study. Results An optimized LC method was used to enable high-resolution separation of selected UOAs (N = 29) and isomers. Total analytical time was 20 min per injection. Both linear and quadratic regressions were used to build the calibration curves. AMR and correlation coefficients of a few representative UOAs were: orotic acid (3.4 to 214.2 mmol/mol creatinine, R^2 = 0.99, linear regression); 2-methylcitric acid (4 to 189 mmol/mol creatinine, R^2 = 0.99, linear regression); 3-methylcrotonylglycine (0.3 to 18.0 mmol/mol creatinine, R^2 = 0.99, linear regression). Repeatability and reproducibility were mostly <=10% CV and no carryover was observed. Spike-recovery study demonstrated recoveries between 80% and 120%, and method comparison study demonstrated no discrepancies with results from Mayo Clinic Laboratory. Conclusions We have developed and validated a novel UPLC-QTOF method for UOA analysis to support the diagnosis of IEMs with acceptable analytical and clinical performances. Compared with the traditional GC-MS method, the UPLC-QTOF method requires a very small specimen volume and does not require laborious and time-consuming sample preparation steps. Continued optimization of the method will be pursued to measure more UOAs to support the diagnosis of more IEMs.

Novel electrochemical sensor based on poly([Cu(H2O)2P2]I2)/GCE for the determination of trimethoprim in clinical samples and cow's milk samples

A Novel bioanalytical detector based on Poly(Diaquabis(1,10-phenanthroline copper (II) Iodide) (poly([Cu(H2O)2P2]I2)/GCE) has been fabricated by potentiodynamic polymerization of [Cu(H2O)2P2]I2. The synthesized complex and the fabricated electrodes showed a promising electrocatalytic behavior towards the electrooxidation of Trimethoprim (TPM). Based on the regression coefficient value of scan rate with peak current and square root of peak current, the oxidation of TMP at poly([Cu(H2O)2P2]I2)/GCE was dominated by diffusion. Under the optimized experimental conditions and square wave voltammetric parameters, the poly([Cu(H2O)2P2]I2)/GCE revealed very wide responses in the range of concentration of 500 nM–160 μM with a limit of detection of 3.91 nM and a Limit of quantification of 13.02 nM with the relative standard deviation below 1.75 %. The valuable applicability of the fabricated sensor for the determination of TMP in real samples, including cow's milk, blood serum, and human urine, was successfully investigated. The electroanalysis results from the spike recovery and interference study provided an excellent range of recovery with an error percent of below 3.0 % in the combination effect with the potential interferents. The sensor exhibited excellent reproducibility, sensitivity, long-term stability, high accuracy, and fast response. The developed bioanalytical sensor revealed the best application prospect for biomedicine and environmental monitoring activities.

Determination of Taurine in Infant Formula and Adult/Pediatric Nutritionals by Ultra High-Performance Liquid Chromatography with Multiple Reaction Monitoring-Mass Spectrometry: Single-Laboratory Validation, First Action 2022.08.

Taurine (2-aminoethanesulfonic acid) is an essential β-amino acid, which is one of the most abundant intracellular amino acid component in humans. The level of free taurine in human milk is higher than in bovine milk. Since taurine is considered important for the development of a newborn, fortification of infant nutrition with taurine is common. This publication describes a rapid method for the determination of taurine in infant formula and adult/pediatric nutritionals. This publication demonstrates the suitability and applicability of the method to determine taurine content in infant formula and adult/pediatric nutritionals. Test portions are deproteinized by acid in an aqueous environment prior to analysis. The level of taurine is analyzed by hydrophilic interaction chromatography (HILIC) coupled with multiple reaction monitoring MS (MRM-MS). Quantitation is accomplished using a stable isotope-labeled internal standard (SILS) for taurine. The accuracy of the method was validated using standard reference materials and spike recovery studies. The average recoveries ranged between 92% and 105%. The relative standard deviation repeatability (RSDr) and relative standard deviation intermediate precision (RSDiR) were in the range of 0.7-4.6% (RSDr) and 1.3-6.7% (RSDiR). The limit of quantitation (LOQ) was below the requirement of 0.5 mg/100 g. The single-laboratory validation (SLV) of the method for determination of taurine content in infant formula and adult/pediatric nutritionals demonstrates that it meets the requirements per AOAC INTERNATIONAL Standard Method Performance Requirements (SMPR) 2014.013. A fast method to selectively determine the content of taurine in infant formula and adult/pediatric nutritionals was evaluated and found to be fit for purpose for routine product compliance testing. Based on the results of this SLV, the method was approved by the AOAC Stakeholder Program on Infant Formula and Adult Nutritionals (SPIFAN) Expert Review Panel for SPIFAN Nutrient Methods for First Action Official MethodsSM status.

Matrix overcompensation calibration: A new strategy to correct matrix effects of carbon origin in multielement analysis by inductively coupled plasma mass spectrometry

BackgroundElemental analysis by inductively coupled plasma mass spectrometry (ICP-MS) may suffer from matrix effects; those caused by organic matrices cannot be corrected by internal standardization. A new strategy, matrix overcompensation calibration (MOC), was developed to correct such matrix effects. ResultsClear fruit juices were diluted 1:50 in 1 % HNO3 (v/v)- 0.5 % HCl (v/v)- 5 % ethanol (v/v). A standard series was treated likewise to construct an external calibration curve. As, Se, Cd, and Pb in juices were determined by dilute-and-shoot ICP-MS based on this MOC strategy. The results agreed with those obtained by standard addition calibration and microwave-aided digestion; data accuracy was validated by spike-recovery studies. SignificanceUnlike standard addition calibration, a single external calibration curve established by MOC can be applicable to juices of diversified fruit, geographical, and manufacturer origins enhancing productivity.

Establishment of an indirect competitive immunoassay for the detection of dicamba based on a highly specific nanobody

Dicamba, a traditional highly effective and low toxicity herbicide, has gained new life with the development of dicamba-tolerant transgenic crops in recent years. However, dicamba is highly volatile and therefore easy to cause drift damage to sensitive crops. The development of efficient and sensitive detection methods is essential for monitoring of trace dicamba in the environment. Nanobody-based immunoassay plays an important role in on-site detection of pesticides. However, now rapid and sensitive immunoassay methods based on nanobody for dicamba detection were lacking. In this study, the nanobodies specifically recognizing dicamba were successfully obtained by immunising camels and phage display library construction, and then an indirect competitive immunoassay based on Nb-242 was constructed with IC50 of 0.93 μg/mL and a linear range of 0.11–8.01 μg/mL. Nb-242 had good specificity with no cross-reactivities against the dicamba analogs other than 2,3,6-trichlorobenzoic acid and the developed immnoassay had a good correlation with the standard HPLC in the spike-recovery studies. Finally, the key amino acid Ala 123, Tyr 55, Tyr 59 and Arg 72 of Nb-242 that specifically recognizing and binding with dicamba were identified by homologous modeling and molecular docking, laying an important foundation for further structural modification of Nb-242. This study has important guiding significance for constructing immunoassay method of dicamba based on nanobody and provides a sensitive, specific, and reliable detection method that is suitable for the detection of dicamba in the environment.

Development of an immunoassay based on a specific antibody for the detection of diphenyl ether herbicide fomesafen

Fomesafen belongs to the diphenyl ether herbicide, and is widely used in the control of broadleaf weeds in crop fields due to its high efficiency and good selectivity. The residual of fomesafen in soil has a toxic effect on subsequent sensitive crops and the microbial community structure because of its long residual period. Therefore, an efficient method for detecting fomesafen is critical to guide the correct and reasonable use of this herbicide. Rapid and sensitive immunoassay methods for fomesafen is unavailable due to the lack of specific antibody. In this study, a specific antibody for fomesafen was generated based on rational design of haptens and a sensitive immunoassay method was established. The half maximal inhibitory concentration (IC50) of the immunoassay was 39 ng/mL with a linear range (IC10–90) of 1.92–779.8 ng/mL. In addition, the developed assay had a good correlation with the standard UPLC-MS/MS both in the spike-recovery studies and in the detection of real soil samples. Overall, the developed indirect competitive enzyme immunoassay reported here is important for detecting and quantifying fomesafen contamination in soil and other environmental samples with good sensitivity and high reproducibility.

Certified reference material (CRM) of tea powder (BARC - D3201) for K, Ca, P, Mg, Mn, Al, Fe, Ba, Zn, Cu, Sr, Pb, As, Cd, and Hg: Method validation and its production

A certified reference material (CRM) for major and trace elements in black tea powder has been developed at National Centre for Compositional Characterization of Materials/Bhabha Atomic Research Centre (BARC), Hyderabad. The study describes an acid assisted microwave digestion of tea powder followed by quantitation of K, Ca, P, Mg, Mn, Al, Fe, Ba, Zn, Cu and Sr by ICP-AES; Pb, Cd by GFAAS and As, Hg by AFS in the digests. The method was validated in terms of selectivity, linearity, limit of detection (LOD), limit of quantification (LOQ), trueness, precision, traceability, ruggedness and combined uncertainty using a CRM of tea powder (NMIJ CRM 7505-a) and spike recovery studies. The obtained values of LOQ ranged from 0.018 % m/m for Ca to 0.35mg kg−1 for Hg. Precision of the method was found to be <5% for all the analytes (K, Ca, P, Mg, Mn, Al, Fe, Ba, Zn, Cu, Sr, Cd and Hg) excepting for Pb and As (6.5 % at 95% confidence level). The obtained LOQ values, trueness and precision established that the validated method could be used for the characterization of candidate tea CRM during homogeneity and stability studies. Candidate tea CRM (∼3.6 Kg) was processed (which included spiking, drying grinding and sieving) and its homogeneity was assessed at various levels with respect to all 15 property values. The assignment of the property values for each element was carried out after an interlaboratory comparison exercise (ILCE), where 11 laboratories from various government and private sectors participated. A total of four techniques inductively coupled plasma mass spectrometry (ICP-MS), inductively coupled plasma atomic emission spectrometry (ICP-AES), graphite-furnace atomic-absorption spectrometry (GFAAS) and atomic-fluorescence spectrometry (AFS) were used during ILCE. Property values were assigned for 14 elements (K, Ca, P, Mg, Mn, Al, Fe, Ba, Zn, Cu, Sr, Pb, Cd, and Hg) along with combined uncertainties. The concentration with expanded uncertainties of the property values were between 0.458 ± 0.025 for Ca (% m/m) to 0.60 ± 0.084 mg kg−1 for Hg. Long-term stability studies suggested that the produced tea CRM is stable at storage temperature (4 °C) for the last two years, whereas, short-term stability studies (at temperatures 4, 25 and 40 °C) established its transportation stability at tested temperatures for a period of three weeks. Preparation and certification of tea CRM (BARC-D3201) was carried out in accordance with ISO Guides 17034 and 35.

Design and Fabrication of a 3D-Printed Microfluidic Immunoarray for Ultrasensitive Multiplexed Protein Detection.

Microfluidic technology has revolutionized device fabrication by merging principles of fluid dynamics with technologies from chemistry, physics, biology, material science, and microelectronics. Microfluidic systems manipulate small volumes of fluids to perform automated tasks with applications ranging from chemical syntheses to biomedical diagnostics. The advent of low-cost 3D printers has revolutionized the development of microfluidic systems. For measuring molecules, 3D printing offers cost-effective, time, and ease-of-designing benefits. In this paper, we present a comprehensive tutorial for design, optimization, and validation for creating a 3D-printed microfluidic immunoarray for ultrasensitive detection of multiple protein biomarkers. The target is the development of a point of care array to determine five protein biomarkers for aggressive cancers. The design phase involves defining dimensions of microchannels, reagent chambers, detection wells, and optimizing parameters and detection methods. In this study, the physical design of the array underwent multiple iterations to optimize key features, such as developing open detection wells for uniform signal distribution and a flap for covering wells during the assay. Then, full signal optimization for sensitivity and limit of detection (LOD) was performed, and calibration plots were generated to assess linear dynamic ranges and LODs. Varying characteristics among biomarkers highlighted the need for tailored assay conditions. Spike-recovery studies confirmed the assay's accuracy. Overall, this paper showcases the methodology, rigor, and innovation involved in designing a 3D-printed microfluidic immunoarray. Optimized parameters, calibration equations, and sensitivity and accuracy data contribute valuable metrics for future applications in biomarker analyses.

A-108 Validation of Plasma Oxalate Measurement on the Roche Cobas c501

Abstract Background Measurement of plasma oxalate is important in patients with renal failure as oxalate may build up due to impaired excretion and lead to organ damage due to precipitation of calcium oxalate crystals. However, the measurement of plasma oxalate is challenging due to the low concentrations present in individuals with healthy kidney function and the pre-analytical steps needed to prevent erroneous oxalate results due to conversion from ascorbate. In order to provide improved turn-around-time for plasma oxalate testing, our objective was to develop and validate a method to measure plasma oxalate on the Roche cobas c501. Methods Samples used in the validation study included both fresh collections and residual plasma from specimens collected in lithium heparin, no-gel tubes. Samples from fresh collections were kept on ice prior to centrifugation, and then frozen if not analyzed immediately. Thawed plasma was acidified by addition of 12.1 M HCl to a pH between 2 and 3 and mixed thoroughly by vortexing. One milliliter of the acidified plasma was added to a Centrifree filter (Millipore). The samples were then centrifuged for 90 min at 1500 × g 20°C in a fixed angle rotor to remove plasma proteins. The resulting filtrate was assayed on the Roche cobas c501. The assay (Trinity Biotech) uses oxalate oxidase to produce an indamine dye through the production of hydrogen peroxide from oxalate in the sample, which is measured spectrophotometrically at 600 nm at a single time point. The analytical measuring range (AMR) was evaluated by performing serial dilutions of a high sample. Interferences from hemolysis, icterus, and lipemia were assessed at both low and high oxalate concentrations. Precision was evaluated using two QC concentrations, and patient samples with low and high concentrations of oxalate. Samples were tested ten times in a single day, or twice per day for ten days to estimate intra- and inter-precision, respectively. Spike recovery studies were used to assess method accuracy. Lastly, reference intervals were verified by assaying samples from twenty healthy volunteers. Total allowable error was ±30% or 2 µmol/L with a maximum 15%CV. Results The assay was validated to be linear across an AMR of 2 to 30 µmol/L, with an average recovery of 105% across five concentrations. The total assay imprecision was &amp;lt;15%. Initial spike recovery studies across the AMR showed an average recovery of 99.4% (n = 12), with a range of 76.6 to 132.4%. In a mixing study, hemolyzed and lipemic sample recovery was on average 82.1% and 76.1% respectively, which was low, but acceptable. Samples mixed with icteric specimens demonstrated unacceptably low recovery (average of 64.1%). The reference interval of &amp;lt;2 µmol/L was successfully verified as all twenty healthy volunteers had results &amp;lt;2 µmol/L. Conclusion Initial validation studies support the clinical utility of the newly developed method. Plasma oxalate methods are documented to have significant inter-method variability and variable recovery, which is hypothesized to be due to variation at the filtration step. This semi-automated assay will require less technologist time and will reduce the possibility for human error affecting the result.

Selective and sensitive measurement of human neutrophil elastase in clinical samples based on a novel assay principle for protease activity measurement

Imbalances between proteases and protease inhibitors have been associated with several pathological conditions including emphysema as seen in α1-antitrypsin deficiency. For this pathological condition, unimpeded neutrophil elastase activity has been ascribed a pivotal role in the destruction of lung tissue and thus in disease progression. Therefore, low, or non-quantifiable neutrophil elastase (NE) activity levels determined in bronchoalveolar lavage solutions indicate the success of α1-antitrypsin (AAT) augmentation therapy as NE activity will be erased. To overcome the known limitations of available elastase activity assays regarding sensitivity and selectivity, we developed a new elastase activity assay, which fundamentally relies on the highly specific complex formation between AAT and active elastase. Plate-bound AAT captured active elastase from the sample undergoing complex formation, followed by the immunological detection of human NE. This assay principle facilitated the measurement of low pM amounts of active human NE. The data of the assay performance check demonstrated adequate accuracy and precision profiles meeting currently accepted best practices for this activity assay, which can be classified as a ligand-binding assay. Furthermore, spike-recovery studies at low human NE levels, carried out for three human bronchoalveolar samples, resulted in recoveries within the 100 ± 20% range, while good linearity and parallelism of the samples’ dilution-response curves was observed. Altogether, complemented by the data of selectivity and robustness studies and the accuracy and precision profile obtained in buffer, this newly developed human NE activity assay was demonstrated to perform accurately and precisely in clinically relevant samples.

Investigation of a failed DNA assay leads to identification of an unexpected contaminant in a commonly used chromatography buffer.

For mammalian cell-derived recombinant biotherapeutics, controlling host cell DNA levels below a threshold is a regulatory requirement to ensure patient safety. DNA removal during drug substance manufacture is accomplished by a series of chromatography-based purification steps and a qPCR-based analytical method is most used to measure DNA content in the purified drug substance to enable material disposition. While the qPCR approach is mature and its application to DNA measurement is widespread in the industry, it is susceptible to trace levels of process-related contaminants that are carried forward. In this study, we observed failures in spike recovery studies that are an integral component of the qPCR-based DNA testing, suggesting the presence of an inhibitory compound in the sample matrix. We generated hypotheses around the origin of the inhibitory compound and generated multiple sample matrices and deployed a suite of analytical techniques including Raman and NMR spectroscopy to determine the origin and identity of the inhibitory compound. The caustic wash step and depth filter extractables were ruled out as root causes after extensive experimentation and DNA testing. Subsequently, 2-(N-morpholino)ethanesulfonic acid (MES), a buffer used in the chromatography unit operations, was identified as the source of the contaminant. A 500-fold concentration followed by Raman and NMR spectroscopy analysis revealed the identity of the inhibitory compound as polyvinyl sulfone (PVS), an impurity that originates in the MES manufacturing process. We have implemented PVS concentration controls for incoming MES raw material, and our work highlights the need for rigor in raw material qualification and control.

Quantitative Determination of Cannabis Terpenes Using Gas Chromatography-Flame Ionization Detector.

Background: Cannabis has a long history of being credited with centuries of healing powers for millennia. The cannabis plant is a rich source of cannabinoids and terpenes. Each cannabis chemovar exhibits a different flavor and aroma, which are determined by its terpene content. Methods: In this study, a gas chromatography-flame ionization detector method was developed and validated for the determination of the 10 major terpenes in the main three chemovars of Cannabis sativa L. with n-tridecane used as the internal standard following the standard addition method. The 10 major terpenes (monoterpenes and sesquiterpenes) are α-pinene, β-pinene, β-myrcene, limonene, terpinolene, linalool, α-terpineol, β-caryophyllene, α-humulene, and caryophyllene oxide. The method was validated according to Association of Official Analytical Chemists guidelines. Spike recovery studies for all terpenes were carried out on placebo cannabis material and indoor-growing high THC chemovar with authentic standards. Results: The method was linear over the calibration range of 1-100 μg/mL with r2>0.99 for all terpenes. The limit of detection and limit of quantification were calculated to be 0.3 and 1.0 μg/mL, respectively, for all terpenes. The accuracy (%recovery) at all levels ranged from 89% to 104% and 90% to 111% for placebo and indoor-growing high THC chemovar, respectively. The repeatability and intermediate precision of the method were evaluated by the quantification of target terpenes in the three different C. sativa chemovars, resulting in acceptable relative standard deviations (less than 10%). Conclusions: The developed method is simple, sensitive, reproducible, and suitable for the detection and quantification of monoterpenes and sesquiterpenes in C. sativa biomass.

Determination of Ethanol Content in Water Kefir Using Headspace Gas Chromatography With Mass Spectrometry Detection: Matrix Extension and Methanol Characterization.

Water kefir is a fermented beverage using water, sugar, and cultured microorganism grains as the primary ingredients. Ethanol may be present at varying levels within the final product due to the fermentation process, so it is vital to have a validated method to meet regulatory, quality, and safety requirements. This study describes using water kefir as a matrix for the evaluation of the previously validated method employing headspace gas chromatography mass spectrometry (HS-GCMS) detection for ethanol in kombucha. The study objective is to demonstrate the method originally using kombucha is also fit for the analysis of water kefir. This method will also evaluate the determination of methanol within the water kefir samples. The matrix extension study was performed as per the AOAC INTERNATIONAL guidance documents outlined in Appendix K: Guidelines for Dietary Supplements and Botanicals using HS-GCMS for ethanol determination. Ethanol determination in each water kefir sample is quantified against an external standard calibration curve. The same instrumentation is used for methanol characterization. RSDr and HorRat values obtained for from the study demonstrated acceptable precision with RSDr values of 1.03 to 6.68% and HorRat values determined to be between 0.23 and 1.52 for ethanol determination within kefir samples. Similarly, acceptable values of RSDr ranging from 1.45 to 3.39% and HorRat ranging from 0.25 to 0.49 were observed with methanol determination. For methanol determination, the limit of detection (LOD) and limit of quantification (LOQ) determined for the method in this study to be 16 and 21 ppm, respectively. The methanol spike recovery study gave overall recoveries ranging from 89 to 91%, demonstrating acceptable method accuracy. The results of this study demonstrate the previously validated HS-GCMS method for ethanol determination in kombucha can also be used to quantify ethanol in water kefir samples. The method is also suitable for the determination of methanol within water kefir samples. A straightforward method has been adapted to include the the quantification of ethanol and methanol in fermented beverages such as Water Kefir samples.

Evaluation of an adenosine deaminase (ADA) assay in serum, pleural, pericardial, peritoneal, and cerebrospinal fluids on the Roche cobas c501 analyzer

ObjectivesAdenosine deaminase (ADA) can be increased in various body fluids during infectious and inflammatory states. The objective of this study was to evaluate the performance characteristics of the Diazyme ADA assay for serum, pleural, pericardial, peritoneal, and cerebrospinal fluids using the Roche cobas c501 analyzer. MethodsAccuracy, linearity, recovery, precision, sensitivity, specificity, reference interval, and stability studies were conducted. Potential interference of hyaluronidase and ultracentrifugation pre-treatment for viscosity on ADA concentrations were further evaluated. ResultsAssay method comparison to two separate external laboratories showed the following results (slope, intercept, %bias): serum (1.053, −0.478, 4.4 %); pleural (1.046, −1.41, 2.6 %). Accuracy (109.6 % recovery) was further demonstrated using a commercially available ADA reference material (BCR647). Linearity and spiked recovery studies showed percent recoveries ranging 94.3–109.3 %. Precision across all specimen types was ≤4.7 %CV. Interference was observed with increasing concentrations of various sources of conjugated and unconjugated bilirubin. Reference intervals were established for serum and pleural fluids, and previously published reference intervals were verified for pericardial, peritoneal, and cerebrospinal fluids. All specimen types were stable for 24 h ambient (8–25 °C), 1 week refrigerated (2–8 °C), and 1 month frozen (−20 °C). Of the two types of hyaluronidase evaluated, one showed positive interference for ADA (Sigma-Aldrich, H3506; 4.59 to 17.90 average % difference from baseline). Ultracentrifugation did not interfere with results (−2.32 to 0.87 average % difference from baseline). ConclusionsThe Diazyme ADA assay was validated for use in our laboratory for all fluid types evaluated. Interference was observed with increasing concentrations of bilirubin and one source of hyaluronidase.

Quality control of cannabis inflorescence and oil products: Response factors for the cost-efficient determination of ten cannabinoids by HPLC

The quality control of medicinal cannabis should include quantification of as many cannabinoids as practicable in a routine analytical lab, to accurately reflect the quality of the product. However, the cost and availability of some cannabinoid standards is an impediment to their routine use. This work seeks to overcome this obstacle by analysing samples using relative retention times (RRT) and relative response factors (RRF), relative to CBD and CBDA reference standards which are readily available. A high-performance liquid chromatography-photodiode array method was developed to quantify ten cannabinoids (Δ9-THC, Δ8-THC, THCA-A, CBN, CBD, CDBA, CBC, CBDV, CBG, and CBGA) in dried cannabis inflorescence and cannabis oil. This method was validated according to ICH guidelines. The proposed method has detection limits ranging from 20 to 78 µg/g, which provided sufficient sensitivity for the panel of cannabinoids. Non-cannabinoid surrogate matrices were used for spike recovery studies to determine method accuracy – analyte recoveries for the inflorescence and oil ranged from 90.1 to 109.3% (inflorescence mean, 100.9%; oil mean, 99.6%). The RRT and RRF values determined independently by three analysts were comparable, indicating the method is robust. The validity of analysis using RRT and RRF was further confirmed by testing six inflorescence samples, as it was found that concentrations above the order of magnitude of the LoQ agreed satisfactorily (range, 95.0 to 111.9%; mean, 100.0%) with the concentrations obtained through the conventional approach of multipoint calibration using pure standards. The proposed method is therefore suitable for the rapid and simple determination of a panel of ten cannabinoids without having to repeatedly purchase every expensive pure standard. Accordingly, analysts in the medicinal cannabis field may explore the use of RRF and RRT for their methods and instruments.

Prospective evaluation of EDXRF for studying the cation exchange membrane separation of Co from Zr in oxalic acid media and comparison with radiotracer experiments

This work focuses on the selective separation of Co from Zr and Nb, for the decontamination of pressure tubes from pressurized heavy water reactor. Initial experiments were carried out using the respective radiotracers. These laboratory scale studies explore the feasibility of using oxalic acid, a benign complexing medium, for the Zr–Nb matrix, with targeted sorption of trace levels of 60Co on cation exchange membranes. Sorption of Co(II) on the membrane followed pseudo second order kinetics and the equilibrium capacity was 1.89 meq g-1. Oxalic acid concentration was a trade-off between the loading of matrix elements and the % sorption of Co(II). Energy dispersive X-ray fluorescence (EDXRF) spectrometry was used for monitoring the sorption of Co on the membrane from inactive sample solutions. Sorption improved the EDXRF detection limits for Co from 50 μg in solution to 1.3 μg on the membrane, where the total concentration of the matrix elements in solution was 50 g L-1. EDXRF method was validated by spike recovery and radiotracer studies.

Development and Validation of a Liquid Chromatography Mass Spectrometry Method for Simultaneous Measurement of 25-OH D3, epi-25-OH D3, 25-OH D2, Vitamin A, α-Tocopherol, and γ-Tocopherol

Abstract Background Circulatory fat-soluble vitamin levels are commonly measured to identify deficiencies that may lead to rickets, osteomalacia, night blindness, and reversible motor and sensory neuropathies. We developed and validated a rapid and robust LC-MS/MS method that simultaneously measures 25-OH D3, epi-25-OH D3, 25-OH D2, vitamin A, α-tocopherol, and γ-tocopherol for clinical use. Method 100 µL of serum was mixed with isotope-labeled internal standard and extracted using a 96-well supported-liquid extraction plate with 1.5 mL of hexanes/isopropanol (90/10) (v/v). Dried eluate was reconstituted with 100 µL of methanol/water (90/10) (v/v) and analyzed by LC-MS/MS with a 10-minute gradient. Accuracy was assessed using NIST Standard Reference Materials SRM972a and SRM968f, patient comparison analysis with a LC-MS/MS method at a reference lab, and spike-recovery studies using patient sera and vitamin D-depleted serum. Analytical measurement range (AMR) was determined by spiking 6 analytes into vitamin D-depleted serum to give 7 specimens at varying concentrations. The lower limit of the measuring interval (LLMI) was assessed using 6 pooled specimens with varying low concentrations of each analyte over 20 days. Precision (repeatability and reproducibility) was assessed using quality control materials. Interference studies were performed using pooled patient specimens spiked with varying concentrations of hemoglobin, bilirubin, or intralipid. Matrix effect was assessed by post-column infusion and by matrix dilution with saline. Results The method was linear covering physiological concentrations with r2 &amp;gt; 0.99. Repeatability and reproducibility were &amp;lt;15% CV at all QC levels. LLMI for 25-OH D3, epi-25-OH D3, 25-OH D2, vitamin A, α-tocopherol, and γ-tocopherol were 4 ng/mL (15% CV), 4 ng/mL (15% CV), 4 ng/mL (18% CV), 1 µg/dL (20% CV), 0.2 µg/mL (20% CV), and 0.2 µg/mL (8% CV). Recoveries for NIST Standard Reference Materials were between 92 - 111% and between 81 - 122% for spike-recovery studies. Passing-Bablok analyses for vitamin D total, vitamin A, and α-tocopherol demonstrated slopes between 1.04 and 1.11 and r2 between 0.94 and 0.96. Minimal matrix effect was observed. Conclusions We have developed and validated a comprehensive and rapid LC-MS/MS method for the simultaneous measurement of 25-OH D3, epi-25-OH D3, 25-OH D2, vitamin A, α-tocopherol, and γ-tocopherol for clinical use.

Thiol–ended polycaprolactone: Synthesis, preparation and use in Pb(II) and Cd(II) removal from water samples

In the present study, thiol–ended polycaprolactone (TPCL) was successfully prepared by “ring opening polymerization” using 2-3 dimercaptopropanol and caprolactone. The synthesized material was employed as a possible adsorbent for removal of Cd(II) and Pb(II) ions from water samples and the influence of essential parameters (pH of 5.0, adsorbent dosage of 10.0 mg and contact time of 30.0 min) on the adsorption process was studied. Different techniques (BET NMR, FTIR, zeta potential, TGA/DTG) were used to characterize physical and chemical properties of the polymer. Various isotherm and kinetic models were employed to understand plausible mechanisms involved in the adsorption. It is revealed that adsorption process fitted very well with the Langmuir isotherm and pseudo-second-order kinetic models for both of the ions. The adsorption capacity decreased slightly (< 10%), demonstrating the thiol–ended polycaprolactone performance even after four consecutive adsorption–desorption cycles. The spike recovery studies with real water samples yielded recoveries of over 90% which indicated the applicability of the method. As a result, thiol–ended polycaprolactone has been demonstrated to be effective sorbent for the removal of Cd(II) and Pb(II) ions from aqueous solution, indicating its potential relevance in water remediation.

Development and Validation of a Liquid Chromatography Mass Spectrometry Method for Simultaneous Measurement of 25(OH)D3, epi-25(OH)D3, 25(OH)D2, Vitamin A, α-Tocopherol, and γ-Tocopherol.

Fat-soluble vitamins are measured to identify deficiencies that may lead to rickets, osteomalacia, night blindness, and reversible motor and sensory neuropathies. We present a rapid liquid chromatography-mass spectrometry (LC-MS/MS) method that simultaneously measures 25-hydroxyvitamin D3 (25[OH]D3), epi-25(OH)D3, 25(OH)D2, vitamin A, α-tocopherol, and γ-tocopherol. We mixed 100 µL serum with internal standard and extracted it by using supported liquid extraction plates. Reconstituted specimens were analyzed by LC-MS/MS with a 10-minute gradient. The method was linear, covering physiological levels with r2 > 0.99, and the total precision was less than 15% at all quality control levels. The lower limit of the measuring intervals for 25(OH)D3, epi-25(OH)D3, 25(OH)D2, vitamin A, α-tocopherol, and γ-tocopherol were 4 ng/mL, 4 ng/mL, 4 ng/mL, 1 µg/dL, 0.2 µg/mL, and 0.2 µg/mL, respectively, with coefficient of variation of 20% or less. Recoveries were between 92% and 111% for National Institute of Standards and Technology reference materials and 81% and 122% for spike-recovery studies. Comparison studies for vitamin D total, vitamin A, and α-tocopherol demonstrated slopes between 1.04 and 1.11 and r2 between 0.94 and 0.96. Minimal matrix effect was observed for all analytes. We developed and validated a rapid LC-MS/MS method for the simultaneous measurement of 25(OH)D3, epi-25(OH)D3, 25(OH)D2, vitamin A, α-tocopherol, and γ-tocopherol.