Validation of an adjusted Evelyn–Malloy method for spectrophotometric assay of methemoglobin and preparation of quality control standards

The aim of this study was to optimize the currently used direct spectrophotometric serum prolidase enzyme activity (SPEA) assay method and compare its diagnostic accuracy with current precipitation and direct spectrophotometric assay methods, AST-to-ALT ratio, age platelet index, AST-to-platelet ratio index, cirrhosis discriminate score, Doha score, FIB-4, FibroQ, fibrosis index, Goteborg University Cirrhosis Index , King's score, and Pohl score for distinguishing Ishak F0 from F1-F3 in patients with chronic hepatitis B (CHB) infection. Liver biopsy results from 112 patients were included in this study. The SPEA values were 529(292-794) U/L, 671(486-927) U/L, and 1077(867-1399) U/L with the precipitation, current, and optimized direct spectrophotometric assay methods, respectively. According to multivariate logistic regression analysis optimized direct spectrophotometric SPEA was the only statistically significant parameter to predict the early stages of liver fibrosis. Optimized direct spectrophotometric SPEA assay method could be used to distinguish early stages of liver fibrosis in patients with CHB infection instead of the currentlyused spectrophotometric SPEA assay methods and other evaluated liver fibrosis indexes.

PSA is probably the most commonly used tumour marker in oncology. The absolute tissue specificity of PSA, as well as its well-established rote in clinical decision making, nave certainly contributed to its widespread use. Nevertheless, the familiarity that comes from the frequent use of the marker prompts the need for the awareness of the impact of the methods on the interpretation of the assay results. Actually, PSA values obtained by different methods may vary. Differences may be of clinical relevance in some instances such as when evaluating a PSA value with reference to a given cut-off point or, mainly, when assessing any variation among serial samples from the same patient. This potential variability related to the method is due to the molecular heterogeneity of PSA as well as to the lack of standard reference material. The problem of the binding of PSA to circulating protease inhibitors is of major relevance. The occurrence of several PSA isoforms should also be taken into account. Both a strict standardization of the method and the preparation of a reference standard are expected to reduce the method-related variability. In the meantime, the same assay method should be used to monitor the same patient. Moreover, dose co-operation between the urologist and the clinical pathologist is mandatory in order to be aware of the methodological clues which eventually may affect the decision-making process.

The effects of single-dose (10 mg) and short-term (10 mg tid) nifedipine treatment on apparent hepatic blood flow, as assessed by indocyanine green (ICG) clearance, were studied in ten healthy male subjects. ICG was measured by both spectrophotometric and high performance liquid chromatography (HPLC) assay methods. Blood clearance of ICG and apparent hepatic blood flow were increased by 30 and 50%, respectively, after single-dose nifedipine, whereas after 4 days' treatment these values were 12 and 30%. The spectrophotometric assay significantly overestimated ICG plasma concentrations from 7 minutes onwards. Although the spectrophotometric and HPLC assay showed marked differences in calculated half-lives and volume of distribution of ICG, the ICG clearance values were similar for the two assay methods.

What's known on the subject? and What does the study add? Today, numerous assays for PSA detection are available from various manufacturers. However, these various assays do not detect PSA equally and several studies have demonstrated variability between them. In order to harmonise PSA results and reduce the discrepancies, reference materials are available for assay calibration. We have demonstrated significantly variability between 6 different assay methods currently in use in 9 hospitals despite assay calibration. Variability in PSA values was reduced with the standardisation of the assay method in 4 hospitals. Our results highlight the dilemma of PSA assay variability and stress the need for nationwide standardisation of PSA testing. To determine whether standardization of total prostate-specific antigen (tPSA) assay methods reduces variability in tPSA measurements. Blood samples from 84 patients attending a single urology department were distributed across nine hospitals selected throughout Ireland for the independent determination of tPSA under the same conditions. The selected hospitals collectively used six different assay methods for tPSA detection: Beckman Hybritech WHO Calibrated (used as reference method), Tosoh AIA 1800, Roche E170 (used in three hospitals), Abbott AxSYM, Immulite 2500 2nd Generation (used in two hospitals) and Siemens ADVIA Centaur. The method of tPSA detection was next standardized in a subset of four hospitals using the same assay method and the measurements were repeated. The difference in mean tPSA in the cohort across the hospitals tested was determined and the Bland-Altman test was used to assess the agreement between each test. Analysis was performed over both the full (0.5-30 µg/L, N = 84) and a narrow (3-7 µg/L, n = 25) tPSA range. The range and the mean tPSA of the full cohort were inflated across the eight test hospitals, when compared with the reference hospital. The poorest agreement between assay methods was associated with a bias of 2.2 ± 2.4 µg/L. The variability in tPSA measurements between assay methods was inconsistent across the range of tPSA values tested and increased with increasing mean tPSA. Agreement in reported tPSA was excellent after standardization of tPSA assay methods (bias <0.2 µg/L). Over the narrow 3-7 µg/L PSA range, 12/25 (48%) patients had a tPSA range of values across all hospitals in excess of 2 µg/L. Following standardization of the tPSA assay method, patient tPSA ranges were <0.5 µg/L for 13/25 (52%) patients. We have shown that the lack of standardization of tPSA assay methods across a panel of Irish hospitals leads to significant variability in the measured tPSA values for the same patient samples. Variability in tPSA values was reduced with the standardization of the assay method in four hospitals. Standardization of PSA testing on a nationwide scale is warranted.

For certification and measurement control, nondestructive assay (NDA) instruments and methods used for verification measurements of special nuclear materials (SNMs) require calibrations based on certified reference materials (CRMs), or working reference materials (WRMs), traceable to the national system of measurements, and adequately characteristic of the unknowns. The Department of Energy Office of Safeguards and Security is sponsoring production of a comprehensive guide to preparation of NDA standards. The scope of the report includes preparation criteria, current availability of CRMs and WRMs, practical considerations for preparation and characterization, and an extensive bibliography. In preparing the report, based primarily on experience at Los Alamos, they have found that standards preparation is highly dependent on the particular NDA method being applied. They therefore include sections that contain information specific to commonly used neutron and gamma-ray NDA techniques. They also present approaches that are alternatives to, or minimize requirements for physical standards.

Microassay for UDP-galactose 4-epimerase activity

1. The oxidation of some para-substituted benzylamines by diamine oxidase produces the corresponding aldehydes. This was studied to develop a spectrophotometric method for following the enzyme reaction, as the aldehydes produced absorb strongly at 250nm where the substrates are almost optically transparent. 2. p-Dimethylaminomethylbenzylamine was the most useful substrate and full details of its preparation are given. The synthesis of its related oxidation product, p-dimethylaminomethylbenzaldehyde, is also described. 3. The effects of variations in pH, ionic strength, temperature and oxygenation on the reaction are described and the usefulness of the method is illustrated by several applications and assessed by comparison with the standard spectrophotometric assay.

The present work was developed to create three rapid, simple, eco-friendly, cheap spectrophotometric methods for concurrent assay of Sofosbuvir (SOF) and Simeprevir (SMV) in their pure, laboratory prepared mixture and pharmaceutical dosage form with high degree of accuracy and precision. Three methods were developed including iso-absorptive point, ratio subtraction and dual wavelength. The linear range of the proposed methods was 3.0–50.0 and 2.0–50.0 µg mL−1 for SMV and SOF, respectively. The proposed methods were validated according to ICH guidelines in terms of linearity, accuracy, precision, limit of detection and limit of quantitation. The proposed approach is highly simple and the procedure is environmentally green making it suitable for the drug analysis in routine works.

Stepwise preparation of calibration standards and quality controls (QCs) is one of the most routine and laborious steps in bioanalysis. An alternative non-contact dispenser using low picoliter digitized dispensing technology is evaluated for its application in non-stepwise preparation of calibration curve and QCs in bioanalysis. Fluorescein was initially used to assess the accuracy and precision of dispense volumes with fluorescent measurement. Various concentrations of MX-1, an in-house proprietary small molecule compound, in neat solution and in dog plasma were prepared manually with calibrated pipettors and digitally by the digital dispenser. The plasma samples were extracted by protein precipitation. The resultant extracted samples and neat solutions of MX-1 were analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) using an electrospray ionization (ESI) source in positive ion mode with selected reaction monitoring (SRM) of the mass transitions. In the three-day precision and accuracy assessment of dispensing volumes between 13 pL to 411.2 nL, the intra-day precision and accuracy ranged from 1.4% to 10.3% and -12.7% to 12.8%, respectively. The inter-day precision and accuracy ranged from 3.5% to 7.8% and -6.6% to 10.4%, respectively. For real analysis of in vivo study samples, all 49 samples analyzed showed a less than 5% difference between calibrations with digital and manual curve preparations. The resultant pharmacokinetic (PK) parameters were physiologically comparable as well. Using the digitized picoliter dispensing technology, high-speed automated precise and accurate dispense of a wide range of volumes can be achieved and tests for bioanalytical standards and QC preparations passed the stringent criteria set forth for regulated bioanalysis using LC/MS/MS-based technology. The digital dispenser has been found to be a useful tool in drug discovery for automatically preparing standards and QCs in seconds with low consumption of stock solutions and blank matrices.

Measurements of blood rheology in clinical studies should incorporate a quality control programme and, for this purpose, we have studied three approaches to the preparation of an erythrocyte standard. Partial fixation of erythrocytes using 0.04% w/v formaldehyde at ambient temperature, followed by storage at 4°C, provided an erythrocyte suspension which gave stable 5 µm pore filtration values for up to 4 weeks. Incubation of erythrocytes with a monoclonal antibody (BRIC 14) to membrane Glycophorin A caused impairment of filterability which was maintained for up to 11 days. A cross-linked acrylic copolymer, comprising soft spheres of 5–8 µm diameter, was also developed as an erythrocyte substitute for filtration studies. This pilot study indicates the potential for development of an international reference standard for rheological studies.

Quality control in environmental radioactivity measurements: Experience of the Central Service for Protection against Ionizing Radiation, acting as international reference center of the World Health Organization

5] β-Lactamase assays

Reverse phase high performance liquid chromatography (HPLC) was used as a rapid means of monitoring the erythromycin and the tetracycline fermentation processes. The sample preparation process for tetracycline in the fermentation broth includes simple dilution and filtration through a Millipore filter prior to injection into the HPLC column. Fermentation growth samples showed no interference, and excellent separation for selective determination of tetracycline, 4-epitetracycline, anhydrotetracycline, chlortetracycline, and 4-epianhydrotetracycline was obtained. The relative standard deviation for the HPLC analysis for tetracycline is about one percent and the correlation coefficient between the HPLC and the spectrophotometric assay methods is better than 0.994. The sample preparation procedure for erythromycin determination in fermentation broth requires solvent cleanup and extraction processes. The chromatographic analysis takes approximately 25 minutes, and the HPLC method is capable of separating and quantifying erythromycins A, B, C, and various epimers and degradation compounds. The correlation coefficient between the HPLC and the microbiological assay method is 0.970.

: A spectrophotometric assay method using concentrated sulfuric acid as the solvent is presented for 1,3,5-triamino-2,4,6-trinitrobenzene. When the purity is 90% or better there is no interference from any of the precursors. In addition to the assay method, a procedure is given for determining water soluble impurities. Qualitative identification of the precursors is afforded by a spectral determination in gamma-butyrolactone or dimethylformamide. The quality of 1,3,5triamino-2,4,6-trinitrobenzene, prepared by amination in benzene and dioxane, is discussed.

The demand for halal foods and beverages is increasing globally. While most halal analysis focuses on porcine, this study focuses on assessing residual organic solvents to ensure their halal compliance and wholesomeness, following several Malaysian standards and guidelines. A significant challenge in this study was the volatility of the residual solvents during the preparation of standards and quality control. To address this issue, a gravimetric technique was employed and effectively minimized the difference between theoretical (1,000 ppm) and actual (710 – 892 ppm) concentrations of the residual organic standard stock solution, except for acetone (588 ppm). The aim of this study was to establish a validated, reliable, and accurate method using SIM-headspace GC-MS to identify and quantify residual organic solvents for halal and wholesomeness analysis. Confirmation of each residual organic solvent was achieved by comparing the obtained spectra with the NIST 11 spectral database, containing 70,832 compounds, with similarity ranging from 80.9% to 96.6%, except for acetonitrile at 52.2%. The validation parameters were carried out according to ISO 17025:2017, the Center for Drug Evaluation and Research, and the European Guidelines. The parameters included recovery ranging from 95.65% to 95.68%, precision from 10.08% to 19.65% RSD, linearity between 0.996 to 0.999, limit of detection from 0.01 to 0.08 ppm, and limit of quantification from 0.02 to 0.24 ppm. Uncertainty considerations were limited to recovery, precision, and linearity, as other uncertainties were negligible based on the bottom-up approach using in-house validation data. This combination of gravimetric and SIM-headspace GC-MS techniques has provided valuable insights for discussions and collaborations among halal authorities worldwide to establish a consensus analytical methodology for halal and wholesomeness assessment.