Examination of the optical purity of voriconazole on the brush-type stationary phase

Validation of a high-performance liquid chromatography method for the measurement of mycophenolic acid and its glucuronide metabolites in plasma

Development and optimization of simplified LC–MS/MS quantification of 25-hydroxyvitamin D using protein precipitation combined with on-line solid phase extraction (SPE)

BackgroundSingle-cell assays of immune function are increasingly used to monitor T cell responses in immunotherapy clinical trials. Standardization and validation of such assays are therefore important to interpretation of the clinical trial data. Here we assess the levels of intra-assay, inter-assay, and inter-operator precision, as well as linearity, of CD8+ T cell IFNγ-based ELISPOT and cytokine flow cytometry (CFC), as well as tetramer assays.ResultsPrecision was measured in cryopreserved PBMC with a low, medium, or high response level to a CMV pp65 peptide or peptide mixture. Intra-assay precision was assessed using 6 replicates per assay; inter-assay precision was assessed by performing 8 assays on different days; and inter-operator precision was assessed using 3 different operators working on the same day. Percent CV values ranged from 4% to 133% depending upon the assay and response level. Linearity was measured by diluting PBMC from a high responder into PBMC from a non-responder, and yielded R2 values from 0.85 to 0.99 depending upon the assay and antigen.ConclusionThese data provide target values for precision and linearity of single-cell assays for those wishing to validate these assays in their own laboratories. They also allow for comparison of the precision and linearity of ELISPOT, CFC, and tetramer across a range of response levels. There was a trend toward tetramer assays showing the highest precision, followed closely by CFC, and then ELISPOT; while all three assays had similar linearity. These findings are contingent upon the use of optimized protocols for each assay.

A novel and precise assay that facilitates high-throughput screening of fibrinolytic agents was developed based on the automated assessment of the euglobulin clot lysis time in microtitre plates. Euglobulin fractions from fresh plasma samples were assessed over 28 days to determine the inter-assay and intra-assay precision. The intra-assay (coefficient of variation range, 0.7-2.6%) and inter-assay precision (coefficient of variation range, 6.8-12.1%) was found to be well within limits required by the Food and Drug Administration. On day 1 and day 28, the results of the microtitre plate euglobulin clot lysis time method were compared with tissue plasminogen activator activity, plasminogen activator inhibitor activity and results produced on fibrin plates. All comparisons were found to correlate significantly. The validity of this method for assaying fibrinolytic agents was assessed by comparing dose-response curves for streptokinase produced using fibrin plates and this method. The critical influence of ambient temperature on the inter-assay reproducibility of this method was established by testing samples over a range of temperatures between 20 degrees C and 40 degrees C.

Theoretical studies of brush-type chiral stationary phases

A new drug combination of phenylephrine hydrochloride (PHE) and doxylamine succinate (DOX) has been introduced for treating allergic rhinitis. Stability testing is critical for uncovering degradation routes and assessing the stability of combined drugs. This study illustrates the application of two eco-friendly chromatographic techniques which are reversed phase high-performance liquid chromatography (RP-HPLC) and high-performance thin-layer chromatography (HPTLC), for assessing PHE and DOX when DOX oxidative degradation product (DOX DEG) is present. Using liquid chromatography- mass spectroscopy to identify DOX DEG. The HPLC method produced the best separation with isocratic elution and a mobile phase consists of ethanol and 0.01 M phosphate buffer pH = 5.0 (30: 70, v/v), and it was pumped at 1.0 mL/min. The analytes were measured at 260.0 nm using diode array detector (DAD), and the Xterra C18 column (100 mm × 4.6 mm × 5 m) used as a stationary phase. The method demonstrated a linear response for DOX and PHE across a concentration range of 5.00 to 100.00 µg/mL. The range for DOX DEG was 5.00 to 30.00 µg/mL. The limits of detection (LOD) were determined to be 1.44 for DOX, 1.59 for PHE, and 0.84 µg/mL for DOX DEG. Correspondingly, the limits of quantification (LOQ) were 4.32, 4.77, and 2.52 µg/mL for DOX, PHE, and DOX DEG, respectively. The separation in HPTLC was accomplished by combining ethanol, methylene chloride, and ammonia 30% in ratio 7:2.5:0.5 (v/v/v) as a developing system. The drugs were then quantitatively determined at wavelengths of 260.0 nm using UV detector. The linearity range was 4.00–26.00 (µg/band) for DOX and PHE while it was 0.50–10.00 (µg/band) for DOX DEG. Values of LOD were 0.65 ,0.76 and 0.16 µg/band for PHE, DOX, DOX DEG, respectively. While1.95,2.28 and 0.48 µg/band were values of LOQ. Per ICH guidelines, two analytical methods were validated and proven to be reliable, reproducible, and selective. Additionally, sustainability assessments confirmed their green credentials and practical applicability.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-22295-6.

Self-monitoring of blood glucose is a key element in diabetes management. Accurate and precise performance of blood glucose monitors (BGMs) ensures that valid values are obtained to guide treatment decisions by patients and physicians. BGStar and iBGStar are hand-held BGMs that use dynamic electrochemistry to correct for potential interferences and thereby minimize system errors. A single-center, in vitro diagnostic device performance evaluation with heparinized oxygenated venous blood samples (intra-assay precision) and control solutions (interassay precision) was performed in a laboratory setting, comparing BGStar and iBGStar with 12 competitors. The primary outcome was the coefficient of variation percent (CV%) of the BGMs investigated. In inter-assay precision analyses, all but GlucoMen LX had a CV <5%, and in intra-assay precision analyses, 10 of the 14 devices tested had CV <5%. BGStar and iBGStar had a CV <5% in both the inter- and intra-assay precision analyses. The smallest variation was found in the near-normoglycemic glucose range (5.3 - 8.0 mmol/l) for both BGStar and iBGStar in the inter-assay precision analysis. BGStar and iBGStar were proven to have very good inter-assay and high intra-assay precision, demonstrating low scattering of replicate measurements with both clinical samples and control solutions.

Optimization and modeling of a green dual detected RP-HPLC method by UV and fluorescence detectors using two level full factorial design for simultaneous determination of sofosbuvir and ledipasvir: Application to average content and uniformity of dosage unit testing

Perindopril and Indapamide, a fixed-dose combination medication, are utilized for the management of hypertension by integrating the properties of an angiotensin-converting enzyme (ACE) inhibitor with a thiazide-like diuretic. The detection of Indapamide, Perindopril, and its active metabolite perindoprilat in human plasma or whole blood was achieved through hyphenated ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS). Internal standards included indapamide-d3, perindopril-d4, and perindoprilat-d4. The chromatography was conducted at a column temperature of 50°C with a flow rate of 0.6 mL/min using isocratic elution, and UV detection was performed at 215 nm. The method demonstrated acceptable linearity, accuracy, and precision across concentration ranges of 1-5μg/mL for Perindopril and 10-15μg/mL for Indapamide. A statistical comparison of the proposed chromatographic method against reference methods was conducted using one-way analysis of variance, with Beer-Lambert’s range established at 0-60 μg/mL. The most commonly used mobile phases are methanol and acetonitrile, whereas the stationary phase used is a Phenomenex Luna C18 column (250 mm × 4.6 mm, 10 µm). Various parameters are collected, including LOD, LOQ, correlation coefficient and linearity, absorbance maxima, and retention time, which were within the limits specified by the ICH guidelines.

Brush Type Stationary Phase with Si-O-C Bonding in Gas Chromatography

Background The objective of this study is to validate Lactate (LAC) measurement in rat plasma using the ADVIA 1800 analyzer. Methods LAC was validated in rat plasma using the ADVIA 1800 analyzer. Validation testing included intra-assay and inter-assay precision, accuracy, linearity of dilution, limit of quantitation (LOQ), limit of detection (LOD), reference interval, carry-over, correlation, recovery, and stability. Samples were collected into tubes containing sodium fluoride. Intra-assay precision was determined by analyzing two biological samples and the two quality control levels of Bio-Rad Lyphochek Assayed Chemistry Control 10 consecutive times. Inter-assay precision was determined by analyzing the two quality control levels in duplicate for six days over a 10-day period. Accuracy was calculated using the data obtained from the inter-assay precision testing. For linearity of dilution, Audit MicroControl General Chemistry Linearity Kit and biological samples were diluted and analyzed in duplicate. The LOQ was determined by diluting the calibrator material to produce a value at the low end of the reportable range. Diluted material was tested six times. The LOD was determined by assaying the appropriate blank 10 times. The reference interval was determined by analyzing 21 plasma samples. Correlation between the two ADVIA 1800 analyzers was determined by assaying 10 samples on both analyzers once. The carry-over of the assay was determined by analyzing the high-level quality control material followed by the low-level quality control material. Recovery was determined by analyzing a pair of test samples in duplicate that have been spiked and diluted. The proportional error between the two test samples was calculated. Stability of plasma samples was tested on wet ice, refrigerated, and frozen (at -70°C) for various durations of storage. Results Measurement of LAC in rat plasma met the acceptance criteria for precision, accuracy, linearity of dilution, limit of quantitation, carry-over, recovery, and stability. For intra-assay precision in specimen and control material, the %CV was within ±20%. The inter-assay precision %CV was within ±20%. The total error observed (TEobs) was within ±20% for accuracy and the obtained mean of the control material was within the manufacturer’s acceptable range. For linearity of dilution, the coefficient of determination was ≥ 0.9000, the slope was within 1.00 ± 0.25, and TEobs for each plasma and linearity kit dilution was ≤20%. The LOQ was set at the lowest concentration for which the TEobs was within ±20%. The LOD was calculated by adding the mean concentration obtained and three times the standard deviation. The reference interval was set to the 2.5th to 97.5th percentile interval using Excel software. The carry-over was acceptable at ≤2%. Correlation between the two ADVIA 1800 analyzers was determined to describe any bias in result interpretation. Recovery was acceptable as the proportional error was within ±20%. Stability at all storage conditions was found to be acceptable with a mean %difference within ±20%. Conclusions LAC in rat plasma met the acceptance criteria for all required validation parameters. The ADVIA 1800 analyzer can be used for preclinical studies to test LAC in rat plasma.

Determination of alosetron in the presence of its degradation products was studied and validated by a novel HPLC method. The separation of the drug and its degradation products was achieved with the Jones Chromatography C18 analytical column (150 mm x 4.6 mm; 3 µm) with a stationary phase in isocratic elution mode. The mobile phase used was 0.01 M ammonium acetate, pH-adjusted to 3.5 with glacial acetic acid and acetonitrile in the ratio of 75:25 (V/V) at a flow rate of 1 ml/min and UV detection was carried out at 217 nm. Further, the drug was subjected to stress studies for acidic, basic, neutral, oxidative, and thermal degradations as per ICH guidelines and the drug was found to be labile in base hydrolysis and oxidation, while stable in acid, neutral, thermal, and photolytic degradation conditions. An MS study has been performed on the major degradation products to predict the degradation pathway of alosetron. The method provided linear responses over the concentration range of 100–1500 ng/ml and regression analysis showed a correlation coefficient value (r2) of 0.994. The LOD and LOQ were found to be 1 ng/ml and 3 ng/ml, respectively. The developed LC method was validated as per ICH guidelines with respect to accuracy, selectivity, precision, linearity, and robustness.

For the determination of artemisinin (ART) and analogs, a reversed-phase high-performance liquid chromatography method using reductive electrochemical detection (ED) was set up with some important modifications as compared to previously published assays. A different technique of deoxygenating resulted in a factor 2-3 lower background current. A Spectroflow 400 liquid chromatograph in combination with a Triathlon autoinjector coupled to a Decade electrochemical detector was used. The detector was operated in the reductive mode as a closed system under chromatography grade helium to exclude any access of oxygen. The Decade has a glassy carbon electrode and a reference Ag/AgCl electrode. Infrequent electropolishing was required implicating a very stable system. By increasing acetonitril or lowering the pH of the mobile phase, the various derivatives could be determined in the same chromatogram. The assay was validated using artemether (ATM) and dihydroartemisinin (DHA) as test substances. In the concentration range seen in people after usual doses (5 to 220 ng/ml), the assay performs with adequate accuracy and precision. The interassay and intraassay precision are < 6% for ATM. For DHA, the interassay and intraassay precision are < 9%. The accuracy expressed as the deviation from the expected concentration varies from -1% to +4.5% for the intraassay ATM-determinations and from +1% to +6.3% for the interassay measurements. For DHA, the accuracy is somewhat less, varying from -0.3% to -9.5% for the intraassay measurements and -0.6% to +2.6% for the interassay measurements. The reproducibility of the assay, measured over a time period of 3 months, is good for ATM and DHA with an interassay precision of < 18% in 70 repetitive samples and an accuracy varying from -0.6% to +7.6%. In a cross-check with two other reference laboratories who used comparable methods of determination, a strong correlation (correlation coefficient > 0.98) was achieved. The method was applied in a study in which artemether was administered orally to healthy white subjects. We consider high-performance liquid chromatography with electrochemical detection an accurate and precise method for quantitative determination of artemisinin derivatives in pharmacokinetic studies.

Introduction/Objective To verify performance after probe replacement on Siemens ADVIA® 1800 analyzer, the vendor recommends running 2 levels of quality control (QC) for each analyte; however, this approach provides only a snapshot of analyzer performance. Additional experiments were conducted to determine if the vendor-recommended protocol adequately verifies analyzer performance. Methods Serum from 20 random patients was mixed, centrifuged at 1000 g for 10 min, and supernatant prepared (PPS). Intra-assay precision was assessed by analyzing 20 replicates of 2 levels of QC, and PPS for the following indicator assays (total allowable error [TEa] in parentheses): alanine aminotransferase (20%), calcium (6%), creatinine (15%), immunoglobulin G (25%), and sodium (5 mEq/L). Inter-assay precision over past 30 days was calculated from the values (90 – 114) of 2 levels of QC. Violation of 13s QC rule is most commonly used for detecting critical error (error &amp;gt; TEa). We used TEa/4 and TEa/3 as the acceptability criteria for intra- and inter-assay precision. Inter-assay precision was also compared to the precision claim by the vendor. Bias between the mean of QC values from intra- and inter- assay precision was calculated and TEa/4 used as the acceptability criterion. Results Intra-assay precision for both levels of QC for all analytes was acceptable. Intra-assay precision (5.48%) for only alanine aminotransferase in the PPS was unacceptable, indicating that patient matrix may impact precision for certain analytes. Inter-assay precision (2.24%) for level 1 QC for calcium was unacceptable. For all analytes, inter- assay precision compared to vendor’s claim was found to be significantly higher (32% - 405%). The bias was unacceptable for level 3 QC for sodium (-2.11). Conclusion Increased imprecision and/or bias may be observed if either probe replacement or alignment was not appropriate; or the verification protocol was not adequate. This study demonstrates the need to conduct experiments beyond vendor-recommended protocol to ensure production of clinically acceptable results.

Background and importanceOn the basis of resolution 189/2018 published by our city health council, the hospital pharmacy service was entrusted with the centralisation of the procedure for the acquisition, compounding,...