Assessment of ∆9-THC and ∆9-THCCOOH Bias, Precision, and Ionization Suppression/Enhancement between Solid Tissue Homogenate and Supernatant by LC-MS/MS.

LC-MS-MS assays are frequently utilized for screening and confirmatory purposes in the forensic toxicology laboratory. While these techniques are excellent for the targeted identification and quantitation of a wide variety of drug classes, validation and determining fit-for-purpose is a requirement for each method. In the United States, ANSI/ASB Standard 036 currently serves as a primary resource in forensic toxicology method validation, and mandates that laboratories evaluate critical performance characteristics to help ensure the production of forensically defensible results. Due to the variability of specimen quality frequently encountered in the discipline of postmortem toxicology, the [Author Information Removed] Office of the Chief Medical Examiner Forensic Toxicology Laboratory routinely analyzes solid tissue specimens as part of the medicolegal death investigation process and evaluates liver as a representative solid tissue matrix during method validation. Authentic postmortem specimens (e.g., liver, kidney, skeletal muscle, and spleen) were used to investigate the effects of analyzing solid tissue homogenate versus solid tissue supernatant on bias, precision, and ionization suppression/enhancement of ∆9-THC and ∆9-THCCOOH. Bias was <20% for Δ9-THC and ∆9-THCCOOH in liver homogenate and supernatant with a single exception of the low QC concentration for Δ9-THC in liver homogenate (-29%). Within-run and between-run CV was <20% for Δ9-THC and ∆9-THCCOOH in liver homogenate and supernatant. Δ9-THC and Δ9-THC-d3 exhibited significant ion suppression in both liver homogenate and supernatant, while ∆9-THCCOOH and ∆9-THCCOOH-d3 showed both ion suppression and enhancement in these matrices. Noticeable quantitative differences were observed in authentic postmortem solid tissue homogenate and supernatant specimens despite evaluating from identical tissue samplings. A brief discussion of the results is presented using a validated LC-MS-MS method for the confirmation and quantitation of ∆9-THC and ∆9-THCCOOH in postmortem casework.

Profiles of volatile sulfur compounds in various vegetables consumed in Korea using HS-SPME-GC/MS technique.

Volatile sulfur compounds (VSCs) are not only important for their therapeutic potential but also significantly influence the flavor profiles of agricultural products. VSCs exhibit various chemical structures due to their stability and volatility, and they may form or be altered as a result of enzymatic and chemical reactions during storage and cooking. This study has focused on profiles of VSCs in 58 different vegetable samples by using HS-SPME-GC/MS technique and chemometric analyses. The validation was carried out using cabbage juice as a vegetable matrix for VSCs analysis, showing satisfactory repeatability (RSD 8.07% ~ 9.45%), reproducibility (RSD 4.22% ~ 7.71%), accuracy and specificity. The established method was utilized on various vegetables, revealing that 21 VSCs such as sulfides, disulfides, trisulfides, isothiocyanates, sulfhydryls, and thiophenes were successfully identified and quantified. These compounds were found in a range of vegetables including Allium species, Cruciferae, Capsicum species, green leafy vegetables, and mushrooms. In particular, isocyanate and allyl groups were abundant in Cruciferae and Allium vegetables, respectively. Cooking conditions were shown to reduce the levels of certain sulfur compounds such as dimethyl sulfide and dimethyl trisulfide in vegetables like broccoli and cabbage, suggesting that heat treatment can lead to the volatilization and reduction of these compounds. The present study provides reliable insights into the compositions of VSCs in various vegetables and examines the changes induced by different cooking methods.

Validation of analytical methods for newly regulated veterinary drug residues in livestock and fishery products with maximum residue limits in Republic of Korea.

Veterinary drugs play a crucial role in the treatment of various animal diseases. However, their residues, stemming from issues, such as withdrawal period lapses, overuse, or abuse, can jeopardize food safety and human health. This study addresses recent regulations in Korea concerning specific veterinary drugs (anacolin, ephedrine, menichlopholan, piperonyl butoxide, and etisazole HCl) and their ongoing discussions. This study aimed to validate two pre-developed methods for quantifying residues in livestock and fishery products using QuEChERS and liquid chromatography-tandem mass spectrometry. Both methods exhibited excellent linearity, recoveries (70.3-119%), and coefficient of variations (1.3-28%), along with low limits of detection and quantification (0.3-4 ng/g and 1-12 ng/g). This study is significant for its contribution to the detection of veterinary drugs in livestock and fishery products, given the limited research available on the methods for analyzing these substances.

Development and validation of a liquid chromatography coupled to a diode array detector (LC-DAD) method for measuring mitotane (DDD) in plasma samples.

INTRODUCTION: Mitotane (o,p'-DDD) is the drug of choice for Adrenocortical Carcinomas (ACC) and its measurement in plasma is essential to control drug administration. OBJECTIVE: To develop and validate a simple, reliable and straightforward method for mitotane determination in plasma samples. METHOD: Drug-free plasma samples were collected in potassium-ethylenediamine tetraacetate (K-EDTA) tubes and spiked with 1.0, 2.5, 10.0, 25.0 and 50.0 µg/mL of mitotane (DDD). The p,p'-DDD was used as an Internal Standard (IS) and was added at 25.0 µg/mL concentration to all samples, standards and controls. Samples were submitted to protein precipitation with acetonitrile and then centrifuged. 50 uL of the supernatant was injected into an HPLC system coupled to a Diode Array Detector (DAD). DDD and IS were detected at 230 nm in a 12 min isocratic mode with a solvent mixture of 60 % acetonitrile and 40 % formic acid in water with 0.1 % pump mixed, at 0.6 mL/min flow rate, in a reversed-phase (C18) chromatographic column kept at 28°C. The sensitivity, selectivity, precision, presence of carry-over, recovery and matrix-effect, linearity, and method accuracy were evaluated. RESULTS: The present study's method resulted in a symmetrical peak shape and good baseline resolution for DDD (mitotane) and 4,4'-DDD (internal standard) with retention times of 6.0 min, 6.4 mim, respectively, with resolutions higher than 1.0. Endogenous plasma compounds did not interfere with the evaluated peaks when blank plasma and spiked plasma with standards were compared. Linearity was assessed over the range of 1.00-50.00 µg/mL for mitotane (R2 > 0.9987 and a 97.80 %‒105.50 % of extraction efficiency). Analytical sensitivity was 0.98 µg/mL. Functional sensitivity (LOQ) was 1.00 µg/L, intra-assay and inter-assay coefficient of variations were less than 9.98 %, and carry-over was not observed for this method. Recovery ranged from 98.00 % to 117.00 %, linearity ranged from 95.00 % to 119.00 %, and high accuracy of 89.40 % to 105.90 % with no matrix effects or interference was observed for mitotane measurements. Patients' sample results were compared with previous measurements by the GC-MS method with a high correlation (r = 0.88 and bias = -10.20 %). CONCLUSION: DDD determination in plasma samples by the developed and validated method is simple, robust, efficient, and sensitive for therapeutic drug monitoring and dose management to achieve a therapeutic index of mitotane in patients with adrenocortical cancer.

Unpacking WHO and CDC Bottle Bioassay Methods: A Comprehensive Literature Review and Protocol Analysis Revealing Key Outcome Predictors.

Background: Resistance monitoring is a key element in controlling vector-borne diseases. The World Health Organization (WHO) and Centres for Disease Control and Prevention (CDC) have each developed bottle bioassay methods for determining insecticide susceptibility in mosquito vectors which are used globally. Methods: This study aimed to identify variations in bottle bioassay methodologies and assess the potential impact on the data that is generated. Our approach involved a systematic examination of existing literature and protocols from WHO and CDC, with a focus on the specifics of reported methodologies, variation between versions, and reported outcomes. Building on this, we experimentally evaluated the impact of several variables on bioassay results. Results: Our literature review exposed a significant inconsistency in the how bioassay methods are reported, hindering reliable interpretation of data and the ability to compare results between studies. The experimental research provided further insight by specifically identifying two key factors that influence the outcomes of bioassays: mosquito dry weight and relative humidity (RH). This finding not only advances our comprehension of these assays but also underscores the importance of establishing precisely defined methodologies for resistance monitoring. The study also demonstrates the importance of controlling bioassay variables, noting the significant influence of wing length, as an indicator of mosquito size, on mortality rates in standardized bioassays. Conclusions: Generating data with improved protocol consistency and precision will not only deepen our understanding of resistance patterns but also better inform vector control measures. We call for continued research and collaboration to refine and build consensus on bioassay techniques, to help bolster the global effort against vector-borne diseases like malaria.

Development and validation of a PMA-qPCR method for accurate quantification of viable Lacticaseibacillus paracasei in probiotics.

The effectiveness of probiotic products hinges on the viability and precise quantification of probiotic strains. This study addresses this crucial requirement by developing and validating a precise propidium monoazide combination with quantitative polymerase chain reaction (PMA-qPCR) method for quantifying viable Lacticaseibacillus paracasei in probiotic formulations. Initially, species-specific primers were meticulously designed based on core genes from the whole-genome sequence (WGS) of L. paracasei, and they underwent rigorous validation against 462 WGSs, 25 target strains, and 37 non-target strains across various taxonomic levels, ensuring extensive inclusivity and exclusivity. Subsequently, optimal PMA treatment conditions were established using 25 different L. paracasei strains to effectively inhibit dead cell DNA amplification while preserving viable cells. The developed method exhibited a robust linear relationship (R 2 = 0.994) between cycle threshold (Cq) values and viable cell numbers ranging from 103 to 108 CFU/mL, with an impressive amplification efficiency of 104.48% and a quantification limit of 7.30 × 103 CFU/mL. Accuracy assessments revealed biases within ±0.5 Log10 units, while Bland-Altman analysis demonstrated a mean bias of 0.058 Log10, with 95% confidence limits of -0.366 to 0.482 Log10. Furthermore, statistical analysis (p = 0.76) indicated no significant differences between theoretical and measured values. This validated PMA-qPCR method serves as a robust and accurate tool for quantifying viable L. paracasei in various sample matrices, including pure cultures, probiotics as food ingredients, and composite probiotic products, thereby enhancing probiotic product quality assurance and contributing to consumer safety and regulatory compliance.

Development of a high-performance liquid chromatography method for simultaneous quantification of sixteen polyphenols and application to tomato.

The main purpose of the current work was to develop a new method to evaluate and quantify sixteen polyphenol compounds from tomato fruit using high-performance liquid chromatography (HPLC). The separation of 16 polyphenols from tomato fruit was achieved in < 60 min by using a Waters Symmetry C18 column (250 × 4.6 mm i. d, 5 µm particle sizes) with a gradient system of ultrapure water (1 % acetic acid) and 100 % methanol, a temperature of 30 °C, an injection volume of 10 µL and a flow rate of 1.1 mL/min, respectively. The analytical characteristics of evaluation method provide sufficient sensitivity for all tomato polyphenols compounds within normal range 0.1-20 µg·mL-1 (R2≥0.999) with 0.069-0.365 µg·mL-1 LOD, and 0.171-1.106 µg·mL-1 LOQ, with good system suitability (<2 % RSD of retention time, peak area, and tailing factor, 6,000-1,336,000 N, and >1.5 peak resolution), <10 % RSD of precision, stability, repeatability, and robustness, and 99.2 - 105.0 % of recovery. The applicability of this method was demonstrated by the determination of polyphenols in nine cultivars of tomatoes. The results showed that '184' possessed the highest content of total polyphenols (1249.53 µg·g-1 DW) followed by 'Disease resistance 184' (1064.93 µg·g-1 DW). The main polyphenol components were rutin, quercetin, gallic acid, chlorogenic acid, 2,5-dihydroxy benzoic acid, caffeic acid and benzoic acid in tomato fruits. In conclusion, this novel HPLC method is useful and acceptable to analyze polyphenols in tomato fruit.

Studying drug excretion into exhaled breath aerosol - A workflow based on an impaction sampling device and LC-HRMS/MS analysis.

BACKGROUND: Exhaled breath (EB) aerosol was in principle shown to be a suitable matrix for bioanalysis of volatile but also non-volatile compounds. This attracted particular interest in the field of drug analysis. However, a big gap still exists in the understanding how and which drugs and/or their metabolites are excreted into exhaled breath and could thus actually be detected. The current study aimed to develop an analytical workflow for the qualitative detection of non-volatile drugs in EB aerosol microparticles. RESULTS: The analyte selection covered different drug classes such as antihypertensives, anticonvulsants or opioid analgesics to investigate and understand the excretion of drugs and their metabolites into EB aerosol. A device for collecting aerosol particles from the lung through impaction was used for the non-invasive sampling procedure. Three expiration cycles per participant and device were collected. The sample preparation consisted of a collector extraction with methanol. Qualitative method development and validation were performed using reversed-phase liquid chromatography (LC) coupled to orbitrap-based high-resolution mass spectrometry (HRMS). Qualitative method validation was done according to published recommendations and international guidelines. Parameters such as selectivity, carry-over, limits of detection and identification, recovery, matrix effects, and long-term stability were evaluated. The limits of detection ranged from 100 pg/collector to 10,000 pg/collector. The procedure was finally used to analyze a total of 31 patient EB samples and demonstrated that e.g., tilidine and its metabolite nortilidine as well as tramadol and its active metabolite O-desmethyltramadol can be detected in EB aerosol. SIGNIFICANCE AND NOVELTY: The work shows a comprehensive workflow for elucidating drug excretion into exhaled breath aerosol. This bioanalytical strategy and the corresponding novel data from this study are the foundation for further method development and to better understand, which drugs and their metabolites can be addressed by exhaled breath aerosol bioanalysis.

Best Practices for Development and Validation of Enzymatic Activity Assays to Support Drug Development for Inborn Errors of Metabolism and Biomarker Assessment.

Aberrant or dysfunctional cellular enzymes are responsible for a wide range of diseases including cancer, neurodegenerative conditions, and metabolic disorders. Deficiencies in enzyme level or biofunction may lead to intracellular accumulation of substrate to toxic levels and interfere with overall cellular function, ultimately leading to cell damage, disease, and death. Marketed therapeutic interventions for inherited monogenic enzyme deficiency disorders include enzyme replacement therapy and small molecule chaperones. Novel approaches of in vivo gene therapy and ex vivo cell therapy are under clinical evaluation and provide promising opportunities to expand the number of available disease-modifying treatments. To support the development of these different therapeutics, assays to quantify the functional activity of protein enzymes have gained importance in the diagnosis of disease, assessment of pharmacokinetics and pharmacodynamic response, and evaluation of drug efficacy. In this review, we discuss the technical aspects of enzyme activity assays in the bioanalytical context, including assay design and format as well as the unique challenges and considerations associated with assay development, validation, and life cycle management.

Validation, measurement uncertainty, and determination of polysorbate-labeled foods distributed in Korea.

This study reports the improvement and validation of a colorimetric method to quantify polysorbates (20, 60, 65, and 80) in food by measuring absorbance at 620 nm using ultraviolet-visible spectrophotometry. The method was validated for linearity, limit of detection (LOD), limit of quantitation (LOQ), precision, accuracy, and measurement uncertainty. The coefficient of determination was linear (r 2 ≥ 0.9991) over the measured concentration range of 50-1000 mg/L. The LOD and LOQ were 2.3-4.9 and 7.0-15.0 mg/kg, respectively. Intra-day and inter-day accuracy and precision were 91.9-104.1% and 0.1-1.1% RSD, and 91.6-103.8% and 0.4-5.0% RSD, respectively. The result of inter-laboratory recovery was 90.9-99.8% and the measurement uncertainty was < 16% with the compliance of the CODEX recommendation. Sauce, bread, whipped cream, rice cake, ice cream, and various other polysorbate-labeled food products (n = 229, detection range; N.D.-16,442.3 mg/kg) distributed in Korea were analyzed to confirm the applicability of the analytical method. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-024-01544-w.

Determination of tetracycline residues in potatoes and soil by LC-MS/MS: Method development, validation, and risk assessment.

A method utilizing liquid-liquid extraction (LLE) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and validated according to the Commission Implementing Regulation (CIR) EU 2021/808 for quantifying four tetracyclines (TCs) in potatoes and soil. The method demonstrated recovery values ranging from 70 to 121% and precision (repeatability and within-laboratory reproducibility), with coefficient of variation (CV) values below 18% for all TCs in both matrices. The limits of quantification (LOQs) for the TCs ranged from 0.90 to 1.87 µg/kg in potatoes and from 0.68 to 1.25 µg/kg in soil. The decision limit (CCα) and detection capability (CCß) ranged from 10.4 to 12.3 µg/kg and 11.9 to 14.3 µg/kg, respectively. Analysis of 538 potato and soil samples from Egyptian farms revealed a 13.2% occurrence of TC residues, with a higher frequency in soil (19.33%) than in potatoes (7.06%). Target hazard quotient (THQ) values indicated that TC residues in potatoes do not pose a health risk to Egyptian consumers.

Evaluation of two real-time PCR methods to detect Yersinia enterocolitica in bivalve molluscs collected in Campania region.

Yersinia enterocolitica (Ye) is a foodborne pathogen isolated from humans, food, animals, and the environment. Yersiniosis is the third most frequently reported foodborne zoonosis in the European Union. Ye species are divided into six biotypes 1A, 1B, 2, 3, 4, and 5, based on biochemical reactions and about 70 serotypes. Biotype 1A is non-pathogenic, 1B is highly pathogenic, and biotypes 2-5 have moderate or low pathogenicity. The reference analysis method for detecting pathogenic Ye species underestimates the presence of the pathogen due to similarities between Yersinia enterocolitica-like species and other Yersiniaceae and/or Enterobacteriaceae, low concentrations of distribution pathogenic strains and the heterogeneity of Yersinia enterocolitica species. In this study, the real-time PCR method ISO/TS 18867 to identify pathogenic biovars of Ye in bivalve molluscs was validated. The sensitivity, specificity and accuracy of the molecular method were evaluated using molluscs experimentally contaminated. The results fully agree with those obtained with the ISO 10273 method. Finally, we evaluated the presence of Ye in seventy commercial samples of bivalve molluscs collected in the Gulf of Naples using ISO/TS 18867. Only one sample tested resulted positive for the ail gene, which is considered the target gene for detection of pathogenic Ye according to ISO/TS 18867. Additionally, the presence of the ystB gene, used as target for Ye biotype 1A, was assessed in all samples using a real-time PCR SYBR Green platform. The results showed amplification ystB gene aim two samples.

Quantitative determination of liposomal irinotecan and SN-38 concentrations in plasma samples from children with solid tumors: Use of a cryoprotectant solution to enhance liposome stability.

Preclinical studies have demonstrated that liposomal irinotecan (CPT-11), a topoisomerase I inhibitor, has broad activity against adult cancers, including pancreatic, gastric, colon, lung, glioma, ovarian, and breast cancer. Encapsulation of irinotecan into liposomes can modify its pharmacokinetic properties dramatically. Also, the pharmacokinetic profiles of liposomal drug formulations are not fully understood; thus, bioanalytical methods are needed to separate and quantify nonencapsulated vs. encapsulated concentrations. In this study, two robust, specific, and sensitive LC-MS/MS methods were developed and validated to separate and quantify the nonencapsulated CPT-11 (NE-CPT-11) from the sum-total CPT-11 (T-CPT-11) and its major metabolite, SN-38, in human plasma after intravenous administration of liposomal irinotecan. NE-CPT-11 and SN-38 were separated from plasma samples by using solid-phase extraction, and T-CPT-11 was measured by protein precipitation. The liposomal CPT-11 formulation was unstable during sample storage and handling, resulting in elevated NE-CPT-11 concentration. To improve the stability of liposomal CPT-11, a cryoprotectant solution was added to human plasma samples prior to storage and processing. CPT-11, SN-38, and their respective internal standards, CPT-11-d10 and SN-38-d3, were chromatographically separated on a reversed-phase C18 analytical column. The drugs were detected on a triple quadrupole mass spectrometer in the positive MRM ion mode by monitoring the transitions 587.3 > 124.1 (CPT-11) and 393.0 > 349.1 (SN-38). The calibration curves demonstrated a good fit across the concentration ranges of 10-5000 ng/mL for T-CPT-11, 2.5-250 ng/mL for NE-CPT-11, and 1-500 ng/mL for SN-38. The accuracy and precision were within the acceptable limits, matrix effects were nonsignificant, recoveries were consistent and reproducible, and the analytes were stable under all tested storage conditions. Finally, the LC-MS/MS methods were successfully applied in a phase I clinical pharmacokinetic study of nanoliposomal irinotecan (Onivyde®) in pediatric patients with recurrent solid malignancies or Ewing sarcoma.

[Validation of a method for measuring the antielastolytic activity of human circulating alpha1-antitrypsin]. / Validation d'une méthode de mesure de l'activité antiélastasique de l'alpha1-antitrypsine circulante humaine.

The existence of alpha-1 antitrypsin variants with apparently unremarkable phenotypes and serum concentrations, contrasting with a clinical picture suggestive of a severe deficiency, led us to investigate whether in these cases there was a reduction or even suppression of the capacity of alpha-1 antitrypsin to inhibit elastase. To this end, in two different laboratories, we adapted and validated a method for measuring the functional activity of alpha-1 antitrypsin, based on spectrophotometric kinetic analysis of the inhibition by serum alpha-1 antitrypsin of the hydrolytic activity of porcine pancreatic elastase on a chromogenic substrate. This method has proved to be robust, reproducible and transferable and made possible to define, on the basis of an analysis of a hospital population, a functionality index with a confidence interval comprised between 0.87 and 1.2, allowing to identify subjects likely to have a functional deficiency of alpha-1 antitrypsin, whether this deficiency being of a genetic origin without any quantitative or phenotypic translation, or whether being acquired under the effect of external agents (cigarette smoke or viruses).

Gentiana capitata Buch.-Ham. ex D.Don Cell Suspension Culture as a New Source of Isosaponarin and 3,7,8-Trimethoxy-9-oxo-9H-xanthen-1-yl 6-O-ß-D-ribopyranosyl-ß-D-allopyranoside and Their Effect on PC-12 Cell Viability.

Some species of the Gentianaceae family are a valuable source of secondary metabolites. However, the phytochemical knowledge of some of these species remains insufficient. Therefore, this work focused on the isolation of the two main secondary metabolites in the methanolic extract from a Gentiana capitata cell suspension using preparative HPLC and the determination of their structure using UHPLC-DAD-IT-MS/MS and NMR methods. Their content in the methanolic extract was quantified using a previously validated HPLC method. The toxicity of the extract and two isolated compounds was also tested on the PC-12 cell line. The structures of the main secondary metabolites were identified as isosaponarin and 3,7,8-Trimethoxy-9-oxo-9H-xanthen-1-yl 6-O-ß-D-ribopyranosyl-ß-D-allopyranoside by comparing the UHPLC-DAD-IT-MS/MS and NMR results with the literature data. The content of isosaponarin was determined to be 0.76 ± 0.04%, and the content of 3,7,8-trimethoxy-9-oxo-9H-xanthen-1-yl 6-O-ß-D-ribopyranosyl-ß-D-allopyranoside was found to be 0.31 ± 0.02% in the dry extract. Additionally, a two-fold increase in the viability of the PC-12 cell line was observed compared to the control after treatment with the methanolic extract at a concentration of 500 µg/mL. These results suggest the potential use of G. capitata cell suspension methanolic extract as a new source of isosaponarin and 3,7,8-trimethoxy-9-oxo-9H-xanthen-1-yl 6-O-ß-D-ribopyranosyl-ß-D-allopyranoside, highlighting their lack of toxicity to the PC-12 (rat pheochromocytoma) cell line.

A Comparative Study of Two Oroxylum indicum (L.) Kurz. Phenotypes Based on Phytochemicals and Antioxidant Effects, and the Anti-Inflammatory Activity of Leaf and Pod Extracts.

Indian trumpet tree Oroxylum indicum (L.) Kurz exhibits a wide range of biological activities in all plant parts, including anti-inflammation, antioxidant, and wound-healing activities. In Thailand, there are tall- and short-stem phenotypes. The latter are preferred for commercial cultivation due to their fast growth and lower harvesting cost. This study aimed to compare the chemical profiles and antioxidant effects of leaves and young pods between two phenotypes using principal component analysis (PCA) and then to evaluate the anti-inflammatory potential of the selected phenotype's plant parts. The biomarker contents were quantified by HPLC. The antioxidants were determined using the DPPH, ABTS, and FRAP models. Nitric oxide (NO) production assays in LPS-induced RAW264.7 macrophages were performed to determine the anti-inflammatory property of the extracts. The PCA revealed that there were no differences in total phenolic content, total flavonoid content, or antioxidant activities between short- and tall-stem phenotypes. Higher potency of the NO-inhibitory effect was achieved from the leaf extract than the pod extract. These results support using the short-stem phenotypes for utilizing the leaf and pod of O. indicum, and suggest choosing the leaf part for further anti-inflammatory product development.

Method Development and Validation of an Aerosol Sampling Technique for the Analysis of Nicotine in Electronic Cigarette Aerosols.

Because of the increasing popularity of e-cigarettes, monitoring the e-cigarette market has become important for national health authorities to guarantee safety and quality. In the EU, the Tobacco Products Directive requires emission studies for e-cigarette products. The absence of industry guidelines for studying these emissions and the lack of proper validation in the literature led us to develop and validate a method using the total error approach for the determination of nicotine in e-cigarette aerosols. A commercial vaping device was used to generate aerosols, which were then collected on Cambridge filter pads and measured for nicotine concentration by UHPLC-DAD after extraction. The method was successfully validated by generating accuracy profiles, which show that the ß-expectation tolerance intervals remained below the acceptance limits of ±20%. Within-run repeatability and intermediate precision were considered acceptable since the highest RSD value obtained was below 5%. The method was applied to 15 commercial e-liquids. A complete validation of a method for the analysis of e-cigarette emissions is presented, including several parameters that impact the accuracy and reproducibility. Similar systematic approaches for method development and validation could be used for other e-cigarette emission analysis methods to ensure the reliability of the measurements.

Sensitivity analysis and quality indicators for an in vitro oral irritation assay.

Biocompatibility testing using in vivo tests is often one of the final evaluations of new dental materials. To reduce the likelihood of failure at this late stage, predictive biocompatibility testing using in vitro methods is needed. In this study, we describe a sensitivity analysis of an oral irritation test by evaluating changes in the viability, using the MTT assay, of 3-D models with EpiOral constructs as a case study. Experiments that tested sources of variability in the assay led to recommendations regarding the storage of the constructs after arrival, pipetting procedure, use of MTT reagents from different vendors, use of transepithelial electrical resistance measurements, and statistical analyses. A statistical model was proposed to evaluate whether test substances yield a positive or negative result and the associated statistical confidence. Testing several test compounds such as the Y-4 polymer, that contains a known irritant, and dentally relevant substances such as sodium dodecyl sulfate (SDS) at varying concentrations revealed statistically significant results as expected. Lastly, a software app was designed to support a multiwell culture plate layout design. Overall, the findings and suggestions documented here will support the further development and potential standardization of this assay system and may be useful for the development of other assays using 3-D constructs.

New in vitro methods can support the development of novel dental materials by yielding information about their biocompatibility. In this study, an in vitro oral irritation assay was investigated to better understand what aspects of the method contribute to its variability. There is a potential that such a method could yield similar information to in vivo experiments and reduce animal testing.

Use of logit transformation within statistical analyses of experimental results obtained as proportions: example of method validation experiments and EQA in flow cytometry.

In laboratory medicine, measurement results are often expressed as proportions of concentrations or counts. These proportions have distinct mathematical properties that can lead to unexpected results when conventional parametric statistical methods are naively applied without due consideration in the analysis of method validation experiments, quality assessments, or clinical studies. In particular, data points near 0% or 100% can lead to misleading analytical conclusions. To avoid these problems, the logit transformation-defined as the natural logarithm of the proportion/(1-proportion)-is used. This transformation produces symmetric distributions centered at zero that extend infinitely in both directions without upper or lower bounds. As a result, parametric statistical methods can be used without introducing bias. Furthermore, homogeneity of variances (HoV) is given. The benefits of this technique are illustrated by two applications: (i) flow cytometry measurement results expressed as proportions and (ii) probabilities derived from multivariable models. In the first case, naive analyses within external quality assessment (EQA) evaluations that lead to inconsistent results are effectively corrected. Second, the transformation eliminates bias and variance heterogeneity, allowing for more effective precision estimation. In summary, the logit transformation ensures unbiased results in statistical analyses. Given the resulting homogeneity of variances, common parametric statistical methods can be implemented, potentially increasing the efficiency of the analysis.

Dissipation and dietary risk assessment of fluoxapiprolin (and its metabolites) residues in cucumber and tomato samples under field conditions.

The present study was undertaken to understand the dissipation behaviour/kinetics of fluoxapiprolin and its metabolites in cucumber and tomato under field conditions. A QuEChERS based extraction method followed by liquid chromatography coupled to mass spectrometry (LC-MS/MS) analysis showed that all method validation parameters were within the acceptable range as per international standards with a limit of quantitation (LOQ) of 0.01 mg kg-1 for all analytes. As significant matrix effects were observed with a few metabolites, matrix matched standards were used for the whole study. Residues of fluoxapiprolin in cucumber at standard dose were steady from 0 to 3 day after application and were below LOQ on the 5th day after application. In cucumber fruit at double dose and in tomato at both the doses the residues followed second-order kinetics and were respectively ≤ LOQ from days 7 and 14 onwards. Pre-harvest intervals (PHI) of 5 days and 14 days are proposed for cucumber and tomato fruits respectively. All the metabolites were ≤ LOQ from day 0 in all the matrices. The consumer risk, assessed as Hazard Quotient (HQ), showed that HQ was ≤1 in all the cases. The results of the present study and earlier studies on other similar fungicides suggest that the use of fluoxapiprolin in cucumber and tomato fruits may not pose health or environmental hazards provided that good agricultural practices are followed and the proposed waiting period is observed. The data from the present study can be used by regulatory bodies in establishing maximum residue limits.