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).

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.

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 (CCβ) 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.

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.

Mitotane (o,p\'-DDD) is the drug of choice for Adrenocortical Carcinomas (ACC) and its measurement in plasma is essential to control drug administration.
To develop and validate a simple, reliable and straightforward method for mitotane determination in plasma samples.
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.
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 %).
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.

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 β-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.

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.

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.

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.

New stability indicating related substances and assay methods for trametinib acetic acid by RP-HPLC have been developed and validated through forced degradation and mass balance studies for the first time. In related substances (RS) method, trametinib acetic acid was successfully separated from its six related substances namely, cyclopropanamide impurity, desiodo trametinib, desacetyl trametinib, trione acetamide intermediate, trione intermediate, and trione PTSA intermediate using YMC-Triart C18 (150 × 4.6 mm, 3 µm) column. Orthophosphoric acid (0.15%) in water was used as buffer. Gradient elution was programmed using mobile phase-A (buffer and acetonitrile mixture in 80:20 v/v ratio) and mobile phase-B (buffer and acetonitrile mixture in 20:80 v/v ratio). Acetonitrile and methanol mixture (1:1 v/v) was used as diluent. Flow rate, injection volume, column temperature, and wavelengths were kept as 0.8 mL/min, 10 µL, 55 °C, and 240 nm, respectively. Desacetyl trametinib and cyclopropanamide impurity were identified as potential degradation impurities in acid and base degradation conditions, respectively. Assay method specific to above six related substances was also developed. Assay of forced degradation samples was also determined, and the mass balance was established by adding total impurities formed in RS method to assay of trametinib acetic acid.