Analytical and Bioanalytical Chemistry最新文献

An increasing number of cannabis-related products have become available and entered the market, particularly those containing cannabidiol (CBD) and Δ⁸-tetrahydrocannabinol (Δ⁸-THC). Analytical methods for cannabinoids in urine have been described extensively in the literature. However, methods providing good resolution for distinguishing interferences from THC positional isomers are needed. The aim of this project was to develop and validate a liquid chromatography with tandem mass spectrometry (LC-MS/MS) method to quantitate a broad panel of cannabinoids in authentic urine specimens. The method was optimized to quantitate Δ⁸-THC and Δ⁹-THC, 11-OH-Δ⁸-THC and 11-OH-Δ⁹-THC, Δ⁸-THC-COOH and Δ⁹-THC-COOH, CBD, 7-COOH-CBD, CBG, and CBN, and validated with the guidance of the American Academy of Forensic Sciences Standards Board (ASB) Standard 036. The validated assay was then used to evaluate urine samples collected over various time points from female patients (N = 69) enrolled in a study assessing prevalence of marijuana/CBD use during pregnancy from November 2022 to May 2024. Δ⁸- and Δ⁹- isomers were chromatographically resolved and successfully separated. For all analytes, the lower limit of quantitation (LLOQ) was determined to be 10 ng/mL, and the upper limit of quantitation (ULOQ) was 1000 ng/mL. In the authentic samples, the most frequently detected analyte was Δ⁹-THC-COOH, with a median concentration of 278 ng/mL (n = 38). Δ⁹-THC and 11-OH-Δ⁹-THC were detected with a median concentration of 42.4 ng/mL (n = 5) and 65.7 ng/mL (n = 34), respectively. Δ⁸-THC-COOH was detected in n = 3 specimens, with a median concentration of 25.5 ng/mL. The study provided a rapid assay for the analysis of cannabinoids in urine.

Free fatty acids (FFAs) are important energy sources and significant for energy transport in the body. They also play a crucial role in cellular oxidative stress responses, following cell membrane depolarization, making accurate quantification of FFAs essential. This study presents a novel supercritical fluid chromatography-mass spectrometry (SFC-MS) method using selected ion recording in negative electrospray ionization mode, enabling rapid quantification of 31 FFAs within 6 min without derivatization. FFAs are identified and quantified using an HSS C18 SB column and a secondary mobile phase consisting of methanol with formic acid by detecting their [M - H]^- ions. Calibration curves showed strong linearity (R² ≥ 0.9910), spanning 1000-12,000 ng/mL for short-chain FFAs and 50-1200 ng/mL for medium- and long-chain FFAs. The method achieves detection limits as low as 1 ng/µL for short-chain FFAs and 0.05 pg/µL for other FFAs per on-column injection. The method demonstrated high accuracy and precision, with bias and coefficients of variation maintained below 15% across five quality control levels. Freeze-thaw and autosampler stability studies confirmed the behavior of matrix-matched standards under optimal storage conditions. The validated method was applied to the analysis of pharmaceutical-grade egg yolk powders, using 13 deuterated FFAs as internal standards (IS) in comparison with heptadecanoic acid (C17:0). Significant variations in FFA quantification using two different IS approaches underscore the importance of selecting an appropriate IS. In summary, this study introduces a reliable and validated SFC-MS method for analyzing FFAs ranging from C4 to C26, requiring minimal sample preparation.

Certified reference materials (CRMs) play a crucial role in ensuring the quality of analytical measurements. Particularly, the EU labelling legislation on genetically modified organisms (GMOs) in food and feed products explicitly requires CRMs for its implementation. The CRMs are used to calibrate and control the quantitative real-time polymerase chain reaction (qPCR) method and support official control laboratories, such as National Reference Laboratories (NRLs), in maintaining their ISO/IEC 17025 accreditation. The Joint Research Centre of the European Commission (EC JRC) is specialized in the production of reference materials and has been producing GMO CRMs since 1998. Together with a validated event-specific qPCR method, these GMO CRMs form the EU reference system for the quantification of EU-authorized GMO events in food and feed products and ensure a common GMO labelling threshold. This article gives a retrospective view on the more than 25 years of GMO CRM production at JRC. It describes requirements for GMO CRMs in view of an EU market authorization under (EC) No. 1829/2003. The evolution and major improvements of the production of GMO CRMs at JRC are summarized as well as the current understanding of the EU's GMO reference system for GMO quantification and its impact on commutability. It provides insights into GMO CRM sales and their worldwide distribution. This information may be useful for policymakers and researchers in understanding the current EU GMO measurement landscape and to anticipate possible future demands related to GMO events based on new genomic techniques (NGTs).

Extracellular vesicles (EVs) have been demonstrated to own the advantages in evading phagocytosis, crossing biological barriers, and possessing excellent biocompatibility and intrinsic stability. Based on these characteristics, EVs have been used as effective therapeutic carriers for drug delivery, but the low drug loading capacity greatly limits further applications. Herein, we developed a drug loading method based on cell-penetrating peptide (CPP) to enhance the encapsulation of therapeutic reagents in EVs, and EVs-based drug delivery system achieved higher killing efficacy to tumor cells. Urinary EVs and chemotherapy reagent doxorubicin (DOX) were used as model. It is easy to conjugate CPP with DOX (CPP-DOX) through the linker N-succinimidyl 3-maleimidopropionate (SMP). CPP-DOX was incubated with EVs under a mild condition, promoting the encapsulation of DOX into EV cavities. CPP-DOX-EVs showed strong anticancer ability since EVs delivery facilitated the uptake by cancer cells. EVs loading of CPP-DOX exhibited higher drug loading efficiency at 37.18%, presenting about 2.5 times increase in efficiency over EVs loading of DOX through passive incubation. Easy operation and controllable condition further reinforce the advantages compared with other loading methods. CPP-based drug loading method provides an effective strategy for EVs-based drug delivery system.

Species identification of botanical products is a crucial aspect of research and regulatory compliance; however, botanical classification can be difficult, especially for morphologically similar species with overlapping genetic and metabolomic markers, like those in the genus Ocimum. Untargeted LC-MS metabolomics coupled with multivariate predictive modeling provides a potential avenue for improving herbal identity investigations, but the current dearth of reference materials for many botanicals limits the applicability of these approaches. This study investigated the potential of using greenhouse-grown authentic Ocimum to build predictive models for classifying commercially available Ocimum products. We found that three species, O. tenuiflorum, O. gratissimum, and O. basilicum, were chemically distinct based on their untargeted UPLC-MS/MS profiles when grown in controlled settings; combined with an orthogonal high-performance thin-layer chromatography (HPTLC) approach, O. tenuiflorum materials revealed two distinct chemotypes which could confound analysis. Three predictive models (partial least squares, LASSO regression, and random forest) were employed to extrapolate these findings to commercially available products; however, the controlled materials were significantly different from external samples, and all three chemometric models were unreliable in classifying external materials. LASSO was the most successful when classifying new greenhouse samples. Overall, this study highlights how growing and processing conditions can influence the complexity of botanical metabolome profiles; further studies are needed to characterize the factors driving herbal products' phytochemistry in conjunction with chemometric predictive modeling.

Chloramphenicol (CAP) is widely used in treating bacteria infection in animals and humans. However, the accumulation of CAP in food and environment caused serious health risk to human. Consequently, sensitive and selective detection of CAP is of great importance in environmental monitoring and food safety. Among various analytical methods, aptamer-based biosensors exhibit great potentials for CAP detection. Here, we developed an aptamer-based biosensor for rapid fluorescence detection of CAP based on pyrene excimer switch by using a newly selected short DNA aptamer with high affinity. The aptamer was labeled with pyrene molecules at both ends. The binding of CAP to the aptamer probe caused two pyrene molecules close to each other and the formation of a pyrene excimer, which induced the increase of the fluorescence signal from the pyrene excimer. CAP detection was achieved by measuring the fluorescence signal changes of the aptamer probes with dual pyrene labels. Under optimized conditions, the developed aptamer biosensor showed a detection limit of 24.4 nmol/L for CAP. The aptamer-based fluorescence sensor could quantify CAP in diluted tap water and lake water, exhibiting potentials for the application in real sample sensing of CAP.

The use of single-guide RNA (sgRNA) for gene editing using the CRISPR Cas9 system has become a powerful technique in various fields, especially with the growing interest in such molecules as therapeutic options in the last years. An important parameter for the use of these molecules is the verification of the correct sgRNA oligonucleotide sequence. Apart from next-generation sequencing protocols, mass spectrometry (MS) has been proven as a powerful technique for this purpose. The protocol and investigations presented in this work show an optimal digestion and 100% sequence coverage of sgRNA, while top-down approaches or other ribonuclease (RNase) digestion strategies obtain a sequence coverage of up to 80-90% utilizing multiple RNases. The results in this publication were obtained by utilizing DNA-RNA hybrid GAPmer-like probes and RNase H, an enzyme which specifically hydrolyzes RNA in DNA-RNA double strands. We assessed the optimal length of the DNA segment of these hybrid probes to maximize the specificity of the RNase H digestion and to achieve complete sequence confirmation by tandem MS analysis of the resulting digestion products. Furthermore, we showed that the approach is applicable for the identification of common synthesis-related impurities, like truncations and elongations. Despite the fact that the accessibility of this approach for highly modified molecules is limited to nucleotides which are not 2'-O-methylated, the optimized sequence coverage makes it a viable method.

Closely related species of Salmonidae, including Pacific and Atlantic salmon, can be distinguished from one another based on nucleotide sequences from the cytochrome c oxidase sub-unit 1 mitochondrial gene (COI), using ensembles of fragments aligned to genetic barcodes that serve as digital proxies for the relevant species. This is accomplished by exploiting both the nucleotide sequences and their quality scores recorded in a FASTQ file obtained via Next Generation (NextGen) Sequencing of mitochondrial DNA extracted from Coho salmon caught with hook and line in the Gulf of Alaska. The alignment is done using MUSCLE (Muscle 5.2) [1], applied to multiple versions of each fragment perturbed according to the nucleobase identification error probabilities underlying the quality scores. The Damerau-Levenshtein distance was used to determine the genetic barcode of the candidate species that is closest to each aligned, perturbed fragment. The "votes" that the sampled fragments cast for the different candidate species are then pooled and converted into identification probabilities, using weights determined by the entropy of the fragment-specific identification probability distributions. This novel approach to quantify the uncertainty associated with measurements made using NextGen Sequencing can be applied to discriminate closely related species, hence to value-assignment for reference materials supporting determinations of the authenticity of seafood, for example, NIST Reference Materials 8256 and 8257 (Coho salmon) [2].