Direct extraction with acetonitrile of hemp seed oil for the analysis of pesticides by using comprehensive two-dimensional gas chromatography-triple quadrupole mass spectrometry.

The method herein described involves a rapid and limited-volume (0.5 mL of acetonitrile) solvent-extraction sample preparation process, for pesticide determination in hemp seed oil. The extraction method was characterized by the absence of both clean-up or pre-concentration steps. The extracts were directly analyzed through cryogenic-modulation comprehensive two-dimensional gas chromatography coupled to triple quadrupole mass spectrometry. The novelty characterizing the present research [compared to a previous one (Arena et al., 2023)] is related to the extension of the number of pesticides (97), and to the investigation of a more challenging matrix, contained in a vegetable oil of increasing interest among consumers. Linearity, limits of detection and quantification, accuracy, precision, recovery, and matrix effect were measured. Particular emphasis was devoted to the matrix effect, with the co-extracted matrix amount defined. Three international regulations (Canada, California, Europe) were considered, and the obtained limits of quantification were found to be too high in five (Canada) and twelve (Europe) cases, for a total number of 15 pesticides. The analysis of ten commercial samples showed the presence of seven pesticide residues in four of them, at concentration levels ranging from 0.02 to 0.98 mg kg-1, with most over the regulation residue limits.

Development of a novel certified reference material for the determination of polycyclic aromatic hydrocarbons (PAHs) in whey protein powder.

Matrix-based certified reference materials (CRMs) play a central role in the analysis of food contaminants for ensuring measurement accuracy and comparability, as they exhibit the same matrix effects during sample preparation and measurement as the food sample under investigation. However, the availability of such CRMs is still limited. This is also true for matrix CRMs containing polycyclic aromatic hydrocarbons (PAHs), for which maximum levels in food are set in the EU by the Commission Regulation (EU) 2023/915 and in Switzerland by the regulation SR 817.022.15. Therefore, a whey protein powder-based certified reference material (WP-CBR001) was developed according to the requirements of ISO 17034 and the recommendations of ISO Guide 35:2017 containing the four PAHs benz[a]anthracene (BaA), benzo[a]pyrene (BaP), benzo[b]fluoranthene (BbF), and chrysene (Chr). We show that the choice of solvent is of crucial importance to extract the PAHs completely from this matrix. Only polar and protic solvents such as methanol or water provided access for complete extraction of the PAHs. In contrast, nonpolar and polar aprotic solvents, such as n-hexane or ethyl acetate, exhibited only very low extraction efficiencies below 20%. The certified mass fractions and expanded uncertainties (k = 2) were (3.17 ± 0.32) µg/kg BaA, (4.18 ± 0.48) µg/kg BaP, (4.73 ± 0.49) µg/kg BbF, and (2.85 ± 0.33) µg/kg Chr. These values were verified by an interlaboratory comparison study and by the gravimetric mass fractions obtained from production data.

The qNMR Summit 5.0: Proceedings and Status of qNMR Technology.

The goal of the qNMR Summit is to take stock of the status quo and the recent developments in qNMR research and applications in a timely and accurate manner. It provides a platform for both advanced and novice qNMR practitioners to receive a well-rounded update and discuss potential qNMR-related applications and collaborations. For over a decade, scientists from academia, industry, nonprofit institutions, and governmental bodies have focused on the standardization of qNMR methodology, as well as its metrological and pharmacopeial utility. This paper reviews key content of qNMR Summits 1.0 to 4.0 and puts into perspective the outcomes and available transcripts of the October 2019 Summit 5.0, with attendees from the United States, Canada, Japan, Korea, and several European countries. Summit presentations focused on qNMR methodology in the pharmaceutical industry, advanced quantitation algorithms, and promising developments.

Development of the first universal mixture for use in system suitability tests for High-Performance Thin Layer Chromatography.

The use of chromatographic methods in routine analysis includes System Suitability Tests (SST). This paper presents a novel approach to SST in HPTLC, which allows qualification of the entire RF range of an HPTLC plate independently of the samples. It is based on the Universal HPTLC mix (UHM), a pre-defined mixture of eight reference substances: guanosine, sulisobenzone, thymidine, paracetamol, phthalimide, 9-hydroxyfluorene, thioxanthen-9-one, and 2-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol, selected to cover a broad range of polarities and functional groups. The chromatographic behavior of the UHM was evaluated for 20 different mobile phases on Silica gel 60 F254. At least three constituents were baseline separated. In a collaborative trial with four laboratories the reproducibility of RF values for three representative mobile phases, was found to be within a confidence interval of 0.040 RF units. The response characteristics of the UHM were assessed with respect to changes in chromatographic conditions, such as variation of the relative humidity, improperly employed saturation, or mistakes in the preparation of the mobile phase. Based on the RF values of the individual constituents significant responses were found for most changes. This qualifies the UHM for use in SST.

Corrigendum: Enzymatic Synthesis of 2-Keto-3-Deoxy-6-Phosphogluconate by the 6-Phosphogluconate-Dehydratase From Caulobacter crescentus.

[This corrects the article DOI: 10.3389/fbioe.2020.00185.].

Enzymatic Synthesis of 2-Keto-3-Deoxy-6-Phosphogluconate by the 6-Phosphogluconate-Dehydratase From Caulobacter crescentus.

The availability of metabolic intermediates is a prerequisite in many fields ranging from basic research, to biotechnological and biomedical applications as well as diagnostics. 2-keto-3-deoxy-6-phosphogluconate (KDPG) is the key intermediate of the Entner-Doudoroff (ED) pathway for sugar degradation and of sugar acid and sugar polymer breakdown in many organisms including human and plant pathogens. However, so far KDPG is hardly available due to missing efficient synthesis routes. We here report the efficient biocatalytic KDPG production through enzymatic dehydration of 6-phosphogluconate (6PG) up to gram scale using the 6PG dehydratase/Entner-Doudoroff dehydratase (EDD) from Caulobacter crescentus (CcEDD). The enzyme was recombinantly produced in Escherichia coli, purified to apparent homogeneity in a simple one-step procedure using nickel ion affinity chromatography, and characterized with respect to molecular and kinetic properties. The homodimeric CcEDD catalyzed the irreversible 6PG dehydration to KDPG with a Vmax of 61.6 U mg-1 and a KM of 0.3 mM for 6PG. Most importantly, the CcEDD showed sufficient long-term stability and activity to provide the enzyme in amounts and purity required for the efficient downstream synthesis of KDPG. CcEDD completely converted 1 g 6PG and a straight forward purification method yielded 0.81 g of stereochemically pure KDPG corresponding to a final yield of 90% as shown by HPLC-MS and NMR analyses.

Recombinant AroL-Catalyzed Phosphorylation for the Efficient Synthesis of Shikimic Acid 3-Phosphate.

Shikimic acid 3-phosphate, as a central metabolite of the shikimate pathway, is of high interest as enzyme substrate for 5-enolpyruvoyl-shikimate 3-phosphate synthase, a drug target in infectious diseases and a prime enzyme target for the herbicide glyphosate. As the important substrate shikimic acid 3-phosphate is only accessible via a chemical multi-step route, a new straightforward preparative one-step enzymatic phosphorylation of shikimate using a stable recombinant shikimate kinase has been developed for the selective phosphorylation of shikimate in the 3-position. Highly active shikimate kinase is produced by straightforward expression of a synthetic aroL gene in Escherichia coli. The time course of the shikimate kinase-catalyzed phosphorylation is investigated by 1 H- and 31 P-NMR, using the phosphoenolpyruvate/pyruvate kinase system for the regeneration of the ATP cofactor. This enables the development of a quantitative biocatalytic 3-phosphorylation of shikimic acid. After a standard workup procedure, a good yield of shikimic acid 3-phosphate, with high HPLC- and NMR purity, is obtained. This efficient biocatalytic synthesis of shikimic acid 3-phosphate is superior to any other method and has been successfully scaled up to multi-gram scale.

Certified Reference Material for Use in 1H, 31P, and 19F Quantitative NMR, Ensuring Traceability to the International System of Units.

In recent years, quantitative NMR (qNMR) spectroscopy has become one of the most important tools for content determination of organic substances and quantitative evaluation of impurities. Using Certified Reference Materials (CRMs) as internal or external standards, the extensively used qNMR method can be applied for purity determination, including unbroken traceability to the International System of Units (SI). The implementation of qNMR toward new application fields, e.g., metabolomics, environmental analysis, and physiological pathway studies, brings along more complex molecules and systems, thus making use of 1H qNMR challenging. A smart workaround is possible by the use of other NMR active nuclei, namely 31P and 19F. This article presents the development of three classes of qNMR CRMs based on different NMR active nuclei (1H, 31P, and 19F), and the corresponding approaches to establish traceability to the SI through primary CRMs from the National Institute of Standards and Technology and the National Metrology Institute of Japan. These TraceCERT® qNMR CRMs are produced under ISO/IEC 17025 and ISO Guide 34 using high-performance qNMR.

Biocatalytic Asymmetric Phosphorylation Catalyzed by Recombinant Glycerate-2-Kinase.

The efficient synthesis of pure d-glycerate-2-phosphate is of great interest due to its importance as an enzyme substrate and metabolite. Therefore, we investigated a straightforward one-step biocatalytic phosphorylation of glyceric acid. Glycerate-2-kinase from Thermotoga maritima was expressed in Escherichia coli, allowing easy purification. The selective glycerate-2-kinase-catalyzed phosphorylation was followed by 31 P NMR and showed excellent enantioselectivity towards phosphorylation of the d-enantiomer of glyceric acid. This straightforward phosphorylation reaction and subsequent product isolation enabled the preparation of enantiomerically pure d-glycerate 2-phosphate. This phosphorylation reaction, using recombinant glycerate-2-kinase, yielded d-glycerate 2-phosphate in fewer reaction steps and with higher purity than chemical routes.

Method development in quantitative NMR towards metrologically traceable organic certified reference materials used as (31)P qNMR standards.

Quantitative nuclear magnetic resonance (qNMR) spectroscopy is employed by an increasing number of analytical and industrial laboratories for the assignment of content and quantitative determination of impurities. Within the last few years, it was demonstrated that (1)H qNMR can be performed with high accuracy leading to measurement uncertainties below 1 % relative. It was even demonstrated that the combination of (1)H qNMR with metrological weighing can lead to measurement uncertainties below 0.1 % when highly pure substances are used. Although qNMR reference standards are already available as certified reference materials (CRM) providing traceability on the basis of (1)H qNMR experiments, there is an increasing demand for purity assays on phosphorylated organic compounds and metabolites requiring CRM for quantification by (31)P qNMR. Unfortunately, the number of available primary phosphorus standards is limited to a few inorganic CRM which only can be used for the analysis of water-soluble analytes but fail when organic solvents must be employed. This paper presents the concept of value assignment by (31)P qNMR measurements for the development of CRM and describes different approaches to establish traceability to primary Standard Reference Material from the National Institute of Standards and Technology (NIST SRM). Phosphonoacetic acid is analyzed as a water-soluble CRM candidate, whereas triphenyl phosphate is a good candidate for the use as qNMR reference material in organic solvents. These substances contain both nuclei, (1)H and (31)P, and the concept is to show that it is possible to indirectly quantify a potential phosphorus standard via its protons using (1)H qNMR. The same standard with its assigned purity can then be used for the quantification of an analyte via its phosphorus using (31)P qNMR. For the validation of the concept, triphenyl phosphate and phosphonoacetic acid have been used as (31)P qNMR standards to determine the purity of the analyte tris(2-chloroethyl) phosphate, and the resulting purity values perfectly overlap within their expanded measurement uncertainties.

Quantitative assessment of preloaded 4-alkoxybenzyl alcohol resins for solid-phase peptide syntheses by 1D and 2D HR-MAS NMR.

The quality of preloaded Wang resins is very important for the success of solid-phase peptide syntheses (SPPS). A critical factor is the capping of remaining hydroxyl groups after loading with the first amino acid, since these free alcohols lead to truncated sequences during the following SPPS steps. Because the detection of hydroxyl groups by color tests is difficult and unreliable, the capping efficiency is often controlled by time-consuming peptide test syntheses. Here, we describe a two-dimensional, high resolution magic angle spinning NMR method for the quantitative determination of remaining 4-alkoxybenzyl alcohols in Fmoc-Xaa-Wang resins with a detection limit of 1 mol-%. The NMR method was validated with samples of known ratios between Fmoc-Ala-Wang and 4-alkoxybenzylalcohol resin. Application to a set of preloaded Fmoc-Ala- and Fmoc-Thr(tBu)-Wang test resins demonstrated that the full range of essential amino acids can be quantified without further spectrometer calibration. Compared to established test synthesis protocols, the NMR method represents not only advantages in terms of time and cost savings but also eliminates all inaccuracies due to further sample treatment like SPPS and cleavage from the resin.

Formation of Fmoc-beta-alanine during Fmoc-protections with Fmoc-OSu.

During the Fmoc-protection of H-alpha-Me-Val-OH, an unknown side product was found and isolated. The characterization using various analytical methods led unambiguously to the result that Fmoc-beta-Ala-OH was formed during the reaction. The reagent Fmoc-OSu was proven to be the source of Fmoc-beta-Ala-OH, following a mechanism that involved many deprotonation and elimination steps and a Lossen-type rearrangement as key sequence. The impurity Fmoc-beta-Ala-OH was found in a variety of reactions in which Fmoc-OSu was applied, either in the reaction mixture or as a contamination of the crude product. Purification of the Fmoc-amino acid derivatives from this impurity incurred high costs and significant reductions in yield.