Evaluation of Individual Variation of d-Amino Acids in Human Plasma by a Two-Dimensional LC-MS/MS System and Application to the Early Diagnosis of Chronic Kidney Disease.

For the discovery of sensitive biomarkers of kidney function focusing on chiral amino acids, a multiple heart-cutting two-dimensional (2D) liquid chromatography-mass spectrometry (LC-MS)/MS system has been designed/developed. As the target analytes, alanine (Ala), aspartic acid, glutamic acid (Glu), leucine (Leu), lysine, methionine, phenylalanine (Phe), proline (Pro), serine (Ser), and valine were selected considering the presence of their d-forms in mammals. The 2D LC-MS/MS system consisted of the nonenantioselective reversed-phase separation of the target amino acids, the separations of the d- and l-enantiomers, and detection using MS/MS. Using the method, the plasma chiral amino acids, precolumn derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole, were isolated from other intrinsic substances, then determined without losing sensitivity by the fully automated whole-peak volume transfer operation from first to second dimension. In all of the tested plasma samples obtained from five healthy individuals and 15 patients with chronic kidney disease (CKD), the target chiral amino acids were determined without interference. In healthy individuals, the levels of all the tested d-amino acids were regulated in the low ranges. In contrast, the % d values of Glu, Leu, and Phe significantly increased with the progress of kidney dysfunction, besides the previously reported values of d-Ala, Pro, and Ser. Concerning Phe, the significant increase of the % d values (p < 0.05) was reported for the first time even in the mild CKD group compared to those of the healthy group; d-Phe might be a more sensitive marker than the previously reported d-forms. These results demonstrated the potential of these d-forms as the sensitive biomarkers of kidney function for the early diagnosis of CKD.

Plasma and milk metabolomics revealed changes in amino acid metabolism in Holstein dairy cows under heat stress.

Our understanding of metabolic alterations triggered by heat stress is incomplete, which limits the designing of nutritional strategies to mitigate negative productive and health effects. Thus, this study aimed to explore the metabolic responses of heat-stressed dairy cows to dietary supplementation with vitamin D3/Ca and vitamin E/Se. Twelve multiparous Holstein cows were enrolled in a split-plot Latin square design with two distinct vitamin E/Se supplementation levels, either at a low (ESe-, n = 6, 11.1 IU/kg vitamin E and 0.55 mg/kg Se) or a high dose (ESe+, n = 6 223 IU/kg vitamin E and 1.8 mg/kg Se) as the main plot. Treatment subplots, arranged in a replicated 3 × 3 Latin square design, comprised heat challenge (Temperature Humidity Index, THI: 72.0-82.0) supplemented with different levels of vitamin D3/Ca: either low (HS/DCa-, 1 012 IU/kg and 0.73%, respectively) or high (HS/DCa+, 3 764 IU/kg and 0.97%, respectively), and a pair-fed control group in thermoneutrality (THI = 61.0-64.0) receiving the low dose of vitamin D3/Ca (TN). The liquid chromatography-mass spectrometry-based metabolome profile was determined in blood plasma and milk sampled at the beginning (day 0) and end (day 14) of each experimental period. The results were analyzed for the effect of (1) TN vs. HS/ESe-/DCa-, and (2) the vitamin E/Se and vitamin D3/Ca supplementation. No group or group × day effects were detected in the plasma metabolome (false discovery rate, FDR > 0.05), except for triglyceride 52:2 being higher (FDR = 0.03) on day 0 than 14. Taurine, creatinine and butyryl-carnitine showed group × day interactions in the milk metabolome (FDR ≤ 0.05) as creatinine (+22%) and butyryl-carnitine (+190%) were increased (P < 0.01) on day 14, and taurine was decreased (-65%, P < 0.01) on day 14 in the heat stress (HS) cows, compared with day 0. Most compounds were unaffected by vitamin E/Se or vitamin D3/Ca supplementation level or their interaction (FDR > 0.05) in plasma and milk, except for milk alanine which was lower (-69%, FDR = 0.03) in the E/Se+ groups, compared with E/Se-. Our results indicated that HS triggered more prominent changes in the milk than in the plasma metabolome, with consistent results in milk suggesting increased muscle catabolism, as reflected by increased creatinine, alanine and citrulline levels. Supplementing with high levels of vitamin E/Se or vitamin D3/Ca or their combination did not appear to affect the metabolic remodeling triggered by HS.

The aromatic profile of wine distillates from Ugni blanc grape musts is influenced by the nitrogen nutrition (organic vs. inorganic) of Saccharomyces cerevisiae.

Although the impact of nitrogen nutrition on the production of fermentative aromas in oenological fermentation is well known today, one may wonder whether the effects studied are the same when winemaking takes place at high turbidities, specifically for the production of wines intended for cognac distillation. To that effect, a fermentation robot was used to analyze 30 different fermentation conditions at two turbidity levels with several factors tested: (i) initial addition of nitrogen either organic (with a mixture of amino acids - MixAA) or inorganic with di-ammonium phosphate (DAP) at different concentrations, (ii) variation of the ratio of inorganic/organic nitrogen (MixAA and DAP) and (iii) addition of different single amino acids (alanine, arginine, aspartic acid and glutamic acid). A metabolomic analysis was carried out on all resulting wines to have a global vision of the impact of nitrogen on more than sixty aromatic molecules of various families. Then, at the end of the alcoholic fermentation, the wines were micro-distilled. A first interesting observation was that the aroma profiles of both wines and distillates were close, indicating that the concentration factor is rather similar for the different aromas studied. Secondly, the fermentation kinetics and aroma results have shown that the nitrogen concentration effect prevailed over the nature of nitrogen. Although the lipid concentration was in excess, an interaction between the assimilable nitrogen and lipid contents was still observed in wines or in micro-distillates. Alanine is involved in the synthesis of acetaldehyde, isobutanol, isoamyl alcohol and isoamyl acetate. Finally, it was demonstrated that modifying the ratio of assimilable nitrogen in musts is not an interesting technological response to improve the aromatic profile of wines and brandies. Indeed, unbalance the physiological ratio of the must by adding a single source of assimilable nitrogen (organic or inorganic) has been shown to deregulate the synthesis of most of the fermentation aromas produced by the yeast. Wine metabolomic analysis confirmed the results that had been observed in micro-distillates but also in the other aromatic families, especially on terpenes. The contribution of solid particles, but also yeast biosynthesis (via sterol management in must) to wine terpenes is discussed. Indeed, the synthesis of terpenes in this oenological context seems to be favored, especially since the concentration of assimilable nitrogen (in addition to the lipid content) favor their accumulation in the medium. A non-negligible vintage effect on the terpene profile was also demonstrated with variations in their distribution depending on the years. Thus, the present study focuses on the metabolism of wine yeasts under different environmental conditions (nitrogen and lipid content) and on the impact of distillation on the fate of flavor compounds. The results highlight once again the complexity of metabolic fluxes and of the impact of nitrogen source (nature and amount) and of lipids. Furthermore, this study demonstrates that beyond the varietal origin of terpenes, the part resulting from the de novo synthesis by the yeast during the fermentation cannot be neglected in the context of cognac winemaking with high levels of turbidity.

Efficient determination of enantiomeric ratios of α-hydroxy/amino acids from fermented milks via ion mobility-mass spectrometry.

Chiral analysis of food components can provide important information for food quality, bioactivity and safety. Determination of enantiomeric ratios in food is a tedious task, due to the poor resolution and insufficient sensitivity for simultaneous discrimination and quantification of trace amounts of d-form metabolites. Herein, a high-throughput, high-sensitive and high-resolution method was developed for simultaneously determining enantiomeric ratios of multiple chiral α-hydroxy/amino acids (HA/AAs) from fermented milks in one-run by [d0]/[d5]-estradiol-3-benzoate-17ß-chloroformate labeling-assisted ion mobility - mass spectrometry. Results revealed extensive variation in chiral HA/AA profiles among 15 fermented milks. A total of 14 D-HA/AAs were identified. d-Lactic acid and d-alanine appeared as the most discriminatory in fermented milks with live lactic acid bacteria (LAB). Results suggested that glycolysis, casein hydrolysis and enantioisomerization of HA/AAs were most likely affected by various starter culture LAB. It may contribute to entail a valuable step forward in food quality control and discovering functional-related chiral biomarkers.

A Practical Method for EPR Dosimetry Using Alanine Powder.

ABSTRACT: This work investigates alanine powder, an inexpensive and versatile material compared to alanine pellets, as a standardized dosimeter for the alanine-EPR system using a Bruker EMX-Micro spectrometer. The feasibility of this method was investigated, and a calibration curve was produced using 40 dosimeters, which were prepared by tightly packing DL-alanine powder in polypropylene microcentrifuge tubes. The dosimeters were irradiated to doses ranging from 0.2-20 Gy using a 60 Co source. A dosimeter handling and measurement protocol was established for all dosimeters. The dosimetric signal was evaluated by measuring the peak-to-peak height of the central resonance peak, and the dose response of alanine powder dosimeters showed a linear behavior in the investigated dose range with relative errors below 13%. Measurement repeatability and reproducibility were tested to show the errors associated with sample placement in the cavity and with the overall measurement method, with both tests showing relative errors below 7%. As an inexpensive material compared to pellet dosimeters, alanine powder has a strong potential to be used as a standardized material for radiation dosimetry applications. The scope of this work is to present an effective and comprehensive methodology with accompanying analysis scripts for dosimetry with alanine powder that is useful in a wide range of applications and dose requirements.

Magnetic mesoporous material derived from MIL-88B modified by l-alanine as modified QuEChERS adsorbent for the determination of 6 pesticide residues in 4 vegetables by UPLC-MS/MS.

More and more attention has been paid to the improved QuEChERS (quick, easy, cheap, effective, rugged and safe) method in dealing complex sample matrices, especially for the study of QuEChERS adsorbents. In this study, a magnetic mesoporous material, which was derived from MIL-88B modified by l-alanine, was synthesized as modified QuEChERS adsorbents for the simultaneous determination of multiple pesticides (Methomyl, Isoprocarb, Carbofuran, 3-Hydroxycarbofuran, Acetamiprid, Imidacloprid) in Chinese cabbage, celery, long bean and leek. The prepared magnetic adsorbents can effectively remove interfering substances from the sample, and the proposed modified QuEChERS method can reduce sample pretreatment time via an external magnetic field. To achieve the best performance of QuEChERS method, the clean-up time and amount of QuEChERS adsorbents were investigated. Under the optimized conditions, a simple, rapid and sensitive method for the determination of 6 pesticide residues in vegetables was established by coupling the modified QuEChERS to ultrahigh-performance liquid chromatography-tandem mass spectrometry. Excellent sensitivity (The limit of detection for the 6 pesticides ranged from 0.001 to 0.020 µg kg-1), satisfactory linearity (r2 ≥ 0.9952), good recovery (73.9-107.7%) and good precision (3.6-16.9% for intraday relative standard deviation, 0.5-15.0% for interday relative standard deviation) were obtained. Compared with traditional QuEChERS method, the proposed method is simple, cost-effective, and efficient, which indicates that the method can be used to detect carbamate and neonicotinoid pesticides in real samples and provide an excellent pretreatment technique for the detection of trace multi-analytes from complex substrates.

Development and Validation of High-Performance Liquid Chromatography Method for the Quantification of Remdesivir in Intravenous Dosage Form.

Corona virus disease 2019 (COVID-19) has posed a mounting threat to public health with worldwide outbreak caused by a novel virus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Recently, remdesivir (RDV) has been approved by Food and Drug Administration (FDA) for treating COVID-19 patients ≥12 years old requiring hospitalization. To the best of our knowledge, a simple method to estimate RDV in the pharmaceutical formulations using high-performance liquid chromatography (HPLC) is still unexplored, highlighting the need for a precise analytical method for its quantification. The prime purpose of the current investigation was to develop and validate a well-grounded HPLC method for quantification of RDV in pharmaceutical formulations. The best chromatogram was obtained by means of an Inertsil ODS-3V column using a mobile phase of milli-Q water modified to pH 3.0 with o-phosphoric acid and acetonitrile (50:50, % v/v) at a flow rate of 1.2 mL/min and wavelength of detector set at 246 nm with retention time being achieved at 6.0 min. The method was validated following International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) Q2 (R1) guidelines for various parameters such as specificity and selectivity, system suitability, linearity, precision, accuracy, limits of detection and quantification, and robustness. The method developed for the quantification of RDV was found to be linear in the concentration range of 25-2,500 ng/mL with limit of detection and limit of quantification of 1.95 and 6.49 ng/mL, respectively. Assay value of 102% ± 1% was achieved for marketed injectable dosage form when estimated by the validated method. Therefore, in this study a simple, rapid, sensitive, selective, accurate, precise, and robust analytical method was developed and validated for the quantification of RDV using HPLC. The established method was successfully employed for quantification of RDV in marketed pharmaceutical formulation.

A flow-cytometry-based assessment of global protein synthesis in human senescent cells.

Senescent cells constantly experience stressful conditions and restrain their protein translation to cope with it. Here, we present a detailed protocol to measure the rate of global protein synthesis using L-azidohomoalanine (L-AHA)-based click chemistry in human senescent fibroblasts. We optimized several aspects of the procedure, including senescence induction, a flow cytometry analysis of senescent cells, and the duration of L-AHA incorporation. This protocol uses senescent human fibroblasts but can be applied to other types of cells or circumstances. For complete details on the use and execution of this protocol, please refer to Lee et al. (2021).

An in silico, structural, and biological analysis of lactoferrin of different mammals.

Lactoferrin (LF) belongs to the family of transferrins having multifunctional roles associated with the immune system of animals. To follow the aims for this study was selected 20 sequences of LF from mammalian species to evaluate the chemical, biological, and structural properties. Bioinformatics approaches used programs such as MAFFT for sequence alignment; PartitionFinder and MrBayes for phylogenetic approaches; I-TASSER, PROCHECK, Molecular Operating Environment (MOE), SWISS Model server, Peptide DB and Expasy ProtParam to estimate the physicochemical properties, to model the protein and predicted secondary structures. A phylogenic analysis shows species with genetic similarities clustered by complexity and unique grouping between Capra hircus, Macaca mulatta, and Myotis lucifugus, since they presented more amino acids but not overall changes in the iron-binding sites or biological aspects. Structural deviations in these clusters obtained in LF from those species were found in residues 46 (position 406-450), that is part of alpha-helix, and 37 (position 295-331), that is part of the beta-sheets. Our predicted model can be used to investigate more about structural aspects of LF and be applied for medicinal research.

Spectral editing of alanine, serine, and threonine in uniformly labeled proteins based on frequency-selective homonuclear recoupling in solid-state NMR.

Spectral editing is crucial to simplify the crowded solid-state NMR spectra of proteins. New techniques are introduced to edit 13C-13C correlations of uniformly labeled proteins under moderate magic-angle spinning (MAS), based on our recent frequency-selective homonuclear recoupling sequences [Zhang et al., J. Phys. Chem. Lett. 2020, 11, 8077-8083]. The signals of alanine, serine, or threonine residues are selected out by selective 13Cα-13Cß double-quantum filtering (DQF). The 13Cα-13Cß correlations of alanine residues are selectively established with efficiency up to ~ 1.8 times that by dipolar-assisted rotational resonance (DARR). The techniques are shown in 2D/3D NCCX experiments and applied to the uniformly 13C, 15N labeled Aquaporin Z (AqpZ) membrane protein, demonstrating their potential to simplify spectral analyses in biological solid-state NMR.

Supercritical fluid chromatography-mass spectrometric determination of chiral fungicides in viticulture-related samples.

Supercritical fluid chromatography (SFC), combined with mass spectrometry (MS), was employed for the determination of five chiral fungicides, from two different chemical families (acylalanine and triazol) in wine and vineyard soils. The effect of different SFC parameters (stationary phase, chiral selector, mobile phase modifier and additive) in the resolution between enantiomers and in the efficiency of compounds ionization at the electrospray source (ESI) was thorougly described. Under final working conditions, chiral separations of selected fungicides were achieved using two different SFC-MS methods, with an analysis time of 10 min and resolution factors from 1.05 to 2.45 between enantiomers. In combination with solid-phase extraction and pressurized liquid extraction, they permitted the enantiomeric determination of target compounds in wine and vineyard soils with limits of quantification in the low ppb range (between 0.5 and 2.5 ng mL-1, and from 1.3 to 6.5 ng g-1, for wine and soil, respectively), and overall recoveries above 80%, calculated using solvent-based standards. For azolic fungicides (tebuconazole, myclobutanil and penconazole) soil dissipation and transfer from vines to wines were non-enantioselective processes. Data obtained for acylalanine compounds confirmed the application of metalaxyl (MET) to vines as racemate and as the R-enantiomer. The enantiomeric fractions (MET-S/(MET-S+MET-R)) of this fungicide in vineyard soils varied from 0.01 to 0.96; moreover, laboratory degradation experiments showed that the relative dissipation rates of MET enantiomers varied depending on the type of soil.

A novel technique for the reduction of pesticide residues by a combination of low-intensity electrical current and ultrasound applications: A study on lettuce samples.

In this study, effects of low-intensity electrical currents (200, 800 and 1400 mA), ultrasound frequencies (24 and 40 kHz) and their combinations were applied at the duration period of 2, 4, 6, 8, and 10 min for the degradation of captan, thiamethoxam and metalaxyl residues in lettuce samples. Residues of the pesticides were determined by gas chromatography with tandem mass spectrometry and electron capture detector. The results indicated that the combination of low-intensity electrical current and ultrasound was found to be effective for the reduction of the pesticides. The most effective combination was obtained to be current of 1400 mA and ultrasound frequency of 24 kHz at 10 min. Under this circumstance, 92.57, 81.99 and 93.09% of captan, thiamethoxam and metalaxyl residues were decreased, respectively. The findings suggest that the combination of low-intensity electrical current and ultrasound applications has an important potential for the degradation of pesticide residues.

Analytical methods for the determination of remdesivir as a promising antiviral candidate drug for the COVID-19 pandemic.

Coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is undoubtedly the most challenging pandemic in the current century. A total of 73,953,702 confirmed cases of COVID-19 and 1,644,416 deaths were reported globally up to December 17, 2020. Therefore, in the absence of a safe and effective vaccine, it is urgent to identify a novel antiviral drug to effectively treat patients with COVID-19. On October 22, the U.S. Food and Drug Administration approved remdesivir, a nucleotide analog prodrug with broad antiviral activity, for adults and children (12 years of age and older and weighing at least 40 kg) who need to be admitted to hospital for covid-19 treatment. In order to monitor the optimization of patient clinical response profile, as well as address the challenges associated with remdesivir metabolism, highly sensitive, selective and accurate analytical methods are necessary. This review clearly covers all the analytical methods developed for the identification and quantitative determination of remdesivir and its metabolites in biological matrices, which helps the researchers in developing new methods for the analysis of remdesivir by considering the pros and cons of the previously reported methods.

Time-resolved non-invasive metabolomic monitoring of a single cancer spheroid by microfluidic NMR.

We present a quantitative study of the metabolic activity of a single spheroid culture of human cancer cells. NMR (nuclear magnetic resonance) spectroscopy is an ideal tool for observation of live systems due to its non-invasive nature. However, limited sensitivity has so far hindered its application in microfluidic culture systems. We have used an optimised micro-NMR platform to observe metabolic changes from a single spheroid. NMR spectra were obtained by directly inserting microfluidic devices containing spheroids ranging from 150 [Formula: see text]m to 300 [Formula: see text]m in diameter in 2.5 [Formula: see text]L of culture medium into a dedicated NMR probe. Metabolite concentrations were found to change linearly with time, with rates approximately proportional to the number of cells in the spheroid. The results demonstrate that quantitative monitoring of a single spheroid with [Formula: see text] 2500 cells is possible. A change in spheroid size by 600 cells leads to a clearly detectable change in the L-Lactic acid production rate ([Formula: see text]). The consumption of D-Glucose and production of L-Lactic acid were approximately 2.5 times slower in spheroids compared to monolayer culture of the same number of cells. Moreover, while cells in monolayer culture were found to produce L-Alanine and L-Glutamine, spheroids showed slight consumption in both cases.

Challenges and stepwise fit-for-purpose optimization for bioanalyses of remdesivir metabolites nucleotide monophosphate and triphosphate in mouse tissues using LC-MS/MS.

Remdesivir is a prodrug of the nucleotide analogue and used for COVID-19 treatment. However, the bioanalysis of the active metabolites remdesivir nucleotide triphosphate (RTP) and its precursor remdesivir nucleotide monophosphate (RMP) is very challenging. Herein, we established a novel method to separate RTP and RMP on a BioBasic AX column and quantified them by high-performance liquid chromatography-tandem mass spectrometry in positive electrospray ionization mode. Stepwise, we optimized chromatographic retention on an anion exchange column, improved stability in matrix through the addition of 5,5'-dithiobis-(2nitrobenzoic acid) and PhosSTOP EASYpack, and increased recovery by dissociation of tight protein binding with 2 % formic acid aqueous solution. The method allowed lower limit of quantification of 20 nM for RMP and 10 nM for RTP. Method validation demonstrated acceptable accuracy (93.6%-103% for RMP, 94.5%-107% for RTP) and precision (RSD < 11.9 % for RMP, RSD < 11.4 % for RTP), suggesting that it was sensitive and robust for simultaneous quantification of RMP and RTP. The method was successfully applied to analyze RMP and RTP in mouse tissues. In general, the developed method is suitable to monitor RMP and RTP, and provides a useful approach for exploring more detailed effects of remdesivir in treating diseases.

Optimization of Alanine Measurements for Fast and Accurate Dosimetry in FLASH Radiation Therapy.

FLASH radiation therapy (FLASH-RT) reference dosimetry to obtain traceability, repeatability and stability of irradiations cannot be performed with conventional dosimetric methods, such as monitor chambers or ionization chambers. Until now, only passive dosimeters have provided the necessary dosimetric data. Alanine dosimetry is accurate; however, to be used for FLASH-RT in biological experiments and for clinical transfer to humans, the reading time needs to be reduced, while preserving a maximum deviation to the reference of ±2%. Optimization of alanine dosimetry was based on the acquisition of electron paramagnetic resonance (EPR) spectra with a Bruker spectrometer. Reading parameters such as the conversion time, the number of scans, the time constant, the microwave power and the modulation amplitude of the magnetic field were optimized as a trade-off between the signal-to-noise ratio (SNR) and the reading time of one measurement using the reference 10.1 Gy alanine pellet. After optimizing the parameters, we compared the doses measured with alanine pellets up to 100 Gy with the reference doses, and then determined the number of measurements necessary to get a difference lower than ±2%. A low-dose alanine pellet of 4.9 Gy was also measured to evaluate the quality of the optimization for doses lower than 10 Gy. The optimization of the Bruker default parameters made it possible to reduce the reading time for one measurement from 5.6 to 2.6 min. That reduction was not at the cost of the SNR because it was kept comparable to the default parameters. Three measurements were enough to obtain a maximum dose deviation to the reference of 1.8% for the range of 10-100 Gy. The total reading time for the three measurements was 7.8 min (3 × 2.6 min). For lower doses such as 4.9 Gy, three measurements led to a deviation greater than 5%. By increasing the number of measurements to five, the average difference to the reference dose was reduced to less than 5% with a total reading time increased to 13.0 min. For doses between 10 Gy and 100 Gy, the optimized acquisition parameters made it possible to keep the average differences between the reference and the measured doses below ±2%, for a reading time of 7.8 min. This enabled an accurate and fast dose determination for biological preparations as part of FLASH-beam irradiations.

Water soluble selenometabolome of Cardamine violifolia.

Low molecular weight selenium containing metabolites in the leaves of the selenium hyperaccumulator Cardamine violifolia (261 mg total Se per kg d.w.) were targeted in this study. One dimensional cation exchange chromatography coupled to ICP-MS was used for purification and fractionation purposes prior to LC-Unispray-QTOF-MS analysis. The search for selenium species in full scan spectra was assisted with an automated mass defect based filtering approach. Besides selenocystathionine, selenohomocystine and its polyselenide derivative, a total number of 35 water soluble selenium metabolites other than selenolanthionine were encountered, including 30 previously unreported compounds. High occurrence of selenium containing hexoses was observed, together with the first assignment of N-glycoside derivatives of selenolanthionine. Quantification of the most abundant selenium species, selenolanthionine, was carried out with an ion pairing LC - post column isotope dilution ICP-MS setup, which revealed that this selenoamino acid accounted for 30% of the total selenium content of the leaf (78 mg (as Se) per kg d.w.).

A Biochemical Lanthanide-Encoding Approach Enables Quantitative Monitoring of the Bacterial Response to Vancomycin Treatment.

A pathogenic bacterium has its own mechanisms for not only pathogenic attack but also exogenous invasion defense, in which the bacterial cell wall is the front line of attack and defense. We developed a biochemical lanthanide-encoding approach to quantify the uncanonical d-amino acid (d-X) that was edited in a small proportion into the terminal acyl-d-Ala-d-X of nascent peptidoglycan UDP-MurNAc-pentapeptides in the bacterial cell wall. This approach overcomes the difficulties regarding quantification and accuracy issues encountered by the popular optical imaging and traditional high-performance liquid chromatography-based methods. Newly synthesized azide-d-Leu and ketone-d-Met were used together with alkynyl-d-Ala for their metabolic assembly and then bioorthogonally encoded by the correspondingly fabricated DBCO-DOTA-Gd, H2NO-DOTA-Eu, and azide-DOTA-Sm tags. This approach allows direct quantification of the d-X in situ in the cell wall using 158Gd, 153Eu, and 154Sm species-unspecific isotope dilution inductively coupled plasma mass spectrometry, avoiding any tedious and complex "cell-broken" pretreatment procedures that might induce racemization of the d-X. The obtained site-specific and accurate in situ information about the d-X enables quantitative monitoring of the bacterial response when Staphylococcus aureus meets vancomycin, showing that the amounts of azide-d-Leu and ketone-d-Met assembled are more important after determining the structure- and composition-dependent bacterial antibiotic resistance mechanisms. In addition, we found that the combined use of vancomycin and d-Ala restores the efficacy of vancomycin and might be a wise and simple way to combat vancomycin intermediate-resistant S. aureus.

Impurity Profiling of a Novel Anti-MRSA Antibacterial Drug: Alalevonadifloxacin.

Alalevonadifloxacin (ALA, formerly described as WCK 2349) is a novel antibacterial drug developed to treat infections caused by Gram-positive bacteria. The current study was aimed to identify and quantify impurities in ALA. Mass spectrometry (MS) compatible reverse phase liquid chromatographic method was developed to identify the impurities in ALA. Three impurities were identified based on the molecular ion peak and their product ions. These impurities were synthesized and characterized using MS and nuclear magnetic resonance spectrometry. However, this method was unable to resolve the diastereomeric impurity, which is likely to be formed during synthesis. Therefore, another method was developed using YMC Chiral NEA-[S] as a chiral stationary phase and validated for quantification of all impurities including diastereomeric impurity. The developed method was employed for quality control and stability studies of the ALA drug substance used in pre-clinical and clinical studies.

Three-dimensional high-performance liquid chromatographic analysis of chiral amino acids in carbonaceous chondrites.

A three-dimensional (3D) HPLC system in combination with fluorescence derivatization has been developed for the highly sensitive and selective analysis of chiral amino acids in extraterrestrial samples. As the targets, alanine (Ala), 2-aminobutyric acid (2AB), valine (Val), norvaline (nVal) and isovaline (iVal), frequently found chiral amino acids in the carbonaceous chondrites, were selected. These amino acids were pre-column derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), and the target analytes were separated from other amino acids and organic compounds by a reversed-phase column in the first dimension. The targets were further separated from interferences by an anion-exchange column in the second dimension, and their enantiomers were separated and determined in the third dimension by a Pirkle-type enantioselective column. The present 3D-HPLC system was validated and applied to the Murchison meteorite and the Antarctic meteorites, and all of the target amino acid enantiomers were clearly observed (0.78-22.33 nmol/g in the Murchison meteorite and 1.79-78.84 nmol/g in the Antarctic meteorites) without severe interferences. The %L values of the non-proteinogenic amino acids were almost 50% in both meteorites, and even the proteinogenic amino acids were almost racemic in the Antarctic meteorites.