ECM derivatized alginate augmenting bio-functionalities of lyophilized mat for skin and liver wound treatment.

Peptides and molecular residues sourced from the fragmentation of the extracellular matrix (ECM) can exacerbate a plethora of cellular functions. We selected a natural ECM-derived complex peptide mixture to functionalize sodium alginate. Three alginate derivatives (sodium alginate conjugated with ECM) SALE-1, SALE-2, and SALE-3 were synthesized using the lowest (10 % w/w), moderate (50 % w/w), and highest (100 % w/w) concentrations of ECM. Thereafter, they were used to fabricate three groups of mat scaffolds EMAT-1 (ECM derivatized alginate thrombin-mat), EMAT-2, and EMAT-3, respectively by the freeze-drying process. To enhance the hemostatic activity, thrombin was loaded onto the scaffolds. Another group, AT, without any derivatized alginate was additionally included in order to comparative analysis. Physical characteristics revealed that the porous mat scaffold showed enhancement in degradation and swelling ability with the increase in ECM content. The higher cell proliferation, migration, and cell viability were noticed in the higher ECM-containing samples EMAT-2 and EMAT-3. In vivo studies using rodent hepatic and rabbit ear models were carried out to ensure the hemostatic ability of the scaffolds. EMAT-2 and EMAT-3 demonstrate excellent liver regeneration ability in rat models. Moreover, the rat cutaneous wound model depicted that EMAT-3 dramatically elevated the skin's healing ability, thereby rendering it an excellent candidate for future clinical application in wound healing.

Discovery of TCP-(MP)-caffeic acid analogs as a new class of agents for treatment of osteoclastic bone loss.

Inhibition of LSD1 was proposed as promising and attractive therapies for treating osteoporosis. Here, we synthesized a series of novel TCP-(MP)-Caffeic acid analogs as potential LSD1 inhibitors to assess their inhibitory effects on osteoclastogenesis by using TRAP-staining assay and try to explore the preliminary SAR. Among them, TCP-MP-CA (11a) demonstrated osteoclastic bone loss both in vitro and in vivo, showing a significant improvement in the in vivo effects compared to the LSD1 inhibitor GSK-LSD1. Additionally, we elucidated a mechanism that 11a and its precursor that 11e directly bind to LSD1/CoREST complex through FAD to inhibit LSD1 demethylation activity and influence its downstream IκB/NF-κB signaling pathway, and thus regulate osteoclastic bone loss. These findings suggested 11a or 11e as potential novel candidates for treating osteoclastic bone loss, and a concept for further development of TCP-(MP)-Caffeic acid analogs for therapeutic use in osteoporosis clinics.

[Determination of the derivatization reactivity between α/ß-dicarbonyl compounds and standard citrullinated peptides based on matrix-assisted laser desorption ionization-time-of-flight mass spectrometry].

Protein citrullination is an irreversible post-translational modification process regulated by peptidylarginine deiminases (PADs) in the presence of Ca2+. This process is closely related to the occurrence and development of autoimmune diseases, cancers, neurological disorders, cardiovascular and cerebrovascular diseases, and other major diseases. The analysis of protein citrullination by biomass spectrometry confronts great challenges owing to its low abundance, lack of affinity tags, small mass-to-charge ratio change, and susceptibility to isotopic and deamidation interferences. The methods commonly used to study the protein citrullination mainly involve the chemical derivatization of the urea group of the guanine side chain of the peptide to increase the mass-to-charge ratio difference of the citrullinated peptide. Affinity-enriched labels are then introduced to effectively improve the sensitivity and accuracy of protein citrullination by mass spectrometry. 2,3-Butanedione or phenylglyoxal compounds are often used as derivatization reagents to increase the mass-to-charge ratio difference of the citrullinated peptide, and the resulting derivatives have been observed to contain α-dicarbonyl structures. To date, however, no relevant studies on the reactivity of dicarbonyl compounds with citrullinated peptides have been reported. In this study, we determined whether six α-dicarbonyl and two ß-dicarbonyl compounds undergo derivatization reactions with standard citrullinated peptides using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Among the α-dicarbonyl compounds, 2,3-butanedione and glyoxal reacted efficiently with several standard citrullinated peptides, but yielded a series of by-products. Phenylglyoxal, methylglyoxal, 1,2-cyclohexanedione, and 1,10-phenanthroline-5,6-dione also derivated efficiently with standard citrullinated peptides, generating a single derivative. Thus, a new derivatization method that could yield a single derivative was identified. Among the ß-dicarbonyl compounds, 1,3-cyclohexanedione and 2,4-pentanedione successfully reacted with the standard citrullinated peptides, and generated a single derivative. However, their reaction efficiency was very low, indicating that the ß-dicarbonyl compounds are unsuitable for the chemical derivatization of citrullinated peptides. The above results indicate that the α-dicarbonyl structure is necessary for realizing the efficient and specific chemical derivatization of citrullinated peptides. Moreover, the side chains of the α-dicarbonyl structure determine the structure of the derivatives, derivatization efficiency, and generation (or otherwise) of by-products. Therefore, the specific enrichment and precise identification of citrullinated peptides can be achieved by synthesizing α-dicarbonyl structured compounds containing affinity tags. The proposed method enables the identification of citrullinated proteins and their modified sites by MS, thereby providing a better understanding of the distribution of citrullinated proteins in different tissues. The findings will be beneficial for studies on the mechanism of action of citrullinated proteins in a variety of diseases.

Measurement of atmospheric amines and aminoamides by column adsorption/extraction and hydrophilic liquid chromatography-electrospray-tandem mass spectrometry.

Sampling and chromatography-mass spectrometry methods were investigated to measure atmospheric amines and aminoamides. Amines and their amide derivatives play significant roles in new particle formation (NPF) in the atmosphere, especially diamines and aminoamides have higher NPF potentials compared to monoamines. For amine sampling, silica gel tube collection and formic acid extraction gave good overall recoveries (>93 ± 8%) for mono-, di-, tri-, tetramines, and aminoamides. Two chromatography methods were subjected to analyze the extracted amines. One involved direct analysis using hydrophilic interaction liquid chromatography with carboxyl or diol group functioned separation column (carboxyl-HILIC or diol-HILIC), and the other utilized derivatization with 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) and subsequent reversed-phase chromatography (HPLC). Separated amines were detected by electrospray ionization and tandem mass spectrometry in both cases. DBD-F-HPLC method provided good sensitivity for mono- and all polyamines (limit of detection (LOD) < 4.6 nM, relative standard deviation (RSD) for 100 nM < 9.2%). However, aminoamides could not be detected by DBD-F-HPLC. Carboxyl-HILIC provided good sensitivities for mono- and diamines and aminoamides (LOD < 1.6 nM, RSD < 4.8%). Forest air measurement was performed and data obtained by carboxyl-HILIC and DBD-F-HPLC showed good agreement for 1,3-diaminopropane, 1,4-diaminobutane (putrescine) and 1,5-diaminopentane (cadaverine) (R2 = 0.9215-0.9739, n = 7-14). Carboxyl-HILIC method was the best for the amine analysis, and combination with silica gel tube sampling provides atmospheric monitoring available. The developed method can be used not only to study atmospheric chemistry of diamines and aminoamides but also to analyze flavor/odor of foods, flowers and wastes.

Tetrodotoxin Derivatization with a Newly Designed Boron Reagent Leads to Conventional Reversed-Phase Liquid Chromatography.

Tetrodotoxin (TTX) is a representative natural toxin causing pufferfish food poisoning, which is especially prominent in East and Southeast Asia, including Japan. TTX has been analyzed through post-column derivatization high-performance liquid chromatography (HPLC), ion-pair LC-MS(/MS), and hydrophilic interaction liquid chromatography (HILIC)-MS(/MS) as alternatives to the mouse bioassay method. However, post-column derivatization requires a system for online derivatization reactions, and with the ion-pair LC-MS approach, it is difficult to remove residual ion-pair reagents remaining in the equipment. Moreover, HILIC-MS provides poor separation compared to reversed-phase (RP) HPLC and requires a long time to reach equilibration. Therefore, we decided to develop a TTX analytical method using pre-column derivatization and RP HPLC for the rapid assessment of outbreak samples, including food remnants. In this study, we focused on the vic-diol moiety of TTX and designed a new derivatization reagent coded as NBD-H-DAB. This NBD-H-DAB was synthesized from 4-hydrazino-7-nitro-2,1,3-benzoxadiazole (NBD-H) and 3-fluoro-2-formylphenylboronic acid (FFPBA) with a simple reaction system and rapidly converted to its boronate form, coded NBD-H-PBA, in an aqueous reaction solution. The NBD-H-PBA demonstrated appropriate hydrophobicity to be retained on the RP analytical column and successfully detected with a UV spectrometer. It was easily reacted with the vic-diol moiety of TTX (C6 and C11) to synthesized a boronic ester. The derivatized TTX could be detected using the RP HPLC-UV, and the limit of detection in the fish flesh samples was 0.06 mg/kg. This novel pre-column derivatization of TTX with NBD-H-PBA proves capable for the analysis of TTX.

Development and evaluation of a liquid chromatography-tandem mass spectrometry method for simultaneous measurement of toxic aldehydes from brain tissue.

Reactive aldehydes are a class of electrophilic low molecular weight compounds that play an essential role in physiological function and lipid peroxidation. These molecules are implicated in many diseases, especially cardiovascular and neurodegenerative diseases, and are potential endogenous markers of lipid peroxidation. However, there are limited options to accurately quantify multiple reactive aldehydes in brain tissue. This study developed and validated a 3-nitrophenylhydrazine derivatization-based LC-MS/MS method to quantify four reactive aldehydes: malondialdehyde, acrolein, 4-hydroxy-2-hexenal and 4-hydroxy-2-nonenal. Method development involved comparing the sensitivity of detection between widely used derivatization reagents: 2,4-dinitrophenylhydrazine and 3-nitrophenylhydrazine. Our data showed that 3-nitrophenylhydrazine resulted in greater sensitivity. Additional method development included evaluation of hydrolysis sample pretreatment, selection of protein precipitation reagent, and optimization of derivatization conditions. The optimized conditions included no hydrolysis and use of 20 % trichloroacetic acid as the protein precipitation reagent. The optimized derivatization condition was 25 mM 3-nitrophenylhydrazine reacted at 20 °C for 30 min. The chromatographic conditions were optimized to reduce matrix effects, ion suppression, and efficient analysis time (<7-minute analytical run). The four aldehyde species were accurately quantified in brain tissue using stable-labeled internal standards. Application of this assay to a traumatic brain injury mouse model revealed significant accumulation of acrolein, 4-hydroxy-2-hexenal, and 4-hydroxy-2-nonenal at 28 days post injury. Overall, a validated method was developed to rapidly quantify the most prominent reactive aldehydes associated with lipid peroxidation during injury progression following acute brain trauma.

Bioactive Peptide Profiling in Collagen Hydrolysates: Comparative Analysis Using Targeted and Untargeted Liquid Chromatography-Tandem Mass Spectrometry Quantification.

The investigation of collagen hydrolysates (CHs) is essential due to their widespread use in health, cosmetic, and therapeutic industries, attributing to the presence of bioactive dipeptides (DPs) and tripeptides (TPs). This study developed a novel targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with propyl chloroformate (PCF) derivatization to measure three bioactive peptides-Hydroxyprolyl-glycine (Hyp-Gly), Glycyl-prolyl-hydroxyproline (Gly-Pro-Hyp), and Prolyl-hydroxyproline (Pro-Hyp)-in CHs, with strong correlation coefficients (0.992, 1.000, and 0.995, respectively) and low limits of detection (LODs) of 1.40, 0.14, and 1.16 µM, respectively. Untargeted data-dependent acquisition (DDA) analyses measured peptide size distribution, while amino acid analysis assessed nutritional content. The analysis of ten commercial CHs revealed similar amino acid profiles but varied peptide lengths, indicating diverse hydrolysis conditions. Products with higher proportions of smaller peptides showed elevated levels of the targeted bioactive peptides, suggesting that a smaller peptide size may increase bioactivity. These findings can inform the optimization of CH supplements, providing consumers with detailed peptide content for more informed choices. Data are available via ProteomeXchange with the identifier PXD051699.

Gas chromatography mass spectrometer determination of dimethylamine impurity in N,N-dimethylformamide solvent by derivatization of N,N-dimethylbenzamide with benzoyl chloride agent.

This study describes the development of a reliable and linear analytical method for precisely determining dimethylamine impurity in N,N-dimethylformamide solvent utilizing a benzoyl chloride derivatization reagent and a gas chromatography mass spectrometer. Benzoyl chloride was used to derivatize dimethylamine. At normal temperature, benzoyl chloride combined with dimethylamine, producing N,N-dimethylbenzamide. This method separated N,N-dimethylbenzamide using Rtx-5 amine (30 m × 0.32 mm × 1.50 µm) as the stationary phase, helium as the carrier gas, argon as the collision gas, and methanol as the diluent. The column flow rate was 2 mL/min. The retention time of N,N-dimethylbenzamide was determined to be 8.5 min. Precision, linearity, and accuracy were tested using ICH Q2 (R2) and USP<1225> guidelines. The percentage coefficient of variation (CV) for N,N-dimethylbenzamide in the system suitability parameter was 1.1%. The correlation coefficient of N,N-dimethylbenzamide was found to be >0.99. In the method precision parameter, the % CV for N,N-dimethylbenzamide was found to be 1.9%, whereas the % CV for N,N-dimethylbenzamide was 1.2% in intermediate precision. The percentage recovery of N,N-dimethylbenzamide was determined to be between 80% and 98%.

Cyanoacetohydrazide as a Novel Derivatization Agent for the Determination of UHPLC-HRMS Steroids in Urine.

The possibility of cyanoacetohydrazide usage as a novel derivatizing agent is demonstrated in the presented article, and a comparison with hydroxylamine as the most commonly used reagent is provided. Optimal conditions for steroid derivatization with cyanoacetohydrazide are provided. According to the collected data, the maximum yield of derivatives was observed at pH 2.8 within 70 min at 40 °C with 5 ng/mL limit of detection for all investigated analytes. It was shown that cyanoacetohydrazide derivatives produces both syn- and anti-forms as well as hydroxylamine, and their ratios were evaluated and shown in presented work. An efficiency enchantment from two to up to five times was achieved with a novel derivatization reagent. Its applicability for qualitative analysis of steroids in urine was presented at real samples. Additionally, the reproducible fragmentation of the derivatizing agent in collision-induced dissociation offers opportunities for simplified non-targeted steroidomic screening. Furthermore, cyanoacetohydrazide increases ionization efficiency in positive mode, which can eliminate the need for redundant high-resolution instrument runs required for both positive and negative mode analyses.

Chromatographic Methods and Sample Pretreatment Techniques for Aldehydes, Biogenic Amine, and Carboxylic Acids in Food Samples.

This review paper critically examines the current state of research concerning the analysis and derivatization of aldehyde, aromatic hydrocarbons and carboxylic acids components in foods and drinks samples, with a specific focus on the application of Chromatographic techniques. These diverse components, as vital contributors to the sensory attributes of food, necessitate accurate and sensitive analytical methods for their identification and quantification, which is crucial for ensuring food safety and compliance with regulatory standards. In this paper, High-Performance Liquid Chromatography (HPLC) and Gas Chromatographic (GC) methods for the separation, identification, and quantification of aldehydes in complex food matrices were reviewed. In addition, the review explores derivatization strategies employed to enhance the detectability and stability of aldehydes during chromatographic analysis. Derivatization methods, when applied judiciously, improve separation efficiency and increase detection sensitivity, thereby ensuring a more accurate and reliable quantification of aldehyde aromatic hydrocarbons and carboxylic acids species in food samples. Furthermore, methodological aspects encompassing sample preparation, chromatographic separation, and derivatization techniques are discussed. Validation was carried out in term of limit of detections are highlighted as crucial elements in achieving accurate quantification of compounds content. The discussion presented by emphasizing the significance of the combined HPLC and GC chromatography methods, along with derivatization strategies, in advancing the analytical capabilities within the realm of food science.

Using Magnetic Molecularly Imprinted Polymer Technology for Determination of Fish Serum Glucose Levels.

In this study, a highly efficient magnetic molecularly imprinted polymer nanocomposite material was prepared using multi-walled carbon nanotubes as carriers. The characterization of the obtained nanocomposite material was conducted using Fourier transform infrared spectroscopy, a vibrating sample magnetometer, a thermogravimetric analyzer, a scanning electron microscope, and a transmission electron microscope. The adsorption properties of the nanocomposite material were evaluated through adsorption experiments, including static adsorption, dynamic adsorption, and selective recognition studies. The prepared nanocomposite material, serving as a selective adsorbent, was applied in magnetic solid-phase extraction. Subsequently, the derivatized samples were analyzed for glucose in fish serum using liquid chromatography-tandem mass spectrometry. Under optimal conditions, the detection limit was 0.30 ng/mL, the quantitation limit was 0.99 ng/mL, satisfactory spiked recovery rates were obtained, and the relative standard deviation was less than 1.1%. Using 2-deoxy-D-ribose as the template molecule and a structural analog of glucose allowed us to eliminate the potential template leakage in qualitative and quantitative analyses, effectively avoiding the issues of false positives and potential quantitative errors, compared to traditional methods. A method for detecting glucose levels in fish serum based on molecularly imprinted polymer technology has been successfully developed to determine the stress and health levels of fish.

Structural characterization of N-acyl-homoserine lactones from bacterial quorum sensing using LC-MS/MS analyses after Paternò-Büchi derivatization in solution.

Bacterial quorum sensing is a chemical language allowing bacteria to interact through the excretion of molecules called autoinducers, like N-acyl-homoserine lactones (AHLs) produced by Gram-negative Burkholderia and Paraburkholderia bacteria known as opportunistic pathogens. The AHLs differ in their acyl-chain length and may be modified by a 3-oxo or 3-hydroxy substituent, or C = C double bonds at different positions. As the bacterial signal specificity depends on all of these chemical features, their structural characterization is essential to have a better understanding of the population regulation and virulence phenomenon. This study aimed at enabling the localization of the C = C double bond on such specialized metabolites while using significantly lower amounts of biological material. The approach is based on LC-MS/MS analyses of bacterial extracts after in-solution derivatization by a photochemical Paternò-Büchi reaction, leading to the formation of an oxetane ring and subsequently to specific fragmentations when performing MS/MS experiments. The in-solution derivatization of AHLs was optimized on several standards, and then the matrix effect of bacterial extracts on the derivatization was assessed. As a proof of concept, the optimized conditions were applied to a bacterial extract enabling the localization of C = C bonds on unsaturated AHLs.

Nitrogen stable isotope analysis of sulfonamides by derivatization-gas chromatography-isotope ratio mass spectrometry.

The continuous introduction of micropollutants into the environment through livestock farming, agricultural practices, and wastewater treatment is a major concern. Among these pollutants are synthetic sulfonamide antibiotics such as sulfamethoxazole, which are not always fully degraded and pose a risk of fostering antimicrobial resistance. It is challenging to assess the degradation of sulfonamides with conventional concentration measurements. This study introduces compound-specific isotope analysis of nitrogen isotope ratios at natural abundances by derivatization-gas chromatography hyphenated with isotope ratio mass spectrometry (derivatization-GC-IRMS) as a new and more precise method for tracing the origin and degradation of sulfonamides. Here, sulfamethoxazole was used as a model compound to develop and optimize the derivatization conditions using (trimethylsilyl)diazomethane as a derivatization reagent. With the optimized conditions, accurate and reproducible δ15N analysis of sulfamethoxazole by derivatization-GC-IRMS was achieved in two different laboratories with a limit for precise isotope analysis of 3 nmol N on column, corresponding to 0.253 µg non-derivatized SMX. Application of the method to four further sulfonamides, sulfadiazine, sulfadimethoxine, sulfadimidine, and sulfathiazole, shows the versatility of the developed method. Its benefit was demonstrated in a first application, highlighting the possibility of distinguishing sulfamethoxazole from different suppliers and pharmaceutical products.

4-Iodobenzonitrile as a fluorogenic derivatization reagent for chromatographic analysis of L-p-boronophenylalanine in whole blood samples using Suzuki coupling reaction.

BACKGROUND: L-p-Boronophehylalanine (BPA) is used in boron neutron capture therapy (BNCT), which is a novel selective cancer radiotherapy technique. It is important to measure BPA levels in human blood for effective radiotherapy; a prompt gamma-ray spectrometer, ICP-AES, and ICP-MS have been used for this purpose. However, these methods require sophisticated and expensive apparatuses as well as experienced analysts. Herein, we propose an HPLC-FL method for the determination of BPA after precolumn derivatization. A new fluorogenic reagent for aryl boronic acid derivatives, namely, 4-iodobenzonitrile, was employed for the fluorogenic derivatization of BPA based on the Suzuki coupling reaction. RESULTS: After the fluorogenic derivatization, a fluorescent cyanobiphenyl derivative is formed with maximum fluorescence at 335 nm after excitation at 290 nm. The developed method showed good linearity (r2=0.997) over the concentration range of 0.5-1000 nmol/L, and the detection limit (S/N = 3) was 0.26 nmol/L. The proposed method is more sensitive than previously reported methods for the determination of BPA, including the ICP-MS. Finally, the proposed method was successively applied to the measurement of BPA in human whole blood samples with a good recovery rate (≥95.7 %) using only 10 µL of blood sample. The proposed method offers a simple and efficient solution for monitoring BPA levels in BNCT-treated patients. SIGNIFICANCE: 4-Iodobenzonitrile was investigated as a new fluorogenic reagent for BPA based on Suzuki coupling. A new HPLC-FL method for BPA in whole blood samples with ultrasensitivity was developed. The developed method is superior in sensitivity to all previously reported methods for BPA. The method requires only a very small sample volume, making it suitable for micro-blood analysis of BPA via fingerstick sampling.

Utility of Green Chemistry for Sustainable Fluorescence Derivatization Approach for Spectrofluorimetric Quantification of Pemetrexed as Antineoplastic Drug in Pharmaceutical Formulation and Spiked Human Plasma.

Pemetrexed is a chemotherapeutic medicine, under the trade name Alimta. Malignant pleural mesothelioma patients. Its application in lung cancer has been studied. Here in, a second spectrofluorimetric method was advanced for quantifying of Pemetrexed in pharmaceutical formulation and spiked human plasma. That method depends on fluorescence derivatization of Pemetrexed with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) at 75 °C in a (pH 9) of borate buffer to produce a fluorescent derivative which can be detected at 520 nm afterwards excitation at 460 nm. The method has been validated using ICH criteria, and it demonstrated linearity in a range of 2-120 ngmL-1. The proposed method was applied precisely and accurately for quantifying Pemetrexed within pharmaceutical formulation and spiking human plasma without any interferences. Moreover, the method's sustainability was evaluated and compared to the published method using two greenness assessment tools termed analytical eco-scale and Analytical GREENness (AGREE). That findings suggest that the method is more sustainable than the published method.

The Pre- and Post-column Derivatization on Monosaccharide Composition Analysis, a Review.

Polysaccharides, as common metabolic products in organisms, play a crucial role in the growth and development of living organisms. For humans, polysaccharides represent a class of compounds with diverse applications, particularly in the medical field. Therefore, the exploration of the monosaccharide composition and structural characteristics of polysaccharides holds significant importance in understanding their biological functions. This review provides a comprehensive overview of extraction methods and hydrolysis strategies for polysaccharides. It systematically analyzes strategies and technologies for determining polysaccharide composition and discusses common derivatization reagents employed in further polysaccharide studies. Derivatization is considered a fundamental strategy for determining monosaccharides, as it not only enhances the detectability of analytes but also increases detection sensitivity, especially in liquid chromatography (LC), capillary electrophoresis (CE), and gas chromatography (GC) techniques. The critical comparison of pre- and post-column derivatization is elaborated in this paper, aiming to serve as a reference for selecting appropriate modes based on the structural characteristics, biological activities of polysaccharides, and reaction systems.

Determination of 3-nitropropionic acid in sugarcane using dispersive solid-phase extraction and gas chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry.

A method for accurately determining 3-nitropropionic acid in sugarcane was established for the first time using gas chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry (GC - APCI-MS/MS). Under acidic conditions, 3-nitropropionic acid is methylated to obtain methyl 3-nitropropionate. The derivative product was purified using dispersive solid-phase extraction (d-SPE) method and analyzed using GC - APCI-MS/MS. The recovery experiments were conducted at three concentrations: low, medium, and high. The recovery rates ranged from 75.1% to 90.2%, the relative standard deviations were <8.2%, and the limit of quantification was 2.0 µg/kg. The method offers the advantage of being accurate, sensitive, and specific, meeting the requirements of the determination of 3-nitropropionic acid in sugarcane.

Quantification of gut microbiome metabolites using chemical isotope derivatization strategy combined with LC-MS/MS: Application in neonatal hypoxic-ischemic encephalopathy rat model.

The gut microbiome plays pivotal roles in various physiological and pathological processes, with key metabolites including short chain fatty acids (SCFAs), bile acids (BAs), and tryptophan (TRP) derivatives gaining significant attention for their diverse physiological roles. However, quantifying these metabolites presents challenges due to structural similarity, low abundance, and inherent technical limitations in traditional detection methods. In this study, we developed a precise and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method utilizing a chemical isotope derivatization technique employing 4-(aminomethyl)-N,N-dimethylaniline-d0/d6 (4-AND-d0/d6) reagents to quantify 37 typical gut microbiome-derived metabolites. This method achieved an impressive 1500-fold enhancement in sensitivity for detecting metabolites, compared to methods using non-derivatized, intact molecules. Moreover, the quantitative accuracy of our chemical isotope derivatization strategy proved comparable to the stable isotope labeled internal standards (SIL-IS) method. Subsequently, we successfully applied this newly developed method to quantify target metabolites in plasma, brain, and fecal samples obtained from a neonatal hypoxic-ischemic encephalopathy (HIE) rat model. The aim was to identify crucial metabolites associated with the progression of HIE. Overall, our sensitive and reliable quantification method holds promise in elucidating the role of gut microbiome metabolites in the pathogenesis of various diseases.

A new chemical derivatization reagent sulfonyl piperazinyl for the quantification of fatty acids using LC-MS/MS.

In our previous study, a chemical derivatization reagent named 5-(dimethylamino) naphthalene-1-sulfonyl piperazine (Dns-PP) was developed to enhance the chromatographic retention and the mass spectrometric response of free fatty acids (FFAs) in reversed-phase liquid chromatography coupled with electrospray ionization-mass spectrometry (RPLC-ESI-MS). However, Dns-PP exhibited strong preferences for long-chain FFAs, with limited improvement for short- or medium-chain FFAs. In this study, a new series of labeling reagents targeting FFAs were designed, synthesized, and evaluated. Among these reagents, Tmt-PP (N2, N2, N4, N4-tetramethyl-6-(4-(piperazin-1-ylsulfonyl) phenyl)-1,3,5-triazine-2,4-diamine) exhibited the best MS response and was selected for further evaluations. We compared Tmt-PP with Dns-PP and four commonly used carboxyl labeling reagents from existing studies, demonstrating the advantages of Tmt-PP. Further comparisons between Tmt-PP and Dns-PP in measuring FFAs from biological samples revealed that Tmt-PP labeling enhanced the MS response for about 80 % (30/38) of the measured FFAs, particularly for short- and medium-chain FFAs. Moreover, Tmt-PP labeling significantly improved the chromatographic retention of short-chain FFAs. To ensure accurate quantification, we developed a stable isotope-labeled Tmt-PP (i.e., d12-Tmt-PP) to react with chemical standards and serve as one-to-one internal standards (IS). The method was validated for accuracy, precision, sensitivity, linearity, stability, extraction efficiency, as well as matrix effect. Overall, this study introduced a new chemical derivatization reagent Tmt-PP (d12-Tmt-PP), providing a sensitive and accurate option for quantifying FFAs in biological samples.

Evaluation of polypropylene microporous membranes as extraction devices for determination of carcinogenic aromatic amines in smoker urine by GC-MS/MS.

Exposure to tobacco smoke is highly correlated to the incidence of different types of cancer due to various carcinogenic compounds present in such smoke. Aromatic amines, such as 1-naphthylamine (1-NA) and 2-naphthylamine (2-NA), are produced in tobacco burning and are linked to bladder cancer. Miniaturized solid phase extraction techniques, such as microporous membrane solid phase extraction (MMSPE), have shown potential for the extraction of aromatic compounds. In this study, a bioanalytical method for the determination of 1-NA and 2-NA in human urine was developed using polypropylene microporous membranes as a sorptive phase for MMSPE. Urine samples were hydrolyzed with HCl for 1 h at 80 °C, after which pH was adjusted to 10. Ultrasound-assisted MMSPE procedure was optimized by factorial design as follows. To each sample, 750 µL of methanol was added, and ultrasound-assisted MMSPE was conducted for 1 h with four devices containing seven 2 mm polypropylene membrane segments. After extraction, the segments were transferred to 400 µL of hexane, and desorption was conducted for 30 min. Extracts were submitted to a simple and fast microwave-assisted derivatization procedure, by the addition of 10 µL of PFPA and heating at 480 W for 3 min, followed by clean-up with phosphate buffer pH 8.0 and GC-MS/MS analysis. Adequate linearity was obtained for both analytes in a range from 25 to 500 µg L-1, while the multiple reaction monitoring approach provided satisfactory selectivity and specificity. Intra-day (n = 6) and inter-day (n = 5) precision and accuracy were satisfactory, below 15 % and between 85 and 115 %, respectively. Recovery rates found were 91.9 and 58.4 % for 1-NA and 2-NA, respectively, with adequate precision. 1-NA was found in first-hand smokers' urine samples in a concentration range from 20.98 to 89.09 µg in 24 h, while it could be detected in second-hand smoker's urine samples, and 2-NA detected in all first and second-hand smokers' urine samples. The proposed method expands the applicability of low cost MMSPE devices to aromatic amines and biological fluids.