Abstract: Metabolomics is a powerful and essential technology for profiling metabolic phenotypes and exploring metabolic reprogramming, which enables the identification of biomarkers and provides mechanistic insights into physiology and disease. However, its applications are still limited by the technical challenges particularly in its detection sensitivity for the analysis of biological samples with limited amount, necessitating the development of highly sensitive approaches. Here, we developed a h ighly s ensitive liquid chromatography tandem mass spectrometry method based on a 3-nitrophenylhydrazine (3-NPH) d erivatization strategy that simultaneously targets c arbonyl, c arboxyl, and p hosphoryl groups for t argeted m etabolomic analysis (HSDccp-TM) in biological samples. By testing 130 endogenous metabolites including organic acids, amino acids, carbohydrates, nucleotides, carnitines, and vitamins, we showed that the derivatization strategy resulted in significantly improved detection sensitivity and chromatographic separation capability. Metabolic profiling of merely 60 oocytes and 5000 hematopoietic stem cells primarily isolated from mice demonstrated that this method enabled routine metabolomic analysis in trace amounts of biospecimens. Moreover, the derivatization strategy bypassed the tediousness of inferring the MS fragmentation patterns and simplified the complexity of monitoring ion pairs of metabolites, which greatly facilitated the metabolic flux analysis (MFA) for glycolysis, the tricarboxylic acid (TCA) cycle, and pentose phosphate pathway (PPP) in cell cultures. In summary, the novel 3-NPH derivatization-based method with high sensitivity, good chromatographic separation, and broad coverage showed great potential in promoting metabolomics and MFA, especially in trace amounts of biospecimens.

Abstract: Molecule-specific techniques such as MALDI and desorption electrospray ionization mass spectrometry imaging enable direct and simultaneous mapping of biomolecules in tissue sections in a single experiment However, neurotransmitter imaging in the complex environment of biological samples remains challenging Our covalent charge-tagging approach using on-tissue chemical derivatization of primary and secondary amines and phenolic hydroxyls enables comprehensive mapping of neurotransmitter networks Here, we present robust and easy-to-use chemical derivatization protocols that facilitate quantitative and simultaneous molecular imaging of complete neurotransmitter systems and drugs in diverse biological tissue sections with high lateral resolution This is currently not possible with any other imaging technique The protocol, using fluoromethylpyridinium and pyrylium reagents, describes all steps from tissue preparation (~1 h), chemical derivatization (1–2 h), data collection (timing depends on the number of samples and lateral resolution) and data analysis and interpretation The specificity of the chemical reaction can also help users identify unknown chemical identities Our protocol can reveal the cellular locations in which signaling molecules act and thus shed light on the complex responses that occur after the administration of drugs or during the course of a disease This protocol describes strategies for in situ chemical derivatization and simultaneous quantitative imaging of multiple neurotransmitters and their precursors and metabolites in brain tissue sections using MALDI and desorption electrospray ionization mass spectrometry imaging

Abstract: Lipids, serving as the structural components of cellular membranes, energy storage, and signaling molecules, play the essential and multiple roles in biological functions of mammals. Mass spectrometry (MS) is widely accepted as the first choice for lipid analysis, offering good performance in sensitivity, accuracy, and structural characterization. However, the untargeted qualitative profiling and absolute quantitation of lipids are still challenged by great structural diversity and high structural similarity. In recent decade, chemical derivatization mainly targeting carboxyl group and carbon-carbon double bond of lipids have been developed for lipidomic analysis with diverse advantages: (i) offering more characteristic structural information; (ii) improving the analytical performance, including chromatographic separation and MS sensitivity; (iii) providing one-to-one chemical isotope labeling internal standards based on the isotope derivatization regent in quantitative analysis. Moreover, the chemical derivatization strategy has shown great potential in combination with ion mobility mass spectrometry and ambient mass spectrometry. Herein, we summarized the current states and advances in chemical derivatization-assisted MS techniques for lipidomic analysis, and their strengths and challenges are also given. In summary, the chemical derivatization-based lipidomic approach has become a promising and reliable technique for the analysis of lipidome in complex biological samples.

Abstract: Matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) is an emerging label-free method for mapping the distribution of diverse molecular species in tissue sections. Despite recent progress in MALDI-MSI analyses of lipids, it is still difficult to visualize minor bioactive lipids including lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P). Here, we have developed a novel on-tissue derivatization method using Phos-tag, a zinc complex that specifically binds to a phosphate monoester group. MALDI-MSI with Phos-tag derivatization made it possible to image LPA and S1P in the murine brain. Furthermore, we were able to visualize other low-abundance lipids containing phosphate monoester, such as phosphatidic acid and ceramide-1-phosphate. Compared with conventional MALDI-MS, this derivatization produced LPA images with high spatial accuracy discriminating LPA artificially produced during MALDI-MS analysis. In mice with deficiencies in enzymes that degrade LPA and S1P, we observed marked S1P and/or LPA accumulation in specific regions of the brain. Thus, the present study provides a simple and optimal way to reveal the spatial localization of potent bioactive lipid phosphates such as LPA and S1P in tissues.

Abstract: The far-ranging occurrence of acrylamide (AA), a Group 2A carcinogen, in extensively consumed processed foodstuffs has aroused worldwide public health concern, and heightened the need for rapid and reliable detection technologies. Current immunoassays for AA are mainly based on antibodies against mercaptobenzoic acid (MBA) derivatized AA, and thus require a relatively complicated pre-analysis sample derivatization using MBA (heating > 50 °C, time>1 h), which limits the analysis efficiency. In this work, two haptens against xanthyl acrylamide (XAA) were synthesized, and a specific and high-affinity polyclonal antibody against XAA (anti-XAA pAb) was obtained. By virtue of this antibody, a fluorescence immunoassay for quantification of acrylamide with a simple sample derivatization by 9-xanthydrol (ambient temperature, 30 min) was developed via introducing the fluorescent signal of carbon dots (CDs) through the facile inner-filter eff ;ect (IFE) strategy. This fluorescence immunoassay displayed a sensitive and specific response to AA with a detection limit of 0.16 μg/L, which was lower than the legal limit in drinking water (0.5 μg/L) set by WHO. Recovery test and method validation by HPLC-MS/MS demonstrated its good accuracy and reliability, suggesting that this fluorescence immunoassay can be a potentially effective tool for the supervision of AA contamination in foods.

Abstract: Derivatization, or chemical structure modification, is often used in bioanalysis performed by liquid chromatography technique in order to enhance detectability or to improve the chromatographic performance for the target analytes. The derivatization process is discussed according to the analytical procedure used to achieve the reaction between the reagent and the target compounds (containing hydroxyl, thiol, amino, carbonyl and carboxyl as the main functional groups involved in derivatization). Important procedures for derivatization used in bioanalysis are in situ or based on extraction processes (liquid-liquid, solid-phase and related techniques) applied to the biomatrix. In the review, chiral, isotope-labeling, hydrophobicity-tailored and post-column derivatizations are also included, based on representative publications in the literature during the last two decades. Examples of derivatization reagents and brief reaction conditions are included, together with some bioanalytical applications and performances (chromatographic conditions, detection limit, stability and sample biomatrix).

Abstract: The improvement of on-tissue chemical derivatization for mass spectrometry imaging (MSI) of low-abundance and/or poorly ionizable functional molecules in biological tissue without delocalization is challenging. Here, we developed a novel hydrogel-assisted chemical derivatization (HCD) approach coupled with airflow-assisted desorption electrospray ionization (AFADESI)-MSI, allowing for enhanced visualization of inaccessible molecules in biological tissues. The derivatization reagent Girard's P (GP) reagent was creatively packaged into a hydrogel to form HCD blocks that have reactivity to carbonyl compounds as well as the feasibility of "cover/uncover" contact mode with tissue sections. The HCD blocks provided a favorable liquid microenvironment for the derivatization reaction and reduced matrix effects from derivatization reagents and tissue without obvious molecular migration, thus improving the derivatization efficiency. With this methodology, unusual carbonyl metabolites, including 166 fatty aldehydes (FALs) and 100 oxo fatty acids (FAs), were detected and visualized in rat brain, kidney, and liver tissue. This study provides a new approach to enhance chemical labeling for in situ tissue submetabolome profiling and improves our knowledge of the molecular histology and complex metabolism of biological tissues.

Abstract: Ambient mass spectrometry (AMS) allows direct desorption and ionization of analytes in real time with minimal-to-no sample preparation. However, it may present inadequate capabilities for differentiating isomers. Here, a reactive flowing atmospheric-pressure afterglow (reactive-FAPA) AMS source is developed for rapid isomer differentiation by derivatization of analytes in real time. The effects of the reactive-FAPA operating conditions on the reagent and product ions were studied and optimized for highly volatile and non-volatile model compounds with different carbonyl functional groups. In addition, two functional isomers of valproic acid (VPA) metabolites, 4-ene VPA and γ-valprolactone, are successfully differentiated for the first time by incorporating methylamine (MA) reagent vapor into the plasma effluent used for desorption/ionization. Reactive-FAPAMS for 4-ene VPA shows only detectable peaks of the protonated acylation product [M + MA-H2O + H]+, while for γ-valprolactone, it shows detectable peaks for both protonated acylation product [M + MA-H2O + H]+ and protonated intermediate [M + MA + H]+. A method for quantitative characterization of mixtures of 4-ene VPA and γ-valprolactone is also developed and validated. In addition, reactive-FAPAMS also shows better detection sensitivity compared to nonreactive-FAPAMS for some larger analyte types, such as UV filters and steroids. The limit of detection (LOD) of pregnenolone acetate in reactive-FAPAMS is 310 ng/mL, which is about 10 times better than its LOD in nonreactive-FAPA.

Abstract: The preparation of bioactive polymeric molecules requires the attention of scientists as it has a potential function in biomedical applications. In the current study, functional substitution of alginate with a benzoyl group was prepared via coupling its hydroxyl group with benzoyl chloride. Fourier transform infrared spectroscopy indicated the characteristic peaks of aromatic C=C in alginate derivative at 1431 cm−1. HNMR analysis demonstrated the aromatic protons at 7.5 ppm assigned to benzoyl groups attached to alginate hydroxyl groups. Wetting analysis showed a decrease in hydrophilicity in the new alginate derivative. Differential scanning calorimetry and thermal gravimetric analysis showed that the designed aromatic alginate derivative demonstrated higher thermo-stability than alginates. The aromatic alginate derivative displayed high anti-inflammatory properties compared to alginate. Finally, the in vitro antioxidant evaluation of the aromatic alginate derivative showed a significant increase in free radical scavenging activity compared to neat alginate against DPPH (2,2-diphenyll-picrylhydrazyl) and ABTS free radicals. The obtained results proposed that the new alginate derivative could be employed for gene and drug delivery applications.

Abstract: Stereospecific recognition of amino acids (AAs) plays a crucial role in chiral biomarker-based diagnosis and prognosis. Separation of AA enantiomers is a long and tedious task due to the requirement of AA derivatization prior to the chromatographic or electrophoretic steps which are also time-consuming. Here, a mass-tagged chiral selector named [d0]/[d5]-estradiol-3-benzoate-17β-chloroformate ([d0]/[d5]-17β-EBC) with high reactivity and good enantiomeric resolution in regard to AAs was developed. After a quick and easy chemical derivatization step of AAs using 17β-EBC as the single chiral selector before ion mobility-mass spectrometry analysis, good enantiomer separation was achieved for 19 chiral proteinogenic AAs in a single analytical run (∼2 s). A linear calibration curve of enantiomeric excess was also established using [d0]/[d5]-17β-EBC. It was demonstrated to be capable of determining enantiomeric ratios down to 0.5% in the nanomolar range. 17β-EBC was successfully applied to investigate the absolute configuration of AAs among peptide drugs and detect trace levels of d-AAs in complex biological samples. These results indicated that [d0]/[d5]-17β-EBC may contribute to entail a valuable step forward in peptide drug quality control and discovering chiral disease biomarkers.

Abstract: During drug development, detailed investigations of the pharmacokinetic profile of the drug are required to characterize its absorption, distribution, metabolism, and excretion properties. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is an established technique for studies of the distribution of drugs and their metabolites. It has advantages over autoradiography, which is conventionally used for distribution studies: it does not require the radiolabeling of drugs and can distinguish between the drug and its metabolites directly in the tissue. However, its lack of sensitivity in certain cases remains challenging. Novel procedures, such as on-tissue chemical derivatization (OTCD), could be developed to increase sensitivity. We used OTCD to enhance the sensitivity of MALDI-MSI for one of the most widely used drugs, acetaminophen, and to study its distribution in tissues. Without derivatization, this drug and some of its metabolites are undetectable by MALDI-MSI in the tissues of treated rats. We used 2-fluoro-1-methylpyridinium p-toluene sulfonate as a derivatization reagent, to increase the ionization yield of acetaminophen and some of its metabolites. The OTCD protocol made it possible to study the distribution of acetaminophen and its metabolites in whole-body sections at a spatial resolution of 400 μm and in complex anatomical structures, such as the testis and epididymis, at a spatial resolution <50 μm. The OTCD is also shown to be compatible with the quantification of acetaminophen by MALDI-MSI in whole-body tissues. This protocol could be applied to other molecules bearing phenol groups and presenting a low ionization efficiency.