Methods for detection and quantification of gelatin from different sources.

Gelatin is a water-soluble protein obtained from the collagen of various animal origins (porcine, bovine, fish, donkey, horse, and deer hide) and has diverse applications in the food, pharmaceutical, and cosmetics industries. Porcine and bovine gelatins are extensively used in food and non-food products; however, their acceptance is limited due to religious prohibitions, whereas fish gelatin is accepted in all religions. In Southeast Asia, especially in China, gelatin obtained from donkey and deer skins is used in medicines. However, both sources suffer from adulteration (mixing different sources of gelatin) due to their limited availability and high cost. Unclear labeling and limited information about actual gelatin sources in gelatin-containing products cause serious concern among societies for halal and fraud authentication of gelatin sources. Therefore, authenticating gelatin sources in gelatin-based products is challenging due to close similarities between the composition differences and degradation of DNA and protein biomarkers in processed gelatin. Thus, different methods have been proposed to identify and quantify different gelatin sources in pharmaceutical and food products. To the best of our knowledge, this systematic and comprehensive review highlights different authentication techniques and their limitations in gelatin detection and quantification in various commercial products. This review also describes halal authentication and adulteration prevention strategies of various gelatin sources, mainly focussing on research gaps, challenges, and future directions in this research area.

Smartphone-Assisted EY@MOF-5-Based Dual-Emission Fluorescent Sensor for Rapid On-Site Detection of Daclatasvir and Nitenpyram.

Fluorescent metal-organic frameworks (MOFs) are promising sensing materials with tunable and robust structural properties and remarkable luminescent capabilities. In this study, a novel dual-emission fluorescent metal-organic framework (EY@MOF-5) composite is synthesized by a one-pot bottle-around-ship approach. Eosin Y (EY) is encapsulated in MOF-5 to enhance its fluorescence properties and selectivity, effectively addressing typical MOF-5 limitations. EY@MOF-5 serves as a versatile dual-functional fluorescent sensor for two different analytes, daclatasvir (DCT) and nitenpyram (NTP), showing an impressive linear range of 10-200 nM and 0.1-300 µM, with detection limits of 233 pM and 65 nM, respectively. The established method is ultrafast, highly sensitive, and extremely selective for DCT and NTP detection in complex biological and food samples. Fluorescence results are compared and validated with the recommended UPLC method. Then, a smartphone-integrated sensing system is introduced for on-site, real-time, and quantitative analysis of DCT and NTP. The smartphone-assisted intelligent sensing method manifests promising results for DCT and NTP monitoring in biological and food samples, demonstrating its promising potential for the on-site detection of biologically and environmentally significant analytes.

Determination of vitamin D metabolites in various biological samples through an improved chemical derivatization assisted liquid chromatography-tandem mass spectrometry approach.

Vitamin D (VD) metabolites are involved in a variety of important metabolic processes and physiological effects in organisms. Profiling of VD metabolites favors a deep understanding of the physiological role of VD. However, VD metabolites are difficult to detect due to their high chemical structural rigidity, structural similarity, and low sensitivities under liquid chromatography-tandem mass spectrometry (LC-MS). Herein, we present a chemical derivatization assisted LC-MS/MS strategy for the detection of VDs, in which 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) is employed to derivatize the conjugated diene of VD metabolites and provides sensitizing reporters for MS detection. After PTAD derivatization, the sensitivities of seven VD metabolites increased by 24-276 folds, with the limits of detection ranging from 3 to 20 pg mL-1. Using this method, we achieved a sensitive and accurate quantification of 7 VD metabolites (vitamin D2, vitamin D3, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D2, 1,25-dihydroxyvitamin D3, and 1,24,25-trihydroxyvitamin D3) of the VD metabolic pathway in different trace biological samples, including human serum, mouse tissues (namely liver, kidney, lung, and spleen), and cells. We believe that the present method can provide a promising tool for an in-depth analysis of VD metabolism.

Enhancement in photocatalytic selectivity of TiO2-based nano-catalyst through molecular imprinting technology.

Improvement in the photocatalytic selectivity is imperative for the effective and efficient utilization of catalysts. In this study, a molecularly imprinted polymer-coated iron-doped titanium dioxide (Fe-TiO2@MIP) nanocomposite was successfully synthesized by precipitation polymerization while using RB-19 as a template. The synthesized nanocomposites (Fe-TiO2@MIP and Fe-TiO2@NIP) were characterized by Fourier transform infrared spectroscopy (FT-IR), atomic force microscopy (AFM), field emission scanning electron microscopy (FESEM) with energy dispersive X-ray (EDX), X-ray diffraction (XRD), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), Brunauer-EMMETT-Teller (BET), and UV-visible spectrophotometry. The optimized binding experiments revealed a high imprinting factor of 5.0 for RB-19. The catalytic degradation efficiency and selectivity of Fe-TiO2@MIP enhanced to almost complete degradation of RB-19 from 70% for the parent Fe-TiO2 and 76% for Fe-TiO2@NIP. An outstanding degradation selectivity of RB-19 was achieved compared to other competitive dyes. Finally, the analysis of the non-degraded and degraded RB-19 by ESI-MS revealed the presence of different intermediates that fits well with the proposed degradation mechanism. The study opens new possibilities of selective photo-degradation of targeted contaminants that may ultimately lead to efficient use of photocatalysts.

Insights into the antibacterial activity and antibacterial mechanism of silver modified fullerene towards Staphylococcus aureus by multiple spectrometric examinations.

Clarifying the antibacterial mechanism of silver (Ag)-based materials is of great significance for the rational design, synthesis, and evaluation of antimicrobials. Herein, detailed description of the antibacterial mechanism of a synthesized silver deposited fullerene material (Ag(I)-C60) towards Staphylococcus aureus was surveyed from the point of view of DNA damage by ultraviolet-visible spectroscopy (UV-vis), inductively coupled plasma mass spectrometry (ICP-MS), and liquid chromatography-mass spectrometry (LC-MS). The model material, Ag(I)-C60, was prepared by liquid-liquid interfacial precipitation method, and characterized by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD), thermos-gravimetric analysis (TGA), and nitrogen adsorption/desorption analysis. Ultra-efficient bacteriostatic rate of Ag(I)-C60 was found to be 88.98% under light irradiation for 20 min. UV-vis measurement of the composition changes of four DNA bases showed that they changed in the presence of Ag(I)-C60 under light irradiation, suggesting Ag(I)-C60 could destroy the cells and genetic material of Staphylococcus aureus and thereby inhibit its growth and reproduction. ICP-MS analysis demonstrated the releasing behavior of Ag+ from Ag-based materials. Finally, the transformation pathway of G, A, C, and T were measured by LC-MS, demonstrating the conversion of Adenine (m/z 136.06) to 8-OH-Ade (m/z 174.04). These collective results suggested that Ag(I)-C60 was a new ultra-efficient antibacterial by slowly releasing Ag+ in water and producing a large amount of ROS under light.

Volume-Corrected Free Energy as a New Criterion for Structural Elucidation in Chemical-Tagging-Based Metabolomics.

Chemical tagging via possible derivatization reagents alters metabolites' retention times, leading to different retention behavior during liquid chromatography-mass spectrometry (LC-MS) analysis. Incorporation of the retention time dimension can dramatically reduce false-positive structural elucidation in chemical-tagging-based metabolomics. However, few studies predict the retention times of chemically labeled metabolites, especially requiring a simple, easy-to-access, accurate, and universal predictor or descriptor. This pilot study demonstrates the application of volume-corrected free energy (VFE) calculation and region mapping as a new criterion to describe the retention time for structure elucidation in chemical-tagging-based metabolomics. The universality of VFE calculation is first evaluated with four different types of submetabolomes including hydroxyl-group-, carbonyl-group-, carboxylic-group-, and amino-group-containing compounds and oxylipins with similar chemical structures and complex isomers on reverse-phase LC. Results indicate a good correlation (r > 0.85) between VFE values and their corresponding retention times using different technicians, instruments, and chromatographic columns, describing retention behavior in reverse-phase LC. Finally, the VFE region mapping is described for identifying 1-pentadecanol from aged camellia seed oil using three proposed steps, including public database searching, VFE region mapping for its 12 isomers, and chemical standard matching. The possibility of VFE calculation of nonderivatized compounds in retention time prediction is also investigated, demonstrating its effectiveness on retention times with different influence factors.

Fluoro-cotton assisted non-targeted screening of organic fluorine compounds from rice (Oryza sativa L.) grown in perfluoroalkyl substance polluted soil.

The toxicity and environmental persistence of perfluorooctanoic acid (PFOA), and perfluorooctane sulfonate (PFOS) are of great concern for food intake in humans. However, PFASs conversion or conjugation to other substances in rice grown on PFASs polluted soil has not been explored clearly. These unknown transformed or conjugated products of PFOA and PFOS could be harmful to human health. The restriction factor in evaluating the possible transformation of PFOA and PFOS is mainly attributed to the lack of an efficient method for screening PFOA and PFOS and their related metabolites. To circumvent this challenge, we established a non-targeted screening method by combining a fluoro-cotton fiber-based solid phase extraction (FC-SPE) and liquid chromatography-high resolution mass spectrometry (LC-HRMS) to monitor the formation of possible organic fluorine compounds from rice (Oryza sativa L.) grown on PFASs. We synthesized fluoro-cotton fibers to serve as the FC-SPE packing material and characterized by field-emission scanning electron-microscope, Fourier transform infrared, and X-ray photoelectron spectroscopy measurements. The optimal extraction conditions for the prepared FC-SPE were investigated. The performance of FC-SPE in LC-MS analysis was validated by linearity, precision, recovery, and matrix effect. Then the FC-SPE combined with LC-HRMS was used to specifically capture organic fluorine compounds from complex matrices via F-F interaction, including rice seedlings grown in PFOA and PFOS polluted soil and soil samples. By the established FC-SPE LC-HRMS method, in total 429 features were found as the possible organic fluorine compounds from rice seedlings grown in PFOA polluted soil among the 1781 features from the rice seedlings. Finally, we employed a13C metabolic tracing analysis of organic fluorine compounds in combination with the FC-SPE LC-HRMS method to further identify the features that detected from rice seedlings grown in PFOA polluted soil. The final result indicated that there were not any new organic fluorine metabolites screened out from rice grown in PFOA or PFOS polluted soil.

Determination of estrogens in human serum using a novel chemical derivatization-assisted liquid chromatography-electrospray ionization-tandem mass spectrometry method.

RATIONALE: Assessing estrogen concentrations in biological systems can provide valuable information on physiological processes, which is crucial for the early diagnosis of many diseases. Because estrogens are present in the human body in low concentrations and in a wide dynamic range, analytical methods with high sensitivity and specificity are required for their determination in complex biological matrices. METHODS: To discover an appropriate derivatization reagent for estrogen mass spectrometry (MS) analysis, we compared five sulfonyl chloride derivatization reagents, namely 3-methyl-8-quinolinesulfonyl chloride (MQSCl) and 8-quinolinesulfonyl chloride (QSCl), 1-methyl-1H-pyrazole-4-sulfonyl chloride, 1,2-methyl-imidazole-5-sulfonyl chloride, and dansyl chloride. By selecting the derivatization reagent with the best performance, we developed and validated a novel chemical derivatization-assisted-liquid chromatography-electrospray ionization-tandem mass spectrometry (CD-LC-ESI-MS/MS) method to simultaneously determine the concentrations of estrone, estradiol, and estriol (E1, E2, and E3) in human serum. RESULTS: It was found that among the five investigated reagents, MQSCl-derivatized estrogens presented the highest sensitivity using LC-ESI-MS/MS. Based on this discovery, MQSCl was chosen to derivatize the analyzed estrogens to assist LC-ESI-MS/MS analysis. The limit of quantification of E1, E2, and E3 was measured as 2.7, 4.6, and 5.1 pg/mL, respectively. Inter- and intra-day precision, expressed as the coefficient of variation, was shown to be lower than 13.2% for all concentrations. The mean recovery was 72.4% overall, with good reproducibility at low, medium, and high concentrations in the calibration range. CONCLUSIONS: The developed method was successfully applied to the quantitative determination of estrogens in clinical human serum from pediatric and adult women, demonstrating the suitability of estrogen analysis in the biological matrix at low concentration (pg/mL).

Quantitative analysis of the relationship of derivatization reagents and detection sensitivity of electrospray ionization-triple quadrupole tandem mass spectrometry: Hydrazines as prototypes.

Chemical derivatization-assisted electrospray ionization-triple quadrupole mass spectrometry (ESI-QqQ-MS) has become an efficient tool for the quantification of low-molecular-weight molecules. Many studies found that the derivatives of the same analytes derivatized by different derivatization reagents with the same reaction group had different detection sensitivity, even under the same conditions of electrospray ionization-mass spectrometry (ESI-MS). This phenomenon was suggested to be caused by the different modifying groups in the derivatization reagents. However, there is still a lack of systematic study on how modifying groups in the derivatization reagents affect the detection sensitivity of their corresponding derivatives of analytes, especially theoretical investigations. In this study, we employed a quantitative structure-activity relationship (QSAR) modeling approach to explore the relationship between modifying group structures and the detection sensitivity of derivatization reagents and their derivatives during ESI-MS detection. A total of 110 derivatization reagents of the hydrazine family and their hexanal derivatives (substituted hydrazones) were selected as the prototypes to construct QSAR models. The established models suggested that several molecular descriptors, related to hydrophobicity, electronegativity, and molecular shape, were related to the detection sensitivity of hexanal derivatives induced by different modifying groups in the derivatization reagents. Besides, we found that the detection sensitivity of compounds detected in selected ion mode (SIM) showed a positive correlation with that obtained in multiple reaction monitoring mode (MRM), and the ionization efficiency was the key factor on the detection sensitivity in both modes.

Simultaneous determination of short-chain fatty alcohols in aged oil and biodiesels by stable isotope labeling assisted liquid chromatography-mass spectrometry.

Short-chain fatty alcohols (SCFAs) are one of the reactants for the production of biodiesels. The SCFA residues at the trace level have a significant impact on biodiesel quality. However, the analysis of SCFAs in aged biodiesels has not been reported so far, which is probably due to the unavailability of an appropriate analytical method for the simultaneous determination of SCFAs. Herein, we developed a novel analytical approach with high sensitivity and selectivity for the simultaneous identification and determination of SCFAs in seed oil and biodiesel samples during the simulated real-time aging by stable isotope labeling assisted liquid chromatography-mass spectrometry (SIL-LC-MS). A pair of isotope labeling reagents, pyridine (Py) and [2H5]pyridine ([2H5]Py), were used to label SCFAs in biodiesels and standards, respectively. The [2H5]Py labeled SCFAs were used as internal standards to compensate for the detection of variances. The simultaneous determination of SCFAs was performed by LC-MS with an improved detection selectivity and sensitivity. The limits of detection (LODs) values were ranged from 0.2 to 0.5 ng mL-1 for the investigated SCFAs. Good linearity was observed in the studied ranges (R2 > 0.99) and good precision with relative standard deviations (RSDs) was in the range of 4.9-18.1%. Average recoveries were obtained in the range of 80.3%-115.4%. The matrix effects were in the range of 70.0-104.3%. The validated SIL-LC-MS method was applied to the simultaneous quantitative analysis of SCFAs in seed oil and biodiesel samples and the LC-MS analysis could be done within 3 min. The formation mechanism of SCFAs in aged oil and biodiesel samples was also investigated by this method. The results suggest that SCFAs were formed and their composition changed during the simulated real-time aging of long-chain fatty acid (LCFA), long-chain fatty acid methyl ester (FAME), seed oil, and biodiesels. Moreover, we found that the formation of 1-pentanol and 1-hexanol was associated with the number and position of double bonds in LCFAs and FAMEs.

A novel charge derivatization-direct infusion mass spectrometry method for the quantitative analysis of C1-C8 fatty acids in rubber seed oil-based biodiesel.

The free fatty acids that contain one to eight carbons (C1-C8) in biodiesel would affect the quality of biodiesel. It is still a matter of challenge to simultaneously determine the composition of C1-C8 fatty acids in seed oil and seed oil-based biodiesel. Herein, a novel method of charge derivatization coupling with direct infusion mass spectrometry (CD-DIMS) was developed for the determination of the C1-C8 fatty acids in biodiesels. A fixed-charge derivatization reagent, 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide (CMCT), was used to convert fatty acids into their cationic derivatives, which significantly improved the sensitivity and selectivity of detection. Good linearity was observed with the limits of detection (LODs) in the range of 0.0002-0.001 µg mL-1 for the investigated fatty acids. The recovery was in the range of 85.1%-101.9% and the matrix effect was within the range of 75.5-93.2%. The developed method was carried out to analyze C1-C8 fatty acids in rubber seed oil (RSO) and RSO-based biodiesels produced by different catalysts, including NaOH, TiO2, and carbodiimide. It was also applied to the dynamic monitoring of C1-C8 fatty acids in RSO and produced RSO biodiesels during the oxidation process. As results, formic acid, acetic acid, and propionic acid were detected in aged RSO and biodiesel samples. The contents of formic acid, acetic acid, and propionic acid all increased in aged RSO and biodiesels, but with different growth rates. These results demonstrated that the developed CD-DIMS method can provide a quick, accurate, and sensitive analysis of C1-C8 fatty acids in seed oil and biodiesel samples.

Hydrogen-Deuterium Scrambling Based on Chemical Isotope Labeling Coupled with LC-MS: Application to Amine Metabolite Identification in Untargeted Metabolomics.

Identification of metabolites at the trace level in complex samples is still one of the major challenges in untargeted metabolomics. One formula in the metabolomic database is always corresponding to more than one candidate, which increases the difficulty and cost in the subsequent process of standard compound matching. In this study, we developed an effective method for amine metabolite identification by hydrogen-deuterium scrambling (HDS) based on chemical isotope labeling coupled with liquid chromatography-mass spectrometry (HDS-CIL-LC-MS). After d4-4-(N,N-dimethylamino)phenyl isothiocyanate (d4-DMAP) labeling, the labeled amine metabolites can produce HDS under collision-induced dissociation (CID). The HDS can effectively reflect the number of labile hydrogen atoms in amine metabolites and thus distinguish amine isomers with different functional groups. The developed HDS-CIL-LC-MS method was applied to the determination of amine metabolites in mice feces, in which the amine candidates obtained by the database based on chemical formula searching were reduced by 64% on average, which greatly reduces the cost of standard compound matching. Taken together, the developed HDS-CIL-LC-MS analysis was demonstrated to be a promising method for untargeted metabolomics and a novel strategy for deciphering tandem mass spectrometry (MS2) spectra.

Sensitive analysis of multiple low-molecular-weight thiols in a single human cervical cancer cell by chemical derivatization-liquid chromatography-mass spectrometry.

Low-molecular-weight (LMW) thiols are important small molecules that regulate or maintain redox homeostasis in physiological and pathological processes. Assessing the concentrations of LMW thiols in biological systems may provide valuable information regarding physiological processes and the early diagnosis of some diseases. Here, we developed a method to simultaneously determine the concentrations of multiple LWM thiols in single cells by chemical derivatization assisted liquid chromatography-mass spectrometry (LC-MS). In this method, we synthesized a pair of stable isotope reagents, N-(acridin-9-yl)-2-bromoacetamide (AYBA) and N-(1,2,3,4-[2H4]-acridin-9-yl)-2-bromoacetamide ([2H4]AYBA). AYBA was used to derivatize LWM thiols in human cervical cancer (HeLa) cells, while [2H4]AYBA was used to derivatize standard LWM thiols to prepare internal standards for the LC-MS method development. The proposed AYBA derivatization greatly enhanced the detection sensitivity of LWM thiols by LC-MS, and thereby achieved the simultaneous detection of multiple LWM thiols by LC-MS in ∼1000 HeLa cells. Finally, the developed method was successfully utilized for the quantitative analysis of multiple LWM thiols in a single HeLa cell and the content changes of LWM thiols in a single HeLa cell before and after oxidative stress treatment. Accordingly, six LMW thiols were detected, including cysteamine, cysteine, glutathione, homocysteine, hydrogen sulfide, and pantetheine.

Simultaneous quantitative analysis of multiple sphingoid bases by stable isotope labeling assisted liquid chromatography-mass spectrometry.

Sphingoid bases (SBs) are one of important components of cell membranes, playing important roles in cellular biology. Meanwhile, SBs are associated with various metabolic diseases such as Type 2 Diabetes mellitus (T2DM). Therefore, simultaneous quantitation of multiple SBs in biological samples could provide crucial information for uncovering underlying mechanisms of SBs related functions and diseases. However, existing methods are difficult to achieve simultaneous quantitation for multiple SBs due to the lack of isotope internal standards (ISs) of corresponding SBs. In the current study, we developed a highly sensitive method for the simultaneous detection of 26 SBs in biological samples by stable isotope labeling coupled with ultra-high performance liquid chromatography tandem mass spectrometry (SIL-UHPLC-MS/MS) analysis. In this respect, a pair of isotope labeling reagents, 3-(N, N-dimethylamino)propyl isothiocyanate (DMPI) and d4-3-(N, N-dimethylamino)propyl isothiocyanate (d4-DMPI), were synthesized and utilized to label SBs in biological samples and SB standards, respectively. The d4-DMPI labeled SB standards were used as ISs to calibrate quantitation deviation in MS analysis from the biological matrix. Using the developed method, we successfully quantitated 19 SBs in cells, 20 SBs in mice feces and 18 SBs in human serum samples. Three C17-SBs used as ISs in many reported works were even found in all prepared samples. In summary, the developed SIL-UHPLC-MS/MS analysis was demonstrated to be a promising method for the simultaneous determination of multiple SBs, which could facilitate the investigation of cellular function of SBs and pathogenesis of related diseases.

A boronic acid modified binary matrix consisting of boron nitride and α-cyano-4-hydroxycinnamic acid for determination of cis-diols by MALDI-TOF MS.

A MALDI-TOF mass spectrometric method is described for the determination of small molecule compounds with cis-diol. It is based on the use of a binary matrix consisting of boron nitride (BN) and α-cyano-4-hydroxycinnamic acid that was modified with the derivatization reagent of (3-(acridin-9-ylamino)phenyl)boronic acid which can recognize cis-diols. The binary matrix is used for desorption/ionization (DI) in the positive ion mode. The mechanism leading to DI enhancement was investigated. The results imply that BN is beneficial for the DI because it induces an enhancement in the positive ion mode. The boronic acid-functionalized binary matrix was successfully applied to capture the glucose, shikimic acid and quinic acid. The method was applied to the determination of 3-chloro-1,2-propanediol in plant oil. Graphical abstract Schematic representation of a method for detecting the cis-diol compounds on matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) using the binary matrix of boron nitride (BN)/α-cyano-4-hydroxycinnamic acid (CHCA) that was modified with (3-(acridin-9-ylamino)phenyl) boronic acid (AYPBA).

Profiling of benzimidazoles and related metabolites in pig serum based on SiO2@NiO solid-phase extraction combined precursor ion scan with high resolution orbitrap mass spectrometry.

Benzimidazoles (BZDs) are widely used veterinary drugs in the domestic food-producing animals, resulting in the metabolism of BZDs and the harmful metabolites residues in some foods. However, some BZD metabolites are unknown and so it is important to discover new metabolites to expand detection targets. For these reasons, a sensitive and selective strategy was designed to identify BZD metabolites in the serum of pigs after oral administration of albendazole, fenbendazole and thiabendazole, respectively. Nickel oxide nanoparticle-deposited silica (SiO2@NiO) composite was used for the enrichment and purification of BZD compounds. High-performance liquid chromatography coupled with precursor ion scan-mass spectrometry (LC-PIS-MS) and high resolution MS/MS analysis (HR-MS/MS) was employed for the BZDs metabolic profiles characterization and metabolites detection. Finally, 18 BZD metabolites were identified, among which 11 metabolites were discovered in pig serum for the first time. Besides, more comprehensive BZD metabolic pathways were presented.

Hydralazine derivative of aldehyde: A new type of [M - H]+ ion formed in electrospray ionization mass spectrometry.

Hydralazine has been widely employed in the development of drugs, derivatization reagents, and ligands. In the present work, we reported a new type of dehydrogenated ion [M - H]+ that was produced from the hydralazine derivative of hexanal in electrospray ionization mass spectrometry (ESI-MS). The formation of [M - H]+ ions in the ESI-MS was found to be independent on the mobile phase composition of the liquid chromatography and ESI source parameters. A series of hydralazine derivatives of aldehyde were investigated to confirm this phenomenon. The results showed that hydralazine derivatives of aldehydes that contained an sp3 hybridization carbon with a hydrogen at the α-position of aldehydes could form the unexpected [M - H]+ ions, whereas hydralazine derivative of acetone could only generate [M + H]+ ion in the ESI-MS. We proposed the possible formation mechanism of [M - H]+ ion for the hydralazine derivatives of aldehydes: the [M - H]+ ion was possibly formed by the loss a hydrogen molecule (H2 ) from the protonated ion [M + H]+ . The results obtained from density functional theory (DFT) calculations supported this proposed formation mechanism of [M - H]+ ion.

Sensitive analysis of trehalose-6-phosphate and related sugar phosphates in plant tissues by chemical derivatization combined with hydrophilic interaction liquid chromatography-tandem mass spectrometry.

Trehalose-6-phosphate (T6P) is an important signaling metabolite that is involved in many physiological processes. However, the mechanism of the biological functions of T6P is not fully understood. Quantification of T6P in plants will be beneficial to elucidate the mechanism. However, it is still a challenge to chromatographically separate and sensitively detect T6P and related sugar phosphates. In the current study, we developed a method for effective separation and sensitive detection of glucose-1-phosphate (G1P), glucose-6-phosphate (G6P), sucrose-6-phosphate (S6P) and T6P in plant tissues by chemical derivatization combined with hydrophilic interaction liquid chromatography-tandem mass spectrometry (ChD-HILIC-MS/MS). With this method, two pairs of isomers (G1P/G6P and S6P/T6P) could be well separated on a HILIC column and sensitively detected by MS with limits of detection (LODs) ranging from 0.1 to 0.6 ng mL-1. The developed method was successfully applied to the detection of endogenous G1P, G6P, S6P and T6P in small amounts of plant tissues, such as 1 mg fresh weight of Oryza sativa shoot.

Spatio-temporal profiling of abscisic acid, indoleacetic acid and jasmonic acid in single rice seed during seed germination.

Abscisic acid (ABA), indoleacetic acid (IAA) and jasmonic acid (JA) are plant hormones that were reported to play indispensable roles during seed germination. However, the interactions between these plant hormones during rice seed germination have still not been explored clearly. A sensitive method for determination of these plant hormones would be beneficial for the exploration of such interactions. Herein, we present a liquid chromatography coupled with mass spectrometry (LC-MS) method for the quantification of ABA, IAA and JA in a single tissue of rice seed to investigate the spatio-temporal distribution of these plant hormones during rice seed germination. To this end, an in silico strategy was developed in order to select a derivatization reagent with an ideal sensitivity of MS detection. This strategy was confirmed with experimental studies on three reagents N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC), N,N-dimethylethylenediamine (DMED), and N-(acridin-9-yl)-2-bromoacetamide (AYBA) and their formic acid derivatives. Our results from the in silico and LC-MS experiments show that AYBA is a good derivatization reagent for ABA, IAA and JA due to its reasonable ionization efficiency in electrospray ionization mass spectrometry (ESI-MS) and excellent hydrophobicity. Finally, a sensitive LC-MS method upon AYBA was established for the determination of ABA, IAA and JA in germinated seeds. Good linearities for ABA, IAA, and JA were obtained with correlation coefficients greater than 0.99. The limits of detection (LODs) were in the range of 0.14-0.16 pg mL-1. The method exhibits good precisions with RSD 1.5%-13.8% (intra-day) and 1.2%-7.3% (inter-day) and acceptable recoveries (88.6%-102.9%, n = 6). Finally, the method was successfully employed in the spatio-temporal profiling of ABA, IAA and JA in a single tissue of rice seed during rice seed germination.

Comprehensive Screening and Identification of Fatty Acid Esters of Hydroxy Fatty Acids in Plant Tissues by Chemical Isotope Labeling-Assisted Liquid Chromatography-Mass Spectrometry.

Fatty acid esters of hydroxy fatty acids (FAHFAs) are a new class of lipid mediators with promising anti-diabetic and anti-inflammatory properties. Comprehensive screening and identification of FAHFAs in biological samples would be beneficial to the discovery of new FAHFAs and enable greater understanding of their biological functions. Here, we report the comprehensive screening of FAHFAs in rice and  Arabidopsis thaliana by chemical isotope labeling-assisted liquid chromatography-mass spectrometry (CIL-LC-MS). Multiple reaction monitoring (MRM) was used for screening of FAHFAs. With the proposed method, we detected 49 potential FAHFA families, including 262 regioisomers, in tissues of rice and Arabidopsis thaliana, which greatly extends our knowledge of known FAHFAs. In addition, we proposed a strategy to identify FAHFA regioisomers based on their retention on a reversed-phase LC column. Using the proposed identification strategy, we identified 71 regioisomers from 11 FAHFA families based on commercial standards and characteristic chromatographic retention behaviors. The screening technique could allow for the discovery of new FAHFAs in biological samples. The new FAHFAs identified in this work will contribute to the in-depth study of the functions of FAHFAs.