Assisted Reductive Amination for Quantitation of Tryptophan, 5-Hydroxytryptophan, and Serotonin by Ultraperformance Liquid Chromatography Coupled with Tandem Mass Spectrometry.

Herein, we used isotopic formaldehyde and sodium cyanoborohydride via reductive amination to label two methyl groups on primary amine to arrange the standards (h2-formaldehyde-modified) and internal standards (ISs, d2-formaldehyde-modified) of tryptophan and its metabolites, such as serotonin (5-hydroxytryptamine) and 5-hydroxytryptophan. These derivatized reactions with a high yield are very satisfactory for manufacturing standards and ISs. This strategy will generate one or two methyl groups on amine to create different mass unit shifts with 14 vs. 16 or 28 vs. 32 in individual compounds for biomolecules with amine groups. In other words, multiples of two mass units shift are created using this derivatized method with isotopic formaldehyde. Serotonin, 5-hydroxytryptophan, and tryptophan were used as examples to demonstrate isotopic formaldehyde-generating standards and ISs. h2-formaldehyde-modified serotonin, 5-hydroxytryptophan, and tryptophan are standards to construct calibration curves, and d2-formaldehyde-modified analogs such as ISs spike into samples to normalize the signal of each detection. We utilized multiple reaction monitoring modes and triple quadrupole mass spectrometry to demonstrate the derivatized method suitable for these three nervous biomolecules. The derivatized method demonstrated a linearity range of the coefficient of determinations between 0.9938 to 0.9969. The limits of detection and quantification ranged from 1.39 to 15.36 ng/mL.

A standard addition method to quantify histamine by reductive amination and hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry.

Histamine is an organic nitrogenous compound that acts as a neurotransmitter in the uterus, spinal cord, and brain and is involved in local immune responses. In this study, we developed a fast and simple derivatization method based on reductive amination that can be used to quantify histamine by hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry. Histamine isotope analogs were synthesized via reductive amination. Histamine was modified with H2-formaldehyde to form N-dimethylated histamine to act as a standard or with D2-formaldehyde to form N-dimethylated histamine-d4 to act as an internal standard. Using this method, we achieved a limit of detection of 3.6 ng/mL, a limit of quantification of 7.9 ng/mL, and a linear calibration curve with a coefficient of determination (R2) of 0.9987. Furthermore, the intra-day relative standard deviations ranged from 0.9% to 3.7% and the inter-day relative standard deviations ranged from 2.0% to 17.6%. After derivatization, N-dimethylated histamine showed 382.5% signal enhancement compared to unmodified histamine in mass spectrometry detection. To demonstrate the applicability of this method for biological samples, we utilized standard addition method to quantify histamine in fetal bovine serum and achieved a recovery of 86.7%.

Reductive amination assistance for quantification of oseltamivir phosphate and oseltamivir carboxylate by HPLC-MS/MS.

Oseltamivir phosphate (OP) is the first line therapy for influenza, and its primary metabolite oseltamivir carboxylate (OC) is the active agent via inhibition of neuraminidase of influenza virus. Dosages of OP and OC might affect human causing nausea and vomiting and it is therefore necessary to evaluate their toxicity and safety. The separation system: liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a powerful technique to monitor OP and OC. However, quantification of OP and OC needs isotopic analogs as internal standards to monitor the stability of the sample pretreatment procedures and instruments. In this study, we demonstrated a modified method (i.e., reductive amination) to synthesize OP and OC deuterated and hydrogenated analogs as internal standards (ISs) and for illustration of calibration curves, respectively. This modification allowed to overcome ISs selection and to enhance the signal intensities via high yield reductive amination in MS detection. We utilized the multiple reaction monitoring (MRM) mode to target m/z values of precursor and product ions. N-dimethylated OP and N-dimethylated OC showed linearity ranging from 1 to 1000 ng/mL with coefficient of determination (R2) values of 0.9995 and 0.9999, respectively. Additionally, the relative standard deviations (RSD) of intra-day ranged from 0.3% to 5.2%, and the RSD of inter-day ranged from 2.0% to 18.8%, respectively. This quantitative method utilized spiked OP and OC at low (20 ng/mL), intermediate (100 ng/mL), and high (500 ng/mL) concentrations in human serum samples. The average recoveries for OP and OC were 84.6%-107.7% and 94.9%-98.5%, respectively.

Reductive amination derivatization for the quantification of garlic components by isotope dilution analysis.

In this work, we synthesized internal standards for four garlic organosulfur compounds (OSCs) by reductive amination with 13C, D2-formaldehyde, and developed an isotope dilution analysis method to quantitate these organosulfur components in garlic samples. Internal standards were synthesized for internal absolute quantification of S-allylcysteine (SAC), S-allylcysteine sulfoxide (alliin), S-methylcysteine (SMC), and S-ethylcysteine (SEC). We used a multiple reaction monitoring (MRM) to detect 13C, D2-formaldehyde-modified OSCs by ultrahigh-performance liquid phase chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) and obtained MS spectra showing different ratios of 13C, D2-formaldehyde-modified and H2-formaldehyde-modified compounds. The resulting labeled and unlabeled OSCs were exhibited correlation coefficient (R2) ranged from 0.9989 to 0.9994, respectively. The average recoveries for four OSCs at three concentration levels ranged from 89% to 105%. By 13C, D2-formaldehyde and sodium cyanoborohydride, the reductive amination-based method can be utilized to generate novel internal standard for isotope dilution and to extend the quantitative application.

A novel reductive amination method with isotopic formaldehydes for the preparation of internal standard and standards for determining organosulfur compounds in garlic.

Garlic (Allium sativum) is a long-cultivated plant that is widely utilized in cooking and has been employed as a medicine for over 4000 years. In this study, we fabricated standards and internal standards (ISs) for absolute quantification via reductive amination with isotopic formaldehydes. Garlic has four abundant organosulfur compounds (OSCs): S-allylcysteine, S-allylcysteinine sulfoxide, S-methylcysteine, and S-ethylcysteine are abundant in garlic. OSCs with primary amine groups were reacted with isotopic formaldehydes to synthesize ISs and standards. Cooked and uncooked garlic samples were compared, and we utilized tandem mass spectrometry equipped with a selective reaction monitoring technique to absolutely quantify the four organosulfur compounds.

A Derivative Method with Free Radical Oxidation to Predict Resveratrol Metabolites by Tandem Mass Spectrometry.

In this study, we demonstrated an oxidative method with free radical to generate 3,5,4'-trihydroxy-trans-stilbene (trans-resveratrol) metabolites and detect sequentially by an autosampler coupling with liquid chromatography electrospray ionization tandem mass spectrometer (LC-ESI-MS/MS). In this oxidative method, the free radical initiator, ammonium persulfate (APS), was placed in a sample bottle containing resveratrol to produce oxidative derivatives, and the reaction progress was tracked by autosampler sequencing. Resveratrol, a natural product with purported cancer preventative qualities, produces metabolites including dihydroresveratrol, 3,4'-dihydroxy-trans-stilbene, lunularin, resveratrol monosulfate, and dihydroresveratrol monosulfate by free radical oxidation. Using APS free radical, the concentrations of resveratrol derivatives differ as a function of time. Besides simple, convenient and time- and labor saving, the advantages of free radical oxidative method of its in situ generation of oxidative derivatives followed by LC-ESI-MS/MS can be utilized to evaluate different metabolites in various conditions.

A proteomics analysis to evaluate cytotoxicity in NRK-52E cells caused by unmodified Nano-Fe3O4.

We synthesized unmodified Fe3O4 nanoparticles (NPs) with particles size from 10 nm to 100 nm. We cultured NRK-52E cell lines (rat, kidney) and treated with Fe3O4 NPs to investigate and evaluate the cytotoxicity of NPs for NRK-52E cells. Through global proteomics analysis using dimethyl labeling techniques and liquid phase chromatography coupled with a tandem mass spectrometer (LC-MS/MS), we characterized 435 proteins including the programmed cell death related proteins, ras-related proteins, glutathione related proteins, and the chaperone proteins such as heat shock proteins, serpin H1, protein disulfide-isomerase A4, endoplasmin, and endoplasmic reticulum resident proteins. From the statistical data of identified proteins, we believed that NPs treatment causes cell death and promotes expression of ras-related proteins. In order to avoid apoptosis, NRK-52E cell lines induce a series of protective effects such as glutathione related proteins to reduce reactive oxygen species (ROS), and chaperone proteins to recycle damaged proteins. We suggested that, in the indigenous cellular environment, Fe3O4 NPs treatment induced an antagonistic effect for cell lines to go to which avoids apoptosis.

Comparative proteomics analysis of degenerative eye lenses of nocturnal rice eel and catfish as compared to diurnal zebrafish.

PURPOSE: The aim of this study was to determine the lens crystallin diversity of degenerative eyes from the rice eel (Monopterus albus) and walking catfish (Clarias batrachus) as compared to that of zebrafish (Danio rerio) by using comparative proteomics methodologies. We endeavored to investigate the evolution of vertebrate lenses particularly concerning the functional loss of lenses in degenerative eyes of rice eels and catfishes living under an environment of perpetual darkness. METHODS: Fish lenses were collected and homogenized to extract total soluble proteins. The protein mixtures were separated by one- and two-dimensional gel electrophoresis (1D or 2D gel), plus the newer gel-free shotgun proteomic strategy, followed by in-gel digestion and subjection of the digested protein bands or spots to liquid chromatography coupled with tandem mass spectrometry. The proteomics data were analyzed and compared based on the proteomics databank of zebrafish. The soluble lens protein solutions of three piscine species were also processed by gel-filtration chromatography and 1D sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the comparison and validation of various crystallin families, e.g., α-, ß-, and γ-crystallins. RESULTS: In zebrafish eye lenses, γ-crystallin constituted about 71% and α- and ß-crystallins comprised 30% of total lens proteins. In rice eel lenses, very little or almost no α-crystallins were detected and ß- and γ-crystallins comprised more than 98% of total lens proteins. In catfish lenses, α- and ß-crystallins comprised about 40% and γ-crystallin constitutes 60% of total lens proteins. It was of interest to find that α-crystallin was totally absent in the rice eel in contrast to the presence, albeit with very low amounts, of α-crystallin in similarly nocturnal catfish. The ratio of α-crystallin subunits (αA/αB) was found to be about 20:1 for the catfish lens, in great contrast to the ratio of about 3:1 found for most mammalian lenses. In contrast, ß- and γ-crystallins were more abundant in lenses of these three piscine species, similar to mammalian lenses. By proteomics analysis, the most abundant ß-crystallins were found to comprise a diverse group of ßA1a, ßA1-2, ßA2a, ßA2-2, ßA4, ßB1, ßB2, and ßB3 subunit crystallins; the monomeric γ-crystallin class contains γB, γD, γM2, γM3, γM5, γM7, γN-A, γN-B, γS1, and γS2 crystallins. CONCLUSIONS: In cave or nocturnal animals, the eye is sometimes reduced or eliminated because of adaptation to life in visual darkness. The comparative proteomics analysis of degenerative and normal lenses forms a firm molecular basis to investigate further the evolution of piscine lenses in the future. The total numbers of α-, ß-, and γ-crystallins in the three fish species as revealed by the current proteomics methodology clearly indicate the complexity and diversity of crystallin species present in the piscine class of vertebrates. The unexpected finding that α-crystallin is absent in the degenerative eye lenses of rice eel may have some bearing on the chaperone function of α-crystallin in regard to its protective role of preventing protein aggregation in diurnal vertebrate lenses to maintain functional transparency.