norHarmane containing ionic liquid matrices for low molecular weight MALDI‐MS carbohydrate analysis: The perfect couple with α‐cyano‐4‐hydroxycinnamic acid

Cinnamic acid derivatives, particularly α‐cyano‐4‐hydroxycinnamic acid (E‐α‐cyano‐4‐hydroxycinnamic acid or (E)‐2‐cyano‐3‐(4‐hydroxyphenyl)prop‐2‐enoate; CHCA), have been extensively used especially for protein and peptide analysis. Together with the introduction of ionic liquid MALDI matrix (ILM) started the study of applications of IL prepared with CHCA and a counter organic base (ie, aliphatic amines) in which CHCA moiety is the chromophore responsible of UV‐laser absorption. Despite the extensive studies of norharmane (9H‐pyrido[3,4‐b]indole; nHo) applications as matrix and its peculiar basic properties in the ground and electronic excited state, nHo containing ILM was never tested in MALDI‐MS experiments. This pyrido‐indole compound was introduced as MALDI matrix 22 years ago for different applications including low molecular weight (LMW) carbohydrates (neutral, acidic, and basic carbohydrates). These facts encouraged us to use it as a base, for the first time, for ILM preparation. As a rational design of new IL MALDI matrices, E‐α‐cyanocinnamic acid.nHo and E‐cinnamic acid.nHo were prepared and their properties as matrices studied. Their performance was compared with that of (a) the corresponding IL prepared with butylamine as basic component, (b) the corresponding crystalline E‐α‐cyanocinnamic and E‐cinnamic acid, and (c) the classical crystalline matrices (2,5‐dihydroxybenzoic acid, DHB; nHo) used in the analysis of neutral/sulfated carbohydrates. The IL DHB.nHo was tested, too. Herein, we demonstrate the outstanding performance for the IL CHCA.nHo for LMW carbohydrate in positive and negative ion mode (linear and reflectron modes). Sulfated oligosaccharides were detected in negative ion mode, and although the dissociation of sulfate groups was not completely suppressed the relative intensity (RI) of [M ? Na]^? peak was quite high. Additionally, to better understand the quite different performance of each IL tested as matrix, the physical and morphological properties in solid state were studied (optical image; MS image).     

2,5‐Dihydroxybenzoic acid solution in MALDI‐MS: ageing and use for mass calibration

2,5‐Dihydroxybenzoic acid (DHB) is one of the most widely used and studied matrix compounds in matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry. However, the influence of ageing of the DHB solution on the MALDI mass spectra has not been yet systematically studied. In this work, the possible changes occurring in the acidified acetonitrile/water solution of the MALDI matrix compound DHB during 1‐year usage period have been monitored with MALDI‐Fourier transform ion cyclotron resonance mass spectrometer (MALDI‐FT‐ICR‐MS) and attenuated total reflectance Fourier transform infrared (ATR‐FT‐IR) spectroscopy. No significant ageing products have been detected. The ability of the aged DHB solution to act as a MALDI matrix was tested with two materials widely used in art and conservation – bone glue (a proteinaceous material) and shellac resin (a resinous material) – and good results were obtained. A number of peaks in the mass spectra measured from the DHB solution were identified, which can be used for internal calibration of the mass axis. Copyright © 2014 John Wiley & Sons, Ltd.     

A convenient purification and preconcentration of peptides with α‐cyano‐4‐hydroxycinnamic acid matrix crystals in a pipette tip for matrix‐assisted laser desorption/ionization mass spectrometry

Peptide samples derived from enzymatic in‐gel digestion of proteins resolved by gel electrophoresis often contain high amount of salts originating from reaction and separation buffers. Different methods are used for desalting prior to matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry (MS), e.g. reversed‐phase pipette tip purification, on‐target washing, adding co‐matrices, etc. As a suitable matrix for MALDI MS of peptides, α‐cyano‐4‐hydroxycinnamic acid (CHCA) is frequently used. Crystalline CHCA shows the ability to bind peptides on its surface and because it is almost insoluble in acidic water solutions, the on‐target washing of peptide samples can significantly improve MALDI MS signals. Although the common on‐target washing represents a simple, cheap and fast procedure, only a small portion of the available peptide solution is efficiently used for the subsequent MS analysis. The present approach is a combination of the on‐target washing principle carried out in a narrow‐end pipette tip (e.g. GELoader tip) and preconcentration of peptides from acidified solution by passing it through small CHCA crystals captured inside the tip on a glass microfiber frit. The results of MALDI MS analysis using CHCA‐tip peptide preconcentration are comparable with the use of homemade POROS R2 pipette tip microcolumns. Advantages and limitations of this approach are discussed. Copyright © 2009 John Wiley & Sons, Ltd.     

Negative ion MALDI‐TOF MS,ISD and PSD of neutral underivatized oligosaccharides without anionic dopant strategies,using 2,5‐DHAP as a matrix

Oligosaccharides represent complex class of analytes for mass spectrometric analysis due to the high variety of structural isomers concerning glycosidic linkages and possible branching. A systematic study of the negative ion mode matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry of various neutral oligosaccharides under selection of an appropriate matrix, like 2,5‐dihydroxyacetophenone (2,5‐DHAP) is reported here, without commonly used anion dopant strategies. Nevertheless, we were able to generate relevant in‐source decay (ISD) cross‐ring fragment ions, typically obtained in the negative ion mode. Data observed indicate that the intrinsic property of the terminal non‐reduced aldose is crucial for this behavior. A systematic study of the post source decay (PSD) of molecular, pseudomolecular and ISD cross‐ring cleavage precursor ions is reported here. A direct comparison of the positive and negative ion mode MALDI MS1 and PSD behavior of neutral oligosaccharides could also be performed under the use of the same matrix preparation, because 2,5‐DHAP is fully compatible with positive ion mode acquisition. We found that PSD spectra of deprotonated neutral oligosaccharides obtained in the negative ion mode are richer, because they contained both glycosidic and cross‐ring fragment ions. However, we also found that cross‐ring fragment ions are readily produced in the positive ion mode when potassiated precursor ions were selected. In addition, we show evidence that non‐anionic dopants and specific instrumental parameters can also significantly influence the ISD fragmentation. Taken together, our results should increase our understanding of oligosaccharide behavior in the negative ion mode as well as increase our knowledge regarding many aspects of in‐source MALDI chemistry. Copyright © 2016 John Wiley & Sons, Ltd.     

A pitfall of using 2‐[(2E)‐3‐(4‐tert‐butylphenyl)‐2‐methylprop‐2‐enylidene]malononitrile as a matrix in MALDI TOF MS: chemical adduction of matrix to analyte amino groups

2‐[(2E)‐3‐(4‐tert‐Butylphenyl)‐2‐methylprop‐2‐enylidene]malononitrile (DCTB) has been considered as an excellent matrix for matrix‐assisted laser desorption/ionization (MALDI) of many types of synthetic compounds. However, it might provide troublesome results for compounds containing aliphatic primary or secondary amino groups. For these compounds, strong extra ion peaks with a mass difference of 184.1 Da were usually observed, which might falsely indicate the presence of some unknown impurities that were not detected by other matrices. On the basis of the possible mechanisms proposed, these extra ions are the products of nucleophilic reactions between analyte amino groups and DCTB molecules or radical cations. In these reactions, an amino group replaces the dicyanomethylene group of DCTB forming a matrix adduct via a ? C?N‐bond. An aliphatic primary amine could react easily with DCTB and the reaction could start once they are mixed in a MALDI solution. For an aliphatic secondary amine, on the other hand, the reaction most likely occurs in the gas phase. Protonation of amino groups by adding acid seems to be a useful way to stop DCTB adduction for compounds with one single amino group, but not for compounds with multiple amino groups. Unlike aliphatic primary or secondary amines, aliphatic tertiary amines and aromatic amines do not yield DCTB adducts. This is because tertiary amines do not have the required transferrable H‐(N) atom to form an extra ? C?N‐bond, while aromatic amines are not sufficiently nucleophilic to attack DCTB. In view of the possible matrix adduction, care should be taken in MALDI time‐of‐flight mass spectrometry (TOF MS) when DCTB is used as the matrix for compounds containing amino group(s). Copyright © 2010 John Wiley & Sons, Ltd.     

Development,validation and comparison of surrogate matrix and surrogate analyte approaches with UHPLC–MS/MS to simultaneously quantify dopamine,serotonin and γ‐aminobutyric acid in four rat brain regions

As biomarkers, endogenous neurotransmitters play critical roles in the process of neuropsychiatric diseases, and neurotransmitter levels in different brain regions can contribute to neurological disease diagnosis and treatment. Due to the lack of a blank matrix for endogenous neurotransmitters, surrogate‐matrix and surrogate‐analyte approaches have been used for the determination of neurotransmitters to solve this problem. In this study, we capitalised on the high accuracy, precision, and throughput of UHPLC‐MS/MS and developed new methods based on the two approaches. Both approaches satisfied FDA and EMA validation criterias after an appropriate parallelism assessment, and they were used to further quantify the three endogenous neurotransmitters, including dopamine (DA), serotonin (5‐HT) and γ‐aminobutyric acid (GABA) in rat brain four regions (cortex, striatum, hypothalamus and hippocampus) which represent the catecholamines, indolamines, and amino acids, respectively. Comparison of the results in the same rats (n = 10) showed there was no significant difference in DA, 5‐HT, or GABA levels between the two approaches (P > 0.05). The concentrations of DA and GABA were highest in striatum and hypothalamus, respectively, and the levels of 5‐HT were paralleled in striatum and hippocampus almost 2‐fold higher than other regions. This is the first study to compare these two approaches in the determination of endogenous neurotransmitter content in the rat brain, and the surrogate‐matrix approach proved to be simple, rapid, and reliable, considering cost, matrix similarity, and practicality.     

Determination of (4‐(1,3‐dioxo‐1,3‐dihydro‐2H‐isoindol‐ 2‐yl)‐N′‐[(4‐ethoxyphenyl) methylidene] benzohydrazide,a novel anti‐inflammatory agent,in biological fluids by UPLC‐MS/MS: Assay development,validation and in vitro metabolic stability study

A thalidomide analog, (4‐(1,3‐dioxo‐1,3‐dihydro‐2H‐isoindol‐2‐yl)‐N ′‐[(4‐ethoxyphenyl) methylidene] benzohydrazide), has been identified as a promising broad‐spectrum anti‐inflammatory agent in previous study. In this study, a sensitive and selective UPLC‐MS/MS assay was developed and validated for its determination in rat plasma samples. The chromatographic separation was performed on an Aquity BEH C₁₈ column using mobile phase comprising of acetonitrile and 10 mm ammonium acetate in the ratio of 85: 15, at flow rate of 0.3 mL/min. The detection and quantification were performed in positive multiple reaction monitoring mode by parent to daughter ion transition of 414.06 ˃ 148.05 for analyte and 411.18 ˃ 191.07 for internal standard (risperidone), respectively using electrospray ionization source. The sample extraction process consisted of liquid–liquid extraction method using diethyl ether as the extracting solvent. The assay was validated by following FDA guidelines and all parameters were found to be within acceptable limits. The linearity was between 10.1 and 2500 ng/mL and the lower limit of quantification was 10.1 ng/mL. The reported results indicate that the assay could meet the requirement for analysis of this compound in amounts expected to the present in actual samples. Further, in vitro metabolic stability study was performed in rat liver microsomes by using the validated assay.