Unsaturated
lipids containing single or more carbon–carbon
double bonds (CC) within tissues are closely associated with
various types of diseases. Mass spectrometry imaging (MSI) has been
used to study the spatial distribution of lipid CC location
isomers in tissue sections. However, comprehensive characterization
of lipid CC location isomers using MSI remains challenging.
Herein, we established an on-tissue charge-switching Paternò–Büchi
(PB) derivatization method using 3-acetylpyridine (3-AP) as a reaction
reagent, which can be used to detect and assign CC location
of glycerophospholipids (GPLs) as well as neutral lipids, such as
fatty acids (FAs), under the same experimental workflow using matrix-assisted
laser desorption/ionization (MALDI)-MSI. High coverage of mono- and
poly-unsaturated CC location isomers among various lipid classes
including FA, phosphatidylcholine (PC), and sulfatide (SHexCer) in
distinct regions of the mouse brain and kidney was visualized using
MALDI-MS/MS imaging. This method has also been applied to map the
spatial distribution of lipid CC location isomers in the Alzheimer’s
disease (AD) mice model for the first time, which provides a new tool
to study the relationships between the distribution of lipid structural
diversity and neurodegenerative diseases.
N-Acyl glycines (NAGlys) are an important class of metabolites in the detoxification system of the human body. They have been used in the diagnosis of several metabolic diseases. Liquid chromatography−mass spectrometry (LC−MS) is the most frequently used NAGlys detection platform. Here, we describe a simple and sensitive method of NAGlys detection by LC−MS in plasma and urine samples. This approach is based on the use of a derivatization reagent, 3-nitrophenylhydrazine. The reaction is quick in aqueous solution, and no quenching step is needed. To expand the coverage of NAGlys when standards are not available, NAGlys were first identified based on high-resolution LC−MS. Quantification was subsequently carried out on triple quadrupole LC−MS. This approach allowed a much broader measurement of NAGlys (41 NAGlys in total), especially when authentic standards are unavailable. Comprehensive analysis of NAGlys with this new method was applied in plasma and urine samples of db/db diabetic and non-diabetic db/m+ control mice. The majority of detected NAGlys were altered with high differentiation ability in plasma and urine samples from diabetic and non-diabetic mice. These identified NAGlys hold the potential to be diagnostic biomarkers for type II diabetes and diabetic complications.
Rationale
Allyl isothiocyanate (AITC) in food products such as wasabi is commonly analyzed by gas chromatography/mass spectrometry (GC/MS), while liquid chromatography/mass spectrometry (LC/MS) is more suitable for the polar metabolites. The development of an effective method for the simultaneous determination of AITC and its phase II metabolites is needed to support drug metabolism and pharmacokinetics (DMPK) studies.
Methods
Derivatization of AITC by reaction with N‐(tert‐butoxycarbonyl)‐L‐cysteine methyl ester (tBocCysME) was used for ultra‐high‐performance liquid chromatography/electrospray ionization mass spectrometry (UHPLC/ESI‐MS) analysis, allowing the simultaneous determination with its phase II metabolites. At room temperature, reaction conditions have been optimized to maximize the reaction of AITC with tBocCysME quantitively. Reaction selectivity was examined by the presence of AITC metabolites. Quantification of AITC was performed by multiple reaction monitoring (MRM) with an external calibration method and applied on the serum of C57BL/6 J mouse for a DMPK study.
Results
The limit of quantification (LOQ) was determined to be 0.842 nM from the derivatization method and by UHPLC/MS/MS analysis. The method accuracy in the mouse serum samples was 75 ± 2% with a relative standard deviation (RSD) of 3.0% of method variation. The UHPLC/MS/MS analysis gave RSDs of 0.2% and 1.8% for intra‐day and inter‐day variations. With the newly developed method, AITC could be detected in mouse serum upon oral administration for the first time.
Conclusions
An analytical method involving a rapid derivatization pre‐treatment and quantitative UHPLC/ESI‐QqQ‐MS/MS analysis has been developed for a biological sample assay of AITC, enabling selective and sensitive detection of the AITC derivative and AITC metabolites simultaneously. The method developed could be applied to the DMPK study of AITC as well.
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