An optimized method for neurotransmitters and their metabolites analysis in mouse hypothalamus by high performance liquid chromatography–Q Exactive hybrid quadrupole-orbitrap high-resolution accurate mass spectrometry

Highlights

• Established a method to detect 10 NTs and their metabolites in mouse hypothalamus.

• LC–QE MS has been proved it can be applied for quantitative analysis of NTs.

• Plain pretreatment was needed for bio-sample in complex matrices with this method.

Abstract

Neurotransmitters (NTs) and their metabolites are known to play an essential role in maintaining various physiological functions in nervous system. However, there are many difficulties in the detection of NTs together with their metabolites in biological samples. A new method for NTs and their metabolites detection by high performance liquid chromatography coupled with Q Exactive hybrid quadruple-orbitrap high-resolution accurate mass spectrometry (HPLC–HRMS) was established in this paper. This method was a great development of the applying of Q Exactive MS in the quantitative analysis. This method enabled a rapid quantification of ten compounds within 18 min. Good linearity was obtained with a correlation coefficient above 0.99. The concentration range of the limit of detection (LOD) and the limit of quantitation (LOQ) level were 0.0008–0.05 nmol/mL and 0.002–25.0 nmol/mL respectively. Precisions (relative standard deviation, RSD) of this method were at 0.36–12.70%. Recovery ranges were between 81.83% and 118.04%. Concentrations of these compounds in mouse hypothalamus were detected by Q Exactive LC–MS technology with this method.

Introduction

Neurotransmitters (NTs) and their metabolites are widely distributed in central nervous system. They are known to play fundamental roles in maintaining various physiological functions. NTs are divided into four classes according to their structures, namely, monoamines, amino acids, peptides and other classes. NTs and their metabolites maintain a delicate balance in health body, and when this balance is broken there will be some diseases such as schizophrenia, depression, Alzheimer’s disease and Parkinson’s disease [1], [2], [3], [4], [5]. Therefore, it is very important for the detection of NTs and metabolites clinically. However, there are many challenges for quantitative analyzing NTs and their metabolites in biological samples, including complex matrix interferences, chemical instability and low concentrations. Structures of all analytes, internal standard (IS) and derived reagent are shown at Fig. 1.

Several methods have been reported recently for NTs and their metabolites detection, such as high-performance liquid chromatography (HPLC) or capillary electrophoresis (CE) coupled with various detection methods, fluorescence detection (FLD) [6], [7], electrochemical detection (ECD) [8], and mass spectrometry (MS) [9], [10], [11], [12], [13], [14]. Among them, liquid chromatography coupled with mass spectrometry is playing an essential role in analyzing several biological samples. Most of them are based on selected reaction monitoring (SRM) or multiple reactions monitoring (MRM) mode. Feng directly detected NTs and their metabolites without derivatization, but MS detection of one sample must be carried out twice in both positive and negative ion modes separately to achieve respective analysis, which was extremely tedious [15]. Zheng used MRM monitoring conditions to detect the derived NTs, and has reported that the LOD was in the range of 0.0005–0.05 nmol/mL, and the LOQ level was between 0.001 nmol/mL and 0.1 nmol/mL. It suggested that method was sufficient to simultaneously monitor a large panel of metabolites with diverse properties [16]. All of them indicated that mass technology provides a good platform for the quantitative detection. However, most of them are based on a triple quadrupole mass spectrometry with SRM or MRM mode, which is in poor resolution. In this paper, we applied high performance liquid chromatography coupled with high-resolution accurate mass spectrometry (HPLC–HRMS) to quantitative analyzing derivative NTs and their metabolites. Reaction formula of NTs with dansyl chloride (DNS-Cl) is shown at Fig. 2. Detection of one sample was only carried out in positive ion modes, which was extremely an easy way and saved time of the experiment.

There are basically two platforms that can be used for liquid chromatography coupled with high-resolution accurate mass spectrometry (LC–HRMS), such as those based on the orbitrap technology and those based on the time-of-flight (TOF) technology [17]. Orbitrap-based mass systems generally provide better selectivity than Q-TOF systems. The Q Exactive hybrid quadruple-orbitrap mass spectrometry (Q Exactive MS) generally provides superior selectivity than triple quadrupole system and Q-TOF. Especially, it is said that higher mass resolution often provides better selectivity in complex sample matrix [18], [19]. The Q Exactive MS technology offers advantages in analytical sensitivity and specificity, because detection is based on molecular mass and chemical structure. Although it is an excellent detection system which is commonly used for qualitative analysis in complex matrixes, but it extremely rarely applies in quantitative analysis.

The aim of this research was to establish a stable, reliable, sensitive and convenient method by Q Exactive LC–MS for simultaneous monitoring NTs and their metabolites. In our knowledge, this method is the first attempt to simultaneously detect NTs and their metabolites in mouse hypothalamus by Q Exactive LC–MS technology.