Abstract
Acetylglutamine (NAG) is the derivative of glutamine, which is the richest free amino acid in the human body. In this work, a novel reliable method of the combination of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and microdialysis (MD) technique for the evaluation of NAG and its metabolites γ-aminobutyric acid (GABA) and glutamic acid (Glu) in rat blood and brain was proposed. A Zorbax SB-C18 column (2.1 × 100 mm, 3.5 μM) was applied to separate the analytes. The mobile phase was acetonitrile-water (70:30, v/v) containing 5 mM ammonium acetate and the flow rate was 0.3 ml/min. Based on the multiple reaction monitoring (MRM) mode of positive ion, the precursors of product ions chosen for NAG, Glu, GABA, and N-carbamyl-L-glutamic (NCG, IS) were (m/z) 189.1→130.0, 148.0→84.1, 104→87.1, and 191.0→130.1, respectively. All the validation data, including precision, accuracy, inter-day repeatability, matrix effect, and stability, were within the acceptable ranges according to the reference of Bioanalytical Method Validation Guidance for Industry (2018). Rats with microdialysis probes inserted into jugular vein and hippocampus were administered the low (75 mg/kg, NAG-L), medium (150 mg/kg, NAG-M), and high (300 mg/kg, NAG-H) doses of NAG and 10 ml/kg Guhong injection (GHI) by tail vein, respectively. In the blood test, the Cmax values of NAG-L group were markedly lower (P < 0.01) than those of NAG-M, NAG-H, and GHI groups, respectively. No differences were observed between NAG-M and GHI groups, while the Cmax values in GHI group were significantly upgraded compared with NAG-H group. There were notable differences in the Cmax values of NAG in brain dialysate after administration of NAG and GHI. The drug distribution coefficients of NAG, Glu, GABA in brain and blood at low, medium, high doses of NAG and GHI groups were 13.99, 27.43, 34.81, 31.37; 11.04, 59.07, 21.69, 2.69%; 212.88, 234.92, 157.59, and 102.65%, respectively. Our investigation demonstrates that NAG and its related metabolites in rat blood and brain can be simultaneously measured according to the above proposed method. Meanwhile, NAG has easy and dose-dependently access to the blood-brain barrier and exhibits a medium retention time in rat.
Highlights
Accounting for about 60% of the total free amino acid in the human body, glutamine has the function to regulate the immune system and treat the gastrointestinal disturbances, clinically (Lopez-Pedrosa et al, 2007)
With the aim to determine the intra-day and inter-day precision (RSD%) and accuracy (RE%), six dialysate quality control samples (QCs) samples at the concentration of 50, 500, 5,000 ng/ml were analyzed within 1 day and 5 consecutive days, respectively
A LC-MS/MS method combined with MD technique was established to quantify the NAG and its metabolites glutamic acid (Glu) and gaminobutyric acid (GABA) in rat blood and brain tissues
Summary
Accounting for about 60% of the total free amino acid in the human body, glutamine has the function to regulate the immune system and treat the gastrointestinal disturbances, clinically (Lopez-Pedrosa et al, 2007). NAG injection is mainly used as a brain function improving agent in the clinical to treat brain trauma, hepatic coma, hemiplegia, and sequelae of cerebral apoplexy (Liu and Wu, 2006; Yan et al, 2009). The major components of Chinese patent medicine like Guhong injection (GHI) are safflower and NAG, and the clinical value of GHI in ameliorating the neurological function of patients with cerebral infarction has been demonstrated. While Glu, as an excitatory neurotransmitter, is closely related to the plasticity of neurons It makes crucial effects on the growth of neurons and the formation of synapses, as well as the improvement of learning
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