Enantioseparation of N‐acetyl‐glutamine enantiomers by LC–MS/MS and its application to a plasma protein binding study

Abstract

A novel chiral method was developed and validated to determine N‐acetyl‐glutamine (NAG) enantiomers by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Enantioseparation was achieved on a Chiralpak QD‐AX column (150 × 4.6 mm i.d., 5 μm) using methanol–water (50 mm ammonium formate, pH 4.3; 70:30, v/v) at a flow rate of 500 μL/min. The detection was operated with an electrospray ionization source interface in positive mode. The ion transition for NAG enantiomers was m/z 189.0 → 130.0. The retention time of N‐acetyl‐l‐glutamine and N‐acetyl‐d‐glutamine were 15.2 and 17.0 min, respectively. Calibration curves were linear over the range of 0.02–20 μg/mL with r > 0.99. The deviation of accuracy and the coefficient of variation of within‐run and between‐run precision were within 10% for both enantiomers, except for the lower limit of quantification (20 ng/mL), where they deviated <15%. The recovery was >88% and no obvious matrix effect was observed. This method was successfully applied to investigate the plasma protein binding of NAG enantiomers in rats. The results showed that the plasma protein binding of NAG enantiomers was stereoselective. The assay method also exhibited good application prospects for the clinical monitoring of free drugs in plasma.

Rat plasma protein binding of kaempferol‐3‐ O ‐rutinoside from Lu’an GuaPian tea and its anti‐inflammatory mechanism for cardiovascular protection

“…Binding of drugs to plasma proteins can impact the pharmacological activities, efficacy, and safety (Liu et al., 2014; Schmidt et al., 2010; Wani & Zargar, 2015). Free drugs can produce pharmacological effects through cell membrane and can be bound to the targets, which is critical for drugs’ bioactivity (Gao et al., 2019; Zhou et al., 2018). Therefore, it is necessary to guide clinical administration by measuring the PPB, especially for adjusting the therapeutic dose of drugs and clarifying blood drug concentration.…”

Characterization of isomeric acetyl amino acids and di‐acetyl amino acids by LC/MS/MS

“…The side‐chain acetyl amino acids were synthesized from respective L‐amino acid by using a well‐known procedure 17 . In brief, alpha‐amino group (–NH 2 ) of the L‐amino acid (serine, threonine, cysteine, tyrosine, arginine, and lysine) was protected with tert‐butyloxycarbonyl (BOC) group followed by acetylation of side chain functional group, and then deprotection of BOC group resulted in side‐chain acetylated amino acid (Scheme 2).…”

“…For structural characterization of N α -acetyl amino acids (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20), the [M + H] + ions product-ion MS/MS spectra were acquired at different collision energies (CEs). The MS/MS spectra at a particular CE that displayed more structure indicative product ions were considered for the study (Table 1).…”

“…As a part of intracellular protein catabolism, the N‐acetyl proteins/peptides are cleaved by an N‐acylpeptide hydrolase to release the corresponding N‐acetyl amino acids 9,16 . N‐ acetyl amino acids may be formed through direct synthesis by specific N‐ acetyltransferases; for example, choline converts into acetylcholine that act as a neurotransmitter 17 …”

“…9,16 N-acetyl amino acids may be formed through direct synthesis by specific N-acetyltransferases; for example, choline converts into acetylcholine that act as a neurotransmitter. 17 N-acetyl amino acids are of great interest because of their wide spectrum of actions and also known markers for several diseases. In arginine biosynthesis, N-acetyl glutamate synthase catalyzes L-glutamate acetylation.…”