title: Challenges in optimizing sample preparation and LC-MS/MS conditions for the analysis of carglumic acid, an N-acetyl glutamate derivative in human plasma.
abstract: This paper describes a systematic approach to overcoming challenges in developing a robust and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for reliable and precise determination of carglumic acid in human plasma. Sample extraction was tested on several reversed-phase solid-phase extraction (SPE) sorbents with different chemistries, such as hydrophobic C18, hydrophilic-lipophilic balance, and mixed-mode cation and anion exchange. The best recovery under the optimized extraction conditions was obtained with Oasis MAX (30 mg, 1cc) mixed-mode anion exchange (~ 50%) cartridge, compared to other sorbents from 100 μL plasma sample. Complete analytical separation of carglumic acid and carglumic acid-13C5 15N as an internal standard (IS) from endogenous plasma components was achieved on ACE 5CN (150 × 4.6 mm, 5 µm) column under isocratic conditions using acetonitrile:methanol (50:50, v/v) - 0.1% acetic acid in water [80:20, v/v] as the mobile phase. The deprotonated precursor → product ion transitions for carglumic acid (189/146) and IS (195/152) were monitored in the negative ionization mode on a triple quadrupole mass spectrometer. The regression curves were linear over a concentration range of 6.00-6000 ng/mL (r(2) ≥ 0.9987). Matrix effect was evaluated in terms of IS-normalized matrix factors, which ranged from 0.95 to 1.01 across four quality control levels. Intra- and inter-batch accuracy and precision, and the stability of carglumic acid in spiked plasma samples were assessed under different conditions. The method was applied to assess the pharmacokinetics of 100 mg/kg body weight carglumic acid in a healthy Indian subject.
authors: Department of Chemistry, School of Sciences, Gujarat University, Ahmedabad, 380009, India.
authors: Primal
authors: P
authors: Sharma
authors: Department of Chemistry, School of Sciences, Gujarat University, Ahmedabad, 380009, India.
authors: Priyanka A
authors: PA
authors: Shah
authors: Chemistry Department, St Xavier's College, Navrangpura, Ahmedabad, 380009, India.
authors: Mallika
authors: M
authors: Sanyal
authors: Department of Chemistry, School of Sciences, Gujarat University, Ahmedabad, 380009, India.
authors: Pranav S
authors: http://orcid.org/0000-0002-1284-1558
authors: PS
authors: Shrivastav
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods; D005971:Glutamates / Q000032:analysis / Q000097:blood* / Q000302:isolation & purification; D006801:Humans; D057230:Limit of Detection; D008297:Male; D008875:Middle Aged; D052616:Solid Phase Extraction / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D005971:Glutamates; C528449:carglumic acid; C016195:N-acetylglutamic acid
keywords: LC-MS/MS; Oasis MAX sorbent; carglumic acid; human plasma; matrix effect