Simultaneous determination of three dipeptides (JBP485, Gly–Sar and JBP923) in the cell lysates by liquid chromatography‐tandem mass spectrometry: application to identify the function of the PEPT1 transfected cell

Abstract

A simple and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of JBP485, Gly-Sar and JBP923 in the cell lysates using methanol as a deproteinization solvent was developed and validated. Detection was performed by turbo ionspray ionization in multiple reaction monitoring mode using the transitions of m/z 147.1 → m/z 90.1 for Gly-Sar, m/z 201.1 → m/z 86.1 for JBP485, m/z 219.1 → m/z 86.1 for JBP923 and m/z 152.0 → m/z 110.0 for paracetamol (internal standard). The analytes were separated on a Hypersil ODS C18 HPLC column using isocratic elution mode with a mobile phase containing 0.1% formic acid in water-methanol (97:3, v/v) at a flow rate of 0.2 mL/min. The calibration curves were demonstrated to be linear over the concentration range of 5.00-5000 nm with coefficient of 0.9968 for Gly-Sar, 0.9975 for JBP485 and 0.9952 for JBP923. The intra- and inter-day precisions were <10.2% for each quality contro; level, and the accuracy was within ±5.6% for each analyte. The matrix effect, the extraction recovery and stabilities of LC-MS/MS analysis were also investigated. This validated method was successfully applied to the simultaneous determination of JBP485, Gly-Sar and JBP923 in the cell lysates for identification of stably transfected HeLa cells with human PEPT1.

Rapid and Sensitive Quantification of Intracellular Glycyl-Sarcosine for Semi-High-Throughput Screening for Inhibitors of PEPT-1

“…Nevertheless, short incubation times may be essential in avoiding extensive non-PEPT-1 specific uptake of Gly-Sar. Previous studies showed that Gly-Sar uptake approaches saturation shortly beyond 10 min [ 26 , 35 ]. However, incubation times shorter than 10 min increase assay intrinsic errors and are not well manageable.…”

“…In addition, it has been demonstrated that Caco-2 cells express considerable levels of human PEPT-1 [ 23 ]. Typically, Caco-2 cells are cultured for approximately 21 d to reach a fully differentiated state exhibiting maximal but, at the same time, heterogeneous transporter expression [ 24 , 25 , 26 ].…”

“…Several quantification methods based on LC-MS for intracellular Gly-Sar have been reported previously [ 20 , 26 , 27 ]. The lowest previously reported lower limit of quantification (LLOQ) was 1 ng/mL in 50 µL cell homogenate relying on cell experiments in formats down to 24-well plates [ 20 ].…”

Dipeptide-modified nanoparticles to facilitate oral docetaxel delivery: new insights into PepT1-mediated targeting strategy

“…Hela cells stably transfected with human PepT1 plasmid (hPepT1-Hela) and vector (mock-Hela) cells were kindly provided by Professor Kexin Liu (Dalian Medical University, Dalian, China). The cells were maintained in high-glucose DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin (Guo et al., 2012 , 2014 ). Porcine ileum Caco-2 cells were obtained from American Type Culture Collection (Rockvile, MD) and cultured in Modified Eagle’s medium (MEM; Invitrogen, Carlsbad, CA) containing 10% FBS, 1 mM sodium pyruvate, 1% non-essential amino acids, 1% L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin.…”