Enrichment and Quantitative Determination of 5-(Hydroxymethyl)-2′-deoxycytidine, 5-(Formyl)-2′-deoxycytidine, and 5-(Carboxyl)-2′-deoxycytidine in Human Urine of Breast Cancer Patients by Magnetic Hyper-Cross-Linked Microporous Polymers Based on Polyionic Liquid

Abstract

5-(Hydroxymethyl)-2'-deoxycytidine (5-hmdC), 5-(formyl)-2'-deoxycytidine (5-fodC), and 5-(carboxyl)-2'-deoxycytidine (5-cadC) are crucial intermediate products of the DNA demethylation pathway, which can also act as potential biomarkers reflecting the diagnosis and prognosis in multiple tumors. Detecting 5-hmdC, 5-fodC, and 5-cadC in human urine has various advantages including readily available samples and being noninvasive to patients. However, few works have reported the detection of 5-fodC and 5-cadC due to their trace amounts. Here we developed a novel magnetic hyper-cross-linked microporous polymer (HMP) material based on polyionic liquid (PIL) for the enrichment of 5-hmdC, 5-fodC, and 5-cadC. These magnetic PIL-HMP materials provided specific enrichment superiority for three modified cytidines. After enrichment, the signal intensity of 5-hmdC, 5-fodC, and 5-cadC increased 10-75-fold with lower limits of quantitation (LLOQ) of 0.049, 0.781, and 0.781 ng/mL, respectively. The recoveries were approximately 86.5-95.2% for 5-hmdC, 95.2-107.8% for 5-fodC, and 99.4-102.4% for 5-cadC under the relative standard deviation (RSD) of 0.2-10.3%. Finally, we successfully applied magnetic PIL-HMP materials coupled with high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) in enrichment and quantitative determination of 5-hmdC, 5-fodC, and 5-cadC in human urine of 10 breast cancer patients and 10 healthy people. We found that the level of 5-hmdC decreased in breast cancer patients ( p < 0.05), while the levels of 5-fodC and 5-cadC increased ( p < 0.05, p < 0.01). Our results demonstrated that the levels of metabolic 5-hmdC, 5-fodC, and 5-cadC in human urine are closely related to breast cancer, which could contribute to the clinical diagnosis and investigation of breast cancer and its occurrence and development mechanisms.

Polymerization retardation isothermal amplification (PRIA): a strategy enables sensitively quantify genome-wide 5-methylcytosine oxides rapidly on handy instruments with nanoscale sample input

“…The detection deviation and the possible nonspecific tagging of other bases in DNA samples might cause the recovery over 100%. However, compared to the previously reported works (36,37), the PRIA method has a good assay accuracy.…”

Advances in detection and quantification of methylcytosine and its derivatives

“…Thus, going forward, urine analysis could be the most convenient approach to assess DNA modifications due to its patient‐friendly nature. In order to detect and quantify 5hmdC, 5fodC, and 5cadC in human urine, Our group developed hyper‐cross‐linked microporous polymer (HMP) with magnetic nanoparticles for enrichment. The allyl bromide, 3‐bromopropyne, and 4‐vinylbenzyl chloride were reacted with vinylimidazole to produce polyenyly/polyalkyl ionic liquid monomer.…”

“…Then, the ionic liquid monomer passed through polymerization with hexanedithiol by thiol‐ene reactions. Finally, the ionic liquid hyper‐cross‐linked microporous polymers (PIL‐HMP) were reacted with magnetic silica (Fe 3 O 4 @SiO 2 ) by (3‐mercaptopropyl)‐trimethoxysilane to get PIL‐HMP via a chemical bonding assembly . The application of the magnetic PIL‐HMP materials not only improves the linearity and recovery of the three compounds but also successfully distinguishes 5hmdC, 5fodC, and 5cadC in urine samples of breast cancer patients and healthy individuals.…”

“…With the enrichment technique together with LC‐MS, all known cytosine modifications have been separated, distinguished and independently quantitated . Importantly, this approach allows DNA modifications to be assessed non‐invasively .…”

A novel malic acid-enhanced method for the analysis of 5-methyl-2′-deoxycytidine, 5-hydroxymethyl-2′-deoxycytidine, 5-methylcytidine and 5-hydroxymethylcytidine in human urine using hydrophilic interaction liquid chromatography-tandem mass spectrometry

“…Furthermore, selective enrichment is another strategy to enhance sensitivity [36,37]. Recently, we successfully enriched and quantified other two intermediates involved in DNA demethylation, 5-formyl-2 ’- deoxycytidine (5-fodC) and 5-carboxyl-2′-deoxycytidine (5-cadC) in human urine by magnetic hyper-cross-linked microporous polymer material [38]. Moreover, mobile phase additive can also enhance the signal response of analytes in MS [28,39], and a recent study demonstrated that NH 4 HCO 3 can significantly improve the determination of 5-mdC, 5-hmdC and 5-fodC [28].…”