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Their simultaneous quantification remains analytically challenging due to their low ionization efficiency and poor chromatographic retention. Here, we synthesized a pair of secondary amine-based chemical isotope labeling reagents, N-(piperidin-4-ylmethyl)benzamide (PMBA) and d5-N-(piperidin-4-ylmethyl)benzamide (d5-PMBA), for the simultaneous labeling of carboxylic metabolites and drugs. The secondary amine moiety on PMBA\/d5-PMBA can efficiently react with carboxyl groups on carboxylic compounds, substantially improving the reversed-phase (RP) chromatographic retention and separation, along with the enhancement of the MS sensitivity. Upon this strategy, the limits of detection (LODs) of 32 carboxylic metabolites and drugs were determined to range from 0.001 to 0.13 ng mL\u20131. The chemical isotope labeling approach with liquid chromatography\u2013mass spectrometry (LC\u2013MS) analysis enabled reliable quantification of diverse carboxylic compounds in serum samples and revealed significant alterations in their abundances during hepatocellular carcinoma progression. This method provides a sensitive and robust analytical platform for the simultaneous quantification of carboxylic metabolites and drugs and facilitates investigations of their biological and pharmacological functions in diseases.<\/jats:p>","DOI":"10.1021\/acs.analchem.6c02580","type":"journal-article","created":{"date-parts":[[2026,8,4]],"date-time":"2026-08-04T13:20:49Z","timestamp":1785849649000},"page":"23032-23040","source":"Crossref","is-referenced-by-count":1,"title":["Secondary Amine-Based\nChemical Isotope Labeling Coupled\nwith Mass Spectrometry for Simultaneous Quantification of Carboxylic\nCompounds during Hepatocellular Carcinoma Progression"],"prefix":"10.1021","volume":"98","author":[{"given":"Jun","family":"Zhang","sequence":"first","affiliation":[{"name":"Anhui Medical University , , ,","place":["Hefei, China, 230032"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yu-Han","family":"Dai","sequence":"additional","affiliation":[{"name":"Anhui Medical University , , ,","place":["Hefei, China, 230032"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yue","family":"Zhu","sequence":"additional","affiliation":[{"name":"Anhui Medical University , , ,","place":["Hefei, China, 230032"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xue-Yi","family":"Zhao","sequence":"additional","affiliation":[{"name":"Anhui Medical University , , ,","place":["Hefei, China, 230032"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Guo-Jia","family":"Song","sequence":"additional","affiliation":[{"name":"Anhui Medical University , , ,","place":["Hefei, China, 230032"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chengjie","family":"Ma","sequence":"additional","affiliation":[{"name":"Zhengzhou Tobacco Research Institute of CNTC , , ,","place":["Zhengzhou, China, 450001"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9538-6772","authenticated-orcid":true,"given":"Fei-Long","family":"Liu","sequence":"additional","affiliation":[{"name":"Anhui Medical University , , ,","place":["Hefei, China, 230032"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,7,30]]},"reference":[{"issue":"1","key":"2026081109100942700_cit1","doi-asserted-by":"publisher","first-page":"55","DOI":"10.1038\/s41579-020-0433-9","article-title":"Gut microbiota in human metabolic health and disease","volume":"19","author":"Fan","year":"2021","journal-title":"Nat. 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