Analysis of carnosic acid metabolites in rats by UHPLC-Q-Exactive MS
(2.Beijing Institution of Tongrentang, Beijing, China 100079)
【Abstract】A method of ultra-high performance liquid chromatography coupled with quadrupole/electrostatic field Orbitrap high-resolution mass spectrometry (UHPLC-Q-Exactive MS) was established to comprehensively identify the metabolites of carnosic acid in rats. After oral gavage of carnosic acid CMC-Na suspension in rats, the urine, plasma, and feces samples were collected and pretreated by solid-phase extraction (SPE). Acquity UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm) was used with 0.1% formic acid solution (A)-acetonitrile (B) as the mobile phase for gradient elution. The biological samples were analyzed by quadrupole/electrostatic field Orbitrap high-resolution mass spectrometry in positive and negative ion modes. Based on the accurate molecular mass, fragment ion information, and related literature reports, a total of 28 compounds (including carnosic acid) were finally identified in rat samples. As a result, the main metabolic pathways of carnosic acid in rats are oxidation, hydroxylation, methylation, glucuronide conjugation, sulfate conjugation, S-cysteine conjugation, glutathione conjugation, demethylation, decarbonylation, and their composite reactions. The study showed that the metabolism of carnosic acid in rats could be efficiently and comprehensively elucidated by using UHPLC-Q-Exactive MS, providing a reference for clarifying the material basis and metabolic mechanism of carnosic acid.
【Keywords】 carnosic acid; UHPLC-Q-Exactive MS; metabolites; metabolic pathways;
 WANG W W, HAN J H, ZHANG H M, et al. 鼠尾草酸对β-淀粉样蛋白损伤的神经干细胞增殖的影响 [J]. Chinese Traditional Patent Medicine, 2017, 39 (7): 1495 (in Chinese).
 AZAD N, RASOOLIJAZI H, JOGHATAIE M T, et al. Neuroprotective effects of carnosic acid in an experimental model of Alzheimer‘’s disease in rats [J]. Cell J, 2011, 13 (1): 39.
 LARA M, SONIA A, RAÚL B, et al. Meat texture and antioxidant status are improved when carnosic acid is included in the diet of fattening lambs [J]. Meat Sci, 2012, 91 (4): 430.
 MARC D L R, RUTHERFORD T J, GUPTA D, et al. An intrinsically labile alpha-helix abutting the BCL9-binding site of β-catenin is required for its inhibition by carnosic acid [J]. Nat Commun, 2012, 3: 680.
 OH J, YU T, CHOI S J, et al. Syk/Src pathway-targeted inhibition of skin inflammatory responses by carnosic acid [J]. Mediat Inflamm, 2012, 2012: 2.
 WEI J, HU W Y, ZHANG L C, et al. Study on the protective effect on neurons damaged by H2O2 and stability of carnosic acid [J]. Pharmacology and Clinics of Chinese Materia Medica, 2016, 32 (2): 44 (in Chinese).
 LUO X F, CHEN Q Y, WANG C, et al. Ultrasonic crushing extraction process of carnosic acid from rosemary leaves and its antioxidant effect on oil [J]. China Oils and Fats, 2019, 44 (5): 118 (in Chinese).
 LUO X F, CHEN Q Y, WANG C, et al. 鼠尾草酸在大鼠体内的药物动力学及代谢研究 [D]. Shenyang: Shenyang Pharmaceutical University, 2008 (in Chinese).
 ZHANG J Y, WANG Z J, ZHANG Q, et al. Rapid screening and identification of target constituents using full scan-parent ions list-dynamic exclusion acquisition coupled to diagnostic product ions analysis on a hybrid LTQ-Orbitrap mass spectrometer [J]. Talanta, 2014, 124: 111.
 ZHANG J Y, WANG Z J, LI Y, et al. A strategy for comprehensive identification of sequential constituents using ultra-high-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometer application study on chlorogenic acids in Flos Lonicerae Japonica [J]. Talanta, 2016, 147 (62): 16.
 ZHANG J Y, CAI W, ZHOU Y, et al. Profiling and identification of the metabolites of baicalin and study on their tissue distribution in rats by ultra-high-performance liquid chromatography with linear ion trap-Orbitrap mass spectrometer [J]. J Chromatogr B, 2015, 985: 91.
 LI X, WANG H H, XU J, et al. Study on active components of Fufang Huangbai Ye for diabetic foot treatment by UPLC-LTQ-Orbitrap-MS and network pharmacology [J]. China Journal of Chinese Materia Medica, 2019, 44 (10): 2110 (in Chinese).
 ZHANG C Y, ZHANG H, REN W G, et al. Pharmacokinetics of four phenolic acids from Danshen-Chuanxiong drug pair in plasma and heart tissue of rats by UPLC-MS/MS [J]. China Journal of Chinese Materia Medica, 2019, 44 (19): 4257 (in Chinese).
 LIU S S, LIU Q, LI P, et al. Simultaneous rapid detection of ten stilbenes in serum of mice by UPLC-MS/MS [J]. China Journal of Chinese Materia Medica, 2020, 45 (9): 2180 (in Chinese).
 REN M M, XIA Y, FENG Z W, et al. Analysis of flavonoids in Coreopsis tinctoria by integrating 2D-TLC and HPLC-IT-TOF-MS [J]. China Journal of Chinese Materia Medica, 2019, 44 (7): 1403 (in Chinese).
 LI Y L, WANG Y, WANG Z, et al. Study on simultaneous determination of six arsenic species by HPLC-ICP-MS [J]. China Journal of Chinese Materia Medica, 2019, 44 (24): 5441 (in Chinese).
 BAI J Q, HUANG J, XU W, et al. Pharmacokinetics of dendrobine in rats by LC-MS/MS [J]. World Chinese Medicine, 2019, 14 (9): 2272 (in Chinese).
 ZHANG M, YU D G, LU H Y, et al. Determination of irbesartan in human plasma by LC-MS/MS and clinical application [J]. Pharmacy Today, 2019, 29 (6): 379 (in Chinese).
 ZHAO W J, HUANG M R, SHANG Z P, et al. Metabolites of tanshinone Ⅰ and tanshinone ⅡA in vivo in rats [J]. China Journal of Chinese Materia Medica, 2018, 43 (1): 175 (in Chinese).
 LIANG Y Y, ZHAO W J, WANG C X, et al. A comprehensive screening and identification of genistein metabolites in rats based on multiple metabolite templates combined with UHPLC-HRMS analysis [J]. Molecules, 2018, 23: 23.
 JIANG Y B, CHAN J H, WANG Y, et al. Application progress of LC-MS/MS technology in drug metabolism research [J]. Chinese Journal of Pharmaceutical Analysis, 2014, 34 (3): 386 (in Chinese).
 MEI X D, WANG Y Q, WANG Z J, et al. Identification of metabolites of Danshensu in vivo in rats [J]. China Journal of Chinese Materia Medica, 2018, 43 (19): 3934 (in Chinese).
 ZHAO W J, LIANG Y Y, WANG Z J, et al. Structural elucidation of genistein metabolites in rats based on UHPLC-LTQ-Orbitrap mass spectrometry [J]. Journal of Chinese Mass Spectrometry Society, 2019, 40 (2): 111 (in Chinese).
 MULINACCI N, INNOCENTI M, BELLUMORI M, et al. Storage method, drying processes and extraction procedures strongly affect the phenolic fraction of rosemary leaves: an HPLC/DAD/MS study [J]. Talanta, 2011, 85 (1): 172.
 SONG Y, YAN H, CHEN J, et al. Characterization of in vitro and in vivo metabolites of carnosic acid, a natural antioxidant, by high performance liquid chromatography coupled with tandem mass spectrometry [J]. J Pharm Biomed Anal, 2014, 89: 194.
 ROMO VAQUERO M, GARCÍA VILLALBA R, LARROSA M, et al. Bioavailability of the major bioactive diterpenoids in a rosemary extract: metabolic profile in the intestine, liver, plasma, and brain of Zucker rats [J]. Mol Nutr Food Res, 2013, 57 (10): 1834.
 ZHANG Y, SMUTS J P, DODBIBA E, et al. Degradation study of carnosic acid, carnosol, rosmarinic acid, and rosemary extract (Rosmarinus officinalis L. ) assessed using HPLC [J]. J Agric Food Chem, 2012, 60 (36): 9310.
 ALMELA L, BLAS S, JOSÉ A, et al. Liquid chromatographic-mass spectrometric analysis of phenolics and free radical scavenging activity of rosemary extract from different raw material [J]. J Chromatogr A, 2006, 1120 (1/2): 226.