Development and Validation of a Sensitive Liquid Chromatography–Tandem Mass Spectrometry Method for the Determination of Naringin and Its Metabolite, Naringenin, in Human Plasma

Journal of Chromatographic Science 2014;52:654– 660 doi:10.1093/chromsci/bmt095 Advance Access publication July 9, 2013 Article Development and Validation of a Sensitive Liquid Chromatography –Tandem Mass Spectrometry Method for the Determination of Naringin and Its Metabolite, Naringenin, in Human Plasma Xin Xiong1,2, Junjie Jiang3, Jingli Duan1*, Yanming Xie3, Jiannong Wang4 and Suodi Zhai1 1 Department of Pharmacy, Peking University Third Hospital, Beijing, 100191, PR China, 2Therapeutic Drug Monitoring and Clinical Toxicology Center, Peking University, Beijing, 100191, PR China, 3Institute of Basic Research in Clinical Medicine, China Academy of Chinese Medical Sciences, Beijing, 100700, PR China, and 4Xiyuan Hospital, China Academy of Chinese Medical Sciences, Beijing 100091, PR China *Author to whom correspondence should be addressed. Email: Received 27 March 2013; revised 19 May 2013 A sensitive and specific method was developed for the simultaneous determination of naringin and its metabolite, naringenin, in human plasma by liquid chromatography –tandem mass spectrometry. Hesperidin was used as the internal standard, plasma samples were extracted with ethyl acetate and the analytes were chromatographically separated by using acetonitrile –0.1% formic acid (gradient elution) as the mobile phase. Detection was performed by electrospray ionization mass spectrometry in negative ion mode with multiple reaction monitoring. The lower limit of quantification was 0.5 ng/mL for naringin and naringenin and the linear calibration curves ranged from 0.5 to 200 ng/mL. The intra-run and inter-run precision values were within 8.6 and 7.7% for naringin and between 13.1 and 10.3% for naringenin. The accuracy ranged from 91.3 to 98.2% for naringin and from 90.2 to 97.6 % for naringenin. The validated method was successfully applied to determine concentrations of naringin and naringenin in clinical patients. method in rat plasma with a lower limit of quantification (LLOQ) of 5 ng/mL for naringin and naringenin in rat plasma; this is not sensitive enough to assay the concentration of naringin in human plasma because of the low content of naringin in Qianggu capsules. Additionally, the matrix effect was not investigated. Therefore, this paper reports a sensitive and specific LC– MS-MS method to determine naringin and its metabolite, naringenin, in human plasma in a low volume (0.2 mL) of human plasma. It was essential to establish a method capable of quantifying naringin and naringenin at concentrations down to 0.5 ng/ mL. The bioanalytical methodology was validated in accordance with Food and Drug Administration (FDA) guidelines (11) considering specificity, sensitivity, linearity, precision, accuracy, matrix effect and stability. Experimental Introduction Traditional Chinese medicine (TCM) has been widely used for thousands of years in China. In recent years, increasing attention has been paid to the efficiency and safety of TCM in clinical use. Qianggu is a Chinese compound formulation, primarily composed of Rhizoma Drynariae. It is a kidney-toning and bonestrengthening formulation that is used for the treatment of osteoporosis. Naringin is known as the primary active constituent of Rhizoma Drynariae (1). Like most flavonoids, naringin has several pharmacological properties such as antimicrobial, antimutagenic, anticancer, anti-inflammatory and antioxidant effects (2 –4). Naringenin, the aglycone of naringin, has also been demonstrated to exhibit anti-ulcer (3) and antioxidant effects (4). Naringin and naringenin have been previously quantified by utilizing a variety of methods including, high-performance liquid chromatography –ultraviolet (HPLC –UV) detection (5 –7) and liquid chromatography–tandem mass spectrometry (LC – MS-MS) (8 –10) in rabbit plasma (5), rat plasma (8, 9), human plasma (6) and human urine (7). Ishii et al. (6) reported that the limit of detection of an HPLC – UV method in human plasma was approximately 5 ng for naringin and naringenin, but this method was unable to concurrently assay naringin and its metabolite, naringenin. Recently, Fang et al. (8) described an LC –MS-MS Chemicals and reagents Naringin and naringenin were purchased from Tauto Biotech (Shanghai, China; purity . 98.0%), whereas the internal standard (IS), hesperidin, was purchased from the National Institute for the Control of Biological Products (Beijing, China). HPLC grade formic acid was commercially obtained from Dikma (Lake Forest, IL) and acetonitrile and methanol (HPLC grade) were purchased from Fisher (Fair Lawn, NJ). Water, purified by a Milli-Q system (Millipore, Bedford, MA), was used throughout the analysis. Instrumentation The compounds were separated by using an Agilent 1200 HPLC system (Agilent Technologies, Palo Alto, CA) consisting of binary pumps, an autosampler and a vacuum degasser. The HPLC system was coupled to an Agilent 6410 triple quadrupole mass spectrometer (Agilent Technologies), under the control of Masshunter software (version B 01.03). An Agilent XDB-C18 (Agilent Technologies) column (50  2.1 mm, 1.8 mm) was employed. The column was maintained at 358C. The gradient mobile phase consisted of 0.1% formic acid as mobile phase A and acetonitrile as mobile phase B. The pump was run at a flow rate of 1 mL/min (split ratio: 1:3) from 10% B to 70% B over 4 min, changed to 100% B at 4.1 min, and remained at 100% B for 1 min. After this, it was returned to the original 10% B at 5.1 min # The Author [2013]. Published by Oxford University Press. All rights reserved. For Permissions, please email: Figure 1. Product ion mass spectra: naringin (A); naringenin (B); hesperidin (C). and remained at 10% B for another 1.9 min. The injection volume was 20 mL. Electrospray ionization (ESI) was performed in negative ion mode with nitrogen as the nebulizer and drying gas. The ion source conditions were set as follows: gas temperature, 3508C; nebulizer gas, 40 psi; gas flow, 11 L/min; capillary voltage, 5,500 V; fragmentor, 200 V for naringin, 120 V for naringenin and 135 V for the IS; collision energy, 30 V for naringin, 12 V for naringenin and 20 V for the IS, and dwell time, Development and Validation of a Sensitive Liquid Chromatography– Tandem 655 Figure 2. MRM chromatograms of human plasma, obtained from: blank human plasma containing 0.5 ng/mL of naringin (A); blank human plasma containing 0.5 ng/mL of naringenin (B); blank human plasma containing 300 ng/mL of the IS (C). 200 ms. The monitored multiple reaction monitoring (MRM) transitions were: m/z 579.2 ! 271.1 (quantification) and m/z 579.2 ! 151.1 (identification) for naringin, m/z 271.0 ! 151.2 (quantification) and m/z 271.0 ! 119.0 (identification) for naringenin and m/z 609.3 ! 301.0 for the IS (see Figure 1). 656 Xiong et al. Preparation of calibration standards and quality control samples Stock solutions of naringin (374 mg/mL), naringenin (388 mg/ mL) and the IS (590 mg/mL) were separately prepared in volu (...truncated)