Development and Validation of a Sensitive Liquid Chromatography ...

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: [email protected] 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

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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 volumetric flasks with methanol and stored at 48C. Combined

Figure 3. Blank human plasma MRM chromatograms: naringin (A); naringenin (B); IS (C).

working solutions were prepared from the stock solutions at concentrations of 10, 20, 40, 100, 400, 2,000 and 4,000 ng/mL for both analytes with methanol as solvent. The IS working solution was prepared by diluting the stock solution with methanol on each day of analysis with a concentration of 300 ng/mL.

Calibration standards were freshly prepared on each day of analysis by adding 10 mL of the appropriate combined standard working solution to a 190 mL aliquot of blank plasma. Standards were prepared at concentrations of 0.5, 1, 2, 5, 20, 100 and 200 ng/mL for both compounds. Validation quality control (QC) Development and Validation of a Sensitive Liquid Chromatography– Tandem 657

samples were prepared in the same way at levels of 1.5, 10 and 160 ng/mL.

Sample preparation Frozen plasma samples were thawed at room temperature and the thawed samples were vortexed for 30 s. Aliquots of 200 mL of plasma were placed in a 1.5 mL polypropylene tube and 40 mL of the IS was added. The mixture was extracted with 1 mL of ethyl acetate. After being vortexed for 2 min and centrifuged for 5 min, the organic layer was transferred to another polypropylene tube. The organic fractions were dried under a gentle stream of nitrogen. The residue was dissolved in 100 mL of the mobile phase and 20 mL of the reconstituted sample was injected into the LC–MS-MS system for analysis. The assay of naringenin with its glucuronide was the same as in previously reported literature (12). Plasma (200 mL) was incubated with 10 mL of 0.78 M sodium acetate –acetic acid buffer ( pH 4.8), 10 mL of 0.1 M ascorbic acid and 10 mL of b-glucuronidase enzyme at 378C for 17 h. Next, 40 mL of the IS were added to the plasma sample and vortexed for 2 min, and the final extracting steps were the same as those described previously.

and the IS were typically 2.9, 3.8 and 2.9 min, respectively. The total analysis time was 7 min for each injection. Although both analytes eluted in , 4.0 min, a gradient mobile phase (described previously) was still adopted; each sample analysis took 7 min to ensure that interfering substances were eliminated from the column Negative detection mode was employed in the experiments. In this mode, the very soft ionization process in the ESI source produced the deprotonated ions [M– H]2. The observed deprotonated ions were m/z ¼ 579.2 and 271.0 for naringin and naringenin, respectively. Each of the deprotonated ions was subjected to collision-induced dissociation to obtain product ions. The collision conditions were optimized and the MS-MS peaks of the product ions for the analytes and the IS were acquired (Figures 1A –C). Based on the product spectra and the mechanisms, the transitions of m/z 579.2 ! 271.1 and 271.0 ! 151.2 were selected for naringin and naringenin, respectively. For the IS, the transition of m/z 609.3 ! 301.0 was selected for quantification.

Validation results Method validation The method was validated with reference to the Guidance for Industry Bioanalytical Method Validation of the FDA. Precision, accuracy, selectivity, extraction recovery, matrix effect and multiple stabilities were evaluated in the method validation. The quantitation ranged from 0.5 to 200 ng/mL for naringin and naringenin.

Results Method development Naringin is a strongly polar compound. In preliminary experiments, methanol and acetonitrile were used to precipitate proteins, but the chromatogram was rather poor as a result of serious matrix effect and high noise. The cost of a solid-phase extraction (SPE) method with cartridges was too high; thus, it was not considered. To obtain clean samples, a liquid –liquid extraction method was used. Different kinds of organic solvents were tried, such as ethyl acetate, methyl tert-butyl diether and ethyl ether. Relatively higher recovery (approximately 40 –45%) was obtained when using ethyl acetate as solvent than with others (30%). Although the recovery of naringin was relatively low when ethyl acetate was used as solvent, it was acceptable because the recovery was consistent over the range of concentrations and demonstrated no significant trending. Several analytical columns of different sizes, brands and different mobile phases were evaluated to find which one produced the sharpest, most symmetrical peaks with appropriate retention times. The Agilent XDB-C18 column (50  2.1 mm, 1.8 mm) and a gradient mobile phase (as described previously) consisting of 0.1% formic acid and acetonitrile with a flow rate of 1 mL/min were found to produce adequate retention and good peak shapes for the analytes and the IS. Representative chromatograms of the analytes and the IS from spiked plasma samples are shown in Figure 2. The retention times of naringin, naringenin 658 Xiong et al.

Selectivity, calibration curve, precision and accuracy A typical chromatogram for the control human plasma (free of analytes) and human plasma spiked with naringin and naringenin at LLOQ along with IS are shown in Figure 3. No interfering peaks from endogenous compounds were observed at the retention times of the analytes and IS. The linearity of the calibration curve was tested over the concentration range of 0.5 –200 ng/mL, using 1/x 2 weighted. The mean equations of linearity for naringin and naringenin are as follows: Y ¼ (4.1145 + 0.16414)X þ (0.0037 + 0.0006) (n ¼ 4); r 2 ¼ 0.9946 (naringin); Y ¼ (5.8775 + 0.2300)X – (0.0015 + 0.0003) (n ¼ 4); r 2 ¼ 0.9925 (naringenin). Data for the precision is presented in Table I. The intra-run and inter-run precision values of naringin ranged from 3.0 to 8.6% and 4.1 to 7.7%, respectively. For naringenin, the intra-run and inter-run precision values were 8.4 to 9.3% and 5.4 to 10.3%, respectively. Accuracy [relative error (RE)] values for naringin and naringenin were –8.7 to –1.8% and – 9.8 to –2.4%, respectively. The LLOQ was 0.5 ng/mL for both naringin and naringenin.

Table I Precision and Accuracy for the Determination of Naringin and Naringenin in Plasma (Four Runs with Five Replicates in Each Run) Added concentration (ng/mL) Naringin 1.5 10 160 Naringenin 1.5 10 160

Found concentration (ng/mL)

Intra-run RSD (%)

Inter-run RSD (%)

Accuracy: RE (%)

1.42 9.13 157

8.6 3.7 3.0

7.7 4.1 6.4

–5.2 –8.7 –1.8

1.43 9.03 156

9.3 8.7 8.4

6.1 5.4 10.3

–5.0 –9.8 –2.4

Table II Stability of Naringin and Naringenin in Plasma under Various Storage Conditions (n ¼ 5) Added concentration (ng/mL)

Naringin 1.5 10 160 Naringenin 1.5 10 160

Found concentration (ng/mL) Room temperature for 19 h

Three freeze-thaw stability cycles

Autosampler at room temperature for 72 h

4 months at –408C

1.37 + 0.07 9.53 + 0.31 170 + 5

1.38 + 0.05 8.84 + 0.38 165 + 13

1.35 + 0.11 9.25 + 0.24 157 + 13

1.36 + 0.06 8.93 + 0.41 167 + 2

1.44 + 0.03 9.19 + 0.22 159 + 12

1.47 + 0.14 8.91 + 0.25 165 + 12

1.35 + 0.06 8.83 + 0.27 144 + 9

1.44 + 0.08 8.67 + 0.16 164 + 17

Extraction recovery The recovery was calculated from the response ratio of QC samples to pure solutions in matrix extracts at the same concentration. The mean recovery values for naringin and naringenin were 41.6 and 82.5%, respectively. Matrix effect The matrix effect (ME) was examined by comparing the peak areas of the analytes and the IS between two different sets of plasmas. In Set 1, analytes were resolved in the reconstituted solutions of blank plasma samples, and the peak areas of analytes were defined as A. In Set 2, analytes were resolved in the mobile phase, and the peak areas of analytes were defined as B. ME was calculated by using the following formula: ME (%) ¼ A/B  100. The ME of the method was evaluated at three concentration levels of 1.5, 10 and 160 ng/mL and the IS concentration level of 300 ng/mL. Five samples were analyzed at each level of the analytes. The results obtained regarding the average MEs for naringin, naringenin and IS were 108.5, 100.3 and 106.0%, respectively. This indicated that the MEs were insignificant and well within measurement errors. Stability The stability of naringin and naringenin in plasma was studied under a variety of storage and handling conditions at low (1.5 ng/ mL), medium (10 ng/mL) and high (160 ng/mL) concentration levels. The short-term temperature stability was assessed by analyzing five aliquots each of low, medium and high concentration samples that were thawed at room temperature and kept at this temperature for 19 h. Freeze-thaw stability (–408C in plasma) was determined through three cycles. Five aliquots at each of the low, medium and high concentrations were stored at –408C for 24 h and thawed unassisted at room temperature. When completely thawed, the samples were refrozen for 24 h under the same conditions. The freeze-thaw cycles were repeated three times and the aliquots were analyzed on the third cycle. The long-term stability was determined by analyzing five aliquots of each of the three concentrations stored at –408C for 4 months. The results in Table II showed no significant degradation of naringin and naringenin under the tested conditions. Applications The method was applied to determine the plasma concentrations of naringin and naringenin following a single 250 mg dose of Qianggu capsules (expressed as the weight of raw materials, containing approximately 25 mg naringin) that were taken orally by

Table III Drug Levels in Plasma of Five Osteoporosis Patients after Oral Administration of 250 mg Qianggu Capsules Series

Time (h)

Naringin (ng/mL)

Naringenin (ng/mL)

1-1 1-2 1-3 2-1 2-2 2-3 3-1 3-2 3-3 4-1 4-2 4-3 5-1 5-2 5-3

1 2 3 1 2 3 1 2 3 1 2 3 1 2 3

0.748 1.97 0.678 1.73 1.80 0.674 1.26 1.62 0.624 1.27 1.72 1.13 0.842 1.13 0.666

37.6 31.2 31.0 61.1 43.4 35.4 51.6 60.4 45.4 42.5 47.4 46.6 33.6 22.2 30.9

osteoporosis patients. This study was approved by the Ethics Committee of Institute of Basic Research in Clinical Medicine China Academy of Chinese Medical Sciences (No. 2010N013). To avoid interference with drug absorption and metabolism, the consumption of citrus (especially grapefruit) and other TCMs (probably containing naringin) was prohibited. The results of drug concentration levels of five osteoporosis patients at several different times (such as 1, 2 and 3 h post-dose) are shown in Table III.

Discussion Thus far, only three LC–MS-MS methods (8 –10) have been published for the simultaneous determination of naringin and naringenin; the lowest reported LLOQ for naringin and naringenin was 5 ng/mL. The preceding data demonstrated that the developed LC–MS-MS method provides good linearity and has sufficient sensitivity, precision and accuracy. This method for the simultaneous determination of naringin and naringenin offers the highest sensitivity (0.5 ng/mL) compared to other methods described in the literature by using a simple liquid – liquid extraction procedure. The LLOQ reported in this study is tenfold lower than the earlier reported LLOQs for naringin and naringenin, respectively. The data in Table III show that the concentrations of naringin are much lower than naringenin in human plasma. As the neohesperidose sugar moiety of naringin is rapidly cleaved off the parent compound in the gastrointestinal tract and liver to leave the aglycone bioflavonoid naringenin, the glucuronic acid conjugates a majority of naringenin to form naringenin glucuronide in the liver (12, 13). Therefore, the results Development and Validation of a Sensitive Liquid Chromatography– Tandem 659

showed that the concentration of naringin is very low in plasma, and it was difficult to directly detect naringenin in plasma. Because the reference standard of naringenin glucuronide was unavailable in the market, samples with enzymatic hydrolysis were utilized to determine the concentration of naringenin by the cleavage action of b-glucuronidase.

Conclusion The results obtained in this study demonstrate that the LC–MS-MS method is suitable for the accurate quantification of naringin and naringenin in human plasma and shows high sensitivity, with an LLOQ of 0.5 ng/mL. No significant interferences or MEs caused by endogenous compounds were observed. The method is useful for screening biologically active components of herbal medicines at low levels in biological fluids and can play a very important role in investigating the action mechanisms of Qianggu capsules.

Acknowledgments This work was supported by the National Major Special Project on Invention and Production of New Drugs, Research on Key Techniques for Postmarketing Reevaluation of Chinese Medicine (2009ZX09502-030).

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