Simultaneous determination of rosuvastatin, naringin and naringenin in rat plasma by RRLC–MS/MS and its application to a pharmacokinetic drug interaction study

Simultaneous determination of rosuvastatin, naringin and naringenin in rat plasma by RRLC–MS/MS... Abstract A rapid resolution liquid chromatography tandem mass spectrometry method was developed and validated for simultaneous determination of rosuvastatin, naringin and naringenin in rat plasma. Chromatographic separation of analytes and internal standard (fluvastatin for rosuvastatin, while isoquercitrin for naringin and naringenin) was performed on Agilent Poroshell 120 EC-C18 column (3.0 × 50 mm, 2.7 μm) using gradient elution with a mobile phase of methanol and water, both with 0.1% formic acid (v/v). The detection was operated in multiple reaction monitoring mode to monitor the precursor-to-product ion transitions of m/z 579.1→270.8 for naringin, m/z 270.9→150.7 for naringenin, m/z 463.1→299.8 for isoquercitrin in negative ionization mode, and m/z 482.2→258.1 for rosuvastatin, m/z 412.1→224.1 for fluvastatin in positive ionization mode. Polarity switch (negative-positive-negative ionization mode) was performed in a total runtime of 5.0 min. The method was validated over a concentration range of 10–2,000 ng/mL for the above three analytes. The intra-day and inter-day precisions and accuracies of the quality control samples at low, medium and high concentration levels exhibited relative standard deviations <10% and the accuracy values ranged from −7.2% to 8.4%. The proposed method was successfully applied to the pharmacokinetic drug interaction study of rosuvastatin combined with naringin in rats. Introduction Rosuvastatin is a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor for the treatment of patients with primary hypercholesterolemia, mixed dyslipidemia and homozygous familial hypercholesterolemia (1). Previous studies had revealed that rosuvastatin is just slightly metabolized by cytochrome P450 (CYP), isoenzyme CYP2C9 (2) and primarily excreted via bile as unchanged drug (3, 4). Transporters, including breast cancer resistance protein (BCRP) (5) and organic anion transporting polypeptide 1B1 (OATP 1B1) (6), play pivotal roles in the absorption and excretion of rosuvastatin. Transport function modulation of BCRP, as well as OATP 1B1, can remarkably affect the cholesterol-lowering efficacy and exposure-dependent toxicity of rosuvastatin therapy (7, 8). Dietary flavonoids, with their antioxidant properties, are regarded as practical supplements to optimize health and reduce the risk of chronic diseases for consumers, including patients with dyslipidemia (9, 10). Naringin, a widespread flavanone glycoside, has been shown to possess extensive bioactivities on health benefits, including anti-inflammatory (11), antioxidant (12), neuroprotection (13) and ameliorating atherogenic dyslipidemia (14). What is more, our preliminary studies revealed that naringin, as well as its active metabolite naringenin, can effectively relieve cough (15) and reduce sputum (16). And now naringin has been approved by China Food and Drug Administration for clinical trials (No. 2013L01586). Several studies have shown that naringin and naringenin could down-regulate BCRP-mediated transport function (17–19). Meanwhile, naringin was considered as an OATP 1B1 inhibitor (20, 21). As hypercholesterolemia (22), cough (23) and inflammation (24, 25) are common ailments among old population, naringin was very likely to be used in clinic combined with rosuvastatin to treat these diseases (26). When used in clinic combined with naringin, the disposition of rosuvastatin may change due to the modulation of BCRP and OATP 1B1. These changes may cause severe rhabdomyolysis which is life-threatening (27). For avoiding or minimizing the transporter-based drug interaction-induced adverse events in clinical application, the identification and quantification of in vivo pharmacokinetics interactions associated with the drug combination of naringin and rosuvastatin in preclinical phase is meaningful. To date, several methods for the quantification of rosuvastatin in plasma were reported (28, 29), as well as the determination of naringin and its metabolite naringenin (30, 31). However, a developed LC–MS/MS-based method for simultaneous determination of rosuvastatin, naringin and naringenin in plasma has not been reported. In the present study, a rapid resolution liquid chromatography tandem mass spectrometry (RRLC–MS/MS) method was established to determine the concentrations of rosuvastatin, naringin and naringenin in rat plasma. The analytical method was validated and successfully applied to evaluate the pharmacokinetic interaction between rosuvastatin and naringin in rats. Materials and methods Chemicals and reagents Rosuvastatin, naringin and fluvastatin reference standards were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Naringenin, isoquercitrin reference standards and β-glucuronidase/sulfatase (Type H-1) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Liquid chromatography–mass spectrometry (LC–MS) grade methanol and formic acid were purchased from Fisher Scientific (Pittsburgh, PA, USA). High-performance liquid chromatography (HPLC) grade methanol and ethyl acetate were purchased from Honeywell B&J (New Jersey, USA). Water was prepared using a Milli-Q purification system (Millipore, Bedford, MA, USA). Rosuvastatin Calcium Tablets (Crestor) were purchased from AstraZeneca Crop. Naringin powder, for oral administration, was extracted from Huajuhong (Citrus grandis “Tomentosa”) with a purity of 98.8%, which was determined by HPLC method with external standard (32). Preparation of calibration standards and quality control samples The stock solutions of rosuvastatin, naringin, naringenin, fluvastatin and isoquercitrin were prepared in 50% methanol. All stock solutions were prepared at 1 mg/mL concentration. Two separate stocks of each analyte were prepared and used for preparation of calibration standards and quality controls (QC). The stock solutions were stored at 4°C and brought to room temperature before use. Working standard and internal standard (IS) spiking solutions were prepared from stock solutions by diluting with 50% methanol. An aliquot of 10 μL working standard solution was added to a 1.5-mL polypropylene tube and evaporated to dryness with N2 at 25°C using a EYELA MG-2200 drying system (Tokyo, Japan). Then 50 μL rat blank plasma was added to the tube and vortex-mixed, to yield calibration standards of 10, 20, 50, 150, 500, 1,000, 1,600 and 2,000 ng/mL. QC samples were prepared at final concentrations of 30, 200 and 1,500 ng/mL in the same manner as the calibration standards. The internal standard spiking solution, with the concentration of 16,800 ng/mL for fluvastatin and 5,000 ng/mL for isoquercitrin, was prepared by diluting the fluvastatin and isoquercitrin stock solution together with 50% methanol. The calibration standards, QC samples and internal standard spiking solution were prepared accompanying each analytical batch. Sample preparation A 50-μL aliquot of plasma was mixed with 10 μL of internal standard spiking solution followed by 1,000 μL ethyl acetate. Then samples were vortex-mixed for 3 min and centrifuged at 10,000 × g for 10 min at 4°C. A 900-μL aliquot of supernatant was transferred into a fresh 1.5-mL polypropylene tube and then evaporated to dryness under a gentle stream of N2 at 37°C. Subsequently, an aliquot of 100-μL mobile phase was added to the tube to dissolve the residue. Samples were ultrasonic extracted for 3 min, vortex-mixed for 3 min and centrifuged at 15,000 × g for 30 min at 25°C. Finally, an aliquot of 10-μL supernatant was injected into the LC–MS/MS system for analysis. The hydrolysis of naringin, which forms its metabolite naringenin, mediated by lactase-phlorizin hydrolase and human intestinal microflora is recognized as the first and determinant step in the absorption of naringin (33). Subsequently, naringenin extensively combines with glucuronide or sulfate and yields its predominant existing form in circulatory system (34–36). To quantitate total naringenin including free and its glucuronide/sulfate conjugates, partial plasma samples were treated with β-glucuronidase/sulfatase as in previous reports (32, 37). As to these samples, 50 μL plasma was mixed with 10 μL β-glucuronidase/sulfatase solution (dissolved in 0.2 mmol/L acetic acid buffer, pH = 5.0, 10 Unit/μL) and incubated at 37°C for 2 h. After that, 10 μL internal standard spiking solution was added to the sample, followed by 1,000 μL ethyl acetate. Other process procedures were the same as above-mentioned. LC–MS/MS condition The LC–MS/MS system consisted of an Agilent 1200 RRLC and an Agilent 6410 triple quadrupole mass spectrometer with an electrospray ionization source (ESI; Agilent Technology, Santa Clara, CA, USA). Chromatographic separation was carried out on an Agilent Poroshell 120 EC-C18 column (3.0 × 50 mm, 2.7 μm) tandem pre-column of Welch Analytical Guard Cartridges Ultimate XB-C18 (4.6 × 10 mm, 5 μm). The column temperature was maintained at 40°C using a thermostatically controlled column oven. The mobile phase was composed of solvent A (0.1% formic acid, v/v) and B (methanol with 0.1% formic acid, v/v). The gradient elution profile consisted of an initial 45:55 ratio of A:B that was maintained for 2.0 min after injection, followed by a linear gradient to 0:100 of A:B over a 0.1-min period (completed at 2.1 min). This solvent composition was maintained for 1.8 min of isocratic hold (until 3.9 min), followed by a linear gradient to 45:55 of A:B over a 0.1-min period (completed at 4.0 min), and subsequently returned to initial conditions of the 45:55 ratio of A:B for a period of 1.0-min, for a total runtime of 5.0-min. The flow rate was kept at 0.3 mL/min. The HPLC effluent was introduced directly to the mass spectrometer without splitting. To obtain higher response of the three analytes, polarity switch (negative-positive-negative ionization mode) was performed in each analytical run. The electrode polarity of the mass spectrometer was switched at 2.9 min and 4.3 min. MS detector was operated in multiple reaction monitoring (MRM) mode at unit mass resolution with a dwell-time of 200-ms for all test compounds. The optimized mass spectrometric parameters, MRM transitions, fragmentors, collision energies are shown in Table I. The ion source parameters were capillary 4,000 V, gas flow 10 L/min, nebulizer 25 psi, gas temperature 350°C for maximum sensitivity. Other MS parameters were adopted from the recommended values for the instrument. Table I. Multiple reaction monitoring transitions and the optimum LC–MS/MS conditions Period  Compound  Q1  Q3  Fragmentor (V)  Collision energy (V)  1  0–1.0 min, To Waste, ESI−          2  1.0–2.9 min, To MS, ESI−          Naringin  579.1  270.8  225  33  Naringenin  270.9  150.7  100  12  Isoquercitrin  463.1  299.8  128  24  3  2.9–4.3 min, To MS, ESI+          Rosuvastatin  482.2  258.1  170  38  Fluvastatin  412.1  224.1  140  33  4  4.3–5.0 min, To Waste, ESI−          Period  Compound  Q1  Q3  Fragmentor (V)  Collision energy (V)  1  0–1.0 min, To Waste, ESI−          2  1.0–2.9 min, To MS, ESI−          Naringin  579.1  270.8  225  33  Naringenin  270.9  150.7  100  12  Isoquercitrin  463.1  299.8  128  24  3  2.9–4.3 min, To MS, ESI+          Rosuvastatin  482.2  258.1  170  38  Fluvastatin  412.1  224.1  140  33  4  4.3–5.0 min, To Waste, ESI−          Table I. Multiple reaction monitoring transitions and the optimum LC–MS/MS conditions Period  Compound  Q1  Q3  Fragmentor (V)  Collision energy (V)  1  0–1.0 min, To Waste, ESI−          2  1.0–2.9 min, To MS, ESI−          Naringin  579.1  270.8  225  33  Naringenin  270.9  150.7  100  12  Isoquercitrin  463.1  299.8  128  24  3  2.9–4.3 min, To MS, ESI+          Rosuvastatin  482.2  258.1  170  38  Fluvastatin  412.1  224.1  140  33  4  4.3–5.0 min, To Waste, ESI−          Period  Compound  Q1  Q3  Fragmentor (V)  Collision energy (V)  1  0–1.0 min, To Waste, ESI−          2  1.0–2.9 min, To MS, ESI−          Naringin  579.1  270.8  225  33  Naringenin  270.9  150.7  100  12  Isoquercitrin  463.1  299.8  128  24  3  2.9–4.3 min, To MS, ESI+          Rosuvastatin  482.2  258.1  170  38  Fluvastatin  412.1  224.1  140  33  4  4.3–5.0 min, To Waste, ESI−          Method validation This method was fully validated with reference to the Guidance for Bioanalytical Method Validation issued by Chinese Pharmacopoeia Commission in 2015. The specificity of the method was evaluated by comparing chromatograms of the standard-spiked plasma samples with the blank plasma from six different sources. The specificity was further confirmed in actual pharmacokinetic study by examining the pre-dosing plasma samples. A calibration curve was constructed from a blank plasma sample, a zero-concentration plasma sample prepared with internal standard and eight concentration levels of samples covering the range of 10–2,000 ng/mL, including lower limit of quantification (LLOQ). Each concentration level was prepared in duplicate. Calibration curves were constructed and fitted by linear least-squares regression analysis to plot the peak area ratio of analyte relative to the internal standard against the analyte concentrations with a weighed of 1/x2 (x= concentration). The acceptance criterion for each measured standard concentration was 15% deviation from the nominal value except LLOQ, which was not more than 20%. Precision and accuracy were evaluated by repeated analyses of QC samples (n = 5) at concentrations of 30, 200 and 1,500 ng/mL on three separate days. Intra-day and inter-day precisions were expressed by the relative standard deviation (RSD, %), while the accuracy was determined by calculating the percentage deviation of the calculated concentrations from the nominal concentrations and expressed as relative error (RE, %). Recoveries of rosuvastatin, naringin and naringenin were determined by comparing the peak area of extracted three levels QC samples to that of the analytes spiked to the blank sample extracts at the corresponding concentration. The recovery of the internal standard was determined in the same way at the working concentration. The matrix effect was measured by comparing the peak area in the analyte-spiked post-extracted sample with that acquired using a neat solution. Blank plasma from six different sources were used to evaluate the matrix effect at low (30 ng/mL) and high (1,500 ng/mL) QC concentration level. Stability studies of analyte were assessed by analyzing three replicates of QC samples at concentrations of 30 and 1,500 ng/mL under the following conditions: long-term stability at −70°C for 90 days, short-term stability at 25°C for 5 h, in processed samples in autosampler vials (25°C) for 24 h, and after four freeze-thaw cycles (−70–25°C), and after incubation with β-glucuronidase/sulfatase at 37°C for 2 h. Pharmacokinetic drug interaction study Male Sprague-Dawley rats (180–220 g) were obtained from Guangdong Medical Laboratory Animal Center (Guangzhou, China) and used to study the pharmacokinetic drug interaction of rosuvastatin and naringin. The animals were acclimated for 1 week prior to the initiation of dosing with feed and water available ad libitum. Environmental conditions were maintained at 20–25°C, 55 ± 15% relative humidity, 12 h light–dark cycles and 12 air change cycles/h. All experimental procedures and protocols were reviewed and approved by the Animal Ethics Committee of the School of Life Sciences in Sun Yat-sen University. Diet was prohibited for 12 h before the experiment but water was freely available. Rats were randomly divided into three groups (n = 6/group). Rats in Group 1 were orally administered rosuvastatin (50 mg/kg) plus drinking water, rats in Group 2 were given naringin (42 mg/kg) plus drinking water and rats in Group 3 were given rosuvastatin (50 mg/kg) plus naringin (42 mg/kg). Blood samples (0.3 mL) were collected from post-orbital venous plexus into heparinized 1.5 mL polythene tubes at 0.5, 1, 1.5, 3, 5, 8, 10, 12 and 24 h after oral administration, respectively. The samples were immediately centrifuged at 5,000 rpm for 10 min. The plasma obtained was stored at −70°C until analysis. Blank plasma was obtained from the rat without any drug administration and was used for the method development and validation. Pharmacokinetic analysis The DAS (Drug and statistics) software (Version 3.0, Shanghai University of Traditional Chinese Medicine, China) was used with a non-compartmental statistical model to determine the pharmacokinetic parameters of the rat plasma samples. The data are expressed as mean ± SD and were evaluated by one-way analysis of variance and Student’s t-tests in SPSS 18.0 (SPSS Inc., Chicago, USA). P-values <0.05 or 0.01 were considered statistically significant. Results Method development and instrumental optimization Both of liquid–liquid extraction and protein precipitation were compared in this study to obtain the optimum extraction method. As a result, liquid–liquid extraction of adding 1,000 μL ethyl acetate to 50 μL plasma showed a high extraction recovery. For the sake of obtaining chromatograms with good separation and strong total ion current, different solutions of acetonitrile in water and methanol in water as mobile phases, with or without 0.1% formic acid, for binary isocratic and gradient elution, were investigated to optimize the chromatographic conditions. Methanol–water both with 0.1% formic acid on the optimized gradient mode exhibited a good separation and abundant signal response. As to the MS conditions, ionization mode of the three analytes was optimized. The mass spectrometer was conducted in the negative ionization mode for the detection of naringin and naringenin, but in the positive ionization mode for the detection of rosuvastatin due to the poor sensitivity of rosuvastatin in negative ionization mode. With the chromatography conditions described above, naringin, naringenin and isoquercitrin were eluted out at 1.2 min, 2.3 min and 1.3 min, respectively. Rosuvastatin and fluvastatin were eluted out at 3.1 min and 3.9 min, respectively. In order to determine rosuvastatin, naringin and naringenin in the same analytical run, polarity switch was performed. The following predominant transitions were selected for quantification: m/z 482.2→258.1 for rosuvastatin, m/z 579.1→270.8 for naringin and m/z 270.9→150.7 for naringenin, respectively. The MS fragment details of these compounds are shown in Figure 1. And other electrospray source and the mass spectrometer parameters were optimized and listed in Table I. Figure 1. View largeDownload slide Product ion spectra of rosuvastatin (A), naringin (B) and naringenin (C). Figure 1. View largeDownload slide Product ion spectra of rosuvastatin (A), naringin (B) and naringenin (C). Method validation Specificity Typical MRM chromatograms of rosuvastatin, naringin and naringenin are presented in Figure 2. Sharp and fine peaks were obtained for naringin, naringenin, isoquercitrin and rosuvastatin, fluvastatin at retention times of 1.2 min, 2.3 min, 1.3 min, 3.1 min and 3.9 min, respectively. No interference was observed, indicating that the developed method was specific for the analytes in plasma samples. Figure 2. View largeDownload slide Representative chromatograms for rosuvastatin, fluvastatin, and naringin, naringenin, isoquercitrin in rat plasma samples: (I) a blank plasma sample; (II) a blank plasma sample spiked with rosuvastatin, naringin, naringenin and ISs at LLOQ, and (III) a rat plasma sample 3 h after a single oral dose of rosuvastatin 50 mg/kg and naringin 42 mg/kg. Figure 2. View largeDownload slide Representative chromatograms for rosuvastatin, fluvastatin, and naringin, naringenin, isoquercitrin in rat plasma samples: (I) a blank plasma sample; (II) a blank plasma sample spiked with rosuvastatin, naringin, naringenin and ISs at LLOQ, and (III) a rat plasma sample 3 h after a single oral dose of rosuvastatin 50 mg/kg and naringin 42 mg/kg. Linearity and LLOQ An acceptable linearity was achieved in the range of 10–2,000 ng/mL for rosuvastatin, naringin and naringenin with correlation coefficients (r) > 0.99 in all three batches. The intra-day assay yielded acceptable precisions of 7.2% RSD for rosuvastatin, 1.8% RSD for naringin and 2.5% RSD for naringenin and accuracies of 8.4% RE for rosuvastatin, −7.2% RE for naringin and −3.2% RE for naringenin (Table II). Table II. Intra- and inter-day accuracies and precisions of rosuvastatin, naringin and naringenin in rat plasma Compound  Concentration (ng/mL)  Intra-day (n = 5)  Inter-day (n = 5*3)  Recovery (%) (n = 5)  RSD%  RE%  RSD%  RE%  Rosuvastatin  10 (LLOQ)  7.2  8.4  16.6  1.7  –  30  5.6  2.1  6.5  −4.1  69.7 ± 1.2  200  9.4  −0.1  6.8  −0.9  63.6 ± 1.1  1,500  8.1  −0.9  7.0  −6.2  71.6 ± 1.3  Naringin  10 (LLOQ)  1.8  −7.2  6.2  2.5    30  4.5  −1.2  4.3  −2.7  60.5 ± 2.2  200  1.3  −0.5  1.7  −1.4  58.6 ± 0.9  1,500  2.1  1.2  2.6  0.1  56.3 ± 2.9  Naringenin  10 (LLOQ)  2.5  −3.2  3.0  3.2    30  1.7  3  2.6  2.3  72.4 ± 1.4  200  2.4  4.5  2.4  4.0  70.2 ± 0.6  1,500  1.4  0.9  2.4  −0.1  71.1 ± 1.9  Compound  Concentration (ng/mL)  Intra-day (n = 5)  Inter-day (n = 5*3)  Recovery (%) (n = 5)  RSD%  RE%  RSD%  RE%  Rosuvastatin  10 (LLOQ)  7.2  8.4  16.6  1.7  –  30  5.6  2.1  6.5  −4.1  69.7 ± 1.2  200  9.4  −0.1  6.8  −0.9  63.6 ± 1.1  1,500  8.1  −0.9  7.0  −6.2  71.6 ± 1.3  Naringin  10 (LLOQ)  1.8  −7.2  6.2  2.5    30  4.5  −1.2  4.3  −2.7  60.5 ± 2.2  200  1.3  −0.5  1.7  −1.4  58.6 ± 0.9  1,500  2.1  1.2  2.6  0.1  56.3 ± 2.9  Naringenin  10 (LLOQ)  2.5  −3.2  3.0  3.2    30  1.7  3  2.6  2.3  72.4 ± 1.4  200  2.4  4.5  2.4  4.0  70.2 ± 0.6  1,500  1.4  0.9  2.4  −0.1  71.1 ± 1.9  Table II. Intra- and inter-day accuracies and precisions of rosuvastatin, naringin and naringenin in rat plasma Compound  Concentration (ng/mL)  Intra-day (n = 5)  Inter-day (n = 5*3)  Recovery (%) (n = 5)  RSD%  RE%  RSD%  RE%  Rosuvastatin  10 (LLOQ)  7.2  8.4  16.6  1.7  –  30  5.6  2.1  6.5  −4.1  69.7 ± 1.2  200  9.4  −0.1  6.8  −0.9  63.6 ± 1.1  1,500  8.1  −0.9  7.0  −6.2  71.6 ± 1.3  Naringin  10 (LLOQ)  1.8  −7.2  6.2  2.5    30  4.5  −1.2  4.3  −2.7  60.5 ± 2.2  200  1.3  −0.5  1.7  −1.4  58.6 ± 0.9  1,500  2.1  1.2  2.6  0.1  56.3 ± 2.9  Naringenin  10 (LLOQ)  2.5  −3.2  3.0  3.2    30  1.7  3  2.6  2.3  72.4 ± 1.4  200  2.4  4.5  2.4  4.0  70.2 ± 0.6  1,500  1.4  0.9  2.4  −0.1  71.1 ± 1.9  Compound  Concentration (ng/mL)  Intra-day (n = 5)  Inter-day (n = 5*3)  Recovery (%) (n = 5)  RSD%  RE%  RSD%  RE%  Rosuvastatin  10 (LLOQ)  7.2  8.4  16.6  1.7  –  30  5.6  2.1  6.5  −4.1  69.7 ± 1.2  200  9.4  −0.1  6.8  −0.9  63.6 ± 1.1  1,500  8.1  −0.9  7.0  −6.2  71.6 ± 1.3  Naringin  10 (LLOQ)  1.8  −7.2  6.2  2.5    30  4.5  −1.2  4.3  −2.7  60.5 ± 2.2  200  1.3  −0.5  1.7  −1.4  58.6 ± 0.9  1,500  2.1  1.2  2.6  0.1  56.3 ± 2.9  Naringenin  10 (LLOQ)  2.5  −3.2  3.0  3.2    30  1.7  3  2.6  2.3  72.4 ± 1.4  200  2.4  4.5  2.4  4.0  70.2 ± 0.6  1,500  1.4  0.9  2.4  −0.1  71.1 ± 1.9  Precision and accuracy The intra-day and inter-day precisions and accuracies for rosuvastatin, naringin and naringenin were showed in Table II. The intra-day and inter-day precisions (RSD) were <10% with accuracies (RE) in the range of −7.2–8.4% for all analytes. The results indicated that the present method was reliable and reproducible for the simultaneous quantitative analysis of rosuvastatin, naringin and naringenin in rat plasma samples. Recovery Mean extraction recoveries of rosuvastatin, naringin and naringenin measured at three different concentrations were 63.6–71.6%, 56.3–60.5% and 70.2–72.4%, respectively (Table II), indicating consistent and reproducible recoveries of analytes. Mean extraction recovery of the fluvastatin and isoquercitrin were 58.1% and 62.1%, correspondingly. Matrix effect The matrix effects of three analytes are presented in Table III. Mean values of rosuvastatin, naringin and naringenin at low (30ng/mL) QC concentration level was 109.4%, 90.9% and 92.4%, respectively. While those at high (1500 ng/mL) QC concentration level 87.1%, 83.1% and 92.8%, respectively. All of these are within the acceptable range of 20%. And RSD for matrix effects of each analyte at different concentration level are < 15%. These data indicated that relative matrix effect for the analytes in rat plasma was negligible under the current conditions. Table III. Matrix effects for rosuvastatin, naringin and naringenin (mean ± SD, n = 5) No.a  Rosuvastatin concentration  Naringin concentration  Naringenin concentration  (ng/mL)  (ng/mL)  (ng/mL)    30  1,500  30  1,500  30  1,500  1  108.1 ± 6.3  86.5 ± 2.1  95.9 ± 3.2  83.7 ± 2.2  93.1 ± 2.5  91.5 ± 0.4  2  110.1 ± 3.7  87.6 ± 1.3  89.5 ± 4.3  82.3 ± 1.3  92.9 ± 1.8  93.2 ± 1.4  3  108.1 ± 1.4  86.9 ± 1.9  86.8 ± 1.9  83.1 ± 4.4  91.7 ± 2.3  92.1 ± 1.6  4  108.3 ± 2.5  86.0 ± 0.7  89.6 ± 1.7  82.6 ± 2.7  92.6 ± 2.9  93.1 ± 1.1  5  111.6 ± 1.1  87.6 ± 1.8  96.6 ± 3.3  81.2 ± 1.1  92.1 ± 2.4  93.3 ± 1.6  6  110.4 ± 5.5  88.0 ± 1.2  86.8 ± 0.4  85.8 ± 4.6  91.8 ± 0.9  93.6 ± 1.9  Mean  109.4 ± 1.5  87.1 ± 0.8  90.9 ± 4.4  83.1 ± 1.5  92.4 ± 0.6  92.8 ± 0.8  RSD%  1.3  0.9  4.8  1.9  0.6  0.9  No.a  Rosuvastatin concentration  Naringin concentration  Naringenin concentration  (ng/mL)  (ng/mL)  (ng/mL)    30  1,500  30  1,500  30  1,500  1  108.1 ± 6.3  86.5 ± 2.1  95.9 ± 3.2  83.7 ± 2.2  93.1 ± 2.5  91.5 ± 0.4  2  110.1 ± 3.7  87.6 ± 1.3  89.5 ± 4.3  82.3 ± 1.3  92.9 ± 1.8  93.2 ± 1.4  3  108.1 ± 1.4  86.9 ± 1.9  86.8 ± 1.9  83.1 ± 4.4  91.7 ± 2.3  92.1 ± 1.6  4  108.3 ± 2.5  86.0 ± 0.7  89.6 ± 1.7  82.6 ± 2.7  92.6 ± 2.9  93.1 ± 1.1  5  111.6 ± 1.1  87.6 ± 1.8  96.6 ± 3.3  81.2 ± 1.1  92.1 ± 2.4  93.3 ± 1.6  6  110.4 ± 5.5  88.0 ± 1.2  86.8 ± 0.4  85.8 ± 4.6  91.8 ± 0.9  93.6 ± 1.9  Mean  109.4 ± 1.5  87.1 ± 0.8  90.9 ± 4.4  83.1 ± 1.5  92.4 ± 0.6  92.8 ± 0.8  RSD%  1.3  0.9  4.8  1.9  0.6  0.9  aNo. represent the six different sources of blank rat plasma. Table III. Matrix effects for rosuvastatin, naringin and naringenin (mean ± SD, n = 5) No.a  Rosuvastatin concentration  Naringin concentration  Naringenin concentration  (ng/mL)  (ng/mL)  (ng/mL)    30  1,500  30  1,500  30  1,500  1  108.1 ± 6.3  86.5 ± 2.1  95.9 ± 3.2  83.7 ± 2.2  93.1 ± 2.5  91.5 ± 0.4  2  110.1 ± 3.7  87.6 ± 1.3  89.5 ± 4.3  82.3 ± 1.3  92.9 ± 1.8  93.2 ± 1.4  3  108.1 ± 1.4  86.9 ± 1.9  86.8 ± 1.9  83.1 ± 4.4  91.7 ± 2.3  92.1 ± 1.6  4  108.3 ± 2.5  86.0 ± 0.7  89.6 ± 1.7  82.6 ± 2.7  92.6 ± 2.9  93.1 ± 1.1  5  111.6 ± 1.1  87.6 ± 1.8  96.6 ± 3.3  81.2 ± 1.1  92.1 ± 2.4  93.3 ± 1.6  6  110.4 ± 5.5  88.0 ± 1.2  86.8 ± 0.4  85.8 ± 4.6  91.8 ± 0.9  93.6 ± 1.9  Mean  109.4 ± 1.5  87.1 ± 0.8  90.9 ± 4.4  83.1 ± 1.5  92.4 ± 0.6  92.8 ± 0.8  RSD%  1.3  0.9  4.8  1.9  0.6  0.9  No.a  Rosuvastatin concentration  Naringin concentration  Naringenin concentration  (ng/mL)  (ng/mL)  (ng/mL)    30  1,500  30  1,500  30  1,500  1  108.1 ± 6.3  86.5 ± 2.1  95.9 ± 3.2  83.7 ± 2.2  93.1 ± 2.5  91.5 ± 0.4  2  110.1 ± 3.7  87.6 ± 1.3  89.5 ± 4.3  82.3 ± 1.3  92.9 ± 1.8  93.2 ± 1.4  3  108.1 ± 1.4  86.9 ± 1.9  86.8 ± 1.9  83.1 ± 4.4  91.7 ± 2.3  92.1 ± 1.6  4  108.3 ± 2.5  86.0 ± 0.7  89.6 ± 1.7  82.6 ± 2.7  92.6 ± 2.9  93.1 ± 1.1  5  111.6 ± 1.1  87.6 ± 1.8  96.6 ± 3.3  81.2 ± 1.1  92.1 ± 2.4  93.3 ± 1.6  6  110.4 ± 5.5  88.0 ± 1.2  86.8 ± 0.4  85.8 ± 4.6  91.8 ± 0.9  93.6 ± 1.9  Mean  109.4 ± 1.5  87.1 ± 0.8  90.9 ± 4.4  83.1 ± 1.5  92.4 ± 0.6  92.8 ± 0.8  RSD%  1.3  0.9  4.8  1.9  0.6  0.9  aNo. represent the six different sources of blank rat plasma. Stability All established stability for the analytes are summarized in Table IV. The data showed that the stability of QC samples after long-term storage at −70°C (90 days), at 25°C for 5 h, in processed samples in autosampler vials (25°C) for 24 h and after four freeze-thaw cycles (−70–25°C) was acceptable. No detectable degradation was observed after a 2-h incubation with β-glucuronidase/sulfatase at 37°C for naringin and naringenin, while rosuvastatin showed partial degradation. Table IV. Stability of rosuvastatin, naringin and naringenin under various conditions (mean ± SD, n = 3) Compound  Conc. (ng/mL)  Long-term (−70°C, 90 days)  Short-term (25°C, 5 h)  Freeze-thaw (four cycles)  Incubation (37°C, 2 h)  Post-preparative (25°C, 24 h)  Rosuvastatin  30  104.8 ± 7.3  105.8 ± 1.3  112.1 ± 9.9  84.5 ± 5.0  107.8 ± 1.6  1,500  98.6 ± 3.1  101.8 ± 5.0  107.4 ± 5.6  80.7 ± 2.7  107.3 ± 2.8  Naringin  30  103.0 ± 1.7  102.4 ± 4.1  97.6 ± 4.0  94.1 ± 2.7  102.0 ± 2.3  1,500  104.4 ± 1.1  102.2 ± 1.5  99.4 ± 0.9  98.3 ± 1.1  103.6 ± 1.0  Naringenin  30  106.7 ± 2.0  104.6 ± 1.6  101.9 ± 2.5  100.2 ± 1.2  105.3 ± 1.9  1,500  102.3 ± 1.6  101.9 ± 0.8  99.7 ± 3.0  95.9 ± 2.3  103.2 ± 2.3  Compound  Conc. (ng/mL)  Long-term (−70°C, 90 days)  Short-term (25°C, 5 h)  Freeze-thaw (four cycles)  Incubation (37°C, 2 h)  Post-preparative (25°C, 24 h)  Rosuvastatin  30  104.8 ± 7.3  105.8 ± 1.3  112.1 ± 9.9  84.5 ± 5.0  107.8 ± 1.6  1,500  98.6 ± 3.1  101.8 ± 5.0  107.4 ± 5.6  80.7 ± 2.7  107.3 ± 2.8  Naringin  30  103.0 ± 1.7  102.4 ± 4.1  97.6 ± 4.0  94.1 ± 2.7  102.0 ± 2.3  1,500  104.4 ± 1.1  102.2 ± 1.5  99.4 ± 0.9  98.3 ± 1.1  103.6 ± 1.0  Naringenin  30  106.7 ± 2.0  104.6 ± 1.6  101.9 ± 2.5  100.2 ± 1.2  105.3 ± 1.9  1,500  102.3 ± 1.6  101.9 ± 0.8  99.7 ± 3.0  95.9 ± 2.3  103.2 ± 2.3  Table IV. Stability of rosuvastatin, naringin and naringenin under various conditions (mean ± SD, n = 3) Compound  Conc. (ng/mL)  Long-term (−70°C, 90 days)  Short-term (25°C, 5 h)  Freeze-thaw (four cycles)  Incubation (37°C, 2 h)  Post-preparative (25°C, 24 h)  Rosuvastatin  30  104.8 ± 7.3  105.8 ± 1.3  112.1 ± 9.9  84.5 ± 5.0  107.8 ± 1.6  1,500  98.6 ± 3.1  101.8 ± 5.0  107.4 ± 5.6  80.7 ± 2.7  107.3 ± 2.8  Naringin  30  103.0 ± 1.7  102.4 ± 4.1  97.6 ± 4.0  94.1 ± 2.7  102.0 ± 2.3  1,500  104.4 ± 1.1  102.2 ± 1.5  99.4 ± 0.9  98.3 ± 1.1  103.6 ± 1.0  Naringenin  30  106.7 ± 2.0  104.6 ± 1.6  101.9 ± 2.5  100.2 ± 1.2  105.3 ± 1.9  1,500  102.3 ± 1.6  101.9 ± 0.8  99.7 ± 3.0  95.9 ± 2.3  103.2 ± 2.3  Compound  Conc. (ng/mL)  Long-term (−70°C, 90 days)  Short-term (25°C, 5 h)  Freeze-thaw (four cycles)  Incubation (37°C, 2 h)  Post-preparative (25°C, 24 h)  Rosuvastatin  30  104.8 ± 7.3  105.8 ± 1.3  112.1 ± 9.9  84.5 ± 5.0  107.8 ± 1.6  1,500  98.6 ± 3.1  101.8 ± 5.0  107.4 ± 5.6  80.7 ± 2.7  107.3 ± 2.8  Naringin  30  103.0 ± 1.7  102.4 ± 4.1  97.6 ± 4.0  94.1 ± 2.7  102.0 ± 2.3  1,500  104.4 ± 1.1  102.2 ± 1.5  99.4 ± 0.9  98.3 ± 1.1  103.6 ± 1.0  Naringenin  30  106.7 ± 2.0  104.6 ± 1.6  101.9 ± 2.5  100.2 ± 1.2  105.3 ± 1.9  1,500  102.3 ± 1.6  101.9 ± 0.8  99.7 ± 3.0  95.9 ± 2.3  103.2 ± 2.3  Pharmacokinetic drug interaction study The developed method was successfully applied to pharmacokinetic drug interaction studies between rosuvastatin and naringin in rats. Naringin was just measured in few plasma samples while naringenin was barely detected without incubation. For the purpose of obtaining a full plasma concentration–time curve, total naringin was used to represent the pharmacokinetic of naringin. Together with the measured naringin, the concentrations of naringenin, which determined in plasma sample with incubation, were converted into equal-molar naringin to make up total naringin. The mean plasma concentration–time profiles of rosuvastatin and total naringin with and without (control) co-administration of the other drug are presented in Figure 3, and the main relevant pharmacokinetic parameters from non-compartment model analysis are listed in Table V. After co-administration of rosuvastatin and naringin, the pharmacokinetic parameters of both rosuvastatin and total naringin showed no significant difference compared with corresponding control group, suggesting that the concurrent use of rosuvastatin and naringin basically would not cause pharmacokinetic drug interaction between above two drugs. Figure 3. View largeDownload slide Mean plasma concentration profiles of rosuvastatin (A) and total naringin (B) in rat plasma with and without (control) co-administration of the other (mean ± SD, n = 6). Figure 3. View largeDownload slide Mean plasma concentration profiles of rosuvastatin (A) and total naringin (B) in rat plasma with and without (control) co-administration of the other (mean ± SD, n = 6). Table V. Pharmacokinetic parameters of rosuvastatin and total naringin with and without (control) co-administration of the other drug in rats (mean ± SD, n = 6) Pharmacokinetic parametersa  Rosuvastatin  Total naringin  (—)  (+Naringin)  (—)  (+Rosuvastatin)  AUC(0−t) (μg · L−1 · h−1)  1385.1 ± 838.1  1068.9 ± 319.3  1835.9 ± 503.6  1165.8 ± 744.0  CL (L · h−1 · kg−1)  35.2 ± 21.3  36.2 ± 15.5  23.4 ± 6.8  29.2 ± 17.1  Cmax (μg · L−1)  164.2 ± 84.4  208.2 ± 199.1  456.0 ± 160.9  325.3 ± 314.1  T1/2 (h)  16.2 ± 7.3  13.8 ± 7.4  1.8 ± 0.6  10.2 ± 16.8  Tmax (h)  0.6 ± 0.2  0.6 ± 0.2  4.5 ± 2.0  4.2 ± 2.3  Pharmacokinetic parametersa  Rosuvastatin  Total naringin  (—)  (+Naringin)  (—)  (+Rosuvastatin)  AUC(0−t) (μg · L−1 · h−1)  1385.1 ± 838.1  1068.9 ± 319.3  1835.9 ± 503.6  1165.8 ± 744.0  CL (L · h−1 · kg−1)  35.2 ± 21.3  36.2 ± 15.5  23.4 ± 6.8  29.2 ± 17.1  Cmax (μg · L−1)  164.2 ± 84.4  208.2 ± 199.1  456.0 ± 160.9  325.3 ± 314.1  T1/2 (h)  16.2 ± 7.3  13.8 ± 7.4  1.8 ± 0.6  10.2 ± 16.8  Tmax (h)  0.6 ± 0.2  0.6 ± 0.2  4.5 ± 2.0  4.2 ± 2.3  aAUC, area under the plasma concentration–time curve; CL, apparent plasma clearance; Cmax, maximum plasma concentration; T1/2, elimination half-life; Tmax, time to Cmax. Table V. Pharmacokinetic parameters of rosuvastatin and total naringin with and without (control) co-administration of the other drug in rats (mean ± SD, n = 6) Pharmacokinetic parametersa  Rosuvastatin  Total naringin  (—)  (+Naringin)  (—)  (+Rosuvastatin)  AUC(0−t) (μg · L−1 · h−1)  1385.1 ± 838.1  1068.9 ± 319.3  1835.9 ± 503.6  1165.8 ± 744.0  CL (L · h−1 · kg−1)  35.2 ± 21.3  36.2 ± 15.5  23.4 ± 6.8  29.2 ± 17.1  Cmax (μg · L−1)  164.2 ± 84.4  208.2 ± 199.1  456.0 ± 160.9  325.3 ± 314.1  T1/2 (h)  16.2 ± 7.3  13.8 ± 7.4  1.8 ± 0.6  10.2 ± 16.8  Tmax (h)  0.6 ± 0.2  0.6 ± 0.2  4.5 ± 2.0  4.2 ± 2.3  Pharmacokinetic parametersa  Rosuvastatin  Total naringin  (—)  (+Naringin)  (—)  (+Rosuvastatin)  AUC(0−t) (μg · L−1 · h−1)  1385.1 ± 838.1  1068.9 ± 319.3  1835.9 ± 503.6  1165.8 ± 744.0  CL (L · h−1 · kg−1)  35.2 ± 21.3  36.2 ± 15.5  23.4 ± 6.8  29.2 ± 17.1  Cmax (μg · L−1)  164.2 ± 84.4  208.2 ± 199.1  456.0 ± 160.9  325.3 ± 314.1  T1/2 (h)  16.2 ± 7.3  13.8 ± 7.4  1.8 ± 0.6  10.2 ± 16.8  Tmax (h)  0.6 ± 0.2  0.6 ± 0.2  4.5 ± 2.0  4.2 ± 2.3  aAUC, area under the plasma concentration–time curve; CL, apparent plasma clearance; Cmax, maximum plasma concentration; T1/2, elimination half-life; Tmax, time to Cmax. Discussion To obtain chromatograms with good separation and strong intensity, mobile phase composition and mass spectrometry parameters were optimized. The final composition of the mobile phase was methanol–water both containing 0.1% formic acid, and three analytes and IS exhibited the best peak shapes and eluted within 5.0 min. Under ESI condition, both naringin and naringenin had good sensitivity in negative ionization mode, while rosuvastatin showed opposite characteristics in the same mode. In consideration of the elution time of analytes and IS, polarity switch (negative-positive-negative ionization mode) was performed to determine above compounds in the same analytical run. Transporter-based drug–drug interactions are important to consider in drug development. BCRP and OATP, expressed in enterocyte and hepatocytes, plays an important role in absorption, distribution, and elimination of various drugs and therefore implicated in transporter-based drug–drug interactions (38, 39). The disposition of rosuvastatin, which is a specific substrate of BCRP and OATP 1B1, is markedly affected by the transport activity modulation of these two transporters. Naringin, as well as naringenin, which could inhibit BCRP and OATP 1B1 in vitro (17–21), were likely to modulate the pharmacokinetic behavior of rosuvastatin in vivo. However, as shown in our results, the plasma pharmacokinetic profile of either rosuvastatin or naringin in rosuvastatin and naringin co-administration group have no significant differences compared with that in corresponding control group. That is to say, in vivo results are inconsistent with in vitro data. As shown in previous studies (34–37), naringenin glucuronide and naringenin sulfate, rather than naringin and naringenin, are the predominant existing form in circulatory system after naringin administration. Structural modification of naringenin may lead to the modulation of inhibitory activities on BCRP and OATP 1B1. What is more, the complexities of in vivo conditions make in vivo results may remarkably different with in vitro data which obtained from relatively simple models. Besides naringin, similar antinomy also appeared in pharmacokinetic interaction study between rosuvastatin and silymarin (40). These results remind us that the extrapolations from in vitro data to in vivo results need great caution. Conclusion A rapid and reproducible validated RRLC–MS/MS method for simultaneous determination of rosuvastatin, naringin and naringenin concentrations in rat plasma was reported in the present paper. The method was found to be accurate, precise and specific, and was successfully applied to the pharmacokinetic drug interaction investigation of rosuvastatin and naringin in rats. Results suggest that the co-administration of rosuvastatin and naringin basically would not result in pharmacokinetic drug interaction between these two drugs. Funding The present work was financial supported by the National Major Scientific and Technical Special Project of China (No. 2015ZX09101014) and Applied Science and Technology R&D Special Fund Project of Guangdong Province (No. 2015B020234004). Conflict of interest statement All the authors declare that there are no known conflicts of interest with this publication. Highlights First simultaneous determination of rosuvastatin, naringin and naringenin in rat plasma. Consistent recoveries with no significant matrix effect for three analytes and corresponding internal standard. The pharmacokinetic interaction between rosuvastatin and naringin in rats is firstly reported. References 1 Cheng, J.W.M.; Rosuvastatin in the management of hyperlipidemia; Clinical Therapeutics , ( 2004); 26( 9): 1368– 1387. 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For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Chromatographic Science Oxford University Press

Simultaneous determination of rosuvastatin, naringin and naringenin in rat plasma by RRLC–MS/MS and its application to a pharmacokinetic drug interaction study

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com
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0021-9665
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1945-239X
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10.1093/chromsci/bmy034
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Abstract

Abstract A rapid resolution liquid chromatography tandem mass spectrometry method was developed and validated for simultaneous determination of rosuvastatin, naringin and naringenin in rat plasma. Chromatographic separation of analytes and internal standard (fluvastatin for rosuvastatin, while isoquercitrin for naringin and naringenin) was performed on Agilent Poroshell 120 EC-C18 column (3.0 × 50 mm, 2.7 μm) using gradient elution with a mobile phase of methanol and water, both with 0.1% formic acid (v/v). The detection was operated in multiple reaction monitoring mode to monitor the precursor-to-product ion transitions of m/z 579.1→270.8 for naringin, m/z 270.9→150.7 for naringenin, m/z 463.1→299.8 for isoquercitrin in negative ionization mode, and m/z 482.2→258.1 for rosuvastatin, m/z 412.1→224.1 for fluvastatin in positive ionization mode. Polarity switch (negative-positive-negative ionization mode) was performed in a total runtime of 5.0 min. The method was validated over a concentration range of 10–2,000 ng/mL for the above three analytes. The intra-day and inter-day precisions and accuracies of the quality control samples at low, medium and high concentration levels exhibited relative standard deviations <10% and the accuracy values ranged from −7.2% to 8.4%. The proposed method was successfully applied to the pharmacokinetic drug interaction study of rosuvastatin combined with naringin in rats. Introduction Rosuvastatin is a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor for the treatment of patients with primary hypercholesterolemia, mixed dyslipidemia and homozygous familial hypercholesterolemia (1). Previous studies had revealed that rosuvastatin is just slightly metabolized by cytochrome P450 (CYP), isoenzyme CYP2C9 (2) and primarily excreted via bile as unchanged drug (3, 4). Transporters, including breast cancer resistance protein (BCRP) (5) and organic anion transporting polypeptide 1B1 (OATP 1B1) (6), play pivotal roles in the absorption and excretion of rosuvastatin. Transport function modulation of BCRP, as well as OATP 1B1, can remarkably affect the cholesterol-lowering efficacy and exposure-dependent toxicity of rosuvastatin therapy (7, 8). Dietary flavonoids, with their antioxidant properties, are regarded as practical supplements to optimize health and reduce the risk of chronic diseases for consumers, including patients with dyslipidemia (9, 10). Naringin, a widespread flavanone glycoside, has been shown to possess extensive bioactivities on health benefits, including anti-inflammatory (11), antioxidant (12), neuroprotection (13) and ameliorating atherogenic dyslipidemia (14). What is more, our preliminary studies revealed that naringin, as well as its active metabolite naringenin, can effectively relieve cough (15) and reduce sputum (16). And now naringin has been approved by China Food and Drug Administration for clinical trials (No. 2013L01586). Several studies have shown that naringin and naringenin could down-regulate BCRP-mediated transport function (17–19). Meanwhile, naringin was considered as an OATP 1B1 inhibitor (20, 21). As hypercholesterolemia (22), cough (23) and inflammation (24, 25) are common ailments among old population, naringin was very likely to be used in clinic combined with rosuvastatin to treat these diseases (26). When used in clinic combined with naringin, the disposition of rosuvastatin may change due to the modulation of BCRP and OATP 1B1. These changes may cause severe rhabdomyolysis which is life-threatening (27). For avoiding or minimizing the transporter-based drug interaction-induced adverse events in clinical application, the identification and quantification of in vivo pharmacokinetics interactions associated with the drug combination of naringin and rosuvastatin in preclinical phase is meaningful. To date, several methods for the quantification of rosuvastatin in plasma were reported (28, 29), as well as the determination of naringin and its metabolite naringenin (30, 31). However, a developed LC–MS/MS-based method for simultaneous determination of rosuvastatin, naringin and naringenin in plasma has not been reported. In the present study, a rapid resolution liquid chromatography tandem mass spectrometry (RRLC–MS/MS) method was established to determine the concentrations of rosuvastatin, naringin and naringenin in rat plasma. The analytical method was validated and successfully applied to evaluate the pharmacokinetic interaction between rosuvastatin and naringin in rats. Materials and methods Chemicals and reagents Rosuvastatin, naringin and fluvastatin reference standards were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Naringenin, isoquercitrin reference standards and β-glucuronidase/sulfatase (Type H-1) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Liquid chromatography–mass spectrometry (LC–MS) grade methanol and formic acid were purchased from Fisher Scientific (Pittsburgh, PA, USA). High-performance liquid chromatography (HPLC) grade methanol and ethyl acetate were purchased from Honeywell B&J (New Jersey, USA). Water was prepared using a Milli-Q purification system (Millipore, Bedford, MA, USA). Rosuvastatin Calcium Tablets (Crestor) were purchased from AstraZeneca Crop. Naringin powder, for oral administration, was extracted from Huajuhong (Citrus grandis “Tomentosa”) with a purity of 98.8%, which was determined by HPLC method with external standard (32). Preparation of calibration standards and quality control samples The stock solutions of rosuvastatin, naringin, naringenin, fluvastatin and isoquercitrin were prepared in 50% methanol. All stock solutions were prepared at 1 mg/mL concentration. Two separate stocks of each analyte were prepared and used for preparation of calibration standards and quality controls (QC). The stock solutions were stored at 4°C and brought to room temperature before use. Working standard and internal standard (IS) spiking solutions were prepared from stock solutions by diluting with 50% methanol. An aliquot of 10 μL working standard solution was added to a 1.5-mL polypropylene tube and evaporated to dryness with N2 at 25°C using a EYELA MG-2200 drying system (Tokyo, Japan). Then 50 μL rat blank plasma was added to the tube and vortex-mixed, to yield calibration standards of 10, 20, 50, 150, 500, 1,000, 1,600 and 2,000 ng/mL. QC samples were prepared at final concentrations of 30, 200 and 1,500 ng/mL in the same manner as the calibration standards. The internal standard spiking solution, with the concentration of 16,800 ng/mL for fluvastatin and 5,000 ng/mL for isoquercitrin, was prepared by diluting the fluvastatin and isoquercitrin stock solution together with 50% methanol. The calibration standards, QC samples and internal standard spiking solution were prepared accompanying each analytical batch. Sample preparation A 50-μL aliquot of plasma was mixed with 10 μL of internal standard spiking solution followed by 1,000 μL ethyl acetate. Then samples were vortex-mixed for 3 min and centrifuged at 10,000 × g for 10 min at 4°C. A 900-μL aliquot of supernatant was transferred into a fresh 1.5-mL polypropylene tube and then evaporated to dryness under a gentle stream of N2 at 37°C. Subsequently, an aliquot of 100-μL mobile phase was added to the tube to dissolve the residue. Samples were ultrasonic extracted for 3 min, vortex-mixed for 3 min and centrifuged at 15,000 × g for 30 min at 25°C. Finally, an aliquot of 10-μL supernatant was injected into the LC–MS/MS system for analysis. The hydrolysis of naringin, which forms its metabolite naringenin, mediated by lactase-phlorizin hydrolase and human intestinal microflora is recognized as the first and determinant step in the absorption of naringin (33). Subsequently, naringenin extensively combines with glucuronide or sulfate and yields its predominant existing form in circulatory system (34–36). To quantitate total naringenin including free and its glucuronide/sulfate conjugates, partial plasma samples were treated with β-glucuronidase/sulfatase as in previous reports (32, 37). As to these samples, 50 μL plasma was mixed with 10 μL β-glucuronidase/sulfatase solution (dissolved in 0.2 mmol/L acetic acid buffer, pH = 5.0, 10 Unit/μL) and incubated at 37°C for 2 h. After that, 10 μL internal standard spiking solution was added to the sample, followed by 1,000 μL ethyl acetate. Other process procedures were the same as above-mentioned. LC–MS/MS condition The LC–MS/MS system consisted of an Agilent 1200 RRLC and an Agilent 6410 triple quadrupole mass spectrometer with an electrospray ionization source (ESI; Agilent Technology, Santa Clara, CA, USA). Chromatographic separation was carried out on an Agilent Poroshell 120 EC-C18 column (3.0 × 50 mm, 2.7 μm) tandem pre-column of Welch Analytical Guard Cartridges Ultimate XB-C18 (4.6 × 10 mm, 5 μm). The column temperature was maintained at 40°C using a thermostatically controlled column oven. The mobile phase was composed of solvent A (0.1% formic acid, v/v) and B (methanol with 0.1% formic acid, v/v). The gradient elution profile consisted of an initial 45:55 ratio of A:B that was maintained for 2.0 min after injection, followed by a linear gradient to 0:100 of A:B over a 0.1-min period (completed at 2.1 min). This solvent composition was maintained for 1.8 min of isocratic hold (until 3.9 min), followed by a linear gradient to 45:55 of A:B over a 0.1-min period (completed at 4.0 min), and subsequently returned to initial conditions of the 45:55 ratio of A:B for a period of 1.0-min, for a total runtime of 5.0-min. The flow rate was kept at 0.3 mL/min. The HPLC effluent was introduced directly to the mass spectrometer without splitting. To obtain higher response of the three analytes, polarity switch (negative-positive-negative ionization mode) was performed in each analytical run. The electrode polarity of the mass spectrometer was switched at 2.9 min and 4.3 min. MS detector was operated in multiple reaction monitoring (MRM) mode at unit mass resolution with a dwell-time of 200-ms for all test compounds. The optimized mass spectrometric parameters, MRM transitions, fragmentors, collision energies are shown in Table I. The ion source parameters were capillary 4,000 V, gas flow 10 L/min, nebulizer 25 psi, gas temperature 350°C for maximum sensitivity. Other MS parameters were adopted from the recommended values for the instrument. Table I. Multiple reaction monitoring transitions and the optimum LC–MS/MS conditions Period  Compound  Q1  Q3  Fragmentor (V)  Collision energy (V)  1  0–1.0 min, To Waste, ESI−          2  1.0–2.9 min, To MS, ESI−          Naringin  579.1  270.8  225  33  Naringenin  270.9  150.7  100  12  Isoquercitrin  463.1  299.8  128  24  3  2.9–4.3 min, To MS, ESI+          Rosuvastatin  482.2  258.1  170  38  Fluvastatin  412.1  224.1  140  33  4  4.3–5.0 min, To Waste, ESI−          Period  Compound  Q1  Q3  Fragmentor (V)  Collision energy (V)  1  0–1.0 min, To Waste, ESI−          2  1.0–2.9 min, To MS, ESI−          Naringin  579.1  270.8  225  33  Naringenin  270.9  150.7  100  12  Isoquercitrin  463.1  299.8  128  24  3  2.9–4.3 min, To MS, ESI+          Rosuvastatin  482.2  258.1  170  38  Fluvastatin  412.1  224.1  140  33  4  4.3–5.0 min, To Waste, ESI−          Table I. Multiple reaction monitoring transitions and the optimum LC–MS/MS conditions Period  Compound  Q1  Q3  Fragmentor (V)  Collision energy (V)  1  0–1.0 min, To Waste, ESI−          2  1.0–2.9 min, To MS, ESI−          Naringin  579.1  270.8  225  33  Naringenin  270.9  150.7  100  12  Isoquercitrin  463.1  299.8  128  24  3  2.9–4.3 min, To MS, ESI+          Rosuvastatin  482.2  258.1  170  38  Fluvastatin  412.1  224.1  140  33  4  4.3–5.0 min, To Waste, ESI−          Period  Compound  Q1  Q3  Fragmentor (V)  Collision energy (V)  1  0–1.0 min, To Waste, ESI−          2  1.0–2.9 min, To MS, ESI−          Naringin  579.1  270.8  225  33  Naringenin  270.9  150.7  100  12  Isoquercitrin  463.1  299.8  128  24  3  2.9–4.3 min, To MS, ESI+          Rosuvastatin  482.2  258.1  170  38  Fluvastatin  412.1  224.1  140  33  4  4.3–5.0 min, To Waste, ESI−          Method validation This method was fully validated with reference to the Guidance for Bioanalytical Method Validation issued by Chinese Pharmacopoeia Commission in 2015. The specificity of the method was evaluated by comparing chromatograms of the standard-spiked plasma samples with the blank plasma from six different sources. The specificity was further confirmed in actual pharmacokinetic study by examining the pre-dosing plasma samples. A calibration curve was constructed from a blank plasma sample, a zero-concentration plasma sample prepared with internal standard and eight concentration levels of samples covering the range of 10–2,000 ng/mL, including lower limit of quantification (LLOQ). Each concentration level was prepared in duplicate. Calibration curves were constructed and fitted by linear least-squares regression analysis to plot the peak area ratio of analyte relative to the internal standard against the analyte concentrations with a weighed of 1/x2 (x= concentration). The acceptance criterion for each measured standard concentration was 15% deviation from the nominal value except LLOQ, which was not more than 20%. Precision and accuracy were evaluated by repeated analyses of QC samples (n = 5) at concentrations of 30, 200 and 1,500 ng/mL on three separate days. Intra-day and inter-day precisions were expressed by the relative standard deviation (RSD, %), while the accuracy was determined by calculating the percentage deviation of the calculated concentrations from the nominal concentrations and expressed as relative error (RE, %). Recoveries of rosuvastatin, naringin and naringenin were determined by comparing the peak area of extracted three levels QC samples to that of the analytes spiked to the blank sample extracts at the corresponding concentration. The recovery of the internal standard was determined in the same way at the working concentration. The matrix effect was measured by comparing the peak area in the analyte-spiked post-extracted sample with that acquired using a neat solution. Blank plasma from six different sources were used to evaluate the matrix effect at low (30 ng/mL) and high (1,500 ng/mL) QC concentration level. Stability studies of analyte were assessed by analyzing three replicates of QC samples at concentrations of 30 and 1,500 ng/mL under the following conditions: long-term stability at −70°C for 90 days, short-term stability at 25°C for 5 h, in processed samples in autosampler vials (25°C) for 24 h, and after four freeze-thaw cycles (−70–25°C), and after incubation with β-glucuronidase/sulfatase at 37°C for 2 h. Pharmacokinetic drug interaction study Male Sprague-Dawley rats (180–220 g) were obtained from Guangdong Medical Laboratory Animal Center (Guangzhou, China) and used to study the pharmacokinetic drug interaction of rosuvastatin and naringin. The animals were acclimated for 1 week prior to the initiation of dosing with feed and water available ad libitum. Environmental conditions were maintained at 20–25°C, 55 ± 15% relative humidity, 12 h light–dark cycles and 12 air change cycles/h. All experimental procedures and protocols were reviewed and approved by the Animal Ethics Committee of the School of Life Sciences in Sun Yat-sen University. Diet was prohibited for 12 h before the experiment but water was freely available. Rats were randomly divided into three groups (n = 6/group). Rats in Group 1 were orally administered rosuvastatin (50 mg/kg) plus drinking water, rats in Group 2 were given naringin (42 mg/kg) plus drinking water and rats in Group 3 were given rosuvastatin (50 mg/kg) plus naringin (42 mg/kg). Blood samples (0.3 mL) were collected from post-orbital venous plexus into heparinized 1.5 mL polythene tubes at 0.5, 1, 1.5, 3, 5, 8, 10, 12 and 24 h after oral administration, respectively. The samples were immediately centrifuged at 5,000 rpm for 10 min. The plasma obtained was stored at −70°C until analysis. Blank plasma was obtained from the rat without any drug administration and was used for the method development and validation. Pharmacokinetic analysis The DAS (Drug and statistics) software (Version 3.0, Shanghai University of Traditional Chinese Medicine, China) was used with a non-compartmental statistical model to determine the pharmacokinetic parameters of the rat plasma samples. The data are expressed as mean ± SD and were evaluated by one-way analysis of variance and Student’s t-tests in SPSS 18.0 (SPSS Inc., Chicago, USA). P-values <0.05 or 0.01 were considered statistically significant. Results Method development and instrumental optimization Both of liquid–liquid extraction and protein precipitation were compared in this study to obtain the optimum extraction method. As a result, liquid–liquid extraction of adding 1,000 μL ethyl acetate to 50 μL plasma showed a high extraction recovery. For the sake of obtaining chromatograms with good separation and strong total ion current, different solutions of acetonitrile in water and methanol in water as mobile phases, with or without 0.1% formic acid, for binary isocratic and gradient elution, were investigated to optimize the chromatographic conditions. Methanol–water both with 0.1% formic acid on the optimized gradient mode exhibited a good separation and abundant signal response. As to the MS conditions, ionization mode of the three analytes was optimized. The mass spectrometer was conducted in the negative ionization mode for the detection of naringin and naringenin, but in the positive ionization mode for the detection of rosuvastatin due to the poor sensitivity of rosuvastatin in negative ionization mode. With the chromatography conditions described above, naringin, naringenin and isoquercitrin were eluted out at 1.2 min, 2.3 min and 1.3 min, respectively. Rosuvastatin and fluvastatin were eluted out at 3.1 min and 3.9 min, respectively. In order to determine rosuvastatin, naringin and naringenin in the same analytical run, polarity switch was performed. The following predominant transitions were selected for quantification: m/z 482.2→258.1 for rosuvastatin, m/z 579.1→270.8 for naringin and m/z 270.9→150.7 for naringenin, respectively. The MS fragment details of these compounds are shown in Figure 1. And other electrospray source and the mass spectrometer parameters were optimized and listed in Table I. Figure 1. View largeDownload slide Product ion spectra of rosuvastatin (A), naringin (B) and naringenin (C). Figure 1. View largeDownload slide Product ion spectra of rosuvastatin (A), naringin (B) and naringenin (C). Method validation Specificity Typical MRM chromatograms of rosuvastatin, naringin and naringenin are presented in Figure 2. Sharp and fine peaks were obtained for naringin, naringenin, isoquercitrin and rosuvastatin, fluvastatin at retention times of 1.2 min, 2.3 min, 1.3 min, 3.1 min and 3.9 min, respectively. No interference was observed, indicating that the developed method was specific for the analytes in plasma samples. Figure 2. View largeDownload slide Representative chromatograms for rosuvastatin, fluvastatin, and naringin, naringenin, isoquercitrin in rat plasma samples: (I) a blank plasma sample; (II) a blank plasma sample spiked with rosuvastatin, naringin, naringenin and ISs at LLOQ, and (III) a rat plasma sample 3 h after a single oral dose of rosuvastatin 50 mg/kg and naringin 42 mg/kg. Figure 2. View largeDownload slide Representative chromatograms for rosuvastatin, fluvastatin, and naringin, naringenin, isoquercitrin in rat plasma samples: (I) a blank plasma sample; (II) a blank plasma sample spiked with rosuvastatin, naringin, naringenin and ISs at LLOQ, and (III) a rat plasma sample 3 h after a single oral dose of rosuvastatin 50 mg/kg and naringin 42 mg/kg. Linearity and LLOQ An acceptable linearity was achieved in the range of 10–2,000 ng/mL for rosuvastatin, naringin and naringenin with correlation coefficients (r) > 0.99 in all three batches. The intra-day assay yielded acceptable precisions of 7.2% RSD for rosuvastatin, 1.8% RSD for naringin and 2.5% RSD for naringenin and accuracies of 8.4% RE for rosuvastatin, −7.2% RE for naringin and −3.2% RE for naringenin (Table II). Table II. Intra- and inter-day accuracies and precisions of rosuvastatin, naringin and naringenin in rat plasma Compound  Concentration (ng/mL)  Intra-day (n = 5)  Inter-day (n = 5*3)  Recovery (%) (n = 5)  RSD%  RE%  RSD%  RE%  Rosuvastatin  10 (LLOQ)  7.2  8.4  16.6  1.7  –  30  5.6  2.1  6.5  −4.1  69.7 ± 1.2  200  9.4  −0.1  6.8  −0.9  63.6 ± 1.1  1,500  8.1  −0.9  7.0  −6.2  71.6 ± 1.3  Naringin  10 (LLOQ)  1.8  −7.2  6.2  2.5    30  4.5  −1.2  4.3  −2.7  60.5 ± 2.2  200  1.3  −0.5  1.7  −1.4  58.6 ± 0.9  1,500  2.1  1.2  2.6  0.1  56.3 ± 2.9  Naringenin  10 (LLOQ)  2.5  −3.2  3.0  3.2    30  1.7  3  2.6  2.3  72.4 ± 1.4  200  2.4  4.5  2.4  4.0  70.2 ± 0.6  1,500  1.4  0.9  2.4  −0.1  71.1 ± 1.9  Compound  Concentration (ng/mL)  Intra-day (n = 5)  Inter-day (n = 5*3)  Recovery (%) (n = 5)  RSD%  RE%  RSD%  RE%  Rosuvastatin  10 (LLOQ)  7.2  8.4  16.6  1.7  –  30  5.6  2.1  6.5  −4.1  69.7 ± 1.2  200  9.4  −0.1  6.8  −0.9  63.6 ± 1.1  1,500  8.1  −0.9  7.0  −6.2  71.6 ± 1.3  Naringin  10 (LLOQ)  1.8  −7.2  6.2  2.5    30  4.5  −1.2  4.3  −2.7  60.5 ± 2.2  200  1.3  −0.5  1.7  −1.4  58.6 ± 0.9  1,500  2.1  1.2  2.6  0.1  56.3 ± 2.9  Naringenin  10 (LLOQ)  2.5  −3.2  3.0  3.2    30  1.7  3  2.6  2.3  72.4 ± 1.4  200  2.4  4.5  2.4  4.0  70.2 ± 0.6  1,500  1.4  0.9  2.4  −0.1  71.1 ± 1.9  Table II. Intra- and inter-day accuracies and precisions of rosuvastatin, naringin and naringenin in rat plasma Compound  Concentration (ng/mL)  Intra-day (n = 5)  Inter-day (n = 5*3)  Recovery (%) (n = 5)  RSD%  RE%  RSD%  RE%  Rosuvastatin  10 (LLOQ)  7.2  8.4  16.6  1.7  –  30  5.6  2.1  6.5  −4.1  69.7 ± 1.2  200  9.4  −0.1  6.8  −0.9  63.6 ± 1.1  1,500  8.1  −0.9  7.0  −6.2  71.6 ± 1.3  Naringin  10 (LLOQ)  1.8  −7.2  6.2  2.5    30  4.5  −1.2  4.3  −2.7  60.5 ± 2.2  200  1.3  −0.5  1.7  −1.4  58.6 ± 0.9  1,500  2.1  1.2  2.6  0.1  56.3 ± 2.9  Naringenin  10 (LLOQ)  2.5  −3.2  3.0  3.2    30  1.7  3  2.6  2.3  72.4 ± 1.4  200  2.4  4.5  2.4  4.0  70.2 ± 0.6  1,500  1.4  0.9  2.4  −0.1  71.1 ± 1.9  Compound  Concentration (ng/mL)  Intra-day (n = 5)  Inter-day (n = 5*3)  Recovery (%) (n = 5)  RSD%  RE%  RSD%  RE%  Rosuvastatin  10 (LLOQ)  7.2  8.4  16.6  1.7  –  30  5.6  2.1  6.5  −4.1  69.7 ± 1.2  200  9.4  −0.1  6.8  −0.9  63.6 ± 1.1  1,500  8.1  −0.9  7.0  −6.2  71.6 ± 1.3  Naringin  10 (LLOQ)  1.8  −7.2  6.2  2.5    30  4.5  −1.2  4.3  −2.7  60.5 ± 2.2  200  1.3  −0.5  1.7  −1.4  58.6 ± 0.9  1,500  2.1  1.2  2.6  0.1  56.3 ± 2.9  Naringenin  10 (LLOQ)  2.5  −3.2  3.0  3.2    30  1.7  3  2.6  2.3  72.4 ± 1.4  200  2.4  4.5  2.4  4.0  70.2 ± 0.6  1,500  1.4  0.9  2.4  −0.1  71.1 ± 1.9  Precision and accuracy The intra-day and inter-day precisions and accuracies for rosuvastatin, naringin and naringenin were showed in Table II. The intra-day and inter-day precisions (RSD) were <10% with accuracies (RE) in the range of −7.2–8.4% for all analytes. The results indicated that the present method was reliable and reproducible for the simultaneous quantitative analysis of rosuvastatin, naringin and naringenin in rat plasma samples. Recovery Mean extraction recoveries of rosuvastatin, naringin and naringenin measured at three different concentrations were 63.6–71.6%, 56.3–60.5% and 70.2–72.4%, respectively (Table II), indicating consistent and reproducible recoveries of analytes. Mean extraction recovery of the fluvastatin and isoquercitrin were 58.1% and 62.1%, correspondingly. Matrix effect The matrix effects of three analytes are presented in Table III. Mean values of rosuvastatin, naringin and naringenin at low (30ng/mL) QC concentration level was 109.4%, 90.9% and 92.4%, respectively. While those at high (1500 ng/mL) QC concentration level 87.1%, 83.1% and 92.8%, respectively. All of these are within the acceptable range of 20%. And RSD for matrix effects of each analyte at different concentration level are < 15%. These data indicated that relative matrix effect for the analytes in rat plasma was negligible under the current conditions. Table III. Matrix effects for rosuvastatin, naringin and naringenin (mean ± SD, n = 5) No.a  Rosuvastatin concentration  Naringin concentration  Naringenin concentration  (ng/mL)  (ng/mL)  (ng/mL)    30  1,500  30  1,500  30  1,500  1  108.1 ± 6.3  86.5 ± 2.1  95.9 ± 3.2  83.7 ± 2.2  93.1 ± 2.5  91.5 ± 0.4  2  110.1 ± 3.7  87.6 ± 1.3  89.5 ± 4.3  82.3 ± 1.3  92.9 ± 1.8  93.2 ± 1.4  3  108.1 ± 1.4  86.9 ± 1.9  86.8 ± 1.9  83.1 ± 4.4  91.7 ± 2.3  92.1 ± 1.6  4  108.3 ± 2.5  86.0 ± 0.7  89.6 ± 1.7  82.6 ± 2.7  92.6 ± 2.9  93.1 ± 1.1  5  111.6 ± 1.1  87.6 ± 1.8  96.6 ± 3.3  81.2 ± 1.1  92.1 ± 2.4  93.3 ± 1.6  6  110.4 ± 5.5  88.0 ± 1.2  86.8 ± 0.4  85.8 ± 4.6  91.8 ± 0.9  93.6 ± 1.9  Mean  109.4 ± 1.5  87.1 ± 0.8  90.9 ± 4.4  83.1 ± 1.5  92.4 ± 0.6  92.8 ± 0.8  RSD%  1.3  0.9  4.8  1.9  0.6  0.9  No.a  Rosuvastatin concentration  Naringin concentration  Naringenin concentration  (ng/mL)  (ng/mL)  (ng/mL)    30  1,500  30  1,500  30  1,500  1  108.1 ± 6.3  86.5 ± 2.1  95.9 ± 3.2  83.7 ± 2.2  93.1 ± 2.5  91.5 ± 0.4  2  110.1 ± 3.7  87.6 ± 1.3  89.5 ± 4.3  82.3 ± 1.3  92.9 ± 1.8  93.2 ± 1.4  3  108.1 ± 1.4  86.9 ± 1.9  86.8 ± 1.9  83.1 ± 4.4  91.7 ± 2.3  92.1 ± 1.6  4  108.3 ± 2.5  86.0 ± 0.7  89.6 ± 1.7  82.6 ± 2.7  92.6 ± 2.9  93.1 ± 1.1  5  111.6 ± 1.1  87.6 ± 1.8  96.6 ± 3.3  81.2 ± 1.1  92.1 ± 2.4  93.3 ± 1.6  6  110.4 ± 5.5  88.0 ± 1.2  86.8 ± 0.4  85.8 ± 4.6  91.8 ± 0.9  93.6 ± 1.9  Mean  109.4 ± 1.5  87.1 ± 0.8  90.9 ± 4.4  83.1 ± 1.5  92.4 ± 0.6  92.8 ± 0.8  RSD%  1.3  0.9  4.8  1.9  0.6  0.9  aNo. represent the six different sources of blank rat plasma. Table III. Matrix effects for rosuvastatin, naringin and naringenin (mean ± SD, n = 5) No.a  Rosuvastatin concentration  Naringin concentration  Naringenin concentration  (ng/mL)  (ng/mL)  (ng/mL)    30  1,500  30  1,500  30  1,500  1  108.1 ± 6.3  86.5 ± 2.1  95.9 ± 3.2  83.7 ± 2.2  93.1 ± 2.5  91.5 ± 0.4  2  110.1 ± 3.7  87.6 ± 1.3  89.5 ± 4.3  82.3 ± 1.3  92.9 ± 1.8  93.2 ± 1.4  3  108.1 ± 1.4  86.9 ± 1.9  86.8 ± 1.9  83.1 ± 4.4  91.7 ± 2.3  92.1 ± 1.6  4  108.3 ± 2.5  86.0 ± 0.7  89.6 ± 1.7  82.6 ± 2.7  92.6 ± 2.9  93.1 ± 1.1  5  111.6 ± 1.1  87.6 ± 1.8  96.6 ± 3.3  81.2 ± 1.1  92.1 ± 2.4  93.3 ± 1.6  6  110.4 ± 5.5  88.0 ± 1.2  86.8 ± 0.4  85.8 ± 4.6  91.8 ± 0.9  93.6 ± 1.9  Mean  109.4 ± 1.5  87.1 ± 0.8  90.9 ± 4.4  83.1 ± 1.5  92.4 ± 0.6  92.8 ± 0.8  RSD%  1.3  0.9  4.8  1.9  0.6  0.9  No.a  Rosuvastatin concentration  Naringin concentration  Naringenin concentration  (ng/mL)  (ng/mL)  (ng/mL)    30  1,500  30  1,500  30  1,500  1  108.1 ± 6.3  86.5 ± 2.1  95.9 ± 3.2  83.7 ± 2.2  93.1 ± 2.5  91.5 ± 0.4  2  110.1 ± 3.7  87.6 ± 1.3  89.5 ± 4.3  82.3 ± 1.3  92.9 ± 1.8  93.2 ± 1.4  3  108.1 ± 1.4  86.9 ± 1.9  86.8 ± 1.9  83.1 ± 4.4  91.7 ± 2.3  92.1 ± 1.6  4  108.3 ± 2.5  86.0 ± 0.7  89.6 ± 1.7  82.6 ± 2.7  92.6 ± 2.9  93.1 ± 1.1  5  111.6 ± 1.1  87.6 ± 1.8  96.6 ± 3.3  81.2 ± 1.1  92.1 ± 2.4  93.3 ± 1.6  6  110.4 ± 5.5  88.0 ± 1.2  86.8 ± 0.4  85.8 ± 4.6  91.8 ± 0.9  93.6 ± 1.9  Mean  109.4 ± 1.5  87.1 ± 0.8  90.9 ± 4.4  83.1 ± 1.5  92.4 ± 0.6  92.8 ± 0.8  RSD%  1.3  0.9  4.8  1.9  0.6  0.9  aNo. represent the six different sources of blank rat plasma. Stability All established stability for the analytes are summarized in Table IV. The data showed that the stability of QC samples after long-term storage at −70°C (90 days), at 25°C for 5 h, in processed samples in autosampler vials (25°C) for 24 h and after four freeze-thaw cycles (−70–25°C) was acceptable. No detectable degradation was observed after a 2-h incubation with β-glucuronidase/sulfatase at 37°C for naringin and naringenin, while rosuvastatin showed partial degradation. Table IV. Stability of rosuvastatin, naringin and naringenin under various conditions (mean ± SD, n = 3) Compound  Conc. (ng/mL)  Long-term (−70°C, 90 days)  Short-term (25°C, 5 h)  Freeze-thaw (four cycles)  Incubation (37°C, 2 h)  Post-preparative (25°C, 24 h)  Rosuvastatin  30  104.8 ± 7.3  105.8 ± 1.3  112.1 ± 9.9  84.5 ± 5.0  107.8 ± 1.6  1,500  98.6 ± 3.1  101.8 ± 5.0  107.4 ± 5.6  80.7 ± 2.7  107.3 ± 2.8  Naringin  30  103.0 ± 1.7  102.4 ± 4.1  97.6 ± 4.0  94.1 ± 2.7  102.0 ± 2.3  1,500  104.4 ± 1.1  102.2 ± 1.5  99.4 ± 0.9  98.3 ± 1.1  103.6 ± 1.0  Naringenin  30  106.7 ± 2.0  104.6 ± 1.6  101.9 ± 2.5  100.2 ± 1.2  105.3 ± 1.9  1,500  102.3 ± 1.6  101.9 ± 0.8  99.7 ± 3.0  95.9 ± 2.3  103.2 ± 2.3  Compound  Conc. (ng/mL)  Long-term (−70°C, 90 days)  Short-term (25°C, 5 h)  Freeze-thaw (four cycles)  Incubation (37°C, 2 h)  Post-preparative (25°C, 24 h)  Rosuvastatin  30  104.8 ± 7.3  105.8 ± 1.3  112.1 ± 9.9  84.5 ± 5.0  107.8 ± 1.6  1,500  98.6 ± 3.1  101.8 ± 5.0  107.4 ± 5.6  80.7 ± 2.7  107.3 ± 2.8  Naringin  30  103.0 ± 1.7  102.4 ± 4.1  97.6 ± 4.0  94.1 ± 2.7  102.0 ± 2.3  1,500  104.4 ± 1.1  102.2 ± 1.5  99.4 ± 0.9  98.3 ± 1.1  103.6 ± 1.0  Naringenin  30  106.7 ± 2.0  104.6 ± 1.6  101.9 ± 2.5  100.2 ± 1.2  105.3 ± 1.9  1,500  102.3 ± 1.6  101.9 ± 0.8  99.7 ± 3.0  95.9 ± 2.3  103.2 ± 2.3  Table IV. Stability of rosuvastatin, naringin and naringenin under various conditions (mean ± SD, n = 3) Compound  Conc. (ng/mL)  Long-term (−70°C, 90 days)  Short-term (25°C, 5 h)  Freeze-thaw (four cycles)  Incubation (37°C, 2 h)  Post-preparative (25°C, 24 h)  Rosuvastatin  30  104.8 ± 7.3  105.8 ± 1.3  112.1 ± 9.9  84.5 ± 5.0  107.8 ± 1.6  1,500  98.6 ± 3.1  101.8 ± 5.0  107.4 ± 5.6  80.7 ± 2.7  107.3 ± 2.8  Naringin  30  103.0 ± 1.7  102.4 ± 4.1  97.6 ± 4.0  94.1 ± 2.7  102.0 ± 2.3  1,500  104.4 ± 1.1  102.2 ± 1.5  99.4 ± 0.9  98.3 ± 1.1  103.6 ± 1.0  Naringenin  30  106.7 ± 2.0  104.6 ± 1.6  101.9 ± 2.5  100.2 ± 1.2  105.3 ± 1.9  1,500  102.3 ± 1.6  101.9 ± 0.8  99.7 ± 3.0  95.9 ± 2.3  103.2 ± 2.3  Compound  Conc. (ng/mL)  Long-term (−70°C, 90 days)  Short-term (25°C, 5 h)  Freeze-thaw (four cycles)  Incubation (37°C, 2 h)  Post-preparative (25°C, 24 h)  Rosuvastatin  30  104.8 ± 7.3  105.8 ± 1.3  112.1 ± 9.9  84.5 ± 5.0  107.8 ± 1.6  1,500  98.6 ± 3.1  101.8 ± 5.0  107.4 ± 5.6  80.7 ± 2.7  107.3 ± 2.8  Naringin  30  103.0 ± 1.7  102.4 ± 4.1  97.6 ± 4.0  94.1 ± 2.7  102.0 ± 2.3  1,500  104.4 ± 1.1  102.2 ± 1.5  99.4 ± 0.9  98.3 ± 1.1  103.6 ± 1.0  Naringenin  30  106.7 ± 2.0  104.6 ± 1.6  101.9 ± 2.5  100.2 ± 1.2  105.3 ± 1.9  1,500  102.3 ± 1.6  101.9 ± 0.8  99.7 ± 3.0  95.9 ± 2.3  103.2 ± 2.3  Pharmacokinetic drug interaction study The developed method was successfully applied to pharmacokinetic drug interaction studies between rosuvastatin and naringin in rats. Naringin was just measured in few plasma samples while naringenin was barely detected without incubation. For the purpose of obtaining a full plasma concentration–time curve, total naringin was used to represent the pharmacokinetic of naringin. Together with the measured naringin, the concentrations of naringenin, which determined in plasma sample with incubation, were converted into equal-molar naringin to make up total naringin. The mean plasma concentration–time profiles of rosuvastatin and total naringin with and without (control) co-administration of the other drug are presented in Figure 3, and the main relevant pharmacokinetic parameters from non-compartment model analysis are listed in Table V. After co-administration of rosuvastatin and naringin, the pharmacokinetic parameters of both rosuvastatin and total naringin showed no significant difference compared with corresponding control group, suggesting that the concurrent use of rosuvastatin and naringin basically would not cause pharmacokinetic drug interaction between above two drugs. Figure 3. View largeDownload slide Mean plasma concentration profiles of rosuvastatin (A) and total naringin (B) in rat plasma with and without (control) co-administration of the other (mean ± SD, n = 6). Figure 3. View largeDownload slide Mean plasma concentration profiles of rosuvastatin (A) and total naringin (B) in rat plasma with and without (control) co-administration of the other (mean ± SD, n = 6). Table V. Pharmacokinetic parameters of rosuvastatin and total naringin with and without (control) co-administration of the other drug in rats (mean ± SD, n = 6) Pharmacokinetic parametersa  Rosuvastatin  Total naringin  (—)  (+Naringin)  (—)  (+Rosuvastatin)  AUC(0−t) (μg · L−1 · h−1)  1385.1 ± 838.1  1068.9 ± 319.3  1835.9 ± 503.6  1165.8 ± 744.0  CL (L · h−1 · kg−1)  35.2 ± 21.3  36.2 ± 15.5  23.4 ± 6.8  29.2 ± 17.1  Cmax (μg · L−1)  164.2 ± 84.4  208.2 ± 199.1  456.0 ± 160.9  325.3 ± 314.1  T1/2 (h)  16.2 ± 7.3  13.8 ± 7.4  1.8 ± 0.6  10.2 ± 16.8  Tmax (h)  0.6 ± 0.2  0.6 ± 0.2  4.5 ± 2.0  4.2 ± 2.3  Pharmacokinetic parametersa  Rosuvastatin  Total naringin  (—)  (+Naringin)  (—)  (+Rosuvastatin)  AUC(0−t) (μg · L−1 · h−1)  1385.1 ± 838.1  1068.9 ± 319.3  1835.9 ± 503.6  1165.8 ± 744.0  CL (L · h−1 · kg−1)  35.2 ± 21.3  36.2 ± 15.5  23.4 ± 6.8  29.2 ± 17.1  Cmax (μg · L−1)  164.2 ± 84.4  208.2 ± 199.1  456.0 ± 160.9  325.3 ± 314.1  T1/2 (h)  16.2 ± 7.3  13.8 ± 7.4  1.8 ± 0.6  10.2 ± 16.8  Tmax (h)  0.6 ± 0.2  0.6 ± 0.2  4.5 ± 2.0  4.2 ± 2.3  aAUC, area under the plasma concentration–time curve; CL, apparent plasma clearance; Cmax, maximum plasma concentration; T1/2, elimination half-life; Tmax, time to Cmax. Table V. Pharmacokinetic parameters of rosuvastatin and total naringin with and without (control) co-administration of the other drug in rats (mean ± SD, n = 6) Pharmacokinetic parametersa  Rosuvastatin  Total naringin  (—)  (+Naringin)  (—)  (+Rosuvastatin)  AUC(0−t) (μg · L−1 · h−1)  1385.1 ± 838.1  1068.9 ± 319.3  1835.9 ± 503.6  1165.8 ± 744.0  CL (L · h−1 · kg−1)  35.2 ± 21.3  36.2 ± 15.5  23.4 ± 6.8  29.2 ± 17.1  Cmax (μg · L−1)  164.2 ± 84.4  208.2 ± 199.1  456.0 ± 160.9  325.3 ± 314.1  T1/2 (h)  16.2 ± 7.3  13.8 ± 7.4  1.8 ± 0.6  10.2 ± 16.8  Tmax (h)  0.6 ± 0.2  0.6 ± 0.2  4.5 ± 2.0  4.2 ± 2.3  Pharmacokinetic parametersa  Rosuvastatin  Total naringin  (—)  (+Naringin)  (—)  (+Rosuvastatin)  AUC(0−t) (μg · L−1 · h−1)  1385.1 ± 838.1  1068.9 ± 319.3  1835.9 ± 503.6  1165.8 ± 744.0  CL (L · h−1 · kg−1)  35.2 ± 21.3  36.2 ± 15.5  23.4 ± 6.8  29.2 ± 17.1  Cmax (μg · L−1)  164.2 ± 84.4  208.2 ± 199.1  456.0 ± 160.9  325.3 ± 314.1  T1/2 (h)  16.2 ± 7.3  13.8 ± 7.4  1.8 ± 0.6  10.2 ± 16.8  Tmax (h)  0.6 ± 0.2  0.6 ± 0.2  4.5 ± 2.0  4.2 ± 2.3  aAUC, area under the plasma concentration–time curve; CL, apparent plasma clearance; Cmax, maximum plasma concentration; T1/2, elimination half-life; Tmax, time to Cmax. Discussion To obtain chromatograms with good separation and strong intensity, mobile phase composition and mass spectrometry parameters were optimized. The final composition of the mobile phase was methanol–water both containing 0.1% formic acid, and three analytes and IS exhibited the best peak shapes and eluted within 5.0 min. Under ESI condition, both naringin and naringenin had good sensitivity in negative ionization mode, while rosuvastatin showed opposite characteristics in the same mode. In consideration of the elution time of analytes and IS, polarity switch (negative-positive-negative ionization mode) was performed to determine above compounds in the same analytical run. Transporter-based drug–drug interactions are important to consider in drug development. BCRP and OATP, expressed in enterocyte and hepatocytes, plays an important role in absorption, distribution, and elimination of various drugs and therefore implicated in transporter-based drug–drug interactions (38, 39). The disposition of rosuvastatin, which is a specific substrate of BCRP and OATP 1B1, is markedly affected by the transport activity modulation of these two transporters. Naringin, as well as naringenin, which could inhibit BCRP and OATP 1B1 in vitro (17–21), were likely to modulate the pharmacokinetic behavior of rosuvastatin in vivo. However, as shown in our results, the plasma pharmacokinetic profile of either rosuvastatin or naringin in rosuvastatin and naringin co-administration group have no significant differences compared with that in corresponding control group. That is to say, in vivo results are inconsistent with in vitro data. As shown in previous studies (34–37), naringenin glucuronide and naringenin sulfate, rather than naringin and naringenin, are the predominant existing form in circulatory system after naringin administration. Structural modification of naringenin may lead to the modulation of inhibitory activities on BCRP and OATP 1B1. What is more, the complexities of in vivo conditions make in vivo results may remarkably different with in vitro data which obtained from relatively simple models. Besides naringin, similar antinomy also appeared in pharmacokinetic interaction study between rosuvastatin and silymarin (40). These results remind us that the extrapolations from in vitro data to in vivo results need great caution. Conclusion A rapid and reproducible validated RRLC–MS/MS method for simultaneous determination of rosuvastatin, naringin and naringenin concentrations in rat plasma was reported in the present paper. The method was found to be accurate, precise and specific, and was successfully applied to the pharmacokinetic drug interaction investigation of rosuvastatin and naringin in rats. Results suggest that the co-administration of rosuvastatin and naringin basically would not result in pharmacokinetic drug interaction between these two drugs. Funding The present work was financial supported by the National Major Scientific and Technical Special Project of China (No. 2015ZX09101014) and Applied Science and Technology R&D Special Fund Project of Guangdong Province (No. 2015B020234004). Conflict of interest statement All the authors declare that there are no known conflicts of interest with this publication. Highlights First simultaneous determination of rosuvastatin, naringin and naringenin in rat plasma. Consistent recoveries with no significant matrix effect for three analytes and corresponding internal standard. The pharmacokinetic interaction between rosuvastatin and naringin in rats is firstly reported. References 1 Cheng, J.W.M.; Rosuvastatin in the management of hyperlipidemia; Clinical Therapeutics , ( 2004); 26( 9): 1368– 1387. 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Journal of Chromatographic ScienceOxford University Press

Published: Apr 26, 2018

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