Pharmacokinetic Comparisons of Naringenin and Naringenin-Nicotinamide Cocrystal in Rats by LC-MS/MS.

Naringenin (NAR), 4′,5,7-trihydroxydihydroflavone, has a wide range of pharmacological activities but shows poor water solubility and low bioavailability. The pharmacokinetics and bioavailability of naringenin-nicotinamide cocrystal (NAR-NCT), which offers improved solubility, were evaluated in this study. Rats were orally administered NAR, a physical mixture of naringenin and nicotinamide (NAR + NCT), and NAR-NCT. The relative bioavailability of NAR-NCT was 175.09% of NAR, Cmax was 8.43 and 2.06 times of NAR and NAR + NCT, respectively, Tmax was advanced from 0.49 h to 0.09 h, CL was decreased from 91.1 L/h/kg to 49.1 L/h/kg, and t1/2 was increased from 5.37 h to 8.24 h, highlighting its rapid absorption and slow elimination. This study showed that NAR-NCT could improve the bioavailability of NAR.

Cocrystal is defined as the crystal of combination with active pharmaceutical ingredient and cocrystal former. It with specific physical and chemical properties can form through weak noncovalent interactions, for example, hydrogen bonding [15]. Compared with the parent compound, active pharmaceutical ingredients in the form of cocrystal can improve different properties [16], such as altered stability [17,18], solubility [19], and bioavailability [20,21]. Previously, we prepared naringenin-nicotinamide cocrystal (NAR-NCT) to improve the solubility of NAR in different pH buffer solutions [22]. Pharmacokinetic comparisons of NAR and NAR-NCT after oral administration in rats were investigated in this work. After oral administration of NAR, the physical mixture (NAR + NCT), and NAR-NCT, and the plasma concentrations of NAR were determined by LC-MS/ MS. NAR-NCT can improve the bioavailability of NAR and result in faster absorption and slower elimination.

Preparation and Characterization of NAR-NCT.
e cocrystal was prepared by solvent evaporation. 50 mg of NAR and 45 mg of NCT (at a molar ratio of NAR and NCT of 1 : 2) were mixed with ethyl acetate (10 mL). After ultrasonic dissolution, the mixtures were heated at 40°C for 4 hours [22]. Differential scanning calorimetry (DSC) was performed on a DSC-TGA-STARe thermal analyzer (Mettler, Toledo, Switzerland). X-Ray powder diffraction (XRPD) was used to analyse the changes of NAR-NCT. XRPD was conducted with a D8 ADVANCE powder diffractometer (Bruker, Karlsruhe, Germany). Infrared spectroscopy (IR) was performed on a Spectrometer 400 Fourier-infrared spectrometer (PerkinElmer, Waltham, America). e NAR-NCT was kept at 60°C, at a relative humidity of 90 ± 5%, under light of 5000 Lx for 10 days and after 6 months at room temperature.
AB SCIEX Qtrap 5500 (MDS-Sciex, Concord, Canada) with a negative mode electrospray ionization (ESI) interface was equipped with Shimadzu UFLC-20AD XR. Multiple reaction monitoring was used to analyse samples. Mass spectrometry parameters are shown in Table 1.
Secondary mass spectrometry enhanced product ion (EPI) scan was used to obtain a high-quality MS/MS spectrum on a specific ion. is scan type was performed on NAR and IS. e product ion mass spectra about NAR and IS are recorded in Figure 2. e ratio of peak area about NAR to IS was used to determine concentrations of NAR. Data acquisition and analysis were calculated by Analyst 1.6 software. After that, to minimize the contamination of the sample in the mass spectrometer, after the peaks of NAR and IS had eluted, the remaining components were sent from the LC to the waste valve instead of proceeding through the MS system.

Standard Working Solutions and Internal Standard
Solution. NAR was dissolved in methanol to obtain a 102.6 μg/mL stock solution. It was diluted with methanol to obtain standard working solutions of 10.3, 30.8, 102.6, 513, 1026, 2052, 4104, 6156, and 8208 ng/mL. Hesperidin was dissolved in methanol (300 ng/mL) as an internal standard solution.

Calibration Standards and Quality Control (QC) Samples.
e calibration standards of NAR at a range of 1.03 to 821 ng/mL, which were prepared from blank plasma and NAR standard working solutions.
QC samples were formulated at low, medium, and high concentrations (LQC, MQC, and HQC) corresponding to NAR of 3.08, 410, and 616 ng/mL, respectively, following the procedure described above. e samples were protected from light.

Pretreatment of the Plasma Samples.
Plasma (100 μL) was combined with 10 μL of 300 ng/mL internal standard solution in a centrifuge tube, and the mixture was vortexed for 5 min. en, samples were mixed with ethyl acetate (300 μL), and mixtures were vortex-mixed for 5 min. After that, mixtures were centrifuged at 8000 r/min for 2 min. All supernatants were pipetted into a new tube and dried at 40°C under flowing nitrogen. And then, residues were reconstituted with methanol (100 μL) and vortex-mixed for 1 min.

Method Validations.
All method validations complied with US Food and Drug Administration guidelines [23].

Specificity.
To demonstrate that substances being measured were intended analytes and endogenous substances and other metabolites do not interfere with the assay, method specificity was established firstly. is analysis involved blank plasma samples (different sources), the mixture of NAR standard solution and blank plasma, and plasma samples after oral administration (n � 6).

e Calibration Curve and Lower Limit of Quantification (LLOQ).
Six blank rat plasma samples were combined with the NAR standard working solutions to give final NAR plasma concentrations of 1.03, 10.3, 51.3, 103, 205, and 821 ng/mL. As described in Section 2.6, the NAR plasma concentration (ng/mL) was taken as the abscissa, and the ratio of the NAR to IS peak areas was taken as the ordinate and by a weighted least-squares linear regression (the weighting factor 1/X 2 ) to obtain the calibration curve. e lowest concentration of analyte that can be reliably quantified in the sample was the LLOQ. Six standard plasma samples containing NAR were analysed as described in Section 2.6. At the LLOQ, the relative standard deviation (RSD, %) was ≤20%, the relative error (RE, %) was required to be within ±20%.

Precision and Accuracy.
NAR plasma standard samples were prepared at the LLOQ, LQC, MQC, and HQC concentrations (1.03, 3.08, 410, and 616 ng/mL), according to Section 2.6. Six samples were prepared at each concentration. e concentration of each sample was determined on the same day and three consecutive days to assess the intraand interday precision and accuracy. e acceptance criteria were RSD (%) was ≤15%, RE (%) was limited to ±15% for the intra-and interday, except at the LLOQ, where RSD (%) should be ≤20% and RE (%) within ±20%.

Extraction Recovery (R)
. NAR plasma standard samples were prepared at the LLOQ, LQC, MQC, and HQC concentrations (1.03, 3.08, 410, and 616 ng/mL). en, internal standard solution (10 μL) was added to the mixture, vortexed for 5 min, and added ethyl acetate (300 μL). After that, the samples were centrifuged at 8000 r/min for 2 min. All supernatant was transferred to a new tube. All of them were concentrated to dryness under flowing nitrogen, and the residue was redissolved in methanol (200 μL) to prepare it for analysis. e peak area of NAR (As) was recorded.
Blank plasma samples (100 μL) from different sources were mixed with ethyl acetate (300 μL). e samples were vortexed for 5 min, then centrifuged for 2 min at 8000 r/min, and then all supernatant was removed for analysis. Aliquots  Journal of Analytical Methods in Chemistry 10 μL of the standard solutions (10.3, 30.8, 4104, and 6156 ng/mL) and 10 μL of 300 ng/mL internal standard solution were added. All of them were concentrated to dryness under flowing nitrogen, and the residues were redissolved in methanol (200 μL) for analysis. e peak area of NAR (As′) was recorded. e extraction recovery (%) of NAR was obtained by substituting As and As′ into the following equation: e recovery of the IS was investigated similarly.
Blank plasma samples (100 μL), from different sources, were each mixed with ethyl acetate (300 μL), and the mixture was vortexed (5 min) and centrifuged (8000 r/min, 2 min). All supernatant of each sample was removed to a new tube, and standard solution (10 μL, at concentrations of 10.3, 30.8, 4104, and 6156 ng/mL) was added to the internal standard solution (10 μL, 300 ng/mL). e sample was dried at 40°C under flowing nitrogen. Methanol (200 μL) was used to reconstitute in the residue. e peak area of NAR (Bs′) was recorded.
e matrix effect (%) on NAR was calculated according to the following formula: e ME of the IS was investigated.

Dilution Reliability.
e dilution reliability of NAR in plasma must be examined because of the high concentrations (beyond the calibration curve) of some drugs in the plasma. e plasma sample (NAR, 6156 ng/mL) was diluted with blank plasma 10 times and analysed according to Section 2.6. After sample analysis, the ratio of peak area of NAR to IS was compared to the calibration curve, and RE and RSD values were also calculated (n = 6). Accuracy of the concentration determined following dilution was required to be between 85% and 115%, and the precision should be within 15%.

Pharmacokinetic Study.
Eighteen SD rats (200 ± 20 g, 8 weeks, half male and half female) were obtained from National Institutes for Food and Drug Control (Beijing, China).
e room environment was about 22 ± 2°C, the humidity of 50 ± 10%, light and dark recycle for 12 h. Before the experiment, the rats can be given water freely but were fasted for 12 h. All experiments were following the National Institutes of Health Guide for the Care and Use of Laboratory Animals [24].
ere were three groups for oral administration. e first group was administered NAR (30 mg/kg). e next group was administered NAR + NCT, in which the molar ratio of NAR and NCT was 1 : 2, in terms of NAR 30 mg/kg. e last group was administered NAR-NCT (57 mg/kg), which was equal to the NAR dose of 30 mg/kg. ese were ground with 0.5% sodium carboxymethyl cellulose, at a dose of 1 mL/ 100 g. At 0.05, 0.083, 0.167, 0.333, 0.5, 1, 2, 4, 8, 10, 12, and 24 h after administration, 0.5 mL serial blood samples were obtained from the ocular vein into centrifuge tubes. e samples were centrifuged at 10000r/min (10 min, 4°C), and after that, all supernatant of each sample was removed to store at −80°C.

Data Analysis.
Noncompartmental modelling was evaluated for pharmacokinetic parameters through DAS Software (China State Drug Administration, version 3.2.4). e following equation was used to calculate the relative oral bioavailability (Fr, %) in rats:

NAR-NCT Cocrystal.
e NAR-NCT cocrystal was prepared and characterized, which can improve solubility in water. e DSC endothermic peak of the NAR-NCT was 134.68°C. e characteristic peaks in the DSC, XRPD, and IR spectra of NAR-NCT were different from the naringeninnicotinamide cocrystal reported in the literature [25]. It is shown that a new NAR-NCT cocrystal was formed. e DSC, XRPD, and IR spectra are shown in Figure 3.

Stability of NAR-NCT.
e stability of NAR-NCT was investigated at 60°C, at a relative humidity of 90 ± 5%, under light of 5000 Lx for 10 days and after 6 months at room temperature. In terms of appearance, NAR-NCT showed no difference under the above conditions. A comparison of the XRPD spectra (Figure 4) showed that the overall peak shape, position, and intensity are not a significant difference, indicating that the NAR-NCT was stable at 60°C, at a relative humidity of 90 ± 5%, under light of 5000 Lx for 10 days and after 6 months at room temperature.

Optimization of the Plasma Sample Preparation.
Protein precipitation and liquid-liquid extraction methods were tested for the removal of interfering species from the sample. Organic solvents, such as methanol, acetonitrile, diethyl ether, ethyl acetate, and n-hexane were investigated, and the results are shown in Figure 5. e extraction effect of ethyl acetate was the best, so the extraction effects of 100, 200, 300, and 400 μL of ethyl acetate were analysed. Results showed that ethyl acetate (300 μL) had the best extraction effect. erefore, ethyl acetate (300 μL) was used to the extracted solvent for pretreatment.

Method Validation.
e blank plasma sample showed no signals interfering with the measurement of the analyte. NAR and IS were well separated from endogenous substances and metabolites, and no interference was observed. e chromatograms are shown in Figure 6. e calibration curve showed good linearity, at the range of 1.03-821 ng/mL, Y � 0.11X + 0.0914, and r � 0.9995. e standard error of slope and intercept was 0.002 and 0.005, respectively. LLOQ was 1.03 ng/mL for the NAR plasma sample. e results of precision and accuracy during intraday and interday were as follows: both RSD (%) values were
After the plasma QC samples were diluted with blank plasma, the RE ranged from −8.1%% to 1.9%, and the RSD was 4.7% (Table 3). ese results met the requirements of methodological validation.     Journal of Analytical Methods in Chemistry

Pharmacokinetics and Bioavailability.
e plasma concentrations of NAR were determined by LC-MS/MS. e concentrations were analysed with DAS Software. e main pharmacokinetic parameters are recorded in Table 4, and the mean plasma concentration versus time curves for NAR is shown in Figure 7.
Compared with NAR, the C max of NAR + NCT was greater by a factor of 2.06, and T max changed from 0.49 h to 0.11 h. Although NAR + NCT accelerated drug absorption, it did not improve drug metabolism. e values of CL and t 1/2 showed that the elimination rates of the two groups were similar. As a result, the relative bioavailability was 77.23% of NAR.
A comparison between NAR-NCT and NAR showed that C max was increased by a factor of 8.43, and T max was advanced from 0.49 h to 0.09 h. In contrast to NAR + NCT, the CL of NAR-NCT decreased from 91.1 L/h/kg to 49.1 L/h/kg, and t 1/2 increased from 5.37 h to 8.24 h, showing the rapid absorption and slow elimination of this formulation. e relative bioavailability of NAR-NCT was 175.09% of NAR, compared with 80.89% reported in the literature [25]. is result showed that NAR-NCT can improve bioavailability and has the advantages of fast absorption and slow elimination. e above phenomenon conformed to the hypothesis [26]. NCT was easily soluble in water, and in aqueous biological environments, NCT was more easily detached from the crystal lattice and readily entered into solution. en, the NAR particles remained in the solution medium. Because the rate of NAR-NCT dissolution was faster than the formation of supramolecular aggregates of the NAR molecule itself, C max increased and T max decreased. After the dissolution of NCT, the amorphous NAR, with high free energy, slowly converted to a metastable state and then to the lowest energy NAR configuration (usually the pure drug). e time required for this process resulted in slower NAR elimination and increased AUC in the body.  0  20  40  60  80  100  120  140  160  180  200  220  240 Intensity, cps Journal of Analytical Methods in Chemistry peak at 0.5 h. And the last points of the pharmacokinetic study in the three groups were less than 1/10 of C max , respectively. After 0.5 h, a longer interval of blood collection was set. e rats could be allowed to drink water freely in longer blood sampling intervals. erefore, the process of blood sampling did not have a serious impact on the pharmacokinetics of NAR.
Only the drug-time curve of NAR-NCT showed the double peaks, which was consistent with enterohepatic circulation [27] and extended NAR action time in the body.

Conclusions
We investigated the differences in pharmacokinetic characteristics and the relative bioavailability of NAR after oral  175.09 * * * * P < 0.0001, * * * P < 0.001, * * P < 0.01, and * P < 0.05.  administration of NAR, NAR + NCT, and NAR-NCT in rats. ese results show that NAR-NCT was a viable method for improving the bioavailability of NAR. ese results provide important references for the application and research of NAR in the future.

Data Availability
All data included in this study are available upon request from the corresponding author.

Conflicts of Interest
e authors declare they have no conflicts of interest.