Journal of Chromatography B, 978–979 (2015) 157–162
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Simultaneous quantification of naproxcinod and its active metabolite naproxen in rat plasma using LC–MS/MS: Application to a pharmacokinetic study Xiaowei Shi a , Weiding Shang a , Shuang Wang a , Na Xue b , Yanxia Hao b , Yabo Wang a , Mengmeng Sun c , Yumin Du a , Deying Cao a , Kai Zhang a,∗ , Qingwen Shi a,∗ a
School of Pharmaceutical Sciences, Hebei Medical University, Shijiazhuang 050017, China Department of Pharmaceutical Engineering, Hebei Chemical and Pharmaceutical College, Shijiazhuang 050026, China c The Second Hospital, Hebei Medical University, Shijiazhuang 050000, China b
a r t i c l e
i n f o
Article history: Received 18 September 2014 Accepted 3 December 2014 Available online 11 December 2014 Keywords: Naproxcinod Naproxen LC–MS/MS Pharmacokinetic study
a b s t r a c t In this study, a liquid chromatography–tandem mass spectrometry method was developed and validated to simultaneously determine naproxcinod and naproxen concentrations in rat plasma for the first time. Plasma samples were prepared by simple one-step extraction with methanol for protein precipitation using only 50 L plasma. Separation was performed on a Synergi Fusion-RP C18 column with a run time of 4 min. Naproxcinod, naproxen and internal standard concentrations were detected in the positive ion mode using multiple reaction monitoring (MRM) of the transitions at m/z 348.2 → 302.2, 231.1 → 185.1 and 271.2 → 203.1, respectively. The calibration curves were linear, with all correlation coefficients being ≥0.9952, in the range of 1.00–400 ng/mL for naproxcinod and 20.0–8000 ng/mL for naproxen. Their accuracy was in the range of −8.1% to 8.7%, and the intra- and inter-day variations were ≤4.53%. The mean extraction recovery of all analytes was more than 93.1% efficient. Stability testing showed that naproxcinod and naproxen remained stable during the whole analytical procedure. After validation, the method was successfully applied to a pharmacokinetic study of naproxcinod and naproxen in rats. The AUC0–∞ of naproxen was 74.6 times larger than that of naproxcinod, which indicated that naproxcinod was rapidly metabolized into naproxen in rats. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Naproxcinod is a CINOD (cyclooxygenase-inhibiting nitric oxide-donating) anti-inflammatory candidate [1,2]. Naproxcinod improved safety profile and provide protection from the effects of naproxen on the gastrointestinal tract and other organs [3]. In October 2013, the European Commission granted the Orphan Drug Designation for the use of naproxcinod in the treatment of Duchenne muscular dystrophy (DMD). DMD, the most common and serious form of muscular dystrophy, is part of a group of inherited diseases causing muscle weakness and muscle loss [4,5]. Long-term treatment with naproxcinod significantly improves the skeletal and cardiac disease phenotype in the mdx mouse model of dystrophy [5].
∗ Corresponding authors. Tel.: +86 31186265624. E-mail addresses: [email protected], [email protected] (K. Zhang), [email protected] (Q. Shi). http://dx.doi.org/10.1016/j.jchromb.2014.12.001 1570-0232/© 2014 Elsevier B.V. All rights reserved.
Naproxcinod is very extensively metabolized to naproxen, nitrate and other metabolites. After administration of a therapeutically relevant oral dose, the plasma concentration of naproxcinod was very low and variable, and no accumulation was apparent. [6]. In clinical pharmacokinetics studies, the oral bioavailability (F) of naproxcinod appears low, with only a few percent at the maximum, and increases by several fold with food intake [7]. Very few methods for the determination of naproxcinod enantiomers concentration by chiral normal-phase liquid chromatography have been reported in the literature [8]. A RP-HPLC method was reported for the separation of naproxcinod and related substances which used a fused core particle technology C8 column [9]. Naproxcinod concentration was also analyzed by LC–MS/MS (LOQ 4 nM, 1.4 ng/mL), and naproxen was analyzed by liquid chromatography with ultraviolet (UV) or fluorescence detection (LOQ 0.5 M, 115.5 ng/mL). These methods were described in the literature without a detailed information and validation [6]. However, until now, no approaches for the simultaneous determination of naproxcinod and its active metabolite naproxen in biological
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samples have been published. In the present study, we developed and validated a rapid and sensitive LC–MS/MS method to simultaneously determine naproxcinod and naproxen concentrations in rat plasma. This method was successfully applied to the pharmacokinetic study of naproxcinod in rats, after intragastric administration. 2. Experimental 2.1. Chemicals and reagents Crude naproxcinod drug and its standards (>99.5% purity) were supplied by the laboratory of medicinal chemistry at the Hebei Medical University. Naproxen (>99.5% purity) and isoimperatorin (internal standard, IS; >98.0% purity) were obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). HPLC-grade methanol and acetic acid were purchased from Dikma Technology (CA, USA). Deionized water was purified using a Milli-Q plot water purification system (MA, USA). 2.2. Instrumentation The LC–MS system consisted of an Agilent 1200 series HPLC system (Agilent Technologies, Palo Alto, CA, USA) equipped with a quaternary solvent delivery system, an autosampler and a column compartment as well as a 3200 QTRAPTM system with an electrospray ionization (ESI) source (AB SCIEX, CA, USA). The AnalystTM software (versions 1.5.2) was used for data acquisition and processing. 2.3. LC–MS/MS analytical conditions Chromatography was performed on a Synergi Fusion-RP C18 column (50 mm × 3.0 mm, 4 m) from Phenomenex (MA, USA). The mobile phase was made of a 0.1% formic acid aqueous solution–methanol mixture (28:72, v/v) and was delivered at a flow rate of 0.5 mL/min. The total run time was 4 min, and the injection size was 10 L. The mass spectrometer was operated in the positive multiple reaction monitoring (MRM) mode. The mass transitions were: naproxcinod, m/z 348.2 → 302.2; naproxen, m/z 231.1 → 185.1; IS, m/z 271.2 → 203.1. The electrospray ion source and MS/MS parameters were set as follows: ion spray voltage, 5500 V; temperature, 650 ◦ C; curtain gas, 25 psi; nebulizer gas (GS1), 60 psi; heater gas (GS2), 65 psi. The adjusted values of declustering potential (DP) and collision energy (CE) were 40 V and 15 eV for naproxcinod, 38 V and 21 eV for naproxen and 25 V and 15 eV for IS. 2.4. Preparation of standard solutions Stock solutions of naproxcinod and naproxen were prepared in methanol at a concentration of 500 g/mL. Stock solutions were then mixed and further diluted with methanol to prepare the mixed standard working solutions, in the concentration range of 2.5–1000 ng/mL for naproxcinod and 50.0–20,000 ng/mL for naproxen. The stock solution of IS prepared in methanol (200 g/mL) was further diluted with methanol to 2 g/mL to prepare the IS working solution. All of the solutions were stored at 4 ◦ C. 2.5. Sample preparation Twenty-microliter aliquots of methanol and 20.0 L of IS solution diluted with 100 L methanol were added to 50.0 L of plasma sample. The mixture was vortexed for 30 s and then centrifuged at
16,654 × g for 10 min. Ten microliters of supernatant was injected in the LC–MS/MS apparatus for analysis. 2.6. Preparation of calibration standards and quality control (QC) samples The calibration standards were prepared by adding 20.0 L of mixed standard working solutions, 20.0 L of IS solution and 100 L of methanol to 50.0 L of blank rat plasma at the following concentrations: 1.00, 2.00, 5.00, 20.0, 50.0, 100, 200 and 400 ng/mL for naproxcinod; 20.0, 40.0, 100, 400, 1000, 2000, 4000 and 8000 ng/mL for naproxen; 800 ng/mL for IS. The QC samples were prepared in 50.0 L blank plasma at four different concentrations (1.00, 2.00, 50 and 320 ng/mL for naproxcinod and 20.0, 40.0, 1000 and 6400 ng/mL for naproxen). 2.7. Method validation The method was validated according to the FDA and EMA guidelines for industry bioanalytical method validation [10–13]. The selectivity of the method was assessed by analyzing 6 blank plasma samples, blank plasma samples spiked with analytes at the lower limit of quantification (LLOQ) level, blank plasma samples spiked with IS and plasma samples obtained 30 min after drug administration using the proposed extraction procedure as well as chromatographic and mass spectroscopic conditions. Carryover was assessed by injecting blank plasma samples directly after an upper LOQ sample during an analytical run. The carryover test was successful when no interfering peaks with areas greater than 20% of the LLOQ level peak area for each analyte and 5% of the IS peak area were detected. Linearity was evaluated by an eight-point calibration curve with plasma, prepared and assayed in duplicate on three consecutive days. Daily calibration curves were constructed using weighed (1/x2 ) least-square linear regression of the ratios between analytes and IS peak areas versus the nominal standard concentrations. Accuracy for the LLOQ should be within ±20%, and its precision should be less than 20% in six replicates on three consecutive days. Precision and accuracy were assessed by analyzing the concentrations of QC samples at four concentrations using six replicates on three consecutive days. Accuracy and precision were expressed by relative error (RE) and relative standard deviation (RSD), respectively. Recovery of analytes was calculated by comparing the peak areas of QC samples (four concentrations in three replicates) throughout the extraction procedure with the post-extraction mean peak area for blank plasma samples spiked at the corresponding concentrations. The recovery of IS was evaluated in a similar way at the concentration of 800 ng/mL. The matrix effect on analytes ionization was assessed by comparing the post-extraction peak areas of blank plasma samples spiked at the concentration of QC samples (A) with the peak areas of the corresponding water-substituted samples (B). The blank plasma used in this study was obtained from six different groups of healthy individuals. The ratio (A/B × 100) is defined as the matrix effect. The matrix effects of internal standards were determined in the same way. In dilution tests, blank plasma sample spiked with 1.00 g/mL naproxcinod and 20.0 g/mL naproxen were diluted 5, 20 and 200fold with blank plasma, in order to prepare the plasma samples containing 200, 50 and 5 ng/mL naproxcinod and 4000, 1000 and 100 ng/mL naproxen separately. Other plasma samples of the corresponding concentrations were prepared directly for comparison. The dilution test was performed by the relative coefficient of variation between the spiked plasma samples peak areas after dilution and the peak areas of samples prepared directly without dilution.
X. Shi et al. / J. Chromatogr. B 978–979 (2015) 157–162
159
Fig. 1. Product ion spectra of naproxcinod, naproxen and IS.
The stability of naproxcinod and naproxen plasma samples were examined under different conditions, such as short-term stability at room temperature for 6 h, post-preparative stability at 4 ◦ C for 24 h, long-term stability at −20 ◦ C for 7 days and after three complete freeze–thaw cycles. Furthermore, the stock solution stability of naproxcinod, naproxen and IS was also assessed at −20 ◦ C for 22 days. 2.8. Application Six male Sprague-Dawley rats (approximately 250 g body weight) were provided by the Laboratory Animal Center of Hebei Medical University. The animal experiment protocols were approved by the Animal Center of Hebei Medical University. Animals were maintained at ambient temperature (22–24 ◦ C) and 60% relative humidity with a 12 h light/dark cycle and kept in an environmentally controlled breeding room for 3 days. Animals were fasted for 12 h (with ad libitum access to water) prior to the experiments. The rats were given an intragastric oral administration of 6.0 mg/kg naproxcinod dissolved in 0.5% sodium carboxymethyl cellulose (CMC). Blood samples (0.3 mL) were collected from the posterior orbital vein using heparinized tubes before (0 h) and at 0.15, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 12, 24, 36 and 48 h following the oral administration. The rats were euthanized immediately after the collection procedure using ethyl ether. The blood was centrifuged at 1359 × g for 10 min to separate red blood cells and plasma. Plasma samples were stored at −20 ◦ C until further analysis. Pharmacokinetic parameters including area under the curve (AUC), mean retention time (MRT), elimination half-life (t1/2 ), plasma clearance (CL) and apparent distribution volume (Vd ) for naproxcinod and naproxen were calculated using the noncompartmental method and the DAS 3.2 pharmacokinetic program (Chinese Pharmacology Society), respectively. Cmax (the maximum peak concentration) and Tmax (the time to Cmax ) were obtained directly from the curves.
sensitivity in positive mode than in negative mode. [M + H]+ were the most abundant ions in the Q1 full-scan spectra for all the analytes, which were used as the precursor ion. The full-scan product ion spectra for each analyte is provided in Fig. 1. Parameters including DP and CE were optimized, and the recommended value of the instrument was used for all other parameters. 3.1.2. Liquid chromatography The Synergi Fusion-RP C18 column (Phenomenex, USA) was optimal compared with the tested C18 columns, including Zorbax SB C18 (Agilent, USA), Luna C18 (Phenomenex, USA), and Diamonsil C18(2) (Dikma, China). The composition of the mobile phase was optimized to achieve satisfactory chromatographic behavior, such as high sensitivity, good peak symmetry, short run time and appropriate ionization. Methanol provided higher responses and lower background noise compared with acetonitrile and was therefore chosen as the organic phase. Sensitivity and peak symmetry of naproxcinod, naproxen and IS were significantly improved by adding formic acid to the mobile phase. The optimal concentration of formic acid in mobile phase was determined empirically (tested range from 0.01% to 0.2%) and the addition of 0.1% formic acid provided a significant sensitivity improvement for naproxcinod, naproxen and IS. Therefore, a solution of methanol–0.1% formic acid in water (72:28, v/v) was selected for the mobile phase which, with a total run time of 4.0 min, met the requirements for high throughput bioanalytical analyses. 3.2. Method validation
3.1. Method development
3.2.1. Selectivity Selectivity was assessed by comparing the chromatograms of six different batches of blank plasma with their corresponding spiked plasma. Typical HPLC–MS/MS chromatograms of naproxcinod, naproxen and IS are presented in Fig. 2. The retention times of naproxcinod, naproxen and IS were 3.1, 1.2 and 2.2 min, respectively. There was no significant interference from endogenous substances observed at the analytes retention times. A carryover experiment was performed by injecting blank samples after an upper LOQ sample. Results showed the absence of carryover.
3.1.1. Mass spectrometry To optimize ESI conditions, naproxcinod, naproxen and IS were dissolved in mobile phase and directly injected into the mass spectrometer. Naproxcinod, naproxen and IS displayed a higher
3.2.2. Linear and LLOQ Linearity was evaluated based on the average of the eight-point calibration curves analyzed in three separate batches. Calibration curves were linear within concentration ranges of 1.00–400 ng/mL
3. Results and discussion
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Fig. 2. Typical LC–MS/MS chromatograms of naproxcinod, naproxen and IS in rat plasma. (A) Blank plasma sample, (B) plasma sample spiked with 1.00 ng/mL of naproxcinod and 20.0 ng/mL of naproxen and (C) plasma sample at 0.5 h after an oral administration of 6 mg/kg naproxcinod.
for naproxcinod and 20.0–8000 ng/mL for naproxen. The typical standard curve was y = 0.00627x + 0.00136 (r = 0.9985) for naproxcinod and y = 0.00357x + 0.00548 (r = 0.9952) for naproxen, where y represents the ratio of the analyte peak area to that of the IS and x represents the plasma concentration of the analyte. The LLOQ for naproxcinod and naproxen are 1.00/20.0 ng/mL, indicating that the method was more sensitive than those of previous studies regarding the determination of naproxcinod (LOQ 4 nM, 1.4 ng/mL) and naproxen (LOQ 0.5 M, 115.5 ng/mL) [6], and the precision and accuracy met the relevant criteria. 3.2.3. Precision and accuracy Intra- and inter-day precision and accuracy values of the QC samples are listed in Table 1. In this assay, the intra- and inter-day precision values were both ≤4.53% for naproxcinod and naproxen and the average intra- and inter-day accuracy values ranged from −8.1% to 8.7%. These results indicated an excellent precision and accuracy for the quantification of naproxcinod and naproxen in plasma using the current method. 3.2.4. Recovery and matrix effect The extraction recovery of naproxcinod ranged from 93.1% to 98.6% at four QC concentrations (Table 2), and the recovery of naproxen ranged from 94.7% to 97.8% at four QC concentrations. The extraction recovery of IS was 96.5% (Table 2).
The matrix effect for each analyte was evaluated by the ratios between the corresponding post-extraction peak areas of spiked plasma and of the corresponding water-substituted references. The matrix effect values for naproxcinod were 94.7–99.5%, 95.6–101.7% for naproxen and 98.2% for IS (Table 2). These results suggested that the matrix effect for naproxcinod, naproxen and IS were negligible under the LC–MS/MS conditions used in the present study.
3.2.5. Dilution tests The RE between the peak areas of spiked plasma samples after dilution (5, 20 and 100-fold) and samples prepared directly without dilution were −2.59%, −0.04% and −3.15%, respectively. The corresponding relative coefficient of variation were −7.65%, −0.59% and −5.68%, respectively.
3.2.6. Stability The stability analysis was performed under similar conditions to those described above, and the results are presented in Table 3. Naproxcinod and naproxen remained stable during the whole analytical period. The stock solutions for all analytes were stable after 22 days of storage at −20 ◦ C, exhibiting a RSD and a RE
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Simultaneous quantification of naproxcinod and its active metabolite naproxen in rat plasma using LC–MS/MS: Application to a pharmacokinetic study Xiaowei Shi a , Weiding Shang a , Shuang Wang a , Na Xue b , Yanxia Hao b , Yabo Wang a , Mengmeng Sun c , Yumin Du a , Deying Cao a , Kai Zhang a,∗ , Qingwen Shi a,∗ a
School of Pharmaceutical Sciences, Hebei Medical University, Shijiazhuang 050017, China Department of Pharmaceutical Engineering, Hebei Chemical and Pharmaceutical College, Shijiazhuang 050026, China c The Second Hospital, Hebei Medical University, Shijiazhuang 050000, China b
a r t i c l e
i n f o
Article history: Received 18 September 2014 Accepted 3 December 2014 Available online 11 December 2014 Keywords: Naproxcinod Naproxen LC–MS/MS Pharmacokinetic study
a b s t r a c t In this study, a liquid chromatography–tandem mass spectrometry method was developed and validated to simultaneously determine naproxcinod and naproxen concentrations in rat plasma for the first time. Plasma samples were prepared by simple one-step extraction with methanol for protein precipitation using only 50 L plasma. Separation was performed on a Synergi Fusion-RP C18 column with a run time of 4 min. Naproxcinod, naproxen and internal standard concentrations were detected in the positive ion mode using multiple reaction monitoring (MRM) of the transitions at m/z 348.2 → 302.2, 231.1 → 185.1 and 271.2 → 203.1, respectively. The calibration curves were linear, with all correlation coefficients being ≥0.9952, in the range of 1.00–400 ng/mL for naproxcinod and 20.0–8000 ng/mL for naproxen. Their accuracy was in the range of −8.1% to 8.7%, and the intra- and inter-day variations were ≤4.53%. The mean extraction recovery of all analytes was more than 93.1% efficient. Stability testing showed that naproxcinod and naproxen remained stable during the whole analytical procedure. After validation, the method was successfully applied to a pharmacokinetic study of naproxcinod and naproxen in rats. The AUC0–∞ of naproxen was 74.6 times larger than that of naproxcinod, which indicated that naproxcinod was rapidly metabolized into naproxen in rats. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Naproxcinod is a CINOD (cyclooxygenase-inhibiting nitric oxide-donating) anti-inflammatory candidate [1,2]. Naproxcinod improved safety profile and provide protection from the effects of naproxen on the gastrointestinal tract and other organs [3]. In October 2013, the European Commission granted the Orphan Drug Designation for the use of naproxcinod in the treatment of Duchenne muscular dystrophy (DMD). DMD, the most common and serious form of muscular dystrophy, is part of a group of inherited diseases causing muscle weakness and muscle loss [4,5]. Long-term treatment with naproxcinod significantly improves the skeletal and cardiac disease phenotype in the mdx mouse model of dystrophy [5].
∗ Corresponding authors. Tel.: +86 31186265624. E-mail addresses: [email protected], [email protected] (K. Zhang), [email protected] (Q. Shi). http://dx.doi.org/10.1016/j.jchromb.2014.12.001 1570-0232/© 2014 Elsevier B.V. All rights reserved.
Naproxcinod is very extensively metabolized to naproxen, nitrate and other metabolites. After administration of a therapeutically relevant oral dose, the plasma concentration of naproxcinod was very low and variable, and no accumulation was apparent. [6]. In clinical pharmacokinetics studies, the oral bioavailability (F) of naproxcinod appears low, with only a few percent at the maximum, and increases by several fold with food intake [7]. Very few methods for the determination of naproxcinod enantiomers concentration by chiral normal-phase liquid chromatography have been reported in the literature [8]. A RP-HPLC method was reported for the separation of naproxcinod and related substances which used a fused core particle technology C8 column [9]. Naproxcinod concentration was also analyzed by LC–MS/MS (LOQ 4 nM, 1.4 ng/mL), and naproxen was analyzed by liquid chromatography with ultraviolet (UV) or fluorescence detection (LOQ 0.5 M, 115.5 ng/mL). These methods were described in the literature without a detailed information and validation [6]. However, until now, no approaches for the simultaneous determination of naproxcinod and its active metabolite naproxen in biological
158
X. Shi et al. / J. Chromatogr. B 978–979 (2015) 157–162
samples have been published. In the present study, we developed and validated a rapid and sensitive LC–MS/MS method to simultaneously determine naproxcinod and naproxen concentrations in rat plasma. This method was successfully applied to the pharmacokinetic study of naproxcinod in rats, after intragastric administration. 2. Experimental 2.1. Chemicals and reagents Crude naproxcinod drug and its standards (>99.5% purity) were supplied by the laboratory of medicinal chemistry at the Hebei Medical University. Naproxen (>99.5% purity) and isoimperatorin (internal standard, IS; >98.0% purity) were obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). HPLC-grade methanol and acetic acid were purchased from Dikma Technology (CA, USA). Deionized water was purified using a Milli-Q plot water purification system (MA, USA). 2.2. Instrumentation The LC–MS system consisted of an Agilent 1200 series HPLC system (Agilent Technologies, Palo Alto, CA, USA) equipped with a quaternary solvent delivery system, an autosampler and a column compartment as well as a 3200 QTRAPTM system with an electrospray ionization (ESI) source (AB SCIEX, CA, USA). The AnalystTM software (versions 1.5.2) was used for data acquisition and processing. 2.3. LC–MS/MS analytical conditions Chromatography was performed on a Synergi Fusion-RP C18 column (50 mm × 3.0 mm, 4 m) from Phenomenex (MA, USA). The mobile phase was made of a 0.1% formic acid aqueous solution–methanol mixture (28:72, v/v) and was delivered at a flow rate of 0.5 mL/min. The total run time was 4 min, and the injection size was 10 L. The mass spectrometer was operated in the positive multiple reaction monitoring (MRM) mode. The mass transitions were: naproxcinod, m/z 348.2 → 302.2; naproxen, m/z 231.1 → 185.1; IS, m/z 271.2 → 203.1. The electrospray ion source and MS/MS parameters were set as follows: ion spray voltage, 5500 V; temperature, 650 ◦ C; curtain gas, 25 psi; nebulizer gas (GS1), 60 psi; heater gas (GS2), 65 psi. The adjusted values of declustering potential (DP) and collision energy (CE) were 40 V and 15 eV for naproxcinod, 38 V and 21 eV for naproxen and 25 V and 15 eV for IS. 2.4. Preparation of standard solutions Stock solutions of naproxcinod and naproxen were prepared in methanol at a concentration of 500 g/mL. Stock solutions were then mixed and further diluted with methanol to prepare the mixed standard working solutions, in the concentration range of 2.5–1000 ng/mL for naproxcinod and 50.0–20,000 ng/mL for naproxen. The stock solution of IS prepared in methanol (200 g/mL) was further diluted with methanol to 2 g/mL to prepare the IS working solution. All of the solutions were stored at 4 ◦ C. 2.5. Sample preparation Twenty-microliter aliquots of methanol and 20.0 L of IS solution diluted with 100 L methanol were added to 50.0 L of plasma sample. The mixture was vortexed for 30 s and then centrifuged at
16,654 × g for 10 min. Ten microliters of supernatant was injected in the LC–MS/MS apparatus for analysis. 2.6. Preparation of calibration standards and quality control (QC) samples The calibration standards were prepared by adding 20.0 L of mixed standard working solutions, 20.0 L of IS solution and 100 L of methanol to 50.0 L of blank rat plasma at the following concentrations: 1.00, 2.00, 5.00, 20.0, 50.0, 100, 200 and 400 ng/mL for naproxcinod; 20.0, 40.0, 100, 400, 1000, 2000, 4000 and 8000 ng/mL for naproxen; 800 ng/mL for IS. The QC samples were prepared in 50.0 L blank plasma at four different concentrations (1.00, 2.00, 50 and 320 ng/mL for naproxcinod and 20.0, 40.0, 1000 and 6400 ng/mL for naproxen). 2.7. Method validation The method was validated according to the FDA and EMA guidelines for industry bioanalytical method validation [10–13]. The selectivity of the method was assessed by analyzing 6 blank plasma samples, blank plasma samples spiked with analytes at the lower limit of quantification (LLOQ) level, blank plasma samples spiked with IS and plasma samples obtained 30 min after drug administration using the proposed extraction procedure as well as chromatographic and mass spectroscopic conditions. Carryover was assessed by injecting blank plasma samples directly after an upper LOQ sample during an analytical run. The carryover test was successful when no interfering peaks with areas greater than 20% of the LLOQ level peak area for each analyte and 5% of the IS peak area were detected. Linearity was evaluated by an eight-point calibration curve with plasma, prepared and assayed in duplicate on three consecutive days. Daily calibration curves were constructed using weighed (1/x2 ) least-square linear regression of the ratios between analytes and IS peak areas versus the nominal standard concentrations. Accuracy for the LLOQ should be within ±20%, and its precision should be less than 20% in six replicates on three consecutive days. Precision and accuracy were assessed by analyzing the concentrations of QC samples at four concentrations using six replicates on three consecutive days. Accuracy and precision were expressed by relative error (RE) and relative standard deviation (RSD), respectively. Recovery of analytes was calculated by comparing the peak areas of QC samples (four concentrations in three replicates) throughout the extraction procedure with the post-extraction mean peak area for blank plasma samples spiked at the corresponding concentrations. The recovery of IS was evaluated in a similar way at the concentration of 800 ng/mL. The matrix effect on analytes ionization was assessed by comparing the post-extraction peak areas of blank plasma samples spiked at the concentration of QC samples (A) with the peak areas of the corresponding water-substituted samples (B). The blank plasma used in this study was obtained from six different groups of healthy individuals. The ratio (A/B × 100) is defined as the matrix effect. The matrix effects of internal standards were determined in the same way. In dilution tests, blank plasma sample spiked with 1.00 g/mL naproxcinod and 20.0 g/mL naproxen were diluted 5, 20 and 200fold with blank plasma, in order to prepare the plasma samples containing 200, 50 and 5 ng/mL naproxcinod and 4000, 1000 and 100 ng/mL naproxen separately. Other plasma samples of the corresponding concentrations were prepared directly for comparison. The dilution test was performed by the relative coefficient of variation between the spiked plasma samples peak areas after dilution and the peak areas of samples prepared directly without dilution.
X. Shi et al. / J. Chromatogr. B 978–979 (2015) 157–162
159
Fig. 1. Product ion spectra of naproxcinod, naproxen and IS.
The stability of naproxcinod and naproxen plasma samples were examined under different conditions, such as short-term stability at room temperature for 6 h, post-preparative stability at 4 ◦ C for 24 h, long-term stability at −20 ◦ C for 7 days and after three complete freeze–thaw cycles. Furthermore, the stock solution stability of naproxcinod, naproxen and IS was also assessed at −20 ◦ C for 22 days. 2.8. Application Six male Sprague-Dawley rats (approximately 250 g body weight) were provided by the Laboratory Animal Center of Hebei Medical University. The animal experiment protocols were approved by the Animal Center of Hebei Medical University. Animals were maintained at ambient temperature (22–24 ◦ C) and 60% relative humidity with a 12 h light/dark cycle and kept in an environmentally controlled breeding room for 3 days. Animals were fasted for 12 h (with ad libitum access to water) prior to the experiments. The rats were given an intragastric oral administration of 6.0 mg/kg naproxcinod dissolved in 0.5% sodium carboxymethyl cellulose (CMC). Blood samples (0.3 mL) were collected from the posterior orbital vein using heparinized tubes before (0 h) and at 0.15, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 12, 24, 36 and 48 h following the oral administration. The rats were euthanized immediately after the collection procedure using ethyl ether. The blood was centrifuged at 1359 × g for 10 min to separate red blood cells and plasma. Plasma samples were stored at −20 ◦ C until further analysis. Pharmacokinetic parameters including area under the curve (AUC), mean retention time (MRT), elimination half-life (t1/2 ), plasma clearance (CL) and apparent distribution volume (Vd ) for naproxcinod and naproxen were calculated using the noncompartmental method and the DAS 3.2 pharmacokinetic program (Chinese Pharmacology Society), respectively. Cmax (the maximum peak concentration) and Tmax (the time to Cmax ) were obtained directly from the curves.
sensitivity in positive mode than in negative mode. [M + H]+ were the most abundant ions in the Q1 full-scan spectra for all the analytes, which were used as the precursor ion. The full-scan product ion spectra for each analyte is provided in Fig. 1. Parameters including DP and CE were optimized, and the recommended value of the instrument was used for all other parameters. 3.1.2. Liquid chromatography The Synergi Fusion-RP C18 column (Phenomenex, USA) was optimal compared with the tested C18 columns, including Zorbax SB C18 (Agilent, USA), Luna C18 (Phenomenex, USA), and Diamonsil C18(2) (Dikma, China). The composition of the mobile phase was optimized to achieve satisfactory chromatographic behavior, such as high sensitivity, good peak symmetry, short run time and appropriate ionization. Methanol provided higher responses and lower background noise compared with acetonitrile and was therefore chosen as the organic phase. Sensitivity and peak symmetry of naproxcinod, naproxen and IS were significantly improved by adding formic acid to the mobile phase. The optimal concentration of formic acid in mobile phase was determined empirically (tested range from 0.01% to 0.2%) and the addition of 0.1% formic acid provided a significant sensitivity improvement for naproxcinod, naproxen and IS. Therefore, a solution of methanol–0.1% formic acid in water (72:28, v/v) was selected for the mobile phase which, with a total run time of 4.0 min, met the requirements for high throughput bioanalytical analyses. 3.2. Method validation
3.1. Method development
3.2.1. Selectivity Selectivity was assessed by comparing the chromatograms of six different batches of blank plasma with their corresponding spiked plasma. Typical HPLC–MS/MS chromatograms of naproxcinod, naproxen and IS are presented in Fig. 2. The retention times of naproxcinod, naproxen and IS were 3.1, 1.2 and 2.2 min, respectively. There was no significant interference from endogenous substances observed at the analytes retention times. A carryover experiment was performed by injecting blank samples after an upper LOQ sample. Results showed the absence of carryover.
3.1.1. Mass spectrometry To optimize ESI conditions, naproxcinod, naproxen and IS were dissolved in mobile phase and directly injected into the mass spectrometer. Naproxcinod, naproxen and IS displayed a higher
3.2.2. Linear and LLOQ Linearity was evaluated based on the average of the eight-point calibration curves analyzed in three separate batches. Calibration curves were linear within concentration ranges of 1.00–400 ng/mL
3. Results and discussion
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Fig. 2. Typical LC–MS/MS chromatograms of naproxcinod, naproxen and IS in rat plasma. (A) Blank plasma sample, (B) plasma sample spiked with 1.00 ng/mL of naproxcinod and 20.0 ng/mL of naproxen and (C) plasma sample at 0.5 h after an oral administration of 6 mg/kg naproxcinod.
for naproxcinod and 20.0–8000 ng/mL for naproxen. The typical standard curve was y = 0.00627x + 0.00136 (r = 0.9985) for naproxcinod and y = 0.00357x + 0.00548 (r = 0.9952) for naproxen, where y represents the ratio of the analyte peak area to that of the IS and x represents the plasma concentration of the analyte. The LLOQ for naproxcinod and naproxen are 1.00/20.0 ng/mL, indicating that the method was more sensitive than those of previous studies regarding the determination of naproxcinod (LOQ 4 nM, 1.4 ng/mL) and naproxen (LOQ 0.5 M, 115.5 ng/mL) [6], and the precision and accuracy met the relevant criteria. 3.2.3. Precision and accuracy Intra- and inter-day precision and accuracy values of the QC samples are listed in Table 1. In this assay, the intra- and inter-day precision values were both ≤4.53% for naproxcinod and naproxen and the average intra- and inter-day accuracy values ranged from −8.1% to 8.7%. These results indicated an excellent precision and accuracy for the quantification of naproxcinod and naproxen in plasma using the current method. 3.2.4. Recovery and matrix effect The extraction recovery of naproxcinod ranged from 93.1% to 98.6% at four QC concentrations (Table 2), and the recovery of naproxen ranged from 94.7% to 97.8% at four QC concentrations. The extraction recovery of IS was 96.5% (Table 2).
The matrix effect for each analyte was evaluated by the ratios between the corresponding post-extraction peak areas of spiked plasma and of the corresponding water-substituted references. The matrix effect values for naproxcinod were 94.7–99.5%, 95.6–101.7% for naproxen and 98.2% for IS (Table 2). These results suggested that the matrix effect for naproxcinod, naproxen and IS were negligible under the LC–MS/MS conditions used in the present study.
3.2.5. Dilution tests The RE between the peak areas of spiked plasma samples after dilution (5, 20 and 100-fold) and samples prepared directly without dilution were −2.59%, −0.04% and −3.15%, respectively. The corresponding relative coefficient of variation were −7.65%, −0.59% and −5.68%, respectively.
3.2.6. Stability The stability analysis was performed under similar conditions to those described above, and the results are presented in Table 3. Naproxcinod and naproxen remained stable during the whole analytical period. The stock solutions for all analytes were stable after 22 days of storage at −20 ◦ C, exhibiting a RSD and a RE
