Research article Received: 19 July 2014,
Revised: 3 September 2014,
Accepted: 2 October 2014
Published online in Wiley Online Library: 6 November 2014
(wileyonlinelibrary.com) DOI 10.1002/bmc.3375
Quantification of an antitumor agent (copen) in rat plasma by liquid chromatography– electrospray ionization tandem mass spectrometry and its application in a preclinical pharmacokinetic study Huili Zhoua, Yunliang Zhenga, Yanan Liub, Xingjiang Hua, Guolan Wua and Jianzhong Shentua* ABSTRACT: Copen is a derivative obtained from the structural modification of osthole, which inhibits tumoral proliferation in many tumor cell lines. A rapid and sensitive liquid chromatography–tandem mass spectrometry (LC-MS/MS) method was established for the quantification of copen in rat plasma. After a simple sample preparation procedure by one-step protein precipitation with methanol, copen and bicalutamide (internal standard, IS) were chromatographed on a Zorbax SB-C18 (4.6×100 mm, 1.8 μm) column with a mobile phase consisting of methanol–5 mM ammonium formate water with 0.1% formic acid (80:20, v/v). MS detection was performed on a triple quadrupole tandem mass spectrometer in the multiple reaction monitoring mode with a positive eletrospray ionization source. The assay was validated in the concentration range of 51.58–20630 ng/mL, with a limit of quantitation (LOQ) of 51.58 ng/mL. The intra- and inter-day precisions (relative standard deviation) were ≤3.21 and ≤11.3%, respectively, with accuracy (%) in the range of 94.66–102.1%. The method was fully validated in a study of the pharmacokinetics of copen (25 mg/kg) after intragastric administration in rats. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: copen; LC-MS/MS; pharmacokinetics; preclinical
Introduction
Biomed. Chromatogr. 2015; 29: 975–980
* Correspondence to: J. Shentu, Research Center for Clinical Pharmacy, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital, Zhejiang University, Hangzhou 310003, China. Email: [email protected] a
Research Center for Clinical Pharmacy, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital, Zhejiang University, Hangzhou 310003, China
b
Guangdong Zonk Drug R&D Limited, Guangdong, Guangzhou, 510730, China Abbreviations used: DP, declustering potential; CE, collision energy; CXP, collision exit potential; MRM, multiple reaction monitoring.
Copyright © 2014 John Wiley & Sons, Ltd.
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Natural products have always been important resources, either as therapeutic agents or as lead compounds in the production of pharmaceutical compounds. Osthole, 7-methoxy-8-(3methyl-2-butenyl) coumarin, an ingredient isolated from the seeds of Cnidium monnieri (L.) cussonLibanotis buchtormensis, has received considerable attention recently because of its significant and diverse pharmacological activities, including in vitro/vivo antitumor effects (Yang et al., 2010; Zhang et al., 2012; Xu et al., 2011, 2012; Kao et al., 2012; Lin et al., 2010) and alleviation of hyperglycemia (Liang et al., 2009) and hypolipidemia (Song et al., 2006), which make it a very promising natural lead compound for new drug discovery. However, osthole exhibits low water solubility, and some structural modifications are required for sufficient bioavailability upon oral administration. Copen is a derivative of osthole obtained by structural modification of the compound. Copen is considered a new antitumor drug candidate (Chinese patent no. ZL200880000690.3; PCT patent application no. CN2008/001089; funded by National Major Scientific and Technological Special Project for ’Significant New Drugs Development’ in China, project no. 2011ZX09302-003-03). It showed higher water solubility and good inhibition effects on 95-D, Bel-7402, MDA-MB-231, PC-3, and HL-60 tumor cells, with IC50 values 99.5%) was provided by Guangdong Zonk Drug R&D Limited, and bicalutamide (purity >99.3%) was kindly provided by Zhejiang Institute for Food and Drug Control (Hangzhou, P.R. China). Their structures are shown in Fig. 1. HPLC-grade reagents (methanol, acetonitrile, formic acid and ammonium formate) were purchased from Merck (Darmstadt, Germany). All other chemicals were of analytical grade and used without further purification. Double-distilled water prepared with a Milli-Q academic water purification system (Milford, MA, USA) was used throughout the study.
LC-MS/MS conditions Calibration standards and quality control sample preparation
Liquid chromatography was performed on a Shimadzu LC-20 AD system (Shimadzu, Tokyo, Japan) equipped with an autosampler, a degasser, a thermostatted column and a binary pump. The system was coupled to an API 4000 triple-quadrupole mass spectrometer (AB Sciex, Ontario, Canada) for mass analysis and detection. Data acquisition was performed with Analyst version 1.4.2 software (AB Sciex). An Agilent ZorbaxSB-C18 column
Accurately weighed solid portions of copen and the IS (25.790 and 12.250 mg, respectively) were dissolved in methanol to prepare stock solutions separately (5.158 mg/mL for copen and 2.450 mg/mL for the IS). Working solutions were prepared by dilution of the stock solution with methanol to obtain series of
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Figure 1. Chemical structures and MS spectra of copen and bicalutamide (IS).
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Copyright © 2014 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2015; 29: 975–980
Quantification of copen in rat plasma in rat plasma by HPLC-MS/MS Table 1. ESI-MS/MS parameters on the parent and daughter ions (m/z) and declustering potential (DP), collision energy (CE) and collision exit potential (CXP) of copen and bicalutamide (IS) Analytes Copen IS
Q1 (m/z)
Q3 (m/z)
DP (V)
CE (V)
CXP (V)
261.2 429.0
217.1 255.0
55 48
28 35
5 25
working concentrations. The IS working solution was finally diluted to 6125 ng/mL with methanol, and all of the solutions were stored at 4 °C until use. The calibration standards (20630, 10320, 5158, 1032, 515.8, 206.3 and 51.58 ng/mL) were prepared by spiking blank plasma with appropriate amounts of the working solutions to yield a final concentration range of 51.58– 20630 ng/mL. Quality control (QC) samples were prepared with blank plasma at three concentration levels of 103.2, 1650.4 and 16504 ng/mL. Sample preparation and extraction The plasma sample was thawed at room temperature, and 40 μL of the plasma were combined with 10 μL of 6125 ng/mL of the IS working solution. After thorough vortex mixing for 30 s, the mixtures were precipitated with 120 μL of methanol, vortex-mixed for 30 s and centrifuged at 10500 g for 5 min. Then, 5 μL of the supernatant were injected into the HPLC-MS/MS system. Method validation
Biomed. Chromatogr. 2015; 29: 975–980
(1)
ERð%Þ ¼ C=B100
(2)
where A is the peak area obtained in neat solution standards; B is the corresponding peak area for standards spiked after extraction into plasma extracts; and C is the peak area for standards spiked before extraction. The stability of the QC samples at the three concentration levels (103.2, 1650.4 and 16,504 ng/mL) was analyzed by exposing them to the following conditions: ambient temperature (20 ° C) for 8 h before sample preparation (room temperature stability), placement in an autosampler (4 °C) for 14 h (postpreparative stability), three freeze–thaw cycles from 80 °C to room temperature (freeze–thaw stability) and storage for 60 days at 80 °C (long-term stability). Animals and treatment Twelve male Sprague–Dawley rats (body weight 200–250 g) were obtained from the Laboratory Animal Center of Zhejiang Academy of Medical Sciences (Hangzhou, China). The animals were maintained in controlled conditions (temperature 24 ± 1 ° C, relative humidity 55 ± 10% and 12 h dark–light cycle), with free access to standard laboratory food and water for 5 days’ acclimation. They fasted the night before and for 4 h after dosing. The rats were randomized to two independent groups (n =6 per group) and treated with 25 mg/kg of copen (dissolved in physiological saline) via the intragastric route. Blood samples (300 μL) were collected from the tail vein at 5, 10, 15, and 30 min and at 1, 1.5, 2, 4, 6, 10 and 24 h after dosing. The collected blood samples were immediately centrifuged at 2500 g at 4 °C for 10 min. The plasma samples were separated and stored at –20 °C until analyzed using LC-MS/MS. The plasma collected from six vehicle-administered rats served as the blank. The animal experiments were carried out according to the Guidelines for the Care and Use of Laboratory Animals, and they were approved by the Animal Ethics Committee of the First Affiliated Hospital, College of Medicine, Zhejiang University.
Results and discussion Optimization of ESI-MS/MS condition To get optimal analytical conditions for this assay, mass spectrometer settings were investigated. Firstly, mass spectrometer parameters were derived from analyte infusion experiment using a syringe pump (typical concentrations were 1.02 and 1.18 μg/mL in water for copen and IS, respectively). ESI sources both with positive and negative ionization were tested for the determination. The results revealed that both copen and IS were more sensitive in negative ionization mode with ESI interface. The deprotonated precursor ion of copen at m/z 261.2 was abundantly generated in the negative ESI mode. Thereafter, the loss of an CO2 portion (44 Da) gave a product ion at m/z 217.1, the loss of –CH3 group (15 Da) led to a product ion of m/z 202.0, and with a further loss of –C3H7 group (43 Da) led to a product ion of m/z 158.9. For IS, the prominent stable product ion was m/z 255.0 [M 174] at the collision energy of 35 eV. The product ion mass spectra and proposed ESI-MS/MS fragmentation processes of copen and IS are shown in Fig. 1. Based on the Q1 and product ion scan of copen, the MRM ion reactions
Copyright © 2014 John Wiley & Sons, Ltd.
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The selectivity was tested by comparison of blank plasma from six rats with the corresponding plasma samples spiked with copen and IS. In addition, chromatograms of typical plasma samples from different rats after intragastric administration of 25 mg/kg of copen for 10 min were analyzed to check for endogenous interference. Linearity was assessed by weighted linear regression (1/x, where x is the concentration of copen in rat plasma) of analyte– internal standard peak area ratios (y) based on analyzing seven spiked standard solutions in the range of 51.58–20630 ng/mL in blank plasma. The limit of detection (LOD) of this method was the concentration of a compound at which its signal-to-noise ratios (S/N) was detected as 3:1; the LOQ was defined as the lowest concentration on the calibration curves of copen measured with acceptable precision and accuracy (i.e., within ±20% bias of the nominal value) and with a response at least 10 times higher than the average noise level. The precision and accuracy of the method were evaluated by analyzing the QC samples at four concentration levels (51.58, 103.2, 1650.4 and 16504 ng/mL) in six replicates of each level on three continuous validation days. The precision was expressed using the relative standard deviation (RSD). The accuracy of the method was determined by comparing the means of the concentrations of copen calculated in the above-mentioned QCs with the nominal concentrations (percentage differences). The extraction recovery (ER, %) and matrix effects (ME, %) of copen in rat plasma of this method were determined in the three QC concentrations (103.2, 1650.4 and 16504 ng/mL, n =6); the recovery and the matrix effect of the IS were evaluated at 612.5 ng/ mL. Results were calculated through the following formulas:
MEð%Þ ¼ B=A100
H. Zhou et al.
Figure 2. Typical chromatograms of (A) mixed blank plasma; (B) blank plasma spiked with copen (51.58 ng/mL) and the IS (612.5 ng/mL); and (C) rat plasma 10 min after oral administration of 25 mg/kg of copen.
Table 2. Matrix effects and extraction recovery of copen and IS in rat plasma Analytes
Spiked concentration (ng/mL)
Copen
Matrix effect (%, mean ± SD, n =6)
103.2 1650.4 16504 612.5
Bicalutamide (IS)
Extraction recovery (%, mean ± SD, n =6)
108.2 ± 10.67 101.9 ± 6.33 104.8 ± 9.45 101.8 ± 3.57%
82.49 ± 12.13 75.76 ± 6.02 71.42 ± 5.19 80.33 ± 5.36
Table 3. Intra- (n =6) and inter-day (n =3 days) precision and accuracy of the method QC concentration (ng/mL) 51.58 103.2 1650.4 16504
Intraday
Accuracy
RSD
Interday
Accuracy
RSD
Mean ± SD (ng/mL)
(%)
(%)
Mean ± SD (ng/mL)
(%)
(%)
48.83 ± 1.57 104.5 ± 3.25 1644.3 ± 27.49 15,865 ± 326.4
94.66 95.81 99.63 96.13
3.21 2.03 1.67 2.06
52.68 ± 5.97 101.8 ± 9.24 1602.7 ± 118.6 16016 ± 1218
102.1 95.43 97.10 97.00
11.3 9.08 7.40 7.60
978
QC, Quality control.
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Copyright © 2014 John Wiley & Sons, Ltd.
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Quantification of copen in rat plasma in rat plasma by HPLC-MS/MS Table 4. Stability of copen in rats plasma under different storage conditions (n =3) QC concentration (ng/mL)
Short-term (8 h, 20 °C)
Autosampler, 4 °C for 14 h
5.39 1.24 5.05
103.2 1650.4 16504
Three freeze–thaw cycles
4.61 5.48 1.25
Long-term (60 days, 80 °C)
12.83 0.63 3.73
10.65 2.61 0.78
Stability values are represented as RE (%) = (found concentration QC concentration)/QC concentration × 100%.
Figure 3. Mean plasma concentration–time curves of copen after intravenous administration of 25 mg/kg of the compound (n =12).
Table 5. Pharmacokinetic parameters of copen (25 mg/kg) after intragastric administration in rats (n =12) Parameters AUC0–t AUC0–∞ MRT0–t MRT0–∞ VRT0–t VRT0–∞ t1/2 Tmax CLz/F Cmax
Units
Mean ± SD
mg/L min mg/L min min min min2 min2 min min L/min/kg mg/L
729.59 ± 437.80 768.02 ± 421.57 199.84 ± 108.14 305.00 ± 208.98 60,519.51 ± 47,153.90 197,852.65 ± 232,393.55 302.16 ± 169.90 10.00 ± 3.69 0.046 ± 0.027 13.39 ± 10.17
were: m/z 261.2→217.1 and 261.2→202.0. Both ion reactions were evaluated for the quantitative analysis of copen in human plasma. Endogenous interferences were obvious, when the ion combination of 261.2→202.0 was chosen for the quantitative analysis copen. The best specificity and sensitivity were obtained with MRM of m/z 261.2→217.1 for copen. Therefore, The precursor-to-production transitions were monitored at m/z 261.2→217.1 for copen, and m/z 429.0→255.0 for IS. The DP, CE and CXP for each transition and ion source parameters were also optimized; the final parameters are shown in the section ’LC-MS/MS conditions’. Optimization of separation conditions
Biomed. Chromatogr. 2015; 29: 975–980
Method validation The typical MRM chromatograms of mixed blank plasma from six copen-free individuals, a spiked plasma sample with copen at LOQ and IS at 612.5 ng/mL, and a plasma sample from 10 min after oral administration of copen are shown in Fig. 2. Under the described chromatographic condition, good separation was achieved and no significant peak was observed in any of the blank plasma samples for copen and the IS. The standard calibration curve for spiked plasma containing copen was linear over the range of 51.58–20,630 ng/mL. Good linearity was observed for the analyte using a weighted (1/x) least squares linear regression analysis with a coefficient of determination of r2 > 0.9990. Typical equations for the calibration curve were as follows: y =0.0863x +5.97 × 100.059 (r =0.9994), where x represents the plasma concentration of copen (μg/mL) and y represents the ratio of the peak area of copen compared with that of the IS. The LOD and LOQ under the optimized conditions was 10.32 ng/mL and 51.58 ng/mL, which is sufficient for the pharmacokinetic study of copen following intragastric and intravenous administration in rats
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To optimize the separation conditions, composition of the stationary phase and mobile phase were investigated in the HPLC domain. The separation of copen and the IS was first investigated
on several different reversed phase columns (Zorbax extend-C18, Hypersil-C18 and Zorbax SB-C18). Comparing to other categories of column, the Zorbax SB-C18 column showed higher sensitivity and better peak shape for copen and IS. Acetonitrile and methanol were considered as optional elution solvents. Methanol was finally adopted for it produced symmetric peak shape and higher detection response of copen and IS than acetonitrile. Then, the organic solvent percentage in the mobile phase was investigated over the range of 60–90%, and 80% was chosen owing to the high response of copen and suitable retention time provided. Formic acid (0.05, 0.1 and 0.2% in aqueous phase) and ammonium formate were considered as additives in the mobile phase to improve the response of copen and IS. Both copen and IS were found to have higher response in the mobile phase with 0.1% formic acid. More test results showed that better peak shapes and higher response could be achieved by adding 5 mM of ammonium acetate into the aqueous portion. According to this, a mobile phase consisting of methanol–5 mM ammonium formate water with 0.1% formic acid (80:20, v/v) was selected in this method. Column temperatures (30, 40, 45, or 50 °C) and the flow rate of the mobile phase (0.4, 0.5 or 0.6 mL/min) were also examined and compared. Finally, plasma samples were separated by HPLC on an Zorbax SB-C18 using a solvent system consisting of methanol–5 mM ammonium formate water with 0.1% formic acid (80:20, v/v) at 40 °C; the flow rate was set at 0.6 mL/min. The retention times for copen and the IS were 3.00 and 2.15 min, respectively (Fig. 2), and the total chromatographic run time was 3.60 min.
H. Zhou et al. The extraction recovery and the matrix effects of copen in rat plasma are shown in Table 2. At the three concentration levels of these analytes, the extraction recovery of copen was >70%, and the extraction recovery of the IS was 80.33%. The matrix effects (percentage nominal concentrations and corresponding standard deviation) determined were 108.2 ± 10.67, 101.9 ± 6.33, and 104.8 ± 9.45 at three different concentrations for copen. The same matrix effect evaluation was performed for the IS and the percentage nominal concentration was 101.8 ± 3.57%. The results indicated that ion from plasma matrix was consistent for this analytical method and would not interfere the measurement of the analyte. Table 3 summarizes the intra- and inter-day precision and accuracy of the method at four different concentration levels (51.58, 103.2, 1650.4 and 16504 ng/mL). As shown in Table 3, the accuracy of the method at the different levels was within the range of 94.66–102.1%. The intra- and inter-day precision (RSD) of copen at the different levels was ≤12.0%. The results demonstrate that the values are all within the acceptable range and that the method is accurate and precise. The stability experiment was performed by using QC samples. The analyte was shown to be stable in rat plasma with the coefficient of variation (relative error, RE)
Revised: 3 September 2014,
Accepted: 2 October 2014
Published online in Wiley Online Library: 6 November 2014
(wileyonlinelibrary.com) DOI 10.1002/bmc.3375
Quantification of an antitumor agent (copen) in rat plasma by liquid chromatography– electrospray ionization tandem mass spectrometry and its application in a preclinical pharmacokinetic study Huili Zhoua, Yunliang Zhenga, Yanan Liub, Xingjiang Hua, Guolan Wua and Jianzhong Shentua* ABSTRACT: Copen is a derivative obtained from the structural modification of osthole, which inhibits tumoral proliferation in many tumor cell lines. A rapid and sensitive liquid chromatography–tandem mass spectrometry (LC-MS/MS) method was established for the quantification of copen in rat plasma. After a simple sample preparation procedure by one-step protein precipitation with methanol, copen and bicalutamide (internal standard, IS) were chromatographed on a Zorbax SB-C18 (4.6×100 mm, 1.8 μm) column with a mobile phase consisting of methanol–5 mM ammonium formate water with 0.1% formic acid (80:20, v/v). MS detection was performed on a triple quadrupole tandem mass spectrometer in the multiple reaction monitoring mode with a positive eletrospray ionization source. The assay was validated in the concentration range of 51.58–20630 ng/mL, with a limit of quantitation (LOQ) of 51.58 ng/mL. The intra- and inter-day precisions (relative standard deviation) were ≤3.21 and ≤11.3%, respectively, with accuracy (%) in the range of 94.66–102.1%. The method was fully validated in a study of the pharmacokinetics of copen (25 mg/kg) after intragastric administration in rats. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: copen; LC-MS/MS; pharmacokinetics; preclinical
Introduction
Biomed. Chromatogr. 2015; 29: 975–980
* Correspondence to: J. Shentu, Research Center for Clinical Pharmacy, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital, Zhejiang University, Hangzhou 310003, China. Email: [email protected] a
Research Center for Clinical Pharmacy, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital, Zhejiang University, Hangzhou 310003, China
b
Guangdong Zonk Drug R&D Limited, Guangdong, Guangzhou, 510730, China Abbreviations used: DP, declustering potential; CE, collision energy; CXP, collision exit potential; MRM, multiple reaction monitoring.
Copyright © 2014 John Wiley & Sons, Ltd.
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Natural products have always been important resources, either as therapeutic agents or as lead compounds in the production of pharmaceutical compounds. Osthole, 7-methoxy-8-(3methyl-2-butenyl) coumarin, an ingredient isolated from the seeds of Cnidium monnieri (L.) cussonLibanotis buchtormensis, has received considerable attention recently because of its significant and diverse pharmacological activities, including in vitro/vivo antitumor effects (Yang et al., 2010; Zhang et al., 2012; Xu et al., 2011, 2012; Kao et al., 2012; Lin et al., 2010) and alleviation of hyperglycemia (Liang et al., 2009) and hypolipidemia (Song et al., 2006), which make it a very promising natural lead compound for new drug discovery. However, osthole exhibits low water solubility, and some structural modifications are required for sufficient bioavailability upon oral administration. Copen is a derivative of osthole obtained by structural modification of the compound. Copen is considered a new antitumor drug candidate (Chinese patent no. ZL200880000690.3; PCT patent application no. CN2008/001089; funded by National Major Scientific and Technological Special Project for ’Significant New Drugs Development’ in China, project no. 2011ZX09302-003-03). It showed higher water solubility and good inhibition effects on 95-D, Bel-7402, MDA-MB-231, PC-3, and HL-60 tumor cells, with IC50 values 99.5%) was provided by Guangdong Zonk Drug R&D Limited, and bicalutamide (purity >99.3%) was kindly provided by Zhejiang Institute for Food and Drug Control (Hangzhou, P.R. China). Their structures are shown in Fig. 1. HPLC-grade reagents (methanol, acetonitrile, formic acid and ammonium formate) were purchased from Merck (Darmstadt, Germany). All other chemicals were of analytical grade and used without further purification. Double-distilled water prepared with a Milli-Q academic water purification system (Milford, MA, USA) was used throughout the study.
LC-MS/MS conditions Calibration standards and quality control sample preparation
Liquid chromatography was performed on a Shimadzu LC-20 AD system (Shimadzu, Tokyo, Japan) equipped with an autosampler, a degasser, a thermostatted column and a binary pump. The system was coupled to an API 4000 triple-quadrupole mass spectrometer (AB Sciex, Ontario, Canada) for mass analysis and detection. Data acquisition was performed with Analyst version 1.4.2 software (AB Sciex). An Agilent ZorbaxSB-C18 column
Accurately weighed solid portions of copen and the IS (25.790 and 12.250 mg, respectively) were dissolved in methanol to prepare stock solutions separately (5.158 mg/mL for copen and 2.450 mg/mL for the IS). Working solutions were prepared by dilution of the stock solution with methanol to obtain series of
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Figure 1. Chemical structures and MS spectra of copen and bicalutamide (IS).
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Copyright © 2014 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2015; 29: 975–980
Quantification of copen in rat plasma in rat plasma by HPLC-MS/MS Table 1. ESI-MS/MS parameters on the parent and daughter ions (m/z) and declustering potential (DP), collision energy (CE) and collision exit potential (CXP) of copen and bicalutamide (IS) Analytes Copen IS
Q1 (m/z)
Q3 (m/z)
DP (V)
CE (V)
CXP (V)
261.2 429.0
217.1 255.0
55 48
28 35
5 25
working concentrations. The IS working solution was finally diluted to 6125 ng/mL with methanol, and all of the solutions were stored at 4 °C until use. The calibration standards (20630, 10320, 5158, 1032, 515.8, 206.3 and 51.58 ng/mL) were prepared by spiking blank plasma with appropriate amounts of the working solutions to yield a final concentration range of 51.58– 20630 ng/mL. Quality control (QC) samples were prepared with blank plasma at three concentration levels of 103.2, 1650.4 and 16504 ng/mL. Sample preparation and extraction The plasma sample was thawed at room temperature, and 40 μL of the plasma were combined with 10 μL of 6125 ng/mL of the IS working solution. After thorough vortex mixing for 30 s, the mixtures were precipitated with 120 μL of methanol, vortex-mixed for 30 s and centrifuged at 10500 g for 5 min. Then, 5 μL of the supernatant were injected into the HPLC-MS/MS system. Method validation
Biomed. Chromatogr. 2015; 29: 975–980
(1)
ERð%Þ ¼ C=B100
(2)
where A is the peak area obtained in neat solution standards; B is the corresponding peak area for standards spiked after extraction into plasma extracts; and C is the peak area for standards spiked before extraction. The stability of the QC samples at the three concentration levels (103.2, 1650.4 and 16,504 ng/mL) was analyzed by exposing them to the following conditions: ambient temperature (20 ° C) for 8 h before sample preparation (room temperature stability), placement in an autosampler (4 °C) for 14 h (postpreparative stability), three freeze–thaw cycles from 80 °C to room temperature (freeze–thaw stability) and storage for 60 days at 80 °C (long-term stability). Animals and treatment Twelve male Sprague–Dawley rats (body weight 200–250 g) were obtained from the Laboratory Animal Center of Zhejiang Academy of Medical Sciences (Hangzhou, China). The animals were maintained in controlled conditions (temperature 24 ± 1 ° C, relative humidity 55 ± 10% and 12 h dark–light cycle), with free access to standard laboratory food and water for 5 days’ acclimation. They fasted the night before and for 4 h after dosing. The rats were randomized to two independent groups (n =6 per group) and treated with 25 mg/kg of copen (dissolved in physiological saline) via the intragastric route. Blood samples (300 μL) were collected from the tail vein at 5, 10, 15, and 30 min and at 1, 1.5, 2, 4, 6, 10 and 24 h after dosing. The collected blood samples were immediately centrifuged at 2500 g at 4 °C for 10 min. The plasma samples were separated and stored at –20 °C until analyzed using LC-MS/MS. The plasma collected from six vehicle-administered rats served as the blank. The animal experiments were carried out according to the Guidelines for the Care and Use of Laboratory Animals, and they were approved by the Animal Ethics Committee of the First Affiliated Hospital, College of Medicine, Zhejiang University.
Results and discussion Optimization of ESI-MS/MS condition To get optimal analytical conditions for this assay, mass spectrometer settings were investigated. Firstly, mass spectrometer parameters were derived from analyte infusion experiment using a syringe pump (typical concentrations were 1.02 and 1.18 μg/mL in water for copen and IS, respectively). ESI sources both with positive and negative ionization were tested for the determination. The results revealed that both copen and IS were more sensitive in negative ionization mode with ESI interface. The deprotonated precursor ion of copen at m/z 261.2 was abundantly generated in the negative ESI mode. Thereafter, the loss of an CO2 portion (44 Da) gave a product ion at m/z 217.1, the loss of –CH3 group (15 Da) led to a product ion of m/z 202.0, and with a further loss of –C3H7 group (43 Da) led to a product ion of m/z 158.9. For IS, the prominent stable product ion was m/z 255.0 [M 174] at the collision energy of 35 eV. The product ion mass spectra and proposed ESI-MS/MS fragmentation processes of copen and IS are shown in Fig. 1. Based on the Q1 and product ion scan of copen, the MRM ion reactions
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The selectivity was tested by comparison of blank plasma from six rats with the corresponding plasma samples spiked with copen and IS. In addition, chromatograms of typical plasma samples from different rats after intragastric administration of 25 mg/kg of copen for 10 min were analyzed to check for endogenous interference. Linearity was assessed by weighted linear regression (1/x, where x is the concentration of copen in rat plasma) of analyte– internal standard peak area ratios (y) based on analyzing seven spiked standard solutions in the range of 51.58–20630 ng/mL in blank plasma. The limit of detection (LOD) of this method was the concentration of a compound at which its signal-to-noise ratios (S/N) was detected as 3:1; the LOQ was defined as the lowest concentration on the calibration curves of copen measured with acceptable precision and accuracy (i.e., within ±20% bias of the nominal value) and with a response at least 10 times higher than the average noise level. The precision and accuracy of the method were evaluated by analyzing the QC samples at four concentration levels (51.58, 103.2, 1650.4 and 16504 ng/mL) in six replicates of each level on three continuous validation days. The precision was expressed using the relative standard deviation (RSD). The accuracy of the method was determined by comparing the means of the concentrations of copen calculated in the above-mentioned QCs with the nominal concentrations (percentage differences). The extraction recovery (ER, %) and matrix effects (ME, %) of copen in rat plasma of this method were determined in the three QC concentrations (103.2, 1650.4 and 16504 ng/mL, n =6); the recovery and the matrix effect of the IS were evaluated at 612.5 ng/ mL. Results were calculated through the following formulas:
MEð%Þ ¼ B=A100
H. Zhou et al.
Figure 2. Typical chromatograms of (A) mixed blank plasma; (B) blank plasma spiked with copen (51.58 ng/mL) and the IS (612.5 ng/mL); and (C) rat plasma 10 min after oral administration of 25 mg/kg of copen.
Table 2. Matrix effects and extraction recovery of copen and IS in rat plasma Analytes
Spiked concentration (ng/mL)
Copen
Matrix effect (%, mean ± SD, n =6)
103.2 1650.4 16504 612.5
Bicalutamide (IS)
Extraction recovery (%, mean ± SD, n =6)
108.2 ± 10.67 101.9 ± 6.33 104.8 ± 9.45 101.8 ± 3.57%
82.49 ± 12.13 75.76 ± 6.02 71.42 ± 5.19 80.33 ± 5.36
Table 3. Intra- (n =6) and inter-day (n =3 days) precision and accuracy of the method QC concentration (ng/mL) 51.58 103.2 1650.4 16504
Intraday
Accuracy
RSD
Interday
Accuracy
RSD
Mean ± SD (ng/mL)
(%)
(%)
Mean ± SD (ng/mL)
(%)
(%)
48.83 ± 1.57 104.5 ± 3.25 1644.3 ± 27.49 15,865 ± 326.4
94.66 95.81 99.63 96.13
3.21 2.03 1.67 2.06
52.68 ± 5.97 101.8 ± 9.24 1602.7 ± 118.6 16016 ± 1218
102.1 95.43 97.10 97.00
11.3 9.08 7.40 7.60
978
QC, Quality control.
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Copyright © 2014 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2015; 29: 975–980
Quantification of copen in rat plasma in rat plasma by HPLC-MS/MS Table 4. Stability of copen in rats plasma under different storage conditions (n =3) QC concentration (ng/mL)
Short-term (8 h, 20 °C)
Autosampler, 4 °C for 14 h
5.39 1.24 5.05
103.2 1650.4 16504
Three freeze–thaw cycles
4.61 5.48 1.25
Long-term (60 days, 80 °C)
12.83 0.63 3.73
10.65 2.61 0.78
Stability values are represented as RE (%) = (found concentration QC concentration)/QC concentration × 100%.
Figure 3. Mean plasma concentration–time curves of copen after intravenous administration of 25 mg/kg of the compound (n =12).
Table 5. Pharmacokinetic parameters of copen (25 mg/kg) after intragastric administration in rats (n =12) Parameters AUC0–t AUC0–∞ MRT0–t MRT0–∞ VRT0–t VRT0–∞ t1/2 Tmax CLz/F Cmax
Units
Mean ± SD
mg/L min mg/L min min min min2 min2 min min L/min/kg mg/L
729.59 ± 437.80 768.02 ± 421.57 199.84 ± 108.14 305.00 ± 208.98 60,519.51 ± 47,153.90 197,852.65 ± 232,393.55 302.16 ± 169.90 10.00 ± 3.69 0.046 ± 0.027 13.39 ± 10.17
were: m/z 261.2→217.1 and 261.2→202.0. Both ion reactions were evaluated for the quantitative analysis of copen in human plasma. Endogenous interferences were obvious, when the ion combination of 261.2→202.0 was chosen for the quantitative analysis copen. The best specificity and sensitivity were obtained with MRM of m/z 261.2→217.1 for copen. Therefore, The precursor-to-production transitions were monitored at m/z 261.2→217.1 for copen, and m/z 429.0→255.0 for IS. The DP, CE and CXP for each transition and ion source parameters were also optimized; the final parameters are shown in the section ’LC-MS/MS conditions’. Optimization of separation conditions
Biomed. Chromatogr. 2015; 29: 975–980
Method validation The typical MRM chromatograms of mixed blank plasma from six copen-free individuals, a spiked plasma sample with copen at LOQ and IS at 612.5 ng/mL, and a plasma sample from 10 min after oral administration of copen are shown in Fig. 2. Under the described chromatographic condition, good separation was achieved and no significant peak was observed in any of the blank plasma samples for copen and the IS. The standard calibration curve for spiked plasma containing copen was linear over the range of 51.58–20,630 ng/mL. Good linearity was observed for the analyte using a weighted (1/x) least squares linear regression analysis with a coefficient of determination of r2 > 0.9990. Typical equations for the calibration curve were as follows: y =0.0863x +5.97 × 100.059 (r =0.9994), where x represents the plasma concentration of copen (μg/mL) and y represents the ratio of the peak area of copen compared with that of the IS. The LOD and LOQ under the optimized conditions was 10.32 ng/mL and 51.58 ng/mL, which is sufficient for the pharmacokinetic study of copen following intragastric and intravenous administration in rats
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979
To optimize the separation conditions, composition of the stationary phase and mobile phase were investigated in the HPLC domain. The separation of copen and the IS was first investigated
on several different reversed phase columns (Zorbax extend-C18, Hypersil-C18 and Zorbax SB-C18). Comparing to other categories of column, the Zorbax SB-C18 column showed higher sensitivity and better peak shape for copen and IS. Acetonitrile and methanol were considered as optional elution solvents. Methanol was finally adopted for it produced symmetric peak shape and higher detection response of copen and IS than acetonitrile. Then, the organic solvent percentage in the mobile phase was investigated over the range of 60–90%, and 80% was chosen owing to the high response of copen and suitable retention time provided. Formic acid (0.05, 0.1 and 0.2% in aqueous phase) and ammonium formate were considered as additives in the mobile phase to improve the response of copen and IS. Both copen and IS were found to have higher response in the mobile phase with 0.1% formic acid. More test results showed that better peak shapes and higher response could be achieved by adding 5 mM of ammonium acetate into the aqueous portion. According to this, a mobile phase consisting of methanol–5 mM ammonium formate water with 0.1% formic acid (80:20, v/v) was selected in this method. Column temperatures (30, 40, 45, or 50 °C) and the flow rate of the mobile phase (0.4, 0.5 or 0.6 mL/min) were also examined and compared. Finally, plasma samples were separated by HPLC on an Zorbax SB-C18 using a solvent system consisting of methanol–5 mM ammonium formate water with 0.1% formic acid (80:20, v/v) at 40 °C; the flow rate was set at 0.6 mL/min. The retention times for copen and the IS were 3.00 and 2.15 min, respectively (Fig. 2), and the total chromatographic run time was 3.60 min.
H. Zhou et al. The extraction recovery and the matrix effects of copen in rat plasma are shown in Table 2. At the three concentration levels of these analytes, the extraction recovery of copen was >70%, and the extraction recovery of the IS was 80.33%. The matrix effects (percentage nominal concentrations and corresponding standard deviation) determined were 108.2 ± 10.67, 101.9 ± 6.33, and 104.8 ± 9.45 at three different concentrations for copen. The same matrix effect evaluation was performed for the IS and the percentage nominal concentration was 101.8 ± 3.57%. The results indicated that ion from plasma matrix was consistent for this analytical method and would not interfere the measurement of the analyte. Table 3 summarizes the intra- and inter-day precision and accuracy of the method at four different concentration levels (51.58, 103.2, 1650.4 and 16504 ng/mL). As shown in Table 3, the accuracy of the method at the different levels was within the range of 94.66–102.1%. The intra- and inter-day precision (RSD) of copen at the different levels was ≤12.0%. The results demonstrate that the values are all within the acceptable range and that the method is accurate and precise. The stability experiment was performed by using QC samples. The analyte was shown to be stable in rat plasma with the coefficient of variation (relative error, RE)
