Research article Received: 17 June 2014,
Revised: 12 August 2014,
Accepted: 28 August 2014
Published online in Wiley Online Library: 21 October 2014
(wileyonlinelibrary.com) DOI 10.1002/bmc.3352
Quantification of heteroclitin D in rat plasma: validation of an LC/MS/MS method and its application in a preclinical pharmacokinetic study Fan Zhanga†, Nan Zhangb†, Li Pangb, Yan Tanc* and Hong Xua* ABSTRACT: A rapid, sensitive and specific liquid chromatography tandem mass spectrometry (LC/MS/MS) method was developed and validated for the quantification of heteroclitin D in rat plasma after using gambogic acid as internal standard (IS). Chromatographic separation was done on a Thermo Hypersil GOLD column (30 × 2.1 mm, 3 μm) using a mobile phase consisting of methanol–water–formic acid (80:20:0.1, v/v/v). The mass spectrometer worked with positive electrospray ionization in multiple reaction monitoring mode, using target ions at [M + H]+ m/z 483.3 for heteroclitin D and [M + H]+ m/z 629.3 for the IS. The standard curve was linear (R2 ≥0.995) over the concentration range 9.98–2080 ng/mL and had good back-calculated accuracy and precision. The intra- and interday precision and accuracy determined on three quality control samples (29.94, 166.4 and 1872 ng/mL) were ≤12.8 and –8.9–3.6%, respectively. The extraction recovery was ≥88.2% and the lower limit of quantification was 9.98 ng/mL. The method was successfully applied to evaluate pharmacokinetics of heteroclitin D in Sprague–Dawley rats following a single intravenous bolus injection of 2.0 mg/kg heteroclitin. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: heteroclitin D; LC/MS/MS; rat plasma; pharmacokinetics
Introduction Kadsurae Caulis (Dian-Jixueteng in Chinese), derived from the dry stems and rattans of Kadsurae interior A. C. Smith, is a member of the Magnoliaceae family and has been used for the treatment of menstrual irregularities, blood deficiencies and other feminine disorders (Jia et al., 2005; Xu et al., 2012; Dong et al., 2013). Dibenzocyclooctene-type lignans are the major active components of the herb and have been extensively investigated (Chen et al., 2002; Liu et al., 2014). They display a variety of biological activities, such as anticolo-rectal tumor-promoting effects, calcium antagonism, antilipid peroxidation and anti-HIV effects (Ding and Luo, 1990; Chen et al., 1997; Hausott et al., 2003; Pu et al., 2008). Moreover, heteroclitin D is the major bioactive lignan used as a phytochemical marker for quality control of Kadsurae Caulis in the Chinese Pharmacopeia (State Pharmacopoeia Commission of P.R. China, 2010). However, no method for the determination of heteroclitin D has previously been reported in any biological matrix and pharmacokinetic data are thus not available. Thus, in this study, we aim to develop an efficient method for the determination of this lignan in rat plasma and further evaluate its pharmacokinetics.
were purchased from the CRM/RM Information Center of China (Beijing, China). HPLC-grade methanol and analytical-grade formic acid were purchased from Tedia Co. (Fairfield, OH, USA). Distilled water was prepared from deionized water. Blank plasma used to prepare daily standard calibration curves and quality control (QC) samples was obtained from male Sprague–Dawley rats.
Instrumentation and analytical conditions The Agilent 1200 rapid resolution HPLC system was interfaced with an Agilent 6460 triple quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source. The column was a Thermo Hypersil Gold column (30 × 2.1 mm, 3 μm). The mobile phase was methanol–water–formic acid (80:20:0.1, v/v/v), and the flow rate was 0.4 mL/min. Quantitation was performed in positive multiple reaction
* Correspondence to: Y. Tan, Tumor Biotherapy Center, Jilin Province People’s Hospital, Changchun 130021, China. Email: tanyan112014@hotmail. com H. Xu, Gastrointestinal Medicine, the First Hospital of Jilin University, Changchun 130021, China. Email: [email protected] †
a
Gastrointestinal Medicine, the First Hospital of Jilin University, Changchun 130021, China
b
Emergency Department, the First Hospital of Jilin University, Changchun 130021, China
c
Tumor Biotherapy Center, Jilin Province People’s Hospital, Changchun 130021, China
Experimental Standards and chemicals
756
Analytical reference standards of heteroclitin D (Fig. 1, MW 482.52 g/mol) and gambogic acid (MW 628.75 g/mol), used as internal standard (IS),
Biomed. Chromatogr. 2015; 29: 756–761
Fan Zhang and Nan Zhang equally contributed to the work.
Abbreviations used: ESI, electrospray ionization.
Copyright © 2014 John Wiley & Sons, Ltd.
Quantification of heteroclitin Recovery and matrix effect. The recovery was determined (n = 6) by comparing processed QC samples with reference heteroclitin D solutions in blank plasma extract at the same concentration level. The matrix effect was assessed by comparing the standard solutions in blank plasma extracts with those of neat solutions at corresponding concentrations.
Figure 1. The chemical structures of heteroclitin D and internal standard gambogic acid.
monitoring (MRM) modes of m/z 483.3 → m/z 355.3, and m/z 629.3 → m/z 573.4 for heteroclitin D and IS, respectively. The values of fragmentation/collision energy were set at 135/15 and 145/20 V for heteroclitin D and IS, respectively. Gas flow rate was 11 L/min; nebulizer pressure was 40 psi; and gas temperature was 350 °C.
Preparation of standard and QC samples Individual stock solutions of heteroclitin D and IS were prepared in methanol at 0.260 and 0.300 mg/mL, respectively. The stock solution of heteroclitin D was then serially diluted with methanol to obtain the working solutions. The IS working solution of 300 ng/mL was also prepared by diluting the stock solution with methanol. Calibration standard samples of heteroclitin D (9.98, 26.62, 66.56, 124.8, 332.8, 832 and 2080 ng/mL) were obtained by spiking 10 μL of the appropriate working solutions to 100 μL of blank plasma. QC samples at low, medium and high concentrations (29.94, 166.4 and 1872 ng/mL) were prepared separately in the same mode.
Preparation of plasma samples Rat plasma sample (50 μL) and 25 μL of IS solution (300 ng/mL) were added to a 1.5 mL Eppendorf tube and vortex-mixed for 15 s, followed by adding 300 μL methanol. The mixture was vortex-mixed for 5 min and then centrifuged at 13,000g for 10 min. The supernatant was transferred into another clean Eppendorf tube and evaporated to dryness at 40°C with nitrogen. The resulting residue was dissolved in 100 μL of mobile phase, and vortex-mixed for 1 min. After centrifugation at 13,000g for 10 min, 5 μL of aliquot was injected into the LC/MS/MS system for analysis.
Method validation Specif icity. Six different sources of rat blank plasma samples were checked for possible endogenous interferences and presence of the analyte. Precision and accuracy. The intra- and interday precision and accuracy were evaluated by determining QC samples on the same day and on three consecutive days, respectively. The intra- and interday precision expressed as relative standard deviation (RSD) should not exceed 15%, and the accuracy expressed as relative error (RE) should be within ±15% (US Food and Drug Administration, 2013).
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Pharmacokinetic study Eight male Sprague–Dawley rats (weighing 220 ± 20 g) were purchased from the Laboratory Animal Center of Jilin University (Changchun, China). All experimental procedures were approved by the Experimental Animal Care and Use Committee of Jilin University (Changchun, China). The rats were housed for 10 days prior to the study and maintained on a 12 h light/12 h dark cycle at 22 ± 2°C and at 50 ± 10% relative humidity. The rats were fasted for 12 h before experiments with free access to water. The dosing solution with heteroclitin D concentration of 0.2 mg/mL was prepared by dissolving appropriate amount of heteroclitin D in saline solution containing 5% DMSO. The actual intravenous dose of the drug was 2.0 mg/kg, and the dose volume was 10.0 mL/kg. After intravenous administration of heteroclitin D through tail vein, approximately 0.2 mL blood samples were collected in heparinized 1.5 mL polythene tubes by orbital bleeding via capillary tubes at 0.083, 0.167, 0.333, 0.667, 1, 1.5, 2, 3, 5, 8, 12 and 24 h postdose. The blood samples were immediately centrifuged at 6000g for 10 min. The plasma was separated and frozen at –20°C until analysis. The plasma concentrations of heteroclitin D were expressed as mean ± SD, and the mean concentration–time curves were plotted. Pharmacokinetic parameters were estimated using DAS 2.1 software package (Chinese Pharmacological Society, China).
Results and discussion LC/MS/MS condition LC/MS/MS operational parameters were carefully optimized for determination of heteroclitin D and IS in rat plasma. The mass spectrometer was tuned in both positive and negative ESI modes. Both heteroclitin D and IS produced much stronger signals in positive ionization mode than in negative ionization mode. In the precursor ion full-scan spectra, the protonated molecules [M + H]+ m/z 483.3 and 629.3 for heteroclitin D and IS were chosen to be the precursor ions, respectively. Other parameters, including capillary voltage, ESI source temperature and fragmentation energy, were further tuned to obtain the optimal intensity of protonated molecules [M + H]+ of the two compounds. It was found that the signal intensity of each precursor ion no longer increased significantly when capillary voltage was higher than 3500 V. Moreover, the optimal fragmentation energy was set at 135 and 145 V for heteroclitin D and IS, respectively. The product ion scan spectra showed highabundance fragment ions at m/z 355.3 and 573.4 for heteroclitin D and IS, respectively. The collision energy was also optimized to obtain for their high signal intensity of product ions. The optimal collision energy was finally set at 15 and 20 eV for heteroclitin D and IS, respectively. Under the above-mentioned optimal conditions, MRM mode using the precursor → product ion
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Calibration curve and sensitivity. Calibration curves were constructed by analyzing spiked calibration samples on three separate days. 2 Standard curves were fitted by weighted (1/x ) least squares linear regression of the peak area ratios of heteroclitin D to IS against the corresponding concentrations in the concentration range of 9.98–2080 ng/mL. The LLOQ was defined as the lowest concentration in the calibration curve at which both precision (RSD) and accuracy (RE) were ≤20%, and signal/noise ratio was >10.
Stability. Freeze–thaw stability was determined by assaying QC samples stored at 20°C and underwent three repeated freeze–thaw cycles. Long-term stability was investigated at three QC levels after storage at 20°C for 9 days. Short-term stability was tested after exposure of QC samples to ambient temperature for 6 h. Postpreparative stability was checked by analyzing samples in autosampler vials for 12 h.
F. Zhang et al. transitions of m/z 483.3 → m/z 355.3, and m/z 629.3 → m/z 573.4, respectively, was employed for quantification of heteroclitin D and IS (Fig. 2). With addition of formic acid to the mobile phase, the signal intensity and peak shape of analytes were improved greatly. When the concentration of formic acid in aqueous phase was optimized from 0.05 to 0.25%, a satisfactory separation of the two analytes was achieved by isocratic elution program using mobile phase consisting of methanol–water–formic acid (80:20:0.1, v/v/v). The isocratic elution used resulted in retention times of 0.6, and 1.1 min for heteroclitin D and IS, respectively.
Method validation Specif icity. The specificity of the method was confirmed by analyzing six different sources of blank rat plasma. The elution of the analytes was rapid and selective, heteroclitin D and IS being eluted within 1.5 min (Fig. 3). No interfering peaks were observed at their retention times, and the peaks were completely resolved from the plasma matrix. Precision and accuracy. Intra- and interday precision and accuracy were evaluated from validation runs performed at
758
+
Figure 2. Full-scan precursor ion and product ion spectra of [M + H] for (A, B) heteroclitin D and (C, D) gambogic acid.
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Copyright © 2014 John Wiley & Sons, Ltd.
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Quantification of heteroclitin
Figure 3. Representative chromatograms of blank plasma (A), blank plasma spiked with the analyte and IS (LLOQ) (B), and a plasma sample 20 min after intravenous administration of heteroclitin D (C).
three QC levels (Table 1). The intraday precision (%RSD) ranged from 3.4 to 6.7% for heteroclitin D, while the accuracy (%RE) was within –4.3–2.6%. Similarly, for the interday experiments, the precision varied from 3.9 to 12.8% for heteroclitin D, while the accuracy was within –8.9–3.6%. Calibration curve and sensitivity. The linear regressions of the peak area ratios vs concentrations were fitted over the concentration range 9.98–2080 ng/mL for heteroclitin D
in rat plasma. A typical equation of the calibration curve was: y = (0.00203 ± 0.00011)x (0.00674 ± 0.01350), r2 = 0.9981, where y represents the ratios of heteroclitin D peak area to that of IS and x represents the plasma concentration. The LLOQ for the determination of heteroclitin D in plasma was 9.98 ng/mL. The intra- and interday precisions at LLOQ were 9.1 and 5.7%, respectively (Table 1). The intra- and interday accuracies were –4.7 and 1.9%, respectively.
Table 1. Accuracy and precision for the analysis of heteroclitin D in rat plasma (n = 3 days, six replicates per day) Nominal concentration (ng/mL)
Interday
Determined concentration (mean ± SD, ng/mL)
RE (%)
RSD (%)
Determined concentration (mean ± SD, ng/mL)
RE (%)
RSD (%)
9.51 ± 0.87 30.25 ± 1.03 159.2 ± 10.7 1921 ± 112
–4.7 1.0 –4.3 2.6
9.1 3.4 6.7 5.8
10.17 ± 0.58 31.02 ± 3.96 162.3 ± 6.4 1705 ± 169
1.9 3.6 –2.5 –8.9
5.7 12.8 3.9 9.9
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9.98 29.94 166.4 1872
Intraday
F. Zhang et al. Table 2. Stability of heteroclitin D under a variety of storage and processing conditions (n = 3) Storage and processing conditions Three freeze–thaw cycles
Long-term (9 days at
20°C)
Short-term (6 h at ambient temperature)
Post-preparative (12 h in autosampler vials)
Nominal concentration (ng/mL)
Determined concentration (mean ± SD, ng/mL)
RE (%)
RSD (%)
29.94 166.4 1872 29.94 166.4 1872 29.94 166.4 1872 29.94 166.4 1872
28.97 ± 1.55 154.3 ± 10.1 1773 ± 153 30.23 ± 2.11 178.9 ± 12.5 1903 ± 202 31.07 ± 0.51 181.9 ± 12.4 1683 ± 191 29.42 ± 1.42 166.2 ± 3.8 1762 ± 51
–3.2 –7.3 –5.3 1.0 7.5 1.7 3.8 9.3 –10.1 –1.7 –0.1 –5.9
5.4 6.5 8.6 7.0 7.0 10.6 1.6 6.8 11.3 4.8 2.3 2.9
for heteroclitin D. The matrix effect of IS was 98.8 ± 1.5%. No significant matrix effect was observed for heteroclitin D and IS, indicating that no co-eluting substance influenced their ionization efficiency. Stability. Heteroclitin D showed good stability in plasma under all the storage and processing conditions evaluated with the bias (%RE) of –10.1–9.3% (Table 2). The method is therefore proved to be applicable for routine analysis.
Pharmacokinetic study
Figure 4. Mean plasma concentration–time curves of heteroclitin D following a single intravenous bolus injection of heteroclitin D at doses of 2.0 mg/kg to rats (n = 8, mean ± SD).
Recovery and matrix effect. The recoveries of the analyte at three QC levels (29.94, 166.4 and 1872 ng/mL) were 89.1 ± 5.7, 93.2 ± 3.8 and 88.2 ± 3.5% for heteroclitin D, respectively. Mean recovery for the IS (300 ng/mL) was 87.6 ± 6.9%. The matrix effects of the analyte at three QC levels (29.94, 166.4 and 1872 ng/mL) were 91.4 ± 4.2, 90.3 ± 7.1 and 92.0 ± 2.2%, respectively
The present method was successfully applied to the pharmacokinetic studies of heteroclitin D in rats after intravenous bolus injection. A plot of plasma concentration vs time for heteroclitin D in rats is presented in Fig. 4. The corresponding pharmacokinetic parameters calculated using noncompartmental analysis are listed as means ± SD in Table 3. The drug concentrations of heteroclitin D in plasma were quantifiable up to 24 h for 2.0 mg/kg intravenous bolus injection. Therefore, the data obtained could be used to investigate pharmacokinetic characterization of heteroclitin D. The area under the plasma concentration vs time from 0 h to the time of last measurable concentration (AUC0–t) or to infinity (AUC0–∞) was 1634.46 ± 151.81 and 1705.13 ± 169.90 μg/L h, respectively. The drug elimination was slow with an elimination half-life (t1/2z) of 5.04 ± 1.84 h. The data obtained from this pharmacokinetic study could give some useful information for further research on this drug.
Table 3. Pharmacokinetic parameters estimated by non-compartmental model analysis following a single intravenous bolus injection of heteroclitin D to rats (n = 8, mean ± SD) Parameters C5min (ng/mL) t1/2z (h) CL (l/h/kg) Vd (l/kg) MRT0–t (h)
Values
Parameters
Values
665.97 ± 29.89 5.04 ± 1.84 1.18 ± 0.12 8.40 ± 2.62 4.29 ± 0.92
MRT0–∞ (h) AUC0–t (μg/L h) AUC0–∞(μg/L h) AUMC0–t (mg h2/mL) AUMC0–∞ (mg h2/mL)
5.34 ± 1.44 1634.46 ± 151.81 1705.13 ± 169.90 7098.11 ± 1977.01 9262.82 ± 3138.51
760
C5min plasma concentration at 5 min; t1/2z, elimination half-life; CL, clearance; Vd, volume of distribution; MRT, mean residence time; AUC, area under plasma concentration; AUMC, area under the first moment curve.
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Quantification of heteroclitin
Conclusions The first full validation of a bioanalytical method was developed for the quantification of heteroclitin D in rat plasma. The sensitive LC/MS/MS assay showed satisfactory accuracy, precision, recovery and stability according to US Food and Drug Administration guidelines. Its application was successfully applied to the pharmacokinetic study of heteroclitin D following a single intravenous bolus injection.
References Chen DF, Zhang SX, Xie L, Xie JX, Chen K, Kashiwada Y, Zhou BN, Wang P, Cosentino LM and Lee KH. Anti-aids agents – XXVI. Structure–activity correlations of Gomisin-G-related anti-HIV lignans from Kadsura interior and of related synthetic analogues. Bioorganic and Medicinal Chemistry 1997; 8: 1715–1723. Chen DF, Zhang SX, Kozuka M, Sun QZ, Feng J, Wang Q, Mukainaka T, Nobukuni Y, Tokuda H, Nishino H, Wang HK, Morris-Natschke SL and Lee KH. Interiotherins C and D, two new lignans from Kadsura interior and antitumor-promoting effects of related neolignans on Epstein–Barr virus activation. Journal of Natural Products 2002; 65: 1242–1245. Ding ZH and Luo SD. Study on lignans from Kadsura interior A. C. Smith. Acta Chimica Sinica 1990; 48: 1075–1079.
Dong K, Pu JX, Du X, Li XN and Sun HD. Two new guaianolide-type sesquiterpenoids from Kadsura interior. Chinese Chemical Letters 2013; 24: 111–113. Hausott B, Greger H and Marian B. Naturally occurring lignans efficiently induce apoptosis in colo-rectal tumor. Journal of Cancer Research and Clinical Oncology 2003; 129: 569–576. Jia ZW, Liao ZX and Chen DF. Two new dibenzocyclooctene lignans from the water extract of Kadsura spp. Helvetica Chimica Acta 2005; 88: 2288–2293. Liu J, Qi Y, Lai H, Zhang J, Jia X, Liu H, Zhang B and Xiao P. Genus Kadsura, a good source with considerable characteristic chemical constituents and potential bioactivities. Phytomedicine 2014; doi: 10.1016/j. phymed.2014.01.015 Pu JX, Yang LM, Xiao WL, Li RT, Lei C, Gao XM, Huang SX, Li SH, Zheng YT, Huang H and Sun HD. Compounds from Kadsura heteroclita and related anti-HIV activity. Phytochemistry 2008; 69: 1266–1272. State Pharmacopoeia Commission of P.R. China. Pharmacopoeia of the P. R. China. Chemical Industry Press: Beijing, 2010; 339–340. US Food and Drug Administration. Guidance for Industry: Bioanalytical Method Validation. US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research: Rockville, MD, 2013. Available from: http://www.fda. gov/CVM Xu LJ, Ma P, Li L, Wang WY, Xiao W, Peng Y and Xiao PG. New collection of crude drugs in Chinese Pharmacopoeia 2010 III. Kadsurae Caulis. Chinese Herbal Medicines 2012; 4: 177–182.
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Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/bmc
Revised: 12 August 2014,
Accepted: 28 August 2014
Published online in Wiley Online Library: 21 October 2014
(wileyonlinelibrary.com) DOI 10.1002/bmc.3352
Quantification of heteroclitin D in rat plasma: validation of an LC/MS/MS method and its application in a preclinical pharmacokinetic study Fan Zhanga†, Nan Zhangb†, Li Pangb, Yan Tanc* and Hong Xua* ABSTRACT: A rapid, sensitive and specific liquid chromatography tandem mass spectrometry (LC/MS/MS) method was developed and validated for the quantification of heteroclitin D in rat plasma after using gambogic acid as internal standard (IS). Chromatographic separation was done on a Thermo Hypersil GOLD column (30 × 2.1 mm, 3 μm) using a mobile phase consisting of methanol–water–formic acid (80:20:0.1, v/v/v). The mass spectrometer worked with positive electrospray ionization in multiple reaction monitoring mode, using target ions at [M + H]+ m/z 483.3 for heteroclitin D and [M + H]+ m/z 629.3 for the IS. The standard curve was linear (R2 ≥0.995) over the concentration range 9.98–2080 ng/mL and had good back-calculated accuracy and precision. The intra- and interday precision and accuracy determined on three quality control samples (29.94, 166.4 and 1872 ng/mL) were ≤12.8 and –8.9–3.6%, respectively. The extraction recovery was ≥88.2% and the lower limit of quantification was 9.98 ng/mL. The method was successfully applied to evaluate pharmacokinetics of heteroclitin D in Sprague–Dawley rats following a single intravenous bolus injection of 2.0 mg/kg heteroclitin. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: heteroclitin D; LC/MS/MS; rat plasma; pharmacokinetics
Introduction Kadsurae Caulis (Dian-Jixueteng in Chinese), derived from the dry stems and rattans of Kadsurae interior A. C. Smith, is a member of the Magnoliaceae family and has been used for the treatment of menstrual irregularities, blood deficiencies and other feminine disorders (Jia et al., 2005; Xu et al., 2012; Dong et al., 2013). Dibenzocyclooctene-type lignans are the major active components of the herb and have been extensively investigated (Chen et al., 2002; Liu et al., 2014). They display a variety of biological activities, such as anticolo-rectal tumor-promoting effects, calcium antagonism, antilipid peroxidation and anti-HIV effects (Ding and Luo, 1990; Chen et al., 1997; Hausott et al., 2003; Pu et al., 2008). Moreover, heteroclitin D is the major bioactive lignan used as a phytochemical marker for quality control of Kadsurae Caulis in the Chinese Pharmacopeia (State Pharmacopoeia Commission of P.R. China, 2010). However, no method for the determination of heteroclitin D has previously been reported in any biological matrix and pharmacokinetic data are thus not available. Thus, in this study, we aim to develop an efficient method for the determination of this lignan in rat plasma and further evaluate its pharmacokinetics.
were purchased from the CRM/RM Information Center of China (Beijing, China). HPLC-grade methanol and analytical-grade formic acid were purchased from Tedia Co. (Fairfield, OH, USA). Distilled water was prepared from deionized water. Blank plasma used to prepare daily standard calibration curves and quality control (QC) samples was obtained from male Sprague–Dawley rats.
Instrumentation and analytical conditions The Agilent 1200 rapid resolution HPLC system was interfaced with an Agilent 6460 triple quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source. The column was a Thermo Hypersil Gold column (30 × 2.1 mm, 3 μm). The mobile phase was methanol–water–formic acid (80:20:0.1, v/v/v), and the flow rate was 0.4 mL/min. Quantitation was performed in positive multiple reaction
* Correspondence to: Y. Tan, Tumor Biotherapy Center, Jilin Province People’s Hospital, Changchun 130021, China. Email: tanyan112014@hotmail. com H. Xu, Gastrointestinal Medicine, the First Hospital of Jilin University, Changchun 130021, China. Email: [email protected] †
a
Gastrointestinal Medicine, the First Hospital of Jilin University, Changchun 130021, China
b
Emergency Department, the First Hospital of Jilin University, Changchun 130021, China
c
Tumor Biotherapy Center, Jilin Province People’s Hospital, Changchun 130021, China
Experimental Standards and chemicals
756
Analytical reference standards of heteroclitin D (Fig. 1, MW 482.52 g/mol) and gambogic acid (MW 628.75 g/mol), used as internal standard (IS),
Biomed. Chromatogr. 2015; 29: 756–761
Fan Zhang and Nan Zhang equally contributed to the work.
Abbreviations used: ESI, electrospray ionization.
Copyright © 2014 John Wiley & Sons, Ltd.
Quantification of heteroclitin Recovery and matrix effect. The recovery was determined (n = 6) by comparing processed QC samples with reference heteroclitin D solutions in blank plasma extract at the same concentration level. The matrix effect was assessed by comparing the standard solutions in blank plasma extracts with those of neat solutions at corresponding concentrations.
Figure 1. The chemical structures of heteroclitin D and internal standard gambogic acid.
monitoring (MRM) modes of m/z 483.3 → m/z 355.3, and m/z 629.3 → m/z 573.4 for heteroclitin D and IS, respectively. The values of fragmentation/collision energy were set at 135/15 and 145/20 V for heteroclitin D and IS, respectively. Gas flow rate was 11 L/min; nebulizer pressure was 40 psi; and gas temperature was 350 °C.
Preparation of standard and QC samples Individual stock solutions of heteroclitin D and IS were prepared in methanol at 0.260 and 0.300 mg/mL, respectively. The stock solution of heteroclitin D was then serially diluted with methanol to obtain the working solutions. The IS working solution of 300 ng/mL was also prepared by diluting the stock solution with methanol. Calibration standard samples of heteroclitin D (9.98, 26.62, 66.56, 124.8, 332.8, 832 and 2080 ng/mL) were obtained by spiking 10 μL of the appropriate working solutions to 100 μL of blank plasma. QC samples at low, medium and high concentrations (29.94, 166.4 and 1872 ng/mL) were prepared separately in the same mode.
Preparation of plasma samples Rat plasma sample (50 μL) and 25 μL of IS solution (300 ng/mL) were added to a 1.5 mL Eppendorf tube and vortex-mixed for 15 s, followed by adding 300 μL methanol. The mixture was vortex-mixed for 5 min and then centrifuged at 13,000g for 10 min. The supernatant was transferred into another clean Eppendorf tube and evaporated to dryness at 40°C with nitrogen. The resulting residue was dissolved in 100 μL of mobile phase, and vortex-mixed for 1 min. After centrifugation at 13,000g for 10 min, 5 μL of aliquot was injected into the LC/MS/MS system for analysis.
Method validation Specif icity. Six different sources of rat blank plasma samples were checked for possible endogenous interferences and presence of the analyte. Precision and accuracy. The intra- and interday precision and accuracy were evaluated by determining QC samples on the same day and on three consecutive days, respectively. The intra- and interday precision expressed as relative standard deviation (RSD) should not exceed 15%, and the accuracy expressed as relative error (RE) should be within ±15% (US Food and Drug Administration, 2013).
Biomed. Chromatogr. 2015; 29: 756–761
Pharmacokinetic study Eight male Sprague–Dawley rats (weighing 220 ± 20 g) were purchased from the Laboratory Animal Center of Jilin University (Changchun, China). All experimental procedures were approved by the Experimental Animal Care and Use Committee of Jilin University (Changchun, China). The rats were housed for 10 days prior to the study and maintained on a 12 h light/12 h dark cycle at 22 ± 2°C and at 50 ± 10% relative humidity. The rats were fasted for 12 h before experiments with free access to water. The dosing solution with heteroclitin D concentration of 0.2 mg/mL was prepared by dissolving appropriate amount of heteroclitin D in saline solution containing 5% DMSO. The actual intravenous dose of the drug was 2.0 mg/kg, and the dose volume was 10.0 mL/kg. After intravenous administration of heteroclitin D through tail vein, approximately 0.2 mL blood samples were collected in heparinized 1.5 mL polythene tubes by orbital bleeding via capillary tubes at 0.083, 0.167, 0.333, 0.667, 1, 1.5, 2, 3, 5, 8, 12 and 24 h postdose. The blood samples were immediately centrifuged at 6000g for 10 min. The plasma was separated and frozen at –20°C until analysis. The plasma concentrations of heteroclitin D were expressed as mean ± SD, and the mean concentration–time curves were plotted. Pharmacokinetic parameters were estimated using DAS 2.1 software package (Chinese Pharmacological Society, China).
Results and discussion LC/MS/MS condition LC/MS/MS operational parameters were carefully optimized for determination of heteroclitin D and IS in rat plasma. The mass spectrometer was tuned in both positive and negative ESI modes. Both heteroclitin D and IS produced much stronger signals in positive ionization mode than in negative ionization mode. In the precursor ion full-scan spectra, the protonated molecules [M + H]+ m/z 483.3 and 629.3 for heteroclitin D and IS were chosen to be the precursor ions, respectively. Other parameters, including capillary voltage, ESI source temperature and fragmentation energy, were further tuned to obtain the optimal intensity of protonated molecules [M + H]+ of the two compounds. It was found that the signal intensity of each precursor ion no longer increased significantly when capillary voltage was higher than 3500 V. Moreover, the optimal fragmentation energy was set at 135 and 145 V for heteroclitin D and IS, respectively. The product ion scan spectra showed highabundance fragment ions at m/z 355.3 and 573.4 for heteroclitin D and IS, respectively. The collision energy was also optimized to obtain for their high signal intensity of product ions. The optimal collision energy was finally set at 15 and 20 eV for heteroclitin D and IS, respectively. Under the above-mentioned optimal conditions, MRM mode using the precursor → product ion
Copyright © 2014 John Wiley & Sons, Ltd.
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Calibration curve and sensitivity. Calibration curves were constructed by analyzing spiked calibration samples on three separate days. 2 Standard curves were fitted by weighted (1/x ) least squares linear regression of the peak area ratios of heteroclitin D to IS against the corresponding concentrations in the concentration range of 9.98–2080 ng/mL. The LLOQ was defined as the lowest concentration in the calibration curve at which both precision (RSD) and accuracy (RE) were ≤20%, and signal/noise ratio was >10.
Stability. Freeze–thaw stability was determined by assaying QC samples stored at 20°C and underwent three repeated freeze–thaw cycles. Long-term stability was investigated at three QC levels after storage at 20°C for 9 days. Short-term stability was tested after exposure of QC samples to ambient temperature for 6 h. Postpreparative stability was checked by analyzing samples in autosampler vials for 12 h.
F. Zhang et al. transitions of m/z 483.3 → m/z 355.3, and m/z 629.3 → m/z 573.4, respectively, was employed for quantification of heteroclitin D and IS (Fig. 2). With addition of formic acid to the mobile phase, the signal intensity and peak shape of analytes were improved greatly. When the concentration of formic acid in aqueous phase was optimized from 0.05 to 0.25%, a satisfactory separation of the two analytes was achieved by isocratic elution program using mobile phase consisting of methanol–water–formic acid (80:20:0.1, v/v/v). The isocratic elution used resulted in retention times of 0.6, and 1.1 min for heteroclitin D and IS, respectively.
Method validation Specif icity. The specificity of the method was confirmed by analyzing six different sources of blank rat plasma. The elution of the analytes was rapid and selective, heteroclitin D and IS being eluted within 1.5 min (Fig. 3). No interfering peaks were observed at their retention times, and the peaks were completely resolved from the plasma matrix. Precision and accuracy. Intra- and interday precision and accuracy were evaluated from validation runs performed at
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Figure 2. Full-scan precursor ion and product ion spectra of [M + H] for (A, B) heteroclitin D and (C, D) gambogic acid.
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Quantification of heteroclitin
Figure 3. Representative chromatograms of blank plasma (A), blank plasma spiked with the analyte and IS (LLOQ) (B), and a plasma sample 20 min after intravenous administration of heteroclitin D (C).
three QC levels (Table 1). The intraday precision (%RSD) ranged from 3.4 to 6.7% for heteroclitin D, while the accuracy (%RE) was within –4.3–2.6%. Similarly, for the interday experiments, the precision varied from 3.9 to 12.8% for heteroclitin D, while the accuracy was within –8.9–3.6%. Calibration curve and sensitivity. The linear regressions of the peak area ratios vs concentrations were fitted over the concentration range 9.98–2080 ng/mL for heteroclitin D
in rat plasma. A typical equation of the calibration curve was: y = (0.00203 ± 0.00011)x (0.00674 ± 0.01350), r2 = 0.9981, where y represents the ratios of heteroclitin D peak area to that of IS and x represents the plasma concentration. The LLOQ for the determination of heteroclitin D in plasma was 9.98 ng/mL. The intra- and interday precisions at LLOQ were 9.1 and 5.7%, respectively (Table 1). The intra- and interday accuracies were –4.7 and 1.9%, respectively.
Table 1. Accuracy and precision for the analysis of heteroclitin D in rat plasma (n = 3 days, six replicates per day) Nominal concentration (ng/mL)
Interday
Determined concentration (mean ± SD, ng/mL)
RE (%)
RSD (%)
Determined concentration (mean ± SD, ng/mL)
RE (%)
RSD (%)
9.51 ± 0.87 30.25 ± 1.03 159.2 ± 10.7 1921 ± 112
–4.7 1.0 –4.3 2.6
9.1 3.4 6.7 5.8
10.17 ± 0.58 31.02 ± 3.96 162.3 ± 6.4 1705 ± 169
1.9 3.6 –2.5 –8.9
5.7 12.8 3.9 9.9
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9.98 29.94 166.4 1872
Intraday
F. Zhang et al. Table 2. Stability of heteroclitin D under a variety of storage and processing conditions (n = 3) Storage and processing conditions Three freeze–thaw cycles
Long-term (9 days at
20°C)
Short-term (6 h at ambient temperature)
Post-preparative (12 h in autosampler vials)
Nominal concentration (ng/mL)
Determined concentration (mean ± SD, ng/mL)
RE (%)
RSD (%)
29.94 166.4 1872 29.94 166.4 1872 29.94 166.4 1872 29.94 166.4 1872
28.97 ± 1.55 154.3 ± 10.1 1773 ± 153 30.23 ± 2.11 178.9 ± 12.5 1903 ± 202 31.07 ± 0.51 181.9 ± 12.4 1683 ± 191 29.42 ± 1.42 166.2 ± 3.8 1762 ± 51
–3.2 –7.3 –5.3 1.0 7.5 1.7 3.8 9.3 –10.1 –1.7 –0.1 –5.9
5.4 6.5 8.6 7.0 7.0 10.6 1.6 6.8 11.3 4.8 2.3 2.9
for heteroclitin D. The matrix effect of IS was 98.8 ± 1.5%. No significant matrix effect was observed for heteroclitin D and IS, indicating that no co-eluting substance influenced their ionization efficiency. Stability. Heteroclitin D showed good stability in plasma under all the storage and processing conditions evaluated with the bias (%RE) of –10.1–9.3% (Table 2). The method is therefore proved to be applicable for routine analysis.
Pharmacokinetic study
Figure 4. Mean plasma concentration–time curves of heteroclitin D following a single intravenous bolus injection of heteroclitin D at doses of 2.0 mg/kg to rats (n = 8, mean ± SD).
Recovery and matrix effect. The recoveries of the analyte at three QC levels (29.94, 166.4 and 1872 ng/mL) were 89.1 ± 5.7, 93.2 ± 3.8 and 88.2 ± 3.5% for heteroclitin D, respectively. Mean recovery for the IS (300 ng/mL) was 87.6 ± 6.9%. The matrix effects of the analyte at three QC levels (29.94, 166.4 and 1872 ng/mL) were 91.4 ± 4.2, 90.3 ± 7.1 and 92.0 ± 2.2%, respectively
The present method was successfully applied to the pharmacokinetic studies of heteroclitin D in rats after intravenous bolus injection. A plot of plasma concentration vs time for heteroclitin D in rats is presented in Fig. 4. The corresponding pharmacokinetic parameters calculated using noncompartmental analysis are listed as means ± SD in Table 3. The drug concentrations of heteroclitin D in plasma were quantifiable up to 24 h for 2.0 mg/kg intravenous bolus injection. Therefore, the data obtained could be used to investigate pharmacokinetic characterization of heteroclitin D. The area under the plasma concentration vs time from 0 h to the time of last measurable concentration (AUC0–t) or to infinity (AUC0–∞) was 1634.46 ± 151.81 and 1705.13 ± 169.90 μg/L h, respectively. The drug elimination was slow with an elimination half-life (t1/2z) of 5.04 ± 1.84 h. The data obtained from this pharmacokinetic study could give some useful information for further research on this drug.
Table 3. Pharmacokinetic parameters estimated by non-compartmental model analysis following a single intravenous bolus injection of heteroclitin D to rats (n = 8, mean ± SD) Parameters C5min (ng/mL) t1/2z (h) CL (l/h/kg) Vd (l/kg) MRT0–t (h)
Values
Parameters
Values
665.97 ± 29.89 5.04 ± 1.84 1.18 ± 0.12 8.40 ± 2.62 4.29 ± 0.92
MRT0–∞ (h) AUC0–t (μg/L h) AUC0–∞(μg/L h) AUMC0–t (mg h2/mL) AUMC0–∞ (mg h2/mL)
5.34 ± 1.44 1634.46 ± 151.81 1705.13 ± 169.90 7098.11 ± 1977.01 9262.82 ± 3138.51
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C5min plasma concentration at 5 min; t1/2z, elimination half-life; CL, clearance; Vd, volume of distribution; MRT, mean residence time; AUC, area under plasma concentration; AUMC, area under the first moment curve.
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Biomed. Chromatogr. 2015; 29: 756–761
Quantification of heteroclitin
Conclusions The first full validation of a bioanalytical method was developed for the quantification of heteroclitin D in rat plasma. The sensitive LC/MS/MS assay showed satisfactory accuracy, precision, recovery and stability according to US Food and Drug Administration guidelines. Its application was successfully applied to the pharmacokinetic study of heteroclitin D following a single intravenous bolus injection.
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