Research article Received: 8 February 2014,
Revised: 5 August 2014,
Accepted: 27 August 2014
Published online in Wiley Online Library: 21 October 2014
(wileyonlinelibrary.com) DOI 10.1002/bmc.3351
Trace quantification of 1-triacontanol in beagle plasma by GC-MS/MS and its application to a pharmacokinetic study Chunfeng Wanga†, Ali Fana†, Xiaojie Zhua, Yang Lub, Shuhua Denga, Wenchao Gaoa, Wei Zhanga, Qi Liua and Xijing Chena* ABSTRACT: 1-Triacontanol (TA), a member of long chain fatty alcohol, has recently been received great attention owing to its antitumor activity. In this study, an accurate, sensitive and selective gas chromatography–tandem mass spectrometry method was developed and validated for the quantification of TA in beagle plasma using 1-octacosanal as the internal standard (IS) for the first time. With temperature programming, chromatographic separation was carried out on an HP5MS column, using helium as carrier gas and argon as collision gas, both at a flow rate of 1 mL/min. TA was analyzed using positive ion electrospray ionization in multiple-reaction monitoring mode, with the precursor to product ion transitions of m/z 495.6 → 97.0 and m/z 467.5 → 97.0 for TA and the IS, respectively. The lower limit of quantitation, linearity, intra- and interday precision, accuracy, stability, extraction recovery and matrix effect of TA were within the acceptable limits. The validated method was successfully applied to a pharmacokinetic study of TA in beagles. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: 1-triacontanol; GC-MS/MS; pharmacokinetics
Introduction
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* Correspondence to: Xijing Chen, Center of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing 210009, China. Email: [email protected] †
These authors contributed equally to the present work.
a
Center of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing 210009, China
b
Division of Pharmacotherapy and Experimental Therapeutics, Eshelman School of Pharmacy, the University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA Abbreviations used: BSTFA, N,O-bis(trimethylsilyl) trifluoroacetamide; MRM, multiple reaction monitoring; TA, 1-Triacontanol.
Copyright © 2014 John Wiley & Sons, Ltd.
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1-Triacontanol (TA, Fig. 1), generally existing in animal and vegetable waxes, is a component of policosanol (Más, 2000; Dullens et al., 2008). Previous studies showed that policosanol has a strong ability to inhibit cholesterol biosynthesis in rats (Noa et al., 1995; Menendez et al., 1996), rabbits (Arruzazabala et al., 1994; Pons et al., 1994), dogs (Mesa et al., 1994), monkeys (Rodríguez-Echenique et al., 1994), healthy volunteers (Hernández et al., 1992) and patients with type II hypercholesterolemia (Más et al., 1998, 2001; Castaño et al., 2000a, 2000b; Castaño et al., 2001, 2003). As an ingredient of policosanol, TA shows a significant effect in inhibiting cholesterol synthesis in cultured rat hepatoma cells (Singh et al., 2006). Recently, the antitumor effect of TA was discovered, especially in the treatment of liver cancer, colon cancer and lung cancer applications (Zhang et al., 2008). Meanwhile, adverse effects on the important immune organs of the tumor-bearing mice were not observed after administration at a dose of 150 mg/kg/day (Fan et al., 2011). Therefore, TA is a promising lead compound candidate for new drug development in cancer treatment owing to its potential antitumor activity and low toxicity. As pharmacokinetic study plays a vital role in the discovery of new drugs, it is necessary to evaluate the pharmacokinetic behavior of TA. Before embarking on the pharmacokinetic study, a reliable analytical method to quantitatively determine TA should be established. Currently, only one method has been reported for the determination of TA in rat plasma (Haim et al., 2009). Despite the fact that an assay for the determination of TA has been developed based on gas chromatography–mass spectrometry (GC-MS),
the limitations of the published literature are obvious and can be summarized as follows: (a) the sensitivity of the reported GC-MS method was 8.4 ng/mL, which is not adequate for a pharmacokinetic study in biological samples; and (b) the oral administrated drug was policosanol, not pure TA. To date, no GC-MS/ MS method has been applied for the determination TA in beagle plasma. As a promising anticancer agent, the development of a highly sensitive, precise and accurate analytical method to monitor the amount of changes in biological specimen is an urgent requirement for pharmacokinetic studies and better understanding of the mechanism of action. In the present study, a reliable gas chromatography–tandem mass spectrometry (GC-MS/MS) method with high sensitivity was established for determination of TA concentration in beagle plasma for the first time. The method is a simple and rapid assessment to support pharmacokinetic studies in beagles.
C. Wang et al. under standard temperature and humidity conditions, and were acclimatized to the housing environment for 1 week before the study. The pharmacokinetic study of TA was performed under the guideline promulgated by the Animals Experimental Ethics Committee of China Pharmaceutical University (Nanjing, China).
Figure 1. The chemical structure of 1-triacontanol (TA) (A) and IS (B).
Experimental Reagents and chemicals 1-Octacosanol (IS, purity ≥ 99%) standard substance was obtained from Sigma-Aldrich Chemical Co. (St Louis, MO, USA). The silylation reagent N,O-bis(trimethylsilyl) trifluoroacetamide (BSTFA, purity ≥ 99%) was purchased from Xiya Reagent Co. Ltd (Chengdu, Sichuan, China). Ethanol and heptane were supplied by Tedia (Fairfield, OH, USA). 1-Triacontanol (purity ≥ 96%) was supplied by Kunming Longjin Inc. (Kunming, Yunnan, China). All other chemicals were of analytical grade.
Animals Beagle dogs (10.0 ± 0.5 kg) of both genders were supplied by Shanghai SIPPR/BK Experimental Animal Co. (Shanghai, China). Animals were kept
Drug preparation methods TA weighing 0.3, 3, 6 and 12 mg was dissolved in 6 mL diethyl ether. Then 50 mg phospholipids were added into each solution and mixed well. The solutions were then evaporated to dryness in the nitrogen, and the residue was reconstituted with 2 mL of glucose. Finally, uniform milky solutions were successfully configured. The doses of TA were 1.5, 15, 30 and 60 mg/kg, respectively.
Instrument and analytical conditions GC (6890A, Agilent Technologies, USA) separation was carried out on a HP-5MS column (30 m × 250 μm, 0.25 μm film thickness, Agilent Technologies, USA) with a temperature program using helium as carrier gas at a 1 mL/min flow rate. The oven temperature gradient was as follows: the initial temperature was 100 °C, then increased to 200 °C at a rate of 40 ° C/min, and maintained for 7.5 min, followed by increasing to 300 °C within 5.0 min and maintained for 5.0 min. Argon was used as a collision
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Figure 2. Precursor/product ion pair for TA (A) and IS (B).
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Copyright © 2014 John Wiley & Sons, Ltd.
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Pharmacokinetics of 1-triacontanol in beagle dogs gas at a flow rate of 1 mL/min. Splitless injection was performed at 300 ° C. The total run time for analysis was 16 min. MS/MS spectrometry (Waters, Milford, USA) with an electron ionization source was operated in positive mode. Mass parameters were as follows: repeller, 10 V; extraction lens, 23 V; Focus lens1, 250 V and Focus lens 3, 27 V; trap, 200 μA; collision energy, 12 eV for TA and 10 eV for IS. The temperature of the interface was 300 °C. The ion source temperature was 250 °C, and electron energy was set at 70 eV. Multiple reaction monitoring (MRM) transitions were used for quantification of all samples by the precursor/product ion pair at m/z 495.6 → 97.0 for TA and m/z 467.5 → 97.0 for IS (Fig. 2). MassLynx V.4.1 was used to control the GCMS/MS system and acquire the data.
Preparation of stock and working solutions, calibration samples and quality controls The standard stock solutions were prepared by dissolving TA (0.1 mg/mL) and 1-octacosanol (0.1 mg/mL) in heptane. The stock solution of TA was successively diluted with heptane to obtain working solutions for calibration with concentrations of 5, 10, 20, 50, 100, 200 and 500 ng/mL. The working solutions for quality control (QC; 10, 100 and 400 ng/mL) samples were prepared in the same way. The stock solution of IS was further diluted with heptane to a final concentration of 100 ng/mL. All working solutions were stored at 4 °C.
Preparation of calibration standards and quality controls samples Calibration samples for TA were prepared in blank dog plasma at concentrations of 5, 10, 20, 50, 100, 200 and 500 ng/mL. The quality control (QC) samples were prepared at low (10 ng/mL), medium (100 ng/mL) and high (400 ng/mL) concentrations in the same way as the plasma samples for calibration, and stored at 20 °C until analysis.
Sample preparation Samples were stored at 20 °C and thawed at room temperature before analysis. Samples were vortexed adequately before pipetting. Aliquots of 50 μL plasma, 50 μL IS (100 ng/mL) and 1 mL ethanolic–NaOH solution (1 mol/L NaOH dissolved in ethanol distilled water; 80/20, v/v) were added to a 10 mL glass tube. After vortex-mixing for 30 s, samples were saponified at 80 °C for 1 h. Then 300 μL HCl (5 M) was added to acidify the solution at 70 °C for 15 min. Then the solution was extracted by adding 2 mL heptane and vortexed for 2 min. The supernatant was then transferred to a new tube and washed with 2 mL ultrapure water. The extraction and washing procedures were conducted three times for each sample. After that, all the supernatant was transferred to a new glass tube to evaporate to dryness by the centrifugal thickener (Centrivap console, Labconco Co., USA). After dryness, samples were derivatized with 300 μL BSTFA at 80 °C for 20 min. Samples were then dried again, and the residue was reconstituted in 50 μL heptane and then 2 μL solution was injected into the GC-MS/MS for analysis.
Method validation
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Application to a pharmacokinetic study in beagle dogs Sixteen healthy adult beagle dogs were fasted for 12 h before dosing with free access to water and were randomized into four groups, each group consisting of four beagle dogs. Plasma samples were collected at 0.5, 0.75, 1, 1.5, 2, 3, 4, 6 and 8 h after a single intragastric administration of TA at a dose of 15, 30 and 60 mg/kg for oral administration to each group. Plasma samples were collected at 2, 5, 10, 15, 30, 60, 120, 240, 360 and 480 min after a single intravenous administration of TA at a dose of 1.5 mg/kg to the last four beagles. Plasma was centrifuged at 8000 rpm for 5 min after harvesting and the supernatant was stored at 20 °C until analysis. All the pharmacokinetic parameters were calculated by noncompartmental analysis using Drug and Statistics software (DAS 2.1.1 version, Mathematical Pharmacology Professional Committee of China), including area under the plasma concentration–time curve (AUC), the mean residence time (MRT), half-life (T1/2), oral clearance (CL/F) and clearance (CL). Based on calculated AUC values, absolute bioavailability (F) was calculated as: F ð%Þ ¼ AUC ig =AUC iv doseiv =doseig 100
Results Assay validation Specificity. Under the described conditions, no endogenous interferences were observed at the retention times of TA
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The method was fully validated for selectivity, matrix effect, linearity, lower limit of quantitation (LLOQ), accuracy and precision, recovery and stability in accordance with the guidelines of the US Food and Drug Administration (2001). The selectivity was evaluated by analyzing blank plasma samples collected from six different sources with plasma samples spiked with TA and IS to investigate the potential interferences at the peak region. A best-fit calibration curve was constructed by plotting the area ratios of the analyte/internal standard (y) vs the concentrations of the analyte (x) in 2 the form of y = a + bx, using weighted (1/x ) least squares linear regression.
The LLOQ was determined as the lowest concentration of the standard. The limit of quantification were determined in quintuplicate by measuring the signal-to-noise (S/N) ratio (10:1) for the analyte at LLOQ. The acceptance criterion of analyte at LLOQ required five replicates with a precision of 20% and an accuracy of 80–120%. The matrix effect was defined as the ion suppression/enhancement on the ionization of analytes, which was evaluated by comparing the peak areas of the analytes in postextracted blank plasma samples spiked with TA at three QC levels with the pure standard solutions with same concentration dried directly and reconstituted with the heptane. The same procedure was performed for the IS. The precision and accuracy of the method were evaluated by analyzing six replicates of spiked beagle dog plasma with known concentrations of TA with QC samples at low (10 ng/mL), medium (100 ng/mL) and high (400 ng/mL) concentration levels. To determine intraday precision and accuracy, the assays were determined on the same day by analyzing five replicates of each concentration level. To determine interday precision and accuracy, the assays were determined on three consecutive days by analyzing three replicates of each concentration level. The precision was expressed as relative standard deviation (RSD) and the accuracy as the relative error (RE).The relative error (RE) and RSD were all within ±15%. Recovery was determined by comparing the peak areas of processed QC samples with those of the pure standards without extraction (n = 5, for each concentration). The stability of analyte in dog plasma was assessed by analyzing replicate (n = 3) QC samples at three concentrations for three freeze–thaw cycles, short-term temperature, postpreparative and long-term temperature stabilities. The stability of TA in plasma was evaluated under various conditions using three levels of QC samples after short-term (pre-processed samples stored at room temperature for 4 h) and long-term storage conditions (stored at 20 ° C for 1 month). The stability of QC samples after three freeze (20 °C) and thaw (room temperature) cycles and postpreparative stability (postprocessed samples stored at 4 °C for 24 h) were also analyzed. Thereafter, samples were analyzed and the resulting values for these samples were the analyte peak area of the postextracted samples vs the analyte peak area of pure standard nominal concentration.
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Figure 3. Representative multiple reaction monitoring chromatograms of TA and IS in beagle plasma: (A) blank plasma sample; (B) beagle plasma spiked with TA at 5 ng/mL and IS; and (C) plasma sample collected 1 h after oral administration of 15 mg/kg TA in beagles.
(14.25 min) and IS (12.13 min). Representative chromatograms of blank beagle plasma, beagle plasma spiked with TA at the 5 ng/mL and test plasma sample obtained at 1 h after the single dose administration of TA (15 mg/kg) are shown in Fig. 3. Linearity and LLOQ. The linearity was evaluated by the calibration curves determined on five separate days. The calibration curve was obtained by plotting the peak-area ratio of TA to the IS vs the TA concentration, showing a good linearity over the concentration range of 5–500 ng/mL. The LLOQ was 5 ng/mL, at which the RSD was 11.0% and the RE was 8.0%. This method was sensitive enough to investigate the pharmacokinetic study of TA with a good S/N (>20) at LLOQ.
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Precision and accuracy. As shown in Table 1, the RSD values of intraday and interday precision were all 85.0% at three QC levels for TA. The mean matrix effects and recovery for IS were 99.0 and 87.5%, respectively. The results suggest that the processing procedure could provide a high extraction efficiency. No ion suppression or enhancement was detected under the current conditions. Stability. Table 3 summarizes the results of short-term, longterm and freeze–thaw stabilities of TA in plasma and postpreparation stability. The high stability property of TA in dog plasma showed that the samples were stable in the preparation and analytical processes. Pharmacokinetic study. The method was successfully applied to the pharmacokinetic study after a single dose of 15, 30 and 60 mg/kg for oral administration and at 1.5 mg/kg for intravenous injection to beagles. The dog plasma concentration–time profiles are shown in Fig. 4 and the corresponding pharmacokinetic
Copyright © 2014 John Wiley & Sons, Ltd.
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Pharmacokinetics of 1-triacontanol in beagle dogs Table 1. Intra- and interday precision and accuracy for 1-triacontanol (TA) in beagle plasma Intraday (n = 5) Nominal concentration (ng/mL) 10 100 400
Interday (n = 15)
Measured
Accuracy,
RSD
Measured
Accuracy,
RSD
concentration
RE (%)
(%)
concentration
RE (%)
(%)
1.00 2.40 3.18
9.60 8.66 6.99
(ng/mL)
(ng/mL)
9.80 ± 0.69 96.0 ± 7.78 423.1 ± 10.70
2.00 4.00 5.75
7.00 8.10 2.53
10.1 ± 0.97 97.6 ± 8.45 412.7 ± 28.86
Table 2. Matrix effect of TA and IS in beagle plasma Compound
TA (n = 6)
IS (n = 18)
Nominal concentration
Matrix effect (%)
Recovery (%)
(ng/mL)
Mean ± SD
RSD
Mean ± SD
RSD
10.0 100.0 400.0 100.0
92.6 ± 14.8 105.6 ± 12.5 106.9 ± 10.1 99.0 ± 12.4
16.0 11.8 9.6 12.5
83.0 ± 7.7 99.2 ± 12.0 82.4 ± 4.4 87.5 ± 0.1
9.3 11.0 5.3 11.2
Table 3. Stability (%) of TA in beagle plasma (n = 5) Storage condition
Room temperature Post-preparative Freeze–thawing Long-term
10 ng/mL
100 ng/mL
400 ng/mL
Mean ± SD
RSD (%)
Mean ± SD
RSD (%)
Mean ± SD
RSD (%)
10.2 ± 1.5 10.5 ± 0.5 9.6 ± 1.1 10.5 ± 1.1
14.4 4.3 11.3 10.8
106.1 ± 8.4 90.3 ± 4.4 90.4 ± 3.9 101.0 ± 3.7
8.0 4.9 4.3 3.6
428.0 ± 16.3 378.6 ± 13.6 398.0 ± 13.9 409.3 ± 7.2
3.8 3.6 3.5 1.8
parameters are listed in Table 4. The values of T1/2 were 1.77 ± 0.05, 2.34 ± 1.14 and 2.12 ± 0.42 h after single oral administration of 15, 30 and 60 mg/kg of TA, and 4.10 ± 3.51 h after intravenous administration of 1.5 mg/kg of TA. After oral administration of TA to beagles, the plasma drug concentration reached the maximum point at about 1.5 h. Then the plasma concentration decreased gradually with T1/2 between 2 and 3 h. The mean T1/2 was 2.1 h, showing that TA was eliminated rapidly in beagles. Peak plasma concentration and AUC were linearly related to the doses, indicating that the pharmacokinetic process of TA was consistent with dose-proportional pharmacokinetics. Based on the pharmacokinetic parameters, the absolute bioavailability (F, %) of TA was calculated to be >8% in beagle dogs.
Discussion
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In the present study, an accurate and highly sensitive GC-MS/MS method was established and fully validated for the determination of TA in beagle plasma. Both TA and IS are compounds with high boiling point and polar hydroxyl groups as well. Therefore, it is difficult for GC to analyze them directly. Thus, we need to carry out structural transformation, such as derivatives, to
analyze them by GC. In this study, we used BSTFA as derivatization reagent. With the addition of one dimethylsilyl group, the molecular weight of TA was increased from 438.8 to 495.6, adding about 57 after the silylation. The initial temperature was 200 °C in the reported GC-MS method (Haim et al., 2009), which was too high for application for high throughput pharmacokinetic studies. Hence, a two-phase temperature gradient program was adopted to protect the column, improve the separation and increase the sensitivity. 1-Octacosanal was chosen as the IS because it is a homologous analog to TA with similar chromatographic behavior and response. In addition, its recovery was satisfactory and it also remains stable during the whole analysis. Based on the full scan of the mass spectrum of TA and IS and their product ion fragments under daughter ion scanning mode, the daughter ions of TA after derivatization were +71, 83, 97, 111, 125 and 137 and those of 1-octacosanal were +75, 83, 97, 111, 125 and 153. By observing the maximum response of the product ions in MRM model, we chose 97 as the qualitative daughter ion. Finally we selected MRM of precursor–product ion transitions with m/z 495.6 → 97.0 for TA and m/z 467.5 → 97.0 for IS. Product ion spectra of TA and IS are shown in Fig. 2.
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Figure 4. The mean plasma concentration–time profile of TA: (A) oral administration of 15, 30 and 60 mg/kg TA in beagles (n = 4). (B) Intravenous administration of 1.5 mg/kg TA in beagles (n = 4).
Table 4. Main pharmacokinetic parameters after oral administration and intravenous injection of TA to beagles (n = 6) i.g.
i.g.
i.g.
i.v.
15 mg/kg
30 mg/kg
60 mg/kg
1.5 mg/kg
1.77 ± 0.05 96.19 ± 9.61 0.013 ± 0.015 3.28 ± 0.13 285.55 ± 32.65 304.28 ± 33.25 8.83 ± 0.96
2.34 ± 1.14 247.16 ± 49.80 0.12 ± 0.038 3.91 ± 0.82 774.24 ± 139.38 866.44 ± 193.34 12.57 ± 2.80
2.12 ± 0.42 460.97 ± 19.41 0.13 ± 0.036 3.74 ± 0.21 1388.57 ± 167.94 1525.78 ± 149.29 11.07 ± 1.08
4.10 ± 3.51 307.38 ± 12.90 0.0004 ± 0.0002 4.46 ± 3.99 275.72 ± 37.03 344.99 ± 120.18 ---
Parameters T1/2 (h) Cmax (ng/mL) Vz/F (L/kg) MRT0–∞ (h) AUC0–t (ng/mL h) AUC0–∞ (ng/mL h) F (%)
T1/2, Half-life; Cmax, peak concentration; MRT, mean residence time; AUC, area under the plasma concentration–time curve; F, absolute bioavailability; Vz, apparent volume of distribution.
In this work, beagles were administered through pure TA, not policosanol. The sensitivity of GC-MS/MS method was 5 ng/mL, which is suitable for the present pharmacokinetic study of TA. The plasma concentration of TA reached its maximum point (120 ng/mL) in 1 h, after oral administration of 100 mg/kg of policosanol (Haim et al., 2009). However, peak plasma concentration was 77.69, 195.90 and 370.28 ng/mL in 2.0 h after a single oral administration of 15, 30 and 60 mg/kg, respectively, indicating that there were differences between oral administration of TA and policosanol. With the calculation of the pharmacokinetic parameters, T1/2 was 2.1 h, AUC was linearly related to the doses, and the F (%) of TA was >8.0% in beagles (Table 4). It might be speculated that TA was highly lipophilic, distributing to various tissues rapidly after administration.
Conclusion A highly sensitive, accurate and selective GC-MS/MS method was developed for the determination of TA in beagle plasma in this study. It was successfully applied to the pharmacokinetic study of TA after single oral administration and intravenous administration to beagles.
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Revised: 5 August 2014,
Accepted: 27 August 2014
Published online in Wiley Online Library: 21 October 2014
(wileyonlinelibrary.com) DOI 10.1002/bmc.3351
Trace quantification of 1-triacontanol in beagle plasma by GC-MS/MS and its application to a pharmacokinetic study Chunfeng Wanga†, Ali Fana†, Xiaojie Zhua, Yang Lub, Shuhua Denga, Wenchao Gaoa, Wei Zhanga, Qi Liua and Xijing Chena* ABSTRACT: 1-Triacontanol (TA), a member of long chain fatty alcohol, has recently been received great attention owing to its antitumor activity. In this study, an accurate, sensitive and selective gas chromatography–tandem mass spectrometry method was developed and validated for the quantification of TA in beagle plasma using 1-octacosanal as the internal standard (IS) for the first time. With temperature programming, chromatographic separation was carried out on an HP5MS column, using helium as carrier gas and argon as collision gas, both at a flow rate of 1 mL/min. TA was analyzed using positive ion electrospray ionization in multiple-reaction monitoring mode, with the precursor to product ion transitions of m/z 495.6 → 97.0 and m/z 467.5 → 97.0 for TA and the IS, respectively. The lower limit of quantitation, linearity, intra- and interday precision, accuracy, stability, extraction recovery and matrix effect of TA were within the acceptable limits. The validated method was successfully applied to a pharmacokinetic study of TA in beagles. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: 1-triacontanol; GC-MS/MS; pharmacokinetics
Introduction
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* Correspondence to: Xijing Chen, Center of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing 210009, China. Email: [email protected] †
These authors contributed equally to the present work.
a
Center of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing 210009, China
b
Division of Pharmacotherapy and Experimental Therapeutics, Eshelman School of Pharmacy, the University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA Abbreviations used: BSTFA, N,O-bis(trimethylsilyl) trifluoroacetamide; MRM, multiple reaction monitoring; TA, 1-Triacontanol.
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1-Triacontanol (TA, Fig. 1), generally existing in animal and vegetable waxes, is a component of policosanol (Más, 2000; Dullens et al., 2008). Previous studies showed that policosanol has a strong ability to inhibit cholesterol biosynthesis in rats (Noa et al., 1995; Menendez et al., 1996), rabbits (Arruzazabala et al., 1994; Pons et al., 1994), dogs (Mesa et al., 1994), monkeys (Rodríguez-Echenique et al., 1994), healthy volunteers (Hernández et al., 1992) and patients with type II hypercholesterolemia (Más et al., 1998, 2001; Castaño et al., 2000a, 2000b; Castaño et al., 2001, 2003). As an ingredient of policosanol, TA shows a significant effect in inhibiting cholesterol synthesis in cultured rat hepatoma cells (Singh et al., 2006). Recently, the antitumor effect of TA was discovered, especially in the treatment of liver cancer, colon cancer and lung cancer applications (Zhang et al., 2008). Meanwhile, adverse effects on the important immune organs of the tumor-bearing mice were not observed after administration at a dose of 150 mg/kg/day (Fan et al., 2011). Therefore, TA is a promising lead compound candidate for new drug development in cancer treatment owing to its potential antitumor activity and low toxicity. As pharmacokinetic study plays a vital role in the discovery of new drugs, it is necessary to evaluate the pharmacokinetic behavior of TA. Before embarking on the pharmacokinetic study, a reliable analytical method to quantitatively determine TA should be established. Currently, only one method has been reported for the determination of TA in rat plasma (Haim et al., 2009). Despite the fact that an assay for the determination of TA has been developed based on gas chromatography–mass spectrometry (GC-MS),
the limitations of the published literature are obvious and can be summarized as follows: (a) the sensitivity of the reported GC-MS method was 8.4 ng/mL, which is not adequate for a pharmacokinetic study in biological samples; and (b) the oral administrated drug was policosanol, not pure TA. To date, no GC-MS/ MS method has been applied for the determination TA in beagle plasma. As a promising anticancer agent, the development of a highly sensitive, precise and accurate analytical method to monitor the amount of changes in biological specimen is an urgent requirement for pharmacokinetic studies and better understanding of the mechanism of action. In the present study, a reliable gas chromatography–tandem mass spectrometry (GC-MS/MS) method with high sensitivity was established for determination of TA concentration in beagle plasma for the first time. The method is a simple and rapid assessment to support pharmacokinetic studies in beagles.
C. Wang et al. under standard temperature and humidity conditions, and were acclimatized to the housing environment for 1 week before the study. The pharmacokinetic study of TA was performed under the guideline promulgated by the Animals Experimental Ethics Committee of China Pharmaceutical University (Nanjing, China).
Figure 1. The chemical structure of 1-triacontanol (TA) (A) and IS (B).
Experimental Reagents and chemicals 1-Octacosanol (IS, purity ≥ 99%) standard substance was obtained from Sigma-Aldrich Chemical Co. (St Louis, MO, USA). The silylation reagent N,O-bis(trimethylsilyl) trifluoroacetamide (BSTFA, purity ≥ 99%) was purchased from Xiya Reagent Co. Ltd (Chengdu, Sichuan, China). Ethanol and heptane were supplied by Tedia (Fairfield, OH, USA). 1-Triacontanol (purity ≥ 96%) was supplied by Kunming Longjin Inc. (Kunming, Yunnan, China). All other chemicals were of analytical grade.
Animals Beagle dogs (10.0 ± 0.5 kg) of both genders were supplied by Shanghai SIPPR/BK Experimental Animal Co. (Shanghai, China). Animals were kept
Drug preparation methods TA weighing 0.3, 3, 6 and 12 mg was dissolved in 6 mL diethyl ether. Then 50 mg phospholipids were added into each solution and mixed well. The solutions were then evaporated to dryness in the nitrogen, and the residue was reconstituted with 2 mL of glucose. Finally, uniform milky solutions were successfully configured. The doses of TA were 1.5, 15, 30 and 60 mg/kg, respectively.
Instrument and analytical conditions GC (6890A, Agilent Technologies, USA) separation was carried out on a HP-5MS column (30 m × 250 μm, 0.25 μm film thickness, Agilent Technologies, USA) with a temperature program using helium as carrier gas at a 1 mL/min flow rate. The oven temperature gradient was as follows: the initial temperature was 100 °C, then increased to 200 °C at a rate of 40 ° C/min, and maintained for 7.5 min, followed by increasing to 300 °C within 5.0 min and maintained for 5.0 min. Argon was used as a collision
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Figure 2. Precursor/product ion pair for TA (A) and IS (B).
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Pharmacokinetics of 1-triacontanol in beagle dogs gas at a flow rate of 1 mL/min. Splitless injection was performed at 300 ° C. The total run time for analysis was 16 min. MS/MS spectrometry (Waters, Milford, USA) with an electron ionization source was operated in positive mode. Mass parameters were as follows: repeller, 10 V; extraction lens, 23 V; Focus lens1, 250 V and Focus lens 3, 27 V; trap, 200 μA; collision energy, 12 eV for TA and 10 eV for IS. The temperature of the interface was 300 °C. The ion source temperature was 250 °C, and electron energy was set at 70 eV. Multiple reaction monitoring (MRM) transitions were used for quantification of all samples by the precursor/product ion pair at m/z 495.6 → 97.0 for TA and m/z 467.5 → 97.0 for IS (Fig. 2). MassLynx V.4.1 was used to control the GCMS/MS system and acquire the data.
Preparation of stock and working solutions, calibration samples and quality controls The standard stock solutions were prepared by dissolving TA (0.1 mg/mL) and 1-octacosanol (0.1 mg/mL) in heptane. The stock solution of TA was successively diluted with heptane to obtain working solutions for calibration with concentrations of 5, 10, 20, 50, 100, 200 and 500 ng/mL. The working solutions for quality control (QC; 10, 100 and 400 ng/mL) samples were prepared in the same way. The stock solution of IS was further diluted with heptane to a final concentration of 100 ng/mL. All working solutions were stored at 4 °C.
Preparation of calibration standards and quality controls samples Calibration samples for TA were prepared in blank dog plasma at concentrations of 5, 10, 20, 50, 100, 200 and 500 ng/mL. The quality control (QC) samples were prepared at low (10 ng/mL), medium (100 ng/mL) and high (400 ng/mL) concentrations in the same way as the plasma samples for calibration, and stored at 20 °C until analysis.
Sample preparation Samples were stored at 20 °C and thawed at room temperature before analysis. Samples were vortexed adequately before pipetting. Aliquots of 50 μL plasma, 50 μL IS (100 ng/mL) and 1 mL ethanolic–NaOH solution (1 mol/L NaOH dissolved in ethanol distilled water; 80/20, v/v) were added to a 10 mL glass tube. After vortex-mixing for 30 s, samples were saponified at 80 °C for 1 h. Then 300 μL HCl (5 M) was added to acidify the solution at 70 °C for 15 min. Then the solution was extracted by adding 2 mL heptane and vortexed for 2 min. The supernatant was then transferred to a new tube and washed with 2 mL ultrapure water. The extraction and washing procedures were conducted three times for each sample. After that, all the supernatant was transferred to a new glass tube to evaporate to dryness by the centrifugal thickener (Centrivap console, Labconco Co., USA). After dryness, samples were derivatized with 300 μL BSTFA at 80 °C for 20 min. Samples were then dried again, and the residue was reconstituted in 50 μL heptane and then 2 μL solution was injected into the GC-MS/MS for analysis.
Method validation
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Application to a pharmacokinetic study in beagle dogs Sixteen healthy adult beagle dogs were fasted for 12 h before dosing with free access to water and were randomized into four groups, each group consisting of four beagle dogs. Plasma samples were collected at 0.5, 0.75, 1, 1.5, 2, 3, 4, 6 and 8 h after a single intragastric administration of TA at a dose of 15, 30 and 60 mg/kg for oral administration to each group. Plasma samples were collected at 2, 5, 10, 15, 30, 60, 120, 240, 360 and 480 min after a single intravenous administration of TA at a dose of 1.5 mg/kg to the last four beagles. Plasma was centrifuged at 8000 rpm for 5 min after harvesting and the supernatant was stored at 20 °C until analysis. All the pharmacokinetic parameters were calculated by noncompartmental analysis using Drug and Statistics software (DAS 2.1.1 version, Mathematical Pharmacology Professional Committee of China), including area under the plasma concentration–time curve (AUC), the mean residence time (MRT), half-life (T1/2), oral clearance (CL/F) and clearance (CL). Based on calculated AUC values, absolute bioavailability (F) was calculated as: F ð%Þ ¼ AUC ig =AUC iv doseiv =doseig 100
Results Assay validation Specificity. Under the described conditions, no endogenous interferences were observed at the retention times of TA
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The method was fully validated for selectivity, matrix effect, linearity, lower limit of quantitation (LLOQ), accuracy and precision, recovery and stability in accordance with the guidelines of the US Food and Drug Administration (2001). The selectivity was evaluated by analyzing blank plasma samples collected from six different sources with plasma samples spiked with TA and IS to investigate the potential interferences at the peak region. A best-fit calibration curve was constructed by plotting the area ratios of the analyte/internal standard (y) vs the concentrations of the analyte (x) in 2 the form of y = a + bx, using weighted (1/x ) least squares linear regression.
The LLOQ was determined as the lowest concentration of the standard. The limit of quantification were determined in quintuplicate by measuring the signal-to-noise (S/N) ratio (10:1) for the analyte at LLOQ. The acceptance criterion of analyte at LLOQ required five replicates with a precision of 20% and an accuracy of 80–120%. The matrix effect was defined as the ion suppression/enhancement on the ionization of analytes, which was evaluated by comparing the peak areas of the analytes in postextracted blank plasma samples spiked with TA at three QC levels with the pure standard solutions with same concentration dried directly and reconstituted with the heptane. The same procedure was performed for the IS. The precision and accuracy of the method were evaluated by analyzing six replicates of spiked beagle dog plasma with known concentrations of TA with QC samples at low (10 ng/mL), medium (100 ng/mL) and high (400 ng/mL) concentration levels. To determine intraday precision and accuracy, the assays were determined on the same day by analyzing five replicates of each concentration level. To determine interday precision and accuracy, the assays were determined on three consecutive days by analyzing three replicates of each concentration level. The precision was expressed as relative standard deviation (RSD) and the accuracy as the relative error (RE).The relative error (RE) and RSD were all within ±15%. Recovery was determined by comparing the peak areas of processed QC samples with those of the pure standards without extraction (n = 5, for each concentration). The stability of analyte in dog plasma was assessed by analyzing replicate (n = 3) QC samples at three concentrations for three freeze–thaw cycles, short-term temperature, postpreparative and long-term temperature stabilities. The stability of TA in plasma was evaluated under various conditions using three levels of QC samples after short-term (pre-processed samples stored at room temperature for 4 h) and long-term storage conditions (stored at 20 ° C for 1 month). The stability of QC samples after three freeze (20 °C) and thaw (room temperature) cycles and postpreparative stability (postprocessed samples stored at 4 °C for 24 h) were also analyzed. Thereafter, samples were analyzed and the resulting values for these samples were the analyte peak area of the postextracted samples vs the analyte peak area of pure standard nominal concentration.
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Figure 3. Representative multiple reaction monitoring chromatograms of TA and IS in beagle plasma: (A) blank plasma sample; (B) beagle plasma spiked with TA at 5 ng/mL and IS; and (C) plasma sample collected 1 h after oral administration of 15 mg/kg TA in beagles.
(14.25 min) and IS (12.13 min). Representative chromatograms of blank beagle plasma, beagle plasma spiked with TA at the 5 ng/mL and test plasma sample obtained at 1 h after the single dose administration of TA (15 mg/kg) are shown in Fig. 3. Linearity and LLOQ. The linearity was evaluated by the calibration curves determined on five separate days. The calibration curve was obtained by plotting the peak-area ratio of TA to the IS vs the TA concentration, showing a good linearity over the concentration range of 5–500 ng/mL. The LLOQ was 5 ng/mL, at which the RSD was 11.0% and the RE was 8.0%. This method was sensitive enough to investigate the pharmacokinetic study of TA with a good S/N (>20) at LLOQ.
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Precision and accuracy. As shown in Table 1, the RSD values of intraday and interday precision were all 85.0% at three QC levels for TA. The mean matrix effects and recovery for IS were 99.0 and 87.5%, respectively. The results suggest that the processing procedure could provide a high extraction efficiency. No ion suppression or enhancement was detected under the current conditions. Stability. Table 3 summarizes the results of short-term, longterm and freeze–thaw stabilities of TA in plasma and postpreparation stability. The high stability property of TA in dog plasma showed that the samples were stable in the preparation and analytical processes. Pharmacokinetic study. The method was successfully applied to the pharmacokinetic study after a single dose of 15, 30 and 60 mg/kg for oral administration and at 1.5 mg/kg for intravenous injection to beagles. The dog plasma concentration–time profiles are shown in Fig. 4 and the corresponding pharmacokinetic
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Pharmacokinetics of 1-triacontanol in beagle dogs Table 1. Intra- and interday precision and accuracy for 1-triacontanol (TA) in beagle plasma Intraday (n = 5) Nominal concentration (ng/mL) 10 100 400
Interday (n = 15)
Measured
Accuracy,
RSD
Measured
Accuracy,
RSD
concentration
RE (%)
(%)
concentration
RE (%)
(%)
1.00 2.40 3.18
9.60 8.66 6.99
(ng/mL)
(ng/mL)
9.80 ± 0.69 96.0 ± 7.78 423.1 ± 10.70
2.00 4.00 5.75
7.00 8.10 2.53
10.1 ± 0.97 97.6 ± 8.45 412.7 ± 28.86
Table 2. Matrix effect of TA and IS in beagle plasma Compound
TA (n = 6)
IS (n = 18)
Nominal concentration
Matrix effect (%)
Recovery (%)
(ng/mL)
Mean ± SD
RSD
Mean ± SD
RSD
10.0 100.0 400.0 100.0
92.6 ± 14.8 105.6 ± 12.5 106.9 ± 10.1 99.0 ± 12.4
16.0 11.8 9.6 12.5
83.0 ± 7.7 99.2 ± 12.0 82.4 ± 4.4 87.5 ± 0.1
9.3 11.0 5.3 11.2
Table 3. Stability (%) of TA in beagle plasma (n = 5) Storage condition
Room temperature Post-preparative Freeze–thawing Long-term
10 ng/mL
100 ng/mL
400 ng/mL
Mean ± SD
RSD (%)
Mean ± SD
RSD (%)
Mean ± SD
RSD (%)
10.2 ± 1.5 10.5 ± 0.5 9.6 ± 1.1 10.5 ± 1.1
14.4 4.3 11.3 10.8
106.1 ± 8.4 90.3 ± 4.4 90.4 ± 3.9 101.0 ± 3.7
8.0 4.9 4.3 3.6
428.0 ± 16.3 378.6 ± 13.6 398.0 ± 13.9 409.3 ± 7.2
3.8 3.6 3.5 1.8
parameters are listed in Table 4. The values of T1/2 were 1.77 ± 0.05, 2.34 ± 1.14 and 2.12 ± 0.42 h after single oral administration of 15, 30 and 60 mg/kg of TA, and 4.10 ± 3.51 h after intravenous administration of 1.5 mg/kg of TA. After oral administration of TA to beagles, the plasma drug concentration reached the maximum point at about 1.5 h. Then the plasma concentration decreased gradually with T1/2 between 2 and 3 h. The mean T1/2 was 2.1 h, showing that TA was eliminated rapidly in beagles. Peak plasma concentration and AUC were linearly related to the doses, indicating that the pharmacokinetic process of TA was consistent with dose-proportional pharmacokinetics. Based on the pharmacokinetic parameters, the absolute bioavailability (F, %) of TA was calculated to be >8% in beagle dogs.
Discussion
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In the present study, an accurate and highly sensitive GC-MS/MS method was established and fully validated for the determination of TA in beagle plasma. Both TA and IS are compounds with high boiling point and polar hydroxyl groups as well. Therefore, it is difficult for GC to analyze them directly. Thus, we need to carry out structural transformation, such as derivatives, to
analyze them by GC. In this study, we used BSTFA as derivatization reagent. With the addition of one dimethylsilyl group, the molecular weight of TA was increased from 438.8 to 495.6, adding about 57 after the silylation. The initial temperature was 200 °C in the reported GC-MS method (Haim et al., 2009), which was too high for application for high throughput pharmacokinetic studies. Hence, a two-phase temperature gradient program was adopted to protect the column, improve the separation and increase the sensitivity. 1-Octacosanal was chosen as the IS because it is a homologous analog to TA with similar chromatographic behavior and response. In addition, its recovery was satisfactory and it also remains stable during the whole analysis. Based on the full scan of the mass spectrum of TA and IS and their product ion fragments under daughter ion scanning mode, the daughter ions of TA after derivatization were +71, 83, 97, 111, 125 and 137 and those of 1-octacosanal were +75, 83, 97, 111, 125 and 153. By observing the maximum response of the product ions in MRM model, we chose 97 as the qualitative daughter ion. Finally we selected MRM of precursor–product ion transitions with m/z 495.6 → 97.0 for TA and m/z 467.5 → 97.0 for IS. Product ion spectra of TA and IS are shown in Fig. 2.
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Figure 4. The mean plasma concentration–time profile of TA: (A) oral administration of 15, 30 and 60 mg/kg TA in beagles (n = 4). (B) Intravenous administration of 1.5 mg/kg TA in beagles (n = 4).
Table 4. Main pharmacokinetic parameters after oral administration and intravenous injection of TA to beagles (n = 6) i.g.
i.g.
i.g.
i.v.
15 mg/kg
30 mg/kg
60 mg/kg
1.5 mg/kg
1.77 ± 0.05 96.19 ± 9.61 0.013 ± 0.015 3.28 ± 0.13 285.55 ± 32.65 304.28 ± 33.25 8.83 ± 0.96
2.34 ± 1.14 247.16 ± 49.80 0.12 ± 0.038 3.91 ± 0.82 774.24 ± 139.38 866.44 ± 193.34 12.57 ± 2.80
2.12 ± 0.42 460.97 ± 19.41 0.13 ± 0.036 3.74 ± 0.21 1388.57 ± 167.94 1525.78 ± 149.29 11.07 ± 1.08
4.10 ± 3.51 307.38 ± 12.90 0.0004 ± 0.0002 4.46 ± 3.99 275.72 ± 37.03 344.99 ± 120.18 ---
Parameters T1/2 (h) Cmax (ng/mL) Vz/F (L/kg) MRT0–∞ (h) AUC0–t (ng/mL h) AUC0–∞ (ng/mL h) F (%)
T1/2, Half-life; Cmax, peak concentration; MRT, mean residence time; AUC, area under the plasma concentration–time curve; F, absolute bioavailability; Vz, apparent volume of distribution.
In this work, beagles were administered through pure TA, not policosanol. The sensitivity of GC-MS/MS method was 5 ng/mL, which is suitable for the present pharmacokinetic study of TA. The plasma concentration of TA reached its maximum point (120 ng/mL) in 1 h, after oral administration of 100 mg/kg of policosanol (Haim et al., 2009). However, peak plasma concentration was 77.69, 195.90 and 370.28 ng/mL in 2.0 h after a single oral administration of 15, 30 and 60 mg/kg, respectively, indicating that there were differences between oral administration of TA and policosanol. With the calculation of the pharmacokinetic parameters, T1/2 was 2.1 h, AUC was linearly related to the doses, and the F (%) of TA was >8.0% in beagles (Table 4). It might be speculated that TA was highly lipophilic, distributing to various tissues rapidly after administration.
Conclusion A highly sensitive, accurate and selective GC-MS/MS method was developed for the determination of TA in beagle plasma in this study. It was successfully applied to the pharmacokinetic study of TA after single oral administration and intravenous administration to beagles.
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