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Yun Li Longshan Zhao Xiaoting Li Bei Guo Juanhang Zhao Xianglin Wang Tianhong Zhang Department of Pharmaceutical Analysis, School of Pharmacy, Shenyang Pharmaceutical University, Shenyang, PR China Received October 6, 2014 Revised November 19, 2014 Accepted November 19, 2014
Short Communication
Quantification of 3-n-butylphthalide in beagle plasma samples by supercritical fluid chromatography with triple quadruple mass spectrometry and its application to an oral bioavailability study A high-throughput, rapid, sensitive, environmentally friendly, and economical supercritical fluid chromatography with triple quadruple mass spectrometry method was established and validated for the first time to determine a cerebral stroke treatment drug named 3-nbutylphthalide in dog plasma. Plasma samples were prepared by protein precipitation with methanol and the analytes were eluted on an ACQUITY UPC2TM HSS-C18 SB column (3 × 100 mm, 1.8 m) maintained at 50⬚C. The mobile phase comprised supercritical carbon dioxide/methanol (90:10, v/v) at a flow rate of 1.5 mL/min, the compensation solvent was methanol at a flow rate of 0.2 mL/min and the total run time was 1.5 min per sample. The detection was carried out on a tandem mass spectrometer with an electrospray ionization source. Calibration curves were linear over the concentration range of 1.02–1021.00 ng/mL (r2 0.993) with the lower limit of quantification of 1.02 ng/mL. The intra- and inter-day precision values were below 15% and the accuracy was from 97.90 to 103.70% at all quality control levels. The method was suitable for a pharmacokinetic study of 3-n-butylphthalide in beagle dogs. Keywords: Beagle plasma/3-n-Butylphthalide / Oral bioavailability / Pharmacokinetic studies / Supercritical fluid chromatography DOI 10.1002/jssc.201401073
1 Introduction 3-n-Butylphthalide (NBP) [(±)-3-butyl-1(3H)-isobenzofuranone] was firstly isolated from Chinese herbs, including Apium graveolens, Ligusticum sinensis, and Liqusticum Chuanxiong Hort [1–3], and has also been obtained by chemical synthesis [4, 5]. NBP is currently used clinically as a preferred drug for the treatment of ischemic stroke by increasing regional cerebral blood flow and inhibiting the release of glutamate and 5-hydroxytryptamine [6, 7]. In addition, recent research has demonstrated that NBP displays beneficial effects by attenuating amyloid-induced cell death in neuronal cultures [8], improving cognitive deficits and preventing neuronal cell death after focal cerebral ischemia in mice [9, 10], Correspondence: Dr. Tianhong Zhang, Department of Pharmaceutical Analysis, School of Pharmacy, Shenyang Pharmaceutical University, No. 103, Wenhua Road, Shenyang 110016, PR China E-mail: [email protected] Fax: +86 2423986321
Abbreviations: Cmax , peak serum concentration; LLOQ, lower limit of quantification; ME, matrix effect; MRM, multiple reaction monitoring; NBP, 3-n-butylphthalide; QC, quality control; sCO2 , supercritical carbon dioxide; SFC, supercritical fluid chromatography; Tmax , the time to reach the peak concentration C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
and protecting cardiomyocytes from ischemia/reperfusioninduced apoptosis [11]. NBP is a highly volatile, oily, and water-insoluble drug which undergoes extensive liver firstpass effect with a low oral bioavailability. To improve the dissolution and oral bioavailability, a new formulation of NBP solid dispersible tablet was prepared in our study. Hereby, a rapid and sensitive analysis method was necessarily developed to investigate the oral bioavailability of the self-prepared NBP solid dispersible tablets and commercial NBP soft capsules. To date, several bioanalytical methods have been developed to determine the NBP concentrations in biological matrices, including HPLC coupled with fluorescence or UV detection [12, 13] and UHPLC–MS/MS [14–16]. However, most of these techniques have various limitations involving low sensitivity, time-consumption (6 min), complex mobile phase composition, laborious sample pretreatment, or clean-up procedures, etc, rendering them unsuitable for highthroughput analysis of NBP concentrations in biological samples. To overcome these drawbacks, we have developed a new analytical method. SFC is a separation technique widely used in food and pharmaceutical analysis [17–19], which is similar to GC and LC but uses a supercritical fluid as the mobile phase [20]. In this study, we used supercritical carbon dioxide (sCO2 ) as the mobile phase, which is non-explosive, non-toxic, and www.jss-journal.com
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non-aggressive chemical nature in regards to that of most organic solvents and has a conveniently achieved supercritical state [19]. On the other hand, the low viscosity, high molecular diffusiveness, and non-polar character of sCO2 favors SFC as a fast and useful chromatographic technique for the analysis of hydrophobic compounds [21] (such as NBP), which leads to greater efficiency in a short analysis time (only 1.5 min for NBP) and shorter time for column equilibration in contrast to UHPLC [20]. These characteristics make SFC a friendly and powerful tool for profiling NBP in plasma. The validated method was successfully applied to a bioavailability study of NBP in beagle dogs for the first time.
2.3 Preparation of standards and QC samples Stock standard solutions of NBP (102.10 g/mL) and IS (101.00 g/mL) were prepared by dissolving each in methanol. Working standard and internal standard solutions (252.50 ng/mL) were prepared by dilution of the stock standards in methanol. Calibration standards were prepared by spiking 200 L blank beagle plasma with 20 L of the appropriate standard solutions. The effective concentrations in plasma samples were 1.02, 2.55, 5.10, 25.53, 51.05, 255.30, 510.50, and 1021.00 ng/mL. The QC samples were prepared at three concentration levels of 2.50, 50.00, and 800.00 ng/mL in a similar way to the calibration standards. All the solutions were stored at −20⬚C until analysis.
2 Materials and methods 2.4 Sample preparation 2.1 Reagents NBP (99.6% of purity) and diazepam (99.9% of purity, IS) were purchased from the National Institutes for Food and R Butylphthalide Soft Drug Control (Beijing, PR China). NBP Capsules were provided by CSPC NBP Pharmaceutical (Shijiazhuang, Hebei, China). NBP solid dispersion tablets were self-prepared preparations. Methanol of HPLC grade was supplied by Fisher Scientific (Pittsburgh, PA, USA). CO2 ( 99.99% of purity) was offered by Shenyang Qianzhen Chemical Gas (Shenyang, Liaoning, China) and high-purity nitrogen (99.999%) was used.
A 20 L aliquot of the IS solution (diazepam, 252.50 ng/mL) was added to 200 L plasma sample, followed by 20 L methanol. The sample was vortexed for 1 min and deproteinized with 300 L methanol, the precipitate was removed by centrifugation at 13 000 rpm for 10 min. Then, the supernatant layer was transferred to an Eppendorf micro tube, and an aliquot of 5 L was injected into the SFC–MS/MS system for analysis after another 10 min centrifugation at 13 000 rpm.
2.5 Method validation
2.2 Equipment and conditions Studies were performed on an ACQUITY UPC2TM system (Waters, Milford, MA, USA). For the chromatographic separations, an ACQUITY UPC2TM HSS C18 SB column (100 × 3 mm, 1.8 m; Waters, Milford, MA, USA) was used and the flow rate was set at 1.5 mL/min, and the backpressure of system was conditioned at 2000 psi while the column temperature was maintained at 50⬚C. The mobile phase comprised carbon dioxide and methanol (modifier) (90:10, v/v) while pure methanol was used as the compensation solvent at a flow rate of 0.2 mL/min. An ACQUITY UPC2TM system was coupled to a Waters Triple Quadrupole mass spectrometer equipped with an ESI source interface (Waters). The MS parameters were as follows: capillary voltage of 3.0 kV, cone voltage of 30 V, the optimized collision energy was 15 V and 28 V for NBP and IS, respectively, source temperature 150⬚C and desolvation temperature 200⬚C. The cone and desolvation gas flow rates were 50 and 600 L/h, respectively. The transitions of NBP and IS were m/z 191.1→145.1 and 285.1→193.0, respectively. All data were acquired and processed using MassLynxTM NT 4.1 software with QuanLynxTM program (Waters, Milford, MA, USA). Under these SFC–MS/MS conditions, the retention time was 0.53 min for NBP and 1.13 min for IS, respectively. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
The validation parameters were selectivity, linearity, precision and accuracy, recovery, matrix effect (ME), and stability. To verify the selectivity of the method, the chromatograms of six different batches of blank plasma with simulated plasma samples (those of corresponding standard plasma samples spiked with NBP and IS) and plasma samples after oral administration of NBP should be compared. Calibration curves were constructed over the range of 1.02– 1021.00 ng/mL and the six different batches of lower limit of quantification (LLOQ) samples were validated with relative error (RE) within ±20% and RSD lower than 20% per sample. The intra-day and the inter-day precision and accuracy were determined by analyzing three consecutive validation runs of the QC samples, and the RSD was used to express the precision and the accuracy as the RE. To achieve the extraction recovery and the ME of NBP, the following formulas (A/B×100)% and (B/C×100)% were used respectively. A, B, C refer to the peak area of blank plasma samples spiked with NBP and IS before extraction, blank plasma samples were extracted before adding analytes and the in vitro samples at corresponding concentrations, respectively. The same method was used for the evaluation of extraction recovery and ME for IS. Stability tests were conducted to evaluate the stability of the analyte in plasma samples under different conditions including stored at ambient temperature for 8 h (room temperature stability), repeated freeze–thaw cycles www.jss-journal.com
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three times (freeze–thaw stability), after pre-treatment sample chamber placed 12 h at 4⬚C (post-preparation stability) and –20⬚C frozen storage for 30 days (long-term stability).
2.6 Pharmacokinetic study The developed method was used to determine the plasma concentrations of NBP in six beagle dogs (Laboratory Animal Center of Shenyang Pharmaceutical University, Shenyang, Liaoning, China). After 24 h fasting (without water deprivation), 5 mL blood samples were collected at preadministration, and at 5, 20, 40, 60, 90, 120, 150, 180, 240, 360, 480, 720 min (12 h) after administration of 200 mg NBP, respectively. The blood samples were centrifuged immediately at 13 000 rpm for 10 min and the plasma samples were labeled and stored at –20⬚C until analysis.
3 Results and discussion 3.1 Optimization of MS and chromatographic conditions To achieve optimal MS/MS parameters for analysis of NBP, ESI source coupled with positive ion mode and negative ion mode made an on-the-spot investigation, respectively. ESI source with positive ion mode was selected to get a higher response, which was consistent with the previous studies [15]. For NBP, two major fragment ions were obtained: m/z 191.1→145.1 and 191.1→173.1, while the former gave a stronger and more stable response and the former was chosen for use in MRM. In this study, to gain a faster and useful chromatographic separation, ACQUITY UPC2TM HSS C18 SB column (100 × 3 mm, 1.8 m) and ACQUITY UPC2TM BEH column (100 × 3 mm, 1.7 m) were firstly investigated. The ACQUITY UPC2TM HSS C18 SB column provided a better chromatographic separation than the latter, which could provide sharper peaks and excellent stability while maintaining satisfactory chromatographic resolution with no influence by MEs. In SFC, three important degrees of freedom can change the elution behavior of the analytes, i.e. temperature, modifier, and system pressure. So, different column temperatures (35, 40, 45, 50, and 55⬚C) were compared to achieve a narrow peak, less analysis time and lower column pressure, and 50⬚C was set as the most suitable temperature with a lower column pressure with shaper peaks (Fig. 1A). As to the composition of mobile phase, two kinds of modifier (methanol and acetonitrile) were compared firstly, it showed a shaper and more symmetric peak with a higher response when methanol was used (Fig. 1B). Then different ratios (95:5, 90:10, and 85:5, v/v) of CO2 /methanol were investigated, a narrow peak and relatively reasonable analysis time were obtained compared with the others when the ratio of CO2 /methanol (90:10, v/v) was used. A series of backpressures (1800, 1900, and 2000 psi) was also investigated but no obvious effect was C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 1. The chromatographic separation of NBP: (a) under different column temperature: 35 (A), 40 (B), 45 (C), 50 (D), and 55⬚C (E); (b) by using different modifier: acetonitrile (A) and methanol (B) at the flow rate of 1.8 mL/min (Rt and CP refer to retention time and column pressure, respectively).
observed on the chromatographic behavior, so the system pressure was set at 2000 psi to maintain the best performance of instrument, which was reviewed by the engineers in Waters. According to our previous results, the mass response of the analytes will be improved when compensation solvent was used [22], therefore different flow rate (0.45, 0.2, 0.1, and 0.05 mL/min) of the compensation solution (methanol) was optimized and finally 0.2 mL/min was chosen with the highest and relatively stable mass response.
3.2 Selection of internal standard In this assay, glipizide, spirolactone, and diazepam were evaluated to achieve a ready available internal standard. The results indicated that the retention time of glipizide was far longer than NBP as it belongs to a different chemical class and has a significantly different chromatographic behavior. Both of spirolactone and diazepam are lactone compounds, so they had a similar retention behavior, but the later had a higher mass response in ESI (+) mode. So, internal standard of diazepam was adopted.
3.3 Sample preparation Since NBP is a highly volatile drug, rapid and simple preparation of the biological samples is of great necessary to gain a high extraction recovery. In our study, a one-step protein www.jss-journal.com
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Figure 2. MRM chromatograms for NBP and IS from (A) a blank plasma sample; (B) a plasma sample with added NBP and IS at LLOQ level; and (C) a plasma sample from a dog at 1.5 h after oral administration of NBP (200 mg).
precipitation procedure was selected for the sample extraction due to its excellent recovery rates, rapid as well as simple and convenient. Furthermore, methanol was used as the more efficient precipitant with superb extraction recovery compared to acetonitrile and acetone.
Table 1. Intra-day and inter-day precision and accuracy for assay of NBP in dog plasma (intra-day: n = 6; inter-day: n = six series per day over three days)
Added (ng/mL)
Found (ng/mL)
Intra-day RSD (%)
Inter-day RSD (%)
Accuracy RE (%)
3.4 Method validation
2.50 50.00 800.00
2.74 ± 0.16 54.17 ± 2.13 819.85 ± 40.78
4.82 4.93 4.04
14.78 8.05 7.70
97.90 102.40 103.70
Selectivity was evaluated by comparing the chromatograms of six different lots of blank beagle plasma with the corresponding spiked plasma at LLOQ level and plasma samples after oral administration. Figure 2 shows that no interference from endogenous substances was observed at the retention time of NBP and IS. The linear regression was fitted over the concentration range of 1.02–1021.00 ng/mL (r2 0.993) in beagle plasma. A typical equation for the calibration curve was as follows: y = 0.0145x + 0.2912, r2 = 0.9973. Good linearity was seen over this concentration range in all analytical runs with the
LLOQ of 1.02 ng/mL, which is sensitive enough to investigate the pharmacokinetic behavior of the drug. The values for the intra- and inter-day precision and accuracy are summarized in Table 1. The results show that the present method was accurate and reproducible to determine the plasma concentrations of NBP in beagle. Mean extraction recoveries of NBP at three concentrations levels were 84.2 ± 4.1, 89.5 ± 11.4, and 91.9 ± 5.1%, respectively (n = 6). The mean recovery of the IS was
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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J. Sep. Sci. 2015, 38, 697–702 Table 2. Stability of NBP in dog plasma under indicated conditions (mean ± SD, n = 3)
Conditions
Added (ng/mL)
Found (ng/mL)
Pretreatment for 12 h
2.50 50.00 800.00 2.50 50.00 800.00 2.50 50.00 800.00 2.50 50.00 800.00
2.30 47.30 859.71 2.51 51.97 874.33 2.50 47.63 836.41 2.47 47.51 860.09
Room temperature for 8h Freeze–thaw stability
−20⬚C Frozen storage for 30 days
± ± ± ± ± ± ± ± ± ± ± ±
0.14 2.54 6.13 0.09 1.32 20.13 0.06 0.56 22.04 0.01 0.90 15.48
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Table 3. Pharmacokinetic parameters of NBP solid dispersion tablets and commercial NBP soft capsules (n = 6, mean ± SD)
RSD (%) 5.99 5.37 0.17 3.74 2.54 2.30 2.38 1.18 2.64 0.35 1.88 1.80
Pharmacokinetic parameters
NBP solid dispersion tablets
Cmax (ng/mL) AUC0–12 (ng·h/mL) AUC0- (ng·h/mL) Tmax (h) T1/2 (h)
814.92 2196.03 2237.36 1.72 2.94
± ± ± ± ±
Commercial soft capsules 83.96 378.51 392.14 0.33 0.68
335.40 658.36 789.24 3.25 3.63
± ± ± ± ±
57.76 237.79 236.99 1.83 0.47
4 Concluding remarks An SFC–MS/MS method for the determination of NBP in beagle plasma and its validation was described for the first time in this paper. Compared to the traditional UHPLC– MS/MS method, it has a short single run time of 1.5 min per sample, which makes it an attractive procedure in the highthroughput analysis of NBP in beagle plasma. The method was highly efficient, had satisfactory sensitivity with the LLOQ of 1.02 ng/mL, superior selectivity and was successfully applied to a preclinical pharmacokinetic study of NBP in beagle dogs. The present work would provide helpful information for further studies.
Figure 3. Mean concentration–time profiles of NBP in beagle plasma after a single oral dose of 200 mg NBP solid dispersion tablets and commercial soft capsules (n = 6).
The authors have declared no conflict of interest.
5 References 92.0 ± 5.1% (n = 6). The relative ME result was RSD 0.05), which indicated that the oral bioavailability of the NBP solid dispersions was increased significantly compared with commercial soft capsules. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
[7] Xu, H. L., Feng, Y. P., Acta Pharmacol. Sin. 1999, 20, 929– 933. [8] Peng, Y., Xing, C., Lemere, C. A., Chen, G., Wang, L., Feng, Y., Wang, X., Neurosci. Lett. 2008, 434, 224–229. [9] Peng, Y., Sun, J., Hon, S., Nylander, A. N., Xia, W., Feng, Y., Wang, X., Lemere, C.A., J. Neurosci. 2010, 30, 8180– 8189. [10] Li, J., Li, Y., Ogle, M., Zhou, X., Song, M., Yu, S. P., Wei, L., Brain Res. 2010, 1359, 216–226. [11] Wang, Y. G., Li, Y., Wang, C. Y., Ai, J. W., Dong, X. Y., Huang, H. Y., Feng, Z. Y., Pan, Y. M., Lin, Y., Wang, B. X., Yao, L. L., Acta Physiol. 2014, 210, 524–533.
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[12] Zhao, C., He, Z., Cui, S., Zhang, R., Biomed. Chromatogr. 2003, 17, 391–395.
[17] Bernal, J. L., Mart´ın, M. T., Toribio, L., J. Chromatogr. A 2013, 1313, 24–36.
[13] Guo, M., Su, X., Kong, L., Li, X., Zou, H., Anal. Chim. Acta 2006, 556, 183–188.
[18] Li, F., Hsieh, Y., J. Sep. Sci. 2008, 31, 1231–1237.
[14] Diao, X., Ma, Z., Lei, P., Zhong, D., Zhang, Y., Chen, X., J. Chromatogr. B 2013, 939, 67–72.
[19] Horia, K., Matsubarab, A., Uchikatab, T., Tsumura, K., Fukusaki, E., Bamba, T., J. Chromatogr. A 2012, 1250, 99–104.
[15] Niu, Z., Chen, F., Sun, J., Liu, X., Wang, Y., Chen, D., He, Z., J. Chromatogr. B 2008, 870, 135–139.
[20] David, F., Medvedovici, A., Sandra, P., Oils, Fats and Waxes: Supercritical Fluid Chromatography. Academic Press, Kortrijk 2000, p. 3567.
[16] Diao, X., Ma, Z., Wang, H., Zhong, D., Zhang, Y., Jin, J., Fan, Y., Chen, X., J. Pharm. Biomed. Anal. 2013, 78–79, 19–26.
[21] Bamba, T., J. Sep. Sci. 2008, 31, 1274–1278.
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[22] Geng, Y., Zhao, L., Zhao, J., Guo, B., Ma, P., Li, Y., Zhang, T., J. Chromatogr. B 2014, 945–946, 121–126.
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J. Sep. Sci. 2015, 38, 697–702
Yun Li Longshan Zhao Xiaoting Li Bei Guo Juanhang Zhao Xianglin Wang Tianhong Zhang Department of Pharmaceutical Analysis, School of Pharmacy, Shenyang Pharmaceutical University, Shenyang, PR China Received October 6, 2014 Revised November 19, 2014 Accepted November 19, 2014
Short Communication
Quantification of 3-n-butylphthalide in beagle plasma samples by supercritical fluid chromatography with triple quadruple mass spectrometry and its application to an oral bioavailability study A high-throughput, rapid, sensitive, environmentally friendly, and economical supercritical fluid chromatography with triple quadruple mass spectrometry method was established and validated for the first time to determine a cerebral stroke treatment drug named 3-nbutylphthalide in dog plasma. Plasma samples were prepared by protein precipitation with methanol and the analytes were eluted on an ACQUITY UPC2TM HSS-C18 SB column (3 × 100 mm, 1.8 m) maintained at 50⬚C. The mobile phase comprised supercritical carbon dioxide/methanol (90:10, v/v) at a flow rate of 1.5 mL/min, the compensation solvent was methanol at a flow rate of 0.2 mL/min and the total run time was 1.5 min per sample. The detection was carried out on a tandem mass spectrometer with an electrospray ionization source. Calibration curves were linear over the concentration range of 1.02–1021.00 ng/mL (r2 0.993) with the lower limit of quantification of 1.02 ng/mL. The intra- and inter-day precision values were below 15% and the accuracy was from 97.90 to 103.70% at all quality control levels. The method was suitable for a pharmacokinetic study of 3-n-butylphthalide in beagle dogs. Keywords: Beagle plasma/3-n-Butylphthalide / Oral bioavailability / Pharmacokinetic studies / Supercritical fluid chromatography DOI 10.1002/jssc.201401073
1 Introduction 3-n-Butylphthalide (NBP) [(±)-3-butyl-1(3H)-isobenzofuranone] was firstly isolated from Chinese herbs, including Apium graveolens, Ligusticum sinensis, and Liqusticum Chuanxiong Hort [1–3], and has also been obtained by chemical synthesis [4, 5]. NBP is currently used clinically as a preferred drug for the treatment of ischemic stroke by increasing regional cerebral blood flow and inhibiting the release of glutamate and 5-hydroxytryptamine [6, 7]. In addition, recent research has demonstrated that NBP displays beneficial effects by attenuating amyloid-induced cell death in neuronal cultures [8], improving cognitive deficits and preventing neuronal cell death after focal cerebral ischemia in mice [9, 10], Correspondence: Dr. Tianhong Zhang, Department of Pharmaceutical Analysis, School of Pharmacy, Shenyang Pharmaceutical University, No. 103, Wenhua Road, Shenyang 110016, PR China E-mail: [email protected] Fax: +86 2423986321
Abbreviations: Cmax , peak serum concentration; LLOQ, lower limit of quantification; ME, matrix effect; MRM, multiple reaction monitoring; NBP, 3-n-butylphthalide; QC, quality control; sCO2 , supercritical carbon dioxide; SFC, supercritical fluid chromatography; Tmax , the time to reach the peak concentration C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
and protecting cardiomyocytes from ischemia/reperfusioninduced apoptosis [11]. NBP is a highly volatile, oily, and water-insoluble drug which undergoes extensive liver firstpass effect with a low oral bioavailability. To improve the dissolution and oral bioavailability, a new formulation of NBP solid dispersible tablet was prepared in our study. Hereby, a rapid and sensitive analysis method was necessarily developed to investigate the oral bioavailability of the self-prepared NBP solid dispersible tablets and commercial NBP soft capsules. To date, several bioanalytical methods have been developed to determine the NBP concentrations in biological matrices, including HPLC coupled with fluorescence or UV detection [12, 13] and UHPLC–MS/MS [14–16]. However, most of these techniques have various limitations involving low sensitivity, time-consumption (6 min), complex mobile phase composition, laborious sample pretreatment, or clean-up procedures, etc, rendering them unsuitable for highthroughput analysis of NBP concentrations in biological samples. To overcome these drawbacks, we have developed a new analytical method. SFC is a separation technique widely used in food and pharmaceutical analysis [17–19], which is similar to GC and LC but uses a supercritical fluid as the mobile phase [20]. In this study, we used supercritical carbon dioxide (sCO2 ) as the mobile phase, which is non-explosive, non-toxic, and www.jss-journal.com
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non-aggressive chemical nature in regards to that of most organic solvents and has a conveniently achieved supercritical state [19]. On the other hand, the low viscosity, high molecular diffusiveness, and non-polar character of sCO2 favors SFC as a fast and useful chromatographic technique for the analysis of hydrophobic compounds [21] (such as NBP), which leads to greater efficiency in a short analysis time (only 1.5 min for NBP) and shorter time for column equilibration in contrast to UHPLC [20]. These characteristics make SFC a friendly and powerful tool for profiling NBP in plasma. The validated method was successfully applied to a bioavailability study of NBP in beagle dogs for the first time.
2.3 Preparation of standards and QC samples Stock standard solutions of NBP (102.10 g/mL) and IS (101.00 g/mL) were prepared by dissolving each in methanol. Working standard and internal standard solutions (252.50 ng/mL) were prepared by dilution of the stock standards in methanol. Calibration standards were prepared by spiking 200 L blank beagle plasma with 20 L of the appropriate standard solutions. The effective concentrations in plasma samples were 1.02, 2.55, 5.10, 25.53, 51.05, 255.30, 510.50, and 1021.00 ng/mL. The QC samples were prepared at three concentration levels of 2.50, 50.00, and 800.00 ng/mL in a similar way to the calibration standards. All the solutions were stored at −20⬚C until analysis.
2 Materials and methods 2.4 Sample preparation 2.1 Reagents NBP (99.6% of purity) and diazepam (99.9% of purity, IS) were purchased from the National Institutes for Food and R Butylphthalide Soft Drug Control (Beijing, PR China). NBP Capsules were provided by CSPC NBP Pharmaceutical (Shijiazhuang, Hebei, China). NBP solid dispersion tablets were self-prepared preparations. Methanol of HPLC grade was supplied by Fisher Scientific (Pittsburgh, PA, USA). CO2 ( 99.99% of purity) was offered by Shenyang Qianzhen Chemical Gas (Shenyang, Liaoning, China) and high-purity nitrogen (99.999%) was used.
A 20 L aliquot of the IS solution (diazepam, 252.50 ng/mL) was added to 200 L plasma sample, followed by 20 L methanol. The sample was vortexed for 1 min and deproteinized with 300 L methanol, the precipitate was removed by centrifugation at 13 000 rpm for 10 min. Then, the supernatant layer was transferred to an Eppendorf micro tube, and an aliquot of 5 L was injected into the SFC–MS/MS system for analysis after another 10 min centrifugation at 13 000 rpm.
2.5 Method validation
2.2 Equipment and conditions Studies were performed on an ACQUITY UPC2TM system (Waters, Milford, MA, USA). For the chromatographic separations, an ACQUITY UPC2TM HSS C18 SB column (100 × 3 mm, 1.8 m; Waters, Milford, MA, USA) was used and the flow rate was set at 1.5 mL/min, and the backpressure of system was conditioned at 2000 psi while the column temperature was maintained at 50⬚C. The mobile phase comprised carbon dioxide and methanol (modifier) (90:10, v/v) while pure methanol was used as the compensation solvent at a flow rate of 0.2 mL/min. An ACQUITY UPC2TM system was coupled to a Waters Triple Quadrupole mass spectrometer equipped with an ESI source interface (Waters). The MS parameters were as follows: capillary voltage of 3.0 kV, cone voltage of 30 V, the optimized collision energy was 15 V and 28 V for NBP and IS, respectively, source temperature 150⬚C and desolvation temperature 200⬚C. The cone and desolvation gas flow rates were 50 and 600 L/h, respectively. The transitions of NBP and IS were m/z 191.1→145.1 and 285.1→193.0, respectively. All data were acquired and processed using MassLynxTM NT 4.1 software with QuanLynxTM program (Waters, Milford, MA, USA). Under these SFC–MS/MS conditions, the retention time was 0.53 min for NBP and 1.13 min for IS, respectively. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
The validation parameters were selectivity, linearity, precision and accuracy, recovery, matrix effect (ME), and stability. To verify the selectivity of the method, the chromatograms of six different batches of blank plasma with simulated plasma samples (those of corresponding standard plasma samples spiked with NBP and IS) and plasma samples after oral administration of NBP should be compared. Calibration curves were constructed over the range of 1.02– 1021.00 ng/mL and the six different batches of lower limit of quantification (LLOQ) samples were validated with relative error (RE) within ±20% and RSD lower than 20% per sample. The intra-day and the inter-day precision and accuracy were determined by analyzing three consecutive validation runs of the QC samples, and the RSD was used to express the precision and the accuracy as the RE. To achieve the extraction recovery and the ME of NBP, the following formulas (A/B×100)% and (B/C×100)% were used respectively. A, B, C refer to the peak area of blank plasma samples spiked with NBP and IS before extraction, blank plasma samples were extracted before adding analytes and the in vitro samples at corresponding concentrations, respectively. The same method was used for the evaluation of extraction recovery and ME for IS. Stability tests were conducted to evaluate the stability of the analyte in plasma samples under different conditions including stored at ambient temperature for 8 h (room temperature stability), repeated freeze–thaw cycles www.jss-journal.com
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three times (freeze–thaw stability), after pre-treatment sample chamber placed 12 h at 4⬚C (post-preparation stability) and –20⬚C frozen storage for 30 days (long-term stability).
2.6 Pharmacokinetic study The developed method was used to determine the plasma concentrations of NBP in six beagle dogs (Laboratory Animal Center of Shenyang Pharmaceutical University, Shenyang, Liaoning, China). After 24 h fasting (without water deprivation), 5 mL blood samples were collected at preadministration, and at 5, 20, 40, 60, 90, 120, 150, 180, 240, 360, 480, 720 min (12 h) after administration of 200 mg NBP, respectively. The blood samples were centrifuged immediately at 13 000 rpm for 10 min and the plasma samples were labeled and stored at –20⬚C until analysis.
3 Results and discussion 3.1 Optimization of MS and chromatographic conditions To achieve optimal MS/MS parameters for analysis of NBP, ESI source coupled with positive ion mode and negative ion mode made an on-the-spot investigation, respectively. ESI source with positive ion mode was selected to get a higher response, which was consistent with the previous studies [15]. For NBP, two major fragment ions were obtained: m/z 191.1→145.1 and 191.1→173.1, while the former gave a stronger and more stable response and the former was chosen for use in MRM. In this study, to gain a faster and useful chromatographic separation, ACQUITY UPC2TM HSS C18 SB column (100 × 3 mm, 1.8 m) and ACQUITY UPC2TM BEH column (100 × 3 mm, 1.7 m) were firstly investigated. The ACQUITY UPC2TM HSS C18 SB column provided a better chromatographic separation than the latter, which could provide sharper peaks and excellent stability while maintaining satisfactory chromatographic resolution with no influence by MEs. In SFC, three important degrees of freedom can change the elution behavior of the analytes, i.e. temperature, modifier, and system pressure. So, different column temperatures (35, 40, 45, 50, and 55⬚C) were compared to achieve a narrow peak, less analysis time and lower column pressure, and 50⬚C was set as the most suitable temperature with a lower column pressure with shaper peaks (Fig. 1A). As to the composition of mobile phase, two kinds of modifier (methanol and acetonitrile) were compared firstly, it showed a shaper and more symmetric peak with a higher response when methanol was used (Fig. 1B). Then different ratios (95:5, 90:10, and 85:5, v/v) of CO2 /methanol were investigated, a narrow peak and relatively reasonable analysis time were obtained compared with the others when the ratio of CO2 /methanol (90:10, v/v) was used. A series of backpressures (1800, 1900, and 2000 psi) was also investigated but no obvious effect was C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 1. The chromatographic separation of NBP: (a) under different column temperature: 35 (A), 40 (B), 45 (C), 50 (D), and 55⬚C (E); (b) by using different modifier: acetonitrile (A) and methanol (B) at the flow rate of 1.8 mL/min (Rt and CP refer to retention time and column pressure, respectively).
observed on the chromatographic behavior, so the system pressure was set at 2000 psi to maintain the best performance of instrument, which was reviewed by the engineers in Waters. According to our previous results, the mass response of the analytes will be improved when compensation solvent was used [22], therefore different flow rate (0.45, 0.2, 0.1, and 0.05 mL/min) of the compensation solution (methanol) was optimized and finally 0.2 mL/min was chosen with the highest and relatively stable mass response.
3.2 Selection of internal standard In this assay, glipizide, spirolactone, and diazepam were evaluated to achieve a ready available internal standard. The results indicated that the retention time of glipizide was far longer than NBP as it belongs to a different chemical class and has a significantly different chromatographic behavior. Both of spirolactone and diazepam are lactone compounds, so they had a similar retention behavior, but the later had a higher mass response in ESI (+) mode. So, internal standard of diazepam was adopted.
3.3 Sample preparation Since NBP is a highly volatile drug, rapid and simple preparation of the biological samples is of great necessary to gain a high extraction recovery. In our study, a one-step protein www.jss-journal.com
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Figure 2. MRM chromatograms for NBP and IS from (A) a blank plasma sample; (B) a plasma sample with added NBP and IS at LLOQ level; and (C) a plasma sample from a dog at 1.5 h after oral administration of NBP (200 mg).
precipitation procedure was selected for the sample extraction due to its excellent recovery rates, rapid as well as simple and convenient. Furthermore, methanol was used as the more efficient precipitant with superb extraction recovery compared to acetonitrile and acetone.
Table 1. Intra-day and inter-day precision and accuracy for assay of NBP in dog plasma (intra-day: n = 6; inter-day: n = six series per day over three days)
Added (ng/mL)
Found (ng/mL)
Intra-day RSD (%)
Inter-day RSD (%)
Accuracy RE (%)
3.4 Method validation
2.50 50.00 800.00
2.74 ± 0.16 54.17 ± 2.13 819.85 ± 40.78
4.82 4.93 4.04
14.78 8.05 7.70
97.90 102.40 103.70
Selectivity was evaluated by comparing the chromatograms of six different lots of blank beagle plasma with the corresponding spiked plasma at LLOQ level and plasma samples after oral administration. Figure 2 shows that no interference from endogenous substances was observed at the retention time of NBP and IS. The linear regression was fitted over the concentration range of 1.02–1021.00 ng/mL (r2 0.993) in beagle plasma. A typical equation for the calibration curve was as follows: y = 0.0145x + 0.2912, r2 = 0.9973. Good linearity was seen over this concentration range in all analytical runs with the
LLOQ of 1.02 ng/mL, which is sensitive enough to investigate the pharmacokinetic behavior of the drug. The values for the intra- and inter-day precision and accuracy are summarized in Table 1. The results show that the present method was accurate and reproducible to determine the plasma concentrations of NBP in beagle. Mean extraction recoveries of NBP at three concentrations levels were 84.2 ± 4.1, 89.5 ± 11.4, and 91.9 ± 5.1%, respectively (n = 6). The mean recovery of the IS was
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J. Sep. Sci. 2015, 38, 697–702 Table 2. Stability of NBP in dog plasma under indicated conditions (mean ± SD, n = 3)
Conditions
Added (ng/mL)
Found (ng/mL)
Pretreatment for 12 h
2.50 50.00 800.00 2.50 50.00 800.00 2.50 50.00 800.00 2.50 50.00 800.00
2.30 47.30 859.71 2.51 51.97 874.33 2.50 47.63 836.41 2.47 47.51 860.09
Room temperature for 8h Freeze–thaw stability
−20⬚C Frozen storage for 30 days
± ± ± ± ± ± ± ± ± ± ± ±
0.14 2.54 6.13 0.09 1.32 20.13 0.06 0.56 22.04 0.01 0.90 15.48
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Table 3. Pharmacokinetic parameters of NBP solid dispersion tablets and commercial NBP soft capsules (n = 6, mean ± SD)
RSD (%) 5.99 5.37 0.17 3.74 2.54 2.30 2.38 1.18 2.64 0.35 1.88 1.80
Pharmacokinetic parameters
NBP solid dispersion tablets
Cmax (ng/mL) AUC0–12 (ng·h/mL) AUC0- (ng·h/mL) Tmax (h) T1/2 (h)
814.92 2196.03 2237.36 1.72 2.94
± ± ± ± ±
Commercial soft capsules 83.96 378.51 392.14 0.33 0.68
335.40 658.36 789.24 3.25 3.63
± ± ± ± ±
57.76 237.79 236.99 1.83 0.47
4 Concluding remarks An SFC–MS/MS method for the determination of NBP in beagle plasma and its validation was described for the first time in this paper. Compared to the traditional UHPLC– MS/MS method, it has a short single run time of 1.5 min per sample, which makes it an attractive procedure in the highthroughput analysis of NBP in beagle plasma. The method was highly efficient, had satisfactory sensitivity with the LLOQ of 1.02 ng/mL, superior selectivity and was successfully applied to a preclinical pharmacokinetic study of NBP in beagle dogs. The present work would provide helpful information for further studies.
Figure 3. Mean concentration–time profiles of NBP in beagle plasma after a single oral dose of 200 mg NBP solid dispersion tablets and commercial soft capsules (n = 6).
The authors have declared no conflict of interest.
5 References 92.0 ± 5.1% (n = 6). The relative ME result was RSD 0.05), which indicated that the oral bioavailability of the NBP solid dispersions was increased significantly compared with commercial soft capsules. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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