Quantitative Detection of Ambroxol in Human Plasma Using HPLC-APCI-MS/MS: Application to a Pharmacokinetic Study

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1 ANALYTICAL SCIENCES OCTOBER 2017, VOL The Japan Society for Analytical Chemistry Original Papers Quantitative Detection of Ambroxol in Human Plasma Using HPLC-APCI-MS/MS: Application to a Pharmacokinetic Study Zhengsheng MAO, Xin WANG, Xin DI, Yangdan LIU, Yanan ZANG, Dongke MA, Youping LIU, and Xin DI Laboratory of Drug Metabolism and Pharmacokinetics, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang , PR China In this study, a rapid and reliable high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method for the determination of ambroxol in human plasma was developed and validated using palmatine as an internal standard (IS). Ambroxol and IS were extracted from 200 μl of human plasma via a simple protein precipitation preparation. Chromatographic separation was achieved on a Platisil C18 column ( mm, 5 μm) using methanol 0.01 formic acid (70:30, v/v) as the mobile phase at a flow rate of 0.6 ml/min under an isocratic condition. The MS acquisition m/z for ambroxol and for IS was performed by atmospheric-pressure chemical ionization (APCI) mass spectrometry in selected reaction monitoring mode. The calibration curve for ambroxol was linear over the concentration range of ng/ml. The matrix effects of ambroxol ranged from to This fully validated method was successfully applied to a pharmacokinetic study of ambroxol in humans after oral administration of ambroxol at a single dose of 75 mg. Keywords Ambroxol, APCI, human plasma, HPLC-MS/MS, pharmacokinetics (Received January 26, 2017; Accepted April 4, 2017; Published October 10, 2017) Introduction Ambroxol, trans-4-(2-amino-3,5-dibromobenzylamino)-cyclohexanol, is an expectorant and mucolytic agent frequently used in the treatment of bronchial asthma and chronic bronchitis. 1,2 Ambroxol has also been reported to have rhinovirus infection inhibition 3 and perioperative lung protection effects. 4 Recently, the increasement of glucosylceramidase activity and reduction of markers of oxidative stress in cells treated with ambroxol suggested that ambroxol may offer a potential treatment for Parkinson s disease. 5 Some bioanalytical methods have been reported for the determination of ambroxol by HPLC-MS/MS analysis In these methods, electrospray ionization (ESI) was most commonly used as an ionization method. However, in our preliminary experiments, ESI had high matrix effects for determining ambroxol in human plasma. Matrix effects mainly led to signal suppression, but signal enhancement was observed on this case. In any case, matrix effects greatly affected the reproducibility and accuracy of method. To overcome this problem, APCI, showing minimal matrix effects in various complex samples, was tried to reduce or eliminate the matrix effects. In the present work, a rapid and reproducible method to determine ambroxol in human plasma was achieved by using an HPLC-APCI-MS/MS. This method was fully validated and successfully applied to a pharmacokinetic study of an ambroxol To whom correspondence should be addressed. dixin63@hotmail.com sustained release capsule preparation in 12 healthy Chinese male volunteers. Experimental Reagents and chemicals Ambroxol hydrochloride (99.9) and palmatine hydrochloride (97.4) were obtained from National Institutes of Food and Drug Control (Beijing, China). The chemical structures of ambroxol hydrochloride and IS are shown in Fig. 1. Methanol and formic acid of HPLC-grade were obtained from Concord Technology Co. Ltd (Tianjin, China) and Kermel Chemical Reagents Co. Ltd (Tianjin, China), respectively. Doubledistilled water was prepared by a double-distilled water distiller (Shanghai, China). Fig. 1 Chemical structures of ambroxol hydrochloride (A) and palmatine (IS) (B).

2 1100 ANALYTICAL SCIENCES OCTOBER 2017, VOL. 33 Preparation of calibration standards and quality control (QC) samples Standard stock solutions of ambroxol (1 mg/ml) and IS (1 mg/ml) were prepared in methanol. The stock solution of ambroxol was successively diluted with methanol water (50:50, v/v) to make standard working solutions at concentrations of 25.00, 75.00, 150.0, 450.0, and 1800 ng/ml. In the same way, QC working solutions were made at 50.00, and 1500 ng/ml. An IS working solution of 1000 ng/ml was prepared by diluting the stock solution of palmatine with methanol water (50:50, v/v). Calibration standards were prepared by spiking 20 μl of standard working solutions into 200 μl of blank human plasma to yield plasma concentrations of 2.500, 7.500, 15.00, 45.00, 90.00, and ng/ml. QC samples were prepared in three concentrations: 5.00, and ng/ml. Sample preparation To a 200-μL of plasma sample, 20 μl of an IS working solution, 20 μl of methanol water (50:50, v/v) and 500 μl of methanol were added. The mixtures were vortex-mixed for 45 s and centrifuged at rpm for 4 min. The supernatant was transferred into the polyethylene autosampler vial, and then 10 μl of the solution was injected into the LC-MS/MS system for analysis. LC-MS/MS conditions Chromatographic analysis was achieved with a Shimadzu HPLC system consisting of a SIL-HTA autosampler and two LC-10AD pumps (Kyoto, Japan). Chromatographic separation was carried out on a Platisil C18 column ( mm, 5 μm) using methanol 0.01 formic acid (70:30, v/v) as a mobile phase under ambient conditions. The flow rate was set at 0.6 ml/min with the isocratic condition, and the injection volume was 10 μl. A Thermo Finnigan TSQ Quantum Ultra triple-quadrupole mass spectrometer (San Jose, CA, USA) was used for sample analysis. APCI source conditions: the vaporizer temperature was set at 400 C and the capillary temperature kept at 270 C. The sheath and auxiliary gasses were set at 30 and 5 arbitrary unit, respectively. The discharge current was set at 4 μa. The selected reaction monitoring (SRM) transitions were monitored at m/z for ambroxol and m/z for IS. ESI source conditions: the electrospray voltage was set at 4.2 kv and the capillary temperature was maintained at 340 C. The SRM transitions were also monitored at m/z for ambroxol and m/z for IS. LCquan quantitation software (Waltham, MA, USA) was used for data acquisition and analysis. Method validation This method developed herein has been fully validated according to the Food and Drug Administration (FDA) guideline for bioanalytical method validation. 15 Pharmacokinetic study Twelve healthy young subjects who participated in this study were given ambroxol at a single dose of 75 mg (75 mg ambroxol sustained release capsule from a Chinese company). Blood samples (2 3 ml) were collected before dosing (0 h) and at 1.0, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 8.0, 10, 12, 24, 36 and 48 h after dosing and placed into heparin-coated tubes. Plasma was harvested after the blood samples following centrifugation at 4000 rpm for 5 min, and then stored at 70 C until analysis. The protocol was approved by local ethics committees, and all participants provided written informed consent. Fig. 2 Matrix effects by ESI and APCI. Results and Discussion Method development Chromatographic condition and sample preparation optimization. Different C18 columns including Phenomenex Synergi C18 column, Hypersil GOLD C18 column, ZORBAX SB C18 column and Platisil C18 were tested. It was found that the Platisil C18 column ( mm, 5 μm) proved to be a suitable column with the optimal peak shape and suitable separation time. The mobile-phase system was tested with various compositions of methanol, acetonitrile, formic acid and ammonium acetate to obtain the optimized response and good peak shape for the analyte. Finally, methanol 0.01 formic acid (70:30, v/v) was chosen as the optimal mobile phase. It was also found that isocratic elution compared with gradient elution could provide stable response and retention time for the analyte and IS. One-step protein precipitation could provide a higher extraction efficiency compared with liquid liquid extraction methods for plasma sample preparation. Therefore, several kinds of protein precipitation agents, such as methanol and acetonitrile, were tested, and finally methanol was chosen as the precipitant for giving less interference. Mass spectrometry optimization. Matrix effects, a pivotal issue for HPLC-MS/MS analysis, directly or indirectly cause varying responses of the analytes in a biological matrix compared to a standard solution. The difference may be described as suppression or enhancement according to whether the response is diminished or magnified. 16 These unpredictable effects are a regular problem for biological sample analysis, and greatly affect the reproducibility and accuracy of bioanalytical method. The common strategies, such as liquid liquid extraction (LLE), solid-phase extraction (SPE) 17,18 and isotope-labeled internal standard normalized matrix factor, 19 were developed to minimize any matrix effects in bioanalytical assays by LC/MS/MS. However, both the LLE and SPE methods suffered from the multiple complicated procedures that are time and cost consuming. Otherwise, it was found that using an isotopelabeled internal standard could also lead to an underestimation of the true concentration. 20 Therefore, it is necessary to find a simple and stable method to reduce any matrix effects. ESI and APCI sources have been widely used as ionization sources in HPLC-MS/MS methods. 21 Even though the absolute response of APCI was usually lower than that of ESI, the mean matrix effects could be significantly reduced when using APCI than ESI. 22 In our experiment, both ESI and APCI sources were tried to test matrix effects of ambroxol in human plasma. The matrix effects of the analyte

3 ANALYTICAL SCIENCES OCTOBER 2017, VOL by ESI, ranged from 147 to 163 (Fig. 2), severely affect the accuracy of quantitative analysis. To overcome this problem, APCI with a positive mode was used, and gave satisfactory matrix effects (less than 112.7). Finally, APCI sources were chosen for the ambroxol determination in human plasma. The SRM was chosen m/z for ambroxol and m/z for IS as precursor-product ion transitions due to its good and sensitivity (Fig. 3). Method validation Selectivity. The selectivity of the assay was checked by comparing six different batches of blank human plasma with blank human plasma spiked with the analyte and IS, and real human plasma sample obtained at 5 h after oral administration of ambroxol at a dose of 75 mg. As the Fig. 4 shows, no interference at the retention times of the analyte and the internal standard was observed. Linearity and lower limit of quantification. A linearity study used six non-zero standard points carried out by a calibration curve in the concentration range of ng/ml. The correlation coefficient (r) values were > for all validated batches. The LLOQ of ambroxol in human plasma was ng/ml with a relative standard deviations (RSD) and relative error (RE) of 10.4 and 5.4, respectively. The results of a linear regression analysis and LLOQ are summarized in Table 1. Precision and accuracy. The precision and accuracy data for the determination of ambroxol at three QC levels are summarized in Table 2. The intra- and inter-day precision for ambroxol were within the range from 4.4 to The accuracy derived from three QC levels was between 4.3 and 4.0. The assay values on both the precision and accuracy were all within the acceptable range. Recovery. The recovery was assessed by calculating the ratio of the peak area of a sample prepared by adding the analyte to a blank matrix extract to the peak area of a sample prepared by adding the analyte to blank matrix prior to extracting. The mean recovery of ambroxol at three QC levels from human plasma ranged from 96.0 to with a maximum RSD of 8.6. The recovery of IS was 97.8 ± 6.9. Table 3 summarizes the recovery for ambroxol and IS. Matrix effect. The matrix effects were assessed by calculating the ratio of the peak area in the presence of the matrix to the peak area in the absence of the matrix. The matrix effects at three QC levels ranged from to (Table 3), indicating that no significant matrix effects were observed from the plasma. Stability. Stability studies were carried out to evaluate the of ambroxol under various sample handling and storage conditions. Ambroxol was found to be stable in human plasma up to 12 h at room temperature (25 C), 24 h after being processed at an auto sampler at 4 C. The freeze-thaw of ambroxol was within 9.4 of the nominal concentrations, which suggested that the analyte in human plasma was stable after three freeze-thaw cycles. Long-term storage values were within 9.2 of the nominal concentrations, indicating that ambroxol human samples could be stored for up to 30 days at 70 C. The results of experiments are summarized in Table 4. Method application. The validated method was successfully applied to a pharmacokinetic study of ambroxol in humans after Fig. 3 Product ion mass spectra of ambroxol (A) and IS (B). Fig. 4 Typical SRM chromatograms of ambroxol and IS in human plasma. (A) Blank plasma. (B) Blank plasma spiked with ambroxol at LLOQ and IS at ng/ml. (C) Real plasma sample obtained from a subject following a single 75 mg oral dose of ambroxol. Table 1 Regression equation, linear range, and LLOQ for the determination of ambroxol in human plasma Compound Regression equation a r Linear range/ LLOQ Concentration/ RSD b, RE, Ambroxol y = x a. y, The area ratio of ambroxol/is obtained from selected reaction monitoring; x, the nominal concentration of ambroxol. b. RSD: relative standard deviations, which calculated by the ratio of the standard deviation to the mean concentration of the analyte.

4 1102 ANALYTICAL SCIENCES OCTOBER 2017, VOL. 33 Table 2 Precision and accuracy for the determination of ambroxol in human plasma (n = 6) Compound Concentration/ Intra-day RSD, Inter-day RSD, RE, Ambroxol Table 3 Matrix effects and extraction recovery for the determination of ambroxol in humans plasma (n = 6) Compound Concentration/ the oral administration of a single dose of an ambroxol sustained capsule at 75 mg. The mean plasma concentration-time curve of ambroxol is shown in Fig. 5. The WinNonlin 6.3 software was applied to calculate the pharmacokinetic parameters of ambroxol by non-compartmental analysis. The pharmacokinetic parameters of ambroxol are summarized in Table 5. After a single-dose administration of an ambroxol sustained capsule, the maximum concentration (C max), time of maximum concentration (T max), half life (t 1/2) and area under the curve (AUC last) of ambroxol were 93.2 ± 18.7 ng/ml, 5.2 ± 0.7 h, 12.1 ± 2.8 h and ± h ng/ml, respectively. These results indicated that the developed method has been successfully applied to a pharmacokinetic study to determine the concentration of ambroxol in human plasma. Conclusions Recovery, Matrix effects APCI ESI Ambroxol ± ± ± ± ± ± 16.1 IS ± ± ± ± 6.9 Table 4 Stability results for ambroxol under different conditions (n = 3) Stability Bench-top Processed sample Freeze and thaw Long term Storage condition Room temperature (12 h) Auto sampler (4 C, 24 h) Concentration/ RE, RSD, After three cycle days at 70 ºC In this study, a simple, sensitive and selective LC-MS/MS method was developed and validated for the determination of ambroxol in human plasma. The matrix effects were eliminated by using atmospheric pressure chemical ionization. The method Fig. 5 Plasma concentration time curve of ambroxol after administration of ambroxol at 75 mg to volunteers (mean ± SD, n = 12). Table 5 Mean pharmacokinetic parameters of ambroxol after oral administration of a 75-mg ambroxol sustained release capsule formulation in 12 healthy Chinese subjects under a fasting condition (n = 12) was successfully applied to a pharmacokinetic study of ambroxol in humans after oral administration of ambroxol at a single dose of 75 mg. This is the first report of a pharmacokinetic study of an ambroxol sustained capsule by the LC-MS/MS method with an atmospheric pressure chemical ionization source. Acknowledgements We thank Ms. Na Zhao and Ms. Yang Liu of Yiling Medical Technology Co., Ltd. for their technical help. References Parameter Ambroxol T max (h) 5.2 ± 0.7 t 1/2 (h) 12.1 ± 2.8 MRT (h) 15.6 ± 1.6 Cl/F (L/h) 51.0 ± 12.1 C max (ng/ml) 93.2 ± 18.7 Vd/F (L) ± AUC last (h ng/ml) ± AUC inf (h ng/ml) ± G. H. Guyatt, M. Townsend, F. Kazim, and M. T. Newhouse, Chest, 1987, 92, J. L. M. Santos, A. Clausse, J. L. F. C. Lima, M. L. M. F. S. Saraiva, and A. O. S. Rangel, Anal. Sci., 2005, 21, M. Yamaya, H. Nishimura, L.K. Nadine, C. Ota, H. Kubo, and R. Nagatomi, Arch. Pharm. Res., 2014, 37, X. Wang, L. Wang, H. Wang, and H. Zhang, Cell Biochem. Biophys., 2015, 73, A. McNeill, J. Magalhaes, C. Shen, K. Y. Chau, D. Hughes, A. Mehta, T. Foltynie, J. M. Cooper, A. Y. Abramov, M. Gegg, and A. H. Schapira, Brain, 2014, 137, T. J. Hang, M. Zhang, M. Song, J. P. Shen, and Y. D. Zhang,

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