Journal of Chromatography B, 967 (2014) 225–234
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Simultaneous determination of bambuterol and its two major metabolites in human plasma by hydrophilic interaction ultra-performance liquid chromatography–tandem mass spectrometry Ting Zhou a,b , Qing Cheng a,b , Chengjuan Zou a,b , Ting Zhao a,b , Shan Liu a,b , Marco Pistolozzi a,b , Evina Tan c , Ling Xu c , Wen Tan a,b,∗ a
School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, 510006, China South China University of Technology-Waters Technology Joint Laboratory, South China University of Technology, Guangzhou, 510006, China c Keypharma Biomedical Inc., Songshan Lake Science & Technology Industry Park, Dongguan, 523808, China b
a r t i c l e
i n f o
Article history: Received 24 May 2014 Accepted 15 July 2014 Available online 21 July 2014 Keywords: HILIC-UPLC-MS/MS Bambuterol Major metabolites Plasma Pharmacokinetics.
a b s t r a c t In this study, a rapid and sensitive hydrophilic interaction ultra-performance liquid chromatography–tandem mass spectrometry (HILIC–UPLC–MS/MS) method was developed for simultaneous determination of bambuterol and its two major metabolites monocarbamate bambuterol and terbutaline in human plasma. All samples were simply precipitated using acetonitrile and separated on a UPLC-HILIC column under gradient elution with a mobile phase consisting of acetonitrile and water with the addition of 10 mm ammonium acetate and 0.1% formic acid at 0.4 mL/min. The analytes were detected by a Xevo TQ-S tandem mass spectrometer with positive electrospray ionization in multiple reaction monitoring mode. The established method was highly sensitive with the lower limit of quantification (LLOQ) of 10.00 pg/mL for each analyte, and the intra- and inter-day precisions were 0.992) for all the analytes. The LLOQ for bambuterol, monocarbamate bambuterol, and terbutaline in plasma was 10.00 pg/mL (Fig. 2, Table 2), and the injection volume was only 2 L while the injection volume in our previous study was 20 L with the same LLOQ for bambuterol and terbutaline. The much smaller injection volume in the current method protected the analytical column and instrument from serious pollution of sample matrix and meant much higher sensitivity. The intra- and inter-run precision and accuracy were summarized in Table 2. The recovery values for bambuterol determined at three concentration levels were 74.7 ± 2.9%, 73.5 ± 3.3%, and 77.4 ± 3.6% (n = 3), respectively, for monocarbamate bambuterol were 78.6 ± 4.1%, 77.1 ± 4.6%, and 79.8 ± 3.2% (n = 3), respectively, and for terbutaline were 72.6 ± 1.7%, 74.1 ± 4.1%, and 77.0 ± 2.9% (n = 3), respectively. The results demonstrated that the method was precise and accurate.
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Fig. 2. Typical MRM chromatograms of blank plasma (A), LLOQ for bambuterol (bam), monocarbamate bambuterol (mono) and terbutaline (ter) in plasma (10.00 pg/mL) and the IS (B), plasma spiked with the three analytes (500.0 pg/mL) and the IS (C), plasma obtained from a volunteer at 1 h after a single oral dose of bambuterol (10 mg) (D).
The matrix effect was evaluated by comparing the peak areas of blank plasma extracts spiked with the analytes with those of neat solutions at the corresponding concentrations. Five different batches of human blank plasma were tested and the results showed that there was no significant matrix effect observed in this study. The carry-over effects were tested by analyzing blank samples just after the plasma samples at ULOQ. No residual signals were observed at the retention times of the analytes or the IS in the chromatograms of blank plasma samples. The stability of the analytes was studied under a variety of storage and handling conditions. The results in Table 3 showed that no significant degradation occurred in plasma maintained at room temperature for 6 h or during the three freeze–thaw cycles. The analytes were stable in plasma at −80 ◦ C for at least 3 months. The stock solutions of all analytes and IS were stable at −20 ◦ C for at least 1 month (data not shown).
healthy volunteers were determined up to 96 h after receiving an oral dose of bambuterol hydrochloride tablets (10 mg). The mean plasma concentration–time curve of the three analytes was shown in Fig. 3. The main pharmacokinetic parameter values in healthy volunteers were calculated and summarized in Table 4. The results showed that Cmax of monocarbamate bambuterol was more than double that of bambuterol or terbutaline, AUC0–96 of monocarbamate bambuterol was triple that of bambuterol and was as much as that of terbutaline. As mentioned above, the active metabolite terbutaline was slowly released by the hydrolysis of both bambuterol and monocarbamate bambuterol catalyzed by BChE. Our data indicated that the slow bioconversion mechanism of bambuterol into its parent drug terbutaline was mainly due to the interaction of BChE with monocarbamate bambuterol, rather than bambuterol as previous reported [4,5].
3.4. Application to a pharmacokinetic study in healthy volunteers
3.5. The effects of the chromatographic conditions on the retention of HILIC
The method described above was successfully applied to a pharmacokinetic study, in which plasma concentrations of bambuterol, monocarbamate bambuterol, and terbutaline in eight
HILIC has become increasingly popular as an alternative to RPLC for the analysis of polar analytes [25]. However, the retention mechanism of HILIC is complex and still poorly understood. In order to
T. Zhou et al. / J. Chromatogr. B 967 (2014) 225–234
231
Table 3 Stability data of bambuterol, monocarbamate bambuterol, and terbutaline in human plasma under various storage conditions (n = 3). Storage conditions Room temperature for 6 h Three freeze–thaw cycles 3 months at −80 ◦ C a
Added Ca (pg/mL)
Bambuterol
30.00 3000 30.00 3000 30.00 3000
Found C (pg/mL) 29.88 3034 27.04 2740 32.31 3249
Monocarbamate bambuterol RE (%) −0.4 1.1 −9.9 −8.7 7.7 8.3
RSD (%) 0.7 1.8 2.2 1.2 2.8 1.6
Found C (pg/mL) 28.72 2981 26.64 2699 33.59 3324
Terbutaline RE (%) −4.3 −0.6 −11.2 −10.0 12.0 10.8
RSD (%) 3.3 2.0 0.9 1.9 1.4 1.0
Found C (pg/mL) 28.97 2992 26.98 2697 32.32 3296
RE (%) −3.4 −0.3 −10.1 −10.1 7.7 9.9
RSD (%) 7.5 1.6 3.4 2.1 1.1 1.2
C, concentration.
Table 4 Main pharmacokinetic parameters of bambuterol, monocarbamate bambuterol, and terbutaline in healthy volunteers after a single oral dose of 10 mg bambuterol hydrochloride (n = 8). Parameters
t1/2 (h) Tmax (h) C max (ng/L) AUC0–96 (h ng/L) AUC0–∞ (h ng/L) CLz /F (L/h) Vz /F (L)
Mean ± SD Bambuterol
Monocarbamate bambuterol
Terbutaline
12.57 ± 6.99 5.06 ± 3.41 1177 ± 1123 13708 ± 4893 16472 ± 7459 721 ± 261 14005 ± 11748
15.15 ± 6.81 2.63 ± 2.66 2588 ± 1973 40715 ± 22065 42984 ± 22046 267 ± 85 5397 ± 2291
23.41 ± 9.15 5.00 ± 1.93 1275 ± 727 46976 ± 18064 51633 ± 22306 223 ± 82 6743 ± 1482
shed some light over the retention mechanism of HILIC stationary phases, the effects of chromatographic conditions on the retention of the four phenylethylamines (the three analytes and the IS) in this study were further investigated. 3.5.1. Effect of the organic phase proportion The mobile phase in HILIC consists of an aqueous–organic mixture. In this study, acetonitrile was used as the organic solvent. The effect of acetonitrile proportion on the retention of the analytes was illustrated in Fig. 4a. The results showed that the retention factor increased as the acetonitrile proportion increased for all the analytes. By increasing the acetonitrile proportion to 95%, the best separation of the four analytes was achieved, and the retention factors followed the order: salbutamol > terbutaline > monocarbamate bambuterol > bambuterol. The polar stationary phases used in HILIC such as silica are known to strongly retain water [26]. A water layer has been proven to be adsorbed onto the surface of the hydrophilic stationary phase
Fig. 3. Mean concentration–time profiles of bambuterol, monocarbamate bambuterol, and terbutaline in plasma after an oral dose of bambuterol hydrochloride tablets (10 mg) administered to the healthy volunteers (n = 8).
in HILIC columns [27]. Thus, in HILIC, the acetonitrile-rich bulk and the water-rich layer of the mobile phase form two liquid phases of different polarities, resembling a liquid–liquid partition system [23]. Therefore, the distribution of the analytes between the two phases bases on their relative partition coefficient. As a consequence, polar hydrophilic analytes are preferentially distributed into the water layer and more strongly retained. The logP of the analytes investigated follows the order: salbutamol (0.83 ± 0.40) < terbutaline (1.02 ± 0.33) < bambuterol monocarbamate (1.20 ± 0.28) < bambuterol (1.5 ± 0.36), which corresponds to the order of the retention factors observed when the acetonitrile proportion was 95%. As the water content in the mobile phase was increased, the difference in polarity between the bulk and the adsorbed layer decreased, so the difference in the retention of the analytes gradually disappeared.
3.5.2. Effect of the buffer concentration Buffers are usually added into the mobile phase to control the electrostatic interaction between the charged analytes and the stationary phase. To improve peak shapes and reproducibility, it is recommended to maintain a minimum buffer concentration in the mobile phase [28]. In this study, ammonium acetate was used due to its good solubility in the acetonitrile-rich mobile phase and compatibility with mass spectrometric detection. The effect of the buffer concentration on the retention of the analytes was illustrated in Fig. 4b, and a decrease in the retention upon increasing the buffer concentration was observed for all analytes. Increasing the buffer concentration has the general effect of weakening the electrostatic interactions between the charged analytes and the charged stationary phases [29]. In the case of electrostatic attraction, the increase of the buffer concentration leads to a decreased retention, whereas in the case of electrostatic repulsions, it leads to increased retention. In the condition of this study, the silica stationary phase was negatively charged while the analytes which have a pKa for the secondary amino group around 9.6 were positively charged. Therefore, the electrostatic interaction between the charged analytes and the charged column in this study was electrostatic attraction, and the increasing buffer concentration led to the decreased retention.
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Fig. 4. The effects of the organic phase proportion (A), the buffer concentration (B), the pH of the mobile phase (C), and the column temperature (D) on the retention of HILIC column.
3.5.3. Effect of pH of the mobile phase The primary effect of the mobile phase pH is on the charged state of the analytes. It is preferable to select the pH to bring the analytes in their charged forms which are usually more hydrophilic and more retained in HILIC than their neutral forms. The effect of pH of the mobile phase on the retention of the analytes was illustrated in Fig. 4c. A marked increase in the retention of the four analytes was observed upon increasing the pH. In the condition employed, the silanol groups on the surface of the silica stationary phase were negatively charged while the four analytes, which have a pKa for the secondary amino group around 9.6, were positively charged. It is probable that as the pH of the mobile phase increased, the silanol groups became more and more deprotonated while the ionization state of the analytes was not significantly affected. As a consequence, the electrostatic interactions of the positive-charged analytes with the stationary phase were dramatically enhanced [30]. 3.5.4. Effect of the column temperature In this study, the effect of the column temperature on the retention of the analytes was investigated in the range of 25–50 ◦ C. As shown in Fig. 4d, the column temperature had a minor impact on the retention of the analytes. However, a little decrease in retention was observed for salbutamol and terbutaline upon increasing the temperature while the retention of bambuterol and monocarbamate bambuterol showed a slight increase passing from
25 to 35 ◦ C and remained stable upon further increase of the temperature. For the compounds with high polarity such as salbutamol and terbutaline, there are several reasons to explain the decrease of the retention. The effect of the temperature was primarily related to the enthalpy of the analyte’s transfer between the mobile phase and the stationary phase. In this case, the transfer of hydrophilic analytes from the acetonitrile-rich bulk to the water layer was an exothermic process, favoring lower temperatures [31]. It has also been suggested that the pKa of basic analytes decreases upon increasing the temperature, resulting in a decrease of polarity [32]. Besides, the electrostatic interactions were postulated to be weakened at elevated temperatures, and the amount of the water layer adsorbed on the silica surface might also tend to thin as the temperature increased, thereby weakening the retention of polar compounds [33]. For the compounds with lower polarity, the situation was more complex. The first slight increase of the retention might be the result of an increase of solutes partition in the water phase adsorbed on the silica surface as the temperature increased. On the other hand, as the temperature increased, the adsorbed water layer on the silica surface became thinner, thus balancing the increase of water solubility at temperatures higher than 35 ◦ C. In summary, according to these results, chromatographic conditions affected the retention of phenylethylamines on the HILIC stationary phase followed the order: organic phase proportion > ammonium acetate concentration > mobile phase
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Fig. 5. The asymmetry factors of the analytes on the HILIC and C18 columns at 0.4 mL/min.
pH > column temperature. The retention of the four phenethylamines on the HILIC column was based on the liquid–liquid partition and the electrostatic interactions between the charged analytes and the stationary phase.
Fig. 6. The number of effective plates (Neff ) of the HILIC and C18 columns at 0.4 mL/min.
3.6. The comparison of HILIC column with C18 column In order to demonstrate the advantages over RPLC offered by the HILIC approach used in this study, an alternative RPLC method based on the same sub-2 m bridged-ethylene hybrid (BEH) material functionalized with C18 chains was optimized and the chromatographic performance of the two methods in the analysis of phenylethylamines was compared. 3.6.1. Comparison of the asymmetry factors The asymmetry factors of the four analytes obtained by using HILIC and RPLC at different flow rates were shown in Figure S1, and the asymmetry factors measured at 0.4 mL/min were shown in Fig. 5. HILIC provided asymmetry factors close to 1 for all the analytes, independently from the flow rate employed (shown in Fig. S1). RPLC provided a worse performance in all the cases (shown in Fig. 5) although the symmetry improved significantly upon increasing the flow rate (shown in Fig. S1). A similar dependence between flow rate and peak symmetry in RPLC was recently observed by Heaton [34] in the analysis of ephedrines, a related class of phenylethylamines. The results indicated that HILIC column could obtain better peak symmetry compared with C18 column in the analysis of phenethylamines. 3.6.2. Comparison of the number of effective plates The number of effective plates (Neff ) represents the column efficiency. As shown in Fig. 6, the number of effective plates was significantly higher in the HILIC column than in the C18 column, except for bambuterol. This is consistent with the lower polarity of bambuterol compared with the other three analytes, and C18 columns have usually good efficiency with medium polarity compounds. In other words, the HILIC column provided better efficiency than the C18 column for the compounds of high polarity. Figure S2 indicated that a reduction of number of effective plates in the HILIC column for all the analytes as the flow rate was increased, reflecting a loss of efficiency at high flow rates. 3.6.3. Comparison of the column back pressure The column back pressure observed for the HILIC and C18 columns was monitored at different flow rates under their optimized isocratic conditions, which were shown in Fig. 7. The results
Fig. 7. The column back pressure of the HILIC and C18 columns monitored at different flow rates.
indicated that the back pressure of both columns increased proportionally with the flow rate, and the back pressure of the HILIC column was much lower than that of the C18 column. At 0.8 mL/min, the back pressure of the C18 column reached almost 10,000 psi, which is the maximum pressure this column could bear while the HILIC column was just above 3000 psi. This difference is related to the higher aqueous content in the mobile phase used in RPLC whose higher viscosity leads to a higher back pressure, especially when sub-2 m particles are used. In contrast, the HILIC separation affords significantly lower mobile phase viscosity and lower column pressure, which permits the use of faster flow rates for increased sample throughput and longer columns for enhanced resolution. 4. Conclusions A sensitive and rapid HILIC–UPLC–MS/MS method for simultaneous determination of bambuterol and its two major metabolites monocarbamate bambuterol and terbutaline in human plasma has been developed for the first time. The established method provided the LLOQ of 10.00 pg/mL and the runtime of 4.0 min per sample, achieving the highest sensitivity and the fastest analysis
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speed compared with the previously reported methods. The calibration curves of all the analytes were linear in the range of 10.00–5000 pg/mL. This method was successfully applied to the clinical pharmacokinetic study of bambuterol in eight healthy volunteers, and the data indicated that the slow bioconversion mechanism of bambuterol into its parent drug terbutaline was mainly due to the interaction of BChE with monocarbamate bambuterol. The investigation for the effects of various chromatographic parameters on the retention of the analytes indicated that liquid–liquid partition and the electrostatic interactions played an important role in the retention of phenylethylamines on the HILIC column used in this study. Moreover, the HILIC approach showed particular advantages over RPLC including a better peak symmetry, higher efficiency, and much lower column pressure. Acknowledgements This work was supported by Major Science and Technology Project of Guangdong ProvinceFunded by Ministry of Science & Technology of Guangdong Province, China (Grant No.: 2012A080204001), the Fundamental Research Funds for the Central Universities (Grant No.: 2013ZB0010), and the Natural Science Foundation of Guangdong Province, China (Grant No.: S2013040015142). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jchromb. 2014.07.022. References [1] [2] [3] [4]
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Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Simultaneous determination of bambuterol and its two major metabolites in human plasma by hydrophilic interaction ultra-performance liquid chromatography–tandem mass spectrometry Ting Zhou a,b , Qing Cheng a,b , Chengjuan Zou a,b , Ting Zhao a,b , Shan Liu a,b , Marco Pistolozzi a,b , Evina Tan c , Ling Xu c , Wen Tan a,b,∗ a
School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, 510006, China South China University of Technology-Waters Technology Joint Laboratory, South China University of Technology, Guangzhou, 510006, China c Keypharma Biomedical Inc., Songshan Lake Science & Technology Industry Park, Dongguan, 523808, China b
a r t i c l e
i n f o
Article history: Received 24 May 2014 Accepted 15 July 2014 Available online 21 July 2014 Keywords: HILIC-UPLC-MS/MS Bambuterol Major metabolites Plasma Pharmacokinetics.
a b s t r a c t In this study, a rapid and sensitive hydrophilic interaction ultra-performance liquid chromatography–tandem mass spectrometry (HILIC–UPLC–MS/MS) method was developed for simultaneous determination of bambuterol and its two major metabolites monocarbamate bambuterol and terbutaline in human plasma. All samples were simply precipitated using acetonitrile and separated on a UPLC-HILIC column under gradient elution with a mobile phase consisting of acetonitrile and water with the addition of 10 mm ammonium acetate and 0.1% formic acid at 0.4 mL/min. The analytes were detected by a Xevo TQ-S tandem mass spectrometer with positive electrospray ionization in multiple reaction monitoring mode. The established method was highly sensitive with the lower limit of quantification (LLOQ) of 10.00 pg/mL for each analyte, and the intra- and inter-day precisions were 0.992) for all the analytes. The LLOQ for bambuterol, monocarbamate bambuterol, and terbutaline in plasma was 10.00 pg/mL (Fig. 2, Table 2), and the injection volume was only 2 L while the injection volume in our previous study was 20 L with the same LLOQ for bambuterol and terbutaline. The much smaller injection volume in the current method protected the analytical column and instrument from serious pollution of sample matrix and meant much higher sensitivity. The intra- and inter-run precision and accuracy were summarized in Table 2. The recovery values for bambuterol determined at three concentration levels were 74.7 ± 2.9%, 73.5 ± 3.3%, and 77.4 ± 3.6% (n = 3), respectively, for monocarbamate bambuterol were 78.6 ± 4.1%, 77.1 ± 4.6%, and 79.8 ± 3.2% (n = 3), respectively, and for terbutaline were 72.6 ± 1.7%, 74.1 ± 4.1%, and 77.0 ± 2.9% (n = 3), respectively. The results demonstrated that the method was precise and accurate.
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Fig. 2. Typical MRM chromatograms of blank plasma (A), LLOQ for bambuterol (bam), monocarbamate bambuterol (mono) and terbutaline (ter) in plasma (10.00 pg/mL) and the IS (B), plasma spiked with the three analytes (500.0 pg/mL) and the IS (C), plasma obtained from a volunteer at 1 h after a single oral dose of bambuterol (10 mg) (D).
The matrix effect was evaluated by comparing the peak areas of blank plasma extracts spiked with the analytes with those of neat solutions at the corresponding concentrations. Five different batches of human blank plasma were tested and the results showed that there was no significant matrix effect observed in this study. The carry-over effects were tested by analyzing blank samples just after the plasma samples at ULOQ. No residual signals were observed at the retention times of the analytes or the IS in the chromatograms of blank plasma samples. The stability of the analytes was studied under a variety of storage and handling conditions. The results in Table 3 showed that no significant degradation occurred in plasma maintained at room temperature for 6 h or during the three freeze–thaw cycles. The analytes were stable in plasma at −80 ◦ C for at least 3 months. The stock solutions of all analytes and IS were stable at −20 ◦ C for at least 1 month (data not shown).
healthy volunteers were determined up to 96 h after receiving an oral dose of bambuterol hydrochloride tablets (10 mg). The mean plasma concentration–time curve of the three analytes was shown in Fig. 3. The main pharmacokinetic parameter values in healthy volunteers were calculated and summarized in Table 4. The results showed that Cmax of monocarbamate bambuterol was more than double that of bambuterol or terbutaline, AUC0–96 of monocarbamate bambuterol was triple that of bambuterol and was as much as that of terbutaline. As mentioned above, the active metabolite terbutaline was slowly released by the hydrolysis of both bambuterol and monocarbamate bambuterol catalyzed by BChE. Our data indicated that the slow bioconversion mechanism of bambuterol into its parent drug terbutaline was mainly due to the interaction of BChE with monocarbamate bambuterol, rather than bambuterol as previous reported [4,5].
3.4. Application to a pharmacokinetic study in healthy volunteers
3.5. The effects of the chromatographic conditions on the retention of HILIC
The method described above was successfully applied to a pharmacokinetic study, in which plasma concentrations of bambuterol, monocarbamate bambuterol, and terbutaline in eight
HILIC has become increasingly popular as an alternative to RPLC for the analysis of polar analytes [25]. However, the retention mechanism of HILIC is complex and still poorly understood. In order to
T. Zhou et al. / J. Chromatogr. B 967 (2014) 225–234
231
Table 3 Stability data of bambuterol, monocarbamate bambuterol, and terbutaline in human plasma under various storage conditions (n = 3). Storage conditions Room temperature for 6 h Three freeze–thaw cycles 3 months at −80 ◦ C a
Added Ca (pg/mL)
Bambuterol
30.00 3000 30.00 3000 30.00 3000
Found C (pg/mL) 29.88 3034 27.04 2740 32.31 3249
Monocarbamate bambuterol RE (%) −0.4 1.1 −9.9 −8.7 7.7 8.3
RSD (%) 0.7 1.8 2.2 1.2 2.8 1.6
Found C (pg/mL) 28.72 2981 26.64 2699 33.59 3324
Terbutaline RE (%) −4.3 −0.6 −11.2 −10.0 12.0 10.8
RSD (%) 3.3 2.0 0.9 1.9 1.4 1.0
Found C (pg/mL) 28.97 2992 26.98 2697 32.32 3296
RE (%) −3.4 −0.3 −10.1 −10.1 7.7 9.9
RSD (%) 7.5 1.6 3.4 2.1 1.1 1.2
C, concentration.
Table 4 Main pharmacokinetic parameters of bambuterol, monocarbamate bambuterol, and terbutaline in healthy volunteers after a single oral dose of 10 mg bambuterol hydrochloride (n = 8). Parameters
t1/2 (h) Tmax (h) C max (ng/L) AUC0–96 (h ng/L) AUC0–∞ (h ng/L) CLz /F (L/h) Vz /F (L)
Mean ± SD Bambuterol
Monocarbamate bambuterol
Terbutaline
12.57 ± 6.99 5.06 ± 3.41 1177 ± 1123 13708 ± 4893 16472 ± 7459 721 ± 261 14005 ± 11748
15.15 ± 6.81 2.63 ± 2.66 2588 ± 1973 40715 ± 22065 42984 ± 22046 267 ± 85 5397 ± 2291
23.41 ± 9.15 5.00 ± 1.93 1275 ± 727 46976 ± 18064 51633 ± 22306 223 ± 82 6743 ± 1482
shed some light over the retention mechanism of HILIC stationary phases, the effects of chromatographic conditions on the retention of the four phenylethylamines (the three analytes and the IS) in this study were further investigated. 3.5.1. Effect of the organic phase proportion The mobile phase in HILIC consists of an aqueous–organic mixture. In this study, acetonitrile was used as the organic solvent. The effect of acetonitrile proportion on the retention of the analytes was illustrated in Fig. 4a. The results showed that the retention factor increased as the acetonitrile proportion increased for all the analytes. By increasing the acetonitrile proportion to 95%, the best separation of the four analytes was achieved, and the retention factors followed the order: salbutamol > terbutaline > monocarbamate bambuterol > bambuterol. The polar stationary phases used in HILIC such as silica are known to strongly retain water [26]. A water layer has been proven to be adsorbed onto the surface of the hydrophilic stationary phase
Fig. 3. Mean concentration–time profiles of bambuterol, monocarbamate bambuterol, and terbutaline in plasma after an oral dose of bambuterol hydrochloride tablets (10 mg) administered to the healthy volunteers (n = 8).
in HILIC columns [27]. Thus, in HILIC, the acetonitrile-rich bulk and the water-rich layer of the mobile phase form two liquid phases of different polarities, resembling a liquid–liquid partition system [23]. Therefore, the distribution of the analytes between the two phases bases on their relative partition coefficient. As a consequence, polar hydrophilic analytes are preferentially distributed into the water layer and more strongly retained. The logP of the analytes investigated follows the order: salbutamol (0.83 ± 0.40) < terbutaline (1.02 ± 0.33) < bambuterol monocarbamate (1.20 ± 0.28) < bambuterol (1.5 ± 0.36), which corresponds to the order of the retention factors observed when the acetonitrile proportion was 95%. As the water content in the mobile phase was increased, the difference in polarity between the bulk and the adsorbed layer decreased, so the difference in the retention of the analytes gradually disappeared.
3.5.2. Effect of the buffer concentration Buffers are usually added into the mobile phase to control the electrostatic interaction between the charged analytes and the stationary phase. To improve peak shapes and reproducibility, it is recommended to maintain a minimum buffer concentration in the mobile phase [28]. In this study, ammonium acetate was used due to its good solubility in the acetonitrile-rich mobile phase and compatibility with mass spectrometric detection. The effect of the buffer concentration on the retention of the analytes was illustrated in Fig. 4b, and a decrease in the retention upon increasing the buffer concentration was observed for all analytes. Increasing the buffer concentration has the general effect of weakening the electrostatic interactions between the charged analytes and the charged stationary phases [29]. In the case of electrostatic attraction, the increase of the buffer concentration leads to a decreased retention, whereas in the case of electrostatic repulsions, it leads to increased retention. In the condition of this study, the silica stationary phase was negatively charged while the analytes which have a pKa for the secondary amino group around 9.6 were positively charged. Therefore, the electrostatic interaction between the charged analytes and the charged column in this study was electrostatic attraction, and the increasing buffer concentration led to the decreased retention.
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Fig. 4. The effects of the organic phase proportion (A), the buffer concentration (B), the pH of the mobile phase (C), and the column temperature (D) on the retention of HILIC column.
3.5.3. Effect of pH of the mobile phase The primary effect of the mobile phase pH is on the charged state of the analytes. It is preferable to select the pH to bring the analytes in their charged forms which are usually more hydrophilic and more retained in HILIC than their neutral forms. The effect of pH of the mobile phase on the retention of the analytes was illustrated in Fig. 4c. A marked increase in the retention of the four analytes was observed upon increasing the pH. In the condition employed, the silanol groups on the surface of the silica stationary phase were negatively charged while the four analytes, which have a pKa for the secondary amino group around 9.6, were positively charged. It is probable that as the pH of the mobile phase increased, the silanol groups became more and more deprotonated while the ionization state of the analytes was not significantly affected. As a consequence, the electrostatic interactions of the positive-charged analytes with the stationary phase were dramatically enhanced [30]. 3.5.4. Effect of the column temperature In this study, the effect of the column temperature on the retention of the analytes was investigated in the range of 25–50 ◦ C. As shown in Fig. 4d, the column temperature had a minor impact on the retention of the analytes. However, a little decrease in retention was observed for salbutamol and terbutaline upon increasing the temperature while the retention of bambuterol and monocarbamate bambuterol showed a slight increase passing from
25 to 35 ◦ C and remained stable upon further increase of the temperature. For the compounds with high polarity such as salbutamol and terbutaline, there are several reasons to explain the decrease of the retention. The effect of the temperature was primarily related to the enthalpy of the analyte’s transfer between the mobile phase and the stationary phase. In this case, the transfer of hydrophilic analytes from the acetonitrile-rich bulk to the water layer was an exothermic process, favoring lower temperatures [31]. It has also been suggested that the pKa of basic analytes decreases upon increasing the temperature, resulting in a decrease of polarity [32]. Besides, the electrostatic interactions were postulated to be weakened at elevated temperatures, and the amount of the water layer adsorbed on the silica surface might also tend to thin as the temperature increased, thereby weakening the retention of polar compounds [33]. For the compounds with lower polarity, the situation was more complex. The first slight increase of the retention might be the result of an increase of solutes partition in the water phase adsorbed on the silica surface as the temperature increased. On the other hand, as the temperature increased, the adsorbed water layer on the silica surface became thinner, thus balancing the increase of water solubility at temperatures higher than 35 ◦ C. In summary, according to these results, chromatographic conditions affected the retention of phenylethylamines on the HILIC stationary phase followed the order: organic phase proportion > ammonium acetate concentration > mobile phase
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Fig. 5. The asymmetry factors of the analytes on the HILIC and C18 columns at 0.4 mL/min.
pH > column temperature. The retention of the four phenethylamines on the HILIC column was based on the liquid–liquid partition and the electrostatic interactions between the charged analytes and the stationary phase.
Fig. 6. The number of effective plates (Neff ) of the HILIC and C18 columns at 0.4 mL/min.
3.6. The comparison of HILIC column with C18 column In order to demonstrate the advantages over RPLC offered by the HILIC approach used in this study, an alternative RPLC method based on the same sub-2 m bridged-ethylene hybrid (BEH) material functionalized with C18 chains was optimized and the chromatographic performance of the two methods in the analysis of phenylethylamines was compared. 3.6.1. Comparison of the asymmetry factors The asymmetry factors of the four analytes obtained by using HILIC and RPLC at different flow rates were shown in Figure S1, and the asymmetry factors measured at 0.4 mL/min were shown in Fig. 5. HILIC provided asymmetry factors close to 1 for all the analytes, independently from the flow rate employed (shown in Fig. S1). RPLC provided a worse performance in all the cases (shown in Fig. 5) although the symmetry improved significantly upon increasing the flow rate (shown in Fig. S1). A similar dependence between flow rate and peak symmetry in RPLC was recently observed by Heaton [34] in the analysis of ephedrines, a related class of phenylethylamines. The results indicated that HILIC column could obtain better peak symmetry compared with C18 column in the analysis of phenethylamines. 3.6.2. Comparison of the number of effective plates The number of effective plates (Neff ) represents the column efficiency. As shown in Fig. 6, the number of effective plates was significantly higher in the HILIC column than in the C18 column, except for bambuterol. This is consistent with the lower polarity of bambuterol compared with the other three analytes, and C18 columns have usually good efficiency with medium polarity compounds. In other words, the HILIC column provided better efficiency than the C18 column for the compounds of high polarity. Figure S2 indicated that a reduction of number of effective plates in the HILIC column for all the analytes as the flow rate was increased, reflecting a loss of efficiency at high flow rates. 3.6.3. Comparison of the column back pressure The column back pressure observed for the HILIC and C18 columns was monitored at different flow rates under their optimized isocratic conditions, which were shown in Fig. 7. The results
Fig. 7. The column back pressure of the HILIC and C18 columns monitored at different flow rates.
indicated that the back pressure of both columns increased proportionally with the flow rate, and the back pressure of the HILIC column was much lower than that of the C18 column. At 0.8 mL/min, the back pressure of the C18 column reached almost 10,000 psi, which is the maximum pressure this column could bear while the HILIC column was just above 3000 psi. This difference is related to the higher aqueous content in the mobile phase used in RPLC whose higher viscosity leads to a higher back pressure, especially when sub-2 m particles are used. In contrast, the HILIC separation affords significantly lower mobile phase viscosity and lower column pressure, which permits the use of faster flow rates for increased sample throughput and longer columns for enhanced resolution. 4. Conclusions A sensitive and rapid HILIC–UPLC–MS/MS method for simultaneous determination of bambuterol and its two major metabolites monocarbamate bambuterol and terbutaline in human plasma has been developed for the first time. The established method provided the LLOQ of 10.00 pg/mL and the runtime of 4.0 min per sample, achieving the highest sensitivity and the fastest analysis
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speed compared with the previously reported methods. The calibration curves of all the analytes were linear in the range of 10.00–5000 pg/mL. This method was successfully applied to the clinical pharmacokinetic study of bambuterol in eight healthy volunteers, and the data indicated that the slow bioconversion mechanism of bambuterol into its parent drug terbutaline was mainly due to the interaction of BChE with monocarbamate bambuterol. The investigation for the effects of various chromatographic parameters on the retention of the analytes indicated that liquid–liquid partition and the electrostatic interactions played an important role in the retention of phenylethylamines on the HILIC column used in this study. Moreover, the HILIC approach showed particular advantages over RPLC including a better peak symmetry, higher efficiency, and much lower column pressure. Acknowledgements This work was supported by Major Science and Technology Project of Guangdong ProvinceFunded by Ministry of Science & Technology of Guangdong Province, China (Grant No.: 2012A080204001), the Fundamental Research Funds for the Central Universities (Grant No.: 2013ZB0010), and the Natural Science Foundation of Guangdong Province, China (Grant No.: S2013040015142). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jchromb. 2014.07.022. References [1] [2] [3] [4]
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