Research article Received: 4 July 2013,
Revised: 6 September 2013,
Accepted: 11 October 2013
Published online in Wiley Online Library: 20 November 2013
(wileyonlinelibrary.com) DOI 10.1002/bmc.3083
High-throughput salting-out-assisted homogeneous liquid–liquid extraction with acetonitrile for determination of baicalin in rat plasma with high-performance liquid chromatography Tingting Lia, Lei Zhanga*, Ling Tongb and Qiongfeng Liaoa ABSTRACT: Baicalin is the main indicator for qualitative and quantitative analysis of Scutellaria baicalensis Georgi and its prescription in vivo and in vitro. Owing to its insolubility and instability, the analysis of baicalin in biological samples is analytically challenging. Although there have been many pharmacokinetic or metabolism studies on baicalin, the current reported sample pretreatment methods are not the optimal choice with regard to absolute recovery and operation procedure. Here we report a high-throughput salting-out-assisted homogeneous liquid–liquid extraction method with acetonitrile and ammonium sulfate. Eight kinds of commonly used salts, preferred salt concentration and auxiliary solvents were investigated. The extraction efficiency in the presence of ammonium salt and auxiliary solvent (methanol) in comparison to that from the salt-free aqueous increased to above 90%. The performance of the developed pretreatment method was further evaluated through testing specificity, linearity, precision, accuracy, extraction recovery and stability. In particular, the stability investigation results proved that the operation at low temperature would no longer necessary be for salting-out-assisted homogeneous liquid–liquid extraction compared with protein precipitation, and the pretreatment method would be valuable if the compounds were unstable within matrices. Copyright © 2013 John Wiley & Sons, Ltd. Keywords: homogeneous liquid–liquid extraction; salting-out; baicalin; LC
Introduction
648
Baicalin (Fig. 1), a flavone compound isolated from Scutellariae Radix, shows activities of antibacterial, antiflammatory, free radical scavenging, antioxidant, anticancer and anxiolytic (Gao et al., 1999; Ikemoto et al., 2000; Ma et al., 2002; Kim et al., 2007, 2009). In the Chinese Pharmacopeia Commission (2010), about 40 kinds of preparations contain baicalin, which is considered as the main indicator for qualitative and quantitative analysis in vivo and in vitro. In vivo analysis, for example determination the content of baicalin in animal or human serum, urine and plasma, has been reported mainly using the techniques of HPLC-UV (Gao et al., 2003; Lai et al., 2003), HPLC-MS (Kim et al., 2006; Ran et al., 2006; Tong et al., 2012) and UPLC-MS (Ju et al., 2007; Huang et al., 2012), and the pretreatment methods are primarily liquid–liquid extraction (LLE), protein precipitation (PPT) and solid-phase extraction (SPE). Most of the reported LLE methods using acetone (Tong et al., 2012) or ethyl acetate (Kim et al., 2006) as extraction regent make the extraction rate between 60 and 70%. However, it evaporation is time-consuming, and large amounts of organic solvent are required. Moreover, nitrogen is necessary for the evaporation process because of the instability of baicalin and thus is consumed in large quantities. Zhang et al. (2009) have explored an analytical method for determination of baicalin in rat plasma by SPE with an HLB cartridge. SPE can obtain a cleaner sample and better extraction recovery (91%). However, the SPE procedure suffers from complicated
Biomed. Chromatogr. 2014; 28: 648–653
operation, poor inter-assay reproducibility and high cost. In the case of unstable compounds, such as baicalin, the relatively long column activation time, sample loading time and elution time make SPE not suitable for high-throughput extraction. There has also been much pharmacokinetic and metabolism research on baicalin adopting PPT as the pretreatment method (Ran et al., 2006; Huang et al., 2012). However, this not only increases the difficulty of chromatographic separation and increases the matrix effect of LC-MS analysis, but also leads to persistent degradation of baicalin in the biological matrices because most nonprotein components of plasma and peptides cannot be precipitated completely and the supernatant still includes many soluble sub-
* Correspondence to: Lei Zhang, College of Chinese Traditional Medicine, Guangzhou University of Traditional Chinese Medicine, Guangzhou 510006, Guangdong, People’s Republic of China. Email: [email protected] a
College of Chinese Traditional Medicine, Guangzhou University of Traditional Chinese Medicine, Guangzhou 510006, Guangdong, People’s Republic of China
b
Institute of Pharmaceutical Analysis, Tianjin Tasly Group Co. Ltd, Tianjin 300410, People’s Republic of China Abbreviations used: BL, baicalin; HLLE, homogeneous liquid–liquid extraction; LLE, liquid–liquid extraction; PPT, protein precipitation; SALLE, salting-out-assisted homogeneous liquid–liquid extraction; SPE, solid-phase extraction; WL, wogonoside.
Copyright © 2013 John Wiley & Sons, Ltd.
Salting-out-assisted homogeneous liquid–liquid extraction of baicalin
Figure 1. Chemical structure of baicalin and wogonoside.
Biomed. Chromatogr. 2014; 28: 648–653
Experimental Chemicals and reagents HPLC-grade methanol and acetonitrile were purchased from Merk (Darmstadt, Germany); Ammonium sulfate and hydrochloric acid were analytical grade and were obtained from Damao Inc. (Tianjin, China). The standard of baicalin was provided by National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Wogonoside (chemical structures shown in Fig. 1), used as internal standard, was provided by Xi’an Rongsheng Biological Technology Co. Ltd (Xi’an, China). Blank plasma samples were collected by centrifuging the blood sample of healthy Sprague–Dawley rats and stored at 20°C. Deionized water was used throughout the experiments.
Apparatus and chromatographic conditions A Shimadzu LC-20 A liquid chromatography system (Shimadzu Co. Japan), equipped with a quaternary solvent delivery system, an autosampler and DAD detector, was used. An Ultimate® XB-C18 column (4.6 × 250 mm i.d., 5 μm) connected to a Phenomenex Luna C18 guard column (4.6 × 2 mm i.d., 5 μm) at ambient temperature was applied for all analyses. UV absorption was set at 275 nm. The mobile phase consisted of (A) acetonitrile and (B) 0.1% aqueous formic acid (v/v) using an isocratic elution of 28% A and 72% B. The flow rate was 1.0 mL/min and aliquots of 20 μL were injected.
Preparation of standard solution The stock solution of baicalin (BL) and wogonoside (WL) were prepared by dissolving a proper amount of BL and WL in methanol to furnish nominal concentrations of 100 and 62 μg/mL. A series of standard working solutions of BL with concentration 0.20–100 μg/mL were obtained by diluting their stock solution with methanol. Saturated aqueous solutions of magnesium sulfate, sodium sulfate, sodium chloride, magnesium chloride, ammonium chloride, ammonium acetate amd ammonium formate were prepared by directly dissolving salts in deionized water and were kept at least 24 h (with occasional shaking) in contact with excess of salts before use. All solutions were kept at 4°C.
Investigation of salting-out reagent Saturated magnesium sulfate, sodium sulfate, sodium chloride, magnesium chloride, ammonium chloride, ammonium acetate and ammonium formate were investigated to select the preferred salting-out reagent. On the basis of the above investigated results, the optimal concentration of the selected salt was further tested.
Copyright © 2013 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/bmc
649
stances which can affect chromatography separation or even the stability of the target compound. Although PPT has the characteristics of easy operation and high extraction recovery for most compounds, it only obtained 66.1 ± 3.1% absolute extraction recovery for baicalin extracted from rat plasma in our previous research, which did not adopt the relative extraction recovery to evaluate the assay as previous literature does (Feng et al., 2012), taking the stability of baicalin into account. In addition, in order to inhibit the biotransformation of baicalin, the chemical reagents and apparatus used for sample preparation are best precooled, and the operation should be done in ice bath. These requirements for the treatment process increase the difficulty of the operation. In conclusion, the current reported sample pretreatment methods are not the optimal choice for extraction of baicalin from biological samples. Homogeneous liquid–liquid extraction (HLLE) is a new extraction method using the phase separation phenomenon to make originally homogeneous mixed solution (generally water and water soluble organic solvent) stratified. Salting-out-assisted homogeneous liquid–liquid extraction method (SALLE) is one of the most important HLLE methods that involves a simple one-step solvent extraction of analytes from biological samples (i.e. whole blood, plasma and urine) followed by salting out the water-miscible organic solvent by adding inorganic salt, like magnesium sulfate, ammonium sulfate, sodium chloride, calcium chloride, potassium carbonate, calcium sulfate or magnesium sulfate (Li et al., 2010). In terms of this method, the use of organic solvent to induce protein precipitation is a simple and effective approach routinely employed in clinical/biomedical laboratories for sample clean-up; meanwhile the addition of inorganic salt for salting out may both greatly enhance the extraction efficiency of the analytes from aqueous solution and further purify the samples. SALLE has the advantages of high extraction rate, good reproducibility and simple operation process, and is particularly suitable for extracting polar and multi-charged compounds. In recent years, SALLE has increasingly attracted researchers’ attention in in vivo drug analysis (Zhang et al., 2009, 2010). Baicalin, a flavanoid glycoside, has the nature of poor solubility, easy biological degradation, pH- and temperature-dependent stability and relatively large polarity. Considering the characteristics of baicalin and SALLE, We speculated that SALLE with the acidic inorganic salt as salting-out regent may be more suitable for extracting baicalin. The characteristics of short processing time, double protein precipitation function to provide a cleaner matrix, and acid salting-out reagent to meanwhile stabilize baicalin could overcome the disadvantages of conventional biological sample
processing method. So far there is only one study reporting the aqueous two-phase extraction method (Zhao et al., 2008), having a similar principle to SALLE, to extract baicalin from Scutellaria baicalensis. However, the processed sample could not be injected into the liquid chromatography (LC) system for analysis because the surfactant of polyethylene glycol was selected as a stratification promotor in the study. Based on the above, a rapid, facile, and convenient pretreatment method for baicalin in biological samples has been developed which provides a higher extraction rate, cleaner matrix and more stable medium. Our study has proved that SALLE, perfectly combining sample clean-up (e.g. acetonitrile deproteinization) with enrichment (via salting-out extraction), is an effective and preferred preparation approach for baicalin extraction from biological samples, and has been applied in LC analysis.
T. Li et al. Sample preparation Acidified blank rat plasma 100 μL (per milliliter rat plasma added with 20 μL concentrated hydrochloric acid) was spiked with 25 μL of IS and 25 μL of BL standard working solutions in a 1.0 mL Eppendorf tube. After adding 200 μL of CH3CN and 150 μL of saturated ammonium sulfate (salting-out solution), the mixture was then vortex mixed for 3 min. Then the Eppendorf tube was centrifuged at 12,000 rpm for 5 min to induce phase separation. The upper acetonitrile phase was transferred to a clean test tube and evaporated to dryness under a stream of nitrogen. The residue was reconstituted in 100 μL methanol and stored at 4°C until use.
chosen as the phase-separation salt. Furthermore, the optimal concentration was tested, and the results (Table 1) showed that the addition of ionic strength promoted the transport of analytes into the organic phase, so the extraction efficiency was increased by increasing (NH4)2SO4 up to saturated concentration. In addition, a rapid and clear phase separation between acetonitrile and aqueous solution could easily be achieved on the condition of saturated (NH4)2SO4 as salting-out reagent.
Addition of auxiliary solvent Preparation calibration of quality control samples Calibration samples were made at concentrations of 0.05, 0.1, 0.5, 2.0, 5.0 and 25.0 μg/mL (equal to plasma concentration). Quality control samples (QC samples) were prepared at low, medium and high concentrations of 0.1, 2.0 and 20.0 μg/mL. The samples were extracted following the procedure described above.
Method validation The method was validated with reference to the Guidance for Industry: Bioanalytical Method Validation (US Food and Drug Administration, 2001). Specificity, matrix effect, linearity, precision, accuracy, extraction recovery and stability were evaluated in the method validation.
Results and discussion Acidification of plasma samples According to the literature reported, maintaining a stable acidic pH in plasma samples is essential when attempting to collect, process and store biological samples of BL (Qiu et al., 2004; Xing et al., 2005). In the present study, in order to make the matrix of simulation plasma samples more similar to the real ones, the blank rat plasma was immediately acidified with concentrated hydrochloric acid to pH 1–2 (per milliliter rat plasma was added 20 μL concentrated hydrochloric acid) and stored at 20°C until use. In addition, acidification of plasma could increase the distribution ratio of BL in acetonitrile and thus improve the extraction efficiency owing to the mechanism that acidic medium could inhibit the ionization of it. Salting-out reagent selection
650
Although it is well known that acetonitrile is miscible with water in any proportion at room temperature, lowering the temperature or adding salt significantly reduces the mutual miscibility, even resulting in phase separation of acetonitrile from aqueous phase. In order to obtain the best phase separation effect and optimal extraction efficiency, the influence of various salts [MgSO4, (NH4)2SO4, Na2SO4, NaCl, MgCl2, NH4Cl, NH4Ac and HCOONH4] and the suitable concentration of selected one were investigated. However, in our present study the complete phase-separation could not be obtained under the condition of employing NaCl, NH4Cl, NH4Ac, Na2SO4 and HCOONH4 as salting-out reagent. The phenomenon resulted from the existence of small percentage of methanol in the homogeneous liquid–liquid extraction system, which made phase separation of acetonitrile from water difficult. On the function of salting-out reagents of MgCl2, MgSO4 and (NH4)2SO4, the extraction efficiency of baicalin from aqueous solutions with acetonitrile was 78.0, 82.3 and 96.0%, respectively. As a result, (NH4)2SO4 was
wileyonlinelibrary.com/journal/bmc
As for LC analysis, acetonitrile was the most commonly used extracted solvent employed by SALLE. Additionally, the baicalin was stable in plasma precipitated by acetonitrile instead of methanol (Xing et al., 2005). However, we found that the extraction recovery of baicalin could only reach 61.2% if the homogeneous system simply consisted of acetonitrile–water (containing salt-out reagent). Therefore we added trace methanol into the homogeneous system (methonal–acetonitrile–water, containing salt-out reagent) to increase the recovery. The results suggested that the extraction efficiency of baicalin could be greatly improved with the addition of methanol. However, when the proportion of methanol in the system exceeded 20% (v/v), phase separation became impossible. Finally, 10% (v/v) contained in the homogeneous system was recommended as the optimal proportion. However, it is worth noting that the addition of methanol also promoted the salt into organic layer, which would result in the mismatching with chromatographic system and broaden the peaks when the extracted layer was directly injected into the LC. Therefore, evaporation to dryness and then reconstitution in organic solvent were indispensable. Because the amount of the final extracted layer was only 1/10–1/20 that processed by LLE, the duration was negligible.
Assay validation Selectivity. In order to investigate whether the method was free from interference of endogenous matrix, a total of six different lots from the rat plasma matrix were used for the interference evaluation. Six lots went through a salting-out-assisted homogeneous liquid–liquid extraction procedure with the addition of baicalin and internal standard. A typical chromatogram is shown in Fig. 2. Under optimum chromatographic conditions, the baicalin and IS peak were well resolved from plasma endogenous peaks, and no interference was observed. Matrix effect. In order to demonstrate that the assay performance is independent from the sample matrix, samples similar in concentration to the low QC were prepared using six different lots of plasma. The concentration of the evaluation samples was measured using the calibration curve. Calculated mean bias was between 2.9 and 5.1%. There was no matrix effect difference observed from SALLE with acetonitrile.
Table 1. Effect of the phase separator reagent concentration (NH4)2SO4 on the extraction efficiency of baicalin Concentration (g/mL) Recovery (%)
Copyright © 2013 John Wiley & Sons, Ltd.
0.3
0.4
0.5
79.37
88.2
92.8
0.6 Saturated 92.1
96.0
Biomed. Chromatogr. 2014; 28: 648–653
Salting-out-assisted homogeneous liquid–liquid extraction of baicalin
Figure 2. Representative chromatograms of baicalin (a) and IS (b) in rat plasma. (A) Blank plasma sample; (B) plasma sample of lower limit of quantitation (50 ng/mL); (C) plasma sample of upper limit of quantitation (25 g/mL).
Calibration curve and LOQ. Calibration standards were prepared by spiking blank rat plasma with the appropriate amount of baicalin and IS solution to yield the final concentrations of 0.05, 0.1, 0.5, 2.0, 5.0 and 25.00 μg/mL for baicalin and 62.00 μg/mL for internal standard in duplicate and analyzed on
Calibration curves
r
y = 0.08478x - 0.00135 y = 0.07891x - 0.00009 y = 0.08491x - 0.00136
0.9963 0.9925 0.9964
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0.1 2 20
Copyright © 2013 John Wiley & Sons, Ltd.
Intra-day (n = 6) Precision (RSD, %)
Accuracy (RE, %)
4.6 6.6 4.7
3.0 4.8 5.5
Inter-day (n = 3) Precision (RSD, %) 12.3 4.3 10.7
Accuracy (RE, %) 5.1 3.8 5.9
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651
1 2 3
Table 3. Validation of the intra-day and inter-day assays Concentration (μg/mL)
Table 2. Calibration curves on 3 different days (n = 2) Days
three consective days. The calibration curves were constructed by plotting the peak area ratio of BL to IS vs their respective concentrations in rat plasma. A weighted (1/x2) linear least-squares
T. Li et al. Table 4. Stability results for baicalin in rat plasma by salting-out-assisted homogeneous liquid–liquid extraction (SALLE) (n = 3) Spiked (μg/mL) 0.10 2.0 20.0
4°C for 24 h
25°C for 24 h
Measured
RE (%)
RSD (%)
Measured
0.107 ± 0.0034 2.10 ± 0.12 20.37 ± 0.35
6.6 5.1 1.9
3.2 5.8 1.7
0.098 ± 0.010 1.74 ± 0.024 17.79 ± 0.32
regression method was used to determine the slope, intercept and correlation coefficient. The LLOQ was determined in accordance to the baseline noise, considering a signal-to-noise ratio of 10:1. The results indicated that the calibration curves showed good linearity over the tested range. The LLOQ for baicalin was 0.05 μg/mL with coefficient of variation 12.3%. The results are shown in Table 2. Accuracy and precision. The accuracy and precision of the assay were evaluated by analyzing QC samples at low (0.1 μg/mL), medium (2 μg/mL) and high (20 μg/mL) concentrations within one day and on three independent days. As Table 3 shows, the accuracy ranged from 6.9 to 5.1% with intra-day and inter-day precision
Revised: 6 September 2013,
Accepted: 11 October 2013
Published online in Wiley Online Library: 20 November 2013
(wileyonlinelibrary.com) DOI 10.1002/bmc.3083
High-throughput salting-out-assisted homogeneous liquid–liquid extraction with acetonitrile for determination of baicalin in rat plasma with high-performance liquid chromatography Tingting Lia, Lei Zhanga*, Ling Tongb and Qiongfeng Liaoa ABSTRACT: Baicalin is the main indicator for qualitative and quantitative analysis of Scutellaria baicalensis Georgi and its prescription in vivo and in vitro. Owing to its insolubility and instability, the analysis of baicalin in biological samples is analytically challenging. Although there have been many pharmacokinetic or metabolism studies on baicalin, the current reported sample pretreatment methods are not the optimal choice with regard to absolute recovery and operation procedure. Here we report a high-throughput salting-out-assisted homogeneous liquid–liquid extraction method with acetonitrile and ammonium sulfate. Eight kinds of commonly used salts, preferred salt concentration and auxiliary solvents were investigated. The extraction efficiency in the presence of ammonium salt and auxiliary solvent (methanol) in comparison to that from the salt-free aqueous increased to above 90%. The performance of the developed pretreatment method was further evaluated through testing specificity, linearity, precision, accuracy, extraction recovery and stability. In particular, the stability investigation results proved that the operation at low temperature would no longer necessary be for salting-out-assisted homogeneous liquid–liquid extraction compared with protein precipitation, and the pretreatment method would be valuable if the compounds were unstable within matrices. Copyright © 2013 John Wiley & Sons, Ltd. Keywords: homogeneous liquid–liquid extraction; salting-out; baicalin; LC
Introduction
648
Baicalin (Fig. 1), a flavone compound isolated from Scutellariae Radix, shows activities of antibacterial, antiflammatory, free radical scavenging, antioxidant, anticancer and anxiolytic (Gao et al., 1999; Ikemoto et al., 2000; Ma et al., 2002; Kim et al., 2007, 2009). In the Chinese Pharmacopeia Commission (2010), about 40 kinds of preparations contain baicalin, which is considered as the main indicator for qualitative and quantitative analysis in vivo and in vitro. In vivo analysis, for example determination the content of baicalin in animal or human serum, urine and plasma, has been reported mainly using the techniques of HPLC-UV (Gao et al., 2003; Lai et al., 2003), HPLC-MS (Kim et al., 2006; Ran et al., 2006; Tong et al., 2012) and UPLC-MS (Ju et al., 2007; Huang et al., 2012), and the pretreatment methods are primarily liquid–liquid extraction (LLE), protein precipitation (PPT) and solid-phase extraction (SPE). Most of the reported LLE methods using acetone (Tong et al., 2012) or ethyl acetate (Kim et al., 2006) as extraction regent make the extraction rate between 60 and 70%. However, it evaporation is time-consuming, and large amounts of organic solvent are required. Moreover, nitrogen is necessary for the evaporation process because of the instability of baicalin and thus is consumed in large quantities. Zhang et al. (2009) have explored an analytical method for determination of baicalin in rat plasma by SPE with an HLB cartridge. SPE can obtain a cleaner sample and better extraction recovery (91%). However, the SPE procedure suffers from complicated
Biomed. Chromatogr. 2014; 28: 648–653
operation, poor inter-assay reproducibility and high cost. In the case of unstable compounds, such as baicalin, the relatively long column activation time, sample loading time and elution time make SPE not suitable for high-throughput extraction. There has also been much pharmacokinetic and metabolism research on baicalin adopting PPT as the pretreatment method (Ran et al., 2006; Huang et al., 2012). However, this not only increases the difficulty of chromatographic separation and increases the matrix effect of LC-MS analysis, but also leads to persistent degradation of baicalin in the biological matrices because most nonprotein components of plasma and peptides cannot be precipitated completely and the supernatant still includes many soluble sub-
* Correspondence to: Lei Zhang, College of Chinese Traditional Medicine, Guangzhou University of Traditional Chinese Medicine, Guangzhou 510006, Guangdong, People’s Republic of China. Email: [email protected] a
College of Chinese Traditional Medicine, Guangzhou University of Traditional Chinese Medicine, Guangzhou 510006, Guangdong, People’s Republic of China
b
Institute of Pharmaceutical Analysis, Tianjin Tasly Group Co. Ltd, Tianjin 300410, People’s Republic of China Abbreviations used: BL, baicalin; HLLE, homogeneous liquid–liquid extraction; LLE, liquid–liquid extraction; PPT, protein precipitation; SALLE, salting-out-assisted homogeneous liquid–liquid extraction; SPE, solid-phase extraction; WL, wogonoside.
Copyright © 2013 John Wiley & Sons, Ltd.
Salting-out-assisted homogeneous liquid–liquid extraction of baicalin
Figure 1. Chemical structure of baicalin and wogonoside.
Biomed. Chromatogr. 2014; 28: 648–653
Experimental Chemicals and reagents HPLC-grade methanol and acetonitrile were purchased from Merk (Darmstadt, Germany); Ammonium sulfate and hydrochloric acid were analytical grade and were obtained from Damao Inc. (Tianjin, China). The standard of baicalin was provided by National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Wogonoside (chemical structures shown in Fig. 1), used as internal standard, was provided by Xi’an Rongsheng Biological Technology Co. Ltd (Xi’an, China). Blank plasma samples were collected by centrifuging the blood sample of healthy Sprague–Dawley rats and stored at 20°C. Deionized water was used throughout the experiments.
Apparatus and chromatographic conditions A Shimadzu LC-20 A liquid chromatography system (Shimadzu Co. Japan), equipped with a quaternary solvent delivery system, an autosampler and DAD detector, was used. An Ultimate® XB-C18 column (4.6 × 250 mm i.d., 5 μm) connected to a Phenomenex Luna C18 guard column (4.6 × 2 mm i.d., 5 μm) at ambient temperature was applied for all analyses. UV absorption was set at 275 nm. The mobile phase consisted of (A) acetonitrile and (B) 0.1% aqueous formic acid (v/v) using an isocratic elution of 28% A and 72% B. The flow rate was 1.0 mL/min and aliquots of 20 μL were injected.
Preparation of standard solution The stock solution of baicalin (BL) and wogonoside (WL) were prepared by dissolving a proper amount of BL and WL in methanol to furnish nominal concentrations of 100 and 62 μg/mL. A series of standard working solutions of BL with concentration 0.20–100 μg/mL were obtained by diluting their stock solution with methanol. Saturated aqueous solutions of magnesium sulfate, sodium sulfate, sodium chloride, magnesium chloride, ammonium chloride, ammonium acetate amd ammonium formate were prepared by directly dissolving salts in deionized water and were kept at least 24 h (with occasional shaking) in contact with excess of salts before use. All solutions were kept at 4°C.
Investigation of salting-out reagent Saturated magnesium sulfate, sodium sulfate, sodium chloride, magnesium chloride, ammonium chloride, ammonium acetate and ammonium formate were investigated to select the preferred salting-out reagent. On the basis of the above investigated results, the optimal concentration of the selected salt was further tested.
Copyright © 2013 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/bmc
649
stances which can affect chromatography separation or even the stability of the target compound. Although PPT has the characteristics of easy operation and high extraction recovery for most compounds, it only obtained 66.1 ± 3.1% absolute extraction recovery for baicalin extracted from rat plasma in our previous research, which did not adopt the relative extraction recovery to evaluate the assay as previous literature does (Feng et al., 2012), taking the stability of baicalin into account. In addition, in order to inhibit the biotransformation of baicalin, the chemical reagents and apparatus used for sample preparation are best precooled, and the operation should be done in ice bath. These requirements for the treatment process increase the difficulty of the operation. In conclusion, the current reported sample pretreatment methods are not the optimal choice for extraction of baicalin from biological samples. Homogeneous liquid–liquid extraction (HLLE) is a new extraction method using the phase separation phenomenon to make originally homogeneous mixed solution (generally water and water soluble organic solvent) stratified. Salting-out-assisted homogeneous liquid–liquid extraction method (SALLE) is one of the most important HLLE methods that involves a simple one-step solvent extraction of analytes from biological samples (i.e. whole blood, plasma and urine) followed by salting out the water-miscible organic solvent by adding inorganic salt, like magnesium sulfate, ammonium sulfate, sodium chloride, calcium chloride, potassium carbonate, calcium sulfate or magnesium sulfate (Li et al., 2010). In terms of this method, the use of organic solvent to induce protein precipitation is a simple and effective approach routinely employed in clinical/biomedical laboratories for sample clean-up; meanwhile the addition of inorganic salt for salting out may both greatly enhance the extraction efficiency of the analytes from aqueous solution and further purify the samples. SALLE has the advantages of high extraction rate, good reproducibility and simple operation process, and is particularly suitable for extracting polar and multi-charged compounds. In recent years, SALLE has increasingly attracted researchers’ attention in in vivo drug analysis (Zhang et al., 2009, 2010). Baicalin, a flavanoid glycoside, has the nature of poor solubility, easy biological degradation, pH- and temperature-dependent stability and relatively large polarity. Considering the characteristics of baicalin and SALLE, We speculated that SALLE with the acidic inorganic salt as salting-out regent may be more suitable for extracting baicalin. The characteristics of short processing time, double protein precipitation function to provide a cleaner matrix, and acid salting-out reagent to meanwhile stabilize baicalin could overcome the disadvantages of conventional biological sample
processing method. So far there is only one study reporting the aqueous two-phase extraction method (Zhao et al., 2008), having a similar principle to SALLE, to extract baicalin from Scutellaria baicalensis. However, the processed sample could not be injected into the liquid chromatography (LC) system for analysis because the surfactant of polyethylene glycol was selected as a stratification promotor in the study. Based on the above, a rapid, facile, and convenient pretreatment method for baicalin in biological samples has been developed which provides a higher extraction rate, cleaner matrix and more stable medium. Our study has proved that SALLE, perfectly combining sample clean-up (e.g. acetonitrile deproteinization) with enrichment (via salting-out extraction), is an effective and preferred preparation approach for baicalin extraction from biological samples, and has been applied in LC analysis.
T. Li et al. Sample preparation Acidified blank rat plasma 100 μL (per milliliter rat plasma added with 20 μL concentrated hydrochloric acid) was spiked with 25 μL of IS and 25 μL of BL standard working solutions in a 1.0 mL Eppendorf tube. After adding 200 μL of CH3CN and 150 μL of saturated ammonium sulfate (salting-out solution), the mixture was then vortex mixed for 3 min. Then the Eppendorf tube was centrifuged at 12,000 rpm for 5 min to induce phase separation. The upper acetonitrile phase was transferred to a clean test tube and evaporated to dryness under a stream of nitrogen. The residue was reconstituted in 100 μL methanol and stored at 4°C until use.
chosen as the phase-separation salt. Furthermore, the optimal concentration was tested, and the results (Table 1) showed that the addition of ionic strength promoted the transport of analytes into the organic phase, so the extraction efficiency was increased by increasing (NH4)2SO4 up to saturated concentration. In addition, a rapid and clear phase separation between acetonitrile and aqueous solution could easily be achieved on the condition of saturated (NH4)2SO4 as salting-out reagent.
Addition of auxiliary solvent Preparation calibration of quality control samples Calibration samples were made at concentrations of 0.05, 0.1, 0.5, 2.0, 5.0 and 25.0 μg/mL (equal to plasma concentration). Quality control samples (QC samples) were prepared at low, medium and high concentrations of 0.1, 2.0 and 20.0 μg/mL. The samples were extracted following the procedure described above.
Method validation The method was validated with reference to the Guidance for Industry: Bioanalytical Method Validation (US Food and Drug Administration, 2001). Specificity, matrix effect, linearity, precision, accuracy, extraction recovery and stability were evaluated in the method validation.
Results and discussion Acidification of plasma samples According to the literature reported, maintaining a stable acidic pH in plasma samples is essential when attempting to collect, process and store biological samples of BL (Qiu et al., 2004; Xing et al., 2005). In the present study, in order to make the matrix of simulation plasma samples more similar to the real ones, the blank rat plasma was immediately acidified with concentrated hydrochloric acid to pH 1–2 (per milliliter rat plasma was added 20 μL concentrated hydrochloric acid) and stored at 20°C until use. In addition, acidification of plasma could increase the distribution ratio of BL in acetonitrile and thus improve the extraction efficiency owing to the mechanism that acidic medium could inhibit the ionization of it. Salting-out reagent selection
650
Although it is well known that acetonitrile is miscible with water in any proportion at room temperature, lowering the temperature or adding salt significantly reduces the mutual miscibility, even resulting in phase separation of acetonitrile from aqueous phase. In order to obtain the best phase separation effect and optimal extraction efficiency, the influence of various salts [MgSO4, (NH4)2SO4, Na2SO4, NaCl, MgCl2, NH4Cl, NH4Ac and HCOONH4] and the suitable concentration of selected one were investigated. However, in our present study the complete phase-separation could not be obtained under the condition of employing NaCl, NH4Cl, NH4Ac, Na2SO4 and HCOONH4 as salting-out reagent. The phenomenon resulted from the existence of small percentage of methanol in the homogeneous liquid–liquid extraction system, which made phase separation of acetonitrile from water difficult. On the function of salting-out reagents of MgCl2, MgSO4 and (NH4)2SO4, the extraction efficiency of baicalin from aqueous solutions with acetonitrile was 78.0, 82.3 and 96.0%, respectively. As a result, (NH4)2SO4 was
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As for LC analysis, acetonitrile was the most commonly used extracted solvent employed by SALLE. Additionally, the baicalin was stable in plasma precipitated by acetonitrile instead of methanol (Xing et al., 2005). However, we found that the extraction recovery of baicalin could only reach 61.2% if the homogeneous system simply consisted of acetonitrile–water (containing salt-out reagent). Therefore we added trace methanol into the homogeneous system (methonal–acetonitrile–water, containing salt-out reagent) to increase the recovery. The results suggested that the extraction efficiency of baicalin could be greatly improved with the addition of methanol. However, when the proportion of methanol in the system exceeded 20% (v/v), phase separation became impossible. Finally, 10% (v/v) contained in the homogeneous system was recommended as the optimal proportion. However, it is worth noting that the addition of methanol also promoted the salt into organic layer, which would result in the mismatching with chromatographic system and broaden the peaks when the extracted layer was directly injected into the LC. Therefore, evaporation to dryness and then reconstitution in organic solvent were indispensable. Because the amount of the final extracted layer was only 1/10–1/20 that processed by LLE, the duration was negligible.
Assay validation Selectivity. In order to investigate whether the method was free from interference of endogenous matrix, a total of six different lots from the rat plasma matrix were used for the interference evaluation. Six lots went through a salting-out-assisted homogeneous liquid–liquid extraction procedure with the addition of baicalin and internal standard. A typical chromatogram is shown in Fig. 2. Under optimum chromatographic conditions, the baicalin and IS peak were well resolved from plasma endogenous peaks, and no interference was observed. Matrix effect. In order to demonstrate that the assay performance is independent from the sample matrix, samples similar in concentration to the low QC were prepared using six different lots of plasma. The concentration of the evaluation samples was measured using the calibration curve. Calculated mean bias was between 2.9 and 5.1%. There was no matrix effect difference observed from SALLE with acetonitrile.
Table 1. Effect of the phase separator reagent concentration (NH4)2SO4 on the extraction efficiency of baicalin Concentration (g/mL) Recovery (%)
Copyright © 2013 John Wiley & Sons, Ltd.
0.3
0.4
0.5
79.37
88.2
92.8
0.6 Saturated 92.1
96.0
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Salting-out-assisted homogeneous liquid–liquid extraction of baicalin
Figure 2. Representative chromatograms of baicalin (a) and IS (b) in rat plasma. (A) Blank plasma sample; (B) plasma sample of lower limit of quantitation (50 ng/mL); (C) plasma sample of upper limit of quantitation (25 g/mL).
Calibration curve and LOQ. Calibration standards were prepared by spiking blank rat plasma with the appropriate amount of baicalin and IS solution to yield the final concentrations of 0.05, 0.1, 0.5, 2.0, 5.0 and 25.00 μg/mL for baicalin and 62.00 μg/mL for internal standard in duplicate and analyzed on
Calibration curves
r
y = 0.08478x - 0.00135 y = 0.07891x - 0.00009 y = 0.08491x - 0.00136
0.9963 0.9925 0.9964
Biomed. Chromatogr. 2014; 28: 648–653
0.1 2 20
Copyright © 2013 John Wiley & Sons, Ltd.
Intra-day (n = 6) Precision (RSD, %)
Accuracy (RE, %)
4.6 6.6 4.7
3.0 4.8 5.5
Inter-day (n = 3) Precision (RSD, %) 12.3 4.3 10.7
Accuracy (RE, %) 5.1 3.8 5.9
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1 2 3
Table 3. Validation of the intra-day and inter-day assays Concentration (μg/mL)
Table 2. Calibration curves on 3 different days (n = 2) Days
three consective days. The calibration curves were constructed by plotting the peak area ratio of BL to IS vs their respective concentrations in rat plasma. A weighted (1/x2) linear least-squares
T. Li et al. Table 4. Stability results for baicalin in rat plasma by salting-out-assisted homogeneous liquid–liquid extraction (SALLE) (n = 3) Spiked (μg/mL) 0.10 2.0 20.0
4°C for 24 h
25°C for 24 h
Measured
RE (%)
RSD (%)
Measured
0.107 ± 0.0034 2.10 ± 0.12 20.37 ± 0.35
6.6 5.1 1.9
3.2 5.8 1.7
0.098 ± 0.010 1.74 ± 0.024 17.79 ± 0.32
regression method was used to determine the slope, intercept and correlation coefficient. The LLOQ was determined in accordance to the baseline noise, considering a signal-to-noise ratio of 10:1. The results indicated that the calibration curves showed good linearity over the tested range. The LLOQ for baicalin was 0.05 μg/mL with coefficient of variation 12.3%. The results are shown in Table 2. Accuracy and precision. The accuracy and precision of the assay were evaluated by analyzing QC samples at low (0.1 μg/mL), medium (2 μg/mL) and high (20 μg/mL) concentrations within one day and on three independent days. As Table 3 shows, the accuracy ranged from 6.9 to 5.1% with intra-day and inter-day precision
