Talanta 131 (2015) 603–608
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Dispersive solvent-free ultrasound-assisted ionic liquid dispersive liquid–liquid microextraction coupled with HPLC for determination of ulipristal acetate Aiqin Gong a,b, Xiashi Zhu a,n a b
College of Chemistry & Chemical Engineering, Yangzhou University, Yangzhou 225002, China Yangzhou Polytechnic Institute, Yangzhou 225127, China
art ic l e i nf o
a b s t r a c t
Article history: Received 20 June 2014 Received in revised form 30 July 2014 Accepted 6 August 2014 Available online 23 August 2014
In this paper, a simple and efficient ultrasound-assisted ionic liquid dispersive liquid–liquid microextraction (UA IL-DLLME) coupled with high-performance liquid chromatography for the analysis of ulipristal acetate (UPA) was developed. UPA could be easily migrated into 1-octyl-3-methylimidazolium hexafluorophosphate [C8mimPF6] IL phase without dispersive solvent. The research of extraction mechanism showed that hydrophobic interaction force played a key role in the IL-DLLME. Several important parameters affecting the extraction recovery were optimized. Under the optimized conditions, 25-fold enrichment factor was obtained and the limit of detection (LOD) was 6.8 ng mL 1 (tablet) or 9.3 ng mL 1 (serum) at a signal-to-noise ratio of 3. The calibration curve was linear over the range of 0.03–6.0 mg mL 1. The proposed method was successfully applied to the UPA tablets and the real mice serum samples. & 2014 Elsevier B.V. All rights reserved.
Keywords: Ulipristal acetate Ionic liquid Liquid–liquid microextraction HPLC
1. Introduction Ulipristal acetate [17α-acetoxy-11β-(4-N,N-dimethyl aminophenyl)-19-norpregna-4,9-diene-3,20-dione] (UPA), a selective progesterone receptor modulator (Fig. 1), can prevent unintended pregnancy by delaying ovulation for up to five days after contraceptive failure. In August 2010, UPA had gained the FDA’s approval for use as an oral emergency contraception tablet in the U.S. with trade name Ella. Until now it is found that Ella may cause serious side effects including abdominal pain, menstrual disorder, headache, nausea and so on [1]. In addition, the study for UPA to treat contraceptive gynecological indications (fibroma uteri, adenomyosis) and Cushing’s syndrome is in progress [2]. In view of safe medication and investigating pharmaceutical dynamics of drugs, a simple, sensitive analytical procedure is needed to determine UPA in pharmaceutical formulation and in biological fluids. HPLC has been used to determine UPA in bulk [3]. But to the best of our knowledge, there were few literatures to analyze UPA in biological samples. An appropriate preconcentration/separation method should be developed due to matrix interference and low concentration of analytes in real biological samples before analysis [4]. In recent years, many preconcentration/separation steps have been oriented
n
Corresponding author. Tel./fax: þ 86 514 7975244. E-mail addresses: [email protected], [email protected] (X. Zhu).
http://dx.doi.org/10.1016/j.talanta.2014.08.021 0039-9140/& 2014 Elsevier B.V. All rights reserved.
toward the fast development of simplification and miniaturization. In particular, the use of alternative non-contaminant and nontoxic solvents instead of high quantities of organic solvents is preferred during preconcentration/separation. Solid phase microextraction (SPME) and liquid phase microextraction (LPME) have been extensively used to preconcentration/separation analytes in complex matrix with their high ability of sample clean up and analyte preconcentration, and low consumption of solvents. SPME would required a specific device loaded with certain adsorption material as well as a high-pressure delivery system that would be relatively expensive [4]. Moreover the operation of LPME is simpler and faster than that of SPME (which includes adsorption progress and desorption progress). Dispersive liquid–liquid microextraction (DLLME), developed by Assadi and co-workers in 2006 [5], is a miniaturized form of liquid phase extraction that employs microliter volumes of extraction solvent. Compared with other microextraction techniques, the advantages of DLLME are simplicity of operation, rapidness, accuracy, and it has been extensively applied in drugs analysis [4,6–8]. Extraction solvents (such as tetrachloroethylene [9], chlorobenzene [10] and carbon tetrachloride [11]) and dispersive solvents (such as methanol [12,13], acetone [9,10] and acetonitrile [11,14]) are usually used in DLLME. Because of the relatively high toxicity of these conventional chlorinated extraction solvents, developing environment-friendly “green” extraction solvents has inspired the great interest of examiners. Ionic liquids (IL) has been more and more used as extraction solvent in DLLME (IL-DLLME)
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Factory, China) was used to accelerate the phase-separation process. The microextraction was assisted by a 40 kHz, 100 W ultrasonic generator (KQ 50E Kunshan Ultrasonic Instrument Co. Ltd., Kunshan, China).
CH3 H3C
N
CH3
H
O CH3 O
H H
CH3 O
O Fig. 1. The structure of ulipristal acetate.
because of its low volatility and low toxicity [4]. Recently ultrasound-assisted (UA) and temperature-controlled (TC) techniques are the most preferred modifications in IL-DLLME [15,16]. However, the technique of dispersive solvent-free UA IL-DLLME has seldom been applied for extraction of drugs in biological samples. In this paper, hydrophobic IL 1-octyl-3-methylimidazolium hexafluorophosphate [C8mimPF6] as extraction solvent of dispersive liquid–liquid microextraction was first time used, which could be completely dispersed into the aqueous sample solution by sonication at 313 K without dispersive solvents, and UPA was easily migrated. The discussion of extraction mechanism showed that hydrophobic interaction force was the main driving force for UPA transfer from water into IL. The proposed method was successfully applied to the real mice serum samples and UPA tablets.
2. Experimental 2.1. Reagents and standards UPA standard (with purity 99%), UPA tablet (30 mg tablet 1) and blank tablet were kindly provided by Jiangsu Lianhuan Pharmaceutical Co., Ltd (Jiangsu, China). Methanol, ethanol, acetonitrile, acetone, ammonia, ammonium chloride, 1-bromobutane, 1-bromohexane, 1-bromooctane and triethylamine were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 1-methylimidazole was obtained from Shanghai Darui Specialty Chemicals Co.,Ltd (Shanghai, China) and ammonium hexafluorophosphate from Shanghai Bangcheng Chemical Co., Ltd (Shanghai, China). Methanol and acetonitrile were chromatographic grade, 1-methylimidazole was chemical pure, and all other materials were analytical reagent grade and water was distilled, deionised. UPA stock solution of 2.0 mg mL 1 was prepared by dissolving 0.20 g of UPA in 100.0 mL of anhydrous ethanol and kept in coolness and darkness. The stock solution was further diluted with anhydrous ethanol to obtain a standard working solution of 0.10 mg mL 1 before using. 2.0 mol L 1 NH3–NH4Cl buffer solution (pH 8.0) was prepared by dissolving appropriate amounts of ammonium chloride and ammonia. 2.2. Apparatus The analysis of UPA was carried by a 1200 series liquid chromatography (Agilent Technologies Inc., USA) equipped with photodiode-array detector (PDA). All absorption spectral recordings and absorbance measurements were performed on a UV 2501 spectrophotometer (Shimadzu, Japan). The pH measurements were done by a pH S-25 pH meter (Shanghai, China). A DK-S22 thermostatic water-bath (Shanghai Jinghong Laboratory Instrument Co., Ltd., China) was used to control temperature. A centrifuge Model 80-2 (Shanghai Pudong Physical Optics Instrument
2.3. Analytical method 2.3.1. Sample preparation For UPA tablet, five tablets of UPA was weighed and crushed, and then sample powder of about one tablet was accurately weighed and placed in a 50 mL of beaker and dissolved with anhydrous ethanol. Insoluble excipient was removed by filtration through a 0.45 μm membrane filter. The filtered solution was diluted to100.0 mL with anhydrous ethanol and kept in coolness and darkness before analysis. For mice serum, abdominal artery blood samples from mice at different time points were collected into heparinized plastic tubes, upon oral administration of 0, 5, 10, 30 mg UPA of 1 kg healthy mice. After placing them at 310 K water bath for 1 h, mice serum samples were obtained after centrifuging the blood samples. According to the method of Chen et al. [17], to eliminate protein, 1.0 mL of serum samples was placed in a 10 mL glass tube and 4.0 mL of acetonitrile was added. The mixture was shaken for 30 s and centrifuged for 10 min at 3000 rpm. Finally the supernatant was determined for UPA.
2.3.2. Synthesis of IL [C4mimPF6], [C6mimPF6] and [C8mimPF6] were synthesized according to Ref. [18], using such materials as 1-bromobutane, 1-bromohexane, 1-bromooctane, 1-methylimidazole and ammonium hexafluorophosphate.
2.3.3. Extraction procedure To a 10.0 mL centrifuge tube, 50.0 mL of [C8mimPF6], 1.0 mL of buffer solution (pH ¼8.0) and adequate UPA standard or sample solutions were added; the solution was diluted to 10.0 mL with distilled water. After shaken, the mixture was ultrasonically extracted for 10 min at 313 K. Then a cloudy mixture was formed. After cooled at 278 K for 15 min, the cloudy solution was centrifuged for 5 min at 2500 rpm and the IL phase was deposited at the bottom of the tube. Then the upper aqueous phase was removed with a syringe. The IL phase was diluted with ethanol to 0.4 mL. The resulting analytical solution was homogenized ultrasonically and filtered with 0.45 mm filter membrane before HPLC analysis.
2.3.4. HPLC measurements Chromatographic separation of UPA was performed on an Apollp C18 column (150 4.60 mm, 5 mm) (Evans Trade Co., Ltd, Shanghai, China). The mobile phase was a mixture of methanol and 0.05% triethylamine (90:10, v/v) at a flow rate of 1.0 mL min 1. The injection volume was 10.0 mL and column temperature was kept at 303 K. The monitoring wavelength was 305 nm and reference wavelength and bandwidth were 350 nm and 4 nm, respectively.
2.3.5. Determination of partition ratio The partition ratio of UPA in ILs (i.e. [C4mimPF6], [C6mimPF6], [C8mimPF6]) and water were determined. The partition ratio DIL/W was calculated according Eq. (1) [19]: DIL=W ¼
C i C f V eq Cf V IL
ð1Þ
A. Gong, X. Zhu / Talanta 131 (2015) 603–608
where Ci and Cf are the concentration of UPA in water phase before and after extraction, Veq is the volume of water phase, and VIL is the volume of IL phase.
3. Results and discussion 3.1. Optimization of extraction conditions Single factor experimental scheme due to its simplicity was used to optimize extraction parameters in this paper (such as types and amount of extraction solvent, solution pH, extraction time and temperature, cooling time and centrifugation time) [20]. All experiments were performed in triplicates (n ¼3). The extraction recovery (ER) was calculated based on Eq. (2): ER% ¼
C ex V ex 100 C0 V 0
ð2Þ
3.1.2. Dispersion solvent free The dispersion degree of extraction solvents plays a crucial role in DLLME. The smaller fine droplet of extraction solvent forms, the higher extraction recovery achieves. Ultrasound energy or high temperature combined with dispersive solvents was usually used in DLLME to improve extraction effect [7,8]. In this paper, the results showed that the ER would be over 95.0% without any dispersive solvents. So in this work, dispersive solvent was free.
3.1.3. Effect of pH In general, pH value of sample solution determines the existential state of analytes, thus affecting extraction recovery. In this paper the effect of sample solution pH value in the range of 2.0–12.0 on the extraction recovery was examined. Fig. 4 showed that the ER of UPA increased with pH from 2.0 to 7.0, and reached the maximum at pH 7.0 (ER495.0%), after that almost unchanged with further increasing pH. The log Dow (Dow, octanol–water 120
The Cex and C0 are the concentration of analyte in the extraction phase and the initial analyte concentration in the sample solution, respectively. Vex and V0 are the volumes of extraction phase and sample solution, respectively.
100
Extraction recovery (%)
3.1.1. Selection of extraction solvent Characteristics of ILs, such as solubility in water, the viscosity and extraction capacity, play a key role in influencing the extraction recovery. When fixing the anion of IL, these characteristics are affected by the cationic part. In this work three hydrophobic ILs, including [C4mimPF6], [C6mimPF6] and [C8mimPF6], were investigated. According to their solubility, 140.0 mL [C4mimPF6], 70.0 mL [C6mimPF6], and 40.0 mL [C8mimPF6] were selected as extraction solvents in the absence of dispersion solvent [7]. Fig. 2 showed that the extraction recoveries (ER) were all over 90.0% in three ILs and a higher ER was obtained in [C8mimPF6]. Furthermore, adsorption capacities of three ILs for UPA were 1.99 mg g 1 ([C4mimPF6]), 9.33 mg g 1 ([C6mimPF6]) and 18.8 mg g 1 ([C8mimPF6]) (Fig. 2), respectively. With its higher adsorption capacity and ER, [C8mimPF6] was chosen for following examination. The effect of volume of [C8mimPF6] on ER was shown in Fig. 3. The highest ER was achieved when 40.0–70.0 mL of [C8mimPF6] was employed. Therefore 50.0 mL of [C8mimPF6] was selected in the work.
605
80
60
40
20
0 20
30
40
50
60
70
V[C mimPF ] ( µ L) 8
6
Fig. 3. Effect of [C8mimPF6] volume on ER of UPA. Extraction conditions: sample volume, 10.0 mL; sample amount, 10.0 mg; IL, [C8mimPF6]; pH, 8.0; ultrasonic temperature, 313 K; ultrasonic time, 10 min; cooling temperature, 278 K; cooling time, 15 min; centrifugation time, 5 min. The error bars were standard deviation.
120
50 120
extraction recovery (%)
100
80
30
60 20 40 10 20
0
-1
Adsorption capacity (mg g )
Extraction recovery (%)
40
100
0
[C4 mimPF6 ]
[C6 mimPF6 ]
[C8 mimPF6 ]
Fig. 2. Effect of the kind of extraction solvents on ER of UPA and adsorption capacity. Extraction conditions: sample volume, 10.0 mL; sample amount, 10.0 mg; pH, 8.0; ultrasonic temperature, 313 K; ultrasonic time, 10 min; cooling temperature, 278 K; cooling time, 15 min; centrifugation time, 5 min. The error bars were standard deviation.
Extraction recovery (%)
-1
adsorption capacity (mg g )
80
60
40
20
0
0
2
4
6
8
10
12
14
pH Fig. 4. Effect of pH on ER of UPA. Extraction conditions: sample volume, 10.0 mL; sample amount, 10.0 mg; [C8mimPF6] volume, 50.0 mL; ultrasonic temperature, 313 K; ultrasonic time, 10 min; cooling temperature, 278 K; cooling time, 15 min; centrifugation time, 5 min. The error bars were standard deviation.
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partition coefficient) values of UPA are higher than 4.0 (log Dow is 4.18 at pH 5.5 or 4.47 at pH 7.4), which means UPA is easily soluble in lipids and hardly miscible with water, and the lipophilic property of UPA is stronger at pH 7.4 than at pH 5.5 [21]. UPA would more easily distribute into the hydrophobic IL in alkaline medium. So a pH of 8.0 was used for all extraction experiments. The effect of ionic strength was tested by adding KCl to UPA solutions. The results showed that ER was significantly decreased when the concentration of KCl was over 0.8 mol L 1. In this study, 1.0 mL of NH3–NH4Cl buffer solution (2.0 mol L 1) added into 10.0 mL sample solution could satisfy the requirement of ionic strength. 3.1.4. Effect of temperature Temperature has a significant effect on IL solubility in water. The solubility and dispersion degree of IL will be improved with increasing temperature that will accelerate the transfer of UPA from water to IL. In this work, the effects of extraction temperature were evaluated in the range of 288–343 K with an ultrasound time of 10 min. The ER was observed to increase with temperature from 288 to 308 K, and then remain constant up to 343 K. Therefore the extraction temperature of 313 K was selected in this study. 3.1.5. Effect of ultrasonic time Dispersion is the key step whether extraction is successfully carried out or not. As a key procedure in IL-USA-DLLME, ultrasound can accelerate the formation of fine dispersive mixtures, and result in higher recoveries [22]. In this work, the sonication time was evaluated in the range of 2 min to 20 min. The experimental results indicated that the maximum ER (95.0%) could be attained within 5 min and longer extraction time would not affect the ER. In examination it was also found that when the ultrasound 24
U
22 20 18
E
16
mAU
14 12 10 8 6
D
4
C B A
2 0 -2 0
1
2
3
4
5
6
t(min) Fig. 5. Chromatograms of blank tablet (A), blank serum (B), standard solution (C), UPA tablet (D) and mouse serum sample (E) (collected at 1 h after oral administration of 10 mg UPA of 1 kg healthy mice). The concentration of standard solution: 0.10 μg mL 1. U: UPA.
irradiation was not applied and the sample solution was intensely shaken for 2 min, the ER could also achieved 95.0%. In order to alleviate manual operation, ultrasound time of 10 min was chosen. 3.1.6. Effect of cooling and centrifugation time Phase separation can be improved with an additional cooling stage due to the decreased solubility of ILs in water [23]. In this work, the effect of the cooling time on the ER was assayed in the range of 5–30 min when fixing cooling temperature 278 K. The results showed a cooling time of 15 min was sufficient to achieve the maximum ER. In the study cooling time of 15 min was chosen. For evaluating the effect of centrifugation on phase separation, the centrifugation time was studied in the range of 1–10 min at a constant rate of 2500 rpm after cooling. 5 min was found to be enough to achieve complete phase separation. 3.1.7. Effect of solution volume The effect of solution volume on ER was examined from 5.0 mL to 30.0 mL when fixing the amount of UPA at 10.0 μg. The results showed that the ER would be less than 85.0% when the dilution volume of sample solution was over 15.0 mL. It is because the dissolved amount of [C8mimPF6] will increase with enhancing solution volume thus affecting its extraction ability. In this work, the sample volume of 10.0 mL was adopted. The IL phase was diluted to 0.4 mL with ethanol after extraction for HPLC determination, and the preconcentration factor (defined as the volume ratio of dilution sample solution and IL phase, i.e. 10.0/0.4) was 25. 3.2. Analytical performance As shown in Fig. 5(C) (chromatogram of UPA standard solution), the retention time of UPA was about at 2.70 min. Comparing with Fig. 5(C) UPA was undetectable and there were no interference peaks in blank tablet (Fig. 5(A)) and blank serum (Fig. 5(B)), therefore blank tablet and blank serum were used for the method validation. 3.2.1. Linearity and limits of detection For evaluating matrix effect, a statistical comparison between the standard curve and working curve was made. The working curves were got with spiking the standard directly into blank tablet and blank serum and extracting under the same conditions. The results were listed in Table 1. The Student’s test was applied and the statistical analysis indicated that the difference between the slope of working curve of blank tablet and standard curve was not obvious, but the difference between the slope of working curve of blank serum and standard curve was significant (P ¼0.95). So working curve was used to determine UPA in UPA tablet and mice serum. As shown in Table 1, the calibration graphs were linear over the concentration ranges of 0.03–6.0 mg mL 1, and the limits of detection (LOD) (the lowest concentration yielding a signal-to-noise ratio of 3) were 6.8 ng mL 1 (blank tablet) and 9.3 ng mL 1 (blank serum). When administered by mouth at a dose of 30 mg, ulipristal acetate is rapidly absorbed. The maximum mean serum concentration (Cmax) 7the standard deviation (SD) of 176 789 ng mL 1 was observed at approximately 1 h [24], which
Table 1 Linearity parameters and LOD of the proposed method in different matrices. Sample
Linear range (mg mL 1)
Slope 7 SD (n ¼3)
Intercept7 SD (n¼ 3)
Correlation coefficient
LOD (ng mL 1)
Standard solution Blank tablet Blank serum
0.02–6.0 0.03–6.0 0.03–6.0
482.1 7 4.2 483.9 7 6.1 495.0 7 5.0
8.92 70.35 12.9 70.58 37.7 71.6
0.9985 0.9963 0.9989
5.7 6.8 9.3
A. Gong, X. Zhu / Talanta 131 (2015) 603–608
means the method can satisfy the determination requirement of UPA in serum. 3.2.2. Precision and repeatability To evaluate intraday and interday precisions, analysis of UPA at three concentration levels (0.50, 1.0, 2.0 μg mL 1) was carried out by performing five experiments on the same day using the same analyte solution and over five consecutive days using different solutions. The intraday and interday RSD values ranged from 2.5% to 3.6% and from 3.9% to 5.1% for blank tablet, and from 1.9% to 3.8% and from 3.6% to 5.5% for blank serum, respectively, reflecting the usefulness of the method in routine use. 3.2.3. Trueness The trueness of the proposed method was evaluated by determining different UPA tablets and mice serum (through standard addition technique). The BIAS% was calculated according to the following equation: (detected content-stated content)/ stated content 100. The data were illustrated in Table 2. Obtained values of BIAS% ranged from þ3.00% to þ 5.30%, from þ 3.50% to þ6.33% for UPA tablet and mice serum, respectively, suggesting the analytical utility of the UA IL-DLLME for UPA determination. The stability of UPA in serum was investigated by extracting and analyzing the serum samples collected at 1 h after oral administration of 10 mg UPA of 1 kg healthy mice. The results indicated that UPA in serum samples was stable for at least 7 days. 3.2.4. Sample determination Comparing the chromatograms of UPA tablet (Fig. 5(D)) and mice serum (Fig. 5(E)) with UPA standard solution (Fig. 5(C)), the reproducibility of the retention time can satisfy analysis requirement. Table 3 showed analytical results for UPA tablet and mice serum using the proposed methodologies. The result of UPA tablet obtained by the proposed method was in good agreement with the label value. The statistical t-test (P¼ 0.95) was used to compare the results, which showed that there was no significant difference between them. From Table 3 it was also known that the UPA concentration in mice serum would rise with increasing intragastric administration dose and the maximum serum concentration was obtained at 1 h, which was consistent with that of the literature [24]. 3.3. Discussion of extraction mechanism
Table 2 The trueness evaluation of the proposed method.
UPA tablet1 UPA tablet2 UPA tablet3 Serum-1b Serum-2b Serum-3b a b
thermodynamic parameters depending on temperature such as enthalpy change, Gibbs energy and entropy change for elaborating extraction mechanism. 3.3.1. Partition ratio DIL/W The partition ratio DIL/W is the concentration ratio of UPA in two immiscible phases at partition equilibrium, and reflects differential solubility of UPA between two phases (ILs-H2O). The DIL/W and ER of UPA in three ILs at 313 K were shown in Table 4. It could be seen from Table 4 that (1) the DIL/W was all over 103 in three ILs, i.e. the concentration ratio of UPA in ILs and in water was 4103, indicating there was stronger interaction between UPA and ILs than that of UPA and water, and UPA was more easily distributed in ILs; (2) the DIL/W of UPA in the ILs followed the order: [C8mimPF6] 4[C6mimPF6] 4[C4mimPF6] and the order DIL/W of was consistent with the order of ER. It is because the hydrophobicity of ILs will enhance with increasing the length of alkyl chains in imidazole ring. The DIL/W and ER were increased from [C4mimPF6] to [C8mimPF6], indicating hydrophobic interaction force played a key role on the partition of UPA between ILs and water. 3.3.2. Extraction thermodynamic parameters From a thermodynamic perspective, the partition of UPA can be regarded as a transfer process of the UPA molecules from water phase to the IL phase. At a given temperature, the changes in Gibbs energy ðΔG0T Þ, enthalpy ðΔH 0T Þ and entropy ðΔS0T Þ of such a transfer process can be calculated from the partition data through following equations: ln DIL=W ¼ C þ ð ΔH 0T =RTÞ
ð3Þ
[25]
ΔG0T ¼ RT½ ln DIL=W þ lnðV m;IL =V m;w Þ
ð4Þ
[19] T ΔS0T ¼ ΔH 0T ΔG0T
ð5Þ
Among them C is constant, R is molar gas constant (8.314 J mol 1 K 1), T is absolute temperature, and Vm,IL and Vm,W are molar volumes of ILs and water. Table 3 Analytical results of UPA in real sample.a
The extraction effect of UPA by ILs depends on the dissolution degree of UPA in IL. The hydrophobic UPA is more likely to be dissolved by hydrophobic ILs than by water based on the hydrophobic interaction. In this work hydrophobic interaction force between UPA and ILs was verified by partition ratio and some
Sample
607
Sample
Label value
Intragastric dose
Collected time (h)
Found7 SDa
Tablet
30 mg tablet 1
/
/ 0.5 1 2 0.5 1 2 0.5 1 2
29.7 7 0.98 mg tablet 1 0.65 7 0.04 mg mL 1 4.617 0.31 mg mL 1 1.46 7 0.04 mg mL 1 1.32 7 0.10 mg mL 1 8.12 70.59 mg mL 1 3.107 0.13 mg mL 1 2.92 7 0.17 mg mL 1 22.8 7 0.99 mg mL 1 6.03 7 0.15 mg mL 1
5 mg kg 1 Mice serum
Stated content (mg mL 1)
Detected content (mg mL 1)a
BIAS (%)
0.10
0.104
þ 4.0
0.20
0.206
þ 3.0
0.30
0.316
þ 5.3
0.10 0.20 0.30
0.105 0.207 0.319
þ 5.0 þ 3.5 þ 6.3
10 mg kg 1
/
30 mg kg 1
a
Mean for three independent determinations. Collected at 1 h after oral administration of 10 mg UPA of 1 kg healthy mice.
Mean for three independent determinations.
Table 4 The DIL/W and ER of UPA in different IL. IL DIL/W, 10 ER (%)
3
[C4mimPF6]
[C6mimPF6]
[C8mimPF6]
3.01 92.3
6.77 94.9
11.5 99.4
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A. Gong, X. Zhu / Talanta 131 (2015) 603–608
Table 5 M volumes of ILs and water, lnDIL/W and thermodynamic parameters of UPA extracted by different IL under different temperature. T (K) Parameters 298.15
lnDIL/W
3
Molar volumes (cm mol
1 a
)
[C4mimPF6] [C6mimPF6] [C8mimPF6] H2O
ΔH 0T (kJ mol 1)
[C4mimPF6] [C6mimPF6] [C8mimPF6]
ΔG0T (kJ mol 1)
[C4mimPF6] [C6mimPF6] [C8mimPF6]
TΔS0T (kJ mol 1)
[C4mimPF6] [C6mimPF6] [C8mimPF6]
a
308.15 7.75 8.18 8.54 209.4 243.1 277.4 18.11
[C4mimPF6] [C6mimPF6] [C8mimPF6] 207.6 241.6 275.7 18.05 21.8 35.9 48.9 25.3 26.7 27.9 47.1 62.6 76.8
26.7 28.5 30.2 48.5 64.4 79.1
318.15 7.96 8.54 9.06 211.2 244.6 279.1 18.18
28.2 30.8 32.9 50.0 66.7 81.8
328.15 8.22 9.04 9.70 213.0 246.2 280.9 18.26
30.0 32.9 35.6 51.8 68.8 84.5
Ref. [19].
It is clear that values of ΔH 0T can be directly obtained from the slope of the linear equation between lnDIL/W and 1/T shown in Eq. (3). Molar volumes of ILs and water (got by Ref. [19]), lnDIL/W, ΔH 0T , and ΔG0T and T ΔS0T calculated with Eqs. (4) and (5) under different temperature were shown in Table 5. From Table 5, the following conclusions were drawn that (1) ΔH 0T 4 0 and partition ratios in three ILs were increased with raising temperature, illustrating that extraction process was a endothermic process and higher temperature was beneficial to extraction; (2) ΔG0T o 0, pointing out extraction was spontaneous; (3) ΔS0T 4 0 and ΔH 0T 〈 T ΔS0T , indicating that the transfer process of UPA from water to IL was driven by entropy change. It is generally recognized that hydrophobic interaction force is main feature of entropy control [26]. Based on these results, it could be deduced that hydrophobic interaction might be the main driving force for the transfer of UPA molecules from water to the IL phases.
Jiangsu Higher Education Institutions and the Foundation of the Excellence Science and Technology Invention Team in Yangzhou University and the Graduate Innovation Project Foundation of Jiangsu province (CXLX13_895).
References [1] [2] [3] [4] [5] [6] [7] [8] [9]
4. Conclusion In this paper a new and environmental friendly ultrasoundassisted ionic liquid dispersive liquid–liquid microextraction coupled with HPLC-PDA was developed for the determination of UPA in dosage form and biological sample. Without any dispersive solvent the ER could be over 95.0% under ultrasonication. The developed method can provide analytical technical support for UPA pharmacokinetics examination. Mechanism discussion of extraction indicated that hydrophobic interaction might be the main driving force for UPA extracted in IL phase.
[10] [11] [12] [13] [14] [15] [16]
Acknowledgements
[17] [18] [19] [20] [21] [22]
The authors acknowledge the financial support from the National Natural Science Foundation of China (21375117, 21155001) and a project funded by the Priority Academic Program Development of
[23] [24] [25] [26]
B.Q. Ma, Shanghai Med. Pharm. J 31 (2010) 315–316. G.F. Yao, Chin. J. Med. Chem. 21 (2011) 78–80. WO 2004/078709. J.N. Sun, Y.P. Shi, J. Chen, J. Chromatogr. B 879 (2011) 3429–3433. Y.F. Zhang, H.K. Lee, Anal. Chim. Acta 750 (2012) 120–126. C. Noelia, P.P. Francisco, I. de la Calle, B. Carlos, L. Isela, Microchem. J. 99 (2011) 246–251. S.Q. Gao, X. Yang, W. Yu, Z.L. Liu, H.Q. Zhang, Talanta 99 (2012) 875–882. A.J. Toraj, F. Nazir, S. Mojtaba, P. Meghdad, J. Pharm. Biomed. Anal. 85 (2013) 14–20. R. Mohammad, A. Yaghoub, M.H. Mohammad-Reza, A. Elham, A. Fardin, B. Sana, J. Chromatogr. A 1116 (2006) 1–9. D. Nagaraju, S.D. Huang, J. Chromatogr. A 1161 (2007) 89–97. M.A. Farajzadeh, M. Bahram, J. Jönsson, Anal. Chim. Acta 591 (2007) 69–79. M. Hatami, Elham Karimnia, Khalil Farhadi, J. Pharm. Biomed. Anal. 85 (2013) 283–287. J.M. Padro´, M.E. Marso´n, G.E. Mastrantonio, J. Altcheh, F. Garcı´a-Bournissen, M. Reta, Talanta 107 (2013) 95–102. X. Liu, R. Fu, M. Li, L.P. Guo, L. Yang, Chin. J. Anal. Chem. 41 (2013) 1919–1922. J.H. Zhang, H.X. Gao, B. Peng, S.Q. Li, Z.Q. Zhou, J. Chromatogr. A 1218 (2011) 6621–6627. M.J. Trujillo-Rodríguez, P. Rocío-Bautista, V. Pino, A.M. Afonso, Trends Anal. Chem. 51 (2013) 87–106. W.Y. Chen, H. Li, W.H. Luo, Chin. J. Mod. Med. 17 (2007) 1665–1673. F.Y. Zhao, J.Y. Wang, H.J. Liu, R.J. Liu, Chem. Reagents 29 (2007) 229–231. Y.C. Pei, Doctoral Dissertation of Lanzhou University (2008). P. Arbeláez, F. Borrull, R.M. Marcé, E. Pocurull, Talanta 125 (2014) 65–71. 〈http://www.chemspider.com/Chemical-Structure.115762.html〉. S.Y. Dong, Q. Hu, Z. Yang, R. Liu, G.Q. Huang, T.L. Huang, Microchem. J. 110 (2013) 221–226. R. Liu, J. Liu, Y. Yin, X. Hu, G. Jiang, Anal. Bioanal. Chem. 393 (2009) 871–883. 〈http://tec.k8008.com/html/124/124073.html〉. X.M. Fu, S.G. Dai, Y. Zhang, Chin. J. Anal. Chem 34 (2006) 598–602. H. Bianco-Peled, S. Gryc, Langmiur 20 (2004) 169–174.
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Dispersive solvent-free ultrasound-assisted ionic liquid dispersive liquid–liquid microextraction coupled with HPLC for determination of ulipristal acetate Aiqin Gong a,b, Xiashi Zhu a,n a b
College of Chemistry & Chemical Engineering, Yangzhou University, Yangzhou 225002, China Yangzhou Polytechnic Institute, Yangzhou 225127, China
art ic l e i nf o
a b s t r a c t
Article history: Received 20 June 2014 Received in revised form 30 July 2014 Accepted 6 August 2014 Available online 23 August 2014
In this paper, a simple and efficient ultrasound-assisted ionic liquid dispersive liquid–liquid microextraction (UA IL-DLLME) coupled with high-performance liquid chromatography for the analysis of ulipristal acetate (UPA) was developed. UPA could be easily migrated into 1-octyl-3-methylimidazolium hexafluorophosphate [C8mimPF6] IL phase without dispersive solvent. The research of extraction mechanism showed that hydrophobic interaction force played a key role in the IL-DLLME. Several important parameters affecting the extraction recovery were optimized. Under the optimized conditions, 25-fold enrichment factor was obtained and the limit of detection (LOD) was 6.8 ng mL 1 (tablet) or 9.3 ng mL 1 (serum) at a signal-to-noise ratio of 3. The calibration curve was linear over the range of 0.03–6.0 mg mL 1. The proposed method was successfully applied to the UPA tablets and the real mice serum samples. & 2014 Elsevier B.V. All rights reserved.
Keywords: Ulipristal acetate Ionic liquid Liquid–liquid microextraction HPLC
1. Introduction Ulipristal acetate [17α-acetoxy-11β-(4-N,N-dimethyl aminophenyl)-19-norpregna-4,9-diene-3,20-dione] (UPA), a selective progesterone receptor modulator (Fig. 1), can prevent unintended pregnancy by delaying ovulation for up to five days after contraceptive failure. In August 2010, UPA had gained the FDA’s approval for use as an oral emergency contraception tablet in the U.S. with trade name Ella. Until now it is found that Ella may cause serious side effects including abdominal pain, menstrual disorder, headache, nausea and so on [1]. In addition, the study for UPA to treat contraceptive gynecological indications (fibroma uteri, adenomyosis) and Cushing’s syndrome is in progress [2]. In view of safe medication and investigating pharmaceutical dynamics of drugs, a simple, sensitive analytical procedure is needed to determine UPA in pharmaceutical formulation and in biological fluids. HPLC has been used to determine UPA in bulk [3]. But to the best of our knowledge, there were few literatures to analyze UPA in biological samples. An appropriate preconcentration/separation method should be developed due to matrix interference and low concentration of analytes in real biological samples before analysis [4]. In recent years, many preconcentration/separation steps have been oriented
n
Corresponding author. Tel./fax: þ 86 514 7975244. E-mail addresses: [email protected], [email protected] (X. Zhu).
http://dx.doi.org/10.1016/j.talanta.2014.08.021 0039-9140/& 2014 Elsevier B.V. All rights reserved.
toward the fast development of simplification and miniaturization. In particular, the use of alternative non-contaminant and nontoxic solvents instead of high quantities of organic solvents is preferred during preconcentration/separation. Solid phase microextraction (SPME) and liquid phase microextraction (LPME) have been extensively used to preconcentration/separation analytes in complex matrix with their high ability of sample clean up and analyte preconcentration, and low consumption of solvents. SPME would required a specific device loaded with certain adsorption material as well as a high-pressure delivery system that would be relatively expensive [4]. Moreover the operation of LPME is simpler and faster than that of SPME (which includes adsorption progress and desorption progress). Dispersive liquid–liquid microextraction (DLLME), developed by Assadi and co-workers in 2006 [5], is a miniaturized form of liquid phase extraction that employs microliter volumes of extraction solvent. Compared with other microextraction techniques, the advantages of DLLME are simplicity of operation, rapidness, accuracy, and it has been extensively applied in drugs analysis [4,6–8]. Extraction solvents (such as tetrachloroethylene [9], chlorobenzene [10] and carbon tetrachloride [11]) and dispersive solvents (such as methanol [12,13], acetone [9,10] and acetonitrile [11,14]) are usually used in DLLME. Because of the relatively high toxicity of these conventional chlorinated extraction solvents, developing environment-friendly “green” extraction solvents has inspired the great interest of examiners. Ionic liquids (IL) has been more and more used as extraction solvent in DLLME (IL-DLLME)
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Factory, China) was used to accelerate the phase-separation process. The microextraction was assisted by a 40 kHz, 100 W ultrasonic generator (KQ 50E Kunshan Ultrasonic Instrument Co. Ltd., Kunshan, China).
CH3 H3C
N
CH3
H
O CH3 O
H H
CH3 O
O Fig. 1. The structure of ulipristal acetate.
because of its low volatility and low toxicity [4]. Recently ultrasound-assisted (UA) and temperature-controlled (TC) techniques are the most preferred modifications in IL-DLLME [15,16]. However, the technique of dispersive solvent-free UA IL-DLLME has seldom been applied for extraction of drugs in biological samples. In this paper, hydrophobic IL 1-octyl-3-methylimidazolium hexafluorophosphate [C8mimPF6] as extraction solvent of dispersive liquid–liquid microextraction was first time used, which could be completely dispersed into the aqueous sample solution by sonication at 313 K without dispersive solvents, and UPA was easily migrated. The discussion of extraction mechanism showed that hydrophobic interaction force was the main driving force for UPA transfer from water into IL. The proposed method was successfully applied to the real mice serum samples and UPA tablets.
2. Experimental 2.1. Reagents and standards UPA standard (with purity 99%), UPA tablet (30 mg tablet 1) and blank tablet were kindly provided by Jiangsu Lianhuan Pharmaceutical Co., Ltd (Jiangsu, China). Methanol, ethanol, acetonitrile, acetone, ammonia, ammonium chloride, 1-bromobutane, 1-bromohexane, 1-bromooctane and triethylamine were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 1-methylimidazole was obtained from Shanghai Darui Specialty Chemicals Co.,Ltd (Shanghai, China) and ammonium hexafluorophosphate from Shanghai Bangcheng Chemical Co., Ltd (Shanghai, China). Methanol and acetonitrile were chromatographic grade, 1-methylimidazole was chemical pure, and all other materials were analytical reagent grade and water was distilled, deionised. UPA stock solution of 2.0 mg mL 1 was prepared by dissolving 0.20 g of UPA in 100.0 mL of anhydrous ethanol and kept in coolness and darkness. The stock solution was further diluted with anhydrous ethanol to obtain a standard working solution of 0.10 mg mL 1 before using. 2.0 mol L 1 NH3–NH4Cl buffer solution (pH 8.0) was prepared by dissolving appropriate amounts of ammonium chloride and ammonia. 2.2. Apparatus The analysis of UPA was carried by a 1200 series liquid chromatography (Agilent Technologies Inc., USA) equipped with photodiode-array detector (PDA). All absorption spectral recordings and absorbance measurements were performed on a UV 2501 spectrophotometer (Shimadzu, Japan). The pH measurements were done by a pH S-25 pH meter (Shanghai, China). A DK-S22 thermostatic water-bath (Shanghai Jinghong Laboratory Instrument Co., Ltd., China) was used to control temperature. A centrifuge Model 80-2 (Shanghai Pudong Physical Optics Instrument
2.3. Analytical method 2.3.1. Sample preparation For UPA tablet, five tablets of UPA was weighed and crushed, and then sample powder of about one tablet was accurately weighed and placed in a 50 mL of beaker and dissolved with anhydrous ethanol. Insoluble excipient was removed by filtration through a 0.45 μm membrane filter. The filtered solution was diluted to100.0 mL with anhydrous ethanol and kept in coolness and darkness before analysis. For mice serum, abdominal artery blood samples from mice at different time points were collected into heparinized plastic tubes, upon oral administration of 0, 5, 10, 30 mg UPA of 1 kg healthy mice. After placing them at 310 K water bath for 1 h, mice serum samples were obtained after centrifuging the blood samples. According to the method of Chen et al. [17], to eliminate protein, 1.0 mL of serum samples was placed in a 10 mL glass tube and 4.0 mL of acetonitrile was added. The mixture was shaken for 30 s and centrifuged for 10 min at 3000 rpm. Finally the supernatant was determined for UPA.
2.3.2. Synthesis of IL [C4mimPF6], [C6mimPF6] and [C8mimPF6] were synthesized according to Ref. [18], using such materials as 1-bromobutane, 1-bromohexane, 1-bromooctane, 1-methylimidazole and ammonium hexafluorophosphate.
2.3.3. Extraction procedure To a 10.0 mL centrifuge tube, 50.0 mL of [C8mimPF6], 1.0 mL of buffer solution (pH ¼8.0) and adequate UPA standard or sample solutions were added; the solution was diluted to 10.0 mL with distilled water. After shaken, the mixture was ultrasonically extracted for 10 min at 313 K. Then a cloudy mixture was formed. After cooled at 278 K for 15 min, the cloudy solution was centrifuged for 5 min at 2500 rpm and the IL phase was deposited at the bottom of the tube. Then the upper aqueous phase was removed with a syringe. The IL phase was diluted with ethanol to 0.4 mL. The resulting analytical solution was homogenized ultrasonically and filtered with 0.45 mm filter membrane before HPLC analysis.
2.3.4. HPLC measurements Chromatographic separation of UPA was performed on an Apollp C18 column (150 4.60 mm, 5 mm) (Evans Trade Co., Ltd, Shanghai, China). The mobile phase was a mixture of methanol and 0.05% triethylamine (90:10, v/v) at a flow rate of 1.0 mL min 1. The injection volume was 10.0 mL and column temperature was kept at 303 K. The monitoring wavelength was 305 nm and reference wavelength and bandwidth were 350 nm and 4 nm, respectively.
2.3.5. Determination of partition ratio The partition ratio of UPA in ILs (i.e. [C4mimPF6], [C6mimPF6], [C8mimPF6]) and water were determined. The partition ratio DIL/W was calculated according Eq. (1) [19]: DIL=W ¼
C i C f V eq Cf V IL
ð1Þ
A. Gong, X. Zhu / Talanta 131 (2015) 603–608
where Ci and Cf are the concentration of UPA in water phase before and after extraction, Veq is the volume of water phase, and VIL is the volume of IL phase.
3. Results and discussion 3.1. Optimization of extraction conditions Single factor experimental scheme due to its simplicity was used to optimize extraction parameters in this paper (such as types and amount of extraction solvent, solution pH, extraction time and temperature, cooling time and centrifugation time) [20]. All experiments were performed in triplicates (n ¼3). The extraction recovery (ER) was calculated based on Eq. (2): ER% ¼
C ex V ex 100 C0 V 0
ð2Þ
3.1.2. Dispersion solvent free The dispersion degree of extraction solvents plays a crucial role in DLLME. The smaller fine droplet of extraction solvent forms, the higher extraction recovery achieves. Ultrasound energy or high temperature combined with dispersive solvents was usually used in DLLME to improve extraction effect [7,8]. In this paper, the results showed that the ER would be over 95.0% without any dispersive solvents. So in this work, dispersive solvent was free.
3.1.3. Effect of pH In general, pH value of sample solution determines the existential state of analytes, thus affecting extraction recovery. In this paper the effect of sample solution pH value in the range of 2.0–12.0 on the extraction recovery was examined. Fig. 4 showed that the ER of UPA increased with pH from 2.0 to 7.0, and reached the maximum at pH 7.0 (ER495.0%), after that almost unchanged with further increasing pH. The log Dow (Dow, octanol–water 120
The Cex and C0 are the concentration of analyte in the extraction phase and the initial analyte concentration in the sample solution, respectively. Vex and V0 are the volumes of extraction phase and sample solution, respectively.
100
Extraction recovery (%)
3.1.1. Selection of extraction solvent Characteristics of ILs, such as solubility in water, the viscosity and extraction capacity, play a key role in influencing the extraction recovery. When fixing the anion of IL, these characteristics are affected by the cationic part. In this work three hydrophobic ILs, including [C4mimPF6], [C6mimPF6] and [C8mimPF6], were investigated. According to their solubility, 140.0 mL [C4mimPF6], 70.0 mL [C6mimPF6], and 40.0 mL [C8mimPF6] were selected as extraction solvents in the absence of dispersion solvent [7]. Fig. 2 showed that the extraction recoveries (ER) were all over 90.0% in three ILs and a higher ER was obtained in [C8mimPF6]. Furthermore, adsorption capacities of three ILs for UPA were 1.99 mg g 1 ([C4mimPF6]), 9.33 mg g 1 ([C6mimPF6]) and 18.8 mg g 1 ([C8mimPF6]) (Fig. 2), respectively. With its higher adsorption capacity and ER, [C8mimPF6] was chosen for following examination. The effect of volume of [C8mimPF6] on ER was shown in Fig. 3. The highest ER was achieved when 40.0–70.0 mL of [C8mimPF6] was employed. Therefore 50.0 mL of [C8mimPF6] was selected in the work.
605
80
60
40
20
0 20
30
40
50
60
70
V[C mimPF ] ( µ L) 8
6
Fig. 3. Effect of [C8mimPF6] volume on ER of UPA. Extraction conditions: sample volume, 10.0 mL; sample amount, 10.0 mg; IL, [C8mimPF6]; pH, 8.0; ultrasonic temperature, 313 K; ultrasonic time, 10 min; cooling temperature, 278 K; cooling time, 15 min; centrifugation time, 5 min. The error bars were standard deviation.
120
50 120
extraction recovery (%)
100
80
30
60 20 40 10 20
0
-1
Adsorption capacity (mg g )
Extraction recovery (%)
40
100
0
[C4 mimPF6 ]
[C6 mimPF6 ]
[C8 mimPF6 ]
Fig. 2. Effect of the kind of extraction solvents on ER of UPA and adsorption capacity. Extraction conditions: sample volume, 10.0 mL; sample amount, 10.0 mg; pH, 8.0; ultrasonic temperature, 313 K; ultrasonic time, 10 min; cooling temperature, 278 K; cooling time, 15 min; centrifugation time, 5 min. The error bars were standard deviation.
Extraction recovery (%)
-1
adsorption capacity (mg g )
80
60
40
20
0
0
2
4
6
8
10
12
14
pH Fig. 4. Effect of pH on ER of UPA. Extraction conditions: sample volume, 10.0 mL; sample amount, 10.0 mg; [C8mimPF6] volume, 50.0 mL; ultrasonic temperature, 313 K; ultrasonic time, 10 min; cooling temperature, 278 K; cooling time, 15 min; centrifugation time, 5 min. The error bars were standard deviation.
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partition coefficient) values of UPA are higher than 4.0 (log Dow is 4.18 at pH 5.5 or 4.47 at pH 7.4), which means UPA is easily soluble in lipids and hardly miscible with water, and the lipophilic property of UPA is stronger at pH 7.4 than at pH 5.5 [21]. UPA would more easily distribute into the hydrophobic IL in alkaline medium. So a pH of 8.0 was used for all extraction experiments. The effect of ionic strength was tested by adding KCl to UPA solutions. The results showed that ER was significantly decreased when the concentration of KCl was over 0.8 mol L 1. In this study, 1.0 mL of NH3–NH4Cl buffer solution (2.0 mol L 1) added into 10.0 mL sample solution could satisfy the requirement of ionic strength. 3.1.4. Effect of temperature Temperature has a significant effect on IL solubility in water. The solubility and dispersion degree of IL will be improved with increasing temperature that will accelerate the transfer of UPA from water to IL. In this work, the effects of extraction temperature were evaluated in the range of 288–343 K with an ultrasound time of 10 min. The ER was observed to increase with temperature from 288 to 308 K, and then remain constant up to 343 K. Therefore the extraction temperature of 313 K was selected in this study. 3.1.5. Effect of ultrasonic time Dispersion is the key step whether extraction is successfully carried out or not. As a key procedure in IL-USA-DLLME, ultrasound can accelerate the formation of fine dispersive mixtures, and result in higher recoveries [22]. In this work, the sonication time was evaluated in the range of 2 min to 20 min. The experimental results indicated that the maximum ER (95.0%) could be attained within 5 min and longer extraction time would not affect the ER. In examination it was also found that when the ultrasound 24
U
22 20 18
E
16
mAU
14 12 10 8 6
D
4
C B A
2 0 -2 0
1
2
3
4
5
6
t(min) Fig. 5. Chromatograms of blank tablet (A), blank serum (B), standard solution (C), UPA tablet (D) and mouse serum sample (E) (collected at 1 h after oral administration of 10 mg UPA of 1 kg healthy mice). The concentration of standard solution: 0.10 μg mL 1. U: UPA.
irradiation was not applied and the sample solution was intensely shaken for 2 min, the ER could also achieved 95.0%. In order to alleviate manual operation, ultrasound time of 10 min was chosen. 3.1.6. Effect of cooling and centrifugation time Phase separation can be improved with an additional cooling stage due to the decreased solubility of ILs in water [23]. In this work, the effect of the cooling time on the ER was assayed in the range of 5–30 min when fixing cooling temperature 278 K. The results showed a cooling time of 15 min was sufficient to achieve the maximum ER. In the study cooling time of 15 min was chosen. For evaluating the effect of centrifugation on phase separation, the centrifugation time was studied in the range of 1–10 min at a constant rate of 2500 rpm after cooling. 5 min was found to be enough to achieve complete phase separation. 3.1.7. Effect of solution volume The effect of solution volume on ER was examined from 5.0 mL to 30.0 mL when fixing the amount of UPA at 10.0 μg. The results showed that the ER would be less than 85.0% when the dilution volume of sample solution was over 15.0 mL. It is because the dissolved amount of [C8mimPF6] will increase with enhancing solution volume thus affecting its extraction ability. In this work, the sample volume of 10.0 mL was adopted. The IL phase was diluted to 0.4 mL with ethanol after extraction for HPLC determination, and the preconcentration factor (defined as the volume ratio of dilution sample solution and IL phase, i.e. 10.0/0.4) was 25. 3.2. Analytical performance As shown in Fig. 5(C) (chromatogram of UPA standard solution), the retention time of UPA was about at 2.70 min. Comparing with Fig. 5(C) UPA was undetectable and there were no interference peaks in blank tablet (Fig. 5(A)) and blank serum (Fig. 5(B)), therefore blank tablet and blank serum were used for the method validation. 3.2.1. Linearity and limits of detection For evaluating matrix effect, a statistical comparison between the standard curve and working curve was made. The working curves were got with spiking the standard directly into blank tablet and blank serum and extracting under the same conditions. The results were listed in Table 1. The Student’s test was applied and the statistical analysis indicated that the difference between the slope of working curve of blank tablet and standard curve was not obvious, but the difference between the slope of working curve of blank serum and standard curve was significant (P ¼0.95). So working curve was used to determine UPA in UPA tablet and mice serum. As shown in Table 1, the calibration graphs were linear over the concentration ranges of 0.03–6.0 mg mL 1, and the limits of detection (LOD) (the lowest concentration yielding a signal-to-noise ratio of 3) were 6.8 ng mL 1 (blank tablet) and 9.3 ng mL 1 (blank serum). When administered by mouth at a dose of 30 mg, ulipristal acetate is rapidly absorbed. The maximum mean serum concentration (Cmax) 7the standard deviation (SD) of 176 789 ng mL 1 was observed at approximately 1 h [24], which
Table 1 Linearity parameters and LOD of the proposed method in different matrices. Sample
Linear range (mg mL 1)
Slope 7 SD (n ¼3)
Intercept7 SD (n¼ 3)
Correlation coefficient
LOD (ng mL 1)
Standard solution Blank tablet Blank serum
0.02–6.0 0.03–6.0 0.03–6.0
482.1 7 4.2 483.9 7 6.1 495.0 7 5.0
8.92 70.35 12.9 70.58 37.7 71.6
0.9985 0.9963 0.9989
5.7 6.8 9.3
A. Gong, X. Zhu / Talanta 131 (2015) 603–608
means the method can satisfy the determination requirement of UPA in serum. 3.2.2. Precision and repeatability To evaluate intraday and interday precisions, analysis of UPA at three concentration levels (0.50, 1.0, 2.0 μg mL 1) was carried out by performing five experiments on the same day using the same analyte solution and over five consecutive days using different solutions. The intraday and interday RSD values ranged from 2.5% to 3.6% and from 3.9% to 5.1% for blank tablet, and from 1.9% to 3.8% and from 3.6% to 5.5% for blank serum, respectively, reflecting the usefulness of the method in routine use. 3.2.3. Trueness The trueness of the proposed method was evaluated by determining different UPA tablets and mice serum (through standard addition technique). The BIAS% was calculated according to the following equation: (detected content-stated content)/ stated content 100. The data were illustrated in Table 2. Obtained values of BIAS% ranged from þ3.00% to þ 5.30%, from þ 3.50% to þ6.33% for UPA tablet and mice serum, respectively, suggesting the analytical utility of the UA IL-DLLME for UPA determination. The stability of UPA in serum was investigated by extracting and analyzing the serum samples collected at 1 h after oral administration of 10 mg UPA of 1 kg healthy mice. The results indicated that UPA in serum samples was stable for at least 7 days. 3.2.4. Sample determination Comparing the chromatograms of UPA tablet (Fig. 5(D)) and mice serum (Fig. 5(E)) with UPA standard solution (Fig. 5(C)), the reproducibility of the retention time can satisfy analysis requirement. Table 3 showed analytical results for UPA tablet and mice serum using the proposed methodologies. The result of UPA tablet obtained by the proposed method was in good agreement with the label value. The statistical t-test (P¼ 0.95) was used to compare the results, which showed that there was no significant difference between them. From Table 3 it was also known that the UPA concentration in mice serum would rise with increasing intragastric administration dose and the maximum serum concentration was obtained at 1 h, which was consistent with that of the literature [24]. 3.3. Discussion of extraction mechanism
Table 2 The trueness evaluation of the proposed method.
UPA tablet1 UPA tablet2 UPA tablet3 Serum-1b Serum-2b Serum-3b a b
thermodynamic parameters depending on temperature such as enthalpy change, Gibbs energy and entropy change for elaborating extraction mechanism. 3.3.1. Partition ratio DIL/W The partition ratio DIL/W is the concentration ratio of UPA in two immiscible phases at partition equilibrium, and reflects differential solubility of UPA between two phases (ILs-H2O). The DIL/W and ER of UPA in three ILs at 313 K were shown in Table 4. It could be seen from Table 4 that (1) the DIL/W was all over 103 in three ILs, i.e. the concentration ratio of UPA in ILs and in water was 4103, indicating there was stronger interaction between UPA and ILs than that of UPA and water, and UPA was more easily distributed in ILs; (2) the DIL/W of UPA in the ILs followed the order: [C8mimPF6] 4[C6mimPF6] 4[C4mimPF6] and the order DIL/W of was consistent with the order of ER. It is because the hydrophobicity of ILs will enhance with increasing the length of alkyl chains in imidazole ring. The DIL/W and ER were increased from [C4mimPF6] to [C8mimPF6], indicating hydrophobic interaction force played a key role on the partition of UPA between ILs and water. 3.3.2. Extraction thermodynamic parameters From a thermodynamic perspective, the partition of UPA can be regarded as a transfer process of the UPA molecules from water phase to the IL phase. At a given temperature, the changes in Gibbs energy ðΔG0T Þ, enthalpy ðΔH 0T Þ and entropy ðΔS0T Þ of such a transfer process can be calculated from the partition data through following equations: ln DIL=W ¼ C þ ð ΔH 0T =RTÞ
ð3Þ
[25]
ΔG0T ¼ RT½ ln DIL=W þ lnðV m;IL =V m;w Þ
ð4Þ
[19] T ΔS0T ¼ ΔH 0T ΔG0T
ð5Þ
Among them C is constant, R is molar gas constant (8.314 J mol 1 K 1), T is absolute temperature, and Vm,IL and Vm,W are molar volumes of ILs and water. Table 3 Analytical results of UPA in real sample.a
The extraction effect of UPA by ILs depends on the dissolution degree of UPA in IL. The hydrophobic UPA is more likely to be dissolved by hydrophobic ILs than by water based on the hydrophobic interaction. In this work hydrophobic interaction force between UPA and ILs was verified by partition ratio and some
Sample
607
Sample
Label value
Intragastric dose
Collected time (h)
Found7 SDa
Tablet
30 mg tablet 1
/
/ 0.5 1 2 0.5 1 2 0.5 1 2
29.7 7 0.98 mg tablet 1 0.65 7 0.04 mg mL 1 4.617 0.31 mg mL 1 1.46 7 0.04 mg mL 1 1.32 7 0.10 mg mL 1 8.12 70.59 mg mL 1 3.107 0.13 mg mL 1 2.92 7 0.17 mg mL 1 22.8 7 0.99 mg mL 1 6.03 7 0.15 mg mL 1
5 mg kg 1 Mice serum
Stated content (mg mL 1)
Detected content (mg mL 1)a
BIAS (%)
0.10
0.104
þ 4.0
0.20
0.206
þ 3.0
0.30
0.316
þ 5.3
0.10 0.20 0.30
0.105 0.207 0.319
þ 5.0 þ 3.5 þ 6.3
10 mg kg 1
/
30 mg kg 1
a
Mean for three independent determinations. Collected at 1 h after oral administration of 10 mg UPA of 1 kg healthy mice.
Mean for three independent determinations.
Table 4 The DIL/W and ER of UPA in different IL. IL DIL/W, 10 ER (%)
3
[C4mimPF6]
[C6mimPF6]
[C8mimPF6]
3.01 92.3
6.77 94.9
11.5 99.4
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A. Gong, X. Zhu / Talanta 131 (2015) 603–608
Table 5 M volumes of ILs and water, lnDIL/W and thermodynamic parameters of UPA extracted by different IL under different temperature. T (K) Parameters 298.15
lnDIL/W
3
Molar volumes (cm mol
1 a
)
[C4mimPF6] [C6mimPF6] [C8mimPF6] H2O
ΔH 0T (kJ mol 1)
[C4mimPF6] [C6mimPF6] [C8mimPF6]
ΔG0T (kJ mol 1)
[C4mimPF6] [C6mimPF6] [C8mimPF6]
TΔS0T (kJ mol 1)
[C4mimPF6] [C6mimPF6] [C8mimPF6]
a
308.15 7.75 8.18 8.54 209.4 243.1 277.4 18.11
[C4mimPF6] [C6mimPF6] [C8mimPF6] 207.6 241.6 275.7 18.05 21.8 35.9 48.9 25.3 26.7 27.9 47.1 62.6 76.8
26.7 28.5 30.2 48.5 64.4 79.1
318.15 7.96 8.54 9.06 211.2 244.6 279.1 18.18
28.2 30.8 32.9 50.0 66.7 81.8
328.15 8.22 9.04 9.70 213.0 246.2 280.9 18.26
30.0 32.9 35.6 51.8 68.8 84.5
Ref. [19].
It is clear that values of ΔH 0T can be directly obtained from the slope of the linear equation between lnDIL/W and 1/T shown in Eq. (3). Molar volumes of ILs and water (got by Ref. [19]), lnDIL/W, ΔH 0T , and ΔG0T and T ΔS0T calculated with Eqs. (4) and (5) under different temperature were shown in Table 5. From Table 5, the following conclusions were drawn that (1) ΔH 0T 4 0 and partition ratios in three ILs were increased with raising temperature, illustrating that extraction process was a endothermic process and higher temperature was beneficial to extraction; (2) ΔG0T o 0, pointing out extraction was spontaneous; (3) ΔS0T 4 0 and ΔH 0T 〈 T ΔS0T , indicating that the transfer process of UPA from water to IL was driven by entropy change. It is generally recognized that hydrophobic interaction force is main feature of entropy control [26]. Based on these results, it could be deduced that hydrophobic interaction might be the main driving force for the transfer of UPA molecules from water to the IL phases.
Jiangsu Higher Education Institutions and the Foundation of the Excellence Science and Technology Invention Team in Yangzhou University and the Graduate Innovation Project Foundation of Jiangsu province (CXLX13_895).
References [1] [2] [3] [4] [5] [6] [7] [8] [9]
4. Conclusion In this paper a new and environmental friendly ultrasoundassisted ionic liquid dispersive liquid–liquid microextraction coupled with HPLC-PDA was developed for the determination of UPA in dosage form and biological sample. Without any dispersive solvent the ER could be over 95.0% under ultrasonication. The developed method can provide analytical technical support for UPA pharmacokinetics examination. Mechanism discussion of extraction indicated that hydrophobic interaction might be the main driving force for UPA extracted in IL phase.
[10] [11] [12] [13] [14] [15] [16]
Acknowledgements
[17] [18] [19] [20] [21] [22]
The authors acknowledge the financial support from the National Natural Science Foundation of China (21375117, 21155001) and a project funded by the Priority Academic Program Development of
[23] [24] [25] [26]
B.Q. Ma, Shanghai Med. Pharm. J 31 (2010) 315–316. G.F. Yao, Chin. J. Med. Chem. 21 (2011) 78–80. WO 2004/078709. J.N. Sun, Y.P. Shi, J. Chen, J. Chromatogr. B 879 (2011) 3429–3433. Y.F. Zhang, H.K. Lee, Anal. Chim. Acta 750 (2012) 120–126. C. Noelia, P.P. Francisco, I. de la Calle, B. Carlos, L. Isela, Microchem. J. 99 (2011) 246–251. S.Q. Gao, X. Yang, W. Yu, Z.L. Liu, H.Q. Zhang, Talanta 99 (2012) 875–882. A.J. Toraj, F. Nazir, S. Mojtaba, P. Meghdad, J. Pharm. Biomed. Anal. 85 (2013) 14–20. R. Mohammad, A. Yaghoub, M.H. Mohammad-Reza, A. Elham, A. Fardin, B. Sana, J. Chromatogr. A 1116 (2006) 1–9. D. Nagaraju, S.D. Huang, J. Chromatogr. A 1161 (2007) 89–97. M.A. Farajzadeh, M. Bahram, J. Jönsson, Anal. Chim. Acta 591 (2007) 69–79. M. Hatami, Elham Karimnia, Khalil Farhadi, J. Pharm. Biomed. Anal. 85 (2013) 283–287. J.M. Padro´, M.E. Marso´n, G.E. Mastrantonio, J. Altcheh, F. Garcı´a-Bournissen, M. Reta, Talanta 107 (2013) 95–102. X. Liu, R. Fu, M. Li, L.P. Guo, L. Yang, Chin. J. Anal. Chem. 41 (2013) 1919–1922. J.H. Zhang, H.X. Gao, B. Peng, S.Q. Li, Z.Q. Zhou, J. Chromatogr. A 1218 (2011) 6621–6627. M.J. Trujillo-Rodríguez, P. Rocío-Bautista, V. Pino, A.M. Afonso, Trends Anal. Chem. 51 (2013) 87–106. W.Y. Chen, H. Li, W.H. Luo, Chin. J. Mod. Med. 17 (2007) 1665–1673. F.Y. Zhao, J.Y. Wang, H.J. Liu, R.J. Liu, Chem. Reagents 29 (2007) 229–231. Y.C. Pei, Doctoral Dissertation of Lanzhou University (2008). P. Arbeláez, F. Borrull, R.M. Marcé, E. Pocurull, Talanta 125 (2014) 65–71. 〈http://www.chemspider.com/Chemical-Structure.115762.html〉. S.Y. Dong, Q. Hu, Z. Yang, R. Liu, G.Q. Huang, T.L. Huang, Microchem. J. 110 (2013) 221–226. R. Liu, J. Liu, Y. Yin, X. Hu, G. Jiang, Anal. Bioanal. Chem. 393 (2009) 871–883. 〈http://tec.k8008.com/html/124/124073.html〉. X.M. Fu, S.G. Dai, Y. Zhang, Chin. J. Anal. Chem 34 (2006) 598–602. H. Bianco-Peled, S. Gryc, Langmiur 20 (2004) 169–174.
