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Hollow fiber based liquid–liquid–liquid microextraction combined with sweeping micellar electrokinetic chromatography for the sensitive determination of second-generation antidepressants in human fluids† Xiaoqing Zhou, Man He, Beibei Chen and Bin Hu* An effective dual preconcentration method involving off-line hollow fiber liquid–liquid–liquid microextraction (HF-LLLME) and on-line sweeping micellar electrokinetic chromatography (sweeping-MEKC) was proposed for the determination of five second-generation antidepressants, including fluoxetine, sertraline, paroxetine, fluvoxamine and citalopram. In HF-LLLME, the analytes were extracted from the sample solution into phenetole impregnated in the pores of the hollow fiber and then back-extracted into 10 µL 0.1 mol L−1 HAc inside the hollow fiber. Then, the acceptor phase was spiked with 2.8 µL isopropanol (IPA) and introduced into CE for sweeping. In the sweeping-MEKC process, five target analytes were separated in less than 15 min with a background electrolyte consisting of 76% (v/v) 50 mmol L−1 citric acid ( pH 2.2) containing 100 mmol L−1 sodium dodecyl sulfate (SDS) and 24% (v/v) IPA. The hydrodynamic injection was performed at 50 mbar for 140 s. Under optimized conditions, the limits of detection were in

Received 1st December 2014, Accepted 6th January 2015

the range of 0.40–1.55 µg L−1 with enrichment factors of 1897- to 5952-fold for target analytes, with a

DOI: 10.1039/c4an02209b

dynamic linear range of 0.6/5.0–200 µg L−1. The developed method demonstrated excellent clean-up ability and high enrichment factors and was successfully applied to the analysis of target analytes in

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human urine and plasma samples.

1.

Introduction

The second generation of antidepressants is a kind of selective serotonin reuptake inhibitor (SSRI), for which fluoxetine (FLU), sertraline (SER), paroxetine (PAR), fluvoxamine (FLUV), citalopram (CIT) and venlafaxine (VEN) are six typical representatives. They have replaced traditional tricyclic antidepressants as the first-line therapeutic drugs in depression treatment due to fewer side effects and satisfactory curative effects. However, depression is a kind of disease requiring long-term medication, which often leads to drug accumulation in the body and psychological dependence on medication. In addition, numerous side effects, including gastrointestinal maladies and sexual dysfunction, have focused increasing attention on the safety and efficacy of these drugs in recent years. Therefore,

Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan 430072, China. E-mail: [email protected]; Fax: +86 27 68754067; Tel: +86 27 68752162 † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c4an02209b

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developing rapid and sensitive methods for the determination of SSRIs in human fluids is of great significance for therapeutic drug monitoring or toxicological purposes. Chromatographic techniques, such as gas chromatography (GC),1–3 high-performance liquid chromatography (HPLC),4–10 and capillary electrophoresis (CE)11–13 have been widely employed for the analysis of SSRIs in environmental and biological samples. Among them, CE offers powerful separation efficiency, rapid analysis and minimal reagent consumption. However, the majority of commercial CE instruments are equipped with diode array detectors (DAD) or ultraviolet detectors (UV), which are severely limited by matrix interference and low sensitivity for trace analysis in real samples. To overcome these limitations, the application of various preconcentration techniques, including on-column preconcentration and off-line sample pretreatment in CE, have been proven to be highly effective.12 On-column preconcentration techniques are powerful approaches for substantially improving the sensitivity of CE-UV with various operation modes available, in addition to being economical without requiring the modification of existing instrumentation. Thus far, several on-column preconcen-

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tration strategies have been developed, such as stacking,14,15 sweeping,16–18 dynamic pH junction (DpHJ),19,20 transient isotachophoresis (t-ITP)21 and some dual12,22–25 or even triple26,27 on-column preconcentration strategies, which combine the various aforementioned techniques. Sweeping, proposed by Quirino et al. in 1998,28 involves interaction between target analytes and the additives, which are usually some kind of surfactant in the micellar electrokinetic chromatography (MEKC) mode or complex reagents in the capillary zone electrophoresis (CZE) mode. Due to its simple operation and high enrichment efficiency, sweeping has been widely used for pharmaceutical analysis, such as voriconazole29 in plasma, TCAs and β-blocker drugs in wastewater30 and steroid hormones in urine31 samples. Moreover, sweeping is easily coupled with other stacking techniques, greatly improving the sensitivity of resultant dual on-column preconcentration techniques. Su et al.12 developed a cation-selective exhaustive injection-sweeping (CSEI-sweeping)-MEKC for the simultaneous analysis of five SSRIs, and the enhancement factors were higher than 5.7 × 104-fold and the limits of detection (LODs) were less than 0.22 μg L−1, whereas in real sample analysis, complicated matrices had a dramatic effect on the precision of the method and the stacking efficiency. Almost all CE on-column preconcentration techniques have rigorous demands for the sample matrix, which means that the extraction and clean-up of samples prior to CE analysis are essential. Several sample pretreatment methods, including solid-phase extraction (SPE),14 solid-phase microextraction (SPME),32,33 stir bar sorptive extraction (SBSE)34 and liquid phase microextraction (LPME),35–37 have been employed as the sample preconcentration and clean-up techniques for CE. Compared with conventional SPE, microextraction techniques, such as SPME, SBSE and LPME are more attractive due to their high enrichment factor (EF), powerful clean-up ability and low consumption of poisonous organic solvents. These microextraction techniques combined with CE on-column preconcentration techniques would not only effectively eliminate sample matrix interference but would also further improve the analytical sensitivity, largely expanding the applications of CE-UV in trace or even ultra-trace analysis. For example, SBSE combined with large-volume sample stacking (LVSS), polymer monolith microextraction combined with field-amplified sample stacking (FASS) and polymer-coated hollow fiber microextraction combined with normal stacking were proposed for the analysis of chemical warfare agent degradation products in water samples,34 sulfonamides33 and quinolone antibiotics32 in chickens, respectively. Dispersive liquid–liquid microextraction (DLLME)35 and single-drop microextraction (SDME)36,37 were coupled with FASS and LVSS/sweeping for the sensitive determination of trace drugs and certain organic pollutants in biological or environmental samples, respectively. Hollow fiber liquid–liquid–liquid microextraction (HF-LLLME)38 has been considered to be a good alternative to SDME or DLLME when coupled with CE on-column preconcentration techniques. In HF-LLLME, the ionized analytes are first extracted from an aqueous sample matrix into an organic solvent, which is

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loaded in the wall pores of hollow fiber and then backextracted into an aqueous acceptor phase inside the fiber. The aqueous acceptor phase can be directly introduced into CE for further concentration with a slight adjustment rather than that of tedious evaporation and resolvation, which is often needed in the coupling of two-phase LPME with CE analysis. Moreover, the special structure of hollow fiber can greatly improve the clean-up ability of the method. In our previous work, HF-LLLME has been combined with LVSS-CE-UV and anionselective exhaustive injection (ASEI)-CE-UV for the trace analysis of organic mercury39 and arsenic speciation40 in fish/hair and soil samples, respectively, and thousands-fold improvements were achieved in the detection sensitivity. Wang et al.41 developed a method by combining an off-line HF-LLLME procedure with an on-line sweeping-MEKC for the trace analysis of two kinds of strychnos alkaloids in urine samples; the obtained low LODs and the successful application to urine samples indicated that LPME-sweeping-MEKC was a promising combination for the analysis of basic drugs at low levels in certain biological samples. In this study, a dual preconcentration method of HF-LLLME-sweeping-MEKC was developed for the sensitive determination of five SSRIs in human fluids. HF-LLLME was used to remove sample matrix and concentrated target analytes, and sweeping-MEKC was then employed to further improve the analytical sensitivity. Moreover, different parameters affecting the extraction process and sweeping procedure were optimized in detail. The proposed method was successfully applied to determine the target analytes in human urine and plasma samples.

2. Experimental 2.1

Reagents and materials

All of the reagents and chemicals were of analytical grade. SER, FLU, VEN, PAR, FLUV and CIT were purchased from the Chinese Food and Drug Inspection Institute (Beijing, China). The structures, pKa and log P values of six target analytes are listed in Table 1. Sodium dodecyl sulfate (SDS) was obtained from Tianjin Chemical Reagent Factory (Tianjin, China). Citric acid, disodium hydrogen phosphate (Na2HPO4·12H2O), sodium hydroxide (NaOH), sodium chloride (NaCl), acetic acid (HAc), isopropanol (IPA), toluene, phenetole, chlorobenzene, n-octanol, n-decane and other organic solvents were all obtained from Sinopharm Chemical Reagent Company (Shanghai, China). High-purity deionized water obtained by a Milli-Q system (18.2 MΩ cm, Millipore, Molsheim, France) was used throughout the experiment. The Q3/2 Accurel polypropylene hollow fiber (600 μm inner diameter, 200 μm wall thickness, and 0.2 μm pore size) used for HF-LLLME was purchased from Membrana (Wuppertal, Germany). The hollow fiber was cut into 3.0 cm segments, which had an internal volume of 10 µL, and one end of the segments was sealed by pressure. Prior to use, the segments were sonicated in acetone for 8 min to remove any contami-

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Paper Table 1

Analyst Structures, log P values and pKa values of six SSRIs

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Analyte

Structure

log P (25 °C)

pKa (25 °C)

Fluoxetine FLU

3.930 ± 0.434

10.05 ± 0.10

Fluvoxamine FLUV

3.713 ± 0.504

9.39 ± 0.10

Venlafaxine VEN

2.475 ± 0.268

9.26 ± 0.28

Paroxetine PAR

3.701 ± 0.436

9.68 ± 0.10

Citalopram CIT

3.475 ± 0.600

9.57 ± 0.28

Sertraline SER

5.079 ± 0.348

9.47 ± 0.40

nants in the fiber. They were then removed from acetone and dried in air. Each piece of porous hollow fiber was discarded after a single extraction.

2.2

CE apparatus and conditions

All separation experiments were performed by a G1600A CE system (Agilent, USA) equipped with a DAD. Separations were performed in 48.5 cm (40 cm effective length) × 50 µm i.d. fused-silica capillary tubes (Yongnian Optical Fiber, Hebei, China), and the capillary temperature was maintained at 25 °C. The detection wavelength was set at 200 nm. The CE sample vial was modified according to our previous research work.40 First, one end of the pipette tip was flame-sealed and cut manually to a proper length; then, this portion of the pipette tip was inserted into a commercial sample vial for

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sample injection. The sample was injected into the CE capillary by the hydrodynamic method. During the duration of the experiment, the capillary was conditioned by successive washings with CH3OH (10 min), 0.1 mol L−1 NaOH (10 min), high-purity water (10 min) and background electrolyte (BGE) for 15 min. Between runs, the capillary was successively rinsed with water (2 min) and the BGE (3 min) when only normal MEKC (50 mbar × 10 s) was employed. When sweeping-MEKC was involved, the capillary was sequentially flushed with CH3OH (2 min), water (3 min) and BGE (5 min). Stock solutions of 0.1 mol L−1 citric acid, 0.2 mol L−1 Na2HPO4 and 0.4 mol L−1 SDS were prepared on a weekly basis and stored at 4 °C. The BGE consisted of 76% (v/v) 50 mmol L−1 citric acid ( pH 2.2, adjusted by 0.2 mol L−1 Na2HPO4 solution) containing 100 mmol L−1 SDS and 24% (v/v) IPA.

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2.3

Sample preparation

Morning urine samples were obtained from both healthy donors and patients undergoing SER therapy (Zoloft, 50 mg day−1). The urine samples collected from healthy donors were filtered through 0.45 μm filters and alkalized to pH 12.8 by NaOH solution without dilution before the HF-LLLME procedure, whereas the two patients’ urine samples were diluted with water in a ratio of 1 : 49 prior to analysis. The healthy human blood samples were supplied by the Hospital of Wuhan University (Wuhan, China). Human plasma samples were collected through the centrifugation of the blood samples at 1500 rpm for 5 min and then frozen for future use. The plasma was 100-fold diluted with water to eliminate the influence of the matrix, adjusted to pH 12.8 and subjected to subsequent HF-LLLME. The donor phase in HF-LLLME was prepared by spiking the target drugs in either high-purity water or the biological samples. It should be stressed that human fluid samples used for the current analysis do not have any identifying information about the participants that were included in this study. The ethics committee of the Hospital of Wuhan University (Wuhan, China) reviewed and approved the informed consent forms provided by all participants according to ethics requirements. 2.4

Procedures

2.4.1 HF-LLLME general procedure. A sample solution (6 mL, adjusted to pH 12.8 with NaOH) was placed in a 7 mL sample vial. 10 µL 0.1 mol L−1 HAc solution (acceptor phase) was first deposited into a 10 µL microsyringe (Gaoge, Shanghai, China) and then introduced into the lumen of hollow fiber, which was fixed on the needle end of the microsyringe. Second, the fiber was immersed into phenetole for about 25 s to impregnate the pores of the fiber with the solvent. Finally, the fiber was immediately placed into the sample solution for extraction. After stirring the sample solution for 40 min, the fiber was removed, and the acceptor phase was drawn back into microsyringe and finally injected into the modified CE sample vial for further analysis. The aforementioned acceptor phase was introduced into CE directly in a normal MEKC mode, whereas it was spiked with 2.8 µL IPA prior to CE analysis in a sweeping-MEKC mode. 2.4.2 Sweeping-MEKC procedure. After flushing the capillary by use of the running buffer, the sample solution was introduced into the capillary immediately for 140 s at a constant pressure of 50 mbar. Once the inlet and outlet vials were switched back to BGE vials, a negative voltage of 20 kV was applied along the capillary for the sweeping and separation of target SSRIs.

3

Results and discussion

3.1

Optimization of HF-LLLME

All of the target analytes in this work are cationic compounds containing different amino groups. In the HF-LLLME process,

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the sample solution was adjusted to alkaline conditions by a strong base to avoid the protonation of SSRIs, favoring their transferring into the organic phase in their molecular forms. On the other hand, the acid acceptor phase would protonize the target analytes and help them be back-extracted from the organic phase into the acceptor phase. The optimization of HF-LLLME parameters was performed in a normal MEKC mode (hydrodynamic injection for 10 s at 50 mbar). Several factors that influence the extraction efficiency, including the organic solvent, pH of sample solution, HAc concentration, stirring rate, salt effect, and extraction time were investigated in detail. 3.1.1 Effect of organic solvents. An appropriate organic solvent is of great importance for determining the extraction efficiency of HF-LLLME. A suitable organic solvent should satisfy the following criteria: water immiscibility, low vapor pressure at room temperature, high-extraction efficiency, good stability and reproducibility, suitable viscosity and ease of operation. Based on these factors, five organic solvents with various polarities (i.e., toluene, phenetole, chlorobenzene, decane and n-octanol) were evaluated for the extraction of six target SSRIs by HF-LLLME under the same conditions. As shown in Fig. 1, decane exhibited the highest extraction efficiency for SER, but it yielded the lowest extraction efficiency for the other five SSRIs among the tested solvents due to its poor polarity. Conversely, n-octanol showed the highest extraction efficiency for VEN, CIT and FLUV but the lowest extraction efficiency for SER among the tested solvents due to its high polarity. Toluene, chlorobenzene and phenetole could simultaneously extract six target SSRIs with relatively high extraction efficiencies, whereas phenetole yielded the highest extraction efficiency for the six target SSRIs among these three types of organic solvents. Hence, phenetole was selected as the extraction solvent for HF-LLLME. 3.1.2 Donor phase pH. The analytes were extracted from the sample solution into organic solvent mainly based on

Fig. 1 Influence of various organic solvents on HF-LLLME. Conditions: donor phase: 6 mL 0.1 mol L−1 NaOH sample solution ( pH 12.8) without salt addition; acceptor phase: 0.2 mol L−1 HAc; stirring rate: 800 rpm, extraction time: 30 min; sample concentration: 200 µg L−1.

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hydrophobic interaction. Therefore, the donor phase was adjusted to alkaline to avoid the protonation of analytes. The effect of NaOH concentration on the extraction efficiency was studied with its concentration varying in the range of 0–0.5 mol L−1. The results in Fig. S1† show that the extraction efficiencies were increased with increasing NaOH concentration from 0 to 0.05 mol L−1, and no obvious enhancement in the extraction efficiency was observed when the concentration of NaOH was higher than 0.05 mol L−1. Therefore, 0.1 mol L−1 NaOH was used as donor media. 3.1.3 Acceptor phase. The acceptor phase of HF-LLLME would be subjected to subsequent sweeping-MEKC, thus the selection of acceptor-phase acidic media is extremely important and rigorous. The employed acid should not only provide excellent extraction efficiency but also meet the requirements of sweeping-MEKC. In the experiment, diluted HCl, H3PO4, HAc and citric acid were preliminarily investigated for the sweeping-MEKC process, and it was found that sweepingMEKC would be deteriorated when diluted HCl and H3PO4 were used as the media, whereas HAc or citric acid had no obvious effect on the sweeping of target analytes. Therefore, HAc and citric acid were tested as the acceptor phase for the extraction of target analytes by HF-LLLME. The experimental results indicated that HAc yielded comparable extraction efficiency with citric acid, but HAc yielded better extraction reproducibility than that of citric acid. Accordingly, HAc was finally selected as the acceptor media in HF-LLLME. The effect of HAc concentration in the range of 0–0.8 mol L−1 on the extraction efficiency of target SSRIs was then investigated. The results in Fig. 2 indicate that the signal intensities of the target analytes were significantly increased with an increase of HAc concentration from 0 to 0.1 mol L−1 and then were kept almost unchanged with a further increase of HAc concentration to 0.8 mol L−1. As a result, 0.1 mol L−1 HAc was chosen as the acceptor phase.

Analyst

3.1.4 Stirring rate. Stirring can increase the mass transfer rate and shorten the extraction time. In this work, the effect of stirring rate on the extraction efficiency of target analytes was examined with the stirring rate varying in the range of 400–1200 rpm. The experimental results show that the peak area of six SSRIs were increased with an increase in the stirring rate from 400 to 1000 rpm, and further increase of the stirring rate resulted in poor reproducibility. Hence, a stirring rate of 1000 rpm was chosen for further works. 3.1.5 Extraction time. Both the extraction and back-extraction involved in HF-LLLME are equilibrium extractions, thus extraction time is of crucial influence. In this work, the effect of extraction time on HF-LLLME was investigated in the time span of 10–50 min. The experimental results in Fig. S2† indicate that except for SER, the signal intensities of other SSRIs were increased with an increase in extraction time from 10 to 30 min and then kept almost unchanged with a further increase in extraction time from 30 to 50 min. A decreased intensity was observed for SER when the extraction time was higher than 30 min. For a simultaneous extraction of six target SSRIs, 40 min was eventually chosen as the extraction time. The extraction time is comparable to that employed in other three-phase LPMEs7,8 and some SBSE-based methods.42–44 However, an extra 15 min was needed for the desorption process in the aforementioned SBSE-based methods. On the other hand, the extraction time is indeed a little longer than that employed in SPE-based method,12 which provided considerably lower EF. 3.1.6 Salt effect. The addition of salts can increase the viscosity of sample solutions, which would affect the mass transfer rate and/or change the partition coefficient. The effect of NaCl concentration in the range of 0–0.2 g mL−1 on the HF-LLLME of target analytes was investigated in this work. The results shown in Fig. S3† indicate that the signal intensities of target analytes were increased slightly when the NaCl concentration was increased from 0 to 0.02 g mL−1 and then decreased slightly with a further increase in NaCl concentration from 0.02 to 0.2 g mL−1. Therefore, 0.02 g mL−1 NaCl was finally selected as the donor phase medium for further experimentation. 3.2

Fig. 2 Influence of HAc concentration on HF-LLLME. Conditions: donor phase: 6 mL 0.1 mol L−1 NaOH sample solution without salt addition; stirring rate: 800 rpm; extraction time: 30 min; sample concentration: 200 µg L−1.

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Optimization of sweeping-MEKC

The optimization of sweeping-MEKC was performed without HF-LLLME, and the sample solution was prepared by spiking standard analytes into non-micellar BGE. 3.2.1 Buffer conditions of sweeping-MEKC. The buffer conditions employed for sweeping-MEKC referred to ref. 12 with some modifications. Fig. 3 shows the effect of IPA content in BGE on sweeping-MEKC. It is apparent that 24% IPA (v/v) resulted in a baseline separation and an appropriate separation time for six target SSRIs. Hence, 24% IPA in the BGE was chosen for further experimentation. The effect of SDS concentration on the separation and sweeping efficiency was also investigated with SDS concentration varying in the range of 50–200 mmol L−1. The results in Fig. S4† show that the signal intensities were decreased with an increase in SDS

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Fig. 3 Influence of IPA content in BGE on sweeping-MEKC. BGE: 50 mmol L−1 citric acid/Na2HPO4 ( pH 2.2) containing 100 mmol L−1 SDS and (a) 0% IPA, (b) 15% IPA, (c) 24% IPA, and (d) 30% IPA; hydrodynamic injection: 50 mbar for 120 s; and peak identification: (1) SER, (2) FLU, (3) PAR, (4) FLUV, (5) CIT, and (6) VEN.

concentration from 50 to 100 mmol L−1. However, the separation time was prolonged to 20 min, and broadened peak shapes were observed when the SDS concentration was less than 80 mmol L−1. Therefore, 100 mmol L−1 SDS was added into the buffer solution for sweeping. Finally, the BGE consisting of 76% (v/v) 50 mmol L−1 citric acid ( pH 2.2, adjusted by 0.2 mol L−1 Na2HPO4 solution) containing 100 mmol L−1 SDS and 24% (v/v) IPA was employed for further experimentation. 3.2.2 Sampling media of sweeping-MEKC. Based on the optimization of HF-LLLME, the acceptor phase was 0.1 mol L−1 HAc. The addition of appropriate IPA into the sampling media would improve the resolution and maintain the sweeping procedure. Therefore, the effect of IPA content in the sample media on the resolution in the sweeping mode was investigated in the range of 0–24% (v/v). It was found that the CE resolution and signal intensity for the target SSRIs were increased with an increase in IPA content from 0 to 22%, and no obvious change was observed for the resolution and signal intensity with a further increase in IPA content in the sample media from 22 to 24% IPA. Fig. S5a and S5b† are the electrophoretograms for the target SSRIs in 0.1 mol L−1 HAc with and without the addition of IPA. It is apparent that 0.1 mol L−1 HAc without the addition of any organic modifier greatly damaged the resolution (Fig. S5a†). Conversely, both the resolution and signal intensities were obviously improved with the addition of IPA in the sample media (Fig. S5b†). Hence, 22% IPA was added into the sampling media for sweeping,

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which means that the 10 μL acceptor phase should be spiked with 2.8 μL IPA prior to sweeping-MEKC. Under this condition, a good resolution for six target SSRIs was obtained (Fig. S5b†). 3.2.3 Injection time. The sample should be injected into the capillary as much as possible to increase the detection sensitivity. The injection time was optimized with time varying in the range of 80–170 s at a constant pressure of 50 mbar. The results in Fig. S6† indicate that the signal intensities of target SSRIs were increased with an increase of injection time from 80 to 170 s, but an overlap between the peak of SER and FLU and a slight delay in the separation time were observed when the injection time was increased to 170 s (electrophoretogram was not shown). Therefore, the injection time was set at 140 s in subsequent experiments.

3.3

Analytical performance

Above all, the optimal conditions for the HF-LLLME included phenetole serving as the organic solvent, the acceptor phase containing 0.1 mol L−1 HAc, the sample solution (donor phase) being adjusted to pH 12.8 with the addition of 0.02 g mL−1 NaCl, the stirring rate at 1000 rpm, and the extraction time at 40 min. The optimal conditions for the sweepingMEKC included the BGE at 76% (v/v) 50 mmol L−1 citric acid ( pH 2.2, adjusted by 0.2 mol L−1 Na2HPO4 solution) containing 100 mmol L−1 SDS and 24% (v/v) IPA, the sample media being the acceptor phase of HF-LLLME with an addition of

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Fig. 4 Electrophoretograms of the analysis of SSRIs by (a) HF-LLLME, (b) sweeping-MEKC and (c) HF-LLLME-sweeping-MEKC; sample concentrations: (a) 200 µg L−1, (b) 2000 µg L−1 and (c) 10 µg L−1 for each analyte, respectively; and peak identification: (1) SER, (2) FLU, (3) PAR, (4) FLUV, (5) CIT, (6) VEN, and (7) phenetole.

2.8 μL IPA, and the hydrodynamic injection performed at 50 mbar for 140 s. Unfortunately, under the aforementioned optimal conditions, the VEN cannot be quantified by HF-LLLME-sweepingMEKC because its peak was overlapped by the solvent peak of phenetole (see Fig. 4c). Therefore, in subsequent experiments, VEN was excluded in the evaluation of analytical performance and application potential of this method. For the other target SSRIs, the analytical performance of the proposed method (HF-LLLME-sweeping-MEKC), including LODs, precision, EFs and linear range, was evaluated, and the results are summarized in Table 2. The relative standard deviations (RSDs) of the peak area (n = 7) were in the range of 7.2–11.6%. High-purity water spiked with target analytes in a series of concentrations (0.6, 1.5, 2.0, 5.0, 10, 50, 100, 150, 200 and 250 μg L−1) was prepared for the evaluation of the linearity of the proposed method. In addition, the linear ranges of 1.5–200 μg L−1 for SER (r2 = 0.9972) and FLU (r2 = 0.9990), 2.0–200 μg L−1 for PAR (r2 = 0.9996), 5.0–200 μg L−1 for FLUV (r2 = 0.9961), and 0.6–200 μg L−1 for CIT (r2 = 0.9998) were

Table 2

obtained. The LODs (S/N = 3) obtained for five SSRIs ranged from 0.40 to 1.55 μg L−1. The obtained high LOD of 1.55 μg L−1 for FLUV presumably resulted from the low EF and poor UV response. The EFs, which were defined as the ratio of LODs obtained before and after dual concentration, ranged from 1897- to 5952-fold. For CIT containing two tertiary amino groups, which have strong alkalinity, back-extraction in the HF-LLLME process was simple, and EF as high as 5952-fold was obtained after dual concentration; and for SER and FLU containing two phenyl groups, which are more hydrophobic, they could be effectively swept by SDS in the sweeping-MEKC procedure, and EFs of 2327- and 2903-fold were obtained, respectively. In addition, both the extraction and sweeping efficiencies for FLUV and PAR were low- to mid-level, leading to EFs of about 1897- and 1923-fold, respectively. A comparison of LODs obtained by the proposed method with those obtained by different strategies for the analysis of SSRIs is shown in Table S1.† The LODs of the corresponding analytes obtained by this method were lower than those obtained by other methods, including DLLME-FASS-CE-UV,35 HF-LLLME followed by HPLC-UV8 and HPLC-fluorescence (FL)7 analysis, and comparable with that obtained by SPME and in-tube SPME followed by GC-mass spectrometry (MS)3 and HPLC-MS10 analysis. SPE-CSEI-sweeping-MEKC method12 exhibited an extremely high improvement in sensitivity (5.7 × 104–1.2 × 105-fold); however, the complicated plasma matrices degrade the reproducibility of the method and even the stacking efficiency with a SPE process for the elimination of complex matrix. Compared with ref. 12, LODs obtained by the developed method were slightly higher for the target analytes. However, the linear range of the developed method was sufficiently wide (0.6/2.0–200 μg L−1) for the analysis of real biological samples (the concentrations of SSRIs in patients’ urine are usually at the level of dozens to thousands of μg L−1 (ref. 1,10)). Moreover, the employed HF-LLLME cannot only remove the sample matrix but can also highly preconcentrate the target analytes. The obtained acceptor phase can be directly introduced into the capillary for sweeping just after spiking with 2.8 μL IPA, instead of tedious evaporation and resolvation. Above all, the developed method greatly enhances the sensitivity of CE-UV detection for the analysis of the target

Analytical performance of the HF-LLLME-sweeping-MEKC method

LOD/µg L−1 Analytes

Linear range/µg L−1

Linear equation/µg L−1

r2

RSDa/% (n = 7)

SIb

Sweeping

This method

EFc

SER FLU PAR FLUV CIT VEN

1.5–200 1.5–200 2.0–200 5.0–200 0.6–200 —

Y = 2.12x − 6.06 Y = 3.84x + 0.63 Y = 3.63x − 1.50 Y = 1.86x − 5.33 Y = 8.74x + 2.43 —

0.9972 0.9990 0.9996 0.9961 0.9998 —

11.6 8.9 10.4 8.8 7.2 —

932 1219 1442 2941 1071 1485

19.7 20.9 35.2 47.3 31.0 36.7

0.40 0.42 0.75 1.55 0.18 —

2327 2903 1923 1897 5952 —

RSD: SER, 4.0 µg L−1; FLU, 4.0 µg L−1; PAR, 6.0 µg L−1; FLUV, 10 µg L−1; CIT, 2.0 µg L−1. b SI: standard injection (normal MEKC), 50 mbar for 3 s. The samples for normal MEKC analysis were prepared directly by spiking certain analytes in 100 mmol L−1 HAc solution, and no IPA was added. c EF = LOD (normal MEKC)/LOD (HF-LLLME-sweeping-MEKC).

a

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five SSRIs and expands the application of CE-UV in real sample analysis.

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3.4

Real sample analysis

Human urine and plasma samples were analyzed for the validation of the proposed method. The external standard method was used for quantification, and the peak area was used for calculation. High purity water spiked with target analytes in various concentrations (2, 5, 10, 30, 100, 150, and 200 μg L−1) was subjected to the proposed procedure and used as the calibration curve. Before the analysis, the effect of human plasma matrix on the HF-LLLME of the target analytes was investigated by direct CE-UV measurement. For this purpose, an appropriate amount of plasma samples collected from healthy people were spiked with a certain amount of target analytes, adjusted to pH 12.8 by NaOH solution, and diluted to a certain volume with different dilution folds (final volume/original plasma sample volume, 5-, 10-, 25-, 50- and 100-folds, respectively). The concentration of each target analyte in the final solution for the subsequent extraction was fixed as 250 μg L−1. For comparison, an aqueous solution sample containing each target analyte also at 250 μg L−1 was prepared by using high purity water instead of the human plasma sample. The aforementioned diluted plasma samples and prepared aqueous solution sample were subjected to the HF-LLLME procedure as described in section 2.4.1, and the results are shown in Fig. S7.† As can be seen, the signal intensity of six target analytes was obviously increased when the dilution fold was increased from 5- to 100-fold. It was presumed that the high viscosity of plasma along with the macromolecular matrix could block the film holes of the hollow fiber and greatly reduce the mass transfer rate of the analytes during the extraction process. A negligible difference in extraction efficiency for the target analytes was observed between 100-fold diluted plasma matrix and the aqueous solution sample without plasma matrix addition, indicating that the matrix effect

Table 3

resulted from plasma could be negligible after 100-fold dilution. Therefore, 100-fold dilution was employed for real plasma sample analysis. The urine matrix exhibited no obvious effect on the HF-LLLME of target analytes and was directly subjected to the proposed method without any dilution. The analytical results of target SSRIs in healthy human urine and plasma samples by the proposed HF-LLLME-sweeping-MEKC method are listed in Table 3, along with the recovery for the spiked samples. It is apparent that no target analyte was detected in urine and plasma samples from healthy donors, and the recoveries for the spiked urine and plasma were in the ranges of 86.8–109% and 87.6–110%, respectively. In addition, the proposed method was used to analyze two patients’ urine samples. As shown in Fig. 5A, an unknown peak was found following the SER peak. It was suspected to be the N-demethylsertraline peak, the main metabolite of SER, which needs further identification and confirmation. The SER concentration found in the diluted urine samples were 26.1 ± 1.2 and 6.63 ± 0.13 µg L−1 (see Table 4), respectively, which means that the SER in the initial patients’ urine samples were 1.31 and 0.332 mg L−1, respectively. In order to validate the accuracy of the results, 10 and 50 µg L−1 SER were spiked into the diluted urine samples, and the recoveries were in the ranges of 84.8–99.3% and 90.8–94.7% for the two patients, respectively. The electrophoretograms of the real samples are presented in Fig. 5, which clearly demonstrated that HF-LLLME remarkably reduced the complex matrix interference in urine samples, and the proposed dual preconcentration method greatly enhanced the signal intensities of the target analytes. Compared with ref. 41, in which the similar strategy of HF-LLLME-sweeping-MEKC was used for the analysis of two kinds of strychnos alkaloids in urine samples, more complete and detailed optimization of both HF-LLLME and sweepingMEKC procedures along with a careful investigation of the

Analysis of the target SSRIs in human urine/plasma samples by the HF-LLLME-sweeping-MEKC method

Urine

Plasma

Analytes

Added (µg L )

Found (µg L )

Recovery (%)

Found (µg L−1)

Recovery (%)

SER

0 5 50 0 5 50 0 5 50 0 10 100 0 5 50

N.D.a 4.70 ± 0.14 50.6 ± 2.5 N.D. 4.74 ± 0.20 48.7 ± 3.9 N.D. 4.58 ± 0.31 43.4 ± 5.9 N.D. 9.15 ± 0.52 99.2 ± 4.7 N.D. 5.44 ± 0.13 47.3 ± 5.2

— 94.0 101 — 94.8 97.4 — 91.6 86.8 — 91.5 99.2 — 109 94.6

N.D. 4.46 ± 0.44 46.8 ± 1.9 N.D. 4.62 ± 0.37 43.8 ± 1.0 N.D. 4.79 ± 0.35 45.7 ± 3.6 N.D. 10.0 ± 0.70 109 ± 6.0 N.D. 5.53 ± 0.38 50.9 ± 2.3

— 89.3 92.2 — 92.3 87.6 — 95.7 91.4 — 100 109 — 110 102

FLU PAR FLUV CIT

a

−1

−1

N.D.: not found.

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Fig. 5 Electrophoretograms of SSRIs in the urine samples of (A) patient 1 and (B) patient 2. Urine samples were analyzed by (a) normal-MEKC (hydrodynamic injection: 50 mbar for 3 s), (b) sweeping-MEKC (hydrodynamic injection: 50 mbar for 140 s), and (c) HF-LLLME-sweeping-MEKC.

Table 4 Analysis of SER in two patients’ urine samples by this developed method

10-fold diluted urine

Added (µg L−1)

Found (µg L−1)

Recovery (%)

Patient 1

0 10 50 0 10 50

26.1 ± 1.2 36.0 ± 2.1 68.5 ± 2.5 6.63 ± 0.13 16.1 ± 0.1 52.0 ± 2.8

— 99.3 84.8 — 94.7 90.8

Patient 2

Total SER concentration in undiluted urine (mg L−1) 1.31 0.332

interference of urine matrix were provided in this work, and therefore, thousands-fold sensitivity improvement (1897- to 5952-fold), was obtained in this work, which is much higher than that obtained (50- to 35-fold) in ref. 41. Additionally, quantification without the use of the spiked blank urine samples as the calibration standards and no internal standard required are undoubtedly the advantages of this work.

4 Conclusions A HF-LLLME-sweeping-MEKC method was developed for the simultaneous and highly sensitive determination of five widespread consumed SSRIs, including SER, FLU, PAR, FLUV and CIT. HF-LLLME was employed for removing sample matrix interference and the selective extraction of the target analytes, and sweeping-MEKC was used to further improve the detection sensitivity of the subsequent CE-UV. The established HF-LLLME-sweeping-MEKC method provides low LODs (0.18–1.55 µg L−1), good reproducibility, a wide linear range, ultrahigh EF and good anti-interference capacity. The combination of HF-LLLME-sweeping-MEKC exhibits good application potential in the analysis of target SSRIs in urine/plasma samples and can be further extended to the analysis of small

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basic organic molecules at very low levels in various biological samples.

Acknowledgements Financial support from the National Natural Science Foundation of China (nos. 20775057, 21175102), the Science Fund for Creative Research Groups of NSFC (nos. 20621502, 20921062) and the Fundamental Research Funds for the Central Universities (114009, MOE China) are gratefully acknowledged.

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