Materials Science and Engineering C 44 (2014) 69–75
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Molecularly imprinted solid-phase extraction of glutathione from urine samples Renyuan Song ⁎, Xiaoling Hu, Ping Guan, Ji Li, Na Zhao, Qiaoli Wang School of Natural and Applied Science, Northwestern Polytechnical University, Xi'an 710072, PR China
a r t i c l e
i n f o
Article history: Received 5 December 2013 Received in revised form 10 June 2014 Accepted 1 August 2014 Available online 7 August 2014 Keywords: Glutathione Molecularly imprinted polymer Living radical polymerization Selectivity extraction Solid-phase extraction
a b s t r a c t Molecularly imprinted polymer (MIP) particles for glutathione were synthesized through iniferter-controlled living radical precipitation polymerization (IRPP) under ultraviolet radiation at ambient temperature. Static adsorption, solid-phase extraction, and high-performance liquid chromatography were carried out to evaluate the adsorption properties and selective recognition characteristics of the polymers for glutathione and its structural analogs. The obtained IRPP-MIP particles exhibited a regularly spherical shape, rapid binding kinetics, high imprinting factor, and high selectivity compared with the MIP particles prepared using traditional free-radical precipitation polymerization. The selective separation and enrichment of glutathione from the mixture of glycyl-glycine and glutathione disulfide could be achieved on the IRPP-MIP cartridge. The recoveries of glutathione, glycyl-glycine, and glutathione disulfide were 95.6% ± 3.65%, 29.5% ± 1.26%, and 49.9% ±1.71%, respectively. The detection limit (S/N = 3) of glutathione was 0.5 mg·L−1. The relative standard deviations (RSDs) for 10 replicate detections of 50 mg·L−1 of glutathione were 5.76%, and the linear range of the calibration curve was 0.5 mg·L−1 to 200 mg·L−1 under optimized conditions. The proposed approach was successfully applied to determine glutathione in spiked human urine samples with recoveries of 90.24% to 96.20% and RSDs of 0.48% to 5.67%. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Glutathione (GSH, 5-L-glutamyl-L-cysteinylglycine) is a tripeptide with three amino acid residues [1–3]. Several methods, including spectrofluorometry [4], capillary electrophoresis [5], and chromatography [1] have been presented to analyze GSH in biological matrices. Although such methods offer more sensitive approaches to GSH analysis, relatively expensive instruments, which are not yet available in all laboratories, are required. In addition, most of the reported determination methods have difficulty in direct separation and enrichment of GSH from complex matrices through sample pretreatment. Generally, GSH is present in complex samples at low concentrations; thus, developing a novel, simple, and fast determination method appropriate for GSH is necessary. Molecularly imprinted polymers (MIPs) are synthetic polymers with specific cavities designed for template molecules [6,7]. MIP applications have attracted significant attention in solid-phase extraction (SPE) as sorbent for generating three-dimensional cross-linked polymeric materials with a “memory” for the shape and functional group positions of template molecules [8]. Molecularly imprinted SPE (MISPE) is a simple and feasible alternative to multi-step SPE method for the preconcentration of target molecules in complex samples, for example, ⁎ Corresponding author. Tel./fax: +86 29 88431639. E-mail address: [email protected] (R. Song).
http://dx.doi.org/10.1016/j.msec.2014.08.005 0928-4931/© 2014 Elsevier B.V. All rights reserved.
extraction of pollutants from river water and various biomolecules from food or vegetables [9–12]. Dinçer et al. [13] synthesized monolith MIPs for GSH via traditional bulk polymerization by using 1-vinyl imidazole as functional monomer. The monolith GSH-imprinted polymers were used as SPE material for detection of GSH in biological samples. A similar method was used by Tong et al. [14]. However, the monolith MIPs prepared through traditional radical polymerization have disadvantages, including broad binding site heterogeneity, relatively low binding affinity, slow kinetics, and low selectivity [15]; such disadvantages are attributed to the uncontrollable chain propagation and termination of the monolith MIPs. Thus, the propagation and termination steps of the reaction should be controlled. The introduction of controlled living radical polymerization (CRP) into imprinted polymerization systems can considerably improve the homogeneous network structures of MIPs and elucidate their structure– property relationship. CRP is a very effective tool for controlling cross-linked polymer structures. Thus far, many different polymer networks with homogeneous structures have been prepared using CRP. Iniferter is an ideal candidate for CRP because of its versatility and simplicity. Iniferter, an initiator-chain transfer molecule, was first discovered by Otzu et al. [16] in 1982. Since then, iniferter has been used to produce linear polymer chains with low polydispersities, specific block copolymers, graft polymers, and cross-linked polymer systems on surfaces [17–22]. Several successful molecular imprinting applications have been reported.
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For example, Shufang et al. [23] prepared sulfamethazine-imprinted core-shell particles by using surface-initiated iniferter-controlled living radical polymerization (IRP) on silica particles. The resultant core-shell MIPs showed high affinity toward the template. A similar method was used by Barahona et al. [24]. Recently, Zhang et al. [25] reported that IRP combined with precipitation polymerization can effectively improve the binding and structural characteristics of MIP particles. Nevertheless, these studies mainly focused on the formation of MIPs on surfaces by using iniferter. By contrast, few studies have been conducted on the direct preparation of functional MIP particles, particularly imprinting of biomolecules, using IRP in the presence of the target molecule. In this study, iniferter-controlled living radical precipitation polymerization (IRPP) method was applied to obtain MIP particles. The apparent morphology, composition, kinetics, adsorption isotherm, and selectivity of the IRPP-MIP particles were characterized with GSH as the model system. The adsorption speed, adsorption capacity, and selectivity of the IRPP-MIP particles improved remarkably because of their uniform distribution and homogenous binding sites compared with the MIP particles prepared through traditional free-radical precipitation polymerization (TRPP). In addition, the obtained IRPP-MIP particles were applied as SPE sorbent material for the selective separation and enrichment of GSH from its structural analogs or human urine samples. 2. Material and methods 2.1. Reagents and chemicals GSH, glutathione disulfide (GSSG), and glycyl-glycine (Gly-Gly) were purchased from Shanghai Jingchun Ke Fine Chemical Corporation (Shanghai, China). Azobisisobutyronitrile (AIBN) was purchased from Tianjin Guangfu Chemical Industry (Tianjin, China). Ethylene glycol dimethacrylate (EGDMA) and 4-vinyl pyridine (4-VP) were purchased from Alfa (Shanghai, China). Tetramethylammonium hydroxide, phenylisothiocyanate (PITC), high-performance liquid chromatography (HPLC)-grade acetonitrile, and triethylamine were provided by Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). All chemicals were of analytical grade and used without further purification. Benzyl N,N-diethyldithiocarbamate (BDC) was prepared following the literature procedure [26]. 2.2. Synthesis of imprinted polymer particles IRPP-MIP particles were synthesized according to the method of Zhang et al. [25]. In a typical synthesis, a mixture of GSH (51.2 mg, 0.167 mmol), 4-VP (0.11 mL, 1 mmol), EGDMA (0.75 mL, 4 mmol), and BDC (35.85 mg, 0.15 mmol) was dissolved in an appropriate porogenic solvent (acetonitrile/water = 9/1, v/v). The mixed solution was sequentially added into a four-neck round-bottom quartz flask (100 mL). After sealing, mixing, and sparging the mixture with nitrogen for 30 min, polymerization was initiated using ultraviolet radiation from a high-pressure mercury lamp (300 W, 365 nm) with stirring at ambient temperature for 12 h. After polymerization, the resulting polymer was extracted with a mixture of ethanol, water, and acetic acid (70/28/2, v/v) by using a Soxhlet apparatus for 48 h. The obtained polymer was washed thrice with ethanol and water and then dried in a vacuum at 40 °C for 36 h. AIBN (17.20 mg, 0.104 mmol) was used as initiator instead of BDC for the MIP particles prepared using TRPP. All other conditions were similar to the preparation of IRPP-MIP. The corresponding nonimprinted polymer (NIP) particles were prepared and purified under the same conditions but without template addition. 2.3. Characterization of chemical structure and morphology The chemical structures of the obtained polymer were identified with a Fourier-transform infrared spectrometer (FT-IR; Shimadzu,
WQF-310) by using KBr compressed pellet method. The surface morphology of the obtained polymer was determined through scanning electron microscopy (SEM, FEI, Quanta 600FEG). 2.4. Static adsorption experiments Static adsorption experiments were carried out to evaluate the adsorption kinetics and adsorption isotherms at 25 °C. In a typical procedure, 10 mg of MIP/NIP was incubated with 10 mL of 0.5 mg·mL−1 GSH solution (pH 7.0) for different times. After filtration using an acetate membrane (0.45 μm pore size), the residual concentration of GSH was determined through HPLC, and the adsorption capacity Q (mg·g−1) was calculated according to the following formula (1): Qe ¼
ðC 0 −C e ÞV m
ð1Þ
where C0 (mg·mL−1) and Ce (mg·mL−1) represent the initial and residual concentrations, respectively. V (mL) is the volume of adsorption solution, and m (g) is the amount of adsorbent. Equilibrium adsorption experiments were performed by incubating MIP/NIP with different concentrations of GSH solution for 8 h. Q is calculated using Eq. (1), and the imprinting factor (α) is obtained according to the following Eq. (2): α¼
Q MIP Q NIP
ð2Þ
where QMIP and QNIP are the adsorption capacities of MIP and NIP, respectively. The effect of initial pH on the recognition property of MIP/NIP toward GSH was determined using batch equilibrium adsorption experiments. The adsorption experiments were performed by incubating MIP/NIP with different pH of the GSH solution for 8 h. 2.5. Selectivity experiments In a typical procedure, 10 mg of MIP/NIP was incubated with 10 mL of GSH and 0.2 mg·mL−1 of the similar structural compounds (pH 7.0) at 25 °C for 8 h. The adsorption capacities of GSH or similar structural analogs were obtained by measuring the residual concentration similar to the batch experiments. The selectivity recognition factor (β) of the MIP was calculated according to the following Eq. (3): β¼
α Tem α Ana
ð3Þ
where αTem and αAna are the imprinting factors of MIP for GSH and its structural analogs, respectively. 2.6. SPE experiments The obtained IRPP-MIP particles (200 mg) were packed into an empty SPE cartridge with two polypropylene upper and lower frits at each end. The entire MISPE cartridge was carefully washed with sufficient methanol and preconditioned with a mixture of acetonitrile and water (7/3, v/v) at a flow rate of 0.5 mL·min− 1 prior to use. Subsequently, 50 mL of the sample solution containing 50 μg·L−1 GSH, GSSG, or Gly-Gly was passed through the preconditioned MISPE cartridge at a specific flow rate. After loading, the MISPE cartridge was first washed with 3.0 mL of a mixture of acetonitrile and water (7/3, v/v) to remove the sample molecules retained by non-specific adsorption from the cartridge. The adsorbed analytes on the MISPE cartridge were eluted using 3.0 mL of a mixture of ethanol, water, and acetic acid (70/28/2, v/v). Finally, the eluate was analyzed through HPLC by using pre-column derivation with PITC at a flow rate of 0.5 mL·min−1
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Fig. 1. Preparation process of GSH-imprinted polymer particles by IRPP.
at 30 °C. By contrast, the C18 cartridge was used under the same procedures with C18 as sorbent material. 2.7. Analysis of GSH and its structural analogs The concentrations of GSH and its structural analogs were determined using HPLC (Shimadzu, LC-2010A) after derivatization with
PITC. In a typical procedure, 200 μL of amino acid solution, 100 μL of PITC (100 mmol·L−1), and 100 μL of the mixture of triethylamine (1 mol·L−1) and acetonitrile were mixed in a tube. The tube was then sealed and allowed to react for 1 h in a 30 °C water bath. About 400 μL of n-hexane was used to extract the remaining PITC and collect the liquid from the lower layer for detection. The chromatography conditions were as follows: mobile phase, acetonitrile and 10 mmol·L− 1
Fig. 2. SEM images of (a) IRPP-MIP/(b) IRPP-NIP and (c) TRPP-MIP/(d) TRPP-NIP.
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Fig. 5. Adsorption kinetics of MIP and NIP toward GSH. Fig. 3. FT-IR spectra of (a) IRPP-MIP and (b) TRPP-MIP.
aqueous solution of phosphate buffer (5/95, v/v); flow rate, 0.5 mL·min− 1; and UV detector wavelength, 254 nm. 3. Results and discussion 3.1. Preparation and characterization of MIP particles The general scheme for the synthesis of IRPP-MIP is illustrated in Fig. 1. BDC was selected as iniferter to improve the recognition properties of the MIP networks in the precipitation polymerization systems. Thus, considering the iniferter characteristics, the obtained MIP particles could have uniform particle size distribution and highly specific recognition performance when prepared through IRPP. In this study, the imprinted polymerization system used GSH, 4-VP, EGDMA, and a mixture of acetonitrile and water as the template, functional monomer, cross-linker, and porogenic solvent, respectively. Polymerization was initiated with an ultraviolet radiation in the presence of an iniferter agent at ambient temperature. Fig. 2 shows that the IRPP-MIP had a smooth surface and fine spherical degree. IRPP-MIP demonstrated a larger particle size and homogeneous distribution (number-average diameter, 0.86 μm; polydispersity index, 1.130) compared with TRPP-MIP (number-average diameter, 0.45 μm; polydispersity index, 1.312). This result revealed the two different types of particle formation mechanisms similar to that of a previously studied traditional precipitation polymerization system [25]. Moreover, the particle sizes of IRPP-MIP and TRPP-MIP were lower
Fig. 4. Effect of pH on the adsorption capacity of MIP and NIP toward GSH.
than those of the corresponding IRPP-NIP and TRPP-NIP, respectively; this finding indicated that the template compound markedly affected the nucleation and growth during precipitation polymerization [27]. Fig. 3 shows the FT-IR spectra of TRPP-MIP (a) and IRPP-MIP (b), which had similar chemical structures. The presence of three significant peaks at 1730 (C_O stretching), 1256, and 1150 cm− 1 (C\O\C stretching) confirmed the existence of poly(EGDMA) in TRPP-MIP and IRPP-MIP. The characteristic peaks corresponding to C_N stretching (1601 and 1640 cm− 1) and C_C stretching (1455 cm− 1) from the pyridine rings were also observed. Thus, the functional monomer 4-VP was successfully polymerized in MIPs. Moreover, compared with the FT-IR spectra of TRPP-MIP, IRPP-MIP demonstrated the characteristic peaks of the thiocarbonyl group from the iniferter groups at 1208 cm− 1 (C_S stretching), as well as at 1421 and 1354 cm− 1 (CH stretching) [27]. This finding revealed that the iniferter was successfully introduced onto the surface of IRPP-MIP.
3.2. Evaluation of adsorption and selectivity of MIP particles 3.2.1. Effect of pH on adsorption properties Molecular recognition is mainly attributed to the binding sites that complement the template molecules in terms of shape, size, and chemical functionality of imprinting cavities [28]. Electrostatic interactions also affect specific template recognition, particularly in peptides or protein molecules. Thus, the effect of pH in 0.05 mol·L−1 phosphate buffer solution on GSH adsorption was evaluated at four different pH (5.0, 6.0, 7.0, and 8.0) by using a specific amount of dilute NaH2PO4 or
Fig. 6. Adsorption isotherms of MIP and NIP toward GSH.
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adsorption behavior of GSH on MIP and NIP can be illustrated by adsorption isotherms (Fig. 6). The saturation capacities to IRPP-MIP and TRPPMIP for GSH were 20.76 and 16.50 mg·g−1, respectively. The adsorption capacity of IRPP-MIP was 25.8% higher than that of TRPP-MIP, indicating that improved binding capacities were achieved in TRPP-MIP. The equilibrium adsorption isotherm that agreed with the experimental data (top inset, Fig. 5) indicated the homogenous distribution of imprinting sites in IRPP-MIP. This finding suggested that the increased binding capacities of IRPP-MIP are mainly caused by their relatively higher binding sites. These results indicated that the homogenous cross-linked network structure of MIP was improved by the intrinsic advantages of IRPP in the process of imprinting, resulting in increased binding properties of MIP. Thus, the forms of IRPP-MIP materials could have an improved binding capacity, kinetics, and site accessibility. The functional groups were randomly distributed, which resulted in low adsorption ability of GSH, because NIP lacked the imprinting process. Thus, nonspecific adsorption of GSH was observed. Therefore, the template absorption of IRPP-MIP and TRPP-MIP was higher than those of the IRPP-NIP and TRPP-NIP because of the imprinting effect.
Fig. 7. Chemical structures of GSH and its analogs.
Na2HPO4 solution for adjustment. Fig. 4 shows that the adsorption capacities of all MIP/NIP evidently increased with increasing pH. The results can be attributed to the decreased pH of the GSH solution from 8.0 to 5.0, which resulted in a more positively charged GSH (pI 5.93) because of protonation. Similarly, IRPP-MIP and TRPP-MIP showed more positive changes caused by the protonation of functional groups (4-VP) in the imprinting cavities. These results indicated that the electrostatic interactions between GSH and MIP enhanced the binding ability at relatively high pH. IRPP-NIP and TRPP-NIP showed lower adsorption capacities than correspond to MIP because of the absence of recognition site. Moreover, IRPP-MIP had higher adsorption capacity than TRPP-MIP because of their homogeneous cross-linked polymer networks and intrinsic IRP advantages. These results indicated that electrostatic interaction had an important function in template recognition. Therefore, pH 7.0 was selected as the optimum pH. 3.2.2. Adsorption kinetic The binding kinetics of GSH with the MIP and NIP prepared through IRPP and TRPP was evaluated using batch adsorption experiments. Fig. 5 shows the adsorption uptake of the polymer versus the incubation time. IRPP-MIP reached the binding equilibrium at 120 min, indicating a relatively fast binding process. In addition, the equilibrium loading capacity of IRPP-MIP was higher than that of TRPP, suggesting that IRPP effectively generated MIP with better mass-transfer properties [29]. By contrast, no specific adsorption was observed for the two types of NIP because no suitable imprinted cavities existed for GSH recognition. 3.2.3. Adsorption isotherm Equilibrium binding experiments were performed to determine the rebinding properties of MIP and NIP. The adsorption capacity and
3.2.4. Selectivity Selectivity test was performed using the structural analogs, such as GSSG and Gly-Gly (Fig. 7) as control compounds. Table 1 shows that MIP exhibited significant binding selectivity for the template molecules. The imprinting factor and adsorption capacity of MIP for GSH were higher than those for the structural analogs, thereby verifying the specific recognition toward the template. In addition to GSH, MIP also adsorbed Gly-Gly and GSSG; this finding demonstrated the presence of a specific cross-binding reactivity. Although the same hydrogen bond can form between the structural analogs and functional groups in imprinting cavities because of the similar structure to the template molecule, the binding amount was lower than that of the template molecule. The results demonstrated that the specific recognition mechanism of MIP was related to the interactional force between the template and functional groups, as well as to the complementary match of imprinting cavities with template molecule in terms of size and shape [30]. The experimental results confirmed this conclusion, and the selectivity coefficients of IRPP-MIP toward GSSG and Gly-Gly were determined to be 1.95 and 2.11, respectively. TRPP-MIP had lower selective coefficients toward the structural analogs than IRPPMIP because IRPP generated more homogeneous binding imprinting cavities. No significant imprinted cavities and sites were observed in IRPP-NIP and TRPP-NIP, and the functional sites of 4-VP in the polymer network were disorderly arranged. Thus, NIP was selective toward these compounds through nonspecific interaction [31]. These results strongly confirmed that the IRPP approach had high binding selectivity for the preparation of IRPP-MIP. 3.3. Solid-phase adsorption behavior of the MIP 3.3.1. Optimization of extraction procedure The potential application of IRPP-MIP for the selective preconcentration of GSH in complex samples was investigated. The composition and volume of elution solution and the loading flow rate were optimized to achieve good sensitivity and precision for this approach.
Table 1 Imprinting efficiency and selectivity coefficients of MIP and NIP. Compound
IRPP
TRPP
Q (mg·g−1)
GSH GSSG Gly-Gly a
MIP
NIP
10.60 ± 0.43 14.56 ± 0.24 3.89 ± 0.30
4.59 ± 0.51 12.36 ± 0.43 3.56 ± 0.22
α
βa
2.30 1.18 1.09
– 1.95 2.11
Q (mg·g−1) MIP
NIP
7.11 ± 0.47 11.53 ± 0.36 3.80 ± 0.14
4.63 ± 0.37 10.69 ± 0.63 3.46 ± 0.25
Selectivity factor, β = αTem/αAna, αTem and αAna are the imprinting factors of MIP/NIP toward the template and its structural analogs, respectively.
α
β
1.54 1.08 1.10
– 1.43 1.40
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The effects of different elution solution on desorption of GSH from the MISPE cartridge were determined with a mixture of methanol and water, as well as with a mixture of methanol, water, and acetic acid. When a mixture of methanol and water (7/3, v/v) was utilized as the elution solution, no GSH signal appeared in the chromatograms using pre-column derivatization with PITC; however, some GSH could be extracted from the MISPE cartridges by eluting with a mixture of methanol, water, and acetic acid. A mixture of methanol, water, and acetic acid (70/28/2, v/v) was selected as the eluent because of its requirement for the adsorption analytes to be completely desorbed from the MISPE cartridge. A series of experiments was performed to confirm the optimum volume of elution solution. The different volumes of elution solution were evaluated while the other conditions were fixed. The volumes ranged from 0 mL to 4.0 mL of the mixture of methanol, water, and acetic acid (70/28/2, v/v). With the increase of the eluent volume from 0 mL to 4.0 mL, the chromatographic peak area of GSH appeared increasing at first (from 0 mL to 2.5 mL) and then tended to a specific value (from 2.5 mL to 4.0 mL). Therefore, 3.0 mL of the eluent volume was selected to ensure the complete desorption of GSH from the MISPE cartridge. Loading flow rate is an important parameter that affects the effective extraction of GSH in the SPE process. The results showed that the chromatographic peak area of GSH increased with decreasing loading flow rate from 1.0 mL·min−1 to 0.5 mL·min−1; however, the variation was not evident. As the loading flow rate decreased from 0.5 mL·min−1 to 0.35 mL·min−1, no variation appeared in the chromatograms. Thus, 0.5 mL·min−1 loading flow rate was used as the optimum condition for further experiments. 3.3.2. HPLC evaluation of selective separation The potential application of IRPP-MIP for the selective preconcentration of GSH in complex samples was evaluated. The MISPE experiment was passed through 50 mL of standard sample solution containing 50 μg·L−1 each of GSH, GSSG, and Gly-Gly at a flow rate of 0.5 mL·min−1. The MISPE cartridge was washed initially with 3.0 mL of the mixture of acetonitrile and water (7/3, v/v) to remove the analytes retained by non-specific adsorption through IRPP-MIP sorbent. The cartridge was then eluted with 3.0 mL of the mixture of methanol, water, and acetic acid (70/28/2, v/v), and the eluate was analyzed through HPLC using pre-column derivation with PITC at flow rate of
Fig. 9. Recoveries of GSH, Gly-Gly, and GSSG on MISPE cartridges.
0.5 mL·min−1 at 30 °C. The mobile phase used for HPLC experiment was a mixture of acetonitrile and 10 mmol·L− 1 aqueous solution of phosphate buffer (5/95, v/v), whereas UV–vis detector was operated at 254 nm. As shown in Fig. 8, peaks 1, 2, and 3 were identified as GSH, Gly-Gly, and GSSG, respectively. An evident peak was observed in the chromatogram (a) after extraction from the MISPE cartridge compared with the results in chromatogram (b); however, the peaks of Gly-Gly and GSSG in the two chromatograms (a, b) were almost the same. These findings demonstrated that IRPP-MIP and IRPP-NIP sorbents had no selectivity for Gly-Gly and GSSG. Moreover, Fig. 8c shows that the C18 cartridge had a remarkable pre-concentration effect without selectivity for all analytes. Thus, the enrichment of GSH, GlyGly, and GSSG was approximately similar, and the IRPP-MIP had highly selective recognition and pre-concentration effect for GSH than Gly-Gly and GSSG (Fig. 8b and c). The recoveries of the MIPSE cartridges for GSH and its structural analogs are shown in Fig. 9. The range of recoveries for GSH was 95.6% ± 3.65%, whereas 29.5% ± 1.26% and 49.9% ± 1.71% for Gly-Gly and GSSG, respectively. Moreover, under optimum experimental conditions with signal-to-noise ratio of 3.0, the linearity of GSH, Gly-Gly, and GSSG was observed within the ranges of 0.5 mg·L−1 to 200 mg·L−1, 0.2 mg·L−1 to 200 mg·L−1, and 0.35 mg·L−1 to 200 mg·L−1, respectively, In addition, the relative standard deviations (RSD) of GSH, GlyGly, and GSSG for 10 replicate extractions of 50 μg·L−1 sample solution were 5.76%, 6.85%, and 5.29%, respectively. These findings further indicated that IRPP-MIP particles were more suitable as an SPE sorbent for scientific separation and pre-concentration. 3.3.3. Analysis of urine samples As shown in Table 2 and Fig. 10, the two sorbents (IRPP-NIP and C18) showed low recoveries for GSH. The interferences and poor recoveries were attributed to the non-specific interactions, including hydrophobic and hydrophilic interactions, between the various components of the sample matrix with the two sorbents [32]. By contrast, the satisfactory Table 2 Assay of GSH in human urine samples by means of the proposed method (N = 3).
Fig. 8. The chromatograms of the 50 μg·L−1 of GSH, Gly-Gly and GSSG standard mixture solution after SPE preconcentration by (a) IRPP-MIP sorbent, (b) IRPP-NIP sorbent and (c) C18 sorbent. Chromatographic conditions of C18 reversed-phase column: mobile phase, acetonitrile and 10 mmol·L−1 aqueous solution of phosphate buffer (5/95, v/v); flow rate, 0.5 mL·min−1; UV detector wavelength, 254 nm; column temperature, 30 °C.
Sorbents
Added (μg⋅L−1)
Founded (μg⋅L−1)
Recovery (%)
IRPP-MIP
10 50 100 10 50 100 10 50 100
9.62 46.93 90.24 3.46 15.7 29.66 4.92 22.35 41.49
96.20 93.86 90.24 34.60 31.40 29.66 49.20 44.70 41.49
IRPP-NIP
C18
± ± ± ± ± ± ± ± ±
0.02 0.08 0.12 0.05 0.10 0.07 0.04 0.09 0.14
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Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 21174111). References
Fig. 10. Chromatogram obtained after SPE pre-concentration of urine sample spiked with 50 μg · L−1 of GSH using (a) IRPP-MIP, (b) IRPP-NIP and (c) C18 sorbent. Chromatographic conditions of C18 reversed-phase column: mobile phase, acetonitrile and 10 mmol · L−1 aqueous solution of phosphate buffer (15/85, v/v); flow rate, 0.5 mL · min−1; UV detector wavelength, 254 nm; column temperature, 30 °C.
recoveries obtained for the MISPE cartridge ranged from 90.24% to 96.20%. In addition, the RSDs of MISPE for 10, 50, and 100 μg⋅L− 1 of GSH were 5.67%, 2.09%, and 0.48% (N = 3), respectively. This result demonstrated the potential applicability of IRPP-MIP for highly effective pre-concentration, separation, and accurate quantification of GSH in actual samples. 4. Conclusions In this study, a simple and effective approach to obtain functional IRPP-MIP particles was developed by considering the iniferter advantages acquired through IRPP. The approach provided IRPP-MIP with regular spherical shape, apparent imprinting effects toward the template, relatively fast template binding process, and appreciable selectivity for structural analogs compared with TRPP-MIP. Furthermore, the successful application of MISPE sorbent in the selective separation and pre-concentration of GSH from its structural analogs or human urine samples implied that the presented method could be an alternative solution for the trace enrichment of specific targets.
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Molecularly imprinted solid-phase extraction of glutathione from urine samples Renyuan Song ⁎, Xiaoling Hu, Ping Guan, Ji Li, Na Zhao, Qiaoli Wang School of Natural and Applied Science, Northwestern Polytechnical University, Xi'an 710072, PR China
a r t i c l e
i n f o
Article history: Received 5 December 2013 Received in revised form 10 June 2014 Accepted 1 August 2014 Available online 7 August 2014 Keywords: Glutathione Molecularly imprinted polymer Living radical polymerization Selectivity extraction Solid-phase extraction
a b s t r a c t Molecularly imprinted polymer (MIP) particles for glutathione were synthesized through iniferter-controlled living radical precipitation polymerization (IRPP) under ultraviolet radiation at ambient temperature. Static adsorption, solid-phase extraction, and high-performance liquid chromatography were carried out to evaluate the adsorption properties and selective recognition characteristics of the polymers for glutathione and its structural analogs. The obtained IRPP-MIP particles exhibited a regularly spherical shape, rapid binding kinetics, high imprinting factor, and high selectivity compared with the MIP particles prepared using traditional free-radical precipitation polymerization. The selective separation and enrichment of glutathione from the mixture of glycyl-glycine and glutathione disulfide could be achieved on the IRPP-MIP cartridge. The recoveries of glutathione, glycyl-glycine, and glutathione disulfide were 95.6% ± 3.65%, 29.5% ± 1.26%, and 49.9% ±1.71%, respectively. The detection limit (S/N = 3) of glutathione was 0.5 mg·L−1. The relative standard deviations (RSDs) for 10 replicate detections of 50 mg·L−1 of glutathione were 5.76%, and the linear range of the calibration curve was 0.5 mg·L−1 to 200 mg·L−1 under optimized conditions. The proposed approach was successfully applied to determine glutathione in spiked human urine samples with recoveries of 90.24% to 96.20% and RSDs of 0.48% to 5.67%. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Glutathione (GSH, 5-L-glutamyl-L-cysteinylglycine) is a tripeptide with three amino acid residues [1–3]. Several methods, including spectrofluorometry [4], capillary electrophoresis [5], and chromatography [1] have been presented to analyze GSH in biological matrices. Although such methods offer more sensitive approaches to GSH analysis, relatively expensive instruments, which are not yet available in all laboratories, are required. In addition, most of the reported determination methods have difficulty in direct separation and enrichment of GSH from complex matrices through sample pretreatment. Generally, GSH is present in complex samples at low concentrations; thus, developing a novel, simple, and fast determination method appropriate for GSH is necessary. Molecularly imprinted polymers (MIPs) are synthetic polymers with specific cavities designed for template molecules [6,7]. MIP applications have attracted significant attention in solid-phase extraction (SPE) as sorbent for generating three-dimensional cross-linked polymeric materials with a “memory” for the shape and functional group positions of template molecules [8]. Molecularly imprinted SPE (MISPE) is a simple and feasible alternative to multi-step SPE method for the preconcentration of target molecules in complex samples, for example, ⁎ Corresponding author. Tel./fax: +86 29 88431639. E-mail address: [email protected] (R. Song).
http://dx.doi.org/10.1016/j.msec.2014.08.005 0928-4931/© 2014 Elsevier B.V. All rights reserved.
extraction of pollutants from river water and various biomolecules from food or vegetables [9–12]. Dinçer et al. [13] synthesized monolith MIPs for GSH via traditional bulk polymerization by using 1-vinyl imidazole as functional monomer. The monolith GSH-imprinted polymers were used as SPE material for detection of GSH in biological samples. A similar method was used by Tong et al. [14]. However, the monolith MIPs prepared through traditional radical polymerization have disadvantages, including broad binding site heterogeneity, relatively low binding affinity, slow kinetics, and low selectivity [15]; such disadvantages are attributed to the uncontrollable chain propagation and termination of the monolith MIPs. Thus, the propagation and termination steps of the reaction should be controlled. The introduction of controlled living radical polymerization (CRP) into imprinted polymerization systems can considerably improve the homogeneous network structures of MIPs and elucidate their structure– property relationship. CRP is a very effective tool for controlling cross-linked polymer structures. Thus far, many different polymer networks with homogeneous structures have been prepared using CRP. Iniferter is an ideal candidate for CRP because of its versatility and simplicity. Iniferter, an initiator-chain transfer molecule, was first discovered by Otzu et al. [16] in 1982. Since then, iniferter has been used to produce linear polymer chains with low polydispersities, specific block copolymers, graft polymers, and cross-linked polymer systems on surfaces [17–22]. Several successful molecular imprinting applications have been reported.
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For example, Shufang et al. [23] prepared sulfamethazine-imprinted core-shell particles by using surface-initiated iniferter-controlled living radical polymerization (IRP) on silica particles. The resultant core-shell MIPs showed high affinity toward the template. A similar method was used by Barahona et al. [24]. Recently, Zhang et al. [25] reported that IRP combined with precipitation polymerization can effectively improve the binding and structural characteristics of MIP particles. Nevertheless, these studies mainly focused on the formation of MIPs on surfaces by using iniferter. By contrast, few studies have been conducted on the direct preparation of functional MIP particles, particularly imprinting of biomolecules, using IRP in the presence of the target molecule. In this study, iniferter-controlled living radical precipitation polymerization (IRPP) method was applied to obtain MIP particles. The apparent morphology, composition, kinetics, adsorption isotherm, and selectivity of the IRPP-MIP particles were characterized with GSH as the model system. The adsorption speed, adsorption capacity, and selectivity of the IRPP-MIP particles improved remarkably because of their uniform distribution and homogenous binding sites compared with the MIP particles prepared through traditional free-radical precipitation polymerization (TRPP). In addition, the obtained IRPP-MIP particles were applied as SPE sorbent material for the selective separation and enrichment of GSH from its structural analogs or human urine samples. 2. Material and methods 2.1. Reagents and chemicals GSH, glutathione disulfide (GSSG), and glycyl-glycine (Gly-Gly) were purchased from Shanghai Jingchun Ke Fine Chemical Corporation (Shanghai, China). Azobisisobutyronitrile (AIBN) was purchased from Tianjin Guangfu Chemical Industry (Tianjin, China). Ethylene glycol dimethacrylate (EGDMA) and 4-vinyl pyridine (4-VP) were purchased from Alfa (Shanghai, China). Tetramethylammonium hydroxide, phenylisothiocyanate (PITC), high-performance liquid chromatography (HPLC)-grade acetonitrile, and triethylamine were provided by Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). All chemicals were of analytical grade and used without further purification. Benzyl N,N-diethyldithiocarbamate (BDC) was prepared following the literature procedure [26]. 2.2. Synthesis of imprinted polymer particles IRPP-MIP particles were synthesized according to the method of Zhang et al. [25]. In a typical synthesis, a mixture of GSH (51.2 mg, 0.167 mmol), 4-VP (0.11 mL, 1 mmol), EGDMA (0.75 mL, 4 mmol), and BDC (35.85 mg, 0.15 mmol) was dissolved in an appropriate porogenic solvent (acetonitrile/water = 9/1, v/v). The mixed solution was sequentially added into a four-neck round-bottom quartz flask (100 mL). After sealing, mixing, and sparging the mixture with nitrogen for 30 min, polymerization was initiated using ultraviolet radiation from a high-pressure mercury lamp (300 W, 365 nm) with stirring at ambient temperature for 12 h. After polymerization, the resulting polymer was extracted with a mixture of ethanol, water, and acetic acid (70/28/2, v/v) by using a Soxhlet apparatus for 48 h. The obtained polymer was washed thrice with ethanol and water and then dried in a vacuum at 40 °C for 36 h. AIBN (17.20 mg, 0.104 mmol) was used as initiator instead of BDC for the MIP particles prepared using TRPP. All other conditions were similar to the preparation of IRPP-MIP. The corresponding nonimprinted polymer (NIP) particles were prepared and purified under the same conditions but without template addition. 2.3. Characterization of chemical structure and morphology The chemical structures of the obtained polymer were identified with a Fourier-transform infrared spectrometer (FT-IR; Shimadzu,
WQF-310) by using KBr compressed pellet method. The surface morphology of the obtained polymer was determined through scanning electron microscopy (SEM, FEI, Quanta 600FEG). 2.4. Static adsorption experiments Static adsorption experiments were carried out to evaluate the adsorption kinetics and adsorption isotherms at 25 °C. In a typical procedure, 10 mg of MIP/NIP was incubated with 10 mL of 0.5 mg·mL−1 GSH solution (pH 7.0) for different times. After filtration using an acetate membrane (0.45 μm pore size), the residual concentration of GSH was determined through HPLC, and the adsorption capacity Q (mg·g−1) was calculated according to the following formula (1): Qe ¼
ðC 0 −C e ÞV m
ð1Þ
where C0 (mg·mL−1) and Ce (mg·mL−1) represent the initial and residual concentrations, respectively. V (mL) is the volume of adsorption solution, and m (g) is the amount of adsorbent. Equilibrium adsorption experiments were performed by incubating MIP/NIP with different concentrations of GSH solution for 8 h. Q is calculated using Eq. (1), and the imprinting factor (α) is obtained according to the following Eq. (2): α¼
Q MIP Q NIP
ð2Þ
where QMIP and QNIP are the adsorption capacities of MIP and NIP, respectively. The effect of initial pH on the recognition property of MIP/NIP toward GSH was determined using batch equilibrium adsorption experiments. The adsorption experiments were performed by incubating MIP/NIP with different pH of the GSH solution for 8 h. 2.5. Selectivity experiments In a typical procedure, 10 mg of MIP/NIP was incubated with 10 mL of GSH and 0.2 mg·mL−1 of the similar structural compounds (pH 7.0) at 25 °C for 8 h. The adsorption capacities of GSH or similar structural analogs were obtained by measuring the residual concentration similar to the batch experiments. The selectivity recognition factor (β) of the MIP was calculated according to the following Eq. (3): β¼
α Tem α Ana
ð3Þ
where αTem and αAna are the imprinting factors of MIP for GSH and its structural analogs, respectively. 2.6. SPE experiments The obtained IRPP-MIP particles (200 mg) were packed into an empty SPE cartridge with two polypropylene upper and lower frits at each end. The entire MISPE cartridge was carefully washed with sufficient methanol and preconditioned with a mixture of acetonitrile and water (7/3, v/v) at a flow rate of 0.5 mL·min− 1 prior to use. Subsequently, 50 mL of the sample solution containing 50 μg·L−1 GSH, GSSG, or Gly-Gly was passed through the preconditioned MISPE cartridge at a specific flow rate. After loading, the MISPE cartridge was first washed with 3.0 mL of a mixture of acetonitrile and water (7/3, v/v) to remove the sample molecules retained by non-specific adsorption from the cartridge. The adsorbed analytes on the MISPE cartridge were eluted using 3.0 mL of a mixture of ethanol, water, and acetic acid (70/28/2, v/v). Finally, the eluate was analyzed through HPLC by using pre-column derivation with PITC at a flow rate of 0.5 mL·min−1
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Fig. 1. Preparation process of GSH-imprinted polymer particles by IRPP.
at 30 °C. By contrast, the C18 cartridge was used under the same procedures with C18 as sorbent material. 2.7. Analysis of GSH and its structural analogs The concentrations of GSH and its structural analogs were determined using HPLC (Shimadzu, LC-2010A) after derivatization with
PITC. In a typical procedure, 200 μL of amino acid solution, 100 μL of PITC (100 mmol·L−1), and 100 μL of the mixture of triethylamine (1 mol·L−1) and acetonitrile were mixed in a tube. The tube was then sealed and allowed to react for 1 h in a 30 °C water bath. About 400 μL of n-hexane was used to extract the remaining PITC and collect the liquid from the lower layer for detection. The chromatography conditions were as follows: mobile phase, acetonitrile and 10 mmol·L− 1
Fig. 2. SEM images of (a) IRPP-MIP/(b) IRPP-NIP and (c) TRPP-MIP/(d) TRPP-NIP.
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Fig. 5. Adsorption kinetics of MIP and NIP toward GSH. Fig. 3. FT-IR spectra of (a) IRPP-MIP and (b) TRPP-MIP.
aqueous solution of phosphate buffer (5/95, v/v); flow rate, 0.5 mL·min− 1; and UV detector wavelength, 254 nm. 3. Results and discussion 3.1. Preparation and characterization of MIP particles The general scheme for the synthesis of IRPP-MIP is illustrated in Fig. 1. BDC was selected as iniferter to improve the recognition properties of the MIP networks in the precipitation polymerization systems. Thus, considering the iniferter characteristics, the obtained MIP particles could have uniform particle size distribution and highly specific recognition performance when prepared through IRPP. In this study, the imprinted polymerization system used GSH, 4-VP, EGDMA, and a mixture of acetonitrile and water as the template, functional monomer, cross-linker, and porogenic solvent, respectively. Polymerization was initiated with an ultraviolet radiation in the presence of an iniferter agent at ambient temperature. Fig. 2 shows that the IRPP-MIP had a smooth surface and fine spherical degree. IRPP-MIP demonstrated a larger particle size and homogeneous distribution (number-average diameter, 0.86 μm; polydispersity index, 1.130) compared with TRPP-MIP (number-average diameter, 0.45 μm; polydispersity index, 1.312). This result revealed the two different types of particle formation mechanisms similar to that of a previously studied traditional precipitation polymerization system [25]. Moreover, the particle sizes of IRPP-MIP and TRPP-MIP were lower
Fig. 4. Effect of pH on the adsorption capacity of MIP and NIP toward GSH.
than those of the corresponding IRPP-NIP and TRPP-NIP, respectively; this finding indicated that the template compound markedly affected the nucleation and growth during precipitation polymerization [27]. Fig. 3 shows the FT-IR spectra of TRPP-MIP (a) and IRPP-MIP (b), which had similar chemical structures. The presence of three significant peaks at 1730 (C_O stretching), 1256, and 1150 cm− 1 (C\O\C stretching) confirmed the existence of poly(EGDMA) in TRPP-MIP and IRPP-MIP. The characteristic peaks corresponding to C_N stretching (1601 and 1640 cm− 1) and C_C stretching (1455 cm− 1) from the pyridine rings were also observed. Thus, the functional monomer 4-VP was successfully polymerized in MIPs. Moreover, compared with the FT-IR spectra of TRPP-MIP, IRPP-MIP demonstrated the characteristic peaks of the thiocarbonyl group from the iniferter groups at 1208 cm− 1 (C_S stretching), as well as at 1421 and 1354 cm− 1 (CH stretching) [27]. This finding revealed that the iniferter was successfully introduced onto the surface of IRPP-MIP.
3.2. Evaluation of adsorption and selectivity of MIP particles 3.2.1. Effect of pH on adsorption properties Molecular recognition is mainly attributed to the binding sites that complement the template molecules in terms of shape, size, and chemical functionality of imprinting cavities [28]. Electrostatic interactions also affect specific template recognition, particularly in peptides or protein molecules. Thus, the effect of pH in 0.05 mol·L−1 phosphate buffer solution on GSH adsorption was evaluated at four different pH (5.0, 6.0, 7.0, and 8.0) by using a specific amount of dilute NaH2PO4 or
Fig. 6. Adsorption isotherms of MIP and NIP toward GSH.
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adsorption behavior of GSH on MIP and NIP can be illustrated by adsorption isotherms (Fig. 6). The saturation capacities to IRPP-MIP and TRPPMIP for GSH were 20.76 and 16.50 mg·g−1, respectively. The adsorption capacity of IRPP-MIP was 25.8% higher than that of TRPP-MIP, indicating that improved binding capacities were achieved in TRPP-MIP. The equilibrium adsorption isotherm that agreed with the experimental data (top inset, Fig. 5) indicated the homogenous distribution of imprinting sites in IRPP-MIP. This finding suggested that the increased binding capacities of IRPP-MIP are mainly caused by their relatively higher binding sites. These results indicated that the homogenous cross-linked network structure of MIP was improved by the intrinsic advantages of IRPP in the process of imprinting, resulting in increased binding properties of MIP. Thus, the forms of IRPP-MIP materials could have an improved binding capacity, kinetics, and site accessibility. The functional groups were randomly distributed, which resulted in low adsorption ability of GSH, because NIP lacked the imprinting process. Thus, nonspecific adsorption of GSH was observed. Therefore, the template absorption of IRPP-MIP and TRPP-MIP was higher than those of the IRPP-NIP and TRPP-NIP because of the imprinting effect.
Fig. 7. Chemical structures of GSH and its analogs.
Na2HPO4 solution for adjustment. Fig. 4 shows that the adsorption capacities of all MIP/NIP evidently increased with increasing pH. The results can be attributed to the decreased pH of the GSH solution from 8.0 to 5.0, which resulted in a more positively charged GSH (pI 5.93) because of protonation. Similarly, IRPP-MIP and TRPP-MIP showed more positive changes caused by the protonation of functional groups (4-VP) in the imprinting cavities. These results indicated that the electrostatic interactions between GSH and MIP enhanced the binding ability at relatively high pH. IRPP-NIP and TRPP-NIP showed lower adsorption capacities than correspond to MIP because of the absence of recognition site. Moreover, IRPP-MIP had higher adsorption capacity than TRPP-MIP because of their homogeneous cross-linked polymer networks and intrinsic IRP advantages. These results indicated that electrostatic interaction had an important function in template recognition. Therefore, pH 7.0 was selected as the optimum pH. 3.2.2. Adsorption kinetic The binding kinetics of GSH with the MIP and NIP prepared through IRPP and TRPP was evaluated using batch adsorption experiments. Fig. 5 shows the adsorption uptake of the polymer versus the incubation time. IRPP-MIP reached the binding equilibrium at 120 min, indicating a relatively fast binding process. In addition, the equilibrium loading capacity of IRPP-MIP was higher than that of TRPP, suggesting that IRPP effectively generated MIP with better mass-transfer properties [29]. By contrast, no specific adsorption was observed for the two types of NIP because no suitable imprinted cavities existed for GSH recognition. 3.2.3. Adsorption isotherm Equilibrium binding experiments were performed to determine the rebinding properties of MIP and NIP. The adsorption capacity and
3.2.4. Selectivity Selectivity test was performed using the structural analogs, such as GSSG and Gly-Gly (Fig. 7) as control compounds. Table 1 shows that MIP exhibited significant binding selectivity for the template molecules. The imprinting factor and adsorption capacity of MIP for GSH were higher than those for the structural analogs, thereby verifying the specific recognition toward the template. In addition to GSH, MIP also adsorbed Gly-Gly and GSSG; this finding demonstrated the presence of a specific cross-binding reactivity. Although the same hydrogen bond can form between the structural analogs and functional groups in imprinting cavities because of the similar structure to the template molecule, the binding amount was lower than that of the template molecule. The results demonstrated that the specific recognition mechanism of MIP was related to the interactional force between the template and functional groups, as well as to the complementary match of imprinting cavities with template molecule in terms of size and shape [30]. The experimental results confirmed this conclusion, and the selectivity coefficients of IRPP-MIP toward GSSG and Gly-Gly were determined to be 1.95 and 2.11, respectively. TRPP-MIP had lower selective coefficients toward the structural analogs than IRPPMIP because IRPP generated more homogeneous binding imprinting cavities. No significant imprinted cavities and sites were observed in IRPP-NIP and TRPP-NIP, and the functional sites of 4-VP in the polymer network were disorderly arranged. Thus, NIP was selective toward these compounds through nonspecific interaction [31]. These results strongly confirmed that the IRPP approach had high binding selectivity for the preparation of IRPP-MIP. 3.3. Solid-phase adsorption behavior of the MIP 3.3.1. Optimization of extraction procedure The potential application of IRPP-MIP for the selective preconcentration of GSH in complex samples was investigated. The composition and volume of elution solution and the loading flow rate were optimized to achieve good sensitivity and precision for this approach.
Table 1 Imprinting efficiency and selectivity coefficients of MIP and NIP. Compound
IRPP
TRPP
Q (mg·g−1)
GSH GSSG Gly-Gly a
MIP
NIP
10.60 ± 0.43 14.56 ± 0.24 3.89 ± 0.30
4.59 ± 0.51 12.36 ± 0.43 3.56 ± 0.22
α
βa
2.30 1.18 1.09
– 1.95 2.11
Q (mg·g−1) MIP
NIP
7.11 ± 0.47 11.53 ± 0.36 3.80 ± 0.14
4.63 ± 0.37 10.69 ± 0.63 3.46 ± 0.25
Selectivity factor, β = αTem/αAna, αTem and αAna are the imprinting factors of MIP/NIP toward the template and its structural analogs, respectively.
α
β
1.54 1.08 1.10
– 1.43 1.40
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The effects of different elution solution on desorption of GSH from the MISPE cartridge were determined with a mixture of methanol and water, as well as with a mixture of methanol, water, and acetic acid. When a mixture of methanol and water (7/3, v/v) was utilized as the elution solution, no GSH signal appeared in the chromatograms using pre-column derivatization with PITC; however, some GSH could be extracted from the MISPE cartridges by eluting with a mixture of methanol, water, and acetic acid. A mixture of methanol, water, and acetic acid (70/28/2, v/v) was selected as the eluent because of its requirement for the adsorption analytes to be completely desorbed from the MISPE cartridge. A series of experiments was performed to confirm the optimum volume of elution solution. The different volumes of elution solution were evaluated while the other conditions were fixed. The volumes ranged from 0 mL to 4.0 mL of the mixture of methanol, water, and acetic acid (70/28/2, v/v). With the increase of the eluent volume from 0 mL to 4.0 mL, the chromatographic peak area of GSH appeared increasing at first (from 0 mL to 2.5 mL) and then tended to a specific value (from 2.5 mL to 4.0 mL). Therefore, 3.0 mL of the eluent volume was selected to ensure the complete desorption of GSH from the MISPE cartridge. Loading flow rate is an important parameter that affects the effective extraction of GSH in the SPE process. The results showed that the chromatographic peak area of GSH increased with decreasing loading flow rate from 1.0 mL·min−1 to 0.5 mL·min−1; however, the variation was not evident. As the loading flow rate decreased from 0.5 mL·min−1 to 0.35 mL·min−1, no variation appeared in the chromatograms. Thus, 0.5 mL·min−1 loading flow rate was used as the optimum condition for further experiments. 3.3.2. HPLC evaluation of selective separation The potential application of IRPP-MIP for the selective preconcentration of GSH in complex samples was evaluated. The MISPE experiment was passed through 50 mL of standard sample solution containing 50 μg·L−1 each of GSH, GSSG, and Gly-Gly at a flow rate of 0.5 mL·min−1. The MISPE cartridge was washed initially with 3.0 mL of the mixture of acetonitrile and water (7/3, v/v) to remove the analytes retained by non-specific adsorption through IRPP-MIP sorbent. The cartridge was then eluted with 3.0 mL of the mixture of methanol, water, and acetic acid (70/28/2, v/v), and the eluate was analyzed through HPLC using pre-column derivation with PITC at flow rate of
Fig. 9. Recoveries of GSH, Gly-Gly, and GSSG on MISPE cartridges.
0.5 mL·min−1 at 30 °C. The mobile phase used for HPLC experiment was a mixture of acetonitrile and 10 mmol·L− 1 aqueous solution of phosphate buffer (5/95, v/v), whereas UV–vis detector was operated at 254 nm. As shown in Fig. 8, peaks 1, 2, and 3 were identified as GSH, Gly-Gly, and GSSG, respectively. An evident peak was observed in the chromatogram (a) after extraction from the MISPE cartridge compared with the results in chromatogram (b); however, the peaks of Gly-Gly and GSSG in the two chromatograms (a, b) were almost the same. These findings demonstrated that IRPP-MIP and IRPP-NIP sorbents had no selectivity for Gly-Gly and GSSG. Moreover, Fig. 8c shows that the C18 cartridge had a remarkable pre-concentration effect without selectivity for all analytes. Thus, the enrichment of GSH, GlyGly, and GSSG was approximately similar, and the IRPP-MIP had highly selective recognition and pre-concentration effect for GSH than Gly-Gly and GSSG (Fig. 8b and c). The recoveries of the MIPSE cartridges for GSH and its structural analogs are shown in Fig. 9. The range of recoveries for GSH was 95.6% ± 3.65%, whereas 29.5% ± 1.26% and 49.9% ± 1.71% for Gly-Gly and GSSG, respectively. Moreover, under optimum experimental conditions with signal-to-noise ratio of 3.0, the linearity of GSH, Gly-Gly, and GSSG was observed within the ranges of 0.5 mg·L−1 to 200 mg·L−1, 0.2 mg·L−1 to 200 mg·L−1, and 0.35 mg·L−1 to 200 mg·L−1, respectively, In addition, the relative standard deviations (RSD) of GSH, GlyGly, and GSSG for 10 replicate extractions of 50 μg·L−1 sample solution were 5.76%, 6.85%, and 5.29%, respectively. These findings further indicated that IRPP-MIP particles were more suitable as an SPE sorbent for scientific separation and pre-concentration. 3.3.3. Analysis of urine samples As shown in Table 2 and Fig. 10, the two sorbents (IRPP-NIP and C18) showed low recoveries for GSH. The interferences and poor recoveries were attributed to the non-specific interactions, including hydrophobic and hydrophilic interactions, between the various components of the sample matrix with the two sorbents [32]. By contrast, the satisfactory Table 2 Assay of GSH in human urine samples by means of the proposed method (N = 3).
Fig. 8. The chromatograms of the 50 μg·L−1 of GSH, Gly-Gly and GSSG standard mixture solution after SPE preconcentration by (a) IRPP-MIP sorbent, (b) IRPP-NIP sorbent and (c) C18 sorbent. Chromatographic conditions of C18 reversed-phase column: mobile phase, acetonitrile and 10 mmol·L−1 aqueous solution of phosphate buffer (5/95, v/v); flow rate, 0.5 mL·min−1; UV detector wavelength, 254 nm; column temperature, 30 °C.
Sorbents
Added (μg⋅L−1)
Founded (μg⋅L−1)
Recovery (%)
IRPP-MIP
10 50 100 10 50 100 10 50 100
9.62 46.93 90.24 3.46 15.7 29.66 4.92 22.35 41.49
96.20 93.86 90.24 34.60 31.40 29.66 49.20 44.70 41.49
IRPP-NIP
C18
± ± ± ± ± ± ± ± ±
0.02 0.08 0.12 0.05 0.10 0.07 0.04 0.09 0.14
R. Song et al. / Materials Science and Engineering C 44 (2014) 69–75
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Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 21174111). References
Fig. 10. Chromatogram obtained after SPE pre-concentration of urine sample spiked with 50 μg · L−1 of GSH using (a) IRPP-MIP, (b) IRPP-NIP and (c) C18 sorbent. Chromatographic conditions of C18 reversed-phase column: mobile phase, acetonitrile and 10 mmol · L−1 aqueous solution of phosphate buffer (15/85, v/v); flow rate, 0.5 mL · min−1; UV detector wavelength, 254 nm; column temperature, 30 °C.
recoveries obtained for the MISPE cartridge ranged from 90.24% to 96.20%. In addition, the RSDs of MISPE for 10, 50, and 100 μg⋅L− 1 of GSH were 5.67%, 2.09%, and 0.48% (N = 3), respectively. This result demonstrated the potential applicability of IRPP-MIP for highly effective pre-concentration, separation, and accurate quantification of GSH in actual samples. 4. Conclusions In this study, a simple and effective approach to obtain functional IRPP-MIP particles was developed by considering the iniferter advantages acquired through IRPP. The approach provided IRPP-MIP with regular spherical shape, apparent imprinting effects toward the template, relatively fast template binding process, and appreciable selectivity for structural analogs compared with TRPP-MIP. Furthermore, the successful application of MISPE sorbent in the selective separation and pre-concentration of GSH from its structural analogs or human urine samples implied that the presented method could be an alternative solution for the trace enrichment of specific targets.
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