TiO2 Nanoparticles Functionalized Monolithic Capillary

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Analytical Chemistry

TiO2 nanoparticles functionalized monolithic capillary microextraction on-line coupled with inductively coupled plasma mass spectrometry for the analysis of Gd ion and Gd-based contrast agents in human urines Xiaolan Liu, Beibei Chen, Lin Zhang, Shiyao Song, Yabing Cai, Man He, Bin Hu* Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan 430072, China * Corresponding author. Tel: 0086-27-68752162; Fax: 0086-27-68754067; Email: [email protected]

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Abstract In this work, a novel method of TiO2 nanoparticles (NPs) functionalized monolithic capillary microextraction (CME) on-line coupling with inductively coupled plasma mass spectrometry (ICP-MS) was developed for the sequential determination of Gd3+ and Gd-based contrast agents in human urine samples. The monolithic capillary was prepared by embedding anatase TiO2 NPS in the poly(methacrylic acid-ethylene glycol dimethacrylate) (MAA-EDMA) framework. The Gd3+ and Gd-based contrast agents (such as

gadolinium-diethylene

triamine

pentaacetic

acid

(Gd-DTPA)

and

Gd-DTPA-bismethylamide (Gd-DTPA-BMA)) display different adsorption behaviors on the prepared monolithic capillary which possesses the adsorption properties of both anatase TiO2 NPS and poly(MAA-EDMA) monolith. Under the optimized conditions, the limits of detection (LODs) were found to be 3.6, 3.2, and 4.5 ng L-1 for Gd3+, Gd-DTPA and Gd-DTPA-BMA, respectively, which are the lowest up to date. The enrichment factor was 25-fold with the sample throughput of 5 h-1. The proposed method was validated by the analysis of Gd3+ and Gd-DTPA in the healthy human urine samples as well as Gd3+ and Gd-DTPA-BMA in patient urine samples. It was found that only a small amount of the free Gd3+ was released from Gd-DTPA-BMA, and accurate results could be obtained since no oxidation/reduction or subtraction is involved in this method. This method is simple, sensitive, rapid and provides a very attractive non-chromatography strategy for the speciation of Gd3+ and Gd-based contrast agents in urine samples. 2


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Keywords: TiO2 nanoparticles, monolithic CME, speciation of gadolinium, ICP-MS, human urine samples

Introduction Magnetic resonance imaging (MRI) allows the generation of noninvasive sectional images and the visualization of detailed internal structures of organs and tissues1, and MRI contrast agents based on paramagnetic metal ions are usually used for the definite differentiation of healthy and diseased tissue. Gd-based contrast agents are the most frequently used in the field of MRI due to the seven unpaired 4f electrons of Gd resulting in a large magnetic momentum. However, Gd3+ is highly toxic, it is prone to inhibit Ca2+ binding and acute toxicity of Gd3+ can cause ataxia, writhing, respiratory problems, sedation, hypotension and death by cardiovascular collapse2. Hence Gd is often applied as contrast agent in a chelated form. Among all the complexes, the commercial linear ligands gadopentetate (Gd-diethylene triamine pentaacetic acid, Gd-DTPA, known as Magnevist in clinical field) are the most commonly used in MRI due to its high stability and

safety.

In

recent

years,

some

Gd-DTPA

based

derivatives

such

as

Gd-DTPA-bismethylamide (Gd-DTPA-BMA, known as Omniscan in clinical field) were also used as MRI contrast agents. However, thirteen years after the introduction of Magnevist (Gd-DTPA), scientists found that the administration of Gd-based MRI contrast agents are related to the 3



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subsequent development of nephrogenic systemic fibrosis (NSF) in several cases3,4. Although the pathomechanisms were not clear, analysts also found that renal insufficient patients would increase the risk of Gd-based contrast agents decomposition as the half-live (t1/2 Gd-DTPA) increases dramatically in these patients (t1/2 of 30-120 h in the patients v.s. t1/2 of 1.5-2 h in healthy people)5,6. In addition, Gd-DTPA is prone to undergo in vivo transmetalation by other endogenously available metal ions such as Fe3+, Cu2+ and Zn2+, which may lead to an accumulation of toxic Gd3+ in patients7. Consequently, monitoring concentration levels of Gd3+/Gd-DTPA in urine samples from the patient who has suffered from MRI is of great importance. The speciation of Gd3+ and Gd-based contrast agents has been achieved by combining an effective separation method with a sensitive detection technique. Among the detection techniques such as ultraviolet-visible spectrometry (UV-Vis)8,9, fluorescence spectrometry10, electrospray ionization mass spectrometry (ESI-MS)7,11, inductively coupled plasma optical emission spectrometry/ mass spectrometry (ICP-OES/ MS)12,13, ICP-MS is the most powerful one for trace element determination because of its extremely low limits of detection (LODs), low mass interference and wide linear range and so on. The separation methods involved in the speciation of Gd include chromatographic methods (such as high performance liquid chromatography (HPLC) and capillary electrophoresis (CE)) and non-chromatographic methods (such as solid phase extraction (SPE)). Compared with CE, HPLC combined with ICP-MS has gained wider 4


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applications because this connection is simple and easy to operate. However, the mobile phase used in HPLC must be compatible with ICP-MS detection system. As an alternative, non-chromatographic method is simpler, cheaper and faster than HPLC method, and is suitable for the separation and enrichment of simple species such as Gd3+/ Gd-DTPA. Up to now, there are few reports on non-chromatographic methods for the separation and preconcentration of Gd3+/ Gd-DTPA. C18 modified with phosphate functional group was used as the SPE sorbent for the preconcentration of Gd3+ and Gd-based contrast agents14, 15. However, both of the methods need to use 6 mol L-1 HNO3 as eluent, as a result the concentrated acid need to be evaporated to dryness and redissolved before ICP-MS detection. In addition, these sorbents showed no selectivity towards Gd3+ and Gd-based contrast agents. Olsina et al.16 used AG50-X8 cation-exchange column to separate Gd3+ from chelated Gd, and then employed a cloud point extraction (CPE) method to preconcentrate Gd3+ from urine samples. The amount of chelated Gd was obtained by subtracting the amount of free Gd3+ from total Gd amount. Due to that the amount of the released free Gd3+ from Gd-DTPA is very small in real biological samples, this kind of subtraction method would cause inaccurate analytical results. Therefore, a highly efficient and easy sample pretreatment technique without subtraction process is needed in urgent to overcome the above-mentioned limitations. Capillary microextraction (CME) was first introduced as a miniaturized sample 5



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pretreatment technique by Bigham et al. in 200217. Because of its inherent merits of low sample/reagent consumption and good compatibility with different analytical instruments, CME has gained widespread applications in trace analysis especially in biological samples. Adsorption materials with high extraction efficiency and good selectivity play great role in the application of CME in elemental speciation18. Zheng et al.19 developed a dual-column CME system for sequential separation/ preconcentration of different As species in human hair extracts. Murko et al.20 developed an anion-exchange monolithic disk for the speciation of Al in human serum. Monolithic column has attracted great interests in CME owing to its unique advantages, such as fast mass transfer, high adsorption capacity and easy preparation. Rigid organic polymer monolithic columns can be modified with different functional sites. Recently, nanoparticles (NPs) are used in the functionalization of monoliths. By dispersing of NPs in monomers and porogens before polymerization21-23 or attaching NPs on the surface of the monolith framework24-31, polymer embedded with NPs exhibits high surface-to-volume ratio and other physico-chemical properties. Based on the above statements, it can be speculated that monolithic CME would be a perfect sample pretreatment technique for Gd speciation in biological samples, and the preparation of a monolithic capillary with proper functional groups is the key point to achieve the separation of Gd3+/Gd-DTPA. TiO2 NPs were known as a good material to extract compounds containing phosphate group32. Because of a strong interaction 6


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between phosphate and Gd3+/Gd-DTPA14,15,33, phosphate can be the intermediate to achieve the adsorption of Gd3+/Gd-DTPA on TiO2 NPs. Besides, carboxylic group modified monolith would selectively adsorb Gd3+ by electrostatic interaction34. Therefore, the aim of this work is to prepare a TiO2 NPs embedded poly(methacrylic acid-ethylene glycol dimethacrylate) (MAA-EDMA) [poly(MAA-EDMA-TiO2 NPs)] monolithic capillary and to develop a new method of on-line poly(MAA-EDMA-TiO2 NPs)-based CME-ICP-MS for the speciation of Gd3+/Gd-DTPA in human urine samples. Extraction performance of the prepared monolith was evaluated and experimental parameters affecting the CME were studied. The developed method was validated by the determination of Gd3+ and Gd-based contrast agents in human urine samples.

Experimental Instrumentation and reagents The experiment was performed by a quadrupole (Q) ICP-MS (Model Agilent 7500a, Hewlett-Packard, Yokogawa Analytical Systems, Tokyo, Japan) with a Babington nebulizer and the optimum operation conditions are summarized in Table S1 in Supporting Information. Fused silica capillary (530 µm i.d. × 680 µm o.d.) was obtained from Yongnian Optical Fiber Factory (Hebei, China). The stock standard solution of Gd (1 g L-1) was obtained by dissolving appropriate amounts of Gd2O3 (The First Reagent Factory, Shanghai, China) in 1 mol L-1 HNO3. The 7



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stock standard solution of Gd-DTPA and Gd-DTPA-BMA (1 g L-1, calculated as Gd) were obtained by dissolving appropriate amounts of Magnevist (Bayer Pharmaceutical Co., Ltd., Germany) and Omniscan (GE Healthcare Co., Ltd., Ireland) in water, respectively. The information on other instruments and reagents is detailed in the Supporting Information.

Preparation of TiO2 NPs embedded monolithic capillary The TiO2 NPs were self-prepared according to the procedure reported in our previous work35 and the details were described in the Supporting Information. The fused silica capillary was activated according to the procedure reported in our previous work 36. The capillary was rinsed sequentially with 1 mol L-1 NaOH for 2 h, water for 30 min, 1 mol L-1 HCl for 2 h, water for 30 min, and then rinsed with ethanol and dried by purging with Ar gas. The inner wall of the fused-silica capillary was vinylized with γ-methacryloxypropyl trimethoxysilane (γ-MAPS) to anchor the polymer monolith on it. Then the vinylized capillary (length of 10 cm) was filled with a mixture containing 17 µL methacrylic acid (MAA), 113 µL ethylene glycol dimethacrylate (EDMA), 375 µL methanol, 125 µL H2O, 1% 2,2-azobisisobutyronitrile (AIBN) (with respect to monomers, w/w), and TiO2 NPs with different quantities. After that the capillary was sealed at both ends with a rubber septum and immersed in a water bath at 60 ºC for 12 h. Then the 8


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capillary was washed with ethanol to remove unreacted components and cut into 2 cm length for further use.

Analytical procedures An aqueous sample solution was prepared in PBS at pH 5. After passing through the prepared monolithic capillary at a flow rate of 100 µL min-1, Gd3+ in the sample solution was retained on the capillary and the raffinate was collected. The retained Gd3+ was eluted with 20 µL 0.5 mol L-1 HNO3 and on-line determined by ICP-MS. After the pH of the raffinate was adjusted to 2.5, it was again passed through the prepared monolithic capillary at a flow rate of 100 µL min-1. Gd-DTPA in the raffinate was adsorbed on the monolithic capillary and eluted by 20 µL 0.5 mol L-1 HNO3 for subsequent on-line ICP-MS determination. The blank solution and the series of standard solutions were subjected to the same procedure of on-line CME-ICP-MS. The whole procedure for each aqueous sample solution was carried out in triplicate.

Sample preparation The urine samples were provided by Tongji Hospital of Wuhan (Wuhan, China). The ethics committee reviewed and approved the informed consent forms provided by all participants according to ethics requirements. Details on preparation are presented in the Supporting Information. 9



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Results and discussion Preparation of TiO2 NPs embedded monolithic capillary The TiO2 NPs are self-prepared, and they were characterized with scanning electron microscopy (SEM), energy dispersive X-ray (EDX) detector and X-ray powder diffraction (XRD), respectively. As can be seen from Figure S1 in the Supporting Information, the TiO2 NPs were quite uniform with an average size of 80 nm. The XRD analytical results in Figure S1 in the Supporting Information show the characteristic peak of anatase TiO2. An optimized polymerization mixture should afford a monolithic capillary with good permeability and extraction performance as well as large adsorption capacity. Porosity of the monolith directly affects the permeability and largely depends on the percentage of monomers. Therefore, the amount of TiO2 NPs in the prepolymerization solution was carefully studied with a TiO2 NPs (mg)/monomer (µL) ratio changing from 0 to 2. Figure 1 shows the SEM of the prepared monolithic capillaries and the corresponding EDX spectra. As can be seen, the prepared monolithic capillaries were uniform and composed of cross-linked micro spheres. The size of the cross-linked micro spheres tend to decrease with the increase of TiO2 NPs/ monomer ratio which results in the decrease of the through pore size. The percentage of Ti increased with the increase of TiO2 NPs/monomer ratio. The effect of the amount of TiO2 in the monolith on the adsorption behavior of Gd3+ 10


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and Gd-DTPA was also investigated, and the results are shown in Figure 2. As can be seen, without TiO2 NPs, Gd-DTPA cannot be extracted by the poly(MAA-EDMA) monolith, while quantitative adsorption (adsorption percentage, calculated as the difference between the concentration of target in original solution and that in raffinate divided by the original concentration, higher than 85%) for Gd-DTPA could be obtained when the TiO2 NPs/monomer ratio ranged from 0.8 to 1.5. On the contrary, embedding TiO2 NPs in the monolith has no remarkable effect on the adsorption of Gd3+. The possible reason could be attributed to the following fact. At pH 5, the adsorption of Gd3+ on the monolith was mainly based on the electrostatic interaction between Gd3+ and carboxylic group of MAA on the monolith as discussed in the section of “Extraction feasibility”. While TiO2 has very little contribution to adsorption of Gd3+ on the monolith because TiO2 exhibits very poorly adsorption performance towards REEs cations when pH is lower than 6.

37

It should be mentioned that increasing the amount of TiO2 NPs in

monolith would lead to an increase in flow resistance due to the decreased cross-linked micro spheres in the monolith (see Figure 1E). Experiments revealed that the pressure of the monolith is too high when the ratio of TiO2 NPs/monomer increased to 2. As the amount of TiO2 NPs directly influences the adsorption capacity of the monolith, a higher amount of TiO2 NPs is favored. To trade off the permeability of the prepared TiO2 NPs embedded poly(MAA-EDMA) monolith and its adsorption performance towards Gd-DTPA, a constant TiO2 NPs/monomer ratio of 1.5 was adopted for the preparation of 11



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TiO2 NPs functionalized monolithic capillary in this work. To justify the uniformity of the TiO2 NPs embedded in the prepared monolith, the cross section of the monolith with TiO2 NPs/monomer ratio of 1.5 was divided into 9 parts for EDX, and the results show that the Wt% of Ti in these 9 parts was varied from 33.1% to 50.5%, which was close to the result of 39.2% (Wt%) for the total cross section.

Extraction feasibility The pH value is one of the key factors to the adsorption behavior of Gd3+ and Gd-DTPA on the poly(MAA-EDMA-TiO2 NPs) monolithic capillary. As described previously, TiO2 NPs are known as a good material to extract compounds containing phosphate group32 and there is a strong interaction between phosphate and Gd3+/Gd-DTPA14,15,33, implying that phosphate can be the intermediate to achieve the adsorption of Gd3+/Gd-DTPA on TiO2 NPs. Therefore, PBS was selected as both the buffer and intermediate for the extraction of Gd3+/Gd-DTPA. To illustrate the adsorption mechanism and the role of PBS, the adsorption behaviors of Gd3+ and Gd-DTPA in the absence of PBS or in the presence of PBS were studied with pH varying in the range of 1-9, and the results are given in Figure 3(a) and (b), respectively. As can be seen in Figure 3(a), no difference of the adsorption behavior of Gd3+ and Gd-DTPA was observed and both Gd3+ and Gd-DTPA can be adsorbed at the pH range of 2-9 in the absence of PBS. In this case, Gd3+ was adsorbed on the poly(MAA-EDMA-TiO2 NPs) monolith mainly based on the electrostatic 12


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interaction between Gd3+ and carboxylic group on the MAA. While the adsorption of Gd-DTPA on poly(MAA-EDMA-TiO2 NPs) monolith was due to the weak intermolecular interaction between Gd-DTPA and TiO2 NPs. This weak interaction could be easily destroyed by adding salts (NaCl) in the solution (data not shown), indicating a weak anti-interference ability. With the use of PBS as the buffer, poly(MAA-EDMA-TiO2 NPs) monolithic capillary exhibited different adsorption behavior for Gd3+ and Gd-DTPA (Figure 3(b)). In the presence of PBS, Gd3+ was adsorbed completely on the poly(MAA-EDMA-TiO2 NPs) monolithic capillary in a relatively wide pH range (2-9), whereas Gd-DTPA could be retained quantitatively on the monolith in the pH range of 2-3 and a sharply decrease of adsorption percentage of Gd-DTPA was observed when pH was higher than 4. These results reveal that PBS played a great role in differentiating the adsorption behaviors of Gd3+ and Gd-DTPA on the poly(MAA-EDMA-TiO2 NPs) monolithic capillary. In the presence of PBS, Gd-DTPA first reacted with PO43- to form a ternary complex (structure shown in Figure 3(c)

33

) because the interaction between

Gd-DTPA and phosphate is stronger than that between Gd-DTPA and TiO2 NPs. This ternary complex could be easily adsorbed by the prepared poly(MAA-EDMA-TiO2 NPs) monolithic capillary due to the good affinity of TiO2 NPs to phosphate group at pH 2-3. When pH is lower than

2, the phosphate group exists as molecular form (the pKa of

H3PO4 is 2.12, 7.20 and 12.36) and could not act as a bridge for adsorption of Gd-DTPA. When pH is higher than 4, due to the isoelectric point of TiO2 NPs at pH 4-5, phosphate 13



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group could not be adsorbed on TiO2 NPs any more. It should be mentioned that the strong interaction between PBS and Gd-DTPA would not destroy the complexation of Gd3+ and DTPA in Gd-DTPA, otherwise the released Gd3+ from Gd-DTPA can also be adsorbed on the monolith (no such phenomenon was observed in Figure 3(b)). Gd3+ can also be complexed with phosphate group, so the adsorption of Gd3+ on the monolith in PBS at pH 2-3 is due to both the electrostatic interaction of Gd3+ and carboxylic group on the MAA and the strong interaction of phosphate group and TiO2 NPs. Accordingly, in order to achieve simultaneous separation/preconcentration of Gd3+ and Gd-DTPA, pH value of 2.5 and 5 were selected for the adsorption of Gd-DTPA and Gd3+, respectively. Specifically, when the sample solution containing Gd3+ and Gd-DTPA was passed through the monolith at pH 5, Gd3+ was adsorbed by the monolith, while Gd-DTPA was not retained by the monolith and passed through the monolith. And then the raffinate was adjusted to pH 2.5 for the adsorption of Gd-DTPA. In order to explore the possibility of the adsorption of other Gd-based contrast agents by the prepared poly(MAA-EDMA-TiO2 NPs) monolithic capillary, the adsorption behaviors of Gd-DTPA-BMA in the absence of PBS or in the presence of PBS was also investigated. The experimental results in Figure 3(a) and (b) demonstrate that Gd-DTPA-BMA shows similar adsorption behavior as Gd-DTPA, indicating that the prepared monolithic capillary is capable to separate Gd3+ and other Gd-based contrast agents, such as Gd-DTPA-BMA. In the subsequent optimization experiments, only 14


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Gd-DTPA was selected as the representative for Gd-based contrast agents. The influence of the concentration of PBS on the adsorption of Gd3+ and Gd-DTPA was investigated and the details were described in Supporting Information. Based on the experimental results in Figure S2, a concentration of 8.67 mg L-1 PBS was employed in the subsequent experiments.

Optimization of CME conditions Taking Gd-DTPA as the model Gd-based contrast agent, we carefully investigated various factors which affect the extraction of Gd3+ and Gd-DTPA, including sample flow rate, sample volume, eluent concentration and eluent volume. The details are shown in Figure S2 to S5 in Supporting Information. The sample flow rate was set as 100 µL min-1. Quantitative adsorption of the targets on the monolithic capillary was obtained within the entire tested volume range from 0.02 to 6 mL, and we choose 0.5 mL for real sample analysis to trade off the enrichment factor, analytical speed, and the limited amount of human fluids available. To ensure a complete elution of the targets from the column, 20 µL 0.5 mol L-1 HNO3 was employed as the eluent with a flow rate of 100 µL min-1. In this case, the enrichment factor is 25 fold, and the sample throughput is 5 h-1. It should be noted that the sample consumption of this method can be reduced to 20 µL, which means the sample throughput of the method can be high to 40 h-1 if we sacrifice the enrichment factor.

15



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Interference study To investigate the potential interference in the proposed method, the effect of coexisting ions prevailing in biological samples on the extraction efficiency of polymer monolithic capillary was studied. For this purpose, 0.5 mL of sample solution containing 1 µg L-1 each target analytes and a certain amount of interfering ions were subjected to the general procedure as described in the above. When the recoveries of the target analytes were kept in the range of 85%-115%, the interference caused by the co-existing ions was considered to be negligible. Table 1 is the tolerance limits of coexisting ions, along with the normal concentration range of each ion in urine samples 38, 39. As can be seen, the concentration of the studied foreign ions in the human urine was all below their tolerance limits. Therefore, it can be concluded that the developed method is highly selective for Gd3+ and Gd-based contrast agents, and has a good application potential in the speciation of Gd3+ and Gd-based contrast agents in complicated biological samples.

Preparation reproducibility, regeneration and adsorption capacity of the prepared polymer monolithic capillary In order to evaluate the preparation reproducibility of the monolithic capillaries (expressed as relative standard deviations (RSDs)), the extraction efficiencies of seven segments of poly(MAA-EDMA-TiO2 NPs) monolith prepared in the same batch and among different batches were investigated with sample solution containing Gd3+ and Gd-DTPA each at 1 µg 16


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L-1 under the optimized conditions. The RSDs in the same batch for Gd3+ and Gd-DTPA were 1.6% and 2.6%, respectively. And the RSDs in different batches for Gd3+ and Gd-DTPA were 1.6% and 4.4%, respectively. These results demonstrated that the preparation of the monolith had a good reproducibility. The regenerability of the self-prepared polymer monolithic capillary was evaluated. It was found that the poly(MAA-EDMA-TiO2 NPs) monolithic capillary could be easily regenerated by passing through 20 µL eluent and 20 µL PBS sequentially after an elution process. After regeneration, the custom-prepared polymer monolithic capillary could be reused more than 30 times without obvious decrease of extraction efficiency. It indicates that the prepared monolith has a good resistance to acid with a long life span. The adsorption capacity is another important factor in evaluating the performance of the extraction capillary. The methodology used for capacity study was adapted based on the method recommended by Hu et al.

40

. The sample solution containing 10 mg L-1

analytes was passed through the poly(MAA-EDMA-TiO2 NPs) monolithic capillary (530 µm i.d., length 2 cm), and the effluent was determined by ICP-MS, respectively. The adsorption capacities of target analytes on the prepared polymer monolithic capillary, defined as the maximum adsorbed amount (µg) of target analytes on 1 m polymer monolithic capillary, were 105 µg m-1 for Gd3+ and 40 µg m-1 for Gd-DTPA, respectively.

Analytical performance 17



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Under the optimized conditions, the analytical performance of the developed on-line poly(MAA-EDMA-TiO2 NPs) monolithic CME-ICP-MS method was evaluated and the results are summarized in Table 2. The limits of detection (LODs), evaluated as the concentration corresponding to three times the standard deviation of 11 runs of the blank solution (3σ), were found to be 3.6, 3.2 and 4.5 ng L−1 for Gd3+, Gd-DTPA and Gd-DTPA-BMA, respectively. The repeatability (expressed as RSDs), based on seven parallel analyses of 0.5 mL aqueous solutions containing the target analytes each at 0.02 µg L-1, were found to be 5.7, 4.5 and 5.5% for Gd3+, Gd-DTPA and Gd-DTPA-BMA, respectively. The enrichment factor for Gd3+, Gd-DTPA and Gd-DTPA-BMA was 25-fold, but when the sample volume increased from 0.5 mL to 6 mL, an enrichment factor of 300-fold will obtained for the targets. And as a result, the LODs for the targets will be much lower. Table 3 is the comparison of LODs obtained in this work with other similar approaches for the determination of Gd3+ and Gd-DTPA. Compared with the LODs reported in Refs 14-16, 41 for trace Gd3+ and Gd-DTPA, the proposed method was the most sensitive method up to now. What is more, compared with the chromatographic method41, the proposed method is fast and simple. Compared with the CPE method, the developed method is an on-line method with high sample throughput and easy operation. Besides, the proposed method is a straight forward method for the speciation of Gd3+ and Gd-based contrast agents, no oxidation/reduction or subtraction is involved. All these 18


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features make the proposed method perfect to be applied in speciation of Gd3+ and Gd-based contrast agents in real biological samples. Compared with general CME method36,42, the eluent volume in the proposed method is quite small, resulting in a relatively high enrichment factor. It achieved the speciation of Gd3+ and Gd-based contrast agents without subtraction methodology which was usually necessary in those CME-based method for elemental speciation19,43, featuring with easy operation and high accuracy. Sample analysis The concentrations of Gd3+ and Gd-DTPA in healthy human urine sample were determined by the developed method, and the analytical results are listed in Table 4. As can be seen, no Gd-DTPA was detected in the healthy human urine, while the concentration of Gd3+ was found to be 0.063 µg L-1. In order to verify the accuracy of the method, the spiked sample was analyzed and good recoveries were obtained as shown in Table 4. As indicated in Figure 3(a) and (b), Gd-DTPA-BMA shows similar adsorption behavior with Gd-DTPA, so the developed method was also applied to the analysis of Gd3+ and Gd-DTPA-BMA in patient urine samples. The analytical results presented in Table 5 show that Gd3+ can be detected in the patient urine samples and the amount of the released free Gd3+ from Gd-DTPA-BMA is less than 10% of the amount of Gd-DTPA-BMA. The results also demonstrate that the amount of Gd3+ and Gd-DTPA-BMA in the patient urine was decreased as time goes on, which was in accordance with the pharmacokinetics. 19



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Conclusions In this paper, a novel poly(MAA-EDMA-TiO2 NPs) monolithic capillary was prepared and an on-line method of poly(MAA-EDMA-TiO2 NPs) monolithic CME-ICP-MS was developed for speciation of Gd3+ and Gd-based contrast agent in human urine samples. The monolith is easy to prepare without further modification, and exhibits high adsorption capacity and good anti-interference ability. The developed method of on-line poly(MAA-EDMA-TiO2 NPs) monolithic CME-ICP-MS is featured with high sensitivity, low sample consumption, efficient separation ability, and provides an attractive non-chromatography strategy for the speciation of Gd3+ and Gd-based contrast agents. What is more, the developed method could be applied for the accurate determination of Gd3+ and Gd-based contrast agents in the human urine without oxidation/reduction or subtraction, which is crucial for the speciation of Gd3+ and Gd-based contrast agents in patient urine samples in which the amount of free Gd3+ released from Gd-DTPA-BMA is very small. Considering the LODs of the CME-ICP-MS method at sub µg L-1 level (without preconcentration) and relative high concentration of Gd-based contrast agents and Gd3+ in patient urine sample (at mg L-1 level), we can sacrifice the enrichment factor to further improve the sample throughput. When reducing sample volume from 0.5 mL to 20 µL, the sample throughput can be high to 40 h-1. Such low sample consumption and high throughput make the proposed method very promising in clinical analysis. 20


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Supporting Information Additional information on operating conditions of ICP-MS (Table S1), instrumentation and reagents, sample preparation, preparation of TiO2 NPs (including Figure S1), influence of PBS (including Figure S2), influence of sample loading conditions (including Figure S3), influence of elution conditions (including Figure S4, S5) as noted in the text. This information is available free of charge via the Internet at http://pubs.acs.org.

Acknowledgements Financial support from the National Nature Science Foundation of China (No. 21205090, 21175102), Science Fund for Creative Research Groups of NSFC (No. 20921062), the National Basic Research Program of China (973 Program, 2013CB933900) and the Fundamental Research Funds for the Central Universities (No. 2015203020209) are gratefully acknowledged.

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Figure Legends Figure 1 SEM of monolithic columns containing TiO2 NPs at a NPs/ monomer ratio of (A) 0/1, (B) 0.8/1, (C) 1/1, (D) 1.5/1, (E) 2/1 (magnification of 5000×) and corresponding EDX spectra. Figure 2 Effect of the amount of TiO2 in the monolith on the adsorption behavior of Gd3+ and Gd-DTPA. Conditions: cGd

3+

=10 µg L-1; sample volume: 0.5 mL; sample pH:

, Gd-DTPA

pH=5 for Gd3+ and pH=2.5 for Gd-DTPA in PBS. The error bar was defined as the standard deviation for the analytical results obtained by triplicate analysis. Figure 3 Effect of pH on the adsorption behavior of Gd3+, Gd-DTPA and Gd-DTPA-BMA in the absence (a) or presence (b) of PBS, and the formation of the deduced ternary complex (c). Conditions: cGd , Gd-DTPA, Gd-DTPA-BMA=10 µg L-1; sample volume: 0.5 3+

mL; sample flow rate: 100 µL min-1. The error bar was defined as the standard deviation for the analytical results obtained by triplicate analysis.

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Figure 1

26


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Figure 2

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Figure 3

28


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Table 1. Tolerance of coexisting ions (CGd3+, Gd-DTPA, Gd-DTPA-BMA= 1 µg L-1) Coexisting ions

Tolerance limits (mg L-1)

Concentration range in

Gd-DTPA/Gd-DTPA-BMA

Gd3+

(at pH=2.5)

(at pH=5)

urine (mg L-1) 38, 39

Na+

40000

50000

~2200

K+

50000

50000

~1.9

Ca2+

1000

5000

~120

Cu2+

1000

5000

4×10-5- 5×10-2

Mg2+

10000

20000

~90

Zn2+

1000

20000

0.27-0.85

Fe3+

50

50

~0.17

Al3+

20

10

2.3×10-3- 0.11

SO42-

20000

20000

~192

NO3-

40000

50000

–

Cl-

50000

50000

~3976

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Table 2. Analytical performance of the developed method for Gd3+, Gd-DTPA and Gd-DTPA-BMA Gd3+

Gd-DTPA

Gd-DTPA-BMA

(at pH=5)

(at pH=2.5)

(at pH=2.5)

0.01-20

0.01-20

0.01-20

y =335143x +

y =388207x +

Elements

Linear range (µg L-1)

Linear equations

y=736210x+87909 25225

34390

Correlation coefficient (R)

0.9906

0.9984

0.9995

LODs (ng L-1)

3.6

3.2

4.5

5.7

4.5

5.5

RSD (%, c=0.02 µg L-1 , n=7) Sample throughput (h-1)

5

Enrichment factors

25

30


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Table 3. Comparison of detection limits reported in literatures for Gd3+ and Gd-DTPA following different analytical methods LODs (ng L-1) Samples

Ref.

Analytical method Gd

3+

Gd-DTPA

Human urine

SEC-ICP-MS

3500

—

41

Human urine

CPE-spectrophotometry

910.6

—

16

River water

SPE-ICP-MS

190

200

14

Water samples

SPE-ICP-MS

45

15

15

Human urine

CME-ICP-MS

3.6

3.2

This work

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Table 4. Analytical results of Gd3+ and Gd-DTPA in healthy human urine (mean±SD, n=3) Gd3+

Gd-DTPA

Added

Found

Recovery

Added

Found

Recovery

(µg L-1)

(µg L-1)

(%)

(µg L-1)

(µg L-1)

(%)

0

0.063±0.005

0

N.D.a

0.2

0.272±0.003

0.2

0.208±0.007

Sample

Urine

a

105

104

, not detected.

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Table 5. Analytical results of Gd3+ and Gd-DTPA-BMA in patient urine (mean±SD, n=3)

a

Sample

Gd3+(mg L-1)a

Gd-DTPA-BMA (mg L-1)b

Urine (8 h)

12.9±1.4

682±4

Urine (12 h)

10.0±2.6

157±12

Urine (16 h)

4.9±0.5

126±3

, urine (8 h), urine (12 h) and urine (16 h) were 5000 times dilution for the detection of

Gd3+ at pH=5; b, urine (8 h), urine (12 h) and urine (16 h) were 50000 times dilution for the detection of Gd-DTPA-BMA at pH=2.5.

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for TOC only

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TiO2 Nanoparticles Functionalized Monolithic Capillary

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