extfieldsofstudy: Chemistry Medicine

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Separation and Purification of Fructo-Oligosaccharide by High-Speed Counter-Current Chromatography Coupled with Precolumn Derivatization

High-speed counter-current chromatography (HSCCC) coupled with precolumn derivatization was developed for isolating and purifying fructo-oligosaccharides (FOSs). Firstly, the total FOSs were precolumn derivatized and then separated by high-speed counter-current chromatography (HSCCC) with two-phase solvent system petroleum ether–n-butanol–methanol–water (3:2:1:4, v/v). Secondly, the obtained compounds were deacetylated and the fructo-oligosaccharides (FOSs) with high purity were obtained. Their structures were identified by mass spectrometry (MS) and nuclear magnetic resonance (NMR). This research successfully established a novel strategy for separation and purification of FOS. There is no doubt that the application of the research will be beneficial for the quantitative and qualitative analysis of products containing FOSs.

Introduction
Prebiotics are 'non-digestible compounds which through metabolisation by gut microbiota, modulate the compositions and/or activities of the gut microorganisms, thereby conferring beneficial physiological effects on host' [1]. In recent decades, the bioactivities of prebiotics have attracted more and more attention, worldwide, because of physiological functions such as improving the gut ecosystem, restoring endothelial dysfunction, increasing heme bioavailability, barrier protective function in human intestinal organoids, modulating lipogenesis, reducing triglyceridemia, immunomodulatory and cerebral protective effects [2][3][4][5][6][7][8]. In addition, they could meet the requirements for both sweet tooths and diabetics, due to their non-digestion and non-absorption in body [9].
Fructo-oligosaccharides (FOSs), one of the most significantly used commercial prebiotics in practice, is a mixture (homopolymer) composed of 2-6 fructose monomers connected via β (2-1) glycosidic bonds linked to a terminal glucose residue. In recent years, FOSs have become one of the most important healthy food ingredients, have great potential to improve the quality of many foods, and have been applied in a wide range of fields such as infant milk powder, health care products, pharmaceuticals and cosmetics. Due to the fact that the newer types of functional food ingredients are increasingly popular, high-quality fructo-oligosaccharides (FOSs) are urgently needed to meet the Molecules 2018, 23, 381; doi:10.3390/molecules23020381 www.mdpi.com/journal/molecules demand for quality control and activity research. To date, a series of methods have been established to purify FOSs, such as fast protein liquid chromatography, size-exclusion chromatography, hydrophilic interaction chromatography, and anion exchange chromatography [10][11][12]. However, due to their low yield, time-consuming processes, and small sample amounts, commercialized reference materials of FOS are expensive. Therefore, it is necessary to develop an efficient method for isolating and purifying FOSs. High-speed counter-current chromatography (HSCCC) is a support-free liquid-liquid partition chromatographic technique, which has been successfully applied in the isolation and purification of many compounds [13][14][15][16][17]. Compared with many traditional liquid-solid separation methods, HSCCC benefits from a number of advantages: (1) no irreversible adsorption; (2) complete recovery of the injected sample; (3) tailing minimized; (4) low risk of sample denaturation; (5) low solvent consumption; and (6) favorable economics, as there are no expensive columns needed, and only common solvents are required once the initial investment in the instrument has been made [18]. Although there are many significant advantages to HSCCC, it is difficult to apply on FOSs because of their high polar. In addition, to the best of our knowledge, no report on the separation of FOSs by HSCCC has been published. In our previous research, the target compounds were eluted close to each other near the solvent front, even the two-phase solvent system containing n-butanol or salt. In this article, the polarity of FOSs was lowered by precolumn derivatization; then, three FOSs were purified by HSCCC. Their structures were identified by mass spectrometry (MS) and nuclear magnetic resonance (NMR).
The aim of the present work was to establish an efficient method for the separation and purification of FOSs, and to provide a novel strategy for the separation and purification of oligosaccharides.

Strategy for Separation and Purification of FOSs
Because of structural complexity, high polarity, and difficulty of detection, it is difficult to isolate and separate FOSs by traditional methods. In addition, separation is difficult to realize by HSCCC due to their strong hydrophilicity, which leads to the target compounds staying in lower phase, even in n-butanol-water solvent systems. Therefore, a modified strategy for preparative separation of FOSs is needed. Firstly, the polarities of FOSs were lowered by means of acetylation, which was able to satisfy the requirements of separation by HSCCC. Then, the obtained compounds were deacetylated, and the separation of FOSs was realized (the roadmap is shown in Figure 1).

Optimization of HPLC for FOSs
HPLC was used to analyze the total FOSs and purified FOS. According to the structure of the target compounds, three kinds of detectors (DAD, ELSD and RID) were tested. The ELSD was selected due to its high sensitivity and the short time required to achieve system balance. Due to the high polarity of FOSs, the resolution of samples eluted with solvent in C18 chromatographic column is poor. The Xamide 100A and NH 2 inersil columns were compared, and the Xamide 100A was selected for its good resolution and short processing time. Several elution systems were evaluated, such as isocratic elution of acetonitrile-water and methanol-water. The results showed that the target compounds were able to achieve suitable separation when the mobile phase consisted of acetonitrile-water (70:30, v/v). The column temperature was 30 • C, the flow rate of the mobile phase was 1.0 mL/min, and the evaporate temperature was 80 • C, the Neb temperature was 90 • C, the gas flow rate was 1.2 SLM. Figure 2 shows that the purities of compounds I, II and III are 98.5%, 98.2%, 98.7%, respectively, as determined by HPLC.

Selection of Precolumn Derivatization Method
In our research, the HSCCC was operated after the precolumn derivatization of FOSs. On suitable method of precolumn derivatization is ether-forming groups, which can be removed easily after completion of the separation process. According to references [19][20][21], the common derivatization methods for hydroxyl groups are methyl, acetyl, and phenyl. Comparing the degree of difficulty of the experiment, the length of operation time, and the degree of hydroxyl group substitutions, acetylation of hydroxyl groups in FOSs was selected.

Selection of Two-Phase Solvent System and Other Conditions of HSCCC
An ideal range of K D value for the applied material is a key factor in successfully selecting a suitable two-phase solvent system, which is also the basis for obtaining a good separation effect of the target compounds using HSCCC. As reference [22] describes, a K D value in the range of 0.5 to 2 is a requirement for selecting a suitable two-phase solvent system. In this experiment, the structures of FOSs were modified by acetylation. After acetylation, the hydroxyls became acetyls, and their polarities were remarkably reduced. According to the experience of separation of low-polar compounds, a series of solvent systems composed of petroleum ether-ethyl acetate-methanol-water with different volume ratios (1:1:1:1, 4:5:4:5, v/v), and petroleum ether-n-butanol-methanol-water with different volume ratios (3:2:2:3, 3:2:1:4, v/v) were designed to achieve an ideal K D for the target compounds. The evaluated K D values for the three compounds are shown in Table 1.

Selection of Two-Phase Solvent System and Other Conditions of HSCCC
An ideal range of KD value for the applied material is a key factor in successfully selecting a suitable two-phase solvent system, which is also the basis for obtaining a good separation effect of the target compounds using HSCCC. As reference [22] describes, a KD value in the range of 0.5 to 2 is a requirement for selecting a suitable two-phase solvent system. In this experiment, the structures of FOSs were modified by acetylation. After acetylation, the hydroxyls became acetyls, and their polarities were remarkably reduced. According to the experience of separation of low-polar compounds, a series of solvent systems composed of petroleum ether-ethyl acetate-methanol-water with different volume ratios (1:1:1:1, 4:5:4:5, v/v), and petroleum ether-n-butanol-methanol-water with different volume ratios (3:2:2:3, 3:2:1:4, v/v) were designed to achieve an ideal KD for the target compounds. The evaluated KD values for the three compounds are shown in Table 1.  As reference [22] describes, when the KD value is smaller than 0.5, the solutes will be eluted close to each other and near the solvent front. As shown in Table 1, the KD values of the two-phase solvent systems consisting of ethyl petroleum ether-n-butanol-methanol-water (3:2:2:3, v/v) was smaller than 0.5, which led to bad peak resolution. When the two-phase solvent systems were used As reference [22] describes, when the K D value is smaller than 0.5, the solutes will be eluted close to each other and near the solvent front. As shown in Table 1, the K D values of the two-phase solvent systems consisting of ethyl petroleum ether-n-butanol-methanol-water (3:2:2:3, v/v) was smaller than 0.5, which led to bad peak resolution. When the two-phase solvent systems were used for separation, the solutes eluted close to each other and near the solvent front. When the two-phase solvent systems composed of petroleum ether-ethyl acetate-methanol-water (4:5:4:5, v/v) were used, the K D value was greater than 2.0. When the two-phase solvent systems were used for separation, the target compounds were able to be isolated; however, the separation time was too long, which led to excessively broad peaks. The K D values of the two-phase solvent systems composed of petroleum ether-ethyl acetate-methanol-water (1:1:1:1, v/v) and petroleum ether-n-butanol-methanol-water (3:2:1:4, v/v) were between 0.5 and 2, which were suitable for separating the target compounds. Therefore, the HSCCC separation was carried out with the above two solvent systems. When the two-phase solvent system composed of petroleum ether-ethyl acetate-methanol-water (1:1:1:1, v/v) was used for isolation, compound I, II and III were able to be eluted in a suitable time, but the separation effect of the target compounds was not perfect. As shown in Figure 3, when the two-phase solvent systems composed of petroleum ether-n-butanol-methanol-water (3:2:1:4, v/v) were used, acceptable separation time and good resolution were able to be obtained. The collected fractions of HSCCC were analyzed by HPLC-ELSD. 25.6 mg of compound I, 29.4 mg of compound II, 12.8 mg of compound III with the purities of 98.5, 98.2%, and 98.7%, respectively, were obtained from 200 mg of the crude sample.

Precolumn Derivatization
The precolumn derivatization of FOS was conducted by the method described in reference [19]. Acetic anhydride (20.4 g, 0.2 mol) was added to a solution of FOSs (5 g) in pyridine (50 mL) at room temperature. After the reaction mixture was stirred overnight, the solution was concentrated under reduced pressure to obtain the raw product as a yellow syrup, which was used for further purification.

Apparatus
Model TBE-300A commercial instrument (Shanghai Tauto Biotech Co., Ltd., Shanghai, China), which comprises a multilayer coil of 150 m in length and 1.6 mm id with a 300 mL total capacity, was employed as the preparative HSCCC instrument in the present study. The β values of this preparative column ranged from 0.5 at the internal to 0.8 at the external (β = r/R, where r is the rotation radius or the distance from the coil to the holder shaft, and R (R = 8 cm) is the revolution radius or the distance between the holder axis and central axis of the centrifuge). A Model HX-1050 constant-flow pump (Beijing Bokang Experimental Equipment Co., Ltd., Beijing, China) was used to pump the solvent into the column. A 30 mL loop manual sample injection (Shanghai Tauto Biotech Co., Ltd., Shanghai, China) was used to inject the sample to the column. Spectra were achieved with a Model 8823A-UV Monitor (Beijing Institute of New Technology Application, Beijing, China) at a wavelength 254 nm. The pH value was monitored by a Model 320 pH meter (Mettler Toledo Instruments, Shanghai, China). The chromatogram was obtained by a portable recorder (Yokogawa Model 3057, Sichuan Instrument Factory, Chongqing, China).

Measurement of Partition Coefficient
The selection of the composition of the two-phase solvent system was based on the distribution constant (K D ) value of the target compounds. HPLC analysis was applied to determine the K D values by the following steps [22]: crude extract (5 mg) was dissolved in a test tube (20 mL) to which each phase (5 mL) of the equilibrated two-phase solvent system was added. The capped tube was shaken vigorously for several minutes to thoroughly equilibrate the sample with two phases. Then, 1 mL upper phase and 1 mL lower phase were evaporated separately to dry by the gentle nitrogen stream. The residues were diluted with methanol (1 mL) and analyzed by HPLC to determine the K D of the target compound. The K D value was calculated by the following equation: K D = A U /A L (A U : the peak area of the upper phase A L : the lower phase).

Preparation of Sample Solution and Two-Phase Solvent
The crude extract was dissolved in the mixed two-phase solvent system (lower phase and upper phase, 1:1, v/v) to obtain the sample solution. Each solvent was added to a separatory funnel and then repeatedly and vigorously shaken at room temperature to obtain the two-phase solvent system. Shortly before use, the two phases were separated, following which the upper phase was used as stationary phase and the lower phase was used as mobile phase.

Separation Procedure
The separation procedure was carried out in accordance with [23]: firstly, the upper phase (stationary phase) was pumped into the multilayer coil column until it was entirely filled at a flow rate of 20 mL/min, while the lower phase (mobile phase) was pumped into the headend of the inlet column at a flow rate of 2 mL/min, and the rotate speed of the apparatus was 800 rpm. After reaching hydrodynamic equilibrium, the sample dissolving a clear mobile phase (200 mg of total FOSs in 8 mL of both phases) was injected into the column via the injection valve. The effluent of the column was continuously monitored with a UV detector at 254 nm, which was collected at 3 min intervals using a fraction collector. After running, the residual solvent in the column was pushed out, and then the retention of the upper phase was measured. The collected fractions were analyzed by HPLC-ELSD. After analysis, the fractions with high purity were collected and dried separately.

Reduction Reaction
The purified acetylated FOSs were reduced by the following method [24]. The target compounds were added to a round-bottom flask while dissolved in MeOH. NaOMe (2 g, 0.037 mol) was added in a single portion and stirred at room temperature for 1 hour. After stirring for 20 min, the reaction was neutralized with Dowex 50WX8 (H + form) and then filtered. The resulting solution was concentrated under reduced pressure till thick oil was obtained. Purification by Sephadex LH-20 column chromatography (eluting with methanol) afforded the target compounds as white powder.

HPLC-ELSD Analysis
The HPLC equipment was an Agilent system, and included an Agilent 1260 TCC, an Agilent 1260 quat pump, an Agilent 385-ELSD (Evaporative Light Scattering Detector), and an Agilent workstation (Agilent, Palo Alto, CA, USA).
The prebiotics and FOSs purified from the HSCCC separation were analyzed by HPLC-ELSD with a Xamide 100A column (250 × 4.6 mm, id) and a column temperature of 25 • C. The mobile phase consisted of acetonitrile-water (75:25, v/v) at a flow rate of 1.0 mL/min.

Structural Identification
The identification of target compounds was carried out by ESI-MS on an Agilent 1100/MSD and by 1 H-NMR, 13 C-NMR spectra on a Varian-600 NMR spectrometer with D 2 O solvent and tetramethylsilane (TMS) as internal standard. Comparing the ESI-MS, 1 H-NMR, and 13 C-NMR data with the literature, the compounds I-III were identified as 1-kestose, 1,1-kestotetraose, and 1,1,1-kestopentaose, respectively.  -6). Compared with the data given in reference [25], peak I was identified as kestose.  -6). Compared with the data given in reference [26], peak II was identified as 1,1-kestotetraose.  Fd-6). Compared with the data given in reference [27], peak III was identified as 1,1,1-kestopentaose.

Conclusions
The present research indicated that a novel strategy had been developed to separate and purify FOS from total FOSs. Firstly, the sample was structurally modified by acetylation. Then, the acetylated target compounds were isolated by HSCCC with two-phase solvent system composed of petroleum ether-n-butanol-methanol-water (3:2:1:4, v/v), and three compounds with high purity were obtained in one step. Finally, the target compounds were obtained by reduction reaction whose structure was identified by ESI-MS and NMR. In the present research, kestose in 98.5% purity, 1,1-kestotetraose in 98.2% purity and 1,1,1-kestopentaose in 98.7% purity were obtained in a single run. Compared with classic methods such as pre-HPLC, HSCCC coupled with precolumn derivatization is characterized by excellent sample loading, simple operation, and as being less time-consuming and more environmentally friendly. This study indicated that it is an efficient method for the separation and purification of FOSs, and provides a novel strategy for the isolation of oligosaccharides.


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Development of a method to quantify total and free irinotecan and 7-ethyl-10-hydroxycamptothecin (SN-38) for pharmacokinetic and bio-distribution studies after administration of irinotecan liposomal formulation

In 2015, liposomal formulation of irinotecan (ONIVYDE) has been approved by FDA and widely applied in the treatment of pancreatic cancer. ONIVYDE is a novel liposome formulation, entrapping CPT-11 in the aqueous core of vesicles using a modified gradient loading method. Due to toxicity concerns, it is essential to explore a rapid and reliable method to effectively isolate and quantify the non-liposomal, namely, free CPT-11and total CPT-11 in plasma. This study focuses on separation of non-liposomal CPT-11, evaluation of the pharmacokinetics of free CPT-11 and total CPT-11 and bio-distribution after intravenous administration of CPT-11 liposome. Free CPT-11 in plasma was separated by solid-phase extraction (SPE). The amount of total CPT-11 and main metabolite 7-ethyl-10-hydroxycamptothecin (SN-38) in plasma was quantified by ultra-performance liquid chromatography–MS/MS. The calibration curves fitted well and lower limit of quantitation for SN-38, free CPT-11, total CPT-11 and CPT-11 in tissue and were 5 ng/ml, 10 ng/ml, 4.44 ng/ml and 25 ng/ml respectively. The recoveries, precision and accuracy of the method appear satisfactory. Using this method, the pharmacokinetics and bio-distribution of CPT-11 liposome formulation after an intravenous dose of 2.5 mg/kg were then investigated.

Introduction
Nanoparticles like small unilamellar liposome have shown their promise for improving the pharmacokinetics and tumor localization of encapsulated anticancer drugs [1] . In 2015, FDA approved ONIVYDE (irinotecan liposome injection) in combination with fluorouracil/folinic acid based on the result of clinical study. Compared with fluorouracil/folinic acid, this dosage regimen significantly pro-longed overall survival of patients with advanced and metastatic pancreatic cancer [2] . ONIVYDE was prepared by a modified active loading method, was able to entrap irinotecan (CPT-11) at an extreme high drug to lipid ratio, retain encapsulated CPT-11 stable in vesicles, and also protect CPT-11 from hydrolysis and metabolic conversion to 7-ethyl-10-hydroxycamptothecin (SN-38) during the systemic circulation [3] . For CPT-11 is an ionizable drug, loaded by active loading method, drug release rate and circulation half-life determined the therapeutic activity of liposomal drug delivery systems. And only if liposomes retain CPT-11 over several hours to days will achieve high drug accumulation in tumor [4] . Therefore, it is important to control the ratio of free drug, monitor the pharmacokinetics (PK) and tissue distribution properties to fully understand and the therapeutic efficacy and side effects of liposome formulation.
To get a clear relationship of pharmacokinetics between total drug and non-liposomal drug, namely the free drug, there is an insistent demand of a sophisticated analytical technique to separate free CPT-11 from liposome in plasma. Meanwhile, the developed separation method should have a consistent recovery and extraction efficiency of free CPT-11. Moreover, formulation integrity should be maintained without leakage or breaking of the liposome during sample preparation and storage. Up to now, there are several methods have been reported to achieve this end for vincristine liposome, amphotericin B liposome and doxorubicin liposome, such as solid-phase extraction (SPE) [5][6][7][8][9][10] , ultrafiltration [11] , ion-exchange chromatography [12] , and capillary electrophoresis [13,14] . Nevertheless, no method has been observed for the separation and detection of free CPT-11 and liposomal CPT-11. Furthermore, as high risk of liposome leakage using ultrafiltration and high sample dilution during gel chromatography, these methods have obvious limitations in applying for CPT-11 liposome determination from biological samples.
Based on previous reports [5][6][7][8][9][10]15] , SPE is the most feasible method for separation of non-liposomal CPT-11 and liposomeencapsulated CPT-11 due to better security and relative rapidness. However, the separation and quantification of nonliposomal, liposomal CPT-11 and metabolite SN-38 was still challenging because carboxylesterase in rat plasma is highly active. Considering that the catalysis of carboxylesterase is related to protein concentration, pH, ionic strength, and these conditions are easily changed under SPE processing [16,17] , the working process especially the solution involved in SPE should be controlled properly. In the present study, we build a method to (i) separate free CPT-11 (F-CPT-11) from liposome by SPE without damaging liposome (ii) achieve total release of encapsulated CPT-11 and extract total CPT-11(T-CPT-11) from liposome using protein precipitation extraction (iii) detect CPT-11 in liver (L-CPT-11), heart, spleen, lung and kidney (OT-CPT-11) of mouse using protein precipitation extraction. And the developed method had been proved to be valid for the separation and quantitation of non-liposomal drug and total drug in plasma and tissue samples according to the FDA guidelines on bioanlytical method [18] , allowing the study of pharmacokinetics and bio-distribution after administration of CPT-11 loaded liposome.

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Materials and methods  LC separations were performed on an ACQUITY UPLC system (Waters Corp., Milford, MA, USA) with UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm; Waters Co., Ltd., USA). Acetonitrile (A) and water containing 0.1% formic acid (B) were used as the mobile phase, and the elution was performed by 27% (A): 73% (B) (v/v) at a flow rate of 0.3 ml/min. The injection volume was controlled at 20 μl. The column temperature and the auto sampler temperature was set to 60 °C and 4 °C respectively.

Instrumentations and UPLC-MS/MS conditions
MassLynx TM Version 4.1 software was applied for data acquisition and analysis.

Plasma sample preparation for CPT-11 and SN-38
Part A: SPE for F-CPT-11 F-CPT-11 in plasma was separated from CPT-1 1 liposome using SPE with an Oasis HLB column (1 cc/30 mg, Waters Corp., Milford, Massachusetts, USA) connected with a vacuum air pump. Previous study has shown that chilling samples at 4 °C could probably sustain the stability of lactone form [19] . Thus, all solvent involved in SPE process was maintained at 4 °C. Firstly, SPE cartridge was preconditioned with 1.0 ml methanol and 1.0 ml water. Then, 75 μl 5% glucose solution were added to 50 μl CPT-11 spiked plasma, the mixture were passed through cartridges without vacuum. Then, 20 μl ZnSO 4 , 75 μl 5% glucose solution and 50 μl IS working solution (5 μg /ml) were added to 50 μl plasma. Samples were mixed by vortex for 1 min and then centrifuged at 7500 rpm for 3 min. Supernatants were then collected, and passed through the conditioned cartridges with vacuum. After complete passage of samples, each cartridge was further rinsed with 2 ml of PBS, 1 ml of 3.75% ammonia. Finally, each cartridge was washed with 1 ml methanol containing 2% acetic acid, target analyte in the rinsed cartridge was collected and evaporated under a stream of nitrogen at 37 °C. Residues were then dissolved with 200 μl methanol/water containing 0.1% acetic acid (70:30, v/v) and centrifuged at 13,000 rpm for 5 min. 20 μl of the supernatant were injected into the UPLC separation system. Part B: Protein precipitation extraction for T-CPT-11 and SN-38 Fifty microliter of IS solution (5 μg/ml in methanol) was firstly added to 50 μl plasma (QC samples and calibration standards were spiked with corresponding working solutions) and vortex mixed for 30 s. Then, 400 μl of methanol containing 0.1% acetic acid was added to the plasma samples, after mixing by vortex for 5 min, the mixture was centrifuged at 15 000 rpm for 10 min. Finally, a 20 μl sample of supernatant were injected into the UPLC column for analysis.

Tissue sample preparation for CPT-11
Firstly, 50 μl IS solution (5 μg /ml in methanol) and 850 μl of methanol /acetonitrile (50:50, v/v) were added to 50 μl tissue samples (QC samples and calibration standards were spiked with corresponding working solutions). Samples were then mixed by vortex for 1 min, and centrifuged at 15,000 rpm for 10 min to precipitate possible particulate matter, and the supernatant was collected and then evaporated under a stream of nitrogen at 37 °C. Residues were then reconstituted in 100 μl of methanol /water containing 0.1% acetic acid (70:30, v/v) and centrifuged at 13 000 rpm for 5 min. Finally, a 20 μl sample of supernatant were injected into the UPLC column for analysis.

Method feasibility
It is necessary to assess the feasibility of SPE method before the method validation, and prove that the separation and quantitation method could be used for pharmaceutical and bio-distribution study. Moreover, the sample preparation methods (both SPE and protein precipitation extraction) must be appropriate for the separation of F-CPT-11 from liposome. Meanwhile, liposome integrity must be ensured. In this research, method feasibility was performed by determining the part of F-CPT-11(F %) and T-CPT-11 (T%) in QC samples (QC samples were spiked with CPT-11 liposomes). And F-CPT-11 (F %) was calculated by the formula: F % = C F /C N × 100, here C F is the F-CPT-11 concentration detected in QC samples and C N is the nominal concentration of the CPT-11 added in QC samples. And C F may compose of originally present F-CPT-11 in liposome formulations as well as the part of released CPT-11 during mixing and SPE separation process. If SPE method could separate F-CPT-11 from liposome, the value of F 1 % after the first SPE process should equal to the encapsulation rate. To further prove the liposome integrity throughout the SPE process, liposome fraction in the first SPE process were collected. The collected liposome fraction was then re-run through a new preconditioned cartridge. Theoretically, if the liposomes remain intact and the CPT-11 was not released from the collected liposome fraction during the second SPE process, the value of F 2 % after the second SPE process should equal to zero. Moreover, the value of T-CPT-11 (T%) could be used to verify the feasibility of protein precipitation extraction process. In the equation: T % = C T /C N × 100, C T is the T-CPT-11 concentration detected in QC samples, and C N is the nominal concentration of the T-CPT-11 added in QC samples. If protein precipitation process could deposit protein completely and ensure a total release of drug molecular from liposomes, the value of T% should be within 85%-115%.

Method validation
In this study, analytical methodology of analytes were built for pharmacokinetics studies of CPT-11 liposome. As liver plays an essential role in drug metabolism and excretion when doing bio-distribution studies, so we build analytical methodology for liver and mixture of other tissues (heart, spleen, lung, kidney) respectively. Moreover, selectivity, linearity of calibration curves, lower limit of quantitation (LLOQ), precision and accuracy, recovery and matrix effect and stability of the analytical methodology were validated. And analytical methodology was run on three consecutive days.

Specificity
Specificity of the methodology was evaluated by comparing the chromatograms of blank plasma, blank liver homogenates, and homogenates mixture of blank tissue (heart, spleen, lung and kidney) with the corresponding analytes spiked plasma or tissue homogenates.

Linearity of calibration curves and LLOQ
Calibration curves of plasma samples were prepared by diluting corresponding stock solutions to seven final concentrations, ranging from 5 ng/ml to 2000 ng/ml for SN-38, 10 ng/ml to 10 000 ng/ml for F-CPT-11, and 4.4 ng/ml to 20 000 ng/ml for T-CPT-11. As for tissue samples, calibration curves achieved by using seven nonzero liver homogenates and seven nonzero tissue homogenates calibration standards from 25 ng/ml to 1000 ng/ml for CPT-11. Calibration curves linearity was analyzed by plotting peak area ratios of CPT-11 and SN-38 versus the nominal concentration. Moreover, least-square regression using a 1/ (concentration) 2 weighting was applied to evaluate linearity relationship. The correlation coefficients (r 2 ) needed to be above 0.99. The LLOQ of the calibration curve was defined by using six lots of blank plasma samples and blank tissue homogenates. Both precision and accuracy are required to be less than 20%.

Precision and accuracy
For all analytes, intra-day precision, inter-day precision and accuracy were estimated by analyzing QC (low, middle and high) samples on three separated days in six replicates. The precision and accuracy was defined as the relative standard deviation (RSD%) and the relative error (RE%). Moreover, the acceptability for precision was within 15% RSD and that of accuracy was within 15% RE of the theoretical values.

Extraction recovery and matrix effect
The extraction recovery of CPT-11 and SN-38 were analyzed by comparing the peak areas of the extracted QC (low, middle and high) samples with average peak area of CPT-11/SN-38 added blank samples after extraction. Moreover, at each QC concentration six replicates were determined. And matrix effect was determined as the ratio of the peak area from extracted with blank plasma/ tissue homogenates to the mean peak area from extracted without plasma/ tissue homogenates at QC concentrations.

Stability
Stability of plasma samples and tissue samples were tested including auto-sampler storage, freeze-thaw cycles, and shortterm, long-term storage stability at three different levels (QC samples). The auto-sampler stability was evaluated by placing the extracted QC samples in the auto-sampler, and keeping them at 4 °C for 24 h. Freeze-thaw stability was evaluated by comparing peak areas of QC samples after 3 cycles of freeze-thaw ( −20 °C to ambient temperature) with peak areas of newly prepared QC samples. Short-term stability of F-CPT-11was invested by keeping the QC samples at 4 °C for 6 h, and that of T-CPT-11, L-CPT-11, OT-CPT-11 and SN-38 was conducted at 25 °C for 12 h for. And the long-term stability was evaluated by comparing the concentration of newly prepared QC samples with that of QC samples stored at − 80 °C for 1 months. Moreover, robustness study of the developed method against small variations of column temperature (from 59 °C to 61 °C) was also conducted.

Preparation of CPT-11 liposome formulation
First, solution of triethylammonium salt sucrose octasulfate (TEA 8 SOS) was prepared. The concentration was adjusted to 0.65 mol/l, and the pH was 5.5-6.0. Then, the mixture of DSPC (6.81 mg/ml), chol (2.22 mg/ml) and mPEG2000-DSPE (0.12 mg/ml) were combined in 50% (w/v) ethanoic solution and then mixed with TEA 8 SOS solution at 65 °C for 30 min. The lipid suspension was then extruded 20 times at 65 °C. Unentrapped TEA 8 SOS were then removed by Sephadex G-75 gel filtration chromatography, and the gel filtration was eluted with pH 6.5 HEPES /NaCl (17/144, mM /mM) buffer. After buffer exchange, CPT-11 HCl was then added to liposome at 4.3 mg/ml. The solution was then incubated at 65 ± 5 °C for 1 h and immediately chilled on ice for 20 min.

Characterization of CPT-11 liposome formulation
The average size and zeta potential of prepared liposomes was analyzed by dynamic light scattering (Zetasizer Nano ZS90; Malvern Instruments Ltd., Malvern, UK). Each sample was measured in triplicate for 14 cycles at 25 °C. Then, ultrafiltration centrifugal method was used to assess the entrapment efficiency of liposome. In this method, 100 μl liposome was added into the ultrafiltration concentrator (10 kDa) and then centrifuged (3000 rpm) for 30 min to make sure the unentrapped CPT-11 follow to the bottom, and the content was the part of the entrapped liposome. The content of F-CPT-11 and liposomal fraction of CPT-11 were then determined by high-performance liquid chromatography (HPLC) on a reverse Agilent 5 TC-C 18 (2) column (250 mm × 4.6 mm, 4.5 μm). The entrapment efficiency was then calculated by the equation: entrapment efficiency % = liposome fraction of CPT-11/ (liposome fraction of CPT-11 + F-CPT-11) * 100.

Pharmacokinetic analysis and bio-distribution study
The developed method was finally applied in the quantification of SN-38, F-CPT-11 and T-CPT-11 for pharmacokinetic study in rat plasma and bio-distribution study (including heart, liver, spleen, lung and kidney) in mice. Animal experiments involved were performed according to the "Guidelines for the Care and Use of Laboratory Animals", and were approved by the Animal Ethics Committee of Shenyang Pharmaceutical University (Shenyang, China). Each rat was given at 2.5 mg/kg CPT-11 liposome by intravenous administration. Then, at 0.083, 0.25, 0.5,1, 2, 4, 6, 8, 12, and 24 h, 0.2-0.3 ml blood samples was obtained through orbital venous plexus and collected in polyethylene tubes. Collected blood samples was immediately centrifuged at 10 000 rpm for 5 min, upper plasma was then stored at −80 °C until analysis.
In bio-distribution study, each mouse (22 ± 2 g) was given 2.5 mg/kg CPT-11 liposome formulation by intravenous administration. Vital organs (heart, liver, spleen, lung and kidney) were then removed at 6 h, 24 h and 48 h and washed with saline thoroughly to remove blood. Then, tissues were dried, weighed and diluted 1:1 by saline and homogenized with saline carefully in an ice bath and stored at − 80 °C until analysis.
In this study, the DAS 2.1.1 was used for pharmacokinetic parameters analysis. All values were expressed as mean ± SD.

Method optimization
As carboxylesterase is in a high active state in rat plasma, inactivating carboxylesterase and preventing the transformation of CPT-11 into metabolite SN-38 during sample processing was a major concern. The lactone forms CPT-11 and SN-38 have a closed α-hydroxy-δ-lactone ring, which play a key role in the interaction between CPT-11 / SN-38 with topoisomerase I [16] . However, it should be noted that this functional lactone ring was liable and probably hydrolyzed to the carboxylate form [20] . The hydrolysis of the lactone ring occurred under a pH-dependent equilibrium, and the rate is dependent on pH [17] , ionic strength [16] , and protein concentration [17] .
In the current study, liposome could stabilize entrapped CPT-11 as active lactone form. And it was also possible to ensure that CPT-11 and SN-38 were quantified as total lactone form by adjusting the pH during pre-treatment and chromatographic separation process [21] . The SPE separation process of F-CPT-11 has to be achieved without breaking the liposome integrity. Previous study has shown that chilling samples at 4 °C could probably sustain the stability of lactone form [19] . Thus, all solvent involved in SPE process was maintained at 4 °C. Moreover, zinc sulfate which has strong capacity of enzyme inactivation and compatibility of mass analyses was added to plasma to inactivate carboxylesterase [22,23] . Mass spectroscopy parameters were optimized in positive ion mode to achieve sensitivity and selectivity. CPT-11, IS and SN-38 spectrometric analysis were scanned in ESI positive, and MRM mode was used for signal acquisition. It was found that CPT-11, IS and SN-38 were more sensitive in positive ion mode, were able to accept proton and generate [M + H] + ions. And the base peak of CPT-11, IS and SN-38 ( Fig. 1 ) was selected at m/z 587.4, 393.2 and 349.2 respectively. In addition, collision energy was further optimized to acquire more abundant product ions of three analytes. The most prominent and stable fragments of CPT-11, SN-38 and IS were selected at m/z 167.1, 349.2 and 305.1 at a collision energy of 37 eV, 34 eV and 35 eV respectively. And the source temperature was set at 120 °C.

Specificity
The specificity of SPE method and protein precipitation extraction were evaluated by analyzing six different batches of blank plasma/tissue versus the corresponding analytes spiked plasma/tissue. The typical chromatograms are shown in Fig. 2 and Fig. S1. The chromatographs demonstrate the sensitivity, selectivity and specificity of both methods. Retention time of 0.67, 1.63, and 1.28 min were observed for CPT-11, IS and SN-38 respectively. Moreover, there was no obvious interference from blank plasma and blank tissue at corresponding retention times.

Precision and accuracy
The intra-day and inter-day precision and accuracy of three analytes were validated by analyzing LLOQ ( n = 6) and QC samples ( n = 6,) at three levels in plasma and tissue. As shown in Table 1 , the established method was accurate and repeatable, tested samples were within the acceptable criteria (RSD% < 15%).

Recovery and matrix effect
SPE and protein precipitation extraction were applied in this study for extracting F-CPT-11 and T-CPT-11 from plasma and tissue. The mean extraction recoveries and matrix effect for two methods were shown in Table 2 . Considering that the extraction recoveries of tissue samples were still reproducible and compatible, the developed method remain sufficient within the detection limit. Moreover, as the matrix effect was also within the acceptable range (below 115%), reproducible and did not alter the precious or accuracy.

Stability
The stability of CPT-11 and SN-38 in plasma and tissue was investigated under a variety of conditions: short-term stability, long-term stability, freeze-thaw stability and auto sampler stability. The results were shown in Table S1. Here, the shortterm stability of the F-CPT-11 samples was investigated at 4 °C to reduce likely occurrence of liposome leakage. As shown, the results indicated that F-CPT-11 was stable at 4 °C for 6 h and at −30 °C for 2 weeks. Other samples were stable under analysis, all RE values were less than 15%. The stability of this UPLC-MS/MS method against column temperature were also evaluated. As shown in Table S2, tested samples were within the acceptable criteria (all RE and RSD values were less than 15%). Moreover, RSD for inter-and intra-batch were 12.32% and 5.04% respectively, all less than 15%. The results indicated that CPT-11 was stable for against small variations of column temperature.

Dilution effect
To ensure the accuracy of quantitation, plasma samples with high concentration of T-CPT-11/SN-38 must be diluted before processing. Therefore, the dilution effect of T-CPT-11 and SN-38 in rat plasma were measured. As shown in Table S1, the established dilution method was accurate and repeatable, tested  samples were within the acceptable criteria (RSD% < 15%, RE % < 15%).

Method feasibility
Protocol for separating F-CPT-11 from plasma was shown in Fig. 3 . And the feasibility results for both SPE and protein precipitation extraction were summarized in Table 3 . To quantify the released F-CPT-11 during the extraction process, QC samples spiked with CPT-11 liposome solution ( n = 3) were extracted at three concentrations by using SPE. After one-step extraction, F-CPT-11 (F 1 %) in LOQ, MOQ, and UOQ were 0, 4.90% and 1.59% respectively. And the corresponding F 2 % value in three QC levels were 0%, 0.92%, 1.34% respectively. Compared with the amount of F-CPT-11 originally present in CPT-11 liposome solution, the value of F 1 % and F 2 % in QC samples were below than 5% and negligible. Thus, this result showed that SPE method could separate the F-CPT-11 from liposomes without changing its percentage and causing liposome damage during the extraction. And the Table 1.

-Precision and accuracy for assay of F-CPT-11, T-CPT-11, SN-38 in rat plasma and L -CPT-11, OT-CPT-11 in mouse
at QC concentrations on three consecutive days (mean ± SD, n = 6).     concentrations of three QC samples spiked with liposomal CPT-11 were determined after extraction using protein precipitation, indicating that the protein precipitation extraction could achieve complete release of encapsulated CPT-11 from liposome.

Preparation and characterization of CPT-11 liposome
For CPT-11 liposome formulation, the entrapment efficiency was 98.23%. The mean diameter and zeta potential of CPT-11 liposomes measured by DLS were 131.4 ± 0.14 nm and − 18.5 ± 8.5 mV, respectively.

Pharmacokinetic and bio-distribution study
The method described above was then used for pharmacokinetic and bio-distribution study after intravenous dose of 2.5 mg/kg CPT-11 liposome. The plasma concentrations-time curves of F-CPT-11, T-CPT-11 and SN-38 were presented in Fig. 4 . And main pharmacokinetic parameters of the analytes were shown in Table 4 . After injection of CPT-11 liposomes, it was anticipated that most of the CPT-11 measured in rat plasma was encapsulated in the liposomal carrier to avoid system toxicity [11] . In this study, the C max of F-CPT-11, T-CPT-11 were 1979.37 ng/ml and 45 812.62 ng/ml respectively, while the latter is about 23 times higher than the former. At 30 min after intravenous administration of CPT-11 liposome, the plasma concentration of F-CPT-11 was 1359.5 ng/ml, and that of T-CPT-11 was 54 207.36 ng/ml. This result indicated that F-CPT-11 represented 2.5% of the total CPT-11 in the plasma at this time point. As the result of method fea-


s2fieldsofstudy: Chemistry Physics

text:
Adsorption of Acetone on Rutile TiO 2 : A DFT and FTIRS Study

: Acetone adsorbed on rutile TiO 2 nanoparticles was investigated with respect to its energetic, vibrational, and chemical properties. Temperature-dependent ultrahigh-vacuum Fourier transform infrared spectroscopy measurements for di ﬀ erent acetone dosages (4.5 − 900 L) give insights into the acetone adsorption behavior. Those experiments indicate thermal-induced reactions of acetone on rutile TiO 2 surfaces yielding new species. Density functional theory calculations were performed to investigate acetone adsorption on rutile TiO 2 (110). Particularly, the importance of sampling the adsorption con ﬁ guration space is shown. Adsorption geometries that are energetically signi ﬁ cantly more favorable than the commonly used high-symmetry con ﬁ gurations are presented. To facilitate the comparability to the experiment, theoretical infrared spectra were computed using density functional perturbation theory for various acetone adsorption geometries using di ﬀ erent exchange-correlation functionals. Additionally, computational spectra were obtained for several species which are potential products from reactions of acetone on TiO 2 surfaces. The investigated species are η 2 -acetate, η 2 -diolate, η 1 -enolate, and mesityl oxide. For η 1 acetone, experimental and calculated spectra ﬁ t well for low temperatures, whereas for elevated temperatures, emerging bands indicate the formation of diolate. Comparison of the energetic and vibrational properties of acetone adsorbed on rutile TiO 2 (110) for various exchange-correlation functionals; computational study on the adsorption of enolate on rutile TiO 2 (110) (PDF)

■ INTRODUCTION
The adsorption and possible subsequent chemical decomposition of acetone on titania (TiO 2 ) surfaces are of broad interest because of their relevance in various processes ranging from photocatalysis 1−6 to the remediation of airborne acetone contaminants. 1,7 At hybrid interfaces between inorganic surfaces and organic linker molecules, acetone may act as a competitor to coupling agents like carboxylic acids. 8−10 Thus, a detailed understanding of the relevant adsorption phenomena is crucial. In general, acetone is present in numerous chemical processes because of its usage as solvent or reagent. 11 Particularly, acetone often occurs in catalytic reactions as a reactant, intermediate, or product. On oxidized TiO 2 surfaces, acetone is photochemically active 12 and associated phenomena have raised widespread interest. 1,4 Numerous studies of the acetone adsorption on TiO 2 exist, e.g., on the site competition during co-adsorption of acetone and other adsorbates, 7,12 on acetone-assisted oxygen vacancy diffusion, 13 as well as on its photochemistry. 1,2,4,5,14,15 In this regard, the well-studied (110) surface of rutile TiO 2 (r-TiO 2 ) serves often as an ideal single-crystal model system for surfacescience studies. 16 The adsorption of acetone on stoichiometric, oxidized, or reduced rutile TiO 2 (110) surfaces was investigated previously by different groups. 12,17−19 In an experimental study, Henderson 19 presented six different species potentially occurring upon the interaction of acetone with a rutile TiO 2 (110) surface. At low temperatures and at a clean surface, acetone is supposed to adsorb as η 1 -acetone. 6,20 Since this species dominates at low temperatures and high coverages, it is the main subject of the computational part of this study. Other species only seem to emerge under specific conditions. 6,20 In the presence of adsorbed oxygen, such conditions are, for example, elevated temperatures for η 2 -diolate formation 1 and UV irradiation for obtaining η 2 -acetate from diolate and η 1enolate directly from acetone. 15 In a computational study, Maŕquez et al. 21 examined the adsorption of acetone on rutile TiO 2 (110) with respect to the influence of adsorption site, coverage, and geometry. They observed a strong dependence of the binding energy on these subjects. The binding energy of acetone is affected strongly by the coverage because of significant intermolecular repulsion at high coverages. The fivefold-coordinated titanium atoms produce a surface electric field, which is able to interact with an acetone molecule via the dipole of its carbonyl group. This interaction makes the adsorption of acetone favorable and advocates for two major adsorption sites. Acetone molecules are supposed to adsorb either on top of the fivefoldcoordinated titanium atoms or in a bridging position between two of those titanium atoms. For each position, the C(CH 3 ) 2 complex can rotate around the C−O axis in a way that the acetone symmetry plane is perpendicular (⊥) to the row of bridging oxygen atoms, parallel (∥), or in between (rot) (see Figure 1).
In this study, we use ultrahigh-vacuum Fourier transform infrared spectroscopy (UHV-FTIRS) to investigate the adsorption and reactions of acetone on the surface of rutile TiO 2 nanoparticles. Such samples are considered more realistic model systems for catalysts than single-crystal surfaces. The IR results allow to identify species formed upon the interaction between acetone and TiO 2 . The interpretation of the experimental results is assisted by theoretical IR spectra calculated within the framework of density functional theory (DFT). Polycrystalline rutile TiO 2 nanoparticles expose predominantly the (110) facet, 22 which is the most stable surface of rutile. 16 Therefore, this surface is investigated in the computational part of this work. The computational results are first validated with respect to published experimental work on single-crystal rutile TiO 2 (110) surfaces, which are mentioned above. In a second step, the calculated results are compared to our experimental results on rutile TiO 2 nanoparticles to see if this allows for new insights. In the literature, the main focus of computational studies was put so far on ⊥and ∥-adsorption orientations of acetone on rutile TiO 2 (110); however, experimental results by Petrik et al. indicated a preference for tilted adsorption geometries for selected coverages. 1 For more details, the configuration space was sampled more exhaustively in this study by investigating numerous additional geometries computationally.
■ METHODS Experimental Methods. For the UHV-FTIR measurements, the polycrystalline rutile TiO 2 (r-TiO 2 ) powder samples (100 m 2 g −1 , Sachtleben) were first pressed onto a gold-plated stainless steel grid (0.5 × 0.5 cm 2 ) and then mounted on a sample holder to record FTIR data in transmission mode. The FTIR measurements on TiO 2 powder samples are done with an UHV apparatus, which combines a state-of-the-art vacuum IR spectrometer (VERTEX 80v, Bruker) with an UHV system. 23 The sample holders, on which the TiO 2 powder samples are mounted after they have been pressed, are specially designed for the FTIR transmission measurements under UHV conditions, allowing a rapid heating and cooling of the samples between 100 and 1000 K. The base pressure in the measurement chamber was 2 × 10 −10 mbar. To achieve a high sensitivity and stability of the measurements, the adsorption of atmospheric moisture was prevented by evacuating the optical path inside the IR spectrometer and the space between the spectrometer and the UHV chamber. By heating to 750 K, the grid and the attached powder particles were cleaned in the UHV chamber to remove all contamination, such as water and hydroxyl groups. After annealing, no significant reduction of the sample was observed. Prior to each exposure to acetone, a spectrum of the clean TiO 2 powder was recorded as a background reference. All UHV-FTIR spectra were collected with 1024 scans at a resolution of 2 cm −1 in the transmission mode. Exposure of the sample to acetone was carried out by back-filling the measurement chamber through a leak valve.
Computational Methods. For a more comprehensive understanding of the adsorption behavior of acetone on a rutile TiO 2 (110) surface, density functional theory (DFT) calculations were performed using the plane-wave code Vienna Ab Initio Simulation Package (VASP) 24−27 with the projector augmented wave (PAW) method. 28,29 Since van der Waals (vdW) interactions can be significant in the adsorption of organic molecules, 9 the exchange-correlation (XC) functional optB88-vdW 30−33 was used for all DFT calculations in this work. This functional belongs to the group of van der Waals density functionals (vdW-DF), based on an approach by Dion et al. 34 Within this approach, vdW interactions are accounted for without the use of external input parameters. Some approximations are, however, done such as that the vdW interactions are considered to be pairwise additive. Klimešet al. introduced several vdW-DFs, the "opt" functionals, 30,31 which have been successfully applied to various systems. 35−41 In particular, the optB88-vdW functional is considered to capture well the subtle contributions of weak interactions in the adsorption of organic molecules. 42−44 For comparisons and consistency checks, the main results were reproduced using various other XC functionals, namely, PBE, 45 PBEsol, 46 and PW91 47 (see the Supporting Information). The Journal of Physical Chemistry C Article All calculations were performed using a 5 × 5 × 1 Monkhorst−Pack set of k-points. 48 The plane-wave expansion was limited by a cutoff energy of 520 eV. During the relaxation process, the atom positions were allowed to adjust until the atomic forces were less than 5 meV Å −1 . For the relaxation process, the systems were preconverged using the conjugate gradient algorithm and then a quasi-Newton algorithm was employed, which is efficient close to a minimum. Finally, the structure was investigated statically to obtain more accurate total energies via the tetrahedron method with Blochl corrections. 49 The surface was modeled using a slab of five O−Ti−O layers in a 2 × 1 surface cell. Atoms in the two lowermost layers were kept fixed in their bulklike positions, whereas the remaining layers were free to relax, thus being able to respond to occurring forces due to surface effects or adsorption. A vacuum region of 23 Å was added above the slab to avoid interaction between the periodic images. 50 A dipole correction is applied to compensate for the slab asymmetry. 51,52 Binding energies E B of adsorbed acetone on rutile TiO 2 (110) were determined as the difference between total energies of the educts, nE adsorbate,gas and E substrate , and the total energy of the product, E subs+ads , divided by the number of adsorbed molecules per cell n adsorbate,gas substrate subs ads (1) To find the ground state of adsorbed acetone on rutile TiO 2 (110), it is not sufficient to investigate a randomly oriented molecule. Large energy barriers and large distances prevent atom movement to the ground-state position during relaxation due to the respective algorithm design in DFT codes. A starting position of the molecule close to the ground state is therefore crucial. To this end, an in-house MATLAB code was used to generate a solution space of distinct acetone molecule geometries on the surface slab. The code includes coarse graining and symmetry check algorithms to minimize the number of initial configurations. In addition to the total energy calculations, vibrational spectra were analyzed using density functional perturbation theory (DFPT). 53,54 In DFPT, the vibrational frequencies in the harmonic approximation are obtained via the dynamical matrix, which is calculated by slightly displacing selected atoms from their ground-state positions and recording the resulting electron density response. Here, the atoms of the adsorbed acetone molecule and the adsorption site Ti atom are allowed to be displaced. Furthermore, it is possible to compute the matrix of Born effective charges (BECs), which refers to the change of an atom's polarization when exposed to an external electric field. Using the formula by Gianozzi and Baroni 55,56 and the procedure proposed by Karhańek et al., 57 the BEC matrix leads to the oscillator strengths, or rather vibrational intensities. Combined with the vibrational frequencies from the dynamical matrix, the infrared (IR) spectra for acetone in the gas phase and for adsorbed species can be calculated. It has to be noted that following this DFT-based method, the calculated vibrational frequencies typically deviate from experimental results by a few percents. 58−60 The vibrational frequencies tend to be underestimated for generalized gradient approximation (GGA)(-based) functionals. The error occurs mainly because of the harmonic and density functional approximation, which, however, partially compensates for GGA(-based) functionals. 61,62 ■ RESULTS AND DISCUSSION Geometries. As mentioned in Introduction, acetone can adsorb on a rutile TiO 2 (110) surface in various geometries. Since the main focus in earlier computational studies 21 was put on high-symmetry geometries, in this study, the configuration space was sampled more exhaustively by generating around 50 η 1 -acetone input structures. These structures were set up with an in-house MATLAB code to cover a high number of distinct orientations. In the following, adsorbed molecules are classified according to the orientation of their molecular symmetry plane with respect to the rows of bridging oxygen atoms of a rutile TiO 2 (110) surface. Except for the geometries with perpendicular (⊥) or parallel (∥) molecular and bridging oxygen planes, all other structures are labeled as rot-geometries. In each 2 × 1 surface supercell, one molecule was placed, resulting in a total coverage of θ = 0.5 monolayer (ML). Deviating coverages are clearly indicated in the following.
In Figure 1, relaxed η 1 -acetone geometries, including their respective calculated binding energies E B , are shown. In addition to the ⊥and ∥-orientated η 1 -acetone molecules, also an energetically more favorable rot-orientation is presented. This rot-geometry exhibits the largest binding energy found in this study and will be referred to as rot max . The computed binding energies range from 0.85 to 1.22 eV when using the optB88-vdW XC functional. A preference for rot-orientations is in agreement with experimental indications in the literature. 1 For comparison and consistency, binding energies using other XC functionals were determined for the main geometries (see the Supporting Information). Even though the binding energies depend sensitively on the employed XC functional, the energetic tendencies and relations between the different geometries remain the same. Since the optB88-vdW functional is expected to adequately represent the energetic impact of the adsorbed molecule, 9 it is used for further computations. Enhanced and more directional interactions of the methyl groups with neighboring bridging oxygen atoms are found for the rot max geometry comparing the differences of electron charge densities for the ⊥ and rot max geometries (see Figure 2). This gives an indication why rot-geometries are favorable in terms of the binding energy. Such differences are obtained by subtracting the charge densities of the isolated molecule and surface from the charge density of the adsorption configuration.
So far, all investigated surface slabs had a coverage of θ = 0.5 ML. However, in a 2 × 1 surface slab, there are two fivefoldcoordinated titanium atoms and thus two possible acetone adsorption sites. To investigate the full coverage (θ = 1 ML) of the rutile TiO 2 (110) surface, two acetone molecules in ⊥-orientation were placed manually on the surface. Due to the steric hindering of neighboring periodic molecules, only ⊥-orientations similar to Figure 1 (left) can lead to high coverages. During the relaxation, however, the distance between the acetone molecules in every second row in the [001] direction and the surface is increased and a second layer of adsorbed molecules is formed as illustrated in Figure 3. This structure is referred to as bilayer in the following. The total binding energy of the bilayer amounts to about 1.3 eV per 2 × 1 surface cell. The relaxed adsorption geometry of acetone molecules close to the surface within the bilayer is almost identical to the ⊥ configuration at θ = 0.5 ML. Consequently, we assume that these acetone molecules are bound to the surface with a similar strength as the ⊥ configuration at 0.5 ML. Comparing the total binding energies, the molecules of the bilayer further away from the surface are associated with only a small binding energy of about 0.34 eV per molecule. Thus, within the bilayer, the second layer binds much weaker than the first layer.
In Table 1, the binding energies per molecule are presented for different acetone coverages on the rutile TiO 2 surface. Here, binding energies for coverages of 1 and 0.5 ML are determined using 2 × 1 supercells, while for a coverage of 0.125 ML, a 4 × 2 supercell was employed. Results show an increase in binding energy per molecule as the coverage decreases. For a coverage of 1 ML, only the ⊥-configuration of adsorbed acetone is possible for geometrical reasons and the average binding energy per molecule amounts to 0.65 eV. For coverages of 0.5 and 0.125 ML, the rot max -configuration remains the energetically most favorable adsorption structure with binding energies per molecule of 1.22 and 1.38 eV, respectively. The variation in the binding energies of the studied configurations is significantly smaller for 0.125 ML than for 0.5 ML. The coverage dependence of the adsorption energy is in qualitative agreement with temperature-programmed desorption experiments in the literature. 19 In that work also, low-temperature desorption peaks occurred for coverages above 0.5 ML, indicating more weakly bound acetone molecules. Although experiments suggest tilted adsorption geometries for coverages of 0.33 and 0.6 ML, for 0.17 ML, a ⊥-configuration is indicated. 1 The latter is not in agreement with our computational results, although we also observe a less pronounced preference for rot-configurations at low coverage. Since, however, the focus of the cited experimental work was not on the orientation, a more dedicated single-crystal study should be performed on this subject in future. Petrik et al. 1 assumed that intermolecular repulsion at increased coverages favors tilted configurations. The decreasing binding energy with increasing coverage (see Table 1) also points toward that explanation. For further clarification, we calculated total energies for acetone molecules in different adsorption geometries (see Figure 1) in the same simulation cell as the previous calculations, however, without the TiO 2 slab. Comparing these results for different geometries and to corresponding calculations including the TiO 2 slab shows an additional stabilization of the rot-geometry with respect to the ⊥-orientation because of interactions with the slab. Such contributions have already been indicated above by the changes in the charge densities (see Figure 2).
The formation of new species upon acetone adsorption and potential temperature-activated reactions can be monitored by UHV-FTIRS performed at different temperatures. To provide a theoretical reference for species determination, DFPT is used to compute the vibrational spectra of several species that are likely products of reactions of acetone on TiO 2 (110), including η 2 -acetate, η 2 -diolate, η 1 -enolate, and mesityl oxide. 19 Relaxed structures of these species are illustrated in Figure 4.
Energy−Geometry Relationship. With regard to the acetone geometries in Figure 1, the relationship between adsorption structure and respective binding energies is not obvious. Despite the fact that the difference of 0.1 eV between the ⊥-orientation (Figure 1, left) and ∥-orientation (Figure 1, middle) corresponds to the literature, 21 rot-orientations reach even higher binding energies. Since also other adsorption  The Journal of Physical Chemistry C Article angles seem to have an influence on the energetics, two adsorption angles, φ and ϑ, are defined and investigated with respect to their effect on the binding energies. For η 1 -enolate on rutile TiO 2 (110), a similar study is presented in the Supporting Information Due to possible hydrogen bonds, the positions of the two methyl groups within the acetone molecule have a large impact on the energetics of the adsorption. First, these positions are further specified using the adsorption angles φ and ϑ. In Figure  5, the locations of the carbon atoms are used to define a plane that is parallel to the symmetry plane of the molecule. Additionally, the surface normal of the (110)-plane serves as a reference. Considering possible interactions of the methyl groups with surface atoms, particularly the bridging oxygen atoms, the angle ϑ between the C 1 −C 2 −C 3 plane and surface normal is of high interest. To facilitate the comparison to the literature, ϑ = 0°corresponds to an upright position of the acetone molecule. The angle describing the ⊥-, rot-, and ∥-orientations of the acetone symmetry plane in relation to the row of bridging oxygen atoms is defined as φ. To this end, the vector between the methyl-group carbon atoms C 2 and C 3 as well as the vector between adjacent bridging oxygen atoms are projected into the (110)-plane. Investigating the influence of φ on the binding energy can also give information about possible methyl-group interaction with the surface atoms. Additionally, it is possible to define two angles between the oxygen atom of the carbonyl group, the adjacent carbon atom C 1 , and the respective carbon atoms C 2 and C 3 of the methyl groups. Considering these two angles, the tilt around the linking carbon atom C 1 in the C 1 −C 2 −C 3 plane can be described. However, investigating all η 1 -acetone adsorption structures for these two angles showed only slight deviations from the symmetric state. After relaxation of the input structures with tilting angles between 0 and 6°, the majority of acetone geometries converged with a tilting angle of around 2°, showing an angle preference of the carbon orbitals.
All energy−angle relations of adsorbed η 1 -acetone are presented in Figure 6. For an angle φ roughly between 20 and 65°(rot-orientation), the highest binding energies (>1.2 eV) were determined, while between 38 and 50°also lower energies of around 1.05 eV occur. Approaching the ⊥or ∥-orientation, the binding energies continue to decline, yielding the minima for the ⊥-orientation and ∥-orientation at 0.96 and 0.85 eV, respectively. However, for two cases with a ∥-like orientation (φ < 10°), the binding energies are also higher than 1.2 eV. For angles ϑ between 20 and 50°, or rather for a shorter distance of the methyl groups to the surface, the resulting binding energies are higher as well (slightly above 1.1  The Journal of Physical Chemistry C Article or 1.2 eV). For completely upright positions (ϑ = 0°), the same minima as for φ = 0 and 90°can be seen. By investigating both angles at the same time a lumped area of high binding energies at 30°< ϑ < 50°and 20°< φ < 60°can be seen, marking an area of energetically favorable and distinct η 1acetone geometries.
Because of the small energy differences between many adsorption geometries, it is expected that numerous configurations in the energetic vicinity of the most favorable one are realized at finite temperatures. Furthermore, continuous transitions between different geometries may occur.
UHV-FTIRS Results. Figure 7 shows the experimental IR spectra recorded for acetone adsorption on the pure, adsorbate-free rutile TiO 2 powder sample at 115 K. After exposure to acetone, a number of IR bands are observed at 2909, 1711, 1695, 1440, 1420, 1363, and 1229 cm −1 , which are related to the adsorption of acetone on the rutile TiO 2 surface. Polycrystalline rutile TiO 2 nanoparticles expose predominantly the (110) facet, 22 which is the most stable surface of rutile. 16 The obtained IR bands fit well to those reported for singlecrystal rutile TiO 2 (110) surfaces. 1 To get deeper insight into the adsorption of acetone on rutile TiO 2 (110), we have carried out temperature-dependent IR measurements. The sample was heated up to elevated temperatures after acetone adsorption at 115 K. The obtained IR spectra are displayed in Figure 8. After annealing the sample to 150 K, most IR bands decreased only slightly in intensity, revealing the chemisorption of acetone molecules on TiO 2 (110). On the basis of the literature, the dominating IR bands at 2909, 1711, 1420, 1363, and 1229 cm −1 are attributed to the ν(C−H), ν(CO), σ as (CH 3 ), σ s (CH 3 ), and ν(C−C) vibrations of acetone, 6,15 respectively. Although it is possible to get a multilayer of acetone on TiO 2 surfaces at 115 K, the IR spectra do not show characteristic bands for multilayer adsorption. This can be explained with the high surface area of the powder samples, which need a much higher amount of acetone for covering the whole surface. Annealing the sample up to 150 K leads to a partial removal of acetone, verified by a decrease of the asymmetry and symmetry vibrational bands of CH 3 , σ as (CH 3 ), and σ s (CH 3 ) at 1420 and 1363 cm −1 , while new vibrational bands appeared at 2974, 1695, 1498, 1434, and 1170 cm −1 and grew in intensity with increasing temperature. Simultaneously, the intensity of molecular acetone-related IR vibrations declined significantly at higher temperatures. These findings (see Figure 8) indicate thermal-induced reactions of acetone on rutile TiO 2 surfaces yielding new species. The 1434 cm −1 band is characteristic for the symmetry mode of O−C−O stretching vibrations, which is formed on the surface when acetone interacts with surface oxygen. The corresponding ν as (OCO) vibration was observed at 1498 cm −1 . At 250 K, the ν(CO) band splits into two peaks (zoom in Figure 8) centered at 1711 and 1695 cm −1 .
Computational Vibrational Analysis. The wavenumbers of the most relevant vibrational modes of acetone are presented in Table 2. Experimental as well as calculated values are given for adsorbed and gas-phase acetone molecules. The computational results for adsorbed acetone are given for the rot max configuration with the largest binding energy (see Figure  1). An isolated acetone molecule is also analyzed computationally to simulate the gas phase before adsorption. Additionally, experimental results for the gas phase and for acetone adsorbed on rutile TiO 2 (110) from the literature 1,63 are presented to show the accuracy and validity of the chosen method. The computed wavenumbers agree very well with the experimental values.  The adsorption of the acetone molecule is indicated by peak shifts to lower frequencies of the CO bonds. Generally, peak shifts to lower frequencies can also occur because of interactions with heavier atoms. Hence, the peak shift to 2897 cm −1 of the C−H stretching bond in η 1 -acetone might indicate hydrogen bonds, explaining the higher binding energy of 1.22 eV in the rot-geometry.
To check the consistency of the employed computational method, the vibrational analysis was performed using various XC functionals. Details can be found in the Supporting Information. Summarizing, the results for C−C and CO modes differ within a range of less than 2% for the tested functionals. For modes including hydrogen, the range shifts up to about 3%. A comparison with the experimental results (see Table 2) shows that the values deviate within about 2%. Although no functional can be considered genuinely superior to the others regarding this vibrational analysis, optB88-vdW yields the smallest average deviations from experimental results. For optB88-vdW, all bands are shifted to lower wavenumbers with respect to the experiment.
Considering the small energy differences between various adsorption geometries, it has to be expected that numerous configurations occur at finite temperatures. Consequently, various geometries contribute to a measured IR spectrum.  The computational and literature values for adsorbed acetone were obtained at the (110) surface for the calculated ones in the rot max configuration (see Figure 1). Figure 9. Calculated (blue) and experimental (brown) infrared spectra of η 1 -acetone adsorbed on rutile TiO 2 (110). The theoretical spectrum is a superposition of the spectra for all calculated configurations exhibiting binding energies within 30 meV to the most favorable rot max geometry (see Figures 1 and 6). The contributions of the different configurations are weighted using a Boltzmann factor according to their differences in binding energy and a temperature of 115 K.

Article
Since the vibrational features depend on the specific configuration, the resulting spectrum will be broadened. Figure  9 shows a comparison between a measured IR spectrum (see Figure 7, curve E) and a calculated spectrum including all investigated adsorption geometries yielding binding energies within 30 meV of the most favorable structure. The calculated spectrum of each structure is first normalized to its highest peak. Then, for a temperature of T = 115 K, following the experimental setup, a Boltzmann factor (exp(−ΔE/k B T)) with the Boltzmann constant k B is used to scale the computed IR peaks for each structure using the energy difference ΔE associated to the most favorable structure. The computed vibrations are in good agreement with the experimental results. In particular, positions and hierarchy of intensities of the ν(CO), σ s (C−H 3 ), and ν(C−C) bands correspond well to the UHV-FTIR measurements in the literature on singlecrystal rutile TiO 2 (110) surfaces 1 as well as to our experimental results on rutile TiO 2 nanoparticles. As expected, the calculated IR bands are slightly shifted toward lower wavenumbers (see Computational Methods). Because of the agreement to various experiments, we believe that we can use our DFT results to learn more about the adsorption on the nanoparticles as well, even though we are missing contributions from minority surface orientations and from features such as edges and kinks.
The vibrational properties of further compounds that may occur upon temperature-or UV-light-induced chemical reactions of the adsorbed acetone molecules 19 were calculated. The possibly relevant compounds are η 2 -acetate, η 2 -diolate, η 1enolate, and mesityl oxide. Along with the experimental UHV-FTIR spectra recorded at elevated temperatures (see Figure 8), the calculated vibrational spectra for all relevant species are presented in Figure 10.
The temperature-induced shifts in the experimental FTIR spectra clearly indicate modifications of the adsorbed acetone molecules. Even though some bands of the calculated IR spectra for η 2 -acetate, η 1 -enolate, and mesityl oxide seem to match areas of the recorded FTIR spectra, they do not account for the major temperature-induced changes of the experimental spectra. In more detail, acetate and enolate share some major IR peaks with acetone because of common vibrational modes.
Both species, however, do not exhibit IR bands that can explain the emerging IR features upon increasing temperature during the experiments. For mesityl oxide, no major agreement between the calculated IR spectra and any experimental one is observed. This allows to rule out the formation of significant amounts of mesityl oxide under specific experimental conditions. Moreover, the formation of large amounts of the three species is rather unlikely considering the experimental conditions. 1,15,19,20 The observed splitting of the ν(CO) band at a temperature of 250 K (1711 and 1695 cm −1 ) fits well to the calculated spectrum of diolate with multiple bands in the same region. Furthermore, bands at 1381 and 1128 cm −1 match peaks in the spectrum for 300 K (G). Since only the major surface orientation, no defects, and only a few adsorption geometries of the possibly relevant compounds were investigated, lacking bands at certain frequencies are expected. Additionally, interactions between acetone and the new species, which could affect the IR spectra, are not investigated. Similar to results from the literature, 1 the formation of a significant amount of diolate seems to be plausible considering the observed IR spectra at the investigated temperatures. The work by Petrik et al. 1 was done on reduced and oxidized surfaces. Diolate formation was shown there for oxidized samples. However, in their IR data, diolate bands seem to appear for high acetone coverages on reduced samples, too. Our current results indicate that diolate also forms at high coverages for neither significantly reduced nor oxidized surfaces. Furthermore, defects such as steps, edges, and kinks, which are more common for nanoparticles, could affect the results as well as adsorption from the residual gas. Thus, subsequent detailed studies should be performed in the future to shed further light on these issues.

■ CONCLUSIONS
In this study, the adsorption behavior of acetone on rutile TiO 2 was investigated with respect to its energetic, vibrational, and chemical properties. A joint approach combining experimental and theoretical techniques was chosen allowing for a detailed analysis of the investigated system. Using UHV-FTIR measurements at 115 K for different acetone dosages (4.5− 900 L) and annealing at various temperatures (120−300 K), insights into the acetone adsorption behavior could be obtained, where the formation of new species with increasing temperatures was indicated by modified IR spectra. Additionally, an extensive DFT study of numerous acetone adsorption geometries and coverages improved the understanding of acetone adsorption on the rutile TiO 2 (110) surface. Complementing previous work in the literature, here the configuration space was sampled more exhaustively. Investigating the binding angles of acetone molecules with respect to the corresponding binding energy indicates a large number of adsorption geometries energetically close to the most favorable calculated configuration. Those geometries exhibit rotated and tilted molecular symmetry planes with respect to the surface features, while highly symmetrically adsorbed acetone molecules correspond to significantly lower binding energies. Considering the small energy differences between various adsorption geometries, continuous transitions between structures may occur. Using DFPT, computed IR spectra for the investigated acetone adsorption geometries as well as for potential acetone reaction products were used to complement the experimental FTIRS results. At low temperatures, the theoretical and experimental IR spectra are in good agreement Figure 10. Comparison of calculated vibrational spectra (blue lines) of four species adsorbed on rutile TiO 2 (110) (see Figure 4) and the experimentally recorded FTIR spectra at 115 K (B) and 300 K (G), as presented in Figure 8.
The Journal of Physical Chemistry C Article with each other and with results from the literature on singlecrystal rutile TiO 2 (110) surfaces. IR bands computed with DFPT are generally shifted toward lower wavenumbers. For higher temperatures, a comparison of the raised IR bands in the UHV-FTIR measurements and theoretical IR computations indicates the formation of significant amounts of η 2diolate.

* S Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcc.8b04222.
Comparison of the energetic and vibrational properties of acetone adsorbed on rutile TiO 2 (110) for various exchange-correlation functionals; computational study on the adsorption of enolate on rutile TiO 2 (110) (PDF)


Modelling the Diclofenac and Indomethacin Recovery by Molecularly Imprinted Polymer

Pharmaceutical compounds in surface waters are an emerging environmental concern due to their biological activity, which consequently present a new challenge for drinking water and wastewater treatment systems. In this work, diclofenac (DCF) and indomethacin (IDM) have been used as target molecules or templates in order to be removed by means of molecularly imprinted polymers (MIP). MIP-DCF and MIPIDM were synthesised and tested using two mathematical models, which are Lagergren and Thomas models. In addition, functional groups of the active functional sites and the target molecules have also been studied by using Fourier transform infrared spectroscopy. Both MIPs followed the Lagergren pseudo first order kinetic model. Comparing the original MIP-DCF and MIP-DCF after loading with DCF, differences in their functional groups were found. In conclusion, MIP-DCF and MIP-IDM work as good sorbents to recover these emerging pharmaceuticals persistent pollutants from water.

INTRODUCTION
A few decades ago, emerging persistent pharmaceutical pollutants (EPPPs) have been introduced as one type of recalcitrant pollutant sources in water. 1 Recent studies have demonstrated that, despite the relatively low concentrations of pharmaceuticals in the environment (typically in sub-parts-per-billion levels), pharmaceuticals are of great concern due to their potential long-term adverse effects on humans and wildlife. 2 Nowadays, there are many methods used for the recovery of pharmaceuticals, such as photocatalysis and sorbents, but they present some drawbacks, e.g., after photocatalysis the by-product generated may be more harmful than the parent compounds. 3 There are many kinds of sorbents that have been studied, but not all the sorbents have the potential to recover the target molecules. In this work, molecularly imprinted polymers (MIPs) as a separation method is proposed and characterised. The polymeric matrices obtained using the imprinting technology are robust molecular recognition elements able to mimic natural recognition entities. 4 MIPs are also good sorbents because of their porosity and their robustness properties. In this study, the removal of diclofenac (DCF) and indomethacin (IDM) have been carried out using MIPs, synthetised via bulk polymerisation with allylthiourea (AT) as the functional monomer and using DCF or IDM as template (MIP-DCF or MIP-IDM, respectively). DCF and IDM were detected using spectrophotometry.
To study the properties of the sorbents, MIPs have been tested in batch mode and continuous flow mode. In the batch mode, Lagergren model was used to describe the adsorption properties. However, in the continuous flow mode, Thomas model was used for the breakthrough study. On the other side, functional groups can be used as a fingerprint for certain MIPs to show if the active functional sites work. However, there are a few articles reporting on this parameter which give important information about MIP development. ATR-FTIR has been used to observe the different types of MIPs and to study their functional sites.

Materials
Indomethacin (IDM) was purchased from Sigma-Aldrich, Spain, with 98%-99% purity (TLC grade). Diclofenac-Na was from Cayman Chemical, United States with purity of more than 99%. Ethanol with 96% purity was acquired from Scharlau Chemical, Spain. Acetic acid was from J. T. Baker, United States with 99.9% of purity, respectively, and HPLC grade. Acetonitrile (99% of purity HPLC grade) from VWR. Grace Alltech "Extract Clean" empty reservoirs with silica frits, 1.5 ml from Fisher Scientific, United States.

Synthesis of MIP-DCF and MIP-IDM
Synthesis of MIP-IDM and MIP-DCF were performed following the procedure of Kugimiya et al. with some modifications. 5

Recovery Study
An amount of 10 mg MIP-IDM was weighed and placed into a 1.5 ml cartridge. Then, 1 ml of ethanol:water (75% v/v) was passed through it twice to activate the MIP-IDM. Next, 1 ml of water was added for 5 times to rinse out the ethanol residue. Afterwards, acetonitrile:water (5% v/v) was added twice for conditioning the MIP-IDM. Later, 1 ml of 15 µg ml −1 of IDM was added twice. The eluted solution was collected and analysed using a UV-vis spectrophotometer at 260 nm. After that, 1 ml of ethanol:water (75% v/v) was loaded twice into the cartridge and the collected solution was also analysed by UV spectrophotometry. The procedure was done in triplicate. The percentage of recovery was calculated using Equation 1, where C i and C f represent the analyte concentration for initial and final respectively: Moreover, the recovery procedure mentioned above was done regenerating the same MIP and reusing it until 10 times (consecutive extractions) in order to evaluate the reuse of the MIP. The same procedure was performed by using MIP-DCF as a sorbent and measuring the eluted solution at 280 nm for DCF analysis.

Kinetic Study
An amount of 10 mg of MIP-DCF was weighed and placed in 5 ml tubes. Then, 2 ml of 5 mg l −1 DCF solution were poured into the tubes and covered using aluminium foil. 6 Solutions were agitated for the required time up to 120 min and then filtered using a syringe filter (0.22 μm). The procedure was performed in triplicate and repeated for MIP-IDM using IDM solution. The calculated capacity (mg of compound/g of MIP) versus time was plotted following the Lagergren model.

Breakthrough Experiment -Continuous Flow Mode
Due to the immiscibility properties of DCF in water, total sorption capacity has been carried out in acetonitrile:water (5% v/v). An amount of 10 mg of MIP-DCF was accurately weighed and placed in a 1.5 ml cartridge. Next, 15 µg ml −1 of DCF was prepared in acetonitrile:water (5% v/v) and the solution was loaded into the cartridge using a peristaltic pump at 1.67 ml min −1 . The eluted solution was collected in fractions of 5 ml until 50 ml, then, in fractions of 50 ml until the absorbance measurements of the eluted solution reached a plateau. All the collected fractions were analysed by UV spectrophotometry. The procedure mentioned above was done in duplicate. The same methodology was repeated in the absence of MIP, in order to measure any other contribution to the elimination of the target compounds, i.e., sorption in cartridge or frits. Similar procedure was performed for IDM removal by MIP-IDM.

Attenuated Total Reflectance Fourier Transform Infrared Analysis
ATR-FTIR has been used in this study to determine the functional groups in the original MIP-DCF and after loading with 2 ml of DCF solution (5 µg ml −1 ) via molecularly imprinted solid phase extraction (MISPE).

Recovery Study and Lagergren Model
The ability to sorb-desorb for the synthetised MIPs were not significantly decreased after 10 cycles reaching almost 100% of recovery ( Figure 1). The average percentage of recovery for the 10 cycles of IDM and DCF using MIP-IDM and MIP-DCF were 98 ± 3% and 97 ± 2% respectively.
From the kinetics, 100% removal was obtained in 3 min in batch mode. It is well accepted that two-parameter adsorption kinetic equations (Lagergren firstorder and second-order) are useful tools to describe the adsorption properties of a sorbent. 7 The Lagergren pseudo first-order equation can be linearly expressed as Equation 2, where Q e and Q t are the adsorption capacities (mg of compound/g of sobent) of MIP at equilibrium and at time t, respectively; k 1 is the first order rate constant. Figure 2 shows that both MIPs fitted to the Lagergren pseudo first order model. For further experimental procedure, MIP-DCF was used as a sorbent.

Breakthrough Study
Breakthrough in column can be identified as the amount of influent solution passing through the bed before a maximum effluent concentration is reached.  the time of the analyte solution loading as seen in Figure 3. As seen in the kinetic study, within 3 min the sorption uptake was higher than 80%, thus resulting in relatively fast attainment of breakthrough. Breakthrough and exhaustion are defined as normal when the ratios of effluent-to-influent concentration are between 10% and 60%, respectively. 8 In this work, the breakthrough point was reached at 6 min and the exhaustion point at 270 min for MIP-DCF and 24 min and 210 min for MIP-IDM respectively. From Figure 3, it is seen that the ratio is about 60% and 80% for MIP-DCF and MIP-IDM respectively. The methodology of adsorption in fixed bed cartridge by the breakthrough curves method proved to be a good option to study the separation of the pharmaceutical compounds in water.

FTIR Analysis
The MIPs, original and after DCF loading, have been differentiated via ATR-FTIR and the spectra obtained could be used as a fingerprint in order to distinguish them as seen in Figures 4(a) and 4(b). N-H stretch functional group at 3000 nm −1 and C-N stretch bond at 1180 nm −1 found in original MIP (Figure 4(a)); OH broad peak at 3300 nm −1 found in MIP loaded with DCF (Figure 4(b)) due to -OH functional group from DCF bonded to MIP-DCF. Probably there is also N-H functional group that might contribute in this peak; C=O bond at 1750 nm −1 found in all MIPs; and C=C bond at 1650 nm −1 found in MIP loaded with DCF.

CONCLUSION
In conclusion, MIP-DCF and MIP-IDM have shown to be highly effective for the removal of the pharmaceutical compounds DCF and IDM, respectively. MIP is a promising analytical method for the removal of pharmaceuticals from water.