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On‐line Trace Enrichment of Three Dipine Species in River Water Using a Modified Epoxy Acrylate Resin‐based Monolith Dan Wei, Haiyan Liu*, Xiaomei Bai, Yamin Ma College of Pharmacy, Hebei University, Hebei Province Key Laboratory of Pharmaceutical Quality Control, Baoding 071002, China; Key Laboratory of Medicinal Chemistry and Molecular Diagnosis, Ministry of Education, Baoding, 071002 weidan0312@126.com; lhy1610@126.com ; 414753471@qq.com; 353240684@qq.com Abstract The modified epoxy acrylate resin‐based monolith was prepared by in situ free‐radical polymerization. The morphology and pressure drop of the monolith has been characterized. The results showed that the monolith had high permeability. Then, felodipine, lercanidipine and nimodiping were enriched by using the prepared monolith as solid phase extraction (SPE) sorbent and tested quantitatively when connected with RP‐C18 column. The linear calibration curves were obtained over a range of 2‐500 ng/mL (r2≥0.998). Precision for intra‐day and inter‐day assay showed acceptable results for quantitative assay with relative standard deviation (RSD) less than 12%. The accuracy and recovery were found to be within the range of 89%‐101%and 89%‐102%. The proposed method was successfully applied to simultaneously screen felodipine, lercanidipine and nimodiping in river water. Keywords On‐line solid phase extraction; Dipine species; Modified epoxy acrylate resin‐based monolith; River water
I. Introduction In recent years, with the improvement of personal life quality, a lot of Ca2+ channel blockers (CCBs) were developed and widely used. CCBs were applied in the treatment of cardiovascular diseases containing coronary heart disease, hypertension and cadiac arrhythmias. But in the past 10 years, CCBs were questioned on the security of long‐term treatment of coronary heart disease and hypertension. It was possible that CCBs could increase the mortality of cardiovascular, the occurrence of cancer, the risk of gastrointestinal bleeding and induce the tendence of suicide[1‐3]. CCBs were divided into dihydropyridine and non‐dihydroridine, according to the chemical structure and the pharmacological effects. Felodipine, lercanidipine and nimodiping (Fig.1), which are dihydropyridine CCBs, are widely used in the treatment hypertension and other cardiovascular. Aquatic ecosystems containing above drugs are harmful to human health. A number of analytical methods by using common sample pretreatment method, combined with HPLC[4‐7], GC[8] ,LC‐MS[9‐11]and other chromatographic analysis had been described for the quantitative determination of dipine species. As we all know sample clean‐up and preconcentration steps are necessary to remove matrix components and enhance sensitivity before chromatographic analysis In recent years, solid phase extraction (SPE) was widely used in the detection of biological and environmental samples[12‐17]. As a popurlar sample pretreatment material, the monolith could meet the requirements of SPE packing in the highly automated condition[18‐20] because of its unique characteristic, containing highly permeability for highly mechanical strength, low‐cost, highly selectivity and easily preparation. In this paper, a novel modified monolith was prepared by co‐condensation of ethylene glycol dimethacrylate (EDMA) and modified epoxy acrylate resin. The monolith was used for the pre‐concentration and determination of dipine species in aquatic system combined with C18 column.
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FIG.1 STRUCTURAL FORMULAR OF NIMODIPING(1), FELODIPINE(2) AND LERCANIDIPINE(3)
II. Experimental 1. Materials 2,2’‐azobisisobutyronitrile (AIBN) was obtained from Shanghai Chemical Plant (Shanghai, China) and purified before use. Methanol was purchased from Tianjin Kemiou Com (Tianjin, China). Polyethylene glycol 200 (PEG200) was provided by Tianjin Guangfu Fine Chemical Research Institute (Tianjin, China). Modified epoxy acrylate resin was produced by Zhongshan Ketian Electronic Materials Co., Ltd (Zhongshan, China). Ethylene glycol dimethacrylate (EDMA) was purchased from Acros (New Jersey, USA). Lercanidipine was obtained from Chongqing Shenghuaxi Pharmaceutical Group Co., Ltd (Chongqing, China). Nimodipine was prepared from Yabao Pharmaceutical Group Co., Ltd (Beijing, China). Felodipine was provided by Hebei Medical University. All reagents were of analytical reagent (AR) grade. Ultrapure water was prepared from a Millipore‐Q water‐purification system (Taiwan, China) and solutions were filtered through a 0.45um membrane before use. In addition, the morphological properties of these monoliths were photographed by S‐4300 scanning electron microscope (SEM). The S‐4300 SEM instrument was purchased from Hitachi (Hitachi High Technologies Tokyo, Japan). In addition, the river water was from moat (Baoding, China). 2. Synthesis and Characterization of Monoliths 0.4g modified epoxy acrylate resin, 0.4 mL EDMA and 0.005g AIBN were dissolved in the mixture of 0.4 mL PEG200 and 1.5 mL methanol. The mixture was ultrasonicated for 15 min to get a homogeneous solution. Then, pouring the reaction solution into a 50 mm × 4.6 mm i.d. stainless‐steel column sealed at one end and then sealed at the other end. The stainless‐steel column was submerged into water bath at 60℃ for 24h. After the reaction had inished, the monolith was connected to an HPLC pump and extensively washed with methanol to remove the unreacted reagents and porogenic solvents. 3. Preparation of Calibration Standards and Samples Felodipine, lercanidipine and nimodiping were dissolved in methanol to get a concentration of 1.0 mg/mL, respectively. The mixed stock solution of felodipine, lercanidipine and nimodipine was prepared at a concentration of 0.5 mg/mL in methanol. Then the solution was diluted with methanol to yield intermediate solutions of 5.0μg/mL, 2.0μg/mL, 1.0μg/mL, 0.5μg/mL, 0.25μg/mL, 0.1μg/mL, 0.05μg/mL, and 0.02μg/mL. These intermediate
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solutions were used to prepare standards at concentration of 500ng/mL, 200ng/mL, 100ng/mL, 50ng/mL, 25ng/mL, 10ng/mL, 5ng/mL, and 2ng/mL by diluting with blank river water for river water samples, respectively. River water filtered through a 0.45 um membrane. All the solutions mentioned above were stored at 4 ℃ until being used.uQuality controlled samples at three different concentration levels of 5ng/mL, 50ng/mL, 200ng/mL were prepared for evaluation of precision, accuracy and recovery in analysis of river water samples. 4. HPLC Analysis These analytes waperformed on a Beijing Heng Tong Innovation HPLC system equipped with P‐3000 pump and a UV‐3000 detector. The observed data were processed by CXTH‐3000 chromatography software. The synthetic monolithic column was used as a pre‐column and a C18 Dikma column (4.6 mm×150 mm I.D.; 5um, Dikma, NY, USA) as the analytical column. The mobile phase for enrichment was triple distilled water; the mobile phase for separation and analysis was methanol‐water, the proportion for nimodiping and felodipine was 85:15 (v/v) and 100% methanol for lercanidipine. The detection wavelength was set at 230 nm. The flow rate was set at 1.0 mL/min. The system was operated at ambient temperature. 5. Investigation of the Pretreatment Ability of the Monolith Ability of drug enrichment on the monolith was investigated by injecting 2.0mL of 1.0μg/mL felodipine, lercanidipine and nimodiping solution into the monolithic column at 230 nm. 6. SPE The monolith, which was used as SPE column for sample enrichment, was equilibrated with triple distilled water at a flow rate of 1.0 mL/min for 5 min. Then, 50 μL of spiked river water standards were directly injected into the SPE column in the “load” position of six‐port injector valve. Then, the SPE column was washed with triple distilled water by the loading pump at a flow rate of 1.0 mL/min to remove impurities in river water for 5 min, meanwhile analytes were enriched on SPE column. As a result, the retained analytes were eluted from the SPE column onto the analytical C18 column with gradient elution of mixture by methanol‐water at a flow rate of 1 mL/min for 16 min. The SPE column was washed with methanol and triple distilled water for the next injection. III. Results 1. Characteristic Features of the Monolith To evaluate the characteristic features of the synthetic monolithic column, the column was flushed with methanol for 1h at a flow rate of 1mL/min. Then, the prepared monolithic columns were cut into a small fragment that were dried in vacuo at 60℃ for 24h. They were characterized by SEM. Fig.2 exhibited the porous skeleton structure. The macropores offered a larger number of channels, which allowed the mobile phase through and led to lower column backpressure. 2. Investigation of the Pretreatment Ability of the Monolith Fig.3 showed that nimodiping, felodipine and lercanidipine could not be eluted when pure water (a) was used as the mobile phase. However, when methanol was used as mobile phase, a, nimodiping, b, felodipine and c, lercanidipine were eluted quickly from the monolithic column. Therefore, the monolithic column could be used as SPE column to completely retain the analytes. 3. SPE‐HPLC The river water samples flowed through the SPE column and then analyzed on the RP‐C18 column. When a gradient elution from 85% to 100% methanol was used at a flow rate of 1 mL/min, nimodiping, felodipine and lercanidipine could be baseline separated on the RP‐C18 column. The total analytical run time was 16 min. Fig. 4 showed the chromatograms of the spiked river water samples (a) containing nimodiping, felodipine and lercanidipine and blank samples (b) using on‐line SPE‐HPLC system. It could be seen that no interfering peaks from endogenous matrix components were observed near the retention time of three dipine drugs.
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FIG.2 SEM IMAGE OF MODIFIED EPOXY ACRYLATE RESIN‐BASED MONOLITH
FIG.3 CHROMATOGRAMS OF THE SAMPLE PREATMENT ABILITY ON THE MONOLITHIC COLUMN: 50MM×4.6 MM I.D.; FLOW RATE: 1.0ML/MIN; 2.0ML OF 1μg/ML a: NIMODIPING, b: FELODIPINE, c: LERCANIDIPINE; MOBILE PHASE: d, DEIONZED WATER, a, b, c, METHANOL; DETECTION WAVELENGTH: 230NM
IV. Discussions 1. Method Validation Selectivity, linearity, accuracy, precision (intra‐and inter‐day), recovery and reproducibility were assessed for the on‐line SPE‐HPLC method. 2. Selectivity The selectivity of the method was evaluated by comparing the chromatograms of the spiked samples containing nimodiping felodipine and lercanidipine with these river water samples. As shown in Fig.4, they were free from significant interfering endogenous substances at the retention times for the selected drugs. These results demonstrated that the developed method was selective and specific. 3. Linearity The calibration curve was constructed from standard solutions at different concentrations (500ng/mL, 200ng/mL, 100ng/mL, 50ng/mL, 25ng/mL, 10ng/mL, 5ng/mL, 2ng/mL) of felodipine, lercanidipine and nimodiping in river water. Each calibration sample of different concentrations was injected at least three times. As could be seen from Table 1, linear relationships were obtained in the selected rang and the correlation coefficients of all the equations were above 0.998. The limit of detection (LOD) and limit of quantification (LOQ) were calculated with
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signal‐to‐noise ratio equal to 3 and 10, respectively. 4. Precision and Accuracy The precision and accuracy of the method were determined by using quality control (QC) samples at low, medium and high levels. The precision included an intra‐day precision and inter‐day precision was expressed as relative standard deviation (RSD) of analytes concentration. The intra‐day precision was selected 5 times in a day that was measured standard‐spiked samples (5, 50 and 200 ng/mL) within the same day; while inter‐day precision was determined by repetitive analysis of standard‐spiked samples over five consecutive days. The accuracy of this method was obtained by the measured concentrations of felodipine, lercanidipine, and nimodiping in river water according to the on‐line SPE to those targeted concentrations. Table 2 showed excellent intra‐and inter‐day precision with RSD and accuracy values less than 11%. The results demonstrated that the reproducibility of method was excellent. 5. Recovery and Reproducibility To investigate the reliability of this method, the method recovery was measured by comparing the theoretical values to the initial concentration of analytes in spiked river water samples. The measured values were obtained by directly injection of nimodiping felodipine, and lercanidipine on a C18 column. The theoretical values were calculated according to the linear equation. The absolute recovery was measured by comparing the peak areas measured after SPE‐LC analysis of spiked river water samples to the measured values. The results were shown in Table 3. The recoveries were satisfactory and the method was acceptable for the analysis of river water sample. Four monoliths were prepared with the same synthesis process as described in Section 2.2 and used for extraction of felodipine, lercanidipine and nimodiping from river water samples. The RSD of the retention time and the peak areas were less than 3.7% and 5.0% , respectively. These data showed that the prepared monoliths could provide good reproducibility and revealed that the prepared monolith was feasible for using as an on‐line SPE sorbent material.
FIG.4 THE CONTRASE CHROMATOGRAMS BETWEEN THE MIXTURE OF SAMPLE IN RIVER WATER AND THE BLANK SAMPLE IN RIVER WATER. a, THE MIXTURE OF NIMODIPINE, FELODIPINE AND LERCANIDIPINE IN THE RIVER WATER SAMPLE AT A CONCENTRATION OF 1.0μg/ML AND b, BLANK RIVER WATER SAMPLE. GRADIENT ELUTION: 0‐8MIN, METHANOL/WATER (85/15, V/V); 8‐16 MIN, 100% METHANOL. FLOW RATE: 1.0 ML/MIN; COLUMN: RP‐C18DIKMA, 150MM×4.6 MM I.D UV DETECTION: 230 NM TABLE 1 CALIBRATION CURVE, LOD AND LOQ OF NIMODIPINE FELODIPINE AND LERCANIDIPINE FROM RIVER WATER SA Analytes
Calibration equations
Correlatin coefficient
LOQ (ng/mL)
LOD (ng/mL)
Nimodiping
Y=2.82×104x－1.63×102
R2=0.9981
1.5
0.5
Felodipine
Y=4.15×10 x－5.37×10
R =0.9999
2.0
0.7
Lercanidipine
Y=1.38×10 x－6.54×10
R =0.9983
1.8
0.5
4 4
2 2
2 2
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TABLE 2 INTRA‐DAY AND INTER‐DAY PRECISIONS AND ACCURACIES OF NIMODIPINE, FELODIPINE AND LERCANDIPINE FROM RIVER WATER SAMPLES AT THREE DIFFERENT CONCENTRATION Analytes (samples)
Concentration(ng/mL)
Precision RSD (%) Intra‐day
Inter‐day
Accuracy (%)
Nimodiping
5
6.74
11.67
100.1
50
4.18
5.62
89.5
200
6.61
3,04
92.3
Felodipine
5
11.11
10.81
98.7
50
7.36
11.36
99.7
200
5.76
2.64
100.4
Lercanidipine
5
8.87
10.22
98.0
50
7.47
3.26
94.5
200
5.38
4.80
97.7
TABLE 3 RECOVERY OF NIMODIPINE, FELODIPINE AND LERCANIDIPINE FROM RIVER WATER SAMPLES Analytes (samples)
Concentration (ng/mL)
Absolute recovery (%)
Method recovery (%)
Nimodiping
5
101.5±3.6
93.6±5.3
50
89.3±3.4
83.7±3.8
200
92.3±4.2
89.6±6.7
Felodipine
5
97.1±5.3
92.1±7.5
50
99.1±3.5
91.3±5.9
200
101.8±4.6
95.7±3.1
Lercanidipine
5
100.1±5.2
98.4±2.9
50
94.5±7.3
89.9±4.5
200
97.0±6.8
90.4±7.1
V. Conclusions The modified epoxy acrylate resin‐based monolith was used as SPE sorbent to simultaneously monitor three dipine drugs in river water. In this process, the sample pretreatment step was embedded into the HPLC chromatographic system and manual intervention was minimized. The preparation of the monolith was simple, convenient and repetitive. The results showed that this method had good linearity, precision, accuracy, recovery and met the analysis requirements. The study provided a re‐confirmation in the application of polymer monolith in the sample pretreatment of complex matrices. ACKNOWLEDGEMENTS
This work was supported by the Nature Science Foundation of Hebei University (no. B2013201082), the funds of Education Department of Hebei province (no. Z2013112), the Natural Science Foundation of Hebei University (No. 2013‐247), the Key Project Foundation of Hebei Province Higher Education (no. ZD2010234), the Key Basic Research Foundation of Hebei Province (no. 11966411D), the National Natural Science Foundation of China (no. 21175031), the Pharmaceutical Joint Research Foundation of the Natural Science Foundation of Hebei Province and China Shiyao Pharmaceutical Group CO.,Ltd. (no. B2011201174) and the issue of second batch of “twelfth Five‐year Plan” of Key Special Project for “significant Drug Discovery” of Ministry of Science and Technology of China (no. 2012ZX09103‐101‐057).
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