| Research Article |
Open Access |
|
| Preparation of a Novel Emulsion-Templated MIP Monolith
and its Application for on Line Assay of Nifedipine in Human
Plasma |
| Gengliang Yang*, Yankun Liu, Haiyan Liu, Chunliu Yang, Ligai Bai, Mingquan Liu and Jia Cheng |
| Key Laboratory of Pharmaceutical Quality Control of Hebei Province, Pharmaceutical Sciences college of Hebei University, China |
| *Corresponding author: |
Dr. Gengliang Yang
Pharmaceutical Sciences college of
Hebei University
Baoding, Hebei, 071002, China
Tel: 0086 312 5971108
Fax: 0086 312 5971107
E-mail: ygl@hbu.edu.cn |
|
| |
| Received September 16, 2010; Accepted October 28, 2010; Published October 30, 2010 |
| |
| Citation:Yang G, Liu Y, Liu H, Yang C, Bai L, et al. (2010) Preparation of a
Novel Emulsion-Templated MIP Monolith and its Application for on Line Assay
of Nifedipine in Human Plasma. J Chromatograph Separat Techniq 1:103. doi:10.4172/2157-7064.1000103 |
| |
| Copyright: © 2010 Yang G, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and
source are credited. |
| |
| Abstract |
| |
| A novel nifedipine molecularly imprinted polymer (MIP) monolith was prepared by radical polymerization of the water
in oil (W/O) emulsions, using vinyl ester resin and methacrylic acid (MAA) as the monomers and nonionic surfactants
such as Polyethylene glycol 400 (PEG 400) and Pluronic F68 (PF 68) as the emulsifying agents. The properties of the
material were investigated. The MIP monolithic column was installed in the high performance liquid chromatography
(HPLC) system and used as the selective sorbent for on line solid-phase extraction of nifedipine in human plasma due
to the good selectivity to nifedipine. For this method, the calibration curve was linear in the concentration range of 5-150
ng/mL (r2=0.998) for nifedipine in human plasma and the limit of detection (LOD) was 2 ng/mL. Accuracy, precisions and
recovery was acceptable for screening nifedipine in plasma samples. The results indicated that the novel material could
be used as pre-column for on line clear-up and screening of nifedipine in plasma samples, which provided a simple and
rapid assay of the drugs in plasma. |
| |
| Keywords |
| |
| Emulsion; MIPs; Monolithic column; Solid-phase
extraction; Nifedipine |
| |
| Abbreviations |
| |
| W/O: Water in oil; MAA: Methacrylic Acid; PEG 400:
Poly Ethylene Glycol 400; PF 68: Pluronic F68; MIPs: Molecularly Imprinted Polymers; SEM: Scanning Electron Microscopy; HPLC: High Performance Liquid Chromatography; LOD: Limit of detection; GMA: Glycidyl Methacrylate; EGDMA: Ethylene Glycol Dimethacrylate; DEA: diethylamine; MISPE: Molecularly Imprinted Solid-Phase Extraction;
BADE: Bisphenol A Diglycidyl Ether |
| |
| Introduction |
| |
| Nowadays, polymeric monoliths hold an impressively strong
position due to excellent properties in comparison with conventional
chromatographic supports and extensive applications such as
biological tissues caffolds [1], catalysis supports [2], ion-exchange
resin [3] and separation media [4]. In recent years, emulsion
templating polymerization technology has been introduced in the
preparation of monolith due to well defined open porous material
that has good mechanical strength and possess favorable stability.
Emulsions are colloidal systems made of liquid droplets dispersed
in another liquid phase, which are produced by shearing these two
immiscible liquids to reach a metastable state through fragmentation
of one phase into the other with various surfactants [5]. Moreover,
the preparation of water-in-oil emulsions containing polar monomers
requires a careful selection of the surfactant and the formulation of
the continuous phase. Krajnc P et al. [6] synthesized high internal
phase emulsion templated monolith using glycidyl methacrylate
(GMA), ethylene glycol dimethacrylate (EGDMA) and surfactant
PEL121. Then the column was modified with diethylamine (DEA) to
separate four standard proteins successfully. |
| |
| The molecular imprinting technique is an effective strategy for
preparing stationary phases with special molecular recognition
properties. Wulff G et al. [7] first synthesized imprinted polymers
by means of free radical copolymerization. After that, molecular
imprinting technique has undergone rapid development in 1990s.
Because of the advantages over biopolymers, such as low cost, good
physical and chemical stability and tailor-made selectivity for a target
molecule, the MIPs materials have been widely applied in solid-phase extraction [8], biosensor [9], chromatographic separation and
analysis [10-12]. |
| |
| Nifedipine (Figure 1), a dihydropyridine calcium channel
antagonist, is widely used in the treatment of hypertension and other
cardiovascular disorders [13]. With the development of modern
technology, new preparation formulation of nifedipine, such as solid
dispersion [14], sustained-release or controlled-release formulation
[15], gradually hold leading position in clinical medication and
expand the clinical application of nifedipine. Consequently, efficient
and fast quantification of nifedipine plays an important part in
analysis of the drug in blood. Numerous methods have been reported
for the quantitative determination of nifedipine in plasma, including
gas chromatography combined with different detectors, HPLC
coupled with varied detectors and recently combined with solid phase extraction [16-18]. Given that nifedipine is a highly unstable
compound, which is rapidly photo-degraded to its nitroso analogue
when exposed to daylight [19] and its plasma protein binding rate
reached 98%, on line solid-phase extraction is suitable for the pretreatment
of plasma sample instead of liquid–liquid extraction and
traditional off-line SPE. But the common material for on line SPE
is a commercially available pre-column, which is not cost-effective
and selective enough to detect nifedipine. Molecularly imprinted
solid-phase extraction (MISPE) offers a higher degree of selectivity
for clean-up and enrichment steps. Nevertheless, Lanza et al. [20]
synthesized nifedipine MIPs, but failed to adopt it in the assay of
nifedipine in plasma. |
| |
|
Figure 1: The structure of nifedipine. |
|
| |
| In this work, an alternative method has been developed
for preparing molecularly imprinted monolithic materials by
polymerization of the W/O emulsions. In the polymerization, the
vinyl ester resin and methacrylic acid were used as monomers. The
properties of the material were investigated and the column exhibited
good stability and selectivity to nifedipine, then the monolith
was used as the SPE pre-column for on line assay of nifedipine in
human plasma coupled to a HPLC-UV system, which overcame the
disadvantages in traditional off -line SPE and enabled clean, fast,
efficient analysis for plasma samples. |
| |
| Materials and Methods |
| |
| Reagents and material |
| |
| Bisphenol A diglycidyl ether (BADE) and tetrabutyl ammonium
bromide were purchased from Tianjin Chemicals Co., Lmt. (Tianjin,
China). EGDMA was purchased from Acros (New Jersey, USA). MAA
and 1, 4-dioxane was purchased from Tianjin Jinli chemicals Co.,
Lmt. (Tianjin, china). PF 68 was obtained from Shenzhen Youpuhui
Chemical Co., Ltd. (Shenzhen, China). PEG 400 was purchased from
Beijing Huaboyuan Science and Technology Development Center
(Beijing, China). Potassium persulfate was purchased from Huadong
Chemical Reagents Factory (Tianjin, China). Anhydrous calcium
chloride was purchased from Tianjin Beifangtianyi Chemical Reagents
Factory (Tianjin, China). Nifedipine reference substance (purity
99.6%) was obtained from CSPC ZhongNuo Pharmaceutical Co., LTD
(Shijiazhuang, China). Methanol (analytical grade) was obtained
from Kermel Chemical Co., Ltd. (Tianjin, China). Water was purified
by a Millipore system. All solutions were filtered through 0.45 µm
membrane filters (Millipore) before use. |
| |
| Human plasma, donated by volunteers, was obtained from the
Hospital of Hebei University and was centrifuged at 4500 rpm for
10 min, then stored under -18°C before use. The institutional review
board approval from Hospital of Hebei University has already been
obtained for the acquisition of plasma from human subjects. |
| |
| Synthesis of vinyl ester resin |
| |
| Vinyl ester resin was used as one of the monomer. First, it was
prepared according to the procedure described in literature [21]. The
process was as follows: 10 g of BADE, 0.2 g of tetrabutyl ammonium
bromide and 10 milliliters of 1, 4-dioxane was put into a three-necked
flask which was heated in a hot up set. 4.3 milliliters of methacrylic
acid was dropped in when the temperature was up to 80°C. Then the
temperature was raised up to 90°C and kept for 4.5 h. The vinyl ester
resin was synthesized. The synthesis scheme was shown in Figure 2. |
| |
|
Figure 2: Synthesis of vinyl ester resin. |
|
| |
| Preparation of molecularly imprinted monolith |
| |
| Five hundred microliters of vinyl ester resin, one hundred
microliters of MAA, eight hundred microliters of EDMA and nine hundred microliters of PEG 400 were placed in a round-bottomed
flask and the mixture was stirred with an overhead stirrer at 400 rpm
to form a homogeneous phase. Then, three hundred microliters of
methanol containing 0.006 g nifedipine was added as the template
dropwise with stir. After that, the aqueous phase consisting 1.5
milliliters of initiator (0.4 % potassium persulfate in deionised water),
375 milliliters of electrolyte (2% anhydrous calcium chloride in
deionised water) and 0.06 g of surfactant PF 68 was added dropwise
in the oil phase slowly. Stirring was continued for another 45 minutes
and a white-milky emulsion was formed. All of the above steps were
conducted in the absence of light. |
| |
| The emulsion was transferred to a stainless-steel tube of
chromatographic column of 50 × 4.6 mm i.d. and baked at 55°C for 36
h. After cooling to room temperature, the column was connected with
the HPLC system to remove the surfactant and soluble compounds by
pumping deionized water (60 mL) and methanol (30 mL) through the
column. Then, methanol- acetic acid (4:1, v/v) was used to wash the
template for 12 hours and methanol was used to wash the acetic acid
subsequently. Thus a novel MIP monolithic column was obtained. |
| |
| For comparison, blank polymer (NIP) was prepared as mentioned
above with the same procedure but in the absence of the template
nifedipine. |
| |
| Characterizations of the MIP monoliths |
| |
| The monoliths, synthesized and washed in the former steps,
were cut into small pieces followed by drying at 60°C overnight.
Morphology of the dried monolith samples was observed by scanning
electron microscopy (SEM). FT-IR spectra were recorded on a FTIR-
8400 spectrometer (Shimadzu, Japan). |
| |
| Chromatography |
| |
| Instruments and conditions: Jasco HPLC system (Jasco Co., Japan) equipped with a PU-1580 pump and a variable-wavelength UV-1570 detector was used for analyses. Data processing was performed with an HW-2000 chromatography workstation (Nanjing Qianpu Software,China). |
| |
| The pre-column was uesd as a pre-column and a C18 diamonsilTMcolumn (150 × 4.6 mm i.d., 5µm, Dikma, NY, USA) was used as the analytical column to detect nifedipine at the temperature 25°C. |
| |
| The mobile phase for separation and analysis was methanol-water
(80:20, v/v) at a flow-rate of 1.0 mL/min. The analytes were monitored
at a wavelength of 235 nm, which was the maximum absorption
wavelength of nifedipine. |
| |
| Standard solutions preparation: First, nifedipine was dissolved with methanol to prepare stock solution of 150 ng/mL. Working solutions of concentration 2.5, 5, 12.5, 25, 37.5, 50, 75µg/mL were prepared from the stock solution. These working solutions were then diluted with blank human plasma to obtain standard solutions with concentration of 5, 10, 25, 50, 75, 100, 150 ng/mL, respectively.If
not used immediately, the stock solution and other samples were all stored in the absence of light and at -20°C before use. |
| |
| On-line SPE: The monolithic column was used as an SPE column, which was placed in the sample-loop position of the injection valve and used for deproteinization and retaining nifedipine in plasma. In the “load” position, 5µL of plasma samples were directly injected into
the SPE column and were washed with 3 mL of water. Then the valve was switched to the “injection” position and nifedipine was desorbed by backflushing with methanol-water (80:20, v/v) and transferred to
the analytical column (C18). |
| |
| Precision, accuracy and recovery studies: Quanlity control
samples at three different concentration levels of 5, 50, 150 ng/mL
(low, medium and high) were prepared for the evaluation of precision,
accuracy and recovery. |
| |
| Precision of the method could be expressed as intra-day and interday
variability in the concentration ranges of nifedipine in plasma
samples and was evaluated by the relative standard deviation (RSD). |
| |
| The accuracy of this method was expressed as relative error (RE).
Relative error = [(found concentration - nominal concentration) / nominal concentration] × 100%. Found concentration was determined by calibration curve according to the procedure on-line SPE. The nominal concentration was the spiked concentration. |
| |
| Intra-day precision and accuracy were determined by five
replicated injections of each three nifedipine plasma samples (5, 50
and 150 ng/mL) within the same day. Inter-day precision and accuracy
were tested over five consecutive days for the same samples in intraday
precision test. |
| |
| The absolute recoveries of nifedipine were determined by
comparing nifedipine peak area obtained by HPLC analysis of
spiked plasma samples after SPE pretreatment to that obtained by
direct injection of nifedipine dissolved in methanol without SPE
pretreatment. |
| |
| Relative value of the concentrations determined by calibration
curve according to the procedure on-line SPE to the real
concentrations was calculated and its percentage form was set as the
relative recovery. All analyses were performed five times. |
| |
| Stability of nifedipine samples |
| |
| The stability of nifedipine stock solution and plasma samples at
experimental conditions were carried out, including freeze and thaw
stability and long-term stability. |
| |
| First, stock solution and spiked plasma samples of 5, 50, 150 ng/
mL nifedipine were analyzed by HPLC before storing. Then they were
freezed at -20°C and thawed at room temperature for three cycles.
Long-term stability was tested after one month stored at -20°C. |
| |
| Results and Discussion |
| |
| Preparation of MIP monolith |
| |
| The formation of stable emulsion was extremely essential in the
whole polymerization process. The factor played important part in
formation of stable emulsion included ratio of oil phase and water
phase, sequence and rate to add liquid, agitating rate and kind of
surfactant. Multiple experiments confirmed the optimal condition
were as followed: 400 rpm agitating, PEG 400 and PF 68 as surfactant.
The emulsion could keep stable for at least 36 hours without
delamination before the polymerization completed. |
| |
| In the polymerization of MIPs, nifedipine was used as the
template molecule, vinyl ester resin and MAA were used as the
dual monomers, among which MAA was the functional monomer
to provide carboxyl group to form hydrogen bonding interactions
with NH group of nifedipine and vinyl ester resin was the functional
monomer to form hydrogen bond with ester group of nifedipine.
However, the recognition ability of nifedipine was low when a single
monomer was used in the preparation of MIPs, but it was improved
when dual monomers were used to the polymerization process, as
reported in [22]. |
|
| |
| In the conventional bulk polymerization of MIPs, the porogenic
solvent governs the strength of non-covalent interactions and
non-polar solvents are favorable for most polymerization of MIPs.
Aqueous solutions can interfere with the imprinting process and the
selectivity is still not of completely satisfactory. Moreover, if the water
content during preparation is further increased, the monolith would
become flexible and provide poor mechanical strength [23]. In this
experiment, emulsion templating polymerization could overcome
the problem. Once the ratio to form a stable milky emulsion was
established, the nifedipine molecularly imprinted monolith could
be synthesized in water containing system with surfactant PEG 400
and PF 68 and the selectivity and mechanical strength were not
weaken correspondingly, which made the MIP monolith capable of
purification in aqueous solution. |
| |
| Characterizations of the MIP monolithic column |
| |
| Morphology and permeability: The morphology of the monolith
was studied by SEM and pictures of different magnifications were
shown in Figure 3. |
| |
| The SEM micrographs showed the multiple micro-pores and
through-pores formed by emulsion templating polymerization
and pores were well-distributed in the monolith.The average pore
diameter of MIPs was 0.65µm. The large pores in monolith facilitated
the polymeric ligand, such as the recognition sites for template,
to access to the surface of the media and might relate to the good
permeability of the monolithic column. |
| |
| The monolith was further characterized using FT-IR spectroscopy
on all samples to confirm their chemical structure (Figure 4). T he
broad peak around 3500 cm-1 indicated the stretching vibration of
-OH belonging to poly (vinyl ester resin) and poly MAA. The strong
absorption observed at 1720 cm-1 was due to the C=O of carboxyl
group. The existing groups of –OH and–COOH provided the hydrogen
bond binding sites for nifedipine in plasma samples. |
| |
|
Figure 3: SEM of the MIPs. |
|
| |
|
Figure 4: FT-IR spectrum of the monolithic material. |
|
| |
| The permeability of the material was tested by connecting it
to the HPLC pump. The backpressure at different flow-rates was
recorded in water and methanol and their relation was studied,
as illustrated in Figure 5. Good linearity (r1=0.9992, r2=0.9996)
between the pressure drop and the velocity of mobile phase proved
that the porous monolith was stable and not compressed at high
flow-rate, which manifested the potential for fast analysis. |
| |
|
Figure 5: Dependence of the back pressure on the flow-rate. |
|
| |
| The tailor-made selectivity of MIPs for nifedipine: The tailor-made
selectivity of the MIPs to nifedipine was studied using the mixture
sample of nifedipine and its analogue nisoldipine at the wavelength
of 235nm.With the increasing of water content, retention time of
nifedipine was prolonged while that of nisoldipine kept invariably at
dead time. When the mobile phase was methanol-water (80:20, v/v),
the baseline separation between nifedipine and nisoldipine could be
achieved and the resolution (Rs) was 2.68 under the condition. Figure
6 was the separation chromatogram of nifedipine and nisoldipine.
At the same time, the retention of the compounds on blank NIP was
also studied. The results showed that both nifedipine and nisoldipine
were eluted quickly and simultaneously and no separation ability was
observed. So the MIPs had significant molecular imprinting effect and
had high affinity and selectivity for nifedipine in water-containing
systems, thus could be used as SPE pre-column. |
| |
|
Figure 6: Separation Chromatogram of nifedipine and nisoldipine |
|
| |
| Stability and reproducibility of the material: In methanol-water
mobile phase system, multiple injections operated on the column seemed to affect the performance scarcely. At the same condition,the migration time and peak area of nifedipine after multiple injections manifested reproducible data for the same column (0.5%,0.8% RSD, respectively, n=11), which indicated the good ruggedness of the monolith. |
| |
| For different columns synthesized by the same processes, the
data, % RSD of migration time and peak area of nifedipine were 1.1 %
and 1.8 % respectively (n=11), showed fine reproducibility between
batches of the material. |
| |
| On line assay of nifedipine in plasma sample |
| |
| Study of plasma sample stability after pretreatment and
chromatography: Firstly, deproteinization ability was tested by
directly injecting 5µL of blank plasma into the SPE column and
eluted with pure water at the wavelength of 280 nm (Figure 7 a). The
elimination of the biological matrix could be considered completely
eluted when the detector signal reached the baseline. The experiment
showed that a washing volume of 3 mL was sufficient for sample
clean-up. Then, 5µL of 50 ng/ml nifedipine solution (nifedipine
dissolved in methanol) was injected directly into the column at the
same condition as above. The chromatogram (Figure 7b) showed that
nifedipine could not be eluted at all when pure water was used as
mobile phase. However, when methanol was used as mobile phase,
nifedipine was eluted quickly from the monolithic column in two
minutes. Therefore, during the washing process (washing liquid was
water), biological matrix was simply removed and nifedipine was
well retained on the SPE column, which illustrated the ability of the
column for plasma sample pretreatment. |
| |
|
Figure 7: Chromatograms of the deproteinization and enrichment of nifedipine
with the monolithic column. |
|
| |
| The chromatographic conditions were also investigated.
Satisfactory separation could be achieved by the use of mixed
solution of methanol and water as mobile phase. When the
proportion of methanol was increased, the drug could be eluted
more quickly. Considering the retention time and separation effect,
methanol-water (80:20, v/v) at a flow-rate of 1.0 mL/min was selected
for analysis. Typical chromatogram resulting from the HPLC-UV
analysis of plasma sample after on line SPE was depicted in Figure 8b.
Compared with chromatogram of standard solution of nifedipine in Figure 8c, the migration time of nifedipine was approximately 5.118
minutes and the peak of nifedipine showed good separation from
matrix peaks. Chromatogram of blank plasma sample (Figure 8a) was
used to determine whether there were any interfering peaks around
the migration time of nifedipine. The result showed that there was
no interference from endogenous compounds around the peak
of nifedipine under the optimized conditions, reflecting the high
specificity and selectivity of the described method. |
| |
|
Figure 8: Chromatograms of nifedipine in plasma samples. |
|
| |
| Calibration: Calibration curve was calculated by linear regression
analysis of the peak area of nifedipine versus its concentrations
(ng/mL) in plasma. Standard plasma samples with seven different
concentrations (5, 10, 25, 50, 75, 100 and 150 ng/mL) of nifedipine
were analyzed to obtain the intra-day calibration curve. Each sample was injected for three times. Each level of the calibration curve was
analyzed in triplicate in the following four days. The peak areaconcentration
showed a linear relationship over the range of 5-150
ng/mL for plasma samples. The mean equation of the calibration
curve obtained from seven points was y = 734.7 x - 745.2 (n = 5
days) with a determination coefficient of r2 = 0.998. The RSD for the
slope, intercept and r2 for linear regression model obtained on five
different days was 2.45, 1.98 and 1.35%, respectively.The limits of
detection (LOD) and quantitation (LOQ) calculated at a signal-to-noise
ratio of 3 and 10 were determined as 2 and 5 ng/mL, respectively. The
back-calculated concentrations of the different levels of samples by
the intra-day calibration curve were within the acceptance criteria. |
| |
| Precision, accuracy and recovery |
| |
| The intra-day (n = 5) and inter-day (n = 5) precision and accuracy
were evaluated from the calibration curve results. The results of
intra-day and inter-day precision and accuracy were given in Table
1 and Table 2, respectively. For plasma quality control samples at
low, medium and high levels of nifedipine investigated, RSD for intraand
inter-day precision was found to be 4.29-6.16%, indicating good
repeatability of this method; Relative error for intra- and inter-day
accuracy was less than 4.83% and it was obvious that the method was
remarkably accurate which ensures obtaining of reliable results. |
| |
| The recoveries determined at three different concentrations (5,50 and 150 ng/mL) were shown in Table 3.The recoveries included
the relative recovery and absolute recovery, among which the relative
recovery was also viewed as method recovery and the absolute
recovery was regarded as extraction recovery. The result for relative
recovery of nifedipine at three concentrations was 95.17, 96.62 and
94.83%, respectively, while that for absolute recovery was 83.92,
85.63 and 88.26%, respectively. |
| |
|
Table 1: The intra-day precision and accuracy of nifedipine in human plasma
samples for validation. |
|
| |
|
Table 2: The inter-day precision and accuracy of nifedipine in human plasma
samples for validation. |
|
| |
|
Table 3: The recovery of nifedipine in human plasma samples. |
|
| |
| Stability of the samples |
| |
| Stability was determined by comparing the nominal
concentration of nifedipine in plasma samples and the test samples.
The result showed that the recovery for tested samples at different
concentrations decreased less than 6.6%, but there was no significant
degradation observed after three freeze/thaw cycles and one month
stored at -20°C, indicating that nifedipine added to plasma were
stable in different storage conditions. |
| |
| Conclusion |
| |
| In summary, an alternative method to prepare molecularly
imprinted monolithic materials was developed. The permeability of
the column was high and the column could be used for rapid analysis
at high flow rate. The MIPs had significant molecular imprinting
effect and had high affinity and selectivity for nifedipine in watercontaining
system. It was successfully used as on-line SPE material
to deproteinization and screening nifedipine in human plasma. The
method required microamount of samples, which could be directly
injected into chromatography system without tedious pretreatment
of samples. The results suggested that on line SPE applying the MIP
monolith as pre-column could be considered as a simple, cheap,
effective and friendly to environment method for assaying drug in
plasma sample. |
| |
| Acknowledgement |
| |
| The authors are grateful for financial support by the National Natural Science Foundation of China (Grant Nos. 20375010 and 20675084) and Hebei Province Programs for Science and Technology Development (Grant Nos. 06276479B and 07276407D). |
| |
|
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