Selective extraction of lamivudine in human serum and urine using molecularly imprinted polymer technique

Journal of Chromatography B, 931 (2013) 50–55

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Selective extraction of lamivudine in human serum and urine using molecularly imprinted polymer technique Maryam Shekarchi a,1 , Mojgan Pourfarzib a,b,1 , Behrouz Akbari-Adergani a , Ali Mehramizi c , Mehran Javanbakht d , Rassoul Dinarvand b,e,∗ a

Food and Drug Laboratory Research Center, Food and Drug Organization, MOHME, Tehran, Iran Department of Pharmaceutics, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran c Tehran Chemie Pharmaceutical Co., Tehran, Iran d Department of Chemistry, Amirkabir University of Technology, Tehran, Iran e Nanotechnology Research Centre, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran 1417614411, Iran b

a r t i c l e

i n f o

Article history: Received 27 January 2013 Accepted 30 April 2013 Available online xxx Keywords: Molecularly imprinted polymer Lamivudine Drug analysis Human serum Urine HPLC

a b s t r a c t In this work, a novel technique is described for determination of lamivudine in biological fluids by molecularly imprinted polymers (MIPs) as the sample clean-up method joint with high performance liquid chromatography (HPLC). MIPs were prepared using methacrylic acid as functional monomer, ethylene glycol dimethacrylate as crosslinker, acetonitrile and tetrahydrofuran as porogen and lamivudine as the template molecule. The new imprinted polymer was used as a molecular sorbent for the separation of lamivudine from human serum and urine. Molecular recognition properties, binding capacity and selectivity of the MIPs were evaluated and the results showed that the obtained MIPs have a high affinity for lamivudine in aqueous medium. HPLC analyses showed that the extraction of lamivudine from serum and urine by MIPs had a linear calibration curve in the range of 60–700 ␮g/L with excellent precisions of 2.73% for serum and 2.60% for urine. The limit of detection and quantization of lamivudine was 19.34 and 58.6 ␮g/L in serum and 7.95 and 24.05 ␮g/L in urine respectively. MIP extraction provided about 10 fold LOQ improvement in serum and 5 fold LOQ improvement in urine samples. The recoveries of lamivudine in serum and urine samples were found to be 84.2–93.5% and 82.5–90.8% respectively. Due to the high precision and accuracy, this method may be the UV-HPLC choice with MIP extraction for bioequivalence analysis of lamivudine in serum and urine. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Lamivudine is an antiviral agent with potent activity against acquired immunodeficiency syndrome (AIDS) and hepatitis B virus through inhibition of nucleoside reverse transcriptase activity. It has been recommended for the treatment of AIDS in combination with other antiviral drugs [1,2]. Lamivudine has been quantified in biological fluids by different methods such as UV–visible spectrophotometry and titrimetric assays [3], radioimmunoassay [4,5] which are low sensitive, difficult and timeconsuming requiring unstable reagents and expensive chemicals and liquid–liquid extraction or heating step. High performance liquid chromatography (HPLC) technique [6–11] has been extensively used for the determination of lamivudine in pharmaceutical or

∗ Corresponding author at: Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran 1714614411, Iran. Tel.: +98 21 66959095; fax: +98 21 66959096. E-mail address: [email protected] (R. Dinarvand). 1 Both authors are treated as first authors with equal responsibility. 1570-0232/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jchromb.2013.04.041

biological samples applying a variety of techniques including plasma protein precipitation [12], solid phase extraction [13] and column switching [14]. Recently, several LC–MS–MS methods with improved sensitivity and specificity have been developed to measure these drugs concentrations in biological matrices. However, mass spectrometers are expensive and not readily available in most laboratories. HPLC-UV instruments are still widely used due to lower cost and greater robustness in bioavailability studies. Since HPLC-UV methods are susceptible to interference from endogenous and exogenous substances, it is necessary to develop a suitable and selective sample clean-up procedure for the analysis of compounds in real samples. Due to insufficient selectivity, the conventional sorbents usually cannot separate analytes proficiently in complex biological samples. Solid-phase extraction (SPE) is nowadays routinely utilized as clean up techniques for the target enrichment and clarification to assist analytical quantification. Compared to liquid–liquid extraction, SPE can decrease the time required, particularly for automated methods, can handle little samples, and consumes small quantity of solvent [15]. Due to factors such as convenience, price, time

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reduction and simplicity SPE is the most common sample pretreatment techniques today [16]. However, the extraction mechanism is mainly based on the development of hydrophobic interactions that lead to the co-extraction of many endogenous compounds having similar physicochemical characteristics. A relatively new improvement in the area of SPE is the use of molecularly imprinted polymers (MIPs) for the sample clean up [17–19]. The main advantages of MIPs, over conventional sorbents used for sample pre-treatment, are the high selectivity and affinity for target analytes. MIPs are synthetic polymers having specific cavities planned for a template molecule which are synthesized by copolymerization of functional and cross linking monomers in the presence of target analyte. In the most common preparation procedure, monomers make a complex with a template in the course of covalent or non-covalent interactions. An ideal MIP adsorbent should have the following characteristics: high binding affinity, specificity and capacity, fast association and dissociation kinetics; broad solvent compatibility; and long-term stability against pH, organic solvents and heating which permit for more adaptability in the analytical methods [20]. The use of MIPs for SPE can include different forms, with conventional SPE where the MIP is filled into columns or cartridges [21,22] and batch mode SPE where the MIP is equilibrated with the sample [23]. Another main benefit of MIPbased SPE, associated to the high selectivity, is the attainment of a well-organized sample clean-up. In a previous work published by members of this group, the technique of molecular imprinting has been applied with success to the preparation of high affinity SPE for bromhexine [24], metoclopramide [25], tramadol [26], dextromethorphan [27], penicillin G [28], carbamazepine [29], and dipyridamole [30]. Herein we present a simple and straightforward method for the performance evaluation of lamivudine based MIPs as selective sorbents for efficient sample clean-up.and further determination of lamivudine from biological matrices by HPLC. However, to the best of our knowledge, there are few reports about MIPs developed for nucleotide prodrugs and nucleoside reverse transcriptase inhibitors (NRTIs). Due to lower values of distribution coefficient (Log P) for lamivudine, owing to its high water solubility and polarity, it was difficult to obtain sufficient recovery using LLE especially in pre-concentration of trace analytes. Protein precipitation method was not found suitable as it gave very low recoveries with frequent clogging of column. A major problem is the inability to make effective imprints in aqueous systems and lamivudine is insoluble in the aprotic organic systems with low polarities generally used in current non-covalent imprinting strategies and Hbond-dominated interactions are usually very weak in water and we test several solvents to determine the optimum recipe for the imprinting of a desired target compound. The aim of this work was to synthesize a selective MIP targeted for lamivudine for its application to the treatment of complex matrices and enrichment in trace analysis. This scheme as MIPs permits the sensitive, uncomplicated and inexpensive separation and determination of the analyte in human serum and urine samples which can be adopted by pharmaceutical laboratories for industrial quality control.

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in chromatography were HPLC grade and obtained from Merck (Darmstadt, Germany). Lamivudine, adefovir, acyclovir, zidovudine reference standards were supplied from Hetero (India). Drug free human serum was obtained from the Iranian Blood Transfusion Organization (Tehran, Iran) and stored at −20 ◦ C until use after gentle thawing. Urine samples were collected from healthy volunteers. The lamivudine stock solution as standard solution (10 mg/L) was prepared in water monthly and stored at 4 ◦ C. Intermediate standard solution (1 mg/L) was prepared weekly by dilution of stock solutions. Working standard of different concentrations (60–700 ␮g/L) for serum and urine were prepared daily by diluting the intermediate standard solution with phosphate buffer pH = 5. 2.2. Instrumentation FT-IR spectra of grounded polymer were recorded on a Bomem MB 155S FT-IR spectrometer (Canada) using KBr pellets in the range of 400–4000 cm−1 . The size of particles was measured by Zetasizer nano ZS (Malvern, UK). In all solutions the pH was adjusted by digital Metrohm pH meter (model 744) equipped with a combined glass-calomel electrode. The HPLC experiment was performed using a Waters Alliance system equipped with a vacuum degasser, quaternary detector. The UV spectra were collected across the range of 200–900 nm, extracting 270 nm for chromatograms. Empower software was utilized for instrument control, data collection and data processing. The column was an ACE 5, phenyl (4.6 mm × 250 mm). The mobile phase was an isocratic mode of methanol:phosphate buffer 0.005 M pH 6.8 (8:92) at flow rate of 1.0 mL/min. The injection volume for all samples and standards was 100 ␮L [6]. 2.3. MIP and NIP preparation with bulk polymerization In order to prepare MIPs, MAA (680 ␮l, 8 mmol), lamivudine (229.3 mg, 1 mmol), acetonitrile (8 mL) and tetrahydrofurane (2 mL) were placed in a glass sample vial and shaken for 2 h. Then EGDMA (7.52 mL, 40 mmol) was added. The mixture was uniformly dispersed by sonication and the reaction initiator AIBN (57 mg, 0.347 mmol) was added. The reaction mixture was purged with N2 for 10 min and the glass tube was sealed under this atmosphere. The polymerization was carried out for 24 h under ultraviolet wave 366 nm and then for 24 h in water bath 40 ◦ C. After the polymerization procedure, the hard polymers, poly(MAA-co-EGDMA) were crushed and dried. Then lamivudine molecules in the MIP were removed by washing with the mixture of methanol and acetic acid (9:1 (v/v), of 98% methanol and glacial acetic acid) for several times and two times with methanol until the absorbance of analyte was no longer detected in the elution by UV spectrophotometer. In order to confirm that retention of template was due to molecular recognition and was not related to any non-specific binding, a nonimprinted polymer (NIP) was prepared with the same procedure without the target molecule (lamivudine). The mean MIP particle size, measured by Zetasizer nano ZS, was about 607 nm. 2.4. Batch rebinding experiments

2. Experimental 2.1. Chemicals and reagents Methacrylic acid (MAA) and ethylene glycol dimethacrylate (EGDMA) from Merck (Darmstadt, Germany) were distilled in vacuum previous to use in order to remove the stabilizers. 2,2Azobis isobutyronitrile (AIBN) purchased from Merck (Darmstadt, Germany) was recrystallized by ethanol. Water was obtained from a Milli-Q purification system (Purelab UHQ Elga). All solvents used

Batch adsorption experiments were used to estimate the binding capacity of the imprinted polymer as reported before [31,32]. In order to extract lamivudine by the MIP, the synthesized polymer particles (40 mg) were added in volumetric flask (5 mL) containing lamivudine at different concentrations (10–800 mg/L) in phosphate buffer pH = 5. The mixtures were shaken overnight, centrifuged for 15 min at 8000 rpm and used the clear supernatant. The free concentration of lamivudine after the adsorption was recorded by UV-HPLC at 270 nm. Three replicate extractions and measurements were carried out for each concentration. After

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Fig. 2. Effect of pH on extraction of MIP and NIP particles in batch experiments.

Fig. 1. Infrared plots of the unleached (A) and leached (B) MIP particles.

washing with the mixture of acetonitrile/tetrahydrofuran (4/1, v/v) the adsorbed lamivudine was desorbed from the MIP by treatment with 5 mL of methanol:acetic acid (9:1, v/v). The imprinted polymer containing lamivudine was placed in the desorption medium and stirred continuously at 500 rpm in room temperature for 15 times. The amount of lamivudine bound to the polymer was calculated by subtracting the concentration of free lamivudine from the initial concentration. The same process was followed for NIP particles. 2.5. Extraction procedure for serum and urine samples Lamivudine standard stock solution (10 mg/L) was prepared in water. Standard solutions were prepared by adding proper volumes of lamivudine solution to 10 mL volumetric flasks and the solutions were diluted to the mark with biological fluids and shaken for 5 min. The spiked serum and urine samples (2 mL) were diluted with buffer pH 5 (3 mL), centrifuged for 30 min at 9000 rpm and then filtered through a cellulose acetate filter (0.20 ␮m pore size, Advantec MFS Inc., CA, USA). Finally 5 mL of the filtrates (60–700 ␮g/L) were directly mixed with the MIP and NIP particles. 3. Results and discussion 3.1. Characterization The IR spectra of the imprinted poly(MAA-co-EGDMA) after and before eluting template are shown in Fig. 1. The IR spectra of the MIPs before and after elution showed similar typical peaks, indicating the characteristics in the backbone structure of the different polymers. The formation of hydrogen binding bonds decreased the electric cloud density of OH and C O and resulted in the decrease in frequency of vibration. As a result of this fact the C O stretching, the O–H stretching and the bending vibrations at 1728, 3425 and 1394 cm−1 in the leached MIP materials were shifted to 1735, 3456 and 1388 cm−1 in the related unleached MIP, respectively. In addition, there were two other separate differences between the IR spectra of the MIPs before and after elution. In the unleached polymer, there were two bands with low and high relative intensities at 1458 cm−1 and 2954 cm−1 that was presented at 1465 and 2947 cm−1 in the same leached MIP, respectively. Other absorption peaks go with both those of MIP: 1636 cm−1 (stretching vibration of residual vinylic C–C bonds), 1257, 1157 cm−1 (symmetric and asymmetric ester C–O stretch bands) and 964 cm−1 (out-of-plane bending vibration of vinylic C–H bond).

3.2. Optimization of MIP formulations As can be seen in Fig. 4, the structure of the lamivudine owns both amino and carbonyl groups which theoretically make it an ideal compound to interact with both basic (4-vinylpyridine, VP) and acidic (methacrylic acid, MAA) monomers. The MIP using methacrylic acid as monomer showed higher recognition ability to target molecule than the MIPs prepared using acrylamide and VP due to MAA stronger electrostatic and hydrophobic interactions with target in polar environment. Owing to the effect of polymerization media polarity on the strength and number of interactions and the recognition of obtained MIPs, the influence of the porogen used in the polymerization was also investigated. Main experiments revealed that the imprinted polymers prepared in chloroform rather than dimethyl formamide, acetonitrile and dichloromethane show better molecular recognition in aqueous environment. As a result of lamivudine poor solubility in chloroform, we preferred to use acetonitrile instead. Generally, correct molar ratios of functional monomer to template are very effective in improving the specific affinity and number of MIPs recognition sites. High ratios of functional monomer to template result in high non-specific affinity, while low ratios create less complexation due to inadequate functional groups [32]. The rebinding (extraction) values and ratio range of MIPs were estimated as shown in Table 1. For the highest specific rebinding of lamivudine, the optimum ratio of the functional monomer to the template was 8:1 (Table 1), which had the best specific affinity and the highest recovery about 92.3%, while the other molar ratio at 2:1, 4:1 and 6:1 were 35%, 43% and 56%, respectively. Therefore, a typical 1:8:40 template:monomer:cross-linker molar ratio was used for more studies. 3.3. Effect of pH, choice of loading, washing and eluent solution The pH of the solution is one of the most important variables affecting retention behavior and selectivity of the MIPs as a consequence of hydrophobic interactions. Especially, the washing and elution conditions need to be carefully optimized in terms of pH, ionic strength and solvent composition in order to fully promote the MIPs ability to recognize lamivudine. Several batch experiments were carried out by equilibrating 40 mg of the imprinted and non-imprinted polymers with 5 mL of solutions containing 100 ␮g/L of lamivudine under the preferred range of pH (3.0–8.0). It was observed that lamivudine undergoes complete rebinding at pH 5.0 (Fig. 2). The lower reactions observed at lower and higher pH, may be produced by the protonation of the amine group of lamivudine and deprotonation of carboxyl groups of the polymer, respectively. Generally, the polymers have binding ability with both specific and non-specific interactions. The

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Table 1 Compositions and evaluations of the extraction of polymers. Polymer

MAA (mmol)

Lamivudine (mmol)

EGDMA (mmol)

AIBN (mmol)

Extraction% ± SDa

MIP1 MIP2 MIP3 MIP4 NIP1 NIP2 NIP3 NIP4

2.0 4.0 6.0 8.0 2.0 4.0 6.0 8.0

1.0 1.0 1.0 1.0 0 0 0 0

40 40 40 40 40 40 40 40

0.34 0.34 0.34 0.34 0.34 0.34 0.34 0.34

35% 43% 56% 92% 25% 28% 20% 12%

a

± ± ± ± ± ± ± ±

1.9 2.4 3.1 2.0 1.7 2.3 1.5 3.2

Average of three determinations.

specific interactions may be created mainly from the imprinting procedure, which produces selective recognition sites for the template. The non specific interactions were evaluated by the binding of the non-imprinted polymer. At first, particles were conditioned with phosphate buffer pH = 5. Standard aqueous solution of lamivudine (100 ␮g/L) was then loaded onto the polymers. The effect of the addition of different organic solvents (i.e. acetonitrile, methanol, acetone, dichloromethane, tetrahydrofuran, and dimethylformamide) was evaluated as possible washing solutions for the removal of non-specifically bound lamivudine compounds on both imprinted polymers. From these possibilities, acetonitrile/tetrahydrofuran (4/1, v/v) was identified as the solvent of choice with the recovery of lamivudine in NIP particles (12.2%) and MIP particles (92.3%). For the recovery of strongly bound lamivudine, the polymers were eluted with three times 1 mL of methanol/acetic acid (9/1, v/v). 3.4. Adsorption capacity The capability of the sorbent is an important factor which establishes how much sorbent is required to extract a specific quantity of drug from the solution quantitatively. Saturation studies of the MIP polymer particles were carried out to estimate their binding capacity. For determination of the adsorption capacity of MIP and NIP particles, the polymers (40 mg) were added into lamivudine solutions (5 mL) at concentrations of 10–800 mg/L. The suspensions were shaken overnight at room temperature, centrifuged and the lamivudine content in the supernatant was measured by HPLC. The isothermal adsorptions of lamivudine are shown in Fig. 3. According to these results, the maximum amount of lamivudine that can be adsorbed by MIP was found to be 256.6 mg/g at pH 5.0. As all the available specific cavities of the MIP are saturated, the retention of the analyte is only due to non-specific interactions which can be approximately identical for MIP and NIP polymers.

3.5. Study of MIP selectivity Chromatographic estimation and equilibrium batch rebinding experiments are the methods most commonly used to consider the selectivity of the imprinted materials [33]. For equilibrium batch rebinding experiments, a solution of template known amount is put into a vial containing a fixed mass of polymer. Once the system has come to equilibrium, the amount of free template in solution and adsorbed to the MIP determined [34]. MIP and NIP particles (50 mg) were loaded by lamivudine, zidovudine, adefovir, acyclovir, nevirapine and efavirenz solutions (100 ␮g/L) in phosphate buffer pH = 5. The distribution ratio (mL/g) of lamivudine between the MIP particles and aqueous solution was evaluated by following equation: KD =

(Ci − Cf )V

(1)

Cf m

where V is the volume of initial solution and m is the mass of MIP materials. Selectivity coefficients for lamivudine ion relative to foreign compounds are defined as: sel Klamivudine =

KDlamivudin

(2)

j

KD j

where KDlamivudin and KD are the distribution ratios of lamivudine and foreign compound, respectively. The relative selectivity coefficient (k ) was also determined by following equation: k =

k(MIP) k(NIP)

(3)

The selectivity tests of MIP were performed using zidovudine, adefovir, acyclovir, nevirapine and efavirenz (Fig. 4). Distribution ratio (KD ), selectivity coefficient (ksel ) and relative selectively coefficient (k ) values of MIP and NIP material for these different drugs are listed in Table 2. The data in Table 2 shows that MIP has high affinity for zidovudine, the relative selectively coefficient is only 4.5. This could be simply clarified by its close correspondence to lamivudine in the way of the arrangement of the functional groups and the size of the three-dimensional structure. For adefovir, MIP showed higher relative selectively coefficient (6.14) than zidovudine. Acyclovir, nevirapine and efavirenz revealed much lower affinity for MIP and k values of 7.06, 7.41 and 9.1 was obtained respectively. Therefore the MIP is specific and selective Table 2 Distribution ratio (KD ), selectivity coefficient (ksel ) and relative selectively coefficient (k ) values of MIP and NIP material for different drugs.

Fig. 3. Effect of lamivudine concentrations on the retention capacities of MIP and NIP particles.

Drug

KD MIP

KD NIP

ksel (MIP)

ksel (NIP)

k

Lamivudine Acyclovir Zidovudine Adefovir Nevirapine Efavirenz

705.5 53.95 100 79.8 46.32 35.56

62.53 33.87 40.05 43.3 30.54 28.66

– 13.07 7.06 8.84 15.23 19.84

– 1.85 1.56 1.44 2.05 2.18

– 7.06 4.52 6.14 7.41 9.10

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Fig. 4. Structures of the drugs used in this study.

for extraction and determination of lamivudine from companion drugs in complex matrices. 3.6. Lamivudine assay in human serum and urine samples To demonstrate the applicability of MIP for the selective clean-up of lamivudine, the MIP was used to the purification of spiked human serum and urine. Aqueous media was used for the loading solution and the washing protocol was estimated for obtaining maximum recovery of the analytes using acetonitrile/tetrahydrofuran (4/1, v/v). The chromatograms obtained from serum and urine samples are shown in Figs. 5 and 6. As shown in these figures, the retention time of lamivudine is about 9.2 and the runtime is 20 min and this well-organized method obtained cleaner extracts and interfering peaks from the complex biological matrices to be removed. Results from the HPLC analyses demonstrated that the MIP extraction of lamivudine for serum and urine samples is linear in the ranges 60–700 ␮g/L with good precision (2.73% and 2.6% for 200 ␮g/L, respectively). The repeatability for 5 mL of spiked serum and urine (200 ␮g/L of lamivudine), expressed

as RSD (n = 6), was lower than 4%. The recoveries for serum and urine were 93.5% and 90.8%, respectively (Table 3). Typical chromatograms were nearby in Figs. 5 and 6 demonstrated that the MIP can be used for the sample clean-up. The limit of detection (LOD) and limit of quantification (LOQ) for lamivudine in serum samples were 19.34 and 58.6 ␮g/L and in urine samples were 7.95 and 24.05 ␮g/L, respectively. Table 3 Assay of lamivudine in human serum and urine by means of the described method. (Recovery% ± SD)a

Sample

Spiked value (ng/mL)

MIP

NIP

Human serum

60.0 200 700

84.2 ± 2.53 93.5 ± 2.73 85.3 ± 3.72

24.8 ± 2.32 10.9 ± 2.39 15.6 ± 3.14

Human urine

60.0 200 700

82.5 ± 2.80 90.8 ± 2.60 85.3 ± 3.26

12.5 ± 2.71 10.5 ± 3.46 13.6 ± 2.15

a

Average of three determinations.

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Fig. 5. HPLC chromatogram obtained from non-extracted sample (A). Chromatograms obtained after clean up a 200 ␮g/L solution of lamivudine in serum samples with NIP(B) and MIP(C) monitored at 270 nm; conditions: column ACE 5 ␮m, phenyl 4.6* 250 mm, eluent: buffer phosphate pH 6.8/methanol(92:8) at flow rate 1.0 mL/min.

Fig. 6. HPLC chromatogram obtained from non-extracted sample (A). Chromatograms obtained after clean up of a 200 ␮g/L solution of lamivudine in urine samples with NIP(B) and MIP(C) experimental conditions are the same as in Fig. 5.

4. Conclusions In this paper, water-compatible molecularly imprinted polymers were synthesized via a non-covalent molecular imprinting approach in acetonitrile and tetrahydrofurane (8/2, v/v) for selective extraction and separation of lamivudine from serum and urine. This proficient method provided cleaner extracts and removed interfering peaks from the complex biological matrices. The method was appropriated to the trace lamivudine determination at three levels, and the recoveries for the spiked human serum and urine samples were 84.2–93.5% and 82.5–90.8% in the 60–700 ␮g/L, respectively. Based on these results, the extraction recoveries of the analytes from the real samples were satisfactory and consequently, the proposed coupled system of MIP-HPLC can be easily employed for the analysis of lamivudine in biological samples with great potential in developing selective extraction. The results presented in this study could be precious for future research toward improvement of new, rapid and low-cost systems useful in clean-up procedures and provide a strategy for expansion of MIPs in products recovery from biological fluids. References [1] A.V. Singh, L.K. Nath, J. Pharm. Anal. 1 (2011) 251–257. [2] S. Strauch, E. Jantratid, J.B. Dressman, H.E. Junginger, S. Kopp, K.K. Midha, V.P. Shah, S. Stavchansky, D.M. Barends, J. Pharm. Sci. 100 (2011) 2054–2063. [3] K. Basavaiah, B.C. Somashekar, J. Sci. Ind. Res. 65 (2006) 349–354. [4] E. Kazue Kano, C.H. Dos Reis Serra, Int. J. Pharm. 297 (2005) 73–79. [5] S.A. Wring, R.M. O Neil, J.L. Williams, W.N. Jenner, M.J. Daniel, J. Pharm. Biot. Med. Anal. 12 (1994) 1573. [6] R.M.W. Hoetelmans, M. Profijt, P.L. Meenhorst, J. Chromatogr. B 713 (1998) 387–394. [7] G. Aymard, K.B. legrand, N. Trichereau, B. Diquet, J. Chromatogr. B: Biomed. Sci. Appl. 744 (2000) 227.

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