piperine , a nitrogenous pungent substance , is an alkaloid found in important and oldest spices , namely , piper nigrum ( black peppers ) and piper longum ( long peppers ) . .   stated that piperine is naturally occurring organic compound that belongs to family piperaceae . the fruits of piperine possess antidepressant effects , hepatoprotective effects , antioxidant activity , antitumour effects , antibacterial effects , and anticonvulsant effects [ 2 , 3 ] . piperine also has the capability of reducing inflammation , relieving pain , improving digestion , and enhancing the bioavailability . piperine is extensively used in medicinal field for years due to various medicinal properties including painkiller , antioxidant , and bioavailability enhancer . molecular imprinting technology ( mit ) is used to design molecular recognition materials because it is capable of mimicking natural recognition entities like antibodies and biological receptors [ 514 ] . the original concept of molecular imprinting is developed by linus pauling in 1940s , but wulff and sarhan stimulated the interest in imprinting materials . according to vlatakis et al . , in the early 1980s , the molecular imprinting polymers ( mips ) were successfully prepared by using noncovalent mit . molecular imprinting is a universal method to produce polymers with high affinity binding sites for organic , inorganic , biological , and chemical molecules or ions . mips allow the functional and crosslinking monomers to copolymerize in the presence of the target compound or known as template . molecular imprinting polymers   can be prepared by various methods such as bulk polymerization , electropolymerization , suspension polymerization , emulsion polymerization , two - step polymerization , and precipitation polymerization . zhou et al .   mentioned that the controlled / living radical polymerization ( crp ) is used to prepare mip microspheres as it permits more precise control over the molecular weight , composition , and end group functionality of the obtained polymers [ 2427 ] . mips show excellent thermal and chemical stability and can be used in aggressive media . according to yan and row , mips have many advantages over their biological counterparts including inexpensive , simple preparation , storage stability , repeated operations without loss of activity , high mechanical strength , durability to heat and pressure , and applicability in harsh chemical media . lai et al .   stated that mips have been used in important application such as chemical sensors [ 30 , 31 ] , capillary electrophoresis and electrochromatography , catalysis , hplc stationary phases [ 3439 ] , and solid - phase extraction ( spe ) . in this research noncovalent imprinting or self - assembly approach the template and a functional monomer interact by noncovalent interactions in the prepolymerization mixture . according to spivak , noncovalent is simpler molecular imprinting method as compared to covalent and semicovalent because it involves synthetic steps toward the prepolymer complex . in this way interaction between the monomer and template this method has been used to produce imprinted polymers of cinnamic acid . in this research as an application these imprinted polymers are used in extraction of piperine from spiked urine sample . ( united states ) , acrylic acid ( aa ) was bought from nippon shokubai co. ltd . ( japan ) , ethylene glycol dimethacrylate ( egdma ) was purchased from sigma - aldrich co. ltd . ( united states ) , acetonitrile ( acn ) was obtained from kunshan yalong trading co. ltd . ( china ) , 2 , 2-azobisisobutyronitrile ( aibn ) was obtained from sigma - aldrich co. ltd . ( united states ) , methanol ( meoh ) was obtained from nuasa kimia sejati co. ( indonesia ) , acetic acid ( ch3cooh ) was purchased from alpha chemika ( india ) , potassium bromide ( kbr ) was obtained from powder pack chem co. ( india ) , and hexane was obtained from seidler chemical co. ( united states ) . scanning electron microscope ( jeol jsm-6390la ) was used to study the morphology of polymer particles . shaker ( nb-101mt ) was used to allow the rebinding of polymer particles with template . shimadzu lc-20a , a reversed - phase high performance liquid chromatography ( rp - hplc ) , was used to evaluate the batch binding of polymer particles . . 0.5  mmol of template ( piperine ) , 2  mmol of monomer ( aa ) , 8  mmol of cross - linker ( egdma ) , 75  ml of porogen ( acn ) , and 0.011  g of initiator ( aibn ) were added into 150  ml conical flask , respectively . the mixture was sonicated for 10 minutes in order to remove bubbles and allow complete dissolution . then , the conical flask containing mixture was placed in a bucket of ice cubes and the reaction mixture was purged with nitrogen gas for 15 minutes . ice cubes were used in this experiment to allow a suitable environment for noncovalent interactions between piperine and acrylic acid . the polymerization was conducted for 6 hours , initially temperature was maintained at 60c for the first three hours , and later temperature was raised up to 80c and maintained for another three hours in order to complete the polymerization . the produced polymer particles were extracted out by using the centrifugation at 5000  rpm for 10  min . the template was removed by washing the mips successively in the mixture of methanol and acetic acid ( 9  :  1 , v / v ) until the template was not detected by rp - hplc at 270  nm . the hplc was conducted by using the c18 column ( 250  4  mm , 5  m ) with the mobile phase consisting of acetonitrile , distilled water , and acetic acid in the ratio of 60  :  39.5  :  0.5 , v / v / v , respectively . the flow rate was set at 0.6  ml / min with uv detection at 270  nm and injection volume was set at 20  l . the nonimprinted polymeric particles ( nips ) were prepared in the same way without the addition of the template molecule . the similar procedure was used for the synthesis of different molecular imprinted polymers of piperine with varying composition of aa and egdma ( table 1 ) by precipitation polymerization , namely , mip 2 , mip 3 , and mip 4 for mips as well as nip . a series of 150  ml five conical flasks containing 0.5  g of the mip ( mip 1 , mip 2 , mip 3 , and mip 4 ) and nip beads were added with a 75  ml of acetonitrile containing 0.5  mmol of piperine . the conical flasks were shaken on the shaker at 100  rpm and the samples were collected at different time intervals ( 0 , 30 , 60 , 90 , 120 , 150 , 240 , and 360 minutes ) . the collected samples were centrifuged at 5000  rpm for 10 minutes in order to remove any suspended particles and supernatant was used for further analysis . the binding capacity of mips and nip of piperine was calculated   by using the following equation:(1)binding capacity%=cicfci100,where ci is the initial piperine concentration in the solution and cf is the final piperine concentration in the solution . a 150  ml conical flask containing 0.5  g of the mip 3 beads was added and a solution of 75  ml of acetonitrile containing equal concentration ( 0.5  mmol ) of piperine and caffeine . both of the conical flasks were shaken on the shaker at 100  rpm and the samples were collected at different time intervals ( 0 , 30 , 60 , 90 , 120 , 150 , 240 , and 360 minutes ) . after shaking at the appropriate time intervals , all the samples were centrifuged at 5000  rpm for 10 minutes . the distribution ratios ( ml  g ) of piperine between the mips or nip in the porogen ( acetonitrile ) were determined   by the following equation:(2)distribution ratio :  kd = cicfvcim , where ci is the initial piperine concentration in the solution , cf is the final piperine concentration in the solution , v is the volume of porogen ( acn ) used , and m is the mass of mip / nip used . the selectivity coefficients for piperine relative to binding competitor caffeine for mip 3 and nip can be calculated   by(3)selectivity coefficient :  kpiperine / caffeinesel = kdpiperinekdcaffeine , where kd is the batch binding assay of mip / nip for piperine and kd is the batch binding assay of mip / nip for caffeine . the relative selectivity coefficient ( k ) was determined   by the following equation:(4)k=kmip  3knip . urine sample was first centrifuged and filtered and then spiked with a piperine to get a concentration of 50  ppm . after this 50  ml of spiked urine sample was added in flask containing 0.5  g of mip 3 . synthesis of microsphere imprinted polymers is a very crucial step in order to produce uniform shape and size of particles . previous studies revealed   that various preparation methods have been carried out for the preparation of polymer microspheres   such as the synthesis of polymer microspheres by dispersion and emulsion polymerization , where the surfactants in aqueous solution   and stabilizers in organic solution   are crucial to stabilize the polymer phase and prevent the aggregation of particles . precipitation polymerization can form polymer microspheres with constant size and shape that can lead to narrow dispersion , without the need for any added surfactant or stabilizer [ 4649 ] . in this research we have successfully produced imprinted polymer microspheres by precipitation method in the acetonitrile ( porogen ) . ir analysis is an important chemical characterization method to detect the functional groups present in a compound . the ft - ir spectra of different mips and nip are shown in figure 1 . based on figure 1 , small peak in the range of 3519.29  cm to 3613.06  cm attributed to the vibration mode of o the bands in the range of 2926.75  cm to 2996.02  cm and 2854.76  cm to 2957.24  cm showed the vibration mode of c h stretching of aliphatic compound as well as asymmetric and symmetric ch2 stretching in mips and nip . strong peaks at 1726.11  cm to 1736.42  cm indicated the presence of c = o of acrylic acid . the vibration mode of c = c stretching of aromatic compound can be found within 1635.37  cm to 1636.84  cm . the ch2 bending at 1450.82  cm to 1452.70  cm indicated the presence of alkane group in mips and nip . n stretching of mips and nips after leaching was detected at 1388.44  cm to 1390.06  cm . peaks of mips and nip at 1253.88  cm to 1259.61  cm showed the vibration mode of o  ch2o symmetric stretching . small band at 1035.88  cm to 1050.59  cm in mips and nip explained the presence of symmetric stretching of = c  o  c . the vibration mode of c h stretching of aliphatic compound can be observed at 2954.09  cm to 2985.19  cm . sem analysis is a very important morphological study for polymer particles that provides the idea about the shape and size . , uniform size of imprinted polymers can be formed by using a noncovalent imprinting approach by precipitation polymerization . arabzadeh and abdouss   stated that interaction between monomer and template could be another factor that contributed to uniform size distribution with clean surfaces . research conducted by park et al .   mentioned that there are various factors that affect the production of uniform polymer microspheres including volume of solvent , reaction of solvent , and presence of template ion . excess solvent or porogen used in the synthesis of polymer particles will produce highly uniform polymer microspheres with imprinted binding sites . rp - hplc was used to evaluate the binding efficiency of mips and nip of piperine . figure 3 depicts the binding capacity of different mips and nip at different time intervals . mip 3 showed the highest binding capacity ( 84.94% ) , followed by mip 2 ( 75.86% ) , mip 1 ( 69.40% ) , and mip 4 ( 60.80% ) . mip 3 contains a higher amount of monomer ratio as compared to mip 1 and mip 2 but mip 4 contains a higher amount of cross - linker . in this study increasing amount of monomer would produce specific interaction sites with the piperine and hence rebinding efficiency was also increased . but the increase in amount of cross - linker has produced a reverse effect as can be seen in mip 4 . if we compare the mips with nip it is clear from figure 3 the binding capacity is low . this can be conferred that nip does not contain any binding site complimentary with the piperine . in order to evaluate the properties of mip of piperine as a sensing material , two compounds ( piperine and caffeine ) were tested using both mip 3 and nip . the distribution ratio of piperine in both mip 3 and nip was higher than the distribution ratio of caffeine in both mip 3 and nip , resulting in higher selectivity coefficient of piperine than that of caffeine in both mip 3 and nip ( table 2 ) . the results indicate that the imprinted polymer has got complimentary binding sites or cavities with the piperine as compared to caffeine . the extensive use of piperine in medicine and spices has generated this idea to first use the selected mip 3 in the extraction of piperine from urine . this will provide us a way forward to expand the application of these imprinted polymer particles . from this study it was found that about 81.18% of piperine was successfully extracted from the spiked urine sample . the binding efficiencies of mips and nip of piperine were evaluated by batch binding assay . mip 3 exhibited the highest binding capacity ( 84.94% ) as compared to nip ( 40% ) .