Synthesis of a molecularly imprinted polymer and its application in selective extraction of fenoprofen from wastewater.

The presence of various classes of pharmaceutical drugs in different environmental compartments has been reported worldwide. In South Africa, the detection of pharmaceuticals especially the non-steroidal anti-inflammatory drugs is recent, and more studies are being done in order to fully understand their fate in the aquatic environment. With considerations for the need of better sample preparation techniques, this study synthesized a molecularly imprinted polymer for the selective extraction of a non-steroidal anti-inflammatory drug, fenoprofen in aqueous environmental samples. Batch adsorption studies showed that adsorption of fenoprofen onto the cavities of the polymer followed a Langmuir isotherm as well as a pseudo second order model implying formation of a monolayer on the surface through chemisorption. The polymer had a maximum adsorption capacity of 38.8 mg g-1 and a Langmuir surface area of 1607 m2 g-1. The imprinted polymer was then used as the selective sorbent for solid phase extraction in the analysis of fenoprofen from wastewater followed by chromatographic determination. The analytical method gave a detection limit of 0.64 ng mL-1 and recovery of 99.6%. The concentration of fenoprofen detected in influent and effluent samples from two wastewater treatment plants ranged from 24 to 58 ng mL-1. The ability of the treatment plants to remove fenoprofen during wastewater processing based on the difference in concentrations in influent and effluent samples was found to be 41%. This work has shown that there is a possibility of release of fenoprofen from wastewater treatment plants into surface water sources.

Spectrophotometric determination of fenoprofen calcium drug in pure and pharmaceutical preparations. Spectroscopic characterization of the charge transfer solid complexes.

Simple, accurate and robust spectrophotometric method was developed for determination of fenoprofen calcium drug (FPC). The proposed method was based on the charge transfer (CT) reaction of FPC drug (as n-electron donor) with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), 2,4,6-trinitrophenol (picric acid, PA) or 1,2,5,8-tetrahydroxyanthraquinone (Quinalizarin, QZ) (as π-acceptors) to give highly colored charge transfer complexes. Different variables affecting the reaction such as reagent concentration, temperature and time have been carefully optimized to achieve the highest sensitivity. Beer's law was obeyed over the concentration ranges of 2-60, 0.6-90 and 4-30µgmL-1 using DDQ, PA and QZ CT reagents, respectively, with correlation coefficients of 0.9986, 0.9989 and 0.997 and detection limits of 1.78, 0.48 and 2.6µgmL-1 for the CT reagents in the same order. Elucidation of the chemical structure of the solid CT complexes formed via reaction between the drug under study and π-acceptors was done using elemental, thermal analyses, IR, 1H NMR and mass spectrometry. X-ray diffraction was used to estimate the crystallinity of the CT complexes. Their biological activities were screened against different bacterial and fungal organisms. The method was applied successfully with satisfactory results for the determination of FPC drug in fenoprofen capsules. The method was validated with respect to linearity, limit of detection and quantification, inter- and intra-days precision and accuracy. The proposed method gave comparable results with the official method.

Hyphenation of ionic liquid albumin glassy carbon biosensor or protein label-free sensor with differential pulse stripping voltammetry for interaction studies of human serum albumin with fenoprofen enantiomers.

A new biosensor or protein label-free sensor composed of 1-butyl-3-methylimidazolium hexafluorophosphates (BMIMPF6)-human serum albumin (HSA) film on glassy carbon electrode (GCE) was produced. Unfortunately, the native proteins themselves are often unstable in physiological conditions. Here, we introduced conjugation with ionic liquid (IL) such as BMIMPF6 which improved the stability and binding affinity of protein onto GCE. A rapid, simple and reliable method for the chiral discrimination and real time protein binding studies of fenoprofen enantiomers with HSA was developed by hyphenating ionic liquid albumin glassy carbon (ILAGC) biosensor with differential pulse cathodic stripping voltammetry under physiological conditions. The electrochemical behavior of chiral fenoprofen was monitored by cyclic voltammetry, from which large response was obtained from l-fenoprofen. The surface coverage of fenoprofen enantiomers was calculated by double potential-step chronocoulometry. The binding constants of chiral fenoprofen with HSA were estimated to be 3.2×10(5)±0.3 L mol(-1) and 0.8×10(4)±0.4 L mol(-1) for L- and D-fenoprofen, respectively giving acceptable precision (SD ≤ 0.4) and good agreement with the literature values. The competitive interactions of ibuprofen with fenoprofen enantiomers-HSA were studied giving a significant decreasing in the binding degrees of analytes to HSA. The reciprocal competitive experiments indicated that L-fenoprofen replaced D-fenoprofen from HSA. The proposed electrochemical biosensor holds great potential for chiral discrimination and real time binding studies of drugs with protein.

Electrokinetic supercharging focusing in capillary zone electrophoresis of weakly ionizable analytes in environmental and biological samples.

Five non-steroidal anti-inflammatory drugs, naproxen, fenoprofen, ketoprofen, diclofenac and piroxicam, were separated and analyzed by electrokinetic supercharging in CZE. Three different setups of the ITP technique were assayed for the separation and preconcentration of these five non-steroidal anti-inflammatory drugs. For the setup that gave the best results, we evaluated the influence of different parameters on separation and preconcentration efficiency such as sample pH, concentration of the leading stacker, BGE composition, electrokinetic injection time, composition and hydrodynamic injection of the solvent plug and of the terminating stacker. In the selected setup, the BGE (10 mM Na(2)B(4)O(7) + 50 mM NaCl in 10% of MeOH aqueous solution) contained the leading electrolyte while the terminating electrolyte, hydrodynamically injected after the sample (50 mbar x 12 s), was 50 mM of CHES. Prior to sample injection at (700 s at -2 kV) a short plug of MeOH (50 mbar x 3 s) was hydrodynamically injected. The results show that this strategy enhanced detection sensitivity 2000-fold compared with normal hydrodynamic injection, providing detection limits of 0.08 µg/L for standard samples with good repeatability (values of relative standard deviation, %RSD < 1.03%). Method validation with river water samples and human plasma demonstrated good linearity, with detection limits of 0.9 and 2 µg/L for river water samples and human plasma samples, respectively (as well as satisfactory precision in terms of repeatability and reproducibility).

Rapid simultaneous determination of four non-steroidal anti-inflammatory drugs by means of derivative nonlinear variable-angle synchronous fluorescence spectrometry.

A rapid, simple, and inexpensive spectrofluorimetric method has been proposed for the simultaneous quantification of diflunisal, salicylic acid, fenoprofen, and 6-methoxy-2-naphthylacetic acid (6MNA). First-derivative nonlinear variable-angle synchronous fluorescence spectrometry has been developed to improve the selectivity of fluorescence measurements without loss of sensitivity. It allows the simultaneous determination of different substances in a mixture from a single spectrum based on a single scan. The analyses were performed in an ethanol-water (70%) medium at a pH of 9.2, adjusted by using ammonium/ammonia (0.5 M) as a buffer solution. The linear concentration ranges are 30.0-100.0, 100.0-600.0, 50.0-150.0, and 30.0-100.0 ng/mL for salicylic acid, fenoprofen, diflunisal, and 6-methoxy-2-naphthylacetic acid, respectively, at lambda(ex)/lambda(em) = 281.1/423.6, 241.2/301.2, 284.1/403.8, and 268.7/339.6 nm, respectively. Analytical parameters of the proposed method were calculated according to the error propagation theory. The sensitivity, repeatability, reproducibility, and limits of detection achieved with the proposed method are adequate for the determination of these anti-inflammatory drugs.

Optimization of gas chromatographic method for the enantioseparation of arylpropionic non-steroidal anti-inflammatory drug methyl esters.

The gas chromatography (GC) method for enantioseparation of well-known non-steroidal anti-inflammatory drugs ibuprofen, fenoprofen and ketoprofen methyl esters mixture was developed. Best enantioseparation was performed on capillary column with heptakis-(2,3-di-O-methyl-6-O-t-butyldimethyl-silyl)-beta-cyclodextrin stationary phase and hydrogen used as a carrier gas. Initial temperature, program rate and carrier pressure were optimized to obtain best resolution between enantiomers.

Using terahertz pulsed spectroscopy to quantify pharmaceutical polymorphism and crystallinity.

Terahertz pulsed spectroscopy (TPS) is a new technique that is capable of eliciting rich information when investigating pharmaceutical materials. In solids, it probes long-range crystalline lattice vibrations and low energy torsion and hydrogen bonding vibrations. These properties make TPS potentially an ideal tool to investigate crystallinity and polymorphism. In this study four drugs with different solid-state properties were analyzed using TPS and levels of polymorphism and crystallinity were quantified. Carbamazepine and enalapril maleate polymorphs, amorphous, and crystalline indomethacin, and thermotropic liquid crystalline and crystalline fenoprofen calcium mixtures were quantified using partial least-squares analysis. Root-mean-squared errors of cross validation as low as 0.349% and limits of detection as low as approximately 1% were obtained, demonstrating that TPS is an analytical technique of potential in quantifying solid-state properties of pharmaceutical compounds.

Application of capillary electrophoresis with different sample stacking strategies for the determination of a group of nonsteroidal anti-inflammatory drugs in the low microg x L(-1) concentration range.

Several on-column sample preconcentration modes--large-volume sample stacking using the EOF pump (LVSEP), LVSEP with anion-selective exhaustive injection (LVSEP-ASEI) and field-amplified sample injection with sample matrix removal using the electroosmotic flow (EOF) pump (FAEP)--were used to analyze some nonsteroidal anti-inflammatory drugs (NSAIDs) by capillary electrophoresis, and then compared. Methanol was the background electrolyte solvent to suppress the EOF. The effect of the type and length of the solvent plug, and the sample injection time were investigated in FAEP to determine the conditions that provided the best response. LVSEP, LVSEP-ASEI, and FAEP improved the sensitivity of the peak area by 100-, 1200-, and 1800-fold, respectively. The methodology developed, in combination with solid-phase extraction (SPE), was applied to the analysis of water samples.

Stereoselective analysis of fenoprofen and its metabolites.

Reversed-phase high-performance liquid chromatographic assays have been developed to quantitate simultaneously fenoprofen and its major metabolites as well as to distinguish between their R- and S- enantiomers following a single oral dose of 600 mg racemic fenoprofen to healthy volunteers. The compounds are extracted from plasma (after precipitation of plasma protein) or assayed directly in diluted urine samples employing a gradient solution on a C18 column and ultraviolet detection. Two internal standards, ketoprofen and flunoxaprofen, are used to allow measurement of very low (0.05 microgram/ml) and high (70 microgram/ml) concentrations in each sample. R- and S-fenoprofen glucuronides can be separated directly; the 4'-hydroxyfenoprofen conjugates are measured via an indirect method by comparing the concentration of 4'-hydroxyfenoprofen before and after hydrolysis. The R- and S-enantiomers of both parent and 4'-hydroxy metabolite are derivatized with L-leucinamide via an ethyl chloroformate intermediate and subsequently analyzed on a C18 column. Concentrations of metabolites found in plasma were low when compared to parent drug. The S/R ratio of fenoprofen in plasma always exceeds 1 and increases with time after dosage while the S/R ratio of its 4'-hydroxy metabolite remains almost unchanged at 1.1. R-Fenoprofen glucuronide disappears rapidly from plasma as compared to its S-antipode; a less pronounced difference is noted between R- and S-4'-hydroxyfenoprofen conjugates. Fenoprofen is almost completely excreted as its S-acyl glucuronides; the renal clearance of unchanged drug is very low.

Identification of some impurities in fenoprofen calcium.

Some impurities in Fenoprofen calcium has been identified using mainly gas-mass method. These impurities are by-products of the synthesis.

Stereospecific high-performance liquid chromatographic (HPLC) assay of fenoprofen enantiomers in plasma and urine.

A new high-performance liquid chromatographic (HPLC) assay suitable for pharmacokinetic studies of enantiomers of fenoprofen (FEN) was developed. Following the addition of internal standard (IS; racemic ketoprofen), the plasma or urine constituents are extracted into a mixture of isooctane:isopropanol (95:5), back extracted into water, and finally, extracted into chloroform. After evaporation of the organic layer, the drug and IS are derivatized with l-leucinamide hydrochloride via ethyl chloroformate intermediate. The formed diastereoisomers are chromatographed on a reversed-phase HPLC with a mobile phase consisting of monopotassium phosphate solution:acetonitrile:triethylamine (65:35:0.02) at a flow rate of 1 ml/min. The detection UV wavelengths are 232 and 275 for the drug and IS, respectively. The suitability of the assay for pharmacokinetic analysis of FEN enantiomers was examined by analysis of the plasma and urine samples taken from a healthy subject, following peroral administration of a single 300-mg dose of racemic FEN.

High-performance liquid chromatographic quantitation of triacylglycerols containing fenoprofen from biological samples.

A normal-phase high-performance liquid chromatographic (HPLC) method has been developed for the quantitation of radiolabelled triacylglycerols containing fenoprofen, synthesized from [3H]glycerol by isolated hepatocytes and adipocytes. The assay consists of extracting the lipids into diethyl ether, separating triacylglycerols from polar endogenous lipids using silica Sep-Pak cartridges and quantitating endogenous triacylglycerols and triacylglycerols containing fenoprofen by HPLC resolution and scintillation counting. HPLC separation is achieved in less than 10 min. Using [14C]tripalmitin as internal standard the assay has a linear relationship between added triacylglycerol and measured endogenous triacylglycerols and triacylglycerols containing fenoprofen with regression coefficients of 0.997 and 0.998, respectively.

Biopharmaceutical investigation of fenoprofen.

The salts of fenoprofen formed with different metals have shown various crystal forms and solubility. The calcium salt has proved the most suitable characteristics for tablet and capsule production. Dissolution and absorption parameters of this substance were studied using in vitro and in vivo methods. The absorption rate and the correlation between pH and membrane diffusion rate constant were investigated in vitro using the "Sartorius" apparatus. The dissolution rate--depending on pH--was investigated by the oscillometric method. The in vivo disintegration of an experimental sample was compared with a commercial preparation. The comparison has been documented by endoscopic photography.

Effects of phenylbutazone and oxyphenbutazone on acidic drug detection in high performance thin layer chromatographic systems.

Interference or "masking" in thin layer chromatography occurs when the presence of one drug on a thin layer plate physically obscures or interferes with the detection of another drug. We investigated the ability of phenylbutazone and oxyphenbutazone to mask or interfere with the detection of acidic drugs of high performance thin layer chromatography. Of 20 acidic drugs called "positive" since 1981 by laboratories affiliated with the Association of Official Racing Chemists, 16 did not comigrate with phenylbutazone or oxyphenbutazone and could not, therefore, be masked by these agents. Three medications (diclofenac, fenoprofen, ibuprofen) were potentially masked by phenylbutazone and one (sulindac) was potentially masked by oxyphenbutazone. These agents were therefore administered to horses to determine whether or not their metabolites would allow their detection. In each case, metabolites of these agents were detectable for at least 24 hr after drug administration and detection was not interfered with by phenylbutazone or oxyphenbutazone. These results suggest that these 20 acidic drugs should be readily detectable in postrace urines of horses in the presence of phenylbutazone either as the parent drug or by virtue of the easily distinguishable metabolites that each agent possesses. There is, therefore, no reason to believe that the agents tested in this study can be effectively masked or interfered with by phenylbutazone or its metabolites in equine urine.

Enantiospecific high-performance liquid chromatographic analysis of 2-phenylpropionic acid, ketoprofen and fenoprofen.

A high-performance liquid chromatographic method has been developed for the quantitation of the R- and S-enantiomers of 2-phenylpropionic acid, ketoprofen and fenoprofen. The assay consists of extracting the arylpropionic acid with an internal standard and measuring the total (R + S) concentration of enantiomers by reversed-phase chromatography, derivatising the chromatographic fraction corresponding to the enantiomers to form R- and S, R-2-phenylethylamide distereoisomers which are resolved by normal-phase chromatography in order to calculate the fraction of each enantiomer. The limits of sensitivity of the assay for 2-phenylpropionic acid, ketoprofen and fenoprofen are 6, 0.2 and 2.5 mg/l, respectively.