Expanding the applicability of magnetic ionic liquids for multiclass determination in biological matrices based on dispersive liquid-liquid microextraction and HPLC with diode array detector analysis.

Monitoring biological samples at trace levels of chemicals from anthropogenic actions such as pesticides, pharmaceuticals, and hormones has become a very important subject. This work describes a method for the determination of eight compounds of different chemical classes in human urine samples. Dispersive liquid-liquid microextraction based on magnetic ionic liquids was used as the sample preparation procedure. The main parameters of the method, such as sample dilution, type, and volume of disperser solvent, amount of magnetic ionic liquids, extraction time, and pH were optimized by univariate and multivariate procedures. Validation was performed using a urine sample of a male volunteer in order to obtain a calibration curve and the main analytical parameters of merit such as limits of detection and quantification. Values varied from 3.0 to 7.5 µg/L and from 10 to 25 µg/L, respectively. Satisfactory precisions of 21% for intraday (n = 3) and 16% for interday (n = 9) were achieved. Accuracy was evaluated by relative recovery assays using different urine samples and ranged from 75 to 130%. Robustness was assured by the Lenth method. The validated procedure was applied to five urine samples from different volunteers and the hormone estrone was found in one sample.

Non-steroidal anti-inflammatory drugs increase urinary neutrophil gelatinase-associated lipocalin in recreational runners.

OBJECTIVES: To study the effects of running with/without the use of pain killers on urinary neutrophil gelatinase-associated lipocalin (uNGAL) and other parameters of kidney function in recreational runners. METHODS: Participants of the 10- and 21.1-km Weir Venloop race were enrolled and their urine samples collected before and after the run. Urine dipstick and other conventional tests used to assess kidney function were performed. The presence of ibuprofen, diclofenac, naproxen, and/or paracetamol was assessed by LC-MS/MS. uNGAL was measured with a two-step chemiluminescent immunoassay. RESULTS: NSAIDs/analgesics were detected in urine of 5 (14.4%) 10-km runners and 13 (28.9%) 21.1-km runners. Only half-marathon participants showed significant increases in uNGAL (pre: 11.7 [7.1-34.3] ng/mL; post: 33.4 [17.4-50.4] ng/mL; P = .0038). There was a significant effect of NSAID/analgesic use on uNGAL increase (F2, 76  = 4.210, P = .004). Post hoc tests revealed that uNGAL increased significantly in runners who tested positive for ibuprofen/naproxen compared to runners who did not use any medications (P = .045) or those who tested positive for paracetamol (P = .033). Running distance had a significant influence on the increase in uNGAL (F1, 53  = 4.741, P < .05), specific gravity (F1, 60  = 9.231, P < .01), urinary creatinine (F1, 61  = 10.574, P < .01), albumin (F1, 59  = 4.888, P < .05), and development of hematuria (χ2 (4) = 18.44, P = .001). CONCLUSIONS: Running distance and use of ibuprofen/naproxen were identified as risk factors for uNGAL increase in recreational runners.

The pharmacokinetics and metabolism of diclofenac in chimeric humanized and murinized FRG mice.

The pharmacokinetics of diclofenac were investigated following single oral doses of 10 mg/kg to chimeric liver humanized and murinized FRG and C57BL/6 mice. In addition, the metabolism and excretion were investigated in chimeric liver humanized and murinized FRG mice. Diclofenac reached maximum blood concentrations of 2.43 ± 0.9 µg/mL (n = 3) at 0.25 h post-dose with an AUCinf of 3.67 µg h/mL and an effective half-life of 0.86 h (n = 2). In the murinized animals, maximum blood concentrations were determined as 3.86 ± 2.31 µg/mL at 0.25 h post-dose with an AUCinf of 4.94 ± 2.93 µg h/mL and a half-life of 0.52 ± 0.03 h (n = 3). In C57BL/6J mice, mean peak blood concentrations of 2.31 ± 0.53 µg/mL were seen 0.25 h post-dose with a mean AUCinf of 2.10 ± 0.49 µg h/mL and a half-life of 0.51 ± 0.49 h (n = 3). Analysis of blood indicated only trace quantities of drug-related material in chimeric humanized and murinized FRG mice. Metabolic profiling of urine, bile and faecal extracts revealed a complex pattern of metabolites for both humanized and murinized animals with, in addition to unchanged parent drug, a variety of hydroxylated and conjugated metabolites detected. The profiles in humanized mice were different to those of both murinized and wild-type animals, e.g., a higher proportion of the dose was detected in the form of acyl glucuronide metabolites and much reduced amounts as taurine conjugates. Comparison of the metabolic profiles obtained from the present study with previously published data from C57BL/6J mice and humans revealed a greater, though not complete, match between chimeric humanized mice and humans, such that the liver humanized FRG model may represent a model for assessing the biotransformation of such compounds in humans.

Single-drop microextraction combined in-line with capillary electrophoresis for the determination of nonsteroidal anti-inflammatory drugs in urine samples.

This study describes a method to determine nonsteroidal anti-inflammatory drugs (NSAIDs) in urine samples based on the use of single-drop microextraction (SDME) in a three-phase design as a preconcentration technique coupled in-line to capillary electrophoresis. Different parameters affecting the extraction efficiency of the SDME process were evaluated (e.g. type of extractant, volume of the microdroplet, and extraction time). The developed method was successfully applied to the analysis of human urine samples with LODs ranging between 1.0 and 2.5 µg/mL for all of the NSAIDs under study. This method shows RSD values ranging from 8.5 to 15.3% in interday analysis. The enrichment factors were calculated, resulting 27-fold for ketoprofen, 14-fold for diclofenac, 12-fold for ibuprofen, and 44-fold naproxen. Samples were analyzed applying the SDME-CE method and the obtained results presented satisfactory recovery values (82-115%). The overall method can be considered a promising approach for the analysis of NSAIDs in urine samples after minimal sample pretreatment.

Design of folding-based impedimetric aptasensor for determination of the nonsteroidal anti-inflammatory drug.

In this work, a novel sensing nanocomposite with highly dispersed platinum nanoparticles (PtNPs) on carbon nanotubes (CNTs) functionalized with polyethyleneimine (PEI) has been developed as a platform for immobilization of diclofenac (DIF) aptamer. PtNPs/PEI/CNTs nanocomposite provided abundant NH2 groups for the immobilization of DIF-specific aptamer. Attachment of DIF-aptamer at the surface of modified electrode was performed through the formation of phosphoramidate bonds between the amino group of PEI and the phosphate group of the aptamer at the 5' end. Nickel hexacyanoferrate (NiHCF) as signal probe was electrodeposited at the surface of nanocomposite by a simple electrodeposition method including two consecutive procedures. Under optimal conditions, DIF was detected by impedance spectroscopy (EIS) quantitatively. By adding DIF as the target at the surface of modified electrode, the aptamer specifically binds to DIF and its end folds into a DIF-binding junction, which leads to retarding the interfacial electron transfer of the probe at the surface of modified electrode. Sensitive quantitative detection of DIF was carried out by monitoring the increase of charge transfer resistance (Rct) by increasing the DIF concentration. The proposed aptasensor showed a good detection range from 10 to 200 nM with an unprecedented detection limit of 2.7 nM.

Hepatic effects of repeated oral administration of diclofenac to hepatic cytochrome P450 reductase null (HRN™) and wild-type mice.

Hepatic NADPH-cytochrome P450 oxidoreductase null (HRN™) mice exhibit normal hepatic and extrahepatic biotransformation enzyme activities when compared to wild-type (WT) mice, but express no functional hepatic cytochrome P450 activities. When incubated in vitro with [(14)C]-diclofenac, liver microsomes from WT mice exhibited extensive biotransformation to oxidative and glucuronide metabolites and covalent binding to proteins was also observed. In contrast, whereas glucuronide conjugates and a quinone-imine metabolite were formed when [(14)C]-diclofenac was incubated with HRN™ mouse liver, only small quantities of P450-derived oxidative metabolites were produced in these samples and covalent binding to proteins was not observed. Livers from vehicle-treated HRN™ mice exhibited enhanced lipid accumulation, bile duct proliferation, hepatocellular degeneration and necrosis and inflammatory cell infiltration, which were not present in livers from WT mice. Elevated liver-derived alanine aminotransferase, glutamate dehydrogenase and alkaline phosphatase activities were also observed in plasma from HRN™ mice. When treated orally with diclofenac for 7 days, at 30 mg/kg/day, the severities of the abnormal liver histopathology and plasma liver enzyme findings in HRN™ mice were reduced markedly. Oral diclofenac administration did not alter the liver histopathology or elevate plasma enzyme activities of WT mice. These findings indicate that HRN™ mice are valuable for exploration of the role played by hepatic P450s in drug biotransformation, but poorly suited to investigations of drug-induced liver toxicity. Nevertheless, studies in HRN™ mice could provide novel insights into the role played by inflammation in liver injury and may aid the evaluation of new strategies for its treatment.

Diclofenac metabolism in the mouse: novel in vivo metabolites identified by high performance liquid chromatography coupled to linear ion trap mass spectrometry.

The metabolism of [(14)C]-diclofenac in mice was investigated following a single oral dose of 10 mg/kg. The majority of the drug-related material was excreted in the urine within 24 h of administration (49.7 %). Liquid chromatographic analyses of urine and faecal extracts revealed extensive metabolism to at least 37 components, with little unchanged diclofenac excreted. Metabolites were identified using a hybrid linear ion-trap mass spectrometer via exact mass determinations of molecular ions and subsequent multi-stage fragmentation. The major routes of metabolism identified included: 1) conjugation with taurine; and 2) hydroxylation (probably at the 4'-and 5-arene positions) followed by conjugation to taurine, glucuronic acid or glucose. Ether, rather than acyl glucuronidation, predominated. There was no evidence for p-benzoquinone-imine formation (i.e. no glutathione or mercapturic acid conjugates were detected). A myriad of novel minor drug-related metabolites were also detected, including ribose, glucose, sulfate and glucuronide ether-linked conjugates of hydroxylated diclofenac derivatives. Combinations of these hydroxylated derivatives with acyl conjugates (glucose, glucuronide and taurine) or N-linked sulfation or glucosidation were also observed. Acyl- or amide-linked-conjugates of benzoic acid metabolites and several indolinone derivatives with further hydroxylated and conjugated moieties were also evident. The mechanisms involved in the generation of benzoic acid and indolinone products indicate the formation reactive intermediates in vivo that may possibly contribute to hepatotoxicity.

Development and implication of a capillary electrophoresis methodology for ciprofloxacin, paracetamol and diclofenac sodium in pharmaceutical formulations and simultaneously in human urine samples.

This work studies the development of a simple and fairly rapid methodology for simultaneous determination/separation of three frequently co-administered drugs; ciprofloxacin (CIP), paracetamol (PCT) and diclofenac sodium (DIC) using capillary electrophoresis (CE) with UV detection at 260 nm. Separation was achieved in only 6.5 min with a simple buffer of sodium tetraborate (50 mM) at pH 9.0. The Parameters affecting the separation and detection were optimized. The calibration curves were linear in the range of 5-500 µg/mL for CIP, 5-250 µg/mL for PCT and 1-125 µg/mL for DIC sodium under the optimized conditions. The lower limit of detection (LOD) was found to be 1 µg/mL for CIP & PCT and 0.5 µg/mL for DIC. The method was successfully used for the analysis of drugs in commercial pharmaceutical formulations and simultaneously from patient's urine sample with RSD 0.5-2.4%. Results obtained with CE method are compared with standard HPLC procedure and were found in good agreement.

In-situ synthesis of nanocubic cobalt oxide @ graphene oxide nanocomposite reinforced hollow fiber-solid phase microextraction for enrichment of non-steroidal anti-inflammatory drugs from human urine prior to their quantification via high-performance liquid chromatography-ultraviolet detection.

The in-situ synthesis and application of nanocubic Co3O4-coated graphene oxide (Co3O4@ GO) was introduced for the first time to present a cost-effective, stable and convenient operation and a simple device for hollow fiber solid-phase microextraction (HF-SPME) of four selected nonsteroidal anti-inflammatory drugs (NSAIDs) including diclofenac, mefenamic acid, ibuprofen and indomethacin. The extracted analytes were desorbed by an appropriate organic solvent and analyzed via high-performance liquid chromatography-ultraviolet detection (HPLC-UV). The prepared sorbent was approved using different characterization methods such as X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX). The variables effective on the Co3O4@GO-HF-SPME method including extraction time, desorption time, desorption solvent volume, sample pH, stirring rate and ionic strength were screened via Plackett-Burman design and then optimized by Box-Behnken design. Under optimal condition, the calibration curves were linear within the range of 1.0-200.0 µg L-1 of analyte concentration with detection limits of 0.18-1.1 µg L-1 and the relative standard deviations less than 10.1%. The limits of quantification (LOQs) were in the range of 0.60-3.67 µg L-1. Matrix effect was not observed with this method; therefore, standard addition is not necessary for quantification of target compounds. The enrichment factors were obtained in the range of 49-68. The relative recoveries of the urine sample analysis were calculated in the range of 93-102%. Finally, the presented method exhibited good sensitivity, excellent repeatability, high reusability and acceptable precision, which will be a promising method to analyze various nonsteroidal anti-inflammatory drugs in urine samples.

[Thin layer chromatography for the analysis of certain anti-inflammatory medicines].

This work was devoted to the elucidation of conditions for isolation of ketorolac and diclofenac from biological fluids. A method is proposed for the extraction of these compounds from solutions with organic solvents at different pH values. Other methods permit to optimize identification of analytes by thin layer chromatography while the densitometric technique may be used for qualitative and quantitative analysis of their composition in biological fluids.

Nanocrystalline cellulose as a biotemplate for preparation of porous titania thin film as a sorbent for thin film microextraction of ketorolac, meloxicam, diclofenac and mefenamic acid.

The present study included the procedure of preparing porous titania thin film using a direct nanocrystalline cellulose templating (NCC) as a bio-template. The microextraction applicability of the porous film was investigated by thin film microextraction (TFME) of the nonsteroidal anti-inflammatory drugs (NSAIDs) including ketorolac, meloxicam, diclofenac and mefenamic acid from different types of urine sample. The extracted NSAIDs were analyzed by HPLC-UV. Under optimum conditions, the calibration curves were linear within the range of 1.0-500 µg L-1 (2.0-200 µg L-1 for ketorolac, 2.0-500 µg L-1 for meloxicam, 1.0-200 µg L-1 for diclofenac and 1.0-200 µg L-1 for mefenamic acid). The limit of detection was found to be between 0.2 and 0.5 µg L-1. The calculated intra- and inter-day relative standard deviations RSDs% (n = 3) at concentration level of 10 µg L-1 were less than 6.3% and 6.0%, respectively. Finally, the method was successfully applied to determine selected NSAIDs in urine samples from different human individuals.

In-situ synthesis of flower like Co3O4 nanorod arrays on anodized aluminum substrate templated from layered double hydroxide as a nanosorbent for thin film microextraction of acidic drugs followed by HPLC-UV quantitation.

The present study is the first report of in-situ growth and application of nanorods-flower like Co3O4 nanosorbent coated on the anodized aluminum substrate for thin film microextraction (TFME) approach. The flower like Co3O4 was successfully fabricated by conversion of Co-Al layered double hydroxide (LDH) precursor to Co3O4 using the simple calcinations process. The cheap and available aluminum foil was electrochemically anodized and used as a porous substrate. Response surface methodology (RSM) was explored for optimization step. Different acidic drugs, including: paracetamol, ibuprofen, aspirin and diclofenac were extracted from biological fluids in order to investigate the capability of the prepared sorbent. The extracted analytes were then analyzed using high performance liquid chromatography-ultraviolet detection (HPLC-UV). Under the optimized conditions, the limits of detection were between 0.2 and 1.7 µg L-1 in different selected matrices. The obtained limits of quantification were also calculated to be between 0.8 and 5.1 µg L-1 in the selected matrices. In addition the enrichment factors were also in the range of 105-169. Batch-to-batch reproducibility at 100 µg L-1 concentration level was also evaluated to be lower than 5.2% (n = 3). Finally, the method was successfully used for analysis of these compounds in the biological fluids.

Dual functional monomers modified magnetic adsorbent for the enrichment of non-steroidal anti-inflammatory drugs in water and urine samples.

According to the molecular properties of non-steroidal anti-inflammatory drugs (NSADs), a new adsorbent for magnetic solid phase extraction (MSPE) was designed and synthesized. Triethyl-(4-vinylbenzyl)aminium chloride and 4-vinylbenzeneboronic acid were utilized as dual functional monomers to copolymerize with divinylbenzene on the surface of pre-modified Fe3O4 nanoparticles. The prepared magnetic adsorbent (Fe3O4@TCVA) was characterized by elemental analysis, Fourier transform infrared, scanning electron microscopy, transmission electron microscopy and vibrating sample magnetometer. Due to the abundant boronic acid, quaternary amine and phenyl groups, the Fe3O4@TCVA displayed satisfactory extraction performance for target NSADs (diclofenac acid, ibuprofen and mefenamic acid) by means of B-N coordination, anion-exchange, π-π and hydrophobic interactions. Under the optimized conditions, the Fe3O4@TCVA/MSPE was combined with high-performance liquid chromatography with diode array detection (HPLC-DAD) to sensitively analyze NSADs in water and human urine samples. Results indicated that the limits of detection for water and urine samples were in the ranges of 0.014-0.031 µg/L and 0.029-0.11 µg/L, respectively. The relative standard deviations for the intra-day and inter-day assay variability were below 10%. The applicability of the proposed Fe3O4@TCVA/MSPE-HPLC-DAD method was demonstrated by the successful extraction and quantification of trace levels of NSADs in real water and human urine samples. Satisfactory spiked recovery and reproducibility were achieved.

Amine modified magnetic polystyrene for extraction of drugs from urine samples.

Polystyrene is one of the best candidates as the extracting medium due to its high stability in different media and acceptable extraction capability. However, the hydrophobic nature and low wettability of polystyrene limits its application to non-polar analytes. To resolve this limitation, in this project, amine groups were chemically attached to the surface of magnetic polystyrene. The resulting hydrophilic magnetic particles were expected to be capable of extracting both polar and non-polar analytes. Non-steroidal anti-inflammatory drugs (NSAIDs) were chosen for testing the applicability of modified magnetic polystyrene according to the importance of their analysis and also their wide polarity range. Major parameters associated with the extraction procedure were optimized using central composite design (CCD) method and pH 3, extraction time of 15 min, desorption volume of 350 µL and desorption time of 4 min were chosen as optimized values. Under these conditions, figures of merit were calculated including: linear dynamic range (0.5-1000 µg L-1), linear equation and limits of detection (0.1-3 µg L-1). To investigate the method precision, inter-day, intra-day and synthesis-to-synthesis relative standard deviation (RSD) were studied (<12%). All studies were conducted using blank urine samples spiked with aspirin, salicylic acid, ibuprofen, diclofenac and mefenamic acid. Naproxen was chosen as the internal standard and high-performance liquid chromatography-UV-Vis (HPLC-UV) was employed for the subsequent determination after extraction. To evaluate the applicability of the method for real sample analysis, urine samples from patients under treatment were analyzed and acceptable results were obtained. The aminated magnetic polystyrene revealed superior extraction efficiency, much higher than polystyrene before functionalization. In addition, hospital wastewater sample was tested and acceptable extraction efficiencies were obtained.

Combustion fabrication of magnetic porous carbon as a novel magnetic solid-phase extraction adsorbent for the determination of non-steroidal anti-inflammatory drugs.

Based on a one-step combustion fabrication approach, a novel magnetic porous carbon (MPC) was fabricated using filter paper as porous carbon source and iron salts as magnetic precursors. The textural properties of the MPC were characterized by transmission electron microscopy (TEM), Fourier transform infrared spectrometry (FT-IR), X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), vibration sample magnetometer (VSM) and nitrogen absorption-desorption isotherms. The as-prepared MPC possessed a high specific surface area, a microstructure comprised of mesopores and strong magnetic response. It was employed as a magnetic solid-phase extraction (MSPE) adsorbent for the determination of three non-steroidal anti-inflammatory drugs (NSAIDs) in environmental water and biological samples coupled with high performance liquid chromatography (HPLC). The main parameters affecting extraction efficiency were investigated in detail and a satisfactory performance was obtained under the optimal conditions. The calibration curves were linear over the concentration ranging from 1 to 1200 µg L-1 for ketoprofen (KET) and 2-1200 µg L-1 for naproxen (NAP) and diclofenac (DCF) with determination coefficients (R2) between 0.9995 and 0.9997. The limits of detection (LODs) were in the range of 0.2-0.4 µg L-1. The intra- and inter-day relative standard deviations (RSDs) were less than 4.03% and 8.72%, respectively. The recoveries ranged from 84.67% to 113.73% with RSDs less than 7.76%. The satisfactory results confirmed the great potential of the novel MPC adsorbent for the extraction of NSAIDs from complex sample matrices.

Relationship between CYP2C8 genotypes and diclofenac 5-hydroxylation in healthy Spanish volunteers.

PURPOSE: CYP2C8 seems to be involved in diclofenac 5-hydroxylation, while, in vitro, the 4'-hydroxylation and 3'-hydroxylation seem to be mediated mainly by CYP2C9. We have demonstrated the relevance of CYP2C9 genotypes for diclofenac 4'-hydroxylation in healthy volunteers, so that the present study was aimed at analyzing the role of both CYP2C8 and CYP2C9 genotypes on diclofenac metabolism, as well as determining the CYP2C8 allele frequencies and their relationship with CYP2C9 variants. METHODS: A group of 142 healthy white Spanish volunteers was studied. Previously, 102 of these subjects had been phenotyped with diclofenac and genotyped for CYP2C9. The CYP2C8 genotypes were determined by allele-specific PCR-RFLP methods. The urinary concentrations of diclofenac and its main metabolites were analysed using an HPLC-UV method after the administration of a single oral dose of diclofenac as described previously for part of the population studied here. RESULTS: The diclofenac/5-hydroxydiclofenac urinary concentration ratio was higher in individuals carrying a CYP2C8*3 or CYP2C8*4 allele than in those homozygous for wild-type allele CYP2C8*1 (P < 0.05). Moreover, approximately 93% of the subjects with a CYP2C8*3 allele also carried a CYP2C9*2, and 80% of the subjects that had CYP2C9*2 variant also carried a CYP2C8*3. In addition, the four CYP2C9*2/*2 individuals were CYP2C8*3/*3. CONCLUSIONS: This is the first study showing the influence of CYP2C8 genotypes on diclofenac metabolism in vivo. The linkage disequilibrium between CYP2C8*3 and CYP2C9*2 alleles was confirmed in this Spanish population.

Isolation and characterization of a new human urinary metabolite of diclofenac applying LC-NMR-MS and high-resolution mass analyses.

The nonsteroidal anti-inflammatory drug diclofenac is widely used. Diclofenac is extensively metabolized to several hydroxylated derivatives and their conjugates. The lactam-dehydrate of 4'-hydroxy diclofenac (4'-OHDD) has now been detected as a new urinary metabolite of diclofenac. Isolation was successfully performed using preparative HPLC in three different steps using water, methanol, and acetonitrile, respectively. The structural characterization of 4'-OHDD was achieved by LC-NMR-MS. In addition, specific mass fragmentation pattern could be obtained using LC-high-resolution MS with both positive and negative electrospray ionization.

Determination of diclofenac sodium by resonance light scattering method using silver nanoparticles as probe.

A sensitive, simple and novel method was developed to determine diclofenac sodium (DS) using silver nanoparticles (AgNPs) as probe by resonance light scattering (RLS) technique. It was found that DS could quench the RLS intensity of AgNPs. Moreover, the decrease in RLS intensity was linearly correlated to the concentration of DS over the range of 0.01-2.0µgmL-1. DS can be measured in a short time (5min) without any complicated or time-consuming sample pretreatment process. Parameters that affect the RLS intensities such as pH, concentration of AgNPs, reaction time, electrolyte concentration, and coexisting substances were systematically investigated and optimized. The results showed that the method had a very good selectivity and could be used conveniently for the determination of DS. The limit of detection (LOD) was 2.85ngmL-1 (3σ), and the relative standard deviation (RSD) was less than 3.6% (n=6). Possible mechanism for the RLS changes of AgNPs in the presence of DS was discussed and the method was successfully applied for the analysis of tablets and urine samples.

Development of electrochemically controlled packed-in-tube solid phase microextraction method for sensitive analysis of acidic drugs in biological samples.

In the present research, for the first time, a novel "packed-in-tube" configuration has been applied to electrochemically controlled in-tube solid phase microextraction, followed by high performance liquid chromatography. In order to prepare a mini packed column, small beads of stainless steel were first placed into the stainless steel column. Then, a nanostructured polypyrrole film was prepared on the internal surface of a stainless steel tube and the surface of stainless steel particles through a facile in-situ electrodeposition method. Filling the column with tiny particles of stainless steel effectively reduces the dead volume of the extraction tube and increases the extraction phase volume. The column was used for separation and preconcentration of diclofenac and mefenamic acid as model analytes from biological samples. Several important factors affecting extraction efficiency, such as extraction and desorption times, flow rates of the sample solution and eluent, and extraction and desorption voltages were investigated and optimized. This method showed good linearity for the drugs in the range of 0.3-200.0 µg L-1, 1.1-200.0 µg L-1, and 1.8-200.0 µg L-1 with coefficients of determination better than 0.9986, 0.9973, and 0.9973 in water, urine, and plasma samples, respectively. Intra- and inter-assay precisions (RSD%, n = 3) were in the range of 2.6-4.8% and 2.9-5.1, respectively, at three concentration levels of 10, 25, and 75 µg L-1. In addition, the limits of detection were in the range of 0.02-0.04 µg L-1. The validated method was successfully applied to the analysis of diclofenac and mefenamic acid in some biological samples. Finally, it is concluded that this method can be a general and reliable alternative to the analysis of ionic compounds in biological matrices.

A new diclofenac molecularly imprinted electrochemical sensor based upon a polyaniline/reduced graphene oxide nano-composite.

A diclofenac (DCF)-imprinted polymer, composed of polyaniline, reduced graphene oxide (rGO) and triphenylamine, as cross linker, was synthetized. This composite was identified by using SEM and FT-IR techniques. The prepared DCF-imprinted polymer (MIP) was used for modification of carbon paste electrodes (CPEs) to fabricate a selective DCF electrochemical sensor. Electrochemical behavior of DCF on the investigated sensor and the optimization of the parameters affecting the DCF determination were screened by cyclic voltammetry (CV). The cyclic voltammogram of DCF showed an anodic peak current at about 0.5 V (vs. SCE). The calibration curve for DCF determination was obtained by applying the investigated sensor as working electrode in differential pulse voltammetry (DPV). A linear increase in the anodic peak current was observed in the range 5-80 mg L-1 of DCF. The corresponding limit of detection was calculated to be 1.1 mg L-1. The relative standard deviations of the inter- and intra-day analysis of DCF presented by the method were found to be as 2.43% and 2.47%, respectively. The selectivity of the investigated sensor was evaluated by its use for determination of DCF in the binary solutions containing DCF/glucose, DCF/urea and DCF/ascorbic acid. It was shown that the fabricated electrode can be successfully used for analysis of DCF in pharmaceutical and urine samples.