A composite adsorbent of graphene quantum dots, mesoporous carbon, and molecularly imprinted polymer to extract nonsteroidal anti-inflammatory drugs in milk.

A composite magnetic adsorbent was developed by embedding graphene quantum dots (GQDs), silica-modified magnetite (Fe3O4-SiO2), and mesoporous carbon (MPC) into a molecularly imprinted polymer (GQDs/Fe3O4-SiO2/MPC/MIP). The adsorbent was applied to extract nonsteroidal anti-inflammatory drugs (NSAIDs) in milk. The MIP was formed via a sol-gel copolymerization using flurbiprofen, diflunisal, and mefenamic acid as template molecules, 3-aminopropyltriethoxysilane as a monomer, and tetraethyl orthosilicate as a cross-linker. GQDs and MPC enhanced affinity binding between NSAIDs and the adsorbent through π-π stacking, hydrogen bonding, and hydrophobic interaction. The Fe3O4-SiO2 nanoparticles embedded in the composite adsorbent enabled its rapid isolation from the sample solution. The extracted NSAIDs were quantified by high-performance liquid chromatography and exhibited good linearity from 1.0 to 100.0 µg L-1 for flurbiprofen and 0.5 to 100.0 µg L-1 for diflunisal and mefenamic acid, respectively. The limits of detection ranged from 0.5 to 1.0 µg L-1. Recoveries of NSAIDs from spiked milk samples ranged from 81.4 to 93.7%, with RSDs below 7%. The reproducibility of the fabricated adsorbent was good and in the optimal conditions, the developed adsorbent could be used for up to six extraction-desorption cycles.

A comprehensive spectroscopic study of the polymorphs of diflunisal and their phase transformations.

Understanding phase transitions in pharmaceutical materials is of vital importance for drug manufacturing, processing and storage. In this paper we have carried out comprehensive high-resolution spectroscopic studies on the polymorphs of the non-steroidal anti-inflammatory drug diflunisal that has four known polymorphs, forms I-IV (FI-FIV), three of which have known crystal structures. Phase transformations during milling, heating, melt-quenching and exposure to high relative humidity were investigated using Raman and terahertz spectroscopy in combination with differential scanning calorimetry and X-ray powder diffraction. The observed phase transformations indicate the stability order FIII>FI>FII, FIV. Furthermore, crystallization experiments from the gas phase and from solution by fast evaporation of different solvents were carried out. Fast evaporation of an ethanolic solution below 70°C was identified as a reliable and convenient method to obtain the somewhat elusive FII in bulk quantities.

Transthyretin stabilization by iododiflunisal promotes amyloid-ß peptide clearance, decreases its deposition, and ameliorates cognitive deficits in an Alzheimer's disease mouse model.

Alzheimer's disease (AD) is the most common form of dementia and now represents 50-70% of total dementia cases. Over the last two decades, transthyretin (TTR) has been associated with AD and, very recently, a novel concept of TTR stability has been established in vitro as a key factor in TTR/amyloid-ß (Aß) interaction. Small compounds, TTR stabilizers (usually non-steroid anti-inflammatory drugs), bind to the thyroxine (T4) central binding channel, increasing TTR tetrameric stability and TTR/Aß interaction. In this work, we evaluated in vivo the effects of one of the TTR stabilizers identified as improving TTR/Aß interaction, iododiflunisal (IDIF), in Aß deposition and other AD features, using AßPPswe/PS1A246E transgenic mice, either carrying two or just one copy of the TTR gene (AD/TTR+/+ or AD/TTR+/-, respectively), available and characterized in our laboratory. The results showed that IDIF administered orally bound TTR in plasma and stabilized the protein, as assessed by T4 displacement assays, and was able to enter the brain as revealed by mass spectrometry analysis of cerebrospinal fluid. TTR levels, both in plasma and cerebrospinal fluid, were not altered. In AD/TTR+/- mice, IDIF administration resulted not only in decreased brain Aß levels and deposition but also in improved cognitive function associated with the AD-like neuropathology in this mouse model, although no improvements were detectable in the AD/TTR+/+ animals. Further, in AD/TTR+/- mice, Aß levels were reduced in plasma suggesting TTR promoted Aß clearance from the brain and from the periphery. Taken together, these results strengthen the importance of TTR stability in the design of therapeutic drugs, highlighting the capacity of IDIF to be used in AD treatment to prevent and to slow the progression of the disease.

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.

Simultaneous determination of the two non-steroidal anti-inflammatory drugs ; diflunisal and naproxen in their tablets by chemometric spectrophotometry and HPLC.

Chemometric spectrophotometry and HPLC were applied to the simultaneous determination of the two non-steroidal anti-inflammatory drugs; diflunisal (I) and naproxen (II). The applied chemometric techniques are multivariate methods including classical least squares (CLS), principal component regression (PCR) and partial least squares (PLS); and the second derivative of the ratio spectra ((2)D(r)) method. To develop the multivariate methods, the UV absorption spectra of the standard solutions of the training and validation sets in methanol were recorded in the range of 242-274 nm at 2 nm intervals. The specificity of the studied multivariate methods has been tested. In the (2)D(r) method, analytical signals at 235 and 259 nm were selected for the determination of (I) and (II), respectively. The HPLC method depends on reversed-phase separation using C18 column. The mobile phase consists of a mixture of acetonitrile - acetate buffer (pH 4.2; 50 mM) (60:40, v/v). The UV detector was set at 255 nm. The developed methods were validated and successfully applied to the simultaneous determination of (I) and (II) in their tablets. The assay results obtained using the chemometric methods were statistically compared to those of the HPLC method and good agreement was observed.

Analysis of diflunisal by electrochemical methods.

A new differential pulse polarographic (DPP) and differential pulse adsorptive stripping voltammetric (DPAdSV) methods for the electrochemical behavior and quantitative determination of diflunisal were described. In these voltammetric methods, the peak potential of diflunisal was found as -0.31 V (vs. Ag/AgCI) with selected Britton--Robinson buffer (BR, pH 7.8) as a supporting electrolyte. The variation of the peak current with the concentration of diflunisal were linear in the 9.0--40.0 and 4.0--30.0 microg ml(-1) concentration ranges for DPP and DPAdSV methods, respectively. The limits of detection (LOD) were found as 5.0 and 0.1 microg ml(-1) for DPP and DPAdSV methods, respectively. The developed methods were validated by evaluation of the validation parameters. The characteristics of the peak current of diflunisal were examined in detail and the results proved that the peak current has an adsorption characteristic. The developed methods were proposed for rapid determination of diflunisal in commercial tablets. The recovery studies showed that developed assays had a good accuracy and precision with mean recoveries 99.92 and 100.02% and mean variation coefficients 0.29 and 0.24% in DPP and DPAdSV methods, respectively.

Characterization of the complexation of diflunisal with hydroxypropyl-beta-cyclodextrin.

The equilibrium dialysis method was applied to the determination of drug cyclodextrin stability constants using diflunisal and 2-hydroxypropyl-beta-cyclodextrin (HPBCD) as a model system. Analysis of the data showed the existence of a linear Scatchard plot, indicative of the formation of a 1:1 diflunisal:HPBCD complex. The mean complexation constant (Kc) +/- S.D. was 3,892 +/- 360 M(-1). The stoichiometry of the complex was verified using the appropriate mass action law equation. The diflunisal:HPBCD complex was also investigated using titration microcalorimetry. A Kc of 3,394 M(-1) was obtained together with an enthalpy change (deltaH) of -20.76 kJ/mol(-1). The Kc values obtained here using the equilibrium dialysis and microcalorimetric methods were comparable to one reported previously using a potentiometric method (5,564 +/- 56 M(-1)).

Simultaneous determination of some multicomponent dosage forms by quantitative thin layer chromatography densitometric method.

In this study, simultaneous determination of naproxen with diflunisal (mixture I), paracetamol with chlorzoxazone (mixture II) and chlorphenoxamine hydrochloride with 8-chlorotheophylline and caffeine (mixture III) in multicomponent mixtures was conducted by a thin layer chromatography densitometric method. The mobile phase ethyl acetate: methanol: ammonia 25% (85:15:5 v/v) was used for the separation of the components of mixtures (I) and (II) with Rf values of 0.16 for naproxen, 0.4 for diflunisal, 0.77 for paracetamol and 0.32 for chlorzoxazone. Efficient separation of the components of mixture (III) was attained using ethyl acetate as mobile phase with Rf values of 0.12, 0.62 and 0.42 for chlorphenoxamine hydrochloride, 8-chlorotheophylline and caffeine, respectively. Linearity ranges, mean recoveries and relative standard deviations in calibration graphs of the proposed method were calculated. The method has been successfully applied to pharmaceutical formulations, sugar-coated tablets, capsules and suppositories. The results obtained were statistically compared with those obtained by applying the reported alternate methods.

Studies on the reactivity of acyl glucuronides--VIII. Generation of an antiserum for the detection of diflunisal-modified proteins in diflunisal-dosed rats.

Acyl glucuronide metabolites of carboxylic drugs such as the salicylate derivative diflunisal (DF) have been shown to react with proteins to produce covalent adducts. To aid in the study of the formation and distribution of these adducts in both humans and rats, we raised an antiserum against human serum albumin modified by covalent attachment of DF via an amide bond, using a carbodiimide reagent. This antiserum had wide reactivity, reacting with all types of DF-modified proteins tested and with free DF (albeit at a lower affinity). It did not cross-react with other salicylates or other non-steroidal anti-inflammatory drugs. The antiserum has been used in immunoblotting to detect proteins covalently modified by DF in the plasma and livers of rats treated with the drug for 7 days. Although some cross-reactivity was apparent on the blots, a series of DF-modified proteins was found in cytosolic, mitochondrial and mixed membrane fractions of hepatocytes, with molecular weights ranging from 28 to 130 kDa.

Reactivity considerations in the analysis of glucuronide and sulfate conjugates of diflunisal.

Reactivity of glucuronide and sulfate conjugates was taken into account in development of a simple isocratic HPLC method for direct assay of diflunisal (DF) and its acyl glucuronide (DAG), phenolic glucuronide (DPG), and sulfate (DS) conjugates. Whereas DPG was stable over the pH range 0-9, DAG was highly labile at neutral to slightly alkaline pH, undergoing rearrangement (isomerisation via acyl migration), hydrolysis, and in the presence of methanol, transesterification to DF methyl ester. The 2-, 3-, and 4-O-acyl positional isomers of DAG appeared as three pairs of peaks. Interconversion between partners of each pair occurred even under acidic conditions inhibitory to acyl migration, implicating mutarotation. DS was stable at neural to slightly alkaline pH, but underwent hydrolysis under relatively strongly acidic conditions. However, this hydrolysis was remarkably catalyzed (e.g., by 1,000-fold) in the presence of solvents (i.e., solvolysis) such as diethyl ether and ethyl acetate. DS (an acid) could not be extracted from aqueous solution because of this acidic solvolysis. Suitable conditions for simultaneous direct analysis (nonextractive, nonconcentrative) of DF and its reactive (DAG and DS) and unreactive (DPG) conjugates were achieved by working at pH of approximately 4.5. The procedure thus developed is suitable for plasma, urine, and bile samples, and has revealed the presence of new, as yet unidentified, metabolites of DF.

Reversed-phase high-performance liquid chromatographic assay for the simultaneous determination of diflunisal and its glucuronides in serum and urine. Rearrangement of the 1-O-acylglucuronide.

A reversed-phase ion-pair high-performance liquid chromatographic assay for the simultaneous determination of diflunisal and its ester and ether glucuronide in urine and serum has been developed. The determination of the ester glucuronide in serum has not been previously reported. The genuine glucuronide conjugates isolated from urine were used as standards. The ester glucuronide is found to be unstable, especially in neutral and basic solutions, and special precautions therefore have to be taken during sampling and sample treatment. Nine rearrangement/degradation products of the ester glucuronide were detected.

High-performance liquid chromatographic analysis of diflunisal in plasma and urine: application to pharmacokinetic studies in two normal volunteers.

A high-performance liquid chromatographic (HPLC) assay with fluorescence detection has been developed for the determination of diflunisal in plasma and urine. The plasma or urine, containing naproxen as the internal standard, was extracted with ether-hexane (1:1). The samples were analyzed on a microparticulate column, and the compounds were eluted using a mobile phase of 0.05 M phosphate buffer (pH 3) and methanol. Plasma samples were analyzed from two healthy male subjects who received a 250- and 750-mg oral dose of diflunisal 3 weeks apart. The data were analyzed according to a two-compartment open model. There was a disproportionate increase in the area under the plasma concentration-time curves (AUC 750 mg/AUC 250 mg was 3.84 for subject A and 4.22 for subject B) and a reduction in plasma clearance after the 750-mg dose of diflunisal. These data suggest that the kinetics of diflunisal may be dose dependent.