Sensors based on ruthenium-doped TiO2 nanoparticles loaded into multi-walled carbon nanotubes for the detection of flufenamic acid and mefenamic acid.

Recent advances to utilize two or more nanoparticles for developing novel sensors with superior sensitivity have spurred advanced detection limits even at low concentrations. In this research, a blend of rutheniumdoped TiO2 (Ru-TiO2) nanoparticles and multiwalled carbon nanotubes (MWCNTs) loaded into carbon paste matrix to fabricate a novel Ru-TiO2/MWCNTs-CPE sensor was used for the detection and quantification of flufenamic acid (FFA) and mefenamic acid (MFA) drugs. The surface morphology of Ru-TiO2 was assessed by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), transmission electron microscopy (TEM), X-ray diffraction (XRD) and atomic force microscopy (AFM). Sensitivity and selectivity of the electrode was improved at the Ru-TiO2/MWCNTs modified CPE compared to nascent CPE due to the amazing surface distinctive characteristics of the modifier at pH 5.0. The effect of concentration of the modifier, pH, pre-concentration time, sweep rate and concentration on signal enhancement of FFA and MFA was studied. The square wave voltammetry (SWV) currents are linearly related in the concentration range of 0.01 µM-0.9 µM with the detection limit values of 0.68 nM for FFA and 0.45 nM for MFA, respectively. The developed electrode assembly was used for the quantification of both the drug analytes in human urine samples.

Influence of the composition of monoacyl phosphatidylcholine based microemulsions on the dermal delivery of flufenamic acid.

Although microemulsions are one of the most promising dermal carrier systems, their clinical use is limited due to their skin irritation potential. Therefore, microemulsions based on naturally derived monoacyl phosphatidylcholine (MAPL) were developed. The influence of the water, oil and surfactant content on dermal delivery of flufenamic acid was systematically investigated for the first time. A water-rich microemulsion led to significantly higher in vitro skin penetration of flufenamic acid compared to other microemulsions. The superiority of the water-rich microemulsion over a marketed flufenamic acid containing formulation was additionally confirmed. Differences in drug delivery could be explained by alterations of the microemulsions after application. Evaporation of isopropanol led to crystal-like structures of MAPL on the skin surface from the surfactant- or oleic acid-rich microemulsions. In contrast, the formation of this additional barrier was hindered in case of the water-rich microemulsion. The skin penetration of MAPL was additionally analyzed by combined ATR-FTIR and tape stripping experiments, where MAPL itself penetrated only into the initial layers of the stratum corneum, independent of the microemulsion composition. Since a surfactant must penetrate the skin to cause irritation, MAPL can be presumed as a skin-friendly emulsifier with the ability to stabilize pharmaceutically acceptable microemulsions.

Sensitivity equation for quantitative analysis with multivariate curve resolution-alternating least-squares: theoretical and experimental approach.

A new equation is derived for estimating the sensitivity when the multivariate curve resolution-alternating least-squares (MCR-ALS) method is applied to second-order multivariate calibration data. The validity of the expression is substantiated by extensive Monte Carlo noise addition simulations. The multivariate selectivity can be derived from the new sensitivity expression. Other important figures of merit, such as limit of detection, limit of quantitation, and concentration uncertainty of MCR-ALS quantitative estimations can be easily estimated from the proposed sensitivity expression and the instrumental noise. An experimental example involving the determination of an analyte in the presence of uncalibrated interfering agents is described in detail, involving second-order time-decaying sensitized lanthanide luminescence excitation spectra. The estimated figures of merit are reasonably correlated with the analytical features of the analyzed experimental system.

Assessing the removal of pharmaceuticals and personal care products in a full-scale activated sludge plant.

PURPOSE: This study aimed to investigate the removal mechanisms of pharmaceutical active compounds (PhACs) and musks in a wastewater treatment plant (WWTP). Biological removal and adsorption in the activated sludge tank as well as the effect of UV radiation used for disinfection purposes were considered when performing a mass balance on the WWTP throughout a 2-week sampling campaign. METHODS: Solid-phase extraction (SPE) was carried out to analyse the PhACs in the influent and effluent samples. Ultrasonic solvent extraction was used before SPE for PhACs analysis in sludge samples. PhAC extracts were analysed by LC-MS. Solid-phase microextraction of liquid and sludge samples was used for the analysis of musks, which were detected by GC-MS. The fluxes of the most abundant compounds (13 PhACs and 5 musks) out of 79 compounds studied were used to perform the mass balance on the WWTP. RESULTS: Results show that incomplete removal of diclofenac, the compound that was found in the highest abundance, was observed via biodegradation and adsorption, and that UV photolysis was the main removal mechanism for this compound. The effect of adsorption to the secondary sludge was often negligible for the PhACs, with the exceptions of diclofenac, etofenamate, hydroxyzine and indapamide. However, the musks showed a high level of adsorption to the sludge. UV radiation had an important role in reducing the concentration of some of the target compounds (e.g. diclofenac, ibuprofen, clorazepate, indapamide, enalapril and atenolol) not removed in the activated sludge tank. CONCLUSIONS: The main removal mechanism of PhACs and musks studied in the WWTP was most often biological (45%), followed by adsorption (33%) and by UV radiation (22%). In the majority of the cases, the WWTP achieved >75% removal of the most detected PhACs and musks, with the exception of diclofenac.

Simultaneous determination of non-steroidal anti-inflammatory drugs in river water samples by liquid chromatography-tandem mass spectrometry using molecularly imprinted polymers as a pretreatment column.

A restricted access media-molecularly imprinted polymer (RAM-MIP) for flufenamic acid has been developed for the simultaneous determination of non-steroidal anti-inflammatory drugs (NSAIDs) in river water samples. The RAM-MIP was prepared using 4-vinylpyridine and ethylene glycol dimethacrylate as a functional monomer and cross-linker, respectively, by a multi-step swelling and polymerization method followed by a surface modification technique. The RAM-MIP for flufenamic acid showed excellent molecular recognition abilities for flufenamic acid and mefenamic acid, and moderate molecular recognition abilities for indomethacin, etodolac and ketoprofen. The simultaneous determination of NSAIDs (mefenamic acid, indomethacin, etodolac and ketoprofen) in river water samples was carried out by LC-MS/MS using the RAM-MIP for flufenamic acid as a pretreatment column. The concentrations of mefenamic acid, indomethacin and etodolac in river water samples were determined to be 0.4, 0.7 and 0.3ng/L, respectively, while ketoprofen was below the limit of quantitation.

Validation of a nonaqueous capillary electrophoretic method for the enantiomeric purity determination of R-flurbiprofen using a single-isomer amino cyclodextrin derivative.

Nonaqueous capillary electrophoresis (NACE) was successfully applied to the enantiomeric purity determination of R-flurbiprofen using 6-monodeoxy-6-mono(2-hydroxy)propylamino-beta-cyclodextrin (IPA-beta-CD) as chiral selector. The nonaqueous BGE was made up of 20 mM IPA-beta-CD, 20 mM ammonium camphorsulfonate and 40 mM ammonium acetate in methanol. Flufenamic acid was selected as internal standard. The CE method was carefully optimized in order to prevent the adsorption of the cationic CD onto the capillary wall, and therefore, to avoid loss of peak efficiency and enantioresolution. To achieve this goal, the addition of ammonium camphorsulfonate was found to be necessary. In the selected conditions, the determination of 0.1% of S-flurbiprofen in R-flurbiprofen could be performed using the method of standard additions. The NACE method was then fully validated by applying a novel strategy using accuracy profiles.

Functionalized magnetic nanoparticles for small-molecule isolation, identification, and quantification.

Functionalized magnetic nanoparticles (MNPs) were synthesized to serve as laser desorption/ionization elements as well as solid-phase extraction probes for simultaneous enrichment and detection of small molecules in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. Two laser-absorbing matrices were each conjugated onto MNP to give MNP@matrix which provided high ionization efficiency and background-free detection in MS leading to unambiguous identification of target small molecules in a complex mixture. MNP@matrix was demonstrated to serve as a general matrix-free additive in MALDI-TOF MS analysis of structurally distinct small molecules. Also, MNP@matrix provides a simple, rapid, and reliable quantitative assay for small molecules by mass without either the use of an internal standard or an isotopic labeling tag. Furthermore, the affinity extraction of small molecules from complex biofluid was achieved by probe protein-conjugated MNP@matrix without laborious purification. We demonstrated that a nanoprobe-based assay is a cost-effective, rapid, and accurate platform for robotic screening of small molecules.

Multicommutated flow-through optosensors implemented with photochemically induced fluorescence: determination of flufenamic acid.

This article describes a multicommutated flow injection-solid phase spectroscopy system implemented with photochemically induced fluorescence for the determination of flufenamic acid (FFA). A strongly fluorescent photoproduct is generated when FFA is irradiated online under UV light in a strong sulfuric medium. The photoproduct generated is retained on C(18) silica gel (which fills the detection area of the flow cell) and directly monitored on the active solid support at 258/442 nm (lambda(ex)/lambda(em)). After maximum signal recording, the sensing zone is regenerated by eluting the retained photoproduct with an appropriate H(2)SO(4)/MeOH solution. The sensor, completely automated, is based on the use of three-way solenoid valves conveniently operated by a homemade multicommutation software written in Java language. The system is calibrated at 10 and 60s for sampling time, showing detection limits of 1.28 x 10(-9) and 5.33 x 10(-10) molL(-1) and sampling rates of 38 and 28 h(-1), respectively, with relative standard deviations of 0.9 and 1.2%. The applicability of the method is demonstrated for the determination of FFA in human serum, human urine, and a pharmaceutical preparation without any pre-treatment. Good recovery levels were achieved between 90.5 and 103.7%.

Application of stable isotope labeled glutathione and rapid scanning mass spectrometers in detecting and characterizing reactive metabolites.

The formation of reactive metabolites from a number of compounds was studied in vitro using a mixture of non-labeled and stable isotope labeled glutathione (GSH) as a trapping agent. GSH was labeled by incorporating [1,2-(13)C(2),(15)N]glycine into the tripeptide to give an overall increase of 3 Da over the naturally occurring substance. Detection and characterization of reactive metabolites was greatly facilitated by using the data-dependent scanning features of the linear ion trap mass spectrometers to give complimentary and confirmatory data in a single analytical run. A comparison was made by analyzing the samples simultaneously on a triple-stage quadrupole mass spectrometer operated in the constant neutral loss mode. The compounds studied included 2-acetamidophenol, 3-acetamidophenol, 4-acetamidophenol (acetaminophen), and flufenamic acid. GSH adducts for each of these compounds produced a characteristic pattern of 'twin ions' separated by 3 Da in the mass spectral data. This greatly facilitated the detection and characterization of any GSH-related adducts present in the microsomal extracts. Furthermore, characterization of these adducts was greatly facilitated by the rapid scanning capability of linear ion trap instruments that provided full-scan, MS/MS and MS(3) data in one single analysis. This method of detecting and characterizing reactive metabolites generated in vitro was found to be far superior to any of the existing methods previously employed in this laboratory. The combination of two techniques, stable isotope labeled glutathione and linear ion traps, provided a very sensitive and specific method of identifying compounds capable of producing reactive metabolites in a discovery setting. The complimentary set of mass spectral data (including full-scan, MS/MS and MS(3) mass spectra), obtained rapidly in a single analysis with the linear ion trap instruments, greatly accelerated identification of metabolically bioactivated soft spots on the molecules. This in turn enabled chemists to rapidly design out the potential metabolic liability from the back-up compounds by making appropriate structural modifications.

In situ monitoring of wet granulation using online X-ray powder diffraction.

PURPOSE: Polymorphic transformations during the wet granulation of a metastable polymorph of flufenamic acid were monitored in situ using online X-ray powder diffraction. The resulting data were used in testing a proposed process induced transformation rate model, which allows the extent and occurrence of polymorphic transformations during wet granulation to be controlled by adjusting the granulation time. METHODS: A small-scale, top mixing granulator was designed for compatibility with novel X-ray powder diffraction equipment (available from X-Ray Optical Systems of East Greenbush, NY). RESULTS: The unique polycapillary optic and X-ray source allowed the transformation of the metastable to the stable polymorph to be followed during the granulation. Following a diffraction peak each for the metastable and stable forms demonstrated that polymorphic transformations during the wetting phase of granulation follow the trends predicted by the model. CONCLUSIONS: The advanced online monitoring may allow real-time control of the process by the adjustment of process parameters, such as granulation time, and clearly qualifies as a PAT (process analytical technology).

Determination of diclofenac sodium, flufenamic acid, indomethacin and ketoprofen by LC-APCI-MS.

A sensitive, selective and accurate high-performance liquid chromatography-mass spectrometry (LC-MS) assay for the determination of selected non-steroidal anti-inflammatory drugs (NSAIDs), namely diclofenac sodium (DIC), flufenamic acid (FLU), indomethacin (IND) and ketoprofen (KET), either individually or in mixtures, was developed. The examined drugs were injected onto Shim-pack GLC-CN column and were eluted with a mobile phase consisting of acetonitrile and 20 mM ammonium acetate solution (5:1 v/v)/pH 7.4 at a flow rate l ml min(-1). The mass spectrometer, operated in the single ion monitoring mode, was programmed to admit the negative ions [M-H] at m/z 295.9 (DIC), 280.1 (FLU), 355.8 (IND) and 252.9 (KET), respectively. The calibration curves were linear (r > or = 0.9993) over the concentration range 50-300 ng ml(-1) (FLU, DIC) and 100-500 ng ml(-1) (KET, IND) with detection limits of 0.5-4.0 ng. The mean predicted concentrations for the analytes were in the range -5.9 and 5.2% of the nominal concentrations. Within-day and between-day precision were in the range of 0.8-9.1% of the R.S.D. Mean recovery percentages of the individual compounds from laboratory-made mixtures and pharmaceutical formulations were (99.5-101.5%) and (100.6-102.2%), respectively.

Simultaneous analysis of anthranilic acid derivatives in pharmaceuticals and human urine by high-performance liquid chromatography with isocratic elution.

A high-performance liquid chromatographic (HPLC) method for simultaneous determination of mefenamic acid (MFA), flufenamic acid (FFA) and tolfenamic acid (TFA) is presented for application to pharmaceuticals and human urine. Isocratic reversed-phase HPLC was employed for quantitative analysis using tetra-pentylammonium bromide (TPAB) as an ion-pair reagent. Urine samples were purified by solid-phase extraction using a silica-based strong anion-exchanger, Bond-Elut SAX cartridge. The HPLC assay was carried out using a Wakosil ODS 5C18 column (5 microm, 150x4.6 mm I.D.). The mobile phase consisted of 1.9 g of TPAB dissolved in 1:1 of a mixture of acetic acid-sodium acetate buffer solution, pH 5.0, and acetonitrile (11:9, v/v). The calibration curves of MFA, FFA and TFA showed good linearity in the concentration range of 33-167 microg/ml with a wavelength of 280 nm for pharmaceuticals, and in the low concentration range (1.7-30.1 microg/ml) with a wavelength of 230 nm for biological fluids. The correlation coefficients were better than 0.9999 in all cases. The lower limits of detection (defined as a signal-to-noise ratio of about 3) were approximately 2 ng for MFA, 3.5 ng for FFA and 2.5 ng for TFA. The procedure described here is rapid, simple, selective and is suitable for routine analysis of pharmaceuticals and pharmacokinetic studies in human urine samples.

Capillary isotachophoretic determination of flufenamic, mefenamic, niflumic and tolfenamic acid in pharmaceuticals.

Anionic capillary isotachophoresis (ITP) with conductimetric detection has been used for determining selected non-steroid anti-inflammatory and analgesic drugs of the phenamate group, namely tolfenamic (I), flufenamic (II), mefenamic (III) and niflumic (IV) acid. Initially the pKa values (proton lost) of I-IV were determined as 5.11, 4.91, 5.39 and 4.31, respectively, by the UV spectrophotometry in aqueous 50% (w/w) methanol. The optimised ITP electrolyte system consisted of 10 mM HCl + 20 mM imidazole (pH 7.1) as the leading electrolyte and 10 mM 5,5'-diethylbarbituric acid (pH 7.5) as the terminating electrolyte. The driving and detection currents were 100 microA (for 450 s) and 30 microA, respectively (a single analysis took about 20 min). Under such conditions the effective mobilities of I-IV varied between 23.6 and 24.6 m2 V(-1) s(-1) (evaluated with orotic acid as the mobility standard). The calibration graphs relating the ITP zone length to the concentration of the analytes were rectilinear (r = 0.9987-0.9999) in the range 10-100 mg l(-1) of the drug standard. The R.S.D.s were 0.96-1.55% (n = 6) when determining 50 mg l(-1) of the analytes in pure test solutions. The method has been applied to the assay of the phenamates in six commercial mass-produced pharmaceutical preparations (Mobilisin gel and ointment, Lysalgo capsules, Nifluril cream, Niflugel gel, and Clotam capsules). According to the validation procedure based on the standard addition technique the recoveries were 98.4-104.3% of the drug and the R.S.D. values were 1.25-3.32% (n = 6).

Voltammetric, spectrofluorimetric and spectrophotometric methods to determine flufenamic acid.

Simple, rapid and sensitive voltammetric, spectrofluorimetric and spectrophotometric methods for determination of flufenamic acid (FF) in bulk powder and capsule dosage form are presented. The methods are based on the cyclisation reaction of FF with concentrated sulphuric acid to produce the corresponding acridone derivative. The voltammetric method is based on the adsorptive stripping differential pulse (DP) technique. The acridone derivative is determined over the concentration range of 8-60 ng ml(-1) using adsorptive preconcentration at the hanging mercury drop electrode (HMDE). The lower detection limit was found to be 1.02 ng ml(-1). The fluorimetric and spectrophotometric methods are based on the measurement of the fluorescence intensity at 450 nm (lambda(ex) = 400 nm)and peak-to-peak measurements of the first- (D1) and second-derivative (D2) curves, respectively. Beer's law is obeyed over the concentration ranges of 2-20 ng ml(-1) and 0.2-8.0 microg ml(-1) for the fluorimetric and spectrophotometric measurements, respectively. The three methods were proved to be accurate and reproducible as indicated by a relative standard deviation of <2%.

Determination of flufenamic, meclofenamic and mefenamic acids by capillary electrophoresis using beta-cyclodextrin.

The possibility of separating flufenamic, meclofenamic and mefenamic acids by capillary electrophoresis was studied. The best approach involved combining a suitable pH of the carrier electrolyte (pH 12.0) with the host-guest complexation effects of beta-cyclodextrin. A running buffer consisting of 30 mM phosphate buffer (pH 12.0), 2 mM beta-CD and 10% (v/v) acetonitrile was found to provide a very efficient and stable electrophoresis system for the analysis of fenamic acids by capillary zone electrophoresis. Responses were linear from 0.4 to 40 microg/ml for the three drugs with detection limits of about 0.3 ng/ml. Intra- and inter-day precision values of about 1-2% R.S.D. (n = 11) and 3-4% R.S.D. (n = 30), respectively, were obtained. The method is highly robust and no breakdowns of the current or capillary blockings were observed for several weeks. The general applicability of this rapid CZE procedure (migration times less than 12 min) is demonstrated for several practical samples, including serum, urine and pharmaceuticals.

Preparative chromatography of furosemide 1-O-acyl-glucuronide from urine using micronized amberiite XAD-2 and its application to other 1-O-acyl-glucuronides.

Furosemide 1-O-acyl glucuronide (Fgnd) was extracted from the urine following oral administration of furosemide. The crude Fgnd was applied to micronized Amberlite XAD-2 column (2.5 cm i.d. x 90 cm length, 75-500 microns particle size). The purified Fgnd was identified by mass spectrometry and beta-glucuronidase treatment. This method was also applicable to the purification of glucuronide of tolmetin (nonsteroidal anti-inflammatory drug, NSAID), suggesting that it was applicable to the other NSAIDs, most of which were known to be metabolized to acyl-glucuronides.

Flow-injection spectrofluorimetric determination of flufenamic and mefenamic acid in pharmaceuticals.

Two sensitive and rapid flow-injection (FI) spectrofluorimetric methods are proposed for the determination of flufenamic acid (FF) and mefenamic acid (MF), based on the formation of complexes of these compounds with A1(III) in an ethanolic medium. The calibration graphs resulting from the measurements of the fluorescence at lambda exc = 351 nm and lambda em = 440 nm, and lambdaexc = 355 nm and lambda em = 454 nm for the complexes with FF and MF, respectively, are linear over the range 0.030-1.20 micrograms ml-1 for FF and 0.30-16.1 micrograms ml-1 for MF. The methods have been applied to the determination of these drugs in pharmaceutical preparations.

Synthesis of the cholesteryl ester prodrugs cholesteryl ibuprofen and cholesteryl flufenamate and their formulation into phospholipid microemulsions.

Phospholipid micoremulsions have been suggested as a drug-delivery system for hydrophobic compounds. In this study hydrophobicity was achieved by derivatizing with cholesterol. Cholesteryl ibuprofen (3) and cholesteryl flufenamate (4) were synthesized. 3 was isolated as an amorphous, white solid with a melting range of 114-120 degrees C. 4 was isolated as a crystalline, white solid with a melting range of 145-148 degrees C. The proposed structures of 3 and 4 were supported by IR, NMR, MS, and organic microanalysis. Phospholipid:cholesteryl ester microemulsions were prepared by the addition of a 1-propanol solution of the cholesteryl ester, other lipids, and phospholipid to a rapidly mixing KCl/KBr solution. The hydrophobic phase was modified by the addition of cholesteryl oleate or triolein to study the effect of the fluidity of the hydrophobic core on the formation of the microemulsions. The results indicated that a molar ratio of 75:25 and a total lipid concentration of 60 mg/mL consistently gave microemulsions with a mean size of 100-150 nm. In addition, the formation of eutectic mixtures of 3 and 4 with cholesteryl oleate were determined to be 16% (w/w) for 3 and 12% (w/w) for 4; melting points were 35.2 and 45.2 degrees C, respectively. The solubilities of 3 and 4 in triolein were determined to be 13.2% (w/w) and 11.5% (w/w), respectively. Other investigators have shown that if the core of a phospholipid:cholesteryl estermicroemulsion exists in a liquid state at physiologic temperature, the turnover of the cholesteryl esters from these microemulsions occurs at a faster rate.(ABSTRACT TRUNCATED AT 250 WORDS)

Physical and chemical changes of medicinals in mixtures with adsorbents in the solid state. IV. Study on reduced-pressure mixing for practical use of amorphous mixtures of flufenamic acid.

Flufenamic acid (FFA) was mixed with magnesium aluminum silicate (MAS) at a reduced pressure of about 10 to 50 mmHg employing a commercial mixer for pharmaceutical production. An amorphous state of FFA in the mixture was efficiently achieved with this equipment, and the dissolution of FFA was enhanced in comparison with that of the physical mixture. Effects of the conditions of mixing, such as pressure, temperature and rotating speed, on dissolution of FFA were determined. Through stability tests at 40 degrees C under both dry and humid conditions, no change in dissolution profiles was recognized in a 5% FFA mixture stored under any conditions. On the other hand, decreases in dissolution behavior were observed in 10% and 20% FFA mixtures when they were stored under humid conditions. These results suggested that humidity should be avoided during the storage of amorphous mixtures of FFA with MAS for production purposes.

Physical and chemical changes of medicinals in mixtures with adsorbents in the solid state. II. Application of reduced pressure treatment for the improvement of dissolution of flufenamic acid.

Flufenamic acid (FFA) was mixed with magnesium aluminum silicate (MAS) and stored at 60 degrees C at a reduced pressure of about 2.5 mmHg. After storage, when its concentration was not more than 20%, FFA was observed by X-ray diffraction and polarizing microscopy to be amorphous. The dissolution of FFA was thus enhanced in comparison with that of a freshly prepared mixture. Furthermore, the dissolution curves showed a typical supersaturation pattern, and the supersaturation state continued longer, the higher the pH value of the dissolution medium. Flufenamic acid, in a mixture with MAS, became amorphous more rapidly at reduced pressure than at atmospheric pressure, and therefore the effect of improved dissolution appeared earlier at reduced pressure. Infrared spectral studies suggested that FFA, after storage at a reduced pressure with MAS, was dispersed monomolecularly in an ionic form. The technique of treating crystalline medicinals, that have poor solubility in water, with adsorbent at reduced pressure may be useful for improving their dissolution characteristics.