Approach for Downscaling of Electromembrane Extraction as a Lab on-a-Chip Device Followed by Sensitive Red-Green-Blue Detection.

A design of electromembrane extraction (EME) as a lab on-a-chip device was proposed for the extraction and determination of phenazopyridine as the model analyte. The extraction procedure was accomplished by coupling EME and packing a sorbent. The analyte was extracted under the applied electrical field across a membrane sheet impregnated by nitrophenyl octylether (NPOE) into an acceptor phase. It was followed by the absorption of the analyte on strong cation exchanger as a sorbent. The designed chip contained separate spiral channels for donor and acceptor phases featuring embedded platinum electrodes to enhance extraction efficiency. The selected donor and acceptor phases were 0 mM HCl and 100 mM HCl, respectively. The on-chip electromembrane extraction was carried out under the voltage level of 70 V for 50 min. The analysis was carried out by two modes of a simple red-green-blue (RGB) image analysis tool and a conventional HPLC-UV system. After the absorption of the analyte on the solid phase, its color changed and a digital picture of the sorbent was taken for the RGB analysis. The effective parameters on the performance of the chip device, comprising the EME and solid phase microextraction steps, were distinguished and optimized. The accumulation of the analyte on the solid phase showed excellent sensitivity and a limit of detection (LOD) lower than 1.0 µg L-1 achieved by an image analysis using a smartphone. This device also offered acceptable intra- and interassay RSD% (<10%). The calibration curves were linear within the range of 10-1000 µg L-1 and 30-1000 µg L-1 ( r2 > 0.9969) for HPLC-UV and RGB analysis, respectively. To investigate the applicability of the method in complicated matrixes, urine samples of patients being treated with phenazopyridine were analyzed.

Application of an HPLC Method for Selective Determination of Phenazopyridine Hydrochloride: Theoretical and Practical Investigations.

HPLC method was developed for the selective determination of phenazopyridine hydrochloride (PAP) in the presence of its computationally selected metabolite. Density functional theory was applied as a computational model to study the energy of PAP metabolites, and the results revealed that 2,3,6-triaminopyridine (TAP) is the most stable metabolite. Good resolution and separation of PAP from TAP was achieved using a reversed-phase BDS Hypersil C18 column with a mobile phase consisting of acetonitrile-water (75 + 25, v/v) at flow rate of 1 mL/min and with UV detection at 280 nm. The linear regression analysis data for the calibration plot of PAP showed a good linear relationship over the concentration range of 5-45 µg/mL, with an LOD of 0.773 µg/mL. Moreover, a theoretical investigation of the relationship between the stationary phase and the studied molecules was performed to confirm the experimental results. The proposed method was successfully applied for the selective determination of PAP in pharmaceutical formulation. In addition, the obtained results were statistically compared to a reported method, with no significant differences found between the investigated method and the reported one with respect to accuracy and precision.

Experimental design of membrane sensor for selective determination of phenazopyridine hydrochloride based on computational calculations.

Computational study has been done electronically and geometrically to select the most suitable ionophore to design a novel sensitive and selective electrochemical sensor for phenazopyridine hydrochloride (PAP). This study has revealed that sodium tetraphenylbarate (NaTPB) fits better with PAP than potassium tetrakis (KTClPB). The sensor design is based on the ion pair of PAP with NaTPB using dioctyl phthalate as a plasticizer. Under optimum conditions, the proposed sensor shows the slope of 59.5 mV per concentration decade in the concentration range of 1.0 × 10(-2)-1.0 × 10(-5) M with detection limit 8.5 × 10(-6) M. The sensor exhibits a very good selectivity for PAP with respect to a large number of interfering species as inorganic cations and sugars. The sensor enables track of determining PAP in the presence of its oxidative degradation product 2, 3, 6-Triaminopyridine, which is also its toxic metabolite. The proposed sensor has been successfully applied for the selective determination of PAP in pharmaceutical formulation. Also, the obtained results have been statistically compared to a reported electrochemical method indicating no significant difference between the investigated method and the reported one with respect to accuracy and precision.

Selective pretreatment and determination of phenazopyridine using an imprinted polymer-electrospray ionization ion mobility spectrometry system.

In this research, selective separation and determination of phenazopyridine (PAP) is demonstrated using molecular imprinted polymer (MIP) coupled with electrospray ionization ion mobility spectrometry (ESI-IMS). In the non-covalent approach, selective MIP produced using PAP and methacrylic acid (MAA) as a template molecule and monomer, respectively. The created polymer is utilized as a media for solid-phase extraction (SPE), revealing selective binding properties for the analyte from pharmaceutical and serum samples. A coupled MIP-IMS makes it possible to quantitize PAP in the range of 1-100 ng mL(-1) and with a 0.2 ng mL(-1) detection limit. Furthermore, the MIP selectivity is evaluated by application of some substances with analogous and different molecular structures to that of PAP. This method is successfully applied for the determination of PAP in pharmaceutical and serum samples.

1H, 13C and 15N NMR assignments of phenazopyridine derivatives.

Phenazopyridine hydrochloride (1), a drug in clinical use for many decades, and some derivatives were studied by one- and two-dimensional (1)H, (13)C and (15)N NMR methodology. The assignments, combined with DFT calculations, reveal that the preferred protonation site of the drug is the pyridine ring nitrogen atom. The chemoselective acetylation of phenazopyridine (2) and its influence on the polarization of the azo nitrogen atoms were evidenced by the (15)N NMR spectra. Molecular calculations of the phenazopyridines 2-4 show that the pyridine and phenyl groups are oriented in an antiperiplanar conformation with intramolecular hydrogen bonding between the N-b atom and the C-2 amino group preserving the E-azo stereochemistry.

Exercise pad testing in continent exercisers: reproducibility and correlation with voided volume, pyridium staining, and type of exercise.

This study was undertaken to determine normal values for a modified pad test to be used for testing outcomes of studies addressing therapy for exercise incontinence. Fourteen asymptomatic volunteers who were continent by history completed three similar exercise sessions wearing pre-weighed pads after ingesting phenazopyridine hydrochloride (Pyridium, Parke-Davis, Sandy, UT). The mean pad weight gain for all exercise sessions combined was 3.19 +/- 3.16 g (range 0.1-12.4). The mean area of pyridium staining was 2.66 +/- 3.14 mm. Mean volume voided after the exercise sessions was 193 +/- 108 cc (range 10-625). The Kendall coefficient of concordance used to test the interest reliability was 0.96 for pad weight gain, 0.76 for area of stain, and 0.60 for volume voided. All but one of the subjects had at least one spot of pyridium stain on at least one of the three pads. The increase in pad weight attributable to sweat and vaginal moisture is reproducible in a given individual performing the same exercise routine, but due to the large variation between subjects, we were unable to establish a cut-off value separating continence from incontinence. Adding pyridium did not improve the specificity of the test, despite instructing subjects to milk the urethra and blot before applying the pad.

Evaluation of phenazopyridine hydrochloride as a tool in the diagnosis of premature rupture of the membranes.

This is a prospective study to determine whether a maternal orally administered azo dye, phenazopyridine hydrochloride, would cross into amniotic fluid, and thus be of potential aid in the diagnosis of rupture of the membranes. Based on anecdotal experience, we hypothesized that this compound would cross the placenta and be excreted in the fetal urine, causing discoloration of the amniotic fluid. Ten patients with uncomplicated pregnancies undergoing elective amniocentesis for obstetric indications received an oral dose of 400 mg of phenazopyridine hydrochloride 4 hours prior to the procedure. Amniotic fluid was also available from five control patients who did not receive phenazopyridine hydrochloride. The typical orange-to-red discoloration of the urine was seen in all study patients, indicating ingestion of the dye. None of the ten patients had evidence of the azo dye in their amniotic fluid by visual inspection or by spectrophotometric absorbance. After the amniotic fluid samples were acidified, the presence of the azo dye was visually demonstrable, and spectrophotometry confirmed measurable concentrations (mean +/- SE: 13.08 +/- 0.72 micrograms/ml). We conclude that although phenazopyridine hydrochloride does cross the placenta into the fetal compartment, its presence causes a visual and spectrophotometric change in the color of amniotic fluid only when the normal basic pH of amniotic fluid is acidified.

Room-temperature luminescence analysis of drugs with application to the Toxi-Lab drug identification system.

A method of analyzing drugs on the Toxi-Lab thin-layer chromatographic system by solid substrate room-temperature luminescence is described. Three pairs of unresolved drugs: quinine/cimetidine, caffeine/chlordiazepoxide, and phenazopyridine/lidocaine, were studied as model compounds. Their luminescence characteristics were obtained and calibration curves were found to be linear over two orders of magnitude. The effect of the nonluminescent component of each pair on the determination of the luminescent component was found to be negligible. Statistical F-tests showed that the observed differences in the slopes and intercepts of the calibration curves were due to random errors. The method was evaluated by determining quinine in urine samples at the ng level.

Amperometric determination of phenazopyridine hydrochloride in a flowing stream at the glassy carbon electrode.

A flow-injection method is described for the determination of phenazopyridine hydrochloride, based on electrochemical oxidation at the glassy carbon electrode. The suggested method is highly specific and can be used to determine phenazopyridine HCl in the presence of most drugs commonly found in pharmaceutical dosage forms or administered therapeutically. Applying a constant potential of +950 mV vs Ag/AgCl/3.5M KCl reference electrode, the calibration curve was linear in the 1-30 micrograms/mL range, with minimum detectability of 0.2 ng (signal-to-noise ratio 2). Good accuracy and precision were obtained when the method was applied to some dosage forms containing phenazopyridine HCl. Although automation was not used in this study, an automated system could be incorporated because the method uses the technique of continuous analysis in a flowing stream.

Effect of diffusion layer pH and solubility on the dissolution rate of pharmaceutical bases and their hydrochloride salts. I: Phenazopyridine.

The pH-solubility profile of phenazopyridine as determined by the addition of HCl or NaOH solutions to its aqueous suspension was identical to that of its hydrochloride salt except during phase transition from base to salt. With the addition of HCl to a suspension of the base, the pH dropped to a certain point and then remained constant until a supersaturated solution was formed. Only after a high supersaturation did precipitation of the hydrochloride salt occur. The solubility of the salt decreased at low pH due to a common ion effect. Unlike solubility profiles, the pH-intrinsic dissolution rate profiles of the base and its salt differed greatly. At low pH, the dissolution rate of the hydrochloride salt decreased with an increase in HCl concentration, whereas the dissolution rate of the base increased. The self-buffering action of the base and the increase in solubility, leading to a supersaturation of the diffusion layer was responsible for the increase in its dissolution rate with a lowering of the pH of the medium. Good conformity with the Noyes-Whitney equation was demonstrated when the solubility values under pH conditions such that the diffusion layer thickness approaches zero (Cs,h = 0) were used rather than solubilities under pH conditions of the bulk media (Cs). Supersaturation of the dissolution medium was observed during dissolution of the hydrochloride salt at pH 7.