Chemiluminescence method for the determination of piroxicam by the enhancement of the tris‐(4,7‐diphenyl‐1,10‐phenanthrolinedisulphonic acid) ruthenium(II) (RuBPS)–cerium(IV) system and its application

A simple, rapid chemiluminescence (CL) method was described for the determination of piroxicam, a commonly used analgesic agent drug. A strong CL signal was detected when cerium(IV) sulphate was injected into tris‐(4,7‐diphenyl‐1,10‐phenanthrolinedisulphonic acid) ruthenium(II) (RuBPS)–piroxicam solution. The CL signal was proportional to the concentration of piroxicam in the range 2.8 × 10^–8–1.2 × 10^–5 mol/L. The detection limit was 2 × 10^–8 mol/L and the relative standard deviation (RSD) was 3.7% (c = 7.0 × 10^–7 mol/L piroxicam; n = 11). The proposed method was applied to the determination of piroxicam in pharmaceutical preparations in capsules, spiked serum and urine samples with satisfactory results. Copyright © 2008 John Wiley & Sons, Ltd.     

Microemulsion System with Improved Loading of Piroxicam: A Study of Microstructure

Formulation of a new oil-in-water (o/w) microemulsion composed of castor oil/Tween 80/ethanol/phosphate buffer for enhancing the loading capacity of an anti-inflammatory drug piroxicam has been accomplished. The pseudo-ternary phase diagram has been delineated at constant surfactant/cosurfactant ratio (1:2). The internal structure of so created four-component system was elucidated by means of an analysis of isotropic area magnitudes in the phase diagram. Conductivity (σ), kinematic viscosity (k _η ), and surface tension (γ) studies with the variation in Φ _w (weight fraction of aqueous phase) show the occurrence of structural changes from water-in-oil (w/o) microemulsion to oil-in-water (o/w). Along with the solubility and partition studies of piroxicam in microemulsion components, the changes in the microstructure of the microemulsion after incorporation of drug have been evaluated using pH, σ, γ, k _η , and density studies. Piroxicam, a poorly water-soluble drug displayed high solubility (1.0%) in an optimum microemulsion formulation using ethanol (55.0%), Tween 80 (26.5%), castor oil (7.5%), and phosphate buffer (11.0%). The results have shown that the microemulsion remained stable after the incorporation of piroxicam. Fluorescence spectra analysis taking pyrene as fluorescent probe was performed, and the results showed that pyrene was completely solubilized in the oil phases of the bicontinuous microemulsions. The fluorescence spectrum of the model drug piroxicam was used to probe the intramicellar region of nonionic microemulsion. The results showed that the piroxicam was localized in the interfacial film of microemulsion systems more deeply in the palisade layer with ethanol as the cosurfactant.     

Measurement of endogenous and TNFα-mediated H2O2 production in supernatants of SK-N-SH neuroblastoma cells with an enhanced chemiluminescence assay

A sensitive peroxidase-dependent luminol-enhanced chemiluminescence (ECL) assay for determination of hydrogen peroxide (H₂O₂) generation by tumour cells was established. This test system allows determination of H₂O₂ in concentrations as low as 25 pmol (50nmol/L) and yields results which are comparable to those obtained using a less sensitive photometric method and a previously described scopoletin flurorescence assay. After 3h incubation time 10⁴SK-N-SH neuroblastoma cells released 60° 5 pmol H₂O₂ in the supernatant and this level was significantly (p<0.025) increased by about 70% in the presence of 5pmol (100 ng/mL) recombinant tumour necrosis factor α (TNFα). In contrast, H₂O₂ production was slightly reduced by TNFα at a very low concentration of 0.5 fmol (0.01 ng/mL).     

Determination of celecoxib in human plasma and rat microdialysis samples by liquid chromatography tandem mass spectrometry

Methods for the determination of celecoxib in human plasma and rat microdialysis samples using liquid chromatography tandem mass spectrometry are described. Celecoxib and an internal standard were extracted from plasma by solid-phase extraction with C₁₈ cartridges. Thereafter compounds were separated on a short narrow bore RP C₁₈ column (30×2 mm). Microdialysis samples did not require extraction and were injected directly using a narrow bore RP C₁₈ column (70×2 mm). The detection was by a PE Sciex API 3000 mass spectrometer equipped with a turbo ion spray interface. The compounds were detected in the negative ion mode using the mass transitions m/z 380→316 and m/z 366→302 for celecoxib and internal standard, respectively. The assay was validated for human plasma over a concentration range of 0.25–250 ng/ml using 0.2 ml of sample. The assay for microdialysis samples (50 μl) was validated over a concentration range of 0.5–20 ng/ml. The method was utilised to determine pharmacokinetics of celecoxib in human plasma and in rat spinal cord perfusate.     

Simultaneous determination of epirubicin, doxorubicin and their principal metabolites in human plasma by high-performance liquid chromatography and electrochemical detection

A high-performance liquid chromatographic method with electrochemical detection has been developed for the simultaneous determination of epirubicin, 13-S-dihydroepirubicin, doxorubicin and 13-S-dihydrodoxorubicin in human plasma. An aliquot of 200 μl plasma, spiked with internal standard, was extracted by solid-phase extraction using polymeric adsorbent columns. Chromatography was performed using a C₁₈ reversed-phase column with a mobile phase consisting of water–acetonitrile (71:29, v/v) containing 0.05 M Na₂HPO₄ and 0.05% v/v triethylamine adjusted to pH 4.6 with citric acid. Linearity of the method was obtained in the concentration range of 1–500 ng/ml for all the analytes. Analytical recoveries of the analytes ranged from 89 to 93%. The assay can be used for the simultaneous determination of the four analytes, or for epirubicin and its metabolite or doxorubicin and its metabolite, using the other parent drug as an internal standard. The method was applied to analyze human plasma samples from patients treated with epirubicin using doxorubicin as an internal standard.     

Extractionless high-performance liquid chromatographic method for the simultaneous determination of piroxicam and 5′-hydroxypiroxicam in human plasma and urine

A simple and rapid (extractionless) high-performance liquid chromatographic method with UV detection, at 330 nm, was developed for the simultaneous determination of piroxicam and its major metabolite, 5′-hydroxypiroxicam, in human plasma and urine. Acidified plasma and alkali-treated urine samples are used and naproxen is added as internal standard. The separation is performed at 40°C on a C₁₈ Spherisorb column with acetonitrile-0.1 M sodium acetate (33:67, v/v, pH 3.3) as mobile phase. The retention time is 2.2 min for 5′-hydroxypiroxicam, 2.6 min for piroxicam and 3.2 min for naproxen. The detection limit is 0.05 μg/ml using a 100-μl loop.     

Determination of saquinavir in human plasma by high-performance liquid chromatography

We developed and characterized a high-performance liquid chromatography (HPLC) assay for the determination of saquinavir, an HIV protease inhibitor, in human plasma samples. Extraction of plasma samples with diethyl ether resulted in quantitative recovery of both saquinavir and its stereoisomer Ro 31-8533 which was used as an internal standard. The assay was performed isocratically using 5 mM H₂SO₄ (pH 3.5) and acetonitrile (75.5:24.5, v/v) containing 10 mM tetrabutylammonium hydrogen sulfate (TBA) as a mobile phase, a Nucleosil 3C8 column kept at 45°C and UV detection at 240 nm. Using this method, saquinavir and Ro 31-8533 can be separated from endogenous substances, and in the concentration range of 5–110 ng/ml the relative standard deviations for the determination of saquinavir were below 5%. The detection limit of saquinavir in human plasma was 1 ng/ml. The usefulness of the method was demonstrated by quantification of saquinavir in plasma of human subjects treated with 600 mg of saquinavir per os or 12 mg intravenously.     

Determination of a novel indolylpiperazine anti-migraine agent in rat, monkey, mouse and rabbit plasma by high-performance liquid chromatography with electrochemical detection

A specific, accurate, precise and reproducible assay for the quantitation of a novel indolylpiperazine anti-migraine agent (I) in plasma from various animal species is described. The method involves addition of internal standard (I.S.) and 1.0 M sodium carbonate to the plasma sample, vortex-mixing and extraction with ethylene dichloride. The organic layer is then back-extracted in a buffer consisting of 0.1 M tetramethylammonium hydroxide (TMAH), pH 3.0 and 0.1 M (NH₄)₂HPO₄, pH 3.0, in water. The aqueous layer is injected on to a Zorbax cyano analytical column with a mobile phase consisting of acetonitrile, methanol and water (15:5:80, v/v/v) with 0.01 M TMAH, pH 3.0 and 0.01 M (NH₄)₂HPO₄, pH 3.0. The eluate is monitored by electrochemical detection at 0.9 V (guard cell), 0.5 V (detector 1) and 0.8 V (detector 2). The retention times of I and I.S. were 7 and 10 min, respectively. In drug-free control plasma, there were no interfering peaks seen at the retention times of I or I.S. The standard curve was linear over the concentration range of 5–500 ng/ml in rat, monkey, mouse and rabbit plasma. The lower limit of quantitation in all four matrices was 5.0 ng/ml. Within- and between-assay variability of quality control samples was less than 9% relative standard deviation and the predicted concentration of the quality control samples deviated by less than 15% from the nominal concentration. The stability of I was established for up to 36 h in the autosampler tray, up to 10 months in plasma at −20°C and up to 2 h in plasma at room temperature. The assay is validated for determination of I in plasma.     

Determination of flunarizine in rat brain by liquid chromatography–electrospray mass spectrometry

A rapid liquid chromatography–electrospray mass spectrometry (LC–ES-MS) assay for the determination of flunarizine (FZ) in rat brain has been developed. A C₁₈ column and an isocratic elution were employed for the separation. Using post-column split, 64% of the eluent was introduced into the ES-MS system for detection. The [M+H]⁺ (m/z 406) and a fragmented ion (m/z 203) were detected using selected ion monitoring. The linear range of this assay was good, ranging from 0.05 to 5 μM (r²=0.99). The intra- and inter-day precisions showed relative standard deviations ranging from 1.4% to 2.0% and 1.3% to 2.9%, respectively. The application of this newly developed method was demonstrated by examining the pharmacokinetics of FZ in rat brain.     

Determination of piretanide and furosemide in pharmaceuticals and human urine by high-performance liquid chromatography with amperometric detection

A high-performance liquid chromatographic method with electrochemical detection (ED) has been developed for the determination of two diuretics: 4-phenoxy-3-(1-pyrrolidinyl)-5-sulfamoylbenzoic acid (piretanide) and 4-chloro-2-furfurylamino-5-sulfamoylbenzoic acid (furosemide). The chromatographic separation was performed on a μBondapak C₁₈ column with a mobile phase of acetonitrile-water (40:60) containing 5 mM KH₂PO₄/K₂HPO₄ and with a flow-rate of 1 ml/min (69 bar). The temperature was optimized at 30 ± 0.2°C. The amperometric detector equipped with a glassy carbon electrode was operated at + 1200 mV versus Ag/AgCl in the direct current mode. The method was applied to the determination of these compounds in two concentration ranges (ppm and ppb), obtaining a reproducibility in terms of relative standard deviations lower than 1% for within-day and 4% for day-to-day and determination limits of 15 ppb for both compounds. Recoveries greater than 90% were obtained for spiked urine samples, using a liquid-liquid extraction method in the sample clean-up procedure. The LC-ED method was applied to commercially available pharmaceuticals (Seguril, furosemide 40 mg, and Perbilén, piretanide 6 mg) and urine samples obtained from healthy volunteers and hypertensive patients.     

Improved gas chromatographic assay for the simultaneous determination of nitroglycerin and its mono- and dinitrate metabolites

A sensitive, specific capillary gas chromatographic—electron-capture detection method for the simultaneous determination of nitroglycerin (GTN), 1,2- and 1,3-glyceryl dinitrate (1,2-GDN and 1,3-GDN, respectively) and 1- and 2-glyceryl mononitrate (1-GMN and 2-GMN, respectively) is reported. The minimum quantifiable concentration for GTN, GDNs and GMNs is 0.4 ng/ml in plasma, with extraction recoveries for GMNs>76% and for GTN and the GDNs>95%. Over the full range of quantifiable concentrations the inter-run assay precision and accuracy were less than 13 and 11%, respectively, for all five nitrates. Similar intra-run assay precision and accuracy values were found. The method was employed in the preliminary in vitro examination of GTN, GDN and GMN kinetics in human blood. Following addition of GTN to human blood, the ratio of 1,2-GDN to 1,3-GDN maximum concentrations (C_max) was ca. 7:1, reflecting preferential denitration of the GTN molecule at the primary positions, while the C_max ratio for 2-GMN to 1-GMN in this system was ca. 6:1, representing a highly selective if not specific primary denitration of the 1,2-GDN molecule. Following the intravenous administration of 1,2-GDN to five healthy male volunteers, 2-GMN/1-GMN C_max ratios averaged 8.8:1, representing a highly selective but not specific formation of 2-GMN from the 1,2-GDN molecule. The assay will find utility in in vitro studies attempting to address the molecular pharmacology of GTN and its metabolites, and in in vivo clinical pharmacology studies attempting to address the relationship between pharmacokinetics and pharmacodynamics of GTN and its metabolites.     

Direct chemiluminescence determination of penicillin G potassium and a chemometrical optimization approach

A highly selective and simple chemiluminescence (CL) method for determination of penicillin G potassium (PGK) was developed. In the proposed method, CL was elicited from PGK upon its oxidation with H₂O₂. The light emission was enhanced in the presence of N‐cetyl‐N,N,N‐trimethylammonium bromide (CTMAB). An experimental design, central composite design (CCD), was used to realize the optimized variables, including pH, surfactant (CTMAB) and H₂O₂ concentrations. Under optimum condition, the calibration graph was linear in the range 3.3 × 10^?3–3.3 × 10^?1 mmol/L, with a detection limit of 8.8 × 10^?4 mmol/L for PGK. The precision was calculated by analysing samples containing 1.6 × 10^?1 mmol/L PGK (n = 5) and the relative standard deviation (RSD) was 1.40%. The utility of this method was demonstrated by determining PGK in pharmaceutical formulations for injection. The proposed method was validated by a reference method. Copyright © 2011 John Wiley & Sons, Ltd.     

Determination of fumagillin in muscle tissue of rainbow trout using automated ion-pairing liquid chromatography

A high-performance liquid chromatographic assay is described as a routine analytical method for the determination of fumagillin in rainbow trout muscle tissue. Muscle tissue samples (1 g) containing fumagillin were deproteinized with 8 ml of an acetonitrile-water mixture (2:6, v/v). The extracts were purified with a Bond Elut Octyl C₈ cartridge column, washed with a water-methanol mixture (95:5, v/v; 4 ml) and fumagillin was eluted with acetonitrile (1 ml). Analytical separations were performed by reversed-phase HPLC with UV detection at 351 nm under gradient conditions. The mobile phase was acetonitrile-0.005 M tetrabutyl ammonium phosphate in water (pH 7.8). The assay is specific and reproducible within the fumagillin range of 20–1000 ng/g and recovery at 20 ng/g was 69.2%. Sample preparation involves the use of a robotic sample preparation system. Gravimetric validation of all operations enabled Good Laboratory Practices to be observed.     

Determination of flunitrazepam and its metabolites in blood by high-performance liquid chromatography–atmospheric pressure chemical ionization mass spectrometry

A selective assay of flunitrazepam (F) and its metabolites 7-aminoflunitrazepam (7-AF), N-desmethylflunitrazepam (N-DF) and 3-hydroxyflunitrazepam (3-OHF) with liquid chromatography–atmospheric pressure chemical ionization mass spectrometry (LC–APCI-MS, positive ions) is described. The drugs were isolated from serum, blood or urine using a solid-phase extraction procedure previously applied to various drugs of abuse. F-d₃ and 7-AF-d₃ were used as internal standards. The drugs were separated on ODS column in acetonitrile–50 mM ammonium formate buffer, pH 3.0 (45:55, v/v). After analysis of mass spectra taken in full scan mode, a selected-ion monitoring detection was applied with following ions: m/z 284 (7-AF and F), 287 (7-AF-d₃ and F-d₃), 314 (F), 300 (N-DF and 3-OHF), 317 (F-d₃), 330 (3-OHF). The limits of detection were: 0.2 μg/l for F and 7-AF, 1 μg/l for N-DF and 3-OHF. The method was linear in the range 1–500 μg/l, the recoveries ranged from 92 to 99%. The method was applied for determination of F and metabolites in clinical and forensic samples. LC–APCI-MS seems to be a method of choice for these compounds.     

Determination of nadolol diastereomers in dog plasma using chiral derivatization and reversed-phase high-performance liquid chromatography with fluorescence detection

A stereospecific high-performance liquid chromatographic method has been developed for the determination of four diastereomers of nadolol in plasma. After the nadolol diastereomers were extracted from plasma using an Extrelut-1 solid-phase extraction cartridge, they were derivatized with (R)-(−)-1-(1-naphthyl)ethylisocyanate to form urea derivatives. These derivatives were then separated on a YMC-AM-303 ODS column using water—acetonitrile (60:40, v/v). The calibration curves of (SR)-, (RS)-, (SS)- and (RR)-nadolol were linear over the range 2.5–200 ng/ml, and the correlation coefficient (r) of the curves were higher than 0.9991 for each diastereomer. The limit of quantification was 2.5 ng/ml for each diastereomer in plasma. This method was used for a pharmacokinetic study in four dogs after oral administration of nadolol (1 mg/kg). The plasma concentrations of nadolol diastereomers showed no significant differences in C_max, T_max or AUC values. The assay appears to be readily applicable to the study of diastereoselective nadolol pharmacokinetics in animals and humans.     

Determination of propofol in rat whole blood and plasma by high-performance liquid chromatography

A simple, accurate and sensitive high-performance liquid chromatographic method was developed for the determination of propofol, an intravenous anaesthetic agent, in rat whole blood or plasma samples. The method is based on precipitation of the protein in the biological fluid sample and direct injection of the supernatant into an HPLC system involving a C₁₈ reversed-phase column using a methanol-water (70:30) mobile phase delivered at 1 ml/min. Propofol and the internal standard (4-tert.-octylphenol) were quantified using a fluorescence detector set at 276 nm (excitation) and 310 nm (emission). The analyte and internal standard had retention times of 6.3 and 10.5 min, respectively. The limit of quantification for propofol was 50 ng/ml using 100 μl of whole blood or plasma sample. Calibration curves were linear (r²=0.99) over a 1–10 μg/ml concentration range and intra- and inter-day precision were between 4–11%. The assay was applied to the determination of propofol whole blood pharmacokinetics and propofol whole blood to plasma distribution ratios in rats.     

A validated high-performance liquid chromatographic assay for the simultaneous determination of denaverine and its N-monodemethyl metabolite in human plasma

An isocratic reversed-phase high-performance liquid chromatographic method for the simultaneous determination of denaverine and its N-monodemethyl metabolite (MD 6) in human plasma is described. The assay involves the extraction with an n-heptane–2-propanol mixture (9:1, v/v) followed by back extraction into 12.5% (w/w) phosphoric acid. The analytes of interest and the internal standard were separated on a Superspher RP8 column using a mobile phase of acetonitrile–0.12 M NH₄H₂PO₄–tetrahydrofuran (24:17.2:1, v/v), adjusted to pH 3 with 85% (w/w) phosphoric acid. Ultraviolet detection was used at an operational wavelength of 220 nm. The retention times of MD 6, denaverine and the internal standard were 5.1, 6.3 and 10.2 min, respectively. The assay was validated according to international requirements and was found to be specific, accurate and precise with a linear range of 2.5–150 ng/ml for denaverine and MD 6. Extraction recoveries for denaverine and MD 6 ranged from 44 to 49% and from 42 to 47%, respectively. The stability of denaverine and MD 6 in plasma was demonstrated after 24 h storage at room temperature, after three freeze–thaw cycles and after 7 months frozen storage below −20°C. The stability of processed samples in the autosampler at room temperature was confirmed after 24 h storage. The analytical method has been applied to analyses of plasma samples from a pharmacokinetic study in man.     

A comparison of two methods for determining titanium dioxide marker content in broiler digestibility studies

The use of inert markers in broiler diets eliminates the need to quantitatively evaluate feed intake and excreta output to determine diet digestibility, and enables nutrient uptake at specific points along the gastrointestinal tract to be examined. Titanium dioxide (TiO₂) is commonly used for this purpose and measured using a UV-spectrophotometric assay. Two experiments were conducted to observe whether an inductively coupled plasma optical emission spectrophotometer (ICP-OES) assay is able to replace the UV-spectroscopy assay for rapid analysis of TiO₂ in broiler feed and ileal digesta samples. In the first experiment, TiO₂ was added at 5 g/kg to 19 broiler diets. Ross 308 male broilers (n=452) fed these diets were involved in a series of digestion studies to determine ileal digesta recovery of TiO₂. In the second experiment, defined amounts of TiO₂ were added to ileal digesta samples from Ross 308 male broilers (n=176) and TiO₂ recoveries were determined. The feed and ileal samples from both experiments were analysed by both UV-spectroscopy and ICP-OES, and relatedness of the findings from the two assays was determined. Overall relatedness of the two assays was strong for determination of TiO₂ concentration in both the broiler diets and ileal digesta samples (r=0.908 and r=0.884, respectively). Overall recovery of supplemented TiO₂ was 97.62% by the UV-spectroscopy assay and 98.77% by the ICP-OES assay. The ICP-OES assay in this study was as accurate as spectrophotometric determination for the quantification of TiO₂ content. The ICP-OES method can also be used to analyse several elements within one assay, with a single preparation step, and thus the measurement of TiO₂ may be incorporated into the analysis of other minerals. Time and resources dedicated to determining diet digestibility in broilers could be minimised by using the ICP-OES assay to replace the UV-spectroscopy assay when measuring TiO₂ concentration.     

A validated silver‐nanoparticle‐enhanced chemiluminescence method for the determination of citalopram in pharmaceutical preparations and human plasma

A simple and sensitive chemiluminescence (CL) method was developed for the determination of citalopram in pharmaceutical preparations and human plasma. The method is based on the enhancement of the weak CL signal of the luminol–H₂O₂ system. It was found that the CL signal arising from the reaction between alkaline luminol and H₂O₂ was greatly increased by the addition of silver nanoparticles in the presence of citalopram. Prepared silver nanoparticles (AgNPs) were characterized by UV–visible spectroscopy and transmission electron microscopy (TEM). Various experimental parameters affecting CL intensity were studied and optimized for the determination of citalopram. Under optimized experimental conditions, CL intensity was found to be proportional to the concentration of citalopram in the range 40–2500 ng/mL, with a correlation coefficient of 0.9997. The limit of detection (LOD) and limit of quantification (LOQ) of the devised method were 3.78 and 12.62 ng/mL, respectively. Furthermore, the developed method was found to have excellent reproducibility with a relative standard deviation (RSD) of 3.65% (n = 7). Potential interference by common excipients was also studied. The method was validated statistically using recovery studies and was successfully applied to the determination of citalopram in the pure form, in pharmaceutical preparations and in spiked human plasma samples. Percentage recoveries were found to range from 97.71 to 101.99% for the pure form, from 97.84 to 102.78% for pharmaceutical preparations and from 95.65 to 100.35% for spiked human plasma. Copyright © 2013 John Wiley & Sons, Ltd.     

Analysis of methylparaben in cosmetics based on a chemiluminescence H2O2−NaIO4−CNQDs system

In this article, a simple, effective chemiluminescence (CL) method for the detection of methylparaben (MP) in cosmetic samples was developed based on an IO₄^?–H₂O₂–carbon nitrogen quantum dots (CNQDs) system without a separation process. The results indicated that the redox reaction between periodate and hydrogen peroxide released hydroxide radicals and superoxide radical anions in the presence of bicarbonate. These two radicals were responsible for the formation of excited luminophor CNQD* with a maximum wavelength at 480 nm. Due to the competitive reaction with hydroxide radicals, CL intensity was markedly diminished in the presence of MP. The relative standard deviation in the intraday assay was below 5.5% (n = 9), and the detection limit was as low as 0.50 μmol/L. The proposed method allowed for the successful, selective determination of MP in cosmetics.