Fast determination of propranolol in urine and pharmaceutical preparations by stopped-flow and micellar-stabilized room-temperature phosphorescence: validation of the method

The stopped-flow mixing technique has been used to study the kinetic determination of propranolol by means of micellar-stabilized room-temperature phosphorescence. This mixing system diminishes the time required for the deoxygenation of micellar medium by sodium sulfite, allowing a kinetic curve that levels off within only 7s to be obtained. The phosphorescence enhancers thallium (I) nitrate, sodium dodecyl sulfate, and sodium sulfite were optimized to obtain maximum sensitivity and selectivity. A pH value of 6.54 was selected as adequate for phosphorescence development. The kinetic curves of propranolol phosphorescence were scanned at lambda(ex)=290 nm and lambda(em)=524 nm. The calibration graphs were linear for the concentration range from 25 to 400 ng mL(-1). The phosphorescence lifetime of propranolol is approximately 1210 micros. The detection limit calculated as proposed clayton was 13.53 ng mL(-1) and by applying the error propagation theory, the detection limit was 8.37 ng mL(-1). The repeatability was studied using 10 solutions of 200 ng mL(-1) of propranolol; if error propagation theory is assumed, the relative error is 1.94%. The standard deviation for a replicate sample was 4.0 ng mL(-1). This method was successfully applied to the determination of propranolol in commercial formulations and in urine. Suitable recovery values were obtained.     

Simplex optimization of the variables affecting the micelle-stabilized room temperature phosphorescence of 6-methoxy-2-naphthylacetic acid and its kinetic determination in human urine

This article reports the kinetic determination of 6-methoxy-2-naphthylacetic acid (6-MNA), the major metabolite of nabumetone, from micelle-stabilized room temperature phosphorescence (MS-RTP) measurements made by using the stopped-flow mixing technique. This methodology allows one to determine analytes in complex matrices without the need for a tedious separation process. It also shortens analysis times substantially. The proposed method uses simplex methodology to optimize the chemical and instrumental variables affecting the phosphorescence. It was applied to the determination of 6-MNA in human urine. The maximum phosphorescence signal is obtained within only 10 s after the sample is prepared. The maximum slope of the kinetic curve, which corresponds to the maximum rate of the phosphorescence development, is measured at lambda(ex)=273 nm and lambda(em)=516 nm. Least-squares regression was used to fit experimental data, and the detection limit, repeatability, and standard deviation for replicate samples were determined.     

Flow-injection chemiluminescence determination of catecholamines based on their enhancing effects on the luminol-potassium periodate system.

A rapid and sensitive chemiluminescence (CL) method using flow injection analysis is described for the determination of four catecholamines, dopamine, adrenaline, isoprenaline and noradrenaline, based on their greatly enhancing effects on the CL reaction of luminol-potassium periodate in basic solutions. The optimized chemical conditions for the chemiluminescence reaction were 1.0 x 10(-4) mol/L luminol and 1.0 x 10(-5) mol/L potassium periodate in 0.2 mol/L sodium hydroxide (NaOH). Under the optimized conditions, the calibration graphs relating the CL signal intensity (peak height) to the concentration of the analytes were curvilinear and they were suitable for determining dopamine, adrenaline, isoprenaline, and noradrenaline in the range 0.1-10 ng/mL, 0.1-100 ng/mL, 1-100 ng/mL and 5-50 ng/mL, respectively, with the relative standard deviations of 0.8-1.7%. The detection limits of the method are 0.02 ng/mL for dopamine, 0.01 ng/mL for adrenaline, 0.1 ng/mL for isoprenaline and 2.0 ng/mL for noradrenaline. The sampling frequency was calculated to be about 60/h. The selectivity of the method was good, because a series of common ions or excipients, such as K(+), Ba(2+), CO(3)(2-), NO(3)(-), SO(4)(2-), PO(4)(3-), sodium citrate, sodium bisulphite, oxidate dopamine, starch, lactose, carbamide and gelatin, could not produce interference when their concentrations were 1000-fold than those of dopamine. The present method was successfully applied to the determination of the four catecholamines in pharmaceutical injections.     

Measurement of amoxicillin in plasma and gastric samples using high-performance liquid chromatography with fluorimetric detection

A rapid, selective and sensitive HPLC assay has been developed for the routine analysis of amoxicillin in rat plasma, gastric juice aspirate and gastric tissue which is applicable to low concentrations of amoxicillin (<1 microg mL(-1)) or small sample volumes. Amoxicillin was converted, via an internal rearrangement, to form a fluorescent product which was subsequently recovered using liquid-liquid extraction. A Kromasil ODS 3 microm (150 x 3.2 mm I.D.) column was maintained at 40 degrees C and used with a mobile phase consisting of methanol-water (55:45, v/v). Fluorimetric detection was at an lambda(ex) of 365 nm and an lambda(em) of 445 nm. The limits of quantitation for amoxicillin were 0.1 microg mL(-1) for gastric juice aspirate (500 microL), 0.5 microg mL(-1) for plasma (50 microL) and 0.075 microg g(-1) for gastric tissue (250 mg). The method was linear up to at least 15 microg mL(-1) in gastric juice aspirate, up to 200 microg mL(-1) in plasma and up to 100 microg g(-1) in gastric tissue, with inter- and intra-day RSDs being less than 19%. The assay has been applied to the measurement of amoxicillin in rat plasma, gastric juice aspirate and gastric tissue for pharmacokinetic studies in individual rats.     

Quantification of meropenem in plasma and cerebrospinal fluid by micellar electrokinetic capillary chromatography and application in bacterial meningitis patients

A high-performance micellar electrokinetic capillary chromatography (MEKC) has been demonstrated for the determination of meropenem in human plasma and in cerebrospinal fluid (CSF) and application in meningitis patients after intravenous (IV) administration. Plasma sample was pretreated by means of solid-phase extraction (SPE) on C(18) cartridge and CSF sample was by direct injection without sample pretreatment, with subsequent quantitation by MEKC. The separation of meropenem was carried out in an untreated fused-silica capillary (40.2 cm x 50 microm I.D., effective length 30 cm) and was performed at 25 degrees C using a background electrolyte consisting of Tris buffer (40 mM, pH 8.0) solution with sodium dodecyl sulfate (SDS) as the running buffer and on-column detection at 300 nm. Several parameters affecting the separation and sensitivity of the drug were studied, including pH, the concentrations of Tris buffer and surfactant. Using cefotaxime as an internal standard (IS), the linear ranges of the method for the determination of meropenem in plasma and in CSF were all over 0.5-50 microg/mL; the detection limits (signal-to-noise ratio=3) of meropenem in plasma and in CSF were 0.2 microg/mL and 0.3 microg/mL, respectively.     

Determination of sulphite using an immobilized enzyme with flow injection chemiluminescence detection

A ?ow injection method is reported for the determination of sulphite‐based on chemiluminescent detection. Hydro‐gen peroxide is produced from sulphite using on‐line covalently bound immobilized sulphite oxidase packed in a mini‐column, which was mixed downstream and detected via cobalt(II)‐catalysed chemiluminescent oxidation of luminol. The limit of detection (2 × standard deviation of the blank) was 1 × 10^?3 mmol/L with sample throughput 60 h^?1. The calibration data was linear over the range of 0.2–1.0 mmol/L with relative standard deviation (n = 4) in the range 0.9–2.0%. Copyright © 2004 John Wiley & Sons, Ltd.     

Fluorescence behavior of tryptophan residues of bovine and human serum albumins in ionic surfactant solutions: A comparative study of the two and one tryptophan(s) of bovine and human albumins

The fluorescence behavior of two tryptophans (Trp-134, Trp-213) in bovine serum albumin (BSA) and a single tryptophan (Trp-214) in human serum albumin (HSA) was examined. The maximum emission wavelength (_max) was 340.0 nm for both proteins. In a solution of sodium dodecyl sulfate (SDS), the _max of BSA abruptly shifted to 332 nm at 1 mM SDS and then reversed to 334 nm at 3 mM SDS. The _max of HSA gradually shifted to 330 nm below 3 mM SDS, although it returned to 338 nm at 10 mM SDS. In contrast to this, in a solution of dodecyltrimethylammonium bromide, the _max positions of BSA and HSA gradually shifted to 334.0 and 331.5 nm, respectively. Differences in the fluorescence behavior of the proteins are attributed to the fact that Trp-134 exists only in BSA, with the assumption that Trp-213 of BSA behaves the same as Trp-214 of HSA. The Trp-134 behavior appears to relate to the disruption of the helical structure in the SDS solution.     

Capillary electrophoresis enantioselective separation of vigabatrin enantiomers by precolumn derivatization with dehydroabietylisothiocyante and UV-vis detection

A simple and reliable capillary electrophoresis (CE) method with UV-vis detection is presented for the enantioselective separation and determination of vigabatrin enantiomers. Dehydroabietylisothiocyante (DHAIC), a novel chiral derivatizing reagent, was used for precolumn derivatization of vigabatrin enantiomers. Optimal separation was obtained with a running buffer consisting of 50 mM Na2HPO4 (pH 9.0), 17 mM sodium dodecyl sulfate (SDS) and 25% acetonitrile. The enantiomeric separation of vigabatrin derivatives was achieved within 25 min, and the resolution was found to be 2.1. Detection was followed by direct UV absorptiometric measurements at 202 nm. A calibration curve ranging from 0.3 to 6.0 microg/ml was shown to be linear, and the limit of detection was 0.15 microg/ml. The developed method has been applied to the determination of vigabatrin enantiomers spiked in human plasma, no interferences were found from endogenous amino acids.     

Simultaneous direct determination of amiloride and triamterene in urine using isopotential fluorometry

A method for the simultaneous fluorometric determination of two diuretics in urine is proposed. The combination of matrix isopotential synchronous fluorescence (MISF) and first derivative techniques provides good analytical results. MISF spectra are obtained by calculating the isopotential trajectory in the three-dimensional fluorescence spectrum for a urine solution. In the spectral contour, the trajectory is taken to be the portion of the line that passes by the fluorescence maxima of both diuretics (lambda(ex) = 365 and lambda(em) = 413 nm for amiloride and lambda(ex) = 365 and lambda(em) = 437 nm for triamterene). Because contour lines connect points of identical intensity and the trajectory is part of a contour line, it is called "isopotential." Analyses was carried out in a 1/1 (v/v) ethanol/water mixture, using an apparent pH of 6.3 provided by 0.01 M sodium/citrate citric acid buffer. Urine samples are diluted 50 times and provide linear calibration plots at amiloride and triamterene concentrations up to 320 and 100 ng mL(-1), respectively. The goodness of the analytical signal was checked by using variance analysis. Signals recorded throughout the calibration range were subjected to three calibrations per each analyte, both in the absence and in the presence of variable amounts of the other analyte. Differences between individual calibrations and slopes were compared with those within individual calibrations. Based on the results, triamterene and amiloride can be accurately quantified in the presence of each other. The limit of detection calculated according to Clayton who uses error propagation throughout the calibration curve and a noncentralized security factor was 16.8 and 2.4 ng mL(-1) for amiloride and triamterene, respectively.     

Resonance Rayleigh scattering method for the determination of chitosan with some anionic surfactants

In weak acidic buffer medium, chitosan binding with an anionic surfactant, such as sodium dodecyl benzene sulphonate (SDBS), sodium lauryl sulphate (SLS) or sodium dodecyl sulphonate (SDS), can result in a significant enhancement of resonance Rayleigh scattering (RRS) intensity. The results showed that under optimum conditions the enhanced RRS intensity is proportional to the concentration of chitosan in the range 0.10–20.0 µg/mL for SDBS, 0.27–15.0 µg/mL for SLS and 0.20–15.0 µg/mL for SDS. Among these, the sensitivity of SDBS is the highest and its detection limit for chitosan is 29 ng/mL, while those of SLS and SDS are 83 and 61 ng/mL, respectively. The method has good selectivity and was applied to the determination of trace amounts of chitosan in practical samples with satisfactory results. Therefore, a simple and convenient method with high sensitivity and selectivity for the determination of chitosan was established. Copyright © 2008 John Wiley & Sons, Ltd.     

Stereospecific analysis of sakuranetin by high-performance liquid chromatography: pharmacokinetic and botanical applications

A stereospecific method for analysis of sakuranetin was developed. Separation was accomplished using a Chiralpak AD-RH column with UV (ultraviolet) detection at 288 nm. The stereospecific linear calibration curves ranged from 0.5 to 100 microg/mL. The mean extraction efficiency was >98%. Precision of the assay was <12% (relative standard deviation (R.S.D.)%), and within 10% at the limit of quantitation (0.5 microg/mL). Bias of the assay was lower than 10%, and within 5% at the limit of quantitation. The assay was applied successfully to pharmacokinetic quantification in rats, and the stereospecific quantification in oranges, grapefruit juice, and matico (Piper aduncum L.).     

Photochemical properties of Yt base in aqueous solution.

Photoreactivity of Yt base [I] has been studied in aqueous solution [pH approximately 6] saturated with oxygen. Two photoproducts (II,III], resulting from irradiation at lambda = 253.7 nm and lambda greater than or equal to 290 nm, were isolated and their structures determined. The quantum yield for Yt base disappearance [zeta dis] is 0.002 [lambda = 313 nm]. It was shown that dye-sensitized photooxidation of Yt base in aqueous solution occurs according to a Type I mechanism, as well as with participation of singlet state oxygen. Quantum yields, fluorescence decay times and phosphorescence of Yt base have been also determined.     

Estimation of p-coumaric acid as metabolite of E-6-O-p-coumaroyl scandoside methyl ester in rat plasma by HPLC and its application to a pharmacokinetic study

A rapid and simple high-performance liquid chromatographic (HPLC) method has been developed for the determination of p-coumaric acid in rat plasma and applied to a pharmacokinetic study in rats after administration of a prodrug, E-6-O-p-coumaroyl scandoside methyl ester, isolated from Hedyotis diffusa (Willd.). Sample preparation involved protein precipitation with acetonitrile. The supernatant was then injected onto a Diamonsil C(18) column (250 mm x 4.6mm i.d., 5 microm). The mobile phase consisted of acetonitrile-water (21:79, v/v) with 1% glacial acetic acid. The UV detector was set at 310 nm. The lower limit of quantification of p-coumaric acid in rat plasma was 0.02 microg/mL. The calibration curves were linear over the concentration range 0.02-5 microg/mL with correlations greater than 0.999. The assay procedure was applied to the study of the metabolite pharmacokinetics of E-6-O-p-coumaroyl scandoside methyl ester in rat.     

A novel HPLC fluorescence method for the quantification of methylphenidate in human plasma

A number of analytical methods have been established to quantify methylphenidate (MPH). However, to date no HPLC methods are applicable to human pharmacokinetic studies without the use of mass spectrometry (MS) detection. We developed a sensitive and reliable HPLC-fluorescence method for the determination of MPH in human plasma using 4-(4,5-diphenyl-1H-imidazol-2-yl) benzoyl chloride (DIB-Cl) as the derivatizing agent. An established GC-MS method was adopted in this study as a comparator assay. MPH was derivatized using DIB-Cl, and separated isocratically on a C18 column using a HPLC system with fluorescence detection (lambda(ex)=330 nm, lambda(em)=460 nm). The lower limit of quantification was found to be 1 ng/mL. A linear calibration curve was obtained over the concentrations ranging from 1 ng/mL to 80 ng/mL (r=0.998). The relative standard deviations of intra-day and inter-day variations were 相似文献   

菓菠萝蛋白酶的分子构象与活力变化的研究

发现CBZ-Lys·pNP能有效地被菓菠萝蛋白酶(Fruit Bromelain E.C.3.4.22.5)作用,测得Km为4.167×10~(-4)mol/L,k_(cat)为742min~(-1)。以荧光和紫外差示光谱为监测手段,对酶分子构象变化进行研究。酶的荧光强度随胍浓度增大而逐渐下降,4mol/L胍变性时,发射峰自332nm红移到353nm,并在310nm处出现新的发射峰。酶的荧光强度都因SDS存在而下降,SDS浓度大于3.47mmol/L有所回升,并出现红移,同时在315nm处出现新的发射肩;紫外差示光谱显示在236nm有一个较显著的员峰,此峰与β-螺旋结构变化有关,278、286和295nm出现三个负峰,260nm有较小正峰,说明酶分子中Tyr、Trp和Phe的微环境发生了明显的变化。测定酶在不同浓度胍和SDS中的变性和失活速度常数,对酶构象变化及催化活力的关系作了比较研究,酶的失活速度均大于变性速度。     

Stereoselective RP-HPLC determination of esmolol enantiomers in human plasma after pre-column derivatization

A stereoselective reversed-phase HPLC assay to determine S-(-) and R-(+) enantiomers of esmolol in human plasma was developed. The method involved liquid-liquid extraction of esmolol from human plasma, using S-(-)-propranolol as the internal standard, and employed 2,3,4,6-tetra-O-acetyl-beta-d-glucopyranosyl isothiocyanate as a pre-column chiral derivatization reagent. The derivatized products were separated on a 5-microm reversed-phase C18 column with a mixture of acetonitrile/0.02 mol/L phosphate buffer (pH 4.5) (55:45, v/v) as mobile phase. The detection of esmolol derivatives was made at lambda=224 nm with UV detector. The assay was linear from 0.035 to 12 microg/ml for each enantiomer. The analytical method afforded average recoveries of 94.8% and 95.5% for S-(-)- and R-(+)-esmolol, respectively. For each enantiomer, the limit of detection was 0.003 microg/ml and the limit of quantification for the method was 0.035 microg/ml (RSD<14%). The reproducibility of the assay was satisfactory.     

The tissue distribution in mice and pharmacokinetics in rabbits of oxaliplatin liposome

A rapid, sensitive, and simple high-performance liquid chromatographic (HPLC) method with an ultraviolet detector (UV) has been developed for the determination of oxaliplatin in the plasma of rabbits and tissues of mice. The sample preparation was carried out by complexation with 0.5?mL of DETC (diethyl-dithiocarbamate) solution and extracted by ether and chloroform. Then, 20?μL of supernatant was injected into the HPLC system with 0.25?mol/L of sodium chloride solution and methanol (30:70 v/v) as the mobile phase at a flow rate of 1.0?mL/min. Separation was performed with a C₁₈ column at 25°C. The peak was detected at 254?nm. The calibration curve was linear (R²?≥?0.9995) in the concentration range of 0.1~200?μg/mL in plasma and tissues. The intra- and interday variation coefficients were not more than 2.61 and 3.83%, respectively. The limit of detection was 20?ng/mL. The mean recoveries of oxaliplatin were ranged from 97.83 to 104.17% in plasma and tissues. The present method has been successfully applied to the pharmacokinetic study of oxaliplatin liposome in mice and rabbits.     

One-step method for plasma determination of ibuprofen by chemiluminescence-coupled ultrafiltration and application in a pharmacokinetic study

A simple one‐step method is established for plasma determination of ibuprofen and its pharmacokinetic study. The method involves simple sample pre‐treatment by dilution, rapid separation by ultrafiltration (UF) and online sensitive detection by chemiluminescence (CL) based on significant intensity enhancement of ibuprofen on the weak CL of potassium permanganate and sodium sulphite in an acidic system. The calibration curve for ibuprofen is linear in the range 0.1–50.0 µg/mL in rat plasma. Average recoveries of ibuprofen at 0.80, 12.0 and 40.0 µg/mL amounted to 98.0 ± 4.2%, 101.2 ± 3.6% and 99.3 ± 5.4%, respectively. Standard deviations of intra‐ and inter‐day measurement precision and accuracy are within ±10.0%. The detection limit for ibuprofen is 10.0 µg/L in plasma samples. Pharmacokinetic study of ibuprofen by the validated method shows that the mean plasma drug concentration–time course confirms to a classical two‐compartment open model with first‐order absorption. The proposed method will be an alternative for pre‐clinical pharmacokinetic study of ibuprofen and other non‐steroidal anti‐inflammatory drugs. Copyright © 2011 John Wiley & Sons, Ltd.     

Synergistic cytotoxic effects of tamoxifen and black cohosh on MCF-7 and MDA-MB-231 human breast cancer cells: an in vitro study

Breast cancer cell cultures were exposed to different concentrations of black cohosh, estradiol (E2), and tamoxifen to examine the effect on cell proliferation; cytotoxicity was assessed by using sulforhodamine B (SRB) dye solution. E2 (10(-10) - 10(-8) mol/L) markedly stimulated the proliferation of MCF-7 cells (p < 0.01). Tamoxifen stimulated MCF-7 cell proliferation at 10(-6) mol/L and 10(-5) mol/L (p < 0.005) but inhibited in a dose-dependent fashion the proliferative effect of E2 (p < 0.001). Black cohosh alone did not show any stimulatory effect, but exhibited a cytotoxic effect, which was significant at 10(3) microg/mL (p < 0.001). Adding black cohosh at 10(0)-10(3) microg/mL to E2 at 10(-9) mol/L also resulted in a dose-dependent inhibition of E2 proliferative effect. Interestingly, the combination of black cohosh (10(0)-10(3) microg/mL) with increasing tamoxifen concentrations further inhibited MCF-7 cell growth. On MDA-MB-231 cells, neither E2 nor tamoxifen displayed any detectable effect. However, black cohosh inhibited MDA-MB-231 cell proliferation at 10(3) microg/mL (p < 0.05), and this inhibitory effect was enhanced by increasing tamoxifen concentrations. This study reveals a cytotoxic effect of black cohosh on both estrogen-sensitive and estrogen-insensitive breast cancer cells and a synergism with tamoxifen for inhibition of cancerous cell growth.     

HPLC method for determination of verapamil in human plasma after solid-phase extraction

A simple, rapid and precise HPLC method has been developed for the assay of verapamil in human plasma. The clean up of the plasma samples was tested using several adsorbents for solid-phase extraction and best recovery was obtained using mixed-mode cartridges (HLB - hydrophilic-lipophilic balance) ranging between 94.70 and 103.71%. HPLC separation was performed with isocratic elution on Lichrospher 60 RP-select B column (250 mm × 4 mm I.D., 5 μm particle size). The mobile phase was 40% acetonitrile and 0.025 mol/L KH₂PO₄ with pH 2.5 at flow rate of 1 mL/min. Diltiazem was used as internal standard and the detection wavelength was 200 nm. The calibration curves were linear in the range of 10–500 ng/mL. The developed method is convenient for routine analysis of verapamil in human plasma.