High-performance liquid chromatographic determination of diuretics in urine by micellar liquid chromatography

The use of micellar liquid chromatography for the determination of diuretics in urine by direct injection of the sample into the chromatographic system is discussed. The retention of the urine matrix at the beginning of the chromatograms was observed for different sodium dodecyl sulphate (SDS) mobile phases. The eluent strengths of a hybrid SDS—methanol micellar mobile phase for several diuretics were compared and related to the stationary phase/water partition coefficient with a purely micellar mobile phase. The urine band was appreciably narrower with a mobile phase of 0.05 M SDS—5% methanol (v/v) at 50°C (pH 6.9). With this mobile phase the determination of bendroflumethiazide and chlorthalidone was adequate. Acetazolamide, ethacrynic acid, furosemide, hydrochlorothiazide and probenecid were overlapped by the urine matrix, and the retention of amiloride and triamterene was too long.     

Treatment of congenital nephrogenic diabetes insipidus with hydrochlorothiazide and amiloride in an adult patient

AIM: The effects of treatment with hydrochlorothiazide (HCTZ) combined with amiloride were elucidated and compared to HCTZ treatment alone and combined with acemetacin or triamterene in a Japanese adult patient with congenital nephrogenic diabetes insipidus. METHODS: The study was divided into seven periods: (1) HCTZ and acemetacin; (2) control period; (3) HCTZ; (4) a second control period; (5) HCTZ and amiloride; (6) a third control period, and (7) HCTZ and triamterene. Fluid intake, urine volume, urinary Na, K, creatinine, and osmolality and serum Na, K, Cl, CO2, and osmolality were measured, and free water clearance and proximal and distal tubular Na reabsorption rates were calculated. RESULTS: Without drug administration, the urine volume was about 8,000 ml/day. The urine volume was reduced to about 6,000 ml/day with HCTZ. A further urine volume reduction to about 5,000 ml/day was obtained with the second drug administration, and the effects were similar among the three regimens. Serum and urinary osmolality and free water clearance were also similar among the three combinations, whereas the urinary potassium excretion was the least, and the serum potassium concentration was the highest with HCTZ plus amiloride. Besides, no alkalosis was observed only with this combination. CONCLUSION: HCTZ plus amiloride may be superior to HCTZ plus acemetacin and HCTZ plus triamterene in preventing hyperkaliuria, hypokalemia, and metabolic alkalosis.     

Direct determination of diuretic drugs in urine by capillary zone electrophoresis using fluorescence detection

Four diuretic drugs banned in sport (amiloride, triamterene, bendroflumethiazide and bumetanide) have been separated by capillary zone electrophoresis (CZE) and detected using conventional fluorescence spectrometry. The effect of pH on electrophoretic parameters such as migration time, peak efficiency and peak height is discussed. Complete separation of the four drugs is achieved in less than 8 min at pH 8. No interference due to endogenous urine components is observed and thus direct urine analysis is feasible. Analytical figures of merit including precision and limits of detection are presented. Limits of detection range between 0.5 fmol for triamterene and 21.6 fmol for bumetanide.     

Determination of methotrexate, several pteridines, and creatinine in human urine, previous oxidation with potassium permanganate, using HPLC with photometric and fluorimetric serial detection

A novel HPLC method, using UV and fluorimetric serial detection, for the simultaneous determination of methotrexate (MTX), five disease marker pteridines, and the reference metabolic subproduct creatinine (CREA) in human urine was established. A previous oxidation process using 10(-3) M KMnO4 (pH 5.0) and 35min of oxidation time was necessary to transform the analytes in the highly fluorescent pteridinic rings. CREA was not affected by the oxidative medium. Using Tris-HCl/NaCl buffer solution (pH 6.6) as mobile phase, MTX and the assayed pteridines were monitored by fluorescence at lambda(em) = 444 nm and lambda(ex) = 280 nm and creatinine was monitored by absorption measurements at lambda(abs) = 230 nm. All components were well resolved in approximately 7 min. Detection limits, according the criteria of Clayton and co-workers, were 10 ng ml(-1) for MTX, less than 1 ng ml(-1) for all of the pteridines, and 4 microg ml(-1) for CREA.     

A coupled fluorometric rate assay for pyruvate dehydrogenase in cultured human fibroblasts

A method for measuring the activity of the pyruvate dehydrogenase complex (PDC) by coupling acetyl-CoA production to acetylation of a fluorescent dye is described. Acetylation of cresyl violet acetate by pigeon liver acetyltransferase results in a shift of its fluorescence spectrum from lambda ex max = 575, lambda em max = 620 nm to lambda ex max = 475, lambda em max = 575 nm. The rate of appearance of acetylated dye was followed fluorometrically and was proportional to PDC activity in extracts of cultured human fibroblasts. The assay showed appropriate substrate and cofactor dependence and had a working range between 0.04 and 70 munits. It is 10 times more sensitive than the spectrophotometric assay on which it is based (working range 0.4-31 munits) and is equally convenient. Unactivated PDC activity in fibroblast extracts was 0.75 (0.60-0.92) munits/mg protein (mean and range for six cell lines).     

Fluorescence-based continuous assay for the aspartyl protease of human immunodeficiency virus-1

5-Dimethylaminoaphthalene-1-sulfonyl-Ser-Gln-Asn-Tyr-Pro-Ile-Val-T rp (Dns-SQNYPIVW) is a fluorescent substrate for the aspartyl protease of human immunodeficiency virus-1. In intact substrate, fluorescence of Trp (lambda ex 290 nm, lambda em 360 nm) was 60% quenched by energy transfer to the dansyl group. Protease-catalyzed cleavage at the Tyr-Pro bond abolished the energy transfer, and the consequent increase in Trp fluorescence was used to follow the enzymatic reaction. At substrate concentrations less than 60 microM, initial reaction velocity increased as a linear function of substrate concentration, with kcat/KM = 9700 M-1 s-1. Limited solubility and internal fluorescence quenching precluded a determination of KM for Dns-SQNYPIVW, but this was clearly greater than 100 microM.     

Dimethyl-and monomethyloxyluciferins as analogs of the product of the bioluminescence reaction catalyzed by firefly luciferase

The absorption and fluorescence spectra of dimethyloxyluciferin (DMOL) and monomethyloxyluciferin (MMOL) were studied at pH 3.0-12.0. In the range of pH 3.0-8.0, the fluorescence spectrum of DMOL exhibits a maximum at lambda(em) = 639 nm. At higher pH values an additional emission maximum appears at lambda(em) = 500 nm (wavelength of excitation maximum lambda(ex) = 350 nm), which intensity increases with time. It is shown that this peak corresponds to the product of DMOL decomposition at pH > 8.0. The absorption spectra of MMOL were studied in the range of pH 6.0-9.0. At pH 8.0-9.0, the absorption spectrum of MMOL exhibits one peak at lambda(abs) = 440 nm. At pH 7.3-7.7, an additional band appears with maximum at lambda(abs) = 390 nm. At pH 6.0-7.0 two maxima are observed, at lambda(abs) = 375 and 440 nm. The fluorescence spectra of MMOL (pH 6.0-9.7, lambda(ex) = 440 or 375 nm) exhibit one maximum. It is shown that decomposition of DMOL and MMOL in aqueous solutions results in products of similar structure. DMOL and MMOL are rather stable at the pH optimum of luciferase. It is suggested that they can be used as fluorescent markers for investigation of the active site of the enzyme.     

HIV-1 protease: characterization of a catalytically competent enzyme-substrate intermediate.

The steady-state and pre-steady-state kinetic parameters for the interaction of E with the fluorogenic substrate 2-aminobenzoyl-Thr-Ile-Nle-Phe(p-NO(2))-Gln-Arg-NH(2) were determined in 1.25 M NaCl, 0.1 M MES-TRIS at pH 6.0 at 25 degrees C. At low concentrations of enzyme, the values of the K(m) and k(cat) calculated from steady-state data were 2.1 microM and 7.4 s(-1), respectively. At high concentrations of enzyme, the time-courses of fluorescence enhancement associated with catalysis were very dependent on the excitation wavelength used to monitor the reaction. Because the absorbance spectrum of the substrate overlapped the fluorescence emission spectrum of the enzyme, these abnormalities were attributed to fluorescence energy transfer between the enzyme and the substrate in an enzyme-substrate intermediate. The kinetic data collected with lambda(ex) = 280 nm and lambda(em) > 435 nm were analyzed according to the following mechanism in which EX was the species with enhanced fluorescence relative to substrate or products: [formula see text]. The values of the kinetic parameters with (1)H(2)O as the solvent were K = 13 microM, k(2) = 150 s(-1), k(-2) = 25 s(-1), and k(3) = 11 s(-1). The values of the kinetic parameters with (2)H(2)O as the solvent were K = 13 microM, k(2) = 210 s(-1), k(-2) = 12 s(-1), and k(3) = 4.4 s(-1). These values yielded solvent isotope effects of 2 on k(cat) and 0.9 on k(cat)/K(m). From analysis of the complete time-course of the fluorescence change (lambda(ex) = 280 nm and lambda(em) > 435 nm) during the course of substrate hydrolysis, the intermediate EX was determined to be 6.3-fold more fluorescent than the product, which, in turn, was 4.5-fold more fluorescent than ES or S. Rapid quench experiments with 2 N HCl as the quenching reagent confirmed that EX was a complex between enzyme and substrate. Consequently, the small burst in fluorescence observed when monitoring with lambda(ex) = 340 nm (0.3 product equiv per enzyme equivalent) was attributed to the fluorescence change upon transfer of substrate from an aqueous environment to a nonaqueous environment in the enzyme. These results were consistent with carbon-nitrogen bond cleavage being the major contributor to k(cat).     

Capillary electrophoresis with laser-induced fluorescence and pre-column derivatization for the analysis of illicit drugs

In the current paper, we report the development of a new capillary electrophoresis method using pre-column derivatization and laser-induced fluorescence detection for the determination of ephedrine and amphetamine drugs. Our new method allows for the identification and quantification of six commonly used illicit drugs namely pseudoephedrine, ephedrine, amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, and 3,4-methylenedioxymethylamphetamine, respectively, as well as propafenone (internal standard). Following derivatization with fluorescein isothiocyanate, a total of six amphetamine drugs and the internal standard could readily be separated using a fused-silica 75 micromID x 60 cm length (effective length: 50.2 cm) capillary column. The mobile phase consisted of buffer containing 20mM borate (pH 12, adjusted with sodium hydroxide). Samples were injected in pressure mode with the capillary being operated at 25kV/25 degrees C, and the detection of the derivatized compounds was sought using a laser-induced fluorescence (LIF) detector (lambda(ex)=488 nm and lambda(em)=520 nm), with a run-time of 20 min. The current method was validated with regard to precision (relative standard deviation, RSD), accuracy, sensitivity, linear range, limit of detection (LOD) and limit of quantification (LOQ). In human blood and urine samples, detection limits were 0.2 ngmL(-1), and the linear range of the calibration curves was 0.5-100 ngmL(-1). The intra-day and inter-day precisions were both less than 13.22%.     

Development of a novel neodymium compound for in vivo fluorescence imaging.

We developed a novel fluorescent probe that contains the neodymium(III) complex moiety and fluorescein moiety. This probe can emit long-lived near-infrared luminescence derived from a Nd ion through excitation of the fluorescein moiety with visible light (lambda(ex) = 488 nm, lambda(em) = 880 nm, lifetime = 2.3 micros). These results indicate the possibility of the probe as a candidate for in vivo fluorescence molecular imaging.     

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   

Effects of thermal denaturation on protein glycation

Protein denaturation occurs at sites of inflammation. We hypothesized that denatured protein may provide a more susceptible target for glycation, which is a known mediator of inflammation. We examined the effects of thermal denaturation on the susceptibility of protein glycation using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and aspartate aminotransferase (AAT) as our target proteins. GAPDH and AAT are ubiquitous proteins that exhibited very different thermal stabilities. Glycating agents, methylglyoxal (MG) and glyceraldehyde (Glyc), caused an increase in the formation of advanced glycation endproducts (AGEs) in native and denatured GAPDH and AAT. The effects of the glycating agents were more pronounced with the denatured proteins. In addition to nitroblue tetrazolium (NBT)- reactivity, our measured endpoints were absorbance (lambda = 365 nm) and fluorescence (lambda(ex) = 370 nm; lambda(em) = 470 nm) properties that are typically associated with protein glycation. We also looked at carnosine's ability to prevent glycation of native and denatured protein. Carnosine, an endogenous histidine dipeptide, exhibits anti-inflammatory activity presumably due to its anti-oxidant and anti-glycation properties. Carnosine prevented Glyc-induced AGE formation in both native and denatured AAT suggesting that carnosine's anti-inflammatory activity may be due in part to carnosine's ability to prevent glycation of denatured protein.     

Fluorescence studies of the interaction of DNA with Hoechst 33258

Absorption and fluorescence measurements of DNA-Hoechst 33258 complexes at high molar ratio of DNA phosphate to dye are consistent with the existence of two types of bound species. One type (Type I) predominates at high ionic strength, whereas the other (Type II) occurs at low ionic strength. The fluorescence peak (lambda fmax) depends on the excitation wavelength (lambda ex); lambda fmax shifts toward longer wavelength with increasing lambda ex. Optical properties obtained are summarized in the following: for Type I, lambda amax (absorption) = 352 nm, lambda fmax at lambda ex of 335 nm = 460 nm, tau (fluorescence lifetime) = 2.0-2.5 ns; for Type II, lambda amax = 360 nm, lambda fmax at lambda ex of 335 nm = 470 nm, tau = 4.0-5.0 ns. This behavior is interpreted in terms of solvent-solute relaxation. Type I corresponds to less hydrated bound species, while Type II to more hydrated bound species.     

A new fluorimetric assay for streptothricins

In strongly acidic medium (70% HClO4) streptothricins form a fluorophore (lambda ex = 312 nm; lambda em = 381 nm) with unknown structure. A fluorimetric determination of pure or crude products and cultures, respectively, was worked out based on this reaction. Concentrations for fluorescence measurements were in the range of 10(-8) - 2 X 10(-7) moles. Interferences of the assay are discussed, a statistical evaluation of results and a comparison between microbiological and fluorimetric findings are given.     

Fluorescence quenching of serum albumin by rifamycin antibiotics and their analytical application.

In neutral medium, rifamycin antibiotics such as rifapentin (RFPT), rifampicin (RFP), rifandin (RFD) and rifamycin SV (RFSV) can bind with human serum albumin (HSA) and bovine serum albumin (BSA) to form complexes, resulting in the quenching of the intrinsic fluorescence (lambda(ex)/lambda(em) = 285/355 nm) of the BSA and HSA. The quenching intensity (DeltaF) is directly proportional to the concentration of the rifamycin antibiotics. Therefore, a new analytical method was established to determine trace rifamycin antibiotics. The method had fairly high sensitivity and the detecting limits (3sigma) for RFPT, RFP, RFD and RFSV were 0.85, 0.98, 1.83, 1.89 ng/mL, respectively, for the HSA system and 0.76, 0.89, 1.55, 1.77 ng/mL, respectively, for the BSA system. All relative standard deviations (RSDs) were <3.8%. In this work, the characteristics of the fluorescence spectra were studied and the optimum reaction conditions and influencing factors were investigated. The influence of coexisting substances was tested and the results showed that the method had good selectivity and could be applied to determine trace rifamycin antibiotics in medicine capsules and urine samples. Taking the RFSV-serum albumin system as an example, the reaction mechanisms, such as binding constants, binding sites, binding distance and the type of fluorescence quenching, were investigated.     

Determination of atenolol by the micelle-stabilized room-temperature phosphorescence methodology.

A micellar-stabilized room-temperature phosphorescence (MS-RTP) method for the determination of atenolol has been developed in micellar solutions of sodium dodecylsulphate (SDS) in the presence of thallium(I) as a heavy atom and sodium sulphite as an oxygen scavenger. The effects of thallium(I) nitrate, SDS and sodium sulphite concentrations on atenolol MS-RTP intensity were studied. Optimized conditions to obtain maximum sensitivity were 0.015 mol/L thallium(I) nitrate, 0.1 mol/L SDS and 0.0075 mol/L sodium sulphite. The maximum phosphorescence signal was completely developed in 10 min and the intensity was measured at lambda(ex) = 272 nm and lambda(em) = 412 nm. The linear range of application obtained was 2.01-16.00 microg/mL. The detection limit estimated from the least-squares regression analysis was 0.86 microg/mL and the relative standard deviation of 10 replicates was 1.7%. The proposed method was applied to the determination of atenolol in a pharmaceutical formulation. The quantitation was carried out by means of standard calibration, standard-additions calibration and Youden calibration. These three experiments were necessary to evaluate the presence of constant and proportional errors due to the matrix.     

Tryptophan fluorescence in electron-transfer flavoprotein:ubiquinone oxidoreductase: fluorescence quenching by a brominated pseudosubstrate

We have studied the intrinsic fluorescence of the 12 tryptophan residues of electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF:QO). The fluorescence emission spectrum (lambda ex 295 nm) showed that the fluorescence is due to the tryptophan residues and that the contribution of the 22 tyrosine residues is minor. The emission maximum (lambda m 334 nm) and the bandwidth (delta lambda 1/2 56 nm) suggest that the tryptophans lie in hydrophobic environments in the oxidized protein. Further, these tryptophans are inaccessible to a range of ionic and nonionic collisional quenching agents, indicating that they are buried in the protein. Enzymatic or chemical reduction of ETF:QO results in a 5% increase in fluorescence with no change of lambda m or delta lambda 1/2. This change is reversible upon reoxidation and is likely to reflect a conformational change in the protein. The ubiquinone analogue Q0(CH2)10Br, a pseudosubstrate of ETF:QO (Km = 2.6 microM; kcat = 210 s-1), specifically quenches the fluorescence of one tryptophan residue (Kd = 1.6-3.2 microM) in equilibrium fluorescence titrations. The ubiquinone homologue UQ-2 (Km = 2 microM; kcat = 162 s-1) and the analogue Q0(CH2)10OH (Km = 2 microM; kcat = 132 s-1) do not quench tryptophan fluorescence; thus the brominated analogue acts as a static heavy atom quencher. We also describe a rapid purification for ETF:QO based on extraction of liver submitochondrial particles with Triton X-100 and three chromatographic steps, which results in yields 3 times higher than previously published methods.     

Antihypertensive and bilateral renal responses to diuretics in 2-kidney, 1-clip Goldblatt hypertensive rats

Experiments were conducted to assess the effect of furosemide or amiloride alone and a combination of both agents on each kidney in anesthetized 2-kidney, 1 clip Goldblatt hypertensive rats (n = 25). Intravenous infusion of furosemide alone (1.02 mg/kg.hr) significantly reduced the blood pressure by 14 +/- 5 mmHg. There were 6- to 10-fold increases in water, absolute sodium and fractional sodium excretions and a 2-fold increase in potassium excretion in the nonclipped kidney. A smaller but significant increase in the excretory function was also observed in the clipped kidney. There was no significant change in GFR of both kidneys. Indomethacin pretreatment (2 mg/kg) failed to significantly alter the vasodepressor and renal responses to furosemide in both hypertensive and normal rats. Removal of the renal artery clip from the hypertensive rats reduced the blood pressure by 12 +/- 3 mmHg and enhanced the function of the ipsilateral, unclipped kidney. Subsequent administration of furosemide further increased the excretory response. Administration of amiloride alone (2.4 mg/kg.hr) or with furosemide into hypertensive rats reduced the arterial pressure and increased excretion rates of urine flow and urinary sodium. Potassium excretion rate decreased bilaterally in amiloride treated rats but did not alter significantly in rats which received a combination of amiloride and furosemide. These results indicate that diuretics ameliorate the excretory function of both the stenotic kidney and the nonstenotic kidney and that the improvement of the kidney function is independent of prostaglandin. Furthermore, removal of the stenosis accentuates the beneficial effect of diuretics on the kidney.     

A direct and simultaneous detection of zinc protoporphyrin IX, free protoporphyrin IX, and fluorescent heme degradation product in red blood cell hemolysates

Fluorescence emission of free protoporphyrin IX (PPIX, em. approximately 626 nm), zinc protoporphyrin IX (ZPP, em. approximately 594 nm) and fluorescent heme degradation product (FHDP, em. approximately 466 nm) are identified and simultaneously detected in mouse and human red cell hemolysates, when excited at 365 nm. A novel method is established for comparing relative FHDP, PPIX and ZPP levels in hemolysates without performing red cell porphyrin extractions. The ZPP fluorescence directly measured in hemolysates (F(365/594)) correlates with the ZPP fluorescence obtained from acetone/water extraction (R(2) = 0.9515, P < 0.0001). The relative total porphyrin (ZPP and PPIX) fluorescence obtained from direct hemolysate fluorescence measurements also correlates with red blood cell total porphyrins determined by ethyl acetate extraction (Piomelli extraction, R(2) = 0.88, P < 0.0001). These fluorescent species serves as biomarkers for alterations in Hb synthesis and Hb stability.     

Novel fluorescent probe for detecting hydroperoxides with strong emission in the visible range

A novel fluorescent probe, a swallow-tailed perylene derivative for detecting hydroperoxides (Spy-HP), containing perylene 3,4,9,10-tetracarboxyl bisimide as the main skeleton in the structure, was developed. Spy-HP reacted rapidly with hydroperoxides such as m-chloroperbenzoic acid (MCPBA) and cumene hydroperoxide to form its oxidized derivative, Spy-HPOx, and emitted an extremely strong fluorescence (phi approximately 1) in the visible range (lambda(ex) = 524 nm and lambda(em) = 535 nm), as the result of canceling the photoinduced electron transfer (PET) effect. The reaction between Spy-HP and hydroperoxides proceeded quantitatively in strict stoichiometry, without being affected by autoxidation or photobleaching. Because of these prominent properties, Spy-HP is expected to be a novel and useful fluorescent probe to 'spy' on hydroperoxides in biosamples.