Validation of an HPLC method for the determination of fleroxacin and its photo-degradation products in pharmaceutical forms

HPLC determination of fleroxacin in dosage forms was carried out using either reversed-phase column YMC pack ODS-AQ or Supelco LC Hisep shielded hydrophobic phase column, with UV detection at 280 nm. The mobile phase for ODS column consisted of 50:50:0.5 v/v/v and for Hisep column 15:85:0.5 v/v/v acetonitrile-water-triethylamine. The pH of the mobile phase was adjusted to 6.30 for ODS column and to 6.85 for Hisep column, with H3PO4. Linear response was obtained in the concentration range of fleroxacin between 0.01 and 1.30 micrograms/mL. Detection limit was 4.8 ng/mL. Recovery test in the determination of fleroxacin in "Quinodis" tablets (Hoffmann La Roche, nominal mass 400 or 200 mg) was 98-101% for both columns. The effect of the composition and pH of the mobile phase on spectra, retention time and dissociation constants of fleroxacin was discussed. The proposed method could be also used for separation of the photo-degradation products of fleroxacin. Ten degradation products were separated on the ODS-AQ column, thus confirming the suitability of the proposed method for stability study of fleroxacin in pharmaceuticals.     

Liquid chromatographic method for the analysis of tocopherols in malt sprouts with supercritical fluid extraction.

A simple, specific and sensitive high-performance liquid chromatographic method has been developed for the determination of tocopherols in malt sprouts. A supercritical fluid extraction (SFE) procedure was used to isolate tocopherols from the vegetal matrix before quantitative analysis. The analytes were separated on a Zorbax reversed-phase column using methanol-water as mobile phase and quantified by measuring its fluorescence at lambda(em)=328 nm after excitation of the analytes at lambda(exc)=303 nm. The limits of detection for alpha-, gamma- and delta-tocopherols were 0.04, 0.05, and 0.05 microg/ml, respectively. The calibration graphs of the method were linear from 0.1 to 1.5, 0.2 to 2.5, and 0.2 to 2.0 microg/ml, for alpha-, gamma- and delta-tocopherols, respectively. This SFE and HPLC procedure is simple, precise and accurate for the determination of tocopherols in malt sprouts.     

Fluorimetric determination of isatin in human urine and serum by liquid chromatography postcolumn photoirradiation

For the fluorimetric determination of isatin in human urine and serum, HPLC-postcolumn photoirradiation using a mobile phase has been developed. Isatin in the urine or serum sample was separated on a Capcell Pak C1 column (250 x 4.6 mm id). The mobile phase consisted of 70 mmol l-1 phosphate buffer (pH 7.2)-tetrahydrofuran (85 + 15% v/v) containing 5 mmol l-1 hydrogen peroxide, which was irradiated with germicidal light to induce fluorescence (lambda ex 302 nm, lambda em 418 nm). The addition of tetrahydrofuran to the mobile phase led to the peaks showing good separation as well as increased sensitivity. The calibration graph for isatin was linear over the range of 0.16-10.7 ng. The pretreatment of the acidified urine or serum samples consisted of diluting steps or deproteinizing steps using perchloric acid, respectively. The mean recovery of isatin from urine and serum was greater than 94%.     

Incorporation of a monolithic column into sequential injection system for drug-protein binding studies

A sequential injection analysis (SIA) manifold was incorporated with a monolithic strong anion-exchanger disk for on-line drug-protein interaction studies. The antibiotic ciprofloxacin (CF) was selected as a model drug compound. The separation principle was based on the strong retention of bovine serum albumin (BSA) on the monolithic strong anion-exchanger and the liberation/release of the free form of the drug. Elution of the retained BSA was easily achieved by delivering a different mobile phase via the SIA manifold. The type of functional group of the monolithic support, the breakthrough volume and the injected volumes of CF and BSA were studied and optimized. The influence of the variation of incubation time was studied in on-line binding assays. Scatchard plot was employed to obtain the number of binding sites and the equilibrium binding constants. For the off-line study of the CF-BSA binding, two binding classes were determined with constants of (3.16+/-0.21)x10(6)M(-1) and (1.27+/-0.48)x10(4)M(-1) and 6.1+/-1.3 and 17.8+/-3.9 binding sites per class, respectively. In non-equilibrium binding experiments the binding rate constant was k(1)=785 M(-1)min(-1). All measurements were monitored with fluorescence (lambda(ext)=300 nm, lambda(em)=460 nm) and spectrophotometric detection (lambda=280 nm). To evaluate the accuracy of the developed method the obtained results were compared versus ultrafiltration experiments and were found in good agreement.     

Molecularly imprinted polymers applied to the clean-up of zearalenone and α-zearalenol from cereal and swine feed sample extracts

A molecularly imprinted polymer prepared using 1-allylpiperazine (1-ALPP) as the functional monomer, trimethyltrimethacrylate (TRIM) as the crosslinker and the zearalenone (ZON)-mimicking template cyclododecanyl-2,4-dihydroxybenzoate (CDHB) has been applied to the clean-up and preconcentration of this mycotoxin (zearalenone) and a related metabolite, alpha-zearalenol (alpha-ZOL), from cereal and swine feed sample extracts. The extraction of ZON and alpha-ZOL from the food samples was accomplished using pressurized liquid extraction (PLE) with MeOH/ACN (50:50, v/v) as the extraction solvent, at 50 degrees C and 1500 psi. The extracted samples were cleaned up and preconcentrated through the MIP cartridge and analyzed using HPLC with fluorescence detection (lambda (exc)=271/ lambda (em)=452 nm). The stationary phase was a polar endcapped C18 column, and ACN/MeOH/water 10/55/35 (v/v/v, 15 mM ammonium acetate) at a flow rate of 1.0 mL min(-1) was used as the mobile phase. The method was applied to the analysis of ZON and alpha-ZOL in wheat, corn, barley, rye, rice and swine feed samples fortified with 50, 100 and 400 ng g(-1) of both mycotoxins, and it gave recoveries of between 85 and 97% (RSD 2.1-6.7%, n=3) and 87-97% (RSD 2.3-5.6%, n=3) for alpha-ZOL and ZON, respectively. The method was validated using a corn reference material for ZON.     

Determination of piceid by photochemically induced fluorescence and second-derivative: response surface methodology for the optimization of a liquid-liquid extraction procedure for its analysis in wine samples

trans-Piceid itself is weakly fluorescent, but the fluorescence signal (lambda(exc/em)=260/361nm) is greatly enhanced by UV-irradiation of its hydroethanolic solutions. Employing the photoinduced emission signal at 361nm or the amplitude of the second-derivative-photoinduced emission spectrum, between 353 and 361nm, a linear relation is found in the assayed range 5.7-31.4ngmL(-1) of trans-piceid and limits of detection of 1.7 and 2.1ngmL(-1), respectively, are obtained. A previous liquid-liquid extraction is necessary for the determination of piceid in wine. Experimental design (Central Composite Design) together with the Response Surface Methodology have been used to find optimum conditions for the extraction procedure. For this purpose, the difference between the photoinduced-fluorescence signal (lambda(exc/em)=260/361nm) of the aqueous phase, before and after being extracted, has been considered as Response Function. A tartrate buffer (pH 5.0) concentration of 0.15molL(-1) and a phase ratio of 1 are determined as optimum conditions. The amplitude of the second-derivative-emission spectrum, corresponding to the evaporated and re-dissolved organic phase, between 353 and 361nm has been employed as analytical signal. Standard addition method has been applied to the analysis of piceid in different wine samples under optimum conditions. Results of wine analysis have been satisfactorily validated by HPLC.     

Deprotonation and dimerization of maleimide in the triplet state: a laser flash photolysis study with optical and conductometric detection

The photochemistry of maleimide in aqueous solution is governed by the coexistence of up to three different triplet states, the keto triplet (lambda(max)=250, 330 nm, lambda(min)=290 nm, pK(a)=4.4+/-0.1, tau=5 micros), the deprotonated or enolate triplet (lambda(max)=360, 260 nm, lambda(min)=320 nm, shoulder at 370-380 nm) and a dimer triplet. This biradical is formed by the addition of the keto triplet to the double bond of a ground state maleimide in competition with electron transfer, (k( (3)MI+MI)=2.6 x 10(9) dm(3) mol(-1) s(-1)). Its spectrum is identical to that of the maleimide H-adduct radical (lambda(max)=370-380 (broad), 255 nm (narrow), lambda(min)=290 nm) and its lifetime is 110 ns. While protolysis is confined to maleimide and aqueous solutions, the dimer triplet is also found in acetonitrile. Dimer triplet formation is also observed with N-ethylmaleimide. Time-resolved conductometry and buffer experiments were used to characterise excited state protolysis. Multi-wavelength "global analysis" of the time profiles allowed the separation of the transient spectra and study of the kinetics of the monomer and dimer triplets. The cyclobutane dimer yield (determined by GC) is independent of maleimide concentration. This indicates that the dimer triplet does not contribute significantly to the initiation of free-radical polymerisation. Time-dependent Hartree-Fock calculations agree with the experimental data and further confirm the proposed mechanisms.     

Development of a quantitative method for the analysis of total L-ascorbic acid in foods by high-performance liquid chromatography

A new method has been developed for the determination of total vitamin C in foods. The method requires less time than the traditional methodologies and uses a radical oxidation of L-ascorbic acid (AA) to obtain dehydro-L-ascorbic acid (DHAA) by means of a peroxyl radical generated in situ by thermal decomposition of an azo-compound, 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). The dehydro-L-ascorbic acid is condensed with benzene-1,2-diamine (o-phenylenediamine, OPDA) to form its highly fluorescent quinoxaline derivative, 3-(1,2-dihydroxyethyl)furo[3,4-b]quinoxaline-1-one (DFQ), which is then separated on a C(18) column eluted with a mobile phase of 80 mM phosphate buffer and methanol at pH=7.8 and detected fluorometrically at lambda(ex)=355 nm and lambda(em)=425 nm. The reaction conditions for the complete conversion of AA to DFQ were 56 degrees C, 36 min and a mumol AAPH/AA ratio of 60. The sample, extracted with an aqueous metaphosphoric acid solution, was analyzed after being filtered through a 0.45 microm membrane. The method has shown good repeatability, sensitivity and accuracy compared to the results obtained with the reference method. The response of the detection system was linear within a range of 0.5-8.0 microg/mL with a correlation coefficient of 0.9997. The limit of detection was 0.27 microg/mL and the limit of quantification was 0.83 microg/mL. The AA contents of some selected foods were analyzed.     

High-performance liquid chromatographic determination of tramadol and its O-desmethylated metabolite in blood plasma. Application to a bioequivalence study in humans

Simultaneous HPLC determination of the analgetic agent tramadol, its major pharmacodynamically active metabolite (O-desmethyltramadol) in human plasma is described. Simple methods for the preparation of the standard of the above-mentioned tramadol metabolite and N1,N1-dimethylsulfanilamide (used as the internal standard) are also presented. The analytical procedure involved a simple liquid-liquid extraction of the analytes from the plasma under the conditions described previously. HPLC analysis was performed on a 250x4 mm chromatographic column with LiChrospher 60 RP-selectB 5-microm (Merck) and consists of an analytical period where the mobile phase acetonitrile-0.01 M phosphate buffer, pH 2.8 (3:7, v/v) was used, and of a subsequent wash-out period where the plasmatic ballast compounds were eluted from the column using acetonitrile-ultra-high-quality water (8:2, v/v). The whole analysis, including the equilibration preceding the initial analytical conditions lasted 19 min. Fluorescence detection (lambda(ex) 202 nm/lambda(em) 296 nm for tramadol and its metabolite, lambda(ex) 264 nm/lambda(em) 344 nm for N1,N1-dimethylsulfanilamide) was used. The validated analytical method was applied to pharmacokinetic studies of tramadol in human volunteers.     

柱前手性衍生化反相高效液相色谱法分离拉贝洛尔对映体

以乙酰葡萄糖异硫氰酸酯（GITC）作柱前手性衍生化试剂,用反相高效液相色谱法成功地分离了拉贝洛尔的两对对映异构体,并以荧光检测和紫外检测作对照,确认了4个衍生物的色谱峰。     

Direct determination of salicylamide in serum by matrix isopotential synchronous fluorimetry

A method for the determination of salicylamide at concentrations between 25 and 350 ng ml(-1) by use of matrix isopotential synchronous fluorescence spectrometry (MISF) in combination with derivative techniques is proposed. The method allows the determination of compounds in samples with unknown background fluorescence without the need for tedious pre-separation. Synchronous scans are performed along a trajectory that connects points of identical intensity in a three-dimensional fluorescence spectrum. The unknown analytical signal of the serum is suppressed from the MISF spectrum, by calculating its first derivative at lambda(exc)=324 nm and lambda(em)=392 nm. In order to ensure maximum sensitivity and adequate selectivity, the experimental variables affecting the fluorescence intensity of the salicylamide band at lambda(exc)=328 nm and lambda(em)=418 nm were studied. Based on the results, the determination was performed in an aqueous medium at pH 12 that was adjusted with a sodium phosphate/hydrogen phosphate buffer. Calibration graphs were subjected to a comprehensive statistical analysis. The error propagation has been considered in order to calculate the detection limit by the criterium of Clayton.     

HPLC-fluorescence determination of equilin and equilenin in postmenopausal women's urine

A high-performance liquid chromatographic (HPLC) method with fluorescence detection (lambda(ex) = 280 nm; lambda(em) = 410 and 312 nm) in combination with a post-column on-line photochemical derivatization is described for the determination of equilin and equilenin in urine from normal postmenopausal women after therapy with conjugated oestrogens. The column effluents were subjected on-line to UV irradiation (254 nm) and the photo-induced modifications were useful for the identification of the analytes. The conjugated (sulphate and glucuronide) forms were analysed after enzymatic or chemical hydrolysis and extracted with chloroform. Solid-phase extraction using strong anion-exchange sorbent was applied to the analysis of unconjugated oestrogen fraction to obtain a practical and reliable sample clean-up. The HPLC separations were achieved using ODS columns with a mobile phase consisting of 0.05 M triethylamine phosphate buffer (pH 4.0)-acetonitrile (64:36, v/v) at a flow rate of 1.0 mL/min. The method was accurate and reproducible; for the equilin and equilenin separation isocratic conditions were satisfactory, allowing a sensitive detection in urine samples with a detection limit of about 50 fmol for equilin (lambda(ex) = 280 nm; lambda(em) = 312 nm, after photoderivatization) and 10 fmol for equilenin (lambda(ex) = 280 nm; lambda(em) = 410 nm).     

Porphyrin, phthalocyanine and porphyrazine derivatives with multifluorenyl substituents as efficient deep-red emitters

The synthesis and photophysical properties are described for a series of porphyrin, phthalocyanine and pyrazinoporphyrazine derivatives which bear four or eight peripheral fluorenyl substituents as antennae. Representative examples are 5,10,15,20-tetra(9,9-dihexyl-9H-fluoren-2-yl)porphyrin (2), 5,10,15,20-tetrakis[4-(9,9-dihexyl-9H-fluoren-2-yl)phenyl]porphyrin (3), 2,3,9,10,16,17,23,24-octakis(9,9-dihexyl-9H-fluoren-2-yl)-29H,31H-phthalocyanine (8) and 2,3,9,10,16,17,23,24-octakis[4-(9,9-dihexyl-9H-fluoren-2-yl)phenyl]-29H,31H-tetrapyrazinoporphyrazine (9). Palladium-mediated Suzuki-Miyaura cross-coupling reactions have been key steps for attaching the substituents. The compounds are deep-red emitters: lambda(max)(em)=659 (3), 737 (8) and 684 nm (9). Their absorption and emission spectra, their fluorescence lifetimes and quantum yields are correlated with the structures of the macrocycles and the substituents. The solution fluorescence quantum yields of porphyrin derivatives substituted with fluorene (2-4) and terphenyl substituents (7) (Phi(f)=0.21-0.23) are approximately twice that of tetraphenylporphyrin. For phthalocyanine derivative 8, Phi(f) was very high (0.88). Specific excitation of the fluorene units of 8 produced emission from both of them (lambda(max)=480 nm) and also from the phthalocyanine core (lambda(max)=750 nm), indicating a competitive rate of energy transfer and radiative decay of the fluorenes. Organic light-emitting devices (OLEDs) were made by spin-coating techniques by using a polyspirobifluorene (PSBF) copolymer as the host blended with 3 (5 wt. %) in the configuration ITO/PEDOT:PSS/PSBF copolymer:3/Ca/Al. Deep-red emission (lambda(max)=663 nm; CIE coordinates x=0.70, y=0.27) was observed with an external quantum efficiency of 2.5 % (photons/electron) (at 7.5 mA cm(-2)), a low turn-on voltage and high emission intensity (luminance) of 5500 cd m(-2) (at 250 mA/ m(2)).     

Sensitive determination of bisphenol A and alkylphenols by high performance liquid chromatography with pre-column derivatization with 2-(4-carboxyphenyl)-5,6-dimethylbenzimidazole

A new and sensitive high-performance chromatographic method for the determination of bisphenol A and 8 alkylphenols with fluorescence detection is reported. Each phenol was derivatized by reaction with 2-(4-carboxyphenyl)-5,6-dimethylbenzimidazole at 40 degrees C for 60 min. The fluorescence derivatives were separated on a Wakosil 5C18 column (4.0 i.d. x 300 mm, 5 microm) with methanol:water (10:90) as mobile phase (detection wavelength: lambda(ex) 336 nm, lambda(em) 440 nm). The detection limits were in the range of 0.1-10.0 pg/mL in serum. The calibration graphs were linear to 1.0 microg/mL. The relative standard deviations were 7.2-8.9%, respectively. The proposed method was applied to the determination of bisphenol A in mother and infant rat serum.     

Kinetic model of energy transfer processes between low-coordinated ions on MgO by photoluminescence decay measurements.

Photoluminescence decay studies of emitting species on MgO nanocubes at room temperature provide evidence of three surface species characterized by an excitation and emission wavelength couple {lambda(exc);lambda(em)}. Species A corresponds to {lambda(exc)=240 nm; lambda(em)=380 nm}, whereas the couple {lambda(exc)=280 nm; lambda(em)=470 nm} is assigned to two species: B and B', the former is involved in energy transfer from excited state A* and the latter in direct emission from excited state B'*. A simple model for energy transfer from species A* to B is proposed. The numerical resolution of equations corresponding to this model is in good agreement with experimental data. This method quantifies the kinetics of intrinsic emission and energy transfer processes. Lifetime values indicate that phosphorescence is taking place, and species A, B and B' are identified as edge O(2-) (4 C), corner O(2-) (3 C) and kink O(2-) (3 C) oxide ions respectively.     

Determination of Fleroxacin in Plasma by Direct Injection High Performance Liquid Chromatography with Column Switching

ABSTRACT

A high-performance liquid chromatography (HPLC) assay was developed for the determination of fleroxacin in plasma. The plasma samples were directly introduced onto a HPLC column after filtering through a MolcutII® membrane filter, which removes high molecular weight proteins. The fleroxacin in filtrate was separated from interfering substances and retained on a pre-column using an ODS stationary phase and then was introduced to an analytical column with an ODS stationary phase by column switching. Fleroxacin and lomefloxacin, as an internal standard, were detected by ultraviolet absorbance at 295 nm. Determination of fleroxacin was possible over the concentration range 50-4000 ng/ml; the limit of detection was 20 ng/ml. The recovery of fleroxacin added to plasma was 97.3-100.4% with a coefficient of variation of less than 2.2%. This method is applicable to drug level monitoring in the plasma of patients being treated with fleroxacin and of healthy volunteers participating in pharmacokinetic studies.     

Lateral phase separation in cholesterol/diheptadecanoylphosphatidylcholine binary bilayer membrane

We investigated the phase behavior of cholesterol/diheptadecanoylphosphatidylcholine (C17:0-PC) binary bilayer membrane as a function of the cholesterol composition (X(ch)) by fluorescence spectroscopy using 6-propionyl-2-(dimethylamino)naphthalene (Prodan) and differential scanning calorimetry (DSC). The fluorescence spectra showed that the wavelength at the maximum intensity (lambda(max)) changed depending on the bilayer state: ca. 440 nm for the lamellar gel ( [Formula: see text] or L(beta)) and the liquid ordered (L(o)) phases and ca. 490 nm for the liquid-crystalline (L(alpha)) phase. The transition temperatures were determined from the temperature dependence of lambda(max) and endothermic peaks of the DSC thermograms. Both measurements showed that the pre- and main transition disappear around X(ch)=0.05 and 0.30, respectively. The constructed temperature-X(ch) phase diagram resembled a typical phase diagram for a eutectic binary mixture containing a peritectic point. The presence of a peritectic point at X(ch)=0.15 suggested that a complex of cholesterol and C17:0-PC is stoichiometrically formed in the gel phase. Consideration based on the hexagonal lattice model revealed that the compositions of 0.05 and 0.15 correspond to the bilayer states where cholesterol molecules are regularly distributed in different ways. The former is nearly equal to the composition for the membrane occupied entirely with Units (1:18), composed of a cholesterol and 18 surrounding C17:0-PC molecules within the next-next nearest neighbor sites. The latter is represented by a Unit (1:6), including a cholesterol and 6 surrounding C17:0-PC molecules. Further, the disappearance of the main transition at X(ch)=0.30 indicates that the pure L(o) phase can exist in X(ch)>0.30. The eutectic behavior observed in the phase diagram was explainable in terms of phase separation between two different types of regions with different types of regular distributions of cholesterol.     

Quantitation of glucosamine from shrimp waste using HPLC

This work presents a high-performance liquid chromatography (HPLC) method for the quantitation of glucosamine in chitin. The method includes an acid hydrolysis of chitin. The chromatographic separation is achieved using a Hypersil ODS 5-microm column (250 x 4.6 mm) at 38 degrees C, with precolumn derivatization with 9-fluorenylmethyl-chloroformate and UV detection (lambda = 264 nm). The mobile phase is a mixture of mobile phase A [30 mM ammonium phosphate (pH 6.5) in 15:85 methanol-water (v/v)], mobile phase B [15:85 methanol-water (v/v)], and mobile phase C [90:10 acetonitrile-water (v/v)], with a flow rate of 1.2 mL/min. The HPLC method proposed showed adequate repeatability (relative standard deviation, 5.8%), accuracy (92.7% recovery), and sensitivity, with a detection limit of 2 microg/mL. The method is successfully applied to the quantitation of glucosamine for the determination of the purity of chitin from shrimp waste.     

Identification and determination of oxytetracycline, tiamulin, lincomycin, and spectinomycin in veterinary preparations by thin-layer chromatography/densitometry

A thin-layer chromatographic/densitometric method was developed for the identification and quantitation of oxytetracycline, tiamulin, lincomycin, and spectinomycin in veterinary preparations. Silica gel-coated thin layer chromatography plates and 2 mobile phases were used to separate these constituents. The appropriate compositions of the suitable mobile phases were established: 10% citric acid solution-n-hexane-ethanol (80 + 1 + 1, v/v) and n-butanol-ethanol-chloroform-25% ammonia (4 + 5 + 2 + 5, v/v). Along with Rf values and spot colors, direct UV and visual densitometric measurements were used for identification. Similar measuring ranges were used for quantitative analysis to obtain repeatable and reliable results for the preparations examined. The results of the quantitative analysis are characterized by a small confidence interval and are close to the declared contents of active constituents: oxytetracycline 30.01 +/- 0.38 g at lambda = 350 nm and 30.24 +/- 0.86 g at lambda = 430 nm; tiamulin, 10.19 +/- 0.86 g at lambda = 450 nm; lincomycin, 2.27 +/- 0.08 g at lambda = 278 nm; and spectinomycin, 2.18 +/- 0.07 g at lambda = 421 nm. The recoveries for all antibiotics ranged from 100.01 to 102.54%.     

Emission analysis of Sm(3+) and Dy(3+):MgLaLiSi(2)O(7) powder phosphors

This paper reports on the structural and emission properties of newly synthesized Sm(3+) and Dy(3+):MgLaLiSi(2)O(7) powder phosphors based on the measurement of their XRD, SEM, FTIR and photoluminescence spectra. Emission spectra of the Sm(3+):MgLaLiSi(2)O(7) phosphors with lambda(exci)=402 nm ((6)H(5/2)-->(4)F(7/2)) and Dy(3+):MgLaLiSi(2)O(7) phosphors with lambda(exci)=385 nm ((6)H(15/2)-->(4)I(13/2)) have been analyzed. Emission mechanisms of these phosphors have also been explained.