Development of a fluorimetric detection method for cinnabarinic acid using ortho‐tolyl hydrazine as the derivatization reagent

A fluorimetric detection method for one of the tryptophan metabolites, cinnabarinic acid (CA), which has recently been reported to have the ability to induce apoptosis in thymocytes, was developed using o‐tolyl hydrazine (TH) as the derivatization reagent. The carbonyl group at position 3 in CA was tagged with the hydrazino moiety of TH at 100°C for 30 min, and the generated derivative, CA tagged with TH, fluoresced at 412 nm with a 316 nm excitation wavelength. The CA tagged with TH was separated on a reversed‐phase HPLC and detected fluorometrically. The relative standard deviation was in the range of 1.1–8.9% (n = 3), and the detection limit was approximately 12?fmol (signal‐to‐noise ratio, 3). The proposed HPLC method can be useful for the sensitive detection of CA. Copyright © 2009 John Wiley & Son, Ltd.     

Determination of amphetamines in human urine by liquid chromatography with fluorimetric detection using a solid-phase extraction procedure

A precise and feasible HPLC method has been developed for the analysis of amphetamine (AMPH), methamphetamine (MAMPH) and methylenedioxymethamphetamine (MDMA, ecstasy) in human urine. A chromatographic run on a C8 Genesis (150 mm x 4.6 mm, 5 microm) column maintained at 30 degrees C lasts about 17 min, using a mobile phase composed of ACN (12%) and a pH 2.5 phosphate buffer (88%) containing 0.3% triethylamine. Mirtazapine was used as the internal standard. Good linearity was found in the 100-2000 ng/mL concentration range for AMPH and MAMPH and in the 12-2000 ng/mL concentration range for MDMA. The pretreatment of urine samples was carried out by means of a careful SPE procedure on C2 cartridges. The extraction yields were very satisfactory for all analytes, with average values greater than 97%. The leading conditions allowed the determination of AMPH, MAMPH and MDMA with satisfactory precision and accuracy. The method has been successfully applied to the determination of the analytes in urine of AMPH users.     

The selection of optimal conditions in HPLC II. The influence of column dimensions and sample size on solute detection

Summary The responding signals of eluted components can be enhanced by using high performance small volume columns with long lengths generating more plates than required for a separation with a preset resolution in combination with instruments which show very small external bandbroadening. The excess number of plates is then consequently erased by maximizing the injection volume of the sample until the preset resolution is reached. Equations, describing the dilution, relative signal enhancement and maximum injection volume as a function of the process parameters are derived and experimentally verified. Theory and experiment are in agreement only if variances of response functions are calculated as their second normalized central moments.     

Simultaneous determination of D- and L-serine in rat brain microdialysis sample using a column-switching HPLC with fluorimetric detection

Both D- and L-serine in rat brain microdialysis sample were simultaneously determined by pre-column fluorescence derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), separation of the derivatives on ODS column, TSKgel ODS-80TsQA, followed by Pirkle type chiral columns, Sumichiral OA-2500 (S), which gave a sufficient enantiomeric separation of NBD-D-serine and NBD-L-serine, and fluorimetric detection at a wavelength of 540 nm with an excitation wavelength of 470 nm. The peaks of NBD-D-serine and NBD-L-serine in the rat brain microdialysis sample were clearly found, and the validation study showed satisfactory results; the precision and accuracy were within 5.14 and 109%, respectively. Using the proposed HPLC method, the time-course profile of D-serine concentration in rat prefrontal cortex following intraperitoneal administration of D-serine was investigated. As a consequence, D-serine appeared to be rapidly distributed in the brain, and then decreased gradually with time in the extracellular fluid of the rat prefrontal cortex. The proposed HPLC method will be useful for in vivo studies on D-serine, which acts as a coagonist for N-methyl-D-aspartate receptor, to the extracellular fluid of rat brain.     

Development and validation of rapid and sensitive LC methods with PDA and fluorescence detection for determination of piribedil in rat plasma and brain tissues and their pharmacokinetic application

Simple, selective and sensitive high‐performance liquid chromatographic (HPLC) bioanalytical methods using fluorescence (FL) and photodiode array (PDA) detectors were developed and validated for determination of piribedil (PBD), an anti‐Parkinson's drug, in rat plasma and brain samples, with telmisartan as internal standard (IS). Protein precipitation technique was used to extract PBD from both biological matrices. Chromatographic separation was achieved on a Phenomenex Kinetex C₁₈ end‐capped column (250 × 4.6 mm, 5 μm), with 38:62 v/v acetonitrile and ammonium acetate buffer (pH 5.0) as mobile phase at 1.0 mL/min flow rate. Linear response in the concentration ranges 5–300 and 150–3000 ng/mL in plasma, and 15–900 and 450–9000 ng/g in brain tissue were achieved in FL and PDA detectors, respectively. The chromatograms were extracted at 239 nm in case of PDA detection and at excitation wavelength of 239 nm and emission wavelength of 385 nm in case of FL detection. FL detection was found to be more sensitive compared with PDA detection. The developed methods were successfully employed in determining the plasma time course, brain distribution and the pharmacokinetic parameters of PBD following intravenous bolus administration of the drug in male Wistar rats.     

Determination of ASP3258, a novel phosphodiesterase type 4 inhibitor,in rat plasma by high‐performance liquid chromatography with fluorescence detection and its application to pharmacokinetic study

The potent phosphodiesterase 4 inhibitor ASP3258 contains a carboxylic acid moiety and a naphthyridine ring and is a novel therapeutic agent for asthma and chronic obstructive pulmonary disease. To support the drug development of ASP3258, we developed and validated a simple method for its determination in rat plasma. Following the addition of the analog AS1406604‐00 as an internal standard, plasma samples were processed using C₁₈‐bonded solid‐phase extraction cartridges under acidic conditions and injected into a high‐performance liquid chromatography system with fluorescence detection. Chromatographic separation was achieved on a Shiseido Capcell Pak C₁₈ UG120 column (3.0 × 150 mm, 5 µm) with a mobile phase consisting of acetonitrile–0.5% acetic acid (50:50, v/v). HPLC eluent was monitored with a fluorescence detector set at a wavelength of 315 nm for excitation and 365 nm for emission. The calibration curve was linear over a range of 2.5–250 ng/mL. Validation data demonstrated that the method is selective, sensitive and accurate. In addition, the present method was successfully applied to rat plasma samples from a pharmacokinetic study. Copyright © 2014 John Wiley & Sons, Ltd.     

高效液相色谱-荧光检测法同时测定大鼠不同脑区中的8种单胺类神经递质

采用高效液相色谱-荧光检测法同时测定了大鼠不同脑区中的左旋多巴、去甲肾上腺素、肾上腺素、多巴胺、二羟基苯乙酸、5-羟色胺、5-羟基吲哚乙酸及高香草酸8种单胺类神经递质的含量。样品经组织裂解液(0.60 mol/L高氯酸、0.50 mmol/L Na2EDTA、0.1 g/L L-半胱氨酸的混合水溶液)处理后,冷冻离心得到上清液;上清液中加入高氯酸沉淀剂(1.20 mol/L K2HPO4、2.00 mmol/L Na2EDTA的混合水溶液)处理后,冷冻离心、过滤。在该优化的色谱条件下,8种单胺类神经递质在1.25～5000 μg/L范围内线性关系良好(r＞0.9999),最低检出限在0.20～5.00 μg/L之间,平均回收率在94.83%～99.19%之间,相对标准偏差在0.08%～2.51%之间。该方法具有操作简便、快捷、回收率高、检出限低、分离度好、结果准确可靠等优点,可以用于体内8种单胺类神经递质的同时检测与分析。     

Chromatographic behavior of cadmium in an ion-pair reversed-phase micro HPLC system and its application to the determination of bio-available cadmium in soil samples

A novel reversed phase ion-pair micro HPLC system with on-line fluorescence detection has been developed systematically and studied for the determination of cadmium in its bio-available fractions of soil samples. In this system, a micro ODS column of 1.0 mm i.d.×150 mm length and a mobile phase containing 6 mmol L^-1 8-hydroxyquinoline 5-sulphonic acid (HQS), 3 mmol L^-1 cetyltrimethylammonium bromide (CTMABr), 10 mmol L^-1 acetic acid-acetate buffer (pH 5.1) as well as 50% acetonitrile at 50 L min^-1 flow rate were employed to determine cadmium with a 2 L flow cell through its fluorescence at 518 nm under 338 nm excitation. Furthermore, the composition of Cd-HQS chelate formed on the column was confirmed to be [Cd(HQS)₂]^2- through a log–log plot method, and then combined with the ion-pair reagent by the electrostatic force under the chromatographic condition proposed. With such a method, the detection limit of cadmium was 8.48 ng mL^-1 (3) with 1 L sample injection, and the linear range for the determination of cadmium was 30–800 ng mL^-1 (R²=0.992). This method has been successfully applied to determination of cadmium in its bio-available fractions of BCR-483 and soil samples without interference from other coexistent metal ions. The RSD (n=6) was less than 7.3%. The results were in agreement with the indicative value for BCR-483 and those for the soil samples obtained by ICP-MS with a pretreatment of bis(1,1,3,3-tetramethylbutyl) phosphinic acid extraction.     

Development and validation of a HPLC method for simultaneous quantitation of gatifloxacin, sparfloxacin and moxifloxacin using levofloxacin as internal standard in human plasma: application to a clinical pharmacokinetic study

A highly selective, sensitive and accurate HPLC method has been developed and validated for the estimation of three fluoroquinolones (FQs) viz., gatifloxacin (GFC), sparfloxacin (SFC) and moxifloxacin (MFC) with 500 microL human plasma using levofloxacin (LFC) as an internal standard (IS). The sample preparation involved simple liquid-liquid extraction of GFC, SFC, MFC and IS from human plasma with ethyl acetate. The resolution of peaks was achieved with phosphate buffer (pH 2.5)-acetonitrile (80:20, v/v) at a flow rate of 1 mL/min on a Kromasil C(18) column. The total chromatographic run time was 18.0 min and the simultaneous elution of GFC, SFC, MFC and IS occurred at approximately 10.8, 12.8, 17.0 and 6.0 min, respectively. The method proved to be accurate and precise at linearity range of 100-10,000 ng/mL with a correlation coefficient (r) of > or =0.999. The limit of quantitation for each of the FQs studied was 100 ng/mL. The intra- and inter-day precision and accuracy values found to be within the assay variability limits as per the FDA guidelines. The developed assay method was applied to a pharmacokinetic study in human volunteers following oral administration of 400 mg GFC tablet.     

Comparison of different sample treatments for the analysis of quinolones in milk by capillary-liquid chromatography with laser induced fluorescence detection

A simple and very sensitive capillary-liquid chromatography method coupled with laser induced fluorescence detection has been developed for the simultaneous determination of seven quinolones of veterinary use in milk. Moreover, a comparison between two different sample treatments (QuEChERS and molecularly imprinted polymer, MIP) has been carried out in terms of efficiency of the extraction (number of analytes to be analysed and absence of interferences), throughput, linear dynamic range in matrix-matches calibrations, detection and quantification limits and accuracy (trueness and precision, by means of recovery assays). The results showed that the QuEChERS procedure was more efficient and faster, showing good recoveries, sensitivity and precision for all the studied compounds. Employing this proposed method, very low detection limits, between 0.4 μg/kg for danofloxacin, and 6 μg/kg for sarafloxacin, have been obtained.     

Determination of N-acetylaspartic acid concentration in the mouse brain using HPLC with fluorescence detection

The concentration of brain N-acetylaspartic acid (NAA) in mice was determined by high-performance liquid chromatography (HPLC) using fluorescence detection after pre-column derivatization with 4-N,N-dimethylaminosulfonyl-7-N-(2-aminoethyl)amino-2,1,3-benzoxadiazole (DBD-ED). Six different brain parts, namely, the prefrontal cortex, olfactory bulb, nucleus accumbens, striatum, cerebellum and hippocampus, of male C57BL6/J mice, were investigated. The NAA concentration (nmol/mg protein) was highest in the olfactory bulb (58.2 ± 4.0, n = 8) and lowest in the hippocampus (42.8 ± 1.6, n = 8). The proposed HPLC method with fluorescence detection was successfully used to determine the NAA concentration in each investigated brain area.     

Optimization of a sensitive method for the determination of nitro musk fragrances in waters by solid-phase microextraction and gas chromatography with micro electron capture detection using factorial experimental design

A solid-phase microextraction method (SPME) followed by gas chromatography with micro electron capture detection for determining trace levels of nitro musk fragrances in residual waters was optimized. Four nitro musks, musk xylene, musk moskene, musk tibetene and musk ketone, were selected for the optimization of the method. Factors affecting the extraction process were studied using a multivariate approach. Two extraction modes (direct SPME and headspace SPME) were tried at different extraction temperatures using two fiber coatings [Carboxen–polydimethylsiloxane (CAR/PDMS) and polydimethylsiloxane–divinylbenzene (PDMS/DVB)] selected among five commercial tested fibers. Sample agitation and the salting-out effect were also factors studied. The main effects and interactions between the factors were studied for all the target compounds. An extraction temperature of 100 °C and sampling the headspace over the sample, using either CAR/PDMS or PDMS/DVB as fiber coatings, were found to be the experimental conditions that led to a more effective extraction. High sensitivity, with detection limits in the low nanogram per liter range, and good linearity and repeatability were achieved for all nitro musks. Since the method proposed performed well for real samples, it was applied to different water samples, including wastewater and sewage, in which some of the target compounds (musk xylene and musk ketone) were detected and quantified. Figure Stardardized Pareto charts for the main effects and interactions     

Organic heterocyclic-based colorimetric and fluorimetric chemosensors for the detection of different analytes: a review (from 2015 to 2022)

Heterocyclic organic compounds, also called heterocycles, are any major class of organic compounds having at least one atom other than carbon in the ring. Due to their excellent electronic and structural features, these compounds exhibit a wide range of biological and nonbiological applications. Among these, indole, benzimidazole, benzothiazole, and benzoxazole are versatile organic heterocyclic compounds widely used in different fields. They show a wide range of applications in polymer, coordination chemistry, pharmacy, dyes, food packages, medicine, and industries. These compounds contain heteroatoms like S-, N-, and O-, through which they interact with metal ions, anions, and neutral species, giving measurable analytical signals that can be used as fluorimetric and colorimetric chemosensors for detecting different analytes in biological, agricultural and environmental samples. This review summarizes indole, benzimidazole, benzothiazole, and benzoxazole-based fluorimetric and colorimetric chemosensors for detecting metal ions, anions, and neutral species. Furthermore, the recognition mechanisms have been discussed in detail, which could help researchers to design efficient, highly selective, and sensitive chemosensors to recognize and determine heavy metal cations.     

Optimizing high-performance liquid chromatography method for quantification of glucosamine using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate derivatization in rat plasma: application to a pharmacokinetic study

A sensitive and reliable HPLC method with fluorescence detection based on the precolumn derivatization of glucosamine with 6-aminoquinolyl-N-hydroxylsuccinimidyl carbamate (AQC) was established for the quantitative determination of glucosamine in rat plasma. The plasma protein was precipitated by acetonitrile, followed by vortex mixing and centrifugation. The supernatant was divided into the organic layer and aqueous layer by adding sodium chloride, and then the aqueous layer was derivatized with AQC in 0.2 M borate buffer of pH 8.8 before the HPLC analysis. An amino acid analysis column (3.9 x 150 mm, 4 microm) was applied, with 140 mM sodium acetate buffer (pH = 5.25) and acetonitrile as mobile phase at a flow rate of 1 mL/min. A linear correlation coefficient of 0.9987 was calculated within the range of 0.1-30 microg/mL of the standard curve for glucosamine. The limit of detection was 30 ng/mL. The intra- and inter-day precisions (as RSD) were less than 7.38 and 12.72%, respectively. The intra- and inter-day accuracy ranged from 91.8 to 110.0%. Extraction recoveries of glucosamine in plasma were more than 90%. The validated method was successfully applied for the quantitative determination of glucosamine in rat plasma and evaluation for pharmacokinetic study of glucosamine. It was also possible to be applied for the quantitative determination of other compounds containing amino group in biological samples.     

Sensitive sequential-injection system for the determination of 2-phenylbenzimidazole-5-sulphonic acid in human urine samples using on-line solid-phase extraction coupled with fluorimetric detection

2-Phenylbenzimidazole-5-sulphonic acid (PBS) is an UV-filter contained in many cosmetics as a sunscreen. A direct, selective and sensitive method to determine traces of PBS is presented. The on-line separation of this compound from urine matrix was directly coupled with fluorimetric detection in a sequential-injection system. The separation was performed using a SAX microcolumn in which the analyte was retained and eluted selectively. The determination is carried out without any derivatization reaction, by directly measuring the intrinsic fluorescence of the analyte. The wavelengths of excitation and emission were 301 and 681 nm, respectively. On-line standard addition calibration is performed into the system, and only one standard solution is required. The limit of detection was 12 ng ml⁻¹. The method was satisfactorily used to determine PBS in both, spiked and unspiked human urine samples, without any pretreatment. The relative standard deviations of the results were in the order of 2-13%. The concentrations of the analyte obtained for unspiked samples taken from sunscreen users were higher than the limit of detection.     

Determination of ascorbic acid in human plasma with a view to stability using HPLC with UV detection

A method for the measurement of ascorbic acid using HPLC with UV detection and investigation into the protein precipitation techniques with regard to stability and recovery are described. The effectiveness of various protein precipitants was tested. Stability of ascorbic acid samples for analysis was investigated over 10 h. Ascorbic acid samples extracted with metaphosphoric acid were stable on a cooled autosampler (4 degrees C) for at least 10 h (with a decline of 1.8% for ascorbic acid solution and 2.8% for plasma). Perchloric acid as protein precipitant for ascorbic acid was unsuitable (with a decline of 36.0% for ascorbic acid solution and 7.3% for plasma). Analytical performance of this method is satisfactory. The intra- and interassay coefficients of variation were 2.1% (n = 10) and 5.8% (n = 12), respectively. The calibration curve was linear with the tested range of 2.0-250.0 micromol/L. The recovery was 96.1% with CV = 4.8% (n = 6) and the LOD was 3 micromol/L. The preliminary reference ranges of ascorbic acid in a group of blood donors are 50.8 +/- 22.4 micromol/L. This assay is a highly sensitive and reproducible HPLC method for the determination of ascorbic acid in human plasma.     

Monitoring arsenic in the environment: a review of science and technologies with the potential for field measurements

This review examines available field assays and other technologies with the potential to measure and monitor arsenic in the environment. The strengths and weaknesses of the various assays are discussed with respect to their sensitivity, ability to detect the chemical states of arsenic, performance in various media, potential interferences, and ease of operation. The state of the science and development efforts of selected technologies is presented.     

Optimization of two methods for the analysis of hydrogen peroxide: high performance liquid chromatography with fluorescence detection and high performance liquid chromatography with electrochemical detection in direct current mode

Two complementary methods were optimized for the separation and detection of trace levels of hydrogen peroxide. The first method utilized reversed-phase high-performance liquid chromatography with fluorescence detection (HPLC-FD). With this approach, hydrogen peroxide was detected based upon its participation in the hemin-catalyzed oxidation of p-hydroxyphenylacetic acid to yield the fluorescent dimer. The second method utilized high performance liquid chromatography with electrochemical detection (HPLC-ED). With this approach, hydrogen peroxide was detected based upon its oxidation at a gold working electrode at an applied potential of 400 mV vs. hydrogen reference electrode (Pd/H(2)). Both methods were linear across the range of 15-300 μM, and the electrochemical method was linear across a wider range of 7.4-15,000 μM. The limit of detection for hydrogen peroxide was 6 μM by HPLC/FD, and 0.6 μM by HPLC/ED. A series of organic peroxides and inorganic ions were evaluated for their potential to interfere with the detection of hydrogen peroxide. Studies investigating the recovery of hydrogen peroxide with three different extraction protocols were also performed. Post-blast debris from the detonation of a mixture of concentrated hydrogen peroxide with nitromethane was analyzed on both systems. Hydrogen peroxide residues were successfully detected on this post-blast debris.