Liquid Chromatography-fluorescence Detection Method Research Articles

High Performance Liquid Chromatography Fluorescence Method for the Determination of Seleno-Amino Acids in Ovine Blood Plasma

This study was performed to validate an effective high performance liquid chromatography-fluorescence detection method for the separation of seleno-amino acids and sulfur amino acids in biological samples using pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. Standard calibration and standard addition procedures were developed, but the quantification of seleno-amino acids was carried out by a second method. The coefficient of variation for precision was < 15% and accuracies were 96 to 98%. Recoveries in the extraction method were between 63 and 99%. This method was applied to the contribution of seleno-amino acids containing methylselenocysteine and selenomethionine in plasma tissue of lambs during infusion study. The method demonstrated good selectivity, sensitivity, precision, and accuracy.

An improved high performance liquid chromatography-fluorescence detection method for the analysis of major phenolic compounds in cigarette smoke and smokeless tobacco products

An improved HPLC method has been developed for the determination of major phenolic compounds in cigarette smoke. A novel reversed phase column with a pentafluorophenylpropyl (PFP) ligand in the stationary phase was chosen to separate the positional isomers (p-, m-, and o-cresols). Methanol instead of acetonitrile was used as the organic mobile phase component to improve the separation of the isomers and cope with the crisis of global acetonitrile shortage in 2009. A shorter analytical column with smaller particle size was used to further increase separation efficiency and reduces solvent consumption. These improvements have led to a new HPLC method that is simpler and faster than the GC–MS method and more sensitive, selective and efficient than the widely used traditional HPLC method. The limit of detection (LOD) and limit of quantification (LOQ) of this method are at the ng/mL level for most of the phenols with good linearity (R2≥0.999) and precision (RSD<15%). The method has been validated using reference tobacco products. The mainstream and sidestream cigarette smoke yields of phenolic compounds obtained by this method are comparable to those obtained by traditional HPLC method with the advantage that p-, m-, and o-cresols can be determined and reported separately by the new method. The method can also be applied for analysis of phenols in smokeless tobacco product.

Pharmacokinetics of Mirtazapine and Its Main Metabolites after Single Oral Administration in Fasting/Fed Horses

Mirtazapine (MRZ) is a human antidepressant drug that is metabolized, predominantly by the cytochrome P450 enzyme system, to 8-OH mirtazapine (8-OH MRZ) and dimetilmirtazapine (DMR) metabolites. In veterinary medicine, this drug is currently administered to cats and dogs with anorexia, although it could also have applications as an antidepressant, antiemetic, and analgesic agent in these species. The aim of this study was to assess the pharmacokinetics of MRZ and its metabolites DMR and 8-OH MRZ in horses. Six healthy female horses were administered MRZ (2 mg/kg) in fasting and fed states according to a balanced crossover study design. Plasma MRZ and metabolite concentrations were evaluated by high-performance liquid chromatography fluorescence detection method. Pharmacokinetic profiles of MRZ and DMR were similar (detected from 0.5 up to 34 and 48 hours, respectively), with an MRZ AUC0-N/DMR AUC0-N ratio range varying between 1.1 and 1.7. Surprisingly, 8-OH MRZ was undetected. Most of the pharmacokinetic parameters were not altered by food, with the exception of the time required to reach maximum concentration; this showed a statistical increase in subjects in the fasting state as compared with the fed state. However, because MRZ is an active substance intended for long-term administration, the slight increase of the time required to reach maximum concentration is not considered to be of any clinical consequence. In conclusion, the pharmacokinetic parameters demonstrated in this study suggest that MRZ is suitable for oral administration in the horse. However, further investigations are required to evaluate both its safety and effectiveness in this animal species.

CONFIRMATION OF THE ABSENCE OF BISPHENOL A IN COPOLYESTERS BY POLYMER HYDROLYSIS IN COMBINATION WITH LC-MS/MS AND LC-FLD

A method was developed to validate that certain polyester and copolyester resins do not contain bisphenol A (BPA). This method satisfies the need for a timely screening method that can be utilized on a timescale that is appropriate for a laboratory in a manufacturing facility. This was accomplished by hydrolyzing the polymer and then analyzing the hydrolysate by either liquid chromatography-tandem mass spectrometry (LC-MS/MS) or liquid chromatography-fluorescence detection (LC-FLD). A hydrolysis procedure offers the unique advantage of a faster sample preparation step as compared to extraction procedures. The hydrolysis was complete after 20 min and the procedure was fully automated with the use of an autosampler. By hydrolyzing the polymer, total BPA is measured rather than only extractable monomeric BPA. Copolyester samples were purposely spiked with polycarbonate to validate the method. The detection limit for the LC-MS/MS method was 0.8 mg kg−1 in the polymer, while the detection limit for the LC-FLD method was 2.6 mg kg−1 in the polymer.

Refinement of AOAC Official MethodSM 2005.06 Liquid Chromatography-Fluorescence Detection Method to Improve Performance Characteristics for the Determination of Paralytic Shellfish Toxins in King and Queen Scallops

AOAC Official Method 2005.06 LC-fluorescence detection (FLD) method is an official alternative to the mouse bioassay for the determination of paralytic shellfish poisoning (PSP) toxins in bivalve shellfish. To validate the method for species of relevance to the UK official control monitoring program, the method performance characteristics were tested for whole king and queen scallops. Validation showed that, while the performance was generally acceptable for the quantitation of non-N-hydroxylated toxins, poor toxin recovery and sensitivity was evident for the analysis of N-hydroxylated toxins following periodate oxidation. These effects occurred in a range of scallop samples with variable temporal and spatial sources. The effects were also noted in other laboratories following a small interlaboratory study. As a result, the method was refined to improve the recovery and sensitivity of analysis following the periodate oxidation step in the PSP method for scallops. Performance improved through alterations to the preparation of the periodate oxidant, use of higher volumes for C18 cleanup, and injection volumes in combination with the use of a king scallop matrix modifier for oxidation of N-hydroxylated toxin calibration standards. A single-laboratory validation of the refined method showed that the selectivity, linearity, sensitivity, recovery, and precision were acceptable and similar to values reported previously for AOAC Official Method 2005.06 in other bivalve species. Results showed the method to be rugged for all parameters investigated, including small changes to the composition of the new periodate reagent utilized in the refined method. The refined scallops LC method was subsequently compared with the European reference method. PSP-positive scallops showed an excellent agreement between the methods for queen and Atlantic scallops, with a small level of positive bias in the LC results for whole king scallops. These differences were related solely to the use of the highest toxicity equivalence factors for toxin epimeric pairs, with gonyautoxin (GTX)1,4 and GTX2,3 in particular present at high concentrations in the king scallops. Overall, the refined LC-FLD method improved the performance characteristics of AOAC Official Method 2005.06 for the determination of PSP toxins in whole king and queen scallops, and showed a good overall agreement between the official methodologies. It is, therefore, recommended as a more appropriate option for the routine monitoring of PSP toxins in these species.

Occurrence and Risk Assessment of Four Typical Fluoroquinolone Antibiotics in Raw and Treated Sewage and in Receiving Waters in Hangzhou, China

A sensitive liquid chromatography-fluorescence detection method, combined with one-step solid-phase extraction, was established for detecting the residual levels of the four typical fluoroquinolone antibiotics (ofloxacin, norfloxacin, ciprofloxacin, and enrofloxacin) in influent, effluent, and surface waters from Hangzhou, China. For the various environmental water matrices, the overall recoveries were from 76.8 to 122%, and no obvious interferences of matrix effect were observed. The limit of quantitation of this method was estimated to be 17 ng/L for ciprofloxacin and norfloxacin, 20 ng/L for ofloxacin, and 27 ng/L for enrofloxacin. All of the four typical fluoroquinolone antibiotics were found in the wastewaters and surface waters. The residual contents of the four typical fluoroquinolone antibiotics in influent, effluent, and surface water samples are 108-1405, 54-429, and 7.0-51.6 ng/L, respectively. The removal rates of the selected fluoroquinolone antibiotics were 69.5 (ofloxacin), 61.3 (norfloxacin), and 50% (enrofloxacin), indicating that activated sludge treatment is effective except for ciprofloxacin and necessary to remove these fluoroquinolone antibiotics in municipal sewage. The risk to the aquatic environment was estimated by a ratio of measured environmental concentration and predicted no-effect concentration. At the concentrations, these fluoroquinolone antibiotics were found in influent, effluent, and surface waters, and they should not pose a risk for the aquatic environment.

High‐performance liquid chromatographic determination of memantine hydrochloride in rat plasma using sensitive fluorometric derivatization

In this study, we investigated a simple, sensitive and reliable liquid chromatography-fluorescence detection method for the determination of memantine hydrochloride in rat plasma which was based on derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl). For the first time, FMOC-Cl was introduced into derivatization of memantine hydrochloride in rat plasma. The amino groups of memantine hydrochloride and amantadine hydrochloride (internal standard) were trapped with FMOC-Cl to form memantine hydrochloride-FMOC-Cl and amantadine hydrochloride-FMOC-Cl compositions, which can be very compatible for LC-FLD. Precipitation of plasma proteins by acetonitrile was followed by vortex mixing and centrifugation. Chromatographic separation was performed on a C(18) column (DIAMONSIL 150 × 4.6 mm, id 5 μm) with a mobile phase consisting of acetonitrile and water at a flow rate of 1.0 mL/min. The retention times of memantine hydrochloride-FMOC-Cl and amantadine hydrochloride-FMOC-Cl compositions were 23.69 and 40.27 min, respectively. Optimal conditions for the derivatization of memantine hydrochloride were also described. The limit of quantification (LOQ) was 25 ng/mL for memantine hydrochloride in plasma, the linear range was 0.025-5.0 μg/mL in plasma with a correlation coefficient (r) of 0.9999. The relative standard deviations (RSDs) of intra-day and inter-day assays were 4.46-12.19 and 5.23-11.50%, respectively. The validated method was successfully applied to the determination of memantine hydrochloride in rat plasma samples.

S100B Transgenic Mice Develop Features of Parkinson's Disease

Parkinson's disease (PD) is one of the most common neurodegenerative disorders. Until now, the cause and mechanism of PD are unknown, making further studies necessary. We undertook this study to establish the brain-specific S100B gene transgenic mice and investigate the role of S100B in the development of PD. The hS100B transgenic vector was constructed by inserting the human S100B gene downstream into platelet-derived growth factor (PDGF) promoter, followed by microinjection to produce transgenic mice. Motor coordination ability of mice in the S100B transgenic group (TG), S100B knockout group (KG) and the non-transgenic control group (CG) were measured by the Rota-rod test. The expressions of dopamine D1 receptor (D1DR), dopamine D2 receptor (D2DR), G protein-coupled receptor kinase2 (GRK2), G protein-coupled receptor kinase5 (GRK5), tyrosine hydroxylase (TH) in the brain tissue, and levels of dioxyphenylalanine (DOPA), dopamine (DA), homovanillic acid (HVA), 5-hydroxytryptamine (5-HT), and 5-hydroxyindoleacetic acid (5-HIAA) in the midbrain tissue were detected by RT-PCR, Western blotting, and high-performance liquid chromatography-fluorescence detection method (HPLC-FLD), respectively. Compared with CG, in TG, the motor coordination ability of mice, expressions ofD2DR and GRK2, and the level of 5-HT visibly decreased, while the levels of DOPA, DA and its metabolic product HVA increased, the expressions of D1DR, GRK5, TH and 5-HIAA were similar. Compared with CG, no obvious change of detection indexes was observed in KG. Overexpression of S100B in the brain resulted in motor coordination impairment, which may have resulted from the downregulation of D2DR and GRK2 expressions, increased DA synthesis and metabolism, and decreased 5-HT level. Therefore, S100B may be a potential cause of pathogenesis in PD.

Temporal and periovulatory changes in ovarian catecholamines in the catfish Heteropneustes fossilis

A high performance liquid chromatography-fluorescence detection method was employed to demonstrate temporal and periovulatory changes in ovarian catecholamines in the catfish Heteropneustes fossilis. Tyrosine, l-DOPA, dopamine (DA), norepinephrine (NE) and epinephrine showed significant seasonal and diurnal changes during the reproductive cycle. A high concentration of tyrosine was detected, the values increased in day and decreased in night during recrudescence from preparatory to spawning phases. Similarly, l-DOPA increased during the breeding phase giving the day value concentration peak in the spawning phase and the night value peak in the postspawning phase. DA activity or turnover index (calculated as a ratio of DA to 3,4-dihydroxyphenylacetic acid, DOPAC) showed a bimodal pattern with the major activity peak in the postspawning phase and the minor one in the prespawning phase, the spawning phase registered the lowest activity. NE activity or turnover index (ratio of NE to normetanephrine, NME) increased during the recrudescent phase to give the peak in the spawning phase and decreased in the quiescent phase. Epinephrine elicited an inverse relationship in the day–night pattern, the day values increased to the peak in the spawning phase. All the study correlates showed significant periovulatory changes after hCG treatment. DA activity dropped to the nadir at 8 h but recovered at 16 and 24 h. NE activity showed only a marginal decrease up to 16 h but decreased drastically at 24 h. Epinephrine levels remained unchanged but only to increase at 24 h. The seasonal patterns and periovulatory changes strongly point to important functions for catecholamines in the ovary. The occurrence of tyramine (an invertebrate neurotransmitter) suggests the presence of alternate catecholamine pathway in fish ovary.

Development of a high-performance liquid chromatography-fluorescence detection method for analyzing nonylphenol/dinonylphenol-polyethoxylate-based phosphate esters

A novel reversed phase HPLC method for the simultaneous analysis of surfactants containing nonylphenol/dinonylphenol-polyethoxylates and their o-phosphate esters is reported, in which eluting substances are detected fluorescently. Their chemical structures were elucidated by direct infusion electrospray-mass spectrometry in positive mode. The limits of quantification and range of the method were determined to be 0.1 mg and 0.1–100 mg surfactant L −1, respectively, with a reproducibility (RSD) at a concentration of 38 mg surfactant L −1 of 5.6%. The accuracy was determined by spiking selected process water samples with known amounts of surfactant, and recoveries were typically in the 82–102% range.

Method performance and multi-laboratory assessment of a normal phase high pressure liquid chromatography–fluorescence detection method for the quantitation of flavanols and procyanidins in cocoa and chocolate containing samples

The quantitative parameters and method performance for a normal-phase HPLC separation of flavanols and procyanidins in chocolate and cocoa-containing food products were optimized and assessed. Single laboratory method performance was examined over three months using three separate secondary standards. RSD (r) ranged from 1.9%, 4.5% to 9.0% for cocoa powder, liquor and chocolate samples containing 74.39, 15.47 and 1.87 mg/g flavanols and procyanidins, respectively. Accuracy was determined by comparison to the NIST Standard Reference Material 2384. Inter-lab assessment indicated that variability was quite low for seven different cocoa-containing samples, with a RSD (R) of less than 10% for the range of samples analyzed.

Simple high-performance liquid chromatography—fluorescence detection method for plasma, kidney and liver of rat as a tool for toxicology studies

A fast and simple HPLC-FLD (high-performance liquid chromatography-fluorescence detection) analytical method has been developed and validated for the determination of ochratoxin A in rat plasma, kidney and liver. The extraction method, calibration curves and chromatographic conditions are common for the three matrices. Plasma and homogenized tissue samples (250 μL) were extracted with ethanol (400 μL) and trichloroacetic acid 20% (w/v) (50 μL). Supernatants were directly injected into the HPLC system, analyzed on a 5-μm (25 cm × 0.4 cm) Tracer Extrasil ODS2 column using FLD (excitation wavelength = 225 nm, emission wavelength = 461 nm). The mobile phase was 29:29:42 (v/v) methanol–acetonitrile–sodium acetate. The small volume of sample needed which allows the obtaining of ochratoxin A levels in individual tissue samples from small animals and the wide range of concentrations that could be analyzed make this method easy to apply in toxicology and toxicokinetic studies of this mycotoxin, even in low dose carcinogenic studies. This method was linear and selective for all the matrices. Precision and accuracy were always <10% and recovery was very efficient in each case. Limits of detection and quantification were also calculated in plasma (1 and 8.4 μg/L), kidney (14.3 and 55.8 μg/kg) and liver (4.1 and 52.8 μg/kg). Stability of the tissue homogenates was assured for at least 10 months at −80 °C. The method has been successfully applied to the analysis of rat samples after 7 days of ochratoxin A (0.5 mg/kg b.w. dissolved in an aqueous NaHCO 3 solution) administration by oral gavage.

Validation of a high-performance liquid chromatographic method with fluorescence detection for the simultaneous determination of tetracyclines residues in bovine milk

A high-performance liquid chromatography-fluorescence detection method was optimized and validated to determine tetracyclines residues in bovine milk. Post-column derivatization using metal complexation in non-aqueous reagent increased the fluorescence of chelates by a factor up to 2.54 compared to water (signal-to-noise ratio enhancement). Overall recoveries ranged from 61 to 115%, with RSD r from 5 to 15% ( n = 54). Detection limits ranged from 5 to 35 μg kg −1. Limits of quantification were established at 50 μg kg −1. Decision limits (CCα) were 109, 108 and 124 μg kg −1 and detection capabilities (CCβ) 119, 117 and 161 μg kg −1 for oxytetracycline, tetracycline and chlortetracycline, respectively. The method was applied successfully in a national monitoring program.

Supercritical fluid extraction and high-performance liquid chromatography–fluorescence detection method for polycyclic aromatic hydrocarbons investigation in vegetable oil

Abstract In spite of the fact that food processes, that involve drying and smoking, may cause polycyclic aromatic hydrocarbon contamination, an extraction clean/up procedure carried out by SFE was developed in order to isolate polycyclic aromatic hydrocarbons from oil vegetable samples for subsequent HPLC–FL determination. The detection and quantification limits obtained were −1 oil and −1 oil, respectively, allowed to check the presence of seven of the eight PAHs with legal limit in olive–pomace oil: benzo[ a ]anthracene, benzo[ e ]pyrene, benzo[ b ]fluoranthene, benzo[ k ]fluoranthene, benzo[ a ]pyrene, dibenzo[ ah ]anthracene and benzo[ ghi ]perilene. In brief, the method permits the evaluation of edible oil safety and, therefore, consumers protection.

High-performance liquid chromatography-fluorescence detection method for endogenous gamma-aminobutyric acid validated by mass spectrometric and gas chromatographic techniques.

High-performance liquid chromatography-fluorescence detection method for endogenous gamma-aminobutyric acid validated by mass spectrometric and gas chromatographic techniques.

Quantitative analysis of methylguanidine and guanidine in physiologic fluids by high-performance liquid chromatography—fluorescence detection method

A rapid and sensitive high-performance liquid chromatographic method has been developed for the quantitative analysis of methylguanidine and guanidine in physiological fluids. These guanidino compounds are separated on a 6 × 0.23 cm cation-exchange column with 0.5 M sodium hydroxide solution. The guanidino compounds are detected with a fluorometer, which monitors the fluorescent guanidine derivatives produced by the reaction of the eluted constituents with 9,10-phenanthrenequinone. Sensitivity to sub-nanomole levels of methylguanidine and guanidine is demonstrated. The method was successfully applied to physiological fluids such as serum and cerebrospinal fluid from uremic patients.