HPLC With Fluorescence Detection Research Articles

Extensive Evaluation of a Method for Quantitative Measurement of Aflatoxins B1 and M1 in Animal Urine Using High-Performance Liquid Chromatography with Fluorescence Detection.

Aflatoxins (AFs) are common feed contaminants and are one of the common causes of toxin-related pet food poisoning and recalls. Currently, there are no validated methods for the detection and quantitation of AFs in biological matrices to diagnose AF exposure in live animals. Following a successful intra-laboratory method development to quantify AFB1 and AFM1 in animal urine by HPLC with fluorescence detection (HPLC-FLD), the present study was conducted to extensively evaluate the method performance in an unbiased manner using blinded samples. The evaluation included two stages. First, the performance was verified in the method-originating laboratory in a single-laboratory blinded method test (BMT-S) trial followed by a multi-laboratory blinded method test (BMT-M) trial. In both trials, accuracy, repeatability, and reproducibility were satisfactory confirming the relatively good ruggedness and robustness of the method and ensuring that it will perform as expected if used by other laboratories in the future. We extensively evaluated the performance of a quantitative method to detect AFB1 and AFM1 in animal urine by HPLC-FLD by two different laboratories in two separate BMT-S and BMT-M trials. Both BMT results demonstrated the satisfactory accuracy and precision of the method. It is now available to be adopted by other diagnostic laboratories for purposes of diagnosing AF intoxication in animals. A simple urine-based diagnostic test method using HPLC-FLD that originated in a single laboratory now has passed a multi-laboratory evaluation and is now available to be shared with other diagnostic laboratories for purposes of diagnosing AF intoxication in animals so better treatment can be rendered.

Identification of potential migrants from epoxy and organosol coatings used in metal food cans

The coatings of metal cans may release complex mixtures of migrants into the contained foods, including non-intentionally added substances (NIAS), such as reaction products. All migrating substances should be studied to demonstrate their safety. In this work, the characterisation of two epoxy and organosol coatings was performed using several techniques. Firstly, the type of coating was identified using FTIR-ATR. Screening techniques based on purge and trap (P&T) and solid-phase microextraction (SPME) coupled to GC-MS were used to investigate volatiles from coatings. For the identification of semi-volatile compounds, an appropriate extraction was performed before analysis by GC-MS. The most abundant substances were compounds with at least one benzene ring and an aldehyde or alcohol group in their structures. Furthermore, a method to quantify some of the identified volatiles was explored. Secondly, HPLC with fluorescence detection (HPLC-FLD) was used to determine non-volatile compounds such as bisphenol analogues and bisphenol A diglycidyl ethers (BADGEs), with subsequent confirmation by LC-MS/MS. Additionally, migration assays were performed by this technique to determine non-volatile compounds migrating into food simulants. Bisphenol A (BPA) and all BADGE derivatives except BADGE.HCl were detected in the migration extracts. Moreover, BADGE-solvent complexes such as BADGE.H2O.BuEtOH, BADGE.2BuEtOH, etc. were also tentatively identified using the accurate mass provided by time-of-flight mass spectrometry (TOF-MS).

Highly Sensitive Determination for Catecholamines Using Boronate Affinity Polymer Monolith Microextraction with In-Situ Derivatization and HPLC Fluorescence Detection

Catecholamines such as dopamine, norepinephrine and epinephrine are important neurotransmitters containing cis-diol moieties, and HPLC with fluorescence detection (HPLC-FD) has been successfully used as highly sensitive detection method for them. However, the complex matrix of biological samples is always a challenge. Boronate affinity microextraction is practical to purify and enrich cis-diol compounds. In this study, we used in-situ derivatization in boronate affinity polymer monolith microextraction (BA/PMME) to combine sample pretreatment and derivatization, then followed by HPLC-FD to develop a highly sensitive and selective determination method for catecholamines. Taking poly(3-acrylamidophenyl boronic acid-co-ethylene dimethacrylate) as BA/PMME material and TMBB-Su as labeling reagent, catecholamines could be derivatizated in polymer monolith in-situ together with the sample enrichment and purification. Comparing with commonly used solid phase extraction followed by derivatization, fewer operation steps are required and higher sensitivity was obtained with LODs as low as 0.06–0.2 nM.

A simple blood microdialysis in freely-moving rats for pharmacokinetic–pharmacodynamic modeling study of Shengmai injection with simultaneous determination of drug concentrations and efficacy levels in dialysate

Microdialysis is a powerful in vivo sampling technique for pharmacokinetic–pharmacodynamic (PK-PD) modeling of drugs in pre-clinical and clinical studies. However, the noticeable limitations of previous studies using microdialysis were that animals anesthesia in the whole experiment and the combination of microdialysis and blood sampling for drug and (or) effect detection, which can obviously influence PK and PD behavior of drugs. In this study, a simple blood microdialysis sampling system in freely-moving rats was established for simultaneous study of PK and PD of Shengmai injection (SMI) effect on inducing real-time nitric oxide (NO) release on isoproterenol (ISO) induced myocardial ischemia rats. The LC–MS/MS and HPLC with fluorescence detection (HPLC-FLD) methods were developed to determine ginsenside Rg1, Rg2, Re, Rf, Rb1, Rd and Rc, the main effective components of SMI, and NOx−, the main oxidation products of NO, in dialysates respectively. Through simultaneous determination of drug concentrations and NO efficacy levels in dialysate, the developed methods were successfully applied to set up concentration-time and effect-time profiles followed by PK-PD modeling of SMI effect on inducing NO release after intravenous administration of 10.8 mL kg−1 SMI in myocardial ischemia rats. The PK-PD modeling characterized the dose-effect relationships of SMI and behaved good prediction ability. The established blood microdialysis in freely-moving rats is an appealing technology for rational PK-PD studies when selecting suitable blood endogenous micromolecule as effect marker.

The occurrence of ochratoxin A in white and parboiled rice

Toman J., Maliř F., Ostrý V., Grosse Y., Dvořak V., Roubal T., Neuchlova L. (2016): The occurrence of ochratoxin A in white and parboiled rice. Czech J. Food Sci., 34: 32-38. The levels of ochratoxin A (OTA), one of the five agricultural and toxicological most important mycotoxins, have been determined by HPLC with fluorescence detection (HPLC-FLD) or enzyme-linked immunosorbent assay (ELISA) in 60 samples of white and parboiled rice, purchased on the Czech food market. In all 60 samples of rice tested by HPLC-FLD. OTA levels were below the limit of quantification (LOQ), i.e. < 0.2 ng/g. The same samples were also tested by ELISA (LOQ = 0.05 ng/g), and 58 samples (96.7%) were found to be positive. OTA levels in white and parboiled rice ranged from 0.05 to 0.17 ng/g; the arithmetic mean for all samples was 0.15 ng/g, the median 0.15 ng/g, and the 90% percen - tile 0.17 ng/g. These findings confirm the necessity of monitoring OTA in different foodstuffs and food, due to the precautionary principle and the uncertainty in the genotoxicity status of OTA.

Influence of Hot Spring Water on Fatty Acid Composition of Skin Surface Lipids in Hairless Mouse Model of Atopic Dermatitis.

When hairless NCN24 mice with atopic dermatitis (AD) were sprayed with a petroleum-containing alkaline salt spring water rich in metaboric acid and sodium bicarbonate, AD symptoms diminished. Reversed-phase HPLC with fluorescence detection (HPLC/FD) and online MS revealed that fatty acid (FA) composition of the skin surface lipids was similar to that in non-AD mice compared with that in AD mice. Strong negative correlations were noted between the levels of total serum immunoglobulin E (IgE) and palmitoleic acid and between the levels of total serum IgE and branched-hexadecanoic acid. Conversely, a strong positive correlation was noted between the levels of total serum IgE and linoleic acid. The present study demonstrates that the petroleum-containing spring water alters the FA composition of skin surface lipids in AD mice, which can be used as an index to evaluate inflammation.

Mycotoxin contamination of animal feedingstuff: detoxification by gamma-irradiation and reduction of aflatoxins and ochratoxin A concentrations

Mycotoxins are fungal secondary metabolites identified in many agricultural products screened for toxigenic moulds. They have been reported to be carcinogenic, teratogenic, tremorogenic, haemorrhagic and dermatitic to a wide range of organisms. With the increasing stringent regulations for mycotoxins imposed by importing countries such as those of the European Union, many cereals that are not safe for human consumption are used in formulations intended for animal feed. Gamma-rays are reported in the scientific literature to destroy ochratoxin A and aflatoxin in food crops and feed. The present study provides preliminary data for establishing the effect of dose of gamma-irradiation, ranging from 0 to 15 kGy, on aflatoxins and ochratoxin A reduction in commercial animal feed. The mycotoxin levels were determined by means of immunoaffinity clean-up (IAC) and HPLC with fluorescence detection (HPLC-FLD). The maximum reductions found at 15 kGy were 23.9%, 18.2%, 11.0%, 21.1% and 13.6% for ochratoxin A, aflatoxin B1, aflatoxin B2, aflatoxin G1 and aflatoxin G2, respectively. Results showed that the gamma-rays even at 15 kGy were not effective in the complete destruction of ochratoxin A and aflatoxins in the tested feed.

Ochratoxin A in traditional dry-cured meat products produced from sub-chronic-exposed pigs

The presence of ochratoxin A (OTA) was determined in traditional dry-cured meat products made from sub-chronically OTA-exposed pigs. The experimental group of pigs (n = 5) was treated with 300 µg OTA kg–1 of feed during 30 days, whereas the control group (n = 5) remained untreated. After the household production of six types of dry-cured meat products based on traditional recipes, OTA residues were determined in final products produced from each treated and untreated animal using an immunoenzymatic technique (ELISA) and HPLC with fluorescence detection (HPLC-FD). The analytical methods showed acceptable analytical performance results and high correlation coefficients. Mean OTA concentrations ranged from 4.51 ± 0.11 µg kg–1 in smoked ham to 6.87 ± 2.01 µg kg–1 in home-made Slavonian sausage. The study demonstrated that pig exposure to OTA leads to the accumulation of OTA residues in muscle and adipose tissue used for the production, and consequently results in contamination of the final meat products.

Assessment of α-tocopherol content in cow and goat milk from the Serbian market

The present paper reports determination of ?-tocopherol in commercial cow, raw cow and goat milk, as well as estimation of its nutritive value based on ?-tocopherol content. Quantification was done by reversed-phase HPLC with fluorescence detection (ex 295 nm, em 330 nm) and with UV detector set at 286 nm and 292 nm. The method of milk sample preparation consisted of alkaline saponification with 30% KOH, denaturation of lipoproteins with methanol followed by liquid-liquid extraction with diethyl-ether. Recovery values of the extraction method were 78.5, 86.7 and 91.0% for three standard addition levels. Analyzed commercial low-fat milk samples had significant lower ?-tocopherol levels than milk with higher fat content. ?-tocopherol concentration below 0.30 ?g/ml in low-fat cow milk and 0.83-0.86 ?g/ml in whole milk were detected. Raw goat milk had much more ?-tocopherol (1.25 ?g/ml) compared with commercial and raw cow milk with similar fat content.

Capillary electrophoresis characterization of molecularly imprinted polymer particles in fast binding with 17β‐estradiol

Molecularly imprinted polymer (MIP) submicron particles were synthesized, using either ethylene glycol dimethacrylate or trimethylolpropane trimethacrylate as a cross-linker, specifically for recognition of 17β-estradiol (E2). HPLC with fluorescence detection (HPLC-FD) results showed that 90(±5)% of E2 bound onto these particles after 2 min of incubation, and 96(±3)% after long equilibrium. The binding capacity was 8(±3) μmol/g for MIP particles prepared using ethylene glycol dimethacrylate, and 33-43(±8) μmol/g for using trimethylolpropane trimethacrylate. CE separation of MIP and non-imprinted polymer particles was successful when 50 mM borate buffer (pH 8.5) containing 0.005% w/v EOTrol™ LN in reverse polarity (-30 kV) was used. The electrophoretic mobilities of MIP and non-imprinted polymer particles, together with dynamic light scattering measurement of particle sizes, allowed for an estimation of their surface charges. Automated injection of E2 and particles in mixture set a lower limit of 20(±1) s on incubation time for the study of fast binding kinetics. The presence of E2 and bisphenol A (BPA) together tested the selectivity of MIP particles, when the two compounds competed for available binding cavities or sites. Addition of E2 after BPA confirmed E2 occupation of the specific binding cavities, via displacement of BPA.

Determination of pamidronate in human whole blood and urine by reversed-phase HPLC with fluorescence detection.

Pamidronate is a bisphosphonate that is effective in treating bone disease including osteopenia and osteoporosis in adults. A sensitive and reliable method for the analysis of pamidronate in whole blood and urine is key to the development of this drug for use in children. A previously described method for pamidronate analysis serum and urine did not consistently detect the drug at satisfactory levels in whole blood. The procedure involves co-precipitation of the bisphosphonates with calcium phosphate, pre-column derivitization with fluorescamine, HPLC utilizing a Nucleosil C(18) column, and fluorescence detection with excitation at 395 nm and emission at 480 nm. Changes to the original protocol included the use of a new internal standard (alendronate), the optimization of the concentration of ethylenediaminetetraacetic acid (EDTA) for dissolving the precipitate, and the elimination of the acidification step prior to deproteinization. The optimum EDTA concentration, which had a significant effect on the labeling capability of fluorescamine, was determined to be 20 mm.A good separation between pamidronate and alendronate was achieved using a heated (40 degrees C ) Nucleosil C(18), 10 micro m particle size column. The mobile phase was an aqueous solution of 1 mm Na(2)EDTA-methanol (97:3, v/v) adjusted to pH 6.5 using a fl ow-rate of 1 mL/min. Fluorescence detection was set at 395 nm for excitation and at 480 nm for emission. The limit of quantitation for pamidronate was 0.5 micro g/mL in whole blood and 0.1 micro g/mL in urine. The method was applied to both whole blood and urine samples from pediatric patients.

Comparison of Plasma Total Homocysteine Measurements in 14 Laboratories: An International Study

Information on interlaboratory variation and especially on methodological differences for plasma total homocysteine is lacking. We studied 14 laboratories that used eight different method types: HPLC with electrochemical detection (HPLC-ED); HPLC with fluorescence detection (HPLC-FD) further subdivided by type of reducing/derivatizing agent; gas chromatography/mass spectrometry (GC/MS); enzyme immunoassay (EIA); and fluorescence polarization immunoassay (FPIA). Three of these laboratories used two methods. The laboratories participated in a 2-day analysis of 46 plasma samples, 4 additional plasma samples with added homocystine, and 3 plasma quality-control (QC) pools. Results were analyzed for imprecision, recovery, and methodological differences. The mean among-laboratory and among-run within-laboratory imprecision (CV) was 9.3% and 5.6% for plasma samples, 8.8% and 4.9% for samples with added homocystine, and 7.6% and 4.2% for the QC pools, respectively. Difference plots showed values systematically higher than GC/MS for HPLC-ED, HPLC-FD using sodium borohydride/monobromobimane (however, for only one laboratory), and EIA, and lower values for HPLC-FD using trialkylphosphine/4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole. The two HPLC-FD methods using tris(2-carboxyethyl) phosphine/ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F) or tributyl phosphine/SBD-F, and the FPIA method showed no detectable systematic difference from GC/MS. Among-laboratory variations within one method can exceed among-method variations. Some of the methods tested could be used interchangeably, but there is an urgent need to improve analytical imprecision and to decrease differences among methods.