Fluorescence Characteristics and Main Fluorescence Component in Burmese 'chameleon' Amber.

One special variety of Burmese amber is "chameleon" amber, named for the bluish-green colour that appears to float on its surface. This material is found in the famous Tengchong market in Yunnan Province, China's largest Burmese amber market. Its bodycolour ranges from golden brown to brownish-red or even red. When exposed to sunlight or strong white light against a black background, its surface shows a uniform green colour. This research presents the gemological properties, spectral characteristics and organic components of Burmese 'chameleon' amber. Three-dimensional (3D) fluorescence spectra showed that Burmese 'chameleon' amber had fluorescence centres near 433, 465 and 470 nm, and the excitation wavelengths of the fluorescence centres of Burmese 'chameleon' amber were shifted from the ultraviolet region (380 ± 10 nm) to the visible region (410 ± 10 nm), with the emission wavelengths concentrated at the bluish-green region. Through the colour simulation and superimposition, the phenomenon of floating bluish-green fluorescence colour of Burmese 'chameleon' amber is not only derived from bluish-green fluorescence centres, but also enhanced by the mixture of surface fluorescence and its bodycolour. Headspace solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS) analysis demonstrated the variety of aromatic compounds in Burmese 'chameleon' amber was related to geological process and the presence of fluorescence components. The high-performance liquid chromatography-fluorescence detector obtained some fluorescent aromatics, particularly benzo[a]anthracene with yellowish-green fluorescence, which is responsible for the fluorescence characteristics of Burmese 'chameleon' amber.

On-site extraction using a 3D printed device coated with Zn/Co-ZIF-derived carbon followed by an on-line SIA-HPLC-FL system for fluoroquinolones determination in wastewater.

A 3D printed device covered with Zn/Co-ZIF-derived carbon allows the on-site extraction of fluoroquinolones (FQs) from wastewater, avoiding the sample transportation to the laboratory, and the subsequent elution, separation and determination using an on-line flow system based on sequential injection analysis (SIA) coupled to HPLC-FL. Several parameters that affect the extraction efficiency and desorption were optimized including the sorption phase immobilization technique on the 3D device, extraction time, pH effect, sample volume as well as the type of eluent, eluent volume, and flow rate. Under optimum conditions, detection limits of 3-9 ng L-1 were achieved for norfloxacin, ciprofloxacin, danofloxacin, enrofloxacin and difloxacin. The precision expressed as relative standard deviation (%RSD, n = 3), showed intraday and interday ranges of 1.5-5.3% and 2.8-5.7%, respectively, demonstrating a good precision of the proposed methodology. To assess matrix effects and accuracy of the proposed method in real samples, recovery studies were performed without and with FQs spiked at different concentrations (0.5-10 µg L-1) to wastewater samples, showing good recoveries in the range of 91-104%. The results allow to confirm the applicability of MOF-derived carbons as adsorbents for on-site extraction, and the satisfactory separation and quantification of FQs by a SIA-HPLC-FL on-line system after their desorption with small eluent volumes.

Accurate determination of Biotinidase activity in serum by HPLC and its utilization as second tier test for the confirmation of initial positive newborn screening results.

Diagnosis of Biotinidase deficiency (BTD) is extremely important to avoid several neurodevelopmental problems in early childhood. Colorimetric and fluorometric methods lack specificity and selectivity due to several interferences resulting in a high number of false positive results. We developed an HPLC method for BTD activity in serum with fluorescent detection. In colorimetric assays, biotinidase attacks the amide linkage of the artificial substrate biotinidyl-4-aminobenzoic acid (B-PABA) and releases p-aminobenzoic acid (PABA), which is converted to a purple dye by diazotization reaction. The newly developed method injects the reaction mixture directly into the HPLC column and quantifies using a six-point calibration curve without coupling and diazotization reaction. The method is linear over the 5-1000 µmol/L range. The detection and quantitation limits were 2.5 µmol/L and 5.0 µmol/L, respectively. When compared with colorimetric assay, the correlation coefficient (R2) was 0.9963. The within-assay and between-assay precision was <10.0% for four levels of quality control samples. No significant variation in BTD activity was detected due to hemolysis, icteric, and lipemic samples. The newly developed method eliminates the potential interference due to the presence of aromatic amines and significantly reduces the false positive results observed with the colorimetric method. It is simple, specific, sensitive, faster in sample preparation, and requires a small sample volume. The newly developed HPLC method was used in our laboratory as a secondary tier test for initial positive BTD samples from newborn screening programs. To our knowledge, no similar HPLC method has been reported to date.

Different Extraction Approaches for the Analysis of Melatonin from Cabernet Sauvignon and Feteasca Neagra Wines Using a Validated HPLC-FL Method.

In recent years, the wine industry has shown a considerable degree of interest in the occurrence of melatonin in wines. Sample pretreatment may be the most important step in trace analysis. Since wine is a complex matrix and melatonin is present in low amounts (ppb), an adequate extraction technique is required. In this study, the effect of several extraction methods, such as solid phase extraction (SPE), Quick, Easy, Cheap, Effective, Rugged, and Safe extraction (QuEChERS), and dispersive liquid-liquid micro-extraction (DLLME) was studied and the variable parameters that can arise throughout the extraction process were optimized to obtain the best results. A high-performance liquid chromatography with fluorescence detector (HPLC-FL) method was adapted and validated, including measurement uncertainty, for the analysis of melatonin in wines and to assess the efficiency of the extraction yield. After comparing the acquired results, the DLLME method was optimized. Extraction recoveries values ranging from 95 to 104% demonstrated that the approach may be successfully applied for the extraction and concentration (enrichment factor of almost eight) of melatonin in wine samples prior to HPLC-FL analysis. The first report of melatonin levels in Feteasca Neagra wines has been made. The data obtained for Cabernet Sauvignon revealed that the final levels of melatonin in the wines are dependent on the winemaking process.

A simple and sensitive HPLC-FL method for bioanalysis of velpatasvir, a novel hepatitis C virus NS5A inhibitor, in rat plasma: Investigation of factors determining its oral bioavailability.

Velpatasvir is a novel inhibitor of hepatitis C virus nonstructural protein 5A that received US Food and Drug Administration approval for the treatment of patients with chronic hepatitis C virus genotypes 1-6. In the present study, a sensitive bioanalytical method for velpatasvir was developed using high-performance liquid chromatography coupled with a fluorescence detector system, which was applied to elucidate the factors determining the oral bioavailability and disposition of velpatasvir. This method offered sufficient sensitivity, with a lower limit of quantification of 0.5 ng/mL, which is comparable to previously reported methods using liquid chromatography coupled with tandem mass spectrometry. Velpatasvir exhibited low oral bioavailability, moderate intestinal permeability, and significant biliary excretion in rats. It was also found to be significantly metabolized in the liver, with a low-to-moderate extraction ratio; however, its intestinal metabolism and enterohepatic circulation did not occur. Thus, our present results demonstrate that the oral bioavailability of velpatasvir is primarily dependent on gut absorption and hepatic first-pass metabolism. The fractions of velpatasvir dose unabsorbed from the gut and eliminated by the liver before reaching the systemic circulation following oral administration were estimated to be 32.8%-58.6% and 4.74%-30.54% of the oral dose, respectively. To our knowledge, this is the first systematic study to investigate the contributory roles of biopharmaceutical and pharmacokinetic factors on the oral bioavailability of velpatasvir, together with a new bioanalytical method for velpatasvir.

Development of An HPLC Method for the Determination of Mesalazine in Human Plasma by Fluorimetric Derivatization and Application to A Prototype Pharmacokinetic Study.

In this study, a new, fast and sensitive HPLC method with fluorometric detection was developed for the determination of mesalazine in human plasma and applied to a pharmacokinetic study. Mesalazine was precolumn derivatized with NBD-Cl and the fluorescent derivative was separated on a C18 (150 × 4.6 mm × 2.6 µm) analytical column at 30 ºC using a mobile phase composed of acetonitrile-0.1% o-phosphoric acid in water (70:30, v/v) by isocratic elution with flow rate of 1.0 mL min-1. The method was based on the measurement of the derivative using fluorescence detection (λex = 280 nm, λem = 325 nm). The retention time of mesalazine is 3.08 ± 0.06 min. Nortriptiline was used as internal standard. This currently developed method was validated according to ICH criteria by evaluating the specificity, linearity, precision, accuracy and robustness. The method was determined to be linear in a concentration range of 0.25-1.5 µg mL-1 with the correlation coefficient of 0.9997. LOD and LOQ were found to be 0.075 and 0.25 µg mL-1, respectively. Intraday and interday RSD values were less than 5.92%. The plasma concentration-time profile and pharmacokinetic parameters such as AUC0-t, AUC0-∞, Cmax, tmax, t1/2, were calculated according to the assays. The presented method can certainly be used for bioequivalence and bioavailability investigations and routine analysis of the drug in plasma.

Synthesis and characterization of new surface modified magnetic nanoparticles and application for the extraction of letrozole from human plasma and analysis with HPLC-fluorescence.

Acetic acid-functionalized magnetic nanoparticles modified by (3-amino-propyl)-tri-ethoxy silane was synthesized and used as a new solid-phases adsorbent. Infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), x-ray diffraction (XRD), energy-dispersive x-ray spectroscopy (EDX), vibrating sample magnetometer (VSM) and Electrophoretic Light Scattering (ELS) were used to characterize the modified nanoparticles. The molecular interaction between letrozole and nanoparticles (NPs) was studied using density functional theory (DFT) calculations. The developed nanoparticles were applied for dispersive solid-phase extraction of letrozole (an anticancer drug) from human plasma. Extracted letrozole was quantified using an isocratic HPLC/FL method. The extraction efficiency was optimized using one experiment at a time optimization method based on the adsorbent quantity, sample pH, adsorption time, desorption time, and elution solvent type/volume. The analysis method was fully validated according to the FDA guideline for bioanalytical method validation. The linear quantification range was 0.01-1 µg/mL and the lower limit of quantification (LLOQ) was 0.01 µg/mL. Plasma samples of 6 patients were analyzed and the measured letrozole concentrations range was 0.04-0.31 µg/mL. The newly synthesized magnetic nanoparticles were used successfully for the extraction of letrozole from spiked and clinical plasma samples. The developed method is a precise and simple method that is suitable for pharmacokinetic studies and clinical applications.

[Examination of Aflatoxin M1 Contamination in Milk and Milk Drinks Available in Fukuyama City].

To determine the relationship between seasons and regions and aflatoxin M1 (AFM1) contamination of milk distributed in Fukuyama City, we conducted a survey once during the summer and once during the winter between June 2018 and January 2019. We compared the AFM1 contamination levels in milk drinks available in Fukuyama City during the same period, to provide more about the factors causing AFM1 contamination. All milk samples examined exhibited AFM1 contamination levels below the standard AFM1 contamination level (0.5 µg/kg). For the comparison based on seasons, one milk sample collected in the summer (0.07 µg/kg) exceeded the EU limit (heat-treated milk: 0.050 µg/kg). However, there was no significant difference in the AFM1 contamination level (p>0.05). For the comparison based on regions, the AFM1 contamination level in the milk sample from the Chugoku Region was significantly higher in the winter and significantly lower in the summer compared to those from other regions. AFM1 contamination of milk did not have a direct relationship with seasons or regions, but was instead influenced by the type, amount, and management of feed supplied to dairy cattle. For the comparison between milk and milk drinks, the AFM1 contamination levels in milk drinks were significantly lower (p<0.01). The highest AFM1 concentration (0.08 µg/kg) was detected in one sample of milk drink sampled during the summer. The AFM1 contamination of milk drinks is likely affected by the level of contamination in raw materials, the proportion of such raw materials in the drinks, and the process type. An increase in non-fat milk solids was assumed to be a factor that increases AFM1 contamination.

Determination of glyphosate, AMPA and glufosinate by high performance liquid chromatography with fluorescence detection in waters of the Santarém Plateau, Brazilian Amazon.

Herbicide use, mainly glyphosate, has been intense in worldwide agriculture, including in the Brazilian Amazon region. This study aimed to validate a method for determining glyphosate and its degradation product, AMPA, and glufosinate by HPLC-FL in 58 water samples collected at the Santarém plateau region (Planalto Santareno), in the western of Pará state, Brazil. The method involves filtration and direct injection in the HPLC-FL for AMPA analysis, or previous concentration (10×) by lyophilization for glufosinate and glyphosate analysis. Analytes were oxidized and complexed with o-phthalaldehyde and 2-mercaptoethanol in a post-column reaction before fluorescence detection. LOQs for AMPA, glyphosate and glufosinate were established at 0.5, 0.2 and 0.3 µg L-1, respectively. A total of 58 samples were collected. Glyphosate and glufosinate were not detected in any of the 30 surface water samples collected in 2015 (

Bioanalytical method for the simultaneous quantification of irinotecan and its active metabolite SN-38 in mouse plasma and tissue homogenates using HPLC-fluorescence.

A simple and rapid bioanalytical method was developed for the simultaneous quantification of irinotecan and SN-38 in mouse plasma and tissue homogenates using High-Performance Liquid Chromatography with Fluorescence detection (HPLC-FL). Camptothecin was used as internal standard and protein precipitation with acetonitrile-methanol (1:1, v/v) followed by acidification with 0.5 M hydrochloric acid was used for sample pre-treatment. The analytes and the internal standard were detected using an excitation and emission wavelength of 368 and 515 nm, respectively. The linearity, selectivity, accuracy and precision, carry-over, limit of detection and lower limit of quantification of the method are described. The method was linear from 7.5 to 1500 ng/mL for irinotecan and from 5 to 1000 ng/mL for SN-38. For all matrices, the accuracy bias and precision variation were within ±15% and ≤15%, respectively. This method was successfully applied to study the pharmacokinetics of irinotecan and SN-38 using in vivo mouse models.

Development of photo-amino acid derivative reactivity assay: a novel in chemico alternative method for predicting photoallergy.

Photoallergy test of cosmetics and several types of pharmaceutical substances is often necessary for obtaining approval from authorities. However, there are no official test guidelines for photoallergy evaluation. Therefore, we tried to establish a photoallergy test by utilizing an in chemico alternative sensitization method, amino acid derivative reactivity assay (ADRA). To determine the criteria for judging the photoallergy potential, photo-ADRA with or without photoirradiation was performed using 60 photoallergenic chemicals, and cysteine and lysine derivatives were detected using high-performance liquid chromatography either by absorbance or fluorescence measurement. The accuracy of prediction was 81.4% (48 of 59) and 80.0% (48 of 60) using the absorbance and fluorescence methods, respectively. However, as chemicals can breakdown into multiple chemicals during photoirradiation, the absorbance method often cannot perform accurate detection due to co-elution, whereas the fluorescence method can do this due to lack of co-elution. Moreover, all eight chemicals that were found to be negative or false-positive for photoirritation in the 3T3 neutral red uptake phototoxicity test were confirmed as positive for photoallergy using this method. Furthermore, we prepared three types of pseudo-mixtures where we added one photoallergen along with five nonphotoallergens and performed the photo-ADRA by the ultraviolet and fluorescence methods. The result of the fluorescence method was almost the same as that obtained with the use of a single photoallergen and hence the outcome was not affected by the mixture. Thus, this study not only showed a method of evaluating the photoallergy potential of a single chemical but also a mixture, making it useful as an in chemico photoallergy alternative test.

Analytical methods for determination of bisphenol A, 4-tert-octylphenol and 4-nonylphenol in herrings and physiological fluids of the grey seal.

•The aim of this work was to develop the methods of determination for phenol derivatives: bisphenol A (BPA), 4-tert-octylphenol (OP) and 4-nonylphenol (NP), in the whole body of herring Clupea harengus and in physiological fluids of the Baltic grey seal Halichoerus grypus grypus (blood and milk).•Methods were based on liquid chromatography coupled with a fluorescence detector (HPLC-FL).•These methods were satisfactorily validated, each showing good recovery (>80%) and precision (RSD < 15%). Regarding the limit of quantification (LOQ), this was established at <2 ng g-1 for herring, <0.07 ng cm-3 for blood and <0.1 ng cm-3 for milk.

Development of a novel ion-pairing HPLC-FL method for the separation and quantification of hydroxychloroquine and its metabolites in whole blood.

Hydroxychloroquine (HCQ) is an old antimalarial drug that has proven to be a safe and effective treatment for systemic lupus erythematosus (SLE) and other autoimmune diseases. Since hematic concentration of HCQ is closely related to the therapeutic response, monitoring the levels of the drug and its metabolites in the blood of HCQ-treated patients helps the clinician in the evaluation of partial or complete unresponsiveness to treatment. We developed and validated a novel ion-pairing HPLC-FL method for the simultaneous dosage of HCQ, and its major metabolites desethylhydroxychloroquine, desethylchloroquine and bisdesethylchloroquine, after extraction from whole blood. This methodological approach was used for the analysis of real samples obtained from patients affected by SLE and undergoing HCQ treatment. The same samples were also analyzed using a previously validated LC/MS/MS method and data obtained with the two approaches were in substantial agreement with each other. Results presented in this work indicate that this approach can be successfully used to monitor the level of HCQ and its metabolites in the blood of various categories of patients (i.e. low and high responders, or those not adhering to the therapy). Comparison of HPLC-FL and LC/MS/MS data confirmed the efficacy of the proposed method for routine clinical analyses.

Development of HPLC with fluorescent detection using NBD-F for the quantification of colistin sulfate in rat plasma and its pharmacokinetic applications.

Colistin sulfate, composed of a mixture of colistin A sulfate (CLA) and colistin B sulfate (CLB), is available for treating life-threatening infections caused by multidrug-resistant Gram-negative bacteria. In this study, the CLA and CLB were quantified separately. Colistin sulfate was extracted from rat plasma with a solid-phase extraction C18 cartridge and reacted with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), and the fluorescent derivatives were subjected to reversed-phase high-performance liquid chromatography analysis and used to investigate the pharmacokinetics of CLA and CLB in rat plasma. The recovery rates of CLA and CLB were 41.2 ± 4.4 and 45.5 ± 3.1%, respectively. The recovery rate calculated from the total area of CLA and CLB was 43.9 ± 3.6%. When 2 mm NBD-F and 10 mm boric acid buffer (pH 9.5) were added to colistin sulfate, the highest recovery rate was obtained. The best heating time was 5 min at 60°C. The lower limits of quantification for CLA, CLB and the total area of CLA and CLB were 0.05, 0.05 and 0.1 µg/mL; the coefficients of variations were 13.5, 14.5 and 14.1%, respectively. This method was found to have acceptable linearity, precision and accuracy, and has been successfully applied to a pharmacokinetic study in rat plasma.

A disposition kinetic study of Tramadol in bile duct ligated rats in perfused rat liver model.

Tramadol hydrochloride is a centrally acting synthetic opioid analgesic drug and is used to treat chronic pain. In this study, the effects of Bile Duct Ligation (BDL) on the pharmacokinetics of tramadol in a liver recirculating perfusion system of male rats were used. Twenty-four Wistar male rats were randomly divided into four groups: control, sham and two weeks BDL and four weeks BDL. Serum levels of liver enzymes were measured before perfusion and the pharmacokinetics of tramadol was evaluated by using liver recirculating perfusion system. Tramadol and metabolites concentrations were determined by HPLC-FL. The sharp increase in liver enzymes level in both BDL groups was observed and significant changes were also observed in liver weight and volume. Tramadol metabolites concentration significantly decreased compared with the control and sham group (P<0.05). The decrease in the hepatic metabolism of tramadol and increase in the half-life of the elimination of tramadol in rats with BDL suggests that personalized treatment and the therapeutic drug monitoring (TDM) data examination are necessary for patients with bile duct diseases and the dose of tramadol should be accordingly adjusted.

Oxidative stress during bacterial growth characterized through microdialysis sampling coupled with HPLC/fluorescence detection of malondialdehyde.

Organisms that grow aerobically are routinely exposed to oxidative stress in the form of reactive oxygen species. Monitoring the dynamic variations of oxidative stress allows us to understand its role in basic cellular function and determine mechanisms of antioxidation. In this study, microdialysis (MD) sampling was employed for continuous monitoring of the formation of malondialdehyde (MDA) in a bacterium-inoculated culture broth. To test the practicality of this approach, oxidative stress was induced by cadmium and then a 60-min interval was selected to collect sufficient amounts of dialysate for high-performance liquid chromatography with fluorescence (HPLC-FL) detection. After optimization of this simple-to-operate, simultaneous, and continuous method for dynamic monitoring of MDA during periods of bacterial growth, a retrodialysis technique and a no-net-flux method were used to assess the probe recovery and analytical performance of the proposed system. The mean probe recovery of MDA was 78.6 ± 0.9%, with intra- and interday precisions of 2.7-6.1 and 3.5-7.6%, respectively. To evaluate the practicality of this method, the dynamic variations in the concentrations of MDA in standardized bacterial species (Staphylococcus aureus, ATCC(®) 29213™) were monitored continuously for 24h. The analytical results confirmed that this MD sampling technique combined with HPLC-FL detection can be used to accurately and continuously monitor the levels of MDA in microbially inoculated culture broths.

Concomitant evaluation of atmospheric levels of polychlorinated biphenyls, organochlorine pesticides, and polycyclic aromatic hydrocarbons in Strasbourg (France) using pine needle passive samplers.

In this study, pine needles were used as cost-effective and reliable passive bio-monitors to concomitantly evaluate atmospheric concentrations of three classes of persistent organic pollutants, polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs), and polycyclic aromatic hydrocarbons (PAHs). The extraction of persistent organic pollutants (POPs) from needle samples was performed. Eleven PCBs, 11 OCPs, and 15 PAHs were detected and followed through time in needle samples from three sites in the Strasbourg region. The urban and rural sites were more exposed to PCBs than the suburban site. The highest concentration of PCBs was found at the urban site, but the largest number of congeners (10) was detected at the rural site. PCB 189 and 156 were the predominant congeners in the rural site and PCB 70 in the urban site. For OCPs, the rural site displayed the highest concentrations (up to 22.9 ng g(-1)) and number of compounds investigated (9). The high concentration of γ- and ß-hexachlorocyclohexane (HCH) at that time in the urban site was the reason for this result. γ- and ß-HCH were the two predominant compounds in all samples. The suburban and urban sites were the most exposed with PAHs with pyrene, phenanthrene, and acenaphthene being the three predominant compounds in these sites. No specific trend in terms of time was apparent for PCBs and OCPs. However, higher concentrations were detected for some compounds in the first sampling, especially for PAHs, and this is attributed to variations in meteorological conditions (e.g., temperature, wind, rain) and variable inputs from both identified and unidentified sources.

Development, validation and clinical application of a HPLC-FL method for CYP2D6 phenotyping in South Brazilian breast cancer patients.

OBJECTIVE: To develop and validate a method for determination of dextromethorphan (DMT) and dextrorphan (DTP) in plasma samples using HPLC-FL and to apply it to CYP2D6 phenotyping of a population from the South of Brazil. METHODS: Samples were prepared by hydrolysis and liquid-liquid extraction. Analysis was conducted in a reversed phase column, with isocratic elution and fluorescence detection. One hundred and forty patients being treated with tamoxifen were given 30 mg of dextromethorphan and their CYP2D6 phenotypes were determined on the basis of [DMT]/[DTP] metabolic ratios in plasma samples collected after 3h. RESULTS: Total chromatography running time was 12 min. Precision (CV%) was below 9.7% and accuracy was between 92.1 and 106.9%. The lower limits of quantification were 1 ng mL(-1) for DMT and 10 ng mL(-1) for DTP. Mean extraction yield of analytes was 86.6%. Mean age of patients was 55.7 years. Phenotype frequencies were as follows: 7.1% poor metabolizers, 13.6% intermediate metabolizers, 77.1% extensive metabolizers and 2.1 ultra-rapid metabolizers. Metabolic ratios for patients on strong (n=11) and weak (n=16) CYP2D6 activity inhibitors were different from each other and also different from ratios for patients not taking enzyme inhibitors (n=113). CONCLUSIONS: A sensitive method for determination of dextromethorphan and its metabolite in plasma samples was developed and successfully applied, providing evidence of the impact that CYP2D6 inhibitors have on the enzyme's metabolic capacity.

Alternative sample treatments for the determination of sulfonamides in milk by HPLC with fluorescence detection.

This paper presents two sample treatments, dispersive liquid-liquid microextraction (DLLME) and QuEChERS for the determination of 9 sulfonamides, regulated by the EU Council in milk samples. Both methods represent useful alternatives to conventional procedures based mainly in solid-phase extraction, in terms of simplicity, reduction of organic solvents, sample throughput and effectiveness for cleaning-up complex samples. They have been evaluated and compared in terms of efficiency, trueness, sensitivity and precision, using HPLC with fluorescence detection employing a previous derivatisation step with fluorescamine. Clean extracts were obtained with recoveries between 90.8-104.7% and 83.6-104.8% for DLLME and QuEChERS, respectively. Matrix-matched calibration curves were established for both methods using milk samples spiked at four concentration levels. LODs (3xS/N) lower than 1.21µgL(-)(1) and 2.73µgL(-)(1) for DLLME and QuEChERS, respectively, were obtained in all cases. The precision, in terms of repeatability and intermediate precision, was lower than 10% in all cases.

Safety evaluation of Whole Algalin Protein (WAP) from Chlorella protothecoides.

Microalgae such as Chlorella spp., were once consumed as traditional human foods; now they are being developed as ingredients for modern diets. Whole Algalin Protein (WAP) from dried milled Chlorella protothecoides was evaluated for dietary safety in a 13-week feeding trial in rodents with genotoxic potential evaluated using in vitro and in vivo assays and the likelihood of food allergy potential evaluated via human repeat-insult patch test (HRIPT). In the subchronic study, rats consumed feed containing 0, 25,000, 50,000 or 100,000 ppm WAP for 92-93 days. No treatment-related mortalities or effects in general condition, body weight, food consumption, ophthalmology, urinalysis, hematology, clinical chemistry, gross pathology, organ weights, and histopathology occurred. Several endpoints exhibited statistically significant effects, but none was dose-related. The no-observed-adverse-effect level (NOAEL) was based on the highest WAP concentration consumed by the rats and was equivalent to 4805 mg/kg/day in males and 5518 mg/kg/day in females. No mutagenicity occurred in Salmonella typhimurium or Escherichia coli tester strains (≤5000 µg/plate WAP) with or without mutagenic activation. No clastogenic response occurred in bone marrow from mice administered a single oral dose (2000 mg/kg WAP). Skin sensitization was not induced by WAP via HRIPT, indicating little potential for food allergy.