High-performance Liquid Chromatography-fluorescence Research Articles

B(a)P adduct levels and fertility: A cross‑sectional study in a Sicilian population.

Benzo(a)pyrene (BaP) is a carcinogenic polycyclic aromatic hydrocarbon for human tissues. Still today it is not fully investigated if BaP can affect negatively the male fertility through the BaP-DNA adducts production. In the present study, BaP Tetrol I-1 (TI-1) and BaP Tetrol II-2 (TII-2) BaP-DNA adducts were investigated in spermatozoa of a Sicilian male population. Semen samples from 86 volunteers in two eastern Sicilian cities (Regalbuto and Melilli) were collected. The quality of semen was evaluated in all samples according to the World Health Organization (WHO) guidelines. We analyzed BaP-DNA adducts in extracted sperm cell DNA using the modified high-performance liquid chromatography-fluorescence method to detects both Tetrols. Differences between Tetrol levels were assessed by the Wilcoxon signed-rank test and the Mann-Whitney U test, as appropriate. Correlation between semen quality parameters and Tetrol concentrations were analyzed using the Spearman's correlation coefficient. Σ(TI-1+TII-2) were significantly higher in spermatozoa of volunteers from Regalbuto. Furthermore, a greater dispersion of the levels of adducts was observed in these specimens. TI-1 adducts were higher than TII-2 in Melilli samples (95% CI) and TII-2 were higher than TI-1 in Regalbuto semen samples (95% CI). A significant inverse correlation between sperm progressive motility and both TI-1 and TII-2 adducts was observed. The present study showed that BaP negatively affects male fertility by TI-1 and TII-2 DNA-adduct production. These results suggest that DNA adducts could be used as biomarker to assess BaP exposure by air pollution. Further studies are needed to confirm if these findings could affect male fertility because of the growing impairment of this function observed in recent years.

Monitoring of Carbendazim and Thiabendazole in Fruits and Vegetables by SiO2@NiO-Based Solid-Phase Extraction Coupled to High-Performance Liquid Chromatography-Fluorescence Detector

This study presents an application of nickel affinity solid-phase extraction (SPE) for the quantitative monitoring of carbendazim and thiabendazole residues in some fruits and vegetables. The nickel oxide nanoparticle was deposited on the silica to obtain an affinity adsorbent (SiO2@NiO) for the selective extraction of the compounds containing imidazole groups from complex matrix. Coupling with high-performance liquid chromatography with a fluorescence detector, the extraction conditions of the SiO2@NiO adsorbent for the carbendazim and thiabendazole were optimized, and a sensitive quantitative method with good linearities (R 2 > 0.998) in the range of 10–1000 ng g−1 was developed. The detection limits for the two analytes were in the range of 2.9–7.5 ng g−1, which are lower than the maximum residue limits established for these compounds. Relative standard deviations (RSDs) of intra-day and inter-day precisions were less than 8.8%, which showed perfect repeatability. The proposed method has been successfully applied to analyze carbendazim and thiabendazole in apples, pears, cucumbers, potatoes, cabbages, and eggplants. Good recoveries of carbendazim and thiabendazole spiked in fruits and vegetables were found to range from 80.0 to 115.4%, demonstrating that the proposed method was reliable in monitoring these two benzimidazoles in fruits and vegetables.

Analgesic effect and central mechanisms of CQ prescription on cancer invasion induced mirror image pain in model mice

This study aimed to analyze the analgesic effect and related central mechanisms of CQ prescription on cancer invasion induced mirror image pain (CIIMIP)in model mice.In the study, male BALB/c mice were randomly divided into normal group, operation control group (injected with 0.2 mL inactivated S180 sarcoma cell sap), model group (injected with 0.2 mL S180 sarcoma cell sap on the right leg near the greater trochanter of femur) and CQ prescription low dose group (intraperitoneally injected with CQ prescription 100 mg•kg⁻¹ on the basis of model mice), CQ prescription middle dose group (intraperitoneally injected with CQ prescription 150 mg•kg⁻¹ on the basis of model mice), and CQ prescription high dose group (intraperitoneally injected with CQ prescription 200 mg•kg⁻¹ on the basis of model mice). Mechanical withdraw threshold (MWT) of the mirror image lateral hind paws were evaluated by Von Frey hairs before modeling and after surgery. The levels of glutamate (Glu), gamma aminobutyric acid (GABA), glycine (Gly), and taurine (Tau) in the L3-L5 spinal cord were measured by the high performance liquid chromatography-fluorescence detector (HPLC-FLD); AimPlex detection technology with multiple factors was used to detect the levels of regulated on activation in normal T-cell expressed and secreted (RANTES), monocyte chemoattractant protein (MCP-3) in the L3-L5 spinal cord. Then we observed the influence of GABAa receptor antagonist (Bicuculline) on analgesic effect of CQ prescription.The results indicated that CQ prescription could remarkably increase MWT of model mice(P<0.01, P<0.05), decrease the level of Glu(P<0.01, P<0.05), improve the levels of GABA, Gly, Tau(P<0.01, P<0.05), lower the ratio of Glu/GABA(P<0.01, P<0.05), and reduce the levels of RANTES, MCP-3(P<0.05) in the L3-L5 spinal cord, and GABAa receptor antagonist significantly blocked the analgesic effect of CQ prescription at two time points(P<0.05).This study showed that CQ prescription had significant analgesic effect on CIIMIP model mice, and its mechanism was associated with regulating the balance between excitability amino acid(EAA) and inhibitory amino acid (IAA) transmitters in central nervous system, partially activating GABAa receptor, and reducing the release of RANTES and MCP-3 in the spinal cord.

Comprehensive N-Glycan Profiling of Cetuximab Biosimilar Candidate by NP-HPLC and MALDI-MS.

Monitoring glycosylation of the mAbs have been emphasized and routinely characterized in biopharmaceutical industries because the carbohydrate components are closely related to the safety, efficacy, and consistency of the antibodies. In this study, the comprehensive glycan profiling of a biosimilar candidate of cetuximab was successfully characterized using Normal phase high-performance liquid chromatography (NP-HPLC) in combination with Matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). The presence of minor N-linked glycans containing sialic acid lactone residues (NeuAcLac) was observed in the biosimilar for the first time, which could influence the quantitative analysis of sialylated glycans and interfere with quantification of neutral glycans when it was analyzed by high performance liquid chromatography fluorescence (HPLC-FL). To overcome this issue, mild alkali treatment was used to hydrolyze lactone of the sialic acid to their neutral formation, which had no impact on the analysis of other glycans before and after the treatment. As a result, the mild alkali treatment might be helpful to obtain quantitative glycan profiling of the mAbs drugs with enhanced accuracy and robustness.

Graphene oxide adsorbent based dispersive solid phase extraction coupled with multi-pretreatment clean-up for analysis of trace aflatoxins in traditional proprietary Chinese medicines.

Graphene oxide (GO)-based dispersive solid phase extraction (D-SPE) method combined with multi-step preparation has been proposed for the evaluation of trace aflatoxins in proprietary Chinese medicines (PCM). After being extracted by methanol, the sample was purified based on multi-step preparation, including dehydration with MgSO4/NaCl and cleanup with neutral alumina. Then GO was used as an adsorbent in D-SPE method for further preconcentration of aflatoxins prior to high performance liquid chromatography-fluorescence detection. The selected conditions were investigated. The Box-Behnken design (BBD) was used to optimize factors affecting adsorption procedure. Under the optimized conditions, good linear relationships had been achieved with the correlation coefficient (R2) varying from 0.9904 to 0.9990. The LODs and LOQs were ranging from 0.020 to 0.041ng/mL and 0.061 to 0.125ng/mL, respectively. The results of the recoveries were 74.0-102.7% for the four aflatoxins, while the precisions from 1.8% to 7.2% were obtained, which indicated that the method was suitable for the analysis of aflatoxins in PCM.

Supramolecular solvent-based direct extraction for the determination of polycyclic aromatic hydrocarbons in water

Supramolecular solvent (SUPRAS) is a nano-structured liquid generated from amphiphiles through a sequential self-assembly process. It is an efficient and excellent solvent for the sample extraction. In this paper, a method to directly extract and rapidly analyze polycyclic aromatic hydrocarbons (PAHs) in water samples by high performance liquid chromatography-fluorescence detection (HPLC-FLD) was developed. The composition and amount of SUPRAS were optimized and practical samples were tested. It indicated that the combination of tetrahydrofuran and 1-octanol was a suitable SUPRAS for the extraction of four PAHs with recoveries between 89.08% and 102.47% and relative standard deviations (RSDs) from 1.38% to 3.92% (n=5). Results showed a good linearity of four PAHs with the correlation coefficients (R2) more than 0.999. The limits of detection (LODs) ranged from 1.26 to 9.23 ng/L. The proposed pretreatment method greatly reduces the analysis time. And the solvent-less approach is in accordance with the development trend of green chemistry and of great application prospects.

Molecular characterization of tocopherol biosynthesisgenes from Olea europaea (L.) cv. Ayvalık

Vitamin E is a group of compounds that includes metabolites known as tocopherols, which have high antioxidant activities. Tocopherols are synthesized in plants, and their beneficial effects on human health have been reported for diseases such as coronary heart disease and cancer. Here we report the full-length transcripts encoding vitamin E biosynthetic enzymes from fruit mesocarp tissues using the rapid amplification of cDNA ends (RACE) method for the first time in olives. We characterized the structure of the genes 4-hydroxyphenylpyruvate dioxygenase (PDS1), homogentisate phytyltransferase 1 (HPT1), vitamin E defective 3 (VTE3), tocopherol cyclase (VTE1), and gamma tocopherol methyltransferase (GTMT), which are responsible for tocopherol biosynthesis in the olive cultivar Ayvalık. Although PDS1 is widespread in all organisms, HPT1, VTE3, VTE1, and GTMT are only present in photosynthetic organisms. We isolated total RNA from the dissected mesocarp tissues of fruit collected at 15-day intervals between October and December 2014. We compared the expression levels of genes using quantitative RT-PCR and determined the tocopherol content using the high-performance liquid chromatography-fluorescence detection (HPLC-FLD) technique during fruit maturation in the Ayvalık cultivar. High OeHPT1, OeVTE3, OeVTE1, and OeGTMT expression was noted in young fruit. However, the OePDS1 mRNA did not exhibit significant expression changes during maturation. The α-tocopherol content varied between 26.78 and 21.05 mg/100 g and was highest at the early stages of fruit development. In addition, expression studies and tocopherol content revealed that tocopherol biosynthesis in olive is more active at the early stages of fruit maturation.

Simultaneous determination and identity confirmation of thiodicarb and its degradation product methomyl in animal-derived foodstuffs using high-performance liquid chromatography with fluorescence detection and tandem mass spectrometry.

A high-performance liquid chromatography-fluorescence detection method was developed for the simultaneous determination of thiodicarb and its degradation product methomyl in animal-derived food products, including chicken muscle, beef, pork, table eggs, and milk. Thiodicarb is known to degrade during analysis; therefore, a thorough investigation was carried out, revealing that thiodicarb degrades to methomyl immediately after spiking into a matrix of animal-derived food products. Consequently, thiodicarb was determined as the sum of the parent compound and methomyl. Samples were extracted with acetonitrile and sodium salts, and purified using solid-phase extraction (SPE). The limits of detection (LODs) and quantification (LOQs) were 0.0013 and 0.004mg/kg, respectively, for both analytes in various matrices. Seven-point external calibration curves were obtained, and they showed excellent linearity with determination coefficients (R2)≥0.999 for all tested matrices. The method was validated at three fortification levels (LOQ, LOQ×2, and LOQ×10) in triplicate with average recoveries ranging from 84.24 to 112.8% (for methomyl) and relative standard deviations (RSDs)≤6.5% in all matrices. The converted recoveries of thiodicarb in various matrices ranged from 74.80 to 107.80% with RSDs≤4.5%. The identities of both compounds in standard solutions and for recovery were confirmed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The developed method was further validated by accurate reproduction at another laboratory. Finally, the method was applied to market samples collected from different areas (and, in the case of milk, different brands), and none of the samples tested positive for thiodicarb or methomyl. In conclusion, the developed method can be successfully applied for a single-run analysis of thiodicarb and methomyl in livestock products.

First evidence of high urinary 1-hydroxypyrene level among rural school children during smoke haze episode in Chiang Mai Province, Thailand

Smoke haze from biomass burning has been a crisis in northern Thailand. There have been plenty of reports on ambient PM10 and PAHs but not on individual exposure during smoke haze episodes. The present study aimed to quantify urinary 1-OHP, a predominant biomarker of PAH exposure, among 200 rural school children in Om Koi and Mae Chaem districts, Chiang Mai Province, northern Thailand. Questionnaires for information regarding environmental exposure and health diaries were given out. Urine samples were collected to measure 1-OHP using high-performance liquid chromatography-fluorescence detection, HPLC-FLD (HP1100 Series/HP1046A). Proficiency testing materials were obtained from the participation of German External Quality Assessment Scheme (G-EQUAS, Erlangen, Germany) and analyzed as part of the method validation of our urinary 1-OHP method. The median urinary 1-OHP level in the school children from Om Koi District was significantly higher than that in Mae Chaem District (0.70 versus 0.24 μmol/mol cr). The present study showed the evidence that urinary 1-OHP level among school children from the selected study sites during haze episode in northern Thailand is higher than those reported in other region of Thailand and other countries.

Simultaneous extraction of polycyclic aromatic hydrocarbons through the complete dissolution of solid biological samples in sodium hydroxide/urea/thiourea aqueous solution

In order to precisely and simultaneously extract polycyclic aromatic hydrocarbons (PAHs) for measurement using a high performance liquid chromatography-fluorescence detector (HPLC-FL), a novel sample preparation method was developed. This method is based on the complete and fast dissolution of biological samples in a new non-alcoholic alkaline medium. A solution composed of NaOH/urea/thiourea at an optimized ratio was used for complete dissolution of approximately 0.25g dried fish samples within 20min. The proposed method was conducted at 10°C and under atmospheric pressure to obtain a stable and highly homogeneous solution, without the need for microwaves or any other apparatus. This process operates at considerably lower temperature than conventional methods and provides an opportunity to simultaneously extract the target analytes from their matrices by adding the extracting solvent in the initial steps of the dissolution; this process greatly reduced the time of analysis and the loss of analytes via vaporization. Several key parameters were identified and their effects on precision and extraction recoveries were investigated. Linearity over a calibration range of 1.0–100 and 2.5–100ngg−1 was achieved, with high coefficients of determination (r2) ranging between 0.9987 and 0.9998. Based on relative standard deviations (n=5), the intra-day and inter-day precisions of the spiked PAHs were found to be better than 3.1% and 3.2%, respectively, at a concentration level of 25ngg−1. The recoveries of PAH from spiked marine fish tissues and shrimp samples were in the range of 90.6%–100.4%. The spiked samples were also treated with the alcoholic alkaline and Soxhlet extraction methods in order to provide a comparison.

Doxorubicin enhances curcumin’s cytotoxicity in human prostate cancer cells in vitro by enhancing its cellular uptake

Doxorubicin (DOX) is a widely used drug in cancer treatment. Despite its popularity, it suffers from systemic side effects and susceptibility to drug resistance. Curcumin (CURC), on the other hand, is a drug that recently gained popularity due to its wide range of biological activities, including anti-inflammatory and anti-cancer activities. Limitations to its clinical translation include its poor water solubility and the need for administration of high doses. Combinatory anti-cancer therapy has been proposed as a common approach to overcome one or more of these challenges. In this work, we propose a combinatory DOX and CURC anti-cancer therapy of prostate cancer cells in vitro. DOX and CURC were administered in the free drug and nanocapsule form, respectively. Cell size and complexity, cytotoxicity and apoptosis were studied by flow cytometry, MTT assay and sub-G1 quantification, respectively. Cellular uptake of CURC nanocapsules (CURC NCs) was quantified by fluorescence microscopy and high-performance liquid chromatography fluorescence detection. Results showed that in vitro treatment with CURC NCs in the presence of subtherapeutic concentrations of DOX, led to significant increase in prostate cancer cells (PC3) apoptosis and death. This was likely due to significantly enhanced CURC uptake by the cells. The study presents a good rationale for pursuing combinatory CURC/DOX therapy in pre-clinical tumor animal models in the near future.

Alkali-based pre-treatment may prevent ochratoxin A in grapes

In traditional raisin production process in Turkey, one of the major raisin producers in the world, grapes undergo an alkali treatment prior to drying. This procedure involves dipping grapes in a potassium carbonate solution for 10-15 seconds to remove the wax layer on the surface to accelerate the drying process. In this study, we aimed to investigate the effect of alkali treatment on ochratoxin A (OTA), an important risk factor for grapes and grape-derived products. First, the stability of the OTA was examined under variable pH (4-12), ion type (Na+, K+), ionic strength (0.1-10%) and temperature (4-60 °C). Then,Aspergillus ochraceus contaminated grapes were treated with an alkaline solution and a subsequent OTA analysis was performed through a high performance liquid chromatography-fluorescence detection. OTA started to decompose at pH values exceeding 10.0. The degradation followed a first order kinetics. Although both sodium carbonate and potassium carbonate solutions were at the same alkali pH and they both triggered the OTA degradation at increasing concentrations, potassium carbonate was more effective even at low concentrations. Elevated temperatures were also found to effect degradation. The degradation rate constant at 40 °C was approximately 5 times greater than the one at 25 °C. Furthermore, OTA levels dropped up to 50% in contaminated grapes after the alkali treatment. Our results confirmed that the amide bond of OTA rapidly hydrolysed to a non-toxic ochratoxin-alpha and phenylalanine under strong alkaline conditions. The results suggest that the alkaline pre-treatment unintentionally offers a great advantage to reduce OTA levels in raisins and it could be a viable approach for other dried fruits through the application of good manufacturing practice.

Vitamin K homologs as potential biomarkers for disease activity in patients with rheumatoid arthritis

The aim of this study was to evaluate the possible role of vitamin K homologs as potential biomarkers for disease activity in patients with rheumatoid arthritis (RA). In this study, 42 patients with RA and 40 healthy controls were enrolled. Serum levels of vitamin K homologs were measured using a high-performance liquid chromatography-fluorescence method. Different biochemical and clinical markers for disease activity were measured and correlated with serum levels of vitamin K homologs. There were no significant differences between RA patients and healthy subjects in demographic data. Patients with RA showed significantly higher levels of biochemical markers compared with healthy subjects (p<0.001). These markers included rheumatoid factor (RF), anticyclic citrullinated polypeptide (anti-CCP), undercarboxylated osteocalcin (ucOC), matrix metalloproteinase (MMP-3), C-reactive protein (CRP), and disease activity score assessing 28 joints with erythrocyte sedimentation rate (DAS28-ESR). In addition, serum levels of vitamin K homologs were reduced in RA patients, and the levels of menaquinone-4 (MK-4) and menaquinone-7 (MK-7) were moderately to strongly inversely correlated with the clinical articular features in RA patients, whereas phylloquinone (PK) levels were weakly correlated. Serum levels of MK-4, MK-7 and PK were strongly inversely correlated with ucOC, MMP-3 and DAS28-ESR in RA patients. In contrast, serum levels of MK-4, MK-7 and PK were weakly correlated with CRP, RF and anti-CCP. These results suggest that serum levels of vitamin K homologs may be considered as potential biomarkers for disease activity. In addition, the results confirm the role of vitamin K deficiency in the etiology of RA.

Intralaboratory development and evaluation of a high-performance liquid chromatography-fluorescence method for detection and quantitation of aflatoxins M1, B1, B2, G1, and G2 in animal liver.

Aflatoxins are potent mycotoxins with effects that include hepatotoxicity, immunosuppression, and suppression of animal growth and production. The etiologic diagnosis of aflatoxicosis, which is largely based on analysis of contaminated feed matrices, has significant disadvantages given the fact that representative feed samples may not be available and feed-based test methods are not confirmatory of an etiologic diagnosis. A tissue-based analytical method for biomarkers of exposure would be valuable for confirmation of aflatoxicosis. We describe in-house development and evaluation of a high-performance liquid chromatographic method with fluorescence detection and precolumn derivatization for determination of aflatoxins M1, B1, B2, G1, and G2 in animal liver. The method demonstrates good selectivity for the tested aflatoxins in the liver matrix. The overall range was 0.03-0.10 ng/g for limit of detection and 0.09-0.18 ng/g for limit of quantitation. The correlation coefficient (R2) of calibration curves was >0.9978 for AFM1, 0.9995 for AFB1, 0.9986 for AFB2, 0.9983 for AFG1, and 0.9980 for AFG2 For fortification levels of 0.2-10 ng/g, repeatability was 10-18% for AFM1, 7-14% for AFB1, 5-14% for AFB2, 6-16% for AFG1, and 10-15% for AFG2 Recovery was 52-57% for AFM1, 54-62% for AFB1, 55-61% for AFB2, 57-67% for AFG1, and 61-65% for AFG2 There was no liver matrix effect found. The method is rugged against minor changes based on the selected factors. The results indicate that the proposed method is suitable for quantitative determination of aflatoxins M1, B1, B2, G1, and G2 in liver.

Determination of Triterpene Acids from 37 Different Varieties of Raspberry using Pre-column Derivatization and HPLC Fluorescence Detection

In this work, we have developed an efficient method for the rapid extraction and separation of triterpene acids from 37 different varieties of raspberry via ultrasound-assisted dispersive liquid–liquid microextraction (UA-DLLME). The triterpene acids were then determined by high-performance liquid chromatography (HPLC) with fluorescence detection using benzimidazo-[2,1-b]quinazolin-12(6H)-one-5-ethyl-p-toluenesulfonate (BQETS) as the labeling agent. Five triterpene acids, including asiatic acid (AA), maslinic acid (MA), corosolic acid (CA), oleanolic acid (OA) and betulinic acid (BA), were extracted by UA-DLLME using chloroform and acetone as the extracting and dispersing solvents, respectively. After extraction and nitrogen flushing, the extracts were simultaneously characterized by HPLC based on pre-column derivatization using BQETS, a new labeling agent synthesized in our laboratory. Several key parameters affecting the extraction efficiency and derivatization yields were investigated and optimized by response surface methodology (RSM) combined with Box–Behnken design (BBD). The method was further validated for linearity (correlation coefficient R 2 > 0.9979), precision (RSD = 0.23–2.45 %), and recovery (RSD = 90–106.5 %). The limits of detection (LODs) and the limits of quantification (LOQs) were determined to be within the range of 1.83–7.69 µg/L and 6.06–25.47 µg/L, respectively. This is the first report of the use of BQETS as a pre-column derivatization agent for the determination of triterpene acids in real samples. The proposed method has been applied to the determination of five triterpene acids in 37 different raspberry varieties with significantly increased sensitivity compared to other methods. The results obtained indicate that the contents of triterpene acids vary significantly across different raspberry varieties.

Hydroxyl radical production by a heterogeneous Fenton reaction supported in insoluble tannin from bark of Pinus radiata.

Fenton reactions driven by dihydroxybenzenes (DHBs) have been used for pollutant removal via advanced oxidation processes (AOPs), but such systems have the disadvantage of DHB release into the aqueous phase. In this work, insoluble tannins from bark can be used to drive Fenton reactions and as a heterogeneous support. This avoids the release of DHBs into the aqueous phase and can be used for AOPs. The production of ·OH was investigated using a spin-trapping electron paramagnetic resonance technique (5-dimethyl-1-pyrroline-N-oxide/·OH) in the first minute of the reaction and a high-performance liquid chromatography-fluorescence technique (coumarin/7-hydroxycoumarin) for 20min. The ·OH yield achieved using insoluble tannins from Pinus radiata bark was higher than that achieved using catechin to drive the Fenton reaction. The Fenton-like system driven by insoluble tannins achieved 92.6±0.3% degradation of atrazine in 30min. The degradation kinetics of atrazine was linearly correlated with ·OH production. The increased reactivity in ·OH production and insolubility of the ligand are promising for the development of a new technique for degradation of pollutants in wastewater using heterogeneous Fenton systems.

Development and Validation of an Analytical Method for Determination of Ergot Alkaloids in Animal Feedingstuffs with High Performance Liquid Chromatography-fluorescence Detection.

A high performance liquid chromatography combined with fluorescence detection (HPLC-FLD) method was developed for determination of five ergot alkaloids (EA): ergometrine, ergotamine, ergocornine, ergocrypine and ergocristine in animal feedingstuffs. The method was based on the application of QuEChERS salts for extraction and modified QuEChERS dispersive SPE for the cleanup step. Alkaloids separation was performed on a C18, 250 mm x 4.6 mm, 5 μm column with the mobile phase containing ammonium carbonate and acetonitrile. The excitation and emission wavelengths were 330 and 420 nm respectively. The method was validated according to the Commission Decision 2002/657/EC and all parameters are in agreement with the requirements of the Decision. Linearity was determined for the concentration range of 25-400 μg/kg. The coefficient of determination (R2) for all curves was from 0.985 to 0.996. The limit of detection (LOD) was in the range 3.23 to 6.53 μg/kg and the limit of quantification (LOQ) from 11.78 to 13.06 μg/kg. The decision limit (CCα) ranged from 29.56 to 43.08 μg/kg and detection capability (CCβ) from 40.65 to 51.01 μg/kg. The highest coefficient of variation (CV) for repeatability was 14.3% and for reproducibility 15.4%.

Sensitive and background-free determination of thiols from wastewater samples by MOF-5 extraction coupled with high-performance liquid chromatography with fluorescence detection using a novel fluorescence probe of carbazole-9-ethyl-2-maleimide

A sensitive and background-free pre-column derivatization method for the determination of thiol compounds using metal-organic framework material (MOF-5) as dispersive solid-phase extraction (DSPE) adsorbent followed by high-performance liquid chromatography fluorescence detection (HPLC-FLD) has been developed. In this paper, a novel labeling reagent, carbazole-9-ethyl-2-maleimide(CAEM), was synthesized and reacted with thiols at 40°C for 10min in the presence of PBS buffer (0.02mol/L, pH 7.5). Interestingly, CAEM itself had no fluorescence, while its derivatives exhibited intense fluorescence with an excitation maximum at λex 274nm and an emission maximum at λem 363nm, which greatly reduced the background interference and improved the sensitivity of the method. Furthermore, the MOF-5 was prepared and used as DSPE adsorbent for the selective adsorption of thiols from wastewater sample. Under the optimized experimental conditions, an excellent linearity for all analytes over their concentration ranges of 0.01–1.0μmol/L (R2>0.9986)were obtained with the limit of detection (LOD) ranging from 8 to 17.1pmol/L for nine tested thiols. The feasibility of this method for the determination of thiols in wastewater samples had been evaluated and satisfactory average recoveries (n=3) were achieved with the range of 86.6–98.5%.

Determination of methylglyoxal in human blood plasma using fluorescence high performance liquid chromatography after derivatization with 1,2-diamino-4,5-methylenedioxybenzene.

Methylglyoxal (MG) is a highly reactive dicarbonyl compound that promotes the non-enzymatic glycation of proteins to yield irreversible advanced glycated end products, leading to the cross-linking or degradation of proteins. The physiological relevance of MG currently remains unclear because its metabolic behavior has not yet been elucidated in detail. Although several labeling methods that require a HPLC system have been developed and used to measure MG, a standard method to analyze the content of MG in biological samples has not been established. We herein present a practical method based on HPLC with fluorescence detection to measure low MG levels. MG concentrations were also measured in human blood plasma using the present method in order to demonstrate its utility. A calibration curve was produced using freshly purified MG at concentrations ranging between 0.05 and 1.0μM. The intra-day and inter-day relative standard diviations of the method were 2.55% and 4.03%, respectively. The limit of detection and limit of quantification were 60fmol and 200fmol, respectively for MG with a 10-μl injection volume of the derivatized sample solution. When the optimized method was applied to human plasma, the resulting concentrations of MG in the plasma of healthy subjects (n=23) ranged between 0.024 and 0.258μM (mean±SD=0.098±0.066). Thus, the method developed herein is simple, sensitive, and easy to operate for the measurement of MG in biological samples.

Molecularly imprinted polymer as efficient sorbent of solid-phase extraction for determination of gonyautoxin 1,4 in seawater followed by high-performance liquid chromatography-fluorescence detection.

A kind of new molecularly imprinted polymer (MIP) was synthesized by bulk polymerization using guanosine as dummy template molecule, α-methacrylic acid as functional monomer and ethylene glycol dimethyl acrylic ester as crosslinker. Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM) showed that the MIP had homogenous and uniform-sized cavities. It was confirmed that the MIP had higher binding affinity and selectivity towards gonyautoxins 1,4 (GTX 1,4) than the non-imprinted polymer (NIP) according to the static equilibrium adsorption. An off-line molecularly imprinted solid-phase extraction (MISPE) method followed by high-performance liquid chromatography with fluorescence detection (HPLC-FLD) was established for the analysis of GTX 1,4. 0.1mol/L acetic acid and 95:5 (v:v) methanol/water were optimized as the washing and elution solutions, respectively. The recoveries of spiked cultured seawater samples were satisfactory, as high as 88%. Using this method, the concentrations of GTX 1,4 from cultured seawater samples of Alexandrium minutum and Alexandrium tamarense were detected to be 1.10μg/L and 0.99μg/L, respectively. Graphical Abstract The synthesis of molecularly imprinted polymer and molecularly imprinted solid-phase extraction analysis for gonyautoxin 1,4.