High-performance Liquid Chromatography With Fluorescence Detection Research Articles

Content of Selected Harmful Metals (Zn, Pb, Cd) and Polycyclic Aromatic Hydrocarbons (PAHs) in Honeys from Apiaries Located in Urbanized Areas.

The chemical composition of honey, and therefore its quality and properties, is influenced by many factors, including its botanical origin and the harvesting conditions-the location of the apiary, access to melliferous plants, the proximity of industrial infrastructure and communication routes, etc. This quality may be reduced by undesirable, toxic compounds that penetrate honey from a contaminated environment, such as heavy metals and residues from other environmental pollutants. Therefore, the aim of our research was to assess the quality of honeys from urbanized areas-in particular, to assess contamination with heavy metals and persistent organic pollutants (PAHs). In total, 35 samples from six different apiaries located in urbanized areas were examined. The content of heavy metals (Cd, Pb, Zn) was determined by atomic absorption spectrophotometry (AAS), and the content of total PAHs as the sum of the concentrations of the compounds benzo(a)anthracene, chrysene, benzo(b)fluoranthene and benzo(a)pyrene was determined by high-performance liquid chromatography with fluorescence detection (HPLC-FLD). The average zinc content ranged from about 2 to 4.5 mg/kg, the average lead content ranged from 3.5 µg/kg to 388 µg/kg and the average cadmium content ranged from 0.5 to 14 µg/kg. It was found that all honeys contained certain amounts of harmful metals, and only lead exceeded the permissible limits. None of the samples tested contained sum content of PAHs exceeding 10 µg/kg of honey. Contrary to our expectations, the results obtained indicate that honeys from urbanized areas do not contain these harmful substances. In general, the presence of harmful metals does not, however, reduce honey's quality or its health value.

Determination of PAH Contamination in Breast Milk Samples from Hungarian Volunteering Mothers, Using HPLC-FLD.

(1) The evidence is mounting that polycyclic aromatic hydrocarbons (PAHs) are a class of hazardous organic compounds with established carcinogenic and toxic properties. Humans may be exposed to PAHs through several different routes, including diet, inhalation, and dermal contact. There is also a possibility that they could transfer into breast milk following maternal exposure, which could potentially endanger breastfeeding infants. (2) The objective of this study was to ascertain the concentration of polycyclic aromatic hydrocarbons (PAHs) in breast milk samples from 50 Hungarian mothers, employing high-performance liquid chromatography with fluorescence detection (HPLC-FLD). An Incremental Life Risk Calculation (IRCL) model estimated the carcinogenic risk to infants. (3) Total PAH concentrations ranged from 0 to 78 ng/mL, with fluorene (5.3 ng/mL), phenanthrene (3.2 ng/mL), and pyrene (2.5 ng/mL) being the most abundant. PAHs were detected in 48 of the 50 samples, with phenanthrene present in 92% of samples. Dibenzo (a,h)anthracene was not detected. (4) According to the model measurements, most of the samples were within acceptable risk levels; however, 2 samples out of 50 posed a higher risk. Statistical analysis of questionnaires completed by the mothers indicated that factors such as diet, residence, and education may influence PAH levels in breast milk.

Presence of Ochratoxin A Residues in Blood Serum of Slaughtered Pigs in Greece.

This study aimed to assess the presence of ochratoxin A (OTA) residues in the blood serum of slaughtered pigs in Greece. Samples were obtained from 1695 healthy slaughtered pigs originating from 113 different farms located in 21 geographic regional units in 8 different geographic regions of Greece and were analyzed using an immunosorbent assay (ELISA) and high-performance liquid chromatography with fluorescence detector (HPLC-FD). OTA contamination assessment showed that 782 (46.1%) and 1233 (72.7%) samples were OTA-positive, with a concentration range of 0.20-5.38 μg/L and 0.15-5.96 µg/L according to ELISA and HPLC-FD analysis, respectively. Also, 88 (77.9%) and 108 (95.6%) of farms were found to be OTA-positive by ELISA and HPLC-FD analysis, respectively. The highest OTA serum positivity rate (>98%) and toxin level (5.96 µg/L) determined by HPLC-FD were observed in the Thessaly region, whereas a high prevalence of up to 100% (range 75-100%) was found on farms in the Crete Island region. The detection of OTA in the serum of slaughtered pigs in different regions in Greece poses a risk for animal and human health and highlights the need for constant OTA monitoring in the swine industry and pork meat production facilities.

Assessment of Polycyclic Aromatic Hydrocarbons in Urban Water Bodies: A Study of Rivers and Lakes in Dhaka, Bangladesh

Polycyclic aromatic hydrocarbons (PAHs) pose significant environmental and health risks due to their toxic properties,making their assessment in urban water bodies crucial. This study aims to evaluate the presence of three notable PAHs—anthracene, fluoranthene, and benzo[a]pyrene—in the surface water of three rivers (Buriganga, Meghna, and Turag) andtwo lakes (Hatirjheel and Gulshan) in Dhaka, Bangladesh. High-performance liquid chromatography with fluorescencedetector (HPLC-FD) was employed and validated for PAH determination, using solid phase extraction (SPE) withpreconditioned C-18 SPE cartridge for sample extraction. Calibration showed excellent linearity with correlation coefficient (R²) ≥ 0.999. The limits of detection (LODs) were 200 ng/L for anthracene and 0.63 ng/L for both fluoranthene and benzo[a]pyrene, with corresponding limits of quantification (LOQs) of 660 ng/L and 2.08 ng/L, respectively. Percent recovery was 91.38% for anthracene, 85.49% for fluoranthene, and 95.72% for benzo[a]pyrene, with relative standard deviations (RSD) of 5.27, 17.55 and 2.84%, respectively. Most water samples had PAH levels below detection limits (bdl) such as anthracene (bdl-1789.57 ng/L), fluoranthene (bdl-1309.23 ng/L), and benzo[a]pyrene (bdl-25.17 ng/L), and the detected concentrations were significantly lower than WHO and USEPA guideline values. This indicates a relatively low level of PAHs contamination in the studied water bodies. Dhaka Univ. J. Sci. 72(2): 75-83, 2024 (July)

Nonclinical Pharmacokinetics Study of OLX702A-075-16, N-Acetylgalactosamine Conjugated Asymmetric Small Interfering RNA (GalNAc-asiRNA).

In this study, the nonclinical pharmacokinetics of OLX702A-075-16, an RNA interference therapeutic currently in development, were investigated. OLX702A-075-16 is a novel N-acetylgalactosamine conjugated asymmetric small-interfering RNA (GalNAc-asiRNA) used for the treatment of an undisclosed liver disease. Its unique 16/21-mer asymmetric structure reduces nonspecific off-target effects without compromising efficacy. We investigated the plasma concentration, tissue distribution, metabolism, and renal excretion of OLX702A-075-16 following a subcutaneous administration in mice and rats. For bioanalysis, high-performance liquid chromatography with fluorescence detection was used. The results showed rapid clearance from plasma (0.5 to 1.5 hours of half-life) and predominant distribution to the liver and/or kidney. Less than 1% of the liver concentration of OLX702A-075-16 was detected in the other tissues. Metabolite profiling using liquid chromatography coupled with high-resolution mass spectrometry revealed that the intact duplex OLX702A-075-16 was the major compound in plasma. The GalNAc moiety was predominantly metabolized from the sense strand in the liver, with the unconjugated sense strand of OLX702A-075-16 accounting for more than 95% of the total exposure in the rat liver. Meanwhile, the antisense strand was metabolized by the sequential loss of nucleotides from the 3'-terminus by exonuclease, with the rat liver samples yielding the most diverse truncated forms of metabolites. Urinary excretion over 96 hours was less than 1% of the administered dose in rats. High plasma protein binding of OLX702A-075-16 likely inhibited its clearance through renal filtration. SIGNIFICANCE STATEMENT: This study presents the first comprehensive characterization of the in vivo pharmacokinetics of GalNAc-asiRNA. The pharmacokinetic insights gained from this research will aid in understanding toxicology and efficacy, optimizing delivery platforms, and improving the predictive power of preclinical species data for human applications.

Determination of sodium cyclamate in Baijiu by pre-column derivatization-high performance liquid chromatography with fluorometric detection

Sodium cyclamate in Baijiu is a key item in the China National Food Safety Supervision and Inspection Plan. A simple, economical, sensitive, and reliable method is urgently needed for routine analysis and internal quality control. A method based on high performance liquid chromatography with fluorescence detection (HPLC-FLD) was developed for the determination of sodium cyclamate in Baijiu by o-phthalaldehyde derivatization. First, the sodium cyclamate in the sample solution was converted into amino compounds using the desulfurization reaction under acidic conditions. Next, 400 g/L sodium hydroxide solution was added to the sample solution for neutralization. The amino compounds in the sample solution were then derivatized with o-phthalaldehyde to produce indole-substituted derivatives that are capable of producing fluorescence signals. Separation was carried out on a C18 column (250 mm×4.6 mm, 5 μm) in isocratic elution mode using a mobile phase consisting of acetonitrile and phosphate buffer. Finally, the eluate was monitored using a fluorescence detector, and an external standard method was used for quantification. A good linear relationship was obtained in the range of 0.1-2.0 mg/L, with correlation coefficients greater than 0.999. The average recoveries of sodium cyclamate spiked at levels of 0.1-1.0 mg/kg in Baijiu samples ranged from 90.7% to 100.9%, with relative standard deviations (RSDs) of 3.5%-5.6% (n=6). The limits of detection and quantification were 0.03 and 0.10 mg/kg, respectively. Nine Baijiu samples collected from the market were tested, and the results demonstrated that the contents of sodium cyclamate detected by the developed method were consistent with those obtained using the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method described in GB 5009.97-2016 (the third method). The proposed method is economical, sensitive, specific, and accurate; thus, it provides a basic approach for the determination of sodium cyclamate in Baijiu samples and has great potential for routine analysis in foodstuffs.

Effects of fermentation time, baking, and storage on ochratoxin A levels in sourdough flat bread.

Ochratoxin A (OTA), which is one of the most important mycotoxins in terms of human health, can be found in cereal products such as bread, "bazlama" (traditional flatbread), and pita bread, as well as cereals such as wheat, barley, and corn. This study aimed to determine the effect of different fermentation times, baking, and storage for various periods on the presence of OTA in sourdough bazlama. Bazlama flour was contaminated with OTA concentrations of 5 and 10 μg/kg. After two different fermentation times (1.5 and 3 h), baking at 300 ± 5°C, and storage at room temperature (25 ± 2°C) for 0, 5, and 10 days, the change in OTA levels of bazlama samples was determined by the high-performance liquid chromatography with fluorescence detector (HPLC-FLD) method. The effect of different storage periods on the presence of OTA is insignificant. Although a general decrease in OTA level has been determined, it has been found that long-term fermentation (at least 3 h) was more effective, especially in flours with a high concentration (10 μg/kg) of OTA contamination. It has been determined that bazlama made from contaminated flours with OTA levels of 5 and 10 μg/kg contained OTA levels exceeding 3 μg/kg when long-term fermentation was not used. This is the maximum permitted limit set by the Turkish Food Codex and the European Commission, indicating that it is not suitable for consumption in this position.

Highly sensitive analysis of fatty aldehydes in vegetable oils using a novel coumarin-based fluorescent probe by HPLC for quality control

A novel fluorescent probe, 3-(4-((hydroxylamino)methyl)-1H-pyrazol-1-yl)-7-methoxyl coumarin (HAPMC), has been designed and synthesized. The compound exhibits exceptional fluorescence properties due to a coumarin-pyrazolyl fluorophore. Moreover, it incorporates an N-hydroxylamine functional group capable of reacting with aldehydes to yield nitrone derivatives under mild conditions. This unique combination renders it an ideal derivatization reagent for aldehyde analysis. A standard method was established for the quantitative analysis of eight fatty aldehydes by utilizing high-performance liquid chromatography with fluorescence detection (HPLC-FLD) and a pre-column derivatization process, which has low detection limits (0.3–1.2 nM), satisfactory reproducibility (intraday and interday relative standard deviations in the range of 1.29 %-2.92 % and 1.73 %-3.45 %), and an extensive linear range (25–5000 nM) with excellent linearity (R2 ≥ 0.9989). The spiked recoveries of the fatty aldehydes range from 89.6 % to 106.0 %. The greenness and sustainability of our approach are reinforced by evaluations conducted with the AGREE and RGB 12 algorithms. The established method was successfully applied to the qualitative and quantitative detection of fatty aldehydes in nine real vegetable oil samples, including peanut, canola, rice, soybean, olive, rapeseed, corn, sunflower, and blended oils. The fatty aldehydes are known to have adverse effects on human health due to their carcinogenic and mutagenic properties. The findings revealed that hexanal and nonanal were the most prevalent saturated fatty aldehydes, with a notable increase in the total aldehyde concentration observed as the temperature and duration of heating increased. Compared to alternative analytical techniques, this pre-column derivatization method utilizing HAPMC displayed simplicity, reliability, and sensitivity. The results demonstrate the potential for broad application of this approach in various important research domains, including food quality control, environmental monitoring, and medical research.

Molecular and aflatoxigenicity analyses of Aspergillus flavus isolates indigenous to grain corn in Malaysia; potentials for biological control.

The present work aimed to distinguish the indigenous Aspergillus flavus isolates obtained from the first (pioneer) grain corn farms in Terengganu, Malaysia, into aflatoxigenic and non-aflatoxigenic by molecular and aflatoxigenicity analyses, and determine the antagonistic capability of the non-aflatoxigenic isolates against aflatoxigenic counterparts and their aflatoxin production in vitro. Seven A. flavus isolates previously obtained from the farms were characterized molecularly and chemically. All isolates were examined for the presence of seven aflatoxin biosynthesis genes, and their aflatoxigenicity were confirmed using high performance liquid chromatography with fluorescence detector (HPLC-FLD). Phylogenetic relationships of all isolates were tested using ITS and β-tubulin genes. Of the seven isolates, two were non-aflatoxigenic, while the remaining were aflatoxigenic based on the presence of all aflatoxin biosynthesis genes tested, and the productions of aflatoxins B1 and B2. All isolates were also confirmed as A. flavus following phylogenetic analysis. The indigenous non-aflatoxigenic isolates were further examined for their antagonistic potential against aflatoxigenic isolates on 3% grain corn agar (GCA). Both non-aflatoxigenic isolates significantly reduced AFB1 production of the aflatoxigenic isolates. The indigenous non-aflatoxigenic A. flavus strains identified in the present work were effective in controlling the aflatoxin production by the aflatoxigenic A. flavus isolates in vitro and can be utilized for in situ testing.

Occurrence and health risk assessment of mineral composition and aflatoxin M1 in cow milk samples from different areas of Sicily, Italy

This study aimed to determine 16 mineral elements (Cd, Pb, As, Na, Mg, Al, Ca, K, Cr, Mn, Fe, Ni, Cu, Zn and Se) using inductively coupled plasma mass spectrometry (ICP-MS) and a direct mercury analyzer (DMA-80) for Hg evaluation. Aflatoxin M1 was determined by high-performance liquid chromatography with fluorescence detection (HPLC-FLD) in cow milk samples. This research considered 180 milk samples, 20 by province (Palermo, Catania, Messina), collected for a period of three years (2020–2022) to assess the potential risks for consumer, the safety status and nutritional quality related to mineral intake by consuming of milk. All samples showed a Pb concentration below the limit reported by European Regulation 915/2023. Cadmium and Hg concentrations were below the Limit Of Quantification (LOQ) in all samples analyzed. The milk samples analyzed proved to be a good source of Ca (up to 44.5 % of the dietary reference values), with well percentages also for Na (up to 7.6 %), K (up to 23.1 %) and Mg (up to 11.1 %). Regarding trace elements, the results reported that chromium requires attention; its value was always higher than 168.8 % in all samples analyzed. Levels of arsenic and lead were up to 20.2 % and up 7.1 % respectively. Aflatoxin M1 concentrations were below the limit of detection (< 0,009 mcg/kg) in all milk analyzed. Therefore, further studies are needed to safeguard consumer health, the quality of the product and to assess the state of animal health.

Evaluation of the safety and quality of Brazil nuts (Bertholletia excelsa) using the tools of dna sequencing technology and aflatoxin profile.

Brazil nuts (BNs) result from sustainable extraction and are widely exploited in the Amazon region. Due to the production characteristics in the forest and the nutritional characteristics of these nuts, the occurrence of fungal contamination and the presence of aflatoxins are extensively discussed in the literature as a great aspect of interest and concern. This study aims to evaluate the microbial profile through DNA sequencing and amplification of 16S and ITS genes for bacterial and fungal analysis, respectively, and the presence of mycotoxins using high-performance liquid chromatography with fluorescence detection (HPLC-FD) from different fractions of the nuts processed. The BN samples, harvest A (HA) and harvest B (HB), from two different harvests were collected in an extractive cooperative in the Amazon region for microbiological analysis (from DNA extraction and amplification of 16S genes, bacteria analysis, and ITS for fungi) and mycotoxins (aflatoxins AFB1, AFB2, AFG1, and AFG2) using HPLC-FD/KobraCell®. The samples showed a very different microbiome and aflatoxin profile. Genera such as Rothia (HA) and Cronobacter (HB) were abundant during the analysis of bacteria; as for fungi, the genera Aspergillus, Fusarium, Penicillium, and Alternaria were also considered prevalent in these samples. Soil microorganisms, including those pathogenic and related to inadequate hygienic-sanitary production practices, as well as aflatoxins, were found in the samples. However, they were within the established limits permitted by Brazilian legislation. Nuts have a diverse microbiota and are not restricted to fungi of the genus Aspergillus. The microbiological and toxicological profile can vary significantly within the same nut in the same extraction region and can be exacerbated by global climate changes. Therefore, it is necessary to advance sanitary educational actions by applying good production practices and inspection programs to ensure the sustainability and quality of the BN production chain.

Anticancer Drug Extraction from Plasma Samples Using Three-Dimensional Polyoxometalate-Based Supramolecular Frameworks as Sorbents.

Four self-assembled inorganic-organic hybrid materials, namely, H{Na(H2O)3[Gd(PDA)(H2O)2]3[BW12O40]}·4H2O (1), H{Na(H2O)3[Tb(PDA)(H2O)2]3[BW12O40]}·3H2O (2), H{Na(H2O)3[Er(PDA)(H2O)3]3[BW12O40]}·H2O (3) (PDA = 1,10-phenanthroline-2,9-dicarboxylate), and [Pr3(H2O)13(pydc-OH)2][BW12O40]·12H2O (4) (pydc-OH = 4-hydroxy-2,6-pyridinedicarboxylate), were hydrothermally synthesized and structurally characterized. Hybrids 1-3 are isostructural and contain a Keggin unit, which is linked to lanthanoids to produce distinct trinuclear lanthanoid building blocks. The fragments are connected by anion-π and hydrogen bonding interactions to create 3D networks. In hybrid 4, a trimeric Pr-organic species bearing a Keggin unit forms a 2D coordination polymer, and then hydrogen bonding interactions between 2D layers lead to the formation of a 3D structure. These polyoxometalate-based frameworks were used as sorbents for the dispersive microsolid-phase extraction (D-μSPE) of two anticancer drugs (doxorubicin and epirubicin) in human plasma samples. Analytes were quantified and separated using high-performance liquid chromatography with fluorescence detection (HPLC-FLD). The method's linearity was between 0.8-500 ng mL-1 and 1.0-500 ng mL-1 for the antineoplastic drugs doxorubicin and epirubicin, respectively. The limits of detection (S/N = 3) were in the range of 0.2-0.3 ng mL-1, while the precision was in the range of 3.5-4.3%. Finally, human plasma samples from patients treated with doxorubicin or epirubicin were analyzed by using the D-μSPE-HPLC-FLD method.

Synthesis of Layer‐By‐Layer Decorated Graphene Oxide with Bismuth Telluride Nanoparticles‐modified Dendrimer: Application for Nonylphenol Removal from Aqueous Solutions

AbstractHerein, for the first time, the bismuth telluride (Bi2Te3) nanoparticles functionalized graphene oxide (GO) were synthesized for the removal of nonylphenol (NP). The surface of the GO‐Bi2Te3 nanocomposite was modified by hyperbranched dendrimer and characterized by different spectroscopic and morphological techniques such as Fourier transform infrared (FT‐IR) spectroscopy, X‐ray diffraction (XRD), field‐emission scanning electron microscope (FE‐SEM), energy dispersive X‐ray spectroscopy (EDX), and thermogravimetric analysis (TGA). In this paper, to enhance the capacity, the surface of GO‐Bi2Te3@GlyTsCl dendrimer was modified by 5‐amino‐2‐hydroxybenzoic acid (5‐ASA). The effects of parameters on the removal of NP were also studied. This study reports the utilization of nanoadsorbent (GO‐Bi2Te3@GlyTsCl/5‐ASA) for the removal of NP by high‐performance liquid chromatography with fluorescence detector (HPLC‐FLD). Under optimal conditions, the removal values for the method were in the range of 90–96 %. Additionally, the Langmuir model was found to be fit with the isotherms data. The best kinetic model fit for the adsorption of NP from nanoadsorbent was the pseudo‐second‐order model. Moreover, GO‐Bi2Te3@GlyTsCl/5‐ASA nanoadsorbent exposed good reusability even after six consecutive cycles (from 100 % to 93 %) with high stability. Consequently, synthesized GO‐Bi2Te3@GlyTsCl/5‐ASA composites could be potentially used as an efficient nanoadsorbent for NP removal in water samples.

Influence of UV light, ultrasound, and heat treatment on the migration of bisphenol A from polyethylene terephthalate bottle into the food simulant

This research examined the impacts of ultrasound, UV light, storage time, and temperature on the leaching of bisphenol A (BPA) from polyethylene terephthalate (PET) drinking water bottles in Turkey. The initial phase of the investigation encompassed the quantification of BPA in two distinct brands of bottled water. Samples were extracted by solid- phase extraction (SPE) and analyzed by high performance liquid chromatography with fluorescence detection (HPLC‐FLD). According to the results in the first part, the highest BPA levels were found in bottled water. In the second part of the study, 10 to 30 min of ultrasound treatment increased the BPA migration with increased time in simulants. In the first and second weeks of storage at 25 °C, the effect of storage on BPA migration was below the detection limit (<LOD) in simulants. However, at 35 °C and 45 °C, BPA concentration increased with increased temperature. According to these results, increasing the storage temperature to 35 °C and 45 °C increased the BPA pass faster than the increase in storage time. When simulants in PET bottles were exposed to UV light for 1 and 6 h, BPA transitions higher from 6 to 1 h was found in simulants. Except for first-brand water samples, the passing of bisphenol A was found to comply with the European Food Safety Authority (EFSA) criteria in this study.

Development, in-house validation and application of a method using high-performance liquid chromatography with fluorescence detection (HPLC-FLD) for the quantification of 12 ergot alkaloids in compound feeds.

Ergot alkaloids (EAs) are toxic substances naturally produced by Claviceps fungi. These fungi infest a wide range of cereals and grasses. When domestic animals are exposed to EAs through contaminated feeds, it is detrimental to them and leads to significant economic losses. For that reason, it is important to monitor feed for the presence of EAs, especially with methods enabling their determination in processed materials. Ergot alkaloids were extracted with acetonitrile, and dispersive solid phase extraction (d-SPE) was used for clean-up of the extracts. After evaporation, the extracts were reconstituted in ammonium carbonate and acetonitrile and subjected to instrumental analysis using high-performance liquid chromatography with fluorescence detection. The developed method was validated in terms of linearity, selectivity, repeatability, reproducibility, robustness, matrix effect, limits of quantification and detection and uncertainty. The EA content of 40 compound feeds was determined. All the assessed validation parameters fulfilled the requirements of Regulation (EU) 2021/808. At least one of the monitored alkaloids was determined in 40% of the samples. The EAs with the highest incidence rate were ergocryptine, ergometrinine and ergocornine. The total concentrations of EAs ranged from under the limit of quantification to 62.3 μg kg-1. The results demonstrated that the developed method was suitable for simultaneously determining twelve EAs in compound feed and could be used for routine analysis.

Synthesis and application of BODIPY-based fluorescent labeling tag for oligosaccharide and N-linked glycan analysis by high-performance liquid chromatography with fluorescence detection

BackgroundGlycosylation analysis is still challenging, not only because of the extreme structure complexity and conjugation diversity of glycans but also because of instrumental aspects such as the sensitivity limits of analyses. Therefore, glycan analysis by chromatographic methods is very often combined with fluorescence detection in addition to MS. The majority of fluorescent labeling employed before LC separation is based on 2-aminobenzamide, which has several disadvantages such as low labeling yield, poor fluorescence properties, and MS ionization efficiency. Therefore, even after several decades of development of new labels, there is still a need for new labeling tags with improved characteristics. ResultsWe present the application of a newly synthesized fluorescent label designed for oligosaccharide and glycan analysis by high-performance liquid chromatography with fluorescence detection (HPLC/FLD). The novel hydrazide derivative of dipyrrometheneboron difluoride (BODIPY) was synthesized from 2,4-dimethylpyrrole, methyl succinyl chloride, and boron trifluoride etherate followed by a reaction with hydrazine. The synthesized label was characterized by several analytical methods including NMR, UV/Vis and fluorescence spectroscopy, and mass spectrometry. The labeling reaction via hydrazone formation chemistry was optimized by labeling of maltooligosaccharide standards. The analysis of maltohexaose labeled by BODIPY-hydrazide followed by HPLC/FLD analysis provided the limit of detection in the low tens of femtomole. The presented method based on fluorescence detection is at least 30 times more sensitive than the standard approach employing labeling by 2-aminobenzamide. In addition, the labeling method by BODIPY-hydrazide was used for N-linked glycan profiling of several glycoproteins (ribonuclease B, immunoglobulin G) by RP-HPLC/FLD as well as HILIC/FLD analysis. SignificanceThis work represents the design, synthesis, and application of a new fluorescent label based on the BODIPY core and hydrazone formation chemistry for oligosaccharide and glycan analysis by HPLC/FLD. The proposed approach significantly improved the oligosaccharide and glycan analysis in comparison to the commonly used procedure employing 2-aminobenzamide.

In Situ Formation of a Relatively Transparent Ion-Associate Liquid Phase from an Aqueous Phase and Its Application to Microextraction/High-Performance Liquid Chromatography-Fluorescence Detection of Bisphenol A in Water.

The design of a simple approach enabling the detection of bisphenol A (BPA) in water samples without the need for large amounts of solvents is of utmost importance. This paper reports a simple method for the separation, concentration, and quantification of BPA in water samples using high-performance liquid chromatography with fluorescence detection (HPLC-FLD) after its microextraction into an in situ formed organic ion-associate (IA) liquid phase (LP). Novel IA phase components without conjugated double bonds, such as benzene rings, were investigated. Ethylhexyloxypropylamine hydrochloride and sodium dodecyl sulfate solutions were added to the water samples to form IAs. The aqueous phase and ion-associate liquid phase (IALP) were separated by centrifugation. The aqueous phase was removed, and the liquid phase was recovered and measured using HPLC-FLD or HPLC-electrochemical detection (ECD). The concentrated phase (IALP) had a relatively low viscosity and could be injected directly into the chromatograph without dissolving it in organic solvents. The detection limits for BPA by HPLC-FLD and HPLC-ECD were 0.009 and 0.3 µg L-1, respectively.

In Vitro Permeability Study of Homotaurine Using a High-Performance Liquid Chromatography with Fluorescence Detection Pre-Column Derivatization Method.

Homotaurine (HOM) is considered a promising drug for the treatment of Alzheimer's and other neurodegenerative diseases. In the present work, a new high-performance liquid chromatography with fluorescence detection (HPLC-FLD) (λex. = 340 nm and λem. = 455 nm) method was developed and validated for the study of substance permeability in the central nervous system (CNS). Analysis was performed on a RP-C18 column with a binary gradient elution system consisting of methanol-potassium phosphate buffer solution (pH = 7.0, 0.02 M) as mobile phase. Samples of homotaurine and histidine (internal standard) were initially derivatized with ortho-phthalaldehyde (OPA) (0.01 M), N-acetylcysteine (0.01 M) and borate buffer (pH = 10.5; 0.05 M). To ensure the stability and efficiency of the reaction, the presence of different nucleophilic reagents, namely (a) 2-mercaptoethanol (2-ME), (b) N-acetylcysteine (NAC), (c) tiopronin (Thiola), (d) 3-mercaptopropionic acid (3-MPA) and (e) captopril, was investigated. The method was validated (R2 = 0.9999, intra-day repeatability %RSD < 3.22%, inter-day precision %RSD = 1.83%, limits of detection 5.75 ng/mL and limits of quantification 17.43 ng/mL, recovery of five different concentrations 99.75-101.58%) and successfully applied to investigate the in vitro permeability of homotaurine using Franz diffusion cells. The apparent permeability (Papp) of HOM was compared with that of memantine, which is considered a potential therapeutic drug for various CNSs. Our study demonstrates that homotaurine exhibits superior permeability through the simulated blood-brain barrier compared to memantine, offering promising insights for enhanced drug delivery strategies targeting neurological conditions.

Highly selective and sensitive high performance liquid chromatography method for determination of fumonisins from feed samples with MIL-101 based dispersed micro-solid-phase extraction and precolumn fluorescence derivatization

A selective and rapid high-performance liquid chromatography with fluorescence detection (HPLC-FLD) method for determining fumonisins was established and validated, which based on dispersed microsolid-phase extraction (D-μ-SPE) and precolumn derivatization. The MIL-101 was prepared as sorbents to selectively enrich two fumonisins from feed samples. The obtained extracts were labeled by a novel derivatization reagent 2,5-dioxopyrrolidin-1-yl 4-(1-methyl-1H-phenanthro[9,10-d]imidazol-2-yl)benzoate (MPIBA-Su). There was no fluorescence from fumonisins itself, while their derivatives exhibited considerable fluorescence with a maximum excitation at λex 255 nm and a maximum emission at λem 457 nm. Key factors affecting extraction efficiency and derivatization efficiency were fully optimized. Excellent linearity was achieved with correlation coefficients > 0.9935 under optimal conditions. The detection limits (LODs) were between 2.4 and 3.5 ng·mL−1, and the quantitation limits (LOQs) were between 8.1 and 11.7 ng·mL−1. With recoveries in the range of 83.8% − 94.3%, the established method has successfully been applied to the quantitative analysis of fumonisins in feed samples.

Development and Comparative Evaluation of Two Different Label-Free and Sensitive Fluorescence Platforms for Analysis of Olaparib: A Recently FDA-Approved Drug for the Treatment of Ovarian and Breast Cancer.

Olaparib (OLA) is a PARP inhibitor drug which has been recently approved by the Food and Drug Administration (FDA) for the treatment of ovarian and breast cancer. A convenient analytical tool for the quantitation of OLA in its dosage form and plasma samples was urgently needed. This study describes, for the first time, the development of two different label-free and sensitive fluorescence-based platforms for the pharmaceutical and bioanalysis of OLA. These platforms were microwell-assisted with a fluorescence microplate reader (MW-FLR) and high-performance liquid chromatography with fluorescence detection (HPLC-FD). Both MW-FLR and HPLC-FD employed the native fluorescence of OLA as an analytical signal. The MW-FLR involved measuring the fluorescence signals in 96-well white-opaque plates. The HPLC-FD involved chromatographic separation of OLA and duvelisib (DUV), as an internal standard on a Nucleosil-CN HPLC column (250 mm length × 4.6 mm i.d., 5 µm particle diameter) with a mobile phase composed of acetonitrile: water (25:75, v/v) pumped at a flow rate of 1.7 mL/min. Elution of OLA and DUV was detected using a fluorescence detector. The optimal conditions of both MW-FLR and HPLC-FD were established, and they were validated according to the guidelines of the International Council for Harmonization for the validation of analytical procedures. The linear ranges of MW-FLR and HPLC-FD were 25-1000 and 5-200 ng/mL, respectively, with limits of detection of 15 and 1.7 ng/mL, respectively. The accuracy and precision of both platforms were confirmed as the recovery values were ≥98.2% and the relative standard deviations (RSD) were ≤2.89%. Both methodologies were satisfactorily applied to the quantitation of OLA in its commercial dosage form (Lynparza® tablets) and plasma samples with high accuracy and precision. The greenness of both MW-FLR and HPLC-FD was assessed using two different multiple parameter-based metric tools, and the results proved their greenness and adherence to the requirements of green analytical approaches. Both platforms have simple procedures and acceptable levels of analytical throughput. In conclusion, the proposed MW-FLR and HPLC-FD are valuable tools for routine use in quality control and clinical laboratories for the quantitation of OLA for the purposes of pharmaceutical quality control, pharmacokinetic studies, and bioequivalence testing.