9-fluorenylmethyl Chloroformate Research Articles

9-Fluorenylmethyl Chloroformate Labeling for O-Glycan Analysis.

Structural analysis of O-glycans from mucins and characterization of the interaction of these glycans with other biomolecules are essential for a full understanding of mucins. Various techniques have been developed for the structural and functional analysis of glycans. While 9-fluorenylmethyl chloroformate (Fmoc-Cl) is generally used to protect amino groups in peptide synthesis, it can also be used as a glycan-labeling reagent for structural analysis. Fmoc-labeled glycans are strongly fluorescent and can be analyzed with high sensitivity using liquid chromatography-fluorescence detection (LC-FD) analysis as well as being analyzed with high sensitivity by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Fmoc-labeled glycans can be easily delabeled and converted to glycosylamine-form or free (hemiacetal or aldehyde)-form glycans that can be used to fabricate glycan arrays or synthesize glycosyl dendrimers. This derivatization allows for the isolation from biological samples of glycans that are difficult to synthesize chemically, as well as the fabrication of immobilized-glycan devices. The Fmoc labeling method promises to be a tool for accelerating O-glycan structural analysis and an understanding of molecular interactions. In this chapter, we introduce the Fmoc labeling method for analysis of O-glycans and fabrication of O-glycan arrays.

Automated method for quantification of20aminoacids in cell culture media during biopharmaceutical development.

Herein, a step-by-step protocol for simultaneous detection of 20 amino acids commonly present incell culture media is described. The protocol facilitates detection of both primary and secondary amino acids through a two-step precolumn derivatization strategy usingortho-phthalaldehyde and 9-fluorenylmethyl chloroformate as derivatizing agents. The separation of derivatized amino acids with varying hydrophobicity is achieved through reverse-phase chromatography. The amino acids are simultaneously detected in a single workflow through the use of Variable Wavelength Detector at 338 and 262nm. The protocol is applicable for both mammalian and bacterial cell culture matrices with an option for automation of precolumn derivatization.

High-performance liquid chromatography with fluorescence detection method for measuring colistin in human serum and its application in critically ill patients treated with colistin methanesulfonate sodium

Colistin, in the form of colistin methanesulfonate sodium (CMS), has a narrow therapeutic window, and colistin levels must be monitored during treatment. The aim of this study was to develop and validate a high-performance liquid chromatography-fluorescence detection (HPLC-FLD) method for measuring colistin in human serum. Colistin was extracted from human serum using trichloroacetic acid and methanol for protein precipitation, followed by in-solid phase extraction derivatization with 9-fluorenylmethyl chloroformate. HPLC-FLD system using a reverse-phase HPLC C18 column and a mobile phase consisting of acetonitrile, tetrahydrofuran, and water (80%:4%:16%) were used for the quantification of colistin A, colistin B, and polymyxin B (internal standard) in human serum. The extraction recoveries were between 71% and 103%. Linear calibration curves were obtained for colistin in concentrations from 0.3 to 8.0 μg/mL, with good fit (r2 = 0.9993). The lower limit of quantitation was 0.3 μg/mL. The intra- and inter-day precision of the assay was 0.5–5% and 3.5–9.4%, respectively. The accuracy ranged from 98% to 100%. This validated method was successfully applied to the analysis of serum-receiving CMS in critically ill patients. This method will be useful to assist colistin dosage adjustment based on individual blood colistin levels for optimization of colistin therapy.

Determination of glyphosate and aminomethylphosphonic acid residues in Finnish soils by ultra‐high performance liquid chromatography–tandem mass spectrometry

Glyphosate [N-(phosphonomethyl) glycine] (GLY) adsorbs strongly in Finnish soils. A new method for GLY and its main degradation product, aminomethylphosphonic acid (AMPA) residues in clay soils (Protovertic Luvisol) was developed and validated. A new method was necessary because the previous one required laborious cleaning pre-treatments, and its recovery was quite poor (<40%–70%). In the new method, the earlier method's extraction solvent, 0.1 M potassium hydroxide (KOH), was replaced by more effective 0.6 M KOH. The old post-column high-performance liquid chromatography and fluorescence (HPLC-FLD) method was replaced by the ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) method. Compounds were identified as their fluorenyl methyl chloroformate (FMOC) derivatives by a multiple reaction monitoring (MRM) technique and quantified by an internal standard method utilising multipoint matrix-matched calibration. Glufosinate-ammonium (GLUF) was used to monitor the effectiveness of extraction with good recovery (80–119%). All calibration curves were found to be linear (R2 ≥ 0.98) in the studied calibration range (0.01–3.31 mg kg−1 in fresh soil). The repeatability and reproducibility were 25% and 28% for GLY, and 20% and 24% for AMPA in real research soil samples. The method was effective throughout the calibration range in all the studied Finnish agricultural soils.•An improved method was created to analyse glyphosate (GLY) and AMPA in Finnish clay soil.•The challenge caused by strong GLY adsorption on soil was solved by using multipoint matrix-matched calibration curve samples which were prepared identically with the research samples.•The method performed well in all tested clay, loam and sandy loam soils.

Removable Dyes—The Missing Link for In-Depth N-Glycan Analysis via Multi-Method Approaches

As the roles of glycans in health and disease continue to be unraveled, it is becoming apparent that glycans’ immense complexity cannot be ignored. To fully delineate glycan structures, we developed an integrative approach combining a set of cost-effective, widespread, and easy-to-handle analytical methods. The key feature of our workflow is the exploitation of a removable fluorescent label—exemplified by 9-fluorenylmethyl chloroformate (Fmoc)—to bridge the gap between diverse glycoanalytical methods, especially multiplexed capillary gel electrophoresis with laser-induced fluorescence detection (xCGE-LIF) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Through the detailed structural analysis of selected, dauntingly complex N-glycans from chicken ovalbumin, horse serum, and bovine transferrin, we illustrate the capabilities of the presented strategy. Moreover, this approach “visualizes” N-glycans that have been difficult to identify thus far—such as the sulfated glycans on human immunoglobulin A—including minute changes in glycan structures, potentially providing useful new targets for biomarker discovery.

Validation and Application of UPLC-MS/MS Method to Analysis of Glyphosate and its Metabolites in Water.

A method was developed to determine glyphosate and their metabolites in water. The widespread use of this herbicide in agricultural activities worldwide, despite the reported adverse effects on both the environment and health, is a cause for concern and makes it necessary to monitor its presence through a method that guarantees the determination at trace levels. A direct extraction of the analytes with phosphate buffer was performed with subsequent derivatization with 9-fluorenylmethyl chloroformate. The quantification was determined by Ultra Performance Liquid Chromatography-tandem mass spectrometer. The method was validated through the following parameters: selectivity, detection and quantification limits, linearity, accuracy, precision and uncertainty. The average recoveries ranged between 94.08 and 103.31%. Additionally, detection limits from 0.396 to 0.433μg/L, and the quantification limit was 5.0μg/L for all the analytes evaluated. In terms of linearity and precision, the results obtained were in the ranges considered adequate (R2 ≥ 0.99 and CV ≤ 20%), the estimated expanded uncertainty was 12.95, 11.15 and 13.83% for glyphosate, aminomethylphosphonic acid and glufosinate, respectively. This method was successfully applied for the determination of the target analytes in irrigation water samples, detecting concentrations of aminomethylphosphonic acid over limit detection for some sampling sites.

Investigation of the performance of activated carbon cloth to remove glyphosate, glufosinate, aminomethylphosphonic acid and bialaphos from aqueous solutions by adsorption/electrosorption.

The present study investigates the removal of glyphosate, glufosinate, aminomethylphosphonic acid and bialaphos herbicides from their 5 × 10-5M aqueous solutions onto activated carbon cloth by adsorption and electrosorption. Analysis of these highly polar herbicides was achieved by UV-visible absorbance measurements, after derivatization with 9-fluorenylmethyloxycarbonyl chloride. The limit of quantification values of glyphosate, glufosinate, aminomethylphosphonic acid and bialaphos were 1.06 × 10-6mol L-1, 1.38 × 10-6mol L-1, 1.32 × 10-6mol L-1 and 1.08 × 10-6mol L-1, respectively. Glyphosate, glufosinate, aminomethylphosphonic acid and bialaphos were removed from their aqueous solutions with higher efficiencies by means of electrosorption (78.2%, 94.9%, 82.3% and 97%, respectively) than of open-circuit adsorption (42.5%, 22%, 6.9% and 81.8%, respectively). Experimental kinetic data were fitted to pseudo-first order and pseudo-second order kinetic models. It was determined that pseudo-second order kinetic model represents experimental data better with satisfactory coefficient of determination, r2 (> 0.985) and normalized percent deviation, P (< 5.15) values. Adsorption isotherm data were treated according to Freundlich and Langmuir isotherm models. Based on the r2 (> 0.98) and P (< 5.9) values, it was found that experimental data well fitted to Freundlich isotherm model. Adsorption capacities of activated carbon cloth for glyphosate, glufosinate, aminomethylphosphonic acid and bialaphos, expressed in terms of Freundlich constant, were calculated as 20.31, 118.73, 239.33 and 30.68mmolg-1, respectively. The results show that the studied ACC can be used in home/business water treatment systems as an adsorbent due to its high adsorption capacity.

Determination of γ-aminobutyric acid in fermented soybean products by HPLC coupled with pre-column derivatization

This study reports a pre-column derivatization HPLC stragety to determine the content of GABA in fermented soybean products. 9-Fluorenylmethyloxycarbonyl chloride (Fmoc-Cl) is employed as the derivative reagent with chromophore linked to GABA, which lacks of chromophore for UV/DAD analysis in its pristine structure. After the detection wavelength was confirmed, the derivatization and analysis conditions were also optimized, including pH and amount of boric acid buffer, amount of derivative reagent, the mobile phases and their gradient. Methodological investigation showed that the derivatized GABA (Fmoc-GABA) has good linearity in the range of 1–100 µg/mL, low limit of quantification (LOQ, at 1 µg/mL) and limit of detection (LOD, at 0.3 µg/mL), and good recoveries of three spiked levels (between 83.8% and 100.8% with RSDs less than 5%). Furthermore, thirty-two commercial fermented soy products in four matrices (soy sauce, soy paste, bean curd, lobster sauce) were analyzed by the proposed method for validation. As a consequence, the concentrations of GABA in these soy products were in the range of 48.4–381 mg/kg.

Determination of amino acids in black garlic using high-performance liquid chromatography after derivatization with 9-fluorenylmethyloxycarbonyl chloride

Purpose: To quantify the amino acids obtained from black garlic (Allium sativum L.) using high-performance liquid chromatography after derivatization with 9-fluorenylmethyloxycarbonyl chloride (FMOC-Cl).&#x0D; Methods: Homogenized black garlic samples were derivatized with 9-fluorenylmethyloxycarbonyl chloride (FMOC-Cl) reagent in borate buffer at 30 °C for 60 min. The black garlic samples were hydrolyzed with 5.0 mL HCl (6 N) and neutralized to pH 5 – 6 by adding 30 mL NaOH (1 N). Amino acids were measured using high-performance liquid chromatography coupled with a fluorescence detector after hydrolysis and derivatization with FMOC-Cl reagent. Gradient elution mixture consisted of acetonitrile, methanol, and water at a flow rate of 1.0 mL/min, 0.2 % ammonium acetate, and 0.1 % formic acid.&#x0D; Results: The ranges of the limits of detection and quantification were 30 and 100 g/mL, respectively. The overall intra- and inter-day variations (%RSD) for the precision findings were smaller than 3.43 and 4.63 %, respectively. The accuracy of the developed method was good, ranging from 90.9 to 106.8 %. The amino acids of six black garlic products from the market were determined using the suggested approach. The findings indicated that glutamic acid, the main amino acid, is present in the maximum concentration (564.64 – 1333.9 mg/100 g) in black garlic.&#x0D; Conclusion: The validated method is for the determination of the amino acids in black garlic and would be a helpful tool for the quality control of products containing black garlic.

Analysis of Methamphetamine in Urine by HPLC with Solid-Phase Dispersive Extraction and Solid-Phase Fluorescence Derivatization.

We have developed a fluorescence detection-liquid chromatography (HPLC-FL) method that involves sample pretreatment by solid-phase dispersive extraction (SPDE) and solid-phase fluorescence derivatization for the simple and rapid analysis of methamphetamine (MA) in urine. This method uses a reversed-phase polymeric solid-phase gel to clean up analytes in SPDE, followed by fluorescence derivatization with 9-fluorenylmethyl chloroformate (FMOC) in the solid-phase. The optimal conditions for SPDE and solid-phase fluorescence derivatization were obtained when J-SPEC PEP was used as the solid-phase gel and 0.5 mmol/L FMOC in 50 mmol/L borate buffer solution (pH 10) was used as the fluorescence derivatization reagent. The recovery experiment of MA in urine yielded a clean chromatogram with no interfering peaks, demonstrating the validity of our method; the recoveries were 83.6% when spiked at a low concentration level (100 ng/mL) and 80.7% when spiked at a high concentration level (1000 ng/mL). Compared with the conventional liquid-phase method, the reaction product (FMOC-MA) of solid-phase fluorescence derivatization had higher stability. Reaction rate constants were calculated by changing the temperature conditions, and physicochemical parameters, including activation energy and activation entropy involved in the degradation reaction, were obtained from the Arrhenius plot and analyzed thermodynamically. Taken together, our results suggest that the HPLC-FL method with SPDE and solid-phase fluorescence derivatization for sample pretreatment provides a simple and rapid means of analyzing MA in urine samples.

Methodical approaches to scale-up measurement of glyphosate concentration using HPLC-FLD

Introduction. Traditionally, the hygienic assessment of working conditions when using glyphosate preparations, an extremely popular herbicide in the world, is based on the use of thin layer chromatography. In this paper, alternative approaches are considered. On the basis of experimental data, the conditions for the analysis of glyphosate content in air by high performance liquid chromatography were optimized. Material and methods. All work with glyphosate solutions was carried out in polypropylene vessels. Sampling from the air medium was carried out on a medium filtration filter (“blue ribbon”). Subsequent extraction was carried out with water. The measurements were performed by high performance liquid chromatography with a fluorimetric detector at an excitation wavelength of 270 nm and an emission wavelength of 313 nm. To transfer glyphosate to a molecule with fluorimetric properties after derivatization of glyphosate in an alkaline medium using 9-fluorenylmethyl chloroformate by heating and washing off the excess of the reagent with toluene. A C18 reverse phase column was used; ammonium acetate with the addition of acetic acid and acetonitrile were used as eluents. Results. Approbation of the developed technique was carried out on real samples taken during ground treatment of fallow fields and lands of industrial territories with glyphosate preparations. The detected levels of glyphosate did not exceed the lower limit of quantitative determination: 0.5 mg/m3 in the air of the working area and 0.025 mg/m3 in the atmospheric air (with maximum allowable concentrations of 1.0 and 0.1 mg/m3, respectively). Limitations. The study considers a limited number of chromatographic columns. The study is performed on 25 model samples of the air of the working area and atmospheric air. Conclusion. Based on the results of the work performed, methodological instructions “Measurement of glyphosate concentrations in the air by high-performance liquid chromatography” were formed and sent for metrological certification in the approved manner.

Stereoselective separation of 4-hydroxyproline by electrokinetic chromatography

A chiral methodology was developed in this work enabling for the first time the separation of the four stereoisomers of the amino acid 4-hydroxyproline (4-Hyp) in the format of capillary electrophoresis (CE). After a screening of different neutral cyclodextrins (CDs) in the electrokinetic chromatography (EKC) mode, methyl-γ-CD was selected as chiral selector to stereoselectively separate 4-Hyp (previously derivatized with 9-fluorenylmethyloxycarbonyl chloride (FMOC-Cl)) in a 75 mM phosphate buffer at pH 7.0. The effect of the concentration of the CD, the separation voltage, and the temperature on the chiral separation was investigated. A concentration of 10 mM for methyl-γ-CD, a voltage of 30 kV, and a temperature of 15 °C allowed the separation of the four stereoisomers of 4-Hyp in less than 21 min with resolutions between consecutive peaks of 1.5, 2.7, and 3.6. The injection of individual standard solutions of each stereoisomer enabled peak identification and the methodology was able to detect up to 0.1 % (1.3 × 10-11 mmol) of each stereoisomer. Analytical characteristics of the developed methodology were adequate to be applied to the analysis of nutricosmetic supplements. A good agreement was observed between the content determined for trans-4-l-Hyp and that indicated in the label for the product. No enantiomeric impurities were detected what shows the great potential of this method in the quality control of these products.

Development and validation of a novel Spectrofluorimetric method of oral anticoagulant Edoxaban via derivatization with 9-fluorenyl methyl chloroformate: green assessment of the method by Eco-Scale and ComplexGAPI

A precise, sensitive eco-friendly, simple, rapid, and derivative spectrofluorimetric method was developed to quantify edoxaban tosylate monohydrate in pure form and pharmaceutical dosage form. Sudden death due to pulmonary embolism as a consequence of coronavirus infection (covid-19) is an emerging problem. As a result, the world health organization introduced new guidelines to treat patients with COVID-19 with oral anticoagulants. Edoxaban tosylate monohydrate is an oral anticoagulant that doesn’t require hospitalization after dose adjustment. This spectrofluorimetric method relies on the derivatization by 9-fluorenyl methyl chloroformate at room temperature in borate buffer pH 9.0. After excitation at 265 nm, the product is highly fluorescent at 309 nm. Many experimental factors influencing the reaction's stability and development were thoroughly investigated and optimized. The method validation was evaluated by using ICH guidelines and showed high precision and accuracy with an average percent recovery of 101.46% ± 1.02. The linear range was 5.0–50.0 ng/mL with a correlation coefficient of 0.9999, the LOD was 1.5 ng/mL, and the LOQ was 4.5 ng/mL. The green assessment of the method was achieved utilizing the eco-scale and the Green Analytical Procedure Index. There was no significant difference between the results of the suggested method and those of the reported method according to Statistical analysis.

Method of Glyphosate, AMPA, and Glufosinate Ammonium Determination in Beebread by Liquid Chromatography-Tandem Mass Spectrometry after Molecularly Imprinted Solid-Phase Extraction.

The aim of this study was to develop a method for the determination of glyphosate, its metabolite aminomethylphosphonic acid (AMPA), and glufosinate ammonium residues in beebread samples, which could then be used to assess bees’ exposure to their residues. The complexity of beebread’s matrix, combined with the specific properties of glyphosate itself, required careful selection and optimization of each analysis step. The use of molecularly imprinted solid-phase extraction (MIP-SPE) by AFFINIMIP glyphosate as an initial clean-up step significantly eliminated matrix components and ensured an efficient derivatization step. Colorless beebread extracts were derivatized by the addition of 9-fluorenylmethyl chloroformate (FMOC-Cl). After derivatization, in order to remove FMOC-OH and residual borate buffer, a solid-phase extraction (SPE) clean-up step using Oasis HLB was carried out. Instrumental analysis was performed by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). The method was validated according to the SANTE/11312/2021 guideline at concentrations of 5, 10, and 100 µg/kg, and satisfactory recovery (trueness) values (76–111%) and precision (RSDr) ≤ 18% were obtained. The limit of quantification (LOQ) was 5 µg/kg for AMPA and glufosinate ammonium and 10 µg/kg for glyphosate. The method was positively verified by the international proficiency test. Analysis of beebread samples showed the method’s usefulness in practice. The developed method could be a reliable tool for the assessment of beebread’s contamination with residues of glyphosate, its metabolite AMPA, and glufosinate ammonium.

Analytical method for simultaneous estimation of voglibose and mitiglinide calcium hydrate by validated RP-HPLC method

A combination of Voglibose and Mitiglinide calcium hydrate is used in Type- 2 diabetes. The method was based on pre-column derivatization of Voglibose with 9-Fluorenylmethyloxycarbonyl chloride due to lack of chromophoric group it cannot directly estimated by UV detector in Reverse Phase High Performance Chromatography (RP-HPLC) method. The chromatographic separation was achieved using phosphate buffer (pH 4.0) and methanol (30:70) as mobile phase. Detection was carried out at 233 nm using UV detector. The retention time of Voglibose and Mitiglinide calcium hydrate were found to be 5.233 min and 3.580 min respectively. The RP-HPLC method for Voglibose and Mitiglinide calcium hydrate was found to be linear over the range of 0.5-1.50 µg/mL and 25-75 µg/mL respectively. Correlation coefficient for Voglibose and Mitiglinide calcium hydrate was found to be 0.998 and 0.999 respectively. Accuracy for Voglibose and Mitiglinide calcium hydrate was 99.79-100.94 % and 100.69-100.88 % respectively. A simple, specific, accurate and precise RP-HPLC method based on pre-column derivatization has been developed. There is a significant advantage in using 9- Fluorenylmethyloxycarbonyl chloride reagent, as the whole derivatization procedure is completed in a few minutes and easy to handle. All validation parameters lie within its acceptance criteria as per International Council for Harmonisation (ICH) guideline. The proposed RP-HPLC method can be highly suitable for analysis of Voglibose and Mitiglinide calcium hydrate without interference.

Development and Validation of a Simple, Selective, and Accurate Reversed-Phase Liquid Chromatographic Method with Diode Array Detection (RP-HPLC/DAD) for the Simultaneous Analysis of 18 Free Amino Acids in Topical Formulations

Even though there are reported methods for the quantification of free amino acids (FAAs) in biological products, no work has been done on the analysis of these substances in formulations. Moreover, further research is required as the reported methods do not fulfill analytical method requirements. The objective of this study was, therefore, to develop and validate a rapid, reliable, and appropriate RP-HPLC/DAD method for the simultaneous determination of 18 FAAs (l-Ala, l-Arg, l-Asn, l-Asp, l-Gln, l-Glu, l-Gly, l-His, l-Ile, l-Lue, l-Lys, l-Met, l-Orn, l-Phe, l-Pro, l-Ser, l-Thr, and l-Val) in topical formulations. After appropriate method development, the technique was validated for selectivity, linearity and range, limit of detection, limit of quantification, precision, and accuracy. The samples were derivatized with 9-fluorenylmethyl chloroformate (Fmoc-Cl). Chromatographic separation was performed on InfinityLab Poroshell 120 E.C 18 (3 × 50) mm, 2.7 μm column at 25 °C. The mobile phase consisting of water and acetonitrile adjusted to appropriate pH was pumped in gradient mode at a flow rate of 0.7 mL/min. Ten microliters were injected and analyte detection was conducted using a DAD. The results indicate that the method was selective for these FAAs. It was linear over the concentration range of 5–80 µM with a correlation coefficient greater than 0.995. Moreover, it was sensitive, precise, accurate, and robust. All the reported drawbacks of RP-HPLC-based analysis of FAAs were resolved, and hence, this new method can be considered appropriate for the analysis of these FAAs in topical formulations.

Determination of the Glyphosate Content in Liquid and Dry Formulations by HPLC-UV: Pre-column Derivation with 9-Fluorenylmethyl Chloroformate (FMOC)

Determination of the Glyphosate Content in Liquid and Dry Formulations by HPLC-UV: Pre-column Derivation with 9-Fluorenylmethyl Chloroformate (FMOC)

Determination of Glufosinate-P-Ammonium in Soil Using Precolumn Derivation and Reversed-Phase High-Performance Liquid Chromatography

This study developed an analytical method to quantify glufosinate-P-ammonium (GLUF-P) in farmland soil using a reversed-phase high-performance liquid chromatography (HPLC) system with a fluorescence detector after derivatization. GLUF-P in farmland soil was extracted with a mixed alkaline solution and was further derivatized with 9-fluorenyl methyl chloroformate (FMOC) at 25 °C for 1 h. The derivatives were separated with an ACE-C18 column, gradient eluted with a mobile phase A of acetonitrile and a mobile phase B of 0.2% phosphoric acid solution, and finally determined by high-performance liquid chromatography (HPLC) with fluorescence detection at an excitation wavelength of 254 nm and an emission wavelength of 279.8 nm. The limits of detection (LODs) in the four types of soil ranged from 0.004 to 0.015 mg/kg, and the limits of quantification (LOQs) ranged from 0.0125 to 0.05 mg/kg. The mean recoveries of GLUF-P ranged from 94% to 119.8%, and the relative standard deviations (RSDs) varied between 2.8% and 9.0% when the spiked concentrations of GLUF-P were 0.1 mg/kg and 1.0 mg/kg, respectively. The coefficients of regression for the linearity equation were more than 0.99. The proposed method had high sensitivity and could be used for the determination of GLUF-P residues in farmland soil.

Effects of salt matrices on the determination of glyphosate, glufosinate, aminomethylphosphonic acid and 2-aminoethylphosphonic acid using reversed-phase liquid chromatography after fluorescence derivatization

This study evaluated the effects of salt matrices on the determination of glyphosate (GLY), glufosinate (GLU), aminomethylphosphonic acid (AMPA) and 2-aminoethylphosphonic acid (2-AEP) in seawater samples, using reversed-phase liquid chromatography–fluorescence detection (LC-FLD) following fluorescence derivatization with 9-fluorenylmethyl chloroformate. Results showed that salinity can clearly improve the derivatization efficiency of GLY and extend the retention time of GLY derivative, while GLU, AMPA and 2-AEP were almost unaffected. In addition, the co-occurrence of metal ions (such as Fe3+, Cu2+, Zn2+, Mn2+, Co2+ and Mo6+) in seawater media did not induce any significant effect on the analysis of GLY, GLU, AMPA or 2-AEP. Calibration curves prepared at the same salinity level as test samples can accurately and effectively correct for any observed salt-matrix effects. The optimized method for the determination of GLY, GLU, AMPA and 2-AEP exhibited wide linear response ranges, with good linearity (R2 > 0.99), high repeatability (relative standard deviation ≤ 4.2%), low limits of detection (0.075–0.75 μg/L) and limits of quantification (0.25–2.5 μg/L) in seawater matrix. The reliability of this method was validated by successfully analyzing seawater medium samples, achieving a satisfactory range of recovery (75–125%).

Determination of Free Amino Acids in Bee Pollen by Liquid Chromatography with Fluorescence Detection

Bee pollen is one of the hive products that is of most interest today due to its multiple beneficial health properties, making it an increasingly popular food supplement. Bee pollen contains many bioactive compounds, such as fatty acids, vitamins, minerals, proteins, and amino acids, among others. In the present study, the free amino acid content was determined in bee pollen by using liquid chromatography coupled to a fluorescence detector. Sample treatment consisted of a solvent extraction of the free amino acids with ultrapure water and a further centrifugation of the extract, which was repeated twice. After that, it was necessary to perform a pre-column derivatization of the amino acids using a combination of two reagents (o-phthalaldehyde and 9-fluorenylmethyl chloroformate) prior to their separation in a Gemini® C18 reverse phase column in gradient elution mode. The analytical performance was evaluated, and several commercial bee pollen samples were analyzed. Significant differences in the free amino acid profile and concentration, which ranged between 19 and 192 mg/g, were observed depending on the botanical origin of the samples.