9-fluorenylmethyl Chloroformate Research Articles

Comparing the selectivity and chiral separation of d- and l- fluorenylmethyloxycarbonyl chloride protected amino acids in analytical high performance liquid chromatography and supercritical fluid chromatography; evaluating throughput, economic and environmental impact

The chiral separation of d- and l- FMOC amino acids was undertaken using the Lux Cellulose-1 polysaccharide based chiral column in HPLC (normal phase and reverse phase) and SFC conditions. This was done to compare the relative selectivity and separation between the three separation modes and to evaluate the potential benefits of SFC separations with regards to resolution, throughput, economic and environmental impact. It was established that the separation of d- and l- FMOC amino acids in SFC displayed behaviours that were similar to both normal phase and reversed phase, rather than distinctly one or the other. Additionally, although reversed phase conditions yielded significantly higher resolution values between enantiomers across the range of amino acids studied, improvements in selectivity in SFC via the introduction of higher concentrations of formic acid in the mobile phase allowed for better resolution per unit of time. Moreover since the SFC mobile phase is composed mostly of recyclable CO2, there is a reduction in organic solvent consumption, which minimises the economic and environmental costs.

Determination of glyphosate in surface water with high organic matter content

In this paper, we investigate the sample preparation and analysis process in order to achieve adequate results for surface water collected from rivers that flow through swamps and are consequently rich in organic matter. We show that matrix effects in glyphosate determination can be reduced by optimizing sample volume, liquid chromatography (LC) mobile phase buffer concentration and pH as well as gradient speed. Also, aspects of derivatization procedure (borate buffer concentration, fluorenylmethyloxycarbonyl chloride concentration) and their influence on accuracy are considered in detail. We encountered a cross-talk effect in the mass spectra, interfering with quantization during analysis, which was removed by optimizing MS parameters. As a result it was demonstrated that isotope-labelled internal standard with just one 13C atom is sufficient for the analysis.All these aspects were found to strongly impact the accuracy of the glyphosate determination but have received little or no attention in earlier works. We propose a reliable solid phase extraction and LC/ESI/MS/MS method for determination of glyphosate in organic-rich waters and demonstrate that LoD can be decreased by about two times using an ESI nebulizer with a modified design.

Mixed-Linkage Glucan Oligosaccharides Produced by Automated Glycan Assembly Serve as Tools To Determine the Substrate Specificity of Lichenase.

The mixed-linkage (1→3),(1→4)-d-glucan (MLG) specific glycosyl hydrolase lichenase is an important biochemical tool for the structural characterization of MLGs. It holds potential for application in the brewery, animal feed, and biofuel industries. Several defined MLG oligosaccharides obtained by automated glycan assembly are used to analyze the substrate specificities of Bacillus subtilis lichenase. Two glucose building blocks (BBs), equipped with a temporary fluorenylmethyloxycarbonyl chloride (Fmoc) protecting group in the C-3 or C-4 position, served to assemble different oligosaccharides by using an automated oligosaccharide synthesizer. Light-induced cleavage of the glycan products from the solid support followed by global deprotection provided seven MLG oligosaccharides of different length and connectivity. After incubation of the MLG oligosaccharides with lichenase, the digestion products were analyzed by HPLC-MS. These digestion experiments provided insights into the enzyme's active site that is in line with other recent evidence suggesting that the substrate specificity of lichenases has to be reconsidered. These results demonstrate that synthetic MLG oligosaccharides are useful tools to analyze mixed-linkage β-glucanases.

Determination of fatty acids by high-performance liquid chromatography and fluorescence detection using precolumn derivatization with 9-fluorenylmethyl chloroformate

ABSTRACTA new high-performance liquid chromatographic method is described for the determination of fatty acids in seed oils. The method was based on precolumn derivatization with 9-fluorenylmethyl chloroformate as a labeling agent and fluorescence detection. Fatty acids were extracted from the samples and subjected to derivatization with the reagent at 60°C for 10 min. The chromatographic separation of 14 fatty acids (C10–C22) was achieved on a combined loading compression octadecyl sulfate (CLC-ODS) column with a run time of 30 min. Three-step gradient elution of a mobile phase consisted of acetonitrile and water was used, and the signal was monitored at excitation and emission wavelengths of 265 and 315 nm, respectively. The method indicated favorable sensitivity and reproducibility for fatty acids’ derivatives. The detection limits, at a signal-to-noise ratio of 3, were 0.01–0.05 µg/ml and relative standard deviations (RSDs) were less than 0.27%. Excellent linear responses were observed with coefficients of 0.9995. This method was applied to quantify fatty acids in white, brown, and black sesame seeds’ oil.

Occurrence of the herbicide glyphosate and its metabolite AMPA in surface waters in Switzerland determined with on-line solid phase extraction LC-MS/MS.

Glyphosate is currently one of the most important herbicides worldwide. Its unique properties provide for a wide range of uses in agriculture but also in non-agricultural areas. At the same time, its zwitterionic nature prevents the inclusion in multi-residue analytical methods for environmental monitoring. Consequently, despite its extensive use, data on occurrence of glyphosate in the aquatic environment is still scarce. Based on existing methods, we developed a simplified procedure for the determination of glyphosate and its main metabolite aminomethylphosphonic acid (AMPA) in water samples using derivatization with fluorenylmethyl chloroformate FMOC-Cl, combined with on-line solid phase extraction and liquid chromatography-tandem mass spectrometry (LC-MS/MS) detection. This method was extensively tested on over 1000 samples of surface water, groundwater, and treated wastewater and proved to be simple, sensitive, and reliable. Limits of quantification of 0.005μg/L were routinely achieved. Glyphosate and AMPA were detected in the vast majority of stream water samples in the area of Zurich, Switzerland, with median concentrations of 0.11 and 0.20μg/L and 95th percentile concentrations of 2.1 and 2.6μg/L, respectively. Stream water data and data from treated wastewater indicated that non-agricultural uses may significantly contribute to the overall loads of glyphosate and AMPA in surface waters. In the investigated groundwater samples, selected specifically because they had shown presence of other herbicides in previous monitoring programs, glyphosate and AMPA were generally not detected, except for two monitoring sites in Karst aquifers, indicating that these compounds show much less tendency for leaching.

Analysis of Amikacin in Human Serum By UHPLC With Fluorescence Detector Using Chloro-Formate Reagent With Glycine

Background: Amikacin belongs to aminoglycosides family, commonly administered in the treatment of systemic infections due to gram negative bacteria. Its narrow therapeutic index results in adverse effects like nephrotoxicity and ototoxicity. Objective: Optimize an ultra-high performance liquid chromatography (UHPLC) based analytical method for the determination of amikacin sulfate in human serum using derivatizaon with FMOCCl and glycine. Methods: Pre-column derivatization reaction of amikacin performed using fluorescence reagent 9-fluorenylmethyl chloroformate (FMOC-Cl) at ambient temperature in the presence of borate buffer (0.2 M). Stabilizing reagent glycine (0.1 M) added into the reaction mixture solution after completion of the derivatization reaction for stabilization of fluorescent complex product. Fluorimetric detection of amikacin was performed at excitation and emission wavelength of 265 nm and 315 nm respectively, using C18 UHPLC column. The reported method was validated by performing linearity, precision, recovery and ruggedness. Results: The optimum mobile phase composition was found to be Acetonitrile:water in the ratio of 70:30 (v/v) at flow rate of 0.4 ml/min. A linear response of amikacin in serum samples ranging from 0.5-10 μg/ml was obtained, with correlation co-efficient of 1.00. The limit of detection (LOD) was found to be 50 ng/ml. Both inter- and intra-day analysis co-efficient values were found to be less than 10%. Conclusion: The developed UHPLC method will be useful for pre-clinical and pharmacokinetic study of amikacin in human serum. Key words: Amikacin Sulphate, Ultra High Performance Liquid Chromatography (UHPLC), 9-Fluorenylmethyl chloroformate (FMOC-Cl) reagent, Borate buffer, Glycine, Fluorescence detector.

New flow injection method for quality control of dietary supplements containing l-carnitine using extraction mediated by sodium taurodeoxycholate coacervate coupled to molecular fluorescence

Carnitine (CAR) is an essential component of the tissue of animals, higher plants and many microorganisms; it plays an important role during the metabolism of the human body. CAR transfers fatty acids to the place of burning-mitochondria and aids in the transformation of fats into energy and in this way supports overweight reduction and immediate physical performance, increases resistance from physical load and protects heart from overload. In this study, a new methodology for the determination of L-CAR by molecular fluorescence is described, after derivatization with a fluorescence active substance, 9-fluorenylmethyl chloroformate. An on-line coacervation process was used in order to isolate the analyte of the complex matrix. The developed methodology was successfully applied to the control of carnitine content in dietary supplements. The detection limit and quantification obtained were 2.4×10−8molL−1 and 7.30×10−8molL−1; respectively. Recoveries between 82 and 103% were obtained. At optimized working conditions, the sample throughput was 30samplesh−1 (without considering the required time for L-CAR derivatization). Thus short analysis and very low running costs compared with the LC/MS conventional methodology can be cited as the main advantages of the on-line coacervation with fluorescence detection proposed methodology.

Determination of amphetamines in hair by integrating sample disruption, clean-up and solid phase derivatization

The utility of matrix solid phase dispersion (MSPD) for the direct analysis of amphetamines in hair samples has been evaluated, using liquid chromatography (LC) with fluorescence detection and precolumn derivatization. The proposed approach is based on the employment of MSPD for matrix disruption and clean-up, followed by the derivatization of the analytes onto the dispersant-sample blend. The fluorogenic reagent 9-fluorenylmethyl chloroformate (FMOC) has been used for derivatization. Different conditions for MSPD, analyte purification and solid phase derivatization have been tested, using amphetamine (AMP), methamphetamine (MET), ephedrine (EPE) and 3,4-methylenedioxymethamphetamine (MDMA) as model compounds. The results have been compared with those achieved by using ultrasound-assisted alkaline digestion and by MSPD combined with conventional solution derivatization. On the basis of the results obtained, a methodology is proposed for the analysis of amphetamines in hair which integrates sample disruption, clean-up and derivatization using a C18 phase. Improved sensitivity is achieved with respect to that obtained by the alkaline digestion or by the MSPD followed by solution derivatization methods. The method can be used for the quantification of the tested amphetamines within the 2.0–20.0ng/mg concentration interval, with limits of detection (LODs) of 0.25–0.75ng/mg. The methodology is very simple and rapid (the preparation of the sample takes less than 15min).

Application of Carbon Nanotubes Modified Coatings for the Determination of Amphetamines by In-Tube Solid-Phase Microextraction and Capillary Liquid Chromatography

In this study, polydimethylsiloxane (PDMS)-coated capillary columns (TRB-5 and TRB-35), both unmodified and functionalized with single-wall carbon nanotubes (SWCNTs) or multiwall carbon nanotubes (MWCNTs), have been tested and compared for the extraction of amphetamine (AMP), methamphetamine (MET) and ephedrine (EPE) by in-tube solid-phase microextraction (IT-SPME). Prior to their extraction, the analytes were derivatized with the fluorogenic reagent 9-fluorenylmethyl chloroformate (FMOC). For separation and detection capillary chromatography with fluorimetric detection has been used. The presence of carbon nanotubes in the extractive coatings enhanced the extraction efficiencies and also significantly improved the chromatographic profiles, thus resulting in a reliable option for the analysis of these drugs. As an example of application, a new method is proposed for the analysis of the tested amphetamines in oral fluid using a TRB-35 capillary column functionalized with MWCNTs. The proposed conditions provided suitable selectivity and reproducibility (CV ≤ 6%, n = 3) at low µg/mL levels, and limits of detection of 0.5–0.8 µg/mL.

Development of simple and sensitive HPLC method for determination of glyphosate residues in soybean

The purpose of this study was to develop a simple and sensitive high performance liquid chromatography (HPLC) method for determination of glyphosate (GP) residues in soybean grains. From soybean matrix, glyphosate was extracted with a mixture of water and methanol (4:1, v/v) from soybean samples followed by protein precipitation with equal volume of methanol. No preconcentration and further clean up of the sample were required. Pre-column derivatization was carried out with excess amount of 9- fluorenylmethyl chloroformate (FMOC-Cl) in the presence of borate buffer. The gradient program developed in this method was successfully applied to a reverse phase HPLC system with a C18 column (ACE 5 μm 4.6 x 250 mm), and eluted with a mobile phase consisting of 50 mM phosphate buffer, pH 2.5, and acetonitrile at the flow rate of 0.8 ml/min and fluorescence detection. Parameters and conditions affecting extraction, derivatization reaction and chromatographic separation were systematically examined. Linearity of the method ranged from 0.005 - 1.0 μg/ml. The correlation coefficient (r2) of calibration curve for glyphosate in soybean sample was found to be 0.99929. The limit of detection (LOD) and limit of quantitation (LOQ) values were determined to be 0.125 mg/kg and 0.25 mg/kg, respectively. Average recovery was 95.2%. Repeatability and intermediate precision calculated on the basis of peak area were excellent and showed relative standard deviation ranged from 0.15 - 1.29% and 1.15 - 3.87%, respectively. The developed method has been successfully applied for determination of glyphosate residues in soybean grains obtained from Thailand and Nepal. Soybean samples (53) from two different lots were analyzed and glyphosate residues ranged from 0.23 mg/kg to 5.06 mg/kg. Almost 50% soybean samples contained nearly consistent residue levels in both lots but in remaining samples there was a significant variation of glyphosate levels between two lots. Relatively higher residues were detected in samples from Thailand (0.27-5.06 mg/kg) compared to Nepal (0.23-0.99 mg/kg). The results suggest that the proposed method can be used to determine glyphosate residues in foods derived from soybean and other crops such as corn, cotton, wheat, etc. where glyphosate is widely applied to these crops.

Residue determination of glufosinate in plant origin foods using modified Quick Polar Pesticides (QuPPe) method and liquid chromatography coupled with tandem mass spectrometry

A sensitive and specific method for the determination of glufosinate in plant origin foods was developed. The method involves extraction using modified QuPPe method, clean-up by multi-walled carbon nanotubes (MWCNTs), derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl) and detection with liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). The method was validated on twelve matrices spiked at 10 or 20, 100 and 500μg/kg. The recovery ranged from 80% to 108% with intra-day RSDs (n=5) of 0.6–9.8% and inter-day RSDs (n=15) of 3.0–9.4%. Good linearities (R2⩾0.9991) were obtained for all matrices. The limit of detection (LOD) and limit of quantification (LOQ) for the selected matrices ranged from 0.3 to 3.3μgkg−1 and from 1 to 10μgkg−1, respectively. The method was demonstrated to be reliable and sensitive for the routine monitoring of glufosinate in plant origin foods.

Simultaneous Detection of Azodicarbonamide and the Metabolic Product Semicarbazide in Flour by Capillary Electrophoresis

In the present work, capillary electrophoresis (CE) was used for the first time for the simultaneous analysis of azodicarbonamide (ADA) and semicarbazide (SEM), and the capillary electrophoresis separation conditions, extraction agents, and derivatization conditions were investigated. In 20 mmol L−1 sodium tetraborate, 30 mmol L−1 β-cyclodextrin (β-CD), 17 % isopropanol (v/v), and 25 mmol L−1 sodium dodecyl sulfate (SDS) running buffer, ADA and SEM previously derivatized with 9-fluorenylmethyl chloroformate (FMOC) were separated in less than 25 min with good sensitivity. The linear ranges were 8.3 × 10−4∼6.6 × 10−2 mmol L−1 and 1.9 × 10−3∼3.4 × 10−2 mmol L−1, and detection limits (S/N = 10) were 0.5 and 0.15 mg kg−1 for ADA and SEM, respectively. The proposed method was successfully applied for the simultaneous analysis of ADA and SEM in five flour samples with satisfactory recovery data from 88.0 to 93.0 % for ADA and 98.0 to 106.0 % for SEM, indicating the valuable potential application of this method for food analysis.

Reversed Phase Chromatographic Analysis of 13 Amino Acids in Honey Samples

A simple method using reversed phase high-performance liquid chromatography (RP-HPLC) was developed for the simultaneous analysis of 13 amino acids. Amino acids were pre-column derivatized with 9-fluorenylmethyl chloroformate (FMOC-Cl) before analysis by RP-HPLC. Experimental parameters affecting the derivatization and chromatographic separation were investigated. Amino acids were derivatized with FMOC-Cl under alkaline condition in 0.1 mol/L borate buffer pH 10.0 at room temperature. The FMOC-amino acid derivatives were separated on an Atlantis C18 column under the gradient elution of 0.05 % trifluoroacetic acid and acetonitrile and UV detection at 265 nm. Linear ranges were 0.2–100.0 μg/mL with the correlation coefficients greater than 0.992. Limits of detection and limits of quantitation were in the range of 0.05–2.0 and 0.2–5.0 µg/L, respectively. The intra-day precision (n = 3) of retention time was less than 1 %, while for the peak area was less than 4 %. The inter-day precision (n = 3 × 3) of retention time was less than 2 % and the peak area was less than 8 %. This method was applied in honey samples and the results showed that proline is the major amino acids in honey samples.

Gentamicin sulphate permeation through porcine intestinal epithelial cell monolayer.

Gentamicin is an aminoglycoside antibiotic widely used in combination with dimethyl sulphoxide (DMSO) in topical drug formulations. It is not known, however, whether DMSO can enhance the permeation of gentamicin through biological membranes, leading to oto- and nephrotoxic side effects. A simple and reliable high-performance liquid chromatographic (HPLC) method was applied for the quantitative determination of gentamicin collected from the apical and basolateral compartments of the porcine intestinal epithelial cell line IPEC-J2 cell monolayer using fluorometric derivatisation of the analyte with fluorenylmethyloxycarbonyl chloride (FMOC) prior to chromatographic run in the presence and absence of 1% DMSO. The lack of change in transepithelial electrical resistance (TER) demonstrated that gentamicin and 1% DMSO did not affect IPEC-J2 cell monolayer integrity via the disruption of cell membranes. Chromatographic data also ascertained that gentamicin penetration across the cell monolayer even in the presence of 1% DMSO was negligible at 6 h after the beginning of apical gentamicin administration. This study further indicates that the addition of this organic solvent does not increase the incidence of toxic effects related to gentamicin permeation.

Simultaneous analysis of N(ε) -(carboxymethyl)Lysine and N(ε) -(carboxyethyl)lysine in foods by ultra-performance liquid chromatography-mass spectrometry with derivatization by 9-fluorenylmethyl chloroformate.

Isotope dilution ultra-performance liquid chromatography-electrospray tandem mass spectrometry with derivatization by 9-fluorenylmethyl chloroformate was successfully applied to quantify N(ε) -(carboxymethyl)lysine (CML) and N(ε) -(carboxyethyl)lysine (CEL) in processed foods. We demonstrate that this analytical method is well validated for the determination of CML and CEL contents in processed foods. Relative standard deviations (RSD) indicate repeatability (RSD < 6% for CML and CEL) and reproducibility (RSD < 6% for CML and < 7% for CEL) in this method. Percent recovery is also good. We obtain recoveries of 102% to 112% for CML and 86% to 114% for CEL. CML levels detected in the samples vary from 2.29 to 480 mg/kg food, whereas CEL is detected in significantly lower concentrations ranging from 0.56 to 107 mg/kg food. These data could help consumers make better food choices by monitoring intake of advanced glycation end-products, which may pose a risk to human health.

Statistical Methods for the Investigation of Solvolysis Mechanisms Illustrated by the Chlorides of the Carbomethoxy Protecting Groups NVOC and FMOC.

The solvolysis of 4,5-dimethoxy-2-nitrobenzyl chloroformate (NVOC-Cl, 1) is followed at 25.0°C in twenty hydroxylic solvents. A comparison with previously published rates for benzyl chloroformate and p-nitrobenzyl chloroformate indicates that the inductive effect of the nitro and the two methoxy groups strongly influences the rate of reaction. For 1, the specific rates of solvolysis are correlated using an extended Grunwald-Winstein (G-W) treatment. A direct comparison with the data for phenyl chloroformate (PhOCOCl) in identical solvents strongly suggests that the addition step within an addition-elimination mechanism is rate-determining for both substrates. A reevaluation of the kinetic data for 9-fluorenylmethyl chloroformate (FMOC-Cl, 2) involves a correlation of log⁡(k/k o)2 versus log⁡(k/k o)PhOCOCl. In this plot, deviations were observed in solvents rich in a hydrogen-bonding fluoroalcohol component. Omitting the aqueous fluoroalcohol rate measurements for 2 in an analysis using the extended G-W equation suggested the occurrence of dual pathways differing in the dependences upon the ionizing power and nucleophilicity of the solvent. In addition, the fluorenyl ring is rotated out of the plane containing the ether oxygen and, as a result, PhOCOCl is found to solvolyze 20 times faster than 2 in ethanol and methanol.

Development and validation of a high-performance liquid chromatography-fluorescence detection method for the accurate quantification of colistin in human plasma.

Recently, colistin has become one of the most important drugs for treating infections caused by multidrug-resistant Gram-negative bacteria. Therapeutic drug monitoring is recommended to ensure the safety and efficacy of colistin and to improve clinical outcomes. This study developed an accurate and sensitive high-performance liquid chromatography-fluorescence detection (HPLC-FLD) method for the quantification of colistin in human plasma. The sample preparation included protein precipitation using trichloroacetic acid (TCA) and methanol, followed by in-solid phase extraction (In-SPE) derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl). A Poroshell 120 EC-C18 2.1×100mm (2.7μm) column was used in the HPLC method with a mobile phase composed of acetonitrile (ACN), tetrahydrofuran (THF), and deionized (DI) water (82%, 2%, 16% (v/v), respectively). Polymyxin B1 was used as the internal standard. The total analysis time was 22min under optimal separation conditions. The HPLC-FLD method was validated over a therapeutic range of 0.3-6.0μgmL(-1). The intra-day and inter-day precisions for colistin A and colistin B were below 9.9% and 4.5% relative standard deviations, respectively. The accuracy test results were between 100.2 and 118.4%. The extraction recoveries were between 81.6 and 94.1%. The method was linear over the test range, with a 0.9991 coefficient of determination. The limit of detection was 0.1μgmL(-1). The validated HPLC-FLD method was successfully applied to quantify the colistin concentrations in 2 patient samples for therapeutic drug monitoring.

Determination of atmospheric amines by on-fiber derivatization solid-phase microextraction with 2,3,4,5,6-pentafluorobenzyl chloroformate and 9-fluorenylmethoxycarbonyl chloride

Alkylamines play an important role in atmospheric chemistry and are of concern for human health. Determining them from the vapor phase is challenging owing to their high polarity and volatility, water solubility, low concentrations, and poor chromatographic properties. We propose on-fiber derivatization solid-phase microextraction (SPME) to increase sensitivity and selectivity for the determination of alkylamines in air samples. SPME fibers coated in head-space with 2,3,4,5,6-pentafluorobenzyl chloroformate (PFBCF, 10min) or 9-fluorenylmethoxycarbonyl (FMOC) chloride (5min) were exposed to the sample for 5–120min, after which the derivatized alkylamines were thermally desorbed in the GC injection port and analyzed by GC-MS. The specific focus of the research was dimethylamine (DMA) but, as well as secondary amines, both coating agents readily react with primary and tertiary amines and with ammonia at ambient temperatures. The fiber coating procedures, sampling times, and analytical conditions were optimized, and methods were tested with natural samples. PFBCF was more selective and almost an order of magnitude more sensitive than FMOC chloride. Both reagents are applicable, however, depending on the requirements. With scan mode and use of molecular ion for quantification, the limit of quantification for DMA was 0.17μgL−1 when derivatized with PFBCF and 3.4μgL−1 when derivatized with FMOC chloride. When selected ion monitoring was used with the most abundant ion, the limit of quantification for DMA was 2.8ngL−1. Intermediate reproducibility expressed as relative standard deviation was around 30% with PFBCF and less than 20% with FMOC chloride. Fibers coated with PFBCF could be used at least up to 24h when stored at 4°C and for 5 to 7h when stored at room temperature. After sampling/derivatization, storage time before analysis should not exceed 48h at 4°C or 24h at room temperature. At maximum, the PFBCF-coated fiber can be used in 100 coating/sampling/analysis cycles.

Determination of glyphosate and aminomethylphosphonic acid in aqueous soil matrices: A critical analysis of the 9‐fluorenylmethyl chloroformate derivatization reaction and application to adsorption studies

The assessment of the environmental fate of glyphosate and its degradation product (aminomethylphosphonic acid) is of great interest given the widespread use of the herbicide. Studies of adsorption-desorption and transport processes in soils require analytical methods with sensitivity, accuracy, and precision suitable for determining the analytes in aqueous equilibrium solutions of varied complexity. In this work, the effect of factors on the yield of the derivatization of both compounds with 9-fluorenylmethyl chloroformate for applying in aqueous solutions derived from soils was evaluated through factorial experimental designs. Interference effects coming from background electrolytes and soil matrices were established. The whole method had a linear response up to 640ng/mL (R(2) > 0.999) under optimized conditions for high-performance liquid chromatography with fluorescence detection. Limits of detection were 0.6 and 0.4ng/mL for glyphosate and aminomethylphosphonic acid, respectively. The relative standard deviation was 4.4% for glyphosate (20ng/mL) and 5.9% for aminomethylphosphonic acid (10ng/mL). Adsorption of compounds on four different soils was assessed. Isotherm data fitted well the Freundlich model (R(2) > 0.97). Kf constants varied between 93 ± 3.1 and 2045 ± 157 for glyphosate and between 99 ± 4.1 and 1517 ± 56 (μg(1-1/) (n) mL(1/) (n) () g(-1) ) for aminomethylphosphonic acid, showing the broad range of applicability of the proposed method.

Determination of 1-deoxynojirimycin in the Larvae of the Silkworm, Bombyx mori, by High-Performance Liquid Chromatography

1-Deoxynojirimycin, known to be one of the most potent α-glycosidase inhibitors, has been used as an antihyperglycemia agent. In this study, a rapid method for the determination of 1-deoxynojirimycin in the larvae of the silkworm, Bombyx mori was established, using high-performance liquid chromatography equipped with fluorescence detection after derivatization with 9-fluorenylmethyl chloroformate. The 1-deoxynojirimycin was efficiently extracted from the larvae using 0.05 mol/L hydrochloric acid, vortexing for 15 s at room temperature, followed by sonication for 30 min. The linear dynamic range extended from 0.7 to 28 µg/mL (r = 0.9999). The average recovery of the 1-deoxynojirimycin was 104% with a relative standard deviation of 3.30%. The limit of detection was 0.05 µg/mL at a signal-to-noise ratio of 3. There were high concentrations of 1-deoxynojirimycin in the larvae, reaching 0.50% of total body mass in the gluttonous stage of the third instar and significant variation in the fifth instar, suggests that the larvae may be a valuable source of 1-deoxynojirimycin for the treatment of carbohydrate-mediated disorders.