Rapid and Simultaneous Determination of Free Aromatic Carboxylic Acids and Phenols in Commercial Juices by GC-MS after Ethyl Chloroformate Derivatization

Natural phenol and phenolic acids are widely distributed in the plant kingdom and the major dietary sources include fruits and beverages derived therefrom. Over the past decades, these compounds have been widely investigated for their beneficial effects on human health and, at the same time, several analytical methods have been developed for their determination in these matrices. In the present paper, 19 different aromatic carboxylic acids and phenols were characterized by GC-MS using ethyl chloroformate as the derivatizing agent. This procedure occurs quickly at room temperature and takes place in aqueous media simultaneously with the extraction step in the presence of ethanol using pyridine as a catalyst. The analytical method herein developed and validated presents excellent linearity in a wide concentration range (25–3000 ng/mL), low LOQ (in the range 25–100 ng/mL) and LOD (in the range 12.5–50 ng/mL), and good accuracy and precision. As a proof of concept, ethyl chloroformate derivatization was successfully applied to the analysis of a selection of commercial fruit juices (berries, grape, apple, pomegranate) particularly rich in phenolic compounds. Some of these juices are made up of a single fruit, whereas others are blends of several fruits. Our results show that among the juices analyzed, those containing cranberry have a total concentration of the free aromatic carboxylic acids and phenols tested up to 15 times higher than other juices.

Derivatizing Reagents for Detection of Organic Compounds By HPLC

Derivatization is the process of chemically modifying a compound to develope a new compound which has properties that are suitable for analysis using HPLC. Derivatization improves the detectability of a target analyte by reaction with suitable derivatizing agent. Derivatization reactions are simple chemical modification of substance that make it compatible with the selected separation method or transforms substance with a low UV- absorption into highly sensitive product. Derivatization reactions in liquid chromatography modify the solutes adding a chomophore for easy UV detection or a fluorophore for sensitive fluorescent detection. The chemical structure of the compound remains same and just modifies the specific functional group for reacting compounds to derivative of deviating chemical and physical properties in order to make them detectable. Introduction of certain elements or groups through chemical derivatization may enhance the detector’s response helpful for the elucidation of structure of analytes. In conclusion, the present review describe various derivatization reagents for pre-column and post column derivatization process in HPLC by UV-visible and fluorescence detection are summarized along with reactions and some practical aspects. The commonly used derivatizing reagents in HPLC are 1-fluoro-2, 4-dinitrobenzene, ninhydrine, 4-N-N-dimethylaminoazobenzene-4’-sulfonyl chloride, benzoyl chloride, phenyl isocyanate for UV-visible detection and o-phthalaldehyde, fluorescamine, 1-dimethylaminonapthalene-5-sulfonyl chloride (DNS-Cl), 9-fluorenylmethyl chloroformate (Fmoc-Cl), benzofurans for Fluorescence detection.

Gas-Diffusion Microextraction (GDME) Combined with Derivatization for Assessing Beer Staling Aldehydes: Validation and Application

In this work, a gas-diffusion microextraction (GDME) methodology was optimized and validated for the analysis of selected staling aldehydes (furfural (FURF), 2-methylpropanal (2-MP), 2-methylbutanal (2-MB), 3-methylbutanal (3-MB), and acetaldehyde (ACET)) during natural and forced aging of beer. The methodology was optimized considering time, temperature of extraction, and derivatizing agent. Using 4-hydrazinobenzoic acid (HBA) as a derivatizing agent, the performance of the method was evaluated by assessing several parameters such as detection limits (ranging from 1.2 to 1857.7 µg/L for 2-MB and ACET, respectively), quantification limits (ranging from 3.9 to 6192.4 µg/L for 2-MB and ACET, respectively), recoveries (higher than 96%), intraday and interday precisions (lower than 3.4 and 9.2%, respectively), and linearity (r2 ≥ 0.995). During beer aging, higher content of Strecker aldehydes and FURF were found, while no significant variations in ACET levels were observed. In general, the aldehydes content assessed for beers stored at 37 ± 1 °C for 7 and 14 days mimics that observed for beers stored at 20 ± 2 °C for 3 and 6 months, respectively. Lower temperatures of storage (4 ± 1 °C) delayed the development of staling aldehydes. Based on PCA analysis, the content of staling aldehydes and beer color were responsible for 91.39% of the variance among the analyzed samples, and it was demonstrated that these are key parameters to discriminate fresh from aged beers. The results herein presented showed that the proposed analytic methodology is a valuable strategy for the characterization and quantification of important staling aldehydes in beer with a potential application in the quality control of beer during storage.

Protein Crystallization in a Microfluidic Contactor with Nafion®117 Membranes

Protein crystallization still remains mostly an empirical science, as the production of crystals with the required quality for X-ray analysis is dependent on the intensive screening of the best protein crystallization and crystal’s derivatization conditions. Herein, this demanding step was addressed by the development of a high-throughput and low-budget microfluidic platform consisting of an ion exchange membrane (117 Nafion® membrane) sandwiched between a channel layer (stripping phase compartment) and a wells layer (feed phase compartment) forming 75 independent micro-contactors. This microfluidic device allows for a simultaneous and independent screening of multiple protein crystallization and crystal derivatization conditions, using Hen Egg White Lysozyme (HEWL) as the model protein and Hg2+ as the derivatizing agent. This microdevice offers well-regulated crystallization and subsequent crystal derivatization processes based on the controlled transport of water and ions provided by the 117 Nafion® membrane. Diffusion coefficients of water and the derivatizing agent (Hg2+) were evaluated, showing the positive influence of the protein drop volume on the number of crystals and crystal size. This microfluidic system allowed for crystals with good structural stability and high X-ray diffraction quality and, thus, it is regarded as an efficient tool that may contribute to the enhancement of the proteins’ crystals structural resolution.

Emerging Contaminants in Aqueous Matrices Determined by Gas Chromatography-Mass Spectrometry

Reports on the determination of emerging contaminants (EC) in aqueous samples have been increasingly common. Due to the low levels of concentration of the analytes as well as the complexity of this matrix, the analysis is done preferably by liquid chromatography (LC). Owing to the polar character of most of the EC determination by gas chromatography is deprecated. One way to overcome this barrier is through derivatization, which, in some cases, can be a lengthy step, presents risks to the analyst as well as to the environment due to the toxicity of the derivatizing agent, and, thus, ends up favoring the use of LC. An analytical protocol was developed in this work to increase the efficiency of derivatization in a shorter reaction time for the determination of ibuprofen, 4-octylphenol, 4-nonylphenol, triclosan, bisphenol A, diclofenac, estrone, 17-β-estradiol, estriol, coprostanol, and cholesterol. The proposal then was to carry out the silylation reaction of the analytes with the aid of a domestic microwave oven. The results indicated that the use of the device provided an increase in the efficiency of the reaction, due to the homogeneous heating of the solution. Besides, there was a significant decrease in the derivatization time of the analytes from 30 min to 5 min. Additionally, through a design of experiments (DOE), it was possible to perceive the influence of some instrumental parameters of GC-MS, such as temperature, pressure intensity, and pressure pulse time in the injector on the detectability of the investigated analytes. This study allowed a satisfactory separation of the analytes and an average increase in their areas of up to 35%. These aspects made it possible to obtain an analytical method with limits for the detection and quantification of EC between 0.03-11.00 ng mL-1 and 0.10-33.35 ng mL-1, respectively, and uncertainties below 9%. The developed method was applied in the determination of the analytes in coastal seawater and the determined concentrations varied from 0.24 ng L-1 for estriol and 43.60 ng L-1 for cholesterol. Thus, the improvement of the silylation reaction, combined with the strategy of instrumental optimization, proved to be simple, efficient, and fast, as well as being a comparable alternative to liquid chromatography.

Evaluation of Different Brands of Cefixime Available in Pakistan by Newly Developed Spectrophotometric Method

A fast, simple and valued method is developed to observe the quality and quantity of different pharmaceutical brands of cefixime. A spectrophotometric method has been developed for analysis of cefixime (CFX) by reacting with 4-dimethylamino benzaldehyde (DAB) as derivatizing agent. The molar absorptivity of CFX-DAB, newly synthesized derivative was calculated as 3.2 x 105 L.mole-1cm-1 and λ maximum was 393 nm. The calibration curve was developed in range of 5-25 µg.mL-1as this concentration followed beers law. The derivatization reaction is stable and didn’t show any difference in absorbance with radiation interaction for up to one day. The percentage recovery of CFX was checked and calculated in different pharmaceuticals was within 95 to 99.5% with RSD value calculated in between RSD 0.69-0.96% (n=3), respectively. This newly developed and validated procedure was proved to be accurate and precise for the analysis of CFX. This method was successfully applied to check amount of CFX from 7 different brands of pharmaceutical preparations commercially available in Pakistan.

Difference spectrophotometric method for the determination of Fluphenazine hydrochloride in tablets using peroxomonosulfate

The oxidative derivatization method using potassium hydrogenperoxomonosulfate for the indirect spectrophotometric determination of Fluphenazine hydrochloride is presented. Potassium hydrogenperoxomonosulfate is introduced as a derivatizing agent for Fluphenazine hydrochloride, yielding the sulfoxide. This reaction product was successfully used for the spectrophotometric determination of the Fluphenazine hydrochloride. The UV spectroscopic detection of the sulfoxide proved to be a more robust and sensitive method. The elaborated method allowed the determination of Fluphenazine hydrochloride in the concentration range of 0.2-30 µg mL-1. The molar absorptivity at 349 nm is 5.6×103 (dm3cm-1mol-1). The limit of quantification, LOQ (10S) is 0.24 µg/mL. A new spectrophotometric technique was developed and the possibility of quantitative determination of Fluphenazine hydrochloride in tablets 5.0 mg was demonstrated. The present method is precise, accurate and excipients did not interfere. RSD for Fluphenazine Hydrochloride 5.0 mg tablets was 1.37 %.

STUDIES USING HPLC-PDA IN GABAPENTIN N-DERIVATIZATION REACTIONS WITH 9-FLUORENYLMETHYL CHLOROFORMATE (FMOC-CL)

FMOC-Cl is a derivatizing agent widely used both in N-protection reactions of a-amino acids as well as in quantification and detection of amino acids and related molecules using HPLC-PDA or fluorescence analysis. In this work we studied the reaction of FMOC-Cl with gabapentin. However, it was observed that the degradation of gabapentin-N-FMOC and FMOC-Cl to FMOC-OH occur simultaneously in presence of the basic reaction medium which is specific for gabapentin derivatization. The degradation reaction is accelerated in temperatures above 45 °C and also by the time of contact of the reactant and product with basic medium. In this study, gabapentin derivatization reactions were shown to be cleaner and more efficient when an FMOC-Cl/gabapentin ratio of 2:1 was used, at 25 °C over a period of 15 minutes.