Selective Analysis of Secondary Amino Acids in Gelatin Using Pulsed Electrochemical Detection

Abstract

A method was developed for selective analysis of the secondary amino acids proline and 4-hydroxyproline from gelatin hydrolysates using anion-exchange high-performance liquid chromatography followed by integrated pulsed amperometric detection (HPLC-IPAD). An extraction scheme was implemented prior to HPLC-IPAD analysis to isolate the secondary amino acids by the removal of primary amino acids through derivatization with o-phthalaldehyde followed by solid-phase extraction with C18 packed columns. The use of the IPAD technique eliminated the need for a second derivatization step to detect secondary amino acids. The removal of interfering primary amino acids prior to chromatographic analysis allowed the use of isocratic mobile-phase conditions to achieve effective and efficient separation of the amino acids. This led to a more precise and accurate quantitation of their content in gelatin hydrolysates. Detection limits approach 10 parts per billion ( approximately 2 pmol/injection) with a chromatographic analysis time under 8 min. The ratios of secondary amino acids, in addition to their abundances, were used to distinguish gelatin manufactured from bovine, porcine, and fish raw material sources.

Use of genetic algorithms with multivariate regression for determination of gelatine in historic papers based on FT-IR and NIR spectral data

“…The detection and quantification of amino acids is routinely performed using high-performance liquid chromatography (HPLC) in combination with a variety of reagents and pre-or post-column derivatisation techniques [14][15][16]. The derivatising compound 9-fluorenylmethyl chloroformate (FMOC) rapidly reacts with primary and secondary amino acids and forms stable derivatives [17].…”

Rapid determination of β2-agonists in urine samples by microchip micellar electrokinetic chromatography with pulsed electrochemical detection

“…The advantages of the use of PED are signal stability, sensitivity comparable to constant potential amperometry and the possibility of performing direct detection of analytes that are dependent on electrocatalysis of metal oxides, such as carbohydrates and amino acids. 39,40 The PED waveform can be optimized so that maximum signal magnitude and stability are achieved. This can be accomplished by generating hydrodynamic voltammograms (multiple injections of the same sample while varying the studied potential and duration time) as a function of each of the potential steps in the waveform.…”

“…As stated previously, the electrochemical oxidation of phenolic compounds can induce the inactivation of electrodes via deposition of electropolymerized lms that are formed when phenoxy radicals attack the electrode surface. 30,[39][40][41][42] This accumulation of reaction products, which leads to the loss of electrode activity, is commonly referred to as poisoning or fouling. In this study, not only was the ability of PED to avoid fouling demonstrated, but also SDS micelle might be involved in the preservation of the signal magnitude.…”

“…It has proven to be effective in overcoming such problems associated with electrode fouling for a large number of analytes, including carbohydrates, amino acids, sulfur-containing compounds, alcohols and phenols. 30,[39][40][41][42] In this study, we describe the application of microchip micellar electrokinetic chromatography (mMEKC) with PED to the analysis of four b 2 -agonists: salbutamol (SAL), terbutaline (TER), cimaterol (CIM) and ractopamine (RAC). These drugs have been frequently chosen as alternatives to clenbuterol, which is strictly banned in any activity of animal feeding.…”

Dual Electrode Detection in LC‐EC Analysis of Sanger‐tagged Amino Acids: Electrochemical Reduction of Aromatic Nitro Groups

“…Selective analysis was achieved by masking primary amines in mixtures of primary and secondary amines with excess amount of OPA followed by secondary amine derivatization with ferrocenecarboxylic acid chloride (FAC) and analysis by liquid chromatography and electrochemical detection (LC-EC) with in-series dual electrodes [14]. A method for selective analysis of amino acids proline and 4-hydroxyproline present in gelatin utilized anion-exchange liquid chromatography and integrated pulsed amperomatric detection (LC-IPAD) [15]. Proline and 4-hydroxyproline were isolated from primary amino acids from gelatin hydrolysate prior to chromatography analysis by extracting OPA derivatized primary amino acids through solidphase extraction with a C18 Sep-Pak ® .…”