High performance liquid chromatography coupled with post - Column derivatization methods in food analysis: Chemistries and applications in the last two decades.

High performance liquid chromatography coupled with post-column derivatization is used for increasing the sensitivity and selectivity of the desirable analytes after the chromatographic separation. The transformation of the analytes can be conducted through the addition of a suitable reagent in the eluted stream or the ultraviolet irradiation of the eluted analytes, forming detectable derivatives for ultraviolet or fluorescence detectors. This review focuses on the developed methods using high performance liquid chromatography coupled with post-column derivatization for the determination of substances in food samples during the last two decades. The significance of the determination of each analyte in foods and the existing guidelines in each case are discussed. Preparation of the samples and the analytical methods are commented. For each analyte, official methods and commercially available systems and reagents are mentioned, as well.

Developments in on-line, post separation sample manipulation in the last 22 years: Pharmaceutical and biomedical applications.

On-line post separation sample manipulation is a powerful approach increasing the sensitivity and selectivity in chemical analysis. Post separation sample manipulation includes the treatment of the analytes after their separation through a suitable separation technique, mainly liquid chromatography and capillary electrophoresis. Typically, post separation approaches include either the addition of a reagent/solvent to derivatize the analyte/enhance the sensitivity, pH change, or the conversion of the analyte through a photochemical/electrochemical system (reagent-free systems). This review focuses on the developed methods using post-column manipulation of sample with pharmaceuticals and biomedical applications, covering the period from 2000 to midle-2023. Chemistries combined with fluorescence, UV-vis and mass spectrometric detection are discussed employing both liquid chromatography and electrophoretic techniques for separation. Noteworthy instrumental modifications are also discussed.

Naphthalene-2,3-dicarboxaldehyde as a pulsed-post column derivatization reagent; comparison with two alternative o-phthalaldehyde based chemistries for the determination of histamine.

In the present study, naphthalene-2,3-dicarboxaldehyde (NDA) was used in on-line post column derivatization (PCD) coupled to liquid chromatography under the new concept of Pulsed-PCD. In Pulsed-PCD, the reagents are introduced into the flowing stream of the mobile phase under precise timing overlapping the eluted analyte. The consumption of the reagents is minimized to a few microliters, resulting in a significant advantage, that is the use of expensive reagents in PCD. For this reason, NDA-CN chemistry was used for the determination of histamine in food samples, such as eggplant and spinach. Two additional methods were developed based on the reaction of histamine with o-phthalaldehyde (OPA), namely the classic OPA - nucleophilic compound reaction and the specific OPA - histamine reaction in alkaline medium. The chromatographic conditions and the Pulsed-PCD conditions were investigated, while the analytical figures of merit were satisfactory. In all three methods, a pulse of 50 µL was used (OPA/NDA + Buffer), reducing dramatically the consumption of PCD reagents.

Pulsed-post column derivatization coupled to green liquid chromatography for the determination of glutathione and cysteine based on thioacrylates formation.

In the present work, we developed a method for the determination of thiols (cysteine and glutathione) in yeast samples under the new concept of Pulsed-post column derivatization (Pulsed-PCD). For the chromatographic separation of the analytes, 100% aqueous mobile phase was used and the eluted compounds reacted on-line with the injected pulses (100 µL) of the derivatizing reagent (ethyl propiolate + Britton-Robinson buffer). Spectrophotometric detection of the derivatives was carried out at 285 nm. The Pulsed-PCD configuration, the selection of the analytical column and the pulsed-PCD reaction conditions were investigated. The method was validated for the determination of endogenous content of the analytes in dry and fresh yeasts, with LODs of 3.0 µmol L-1. The percent recovery ranged between 85.2 and 114.4% in all cases and the results were compared with a corroborative method based on classical PCD. The analytical greenness of the proposed method was evaluated using two tools; Analytical Eco-Scale and Green Analytical Procedure Index (GAPI). The greenness score of the HPLC-Pulsed-PCD method (score = 77) was compared with that of the corroborative HILIC-PCD method (score = 71) and was found to be greener in terms of the amount of chemicals used and the produced wastes.

Determination of Free Histidine in Complex Hair Care Products with Minimum Sample Preparation Using Cation-Exchange Chromatography and Post Column Derivatization.

In this communication, we describe the first analytical method for the determination of free histidine in hair care products (shampoos and conditioners). Cation-exchange chromatography combined with postcolumn derivatization and fluorimetric detection enabled the accurate (recovery: 83.5-114.8%) and precise (2.4-5.6% RSD) determination of free histidine without matrix interferences at concentration levels down to 1.5 mg kg-1. Real commercially available samples were found to contain the amino acid at levels ranging between 70 and 535 mg kg-1.

Development and evaluation of pulsed - Post column derivatization in liquid chromatography as a concept to minimize reagent consumption.

In the current article, we propose an alternative approach to reduce the consumption of the reagents in liquid chromatography coupled to on-line post column derivatization. In our proposal post column reagents do not flow continuously but they are instead introduced as well-defined pulses (at microliter levels) that are merged on-line with the eluted analytes through precise tuning (Pulsed-Post Column Derivatization, Pulsed-PCD). The profiles of the pulses in terms of time and flow rate were investigated "visually" using caffeine as model compound (at 274 nm). The robustness of the procedure was evaluated by Monte Carlo simulations and was verified taking into account the precisions of typically used propulsion systems. As a proof of concept, we selected the determination of histidine in human urine after separation by cation exchange chromatography and Pulsed-PCD derivatization with o-phthalaldehyde. The consumption of the derivatizing reagent was downscaled to the microliter level per run, while the analytical results were within the expected ranges (110 - 1520 µmol L-1) and with good agreement with the corroborative method based on classic HPLC-PCD.

Development of an equipment free paper based fluorimetric method for the selective determination of histidine in human urine samples.

A direct fluorimetric method, employing µicro-analytical paper-based devices (µ-PADs) for the selective determination of histidine (HIS) is described. The suggested method exploits the fluorescence emission of histidine after its rapid reaction with o-phthalaldehyde (OPA) at a basic medium (pH = 10) on the surface of a paper device with the application of a UV lamp at 354 nm. The devices are inexpensive and are composed of chromatographic paper and wax barriers. The analytical protocol is easily applicable with minimal technical expertise and without the need of expensive experimental apparatus. The user has to add a test sample, illuminate the device with a UV lamp, and read the fluorescence of the sensing area using a simple imaging device such as a cell-phone camera. The method is free from common interferences likely to affect the measurement of histidine and is selective among all other amino acids. This analytical procedure was optimized and validated, paying special attention to its intended application. The detection limits are as low as 1.8 µM with very satisfactory precision ranging from 6.4% (intra-day) to 8.9% (inter-day). Random urine samples from adult volunteers (n = 5) were successfully analyzed and HIS content ranged between 260 and 1114 µmol L-1 with percentage recoveries in the range of 78.2 and 124.6%.

HPLC Determination of Colistin in Human Urine Using Alkaline Mobile Phase Combined with Post-Column Derivatization: Validation Using Accuracy Profiles.

In this study, the development, validation, and application of a new liquid chromatography post-column derivatization method for the determination of Colistin in human urine samples is demonstrated. Separation of Colistin was performed using a core-shell C18 analytical column in an alkaline medium in order (i) to be compatible with the o-phthalaldehyde-based post-column derivatization reaction and (ii) to obtain better retention of the analyte. The Colistin derivative was detected spectrofluorometrically (λext/λem = 340/460 nm) after post-column derivatization with o-phthalaldehyde and N-acetyl cysteine. The post-column derivatization parameters were optimized using the Box-Behnken experimental design, and the method was validated using the total error concept. The ß-expectation tolerance intervals did not exceed the acceptance criteria of ±15%, meaning that 95% of future results would be included in the defined bias limits. The limit of detection of the method was adequate corresponding to 100 nmol·L-1. The mean analytical bias (expressed as relative error) in the spiking levels was suitable, being in the range of -2.8 to +2.5% for both compounds with the percentage relative standard deviation lower than 3.4% in all cases. The proposed analytical method was satisfactorily applied to the analysis of the drug in human urine samples.

Single-Step Hydrolysis and Derivatization of Homocysteine Thiolactone Using Zone Fluidics: Simultaneous Analysis of Mixtures with Homocysteine Following Separation by Fluorosurfactant-Modified Gold Nanoparticles.

Herein, we report a new automated flow method based on zone fluidics for the simultaneous determination of homocysteine and homocysteine thiolactone using fluorimetric detection (λext = 370 nm/λem = 480 nm). Homocysteine thiolactone is hydrolyzed on-line in alkaline medium (1 mol L−1 NaOH) to yield homocysteine, followed by reaction with o-phthalaldehyde in a single step. Derivatization is rapid without the need of elevated temperatures and stopped-flow steps, while specificity is achieved through a unique reaction mechanism in the absence of nucleophilic compounds. Mixtures of the analytes can be analyzed quantitatively after specific separation with fluorosurfactant-capped gold nanoparticles that are selectively aggregated by homocysteine, leaving the thiolactone analogue in solution. As low as 100 nmol L−1 of the analyte(s) can be quantified in aqueous solutions, while concentrations > 2 µmol L−1 can be analyzed in artificial and real urine matrix following 20-fold dilution. The percent recoveries ranged between 87 and 119%.

Development and validation of a direct HPLC method for the determination of salivary glutathione disulphide using a core shell column and post column derivatization with o-phthalaldehyde.

Glutathione disulfide (GSSG) has been monitored in human saliva samples by an optimized and validated method that is based on liquid chromatography coupled to on-line post column derivatization. The analyte was separated from the sample matrix using a 100% aqueous mobile phase through a core-shell reversed phase column. Following optimization of the reaction using Box- Behnken experimental design and validation, GSSG was quantified accurately and selectively in the range of 100-2000 nmol L-1 with a LOD of 20 nmol L-1. GSSG was quantified in 15 out of 20 human saliva samples (75%) with a mean value of 860 nmol L-1 (150-4600 nmol L-1). Blocking of reduced Glutathione with N-ethylmaleimide ensured stability of the samples for at least 72 h at all temperatures examined.

HPLC method with post-column derivatization for the analysis of endogenous histidine in human saliva validated using the total-error concept.

Histidine (His) is an essential amino acid that plays an important biological role and associated with various pathological conditions. A simple and reliable method for the determination of endogenous histidine in human saliva was optimized and validated. The analyte was separated from the saliva matrix by cation exchange chromatography and detected fluorimetrically (λex/λem = 360/440 nm) after online, specific post-column derivatization (PCD) reaction with o-phthalaldehyde. The chemical and instrumental variables of the post-column reaction were optimized using Box-Behnken experimental design to achieve maximum sensitivity. Method validation was carried out employing the total-error concept. Histidine could be analyzed reliably in the range of 0.5-5.0 µΜ, with an LOD (S/N = 3) of 50 nM. Monte Carlo simulations and capability analysis were used to investigate the ruggedness of the PCD reaction. The sampling strategy, sample preparation and stability were also investigated. Seventeen saliva samples were successfully analyzed with histidine levels being in the range of 2.7-19.5 µΜ.

Simple and Reliable Determination of the Histamine Content of Selected Greek Vegetables and Related Products in the Frame of "Low Histamine Diet".

The determination of histamine in Greek foods that should potentially be avoided during a "low histamine diet" is reported herein. Cation exchange chromatography combined to selective post column derivatization proved to be an excellent tool for this type of analysis as well, offering accurate results following minimal sample preparation. Tomato-, eggplant- and spinach-related products have been successfully analyzed and were all found to contain histamine. Higher amounts were quantified in eggplants, eggplant salads and spinach in the range of 15.4-34.2 mg kg-1 and lower in fresh tomatoes and related products (0.8-10.6 mg kg-1). The method is capable of determining as low as 0.5 mg kg-1 histamine without matrix effects, with percent recoveries ranging between 87 and 112% (tomatoes and related products), 95 and 119% (eggplants and related products) and 90 and 106% (fresh and frozen spinach).

Single run analysis of glutathione and its disulfide in food samples by liquid chromatography coupled to on-line post-column derivatization.

Glutathione and its disulfide were determined in a single run using liquid chromatography with on-line post-column derivatization and fluorimetric detection (340 nm/425 nm). The analytes were separated using a reversed-phase column capable of operating at 100% aqueous mobile phase and detected following direct on-line reaction with o-phthalaldehyde (7.5 mmol L-1) in highly basic medium (0.37 mol L-1 NaOH). The instrumental and chemical variables were carefully investigated towards high sensitivity and throughput, while special attention was paid to validating potential matrix effects. Glutathione and its disulfide could be selectively determined with respective LODs of 0.10 and 0.30 µmol L-1 in the absence of matrix effect (<6%). The endogenous content of the analytes was accurately determined in various food samples with recoveries ranging between 80 and 120% in all cases. The proposed method is reliable and promising as a generic analytical tool for the convenient estimation of the redox status of glutathione in various food matrices.

Determination of histidine in human serum and urine by cation exchange chromatography coupled to selective on-line post column derivatization.

A reliable and highly selective method for the determination of histidine in human serum and urine is described. Histidine was separated from the matrix by cation exchange chromatography and detected selectively using on-line post column derivatization and fluorimetric detection. Unique reaction of histidine with o-phthalaldehyde in the absence of nucleophilic compounds offered specific detection in the complex biological substrate. Linearity was obeyed in the range of 0.5 - 25 µmol L-1 with a limit of detection of 0.160 µmol L-1. The absence of matrix effect (<5%) enabled the processing of real samples after minimal pretreatment. Endogenous histidine has been determined in human serum in the range of 78 - 119 µmol L-1 and random human urine in the range of 266 - 2034 µmol L-1. The percent recoveries were satisfactory in all cases, ranging between 89 and 114%.

Selective post-column derivatization coupled to cation exchange chromatography for the determination of histamine and its precursor histidine in fish and Oriental sauce samples.

Histamine is a biogenic amine that is formed from histidine by action of the enzyme histidine decarboxylase and can be toxic at high intakes. Thus, the quantification of these analytes in foods constitutes a significant axis of food safety. In this study we present the development, validation and application of a new method for the determination of histamine and its precursor histidine in fish products and oriental sauces. The analytes were separated rapidly through a cation exchange column using an acidic mobile phase (7 mmol L-1 nitric acid) and reacted downstream with o-phthalaldehyde in post-column mode in the absence of nucleophilic reagents. The derivatives were detected spectrofluorimetrically at λex/λem. = 360/440 nm. Following investigation of the chromatographic and post-column conditions, the method was validated as for its intended applications. The limits of detection were 0.16 and 0.17 µmol L-1 for histidine and histamine respectively (ca. 0.1 mg kg-1) and the precision was better than 5%. Various food samples were successfully analyzed without matrix interferences following minimal pretreatment. The percent recoveries ranged between 91.3 and 117.9%.

Automated fluorimetric sensor for hydrazine determination in water samples based on the concept of zone fluidics.

In the present study, we report an automated fluorescence sensor for the determination of hydrazine in various water samples based on the concept of zone fluidics. Hydrazine and O-phthalaldehyde react through a unique mechanism in acidic medium (pH = 1.5) and without the presence of additional nucleophilic compounds. Another interesting feature of the proposed reaction is that it is not based on the Red/Ox properties of hydrazine, enhancing further the selectivity of the analytical procedure. The produced hydrazone exhibits high fluorescence at Ex = 318/Em = 376 nm. Using 120 s as a stopped flow step and temperature of 70 °C, we achieved satisfactory sensitivity for the determination of the analyte at a microgram per liter level with a limit of detection of 1.4 µg L-1 and an analysis rate of 12 h-1. The absence of matrix effect enabled the direct analysis of drinking (tap, mineral, table) and boiler feedwater samples with percent recoveries in the range of 91-111%.

Determination of glutathione and glutathione disulfide using zone fluidics and fluorimetric detection.

In the present study we report the simultaneous determination of glutathione (GSH) and glutathione disulfide (GSSG) by an automated flow method based on the concept of zone fluidics. GSH is quantified selectively in a first run by reaction with o-phthalaldehyde at a mildly basic pH = 8, without interference from GSSG. The latter was also found to react with o-phthalaldehyde but in highly basic medium (0.2 mol L-1 NaOH) and was determined after masking of GSH with N-ethyl-maleimide. Detection was carried out fluorimetrically at 340/425 nm. The flow procedure was optimized and validated, paying special attention to its selectivity. The LOD was 60 nmol L-1 for GSH and for 53 nmol L-1 for GSSG, while the within-day and day-to-day precisions were better than 1.5% and 3.7% respectively. Real yeast samples were successfully analyzed without matrix effect (-2.0 to +4.1%) and with percent recoveries being in the range of 87.0 and 103.3%.

Selective reaction of homocysteine with o-phthalaldehyde under flow conditions in highly alkaline medium: fluorimetric determination using zone fluidics.

In the present study we report the reaction between homocysteine and o-phthalaldehyde under flow conditions. Homocysteine reacts on-line with the derivatization reagent in a strong alkaline medium and in the absence of nucleophilic reagents to yield a fluorescent derivative (λex /λem = 370/480 nm). The reaction variables were investigated using the concept of zone fluidics. Selectivity factors against other compounds were calculated at 10-fold excess. The findings formed the basis of an automated proposed method that was found to be linear in the range 0.1-1.5 µmol L-1 , with a limit of detection of 20 nmol L-1 and relative standard deviation < 0.5% (within-day) and 3.2% (between-day). The method proved to be rapid, offering a practical sampling rate of 24 h-1 and accurate following application to an artificial urine matrix with minimum dilution.

Fluorimetric Method for the Determination of Histidine in Random Human Urine Based on Zone Fluidics.

In the present study, the determination of histidine (HIS) by an on-line flow method based on the concept of zone fluidics is reported. HIS reacts fast with o-phthalaldehyde at a mildly basic medium (pH 7.5) and in the absence of additional nucleophilic compounds to yield a highly fluorescent derivative (λex/λem = 360/440 nm). The flow procedure was optimized and validated, paying special attention to its selectivity and sensitivity. The LOD was 31 nmol·L-1, while the within-day and day-to-day precisions were better than 1.0% and 5.0%, respectively (n = 6). Random urine samples from adult volunteers (n = 7) were successfully analyzed without matrix effect (<1%). Endogenous HIS content ranged between 116 and 1527 µmol·L-1 with percentage recoveries in the range of 87.6%-95.4%.

Automated fluorimetric sensor for glutathione based on zone fluidics.

A zone-fluidics (ZF) based automated fluorimetric sensor for the determination of glutathione (GSH) is reported. Discrete zones of GSH and o-phthalaldehyde (OPA) mix and react on-line under mild basic pH without the need of additional nucleophillic reagents, to yield a fluorescent isoindole derivative (λex/λem = 340/425 nm). The proposed ZF sensor was optimized (pH, c(OPA), time, instrumental variables) and validated. Cysteine, glutamate, glycine and ammonium were representatively examined in terms of selectivity and were found not to react in 10-fold excess. Linearity was proved in the range of 5-100 µmol L-1 GSH, with an LOD of 1 µmol L-1 at a practical sampling rate of 20 h-1 and RSD < 0.5% (within-day) and 4.2% (day-to-day). The dosage uniformity of commercially available GSH - containing nutraceuticals was evaluated.