Validation and optimisation of reduced glutathione quantification in erythrocytes by means of a coulometric high‐performance liquid chromatography analytical method
Fuente:
Wiley Open Access
Saved in:
| Main Authors: | , , , , , , |
|---|---|
| Format: | Artículo Open Access |
| Published: |
Wiley
2024
|
| Subjects: | |
| Online Access: | |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1867010859883036672 |
|---|---|
| author | Stef Lauwers Maxim Van Herreweghe Kenn Foubert Mart Theunis Annelies Breynaert Emmy Tuenter Nina Hermans |
| author_facet | Stef Lauwers Maxim Van Herreweghe Kenn Foubert Mart Theunis Annelies Breynaert Emmy Tuenter Nina Hermans Stef Lauwers Maxim Van Herreweghe Kenn Foubert Mart Theunis Annelies Breynaert Emmy Tuenter Nina Hermans |
| collection | Wiley Open Access |
| contents | Validation and optimisation of reduced glutathione quantification in erythrocytes by means of a coulometric high‐performance liquid chromatography analytical method Stef Lauwers Maxim Van Herreweghe Kenn Foubert Mart Theunis Annelies Breynaert Emmy Tuenter Nina Hermans Biomedical Chromatography AbstractGlutathione (GSH), a tripeptide that consists of cysteine, glutamate and glycine, is present in all mammalian tissues in the millimolar range. Besides having numerous cellular functions, GSH is an important antioxidant and is considered a valuable biomarker in evaluating oxidative stress. This paper provides a sensitive analytical method using HPLC‐ECD to quantify GSH in erythrocytes, validated using the ICH guidelines for Bioanalytical Method Validation. The sample preparation was optimised using centrifugal filtration and a hypotonic phosphate buffer for extracting GSH from erythrocytes. HPLC‐ECD parameters were adjusted to allow a fast, reversed phase, isocratic separation in 10 min. The detector response was linear between 0.3 and 9.5 μg/mL with a satisfactory regression coefficient and a LOQ of 0.11 μg/mL. Intra‐ and inter‐day repeatability ranged between 1.10% and 8.57% with recoveries ranging from 94.3% to 106.0%. Dilution integrity, benchtop, freeze–thaw and long‐term stability were investigated. Samples were stable for up to 6 months at −80°C. This method has a good linear response and is repeatable, precise and accurate. It minimises GSH auto‐oxidation using a centrifugal filter during sample preparation, instead of acidification. Therefore, this analytical method is suitable for quantifying GSH in erythrocytes as a marker of oxidative stress. 10.1002/bmc.6021 http://creativecommons.org/licenses/by-nc-nd/4.0/ |
| doi_str_mv | 10.1002/bmc.6021 |
| format | Artículo Open Access |
| id | wiley_oa_10_1002_bmc_6021 |
| institution | Wiley Open Access |
| license_str_mv | http://creativecommons.org/licenses/by-nc-nd/4.0/ |
| publishDate | 2024 |
| publisher | Wiley |
| record_format | wiley_oa |
| spellingShingle | Validation and optimisation of reduced glutathione quantification in erythrocytes by means of a coulometric high‐performance liquid chromatography analytical method Stef Lauwers Maxim Van Herreweghe Kenn Foubert Mart Theunis Annelies Breynaert Emmy Tuenter Nina Hermans Biomedical Chromatography Validation and optimisation of reduced glutathione quantification in erythrocytes by means of a coulometric high‐performance liquid chromatography analytical method Stef Lauwers Maxim Van Herreweghe Kenn Foubert Mart Theunis Annelies Breynaert Emmy Tuenter Nina Hermans Biomedical Chromatography AbstractGlutathione (GSH), a tripeptide that consists of cysteine, glutamate and glycine, is present in all mammalian tissues in the millimolar range. Besides having numerous cellular functions, GSH is an important antioxidant and is considered a valuable biomarker in evaluating oxidative stress. This paper provides a sensitive analytical method using HPLC‐ECD to quantify GSH in erythrocytes, validated using the ICH guidelines for Bioanalytical Method Validation. The sample preparation was optimised using centrifugal filtration and a hypotonic phosphate buffer for extracting GSH from erythrocytes. HPLC‐ECD parameters were adjusted to allow a fast, reversed phase, isocratic separation in 10 min. The detector response was linear between 0.3 and 9.5 μg/mL with a satisfactory regression coefficient and a LOQ of 0.11 μg/mL. Intra‐ and inter‐day repeatability ranged between 1.10% and 8.57% with recoveries ranging from 94.3% to 106.0%. Dilution integrity, benchtop, freeze–thaw and long‐term stability were investigated. Samples were stable for up to 6 months at −80°C. This method has a good linear response and is repeatable, precise and accurate. It minimises GSH auto‐oxidation using a centrifugal filter during sample preparation, instead of acidification. Therefore, this analytical method is suitable for quantifying GSH in erythrocytes as a marker of oxidative stress. 10.1002/bmc.6021 http://creativecommons.org/licenses/by-nc-nd/4.0/ |
| title | Validation and optimisation of reduced glutathione quantification in erythrocytes by means of a coulometric high‐performance liquid chromatography analytical method |
| topic | Biomedical Chromatography |
| url | https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/10.1002/bmc.6021 |