Simultaneous Determination of Lyso-Gb1 and Lyso-Gb3 in Plasma Using Salt-Assisted Liquid-Liquid Extraction Combined with LC-MS/MS

Q: What are Lyso-Gb1 and Lyso-Gb3 used for in this study?

They are treated as key biomarkers for Gaucher type 1 and Fabry diseases, respectively, supporting diagnosis and disease monitoring using plasma measurements.

Q: How does the method prepare samples for LC-MS/MS quantification?

It uses salt-assisted liquid-liquid extraction (SALLE), where extraction conditions (solvent type/volume and ammonium acetate concentration) are optimized to maximize recovery and minimize matrix effects.

Q: What performance results did the study report for the biomarkers?

The method showed excellent linearity with low LLOQs, high recoveries with negligible matrix effects, and ROC analysis that achieved perfect discrimination (AUC = 1.000) with high sensitivity and specificity.

Sep 28, 2026

Uploaded by Alexandar

Lyso-Gb1 and Lyso-Gb3 serve as key biomarkers for Gaucher type 1 and Fabry diseases, enabling accurate diagnosis and monitoring. However, standard LC-MS/MS quantification often uses large volumes of organic solvents, produces hazardous waste, and involves time-intensive preparation that limits routine use. The study develops, validates, and applies a rapid, sensitive, and greener SALLE-based LC-MS/MS method for simultaneous plasma determination of both biomarkers.

LanguageEnglish

CategoryHealthcare

FormatPDF

Pages12

Reading Time30 min

AccessFree to download

Abstract

Introduction

Methods and Optimization

Method Validation (ICH M10)

Clinical Applicability and ROC Analysis

Conclusion

This article is licensed under CC-BY-NC-ND 4.0 [http://pubs.acs.org/journal/acsodf](http://pubs.acs.org/journal/acsodf) Article Simultaneous Determination of Lyso-Gb1 and Lyso-Gb3 in Plasma Using Salt-Assisted Liquid−Liquid Extraction Combined with LC−MS/MS Erhan Canbay, * etin Uzun, Yasemin Akay, Ebru Canda, Havva Yazıcı, Sema Kalkan Uar, Mahmut oker, and Eser Yıldırım Sözmen Cite This: ACS Omega 2025, 10, 63072−63083 Read Online | | | | | | --- | --- | --- | --- | | ACCESS | Metrics & More | | Article Recommendations | ABSTRACT: Background: Lyso-Gb1 and Lyso-Gb3 are key biomarkers for Gaucher type 1 and Fabry diseases, offering high diagnostic accuracy and utility in disease monitoring. However, conventional sample preparation workflows for their LC−MS/MS quantification often require large volumes of organic solvents, generate hazardous waste, and involve time-consuming steps, limiting routine applicability. Objective: This study aimed to develop, validate, and apply a rapid, sensitive, and environmentally friendly LC−MS/MS method using salt-assisted liquid−liquid extraction (SALLE) for the simultaneous determination of LysoGb1 and Lyso-Gb3 in human plasma. Methods: The SALLE procedure was optimized for extraction-solvent type and volume, salt type, and concentration, with heptane (450 μL) and ammonium acetate (5 M) selected as optimal conditions to achieve high recovery and minimal matrix effects. Method validation was performed according to ICH M10 guidelines, evaluating selectivity, linearity, accuracy, precision, recovery, matrix effect, carryover, dilution integrity, stability, and reinjection reproducibility. Clinical applicability was assessed in two independent real-sample applications: (i) 31 healthy individuals and 20 Gaucher type 1 patients for Lyso-Gb1, and (ii) 29 healthy individuals and 13 Fabry disease patients for Lyso-Gb3. Result: The method demonstrated excellent linearity for Lyso-Gb1 (0.380−200 ng/mL, R2 = 0.9890) and Lyso-Gb3 (0.756−200 ng/mL, R2 = 0.9982), with low LLOQs (0.380 and 0.756 ng/mL, respectively), recoveries of 92.5 − 109.9%, and negligible matrix effects. In clinical samples, Lyso-Gb1 and Lyso-Gb3 concentrations were significantly higher in their respective patient groups compared to healthy controls (p < 0.0001). ROC analysis showed perfect discrimination for both biomarkers (AUC = 1.000), with optimal cut-offs of 5.80 ng/mL for Lyso-Gb1 and 3.334 ng/mL for Lyso-Gb3, each yielding 100% sensitivity and specificity. Conclusion: The developed SALLE−LC−MS/MS workflow is robust, high-throughput, and fully compliant with green-chemistry principles, using minimal organic solvent, reducing hazardous waste, and shortening the preparation time. Its excellent analytical performance and perfect clinical discrimination support its integration into routine diagnostic laboratories for the efficient diagnosis and monitoring of Gaucher type 1 and Fabry diseases. 1. INTRODUCTION Lysosomal storage disorders (LSDs) are a heterogeneous group of inherited metabolic diseases caused by lysosomal enzyme deficiencies, leading to substrate accumulation and multisystemic clinical manifestations. Fabry disease, an X-linked disorder due to α-galactosidase A deficiency, results in the progressive accumulation of globotriaosylceramide (Gb3) and lyso-Gb3, predominantly affecting the kidneys, heart, and vascular endothelium, and manifests with symptoms ranging from angiokeratomas and neuropathic pain to severe renal and cardiovascular complications. 1−6 Genetic testing plays a pivotal role in early and definitive diagnosis, which is critical as many cases remain undetected until advanced disease stages.5,7 Gaucher disease, the most common LSD, is caused by glucocerebrosidase deficiency, leading to glucosylceramide accumulation and Gaucher cell formation, and presents with hepatosplenomegaly, bone pain, thrombocytopenia, and anemia, with variable clinical phenotypes that may cause delay

FAQs

What are Lyso-Gb1 and Lyso-Gb3 used for in this study?

They are treated as key biomarkers for Gaucher type 1 and Fabry diseases, respectively, supporting diagnosis and disease monitoring using plasma measurements.

How does the method prepare samples for LC-MS/MS quantification?

It uses salt-assisted liquid-liquid extraction (SALLE), where extraction conditions (solvent type/volume and ammonium acetate concentration) are optimized to maximize recovery and minimize matrix effects.

What performance results did the study report for the biomarkers?

The method showed excellent linearity with low LLOQs, high recoveries with negligible matrix effects, and ROC analysis that achieved perfect discrimination (AUC = 1.000) with high sensitivity and specificity.

Sep 28, 2026

Uploaded by Alexandar