Extraction of Vitamin B7 (Biotin) from Serum Using EVOLUTE® EXPRESS ABN Prior to LC-MS/MS Analysis

Importance of the Topic

Accurate quantification of vitamin B7 (biotin) in serum is essential for clinical diagnostics, nutritional studies and therapeutic monitoring. Its low endogenous concentration and the complex serum matrix demand a robust sample preparation workflow to ensure high sensitivity, reproducible recovery and minimal matrix effects.

Objectives and Overview

The application note describes an optimized solid-phase extraction (SPE) protocol using the EVOLUTE EXPRESS ABN 96-well plate to isolate biotin from human serum prior to LC-MS/MS analysis. Key aims were:

• Maximize extraction recovery and reproducibility for biotin and its deuterated internal standard.
• Minimize co-extracted proteins, lipids and phospholipids.
• Demonstrate compatibility with both manual and automated (Biotage Extrahera) workflows.
• Validate chromatographic performance, calibration linearity and matrix cleanliness.

Methodology

Sample Preparation:

• Add 250 pg/mL biotin-[2H2] internal standard to 200 µL serum and dilute with 200 µL 1% formic acid.
• Load 400 µL pretreated serum onto conditioned EVOLUTE EXPRESS ABN wells.
• Wash sequentially with 500 µL water, then 500 µL water/methanol (95:5).
• Elute analytes with 200 µL 0.1% NH4OH in water/methanol (90:10).
• Evaporate eluates at 40 °C under air or nitrogen, then reconstitute in 200 µL water/acetonitrile (90:10).

Instrumentation Used

• SPE media: EVOLUTE EXPRESS ABN 10 mg 96-well plate.
• Automation: Biotage Extrahera platform (optional).
• UPLC: Waters ACQUITY I-Class with ACE Excel C18-PFP column (100 × 2.1 mm, 1.7 µm).
• LC mobile phase: A) 1 mM ammonium fluoride; B) acetonitrile; gradient 90:10 to 20:80 and back.
• MS: Waters Xevo TQ-S triple quadrupole, negative MRM mode (biotin transitions 243.1→200.0 and 243.1→166.0; IS 245.1→168.0).

Key Results and Discussion

Recovery and Precision:

• Recoveries exceeding 80% across 25–5000 pg/mL with RSDs below 10%.
• Linear calibration (25–1000 pg/mL) with r² >0.99; low endogenous biotin background.

Chromatography and Sensitivity:

• Retention time ~0.8 min; sharp peak shape at 25 pg/mL quantifier level.
• Negative ionization yielded superior selectivity and signal-to-noise compared to positive mode.

Matrix Cleanliness:

• Phospholipid MRM monitoring showed significant removal relative to protein precipitation.
• Post-column infusion demonstrated minimal ion suppression at analyte elution window.

Benefits and Practical Applications

The protocol delivers a fast, high-throughput SPE workflow compatible with 96-well plates and automation. It ensures reproducible biotin quantitation in clinical and research laboratories, supporting quality control and diagnostic assays with low matrix interference and high assay sensitivity.

Future Trends and Opportunities

Potential developments include:

• Direct injection strategies using pH-stable columns to eliminate evaporation steps.
• Micro-SPE formats or online SPE-LC integrations for further throughput gains.
• Expanded use of mixed-mode sorbents and tailored elution chemistries to improve analyte coverage and matrix removal.

Conclusion

The EVOLUTE EXPRESS ABN SPE method provides an efficient, reproducible and automatable approach for isolating vitamin B7 from serum. Coupled with sensitive LC-MS/MS detection, it delivers robust quantitation with minimal matrix effects, making it highly suitable for routine bioanalytical applications.