A Rapid and Simple Clinical Research Method for Vitamin C in Serum by UPLC- QDa

Importance of the Topic

Accurate measurement of vitamin C in human serum is critical for clinical research and nutritional status assessment. Traditional HPLC methods with electrochemical or UV detection often involve lengthy sample preparation and run times, limiting throughput and routine application in clinical laboratories. A streamlined, sensitive assay accelerates data acquisition, supports patient monitoring, and enhances research into oxidative stress and metabolic disorders.

Objectives and Study Overview

This work aimed to establish a rapid, simple, and reliable ultra-performance liquid chromatography method coupled to a single quadrupole mass detector (UPLC-QDa) for quantifying vitamin C in serum. Key goals included minimizing sample volume, simplifying preparation, and achieving high analytical performance within a shortened analysis time.

Methodology and Instrumentation

Sample Preparation

• Serum volume: 200 µL.
• Internal standard: 13C6-labeled vitamin C at 1.67 µmol/L in 12% trichloroacetic acid (TCA).
• Protein precipitation: Vortex mixing (1 000 rpm, 5 min) and centrifugation (11 000 rpm, 5 min).
• Injection: 3 µL of supernatant.

Chromatographic Conditions

• Column: Atlantis Premier BEH C18 AX, 2.1×100 mm, 1.7 µm.
• Mobile phases: A – 20 mM ammonium formate in water; B – 20 mM ammonium formate in methanol.
• Gradient: 95% A to 5% A over 6 min; flow rate 0.40 mL/min.
• Column temperature: 40 °C; sample temperature: 10 °C.

Instrumental Setup

• UPLC system: ACQUITY UPLC H-Class PLUS.
• Mass detector: ACQUITY QDa single quadrupole MS with ESI− in SIR mode.
• Software: MassLynx 4.2.

Results and Discussion

The method enabled quantification of vitamin C down to 0.5 µmol/L with signal-to-noise ratio >10, accuracy bias <15%, and precision (%RSD) <20%. Calibration curves were linear from 0.5 to 20 µmol/L (r2>0.995). Repeatability and intermediate precision across five days (n=25) showed CV≤6% at three quality control levels (0.80, 3.02, 15.11 µmol/L). Matrix effects were assessed across six individual serum samples, demonstrating consistent suppression or enhancement offset by the internal standard (adjusted matrix factor ~1).

Benefits and Practical Applications

• Low serum requirement (200 µL).
• Rapid sample preparation and 6-minute runtime.
• High sensitivity and selectivity with mass confirmation.
• Suitable for clinical research, nutritional monitoring, and high-throughput laboratories.

Future Trends and Potential Applications

Ongoing developments may extend this approach to multiplex vitamin panels, integrate automated sample handling, and apply high-throughput screening in population studies. Advances in compact mass detectors could further democratize access to mass-selective assays in point-of-care and remote settings.

Conclusion

The UPLC-QDa assay for serum vitamin C combines streamlined sample processing with rapid, sensitive detection, offering clear advantages over conventional HPLC approaches. Its performance and workflow make it a robust tool for clinical research and routine laboratory analysis.

References

• Waters Corporation. Analysis of Vitamin A and E in Serum by UPLC-QDa for Clinical Research, 2021.
• Gazdik Z, Zitka O, Petrlova J, et al. Determination of Vitamin C Using HPLC with Electrochemical Detection. Sensors, 2008, 8:7097–7112.
• Robitaille L, Hoffer LJ. A Simple Method for Plasma Total Vitamin C Analysis Suitable for Routine Clinical Laboratory Use. Nutrition Journal, 2016, 15:40.