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

We describe a clinical research method for the analysis of vitamin C in serum by ACQUITY UPLC H-Class PLUS and a single quadrupole mass detector, the ACQUITY QDa. A simple protein precipitation by Trichoroacetic acid (TCA) provides sufficient selectivity and analytical sensitivity for vitamin C to be analyzed by UPLC-QDa. From fat-soluble¹ to water-soluble vitamins, UPLC QDa is suitable as a vitamin analytical system for clinical research purposes.

Benefits

• Requires only 200 µl of serum
• Simple sample preparation
• An accurate and sensitive method for the analysis of vitamin C in serum with a total analysis time of 6 minutes

Vitamin C or L-ascorbic acid is a water-soluble vitamin and essential nutrient for humans. Research analysis methods for vitamin C blood levels have been developed; HPLC with Electrochemical Detector (ECD) or Ultra-violet (UV) detection are commonly recommended as the method of choice for plasma vitamin C analysis.^2,3 Electrochemical detection is an attractive method for detection of electroactive species because of its inherent advantages of simplicity, ease of miniaturization, high analytical sensitivity, and relatively low cost.¹ However, both ECD and UV detection methods involve complicated sample preparation and mobile phase, dedicated column, and long analysis time. QDa detection not only provides analytically sensitive and selective analysis, but also with UPLC compatible speed and mass information for confirmation.

200 µL of sample (calibrator, control, or unknown) was pipetted into a clean microtube. 600 µL of working internal standard (1.67 µg/ml of vitamin ¹³C₆ in 12 % TCA (v/v)) was added. The tubes were capped and placed on a multi-tube vortex at 1000 r.p.m. for 5 minutes before centrifugation at 11000 rpm for 5 minutes. 400 µL supernatant was transferred into the maximum recovery vial for the LC-MS analysis. Using an ACQUITY UPLC H-Class PLUS System with an Atlantis Premier BEH C₁₈ AX Column (1.7 µm, 2.1 x 100 mm, P/N: 186009368), a gradient elution of 95% mobile phase A (MP A) to 5% MP A was run to achieve separation. The injection to injection time was 6 minutes.

Figure 1 shows the chromatograms of 0.5 µg/mL of vitamin C in the stripped serum. The analytical sensitivity was investigated, demonstrating that quantitation of 0.5 µg/mL of vitamin C in serum is achievable (% RSD <20%, Bia <15%, S/N> 10). Figure 2 shows the calibration curve of the method. The method was shown to be linear in the range from 0.5 µg/ml to 20 µg/ml. Correlation coefficients (r²) were >0.995 with all calibrator residuals <15%. 

Precision was assessed by extracting and analyzing 5 replicates from low to high concentration in the stripped serum over 5 days (n=25). Repeatability and total precision were ≤6% CV at all concentration levels tested and are summarized in Table 1. All calculated percentage recoveries were within ±15% for typical endogenous interferences tested when comparing test and control samples.

Matrix factor results were within ±15% between test samples from six individuals and control samples compensated for by using an internal standard (Table 2). Significant matrix enhancement was observed for vitamin C, but it was also compensated for by the internal standard (adjusted matrix factor close to 1).

We explore the application of a UPLC-single quadrupole MS system, the ACQUITY QDa Mass Detector, on a clinical research from fat-soluble vitamins to water-soluble vitamins. The simple preparation and short running time provide significant advantages over traditional HPLC methods. It well demonstrates that UPLC-QDa is suitable for use as a vitamin analytical system in clinical research analysis.

1. Analysis of Vitamin A and E in Serum by UPLC-QDa for Clinical Research. Waters Corporation, 2021 https://www.waters.com/webassets/cms/library/docs/720007164en.pdf.
2. Gazdik, Z.; Zitka, O.; Petrlova J.; Adam, V.; Zehnalek, J.; Horna, A.; Reznicek V.; Beklova, M.; Kizek, R. Determination of Vitamin C (Ascorbic Acid) Using High Performance Liquid Chromatography Coupled with Electrochemical Detection. Senors, 2008,8, 7097–7112.
3. Robitaille, L; Hoffer, L, J. A Simple Method for Plasma Total Vitamin C Analysis Suitable for Routine Clinical Laboratory Use. Nutrition Journal, 2016, 15:40.