B-174 Quantification of vitamin C in serum as a biomarker using the Laser Diode Thermal Desorption technique “LDTD”/Mass Spectrometry (Luxon)
Authors: Correa Wengerkievicz Lopes A , Gomes D M , Argolo S V , Maia C A , dos Santos S A , Gazda V E , Muricy R C , Verdan A d , Calabria C C , Alessandro L d 
Published: October 2024

Background

Ascorbic acid (AA) or vitamin C, is a crucial substance that is involved in numerous physiological processes in living beings, including collagen production and antioxidant activity. When ascorbic acid is exposed to moisture, air, heat, light and oxygen, it oxidizes and turns into dehydroascorbic acid (DHAA). Our objective is to describe the sample preparation procedure and total quantification of AA and DHAA in human serum sample based on LDTD-MS/MS, and demonstrate its efficiency in quantification, combined with analysis selectivity and rapidity.

Methods

The Luxon is a sample introduction and ionization source that used a fiber-coupled laser diode to thermally desorb neutral molecules and charge them using a corona needle so they can be analyzed by mass spectrometry. Due to the instability of ascorbic acid, plasma samples were enriched and stabilized with an acidic solution. Sample preparation utilized a phosphate-containing buffer to minimize interferents and help volatize the analyte, followed by a precipitation step using acetonitrile to further clean the extract. Stable isotope-labeled internal standard (13C6 ascorbic acid) and homemade calibration curve and water quality controls were used for quantification. The analysis was performed using a Sciex 6500+ Triple Quad in APCI (Atmospheric Pressure Chemical Ionization) negative mode. Calibration curves ranging from 1 to 100 μg/mL and QCs were prepared in ascorbic acid-depleted serum (kept at room temperature/exposed to light for 4 days). The peak area against the internal standard (IS) ratio was used to normalize the signal. The calibration curves were plotted using the peak area ratio and the nominal concentration of standards. The following parameters were assessed for quantitative analysis: linearity, precision, accuracy, carryover and parallelism. A linear regression model with 1/X2 weighting was used to obtain the calibration curve equation and determine the correlation coefficient (r2). Parallelism was assessed by comparing the correlation coefficient of calibration curves prepared in water to six curves prepared with biological matrixes. Intra and inter assay precision and accuracy were determined by measuring QC samples ranging from low to high concentration in five replicates for 3 consecutive days.

Results

Calibration curves (n=3) showed excellent linearity. The calibration curve resulted in a linear response throughout the concentration range, the correlation coefficient was 0.99. For each quality control concentrations CVs were &lt;10% complying with desirable specifications for imprecision and accuracy. Intra-assay CVs were 3.0-8.89% and inter-assay CVs were 7.64-9.79% for precision which reflects the agreement between repeated measurements. The results for accuracy showed similar results, intra-assay CVs were 3.83-9.20% and inter-assay CVs were 5.0-9.7%.

Conclusions

The new LDTD-MS/MS method described here has been validated as an effective technique to reliably and rapidly quantify ascorbic acid. Simple sample preparation, rapid acid stabilization combined with high yield allow analysis without degradation responses.