abstract

Abstract

In this technical note, a rapid protein precipitation sample preparation procedure and a robust and sensitive LC-MS/MS method using the SCIEX QTRAP 6500+ enabled the accurate quantitation of 6 fat-soluble vitamins in human serum. Excellent linearity was observed across clinically relevant concentrations. Low-ng/mL level sensitivity was achieved at the lowest calibrator with signal-to-noise ratios (S/N) of 15:1, 34:1, 55:1, 260:1, 1040:1, and 41:1 for retinol, β-carotene, cholecalciferol, ergocalciferol, α-tocopherol, and phylloquinone, respectively. In addition, the method showed excellent precision (<3.1%CV) at low-level concentrations, demonstrating the quantitative performance of the assay.

Figure 1. Chromatogram of 6 fat-soluble vitamins extracted from serum matrix. Chromatogram of calibration standard for fat-soluble vitamins using the SCIEX QTRAP 6500+ system following the sample preparation and LC-MS/MS conditions.

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Keybenefits

Key benefits of fat-soluble vitamin analysis from human serum using the QTRAP 6500+ system

Low-ng/mL level sensitivity and excellent quantitative performance: Sensitive quantitation of fat-soluble vitamins was performed with excellent precision at the lowest calibrator levels (2.0%, for retinol at 10 ng/mL, 2.7% for β-carotene at 12 ng/mL, 2.3% for cholecalciferol at 1 ng/mL, 3.1% for ergocalciferol at 4 ng/mL, 1.0% for α-tocopherol at 200 ng/mL and 1.3% for phylloquinone at 0.064 ng/mL

Rapid sample preparation: Fat-soluble vitamin complexes were extracted from human serum samples using a liquid-liquid extraction (LLE) with 50:50 (v/v) hexane/ethyl acetate

Chromatographic separation: Optimized LC conditions enabled fast, 5 min chromatographic separation of 6 fat-soluble vitamins

Excellent linearity: Calibration curves for the 6 fat-soluble vitamins showed r² values above 0.99 across the calibration ranges

introduction

Introduction

Fat-soluble vitamins, including vitamins A, D, E, and K, play crucial roles in maintaining vision, bone health, antioxidant defence, and blood coagulation. Unlike water-soluble vitamins, these nutrients are stored in the body's fatty tissues and liver, making their regulation and  assessment particularly important to avoid both deficiencies and toxicities. Therefore, accurate quantification of fat-soluble vitamins in biological samples is essential to diagnose deficiencies and toxicities.

Methods

Methods

Sample preparation: Fat-soluble vitamins were extracted from human serum using a liquid-liquid extraction (LLE) with 50:50 (v/v) hexane/ethyl acetate, evaporated to dryness under nitrogen, and reconstituted in methanol.

Liquid chromatography: Chromatographic separation was achieved using a Phenomenex Kinetex Phenyl-Hexyl column (50 × 4.6 mm, 2.6 µm, 00B-4495-E0). Mobile phase A was water with 0.1% (v/v) formic acid, and mobile phase B was methanol with 0.1% (v/v) formic acid. The LC flow rate was 400 μL/min, and the total run time was 5 minutes. The injection volume was 5 μL.

Mass spectrometry: Data was collected using a QTRAP 6500+ system with an IonDrive Turbo V ion source and operated in electrospray ionization (ESI) positive mode. The Scheduled MRM algorithm was used in SCIEX OS software (version 3.1.6) to collect 10-12 data points for quantifiable data. Compound-dependent parameters were optimized by infusion.

Data processing: Data processing was performed using SCIEX OS software (version 3.1.6). Peak integration was achieved using the MQ4 algorithm. Quantitative analysis was conducted in the Analytics module of SCIEX OS, where calibration curves, concentration calculations, and assay precision statistics were automatically generated.

results

Results and discussion

Figure 1 shows the chromatographic separation of 6 fat-soluble vitamins in a control human serum sample. The 5 min gradient, in combination with the column selection and mobile phase composition, resulted in baseline separation of the 6 fat-soluble vitamins. The extracted ion chromatograms showed S/N of S/N of 15:1, 34:1, 55:1, 260:1, 1040:1, and 41:1 for retinol, βcarotene, cholecalciferol, ergocalciferol, α- tocopherol, and phylloquinone, respectively, at the lowest matrix calibrator measured, calculated using the peak-to-peak algorithm in SCIEX OS.

The quantitative performance of the method was investigated by injecting a series of calibrator samples spiked at different concentrations from 10-2000, 12-1200, 1-100, 4-100, 200-20000, 0.064-20 ng/mL for retinol, β-carotene, cholecalciferol, ergocalciferol, α-tocopherol, and phylloquinone, respectively. Linearity and precision were assessed across the calibration ranges for each of the six analytes. Figure 2 shows the calibration curves for 6 fat-soluble vitamins over the analytes’ respective calibration ranges. The plots show excellent linear responses across  the calibration series, with r² values greater than 0.99 for analytes.

The precision values were calculated from 3 replicates of the lowest matrix calibrators analyzed. The precision (%CV) were 2.0%, 2.7%, 2.3%, 3.1%, 1.0%, and 1.3% for retinol, β-carotene, cholecalciferol, ergocalciferol, α-tocopherol, and phylloquinone, respectively.

Figure 2. Linear calibration curves for the 6 fat-soluble vitamins extracted from serum matrix. The calibration curves were run across the measured ranges shown in Table 1. The curves were generated using linear regression and 1/x weighting for fat-soluble vitamins in serum, resulting in r² values >0.99 for all the analytes.

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Conclusion

Conclusion

A fast and sensitive LC-MS/MS method for the detection of fat-soluble vitamins extracted from human serum samples was
developed. The method demonstrated:

• Fast sample preparation which consisted of a simple protein deproteination
• Chromatographic separation of 6 fat-soluble vitamins
• Excellent linear responses across the calibration series, with r² values greater than 0.99 for all analytes
• High quantitation performance of the method, resulting in excellent precision at the lowest calibrator levels