abstract

Abstract

In this technical note, a rapid immunopurification sample preparation procedure and a robust and sensitive LC-MS/MS method using the SCIEX QTRAP 6500+ system enabled accurate quantitation of 1,25-dihydroxyvitamin D₂ and D₃ in human serum. Excellent linearity was observed from 5 to 200 pg/mL. Low-pg/L level sensitivity was achieved at the lowest calibrator with signal-to-noise ratios (S/N) of 20:1 for both 1,25-dihydroxyvitamin D₂ and D₃ at 5 pg/mL. In addition, the method showed excellent precision (4.8% for 1,25-dihydroxyvitamin D₂ and 1.2% for 1,25-dihydroxyvitamin D₃) at the lowest calibrator level (5 pg/mL), highlighting the assay's strong quantitative performance.

Figure 1. Overland extracted ion chromatograms (XICs) of 1,25-dihydroxyvitamin D2 and D3 extracted from serum matrix. Chromatogram of calibration standard for 1,25-dihydroxyvitamin D₂ (1,25-DHVD2) and 1,25-dihydroxyvitamin D₃ (1,25-DHVD3) at 5 pg/ mL using the QTRAP 6500+ system following the sample preparation and LC-MS/MS conditions.

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Keybenefits

Key benefits of 1,25-dihydroxyvitamin D₂ and D₃ analysis from human serum using the QTRAP 6500+ system

Chromatographic separation: Optimized LC conditions enabled chromatographic separation of 1,25-dihydroxyvitamin D₂ and D₃

Excellent linearity. Calibration curves for 1,25-dihydroxyvitamin D₂ and D₃ showed r² values above 0.99 across the calibration range (5-200 pg/mL)

Low-pg/L level sensitivity and excellent quantitative performance. Sensitive quantitation of 1,25-dihydroxyvitamin D2 and D3 was achieved with excellent precision (4.8% for 1,25-dihydroxyvitamin D₂ and 1.2% for 1,25-dihydroxyvitamin D₃) at the lowest serum calibrator level (5 pg/mL)

introduction

Introduction

1,25-Dihydroxyvitamin D₂ and D₃ are the active forms of vitamin D that regulate calcium and phosphate metabolism, playing a crucial role in bone health and immune function.

Methods

Methods

Sample preparation: 1,25-dihydroxyvitamin D2 and D3 were extracted from human serum using immunopurification. 500 µL of blank serum spiked with 1,25-dihydroxyvitamin D₂ and D₃ were used for the procedure. The purified mixture was derivatized prior to analysis.

Liquid chromatography: Chromatographic separation was achieved using a Phenomenex Kinetex Luna Omega Polar C18 column (50 x 2.1 mm, 1.6 µm, 00B-4748-AN). Mobile phase A was formic acid in water and mobile phase B was formic acid in methanol. The total run time was 10 min at a flow rate of 600 μL/min.

Mass spectrometry: Data was collected using a QTRAP 6500+ system with an IonDrive Turbo V 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, assay precision, and accuracy statistics were automatically generated.

Results and discussion

Figure 1 shows the chromatographic separation of 1,25-dihydroxyvitamin D₂ and D₃ (zoomed in view) in a control human serum sample at a final concentration of 5 pg/mL. The extracted ion chromatograms showed a S/N of 20:1 for both 1,25-dihydroxyvitamin D₂ and D₃, at the lowest matrix calibrator measured (5 pg/mL), 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 concentrations ranging from 5 to 200 pg/mL. Linearity, accuracy and precision were assessed across the calibration ranges for the two analytes. Figure 2 shows a representative calibration curve for 1,25-dihydroxyvitamin D₂ and D₃ over the defined concentration range (5-500 pg/mL). The plot shows excellent linear responses across the calibration series. The r² values were 0.9995 for 1,25-dihydroxyvitamin D₂ and 0.9994 for 1,25-dihydroxyvitamin D3, respectively.

The precision values were calculated by 3 replicates in the lowest matrix calibrators measured (5 pg/mL). The precision (%CV) was 4.8% for 1,25-dihydroxyvitamin D₂ and 1.2% for 1,25-dihydroxyvitamin D₃, respectively.

Figure 2. Linear calibration curve for 1,25-dihydroxyvitamin D₃ extracted from serum matrix using the primary MRM transition. The calibration curve using the matrix calibrators was analyzed across the concentration range (5-200 pg/mL). The curve was generated using linear regression and 1/x weighting, resulting in a r² value of 0.9994 for 1,25-dihydroxyvitamin D3.

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results

Conclusion

Conclusion

A fast and sensitive LC-MS/MS method for the detection of 1,25-dihydroxyvitamin D2 and D3 extracted from human serum samples was developed using the 4500 system. The method demonstrated:

• Chromatographic separation of 1,25-dihydroxyvitamin D₂ and D₃
• Excellent linear responses across the calibration series consisting of 6 calibrators, with r² values greater than 0.999 for both analytes
• Good sensitivity resulting in S/N of 20:1 for both 1,25-dihydroxyvitamin D₂ and D₃ at the lowest calibrator level (5 pg/mL)
• High quantitation performance of the method, resulting in excellent precision (4.8% for 1,25-dihydroxyvitamin D₂ and 1.2% for 1,25-dihydroxyvitamin D₃) at the lowest calibrator level (5 pg/mL)