Table 1:
Accuracy, precision, recovery, matrix effects and process efficiency values of each vitamin D metabolite in the developed LC-MS/MS method.
| Compound | Concentration, ng/mL | Accuracy and precision (CV) | Matrix effects and recovery | ||||
|---|---|---|---|---|---|---|---|
| Within-day, % n=6 |
Between-day, % 6+6+6 |
Accuracy, % n=6 |
Matrix effects,% n=6 |
Recovery, % n=6 |
Process efficiency,% n=6 | ||
| 25(OH)D3 | 2.0 | 5.4 | 6.6 | 104.5 | 94.4 | 82.8 | 87.7 |
| 20 | 1.4 | 2.0 | 99.1 | 90.4 | 74.4 | 82.3 | |
| 100 | 2.1 | 2.1 | 97.5 | 85.0 | 73.8 | 86.9 | |
| 25(OH)D2 | 0.250 | 6.1 | 6.3 | 97.5 | 84.7 | 77.3 | 91.3 |
| 3.0 | 2.7 | 4.3 | 94.4 | 85.1 | 72.2 | 84.9 | |
| 30 | 1.7 | 3.7 | 104.9 | 86.1 | 76.4 | 88.7 | |
| 3-Epi-25(OH)D3 | 0.250 | 2.5 | 4.1 | 99.8 | 88.2 | 79.1 | 89.6 |
| 3.0 | 3.8 | 4.6 | 98.0 | 96.0 | 76.2 | 79.4 | |
| 30 | 4.3 | 6.0 | 97.3 | 95.2 | 85.2 | 89.6 | |
| 3-Epi-25(OH)D2 | 0.200 | 3.7 | 5.3 | 99.2 | 84.5 | 78.1 | 92.4 |
| 1 | 3.1 | 4.2 | 91.9 | 94.1 | 64.7 | 68.7 | |
| 10 | 4.0 | 4.6 | 90.4 | 99.9 | 87.7 | 87.8 | |
| 20(OH)D3 | 0.200 | 3.8 | 6.4 | 109.3 | 88.7 | 67.6 | 76.3 |
| 1.0 | 3.8 | 4.3 | 104.9 | 90.9 | 77.8 | 85.6 | |
| 10 | 2.4 | 5.0 | 100.8 | 90.4 | 79.2 | 87.6 | |
| 22(OH)D3 | 0.200 | 2.0 | 5.5 | 108.8 | 88.8 | 74.1 | 83.4 |
| 2.50 | 3.7 | 3.9 | 101.0 | 82.4 | 69.9 | 84.8 | |
| 10 | 1.0 | 5.2 | 108.3 | 96.1 | 83.1 | 86.4 | |
| 24,25(OH)2D3 | 0.250 | 6.9 | 7.2 | 94.7 | 86.8 | 75.3 | 86.7 |
| 3.0 | 2.2 | 2.4 | 99.7 | 88.7 | 83.7 | 94.3 | |
| 30.0 | 3.1 | 2.3 | 102.0 | 97.6 | 85.9 | 88.0 | |
| 1α,25(OH)2D3 | 20 pg/mL | 6.6 | 5.4 | 96.8 | 90.8 | 90.5 | 99.6 |
| 75 pg/mL | 1.0 | 3.7 | 101.4 | 83.5 | 79.0 | 94.6 | |
| 250 pg/mL | 1.2 | 2.9 | 102.0 | 88.4 | 87.9 | 99.4 | |
| 1α,25(OH)2D2 | 10 pg/mL | 11.5 | 8.1 | 93.9 | 96.9 | 93.8 | 96.8 |
| 40 pg/mL | 2.5 | 4.7 | 106.3 | 91.6 | 87.3 | 95.3 | |
| 150 pg/mL | 3.2 | 5.2 | 112.3 | 90.0 | 89.0 | 98.9 | |
| 20S,24R(OH)2D3 | 1.0 ng/mL | 9.2 | 8.8 | 107.4 | 84.3 | 76.3 | 90.5 |
| 4.0 ng/mL | 0.9 | 1.7 | 106.3 | 90.7 | 82.9 | 91.4 | |
| 20.0 ng/mL | 3.2 | 5.8 | 102.0 | 96.2 | 83.5 | 86.8 | |
| 1a,20S(OH)2D3 | 100 pg/mL | 9.2 | 6.1 | 92.1 | 92.8 | 90.8 | 97.8 |
| 350 pg/mL | 2.4 | 6.6 | 88.5 | 92.5 | 88.1 | 95.2 | |
| 2000 pg/mL | 12.8 | 9.8 | 118.4 | 93.2 | 92.4 | 99.1 | |
| 20,22(OH)2D3 | 50 pg/mL | 1.8 | 5.4 | 102.7 | 82.7 | 65.8 | 79.5 |
| 300 pg/mL | 4.3 | 3.1 | 101.9 | 88.8 | 77.7 | 87.5 | |
| 600 pg/mL | 5.9 | 3.6 | 105.2 | 86.5 | 78.9 | 91.2 | |
| 20,22(OH)2L3 | 100 pg/mL | 4.9 | 4.7 | 104.1 | 88.1 | 70.7 | 80.3 |
| 500 pg/mL | 1.0 | 1.8 | 100.0 | 93.3 | 82.6 | 88.5 | |
| 2,000 pg/mL | 3.8 | 2.9 | 103.3 | 94.8 | 84.4 | 89.0 | |
| 1,24,25(OH)3D3 | 50 pg/mL | 6.9 | 5.1 | 96.3 | 81.4 | 75.0 | 92.1 |
| 200 pg/mL | 2.4 | 7.8 | 105.2 | 86.0 | 78.3 | 91.0 | |
| 2,000 pg/mL | 5.1 | 9.5 | 103.9 | 88.5 | 83.8 | 94.7 | |
The recoveries of the six monohydroxyvitamin D analysed ranged from 67.6 and 87.7% and that of the six dihydroxyvitamin D metabolite ranged from 65.8 and 93.8%. There were no significant signal enhancements or suppression across the vitamin D analytes based on matrix effects values. A minor ion suppression (15–20%) was observed for the following metabolites (QC concentration); 25(OH)D2 (0.250 ng/mL), 3-epi-25(OH)D2 (0.200 ng/mL), 22(OH)D3 (2.5 ng/mL), 1,25(OH)2D3 (75 pg/mL), 20S,24R(OH)2D3 (1 ng/mL), 20,22(OH)2D3 (50 pg/mL) and 1,24,25(OH)3D3 (50 pg/mL). Furthermore, the accuracy and precision values for these analytes was <15% at high, medium and low QC levels. Any ion suppression from these analytes is therefore unlikely to interfere with the accurate quantitation in samples. LC-MS/MS, liquid chromatography-tandem mass spectrometry; CV, coefficient of variation.