Bio-Analytical SPE Method: Vitamin D3
Estimation of metabolite of Vitamin D3 (1,25-Dihydroxyvitamin D3, calcitriol) from plant extracts using SPE-LC/MS-MS
Vitamin D3
Vitamin D3 can be sourced from certain plants, specifically lichens. These plants produce Vitamin D3 (cholecalciferol) when exposed to UV light, similar to how animals synthesize it. This plant-based Vitamin D3 is a vegan alternative to D3 derived from lanolin (sheep's wool) or other animal sources.
Cholecalciferol (Vitamin D3): Found in various plant species, including those from the Solanaceae family (like tomatoes and potatoes) and others.
25-Hydroxyvitamin D3 (25-OH D3): A common metabolite of vitamin D3 found in some plants, such as tomatoes and Capsicum.
1,25-Dihydroxyvitamin D3 (calcitriol): The biologically active form, found in some plants like Solanum glaucophyllum, often in glycosylated forms (e.g., 1α,25-(OH)2D3-glycoside).
Method Details
1,25-Dihydroxyvitamin D3 (Calcitriol)
| Parameters | Details |
|---|---|
| Chemical structure |  |
| Molecular weight | 416.64 g/mole |
| Sample type | Plant extract homogenate |
| LLOQ | NA |
Chromatographic Conditions
| Parameters | Details |
|---|---|
| Mobile phase A | Lithium acetate (0.378 mM) aqueous solution - 30% |
| Mobile phase B | Methanol - 70% |
| Column | Acquity UPLC BEH C18 (100 × 2.1 mm, 1.7 μm or similar alternate) |
| Flow rate | 0.3 mL/min |
| Column temp | 40 °C |
Mass Spectrometry Conditions
| Parameters | Details |
|---|---|
| Mass spec | API-4000 |
| 1,25-Dihydroxyvitamin D3 m/z | 423.3 > 369.3 (Lithium Adduct) |
| 1α,25(OH)2D3-d6 | 429.3 > 374.3 (Lithium Adduct) |
| Ion source | ESI spray |
| Mode | Positive ionization mode |
Sample Preparation-1 (SPE, Affinity HLB Pro /C18 30mg, 1 cc cartridges)
| Step | Procedure |
|---|---|
| Sample pretreatment | Sample matrix (100 uL) + drug + IS- mix well. Add 200 ul 0.1% formic acid mix well. Add 300 ul Acetonitrile and vortex for 5 mins. Centrifuge the samples at 10000 rpm for 5 mins. |
| Condition | 1 ml Methanol |
| Equilibration | 1 ml water |
| Sample Loading | Load the sample (supernatant) |
| Washing | Wash-1: 1 ml water Wash-2: 1 ml 10% to 30% methanol in water (optimize the washing) |
| Elution | 1 ml methanol or Acetonitrile. Optimize the elution |
| Evaporation | Evaporate to dryness at 50°C |
| Reconstitution | 200 ul mobile phase |
| Direct Injection | 10-20 uL |
Sample Preparation-2 (SPE, Affinity HLB Pro /C18 30mg, 1 cc cartridges)
| Step | Procedure |
|---|---|
| Sample pretreatment | Sample matrix (100 uL) + drug + IS- mix well. Add 200 μL of 0.2 mol/L zinc sulfate mix well. Add 600 ul methanol and vortex for 5 mins. Centrifuge the samples at 10000 rpm for 5 mins. |
| Condition | 1 ml Methanol |
| Equilibration | 1 ml water |
| Sample Loading | Load the sample (supernatant) |
| Washing | Wash-1: 1 ml water Wash-2: 1 ml 10% to 30% methanol in water (optimize the washing) |
| Elution | 500 ul to 1 ml methanol: IPA (90:10) Optimize the elution (can also inject directly by adding 100 ul water) |
| Evaporation | Evaporate to dryness at 50°C |
| Reconstitution | 200 ul mobile phase |
| Direct Injection | 10-20 uL |