Introduction. Cholecalciferol is a precursor of the calcium regulating hormone calcitriol. (Figure 1.). Dosing with a precursor to calcitriol can extend the.
DEVELOPMENT OF A HIGH THROUGHPUT METHOD FOR THE QUANTIFICATION OF CHOLECALCIFEROL IN HUMAN PLASMA WITH DERIVIATIZATION AND LC-MS/MS DETECTION Lee Winchester; Anthony Podany; Corey Ohnmacht; Wei Sun; Chad Briscoe MDS Pharma Services, Lincoln, NE
Introduction
Methods and Materials
Cholecalciferol is a precursor of the calcium regulating hormone calcitriol (Figure 1.). Dosing with a precursor to calcitriol can extend the bioavailability.
Standard calibrators were prepared fresh daily by spiking methanolic solutions of cholecalciferol into 20% human plasma in phosphate buffered saline mixture. Plasma lots screened for levels of cholecalciferol low enough not to affect the prepared concentrations were used to make quality control samples.
Currently, cholecalciferol is dosed with alendronate sodium for the treatment of osteoporosis. Endogenous analytes such as cholecalciferol present specific challenges in bio-analysis. A suitable surrogate matrix is required for the preparation of calibrators to ensure accurate quantification of unknowns at or near basal concentrations. Also, many natural products produced in-vivo or ingested may have similar mass, structure and polarity to cholecalciferol. These are factors that must be addressed when developing a selective and sensitive assay for the measurement of cholecalciferol. Figure 1. Cholecalciferol (precursor) and Calcitriol (product)
Human plasma standards, quality controls and unknown samples are mixed with acetonitrile and allowed to incubate for at least 1 hour to ensure complete protein precipitation. A Zymark/Caliper Sciclone was used as an automated liquid handling device to perform each step of a 96-well solid phase extraction (SPE). A Varian® C18 96-well plate is conditioned with methanol and water prior to direct loading of the supernatant from the centrifuged samples. After washing and eluting the samples from the SPE plate a derivatization reagent is used to prepare the samples for LC-MS/MS detection. ®
Samples are injected to an offline Thermo Electron Corporation™, Hypersil BDS C18, 10 x 2.1 mm, 3 µm pre-column and “heart-cut” onto a Thermo Electron Corporation™, Hypersil BDS C18, 50 x 3.0 mm, 5 µm analytical column (Figure 2.). Figure 2. System Set-up
Electrospray ionization of the derivatized products are monitored on an AB | MDS Sciex API 5000 in the positive ion mode.
Results/Data
Figure 4. ACN Washes
Figure 9. Blank Human Plasma (EDTA) Sample
Validation Summary
Discussion
Three successive washes of acetonitrile were collected and analyzed after loading cholecalciferol to the C18 sorbent (Figure 4.).
Cholecalciferol
Internal Standard (IS)
d6 - Cholecalciferol
Average Recovery of Drug (% Mean)
86% at 0.200 ng/mL 86% at 3.00 ng/mL 83% at 12.0 ng/mL
Average Recovery of IS (% Mean)
87%
QC Concentrations (ng/mL)
LLOQ QC, 0.227, 1.50, 4.50, and 12.0 ng/mL
Bench-top Stability (Hrs)
Short-term Stability: 28 hours in polypropylene tubes at ambient temperature under white light
Processed Stability (Hrs)
Post-preparative Stability: 166 hours in a polypropylene 96 well plate at 5°C
Freeze-thaw Stability (Cycles)
Freeze and Thaw Stability: 6 cycles in polypropylene tubes at 20°C under UV-shielded light
Long-term Storage Stability (days)
Long-term Stability: 158 days in polypropylene tubes at 20°C (0.227 and 25.0 ng/mL)
Assay Volume Required
0.200 mL
Co-administered Compound Evaluation Calcitriol (0.400 ng/mL)
Figure 10. LLOQ (0.100 ng/mL) Sample
Quality Control Samples Inter-batch
LLOQ Low Medium (QC B) Medium (QC C) High
Batch Size
Figure 3. Sample Preparation
Precision (% CV)
Accuracy (% Bias)
4.1 4.7 4.5 5.3 3.7
-0.1 0.0 -6.0 -9.3 -10.8
131 injections
Figure 6. Sample Pre Heart-Cut
Conclusion
The derivative provided better fragmentation and increased sensitivity of both the precursor and product ions. Initial chromatographic testing demonstrated a loss of sensitivity over time while samples were separated on a C18 column without column switching. It was discovered that a build-up of late eluting matrix suppression was causing the signal loss after multiple injections. A heart-cut/pre-column stripping routine was incorporated to solve the problem (compare Figures 6 and 7). A pre-column heart-cut was used to minimize the sample introduction to the analytical column while the highly retained sample material was eluted from the pre-column to waste prior to the next injection.
Analyte
Standard Curve Concentrations (ng/mL) 0.100, 0.200, 0.600, 1.00, 2.00, 3.00, 4.80, 6.00, 13.0, and 15.0 ng/mL
Acetonitrile is typically a strong solvent in reversed phase applications. However, while profiling wash solutions it was observed that acetonitrile was a relatively weak solvent for the elution of cholecalciferol on a C18 SPE column.
A dienophile, 4-Phenyl-1,2,4-triazoline-3,5-dione (PTAD), was used for derivatizing the cholecalciferol and d6-cholecalciferol (IS) by means of a Diels-Alder reaction to enhance positive ionization for mass spectrometric detection (Figure 5.).
Table 1. Validation Data Summary
Figure 11. ULOQ (15.0 ng/mL) Sample
Figure 7. Sample Post Heart-Cut
Figure 5. Derivatization
Strong solvent sample preparation, Diels-Alder derivatization and sophisticated but reliable automation allow a rapid, selective and sensitive method for cholecalciferol quantification
