Xuejun Zang (Presenter)
Orochem Technologies Inc
Authorship: Xuejun (June) Zang, Chhavi Bhardwaj, Slobodan Milasinovic, Anil Oroskar, Asha Oroskar
Orochem Technologies Inc
| Short Abstract LC-MS/MS method has emerged as a reliable method of choice for vitamin D metabolite analysis. However, it ‘s easily affected by phospholipids content in the serum samples. We present two different vitamin D metabolites extraction methods. One is using Vitamin D extraction plate. The process is simple and high throughput. For 96-well plate , the process time is less than 10 minutes. Most matrix interferences are removed, while the recoveries of mono- and di- hydroxyl vitamin D are good. Another procedure is using polymeric SPE extraction product. The final recoveries of vitamin D metabolites are all above 86%. |
Long Abstract
Introduction
Increased clinical awareness of the prevalence of vitamin D deficiency and insufficiency has led to a need for large volume vitamin D metabolite assay. Vitamin D and its metabolites circulate in blood bound to serum proteins. Thus, to be able to analyze the metabolites, one will need to dissociate them from the serum proteins. LC-MS/MS method has emerged as a reliable method of choice for vitamin D metabolite analysis due to its high specificity and high accuracy at low ng/mL level. However, LC-MS/MS is easily affected by phospholipids content in the serum samples. We present two different approaches here , one is using vitamin D metabolites extraction plate. It is simple and fast, while provides reasonable recoveries; the other one using Sagacity HL SPE products, provides high recoveries and reproducibility of mono- and di- hydroxyl vitamin D.
Instruments and Materials
All automated extractions were carried out using Orochem’s Oroflex Personal Pipettor robot. All LC-MS/MS method used a Shimadzu HPLC system coupled to an API 3000 mass spectrometer with a turbo spray ESI source operated in positive mode.
Orochem’s 96-well Vitamin D Metabolite Extraction plate, and Sagacity HL SPE plate were used for all extractions. The 25-hydroxyvitamin D2 and D3, 1,25-dihyroxyvitamin D3 standards were purchased from Sigma-aldrich . HPLC grade solvents such as 2-propanol, acetonitrile, water and methanol were purchased from Pharmco-Aaper . An EZYPRESS HT 96-well plate positive pressure manifold unit was used for conditioning, washing and processing the Vitamin D metabolite extraction plate and Sagacity HL SPE plate. Vitamin D and metabolites free human serum purchased from Golden West Biologicals (Temecula, CA)
Methods
For vitamin D metabolites extraction plate, first aliquot 0.4 mL of acetonitrile and methanol mixture into each well, then add 0.1 mL of fortified serum sample. Wait about 1-2 minutes, then positive pressure was applied to the plate such that fluid flowed through the extraction plate drop by drop, total process is less than 10 minutes for 96-well plate. The vitamin D eluate was evaporated and reconstituted with 0.1 mL of a solution of 80% methanol in water.
For Sagacity HL SPE plate, first condition the plate with 1 mL of acetonitrile followed by 1m of water. Pretreat the fortified serum 0.1 mL with 50 µL of isopropyl alcohol first, then add 70 µL of water. Load pretreated serum to the SPE plate, and wash with 1 mL of water followed by 1mL of 30% acetonitrile in water. The metabolites were eluted with 1 mL of acetonitrile. The vitamin D eluate was evaporated and reconstituted with 0.1 mL a solution of 50% methanol in water.
For LC-MS/MS analysis Orochem’s Reliasil C18 column, 3 µm, 50 x 4.6 mm with a 87% Methanol (0.1% Formic acid, 15 mM Ammonium Acetate) mobile phase was used.
Results
Reasonable recoveries (> 68%) obtained for all three metabolites using vitamin D metabolites extraction plate. In the meantime, almost all the phospholipids were removed from the extract. We tested several different precipitation reagents, methanol/acetonitrile 1:1 ratio provides the best recovery and phospholipids removal rate. Compared to some commercial phospholipid depletion plate, vitamin D metabolites gave the best recoveries.
When using Sagacity HL SPE product, recoveries for all three metabolites were all above 86%.
We tested several different pretreatment method, found out 2:1 serum/IPA ratio could sufficiently release protein bounded vitamin D metabolites, while not enough precipitation to block the SPE. Additionally, certain volume of water needed to obtain 1,25 dihydroxy vitamin D on the Sagacity HL sorbent.
Conclusions
Two different types of vitamin D metabolite extraction methods have been established. Vitamin D extraction plate provides reasonable recovery while it is fast and simple. It is very easy to operate by clinical technicians. It is great for 25-hydroxy vitamin D test.
Sagacity HL SPE plate method is high recovery, and still high through put. It could be used for testing low concentration 1,25-dihydroxy vitamin metabolites.
References & Acknowledgements:
1. Free 1,25-Dihydroxyvitamin D levels in Serum from Normal Subjects, Preganant Subjects, and Subjects with Liver Disease. Bikle, Daniel D., et al. December 1984, Journal of Clinical Investigation, Inc., Vol. 74, pp. 1966-1971.
2. Plasma 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, and parathyroid hormone in familial hypocalciuric hypercalcemia and primary hyperparathyroidism. Christensen, SE, et al. 6, Dec 2008, Eur J Endocrinol., Vol. 159, pp. 19-27.
3. Serum Levels of 1,25-dihydroxyvitamin D, 24,25-dihydroxyvitamin D and 25-hydroxyvitamin D in nondialized patients with chronic renal failure. Ishimura, Eiji, et al. 1999, Kidney International, Vol. 55, pp. 1019-1027.
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