Berit P Jensen1, Rajneeta Saraf2, Jing Ma3, Sarah Berry2, Cameron C Grant4, Carlos A Camargo5, Christiaan W Sies3. 1. Specialist Biochemistry, Canterbury Health Laboratories, PO Box 151, Christchurch, New Zealand. Electronic address: berit.jensen@cdhb.health.nz. 2. Centre for Longitudinal Research-He Ara ki Mua and Growing Up in New Zealand, The University of Auckland, New Zealand. 3. Specialist Biochemistry, Canterbury Health Laboratories, PO Box 151, Christchurch, New Zealand. 4. Centre for Longitudinal Research-He Ara ki Mua and Growing Up in New Zealand, The University of Auckland, New Zealand; Department of Paediatrics: Child & Youth Health, The University of Auckland, New Zealand. 5. Massachusetts General Hospital and Harvard Medical School, Boston, United States.
Abstract
BACKGROUND: Demand for measurement of 25-hydroxyvitamin D (25OHD) is growing and dried blood spot (DBS) sampling is attractive as samples are easier to collect, transport and store. METHODS: A 2D LC-MS/MS assay without derivatization was developed. DBS punches (3.2 mm) were ultrasonicated with d6-25OHD3 in 70% methanol followed by hexane extraction, dry-down and reconstitution. The assay was validated and applied to two studies comparing whole blood adult DBS with serum samples (n = 40) and neonatal whole blood DBS with cord serum samples (n = 80). RESULTS: The assay was validated in whole blood DBS over the range 13-106 nmol/L 25OHD3 and 11-91 nmol/L 25OHD2 with a limit of detection of 3 nmol/L. Intra- and inter-day imprecision was <13% CV and bias <12%. The assay had high recovery and minimal matrix effects. Triplicate DBS study samples had a mean CV of ≤13% for 25OHD3. No 25OHD2 was detected. DBS calculated serum 25OHD3 concentrations correlated strongly with serum concentrations in the adult DBS/serum study (r = 0.94) and moderately in the neonatal DBS/cord serum study (r = 0.69). CONCLUSIONS: Direct quantitation of 25OHD in DBS by 2D LC-MS/MS without derivatization was found to be an alternative to serum quantitation applicable to clinical research studies on adult DBS samples.
BACKGROUND: Demand for measurement of 25-hydroxyvitamin D (25OHD) is growing and dried blood spot (DBS) sampling is attractive as samples are easier to collect, transport and store. METHODS: A 2D LC-MS/MS assay without derivatization was developed. DBS punches (3.2 mm) were ultrasonicated with d6-25OHD3 in 70% methanol followed by hexane extraction, dry-down and reconstitution. The assay was validated and applied to two studies comparing whole blood adult DBS with serum samples (n = 40) and neonatal whole blood DBS with cord serum samples (n = 80). RESULTS: The assay was validated in whole blood DBS over the range 13-106 nmol/L 25OHD3 and 11-91 nmol/L 25OHD2 with a limit of detection of 3 nmol/L. Intra- and inter-day imprecision was <13% CV and bias <12%. The assay had high recovery and minimal matrix effects. Triplicate DBS study samples had a mean CV of ≤13% for 25OHD3. No 25OHD2 was detected. DBS calculated serum 25OHD3 concentrations correlated strongly with serum concentrations in the adult DBS/serum study (r = 0.94) and moderately in the neonatal DBS/cord serum study (r = 0.69). CONCLUSIONS: Direct quantitation of 25OHD in DBS by 2D LC-MS/MS without derivatization was found to be an alternative to serum quantitation applicable to clinical research studies on adult DBS samples.
Authors: Barbara Altieri; Etienne Cavalier; Harjit Pal Bhattoa; Faustino R Pérez-López; María T López-Baena; Gonzalo R Pérez-Roncero; Peter Chedraui; Cedric Annweiler; Silvia Della Casa; Sieglinde Zelzer; Markus Herrmann; Antongiulio Faggiano; Annamaria Colao; Michael F Holick Journal: Eur J Clin Nutr Date: 2020-01-06 Impact factor: 4.016