Takayuki Ishige1, Mamoru Satoh2, Shoujiro Ogawa3, Motoi Nishimura4, Kazuyuki Matsushita4, Tatsuya Higashi3, Fumio Nomura2. 1. Division of Laboratory Medicine, Chiba University Hospital, Chiba, Japan. Electronic address: ishige-t@chiba-u.jp. 2. Division of Clinical Mass Spectrometry, Chiba University Hospital, Chiba, Japan. 3. Faculty of Pharmaceutical Sciences, Tokyo University of Science, Chiba, Japan. 4. Division of Laboratory Medicine, Chiba University Hospital, Chiba, Japan.
Abstract
BACKGROUND: Although immunoassays have several limitations such as the cross-reactivities of antibodies, such techniques are widely used for serum/plasma 1,25(OH)2D quantification. An accurate method is required for the determination of the 1,25(OH)2D status. METHODS: We designed a serum/plasma 1,25(OH)2D quantification method using LC-MS/MS. Immunoaffinity extraction (IE) and the recently developed Cookson-type reagent 4-(4'-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD) were used for sample preparation and derivatization, respectively. Analytical and pre-analytical validations were performed. Serum 1,25(OH)2D3 concentrations were determined in 232 healthy Japanese individuals. RESULTS: The intra- and inter-assay CVs for 1,25(OH)2D3 were 5.2% and 7.0%, respectively. The limit of quantification for 1,25(OH)2D3 was 7.1pg/ml. Rheumatoid factor (RF) at concentrations below 517IU/ml did not affect serum 1,25(OH)2D analysis. No significant differences were observed for various blood collection tubes, repeated freeze-thaw cycles, whole blood standing time, or serum storage time. A strong correlation between LC-MS/MS and radioimmunoassay (RIA) was observed (r=0.786), but serum 1,25(OH)2D concentrations obtained from RIA were 2-fold higher than those obtained from LC-MS/MS. Serum 1,25(OH)2D3 concentrations by LC-MS/MS were 18.7-53.9pg/ml. CONCLUSION: A highly sensitive and selective LC-MS/MS-based serum/plasma 1,25(OH)2D quantification method was developed using IE and DAPTAD derivatization. This method will enable the accurate determination of serum/plasma 1,25(OH)2D concentrations in the clinical setting.
BACKGROUND: Although immunoassays have several limitations such as the cross-reactivities of antibodies, such techniques are widely used for serum/plasma 1,25(OH)2D quantification. An accurate method is required for the determination of the 1,25(OH)2D status. METHODS: We designed a serum/plasma 1,25(OH)2D quantification method using LC-MS/MS. Immunoaffinity extraction (IE) and the recently developed Cookson-type reagent 4-(4'-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD) were used for sample preparation and derivatization, respectively. Analytical and pre-analytical validations were performed. Serum 1,25(OH)2D3 concentrations were determined in 232 healthy Japanese individuals. RESULTS: The intra- and inter-assay CVs for 1,25(OH)2D3 were 5.2% and 7.0%, respectively. The limit of quantification for 1,25(OH)2D3 was 7.1pg/ml. Rheumatoid factor (RF) at concentrations below 517IU/ml did not affect serum 1,25(OH)2D analysis. No significant differences were observed for various blood collection tubes, repeated freeze-thaw cycles, whole blood standing time, or serum storage time. A strong correlation between LC-MS/MS and radioimmunoassay (RIA) was observed (r=0.786), but serum 1,25(OH)2D concentrations obtained from RIA were 2-fold higher than those obtained from LC-MS/MS. Serum 1,25(OH)2D3 concentrations by LC-MS/MS were 18.7-53.9pg/ml. CONCLUSION: A highly sensitive and selective LC-MS/MS-based serum/plasma 1,25(OH)2D quantification method was developed using IE and DAPTAD derivatization. This method will enable the accurate determination of serum/plasma 1,25(OH)2D concentrations in the clinical setting.
Authors: Ali K Alshabrawy; Yingjie Cui; Cyan Sylvester; Dongqing Yang; Emilio S Petito; Kate R Barratt; Rebecca K Sawyer; Jessica K Heatlie; Ruhi Polara; Matthew J Sykes; Gerald J Atkins; Shane M Hickey; Michael D Wiese; Andrea M Stringer; Zhaopeng Liu; Paul H Anderson Journal: Biomolecules Date: 2022-07-08