A Method for Simultaneous Determination of 25-Hydroxyvitamin D₃ and Its 3-Sulfate in Newborn Plasma by LC/ESI-MS/MS after Derivatization with a Proton-Affinitive Cookson-Type Reagent

Abstract

A method for the simultaneous determination of 25-hydroxyvitamin D 3 [25(OH)D 3 ] and its 3-sulfate [25(OH)D 3 S] in newborn plasma, which is expected to be helpful in the assessment of the vitamin D status, using stable isotope-dilution liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) has been developed and validated. e plasma was pretreated based on the deproteinization and solid-phase extraction, then subjected to derivatization with 4-(4-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD). e derivatization enabled the accurate quanti cation of 25(OH)D 3 without interference from 3-epi-25(OH)D 3 and also facilitated the simultaneous determination of the two metabolites by LC/positive ESI-MS/MS. Quanti cation was based on the selected reaction monitoring with the characteristic fragmentation of the DAPTAD-derivatives during MS/MS. is method was reproducible (intra-and inter-assay relative standard deviations of 7.8% or lower for both metabolites) and accurate (analytical recovery, 95.4-105.6%). e limits of quanti cation were 1.0 ng/mL and 2.5 ng/mL for 25(OH)D 3 and 25(OH)D 3 S, respectively, when using a 20-µL sample. e developed method was applied to the simultaneous determination of plasma 25(OH)D 3 and 25(OH)D 3 S in newborns; it was recognized that the plasma concentration of 25(OH)D 3 S is signi cantly higher than that of 25(OH)D 3 , and preterm newborns have lower plasma 25(OH)D 3 S concentrations than full-term newborns.

Simultaneous quantification of 25-hydroxyvitamin D3-3-sulfate and 25-hydroxyvitamin D3-3-glucuronide in human serum and plasma using liquid chromatography–tandem mass spectrometry coupled with DAPTAD-derivatization

“…The sensitivity of the current method for quantitation of 25-OHD 3 -S is comparable to a previous method in which the LLOQ of 2.5 ng/mL (~5 nM) was achieved by using 20 µL of serum [19]. The current method for quantification of 25-OHD 3 -G gave markedly improved sensitivity and reproducibility compared to what we reported previously, in which the LLOQ of ~1 nM was achieved by using 500 µL of plasma [12].…”

“…In addition, both 25-OHD 3 -S [16, 17] and 25-OHD 3 -G [12] are found in human plasma and bile, suggesting that conjugates formed in hepatocytes are transported out of the cells by as yet unknown mechanisms [12, 18]. Indeed, the relatively high concentration of 25-OHD 3 -S in plasma [11, 16, 19] suggests that it is a major metabolite of 25-OHD 3 . Although the biological roles of 25-OHD 3 -S and 25-OHD 3 -G are still not fully understood, the two conjugative metabolites could serve as reservoirs for 25-OHD 3 through metabolic deconjugation [12, 20].…”

“…Methods using high-performance liquid chromatography coupled with ultraviolet detection (HPLC-UV) or mass spectrometry (LC-MS/MS) have been reported for measurement of 25-OHD 3 -S and 25-OHD 3 -G in human serum or plasma[12, 16, 17, 19]; however, thus far reliable quantification of 25-OHD 3 -G is limited by analytical sensitivity. The objective of this study was to develop and validate a LC-MS/MS method for simultaneous, sensitive and accurate determination of 25-OHD 3 -S and 25-OHD 3 -G in human serum or plasma to enhance experimental and clinical monitoring of the two conjugative metabolites.…”

Identification of conjugation positions of urinary glucuronidated vitamin D3 metabolites by LC/ESI–MS/MS after conversion to MS/MS‐fragmentable derivatives

“…From the derivatized 24,25(OH) 2 D 3 ‐24G (Figure b), a characteristic product ion was observed at m / z 341. As previously described, the C6–7 bond of the vitamin D skeleton became fragmentable after the DAPTAD derivatization, then the product ion containing the A‐ring of the vitamin D skeleton and DAPTAD moiety was observed with a satisfactory intensity (Higashi et al, ; Ogawa et al, ). The derivatized 25(OH)D 3 ‐25G and 24,25(OH) 2 D 3 ‐25G also provided the product ion of m / z 341, and their mass spectra (Supplementary Figure S1) were very similar to that of the derivatized 24,25(OH) 2 D 3 ‐24G.…”

“…The proton‐affinitive moiety ( N , N ‐dimethylamino group) of DAPTAD facilitates ionization of the vitamin D 3 metabolites in the positive‐ion mode. Furthermore, the C6–7 bond of the vitamin D skeleton becomes fragmentable by DAPTAD derivatization (Higashi et al, ; Higashi & Shimada, ; Ogawa et al, ); therefore, characteristic product ions which have information regarding the conjugation positions could be formed from the resulting derivatives during MS/MS.…”