A novel caged Cookson‐type reagent toward a practical vitamin D derivatization method for mass spectrometric analyses

Abstract

Rationale 25‐Hydroxylated vitamin D is the best marker for vitamin D (VD). Due to its low ionization efficiency, a Cookson‐type reagent, 1,2,4‐triazoline‐3,5‐dione (TAD), is used to improve the detection/quantification of VD metabolites by liquid chromatography/tandem mass spectrometry (LC/MS/MS). However, the high reactivity of TAD makes its solution stability low and inconvenient for practical use. We here describe the development of a novel caged Cookson‐type reagent, and we assess its performances in the quantitative and differential detection of four VD metabolites in serum using LC/MS/MS. Methods Caged 4‐(4′‐dimethylaminophenyl)‐1,2,4‐triazoline‐3,5‐dione (DAPTAD) analogues were prepared from 4‐(4′‐dimethylaminophenyl)‐1,2,4‐triazolidine‐3,5‐dione. Their stability and reactivity were examined. The optimized caged DAPTAD (14‐(4‐(dimethylamino)phenyl)‐9‐phenyl‐9,10‐dihydro‐9,10‐[1,2]epitriazoloanthracene‐13,15‐dione, DAP‐PA) was used for LC/MS/MS analyses of VD metabolites. Results The solution stability of DAP‐PA in ethyl acetate dramatically improved compared with that of the non‐caged one. We measured the thermal retro‐Diels‐Alder reaction enabling the release of DAPTAD and found that the derivatization reaction was temperature‐dependent. We also determined the detection limit and the lower limit of quantifications for four VD metabolites with DAPTAD derivatization. Conclusions DAP‐PA was stable enough for mid‐ to long‐term storage in solution. This advantage shall contribute to the detection and quantification of VD in clinical laboratories, and as such to the broader use of clinical mass spectrometry.

Development of free 25-hydroxyvitamin D3 assay method using liquid chromatography-tandem mass spectrometry

“…After 5 min of incubation, 25(OH)D 3 was eluted using 600 μl of ethyl acetate/hexane (50/50 v/v) three times followed by evaporation at 60°C using Centrifugal Evaporator CVE-3110 ® (TOKYO RIKAKIKAI CO., LTD, Tokyo, Japan). The residue was then derivatized using 14-(4-(dimethylamino)phenyl)-9-phenyl-9,10-dihydro-9,10-[ 1 , 2 ]epitriazoloanthracene-13,15-dione (DAP-PA) ethyl acetate solution as described previously [ 23 ]. After 15 min of derivatization and evaporation at 60°C, the residue was dissolved in 25 μl of 50% (v/v) acetonitrile, and 5 μl of aliquot was injected into the LC-MS/MS system.…”

Analysis of vitamin D metabolic markers by mass spectrometry: Recent progress regarding the “gold standard” method and integration into clinical practice

“…The diene moiety is mostly used for derivatization because of the higher specificity. In this case, Diels–Alder reagents are utilized, where most studies implemented Cookson‐type reagents (4‐substituted 1,2,4‐triazoline‐3,5‐dione) such as 4‐phenyl‐1,2,4‐triazoline‐3,5‐dione (PTAD) (Ding et al, 2010; Lyu et al, 2020), 4‐(4′‐dimethylaminopheyl)‐1,2,4‐triazoline‐3,5‐dione (DAPTAD) (Ogawa et al, 2013), (14‐(4‐(dimethylamino)phenyl)‐9‐phenyl‐9,10‐dihydro‐9,10[1,2]epitriazoloanthracene‐3,15‐dione (DAP‐PA), which is a stabilized version of DAPTAD by 9‐phenylanthracene, activated through retro‐Diels–Alder reaction in situ by heating it up to 80°C (Figure 3A) (Seki et al, 2020), 4‐[2‐(6,7‐dimethoxy‐4‐methyl‐3‐oxo‐3,4‐dihydroquinoxalyl) ethyl]‐1,2,4‐triazoline‐3,5‐dione (DMEQTAD) (Ogawa et al, 2014), 4‐[4‐(6‐methoxy‐2,2‐benzoxazolyl)phenyl]‐1,2,4‐triazoline‐3,5‐dione (MBOTAD) (Ogawa et al, 2013), 4‐ferrocenylmethyl‐1,2,4‐triazoline‐3,5‐dione (FMTAD) (Murao et al, 2005), 4‐(4‐nitrophenyl)‐1,2,4‐triazoline‐3,5‐dione (NPTAD) (Ogawa et al, 2013), 4‐(4‐diethylaminophenyl)‐1,2,4‐triazoline‐3,5‐dione (DEAPTAD), 4‐(6‐quinolyl)‐1,2,4‐triazoline‐3,5‐dione (QTAD) (Ogawa et al, 2016), SecoSET (Ahn et al, 2020; Kim et al, 2013), or commercial Amplifex Diene reagent (Hedman et al, 2014).…”

“…(A) in situ activation of DAPTAD out of DAP‐PA by retro‐Diels–Alder and (B) C‐6‐C‐7 cleavage. Source : Reprinted with permission from Jon Wiley & Sons; Ogawa et al (2013) and Seki et al (2020)…”

Determination of vitamin D metabolites in various biological samples through an improved chemical derivatization assisted liquid chromatography-tandem mass spectrometry approach

“…Chemical derivatization can introduce easily ionizable groups into the metabolite structure, thereby enhancing the ionization efficiency of metabolites in ESI sources and improving their CID fragmentation. 25–28 One notable dienophile, 1,2,4-triazoline-3,5-dione (TAD), can readily undergo a [4 + 2] cycloaddition reaction named Diels–Alder reaction with conjugated diene structures, 29–33 making it an excellent choice for derivatization reactions of VD. However, these methods are only applicable to a limited range of biological samples and restricted number of VD metabolites.…”