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

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.

accounts for most of serum 25(OH)D, differential determination of 25-hydroxyvitamin D 2 (25(OH)D 2 ) is desirable when VD 2 -containing supplements are used by the subject (patient). Because significant levels of the C-3 epimer of 25(OH)D 3 , 3-epi-25(OH)D 3 , are present in the serum in both infants and adults, 3 its presence may result in the overestimation of 25(OH)D if it is not properly resolved by chromatography. Furthermore, the accurate quantification of 24R,25-dihydroxyvitamin D 3 (24,25(OH) 2 D 3 ) is essential for the differential diagnosis of infantile hypercalcemia of unknown etiology. 4 Thus, there is an increasing demand for the quantitative and routine mass spectrometric measurement of differential VD metabolites. 5 Because VD metabolites exhibit a low ionization efficiency under the conditions used in VD analysis by liquid chromatography/tandem mass spectrometry (LC/MS/MS), attempts to improve ionization efficiency by derivatization are being reported. 6 VD metabolites have a particular structural feature: a conjugated s-cis diene. Consequently, VD selective derivatization reagents that take advantage of the reactive dienophile, 1,2,4-triazoline-3,5-dione (TAD), which is known as a Cookson-type reagent, have been developed for enhancing their detection limits in mass spectrometric analyses. 7 DAPTAD solution without purification needs to be stored at −18°C and is recommended for use within 2 months. 10 In addition, on-site preparation of the chemicals is inherently associated with a risk of contamination, which may affect the yield of the derivatization reaction. Furthermore, water condensation can also disturb or hamper the derivatization reaction, and ambient or near-ambient temperature (refrigerator level) storage of the reagent solution is thus desirable, which, on the other hand, will affect the reagent's stability. Overcoming these limitations would enable not only an easier operational handling, but may also open the way to the automated and routine detection/quantification of VD metabolites.
Effective TAD reagents, including DAPTAD, for VD metabolites have been reported; 13,14 however, their solution stability were often low or in many cases not investigated. Although 2-nitrosopyridine 15 might be a better derivatization reagent for VD metabolites than TAD reagents, popularity and accumulated knowledge of 2nitrosopyridine as a derivatization reagent are still limited compared with those of well-known and widely used TAD reagents. In addition, long-term solution stability of 2-nitrosopyridine has not yet been confirmed. These factors motivated us to use TAD in our experiment. The stability of TAD reagents is dependent on the TAD group and not on the conjugated aromatic functional group.
Therefore, we focused on DAPTAD as a representative TAD molecule.

| Analytical high-performance liquid chromatography (HPLC)
The chromatographic analysis of the RDA reaction was performed using an

| Materials for LC/MS/MS
The

| Solution stability of DAPTAD and DAP-PA
Although TAD is one of the most reactive dienophiles and provides an excellent derivatization tag, its solution instability has been reported. [17][18][19][20] In nucleophilic solvent systems (alcohols or water), it undergoes a nucleophilic attack of its oxygen functional groups or a loss of nitrogen-yielding dimeric compounds. We first examined the solution stability of DAPTAD, which was synthesized according to the published protocol, 10 using NMR. The amount of residual DAPTAD was estimated by the integration of aromatic proton peaks (Figures 5 and S1, supporting information). As expected, a timedependent decrease in the proton signals was observed in ethyl acetate, which is a common solvent for DAPTAD derivatization. 10 The addition of molecular sieves 4A at 4°C increased the solution stability of DAPTAD, which strongly suggested that a nucleophilic attack by residual moisture occurred in the non-caged TAD. On the other hand, the solution stability of DAP-PA in ethyl acetate dramatically improved ( Figure 6) in comparison with that of the noncaged TAD, which indicated that a DA-type protection (Figure 3) of the dienophile, TAD, was effective.

| RDA reaction of caged DAPTAD
The RDA reaction is a reversible reaction, which can be controlled by thermal regulation (Figure 3). 21,22 This dynamic, reversible covalent bond formation and cleavage makes it possible to consider thermal protection and deprotection. The thermal RDA reaction enabling the release of DAPTAD was examined using analytical HPLC. DAPTAD itself was difficult to detect using reversed-phase HPLC due to its instability in aqueous solvents, and we thus monitored the presence of 1,4-diphenyl-1,3-butadiene and 9-phenylanthracene at 70°C in  ethyl acetate, which showed that the reaction with 1,4-diphenyl-1,3butadiene was irreversible (Figure 3). On the other hand, the release of 9-phenylanthracene was observed using the irreversible diene tag, 1,4-diphenyl-1,3-buthadiene (Figures 4 and S8, supporting information). The release rate of 9-phenylanthracene from DAP-PA in ethyl acetate was temperature-dependent (Table 2). 21,22 The reaction temperature (Table S9, supporting information), time (Table   S10, supporting information), solvent (Table S2, supporting information), and concentration ( Figure S11, supporting information) were also investigated using LC/MS/MS. The reaction was saturated in about 15 min. Although the reaction with aromatic solvents (toluene, anisole, and o-dichlorobenzene) gave better rate constants than that with ethyl acetate (Table S2, supporting information), the peak area in the SRM chromatogram of derivatized VD metabolites produced in ethyl acetate was larger than that produced in toluene ( Figure S11, supporting information). Considering their boiling points (indicating the easiness to remove), we concluded that ethyl acetate at 80°C was the best reaction condition.

| LC/MS/MS analyses of VDs without serum
Four VD metabolites with and without DAPTAD derivatization in the absence of serum were detected using the previously reported procedure. 12 Their retention time (t R ), LOD values, and sensitivity increase are summarized in Table 3.

| LC/MS/MS analyses of VDs in SRM972a level 2 serum
LLOQs of four VD metabolites in SRM972a level 2 serum with DAPTAD derivatization are summarized in Table 4. Their SRM chromatograms are shown in Figure 7. In our study 1α, 25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) could not be detected quantitatively due to its extremely low concentration and the difficulty in chromatographic separation of 1,25(OH) 2 D 3 and other dihydroxylated VD metabolites such as 4β, 25-dihydroxyvitamin D. 23 This problem could be solved by combining our present protocol with immunoaffinity extraction. 23

| CONCLUSIONS
We screened several diene groups to develop the caged Cooksontype reagent, DAP-PA, which was stable enough for mid-to longterm storage in solution. Highly stable reagents are essential for data reproducibility in clinical laboratories. The stability of the reagents in a solution is guaranteed by the production of pure products, which is generally achieved by crystallization, and the caged DAPTAD is easy to crystallize, which is a strong advantage in terms of quality control. In addition, from a practical viewpoint, the caged DAPTAD is available in large quantities, and thus market supply is stable and ample. This advantage will contribute to the field of VD detection and quantification in clinical laboratories, and thus to the broader use of clinical mass spectrometry. 24