Determination of 24,25‐dihydroxyvitamin D₃ in human plasma using liquid chromatography‐mass spectrometry after derivatization with a Cookson‐type reagent

Abstract

A practical LC-MS method for determination of (24R)-24,25-dihydroxyvitamin D(3) [24,25(OH)(2)D(3)] in human plasma has been developed using derivatization with a Cookson-type reagent, 4-[4-(6-methoxy-2-benzoxazolyl)phenyl]-1,2,4-triazoline-3,5-dione (MBOTAD). The derivatization with MBOTAD significantly improved the ionization efficiency of the analyte with a detection limit of 18 fmol [equivalent to 7.5 pg of 24,25(OH)(2)D(3)] per injection. The method employed protein precipitation with acetonitrile, purification with OASIS HLB cartridge and silica gel column, derivatization with MBOTAD and atmospheric pressure chemical ionization MS detection. The mass spectrometer was operated in the positive-ion mode of mass chromatography and [26,26,26,27,27,27-(2)H(6)]-24,25(OH)(2)D(3) was used as an internal standard. The intra- and inter-assay coefficients of variation were below 3.4 and 2.5%, respectively, and the analytical recovery of 24,25(OH)(2)D(3) was quantitative. Assay linearity was obtained in the range of 0.05-1.2 ng per tube and the limit of quantitation was 0.23 ng/mL for a 0.3 mL plasma aliquot. The developed method was applied to plasma samples obtained from volunteers and gave satisfactory results.

Simultaneous Determination of 25-Hydroxyvitamin D2 and 25-Hydroxyvitamin D3 in Human Plasma by Liquid Chromatography-Tandem Mass Spectrometry Employing Derivatization with a Cookson-Type Reagent.

“…8) In the study, 4-[4-(6-methoxy-2-benzoxazolyl)phenyl]-1,2,4-triazoline-3,5-dione (MBOTA-D), 9) which is rich in proton-affinitive atoms (oxygen and nitrogen atoms), was very effective in increasing the ionization efficiency of the resulting derivative in APCI operating in the positive-ion mode. These data indicate that the derivatization with a Cookson-type reagent having a different substituent at the 4-position, for example, one having more proton-affinitive atoms is capable of providing higher sensitivity in the positive APCI-MS.…”

Electron-capturing Derivatization of Neutral Steroids for Increasing Sensitivity in Liquid Chromatography/Negative Atmospheric Pressure Chemical Ionization-Mass Spectrometry

“…As mentioned above, we have developed a practical LC/positive APCI-MS method for the determination of the metabolite in human plasma as the derivative with the proton-affinitive Cookson-type reagent. 7 In the present study, negative APCI-MS of 24,25(OH)2D3 was examined, which is expected to exceed the previous method in sensitivity.…”

“…In the previous method using the Cookson-type reagent, 7 LOD was 18 fmol and 300 µL of plasma was required for the analysis. Furthermore, because some endogenous substances lowered the derivatization rate, the method required two steps of chromatographic purifications to remove them prior to derivatization.…”

“…In positive APCI-MS, the introduction of proton-affinitive atoms, such as oxygen and nitrogen, to the analyte is very effective in increasing sensitivity of the resulting derivative. Based on this information, we have developed the LC/positive APCI-MS methods for the determination of vitamin D compounds 7,8 and neurosteroids 9 after conversion to their Diels-Alder adducts with Cookson-type reagents and methyloximes, respectively. On the other hand, APCI operating in the negative-ion mode is expected to provide greater sensitivity, because background noise is relatively low in this mode.…”

“…]-24,25(OH)2D3} was prepared in our laboratories. 7 2,4-Dinitrophenylhydrazine (DNPH) was obtained from Kanto Chemical (Tokyo). OASIS HLB cartridges (60 mg adsorbent; Waters, Milford, MA, USA) were successively washed with AcOEt (2 mL), MeOH (2 mL) and H2O (2 mL) prior to use.…”