LC/MS/MS of Steroids Having Vicinal Diol as Electrospray-Active Boronates

Abstract

A derivatization procedure with (3-dimethylaminophenyl)dihydroxyborane (DAPB) was introduced to enhance the detectability of steroids having a vicinal diol in LC/electrospray ionization (ESI)-MS/MS. DAPB reacted with the vicinal diol on the steroids [4β-hydroxycholesterol (4-HCh), pregnanetriol (PT) and 20R,22R-dihydroxycholesterol] in pyridine at 50°C within 1 h. The resulting DAPB-derivatives were highly responsive in ESI-MS operating in the positive-ion mode and gave characteristic product ions during MS/ MS, which enabled sensitive detection using a selected reaction monitoring mode; the detection responses of the DAPB-derivatives were increased by 20-160-fold over those of the intact steroids and the limits of detection were in the low femtomole or attomole range. The derivatization procedure was successfully applied to biological sample analysis; the derivatization followed by LC/ESI-MS/MS enabled the specific detection of trace amounts of 4-HCh in human plasma and PT in human urine with a small sample volume, simple pretreatment and short chromatographic run time.Key words derivatization; electrospray ionization-MS/MS; steroid; vicinal diol; detectability A specific and sensitive method for the characterization and determination of endogenous steroids in biological fluids and tissues is useful for the elucidation of the nature, diagnosis and treatment of diseases. LC coupled with electrospray ionization (ESI)-MS/MS is widely used for steroid analysis due to its specificity, versatility and simultaneous multi-analyte quantification capability.1-3) However, not all steroids are suitable for ESI-MS/MS. One of the limitations in ESI-MS/MS for some steroids is the poor ionization and fragmentation behavior, leading to low sensitivity; current MS/MS instruments are extremely sensitive, but still their absolute sensitivity is analyte-dependent. To overcome this limitation, derivatization of the steroids has often been employed in LC/ESI-MS/ MS [4][5][6] ; a suitable derivatization can enhance the ionization efficiency of a poorly ionizable steroid in ESI-MS and assist fragmentation suitable for the selected reaction monitoring (SRM) mode.In most of the derivatization procedures reported until now, an ESI-active (proton-affinitive or permanently-charged) moiety is introduced to the target steroid through a hydroxyor an oxo-group. [4][5][6] These procedures have been proven to be very useful for LC/ESI-MS/MS of some steroids, but the derivatization utilizing a vicinal bifunctional group, such as vicinal diol and vicinal diketone, has not been fully examined. By such a derivatization, more specific detection and quantification of the target steroid may become possible. Yamashita et al. reported a sensitive and specific assay method for estrogen o-quinones using LC/ESI-MS/MS combined with the o-phenylenediamine-derivatization.7) Brassinosteroids (vicinal diol-containing plant hormones) were also analyzed by a derivatization followed by LC/ESI-MS. 8) However, to the best of our knowledge, a practical derivatization p...

Current trends in isotope‐coded derivatization liquid chromatographic‐mass spectrometric analyses with special emphasis on their biomedical application

“…Such a bifunctional group is characteristic for certain biomolecules such as ribonucleosides and some steroids. For example, Higashi et al () designed an ICD reaction for vicinal diol‐containing steroids (4β‐hydroxycholesterol, pregnanetriol, 20R,22R dihydroxycholesterol) using the boronic acid derivative, 2 H 0 / 2 H 6 ‐3‐dimethylaminophenyl)‐dihydroxyborane ( 2 H 0 / 2 H 6 ‐DAPB), coupled with LC–MS/MS. The derivatization reaction was carried out at 50°C for 60 min.…”

“…Such a bifunctional group is characteristic for certain biomolecules such as ribonucleosides and some steroids. For example, Higashi et al (2013) (Li, Jin, et al, 2015). The same derivatization reaction was utilized in combination with cerium dioxide-based dispersive solid-phase extraction and double neutral loss scan-mass spectrometry for profiling of urinary ribonucleosides as cancer biomarkers (Chu et al, 2015).…”

Hydrolytic dynamic kinetic resolution of racemic 3-phenyl-2-oxetanone to chiral tropic acid

“…The results revealed that depending on the class of the nucleophiles, the hydrolysis reaction can occur through a substitution reaction either in an addition-elimination fashion at the carbonyl carbon or in an S N 2-fashion at the b-carbon. 12,17 Therefore, to identify whether the hydroxide ions attack the carbonyl carbon (path A) or the b-carbon center (path B) of 4 (Scheme 6), an isotope labeling experiment with H 2 18 O was performed.…”

“…A mixture of 18 O-incorporated tropic acid ( 18 O-3) and 3 was obtained by the hydrolysis of (RS)-4 with KOH (1 M) in H 2 18 O under general biphasic conditions using 11. The negative ESI mass spectrum revealed that the 18 O content of the hydrolyzed mixture was 85%.…”

“…A mixture of 18 O-incorporated tropic acid ( 18 O-3) and 3 was obtained by the hydrolysis of (RS)-4 with KOH (1 M) in H 2 18 O under general biphasic conditions using 11. The negative ESI mass spectrum revealed that the 18 O content of the hydrolyzed mixture was 85%. Then, the mixture of 18 O-3 and 3 was reduced to 2-phenyl-1,3-propanediol ( 18 O-21 and 21) by LiAlH 4 and then converted to the corresponding cyclic boronates ( 18 O-23 and 23) of 3-dimethylaminophenylboronic acid (22) because the detection sensitivity of 18 O-21 and 21 in mass spectrometry was extremely poor.…”