A simple liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) method for determination of the eicosapentaenoic acid (EPA) concentration to arachidonic acid (AA) concentration ratio in human saliva has been developed. The EPA/AA ratio in serum or plasma is widely recognized as a useful indicator in identifying the risk of cardiovascular disease, especially atherosclerosis. The salivary EPA/AA ratio is expected to be a convenient alternative to the serum or plasma EPA/AA ratio, because saliva offers the advantages of easy and noninvasive sampling. The saliva was deproteinized with acetonitrile, purified using an Oasis HLB cartridge, and derivatized with 1-[(4-dimethylaminophenyl)carbonyl]piperazine (DAPPZ). The derivatized EPA and AA were subjected to LC/ESI-MS/MS, and the EPA/AA ratio was determined using the selected reaction monitoring mode. The DAPPZ-derivatization increased the ESI sensitivity by 100- and 300-fold for EPA and AA, respectively, and enabled the detection of trace fatty acids in saliva using a 200 μL sample. The assay reproducibility was satisfactory (relative standard deviation, <5.0%). The method was successfully applied to the measurement of the salivary EPA/AA ratios of healthy Japanese subjects and their changes owing to the supplementation of EPA.

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...