Yoshiharu Ohyama

Microdetermination of 2,3-dinor-6-ketoprostaglandin F1α in human urine using gas chromatography-high-resolution selected-ion monitoring

The microdetermination of 2,3-dinor-6-ketoprostaglandin F1 alpha (I) in human urine is described. Samples to which the [2H4]-analogue was added as an internal standard were extracted by chromatographic sample preparation using a Bond Elut C18 cartridge and a silica gel column. Conversion of the extracted I into the 1-methyl ester-6-methoxime-9,11,15-trisdimethylisopropylsilyl ether derivative was followed by gas chromatography-high-resolution selected-ion monitoring (GC-HR-SIM). Interfering substances from the urine matrix were eliminated during GC-HR-SIM analysis using a DB-1 column. A good linear response over the range 10 pg-100 ng per tube was demonstrated. Compound I could be detected in the range 26-375 pg/ml of human urine. The proposed method can be applied to the determination of I in human urine.

Novel derivatization and immunoextraction to improve microanalysis of 11-dehydrothromboxane B2 in human urine.

A new, highly selective procedure for the determination of 11-dehydrothromboxane B2 (11-dehydro-TXB2) in human urine is described. Following the addition of [19,19,20,20-2H4]11-dehydro-TXB2 as an internal standard, samples were extracted with an affinity column of anti-11-dehydro-TXB2. Conversion of the immunoextracted 11-dehydro-TXB2 into its 1-methyl ester-11-n-propylamide-9,12,15-tris-dimethylisopropylsilyl ether derivative was followed by gas chromatography-selected-ion monitoring. The mass spectrum of the 11-dehydro-TXB2 derivative was dominated by the base peak ion of [M-C3H7]+ at m/z 698, which accounted for more than 10% of the total ion current. A typical result showed that the immunoaffinity purification procedures provided an extremely clean alternative to more conventional methods of chromatographic fractionation, and that interfering substances from the urine matrix were almost entirely eliminated during the microanalysis.

Analysis of the eicosapentaenoic acid (EPA) metabolite Δ17-6-keto-PGF1α in human plasma by GC/SIM using [18O] Δ17-6-keto-PGF1α

Abstract A method of the microdetermination of Δ 17 -6-keto- PGF 1 α , a hydrolyzed metabolite of PGI 3 , is described. An authentic Δ 17 -6-keto- PGF 1 α (120 mg) was prepared from eicosapentaenoic acid (EPA) incubated with homogenate of bovine aortic intima. [ 18 O] Δ 17 -6-Keto- PGF 1 α was synthesized by repeating base-catalyzed hydrolysis of methyl ester derivatives in [ 18 O]water, to obtain an internal standard in gas chromatography/selected ion monitoring (GC/SIM) of Δ 17 -6-keto- PGF 1 α . Good linear response over the range of 10 pg-10ng was demonstrated. Chromatographic conditions using a MP-65HT column presented nearly baseline separation of Δ 17 -6-keto- PGF 1 α and 6-keto- PGF 1 α . We were able to detect Δ 17 -6-keto- PGF 1 α in the range from 6 to 26 pg/ml of the human plasma. The present method can be applied to the determination of Δ 17 -6-keto- PGF 1 α in the human urine and plasma.

Microdetermination of the prostaglandin B1 in human plasma by gas chromatography/selected ion monitoring using [18O]prostaglandin B1 as an internal standard

We devised a simple and effective purification for the microdetermination of prostaglandin B1 (PGB1), a metabolite of PGE1. [18O]PGB1 was synthesized by the repeated base-catalyzed hydrolysis of methyl ester derivatives in H(2)18O, to obtain an internal standard for the gas chromatography/selected ion monitoring (GC/SIM) of PGB1. The methyl ester-methoxime-dimethylisopropylsilyl ether derivative was prepared, then GC/SIM was carried out by monitoring the ion at m/z 448.3 for PGB1 and that at m/z 452.3 for internal standard. A good linear response over the range of 10 pg to 100 ng was demonstrated. We detected PGB1 to a level of about 40 pg/mL in human plasma. This method can be used to determine PGB1 in biological samples.

Separation and concentration of Δ17-6-keto-PGF1α using monoclonal antibody to ω3-olefin structure of trienoic prostanoids

Abstract A monoclonal antibody against cis-3-hexen-1-ol was prepared and used to separate and/or concentrate Δ17-6-keto-prostaglandin F1α (PGF1α) in the human sera. cis-3-Hexen-1-ol was conjugated with the human serum albumin (HSA) according to the N-succinimidylester method and hyperimmunized to BALB/c mouse. The monoclonal afntibodies were obtained from hybridoma clones established by a fusion between SP2/0-Ag14-k13 mouse myeloma cells and splenocytes of a mouse. A monoclonal antibody, named 4G9-12B, recognized the epitope characteristic for ω3-olefin structure. The 4G9-12B antibody became more specific for Δ17-6-keto-PGF1α than 6-keto-PGF1α by applying inhibition ELISA using amino-residue coating plates. Using the prepared immunoaffinity columns of this antibody, Δ17-6-keto-PGF1α was clearly detected in 6 pg/ml of the human blood sera by GC/MS analysis. These results suggest that the monoclonal antibody to the partial structure of trienoic prostanoid, ω3-olefin unit, and that its immunoaffinity columns are useful in separating and concentrating Δ17-6-keto-PGF1α in the human blood or urine.