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Gas chromatography/high-resolution selected ion monitoring (GC/HR-SIM) for the determination of 11-dehydrothromboxane B2 (11-dehydro-TXB2) in human urine is described. Combined use of the lactone ring opening reaction and dimethylisopropylsilylation has been adopted for the analysis of 11-dehydro-TXB2, to give a new chemically stable derivative. The feasibility of using this derivative for the analysis of 11-dehydro-TXB2 in human urine has been evaluated. Following the addition of 18O2-labelled analogue, for use as an internal standard, the samples were extracted using chromatographic separation with Bond Elut C18 and silica gel. Derivatization of the extracted 11-dehydro-TXB2 was followed by GC/SIM at resolution of 5000. This method enables selective detection of 11-dehydro-TXB2 in human urine without serious interference from other endogenous substances. In 11 volunteers, the mean urinary excretions of 11-dehydro-TXB2 was 547.6 +/- 217.6 ng per day.

In order to identify vitamin D2 contained in shiitake mushroom (Lentinus edodes), which is taken routinely in Japan, vitamin D2 was isolated by thin-layer chromatography and high-peformance liquid chromatography and identified by thermospray-interface mass spectrometry; this procedure prevents the decomposition of vitamin by heat, which is a common problem in gas chromatography—mass spectrometry of vitamin D2.

The microdetermination of 2,3-dinor-6-ketoprostaglandin F 1α ( I ) in human urine is described. Samples to which the [ 2 H 4 ]-analogue was added as an internal standard were extracted by chromatographic sample preparation using a Bond Elut C 18 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.

Gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS) properties of 11-dehydrothromboxane B2 (11-dehydro-TXB2) methyl ester-11-n-propylamide-15-diethylhydrogensilyl (DEHS)-9, 12-cyclic diethylsilylene (DES) derivatives were studied. The methylene unit (MU) value of this derivative was 35.17, being 5.7 higher than that of the 11-dehydro-TXB2 methyl ester-bis-trimethylsilyl ether derivative. The mass spectrum of this derivative was characterized by the ion at m-z 368, which consisted of the fragment of the DES ring and the α-side chain. The major fragmentations were directed by fission of the DES group to give silicon atom-containing ions, inducating that the DES ring takes a leading part in the formation of characteristic ions. Other fragmentations common to the prostanoid derivatives were losses of an ethyl radical from the DAHS or DES group, a C5H11 hydrocarbon fragment and diethylhydrogensilanol. The fragmentation mechanisms are briefly discussed.

The mass spectrum exhibited a series of ions characteristic of the expected 11-dehydrothromboxane B2 (11-dehydro-TXB2) derivative. Many of the fragmentation products could be explained in terms of simple bond fission mechanisms. A further series of fragmentations proceeded via a six-membered ring transition state or by formation of the cyclic dimethylsilylene ring system. These produced ions in the low mass region containing silicon atoms, characteristic of the protected C-11 carboxylic and moiety. The appearance of these ions may be used for the identification of 11-dehydro-TXB2 and its metabolites having a lactone ring. Six homologue and analogue derivatives were prepared for investigation of the fragmentation mechanism. The proposed fragmentation mechanisms have been supported by accurate mass measurement and mass analyzed ion kinetic energy mass spectrometry.

2, 3-Dinor-6-keto-prostaglandin F (I), extracted from its acidic aqueous solution, was converted to its 1-m__--propylamide-6-methyloxime-9, 11, 15-tris-dimethylisopropylsilyl (DMIPS)-ether derivative by treating it with n__-propylamine. O-methylhydroxylamine-HCI and then with DMIPS-imidazole. The chromatogram of the reaction product showed a single broad peak accompanying a shoulder, which was considered to be the unresolved structural syn- and anti-isomer pair. The methylene unit value of this derivative was 34.28, being about 7.5 higher than that of the trimethylsilyl ether derivative of I methyl ester-methyloxime. The reaction product gave a mass spectrum with the ion of [M-43]+ at m/z 669 as a base peak. The molecular ion was observed at m/z 712 with low intensity, and many characteristic ions reflecting the structure with moderate or low intensity. The present I derivative concentrated more than 5% of the total ion current (above m/z 100) into the base peak ion. When Gas chromatography/high resolution selected ion monitoring was carried out at the resolution of 10000 monitoring the ion at m/z 669.4512, specific for the structural integrity of I, the selected ion recording showed a single broad peak with signal-to-noise ratio of more than 10 : 1 after injection of 2 picograms of the derivative.

A gas chromatography/selected ion monitoring (GC/SIM) method for a simultaneous determination of cortisol (Kendall's compound 'F' : F) and its hydroxylated metabolite, 6β-hydroxycortisol (6OHF) in human urine has been developed using [2H3] cortisol as an internal standard. These compounds were analyzed as their methyloxime-diethylhydrogensilyl-cyclicdiethylsilylene derivatives. Gas chromatography of the F derivative showed a fairly broad single peak, whereas the 6OHF derivative exhibited a well-resolved doublet peak corresponding to the structral syn- and anti-isomers. The mass spectra were characterized by their intense molecular ion peaks and by their inherent ions of [M-OCH3]+, [M-diethyl-hydrogensilanol (DEHSOH)]+., and [M-OCH3-DEHSOH]+. When GC/SIM was carried out using their base peak ions at m/z 559 for F and 557 for 6OHF, the sensitivity for F was about 10 pg with signal-to-noise ratio of more than 10, and this was about a 5 times higher response factor than that of 6OHF. The calibration graphs were found to be linear in the observed range covering the concentrations found in healthy human urine.

The mass spectrum of the 2,3-dinor-11-dehydrothromboxane B2 (DNDTXB2) 1-methyl ester-11-n-propylamide-9,12,15-tris-dimethylisopropylsilyl ether derivative was characterized by the base peak ion [M C3H7]+ (m/z 670), and it exhibited a series of ions typical of the expected derivative. Many of the fragmentation products could be explained in terms of their simple bond fission mechanisms. Ions containing silicon atom in the low-mass region were produced via a six-membered ring transition or by formation of the cyclic dimethylsilylene ring system, which were common to the TXB metabolites having a δ-lactone ring system. These results revealed that this mass spectrum was similar to that of the corresponding 11-dehydro-TXB2 derivative except for the obvious shift produced by the lack of C2/C3 hydrocarbon units. The presence of DNDTXB2 in the urine of a healthy adult was confirmed by gas chromatography/selected ion monitoring.