Methoxime-TMS derivative

Add 100 pi 1 of MSTFA reagent to less than 1 mg of dry extract. Heat at 60° for 15-20 min. If necessary, add 250 /a 1 of acetonitrile or other suitable solvent. For additional structural information, prepare the methoxime-TMS derivative to determine if one or more carbonyl groups are present. 

System (4) has been reported for the simultaneous determination of cortisol and cortisone in human plasma by stable-isotope dilution-MS [178]. For the determination, capillary GC-MS with H5 -cortisol and H5 -cortisone (as internal standards) was used. The method used a SPB-1 fused-silica capillary column (7 m x 0.25 mm) and helium as the carrier gas (at 29.4 Pa), with 70 eV EIMS being used for selective-ion monitoring. The concentrations were determined from the peak height ratios of the [M-31] fragment ions of the methoxime-TMS derivatives of cortisol, cortisone, and their internal standards. The sensitivity of the method was 200 pg per injection for both cortisol and cortisone. 

For low molecular weight aliphatic acids, try TMSDEA reagent. Otherwise, use MSTFA, BSTFA, or TRI-SIL BSA (Formula P). For analysis of the keto acids, methoxime derivatives should be prepared first, followed by the preparation of the trimethylsilane (TMS) derivatives using BSTFA reagent. This results in the meth-oxime-TMS derivatives. 

B. Preparation of Methoxime (MO)-TMS Derivatives (especially for hydroxyketosteroids, which may decompose under the given GC conditions unless the MO-TMS derivatives are prepared.) Add 0.25 ml of methoxime hydrochloride in pyridine to the dried extract. Let stand for 3 hours at 60° or overnight at room temperature. Evaporate to dryness with clean, dry nitrogen. Add 0.25 ml of TRI-SIL TBT and 0.25 ml of pyridine to the dried reaction mixture. Cap tightly and heat at 60° for a few hours or overnight at room temperature. 

Mass spectra of methoxime (MO)-TMS derivatives of steroids typically give the following fragments M-31 (loss of oxime), sequential 90 losses (trimethylsilanol), loss of the primary TMS group (M-103), and combinations thereof. Often the ion chosen for monitoring is one formed by the above fragmentations. A detailed de-... 

If silylation alone (0.2 ml of BSTFA + 0.05 ml of TMCS) without the preceding methoximation is carried out, TMS enol ether—TMS esters are produced from keto acids. Using the procedure described, methoxime-TMS esters of keto acids and TMS ether—TMS esters of hydroxy acids are produced. Unsubstituted acids give TMS esters. The procedure eliminates possible losses of the derivatives, which can be caused by, e.g., evaporation of the solvent between the esterification and the silylation steps, and is quantitative. SE-30, OV-17 and OV-22 can be used and retention data on these stationary phases have been reported for 15 acids [159]. An example of the separation of the derivatives of some acids prepared by this procedure is illustrated in Fig. 5.12. 

The use of molar equivalents of methylboronic add and steroid diol affords only the cyclic ester. Acyclic boronate esters will be formed at remote hydroxyl groups if excess reagent is used. While these latter derivatives display undesirable GC properties, the acyclic boronate groups are readily displaced by trimethylsilyiation the cyclic methaneboronate will be unaffected under the mild silylation conditions described above. n-Butyl boronate—methoxime—TMS derivatization has been... 

TBDMS derivatives have been applied to the analysis of organic acids in urine as oxime-TBDMS [91] and in tissues [92] as methoxime-TBDMS derivatives. The intense [M —57]" ions found in the spectra of the latter were shown to contain all the original hydrogen atoms. Thus, the derivatization should find application in quantification by isotope dilution and in metabolic studies with deuterium-labelled compounds. TMS and oxime-TMS derivatives, however, still remain the more popular derivatives for general screening, a consequence of the more structurally informative mass spectra, shorter analysis times and the ready availability of reference spectra. 

Formation of the methyl ester methoxime TMS-ether derivative... 

A comprehensive collection of spectra from a number of sources of trimethylsilyl derivatives of organic acids of biological interest was compiled by Markey et al (1972) and subsequently included in a larger reference collection (Markey et al., 1974b) and in other library files (e.g. Mass Spectrometry Data Centre Collection, The University, Nottingham, England). In addition to spectra of trimethylsilyl (TMS) derivatives are spectra of free acids, methyl esters, methyl ester-trimethylsilyl ethers (hydroxy acids), and trimethylsilyl esters-trimethylsilyl ethers-methoximes (oxo acids or oxo hydroxy acids). 

Chalmers and Watts [161] preferred ethoxime-TMS esters for the analysis of some physiologically important keto- and aldo-carboxylic acids. These derivatives were said to be more stable than their methoxime analogues and had good chromatographic properties on OV-101. 

Fig. 5.18 includes the spectra of 2-oxoglutaric-MO TMS and -EO-TMS. As methoxime and ethoxime derivatives fragment in closely similar ways, this can be useful for elucidating the composition of certain ions (Brooks and Harvey,... 

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