Trifluoroacetamides, derivatizing

In a study by Stresser and co-workers, the effect on tumor modulation by 227 has been investigated. HPLC on liver extracts from Fisher 344 rats revealed two major compounds, 3,3 -bisindolylmefliane (133) and a linear trimer, together with a < l(KX)-fold lower content of 4 in comparison with the two major substances. The HPLC isolate was derivatized with /V-methyl-/V-bis(trifluoroacetamide) that, upon MS detection, gave a compound identical to /V,W -ditrifluoroacetylindolo-[3,2-()]carbazole. The content of 4 in this system was estimated to be 0.(XKX)13% of the total dose of 227 given. Thus, it was concluded that the beneficial effect of oral distribution of 227 is due to the total content of derivatives formed (95MI5). 

The many derivatizing reagents commercially available include methanolic HC1 and diazomethane for methylation, and iV,(9-bis(trimethylsilyl)trifluoroacetamide (BSTFA), with or without 1% trimethylchlorosilane (TMCS), for silylation. Using BSTFA, hydroxyl moieties also can be silylated, giving the corresponding trimethylsilyl ethers. 

Figure 7.3 Derivatization of organic acid and alcohol compounds by diazomethane (CH2N2 top two reactions) by BSTFA (N, O-bis(trimethylsilyl) trifluoroacetamide middle two reactions), and transmethylation of fatty acid esters by saponification using methanolic sodium hydroxide.

The advantage of trimethylsilyl (TMS) derivatives lies in the simplicity of the derivatization procedure, which is carried out by the addition of N,0-bis(trimethylsilyl)trifluoroacetamide (BSTFA) in acetonitrile and heating for approximately 2 h at 150 °C under anhydrous conditions in a sealed tube. However, there may be problems owing to the formation of multiple derivatives of each amino acid. Another technique involves the formation of n-butyl esters of the amino acids and their subsequent trimethylsilylation by a similar procedure. The n-butyl esters are formed by heating the amino acids for 15 min in n-butanol and HC1 and these are then converted to the A-TMS-n-butyl ester derivatives. A-acyl amino acid alkyl esters are commonly used. Acetylation of the butyl, methyl or propyl esters of amino acids,... 

The analysis of codeine, morphine, 6-monoacetylmorphine (6-MAM, a metabohte of heroin), and cocaine is important for many toxicology labs to determine illicit drug use. When analyzing opiates in urine samples, frequently the matrix chosen for drug screening, the conjugated metabolites must be hydrolyzed however, this process can break down 6-MAM (Christophersen et al., 1987). These compounds can be derivatized to increase sensitivity, and both BCD and NPD are used for these assays. Derivatizations used include reaction with N-methyl-N-trimethylsilyltrifluoroacetamide followed by GC-FID (Lin et al., 1994) or with N,0-bis(trimethylsilyl)trifluoroacetamide (Christophersen et al., 1987 Lee and Lee, 1991), PFPA (Christophersen et al., 1987), or heptafluorobutyric anhydride (HFBA) followed by GC-ECD. All these methods show good sensitivity and selectivity. 

Gas chromatographic separation has not gained wide acceptance in spite of being quite sensitive and specific. This mode of separation is complicated by the need for derivatization of sulfonamide residues before gas chromatographic analysis. These drugs are subjected to derivatization via methylation with diazomethane (223, 224, 253, 254, 271), or double derivatization via methylation followed either by silylation with Ai-methyl-Ai-trimethylsilytrifluoroacetamide (261) or by acylation with A-methyl-bis(trifluoroacetamide) (256). This derivatization step is required not only to form the volatile derivatives of the sulfonamides but also to improve their chromatographic properties (thermal stability and decreased polarity). 

Gas chromatographic separation of -agonist residues is generally complicated by the necessity of derivatization of their polar hydroxyl and amino functional groups. Silyl derivatives are preferentially prepared by treating sample extracts with N,0-bis(trimethylsilyl)trifluoroacetamide (470,471,473,475,483, 487), N-metliyl-N-(trimethylsilyl)trifluoroacetamide (482) or N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (473, 487). Pentafluoropropionic anhydride (481), phosgene (484), trimethylboroxine (480), methyl- and butylboronic acid (489), or a combination of N,O bis(trimethylsilyl)trifluoroacetamide with... 

Chemical derivatization is a standard procedure for GC-MS analysis of steroid hormones, because steroid hormones are not volatile to go through GC column [1, 6], The major concerns of derivatization reagents for GC-MS analysis of steroid hormones are the completeness of the derivatization reaction and the volatility of hormone derivatives. The typical derivatization reagents for GC-MS samples are silylation reagents, e.g., Af-methyl-Af-trifluorotrimethyl acetamide (MSTFA, [37, 39]) and A,0-bis(trimethylsilyl) trifluoroacetamide (BSTFA, [33]), which react with both alcoholic and phenolic hydroxy groups on steroid hormone molecules. 

The most widely used procedures for environmental analysis of alkyl MPAs use conversion to trimethylsilyl (TMS) or ferf.-butyldimethylsilyl (TBDMS) esters. Both have also been applied to samples associated with the terrorist use of sarin or VX in Japan, although they are less sensitive than methods employing pentaflu-orobenzyl esters. Derivatization conditions for most applications typically involve treatment with /V,<9-bis(trimethylsilyl)trifluoroacetamide (BSTFA) +1—1% trimethylsilyl chloride (TMSC1) (60 °C, 30 min) or A-methyl-A-(ferf.-butyldimethylsilyl) trifluoroacetamide MTBSTFA + 1 % tert. -butyldim-ethylsilyl chloride (TBDMSC1) (60 °C, 30-60 min). 

Depending upon the chromatographic system utilized, cocaine analytes (especially polar metabolites) may require derivatization prior to GC/MS analysis. Commonly utilized derivatization reagents include N,0-bis(trimethylsilyl)trifluoroaceta-mide (BSTFA), pentafluoropropionic acid anhydride/hexafluoroisopropanol (PFPA)/(HFIP), N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA), pentafluoropropionic acid anhydride/pentafluoropropanol (PFPA)/(PFP), iV,0-bis(trimethylsi-lyDacetamide (BSA) and N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide (MTBSTEA). For ease of analysis and stability of the derivatives, the authors prefer BSTFA with 1% trimethylchlorosilane (TMCS). 

A GC-MS method was developed for the determination of hyoscyamine and scopolamine in blood semm [91,92]. Extraction was carried out using aqueous basic solution followed by a purification step on an Extrelut column. Derivatization was done with N,0-bis(trimethylsilyl)trifluoroacetamide/trimethylchlorosilane (99 1). GC-MS was performed on a HP-5 MS column (30m x 0.25 mm i.d. with a 0.25 p,m film thickness). The linearity was good between 10 and 5000ng/mL. The limit of detection (LOD) was 5ng/mL for each compound. 

TMS production involves one specific functional group (-OH, -COOH, =NH, -NH2, or -SH), which loses an activated hydrogen and is replaced by a trimethylsilyl group (Proestos et ah, 2006). To achieve silylation, some authors have used BSTFA (N,0-hA(trimethyl-silyl)trifluoroacetamide) and TMCS (trimethylchlorosilane) successfully in several matrices (e.g. aromatic plants, cranberry fixiit) (Zuo et ah, 2002 Proestos et ah, 2006). Using silylated derivatives is advantageous for several reasons phenols and carboxylic acids are prone to silylation, these compounds can be derivatized in the same part of the process, and the minor products do not impede analysis and are well documented (Little, 1999 Stalikas, 2008). A two-step methylation procedure was used to analyze catechins and tannins in plant extracts. The first step used trimethylsilyl diazomethane (TMS-diazomethane) to pre-methylate the sample, and the second step used thermally assisted hydrolysis and methylation (THM). The pre-methylation step with TMS-diazomethane stabilized the dimer molecule m/z 540) by minimizing isomerization and reducing reactivity. (Shadkami et ah, 2009). 

In order to identify less volatile compounds, the pyrolysis of cellulose acetate sample (at 590° C) can be followed by an off-line derivatization with bis-(trimethylsilyl)-trifluoroacetamide (BSTFA). Typically the derivatized pyrolysate is analyzed by GC/MS on a non-polar column. The results of this type of analysis (separation on a 60 m DB-5 column 0.32 mm i.d. and 0.25 p film thickness) is shown in Figure 7.3.2 and the peak identification is given in Table 7.3.3. 

One of the most popular methods of single-stage amino acid derivatization at present is their conversion to N,0 S) err-butyldimethylsilyl derivatives [the reagent rert-butyldimethylsilyl trifluoroacetamide (MTBSTFA) or its A-Me analog]. Another way, which was proposed at the beginning of the 1970s is based on amino acid interaction with dimethylformamide di-alkylacetals (CH3)2NCH(OR )2 (R = Me, Et, Pr, iso-Pr, Bu, Am) with formation of A-dimethylaminometh-ylene derivatives of amino acids esters ... 

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