Methylation, on-column

EtOAc extn, liq-liq partns, on column methylation with trimethylanilinium... 

A brief comment should be made concerning the technique of "on-column" methylation. This is a technique wherein a methylating agent is injected along with the drug into the gas chromatograph whose temperatures initiate and complete derivatization. Trimethylanilinium hydroxide is the most commonly employed reagent and has been used to form the 1,3-dimethyl derivatives of barbiturates which do not form stable TMS derivatives (29). 

However, two other areas are of interest (Fig. 9) namely, a) derivatisation of phenols or amines to phosphorus-containing compounds and b) on-column methylation. In the former, phosphorylation of 7 alcohols and 12 phenols with diethyl chlorophosphate in the presence of triethylamine at 60-70 C in benzene has been reported (35). Aliphatic alcohols, such as methanol, butanol or isoamyl alcohol react instantaneously while phenol and cresols require ca 1-1 1/2 hrs. and xylenols 3-4 hrs. for best results. 

Similarly, on-column methylation has been applied to carbamate pesticides containing an active N-H group. Wien and Tanaka (41) showed that N-aryl carbamates are methylated on-column with tri-methylanilinium hydroxide-methanol to give the intact N-methyl and N-aryl derivatives. On the other hand N-methyl, 0-aryl carbamates such as carbaryl or carbofuran yielded only the methyl ethers of their respective phenols. This work has now been extended to sulfur-containing carbamates such as methomyl, methio-carb, aldicarb, etc. (42-43). Here the oxime hydrolysis products of these carbamates are chromatographed as the 0-methyl oximes. 

However, it must be pointed out that to date, the application of the phosphorylation and on-column methylation techniques have had only limited application. 

Figure 10. Examples of on-column methylation reactions of organophosphorus...

On-column methylation of barbiturates, hydan-toins, and some carboxylic acids can be achieved by injecting the sample mixed with 0.2M trimethyl-anilinium hydroxide in methanol. For carboxylic acids, the sample can be dissolved in 14% boron trifluoride in methanol and heated at 60 to 100° for 30 minutes. After evaporation of the bulk of the methanol, the mixture is diluted with a few millilitres of water and the methyl derivative extracted with a small volume of hexane. 

Valentine et al. applied, in principle, the method of Midha and Charette for a quantitative assay of quinidines (quinidine and hydroquinidine) in plasma, using methylene chloride instead of benzene for the extraction of a small volume of plasma after basification. To the extract was added the internal standard, cinchonine evaporation to dryness and reconstituting in a methanolic solution of trimethylanilinium hydroxide followed. An aliquot of this solution was analyzed by GLC via on-column methylation reaction. Evaluation of the method over the range 0.5 - 10 ug/ml in human plasma gave a precision and accuracy overall of 4.5 %... 

Because the peak shape of theophylline can be poor in gas chromatography, as also can be the sensitivity of the method, Dusci et al.17 preferred to convert theophylline with on-column methylation by means of trimethylanilinium hydroxide to caffeine. In that manner they... 

Moody RP, Franklin CA, Riedel D, Muir NI, Greenhalgh R, Hladka A. A new GC-on-column methylation procedure for analysis of DTMP ((9,O-dimethyl phosphorothioate) in urine of workers exposed to fenitrothion. J Agric Food Chem 1985 33 464—7. 

An on-column methylation of dimethylphosphorothioate in urine samples used trimethylanilinium hydroxide in acetone (Moody et al. 1985). 

The method (27) describes the measurement of clonidine in human plasma and urine by combined gas chromatography-mass spectrometry with ammonia chemical ionization. Addition of [ 84] clonidine to plasma or urine is followed by ethylacetate extraction of clonidine from alkaline medium, back extraction into acid extraction into ethyl ether from alkaline medium and evaporation of the extract to dryness. Trimethylanilinium hydroxide is added to the residue, and dimethyl derivatives of clonidine are formed by on column methylation with an injection-port temperature of 250 for g.c. -70-eV m.s., the glass column (1.8 m X 2 mm) packed with 3% of OV-17 on Gas-Chrom Q (100 to 120 mesh) is operated at 245 . With He s carrier gas (15 ml min" ) NH3 is admitted to an ion-source pressure of 0.2 Torr, and ions are monitored at m/e 258 and 264. Graphs of peak height ratios (m/e 258 to 264) vs amounts of clonidine in urine (up to 40 ng ml-i) and in plasma (up to 5 ng ml i) are rectilinear. The precision for assay of clonidine in plasma is 11% at 0.25 ng ml" and 5% at 0.5 ng ml" and the lower limit of determination is 0.1 ng ml. ... 

Trialkylanilinium hydroxide 2.13 Af-Alkylation of barbiturates [841, other sedatives, phenolic alkaloids and related substances Convenient flash heater (or on-column) methylation for GLC analysis the injector temp, should be set at 250-300 °C ... 

Another class of esterification reagents are halogenated compounds (alkyl iodides, substituted benzyl, and phena-cyl bromides), which need basic media for their reaction [K2CO3 or DMFA (dimethyl formamide) is normally used for the neutralization of HHal as acid by-product]. For methy-lation of carboxylic acids, some tetra-substituted ammonium hydroxides or halides can be used, e.g., tetramethylammo-nium hydroxide (in aqueous solutions) or trimethylanilinium hydroxide (in methanol solution). The intermediate ammonium carboxylates are thermally unstable and produce methyl alkanoates when the reaction mixtures are heated or when the carboxylates are introduced into the hot injector of the gas chromatograph (flash or on-column methylation) ... 

Multiple Internal Standards in Determination of Antiepileptic Drugs by a Gas Chromatographic on-Column Methylation Technique Antiepileptic Drugs Quant. Anal. Interpret. 1978 35-41 CA 88 130638s... 

Phenytoin, Phenobarbital, Primidone, and Ethosuximide Simultaneous Determination with On-Column Methylation (TMAH) Antiepileptic Drugs Quant. Anal. Interpret. 1978 338-342 CA 88 130642p... 

An Alternative Internal Standard for Analysis of Ethosuximide by On-Column Methylation and Gas Chromatography... 

Use of Trimethylanilinium Hydroxide-Tetramethylaramonium Hydroxide as On-Column Methylating Agent for GLC Analysis of Phenytoin J. Pharm. Sci. 66(10) 1510-1511 (1977) CA 87 193487s... 

The latter authors used trimethyl an ilinium hydroxide for on column methylation of the phenolic group and relied on the trident erome thy 1 ether as internal standard in the last step. 

---