Barbiturates derivatization

Fernandez Garcia-Borregon, R, Lores, M., and Cela, R., Analysis of barbiturates by micro-high-performance liquid chromatography with post-column photochemical derivatization, /. Chromatogr. A, 870 (1 2), 39, 2000. 

Greeley, D.H., New approach to derivatization and gas chromatographic analysis of barbiturates, Clin. Chem., 20,192,1974. 

Barbiturates were among the first classes of chiral compounds investigated in the early stages of the application of macrocyclic antibiotics as chiral selectors they were screened on a vancomycin CSP under RP [7] and NP mode systems [157], and on 3,5-dimethylphenyl-derivatized vancomycin CSP [7],... 

This technique can be also applied when the water-miscible acetonitrile is added to the serum sample for extracting the analytes. Following the addition of acetonitrile, the solution is saturated with potassium chloride so that the water and acetonitrile phases separate and derivatization is performed in the acetonitrile layer. Such alkylating procedures have been applied for prechromatographic derivatization of various drug residues with carboxylic groups, including penicillins (252) and barbiturates (253). 

The GC of barbiturates has made significant progress since Janak first tried 800°C pyrolysis in 1960 (33). The newer liquid phases have reduced problems with tailing and adsorption generally associated with the gas chromatographic analysis of the barbiturates. These phases allow the separation of the barbiturates without derivatization, at concentrations of 0.1 pg/yl. 

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). 

The fluorimetric determination of barbiturates has been examined by derivatization with DNS-C1 followed by HPLC [125]. The derivatives are formed under similar conditions to those used for the alkaloids. Therapeutic amounts (1.0 ppm) of barbiturates can be detected in as little as 20 Ail of blood. 

For the preparation of derivatives on a micro scale (at the microgram and picogram levels) reaction with methyl iodide was recommended [510] 100—200 /il of an acetone solution of barbituric acids was mixed with a 3- to 1000-fold molar excess of methyl iodide and refluxed with 3—5 mg of potassium carbonate. After about 30 min, 0.2 /A of the supernatant was injected directly into a column with 3% of OV-225, with temperature programming at 8°C/min from 100 to 240°C. The advantage of the method is that no toxic and hazardous chemicals are used. The method was further modified for the micro-determination of barbiturates in blood [511]. Only 20-25 /d of blood were necessary for the determination, and with the use of a detector sensitive to nitrogen down to 1 ng of barbiturate could be determined in this sample. The derivatization reaction was performed in a micro-refluxer (see Fig. 2.6). 

Due to their highly polar nature, barbiturates require derivatization prior to analysis by GC-MS. In such cases, the sample is dissolved in methanol, centrifnged, the supernatant recovered and placed in a derivatizing vial and is then blown down under nitrogen. The derivatization procednre, using 0.2 M trimethylanilin-ium hydroxide in methanol, is, in principle, the same as that used for other pre-column derivatizations. However, with this system the reaction does not occnr immediately because insufficient activation energy is available at ambient temperature for this to take place. Direct transfer of the reaction mixture onto the heated injection block of the gas chromatograph overcomes this problem and the derivatization reaction can then proceed. Snch a reaction, an example of flash alkylation is illustrated in Scheme 9.1. 

In some cases, the forensic scientist will be required to quantify barbiturates and benzodiazepines in drug samples. Due to the problems associated with sample derivatization, quantification of barbiturates can best be achieved by using HPLC. Benzodiazepines are thermally labile compounds and thus HPLC is again the method of choice for qualitative analysis. 

Gas chromatographic methods have been developed to solve this problem. Theophylline has been gas chromatographed and determined as such, but in most cases derivatization has been carried out to obtain better gas chromatographic properties and better detection possibilities. For quantitative work several substances have been used as internal standard, mainly barbiturates (alphenal, amobarbital, aprobarbital, heptabarbital, hexabarbital, thiobarbital), but also other substances have been used (fluoranthene, codeine, theobromine, medazepam, pramoxine hydrochloride, cyheptamine, probenecid propylester). Recently 3-isobutyl-1-methyl... 

The substance is dissolved in a 0.2 M alcoholic solution of trimethylanilinium hydroxide (1 5 w/v for the effect of concentration of the derivatizing agent upon the quantitation of barbiturates see Note and Ref. 22) and, after 2 min, the solution is injected into the gas chromatography for on-column reaction and GC analysis. 

Proper selection of derivatizing agents can also help determine the molecular weight of a compound. Compared to parent compounds, TMS derivatives of N-substituted barbiturates are found to generate less olefin radical elimination ([M-41]" and [M-55] ). Instead, the formation of the [M-15]ion is favored, making it easier to recognize the molecular weight of the compound under examination. 

Figure 8 Two-component gel based on complementary hydrogen-bond interactions between derivatized barbituric acid (2) and pyrimidine (3) building blocks.

Glass Capillary Column Gas Chromatography of Barbiturates After Flash-Heater Derivatization with... 

Zhao, H. Wang, L. Qiu, Y. Zhou, Z. Zhong, W. Li, X. (2007). Multiwalled carbon nanotubes as a solid-phase extraction adsorbent for the determination of three barbiturates in pork by ion trap gas chromatography-tandem mass sp>ectrometiy (GG/MS/MS) following microwave assisted derivatization. Analytica Chimica Acta,... 

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