Barbiturates, labelled

Cais, who coined the term metalloimmunoassay, prepared a series of organometallic markers (metallohaptens) with the potential for use in immunoassay. These are derivatives of steroids (estradiol, estriol), mood-altering drugs (amphetamine and cocaine) or medications (morphine and barbiturates), labeled with ferrocene, cobaltocenium and cymantrene [57-59]. He then assessed the use of a Sepharose 4B column to separate the free and bound fractions of the tracer and quantification of the tracer by atomic absorption spectrometry [58]. 

Part—VI has been solely devoted to Miscellaneous Assay Methods wherein radioimmunoassay (RIA) (Chapter 32) has been discussed extensively. Various arms of theoretical aspects viz., hapten determinants and purity importance of antigenic determinants and analysis of competitive antibody binding of isotopically labeled compounds. The applications of RIA in pharmaceutical analysis, such as morphine, hydromorphone and hydrocordone in human plasma clonazepam, flurazepam in human plasma chlordiazepoxide in plasma barbiturates, flunisolide in human plasma have been described elaborately. Lastly, the novel applications of RIA-techniques, combined RIA-technique-isotope dilution and stereospecificity have also been included to highlight the importance of RIA in the analytical armamentarium. 

Hulshoff, A., Roseboom, H., and Renema, J., Improved detectability of barbiturates in high-performance liquid chromatography by pre-column labelling and ultraviolet detection, J. Chromatogr., 186, 535, 1979. 

The sorption efficiency of MC was determined as the ratio of the quantity of the adsorbed substance to its initial amount (w / w), expressed in % for a certain ratio (w / w) of adsorbent to substance. Optimal ratios of adsorbent to substance equal 15-20 for barbiturates, 20 - 25 for cyanocobalamin and bilirubin, and 40 - 50 for hemoglobin. The initial concentration of absorbed substances was 100 - 200 pg/ml. The substances were incubated for lmin with MC either in physiological solution or in donor plasma and donor blood at room temperature (pH 7.4). The concentration of substances in the solutions was measured by differential visual and UV-spectroscopy. Concentrations of substances in blood and plasma and adsorption of total plasma proteins was determined by thin-layer chromatography with a fluorescent label. 

Optical purity of barbituric acid enantiomers was determined by the isotope dilution method using 2-14C-labeled compounds 215 or by an NMR... 

L-[ N]Alanine has been administered to rabbits, which were killed 5 min after injection. Pancreas/liver concentration ratios of 1.8 and 2.3 were reported for barbiturate-anesthetized and unanesthetized animals, respectively. The kidneys contained about 20% higher concentrations than did the liver (45). Label content of the liver itself was not reported. In gamma camera studies of hmnans, imaging of the heart and pancreas was reported with l-[ N]alanine (46). l-[ N]alanine has also been used to image the human myocardium and pancreas by positron emission tomography (47). It should be noted, however, that radioactivity derived from dl-[1- C] alanine did not localize in either organ in human subjects (48). 

N-Chloromethylphthalimide (ClMPI), N-chloromethyl-4-nitrophthalimide (ClMNPl) and N-chloromethylisatin (ClMIS) react quantitatively with fatty acids, dicarboxylic acids and barbiturates (Figure 11) [105]. The reactivity of these labels is due to the high mobility of the chlorine atom. This reaction is similar to those with phenacyl bromide. For a complete reaction it is necessary to convert the acids to alkali metal or ammonium salts. Triethylamine or a crown ether is used as catalyst. Aprotic solvents such as acetonitrile, methanol and diethyl ether are suitable reaction media. The reaction is complete within 30—40 min at 60 °C. The disadvantage of these labels is reactivity to alcohols and primary and secondary amines, and as a result the selectivity is limited. HPLC separation of phthalimidomethyl esters was performed on a reversed phase column (Cg) with acetonitrile/water in various proportions as the mobile phase. Detection was at 254 nm. 

The electrophoretic mobilities of C-labeled cholic, deoxycholic, and chenodeoxycholic acid and their corresponding taurine and glycine conjugates were determined by Norman (42). The paper electrophoresis was performed in barbiturate buffer of ionic strength 0.1, pH 8.6, in an electric field of 7.5 V/cm for 3 hr. When 1 pg of each acid, as the sodium salt dissolved in 25 pi of water, was applied to the paper strips, the isotope determination after electrophoresis showed broad peaks all with a mean mobility similar to that of albumin or slightly lower. The electrophoretic mobilities of all of the bile acids were influenced by the concentration in the solution applied and presented difficulties in identifying bile acids in natural extracts. The migration of bile salt-lecithin micelles on paper electrophoresis has been reported by Shimura (43). The micelles were prepared by addition of lecithin to mixed bile salts, which may have also contained cholesterol. 

Labeling of phenobarbital, a barbiturate used in the treatment of convulsions, was carried out by acylation of p-aminophenobarbital 15 by the acid chloride of cymantrene 11 (Scheme 8.8). Synthesis of 15 was performed by nitration of the benzene ring of phenobarbital [35] to give a mixture of m- andp-nitrophenobarbital 13 and 14 followed by reduction of p-nitrophenobarbital by hydrogenation in the presence of palladium on charcoal [36]. 

Forensically, these drugs are encountered as diverted pharmaceuticals, simplifying the analysis. In such cases, and in any case in which commercial labeling or marking remains, the first step is usually a check of the Physician s Desk Reference (PDR) to determine the likely identity and constituents of the preparation. Rather than identifying a complete unknown, the task then becomes one of confirmation with instnimental techniques. In effect, the PDR becomes a presumptive test. While there are cases in which commercial preparations are faked, such cases are rare. Fortunately, abuse of these new-generation medications is far less prevalent than was abuse of earlier medications, such as the barbiturates. 

Further labeled pyrimidines such as [2- C]barbituric acid (22, R, R = H) and its derivatives such as [2- C]barbital (22 R R = Et), and [2- C]pentobarbital (22 R = Et, R = 1-methylbutyl) are readily available from cyclocondensation of [ CJurea and the respective diethyl malonates in the presence of sodium ethoxide or butoxide. For phenobarbital (R = Et, R = Phe), decarboxylation of the malonate component was observed as a main side reaction, so that the respective malonyl dichloride had to be employed in order to achieve satisfactory yields. Analogous syntheses using unlabeled urea and labeled malonate derivatives are described in Chapter 6, Section 6.5. 

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