Caffeine assay

There are numerous methods in the literature for the determination of caffeine, theobromine, and theophylline in food matrices, including coffee, tea, and cocoa. Until recently, methods have emphasized the determination of the major methylxanthines in a commodity, for example, caffeine in coffee or theobromine in cocoa. Present methods range from being specific for one of the compounds in a single matrix to being an all-encompassing assay of major and minor methylxanthines in food products. 

HPLC allows a quantitative determination with relatively simple extractions. In many cases, extraction only involves a heating of the commodity with water, followed by filtration and injection onto an HPLC column. In the determination of caffeine, theobromine, and theophylline in cocoa, coffee, or tea, as well as in other foods, there is scarcely a month that passes without a new paper on this assay. Kreiser and Martin provide typical conditions for analysis.28 In their studies, samples were extracted in boiling water and filtered prior to injection onto the HPLC column. The HPLC conditions used a Bondapak reversed phase column and a mobile phase of water methanol acetic acid (74 25 1) with detection at 280 nm. This method is accurate, precise, and conserves time. It has also been adopted by the AOAC as an official method for the determination of theobromine and caffeine in cocoa beans and chocolate products.29... 

Kali, M.A. and Clausen, J. (1995) Dietary effect on mixed function P450 1A2 activity assayed by estimation of caffeine metabolism in man. Human and Experimental Toxicology, 14 (10),... 

The suggested derivative spectrophotometric method and PLS-1 were applied to determirration of caffeine in energy drirrks (Table 31.2). The assay results obtained by both methods were statistically compared at the 5% level. As shown in Table 31.2 there was no sigrrifrcant differences between the mean values and precisiorrs of two methods. 

After terminating the coupling reaction with ascorbic acid, reaction mixture clarified by precipitating protein with ethanol Turbidity decreased by final addition of caffein, sodium benzoate, and Teepol micromethod After the coupling reaction with ascorbic acid is terminated, azo color is extracted and read low blank the extract can be applied directly to a thin-layer plate and be separated rapidly to determine the ratio BMC BDC Micro versions assay artificially activated enzyme... 

A variety of xanthines including caffeine, theobromine, and theophylline have been found from food materials including.coffee, chocolate, and tea (419-420). Theophylline determination in sera has been much studied. The technique allows the determination of theophylline at serum levels of 1.5-20 mg/liter theophylline with sample sizes ranging from 50 to 10 /xl (42 -425). Hill (426) assayed theophylline using 50 /xl of serum and an analysis time of 8 min with good interbatch precision and accuracy. Alternative methods which allow the determination of as little as 0.1 mg/ml (427) or 20 ng theophylline in 10 ml serum have been described (428). 

Affinity of MIP towards the target analyte should be examined prior to fabrication of the chemosensor. Batch binding assays are used to test selectivity of suitable MIPs. Especially, affinity of MIP to compounds, which are structurally related to the target analyte, should be tested. If MIP binds similarly with these compounds as the template, then cross-reactivity is manifested [156], This effect was exploited for determination of adenine and its derivatives with the use of MIP templated with 9-ethyladenine. Nevertheless, the cross-reactivity, if undesired, can be avoided by suitable sample pretreatment, e.g. by interferant extraction with a supported liquid membrane (SLM) coupled to the MIP-PZ chemosensor. The Fluoropore membrane filter of submicrometre porosity can serve that purpose. That way, this membrane holds interferants, thus eliminating the matrix effect. The SLM-involving determination procedure is cheaper than traditional laborious sample pretreatment used to remove the interfering substances. For instance, caffeine [143] and vanillin [157] in food samples have been determined using this procedure. 

LAI E P C, FARFARA A, VANDERNOOT V A, KONO M and POLSKY B (1998), Surface plasmon resonance sensors using molecularly imprinted polymers for sorbent assay theophylline, caffeine and xanthan , Can J Chem, 76, 265. 

Crowther and Henion have reported the SFC assay of polar drugs on packed columns using mass spectrometric detection [10]. Their method was shown to be suitable for a multicomponent mixtures containing nonpolar and polar analytes. They mentioned that one of the advantages of SFC versus HPLC was the faster column reequilibration time. In this method, silica, amino, nitrile, and diol packed columns (20 cm x 2.1 mm ID) were used. Cocaine, codeine, caffeine, methocarbamol, phenylbutazone and ox-yphenbutazone were separated on one or more of these columns and mass spectra were obtained. 

X. G. Fang, P. Shen, and S. Ghodbane, Mixed ion pair liquid chromatography method for the simultaneous assay of ascorbic acid, caffeine, chlorpheniramine maleate, dextromethorphan HBr monohydrate and paracetamol in Frenadol sachets, J. Pharm. Biomed. Anal., 72 85 (1994). 

S. L. Yang, L. O. Wilken, and C. R. Clark, A high performance liquid chromatographic method for the simultaneous assay of aspirin, caffeine, dihydrocodeine bitratrate, and promethazine hydrochloride, Drug Dev. Ind. Pharm., 77 799(1985). 

Kalow W, Tang BK. The use of caffeine for enzyme assays a critical appraisal. Clin Pharmacol Ther 1993 53 503-514. 

There are two commonly used and robust methods for phenotyping. The first one measures caffeine (1,3,7-methylxanthine) and its N-demethylated metabolite 1,7-dimethylxanthine (paraxanthine) in a plasma or saliva sample collected within 5 to 7 hours post caffeine dosing (Fuhr and Rost 1996). The second one uses the assay of the metabolites 1-methylurate... 

Commonly used methods for caffeine and metabolite ) assay in plasma or urine involve an extraction step followed by HPLC with UV detection (Krul and Hageman 1998a Rasmussen and Bosen 1996 Schreiber-Deturmeny and Bruguerolle 1996). Urine needs to be acidified (pH 3.0-3.5) before sample freezing. 

Recent drug assay development involved LC-MS methods (Caubet et al. 2004 Kanazawa et al. 2000). A less practical breath test, using 13C or 14C labeled caffeine, can also be used (Kalow and Tang 1991). 

The most common methods to assay caffeine and its metabolite in urine used HPLC with UV detection (Grant 1984 Krul 1998b) or mass spectrometry (Baud-Camus 2001). 

The urinary caffeine test is not based on assays of specific substrates and products of NAT2 ( including other metabolism pathways involving at least xanthine-oxidases), and is affected by diet habits, xanthine-oxidase inhibitors such as allopurinol (Fuchs 1999), or other drugs (Klebovitch 1995). NAT activities are affected by anti-inflammatory drugs. Of note, acetominophen is an inhibitor of NAT2 in vivo (Rothen 1998). 

The antioxidant activity of individual tea polyphenols in different model assays showed a proportional relationship to the number of hydrogen radical donors of catechins. A synergistic effect was observed between tea catechins and caffeine, ascorbic, citric, malic, and tartaric acids and tocopherols (153). Formation of oxidation products of (+)-catechin dirring the antioxidative process has been observed in oxidation model studies. According to the proposed mechanism, (+)-catechin can scavenge four radicals per molecule (154, 155). Yamamoto et al. (156) have summarized the chemistry and application aspects of green tea, especially in relation to using their catechins. 

GC-MS methods provide greater specificity and in many cases sensitivity when compared with more conventional techniques. They offer increased scope for the study of pharmacokinetics and of plasma concentration in relation to biological effect. SIM assay has been applied to the investigation of placental transfer of lipid soluble drugs and their subsequent elimination in the newborn (barbiturates, diphenylhydantoin, caffeine, pethidine and diazepam [122,408] diphenylhydantoin [411] amylobarbitone and 3 -hydroxyamylobarbitone [83,423]). 

For the assay of adenylate cyclase activity of washed dog heart particles, a medium has been employed which contained 40 mM Tris or 20 mM potassium phosphate buffer (pH 7.5) with 4 mM ATP, 6.6 mM magnesium sulphate, 13 mM caffeine, and 2-5 ixglml of bovine serum albumin in a volume of 1.8 ml [95]. The reaction was stopped by heating the vessels for 3 min in a boiling water bath. After centrifugation, the supernatant fluid was assayed for cyclic AMP by the dog liver phosphorylase-activation method. 

As with DNA aptamers, there is a trend towards applying RNA aptamers to a particular application. Allosteric ribozyme sensors have been developed which are specific for caffeine and aspartame. " Using a fluorescence-based assay, caffeine or aspartame may be detected in solution over a 0.5 5mM concentration range. Aptamers designed to malachite green (151) or other triphenylmethane dyes have been developed that enhance the fluorescence of the dye up to 2300-fold. " A further fluorescence-based assay has been... 

Kobayashi T, Murawaki Y, Reddy PS, Abe M, and Fujii N. Molecular imprinting of caffeine and its recognition assay by quartz-crystal microbalance. Anal. Chim. Acta 2001 435 141-149. 

Content Uniformity/Assay. The question most often asked is when NIR will be able to be used as a release test. In the earliest NIR assays, tablets and capsules were not analyzed intact. Prior to scanning, the active was extracted and scanned in a clear liquid. The first use of NIR for tablets was reported in 1968. Sherken assayed the meprobamate content in tablet mixtures and commercial preparations. Allen used NIR to analyze a three-component mix carisoprodol, phenacetin, and caffeine. The powder was extracted with chloroform and scanned in the NIR. Several other publications took advantage of the dissolve and scan approach.t ... 

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