Theophylline separation from caffeine

In a further application of MI-SPE, theophylline could be separated from the structurally related caffeine by combining the specific extraction with pulsed elution, resulting in sharp baseline-separated peaks, which on the other hand was not possible when a theophylline imprinted polymer was used as stationary phase for HPLC. A detection limit of 120 ng mb1 was obtained, corresponding to a mass detection limit of only 2.4 ng [45]. This combination of techniques was also used for the determination of nicotine in tobacco. Nicotine is the main alkaloid in tobacco and is the focus of intensive HPLC or GC analyses due to its health risk to active and passive consumers. However, HPLC- and GC-techniques are time-consuming as well as expensive, due to the necessary pre-purification steps required because the sample matrices typically contain many other organic compounds besides nicotine. However, a simple pre-concentration step based on MI-SPE did allow faster determination of nicotine in tobacco samples. Mullett et al. obtained a detection limit of 1.8 jig ml 1 and a mass detection limit of 8.45 ng [95]. All these examples demonstrate the high potential of MI-SPE to become a broadly applicable sample pre-purification tool. 

Separation of theophylline and caffeine by column containing polymer imprinted with theophylline. Caffeine washed right through with CHCI3 solvent. Addition of 20 id. of CH3OH to the solvent breaks hydrogen bonds between theophylline and the polymer and elutes theophylline in a volume of t mL [From W. M. Mulleti and P C. Lai. Anal. Chem. 1998, 70.3636.]... 

FIGURE 6-1. Chromatograms for separation of theophylline and caffeine. Mobile phase 95/5/0.2 water/acetonitrile/glacial acetic acid. Detector 280 nm, 0.02 AUFS. (Reprinted from reference 1 with permission.)... 

Precipitation of the extract is achieved by pressure reduction and/or absorption of the extracted compounds in an absorbing liquid. For the extraction of xanthines (caffeine, theobromine and theophylline) the absorbing liquid is water. The supercritical gas. loaded with the extract, enters the precipitator at the lower end and flows upwards against the absorbing water, which is sprayed from the top of the vessel. The regenerated gas leaves the vessel at the top after passing a liquid/gas separator for removing droplets. 

FIGURE IS Electrochromatograms obtained for the separation of basic drugs spiked in a human serum compared with a blank in a hydrophobic interaction CEC. Column 5 pm 300 A polysulfoethyl A particles, 20 cm packed length, 50 pm ID mobile phase ACN/TEAP buffer (80 20) applied voltage, 10 kV detection at 214 nm. Drugs (I) amobarbital (2) phenobarbital (3) barbital (4) caffeine (5) sulfanilamide (6) theophylline (7) 2,4-dimethylquinoline (8) propranolol. (Reproduced with permission from reference 76.)... 

The adjustment of die pH is critical for the separation of 8-chlorodieophylline. UV detection is at 280 nm. Other xandiines and xandiine metabolites also elute from this system. Retention times relative to theophylline are 3-methylxanthine, 0.49 theobromine, 0.63 paraxanthine, 0.88 caffeine, 1.36 ... 

Figure 18-7. Effect of temperature on the separation of caffeine derivatives on a Hypercarb column (1 mm x 100mm). (a) Column at 100°C, mobile phase acetonitrile (b) Colnmn at 180°C, mobile phase water/acetonitrile 70/30. Samples 1, hypoxantine 2, theobromine 3. theophylline 4, caffeine 5, P-hydroxyethyltheophylline. (Reproduced from reference 35, with permission.)...

Fig. 11.1. Separation of some xanthine derivatives Column pBondapak C18 (300x4 rim ID), mobile phase acetonitrile - 0.01 M sodium acetate buffer (pH 4.0) (7 93), flow rate 2.0 ml/min, detection UV 254 nm. Peaks 1, 1-methyluric acid 2, 3-methyl xanthine 3, 1,3-dimethyl-uric acid 4, theobromine 5, theophylline 6, B-hydroxy-ethyltheophylline 7, phenobarbital 8, caffeine 9, 8--chlorotheophyl1ine. (reproduced with permission from ref. 56, by the courtesy of Clinical Chemistry)...

Xanthines. It was found that the capacity factors and the separation ratios were both functions of the identity of the modifier as well as of the quantity of the modifier in the carbon dioxide. In Figure 6, it can be seen that with 9-5% 2-methoxyethanol in carbon dioxide the first two components of a xanthine mixture, caffeine and theophylline, are not well-separated but the elution order is caffeine, theophylline, theobromine, and xanthine. At 6.5f 2-methoxyethanol in carbon dioxide, there is essentially baseline resolution of the first three components in less than one minute xanthine (not very soluble in liquid 2-methoxyethanol and not at the usual 1 mg/mL sample concentration but at some undetermined saturation concentration) at a k of over 5 has begun to tail and to disappear into the baseline. While it is obvious that the capacity factors, k, increase with a decrease in modifier concentration from 9-5 to 6.5% 2-methoxyethanol in C02, less obviously the separation ratios (for example, with respect to caf-... 

The molecular recognition capabilities of polyelectrolyte multilayers have also been investigated by Laschewesky [55], while imprinted films have been grown on membrane surfaces in approaches similar to the phase inversion method for preparing a membrane imprinted with theophylline. Wang et al. [56] adopted an acrylonitri-le/dithiocarbamoyl-methylstyrene copolymer (Fig. 16) to effect separation of caffeine from the theophylline-imprinted membrane. 

Fig. 3 Hydrophilic interaction CEC separation of basic drugs spiked in human serum. Colunm fused-silica capillary, 27 cm (20 cm packed with 5 p.m, 300 A PolySULFOETHYL A particles). Mobile phase 80% v/v acetonitrile in 100 mM TEAP buffer. TEAP buffer at pH 2.8. Applied voltage 10 kV. Injection 0.5 psi for 90 sec. Temperature 25°C. Detection UV at 214 nm. Concentration of drugs 40 p,g/ml. Solutes 1, amo-barbital 2, phenobarbital 3, barbital 4, caffeine 5, sulfanilamide 6, theophylline 7, 2,4-dimethylquinoline 8, propranolol. Source From Determination of basic pharmaceuticals in human serum by hydrophilic interaction capilary electrochromatography, in Electrophoresis.

Nine xanthines (xanthine, I-, 7-, and 3-methylxanthine, isocaffeine [IS], theobromine, paraxanthine, theophylline, caffeine) were extracted from serum and urine separated on a 32°C Cg column (A = 270 run) using a 20-min 90/10 70/30 water... 

Figure 20 Separation of some xanthines in tea extract by HPLC. 1. theophylline 2, caffeine 3, theobromine each 1 pg/10 pi eluent flow rate, 0.75 cm3/min. (a) Using direct injection sample volume, 10 pi. (b) Using preseparation in a tank and analyte transfer by means of the OPLC interface sample volume 10 pi, applied on to the layer, developed twice with ethyl acetate prior to analysis. (Reproduced by permission from Ref. 71.)...

Figures Chromatograms on MIL-53(Al)-packed column (7-cm longx4.6-mm i.d.) for RP HPLC separation using CHjCN/HjO as mobile phase at l.OmLmin b (a) ethylbenzene and toluene (b) PAHs (c) thiourea (1), phenol (2), aniline (3), benzaldehyde (4), bromobenzene (5), naphthalene (6) (d) o-benzenediol, m-benzenediol, and p-benzenediol (e) aniline, N,N-dimethylaniline, and m-nitroaniline (f) xanthine, theophylline, and caffeine. Mobile phase composition (CHjCN/HjO, v/v) (a) 7 3 (b) 10 0 (c) 6 4 (d) 1 9 (e) 9 1 (f) 5 5. UV detection at 210 run (a, c), 256 run (b), 70 run (f), and 280 nm (d, e). (Reproduced from Ref 69 with permission of The Royal Society of Chemistry. DOI 10.1039/C2AN15925B.)...

FIGURE 17 Reversed-phase separation of beverage additives on 10-fjim C-18 silica (Partisil-10 ODS-2). Column 4.6 mm x 25 cm, mobile phase 50 50 methanol-water, flow rate 0.6 ml/min., pressure 529 psi, UV detection at 254 nm. Peaks (a) Saccharin, (b) Theobromine, (c) Theophylline, and (d) Caffeine. [Reprinted by permission from Whatman, Inc.]... 

Important kinetic differences and variations in the quantitative as well as qualitative metabolic profiles have been shown between species, thus making extrapolation from one species to another very difficult. All of the metabolic transformations include multiple and separate pathways with demethylation to dimethyl- and monomethylxanthines, formation of dimethyl- and monomethylurates, and ring opening yielding substituted diaminouracils (Figure 2). The reverse biotransformation of theophylline to caffeine is demonstrated not only in infants but also in adults. 

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