thin-layer chromatographic method for the determination of theophylline in human plasma

Kurze Mitteilungen 239

metriert. Die Konzentration der zwei Farbstoffzwischenpro- dukte wird anhand yon aufgestellten Eiehkurven ermittelt.

Ergebnisse. Die Trennung erfolgt am besten mit einer mobilen Phase bestehend aus i-Octanol-Methanol-l,5 %igem w~iBri- gem Ammoniak (30: 17:5). Nach 4 h erscheinen die Flecken durch eine kaum verf'firbte 2 - 3 mm breite Zone klar ge- trennt. Methanol kann durch Athanol ersetzt werden, das Ammoniak bis zu 4,5 % ohne merkliche ,gmderung erh6ht werden. Mehr Methanol ergibt h6here RrWerte aber zum Schaden der Trennsch/irfe. H6here aliphatische Alkohole st6ren.

Mit n-Octanol steigen die Rf-Werte etwas an, die Flecken werden aber breiter und folgen unmittelbar nacheinander, wobei AAS (Aminoanthrachinonsulfons~iure) am besten durch ihre UV-Fluorescenz differenziert werden kann. Buta- nol, n-Amyl- und n-Hexylalkohol verhalten sich ~ihnlich.

In 0,2M NaHCO 3 weisen AAS und ABAS (Aminobrom- anthrachinonsulfons~iure) Absorptionsmaxima bei 470 bzw. 485nm auf. Das Lambert-Beer-Gesetz wird bis zu 50 bzw. 100 ~tg/ml erfiillt. Die Lichtabsorption steigt linear mit der Konzentration gem/iB ~lg/ml = k �9 m. Die entsprechenden Steilheitsfaktoren betragen k47o = 61,1 bzw. k48 s = 66,7.

Einige Kontrollanalysen sind in Tabelle 1 angef/Jhrt. Nach Entwickeln und Eluieren wurden 9 0 - 9 5 % AAS

bzw. 8 5 - 89 % ABAS wiedergefunden.

Tabelle 1

Ein- Gefunden waage

~tg Rf m lag %

AAS 90,7 0,26 0,270 82,5 91 ABAS 45,6 0,35 0,120 40 87

AAS 134,9 0,27 0,420 128 95 ABAS 27,4 0,36 0,071 23,7 86

AAS 122 0,30 0,377 115 94 ABAS 45,6 0,38 0,122 40,7 89

AAS 88,5 0,28 0,260 79,4 90 ABAS 136,8 0,36 0,350 116,7 85

Literatur

1. Gemzovfi, I., Gasparic, J. : Collect. Czechoslov. Chem. Commun. 34, 3075 (1969)

2. Takumitsu, T., Hara, T., Hayashi, T. : Kogyo Kagaku Zasshi 67, 197 (1964)

Eingegangen am 20. Dezember 1977

Fresenius Z. Anal. Chem. 292, 239-240 (1978) - �9 by Springer-Verlag 1978

Thin-Layer Chromatographic Method for the Determination of Theophylline in Human Plasma

H. Roseboom*, H. Lingeman, and G. Wiese

Vakgroep Analytische Farmacie, Farmaceutisch Laboratorium der Rijksunjversiteit, Catharijnesingel 60, Utrecht, The Netherlands

DUnnsehieht-ehromatographisehes Verfahren zur Bestimmung yon Theophyllin im menschlichen Plasma

Key words: Best. von Theophyllin in Plasma; Chromatogra- phic, Dtinnschicht/Spektralphotometrie

Theophylline (1,3-dimethylxanthine) is one of the numerous drugs for which monitoring drug plasma concentrations is necessary to ensure effective and safe therapy. Several methods for its determination in biological fluids have been described. The classical spectrophotometric method [3] has been largely replaced by gas-chromatographic [2,4] and H.P.L.C. [1,5] techniques, because of potential interferences from other drugs and theophylline metabolites. A thin-layer chromatographic method has been described [6] but it has the disadvantage, that it needs an expensive spectrodensitometer, which is not generally available.

* Address for correspondence

This paper describes a simple, specific method for the quantitative determination of theophylline in human plasma. It consists of extraction of the drug from plasma and isolation by TLC. After that the spots are cut out from the plate and eluted with 0.1 N sodium hydroxide solution and the absorbance of the alkaline solution is measured at 275 nm.

Experimental

Reagents. Theophylline, theobromine and caffeine were ob- tained from Brocacef(Maarssen, Holland). 3-methylxanthine was obtained from Aldrich-Europe (Janssen Pharmaceutica, Beerse, Belgium). All other reagents and solvents were reagent grade (E. Merck, Darmstadt, G.F.R.).

Apparatus. A Perkin-Elmer 139 UV-Vis spectrophotometer was used. Thin-layer chromatography was carried out on precoated silica gel 60 F254 plastic sheets (Merck). Samples were spotted onto the plates using an automatic spotter (Kontes Chromaflex Spotter, van Oortmerssen, Holland). Phaseseparating paper (Whatman 1 PS) was obtained from Tamson (Zoetermeer, Holland).

Standard Solutions. Theophylline was dissolved in chloro- form to a concentration of 1 mg/ml. Plasma standards were prepared by adding small volumes of this solution to 3 ml of plasma, just prior to extraction.

Procedure. To 3 ml of plasma is added 0.5 ml 0.5 N hydrochloric acid and 20 ml of a chloroform-isopropanol mixture (95 + 5 by volume). After shaking for 1 min the phases are separated by means of filtration through phase separating paper and the walls of the tube are rinsed twice

240 Fresenius Z. Anal. Chem., Band 292 (1978)

with an additional 2 ml of chloroform. The organic phase is evaporated under nitrogen in a metal heating block at 60 ~ C to a final volume of 0.5 ml. This solution is spotted quantitative- ly onto a thin-layer plate, using the automatic spotter. This procedure allows to spot six samples on one 20 x 20 cm plate. After equilibrating the plate during 10 min the chromatogram is developed over a distance of 15 cm using ethylacetate- methanol-ammonia (80 + 20 + 15) as a solvent. The spots are visualized under UV-light (254 nm), cut out and transferred into a small centrifuge tube. To this tube is added 3 ml of 0.1 N sodium hydroxide solution and after shaking and centrifuga- tion the absorbance of the alkaline solution is measured in a I cm cell at 275 nm.

Results and Discussion

Thin-layer chromatography of the methylxanthines in diffe- rent s~stems revealed that a good separation is obtained between theophylline, its metabolites and the dietary xanthi- nes caffeine and theobromine on silica gel using ethylacetate- methanol-ammonia (80 + 20 + 15) as a solvent (Table 1). Uric acid was also spotted because some of the uric acids are theophylline metabolites and their UV spectra are similar to that of theophylline. TLC of blank plasma showed no spots with a Rf-value similar to that of theophylline.

Recovery studies showed that with the extraction proce- dure described 90 + 2 % of the theophylline is extracted, the

Table 1. Rf-values of methylxanthines and uric acid on silicagel Solvent: ethylacetate-methanol-6 N ammonia (80 +20+15)

Compound R f-value

Caffeine 0.75 Theobromine 0.62 Theophylline 0.43 3-Methylxanthine 0.19 Uric acid 0.00

recovery of the chromatographic procedure is 97 _+ 1.5 %. A calibration curve was made by adding known amounts of theophylline to blank plasma and treating these samples according to the procedure described. This procedure proved to be linear from 1 - 20 gg/ml with a standard deviation of 0.3 gg/ml over the whole range. Plasma samples obtained from donors who had not abstained from coffee et cetera showed blank values of up to 5 gg/ml when treated according to the Schack and Waxler procedure, the blank values for the same samples were below 0.3 lag/ml when treated as described above.

The use of the automatic spotter allows the application and separation of six samples simultaneously, so that a single technician can easily handle up to 24 samples a day, which is quite comparable to G. C. or L. C. methods. The biggest advantage of the method is its low cost and ease of operation with generally available equipment.

By using spectrophotometric cells with a pathlength of 10mm and a volume of 1 ml the sensitivity of the method can be easily increased or the volume of plasma needed decreased by a factor of 3. In that case less than I ml of sodium hydro- xide solution is sufficient to elute the theophylline from the chromatogram.

References

1. Franconi, L. C., Hawk, G. L., Sandmann, B. J., Haney, W. G.: Anal. Chem. 48, 372 (1976)

2. Lear, E., Perrier, D.: Clin. Chem. 22, 898 (1976) 3. Schack, J. A., Waxler, S. H. : J. Pharmacol. Exp. Ther. 97,

283 (1949) 4. Shah, V. P., Riegelman, S. : J. Pharm. Sci. 63, 1283 (1974) 5. Thompson, R. D., Nagasawa, H. T., Jenne, J. W. : J. Lab.

Clin. Chem. 84, 584 (1974) 6. Wesley-Hadzija, B., Mattocks, A. M.: J. Chromatogr.

115, 501 (1975)

Received March 5, 1978

Fresenius Z. Anal. Chem. 292, 240 (1978) - �9 by Springer-Verlag 1978

Use of Molecular Sieve 3A as Adsorbent for Water in Elemental Analysis

S. S. Roy and Kashmir Singh

Indian Institute of Petroleum, Dehradun 248005, India

Verwendung von Molekularsieb 3A als Wasseradsorbens in der Elementaranalyse

Key words: Verw. von Molekularsieben; Elementaranalyse; Sieb 3A, Wasseradsorbens

Various substances have been described in literature as water adsorbents for the hydrogen determination in organic corn-

pounds by the combustion method. All these reagents do not permit regeneration and are uneconomically in that regard. Therefore, we looked for a new reagent and found molecular sieve 3A to be most satisfactory. Sieves of other diameters were not sufficient enough and only the 3A sieve did adsorb water alone and not carbon dioxide. We used the material supplied by Associate Cement Company of India, 3/6 pellet size, sieved to 14-22mesh. The material was activated at 250~ in a current of dry nitrogen and normally about 50 determinations were safely carried out without regeneration. Tests with the regenerated substance proved that there was no change in accuracy. Hydrogen (and carbon) determinations were carried out in a variety of different organic compounds. Standard deviations did not exceed 0.07 70 for H contents between 4 and 15 %.

Received January 12, 1978; revised March 22, 1978