Anal. Chem. 1982, 54, 2612-2613
2812
TO
THERMOCOUPLE GAUGE
DIFFUSION PUMP
7,( ),
1
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through the volumes for 30 min is sufficient to eliminate measurable isotopic inhomogeneities in the system. For preparation of hydrogen and nitrogen gases it is desirable that both the volume of the manifold and the volume between each tube and its associated stopcock (item D in Figure 1) be as small as possible. Hydrogen and nitrogen are sealed in 9-mm 0.d. glass tubes with a hand torch without freezing or sorbing the gas. This line has now been used to prepare hydrogen, nitrogen, and carbon dioxide reference samples that are isotopically homogeneous as measured by state-of-the-art isotope ratio mass spectrometry.
1 ~
l l
ACKNOWLEDGMENT
I '
We thank John Laughlin, Jessica Hopple, Vicki Ober, Judy Rowe, and Freddie Leighty for sample preparation and analysis of these reference samples. We appreciate the reviews of this work by our colleagues I. Lynus Barnes and P. Morales.
d -
TOTAL OF 20 RESERVOIRS AND GAS EXTRACTORS
LITERATURE CITED
Glass vacuum preparatlon line for preparation of homogeneous carbon dioxide reference samples: (A) Hoke 500-cm3stainless steel sampling cylinder with a burst pressure of at least 13 MPa; (B) Nupro 4BK-series stainless steel valve with KeCF stem tip; (C) 5 0 k m 3 ghss sampling volumes (total of 20); (D) 100-cm3glass volumes (total of 20); (E) size 7 Ace-Thread connector with FETFE O-ring (Ace Glass Inc., Vlneland, NJ) for 6-mm 0.d. tubing or size 11 Ace-thread connector for 9-mm 0.d. tubing (CaJonO-ring Ultra-torr unions are also satisfactory); (F) 6"or 6%" ('I4 In.) glass tube about 40 cm long. Figure 1.
volumes was 1%. Consequently, subliming the carbon dioxide in the stainless steel cylinder is a critical step in preparing homogeneous reference samples. Although an extra expense is involved, some users may wish to install a stainless steel bellows pump in the system to cycle gas through it. Then additional stopcocks and interconnections are needed so that all 500-cm3 volumes can be placed in series with each other and with the pump. Cycling gas
Gonflantlnl, R. Nature (London) 1079, 271, 534-536. Cralg, H.; Gordon, L. I. In "Stable Isotopes In Ocenaographlc Studies and Paleotemperatures"; Tonglorgl, E., Ed.; V. Lischl: Plsa, 1965; pp 9-130. Coplen, T. B. Anal. Chem. 1981, 53, 940-942. Des Marais, D. J.; Hayes, J. M. Anal. Chem. 1976, 4 8 , 1651-1652. Coleman, D. D. Anal. Chem. 1981, 53, 1962-1963. Coplen, T. B. Int. J . Mass Spectrom. Ion Phys. 1973, 1 1 , 37-40. Friedman, I.; O'Neil, J. R. Geol. Surv. Prof. Pap. ( U . S ) 1977, 440KK, 12 p. Craig, H. Geochlm. Cosmochim. Acta 1957, 12, 133-149. Mook, W. G.; Grootes, P. M. Int. J . Mass Spectrom. Ion Phys. 1973, 72, 273-298. Coplen, T. 8.; Kendall. C.; Hopple, J., submitted for publlcatlon in Nature (London).
RECEIVED for review July 13, 1982. Accepted September 3, 1982. Use of trade names and trademarks in this publication is for descriptive purposes only and does not constitute endorsement by the U.S. Geological Survey.
Elution of Disposable Octadecylsilane Cartridges with Hydrophobic Organic Solvents Sharon L. Pallante," Martln Stognlew,' Michael Colvln, and Danlel J. Llberato DepaHment of Pharmacology and Experimental Therapeutics, Johns Hopkins School of Medicine, 7 2 5 North Wolfe Street, Baltimore, Maryland 2 1205
The determination of various xenobiotic substances in biological fluids or other complex matrices usually involves protein precipitation, filtration, and solvent extraction in order to prepare the samples for analysis by either spectrometric or chromatographic techniques. These cleanup procedures are required to remove a multitude of interfering compounds. The removal of these interfering substances is essential in order to obtain a pure sample by subsequent procedures or, if quantitative studies are required, a signal-to-noise ratio of sufficient intensity to ensure accurate and reproducible measurements. Recently, prepacked octadelylsilane columns or cartridges (ODSC) have seen wide use in sample cleanup. Organic substances are adsorbed onto the ODSC from biological fluids and subsequently recovered by elution with water or mixtures of water and water misible solvents (e.g., methanol and acetonitrile) (1). In use, the ODS column or cartridge has already proven its superiority over liquid-liquid extraction for the removal of organic salts and sugars from biological samples. Present address: Pharmaceutical Division, Ciba-Geigy Suffern, NY 10901.
Corp.,
0003-2700/82/0354-2612$01.25/0
While this procedure is extremely useful and provides mixtures of organic substances free of the biological matrix, it would be highly desirable to have a technique which would also remove the less polar components prior to the more polar ones and thus provide partial purification as well as recovery from the biological or environmental matrix. In the process of isolating polar conjugates of drugs from urine, blood, and enzymatic incubations (all of which contain compounds with a broad range of solubilities), we devised such a procedure based on the use of the ODS cartridges. This system involves aqueous adsorption onto the ODSC followed by selective removal of compounds with a water immiscible organic solvent. An elution with ether, for example, is capable of removing those ether soluble compounds which have, in the past, been eliminated only by liquid-liquid extraction. In conventional ODSC procedures (elution with water-MeOH) these same compounds have coeluted in our glucuronide-containing methanol fraction. This technique therefore has the advantage of combining filtration, extraction, and adsorption into one step resulting in faster, less expensive, and in some cases a more efficient cleanup method. The method will be illustrated here by the 0 1982 American Chemical Soclety
ANALYTICAL CHEMISTRY, VOL. 54, NO. 14, DECEMBER 1982
2613
Thin-layer chromatographic analysis was performed on E. Merck Silica Gel 60 F254 plates, 20 X 5 cm. The solvent system was butano1:benzene:water:methanol (200:100:100125). Glucuronides were visualized on TLC by reaction with naphthoresorcinol as previously described (2). The HPLC analysis was performed on an Altex HPLC using a Zorbax 250 X 4.6 mm ODS (Du Pont, Wilmington, DE) column with a solvent system of acetonitri1e:water:acetic acid (200:792:8). The flow rate was 1 mL/min and the detector wavelength was 280 nm.
1
ODSC-MeOH
11
0.016+
Omin
5
IO
15
20
Flgure 1. A comparison of the HPLC chromatograms from the parallel ODSC-extraction chloramphenicol experiment: detector at 280 nm, flow rate 1 mL/min; injection volumes = 10 pL wlth changing absorbance ranges as noted; (a) chloramphenicol glucuronide, (b) un-
identified metabolite, (c) chloramphenicol. Total sample volumes are equal. purification of several milligrams of chloramphenicol glucuronide produced in vitro by enzymatic synthesis. We have also successfully applied this technique to the analysis of the hypocholesterolamic drug clofibrate and its metabolites in urine. EXPERIMENTAL SECTION Use of Disposable Cartridge (ODSC). Disposable cartridges (Sep-PAK CI8,Waters Associates, Inc., Milford, MA) were washed with diethyl ether (3 mL), imethanol(3 mL), and water (10 mL). Aqueous samples of urine, plasma, water, or phosphate buffer, pH 7.8,O.l M, ranging in volume from 25 to 100 mL dependent upon the amount of metabolite present, were passed through the prewashed ODSC with a syringe at a flow rate of 1 drop/s. A 30 mL distilled water wash at the same flow rate was applied to remove salts and any water-soluble compounds present and then the cartridges were dried by forcing air through them with a syringe until no further water was removed. Diethyl ether (3-6 mL) and Baker Analyzed HPLC grade methanol (3-6 mL) were used as elution solvents and collected separately. (Other i m miscible organic solvents such as ethyl acetate may be used in place of ether if necessary.) These samples were dried under a stream of nitrogen and analyzed in an appropriate manner. Synthesis of Chloramphenicol Glucuronide. Chloramphenicol glucuronide was synthesized with glucuronosyl transferase immobilized on Sepharose-a technique previously described (2). Chloramphenicol (24 mg), immobilized enzyme (15 mL), and uridine-5’-diphosphoglucuronicacid (UDPGA. NH3.2H20,56 mg) were incubated in phosphate buffer pH 7.8 overnight at room temperature. The Sepharose-bound enzyme was removed by filtration. Workup A. Half of t h e aqueous phase was extracted with diethyl ether (3 X volume) followed by acidification to pH 3 and extraction with 1-butanol (3 X volume). The organic extracts were evaporated under reduced pressure and analyzed by HPLC and TLC. Workup B. The remaiining half of the aqueous phase was treated as described under “Use of Disposable ODSC”. The ether and methanol eluates were dried under a stream of nitrogen and analyzed by HPLC and TIE.
RESULTS AND DISCUSSION The separations of chloramphenicol from chloramphenicol glucuronide using both the liquid-liquid extraction method and the ODSC adsorption procedure were quite similar (Figure 1). A comparison of the two methods by HPLC showed the ether extract and the ODSC ether eluent both containing virtually all of the chloramphenicol (ODSC ether 95%, ether extract 98%). The butanol extract and the ODSC methanol eluent both contained the glucuronide metabolite, but the sample in the methanol eluent was much cleaner and has a glucuronide recovery twice that of the butanol extraction. The adsorption of the chloramphenicol glucuronide on the ODSC and its subsequent elution were unaffected by the pH of the aqueous phase, whereas extraction of the same glucuronide by 1-butanol was accomplished only after acidification of the aqueous phase to pH 3 with 12 N HC1. This ODSC method can therefore be an advantage when working with acid-labile compounds. The volumes of ether and the final elution solvent from the cartridge method are quite small and can be rapidly dried with nitrogen, totally eliminating the need for large volumes of extracting solvents, evaporation under reduced pressure, and sample transfer where often significant sample losses occur. In addition, thin-layer chromatography showed the presence of glucuronic acid and other W active materials (not detected by HPLC) in the butanol extract which are absent in the ODSC methanol eluent. The literature procedure for purification of clofibrate and its metabolites (3) required an acidification and ether extraction of the parent drug clofibrate prior to purification of the metabolites by HPLC. A parallel experiment involving an ether elution from an ODS cartridge led to HPLC chromatograms very similar to those obtained by the liquid-liquid literature technique. After removal of clofibrate by the ether wash, the cartridge was eluted with acetonitrile to separate a conjugated clofibrate metabolite. The use of water immiscible organic solvents to elute material from an ODS cartridge or column thus seems to mimic liquid-liquid extractions but has the added advantages of neutral pH, reduced analysis time, obviated need for copious amounts of potentially dangerous solvents, improved purification, and increased flexibility. ACKNOWLEDGMENT We are very grateful to Catherine Fenselau for support and guidance. LITERATURE C I T E D (1) Shackleton, C. H. I..; Whitney, J. 0. Clh. Chim. Acta 1980, 107,
231-243. (2) Fenselau, C. C.;Pallante, S. L.; Parikh, I . J . Med. Chem. 1976, 19, 879-883. (3) Veenendaal, J. R.; Meffin, P. J. J . Cbromatogr. 1981, 223,147-154.
RECEIVED for review August 2,1982. Accepted September 16, 1982. This work was funded in part by a grant from the National Institutes of Health, Grant No. GM21248.
