of uneven distribution of sample material on the electrode and the constant change of electrode configuration resulting from the movement of the sample electrode during sparking. The precision for this method is normally *25 to 35%. Method B should be a very useful method for quickly surveying elements in biological materials, solutions, and water. The method described can be considered quite specific for bromine- or fluorine-containing pesticide compounds. This is true because the hexane extract which has been backwashed with water would contain only organic pesticides and certain nonhalogenated natural organic compounds from soil. Furthermore, of the total number of compounds in agricultural use there are very few pesticides and herbicides that contain bromine and fluorine. Thus, a knowledge of the spray history of the biological sample to be analyzed would permit the analyst to attribute the bromine or fluorine found to those few specific compound(s) which may have been applied. Although many chlorinated pesticides might be present in the sample, they would not interfere.
Attempts were made to analyze chlorinated and iodinated herbicides in soil by the same technique. Unfortunately, the background for chlorine is usually too high for meaningful results. At present the detection limit is about 1 ppm, even though the sensitivity is below 0.1 ppb. For iodinecontaining herbicides, difficulties were encountered due t o light sensitivity of some of the compounds and vaporization losses in the vaccum. If the iodine compounds can be stabilized under vacuum, the sensitivity should be below 0.1 PPb. ACKNOWLEDGMENT The authors thank R. J. Hicks for his technical assistance and Corning Glass Works for use of their mass spectrometer for this study. RECEIVED for review October 12, 1971. Accepted December 22, 1971.
Comparison of Cell Extraction Procedures for Use with High Pressure Liquid Chromatography P. R. Brown and R. P. Miech Section of Biochemical Pharmacology, Dicision of Biological and Medical Sciences, Brown University, Procidence, R.I. 02912 USEOF HIGH PRESSURE liquid chromatography for determining free nucleotides in tissues ( I , 2) has made it necessary to reinvestigate the various procedures used in extracting free nucleotides from cells. Excellent reviews have been written on extraction procedures (3-5), but because of the high resolving power and high sensitivity of the nucleic acid analyzers, special care must be taken so that the extraction techniques will not cause sizeable errors in the quantitative results. An analysis by high pressure liquid chromatography can be made in a fairly short time. Therefore, metabolic, pharmacological, and clinical studies of the changes in naturally occurring nucleotides or the formation of various metabolites are amenable to analysis. Since a large number of samples are usually involved, speed of preparation as well as complete extraction and accuracy are important. The ease of preparation of extracts is essential if this method is to be used clinically. Each additional step can compound errors and the simplicity of procedure lessens the probability of inaccuracies. Another factor to be considered is the concentration of the nucleotides in solution since a very small volume (1-20 pl) is used on the analyzer. In addition, the solutions must be stable on storage since a backlog of samples may develop with the routine use of a high pressure liquid chromatographic system. Standard solutions of nucleotides or nucleotide pools extracted from cells using the trichloro(1) C. G. Horvath, B. A. Preiss. and S. R. Lipsky, ANAL.CHEM., 39, 1422 (1967). f2) P. R. Brown. J . Chromatoar.. 52, 257 (1970). (3j W. C. Hutchinson and H-N. Munro, Analyst, 86, 768 (1961); 87,303 (1962). (4) H. N. Munro and A. Fleck, Methods Biochem. Anal., 14, 113 (1964). (5) P. Mandel, Progr. Nucl. Acid Res. Mol. Biol., 3, 305 (1964). .
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acetic acid-ether procedure showed little deterioration when stored a t a neutral p H a t - 20 or - 80 "C for two months (6). The following extraction procedures were considered : Perchloric acid (PCA) extraction followed by neutralization with potassium hydroxide. Perchloric acid extraction followed by neutralization with potassium hydroxide, lyophilization, and the dissolving of the dried sample in a minimum volume of solvent. Trichloracetic acid (TCA) extraction with the acidic sample used directly on the analyzer. Trichloracetic acid extraction followed by the extraction of the TCA with water saturated diethyl ether. Trichloracetic acid extraction followed by neutralization with tris (hydroxymethy1)aminomethane (TRIS) to p H 7.5. Since the PCA extraction procedures did not meet the criteria for the ease and simplicity of preparation, special studies were not carried out on these extraction techniques at this time. Both involved several steps and the second necessitated the use of a lyophylizer which is not available in every laboratory. Preliminary studies, however, showed that the PCA extraction procedures gave results comparable to those obtained with TCA. In the TCA extraction procedures, 1 ml of freshly drawn, heparinized, human whole blood was vortexed with 2 ml of cold 12% TCA as previously described (7) to ensure complete extraction. The TCA extract obtained after centrifugation in the cold was immediately subjected to three different procedures before analysis by high pressure liquid chromatog( 6 ) P. R. Brown, Anal. Biochem., 43, 305 (1971). (7) R. P. Miech and M. C. Tung, Biochem. Med., 4,435 (1970).
Table I. Comparison of Extraction Procedures of ADP and ATP from Whole Blood. Extraction Procedure ADP ATP 0.35 0.86 TCA TCA extracted with EtzO 0.27 0.87 0.23 0.88 TCA TRIS a Concentrations of ADP and ATP in millimoles of nucleotide/ liter packed erythrocytes. Nucleotide analysis: instrument, Varian LCS 1000; column, pellicular anion exchange resin, 1 X 300 mm; eluents, 0.015M KHzPOa, and 0.25M KHzPO~ in 2.2M KC1; flow rates, 12 ml/hr, 6 ml/hr; UV output, 0.08 O.D. unit; sample size, 20 pl.
+
Table 11. Effect of Storage of TCA Extract Which Was Not Neutralized. Time of storage ADP ATP 0 0.35 0.86 1 week 0.26 0.55 1 month 0.67 0.31 See footnote a, Table I, for conditions.
raphy. One aliquot of the TCA extract was untreated (I); one was extracted three times with five volumes of water saturated diethyl ether (11), and one was neutralized by the addition of solid TRIS (111). The total time that elapsed from withdrawal of the blood sample through the initial analysis by high pressure liquid chromatography did not exceed eight hours for all three aliquots. Aliquots of the three solutions were stored at -20 "C and were analyzed after one week and one month. Aliquots from solution I11 were also analyzed at intervals up to four months. Solution I was discarded because of the obvious deterioration of the nucleotides. The quantity of adenine nucleotides extracted was not dependent on the extraction procedure if the analysis were carried out immediately (Table I). However, the initial levels of adenine nucleotides changed dramatically when the acidic solutions were not neutralized even though they were stored at -20 "C (Table 11). With the ether extraction of TCA, this deterioration was not seen (6). However, the ether extraction of the TCA was time consuming. In addition, any change in the sample volume
Table 111. Effect of Storage on TRIS Neutralized TCA Extract Time of storage ADP ATP 0.23 0.88 0 1 week 0.21 0.89 1 month 0.23 0.86 4 months 0.27 0.84 See footnote a, Table I, for conditions.
during the ether extraction can cause the quantitative results to vary from 10-20z. Unfortunately, the accuracy of the results obtained using this extraction procedure is very dependent on a technician's knowledge of the method and skill in carrying it out. For example, failure to saturate the ether solution or to remove the last traces of ether may cause volume changes. On the other hand, errors also can be caused by removal of cell extract solution along with the ether. Therefore, if the volume is increased because excess water from the ether solution has been added to the extract, the quantitative results are low. If the volume is decreased, the results, in all probability, are high. Moreover, even if the volume remains the same, it is difficult to determine whether any solute was removed during the extraction procedure and was replaced by water from tho ether solution. According to the Varian Manual and our experience, the coefficient of variation for sampling, instrument, and calculation errors is approximately 2%. When the TCA solutions were neutralized with solid TRIS, the change in concentration on storage for a month was within this 2% coefficient of variation. When the adenine nucleotide levels of samples stored for four months were compared to initial values, only a 4 % change was detected (Table 111). Since this method offers the advantages of simplicity, rapidity, and stability of samples, it best meets the criteria for sample preparation prior to analysis by high pressure liquid chromatography. It must be noted, however, that for this procedure to be successful the TCA must be free of UV-absorbing impurities. RECEIVED for review October 20, 1971. Accepted December 27, 1971. This work has been supported by Grant No. GM 16538 from the United States Public Health Service and by Grant No, GB 5990 from the National Science Foundation.
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