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Anal. Chem. 1985, 57,472-474
Study of the Effect of Decomposition Methods on the Accurate Determination of Zinc in Biological Samples by Electrophoresis J. Y. Yang and M. H. Yang* Institute of Nuclear Science, National Tsing Hua University, Hsinchu, Taiwan 300, Republic of China
S. M. Lin Graduate Institute of Medicine, Kaohsiung Medical College, Kaohsiung, Taiwan 800, Republic of China
A method combining radiotracer techniques with paper electrophoresis to investigate the optimal decomposition conditions for the biological material is developed. After administration of "Zn solution to the tested rats, the livers are removed and decomposed with both wet and dry methods, and the %+containing species in the decomposed samples are finally Investigated by electrophoresis analysis. The results reveal that aside from the =Zn(II) ion, there appears on the electrophoresis histogram also one s5Zn-containingorganic species under certain specific decomposition conditions, indicating the incomplete decomposition of the sample matrix. The fate of the flZntontaining organic species In the course of Separation by solvent extraction is investigated, and the possible connection of decomposition conditions with the analytical errors is also discussed.
In recent years there has been an increasing interest in evaluating the role and/or effect of trace elements in the field of environmental and biomedical research. Often, researchers have compared their results with those of other laboratories and found wide discrepancies that lead to serious confusion about the interpretation of the results ( 1 ) . For example in biomedical research, some investigators consider a serum chromium level of 0.5 ng/mL as the upper limit in normal individuals (2); others accept a value of 5 ng/mL as definite proof of chromium deficiency (3). Furthermore, the values found in apparently healthy subjects in some laboratories may vary considerably (1). The frequent inconsistency in the published data in environmental and biomedical studies is attributed mainly to analytical errors and sample variations used for analysis. In some studies it reveals that the error that arose from the analytical process is more important than that from the variation of sample ( 1 ) . The analytical process most commonly used for the determination of trace elements in biological sample is by digestion (ashing) of the sample matrix followed by direct instrumental determination of the trace elements in the decomposed sample or, alternatively, by separation and/or concentration of the trace elements from the decomposed sample and finally by instrumental determination of the isolated elements. The sources of error most probably encountered in this analytical procedure, aside from systematic errors inherent in the trace analysis, may be due to the matrix effect by the undecomposed organic substance in the instrumental determination step or to the incomplete recovery of trace elements in the preconcentration step from the decomposed sample solution. The decomposition process undoubtedly constitutes an important step which vitally influences the analytical accuracy of trace elements in the biological samples. Though the completeness of destruction of sample matrix by a specific decomposition process may be verified
from the consistent agreement of the analytical results obtained under various decomposition conditions, it is desirable to develop an analytical technique capable of detecting the extent of destruction of sample matrix in order to better assure the accuracy of analysis. The present study is attempted to explore a possible way to find out the species distribution of trace elements in the decomposed sample solution using paper electrophoresis analysis. In order to increase detection sensitivity, the internal radioisotope labeling technique is employed. Rat liver is used as a typical sample for this investigation. After injection of 65Znsolution to the tested rats, the livers are removed and decomposed with both wet and dry methods, and the 65Zncontaining species in the digested samples are finally investigated by electrophoresis analysis. I t seems reasonable to assume that the appearance of only one peak corresponding to the &Zn(II) ion in the electrophoresis histogram may imply total destruction of the sample matrix in the decomposition process, while additional appearance of &Zn-containingspecies other than @Zn(II)ion may therefore indicate incomplete destruction of the sample. The species other than 65Zn(II) ion that appeared on the histogram may be attributed to the undecomposed biological material or possibly to the recombination product of the once-released &Zn(II) ion reacting with the organic residual in the ashed sample. In any case, the existence of any organically bonded species other than 65Zn(II) in the decomposed sample solution might decrease the recovery of Zn in the separation step and thus result in analytical error. EXPERIMENTAL S E C T I O N Labeling with Radioactive @Zn. High specific activity (-80 pCi/pg) of 65Znwas obtained by irradiating Zn metal (50 mg) in the Taiwan Research Reactor at a flux of 2 X 1013g cm-* s-l for 14 days. The irradiated metal was dissolved in concentrated HN03, evaporated to dryness, diluted, and finally adjusted to pH 2 to 3. About 0.1 mL of 65Znsolution which contains about 6 pg of Zn was given intraperitoneally to the tested rats. After 1 day, the animals were killed and the liver samples were dissected for decomposition. Decomposition Procedure. The 65Zn-labeledliver samples were digested with a wet-oxidation method by refluxing with the mixture of concentrated HN03 and H2S0, solution. A Sjostrand type of wet-oxidation reflux apparatus was used ( 4 ) . About 0.3-g sample was put into the digestion flask (150 mL) and a mixture of 3 mL of HNOBand 2 mL of H,SO, was added to start the digestion. In the intial stage reflux was continued with the condensate returning to the flask for about 15 min. Then the tap on the condensate reservoir was closed, and water and HNOB were collected in the condensate reservoir. When a slight darkening of the digest occurred, small amounts of condensate were allowed to drain into the flask t o continue the oxidation and remove the darkening. The operation was continued until darkening no longer occurred and a white fume of sulfur trioxide was observed ( 5 ) . The whole process described above constitutes the one-cycle digestion. Repeat the process two to three times
0003-2700/85/0357-0472$01.50/0 1985 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 57, NO. 2, FEBRUARY 1985
300{
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Distance, cm Figure 1. Electrophoresis histogram of various 65Zn-containingsamples: (a) Zn(I1) ion: (b) wetdigested liver sample; (c) low-temperature ashed liver sample.
constitutes the so-called two-cycle and three-cycle digestion processes as indicated in Figure 2. Dry ashing was performed in the low-temperature oxygen plasma asher (Product of Branson/IPC 100). The 65Zn-labeled samples (each about 0.5 g) were first dried at 105 "C and then placed in the plasma asher for 3 h of ashing. The ashing power was fixed at 80 W which had been previously selected for liver sample (6). The ashed sample was dissolved in 0.1 N "03 and parts of it were adjusted to about pH 6.5 for electrophoresis analysis. Electrophoresis Analytical Procedure. Paper electrophoresis was conducted with a CAMAAG high-voltage electrophoresis apparatus. The decomposed sample solution which had been adjusted t o pH 6.5 was spotted in 50-pL aliquots on 2 X 48 cm strips of Whatman 3 MM filter paper. The paper strips were wetted in the acetate buffer solution (pH 6.5,0.25 M NaOAA.25 M HOAc) prior to electrophoresis and were then placed between two sheets of chemically resistant Plexiglas. The paper tails projected beyond the Plexiglas and dipped into the tanks of electrolyte. A potential of 1000 V was applied for 30 min. The paper strips were then dried and cut into numbered 1-cm pieces. The 65Zn distribution was measured with a well-type NaI(T1) sintillation counter. Solvent Extraction Procedure. Parts of the decomposed sample solutions were subjected to solvent extraction for separating 65Zn(II) using APDC/CHC13 (ammonium pyrrolidinecarbodithioate in chloroform) as extractant at pH between 2 and 3.
RESULTS AND DISCUSSION The trace elements in biological material exist in chemically bonded form. The role of decomposition is primarily for macroscopic destruction of the sample matrix and breaking of the chemical combination of trace elements with the bulk substance. In the completely decomposed sample solution the trace elements should basically be present in free chemical forms which can further be separated from the residual matrix and subsequently determined by instrument. The main interest of the present study is to establish an electrophoresis technique to investigate the species distribution of trace elements in the decomposed samples and to correlate the fraction obtained by solvent extraction using APDC/CHCl, as an extractant. T o begin this study, the paper electrophoretic behavior of Zn(I1) ion is first investigated. Figure 1 shows the histogram
Distance , c m Flgure 2. Electrophoresis histograms of liver samples subjected to varying extents of wet-oxidation digestion.
of Zn(I1) in an acetate buffer solution of p H 6.5. I t is seen that only one peak appeared on the negative side about 8 cm from the zero point. The established electrophoretic condition can be used as a basis for the following study of species distribution of ashed sample. Decomposition processes including wet-oxidation digestion and low-temperature plasma ashing are employed in this study. Both decomposition processes are well-established techniques and are commonly used for the determination of trace elements in biological samples. The typical electrophoresis histograms for the liver samples which are treated by wet-oxidation digestion (one-cycle process) and low-temperatwe plasma ashing (3 h duration) are respectively shown in b and c of Figure 1. I t indicates clearly that aside from a peak corresponding to Zn(II), there appears also a peak, a t about 2 cm on the same side as Zn(II), corresponding to a slightly positively charged species which can be assumed to be an incompletely dissociated Zn-containing species. The presence of positively charged organic species in the decomposed sample may imply the fact that the decomposition conditions used above are not sufficiently effective to destruct completely the liver samples. One question that remained to be clarified is whether the positively charged organic species is really an incompletely decomposed species in the decomposition process or a species resulting from recombination of once-free Zn(I1) ion with the organic residue in the subsequent treatment of decomposed sample solution. In order to verify the origin of this species, an experimental design is made by mixing 65Zn(II)with the decomposed liver sample solution which contains no 65Zn tracer and then subjecting the mixture to electrophoresis analysis. The result so obtained indicates that aside from the peak of &Zn(II) there is no perceptible species corresponding to the peak found as in (b) and (c) of Figure 1. From the experimental fact it can be sure that the slightly positively charged species should result directly from the undecomposed biological material. The results obtained so far indicate clearly the feasibility of using electrophoresis analysis to study the optimal decomposition conditions of the biological sample. Figure 2 shows the results of a typical wet-oxidation process for the liver sample with varying extents of digestion. I t is seen that the percentage yields of "Zn-containing organic species decrease
474
Anal. Chem. 1985, 57,474-481
with increasing the digestion cycle, they are 14.2%, 5.7 %, and 0.8% corresponding respectively to one-cycle, two-cycle, and three-cycle digestion processes. It is of interest to further explore the fate of the undissociate species in the course of separation by the commonly employed solvent extraction method. In this study APDC/CHCl, is used as an extractant aiming to separate Zn(I1) ion from the digested sample solution. Interestingly, the percentage of &Zn extracted into the organic phase very closely agreed with the fraction of 65Zn(II) shown in the histogram of Figure 2, and that remaining in the aqueous phase is found equivalent to the fraction of 65Znorganic species. The percentage of extraction for those three samples with different extents of decomposition is also shown in the same figure for ease of comparison. From the experimental facts it is clear that one of the sources of analytical error is attributed to the incomplete dissociation of sample matrix, as is obvious from the example shown in Figure 2 that about 14% of error might be expected for the one-cycle digested sample due to the apparently low recovery of Zn by the solvent extraction process. The method combining radiotracer techniques with paper electrophoresis provides a unique possibility to study the effectiveness of the decomposition process of biological samples. In view the importance of the decomposition process
in relating to the attainable analytical accuracy, further investigation to explore the behavior of trace elements other than Zn in various biological matrices under different decomposition conditions might prove to be valuable in environmental and biomedical studies.
ACKNOWLEDGMENT This work was supported by a grant from the National Science Council of the Republic of China, to which the authors wish to express their thanks. Registry No. Zn,7440-66-6. LITERATURE CITED (1) Versieck, J.; Cornelis, R. Anal. Chem. 1980, 776,217-254. (2) Seeling, W.; Dolp, R.; Ahnefeld, F. W.; Dick, W. Infusionsther. Klin. Ernaehr.-Forsch. Prax. 1975, 2 , 144. (3) Freund, H.; Atamian, S.;Fisher, J. E. J . A m . M e d . Assoc. 1979, 247, 496. (4) Mercury Analysis Working Party of BITC (Belgium) Anal. Chim. Acta 1970. 8 4 . 231-257. (5) LO, J. M.; We, J. C.; Yang, M. H.; Yeh, S. J. J . Radioanal. Chem. 1982, 72,571-585. (6) Sun, H. J.; Lin, H. M.; Yang, M. H. Chemistry (Taipei) 1984. 42, 51-60.
RECEIVED for review August 20, 1984. Accepted October 17, 1984.
Thermospray Liquid Chromatography/Mass Spectrometry Determination of Drugs and Their Metabolites in Biological Fluids Tom R. Covey, Jonathan B. Crowther, Elizabeth A. Dewey, and Jack D. Henion* Equine Drug Testing and Toxicology Program, N Y S College of Veterinary Medicine, Cornell University, 925 Warren Drive, Ithaca, New York 14850
A revised, simplified thermospray LC/MS probe and ion source is constructed to fit a standard, commercially available GC/MS instrument. This system offers a significant improvement in reliability and versatiitty of the GC/MS because the GC remains interfaced to the MS. The system provides stable ion current profiles and low picogram sensitivity for certain organic compounds. I t is utilized to analyze thermally labile promazine N-oxide and N-oxide sulfoxide metabolites in addition to major urinary imipramine metabolites isolated from alkaline extracts and enzyme hydrolysis extracts of equine urine. The use of short, 3-pm reversed-phase HPLC columns allows rapid thermospray LC/MS analyses of complex biological extracts with abundant molecular weight information. The GC/MS system can be easily converted back to Its conventional operational modes by simply removing the thermospray ion source block.
The on-line combination of high-pressure liquid chromatography (HPLC) with mass spectrometry (MS) to provide routine LC/MS remains a desirable goal. The number of reported new approaches to combined LC/MS continues to increase (1-12), but only a limited number of problem solving applications for LC/MS have been reported. The application
of LC/MS to complex samples containing trace levels of targeted and unknown analytes must be demonstrated on a variety of compound types before the technique is accepted as a useful analytical tool. Thermospray LC/MS offers a new approach to combining conventional HPLC flow rates with MS (11-14). These recent applications of thermospray LC/MS suggest this technique has some unique advantages. These include the ability to (a) introduce up to 2 mL/min of aqueous HPLC eluent into the MS, (b) use HPLC eluents containing high percentages of water, (c) use volatile ionic modifiers in the HPLC eluent, and (d) provide very mild ionization conditions for labile or intractable organic compounds. These features differ somewhat from other approaches to LC/MS which impose limitations upon conventional HPLC with respect to eluent flow rate, water content, and difficulties obtaining molecular weight information from some labile compounds (1-8, 10). In fact, thermospray ionization conditions actually prefer high percentages of water in the HPLC eluent, a volatile modifier such as ammonium acetate, and an eluent flow rate of about 1.5 mL/min (11). We have continued our earlier thermospray LC/MS work (12) in an effort to develop the hardware and the technique to provide routine capability for solving “real world” analytical problems. The modified conventional chemical ionization (CI)
0003-2700/85/0357-0474$01.50/0 1985 American Chemical Society
