Enzymic Digestion–High-pressure Homogenization Prior to Slurry Introduction Electrothermal Atomic Absorption Spectrometry for the Determination of Selenium in Plant and Animal Tissues Yanxi Tan and William D. Marshall* Department of Food Science and Agricultural Chemistry, Macdonald Campus of McGill, 21 111 Lakeshore Road, Ste.-Anne-de-Bellevue, Qu�ebec, Canada H9X 3V9. E-mail: marshall@agradm.lan.mcgill.ca Homogenization, in combination with partial enzymic digestion with a crude protease alone or admixed with lipase or cellulase, was investigated as a means of releasing Se residues from zoological and botanical matrices prior to slurry introduction ETAAS.Preliminary timed trials with two zoological certified reference materials (CRMs), one botanical CRM and one animal feed indicated that Se release became quantitative with 4–8 h of digestion, that homogenization prior to digestion increased the initial rate of analyte release, but that homogenization post-digestion and immediately prior to ETAAS did not significantly improve the precision of replicate digestions.Storage of the crude botanical digests at 4 °C for 5 d resulted in quantitative recoveries of Se from each of the digests. Storage at 4 °C for 10 d of 4 and 8 h lipase/protease digests of six other CRMs resulted in quantitative recoveries of Se unless their certified concentrations were appreciably less than the levels determined in control digests containing the enzyme(s) alone.Apparently, Se residues were transferred virtually quantitatively to the liquid phase of the digested suspension and showed no tendency to segregate during the 10 d of storage. Eight other mixtures of ground plant matter (0.13 @ [Se] @ 1.31 mg g21), formulated as animal feed supplements, behaved identically when stored post-digestion. The technique was also applied successfully to freeze-dried fresh and boiled fish tissues The principal advantages of the enzymic digestion procedure are its simplicity and lack of operator intervention.Keywords: Selenium determination; enzymic digestion; slurry introduction electrothermal atomic absorption spectrometry; botanical and zoological certified reference materials Conventional sample preparation of biological materials prior to atomic spectrometry involves complete solubilization of the analyte and matrix, which is achieved typically by oxidative mineralization of the organic matter and solubilization of the residue in a suitable solvent.1–4 Even for microwave-assisted digestions, whereas complete dissolution can usually be achieved by a suitable choice of digestion conditions, complete decomposition of the organic matrix in biological/botanical samples is appreciably more difficult.Often complete mineralization is achieved only with supplemental treatment of the digested matrix with H2O2 or even HF.5 These digestion procedures can be labour intensive, time consuming and prone to contamination errors.In consequence, there is a continuing interest in the development of simplified sample preparation techniques. The preparation/introduction of slurried samples continues to attract considerable attention because of the ease with which quasi-stable preparations can be generated and their compatibility with conventional liquid handling techniques. Within the general field of solid sampling analysis,6–11 it is the use of slurried samples which has become the most popular approach to trace element determination.Direct atomization from the solid state can provide excellent sensitivity, but the interpretation of results can be complicated by molecular absorptions and/or scattering from the matrix, which can produce sufficiently large background signals to overwhelm the compensation capabilities of common deuterium background correction systems. Additional difficulties can include sample inhomogeneities, the requirement for repeated microweighings and the lack of suitable calibration standards and techniques.A variety of sample pre-treatment procedures and additives12 –18 have been described and evaluated for the production of quasi-stable suspensions of samples prior to analysis by atomic spectrometry. Alternatively, suspensions with a tendency to segment rapidly have been reproducibly sampled by using ultrasonic agitation,19 air or argon20 bubbling, vortex mixing21 or magnetic stirring.22 Partial digestion procedures to produce carbonaceous slurries have also been successfully applied to the analysis, by ICP-AES, of a series of standard reference materials of biological origin.23 Various alkylammonium hydroxide formulations have been used extensively to solubilize tissues,24–27 particularly those of zoological origin. Recently, high-pressure homogenization has been evaluated for the preparation of quasi-stable dispersions suitable for FTIR28 or ETAAS.29,30 The advantages of this approach to sample preparation were the ease and speed of the slurry preparation, which required less than 1 min, and the fact that analyte metals were quantitatively extracted into the liquid phase during the preparation so that no analyte segmentation was detected within the slurry even after standing for several days.Certified reference materials (CRMs), frozen liver and kidney and dried animal feeds of botanical origin were analysed successfully for Cd, Cr, Cu, Mn, Ni and Pb but not for Se.The principal limitation of the high-pressure homogenization technique was the amounts of contaminating analyte metals introduced into the sample by the homogenization operation. Contamination was reduced appreciably, but not eliminated entirely, by capping the flat face of the stainless-steel homogenizing valve with a ruby disc.30 The objectives of this work were (i) to evaluate the efficiencies of other materials as caps to reduce further the levels of contamination introduced by the high-pressure processing and (ii) to develop efficient alternative slurry preparation techniques for the determination of Se in biological materials.Although there have been few reports of the determination of Se in slurried samples,31–34 recent reports35,36 indicate that the approach is promising for this analyte. Prolonged enzymic digestion with a crude protease fraction has been used37 to liberate component selenoamino acids from proteins.This approach seemed promising as a pre-homogenization sample preparation. Analyst, January 1997, Vol. 122 (13–18) 13Experimental Reagents TRIS was purchased from Aldrich (Milwaukee, WI, USA) and aqueous Se solution (1000 mg ml21, traceable to NIST primary standard) was purchased from SCP Chemical (St.-Laurent, Qu�ebec, Canada). Samples CRMs were purchased from the National Research Council of Canada (NRCC) or the US National Institute of Science and Technology (NIST).Samples of animal diet mixtures destined for a zoo were chosen to contain a variety of plant and animal materials, including timothy grass, bamboo leaves, whole smelts, cricket chow and a mixture (contents unspecified) formulated for panda bears. Animal feed supplements were composed of mixed forage crops. Sample Preparation An accurately weighed sample (approximately 0.2 g) of CRM, dried feed or supplements (ground, to pass a 0.5 mm screen, in a Tecator Cyclotec sample mill; Tecator, H�ogan�as, Sweden) was added directly to 10 ml of ethanol–0.03 m TRIS (1 + 19 v/v, pH 7.5) containing either 20 mg of crude protease (Type XIV; Sigma St.Louis, MO, USA), 20 mg of protease plus 20 mg of lipase (Type VII; Sigma) or 20 mg protease plus 20 mg of cellulase (Cellulysin; Calbiochem–Novabiochem, La Jolla, CA, USA). The resulting suspension was then processed through the 20 ml capacity flat valve homogenizer (EmulsiFlex Model EFB3; Avestin, Ottawa, ON, Canada), capable of developing 138 MPa when provided with compressed air (689.4 kPa).Each slurried sample was re-processed through the homogenizer three more times. The homogenates, in 50 ml Erlenmeyer flasks, were then digested at 37 °C with gentle agitation for 4–8 h. Homogenizer The valve stem of the screw-cap assembly of the homogenizer was modified by gluing a polished 4 mm diameter 32 mm thick disctured from a 6–12 mm diameter sphere of tungsten carbide (Spex, Metuchen, NJ, USA), zirconia (Optimize Technologies, Portland, OR, USA), sapphire (from an HPLC piston) or polymethacrylate (Spex).Sample + enzyme suspension was transferred to the sample chamber via the inlet port, which was then sealed with a fine-threaded screw-cap. The stainless-steel piston (connected to a pneumatic multiplier) then forced the fluid through an aperture and the homogenate was collected from the sample outlet. Each sample was re-processed three more times with the valve stem retracted slightly to provide a slightly larger gap setting.Selenium Determinations (Hydride Generation or Fluorescence) Feed samples were dried to constant mass and ground to pass a 1 mm screen. Accurately weighed aliquots of ground feed or freeze-dried fish tissue (approximately 2 g) were digested at room temperature in a perchlorate fume-hood with 25 ml of 70% HNO3–HClO4 (4 + 1 v/v) until gas evolution had ceased, then heated at 80 °C until a clear yellow solution was obtained.The resulting strong acid digests were analysed by HGAAS38 or fluorescence of the piazselenol derivative39 after conversion of the analyte residues in to SeIV. ETAAS Selenium determinations were performed using a hot injection technique on a Varian (Palo Alto, CA, USA) Model 300 ETAAS system equipped with an autosampler, pyrolytic graphite-coated platform graphite tubes, a conventional Se hollow-cathode lamp and Zeeman-effect background correction.Ashing–atomization curves were generated for Se standard in the presence and absence of co-injected biological sample. At temperatures < 2300 °C, the Se atomization signal was broadened by the presence of biological materials but was sharpened (and did not tail) for atomizations at 2400 °C. In the presence of the palladium–citric acid modifier, no loss in the Se signal was observed at an ashing temperature @1400 °C. Analytical operating parameters are presented in Table 1.Calibration ETAAS quantification was performed by both the method of external standards (ES) and by standard additions (SA). ES consisting of appropriately diluted processed reagent blank and up to four levels of standard were prepared automatically by the sample introduction device. The background-corrected peakarea response, resulting from three replicate injections of each diluted standard, was used to define the best-fit regression equation. For SA calibrations, 10 ml aliquots of processed fluid were amended with 2, 5 or 10 ml of aqueous standard chosen to result in a range of peak areas including signals which were half and at least twice the signal for the unamended sample.The data were modelled by least-squares linear regression. Quantification was performed by dividing the intercept on the ordinate of the regression equation by the slope of the equation and the overall standard error of the estimate (SEest) was calculated from SEest = (SE2 y-int + SE2 slope)1/2 Results and Discussion Preliminary experiments were directed to evaluating the influence of different capping materials on the levels of contaminating metals introduced into the homogenates during processing. It was postulated that exposed stainless-steel surfaces within the valve homogenizer, particularly the flat face of the demountable valve head, were the principle sites responsible for the contamination.Further, capping the valve head with an inert surface capable of withstanding the impact of the jet of fluid exiting the homogenizing orifice might reduce the levels of contamination appreciably.It has been reported40 previously that zirconium oxide beads used to reduce the particle size and to mix particulate solids introduced appreciable levels of Fe, Cr and Al but that silicon nitride or boron carbide provides good abrasion resistance and offers little likelihood of Table 1 Furnace operating parameters for determinations of selenium Parameter* Value Wavelength/nm 196.0 Lamp current/A 10 Slit width/nm 1.0 Injection temperature/°C 60 Pre-injection Yes Temperature of last dry step (10 s)/°C 250 Charring sequence 10 s ramp to 1400 °C, 40 s hold Cooling None Atomization 0.6 s ramp to 2400 °C, 5.0 s hold Measurement time/s 5.6 Chemical modifier 5 ml (0.5% m/m Pd + 2.5% m/m critic acid) for 10 ml sample * Each step of the furnace programme (with the exception of the read step) was performed in the presence of argon purge gas (3 l min21). 14 Analyst, January 1997, Vol. 122contamination. However, even for the relatively lower pressure requirements of pistons and check valves for HPLC, sapphire, ruby and zirconium oxide are preferred over other ceramic materials for their superior resistance. Separate discs composed of zirconia, tungsten carbide or polymethacrylate, which had been manufactured by grinding and polishing a 6–12 mm diameter grinding ball, were glued temporarily to the flat face of the demountable valve head.Similarly, the sapphire disc was generated from a used HPLC piston. Solvent mixture (20 ml) was homogenized four successive times (in the presence or absence of the test disc) prior to ETAAS analysis for Al, As, Cd, Cr, Cu, Fe, Mn, Ni, Pb and Se. Analyte concentrations (Table 2) were expressed as if the solvent had contained 0.100 g of sample. The heavy metal content of the homogenized fluid was lowered appreciably in all cases.Nonetheless, contamination remained appreciable for several elements, even in the presence of the polymethacrylate or the sapphire cap. Previous attempts to determine Se in biological materials by slurry introduction ETAAS of high-pressure homogenates were not successful in our hands using a variety of furnace programmes, yet there was no evidence of any analyte loss prior to the atomization stage as judged by the signal graphics software, which provided a continuous display of the Se signal over the course of the furnace programme. Since a high proportion of the analyte element in biological materials is considered to be protein bound, it was decided to evaluate partial enzymic hydrolysis as a means of liberating bound analyte residues.Arbitrarily, it had been decided initially to attempt to develop a single combination of mixed enzymes which it was hoped would be applicable to all sample matrices. Previous studies41 had indicated that a combination of crude proteases and lipases efficiently hydrolysed avian egg yolk.Initial studies were limited to this combination of enzymes. The mixture of crude enzymes was suspended in 10 ml of TRIS buffer (pH 7.5), then passed sequentially four times through the polymethacrylate-capped homogenizer to furnish a digestion control homogenate. Relative to a distilled, de-ionized water blank, this ‘zoological’ control sample contained 0.11 mg g21 ± 12.6% and 0.10 mg g21 ± 11.3% after 4 and 8 h of digestion respectively (Table 3), when it was assumed that the digests had contained 0.200 g of sample.Similarly, a control homogenate composed of protease alone contained 0.044 mg g21 ± 12.6% after 4 h. A crude cellulase was substituted for the lipase in the enzyme mixture and the digestions were performed in analogous fashion to furnish alternative enzymic digestion control samples. The ‘botanical’ control samples contained 0.048 mg g21 ± 14.2% and 0.051 mg g21 ± 12.0% after 4 and 8 h of digestion respectively (Table 3), and the solvent blank + protease alone contained 0.044 mg g21 ± 12.6%, again assuming that the digests had contained 0.200 g of sample.Thus, virtually all of the Se in control digests originated with the lipase and/or the protease. Based on a 3 RSD criterion, the corresponding method limit of detection (LOD) for digestions with mixed protease lipase, with protease cellulase and with protease alone were 0.020, 0.010 and 0.010 mg g21, respectively.In preliminary experiments, three biological CRMs and one animal feed, suspended in 10 ml TRIS buffer, were digested with a combination of protease and lipase for up to 16 h at 37 °C. Table 2 Apparent analyte concentrations (mg g21 sample) in 20 ml of solvent mixture following various mixing treatments. Concentrations in the homogenized fluid are expressed as if the solvent mixture had contained 0.100 g of sample Treatment Al As Cd Cr Cu Fe Pb Mn Ni Se Unhomogenized solvent blank 0.32 n.d.* n.d.n.d. n.d. 0.40 n.d. n.d. n.d. n.d. Four successive homogenizations with: Stainless-steel head 42.12 0.02 4.53 15.0 56.94 1.38 2.31 3.57 n.d. Polymethacrylate cap 7.92 n.d. 0.02 1.80 n.d. 5.20 n.d. 0.10 n.d. n.d. Ruby cap 21.52 n.d. 0.03 4.02 0.70 13.99 0.28 0.39 0.11 n.d. Sapphire cap 3.64 n.d. n.d. 3.65 0.80 4.90 n.d. 0.10 n.d. n.d. Tungsten carbide cap 15.32 n.d. 0.04 4.40 1.40 38.6 2.00 0.40 0.10 n.d. Zirconia cap 0.42 n.d. 0.02 4.00 1.20 15.7 n.d. 0.20 0.10 n.d. * n.d. = None detected above the mean background signal for ethanol–0.03 m TRIS (1 + 19 v/v, pH 7.5) solvent. Table 3 Selenium concentrations (mg g21) (±1 RSD based on three replicate samples) in certified reference materials determined immediately after 4 or 8 h of enzymic digestion or following digestion plus 10 d of storage 4 h digestion + 8 h digestion + Certified Matrix 4 h digestion 8 h digestion 10 d storage 10 d storage concentration Solvent blank + protease + lipase 0.11 ± 12.6% 0.10 ± 11.3% 0.12 ± 10.2% 0.10 ± 13.6% Solvent blank + protease 0.044 ± 12.6% Solvent blank + protease + cellulase 0.048 ± 14.2% 0.051 ± 12.0% 0.040 ± 23.2% 0.045 ± 12.2% Oyster Tissue* 2.18 ± 12.7% 2.27 ± 11.6% 2.16 ± 11.4% 2.15 ± 12.6% 2.21 ± 0.24 DORM-2* 1.38 ± 9.2% 1.35 ± 11.6% 1.34 ± 6.6% 1.33 ± 11.0% 1.40 ± 0.090 Bovine Muscle* 0.067 ± 22.6% 0.066 ± 20.5% 0.067 ± 20.2% 0.070 ± 21.4% 0.076 ± 0.010 Apple Leaves† 0.043 ± 12.7% 0.045‡ ± 19.9% 0.047 ± 12.7% 0.041 ± 12.7% 0.050 ± 0.009 Corn Bran† 0.034‡ ± 12.5% 0.036 ± 14.9% 0.033 ± 15.3% 0.036 ± 16.5% 0.045 ± 0.008 Corn Stalk† n.d.§ n.d.§ 0.025‡ ± 47.0% 0.011‡ ± 52.2% 0.016 ± 0.008 * Reported concentrations have been corrected for the [Se] in the protease + lipase control homogenate.† Reported concentrations have been corrected for the [Se] in the protease + cellulase control homogenate. ‡ No [Se] above the LOD (0.010 mg g21) was detected in one of the three aliquots. § No [Se] above the LOD (0.010 mg g21) was detected in any of the three aliquots.Analyst, January 1997, Vol. 122 15Each suspension was homogenized immediately prior or directly after digestion, then analysed by ETAAS. The results are presented in Figs. 1 and 2. The TORT-1 results and the animal feed results (triangular symbols in Fig. 1 and Fig. 2, respectively) have been displaced by 0.4 h for clarity of presentation. For all four substrates, homogenization prior to digestion (closed symbols) generally resulted in higher recoveries relative to homogenization post-digestion (open symbols), although the differences were only rarely statistically significant.Moreover, the differences tended to decrease with longer digestion times. Presumably, homogenization initially exposed more of the protein component to the enzyme. On the other hand, homogenization post-digestion and immediately prior to ETAAS did not significantly improve the precision of the determination (as judged by the RSD associated with three replicate measurements performed on each of three digests). In general, there was a gradual but small improvement in precision with increased length of digestion (more evident with the plant samples in Fig. 2). For both the DORM-1 and the TORT-1 marine tissue samples in Fig. 1, 4 h of digestion at 37 °C were sufficient to liberate the Se quantitatively, whereas recoveries from the plant samples were quantitative only after 8 h of digestion.After sampling for ETAAS, the plant digests were stored at 4 °C for 5 d and then re-analysed. No effort was made to resuspend solid materials; instead, a portion of each supernatant fraction was transferred directly to the sampling cup. The recovery of Se from each of the supernatant fractions was quantitative (Fig. 3), indicating that the crude protease was active at the storage temperature and that there was no apparent segmentation of the Se residues between the liquid and solid phases of the crude digest.Surprisingly, the short-term repeatability of the procedure was not improved by the storage, as evidenced by the RSD associated with replicates. Repeatability continued to be improved for longer digestions at 37 °C. Three replicate aliquots of each of six other certified reference materials were homogenized and then digested for 4 or 8 h prior to ETAAS. The results, corrected for the Se concentration in the appropriate zoological or botanical control digest, are presented in Table 3.Whereas digestion of the three marine CRM homogenates provided estimates which were not significantly different from the certified Se concentrations (despite the higher Se concentration in the zoological control) the lower concentrations in the botanical CRMs resulted in estimates which, occasionally, were not different from the control concentrations. In the latter cases, the certified Se concentrations were less than the Se concentrations in control digests.Likewise, three aliquots (approximately 0.2 g) of each of eight dried, ground feed supplements consisting of mixed forage crops were suspended in 10 ml of TRIS buffer, homogenized and then digested with the protease–cellulase combination for either 4 or 8 h prior to Se determination by ETAAS. Again, there was good agreement between the results (Table 4) for slurry introduction ETAAS following 4 or 8 h of enzymic digestion and a single fluorescence determination Fig. 1 Variation in percentage recovery of Se (±1 RSD based on three replicate samples) from DORM-1 or TORT-1 certified reference materials versus hours of enzymic digestion with protease plus lipase prior to (open symbols) or after (closed symbols) high-pressure homogenization. For clarity of presentation, the TORT-1 results have been displaced by 0.4 h. Fig. 2 Variation in percentage recovery of Se (±1 RSD based on three replicate samples) from Durham wheat flour CRM or a ground animal feed sample versus hours of enzymic digestion with protease plus cellulase prior to (open symbols) or after (closed symbols) high-pressure homogenization. For clarity of presentation, the animal feed results have been displaced by 0.4 h.Fig. 3 Influence of storage subsequent to slurry preparation on percentage recovery of Se (±1 RSD based on three replicate samples) in flour CRM or a ground animal feed sample.For clarity of presentation, the animal feed results have been displaced by 0.4 h. 16 Analyst, January 1997, Vol. 122following strong acid digestion and piazselenol formation. Storage of the digests for 10 d at 4 °C did not change the measured concentrations of analyte (Table 4). In only one feed supplement (in which the Se concentration was appreciably less than that in the botanical control digest) were the results of the two methods discordant. No matrix effects for Se determinations in any of the samples were detected.The slopes of the best-fit regression lines for standard additions to homogenized protease–lipase or protease– cellulase enzyme suspensions in TRIS buffer, in the presence or absence of DORM-1, TORT-1, wheat flour, corn bran or apple leaf CRM, or to five of the feed supplements varied by less than 11% (RSD) provided that calibrations and determinations were performed on the same day. This observation suggested that a single calibration by standard addition(s) to the enzyme mixtures would suffice for the determination of Se in any of the samples.A single calibration curve generated by adding aqueous Se standard to the botanical control homogenate was then used to determine the Se content of freeze-dried freshwater fish fillets which had been frozen fresh or boiled to simulate cooking following common native practice. Aliquots of the freeze-dried materials were digested enzymically for 4 or 8 h and then analysed by ETAAS or digested with strong acids and then analysed by HGAAS (Table 5).Boiling the fillet prior to freezedrying did not inhibit the enzymic release of Se residues from the matrix but apparently lowered the Se concentration in the cooked product. There were no significant differences between the results after 4 and 8 h of digestion or between ETAAS and HGAAS results. However, the precision associated with replicate enzymic digestion–ETAAS Se determinations (mean RSD nearly 15 ± 2%) was appreciably worse than the precision associated with hydride generation determinations (mean 7 ± 4%) but typical of the replicate determinations of other experiments (mean RSD for the 36 determinations in Table 3 14.4 ± 0.3% and for 29 of the determinations in Table 4 12.6 ± 0.5%).Thus, the short-term repeatability (i) was not adversely affected by the use of the single calibration curve but (ii) can be expected to be degraded relative to other conventional procedures for Se determination.The principal advantages of the enzymic digestion procedure are the simplicity and speed relative to conventional unassisted acid digestions and that they can be performed unattended. The conditions of digestion do not appear to be critical and there was no tendency for the liberated Se residues to segregate within the resulting suspensions. Feed samples and feed supplements and determinations of their Se content by fluorescence of their piazselenol derivatives were generously supplied by E.R. Chavez, McGill University. Samples of fish fillets and determinations of their Se content by HGAAS were kindly donated by H. M. Chan, McGill University. Financial support in the form of an operating grant from the Natural Science and Engineering Research Council of Canada (NSERC) is gratefully acknowledged. References 1 Novozamski, I., van der Lee, H. J., and Houba, V. J. G., Microchim. Acta, 1995, 119, 183. 2 Sansoni, B., and Panday, V. K., in Analytical Techniques for Heavy Metals in Biological Fluids, ed. Fachetti, S., Elsevier, Amsterdam, 1983, p. 91. 3 Mincwewski, J., Chwastowska, J., and Dybczynski, R., Separation and Preconcentration Methods in Inorganic Trace Analysis, Ellis Horwood, Chichester, 1982. 4 Matusiewicz, J., and Sturgeon, R. E., Prog. Anal. Spectrosc., 1989, 12, 21. 5 Reid, H. J., Greenfield, S., and Edmonds T. E., Analyst, 1995, 120, 1543. 6 Langmyhr, F.J., Analyst, 1979, 104, 993. 7 Langmyhr, F. J., Prog. Anal. At. Spectrosc., 1985, 8, 193. 8 Miller-Ihli, N. J., Anal. Chem., 1992, 64, 965A. 9 de Benzo, Z. A., Velosa, M., Ceccarelli, C., de la Guardia, M., and Salvador, A., Fresenius’ J. Anal. Chem., 1991, 339, 235. 10 Bendicho, C., and de Loos-Vollebregt, M. T. C., J. Anal. At. Spectrom., 1991, 6, 353. 11 Miller-Ihli, N. J., Fresenius’ J. Anal. Chem., 1993, 345, 482. 12 Stephen, S. C., Littlejohn, D., and Ottaway, J.M., Analyst, 1985, 110, 1147. 13 Thompson, D. D., and Allen, R. J., At. Spectrosc., 1981, 2, 53. 14 Madrid, Y., Bonilla, M., and Camara, C., J. Anal. At. Spectrom., 1989, 4, 167. 15 L�opez Garc�ýa, I., Ortiz Sobejano, F., and Hern�andez C�ordoba, M., Analyst, 1991, 116, 517. 16 Hoenig, M., and Hoeyweghen, P. V., Anal. Chem., 1986 58, 2614. 17 Albers, D., and Sacks, R., Anal. Chem., 1987, 59, 593. Table 4 Selenium concentrations (mg g21) in dried ground plant materials as determined by fluorescence (single measurement) or by ETAAS* (±1 RSD for triplicate determinations of three replicate samples) immediately after 4 or 8 h of enzymic digestion or following enzymic digestion plus 10 d of storage Strong acid 4 h digestion + 8 h digestion + digestion + Sample 4 h digestion 8 h digestion 10 d storage 10 d storage fluorescence Solvent blank + protease + cellulase 0.048 ± 14.2% 0.051 ± 12.0% 0.040 ± 23.3% 0.045 ± 12.2% 9–3 1.29 ± 10.6% 1.24 ± 9.9% 1.28 ± 8.1% 1.25 ± 9.0% 1.31 6–3 1.31 ± 12.3% 1.19 ± 9.0% 1.25 ± 9.1% 1.20 ± 8.9% 1.23 7–3 1.04 ± 12.3% 1.15 ± 9.0% 1.11 ± 6.6% 1.18 ± 6.5% 1.17 546–3 0.79 ± 8.2% 0.79 ± 11.1% 0.80 ± 9.6% 0.79 ± 10.5% 0.81 158–5 0.53 ± 9.2% 0.57 ± 8.9% 0.49 ± 11.0% 0.55 ± 7.2% 0.59 299–5 0.44 ± 13.7% 0.42 ± 8.2% 0.45 ± 18.2% 0.40 ± 10.7% 0.43 314–5 0.14 ± 16.0% 0.14 ± 13.2% 0.14 ± 20.8% 0.13 ± 10.7% 0.13 307–5 0.021 ± 23.5% 0.022 ± 24.1% 0.022 ± 24.2% 0.021 ± 20.9% 0.03 * Reported concentrations have been corrected for the [Se] in the protease + cellulase control homogenate.Table 5 Selenium concentrations (mg g21) (±1 SEE*) in freeze-dried fresh or boiled fish fillet following 4 or 8 h of enzymic digestion with protease and lipase and ETAAS Hydride Sample 4 h digestion 8 h digestion generation† Lake trout (boiled) 3.56 ± 20.9% 3.48 ± 14.1% 3.60 ± 8.0% Lake trout (fresh) 1.86 ± 11.9% 2.12 ± 13.2% 1.69 ± 10.1% Northern pike (fresh) 2.48 ± 16.6% 2.51 ± 12.5% 2.75 ± 2.9% * SEE, standard error of estimate based on three replicate determinations of three separate digests.† ±1 RSD based on duplicate determinations. Analyst, January 1997, Vol. 122 1718 Tsalev, D. L., Slaveykova, V. I., and Mandjunkov, P. B., Spectrochim. Acta, Rev., 1990, 13, 225. 19 Miller-Ihli, N. J., J. Anal. Atom. Spectrom., 1989, 4, 295. 20 Bendicho, C., and de Loos-Vollebregt, M. T. C., Spectrochim. Acta, Part B, 1990, 45, 679. 21 Miller-Ihli, N. J., J. Anal. At. Spectrom., 1988, 3, 73. 22 Lynch, S., and Littlejohn, D., J. Anal. At. Spectrom., 1989, 4, 157. 23 Fagioli, F., Landi, S., Locatelli, C., Righini, F., Settimo, R., and Magarini, R., J. Anal. At. Spectrom., 1990, 5, 519. 24 Hansen, D. L., and Bush, E. T., Anal. Biochem., 1967, 18, 320. 25 Jackson, A. J., Michael, L. M., and Schumacher, H. J., Anal. Chem., 1972, 44, 1064. 26 Murthy, L., Menden, E. E., Eller, P. M., and Petering, H. G., Anal. Biochem., 1973, 53, 365. 27 Uchida, T., Isoyama, H., Yamada, K., Oguchi, K., Nakagawa, G., Sugie, H., and Iida, C., Anal.Chim. Acta, 1992, 256, 277. 28 Dion, B., Ruzbie, M., van de Voort, F. R., Ismail, A. A., and Blais, J. S., Analyst, 1994, 119, 1765. 29 Tan, Y., Marshall, W. D, and Blais, J.-S., Analyst, 1996, 121, 483. 30 Tan, Y., Blais, J.-S., and Marshall, W. D., Analyst, 1996, 121, 1419. 31 Ebdon, L., and Perry, H. G. M., J. Anal. At. Spectrom. 1988, 3, 131. 32 Bradshaw, D., and Slavin, W., Spectrochim. Acta Part B, 1989, 44, 1245. 33 Wagley, D., Schmiedel, G., Mainka, E., and Ache, H. J., At. Spectrosc., 1989, 10, 106. 34 Bendicho, C., and Sancho, A., At. Spectrosc., 1993, 14, 187. 35 Cabrera, C., Lorenzo, M. L., and Lopez, M. C., J. AOAC Int., 1995, 78, 1061. 36 L�opez-Garc�ýa, I., Vi�nas, P., Campillo, N., and Hern�andez- C�ordoba, M., J. Agric. Food Chem.. 1996, 44, 836. 37 Gilon, N., Astruc, A., Astruc, M., and Potin-Gautier, M., Appl. Organomet. Chem., 1995, 9, 623. 38 Dedina, J. and Tsalev, D.L., Hydride Generation Atomic Absorption Spectrometry (Chemical Analysis, vol. 130), ed. Wineforder, J. D., and Kolthoff, I. M., Wiley, Chichester, 1995. 39 Johansson, K., Luo, X., and Olin, A., Talanta, 1995, 42, 1979. 40 Miller-Ihli, N. J., At. Spectrosc., 1992, 13, 1. 41 Forsyth, D. S., and Marshall, W. D., Environ. Sci. Technol.. 1986, 20, 1033. Paper 6/05880I Received August 27, 1996 Accepted October 15, 1996 18 Analyst, January 1997, Vol. 122 Enzymic Digestion–High-pressure Homogenization Prior to Slurry Introduction Electrothermal Atomic Absorption Spectrometry for the Determination of Selenium in Plant and Animal Tissues Yanxi Tan and William D.Marshall* Department of Food Science and Agricultural Chemistry, Macdonald Campus of McGill, 21 111 Lakeshore Road, Ste.-Anne-de-Bellevue, Qu�ebec, Canada H9X 3V9. E-mail: marshall@agradm.lan.mcgill.ca Homogenization, in combination with partial enzymic digerotease alone or admixed with lipase or cellulase, was investigated as a means of releasing Se residues from zoological and botanical matrices prior to slurry introduction ETAAS.Preliminary timed trials with two zoological certified reference materials (CRMs), one botanical CRM and one animal feed indicated that Se release became quantitative with 4–8 h of digestion, that homogenization prior to digestion increased the initial rate of analyte release, but that homogenization post-digestion and immediately prior to ETAAS did not significantly improve the precision of replicate digestions. Storage of the crude botanical digests at 4 °C for 5 d resulted in quantitative recoveries of Se from each of the digests.Storage at 4 °C for 10 d of 4 and 8 h lipase/protease digests of six other CRMs resulted in quantitative recoveries of Se unless their certified concentrations were appreciably less than the levels determined in control digests containing the enzyme(s) alone. Apparently, Se residues were transferred virtually quantitatively to the liquid phase of the digested suspension and showed no tendency to segregate during the 10 d of storage.Eight other mixtures of ground plant matter (0.13 @ [Se] @ 1.31 mg g21), formulated as animal feed supplements, behaved identically when stored post-digestion. The technique was also applied successfully to freeze-dried fresh and boiled fish tissues The principal advantages of the enzymic digestion procedure are its simplicity and lack of operator intervention.Keywords: Selenium determination; enzymic digestion; slurry introduction electrothermal atomic absorption spectrometry; botanical and zoological certified reference materials Conventional sample preparation of biological materials prior to atomic spectrometry involves complete solubilization of the analyte and matrix, which is achieved typically by oxidative mineralization of the organic matter and solubilization of the residue in a suitable solvent.1–4 Even for microwave-assisted digestions, whereas complete dissolution can usually be achieved by a suitable choice of digestion conditions, complete decomposition of the organic matrix in biological/botanical samples is appreciably more difficult.Often complete mineralization is achieved only with supplemental treatment of the digested matrix with H2O2 or even HF.5 These digestion procedures can be labour intensive, time consuming and prone to contamination errors.In consequence, there is a continuing interest in the development of simplified sample preparation techniques. The preparation/introduction of slurried samples continues to attract considerable attention because of the ease with which quasi-stable preparations can be generated and their compatibility with conventional liquid handling techniques. Within the general field of solid sampling analysis,6–11 it is the use of slurried samples which has become the most popular approach to trace element determination.Direct atomization from the solid state can provide excellent sensitivity, but the interpretation of results can be complicated by molecular absorptions and/or scattering from the matrix, which can produce sufficiently large background signals to overwhelm the compensation capabilities of common deuterium background correction systems. Additional difficulties can include sample inhomogeneities, the requirement for repeated microweighings and the lack of suitable calibration standards and techniques.A variety of sample pre-treatment procedures and additives12 –18 have been described and evaluated for the production of quasi-stable suspensions of samples prior to analysis by atomic spectrometry. Alternatively, suspensions with a tendency to segment rapidly have been reproducibly sampled by using ultrasonic agitation,19 air or argon20 bubbling, vortex mixing21 or magnetic stirring.22 Partial digestion procedures to produce carbonaceous slurries have also been successfully applied to the analysis, by ICP-AES, of a series of standard reference materials of biological origin.23 Various alkylammonium hydroxide formulations have been used extensively to solubilize tissues,24–27 particularly those of zoological origin.Recently, high-pressure homogenization has been evaluated for the preparation of quasi-stable dispersions suitable for FTIR28 or ETAAS.29,30 The advantages of this approach to sample preparation were the ease and speed of the slurry preparation, which required less than 1 min, and the fact that analyte metals were quantitatively extracted into the liquid phase during the preparation so that no analyte segmentation was detected within the slurry even after standing for several days.Certified reference materials (CRMs), frozen liver and kidney and dried animal feeds of botanical origin were analysed successfully for Cd, Cr, Cu, Mn, Ni and Pb but not for Se.The principal limitation of the high-pressure homogenization technique was the amounts of contaminating analyte metals introduced into the sample by the homogenization operation. Contamination was reduced appreciably, but not eliminated entirely, by capping the flat face of the stainless-steel homogenizing valve with a ruby disc.30 The objectives of this work were (i) to evaluate the efficiencies of other materials as caps to reduce further the levels of contamination introduced by the high-pressure processing and (ii) to develop efficient alternative slurry preparation techniques for the determination of Se in biological materials.Although there have been few reports of the determination of Se in slurried samples,31–34 recent reports35,36 indicate that the approach is promising for this analyte. Prolonged enzymic digestion with a crude protease fraction has been used37 to liberate component selenoamino acids from proteins.This approach seemed promising as a pre-homogenization sample preparation. Analyst, January 1997, Vol. 122 (13–18) 13Experimental Reagents TRIS was purchased from Aldrich (Milwaukee, WI, USA) and aqueous Se solution (1000 mg ml21, traceable to NIST primary standard) was purchased from SCP Chemical (St.-Laurent, Qu�ebec, Canada). Samples CRMs were purchased from the National Research Council of Canada (NRCC) or the US National Institute of Science and Technology (NIST).Samples of animal diet mixtures destined for a zoo were chosen to contain a variety of plant and animal materials, including timothy grass, bamboo leaves, whole smelts, cricket chow and a mixture (contents unspecified) formulated for panda bears. Animal feed supplements were composed of mixed forage crops. Sample Preparation An accurately weighed sample (approximately 0.2 g) of CRM, dried feed or supplements (ground, to pass a 0.5 mm screen, in a Tecator Cyclotec sample mill; Tecator, H�ogan�as, Sweden) was added directly to 10 ml of ethanol–0.03 m TRIS (1 + 19 v/v, pH 7.5) containing either 20 mg of crude protease (Type XIV; Sigma St.Louis, MO, USA), 20 mg of protease plus 20 mg of lipase (Type VII; Sigma) or 20 mg protease plus 20 mg of cellulase (Cellulysin; Calbiochem–Novabiochem, La Jolla, CA, USA). The resulting suspension was then processed through the 20 ml capacity flat valve homogenizer (EmulsiFlex Model EFB3; Avestin, Ottawa, ON, Canada), capable of developing 138 MPa when provided with compressed air (689.4 kPa).Each slurried sample was re-processed through the homogenizer three more times. The homogenates, in 50 ml Erlenmeyer flasks, were then digested at 37 °C with gentle agitation for 4–8 h. Homogenizer The valve stem of the screw-cap assembly of the homogenizer was modified by gluing a polished 4 mm diameter 32 mm thick disc manufactured from a 6–12 mm diameter sphere of tungsten carbide (Spex, Metuchen, NJ, USA), zirconia (Optimize Technologies, Portland, OR, USA), sapphire (from an HPLC piston) or polymethacrylate (Spex). Sample + enzyme suspension was transferred to the sample chamber via the inlet port, which was then sealed with a fine-threaded screw-cap.The stainless-steel piston (connected to a pneumatic multiplier) then forced the fluid through an aperture and the homogenate was collected from thle outlet. Each sample was re-processed three more times with the valve stem retracted slightly to provide a slightly larger gap setting.Selenium Determinations (Hydride Generation or Fluorescence) Feed samples were dried to constant mass and ground to pass a 1 mm screen. Accurately weighed aliquots of ground feed or freeze-dried fish tissue (approximately 2 g) were digested at room temperature in a perchlorate fume-hood with 25 ml of 70% HNO3–HClO4 (4 + 1 v/v) until gas evolution had ceased, then heated at 80 °C until a clear yellow solution was obtained. The resulting strong acid digests were analysed by HGAAS38 or fluorescence of the piazselenol derivative39 after conversion of the analyte residues in to SeIV. ETAAS Selenium determinations were performed using a hot injection technique on a Varian (Palo Alto, CA, USA) Model 300 ETAAS system equipped with an autosampler, pyrolytic graphite-coated platform graphite tubes, a conventional Se hollow-cathode lamp and Zeeman-effect background correction.Ashing–atomization curves were generated for Se standard in the presence and absence of co-injected biological sample. At temperatures < 2300 °C, the Se atomization signal was broadened by the presence of biological materials but was sharpened (and did not tail) for atomizations at 2400 °C. In the presence of the palladium–citric acid modifier, no loss in the Se signal was observed at an ashing temperature @1400 °C. Analytical operating parameters are presented in Table 1.Calibration ETAAS quantification was performed by both the method of external standards (ES) and by standard additions (SA). ES consisting of appropriately diluted processed reagent blank and up to four levels of standard were prepared automatically by the sample introduction device. The background-corrected peakarea response, resulting from three replicate injections of each diluted standard, was used to define the best-fit regression equation. For SA calibrations, 10 ml aliquots of processed fluid were amended with 2, 5 or 10 ml of aqueous standard chosen to result in a range of peak areas including signals which were half and at least twice the signal for the unamended sample.The data were modelled by least-squares linear regression. Quantification was performed by dividing the intercept on the ordinate of the regression equation by the slope of the equation and the overall standard error of the estimate (SEest) was calculated from SEest = (SE2 y-int + SE2 slope)1/2 Results and Discussion Preliminary experiments were directed to evaluating the influence of different capping materials on the levels of contaminating metals introduced into the homogenates during processing.It was postulated that exposed stainless-steel surfaces within the valve homogenizer, particularly the flat face of the demountable valve head, were the principle sites responsible for the contamination. Further, capping the valve head with an inert surface capable of withstanding the impact of the jet of fluid exiting the homogenizing orifice might reduce the levels of contamination appreciably.It has been reported40 previously that zirconium oxide beads used to reduce the particle size and to mix particulate solids introduced appreciable levels of Fe, Cr and Al but that silicon nitride or boron carbide provides good abrasion resistance and offers little likelihood of Table 1 Furnace operating parameters for determinations of selenium Parameter* Value Wavelength/nm 196.0 Lamp current/A 10 Slit width/nm 1.0 Injection temperature/°C 60 Pre-injection Yes Temperature of last dry step (10 s)/°C 250 Charring sequence 10 s ramp to 1400 °C, 40 s hold Cooling None Atomization 0.6 s ramp to 2400 °C, 5.0 s hold Measurement time/s 5.6 Chemical modifier 5 ml (0.5% m/m Pd + 2.5% m/m critic acid) for 10 ml sample * Each step of the furnace programme (with the exception of the read step) was performed in the presence of argon purge gas (3 l min21). 14 Analyst, January 1997, Vol. 122contamination. However, even for the relatively lower pressure requirements of pistons and check valves for HPLC, sapphire, ruby and zirconium oxide are preferred over other ceramic materials for their superior resistance. Separate discs composed of zirconia, tungsten carbide or polymethacrylate, which had been manufactured by grinding and polishing a 6–12 mm diameter grinding ball, were glued temporarily to the flat face of the demountable valve head.Similarly, the sapphire disc was generated from a used HPLC piston. Solvent mixture (20 ml) was homogenized four successive times (in the presence or absence of the test disc) prior to ETAAS analysis for Al, As, Cd, Cr, Cu, Fe, Mn, Ni, Pb and Se. Analyte concentrations (Table 2) were expressed as if the solvent had contained 0.100 g of sample. The heavy metal content of the homogenized fluid was lowered appreciably in all cases.Nonetheless, contamination remained appreciable for several elements, even in the presence of the polymethacrylate or the sapphire cap. Previous attempts to determine Se in biological materials by slurry introduction ETAAS of high-pressure homogenates were not successful in our hands using a variety of furnace programmes, yet there was no evidence of any analyte loss prior to the atomization stage as judged by the signal graphics software, which provided a continuous display of the Se signal over the course of the furnace programme.Since a high proportion of the analyte element in biological materials is considered to be protein bound, it was decided to evaluate partial enzymic hydrolysis as a means of liberating bound analyte residues. Arbitrarily, it had been decided initially to attempt to develop a single combination of mixed enzymes which it was hoped would be applicable to all sample matrices. Previous studies41 had indicated that a combination of crude proteases and lipases efficiently hydrolysed avian egg yolk.Initial studies were limited to this combination of enzymes. The mixture of crude enzymes was suspended in 10 ml of TRIS buffer (pH 7.5), then passed sequentially four times through the polymethacrylate-capped homogenizer to furnish a digestion control homogenate. Relative to a distilled, de-ionized water blank, this ‘zoological’ control sample contained 0.11 mg g21 ± 12.6% and 0.10 mg g21 ± 11.3% after 4 and 8 h of digestion respectively (Table 3), when it was assumed that the digests had contained 0.200 g of sample.Similarly, a control homogenate composed of protease alone contained 0.044 mg g21 ± 12.6% after 4 h. A crude cellulase was substituted for the lipase in the enzyme mixture and the digestions were performed in analogous fashion to furnish alternative enzymic digestion control samples. The ‘botanical’ control samples contained 0.048 mg g21 ± 14.2% and 0.051 mg g21 ± 12.0% after 4 and 8 h of digestion respectively (Table 3), and the solvent blank + protease alone contained 0.044 mg g21 ± 12.6%, again assuming that the digests had contained 0.200 g of sample.Thus, virtually all of the Se in control digests originated with the lipase and/or the protease. Based on a 3 RSD criterion, the corresponding method limit of detection (LOD) for digestions with mixed protease lipase, with protease cellulase and with protease alone were 0.020, 0.010 and 0.010 mg g21, respectively.In preliminary experiments, three biological CRMs and one animal feed, suspended in 10 ml TRIS buffer, were digested with a combination of protease and lipase for up to 16 h at 37 °C. Table 2 Apparent analyte concentrations (mg g21 sample) in 20 ml of solvent mixture following various mixing treatments. Concentrations in the homogenized fluid are expressed as if the solvent mixture had contained 0.100 g of sample Treatment Al As Cd Cr Cu Fe Pb Mn Ni Se Unhomogenized solvent blank 0.32 n.d.* n.d.n.d. n.d. 0.40 n.d. n.d. n.d. n.d. Four successive homogenizations with: Stainless-steel head 42.12 0.02 4.53 15.0 56.94 1.38 2.31 3.57 n.d. Polymethacrylate cap 7.92 n.d. 0.02 1.80 n.d. 5.20 n.d. 0.10 n.d. n.d. Ruby cap 21.52 n.d. 0.03 4.02 0.70 13.99 0.28 0.39 0.11 n.d. Sapphire cap 3.64 n.d. n.d. 3.65 0.80 4.90 n.d. 0.10 n.d. n.d. Tungsten carbide cap 15.32 n.d. 0.04 4.40 1.40 38.6 2.00 0.40 0.10 n.d.Zirconia cap 0.42 n.d. 0.02 4.00 1.20 15.7 n.d. 0.20 0.10 n.d. * n.d. = None detected above the mean background signal for ethanol–0.03 m TRIS (1 + 19 v/v, pH 7.5) solvent. Table 3 Selenium concentrations (mg g21) (±1 RSD based on three replicate samples) in certified reference materials determined immediately after 4 or 8 h of enzymic digestion or following digestion plus 10 d of storage 4 h digestion + 8 h digestion + Certified Matrix 4 h digestion 8 h digestion 10 d storage 10 d storage concentration Solvent blank + protease + lipase 0.11 ± 12.6% 0.10 ± 11.3% 0.12 ± 10.2% 0.10 ± 13.6% Solvent blank + protease 0.044 ± 12.6% Solvent blank + protease + cellulase 0.048 ± 14.2% 0.051 ± 12.0% 0.040 ± 23.2% 0.045 ± 12.2% Oyster Tissue* 2.18 ± 12.7% 2.27 ± 11.6% 2.16 ± 11.4% 2.15 ± 12.6% 2.21 ± 0.24 DORM-2* 1.38 ± 9.2% 1.35 ± 11.6% 1.34 ± 6.6% 1.33 ± 11.0% 1.40 ± 0.090 Bovine Muscle* 0.067 ± 22.6% 0.066 ± 20.5% 0.067 ± 20.2% 0.070 ± 21.4% 0.076 ± 0.010 Apple Leaves† 0.043 ± 12.7% 0.045‡ ± 19.9% 0.047 ± 12.7% 0.041 ± 12.7% 0.050 ± 0.009 Corn Bran† 0.034‡ ± 12.5% 0.036 ± 14.9% 0.033 ± 15.3% 0.036 ± 16.5% 0.045 ± 0.008 Corn Stalk† n.d.§ n.d.§ 0.025‡ ± 47.0% 0.011‡ ± 52.2% 0.016 ± 0.008 * Reported concentrations have been corrected for the [Se] in the protease + lipase control homogenate.† Reported concentrations have been corrected for the [Se] in the protease + cellulase control homogenate. ‡ No [Se] above the LOD (0.010 mg g21) was detected in one of the three aliquots.§ No [Se] above the LOD (0.010 mg g21) was detected in any of the three aliquots. Analyst, January 1997, Vol. 122 15Each suspension was homogenized immediately prior or directly after digestion, then analysed by ETAAS. The results are presented in Figs. 1 and 2. The TORT-1 results and the animal feed results (triangular symbols in Fig. 1 and Fig. 2, respectively) have been displaced by 0.4 h for clarity of presentation. For all four substrates, homogenization prior to digestion (closed symbols) generally resulted in higher recoveries relative to homogenization post-digestion (open symbols), although the differences were only rarely statistically significant.Moreover, the differences tended to decrease with longer digestion times. Presumably, homogenization initially exposed more of the protein component to the enzyme. On the other hand, homogenization post-digestion and immediately prior to ETAAS did not significantly improve the precision of the determination (as judged by the RSD associated with three replicate measurements performed on each of three digests).In general, there was a gradual but small improvement in precision with increased length of digestion (more evident with the plant samples in Fig. 2). For both the DORM-1 and the TORT-1 marine tissue samples in Fig. 1, 4 h of digestion at 37 °C were sufficient to liberate the Se quantitatively, whereas recoveries from the plant samples were quantitative only after 8 h of digestion.After sampling for ETAAS, the plant digests were stored at 4 °C for 5 d and then re-analysed. No effort was made to resuspend solid materials; instead, a portion of each supernatant fraction was transferred directly to the sampling cup. The recovery of Se from each of the supernatant fractions was quantitative (Fig. 3), indicating that the crude protease was active at the storage temperature and that there was no apparent segmentation of the Se residues between the liquid and solid phases of the crude digest.Surprisingly, the short-term repeatability of the procedure was not improved by the storage, as evidenced by the RSD associated with replicates. Repeatability continued to be improved for longer digestions at 37 °C. Three replicate aliquots of each of six other certified reference materials were homogenized and then digested for 4 or 8 h prior to ETAAS. The results, corrected for the Se concentration in the appropriate zoological or botanical control digest, are presented in Table 3.Whereas digestion of the three marine CRM homogenates provided estimates which were not significantly different from the certified Se concentrations (despite the higher Se concentration in the zoological control) the lower concentrations in the botanical CRMs resulted in estimates which, occasionally, were not different from the control concentrations. In the latter cases, the certified Se concentrations were less than the Se concentrations in control digests. Likewise, three aliquots (approximately 0.2 g) of each of eight dried, ground feed supplements consisting of mixed forage crops were suspended in 10 ml of TRIS buffer, homogenized and then digested with the protease–cellulase combination for either 4 or 8 h prior to Se determination by ETAAS.Again, there was good agreement between the results (Table 4) for slurry introduction ETAAS following 4 or 8 h of enzymic digestion and a single fluorescence determination Fig. 1 Variation in percentage recovery of Se (±1 RSD based on three replicate samples) from DORM-1 or TORT-1 certified reference materials versus hours of enzymic digestion with protease plus lipase prior to (open symbols) or after (closed symbols) high-pressure homogenization.For clarity of presentation, the TORT-1 results have been displaced by 0.4 h. Fig. 2 Variation in percentage recovery of Se (±1 RSD based on three replicate samples) from Durham wheat flour CRM or a ground animal feed sample versus hours of enzymic digestion with protease plus cellulase prior to (open symbols) or after (closed symbols) high-pressure homogenization.For clarity of presentation, the animal feed results have been displaced by 0.4 h. Fig. 3 Influence of storage subsequent to slurry preparation on percentage recovery of Se (±1 RSD based on three replicate samples) in flour CRM or a ground animal feed sample.For clarity of presentation, the animal feed results have been displaced by 0.4 h. 16 Analyst, January 1997, Vol. 122following strong acid digestion and piazselenol formation. Storage of the digests for 10 d at 4 °C did not change the measured concentrations of analyte (Table 4). In only one feed supplement (in which the Se concentration was appreciably less than that in the botanical control digest) were the results of the two methods discordant.No matrix effects for Se determinations in any of the samples were detected. The slopes of the best-fit regression lines for standard additions to homogenized protease–lipase or protease– cellulase enzyme suspensions in TRIS buffer, in the presence or absence of DORM-1, TORT-1, wheat flour, corn bran or apple leaf CRM, or to five of the feed supplements varied by less than 11% (RSD) provided that calibrations and determinations were performed on the same day. This observation suggested that a single calibration by standard addition(s) to the enzyme mixtures would suffice for the determination of Se in any of the samples.A single calibration curve generated by adding aqueous Se standard to the botanical control homogenate was then used to determine the Se content of freeze-dried freshwater fish fillets which had been frozen fresh or boiled to simulate cooking following common native practice. Aliquots of the freeze-dried materials were digested enzymically for 4 or 8 h and then analysed by ETAAS or digested with strong acids and then analysed by HGAAS (Table 5).Boiling the fillet prior to freezedrying did not inhibit the enzymic release of Se residues from the matrix but apparently lowered the Se concentration in the cooked product. There were no significant differences between the results after 4 and 8 h of digestion or between ETAAS and HGAAS results. However, the precision associated with replicate enzymic digestion–ETAAS Se determinations (mean RSD nearly 15 ± 2%) was appreciably worse than the precision associated with hydride generation determinations (mean 7 ± 4%) but typical of the replicate determinations of other experiments (mean RSD for the 36 determinations in Table 3 14.4 ± 0.3% and for 29 of the determinations in Table 4 12.6 ± 0.5%).Thus, the short-term repeatability (i) was not adversely affected by the use of the single calibration curve but (ii) can be expected to be degraded relative to other conventional procedures for Se determination. The principal advantages of the enzymic digestion procedure are the simplicity and speed relative to conventional unassisted acid digestions and that they can be performed unattended.The conditions of digestion do not appear to be critical and there was no tendency for the liberated Se residues to segregate within the resulting suspensions. Feed samples and feed supplements and determinations of their Se content by fluorescence of their piazselenol derivatives were generously supplied by E.R. Chavez, McGill University. Samples of fish fillets and determinations of their Se content by HGAAS were kindly donated by H. M. Chan, McGill University. Financial support in the form of an operating grant from the Natural Science and Engineering Research Council of Canada (NSERC) is gratefully acknowledged. References 1 Novozamski, I., van der Lee, H. J., and Houba, V. J. G., Microchim.Acta, 1995, 119, 183. 2 Sansoni, B., and Panday, V. K., in Analytical Techniques for Heavy Metals in Biological Fluids, ed. Fachetti, S., Elsevier, Amsterdam, 1983, p. 91. 3 Mincwewski, J., Chwastowska, J., and Dybczynski, R., Separation and Preconcentration Methods in Inorganic Trace Analysis, Ellis Horwood, Chichester, 1982. 4 Matusiewicz, J., and Sturgeon, R. E., Prog. Anal. Spectrosc., 1989, 12, 21. 5 Reid, H. J., Greenfield, S., and Edmonds T. E., Analyst, 1995, 120, 1543. 6 Langmyhr, F. J., Analyst, 1979, 104, 993. 7 Langmyhr, F. J., Prog. Anal. At. Spectrosc., 1985, 8, 193. 8 Miller-Ihli, N. J., Anal. Chem., 1992, 64, 965A. 9 de Benzo, Z. A., Velosa, M., Ceccarelli, C., de la Guardia, M., and Salvador, A., Fresenius’ J. Anal. Chem., 1991, 339, 235. 10 Bendicho, C., and de Loos-Vollebregt, M. T. C., J. Anal. At. Spectrom., 1991, 6, 353. 11 Miller-Ihli, N. J., Fresenius’ J. Anal. Chem., 1993, 345, 482. 12 Stephen, S. C., Littlejohn, D., and Ottaway, J.M., Analyst, 1985, 110, 1147. 13 Thompson, D. D., and Allen, R. J., At. Spectrosc., 1981, 2, 53. 14 Madrid, Y., Bonilla, M., and Camara, C., J. Anal. At. Spectrom., 1989, 4, 167. 15 L�opez Garc�ýa, I., Ortiz Sobejano, F., and Hern�andez C�ordoba, M., Analyst, 1991, 116, 517. 16 Hoenig, M., and Hoeyweghen, P. V., Anal. Chem., 1986 58, 2614. 17 Albers, D., and Sacks, R., Anal. Chem., 1987, 59, 593. Table 4 Selenium concentrations (mg g21) in dried ground plant materials as determined by fluorescence (single measurement) or by ETAAS* (±1 RSD for triplicate determinations of three replicate samples) immediately after 4 or 8 h of enzymic digestion or following enzymic digestion plus 10 d of storage Strong acid 4 h digestion + 8 h digestion + digestion + Sample 4 h digestion 8 h digestion 10 d storage 10 d storage fluorescence Solvent blank + protease + cellulase 0.048 ± 14.2% 0.051 ± 12.0% 0.040 ± 23.3% 0.045 ± 12.2% 9–3 1.29 ± 10.6% 1.24 ± 9.9% 1.28 ± 8.1% 1.25 ± 9.0% 1.31 6–3 1.31 ± 12.3% 1.19 ± 9.0% 1.25 ± 9.1% 1.20 ± 8.9% 1.23 7–3 1.04 ± 12.3% 1.15 ± 9.0% 1.11 ± 6.6% 1.18 ± 6.5% 1.17 546–3 0.79 ± 8.2% 0.79 ± 11.1% 0.80 ± 9.6% 0.79 ± 10.5% 0.81 158–5 0.53 ± 9.2% 0.57 ± 8.9% 0.49 ± 11.0% 0.55 ± 7.2% 0.59 299–5 0.44 ± 13.7% 0.42 ± 8.2% 0.45 ± 18.2% 0.40 ± 10.7% 0.43 314–5 0.14 ± 16.0% 0.14 ± 13.2% 0.14 ± 20.8% 0.13 ± 10.7% 0.13 307–5 0.021 ± 23.5% 0.022 ± 24.1% 0.022 ± 24.2% 0.021 ± 20.9% 0.03 * Reported concentrations have been corrected for the [Se] in the protease + cellulase control homogenate.Table 5 Selenium concentrations (mg g21) (±1 SEE*) in freeze-dried fresh or boiled fish fillet following 4 or 8 h of enzymic digestion with protease and lipase and ETAAS Hydride Sample 4 h digestion 8 h digestion generation† Lake trout (boiled) 3.56 ± 20.9% 3.48 ± 14.1% 3.60 ± 8.0% Lake trout (fresh) 1.86 ± 11.9% 2.12 ± 13.2% 1.69 ± 10.1% Northern pike (fresh) 2.48 ± 16.6% 2.51 ± 12.5% 2.75 ± 2.9% * SEE, standard error of estimate based on three replicate determinations of three separate digests. † ±1 RSD based on duplicate determinations. 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M., Wiley, Chichester, 1995. 39 Johansson, K., Luo, X., and Olin, A., Talanta, 1995, 42, 1979. 40 Miller-Ihli, N. J., At. Spectrosc., 1992, 13, 1. 41 Forsyth, D. S., and Marshall, W. D., Environ. Sci. Technol.. 1986, 20, 1033. Paper 6/05880I Received August 27, 1996 Accepted October 15, 1996 18 Analyst, Janu