Fresenius Zeitschrift flit Fresenius Z Anal Chem (1988) 332:652-656

�9 Springer-Verlag 1988

Neutron activation analysis of the NIST Bovine Serum Standard Reference Material using chemical separations Robert R. Greenberg, Rolf Zeisler, Howard M. Kingston, and Theresa M. Sullivan

Center for Analytical Chemistry, National Institute of Standards and Technology (formerly National Bureau of Standards), Gaithersburg, MD 20899, USA

Neutronenaktivierungsanalyse des Standardreferenzmaterials NIST Bovine Serum mit Hilfe chemischer Trennungen

Summary. The US National Institute of Standards and Tech- nology is currently in the process of certifying a Bovine Serum Standard Reference Material. In addition to elements normally considered to be of clinical interest, a number of other elements, which are analytically more difficult to determine yet are of importance from either a nutritional or toxicological viewpoint, are being determined by a variety of analytical techniques. Neutron activation analysis in com- bination with appropriate pre- or post-irradiation chemical separations, has been used to determine many of these diffi- cult elements.

Introduction

As bioanalytical methods for trace element determinations have become more sensitive, biological and medical trace element research has included studies of many of the "difficult" elements such as A1, As, Cd, Cr, Mo, Mn, Sb and V. These elements are currently of interest, for example, in human blood serum where they typically occur at the low ng/g level or below. The accurate determination of trace elements at these levels usually requires certified reference materials for method evaluation and for quality assurance purposes. This need for low-level reference materials for accurate analyses of human blood serum and related ma- terials has been well documented [1]. To help meet this need the US National Institute of Standards and Technology, and the US Department of Agriculture have cooperated in the production and certification of a frozen Bovine Serum Stan- dard Reference Material (SRM) with extremely low levels of a wide variety of trace elements.

The production of certified reference materials intended for quality assurance use when determining trace and ultra- trace level elemental concentrations in biological and medi- cal samples is particularly challenging. Extreme precautions have been taken during the collection, bottling, storage and analysis of the Bovine Serum SRM to prevent, or at least minimize, contamination. This material will be a good sub- stitute for human blood serum because the trace element levels in human and bovine sera are nearly identical [2]. In

Offprint requests to: R. R. Greenberg

addition, bovine serum can be handled without the problems associated with actual human serum such as the potential transmission of hazardous human viruses. It thus fulfills the same quality assurance role for trace element analysis without adding the danger of handling human serum. Another advantage of using bovine serum instead of human serum for an SRM, is that the former can be obtained, in much larger quantities, using procedures which lend themselves to the high degree of contamination control required for a non-contaminated material.

The certification of trace elements in this Bovine Serum (SRM 1598) was particularly difficult due to the very low levels of many of the elements of interest. The certification process usually involves the determination of elemental con- centration by two or more independent reference methods, or alternatively, by one (or more) "'definitive methods." A variety of analytical techniques has been used for the Bovine Serum certification. For several of these techniques, an ex- tension of procedures beyond previously demonstrated mea- surement competence has been required. One of the analyti- cal techniques used for the Bovine Serum was neutron activation analysis (NAA), which was able to determine the concentrations of most of the elements desired for certifica- tion. Many of the difficult, low-level elements have been determined by NAA with appropriate pre- or post-irradia- tion chemical separations. This paper describes the pre- and post-irradiation chemical separation procedures used for these determinations, as well as presents the data obtained for the new Bovine Serum SRM.

Chemical separations, in this work, have been used to reduce interferences from matrix activities such as from Na, C1, Br and P, or in some cases to totally isolate the element of interest from essentially all other radionuclides. These separation procedures thus enhance the specificity and sensitivity of the method. Post-irradiation separations are particularly useful for low-level determinations since no re- agent blank has to be considered. Two radiochemical separa- tion procedures have been applied to the Bovine Serum. Arsenic, along with Mo, has been determined using the inorganic ion exchanger H M D (hydrated manganese dioxide), and Cd along with Cu, have been determined in the eluent from the same samples using sequential extrac- tions into solutions of bismuth and zinc diethyldithiocar- bamates in chloroform. Chromium has been determined in an additional set of samples following extraction into a solution of tribenzylamine in chloroform.

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Aluminum, Cu, Mn and V have been determined following a pre-irradiation separation using Chelex-100. All dissolution and chemical manipulation of the samples were done under class 100 clean air conditions. Although the post-irradiation advantage of blank-free chemistry was lost, separating before irradiation allowed complete sample dis- solution, as well as a relatively slow, but highly quantitative and selective, separation procedure to be used. This would not be possible after irradiation in view of the short half- lives of the activation products of A1 and V.

Experimental Reagents. Hydrated manganese dioxide (HMD) inorganic- ion exchanger was obtained from Carlo Erba, Milan (Italy). Bismuth and zinc diethyldithiocarbamates (DDC) were pre- pared from aqueous solutions of NaDDC and bismuth (or zinc) nitrates as described elsewhere [3]. All other reagents used were ACS reagent grade or better.

Chromatographic columns. The polyethylene chromato- graphic columns (7 mm i.d.) were also obtained from Carlo Erba. Three ml of the inorganic-ion exchanger could be packed into the columns with the reservoir on top of the columns holding up to 15 ml of liquid.

Microwave digestion unit and dissolution vessels. The microwave digestion unit used for pre-digestion of some samples was a model MDS 81D (CEM, Inc.). The 60-ml Teflon PFA digestion vessels were obtained from Savellex Corp.

Irradiations. Irradiations for Cr determination were carried out in the RT-4 pneumatic tube facility of the NBS reactor at a thermal neutron flux of 2.7 x 1013 n/cm2-s, while deter- mination of other elements were carried out in the RT-3 facility at a flux of 1 x 1014 n/cm2-s.

Counting equipment. Two different Ge(HP) detectors were used for the radiochemical determinations: one having an efficiency of approximately 30% and a resolution (FWHM) of approximately 0.8 keV at 122 keV, and a second with an efficiency of approximately 50% and a resolution (FWHM) of approximately 1.2 keV at 122 keV. Resolution figures are specified at low energies as the elements determined radiochemically in this work had energies below 600 keV. A third detector, with an efficiency of approximately 10% and a resolution (FWHM) of approximately 2.5 keV at t332 keV (at an amplifier shaping time of I gs) was used for the analyses involving the pre-irradiation separation. This short amplifier time constant was selected to minimize pulse- pileup events. The detectors were connected either directly to an ND-6620 or ND-6700 computer-based analyzer system, or indirectly through an ND-66 analyzer. Peaks of interest were integrated via a channel-by-channel summation routine using peak and background regions specified by the analyst, and were compared as a QA measure to areas determined by a computer-run peak search routine.

Radiochemical procedure for As, Cd, Cu, Mo and Sb. In view of the very low levels, and the relatively short half-

lives of most of the elements of interest, no more than two samples and/or blanks could be processed together. Three of the samples analyzed were lyophilized prior to irradiation and a fourth was irradiated in the wet state to check for any differences due to contamination, or losses of volatile elements during lyophilization; no differences were ob- served. Only one sample was irradiated in the wet state due to the considerable amount of difficulty in removing the wet samples from the quartz vial after irradiation. A pre- digestion procedure using a microwave oven was employed to reduce total dissolution time while maintaining complete destruction of the organic matrix.

Multiple Bovine Sermaa samples containing approx- imately 0.5 g (fresh weight) were transferred to cleaned, high-purity quartz vials and irradiated for 2 or 3 h in RT-3 along with liquid standards also sealed in quartz. Several days after irradiation, the quartz vials were washed in aqua regia and opened. Composite samples ( 1 - 2 g each) were prepared by transferring the contents of 2 to 4 of the vials to a single PFA microwave dissolution vessel. The nearly- empty quartz vials for each composite sample were trans- ferred to a small beaker with 25 gg of As, Mo and Sb carriers, 275 gg of Cd and Cu carriers, and 6.5 ml of concen- trated nitric acid was added to loosen residual material sticking to the walls of the vials. Blanks were prepared by leaching empty (irradiated) quartz vials in the same manner. After approximately 30 min, the acid and residual material were transferred to the PFA dissolution vessels, which were closed and heated in the microwave dissolution unit (in pairs) for 8 min at 35% power (164 watts). The dissolution vessels were cooled and opened, and the contents transferred to TFE Teflon or Pyrex beakers. Additional nitric acid was used to rinse the empty quartz vials and the dissolution vessels, and this rinse was added to the appropriate dissolved sample. The samples were then heated at low temperature (open) on a hot plate for approximately 30 rain, after which 5 ml of perchloric acid was added. The samples were heated, gradually increasing the temperature, until approximately 0.5 ml of perchloric acid remained. The samples were then cooled and 15 ml of 1 mol/1 nitric acid was added. Arsenic, Mo and Sb were then separated with the inorganic-ion ex- changer H M D using the same procedure previously applied to the Milk Powder SRM and described in detail elsewhere [4]. Basically this procedure involved pre-conditioning the resin with 15 ml of a solution containing 1 mol/1 of both nitric and phosphoric acids, washing the resin three times with 15 ml of I mol/1 nitric acid, passing the sample (in 15 ml of 1 mol/1 nitric acid) through the resin, and washing the resin twice with a solution containing I mol/1 nitric acid and 0.0025 mol/1 phosphoric acid. After separation, the HMD was transferred to a scintillation vial, centrifuged, and the supernatant liquid was discarded. The samples were counted immediately to determine As, and again after at least 48 h to determine Mo. This decay was necessary to allow equili- bration between Mo-99 and its Tc-99m daughter (half-life = 6.0 h). Antimony was determined in both counts.

The combined eluent and washes for each sample were next processed to separate Cu and Cd. Details of this proce- dure are given elsewhere [4]. Basically the procedure involves the addition of 100 mg of Zn holdback carrier, adjusting the pH to 1.5 with amminia, extracting Cu into 25 ml of 0.003 mol/1 [Bi(DDC)3] in chloroform (30min shaking time), extracting Cd from the remaining aqueous fraction into 25 ml of 0.005 mol/1 Zn(DDC)2 in chloroform (2 min),

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washing the organic fraction with 25 ml of an aqueous solu- tion, at pH 1.5, containing 100 mg of Zn carrier, and back- extracting Cd into 20 ml of 2 tool/1 HC1. The Bi(DDC)a/ chloroform fraction containing Cu was counted immediately, while the HC1 solution containing Cd was allowed to decay for 24 h prior to counting to allow equilibration between Cd-l l5 and its daughter, In - l l5m (half-life=4.5 h), which was the nuclide used for Cd quantitation. There was one modification to the published procedure; the Zn(DDC2) organic fraction containing Cd was washed once instead of twice with the aqueous Zn carrier. With this separation procedure, recovery of As, Cu, Mo and Sb is quantitative ( > 99.5%), while recovery of Cd is nearly quantitative (96.8 + 0.3 %), and is highly reproduc- ible.

Table 1. Elemental concentrations observed in Bovine Serum, SRM 1598 (corrected for blanks) with estimated 95 % confidence intervals

A1 (ng/g) 4.1 • 0.7 As (pg/g) 148 • 16 Cd (pg/g) 93 • 24 Cr (pg/g) 177 _+ 21 Cu (ng/g) 715 • 14" Mn (ng/g) 3.72 • 0.15 Mo(ng/g) 11,2 _+ 0.6 Sb (pg/g) < 200 b V (pg/g) 55 • 34

a Cu concentration is a weighted mean of 705 + 19 ng/g determined via the Chelex 100 procedure, and 722• determined radiochemically b Value listed for Sb corresponds to a "less than or equal to value"

Radiochemicalprocedurefor Cr. Approximately 1 g samples of Bovine Serum were poured into weighed quartz vials which were stoppered with a Teflon plug under class 100 laminar flow in a clean room. The samples were then weighed and lyophilized in the clean room. Two samples of SRM 1549 (Milk Powder) were packaged similarly as controls. The samples were irradiated with standards for 16 h in RT-4. Approximately one month after irradiation, the samples were transferred to Teflon vessels along with 1 mg of Cr carrier. The samples were dissolved and Cr separated using a previously described procedure [5]. A sum- mary of the procedure is as follows: the samples were dis- solved using a two-stage dissolution procedure involving nitric and sulfuric acids followed by perchloric and sulfuric acids. Chromium was then oxidized and maintained in the + 6 state with KMnO4, and extracted into a 5% solution of tribenzylamine (TBA) in chloroform. The Cr was then back- extracted into an aqueous solution containing 2 mol/1 NaOH. The pH was adjusted to 6.5 _+ 0.5 with acetic acid and barium acetate was added to precipitate barium chromate, which was filtered, washed and counted. The overall yield for this procedure has been determined pre- viously to be 98.1 _+ 0.5%.

Pre-irradiation procedure for AI, Cu, Mn and V. Five samples containing approximately 20 g of Bovine Serum were trans- ferred to PFA Teflon containers and dissolved (closed) with nitric acid, and then perchloric acid (open). The large sample size was selected in order to minimize the effect of the A1 blank introduced during sample irradiation (discussed be- low). One 15 g sample of the old Bovine Serum (RM 8419) was processed in the same manner as a control. A series of reagent blanks was also prepared in a similar manner. One additional control sample of Trace Elements in Water (SRM 1643a) was also prepared, although no dissolution step was required. The pH of each sample was adjusted to 5 . 3 - 5.9 and the samples were separated utilizing a Chelex-100 separation [6]. Several changes in the published procedure were made, including: closed vessel decomposition to reduce blank and increase efficiency; ultra-purification of the I tool/1 ammonium acetate accomplished by passing this reagent through a Chelex-100 column prior to use in the separation procedure, and as the final step in the separation procedure, transfer of the Chelex-100 resin, while moist, to an acid-cleaned piece of parafilm, which was folded and placed in a second such container. The encapsulated sample was then sealed in a polyethylene bag for irradiation. All

Table 2. Blank concentrations observed for the Bovine Serum with estimated 95% confidence intervals

A1 (ng/g) 1.47 • 0.45 As (pg/g) 3.5 • 0.8 Cd (pg/g) 50 • 16 Cr (pg/g) < 10 Cu (rig/g) < 4 (Chelex 100) Cu (ng/g) 1.4 ___ 0.5 (RNAA) Mn (ng/g) < 0.004 Mo (rig/g) 0.09_ 0.03 Sb (pg/g) 300 • 300 V(pg/g) - 1 3 . 8 -I- 17.7"

a This value corresponds to a negative signal blank due to a non- linear background under the peak of interest - see text

sample preparation prior to irradiation was done under class 100 clean air conditions.

The samples, controls, multielemental standards, and blanks were individually irradiated for I min in RT-3. After irradiation, the samples were removed from the packaging material and washed into a 60 ml polyethylene vial with deionized water. The samples were then counted for 10 min to determine A1, Cu and V. A counting geometry of 10 cm was used. Approximately 2 h after irradiation, the samples were recounted to determine Mn after the major activity of the spectrum, C1-38, had decayed away. Some samples were counted a third time. Standards, blanks and check samples were counted in the same manner as the samples.

Results and discussion

The results for Bovine Serum analyses are listed in Table 1, blank concentrations are given in Table 2, and concentra- tions for control samples are in Table 3. Copper was deter- mined in the Bovine Serum using two nearly independent methods, i.e., measurement of Cu-66 after a pre-irradiation separation and measurement of Cu-64 after a post-irradia- tion separation. The agreement between the two sets of Cu concentrations (see footnote at the end of paper) gives added confidence to the results observed for the other dements determined with Cu using the group separations, since the possibility of any sources of error which affect the entire sample, such as irradiation geometry errors, weighing errors, loss of transferred material etc., can be eliminated.

Table 3. Elemental concentrations (ng/g) and estimated 95% confidence intervals observed for control samples

A1 Cr Cu Mn V

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Bovine serum, RM 8419 -- Chelex 100 procedure This work 9 • 2 Recommended values [2] 13 _+ 5

Trace Elements in Water, SRM 1643a - Chelex 100 procedure This work 118 • 8 Certified values Previous NAA 129 • 10 results 121 • 8

Milk powder, SRM 1549 - Radiochemical procedure This work Certified value

2.6 -+ 0.2 2.6_+0.7

696 • 32 2 . 2 4 _ + 0 . 1 6 0.60• 730 • 2.5 _+0.5 <2

<40 30.4 +1.4 54.2 • 18 • 2 31 _+ 2 53 • 3 19.1 • 0.6 30.9 • 52 __ 1

The uncertainties given in Tables 1 - 3 correspond to estimated 95% confidence intervals and include allowances for systematic as well as random errors. Due to the high and variable nature of the Sb blank, an actual concentration could not be determined, and a value of less than or equal to 200 pg/g is given. The concentrations given for each of the elements in these tables are the weighted means for the individual samples analyzed. Weighting factors were the inverse of the variances due to counting statistics combined in quadrature with an additional 1% random error (estimated) to account for variations in counting geometry. The estimated 95% confidence levels were calculated by combining in quadrature the random errors for the samples and blanks, and adding linearly an estimate of the possible systematic error for this analysis. For the concentrations of A1, As, Cd, Cr, Cu (Chelex-100), Mn, Mo, and V in the Bovine Serum samples the random error was considered to be 1.96 times the a priori standard deviations of the mean which were calculated by combining in quadrature the counting statistics with an additional 1% error to account for variations in counting geometry. This method of calculating random error was chosen as the observed pre- cision was consistent with the counting statistics (probability between 5 and 95%). The same method was used for As, Cd, Mo and V in the blanks. Since the a postieri uncertainties for the concentrations of Cu both in the Serum samples and in the blanks (via RNAA), as well as A1 in the blanks, were considerably larger than the a priori value, the a postieri uncertainties [ts/sq.rt.(n)] were used as the random errors. The estimated systematic error in this analysis was considered to be 1%, at the 95% confidence level, for the radiochemical analyses, and 2% for the pre-irradiation separations. This systematic error included allowances for irradiation geometry differences between samples and standards, as well as potential errors in preparation of the standards.

In view of the extremely low levels of the elements deter- mined in this work, the blanks were highly significant for many elements, even those determined radiochemically. The A1 blank was due to transfer of activated A1-28 from the parafilm used to hold the sample, to the Chelex-100 resin (as a result of nuclear recoil during the irradiation). An apparent V blank resulting from a small amount of C1 re- maining on the resin, was also significant and is discussed below. Since the blank concentrations for these two elements

represented approximately 30% of the sample content, a series of ten blanks were analyzed for A1 and V.

The separated samples each contained approximately 100 gg of C1, possibly as a result of heavy HC1 usage in the clean room where the samples were separated and partially dried. The HC1 appears to have bound to the ammonium acetate solution remaining on the Chelex-100 resin, and interferred indirectly during the analysis by elevating the background levels under the peaks of interest. This signifi- cantly worsened the detection limit for V (and to a lesser extent A1) as the 1434 keV gamma-ray from V-52 lies just above (slightly higher energy) the Compton edge of the 1642 keV gamma-ray from C1-38. Due to this proximity to the Compton edge, the background under the V-52 peak had a non-linear (upwardly curving) shape. Since a linear background was assumed (and subtracted), the actual background under the V-52 peak was overestimated, re- sulting in an underestimation of the actual number of counts in this peak. This bias was removed by subtracting a negative signal blank (equivalent to a V concentration of - 13.8 pg/g) from the V concentrations in the samples. This signal blank was determined by using the same channel-by- channel summation to integrate the 1434 keV peak region of V in the Bovine Serum samples, and in 10 blanks. Since the serum samples and blanks all had the same amount of C1, this procedure was equivalent to subtracting an average C1 background (blank) spectrum from each serum sample prior to peak integration.

A true blank value for Cr could not be determined since the irradiated sample material was transferred, after irradia- tion, from the quartz vials without the use of acid to dissolve residual material sticking to the walls. This loss of sample was corrected for by reweighing the transferred material. Thus leaching the walls of empty quartz vials with acid would overestimate any Cr blank. Since some method of transfer was necessary to estimate a blank contribution, irradiated quartz vials were filled with deionized water and allowed to sit for several hours. The water was then removed and submitted to gamma spectrometry. No Cr-51 was ob- served.

Conclusions

The concentrations of seven biologically important elements at the ng/g level, as well as Cu at the gg/g level, have been

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determined in the new Bovine Serum S R M utilizing N A A combined with pre- and post - i r radia t ion chemical separa- tions. These concentrations, when combined with the results from other analytical techniques, will al low the cerification of many of these difficult elements in a natural matr ix mate- rial. The new Bovine Serum SRM should be of great value for method development, and for quali ty assurance purposes, when researchers analyze b lood serum or related materials.

Note. Certain commercial equipment, instruments or materials are identified in this paper in order to adequately specify the experimen- tal procedure. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Tech- nology, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose.

References

1. Versieck J, Vanballenberghe L, De Kesel A, Hoste J, Wallaeys B, Vandenhaute J, Baeck N, Steyaert H, Byrne A, Sunderman F (1988) Anal Chim Acta 104:63-75

2. Veillon C, Lewis S, Patterson K, Wolf W, Harnly J, Versieck J, Vanballenberghe L, Cornelis R, O'Haver T (1985) Anal Chem 57:2106-2109

3. Gallorini M, Greenberg RR, Gills TE (1978) Anal Chem 50:1479-1481

4. Greenberg RR (1986) Anal Chem 58:2511-2516 5. Greenberg RR, Zeisler R (1988) J Radioanal Nucl Chem, in

press 6. Greenberg RR, Kingston HM (1983) Anal Chem 55:1160-1165

Received August 23, 1988