Microwave digestion methods for the determination of trace elements

( Springer-Verlag 1996

Fresenius J Anal Chem (1996) 355: 120—128

OR I G I N A L P AP E R

Michael Krachler · Herbert Radner · Kurt J. Irgolic

Microwave digestion methods for the determination of trace elements in brain and liver samples by inductively coupled plasma mass spectrometry

Received: 16 June 1995/Revised: 21 July 1995/Accepted: 25 July 1995

Abstract Two microwave digestion systems (openfocused and closed-pressurized) were tested for the mineralization of human brain and bovine liver (NIST SRM 1577a) as dissolution steps prior to the determination of 16 trace elements (Bi, Cd, Co, Cs, Cu, Fe, Hg, Mn, Mo, Pb, Rb, Sb, Sn, Sr, Tl, and Zn) by inductively coupled plasma mass spectrometry (ICPMS). Digestion parameters (mass of sample, digestion mixture, and power/time steps) were optimized using temperature and pressure sensors. Digestions with the open-focused microwave system require larger volumes of conc. HNO and 30% H O than digestions with 3 2 2 the closed-pressurized system. Both systems produce correct results for the bovine liver samples. The concentrations obtained for the digests of the open-focused system tend to be less precise than the concentrations from the ‘‘closed-pressurized’’ digests. Because the ‘‘open-focused’’ digests must be diluted to 50 mL to bring the acid concentration to 0.7—2.0 mol/L required by the ICP-MS (closed-pressurized digests need to be diluted to only 20 mL), the detection limits for the system with the open-focused digestion are higher than for the system with the closed-pressurized digestor. The open-focused digestor cannot handle more than 150 mg brain tissue, whereas the closed-pressurized system can mineralize 470 mg. The latter method gave better results with brain tissue than the open-focused system. The preparation of brain tissue as reference material for the determination of trace elements in brain samples is described.

M. Krachler · K.J. Irgolic ( ) Institut fu¨r Analytische Chemie, Karl-Franzens-Universita¨t Graz, Universita¨tsplatz 1, A-8010 Graz, Austria H. Radner Institut fu¨r Pathologie, Karl-Franzens-Universita¨t Graz, Auenbruggerplatz 25, A-8036 Graz, Austria

Introduction Inductively coupled plasma mass spectrometry (ICPMS) is a relatively new, and very powerful technique for the simultaneous determination of trace elements with very low detection limits and linear calibration curves over five orders of magnitude. ICP-MS has been used for the determination of trace and ultratrace elements in many biological samples. A review of the capabilities of ICP-MS for the quantification of trace elements in body fluids and tissues was published in 1993 by Vanhoe [1]. The necessity to completely oxidize the organic matrix of biological samples prior to the determination of trace elements by instrumental methods to achieve accurate and reproducible results is widely recognized [2, 3]. The very low detection limits claimed for ICP-MS can only be reached when the concentration of total dissolved solids in the solution to be analyzed is kept to a minimum. High concentrations of inorganic and organic solids change the properties of the argon plasma, cause deposits to form on the central tube of the plasma torch, may lead to clogged orifices in the cones of the ICP-MS, and will certainly increase the detection limits. The organic (but not the inorganic) matrix of the sample can be removed by oxidative conversion to carbon dioxide and water. During the past decade microwave digestion methods have become popular because they are more reproducible, more accurate, and less time-consuming than conventional digestions on hot plates in open beakers [4]. Microwave systems keep blank levels low because only small volumes of reagents are required, and allow more samples to be processed per hour than conventional digestion systems. Three different approaches to microwave digestion were developed for the decomposition of biological materials: openfocused microwave systems [5—8], closed-pressurized microwave digestion systems [9—19], and a combination of both systems [20]. .

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The aim of this work was to test the closed-pressurized and open-focused microwave systems with lipidrich brain and liver samples and to check the suitability of the digests for the determination of trace elements by ICP-MS.

Table 1 Operating conditions for the ICP-MS Plasma:

rf power

Argon gas flows:

cooling gas auxiliary gas nebulizer gas nebulizer

Nebulization:

Experimental Apparatus MLS 1200 MEGA digestion system. The high-performance digestion unit MLS (Microwave Laboratory System) 1200 MEGA from Milestone, Leutkirch, Germany, with a rotor for ten Teflon digestion vessels designed for pressures up to 30 bar (1 bar B1]105 PaB750 Torr), served as the closed-pressurized microwave system. The vessels are equipped with a pressure release system to prevent explosions. A specially designed vessel, that allows a temperature sensor and a pressure sensor to be connected and the progress of the digestion to be monitored, can be substituted for one of the ten vessels. The temperature and pressure data are displayed on the instrument and can be stored and visualized with a dedicated computer and software. The magnetron mounted on the ceiling of the microwave oven can operate at powers of 250 or 1000 W. The 1000 W stage can be pulsed to deliver time-averaged power in 10 W steps in the range from 10 to 1000 W. Prolabo Microdigest A 301. The open-focused microwave system, a Microdigest A 301 from Prolabo (Paris, France), has one well for the irradiation of a single digestion vessel. From a carousel with a capacity of 16 vessels the well is loaded automatically by a robot arm with the vessel to be irradiated. When a vessel is in the well the reagents are added with a three-way pump. Then the magnetron is turned on and the digestion is carried out according to a preselected program. When the program is completed, the vessel is returned to the carousel and replaced by the next vessel. The digestions were carried out in 50 mL, flat-bottomed Pyrex vessels each fitted with a reflux condenser. The energy of the microwaves (2.45 GHz, wavelengthB10 cm) is transferred to the digestion mixture via a wave guide. The magnetron delivers 200 W continuously. The continuous (unpulsed) power can be regulated from 20 to 200 W in 10 W steps. ICP-MS. Inductively coupled plasma mass spectrometric measurements were performed with a VG PlasmaQuad Turbo Plus (VG Elemental Ltd., Winford, UK) equipped with a Meinhard concentric glass nebulizer, a double-pass, Scott-type spray chamber (water cooled, 0 °C), and a Gilson Minipuls-3 peristaltic pump. The operating conditions for the ICP-MS are summarized in Table 1. Freeze-drier. The Alpha 1—4 freeze dryer (Christ, Osterode, Germany) equipped with a control panel LDC-1M, a temperature sensor, and a pressure sensor, was operated at 0.05 mbar and 20 °C.

Reagents and materials Stock solutions (1000 mg element/L) for each of the 16 elements to be determined in the digests by ICP-MS were prepared by diluting the content of Titrisol ampoules (Merck, Darmstadt, Germany) containing solutions of element nitrates or oxides in water, dilute nitric acid, or dilute hydrochloric acid to one liter with NANOpure water. By combination and appropriate dilution of aliquots of these stock solutions with 0.29 mol/L high-purity nitric acid two multielement standard solutions with concentrations of 10 mg/L for each element were obtained. The first solution contained Cu, Fe, Rb, and

Ion sampling:

sample cone skimmer cone

Vacuum:

expansion intermediate analyzer channels/amu dwell time data aquisition

Measurement:

forward 1.40 kW reflected(5 W 13.5 L min~1 1.1 L min~1 0.84 L min~1 Meinhard concentric glass nebulizer type: SB-30-A3, uptake+1.0 mL min~1 Nickel, orifice 1 mm diameter Nickel; orifice 0.7 mm diameter 1.6 mbar 1.0]10~4 mbar 2.1]10~6 mbar 20 320 ls scanning mode

Zn (Standard 1), the second Bi, Cd, Co, Cs, Hg, Mn, Mo, Pb, Sb, Sn, Sr, and Tl (Standard 2). A third solution containing Ga (GaCl in 3 1 M HCl), In (In O in dilute HCl), and Re (Aldrich No. 20,740-3) at 2 3 concentration of 10 mg/L for each element was prepared similarly (Standard 3). Aliquots of Standard 1 were diluted with 0.29 mol/L nitric acid to produce solutions of 50, 100, or 150 lg/L for each element. Before the volumetric flasks were filled to the mark, aliquots of Standard 3 were added to achieve a final concentration of 50 lg/L for each element. The standard 2 solution was also spiked with Ga-In-Re to 50 lg/L and then diluted to achieve concentrations of 2, 5 or 10 lg/L. The calibration curves were established with these most dilute solutions containing the Ga-In-Re spikes that are needed for the internal calibration of the ICP-MS response. The digestions were carried out with high purity hydrogen peroxide (30%, Suprapur', Merck) and conc. nitric acid purified by subboiling distillation. All glassware was cleaned by soaking for 24 h in conc. HNO followed by rinsing three times with high resistivity 3 water (18 M) cm, Barnstead NANOpure, Boston, USA). Bovine liver SRM 1577a was purchased from the National Institute of Standards and Technology (Gaithersburg, Maryland, USA) and dried at 0.05 mbar and 20 °C for 24 h in a lyophilizer before digestion. Brain reference material. The brain collected 8 h post mortem at the time of autopsy from a 58-year old male, who had died from a myocardial infarction, was rinsed with high-purity water. All further dissections of the brain were carried out with Teflon or ceramic tools. After removal of the leptomeninges, the surface of the brain was washed with NANOpure water by pouring a stream of water over the brain resting on a plastic-covered board. The cerebellum was removed and the remaining brain (B1000 g) was cut into B1 cm thick slices, which were dissected into B2 cm pieces. Each piece was rinsed with high-purity water. The pieces were placed into six 200 mL, wide-mouthed polyethylene bottles. The bottles were tightly closed with screw caps, placed immediately into a freezer kept at !30 °C, and stored in the freezer. To remove most of the water from the brain slices, the open bottles were kept for three days in a freeze-drier at 0.05 mbar and 20 °C. The relatively dry material was broken into smaller (B0.5 cm) pieces on a quartz dish with a titanium fork. The resulting pieces were kept for two days in the freezedrier at 0.05 mbar and 20 °C. The dry material was then ground in an agate mortar with an agate pestle. The resulting powder was passed through a Nylon sieve (1 mm). The sieved, coarse-grained brain powder was freeze-dried for another day. Before the powder was introduced into an ultracentrifugal mill (Type ZM 1000, Retsch company, Haan, Germany) equipped with a titanium ('99.4%)

122 sieve and rotor, the powder was cooled to !30 °C. The pulverization in the mill was more effective, when the brain powder was frozen. The fine powder was then dried at 80 °C to constant mass in a clean, uncontaminated drying oven (Heraeus, Germany). From the 1000 g wet brain 139 g of dry powder were obtained. For homogenization the powder was transferred into a polyethylene flask (250 mL). The flask was placed on a home-made rotary mixer and turned for 24 h.

Digestion procedures For the digestion only concentrated nitric acid and 30% hydrogen peroxide could be used, because potential reagents such as sulfuric acid or perchloric acid give rise to serious spectral interferences in the ICP-MS measurements. Optimization of digestions with the open-focused microwave system. The dry brain samples (not more than 150 mg) or the dry bovine liver (not more than 250 mg) were weighed to 0.1 mg into each of the 16 Pyrex digestion flasks. The 16 flasks, all equipped with a reflux condenser, were placed into the automated carousel, from which the robot arm transferred each flask into the mircrowave well. The digestion reagents were measured automatically into the flasks by a 3-way pump. Nitric acid (volumes from 7 to 10 mL) was added in steps 1 and 7 of the digestion program, hydrogen peroxide (volumes 2 to 5 mL) in steps 4 and 9. The microwave power applied during the steps of the digestion program (Table 2) was varied from 40 to 120 W. The durations of the digestion steps were chosen between 2 and 10 min. After completion of a program the flask was transferred from the microwave well back to the carousel. The next flask was readied for digestion under different conditions. The solutions were quantitatively transferred into 50-mL volumetric flasks. Aliquots of the Standard 3 solution were added and the flask was filled to the mark with NANOpure water. Optimization of digestions with the closed-pressurized microwave system. For the development and optimization of the digestion procedure for brain tissue the rotor with ten Teflon digestion vessels was employed. Each of the nine vessels was filled with 3 mL of water. A specially designed lid on the tenth Teflon vessel allowed the connection of a temperature and a pressure sensor. Into this vessel were placed about 150 mg (range 145—155 mg) of the dry brain. The digestion mixtures with varying amounts of conc. nitric acid (1.5 or 2.0 mL) and 30% hydrogen peroxide (0.5, 1.0, 1.5 mL) were added.

Table 3 Optimized digestion program for the closedpressurized microwave digestion

The rotor with the closed vessels was placed on the turntable that oscillated through 180 ° during the entire digestion procedure. The first digestions were performed with the standard digestion program (Table 3) recommended by the manufacturer (no 800 W step, shorter 600 W step) that is claimed to be sufficient for more than 90% of all biological samples. The digestions under the recommended program yielded turbid solutions. Therefore, the 600 W step was prolonged and an additionally 800 W step was added. Before the rotor was taken from the microwave, a two-minute ventilation step (no microwave power) cooled the vessels sufficiently to protect the operator from burns. Then, the rotor was transferred into a water bath and allowed to cool for about half an hour to reduce the pressure inside the vessels to ambient values. The solutions were quantitatively transferred into 20 mL volumetric flasks. Aliquots of the Standard 3 solution were added and the flasks were filled to the mark with NANOpure water. Homogeneity testing of the brain reference material. Before analysis the powdered brain was freeze-dried at 5 mbar and 25 °C for 24 h. Twenty samples (240 to 470 mg) from different locations within the bottle were taken and digested with the closed-pressurized microwave system under the optimized program (Table 3). The digests were analyzed by ICP-MS.

Table 2 Optimized conditions for the open-focused microwave digestions Step

Reagent addition

Volume [mL]

Power [W]

Time [min]

1 2 3 4 5 6 7 8 9 10 11 12

conc. HNO 3 — — 30% H O 2 2 — — conc. HNO 3 — 30% H O 2 2 — — —

7 — — 3 — — 4 — 1 — — —

— 40 50 — 40 60 — 50 — 50 60 80

— 10 3 — 5 2 — 10 — 3 5 7

Total volume

15 mL

Total time

45 min

Step

Reagent addition

Volume [mL]

Microwave power [W]

Average power [W]

Time [min]

1 2 3 4 5 6 7 8 9 10 11

conc. HNO 30% H O 3 2 2 — — — — — — — — —

2.0 1.5 — — — — — — — — —

— — 250 0 250 0 1000! 0 1000! 1000! 0

— — 250

— — 3.0 0.5 5.0 0.5 5.0 0.5 5.0 (3.0)" 5.0 2.0

Total volume

3.5 mL

! Pulsed 1000 W " Recommended Standard program; no step 10

250 450 600 800 Total time

26.5 min

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Results and discussion The determination of trace elements in human samples (blood, serum, urine, hair) has gained in importance during the past decades [21]. Concentrations of toxic and essential elements in such samples provide information about the trace element status and can be the basis of appropriate clinical interventions. Improved analytical instruments allow now the simultaneous quantification of elements even in small samples. To establish normal concentrations of trace elements in human brain centers, a reproducible, reliable, accurate, and rapid method for the mineralization of the lipidrich brain tissues was needed that must produce solutions analyzable without interferences by ICP-MS. Conditions imposed by this simultaneous, multi-element instrument include: high purity for all digestion reagents to keep the blank levels as low as possible; the oxidizing agents must not produce fragments that interfere with the mass spectrometric determination of the elements; the concentrations of acids in the solution to be aspirated should not be too high. The ideal digestion method should allow the mineralization of many samples in the shortest possible time, because each brain will be divided into approximately sixty centers and statistically valid, normal concentrations of trace elements in these centers can be obtained only, when at least 30 brains are analyzed. Among the potential digestion methods [22], mineralization in open beakers cannot be used because they require too much time and are threatened by contamination. Microwave digestion systems were found to be rapid, almost contamination free, and especially suited for samples that are difficult to mineralize [4]. Two microwave systems (Prolabo, open-focused; MLS, closed-pressurized) were available for evaluation with the lipid-rich (freeze-dried brain 60%, liver 10% lipids), difficult-to-digest brain samples.

hamper the determination of elements forming lowsolubility sulfates [5]. Perchloric acid gives rise to 40Ar35Cl`, 40Ar37Cl`, CCl` 2 , and 35Cl16O` which interfere with 75As, 77Se, 82Se, and 51V. The determination of trace amounts of chromium with ICP-MS is prone to interferences from 40Ar12C`, when organic substances are in the nebulized solution, and in a minor way by 35Cl16OH`. For the determination of trace amounts of chromium the minor isotope of chromium, 53Cr, cannot be used, because 53Cr with only 9.55% natural abundance produces a low-intensity signal at the same m/z as 40Ar37Cl`. Nitric acid produces only one major isobaric interference 40Ar14N` with 54Fe. This interference is not serious, because iron can be determined using 57Fe. Although 57Fe is a minor isotope of iron (2.15% natural abundance), the high concentrations of iron in the samples allow the use of this isotope. Interferences by combinations of nitrogen with other argon isotopes (36Ar14N`, 38Ar14N`) are not serious because vanadium is not determined at m/z 50 because of the low natural abundance of 0.24% for 50V and the interference with the determination of chromium by 38Ar14N` is negligible compared with 40Ar12C. Although conc. nitric acid would completely oxidize the organic matrix, if sufficiently large volumes of acid were used, such large volumes cannot be employed when the resulting solutions are to be analyzed by ICP-MS. Nitric acid concentrations in the solutions to be nebulized higher than approximately 1.5 mol/L cause rather rapid and severe corrosion of the sampler and the skimmer cones. Dilution of the digests with distilled water to sufficiently large volumes and

Selection of digesting reagents The very low detection limits of the ICP-MS (0.01—1 ng/mL) can only be realized, when the concentrations of dissolved organic and inorganic concomitants are not too high. The organic components can be oxidized to carbon dioxide and water. Oxidizing reagents used for digestions must not produce fragments that have the same m/z values as trace element ions of interest. Although sulfuric acid is frequently used for the decomposition of the organic matrix of biological samples [5—8] it cannot be used because of serious interferences with several elements of interest. Sulfuric acid decomposes in the plasma to sulfur-oxygen species interfering with copper at mass 65 (63Cu is disturbed by 40Ar23Na freeze-dried brain \8000 mg/kg Na) and zinc at mass 64—70. Furthermore, sulfuric acid will

Fig. 1 Influence of the nitric acid concentration on the signals generated by solutions of Re (50 lg/kg), Co, Tl, La, Cs, Pb and Sr (20 lg/kg)

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concomitantly low concentrations of nitric acid (0.1 to 1 mol/L) are possible. However, such dilutions will lower the concentrations of several trace elements in the final solutions to levels approximately at or below the detection limits. The signals are dependent on the nitric acid concentration. They appreciably decrease in the range from 0 to 0.7 mol/L to nitric acid (for instance lead is reduced by 25%), but in the range from 0.7 to 2.0 mol/L to a smaller extent (Fig. 1). Therefore, solutions with a nitric acid concentration of approximately 1 mol/L provide signals that vary little with the nitric acid concentration and are not too corrosive to the cones. To minimize the volume of conc. nitric acid, but to increase the oxidizing power of the digestion mixture, 30% hydrogen peroxide was added. Because hydrogen peroxide is reduced to water, no additional interferences are introduced. Therefore, mixtures of conc. nitric acid and suprapur 30% hydrogen peroxide were used for all further experiments. The conc. nitric acid (pro analysi, Merck, Darmstadt) was purified in all-quartz sub-boiling distillation unit to keep blank levels low. Digestion with the open-focused microwave system To reach complete mineralization of a brain sample, the power setting, the duration of each digestion step and the volumes of the digestion reagents can be adjusted. Because solutions nebulized into the ICP-MS should have acid concentrations of approximately 1 mol/L, small volumes (4 mL) of conc. nitric acid were employed for the digestion of 150 mg samples. With this volume of acid the organic material was not completely decomposed. Under these conditions partially decomposed organic material was deposited on the inside walls of the digestion flask. Consequently, 30% hydrogen peroxide (3 mL) was added after the initial reaction with nitric acid had subsided. The resulting digests still contained suspended solids. A larger volume of nitric acid (7 mL) for the first digestion step followed by hydrogen peroxide (3 mL) produced improvements, but homogenous solutions were still not obtained. Further addition of conc. nitric acid (4 mL) and hydrogen peroxide (1 mL) finally produced homogenous solutions that were yellowish after dilution with NANOpure water to 50 mL and 1.5 mol/L with respect to nitric acid. Half of the nitric acid (80 mmol) was used and lost in the open system. Further dilution would unacceptably increase the detection limits of the method expressed in lg/g brain tissue. The power at the beginning of the digestion must be set rather low to prevent an overheating of the mixture that might be favoured by the sudden onset of exothermic oxidation reactions. After the initial reaction has subsided, the power settings must be chosen to heat the mixture not more than required to achieve very gentle boiling. Although most of the acid fumes are liquefied

in the air-cooled reflux condenser even under more vigorous boiling, some of the acid fumes nevertheless reach the injection head and condense there. Although the system is located in a ventilated hood, the acid vapours leaving the condenser corroded the mechanical parts of the system. Several flasks developed cracks close to the bottom of the flasks under normal operating conditions. The cracks are very likely caused by local high temperatures generated through the efficient absorption of microwave energy by black carbonaceous material formed on the walls of the digestion flasks, whenever more than 150 mg of brain tissue were digested. The duration of each digestion step (Table 2) can be varied at will. However, the necessity to digest as many samples as possible per unit time demands that the total time for one digestion shall be as short as feasible. Because the rate of the decomposition reaction in the presence of a large excess of the oxidizing agent is influenced only by the temperature, and the temperature cannot exceed the boiling temperature of the digestion mixture, the time allocated for each digestion step must be sufficiently long to obtain complete oxidization of the organic material. The times listed in Table 2 for each digestion step are adequate to reach this goal. Unfortunately, one digestion requires 45 min. These optimized conditions (Table 2) produce homogenous solutions only when not more than 150 mg of freeze-dried brain or not more than 250 mg of bovine liver are digested. Digestion with the closed-pressurized microwave system The MLS system with closed vessels allows the mineralization to be carried out at temperatures above the normal boiling point of the digestion reagents but not above 220 °C. At these higher temperatures the oxidation reactions proceed faster, resulting in short digestion times. The power settings and durations for each step must be chosen to keep the temperature of the digestion mixture below 220 °C and below a pressure of 30 bar. The vessel equipped with the temperature and pressure sensors allows the monitoring of the reaction. Initial attempts to digest 150 mg of freeze-dried brain with 1.5 mL of conc. nitric acid and 1.0 mL of 30% hydrogen peroxide under the conditions recommended by the manufacturer (Table 3) produced turbid solutions. Increasing the duration of the 600 W step from 3 to 5 min and adding an additional 5 min, 800 W step gave an almost clear solution with suspended particles. The temperatures and pressures during the digestion never exceeded the limits (Fig. 2). To obtain a completely homogenous digest, 2 mL conc. nitric acid and 1.5 mL 30% hydrogen peroxide were employed. Initially, pressure and temperature (Fig. 3) differed little from the values observed with 1.5 mL HNO3/l mL H2O2. However, at the beginning

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Fig. 2 Temperature and pressure during the digestion of 150 mg of freeze-dried brain with the MLS digestor using 1.5 mL conc. HNO 3 #1 mL 30% H O . Bold line: temperature; light line: pressure in the 2 2 digestion vessel; dotted line: power steps of the microwave program

Fig. 4 Temperature and pressure during the digestion of 470 mg of dry brain sample with the MLS digestor using 2 mL conc. nitric acid and 1.5 mL 30% H O 2 2

activate the pressure release system, the pressure limit was set at 20 bar. The instrument automatically turned off the magnetron at 20 bar and returned power to the system when the pressure had fallen below 20 bar (Fig. 4). Under these conditions (Table 3) brain samples up to 470 mg could be completely digested in 30 min. The MLS digests had to be diluted only to 20 mL. After these dilutions the solutions were 0.9 molar with respect to nitric acid. This acid concentration is well suited for the determinations of trace elements by ICP-MS. Comparison MLS-Prolabo

Fig. 3 Temperature and pressure during the digestion of 150 mg of dry brain with the MLS digestor using 2 mL conc. HNO #1.5 mL 3 30% H O 2 2

of the 600 W stage the pressure began to increase, to a maximum of 11.5 bar during the 800 W stage. The temperature reached 205 °C at the end of the 800 W stage. Under these conditions completely clear, colourless, homogenous digests were obtained. When larger amounts of brain were digested, the solutions were no longer colourless. With 470 mg a deep yellow, but still homogenous digest was obtained. However, the pressure in the vessels exceed 20 bars. In order not to

The experiments with bovine liver and human brain tissue indicate that both systems produce correct results for bovine liver. The Prolabo concentrations have a trend to less precision (Table 4). The same trend in precision is present in the values for brain tissue. The concentrations in brain tissue obtained with the Prolabo digests tend to diverge considerably from the MLS values, when the concentrations are low (Table 5). The higher standard deviations for the Prolabo values are not caused by the smaller number (n"7) of samples, analyzed with the open-focused system than with the closed-pressurized system (n"20). The standard deviations of several sets of seven results selected randomly from the ‘‘MLS’’-concentrations were not significantly larger than the average deviation for all 20 determinations. To produce solutions suitable for ICP-MS measurements (acid concentration 0.7 to 2 mol/L) Prolabo

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digests have to be diluted to 50 mL, MLS digests to 20 mL. A smaller amount (\150 mg) of freeze-dried brain is digestible with the Prolabo than with the MLS digestion system (470 mg) under the optimized conditions. Consequently, the detection limits of the method are higher with the Prolabo digestion than with the MLS digestion. These observations indicate that the MLS procedure has advantages over the Prolabo method for the digestion of brain tissue, when trace elements are to be quantified in the digests by ICP-MS.

Table 4 Results of the ICP-MS analysis of SRM 1577a bovine liver, after digestion with the Prolabo and the MLS microwave systems Element

209Bi 114Cd 59Co 133Cs 65Cu! 57Fe! 202Hg 55Mn! 98Mo! 208Pb 85Rb! 121Sb 120Sn 88Sr 205Tl 66Zn!

Concentration$standard deviation [lg/kg or mg/kg] MLS (n"20)

Prolabo (n"7)

Vanhoe [1]

Certified

(1.6 355$4 225$2 13.0$0.4 159$4 185$1 (6.4 9.39$0.02 3.48$0.04 112$6 12.4$0.3 (4.8 22.6$2.0 128$6 2.98$0.16 119$2

(4 415$19 235$16 14.3$1.6 141$3 183$9 (16 9.97$0.15 3.75$0.08 125$23 12.5$0.2 (12 — 133$4 (4 113$2

— 363$13 — 12$1 — — (20 — 3.82$0.06 138$8 12.61$0.23 3.3$0.5 (2 147$7 — —

— 440$60 210$50 — 158$7 194$20 4$2 9.9$0.8 3.5$0.5 135$15 12.5$0.1 (3)" — 138$3 (3)" 123$8

! mg/kg dry weight " Information value only Table 5 Results of the ICP-MS analysis of freeze-dried human brain tissue

Element

209Bi 114Cd 59Co 133Cs 65Cu 57Fe 202Hg 55Mn 98Mo 208Pb 85Rb 121Sb 120Sn 88Sr 205Tl 66Zn

SRM bovine liver The conditions optimized for the digestion of brain tissue with the Prolabo and MLS microwave systems were evaluated with the NIST SRM 1577a (bovine liver). Aliquots (B250 mg) of bovine liver produced clear, colourless, homogeneous solutions upon digestions with the MLS system and slightly yellow solutions containing a few white particles upon digestion with the Prolabo system. The average concentrations of 16 elements determined in the digests with ICP-MS are listed in Table 4. Most of the concentrations (exception Cd, Pb for MLS; Cu, Zn for Prolabo) are well within the certified averages and their standard deviations. The relative standard deviations for the certified concentrations are rather high (1 to 50%). The ICP-MS values for both types of digests have much lower relative standard deviations than the certified concentrations. The cadmium value for MLS-digests (355$ 4 lg/kg) was much lower than certified (440$60 lg/kg), but a similar low Cd-value (363$13 lg/kg) was also given by Vanhoe [1]. The not certified Cs concentration (12$1 lg/kg) given by Vanhoe [1] is in good agreement with our values (13.0$0.4, MLS; 14.3$ 1.6 lg/kg, Prolabo). The results in Table 4 clearly indicate that both digestion systems produce, in general, acceptable results. Brain tissue Digestion of freeze-dried human brain powder with the MLS system (250 to 470 mg) gave completely clear, homogenous, slightly yellow (when sample'430 mg) solutions. The Prolabo system allowed only to digest

Concentration$standard deviation [lg/kg] (RSD, %)

Limit of detection!

MLS (n"20), f"802

Prolabo (n"7), f"330"

Solution [lg/L]

MLS [lg/kg]

Prolabo [lg/kg]

(1.6 125$11 12.8$1.3 65.8$0.8 22,200$400 224,000$5,000 (6.4 940$20 53$11 20$10 11,700$300 (4.8 39.2$1.7 80.3$5.6 1.6$0.3 45,200$1,500

(6.6 112$19 30.2$4.9 76.1$3.0 23,900$200 266,000$11,000 (26.4 880$30 131$20 (13 11,800$200 (19.8 (10 116$19 (6.6 46,200$1,100

0.02 0.08 0.04 0.02 0.10 30 0.08 0.08 0.03 0.04 0.05 0.06 0.03 0.02 0.02 0.10

1.6 6.4 3.2 1.6 8.0 2,400 6.4 6.4 2.4 3.2 4.0 4.8 2.4 1.6 1.6 8.0

6.6 26.4 13.2 6.6 33 9,900 26.4 26.4 9.9 13.2 16.5 19.8 9.9 6.6 6.6 33

(8.8) (10) (1.2) (1.8) (2.2) (2.2) (21) (50) (2.6) (4.3) (7.0) (19) (3.3)

(17) (16) (3.9) (0.8) (4.1) (3.4) (15) (1.7) (16) (2.4)

! Limit of detection (LOD) is equivalent to 3r of the blank " f . . . . dilution factor (mL diluted digest/g brain tissue digested; 250 mg for MLS; 150 mg for Prolabo)

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about 150 mg resulting in yellow solutions sometimes containing particles. Because brain tissue is richer in lipids than bovine liver and lipids are difficult to mineralize under ambient pressure, the Prolabo openfocused digestion system was able to digest only smaller amounts of brain tissue than the pressurized system. Recently, Andra´si [23] employed a closed-pressurized microwave system (CEM MDS-81D) for the mineralization of brain samples. Their method allowed the decomposition of 100 mg dry sample with 3 mL concentrated nitric acid in 25 min. The average concentrations for 16 elements measured in the MLS and Prolabo digests and the corresponding standard deviations are summarized in Table 5. The averages were calculated from results of 20 experiments with the MLS and 7 with the Prolabo system, with each experiment using a separate aliquot of the brain tissue. Generally, the Prolabo concentrations are higher (up to 150%) than the MLS concentrations. The differences are particularly pronounced at low concentrations ((100 lg/kg). The standard deviations are considerably higher for the Prolabo than for the MLS concentrations. The detection limits for the method (digestion, ICPMS) are a composite of the low detection limits obtainable with the ICP-MS, the required dilution for the digests, and the mass of the samples digested. For instance, the nebulized solution must have 30 lg/L iron to produce a signal 3 r above the noise at m/z 57 (56Fe with 91,7% abundance at m/z 56 cannot be used because of interference from 40Ar16O). Thus, the 20 mL of diluted digest obtained from 250 mg brain tissue must at least contain 0.6 lg. The detection limit for Fe under these conditions is then 2,400 lg iron per kg of brain tissue. Because of the limits on the acid concentration in the solutions imposed by the ICP-MS, the dilution factor of 330 for the Prolabo experiments is much larger than for the MLS experiments (f"80). In addition, the Prolabo system can digest only 150 mg brain tissue; the MLS up to 470 mg. The higher dilution factor and the smaller amount that can be digested lead to detection limits for the Prolabo method about 4-times higher than for the MLS method. Consequently, lead, tin, and thallium cannot be determined with the Prolabo method. The concentrations of other elements in brain tissue (Cd, Co) are uncomfortably close to the Prolabo detection limits leading to high relative standard deviations. Values for lead in the MLS-digests ranged from 4.4 to 38 lg/kg resulting in a mean value of 20$10 lg/kg. This high standard deviation (50%) is not explainable on the basis of the detection limit (3.2 lg/kg). Each of the 20 repetitions (digestion, ICP-MS) gave precise results with standard deviations not larger than 10%. Lead concentrations in certified biological reference materials much higher than in brain tissue (Lobster LUTS-1, Dogfish DORM-1, both from the National

Research Council Canada) are also associated with high standard deviations (20 and 30%). Iron and strontium concentrations from the Prolabo digests were appreciably higher than from the MLS digests. For all other elements (Cs, Cu, Fe, Mn, Rb, Zn) comparable concentrations were found with both digestion methods. Homogeneity of the brain reference material Reference materials are important for quality control of analytical measurements and are needed for the development and optimization of analytical procedures. A certified reference material for brain is not available. Most investigators involved with the determination of trace elements in brain tissue use bovine liver for quality control, because bovine liver with 10% lipids comes closest among the commercially available reference materials to the lipid concentration in brain (60%). However, bovine liver is not the ideal reference material for brain. Consequently, a human brain without cerebellum was freeze-dried, powdered, and homogenized. The homogeneity of the dried powder was tested by analyzing 20 aliquots from various locations within the storage bottle after digestion with the MLS system by ICP-MS. With exception of lead (discussed above), thallium (concentration \ detection limit), and molybdenum, the relative standard deviations are in the range 1.2 to 10% (Table 5). Therefore, the brain material can be considered to be homogenous at least for the elements with relative standard deviations of (10% for their concentrations. This powdered brain (\140 g) will be used as a reference material, when brain centers are analyzed for the 13 elements listed in Table 5 by ICP-MS. Acknowledgement The MLS-1200 microwave digestion system was purchased with funds provided by the ‘‘Jubila¨umsfonds’’ der O®sterreichischen Nationalbank.

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