@Copyright 1987by The Humana Press Inc. All rights of any nature, whatsoever, reserved. 0163-4984/87/1300~363502.00
A Hydrogen-Peroxide Digestion System for Tissue Trace-Metal Analysis NANCY W. ALCOCK
Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021
ABSTRACT Tissue digestion prior to analysis for trace metals is usually carried out with strong acids. Nitric acid, alone or in combination with perchloric acid, is most commonly used. In addition to the laborious acid washing of all glassware prior to use, the digestion necessitates exposure to potential environmental contamination. Use of perchloric acid mandates a specially constructed hood with facilities for washing to remove acid deposits. A simple digestion procedure using 30% hydrogen peroxide in polyethylene vials in an oven at approximately 75~ has been previously described for the measurement of zinc in tissues using flame or flameless atomic absorption spectrophotometry and of selenium in liver by flameless atomic absorption. Readings for reagent blanks were insignificant. The technique has been further developed with a reduction in digestion time using 50% H202. Analysis of liver has been extended to include copper, manganese, and arsenic. Although the level of arsenic present was too low to be detected, 50 and 100 ng of this element added to the liver powder was completely recovered. The digest obtained when dissolved in appropriate solvent is suitable for analysis for multiple trace metals. Index Entries: Hydrogen peroxide digestion; trace metal analysis; zinc; copper; selenium; manganese; arsenic; liver; flame atomic absorption; flameless atomic absorption.
INTRODUCTION Trace metals in tissues may be measured by flame or flameless atomic absorption spectrophotometry following tissue digestion to de*Authorto whom all correspondence and reprint requests should be addressed. Biological Trace Element Research
363
VoL 13, 1987
Alcock
364
stroy organic matter and dissolution of the resulting ash in acid. The destructive decomposition of the tissue is usually carried out in strong acids in glass vessels. Nitric acid alone or in combination with perchloric acid are usually used at temperatures of 100~ or higher (1). Alternatively, dry ashing may be used at 400-500~ (2). Contamination during the preparative steps may arise from the environment or from the glassware or reagents used. For metals such as zinc, which is ubiquitous, and for those present in ultratrace amounts, such as arsenic, contamination can be of such high magnitude that the results obtained may exceed the actual amount present by orders of magnitude. It appears certain that contamination accounts for the extremely high range of values reported for normal serum chromium (3). A wide range of values have also been reported for the zinc content of various regions of brain (4-7). In addition, more volatile elements, such as zinc, arsenic, and selenium, may be lost when high temperatures are employed. The procedure described for tissue preparation for calcium and magnesium (8) seemed ideal for trace metal analysis. Hydrogen peroxide (30%) was used to oxidize organic matter at 75~ in a polyethylene vial. The digestion may be carried out in an oven. Application of the technique to the measurement of zinc in liver, and in milligram amounts of brain tissue using either flame or flameless atomic absorption spectrophotometry has previously been described (9). No loss of selenium occurred when liver was subjected to the digestion procedure (10) and recovery of arsenic added to the liver sample was complete (11). The procedure has been extended to include manganese, and both digestion time and temperature were reduced by employing 50% hydrogen peroxide.
MATERIALS AND METHODS Materials Liver powder: Vials: Tubes: Automatic pipets, various capacity: Hydrogen Peroxide: Reference standards (Fisher Scientific):
Lot #1577a (National Bureau of Standards, Gaithersburg, MD). high-density polyethylene, 20 mL capacity (Fisher Scientific, Cat. #3-337-12). polypropylene translucent tubes, 16 and 7 mL capacity, (Fisher Scientific, Cat. #14-959A and B). 20-1000 b~L capacity (Eppendorf) and 1.0-5.0 mL capacity (Oxford). 30% (Fisher Scientific, Cat. #H-325) and 50% (Fisher Scientific, Cat. #H-341). containing 1.0 mg/mL zinc (Cat. #SO-Z-13), 1.0 mg/mL copper (Cat. #SO-C-194), 1.0 mg/mL sele-
Biological Trace Element Research
Vol. 13, 1987
Peroxide Digestion for Trace Metals
Nitric acid: Magnesium nitrate hexahydrate: Nickelous nitrate hexahydrate: Water:
Parafilm: Sample cups for autosampler:
36.5
nium (Cat. #SO-S-464), 1.0 mg/mL arsenic (Cat. #SO-A-449), or 1.0 mg/mL manganese (Cat. #SO-M-81). double distilled from Vycor (G. Fred. Smith Chemical Co., Cat. #621). (Fisher Scientific, Cat. #M-46). Certified A.C.S. (Fisher Scientific, Cat. #N-63). Certified A.C.S. distilled water further purified by passage through ion exchange resin and activated charcoal cartridges (Millipore Corporation, MilliQ system). American Can Co. polystyrene, 2.0 and 0.5 mL capacity (American Scientific Products, Cat. #B-2713-2 and B-2713-ST, respectively).
Instruments and Other Equipment The following Perkin-Elmer instruments were used: Model 372 Atomic Absorption Spectrophotometer with a single-slit burner for air-acetylene flame; Model 5000 Spectrophotometer; Model 500 Graphite Furnace and AS-40 autosampler, with Recorder Model 56 and Printer Model P-10; Model 5000 Zeeman Furnace with AS-40 autosampler, dual pen recorder Model 56 and printer Model Pl00; dual electrodeless discharge lamp power source. Pyrolytically coated graphite tubes (Perkin-Elmer, Cat. #10932) and pyrolytic platform (Perkin-Elmer, Cat. #290-2311) were used. Also used were hollow cathode zinc, copper, manganese, and arsenic lamps (Perkin-Elmer); and electrodeless discharge selenium lamp (PerkinElmer). The oven was a thermostatically controlled oven to give selected temperatures of 55-80~ Methods
Digestion Dried liver powder (approximately 50 rag) was weighed into a polyethylene vial. The vial was placed on a glass plate in an oven maintained at a preselected temperature (55-75~ to reach temperature equilibration, and approximately 1 mL of hydrogen peroxide was added using a 1-mL automatic pipet with plastic tip. The vial was gently rotated and a few drops of hydrogen peroxide added to wash down particles that adhered to the wails of the vial. The vials were placed on a glass plate in the oven and inspected periodically over the next 2 h. The contents were
Biological Trace Element Research
Vol. 13, 1987
Alcock
366 TABLE 1 Program for Flameless Atomic Absorption of Zinc''~' Step 1 Temp., ~ Ramp time, s Hold time, s Recorder Read Int. flow
100-120' 20 40
300
2
3
4
5
6
1000 10 40
1800 0 5 -2 0 300
2600 1 6 0
20 20 20
300
300
D E L E T E
300
'Spectrophotometric settings: X = 213.9 n m ; low slit = 0.7 n m ; ZAA, on; peak area
absorbance; t = 5.0 s; average, 3 readings; hollow cathode lamp; current = 15mA; sample vol = 15 fxL; alt. vol = 7 btL [3.5 #g Mg as Mg(NO3)2]. "Pyrolyticallycoated tubes were used with a pyrolytic platform. 'Actual temperature must be determined by observation of specimen.
mixed at each inspection and frothing dispersed by tapping the vial on the bench. Digestion was considered to be complete w h e n a white resid u e r e m a i n e d on complete evaporation of solution. Further addition(s) of small volumes of h y d r o g e n peroxide was a d d e d as required for complete digestion. The time required was approximately 8 h using 30% H 2 0 2 and 4-6 h using 50% H202. The dry residue was dissolved in 0.5N HNO3, 1.0 mL. Prior to use, the solution was left at room temperature for at least 2 h with occasional agitation to facilitate dissolution.
Atomic Absorption Spectrophotometry FLAME. Zinc, copper, and m a n g a n e s e were m e a s u r e d by flame atomic absorption spectrophotometry. Hollow cathode lamps were used to m e a s u r e absorption at 213.9, 324.7, and 279.5 nm, respectively. OptiTABLE 2 Program for Flameless Atomic Absorption of Manganese"" Step 1 Temp., ~ Ramp time, s Hold time, s Recorder Read Int. flow
2
1 0 0 - 1 2 0 ' 1150 15 5 60
45
300
300
3
4
5
6
2100 0
2600 1
20 20
5 -2 0 50
6 0
20
D E L E T E
300
300
"Spectrophotometric settings: X 279.5 nm; low slit - 0.2 n m ; ZAA, on; peak area absorbance; t - 5.0 s; average, 3 readings; hollow cathode lamp; current = 20mA; s a m p l e vol = 15 ILL; Alt. vol - 7 txL [3.5 txg Mg as Mg(NO3)2]. Pyrolytically coated tubes were used with a pyrolytic platform. Actual t e m p e r a t u r e m u s t be d e t e r m i n e d by observation of s p e c i m e n .
Biological Trace Element Research
Vol. 13, 1987
Peroxide Digestion for Trace Metals
367
TABLE 3 Program for Flameless Atomic Absorption of Arsenic
A Hydrogen-Peroxide Digestion System for Tissue Trace-Metal Analysis NANCY W. ALCOCK
Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021
ABSTRACT Tissue digestion prior to analysis for trace metals is usually carried out with strong acids. Nitric acid, alone or in combination with perchloric acid, is most commonly used. In addition to the laborious acid washing of all glassware prior to use, the digestion necessitates exposure to potential environmental contamination. Use of perchloric acid mandates a specially constructed hood with facilities for washing to remove acid deposits. A simple digestion procedure using 30% hydrogen peroxide in polyethylene vials in an oven at approximately 75~ has been previously described for the measurement of zinc in tissues using flame or flameless atomic absorption spectrophotometry and of selenium in liver by flameless atomic absorption. Readings for reagent blanks were insignificant. The technique has been further developed with a reduction in digestion time using 50% H202. Analysis of liver has been extended to include copper, manganese, and arsenic. Although the level of arsenic present was too low to be detected, 50 and 100 ng of this element added to the liver powder was completely recovered. The digest obtained when dissolved in appropriate solvent is suitable for analysis for multiple trace metals. Index Entries: Hydrogen peroxide digestion; trace metal analysis; zinc; copper; selenium; manganese; arsenic; liver; flame atomic absorption; flameless atomic absorption.
INTRODUCTION Trace metals in tissues may be measured by flame or flameless atomic absorption spectrophotometry following tissue digestion to de*Authorto whom all correspondence and reprint requests should be addressed. Biological Trace Element Research
363
VoL 13, 1987
Alcock
364
stroy organic matter and dissolution of the resulting ash in acid. The destructive decomposition of the tissue is usually carried out in strong acids in glass vessels. Nitric acid alone or in combination with perchloric acid are usually used at temperatures of 100~ or higher (1). Alternatively, dry ashing may be used at 400-500~ (2). Contamination during the preparative steps may arise from the environment or from the glassware or reagents used. For metals such as zinc, which is ubiquitous, and for those present in ultratrace amounts, such as arsenic, contamination can be of such high magnitude that the results obtained may exceed the actual amount present by orders of magnitude. It appears certain that contamination accounts for the extremely high range of values reported for normal serum chromium (3). A wide range of values have also been reported for the zinc content of various regions of brain (4-7). In addition, more volatile elements, such as zinc, arsenic, and selenium, may be lost when high temperatures are employed. The procedure described for tissue preparation for calcium and magnesium (8) seemed ideal for trace metal analysis. Hydrogen peroxide (30%) was used to oxidize organic matter at 75~ in a polyethylene vial. The digestion may be carried out in an oven. Application of the technique to the measurement of zinc in liver, and in milligram amounts of brain tissue using either flame or flameless atomic absorption spectrophotometry has previously been described (9). No loss of selenium occurred when liver was subjected to the digestion procedure (10) and recovery of arsenic added to the liver sample was complete (11). The procedure has been extended to include manganese, and both digestion time and temperature were reduced by employing 50% hydrogen peroxide.
MATERIALS AND METHODS Materials Liver powder: Vials: Tubes: Automatic pipets, various capacity: Hydrogen Peroxide: Reference standards (Fisher Scientific):
Lot #1577a (National Bureau of Standards, Gaithersburg, MD). high-density polyethylene, 20 mL capacity (Fisher Scientific, Cat. #3-337-12). polypropylene translucent tubes, 16 and 7 mL capacity, (Fisher Scientific, Cat. #14-959A and B). 20-1000 b~L capacity (Eppendorf) and 1.0-5.0 mL capacity (Oxford). 30% (Fisher Scientific, Cat. #H-325) and 50% (Fisher Scientific, Cat. #H-341). containing 1.0 mg/mL zinc (Cat. #SO-Z-13), 1.0 mg/mL copper (Cat. #SO-C-194), 1.0 mg/mL sele-
Biological Trace Element Research
Vol. 13, 1987
Peroxide Digestion for Trace Metals
Nitric acid: Magnesium nitrate hexahydrate: Nickelous nitrate hexahydrate: Water:
Parafilm: Sample cups for autosampler:
36.5
nium (Cat. #SO-S-464), 1.0 mg/mL arsenic (Cat. #SO-A-449), or 1.0 mg/mL manganese (Cat. #SO-M-81). double distilled from Vycor (G. Fred. Smith Chemical Co., Cat. #621). (Fisher Scientific, Cat. #M-46). Certified A.C.S. (Fisher Scientific, Cat. #N-63). Certified A.C.S. distilled water further purified by passage through ion exchange resin and activated charcoal cartridges (Millipore Corporation, MilliQ system). American Can Co. polystyrene, 2.0 and 0.5 mL capacity (American Scientific Products, Cat. #B-2713-2 and B-2713-ST, respectively).
Instruments and Other Equipment The following Perkin-Elmer instruments were used: Model 372 Atomic Absorption Spectrophotometer with a single-slit burner for air-acetylene flame; Model 5000 Spectrophotometer; Model 500 Graphite Furnace and AS-40 autosampler, with Recorder Model 56 and Printer Model P-10; Model 5000 Zeeman Furnace with AS-40 autosampler, dual pen recorder Model 56 and printer Model Pl00; dual electrodeless discharge lamp power source. Pyrolytically coated graphite tubes (Perkin-Elmer, Cat. #10932) and pyrolytic platform (Perkin-Elmer, Cat. #290-2311) were used. Also used were hollow cathode zinc, copper, manganese, and arsenic lamps (Perkin-Elmer); and electrodeless discharge selenium lamp (PerkinElmer). The oven was a thermostatically controlled oven to give selected temperatures of 55-80~ Methods
Digestion Dried liver powder (approximately 50 rag) was weighed into a polyethylene vial. The vial was placed on a glass plate in an oven maintained at a preselected temperature (55-75~ to reach temperature equilibration, and approximately 1 mL of hydrogen peroxide was added using a 1-mL automatic pipet with plastic tip. The vial was gently rotated and a few drops of hydrogen peroxide added to wash down particles that adhered to the wails of the vial. The vials were placed on a glass plate in the oven and inspected periodically over the next 2 h. The contents were
Biological Trace Element Research
Vol. 13, 1987
Alcock
366 TABLE 1 Program for Flameless Atomic Absorption of Zinc''~' Step 1 Temp., ~ Ramp time, s Hold time, s Recorder Read Int. flow
100-120' 20 40
300
2
3
4
5
6
1000 10 40
1800 0 5 -2 0 300
2600 1 6 0
20 20 20
300
300
D E L E T E
300
'Spectrophotometric settings: X = 213.9 n m ; low slit = 0.7 n m ; ZAA, on; peak area
absorbance; t = 5.0 s; average, 3 readings; hollow cathode lamp; current = 15mA; sample vol = 15 fxL; alt. vol = 7 btL [3.5 #g Mg as Mg(NO3)2]. "Pyrolyticallycoated tubes were used with a pyrolytic platform. 'Actual temperature must be determined by observation of specimen.
mixed at each inspection and frothing dispersed by tapping the vial on the bench. Digestion was considered to be complete w h e n a white resid u e r e m a i n e d on complete evaporation of solution. Further addition(s) of small volumes of h y d r o g e n peroxide was a d d e d as required for complete digestion. The time required was approximately 8 h using 30% H 2 0 2 and 4-6 h using 50% H202. The dry residue was dissolved in 0.5N HNO3, 1.0 mL. Prior to use, the solution was left at room temperature for at least 2 h with occasional agitation to facilitate dissolution.
Atomic Absorption Spectrophotometry FLAME. Zinc, copper, and m a n g a n e s e were m e a s u r e d by flame atomic absorption spectrophotometry. Hollow cathode lamps were used to m e a s u r e absorption at 213.9, 324.7, and 279.5 nm, respectively. OptiTABLE 2 Program for Flameless Atomic Absorption of Manganese"" Step 1 Temp., ~ Ramp time, s Hold time, s Recorder Read Int. flow
2
1 0 0 - 1 2 0 ' 1150 15 5 60
45
300
300
3
4
5
6
2100 0
2600 1
20 20
5 -2 0 50
6 0
20
D E L E T E
300
300
"Spectrophotometric settings: X 279.5 nm; low slit - 0.2 n m ; ZAA, on; peak area absorbance; t - 5.0 s; average, 3 readings; hollow cathode lamp; current = 20mA; s a m p l e vol = 15 ILL; Alt. vol - 7 txL [3.5 txg Mg as Mg(NO3)2]. Pyrolytically coated tubes were used with a pyrolytic platform. Actual t e m p e r a t u r e m u s t be d e t e r m i n e d by observation of s p e c i m e n .
Biological Trace Element Research
Vol. 13, 1987
Peroxide Digestion for Trace Metals
367
TABLE 3 Program for Flameless Atomic Absorption of Arsenic
