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Atomic absorption spectrometric determination of aluminium in whole blood
Anclyticu oE!swier
Chimica Ada,
92 (1977)
79-83
Scientific Publishing Company. Amsterdam - Printed in The NetherlanA
ATOMIC ABSORPTION SPECTROMETRIC ~~~rN~~~ fN WHOLE BLOOD
F. J. LANCMYHR’
OF
and D. L. TSALEV*
of Chemistry. Unicersity (Received 15th February 1977) Dcportmerlt
DETERMINATION
of
Oslo,
Oslo
3 (Norway)
SUMMARY TWO methods are described for atomic absorption spectrometric determinations of aluminium in heparinized and haemolyzed samples of undiluted whole human blood. In the direct method 2 4 of blood are pipetted into a graphite cup atomizer; after a drying and two shing steps aluminium is determined by atomization at 2500°C. In the second method. 15 ul of blood are decomposed by nitric acid in polytetrafluoroethylene tubes; 2-rrl portions of the so’lution are then analyzed with the mphite tube atomizer. The direct method was applied to the determination of aluminium in whole blood from 48 Norwegian workers occupationally unexposed to the element; the concentrations of aluminium ranged from 0.05-Q.59 p.p.m. (mean value, 0.20 p.p.m.)_ For 0.35 p.p.m. duminium. the relative standard deviation of both methods wiu 8%. The detection limit of the direct method is 0.05 p.p.m. aluminium.
During recent years the universal interest in trace elements has stimulated a number of studies of their concentration and distribution in the human body, with the purpose of establishing the normal values. and to detect illnesses, occupational diseases and toxic effects. Whole human blood contains small arrounts of ~uminium (of the order of one or more tenths of 1 p.p.m.); the concentration has been found to vary during certain diseases [ 11. At present, nothing definite can be said about the toxicity of aluminium, for this question is still being debated. The determination of aluminium in biological materials is complicated by the low concentration of the element, the limited amounts of sample available, and the large number of analyses required in clinical situations. Various methods, such as spectrophotometry [ 2 1, emission spectroscopy 131, neutron activation analysis (4. 51, and atomic absorption spectrometry (a.as.j j6-111, have been used for the determination of aluminium in biological materials. The furnace a.a.s. technique has been applied to the determination of aluminium m serum [ll), but, to the authors’ best knowledge, it has not been used for determining the metai in undiluted whole blood. *On leave from Faculty of Chemistry, Unircrsity of Sofia, Sofia 26, Bulgaria.
CO The purpose of the present method for the determination
work was to develop 2 direct, rapid and simple of aluminium in undiluted whole human blood.
SXPEFU3lENTAL
Appamtus The measurements mere made with 2 Perkin-Elmer 400 S atomic absorption spcctrophotometer, the cup and tube version of the VarianTcchtron CRA-63 resistance-heated graphite furnace and a Perkin-Elmer single-channel recorder. The instrument was equipped with two lamps for background correction; however, as apparent from the preliminary investigations (vide infra), the analyses can be made without using a background corrector.
The furnace
was heated
by a four-step
power
supply
constructed
in the
laboratory. Temperature calibration curves for the furnaces were recorded with an Ircon radiation thermometer. Sample and standard solutions were introduced into the cup with 2 2-~1 Lntra-Micro pipette (Oxford Labs. Intern.), and a 5--50~~1 adjustable Finnpipette (Kemistien OY, Finland). For the decomposition of whole blood, 100~~1 tubes with stoppers were made from rods of a dense quality of polytetrafluoroethylene (F’TFE). During heating the tubes were placed in holes drilled in a circular steel plate, and the stoppers xere pressed into the tubes by a second plate fastened to the perforated plate with 2 screw in the centre. The tubes were heated in a drying oven.
Reagents
and standard
solutions
A primary lOOO-p.p_m. aluminium standard solution was prepared by dissolving 1 g of high-purity metal (Johnson blatthey, London) in 200 ml of (1 + 9) sulphuric acid (Suprapur, Merck), and diluting the solution to 1 1 with deionized water. Secondary standard solutions were prepared daily by dilution. Concentrated nitric acid (Suprapur, Merck) was employed for the decomposition of blood. The graphite furnace was purged with argon of purity 99.9% (by volume).
Samples The feasibility
of the proposed direct method was demonstrated by the analysis of whole b!ood samples from 48 Norwegian workers who were not occupationally exposed to aluminium. The samples were collected in heparinized “Vacutiner” tubes and were haemolyzed by the addition of four drops of Triton X-100.
Preliminary work Some introductory measurements were made to establish the best conditions for the ashing and atomizaticn steps. It was found that aluminium is not lost by ashing whole blood samples in the furnace at 1200°C. When the proposed direct method was used, the peak heights were the same whether the background corrector was used or not; consequently, the non-specific absorption of radiation during atomization of aluminium can be considered to be negligible. When calibration curves were plotted for aluminium in a whole blood matrix, and from aqueous aluminium standard solutions, it was observed that t.% slope of the curves varied considerably. In the direct method, the measurc:x.~-l~ts should therefore be based either on the tedious and time-consuming standard addition technique, or on employing one or more carefully analyzed whole blood samples as the standard. The latter approach was used in the present direct anaIyses. After the wet-ashing of whole blood by nitric acid, the matrix consists of a dilute solution of simple salts; in such cases aqueous aluminium solutions can be used as standards. Procedures Before the start of the measurements, the hollow-cathode lamp was heated for about 15 min. The flow of argon was adjusted to 4 1 min-‘. The measurements were made at 309.3 nm. All analyses were based on measuring peak hei&ts. Direct annlysis. Undiluted whole blood (2 ~1) was transferred to the graphiti cup of the atomizer; during the introduction of the sample, the pipette tip should not touch the interior walls of the cup. The samples were analyzed with the following heating program: drying at 90°C for 40 s, first ashing at 400°C for 30 s, second ashing at 1200°C for 30 s, and atomization at 2500°C for 5 s. Two blood samples were employed as standards, the content of aluminium in these being established by the standard addition technique. To three of a series of four l-ml portions of the blood to be used as standard, 10 ~11of 10, 20 or 50 p.p.m. aluminium standard solutions were added; the concentrations in the blood of the aluminium added thus corresponded to 0.1, 0.2 and 0.5 p_p.m., respectively. Analysis by wet-asking. In addition to the analyses by the above direct method, aluminium was also determined in some samples decomposed by wet-ashing. Portions (15 ~1) of whole blood were transferred to lOO+l PI’FE tubes, 15 ~1 of nitric acid were added, and the tubes were stoppered. The contents were mixed vigorously and heated at 90°C for 2 h. After being cooled to room temperature, the contents were again mixed by shaking. Portions (2 ~1) of the solutions were pipetted into the graphite tube atomizer, and aluminium was determincul as described in the above procedure. The analyses were made with aqueous aluminium solutions as standards.
Other analyses For comparison some samples were also analyzed in another laboratory (Institute of Occupational Health, Oslo). The equipment employed was a Perkin-Elmer 300 atomic absorption spectrophotometer with a HGA-76 graphite furnace. Portions of diluted whole blood sampies (1 + 10) were analyzed by the standard addition technique, and with the following heating program: drying at 90°C. rate 2, for 30 s; first ashing at 5OO”C, rate 2, for 30 s; second ashing at 1200°C. rate 1, for 30 s; and atomization at 27OO”C, rate 0, for 10 s with gas stop for 7 s. RESULTS
The above direct procedure was employed in the analysis of 48 undiluted whole blood samples. The results ranged from 0.05 to 0.59 p.p_m_ with a mean value of 0.20 p.p.m. The distribution of the results \\RSas follows: 23% in the range 0.05-0.10 p.p.m.; 42% in the range 0.11-0.20 p.p.m.; 12% in the range 0.21-0.30 p.p.m_: 15% in the range 0.51-0.40 p.p.m.; p.p.m. 4% in the range 0.41-0.50 p.p.m.; and 4 % in the range 0.51-0.60 Two of the samples were analyzed by three different methods. Tine data from these analyses are listed in Table 1. DISCUSSION
The results of the present investigation lie in the range covered by the data from the following previous studies: 0.13 p-p-m. [12], 0.21-0.94 p.p_m. (mean value 0.53 p.p.m.) [3], a-.d 1.40 p_p.m. [13]. As is apparent from Table 1, the data obtained by the direct method are in good agreement with those of the two other procedures. Statistical calculations confmed that the differences between the mean values are explained by the presence of random errors alone. TABLE
1
Analytical results (in p.p.m.) for alumirium in two san?plcs of whole human blood analyzed by three atomic absorption spectrometric methods. The figures tabulafed are the mean values I one stndxd deviation as calculated from N analyses Sample
Method
A
0.35 + 0.03 l = 6
0.36 f 0.03 s = 6
0.39 * 0.03 N 1 6
B
0.16 : 0.03 .v = 10
0.14 2 0.04 1%.= 4
0.13 : 0.0.: N = 4
-‘Method I bJlcthod II =X!ethod III Health. Oslo).
I=
Me:hod
IIb
Method
III=
Direct analysis of undiluted whole blood. Analysis by wet-ashing. Direct analysis of diluted whole blood (1 + lo),
(Ir~titutc
of Occupational
a3
The precision of the direct method is satisfactory. It was found to be 20.03 p.p.m., i.e. a relative standard deviation of 8% at a concentration 0.35 p.p.m. aluminium. The detection limit of the direct method is approximately 0.05 p.p.m. aluminium. The main advantages of the proposed direct method are that the analyses are made without the addition of reagents and without any separation and/or concentration steps; the risks of introducing contaminations and of losing aluminium are thus considerably reduced. The authors are indebted Health, Oslo, for providing the manuscript.
to Dr. N. Gundersen, The Institute the blood samples and for critical
of Occupational comments on
REFERENCES 1 J. R. J. Sorenson, 1. R. Campbell, L. B. ‘Pepper and R. D. Lingg. Environ. Health PC=PCC~~VCS, a (1974) 3. 2 %I. Seilbold, Klin. Wochschr., 38 (1960) 117. 3 H. Wolff, Biochcm. Z.. 319 (1948) 1. 4 K. Fritze and R. Robertson, J. Radioanal. Chcm., 7 (1971) 213. 5 H. Galenkamp. J. van Leeuwen and H. A. Das. Reactor Cent. Ncdcrland Rep., RCN-208 (19i4) 19. 6 E. Berman. ApeI. Spectrosc.. 29 (1975) 1. 7 F. Dolins’ck. J. Stupar and M. Spenko. Analyst, 100 (1975) 884. R S. S. Krishnan. K. A. Gillespie and D. R. Crapper, Anal. Chem.. 44 (1972) 1469. 9 J. R. McDermott and J. Whitehill, Anal. Chim. Acta. 65 (1976) 195. 10 H. A. Schroeder and A. P. NJ-son. Clin. Chem.. 17 (1971) 461. 11 C. Fuchs, M. Brasche, K. Paschcn, H. Nordbeck and E. Quellhorst, Clin. Chim. Acta. 52 (1974) 71. 12 R. A. Kehoe, J. Cholak and R. V. Story, J. Nutr.. 19 (1940) 579. 13 F. Burriel Marti, C. Alvrrrcz Hcrrcro and hf. P. Martin hiatcw. Rev. Clin. Fsp.. 113 (1969) 413.
Chimica Ada,
92 (1977)
79-83
Scientific Publishing Company. Amsterdam - Printed in The NetherlanA
ATOMIC ABSORPTION SPECTROMETRIC ~~~rN~~~ fN WHOLE BLOOD
F. J. LANCMYHR’
OF
and D. L. TSALEV*
of Chemistry. Unicersity (Received 15th February 1977) Dcportmerlt
DETERMINATION
of
Oslo,
Oslo
3 (Norway)
SUMMARY TWO methods are described for atomic absorption spectrometric determinations of aluminium in heparinized and haemolyzed samples of undiluted whole human blood. In the direct method 2 4 of blood are pipetted into a graphite cup atomizer; after a drying and two shing steps aluminium is determined by atomization at 2500°C. In the second method. 15 ul of blood are decomposed by nitric acid in polytetrafluoroethylene tubes; 2-rrl portions of the so’lution are then analyzed with the mphite tube atomizer. The direct method was applied to the determination of aluminium in whole blood from 48 Norwegian workers occupationally unexposed to the element; the concentrations of aluminium ranged from 0.05-Q.59 p.p.m. (mean value, 0.20 p.p.m.)_ For 0.35 p.p.m. duminium. the relative standard deviation of both methods wiu 8%. The detection limit of the direct method is 0.05 p.p.m. aluminium.
During recent years the universal interest in trace elements has stimulated a number of studies of their concentration and distribution in the human body, with the purpose of establishing the normal values. and to detect illnesses, occupational diseases and toxic effects. Whole human blood contains small arrounts of ~uminium (of the order of one or more tenths of 1 p.p.m.); the concentration has been found to vary during certain diseases [ 11. At present, nothing definite can be said about the toxicity of aluminium, for this question is still being debated. The determination of aluminium in biological materials is complicated by the low concentration of the element, the limited amounts of sample available, and the large number of analyses required in clinical situations. Various methods, such as spectrophotometry [ 2 1, emission spectroscopy 131, neutron activation analysis (4. 51, and atomic absorption spectrometry (a.as.j j6-111, have been used for the determination of aluminium in biological materials. The furnace a.a.s. technique has been applied to the determination of aluminium m serum [ll), but, to the authors’ best knowledge, it has not been used for determining the metai in undiluted whole blood. *On leave from Faculty of Chemistry, Unircrsity of Sofia, Sofia 26, Bulgaria.
CO The purpose of the present method for the determination
work was to develop 2 direct, rapid and simple of aluminium in undiluted whole human blood.
SXPEFU3lENTAL
Appamtus The measurements mere made with 2 Perkin-Elmer 400 S atomic absorption spcctrophotometer, the cup and tube version of the VarianTcchtron CRA-63 resistance-heated graphite furnace and a Perkin-Elmer single-channel recorder. The instrument was equipped with two lamps for background correction; however, as apparent from the preliminary investigations (vide infra), the analyses can be made without using a background corrector.
The furnace
was heated
by a four-step
power
supply
constructed
in the
laboratory. Temperature calibration curves for the furnaces were recorded with an Ircon radiation thermometer. Sample and standard solutions were introduced into the cup with 2 2-~1 Lntra-Micro pipette (Oxford Labs. Intern.), and a 5--50~~1 adjustable Finnpipette (Kemistien OY, Finland). For the decomposition of whole blood, 100~~1 tubes with stoppers were made from rods of a dense quality of polytetrafluoroethylene (F’TFE). During heating the tubes were placed in holes drilled in a circular steel plate, and the stoppers xere pressed into the tubes by a second plate fastened to the perforated plate with 2 screw in the centre. The tubes were heated in a drying oven.
Reagents
and standard
solutions
A primary lOOO-p.p_m. aluminium standard solution was prepared by dissolving 1 g of high-purity metal (Johnson blatthey, London) in 200 ml of (1 + 9) sulphuric acid (Suprapur, Merck), and diluting the solution to 1 1 with deionized water. Secondary standard solutions were prepared daily by dilution. Concentrated nitric acid (Suprapur, Merck) was employed for the decomposition of blood. The graphite furnace was purged with argon of purity 99.9% (by volume).
Samples The feasibility
of the proposed direct method was demonstrated by the analysis of whole b!ood samples from 48 Norwegian workers who were not occupationally exposed to aluminium. The samples were collected in heparinized “Vacutiner” tubes and were haemolyzed by the addition of four drops of Triton X-100.
Preliminary work Some introductory measurements were made to establish the best conditions for the ashing and atomizaticn steps. It was found that aluminium is not lost by ashing whole blood samples in the furnace at 1200°C. When the proposed direct method was used, the peak heights were the same whether the background corrector was used or not; consequently, the non-specific absorption of radiation during atomization of aluminium can be considered to be negligible. When calibration curves were plotted for aluminium in a whole blood matrix, and from aqueous aluminium standard solutions, it was observed that t.% slope of the curves varied considerably. In the direct method, the measurc:x.~-l~ts should therefore be based either on the tedious and time-consuming standard addition technique, or on employing one or more carefully analyzed whole blood samples as the standard. The latter approach was used in the present direct anaIyses. After the wet-ashing of whole blood by nitric acid, the matrix consists of a dilute solution of simple salts; in such cases aqueous aluminium solutions can be used as standards. Procedures Before the start of the measurements, the hollow-cathode lamp was heated for about 15 min. The flow of argon was adjusted to 4 1 min-‘. The measurements were made at 309.3 nm. All analyses were based on measuring peak hei&ts. Direct annlysis. Undiluted whole blood (2 ~1) was transferred to the graphiti cup of the atomizer; during the introduction of the sample, the pipette tip should not touch the interior walls of the cup. The samples were analyzed with the following heating program: drying at 90°C for 40 s, first ashing at 400°C for 30 s, second ashing at 1200°C for 30 s, and atomization at 2500°C for 5 s. Two blood samples were employed as standards, the content of aluminium in these being established by the standard addition technique. To three of a series of four l-ml portions of the blood to be used as standard, 10 ~11of 10, 20 or 50 p.p.m. aluminium standard solutions were added; the concentrations in the blood of the aluminium added thus corresponded to 0.1, 0.2 and 0.5 p_p.m., respectively. Analysis by wet-asking. In addition to the analyses by the above direct method, aluminium was also determined in some samples decomposed by wet-ashing. Portions (15 ~1) of whole blood were transferred to lOO+l PI’FE tubes, 15 ~1 of nitric acid were added, and the tubes were stoppered. The contents were mixed vigorously and heated at 90°C for 2 h. After being cooled to room temperature, the contents were again mixed by shaking. Portions (2 ~1) of the solutions were pipetted into the graphite tube atomizer, and aluminium was determincul as described in the above procedure. The analyses were made with aqueous aluminium solutions as standards.
Other analyses For comparison some samples were also analyzed in another laboratory (Institute of Occupational Health, Oslo). The equipment employed was a Perkin-Elmer 300 atomic absorption spectrophotometer with a HGA-76 graphite furnace. Portions of diluted whole blood sampies (1 + 10) were analyzed by the standard addition technique, and with the following heating program: drying at 90°C. rate 2, for 30 s; first ashing at 5OO”C, rate 2, for 30 s; second ashing at 1200°C. rate 1, for 30 s; and atomization at 27OO”C, rate 0, for 10 s with gas stop for 7 s. RESULTS
The above direct procedure was employed in the analysis of 48 undiluted whole blood samples. The results ranged from 0.05 to 0.59 p.p_m_ with a mean value of 0.20 p.p.m. The distribution of the results \\RSas follows: 23% in the range 0.05-0.10 p.p.m.; 42% in the range 0.11-0.20 p.p.m.; 12% in the range 0.21-0.30 p.p.m_: 15% in the range 0.51-0.40 p.p.m.; p.p.m. 4% in the range 0.41-0.50 p.p.m.; and 4 % in the range 0.51-0.60 Two of the samples were analyzed by three different methods. Tine data from these analyses are listed in Table 1. DISCUSSION
The results of the present investigation lie in the range covered by the data from the following previous studies: 0.13 p-p-m. [12], 0.21-0.94 p.p_m. (mean value 0.53 p.p.m.) [3], a-.d 1.40 p_p.m. [13]. As is apparent from Table 1, the data obtained by the direct method are in good agreement with those of the two other procedures. Statistical calculations confmed that the differences between the mean values are explained by the presence of random errors alone. TABLE
1
Analytical results (in p.p.m.) for alumirium in two san?plcs of whole human blood analyzed by three atomic absorption spectrometric methods. The figures tabulafed are the mean values I one stndxd deviation as calculated from N analyses Sample
Method
A
0.35 + 0.03 l = 6
0.36 f 0.03 s = 6
0.39 * 0.03 N 1 6
B
0.16 : 0.03 .v = 10
0.14 2 0.04 1%.= 4
0.13 : 0.0.: N = 4
-‘Method I bJlcthod II =X!ethod III Health. Oslo).
I=
Me:hod
IIb
Method
III=
Direct analysis of undiluted whole blood. Analysis by wet-ashing. Direct analysis of diluted whole blood (1 + lo),
(Ir~titutc
of Occupational
a3
The precision of the direct method is satisfactory. It was found to be 20.03 p.p.m., i.e. a relative standard deviation of 8% at a concentration 0.35 p.p.m. aluminium. The detection limit of the direct method is approximately 0.05 p.p.m. aluminium. The main advantages of the proposed direct method are that the analyses are made without the addition of reagents and without any separation and/or concentration steps; the risks of introducing contaminations and of losing aluminium are thus considerably reduced. The authors are indebted Health, Oslo, for providing the manuscript.
to Dr. N. Gundersen, The Institute the blood samples and for critical
of Occupational comments on
REFERENCES 1 J. R. J. Sorenson, 1. R. Campbell, L. B. ‘Pepper and R. D. Lingg. Environ. Health PC=PCC~~VCS, a (1974) 3. 2 %I. Seilbold, Klin. Wochschr., 38 (1960) 117. 3 H. Wolff, Biochcm. Z.. 319 (1948) 1. 4 K. Fritze and R. Robertson, J. Radioanal. Chcm., 7 (1971) 213. 5 H. Galenkamp. J. van Leeuwen and H. A. Das. Reactor Cent. Ncdcrland Rep., RCN-208 (19i4) 19. 6 E. Berman. ApeI. Spectrosc.. 29 (1975) 1. 7 F. Dolins’ck. J. Stupar and M. Spenko. Analyst, 100 (1975) 884. R S. S. Krishnan. K. A. Gillespie and D. R. Crapper, Anal. Chem.. 44 (1972) 1469. 9 J. R. McDermott and J. Whitehill, Anal. Chim. Acta. 65 (1976) 195. 10 H. A. Schroeder and A. P. NJ-son. Clin. Chem.. 17 (1971) 461. 11 C. Fuchs, M. Brasche, K. Paschcn, H. Nordbeck and E. Quellhorst, Clin. Chim. Acta. 52 (1974) 71. 12 R. A. Kehoe, J. Cholak and R. V. Story, J. Nutr.. 19 (1940) 579. 13 F. Burriel Marti, C. Alvrrrcz Hcrrcro and hf. P. Martin hiatcw. Rev. Clin. Fsp.. 113 (1969) 413.