Comparison of procedures for determination of copper and zinc in serum by atomic absorption spectroscopy

83

Clinica

Chimica

Acta,

110 (1981) 83-90 Biomedical Press

@ Elsevier/North-Holland

CCA 1646

COMPARISON OF PROCEDURES FOR DETERMINATION OF COPPER AND ZINC IN SERUM BY ATOMIC ABSORPTION SPECTROSCOPY

ANDREW

TAYLOR

a** and TREVOR

N. BRYANT

b

a Robens Institute of Industrial and Environmental Health and Safety and b Department Microbiology, University of Surrey, Guildford, Surrey GU2 5XH (U.K.) (Received

July llth,

of

1980)

Summary Methods used to prepare serum samples for the determination of copper and zinc by atomic absorption spectroscopy have been compared using the results obtained from an external quality control scheme. The methods most commonly employed were dilution with water, dilution with dilute butan-l-01 or propan-l-01, removal of protein by trichloroacetic acid (TCA) precipitation and electrothermal atomization (ETA). The effects of the inclusion of sodium and potassium ion upon results has been examined. Low results for serum copper are given by the technique of ETA. High zinc values are found following TCA precipitation. None of the methods showed superior between-batch precision. Ionization interference was variable and the addition of sodium and potassium to standard solutions is not always necessary.

Introduction Measurements of copper and zinc in serum are relevant to the investigation of a variety of clinical conditions. However, provision of accurate results with good between-batch precision is complicated by contamination during sample collection and analysis, by the paucity of quality control material available to provide external checks and by the different analytical procedures employed. Kelson and Shamburger [l] compared two methods for sample preparation for the determination of zinc in serum prior to atomic absorption spectroscopy. At about the same time we initiated an external trace element quality control scheme to allow an independent evaluation of performance. While such quality control schemes identify good and poor performers, they should give data which can be used to evaluate analytical techniques [2]. We * To whom correspondence

should be addressed.

therefore examined results from this quality control scheme according to the method of sample preparation to determine which were the most reliable. Materials and methods Quality control scheme Donkey serum with low levels of copper and zinc was prepared by mixing with Chelex 100 cation exchange resin. Known amounts of copper and zinc were added, the samples mixed, dispensed and stored at -20°C. Each sample was distributed to participants on two separate occasions and nine samples were included in this study. The mean and standard deviation of all results were calculated for each sample. Results outside the range of mean + 2 S.D. were omitted from subsequent evaluations. Sample preparation procedures represented in this scheme are given in Table I. The figures in parentheses show the number of laboratories, which include sodium with or without potassium in standard solutions. Not all participants in the programme described their methods in sufficient detail and their results were not included in any comparisons, although they were used to calculate the concensus mean for each sample as given in Table II. The data was analysed using the Statistical Package for the Social Sciences, version 7.2 of Nie et al. [ 31. Additional experiments Samples from a serum pool were prepared for analysis by dilution with water, by dilution with 6% v/v butan-l-01 (butanol) and by protein precipitation using 10% w/v trichloroacetic acid (TCA). The aspiration rates of the samples and diluents were then measured using available atomic absorption spectrophotometers. Copper and zinc standard solutions were similarly diluted and the atomic absorption signals compared with those from standards prepared in the same way but containing 140 mmol/l sodium and 5.0 mmol/l potassium.

TABLE

I

TECHNIQUES

FOR SAMPLE

PREPARATION

Showing the number of laboratories using each procedure. Figures laboratories who include sodium and potassium in standard solutions. copper

Dilution with water Dilution with water, glycerol added to standards Dilution with butan-l-o1 or propan-l-01 Protein precipitated by tricbloroacetic acid Protein precipitated by TCA followed by heating Electrothermal atomization Acid digestion

in brackets

Zinc

6 (2) l-

13 (7) 2-

13 (7) 6 (1) 1 (1) 4-

15 (8) 2-

1 (1)

I (1)

1 (1) l-

indicate

the number

of

85

Results The mean values for copper and zinc concentrations of the nine samples are presented in Table II. The spread of results for zinc was wider than that of copper. The number of extreme values which were excluded from further calculations was also greater for zinc. Differences between the concensus means and the method means for each of the nine samples are shown in Fig. 1; these are expressed as standard deviations from the concensus mean. There were no significant differences between the duplicate samples for any of the methods. For the measurement of copper, dilution of samples with water gave results which were significantly greater (p < 0.001) than the overall mean (mean difference = +3.3%). By contrast the use of electrothermal atomization (ETA) produced lower results (p < 0.001) than the overall mean (mean difference = -3.9%). Of the analytical procedures used for zinc estimation, TCA precipitation gave significantly higher results @ < 0.01, mean difference = +10.9%). The recovery of copper added to serum is shown in Fig. 2. ETA methods gave significantly low rates of recovery (p < 0.05). No significant variations were observed with the procedures used for the analysis of zinc (Fig. 3). The effect of sodium and potassium in standard solutions on some of the procedures for estimating copper and zinc is illustrated in Figs. 4 and 5. The presence of these cations gave significantly lower results for copper Gr, < 0.001) with water dilution and with butanol/propanol dilution procedures; their mean differences were 1.71 and 1.81 pmol/l respectively, whereas with the TCA precipitation method, the presence of sodium and potassium had the opposite effect and results were 0.97 pmol/l higher (p < 0.05). The butanol/propanol method for zinc, as with copper, gave lower results, 1.07 ymol/l (p < O.Ol), in the presence of sodium and potassium. The effects of sodium and potassium upon the absorption signal of aqueous standards are shown in Fig. 6. There was no difference between the standards prepared to contain these ions and those with them omitted.

TABLE

II

RESULTS

FROM

(Summaries Copper

N

QUALITY

of mean,

CONTROL

standard

deviation

PROGRAMME and

/Jmol/l

coefficient

of variation Zinc

Mean

S.D.

C.V.%

for

9 samples.)

wmol/l

N

Mean

S.D.

C.V.%

59

24.2

2.3

9.5

70

11.9

3.2

26.9

52

34.6

2.4

6.9

67

25.4

4.1

16.1

57

19.0

2.0

10.5

66

4.4

1.8

40.9

58

20.2

2.0

9.9

68

2.2

19.8

55

19.9

2.3

11.5

63

8.4

1.9

22.6

51

33.6

2.6

7.7

65

16.4

1.6

64

29.0

2.1

9.3

75

18.7

2.5

67

12.5

1.4

11.2

83

67

23.1

1.8

1.8

84

11.1

3.5 13.7

9.8 13.4

1.8

51.4

2.8

20.4

86 RECAPPER

all

VI

V

IV

IV

VI

Fig. 1. Difference between concensus mean and method mean. The diagram shows the difference for (a) copper determinations and (b) zinc determinations using 6 analytical methods; (I) water dilution. (II) butanol dilution, (III) TCA precipitation. (IV) TCA precipitation and heating, (V) electrothermal atomization, (VI) acid digestion. The difference between the concensus mean and the method mean for 9 Samples is expressed as multiples of the concensus standard deviation.

5

COPPER

10 ADDED

15

20

rmol,,

Fig. 2. Recovery of added copper. The graph shows the amount of copper recovered plotted against the amount of copper added for 4 analytical methods (0. water dilution, 9, butanol dilution, 0, TCA precipitation, a. electrothermal atomization). The line indicates 100% recovery.

ZINC

ADoeR

pmol/l

Fig. 3. Recovery af added zinc. The graph shows the amount of lint recovered plotted against the amount of zinc added for 3 analytical methods (0, water dilution, a, butanol dilution. 0, TCA precipitation). The line indicates 100% recovery.

water

buranol

dilution

dilution

pe.001

ps-007

40

20

0

Fig. 4. Effect of sodium and potassium upon copper results. -The diagram shows the mean vaiues for swum copper determinations with 3 different methods: water diiutio% butanol difution and TCA precipitation, when read against standards containing (“) and not containfng (a) sadium and potassium ions. Significant differences are indicated.

water

dilution

n

s

30

20

10

0

I

butanol

dilution

P c .Ol

Fig. 5. Effect of sodium and potassium upon serum zinc results. The diagrams show the mean values for serum zinc determinations for water dilution and butanol dilution when read against standards containing (p) and not containing (0) sodium and potassium ions. Significant differences are indicated.

150

100

pmOl/l

p

a

m

I/,

0

b

Fig. 6. Effect of sodium and potassium ions on absorption signal. The diagrams show the effect of the presence or absence of sodium and potassium ions on the absorption signal for (a) copper and (b) zinc determinations for 3 analytical methods. (A water dilution, 0 butanol dilution and 0 TCA precipitation. Shaded symbols represent the above methods with the inclusion of sodium and potassium ions).

89

Discussion Sufficient data were available from this study to allow comparison of ETA, the dilution procedures and TCA precipitation for copper analysis, and of dilution and TCA precipitation for determination of zinc. The significant difference between concensus mean and method mean (Fig. la) and the reduced recovery (Fig. 2) indicates that with ETA, copper in serum is not atomized as efficiently as in inorganic standards. The higher copper concentrations measured following dilution with water are not due to differences in aspiration rate between sample and standards, since recovery of added copper was not increased (Fig. 2) and sample uptake was not faster than that of the standards (Table III). Elimination of ionization interference by the addition of sodium and potassium to standards produces lower results (Fig. 4) and of the six laboratories measuring copper after dilution with water, only two included these ions in the standards. Results from this group might therefore be expected to be positively biased. Higher results for serum zinc measured following TCA precipitation compared with dilution (Fig. lb) is in agreement with the observations of Kelson and Shamburger [l]. These authors inferred that a non-dialysable component of serum interfered with atomization of zinc in the flame and that dilution methods, which do not remove this component, produce low results. However, recovery of zinc added to ion-exchange stripped serum is not decreased (Fig. 3), thus failing to demonstrate the proposed interference. Kelson and Shamburger [l] proposed that similar differences should be observed with copper determinations; this is not supported by our data (Fig. 1). High results found with TCA precipitation methods probably reflect contamination consequent upon the several steps involved, compared with a simple dilution procedure. The presence of sodium and potassium in standard solutions gave lower results for copper and zinc with butanol/propanol dilution and for copper with water dilution. These cations will act as suppressors, in serum, where the analyte is liable to ionise during analysis. When this occurs it is necessary to match the sodium and potassium content of the standards so that an equivalent atomic absorption signal is produced by both test and standard solutions. Therefore, where analyte ionisation is significant, measured concentrations in the unknowns will appear to be higher if suppressants such as sodium and potassium are absent from standard solutions. The effects of these cations are not always apparent and may be variable. The TCA precipitation procedure gave paradoxically high

TABLE III (Rates of sample aspiration through the pebulisers. expressed as the ratio of diluent to diluted serum.) Instrument

IL 353

PE 103

PYE SP9

1.06 1.06 1.02

1.02 0.98 1.05

1.04 1.04 0.98

Diluent Distilled water Butan-l-ol TCA

90

copper results in the presence of sodium and potassium while identical responses were obtained for standards prepared with or without these cations (Fig. 6). Other investigators [5,6] have shown variable response, suggesting that other factors, e.g. nebulizer design and efficiency, flame conditions, etc., must also be involved. The results from this quality control scheme have indicated that the measurement of serum zinc is technically difficult compared with serum copper, which is probably due to contamination. The wide variance of zinc results obtained for each sample, therefore, hinders the identification of any methodological features which have a significant effect on the analysis of this element. References 1 K&on, J.R. and Shamburger, R.J. (1978) Methods compared for determining zinc in serum by flame atomic absorption. Clin. Chem. 24, 240-244 2 Buttner, J., Barth, R., Boutweli. J.H., Broughton. P.M.G. and Bowyer, R.C. (1978) Provisional recommendations on quality control in clinical chemistry. Part 5. External quality control. Clin. Chim. Acta 83.191F-202F 3 Nie. N.H., Hull, C.H.. Jenkins, J.G., Steinbrenner, K. and Bent, D.H. (19’78) Statistical Package for the Social Sciences. 2nd Edn., McGraw-Hill, New York 4 Olson. A.D. and Hamlin, W.B. (1968) Serum copper and zinc by atomic absorption spectrophotometry. At. Absorpt. New& 7.69-71 5 Dawson, J.B. and Walker, B.E. (1969) Direct determination of zinc in whole blood, plasma and urine by atomic absorption spectroscopy. Clin. Chim. Acta 26. 465-475 6 Momcilovic. B.. Belonje, B. and Shah, B.G. (1976) Effect of matrix of the standard on results of atomic absorption spectrophotometry of zinc in serum. Clin. Chem. 21, 688-690