Mikrochimica Acta [Wien] 1974, 65--72 9 by Springer-Verlag 1974

Clinical Biochemistry Laboratories, Government Department of Pathology, Sepoy Lines, Singapore, 3

Determination of Serum Iron and Zinc by Atomic Absorption Spectroscopy

By

Kum-Tatt Lee* and Edward Jacob (Received September 5, 1972) Introduction

The use of atomic absorption spectroscopy (AAS) as an analytical tool in the clinical laboratory has introduced precision and reliability in the determination of metals in biological fluids and tissues. The method is entirely specific for a given element. It has the distinct advantage over existing methods in that interference from other metals is almost completely negated, as no two metals are known, which have the same resonance lines. While success has been achieved in this field with serum calcium and magnesium determinations, the application of this technique to serum iron has shown that the direct aspiration of serum into the flame as described by Rodgerson and Helfer 1 and by Sprague and Slavin ~ is not as effective as the chelation of iron and subsequent extraction of the complex into an organic solvent, which is then analysed by AAS. In the former case, that of using diluted serum or plasma, possible contamination due to the presence of haemoglobin iron can contribute significant error to the iron value exhibited in normal serum. Furthermore, the high protein content tends to clog the burner head, resulting in unstable readings. Zettner 3 found that the direct aspiration of serum into the flame gave sufficiently accurate data in the range above 200/~g iron per 100 ml serum, but unreliable results were encountered in the lower ranges because of matrix interference ~" Singapore Institute of Standards and Industrial Research, 179 River Valley Road, Singapore-6, Republic of Singapore.
Mikrochim. Acta 1974/1 5

66

K.-T. Lee and E. Jacob:

and haemolysis. They successfully employed bathophenanthroline as the complexing agent and extracted the metal complex into methylisobutyl ketone (MIBK). Allan 4 used ammonium pyrrolidine dithiocarbamate for complexing iron in aqueous solution and showed the favourable extraction ratio of the complex into MIBK. We investigated the use of the compound 1,3-bis(2-pyridyl)-l,2-diazaprop-2-ene (pyridine-2-aldehyde-2-pyridil hydrazone; "PAPHY"; I) as a chelator of iron and zinc in serum, for AAS.

~ ix~l CHN~N/NH/'N~l ~N~ I

The tridentate ligand, PAPHY, and its complexes with a metal coordinated to the tertiary nitrogen atoms were first described by Lions and Martin 5. It was later shown by Geldard and Lions% that the cationic bis complexes of PAPHY with transition metals in the + 2 oxidation state could easily be converted by the loss of two protons to highly coloured uncharged bis complexes, soluble in organic solvents, but almost insoluble in water. This sequestering action of PAPHY, coupled with the preferential solubility of the uncharged complexes in organic solvents, suggested an effective means of removing iron and zinc from serum. This unique property of PAPHY has been utilized to develop a method for the simultaneous determination of iron and zinc in serum.

Experimental
Materials

Reagents used are as follows: (1) Trichloroacetic acid, 10%. (2) Sodium sulphite, 0.1 M. (3) Hydrochloric acid, 1.0N. (4) Pyridine-2-aldehyde-2-pyridyl hydrazone, 100 mg per 100 ml 0.16N hydrochloric acid. (5) Potassium hydroxide, 40%. (6) Ferric ammonium citrate, 5.0 mg Fe per 100 ml. (7) Amberlite IRA 410 resin. (8) Buffer, pH 7.5 containing 0.11 M sodium chloride and 0.04M barbital. Solutions 6 and 8 were prepared as described by Peters and co-workers7. All reagents used were of Analytical Reagent grade. PAPHY was prepared by the method of Lions and Martin 5.

D e t e r m i n a t i o n of S e r u m Iron a n d Z i n c

67

Assay Procedure
Into glass stoppered centrifuge tubes (15 ml) are transferred 1.0 ml of serum; 1.0 ml water ("blank" sample); and 1.0 ml of working iron and zinc standard solution (0.2, 0.5, 1.0, 1.5, 2.0 #g of metal per ml). 4 ml of water, 1.0 ml of 1.0 N hydrochloric acid, 2.0 ml of 0.1 M sodium sulphite solution and 2.0 ml 10 ~ trichloroacetic acid are added to each tube. The contents are mixed and the tubes placed in a boiling water bath for 5 minutes. The contents of each tube are then stirred with a fine glass rod, and after cooling, the tubes are centrifuged and 8.0 ml of the clear supernantant from each tube are transferred to 15 ml glass stoppered test tubes. One ml of PAPHY solution is added to each tube, the contents mixed and allowed to stand for 5 minutes. This is followed by the addition of 1.0 ml of 40 ~ potassium hydroxide and 1.0 ml of amyl alcohol. The tubes are stoppered and the contents mixed vigorously for a few seconds. The two layers are allowed to separate and the organic layer is then aspirated into the air-acetylene flame. Determination o[ total iron-binding capacity. The method of Peters and coworkers 7 was adopted with slight modifications. 0.05 ml of the ferric ammonium citrate solution is added to 0.5 ml of serum in a test tube, and allowed to react for 5 minutes. 0.2 ml of the resin is then added and the contents of the tube mixed continuously for 5 minutes. Buffer (2.5 ml) is added and after mixing well, the tube is centrifuged. To 2.0 ml of the clear supernatant is added 3.0 ml of water and the 5.0 ml aliquot is then treated as described for serum iron. Instrumentation. A Unicam SP 90 atomic absorption spectrophotometer, fitted with cathode lamps of iron and zinc, was used.
T a b l e I. I n s t r u m e n t a l P a r a m e t e r s for M e a s u r e m e n t s of Iron a n d Z i n c by AAS Parameter Measurement Iron 15 m A m p 248.3 n m 2/~m 40 psi 5.5 1 p e r min. 8.0 psi 1.2 1 per min. 2.5 ml per min. Zinc 12 m A m p 213.9 n m 2#m 40 psi 5.5 1 p e r rain. 8.0 psi 1.3 1 per min. 3.0 ml per min.

Lamp Current .................... Absorption Wavelength ............ E n t r a n c e slit . . . . . . . . . . . . . . . . . . . . . Air p r e s s u r e . . . . . . . . . . . . . . . . . . . . . . Air flow': . . . . . . . . . . . . . . . . . . . . . . . . Acetylene p r e s s u r e . . . . . . . . . . . . . . . . Acetylene flow* . . . . . . . . . . . . . . . . . . . S a m p l e a s p i r a t i o n rate . . . . . . . . . . . . .

* M e a s u r e m e n t s of air a n d acetylene f l o w are e x p r e s s e d in a r b i t r a r y u n i t s i n d i c a t e d on the f l o w m e t e r s of t h e gas r e g u l a t o r u n i t s of t h e U n i c a m SP 90 a t o m i c absorption spectrophotometer. 5*

68

K.-T. Lee and E. Jacob:

The absorption line of 248.3 nm and 213.9 nm for iron and zinc respectively, were used throughout the study. Stability of the burner head, optical and electronic systems is verified by monitoring the zero-point for several minutes while amyl alcohol is continuously aspirated into the flame. The instrument is adjusted to the operation conditions as listed in Table I. Measurements of atomic absorption. The blank, standards and the unknown samples are aspirated into the flame, and the absorption of the samples is directly read on the scale of the instrument. The concentration of iron and zinc in the unknown is then computed from the calibration curves.
Results and Discussion

Most published methods for the determination of serum iron utilize an initial splitting of the iron from its protein combination by exposure to acid followed by precipitation of the serum protein. Bothwell and Mallett s and Peters and co-workers~ have shown that the use of strong (2 to 6 N) hydrochloric acid gives complete extraction of the iron from protein. Barkan 9 introduced the use of dilute (0.2 to 0.3 N) hydrochloric acid, while Peters and co-workers v combined dilute hydrochloric acid with a reducing agent (thioglycolic acid) and obtained satisfactory iron extraction. Ramsay 1~ used sodium sulphite to release the iron from its combination. We have combined successfully the use of dilute hydrochloric acid and sodium sulphite. During the initial deproteinisation with trichloroacetic acid, release of the iron and zinc from protein is facilitated by boiling in the presence of hydrochloric acid. The iron is simultaneously reduced by the acid and sodium sulphite. In order to avoid any trapping of the metallic ions in the interstices of the precipitate, it was necessary to stir the mixture well with a fine glass rod. The iron and zinc are evenly distributed throughout the aqueous phase. This is borne out by the results of the recovery experiments, which indicate that neither metal is lost in the protein precipitate. A distinct advantage of this atomic absorption method is that the concentration of iron and zinc in the amyl alcohol layer which is aspirated into the flame for analysis, is identical to that in the original serum. If it is desired, a smaller volume of organic solvent can be employed in the final extraction. This would result in a concentration of the serum constitutents being investigated, and is a great advantage when low values are encountered. With most

Determination of Serum Iron and Zinc

69

c o t o r i m e t r i c p r o c e d u r e s , the s e r u m c o n s t i t u e n t s u n d e r g o a d i l u t i o n b e f o r e t h e final a n a l y s i s is c a r r i e d out. R e p r o d u c i b i l i t y . T h e results of 10 a n a l y s e s for s e r u m i r o n a n d zinc o n each of 2 p o o l e d s e r a b y t h e p r o c e d u r e d e s c r i b e d are s h o w n in T a b l e II. T h e p r e c i s i o n of t h e m e t h o d f o r s e r u m i r o n in the r a n g e Table II. Precision of Repeated Analyses Iron /~g % Serum A 24.0 25.0 26.5 24.5 26.0 24.0 25.5 26.5 24.5 25.0 25.2 0.94 3.75 Serum B 54.0 53.5 57.0 55.5 55.5 54.0 53.5 55.5 55.5 54.0 54.8 _+1.16 2.12 Serum A 80.0 82.0 81.5 83.0 82.5 81.0 80.5 83.5 80.5 81.5 81.6 1.15 1.41 Zinc /~g % Serum B 143.0 145.0 143.0 145.5 145.0 144.0 144.5 145.0 143.5 144.5 144.3 0.88 0.62

Mean S.D. C.V.

b e l o w 60 # g / 1 0 0 ml, t h e clinically i m p o r t a n t r a n g e , is s h o w n to be g o o d , as, w i t h t h e s e r u m p o o l s of l o w i r o n c o n t e n t , 10 r e p e a t e d a n a l y s e s o n t h e t w o s a m p l e s g a v e m e a n s of 25.2 0.94 (S. D.) a n d 54.8 + 1.16 (S. D.). T h e coefficients of v a r i a t i o n w e r e 3.75 ~ a n d 2.12 ~ respectively. R e c o v e r y e x p e r i m e n t s . T h e results of a series of r e c o v e r y e x p e r i m e n t s a r e s h o w n in T a b l e III. T h e s e w e r e c a r r i e d o u t b y t h e a d Table III. Recovery of Iron and Zinc Added to Serum Serum Iron Iron Content in #g/100 ml Serum Value 28 33 60 85 108 230 Added 50 50 100 50 50 50 Found 76 82 167 136 160 280 Expected 78 83 160 135 158 280 Serum Zinc Zinc Content in/~g/100 ml Serum Value 80 87 109 125 127 153 Added 50 50 50 100 100 50 Found 130 135 161 223 224 201 Expected 130 137 159 225 227 203

d i t i o n of 20/~1 of a d i l u t e d s t a n d a r d s o l u t i o n of i r o n a n d zinc to 1 m l of s e r u m , to give final a d d e d i r o n a n d zinc c o n c e n t r a t i o n s of

70

K.-T. Lee and E. Jacob:

50 ,ug or 100/~g per 100 ml of serum. The recoveries were calculated by comparing the results obtained before and after the addition of the standard solutions. The sera chosen for the recovery experiments had a wide concentration range of iron and zinc, and the observed consistent recoveries of added metal demonstrates the precision of the method over a wide range.

Comparison with a colorimetric procedure. Iron analyses were carried out on the same serum samples by the ASS procedure and Ramsay's i~ method. The results obtained are shown in Table IV.
Table IV. Comparison of Iron Values by Colorimetry and AAS Sample No. 1 2 3 4 5 6 7 8 9 10 Serum Iron,/*g per 100 ml Colorimetry AAS 62 72 35 60 80 75 70 72 250 117 62 69 25 59 71 69 71 66 240 109 T.I.B.C., ~g per 100 ml Colorimetry AAS 388 241 316 292 270 241 324 238 392 327 398 233 327 270 251 232 312 229 386 319

There is reasonable agreement between the methods. Colorimetric procedures in general, are exposed to interference from pigments, other serum constituents and turbidity of the final coloured solutions. These drawbacks are of no consequence to atomic absorption methods, which are specific for the metal being investigated. Interference from turbidity might explain the higher values obtained by Ramsay's procedure. Table V. Normal Values with New Procedure No. of persons Serum Iron . . . . . . . . . . . . . . . . Total Iron-binding Capacity Serum Zinc . . . . . . . . . . . . . . * Observed range. 57 57 57 Contents in /~g per 100 ml serum Range Mean S.D. 76 --181 230"--410 99 --146 128.0 326.0 122.0 _+26.0 -+ 24.0

Normal values. The analyses of 57 morning samples from healthy normal individuals for serum iron, the total iron-binding capacity and serum zinc levels yielded the results shown in Table V.

Determination of Sermn Iron and Zinc

71

The mean value for serum iron is 128/~g per 100 ml, with a range of 76 to 181 #g per 100 ml, while the mean for TIBC is 326 #g per 100 ml, with a range of 230 to 410/~g per 100 ml. The range obtained for serum zinc is 99 to 146/~g per 100 ml, with a mean value of 122 #g per 100 ml. The skewness of the distribution of the values suggest that a non-normal distribution may be characteristic of a group of subjects who are heterogenous with respect to race, age, dietary habits and other characteristics. Table V summarizes the normal values obtained with the present procedure.

Summary
Determination o[ Serum Iron and Zinc by Atomic Absorption Spectroscopy
This paper presents a rapid sensitive atomic absorption spectroscopic method for the simultaneous determination of serum iron and zinc levels. It is also applicable to the detection of these metals in urine. The technique is based on the ability of pyridine-2-aldehyde-2-pyridyl hydrazone (PAPHY) to form stable cationic bis complexes with iron or zinc, in the + 2 oxidation state, while in an aqueous medium. When the pH of the medium is shifted towards the alkaline side, the charged complexes attain neutrality and are quantitatively extractable into an organic solvent. Thus, the metals are free of the interfering effects of serum constituents, and are easily estimated by atomic absorption spectroscopy.

Zusammenfassung
Eine rasche und empfindliche Methode zur gleichzeitigen Bestimmung von Eisen und Zink im Serum durch Messung der Atomarabsorption wurde angegeben. Sie eignet sich auch zum Nachweis der beiden Metalle im Ham. Sie beruht auf der Bildung stabiler kationischer Bis-Komplexe des zweiwertigen Eisens oder Zinks in w~if~rigerPhase mit Pyridin-2-aldehyd-2-pyridylhydrazon (PAPHY). Wird das pH nach der alkalischen Seite verschoben, so verlieren die Komplexe ihre Ladung und werden mit einem organischen L6sungsmittel extrahierbar. Sonstige Serumbestandteile st6ren nicht. References 1 D. O. Rodgerson and R. E. Helfer, Clin. Chemistry 12, 338 (1966). s S. Sprague and W. Slavin, Atomic Absorption Newsletter 4, 228 (1965) Perkin Elmer Corp. Norwalk Conn.

72

K.-T. Lee and E. Jacob: Determination of Serum Iron and Zinc 3 A. Zettner, L. C. Sylvia, and L. Capacho-Delgado, Amer. J. Clin. Path.

45, 533 (1966).

4 j. E. Allan, Spectrochim. Acta 17, 476 (1961). 9 5 F. Lions and K. V. Martin, J. Amer. Chem. Soc. 80, 3858 (1958). 6 j. F. Geldard and F. Lions, J. Amer. Chem. Soc. 84, 2262 (1962). 7 T. Peters, T. Giovanniello, L. Apt, and J. F. Ross, J. Lab. Clin. Med. 48, 274 (1956). 8 T. H. Rothwell and B. Mallett, Clin. Sci. 14, 235 (1955). 9 G. Barkan, Klin. Wschr. 6, 1615 (1927). 10 W. N. M. Ramsay, Clin. Chim. Acta 2, 214 (1957).