Determination of Ca Ici um, Magnesium, Copper, and Zinc in Red Blood Cells by Emission Spectrometry LESLIE S. VALBERG, JOHN M. HOLT, and JOHN SZIVEK Department of Medicine, Queen's University, Kingston, Ontario, Canada
b A method i s described for the determination of calcium, magnesium, zinc, and copper in red blood cells, the latter in a range of 0.1 to 3 pg. per gram. Blood collected carefully in plastic syringes fitted with stainless steel needles i s centrifuged in the syringe to separate the blood cells. The red blood cells are ejected from the base of the syringe and ashed in platinum dishes at 450" C. The salts are converted to their chloride form and strontium i s added as the internal standard. The samples are applied to the surface of platform electrodes coated with plicene. The elements are determined by a direct reading emission spectrograph with a high voltage spark technique. The reproducibility o f the technique i s of the order of 10%. Normal values for erythrocyte calcium, magnesium, zinc, and copper obtained with this technique are given and compared with values reported in the literature.
T
in the role of trace elements in metabolism and the production of disease requires methods for the measurement of metals at concentrations of a few parts per million that are both easy and precise. Emission spectrometry permits the simultaneous determination of many elements in tissues and the direct reading spectrograph shows promise of making this task less tedious and even more precise ( 7 ) . HE GROWING INTEREST
EXPERIMENTAL
Instruments. A Jarrell Ash 3.4meter Ebert mount spectrograph with direct reading attachment was used. Exit slits located on analytic lines for magnesium at 2852.1 A., copper a t 3247.5 A,, zinc at 3345.0 A., calcium at 4226.7 -4., and strontium at 4607.3 A,, were 86, 125, 88, 88, and 83.5 microns in width, respectively. Reagents and Chemicals. Ultrapure water was prepared from raw water by a system t h a t employed a still in combination with mixed-bed ion exchange resins ( 6 ) . The resistivity of the water was 10 X 106 ohms or greater. Hydrochloric acid was prepared from commercial anhydrous hydrogen chloride gas (81) and commercial sodium heparin was purified by extraction with dithizone (8). Spectrographically standardized pure salts obtained
790
ANALYTICAL CHEMISTRY
from Johnson and Matthey Co. Ltd., London, were used in the preparation of standard solutions. Cleaning of Apparatus. Glassware and polyethylene ware were cleaned in a dilute solution of detergent, placed in a 1 :1 mixture of concentrated sulfuric acid and concentrated nitric acid for 8 to 12 hours and then rinsed thoroughly with ultra-pure water. Plastic syringes were dismantled, immersed for 24 hours in 1N sodium ethylene-diaminetetraacetate adjusted to p H 11 with sodium hydroxide and then rinsed thoroughly with ultra-pure water. Platinum crucibles were cleaned with sodium bisulfate and by immersion in boiling nitric acid (21). Preparation of Samples. With a 30ml. plastic syringe (Stylex Syringe, J. F. Hartz Co., Toronto, Ont.) fitted with a stainless steel needle (Becton, Dickinson Co. Ltd., Toronto, Ont.) blood was taken from patients after an overnight fast. Purified heparin (0.1 mg.) was placed in each syringe before the blood was drawn to prevent coagulation of the blood. The red blood cells were separated from the blood plasma and other cellular constituents by centrifugation of the blood in the syringe at 2000 G a t 4" C. for 60 min. The plasma was removed from the upper part of the syringe for the determination of plasma calcium (11). The plunger was re-inserted in the barrel, the red blood cells were ejected from the base of the syringe and 1 gram was weighed into each of two platinum crucibles for the determination of calcium, magnesium, copper, and zinc. The samples were dried in a dust cover (21) under a n infrared lamp and this was augmented by heat from a n underlying hotplate. When charring was complete, the samples were transferred to a muffle furnace where they were heated at 450" C. to completely destroy the organic matter. After about 48 hours the samples were removed from the furnace and the reddish-brown residue in each dish was dissolved in 0.5 ml. of 6N HCl. The samples were evaporated to dryness in a dust cover under an infrared lamp and the chloride salts were dissolved in 1 ml. of 2W HC1 which contained 15 pg. of strontium as the internal standard. The solutions were transferred to polyethylene containers and stored at 4" C. until they were analyzed. The red blood cells prepared by this technique contained an average of 1.9% trapped plasma as determined by the Evans blue dye dilution technique (S). A correction was applied t o the results
of red cell calcium because of the relatively high concentration of calcium in plasma compared to red blood cells. Preparation of Standards. Six standards were made from stock solutions which were prepared b y dissolving in 2.47 HCl spectrographically standardized chemicals previously dried in a vacuum oven a t 45' C . for 48 hours. The matrix of each of six standards solutions contained potassium, 3500 pg. /ml,; sodium, 400 pg./ml.; iron, 510 pg./ml.; and phosphorus, 230 pg./ml. Various amounts of calcium, magnesium, copper, and zinc were added to each solution in a random manner ( 5 ) to provide concentrations ranging from 1 to 45 pg./ml., 5 to 125 pg./ml., 0.1 to 3.0 pg./ml., and 5 to 35 pg./ml., respectively. Strontium, 15 pg./ml.> was added as the internal standard. Application of Samples to Electrodes. -4 rotating platform technique was used (16) and graphite elec-
trodes (United Carbon Co., Ultra High Purity, No. 106 or 1907) treated with 294 plicene (4) to prevent seepage of the sample into the graphite were used as platform electrodes. Ten microliters of 4% Photo Flo (Eastman KodakCo., Ltd.) was added to 1 ml. of ashed red blood cells to facilitate uniform drying of the sample on the electrodes. One hundred pl. of sample roughly equivalent to 1 mg. of ash were applied to the inner part of each electrode with a micropipet and evaporated to dryness a t 80" C. on a hotplate. Excitation Conditions. Excitation conditions originally described b y Morris and Pink ( I d ) were modified to give greater sensitivity. The conditions were as follows: amperage, 2.0.; voltage, 26,000. ; breaks per half cycle, 8 . ; capacitance, 0.005 If.; inductance, 625 ph.; secondary resistance, 3 ohms.; analytical gap, 2.0 mm.; entrance slit width, 25 microns.; entrance slit height, 12 mm.; and revolutions of platform electrode, 15 per min. The strontium dynode was adjusted to give a n exposure time of about 24 seconds and no pre-excitation of the sample was made. Calibration Curves. Calibration curves for calcium, magnesium, copper, and zinc were prepared from t h e standard solutions b y plotting the intensity ratio obtained from t h e spectrometer against t h e concentration of the element in the standard. The shift in curves over a period of six months is shown by the following data: relative standard deviation for calcium at 5.0
pg. per ml.'is 10%); magnesium at 50 pg. per ml. is 24%; copper at 1.0 pg. per rnl. is 10%; and zinc a t 10.0 g.per ml. is 11%. There was no :appreciable change in the shape of the calibration curves during this period. RESULTS
Evaluation of Me.:hod.
T h e rotating platform technic ue was compared with other methods Eor the analysis of solutions. ;1 solution of ashed red blood cell? waq analyzed with a rotating disk electrode ( I d ) , vacuum cup electrode (26), flat top electrode (121, and with the rotatiqg platform technique that has just keen described. It will be seen from T ~ b l eI that the rotating platform technique was the most sensitive of the four riethods and it u-as the only one that provided sufficient sensitivity for the analysis of copper and zinc in red blood cel s. The results of reproducibility studks with the platform technique and with methods employing. flat top electrodes shown in Table I indicate that reproducibility with the rotating platform technique was better for each of the elements. Results of a recover)' study are shown in Table 11. Known amounts of calcium, magnesium. copper, and zinc were added to samples of red blood cells of known composition and the samples were analyzed in duplicate. Reproducibility of the method over a period of six months is given in Table 111. A pooled sample of packed cells was analyzed in duplicate with each batch of specimens. Normal Values in Control Subjects. T h e mean, range, and standard deviation of normal values for erythrocyte calcium, magnesium copper, and zinc in 27 healthy men and 30 healthy women who ranged in age from 16 t o 75 years are shown in Table IV. There was no difference between the results in men and those in wornen and there was no relation between metal concentration and age of the subjects. DISCUSSION
Emission spectrochemical methods employing a photographic process for measuring the relai ive intensity of spectrum lines have been widely applied in the analysis of biological samples (18, 1 9 , 22) but the direct reading spectrograph has had limited use to date. Recently Nusbaum a i d coworkers ( I S ) described the use of 1 he Industrial Research Quantometer manufactured b y Applied Research Laboratories for the determination of 12 elements simultaneously in human tissue by a d.c. arc technique. The relative standard deviation of the method, determined from thirtysix runs on a synthetic sample varied from a low of 1.4% fo. lead at a concentration of 21 pg. per gram to a high of 13.4y0 for aluminum a t a level of 186 pg.
Table I.
Sensitivity and Reproducibility of Different Solution Techniques
Element Calcium Magnesium Copper Zinc Table II.
Concn., Mg./g. 4 55 1.0
10
Signal: background ratio Rotating T'acuum Flat Rotating disk cup top platform 4 6 5 5 3 8 147 11.7 11.9 10.8 19.2 3.4 1.6 1.3 1.7 1.6 1.2 1.7 4.6
_Rel. std. _ dev._
Flat top 4 0 10.1 20 5 21.9
Rotating platform 1.4 4 0 4.5 4 3
Recovery of Known Additions of Metals to a Sample of Erythrocytes erg. per g.
Element Calcium
a
In sample 12.2 12.2 12.2 52.0 52.0 52.0 0.8 0.8 0.8
Recovery,
Added Total Found 8.0 20.2 18.9 16.0 28.2 29.5 ... 32.0 42.0 Magnesium 30.0 82.0 81.0 60.0 112.0 108.0 120.0 172.0 181.0 Copper 0.5 1.3 1.2 1. 0 1.8 2.0 2.0 2.8 2.7 Zinc 8.8 4.0 12.8 11.0 8.8 8.0 16.8 17.0 8.8 16.0 24.8 24.5 Unable to determine accurately from calibration curve.
Table 111.
7c
93.6 104.6 ... 98.8 96.4 105.2 92.3 111.1 96.4 85.9 101.2 98.8
Results of Analysis of a Control Sample of Red Blood Cells over a Six-Month Period
pg./g. No. of
Metal analyses Mean Range 8.5-11.9 Calcium 13 10.2 57-78 Magnesium 13 67 12a 0.89 0.72-1.01 Copper 6.9-10.1 Zinc 13 8.7 a One result discarded because of contamination by copper. Table
IV.
Std. dev. 0.72 7.3 0.07 1.0
Rel. std. dev., 74 7 11
8
11
Comparison of Erythrocyte Calcium, Magnesium, Copper, and Zinc in 57 Control Subjects with Results in the Literature
Jlean level, Fg./g. Element cells 2.4 Calcium 15.0 18.0 20.0 Magnesium 56.5 61.2 64.8 48.6 49.0 Copper 0.92 0.98 0.82 0.92 Zinc 10.6 15.7 11.8 11.4
Range d g .
1.0-3.7 10-20 ... , . .
40-73 26-11 2 39-58 ... 0.69-1.17 0.69-1.18 0.22-2.80 0.23-2.16 7.7-14.0 8 . o-'i4.9
3.8-16.6
Method Spectrochemical Spectrophotometric Spectrophotometric Spectrophotometric Spectrochemical Spectrophotometric Spectrochemical Spectrophotometric Spectrophotometric Spectrochemical Spectrophotometric Spectrochemical Spectrophotometric Spectrochemical Spectrophotometric Spectrophotometric Spectrochemical
per gram. It is not clear from the description of their results if this represents the variation obtained from runs on the same d a y or over a period of days or weeks. The sensitivity of the present technique with the direct reading spectrograph was less than that obtained with Eastman Kodak Yo. 103-0 photographic
Reference Present work Albritton ( 1 ) Wallach et al. ( 6 5 ) Keitel (9) Present work rllbritton ( 1 ) Paixao and Yoe ( 1 5 ) Wallach et al. ( 2 4 ) Carubelli et al. ( 2 ) Present work Shields et al. ( 17 ) Paixao and Yoe ( 1 5 ) Koch et al. (IO) Present work S'allee and Gibson ( 2 3 ) Talbot and Ross ( 2 0 ) Paixao and Yoe ( 1 5 )
plates by a factor of 3 to 5 , but nevertheless it still permitted the precise measurement of 0.20 pg. of copper, 2.0 pg. of zinc, and even smaller amounts of calcium and magnesium per gram of red blood cells. The use of direct reading analysis in place of the tedious procedure of measuring relative intensities b y photoVOL. 3 6 , NO. 4 , APRIL 1964
* 791
~
graphic methods has greatly facilitated our analytical work and it has provided a precise and sensitive method applicable to the anal) ber of samples. The mean and range of results obtained by this method are compared with values reported by other. for normal subjects in Table 11-. It will be seen that the mean values for magnesium. copper, and zinc are very qimilar to result. that have been obtained by other.. The normal range for magnesium, coppw. and zinc is smaller than that reported by Paixao and Yoe who employed an emiksion spectrographic method for the analysis of blood cells. The mean calcium concentration is much lower than the values reported preT. ioii4y. It would appear that previous workers have neglected to correct their results for the relatively high concentration of calcium in trapped plasma. ACKNOWLEDGMENT
We are grateful to G. AI. I3rown for his helpful advice and criticism and we are indebted to Eleanor Paulson and Miriam I3enson for their assistance.
LITERATURE CITED
(1) Albritton, E. C., Standard Talues in Blood, p. 119, USAF Tech. Report 6039, 19.51. (2) Carubelli, R., Smith, W. 0.)Hammernten, J. F., J . Lab. Cltn. N e d . 51, 964 (1958). (3) Chaplin, H., Mollison, P. L., Blood 7, 1227 (1952). (4) Feldman, C., Ellenburg, J. Y., ANAL. CHEM.30, 418 (1958).
( 5 ) Fisher, R. A., “The Design of Experiments,” p. 49. Oliver and Boyd, Lon-
don, 1951. ( 6 ) Holt, J. M., Lux, IT.,T-alberg, L. S., Can J . Biochem. Physaol. 41, 2029 (1963). (7) Jarrell. It. .4., “Encyclopedia of Spectroscopy,” p. 138, Reinhold, Sew York, 1960.
(8) Kagi, J. H. It. It., TTallee,B. I,., ASAI,. CHEW.30, 1951 (1958). (9) Keitel, H. G., Brrman, H., Jones, H., MacLachlan, E.. MacLachlan. E., Blood 10, 370 (1955 1 1955 jJ,. (10) Koch, H. J., Smith, E. R.,Shimp, N. F., Connor, J., Cancer 9, 49b 499 (1956). (11) MacIntyre, I., Biochem. J . 67, 164 i 1 M-T \/. \ -
M.,Pink, F. S..ASTM Special Technical Publications S o . 221, p. 39, Am. Soc. Testing ?*later., Philadelohia. 1957. (13) husbaum, R. E., Butt, E. hl., Gilmour, T. C., Dinio, S.L., Am. J . (“Zin. (12) Morris, J.
Pathol. 3 5 , 44 (1961). (14) Pagliassoti, J. P.,i l p p l . Spectry. 9,153 (195.5).
5 ) Paixao, L. ?*I., Yoe, J. H., Clin.Chim. Acta 4, 507 (1959). 6 ) Rozsa, J. T., Zeeb, L. E., Petrol. Process. 8 , 1708 (1953). 7) Shields, G. S., Markowitx, H., Klas-
sen, W. H., Cartwright, G. E., Wintrobe, 11. ?VI.,J . Clzn. Incest. 40, 2007 i l c ). 6.l \. j .
(18) Smith, I. L., Yaeger, E., Kaufman, 3 ., Hovorka, F., Kinney, T. I)., A.M.A. Arch. of Pathol. 5 2 , 321 (1951). (I!)) Stitch, S. R., Biochem. J . 67, 97 i19,i7) (20) Talbot, T R , Ross, J. F , Lab Invest. 9, 174 (1060) 121) Thiers. R E , Methods Baochem .Inaly. 5 , 273 (1957). (22) Tipton, I. H., “PIIetal-Binding in Medicine,” p. 27, J. B. Lippincott Co., Philadelphia, 1960 (23) Yallee, B. I,., Gibson, J. G., J . Rzol. Cheni. 176, 435 (1948). (24) IVallach, S., Cahill, L. X., Rogan,
F. H., Jones, H. L., J Lab. Clin. Jfed.
59, 195 (1962). (2,i) Wallacah, S.,Zemp, J.
IT-.,Cavins, J. A , ,Jenkins, L. J., Bethea, M., Freshette, L., Haynes, L. L.. Tullis, J. L., Blood 2 0 , 344 11962). (26) Zink, T. H., Appl. Spectry. 1 3 , 94 (1959).
RECEIVEIIfor review October 7, 1963. Accepted January 17, 1964. This nork was supported by a grant from the Medical Researrh Council of Canada.
Spectrophotometric Determination of Traces of Peroxides in Organic Solvents DlLlP K. BANERJEE and CLIFFORD C. BUDKE Research Division,
U. S.
Industrial Chemicals Co., Division of Nafional Distillers and Chemical Corp., Cincinnati, Ohio
b A sensitive spectrophotometric method for the determination of traces of organic peroxides in organic solvents is described. The sample is diluted with a mixture of acetic acid and chloroform and treated with potassium iodide after deaeration. The iodine liberated is measured spectrophotometrically a t 470 mp in 1 -cm. cells. Active oxygen in the range of 5 to 80 p.p.m. can be determined. By using 1.5-cm. cells and a wavelength of 410 mp for the absorbance measurements, the range of the method can be extended to cover 0 to 5 p.p.m. of active oxygen. Quantitative results have been obtained with 17 commercial peroxides of varying reactivity. N o reaction was obtained with di-ferf-butyl peroxide and dicumyl peroxide. The method was satisfactory for the determination of peroxides in benzene, chloroform, 2-propanol, methanol, pentane, hexane, toluene, ethyl ether, acetone, vinyl acetate, and ethyl acetate. It should b e applicable to organic solids which are soluble in a mixture of acetic acid and chloroform. 792
ANALYTICAL CHEMISTRY
colorimetric procedures have been deqcribed for the determination of organic peroxides. Martin (,9) reviewed a large number of these and pointed out their limited applicability. Kolthoff and Medalia (7’) summarized the disadvantages of ferrous ion methods which have been widely used for the determination of peroxides in gasoline, fats and oils, ethers, and rubber. The leucomethylene blue method of Sorge and Ueberreiter (14) has been used for hydroperoxides and diacyl peroxides, but the reagent is difficult to prepare and relatively unstahle. Eiss and Geisecke (5) recommended benzoyl leuco-methylene blue as an alternative reagent because of its stability in air. However, it has to be protected from light and its reaction rate with peroxides is relatively slow without the use of metal accelerators. I n the past three years there has been considerable interest in the qualitative identification and determination of traces of peroxides in solvents and other organic materials. Dugan (2. 3) used S,N-p-phenylenediamine sulfate to determine traces of UMEROUS
lauroyl and benzoyl peroxide in polymers and for the qualitative detection of peroxides in ethers. The possibility of extending this rapid method to the determination of other peroxides was mentioned, but its range of applicability \\as not determined. I n his most recent nork (+$) Dugan demonstrated that the reagent reacted with several other peroxides in benzene solution but also noted that a specific peroxide tended to react a t different rates and occasionally yielded a different end color a i t h a change in solvent. Ryland ( 1 1 ) investigated the use of N,Ndiphenyl-p-phenylenediamine, A‘,A’-dimethyl-p-phenylenediamine sulfate, and N , S , S ’ , X ’- tetramethyl - p - phenylenediamine hydrochloride as colorimetric reagents for peroxides and pointed out their advantages and disadvantages. No quantitative data were obtained. Hydroperoxides have been determined by measuring the absorbance of the colored complex formed between titanium and the hydrogen peroxide produced by the strong acid hydrolysis of the hydroperoxide (IO,15). I n general. the reagents used in the titanium
