Direct determination of chromium in urine by electrothermal atomic absorption spectrometry.

Direct determination of chromium in urine by electrothermal atomic absorption spectrometry. Scand. j. work environ. & health 5 (1979) 404-410. A simple, direct method for the determination of chromium levels in urine is described, electrothermal atomic absorption spectrometry having been used after the urine samples had been acidified. The optimal conditions at the ashing and atomization stages was determined. The detection limit of this method is 5 nmol/l, and the coefficient of variation (within day) is about 3 % in the con centration range 20-400 nmol/l. Use of a low temperature asher with electrically excited oxygen before analysis gave results similar to those of the direct procedure. Good correlation was obtained from two other laboratories using different instru ments and pretreatments.

of malfunc:tions, e.g., artery disease (9,13). It occurs in high concentrations in soil, vegetables, and water, which in certain areas constitute important sources of supply for humans. Extensive reviews about the occurrence and function of chromium in biological systems have been published (8,10).
Because chromium taken up in the body is excreted predominantly in urine (10), one would expect the analysis of urine to provide an estimate of the absorbed dose. Being able to measure urinary chromium levels would be especially relevant to the evaluation of occupational exposure, but it is also important for the estimation of uptake from food and in studies of dietary chromium deficiency.
Many analytical methods for the determination of chromium in urine have been published, some of which depend on the use of gas chromatography after derivatization (13), while others use atomic absorption spectrometry (AAS) (3,5). Gener-ally the methods described earlier require considerable sample preparation, and they are quite laborious. One recently published, more direct procedure makes use of a one part in ten dilution of urine, which, however, results in a low precision at low concentrations and a detection limit above the normal concentration (17). Other procedures have used a direct injection of urine and calibration against standards prepared in water (14). But due to the variable composition of urine there have been problems with high background absorption, and the results have been unreliable. Furthermore, the use of temperatures above 1250°C at ashing may result in losses of chromium (15,18). Difficulties with the analysis of chromium in various materials have been illustrated in an international laboratory control program (11).
We have examined possibilities of analyzing chromium in urine with the flameless atomic absorption of acidified samples following a careful optimization of the parameters at ashing and atomization and using strictly controlled conditions.

MATERIAL AND METHODS
Urinary samples were taken from welders and people with no known occupational exposure to chromium. The samples were stored at -20°C until analysis.

Chemicals
A chromic chloride standard containing 1 mg of chromium per milliliter in 1 mol of nitric acid per liter (BDH, Poole, England) was diluted in 0.01 mol of nitric acid per liter upon use. The nitric acid, Suprapure 85 0/0 (w/v), was from Merck, Darmstadt, the Federal Republic of Germany. The water used was distilled and deionized.

Instrumentation
An atomic absorption spectrophotometer model AA-6 (Varian Tectron, Melbourne, Australia) with a carbon rod CRA-63, a workhead model 90, and a background corrector was used, as well as a temperature control unit according to Lundgren (7), which measures the temperature of the carbon rod with a photodiode. Other details and instrument settings are given in table 1.
A recorder model TE 220/2 (Tekman Electronics Ltd., Oxfordshire, England) with a full-scale deflection response time of less than 0.3 s was used. The recorder was modified so that when no atomizing signal was expected it could be shortcircuited.
The electronic signals were also evaluated by use of a Peak Reader Module (PRM 6; LL-Electronic, Umea, Sweden) which gives a digital readout of the absorbency and integrated area of each peak.
When many samples were to be analyzed, an Automatic Sample Dispenser ASD-53 (Varian) was used.
The instrument was modified by changing the damping of the output signal from 0.35 s to 50 ms to obtain a fast response (6). This procedure increased the range of absorbency with a linear response. A digital printer (Kontron, Switzerland) was also used. An electromagnetic high-frequency low-temperature vacuum asher, Typ 500 R (Uppsala Elektronik, Stackmastarv. 4, Uppsala, Sweden) producing excited oxygen (4) was used in some experiments according to instructions from the manufacturer. Brussels, Belgium) and then thoroughly rinsed in metal-free water prior to use. After the addition of 0.10 ml of concentrated nitric acid to 10.0 ml of urine and thorough mixing, the test tubes were allowed to stand for at least 1 h before analysis. From this mixture 1.0 ml portions were pipetted into three test tubes, two of which also received standard additions at different levels. Usually 5.0-,u1 samples were pipetted into the carbon tubes either manually with a Micropipette (Oxford Lab. Int. Corp., Ireland) with disposable teflon tips or with an automatic sample dispenser. Three pipettings were made of each solution. In order to obtain the best reproducibility, threaded carbon tubes were used. Urine samples containing a high chromium concentration were diluted with nitric acid (0.01 mol/I) before analysis. The peak heights of the recorded signal or the peak absorbencies from the PRM-6 were used for the calculation of the chromium content in the urine samples. Test tubes of polystyrene (size 12 ml, Haeger Plastics, Stallarholmen, Sweden and size 4 ml, Nunc, Roskilde, Denmark) were washed in metal-free water containing the detergent RBS-25 (Chern. Prod.,

RESULTS
Various temperatures have been used at the ashing stage. As can be seen in fig. 1, an approximately constant absorbency was obtained for chromium in nitric acid (0.01 mol/I) in the range 600-1500°C. Decreased absorbency was obtained at temperatures above this range, and losses in the ashing sequence were therefore indicated at elevated temperatures. The corresponding studies for urine samples are shown in fig. 2, where it can be seen that temperatures up to 1250°C may be used without significant losses in order to obtain a low background. During the study the use of ashing aids was investigated in order to improve the ratio of the chromium signal to the background noise. Ammonium sulfate, magnesium nitrate, ammonium nitrate, and ascorbic acid were added in different experiments to obtain final concentrations of 0.5 to 5 Ofo (w/v). A somewhat higher sensitivity was achieved with the addition of ammonium sulfate and ammonium nitrate, but the background noise  also became higher. Ascorbic acid lowered the background but also gave a reduced chromium signal. Therefore, for the final method we added nitric acid only.
The influence of different atomization temperatures has also been studied in some detail. As can be seen in fig. 3, a satisfactory temperature was obtained at 2500°C. There was no difference when calculations of the chromium concentrations in urine samples were made from peak heights or integrated values. It was very important to know whether different urine samples would give rise to different calibr.ation slopes of their standard curves. For this purpose we analyzed many urine samples with and without additions of chromium at four levels and with three determinations on each level. Most urine samples gave very similar calibration slopes. However, among ten samples there were about three which produced rather aberrant slopes; they give rise to concentration values differing up to 25 Ofo from that obtained with the standard addition method.
To decrease the error of single samples, we therefore used the method of standard addition instead of evaluation against a standard curve. The precIslOn and reliability of this method was studied by determination of the recovery of additions of chromium as can be seen in table 2.
Precision data are presented in table 3 (within day and between days). The detection limit was 5 nmolll when calculated with the formula (2 X SD X Cr concentration)/x, where x is the peak height average and SD the standard deviation, from 15 injections of 10 III of a urine sample low in chromium. Similar results were obtained with two other urine samples. The blank was mostly negligible.
It is well known that precipitates may occur in urine samples, especially during storage. We therefore determined the chromium in the supernatant after centrifugation and in homogenized samples. For the ten samples examined after storage for three weeks at +4°C, no difference was found; therefore no significant enrichment of chromium in the precipitates was indicated. Nine urine samples were also analyzed with Perkin-Elmer instruments and different pretreatment procedures in two other laboratories. As can be seen in table 4, good agreement, at least above 300 nmol/l, was obtained between the different laboratories. We also analyzed the the urine samples after low temperature ashing at < 200°C by using a method similar to the one described earlier (18). No statistically significant difference between the two methods was obtained, as can be seen in time consuming, these procedures impart Tabte 4. Results of the analyses of chromium in urine (nmol/l) by electrothermal atomic absorption spectrometry, as obtained by different laboratories. Laboratory A was the Institute of Occupational Health, Oslo (N. Gundersen); a Perkin-Elmer AAS Model 300 with an HGA 76 and direct injection of 50 ftl of undiluted urine were used with two standard additions to each sample. (This analytical procedure was found to be critical and required, among other things, a very careful alignment of the lamps.) Laboratory B was the Institute of Occupational Health, Helsinki (J. Kilpia) a 1/10 dilution of urine was used as described earlier (17). Laboratory C was our laboratory, column I being the method described in this paper and column II the same method after low temperature ashing.

Sample Laboratory
A B C (I) C (II)   1  110  75  85  90  2  640  635  660  505  3  280  290  260  305  4  680  705  695  695  5  645  680  630  585  6  750  720  680  690  7  625  560  535  525  8  45  60  20  25  9 400 415 360 400 the risks of variable recovery and contamination. Pitfalls encountered in flameless atomic absorption have been described in a recently published critical study (16). Explanations for failure using direct injections of urine samples may be that undiluted urine has been used and/or that the parameters for drying, ashing and atomization as well as the background correction have not been evaluated. We have found that these conditions may be criti-caI.2 It has been reported that part of the chromium in urine occurs in the form of a complex, which is said to be much more volatile than chromium ions in water solutions (15). Some earlier published methods can be criticized for not completely measuring total chromium due to losses arising during sample preparation, caused by excessive temperatures being used during the dry ashing or in the flameless atomic absorption. To diminish these possibilities, we used a lower ashing temperature (1050°C) than the ones used in earlier reports [e.g., 1300 0 C (15) or 1600 0 C (14)]. Secondly, we treated the samples with nitric acid prior to analysis. We believe that this procedure eliminates the problem of volatile chromium because acidic and oxidizing conditions are known to destroy complexes of most organic compounds and metals.

Regression equations
The concentration levels of chromium found in the urine of persons not occupationally exposed have been reported in the literature to range down to about 20 nmol/I. The detection limit (5 nmol/l) of the method reported in this paper is well below this level, and accordingly we have found some samples with chromium concentrations in this range. Thus, earlier published procedures do not have a sufficiently low detection limit. We can lower 2  our detection limit somewhat by increasing the sample volume injected into the graphite tube. Furthermore, the precision obtained with our procedure is better than that reported for earlier published methods. Because no certified method exists for quantifying chromium levels in urine, it is difficult to ascertain the accuracy of the new method. However, the analytical recovery of chromium added to urine was close to 100 %. Furthermore, the good agreement between different laboratories (table 4) using different instruments and procedures implies a good accuracy.
In comparison to other procedures using extraction, ashing and extraction, and dry ashing and dissolution of the ash the procedure described in this report is straightforward and simple, involving less risk of contamination by obviating the use of many chemicals, pipettings, and vessels. When evaluating several urine samples against one standard curve, one person can analyze about 40 urine samples in 8 h, including the calculation of results. When the method involving the addition of standards is used for each sample, at least 20 urine samples may be analyzed. This rate is faster than analysis after extraction and less elaborate than other procedures which depend, for example, on dry or wet ashing. With automatic injection with the autosampler, about 30 urine samples may be evaluated in 1 d with the method of added standards.