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Total Protein-Bound. Alpha-2-globulin

Solution. Cr% SD. Cr% SD. Phosphate buffer. 7.0 2.2 2.4 0.5 Water wash. 2.4 0.3 2.1 0.3 Petroleum spirit. 1 .5 1 .2 2.5 1 .3 Ammonia EDTA. 73.3 2.2 80.5 2.9 Protein mass. 12 6.3 5.3 1 .0 Total. 96.2 / 92.8 / Cr unaccounted for. 3.8 / 7.2 /

The standard pretreatment process CDIII gave recoveries of 73% for the total protein-bound chromium and 81% for the alpha-2-globulin-bound chromium. The major identified location of

unrecovered chromium is the protein mass in both cases. However the chromium left in the less penetrable precipitates from the total protein-bound chromium series at 12% is slightly more than twice the amount left in the alpha-2-globulin-bound precipitates at 5%. The chromium loss in the phosphate buffer wash is also higher in the total protein-bound series, presumably due to the higher acidity of the petroleum spirit extract from the alpha-2-globulin-bound stream. The higher value of the "lost" chromium in the alpha-2-globulin-bound process is due to the less well demarcated petroleum spirit/aqueous phase boundaries at the wash stages leading to less complete transfer of .the organic phase, particularly from the wide diameter tuftainers. Haemolysis and high serum iron levels can both reduce the recovery with the total protein-bound chromium process.

4.13.2. AAS/ETA. Relative Response.

The AAS/ETA conditions are discussed in the next chapter,however the relative signals given by chromium in the processed materials compared with unprocessed material are relevant to the pretreatment process, as discussed in the section on excess hfacac.

The AAS/ETA relative response is defined as :- Peak height x 100 / Expected peak height.

Expected peak height is the peak height given by an equal concentration of chromium in unprocessed ammonia/EDTA/acetic acid solution. The concentrations of chromium in the solution derived from processed samples were calculated from radiometric recoveries. Incremental peak height values were used for the relative responses. The reference values for the increments were reagent blanks and unspiked serum values respectively for determinations on standards and serum samples. Spike values were equivalent to 0.125 p.g Cr/L in a specimen.

Standard AAS/ETA conditions as described in E494 were used, with uncoated profile graphite tubes and 25 /iL injections. A summary of the results is given in table 4.25 below.

TABLE 4.25.

Relative Responses, Processed and Unprocessed Materials. i) Unprocessed standards. iij Processed standards.

Response 100%, RSD 4.5%, n=4 Response 94%, RSD 3.9%, n=12 iii) Total Protein-Bound Serum Cr. iv) Alpha-2-globulin-Bound Cr.

The relative responses of processed material on AAS/ETA compared with unprocessed standards was subjected to Student's t test. The material from the total protein-bound chromium process did not differ significantly from the unprocessed material (95% confidence). However the processed standards and the alpha-2-globulin-bound chromium material both showed significantly different responses from unprocessed material, the confidence limits being 95% and 99% respectively.

4.13.3. Matrix Simplification Achieved.

The weight of the residue from 4 mL of serum processed for total protein-bound chromium using method CDIII was compared with the weight of the total dried solids from 4 mL of the same human serum pool. The mean weight of the processed material was 2.9 mg representing only

0.82% of the solids in the original specimen.

The chromium concentration factors are 24 and 27 for the total protein-bound chromium and alpha-2-globulin-bound chromium processes respectively.

The matrix simplification factor is 3.7 based on 8.8% dried solids in the human pool serum tested compared with 2.4% serum derived solids in the solution for AAS/ETA. However the chromium concentration factor multiplied by the matrix simplification factor is (27 X 3.7) =

1 0 0

.

Metals other than those present at an oxidation stage capable of forming "inert" complexes will be eliminated at the phosphate wash stage as shown by the recoveries of Cu, Fe and Zn, all well below 1%. Iron and nickel are the only metals forming "inert" complexes which are probably normally present in human serum at levels higher than 1 .0 ,ug/L. However the serum iron is present as Fe(III) (Lemberg et al (8)),

and observation has indicated that the possibly inert Fe(II) complex is oxidised to the Fe(III) at the heating stage. The serum nickel is probably less than 5 pg/L and problems due to the presence of metals other than Cr in the test extracts are extremely improbable.

4.13.4. Detection Limit.

The detection limit is clearly governed in this method by the precision of the blank. A gross signal > blank + (4 x blank SD) , can be detected with 95% confidence. Thus a sample chromium level equal to 4 x the blank standard deviation in pg Cr/L equals the detection limit. Table 4.26 below lists the blanks and detection limits of four batches.

TABLE 4.26.

Blank values, and derived Detection Limits.

Blank jig Cr/L. Detection Limit.

Batch. Mean. Standard Deviation. >ug Cr/L.

I. 0.08 0.0076 0.030 II. 0.08 0.0078 0.031 III. 0.05 0.0086 0.034 IV. 0.09 0.0088 0.035 Means. 0.075 0.0082 0.033 4.13.5. Summary.

A pretreatment has been developed that will extract and concentrate 75% of the total protein— bound chromium, or 80% of the alpha-2-globulin— bound chromium into 120 jiL of final solution containing less than 1% of the solids present in the original sample. The final solution appears to be suitable for the determination of chromium by conventional AAS/ETA without background correction. The detection limit using a sample volume of 4 mL is about 0.033 /ig Cr/L. However individual specimens need to have a radioactive chromium spiked aliquot processed in parallel to monitor the recovery matrix modified AAS/ETA response.

The batch of tests cannot be processed in under three days, and the maximum number of tests in a batch is probably 24, representing duplicate determinations for both serum Cr parameters from a single glucose tolerance test. The radioactive chromium is not expensive, the hfacac is easily the most costly consumable.