Ruth Watkins

Interesting interferences in a direct serum creatinine reaction

Abstract A spectrophotometric study on a direct picric acid reaction for creatinine in severely jaundiced serums is described. A problem appears to be caused by the oxidation of bilirubin which minimizes rising absorbance when using continuous measurement. Simple examples of interferences with the kinetic mode are shown along with the hitherto unreported interference of the drug, Cephalothin, which also undergoes a picric acid reaction. A procedure in which a delta absorbance is obtained after decolorization of the Jaffe complex by acidification is shown as one available means for obviating the bilirubin effect. However, the theory that Jaffe-reactive interferences do not decolorize with the same acid treatment is not totally applicable when the drug, Cephalothin, is present.

Determination of copper, iron, and zinc from a single small sample☆

Abstract Procedures have been described for the sequential determination of copper and iron in one aliquot of a trichloroacetic acid-ascorbic acid filtrate followed by the determination of zinc in a second aliquot. Alternatively, copper, iron, and zinc can be determined individually on three aliquots of the same solution or either can be determined separately on a smaller sample. All reactions are selective for the three metals under the described conditions. The various parameters of the procedure are shown to be optimum for color formation and without interference or interaction for any metal by either of the other two metals.

Spectrophotometric study of bilirubin and hemoglobin interactions in several hydrogen peroxide generating procedures

Abstract The interference effects of bilirubin and hemoglobin have been described for the peroxidase-hydrogen peroxide oxidation of a hydrogen donor and the catalase-hydrogen peroxide oxidation of methanol to formaldehyde. A competition between bilirubin and the intended hydrogen donor is shown for the substitute analyte, hydrogen peroxide, with a resultant diminution of color due to the loss of intended reaction. No inhibition of peroxidase action appears to take place; its action when complexed with hydrogen peroxide is directed toward the competing hydrogen donor, bilirubin. The final color measured appeared to be partially compensatory, that is the sum of intended color plus the color of residual bilirubin. The subtraction of a serum blank representing a static system will result in a lowered value and a larger error. Hemoglobin, with its strong Soret band can, if its concentration is excessive, cause a major interference in reactions such as the Hantzsch reaction which result in overlapping bands at the reaction wavelength. Samples which are both hemolyzed and jaundiced would present as formidable blanking problems. Further studies on bilirubin and its glucuronide and their individual effect on the peroxidase-peroxide reaction are presently in progress.

Essential serum trace metals: I. Determination of iron

Abstract Studies of serum iron determination have been described using sensitive color reagents of high molar absorptivities. Each selective reaction for iron is strengthened by means of complexing molecules which eliminate trace metal interferences. One ligand. 4-(2-pyridylazo)-resorcinol, exhibits two sensitive peaks, either of which can be used to determine iron quantitatively if an interference is present at one wavelength, or to qualitatively help ensure by ratio measurement that iron is the only metal determined. The second ligand, 2,4-bis(5,6-diphenyl-1,2,4-triazin3-yl) pyridinetetrasulfonic acid, is not only sensitive but has the capability of measuring iron, copper, or both by a judicious choice of selective conditions. Each compound has a useful potential in either discrete sampling or on-stream automation.

Sensitive microreacting ligand for serum copper

Abstract A rapid and simple filtrate procedure for the determination of serum copper is described. It involves only 2 chemical steps and is quite sensitive with a molar absorptivity of 33,300. The color formed is stable for more than 30 minutes and the results indicate that the reaction is quite applicable to the determination of copper found in serum.

Essential serum trace metals: II. Determination of copper and zinc☆

Abstract Several complexing ligands for copper and zinc were tested for their feasibility as analytical reagents. Of these, tetraethylthiuram disulfide and its analog, bis (1-piperi-dylthiocarbonyl) disulfide were selected for copper with some advantages determined to accrue to each. Two others, 1-(2-pyridylazo)-2-naphthol and 1-[(5-chloro-2-pyridyl) azo]-2-naphthol were selected for zinc with the preference given to the former for stability of color formed and accessibility of the reagent although the chloro analog is inherently the more sensitive of the two reagents. For those who do not have atomic absorption spectrophotometry at their disposal, the molecular absorption approach still appears to be an excellent one with good selectivity for the two metals described.

Postulated flaw in densitometry

Abstract An effort was made to explain the reported large discrepancies between comparative spectrophotometric measurements for two separated hemoglobins. This is the simplest quantification requirement following electrophoretic separation involving either integrating densitometry or elution followed by spectrophotometry. A graphic simulation of several exaggerated separation conditions with known areas and assigned absorbances was postulated from which the results obtained with inadequate slit geometry were calculated. The relative concentrations found were always in marked nonlinear disagreement with the linear theoretical values present. From this one could infer that true relationships between the volume relationships of separated zones are not realized if one neglects one dimension of a three dimensional measurement. Comparison between elution followed by spectrophotometry and direct spectrophotometry (densitometry) showed a smaller difference in means than did the work which stimulated this study (12). There is no way in which the differences in these two findings can be easily defined. However, one could infer that neglect of a zone dimension during the measurement process could be considered one source of such a measurement discrepancy. But even if nonproportional measurement of different zones to obtain relative areas of each does not describe the total problem, it should still be an important factor to keep in mind. A further study of other factors such as translucence or opacity with its Lambert effect on measurement, and the related severe turbidity factor in solution measurements is in progress.

Sensitive micro determination of nonesterified fatty acids

Abstract A procedure is described in which an extremely sensitive copper ligand, TETD, is used as a substitute for DDC in the indirect determination of NEFA. The latter was isolated by extraction into chloroform as a copper soap followed by reaction between TETD and the copper in acid solution. This resulted in an approximate 3-fold increase in absorbance enabling determinations of NEFA to be carried out with a greater reaction sensitivity.

The Zimmermann reaction and a correction for irrelevant absorption

Abstract Two similar modifications of a previously described aqueous Zimmermann reaction for 17-ketosteroids were studied to see whether the Allen correction for irrelevant background absorption could be accurate when pure steroids were compared against a single steroid as a calibrator. Dehydroisoandrosterone was selected as the standard because most procedures use it for that purpose. In one procedure in which varied spectral shapes, wide-ranging peak maximum values, and molar absorptivities were evident, accuracy of recovery was poor. In a second procedure, in which the primary variation was in band width rather than in either fine structures or molar absorptivities, the results were more accurate though still inaccurate as compared to peak measurement calculations. It would seem that this correction could only be valid for those procedures in which calibrator and analyte yield spectra which show better uniformity than the two procedures described here.

Comparative chromogenicities and discrepant spectra obtained between a heterogeneous and homogeneous Zimmermann reaction

Abstract A spectrophotometric study is shown for two modifications of a 17-KS procedure in which nearly all aqueous reaction media were used. Although the apparent differences in matrices of reaction between the two procedures appear to be minute, a small volume of ethanol for one is substituted for the same volume of methanol in the other, the final alkalinity of the latter procedure is stronger, and the reaction temperature was increased, wide differences in spectral results are found between the two methods. The ethanolic, lower alkalinity procedure is a homogeneous, single-phase reaction while the methanolic, higher-alkalinity procedure is a heterogeneous system in which a precipitation of detergent occurs and the precipitate becomes an integral part of the color reaction. The rate of formation of the ethanolic procedure is variable for the different 17-KS but it is constant in the methanolic procedure. The chromogenicities of color formation between the two procedures differ in that the methanolic system provides uniform and predictable spectra with more equivalent molar absorptivities. Lastly, the methanolic system shows a much greater sensitivity owing, apparently, to a more complete final reaction. This inability to form full color for several compounds in the homogeneous system can be related in some way to the large variations in ability to form that color owing to matrix composition.