Correction for relative interferences in a cholesterol determination

MICROCHEMICAL

JOURNAL

21, 485-495

(1976)

Correction for Relative Interferences Cholesterol Determination’

in a

M. T. PERLSTEIN* Department

of Chemistry,

University Canada N9B

of Windsor,

Windsor,

Ontario,

3P4

A. MANASTERSKI Department

of Pathology.

Mt.

Carmel Mercy 48235

R. J. THIBERT Department of Chemistry, Windsor, Ontario,

Hospital.

Detroit.

Michigan

AND B. ZAK University of Windsor, Canada N9B 3P4

and Departments of Pathology, Wayne State University School of Medicine and Detroit General Hospital, Detroit, Michigan 48201 Received

July

24. 1976

INTRODUCTION

Bromide is an anion which is eliminated slowly from the circulation, and therefore, bromide contaminated serums are encountered for some time after ingestion has ceased. Thiouracil and its therapeutic analog, propylthiouracil, are used as medications which thereby become in vivo constituents of serum. In addition, thiouracil has been suggested as an in vitro serum preservative in butanol-extractable iodine determinations (3) where an aliquot of the same sample could be used for a cholesterol determination. Both the uracils and bromide have been observed to interfere with modifications of iron color reactions which are used for the determination of serum cholesterol (2, 3). As an example, thiouracil and bromide both enter into the chemical reaction taking place between cholesterol and ferric perchlorate in an ethyl acetate-sulfuric acid reaction medium (4) as evidenced by the fine structure spectral changes which occur in the presence of these compounds (4). In addition, when bromide interacts with cholesterol and reagents, it causes a hyperchromic effect

’ Supported ration. z In partial of Laboratory

in part by a Grant-in-Aid fulfillment Medicine,

from

the Detroit

Hospital

of the Ph.D. in Clinical Biochemistry. Present Sinai Hospital, Detroit, Mich. 4823.5. 48s

Copyright All rights

General

@ 1976 by Academic Press. Inc. of reproduction in any form reserved.

Research

address:

Corpo-

Department

486

PERLSTEIN

ET AL.

even though it does not itself react to exhibit a blank color at 560 nm, the peak maximum of the chromogenic reaction, indicating that this enhancement occurs only in the presence of cholesterol. In like manner, the uracils do not react to generate a blank color with this iron perchlorate reagent. They also have the ability to interact, but they reduce the intensity of color obtained when they are added into the reaction. Yet if they, or bromide, are added after the reaction with cholesterol is complete, they do nothing to perturb the spectra obtained. This evidence, and the linear fanning effects obtained with contaminated and uncontaminated calibration curves on the addition of these compounds into the reaction, lead to the inference that the manner in which bromide and the uracils behave must be as relative interferences of an enhancing and inhibiting type, respectively. This type of interaction depends both on the concentration of the interference as well as on the concentration of cholesterol, unlike absolute interferences which depend only on the concentration of the offending compound and which usually react simultaneously and independently from the desired analyte. When other modifications of the iron reaction for cholesterol were studied for bromide interference, it was shown that its effect could be eliminated or obviated by treatment of the sample with an ion exchanger to remove bromide ions (I), by metathetical exchange of bromide with the iodate of insoluble silver iodate (2), by the addition of bromide into a plateau region where the observed enhancement becomes independent of the bromide concentration (4), or most simply, by a change in the solvent character of the reaction media from acetic acid to ethanol, wherein bromide did not distort the peak maximum (1, 7). Thiouracil, like bromide, has also been removed as a reaction interference by means of the silver iodate technique (1). One advantage of a relative over an absolute error is that the method of internal standardization can be applied where the standard can react within the sample matrix, undergo the same interaction effect and thereby eliminate the need for separate chemical treatment of the sample to remove the interference prior to analysis. Another possible alternative might be to dilute the interfering substance down to a concentration at which its interaction with reagent and analyte is minimized (3). However, in the final analysis, that approach may lead to less favorable comparative absorbances at the dilution required to obviate the interaction effect. The intent of the present manuscript is to show how the use of the recovery as a standard in the method of internal standardization is a simpler alternative for overcoming a relative interference, whether it be enhancing or inhibiting, than is the purposeful in vifro treatment of the sample to either remove the interfering substance, or dilution of the chemical protagonists in order to minimize the interference (3). Evidence to this effect will be offered for bromide and thiouracil as an alternative to

CHOLESTEROL

DETERMINATION

487

sample pretreatment according to several earlier descriptions (2, 3), and for propylthiouracil which has not previously been described in the same manner even though it interferes with iron reactions for cholesterol just as thiouracil does. Presumably, other substances which result in similar enhancement or inhibition of this cholesterol reaction might be obviated more conveniently as interferences by the use of the recovery as a standard than would be possible using any of the several previously described options. MATERIALS

AND METHODS

Reagents Cholesterol standard solution, 2000 mg/liter in glacial acetic acid. Ferric perchlorate color reagent was prepared to contain 520 mg of ferric perchlorate, 600 ml of ethyl acetate and 400 ml of concentrated sulphuric acid. Propylthiouracil and thiouracil aqueous stock solutions (100 mg/lOO ml) were prepared and diluted as needed. Sodium bromide stock solution was prepared to contain 100 mEq/l, and dilutions made as necessary. Propylthiouracil, thiouracil, and bromide were aqueous solutions. Therefore, dilutions were made from the corresponding stock solution and 50 ~1 was added to 5.0 ml of the color reagents, and mixed well before the addition of the cholesterol solution. The blank solution, in this case, consisted of the color reagent (5.0 ml) glacial acid (50 ~1) and water (50 ~1). Water (50 ~1) was added to the cholesterol standard reaction. Otherwise these reactions were performed as described (5). Methods The experimental procedures used here have been previously described (5). Briefly, 50 ~1 of a sample or standard containing O-5000 mg/liter of cholesterol was added to 5.0 ml of a color reagent containing ferric perchlorate dissolved in a solvent medium of ethyl acetate, and sulfuric acid. The well-mixed solution was heated for 90 set in a heating block, cooled and then scanned across the visible range from 700-400 mm against its blank in a double-beam recording spectrophotometer. DISCUSSION AND RESULTS

It is commonly accepted that recovery of an addition of analyte which had been added to the base value of a sample bears some relationship to the accuracy of the reaction employed to determine that recovery. It should also be just as well accepted that recovery of an addition by itself cannot be used to prove that a base value was accurately determined, if an error exists for the reaction owing to any one of several potential factors, and evidence of this latter fact will be described. However, even though accurate determination of an amount added may not truly be representative of the accuracy of determination of the analyte in an original sample,

488

PERLSTEIN

ET AL.

recoveries can have at least one other important function aside from a possible demonstration of losses encountered during procedural handling. If recovery of an addition is accurate, it is convincingly clear that the matrix of the sample contains nothing that influences the reaction in a manner which is either of an inhibiting or enhancing nature. But if recovery of an addition is inaccurate the increasingly either high or low with increasing concentration of analyte, it may be clearly evident that the matrix of the sample contains something that is probably influencing the reaction for the analyte in a relative manner. In the case of an interfering compound which may be present to cause an absolute error, no concentration of addition of analyte per se would reveal that the interference existed, because both analyte and interfering compound may react independently from each other with the reagent. However, unlike the compounds which cause absolute errors, a fixed concentration of a matrix substance which exerts an influence upon a reaction to cause a constant percentage of enhancement or inhibition at different concentrations of an analyte can be shown to demonstrate that there is an interaction of that interference in the reaction between reagent and analyte. In effect, this can be likened to having a new reagent which yields a different molar absorptivity with the analyte. It is this latter phenomenon with which the remaining discussion is concerned along with the effective use of these relative interference characteristics as a calibration device. An intimate picture of absolute versus relative errors caused by interferences provides some clarification of these views, as follows. Two common classifications for reaction interferences are those in which either absolute or relative errors occur. When the error is absolute, it can be expressed as a fixed value whose percentage as an error decreases as the concentration of the analyte increases. When the error is relative, it can be expressed as a fixed percentage whose concentration value as an error increases as the concentration of the analyte increases. Therefore, on the one hand, if the concentration of an absolute interference is constant at all concentrations of analyte encountered, the slope of the results will be parallel to that slope obtained with the uncontaminated analyte, but the interference is positive or negative in its action. On the other hand, if the concentration of a relative interference causing such errors is constant at all concentrations of an analyte encountered, the slope of the results obtained will be greater or less than that of the uncontaminated analyte, depending on whether the effect is enhancing or inhibiting, and all representative curves will originate from a common coordinate set. It might be restated then that an interference which results in an absolute error usually reacts with the reagents or with the analyte independent of the reaction of the desired constituent, while in effect, the relative error is caused by an interfering compound whose reaction is dependent upon the presence of the analyte

CHOLESTEROL

DETERMINATION

489

before the interaction of the substance manifests itself. Figures 1 and 2 graphically illustrate these phenomena. In Fig. 1, absolute errors of a negative or positive nature, fixed at one concentration of the interference but added to several concentrations of desired constituent are shown in a comparison to those same concentrations of uncontaminated analyte. The exemplification of a positive absolute interference is shown by the action of bilirubin in the presence of the simultaneous action of cholesterol in a cholesterol determination in which each reaction can be shown to be taking place independently of the other with the reagents (4). A mode1 calibration curve for cholesterol can be constructed as shown in the figure in the presence (curve B) and in the absence (curve A), of a constant amount of bilirubin. In this circumstance, the slope of the line will be identical to that of the true standard curve, but it would be displaced by the absolute amount caused by the interference. Since the slopes of the curves are identical when an absolute error occurs, recovery of an addition is not an appropriate measure of accuracy, because the fixed error is still there. This finding reinforces the concept that recovery of an addition can be accurate even though determination of the base value to which it is made may not necessarily be accurate. Therefore accuracy of recovery should not indicate that base values are accurately obtained. A measure of this independence as reactants has already been shown for both compounds in several cholesterol reactions (4). The flat curve at the bottom of the graph, curve (B-A), demonstrates the reaction of interference for this fixed quantity of bilirubin, and this is in effect the result obtained when the standard curve representing cholesterol is subtracted from the curve containing bilirubin. Exemplifying the negative absolute interference could be shown in similar fashion if a fixed amount of EDTA were present for several concentrations of calcium in a calorimetric determination of the latter metal. In this case, the interference does not react with the reagents but with the analyte, making it unavailable for reaction. If no analyte were present, there would appear to be no reacting interference present, and a flat curve superimposing the zero abscissa would result. If one used curve A as the uncontaminated calibration line and curve C as the EDTA contaminated standards, then (C-A) would be the flat resultant curve representing the negative absolute interference. In either case, when positive or negative absolute interferences are present, the errors are indicated by the parallel nature of the curves obtained and the residual interference between slopes will parallel the abscissa of concentration. An example of a positive relative interference can be shown by the absorbance versus concentration data obtained with the interaction effect of bromide in the iron perchlorate reaction with several concentrations of cholesterol, which is graphed as the enhanced line of Fig. 2. When compared to the normal uncontaminated curve it can be seen that a linear

490

PERLSTEIN

ET AL.

CONCENTRATION 1. Curve A is a simulated uncontaminated cholesterol (and calcium) calibration curve. Curve B represents the same standard solutions to which a fixed concentration of bilirubin is added. Curve (B-A) is the positive difference between the two curves. Curve C shows the same standards (calcium) to which a fixed concentration of EDTA is added. Curve (C-A) is the negative difference between the two curves. FIG.

Rt’l.WI\E

CHOLESTEROL

t:RROR

(MG/L)

FIG. 2. A normal uncontaminated calibration curve of cholesterol is compared to the same standards enhanced by a fixed concentration of bromide or inhibited by a fixed concentration of thiouracil or propylthiouracil.

CHOLESTEROL

DETERMINATION

491

fanning effect is achieved from a common origin and that the percentage of the error remains constant, while the concentration of cholesterol represented by the error increases with increasing concentration of cholesterol. In like but opposite manner as graphed in the inhibited curve, thiouracil causes a negative error in the measurement slope by its interaction in the reaction of iron with cholesterol and also results in a linear fanning effect in the measured slope for cholesterol. In fact, this fanning effect is one way in which a relative error manifests itself. An illustration of the interaction with both cholesterol and reagent rather than reaction of interference with the reagent alone can be shown by the spectra of Figs. 3 and 4 for bromide and thiouracil, respectively. Figure 3 graphically depicts the spectra obtained with both uncontaminated specimens of cholesterol standards as well as with those same standards to which a fixed amount of bromide was added. Two facts are discernibly evident in comparing the paired spectra. The contaminated standards show a change in the fine structure of their spectra with a shoulder generated at 625 nm. At the same time a fixed percentage increase in peak absorbance occurs because of this interaction of bromide with reagent and cholesterol. The hyperchromic effect at this concentration of contaminant is 12%. There is a blank reaction between bromide and the iron reagent near the edge of the ultraviolet range which shows some qualitative and quantitative characteristics for bromide, that is, if its concentration was

FIG. 3. Spectra of three concentrations of cholesterol are shown when reacted with iron reagent and then interacted with a fixed quantity of bromide.

492

PERLSTEIN

450

ET AL

550

650

FIG. 4. Spectra of three concentrations of cholesterol are shown when reacted with iron reagent and then interacted with a fixed quantity of thiouracil.

varied, its spectra would reflect the change in concentration. Interestingly, when one sums up the blank reaction involving bromide plus the cholesterol reaction by itself, and compares it to the spectrum of the reaction containing both cholesterol and bromide, it becomes evident that the spectra are not additive and that the effect of bromide is to interact with reactants to obtain a new spectrum with a different molar absorptivity and a modification in fine structure. Obviously, unless the blank solution could change its spectrum at each concentration of cholesterol, a seemingly impossible phenomenon, then it could never sum up adequately anyway, because percentage changes with analyte automatically imply that each increased concentration tested would result in an increased interaction effect on that concentration over the lower concentration. In addition, if one adds bromide after the reaction of iron with the cholesterol is complete, there is no effective alteration of the spectrum, for it remains unchanged. In like but opposite manner, a hypochromic effect of 12% in peak value and an increase in fine structure with a shoulder at 625 nm results from the addition of a fixed concentration of thiouracil to several concentrations of cholesterol. Both pure and contaminated spectra are shown in Fig. 4 for 200 mg/liter of thiouracil (1.56 mmol/liter) added to 1000, 2000, and 3000 mg/liter of cholesterol (2.6, 5.2, and 7.8 mmol/liter). There is no blank reaction color for thiouracil, and as described for bromide, if this interfering compound is added after the reaction with cholesterol has already taken place and the final chromogen has formed there is likewise no evidence of any change in the spectra at

CHOLESTEROL

493

DETERMINATION

any concentration, again indicating the interacting nature rather than the direct reacting nature of that interference. The use of the method of standard additions to correct the interaction effects of bromide and thiouracil was applied to bromide and thiouracil contaminated reactions containing either standard cholesterol solutions or serums. The results of this study are shown in Table 1. Another iron reaction for cholesterol (6) where these two compounds have also been reported to interfere is also shown here to illustrate the versatility of this technique. The method of standard additions is fast, simple, and produces accurate values. Since it is not impossible to encounter both kinds of interferences in the same sample, an experiment was designed to indicate what would happen in such a circumstance. Figure 5 shows the competitive interaction effect obtained on adding thiouracil and bromide at equivalent concentrations into the reaction of iron with cholesterol and the spectrum generated then compared to the individual interaction spectra and the uncontaminated spectrum. The observed cholesterol value in the presence of this combination reasonably approximates the true value. It is interesting that this combination gives rise to compensating reactions where each interference exercises an equal but opposite effect at what must be the same reaction and interaction velocities. In this circumstance, the interferences null each other out, and the measured absorbance value of the doubly conTABLE

1

Cholesterol Thiouracil (mg/liter)

Bromide (mEq/liter)

Known

Corrected 199 198. I 98.9 199.2 93 201 109.3 229 92.4 92.7 117 210

99.6 99. I 99.6 99.6 91 104. I III I08 95.6 95.1 115 105

172.2 90.4 207.4

98.4 93.4 102

-

-

IO 10 IO

174.1 185.7 71.3 143.4 76.3 160.0 147.5 282 133.0

-

10

133.4

IS I5 poisoned

160.7 295

200 200 99.3 200 102 193 98.4 212 96.7 97.5 101.4 199.0

193.2 108.6 235.5

175.0 96.8 212

In viva serum

Percent of known value

Found

I5 I5 20 20

-

(mgiliter)

494

PERLSTEIN

ET AL.

NANOMETERS 5. Spectrum obtained when uncontaminated cholesterol was reacted with iron reagent is shown (std). Spectra for the same concentration of cholesterol are shown when enhanced (Br-), inhibited (Thio), and then finally treated with both enhancer and inhibitor (Thio + Br-) in a nulling effect on interfering contaminants. FIG.

taminated specimen is similar to that of the uncontaminated specimen even though it is demonstrable that the spectrum obtained has been altered from the one obtained with cholesterol standard only. In this example some obviously interesting and complex chemical events must have taken place. To reiterate, while the bromide was interacting to enhance color formation, thiouracil appeared to be interacting to depress color formation so that the overall effect of the two compounds cancelled each other out. SUMMARY The detrimental influences of thiouracil, propylthiouracil, and bromide on the ferric perchlorate reaction for cholesterol have been previously reported (2,3). On the basis of recovery studies it can be shown that their role in the reaction results in a relative rather than an absolute error of cholesterol measurement. Because a relative error causes a linear fanning effect on increasing concentrations of analyte which depends for its slope on both the concentration of the interference as well as on the concentration of constituent being measured, the method of standard additions can be used to correct a matrix effect on the reaction. A system in which the standard is used internally can eliminate the need to pretreat the sample in order to separate the interfering compound from the analyte. Therefore, both the interference and its obviation by this method of internal standardization are discussed here in a procedure which results in a means for simpler rescue of a contaminated sample than is afforded by the more laborious and time consuming pretreatment of previous workers.

REFERENCES I. Manasterski, A., and Zak, B., Spectrophotometric study of thiouracil interference in a serum cholesterol determination. Microchem. J. 18, 240-248 (1973). 2. Rice, E. W., and Lukasiewica, D. B., Interference of bromide in the Zak ferric chloride

CHOLESTEROL

3. 4.

5.

6. 7.

DETERMINATION

495

sulphuric acid cholesterol method and means of eliminating this interference. C/in. Chem. 3, 160-162 (1957). Rice, E. W., Interference of thiouracil in the ferric chloride sulphuric acid cholesterol reaction. C/in. Chem. 10, 1025-1027 (1964). Pertstein, M. T., Thibert, R. J., and Zak, B., Spectrophotometric study of influences of the direct ferric perchlorate method for the determination of serum cholesterol. Microchem. J. 20, 428-439 (1975). Wybenga, D. R., Pileggi, V. J., Dirstine, P. H., and DiGeorgio, J., Direct manual determination of serum total cholesterol with a single stable reagent. Clin. Chem. 16, 98CL984 (1970). Zak, B., and Epstein, E., A study of several color reactions for the determination of cholesterol. C/in. Chim. Acta 6, 72-78 (196 I). Zak, B., Epstein, E., and Baginski, E. S., Review and critique ofcholesterol methodol-

ogy. Ann. C/in. Lab. Sci. 2, 101-125 (1972).