S. 0. Farwell R. A. Kagel Depafiment of Chemistry
University of Idaho MOSCOW, Idaho 83843
Edited by Jeanette G. Grasselli
S. K. Gutenberger D. P. Olson Department of Veterinary Medicine University of Idaho Moscow, Idaho 83843
wakcdfsyndromeandthe DeterminationofCortisol: Adapting LiteratureMethodsto Real-Life Problems The work described in this article began as a result of a study a t the Uuiversity of Idaho, Department of Veterinary Sciences, on the cause of weak calf syndrome (WCS). In western states such as Idaho where spring calving is practiced, the cold and wet weather causes an abnormally large number of cows to give birth to calves afflicted with WCS. The afflicted calves typically show few clinical symptoms until they are three to seven days old, at which time weakness, lameness, diarrhea, dehydration, and secondary infections signal the onset of WCS. Approximately 80% of these calves die, and those calves that survive show ronsiderable growth reductions ( 1 . 2 ) . A clue to the possible cause of WCS
0003-2700/83/0351-985A501.50/0 C 1983 American Chemical Society
, comes from investigations of cortis,. levels in plasma samples from newborn calves. Immediatelv after birth. calves have extremely high levels of plasma cortisol, but these initial levels rapidly decrease during the next few days. It isalso known that pathological hyperserretion of cortisol can in-
duce symptoms quite similar to those of WCS, e.g., inhibition of skeletal maturation, collagen production, and wound healing. Since cortisol is actively involved in strestresponse mechanisms, it has been postulated that abnormally high levels of cortisol, sustained for relatively long periods of time and induced by cold stress, may be the biological cause of WCS. Hence, a cooperative interdepartmental research project was initiated to measure plasma cortisol levels in coldstressed and unstressed newborn calves. First attempts to monitor these plasma cortisol levels were based un a competitive protein binding (CPBJ method ( 3 ) .However. the CPB methcd proved inadequate for detecting
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small cortisol level differences because of poor overall precision and poor cortisol specificity. A literature review subsequently suggested high-performance liquid chromatography (HPLC) as an alternative method for determining plasma cortisol (4-7). Unfortunately, HPLC-based determinations on real-world samples are often subject to “ruggedness” problems. Nonselective detectors and variations in columns, sample matrices, potential contaminants, equipment performance, and analyst expertise contribute to this irreproducibility in HPLC literature methods. To obtain reliable results, these various experimental parameters must be either duplicated, proved unimportant, or controlled. The literature-to-laboratory process can be divided into four basic steps: performance definitions, development of separationlsample preparation system, quantification1 validation, and optimization and control.
Performance Definitions At the start of a project an estimate of the minimum required performance can aid in selecting the proper analytical method. This selection should be based on considerations such as the required detection range, the required accuracy and precision, the equipment available, the number of samples, the
allowable time, and the casts. In the cortisol problem, literature reports indicated that typical bovine plasma cortisol concentrations range from &ZOO ng/mL and that they could vary by approximately i15% within a n y one control group. Based on this information, we adopted a preliminary performance precision goal that allowed two plasma samples to be statistically resolved if their respective cortisol concentrations differed by more than
*lo%. This first approximation of the minimal precision requirement was chosen to allow likely variations within a group to be just detectable while maximizing the method‘s ability to resolve svstematic variations from the controi group. The availahilitv of a Varian Model 5000 HPLC and our experience in HPLC procedures were also important factors in the method selection. A search of the HPLC literature revealed several methods for plasma or serum cortisol. Four of these puhlished methods reporting low ppb detectability, approximately *6% precision, and a linear dynamic range of a t least 103 appeared to be particularly applicable to our problem. However, a closer look a t these methods revealed that several factors that could potentially affect the accuracy, and hence the subsequent validity of the mea-
surement datq, had been neglected. For example, the identification of cortisol in the plasma or serum samples was apparently based solely on equivalent chromatographic retention times on one column. Two of the literature methods described the use of an internal standard, but neither mentioned the possible effects sample extraction might have on the cortisolfinternal standard ratio. One report described the preparation of a standard analytical curve by the addition of known amounts of cortisol along with an internal standard to aliquots of a serum pool. However, no mention was made of the native cortisol that may already have been in the serum before the additions. These observations suggested that the previous literature methods could not be immediately applied to our cortisol-WCS problem. Nevertheless, the literature methods did supply an adequate sample preparation method as well as a starting point for the proper HPLC conditions.
Development of Separation/ Sample PreparationSystem Literature methods (4-7) for CH2C12 extraction of plasma samples followed by reversed-phase separation and UV detection adequately resolved the analyte components from the bovine plasma sample matrices. The specific volumes of plasma sample, extraction solvent, wash solvent, sample injection, and the evaporation time1 temperature were adjusted along with the particular HPLC conditions to meet the requirements of the bovine plasma matrix and concomitant analyte levels. We examined several columns and mobile phase compositions
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Figure 1. Standard curves using equlienln (100 ng/mL) fcf the internal standard (a) Prepareddirectly in meIhanol: lbl extracted ham aqwws samples belore injection
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Figure 2. Standard curves using dexamethasone (100 ng/mL) for the internal standard (a) Prepareddirectly in methanol: (b)extracted from aqueous samples betwe injection
and rhose an Altex Ultrasphere C - 8 (15 cm) column with S7°0 methanolwater as eluent lor the final system. Sample preparation involved the extraction of 3 mL of plasma with 12 mL of CH&, centrifugation, and subsequent washing of the CH2C11with 0.1 N NaOH. The CH2Ch extract was then evaporated to dryness and redissolved in 300 PL of methanol. A 100-~Laliquot(Le., I.mi. plasma equivalent) ol this final solution was used fur the HP1.C determination.
Quantification/Valldatlon Three basir methods of HPLC quantification are available-external standardization, internal standardizatiun, and standard addition. If the comhined variability in sample recuvery due to the sample pretreatment and injection procedure is greater than the acceptable variance for the overall analyrk. external standardization can be eliminated. Table I shows the rerovery and standard deviation data from replicate cortisol extractions on three different sample pools of plasma obtained with radiolabeled cortisol spikes. Since typical extrac. tion recovery variations are in excess of i15% over the roncentration range of interest, the iIWo precisiongoal could not be attained with external calibration. Internal standardization is often the choice for trace-level determinations and has the advantage of compensating for variations in analyte recoveries and injection volumes. However, there are three major drawbacks to internal standardization: Complete chromatographic resolution must be obtained for another component in the samples; the need for quantitative determination of responses for two components instead of one often results in additional measurement variance; and there in a potential for misuse due to improper validation of various factors that may be assumed true by oversight or inexperience. Figure 1 illustrates this third drawback. The left portion of Figure 1 shows the difference hetween an analytical curve obtained by duplicating the procedure used in the literature (a) and the more appropriate curve (h). Curve tal was obtained by injecting cortisol standards prepared in methanol and adding eyuilenin internal standard. Curve (b) was obtained by extracting aqueous samples of standard cortisol plus equilenin in a manner identical to the actual sample preparation procedure. These data demonstrate that equilenin is preferentially extracted from aqueous samples. For typical samples an error of approximately -56% (at a peak height ratio of one, is intruduced if the analyst assumes the cortiso1:equilenin ratiii to be constant!
Figure 2 shows the HPLC results when dexamethasone rather than equilenin was used as the internal standard. Dexamethasone clearly mimics cortisol through the sample preparation procedure better than equilenin. Nevertheless, either equilenin or dexamethasone could be used as the internal standard for this bovine plasma method without introducing related bias as long as the calibration and internal standards are treated exactly like the samples. It is also possible that the two curves labeled (b) in Figures 1 and 2 would be different if the samples had been extracted from bovine plasma rather than water. Such extraction data are difficult to obtain due to the lack of a cortisol-free plasma. Fortunately, the method of standard addition is a solution to this problem of matrix dependent variability and can be combined with internal standardization to express all responses as peak height ratios. Unfortunately, conventional standard additions are impractical for routine determinations of this type because of the large number of additional samples that must be run. Hence, we made an adequate compromise that used a n internal standard method, but only after the analytical calibration curve was shown to have the same slope as the curve obtained by making standard additions to several plasma sample pools (i.e., after correcting for native cortisol). By using dexamethasone and the foregoing sample treatment procedures, the cortisol extraction efficiency was improved from 67 15% to an internally compensated recovery of 96 i 5%. If the sample type of interest can be stored without the analyte concentration changing, sample pools can be very useful in the validation of the analytical measurement procedure. In this work, three plasma sample pools containing low, normal, and high concentrations &e., 8,65, and 120 ng/mL, respectively) of cortisol were obtained. These sample pools were repeatedly quantified using a n internal standard plus standard additions. This procedure verified that the determined quantity would be accurate if the responses measured as cortisol and dexamethasone were in fact due only to these compounds. Then, the determined cortisol concentration values would be valid once the identity and purity of the cortisol and internal standard HPLC peaks in the previouslv auantified samde . .DOOIS had heen established. The feasibilitv of obtainine a confirmatory identification and the relative purity of the suspected cortisol chromatographic peak were initially established with an authentic cortisol standard. A liquid fraction corresponding
Figure 3. Typical separation of a normal pool sample la) Ccftisol: (b) dexamethasone.UV detectionat 242 nm
to the cortisol peak was collected, and the cortisol was extracted with CHClz. The extract was evaporated on the direct insertion probe, and its mass spectrum was identical to that of pure cortisol. Once a sample preparation procedure had been developed and the cortisol peak had been chromatographically resolved from other obvious component peaks, the same fractionation and MS study were performed on several real plasma samples. OplimizalionlControl Replicate analyses of the reference sample pools and the corresponding analyte-spiked pools were used to estimate the analytical method’s linear dynamic range, slope sensitivity and zero intercept, detection limit, precision, and accuracy. These figures of merit were monitored as a guide during optimization of the analysis system. Final optimization procedures included minor adjustments to the chromatographic conditions, injection volume, and detector wavelength plus the addition of a guard column. For instance, UV detection at 2.54 nm was employed during the development period; however, later optimization studies showed that detection of the cortisol peak at 242 nm resulted in a doubling of the S/N ratio. Figure 3 shows a typical HPLC separation of a normal pool sample using our optimized plasma cortisol method and also illustrates the necessity for a continuous program of control samples after initial validation of the
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method. In this chromatogram, cortisol and dexamethasone are quite well resolved from the other compounds in the plasma matrix and from each other. However, compound selectivity available from HPLC with fixedwavelength UV detection is often inadequate for trace determinations on real samples, where the probability for interferences can be quite high (6).In fact, interferants were found in some bovine plasma samples a t two different times during the first months of application with the HPLC cortisol method. Furthermore, these interferants had identical retention times to cortisol. A combination of control samples signaled the presence of the interferences and allowed us to identify them. The control samples used in this work comprised about 15% of the total samples analyzed and included the low, normal, and high reference sample pools; distilled/deionized water (i.e., the reagent blank); and cortisol standards and real sample extracts where both were prepared without the addition of dexamethasone. The interference problems were initially indicated by high, incorrect values on the sample pools. The source of the problem was identified when the reagent blanks showed a significant level of “cortisol.” Further investigations showed that in one case, the interferant was a compound extracted from one particular type of lid liner from the sample vials. In another incident, the interferant was traced to a compound present in an inferior reagent grade of CH2C12 extraction solvent. Note that neither internal standards nor standard additions would have caught this type of interference problem. In a few bovine plasma samples an additional interferant coeluted with the internal standard. If undetected, this interference would have caused a I
As seen in Table 11, the HPLC method yielded better precision than the CPB method, specifically f9% vs. i17%, and the mean values revealed a significant difference between results ohtained on plasma samples via the two methods. Figure 4 illustrates the effect I the plasma matrix had on these com) parison results. Curve (a) demon.9 strates that an excellent correlation ? exists between plasma matrix free cortisol standards throughout the concentration range of interest. The slope of the regression line for these data is negative rather than a positive error in 1.01with a correlation coefficient of the measured analyte concentrations. 1.0. However, as shown in curve (b), Its presence was identified with the the results obtained on the 40 calf third type of control sample, Le., those plasma samples revealed a systematic with no internal standard. Once dedifference between corresponding values determined by the two methods. tected, an adjustment in the composition of the HPLC mobile phase adeThe slope of this regression line is quately resolved the interferant peak 0.81. Although the precision about from the dexamethasone peak. this line was not as good as that ohOne final, useful control included tained with pure standards, a highly significant difference was still indicatperiodic monitoring of the perfored. Subsequent experiments using anmances of the HPLC column and asalyte-spiked samples and HPLC-purisociated equipment with a test mixture for which plate heights, selectivified plasma cortisol fractions suggest that a positive interference exists in ties, and detector responses were wellreal samples for this CPB method. known. Similar observations with the CPB Concluslon method as well as a popular radioimA common approach to checking re- munoassay for cortisol have been noted by other workers ( 4 ) .These inliability of a particular determination terferences are apparently due to nonis to compare the results obtained On specific binding of plasma matrix the same samples by two or more incomponents to the corticosteroid globdependent methods. For our project, ulin and, consequently, they do not af40 different calf plasma samples as fect the HPLC results. well as analyte-spiked samples, samOver 350 different calf and cow ple pools, and pure standards were plasma samples have been analyzed split and analyzed in triplicate by with the HPLC method. The assay both the HPLC and CPB methods. system is reliable and has remained The means of the triplicate values ohwithin analytical control over a period tained from these two different methof several months. At this point in the ods were compared for each sample WCS project, an extensive survey of with respect to the precision within plasma cortisol levels in cold-stressed each set of replicates. The occurrence and unstressed newborn calves using of a significant difference between the this HPLC assay system is planned. It results obtained with the two methods is hoped that this successive study will w a tested ~ at the 95% confidence level. vield information as to the signifi1 cance of cortisol in the weak calf syndrome. The HPLC used in this work was purchased on an earlier Natianhl Science Foundation Grant (No. SER-7913526)toS. 0. Farwell.
References (1) Card, C. S.;Spencer, G. R.; Stauber,
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Flgure 4. Correlation plots 01 HPLC vs. CPB conisol results on (a)aqueous, matrixfree standard samples and (b) bovine plasma samples @88A
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E. H.; Frank, F. W.; Hall, R. F.; Ward, A. United Stoles Animal Health Assoeiation Proceedings 1974,77,67. (2) Ivanoff,M. S.; Renshaw, H. W.Am. J. Vel. Res. 1975,36,1129. (3) Murphy, B.E.P. J. Clin. Endacrinol. 1967,27,973. (4) Reardon, G. E.; Caldarella, A. M.; Canalis, E. Clin. Chem. 1979,25, 122. ( 5 ) Canalis, E.; Caldarella, A. M.; Reardon, G. E.Clin. Chem. 1979,25.1700. (6) Schoneshofer,M.; Skobolo, R.; Duke, H. J. J. Chmmatogr. 1981,222,478. (I) Kabra, P.M.;Tasi, L.; Marton, L. J. Clin. Chem. 1979,2S,1293.
