The Analytical Approach
S. Ο. Farwell R. A. Kagel Department 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
Weak CalfSyndromeand the Determination of Cortisol: Adapting Literature Methods to Real-Life Problems The work described in this article began as a result of a study at the Uni versity of Idaho, Department of Veter inary 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 weak ness, lameness, diarrhea, dehydration, and secondary infections signal the onset of WCS. Approximately 80% of these calves die, and those calves that survive show considerable growth re ductions (1, 2). A clue to the possible cause of WCS
0003-2700/83/0351-985AS01.50/0 © 1983 American Chemical Society
comes from investigations of Cortisol levels in plasma samples from new born calves. Immediately after birth, calves have extremely high levels of plasma Cortisol, but these initial levels rapidly decrease during the next few days. It is also known that pathologi cal hypersecretion 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 active ly involved in stress-response mecha nisms, it has been postulated that ab normally high levels of Cortisol, sus tained for relatively long periods of time and induced by cold stress, may be the biological cause of WCS. Hence, a cooperative interdepartmen tal 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 on a competitive protein binding (CPB) method (3). However, the CPB meth od proved inadequate for detecting
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Table I.
Percentage Cortisol Recoveries from Plasma Pools Replicate extractions
High pool Medium pool Low pool
68 64 67
75 70 64
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 separation/sample preparation system, quantification/ 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
61 70 69
83 53 74
Mean SD
72 62 62
72 ± 8 64 ± 7 67 ± 5
allowable time, and the costs. In the Cortisol problem, literature reports indicated that typical bovine plasma Cortisol concentrations range from 5-200 ng/mL and that they could vary by approximately ±15% within any 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 ±10%. 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 systematic variations from the control group. The availability 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 published methods reporting low ppb detectability, approximately ±5% precision, and a linear dynamic range of at least 103 appeared to be particularly applicable to our problem. However, a closer look at these methods revealed that several factors that could potentially affect the accuracy, and hence the subsequent validity of the mea-
surement data, 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 cortisol/internal 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 Preparation System
Literature methods (4-7) for CH2CI2 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 time/ 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
Figure 1. Standard curves using equiienin (100 ng/mL) for the internal standard
Figure 2. Standard curves using dexamethasone (100 ng/mL) for the internal standard
(a) Prepared directly in methanol; (b) extracted from aqueous samples before injection
(a) Prepared directly in methanol; (b) extracted from aqueous samples before injection
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and chose an Altex Ultrasphere C-8 (15 cm) column with 57% methanolwater as eluent for the final system. Sample preparation involved the ex traction of 3 mL of plasma with 12 mL of CH2C12, centrifugation, and subse quent washing of the CH 2 C1 2 with 0.1 Ν NaOH. The CH2C12 extract was then evaporated to dryness and redissolved in 300 μ-L of methanol. A lOO^L aliquot (i.e., 1-mL plasma equivalent) of this final solution was used for the HPLC determination. Quantification/Validation
Three basic methods of HPLC quantification are available—external standardization, internal standardiza tion, and standard addition. If the combined variability in sample recov ery due to the sample pretreatment and injection procedure is greater than the acceptable variance for the overall analysis, external standardiza tion can be eliminated. Table I shows the recovery and standard deviation data from replicate Cortisol extrac tions on three different sample pools of plasma obtained with radiolabeled Cortisol spikes. Since typical extrac tion recovery variations are in excess of ±15% over the concentration range of interest, the ±10% precision goal could not be attained with external calibration. Internal standardization is often the choice for trace-level determinations and has the advantage of compensat ing for variations in analyte recoveries and injection volumes. However, there are three major drawbacks to internal standardization: Complete chromato graphic resolution must be obtained for another component in the samples; the need for quantitative determina tion of responses for two components instead of one often results in addi tional measurement variance; and there is 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 be tween an analytical curve obtained by duplicating the procedure used in the literature (a) and the more appropri ate curve (b). Curve (a) was obtained by injecting Cortisol standards pre pared in methanol and adding equilenin internal standard. Curve (b) was obtained by extracting aqueous sam ples 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 introduced if the analyst assumes the cortisohequilenin ratio 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 equile nin or dexamethasone could be used as the internal standard for this bo vine plasma method without introduc ing related bias as long as the calibra tion and internal standards are treat ed 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. Fortu nately, the method of standard addi tion is a solution to this problem of matrix dependent variability and can be combined with internal standard ization to express all responses as peak height ratios. Unfortunately, conven tional standard additions are imprac tical for routine determinations of this type because of the large number of additional samples that must be run. Hence, we made an adequate compro mise that used an 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 sev eral plasma sample pools (i.e., after correcting for native Cortisol). By using dexamethasone and the forego ing sample treatment procedures, the Cortisol extraction efficiency was im proved from 67 ± 15% to an internally compensated recovery of 96 ± 5%. If the sample type of interest can be stored without the analyte concentra tion changing, sample pools can be very useful in the validation of the an alytical measurement procedure. In this work, three plasma sample pools containing low, normal, and high con centrations (i.e., 8, 65, and 120 ng/mL, respectively) of Cortisol were obtained. These sample pools were repeatedly quantified using an internal standard plus standard additions. This proce dure verified that the determined quantity would be accurate if the re sponses measured as Cortisol and dexamethasone were in fact due only to these compounds. Then, the deter mined Cortisol concentration values would be valid once the identity and purity of the Cortisol and internal standard HPLC peaks in the previous ly quantified sample pools had been established. The feasibility of obtaining a confir matory identification and the relative purity of the suspected Cortisol chro matographic peak were initially estab lished with an authentic Cortisol stan dard. A liquid fraction corresponding
Figure 3. Typical separation of a nor mal pool sample (a) Cortisol; (b) dexamethasone. UV detection at 242 nm
to the Cortisol peak was collected, and the Cortisol was extracted with CH 2 C1 2 . The extract was evaporated on the direct insertion probe, and its mass spectrum was identical to that of pure Cortisol. Once a sample prepara tion procedure had been developed and the Cortisol peak had been chromatographically resolved from other obvious component peaks, the same fractionation and MS study were per formed on several real plasma sam ples. Optimization/Control
Replicate analyses of the reference sample pools and the corresponding analyte-spiked pools were used to esti mate the analytical method's linear dynamic range, slope sensitivity and zero intercept, detection limit, preci sion, and accuracy. These figures of merit were monitored as a guide dur ing optimization of the analysis sys tem. Final optimization procedures in cluded minor adjustments to the chro matographic conditions, injection vol ume, and detector wavelength plus the addition of a guard column. For in stance, UV detection at 254 nm was employed during the development pe riod; however, later optimization stud ies showed that detection of the Corti sol peak at 242 nm resulted in a dou bling of the S/N ratio. Figure 3 shows a typical HPLC sep aration of a normal pool sample using our optimized plasma Cortisol method and also illustrates the necessity for a continuous program of control sam ples 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, interférants were found in some bovine plasma samples at two different times during the first months of application with the HPLC Cortisol method. Furthermore, these interférants 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 interférant was a compound extracted from one particular type of lid liner from the sample vials. In another incident, the interférant was traced to a compound present in an inferior reagent grade of CH2CI2 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 interférant coeluted with the internal standard. If undetected, this interference would have caused a
Table II. Typical Comparative Calf Plasma Cortisol Levels (ng/mL ± SD) Sample No.
HPLC
10 51
61.7 ± 0 . 5 86.3 ± 4 . 0 99.1 ± 2 . 9 84.1 ± 2 . 5 77.9 ± 1 . 0
26 25 38
CPB
62.9 105.9 108.4 90.8 90.2
±0.1 ±2.0 ±10.9 ±4.7 ±7.3
negative rather than a positive error in the measured analyte concentrations. Its presence was identified with the third type of control sample, i.e., those with no internal standard. Once detected, an adjustment in the composition of the HPLC mobile phase adequately resolved the interférant peak from the dexamethasone peak. One final, useful control included periodic monitoring of the performances of the HPLC column and associated equipment with a test mixture for which plate heights, selectivities, and detector responses were wellknown. Conclusion
A common approach to checking reliability of a particular determination is to compare the results obtained on the same samples by two or more independent methods. For our project, 40 different calf plasma samples as well as analyte-spiked samples, sample pools, and pure standards were split and analyzed in triplicate by both the HPLC and CPB methods. The means of the triplicate values obtained from these two different methods were compared for each sample with respect to the precision within each set of replicates. The occurrence of a significant difference between the results obtained with the two methods was tested at the 95% confidence level.
As seen in Table II, the HPLC method yielded better precision than the CPB method, specifically ±9% vs. ±17%, and the mean values revealed a significant difference between results obtained on plasma samples via the two methods. Figure 4 illustrates the effect the plasma matrix had on these comparison results. Curve (a) demonstrates 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 1.01 with a correlation coefficient of 1.0. However, as shown in curve (b), the results obtained on the 40 calf plasma samples revealed a systematic difference between corresponding values determined by the two methods. The slope of this regression line is 0.81. Although the precision about this line was not as good as that obtained with pure standards, a highly significant difference was still indicated. Subsequent experiments using analyte-spiked samples and HPLC-purified plasma Cortisol fractions suggest that a positive interference exists in real samples for this CPB method. Similar observations with the CPB method as well as a popular radioimmunoassay for Cortisol have been noted by other workers (4). These interferences are apparently due to nonspecific binding of plasma matrix components to the corticosteroid globulin and, consequently, they do not affect the HPLC results. Over 350 different calf and cow plasma samples have been analyzed with the HPLC method. The assay system is reliable and has remained within analytical control over a period of several months. At this point in the WCS project, an extensive survey of plasma Cortisol levels in cold-stressed and unstressed newborn calves using this HPLC assay system is planned. It is hoped that this successive study will yield information as to the significance of Cortisol in the weak calf syndrome. The HPLC used in this work was purchased on an earlier National Science Foundation Grant (No. SER-7913526) to S. O. Farwell. References
Figure 4. Correlation plots of HPLC vs. CPB Cortisol results on (a) aqueous, matrixfree standard samples and (b) bovine plasma samples 988 A · ANALYTICAL CHEMISTRY, VOL. 55, NO. 9, AUGUST 1983
(1) Card, C. S.; Spencer, G. R.; Stauber, E. H.; Frank, F. W.; Hall, R. F.; Ward, A. United States Animal Health Association Proceedings 1974, 77, 67. (2) Ivanoff, M. S.; Renshaw, H. W. Am. J. Vet. Res. 1975,36,1129. (3) Murphy, B.E.P. J. Clin. Endocrinol. 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. Chromatogr. 1981, 222, 478. (7) Kabra, P. M.; Tasi, L.; Marton, L. J. Clin. Chem. 1979,25, 1293.
