Clinical Cbemistry
show around 8-10-fold higher sensitivities for both the intact and des-3 1,32-proinsulin assays compared with thosepossible in methods utilizing the aminocellulose-bound antibody, despite improvements in sensitivity in the latter methods. The assays can measure fasting concentrations of these molecules in nondiabetic subjects as well as in clinical conditions where their concentrations are higher [6]. In the intact proinsulin assay the cross-reactivity with des-64,65-promnsulin was slightly higher than published for the cellulose IRMA (78% vs 66%, respectively). On the other hand, in the des-31,32-proinsulin assay the cross-reactivity with intact proinsulin was lower (59% vs 84%, respectively), whereas that with des-64,65 was similar(54% and 60%, respectively). This methodology is very easy to set up and allows the processing of large numbers of samples. It also lends itself to automation. The detection system can be modified by replacing the radiolabel with an enzyme label. This may be particularly relevant for the des-64,65-proinsulin assay, for which improvements in sensitivity are necessary. The comparison of results obtained by the microplate IRMA with those by the cellulose IRMA and HPLC methods, and in fastingas well as stimulated samples, shows good correlation. Some of the discrepancies may be attributable to differing lengths of storage of the samples: The microplate IRMA was carried out a year after the cellulose IRMA and HPLC assays. The effects of prolonged freezing on microplate IRMA results are not known, but do not appear to have adversely affected the correlation. In conclusion, this microplate IRMA for proinsulin and des-3 l,32-proinsulin is simple and sensitive and appears to be applicable for use with large numbers of samples. References 1. Temple RC, Clark PMS, Nagi DK, Schneider AE, Yudkin JS, Hales CN. Radioimmunoassay may overestimate insulin in non-insulin-dependent diabetics. Clin Endocrinol 1990;32:689-93. 2. Dhahir F, Cook DB, Self CH. An amplified enzyme-linked immunoassay for human proinsulin. Clin Chem 1992;38:227-32. Lundkjerns L. R#{248}der ME, Dinesen B, Hartling 5G. J#{248}rgensen PN, Binder C. Highly sensitive enzyme immunoassay of proinsulin immunoreactivity with use of two monoclonal antibodies. Clin Chem 1993;10:2146-50. 4. Sobey WI, Beer SF, Carrington CA, Clark PMS, Frank BH, Gray IP, et al. Sensitive and specific two-site immunoradiometric assays for human insulin, proinsulin, 65-66 split, and 32-33 split proinsulin. Biochem J 1989; 260:535-41. 5. Ostrega D, Polonski K, Nagi DK, Yudkin J, Cox U, Clark PMS, Hales CN.
3.
Measurement of proinsulin and intermediates: validation of immunoassay methods by high-performance liquid chromatography. Diabetes 1995;44: 437-40. 6. Mohamed-Ali V. Gould MM, Goubet S. Yudkin JS. The relationship between insulin, intact proinsulin and des 31,32 proinsulin and other cardiovascular risk factors in non-diabetics [Abstract]. Diabet Med 1993;10(Suppl 3):P54.
Improved HPLC Method for Simultaneous Analysis of Cortisol, I 1-Deoxycortisol, Prednisolone, Methylpredmsolone, and Dexamethasone in Serum and Urine, BrettC. McWhinney, * Gregory Ward, and Peter E. Hickman (Dept. of Chem. Pathol., Princess Alexandra Hosp., Woolloongabba, QLD, Australia 4102; *author for correspondence: fax 61 7 32407070, e-mail
[email protected])
Assay of steroid
hormones is a major part of any general endocrinology laboratory. Routine analysis of serum and urine cortisol is used in the differential diagnosis of Cushing syn-
42, No. 6, 1996
979
drome. The assay of other steroids such as 11 -deoxycortisoluseful in the 11 (3-hydroxylasedeficiency form of congenital adrenal hyperplasia [1]and after giving metyrapone or ketoconazole, which blocks cortisol synthesis [2]-prednisolone and methylprednisolone (for elucidating pharmacokinetics in organtransplant patients and people with asthma), and dexamethasone (which is metabolized in Liddle’s test) is also of importance. These steroids can all be measured separately by RIA but not without significant potential for cross-reactivity [3]. HPLC methods have been described for these steroids, but no technique has yet been described for the analysis of all these steroids in one run. We have developed an HPLC procedure with fludrocortisone asan internalstandard that both simultaneously measures these steroids at clinically useful concentrations and eliminates the problem of cross-reactivity. We purchased cortisol, prednisolone, methylprednisolone, 11-deoxycortisol, dexamethasone, and fludrocortisone from Sigma Chemicals (St. Louis, MO). Tetrahydrofuran from Merck (Sydney, Australia) was of HPLC grade, and water was processed by a water purification system (Nanopure Barnstead, Sydney, Australia). All other chemicals were of analytical reagent grade. Stock calibration solutions (500 molil) of cortisol and fludrocortisone were prepared in methanol. The working internal standard solution, prepared by diluting the stock fludrocortisone solution 200-fold with mobile phase, was stored at 4 #{176}C. Urine cortisol calibrators were prepared from a urine sample in which the concentration of endogenous cortisol had been determined by the use of mobile phase-based calibrators. The working solutions of the urine-based calibrators were prepared by adding the appropriate volume of the stock solution to this urineto give concentrations of 500 and 1500 nmoVL; aliquots (2 mL) were then stored frozen at -20 #{176}C until analyzed. Calibrators for the other steroids were prepared in similar manner. For HPLC we used a Waters (Milford,MA) Model 712 WISP autosampler, a Model 510 solvent-delivery system, and a Model 481 UV/VIS detector. The system was controlled by Millennium 2010 Chromatography Manager (Waters). The analytical column was 250 X 4.6 mm (i.d.) Spherex C18 5-gm particles Phenomenex (Torrance, CA). The mobile phase was methanol, tetrahydrofuran, and water (3:25:72 by vol), which we filtered and degassed by passing the mixture through a 0.45-sm pore-size Millipore (Bedford, MA) filter before use. The system was run isocratically at a flow rate of 1.0 mL/min and the absorbance peaks were detectedat 254 nm. Total analysis time for allsteroids was 32 mm. To analyze serum samples, we added 500 j.tL of 0.2 moltL acetate buffer (pH 3.85) to 1 mL of serum before extraction; urinerequired no pretreatment. Fludrocortisone (2.5 p.moVL in mobile phase) was added (400 L) to all samples before extraction. If sample volume was limited (e.g., pediatric), we used a smaller sample volume with a corresponding reduction in the amount of internal standard added. For example, reducing sample volume to 0.5 mL increases the detection threshold to
