Michael G. Bissell

Development of a urinary free cortisol assay using solid-phase extraction-capillary electrophoresis.

In clinical practice, the measurement of urinary free cortisol (UFC) provides the most sensitive and specific diagnostic information for excess adrenal production of cortisol. The existing methodologies (RIA and HPLC) are time consuming, costly, involve tedious extractions, derivatizations and problems with non-specific interactions with cortisol metabolites in urine. In the present study, we describe the development of an SPE-CE method for the rapid analysis of UFC. UFC was concentrated using SPE C18 cartridges (3M Empore) under a vacuum and eluted with acetonitrile-SDS. The use of 10% acetone to wash cartridges before final elution with acetonitrile-SDS showed significant improvements in the free cortisol recovery. The complete extraction was accomplished in 10-15 min with a recovery of 89-94%. CE analysis was done on a Beckman P/ACE 5010 with detection at 254 nm using a neutral capillary. Detection limits of free cortisol in urine was improved to 10 microg/l with SPE compared to 500 microg/l without SPE. No interferences either from BSA or other urinary cortisol metabolites affected the free cortisol determinations. The results showed the feasibility of a rapid UFC detection with improved sample handling capacity.

Micellar electrokinetic capillary chromatographic separation of steroids in urine by trioctylphosphine oxide and cationic surfactant.

Separation of the six structurally similar and hydrophobic neutral steroids, testosterone, dimethyltestosterone, testosterone propionate, cortisone, hydrocortisone and 17-deoxycorticosterone, was achieved by hydrophobic micellar electrokinetic chromatography. A triphasic separation involving micellar dodecyltrimethylammonium bromide (DTAB), a dynamic bilayer formed due to electrostatic interaction between the silica surface and DTAB, and aqueous phase is proposed to account for the observed separation of the steroids. The running buffer consisted of 0.05 M DTAB and 0.0052 M trioctylphosphine oxide in 0.01 M of phosphate buffer pH 7.4. A detection limit of 500 ng/ml was achieved for each steroid and the application of the method to urine samples is described.

Anionic-zwitterionic mixed micelles in the micellar electrokinetic separation of clinically relevant steroids on a fused-silica capillary

Abstract The structural similarities of different clinically relevant steroids make their routine determination in biological samples an analytical challenge. Determination of steroidal hormones in urine and serum is of great interest in clinical endocrinology. A large number of disorders is associated with hypo- or hypersecretion of one or more steroids, for example, Addisons disease, Cushings syndrome, hirsutism and virilism, adrenal adenomas, hypertension, congenital adrenal hyperplasia etc. A SDS/SB3-12 mixed micellar system was used to achieve baseline separation of eight steroids in less than 10 min. Five different zwitterionic surfactants were evaluated at different concentrations, pH and temperature conditions. The SB3-12/SDS mixed micellar system gave highest resolution and precision. No significant capillary wall interaction or coating effects were observed with the zwitterionic surfactants as is evident from the reproducible migration times (relative standard deviation ≤2%). A detection limit of 1–5 μm/ml was obtained for pure standards.

Laser induced resonance energy transfer - A novel approach towards achieving high sensitivity in capillary electrophoresis I. Clinical diagnostic application

Most of the procedures currently performed by capillary zone electrophoresis (CZE) with laser induced fluorescence detection requires prior derivatization. This increases cost, the turn-around-time and chances of extraneous contaminations. CZE with laser induced resonance energy transfer is demonstrated as a viable alternative for detecting non-fluorescent compounds with no prior derivatization. The feasibility of this approach is demonstrated by separating and directly detecting salicylic acid (2,4-dihydroxybenzoic acid), gentisic acid (o-methoxybenzoic acid), salicyluric acid (o-hydroxyhippuric acid) and 4-aminosalicylic acid in urine. The detection of salicylate in serum is also shown. The method is highly sensitive with detection limits in the 1.10(-7) M range. Importantly it requires no prior preconcentration or sample preparation and can be used with complex sample matrices such as serum and urine.

Laser induced resonance energy transfer — a novel approach towards achieving high sensitivity in capillary electrophoresis Part B: Applications for environmental testing☆

Abstract Capillary electrophoresis with laser induced resonance energy transfer detection is used for direct determination of ethylenediamine tetraacetic acid (EDTA), 1,2-diaminopropane-N,N,N′,N′-tetraacetic acid (DPTA) and (±)- trans -1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid (DCTA) in aqueous samples. The method involves CZE separation of salicylate-Tb-EDTA, salicylate-Tb-DPTA and salicylate-Tb-DCTA ternary complexes at alkaline pH. The highly efficient transfer of energy from salicylate to terbium in these complexes facilitated a sensitive detection. Detection limits in the 1·10 −7 M range are easily achieved. A 325 nm HeCd laser is used for excitation and terbium luminescence is monitored at 547 nm. The potential application of this method in analyzing these acids in a complex radioactive waste is discussed.

Evaluation of Immuno-1 toxoplasma IgG assay in the prenatal screening of toxoplasmosis

We evaluated the Immuno-1 (Bayer Diagnostics, Tarrytown, New York, USA) and IMx (Abbott Laboratories, Chicago, Illinois, USA) toxoplasma IgG assays in 298 (223 fresh in-house prenatal + 75 supplied by Bayer) specimens over 15 days. Discordant results were resolved by indirect fluorescence assay (Gull Laboratories, Salt Lake City, Utah, USA). The performance of Immuno-1 assay was found to be comparable to the IMx assay. Immuno-1, being a random access analyzer with minimum hands-on time requirements may have an advantage in the overall laboratory efficiency.