Steroids separation methods

Much effort has been made to detect steroids in biological fluids. Even simple TLC methods have been used for qualitative analysis [38], One method that been used for quantification involves an immunoassay, but several problems exist with that method, most notably cross-reactions and interference with other substances [39], On the other hand, a number of chromatographic methods have been developed to overcome these problems. The majority of analytical methods involved GC, which has good detection limits, but requires previous derivatization [40] of the steroids to accomplish volatilization. Many methods have also been reported using HPLC with UV detection or LC-MS [40, 41], Previously used stationary phases for LC was e.g., Sephadex LH-20, Celite and Lipidex, but they could not be operated with high pressure [42], These columns were therefore slow to run and the separation of steroids was very time-consuming [43], Nowadays applications mainly use HPLC as a separation method with both normal-phase and re-versed-phase chromatography. 

Touchstone (1986) provided extensive information on the chromatographic analyses of the aforementioned steroids and outlined methods for their extraction and sample preparation. He also provided extensive tabular data on numerous steroids separated by GC, HPLC, and TLC. 

In comparison with the literature on other methods, e.g., HPLC or in other fields of pharmaceutical analysis, there are few reports on the application of TLC to steroid separation. The practical examples which follow serve to demonstrate the newest developments in the field of steroid analysis. 

Nicotinic acid (vitamin B3), 1048 Nondestructive detection methods for carbohydrates, 499-501 Nonpolar extraction, 13 Nonpolar steroids, separation of, 974 Normal chamber RPC (N-RPC), 325-326 Nuclear magnetic resonance (NMR), 4,33 Nucleic acids and their derivatives, 921-969 detection general methods, 931-935 reliability, 934 sensitivity, 934... 

Table 7. Methods for Chromatographic Separation of Vitamin D and Related Steroid ...

The release of steroids such as progesterone from films of PCL and its copolymers with lactic acid has been shown to be rapid (Fig. 10) and to exhibit the expected (time)l/2 kinetics when corrected for the contribution of an aqueous boundary layer (68). The kinetics were consistent with phase separation of the steroid in the polymer and a Fickian diffusion process. The release rates, reflecting the permeability coefficient, depended on the method of film preparation and were greater with compression molded films than solution cast films. In vivo release rates from films implanted in rabbits was very rapid, being essentially identical to the rate of excretion of a bolus injection of progesterone, i. e., the rate of excretion rather than the rate of release from the polymer was rate determining. 

Electron impact ionization, also known as particle beam ionization, has been applied to the online determination of steroids such as hydrocortisone, cortisone, prednisolone and prednisone. Polymer additives such as NC-4, Irga-nox 1076,1-octadecanol and Naugard -XL were identified and quantitated online by electron impact and, separately, by atmospheric pressure chemical ionization methods.78... 

Toasaksiri, S., Massart, D. L., and Vander Heyden, Y. (2000). Study of method validation criteria in a capillary electrophoresis method for the separation of non-steroidal anti-inflammatory drugs. Anal. Chim. Acta 416, 29-42. 

At higher temperatures, zirconium dioxide and titanium dioxide supports gave much greater stability along with polymer-based supports [100,101] based on polystyrene-divinyl benzene (PS-DVB) such as PLRP-S noted in Table 9.5. PS-DVB supports have been reported to give a serious column bleed at 250°C [66]. Polybutadiene (PBD) modified zirconia columns have been used at temperatures up to 300°C and carbon-coated zirconia has been used at temperatures up to 370°C [66]. Applications have included the separation of steroids [73] and herbicides [102].The specific order of column bleed varied depending on the detection method as shown in Table 9.5. 

The format of these Reports has remained relatively constant from year to year to facilitate the location of subject matter. However, this year two changes have occurred. Firstly, as an experiment, the information on biosynthesis is contained within the chapters dealing with the individual group of terpenoids rather than as a separate chapter. Secondly, the steroid section has been recast to minimize overlap. A report of the application of physical methods to steroids forms one chapter whilst steroid reactions and partial syntheses are reported in the second. Total synthesis will be reviewed biennially. 

Further methods of isolation were later developed [20-23]. The isolation was mostly effected by the use of Girard s reagent T to separate the keto steroids from the non-keto compounds, and each fraction was then separated by adsorption or partition chromatography. 

Despite the distinct advantages of pneumatic nebulizers, ultrasonic nebulizers may alternatively be used, in some instances, with success. In a recent application, a variation of ultrasonic nebulizer called spray nozzle-rotating disk FTIR interface was successfully applied to confirm the presence of methyltestosterone, testosterone, fluoxymesterone, epitestosterone, and estradiol and testosterone cyp-ionate in urine, after solid-phase extraction and reversed-phase LC separation (151). Using a commercial infrared microscopy spectrometer, usable spectra from 5 ng steroid deposits could be readily obtained. To achieve success with this interface, phosphate buffers in the mobile phase were not used because these nonvolatile salts accumulate on the collection disk and their spectra tend to swamp out small mass deposits. Another limitation of the method was that only nonvolatile analytes could be analyzed because volatile compounds simply evaporated off the collection-disk surface prior to scanning. 

Supercritical-fluid chromatography has been applied by Ramsey et al. (213) for the determination of trimethoprim, along with three steroid hormones, in swine kidney. Separation was performed on a Spherisorb 5 amino-bonded column, using carbon dioxide with methanol modifier as the mobile phase. Detection at levels greater than 10 ppm was accomplished by tandem mass spectrometry using thermospray interface. However, this method lacks the sensitivity required to detect the low ppb levels likely to occur in milk and tissues. 

---