Liquid chromatographic method development for steroids determination (corticoids and anabolics)

A LC method for corticosteroids (CC) determination in poultry feed using a Chromolith column and UV detection has been developed and validated. The method development involved the optimization of different hydro-organic mobile phases using methanol or ACN as organic modifiers, flow rate, and temperature. The optimum separation was achieved at 40 degrees C using ACN/water (21:79 v/v) as mobile phase and 3 mL/min flow rate, allowing the separation to baseline of four out of seven CC in about 10 min. Prior to LC, a sample preparation procedure previously assayed for anabolics was used. It includes a leaching process, saponification of the esters from fatty acids, and SPE. Method validation was carried out according to the EU criteria established for quantitative screening methods. The extraction efficiencies, decision limits (CCalpha), and detection capabilities (CCbeta) for these compounds were in the ranges of 86-92%, 27-36 microg/kg, and 33-43 microg/kg, respectively. The repeatability and the within-laboratory reproducibility at 1, 1.5, and 2 CCbeta concentration levels were smaller than 9.0, 5.0, and 4.2% and 9.4, 6.4, and 4.9%, respectively. The CV values of the robustness test were less than 3.8% and the accuracy was in the range of 98-103%. The proposed method was applied to other feed with satisfactory results.

Section: Methods Developmentmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 97%

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Method development validation for corticoids in animal feed samples by liquid chromatography using a monolithic column

Muñiz‐Valencia

Gonzalo-Lumbreras

Santos-Montes

et al. 2007

“…The dry extracts were derivatized and the chromatograms obtained indicate the presence of AAS and interferences mainly due to fatty acids from seeds contained in piglet feed samples such as myristic, rinoleic, oleic, lauric, palmitic, palmitoleic, stearic, decanoic, phthalic, nonanoic, capric, araquidic, and linoleic. In order to eliminate these interferences a clean-up procedure using silica cartridges (Si) was tested [9]. These assays indicate that ACN extract results in cleaner chromatograms.…”

Section: Extraction Of Aas From Feed Samples and Clean Upmentioning

confidence: 99%

“…Boldenone has also been determined in feed samples for veal calves by a leaching process using acetate buffer/MeOH, SPE using C18 and NH 2 cartridges and LC fractionation of the extracts on a C18 column before LC-MS-MS analysis [6]. A LC-UV method for anabolics and corticoids in piglet feed samples including a leaching process using diethyl ether (DEE) and SPE using silica cartridges has also been reported [9].…”

Section: Introductionmentioning

confidence: 98%

GC‐MS method development and validation for anabolic steroids in feed samples

Muñiz‐Valencia

Ceballos‐Magaña

Gonzalo-Lumbreras

et al. 2008

A GC-MS method for the determination of AAS used as growth promoting agents using SIM in piglet feed samples has been developed and validated, using testosterone as internal standard. The formation of volatile steroid derivatives was carried out by derivatization with N-methyl-N-(trimethylsilyl)trifluoroacetamide. The optimum separation was achieved using a Zebron ZB-5 column under a gradient temperature elution, allowing the separation of steroids in 18 min. The required sample treatment process was discussed. A leaching using ACN, saponification using a binary NaOH/MgCl2 solution, and LLE using ethyl acetate were finally selected. Method validation has been carried out according to the Commission Decision 2002/657/EC criteria established for quantitative confirmatory methods. The extraction efficiencies, CCalpha and CCbeta for these compounds were in the ranges 78-98%, 10-21 and 18-35 mug/kg, respectively. The repeatability and the within-laboratory reproducibility at 1, 1.5, and 2 CCbeta concentration levels were smaller than 8.2, 7.5, and 5.8% and 12.2, 9.5, and 7.5%, respectively. Accuracy was in the 99-103% range. The robustness was evaluated using the Youden robustness test. The proposed method was applied to the analysis of steroids spiked in different kinds of animal feed samples with satisfactory results.

Quantitative screening for steroids in animal feeding water using reversed phase LC with gradient elution

Muñiz‐Valencia¹,

Gonzalo-Lumbreras²,

Santos-Montes³

et al. 2008

Several isocratic separations for the determination of 20 steroids (STER) in animal feeding water samples (AFWS) from drinking-trough by LC using a mobile phase ACN/H(2)O (35:65 v/v) and different RP columns (Hypersil C18, Gemini C18 (GM), Purospher Star C18, Synergi Max C12, and Synergi Fusion) and UV detection were obtained. The elution order was the same: a first group of corticoids (CC) was early eluted, a second group of CC and anabolics (AAS) exhibited intermediate retention, and a third group constituted by AAS was strongly retained. To improve the separation performances of the isocratic separations an ACN gradient elution optimization was carried out for each column. The most satisfactory results were obtained using a Purospher column which allowed the separation of 19 STER in an analysis time close to 26 min. After sample preparation using SPE, method validation was performed in an AFWS spiked with STER according to the EC decision criteria established for quantitative screening methods. For this purpose calibration graphs, extraction efficiencies, decision limits, detection capabilities, precision (repeatability and within-laboratory reproducibility), accuracy, selectivity, and robustness were evaluated. The proposed method was applied to other AFWS with satisfactory results.