Derivatization and fragmentation pattern analysis of natural and synthetic steroids, as their trimethylsilyl (oxime) ether derivatives by gas chromatography mass spectrometry: analysis of dissolved steroids in wastewater samples.

This paper reports the extension of our multiresidue analysis (MA) procedure with 18 natural and synthetic steroids; permitting the identification and quantification, in total of 81 pollutants from one solution, by a single injection, as their trimethylsilyl (TMS)-oxime ether/ester derivatives, by gas chromatography-mass spectrometry (GC-MS), within 31 min. As a novelty to the field, basic researches, such as fragmentation pattern analysis and derivatization optimization studies were performed for androsterone, transdehydroandrosterone, transandrosterone, mestranol, dihydrotestosterone, ethinylestradiol, testosterone, norethisterone, estriol, 4-androstene-3,17-dione, gestodene, levonorgestrel, etonogestrel, coprostanol, progesterone, cholesterol, medroxy-progesterone-acetate, stigmasterol and ß-sitosterol. Results confirmed that (i) the TMS oxime-ether derivatives of the keto steroids provide from 1.40 times (gestodene) up to 4.25 times (norethisterone) higher responses compared to their TMS-ether ones, and (ii) the distribution of syn/anti oximes is characteristic to the ketosteroid species examined. Based on our optimized mass fragmentation, solid phase extraction (SPE) and derivatization studies separations have been performed in the total ion current (TIC) mode, identification and quantification of compounds have been carried out on the basis of their selective fragment ions. Responses, obtained with derivatized standards proved to be linear (hydroxysteroids), or have been calculated from calibration curves (ketosteroids) in the range of 1.88-750ng/L levels. Limit of quantitation (LOQ) values varied between 1.88ng/L and 37.5ng/L concentrations. The most important practical messages of this work are the high androsterone (0.744-4.28µg/L), transandrosterone (0.138-4.00µg/L), coprostanol (2.11-302µg/L), cholesterol (0.308-41µg/L), stigmasterol (1.21-8.40µg/L) and ß-sitosterol (1.12-11.0µg/L) contents of influent wastewaters. ß-Estradiol (100ng/L) and estriol (54ng/L) were found in one influent sample, only. Reproducibilities, characterized with the relative standard deviation percentages (RSD%) of measurements, varied between 1.73 RSD% (ß-estradiol) and 5.4 RSD% (stigmasterol), with an average of 4.82 RSD%.

Retention/quantitation properties of the o-phthaldialdehyde-3-mercaptopropionic acid and the o-phthaldialdehyde-N-acetyl-L-cysteine amino acid derivatives in reversed-phase high-performance liquid chromatography.

The separation/identification of 25 amino acids as their o-phthaldialdehyde-3-mercaptopropionic acid (OPA/MPA) and o-phthaldialdehyde-N-acetyl-L-cysteine (OPA/NAC) derivatives have been optimized [paying particular attention to those amino acids which elute with more than one derivative (glycine, histidine, gamma-aminobutyric acid, beta-alanine, ornithine, lysine) and that are expected to be present in apples in their free form]. Optimum separation conditions are reported on six reversed-phase columns: Nucleosil 3 and 5 microm, 150(+20 guard)x4.0 mm; Gromsil 3 microm, 150(+10 guard)x4.0 mm; Hypersil 5 microm, 150(+20 guard)x4.0 mm and 200(+20 guard)x4.0 mm; and Hypersil 3 microm, 150(+20 guard)x4.0 mm. Elutions were followed, simultaneously, with photodiode array and fluorescence detectors connected in line. Optimization studies carried out in model solutions as a function of temperature (30-55 degrees C) and eluent flow-rate (0.8-2.5 mL/min) demonstrated that optimum resolutions are obtained with the highest flow-rate applicable (remaining on the safe side with a column pressure of << 3500 p.s.i.; 1 p.s.i.=6894.76 Pa) in the temperature range 30-50 degrees C. Twenty-five amino acids, eluting in 31 separate, characteristic derivatives, were determined on all six columns (the main component, asparagine, present in overwhelming excess, together with the minor constituents glutamine, beta-alanine, gamma-aminobutyric acid, homoserine, and homoarginine). Optimum conditions in the case of both derivatives were obtained on the same type of column (Hypersil, 5 microm), as follows: for the OPA/MPA amino acids with programmed flow-rate [1.3-2.3 ml/min; column, 200(+20 guard)x4 mm], at 50 degrees C, while, for the OPA/NAC amino acids at 2.1 ml/min flow rate, at 30 degrees C [column, 150(+20 guard)x4 mm], with 40 and 37 min run times, including equilibration. Responses of the corresponding amino acids proved to be independent of the column used; reproducibility in the concentration range 6-12,000 pmol, related to the injected amount of amino acids, was <3.4% RSD (average relative standard deviation percentage). The utility of the protocol was demonstrated in the quantitation of the free amino acid content of five apple varieties (Jonagored, Idared, Jonica, Florina, Freedom) on various harvesting dates and after different storage times. Derivatization of the apple pulp was performed with filtered samples, applying any special isolation processes.

Temperature, eluent flow-rate and column effects on the retention and quantitation properties of phenylthiocarbamyl derivatives of amino acids in reversed-phase high-performance liquid chromatography.

The separation and identification possibilities of 27 PTC-amino acids (with particular attention to those present in apples in free forms), are reported on seven RP columns such as, Nucleosil, 3 and 5 microns: 150(+20 guard) x 4.0 mm; Gromsil 3 microns; 150(+10 guard) x 4.0 mm; Hypersil 5 microns: 130(+20 guard) x 4.0 mm, 150(+20 guard) x 4.0 mm and 200(+20 guard) x 4.0 mm, as well as, Hypersil 3 microns: 150(+20 guard) x 4.0 mm: a UV range photodiode array (PDA) detection was employed. Optimization studies carried out in model solutions, as a function of the temperature (30-55 degrees C) and flow-rate (0.8-2.5 ml/min) of eluents proved that optimum resolutions are associated with the highest flow-rate applicable, (remaining on the safe side with a column pressure of < 3500 p.s.i., 1 p.s.i. = 6894.76 Pa), in the temperature range of 30-50 degrees C. Twenty-seven amino acids, characteristic in apples in free forms, have been separated and determined on all seven columns, performing the same gradient program, (the main component asparagine, present in overwhelming excess, and the minor constituents glutamine, beta-alanine, gamma-aminobutyric acid, homoserine, homoarginine and 1-aminocyclopropane-1-carboxylic acid). Optimum conditions, at 2.1 ml/min, at 50 degrees C, with 40 min run time, including equilibration, have been obtained with the Hypersil, 150(+20 guard) x 4 mm column, performing elutions. Responses of the corresponding amino acids proved to be independent of the column used; reproducibility in the concentration range of 15-1500 pmol was < 4.0% R.S.D. (relative standard deviation). Detailed study of the PDA spectra revealed that in addition to the identification/peak purity possibilities further characteristics can be obtained taking advantage of the difference in maximum values and of those of their special ratio values, respectively. The utility of the protocol was shown in the quantitation of the free amino acid content of three apple varieties.