Simultaneous analysis of four sulfonamides in chicken muscle tissue by HPLC.

The aim of this study was to develop a simple high-performance liquid chromatography (HPLC) with UV detection method for the determination of four sulfonamides in chicken muscle tissue. The sulfonamides were extracted with acetonitrile, acetone and dichloromethane. Separation was carried out on a C18 column using as the mobile phase a mixture of 6‰ disodium hydrogen phosphate and methanol. The analytes were detected by UV in one run. Calibration curves were linear with very good correlation coefficients for concentration ranging from 30 to 150 µg kg(-1). The limits of detection (LOD) for sulfonamides ranged from 6.5 to 0.14 µg kg(-1). The recovery for spiked chicken muscle with 50-150 µg kg(-1) was more than 70%. The relative standard deviations (RSDs) of the sulfonamides for six measurements at 50, 100 and 150 µg kg(-1) were less than 15%. These parameters met the European Union criteria for method validation. The results were confirmed by LC-MS/MS using multiple reaction monitoring as the operating mode. Confirmation required the retention times of the analytes to be within ±2.5% of the retention times of the standards, the presence of the parent ion and two characteristic fragment ions (product ions) per analyte, as well as the relative ion abundance ratios of the fragment ions corresponding to ratios obtained for the standards, within permitted limits. The transition of two common product ions at m/z 155.7 and 107.5 were monitored for all sulfonamides. Each of the analytes, in all tested samples, met the confirmation criteria. Thus, the applicability of the HPLC-UV method for routine analysis of chicken muscle tissue was demonstrated.

Total and free amino acids in the "Crinofizin" pineal extract.

In order to establish the chemical nature of Crinofizin, a protidic extract obtained from bovine pineal, the amino acids content was analysed using two analytic programs: one for biologic fluids, the other for protein hydrolysates. The results showed that the pineal extract contains besides the common protein amino acids reported earlier, others, rare in proteins, such as hydroxyproline, hydroxylysine and 1-methylhistidine as well as non-protein amino acids. From among the latter, the alpha-aminoadipic acid, beta- alanine, O-cysteine, are found only in a bound form, and are detected only by hydrolysis. Other amino acids, such as taurine, glutamine, citruline are present only in free form whereas the alpha-amino-n-butyric acid, GABA, ethanolamine and ornithine are present in both forms, free or bound. The paper discusses the physiologic significance of some amino acids.

Purification and physico-chemical characterization of the antigonadotropic fractions obtained by ultrafiltration from the pineal extract.

In order to isolate the antigonadotropic factor from a pineal extract prepared from bovine pineals the authors first applied ultrafiltration, which yielded three fractions: F1 greater than 10 kDa, 1 kDa less than less than F2 less than 10 kDa, and F3 less than 1 kDa. Examination of the biologic activity showed that only F1 and F2 have a statistically significant antigonadotropic activity. Ion exchange chromatography of F1 yielded two subfractions: one, F11, which was not retained, and another one, F12, which was retained by the exchanger. Both subfractions showed biologic activity. While F11 is closer to F2 less than 10 kDa, F12 has a molecular weight around 13.7 kDa, as resulted after applying the SDS-PAGE procedure. The F1 fraction has a proteic content of 903 micrograms/ml, F11 = 308 micrograms/ml and F12 = 247 micrograms/ml. Amino acids assay showed that the F12 subfraction has the same composition as F1 and the F11 subfraction also has free amino acids which indicates a process of adsorption of small molecules on large molecules. The F2 less than 10 kDa fraction, biologically active, has a proteic content of 433 micrograms/ml and an amino acids composition similar to F11 except for cysteine and methionine. The F3 less than 1 kDa fraction with a proteic content of 79 micrograms/ml having an amino acid composition in which prevails the glutamic acid shows poor antigonadotropic activity. The results of this study, as well as the fact that the antigonadotropic activity is concentrated in fractions both above and under 10 kDa, suggests at least two hypotheses: either that the pineal antigonadotropic factor exists under various molecular forms in which the activity is preserved, or that the antigonadotropic factor is a peptide with strong aggregation properties as demonstrated in our study.

The antigonadotropic activity and amino acid composition of a total and dialyzed melatonin-free pineal extract.

The antigonadotropic activity of a melatonin-free pineal extract before and after a 24 h dialysis was assessed by the mouse uterine-weight test. In both cases, the uterus-stimulating effect of HCG was inhibited, showing that after dialysis the extract not only retains its effect but that the latter is even higher. The amino acids content established by an amino acid analyzer was the same in two batches of extract obtained from two different stores of pineal powder. The analyzer also showed that the glutamic acid is the prevailing component as a free and bound amino acid in the total extract. It was also found that methionine is absent from the dialyzed extract. Qualitative analysis of the pineal extract revealed 16 amino acids and a concentration of proteins of 1.90-1.96 mg/ml. Quantitatively, the glutamic and aspartic acids, proline and glycine prevail over the other constituents. The glutamic acid concentration was over two times higher than the latter three.