3 ml/min. Prior to injection, the column was equilibrated with 15% B. After injection of sample, this proportion
was modified to 23% B in 1 min, kept constant until 23 min and increased to 50% B until the end of the 35 min run. Between injections, 20 min intervals were used to re-equilibrate the column with 15% B. Isoflavones were monitored by DAD between 190 and 370 nm and soyasaponins were monitored by MS using positive ionisation, with a nebuliser gas (N2) flow of 3.0 L/min, operated in the single ion monitoring (SIM) mode to detect pseudomolecular ions. Identification of compounds was performed by comparison with retention time and molecular weight of the respective standard. Malonylglycosilated and acetylglycosilated isoflavones, for which commercial standards http://www.selleckchem.com/products/PD-0332991.html were unavailable, were identified by their pseudomolecular ions in the MS. Quantification
was performed by external standardisation. Isoflavones were quantified by their DAD peak areas (250 nm). The contents of malonylglycosilated and acetylglycosilated isoflavones were determined from the calibration curve of the corresponding β-glycosylated isoflavone, correcting for differences in molecular weight. Soyasaponins (B-I, B-II and JNJ-26481585 B-III) and soyasapogenol were quantified by their MS fragment ions, m/z 423 and m/z 223, respectively. Although soyasaponins B-II and B-III isolated standards were available, these compounds were quantified together, as it was not possible to chromatographically separate these compounds. Data were acquired by LCMSsolution software (Shimadzu Corp., version 2.00, 2000) for the mass spectrometer. Recovery values were taken into consideration for calculating the contents of these compounds in the samples. The daily intake of soy isoflavones and soyasaponins according to infant’s age, expressed per kilogram of body weight, was estimated from the total content of these classes of bioactive compounds found in the analysed infant formulas. We considered the recommended use according to the manufacturer’s directions (number for of feeding bottles given per day and amount of powder used to prepare each feeding bottle) and the mean body weight (50th percentile) of infants
of both sexes, according to age (1–2 weeks: 3.3 kg; 3–4 weeks: 4.3 kg; 2 months: 5.3 kg; 3 months: 6.1 kg; 4 months: 6.7 kg; 5 months: 7.2 kg; 6 months: 7.6 kg) (WHO, 2006). Data are presented as mean ± standard deviation. The contents of isoflavones and soyasaponins in the analysed soy-based infant formulas were compared using analysis of variance (one-way ANOVA), followed by Tukey’s multiple comparison post-test. All statistical analyses were performed using GraphPad Prism software for Windows, version 5.04 (GraphPad Software, San Diego, CA). Differences were considered significant when p < 0.05. The method showed good linearity (R2 > 0.994) in the concentration range of 0.1–5.0 μg/ml and 1.0–20.0 μg/ml for isoflavones and soyasaponins, respectively ( Table 1). Rostagno et al.