Influence of Hydrolysis, Purification, and Calibration Method on Furosine Determination Using Ion-Pair Reversed-Phase High-Performance Liquid Chromatography

Abstract

The influence of HCI concentration (6M, 8M, and 10M) and the ratio of sample protein to acid (1 or 5 mg of protein per mL of acid) on furosine formation during sample hydrolysis is studied. The conditions that maximize furosine formation are 10M HCI in the ratio of 1 mg of protein to 1 mL of acid. Purification of the hydrolysate by solid-phase extraction is also considered by examining the effect of hydrolysate volume and volume of 3M HCI used to elute the furosine. Furosine quantitation is carried out using the standard additions and external standard methods. The results indicate that there is no interference by the sample matrix and that external calibration is adequate.

RP-HPLC analysis of furosine and acid-soluble β-lactoglobulin to assess the heat load of extended shelf life milk samples in Austria

“…However, as furosine and β-Lg contents of many milk samples were to be analysed in alternating series, an effort was made to develop an RP-HPLC method for the analysis of furosine using the same column as for β-Lg, to avoid recurrent replacement of column during this study. Figure 3 shows the chromatogram of a furosine standard using a Symmetry 300™ C 18 column (Waters), which enabled an excellent separation of furosine within 8 min superior to that reported in the IDF standard procedure within 22 min [13], and comparable to that of other authors [8,16,26]. However, in contrast to all available reports [7,8,16,26,27], two separated mobile phases were used, which were continuously mixed by the used HPLC multisolvent delivery system during isocratic separation of furosine.…”

“…Figure 3 shows the chromatogram of a furosine standard using a Symmetry 300™ C 18 column (Waters), which enabled an excellent separation of furosine within 8 min superior to that reported in the IDF standard procedure within 22 min [13], and comparable to that of other authors [8,16,26]. However, in contrast to all available reports [7,8,16,26,27], two separated mobile phases were used, which were continuously mixed by the used HPLC multisolvent delivery system during isocratic separation of furosine. Continuous mixing of two separated solvents (solvent A was 0.2% formic acid in 5 mmol·L −1 sodium heptane sulphonate and solvent B was 100% acetonitrile) was of utmost importance to get a stable baseline, a proper resolution, and constant retention time of furosine peaks in different samples.…”

“…The hydrolysate was filtered (Schleicher & Schuell 595½) and applied to SPE to minimize contamination: 0.5 mL of hydrolysate was added to a pre-wetted (5 mL methanol and 10 mL water) Sep-Pak ® Vac 3 cc (500 mg) C 18 cartridge (Waters); the eluted liquid was discarded, and furosine was then eluted with 3 mL of 3 mol·L −1 HCl. In contrast to the IDF standard procedure [13], samples were not directly used for HPLC analyses, but were dried as reported by some authors [8,16,26]. Purified eluates (200 μL) were gently vacuum-dried using a Waters Pico·Tag™ workstation, and dried samples were dissolved in 200 μL of a freshly prepared mixture of water, acetonitrile, and formic acid (94.8:5:0.2) before HPLC analysis.…”

“…Column eluates were monitored at 280 nm using a Waters 2489 UV/VIS Detector. In contrast to other authors [7,8,16,26,27], two separated mobile phases were used, which were continuously mixed by the used HPLC multisolvent delivery system during isocratic separation of furosine. Solvent A consisted of 0.2% formic acid in 5 mmol·L −1 sodium heptane sulphonate (Sigma) solution (prepared from a 50 mmol·L −1 stock solution), and solvent B was 100% acetonitrile.…”

“…A specific "Furosine dedicated" RP-HPLC C 8 column had been proposed by the FIL/IDF standard [13], whereas different types of C 18 columns were reported for RP-HPLC by other authors [7,8,16,23,26,27]. However, as furosine and β-Lg contents of many milk samples were to be analysed in alternating series, an effort was made to develop an RP-HPLC method for the analysis of furosine using the same column as for β-Lg, to avoid recurrent replacement of column during this study.…”