Directly coupled sample treatment-high-performance liquid chromatography for on-line automatic determination of liposoluble vitamins in milk.

We developed an on-line system for the determination of liposoluble vitamins A, D3 and E in milk, both liquid and in powder form, using an automated sample treatment system coupled to chromatographic determination. For this, C18 cartridges were used because of the strong capacity of this material for the extraction and preconcentration of such vitamins, its ease in handling and the possibility of coupling it with automatic analysis systems. Alkaline hydrolysis of the samples was performed in an on-line system comprising two confluent channels through which the sample solution and alcoholic sodium hydroxide plus ascorbic acid flowed for a given period of time. A third channel merged with the other two to neutralize the solution before it arrived at the C18 cartridge. The latter, inserted into a loop with a six-port injection valve, retained the soluble vitamins. The vitamins were eluted with a stream of methanol and the eluate was automatically injected into the chromatographic system. Variables affecting the on-line system were optimized: sample size, flow-rate, preconcentration, washing and elution times, etc. The recoveries for powdered and liquid milk for the three vitamins assayed ranged between 80 and 105% (n = 10). Additionally, a day-to-day precision (10 days) of the method of 4.5% was obtained for the different vitamins.

Determination of fat-soluble vitamins in yogurt by HPLC with electrochemical detection.

A method employing HPLC with electrochemical detection for the rapid and simultaneous determination of vitamins A, D(3) and E is described. The method uses a C-18 reverse phase column and 2.5 mM HAcO-NaAcO in methanol-water (99:1, v/v) solution as the mobile phase. The compounds are quantified using amperometric detection with a glassy carbon electrode at a potential of + 1300 mV (vs. Ag/AgCl) and the results are compared with those obtained using UV detection at a wavelength of 280 nm. The method was successfully applied to the analysis of vitamins A, D(3) and E in yogurt samples. After saponification, fat-soluble vitamins were extracted and the methanolic solution of the extracts was injected directly into the chromatographic system, avoiding the clean-up step which is necessary when no electrochemical detection is used. Good recovery percentages were obtained.

Determination of vitamins A, E and K1 in milk by high-performance liquid chromatography with dual amperometric detection.

A high-performance liquid chromatographic method with dual-electrochemical detection (HPLC-DEC) for the simultaneous determination of vitamins A, E and K1 is described. Separation was carried out using a C18 reversed-phase column and 2.5 mmol l-1 acetic acid-sodium acetate in methanol-water (99 + 1, v/v) as the mobile phase. The compounds eluted with good resolution in the above mentioned order within about 20 min and were quantified by dual-amperometric detection with a glassy carbon electrode at -1100 mV (E1) and + 700 mV (E2) (versus Ag/AgCl). This reductive-oxidative detection mode gave reproducible results and the detection limits were of the order of 0.06, 0.19 and 3.1 ng for vitamins A, E and K1, respectively. The HPLC-DEC method was successfully applied to the analysis of vitamins A, E and K1 in liquid cows' milk and infant-formula powdered-milk samples after applying alkaline hydrolysis of the fatty material and extraction of the vitamins with hexane for the analysis of vitamins A and E. In the case of vitamin K1 it was necessary to carry out enzymic hydrolysis, since this vitamin is degraded in basic medium, followed by cleaning with a Sep-Pak silica cartridge to isolate it from the other vitamins. Good recoveries (between 81 and 106%) were obtained.