Analysis of High- and Low-Dosed Vitamins in a Single Run Using the Agilent 1200 Infinity Series High Dynamic Range Diode Array Detector Solution

July 2, 2013
Angelika Gratzfeld-Huesgen

The Application Notebook

The Application Notebook, The Application Notebook-07-02-2013, Volume 0, Issue 0
Page Number: 415

Agilent Technologies, Inc

Angelika Gratzfeld-Huesgen, Agilent Technologies Inc.

The Agilent 1200 Infinity Series High Dynamic Range Diode Array Detector (HDR-DAD) solution expands the linear dynamic range by a factor of 30. Vitamins were separated and high-dosed vitamin C and low-dosed vitamin B12 were quantified in a single run.

In vitamin tablets, some vitamins are present at high concentrations; however, vitamin B12 is present in very low concentrations. In the literature for vitamin B12, different sample preparation procedures and different chromatographic methods are able to detect and quantify vitamin B12 in vitamin preparations. Until now, it was not possible when applying one sample preparation and one chromatographic method to detect and quantify all water-soluble vitamins present in a vitamin tablet. The combination of an Agilent 1200 Infinity Series LC with the 1200 Infinity Series HDR-DAD solution offers the perfect answer.

Experimental Conditions

Column: Agilent ZORBAX RRHT Eclipse plus C18, 4.6 × 150 mm, 1.8 μm (p/n 959994-902)

Mobile phases: A = water + 4 g/L K2HPO4 +0.5 g/L hexanesulphonate, pH 3 with phosphoric acid, B = methanol

Gradient: At 0 min 3% B, at 1 min 3% B, at 10 min 34% B, at 11 min 50% B

Flow: 1.2 mL/min

Stop time: 14 min

Post time: 5 min

Injection volume: 2 μL, sample temperature was kept at 4 °C

UV: 220/10 nm for pantothenic acid and vitamin B6; 265/10 nm for vitamin C, niacinamid, and vitamin B1; 360/10 nm for vitamin B12 and B2; Ref: off, 20 Hz

Column temperature: 40 °C

Sample Preparation

  • Two vitamin effervescent tablets were placed in 20 mL water and 20 mL ethanol. Ethanol is the optimum solvent for niacinamid and vitamin B12.

  • The solution was stirred for 10 min in the dark.

  • A sample of 20 mL was filtered using two Agilent Captiva Premium Syringe Filters, regenerated cellulose, p/n 5190-5111.

  • A sample of 1.5 mL was transferred into an autosampler vial and the sequence was started immediately.

Table 1: Concentration and injected amount.


Quantitation of all vitamins was possible because of the enhanced linear range of the 1260 Infinity HDR-DAD. One injection was sufficient to analyse and evaluate low-dosed and high-dosed vitamins, see Figure 1.

Figure 1: Analysis of vitamins with an Agilent 1260 Infinity HDR-DAD.


The Agilent 1260 Infinity High Dynamic Range DAD (HDR-DAD) allowed the analysis of main and trace compounds in one single run because of an increased linear dynamic range by a factor of >30. The analysis of all water-soluble vitamins in vitamin tablets was possible by using the Agilent 1260 Infinity HDR-DAD. Vitamin B12 with a concentration of 2 μg/tablet was quantified as well as vitamin C with a concentration of 60 mg/tablet in one run.

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