LC/MS/MS Can Help Screen Cereal

Water-soluble vitamins, which are very polar, have poor retention on reverse phase columns. The presence of ion pair reagents such as heptafluorobutyric acid in the mobile phase has been shown to improve the separation and retention of these compounds. The drawback of using such ion pair reagents, however, is the high background levels generated inside the mass spectrometer (MS). Therefore, we have developed a rapid and sensitive method with ammonium formate in the mobile phase solvent using a column with a bonded phase designed to retain hydrophilic compounds.

The 1200 Series LC (liquid chromatography) system (Agilent Technologies, Inc.; Santa Clara, Calif.) used in this work was designed to take advantage of sub-2 micron particle columns for rapid, high-resolution separations. Included in the LC design were decreased delay volume, increased pressure range, and increased column temperature. This LC system was coupled with the Agilent 6410 Triple Quadrupole Mass Spectrometer by way of the G1948B electrospray ionization source. Target compound separation was achieved on a Zorbax AQ 1.8-micron column using a water and methanol gradient with ammonium formate.

Typical LC/MS methods for water-soluble vitamins have shown analysis times as high as 30 minutes with heptafluorobutyric acid ion pairing reagent in the mobile solvent. We have developed a rapid and sensitive method for the LC/MS/MS analysis of water-soluble vitamins by employing a high efficiency 1.8-micron column in a low dispersion, 600 bar LC/MS configuration that allows screening and quantitation with a run-to-run cycle time as low as 10 minutes.

Linearity of the MS response was observed over three orders of magnitude with limits of quantitation in the 0.5 pg/mL range for all of the analytes except for pyridoxine. In the case of pyridoxine, good sensitivity was demonstrated, but good linearity was limited to just under three orders of magnitude. Calibration curves and chromatograms for the vitamins between 0.5 and 500 pg/mL were generated for all compounds with the exception of pyridoxine, which was 0.5 – 250 pg/mL.

Sample Preparation

A standard mix containing all eight compounds in methanol was provided by ConAgra Foods (Omaha, Neb.) and diluted in 90% water/10% methanol with 20 mM ammonium formate and 0.1% formic acid to the following concentrations: 500, 250, 100, 50, 5, and 0.5 pg/mL. These dilutions were used for the quantitation of unknown samples.

One B vitamin-fortified sample was also provided, using the following sample preparation procedure:

1. Grind and homogenize breakfast cereal in a blender.
2. Weigh one gram of homogenized sample into a 50 mL vial.
3. Add 25 mL of 0.1M HCl and heat in a water bath at 100°C for 20 minutes. This solubilizes the vitamins.
4. Cool to ambient temperature.
5. Adjust volume to one liter with deionized water.
6. Filter with 0.45 mm glass microfiber membrane.

It should be noted that the fortified sample was created to test the sensitivity of the instrument for customer demonstration purposes. A typical unfortified sample extraction consists of one gram of homogenized sample, treated with enzymatic digestion in order to release naturally occurring vitamins from their conjugated forms and volume adjusted to 10 mL, which is 1/100th the volume used in the fortified sample analyzed in this work. At the higher concentration, salts and other matrix contributions cause interference in the analysis of some vitamins. Further dilution may be used to accommodate the matrix effect in these samples.

LC/MS Method

• LC conditions: Agilent 1100 series binary pump, degasser, wellplate sampler, and thermostatted column compartment.
• Column: Agilent Zorbax RRHT SB-Aq, 3.0 x 100 mm, 1.8 mm (PN: 828975-314).
• Column temperature: 35°C.
• Mobile phase: A = 20 mM ammonium formate and 0.1% formic acid in water. B = 20 mM ammonium formate and 0.1% formic acid in methanol.
• Flow rate: 0.5 mL/min; injection vol: 10 mL.
• Gradient:
  Time (min) %B
  0.0 10
  8.0 55 Stop time: 10 min.
  8.1 10
  Needle wash (75:25 methanol/water) – flush port 20 seconds.

MS Conditions

• Mode: Positive ESI using the Agilent G1948B ionization source.
• Nebulizer: 30 psig; drying gas flow: 10 L/min; drying gas temp: 350°C.
• Vcap: 1850 V.
• Resolution (FWHM): Q1 = low res; Q2 = low res.
• Dwell time for all multiple reaction monitoring (MRM) transitions: = 200 msec (for more on MRM mode parameters.

The precursor ion mass for cyanoco-balamin (m/z 678.6) is about half of the expected value in which the empirical formula for this compound is C63H88CoN14O14P. It is believed that the structure of this compound is not very stable and breaks apart during the ionization process.

Results and Discussion

We determined the calibration curves for all eight compounds. Of all the compounds, the 500 parts per billion (PPB) level needed to be removed for good linearity only in the case of pyroxidine. No internal standard is included. All line fits to data are carried out as linear with the origin ignored and a 1/x weighting.