A More Versatile C18 Column: The Aqueous C18 - a single phase solution for multiple Dietary Supplement Applications

• Extends the analytical range of your C18.
• Better retention of hydrophilic compounds than on a standard C18.
• Compatible with 100% aqueous mobile phases.

Method development continually ranks in surveys as one of the most challenging and time-consuming issues in HPLC. Choosing the right analytical column is the cornerstone of developing sound methods, but with the number and complexity of columns available on the market today, this can be a difficult process. Evaluating multiple columns quickly becomes expensive and laborious, so it is good practice to begin method development with a versatile column. Conventional alkyl (C18) columns are frequently used, but limited by extremely poor retention of hydrophilic compounds. In comparison, Aqueous C18 phases are more versatile than standard C18 phases, due to much higher hydrophilic retention. In this article we demonstrate the broad utility of Aqueous C18 columns across a range of analytes relevant to dietary supplement testing.

Ideal for Multivitamin Analyses—Easily Retains Water-Soluble Vitamins

Many consumers are augmenting their diets with multivitamins. These supplements usually contain multiple water-soluble vitamins in a variety of chemical forms and concentrations. While water-soluble vitamins can be analyzed by HPLC, obtaining adequate retention of hydrophilic analytes is often problematic. As shown in Figure 1, the Ultra II® Aqueous C18 column provides excellent retention and completely resolves a test mix of seven B vitamins.

Also, due to the column's unique bonding chemistry, 100% aqueous mobile phases can be used to elute highly polar compounds, such as ascorbic acid (vitamin C), which are not retained on a conventional C18 (Figure 2). Organic acids can also be retained, further demonstrating column versatility (Figure 3). The Aqueous C18 phase provides greater retention of hydrophilic compounds than conventional C18 columns, while still supplying the hydrophobic retention expected from a standard C18.

Maximum Versatility—Use the Same Phase for cGMP Pesticide Screens

The biggest challenge facing many dietary supplement laboratories today is becoming compliant with FDA current Good Manufacturing Practices (cGMP) requirements. One of the new hurdles presented by cGMP is implementing a comprehensive screening method for pesticide residues in articles of botanical origin. This testing requires either GC/MS or LC/MS/MS, which is a technology change from traditional HPLC assays. Aqueous C18 columns are ideal for LC/MS/MS screening of pesticides residues, as hundreds of pesticides can be resolved and quantified in the same run (Figure 4). Multiresidue pesticide analysis is simplified by the unique selectivity and excellent resolution of the Ultra Aqueous C18 column, which can retain even difficult polar species.

The Aqueous C18 phase, available on several silicas, is an excellent general purpose phase that performs well for many analyses in today's dietary supplement laboratory. Columns are available in a full range of scalable particle sizes, from 1.9µm UHPLC columns to traditional 5µm HPLC columns. Regardless of the instrumentation used, the unique selectivity and enhanced hydrophilic retention of Aqueous C18 columns makes them more versatile than a standard C18 and ideal for method development.

Figure 1: A selective separation of seven B vitamins using a simple buffer:acetonitrile gradient on an Ultra II® Aqueous C18 column.

Peaks RT (min.) 1. Vitamin B3 2.472 2. Vitamin B6 2.764 3. Vitamin B5 3.804 4. Vitamin B9 4.126 5. Vitamin B12 4.261 6. Vitamin B2 4.329 7. Vitamin B7 4.472 Column Ultra II® Aqueous C18 (cat.# RE9608565) Dimensions: 150 mm x 4.6 mm ID Particle Size: 5 µm Pore Size: 100 Å Temp.: 40 °C Sample Diluent: water:methanol (70:30) Conc.: 100 µg/mL each component Inj. Vol.: 1 µL Mobile Phase A: 10mM potassium phosphate (pH 2.5) B: acetonitrile Time (min.) %B 0 2 1.5 2 5 75 Flow: 1.5 mL/min. Max Pressure: 105 bar Detector UV/Vis @ 210 nm Instrument Shimadzu UFLCXR LC_PH0503

Figure 2: Retention of the polar compound ascorbic acid (vitamin C) using a 100% aqueous mobile phase on an Ultra II® Aqueous C18 column.
Peaks RT (min.) 1. Ascorbic acid (vitamin C) 1.759 Column Ultra II® Aqueous C18 (cat.# RE9608565) Dimensions: 150 mm x 4.6 mm ID Particle Size: 5 µm Pore Size: 100 Å Temp.: 40 °C Sample Diluent: water Conc.: 100 µg/mL Inj. Vol.: 2 µL Mobile Phase 100% 10mM potassium phosphate (pH 2.5) Flow: 1.5 mL/min. Max Pressure: 95 bar Detector UV/Vis @ 242 nm Instrument Shimadzu UFLCXR LC_PH0504

Figure 3: The analysis of organic acids using a 100% aqueous mobile phase and an Ultra II® Aqueous C18 column.

Peaks RT (min.) 1. Tartaric acid 2.061 2. Quinic acid 2.210 3. Malic acid 2.596 4. Citric Acid 4.177 5. Fumaric Acid 5.853 Column Ultra II® Aqueous C18 (cat.# RE9608565) Dimensions: 150 mm x 4.6 mm ID Particle Size: 5 µm Pore Size: 100 Å Temp.: 30 °C Sample Diluent: Water Conc.: 10 µg/mL fumaric acid; 2,000 µg/mL each other acids Inj. Vol.: 5 µL Mobile Phase 100% 20mM potassium phosphate (pH 2.5) Flow: 1.0 mL/min. Detector UV/Vis @ 226 nm Instrument Shimadzu UFLCXR LC_PH0498

Figure 4: Comprehensive LC/MS/MS pesticide screen using an Ultra Aqueous C18 column.

A. Pesticides in positive ion mode. See Table 1 for compound identification and MS details. B. Pesticides in negative ion mode. See Table 2 for compound identification and MS details. C. Improved retention of difficult pesticides. See Table 1 for compound identification and MS details. Column: Ultra Aqueous C18 Cat. #: RE9178312 Dimensions: 100mm x 2.1mm Particle size: 3µm Pore size: 100Å Sample: multicomponent pesticide standard Inj.: 10µL Conc.: 1ppb each pesticide Sample diluent: acetronitrile Conditions: Instrument: Shimadzu Prominence® UFLCXR Mobile phase: A: 10mM NH4OAC in water B: 10mM NH4OAC in methanol Time (min.) %B 0.0 20 8.0 90 12.0 100 14.8 100 14.9 20 Flow: 500µL/min. Temp.: 35°C Det.: Applied Biosystems 4000 QTRAP® LC/MS/MS system Ion Source: TurboIonSpray® A&C: ESI+ B: ESI- IonSpray Voltage: 5kV (ESI+), -4.2kV (ESI-) Gas 1: 50psi Gas 2: 60psi Source Temp.: 600°C A. LC_FF0490 B. LC_FF0490A C. LC_FF0490B

Data for 3µm Ultra Aqueous C18 column are shown.

Data for 3µm Ultra Aqueous C18 column are shown.