Develop More Productive PPCP Methods by Replacing C18 and HILIC Columns with a Single Ultra II® Aromax Column

• Use 1 column instead of 2—no need for separate HILIC analysis.
• Better response than on a C18; higher retention reduces ion suppression from coeluting compounds.
• Lower detection limits—highly organic mobile phase improves sensitivity.

Media reports of pharmaceuticals and personal care products (PPCPs) occurring in the environment have raised public concern and increased demand for testing. Although PPCP levels in drinking and waste water are currently unregulated, and reports of part-per-billion levels have not been tied specifically to negative health impacts, public concern has prompted the US EPA to release Draft Method 1694 to assist labs in developing testing procedures. The draft method is time-intensive and splits the target compounds into 4 groups, which are analyzed separately by LC/MS/MS using 2 columns. Groups 1-3 are acid extractions run on a C18 column, and Group 4 is a basic extraction run on a HILIC column. While the draft method is a plausible initial approach, labs are encouraged to develop alternative procedures. Here we demonstrate a simpler strategy that can improve lab productivity by replacing both the C18 and HILIC columns with a single, highly selective Ultra II® Aromax column.

PPCP testing is complicated by the number of analyses required to determine the target compounds. Two columns are used because the Group 4 compounds are not retained at all on a C18 and elute in the void volume. Further, retention of Group 1-3 compounds on a C18 is suboptimal. While C18 columns work well for compounds that can be separated based on hydrophobicity, they are not ideal for PPCPs, as many have a low carbon:heteroatom ratio and contain aromatic rings or polarizable groups. Phases that exhibit greater aromatic selectivity can provide increased retention and better resolution, possibly eliminating the need for a second column. To determine if a single, highly retentive column could be used for PPCP analysis, a subset of Draft Method 1694 compounds was chosen based on report frequency and analyzed on a variety of stationary phase chemistries.

Ultra II® Aromax columns were determined to be the most selective for PPCPs and offered several benefits over the approach presented in Draft Method 1694. Most importantly, Group 4 compounds—can be retained and resolved on an Ultra II® Aromax column, eliminating the need for separate HILIC analysis (Figure 1). This offers analysts a chance to develop PPCP methods on a single column, and possibly analyze Group 4 compounds in the same injection as Group 1 compounds, saving time and increasing productivity.

In addition to retaining Group 4 analytes and eliminating the need for HILIC columns, Ultra II® Aromax columns offer several advantages compared to C18 columns. Greater retention results in increased separation of target analytes and unretained matrix interferences, thus minimizing ionization suppression (Figure 2). This assures good low level detection and protects against false negatives.

Another benefit to developing PPCP methods on Ultra II® Aromax columns is that, since the column is more retentive than a C18, the target analytes elute when the mobile phase is more organic. This increases desolvation efficiency, leading to better ionization, which can improve response, and lower detection limits (Figure 3).

In conclusion, making strategic column choices during method development can pay big dividends by simplifying routine analysis. Using a selective Ultra II® Aromax column with good retention for aromatic compounds can significantly improve PPCP resolution and eliminate the need for a time-consuming two column approach. Labs interested in more sensitive, efficient PPCP methods should consider Ultra II® Aromax columns during method development.

Figure 1: Ultra II® Aromax columns retain and resolve Group 4 PPCP compounds, eliminating the need for separate HILIC analysis.

Peaks EIC(plotted m/z) 1. Albuterol 240.1 2. Cimetidine 253.1 3. Ranitidine 315.1 Column Ultra II® Aromax (cat.# RE9607312) Dimensions: 100 mm x 2.1 mm ID Particle Size: 3 ?m Pore Size: 100 Å Temp.: 40 °C Sample Diluent: 0.1% formic acid in methanol:water (75:25) Conc.: 25 µg/mL Inj. Vol.: 10 µL Mobile Phase A: 0.3% formic acid and 0.1% ammonium formate in water B: acetonitrile:methanol (1:1) Time (min.) %B 0 5 10 25 15 30 Flow: 0.3 mL/min. Detector Shimadzu 2010EV Acquisition Type: Scan Probe Voltage: 1.5 kV Interface: ESI+ Scan Range: 200-350 amu Event Time: 0.4 sec. Instrument Shimadzu UFLCXR LC_EV0516