Fast Screening Methods for Analgesics and Non-Steroidal Anti-Inflammatory (NSAIDS) Drugs by HPLC with Agilent Poroshell 120 Columns

Jun 01, 2014

Using Selectivity to Enhance Separation of Analgesics

Selectivity is the most powerful tool to optimize separations in HPLC. This parameter is changed by using different bonded phases, including C18, polar embedded, phenyl bonded phases and perflorophenyl, or by changing the mobile phase. In this work, 4.6 × 50 mm Poroshell 120 columns are used to quickly evaluate method development choices for the analysis of non-steroidal anti-inflammatory drugs (NSAIDS). The short column length and high efficiency provide short analysis times and rapid equilibration, leading to fast investigations of selectivity.

Experimental Conditions

Instrument: Agilent 1260 Infinity Binary LC System
Columns: Noted below
Flow rate: 2 mL/min
Mobile Phase: A: 20 mM NH4HCO2pH 3.0 B: Acetonitrile
Temperature: 40 °C
Detection: UV, 254 nm

Time     % Organic
0           8
6           100
7           100
8           8

The Agilent 1260 Infinity Binary LC System was configured as follows:

  • G1312B Binary Pump SL, capable of delivering up to 600 bar
  • G1316C Thermostatted Column Compartment (TCC)
  • G1376D High Performance Autosampler SL Plus
  • G4212A Diode Array Detector equipped with a G4212-60008 10 mm path length, 1 µL volume flow cell

The following columns were used in this study.

  • Agilent Poroshell 120 PFP, 4.6 × 50 mm, 2.7 µm (p/n 699975-408)
  • Agilent Poroshell 120 EC-C18, 4.6 × 50 mm, 2.7 µm (p/n 699975-902)
  • Agilent Poroshell 120 Bonus-RP, 4.6 × 50 mm, 2.7 µm (p/n 699968-901)
  • Agilent Poroshell 120 Phenyl-Hexyl, 4.6 × 50 mm, 2.7 µm (p/n 699975-912)

Table I: Retention time, Log P, and pKa data for selected analgesics
A generic gradient separation was used to evaluate these columns consisting of ammonium formate (20 mM NH4HCO3pH 3.0) using either methanol or acetonitrile.

Figure 1: Structures of selected analgesics.
The analgesic materials all possess a wide variety of functional groups including fluorine (sulindac and diflunisal) and chlorine (diclofenac). The structures of the compounds examined are shown in Figure 1 and Table I. All samples were prepared at 10 mg/mL in acetonitrile and were diluted in water to a final concentration of 0.1 mg/mL.

Column Choice to Enhance Selectivity

Figure 2: Separation of analgesics using Agilent Poroshell 120 columns using methanol.
The columns were chosen to improve selectivity in the separation. They included a highly end capped C18 column recommended as a first choice in method development (Poroshell120 EC- C18).

Poroshell 120 Bonus-RP can be used for many of the same separations as a C18 column while avoiding some of the disadvantages of C18, such as poor wettability in high aqueous mobile phases. In addition, it is much more retentive for those molecules that can interact by hydrophobic interactions and also by H-bonding with the amide group. Compared to alkyl only phases, Bonus-RP has enhanced retention and selectivity for phenols, organic acids, and other polar solutes due to strong H-bonding between polar group (H-bond acceptor) and H-bond donors, like phenols and acids. Bonus-RP gives retention slightly less than a C18 allows, for easy column comparison without the need to change mobile phase conditions. The Bonus-RP phase gives different selectivity than C18 for polar compounds. It is also compatible with 100% water.

Poroshell 120 Phenyl-Hexyl columns deliver unique selectivity for compounds with aromatic groups, providing superior resolution for these samples. Poroshell 120 Phenyl-Hexyl can also provide optimum separations of moderately polar compounds where typical alkyl phases (C18 and C8) do not provide adequate resolution. Acetonitrile tends to decrease the π–π interactions between aromatic and polarizable analytes and the phenyl-hexyl stationary phases, but methanol enhances those same interactions, giving both increased retention and changes in selectivity. This does not mean that acetonitrile should not be used with a phenyl bonded phase or that it might not provide an acceptable separation, but methanol is more likely to deliver the different selectivity that is desired from a phenyl phase.

Poroshell 120 PFP columns possess a pentafluorophenyl ligand. This can provide an orthogonal separation mechanism to traditional reverse phase columns. By specifically targeting many polar retention mechanisms, PFP phases can separate analytes based on small differences in structure, substitution, and steric access to polar moieties. The resulting selectivity for positional isomers, halogenated compounds, and polar analytes is particularly useful in the analysis of complex mixtures, and small molecule pharmaceuticals

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