Pharmaceutical Article

RP-HPLC Analysis of Selective Serotonin Reuptake Inhibitors

Using Polar Stationary Phases

By Rick Lake, Pharmaceutical Innovations Chemist

Good retention and selectivity without ion-pairing chromatography.
Practical at acidic pH (Ultra PFP phase) or neutral pH (Allure™ Basix phase).
Excellent peak shape for basic compounds.

Background

An estimated 9.5 percent of the U.S. population — nearly 19 million American adults — suffer from depressive illness.(1) Probably the most common treatment for depression is prescription of antidepressant drugs. Selective serotonin reuptake inhibitors (SSRIs) are a newer, highly prescribed class of antidepressants that have gained much acceptance in the medical community. Although these compounds are no more effective than the traditionally used tricyclic antidepressants, they produce fewer side effects, which give them a clear medicinal advantage.

Operation of SSRIs

Symptoms of depression are related to an imbalance of neurotransmitters in the brain. Information is passed between two neurons, or nerve cells, across a synapse — a small gap between the two cells — when the sending neuron releases neurotransmitters into the synapse, serotonin among them, and these are taken up in receptor sites in the recipient neuron. This triggers the recipient neuron to release neurotransmitters, thus relaying a signal along a nerve cell pathway. Depression is believed to be caused by a lack of stimulation in the recipient neuron, dulling the transmittance. Roughly 10% of the neurotransmitter molecules are lost during the relay process, and 90% are released from the receptor sites and taken up again by the sending cell — thus “reuptake.” In general, SSRIs help cure depression by inhibiting the reuptake of serotonin by the sending cell, keeping it in the synapse longer, and giving it repeated opportunity to be recognized by the recipient neuron. The end result is an increase in recipient cell stimulation, a stronger transmittance along the nerve cell pathway, and a reduction in the symptoms of depression.

Ion Pairing Chromatography versus a Polar Stationary Phase

SSRIs are polar bases containing amine and halogenated functionalities (Figure 1) and, as such, historically have been analyzed through ion-pairing chromatography (IPC). IPC increases the retention of ionic compounds on hydrophobic alkyl stationary phases (e.g., C18) through the “pairing” or neutralization of the ionic analytes with a counter-ionic reagent that also possesses a hydrophobic portion capable of retention on non-ionic reversed phase packings. For example, in an IPC analysis of bases, hexane sulfonate often is used as the ion-pairing reagent — its SO3- anion is attracted to the protonated basic analyte (analyte-H+), and the reagent-analyte ion pair gains retention through attachment of the alkyl portion (C6) of the reagent to the hydrophobic stationary phase. IPC is a good alternative when reversed phase chromatography (RPC) on an alkyl phase cannot provide adequate separation. IPC has its disadvantages, however, including artifactual peaks, slow column equilibrium, poor peak shape, poor reproducibility, and incompatibility with mass spectrometry.

Because of the downsides to IPC, we evaluated using polar cyanopropyl and pentafluorophenyl stationary phases, rather than alkyl phases and ion-paring reagents, for a simplified analysis of SSRIs. The compounds should be separable through interaction between the polar functional groups embedded on these stationary phases and the amine or halogenated moieties in the target compounds.

Figure 1 Structures of selective serotonin reuptake inhibitors (SSRIs) make their chromatographic analysis a challenge.

fluoxetine	fluvoxamine maleate	sertraline HCl

Analysis Using a Cyanopropyl Phase

According to acid-base equilibria, as pH decreases, bases gain a proton (ionize), becoming more hydrophilic and less retained by RPC. Conversely, as pH increases, acids lose a proton (ionize), becoming more hydrophilic and less retained by RPC. This can be demonstrated best by a plot of the capacity factor for acids, bases and neutrals versus mobile phase pH (Figure 2). Therefore, retention of basic SSRI compounds is greatest at an increased, or basic, pH. Complete ionization is ensured by using a mobile phase pH approximately 2 units from the analyte’s pKa. The high pKa values for SSRIs (e.g., fluvoxamine maleate: 8.7, fluoxetine: 9.1, sertraline HCl: 9.5), however, puts such mobile phases above the operating range of silica-based columns (pH 2-8). Consequently, a neutral or acidic pH must be employed when using conventional silica-based packing for analyzing SSRIs.

After ruling out basic mobile phases, we observed the greatest retention of SSRIs using an Allure™ Basix column and a mobile phase at neutral pH. The highly retentive cyanopropyl stationary phase provides good retention, selectivity, and peak shape for the SSRIs at neutral pH (Figure 3). The Allure™ Basix column is an excellent choice for analytes that contain amine groups and, if optimum retention of SSRIs is desired, the Allure™ Basix column, used at neutral pH, has proven to be a good candidate.

Overall, for basic analytes, polar stationary phases provide better peak shape than alkyl phases. Often, an amine modifier is added to the mobile phase in an RPC analysis of basic analytes, to enhance peak shape and to produce alternate selectivity. As the concentration of modifier increases, retention decreases, and peak shape sharpens. Under the conditions used here, adding triethylamine (TEA) to the mobile phase only slightly improved peak shape, but reversed the elution order of fluoxetine and sertraline HCl (Figure 4). This could be an effective way to produce alternate selectivity or, possibly, to enhance peak shape, but note that amine additives work by blocking ionization of silanols on the surface of the packing material, and concentrations of these groups can vary from column to column. If you use an amine modifier, be careful to ensure that the amine concentration is high enough to suppress all possible silanol effects.

Figure 2 Relationship of retention capacity to mobile phase pH for an acidic (blue), neutral (pink), or basic (green) test probe.

Column:	Allure™ Basix 150mm x 4.6mm, 5µm (cat.# 9161565)
Mobile Phase pH 3:	20mM potassium phosphate monobasic in water:acetonitrile, 40:60
Mobile Phase pH 4:	20mM ammonium acetate in water:acetonitrile, 40:60
Mobile Phase pH 7:	20mM potassium phosphate dibasic in water:acetonitrile, 40:60
Flow rate:	1.0mL/min.
Det.:	UV @ 230nm
Temp.:	30°C

Figure 3 SSRIs are well separated, with good peak shape, on an Allure™ Basix column at neutral pH.

Elution Order		USP Tailing
6.	uracil
7.	impurity
8.	fluvoxamine maleate	1.13
9.	sertraline HCl	1.33
10.	fluoxetine	1.15

Column:	Allure™ Basix 150mm x 4.6mm, 5µm (cat.# 9161565)
Mobile Phase:	20mM potassium phosphate dibasic in water (pH 7):acetonitrile, 40:60
Flow rate:	1.0mL/min.
Det.:	UV @ 230nm
Temp.:	30°C

Figure 4 An amine modifier improves peak shape and changes selectivity for SSRIs on an Allure™ Basix column.

Elution Order		USP Tailing
1.	uracil
2.	impurity
3.	fluvoxamine maleate	1.17
4.	fluoxetine	1.15
5.	sertraline HCl	1.22

Column:	Allure™ Basix 150mm x 4.6mm, 5µm (cat.# 9161565)
Mobile Phase:	1% triethylamine (TEA) in water (pH 6): acetonitrile, 50:50
Flow rate:	1.0mL/min.
Det.:	UV @ 230nm
Temp.:	30°C

Figure 5 SSRIs also are separated well at low pH on an Ultra PFP column.

Elution Order		USP Tailing
1.	uracil
2.	impurity
3.	fluvoxamine maleate	1.02
4.	sertraline HCl	1.10
5.	fluoxetine	1.01

Column:	Ultra PFP 150mm x 4.6mm, 5µm (cat.# 9176565)
Mobile Phase:	20mM potassium phosphate monobasic in water (pH 3):acetonitrile, 70:30
Flow rate:	1.0mL/min.
Det.:	UV @ 230nm
Temp.:	30°C

Analysis Using a Pentafluorophenyl Phase

Pentafluorophenyl phases typically exhibit heightened retention of basic analytes or analytes containing amine or halogenated functionalities. In contrast to the results obtained with the Allure™ Basix column, a column containing a pentafluorophenyl stationary phase, the Ultra PFP column, performed best at an acidic pH (Figure 5). The structural difference between the Ultra PFP phase and another pentafluorophenyl phase, Allure™ PFP Propyl, is a propyl spacer in the Allure™ PFP Propyl phase, between the functional group and the surface silica. As it does in the Allure™ Basix phase, the propyl spacer produces a sterically favorable advantage that, in most applications, can enhance retention capacity. The very high retention capacity of the Allure™ PFP Propyl stationary phase makes it appropriate for gradient separations of chemically similar compounds when neither a cyanopropyl column nor a pentafluorophenyl column is effective. We tested an Allure™ PFP Propyl column for the analysis of SSRIs, but found it to be too highly retentive at any mobile phase pH: the amount of organic solvent needed was so high that selectivity under isocratic conditions was diminished.

Conclusion

These investigations revealed that polar stationary phases and reversed phase chromatography can replace ion-pairing chromatography for analyses of SSRIs, and stationary phases containing a spacer between the functional group and the silica surface provide greater retention. If the analysis is to be conducted at neutral pH, an Allure™ Basix column, containing a cyanopropyl phase, is a good choice. If the analysis is to be conducted at an acidic pH, however, the Ultra PFP column would be the better candidate.