UHPLC Analysis of Structurally Similar Compounds: A Case Study of Ephedrine and Related Analogs on a Carbon Column

Abstract

The Supel™ Carbon LC column was used to baseline resolve polar structural similar compounds, ephedrine and related compounds, under normal phase conditions and is compared to another carbon based column available in the market. The results indicate that the Supel™ Carbon LC column is a suitable option for forensic and pharmaceutical analysis in counterfeit drug detection.

Section Overview

• Introduction
• Experimental Conditions
• Results
• Conclusion
• Related Products

Introduction

Ephedrine and related analogs are used as the active components of many decongestants and bronchodilators. However, counterfeit versions of these products may have dangerous amounts of one or more analogs of ephedrine. Therefore, it is important to have analytical methods available to resolve these structurally similar compounds. The use of HPLC columns packed with carbon particles can fill this analytical need, as the polar retention effect on graphite (PREG) mechanism can discriminate compounds by differences in their three-dimensional structure. This application demonstrates the use of the Supel™ Carbon LC column to resolve ephedrine and its three structural analogs - norephedrine, pseudoephedrine, and synephrine, and compares it to another carbon based column available in the market.

Figure 1. Chemical structures of compounds used in the study.

Experimental Conditions

Table 1. Chromatographic conditions for the analysis of ephedrine compounds

Column(s):	Supel™ Carbon LC, 10 cm x 2.1 mm I.D., 2.7 µm (59986-U); Competitor T, 10 cm x 2.1 mm I.D., 3.0 µm
Mobile Phases:	[A] Water (0.1% (v/v) DFA); [B] Acetonitrile (0.1% (v/v) DFA)
Gradient:	Time (min) %A %B 0 95 5 1 95 5 6 85 15 10 95 5
Flow rate:	0.30 mL/min
Temperatures:	Column 35 oC; Autosampler 10 oC
Detector:	UV: 254 nm
Injection:	2 µL
Sample:	Ephedrine Test Mix, varied concentration, water (0.1% (v/v) DFA) - see Table 2

Table 2. Composition of Test Mix used in the study

	Test Mix Analyte/Concentration
1	Norephedrine (200 µg/mL)
2	Pseudoephedrine (150 µg/mL)
3	Ephedrine (200 µg/mL)
4	Synephrine (200 µg/mL)

Results

Figure 2. Analysis of ephedrine compounds on Supel™ Carbon LC.

Figure 3. Analysis of ephedrine compounds on a competitor carbon column.

Table 3. Retention time parameters for compounds seen in Figure 2

Peak	Compound	Ret. Time (min)
1	Norephedrine (200 µg/mL)	2.646
2	Pseudoephedrine (150 µg/mL)	3.166
3	Ephedrine (200 µg/mL)	3.665
4	Synephrine (200 µg/mL)	4.668

Table 4. Retention time parameters for compounds seen in Figure 3

Peak	Compound	Ret. Time (min)
1	Norephedrine (200 µg/mL)	2.406
2	Pseudoephedrine (150 µg/mL)	2.662
3	Ephedrine (200 µg/mL)	3.128
4	Synephrine (200 µg/mL)	4.447

Conclusion

This application has demonstrated the use of the Supel™ Carbon LC column in resolving ephedrine and related compounds. By employing the unique PREG effect, these compounds were baseline separated with excellent peak shape in under five minutes. In addition, the narrower particle size distribution and unique surface properties of the Supel™ Carbon LC column led to the generation of narrower peaks and better resolution compared to a column from an alternative vendor. The Supel™ Carbon LC column is an ideal choice for the resolution of mixtures of structurally similar compounds.