Increased Sensitivity, Improved Resolution and Faster Analysis Times For Compendial Methods: Determination of Impurities and Related Substances Using Newly Developed 2.6 µm Core-Shell Kinetex LC Columns

Introduction

Recently a newly developed Kinetex 2.6 μm core-shell chromatographic particle has been commercialized that offers the performance benefits of sub-2 μm fully-porous particles but at substantially lower operating pressures. To demonstrate the performance benefits of this new core-shell technology, a Kinetex 2.6 μm Core-Shell C18 column was compared with a fully-porous 5 μm C18 column referenced in EP [European Pharmacopoeia (Ph. Eur.)] Monograph 0703 for atenolol and related substances on a conventional HPLC instrument with an upper pressure limit of 400 bar.

First, to demonstrate equivalency, a Kinetex column of similar dimension to the column referenced was operated under the conditions specified in the monograph. Then the Kinetex column was operated at a higher flow-rate still within the 50% adjustment allowed by the EP for meeting system suitability. The Kinetex column achieved a 65% faster analysis time (3× productivity improvement) and significantly improved resolution and sensitivity versus the EP referenced fully-porous 5 μm column.

Atenolol and Related Substances: European Monograph 0703

Experimental

Columns used: A fully-porous 5 μm C18 125 × 4.0 mm column (as specified by the monograph) was compared with a Kinetex 2.6 μm C18 100 × 4.6 mm column (the closest available dimension).

Instrumentation: Agilent 1100 LC system (Agilent Technologies Inc., Palo Alto, California, USA) equipped with a Quaternary gradient pump, autosampler, column oven and variable wavelength detector.

Mobile phase preparation:

A: Dissolve 3.4 g of potassium dihydrogen phosphate in 1 L of DI water, adjust the pH to 3.0 using phosphoric acid.

B: Mix 180 mL of methanol and 20 mL of tetrahydrofuran and 800 mL of A.

C: Dissolve 1.0 g of sodium octanesulphonate and 0.4 g of tetrabutyl ammonium hydrogen sulphate in 1 L of B.

Sample preparation:

Atenolol certified reference standard (CRS) for system suitability (containing atenolol and impurities B, F, G, I and J) was obtained from the European Pharmacopoeia. 5 mg of atenolol CRS was dissolved in 2.5 mL of the mobile phase.

Atenolol analysis method:

The monograph calls for isocratic elution with 100% of mobile phase as prepared above at 0.6 mL/min. Column temperature kept at ambient and UV detection wavelength set at 226 nm.

Results and Discussion

Following the methodology described in European Pharmacopoeia Monograph 0703 and using a fully-porous 5 μm C18 125 × 4.0 mm column as referenced in the method, a chromatogram similar to that of the specimen chromatogram provided with the atenolol CRS was obtained (Figure 1).

Figure 1: Atenolol CRS: Fully-porous 5 μm C18 125 × 4.0 mm at 10 μL injection 0.6 mL/min.

A Kinetex 2.6 μm C18 100 × 4.6 mm column (the closest available dimension) was used according to the conditions specified in the monograph. The resulting chromatogram demonstrated equivalency for selectivity and also demonstrated significantly improved sensitivity (Figure 2).

Figure 2: Atenolol CRS: Kinetex 2.6 μm C18 100 × 4.6 mm at 10 μL injection 0.6 mL/min.

Table 1 summarizes the data comparing the Kinetex column to the fully-porous 5 um column at the specified flow-rate of 0.6 mL/min. The monograph requires resolution between impurities I and J of at least 1.4. Because of the significantly narrower peaks generated by the higher efficiency Kinetex column, a substantial improvement in resolution between impurities I and J was achieved with Kinetex.

Table 1: Equivalency study.

Sensitivity was also significantly improved for all impurities as a result of the Kinetex column generating narrower and taller peaks. Signal-to-noise ratios for the early eluting impurities were increased by roughly a factor of 2 and by roughly a factor of 3 for later eluting impurities.

With 10 μL of the atenolol CRS injected, the fully-porous 5 μm C18 column generated a signal-to-noise ratio of 12.3 for impurity I. Multiple 10 μL injections were performed on the fully-porous 5 μm C18 column and the resulting %RSD value for peak area of impurity I was 2.77.

In comparison, with 10 μL of the atenolol CRS sample injected on the Kinetex 2.6 μm core-shell C18 column, a signal-to-noise ratio of 28.9 was observed for impurity I. Multiple 10 μL injections were performed on the Kinetex column and the resulting %RSD value for peak area of impurity I was 1.61.

The higher signal-to-noise ratios and subsequently lower %RSD values observed with the Kinetex core-shell technology represent a significant performance advantage. It should be noted that the Kinetex column generated a system pressure comparable to the 5 μm column under these conditions.

Significantly Faster Analysis Times with Kinetex

Kinetex 2.6 μm core-shell particles are capable of maintaining high efficiencies (low plate heights) with increasing mobile phase flow-rates. This is because of favourable physical, kinetic and thermodynamic properties attributed to core-shell particles. Shorter analysis times may be achieved with Kinetex either by reducing the length of the column or increasing the mobile phase flow-rate (or a combination of both) without significantly compromising chromatographic performance.

Table 2: Acceptable modifications for meeting system suitability.

Following European Pharmacopoeia guidelines, the extent to which the various parameters of a chromatographic test may be adjusted to satisfy system suitability (when replacing one column with another of the same type, for example) is summarized in Table 2. Staying within these guidelines, the Kinetex 2.6 μm core-shell C18 column was run according to the conditions specified in the monograph, but with 50% increase in the flow-rate (from 0.6 mL/min to 0.9 mL/min). Total analysis time was shortened from over 33 minutes to just under 12 minutes (Figure 3).

Figure 3: Atenolol CRS: Kinetex 2.6 μm C18 100 × 4.6 mm at 10 μL injection 0.9 mL/min.

Conclusions

Laboratories performing routine API and related substance analysis with traditional fully-porous LC columns can benefit from the increased speed, resolution and sensitivity that Kinetex 2.6 μm core-shell columns provide without having to replace existing instrumentation with ultra-high pressure capable LC systems. Faster analysis times resulting in higher throughput and productivity can be achieved with Kinetex columns with minimal changes to validated methods by employing shorter length columns and/or higher mobile phase flow-rates without sacrificing performance. Improved resolution and higher sensitivity resulting from narrower and taller chromatographic peaks generated by Kinetex columns allow for more precise detection and quantification of low level impurities in routine operation.

Table 3: Improvements to the monograph.

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