• Application Note

Assessing the Impact of Increased Pre-Column System Volume on Peak Shape for High Organic Diluent Samples Using UHPLC

Assessing the Impact of Increased Pre-Column System Volume on Peak Shape for High Organic Diluent Samples Using UHPLC
  • Chris Henry
  • Paul D. Rainville
  • Patricia R. McConville
  • Paula Hong
  • Waters Corporation
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Abstract

The Ability to successfully run compendia methods on an ACQUITY UPLC H-Class System is described utilizing high percentage organic solvent as diluent without peak distortion.

To investigate the means for a simple solution to overcome the effect of strong solvents in the sample diluent, five United States Pharmacopeia (USP) methods were selected (i.e. acetaminophen, itraconazole, ketoconazole, loratidine, and bicalutamide) which require sample diluent organic levels ranging between 67%–100% organic.

All methods were conducted on the ACQUITY UPLC H-Class System with dispersion volumes calculated for every configuration used. These were combined with structured and iterative modifications to increase pre-column volume to assess the impact of additional pre-column volume on peak symmetry problems brought on as a result of high organic diluents.

Benefits

Ability to successfully run compendial methods on an ACQUITY UPLC H-Class System utilizing high percentage organic solvent as diluent without peak distortion.

Introduction

Ideally when running a chromatographic method, the sample diluent composition should be as close to the method starting conditions as possible. This is done in order to minimize the possibility of band spreading and peak distortion from sample solvent effects, which can lead to poor peak symmetry, peak splitting, or unusable data.

The cause for these effects is a difference in elutropic strength between the diluent and the mobile phase. Peak broadening and shape abnormalities generally get worse as the diluent becomes stronger than the mobile phase.1-2

In fact, it is widely recognized that the sample injected should ideally be dissolved in the starting mobile phase conditions. However the pre-treatment of a given sample often ends in the analytes dissolved in a solvent composition very different from that used in the mobile phase. In order to prevent solubility and poor peak shape problems, many protocols require evaporation of the sample solvent in pre-treatment and reconstitution in mobile phase. However, this added step is a time consuming process that often takes longer than the HPLC analysis.1

The practice widely recommended is to avoid stronger solvents than the mobile phase to dissolve samples and standards. The underlying assumption is that an injection solvent stronger than the mobile phase can interfere with the adsorption of the sample at the column head, especially when large injection volumes are used.2 Unfortunately, in practice this is not always possible as sample solubility often dictates the amount of organic content needed to ensure complete dissolution.

With older, higher dispersion volume LC systems, this phenomenon is less problematic due to sufficient pre-column sample/solvent/mobile phase mixing which mitigated peak problems brought about by solvent effects.

However, for modern lower dispersion UHPLC systems, high organic diluents can be problematic when injected in larger volumes and can result in poor peak symmetry or splitting.

To understand this phenomenon and investigate the means for a simple solution to overcome the effect of strong solvents in the sample diluent, five United States Pharmacopeia (USP) methods were selected (i.e. acetaminophen, itraconazole, ketoconazole, loratidine, and bicalutamide) which require sample diluent organic levels ranging between 67%–100% organic.

All methods were conducted on the ACQUITY UPLC H-Class System with dispersion volumes calculated for every configuration used. These were combined with structured and iterative modifications to increase pre-column volume to assess the impact of additional pre-column volume on peak symmetry problems brought on as a result of high organic diluents.

Experimental

The methods were selected from the USP and the standards were analysed six times (n=6). The standards were prepared as per USP methods (Table 1).

The methods were run on an ACQUITY UPLC H-Class System with and without a 50 µL loop inserted between position six of the injector pod located in the sample manager and the column inlet tubing as shown in (Figure 1). Under both conditions, the chromatography was appraised visually, the mean of the peak asymmetry at 4.4% peak height and the % RSD of the peak area were compared as indicators of chromatographic performance in this study.

All methods with the exception of loratidine utilized the column manager (CM-A). Loratidine, required the CH30-A ACQUITY Column Heater to accommodate the 25 cm HPLC column detailed in the USP method. 

Table 1. Diluent content of standards analyzed.
Figure 1. Additional 50 µL pre-column loop.

The materials used for analysis include (analytical standards in bold):

  • Ketoconazole: Sigma-Aldrich Lot SLBR1290V
  • Itraconazole: EP reference standard Y0001100 Batch 2.0
  • Bicalutamide: USP reference standard – Lot G01298
  • Loratidine: Sigma-Aldrich – Lot LRAA9165
  • Haloperidol: Sigma-Aldrich – Lot LRAA7399
  • Acetaminophen: Sigma-Aldrich – Lot SLBM5923V
  • Water: ELGA Purelab
  • Acetonitrile: Fisher Chemicals Optima LC-MS grade – Lot 1731013
  • Methanol: Honeywell LC-MS Chromasolv – Lot SZBG246C
  • Trifluoroacetic acid: Sigma-Aldrich – Lot 6942V
  • Monobasic sodium phosphate: Sigma-Aldrich – Lot BCBV1183
  • Dibasic sodium phosphate (anhydrous): Fisher Chemicals – Lot 164693
  • Tetrabutylammonium hydrogen sulfate: ACROS Organics – Lot A0375G05
  • Pentane sulphonic acid: ACROS Organics – Lot A0378319

Prior to analysis, the dispersion volume for all configurations were calculated using a blank union (P/N 700002636) under the following conditions (Table 2).

Table 2. Method for calculating extra-column volume in LC systems.

Extra column dispersion (μL) was calculated as the peak width (minutes) at 4.4% peak height (5σ) multiplied by the flow rate (μL per minute).

Figure 2. Overlay of caffeine standard using all configurations.
Table 3. Calculated peak width and dispersion for all configurations using caffeine standard.

Results and Discussion

The inclusion of additional pre-column volume, i.e. a 50 μL loop on the ACQUITY UPLC H-Class System, has significantly improved the peak shape of acetaminophen (Figures 3–4/Tables 5–6), itraconazole (Figures 5–6/Tables 8–9), haloperidol (Figures 7–8/Tables 11–12) and loratidine (Figures 9–10/Tables 15–16). Peak symmetry improved noticeably for acetaminophen (0.2 to 1.0) and haloperidol (0.1 to 1.0). These compounds also exhibited an improvement in peak area % RSD (Table 22).

USP Methods Improved Using Additional Pre-Column Volume

Example 1: Acetaminophen (assay) – Diluent: 100% MeOH

Table 4. Acetaminophen USP method.
Figure 3. Acetaminophen USP method without additional pre-injection volume. Acetaminophen overlay (n=6) chromatogram and result table without loop fitted.
Table 5. Acetaminophen USP method without additional pre-injection volume. Acetaminophen overlay (n=6) chromatogram and result table without loop fitted.
Figure 4. Acetaminophen USP method with additional pre-injection volume. Acetaminophen overlay (n=6) chromatogram and result table with loop fitted.
Table 6. Acetaminophen USP method with additional pre-injection volume. Acetaminophen overlay (n=6) chromatogram and result table with loop fitted.

Example 2: Itraconazole

Itraconazole (imps) – Diluent: 0.4% HCl in MeOH    

Table 7. Itraconazole USP method.
Figure 5. Itraconazole USP method without additional pre-injection volume. Itraconazole overlay (n=6) chromatogram and result table without loop fitted.
Table 8. Itraconazole USP method without additional pre-injection volume. Itraconazole overlay (n=6) chromatogram and result table without loop fitted.
Figure 6. Itraconazole USP method with additional pre-injection volume. Itraconazole overlay (n=6) chromatogram and result table with loop fitted.
720006242en-t9-with-loop

Example 3: Haloperidol

Haloperidol USP method (imps) – Diluent 100% MeOH    

Table 10. Haloperidol USP method.
Figure 7. Haloperidol USP method without additional pre-injection volume. Ketoconazole overlay (n=6) chromatogram and result table with loop.
Table 11. Haloperidol USP method without additional pre-injection volume. Ketoconazole overlay (n=6) chromatogram and result table with loop.
Figure 8. Haloperidol USP method with additional pre-injection volume. Ketoconazole overlay (n=6) chromatogram and result table with loop.
720006242en-t12-with-loop

Example 4: Loratidine

Loratidine USP method (imps) – Diluent 100% MeOH

Table 13. Loratidine USP method.
Figure 9. Loratidine USP method without additional pre-injection volume. Loratidine overlay (n=6) chromatogram and result table without loop fitted.
Table 14. Loratidine USP method without additional pre-injection volume. Loratidine overlay (n=6) chromatogram and result table without loop fitted.
Figure 10. Loratidine USP method with additional pre-injection volume. Loratidine overlay (n=6) chromatogram and result table with loop fitted.
720006242en-t15-with-loop

Bicalutamide (Figures 11–12/Tables 17–18) showed no significant peak distortion without the loop and no significant impact (positive or negative) with the use of the additional pre-column volume. This may be due to the relatively low concentration of organic diluent (i.e. 67%).

USP Methods Unaffected Using Additional Pre-Column Volume

Example 5: Bicalutamide

Bicalutamide USP method (imps) – Diluent 67% MeOH/0.01%TFA

Table 16. Loratidine USP method.
Figure 11. Bicalutamide impurities USP method without additional pre-injection volume. Bicalutamide overlay (n=6) chromatogram and result table  without loop fitted.
Table 17. Bicalutamide impurities USP method without additional pre-injection volume. Bicalutamide overlay (n=6) chromatogram and result table  without loop fitted.
Figure 12. Bicalutamide impurities USP method with additional pre-injection volume. Bicalutamide overlay (n=6) chromatogram and result table  with loop fitted.
720006242en-t18-with-loop

Example 6: Ketoconazole

Ketoconazole USP method (imps) – Diluent 100% MeOH

Table 19. Ketoconazole USP method.

Ketoconazole (Figures 13–14/Tables 20–21) also showed no significant peak distortion without the loop and no significant impact (positive or negative) w ith the use of the additional pre-column volume despite having 100% methanol as diluent. This may be explained by the presence of organic component in both Mobile phase A and B, facilitating sufficient mixing prior to injection onto the column. Further work would be required to verify this theory.

Figure 13. Ketoconazole USP method without additional pre-injection volume. Ketoconazole overlay (n=6) chromatogram and result table  without loop fitted.
Table 20. Ketoconazole USP method without additional pre-injection volume. Ketoconazole overlay (n=6) chromatogram and result table  without loop fitted.
Figure 14. Ketoconazole USP method with additional pre-injection volume. Ketoconazole overlay (n=6) chromatogram and result table with loop.
Table 21. Ketoconazole USP method with additional pre-injection volume. Ketoconazole overlay (n=6) chromatogram and result table with loop.

All results are summarized in Table 22.

Table 22. Result summary of all compounds test with and without 50 µL increase in  pre-column volume.

Conclusion

The addition of pre-column volume by utilizing a 50 µL loop has had a significant impact on the peak shape and peak area %RSD for acetaminophen, itraconazole, haloperidol, and loratidine indicating  that it is a valid strategy for overcoming peak distortions when working with high organic diluent on a low dispersion LC system.

This solution would enable customers bound by compendial method parameters to overcome unsatisfactory/non-usable data with a  simple fix that would be fully supported in a regulatory compliant  laboratory environment. 

References

  1. Eric Loeser, Patrick Drum: Using strong injection solvents with 100% aqueous mobile phase in RP-LC. J.Sep.Sci 2006, 29, 2847–2852.
  2. Sonia Keunchkarian, Mario Reta, Lilian Romero, Cecilia Castells: Effect of sample solvent on the chromatographic peak shape of analytes eluted under reversed-phase liquid chromatographic conditions. Journal of Chromatography A.

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