Evaluation of Waters’ Acquity UPLC

Amanda Plumb, BASi UK

Introduction

The Waters’ UPLC has been evaluated at BASi UK over the period 23rd November to 10th December, 2009. All the experimental work detailed in this evaluation was conducted by Amanda Plumb. This equipment exists in BASi’s West Lafayette laboratories.

Per Waters’ Aquity web site:

The ACQUITY UPLC® System will eliminate significant time and cost per sample from your analytical process while improving the quality of your results. By outperforming traditional or optimized HPLC, the system allows chromatographers to work at higher efficiencies with a much wider range of linear velocities, flow rates, and backpressures.

UPLC® Technology, which has been adopted successfully in laboratories around the world for the most demanding separations, is a highly robust, dependable, and reproducible system. What differentiates the system’s holistic design is Waters’ patented sub-2-μm hybrid particle chemistry, which offers significant benefits over today's HPLC systems equipped with standard 5-μm particle chemistries.

WATERS ACQUITY UPLC

An evaluation plan was prepared, discussed and agreed with Waters. The plan was to assess various aspects of the chromatography by preparing laboratory samples from different assays

Summary

Three methods that had been previously developed for HPLC were transferred to the Acquity. Diltiazem was chosen because it is a liquid/liquid extraction, Resveratrol because of resolution problems and Cortisol was chosen because of the long run time and suppression problems of 6 β- hydroxycortisol. Although the software is not compatible with Analyst 1.4.1 the Acquity has the ability to improve efficiency and productivity by:

• Shorter run times.
• Better resolution.
• Increased sensitivity.
• Increased sample through put.
• Reduced method development time.
• Reduced costs of laboratory consumables as sample protein crash is preferred.

Diltiazem Human Plasma Assay

The current method is a robust liquid/liquid extraction with no known problems.

Points to Note

• Peak shape.
• Overall run time 4 min.

The Assessment of this Assay on the ACQUITY Was to

• Introduce ‘dirty’ samples from a protein crash experiment. This would be the preferred option, as this decreases the sample preparation time.
• Reduce overall run time.
• Improve peak shape.
• Assess carryover.

Findings
Injection of ‘dirty’ samples from a protein crash preparation did not have any effect on the performance of the ‘narrow bore system’ and therefore would be the preferred technique reducing laboratory sample preparation time.

The overall run time was reduced to 1.5 minutes and the peak shape was improved, which ultimately would improve the sensitivity.

Diltiazem 1ng/mL using HPLC

Diltiazem 1ng/mL using UPLC

Resveratrol  Human Plasma Assay

The current method detects cis- and trans- resveratrol and sulphate and glucuronide metabolites.

Points to Note

• Resolution of cis- and trans- resveratrol was a problem during HPLC development.
• Overall run time 8 min.

The assessment of this assay on the ACQUITY was to

• Improve resolution of cis- and trans- resveratrol.
• Reduce overall run time.
• Improve peak shape for all compounds.
• Assess carryover.

Findings
The resolution was improved 1.65 minutes and 2.15 minutes from 3.06 minutes and 3.58 minutes for cis and trans respectavly.

The overall run time was reduced to 6 minutes, however the glucuronide peak was wider than previously developed.

There was a limited amount of time available to further develop the UPLC chromatographic method. However it is anticipated that good chromatography could be produced with the columns and mobile phases available.

No carryover was seen with this method (steel needle used).

Resveratrol 250ng/mL using HPLC

Resveratrol 250ng/mL using UPLC

Cortisol and 6 β-hydroxycortisol Urine Assay

This current method involves aliquots of samples diluted in methanol/water prior to injection.

Points to Note

• The chromatographic method involves a gradient elution with a run time of 8 minutes and an overall time of 15 minutes between injections.
• The additional 7 minutes is required to allow the system to equilibrate between injections.
• On occasions suppression is noted during the retention time of 6 β-hydroxycortisol resulting in low results for mid and high QCs.

The assessment of this assay on the ACQUITY was to

• Reduce the overall run time.
• Assess the suppression.
• Assess carryover.

Findings
The chromatographic run time was vastly improved from 15 minutes to 3 minutes. This was sufficient time to allow equilibrium between injections.

The cortisol quality controls however gave inconsistent results, which may be attributed to suppression as there is some peak interference. The retention time of the cortisol was further decreased to reduce interference but sensitivity was poor.

There was a limited amount of time available to further develop the UPLC chromatographic method. However it is anticipated that good chromatography could be produced with the columns and mobile phases available. In addition, as the run time has been dramatically reduced, developing an analytical method takes less time as more injections can be undertaken throughout the working day.

There were problems initially with carryover for both analytes. The weak injection wash was changed from 5% CH3CN to 100% CH3CN and the ETFE needle was changed to a steel needle. No carryover was seen after these changes.

Cortisol 1ng/mL using HPLC
A further 7 minutes is required to allow the mobile phase to equilibrate using the HPLC method.

Cortisol 1ng/mL using UPLC
This chromatogram includes the equilibration time.

Conclusion

The Acquity is capable of injecting samples that have been prepared for injection using protein crash techniques, which is the preferred option in other laboratories using this system. This dramatically reduces the laboratory preparation time and therefore reduces overheads. The reduced run times increases capacity during production and also greatly assists in what can be achieved during method development in a working day.