When high performance is not good enough

Could Ultra High performance liquid chromatography be the answer for time pressured analytical labs who find that HPLC is not up to the grade?

High performance liquid chromatography (HPLC) has been the workhorse technique in liquid phase separations for the past 30 years. One of the primary drivers for its continued use has been the evolution of the packing materials used to effect the separation.

An underlying principle of HPLC dictates that as column packing particle size decreases, efficiency and thus resolution also increases. However, HPLC system designs have always struggled with the ability to take advantage of the smallest particle sizes. Not only have small particle sizes required systems to operate at backpressures above current limitations (e.g. 6000 psi/400 bar) and possess much smaller system dwell volumes that do not compromise gradient performance but have also required high speed detectors capable of working with peaks that may be only a few seconds wide.

However, there has been a major development in packing materials that lets the chromatographer work at higher efficiencies with a much wider range of linear velocities, flow rates and backpressures to obtain superior results in speed and resolution.  This new development is termed sub 2-micron particle liquid chromatography and provides the kind of quality results and throughput that laboratories often need to meet their productivity requirements.

Sub 2-micron particle liquid chromatography is based on smaller particles, so that speed and peak capacity (number of peaks resolved per unit time) can be extended to new limits for faster method development. The method makes it possible to run separations on shorter columns and/or with higher flow rates for superior resolution and sensitivity with increased speed.
The ACQUITY UPLC system achieves
narrower and sharper peaks and greater
peak capacity.

Sub 2-micron particle liquid chromatography achieves 10X better throughput, almost 2X better resolution, and 3X better sensitivity over traditional HPLC.

The analytical challenge
Case in point is a high throughput veterinary drug residue testing laboratory in the UK that took a 25-minute LC/MS run down to four to five minutes resulting in a five-fold improvement in overall run time and a tripling of the lab’s output using sub 2-micron particle liquid chromatography. The laboratory tests domestically-grown poultry and livestock as well as imported food of animal origin for banned substances and residues of legal veterinary drugs and admits approximately 35,000 samples annually.

Farm-raised poultry and livestock regularly receive drugs, both licensed and illegal, to protect them from disease and spur their growth. Through ignorance, carelessness or deceit on the part of growers, poultry and livestock prepared for export can contain banned substances or legal drugs at concentrations that exceed maximum allowable European Union levels.

Whenever possible, inexpensive, rapid screening tests are employed. A positive result for one of these screening tests (a presumptive positive) means that the sample may contain a banned drug or a licensed drug at a concentration above the maximum allowable level. 
                                                                         
Livestock prepared for export
can contain banned substances
and samples kept for long
peroids can spoil
When the lab suspects that a farmer is using veterinary drugs improperly, it has the authority to detain a shipment of marketable product until the tests have been completed. For these samples, results must be delivered within five days. If a test takes more than five days, and the results come back negative—and the carcass spoils—the grower can sue the laboratory for economic damages.

If the lab knows it won’t meet the deadline, it has no recourse but to release the shipment and allow potential health risks into the food supply. At times the backlog on an instrument can exceed 10 days, from which, the lab manager admits, it can be very difficult to recover. “Nothing is as infuriating as to be sitting on a bunch of samples coming up to their deadline, and knowing that you’re not going to be able to get them analysed in time.”

UPLC solution
In 2005, the lab acquired a sub 2-micron particle liquid chromatography instrument - a Waters Corporation Ultra Performance LC (UPLC) - that was installed on the front end of a Waters tandem quadrupole mass spectrometer. According to the lab director, it took about three hours to transfer the 24-minute nitrofuran HPLC separation to a UPLC 4.5-minute separation—one that he says gives him “much better resolution.”

The speed of the UPLC instrument combined with the tandem quadrupole mass spectrometer’s exceptionally fast scanning and rapid switching between ionization modes allows the lab to complete runs in less time —and to expand the scope of its multi-residue testing by being able to detect a much larger number of drugs per analytical run. For example, 53 MS/MS transitions can be measured in a single 4.5-minute UPLC run.

UPLC business benefit
The lab can now conduct two or three batches of samples in an eight-hour period —tripling it output. The laboratory is also developing new methods for UPLC based on the speed of the technology and the versatility of the stationary phase. For example, two corticosteroids—dexamethansone and hexamethasone—are notoriously difficult to separate with conventional C18  HPLC. They have the same mass and produce the same fragment ions, and cannot be distinguished even by MS/MS.

The lab manager performed the separation using UPLC technology and achieved baseline resolution of the two compounds in less than four minutes. In his opinion, “these columns can be used for a fairly wide range of compounds, which is very useful because I don’t have to chain the methods together for overnight scheduling, and I can analyze chloramphenicol, nitrofurans and steroids in one overnight run.”

Probably the largest benefit is in methods development. In developing any HPLC method, a typical reversed-phase HPLC gradient method tends to take anywhere from 25 to 30 minutes. With 40-minute run times (allowing for re-equilibration), chromatographic method development can be a very slow process.

“If UPLC runs are four to five minutes, essentially you don’t have time for a cup of tea before you know whether your run worked or not and you are ready to do your next injection. At least with UPLC, you know very quickly. What we’ve found is that methods development is much more rapid with UPLC than with traditional HPLC,” according to the lab manager.

By
Kelly A. Johnson, Pharmaceutical Marketing Manager, Waters Corporation