Facilitating Fast Sample Preparation

BY L.P. RAMAN AND OKSANA PENEZINA, PHD

Technologies that work to improve sample preparation efficiency are always a welcome addition to any laboratory. This is particularly true when large numbers of samples are awaiting analysis, a situation that can prove quite tedious for manual operations. An individual using a standard protein precipitation protocol, for example, may have to follow six different steps before getting the sample ready for injection into an HPLC system. Moreover, net sample number is invariably lower when procedures are carried out manually. Our anecdotal experience suggests an operator can prepare about 50 HPLC samples in a working day.
Auto samplers have not only reduced the need for tedious sample preparation, they have also vastly increased sample preparation productivity. In fact, these technologies now permit HPLC systems to function around the clock.
Users looking for even further improvement on sample preparation efficiency can turn to a technology that combines two key steps into a single device that can be integrated with auto samplers.
Clifton, N.J.-based Whatman® Inc. has devised the Mini-UniPrep™ as a syringeless filter that permits sample filtration and storage in one unit (Fig. 1). The technology is designed as a preparation device that is used for clarification, particle removal or purification of samples for small volumes of liquids. The device also reduces the need for disposable lab supplies such as pipettes and transfer vials, while decreasing chemical usage.

Mechanism of Action and Applications
The syringeless filter consists of a chamber that can hold up to 500 µl of sample and a plunger that holds the filtration media. Sample preparation involves two steps. Users first place an unfiltered sample into the chamber and then compress the filter plunger. Clean filtrate then fills the reservoir from bottom up.
The fully assembled filter has the same dimensions and physical appearance of a 1.8-ml (12-mm) auto-sampler vial, and is designed to fit into most common auto samplers (Fig. 2). Accelerating sample preparation by 300 per cent, the filter features a pre-assembled construction, including a pre-attached septum. During filtration, air is displaced and upon operation completion, an evaporation seal is locked in.
The filter can be used for various sample preparation applications including:
l     Particle removal and clarification prior to HPLC injection
l     Content uniformity of pharmaceutical samples
l     Removal of protein precipitates from biological samples
l     Dissolution testing of tablets as required by regulatory agencies
l     Composite assays in developing new drug candidates
l     Solubility studies of pharmaceutical products

Case Study — Analysis of Capsaicin Cream
Background
Capsaicinoids are what make peppers hot. The chemical name is N-Vanillyl-8-methyl-6-(E)-nonemaide. There are two forms of capsaicinoids — capsaicin and di-hydrocapsaicin (DH). Capsaicin causes increased localized blood circulation and inhibits neurons that transmit sensation of pain. Hence, it is used in arthritis topical creams.
Whatman’s syringeless filter has been validated for quantitative measurement of capsaicin and capsaicin DH content in OTC arthritis cream. Capsaicin cream was chosen because capsaicin had been previously validated by HPLC testing, and it was felt that the sample preparation process could be adapted to the syringeless filter.
Work Flow Schematic
Figure 3 shows the protocol developed for analysis of capsaicin cream. In the experimental work, 50 or 100 mg of the sample was placed in the filter chamber, into which 400 µl of methanol was added and the unit’s plunger was inserted about one-fourth of the overall chamber length. This assembly was vortexed to mix the sample completely and was then incubated at 50 C for 30 minutes. The sample was allowed to cool down and the plunger was completely compressed. Filtered sample filled the device’s chamber— which bore a 0.45-µm polypropylene filter membrane — and the entire unit was placed in the auto sampler for analysis.
Using conventional protocol, the operator would have to place the capsaicin cream into a vial, add methanol and vortex. After sample incubation, the vial’s contents would be filtered through a syringe bearing a filter and the filtrate would be placed into an auto-sampler vial and analysed.

Results and Discussion
The cream was analysed at two different sample sizes (50 mg and 100 mg) and the tests were done in triplicate. The results are shown in Table 1.
A sample HPLC chromatogram from this study is shown in Figure 4. It shows the peaks for both capsaicin and di-hydrocapsaicin with a retention time of 6.2 and 9.7 minutes, respectively.
The filter could be easily adapted to analyse capsaicin and topical creams and the protocol provides repeatable results. While the label claim on the cream was a minimum of 0.075% capsaicin, the actual capsaicin content was 0.100% capsaicin by weight. The label claim simply stated 0.075% capsaicin, did not specify the units of measurement and did not state whether the measurement was just for capsaicin or if it included the di-hydrocapsaicin.
In the capsaicin cream analysis, only 400 µl of methanol was required for each test. Hence, the sample preparation uses fewer chemicals, and results in lower waste disposal costs for a laboratory because just the right amount of methanol is used to fit the auto sampler.
The syringeless filter is widely accepted within the HPLC field because it is compatible with most major auto samplers. There is less need for acquisition of supplies, storage of samples and disposal of chemicals and lab supplies.

L.P. Raman is the technical marketing manager for the Lab Sciences Business and Oksana Penezina, PhD is a senior scientist, Research and Development for Whatman® Inc.