Advances in Bioanalytical Applications of HPLC and LC-MS

Life sciences applications are a major driving force for the high performance liquid chromatography (HPLC) and the liquid chromatography/mass spectrometry (LC-MS) market. Liquid chromatography is commonly used in biotechnology. The LC-MS technique, which combines the separation capabilities of liquid chromatography with the mass analysis capabilities of mass spectrometry, is a powerful tool for pharmacokinetics and proteomics.At Pittcon 2006, the Monday afternoon poster session titled "Bioanalytical Applications of LC and LC-MS" showcased many different research advances in the use of these techniques.

RP-HPLC in Proteomics

Reversed phase HPLC is by far the most common form of HPLC, and is an important technique for the analysis and purification of proteins and peptides. While small organic molecules can be eluted by the distribution mechanism, large molecules such as proteins and peptides are eluted by adsorption-desorption. This means that pore size of the column packing material plays a key role in determination of the resolution.

Users face limitations when analyzing samples containing complex mixtures of analytes that are similar or closely related. While wide-pore silica columns provide the resolution needed for these applications, only silica materials with the majority of pore diameters in the 250-350 angstrom range work for analytes of molecular weight more than 3000. Cathy Gross and her colleagues from Restek Corporation (Bellefonte, Pennsylvania) have developed a 300-angstrom silica column that ranked highest in retention and selectivity and produced the best peak symmetry when compared with four commercially available C18 wide-pore HPLC columns. The researchers attribute their column's performance to "good effective resolution, thorough base deactivation … large available surface area, and a highly desirable pore volume and narrow pore diameter distribution."

Researchers at YMC Co., Ltd. (Kyoto, Japan) demonstrate the use of a dynamic axial compression column with alkyl-bonded silica gel packing products to purify proteins and peptides. Using the column, they say the compounds are easily fractioned by changing the separation conditions, such as packing material, column length and volume. Testing the column for separation of insulin and other proteins, they report a good peak shape and separation for an optimum pore size of the gel.

Affinity chromatography in proteomics

Immobilized metal affinity chromatography (IMAC), a technique based on covalent binding between specific amino acids and various immobilized metal ions, is used routinely to purify proteins. But fractions collected from IMAC cartridges usually contain impurities, highlighting the need for a high resolution IMAC-HPLC column. Patrick McCarthy and his colleagues at Dionex Corporation (Sunnyvale, California) engineered the surface of 10 micron nonporous polymeric beads with isolated, metal-containing nanoparticles that act as IMAC interaction sites. The column was able to separate prion peptides differing in number of octapeptide repeat units. Unexpectedly, it also separated prion-related peptide mixtures where the peptides contained mutation. The researchers conclude that their IMAC column "is stable at high pressure, supports multiple injections without recharging … and can be recharged with metal for extended use."

Hydrophobic interaction chromatography in proteomics

Yet another technique popular for protein purification is hydrophobic interaction chromatography. A second poster from Dionex, authored by Srinivasa Rao, describes a new silica based HIC stationary phase with multifunctional amide group attachments. The researchers modify the chemistry of this phase by including hydrophobic and hydrophilic ligands, which improves the hydrolytic stability of silica support in the aqueous media. They have demonstrated the use of this column on mixtures of peptides, various proteins, monoclonal antibodies, and human skeletal muscle cell extracts.

LC-MS-MS for in-vivo trace level drug detection

In the past, researchers have used ion trap based mass spectrometry under multiple reaction monitoring to detect low levels of drugs present in the body. Researchers at the Nicholas Piramal Research Center (Mumbai, India), have now used LC-MS-MS to detect trace levels of the anti-cancer drug P276-00 and its pro-drugs P320A-04 and P1275B-05 in tumors. Venkat Manohar and his colleagues administered the drugs orally to mice with non-small-cell lung carcinoma for 12 days. They then sacrificed the mice and analyzed their plasma and tumors. Their study shows that the pro-drugs were converted to the parent drug P276-00. This will help in fine-tuning the dose of anti-cancer drugs, write the researchers, which means that they can maximize the drug efficiency while minimizing its toxicity.