Agilent in Pharmaceutical Analysis, Issue 25 

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Home – Special Offers CUSTOMER STORY: Fast analysis with the Agilent 1200 Series RRLC system cuts method development time by 50 percent. FDA releases draft guidance for residual solvent analysis (USP<467>). HPLC-Chip/MS – Unprecedented sensitivity improves data quality and substantially reduces cost in animal PK studies.

Higher sensitivity, robustness, and speed with new 1200 Series variable wavelength detectors. CUSTOMER STORY: Compound repository integrates with BioCel Automation System to accelerate drug discovery in Australia.

HPLC-Chip/MS – Unprecedented sensitivity improves data quality and substantially reduces cost in animal PK studies

By Stephan Buckenmaier
Agilent Application Scientist

The creation of a new pharmaceutical drug consumes vast amounts of capital. The competition is intense and cost-savings may become essential, even for leading pharmaceutical companies. Considerable savings can be achieved by the miniaturization of laboratory processes. A prime example for this situation exists in the drug metabolism and pharmacokinetics (DMPK) laboratory. Here, reductions in rodent size and numbers enable you to use less of the active pharmaceutical ingredient per PK study, which ultimately leads to substantial cost-savings. Yet, small animals can only donate a limited sample volume, in particular when serial sampling studies are performed to increase PK data quality.

This puts the burden on the analytical chemist to choose the appropriate analytical platform that provides the detection sensitivity to handle smallest sample quantities – a challenge that drove Agilent to develop the HPLC-Chip/MS system, first launched in 2005, for high-sensitivity analyses. the HPLC-Chip Cube (MS interface) can be mounted on any of the Agilent LC/MS systems. For instance, Figure 1 shows the 1200 Series Nanoflow HPLC-Chip/6410 Triple Quadrupole LC/MS system.

	Figure 1. Agilent 1200 Series Nanoflow HPLC-Chip/6410 Triple Quadrupole LC/MS system.

	Figure 2. Chip schematic (A) and blueprints of ultra-high capacity small molecule HPLC-chip (UHC-chip) obtained using light microscopy (B). (Click here to see this image larger.)

In 2008, the ultra-high capacity small molecule HPLC-chip (UHC-chip) was added to the HPLC-chip portfolio. It was especially designed for analyses of small molecules covering a broad range of polarities. Figure 2 shows a schematic. The UHC-chip incorporates a 500 nL enrichment column, a 150 mm x 75 µm analytical column, and the nanoelectrospray emitter. The insertion into the system (Figure 1) is fully automated: First the HPLC-Chip/MS interface establishes all connections between HPLC-chip, nanoflow pump, and autosampler; then the chip automatically moves into spraying position and is ready to use within minutes, for automated pre-concentration and analysis of your samples. View the online video that shows how the entire system works.

High sensitivity quantitation of compounds covering a wide range of polarities

A recent Application Note co-authored by Novartis AG describes the technical evaluation of this new UHC-chip for the quantitative analysis of pharmaceuticals using an Agilent 6410 Triple Quadrupole LC/MS.

	Figure 3. Chromatograms obtained for a four-compound mix covering a hydrophilicity range logP 0 to 4.2 using narrow bore LC/MS (A) and UHC-chip/MS (B). (Click here to see this image larger.)

Quantitative pre-concentration on the chip-integrated enrichment column was demonstrated using pharmaceuticals covering a broad range of hydrophilicities. Figure 3 shows illustrative chromatograms obtained from the analysis of a four-compound mixture covering logP values from 0 to 4.2. Figure 3A was obtained using narrow-bore LC and an atmospheric pressure ionization (API) electrospray source, and Figure 3B was obtained with the UHC-chip. Both analyses used the same 6410 Triple Quadrupole. The 100-fold increase in sensitivity of the UHC-chip/MS system allowed for detection of 10 fg/µL levels of drugs in blood plasma. The Application Note further shows excellent retention time and peak area precision, chip-to-chip reproducibility and chip lifetime with aqueous and blood plasma samples. (Read the full Application Note: 5989-7967EN.)

Further work demonstrates the quantification of a test drug extracted from only 10 µL of whole blood. After simple sample preparation, the analysis employed the UHC-chip/6410 Triple Quadrupole. Figure 4 shows a plot of the proportional relationship between peak area and concentration of the test compound nortriptyline over the range 0.05 to 15 ng/mL of whole blood. (Read the full Application Note: 5989-9896EN.)

	Figure 4. Plot of peak area versus concentration obtained for nortriptyline in the range 0.05 to 15 ng/mL of whole blood following extractions from 10 µL blood volumes (A). Chromatograms for blank extract and lowest concentration analyzed (B). (Click here to see this image larger.)

The HPLC-Chip/6410 Triple Quadrupole system opens up the possibility to quantify drugs from tremendously reduced sample volumes. This can increase PK data quality due to the ability to serially sample blood from the same small-size rodent without jeopardizing its physiological welfare and without increasing recovery periods. The use of small sample volumes aligns with ethical arguments, can be important in other areas (such as pediatric studies), and results in substantial cost savings. For more information, visit our product page for the 1200 Series HPLC-Chip/MS system.