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Mar 1, 2012 (Vol. 32, No. 5)

Optimizing LC/MS for Drug Bioanalysis

  • Click Image To Enlarge +
    Shimadzu’s 2D-LCMS-IT-TOF system: The pink dotted line region is the first dimension LC system, and the green dotted line region is the second dimension LC/MS system.

    Bioanalysis of drugs in development is shifting toward high-sensitivity and high-throughput methodologies based on LC/MS platforms capable of drug and metabolite identification and quantification, with the ability to detect low-volume analytes and to discriminate between closely related molecules in complex biological samples. At the upcoming “Pittcon” conference, several presentations will focus on strategies for optimizing the use of LC/MS for analyzing small molecule and biological drugs in development. The tools and techniques described all aim for a simplified and automated workflow, increased accuracy and reproducibility, and optimal detection of the analyte of interest with minimal interferences and background noise.

    Robert Ellis, Ph.D., R&D director at AB Sciex, will focus his talk on four main trends in LC-MS/MS analysis of small molecule analytes: improvements in selectivity, capacity, confidence, and sensitivity.

    LC-MS/MS provides time- and chemistry-based (on LC separation) and mass-based (on MS detection) selectivity. However, background interference and the presence of isobaric molecules can still compromise the ability to discriminate and quantify the target analyte.

    Dr. Ellis describes method development work at AB Sciex that exploits differences in ion mobility to design an orthogonal sample- prep method that makes it possible to separate out isobaric or background interferences and to distinguish between isomeric forms of a compound. By passing samples through an asymmetric waveform field before they enter the mass spectrometer, this technique differentiates analytes based on their trajectory, which depends on their size and shape.

    For the second trend described by Dr. Ellis, enhancing capacity of LC-MS/MS analysis, strategies may include increasing the throughput of sample processing or extracting more information from each experiment. He will discuss the benefits of the SWATH™ technique, in which groups of ions are scanned sequentially across defined time periods and across the full mass range.

    With SWATH, instead of focusing MS analysis on the detection of a particular target analyte, all of the ions are detected, generating a complete dataset that can then be processed and analyzed to obtain various types of information and to test multiple hypotheses without the need to rerun a sample. The main challenge with this approach is the need for complex algorithms and a high-capacity data-management system to collect, store, and analyze the data.

    Improving confidence in bioanalytical results will require optimizing the speed and accuracy of existing systems, doing multiple reaction monitoring (MRM) more efficiently, and collecting more data points per peak.

    “In R&D you have to hit the sweetspot,” says Dr. Ellis. “You need to recognize that optimizing one parameter might compromise another.”

    The goal of increased sensitivity depends mainly on improving the signal-to-noise ratio and being able to achieve accurate analyte detection in a smaller sample.

    On the LC side, improvements in sensitivity are being achieved using techniques such as microflow-LC, which enables separation of small molecules at rates of 10–15 µL/min, according to Dr. Ellis. As both sampling efficiency and the efficiency of MS ion sources improve, more ions will be generated in a shorter amount of time, yielding a better signal.

    Ichiro Hirano, manager of the mass spectrometry business unit at Shimadzu, will present data related to the development of an automated LC/MS method for assessing the purity of the active pharmaceutical ingredient (API) atorvastatin calcium hydrate, sold under the trade name Lipitor (Pfizer), which could be applied in the development of a generic form of the drug.

    Conventional methods development for purity analysis of small molecule drugs tends to rely on nonvolatile buffers. However, as the bioanalytical workflow shifts toward the use of LC/MS, a volatile buffer is needed for the mobile phase, which introduces uncertainties as the same chromatographic pattern cannot be reproduced.

    Hirano described a 2D-LC/MS-IT-TOF approach in which a nonvolatile buffer is used in the first-dimension LC process to separate the impurities in an atorvastatin sample. The impurity peaks are collected and fractionated by passing them through a loop module controlled by a high-pressure valve system. The impurity fractions are automatically captured based on the known retention times of each impurity, which are input into the system by the operator.

    The system uses the retention time information to create a time program for fractionating the peaks. The fractionated impurities are then injected onto the second-dimension LC/MS system for accurate mass analysis using a volatile buffer.

    In the study presented, the atorvastatin impurities are analyzed using ion trap (IT) time-of-flight (TOF) mass spectrometry, using both positive and negative ion polarity. Hirano provides spectral data showing that the 2D-LC/MS method yields individual peaks for each of the unknown impurities and enables the determination of their empirical formulas.

    With this approach, explains Hirano, drug developers can continue to use established LC separation methods with nonvolatile buffers (as accepted by regulatory bodies such as Japanese Pharmacopeia) in the first dimension, and then introduce an MS-friendly volatile buffer on injection of the fractionated impurity peaks onto the mass spectrometer in the second dimension.


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