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How to Develop Validated HPLC Methods

Michael Rummel has held the position of chief operating officer at InSource Diagnostics, an independent laboratory that focuses on medication monitoring and compliance testing, for several years. Since graduating from the University of Wisconsin-Madison in 2005, Michael has pursued a career working in the analytical and clinical sciences. He has expertise in analytical chemistry assay development specifically with liquid chromatography/mass spectrometry, assay optimization, and sample preparation optimization and development.

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In his current position at InSource Diagnostics, he has become intimately involved in clinical workflows, regulatory requirements, and enforcement of strict quality control/quality assurance for LC/MS clinical assays. He has future aspirations of leveraging his knowledge to help develop a next generation of clinical diagnostics assays that involve less invasive sampling techniques and greater accessibility for patients.

Q: What specifically do you use HPLC for in your laboratory?

A: We specialize in illicit drug testing and medication monitoring, so we use HPLC in tandem with mass spectrometry, specifically triple quad tandem mass spectrometry (LC/MS/ MS). It’s used to confirm immunoassay screen results as well as perform direct quantitation of medications on human specimens.

Q: What are the most important things an HPLC method must have for the work you do?

A: HPLC is used to separate out target analytes away from other drugs and interferences that may be encountered in human matrices. One can imagine that from urine, to blood, to oral fluid—there are many different compounds, both endogenous and exogenous—that must be separated from our target analytes. Even more critical, HPLC is utilized to separate analytes from other target analytes with the exact same chemical mass. These are referred to as isobaric compounds which a mass spectrometer alone cannot distinguish between. HPLC adds another dimension in order to resolve isobaric compounds in order to achieve accurate identification and quantitation. A prime example of a critical isobaric separation is discerning methamphetamine and phentermine. Street methamphetamine is an illicit substance, (there is a prescription medication for methamphetamine but it’s rarely prescribed) and phentermine is a drug prescribed for weight management. There are obviously rather large implications in accurately identifying methamphetamine versus phentermine. Both compounds have the same exact mass and would look the same to a mass spectrometer so it is critical these two drugs are resolved using HPLC in order to distinguish one from the other.

Q: How do you develop validated HPLC methods?

A: It’s a very laborious process. CLIA [Clinical Laboratory Improvement Amendments] is our governing body as InSource Diagnostics is a high complexity clinical laboratory. An LC/MS-based assay is considered a laboratory-developed test so there are many aspects of assay validation that are performed in order to ensure it is stress-tested adequately. As part of the complete validation, accuracy, precision, linearity, carryover, interferences, sensitivity, dilution integrity, analyte stability, and correlation studies against validated methods are evaluated. Additionally, because it is a mass spectrometry method, matrix contributed ion suppression/enhancement must be assessed as well. The length of a typical validation can take anywhere from two to four months, sometimes longer depending on the complexity of the assay. Since clinicians are making medical decisions that direct patient care based on our test results, utmost accuracy is imperative. There’s no room for error. We have to ensure high quality and that begins at the validation process. Taking the time up front during validation definitely translates to a more rugged and robust assay upon going live with patient samples.

Q: You mentioned time is a key challenge in developing such methods, what are some of the other major hurdles you face? How do you overcome those challenges?

A: One of the biggest challenges with clinical samples is that they vary significantly based on disease state, collection device, geographic origin, patient diet, genetics, etc. When developing a robust analytical method, you must ensure you are able to get the right answer regardless of the sample you are given. When we develop a new assay, we spend a significant amount of time evaluating different samples looking for matrix interferences or other endogenous or exogenous compounds that will present a challenge when the assay goes live. We then check our results against reputable reference laboratories to ensure that we are getting the right answer. At the end of the day, the more time and effort a lab spends during this phase of method development, the more robust the assay will be when it goes into production.

Q: What have been some recent changes or trends in validated method development for HPLC over the last few years?

A: Within the clinical industry LC/MS is becoming more rapidly adopted. It can be an incredibly powerful tool to have in the clinical laboratory, so more hospitals and independent laboratories, even some physician laboratories, are adopting LC/MS. This has especially been evident over the last five to ten years. With LC/MS becoming more prevalent in clinical laboratories, there is more opportunity to collaborate and share information. As a result, there is impetus for standardization of LC/ MS/MS laboratory tests. For example, the CDC (U.S. Centers for Disease Control and Prevention) orchestrates a program for hormone and vitamin D standardization. The idea is increased consistency of results whether it’s a laboratory such as InSource Diagnostics in California, or a laboratory in New York or Pennsylvania. I believe the trend for standardization and harmonization will continue.

Q: Where does validated HPLC method development appear to be moving for the future?

A: On the same note of increased standardization and harmonization, CLSI, the Clinical and Laboratory Standards Institute, released a guidance document to address the specific needs of an LC/MS/MS-based clinical assay validation. The publication was a collaborative effort from many different scientists and lab professionals throughout the country.

Q: What advice do you have for lab managers or professionals who are new to validated HPLC method development?

A: Do not underestimate the complexity of HPLC method development. It’s important to have qualified personnel with the proper training and experience to perform HPLC or LC/ MS/MS validations. It’s not something you can just apply knowledge from a textbook and accomplish. It takes extensive training and experience and the person tasked with validation should have, at a minimum, four years of LC/MS experience.