USP 621 Allowable Adjustment to Chromatography HPLC Methods

Effective August 1st 2014, the United States Pharmacopoeia (USP) published the latest revision to General Chapter <621> mapping out the "allowable adjustments" that can be made to USP methods without having to re-validate these methods. Articles provided by industry leaders in separation sciences, pharmacology and chemistry.

1. About Our Sponsors Alkemist Labs Alkemist Labs is an ISO 17025 accredited contract testing laboratory specializing in plant authentication, botanical ingredient identification and quantitative analytical services to the Food & Beverage, Nutraceutical and Cosmeceutical industries. Located in Costa Mesa, California, Alkemist Labs offers clients a wide range of specialty research services to evaluate the identity, purity and quality of botanical raw materials, dietary ingredients and finished products. Alkemist Labs produces a complete line of Composite Reference Botanicals (CRBs), critical tools for botanical identity verification, and also distributes phytochemical reference standards from Extrasynthese of Lyon, France. Alkemist Labs has become the ideal “Partner for Quality” to companies interested in producing high quality natural products requiring independent, third-party analysis, Stability/Shelf-life studies, Certification of Analysis and cGMP compliance. www.alkemist.com Phenomenex Phenomenex, headquartered in the United States, is a global leader in the research and manufacture of advanced technologies for the separation sciences. Phenomenex is committed to developing novel analytical chemistry solutions that solve the separation and purification challenges of researchers in pharmaceutical, industrial, clinical research, food testing, environmental, government and academic labs. With over 700 employees in 16 companies around the world, three manufacturing sites located in the United States, and a network of 60 distributors, the company is able to serve tens of thousands of customers in over 100 countries each year. www.phenomenex.com   United States Pharmacopeial Convention The U.S. Pharmacopeial Convention (USP) is a scientific nonprofit organization that sets standards for the identity, strength, quality, and purity of medicines, food ingredients, and dietary supplements manufactured, distributed and consumed worldwide. USP’s drug standards are enforceable in the United States by the Food and Drug Administration, and these standards are used in more than 140 countries. www.usp.org

2. About Our Speakers Philip Koerner Ph.D Senior Technical Manager, Phenomenex Dr . Koerner has more than 27 years of industry experience in LC, GC, and sample preparation techniques, 11 years of which were at DuPont followed by over 15 years at Phenomenex. Among his various degrees are a Masters in Science in Chemistry and a Ph.D.in Analytical Chemistry from University of Illinois, Urbana-Champaign. He prides himself as being a food geek and has spent the most part of the last few years traveling and visiting various laboratories globally to provide seminars , hands-on trainings , and technical assistance. Dr. Koerner is also a member of the AOAC International Expert Review Panel (ERP) for Dietary Supplements. Questions : Email at [email protected] Zeshan Aqeel Application Scientist, Phenomenex   Zeshan Aqeel  has over 10 years of experience in LC, GC, and Sample Preparation.   He has a degree in Biochemistry from California State University of Northridge and is currently an Applications Scientist at Phenomenex .  Prior to Phenomenex, Zeshan worked as an analytical chemist at Amgen and Baxter Bioscience focusing on the optimization of LC and LC/MS methods.  He has played an integral role in supporting our teams with hands-on  lab trainings, problem solving, troubleshooting , and applications support around the world. In addition he has developed and presented many technical seminars globally . Zeshan has assisted in the developments of many applications using the Kinetex ® Core-shell technology, including various dietary supplements applications. Q uestions : Email at [email protected]

3. About Our Speakers cont. Holly E. Johnson Ph.D Laboratory Director, Alkemist Labs Dr . Johnson took her Ph.D. in Pharmacognosy at the College of Pharmacy , University of Illinois - Chicago, under renowned Pharmacognosist and researcher Dr. Norman Farnsworth. Holly was awarded a National Institutes for Health NRSA Fellowship and worked in the NIH Center for Botanical Dietary Supplements for Women's Health. Additional education includes a B.S. in Environmental Biology and a B.S. in Botany, Department of Biological Sciences, Humboldt State University, Arcata , CA. In her ethnobotanical research Holly led myriad field expeditions collecting plants, mostly in Caribbean and South Pacific island nations, and is currently a Research Associate with the National Tropical Botanical Garden. Her research appears in peer reviewed publications such as Journal of Natural Products & Phytochemistry . She also worked for Waters Corporation conducting technical training courses and consulting on chemistry applications and regulatory compliance for pharmaceutical and supplements companies. Holly has 20 years experience in natural products chemistry with botanicals and spent many happy years conducting research on medicinal plants and giving courses at the University of Hawaii. Holly E. Johnson Ph.D., joined Alkemist in 2014 leading project teams in analytical testing of natural products and botanical ingredients as well as performing specialized research. Questions : Email at [email protected]

4. About Our Speakers cont. Anton Bzhelyansky Scientific Liaison, United States Pharmacopeial Convention Anton Bzhelyansky holds a Master’s in analytical chemistry from the University of Maryland Baltimore County.  From his academic advisor , professor George M. Murray, he inherited a conviction that the unbiased technical versatility is an analytical chemist’s imperative .  His thesis was on uranyl-templated polymers .  Subsequent four-and-a-half years’ worth of method development in generic pharma sufficiently prepared him for the challenges of dietary supplement analysis; or so he thought.  For the next eight years, Anton had been developing methods for a wide range of DS: from marine oils to chondroitin sulfate to botanicals.   At Nutramax Laboratories, he implemented total inspection of incoming raw materials by NIR, established ICP-OES methodology, investigated sampling of incoming ingredients and in-process blends, worked on formulation of enteric-coated DS dosage forms, and served as a lab Empower administrator.  An AOCS Approved Chemist in 2011-2012, Anton developed a 20-minute marine oil GC method and optimized Peroxide and Anisidine Value protocols.  His most memorable analytical work, however, remains the suite of methods that monitored glucosinolates and isothiocyanates in Cruciferae , as well as their enzymatic conversion rate.     Anton has been with USP for three-and-a-half years.  He is responsible for the majority of botanical monographs in the USP-NF Dietary Supplement section.  Anton dedicates a significant effort to development of the General Chapter <2251> Screening For Undeclared Drugs and Drug Analogues, and is currently compiling the USP Adulterants database.  In line with the USP's “Up-To-Date" policy, he is continuously looking to improve compendial analysis.  Anton is interested in implementing advanced methodologies for challenging analytes such as proanthocyanidins and complex polysaccharides, as well as devising a practical route for adoption of chemometrics in DS monographs.  He is a member of AOAC and AOCS . Questions : Email at [email protected]

5. Effective 1 August 2014, the United States Pharmacopeia (USP) published the latest revision to General Chapter <621> on Chromatography that provides clear guidance regarding what “allowable adjustments” can be made to USP methods without having to revalidate these methods. Understanding this latest revision can prove beneficial for improving productivity and cutting costs in any lab environment . Generally USP monographs were developed for generic drugs and dietary supplements, and many of these methods were developed on older materials and are time consuming to run. With the availability of much higher efficiency HPLC and UHPLC columns, many of these methods could be improved to increase productivity in the lab and reduce the lengthy run times often associated with these methods, while maintaining or improving chromatographic resolution and meeting the system suitability requirements defined in the monograph . Previous versions of USP <621> simply said that adjustments could be made to help meet system suitability; however, the extent of an adjustment was not always clearly defined. Following several further revisions, the 2014 revision (USP37-NF32) now provides much clearer definition of “allowable adjustments ’ Allowable Adjustments and System Suitability Before we review the allowable adjustments, let’s summarize some of the key points of USP <621>: Defines chromatographic terms and procedures Allows for adjustments to methods “in order to comply with system suitability” when “it may be desirable to use a column with different dimensions” “only when suitable standards (including Reference Standards) are available for all compounds used in the suitability test and the adjustments yields a chromatogram that meets all the system suitability requirements specified in the official procedure.” “are not to be made in order to compensate for column failure or system malfunction” Defines the maximum allowable adjustments Adjustments may require additional validation Multiple adjustments should be considered carefully Addresses the continued trend toward <3 μm particles, superficially porous (core-shell) particles, and fast LC / UHPLC Featured Article Allowable Adjustments to Pharmacopoeia HPLC Methods Philip Koerner Ph.D, Senior Technical Manager, Phenomenex Inc.

6. The maximum allowable adjustments are outlined in Figure 1 for isocratic methods, and Figure 2 for gradient methods (more limited). Adjustments may require verification, and multiple  adjustments should be considered carefully as they can have a cumulative effect on system performance. Changes in chemical characteristics of stationary phase, such as a change from L1 (C18) to L7 (C8), are considered a modification and will require revalidation. Featured Article cont. Allowable Adjustments to Pharmacopoeia HPLC Methods

7. Adjustments to Column Length and Particle Size For isocratic separations only ( not allowed for gradient separations ), the ratio of column length (L) to the particle size ( dp ) must remain constant or within range between -25% to +50% of the prescribed L/ dp ratio. Alternatively, other combinations of L and dp (e.g. for the application of particle size adjustment to superficially porous particles (aka core-shell particles)) can be used provided that the number of theoretical plates (N) is within - 25% to +50%, relative to the prescribed column .   USP <621> and Guard Columns The use of guard columns will protect valuable analytical columns by removing particulates and strongly retained sample components that may accumulate on column, increase columns lifetimes, and help maintain column performance (efficiency, resolution, and peak shape). This latest revision clearly addresses the use of guard columns. Specifically, a guard column may be used with the following requirements, unless otherwise indicated in the individual monograph: Guard column length must be ≤15% the length of analytical column, Inner diameter must be the same or smaller than the analytical column, and The packing material should be the same as the analytical column (e.g. silica) and contain the same bonded phase (e.g. C18). When using a guard column all system suitability requirements specified in the official procedure must be met .   In conclusion, USP General Chapter <621> defines the maximum “allowable adjustments” to compendial methods. These adjustments permit flexibility for users of compendial  methods to greatly increase productivity in the lab by ultimately reducing run times while also minimizing solvent usage and cutting costs. System suitability must be met or revalidation will be required. Finally, USP <621> also allows for guard columns as long as stated requirements for their use are met . To learn more about USP <621> and how the allowable adjustments can improve your compendial methods, please contact Phil Koerner at [email protected] or watch our on demand webinar for complete presentation. https :// www.phenomenex.com/Account/LogOn/form~16lcuspwebinar Featured Article cont. Allowable Adjustments to Pharmacopoeia HPLC Methods