Technology Forum: Pharmaceutical Analysis

Pharmaceutical analysis is the cornerstone of chromatographic research, with the majority of practitioners (and LCGC subscribers) performing research in this critical application area. Lives are directly affected by the work being done in separations laboratories around the world each day, as analytical chemistry attempts to improve our world with life-saving and life-improving drugs.

Joining us for this discussion are Jim Koers and Nick Duczak of Thermo Fisher Scientific; Phillip DeLand of Dionex Corporation; Roger N. Bardsley of Teledyne Tekmar; and Atis Chakrabarti of Tosoh Bioscience LLC.

What are the main techniques for Pharma analysis and why?

Koers and Duczak:The predominant analytical technique for pharmaceutical analysis, be it qualitative or quantitative, is LC–MS. Liquid chromatography, either standard LC or UHPLC, provides for extremely high levels of analyte selectivity and the mass spectrometer gives superior specificity and sensitivity compared to other detection methods.

DeLand: Because of its qualitative and quantitative capability, isocratic and gradient HPLC separations, using quaternary pumping systems with photodiode array detection, are still the most common analytical tools used in the pharmaceutical industry. LC–MS continues to grow, enabling pharmaceutical scientists to better characterize potential drugs at earlier stages of discovery and development. UHPLC separations using small particles and high linear velocities are gaining acceptance in many areas other than high-end R&D. Chiral separations are another area where interest has grown over the past several years.

Bardsley: The production of chemical pharmaceutical products can be characterized by four basic manufacturing steps. These are raw materials testing, in-process testing, finished product testing, and stability testing. These manufacturing steps have some common analytical techniques. Yet some of the steps have analytical techniques that are only used for that process.

Liquid chromatography is one of the analytical techniques that can be common to all four steps and is most prevalent in the pharmaceutical laboratory today. The various liquid chromatography separation techniques used can be based on reversed phase, normal phase, GPC, SEC, ion, SCX, and SAX chromatography. The separated compounds can be detected by UV-Vis, fluorescence, electrochemical, ELSD, and mass spectrometry detectors. The sample preparation can be traditional volumetric preparation of the sample, or can be based on dissolution of the sample in a dissolution step.

Raw materials testing will use LC to determine the identity and strength of API’s and the excipients used to manufacture the finished product. The excipients can be polymers that must meet certain molecular weight requirements for the manufacture of the finished product. The API could be a protein like Amylin’s Byetta, which cannot be adequately characterized by reversed phase chromatography.

In-process testing will use LC to determine the strength of the in-process material. When a tablet is in the final dosage form, LC is used to confirm that the batch has the proper concentration of API and that it is evenly distributed prior to tableting or vial filling. The tableting or vialing step in the manufacturing process has a greater cost associated with it than the blending step.

Finished product and stability testing will use LC to confirm that the product has the correct dosage strength and that it has not degraded in the manufacturing process or with age. The testing can also be of the excipients used for the manufacture of the finished product. GPC is used to monitor polymers used in the manufacturing of drugs to ensure that the polymer has not degraded, causing the drug to not release or be released too fast into the body.

The second technique that is also common to all four steps is gas chromatography. Gas chromatography uses polar and non-polar columns. The column effluent can be detected with FID, ECD, FPD, NPD, and mass spectrometry detectors. The sample can be introduced by traditional volumetric preparation of the sample for injection and headspace or purge-and-trap introduction of the sample for injection.

Raw materials testing will use GC to determine the identity and strength of the API’s and excipents used to manufacture the finished product. Headspace GC is used for the determination of residual solvents and impurities in these materials.

In-process, finished product, and stability testing will use GC to determine the strength of the in-process or final material. This could be either the active drug or an excipient. An injectable solution that has a preservative will use GC to determine that the correct amount of preservative has been added. Headspace GC is frequently used to ensure that solvents used in the manufacturing process have been adequately removed from the product. These could be solvents used to spray-coat a tablet or to dissolve a polymer for encapsulation of the API.

Numerous other techniques are used in the pharmaceutical industry that are not related to chromatography. TOC and TON analyzers have become common in the pharmaceutical industry for ensuring the purity of water for injectable solutions, cleaning of equipment, and cleaning validation.

X-ray diffraction analyses are used for raw materials and in-process testing to ensure the correct crystalline form is used. Particle size testing is used for raw material, in-process testing, and finished-product testing to ensure the correct manufacturing properties of the excipients and API and the proper release profile of the finished product.

Chakrabarti: Different types of liquid chromatography are the main techniques used. Size exclusion and reversed phase chromatography are very important.

What are some benchmark breakthroughs in the past few years regarding pharmaceutical analysis?

Koers and Duczak:The past few years have brought many technological innovations that have greatly increased both the sensitivity and speed of pharmaceutical analysis. One of our company’s instruments allows for samples to be directly desorbed into the mass spectrometer. The combination of techniques will allow for a discovery laboratory to analyze most any pharmaceutical sample quantitatively in a matter of seconds with no instrument optimizations needed.

DeLand:Because of continuous pressure to increase productivity, the pharmaceutical chemist is forced to spend more time crunching data and producing reports and less time on improving the chromatography. Therefore, any development which can accelerate data processing becomes extremely attractive. Over the past 1-2 years, the biggest breakthrough has probably been in the area of improving the ease of use of chromatography data systems. Operators can select a system, submit samples, and have reports emailed to their desk. Multiplexed systems (for tandem or parallel separations) have gained in popularity for increasing analytical throughput. UHPLC was introduced more than five years ago and has experienced continuous refinement since that time, but nothing really big has changed. However, there is now a much wider range of sub-3µm stationary phases available.

Bardsley:One of the benchmark breakthroughs is the change from traditional chromatography techniques to other chromatography techniques. One example is the acceptance of headspace GC for the determination of residual solvent instead of the direct injection of the dissolved samples. Another is the acceptance of other LC chromatography techniques other than reversed phase chromatography for some of the newer drug entities on the market.

Chakrabarti:New chromatographic capabilities with shorter analytical time and stringent reproducibility are important. High-throughput columns, reduction in particle size, development of a number of new types of stationary phases, and the emergence of UHPLC have reduced the chromatographic analysis time a lot.

What obstacles stand in the way of furthering pharmaceutical analysis?

Koers and Duczak:The largest obstacle is the need for better data processing software. The hardware has continued to push forward by analyzing faster and collecting more detailed data. The bottleneck is now firmly in the hands of software teams to deliver applications that can more rapidly quantify and identify pharmaceutical products and metabolites.

DeLand:Many chemists performing pharmaceutical analyses want the improved performance that comes with UHPLC, but need to retain the ability to run legacy HPLC methods. Budgets are tight and inhibit the purchase of additional systems to develop and run new UHPLC methods. But system utilization is only one issue. The original UHPLC system designs incorporated binary solvent delivery systems. Therefore, these systems had difficulty running legacy methods, which were developed on older ternary and quaternary HPLC systems without some modification to the method. However, method validation is a long and tedious process and this then becomes an obstacle to adopting the new technology. Fortunately, there are now ternary and quaternary solvent delivery systems available, which can run both older HPLC and newer UHPLC methods. This not only increases system utilization, but also offers greater flexibility in developing new UHPLC methods.

Bardsley:The harmonization of analysis between the different pharmacopeia’s around the world. Country X may accept direct inject of sample for residual solvent and not headspace, while country Y prefers headspace GC of samples for residual solvent.

Chakrabarti:Demand and competition for faster and cleaner separation requires integration of the new techniques. New techniques need to overcome the engineering obstacles with the multidisciplinary approach of the scientist from different fields of engineering, chemists, and biologists. The recentacetonitrile shortage also created problems for the methods already validated. Revalidation of the new methods without the use of acetonitrile has become a necessity.