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Pharmaceutical Analysis
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HPLC and mass spectrometry each account for only about $5 billion of global sales. In
contrast, pharmaceutical global sales are about $1.3 trillion. Even though the value of
HPLC equipment sales is minuscule compared to pharmaceutical sales, HPLC is very
important in affecting health and well-being when it comes to the drug development
process and production.
Before a drug can be marketed it needs to have proven safety and efficacy - both
depend on the purity of the drug. Impurities can make the drug unsafe and the level of
drug potency depends on how much of the active pharmaceutical ingredient (API) is
present. In order for pharmaceutical companies to be certain of the purity of their drugs
they conduct numerous quality control analyses.
Acceptable potency is specified for each drug using the HPLC stability-indicating assay,
which ensures that each batch of drug product is within 5-10% of the target level (label
claim). Similarly, it must confirm that the level of impurities is acceptably low, for
example, a maximum of 0.1 - 1% of the total drug. HPLC is a primary tool for the quality
control of pharmaceuticals, for both large and small molecules.
The stability-indicating assay for drug purity is important because the purity of drugs is
equated with quality and safety. Stability studies are required by regulatory bodies such
as the US FDA (United States Food and Drug Administration), or the EMA (European
Medicines Agency) in Europe. The purity of the drug must be within specification before
it can be sold (release testing) and within the stated expiration date under the
recommended storage conditions. Consequently, HPLC is pivotal to the pharmaceutical
industry and hundreds of thousands of HPLCs are being used for this purpose alone.
More than 50% of HPLC is in the pharmaceutical industry.
In order to avoid contamination of the column when compounds with particularly strong
absorption to silica are present, reverse-phase chromatography was developed. This
involves modifying the silica surface to make it hydrophobic and diluting the sample in
aqueous solution. It is the reversed of normal phase as the mobile phase is polar and
the stationary phase is nonpolar.
Regulations stipulate that the HPLC method must be able to separate the main
component, the API, and all impurities including degradation products. All the small
molecule pharmaceutical drugs are made by a synthetic process to obtain a highly-
purified compound. However, there will always be some trace impurities and each
impurity will form a distinct band or peak on the chromatogram. The area under the
different peaks in the HPLC chromatograms are proportional to the concentration of the
trace impurities.
Pharmaceuticals, even though they are highly stable, do degrade forming degradation
products. These too are measured using the stability-indicating method. The
chromatography method may need to be tweaked many times in order to achieve a
separate peak or zone from the API, the excipients, each impurity and the degradation
product, which is essential to accurately quantify each of these components. Good
sensitivity, down to at least 0.05% is also needed.
Once the HPLC method has been developed, it must be reproducible for many
laboratories around the world as most companies do global manufacturing. For
example, in a stability study, a drug may be in the chamber for two to three years, and
needs to be measured every three months, year after year. The bar for method
performance, accuracy, and reproducibility, is thus very high. So, not only is it HPLC
challenging, it is also under very stringent regulatory compliance guidance.
The challenges for HPLC assay are increasing as newer drugs get more complex. More
than 50% of newly developed pharmaceuticals are chiral-compounds with many having
multiple chiral centers. Consequently, it is not unusual to have 40-60 different peaks in
the HPLC impurity profiles of a complex drug. These more complex drugs require
greater separation power in the HPLC column. Development of such methods are time-
consuming and arduous, and the analysis time keeps getting longer.
High performance liquid chromatography uses a column with smaller particles and the
whole process is automated using a high-pressure pump. For about 50 years, the
pressure limit was 6,000 psi (400 bars). As more complex separations were needed, for
example to analyse complex drugs, environmental samples, physiological fluid bloods
and serum, or in proteomic studies, there was a need for greater separation power and
faster analysis.
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In 2004 the first commercial UHPLC instrument was developed by Waters Corporation,
who you will be able to visit in the exhibition floor at Pittcon 2019. HPLC conducted at
pressures above 15,000 psi is generally referred to as ultra-high-pressure liquid
chromatography (UHPLC).
HPLC can separate a maximum of about 200 peaks, whereas UHPLC can separate
almost 1,000. This makes it possible to quantitate very complex drugs much more
confidently and accurately.
There are also other features of UHPLC that make it better. Typically, they have a
smaller flow path (system dispersion). The UV detector flow cell is smaller; less than
one microliter, compared with 8-10 microliters in HPLC. A smaller flow cell controls
dispersion or band broadening so that narrow peaks are maintained.
At that time, I was supporting an oncology project for a drug for prostate cancer and
gastric cancer. The molecule is quite complex and has 3 chiral canters, and many
methods must be developed quickly to support the synthetic process to control the
purities of the starting materials, intermediates and the API. I applied the three-pronged
template approach in this project and developed 40+ methods to support this very
complex project efficiently.
The first one is a HPLC stability-indicating method for an API with 3 chiral centers
mentioned before. Figure 1a is the structure of the NCE; 1b is the regulatory HPLC
method, while 1c and 1d are UHPLC methods used to support phase 2 drug product
development which are faster and has higher resolution.
The second case study illustrates the high-resolution capability of a well-developed
UHPLC method for a drug product with 4 APIs using a 5-gradient-segment method
capable of separating up to 60+ impurities in a robust manner.
He holds a Ph.D. in Analytical Chemistry from the City University of New York, and a
certificate in Biotechnology from U. C. Santa Cruz. He has 120+ publications including a
bestselling book on chromatography (Modern HPLC for Practicing Scientists, Wiley). He
is an editorial advisory board member of LCGC magazine, American Pharmaceutical
Review, and Chinese American Chromatography Association.
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