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Jun 1, 2011 (Vol. 31, No. 11)

Versatility of HPLC Sets Technology Apart

Automation and Spatial Resolution Improvements Drive Broader Usage

  • Detection of Organic Contamination

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    According to EMD Millipore, high-purity water plays a critical role in reversed-phase HPLC separations. Contaminants in the water could be responsible for high background noise and other interferences.

    Water supplies throughout the U.S. are showing unprecedented levels of contamination from drugs, antibiotics, hormones given to farm animals, and personal-care products such as lotions, beauty products, soaps, and hair dyes.

    According to Maricar Tarun, Ph.D., an application scientist at EMD Millipore, at least 46 million Americans are supplied with drinking water that tested positive for at least one of these substances. Contaminants run the gauntlet, including acetaminophen, caffeine, codeine, nicotine, and sulfamethoxazole.

    With water supplies deeply compromised, the question arises as to what extent these molecules wend their way into the high-purity water so essential to the operation of scientific laboratories.

    Using reversed-phase HPLC with a quadrupole ion trap mass spectrometry system, Dr. Tarun and her co-workers examined water that had been subjected to a number of different purification regimes. They detected six pharmaceutical molecules in tap water, between 2 and 76 ng/L.

    After reverse osmosis and deionization, Dr. Tarun was able to detect low nanogram levels of all the molecules except acetaminophen. In the HPLC-grade water tested, most of the molecules that were examined yielded levels in the range of 200 pg/L, yet one molecule, caffeine, was much higher, giving values of about 11 ng/L. In LC/MS grade bottled water, half of the antibiotics and drugs were undetectable, although caffeine still weighed in at ca. 10 ng/L. And this was even after an elaborate series of purification steps such as reverse osmosis, ion-exchange resins, UV photo-oxidation, and treatment with activated carbon.

    Dr. Tarun noted that high-purity water plays a critical role in reversed-phase HPLC separations. Contaminants in the water used to prepare the aqueous mobile phase accumulate in the column and could be responsible for high background noise and other interference, rendering meaningless studies on such pressing questions as environmental contamination. Without a legitimate baseline, it will be difficult to rely on values obtained from samples.

    Dr. Tarun’s work points out the difficulties that environmental scientists are faced with in an era when extremely powerful technology is available that can detect contaminants as rare as one in a billion of even one in a trillion. The amount of caffeine in one cup of coffee is 400 million times higher than what she detected in her water samples. Assigning health risks to such miniscule levels of contaminants would be virtually impossible. So while the health risks may be negligible or undetectable, clearly it is essential to monitor the water used in scientific investigations.

  • Superficially Porous Columns

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    Researchers from Agilent Technologies are evaluating a range of HPLC methodologies in order to facilitate productivity enhancements in pharmaceutical quality control applications.

    “Higher productivity is the new catch phrase in the pharmaceutical industry,” says William J. Long, Ph.D., research scientist at Agilent Technologies. In order to move productivity forward, companies are examining their options, including investment in ultra high pressure liquid chromatography instruments as well as alternative column technologies with existing equipment. Dr. Long notes that there is now more flexibility in FDA/USP regulations.

    The United States Pharmacopeial Convention (USP) is a volunteer-driven, not-for-profit, scientific organization that sets standards that are enforced by the FDA. In recent years new flexibility has been introduced so that today new methods validation is not always necessary, and many productivity enhancements can be made with simple adjustments to the methods. Additionally, there is more focus on cost-per-analysis, so that a high-use method can be modified to achieve large savings, taking into account physical time, reagents, and reduced instrument usage.

    To meet these challenges, Dr. Long has been evaluating superficially porous HPLC columns for use in pharmaceutical quality control applications. Superficially porous column technology is based on particles with a solid core and a superficially porous shell. These particles consist of a 1.7 μm solid core with a 0.5 μm porous silica shell. In total, the particle size is about 2.7 μm. They provide 40–50% lower back pressure and 80–90% of the efficiency of a sub-2 μm totally porous particle or approximately twice the efficiency of a 3.5 micron particle.

    Dr. Long reviewed the adaptation of the Poroshell 120 columns to the analysis of naproxen, ibuprofen, and cefipime in accordance with the requirements for method adjustments under USP Chapter 621. Long indicated that these adjusted methods can meet the requirements using lower amounts of solvent and substantially less time. Since these columns are constructed with 2 μm frits, no changes to sample preparation are needed. Lifetime of these columns and the adjusted methods was demonstrated using over 5,000 injections using a prepared ibuprofen sample, Dr. Long explained.

    “We have demonstrated that the use of the Poroshell 120 column meets or exceeds the performance requirements for Compendial methods (those that are reviewed in the USP compendium) while staying within adjustment guidelines. In addition, the potential for even higher productivity is demonstrated within guidelines of proposed changes. Substantial improvements in throughput and reduction of analysis time can be accomplished using instrumentation already in the laboratory with even higher productivity by using newer equipment.”

  • Chiral Compounds

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    Scientists at Sigma-Aldrich have developed an HPLC-based method for purifying D-luciferin that utilizes a Chrom Tech AGP column.

    Chiral chemistry is based on the phenomenon of isomerism, in which two compounds may have the same composition, but the arrangement of the atoms is different, resulting in molecular structures which are the mirror image of one another. Because living systems use one form exclusively, the same enzymatic machinery cannot process both forms, or enantiomers. Ettigounder Ponnusamy, Ph.D., and Mark Nowlan, research scientists at Sigma-Aldrich, have taken this issue into account in developing an HPLC-based method for D-luciferin, the biologically active form of this commercially important compound.

    D-luciferin is found in many insects, where it acts as a sexual attractant. It is the natural substrate for luciferase, catalyzing the production of the typical yellow-green light of fireflies. The bioluminescence of this substance is widely applied as an important marker throughout the biological sciences for such purposes as in vivo luminescence monitoring, characterizing gene expression, and detection of the level of cellular ATP in cell viability assays.

    Dr. Ponnusamy and Nowlan designed a chemical synthesis of D-luciferin by reacting 2-Cyano-6-hydroxybenzothiazole and D-cysteine in aqueous methanol, ending with a high-quality product generated in large amounts. A chiral HLPC method suitable for luciferin was developed. The optical purity of the D-luciferin was determined by chiral HPLC.

    The reversed-phase chiral HPLC method took advantage of a Chrom Tech AGP (α1-acid glycoprotein) column, preferred because of in-house availability and smaller particle size, Dr. Ponnusamy explained. The α1-acid glycoprotein is immobilized onto 5 µm spherical silica beads capable of withstanding organic solvents and higher temperatures within a pH stability range of 4–7. It provides both chiral and general reversed-phase separation power, which can be modulated through shifts in buffer ionic strength and pH, as well as type and level of organic modifier.

    The HPLC chiral methodology offers a versatile approach in terms of alternative UV wavelengths, detection modes (ultraviolet and fluorescence), chiral phases (AGP and Dacel Chiralpak WH), and adjustable conditions for enantiomeric purity screening of synthetic formulations. “While we are pleased with the performance of this method,” said Dr. Ponnusamy, “there are a number of areas that we are following including impurity characterization, stability studies, in-process monitoring, and faster, smaller-particle methods.”

  • An Advancing Frontier

    As improvements in HPLC technology move forward, the precision of spatial resolution and automation of methods allows broader application and more rapid processing of samples and increases in the number of compounds detectable. Widely adopted analytical techniques that are often paired with liquid chromatography include capillary electrophoresis, enzyme assays, sensors, and mass spectrometry. The advances profiled here are only a part of a constantly changing industry.


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