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An impurity in cold medicine Researchers in Madrid have identified a major degradation product in several pharmaceutical preparations that target the common cold.
he same peak kept showing up in chromatograms, one after another. As time went on, the peak got bigger. Researchers at the Universidad San Pablo–CEU and the pharmaceutical company Lilly S.A. (both in Spain) became concerned. They were developing an assay to test the short-term stability of common over-the-counter cold medicines. They knew that the mysterious peak had to belong to an unidentified degradation product. Once the amount of the impurity was >0.1%, the level deemed unacceptable by regulatory agencies, the detective work began. The team of analytical chemists, led by Coral Barbas, set out to isolate and identify the unknown product. After months of arduous work, which is described in the January 15 issue of Analytical Chemistry (pp 471–477), the researchers put together numerous clues and narrowed the possibilities down to just one compound. The first clue came from forced degradation assays that were performed before the study began. Under extremely harsh conditions, including highly acidic, basic, and oxidizing environments, the degradation product did not appear, says Barbas. In addition, the amount of the compound increased with temperature. But at temperatures >80 °C, it did not appear, says Barbas. “It appears in common conditions, not in very stressing conditions,” she emphasizes. Unfortunately, those common conditions are the same ones that a pharmaceutical is exposed to on drugstore shelves. 94 A
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What’s in that drug? The next time you reach into the medicine cabinet for relief from the common cold, you may want to read the label carefully. Researchers have discovered an impurity in several over-the-counter cold medicines.
The pharmaceutical formulation under investigation contained three active compounds—the pain reliever acetaminophen, the decongestant phenylephrine hydrochloride, and the antihistamine chlorpheniramine maleate. When the formulation was separated by HPLC with UV detection, an unidentified peak similar to that of phenylephrine appeared in the chromatogram. Over time, the peak grew, while those of phenylephrine and maleate decreased. The researchers speculated that the two active ingredients were interacting with each other to form an unknown degradation product. But they needed more evidence to prove it. They decided to turn to CE. To get as much information as possible, they developed two different CE methods—one
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that used normal polarity and another that used reversed polarity. When they applied the two methods to study the stability of the drug capsules, they observed a new peak with a spectrum similar to that of phenylephrine. With the normal-polarity method, they observed a decrease in the peak of phenylephrine, whereas with the reversed-polarity method, they detected a decrease in the peak of maleate. In reversed-polarity mode, they also observed a new peak similar to that of maleate, which they later attributed to the impurity fumarate. The researchers were not very concerned about the occurrence of fumarate, which is the trans isomer of maleate, because the levels were relatively low. “It is a common compound in body fluids. We know that it presents no problem,” says Barbas. Although the CE results were similar to those obtained with HPLC, they did provide the researchers with a few additional clues about the nature of the unknown degradation product. At neutral pH, the compound moved toward the positive end of the capillary, which suggested that it is negatively charged under those conditions. At low pH, this did not happen. This behavior led the researchers to believe that the compound was acidic and possibly contained a carboxylic acid group. In the next step of their investigation, the researchers used LC/MS to obtain the molecular weight of the degradation product. The impurity had a m/z of 284, which could result from © 2005 AMERICAN CHEMICAL SOCIETY
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the addition of phenylephrine and maleate. The researchers proposed two possible structures for the unknown product. The two structures differed only in the position of a hydroxyl group. They then performed MS/MS experiments using an ion trap to confirm the possible structures. Other structures were also possible, but these two were the most probable, says Barbas. To obtain detailed structural information for the compound, the researchers needed a new technique, and “NMR seemed to be the best option,” says Barbas. But first, they had to isolate the compound in pure form. Because the degradation product was only a minor component (∼0.20% in total weight) of the cold medicine, the researchers had to develop two different purification steps. First, they used solidphase extraction to remove acetaminophen, which was the most abundant compound in the sample. Then, they
used a more refined HPLC separation to remove phenylephrine, which is very similar in polarity to the degradation product and therefore would interfere with the NMR signal. Once they isolated the unknown compound, they characterized it using several NMR techniques, including 1H NMR, 13C NMR, correlation spectroscopy, phase-sensitive heteronuclear single-quantum correlation, and heteronuclear multiple-bond correlation (HMBC). Each technique provided clues to the structure, but HMBC provided the key piece of information. On the basis of those data, the researchers could determine which of the two structures was correct. They concluded that the unknown degradation product was 3-hydroxy-N-[2-hydroxy-2-(3-hydroxyphenyl)ethyl]-N-methylsuccinamic acid. Identification of the unknown compound allowed the researchers to confirm how it forms. They believe it is due
to an interaction between phenylephrine and maleate. Several commercial cold medicines contain the active ingredients phenylephrine hydrochloride and chlorpheniramine maleate, and all of those tested revealed the presence of the degradation product. It is unclear whether the newly identified impurity is harmful. Toxicity tests must still be performed. But now that pharmaceutical companies are aware of the problem, some are likely to modify their cold medicine formulations. A few have already begun to make changes, such as eliminating chlorpheniramine maleate, says Barbas. Still others have a different attitude, she says. They believe that because the product has been on the market for so long and no one has become ill or died from it, it must be safe. Even so, consumers may not be getting what they think they are. Let the buyer beware. a —Britt E. Erickson
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