Laboratory Testing Blog

Impure substances present in pharmaceutical products have to be identified to make sure no mutagenic or toxic substances will be administered to patients.  Impurity profiling of pharmaceutical products must be established to guide stable formulation development and provide suitable drug shelf life assessment.  Impurity profiling of pharmaceuticals i.e., the identification, isolation, characterization and purification of these impurities is required.  The traditional approach in impurity identification involves isolation and purification by semi-preparative HPLC, followed by characterization using NMR spectroscopy, accurate MS or LC-MS/MS methods.

The most important concern in the drug development process is safety of a drug and its impurities.  The presence of impurities in trace quantities in a drug substance or drug products is inevitable.  Therefore, the level of impurities in drug substances and drug products should be controlled and monitored.

As drug candidates, there is currently great interest in oligonucleotides due to the reduced time required to achieve lead molecules and to their potential for treating previously untractable diseases.  Synthetic oligonucleotides are used extensively in the field of molecular biology, clinical diagnosis and the development of new therapeutic agents.

Typical oligonucleotides used as therapeutics or potential therapeutic compounds are 15 to 30 nucleotides (nt) long.  Oligonucleotides <15 nt can be readily resolved by HPLC technology.  However, the separation of longer sequences are more challenging.  Ion-pair reversed-phase (IP-RP) HPLC has been traditionally used for oligonucleotides analysis.  The traditional IP-RPLC eluent system typically uses TEA or TEAA or HFIP and the ion-pairing agent triethylammonium ion and a C18, column at 60^oC.  Under such conditions, the HPLC column's hydrolytic stability becomes crucial and is important to prevent the potential contribution of the oligonucleotide secondary structure from impacting retention.  To overcome this, Waters has an ACQUITY UPLC BEH column that has demonstrated excellent performance for oligonucleotides of similar structures.

Modification of low density lipoproteins (LDL) by oxidation of their polyunsaturated lipid components has been implicated in the etiology of atherosclerosis.  Oxidation proceeds by interaction of polyunsaturated fatty acids (PUFA) with active oxygen species or with lipid oxidation products.  Moreover, there are indications that lipid oxidation promotes pre-mature coronary atherosclerosis since susceptibility to LDL oxidation has been associated with the severity of coronary heart disease.  Protection against oxidation can come from antioxidants, antioxidant enzymes and co-factors. The use of antioxidants to inhibit LDL oxidation has been reviewed extensively. Under in vivo conditions, there exists a competition between oxidative and protective processes that depend on PUFA composition and on antioxidant levels.  Therefore, it is important to properly assess the oxidation of the fatty acids in marine oils.

The purpose of dermal absorption studies of topical formulations is to obtain quantitative information on the amounts that can enter, under in-use conditions, into the systemic compartment.  These quantities can then be taken into consideration to calculate a safety factor.  Justification for the use of in vitro dermal absorption studies on isolated skin is based on the fact that the epidermis, in particular the stratum corneum, forms the principal in vivo barrier of the skin against the penetration and uptake of xenobiotics in the body.

First introduced over 100 years ago, USP<231> is a colorimetric procedure based on the precipitation of insoluble metal sulfides.  This test for heavy metals is qualitative rather than quantitative.  It is not an element specific method, nor is it equally sensitive to each metal.  The limits specified by the test are based on the ability to observe the precipitate, rather than on the analysis of toxicological data.  The procedure does not necessarily detect all potential forms and/or valences of elements of concern when they are present as the oxo ions or in the organometallic form.  Chromium and nickel are potential contaminants from modern stainless steel processing equipment and are not detected by the USP<231> test method.  Other studies indicate inconsistent recoveries of monitor and standard solutions using USP<231> method II.2, 3

The efficient characterization of antibody drugs is important to both regulatory agencies and pharmaceutical companies to ensure the safety and efficacy of biopharmaceutical products.  Proteins with different sizes may show similar hydrophobicity and are therefore difficult to separate by traditional reversed-phase chromatography (RP).  

Plants respond to several environmental stresses such as pathogen attack, mechanical or herbivorous insect-driven wounding and the presence of pollutants by the synthesis of secondary metabolites that mediate interplant communication for defense responses.  Among these signalling molecules are phytohormones: jasmonic acid (JA), salicylic acid (SA) and abscisic acid (ABA).  They are considered to be the major regulators of plant defense responses.  The role of salicylic acid in influencing plant resistance to pathogens (aphids, bacteria, fungi) is well documented, whereas jasmonic acid is commonly believed to play an important role in plant responses to insect herbivores and abiotic stress.  The soluble jasmonic acid acts as a direct defense to herbivores or others injuries, whereas the volatile form, methyl jasmonate, represents a plant signal molecule, which serves as an indirect defense mechanism.

In moving from a chemical to an instrument-based methodology, the USP has taken great care to allow for a flexible approach and is working closely with both the FDA and industry to ensure widespread agreement on interpretation of the revisions.  Following are brief descriptions of the techniques and methodologies being proposed by USP: