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Pharmacopoeial

Along with being key products for the food industry, amino acids share a similarly strong relevance in pharmaceutical applications. These natural building blocks of peptides and proteins cover a variety of functions: they are direct components of parenteral nutrition, serve as important precursors during synthesis of pharmaceutical products, or may even represent the active pharmaceutical ingredient within a drug itself. Due to their usually natural origin and widespread functionality, extremely high quality standards have to be maintained by their suppliers and downstream processers.

 

The quality standards and requirements of materials for pharmaceutical use are in most part dictated by the compendia of the major pharmacopoeia: the U.S. Pharmacopeia (USP) and its European counterpart the European Pharmacopoeia (Ph. Eur.). Their guidelines and specifications include legally binding analytical procedures which have to be met in order to officially manufacture and release any pharmaceutical product.

Chemical analytics of amino acids encompass a variety of substantially different methods and techniques which are - at least in part - attributed to the chemical heterogeneity of this substance class. One specific subset of the methods employed in amino acid analysis focuses on the detection of impurities which share chemical similarity to the actual amino acid itself. Presence of such compounds, which share a primary (or secondary) amino function, may significantly alter the efficacy of the drug or, even worse, may lead to undesirable side effects. Consequently, even minute levels of these related compounds should be monitored to ensure proper quality of any amino acid-derived pharmaceutical product.

For the past decades, detection and quantitation of primary and secondary amines have been covered in the compendial monographs by thin-layer chromatography (TLC) followed by subsequent derivatization and visualization using the amino-reactive reagent Ninhydrin. Quantitation of these so-called Ninhydrin-positive substances (NPS) is usually performed by visual comparison to solutions of known quantities of reference amino acids. This somewhat outdated method suffers from low accuracy and doubtful significance, since it basically represents a visual limit test, with minor semi-quantitative properties, which may only be used as rough estimation of impurity levels. Additionally, visual evaluation of colored spots on a TLC plate inherently involves a substantial level of subjectivity which further adds to this method’s imprecision (Figure 1). In short, the detection and evaluation of NPS via TLC yields a considerably less reliable result because the method cannot compare with the separation capabilities of modern liquid chromatographic systems.

 

Adaptation of the compendia to more up-to-date methods usually represents a protracted process, whereby antiquated techniques are replaced by more current applications on a slow but constant basis within the pharmacopoeial text. The Ph. Eur. began introducing liquid chromatography-based analysis called Amino Acid Analysis (AAA) of NPS in July of 2013, and gradually increased the number of altered monographs since then. In its current edition 8.4 (January 2014), 16 out of 28 amino acid monographs contain the new procedure for NPS as an offical requirement, with more updates likely to come. While the USP monographs still rely on the TLC method, the Ph Eur began its updates with the first amino acids over one and half year ago, and the trend would indicate that harmonization of pharmacopeial requirements is possible.

We recently have implemented amino acid analysis in full compliance with the revised Ph. Eur. monographs in our laboratories. The adapted methods for determining NPS and ammonium cations are performed using a fully automated amino acid analyzer (ARACUS Amino Acid Analyzer, membraPure). Any inherent chemical variabilty of the amino acid sample is compensated by the use of post-column derivatization and subsequent detection of a released chromophore. The sample contents are separated by ion-exchange high-pressure liquid chromatography, using a six eluent step-gradient specifically designed for the separation of amino acids and similar compounds. After successful separation, each compound is derivatized in succession with a temperature-controlled reaction coil using Ninhydrin, a process in which the variable fraction of each amino acid is liberated and the dye Ruhemann’s Purple is formed. Secondary amines react with Ninhydrin to form a different colored product, which can both be detected and quantified using a spectrophotometer at their respective wavelengths of 570 nm (primary amines) and 440 nm (secondary amines).
Common NPS’s are comprised of a rather heterogeneous group of known and unknown impurities. Since multiple specimen may be present in often minimal quantities within the same sample, sufficient separation power and sensitivity are basic requirements for a fully quantitative NPS technique.

The AAA method performed in our laboratories is capable of separating a mixture of 43 physiological amino acids with sufficient resolution to specifically quantify each of the reference amino compounds and every other unknown NPS in the sample (Figure 2). For improved separation of non-proteinogenic amino acids and unknown NPS, a lithium-based eluent system is used rather than the widespread and more common sodium-based eluents. Although not all analytes achieve baseline separation, this AAA implementation clearly surpasses the resolution requirements of the Ph. Eur.

Visual detection using the classic TLC procedure usually hits a detection barrier at approximately 0.1 % impurity threshold. Robust and reliable detection at these absolute levels requires an increased method sensitivity. Liquid chromatographic analysis of NPS yields a fully quantitative response with reference amino acids and achieves the Ph. Eur. reporting threshold requirement of 0.05 % (Figure 3).

The described method’s capability to separate the subset of 43 physiological amino acids in a single chromatographic run in combination with a required reporting sensitivity of 0.05% render post-column AAA the optimal choice for evaluating NPS content in test samples. Our laboratory can provide full ICH Q2(R1) validation/verification support of these AAA methods to demonstrate their suitability with respect to client-specific matrices in a qualified GMP environment. Each revised monograph is covered by a basic set of verification data, which already contains additional elements of a more extensive validation due to the rather indistinct pharmacopoeial method description. Accessory client-adapted validation parameters may be included to further enhance the method’s validation status.

References:

1. European Pharmacopoeia, Ph. Eur., Supplement 8.3 to Eight Edition, Council of Europe, EDQM
2. U.S. Pharmacopoeia, USP 37 - NF 32, Second Supplement, The United States Pharmacopeial Convention

For further information, please contact:

Benjamin Rietschel
Project Manager
SGS Life Science Services
Im Maisel 14
Taunusstein
Germany
t: +49 6128 744 463