An Amino Acid Analysis According to European Pharmacopoeia 8.0

The European Pharmacopoeia (Ph. Eur.) defines requirements for the qualitative and quantitative composition of medicines, as well as the tests to be carried out on medicines and on substances and materials used in their production.

It covers active substances, excipients, and preparations of chemical, animal, human or herbal origin, homoeopathic preparations and homoeopathic stocks, and antibiotics, as well as dosage forms and containers. It also includes tests on biologicals, blood and plasma derivatives, vaccines, and radiopharmaceutical preparations. The European Pharmacopoeia and its requirements are legally binding in the member states of the European Pharmacopoeia Convention and the European Union.

Figure 1: Sodium chromatogram of amino acids analyzed using Pharmacopoeia 8.0 methods (3 µg/mL each, 50 µL injection).

All manufacturers of medicines or substances for pharmaceutical use therefore must apply the Ph. Eur. quality standards in order to be able to market and use these products in Europe.

Amino acids analysis can be used for:

• identification tests on biopharmaceutical active ingredients (such as peptides and proteins) by means of amino acids composition analysis;

• impurities and related substances determination on active pharmaceutical ingredients (APIs) (such as free amino acids) and intermediates;

• single or total amino acids quantification in drug products, including markers determination in complex matrixes (such as phytopharmaceuticals).

The following Ph. Eur. monographs have already officially introduced the amino acid analysis method with post-column ninhydrin derivatization as the analytical procedure required for the determination of the ninhydrin-positive substances, and additional papers are expected to be published in upcoming months: Cysteine HCl Monohydrate 01/2014:0895, Isoleucine 07/2013:0770, Leucine 07/2013:0771, Lysine HCl 07/2013:0930, Serine 01/2014:0788, Proline 01/2014:0785, Threonine 01/2014:1049, Valine 01/2014:0796, and Arginine 07/2014:0806

Figure 2: Sodium chromatogram of alternative amino acids analyzed using European Pharmacopoeia 8.0 methods (3 µg/mL each, 50 µL injection).

Pickering Laboratories, Inc. offers a complete solution for amino acids analysis according to European Pharmacopoeia 8.0. This includes the Pinnacle PCX post-column derivatization instrument, analytical columns and GARDs, buffers, and Trione® Ninhydrin reagent. The Pinnacle PCX is capable of performing column temperature gradients that allow easily modified conditions and improved run times and amino acids separations. The methods presented in this application note were optimized to comply with system suitability requirements of European Pharmacopoeia 8.0 methods.

Each European Pharmacopoeia monograph describes the preparation of the test and reference solutions specific for each amino acid. The solutions are used for calculations of percentage contents, impurity levels as well as parameters of system suitability. Resolution of 1.5 is required between leucine and isoleucine peaks.

For all amino acids, except cysteine, sodium-based and lithium-based methods are available. For cysteine analysis, only lithium-based methods are suitable. Sodium-based methods have shorter run times and are preferable for all amino acids except cysteine.

Equipment:

Quaternary HPLC pump, autosampler, UV-vis detector, and Pinnacle PCX post-column derivatization system

Analytical columns and Eluents:

For Sodium-based methods: High-efficiency Sodium cation-exchange column, 4.6 × 110 mm, Catalog Number 1154110T. Eluents: Na315, Na425, Na640, and RG011.

Figure 3: Lithium chromatogram of amino acids used as reference solutions for cysteine analysis (3 µg/mL each, 50 µL injection).

For lithium-based methods: High-efficiency lithium cation-exchange column, 4.6 × 75 mm, Catalog Number 0354675T. Eluents: 1700-1125, Li365, Li375, and RG003.

Figure 4: Lithium chromatogram of amino acids analyzed using European Pharmacopoeia 8.0 methods (3 µg/mL each, 50 µL injection)

Post-column reagent

Trione Ninhydrin Reagent

To make it easier to start using Pickering Laboratories methods, we offer chemistry kits that include: analytical column, GARD, buffers, and reagents for amino acids analysis. All parts of the kit can be ordered individually if needed. Please contact Pickering Laboratories if you have any questions regarding this application.

Pickering Laboratories will keep updating its European Pharmacopoeia 8.0 methods as new monographs are released. Please contact support@pickeringlabs.com for the latest methods and chromatograms.

Pickering Laboratories, Inc.
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