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Method Abstract 391

MA 391 / 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, 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.

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 (e.g. peptides, proteins) by means of amino acids composition analysis;
  • Impurities and related substances determination on APIs (Active Pharmaceutical Ingredients, e.g. free amino acids) and intermediates;
  • Single or total amino acids quantification in drug products, including markers determination in complex matrixes (e. g. 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
  • Arginine 07/2014:0806

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 Pharmacopoeia 8.0 methods.

Each 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.

Table 1 summarizes the solutions used in each monograph.
 

Table 1. Reference guide for Pharmacopoeia 8.0 methods

Amino acids

Test solutions ug/mL

Cys

CSSC Lys Ser Thr Val Ala Arg Leu Ile Ile & Leu Pro NH4

Valine

600

          1.2       1.2 3 each 1.2 0.12

Proline

600

            0.6       3 each 1.2 0.12
Leucine 600 & 24                 1.2 0.12 3 each 1.2 0.12
Threonine 600         1.2           3 each 1.2 0.12
Serine 600       1.2             3 each 1.2 0.12
Lysine 600     1.2               3 each 1.2 0.12
Isoleucine 600           1.2     1.2 1.2 3 each 1.2 0.12
Arginine 600             1.2 1.2     3 each 1.2 0.12
Cysteine 600 1.2 1.2                 3 each 1.2 0.12

Cys = Cysteine, CSSC = Cystine, Lys = Lysine, Ser = Serine, Thr = Threonine, Val = Valine, Ala = Alanine, Arg = Arginine, Ile = Isoleucine, Leu = Leucine, Pro = Proline, NH4 = ammonia

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

Methods using High-efficiency Sodium column for analysis of following Amino Acids:
Valine, Proline, Leucine, Isoleucine, Serine, Threonine, Lysine, Arginine
     
METHOD

Analytical Conditions

Column: High-efficiency Sodium cation-exchange column,
       4.6 x 110 mm, Catalog Number 1154110T
Flow Rate: 0.6 mL/min
Mobile Phase: See method in Table 2
Injection Volume: 50 uL

Post-column Conditions

Post-column System: Pinnacle PCX
Reactor Volume: 0.5 mL
Reagent: Trione®
Reagent Temperature: 130 °C
Column Temperature: See method in Table 3
Flow Rate: 0.3 mL/min
Detection: UV/VIS 570 nm for primary amino acids, 440 nm for secondary amino acids

 
 

 

Table 2. HPLC Program for Column 115411T

TIME

NA315

NA425 

NA640 RG011

0

100

0

0 0

4

100

0

0 0
23 10 90 0 0
24 0 0 100 0
42 0 0 100 0
42.1 0 0 0 100
45 0 0 0 100
45.1 100 0 0 0
55 100 0 0 0

Table 3. COLUMN OVEN PROGRAM

TIME

Temp, ºC

0

42
4 42
12 60
23 60
35 70
43 70
44 42

391-a
391b
391-c
391-f

Due to the poor separation of Cysteine and Proline peaks with the Sodium-based method, the Lithium High-efficiency column needs to be used for Cysteine analysis according to Pharmacopeia 8.0. The same Lithium method can be used for all other amino acids, though analysis time is longer than with the Sodium method.

Methods using High-efficiency Lithium column for analysis of following amino acids:
Cysteine, Valine, Proline, Leucine, Isoleucine, Serine, Threonine, Lysine, Arginine
 

METHOD

Analytical Conditions

Column: High-efficiency Lithium cation-exchange column,
       4.6 x 75 mm, Catalog Number 0354675T
Flow Rate: 0.55 mL/min
Mobile Phase: See method in Table 4
Injection Volume: 50 uL

Post-column Conditions

Post-column System: Pinnacle PCX
Reactor Volume: 0.5 mL
Reagent: Trione®
Reagent Temperature: 130 °C
Column Temperature: See method in Table 5
Flow Rate: 0.3 mL/min
Detection: UV/VIS 570 nm for primary amino acids,
       440 nm for secondary amino acids
   

Table 4. HPLC Program for column 0354675T

TIME

1700-1125

LI365

LI375 RG003

0

100

0

0 0

15

100

0

0 0
35 40 60 0 0
38 0 100 0 0
43 0 100 0 0
43.1 0 0 100 0
57 0 0 100 0
57.1 0 0 0 100
60 0 0 0 100
60.1 100 0 0 0
72 100 0 0 0

Table 5. Column Oven Program

TIME

Temp, ºC

0

34
6 34
30 45
32 70
59 70
60 34

 

391-g

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 could be ordered individually if needed. Please contact Pickering Laboratories if you have any questions regarding this application.
 

PART NUMBER

DESCRIPTION

00352-0057

30-Minute High Efficiency Protein Hydrolysate Kit (for Sodium methods):

1154110T

30-Minute Sodium Cation-exchange Column 4.6 x 110 mm &
1700-0070 amino acid test mixture

1700-3102 Cation-exchange GARD™ assembly: Holder w/ 2 replaceable GARD™
Na220 Sodium Diluent, pH 2.20, 4 x 250 mL
Na315 Sodium Eluant, pH 3.15, 4 x 950 mL
Na425 Sodium Eluant, pH 4.25
Na640 Sodium Eluant, pH 6.40
RG011 Sodium Column Regenerant, 950 mL
T100C TRIONE® Ninhydrin Reagent, 4 x 950 mL (4-month shelf life)
012506H Sodium Calibration Standard, for protein hydrolysate, 0.25 μmole/mL, 5 mL
0352-0058 Kit Identical to 0352-0057 with T200 replacing T100C:
T200 TRIONE® Two-part Ninhydrin Reagent, prepares 4 x 900 mL (12-month shelf life)
0352-0006 70-minute Physiologic Fluid/Native Sample Kit (for Lithium methods):
0354675T Lithium Ion-exchange Column, 4.6 x 75 mm (with test mixture 1700-0070)
1700-3102 Cation-exchange GARD™ assembly: Holder w/ 2 replaceable GARD™
1700-1125 Lithium Eluant, pH 2.80, 4 x 950 mL
Li220 Lithium Diluent, pH 2.20, 4 x 250 mL
Li365 Lithium Diluent, pH 3.65, 4 x 950 mL
Li375 Lithium Diluent, pH 3.75, 4 x 950 mL
RG003 Lithium Column Regenerant, 950 mL
1700-0170 Lithium Calibration Standard, without Norleucine and AGPA, 0.25 μmole/mL, 5 mL
SP100 SERAPREP™, 250 mL
UP100 URIPREP™, 250 mL
T100C TRIONE® Ninhydrin Reagent, 4 x 950 mL (3-month shelf life)
0352-0007 Kit Identical to 0352-0006 with T200 replacing T100C:
T200 TRIONE® Two-part Ninhydrin Reagent, prepares 4 x 900 mL (12-month shelf life)

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