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Hitachi High-Technologies GLOBAL

The Japanese Pharmacopoeia Sixteenth Edition, effective as of April 2011, specifies that HPLC analysis with a conventional column is to be used for the identification of isoleucine, leucine, and valine granules. It also specifies that the post -column ninhydrin method should be used for the assay.
In this publication, the Chromaster, a Hitachi high speed liquid chromatography instrument, and the Hitachi L-8900 amino acid analyzer were used to perform the identification test and assay under the analysis conditions conforming to the Pharmacopoeia. The specified values and measured values related to the system suitability, etc. were compared.

Identification by Chromaster

Concentration of Each Component

Component Concentration (mg/mL)
Val: Valine 1.10
Ile: Isoleucine 0.92
Leu: Leucine 1.84

Analysis Conditions

Column HITACHI LaChrom C18(3 µm) (4.6 mm I.D. × 150 mm)
Eluent (A)/acetonitrile = 970/30 (v/v)
Flow rate 0.9 mL/min
Column temperature 40°C
Detection wavelength: UV 210 nm
Injection volume 20 µL
*(A)
Add 31.2 g of sodium dihydrogen phosphate dihydrate to 1,000 mL of water to dissolve and then add phosphoric acid to adjust the pH to 2.8.
*
Adjust the flow rate so that the retention time of valine is about 2.5 minutes.

Sample Preparation Method:

Weigh 92 mg of L-isoleucine, dissolve in eluent, and adjust volume to 100 mL. The solution is filtered through a 0.45 µm filter prior to injection.

Identification:

Shown below are the system suitability requirements for the identification of isoleucine, leucine, and valine, as well as the analytical results. This comparison shows that the results meet the requirements for "peak retention times that are the same."

System Suitability for Standard

Item Requirement for system suitability Measurement result
System performance Elution order In order of Val, Ile, and Leu OK
Resolution for Ile -Leu NLT 1.5 2.55
System repeatability Relative standard deviation of each peak retention time (n = 6) NMT 1.0% 0.05% (Va)
0.09% (Iie)
0.09% (Leu)

Result for Identification

Component Requirement Retention time of standard (min) Retention time of sample (min)
Val: Valine Peak retention times are the same 2.51 2.50
Ile: Isoleucine 4.11 4.09
Leu: Leucine 4.51 4.50
*
The sample for this analysis was provided by Division of Physical Pharmaceutical Chemistry, Faculty of Pharmacy at Keio University

Assay Method by L-8900

Concentration of Each Component

Component Concentration in the sample for injection(ng/20 µL)
Gly: Glycine (internal standard) 400
Val: Valine 960
Ile: Isoleucine 800
Leu: Leucine 1,600

Analysis Conditions for Standard Analysis Method

Column #2622PH 4.6 mm I.D. × 60 mm
#2650L 4.6 mm I.D. × 40 mm
Ammonia filter column: PH Buffer Kit
Eluent 0.4 mL/min
Flow rate 57°C
Column temperature Ninhydrin coloring solution kit for HITACHI
Reaction reagent flow rate 0.35 mL/min
Reaction temperature 130°C
Detection wavelength VIS 570 nm
Injection volume 20 µL

Sample Preparation Method

Weigh 0.95 g of L-isoleucine. Add 10 mL of the internal standard solution (glycine)*, dissolve in 0.1 mol/L hydrochloric acid reagent, and adjust volume to 250 mL. Dilute 2 mL of the above solution to a volume of 200 mL with 0.02 mol/L hydrochloric acid. Filter the solution through a 0.2 µm filter.

*
Internal standard solution: Glycine in 0.1 mol/L hydrochloric acid reagent solution (1 g/20 mL)

Assay:

Shown below are the system suitability requirements for the assay as well as the analytical results, which meet the requirements.

System Suitability for Standards

Item Requirement for system suitability Measurement result
System performance Elution order In order of Val, Ile, and Leu OK
Resolution for Ile -Leu NLT 1.2 1.37
System repeatability Relative standard deviation for area ratio of the internal standard (Gly) peak to each peak (n = 6) NMT 1.0% 0.09% (Va)
0.17% (Iie)
0.09% (Leu)

Assay Result

Component Content/Label claim x 100 (%)
Required value Measurement result
Val: Valine 93.0 - 107.0 100.3
Ile: Isoleucine 98.4
Leu: Leucine 100.4
*
The sample for this analysis was provided by Division of Physical Pharmaceutical Chemistry, Faculty of Pharmacy at Keio University.

NOTE:
These data are an example of measurement; the individual values cannot be guaranteed.
The system is for research use only, and is not intended for any animal or human therapeutic or diagnostic use.

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