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  2. Analytical Chemistry
  3. Amino Acid, Peptides & Proteins
  4. Amino Acid Analysis (Quantitative/Compositional Analysis)
  5. (R)-BiAC Method (Chiral Amino Acid LC/MS Analysis)
Derivatization reagent for LC/MS analysis of D/L-amino acids (chiral amino acids)

(R)-BiAC Method (Chiral Amino Acid LC/MS Analysis)

Amino acids are compounds that occur widely in living organisms and in nature. Amino acids include the enantiomers L-amino acid and D-amino acid, most of which exist as L-amino acids. In recent years, it has been revealed that D-amino acids, which exist in trace amounts, have functionalities such as involvement in memory and learning ability1), and it has become increasingly important to analyze them separately from L-amino acids. (R)-BiAC is a derivatization reagent for LC/MS analysis of D/L-amino acids (chiral amino acids) using a pre-column derivatization method. By using (R)-BiAC and the Derivatization Reagents Set, chiral amino acid analysis can be performed with high resolution and in a short time (19 min.), without the use of specialized equipment.

  1. Hashimoto, A., Oka, T.: Prog. Neurobiol., 52, 325 (1997).

Features

03157_img01.png
  • Chiral amino acids (D-amino acids / L-amino acids) can be analyzed by LC/MS without using a dedicated equipment!
    Derivatization with (R)-BiAC on a pre-column allows chiral amino acids (D-amino acids / L-amino acids) to be measured by LC/MS without the use of dedicated equipment.
  • Simultaneous analysis of 20 D/L-amino acids in about 19 minutes!
    Simultaneous analysis of multi-chiral amino acids (D-amino acids / L-amino acids) can be performed in a short time.
  • Chiral amino acids can be detected with high sensitivity!
    The high ionization efficiency of (R)-BiAC derivatized amino acids in MS allows detection of chiral amino acids (D-amino acids / L-amino acids) with a sensitivity at the atto mol level.
  • A dedicated reagents set for derivatization is available!
    The use of the pre-prepared (R)-BiAC Derivatization Reagents Set makes the derivatization process easier.

Derivatization Procedure Using (R)-BiAC Derivatization Reagents Set

Content of the (R)-BiAC Derivatization Reagents Set

Code No. Product Name Volume Amount
293-86011 Buffer for (R)-BiAC 3 mL 1
290-86021 Reaction Terminator 10 mL 1

Complementary products in addition to the (R)-BiAC Derivatization Reagents Set

Code No. Product Name Grade
012-19851 Acetonitrile for LC/MS
025-19761 (R)-BiAC for Amino Acid Analysis
235-64051 Wakopak® Ultra APDS TAG® φ2.1 mm × 100 mm (D) -

Preparation of reagent

03157_img02.png 1. (R)-BiAC solution
Rinse the entire bottle of (R)-BiAC with 1-2 mL of Acetonitrile and transfer the solution into another container. Add Acetonitrile until the total volume becomes 5 mL and mix until it completely dissolved.*
*After dividing the prepared solution into the required number, store it at -20℃, avoid repeated freezing and thawing, and use it as soon as possible. It can be stored at -20℃ for about a week.
03157_img03.png 2. Buffer for (R)-BiAC-acetonitrile solution
Add 3 mL of Acetonitrile to the Buffer for (R)-BiAC container and mix.

Derivatization

Preparation

  1. Sample 15 μL + Buffer for (R)-BiAC-acetonitrile solution 30 μL + (R)-BiAC solution 10 μL (Derivatized solution)
  2. Pipetting x 5

Reaction

  1. Stirring
  2. Heating (55℃, 10 min.)
  3. Cooling (r.t.)

Stop the reaction

  1. Derivatized solution + Reaction Terminator 100 μL

LC/MS measurement

Analysis Example of Chiral Amino Acids in Human Urine*

Pretreatment conditions

  1. Sample/Standard solution 20 μL + Internal standard solution 20 μL + Acetonitrile 40 μL
  2. Stirring
  3. Certrifugation (20,000 × g, 10 min.)

Collection of supernatant

Derivatization

Analysis example of 13 chiral amino acids (HPLC column: Wakopak® Ultra APDS TAG®)

LC/MS/MS Chromatogram (standard solution (D-amino acid: 5 μM L-amino acid: 50 μM))

03157_img11.png

LC/MS/MS Chromatogram (human urine sample)

03157_img12.png
Constituent Monitoring Ions (m/z) Constituent Monitoring Ions (m/z) Constituent Monitoring Ions (m/z)
Precursor Product Precursor Product Precursor Product
D-Asn 427 295.1 L-Glu 443 295.1 L-Met 445 295.1
L-Asn 428 295.1 D-allo-Thr 414 295.1 D-allo-Ile 426 295.1
D-Gln 441 295.1 L-Thr 415 295.1 L-Ile 427 295.1
L-Gln 442 295.1 D-Ala 384.1 295.1 D-Leu 426 295.1
D-Ser 400 295.1 L-Ala 385.1 295.1 L-Leu 427 295.1
L-Ser 401 295.1 D-Tyr 476 295.1 D-Phe 460.1 295.1
Gly 371 295.1 L-Tyr 477 295.1 L-Phe 461.1 295.1
D-Asp 428.2 295.1 D-Val 412 295.1 D-Trp 499 295.1
L-Asp 429.2 295.1 L-Val 413 295.1 L-Trp 500 295.1
D-Glu 442 295.1 D-Met 444 295.1
[Instrument]

Shimadzu Nexera X2 HPLC System / Sciex Triple QuadTM 6500

[HPLC]
Column
： Wakopak® Ultra APDS TAG® φ2.1 mm × 100 mm (D)
Column temperature
： 40℃
Eluent
： A) 0.1% HCOOH in 10 mM HCOONH3 aq., B) 95% CH3CN in H2O
Gradient
：
Time (min.) B conc. (%)
0-3 14-16
3-14.3 16-33
14.3-17 33-45
17-17.1 45-90
17.1-18 90
18-18.1 90-14
18.1-20 14
Flow rate
： 0.4 mL/min.
[MS]
Ionization
： ESI
Mode
： SRM

Analysis Example of Chiral Amino Acids in Lactic Acid Beverages*

Pretreatment conditions

  1. Sample/Standard solution 20 μL + Internal standard solution 20 μL + Acetonitrile 40 μL
  2. Stirring
  3. Certrifugation (20,000 × g, 10 min.)

Collection of supernatant

Derivatization

Analysis example of 13 chiral amino acids (HPLC column: Wakopak® Ultra APDS TAG®)

LC/MS/MS Chromatogram (standard solution (D-amino acid: 5 μM L-amino acid: 50 μM))

03157_img13.png

LC/MS/MS Chromatogram (lactobacillus acid beverage sample)

03157_img14.png
Constituent Monitoring Ions (m/z) Constituent Monitoring Ions (m/z) Constituent Monitoring Ions (m/z)
Precursor Product Precursor Product Precursor Product
D-Asn 427 295.1 L-Glu 443 295.1 L-Met 445 295.1
L-Asn 428 295.1 D-allo-Thr 414 295.1 D-allo-Ile 426 295.1
D-Gln 441 295.1 L-Thr 415 295.1 L-Ile 427 295.1
L-Gln 442 295.1 D-Ala 384.1 295.1 D-Leu 426 295.1
D-Ser 400 295.1 L-Ala 385.1 295.1 L-Leu 427 295.1
L-Ser 401 295.1 D-Tyr 476 295.1 D-Phe 460.1 295.1
Gly 371 295.1 L-Tyr 477 295.1 L-Phe 461.1 295.1
D-Asp 428.2 295.1 D-Val 412 295.1 D-Trp 499 295.1
L-Asp 429.2 295.1 L-Val 413 295.1 L-Trp 500 295.1
D-Glu 442 295.1 D-Met 444 295.1
[Instrument]

Shimadzu Nexera X2 HPLC System / Sciex Triple QuadTM 6500

[HPLC]
Column
： Wakopak® Ultra APDS TAG® φ2.1 mm × 100 mm (D)
Column temperature
： 40℃
Eluent
： A) 0.1% HCOOH in 10 mM HCOONH3 aq., B) 95% CH3CN in H2O
Gradient
：
Time (min.) B conc. (%)
0-3 14-16
3-14.3 16-33
14.3-17 33-45
17-17.1 45-90
17.1-18 90
18-18.1 90-14
18.1-20 14
Flow rate
： 0.4 mL/min.
[MS]
Ionization
： ESI
Mode
： SRM

* These data is an example of analysis. Ion source parameters, etc. may differ depending on the instrument used.

References

  1. Harada, M. and Karakawa, S. et al.: J. Chromatogr. A, 1593, 91 (2019).
  2. Harada, M. and Karakawa, S. et. al.: Wako Junyaku Jiho, 87 (1), 5 (2019).
  3. Harada, M. and Karakawa, S. et. al.: Symmetry, 12, 913 (2020).

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Derivatization reagents

HPLC Column

Mixture Standard Solutions

*For composition information, please click here.

Internal Standard Mixture Solution

For research use or further manufacturing use only. Not for use in diagnostic procedures.

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