(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.
- Hashimoto, A., Oka, T.: Prog. Neurobiol., 52, 325 (1997).
Features
- 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
 |
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. |
 |
2. Buffer for (R)-BiAC-acetonitrile solution Add 3 mL of Acetonitrile to the Buffer for (R)-BiAC container and mix. |
Derivatization
Preparation
- Sample 15 μL + Buffer for (R)-BiAC-acetonitrile solution 30 μL + (R)-BiAC solution 10 μL (Derivatized solution)
- Pipetting x 5
Reaction
- Stirring
- Heating (55℃, 10 min.)
- Cooling (r.t.)
Stop the reaction
- Derivatized solution + Reaction Terminator 100 μL
LC/MS measurement
Analysis Example of Chiral Amino Acids in Human Urine*
Pretreatment conditions
- Sample/Standard solution 20 μL + Internal standard solution 20 μL + Acetonitrile 40 μL
- Stirring
- Certrifugation (20,000 × g, 10 min.)
Collection of supernatant
Derivatization
Analysis example of 15 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))
LC/MS/MS Chromatogram (human urine sample)
| 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 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
- Sample/Standard solution 20 μL + Internal standard solution 20 μL + Acetonitrile 40 μL
- Stirring
- Certrifugation (20,000 × g, 10 min.)
Collection of supernatant
Derivatization
Analysis example of 15 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))
LC/MS/MS Chromatogram (lactobacillus acid beverage sample)
| 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 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
- Harada, M. and Karakawa, S. et al.: J. Chromatogr. A, 1593, 91 (2019).
- Harada, M. and Karakawa, S. et. al.: Wako Junyaku Jiho, 87 (1), 5 (2019).
- Harada, M. and Karakawa, S. et. al.: Symmetry, 12, 913 (2020).
Product List
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Derivatization reagents
HPLC Column
Mixture Standard Solutions
Internal Standard Mixture Solution
For research use or further manufacturing use only. Not for use in diagnostic procedures.
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