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.

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

Features
Derivatization Procedure Using (R)-BiAC Derivatization Reagents Set
Analysis Example of Chiral Amino Acids in Human Urine*
Analysis Example of Chiral Amino Acids in Lactic Acid Beverages*
Product List
Related Information

Features

Chiral 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 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 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 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

Analysis Example of Chiral Amino Acids in Human Urine*

Pretreatment conditions

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)
Constituent	Precursor	Product	Constituent	Precursor	Product	Constituent	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