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DL-Amino Acid Labeling Kit

(D)FDLDA; Amino acid labeling agent for HPLC analysis

This product is an amino acid labeling kit for HPLC analysis. Due to their high polarity and insufficient UV absorbance, amino acids need to be labeled prior to HPLC. In addition, research on D-amino acids has recently been gaining prominence. With conventional labeling reagents, such as phenyl isothiocyanate and dansyl chloride, costly chiral columns are required. This product is designed to label amino acids without intensive labor and achieve chiral separation with achiral columns, such as C18.

  • Simple protocol
  • Chiral separation with C18 column
  • Compatible with high-sensitivity MS analysis

HPLC application

DL-amino acid separation (protocol 1)

19 DL-amino acids are separable (glycine is not, as it is achiral).

DL-amino acid chromatograms

* (D)FDLDA (Hydrolysed), ** (D)DLDA-S-C6H12OH: Both peaks are derived from the labeling agent itself. See figure 1 on page 2 for details.

(Condition)
ColumnCOSMOSIL 5C18-AR-II 4.6 mm I.D. x 150 mmFlow rate1.0 mL/min
Mobile phaseA: 0.1 % Formic Acid - 20 % Acetonitrile
B: 0.1 % Formic Acid - 50 % Acetonitrile
B conc. 0 → 100% 20 min Linear gradient
Temperature30°C
DetectionUV 340 nm
Sample(DL)Amino Acid-(D)DLDA derivatives

Kit components

  Name PKG size Required volume / assay
1 Sample*1 User-supplied 100 μL
2 Labeling solution*2 10 mL 100 μL
3 Initiator solution 10 mL 100 μL
4 Delabeling solution
(for side chain)*3
10 mL 100 μL
5 Stop solution 10 mL 100 μL
6 Methanol or acetonitrile User-supplied 500 μL or 600 μL

*1 Total amount of functional groups to be reacted should be less than 1.0 μmol. If higher, dilute or reduce sample amount.
*2 Uses (D)FDLDA as the labeling reagent.
*3 Contains 6-mercapto-1-hexanol [M.W.: 134.24 (C6H14OS)]

structure of (D)FDLDA

Protocol

Two protocols are available

The labeling reagent reacts with amino groups. In addition, some side chains, such as the phenolic hydroxyl group of tyrosine and the thiol group of cysteine, are also labeled. Protocol 1 (step 2) achieves removal of labeling agents on side chains (except for Lys).
Protocol 1
Step 1: Add 100 µL each of sample solution, labeling agent solution, and start solution to a glass vessel, seal the vessel, mix in a vortex mixer for 5 seconds, and then react at 50°C for 2 hours.
Step 2: Add 100 µL of the delabeling agent solution for side chain, mix in a vortex mixer for 5 seconds, and then react at 50°C for 15 minutes.
Step 3: Add 100 µL of stop solution and 500 µL of acetonitrile or methanol, then analyze by HPLC (after filtration, if necessary).
Protocol 2
Step 1: Add 100 µL each of sample solution, labeling agent solution, and start solution to a glass vessel, seal the vessel, mix in a vortex mixer for 5 seconds, and then react at 50°C for 2 hours.
Step 2: Add 100 µL of stop solution and 600 µL of acetonitrile or methanol, then analyze by HPLC (after filtration, if necessary).
  
 
reaction diagram

mono: Only the amino group of the α-carbon is labeled.
di: In addition to the α-carbon, the functional groups on the side chains are also labeled.

Figure 1: HPLC peaks derived from labeling reagent
Name Molecular weight Description
(D)FDLDA 385.39 (C16H24FN5O5) Unreacted labeling reagent (only appears with protocol 2)
(D)FDLDA(Hydrolysed) 383.40 (C16H25N5O6) Hydrolysate of labeling reagent
(D)FDLDA-S-C6H12OH 499.63 (C22H37N5O6S) Reaction product of labeling reagent and delabeling reagent (only appears with protocol 1)

It is strongly recommended that a blank analysis is performed prior to analyzing your sample.

LC/MS application

Comparison with other labeling reagents

For chiral separation of amino acids by achiral columns, (L)FDAA or (L)FDLA are well-known labeling reagents based on Marfey's method. This kit’s labeling reagent (D)FDLDA is easily ionized at the red circle in the structure below, so MS sensitivity is higher compared to the conventional method, while UV sensitivity is about the same. Since the D-form labeling reagent is used, elution order is reversed compared to the conventional method.

MS-ESI(+) chromatograms showing improved sensitivity with the labeling kit
Condition
ColumnCOSMOCORE 2.6C18 2.1 mm I.D. - 100 mmFlow rate0.2 mL/min
Mobile phaseA: 0.1 % Formic Acid - 20 % Acetonitrile
B: 0.1 % Formic Acid - 70 % Acetonitrile
B conc. 0 → 100% 20 min Linear gradient
Temperature40°C
DetectionUV 340 nm, MS-ESI (+)
SampleLabeled Leucine
1. D form
2. L form

DL -amino acid separation (protocol 2)

separation of various DL-amino acids using protocol 2
Condition
ColumnCOSMOCORE 2.6C18 2.1 mm I.D. - 100 mmFlow rate0.2 mL/min
Mobile phaseA: 0.1 % Formic Acid - 20 % Acetonitrile
B: 0.1 % Formic Acid - 50 % Acetonitrile
B conc. 0 → 100% 20 min Linear gradient
Temperature40°C
DetectionMS-ESI (+)
Sample(DL)Amino Acid-(D)DLDA derivatives

Quantitative performance

Different concentrations of amino acids labeled with this kit were analyzed by HPLC. The calibration curve shows high linearity over the tested concentrations.

calibration curve for various L-amino acids

Labeling reaction

Protocol 1 : (L)Tyr (mono)
Protocol 2 : (L)Tyr (di), (L)Val, (L)Pro

Other application data

Commercial sports drink (protocol 2 with HPLC)

chromatogram of various L-amino acids

 

MEM non-essential amino acids solution (protocol 1 with LC/MS)

chromatogram of MEM non-essential amino acids

 

Commercial amino acid soft drink (protocol 2 with HPLC)

chromatogram of L-ornithine

 

Histamine (protocol 1 with HPLC)

chromatogram of histamine

In addition to amino acids, this product is usable for other amine and thiol compounds.

Comparison of DL-Amino Acid Labeling Kit and Achiral Labeling Reagents

A comparison of the below labeling reagents was performed using pre-column derivatization.

  1. DL-Amino Acid Labeling Kit (Cat. No.: 19942-74)
  2. Dabsyl Chloride (Cat. No.: 10427-91)
  3. o-Phthalaldehyde (cat. No.: 27810-44)

Optical separation of DL enantiomers

Labeled DL-phenylalanine was analyzed using a C18 column. The labeling reagent in the DL-Amino Acid Labeling Kit has a chiral carbon, resulting in the labeled D and L forms being diastereomers. This makes them separable on C18 columns.

* Generally, fluorescence detection is used for amino acids labeled with o-phthalaldehyde.

 

Detection sensitivity with mass spectrometry (MS)

Detection sensitivity was compared using labeled L-phenylalanine as the sample. Both the DL-Amino Acid Labeling Kit and dabsyl chloride had high MS sensitivity, due to their easily ionizable structures.

Stability of labeled samples

Labeled L-phenylalanine was analyzed by HPLC 1, 3, and 7 days after labeling. The sample labeled with o-phthalaldehyde was not stable, and therefore should not be labeled manually, but rather using online analysis within the instrument.

Days after labelingDL-Amino Acid Labeling KitDabsyl chlorideo-Phthalaldehyde
1100%100%100%
3113%95%0%
7106%96%0%

* Analysis conditions are as in "Optical separation of DL enantiomers". The peak area measured after 1 day was taken as 100%. Samples were stored refrigerated.

Alternative selectivity with Cholester

Cholester offers alternative selectivity, which can resolve overlapping peaks caused by the labeling reagents. This can be useful for quantification with a UV detector.

When used together with a C18 column, the widest range of selectivity is achieved.

Condition
ColumnCOSMOSIL 5C18-AR-II 4.6 mm I.D. x 150 mmFlow rate1.0 mL/min
Mobile phaseA: 0.1 % Formic Acid - 20 % Acetonitrile
B: 0.1 % Formic Acid - 50 % Acetonitrile
B conc. 0 → 100% 20 min Linear gradient
Temperature30°C
DetectionUV 340 nm
Sample(DL)Amino Acid-(D)DLDA derivatives

Downloads / Reference

 DL-Amino Acid Labeling Kit (Instruction)

 DL-Amino Acid Labeling Kit (Brochure)

 

Reference

  • Kuranaga, T.; Minote, M.; Morimoto, R.; Pan, C.; Ogawa, H.; Kakeya, H. Highly Sensitive Labeling Reagents for Scarce Natural Products. ACS Chem. Biol. 2020, 15(9), p. 2499-2506.

Ordering Information

Product Cat.No. Storage PKG Size Price(US$)  
DL-Amino Acid Labeling Kit 19942-74 15-25℃ 100 tests 330.00 Buy

Storage: Avoid extreme heat (store at 15-25℃)
Shipping at room temperature