Home Small Molecule HPLCAnalysis of 20 Underivatized Amino Acids

LC-MS/MS Analysis of Underivatized Amino Acids on Supel^™ Carbon LC Column

Amino acids are the building blocks of proteins and peptides in biological systems. Amino acids have also been used as supplements to support health and as indicators for certain diseases. The challenge with amino acids analysis lies in the wide- ranging polarities across all 20 amino acids (Figure 1). Due to this complexity, amino acid analysis methods in the past have relied on derivatization of amino acids; however, derivatization can lead to further complexity due to the presence of derivatized/underivatized amino acids and interferences from the derivatizing reagent itself.

This application outlines an LC-MS/MS method (Table 1) for analyzing all 20 amino acids, without derivatization, utilizing the Supel^™ Carbon LC column. All 20 amino acids are retained on the column under reversed phase conditions, with good peak shape.

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Experimental
Results
Conclusion

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Chemical structures of 20 amino acids are presented in 5 rows, each with their three-letter codes. The first row displays Gly, Ser, Ala, Pro, Val, and Asp from left to right. The second row shows Gln, Glu, Lys, and Cys. The third row includes Arg, Leu, Ile, and His. The fourth row features Tyr, Met, Phe, and Trp. The fifth and final row has Asn on the left and Thr on the right.

Figure 1.Structures and three letter codes of amino acids.

Experimental

Table 1.LC Conditions used for analysis of underivatized amino acids
LC Conditions
Column:	Supel^™ Carbon LC, 2.7 µm, 10 cm x 2.1 mm I.D. (59986-U)
Mobile phase:	[A] Water (0.1% (v/v) DFA); [B] Acetonitrile (0.1% (v/v) DFA)
Gradient:	Hold at 0% B for 7 min; 0% B to 5% B in 5 min; 5% B to 100% B in 10 min
Flow rate:	0.2 mL/min
Column temp.:	12 °C
Detector:	MSD, ESI(+), MRM (Table 2 & 3)
Injection:	1.0 μL
Sample:	Amino Acid Mix, varied concentrations (Table 4) in water (0.1% (v/v) DFA)

Table 2.MS conditions for the detection of amino acids
Ion Source Type:	Turbo Spray
Polarity:	ESI(+)
Curtain Gas:	25
Ion Spray Voltage:	4000 V
Temperature:	300 °C
Ion Source Gas 1:	20
Ion Source Gas 2:	30
Interface Heater:	On

Table 3.MRM fragmentation parameters and ions used for the 20 amino acids
Name	Q1	Q3	DP	EP	CEP	CE	CXP
Lysine	147.1	84.0	21.7	7.0	5.3	21.0	3.0
Proline	116.1	70.1	22.2	7.9	6.2	19.1	4.1
Aspartic Acid	134.1	74.0	17.0	8.3	12.8	17.0	3.7
Serine	106.1	60.0	13.0	4.0	8.8	15.0	3.1
Glycine	76.1	30.0	7.0	7.0	4.9	17.0	5.5
Alanine	90.1	44.0	16.0	6.0	4.9	16.9	5.6
Threonine	120.1	56.0	19.0	5.3	7.0	22.0	3.3
Asparagine	133.1	87.0	17.0	3.3	8.2	14.0	2.6
Valine	118.1	72.0	14.5	6.0	7.5	14.0	3.2
Glutamine	147.1	84.0	12.0	6.8	6.1	23.0	3.5
Leucine	132.1	86.0	16.0	7.4	10.2	14.0	3.2
Glutamic Acid	148.1	84.0	24.0	6.1	9.0	20.0	3.0
Isoleucine	132.1	86.0	16.0	7.4	10.2	14.0	3.2
Methionine	150.2	104.0	16.0	5.5	11.8	13.6	3.9
Histidine	156.1	110.0	24.0	3.5	11.0	19.0	3.7
Arginine	175.2	70.0	25.2	6.7	10.0	37.7	3.0
Phenylalanine	166.2	120.2	18.0	7.0	9.1	16.8	3.2
Tryptophan	205.2	146.2	19.4	4.8	17.7	23.0	4.0
Tyrosine	182.2	136.2	19.2	7.7	6.4	18.04	4.0
Cystine	241.2	120.0	26.0	7.8	9.8	26.0	4.1

Results

Figure 2 displays the MS spectral results for the analysis of 20 underivatized amino acids while Table 2 displays the optimized MS conditions for the separation, and Table 3 displays the fragment ions and fragmentation parameters for the amino acids.

A plot with intensity on the y-axis and time in minutes on the x-axis shows separated peaks for 20 underivatized amino acids of varying heights. The x-axis ranges from 1 to 22 minutes in 1-minute intervals, and the y-axis has major tick marks at 20,200, 40,200, 60,200, 80,200, 100,200, and 120,200. The top left shows a blown up version of the chromatogram measured between 1 to 3 minutes showing a clear peak for the amino acid glycine in purple color.

Figure 2.Separation of 20 underivatized amino acids by LC-MS/MS at various concentrations (Table 4).

Table 4.Twenty underivatized amino acids determined by LC/MS - Elution order, concentrations and retention times
Peak	Compound	Concentration (μg/mL)	Retention Time (min)
1	Glycine	20	1.27
2	Serine	2	1.43
3	Alanine	2	1.45
4	Lysine	1.5	1.54
5	Threonine	2	1.75
6	Proline	0.5	2.03
7	Asparagine	2.5	2.29
8	Aspartic Acid	2.5	2.65
9	Valine	0.5	2.85
10	Glutamine	1.5	3.69
11	Glutamic Acid	1	5.13
12	Leucine	0.5	5.18
13	Cystine	7.5	7.34
14	Isoleucine	0.75	8.93
15	Methionine	1.5	12.61
16	Histidine	0.5	12.81
17	Arginine	1.5	13.30
18	Phenylalanine	0.25	16.03
19	Tyrosine	5	16.29
20	Tryptophan	5	18.42

Conclusion

This application demonstrated the effectiveness of the Supel^™ Carbon LC column in the resolution of amino acids without the need for a derivatization reagent. Qualitative analysis of amino acids is typically done with specialty, silica-based columns, ion exchange columns, or normal phase columns. However, by utilizing tandem MS/ MS detection, analyses of amino acids can be accomplished on Supel^™ Carbon LC column with fast run times compared to most commercial approaches. Due to porous graphitic carbon’s unique ability to discriminate three- dimensional differences between compounds, leucine and isoleucine can be resolved with exceptional resolution

Amino Acid Standards

LC-MS/MS Analysis of Underivatized Amino Acids on Supel™ Carbon LC Column

Experimental

Results

Conclusion

LC-MS/MS Analysis of Underivatized Amino Acids on Supel^™ Carbon LC Column