Fast, Direct Analysis of Underivatized Amino Acids in Infant Formula

• Simple, one-step sample preparation for direct analysis of underivatized amino acids.
• Simultaneous analysis of nonpolar, polar, positively charged, and negatively charged amino acids in a short 10-minute run.
• Well-designed gradient separates contaminants from target analytes.

To ensure that nutritional requirements are met, reliable analytical methods are needed for the accurate determination of free amino acids in infant formulas. Precolumn derivatization followed by reversed-phase LC analysis is a typical approach, with derivatization being necessary due to the lack of chromatographic retention and poor sensitivity for free amino acids. However, these methods are often time-consuming and labor intensive. The use of perfluorinated acids as ion-pairing reagents to improve the retention of underivatized amino acids on a C18 column is another common technique, but this practice can negatively impact the chromatographic system and mass spectrometer.

In the simpler approach shown here, underivatized amino acids can be measured directly—following a single-step sample preparation procedure—using a Raptor Polar X column paired with an MS/MS detector. Raptor Polar X columns feature a hybrid phase chemistry (HILIC and ion exchange) that provides the balanced retention needed for simultaneous analysis of a wide range of analyte chemistries. As demonstrated, underivatized amino acids with nonpolar, polar, positively charged, and negatively charged side chains were adequately retained and then quickly eluted using a 10-minute gradient. This gradient also separated amino acid system contaminants from target analytes, adding ruggedness to the method. By following this method, time- and labor-intensive sample preparation procedures, which can include expensive derivatization kits, are replaced by a simple protein precipitation and direct analysis of the resulting extract. Direct analysis of underivatized amino acids on a Raptor Polar X column provides excellent results in a fast, easy workflow, making this a beneficial alternative to traditional methods.

	Peaks	tR (min)	Precursor Ion	Product Ion
1.	Tryptophan	1.17	205.07	146.08
2.	Phenylalanine	1.26	166.13	120.10
3.	Leucine	1.41	132.13	86.10
4.	Isoleucine	1.55	132.13	86.10
5.	Methionine	1.62	150.07	104.10
6.	Tyrosine	1.69	182.10	136.08
7.	Taurine	1.91	126.07	108.07
8.	Valine	1.98	118.13	72.11
9.	Proline	2.29	116.13	70.09
10.	Alanine	3.00	90.03	44.10

	Peaks	tR (min)	Precursor Ion	Product Ion
11.	Threonine	3.12	120.13	74.08
12.	Glycine	3.62	76.10	30.11
13.	Glutamine	3.87	147.13	84.07
14.	Serine	3.93	106.07	60.09
15.	Asparagine	4.08	133.13	74.07
16.	Arginine	4.47	175.17	70.09
17.	Histidine	4.66	156.07	110.16
18.	Lysine	4.97	147.13	84.13
19.	Glutamic acid	5.89	148.10	84.10
20.	Cystine	6.10	241.13	152.00
21.	Aspartic acid	7.12	134.07	74.06

Column	Raptor Polar X (cat.# 9311A12)
Dimensions:	100 mm x 2.1 mm ID
Particle Size:	2.7 µm
Temp.:	30 °C
Standard/Sample
Diluent:	20:80 Water:acetonitrile, 0.01 N HCl
Conc.:	Endogenous amino acids
Inj. Vol.:	5 µL
Mobile Phase
A:	Water, 0.5% formic acid
B:	Mobile Phase B: 9:1 Acetonitrile:20 mM ammonium formate in water (pH 3.0) (The ammonium formate concentration is 20 mM relative to the total volume of mobile phase B. See preparation notes for instructions on diluting a 200 mM aqueous starting solution.)
	Time (min) Flow (mL/min) %A %B 0.00 0.5 12 88 3.50 0.5 12 88 8.00 0.5 70 30 8.01 0.5 12 88 10.0 0.5 12 88

Detector	MS/MS
Ion Mode:	ESI+
Mode:	MRM
Instrument	UHPLC
Sample Preparation	A 200 µL aliquot of protein hydrolysate formula (Similac ALIMENTUM) was mixed with 800 µL of acetonitrile and 10 µL of 1 N HCl. After centrifugation at 4000 rpm for 5 minutes, the supernatant was diluted 20-fold with 20:80 water:acetonitrile (0.01 N HCl) and injected for analysis.
Notes	Mobile Phase B Preparation: To make 500 mL of mobile phase B, measure ~45 mL of water into a small beaker and add 1 mL of 10 M ammonium formate solution. Adjust pH to 3.0 by adding formic acid and then bring the volume to 50 mL with water. Combine this 50 mL ammonium formate solution (pH 3.0) with 450 mL of acetonitrile to complete the preparation.

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