AL
Table 4 – Curve fi t and LOD/LOQ results Amino acid R2 Ala
values
Arg Asp Cys Glu Gly His Ile
Leu Lys
Met Phe Pro Ser Thr Tyr Val
Figure 3 – Results of fi ve-level calibration suite for aspartic acid, glycine, histidine and methionone (n = 3).
In addition, the results demonstrate exceptional linearity and retention time repeatability over the tested concentration range.
The R2
coeffi cients for the 17 amino acids are seen in Table 4, which dem- onstrates that all values exceeded 0.999 (n = 3 at each level).
As shown in Table 4, limit of detection (LOD) and limit of quantitation (LOQ) levels were established for each analyte, based on a signal-to-noise (S/N) of >3/1 for the LOD and >10/1 for the LOQ. The considerably higher values for alanine are due to its lower ionization effi ciency.
Conclusion
Apart from the higher limit for alanine, which was due to its lower ioniza- tion effi ciency, the LODs for all other analyzed amino acids ranged from 0.12 to 1.44 nmol/mL. Also, with the exception of alanine, all LOD/LOQ values were more than fi vefold lower than historical values obtained for OPA-derivatized amino acids.3
This work demonstrates a simple, rapid and direct method using HPLC coupled with MS detection for the determination of amino acids, off ering high sensitivity and selectivity without pre- or post-column derivatization.
AMERICAN LABORATORY 25
References 1. Agrafi otou, P.; Sotiropoulos, S. et al. Direct RP-HPLC determination of underivatized amino acids with online dual UV absorbance, fl uores- cence and electrochemical detection. J. Sep. Sci. 2009, 32, 949–54.
2.
www.perkinelmer.com/Content/EHApplicationNotes/012533_01%20 The%20Analysis%20of%20Underivatized%20Amino%20Acids%20 by%20HPLC%20with%20MS%20Detection.pdf
3. Bartolomeo, M.P. Maisano, F. Validation of a reversed-phase HPLC method for quantitative amino acid analysis. J. Biomol. Tech. 2006, 17, 131–7.
Chi Man Ng, at the time this article was written, was a strategic applications specialist with PerkinElmer Inc. Wilhad M. Reuter is currently a strategic appli- cations leader with PerkinElmer, 710 Bridgeport Ave., Shelton, Conn. 06484, U.S.A.; tel.: 203-925-4602; e-mail:
wilhad.reuter@
perkinelmer.com; www.
perkinelmer.com
APRIL 2016
0.99967 0.99908 0.99998 0.99982 0.99987 0.99989 0.99983 0.99998 0.99996 0.99915 0.99985 0.99995 0.99997 0.99982 0.99983 0.99989 0.99983
LOD
(nmol/mL) 35.07
0.360 1.104 1.439 0.129 0.631 0.428 0.157 0.155 0.779 0.147 0.198 0.121 0.246 0.549 0.261 0.199
LOQ
(nmol/mL) 116.9
1.200 3.680 4.798 0.430 2.104 1.426 0.524 0.516 2.596 0.490 0.659 0.404 0.820 1.830 0.870 0.662
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