AL
Table 2 – MS and ionization method parameters
Ionization mode Electrospray (+) Capillary voltage 3.50 kV Source temp Cone voltage
150 ºC 35 V
Desolvation temp 400 ºC Desolvation gas 700 L/hr Cone gas fl ow 25 L/hr
Empower 3 chromatography data software (CDS) platform.2
The chromatographic sep-
aration system combines the flexibility of quaternary solvent along with a flow-through, direct sampling needle injector, allowing low dispersion, high resolution, high sensitivity and improved throughput. The MS detector features a single, high-resolution quadrupole mass analyzer utilizing an electrospray ioniza- tion (ESI) source and is fully integrated into the CDS platform. This enables the user to set up a single method, covering chromatographic separation and MS conditions, together with comprehensive reporting capabilities that permit full traceability and data integrity.
analyze underivatized amino acids provides the advantages of increased convenience, simplicity and repeatability, while still provid- ing the desired sensitivity and separation speed. Eliminating the derivatization steps also reduces the possibility of side reactions, interferences from reagents and sample con- tamination or degradation.1
Experimental An Altus HPLC system was used for the chromatographic separations, and included the solvent and sample module, integrated vacuum degasser/column oven and an MS detector (all from PerkinElmer Inc., Shelton, Conn.). Instrument control, analysis and data processing was performed using the Waters
Method parameters HPLC method parameters are shown in Table 1; Table 2 lists the MS and ionization method parameters.
Solvents, standards and samples All solvents and diluents used were HPLC grade and fi ltered via 0.45-μm fi lters. The diluent was 0.1N HCl. The Pierce 17-Amino Acid Standard H Mix (protein hydrolysate) was obtained from Thermo Fisher Scientifi c (Rockford, Ill.). This included alanine (Ala), arginine (Arg), aspartic acid (Asp), cysteine (Cys), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tyrosine (Tyr) and va- line (Val). The concentration for each amino
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Figure 1 – Overlay of SIRs for 17 analytes: 1) Phe, 2) Tyr, 3) Leu, 4) Met, 5) Ile, 6) Val, 7) Glu, 8) Pro, 9) Thr, 10) Asp, 11) Ala, 12) Ser, 13) Gly, 14) Cys, 15) His, 16) Lys, 17) Arg.
AMERICAN LABORATORY 23 APRIL 2016
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