LC-MS Analysis of Amino Acids on a Novel Mixed-Mode HPLC Column

Transcription

1 Liquid Chromatography Mass Spectrometry SSI-LCMS-022 LC-MS Analysis of Amino Acids on a ovel Mixed-Mode PLC Column LCMS-8040 Background There are four established methods for analyzing amino acids: prelabeled, post-labeled, ion-pairing reversed-phase, and normal-phase, but each of these methods has disadvantages. The pre-labeled method has problems with derivatization efficiency and cost, while the post-labeled method is usually not compatible with LC-MS due to non-volatile mobile phases. The ion-pairing reversed-phase method has difficulty separating polar amino acids, whereas, the normal-phase mode has problems separating all the compounds, especially the Leucine and Isoleucine isomers. To alleviate these challenges, Imtakt developed the Intrada amino acid separation column for LC-MS/MS which can separate 55 amino acids in 10 minutes using a mixed-mode stationary phase without utilizing either pre- or postcolumn labelling methods. Additionally, the column can separate gamma-aminobutyric acid (GABA) isomers and dipeptides also without derivatization. Imtakt has optimized separation and detection characteristics utilizing the Shimadzu LCMS-8040 and LCMS Method This mixed-mode stationary phase for LC-MS analysis of amino acids consists of 3µm silica particles modified with ion ligands which couples ion exchange with normal-phase interactions. All amino acid and bovine sera standards were purchased commercially. Each figure below provides instrument parameters specific to each acquisition. 1

2 LCMS 2020 Analysis of 20 Amino Acids Standard Arg (m/z 175.1) Lys (m/z 147.0) is (m/z 156.0) (Cys)2 (m/z 241.0) Asn (m/z 133.0) Gly (m/z 76.4) Gln (m/z 147.0) Ser (m/z 106.1) Ala (m/z 90.4) Asp (m/z 134.0) Thr (m/z 120.1) Pro (m/z 116.0) Glu (m/z 148.0) Val (m/z 118.0) % Leu, Ile (m/z 132.0) Met (m/z 150.0) A: acetonitrile / tetrahydrofuran / 25 mm ammonium formate / formic acid Tyr (m/z 182.0) = 9 / 75 / 16 / 0.3 B: acetonitrile / 100 mm ammonium formate = 20 / 80 Phe (m/z 166.0) 0 %B (0-2.5min), 0-17 %B ( min), 100 %B ( min) 0.6 ml/min (6MPa), 35 deg.c, 5 ul, Trp (m/z 205.0) Figure 1: 20 standard amino acids separated and detected on LCMS Leucine and Isoleucine isomers were also separated in several minutes. Pro (Q1/Q /70.1) Glu (Q1/Q /84.1) Val (Q1/Q /72.05) Leu, Ile (Q1/Q /86.15) Met (Q1/Q /56.1) Tyr (Q1/Q /136.25) Phe (Q1/Q /120.1) Trp (Q1/Q /188.1) Lys (Q1/Q /84.1) is (Q1/Q /110.1) (Cys) 2 (Q1/Q /151.95) Asn (Q1/Q /74.05) Gly (Q1/Q3 75.8/30.1) Gln (Q1/Q /84.1) Ser (Q1/Q /60.2) Ala (Q1/Q3 90.1/44.1) Thr (Q1/Q /74.0) Asp (Q1/Q /73.95) Arg (Q1/Q /70.1) A: AC / TF / 25 mm C4 /C = 9 / 75 / 16 / 0.3 B: AC / 100 mm C4 = 20 / 80 0 %B (0-3 min) 0-17 %B (3-6.5 min) 100 %B ( min) 0.6 ml/min, 40 deg.c, 1 ul (0.1 Cl) LCMS-8040, ESI (MRM, positive) 100 nmol/ml amino acids standard mixture min Figure 2: 20 standard amino acids were separated and detected using the LCMS

3 Calibration Standard Curve for Various Amino Acids x Aromatic Amino Acid R2 = Trp x Acidic Amino Acid R2 = Glu nmol/ml nmol/ml x Aliphatic Amino Acid R 2 = x Basic Amino Acid R2 = Leu Arg nmol/ml nmol/ml Figure 3: Typical standard (aromatic, aliphatic, acidic and basic) amino acids showed good results for linearity and sensitivity. Glu (m/z 148.0) Thr (m/z 120.1) Ala (m/z 90.4) Val (m/z 118.0) Tyr (m/z 182.0) Pro (m/z 116.0) Met (m/z 150.0) Leu, Ile (m/z 132.0) Trp (m/z 205.0) Phe (m/z 166.0) Arg (m/z 175.1) Lys (m/z 147.0) is (m/z 156.0) (Cys)2 (m/z 241.0) Asn (m/z 133.0) Gln (m/z 147.0) Ser (m/z 106.1) Gly (m/z 76.4) Asp (m/z 134.0) A: acetonitrile / formic acid = 100 / 0.1 B: 100 mm ammonium formate 14 %B (0-3min), %B (3-10 min), 14 %B (10-12 min) 0.6 ml/min (4MPa), 35 deg.c, 5 ul 0 min Figure 4: Simple gradient elution condition was also successful for standard amino acids separation in 10min with low pressure. 3

4 5min igh Throughput Analysis Arg (m/z 175.1) Lys (m/z 147.0) is (m/z 156.0) Intrada Amino Acid, 150 x 2 mm A: tetrahydrofuran / 20 mm ammonium formate / formic acid = 75 / 25 / 0.3 B: 200 mm ammonium formate / acetonitrile = 80 / 20 0 %B (0-2.5 min), 100 %B (2.5-5 min) 0.6 ml/min (45 MPa), 37 deg.c, 5 ul Gly (m/z 75.8) Asn (m/z 133.0) Gln (m/z 147.0) Ala (m/z 89.9) Ser (m/z 105.9) Thr (m/z 119.9) Pro (m/z 116.0) Val (m/z 118.0) (Cys)2 (m/z 241.0) Asp (m/z 133.9) Glu (m/z 148.0) Leu, Ile (m/z 132.0) Met (m/z 150.0) Phe (m/z 166.0) Tyr (m/z 182.0) Trp (m/z 205.0) min Figure 5: igh throuput analysis using exara UPLC LCMS-2020 system was achieved in 5min. Application for Amino Acids in Serum Bovine Serum (0.4 Cl4 treatment) Arg (m/z 175.1) Lys (m/z 147.0) is (m/z 156.0) (Cys) 2 (m/z 241.0) Asn (m/z 133.0) Gly (m/z 76.4) Gln (m/z 147.0) Ser (m/z 106.1) Ala (m/z 90.4) Asp (m/z 134.0) Thr (m/z 120.1) Pro (m/z 116.0) Glu (m/z 148.0) Val (m/z 118.0) A: AC / TF / 25 mm C4 /C Leu, Ile (m/z 132.0) = 9 / 75 / 16 / 0.3 B: AC / 100 mm C4 = 20 / 80 Met (m/z 150.0) 0 %B (0-2.5 min) 0-17 %B ( min) Tyr (m/z 182.0) 100 %B ( min) Phe (m/z 166.0) 0.6 ml/min(6mpa), 35 deg.c, 1 ul (0.1 Cl) Trp (m/z 205.0) min Figure 6: The novel amino acid analysis column was applied for amino acids in serum and several amino acids were detected using the LCMS

5 SSI-LCMS-022 Dipeptide Analysis 2 S S 2 L-cystine ((Cys)2) Intrada Amino Acid, 75 x 2 mm A: acetonitrile / formic acid = 100 / 0.3 B: 100 mm ammonium formate %B (0-10 min), 100 %B(10-12 min) 0.3 ml/min (5 MPa), 35 deg.c 2 ul ( ug, 0.1-Cl aq.) 2 -(L-alpha-glutamyl) -L-glutamic acid (Glu-Glu) 2 L-anserine 2 L-carnosine -L-histidyl-L-leucine (is-leu) min Figure 7: The amino acid analysis column could be applied for dipeptides using the LCMS aminobutyric acid (beta-aminobutyric acid) m/z aminobutyric acid (alpha-aminobutyric acid) m/z aminobutyric acid (GABA, gamma-aminobutyric acid) m/z Intrada Amino Acid, 75 x 2 mm A: acetonitrile / formic acid = 100 / 0.3 B: 100 mm ammonium formate %B (0-8 min), 15 %B (8-10 min) 0.3 ml/min (4 MPa), 35 deg.c 5 ul (10 ug/ml, 0.1-Cl aq.) ESI (SIM, positive, m/z 104.1) min Figure 8: ot only alpha-amino acids but also beta- and gamma-amino acids isomers were well separated. 5

6 Intrada Amino Acid, 10 x 2 mm A: acetonitrile / formic acid = 100 / 0.1 B: 100mM ammonium formate %B (0-0.8 min), 100 %B ( min) 0.4 ml/min (1.6 MPa), 35deg.C, 1 ul (0.1 Cl) C2 + C m/z (240 nmol/ml) ornithine m/z (170 nmol/ml) m/z (300 pmol/ml) m/z (1.6 umol/ml) citrulline carnitine S taurine sec Figure 9:Amino acid related compounds were analyzed by one-munite high throuhput separation with 10mm column using the LCMS L-leucine allo-l-isoleucine 2 L-isoleucine Intrada Amino Acid, 150 x 3 mm A: Me / Water / Formic acid = 85 / 15 / 0.3 B: AC / 100mM Ammonium formate = 20 / 80 Gradient Conditions: %B (0-12 min) 100 %B (12-15 min) 0.4 ml/min (11 MPa) 35 C, 5 µl (0.1 Cl) ESI (positive, m/z 132.0) min Figure 10: Separation of Leucine (131Da) isomers, detected by the LCMS

7 a) a-aminobutyric acid ( a-abu) b) b-aminobutyric acid ( b-abu) c) b-aminoisobutyric acid ( b-aiba) d) g-aminobutyric acid (GABA, m/z 104.0) a b a b a) leucine (m/z 132.0) b) isoleucine allo-isoleucine (m/z 132.0) norleucine (m/z 132.0) methionine (m/z 150.0) tyrosine (m/z 182.0) phenylalanine (m/z 166.0) tryptophan (m/z 205.0) thioproline (m/z 134.0) cysteic acid (m/z 170.0) a b c d a b a b c aspartic acid (m/z 134.0) threonine (m/z 120.1) proline (m/z 116.0) taurine (m/z 126.0) glutamine (m/z 147.0) serine (m/z 106.1) methionine sulfone (m/z 182.0) glutamic acid (m/z 148.0) lysine (m/z 147.0) histidine (m/z 156.0) 2-aminoethanol (m/z 62.1) cystathionine (m/z 223.1) 5-aminolevulinic acid (m/z ) cystine (m/z 241.0) o-phosphoserine (m/z 186.0) citrulline (m/z 176.1) asparagine (m/z 133.0) glycine (m/z 76.4) c a) o-methylserine (m/z 120.1) b) allo-threonine c) homoserine hydroxyproline (m/z 116.0) a-aminoadipic acid (m/z 162.1) theanine (m/z 175.1) cysteine (m/z 122.0) a) norvaline b) valine (m/z 118.0) a-aminopimelic acid (m/z 162.1) arginine (m/z 175.1) ornithine (m/z 133.1) hydroxylysine (m/z 163.0) carnosine (m/z 227.1) anserine (m/z 241.1) b a) 1-methylhistidine (m/z 170.1) b) 3-methylhistidine o-phosphoethanolamine (m/z 142.0) 5-aminovaleric acid (m/z 118.1) glycylproline (m/z 173.1) a) alanine (m/z 90.0) b) sarcosine c) b-alanine min Figure 11: 55 Amino Acids (standard samples) separated and detected utilizing the LCMS a A: AC / TF / 25mM Ammonium formate / Formic acid = 9 / 75 / 16 / 0.3 B: AC / 100mM Ammonium formate = 20 / 80 Gradient Conditions: 0 %B (0-2.5min) 0-17 %B ( min) 100 %B ( min) 0.6 ml/min (6MPa), 35 C, 5 µl (1µmol/mL) 7

8 Results and Discussion Figures 1 and 2 demonstrate methods utilizing the LCMS-2020 single quadrupole mass spectrometer and LCMS-8040 triple quadrupole mass spectrometer to characterize a separation of the 20 standard amino acids including Leucine and Isoleucine without need for derivatization. Figure 3 provides calibration curves of amino acids representing the four categories of these molecules, aromatic, aliphatic, basic, and acidic to demonstrate the ability of the mixed-mode column and the mass spectrometer to accurately quantitate these four classes of analytes. Figure 4 provides a simple gradient procedure for separating a mixture of amino acids in 10 minutes. A 5 minute separation of the 20 standard amino acids was able to maintain resolution between Leucine and Isoleucine, (Figure 5). Figure 6 demonstrates measurement of free amino acids over 10 minutes in bovine serum utilizing the LCMS Additionally, dipeptides and beta and gamma amino acids were measured with the LCMS-2020 as charted in Figures 7 and 8. rnithine, citrulline, carnitine, taurine, and other related compounds are also candidate analytes for separation using this strategy. Method conditions have been optimized to separate these compounds in less than 60 seconds. Isobaric Leucine isomers are readily resolved using the separation strategy provided in Figure 10. Figure 11 demonstrates the separation of 55 amino acid analytes over 10 minutes. Conclusion Label free amino acid separations and detection utilizing a mixed-mode stationary phase coupled with the single and triple quadrupole mass spectromters, LCMS-2020 and LCMS-8040 has been demonstrated. Amino acid samples of varying complexity and from various matrices, including isomers, dipeptides, and related compounds are separated using optimized methods spanning from 10 minutes to as short as one minute. This combination of column, UPLC, and MS yields a powerful tool for amino acid analysis for many different biochemical applications. 8

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