MASS SPEC 07, (Sep, 07, Boston, USA) Amino acids and metabolites/derivatives analysis without derivatization using a novel mixed-mode column
ITRDUCTI Amino acids and derived metabolites/derivatives are usually difficult to analyze without derivatization procedure due to poor retention on conventional reversed-phase columns. Also it is difficult to use non-volatile ion-pairing reagent for LC- MS.Therefore, it is important to break-through this difficult separation issue by creating a new stationary phase for underivatized polar compounds. We have focued attention on ion-exchange (IEX) mode due to the compounds structure which has positive charge under acidic conditions, and we have succeeded to develop a specialized amino analysis column for LC-MS using normal-phase(p) and IEX mixed mode. Also we found that this mixed-mode column can analyze not only amino acids but also derived metabolites. METD We used a P+IEX column, Intrada Amino Acid (Imtakt Corp.), to analyze amino acids and metabolites/derivatives using LCMS-00 (Shimadzu Corp.).
RESULTS AD DISCUSSI We found that not only amino acids but also polar metabolites and derivatives were directly analyzed using developed columns with various column dimensions. We got good results about the following analytical subjects: Fig. Fig. 6, 7 Fig. 8 Fig. 9 Fig.0 Fig. Fig. Fig. Fig.4 Fig. Fig.6 0 free amino acids Trp, Tyr, Glu, Gly Glu Phe, Tyr, Trp is, rn, Lys, Arg Tyr Lys, Met is Met Met Trp CCLUSI Serum amino acids eurotransmitters GABA Isomers Biogenic Amines Biogenic Amines Parkinson's Disease Carnitine Biosynthesis Skeletal Muscle Breakdown yperhomocysteinemia SAM (S-Adenosyl methionine) Kynurenine Pathway This novel separation column will be a powerful tool for amino acids, metabolites and derivatives LC-MS analysis in many biochemistry applications.
Amino acid charge pi : Isoelectric Point Positive Charge eutral Side Chain pi 6 glycine (Gly / G) C C C C C C Low p igh p pi. C (C) C C C (C) Acidic Amino Acid C C L-glutamic acid (Glu / E) C (C) C C Basic Amino Acid pi 9.8 (C) 4 C C (C) 4 C C L-lysine (Lys / K) (C) 4 C C
Amino Acid Analysis Column for LC-MS Mixed-mode ormal Phase + Ion Exchange Adsorption rganic Solvent (Low Polarity) Desorption igh p igh Ionic Strength R C C R C C Low p Low Ionic Strength Time igh Polarity
LC-MS analysis for Amino Acids Intrada Amino Acid, 0 x mm A: AC /TF /mm C4 /C = 9 / 7 / 6 / 0. B: 00mM C4 / AC = 80 / 0 0 %B (0-. min), 0-7 %B (.-6. min), 00 %B (6.- min) 0.6 ml/min (0 MPa), deg.c, ul (umol/ml) ESI (SIM, Positive) a) -aminobutyric acid ( -Abu) a b) -aminobutyric acid ( -Abu) c) -aminoisobutyric acid ( -Aiba) d) -aminobutyric acid (GABA, m/z 04.0) a b glycylproline (Gly-Pro, m/z 7.) o-phosphoserine (Ser (P), m/z 86.0) citrulline (Cit, m/z 76.) asparagine (Asn, m/z.0) glycine (Gly, m/z 76.4) glutamine (Gln, m/z 47.0) serine (Ser, m/z 06.) a b c a) alanine (Ala, m/z 90.0) b) sarcosine (Sar) c) -alanine ( -Ala) aspartic acid (Asp, m/z 4.0) threonine (Thr, m/z 0.) a b c a) o-methylserine (m/z 0.) b) allo-threonine (allo-thr) c) homoserine hydroxyproline (yp, m/z 6.0) proline (Pro, m/z 6.0) taurine (Tau, m/z 6.0) methionine sulfone (m/z 8.0) glutamic acid (Glu, m/z 48.0) -aminoadipic acid ( -Aaa, m/z 6.) theanine (m/z 7.) cysteine (Cys, m/z.0) a) norvaline b) valine (Val, m/z 8.0) -aminopimelic acid (Apm, m/z 6.) a b a) leucine (Leu, m/z.0) b) isoleucine (Ile) allo-isoleucine (m/z.0) norleucine (m/z.0) methionine (Met, m/z 0.0) tyrosine (Tyr, m/z 8.0) phenylalanine (Phe, m/z 66.0) tryptophan (Trp, m/z 0.0) thioproline (m/z 4.0) cysteic acid (Cys(), m/z 70.0) arginine (Arg, m/z 7.) ornithine (rn, m/z.) hydroxylysine (ylys, m/z 6.0) lysine (Lys, m/z 47.0) carnosine (Car, m/z 7.) anserine (Ans, m/z 4.) a b a) -methylhistidine (-Mehis, m/z 70.) b) -methylhistidine (-Mehis) histidine (is, m/z 6.0) -aminoethanol (Et, m/z 6.) cystathionine (Cysthi, m/z.) -aminolevulinic acid (m/z.0 ) cystine ((Cys), m/z 4.0) o-phosphoethanolamine (m/z 4.0) -aminovaleric acid (m/z 8.) 0. 7. 0min b c d
4 Amino Acid Analysis on LC-MS/MS Thermo Scientific: UltiMate 000 LC System - TSQ Endura (Triple-Stage Quadrupole Mass Spectrometer ) Intrada Amino Acid, 00 x mm A: acetonitrile / formic acid = 00 / 0. B: acetonitrile / 00mM C4 = 0 / 80 0%B (0-4 min) 0-00%B (4-4 min) 00%B (4-6 min) 0.6 ml/min, 7 C, µl (0.M Cl) Triple Quad. MS: ESI (SRM, positive) 0 0 min Courtesy of Dr. Kuniko MITAMURA, KIDAI UIVERSITY, JAPA 89
Amino acids in human serum (SSA) uman Serum (commercial reagent) %.-sulfosalicylic acid (:, vol) mixing centrifugation (>00 rpm, > min) Supernatant (Injection Sample) Arg (m/z 7.) Lys (m/z 47.0) is (m/z 6.0) (Cys) (m/z 4.0) Asn (m/z.0) Gln (m/z 47.) Ser (m/z 06.0) Gly (m/z 76.0) Asp (m/z 4.0) Glu (m/z 48.0) Thr (m/z 0.) µm 00 00 0 Leu Ala (m/z 90.0) Val (m/z 8.) Pro (m/z 6.) Tyr (m/z 8.) Ile Met (m/z 0.0) (m/z.) Trp (m/z 0.) Phe (m/z 66.) Intrada Amino Acid, 00 x mm A: AC / C = 00 / 0. B: AC / 00mM C4 = 0 / 80 0 %B (0-4 min) 0-00 %B (4-4 min) 00 %B (4-6 min) 0.6 ml/min (MPa), 7 C, µl (.% -slufosalicylic acid) Single Quad. MS: ESI (SIM, positive) 0 4 6 8 0 4 6 min 8
6 Phe, Trp, Tyr, Glu, Gly - eurotransmitters Intrada Amino Acid, 00 x mm A: methanol / water / formic acid = 80 / 0 / 0. B: methanol / 00mM ammonium formate = 0 / 70 6 0 %B (0-min), 0-00 %B (-0min), 00 %B (0-0min) 0.6 ml/min ( MPa), 7 C, 0 µl (0µg/mL, % C) Single Quad. MS (ESI, positive, negative) [M+] + m/z 9.0 L-tryptophan (Trp) L-tyrosine (Tyr) L-phenyl alanine (Phe) µg/ml 0 0 7 4 6 8 9 0 [M+] + m/z 48.0 [M+] + m/z 66.0 [M+] + m/z.0 [M+] + m/z 8.0 [M+] + m/z 0.0 [M-] - m/z 96.0 [M+] + m/z 04.0 [M+] + m/z 46.0 [M+] + m/z 84.0 [M-] - m/z 68.0 [M+] + m/z 4.0 [M+] + m/z 77. 4 9 -hydroxyindole -- acetic acid (-IAA) serotonin (-T) -hydroxy-l- tryptophan (-TP) C Cl- + C C acetylcholine chloride 7 0,4-dihydroxy- L-phenylalanine (L-DPA) L-noradrenaline (orepinephrine) dopamine L-adrenaline (Epinephrine) 8 L-glutamic acid (Glu) 4-aminobutyric acid (GABA) 0 0 0 min 80
7 LC-MS Analysis for amino acids and catecholamines in rat pituitary Determination of euronal Peptides and Catecholamines and Effects of Diethylstilbestrol on Male Rat Pituitary aoyuki Maeda,, Emi Tanaka, Kanae Masu, Kanako kumura Yuki Ikeda, Taku Miyasho, Satoko aeno and iroshi Yokota. ) Japan Meat Science & Technology Institute ) Rakuno Gakuen University The separation was achieved using an Intrada Amino Acid (00 x mm µm particle size, Imtakt) (A) CC / TF / mm C4 / C=0 / 80 /0 / 0.4 (B) CC/00mM C4 = 0/80 0 %B (0- min) 0-7 %B (-6. min) 00 %B (6.-0 min) 00 %B hold (0-8 min) 400 µl/min flow rate at C µl injection orepinephrine (EP) Alanine(Ala) Threonine (Thr) Aspartic acid (Asp) Proline (Pro) Serine (Ser) Glycine (Gly) Glutamine (Gln) Glutamic acid (Glul) aminobutyric acid d4 (GABA d4) Aminobutyric acid (GABA) Dopamine d4 (DP d4) Dopamine (DP) Cystine (Cys) Epinephrine (EP) Serotonin (/T) Serotonin d4 (/T d4) Asparagine (Asn) Arginine(Arg) Lysine(Lys) istidine (is) ACMS0 (6st), Sept 0, 0, Tsukuba, Japan Diethylstilbestrol Methionine(Met) Isoleucine (Ile) Leucine (Leu) Valine(Val) Dioxyphenylalanine (L/DPA) Tyrosine (Tyr) Phenylalanine (Phe) /ydroxytryptophan (/TP) 90 Tryptophan (Trp) /hydroxyindoleacetic acid /IAA) % Melatonin -0 0.00.00 4.00 6.00 8.00 0.00.00 4.00 6.00 Time Determination procedure for neuronal peptides and catecholamines were developed by MS analysis. The regulatory factors for prolactin induction mediated by diethylstilbestrol were analyzed by LC-MS analysis using the selected reaction monitoring (SRM). Dopamine suppressing prolactin secretion did not decreased, on the other hand, vasoactive intestinal peptide (VIP) which mediates the acute release of prolactin was increased in the pituitary glands of the DES- treated rats. Simultaneous Determination of the eurotransmitters and Free Amino Acids in Rat rgans by LC-MS Analysis aoyuki Maeda, Michiko Sato, Satoko aeno and iroshi Yokota Science Journal R, March, 0 Courtesy of Dr. Maeda, Safety Research Institute for Chemical Compounds Co.,Ltd,, Japan
8 Glu - GABA Isomers -aminobutyric acid C (alpha-aminobutyric acid) 4 4 C -aminobutyric acid (beta-aminobutyric acid) 0 x mm C -aminoisobutyric acid (beta-aminoisobutyric acid) 4-aminobutyric acid (GABA, gamma-aminobutyric acid) (m/z 04.) (m/z 48.) L-glutamic acid 0.0..0 7. 0.0 min Intrada Amino Acid, 0 x mm A: AC /C = 00 / 0. B: 00mM C4 0- %B (0- min) 00 %B (-min) 0. ml/min (6MPa) 60 deg.c, ul (mm, 0. Cl ) ESI (SIM, positive) 74
9 Phe, Tyr, Trp - Biogenic Amines (%, 00u M) 00 0 0 0.0..0 7. 0.0 min 4 6 m/z 66. m/z. m/z 8. m/z 8. m/z 0. m/z 6. L-phenylalanine L-tyrosine L-tryptophan 4 phenethylamine tyramine tryptamine Intrada Amino Acid, 00 x mm A: acetonitrile / formic acid = 00 / 0. B: acetonitrile / 00 mm ammonium formate = 0 / 80-00 %B (0-0min), 00 %B (0-min) 0.4 ml/min (4 MPa), 7 C ul (00nmol/mL, 0. Cl) Single Quad. MS (ESI, positive) 6 78
0 is, rn, Lys, Arg - Biogenic Amines m/z 6. L-histidine histamine 4 m/z. m/z. m/z 89.4 4 L-ornithine putresine 6 L-lysine cadaverine 7 6 m/z 47. m/z 0. 7 L-arginine 8 agmatine m/z 7. m/z. 0.0..0 7. 0.0 min 8 Intrada Amino Acid, 0 x mm A: methanol / formic acid = 00 / 0. B: 80 mm ammonium formate 40-00 %B (0-min), 00 %B (-min) 0.4 ml/min ( MPa), 7 C, ul (00nmol/mL, 0. Cl) Single Quad. MS (ESI, positive) 78
Tyr - Parkinson's Disease L-tyrosine 4,4-dihydroxy-L-phenylalanine (L-DPA) 6 dopamine C -methoxytyramine [M+C] - m/z 7.0 [M-] - m/z 67.0 4 [M+] + m/z 8.0 [M+] + m/z 98. 6 [M+] + m/z 68. [M+] + m/z 4. 0.0.0 0.0.0 min,4-dihydroxyphenyl acetic acid (DPAC) C homovanillic acid (VA) Intrada Amino Acid, 0 x mm A: acetonitrile / formic acid = 00 / 0. B: 00mM ammonium formate 0-00 %B (0-0min), 00 %B (0-min) 0. ml/min (6 MPa), 7 C 0 µl (0µg/mL, % C) Single Quad. MS (ESI) 87
Lys, Met - Carnitine Biosynthesis S C L-methionine + L-lysine C C + C L-carnitine µm 00 0 0 [M+]+ m/z 47. [M+]+ m/z 6. [M+]+ m/z 0.0 0.0.0.0.0 4.0.0 6.0 min Intrada Amino Acid, 0 x mm A: acetonitrile / formic acid = 00 / 0. B: 00mM ammonium formate -00 %B (0-6min), %B (6-0min) 0.6 ml/min ( MPa), 7 C µl (00nmol/mL, % C) Single Quad. MS (ESI, positive) 84
is - Skeletal Muscle Breakdown L-histidine C -methyl-l-histidine C -methyl-l-histidine (m/z 6.0) Intrada Amino Acid, 00 x mm A: acetonitrile / water / formic acid = 8 / / 0. B: 00mM ammonium formate -60 %B (0-0 min), 00 %B (0- min) 0.4 ml/min(mpa), 0 C, ul (mm in 0. Cl) Single Quad.MS (ESI, positive) (m/z 70.) 0.0..0 7. 0.0 min 74
4 Met - yperhomocysteinemia (%, 00u M) 00 C S L-methionine 0 (00nmol/mL, 0. Cl) 0 m/z 69.0 m/z 0.0 0.0.0 4.0 6.0 8.0 0.0.0 (%, 00u M) 00 0 0 (00nmol/mL, 0. Cl + 00mM TCEP*) * tris (-carboxyethyl) phosphine m/z 6.0 0.0.0 4.0 6.0 8.0 0.0.0 min min S L-homocysteine S L-homocystine Intrada Amino Acid, 0 x mm A: acetonitrile / formic acid = 00 / 0. B: 00mM ammonium formate 0-60 %B (0-0 min), 00 %B (0- min) 0.6 ml/min( MPa), 7 C, ul (0. Cl) Single Quad. MS (ESI, positive) S 776
Met - SAM, SA C S L-methionine [M+]+ m/z 0. S + C S-('-adenosyl)- L-methionine (SAM) S [M+]+ nm/z 8. [M+]+ m/z 99. 0 0 min S-('-adenosyl)- L-homocysteine (SA) Intrada Amino Acid, 00 x mm A: acetonitrile / formic acid = 00 / 0. B: acetonitrile / 00mM C4 = 0 / 80 0-00 %B (0-0min), 00 %B (0-min) 0.4 ml/min ( MPa), 7 C 0 µl (µg/ml, % C) Single Quad. MS (ESI, positive) 86
6 Trp - Kynurenine metabolism pathway Intrada Amino Acid, 00 x mm A: Me / water / C = 60 / 40 / 0. B: 00mM C4 0-00%B (0-0 min) 0.4 ml/min (9 MPa), 7 C 0 µl (0µg/mL, % formic acid ) Single Quad. MS (ESI) [M+] + m/z 68.0 [M+]+ m/z 90.0 4 6 L-tryptophan L-kynurenine kynurenic acid [M+] + m/z 4. 4 [M+] + m/z 4. 6 -hydroxyanthranilic acid µg/ml [M+] + m/z 09.0 [M+] + m/z 0.0 picolinic acid quinolinic acid 0.0.0 4.0 6.0 8.0 0.0 min 846
7 min igh Throughput Analysis Intrada Amino Acid, 0 x mm A: AC /C = 00 / 0. B: 00mM C4-00 %B (0-0.8 min), 00 %B (0.8-.0 min) 0.4 ml/min (.6 MPa), deg.c, ul (0. Cl) ESI (SIM, positive) C C + C - 0 x mm m/z. (40 nmol/ml) ornithine m/z 76. (70 nmol/ml) m/z 6. (00 pmol/ml) citrulline carnitine m/z 6. (.6 umol/ml) taurine S 0 4 6 48 60 sec