PITTCON 2009 Chicago, IL
March 2009
LC/MS of Metabolites using a Silica Hydride-Based Stationary Phase Joseph J. Pesek Department of Chemistry San Jose State University San Jose, CA, 95192 Maria T. Matyska Microsolv Technology Corporation Eatontown, NJ 07724
The Essential Feature of the Aqueous Normal Phase Mode is the Presence of the Hydride Surface
ORDINARY SILICA
O O Si O O Si O O Si O
HYDRIDE SILICA
O OH OH OH
O Si O O Si O O Si O
O O O O
Si O Si O Si O
H H H
ANP RETENTION ON DIAMOND HYDRIDE COLUMN AMINO ACIDS - PHENYLALANINE 70:30 ACN/water + 0.1% FA tR = 2.96 min
NH 2
t0 = 0.95 min
HO 2 C
CH
CH 2
Ph
80:20 ACN/water + 0.1% FA tR = 5.22 min
90:10 ACN/water + 0.1% FA tR = 18.27 min Diamond Hydride: essentially an unmodified hydride surface
Detection: Agilent TOF-MS m/z = 166
L-Alanine L-Arginine L-Aspartic acid L-Cystine L-Glutamic acid L-Glycine L-Histidine L-Isoleucine L-Leucine L-Lysine L-Methionine L-Phenylalanine L-Proline L-Serine L-Threonine L-Tyrosine L-Valine
9 8
Retention time (min)
7 6 5 4 3 2
Retention time vs. % acetonitrile in the mobile phase
1 0 40
50
60
70
% ACN
80
90
100
Retention Time With Acetonitrile L-Alanine L-Arginine L-Aspartic acid L-Cystine L-Glutamic acid L-Glycine L-Histidine L-Isoleucine L-Leucine L-Lysine L-Methionine L-Phenylalanine L-Proline L-Serine L-Threonine L-Tyrosine L-Valine
9 8
Retention time (min)
7 6 5 4 3 2
Retention time vs % acetone in the mobile phase
1 0 40
50
60
70
80
% Acetone
Retention Time With Acetone
90
100
x10 2 + EIC(120.00000-120.20000) Scan Mix4_Gradient09A_Temp25_02.d
Leu
1 0.95 0.9 0.85
Gln
Ile
Pro
0.8 0.75 0.7
Phe
0.65 0.6
Retention Time L-Tryptophan 11.07 L-Leucine 11.22 L-Phenylalanine 11.25 L-Isoleucine 11.31 L-Tyrosine 11.33 L-Methionine 11.55 L-Valine 11.61 L-Aspartic acid 11.83 L-Glutamic acid 11.83 L-Alanine 12.05 Amino Acid
Amino Acid L-Threonine L-Glycine L-Serine L-Proline L-Asparagine L-Glutamine L-Arginine L-Histidine L-Lysine
Retention Time 12.29 12.32 12.52 12.94 13.20 13.35 16.63 16.67 17.01
0.5
His
Arg
0.55
0.45
0.35
Val
Trp
0.4
Asp
0.1 0.05 0.5
11
11.5
12
Lys Asn
Ser
Thr
0.15
Ala
Glu
0.2
Gly
0.25
Met
Tyr
0.3
12.5
13
13.5 14 14.5 Counts (%) vs. Acquisition Time (min)
15
15.5
16
16.5
17
Extracted Ion Chromatogram Of Nineteen Amino Acid Separation All of the critical amino acid pairs (those that are isobaric or have masses within one mass unit ) are separated under these conditions except for the Leucine / Isoleucine pair. A different gradient is needed for the separation as shown on the next slide.
Separation of Leucine and Isoleucine
x10 4 7
2
+ EIC(132.00000-132.20000) Scan Ileu_dil_Gr16D_N_02.d 1
1
6.5 6
1
5.5 5 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 0.5
1
1.5
Figure 2.
2
2.5
3
3.5
4
4.5
5 5.5 6 6.5 7 Counts vs. Acquisition Time (min)
7.5
8
8.5
9
1 = leucine 2 = isoleucine
9.5
10
10.5
11
11.5
12
EXTRACTED ION CHROMATOGRAMS OF ORGANIC ACIDS
maleic acid (1) aconitic acid, trans (2) aconitic acid, trans stereoisomer (3) aconitic acid, cis (4) fumaric acid (5) citric acid (6) oxaloacetic acid (7)
Metabolites in Urine at m/z 133
1 = methyl succinic acid; 2 = glutaric acid; 3 = oxaloacetic acid; 4 = creatine; 5 = 4-hydroxyproline; 6 = asparagine; 7 = ornithine.
GRADIENT REPRODUCIBILITY
The table shows retention time reproducibility for nine amino acids at two temperatures. Four replicates were performed at each temperature. The reproducibility was 0.28% or better for the amino acids. This is a significant improvement over what is usually observed for most HILIC analyses, especially considering this is gradient data with only a 5 minute re-equilibration time between runs.
Isocratic separation of seven component mixture of nucleotides on the undecenoic acid colum
85:15 90% acetonitrile/10% DI water + 0.1% ammonium formate/ DI water + 0.1% ammonium formate. Column: 4.6 x 75 mm. Flow rate 1 mL/min. Detection at 254 nm.
Solutes: 1 = adenosine-3’,5’-cyclic monophosphate; 2 = adenosine-5’monophosphate; 3 = adenosine-5’-triphosphate; 4 = thymidine-5’triphosphate; 5 = uridine-5’- triphosphate; 6 = cytosine-5’-triphosphate; 7 = guanosine-5’-triphosphate.
Gradient separation of five component mixture of nucleotides on the undecenoic acid column
Gradient for: 0.0 min 90% B, 0.0 – 10.0 min to 70% B.
Mobile phase: A, DI water + 0.1% ammonium formate; B, 90% acetonitrile/10% DI water + 0.1% ammonium formate
Solutes: 1 = adenosine-3’,5’-cyclic monophosphate; 2 = adenosine-5’monophosphate; 3 = adenosine-5’-triphosphate; 4 = thymidine-5’-triphosphate; 5 = uridine-5’- triphosphate
LC-MS EIC for isobaric nucleotide species on the DH column
A), m/z = 506 solutes: 1 = adenosine triphosphate and 2 = deoxycytidine-5’monophosphate; (B), m/z = 346 solutes: 1 = adenosine-3’-monophosphate and 2 = adenosine-5’-monophosphate. Column: 2.1 x 150 mm. Flow rate 0.4 mL/min. Mobile Phase: A, DI water + 15 mM ammonium acetate and B, 90% acetonitrile + 10% water + 15 mM ammonium acetate. Gradient: 0.00 min 95% B; 0.00 to 1.00 min to 90% B; 1.00 to 3.00
Extracted ion chromatograms from synthetic urine sample (A) m/z 145 negative ion mode. Solutes: 1 = adipic acid (145.0506) and 2 = 2-oxoglutaric acid (145.0142
(B) m/z 176 positive ion mode. 4 = citruline, 5 = Arginine (M+1), 1,2,3 not identified
Column: DH 2.1 x 150 mm. Flow rate 0.4 mL/min. Injection volume = 1 µL. Mobile Phase, A = DI water +0.1% formic acid; B = acetonitrile+0.1% formic acid. Gradient: 00.2 min 95% B; 0.2-30 min to 50%B; 30-35 min 50%B.
Mass chromatograms in the positive ion mode of a human saliva sample using the DH column. (A) total ion chromatogram
(B) m/z 166, phenylalanine
(C) m/z 182, tyrosine
(D) m/z 118, valine
(E) m/z 181, glucose
Flow rate 0.4 mL/min. Mobile Phase, A = DI water + 10 mM ammonium acetate; B = 98:2 acetonitrile/DI water + 10 mM ammonium acetate. Gradient: 0.0 min 100% B; 0.0 to 14.0 min to 60% B; 14.1 min to 5% B.
Extracted ion chromatograms for amino acids in saliva on the DH column
m/z 175, arginine
m/z 147, glutamine (1) and lysine (2)
DETECTION OF CARBOHYDRATES IN SALIVA SAMPLES
Solutes: 1 = glucose, 2 = sorbitol
Column: Diamond Hydride. Flow rate 0.4 mL/min. Mobile Phase, A = DI water + 10 mM ammonium acetate; B = 98:2 acetonitrile/DI water + 10 mM ammonium acetate. Gradient: 0.0 min 100% B; 0.0 to 14.0 min to 60% B; 14.1 min to 5% B.
EICs for organic acids from saliva samples on the DH column
Peak identity: (1) aconitic acid (m/z 173); (2) succinic acid (m/z 117); and (3) adipic acid (m/z 145).
EICs at m/z 147 for glutamine and lysine from saliva samples on the DH column
Sample from Cancer Patient
Sample from Normal Patient
Sample from Pancreatitis Patient
Peak Identity: 1 = glutamine, 2 = lysine
Composite EICs for various metabolites in human urine from two donors Positive Ion Mode Donor 1
Donor 2 Flow rate 0.4 mL/min. Mobile Phase, A = DI water + 0.1% formic acid; B = 90:10 acetonitrile/DI water + 0.1% ammonium acetate. Gradient: 0.0 to 1.00 min 98% B; 1.00 to 16.00 min to 20% B.
Solutes: 1 = glucose; 2 = methionine; 3 = leucine; 4 = tryptophan; 5 = sucrose
Composite EICs for various metabolites in human urine from two donors Negative Ion Mode
Donor 1
Donor 2
Solutes: 1 = fumaric acid; 2 = adipic acid; 3 = oxoglutaric acid; 4 = succinic acid
Creatinine and other metabolites in urine
Solutes: 1 = Creatinine - M+H 114.0662; 2 = Creatine – M+H 132.0768; 3 = 4-Hydroxyproline M+H 132.0655
Mobile Phase, A = DI water + 0.1% formic acid; B = acetonitrile = 0.1% formic acid. Gradient: 0.0 to 0.2 min 95%B; 0.2 to 30 min to 50% B; 30 to 35 min hold 50% B.
FOR MORE INFORMATION ON:
THE DIAMOND HYDRIDE FOR THE ANALYSIS OF METABOLITES AND OTHER TYPE C SILICA HYDRIDE PHASES
GO TO THE MICROSOLV TECHNOLOGY BOOTH #1974
