LC/MS Analysis of Various Low-Molecular Weight Cationic Compounds Using HILIC Mode
Shodex Technical Article
Introduction
Most low-molecular weight cationic compounds are hydrophilic and hardly retained under reversed phase mode. To overcome the problem, pre-column derivatization or ion-pair reagents are often used, however they add additional steps in the analysis. The use of derivatization adds additional steps in the analysis while the use of ion-pair reagent increases background level from the ion-pair reagent residues on the column and the flow lines.
The Shodex HILICpak VC-50 2D column used in this application is packed with multi-porous polyvinyl alcohol polymers modified with carboxyl functional groups. The 2.0 mm internal diameter of the column is designed to work well with LC/MS. The method using the VC-50 2D column provides a highly sensitive analysis of various low-molecular weight cationic compounds, while requiring simpler analytical conditions than previously available methods.
Fig. 1. Schematic diagram of VC-50 packing material showing PVA polymer with COOH functional groups
Figure 1 shows the schematic diagram of the packing material used in the VC-50 2D. Because of its hydrophilicity, increasing the percentage of acetonitrile in the eluent promotes HILIC mode, enhancing the retention of hydrophilic compounds. In addition, the low-molecular weight cationic compounds are retained in the column by cationic ion-exchange effects.
This technical article introduces LC/MS analysis methods using the VC-50 2D for various cationic compounds such as choline and acetylcholine, neurotransmitters, oral anti-diabetic drugs, salacinol related compounds, and aminoglycoside antibiotics.
Experimental
The LC/MS system used was Shimadzu Nexera/LCMS-8030 Plus. The column used was Shodex HILICpak VC-50 2D (2.0 mm I.D. × 150 mm; particle size 5 μm; pore size 100 Å). High pressure isocratic or linear gradient elution with formic acid and acetonitrile eluents were used for most analysis. The flow rate was set at 0.2 - 0.3 mL/min and the column temperature was set at 30 - 40 °C. An ESI was used as a means of ionization and SIM(-) or MRM(+) mode was used for the detection.
Results and Discussion
1. Analysis of Neurotransmitters
Neurotransmitters are low-molecular weight chemicals formed in neurons and transmit signals from the synapse to the target cells to excite or suppress their functions. They take major roles in many and various mental diseases. Some known neurotransmitters include monoamine type, e.g., acetylcholine and dopamine, and amino acid type, e.g., glutamic acid.
1.1 Analysis of Choline and Acetylcholine
There are reports that patients with Alzheimer's disease show low levels of the brain neurotransmitter acetylcholine. Since choline is the source of acetylcholine, foods containing choline has been drawing attention for the prevention of Alzheimer's disease. Figure 2 shows the LC/MS chromatograms of choline and acetylcholine in a mixed standard. The method demonstrated its feasibility in detecting the analytes in 1-nM level.
Fig. 2. LC/MS chromatograms of choline and acetylcholine
| Sample | 5 nM each (in H2O), 5 µL |
| Column | Shodex HILICpak VC-50 2D |
| Eluent | (A) 200 mM HCOOH aq./(B) CH3CN = 40/60 |
| Flow rate | 0.3 mL/min |
| Detector | ESI-MS MRM(+) |
| Column temp. | 40 °C |
As a next step, we quantified choline in the serum of a guinea pig. Figure 3 shows the LC/MS chromatogram of choline in the sample. The sample preparation used here was very simple - The sample was deproteinized through the addition of acetonitrile and the following centrifugation.
Fig. 3. LC/MS analysis of choline in guinea pig serum
- Dilute guinea pig serum with H2O in 10 fold.
- Add × 3 volume of CH3CN.
- Centrifuge the sample.
- Dilute the supernatant with H2O in 10 fold.
- Inject 20 µL.
| Column | Shodex HILICpak VC-50 2D |
| Eluent | (A) 100 mM HCOOH aq./(B) CH3CN = 50/50 |
| Flow rate | 0.3 mL/min |
| Detector | ESI-MS MRM(+) |
| Column temp. | 40 °C |
1.2 Analysis of Monoamine Neurotransmitters
Figure 4 shows the LC/MS chromatograms of five monoamine neurotransmitters and acetylcholine. Histamine is a highly basic and strongly retained under isocratic method. By using a gradient method with increased formic acid ratio enabled simultaneous analysis of six compounds.
Fig. 4. LC/MS chromatograms of five monoamine neurotransmitters and acetylcholine (noradrenaline, adrenaline, dopamine, serotonin, acetylcholine, and histamine)
| Sample | 0.1 µM each (in H2O), 5 µL |
| Column | Shodex HILICpak VC-50 2D |
| Eluent | (A) 200 mM HCOOH aq./(B) CH3CN High pressure linear gradient 60% B (0 - 5 min) → 10% B (6 - 20 min) |
| Flow rate | 0.3 mL/min |
| Detector | ESI-MS MRM(+) |
| Column temp. | 40 °C |
1.3 Analysis of Amino Acid Neurotransmitters
Figure 5 shows the LC/MS chromatograms of four amino acid neurotransmitters. Two acidic amino acids, aspartic and glutamic acids, tend to elute earlier than expected. This was probably because of the electrostatic repulsion effects between the amino acids and the carboxyl functional group on the surface of the packing material. Meanwhile, the retention mechanism that works between GABA (γ-amino butyric acid) and the packing material seemed to be different from that of other amino acids: Rather than HILIC mode, hydrophobic interaction is mostly responsible for the retention of GABA.
Fig. 5. LC/MS analysis of four amino acid neurotransmitters (Asp, Glu, Gly, and GABA)
| Sample | 5 nM each (in H2O), 5 µL |
| Column | Shodex HILICpak VC-50 2D |
| Eluent | (A) 40 mM HCOOH aq./(B) CH3CN = 50/50 |
| Flow rate | 0.2 mL/min |
| Detector | ESI-MS MRM(+) |
| Column temp. | 40 °C |
2. Simultaneous Analysis of 21 Amino Acids
Figure 6 shows the LC/MS chromatograms of cystine and 20 amino acids that compose proteins. The basic amino acids, histidine, lysine, and arginine, are strongly retained by cationic ion-exchange effects. Elution of these compounds required increasing the formic acid ratio in the gradient elution, which weakened the cationic ion-exchange effects. The method was also effective separating the two isomeric compounds, leucine and isoleucine.
Fig. 6. LC/MS chromatograms of 21 amino acids including Asp, Glu, Cys, Thr, Ser, Pro, Tyr, Asn, Met, Gln, Ala, Gly, Phe, Val, Leu/Ile, Trp, Cys2 (derived from Cys), His, Lys, and Arg
| Sample | 100 ng/mL each (in H2O), 5 µL |
| Column | Shodex HILICpak VC-50 2D |
| Eluent | H2O/100 mM HCOOH aq./CH3CN Low pressure linear gradient 30/20/50 (0 min) → 0/90/10 (5 - 30 min) |
| Flow rate | 0.2 mL/min |
| Detector | ESI-MS SIM(+) |
| Column temp. | 30 °C |
3. Analysis of Low-Molecular Weight Peptides
Recently, medical, pharmaceutical, and food industries are finding potentials in functionalities of the low-molecular weight peptides. Figure 7 shows the LC/MS chromatograms of five dipeptides and three tripeptides. The results demonstrated that VC-50 2D is suitable for the analysis of low-molecular weight peptides.
Fig. 7. LC/MS chromatograms of dipeptides and tripeptides including Ser-Glu, Gly-Tyr, Gly-Gly-Leu, Thr-Gln, Gly-Gly-Ala, Gly-Gln, Arg-Asp, and Gly-Gly-His
| Sample | 50 ng/mL each (in H2O), 5 µL |
| Column | Shodex HILICpak VC-50 2D |
| Eluent | (A) 100 mM HCOOH aq./(B) CH3CN High pressure linear gradient 60% B (0 - 5 min) → 10% B (6 - 15 min) |
| Flow rate | 0.3 mL/min |
| Detector | ESI-MS SIM(+) |
| Column temp. | 40 °C |
4. Analysis of Histamine and Histidine
Figure 8 shows the LC/MS chromatograms of histamine and histidine. Histamine is known for its neurotransmission role, but it can be a cause of food poisoning. Thus, simultaneous analysis of histamine and its precursor, histidine, is sometimes required. The result shows that the VC-50 2D is applicable for the simultaneous analysis of an amino acid and an amine.
Fig. 8. LC/MS chromatograms of histamine and histidine
| Sample | 5 ng/mL each (in H2O), 20 µL |
| Column | Shodex HILICpak VC-50 2D |
| Eluent | (A) 250 mM HCOOH aq./(B) CH3CN = 70/30 |
| Flow rate | 0.3 mL/min |
| Detector | ESI-MS MRM(+) |
| Column temp. | 40 °C |
5. Analysis of Oral Anti-Diabetic Drugs
Figure 9 shows the LC/MS chromatograms of four oral anti-diabetic drugs. Metformin falls under the biguanide drug family and it is highly basic. By increasing formic acid ratio in the gradient elution let the metformin to elute and enabled a simultaneous analysis of metformin and three α-glucosidase inhibitor type drugs.
Fig. 9. LC/MS chromatograms of oral anti-diabetic drugs including Acarbose (MW 645), Miglitol (MW 207), Voglibose (MW 267), and Metformin (MW 129)
| Sample | 5 ng/mL each (in H2O), 5 µL |
| Column | Shodex HILICpak VC-50 2D |
| Eluent | (A) 200 mM HCOOH aq./(B) CH3CN High pressure linear gradient 60% B (0 - 5 min) → 20% B (6 - 15 min) |
| Flow rate | 0.3 mL/min |
| Detector | ESI-MS MRM(+) |
| Column temp. | 40 °C |
6. Analysis of Ribavirin
Figure 10 shows an LC/MS chromatogram of ribavirin, a nucleoside analog inhibitor of hepatitis C. Its retention by HILIC mode was increased by using 90% acetonitrile in the eluent. The high percentage of the acetonitrile also contributed to increase the ESI-MS detection sensitivity.
Fig. 10. LC/MS chromatogram of ribavirin
| Sample | 0.5 ng/mL each (in H2O/CH3CN = 1/4), 5 µL |
| Column | Shodex HILICpak VC-50 2D |
| Eluent | (A) 50 mM HCOOH aq./(B) CH3CN = 10/90 |
| Flow rate | 0.25 mL/min |
| Detector | ESI-MS MRM(+) |
| Column temp. | 40 °C |
7. Analysis of Salacinol Related Compounds
The salacinol compounds, neokotalanol and neosalacinol, are sometimes used in functional foods, as they have blood sugar suppression effects. Figure 11 shows the LC/MS chromatograms of neokotalanol and neosalacinol in the extract of a commercial supplement. The result demonstrated that the VC-50 2D is suitable for analyzing the sulfonium-containing cationic compounds.
Fig. 11. LC/MS chromatograms of neokotalanol (MW 344) and neosalacinol (MW 254) in the extract of a commercial supplement
- Grind a tablet.
- Measure 0.1 g of the sample, add 1-mL ultra-pure water.
- Sonicate 10 min, centrifuge.
- Add an equal portion of CH3CN to supernatant, centrifuge.
- Dilute supernatant with H2O/CH3CN = 1/1 in × 10,000 fold.
- Inject 5 µL.
| Column | Shodex HILICpak VC-50 2D |
| Eluent | (A) 50 mM HCOOH aq./(B) CH3CN = 50/50 |
| Flow rate | 0.3 mL/min |
| Detector | ESI-MS MRM(+) |
| Column temp. | 40 °C |
8. Analysis of Aminoglycoside Antibiotics
Streptomycin and its derivative, dihydrostreptomycin, are aminoglycoside antibiotics. They were analyzed under an acidic condition containing formic acid. Figure 12 shows the LC/MS chromatograms of streptomycin and dihydrostreptomycin.
Fig. 12. LC/MS chromatograms of streptomycin and dihydrostreptomycin
| Sample | 50 ng/mL each (in H2O), 20 µL*Both are sulfates |
| Column | Shodex HILICpak VC-50 2D |
| Eluent | (A) 250 mM HCOOH aq./(B) CH3CN = 90/10 |
| Flow rate | 0.3 mL/min |
| Detector | ESI-MS SIM(+) |
| Column temp. | 40 °C |
On the other hand, kanamycin, tobramycin, gentamicin, neomycin, and amikacin were retained in the column even when the concentration of the formic acid was increased to 500 mM. Aminoglycoside antibiotics have more than four amino functional groups, resulting in a strongly cationic structure retaining on the column. To overcome this hurdle, we added ammonium to the eluent making it alkaline allowing the five compounds to elute. Figure 13 shows the LC/MS chromatograms of aminoglycoside antibiotics. It was assumed that using the alkaline eluent suppressed the dissociation of amino groups in the aminoglycoside molecules, and weakened the ionic interactions between the analytes and the packing material. The pH of the 1.5% ammonium water used in the experiment was about 12. Use of the basic condition was possible because of the polymer-based materials packed in the VC-50 2D.
Fig. 13. LC/MS chromatograms of kanamycin (MW 484), tobramycin (MW 467), gentamicin (MW 477), neomycin (MW 614), and amikacine (MW 585)
| Sample | 100 ng/mL each (in H2O), 20 µL*Amikacine is dehydrate, others are sulfates |
| Column | Shodex HILICpak VC-50 2D |
| Eluent | (A) 1.5% mM NH3 aq./(B) CH3CN High pressure linear gradient 30% B (0 min) → 10% B (5 - 15 min) |
| Flow rate | 0.3 mL/min |
| Detector | ESI-MS SIM(+/-) |
| Column temp. | 40 °C |
Conclusions
In this technical note, the polymer-based carboxyl HILIC column, Shodex HILICpak VC-50 2D demonstrated simple LC/ESI-MS methods for the analysis of various low-molecular weight cationic compounds using formic acid/acetonitrile eluents. The use of gradient condition adds another advantage to simultaneously analyze multiple compounds having large differences in their cationic strengths. Also, the use of alkaline condition, with an addition of ammonium to the eluent, is useful for the analysis of cationic compounds that are normally retained strongly in the column under the acidic conditions.