LC/MS Analysis of Various Hydrophilic Compounds Using HILIC Mode and Alkaline Eluent
Shodex Technical Article
Introduction
Components of pharmaceutical and food products often include high polar compounds. Those compounds are hardly retained under reversed phase mode, the most often used HPLC separation mode. To overcome the problem, pre-column derivatization or ion-pair reagents are often used. However, 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.
Shodex HILICpak VG-50 series used in this application is a set of polymer-based amino columns which effectively separate various saccharides. The packing material consists of a polyvinyl alcohol base with a hydrophilic functional group, a modified tertiary amine (Fig. 1).
Fig. 1. Schematic diagram of VG-50 packing material showing PVA base with hydrophilic group and modified tertiary amine.
Key Advantages of HILICpak VG-50 Series
- High Recovery Rate: Reducing sugars do not form Schiff base with the packing material, leading to excellent recovery rates
- Minimal Column Bleeding: Unlike silica-based amino columns, column bleeding is rarely observed, reducing background noise and ion suppression in MS
- Wide pH Range: Can be used under alkaline conditions (working pH range: 2 to 13), enabling high sensitivity analysis of saccharides using negative mode ESI-MS
- Analysis of Anionic Compounds: Alkaline conditions allow elution of organic saccharides that tend to be retained with previous methods
This application introduces analysis of not only saccharides, but includes simultaneous analysis of saccharides, organic acids, and amino acids using a semi-micro size column, Shodex HILICpak VG-50 2D, with LC/MS alkaline gradient conditions.
Experimental
The LC/MS system used was Shimadzu Nexera/LCMS-8030 Plus. The column used was Shodex HILICpak VG-50 2D (2.0 mm I.D. x 150 mm; particle size 5 μm; pore size 100 Å). Specific analytical conditions used for each analysis are mentioned with their results. It should be noted that the pH of 0.5% ammonia water used as an eluent is about 11.5. The LC/MS system used in the experiment was durable against the alkaline condition up to pH 13.
Results and Discussion
1. LC/MS Analysis of Sugars
1.1 Neutral Saccharides
Figure 2 shows the chromatograms for meso-erythritol, arabinose, xylose, fructose, mannose, glucose, sucrose, lactose, and maltose. Gradient elution of 0.1% ammonia water/acetonitrile was used. The neutral saccharides analyzed in this experiment can also be separated using water/acetonitrile as an eluent and results in similar resolution.
However, addition of ammonia (analyzing under alkaline condition) promotes deprotonation during ESI which increases the sensitivity of negative ion detection. The peak height observed using ammonia water/acetonitrile eluent was three times higher than that of using water/acetonitrile eluent. The pH of 0.1% ammonia water is about 11, thus most silica-based LC columns cannot be used under this condition. This emphasizes an advantage of VG-50 2D, packed with polymer-based packing material, as it well-tolerates the high pH eluent like the one used in this experiment.
Fig. 2. Chromatograms of various neutral saccharides including meso-erythritol, arabinose, xylose, fructose, mannose, glucose, sucrose, lactose, and maltose.
| Sample | 50 ng/mL each in H2O/CH3CN = 1/9, 5 μL |
| Column | Shodex HILICpak VG-50 2D |
| Eluent | (A) 0.1% NH3 aq./(B) CH3CN Linear gradient: 95% B (0-5 min) → 80% B (15-20 min) |
| Flow rate | 0.3 mL/min |
| Detector | ESI-MS MRM(-) |
| Column temp. | 60°C |
1.2 Acidic Saccharides
The use of water/acetonitrile eluent causes ionic adsorption effects, and thus retains the acidic saccharides in the column. The use of alkaline eluent prevents the dissociation of amino function groups on the stationary phase, and this prevents the saccharides from being kept retained in the column. Figure 3 shows the chromatograms of glucuronic acid and galacturonic acids. The degree of separation achieved here was better than that of previously available method using ion-exclusion chromatography. Using a high acetonitrile ratio is also advantageous for achieving a high sensitivity MS result.
Fig. 3. Chromatograms of glucuronic acid and galacturonic acid showing excellent separation under alkaline conditions.
| Sample | 10 ng/mL each in H2O/CH3CN = 1/4, 5 μL |
| Column | Shodex HILICpak VG-50 2D |
| Eluent | (A) 0.5% NH3 aq./(B) CH3CN = 25/75 |
| Flow rate | 0.2 mL/min |
| Detector | ESI-MS SIM(-) |
| Column temp. | 30°C |
1.3 Glucose and Gluconic Acid
Gluconic acid is generally converted from glucose, and thus simultaneous analysis of those two compounds is sometimes required. However, previously available method using ion-exclusion chromatography under acidic conditions did not provide an effective separation. Figure 4 shows chromatograms demonstrating a good separation of the two compounds. An additional advantage of the method described is that while acidic condition lactonizes the gluconic acid which causes the peak tailing, but analysis under alkaline condition will not let the lactonization, and thus prevents tailing to occur. Thus, the use of an alkaline eluent improves the quantification of gluconic acid.
Fig. 4. Chromatograms showing excellent separation of glucose and gluconic acid with calibration curves demonstrating good linearity.
| Sample | 10 ng/mL each in H2O/CH3CN = 1/4, 5 μL |
| Column | Shodex HILICpak VG-50 2D |
| Eluent | (A) 0.5% NH3 aq./(B) CH3CN = 25/75 |
| Flow rate | 0.2 mL/min |
| Detector | ESI-MS SIM(-) |
| Column temp. | 40°C |
1.4 Amino Acids
Figure 5 shows chromatograms of N-acetylglucosamine and glucosamine. Since glucosamine contains an amino function group, higher sensitivity was achieved by monitoring protonated compound than monitoring its deprotonated compound. Amino sugars and their acetylated metabolites can also be analyzed under the alkaline condition (data not shown).
Fig. 5. Chromatograms of N-acetylglucosamine and glucosamine showing clear separation.
| Sample | 100 ng/mL each in H2O/CH3CN = 1/4, 5 μL |
| Column | Shodex HILICpak VG-50 2D |
| Eluent | (A) 0.5% NH3 aq./(B) CH3CN = 25/75 |
| Flow rate | 0.2 mL/min |
| Detector | ESI-MS SIM(+/-) |
| Column temp. | 40°C |
2. Simultaneous Analysis of Saccharides, Organic Acids, and Amino Acids
The successful method developed for the analysis of acidic saccharides in the alkaline condition was extended for the separation of organic acids and amino acids. Figure 6 shows the chromatograms of a mixture containing 14 saccharides, 9 organic acids, and 20 amino acids. A gradient method was used for the analysis.
The result demonstrates the VG-50 column's capability of analyzing organic acids. The elution order of the organic acids was mono, di, and tribasic acids. It required to use approximately 0.5% ammonia to make the citric acid (a tribasic acid) to elute. Oxalic acid and citric acid are not retained well by ion-exclusion chromatography. The method developed here retains the acid peaks well, and allows the peaks to be less affected from early eluting impurities.
It also demonstrated the feasibility of analyzing amino acids. The chromatograms showed hydrophobic amino acids to have tendencies of eluting earlier while acidic amino acids to elute later.
Fig. 6. Chromatograms showing the simultaneous LC/MS analysis of a mixture containing 14 saccharides, 9 organic acids, and 20 amino acids.
| Sample | 1 μg/mL each in H2O/CH3CN = 1/4, 5 μL |
| Column | Shodex HILICpak VG-50 2D |
| Eluent | (A) 0.5% NH3 aq./(B) CH3CN Linear gradient: 80% B (0-2 min) → 10% B (12-15 min) → 80% B (15.01-20 min) |
| Flow rate | 0.2 mL/min |
| Detector | ESI-MS MRM(-) |
| Column temp. | 40°C |
3. Application of the Method for Analyzing Commercial Energy Drink
Figure 7 shows LC/MS analysis result of a commercially available energy drink. It demonstrated an effective simultaneous analysis of saccharides (fructose, glucose, sucrose), citric acid, and amino acids (isoleucine, phenylalanine, threonine, glutamic acid). Also, the method was feasible analyzing caffeine and water-soluble vitamins (nicotinamide, riboflavin, pyridoxine) present in the energy drink.
Fig. 7. LC/MS analysis result of a commercial energy drink showing detection of multiple compound classes including saccharides, amino acids, vitamins, and caffeine.
| Sample | Commercial energy drink x100 dilution in H2O/CH3CN = 1/1, 2 μL |
| Column | Shodex HILICpak VG-50 2D |
| Eluent | (A) 0.5% NH3 aq./(B) CH3CN High pressure linear gradient: 70% B (0 min) → 10% B (5-15 min) |
| Flow rate | 0.2 mL/min |
| Detector | ESI-MS MRM(-) |
| Column temp. | 40°C |
Conclusions
A polymer-based amino column, Shodex HILICpak VG-50 2D, provides many advantageous analytical features when used under alkaline conditions. LC/MS with an ammonia water/acetonitrile gradient elution is effective in providing good separation and high sensitivity analysis of various hydrophilic compounds. The method is feasible for the analysis of saccharides, organic acids, and amino acids simultaneously, which was difficult by using previously available methods. This can be achieved without using pre-column derivatization nor addition of ion-pairing reagent.
The alkaline conditions promote deprotonation of saccharides which make it possible to monitor negative ions and contributes to the enhanced high sensitivity detection. The developed method showed its effectiveness in analyzing commercial energy drink. Not only monitoring saccharides, citric acid, and amino acids, the method demonstrated its ability to monitor caffeine and water soluble vitamins simultaneously.