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HomeSmall Molecule HPLCUHPLC Analysis of Nucleosides on Supel™ Carbon LC Column: Performance Comparisons with Competitor

UHPLC Analysis of Nucleosides on Supel™ Carbon LC Column: Performance Comparisons with Competitor

William L. Maule III, Clint Corman, Michael Ye, Cory Muraco, Curtis Frantz

Merck Bellefonte, PA

Introduction

Nucleosides are an important class of small molecules that are the building blocks of nucleic acids. In addition, nucleosides have found interest as active pharmaceutical ingredients (APIs) in antiretroviral drugs and as biomarkers for certain diseases. However, due to the structural similarity between these analytes, developing an analytical method to characterize a series of these compounds can be challenging. This application demonstrates the use of the SupelTM Carbon LC column to resolve a set of 12 nucleosides.

2-D chemical structures (Bond line structure) of nucleosides used in the study to resolve 12 nucleosides by Ultra high performance liquid chromatography (UHPLC)

Figure 1.Chemical Structures of Compounds Used in this Study.

Experimental conditions for Nucleoside analysis

Experimental Conditions
Column(s)Supel™ Carbon LC, 10 cm x 2.1 mm I.D., 2.7 µm
 Competitor T, 10 cm x 2.1 mm I.D., 3.0 µm
DetectionUV; 260 nm
Buffer5 mM Ammonium formate in water, pH adjusted to 5.3
Mobile phase[A] 5 mM Ammonium formate, pH 5.3; [B] Acetonitrile
GradientTime (min)%A%B
 09010
 404060
 509010
Test MixNucleosides Test Mix, varied concentration, 1% sodium formate.
Injection Volume10 µL
Flow Rate0.25 mL/min
TemperatureColumn: 55 °C
Table 1.Chromatographic Conditions for Analysis of Nucleosides.
 Test Mix Analyte/Concentration
1Cytidine (50 µg/mL)
2Guanosine (25 µg/mL)
3Inosine (25 µg/mL)
41-Methyladenosine (25 µg/mL)
55-Methylcytidine (100 µg/mL)
62'-O-Methylcytidine (20 µg/mL)
73-Methylcytidine methosulfate (100 µg/mL)
87-Methylguanosine (25 µg/mL)
95-Methyluridine (50 µg/mL)
10ß-Pseudouridine (25 µg/mL)
112-Thiocytidine dihydrate (10 µg/mL)
12Uridine (25 µg/mL)
Table 2.Components of Test Mix Used in Study.

Performance results and comparison

Chromatograms showing peaks obtained for the analysis of Twelve Nucleosides on Supel™ Carbon LC

Figure 3.Analysis of Twelve Nucleosides on Supel™ Carbon LC.

PeakCompoundRetention Time (min)
1ß-Pseudouridine (25 μg/mL)5.346
23-Methylcytidine methosulfate (100 µg/mL)5.791
3Cytidine (50 µg/mL)5.982
4Uridine (25 µg/mL)7.328
52'-O-Methylcytidine (20 µg/mL)8.283
65-Methylcytidine (100 µg/mL)9.307
71-Methyladenosine (25 µg/mL)9.530
85-Methylcytidine (100 µg/mL)11.641
9Inosine (25 µg/mL)12.130
107-Methylguanosine (25 µg/mL)12.725
112-Thiocytidine dihydrate (10 µg/mL)13.571
12Guanosine (25 µg/mL)14.203
Table 3.Elution Order for Nucleosides on Supel™ Carbon LC.
Chromatograms showing peaks obtained for the analysis of Twelve Nucleosides on Competitor Column

Figure 4.Analysis of Twelve Nucleosides on a Competing Carbon Column.

PeakCompoundRet. Time (min)
1ß-Pseudouridine (25 µg/mL)5.242
2Cytidine (50 µg/mL)6.258
3Uridine (25 µg/mL)6.804
42'-O-Methylcytidine (20 µg/mL)8.652
55-Methylcytidine (100 µg/mL)9.705
65-Methyluridine (50 µg/mL)10.971
73-Methylcytidine methosulfate (100 µg/mL)11.616
8Inosine (25 µg/mL)12.199
92-Thiocytidine dihydrate (10 µg/mL)14.040
101-Methyladenosine (25 µg/mL)N/A
11Guanosine (25 µg/mL)17.366
127-Methylguanosine (25 µg/mL)N/A
Table 4.Elution Order for Nucleosides on Competing Carbon Column.

Conclusion

This application has demonstrated the use of SupelTM Carbon LC column in resolving 12 nucleosides in under 15 minutes. The SupelTM Carbon LC column also outperforms a competitor column where two co-elutions occur and is an excellent choice for use in nucleoside biomarker identification and quantitation.

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