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 Supel^TM Carbon LC column to resolve a set of 12 nucleosides.

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

Experimental conditions for Nucleoside analysis

Table 1. Chromatographic Conditions for Analysis of Nucleosides.

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
Detection	UV; 260 nm
Buffer	5 mM Ammonium formate in water, pH adjusted to 5.3
Mobile phase	[A] 5 mM Ammonium formate, pH 5.3; [B] Acetonitrile
Gradient	Time (min)	%A	%B
	0	90	10
	40	40	60
	50	90	10
Test Mix	Nucleosides Test Mix, varied concentration, 1% sodium formate.
Injection Volume	10 µL
Flow Rate	0.25 mL/min
Temperature	Column: 55 °C

Table 2. Components of Test Mix Used in Study.

	Test Mix Analyte/Concentration
1	Cytidine (50 µg/mL)
2	Guanosine (25 µg/mL)
3	Inosine (25 µg/mL)
4	1-Methyladenosine (25 µg/mL)
5	5-Methylcytidine (100 µg/mL)
6	2'-O-Methylcytidine (20 µg/mL)
7	3-Methylcytidine methosulfate (100 µg/mL)
8	7-Methylguanosine (25 µg/mL)
9	5-Methyluridine (50 µg/mL)
10	ß-Pseudouridine (25 µg/mL)
11	2-Thiocytidine dihydrate (10 µg/mL)
12	Uridine (25 µg/mL)

Performance results and comparison

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

Table 3. Elution Order for Nucleosides on Supel™ Carbon LC.

Peak	Compound	Retention Time (min)
1	ß-Pseudouridine (25 μg/mL)	5.346
2	3-Methylcytidine methosulfate (100 µg/mL)	5.791
3	Cytidine (50 µg/mL)	5.982
4	Uridine (25 µg/mL)	7.328
5	2'-O-Methylcytidine (20 µg/mL)	8.283
6	5-Methylcytidine (100 µg/mL)	9.307
7	1-Methyladenosine (25 µg/mL)	9.530
8	5-Methylcytidine (100 µg/mL)	11.641
9	Inosine (25 µg/mL)	12.130
10	7-Methylguanosine (25 µg/mL)	12.725
11	2-Thiocytidine dihydrate (10 µg/mL)	13.571
12	Guanosine (25 µg/mL)	14.203

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

Table 4. Elution Order for Nucleosides on Competing Carbon Column.

Peak	Compound	Ret. Time (min)
1	ß-Pseudouridine (25 µg/mL)	5.242
2	Cytidine (50 µg/mL)	6.258
3	Uridine (25 µg/mL)	6.804
4	2'-O-Methylcytidine (20 µg/mL)	8.652
5	5-Methylcytidine (100 µg/mL)	9.705
6	5-Methyluridine (50 µg/mL)	10.971
7	3-Methylcytidine methosulfate (100 µg/mL)	11.616
8	Inosine (25 µg/mL)	12.199
9	2-Thiocytidine dihydrate (10 µg/mL)	14.040
10	1-Methyladenosine (25 µg/mL)	N/A
11	Guanosine (25 µg/mL)	17.366
12	7-Methylguanosine (25 µg/mL)	N/A

Conclusion

This application has demonstrated the use of Supel^TM Carbon LC column in resolving 12 nucleosides in under 15 minutes. The Supel^TM 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.