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2026
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08
Analysis of Inosine, referring to the Chinese Pharmacopoeia (2025 edition), Part II
Inosine is a nucleoside compound formed by the combination of hypoxanthine and ribose. It has a wide range of clinical applications and can be used for leukopenia caused by various reasons, acute and chronic hepatitis or cirrhosis, and various heart diseases.
According to the testing methods for substances and content measurement in the inosine section of the Chinese Pharmacopoeia (2025 edition, Part II), chromatographic columns were screened for testing.
Considering that the mobile phase condition of this method is 5% methanol, CAPCELL PAK C18 AQ and DAISOPAK SP-ODS-BP columns, which can withstand 100% aqueous conditions, were chosen for the analysis.
Under the pharmacopoeia conditions, both columns meet the pharmacopoeia requirements, with a separation degree of over 3 between the inosine peak and the guanosine peak, and more than 10,000 theoretical plates.
01
CP C18 AQ Analysis
About matter
According to the analysis conditions for the relevant substance, the results using a CAPCELL PAK C18 AQ S5; 4.6 mm i.d. × 250 mm column are shown in Figures 1 and 2.
In the system suitability solution, the retention time of inosine is 14.08 min, with a resolution of 4.02 from the guanosine peak, meeting the pharmacopeia requirement of not less than 3.0. The theoretical plate number is 11,040, meeting the pharmacopeia requirement of not less than 3,000, and the asymmetry factor is 1.10, giving a good peak shape.
Figure 1 System Applicability Solution Analysis Spectrum
The numbers shown in the diagram are the separation degree and the number of theoretical plates.
Table 1 Results of Peak Integration
Figure 2 Test Sample Analysis Results
【Chromatography conditions】
Column：CP C18 AQ S5; 4.6×250 mm
Mobile phase: methanol / water = 5 / 95
Flow rate：1.0 mL/min
Coulmn Temperatrue：25 ℃
Detector：UV 248 nm
Injection Volumn：10 μL
System suitability solution: Prepared according to pharmacopoeia methods, with 0.5 µg/mL hypoxanthine, 1.5 µg/mL guanosine, and 0.5 mg/mL inosine (water-soluble)
Test item: 0.5 mg/mL (water-soluble)
Content measurement
Using the method under the content determination item, we can also get good analysis results. As shown in Figure 3, the inosine peak shape is good, with an asymmetry factor of 1.02.
Figure 3 Content Measurement Analysis Results
【Chromatography Conditions】
Column：CP C18 AQ S5; 4.6×250 mm
Mobile phase：Methanol / Water = 5 / 95
flow rate：1.0 mL/min
Column temperature：25 ℃
Check：UV 248 nm
Injection Volumn：10 μL
Test item: 20 μg/mL (water-soluble)
02
DP SP-ODS-BP Analysis
About matter
According to the analysis conditions for the relevant substance, the DAISOPAK SP-120-5-ODS-BP S5; 4.6 mm i.d. × 250 mm column was used, and the results are shown in Figures 4 and 5.
In the system suitability solution, the retention time for inosine was 15.78 min, with a resolution of 3.98 from the guanosine peak, meeting the pharmacopeia requirement of not less than 3.0. The theoretical plate number was 13,328, above the pharmacopeia minimum of 3000, with an asymmetry factor of 1.11, indicating a good peak shape.
Figure 4 System Applicability Solution Analysis Results
The numbers shown in the diagram are the separation degree and the theoretical number of plates.
Table 2 Points Table
Figure 5 Test Sample Analysis Results
【Chromatography conditions】
Coumn：DP SP-ODS-BP S5; 4.6×250 mm
Mobile phase：Methanol / Water = 5 / 95
Flow rate：1.0 mL/min
Column Temperature：25 ℃
Detector：UV 248 nm
Injection Volumn：10 μL
System suitability solution: prepared according to pharmacopoeia methods, with 0.5 µg/mL hypoxanthine, 1.5 µg/mL guanosine, and 0.5 mg/mL inosine (water-soluble)
Test item: 0.5 mg/mL (water-soluble)
Content measurement
Using the method under the content determination item, we can also get good analytical results. As shown in Figure 6, the inosine peak shape is good, with an asymmetry factor of 1.10.
图6 含量测定分析结果
【Chromatography conditions】
Column：DP SP-ODS-BP S5; 4.6×250 mm
Mobile phase: methanol / water = 5 / 95
Flow rate：1.0 mL/min
Column Temperature：25 ℃
Detector：UV 248 nm
Injection Volumn：10 μL
Test item：20 μg/mL （Water-soluble）
In summary, according to the methods for substances and content determination under the Inosine section of the 2025 edition of the Chinese Pharmacopoeia (Part II), using CAPCELL PAK C18 AQ S5; 4.6 mm i.d.×250 mm and DAISOPAK SP-120-5-ODS-BP S5; 4.6 mm i.d.×250 mm columns that can withstand 100% aqueous phase, good detection results can be obtained.
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