adenosine and adenine were obtained as gift samples from defence institute of physiology and allied sciences ( delhi , india ) . milli - q water , used throughout the experiment , was prepared using millipore water purification system . chromatographic experiments were conducted on a yl9100 hplc system ( south korea ) that comprised quaternary yl9110 pumps , a variable wavelength programmable yl9120 ultraviolet ( uv)-visible detector , yl9130 column oven and a system controller . the instrument was controlled by use of yl - clarity software installed with the equipment . samples were injected by using a rheodyne injector fitted with a 20 l fixed loop . the separation was achieved by using c18 reversed phase column ( merck lichrocart 250 - 4 , lichrosphere 100 rp 18e , 5.0 m , sorbent lot number l57020637 ) . the mobile phase consisted of methanol and potassium dihydrogen phosphate ( kh2 po4 ) as the mobile phase in isocratic elution method at a flow rate of 1.0 ml / min . all the analysis was performed at room temperature and detection was carried out at a wavelength of 254 nm using a uv - visible detector . g. lucidum and c. sinensis were procured from ethno - botany division of field research laboratory , leh , india , and defence institute of bio - energy research , haldwani , respectively , where the voucher specimen of the plant was preserved in the herbarium . fruit body of c. sinensis , and g. lucidum were cleaned and washed thoroughly with water and re - washed with distilled water and dried under shade in a clean , dust - free environment and crushed using a laboratory blender . aqueous extract of c. sinensis and g. lucidum were prepared by the accelerated solvent extraction system equipped with a solvent controller unit ( ase 350 , dionex corporation , sunnyvale , ca , usa ) . extractions were carried out using distilled water . the sample was prepared by taking 20 mg of previously dried g. lucidum aqueous extract and 20 mg of c. sinensis extract in 2.0 ml of methanol each . the solutions were filtered using minigen mg25020ny 0.2 m syringe filter and used for analysis . adenosine and adenine were obtained as gift samples from defence institute of physiology and allied sciences ( delhi , india ) . milli - q water , used throughout the experiment , was prepared using millipore water purification system . chromatographic experiments were conducted on a yl9100 hplc system ( south korea ) that comprised quaternary yl9110 pumps , a variable wavelength programmable yl9120 ultraviolet ( uv)-visible detector , yl9130 column oven and a system controller . the instrument was controlled by use of yl - clarity software installed with the equipment . samples were injected by using a rheodyne injector fitted with a 20 l fixed loop . the separation was achieved by using c18 reversed phase column ( merck lichrocart 250 - 4 , lichrosphere 100 rp 18e , 5.0 m , sorbent lot number l57020637 ) . the mobile phase consisted of methanol and potassium dihydrogen phosphate ( kh2 po4 ) as the mobile phase in isocratic elution method at a flow rate of 1.0 ml / min . all the analysis was performed at room temperature and detection was carried out at a wavelength of 254 nm using a uv - visible detector . g. lucidum and c. sinensis were procured from ethno - botany division of field research laboratory , leh , india , and defence institute of bio - energy research , haldwani , respectively , where the voucher specimen of the plant was preserved in the herbarium . fruit body of c. sinensis , and g. lucidum were cleaned and washed thoroughly with water and re - washed with distilled water and dried under shade in a clean , dust - free environment and crushed using a laboratory blender . aqueous extract of c. sinensis and g. lucidum were prepared by the accelerated solvent extraction system equipped with a solvent controller unit ( ase 350 , dionex corporation , sunnyvale , ca , usa ) . the sample was prepared by taking 20 mg of previously dried g. lucidum aqueous extract and 20 mg of c. sinensis extract in 2.0 ml of methanol each . the solutions were filtered using minigen mg25020ny 0.2 m syringe filter and used for analysis . a variety of mobile phases was investigated for the development of the hplc method suitable for analysis of adenine and adenosine in crude drug extract . the investigated mobile phases included methanol : water , 50:50 ( % v / v ) , acetonitrile : phosphate buffer ( ph 3.5 adjusted with orthophosphoric acid ) , 80:20 ( % v / v ) , acetonitrile : phosphate buffer ( ph 3.5 with orthophosphoric acid ) , 60:40 ( % v / v ) , acetonitrile : water , 50:50 ( % v / v ) and acetonitrile : water , 60:40 ( % v / v ) . the methanol : kh2 po4 at 10:90 resulted in sharp peaks with a good resolution [ figure 1 ] . high - performance liquid chromatography chromatogram of adenosine and adenine at 254 nm : standard ( a ) ; cordyceps sinensis ( b ) ; ganoderma lucidum ( c ) linear regression analysis data for the calibration plot showed a good linear relationship between response and concentration in the range of 25200 g / ml for adenosine and 100800 g / ml for adenine with regression coefficient of 0.999 and 0.996 , respectively ; the regression equation is y = 239.64x  244 and y = 28.936x  82.86 , respectively . the slope  standard deviation ( sd ) were found to be 238.98  0.52 and 28.23  0.43 , respectively . intercept  sd were found to be 244  1.2 and 82.86  1.1 , respectively [ figure 2 ] . calibration curve for ( a ) : standard adenosine ( b ) : standard adenine the quantitative estimation of adenine and adenosine content was carried out by hplc by using calibration curve with respect to the peak area of standard peaks . the adenine was found to be 0.16% and 0.71% w / w in g. lucidum and in c. sinensis , respectively , and adenosine was found to be 0.14% w / w in g. lucidum whereas absent in c. sinensis . a variety of mobile phases was investigated for the development of the hplc method suitable for analysis of adenine and adenosine in crude drug extract . the investigated mobile phases included methanol : water , 50:50 ( % v / v ) , acetonitrile : phosphate buffer ( ph 3.5 adjusted with orthophosphoric acid ) , 80:20 ( % v / v ) , acetonitrile : phosphate buffer ( ph 3.5 with orthophosphoric acid ) , 60:40 ( % v / v ) , acetonitrile : water , 50:50 ( % v / v ) and acetonitrile : water , 60:40 ( % v / v ) . the methanol : kh2 po4 at 10:90 resulted in sharp peaks with a good resolution [ figure 1 ] . high - performance liquid chromatography chromatogram of adenosine and adenine at 254 nm : standard ( a ) ; cordyceps sinensis ( b ) ; ganoderma lucidum ( c ) linear regression analysis data for the calibration plot showed a good linear relationship between response and concentration in the range of 25200 g / ml for adenosine and 100800 g / ml for adenine with regression coefficient of 0.999 and 0.996 , respectively ; the regression equation is y = 239.64x  244 and y = 28.936x  82.86 , respectively . the slope  standard deviation ( sd ) were found to be 238.98  0.52 and 28.23  0.43 , respectively . intercept  sd were found to be 244  1.2 and 82.86  1.1 , respectively [ figure 2 ] . the quantitative estimation of adenine and adenosine content was carried out by hplc by using calibration curve with respect to the peak area of standard peaks . the adenine was found to be 0.16% and 0.71% w / w in g. lucidum and in c. sinensis , respectively , and adenosine was found to be 0.14% w / w in g. lucidum whereas absent in c. sinensis . the developed hplc method was developed and validated and found to be simple , sensitive , and accurate in wide linearity range , which can be used for the quantification of adenosine in crude drugs and formulations . it can also be used for quality control and standardization of crude drugs for in which adenine and adenosine are present as a major constituent .
background : nucleosides are supportive in the regulation and modulation of various physiological processes in body , they acts as precursors in nucleic acid synthesis , enhance immune response , help in absorption of iron and influence the metabolism of fatty acids . 
 cordyceps sinensis and ganoderma lucidum are well - known for its use in traditional medicine of china , nepal and india . 
 they are rich in nucleosides such as adenine , adenosine , cordycepin , etc . 
 hence , a simple , economic and accurate high - performance liquid chromatography ( hplc ) analytical method was proposed for determination of adenine and adenosine for the quality control of plants.materials and methods : chromatographic experiments were conducted on yl9100 hplc system ( south korea ) . 
 reversed - phase chromatography was performed on a c18 column with methanol and dihydrogen phosphate as the mobile phase in isocratic elution method at a flow rate of 1.0 ml / min . 
 detection was carried out at 254 nm , which gives a sharp peak of adenine and adenosine at a retention time of 6.53  0.02 min and 12.41  0.02 , respectively.results and discussion : linear regression analysis data for the calibration plot showed a good linear relationship between response and concentration in the range of 25200 g / ml for adenosine and 100800 g / ml for adenine with regression coefficient of 0.999 and 0.996 , respectively . 
 the adenine was found 0.16% and 0.71% w / w in g. lucidum and in c. sinensis , respectively , and adenosine was found to be 0.14% w / w in g. lucidum whereas absent in c. sinensis.conclusion:the developed hplc method for the quantification of adenosine and adenine can be used for the quality control and standardization of crude drug and for the different herbal formulations , in which adenine and adenosine are present as major constituents . the wide linearity range , sensitivity , accuracy , and simple mobile phase imply the method is suitable for routine quantification of adenosine and adenine with high precision and accuracy .