ergosterol is one of the best - known steroids , which exists widely in many medicinal fungi such as polyporus umbellatus , cordyceps sinensis , and hypsizigus marmoreus [ 13 ] . ergosterol has also been reported to possess cytotoxic activity   and anti - inflammatory activity . despite the fact that ergosterol showed multiple pharmacological activities , several pharmacokinetic and biochemical aspects of this compound remain unclear . however , the final effect of the drug in vivo might be influenced by many factors , such as body - and / or cell - compartment distribution , drug metabolism , lipophilicity , membrane permeability , and protein binding . so , these multiple pharmacological activities of ergosterol make it worth carrying out further study on pharmacokinetic properties and elimination pathway of ergosterol . a number of methods have been reported for the quantification of ergosterol in raw materials , such as high performance liquid chromatography - ultraviolet detection ( hplc - uv ) [ 1 , 611 ] , high performance liquid chromatography - tandem mass spectrometry ( hplc - ms / ms ) [ 12 , 13 ] , and gas chromatography - mass spectrometry ( gc - ms ) [ 1416 ] . to the best of our knowledge , there is no information describing the quantification of ergosterol in biological samples such as rat or human plasma , urine , and faeces . in addition , the effects on the elimination pathway of ergosterol have not been reported . in general , preclinical research including metabolism and pharmacokinetics of herbal medicine components are of great importance in understanding their biological effects and safety [ 17 , 18 ] . in order to investigate the pharmacokinetic properties and elimination pathway of ergosterol , a simple and reproducible analytical method for quantification of ergosterol in rat plasma , urine , and faeces recently , the cloud - point extraction ( cpe ) method has aroused much attention as a convenient alternative to the conventional extraction systems . compared with classical organic solvents , it offers the advantages of safety , low cost , high - concentration efficiency , easy disposal of surfactants , low toxicity , less environmental pollution , and simple procedure . cpe has been increasingly applied for the selective extraction of various analytes from biological and environmental samples ( proteins , vitamins , and metal ions ) , but only a few reports involve the extraction of drugs from plasma for pharmacokinetic studies [ 20 , 21 ] . in this paper , a simple , specific , and reproducible hplc - uv method with a simple protein precipitation procedure was described to determine ergosterol in rat plasma , urine , and faeces for the first time . the method was validated and successfully applied to the pharmacokinetic study of ergosterol in rat plasma , urine , and faeces after oral administration of ergosterol at a dose of 100  mg / kg . the standard of ergosterol ( figure 1(a ) ) was isolated by the authors from polyporus umbellatus . its structure was characterized by chemical and spectroscopic methods ( h nmr , c nmr , and ms ) and compared with those found in the literature . ergosta-4,6,8(14),22-tetraen-3-one ( ergone , figure 1(b ) ) was used as internal standard ( is ) which was one of the components of polyporus umbellatus purified in our laboratory with 99% purity as determined by hplc . ultrahigh purity water was prepared by a millipore - q sas 67120 molsheim ( france ) . standard stock solutions of ergosterol ( 5  g / ml ) and ergone ( 1 , 5 , and 100  g / ml ) were prepared by dissolving suitable amounts of pure substance in acetone and were stored in darkness at 4c . faecal samples were homogenized with acetone in the ratio 1  :  10 ( g  :  vol = faeces  :  acetone ) to obtain faecal homogenate . for hplc determination , calibration standards of ergosterol at concentration levels of 0.09 , 0.25 , 0.50 , 0.75 , 1.00 , 2.00 , and 2.50  g / ml were prepared by spiking appropriate amount of the standard solutions in blank plasma and urine obtained from healthy rats . calibration standards of ergosterol at concentrations of 0.08 , 0.25 , 5 , 25 , 50 , 100 , and 125  g / ml were also prepared by spiking appropriate amount of the standard solutions in faecal homogenate obtained from healthy rats . the quality control ( qc ) samples were separately prepared in a similar manner as those used for the calibration curve . concentrations of 0.25 , 0.75 , and 2.00  g / ml were used for plasma and urine calibration standards , whereas concentrations of 0.25 , 25 , and 100  g / ml were used for faecal homogenate corresponding to the low qc , medium qc , and high qc , respectively . hplc was performed with a waters 2695 instrument composed of a quaternary pump , a column oven , and a waters 2487 dual wavelength absorbance detector , and empower was used for data collection . the chromatographic separation was performed on an inertsil ods-3 analytical column ( 250  mm  4.6  mm i.d . , 5  m , japan ) with the column temperature set at 30c . an isocratic elution of methanol ( a ) and water ( b ) was 98  :  2 ( v / v ) in 23  min . the flow rate was 1.0  ml / min with detector wavelength set at 283 nm ( maximum absorption wavelength of ergosterol ) , and the injection volume was 20  l . the plasma or urine was prepared by a cloud - point extraction method . in a 1.5  ml capped centrifugal tube , 50  l of plasma or urine was spiked with 15  l ( 5  g / ml for plasma , 1  g / ml for urine ) of is working solution and 135  l of 0.3  mol / l sodium chloride solution . the contents were mixed for 2  min , and then followed by the addition of 100  l of 5% triton x-114 ( v / v ) aqueous solution . after that , the obtained contents were well mixed again for 5  min and then incubated in the thermostatic bath at 40c for 20  min . after the phase separation was formed by centrifugation at 5000  rpm for 10  min , the surfactant - rich phase was obtained in the bottom of the tube by the removal of the water phase . then , the contents were mixed and centrifuged at 16,000  rpm for 5  min , respectively . most of the surfactants and coextractants such as hydrophobic proteins were precipitated in the bottom of the tube , and 20  l of supernatant fluid was injected into the hplc system for analysis . a 100  l volume of faecal homogenate standard or sample was transferred to a 1.5  ml centrifuge tube , and then 30  l of is working solution was spiked and vortex - mixed for 1  min . 70  l of acetone was added to the solution and vortex again for 2  min . a 20  l of supernatant of the solution was injected into the hplc for analysis . hplc - uv method was applied for the quantification of ergosterol from rat plasma , urine , and faeces . calibration standards of seven concentrations of ergosterol for plasma and urine ( 0.09 , 0.25 , 0.50 , 0.75 , 1.00 , 2.00 , and 2.50  g / ml ) and calibration standards for faecal homogenate ( 0.08 , 0.25 , 5 , 25 , 50 , 100 , and 125  g / ml ) were extracted and assayed . the linearity of the calibration curve was confirmed by plotting the peak - area ratios of ergosterol to is versus the ergosterol concentrations with a 1/x - weighted least - squares linear regression analysis . the limit of detection ( lod ) was considered as the final concentration producing a signal - to - noise ratio of 3 . the lower limit of quantification ( lloq ) was considered as the lowest concentration on the calibration curve where precision was within 20% and accuracy was within 100  20% . the accuracy and intra- and interday precisions of the method were determined for ergosterol according to fda guidance for bioanalytical method validation . accuracy and precision were assessed by the determination of qc samples at three concentration levels in three different validation assays . accuracy was calculated by the percentage difference between the concentration of drug measured from calibration curve and that of drug added to the blank plasma , urine , and faecal homogenate . selectivity of the method was assessed by analyzing five independent sources of blank plasma , urine , and faecal homogenate or plasma , urine , and faecal homogenate samples spiked with ergosterol and the is , and observing the extent to which interferent from plasma , urine , and faecal homogenate may interfere with the analyte or is . the extraction recovery for plasma , urine , and faecal homogenate at three different concentrations of ergosterol was determined . the analyte / is peak - area ratios were compared to those obtained from the direct injection of the compounds dissolved in the solutions at the same theoretical concentrations . short - term stability was assessed by analyzing qc plasma , urine , and faecal homogenate samples kept at room temperature for 6  h that exceeded the routine preparation time of samples . long - term stability was determined by assaying qc plasma , urine , and faecal homogenate samples after storage at 20c for 30 days . freeze - thaw stability was investigated after three freeze-(20c ) thaw ( room temperature ) cycles . to validate the method with real samples , a trial was undertaken to determine ergosterol in the plasma and excretion samples of healthy rats , which were administered a single dose of ergosterol orally . the study was conducted in accordance with the regulations of experimental animal administration issued by the state committee of science and technology of china . all procedures and the care of the rats were in accordance with institutional guidelines for animal use in research . rats were housed in individual metabolic cages on standard laboratory food bedding in a temperature controlled room ( 22  2c ) with a 12  h light / dark cycle . male sprague - dawley ( sd ) rats , 200  10  g , fasted overnight with free access to water for at least 12  h , were dosed orally by gavages with 100  mg / kg body weight of ergosterol dissolved in plant oil as a vehicle . rats were divided into three groups ( n = 6 ) based on the time of blood sampling with two animals each . the control groups received the vehicle only . the blood samples ( approximately 300  l ) were collected from vena orbitalis in heparinized tubes at control and 1 , 2 , 3 , 4 , 5 , 6 , 8 , 10 , 12 , 14 , 16 , 24 , and 36  h after the administration . samples were immediately centrifuged at 5,000  g for 10  min and the plasma was frozen at 20c and stored until analysis . the estimation of ergosterol in all the samples was undertaken within 36  h of blood collection by the method described previously . excretion was studied in another four groups ( n = 6 ) based on the time of urine and faeces sampling with three animals each . rats were housed with free access to food and water in individual metabolic cages , except for the final 12  h before a single oral administration of 100  mg / kg of ergosterol ( access to water was ad libitum during the experiment ) . faeces and urine were collected after administration in different periods ( 02 , 24 , 46 , 68 , 810 , 1012 , 1214 , 1416 , 1618 , 1824 , and 2436  h ) . the amount of faeces and urine collected over each period was recorded , respectively , and then urine and faeces were stored at 20c until analysis . pharmacokinetics analysis was carried out by noncompartmental method with the aid of the software das 2.0 ( issued by the state food and drug administration of china for pharmacokinetic study ) and pharmaceutical parameters were obtained . the percentage of ergosterol eliminated in faeces was calculated with the accumulated ergosterol eliminated in all periods divided by administration amount ( ergosterol eliminated over each period was calculated using the amount of faeces multiplied by the concentration of ergosterol in it ) . the characteristics of ergosterol are h nmr ( cdcl3 , 500 mhz ) 3.63 ( 1h , m , h-3 ) , 5.39 ( 1h , m , h-6 ) , 5.58 ( 1h , m , h-7 ) , 0.63 ( 3h , s , h-18 ) , 0.95 ( 3h , s , h-19 ) , 1.04 ( 3h , d , j = 6.6  hz , h-21 ) , 5.18 ( 1h , dd , j = 15.2 , 8.0  hz , h-22 ) , 5.22 ( 1h , dd , j = 15.2 , 8.0  hz , h-23 ) , 0.83 ( 3h , d , j = 6.7  hz , h-26 ) , 0.84 ( 3h , d , j = 6.4  hz , h-27 ) , 0.92 ( 3h , d , j = 6.8  hz , h-28 ) ; c nmr ( cdcl3 , 125  mhz ) 38.3 ( c-1 ) , 32.1 ( c-2 ) , 70.6 ( c-3 ) , 40.8 ( c-4 ) , 139.9 ( c-5 ) , 119.7 ( c-6 ) , 116.4 ( c-7 ) , 141.5 ( c-8 ) , 46.4 ( c-9 ) , 37.1 ( c-10 ) , 21.3 ( c-11 ) , 39.0 ( c-12 ) , 42.8 ( c-13 ) , 54.7 ( c-14 ) , 23.1 ( c-15 ) , 28.4 ( c-16 ) , 55.8 ( c-17 ) , 12.2 ( c-18 ) , 17.7 ( c-19 ) , 40.5 ( c-20 ) , 21.2 ( c-21 ) , 135.5 ( c-22 ) , 132.1 ( c-23 ) , 42.9 ( c-24 ) , 33.2 ( c-25 ) , 19.8 ( c-26 ) , 20.1 ( c-27 ) , 16.4 ( c-28 ) . the data of mass spectrum was published in our previous paper [ 25 , 26 ] . chromatographic optimisation of hplc conditions was carried out with respect to mobile phase conditions ( methanol - water or acetonitrile - water ) , stationary phase ( inertsil ods-3 column , 250  mm  4.6  mm i.d . , 5  m or diamonsil c18 column , 250  mm  4.6  mm i.d . , 5  m ) , temperature ( 30 or 35c ) , and peak shape . the test results showed that the peak shapes of ergosterol were improved by the solvent system of methanol and water . the previously - mentioned method was demonstrated by comparing chromatograms of six independent biological samples ( plasma , urine , and faecal homogenate ) from blank rats , each as a blank sample and a spiked sample . the chromatograms of ergosterol and the is are showed in figures 2(a1)2(a3 ) , 2(b1)2(b3 ) , and 2(c1)2(c3 ) , respectively . figures 2(b1)2(b3 ) indicate no significant interferences at the retention times of ergosterol and the is . the ergosterol and the is of the retention times were 18.8 and 15.7  min , respectively , with a total run time of less than 23  min . system suitability parameters for the method were as follows : the theoretical plates for ergosterol and the is were 4900 and 5400 , respectively . tailing factor was less than 1.1 for all ergosterol and the is and resolution among ergosterol and the is was more than 1.5 . ergosterol was chosen as is because of its similarity to the analytes with respect to polarity , high recovery , and suitable retention time . calibration curves of ergosterol were linear over the concentration range of 0.092.50  g / ml for plasma and urine and 0.08125  g / ml for faecal homogenate . the typical equation was y = 0.8251  x + 0.0357 ( r = 0.9996 ) for plasma , y = 2.9189  x + 0.1265 ( r = 0.9995 ) for urine , and y = 0.0262  x + 0.0104 ( r = 0.9994 ) for faecal homogenate . y = peak - area ratio ( ergosterol / is ) and x = ergosterol concentration . during routine analysis , each analytical run included a set of calibration samples , a set of qc samples , and the unknowns . the lod of ergosterol in rat plasma , urine , and faecal homogenate were 42 , 50 , and 49  ng / ml , respectively . the loq of ergosterol in rat plasma , urine , and faecal homogenate was 90 , 90 , and 80  ng / ml respectively . table 1 shows the intra- and interday precision and accuracy for ergosterol from biological matrices qc samples . the intra- and interday precisions were measured below 9.6% and 7.8% , respectively , with relative recovery from 95.1% to 103.6% . the selectivity of the method was demonstrated by comparing chromatograms of five independent biological samples ( plasma , urine , and faecal homogenate ) from blank rats , each as a blank sample and a spiked sample . figure 2 indicates no significant interferences at the retention times of ergosterol and is in plasma , urine , and faeces samples . the ergosterol and is of the retention times were 18.8 and 15.7  min , respectively . the mean extraction recovery of ergosterol from plasma , urine , and faecal homogenate was 90.9  2.9% , 92.4  2.6% , and 93.7  3.3% , respectively . the mean relative recovery for is was 94.5  3.2% ( n = 5 ) . table 3 shows the results of short - term , long - term , and freeze - thaw stability of ergosterol in plasma , urine , and faeces . ergosterol was found to be stable in frozen plasma , urine , and faeces after three freeze - thaw cycles . long - term stability studies of ergosterol in three rat matrices showed appreciable stability over 30 days . furthermore , ergosterol in three rat matrices was found to be stable at room temperature for a period of 6  h. all the results well met the criterion for stability measurements . pharmacokinetic studies of this drug are important for further biological and biochemical research and for future clinical trials . after a single oral administration of ergosterol ( 100  mg / kg ) to rats , the concentrations of ergosterol in plasma , urine , and faecal the ergosterol was detected in rat plasma , urine , and faeces samples collected from 0 to 36  h after an oral administration . the results indicate that the ergosterol levels in faeces are much higher than those in plasma and urine of the rat ( figure 4(a ) ) . almost 62.5% of loading dose is cumulative in the faeces within 36  h after an oral dose with a dosage of 100  mg / kg ( figure 4(a ) ) , but the lower level of ergosterol was found in urine ( figure 4(b ) ) . the cloud - point extraction technique has been successfully applied for the first time as an effective method for the extraction and preconcentration of ergosterol from rat plasma samples . it was shown that this method is suitable for the analysis of ergosterol in rat plasma samples collected for pharmacokinetic study . the hplc - uv method is simple , specific , and reproducible for quantitative determination of ergosterol in rat plasma , urine , and faeces . the small amount of biological matrices required ( 0.3  ml per determination ) made this method suitable for routine analysis in preclinical pharmacokinetic studies , and the method was helpful in clinical pharmacokinetic studies .
ergosterol from many medicinal fungi has been demonstrated to possess a variety of pharmacological activities in vivo and in vitro . 
 a new method based on cloud - point extraction has been developed , optimized and validated for the determination of ergosterol in rat plasma , urine and faeces by liquid chromatography . 
 the non - ionic surfactant triton x-114 was chosen as the extract solvent . 
 the chromatographic separation was performed on an inertsil ods-3 analytical column with a mobile phase consisting of methanol and water ( 98  :  2 , v / v ) at a flow rate of 1  ml / min . 
 the methodology was validated completely . 
 the results indicated good performance in terms of specificity , linearity , detection and quantification limits , precision and accuracy . 
 the method was successfully applied to the pharmacokinetic studies of ergosterol in rats . 
 the results indicate that the ergosterol levels in feces are much higher than those in plasma and urine of the rat .