over the last two decades , immunosuppression associated with human immunodeficiency virus , organ transplantation , and anticancer chemotherapy has resulted in a dramatic increase in the incidence of serious mucosal and systemic fungal infections . posaconazole ( psz ) ( figure 1 ) , chemically known as 4-{p-[4-(p-{[(3r,5r)-5-(2,4-difluorophenyl)tetrahydro-5-(1h-1,2,4-triazol-1-ylmethyl)-3-furyl]methoxy}phenyl)-1-piperazinyl]phenyl}-1-[(1s,2s)-1-ethyl-2-hydroxypropyl]--1,2,4-triazolin-5-one , is a structural analogue of itraconazole . it is commercially available in the form of oral suspension ( 40  mg / ml ) and delayed release ( 100  mg ) tablets . both dosage forms can be used in the prophylactic treatment of invasive aspergillus and candida infections in severely immunocompromised patients thirteen years old and older . those immunocompromised patients include those with hematopoietic stem cell transplantation with graft - versus - host disease or hematologic malignancies with prolonged neutropenia from chemotherapy . however , only the oral suspension has been recently indicated for the treatment of oropharyngeal candidiasis refractory to itraconazole and/or fluconazole . despite that psz oral suspension is commercially available since 2005 and delayed release 100  mg tablets since november 2013 , it is not yet an official drug in any of the famous pharmacopoeias , including the british pharmacopoeia , european pharmacopoeia , and united states pharmacopoeia . nevertheless , several methods can be found in the scientific literature for its analysis in biological fluids . most of these methods are based on liquid chromatography using different types and detection modes such as hplc with uv detection [ 46 ] , hplc with fluorescence detection [ 7 , 8 ] , hplc - tandem mass spectrometry ( lc - ms / ms ) [ 912 ] , uplc - uv detection , and uplc - ms / ms . recently , hplc with dad detection was applied for the stability indicating determination of psz in bulk form . moreover , other separation techniques such as capillary electrophoresis   and micellar electrokinetic chromatography ( mekc )   were utilized for the quantification of psz in human plasma samples . ultra high performance liquid chromatography ( uhplc ) is a relatively new technique which has been introduced in 2004 . uhplc is a derivative of hplc showing a dramatic enhancement in speed , resolution , and the sensitivity of analysis due to the use of column particle size less than 2  m . this advantageous system can operate at higher pressure with the mobile phase running at greater linear velocities as compared to hplc . it is considered a new focal point in field of liquid chromatographic studies with significant reduction in analytical time , sample volume , and solvent consumption along with superior chromatographic separation [ 18 , 19 ] . thus it has been adopted by pharmaceutical industry for routine quality control purposes . the fact that up till now the analysis of this drug formulation has not been tackled yet encouraged us to develop and optimize two simple , advanced , and reliable hplc - dad and uhplc - uv procedures for quality control purposes . the structurally related itraconazole ( figure 1 ) was used as an internal standard ( is ) in both methods . , loughborough , leicestershire , uk ) , analytical grade potassium dihydrogen orthophosphate ( riedel - de - han , germany ) , and high purity distilled water were used . noxafil 40  mg / ml oral suspension ( patheon inc . , ontario , canada , bn 3005a ) was purchased from schering - plough s.a . for the hplc - dad assay , the chromatography system consisted of agilent 1200 series ( quaternary pump , vacuum degasser , diode array , and multiple wavelength detector g1315 c / d and g1365 c / d ) connected to a computer loaded with agilent chemstation software ( agilent technologies , santa clara , ca , usa ) . the chromatographic separation of psz and itraconazole ( is ) was accomplished using a zorbax sb - c18 ( 4.6  250  mm , 5  m ) column ( agilent technologies , santa clara , ca , usa ) . the gradient elution composed of the mobile phase acetonitrile : 15  mm potassium dihydrogen orthophosphate ( 30  :  70 to 80  :  20 , linear over 7 minutes ) pumped at 1.5  ml / min . injection volume was between 20 and 50  l . all determinations were performed at 25c . for the uhplc assay , the chromatography system consisted of agilent 1290 infinity binary pump lc combined with a uv detector ( agilent technologies , santa clara , ca , usa ) . the chromatographic separation of psz and itraconazole ( is ) was accomplished using a kinetex - c18 ( 2.1  50  mm , 1.3  m ) column ( phenomenex , torrance , ca , usa ) . chromatographic data were collected and compiled by use of chemstation software ( agilent technologies , santa clara , ca , usa ) . the mobile phase composed of acetonitrile : 15  mm potassium dihydrogen orthophosphate ( 45  :  55 ) pumped isocratically at 0.4  ml / min . injection volume was 5  l . a 100  g / ml stock solution of psz was prepared by dissolving 10  mg of psz in 100  ml methanol . a 100  g / ml stock solution of is was prepared by dissolving 10  mg of itraconazole in 100  ml methanol . to prepare samples for the calibration curve and validation assessment , three working solutions of 0.1 , 1 , and 10  g / ml of psz were prepared by successive 1/10 dilutions of the stock solution with methanol . is ( 10  g / ml ) was added to psz sample in a 2.5  ml polypropylene microcentrifuge tube . a volume of 0.1  ml of the oral suspension ( 40  mg / ml ) was diluted to 10  ml methanol ( s1 ) . 10  g / ml is was added to 0.1  ml of s1 supernatant and diluted with methanol to a final volume of 1  ml ( s2 ) in a 2.5  ml polypropylene microcentrifuge tube . four replicates of s2 were freshly prepared on the day of the experiment of which 20 and 5  l were injected and chromatographed according to the previously described hplc and uhplc methods , respectively . intraday accuracy and precision of the assay were determined using three sample replicates of 5 , 20 , and 50  g / ml of psz . to permit the assessment of interday accuracy and precision concentrations were determined by comparison with a calibration curve prepared on the day of the analysis . precision was determined using percentage coefficient of variation ( cv% ) and bias was assessed using mean intra- or interday percentage error of the mean . limits of detection and quantitation were determined , at the signal to noise ratio of 3  :  1 and 10  :  1 , respectively . during the development of the gradient liquid chromatographic method coupled with diode array detection for the routine quality control analysis of psz in its suspension dosage form , several experiments were carried out in order to optimize the choice of both the stationary and mobile phases . for optimization of the stationary phase , several reversed phase columns zorbax sb - c8 ( 4.6  250  mm ) , zorbax sb - c18 ( 4.6  250  mm ) , zorbax eclipse xdb - c18 ( 4.6  150  mm ) , and brownlee spheri-5 c18 ( 4.6  220  mm , 5  m ) were tested . the best clear separation between the eluting peaks , sharper symmetric psz peak , and relatively shorter retention times was attained by using the zorbax sb - c18 column ; hence , it became the column of choice for this study . several mobile phases were also evaluated using various proportions of different aqueous phases and organic modifiers . the best mobile phase combination was acetonitrile and 15  mm potassium dihydrogen phosphate solution . methanol was tried as an organic modifier and phosphate solution was substituted by other aqueous phases such as phosphoric acid solution . in these trials , chromatograms showed poor peak shape and/or inadequate separation between the eluting peaks . isocratic elution of different proportions of acetonitrile and 15  mm potassium dihydrogen phosphate did not provide satisfactory separation between psz and is peaks ; in addition broad peaks were frequently observed . to overcome these complications , several gradient programs were tried and the best compromise between adequate resolution and reasonable retention times was achieved using a gradient system starting with 30% ( by volume ) acetonitrile ramped up linearly to 80% in 7  min and then kept at this percentage till the end of the run . flow rate was kept constant at 1.5  ml / min all over the run , and temperature was adjusted at 25c . it is noteworthy to mention that the applied gradient program produced stable baseline without any drift or deformation . both psz and is ( itraconazole ) show considerable uv absorption with prominent maximum at 262  nm which was found suitable to record all chromatograms in this study . during the optimization of the uhplc method , efficient separation with nondrifting baseline was achieved using a kinetex - c18 ( 2.1  50  mm , 1.3  m ) column . again , the uv detector was set at 262  nm and a combination of acetonitrile and 15  mm potassium dihydrogen orthophosphate ( 45  :  55 , by volume ) was used as a mobile phase ; however an isocratic elution was found optimum . flow rate was adjusted at 0.4  ml / min , and temperature of the column was kept at 40c . in the hplc - dad assay , psz and the is ( itraconazole ) eluted at approximately 7.7 and 9.6  min , respectively with a total analytical run time of 11 minutes ( figure 2 ) , while in the uhplc - uv assay , psz and the is eluted at about 1.0 and 2.4  min , respectively with a total analytical run time of 3 minutes ( figure 3 ) . the column capacity factors ( k ) for psz and is were calculated to be 3.88 and 5.13 in hplc - dad and 4.19 and 11.4 in uhplc - uv , respectively . the column separation factor (  ) was 1.25 and 2.66 for the hplc - dad and uhplc - uv assays , respectively . the proposed hplc and uhplc methods were validated according to the international conference on harmonization ( ich ) guidelines on validation of analytical procedures . both assays showed highly linear relationships between the analyte / is peak height or area ratios and concentrations ranging from 5 to 50  g / ml . the mean r for the standard curves were 0.9997 and 0.9995 for the hplc - dad and uhplc - uv assays , respectively . a representative standard curve using peak area ratios yielded slopes of 0.0797 and 0.0874 for hplc - dad and uhplc - uv assays , respectively . the validation data showed the assay to be sensitive , accurate , and precise , with the intraday and interday assessment cv% less than 2% and 3% for hplc - dad and uhplc - uv assays , respectively ( table 1 ) . the mean interday error was less than 2% and 3% for hplc - dad and uhplc - uv assays , respectively . the calculated limit of detection and limit of quantitation were 0.82 and 2.73  g / ml for hplc - dad and 1.04 and 3.16  g / ml for uhplc - uv , respectively . the optimized hplc - dad procedure was applied for the assay of psz in its bulk powder and its pharmaceutical formulation ( noxafil suspension ) . the active ingredient was extracted with the same solvent used for the preparation of the standard stock solutions ( hplc grade methanol ) and then dilution was made to reach concentration levels within the specified calibration curve range . the active ingredient eluted at its specific retention time and no interfering peaks were observed from any of the inactive ingredients , as polysorbate 80 , sodium benzoate , artificial cherry flavor containing benzyl alcohol , xanthan gum , and so forth ( figures 2(b ) and 3(b ) ) . the diode array detection enabled peak purity verification where no signs of coelution from any of the above excipients were detected . the assay results revealed satisfactory accuracy and precision as indicated from % recovery , sd , and rsd% values ( table 2 ) . recovery data obtained from both methods were statistically compared together using the student 's t- and the variance ratio f - tests . in both tests , the calculated values did not exceed the theoretical ones at the 95% confidence level ( table 2 ) . two novel , simple , and selective methods have been developed for the determination of psz in its bulk powder and suspension dosage form . the adequate recovered concentrations in addition to the low values of coefficient of variation ( cv% ) and percentage relative error ( er% ) gathered in tables 1 and 2 confirm the precision and accuracy of the developed methods for the assay of psz in its pure and suspension forms . in addition , the obtained t and f values indicate that there are no significant differences between the recoveries obtained from both methods . to the best of our knowledge these are the first two methods described for the determination of the drug in its dosage form . the patency and the expensive price of the drug powder and dosage form might be a reason behind that . our uhplc - uv method showed similar precision and accuracy compared to the hplc - dad method ( tables 1 and 2 ) ; however , as previously discussed , it showed > 3 fold reductions in analytical time , turnaround time , and 4 fold reduction in sample volume and solvents consumption with superior chromatographic separation as shown in the chromatographic parameters above . despite such great advantages , the capital investment in such new apparatuses and the availability of the hplc in a lot of the research labs make our simple relatively quick hplc assay an acceptable alternative as well . previous uhplc methods for determination of psz in biological samples showed elution times ranging from 1.09 to 4.04 minutes [ 13 , 14 ] , whereas previous hplc methods reported elution times ranging from 9 to 19 minutes [ 19 , 2124 ] . the only hplc - dad method for the determination of psz in bulk powder reported elution time of 8.36 minutes . as such our two methods show comparable retention / run times that fall on the lower range border . the only reported method for the determination of psz in bulk powder was an hplc - dad method using a c8 column and isocratically pumped mobile phase made of methanol  :  water ( 75  :  25 ) , used only external standard quantitation method , had longer psz elution time and showed equivalent accuracy and precision . unlike fluconazole and voriconazole , the extraction of the drug occurred with no interferences from the inactive ingredients by simple dilution . among the inactive ingredients that have uv absorption properties however , they were tested and proven undetected at such concentrations using our current assays . this was further confirmed using the diode - array detector whose peak purity verification of the analyte showed no signs of coelution from any of the inactive components . the paper presents the first two analytical assays to determine posaconazole in its pharmaceutical dosage form . both assays were found to be accurate , reproducible , selective , and easily applied ; therefore they can be applied for the routine analysis of posaconazole in its suspension dosage form .
objective . to develop and compare hplc - dad and uhplc - uv assays for the quantitation of posaconazole in bulk powder and suspension dosage form 
 . methods . 
 posaconazole linearity range was 550  g / ml for both assays . 
 for hplc - dad assay , samples were injected through zorbax sb - c18 ( 4.6  250  mm , 5  m ) column . 
 the gradient elution composed of the mobile phase acetonitrile : 15  mm potassium dihydrogen orthophosphate ( 30  :  70 to 80  :  20 , linear over 7 minutes ) pumped at 1.5  ml / min . 
 for uhplc - uv assay , samples were injected through kinetex - c18 ( 2.1  50  mm , 1.3  m ) column . the mobile phase composed of acetonitrile : 15  mm potassium dihydrogen orthophosphate ( 45  :  55 ) pumped isocratically at 0.4  ml / min . 
 detection wavelength was 262  nm in both methods . 
 results . 
 the run time was 11 and 3 minutes for hplc - dad and uhplc - uv assays , respectively . both assays were linear ( r2 > 0.999 ) with cv% and % error of the mean < 3% 
 . limits of detection and quantitation were 0.82 and 2.73  g / ml for hplc - dad and 1.04 and 3.16  g / ml for uhplc - uv , respectively . the methods quantitated psz in suspension dosage form with no observable interferences . conclusions . 
 both assays were proven sensitive and selective according to ich guidelines . 
 uhplc - uv assay exhibited some economic and chromatographic separation superiority .