tuberculosis ( tb ) is still the infectious disease with the highest lethality , and china has the second highest tb burden globally . in 2013 , there were notifications of 136,000 people with multidrug - resistant ( mdr)-tb or rifampicin ( rfp)-resistant tb ( tb ) who were eligible for mdr - tb treatment worldwide . the treatment of tb requires more than two drugs to prevent the emergence of resistant strains . fixed - dose combinations ( fdcs ) are combinations of two or more drugs in a single pill . the first - line anti - tb drugs , including isoniazid ( inh ) , rfp , pyrazinamide ( pza ) , and ethambutol ( emb ) are often the components of fdcs . according to the published research studies have reported differences in rfp bioavailability between products containing rfp alone and fdc products . the use of qualified fdc formulations in the treatment of tb can increase patients compliance compared with free combinations of the same drugs , and reduce the risk of treatment failure and emergence of drug - resistant strains . the who has thus recommended the use of fdcs in the treatment of tb and has published a list of prequalified products . the aim of the present study was therefore to determine the bioequivalence of rfp in fdc formulations compared with rfp in free combinations in healthy chinese male volunteers . 1102041 ) were supplied by hongqi pharmaceutical company ( shenyang , china ) as reference drugs . the four - drug fdc formulation was a tablet containing 75 mg inh , 150 mg rfp , 275 mg emb , and 400 mg pza ( test formulation a , batch no . the two - drug fdc test formulations were tablet b ( 150 mg inh , 300 mg rfp , batch no . 110204 ) , capsule c ( 75 mg inh , 150 mg rfp , batch no . 110501 ) and capsule d ( 150 mg inh , 300 mg rfp , batch no . the fdc formulations were from different manufacturers except that formulation c and d were from the same manufacture . the drug content and dissolution profiles of all formulations were determined in accordance with chinese pharmacopeia protocols . the dissolution study was conducted at 75 r / min and 37c using 900 ml phosphate buffer saline . dissolved rfp was assayed at 254 nm using high - performance liquid chromatography ( hplc ; agilent 1200 , usa ) . three bioavailability studies were conducted as open , two- or three - period , two- or three - sequence crossover trials . two two - drug fdcs ( b , d ) containing inh and rfp were administered to a group of 18 volunteers with washout periods of 1 week between treatments . one two - drug fdc ( c ) containing inh and rfp was administered to a group of 20 volunteers with a washout period of 1 week . one four - drug fdc ( a ) containing inh , rfp , emb , and pza was administered to a group of 18 volunteers with a washout period of 1 week . all volunteers underwent medical history screening , physical examination , blood pressure measurement , hemogram test , liver function test , hepatitis b virus test , chest radiography , electrocardiography , and routine urine analysis . volunteers with a history of allergy to drugs related to the study medications or metabolic diseases , and those who had taken any drugs in the 2 weeks before the start of the study were excluded . the volunteers had no history of tobacco or alcohol use , or heart , liver , kidney or gastrointestinal disorders . the volunteers were aged between 20 and 40 years , and had a body mass index ranging from 19 to 24 kg / m . after overnight fasting , each volunteer received either a fdc formulation or the reference product ( containing a single dose of 600 mg rfp ) with 200 ml water [ table 1 ] . blood samples ( 3 ml each ) were collected in heparinized tubes at 0 , 10 , 20 , and 40 min and 1 , 1.5 , 2 , 3 , 4 , 6 , 9 , 12 , and 24 h postdose . details of the bioequivalence trials of fdc formulations versus free combinations of anti - tb drugs fdc : fixed - dose combination ; tb : tuberculosis ; rfp : rifampicin ; inh : isoniazid ; pza : pyrazinamide ; emb : ethambutol . high - performance liquid chromatography - tandem mass spectrometry apparatus and conditions an agilent hplc systems apparatus ( agilent technologies , usa ) equipped with a model g1329a als and a column heater ( g1316a ) was used . the mass spectrometer ( ms ) was a 6410b triple quadrupole ms ( agilent technologies , usa ) . the plasma concentration of rfp was measured using a hplc - ms / ms method developed in our laboratory . the mixture was vortexed for 10 s and centrifuged for 10 min at 12,000 g ; 2 l of the supernatant was injected into the chromatographic system . analysis was performed using a zorbax sb c-18 ( 2.1 mm  50 mm , 1.8 m ; agilent ) . the mobile phase was a mixture of water ( 0.1% formic acid ) : acetonitrile ( 40:60 v / v ) . the assay was linear over the range of 0.228 g / ml ( r = 0.999 ) . the limits of quantification and detection of rfp were 0.2 g / ml and 0.01 g / ml , respectively . pharmacokinetic ( pk ) parameters were analyzed by drug and statistics software ( das 3.1.5 , mathematical pharmacology professional committee of china , shanghai , china ) using noncompartmental analysis . peak plasma concentration ( cmax ) and time to reach cmax ( tmax ) were directly obtained from individual plasma concentration - time profiles . the area under the concentration - time curve to the last measurable concentration ( auc024 h ) was calculated by the linear trapezoidal method . bioavailability equivalence was accepted if the 90% confidence intervals ( 90% cis ) calculated for the test / reference formulation mean ratios were within the ranges of 80%125% for auc024 h and 70%143% for cmax . the tmax between two treatment arms was analyzed using the wilcoxon test , and differences were considered significant at p < 0.05 . 1102041 ) were supplied by hongqi pharmaceutical company ( shenyang , china ) as reference drugs . the four - drug fdc formulation was a tablet containing 75 mg inh , 150 mg rfp , 275 mg emb , and 400 mg pza ( test formulation a , batch no . the two - drug fdc test formulations were tablet b ( 150 mg inh , 300 mg rfp , batch no . 110204 ) , capsule c ( 75 mg inh , 150 mg rfp , batch no . 110501 ) and capsule d ( 150 mg inh , 300 mg rfp , batch no . the fdc formulations were from different manufacturers except that formulation c and d were from the same manufacture . the drug content and dissolution profiles of all formulations were determined in accordance with chinese pharmacopeia protocols . the dissolution study was conducted at 75 r / min and 37c using 900 ml phosphate buffer saline . dissolved rfp was assayed at 254 nm using high - performance liquid chromatography ( hplc ; agilent 1200 , usa ) . three bioavailability studies were conducted as open , two- or three - period , two- or three - sequence crossover trials . two two - drug fdcs ( b , d ) containing inh and rfp were administered to a group of 18 volunteers with washout periods of 1 week between treatments . one two - drug fdc ( c ) containing inh and rfp was administered to a group of 20 volunteers with a washout period of 1 week . one four - drug fdc ( a ) containing inh , rfp , emb , and pza was administered to a group of 18 volunteers with a washout period of 1 week . all volunteers underwent medical history screening , physical examination , blood pressure measurement , hemogram test , liver function test , hepatitis b virus test , chest radiography , electrocardiography , and routine urine analysis . volunteers with a history of allergy to drugs related to the study medications or metabolic diseases , and those who had taken any drugs in the 2 weeks before the start of the study were excluded . the volunteers had no history of tobacco or alcohol use , or heart , liver , kidney or gastrointestinal disorders . the volunteers were aged between 20 and 40 years , and had a body mass index ranging from 19 to 24 kg / m . after overnight fasting , each volunteer received either a fdc formulation or the reference product ( containing a single dose of 600 mg rfp ) with 200 ml water [ table 1 ] . blood samples ( 3 ml each ) were collected in heparinized tubes at 0 , 10 , 20 , and 40 min and 1 , 1.5 , 2 , 3 , 4 , 6 , 9 , 12 , and 24 h postdose . details of the bioequivalence trials of fdc formulations versus free combinations of anti - tb drugs fdc : fixed - dose combination ; tb : tuberculosis ; rfp : rifampicin ; inh : isoniazid ; pza : pyrazinamide ; emb : ethambutol . high - performance liquid chromatography - tandem mass spectrometry apparatus and conditions an agilent hplc systems apparatus ( agilent technologies , usa ) equipped with a model g1329a als and a column heater ( g1316a ) was used . the mass spectrometer ( ms ) was a 6410b triple quadrupole ms ( agilent technologies , usa ) . the plasma concentration of rfp was measured using a hplc - ms / ms method developed in our laboratory . the mixture was vortexed for 10 s and centrifuged for 10 min at 12,000 g ; 2 l of the supernatant was injected into the chromatographic system . analysis was performed using a zorbax sb c-18 ( 2.1 mm  50 mm , 1.8 m ; agilent ) . the mobile phase was a mixture of water ( 0.1% formic acid ) : acetonitrile ( 40:60 v / v ) . data were collected and processed using g3335aa - masshunter _ software-78 ( agilent technologies , usa ) . the assay was linear over the range of 0.228 g / ml ( r = 0.999 ) . the limits of quantification and detection of rfp were 0.2 g / ml and 0.01 g / ml , respectively . pharmacokinetic ( pk ) parameters were analyzed by drug and statistics software ( das 3.1.5 , mathematical pharmacology professional committee of china , shanghai , china ) using noncompartmental analysis . peak plasma concentration ( cmax ) and time to reach cmax ( tmax ) were directly obtained from individual plasma concentration - time profiles . the area under the concentration - time curve to the last measurable concentration ( auc024 h ) was calculated by the linear trapezoidal method . bioavailability equivalence was accepted if the 90% confidence intervals ( 90% cis ) calculated for the test / reference formulation mean ratios were within the ranges of 80%125% for auc024 h and 70%143% for cmax . the tmax between two treatment arms was analyzed using the wilcoxon test , and differences were considered significant at p < 0.05 . , the percentage of rfp dissolved after 30 min was 84.9%  1.32% , while for formulations a and c it was above 80% ( 96.2%  0.50% and 97.2% 1.64% , respectively ) , and for formulation b and d it was 33.7%  0.48% and 65.8%  1.05% . dissolution profiles of rfp from 4 fdcs ( a , b , c , d ) and free combination ( reference ) in pbs solution . fdc : fixed - dose combination ; rfp : rifampicin ; sd : standard deviation ; pbs : phosphate buffer saline . mean concentration - time profiles of rfp for the four fdc formulations are presented in figure 2 . the pk parameters ( auc024 h , cmax , tmax and t ) of rfp are given in table 2 . out of four fdc formulations tested , only one , fdc a , had comparable auc024 h and cmax values of rfp to those for the corresponding free combinations and hence passed the bioequivalence test . for both the fdc and free combination , a 600-mg dose of rfp produced auc024 h values of 55.5108.3 gh ml and cmax values of 7.916.2 g / ml . mean concentration - time profiles of rfp after administration of free combination and fdcs in three bioequivalence trials ( formulation a  d ) . y - error bars indicate sd ( for sample sizes refer to table 1 ) . fdc : fixed - dose combination ; rfp : rifampicin ; sd : standard deviation . pharmacokinetic parameters of fdc formulations a , b , c and d for rfp resulted from different bioequivalence trials all the values are given as mean  sd . fdc : fixed - dose combination ; auc : area under the curve ; sd : standard deviation ; rfp : rifampicin . test / reference ratios and 90% ci derived from pharmacokinetic parameters of rfp for the assessment of bioequivalence statistical evaluation was done by das 3.1.5 . fdc : fixed - dose combination ; ci : confidence interval ; auc : area under the curve ; rfp : rifampicin . the concentration of rfp in fdc formulation b was considerably lower compared with the free combination , at approximately 50% throughout the full sampling time interval of the reference . the 90% cis for the cmax and auc024 h ratios were clearly outside the 70%143% and 80%125% ranges for the free combination , indicating the bioinequivalence of formulation b compared with the reference product . the differences between the cmax of the fdc and free combination were not apparent for formulations c and d , shown in figure 2 . however , table 3 shows that formulations c and d were not equivalent to the reference formulations . about 90% ci of auc024 h ( 76.9%91.7% ) and cmax ( 67.5%87.6% ) in formulation c were both inequivalent compared with a reference . of the 20 volunteers , 10 had auc024 h values that were less than 80% of the reference and seven had cmax values that were < 70% of the reference . the auc024 h of formulation d was within the range of 79116% of the reference . seven volunteers had auc024 h values that were less than 80% of the reference in formulation d ( 40.85 , 58.49 , 63.00 , 76.94 , 81.18 , 81.63 , and 84.78 gh ml , respectively ) . the t of the reference product ( 3.384.46 h ) and test formulations ( 3.313.81 h ) were within the interval reported by other authors . the tmax for the test product was not significantly different from that of the reference product except for formulation b , for which the tmax was 2.78  1.53 h ( p < 0.05 ) while the reference was 1.54  0.59 h. , the percentage of rfp dissolved after 30 min was 84.9%  1.32% , while for formulations a and c it was above 80% ( 96.2%  0.50% and 97.2% 1.64% , respectively ) , and for formulation b and d it was 33.7%  0.48% and 65.8%  1.05% . dissolution profiles of rfp from 4 fdcs ( a , b , c , d ) and free combination ( reference ) in pbs solution . fdc : fixed - dose combination ; rfp : rifampicin ; sd : standard deviation ; pbs : phosphate buffer saline . mean concentration - time profiles of rfp for the four fdc formulations are presented in figure 2 . the pk parameters ( auc024 h , cmax , tmax and t ) of rfp are given in table 2 . out of four fdc formulations tested , only one , fdc a , had comparable auc024 h and cmax values of rfp to those for the corresponding free combinations and hence passed the bioequivalence test . for both the fdc and free combination , a 600-mg dose of rfp produced auc024 h values of 55.5108.3 gh ml and cmax values of 7.916.2 g / ml . mean concentration - time profiles of rfp after administration of free combination and fdcs in three bioequivalence trials ( formulation a  d ) . y - error bars indicate sd ( for sample sizes refer to table 1 ) . fdc : fixed - dose combination ; rfp : rifampicin ; sd : standard deviation . pharmacokinetic parameters of fdc formulations a , b , c and d for rfp resulted from different bioequivalence trials all the values are given as mean  sd . fdc : fixed - dose combination ; auc : area under the curve ; sd : standard deviation ; rfp : rifampicin . test / reference ratios and 90% ci derived from pharmacokinetic parameters of rfp for the assessment of bioequivalence statistical evaluation was done by das 3.1.5 . fdc : fixed - dose combination ; ci : confidence interval ; auc : area under the curve ; rfp : rifampicin . the concentration of rfp in fdc formulation b was considerably lower compared with the free combination , at approximately 50% throughout the full sampling time interval of the reference . the 90% cis for the cmax and auc024 h ratios were clearly outside the 70%143% and 80%125% ranges for the free combination , indicating the bioinequivalence of formulation b compared with the reference product . the differences between the cmax of the fdc and free combination were not apparent for formulations c and d , shown in figure 2 . however , table 3 shows that formulations c and d were not equivalent to the reference formulations . about 90% ci of auc024 h ( 76.9%91.7% ) and cmax ( 67.5%87.6% ) in formulation c were both inequivalent compared with a reference . of the 20 volunteers , 10 had auc024 h values that were less than 80% of the reference and seven had cmax values that were < 70% of the reference . the auc024 h of formulation d was within the range of 79116% of the reference . seven volunteers had auc024 h values that were less than 80% of the reference in formulation d ( 40.85 , 58.49 , 63.00 , 76.94 , 81.18 , 81.63 , and 84.78 gh ml , respectively ) . the t of the reference product ( 3.384.46 h ) and test formulations ( 3.313.81 h ) were within the interval reported by other authors . the tmax for the test product was not significantly different from that of the reference product except for formulation b , for which the tmax was 2.78  1.53 h ( p < 0.05 ) while the reference was 1.54  0.59 h. resistance to inh and rfp often results in incurable tb . the global spread of tb is further complicated by the ubiquitous appearance of mdr strains defined as those , which are resistant to at least inh and rfp . the who has suggested that qualified fdcs be used for the treatment of tb . a clinical trial conducted at11 sites in 9 countries supported the who recommendations for the use of fdcs because of the potential advantages compared with free combinations of the component drugs . fewer previous studies have been conducted in china comparing the bioequivalence of the anti - tbfdc products available there . because there was no certified reference fdc product available in china at the time of this study , we used separate drugs as the reference in this bioequivalence study . suggested that the rfp bioavailability problem is more attributable to extrinsic factors such as formulation or rfp bulk material rather than intrinsic variability of rfp absorption . our in vitro dissolution test showed that , although formulation c meets the dissolution requirement , it failed the bioequivalence study . the test showed that , even when rfp formulations meet dissolution requirements , the drug is not always well absorbed . other authors have also found that the relationship between rfp dissolution properties and bioavailability is poor . we chose sample sizes of 1820 volunteers and a sampling time of 24 h for our study because previous studies have shown these to be sufficient to demonstrate bioinequivalence . in the relative bioavailability study , the rfp concentration - time profile of formulation b clearly differed from the reference , while those of the other three fdcs were similar to the reference [ figure 2 ] . the cmax and auc024 h of formulations a , c , and d also had no obvious difference from the reference . the rfp cmax value of formulations a , c , and d were above the minimum therapeutic plasma concentration ( 10 g / ml ) , and this was comparable to the results from an indian study , which used a dose of 600 mg . we examined the data of each volunteer and found substantial inter - subject variability in the data . for formulation c , half of the volunteers auc024 h values were below the 80% mean value of the reference and one - third of the volunteers cmax values were below the 70% mean value of the reference . the other volunteers had higher values of the auc024 h and cmax , indicating that the data were polarized . the situation was similar for formulation d , but only the auc024 h was slightly lower than 80% value of the reference . based on this consideration , although sample sizes of 18 volunteers are typical for bioequivalence studies , a small sample from a single center was a limitation of this study . furthermore , healthy male volunteers are not representative of patient populations . out of four fdc formulations evaluated , only one formulation was bioequivalent for rfp to free combinations of the component drugs , whereas the other three formulations were below the limits of bioequivalence . our studies showed the importance of conducting bioequivalence studies before the distribution of fdc products within the chinese health system . any reduction in the bioavailability of anti - tb drugs such as rfp could have serious consequences , including potential treatment failure and selection of drug - resistant mutants . the poor bioavailability of rfp represents a serious problem for the success of tb treatment , which must be addressed by the chinese health authorities .
background : decreases in the bioavailability of rifampicin ( rfp ) can lead to the development of drug resistance and treatment failure . 
 therefore , we investigated the relative bioavailability of rfp from one four - drug fixed - dose combination ( fdc ; formulation a ) and three two - drug fdcs ( formulations b , c , and d ) used in china , compared with rfp in free combinations of these drugs ( reference ) , in healthy volunteers.methods:eighteen and twenty healthy chinese male volunteers participated in two open - label , randomized two - period crossover ( formulations a and c ) or one three - period crossover ( formulations b and d ) study , respectively . 
 the washout period between treatments was 7 days . 
 bioequivalence was assessed based on 90% confidence intervals , according to two one - sided t - tests . 
 all analyses were done with das 3.1.5 ( mathematical pharmacology professional committee of china , shanghai , china).results : mean pharmacokinetic parameter values of rfp obtained for formulations a , b , c , and d products were 11.42  3.41 g / ml , 7.86  5.78 g / ml , 13.05  6.80 g / ml , and 16.18  3.87 g / ml , respectively , for peak plasma concentration ( cmax ) , 91.43  30.82 gh1 ml1 , 55.49  37.58 gh1 ml1 , 96.50  47.24 gh1 ml1 , 101.47  33.07 gh1 ml1 , respectively , for area under the concentration - time curve ( auc024 h).conclusions : although the concentrations of rfp for formulations a , c , and d were within the reported acceptable therapeutic range , only formulation a was bioequivalent to the reference product . 
 the three two - drug fdcs ( formulations b , c and d ) displayed inferior rfp bioavailability compared with the reference ( chinese clinical trials registration number : chictr - ttrcc-12002451 ) .