appropriate management of moderate postoperative pain is well achieved associating several analgesic drugs with adjuvant agents through synergistic interaction .
the potential advantage of this combined therapy is related to the minimization of the incidences of adverse effects of each drug and to the improvement of the outcome .
postoperative clinical situations , such as difficult patient extubation , respiratory depression due to opioids , and cardiovascular side effects , require a combined therapy to improve analgesia quality and decrease adverse effects of each drug when used alone .
this has led to administering pain drugs in continuous intravenous ( iv ) infusion with different mechanisms and sites of action , together with adjuvant agents .
tramadol hydrochloride ( hcl ) and ketorolac tromethamine are analgesic drugs commonly used in combination in postoperative pain management .
tramadol hcl is a synthetic , centrally acting analgesic with no anti - inflammatory activity and one of the most interesting and useful weak opioids for treatment of moderate to moderately severe pain with weak -receptor agonist properties and noradrenergic and serotonergic neurotransmission effects.[39 ] ketorolac tromethamine is a potent nonsteroidal anti - inflammatory drug ( nsaid ) with analgesic efficacy similar to opioids.[289 ] this drug is administered to treat moderate pain or , combined with reduced opioid doses , for severe pain . according to some studies , metoclopramide hcl as an adjuvant agent can improve analgesia and decrease the need for other pain drugs ; it is a dopamine and 5-ht receptor antagonist , commonly used as a prokinetic and antiemetic .
also , this has been recently investigated as an agent that can enhance the efficacy of analgesic drugs.[91113 ] some studies have also suggested a role of magnesium sulfate ( mgso4 ) as an adjuvant agent , an n - methyl - d - aspartate receptor antagonist , in the management of postoperative pain . the perioperative administration of iv mgso 4 is associated with smaller analgesic requirements , and magnesium sulfate could be of interest as an adjuvant to postoperative analgesia.[1416 ] the aim of this study was to investigate the chemical stability of tramadol hcl combined with ketorolac tromethamine and metoclopramide hcl in solution for 48 h ( 25c ) and 5 days ( 5c ) , with the presence of mgso4 .
milan , italy ) 2 mg / ml , tramadol hcl ( contramal ; grnenthal , gmbh , stolberg , germany ) 20 mg / ml , and ketorolac tromethamine ( lixidol ; roche s.p.a . , milan , italy ) 6 mg / ml stock solutions were prepared in high - performance liquid chromatographic ( hplc)-grade deionized water and stored at 4c during experimental assay .
well - defined volume samples were drawn from each stock solution , joined , and diluted in deionized water to produce 5 working standard solutions , with concentrations in the range of 0.51.5 g / ml for metoclopramide hcl , 2060 g / ml for tramadol hcl , and 39 g / ml for ketorolac tromethamine .
every 48 h the working standard solutions were prepared from stock solutions and stored at 4c .
limit of quantitation ( loq ) values were found to be 1 , 0.5 , and 0.1 g / ml for tramadol , ketorolac , and metoclopramide , respectively .
limit of detection ( lod ) values were 0.05 , 0.2 , and 0.1 g / ml for tramadol , ketorolac , and metoclopramide .
precision and accuracy were determined on spiked samples at 4 concentrations with respect to a calibration graph prepared every day ( n = 3 ) .
the precision of the method was evaluated as the intra- and interday relative standard deviation ( rsd ) of the measured peak areas by assaying spiked samples at 4 different concentrations .
all samples for these purposes were freshly prepared , including preparing the standard solution from the same stock solution .
six drug admixtures were prepared by transferring the contents of one ampule of metoclopramide hcl , 4 ampules of tramadol hcl , 2 ampules of ketorolac tromethamine , and 4 ampules of mgso4 ( 1 g/10 ml ampule , magnesio solfato monico ; monico s.p.a . , venezia mestre , italy ) to give each a final volume of 52 ml .
all the solutions were prepared on different days , stored at 25c and assessed over a period of 48 h. solutions were stored in amber colored glass bottles with air - tight caps to protect them from direct light exposure .
the nominal concentration of each drug in all prepared solutions was 0.19 mg / ml for metoclopramide hcl , 7.69 mg / ml for tramadol hcl , and 1.15 mg / ml for ketorolac tromethamine .
all the drug admixtures were visually inspected immediately after preparation ( 0 h ) and at 24 and 48 h against a black and white background to ensure any physical changes ( eg , color , phase separation , precipitation ) .
a 100 l sample was drawn from each solution at intervals of 0 ( initial ) , 24 , and 48 h and diluted to 20 ml with deionized water ; we prepared solutions in triplicate at each time interval to have 54 samples .
twenty microliters of each sample was injected into the hplc system to determine concentration of each drug .
as previously described , 6 drug admixtures were prepared on different days , stored at 5c ( refrigerated ) , assessed , and visually inspected at intervals of 0 ( initial ) , 1 , 3 , and 5 days .
a 100 l of sample was drawn from each solution and diluted to 20 ml with deionized water ; we prepared solutions in triplicate at each time interval to have 72 samples .
twenty microliters of each sample was injected into the hplc system to determine concentration of each drug . the hplc method developed by kk et al
was modified for use in this study.[1718 ] the instrumentation included a binary pump ( model g1312a ; agilent technologies ; hewlett - packard , waldbronn , germany ) and an ultraviolet ( uv)variable wavelength detector ( model g1314a ; agilent ) . at the beginning of our study
, we looked for the maximum uv absorption wavelength for each drug solution on the basis of uv spectra for each solution . as we used uv 
variable wavelength detector , and metoclopramide and tramadol retention times were very close reciprocally , we found that 271 nm was the best wavelength for both drugs to have good sensitivity and maximum signal / noise ratio . for ketorolac drug solutions
separation was achieved using a reversed - phase c18 5 m particle size column ( luna c18 ( 2 ) 100a , 150  4.6 mm ; chemtek analytica srl , anzola emilia ( bo ) , italy ) , equipped with a 2-cm precolumn , which was maintained at 25c with a column temperature controller ( thermosphere ts-130 ; phenomenex , torrance , california ) .
hplc - gradient analysis were performed using the following mobile phase : 0.01 m phosphate buffer , potassium dihydrogen phosphate [ kh2po4 ] ultra for molecular biology ( fluka and riedel - de han , buchs sg , switzerland ) and acetonitrile ( sigma - aldrich supelco , bellefonte , pennsylvania ) ( 75:25 , v / v ) with the addition of 0.1% triethylamine , adjusted to ph 3 with phosphoric acid ( solvent a ) and h2o  acetonitrile ( 50:50 , v / v , solvent b ) .
0% b , 610 min 80% b , 1015 min 0% b [ table 1 ] .
the elution - gradient program each mobile phase was prepared fresh daily , filtered through a 0.45 m  47 mm nylon membrane filter ( supelco , bellefonte , pennsylvania nylon 66 filter membranes pore size 0.20 m , diameter 47 mm ) , and degassed ultrasonically for 20 min before use .
the flow rate was 1 ml / min , and the injected volume was 20 l .
the run time was 15 min and the approximate retention times for metoclopramide , tramadol , and ketorolac were 2.8 , 3.4 , and 13.4 min , respectively [ figure 1 ] .
uv chromatogram of metoclopramide hydrochloride ( a ) , tramadol hydrochloride ( b ) , and ketorolac tromethamine ( c ) , eluted at 2.8 , 3.4 , and 13.4 min , respectively .
calibration curves were produced using linear regression of the peak area against concentration of each drug [ table 2 ] .
we obtained 5 calibration curves for each drug and all the curves were linear over the concentration ranges considered for each drug : metoclopramide hcl r = 0.991 ; tramadol hcl r = 0.995 ; ketorolac tromethamine r = 0.961 .
the results were averaged and analyzed by linear simple regression model of y = mx + q by the least - squares method .
the t test was used to examine the concentration difference at each time , and the significance level of  error was less than 0.001 .
linear simple regression model ( y = mx + q ) by the least - squares method all the data reported in tables 3 and 4 were submitted to anova and differences analyzed by tukey 's honestly significant differences test .
no significant admixture and time - dependent effect interactions were observed ( p  0.05 ) .
percentage  % sd of metoclopramide hydrochloride , tramadol hydrochloride , and ketorolac tromethamine remaining in the admixtures with magnesium sulfate after storage at 25c for 48 h percentage  % sd of metoclopramide hydrochloride , tramadol hydrochloride , and ketorolac tromethamine remaining in the admixtures with magnesium sulfate stored at 5c for 5 days
milan , italy ) 2 mg / ml , tramadol hcl ( contramal ; grnenthal , gmbh , stolberg , germany ) 20 mg / ml , and ketorolac tromethamine ( lixidol ; roche s.p.a . , milan , italy ) 6 mg / ml stock solutions were prepared in high - performance liquid chromatographic ( hplc)-grade deionized water and stored at 4c during experimental assay .
well - defined volume samples were drawn from each stock solution , joined , and diluted in deionized water to produce 5 working standard solutions , with concentrations in the range of 0.51.5 g / ml for metoclopramide hcl , 2060 g / ml for tramadol hcl , and 39 g / ml for ketorolac tromethamine .
every 48 h the working standard solutions were prepared from stock solutions and stored at 4c .
limit of quantitation ( loq ) values were found to be 1 , 0.5 , and 0.1 g / ml for tramadol , ketorolac , and metoclopramide , respectively .
limit of detection ( lod ) values were 0.05 , 0.2 , and 0.1 g / ml for tramadol , ketorolac , and metoclopramide .
precision and accuracy were determined on spiked samples at 4 concentrations with respect to a calibration graph prepared every day ( n = 3 ) .
the precision of the method was evaluated as the intra- and interday relative standard deviation ( rsd ) of the measured peak areas by assaying spiked samples at 4 different concentrations .
all samples for these purposes were freshly prepared , including preparing the standard solution from the same stock solution .
six drug admixtures were prepared by transferring the contents of one ampule of metoclopramide hcl , 4 ampules of tramadol hcl , 2 ampules of ketorolac tromethamine , and 4 ampules of mgso4 ( 1 g/10 ml ampule , magnesio solfato monico ; monico s.p.a . , venezia mestre , italy ) to give each a final volume of 52 ml .
all the solutions were prepared on different days , stored at 25c and assessed over a period of 48 h. solutions were stored in amber colored glass bottles with air - tight caps to protect them from direct light exposure .
the nominal concentration of each drug in all prepared solutions was 0.19 mg / ml for metoclopramide hcl , 7.69 mg / ml for tramadol hcl , and 1.15 mg / ml for ketorolac tromethamine .
all the drug admixtures were visually inspected immediately after preparation ( 0 h ) and at 24 and 48 h against a black and white background to ensure any physical changes ( eg , color , phase separation , precipitation ) .
a 100 l sample was drawn from each solution at intervals of 0 ( initial ) , 24 , and 48 h and diluted to 20 ml with deionized water ; we prepared solutions in triplicate at each time interval to have 54 samples .
twenty microliters of each sample was injected into the hplc system to determine concentration of each drug .
as previously described , 6 drug admixtures were prepared on different days , stored at 5c ( refrigerated ) , assessed , and visually inspected at intervals of 0 ( initial ) , 1 , 3 , and 5 days .
a 100 l of sample was drawn from each solution and diluted to 20 ml with deionized water ; we prepared solutions in triplicate at each time interval to have 72 samples .
twenty microliters of each sample was injected into the hplc system to determine concentration of each drug .
the hplc method developed by kk et al was modified for use in this study.[1718 ] the instrumentation included a binary pump ( model g1312a ; agilent technologies ; hewlett - packard , waldbronn , germany ) and an ultraviolet ( uv)variable wavelength detector ( model g1314a ; agilent ) . at the beginning of our study
, we looked for the maximum uv absorption wavelength for each drug solution on the basis of uv spectra for each solution . as we used uv 
variable wavelength detector , and metoclopramide and tramadol retention times were very close reciprocally , we found that 271 nm was the best wavelength for both drugs to have good sensitivity and maximum signal / noise ratio . for ketorolac drug solutions , we found that 323 nm was the best absorption wavelength .
separation was achieved using a reversed - phase c18 5 m particle size column ( luna c18 ( 2 ) 100a , 150  4.6 mm ; chemtek analytica srl , anzola emilia ( bo ) , italy ) , equipped with a 2-cm precolumn , which was maintained at 25c with a column temperature controller ( thermosphere ts-130 ; phenomenex , torrance , california ) .
hplc - gradient analysis were performed using the following mobile phase : 0.01 m phosphate buffer , potassium dihydrogen phosphate [ kh2po4 ] ultra for molecular biology ( fluka and riedel - de han , buchs sg , switzerland ) and acetonitrile ( sigma - aldrich supelco , bellefonte , pennsylvania ) ( 75:25 , v / v ) with the addition of 0.1% triethylamine , adjusted to ph 3 with phosphoric acid ( solvent a ) and h2o  acetonitrile ( 50:50 , v / v , solvent b ) .
the elution - gradient program was 06 min 0% b , 610 min 80% b , 1015 min 0% b [ table 1 ] .
the elution - gradient program each mobile phase was prepared fresh daily , filtered through a 0.45 m  47 mm nylon membrane filter ( supelco , bellefonte , pennsylvania nylon 66 filter membranes pore size 0.20 m , diameter 47 mm ) , and degassed ultrasonically for 20 min before use .
the flow rate was 1 ml / min , and the injected volume was 20 l .
the run time was 15 min and the approximate retention times for metoclopramide , tramadol , and ketorolac were 2.8 , 3.4 , and 13.4 min , respectively [ figure 1 ] .
uv chromatogram of metoclopramide hydrochloride ( a ) , tramadol hydrochloride ( b ) , and ketorolac tromethamine ( c ) , eluted at 2.8 , 3.4 , and 13.4 min , respectively .
calibration curves were produced using linear regression of the peak area against concentration of each drug [ table 2 ] .
we obtained 5 calibration curves for each drug and all the curves were linear over the concentration ranges considered for each drug : metoclopramide hcl r = 0.991 ; tramadol hcl r = 0.995 ; ketorolac tromethamine r = 0.961 .
the results were averaged and analyzed by linear simple regression model of y = mx + q by the least - squares method .
the t test was used to examine the concentration difference at each time , and the significance level of  error was less than 0.001 . linear simple regression model ( y = mx + q ) by the least - squares method all the data reported in tables 3 and 4
no significant admixture and time - dependent effect interactions were observed ( p  0.05 ) .
percentage  % sd of metoclopramide hydrochloride , tramadol hydrochloride , and ketorolac tromethamine remaining in the admixtures with magnesium sulfate after storage at 25c for 48 h percentage  % sd of metoclopramide hydrochloride , tramadol hydrochloride , and ketorolac tromethamine remaining in the admixtures with magnesium sulfate stored at 5c for 5 days
at the mediterranean institute for transplantation and highly specialized therapies ( ismett , palermo , italy ) is already using an experimental protocol for the treatment of moderate to severe pain , which involves the administration of intravenous mixture of tramadol ( 7.69 mg / ml ) , metoclopramide ( 0.19 mg / ml ) , ketorolac ( 1.15 mg / ml ) , and magnesium sulfate ( 77 mg / ml ) . to use this drug combination
is necessary that the drugs are compatible with each other and the mixture is stable over time .
table 3 shows drugs solution concentration after storage period up to 48 h. the data obtained are in agreement with data reported in the literature regarding a mixture of tramadol , ketorolac , metoclopramide , and ranitidine in a solution for intravenous perfusion .
data reported in table 4 show the remaining percentage of drug concentrations in all admixtures prepared and assessed over a period of 5 days .
it is possible to observe constant percentage concentrations remaining for all the 3 drugs at 1 , 3 , and 5 days when stored at 5c .
we believe that the information has clinical utility in facilitating the preparation and dispensing of the tramadol , metoclopramide , ketorolac , and magnesium sulfate mixture in postoperative pain management . on the basis of our results ,
the mixture may be considered for a possible commercial formulation , at the concentrations listed .
in this study , precise and accurate hplc method suitable for stability evaluation of tramadol hcl combined with ketorolac tromethamine and metoclopramide hcl is described .
the physical appearance of the solutions remained constant during the study period , without the formation of any visible discoloration , cloudiness , or precipitation . in admixtures containing mgso4 and stored at 25c , each drug , at the same time , reached at least 98% of the initial concentration during 48 h. solutions prepared adding mgso4 assessed over a period of 5 days at 5c show the lower confidence limit of the estimated regression line of the concentration at 98% of the initial concentration . within these limits , for the purpose of pre - preparing drug admixtures to use with confidence , tramadol hcl infusions may be prepared in advance and then thawed before use in clinical units .
moreover , information about the chemical stability of the drugs could be utilized in further investigations focused on iv infusion solutions .
 background : tramadol hydrochloride ( hcl ) and ketorolac tromethamine are analgesic drugs , which are commonly used in combination in postoperative pain management . according to some studies , metoclopramide and magnesium sulfate ( mgso4 ) as adjuvant agents can improve analgesia and decrease the need for other pain drugs.materials and methods : the chemical stability of tramadol hcl combined with ketorolac tromethamine and metoclopramide hcl has been studied using a stability - indicating high - performance liquid chromatographic assay method . 
 calibration curves were produced using linear regression of the peak area against concentration of each drug , with an r2 value  0.96 . 
 our aim was to investigate the stability of admixture solution of tramadol hcl combined with ketorolac tromethamine and metoclopramide hcl for 48 h ( 25c ) and 5 days ( 5c ) , with mgso4 , which has never been assessed.results:data obtained for admixtures prepared and stored at temperatures of 25c and 5c , show that all drugs have reached at least 98% of the initial concentration.conclusions:for the purpose of pre - preparing drug admixtures to use with confidence , tramadol hcl infusions may be prepared in advance and then thawed before use in clinical units . on the basis of our results , the intravenous mixture of tramadol ( 7.69 mg / ml ) , metoclopramide ( 
 0.19 mg / ml ) , ketorolac ( 1.15 mg / ml ) , and magnesium sulfate ( 77 mg / ml ) may be considered for a possible commercial formulation . 