Iraqi J Pharm Sci, Vol.19(2) 2010 MMooddiiffiiccaattiioonn ooff GGSSAAMM 31 VVaalliiddiittyy ooff GGeenneerraalliizzeedd SSttaannddaarrdd AAddddiittiioonn MMeetthhoodd ffoorr aa MMiixxttuurree ooff AAmmiinnoo AAcciidd AAnnaallyyssiiss AAzzhhaarr MM.. JJaassiimm** ,,11 **DDeeppaarrtteemmeenntt ooff PPhhaarrmmaacceeuuttiiccaall CChheemmiissttrryy,,College of Pharmacy,University of Baghdad,Baghdad, Iraq. AAbbssttrraacctt AA MMooddiiffiieedd vveerrssiioonn ooff tthhee GGeenneerrlliizzeedd ssttaannddaarrdd aaddddiittiioonn mmeetthhoodd (( GGSSAAMM)) wwaass ddeevveellooppeedd.. TThhiiss mmooddiiffiieedd vveerrssiioonn wwaass uusseedd ffoorr tthhee qquuaannttiittaattiivvee ddeetteerrmmiinnaattiioonn ooff aarrggiinniinnee ((AArrgg)) aanndd ggllyycciinnee (( GGllyy)) iinn aarrggiinniinnee aacceettyyll ssaalliiccyyllaattee –– ggllyycciinnee ccoommpplleexx .. AAccccoorrddiinngg ttoo tthhiiss mmeetthhoodd ttwwoo lliinneeaarr eeqquuaattiioonnss wweerree ssoollvveedd ttoo oobbttaaiinn tthhee aammoouunnttss ooff ((AArrgg)) aanndd ((GGllyy)).. TThhee ffiirrsstt eeqquuaattiioonn wwaass oobbttaaiinneedd bbyy ssppeeccttrroopphhoottoommeettiicc mmeeaassuurreemmeenntt ooff tthhee ttoottaall aabbssoorrbbaannccee ooff ((AArrgg)) aanndd ((GGllyy)) ccoolloorreedd ccoommpplleexx wwiitthh nniinnhhyyddrriinn .. TThhee sseeccoonndd eeqquuaattiioonn wwaass oobbttaaiinneedd bbyy mmeeaassuurriinngg tthhee ttoottaall aacciidd ccoonnssuummeedd bbyy ttoottaall aammiinnoo ggrroouuppss ooff ((AArrgg)) aanndd (( GGllyy)).. TThhee ttiittrraattiioonn wwaass ccaarrrriieedd oouutt iinn nnoonn-- aaqquueeoouuss mmeeddiiaa uussiinngg ppeerrcchhlloorriicc aacciidd iinn ggllaacciiaall aacceettiicc aacciidd aass aa ttiittrraanntt.. TThhee ddeevveellooppeedd mmeetthhoodd iiss aaccccuurraattee,, pprreecciissee,, aanndd ffrreeee ffrroomm iinntteerrffeerreenncceess aanndd mmaayy pprroovviiddee aa uusseeffuull aapppprrooaacchh ttoo ccaalliibbrraattee iinn tthhee ddiirreecctt aannaallyyssiiss ooff ssoolliidd ssaammppllee aanndd iitt iiss ssuuiittaabbllee mmeetthhoodd ttoo bbee uusseedd aass aa qquuaalliittyy ccoonnttrrooll pprroocceedduurree.. KKeeyy wwoorrddss:: GGSSAAMM ,, aammiinnoo aacciidd aannaallyyssiiss .. الخالصةالخالصة بشمممه هلممٕه دسممخ خٌٕط وتممطن خوخميمميُ خومم مٍ وى ممٗخٌ GSAMطفت خو يطسمميت خومطٌممت حممً خالممطن ح ممٗ ط يىمما ضط ممت خ مم لال لمميُ ٖوى لممٗن يىمما ٌمممطاوخيُ ل يخمميُ حممً اٌمم خو ط مم ت –خ ٌيّيمت خومم خلىت فمم حطليممـ خو ٍممم دسي ّيمم ُ دسيخ ط مم ه سطومم يليىيج ٖوممما ٖضط مممت خوخلممم ي فممم ٌ ممميد مٌمممط أ بخسمممخ خي مممطٌ خوىممم ِٗيت أ بخسمممخ خي ٌ ىمممٗن خوّّٕممم س ُ وى لمممٗن يىممما خوٍمطاومممت خ ٖذوممك بممم يممين خ سمميخط ه سطويلمميىك دسممي ٌممُ خوٍم مم بخ ممطفت ممطٌ ٬خوبيطلىٗس ممك لٍ ىممٗن ٌلمم وى لممٗن يىمما خوٍمطاوممت خو طِيممت ٖدسممخ الا خ سميخط ه سطويلميىك دسممي خوٍخ مطس بٗخسم ت خوٍمم ـ خومومٗ خ مط ٖح مم ط لٍيخمٓ ب ط ممت )ييمطس ٥٫٠خوٕيم سٖلىٗس كأ ُ خوخل ي أ خوخل ي خوٍبطشط دٖخ سيطي . خوّخط خوخ حً خوخٗصه خويٕط اوج يىا لَٗ ٔ ْ خو ط ت سط مت ٖخوّخط اقي ت ٖلطويمت ٌم ٌم ة . خوخ خلالث خوميٍيطٖ ت ٖم ح خطج يٕية IInnttrroodduuccttiioonn NNuummeerroouuss mmeetthhooddss ffoorr tthhee aannaallyyssiiss ooff aacceettyyllssaalliiccyylliicc aacciidd ccoouulldd bbee ffoouunndd iinn tthhee lliitteerraattuurree ((11--1100)) .. HHoowweevveerr,, tthheessee mmeetthhooddss aarree nnoott ssuuiittaabbllee ffoorr tthhee aannaallyyssiiss ooff aacceettyyllssaalliiccyylliicc aacciidd mmiixxttuurree wwiitthh aarrggiinniinnee aanndd ggllyycciinnee.. AArrggnniinnee aanndd ggllyycciinnee iinntteerrffeerree wwiitthh ddiirreecctt ttiittrriimmeettrriicc ddeetteerrmmiinnaattiioonn ooff aacceettyyllssaalliiccyylliicc aacciidd tthheerreeffoorree,, iitt wwaass iinntteennddeedd ttoo sseeppaarraattee aacceettyyllssaalliiccyylliicc aacciidd qquuaannttiittaattiivveellyy ffrroomm iittss ssaallttss bbeeffoorree iitt iiss ddeetteerrmmiinneedd bbyy ttiittrriimmeettrriicc mmeetthhoodd.. IItt wwaass iinntteennddeedd ttoo uussee ssppeeccttrroopphhoottoommeettrryy oorr ttiittrriimmeettrryy ffoorr ddeetteerrmmiinnaattiioonn ooff aarrggiinniinnee aanndd ggllyycciinnee .. TThheessee ttwwoo mmeetthhooddss aarree ssiimmppllee aanndd tthheerreeffoorree tthhee mmoosstt wwiiddeellyy uusseedd iinn rroouuttiinnee ddrruugg aanndd mmaannyy ccoommppoouunnddss aannaallyyssiiss aass ccoouulldd bbee sseeeenn ffrroomm tthhee llaatteesstt vveerrssiioonn ooff BBrriittiisshh ,, SSwwiissss aanndd UUnniitteedd SSttaattee PPhhaarrmmaaccooppeeiiaass.. HHoowweevveerr,, ddiirreecctt aapppplliiccaattiioonn ooff tthheessee mmeetthhooddss ffoorr ddeetteerrmmiinnaattiioonn ooff aarrggiinniinnee aanndd ggllyycciinnee iinn aa mmiixxttuurree ccoonnttaaiinniinngg bbootthh ooff tthheemm iiss nnoott ppoossssiibbllee bbeeccaauussee tthheeyy iinntteerrffeerree wwiitthh eeaacchh ootthheerr.. AAss aa rreemmeeddyy ffoorr tthhiiss ssiittuuaattiioonn iitt wwaass ddeecciiddeedd ttoo uussee tthhee GGSSAAMM wwhhiicchh hhaass wwiiddee aapppplliiccaattiioonnss ((1111--1177)) .. IItt iiss bbaasseedd oonn tthhee pprriinncciippllee ooff vvaarryyiinngg bbootthh ooff tthhee ssaammppllee mmoollaarr ccoonncceennttrraattiioonn aanndd ppHH ooff ssoolluuttiioonn.. AAiimmeedd aatt tthhee vvaalliiddaattiioonn aanndd ssttaannddaarrddiizzaattiioonn ooff aannaallyyttiiccaall pprroocceedduurreess wwiitthh ddiirreecctt ssoolliidd ssaammppllee ccoonnttaaiinn ttwwoo ttyyppeess ooff aammiinnoo aacciiddss wwiitthhoouutt pprreevviioouuss iissoollaattiioonn.. MMaannyy mmeetthhooddss wweerree uusseedd ffoorr tthhee ddeetteerrmmiinnaattiioonn ooff aarrggiinniinnee aanndd ootthheerr aammiinnoo aacciidd iinnvvoollvveedd pprreevviioouuss iissoollaattiioonn lliikkee tthhee ccllaassssiiccaall aannaallyyttiiccaall tteecchhnniiqquuee uusseess aauuttoommaatteedd aammiinnoo aacciidd aannaallyyzzeerrss,, HHoowweevveerr tthheessee mmeetthhooddss rreeqquuiirree eexxppeennssiivvee ddeeddiiccaatteedd eeqquuiippmmeenntt dduuoo ttoo tthhee ppoosstt-- ccoolluummnn ddeerriivvaattiizzaattiioonn ooff tthhee aammiinnoo aacciiddss ,, lloonngg aassssaayy ttiimmeess aanndd llaarrggee ssaammpplleess vvoolluummeess (( 1188 )) ..AAllssoo tthhee ssttuuddiieess ooff PPIITTCC aass tthhee ddeerriivvaattiizziinngg aaggeenntt,, ffoolllloowwiinngg tthhee PPiiccoo--TTaagg mmeetthhoodd uusseedd ffoorr tthhee ddeetteerrmmiinnaattiioonn ooff aarrggiinniinnee && ootthheerr aammiinnoo aacciidd ,, tthhoossee ssttuuddiieess rreeqquuiirree tthhee eessttaabblliisshhmmeenntt ooff ssttaannddaarrdd––aaddddeedd ccuurrvvee ((bbyy aapppplliiccaattiioonn ooff tthhee ssttaannddaarrdd aaddddiittiioonn mmeetthhoodd ))ttoo aavvooiidd pprrooppoorrttiioonnaall eerrrroorrss ((1199)) .. TThhee nnoonn--aaqquueeoouuss ttiittrraattiioonn wwaass uusseedd ffoorr ddeetteeccttiioonn ooff ffrreeee aammiinnoo ggrroouupp iinn aammiinnoo aacciidd ((2200)) ..TThhiiss mmeetthhoodd uusseedd ffoorr tthhee qquuaannttiittaattiivvee ddeetteerrmmiinnaattiioonn ooff mmaannyy cchheemmiiccaall ccoommppoouunnddss aanndd ddrruuggss iinn pphhaarrmmaacceeuuttiiccaall ffoorrmmss,, pprroovviiddiinngg pprreecciissee aanndd aaccccuurraattee rreessuullttss,, wwhhiicchh ccoouulldd bbee vveerriiffiieedd bbyy ssttaattiissttiiccaall mmeetthhooddss ((2211--2233)) .. AA ssppeeccttrroopphhoottoommeettrriicc mmeetthhoodd aarree wwiiddeellyy uusseedd ffoorr ddeetteerrmmiinnaattiioonn ooff aammiinnoo aacciidd bbaasseedd oonn tthhee rreeaaccttiioonn wwiitthh ccoolloorriinngg aaggeenntt aatt ddiiffffeerreenntt ppHH ffoorrmmiinngg ccoolloorreedd ccoommpplleexx tthhaatt mmeeaassuurreedd aatt ssppeecciiffiicc wwaavveelleennggtthh ((2244--2277)) .. 1Corresponding author E- mail : aazzhhaarrmmjjkk@@yyaahhoooo..ccoomm Received : 11/5/2010 Accepted : 27/6/2010 mailto:azharmjk@yahoo.com Iraqi J Pharm Sci, Vol.19(2) 2010 MMooddiiffiiccaattiioonn ooff GGSSAAMM 32 EExxppeerriimmeennttaall MMaatteerriiaall aanndd MMeetthhooddss Glacial acetic acid (99.8 %) was obtained from sigma. Standard perchloric acid (0.1 ml) in glacial acetic acid was prepared by diluting (4.25 ml) of perchloric acid (72%) to (500 ml) with glacial acetic acid containing (10 ml) acetic anhydride. The prepared solution was standardized by titration with standard anhydrous sodium carbonate (0.106 gm) in glacial acetic acid (50 ml) the titration end point was determined potentiometrically with potentiometric titration equipped with glass – calomel combined electrode. The prepared standard was used in the titrimetric procedure for the determination of arginine and glycine in arginine acetylsalicylate–glycine complex. Anhydrous sodium carbonate (99.5 %) , sodium hydroxide (0.5 M) and hydrochloric acid (0.5 M) were Aldrich standards . Arginine (98%, sigma) was standardized to determine the exact percentage of arginine by titration with standard hydrochloric acid (0.5 M). Glycine (99%, fluka) was used after drying in an oven for 1hr at (105ºC). Acetylsalicylic acid was purified by recrystilization from ethanol/ water mixture and standardized with standard (0.5 M) sodium hydroxide (10) . The determined percentage of acetylsalicylic acid was (99.4 %).The standards arginine , glycine and acetylsalicylic acid were used to prepare arginine acetylsalicylate – glycine complex . The later was used to test the validity of the proposed method. Sodium acetate buffer (4M, pH 5.5) was prepared by dissolving sodium acetate trihydrate NaOAc.3H2O (BDH “Analar “99.9) in 200 ml of deionized water. The solution was heated with stirring at (60ºC ) until a clear solution was obtained. Glacial acetic acid (50 ml) was added and the volume was completed to (500 ml) with distilled water. The pH was adjusted by a drop wise addition of sodium hydroxide (4M) to pH (5.5). Ninhydrin reagent was prepared as following : ninhydrine ( I gm) in 2 - methoxy ethanol (25 ml) and stannous chloride SnCl2.2H2O (0.07 g) with continuous stirring until completely dissolved (28) . Sodium acetate buffer (8.3 ml) was added and the resulting solution was immediately transferred to dark glass reservoir bottle and a steam of nitrogen gas was babbled into the reagent solution for approximately (20 min). Titration was made with potentiometric titrator (Metrohm , Switzerland ) titro process or equipped with(Metrohm).Dosimate and glass calomel combined electrodes. Spectrophotometric measurement was performed on carry (100 conc) uv- visible spectrophotometer using (1 cm) cells. Determination of acetyl salicylic acid in this complex (1gm) of arginine acetylsalicylate– glycine complex was weighed and dissolved in (25 ml) of distilled water. The salt was converted to free acetyl salicylic acid by acidification with hydrochloric acid (3 M) until pH (2.7) was reached. The acetyl Salicylic acid was extracted by ether (3 × 20 ml). The ether portions were pooled together and ether was evaporated by a rotary evaporator under vacuum. The acetyl salicylic acid was collected to be determined quantitatively by back titration method or by direct titration method (5, 10) . The result obtained for acetyl salicylic acid was corrected for the presence of salicylic acid as an impurity during liberating and separating acetyl salicylic acid form the complex. Salicylic acid was then determined in the liberated materials (29) . Determination of arginine and glycine in this complex This was achieved by constructing and solving equations (2) and (4) using ninhydrin spectrophotometric and titrimetric method in non – aqueous media respectively. Spectrophotometric Method (36 mg) of the complex was precisely weighed and dissolved in (50 ml) distilled water to prepare a stock solution. An aliquot (1 ml) of this stock solution was diluted to (25 ml) with distilled water. An aliquot ( 1 ml ) of this solution was mixed with ( 1ml ) of ninhydrin reagent in a stoppered test tube , shacked and placed in a boiling water bath for ( 15 min ). Ethanol (2.5 ml) and sodium acetate buffer (2.5 ml) were added to the mixture which was then cooled below (30 ºC ). The sample was shacked thoroughly for (30 second) and absorbance was measured at (570 nm) against a reagent blank using (1 cm).Standard curves for arginine and glycine were constracted using (0.05 = 0.2 mM) standard solution for each. The values of the molar absorpitivity coefficient aA and aG in equation (2) were determined from slops of these calibration curves. Titrimetric Method in non – aqueous media (0.3 gm) of the complex was dissolved in glacial acetic acid (50 ml) in a dry flask. The glass electrode of the titroprocessor was immersed in the solution and the mixture was titrated against standard perchloric acid (reagent 1). A plot of pH against the titrant volume was constructed to obtain the titration end point. Stability study Identical aqueous solution (0.1 g/ 100 ml) of the complex was made. The stability was determined at different temperatures (25, Iraqi J Pharm Sci, Vol.19(2) 2010 MMooddiiffiiccaattiioonn ooff GGSSAAMM 33 40, 50, 60 & 70 ºC ) incubated in ovens for certain intervals. The decomposition of arginine acetylsalicylate – glycine complex was indicated by the release of acetylsalicylic acid , arginine and glycine as appears on TLC using a solvent system of ( n-propanol : 34% ammonia 7 :3 ) . The appearance of three spots on TLC plates for acetyl salicylic acid, arginine and glycine when the samples were incubated at (50, 60 & 70 ) ºC . While incubation at (25 and 40) ºC showed only one spot for arginine acetylsalicylate – glycine complex. Effect of Buffer Glycine was used as a buffer to stabilize arginine acetylsalicylat complex. The concentration of glycine is (0.5 M) in respect to arginine (1M) to maintain the pH of the preparation at (4.7). RReessuulltt aanndd DDiissccuussssiioonn Method of calculation The GSAM is a traditional vector – matrix notation to construct and to solve a system of (n) linear equation in order to determine the concentration of a mixture of (n) substances. The mixture of compounds interferes with each other when measured by (n) different sensors that belong to any suitable analytical technique. According to GSAM, the following system of two linear equations is required to determine arginine and glycine: AA11 == aa1111%%AA++ aa1122 %% GG AA22 == aa2211%%AA++ aa2222 %% GG Where ; A1 and A2 are the total responses of two analytical sensors ( 1 and 2 ) to the percentage of arginine ( % A ) and glycine ( % G ) in the sample the factors a11 – a22 are absorptivity constant multiplied by the optical path length ( i.e of the sample ) .Theory of GSAM equations (1) gives precise result for %A and %G when the following two conditions are fulfilled: Firstly there should be a large difference in magnitude between the ratio a11/a12 and the ratio a21/a22. Secondly the precision is measuring A1 and A2 should be high. These requirements arise from the fact that the mathematical manipulation magnify the random error in measurement , so that large random error is produced in the calculated concentration of the measured substances ( i.e %A and %G ) (11,12) .According to the requirements stated in these two conditions, it was decided to construct one of the equations of the system by using spechtrophotometric technique, while the second equation is to be constructed using titrimetic method. Ninhydrin was chosen as a color developing reagent in the spechtrophtometric procedure, as it is the most selective among other coloring agent for spechtrophotometric ddeetteerrmmiinnaattiioonn for amino acids (30-31) .The titrimetric method was used to obtain the second equation which could not be performed in aqueous medium because of the neutral behavior of salts in aqueous medium that prevent the occurrence of a clear titration end point . Accordingly, a titrimetric method in non- aqueous media was adopted using glacial acetic acid as a solvent and a solution of perchloric acid in glacial acetic acid as a titrant. This method was used for quantitative determination of salt of amines with carboxylic acids (32-33) .The presence of an acidic salts in non-aqueous solvent behave as bases and therefore produce a clear titration end point when titrated with strong acid (34) .This experiment of procedure was intended to produce two linear equations in which the ratio a11/a12 and a21/a22 are very different in magnitudes. This was expected from the fact that arginine and glycine have nearly the same efficiency to produce colored complexes with ninhydrin reagent in spectrophotometric procedure (31) .While in the titrimetric procedure arginine molecules have two titratable amino groups in contrast to the glycine molecules which have one titratable group. The first equation which is obtained from the spectrophotometric procedure was derived starting from Lambert – beer law as follows : … (2) Equation (2) can be abbreviated as follows: A = a11 %A + a12 %G ……… (3) In equation (2) Wasg gm of arginine acetyl salicylate – glycine complex sample was dissolved to prepare (vo ml) of stock solution. This stock solution was then subjected to serial dilutions (n) with f1,f2…fn dilution factors to reach the required final concentration. Mw A and Mw G are the molecular weight of arginine and glycine respectively. aA and aG are the molar absorpitivity constants of arginine and glycine respectively.The use of a ……… (1) زز.…… Iraqi J Pharm Sci, Vol.19(2) 2010 MMooddiiffiiccaattiioonn ooff GGSSAAMM 34 combination of titrimetry and spechtrophotometry satisfies the requirements stated in (2). The precision of the spectrophotometric method is moderate in general.While; the precision of the titrometric method is very high. Therefore, the use of non- aqueous titration method improves the overall precision of the results obtained from such combination of methods. The second equation, was obtained from titrimetric procedure as follows: …(4) Equation (4) can be abbreviated as below: MpVp = a21%A + a22%G …….. (5) Equation (4) V ml of glacial acetic acid containing V asg of the analyzed arginine acetyl Salicylic-glycine complex was titrated with Mp molar standard of perchloric acid, so that Vp ml of the standard was required to reach the end point . Equations (3) and (5) are mathematically compatible and can be solved linearly. The ratio a11/a12 and a21/a22 have large differences in their magnitudes since a11/a12 ≈ 2 while a 21/a 22 ≈ 1. Development of Titrimetric Procedure for Acetyl salicylic acid determination The complex sample solution must be acidified to liberate acetyl salicylic acid form its salt with arginine before extraction by ether. The optimum pH of the acidified aqueous solution was determined experimentally by applying the sample complex at different pH values of the extracted sample solution. The result of this study indicates that pH (2.7) is the optimum pH for acetyl salicylic acid extraction. At pH higher than (2.7) low result for acetyl salicylic acid were obtained due to incomplete liberation acetyl salicylic acid from its salt with arginine.The extraction step is vital in the development procedure because an attempt to perform direct titration with hydrochloric acid without performing apparent titration end point as could be seen from (Figure 1). Figure 1: Titration curve of arginine acetylsalicylate -glycine solution with standard hydrochloric acid (0.5). Analysis of Arginine Acetyl salicylate – Glycine complex Quantitative Determination of Acetyl salicylic acid. Acetyl salicylic acid was determined quantitatively by hydrolysis and back titration method (29) . The result of the two methods was summarized in table (1). Table 1: result of acetylsalicylic acid, arginine and glycine in arginine acetyl salicylate – glycine complex * Hydrolysis and back titration method. * *Direct titration method. Quantitative determination of arginine and glycine in the complex The quantitative determination of arginine and glycine in the complex using a modified version of the GSAM was achieved by a combination of colorimetric method ( to obtain the first equation ) and non-aqueous titration method ( to obtain the second equation).The colorimetric method using the colored complex of arginine and glycine with ninhydrin was applied once at different pH media and once by using different wavelengths; the results of these experiments were summarized in table (2) and (3) and in figure (2) and (3) respectively. Table 2: Molar absorptivity constant a* of arginine and glycine colored complex with ninhydrin at different pH values measured at 570nm *Represent the Molar absorptivity multiplied by the cell width C.V Calculated Percentage ( w/w ) ± SD Expected Percentage W/W Item 0.4 % 0.6 % 49.6 ± 0.2 * 49.6 ± 0.3 * 50.0 Acetylsalicylic Acid 1.5 % 40.3 ± 0.6 40.0 Arginin 2.0 % 9.7 ± 0.2 10.0 Glycine pH of ninhydrin solution aA aG aA / aG 5.5 0.83 0.62 1.34 7.0 1.07 0.78 1.37 9.0 1.73 0.97 1.41 Iraqi J Pharm Sci, Vol.19(2) 2010 MMooddiiffiiccaattiioonn ooff GGSSAAMM 35 Table 3: Molar absorptivity constant a* of arginine and glycine colored co mp lex with ninhydrin at different wavelengths. pH of ninhydrin solution Wavelength hs nm aA aG aA/aG 5.5 244 1.19 0.92 1.29 409 0.74 0.54 1.37 570 0.83 0.63 1.32 7.0 244 1.28 0.97 1.32 409 0.87 0.62 1.40 570 1.02 0.75 1.36 9.0 244 1.40 1.08 1.30 409 0.92 0.67 1.37 570 1.32 1.00 1.32 *Represent the Molar absorptivity multiplied by the cell width Figure 2: standard curve of arginine and glycine colored complexes with ninhydrin at different pH values measured at 570 nm Figure 3: uv-visible spectra of arginine and glycine colored complexes with ninhydrin at different pH values According to the non-aqueous titration method; the titration end point of the titration curve between standard perchloric acid with the complex was determined potentiometrically. A typical plot for the titration curve was exhibited in figure ( 4 ) Iraqi J Pharm Sci, Vol.19(2) 2010 MMooddiiffiiccaattiioonn ooff GGSSAAMM 36 Figure 4: titration curve between standard perchloric acid (0.1N) and arginine acetylsalicylate –glycine complex. The result of stability study indicates the appearance of three spots on TLC at (50, 60, 79)ºC and only one spot at (25, 40) ºC as summarized in table (4). Table 4: *Rf values of the arginine acetylsalicylate – glycine complex at different temperatures for certain time intervals C Intial 1 st 2 nd 4 th 25 0.42 0.42 0.42 0.41 40 0.42 0.42 0.42 0.41 50 0.42 0.43 0.46 0.55 60 0.42 0.44 0.45 0.57 70 0.42 0.45 0.55 0.56 *The solvent system is (n-propanol : 34% ammonia 7 :3 ) IInntteerrffeerreennccee As mentioned earlier the degradation product of arginine acetylsalicylate – glycine complex might interfere with the determination of arginine acetyl Salicylic acid and glycine by the proposed method. The evacuation step in the acetylsalicylic acid analysis procedure ensures complete removal of acetic acid thus prevents its influence on the titration of acetyl salicylic acid with sodium hydroxide. Beside, the determination of the amount of salicylic acid (20) and the subsequent correction eliminates its interference on the acetyl Salicylic acid determination. The non – aqueous titration procedure is not affected by all the degradation procedure as this procedure is selective for basic groups such as the amino group. CCoonncclluussiioonn The proposed procedure is suitable to be used as a quality control procedure for the determination of arginine acetylsalicylate – glycine complex, as well as many formulas that contain essential and non essential amino acids (free or in combination)in different preparation like food supplement or other pharmaceutical preparation.This proposed procedure is simple, fast and accurate in compares with other procedures. RReeffrreenncceess 1. 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