12 Jour.Chem-Soc.Pak. Vol. 24, No. 2, 2002 Assay of Urea with p-Dimethylaminobenzaldehyde L HUSSAIN®, Z. MAHMOOD, R. YASMEEN, M. JAHANGIR, R. HAMMED AND R. NASIR Institute of Chemistry, University of the Punjab, Lahore, Pakistan (ern 1% Aug, 210, reid 1" anomy, 20002) Summary: pDinelytminebealddde and ure ia cid wade ge yelow soloution ‘he cromegn mpl fr te yw claw m Sehlfbee). Tee wnat of cranes ‘sated wi chenicel wd qecrowopic went The yellow ccow abot ot i 29 mm. The fcastion of yo colour aberbuce dpedh upon Ge seagih of wid Sever ‘aston media bs, he CHLOHSO, matte appeal be te bot sed good rele ‘The opium snout of H3S0, GN) 02 ~ 03 wl CHLOH i fund wo beth bt ration Introduction. Urea is one of the end products of the reactions that are occurring in « human body. It is also a chief nitrogen containing end product of protein metabolism. For diegnoss of various diseases, connected with variation of urea level in blood and urine serum, exact determination of urea in biological fluids is required. Early method (1-5) for urea estimation rely on the specific hydrolysis of urea to ammonia and carbon dioxide by the enzyme urease, produced in moukls, bacteria and plans (.e Jack bean) followed by measurement of one of these end-products by nesslrisation or by the Berthelot reaction, The optimum parameters in the Berthelot feaction have been suggested by various workers [5- 10}, More recently it has been superceded by the shutamate dehydrogenase method, currently one of the most commonly used wrea method However, disadvantages in the urease method aise from the facts that ammonia from other sources. ‘may already be present in plasma, and that urease is subjected to inhibition or inactivation, The presence of fluoride ions in serum further aggravate analytical procedure, Direct chemical estimation is relatively new and is rather less specific than the enzymic one, Urea may be measured without conversion to ammonia with some a-diketones and other chemicals, They yield various colorations with urea. These colours hhave been used for the colorimetric determination of ‘urea concentration in biological fluids for the last many year [10-12]. Of three avdiketones, diacetyl (butane-2,3-dione) has been extensively used and is ‘oflen prefered tothe urease method. As stated above, the analytical procedure for the determination of urea in biological fuids depends ‘upon the colour that forms and the nature of the coloured products has remained » mystery for the last ‘many years. Recently, we have been involved in the investigation of the reactions of urea with a= diketones. Also the merits and demerits of the chemicals that are being used in hospital and private Tinical laboratories have been studies [13-16] Several workers (17-33] made use of p- dimethylaminobenzaldehyde as a diagnostic reagent for the estimation of urea and other compounds present in biological as well as non-biological fhuids and compared results with dietylmono-oxime and Berthelot methods. They could not generalise analytical procedure. Moreover, they were unable 10 determine the exact parameters and role of interfering substances involved inthis reaction. Recently [17], we have isolated all possible hromogenes resuling fiom the reactons of p- imethylaminobenzaldehyde with rea, thiourea ond their allyVeryl derivatives. This reaction appears to be acid sensitive and the yellow colour results fom the teacion of p-dimethylaminobenzaldehyde with ue absorbs at Pa 420 nm nd obeys Bea’ law at a definite pH. We ave used this yellow colour as an snaytical tool for the estimation of urea present in tne serum, The results seem to be very encourging “To whan all conespondce saul be addenLHUSSAIN eal, ‘when compared with diacetytmono-oxime and urease methods. Results and Discussion When equimolar quantities of ures and ‘aldehyde suspended in benzene containing TFA. were stimed at room temperature, a yellow solid was obiained. It showed molecular jon peak at 191 and elemental analysis corresponded to an emperical formula of CjgyNjO. In proton NMR specie it gave two doublets at § 6,83 and 7.73 ppm. They were assigned to protons Ha and Hb. The amide NH and CH = N- protons absorbed at 6 8.72 and 9.74 ppm respectively. The integration ratio of. absorption peaks well matched with the mumber of protons present in the compound (I). Hence, we suggested structure (1) to CisHyN3O. Is soluble in water and imparts yellow colour. In UV/Visible spectra it absorbed at mm 420 nm. The yeliow colour disappeared when treated with an acid or base It also ecolourized KMnO, and bromine water suggesting the presence of C = IN in the compound. Moreover, its infared spectra confirmed the presence of C=O and C=N groups in compound (1). After establishing the structure of chromogen responsible for yellow colour, we have diverted our attention to find the exact parameters of colour-development and intends to use the colour as an analystical tool for the estimation of urea. present in biological fhuids. Honsey and Finney (27] have used p-dimethylamino benzaldehdye for the determinsion of urea in acid media, but they could not reach at final conclusion We have tried our best to find out the optimum parameters of this reaction, where the yellow colour should remain stable, sensittve end specific. We hhave discovered that’ the yellow colour is highly sensitive and fluctuates with acid strength. It should bbe possible that degree of protonstion of the chromogene isthe eause of absorbance fluctuation. In ‘addition, we have also focused our attention to study the pH of the reaction media where the yellow colour remains stable and helps in shifting the equilibria from left to right, We believe that actual chromogen ‘which is responsible for the yellow colour is Schiff- ‘base (1). The formation of the Schif-base in acidic ‘media depends upon the acid strength. A reacion mechanism as shown in Scheme-1 is suggested Schiff-base with quinoid structure (1a) has light obsorbing property and it absorbs at dew 420 nm, The decrease end inerease in the sbsorption intensity depend upon the quantity of compound () tnd amount of the acid used. Greater the formation of ‘Jour. ChemSoc-Pak. Vol. 24,No.2,2002 123 compound (1), greater the absorption intensity. We have tied CH,OH - HCl, CH,OH - HyPO,, CH,OH - ~ FESO, HyPO, and CHOH N:SO, mixtures in the development of yellow colour. The CHsOH - HSO, mixture appears to be the best reaction modia which wwe report here. In the study reactants and products remain in solution and do not got precipitated at any stage, The absorbance of standard solutions of urea are recorded using reference solutions as given in Tables (HHI), In these experiments we have used CELOH ~ HS, and Blank solutions (without urea) as references solutions. In this way, the absorbance of yellow colour if a any stage, produced by p-dimethyl ‘minobenzaldehyde in the absence of urea is itself electronically subtracted, p-Dimethylamino-benzal- dehyde in methanol absorbs at Zee 320 num and in acid media its absorbance is further suppressed. Hence, the fear of its interference with yellow colour Gena 420 nm) solution resulting from urea snd p- DMAB is itself eliminated. We have focused our attention to find a ‘suitable amount of SO, which can be used in urea P-DMAB reaction. It is observed that asthe ‘concentration of sulphuric acid is increased, the abosrbances of the resulting chromogen are decreased. Two to ten ml H;SO, (36N) in 70 ml ‘methanol has been used. The pH of the reaction found to be below zero. In these cases, the absorbances range is found to be 0.519 to 0.909. It reflects that chromogen () which is responsible for the yellow colour is heavily protonated resulting in the depression of the absorbances in each case Contrarily, when H,SO, ranging from 1.0 ml to 0.25 ‘ml in 70 ml CHyOH is used, the absorbances of yellow solution are considerably increased, ie., from 1.725 to 2.832. Thereafter, when the amount of 1H,SOx ranging from 0.2 to 0.05 ml in 70 ml CHsOH is used, the absorbances of yellow colour stats decreasing, After thorough ‘studies of the reacion of p- imethylaminobervaldehyde and urea, it has seen that 0.2 to 0.3 ml HsSOx 36N) in 70 mi CHSOH is « suitable quantity where reliable results canbe achieved. Moreover, a brief look at Tables. (Ll) reflects that 0.25 mi HSO, in 70 ml HS0, is the most favourable amount for the reaction to proceed ‘where the protonation of the reactants and reacton products are productive, do not precipitate and exist in very congenial environment. All the absorbance points in Graphs (Ll) fall in a straight line but the Graph IL appears to be the best and reliable as compare to other graphs124 Jour.ChemSoe-Pak. Vol. 24,No. 2,2002 ASSAY OF UREA WITH P.DIMETHYLAMINOBENZALDEHYT ‘Table 1: Absorbance using CH,OH-H,SO (70:0.2ml v/v) solution at £20 nm. ‘Seuple Vol ofwen VoLof Vol of pDiautiy+ Volane of ‘Abeaenes or Reinga NSC Ne sain Lal vate) aminotennldse CHLOHSO, Referee Vel=Dagias) soe. (O02 W9(a) Ser Goo2v) solion T oa aE ‘062 Tae 2 om 40 56 095 os 3 02 36 Lat 110 ‘ 026 36 1386 130 5 020 36 163 15s ‘ os 4 56 1a 738 7 on 40 36 2s 200 ‘ om 40 36 an 20 ° om 40 36 2360 asia 10 000 40 56 2m. 270 t A oO. we 0 ono Le oe tot ae ee 40 (Urea come. lawl, —P Graph IA: Asborbances using CH,OH-H,S0,(70:0.2 Graph IB: Asborbanoes using CHsOH-H;S0, (70.0.2 IV) soln. References: CHsOH-H,S0, 'VIV) soln. References: Blank soln soln ‘Table It Absorbances using CH:OH-H,SO, (10: 0.25ml vA) solution st 420 nm Semple Vol ofwee Vol of Vol of Vola of nrc ter ig Fir DS mE Nar tla mie water (el) pDimtlylmine: HOHE SO vale Telos Teed Keecalsiyee COW Cal) CHROHEESO, ink soon Yei-20 m6 conv) eb 1 tor 33640 a ro Ts 2 om 32d as 935 5m 3 a corey ss ue tile 4 te om 4a 36 tha 1 5 ot Sa 1459 vane ‘ ois to Se 19s 1756 7 62 40 36 2981 ibis : cor 40 36 228s zai $ oo 40 56 2308 232 0 e040 56 2607 2550 1 te ios A aS ot ioaniaero os onbuouw mew GraphIIA: Asborbences ~ using CHSOH-H:SO. Heeaeepaeneloaee (70:0.25 V/V) soln, References: CH,OH- Graph IIB: Asborbances using CH,OH-H;S0, (70:02 HS. soln VIM) soln. References: Blank solaLLHUSSAIN a at, ‘Jour.Chem-Soe-Pak. Vol. 24,No. 2, 2002 125 ‘Table-IIl: Absorbances using CHjOH-H;S0, (70:0.3 mi viv) solution at 420 nm Vol ofrea Volof soln 10mg water (al) @ Sample Xe Vola of CHOHHSO, soln M02) ob ‘Aberbance After Heating or 20 Minn At 50°C "Refermoe GDS, —Biacsiaion — D0 008 on 016 02 02 02 032 036 a0 t= oun wm oe Urea cone. in i, —> Graph ILA: Asborbances using CH,OH-H,S0, (70:3 VAY) soln, References: CH,OH-H;S0. soln In addition, it was also found that time is needed forthe reacion to complete. The absorbances ‘of the yellow colour after immediate mixing of p- DMAB and urea do not yield a straight line. On the ‘other hand, when the yellow solution was heated for 20 minutes at 50°C, al the absorbances points come ‘up in one line. Hence, it was concluded that this reaction is not instantaneous but needs time for its ‘completion. In the present work we used our findings in the determinaton of urea present in bicloguical samples In this regard, patient samples sustaining various diseases were examined and compared with other existing methods, Diacetylmono-oxime (DAM) and urease (enzymatic) methods [1-4,10) are being widely used in clinical Ieboralories for the estimation of urea. The ‘urea concentrations oblained by p-dimethylamino- benzaldehyde are found to be high and in some cases Jow as compare to DAM and urease methods. But, in some cases they are very close to each other. (Table IV). The chemistry of DAM method has been co 56 56 56 56 36 s6 36 ss ry om 1.000 1301 15362 1903 1089 2409 230 2398 Urea come. ia i, > Graph IIB: Asborbances using _CH,OH-H,SO, 003 VN) woh, Refrences: Bia sol thoroughly discovered by Butler and Hussain [14,16]. Keeping in view the background of DAM end urease ‘methods one can say that p-dimethylamino- benzaldehyde method seems to be a beter method, It was also found that p-dimethylamino- benzaldehyde did not react with any of commonly biological substances present in urine except those ‘which contain free NH, group. It is reported [4, 27) that typtophane, indoles, Kynurenine, citrulline, indowyl sulfate, phorphobilinogen and urobilinogen ‘impart coloration with p-timethylaminobenzaldchyde and absorb at different wavelengths as compared to _p-dimethytaminobenzaldehyde-urea reaction. tis reported that urobilinogen does not have ‘roe amino (NH) group, but gives fed coloration with ‘P-dimethylaminobenzaldehyde [4]. The red color ab30rbS a Jou 530 and this technique is being used fot the determination of urobilinogen present in blood. The chemistry of this reaction anmears to be126 Jour.Chem.Soe.Pak. Vol 24, No. 2,2002 ASSAY OF UREA WITH P-DIMETHYLAMINOBENZALDEHYDE ‘Teble-IV: Comparison of results using patients urine _samples sustaining various diseases ‘Searle AMOUNT OF UREA (mg) Now PDMAB Dan Urewe Dine ‘Metod Mebod_Mattoé Tans Toso — Rigi Urea ca 2 28 200s BPE 3 280 300 220 Haein 2 256 228140 Chronos fhe $a 3a 2a) Relat & 260206280 Oharaive Uroptly 7 os 72 om BP 2 mie us 35a BP 3 250352 220 Bladder Out chron oss mL rr ee ed 12 tas a2 La Rel Soe 1132 12801000 Renae 2 2a aso BPA 1s 23020561360 LeU Oberon 16 1087 9201080 Renal Cab 17 30 SBP 1 St HS 500 Re Same 1 7H 1600 Vale Cae MOHD 1060 Rem Some sam tomd BAL 2250 W229 Lert Oeewticn 190013251980 Rental Sene 24 ‘3001S 300. Chrnievoa ire 2% sos et) BPE 2% Tena 7 1 10 1 Heenan 2256150300 Cab ee T9260 Ren Calo 3S isa 1000 BP 30 HS 0 Hema 32 6 G58 Right reer Oberason BB 200 Rem Cala dso 20 20 BP 3s aslo 27202505 Real Cle 3614001281580 Tramai Uren Oban 37300265210 Caen Urinry Blader Mt 80S 900 PHL Rel fe 39300 BPH % ms os sms BP 42S 250 300 Cio Real bre 238 300 Seiwe etre ambigous, It is feared that urobilinogen es well as orphobilinogen may not interfere inthe 2NH, + CO; Salicylate and hypochlorite in the reagent react with the ammonium ions to form a green ‘complex (22 dicarboxylindephene) Reagent ‘A. Urease 50,000 whit B Phosphstebufer 120 mmol, pH1=7.0 Sodium salicylate 625 mmoVfit Sodium nitroprusside 5.0m molt EDTA 148m molt CC. Sodium hypochlorite 18 mmollt Sodium hyuroxide 450 mmoViit Standard 50 mg/100 ‘Urine sample disiled water (1:100) Procedure First of all, the reagent "B* was dissolved in 100 ml water and reagent "A" was mixed in it It was called “working reagent” ‘Three test tubes were marked as T,, Ts and Ty In test tube T, urine solution (10 pL) and in test ‘tube T; standard solution (10 iL) were added, In ‘each test tube “working reagent” (1.0 ml) was added. Incubsted at 37°C for 3 minutes. 3 = Testsolution Ty = Staidard solution Ty = Blank solution In cach test tube reagent *C* (200 lL) was ‘added. Mixed thoroughly and incubated at 37°C for S ‘minutes, Bluish green colour developed in all cases. Absorbances were recorded at Je 600 mm, using ‘blank solution as reference. The amount of urea was caloulated as: Concentration of urea (mg/100m))= ‘Abeothance of standard soln, ASSAY OF UREA WITH P.DIMETHYLAMINOBENZALDEHTYDE ‘Note: All other urine samples were treated in the same manner and their results are recorded in Table- Vv. References 1. EDP. Wooton, ‘Microanalysis in Medical Biochemisty’, '5* edit, p74, Churchil Livingstone, Edinburgh(1979). 2 RJ. Henry, Cannon D.C. and Winkelman JW; Clin, Chemisty, "Principles and Technic! Harper and Row, New York, 2™ Bait, P51 974), 3. D. Jung, H. Biggs J, Erikson and PLU, Ledyard lin. Chem, 21,8 (1975), 4. AIL Gowenlock, JR. MeMuray and DM. Melmchlan "Varley Practical Clinical Biochemisty*, 6* edit, P362, Heinemann Professional publishing Lid; Halley Cour, Jordan Hil, Oxford OX2 8EJ Britain (1988) 5. AL Chaney and EP. Marbach, Clin Chem. 8, 130 (1962), RG. Martinek, JAmer. Technol 31,678 (1969). JAF Napier. and JG. Laines, Ann Clin Biochem, 6,59 (1969), 8 SA. Gordon, A. Fleck and J. Bel, Ann. 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