Europa,schesP_ MM II II II MM I MM II I Ml II I II J European Patent Office i-o >i © Publication number: 0 396 584 B1 Office_„. europeen des brevets © EUROPEAN PATENT SPECIFICATION © Date of publication of patent specification: 25.01.95 © Int. CI.6: C12Q 1/00, C12Q 1/26 © Application number: 89900286.9 @ Date of filing: 02.12.88 © International application number: PCT/GB88/01063 © International publication number: WO 89/05356 (15.06.89 89/13) C54) ASSAY OF SALICYLATES OR REDUCED PYRIDINE NUCLEOTIDES. ® Priority: 03.12.87 GB 8728296 © Inventor: ATKINSON, Anthony "Twlngley", Mill Corner, Wlnterbourne Gun- @ Date of publication of application: ner 14.11.90 Bulletin 90/46 Salisbury, Wiltshire SP4 6JJ (GB) Inventor: CAMPBELL, Robert Stewart © Publication of the grant of the patent: 26a Millington Road 25.01.95 Bulletin 95/04 Cambridge CB3 9HB (GB) Inventor: HAMMOND, Peter Michael © Designated Contracting States: 46 Oakwood Grove AT BE CH DE FR GB IT LI LU NL SE Alderbury SP5 3BN (GB) Inventor: MORRIS, Helen Christine © References cited: 165 Hills Road EP-A- 124 909 Cambridge CB2 2BJ (GB) EP-A- 138 530 Inventor: RAMSAY, John Richard 3 South Front, Dews Road CLINICAL CHEMISTRY, vol. 30, no. 9, Septem- Salisbury SP2 7SL (GB) ber 1984, Winston-Salem, NC (US); KWAN-SA Inventor: PRICE, Christopher Philip YOU et al., pp. 1549-1 551 # Church Eng, Mingle Lane Stapleford © Proprietor: The Microbiological Research Au- Cambridge CB2 5BG (GB) 00 thorlty CAMR, Porton Down 00 Salisbury, m Wiltshire SP4 0JG (GB) CO Oi Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid (Art. 99(1) European patent convention). Rank Xerox (UK) Business Services (3. 10/3.09/3.3.3) EP 0 396 584 B1 0 Representative: Lockwood, Peter Brian et al Ministry of Defence (PE), Directorate of Intellectual Property Rights, Room 2012, Empress State Building, Lillie Road London SW6 1TR (GB) 2 EP 0 396 584 B1 Description This invention relates to a method for the estimation of salicylates or reduced pyridine nucleotides, and to a diagnostic kit adapted to facilitate the routine performance of said method. 5 There is a requirement to estimate the level of salicylate compounds, particularly the drugs ortho-acetyl salicylic acid (aspirin) and salicylic acid (which is commonly used as its sodium salt) in a biological fluid. In such cases, and especially where a patient is suffering from a drug overdose, it is important that the drug level in the body may be assessed rapidly so that an antidote or other treatment may, if necessary, be administered. io A number of methods for estimating salicylate in a sample or in a solution are known. For example, the salicylate ion can be reacted with the Folin-Ciocalteau reagent in a strongly alkaline solution to produce a blue colour which is then analysed colorimetrically (Smith & Talbot 1950, Brit J Exp Path. 3j[ 65). This method, however, gives high and variable blank values in samples where salicylates are absent. Alternatively, the salicylate ion can be reacted with ferric salts in acidic solution and the purple colour 75 produced analysed by a colorimetric means. (Trinder 1954, Biochem J. 57, 301). This method too has disadvantages, and is subject to non-specific interference. In another method of salicylate estimation, the salicylate is first converted enzymically to form pyrocatechol. The latter compound is then reacted with hydrazone and molecular oxygen in the presence of a second enzyme, tyrosinase, to form an azine dye. (Roder et al 1980, EP 0 054 146). The enzyme 20 tyrosinase is known to oxidise monophenols other than pyrocatechol, and hydrazone may couple oxidatively with any resulting quinones to form a coloured product. Furthermore, the presence of other monophenols and particularly of tyrosine in unknown quantities may result in competition for binding sites on the tyrosinase enzyme, reducing the efficiency of the system. In yet another method of salicylate estimation as disclosed in European patent application number 25 84306810.7, published as EP 0138530A, the salicylate is first converted enzymically to catechol. The catechol is then reacted with aminophenol under alkaline conditions to form an indophenol which is capable of quantitative estimation by colorimetric techniques. The aminophenol is however, unstable in alkaline conditions and it is therefore necessary to store it before reaction with catechol under acidic conditions. Furthermore unless the reaction of aminophenol with catechol is rapid, the alkaline conditions under which 30 the reaction of aminophenol with catechol takes place can result in degradation of the aminphenol or side reactions of the aminophenol, which can give rise to inaccurate results. A further problem with this technique is that it is not practical to provide it in the form of an efficient all in one reagent kit as the aminophenol would need to be kept under acidic conditions prior to assay and the indophenol product requires neutral and preferably alkaline conditions for its formation. 35 Ideally the components of a salicylate estimation method will be soluble in water, have a high speed of colour development when reacted and give good colour yield and high stability of the final colour. It is one object of the present invention to provide a method for the estimation of salicylate by which the above disadvantages are overcome or at least mitigated, but which still produces an estimation of salicylate level sufficiently quickly to allow the method to be used in the determination of drug levels in the biological 40 fluids of suspected overdose patients. According to the first aspect of the present invention, therefore, there is provided a method for the estimation of salicylate in a sample characterised by enzymically converting the salicylate to a catechol by the action of a salicylate mono-oxygenase enzyme on the salicylate in the presence of a reduced pyridine nucleotide (especially NADH or NADPH) and reacting the catechol with a compound selected from those of 45 formula I or amine or phenolic compounds of formulae II, III and IV below: 50 55 3 EP 0 396 584 B1 Formula I Formula II Formula III Formula IV wherein Ri , R2 and R3 are independently H, -NH2 or Ci-Cg alkyl, R+ to R10 are independently selected from H, alkyl, -NH2, -NRn Ri2,-OH, -CH2OH, -CHO, -COOH, -CO(CH2)nH, -CONH(CH2)nCOOH or -NH2.HOOCCOOH.H2NCgH5 where n is an integer from 1 to 5 inclusive and Rn, Ri2 are independently selected from C1-C5 alkyl provided that where in formula II one of the groups R+ to Rg is -NH2 or NRnRi2 20 then at least one of the other groups R+ to Rg is selected from Ci-Cg alkyl, -NH2, -NRnRi2, -CHO, -COOH, -CO(CH2)nH, -CONH(CH2)nCOOH or -NH2.HOOCCOOH.H2NCGH5, wherein formula II does not include compounds falling into the scope of the term 'aminophenols', to form a dye the quantity of which can be estimated colorimetrically. The quantity of the dye is then related to the quantity of the salicylate in the sample. Preferably the dye 25 is estimated spectrophotometrically. In formula I preferably R1 is methyl, R2 is H or methyl and R3 is -NH2. In formula II, preferably when R+ is -COOH, R5 is -NH2 and RG to Rg are H; preferably when R+ is -CONH(CH2)nCOOH then n is 1, R7 is NH2 and R5, Rg, Rs and Rg are H; preferably when R+ is -CO(CH2)nH then n is 1, R7 is NH2 and R5, Rg, Rs and Rg are H. In formula III R7 is preferably NH2 and R+ to Rg and Rs to R10 are preferably H. In formula IV 30 preferably R1 and R2 are methyl and R3 is -NH2. The word dye as used herein refers to a compound which absorbs electro-magnetic radiation over one or more of the ultra-violet (UV) visible or infra-red (IR) spectral regions. The colour reaction will occur when using a range of compounds selected from formula I to IV. Preferably concentrations of 0.5 -50 mmol/L but more preferably concentrations of 1.3-2.9 mmol/L are used. 35 The invention is based on the following reactions (using (NADPH) or NADH to exemplify the reduced pyridine nucleotide). Salicylate Mono - oxygen as e 40 (i) Salicylate + (NADP H) + 02 , (or NADH) EC: l.lU.13.1 Catechol + C02 + H2= + (NADP) ... (A) (or NAD) 45 (ii) Catechol + compound ^ (I), (II), (III) or (IV) dye ...(B) 50 In reaction A above, the reduced pyridine nucleotide comprising either reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADP H) is converted to its respective oxidised form nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NAD P). 55 This method of salicylate detection preferably uses an NADH or NADPH concentration in excess of the concentration of the highest salicylate standard for which the assay is linear. This excess means that this method can also be used for the estimation of NADH or NADPH by including salicylate, in excess, in the enzyme reaction. This method can therefore be used to detect the NADH or NADPH produced by the 4 EP 0 396 584 B1 action of dehydrogenase enzymes on substrates such as glucose or lactates. Conveniently the two reactions A and B, of the salicylate assay method can either be performed simulataneously as a one step reaction where all the reagents are present together.