This invention relates to a method of measuring nicotine metabolites in urine as a way of assessing smoking habit.

The degree of dilution of a urine sample is important when measuring an analyte, particularly drugs of abuse, eg nicotine or a metabolite thereof such as cotinine. Variations in the fluid intake will modify the concentration of urine produced, and this can alter validity of the result. This can lead to misclassification as false positives or false negatives. To overcome this problem, various correction factors can be employed. These usually involve the measurement of specific analytes in the urine sample. But all these procedures are laboratory based tests, requiring reagents and equipment, and specially trained staff.

There is a growing need to perform simple, rapid biochemical tests at the time the samples are obtained, rather than sending them away to specialised laboratories for analysis. Cigarette smoking is a major health-care problem, and is without a satisfactory method for extralaboratory assessment. Cigarette smoking is the most important cause of preventable mortality from cardiovascular disease, respiratory diseases and various forms of cancer. For treatment to be effective, accurate information regarding smoking habit is required. However, the patient cannot always be relied upon to give reliable details.

Smoking habit can be monitored in a number of ways. Various products of tobacco combustion can be measured, either in the breath or

in bodily fluids. Specific compounds can be determined with great precision, but only with specialised equipment. Alternatively, simpler, less specific colorimetric assays can be employed, but all are laboratory- based tests. Near patient testing can be performed with portable expired- air carbon monoxide monitors, but these have a number of drawbacks, including lack of specificity, a short life of an analyte in the body, and the presence of interfering substances.

Several techniques have been previously proposed to measure the degree of dilution of a urine sample. These include measurement of urea clearance, creatinine clearance, specific gravity, and osmolarity. Of these, osmolarity and creatinine are routinely used, with creatinine being the method of choice. Creatinine can be measured either by enzymatic methods or by a colorimetric reaction based on the Jaffe reaction. The latter although cheap, does involve a toxic colour reagent, which can be explosive under certain circumstances. Osmolarity is determined by measuring changes to the freezing point of a sample, which is influenced by the amount of fluid. These types of measurements require specialised instruments and trained staff.

It is an object of the present invention to provide a method of measuring the absolute concentration of nicotine metabolites in a sample of urine which utilises a simple and convenient way of measuring the degree of dilution of urine (hereinafter called "urine concentration") in order to arrive at the absolute concentration of nicotine metabolites in the urine.

According to the present invention, there is provided a method of measuring the absolute concentration of nicotine metabolites in urine,

comprising measuring the urine concentration by measuring the absorbance or reflectance of light by the urine sample at at least one wavelength between 400 nm and 650 nm, measuring the actual concentration of said nicotine metabolites in a sample of urine, and comparing said actual concentration with the urine concentration so as to derive the absolute concentration of said nicotine metabolites.

According to a further aspect of the present invention, there is provided a method of measuring the urine concentration of a sample, comprising measuring the absorbance or reflectance of light by the urine sample at at least one wavelength between 400 nm and 650 nm. Such a method enables urine concentration to be determined by reference to the creatinine concentration thereof, and is useful as a method of measuring the absolute concentration of another analyte of urine. Such a method preferably involves measuring the actual concentration of said another analyte (eg a drug or drug metabolite) in the sample, and comparing said actual concentration with the urine concentration determined in accordance with said further aspect of the present invention, to give an absolute concentration of said another analyte.

Most preferably, the method of the present invention involves measuring the absorbance of the sample at a wavelength of 400 to 550 nm. Absorbance at this wavelength has been found to give a particularly close correlation with creatinine concentration in the urine sample. Thus, the absolute concentration of the nicotine metabolite in the urine can be obtained by reference to the creatinine concentration in a simple and reliable way.

The combustion of tobacco produces a multitude of different chemicals. Measurement of some of these compounds in the body is employed to assess smoking habit. Carbon monoxide (CO) is inhaled from burning tobacco. Some combines with the blood, and is then exhaled. The amount of expired-air CO correlates well with cigarette consumption, but CO is not specific to tobacco smoke, and has a short life in the body (half-life 3-4 hours). Serum thiocyanate comes from cyanide generated by tobacco burning. Although it lasts much longer in the body (half-life 14 days), and correlates with cigarette smoking, it is again non-specific to tobacco smoke.

Nicotine and its metabolites are regarded as the best indicators of smoking habit. Specific breakdown products, such as cotinine, can be measured with great specificity and precision with techniques such as gas chromatography. But they can only be measured by specialised laboratories with sophisticated instruments.

Colorimetric assays based on the Kόnig reaction for the measurement of nicotine metabolites in urine are available. These rely on the detection of the pyridine ring by reaction with a condensing agent, barbituric acid, 1 ,3-diethyl-2-thiobarbituric acid (DETB) or isopropylidene malonate (Meldrum's acid), to produce an orange or red/pink colour respectively. The assays can be used qualitatively or semi-quantitatively if assessed by eye, or quantitatively by measurement of optical density, in the case of barbituric acid test at 506 nm, for DETB at 532 nm and at 492 nm for Meldrum's acid. Such test procedures involve sequential and timed addition of the following liquid reagents to urine: 4 mol/l acetate buffer pH 4.7, 10% (1.54 mol/l) aqueous potassium cyanide, 10% (0.44 mol/l) aqueous chloramine-T, and 1 % (78

mmol/l) barbituric acid, (50 mmol/l) diethylthiobarbituric acid or 10% Meldrum's acid, all dissolved in acetone water (1 :1 v/v). In addition, to allow for variations in diuresis, urinary creatinine measurements are performed as described above, and the final results expressed as the ratio of nicotine metabolites to creatinine. Such existing procedure is unsuitable for extralaboratory use. An example of a test procedure using Meldrum's acid is described by O'Doherty, S et al in "Enhancing the LC Analysis of Nicotine and its Metabolites in Urine using Meldrum's Acid as a Complexing Agent", Journal of High Resolution Chromatography, vol.13, January 1990, 74-77.

It is an object of a preferred aspect of the present invention to provide an improved reagent for determining nicotine metabolites in a biological fluid such as urine. According to said preferred aspect of the present invention, there is provided a reagent composition for measuring nicotine metabolites in a biological fluid (eg urine or saliva), said reagent composition comprising, in dried particulate form:

(a) a suitable source of CN ^' , eg a soluble cyanide (preferably potassium cyanide),

(b) a suitable source of Cl\ eg a mild bleaching agent such as chloramine-T,

(c) a suitable acid, eg diethylthiobarbituric acid, barbituric acid or Meldrum's Acid, and (d) a buffering agent (eg citric acid/citrate), wherein component (b) is maintained separate from components (a), (c) and (d) for mixing therewith immediately prior to use of the reagent composition.

Components (a) and (b) are chosen so that, in use, they interact to form cyanogen chloride.

The method of the present invention prefereably uses an assay device comprising a reaction chamber, a reagent composition according to said preferred aspect as above defined, and means for separating component (b) from components (a), (c) and (d). Preferably, the device is constructed so that introduction of the sample to be assayed into the reaction chamber automatically causes the reagents (a) to (d) to be brought into mutual contact.

In a particularly preferred assay device used in the method of the present invention, the chloramine-T is retained between two layers of a membrane in an assay device as described and shown in WO 93/09431 published on 13 May 1993 (PCT Application GB92/01981) based on Fig. 1 c of British Patent Application No. 9214457.5 filed on 18 July 1992, the disclosure of which is incorporated herein by reference.

An embodiment of the present invention will now be described in the following Example.

1. Sample Concentration

Measurement of the absorbance of light between the wavelengths 400 nm and 650 nm has been shown to correlate significantly with measurement of creatinine by the Jaffe reaction (p< 0.01). The degree of correlation has been shown to vary with the wavelength (Figure 1), with the best relationship found at 400 nm (Figure 2). Urine samples (n = 14) were collected and assayed for creatinine by the picrate method (Sigma kit). The absorbance of light by unprocessed urine was measured using a bench-top CE 393 digital spectrophotometer with manual wavelength change (Cecil Instruments) at various wavelengths from 320 nm to 700 nm (with correction for the polystyrene

cuvette used). The correlation coefficient (CC) between the creatinine measurement and the absorbance at each wavelength was calculated. Although the best CC was achieved at 400 nm (0.97, figures 1/2) the optium wavelength for the nicotine test was 510 nm (CC = 0.93), and thus it more convenient to perform both at the same wavelength.

2. Assay for Nicotine and its Metabolites

2.1 Fixed amounts of the potassium cyanide (6 mg) and 1 ,3-diethyl-2-thiobarbituric acid (2 mg) are held, together with a buffering agent (consisting of a fixed ratio (1 :1.31 w:w) of solid citric acid (18 mg) and sodium citrate (20 mg) to produce a low final pH of approximately 4.7) in a dried solid particulate form in the reaction chamber 12 of an assay device of the type described with reference to Fig. 1c of WO 93/09431 published on 13 May 1993 (PCT Application No. GB 92/01981 filed on 29 October 1992) and entitled "Assay Device", the disclosure of which is incorporated herein by reference.

2.2 A fixed amount of chloramine-T (11 mg) is contained in chamber 17 of such assay device, whereby it is physically isolated from the above-described mixture by an airtight barrier constituted by lower membrane 16c of the assay device. This arrangement allows the necessary chemicals for the reaction to be held in a stable form prior to use.

2.3 2 ml of a urine sample to be assayed for nicotine is introduced into the assay device through by means of sample collector/dispenser 20 of the assay device. In

so doing, the membrane 16c is pierced and causes the ch'oramine-T to be placed in contact with the other mixed chemicals in the reaction chamber 12 along with the urine sample whilst keeping the assay device as steady as possible so as to result in a minimum amount of mixing at this stage. The initial absorbance reading at 510 nm taken by recording the lowest optical density reading (A).

2.4 The urine and chemicals are then thoroughly mixed together to start the reaction. The presence of nicotine will cause an increase in absorbance (at 510 nm). The first order kinetics allow the rate of increase to be compared with that from a standard nicotine to obtain a value of "Nicotine equivalents" in a given volume.

2.5 Calculation of the "Nicotine equivalents'VA ratio provides an accurate method for measuring the absolute amount of tobacco products in the urine sample.

In another preferred aspect, there is provided an improved assay device comprising an elongate absorbent element having a sample- receiving region, a first measuring region downstream of said sample- receiving region, a reagent region downstream of said first measuring region and containing at least one assay reagent for reaction with a sample which has passed along said element, in use, from said sample- receiving region and through said first measuring region, and a second measuring region disposed downstream of the reagent region.

The reagent region may be provided by one or more bands of reagent extending across the elongate element intermediate its ends. However, in a particularly preferred embodiment, the elongate absorbent element has one or more absorbent members secured to the elongate absorbent element at a location which is disposed downstream of the sample-receiving region and first measuring region, said reagent region being separated from the element by an impermeable element having at least one aperture therethrough.

In a particularly convenient embodiment, the elongate absorbent element comprises a strip having said reactant region formed by one or more absorbent members which are secured to the strip with the intervening apertured impermeable material arranged so that there is a single aperture which provides communication between the strip and a location at or adjacent the centre of the or one of the absorbent members.

The assay device is particularly suitable for the method of measuring nicotine metabolite concentration in a sample of urine in accordance with second and first aspects of the present invention.

In Figs 3 to 5 of the accompanying drawings, Fig. 3 is a schematic perspective view of one embodiment of assay device for use in the present invention, Fig. 4 is a front view of another form of assay device for use in the present invention, and Fig. 5 is a side view of the device of Fig. 4.

Referring now to Fig. 3 of the drawings, the assay device comprises an elongate body 10 formed of a compressed fibrous material

(eg bleached wood pulp fibres such as Whatman AP25 filter paper). In this embodiment, the body 10 is of rectangular shape, is absorbent to liquid and is white in colour. The body 10 has a sample-receiving end region 12 which is dipped, in use, into a sample of urine 14 to be tested. A region 16 of the body 10 which is spaced downstream of the sample- receiving region 12 defines a first measuring region. A reagent region 18 is provided in the body 1 downstream of the region 16 and is defined by a pair of absorbent pads 18a and 18b extending completely across the cross-sectional area of the body 10.

Pad 18a is loaded with potassium cyanide, 1 ,3-diethyl-2- thiobarbituric acid, citric acid and sodium citrate, whilst pad 18b is loaded with chloramine-T.

Downstream of the reagent region 18 there is defined a second measuring region 20.

In use, when the end region 12 of the body 10 is dipped into the sample of urine 14, the urine is drawn along the body 10 by the natural wicking action of the material of which the body 10 is formed. At the first measuring region 16, the reflectance of light of the urine is measured between the wavelengths 400nm to 600nm using a reflectance photometer. The urine continues to wick along the body 10 until it enters the pad 18a. This causes the chemicals in the pad 18a to be activated and transported to the pad 18b where the desired reactions take place to produce a pink colour whose intensity is measured at the second measuring region 20 in order to enable the nictone metabolite concentration of the urine to be assayed in accordance with the previously described method.

The assay device of Fig 3 suffers from a potential disadvantage that the reagents in pads 18a and 18b can remain separate and the elongated extent of the body is such that mixing of the reagents with the sample may be inadequate and a poor or non-representative colour may be produced. These problems can be mitigated in the embodiment of Figs. 4 and 5 where similar parts are accorded the same reference numerals. In this embodiment, pads 18a and 18b take the form of circular discs which are secured to the top of body 10 which takes the form of a thin plate. A circular washer is interposed between pad 18a and the body 10 and this assembly is secured in place and held tightly together using a suitable cover (not shown). In use, urine 14 is drawn along the body 10, the nicotine concentration thereof is assessed using a reflectance photometer at the first measuring region 16. The urine then passes through the relatively small central hole in washer 22 and flows radially into the pad 18a and then passes into pad 18b to produce a distinctive colour change which is measured using reflectance photometry at the second measuring region 20 which, in this embodiment, is defined by the exposed face of pad 18b. Because diffusion of the urine with reactants is limited to the thickness of the porous pads 18a and 18b, there is an improvement in mixing and a consistent colour reaction can be achieved.