Abstract:
The invention describes a practical and robust multi-antibody approach to the sensitive immunodetection and determination of the drug of abuse m-chlorophenyl piperazine (mCPP). The invention also describes methods and kits for mCPP detection in an in vitro sample.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application claims benefit of priority from PCT application PCT/GB2010/050,874 filed May 26, 2010, and from GB Patent Application No. 0910031.4 filed Jun. 11, 2009. Each of these applications is incorporated herein by reference in its entirety. 
     FIELD OF THE INVENTION 
     The invention relates to a novel, practical and robust method for the unequivocal identification of the drug of abuse mCPP. 
     BACKGROUND TO THE INVENTION 
     1-(3-chlorophenyl)piperazine, common name m-chlorophenylpiperazine (mCPP), is the main metabolite of the widely used antidepressant drug, trazodone, and its less commonly-used analogue nefazodone. It is a serotonin receptor agonist that affects hormone levels, physiology and behaviour, and is known to be a mild hallucinogen with weak ecstasy-like effects. A report by the European Union-funded European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) and Europol describes its increasing abuse across Europe and it is described as the most widely encountered new psychoactive substance since the inception of a European drug monitoring early warning system in 1997 (Europol-EMCDDA Active Monitoring Report on a new psychoactive substance). It is scheduled in several European countries as well as New Zealand, the apparent origin of its use as a “party pill”. 
     An impediment to testing for mCPP intake is the metabolic production of mCPP by the antidepressant drugs trazodone and nefazodone. Trazodone, systematic name 2-(3-[4-(3-chlorophenyl)piperazin-1-yl]propyl)-[1,2,4]triazolo[4,3-α]pyridin-3(2H)-one, is an antidepressant drug with anxiolytic and hypnotic activity which is metabolized in the liver by hydroxylation, dealkylation and N-oxidation. Staack et al (2007) developed a technique to overcome the possibility of a false-positive test for mCPP intake using gas chromatography and mass spectroscopy (GC-MS). The test rules out trazodone intake by detecting either the parent molecule trazodone or the metabolite hydroxytrazodone. Nefazodone is eliminated as a possible mCPP source by detection of the major metabolites hydroxyl nefazodone and hydroxyethyl deamino hydroxyl nefazodone, both found at higher levels than mCPP. It is reported that mCPP is a minor urinary metabolite of nefazodone in humans, representing less than 1% of total metabolites (Mayol et al. 1994). The drawbacks of the Staack test for mCPP intake are the protracted pre-treatment steps required to prepare derivatives that are amenable to gas chromatography, the expensive and highly-specialised equipment required which is unsuitable and impractical for application outside of the laboratory, and the requirement of an external reference standard of trazodone to confirm the retention time to support an accurate examination of the GC-MS spectra. Furthermore, the requirement of ‘careful screening’ to differentiate between mCPP intake and trazodone implies a method that is non-robust. 
     Specific binding reactions, such as antibody-antigen interactions, have been used extensively in immunoassays to detect a variety of substances present in tissue extracts. Thus, for example, radioimmunoassays (RIAs) could be used for the determination of mCPP and drugs that produce mCPP as a metabolic product. Radioimmunoassays are very sensitive, but do require radionuclide tracers, for example  125 I and  3 H. There are no known RIAs for mCPP and drugs that produce mCPP as a metabolic product such as trazodone. Enzyme-linked immunosorbent assays (ELISAs) are a non-radioactive alternative that could be used for the qualitative and quantitative determination of mCPP and drugs that produce mCPP as a metabolic product. 
     To enable drug screening of mCPP for clinical and forensic toxicology purposes, an economically viable, practical, sensitive and robust test is required. The invention described herein, based on the antibody-antigen interaction, possesses these attributes. 
     SUMMARY OF THE INVENTION 
     The invention provides a solution to the problem of the unequivocal and practical analytical detection and determination of mCPP intake. This solution requires the use of antibodies that bind to mCPP together with antibodies specific for drugs that produce mCPP as a metabolic product. 
     A first aspect of the invention is an antibody or antibodies specific for a drug or drugs that produce mCPP as a metabolic product together with an antibody that binds mCPP for use in an in vitro test for mCPP intake. The drug that produces mCPP as a metabolic product is preferably trazodone or nefazodone. The drugs that produce mCPP as a metabolic product are preferably trazodone and nefazodone. 
     In a second aspect, the present invention describes a method of detecting or determining mCPP intake in an individual. The method comprises contacting an in vitro sample taken from the individual with two or more conjugates and one or more antibodies specific for a drug that produces mCPP as a metabolic product and at least one antibody that binds an epitope of mCPP. The bound conjugates are detected and the presence of or amount of non-metabolic mCPP deduced using calibration values. The drug that produces mCPP as a metabolic product is preferably trazodone or nefazodone. The drugs that produce mCPP as metabolic products are preferably trazodone and nefazodone. For example, an assay to distinguish between trazodone and mCPP intake would incorporate a trazodone-specific antibody, an antibody sensitive to mCPP and appropriate conjugates for their detection and quantification. An assay to distinguish between trazodone, nefazodone and mCPP intake could incorporate a trazodone-specific antibody, a nefazodone-specific antibody and an antibody sensitive to mCPP and appropriate conjugates for their detection and quantification. The meaning of specific herein is as would be interpreted by the skilled person in the art of immunoassays, in which the binding of non-target analytes by an analyte-specific antibody used in an assay is at a low or non-measurable level so as not to compromise the validity of the assay. The sample can be any peripheral biological fluid but is preferably urine. In the context of the invention reference to a sample implies one or more samples. The conjugates of the method are made up of haptens attached to labelling agents. The haptens of the conjugates are molecules that can bind to the antibodies of the method. The use of haptens, conjugates and antibodies in the context of immunoassays is well known in the art. Preferably, the labelling agent of the conjugates is selected from an enzyme, a luminescent substance, a radioactive substance, or a mixture thereof. More preferably, the labelling agent is an enzyme, preferably a peroxidase, most preferably horseradish peroxidase (HRP). 
     Alternatively, or additionally, the luminescent substance may be a bioluminescent, chemiluminescent or fluorescent material. 
     A further aspect of the invention is a kit for detecting or determining mCPP intake, the kit including one or more antibodies specific for a drug or drugs that produce mCPP as a metabolic product and an antibody that binds mCPP. Another embodiment of the invention is a kit for detecting or determining mCPP intake that includes at one or more antibodies specific for a drug or drugs that produce mCPP as a metabolic product and an antibody that binds an epitope of mCPP. The drug that produces mCPP as a metabolic product is preferably trazodone or nefazodone. Alternatively, the drugs that produce mCPP as a metabolic product are trazodone and nefazodone. The kit may optionally include instructions for the use of said conjugates and said antibodies for detecting or determining mCPP and drugs that produce mCPP as a metabolic product. 
    
    
     
       DESCRIPTION OF DRAWINGS 
         FIG. 1  Structures of trazodone and mCPP 
         FIG. 2  Synthesis of hapten A 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Methods, Examples and Results 
     Preparation of Haptens, Immunogens and Conjugates 
     Although haptens provide defined structural epitopes, they are not in themselves immunogenic and therefore need to be conjugated to carrier materials, which will elicit an immunogenic response when administered to a host animal. Appropriate carrier materials commonly contain poly(amino acid) segments and include polypeptides, proteins and glycoproteins. Illustrative examples of useful carrier materials are bovine serum albumin (BSA), egg ovalbumin, bovine gamma globulin, bovine thyroglobulin (BTG), keyhole limpet haemocyanin (KLH) etc. Alternatively, synthetic poly(amino acids) having a sufficient number of available amino groups, such as lysine, may be employed, as may other synthetic or natural polymeric materials bearing reactive functional groups. In particular, carbohydrates, yeasts or polysaccharides may be conjugated to the hapten to produce an immunogen. The haptens can also be coupled to a detectable labelling agent such as an enzyme (for example, horseradish peroxidase), a substance having fluorescent properties or a radioactive label for the preparation of conjugates (or detection reagents) for use in the immunoassays. The fluorescent substance may be, for example, a monovalent residue of fluorescein or a derivative thereof. Immunogen formation involves conventional conjugation chemistry in which the oxygen of the hydroxyl group of Hapten A ( FIG. 2 ) combines first with DCC and then NHS to form an ester with a powerful leaving group. Nucleophilic attack on the carbonyl of the ester functionality by a amine group on the protein (BSA or BTG), results in an amide bond and formation of the target immunogen. In order to confirm that adequate conjugation of hapten to carrier material has been achieved, prior to immunisation, each immunogen is evaluated using matrix-assisted UV laser desorption/ionisation time-of-flight mass spectroscopy (MALDI-TOF MS). 
     General Procedure for MALDI-TOF Analysis of Immunogens. 
     MALDI-TOF mass spectrometry was performed using a Voyager STR Biospectrometry Research Station laser-desorption mass spectrometer coupled with delayed extraction. An aliquot of each sample to be analysed was diluted in 0.1% aqueous trifluoroacetic acid (TFA) to create 1 mg/ml sample solutions. Aliquots (1 μl) were analysed using a matrix of Sinapinic acid and bovine serum albumin (Fluka) was used as an external calibrant. 
     Preparation of Antisera 
     In order to generate polyclonal antisera, the immunogen of the present invention is mixed with Freund&#39;s Adjuvant and the mixture is injected into a host animal, such as rabbit, sheep, mouse, guinea pig or horse. Further injections (boosts) are made and serum is sampled for evaluation of the antibody titre. When the optimal titre has been attained, the host animal is bled to yield a suitable volume of specific antiserum. The degree of antibody purification required depends on the intended application. For many purposes, there is no requirement for purification, however, in other cases, such as where the antibody is to be immobilised on a solid support, purification steps can be taken to remove undesired material and eliminate non-specific binding. 
     Immunoassay Development 
     The process of developing an immunoassay is well known to the person skilled in the art. Briefly, for a competitive immunoassay in which the target analyte is a non-immunogenic molecule commonly referred to as a hapten, the following process is conducted: antibodies are produced by immunising an animal, preferably a mammalian animal, by repeated administration of an immunogen. The serum from the immunised animal is collected when the antibody titre is sufficiently high. A conjugate is added to a sample containing the target analyte and the raised antibodies, and the conjugate and analyte compete for binding to the antibodies. The process may comprise fixing said serum antibodies to a backing substrate such as a polystyrene solid support or a biochip. The antibodies can be polyclonal or monoclonal. The signal emitted in the immunoassay is proportionate to the amount of conjugate bound to the antibodies which in turn is inversely proportionate to the analyte concentration. The signal can be detected or quantified by comparison with a calibrator. 
     Example 1 
     Preparation of 2-(γ-chloropropyl)-1,2,4-triazolo[4,3-a]pyridin-3(2H)-one (Structure 1, FIG.  2 ) 
     To a preheated solution at 80° C. of 1,2,4-triazolo[4,3-a]pyridin-3(2H)-one (25 g, 0.18 mol) in DMF (200 ml) under nitrogen was added sodium hydride (60%) (7.37 g). The mixture was heated at 80° C. for 1 hour under stirring, then 1-chloro-3-iodopropane (37.26 g, 0.18 mol) was added. The mixture was heated under stirring at 100° C. for 6 hours, and left at room temperature overnight. The solid formed was filtered off, washed by DMF (10 ml) and the filtrate was concentrated to dryness. The crude product obtained was diluted with saturated NaHCO 3  (300 ml) and the solution extracted with ethyl acetate (2×300 ml). The combined organic layers were washed by water (200 ml), brine (200 ml), dried over Na 2 SO 4 , filtered and concentrated to dryness to give the title compound 1 (20.1 g) as a viscous oil. 
     Example 2 
     Preparation of 3-methoxytrazodone (Structure 2, FIG.  2 ) 
     To a solution of the compound 1 (12 g, 0.057 mol) in anhydrous toluene (200 ml) was added 1-(3-methoxyphenyl)piperazine hydrochloride (13.0 g, 0.057 mol) and triethylamine (TEA) (15.9 ml, 0.114 mol). The resulting mixture was heated at reflux for 6 hours. The solution was cooled at room temperature, washed with water (150 ml), brine (100 ml), dried over Na 2 SO4, filtered and the solvent removed under reduced pressure. The residue obtained was purified by column chromatography (5% methanol/95% chloroform) to give 3-methoxytrazodone (10.6 g) as a white solid.  13 C NMR (δ: ppm): 162.44, 154.44, 150.63, 143.63, 132.24, 131.11, 124.96, 116.52, 112.98, 110.52, 106.51, 104.09, 57.24, 55.95, 54.69, 50.64, 45.95, 27.03. 
     Example 3 
     Preparation of 3-hydroxytrazodone (Structure 3, FIG.  2 ) 
     To a solution of hydrobromic acid 48 wt % in water (200 ml) was added 3-methoxytrazodone (9.5 g, 0.026 mol) and the mixture was heated at reflux for 3 hours. The solution was cooled to room temperature and concentrated to dryness. Water was then added (200 ml), neutralized to pH 7-8 and the solution extracted with ethyl acetate (3×150 ml). The combined organic layers were washed by water (150 ml), brine (150 ml), dried over Na 2 SO4, filtered and concentrated to dryness. The crude product was recrystallized from ethyl acetate/hexane to give 3-hydroxytrazodone (6.5 g) as a white solid. 
     Example 4 
     Preparation of 3-(ethoxycarbonylmethoxy)trazodone (Structure 4, FIG.  2 ) 
     To a suspension of sodium hydride (NaH) (685 mg, 0.02 mol) in DMF (50 ml) under nitrogen was added a solution of 3-hydroxytrazodone (6.0 g, 0.017 mol) in DMF (100 ml) over a period of 15 minutes and the mixture heated at 60° C. for 1 hour. After cooling the mixture to room temperature a solution of ethyl bromoacetate (3.4 g, 0.02 mol) in DMF (50 ml) was added to the mixture. The mixture was then heated again at 60° C. for 1 hour and stirred at room temperature overnight. The solution was concentrated to dryness, water (200 ml) was added and the pH of the solution was adjusted to 10 by NaOH (1N). The aqueous solution was then extracted with ethyl acetate (2×200 ml), washed with water (100 ml), brine (100 ml), dried over Na 2 SO4, filtered and concentrated to dryness. The crude product was purified by chromatography on silica gel using chloroform/methanol (9/1) to give 3-(ethoxycarbonylmethoxy)trazodone (5.06 g) as a white solid.  13 C NMR (δ: ppm): 168.06, 157.82, 151.7, 147.63, 140.43, 128.74, 122.72, 114.37, 109.46, 108.71, 103.44, 102.29, 64.49, 60.29, 54.60, 52.06, 47.81, 43.44, 29.91, 29.02, 13.17. 
     Example 5 
     Preparation of 3-(carboxymethoxy)trazodone (Hapten A, FIG.  2 ) 
     3-(Ethoxycarbonylmethoxy)trazodone (4.5 g, 0.0102 mol) was dissolved in THF (100 ml) and water (100 ml). Potassium hydroxide (4.37 g, 0.032 mol) was added and the mixture was stirred overnight at room temperature. The THF was removed under reduced pressure and the aqueous solution acidified to pH 2 by HCl (2N). The resultant white solid was filtered, washed with water and dried. Recrystallization with methanol gave 3-(carboxymethoxy) trazodone (3.9 g) as a white solid.  13 C NMR (δ: ppm): 171.14, 161.46, 156.24, 150.65, 143.66, 132.27, 131.03, 124.97, 116.54, 112.98, 110.62, 107.51, 105.01, 68.89, 57.12, 54.51, 50.39, 45.84, 26.83. 
     Example 6 
     Conjugation of Hapten A to Bovine Serum Albumin (BSA) 
     To a solution of Hapten A (30.8 mg, 0.075 mM) in pyridine (2.5 ml) was added N,N-dicyclohexylcarbodiimide (DCC) (15.5 mg, 0.075 mM) and N-hydroxysuccinimide (8.63 mg, 0.075 mM) and the mixture was stirred at room temperature overnight. The dicyclohexylurea formed was removed by filtration and the solution was added drop-wise to a solution of BSA (100 mg, 1.5 μmol) in 50 mM sodium bicarbonate solution (5 ml). The mixture was stirred overnight at 4° C. The solution was dialysed with 50 mM phosphate buffer pH 7.2 (3 changes) for 24 hours at 4° C., and freeze-dried. MALDI results showed 14.77 molecules of hapten A had been conjugated to one molecule of BSA. 
     Example 7 
     Conjugation of Hapten A to BTG 
     To a solution of Hapten A (55.54 mg, 0.1325 mM) in pyridine (2.5 ml) was added N,N-dicyclohexylcarbodiimide (DCC) (55.62 mg, 0.27 mM) and N-hydroxysuccinimide (31.07 mg, 0.27 mM) and the mixture was stirred at room temperature overnight. The dicyclohexylurea formed was removed by filtration and the solution was added dropwise to a solution of BTG (150 mg, 2.25 μmol) in 50 mM sodium bicarbonate solution (10 ml). The mixture was then stirred overnight at 4° C. The solution was then dialysed with 50 mM phosphate buffer pH 7.2 (3 changes) for 24 hours at 4° C., and freeze-dried. 
     Example 8 
     Conjugation of Hapten A to HRP 
     EDC hydrochloride (10 mg) was dissolved in water (0.5 ml) and immediately added to a solution of Hapten A (2 mg) in DMF (0.2 ml). After mixing, the solution was added drop-wise to a solution of HRP (20 mg) in water (1 ml). Sulfo-NHS (5 mg) was added and the reaction mixture was incubated in the dark at room temperature overnight. Excess hapten was removed with double PD-10 columns (Pharmacia) in series, pre-equilibrated with PBS at pH 7.2. The hapten-HRP conjugate was then dialysed overnight with 10 L of PBS at pH 7.2 at 4° C. 
     Example 9 
     Preparation of Ethyl 6-[N′-(3-chlorophenyl)-N-piperazinyl]hexanoate 
     To a solution of 1-(3-chlorophenyl)piperazine hydrochloride (12 g, 0.051 mol) in anhydrous toluene (250 ml) was added triethylamine (TEA) (15.6 ml, 0.112 mol) and ethyl 6-bromohexanoate (13.6 g, 0.061 mol) and the mixture heated at reflux for 4 hours. The mixture was then cooled to room temperature, washed with water (2×100 ml), brine (100 ml), dried over Na 2 SO4, filtered and concentrated to dryness. The residue obtained was purified by flash chromatography on silica gel using ethyl acetate/hexane (1/1) to give ethyl 6-[N′-(3-chlorophenyl)-N-piperazinyl]hexanoate (10.3 g) as a clear oil. 
     Example 10 
     Preparation of 6-[N′(3-chlorophenyl)-N-piperazinyl]hexanoic Acid 
     To a solution of [N-(carboethoxypentyl)-N-(3-chlorophenyl)]piperazine 5 (10 g, 0.029 mol) in a mixture of THF/water (1/1) was added potassium hydroxide (12 g, 0.087 mol) and the mixture was stirred at room temperature overnight. The THF was removed under vacuum and the solution was acidified with HCl (2N). The precipitate was filtered, washed by water and dried. Recrystallization with methanol gave 6-[N′-(3-chlorophenyl)-N-piperazinyl]hexanoic acid (6.5 g).  13 C NMR (δ: ppm): 177.61, 152.78, 136.6, 132.15, 122.18, 118.15, 116.29, 58.21, 53.3, 47.86, 34.83, 27.43, 25.77, 25.18. 
     Example 11 
     Conjugation of 6-[N′-(3-chlorophenyl)-N-piperazinyl]hexanoic Acid to BSA 
     To a cooled solution at 0° C. of 6-[N′-(3-chlorophenyl)-N-piperazinyl]hexanoic acid (37.28 mg, 0.12 mmol) in DMF (3 ml) under nitrogen was added tri-n-butylamine (31.42 μl, 0.132 mmol) and isobutyl chloroformate (IBCF) (17.02 μl, 0.132 mmol). The mixture was stirred at 0° C. for 15 minutes and then added drop-wise to a cooled solution of BSA (100 mg) in sodium bicarbonate (100 mM, 10 ml) and the mixture was stirred at 4° C. overnight. The solution was then dialysed against 50 mM phosphate buffer pH 7.2 (3 changes) for 24 hours at 4° C., and freeze-dried. MALDI results showed 22.02 molecules of 6-[N′-(3-chlorophenyl)-N-piperazinyl]hexanoic acid had been conjugated to one molecule of BSA. 
     Example 12 
     Conjugation of 6-[N′-(3-chlorophenyl)-N-piperazinyl]hexanoic Acid to BTG 
     To a cooled solution at 0° C. of 6-[N′-(3-chlorophenyl)-N-piperazinyl]hexanoic acid (65.09 mg, 0.203 mmol) in DMF (3 ml) under nitrogen was added tri-n-butylamine (53.1 μl, 0.223 mmol) and isobutyl chloroformate (IBCF) (28.78 μl, 0.223 mmol). The mixture was stirred at 0° C. for 15 minutes and then added drop-wise to a cooled solution of BTG (150 mg) in sodium bicarbonate (100 mM, 10 ml) and the mixture was stirred at 4° C. overnight. The solution was then dialysed with 50 mM phosphate buffer pH 7.2 (3 changes) for 24 hours at 4° C., and freeze-dried. 
     Example 13 
     Conjugation of 6-[N′-(3-chlorophenyl)-N-piperazinyl]hexanoic Acid to HRP 
     To a cooled solution at 0° C. of 6-[N′-(3-chlorophenyl)-N-piperazinyl]hexanoic acid (2 mg) in DMF (200 μl) under nitrogen was added tri-n-butylamine (38 μl) and isobutyl chloroformate (IBCF) (2 μl). The mixture was stirred at 0° C. for 10 minutes and then added drop-wise to a cooled solution of HRP (200 mg) in water (800 μl) and the reaction mixture was incubated in the dark at room temperature overnight. Excess hapten was removed with double PD-10 columns (Pharmacia) in series, pre-equilibrated with PBS at pH 7.2. The hapten-HRP conjugate was then dialysed overnight with 10 L of PBS at pH 7.2 at 4° C. 
     Example 14 
     Development of ELISAs for Trazodone and mCPP 
     Trazodone and mCPP were coupled by way of a crosslinker to bovine thyroglobulin (Examples 7 and 12). The resulting immunogens were administered separately to adult sheep on a monthly basis to provide target-specific polyclonal antisera. IgG was extracted from the antisera via caprylic acid/ammonium sulphate precipitation of immunoglobulin. Microtitre plates (Thermo Scientific, 95029180) were coated with antibody (125 μl) in coating buffer (10 mM Tris pH 8.5) at 37° C. for 2 hours. Antibody raised from the mCPP-derived immunogen was coated at 2.5 μg/ml and antibody raised from the trazodone-derived immunogen was coated at 0.625 μg/ml. The plates were then washed. 50 μl of sample/standard (trazodone, Sigma T6154-1 g; mCPP, Alfa-Aesar L01772; nefazodone, Sigma N5536) was added to the appropriate wells in triplicate, followed by 75 μl of conjugate from Example 13 for the generic antibody, and 75 μl of conjugate from Example 8 for the trazodone-specific antibody) and incubated at 25° C. for 1 hour. The plates were then washed and 125 μl of TMB (Randox, 4380-15) was added to each well and left at room temperature for 20 minutes in the dark. The reaction was stopped using 125 μl of 0.2M sulphuric acid. The absorbances were then read at 450 nm with an ELISA microplate reader (BIO-TEK Instruments, EL340) and the means calculated. Antibody specificity and sensitivity were then determined. 
     Results 
     Competitive ELISA results in Tables 1 &amp; 2 highlight a mCPP generic antibody and a trazodone-specific antibody, respectively. An assay that makes use of both these antibodies can confirm mCPP abuse by verifying the presence of mCPP and the absence of trazodone. 
     
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Generic antibody cross-reactivity and sensitivity profile 
               
             
          
           
               
                   
                 Trazodone 
                 mCPP 
                 Nefazodone 
               
             
          
           
               
                 Standard 
                   
                 % 
                   
                 % 
                   
                 % 
               
               
                 conc n  ng/ml 
                 A 450   
                 B/B 0   
                 A 450   
                 B/B 0   
                 A 450   
                 B/B 0   
               
               
                   
               
             
          
           
               
                 0.000 
                 2.184 
                 100 
                 2.133 
                 100 
                 2.023 
                 100 
               
               
                 0.156 
                 1.456 
                 67 
                 1.821 
                 85 
                 1.850 
                 91 
               
               
                 0.313 
                 1.271 
                 58 
                 1.679 
                 79 
                 1.671 
                 83 
               
               
                 0.625 
                 1.041 
                 48 
                 1.534 
                 72 
                 1.467 
                 73 
               
               
                 1.250 
                 0.763 
                 35 
                 1.380 
                 65 
                 1.168 
                 58 
               
               
                 2.500 
                 0.551 
                 25 
                 1.183 
                 55 
                 0.923 
                 46 
               
               
                 5.000 
                 0.398 
                 18 
                 1.027 
                 48 
                 0.683 
                 34 
               
               
                 10.000 
                 0.287 
                 13 
                 0.888 
                 42 
                 0.494 
                 24 
               
             
          
           
               
                 IC 50   
                 0.532 
                 8.476 
                 3.901 
               
               
                 % CR 
                 733 
                 46 
                 100 
               
               
                   
               
               
                 A 450  = absorbance at 450 nm; 
               
               
                 B = absorbance at 450 nm at x ng/ml standard concentration 
               
               
                 B 0  = absorbance at 450 nm at 0 ng/ml standard concentration; 
               
               
                 IC 50  = standard concentration which produces 50% B/B 0 ; 
               
               
                 % CR = percentage cross-reactivity based on Nefazodone (100%) 
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Trazodone antibody cross-reactivity and sensitivity profile 
               
             
          
           
               
                   
                 Trazodone 
                 mCPP 
                 Nefazodone 
               
             
          
           
               
                 Standard 
                   
                 % 
                   
                 % 
                   
                 % 
               
               
                 conc n  ng/ml 
                 A 450   
                 B/B 0   
                 A 450   
                 B/B 0   
                 A 450   
                 B/B 0   
               
               
                   
               
             
          
           
               
                 0.000 
                 1.744 
                 100 
                 1.759 
                 100 
                 1.674 
                 100 
               
               
                 0.156 
                 0.816 
                 47 
                 1.721 
                 98 
                 1.631 
                 97 
               
               
                 0.313 
                 0.570 
                 33 
                 1.755 
                 100 
                 1.656 
                 99 
               
               
                 0.625 
                 0.375 
                 21 
                 1.756 
                 100 
                 1.621 
                 97 
               
               
                 1.250 
                 0.237 
                 14 
                 1.759 
                 100 
                 1.677 
                 100 
               
               
                 2.500 
                 0.151 
                 9 
                 1.782 
                 101 
                 1.663 
                 99 
               
               
                 5.000 
                 0.093 
                 5 
                 1.814 
                 103 
                 1.765 
                 105 
               
               
                 10.000 
                 0.057 
                 3 
                 1.857 
                 106 
                 1.799 
                 107 
               
             
          
           
               
                 IC 50   
                 0.872 
                 — 
                 — 
               
               
                 % CR 
                 100 
                 0.68 
                 1.38 
               
               
                   
               
               
                 A 450  = absorbance at 450 nm; 
               
               
                 B = absorbance at 450 nm at x ng/ml standard concentration 
               
               
                 B 0  = absorbance at 450 nm at 0 ng/ml standard concentration; 
               
               
                 IC 50  = standard concentration which produces 50% B/B 0 ; 
               
               
                 % CR = percentage cross-reactivity based on Trazodone (100%) 
               
             
          
         
       
     
     BIBLIOGRAPHY 
     
         
         Staack R. F. et al. (2007).  J. Chromatog. B,  855: 127-133. 
         Mayol R. F. et al (1994).  Drug Metab. Dispos.,  22: 304-311.