Assays cotinine

Principles and characteristics of lateral flow strip assays are reviewed. Recent technology developments permit the use of inexpensive electronic readers for interrogating lateral flow strip test results, thus avoiding the inevitable variation and subjectivity of visual inspection to assess the capture of reporter-labeled analyte on test lines of the strip. Protocols for developing lateral flow assays are described, including two specific case studies for assaying cotinine (a small-molecule metabolite of nicotine) in a competitive format, and assaying HIV antibodies in a sandwich-type assay format. 

Dawson DA et al (1988) Evaluation of the developmental toxicity of nicotine and cotinine with frog embryo teratogenesis assay Xenopus. Teratog Carcinog Mutagen 8 329-338... 

Although 1-nicotine is the predominant natural isomer, the occurrence of some d-nicotine makes it of interest to develop enantioselective assays for this compound. A review of nicotine and cotinine assays has been given (42). An early report of an RIA for 1-nicotine described an immunogen prepared from 6-aminonicotine. This compound was converted to 6-(p-aminobenzamido)nicotine and conjugated to albumin (Fig. 4 [8b]) by diazotization (43). The enantiomeric purity of the 6-aminonicotine was not discussed, and the synthesis of 6-aminonicotine is reported to cause racemization (44). However, the antiserum eventually obtained showed only 6% cross-reaction with d-nicotine (43). 

A racemic nicotine analog, fra s-3-hydroxymethyl-nicotine, was converted to the hemisuccinate, which was conjugated to protein to form an immunogen (Fig. 4 [8c]). The resulting antiserum was used with tiitiated 1-nicotine as radioligand. With this radioligand the assay was highly selective for 1-nicotine, with less than 0,01% cross-reaction with d-nicotine. Similar enantioselectivity is claimed for 1-cotinine (42). 

R. J. Bjercke, G. Cook, N. Rychlik, H. B. Gjika, H. Van Vunakis, and J. J. Langone, Stereospecific monoclonal antibodies to nicotine and cotinine and their use in enzyme-linked immunosorbent assays, J. Immunol. Meth., 90 203-213 (1986). 

Beckett and Triggs were interested in the determination of nicotine and its main metabolite cotinine in urine. An acidified urine was extracted with diethyl ether to remove impurities, the urine was made alkaline with sodium hydroxide and the nicotine extracted with diethyl ether. Cotinine was extracted from urine with methylene chloride after basification of the sample with ammonia. On concentration of the solution, the gas chromatography was carried out on a packed column treated with KOH using Carbowax 20 M as stationary phase. A relative recovery of 95-100 % for the two alkaloids was obtained with respect to their internal standard, chlorophentermine for nicotine and lignocaine for cotinine, which were added to the urine at the start of the assay procedure. Typical chromatograms are given in Figure 4. 

Dumas et al. also developed a micro-method for the same determination in urine and blood. Samples of 3 ml blood were treated with ammonium oxalate and filtered. 1 ml plasma was made alkaline and extracted with chloroform three times, the chloroform extract was evaporated and the residue solved in 10 yl of a solution of 5 ng diphenylamine/ul (internal standard) in ethyl acetate. The solution was used for the gas chromatographic assay. A specific N-detector was used and the sensitivity was 0.2 ng for nicotine and 1 ng for cotinine. The recovery is given in Table 5.9. 

Our laboratory has been using an Avagotech reader for evaluation of reagents for HIV and cotinine lateral flow assays. Figure 2. shows an example of results for a sample diluted in our prototype HIV antibody detection lateral flow test strips. The Avagotech reader allows the data to be plotted and coefficients of variation to be determined, making the screening more quantitative and less subjective. 

Fig. 2. This shows the results from a series of assays for HIV antibodies or cotinine, using the Avagotech RDx reader. Each assay is placed in the reader following test development. The absolute reflectance values were plotted for each assay standard tested.

The limit of detection is determined as the lowest concentration of cotinine on the standard curve for which the following condition is true mean sample signal plus three standard deviations (3SD) < mean blank signal minus 3SD (the 3SD error bars should not overlap). The 3SD rule can be modified depending on the exact requirements for the assay. The 3SD rule provides 99.7% confidence, 2SD - 95%, and 4SD - 99.99%. Protocol (Number 3) outlines steps for determining the assay detection limit as follows ... 

After the antibodies have demonstrated acceptable sensitivity, one should proceed to testing for specificity (cross-reactivity) with structurally similar compounds or compounds that are most likely to be present in the assay matrix. The assay specificity was determined by running the cotinine assay in the presence of compounds structurally similar to cotinine or likely to be present in human samples. Examples included caffeine, nicotinic acid, nicotinamide, and aspirin. Protocol (Number 4) provides testing for assay specificity with the following steps ... 

Assay results for 0 and lOng/mL cotinine samples are used to calculate % displacement as a quality control step. 

Nicotine produced teratogenic effects in test animals, causing postimplantation mortality, fetal death, and developmental abnormalities. Nicotine and its primary metabolite cotinine exhibited teratogenic potential with Xenopus frog embryo teratogenesis assay (Dowson et al. 1988). 

The literature abounds with assay statistics but perhaps the most statistically interesting publication, based solely on the vast number of samples analyzed, is an epidemiological study of the effects of exposure to secondhand cigarette smoke conducted by the U.S. Center for Disease Control. This assay employed APCI LC/MS/MS for the determination of cotinine, a nicotine metabolite, in semm using a stable isotope internal standard. At the time of the publication, data on >32,000 samples utilizing a single mass spectrometer had been amassed. Precision and accuracy of the method was approximately 6% except at the limit of quantitation (25 pg/mL), which had a C Vof 12%. It is appropriate to point out here that these statistics provide the variation in the entire method. The previous discussion on reproducibility (see Section 13.3.1 and 13.3.2) dealt only with variabihtyhmits imposed by the mass spectrometer based on ion current stability measurements that establish the theoretical limits. 

Electrospray has demonstrated results similar to those in the cotinine example and, as described in the sensitivity section, is more widely used and is frequently a more sensitive approach. But it is not always the method of choice when considering targeted specific assays. The ease of mning APCI, its routine use with 4.6-mm columns, and more predictable susceptibility to suppression effects explain its continued acceptance. In some bioanalytical laboratories, APCI is utilized in up to 30% of the assays developed. ... 

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