Quantitative Confirmatory Tests

Other simple tests include the soil burial test used to demonstrate the biodegradabiUty of polycaprolactone (25), following its disappearance as a function of time, and the clear 2one method which indicates biodegradation by the formation of a clear 2one in an agar medium of the test polymer or plastic as it is consumed (26). The burial test is still used as a confirmatory test method in the real-world environment after quantitative laboratory methods indicate bio degradation. 

The biological determinant is an indicator of exposure to the chemical, but the quantitative interpretation of the measurement is ambiguous. These determinants should be used as a screening test if a quantitative test is not practical or as a confirmatory test if the quantitative test is not specific and the origin of the determinant is in question. 

The nonspecific initial tests in a series are valuable for determining the presence or absence of a particular class of compounds. Colorimetric tests to detect the presence of phenothiazines would give initial information about a drug class present. This would be followed by more specific tests to identify the actual compound as well as provide quantitative data. Another example of a type of initial test would be an immunoassay that determines the presence of barbiturates. Confirmatory tests are mandatory to identify the particular drug within the class detected. 

In terms of traditional quantitative validation, the most efficient, albeit slowest, approach was to validate by disease and metabolites. It was also helpful to focus on key metabolites in disease and not every marker potentially detectable from a quantitative perspective. Other metabolites that did not have a standard could be considered supportive qualitative markers. This approach could then be incorporated into a method for interpretation, a process that continues to refine itself based on the screening experience for rare disorders, method improvement, and secondary or confirmatory test. 

A variety of methods has been devised for the confirmation of heptachlor residues (Table II). The presence in the heptachlor molecule (Figure 1) of a reactive allylic chlorine atom has been the basis of three confirmatory tests based on its ease of replacement. Reaction with a silver acetate-glacial acetic acid mixture produced 1-acetoxychlordene which, with the GLC conditions used, had a retention time close to heptachlor epoxide 44). Of the common organochlorine pesticides, only heptachlor reacted quantitatively. Endrin reacts to a small extent with the glacial acetic acid to give a secondary endrin ketone peak. When the reaction of heptachlor with silver salts was extended to silver carbonate in aqueous alcohol, 1-hydroxychlordene was obtained which can easily be converted to the more volatile and GG-responsive silyl ether. Unfortunately, this silyl ether has a Rt identical to aldrin. With silver carbonate, hepta-... 

In this example, urine specimens from six healthy male subjects with a history of marijuana use, who were undergoing medical treatment, were initially tested using eight semiquantitative cannabinoid immunoassays (Table 16.7). In these assays, the metabolite 1 l-nor-9-carboxy-A9-tetrahydrocannabinol (THCCOOH) was detected. The effect of two different cutoff values (100- and 50-pg/L THCCOOH) were investigated with respect to sensitivity, specificity and efficiency, as defined previously in Eq. 16.31-16.33. In this study, all the specimens were also assayed by GC-MS, as a quantitative confirmatory method, using a cutoff value of 15 pg/L this value was used to define positive and negative test results (>15 and <15 pg/ L, respectively). 

During the second year of UG, simple one step organic synthesis (e.g. preparation of bromo or nitro derivative) and quantitative estimations based on specific reactions (e.g. estunation of phenol/aniline by bromination) are introduced along with qualitative analysis. The qualitative analysis is at micro-scale level and is more complex in UG second year, as compounds with multifunctional groups arc given for analysis. In addition to characterisation, students have to identify the compound by performing the confirmatory tests. 

Confirmatory test (GCMS, HPLCMS, IR, or Raman spectrophotometry) Quantitative analysis (if needed)... 

The system can be highly multiplexed to test for numerous biological agents simultaneously, to provide confirmatory tests for different unique sequences from a target organism, and to provide highly accurate and quantitative results. 

Preparation of Derivatives.—Apply confirmatory tests by preparing one or more characteristic derivatives (Chapter X) and determine the physical constants of these derivatives. A color reaction, although of value as an indication, cannot be accepted as a confirmatorA test. Neutral equivalents, saponification equivalents, volatilitj constants of certain aliphatic acids, and quantitative estimation of groups, are occasionally equivalent to a derivative. Usually one typical derivative is sufficient but the amount of confirmatory work will depend upon the requirements for the differentiation between the individual compounds that are accepted as possibilities after completion of the work in the preceding sLx sections. 

If a positive qualitative reaction has been obtained, the confirmatory test must be applied. The B,P, confirmatory test is a qualitative modification of Evers quantitative method, in which the arachidic acid is... 

TLC-bioautography has been used in Canada since 1984 for the confirmation of tetracycline-positive in plant tests (65). However, TLC-bioautography is not quantitative and only gives direction to the analyst as to what confirmatory method of analysis should be used. Therefore, presumptive positives must be confirmed by physicochemical techniques that have been validated in terms of detection limit, precision, and accuracy. 

Coupling chromatographic procedures with immunochemical techniques can also provide a very sensitive and specific analytical system for either determinative or confirmatory analysis. If the antibody used is very specific for the analyte of interest and the antibody reactivity is known to be sensitive to small variations in the structure of the analyte tested, positive reactions with the method are strongly indicative that an analyte of defined structural characteristics is present in the sample. Full rigorous confirmation, however, would depend on further analysis by mass spectrometry, which is the method of choice in confirmatory analysis. Mass spectrometry gives specific information on the identity and structure of the compound of interest. Coupled with chromatographic techniques it becomes a very powerful confirmatory tool for both quantitative and qualitative assessment of drug residues in foods. 

If the specimen provided is a trace sample, sufficient material should be recovered to allow an instrumental analysis directly. The nature of the sample will often provide a clue as to the drug(s) involved and direct comparison can be made by using gas chromatography-mass spectrometry (GC-MS), for example. If the specimen is a bulk sample, presumptive (colour) tests are undertaken to determine the class or classes of drugs which the sample contains. Thin layer chromatography (TEC) is used to determine which members of the classes are present and it might also be possible to make a semi-quantitative estimate of the amount(s) of drug(s) present. Standard mixes can then be prepared for use in the confirmatory techniques. 

When drugs of abuse or other banned substances are determined, a two-step procedure is often used. In the first step, specimens are tested by a semiquantitative assay specimens determined positive in this step are examined by a confirmatory, quantitative assay in a second step. The second method is generally based on a... 

A two-tier approach is often utilized by residue control laboratories whereby samples are first screened to identify the suspected positive (non-compliant) samples, which are subject to further quantitative and confirmatory analysis. Screening methods should be inexpensive and rapid and permit a high sample throughput. The basic criteria that should be met are a detection capability below the RL, a low incidence of false-negative (compliant) results (<5%), and a high degree of repeatability, reproducibility, and robustness. A low incidence of false-positive results is also important to reduce the costs incurred by additional confirmatory analysis. False-positive results in screening analysis can occur for a number of reasons, such as if the test is sensitive to other structurally related compounds naturally present in the matrix or to co-contaminants. 

---