Analysis Methods

As with most toxicological analyses, screening technologies as well as confirmatory technologies are extensively used. Immunoassay-based screening technologies such as enzyme multiplied immunoassay technique EMIT; (Dade-Behring Diagnostics) fluorescence polarization immunoassay (FPIA – Abbott Diagnostics) and cloned enzyme donor immunoassay (CEDIA) all offer varying cross-reactivity to the morphine-like compounds. All of these kits can be used on blood, urine, or other matrices. However, blood and hair analyses usually require additional preparation of the samples E.g., precipitation of protein or digestion and extractions and often fall outside of the manufacturer’s specifications on the kits. As there is greater demand for screening and confining other Opoids, newer kits that are more specific to other Opoids have become available. Some of these kits are heterogeneous assays in 96-well ELISA formats. Again, many of these assays are used in more specialized populations where there is greater likelihood of abuse of other Opoids.

Table 10.3 compiles information about currently available Opoid related immunoassay kits. It is apparent from this table that a wide array of kits is now available for many Opoids. The table includes the reported cross-reactions of the kit to Opoids other than the designed target. Some kits have far broader cross-reactions than other kits and care should be taken to match the cross-reactivity profile of the assay to the screening needs. If a very narrow selection of Opoids will be confirmed a widely cross-reactive kit may produce a low confirmation rate. A variety of studies have evaluated the efficacy of immunoassay kits over the years. Screening cutoffs for morphine/codeine Opoid assays are 2000ng/mL for federally regulated testing, with many manufacturers supporting a 300ng/mL cutoff as well.

Obviously, the 300 ng/mL type cutoffs are not eligible to be used in regulated testing, but the 300 ng/mL cutoffs have applicability in programs where the detection of other Opoids is desirable. The 2,000 ng/mL cutoff was adopted largely to address concerns about potential positive urine results from consuming poppy seeds rather than in appropriate drug usage. A wide variety of confirmatory testing methods predominantly gas chromatography-mass spectrometry GC-MS, are used. New methods using high-performance liquid chromatography-mass spectrometry (LCMS) are being developed.

As many of the compounds are present in both blood and urine as conjugates hydrolysis of these conjugates is often necessary before extraction. Hydrolysis is often accomplished by the addition of strong acid and pressurized heat treatment or the addition of β-glucuronidase. Acid hydrolysis is faster; however, compounds such as 6 acetylmorphine are not stable under these conditions. Enzymatic hydrolysis occurs under gentler conditions, but usually takes longer. Additionally, enzyme sources are usually derived from bacteria and have varying degrees of effectiveness in hydrolyzing the conjugates of interest and variation from lot to lot. Confirmation testing usually requires the extraction of compounds from the biological matrix either by solid-phase extraction (SPE) or liquid-liquid extraction.

A wide range of methods has been reported for primarily cationic and mixed-bed type SPE methods. Liquid-liquid extractions classically use an initial basic exaction into an organic solvent followed by subsequent acidic back extraction clean-up steps and final extraction into an organic solvent. For GC-MS, derivatization is often necessary for acceptable chromatographic performance and fragmentation. Silation with agents such as N,O-bis(trimethylsilyl)trifluoroacetamide [BSTFA], and acylation with agents such as propionic anhydride and pentafluoropropionic anhydride, are common means of derivatization.

Care should be exercised if derivatizing with acetic anhydride, understanding that diacetylmorphine will be formed from 6-acetylmorphine and morphine present in the sample. If the analysis needs to maintain this information, deuterated acetic anhyride is available so that diacetylmorphine formed during derivatization can be distinguished, as can the derivatized 6-acetylmorphine from morphine. A wide range of sensitivities are obtained by various methods, and the specific limits of detection and linearity pertinent to the individual analysis should be considered in any interpretation. Other matrices may also be utilized in the workplace testing setting with differing strengths and weaknesses for workplace testing. In the case of hair analysis, an additional step to digest the hair matrix is required. Normally, this is accomplished by the addition of strong base, strong acid, enzymes, or sodium sulfide.

These conditions result in varying degrees of digestion of the hair matrix and do not completely digest melanosomes present in the hair. This may result in the incomplete liberation of the target compounds from the hair (especially in the case of enzymatic digestion) for extraction and analysis. Likewise the relative impermeability of melanosomes to digestion may result in the sequestration of target compounds in melanosomes. As much of the stored drug in hair may be associated with melanin even as a covalent adduct, this sequestration may dramatically reduce the sensitivity of the testing.

Even with the potential difficulties in the use of hair, many Opoids have been reported as detected in hair. The biology of hair growth and the potential for surface contamination have confounded the relation of hair concentrations to serum concentrations. Additionally, hair concentrations may saturate at relatively low concentrations for many drugs and even for some Opoids. Saliva has also become a novel alternative testing method that may help provide rapid roadside drug-testing results.

Many compounds have been detected in saliva. Screening and confirmatory technologies typical of other matrices are also used in saliva testing. Confounding the relationship of saliva concentrations to serum levels is the potential for oral contamination. Most of these methods are based on tandem mass spectrometry methods because of the sensitivity needs driven by the small sample sizes.  Other matrices such as fingernails, sweat, breast milk, and sebum have all been examined for their content of various Opoids. Most of these analyses are again conducted by immunoassays and GGMS or LC-MS methods. Depending on the matrix, additional steps may be necessary to break down the matrix to facilitate extraction.

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