What Drug Give Negative And Positive Result With Trinder Test

As required

Immunoassays imply the use of urine; however, if a urine specimen is unavailable, or "indicates the presence of" a drug, a blood analysis should follow. At a minimum, perform amphetamines, barbiturates, benzodiazepines, cocaine, opiates, PCP, and others as required by case type and history.

CO (carbon monoxide): Between the months of September and May, request on all MEO (medical examiner office) cases involving apparent natural death and pending cases occurring indoors.

MCTs: Require urine and gastric contents.

Key: IA = immunoassays; B/U = blood and/or urine; CO/CN = carbon monoxide and cyanide; BDS = basic drug screen; WAN = weak acid neutral drug screen; TOXI-A/B = commercial thin-layer chromatography acids and bases; MCT = microcolor tests; MVA = motor vehicle accident; COD = cause of death; SIDS = sudden infant death syndrome; APAP = acetaminophen; ASA = salicylates.

4.3.1 Amphetamines

The amphetamines are relatively volatile, and can be lost by evaporation if the extract is not first acidified. In addition, they tend to give tailing peaks unless the gas chromatography (GC) columns are well maintained, or the amphetamines are derivatized. However, ToxiLab can effectively detect and differentiate the sympathomimetic amines in urine, so the amphetamines immu-noassay could be omitted if ToxiLab is used. If an amphetamines immunoassay is used, it should be one of broad class selectivity, not one of the newer and more specific monoclonal antibody assays, so that is has the chance of detecting the maximum number of amphetamines and related drugs (e.g., ephedrine, pseudoephedrine).96-98

4.3.2 Barbiturates

An immunoassay for barbiturates can eliminate the need for labor-intensive chromatographic screens of weakly acidic and neutral drugs in the majority of cases. Only those cases testing positive, or where phenytoin is indicated, need the chromatographic screen. The ToxiLab B system can help to differentiate barbiturates in urine, or the testing can proceed directly to a GC, HPLC, or GC-MS screen on an acidic drug extract of blood to identify the specific barbiturate(s) present in the body.99-104

4.3.3 Benzodiazepines

The benzodiazepines comprise a large class of drugs that are often detected in postmortem cases. ToxiLab does not ordinarily detect them in urine at therapeutic doses, and a basic drug GC screen rarely detects the more potent drugs in this class, except in cases of overdose. Available immunoassays can yield positive results in urine for metabolites from most of the common benzodiazepines. Exceptions are lorazepam and flunitrazepam. GC with electron capture detection, or GC/mass spectrometry (GC/MS), may be applied to urine or blood to differentiate benzodiazepines and to detect those that are missed by immunoassays.105-117 Whether or not to use these more labor intensive chromatographic tests in the absence of a positive immunoassay depends upon the potential significance of a benzodiazepine, if it were to be found, and on any indications from the investigation that one may have been taken. Several methods have also been published for the detection of a wide range of benzodiazepines by LC/MS or LC/MS/MS.

4.3.4 Cocaine

Cocaine should always be included in a postmortem toxicology screen. Even infants and elderly individuals occasionally test positive. The most efficient way to screen for cocaine is to use an immunoassay for benzoylecgonine.118-121 Although both ToxiLab A and basic drug GC screens can actually detect cocaine itself, special procedures are required for the chromatographic detection of benzoylecgonine. Cocaine may not always be found in the urine with benzoylecgonine, although benzoylecgonine is almost always found whenever cocaine is present.

4.3.5 Opiates

The common opiates include morphine, 6-monoacetylmorphine, codeine, hydromorphone, hydrocodone, and oxycodone. Routine ToxiLab A or basic drug GC screens detect codeine, hydro-codone, and oxycodone. These methods are less effective for morphine and hydromorphone, which are usually found in the urine as water-soluble conjugates of glucuronic acid. Immunoassays can detect both parent drug and metabolites. Differentiation of opiates is usually accomplished by GC/MS, using procedures that include hydrolysis of conjugates and derivatization for maximum sensitivity and specificity, or by LC/MS.122-124

4.3.6 Phencyclidine

Phencyclidine (PCP) is more commonly encountered in some regions, while not in others. It is a powerful dissociative anesthetic with significant effects on behavior at low doses, and should be included as part of the postmortem drug screen. Both thin-layer chromatography (TLC) and GC detect PCP, although they may be insufficiently sensitive to reliably rule it out. Immunoassays capable of detecting PCP in urine down to 25 ng/mL provide a reliable screening test.125,126

4.3.7 Immunoassays for Other Illegal Drugs

Immunoassays have been developed to screen for the presence of other illegal drugs, including cannabinoids, LSD, and prescription drugs such as propoxyphene, methadone, and methaqualone. With the exception of cannabinoids and LSD, all of the analytes are readily detected by either ToxiLab A or GC basic drug screens. Cannabinoid and LSD assays may be included in a standard screening protocol or may be reserved for cases involving an issue of possible behavioral toxicity, since those are not known to contribute to either fatal intoxications or deaths attributable to natural causes.

4.3.8 Chromatographic Methods

Chromatographic methods are used to expand the range of a drug screen beyond those drugs detectable by the immunoassays. The extraction system should be selective for basic drugs, and neutral substances will also be extracted.47,58,62,71-76,127-130 ToxiLab A for Urine and Gastric Contents

The ToxiLab A system for basic drug detection is a powerful tool for drug screening in urine or gastric contents.55-57,131 The four-stage visualization process, combined with Rf and detection of metabolite patterns, adds considerably to the confidence in drug identification. Sensitivity for many drugs is on the order of 0.5 to 1.0 mg/L, which is satisfactory for screening urine or gastric samples. The ToxiLab A can detect nearly 150 drugs and their metabolites. Alternatives to ToxiLab

A screen for basic and neutral drugs by CG with a nitrogen/phosphorus detector (NPD) is an alternative to ToxiLab TLC for a laboratory having sufficient GC capacity.47,71,74-76,82,127 GC is much more sensitive than TLC, having limits of detection on the order of 0.01 to 0.10 mg/L from 1 mL of sample. GC can be calibrated to presumptively identify hundreds of drugs and drug metabolites, and the NPD data can reveal other unidentified nitrogen-containing substances that may be characterized by additional analysis of the extract by GC/MS.

The Trinders test is added to the screen to detect salicylate from aspirin, a common drug and one that is sometimes taken for suicidal purposes. It may be eliminated if the screen is intended only to evaluate potential behavioral toxicity. The chemical tests for ethchlorvynol and chloral hydrate fill another gap in the screening protocol, since these drugs are not detected by immunoassay or in chromatographic screens for basic drug or volatiles. Figure 4.2 illustrates the analytical scheme for routine cases with both urine and blood submitted to the laboratory.

4.3.9 Gastric Contents vs. Urine

Often, urine is not available for testing because the bladder was empty at the time of autopsy. In such cases, an extract or filtrate of gastric contents may be substituted. However, a gastric drug screen alone may not be sufficient to exclude all potentially relevant substances. Drugs that are typically administered parenterally, smoked, or insufflated may not diffuse into the stomach in detectable amounts, so blood or plasma analysis with immunoassays for opiates and benzoylecgo-nine is often required to detect those substances.

Drugs taken hours before death may not remain in the stomach. However, some quantity of a basic drug will diffuse into the stomach from blood and become ionized, and thus remains there. The resulting concentration in the stomach contents is determined in part by the drug's concentration in blood and in part by its pKa. For optimum sensitivity, a gastric content drug screen may be combined with immunoassays on plasma, vitreous humor, or blood for barbiturates, benzodiaz-epines, benzoylecgonine, opiates, and a basic drug screen of blood by GC-NPD. The following protocols, which employ tests on gastric contents and blood (or plasma), can be used in place of a urine drug screen. The same blood test panel may be used when gastric contents are not available, or applied to tissue homogenates when blood is not available.

Immunoassays can usually be performed directly on unhemolyzed plasma or vitreous humor. Even hemolyzed plasma and postmortem whole blood can be tested directly if FPIA or RIA is

Figure 4.2 Flowchart for routine toxicology cases.

used.5052 132-145 If results are unsatisfactory, simple dilution with an equal volume of assay buffer is often sufficient to render an analyzable sample. If FPIA or RIA is not available in the laboratory, blood may be screened with spectrophotometry-based immunoassays, although hemoglobin and other proteins must first be precipitated by addition of acetone, methanol, or acetonitrile. The centrifuged supernatant from such treatment can be assayed against similarly prepared calibra-tors.49-52146-154 When applied to blood, plasma, or vitreous humor, immunoassays should be calibrated to a lower cutoff threshold than that used for urine screening. Cutoffs, on the order of 50 ng/mL (500 ng/mL for barbiturates), will yield some false positives. Considering the variations in cross-reactivity for analytes of interest (e.g., benzodiazepines), some false-positive immunoassay results may be acceptable in the interest of minimizing false negatives. Any positive immunoassay result must, of course, be confirmed if it is to be reported. If the case may involve lethal toxicity, immunoassays for acetaminophen and salicylate should be added to the panel.

4.3.10 Screening with Gas Chromatography

GC for basic drugs in blood is more sensitive than a basic drug screen on gastric contents, but is also more labor intensive. It may be included in a general drug screen whenever urine is not available or it may be reserved for cases where intoxication is indicated by the investigation, and gastric contents yield negative screening results. Figure 4.1 illustrates general screening protocols for various combinations of postmortem samples.

When initial chromatographic screening tests reveal an unidentified spot on TLC or a response from GC that does not match any standard, extracts may be further screened by GC/MS in the full scan electron-impact ionization mode.80,82,83,100,155-163 Reconstructed ion chromatograms are inspected for spectra that indicate exogenous (i.e., xenobiotic) substances. Suspect spectra are compared with the instrument's computerized libraries of drugs, poisons, and their metabolites. In addition, spectra may be visually compared with published compilations of mass spectral data.164-169 Whenever a tentative identification is made, the unknown and reference spectra must be visually compared to verify their identity. GC/MS is the most complex method of screening for drugs and poisons, and also the most expensive. The analyst must have a considerable amount of training and experience to reliably perform GC/MS screening.

To provide adequate support for a medical examiner's office the toxicology laboratory should periodically assess the prevalence of drugs in the population served, and adjust its offering of routine tests accordingly. Certain drugs are more prevalent in various localities due to supply routes, ethnic practices, and demand. Changing patterns of drug use may be identified through crime laboratory statistics and various epidemiological monitoring programs, such as the Drug Abuse Warning Network (DAWN) and the community-based Drug Epidemiology Network. In Europe, the www.emcdda Web site offers similar information for the EU countries.

4.3.11 Screening with Liquid Chromatography

Many drugs that cannot be detected by GC can be detected by high-performance liquid chro-matography (HPLC). HPLC separates analytes in solution, at or near ambient temperature, and therefore can be used for drugs that are too thermolabile or polar to be analyzed by GC. Furthermore, chemical derivatization is rarely, if ever, required prior to analysis of drugs by HPLC. While most, but not all, drugs absorb ultraviolet (UV) light sufficiently well to be detected by a UV detector, a UV spectrum usually does not give sufficient information on its own for a forensically valid identification. Therefore, the preferred detector increasingly is becoming the mass spectrometer. However, LC/MS has not yet become widely accepted as a useful screening technique for two reasons. First, LC/MS instrumentation continues to cost approximately twice as much as a GC/MS instrument. Second, and more important, it has been very difficult to generate reference libraries that are universally useful. Although, perhaps arguably more useful than UV spectra, most LC/MS spectra lack the detail of typical electron impact (EI) GC/MS spectra, and worse, relative abundances across the spectrum can vary with pH and ionic strength.

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  • fre-swera
    What drug give negative and positive result with trinder test?
    3 years ago

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