Different types of evidence may be presented to a forensic chemistry laboratory for analysis. This includes solid or liquid material such as powders, tablets, capsules, plant material or solutions and paraphernalia such as syringes, cookers, spoons, cigarettes and pipes. Although some of the components of the evidence may be suggested by external appearance, the forensic chemist is still required to perform analytical testing on the submitted evidence. The material may contain ‘active’drug or drugs as well as inactive substances used to dilute the potency of the preparation. As workload for the forensic chemist increases, it would be impractical to subject all submitted evidence to sophisticated and time-consuming extraction and instrumental techniques. Therefore, there is a need for a battery of relatively simple screening tests that the chemist can perform to assist in directing toward an ultimate identification.
The forensic toxicologist frequently faces the same challenges that the forensic chemist encounters. For instance, in postmortem cases, death may occur from exposure to one or more toxic substances. Most of the cases involve alcohol and therapeutic or abused drugs, but other cases may involve gases, volatile substances, pesticides, metals or environmental chemicals. The postmortem forensic toxicologist must be equipped to assist the medical examiner or coroner in the investigation of death due to any of the above substances. In a ‘general unknown’case, most toxicology laboratories perform a series of tests designed to screen for a large cross-section of substances that would reasonably be encountered. Many of these tests are designed to detect or rule out a class or classes of drugs.
One of the oldest analytical procedures used by forensic chemists or forensic toxicologists is broadly classified as ‘color tests’. Color tests or spot tests are chemical tests which involve the reaction of a sample with a reagent or a series of reagents to produce a color or change in color. The biggest advantages to color tests are their simplicity and ease of use. No sophisticated equipment is required and the time needed to train analysts is short. A negative result for a color test is helpful in excluding a drug or a class of drugs, depending on the test performed. It is interesting that many of these color tests were developed empirically. The chemistry behind these color tests is often complex or unknown.
A chemical test with greater specificity than the color test is the microcrystalline test. To perform a microcrystalline test, a drop of reagent is added to a small quantity of sample on a microscope slide. After the reaction is completed, crystals with unique size and shape develop in relation to the substance present. Although these tests may be performed directly on the powder, the presence of dilution agents may change the structure of the crystals.
It must be emphasized that all chemical tests must be confirmed by an alternate analytical technique based on a different chemical principle. In the forensic chemistry laboratory, the confirmatory test usually includes infrared spectrophotometry or mass spectrometry. In the forensic toxicology laboratory, gas chromatography/mass spectrometry is currently the method of choice for confirming the presence of most drugs.
This article is divided into two sections; the first section deals with chemical tests that are often used or are otherwise available to the forensic chemist to test evidence such as material or paraphernalia. The second section discusses chemical tests employed by the forensic toxicologist to screen biological specimens for toxic substances. It is beyond the scope of this article to include all possible chemical tests available to the forensic chemist or toxicologist. Instead, many of the currently used or historically important tests are covered.
Chemical Tests on Evidence Color tests
There are a large number of color tests that have been developed over the years to screen for the presence of certain drugs or drug classes. The following are some of the more common color tests.
Dille-Koppani test Two reagents are required for this reaction: 1% cobalt nitrate in methanol and 5% isopropylamine in methanol. After sequential addition of the two reagents, a violet color is obtained with imides in which the cabonyl and amine are adjacent in a ring. This would include barbiturates, glutethimide and saccharin. No color reaction occurs when there are substituent groups on the nitrogen atom.
Duquenois-Levine test Two reagents and chloroform comprise this reaction. The first reagent added is a mixture of 2% vanillin and 1% acetaldehyde in ethanol; the second reagent is concentrated hydrochloric acid. A blue-purple color indicates cannabis, coffee or tea. Cannabis may be differentiated from the other substances by extracting with chloroform. If the purple color enters the chloroform layer, then the test is presumptively positive for cannabis.
Erlich test The addition of 1% p-dimethylamino-benzaldehyde in 10% hydrochloric acid in ethanol to a sample containing ergot alkaloids yields a violet color. This serves as a rapid screening test for LSD.
Liebermann test The reagent is prepared by adding 5 g of sodium nitrite to 50 ml sulfuric acid. A number of colors, from orange to orange-brown to yellow are produced. Different structures are suggested by the color. The test needs to be repeated with sulfuric acid alone since many compounds produce color changes with the acid.
Mandelin test This is another reaction that produces a wide array of colors and must be interpreted in light of the Liebermann test. The reagent is 0.5% ammonium vanadate in sulfuric acid.
Table 1 lists the colors produced by common drugs with both the Liebermann and the Mandelin tests.
Marquis test This reagent is prepared by mixing one part formaldehyde to nine parts sulfuric acid. By adding one drop of this reagent to the sample, a multitude of colors may arise. Structures that give violet colors include a ring sulfur, a ring oxygen or aromatic compounds consisting entirely of carbon, hydrogen and oxygen. Heroin, morphine and most opioids produce a violet color. Amphetamine and methamphetamine produce an orange-brown color with this reagent. Table 2 lists a number of drugs and the colors generated with this reagent.
Table 1 Color reactions of some common drugs with Liebermann’s and Mandelin’s reagents
|Methadone||Brown-orange||Green to blue|
|MDA||Black||Green to blue|
Scott test This is a series of color reactions used to screen for the presence of cocaine. Two percent cobalt thiocyanate in water and glycerine (1:1) will cause a powder containing cocaine to turn blue. The addition of concentrated hydrochloric acid changes this color to a clear pink. The blue color reappears on extraction with chloroform.
It is recommended that a polarizing microscope be used for the microcrystalline tests. The best magnification for observing crystals is approximately 100 x . The description and classification of microcrystals can be a complex process. When a reagent is added to different substances, differences in shape, color or dichroism may result. The following is a general description of some of the shapes that may be encountered when performing microcrystalline tests. These descriptions are based on the classification of Fulton (1969) in Modern Microcrystal Tests for Drugs.
• Bars: Solid appearing crystals with three unequal dimensions.
• Blades: Thin flat crystals with much greater length than width.
• Grains: Granular precipitate without distinction at 100 x magnification, or crystals that have approximately the same length, width and thickness.
• Needles: Crystal with little width or thickness in comparison to length.
• Plates: Thin flat crystals with width comparable to length.
• Rods: Solid appearing crystals with greater length than width or thickness; width and thickness significant and equal in magnitude.
Table 2 Drugs that react with the Marquis reagent
• Rosettes: Aggregate of crystals that grow out from a central point in all directions.
• Tablets: Flat crystals with similar length and width greater than thickness
Beside a general shape, crystals will often have a characteristic angle of form that may be measured. In addition, crystals will often display dichroism or two different colors with different orientation of polarized light. Although additional colors may be seen, they are usually gradations of the two extreme colors. Undoubtedly, the best description of crystal forms for a particular test is via a photograph. Furthermore, the unknown crystals should be compared to crystals obtained with actual standard material.
Another factor in the type of crystals formed is the reagent or reagents used to generate the crystals. Table 3 lists some of the most common reagents used. The solvent in which these reagents are dissolved is also a significant factor in the types of crystals produced. The general medium for these tests is water. Several aqueous acids have been used most commonly as solvents, including phosphoric acid, sulfuric acid, hydrochloric acid and acetic acid. These acids alter the solubility characteristics of the crystals. For example, phosphoric acid produces the greatest insolubility whereas acetic acid yields the greatest solubility.
Table 3 Common microcrystalline reagents
Although the use of microcrystalline tests in forensic chemistry has diminished over the years with the availability of more advanced instrumentation, there are still a number of tests which are still in use for commonly seized substances. Table 4 lists a common test for some of these substances.
Chemical Tests on Biological Specimens
Some biological specimens are amenable to presumptive chemical tests without any specimen pretreatment. For instance, color tests can often be performed directly on urine or stomach contents. Other specimens, such as blood, bile, liver and kidney require some type of pretreatment prior to the chemical test. Although liquid-liquid extraction or solid-phase extraction can precede the chemical test, there are simpler separation techniques that are more ‘appro-priate’given the presumptive screening nature of these tests. Two such separation techniques are Con-way microdiffusion and protein precipitation.
A Conway microdiffusion cell is a porcelain dish containing either two or three concentric reservoirs with an accompaning glass cover. A trapping reagent is added to the center well. As the name implies, the trapping reagent captures the analyte of interest for the subsequent chemical reaction. In some situations, the trapping reagent is the chemical reagent. In a two-reservoir well, the blood or tissue specimen is added to the outer well, followed by the releasing agent. This releasing agent causes the analyte to leave the biological sample. A glass cover is rapidly placed over the cell, which is then swirled for a brief time. The reaction then proceeds at room temperature for several hours. The glass cover is usually greased to prevent release of analyte from the cell. Alternatively, a three well cell is used. The sample and releasing reagent are added to the middle well while a sealing agent is added to the outer well. After the reaction is complete, the center well contains the trapped analyte.
Table 4 Examples of microcrystalline tests for commonly abused drugs
|d-Amphetamine||5% HAuBr4in (1 + 2) H3PO4 + 1(2 + 3) H2SO4||Light colored trapezoidal blades|
|Cocaine||(1) 20% HOAc
(2) 5% HAuCL4 in H20
|Long rods with short arms at right angles|
|Heroin||5% HgCl2 in H20||Rosettes of needles|
|d-Methamphetamine||H3BM6 in (1+7) H2SO4, hanging drop||0range needles|
|Phencyclidine||10% Kl in water||Branching needles|
Protein precipitation is another simple pretreatment technique. The protein content of human body fluids and tissues is considerable, from about 6% by weight in plasma to greater than 50% by weight in liver and other organs. Numerous reagents have been developed to precipitate protein; two that are commonly used in drug analysis are trichloroacetic acid (10-15% in water) and tungstic acid (10% sodium tungstate in water and used in conjunction with 3n sulfuric acid). Once proteins have been precipitated, separation of aqueous and solid protein must occur by filtration or centrifugation.
Common color tests: classes of substances
A number of color reactions have been devised to identify a number of drugs within a particular drug class. Many of the color reactions on drug classes are used in conjunction with thin layer chromatography. Once the individual substances have been separated on the thin layer plate, these color reagents are applied to the plate for visualization of color. Interpretation of color tests for drug classes must be done with caution. Drugs within a class may have therapeutic ranges that differ by orders of magnitude. This means that a negative result may not exclude the presence of a drug whereas a positive test does not necessarily mean that a toxic amount of drug is present.
Barbiturates Mercuric nitrate and diphenylcarba-zone will react with a chloroform extract of a biological specimen to produce a purple color. The specific barbiturate is not identified by this method. Pheny-toin will also produce a purple color.
Benzodiazepines The Bratton-Marshall test is a classic screening method for identifying benzodiaze-pines. Benzodiazepines are converted into benzophe-nones by acid hydrolysis and heating. After an extraction, the following color reagents are added: 10% sulfuric acid, 0.1% sodium nitrite, 0.5% sulpha-mic acid and 0.1% N-1-naphthylethylene diamine. A purple color will ensue if a benzophenone is present.
Carbamates After an alkaline extraction and concentration, the residue is treated with 10% furfural in ethanol and exposed to fumes of concentrated hydrochloric acid. A blue-black color indicates the presence of carbamates such as carisoprodal, meprobamate or methocarbamol.
Chlorinated hydrocarbons The Fujiwara test is a classic assay to identify trichlorinated compounds such as trichloroethanol. To urine or a tungstic acid precipitate of blood or tissue is added concentrated sodium hydroxide and pyridine. The mixture is placed in a boiling-water bath for several minutes; the presence of a pink color in the pyridine layer is a positive test. It is especially critical to run a reagent blank when performing the Fujiwara test as some of the reagents used may have contaminants which yield an apparent positive result.
Heavy metals This screening test was developed by the German chemist Reinsch and is still used as a qualitative screening test for an overdose of arsenic, antimony, bismuth, or mercury. The method is based on the fact that copper will displace from solution elements below it in the electromotive series. This method involves boiling a small copper coil in an acidified solution of urine, liver or stomach contents. If any of the four mentioned metals are present in the specimen, they will replace the copper on the coil as a dark film. A black- or purple-colored deposit may be due to arsenic, antimony or bismuth; a silver or gray deposit may be due to mercury. Sulfur compounds can interfere with this test by producing a sulfide salt. This method lacks sensitivity but can usually determine an overdose.
Phenothiazines In the presence of an appropriate oxidizing agent, phenothiazines will produce a color. One such reagent is known as the ‘FPN’ reagent and is a combination of ferric chloride, perchloric acid and nitric acid. A variety of colors may be produced, depending on the phenothiazine present. This test may be useful in the identification of an intoxication of some phenothiazines.
Common color tests: specific substances
Acetaminophen The test for acetaminophen is performed on urine or a protein-free filtrate of blood and requires heating at 100°C after the addition of hydrochloric acid. A blue color after the addition of 1% o-cresol in water and ammonium hydroxide constitutes a positive test for acetaminophen. Therapeutic or toxic use of aceteminophen can be identified using this color reaction.
Carbon monoxide (CO) One of the early methods of CO analysis involved microdiffusion using a Con-way cell. The specimen is placed in the outer well and sulfuric acid is added to release the CO from hemoglobin. A solution of palladium chloride is added to the center well. The cell is sealed and incubated at room temperature for one to two hours. As the reaction proceeds, the palladium chloride is reduced to metallic palladium, forming a black or a silver mirror in the center well and the CO is changed to carbon dioxide. This method is capable of distinguishing between normal carboxyhemoglobin saturation levels (< 10%) and elevated carboxyhemoglobin saturation levels.
Cyanide In a two-reservoir well of a Conway micro-diffusion cell, the blood or tissue specimen is added to the outer well, followed by the releasing agent. This releasing agent may be a dilute mineral acid such as sulfuric acid or may be an organic acid such as tartaric acid. The releasing agent causes the formation of the gaseous hydrocyanic acid (HCN). To the center well is added dilute base. This serves as a trapping reagent for the released HCN. The glass cover is rapidly placed over the cell, which is then swirled for a brief time. The reaction then proceeds at room temperature for several hours Alternatively, a three-well cell is used. The sample and acid are added to the middle well and a more dilute acid is added to the outer well. This acts as a sealing agent. After the reaction is complete, the center well contains the trapped cyanide and is available for detection.
A number of colorimetric reactions have been developed to detect cyanide from the trapping agent. One classical color reaction uses chloramine-T to convert cyanide in to cyanogen chloride. A color reagent containing pyridine and barbituric acid is then added to produce a red color. Another common color reagent uses p-nitrobenzaldehyde and o-dini-trobenzene which, when added to the trapping agent produces a violet color.
Ethanol Most of the early methods for ethanol analysis in biological fluids and tissues used wet chemical methods. Due to its volatility, ethanol can be easily separated from a biological matrix by distillation or microdiffusion. Microdiffusion is more amenable to batch analysis than is distillation. Potassium dichromate and sulfuric acid is a common color reagent which is placed in the center well of a Con-way microdiffusion cell. In the presence of ethanol or any other volatile reducing substance, dichromate is converted to the chromic ion, with a change of color from yellow to green. This color test can be observed visually, making this a simple screening method. The main drawback to this method is that it is nonspecific for ethanol; other aldehydes and ketones will oxidize and interfere with the ethanol quantitation.
Ethchlorvynol Although ethchlorvynol is rarely used today as a sedative-hypnotic, a specific and sensitive color test had been developed during a period of greater use. Diphenylamine is added to urine or a protein-free filtrate of blood. Concentrated sulfuric acid is gently poured down the side of the tube and a red color at the interphase represents a positive test.
Imipramine The Forrest reagent can be used to identify toxic concentrations of imipramine, desipra-mine, clomipramine or trimipramine. The reagent consists of equal portions of 0.2% potassium dichro-mate, 30%, v/v, sulfuric acid, 20% perchloric acid and 50% nitric acid. A green color is a positive test for any of the mentioned drugs. Phenothiazines will also produce a color with this reagent.
Methanol A color test specific for methanol involves the oxidation of methanol to formaldehyde by 3% potassium permanganate. Sodium bisulfite and chromotropic acid are then added, followed by a layering of concentrated sulfuric acid. A purple color at the acid/filtrate interface is a positive test. The test needs to be repeated without the addition of permanganate to rule out the presence of formaldehyde in the specimen.
Nitrite Although nitrites have been used for many years as antihypertensive agents, the need to test for nitrites in urine specimens has taken on greater importance over the past several years. Nitrite adulteration causes a significant reduction in the recovery of the major urinary metabolite of marijuana when tested by gas chromatography/mass spectrometry. Urine specimens can be screened for the presence of nitrites by adding 0.6% sulfanilic acid in 20% hydrochloric acid and 0.48% naphthylamine in 20% hydrochloric acid. A red color is a positive screening test.
Paraquat Paraquat is reduced by an alkaline solution of 1% sodium dithionite to produce a purple color. Diquat will yield a green color under similar conditions.
Salicylate Salicylate, the metabolite of aspirin, reacts with an acidic solution of ferric chloride to produce a purple color. This color reaction requires the presence of both the free phenolic group and the free carboxylic acid group that appears on the salicy-late molecule. Therefore, aspirin itself will not produce a positive result prior to hydrolysis to salicylate. This color test has sufficient sensitivity to detect therapeutic use of salicylate.