1.7.1 screening tests
No other topic related to the identification of controlled substances causes as much controversy as testing specificity. Forensic science laboratories conduct two different categories of tests. Tests in the first category are called "screening tests". They include a series of tests used to make a preliminary determination of whether a particular drug or class of drugs is present. It must be emphasized that screening tests are not used to positively identify any drug. At best, screening tests can only be used to determine the possibility that members of a particular class of drug may be present. Some say that screening tests can result in "false positives", meaning that either the test indicates the possible presence of a controlled substance when none is present or that the test indicates the possible presence of one controlled substance when a different controlled substance is present. That should not be a problem, so long as it is understood that screening tests have very little if any specificity, and that a false positive test will only lead to more testing, not a false conclusion. The identification of any drug by a chemical analysis is a systematic process involving a progression from less specific methods to more specific methods. The most specific methods involve instrumental analyses. Properly trained scientists should know when a false positive is possible, and how to take steps to narrow the focus of the testing. The more tests used, the fewer the chances for error.
False negative screening tests also occur. Very weak or diluted samples containing controlled substances may yield a negative screening test. An example of this situation would be a 1% heroin sample cut with a brown powder. Testing this sample with Marquis Reagent, which contains sulfuric acid and formaldehyde, may result in a charring of the brown powder and subsequent masking of the bleeding purple color characteristic of an opium alkaloid. Weak or old reagents may also yield false negatives. Examiner fallibility or inexperience in discerning colors may also result in false negatives. The possibility of a false negative leads many examiners to conduct a series of screening tests or, when warranted, to progress directly to more narrowly focused screening tests.
Specificity is the key to the forensic identification of controlled substances. There is no one method that will work as a specific test for any and all exhibits at any and all times. The choice of which specific method one utilizes must be determined by the type of controlled substance, the concentration of the controlled substance in the sample, the nature of the diluents and adulterants, the available instrumentation, and the experience of the examiner. There is an ongoing debate as to whether one can achieve this scientific certainty by combining a series of non-specific tests. This will be discussed later in this section.
Occassionaly an experienced forensic analyst can just look at an exhibit in a drug case and determine the probable nature of the substance. However, "probable natures" are not enough for an identification, and most examiners will usually conduct more than one test before reporting the presence of a controlled substance. The morphology of botanical substances such as marijuana and the peyote cactus are familiar enough to many laboratory analysts. Marijuana is one of those controlled substances which is examined with such frequency in the laboratory that a preliminary identification is probable based on the morphology of the botanical substance, gross physical appearance, texture, and odor. However, even after a microscopic examination of the cystolithic hairs using a micrcoscope, the modified Duquenois-Levine test is usually run to corroborate the identification. The peyote cactus with its button-like appearance is also unique. In a like manner, the identification of the opium poppy requires a confirmation of the morphine; and the identification of the pysilocibin mushroom requires an identification of the psilocybin or the psilocin.
The physical characteristics of these four agronomic substances might enable an expert witness with a background in plant taxonomy and botany to make an identification based solely on these characteristics. The forensic analyst relies on the physical characteristics and corroborating chemical examinations to identify these materials as controlled substances.
The color test is usually the first chemical examination examiners conduct after a package suspected of containing controlled substances is opened and weighed. Small amounts of the unknown material are placed in depressions in a porcelain spot plate or a disposable plastic or glass spot plate. Chemical reagents are then added to the depressions and the results noted: color changes, the way in which the color changes take place (flashing or bleeding), the rate at which the color changes take place, and the intensity of the final colors. The most common color reagents are the Marquis reagent for opium alkaloids, amphetamines, and phenethylamines such as MDA or MDMA; cobalt thyocyanate reagent for cocaine and phencyclidine (PCP); Dille-Koppanyi reagent for barbiturates; Duquenois reagent for marijuana; and Ehrlich's reagent for LSD. A more complete listing of these tests is available in the literature.1 Many of these tests are multi-step and multi-component.
These color tests are designed as a starting place for the examiner in deciding how to proceed as the pyramid of focus narrows in forming a conclusion. Adulterants and diluents can also cause color changes and are sometimes said to be responsible for "false positives." The resulting color changes are not really false. They simply reflect the presence of a substance which is not the primary focus of the analytical scheme. Problems of "false negatives" and "false positives" are usually recognized very early in the analytical scheme, and they are resolved logically and rationally.
Thin-layer chromatography (TLC) is a separation technique. The method utilizes a glass plate which is usually coated evenly with a thin layer adsorbant. The most commonly used adsorbant is silica gel. A small amount of the sample is put into solution with a chemical solvent. A capillary pipet is then used to place a small amount of the liquid onto the TLC plate approximately 2 cm from the bottom of the plate. A second capillary pipet containing a small amount of a known controlled substance in solution is used to place a second spot on the plate usually next to, but not overlapping, the first spot.
The plate is then placed into a tank containing a solvent system which rises about 1 cm from the bottom of the tank. Through capillary action, the solvent will migrate up the plate, and the components of the unknown will usually separate as the solvent migrates. The separated components can usually be visualized using longwave or shortwave ultraviolet light, a chemical spray, or some combination of both. The distance each sample migrates is then divided by the distance the solvent in the tank migrates up the plate (know as the Rf value). The result is then compared to published values that have been established for pure samples of the abused drugs. If one of the components of the unknown migrates the same distance up the plate as the known, the examiner has another piece of corroborating information. If the unknown does not contain a component that migrates the same distance as the known, there are many explanations. Perhaps the known and unknown are not the same. Perhaps there is a component in the unknown solution which is binding the chemical of interest to the silica gel. The explanations for matches are numerous. The explanations for non-matches are just as numerous.
The literature is replete with values for drug/solvent migration ratios. However, these values can be affected by many factors, including the storage conditions of the TLC plates and solvent temperature. It is not uncommon for the Rf values in the laboratory to differ from those in the literature. The importance of a TLC analysis lies in its ability to separate components in a mixture. A match is another piece of corroborating information. A non-match can usually be explained.
Using TLC to identify marijuana, hashish, or hash oil is a much more complicated process than using it to identify other controlled substances.2 The TLC analysis of cannabis exhibits results in a series of bands on the thin-layer plate. Depending on the solvent system, the number of bands can range from at least three to at least six bands.3 Each band will have a specific color and lie at a specified place on the plate corresponding to the known cannabinoids in a standard marijuana, hashish, or THC sample.4 The key point here is that this type of identification involves a specific chromatographic pattern as opposed to one spot where a known is compared directly with an unknown. Even with the increased specificity of a TLC analysis in the examination of cannabis or a cannabis derivative, a modified Duquenois-Levine test is suggested.
1.7.2 confirmatory chemical tests
Microcrystal tests are conducted using a polarized light microscope and chemical reagents. These microscopic examinations are not screening tests. The analyst will usually place a small amount of the sample on a microscope slide and add a chemical reagent and note the formation of a specific crystal formation. These crystals are formed from specified reagents. There should be very little subjectivity in evaluating a microcrystal test.5 Either the crystal forms or it does not form. If the appropriate crystal forms in the presence of the reagent, the drug is present. If the crystal does not form and the drug is present, the problem is usually one in which the drug concentration is too dilute, or the reagent has outlived its shelf life.
One disadvantage of microcrystal tests is the absence of a hard copy of what the analyst sees. Unless a photograph is taken of the crystal formation, the examiner cannot present for review documentation of what he saw under the microscope. Microcystal tests are an excellent way of evaluating the relative concentration of a drug in a sample to determine the kind of extraction technique for separation and further confirmation.
Gas chromatography (GC) has been a standard operating procedure in forensic science laboratories for the past three decades. In this technique, a gaseous mobile phase is passed through a column containing a liquid coated, stationary solid, support phase. The most common form of GC uses a capillary column of a very fine diameter for separating the component of a mixture. The sample is usually put into solution using an organic solvent such as methanol. The liquid is then introduced into the injection port of the gas chromatograph using a fine needle syringe capable of delivering microliter quantities of the solution. The amount injected depends on the concentration of the sample. One microliter (one-one hundreth of a milliliter) of 1 mg of solute per l ml of solvent is a typical injection amount. The sample is vaporized in the heated injection port, and with the aid of a carrier gas travels through the long capillary column where the different components are separated. There are many different kinds of capillary columns with different internal coatings, lengths (which can vary from one foot up to tens of meters), and diameters (measured in micrometers). This separation is determined by the polarity and molecular size of each component. Each component exits the column onto a detector. A flame ionization detector (FID) is the most common detector used in most laboratories. Other types of less frequently encountered detectors include the nitrogen phosphorous detector and the electron capture detector.
As each component elutes from the column through the FID, a signal is generated which results in a "peak" on a recording device. The recorder is used to document the resulting data. This recorder is usually a part of a data station that not only generates a representation of the chromatogram on a monitor, but also controls instrument parameters and ensures the consistancy of the analysis. The peaks of interest are evaluated by their retention times (RT's) and by the areas under the peaks. The retention time data can be used either as confirmation of the probable identity of the substance generating the peak, or the data can be evaluated as screening information to determine the possible presence of a controlled substance . This RT data is compared to the retention time of a known standard injected onto the same column in the same instrument at the same temperature and rate flow conditions. The RTs of the known and the unknown should be almost the same within a very narrow window. The area under the peak can be used to quantitatively determine the relative concentration of the substance.
There are some disadvantages of GC. Retention times are not absolute and usually fall within a narrow window. Other compounds may fall within this same RT window. One way to overcome this problem is to analyze the same sample using a second capillary column with a different internal coating and to note its retention time as compared to the known standard. The values should be the same within a narrow RT window. A second disadvantage of GC is that some samples degrade in the injection liner at high temperatures and must be evaluated by using a derivatizing agent. This derivatizing agent is added to the drug and forms a molecular complex. The molecule complex remains intact as it passes from the injection port, through the column, and onto the detector.
GC by itself is a very powerful tool for the forensic analyst. Its most useful application today remains one in which it is interfaced with a mass spectrometer (mass selective detector) which serves as a detector and separate instrumental identification method unto itself. Gas chroma-tography/mass spectrometry will be discussed later in this section.
188.8.131.52 High Performance Liquid Chromatography (HPLC)
This chromatographic technique is also a separation technique, but with a bit more selectivity than GC. In HPLC, the mobile phase is a liquid and the stationary phase is a solid support or a liquid-coated solid support. In GC, a carrier gas is used to carry the sample through the chromatography column. In HPLC, a high pressure pump is used to carry the solvent containing the compound of interest through the column. Separation results from selective interactions between the stationary phase and the liquid mobile phase.6 Unlike GC, the mobile phase plays a major role in the separation. HPLC can be used for the direct analysis of a wide spectrum of compounds and is not dependent on solute volatility or polarity. The operator need not worry about chemical changes in the molecule which can occur in GC due to thermal degradation.
HPLC chromatograms are evaluated based on retention time and area under the peak of interest. Retention time is not an absolute value, but a time within a narrowly defined window. The five basic parts of the liquid chromatograph include the solvent reservoir, the pump, the sample injection system, the column, and the detector. A recorder is used to document the resulting data. This recorder is usually a part of a data station which controls instrument parameters and ensures the consistancy of the analysis. The most common detectors are the ultraviolet/visible detector (UV/VIS), the florescence detector, the electrochemical detector, the refractive index detector, and the mass spectrometer. The UV/VIS detector is the most widely used device, and it is dependent on the solute's ability to absorb ultraviolet or visible light. The variable wavelength detector allows the analyst to select any wavelength in the ultraviolet or visible range. The diode array or rapid scan detector is also used which allows a rapid scan of the entire UV spectrum to identify the components eluting from the column.
Because the components elute from the UV detector in solution, they do not undergo degradation or destruction. This one very useful characteristic of HPLC affords the analyst the option of collecting fractions of the eluent for further analysis. This is not possible in GC because the eluent is destroyed by the FID.
184.108.40.206 Capillary Electrophoresis (CE)
Capillary electrophoresis is a technique that separates components on the basis of charge-tomass ratios under the influence of an electrical field. It uses high voltage for fast separations and high efficiencies. Osmotic flow is the main driving force in CE, especially at higher pH values, and results primarily from the interaction of positive ions in solution with the silanol groups on the capillary in the presence of an applied field. Narrow bore capillary columns of uncoated fused silica are used for heat dissipation during the separation process. The detector is normally an ultraviolet detector.
Micellar electrokinetic capillary chromatography (MECC) is a form of CE which allows for the separation of cations, neutral solutes, and anions.
CE has several advantages over HPLC and GC. The method can be used with ionic and neutral solutes which present problems in GC. There is a higher efficiency, resolving power, and speed of analysis compared to HPLC. From a cost perspective, CE requires much less solvent than HPLC, and the CE capillary column is much less expensive than the HPLC or GC capillary columns. Two disadvantages of CE are the limited sensitivity for UV detection (30 to 100 times less than that of HPLC); and fraction collection is troublesome because of mechnical problems and small sample size. This technique uses a micelle as a run buffer additive to give separations that are both electrophoretic and chromatographic.
One of the advantages of MECC is the ability to separate racemic mixtures of compounds into the d- and l-isomers. This is an ability that is extremely valuable when identifying compounds where one isomer is controlled (dextropropoxyphene) and the other isomer is not controlled (levopropoxyphene). This is usually accomplished by adding cyclodextrins to the run buffer.
220.127.116.11 Infrared Spectrophotometry (IR)
Infrared spectrophotometry is one of the most specific instrumental methods for the identification of a controlled substance. A pure drug as a thin film on a KBr salt plate, or as crystals mounted in a KBr matrix are placed into the sample compartment of the infrared spectropho-tometer. A source of electromagnetic radiation in the form of light from a Nernst glower passes light through the sample. The instrument, through a mechanical means, splits the beam into a reference beam and an incident beam. The reference beam passes unobstructed through a monochrometer to a photometer; the incident beam passes through the mounted sample through the same monochrometer to the photometer. The reference beam passes 100%
unobstructed to the photometer. The incident beam passing through the sample has some of its enegy absorbed by the sample. This energy is absorbed at differenct wave lengths across the infrared spectrum from 4000 cm-1 down to 250 cm-1. The amount of relative absorption and where on this spectrum the absorption takes place is dependent upon the molecular structure and, more specifically, the functional groups of the drug. Different functional groups and molecular interactions brought on by symmetrical and assymetrical molecular stretching vibrations and in-plane and out-of-plane bending vibrations result in a number of peaks and valleys on the IR chart. The resultant spectrum is usually formed on an x/y coordinate axes. The wavelength (|) or wave number (cm-1) where the absorption occurs is depicted on the x-axis, and a measure of the amount of light absorbed by the sample, but usually referenced by transmittance units from 0 to 100%, is depicted on the y-axis
The infrared spectrum of a suspected drug results in a specific pattern that can be used to positively determine the identity of the substance. For most controlled substances, the resulting spectrum consists of 20 to 70 peaks. These peaks form a pattern that is unique to the chemical structure of the drug. This pattern can then be compared with a reference IR spectrum of a primary drug standard. If the analyst determines that the two spectra match within the limits of scientific certainty, an identification is possible. It is rarely, if ever, possible to overlay the reference spectra with the spectra of the unknown and have a "perfect match". The analyst is looking for a match in the patterns. Any shifts in peak intensity or wave number must be evaluated in conjunction with the pattern. Small shifts of 1 or 2 cm-1 and minor intensity variations of individual peaks are expected. However, major variations must be evaluated on a case by case basis. Some authors refer to IR as a "fingerprint" identification method. This implies an ability to overlay two spectra and obtain a perfect match in every way. This degree of perfection is rarely, if ever, possible.
Another factor that must be considered is that when two spectra are being compared peak-by-peak as opposed to pattern-by-pattern, they ideally should be from the same instrument and collected at about the same time. Comparing a literature reference spectrum with an unknown for a pattern match is acceptable. Comparing the same literature reference spectrum wave number by wave number, absolute transmittance value by absolute transmittance value will probably result in minor differences.
IR does have limitations. In order to obtain an acceptable spectrum, the sample must be very clean and dry. For forensic exhibits, this usually means that most samples must go through extraction processes to remove impurities. In the past, sample size was a problem. However, because of advances in Fourier transform IR technology and the interfacing of an IR spectro-photometer with a microscope, evaluting microgram quantities of a sample results in excellent spectra which are conclusive for the identification of a controlled substance. IR has very definite limitations in its ability to quantitate controlled substances, and differentiating some isomers of controlled substances can pose problems.
18.104.22.168 Gas Chromatography/Mass Spectroscopy (GC/MS)
Gas chromatography/mass spectrometry is by far the most popular method of identifying controlled substances in the forensic science laboratory. In this method, a gas chromatograph is interfaced with a mass selective detector (MSD). The sample undergoing an examination is placed into solution with a solvent such as methanol. A very small injection volume of 1 or 2 |l is injected into the GC injection port. It then travels through the column where the different components of the sample are separated. The separated components can then be directed into the ionization chamber of MSD where they are bombarded by an electron beam. In electron impact gas chromatography/mass spectrometry (EI MS), high energy electrons impact the separated component molecules. The resulting spectrum of each component is typically complex with a large number of mass fragments. These fragments are represented as peaks of varying intensity that provide the basis for comparison with a primary reference standard. The components are then ionized and positively charged. This ionization also results in a fission, or fragmentation process. The molecular fragments traverse into a magnetic field where they are separated according to their masses. In this magnetic field, larger mass fragments are less affected by the magnetic field, and smaller fragments are more affected and undergo a deflection. Upon exiting the magnetic field, these fragments impact a detector losing the charge generated by the beam of electrons impacting the sample. The result of this fragmentation process is a pattern unique for the substance that is being analyzed.
The resulting mass spectrum consists of an x/y coordinate axis. The numerical value on the x-axis represents the mass number determined by the number of neutrons and protons in the nucleus. It is usually the molecular weight of a specific fragment. The largest magnitude peak on the x-axis will often be the molecular ion and will represent the molecular weight of the unfragmented compound. There will usually be a very small peak to the right of the molecular ion which represents the molecular weight plus 1. The y-axis represents the relative abundance of each peak comprising the mass spectrum. The tallest peak on the y-axis is the base peak and represents that part of the molecule which is the most stable and undergoes the least amount of fragmentation. The base peak is assigned a relative abundance value of 100. The other peaks in the resulting spectrum are assigned relative values along the y-axis.
The numerical values on the x- and y-axis are calculated and assigned by the data station which is interfaced with the mass spectrometer. The accuracy of these numbers is predicated on the fact that the instrument has been properly tuned. This tuning process can be compared to checking the channel tuning on a television set. This might be accomplished by opening a television guide to determine what programs are scheduled at a particular hour. The television is then turned on and the program for each channel checked. If the programs cited in the televison magazine appear on corresponding channels at the proper times, the television has been proven to be properly tuned. The tuning of a mass analyzer presents an analagous situation.
The tuning process of a mass analyzer involves a procedure in which a chemical of a known molecular weight and fragmentation pattern is analyzed and the resulting data evaluated. This process includes verifying instrument parameters and the resulting spectrum. If the response of the tuning process falls within specified limits, the mass spectrometer is deemed operationally reliable, and the resulting data can be considered reliable. One such chemical used to tune mass spectrometers is perflurotributylamine (PFTBA).
Fragmentation patterns of controlled substances are typically unique. Once a fragmentation pattern has been obtained, the forensic analyst should be able to explain the major peaks of the spectrum and relate them to the molecular structure. If properly evaluated, mass spectral data can usually be used to form a conclusion as to the identity of a controlled substance.
GC/MS has many advantages in the analysis of controlled substances. The sample being analyzed need not be pure. Multi-component samples are separated and each soluble organic component can be individually identified. The analyst must be aware of isomeric compounds that have very similar chemical structures and similar fragmentation patterns. These kinds of situations can usually be handled by noting the GC retention time data to discriminate between similar compounds. Possible coelution of compounds from the capillary GC column and thermal degradation as noted in the gas chromatography section of this chapter should also be recognized. GC/MS does not allow the forensic analyst to directly identify the salt form of the drug. This task can be accomplished by considering the solubility properties of the drug being analyzed. In using this knowledge and performing extractions prior to injection onto the GC column, the salt form can be determined indirectly.
When all methods of instrumental analysis of controlled substances are considered, GC/ MS is recognized in most instances as one of the efficient analytical techniques. If the analyst is cognizant of maintaining instrument reliability standards and the guidelines of mass spectral interpretation, GC/MS affords one of the highest degrees of specificity in the identification of controlled substances.
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