Drug Testing Technologies and Applications

Jane S-C. Tsai and Grace L. Lin

Summary

Over the past few decades, a remarkable gamut of increasingly sophisticated technologies has been employed for the development of drug-testing applications. Recent advancements in analytical instrumentation and computer technologies have further expanded the capabilities and dimensions for drug testing and toxicological analysis. Technologies of different chemical principles can be used sequentially or in combination to accomplish the specific goals and requirements of the drug analysis programs. Ligand-binding assays such as immunoassays are commonly used for screening. Separation techniques such as chromatography or electrophoresis, as well as their coupling with powerful detectors such as mass spectrometry, can be effectively used for confirmatory testing of preliminary positive results or systematic analysis of generally unknown toxic compounds. Each of these technology categories can be further broken down into multiple selections for instrumentations and methodologies. This chapter presents a general overview of the commonly used analytical technologies and their utilities in drug testing. The analytical technologies afford a powerful means toward the detection, identification, and quantification of the presence of abused drugs in biological specimens. However, the overall interpretation of analytical results ought to take into consideration the reasons for testing and the performance characteristics of the applied technologies.

1. Introduction

"Drugs-of-abuse testing" is a simple term but comprises diverse fields of drug testing, with manifold medico-legal and socioeconomic implications.

From: Forensic Science and Medicine: Drugs of Abuse: Body Fluid Testing Edited by R. C. Wong and H. Y. Tse © Humana Press Inc., Totowa, NJ

Although the objectives of drug testing are mainly the detection, identification, and/or deterrence of substance abuse or misuse, the processes and regulations related to this topic may vary among different drug-testing sectors. The scopes of different drug-testing applications can vary, depending on the types of specimen tested, the regulations for testing procedures and programs, the drug-use prevalence in the population tested, the desired menu of drugs for analysis, the choice of testing technologies, and the interpretation and reporting of testing results. Depending on the goals and requirements of the drug-testing programs, technologies of different chemical principles can be employed sequentially or in combination to accomplish the detection, identification, and quantification of the drugs present in a biological specimen.

In general, the choice of analytical drug-testing technologies can be grouped into three major categories of general analytical techniques. Each of the three categories listed below can be further broken down into multiple selections for instrumentations and methodologies.

1. Assays that are based on molecular recognition and ligand binding, with immunoassays being the most popular techniques for drugs-of-abuse screening. Depending on the circumstances and requirements of testing, both instrumented immunoassays for laboratory testing and noninstrumented immunoassays for point-of-collection testing (POCT) are widely employed as initial tests for drugs of abuse (1-17).

2. Separation methodologies, such as various chromatographic or electrophoresis techniques, which physically separate the analyte(s) of interests from the other sample components. Examples include gas chromatography (GC), high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), capillary electro-phoresis (CE), and capillary electrochromatography. A variety of detection methods are available for each of the separation techniques (18-34).

3. Mass spectrometry (MS), which identifies, quantifies, and/or elucidates the structure of substances in diverse types of sample matrix. The mass spectrometer is one of the most powerful detectors for various separation techniques, and is used for the majority of the "hyphenated" or "coupled" techniques. For example, GC-MS has become an integral part of forensic toxicology and abused drug confirmation. Notable progress has been made in the development of the interface for LC-MS and CE-MS in recent years. Comprehensive two-dimensional gas chromatography (GC x GC) with MS has recently been applied for drug screening and confirmation. Moreover, the advances in tandem MS (MS-MS) or multi-stage MS provide additional dimensions in the analysis of minute concentrations of compounds in various biological matrices (35-64).

To date, the most common drug-testing practices are based on a two-tier approach of initial immunoassay screening followed by confirmatory testing of preliminary positive screen results. Various separation methodologies or GC-MS in scan mode have also been employed as initial drug-screening tools. The hyphenated techniques are effective tools for confirmatory testing of presumptive positive results or for systematic analysis of generally unknown compounds. In the field of behavioral toxicology, the initial steps of drug recognition examination involve a series of physiological evaluations, including psychomotor tests and the examination of the eye (65-67). Nevertheless, the evaluation often culminates in the collection of biological specimens for chemical analysis.

This chapter presents a general overview of drugs-of-abuse testing and analytical drug-testing technologies. The applications and general considerations of drugs-of-abuse testing are discussed first, because the analytical results are often interpreted in the context of the drug-testing applications. Then the principles of each of the technologies are compendiously reviewed, and the more commonly used drug-testing techniques are discussed. In addition to the abundance of methodology choices, there are myriad existing literature sources on all aspects of the technologies. Therefore, the aim of this chapter is to provide a synopsis of the technologies and their utilities in drugs-of-abuse testing.

2. The Essence of Drugs-of-Abuse Testing

To understand the principles underlying the array of drug-testing technologies, some basic knowledge is required in terms of the drug metabolism and the intent of the particular drug-testing program. The route and method of drug administration, drug dose, and drug metabolism are closely related to the presence and amounts of the drug and/or its metabolites in the testing specimen at the time of specimen collection.

Over the past several decades, significant efforts have been devoted to the research of drug pharmacokinetics (i.e., the process by which a drug is absorbed, distributed, metabolized, and eliminated by the body), pharmacodynamics (i.e., the mechanism of drug action and effect on the body), as well as toxicological and epidemiological investigations. Advances have also been made in understanding the intra-individual and inter-individual variabilities of drug metabolism and excretion.

It should always be noted that the actual testing of abused drugs in any given biological specimen is only one component of a specific drug-testing program or application. There are legal, legislative, social, forensic, and/or medical elements of drug testing. Modern analytical technologies allow the pursuit of detecting and measuring the identified analyte(s) in a biological specimen with proper accuracy and precision. The interpretation and judgment of analytical measurement results, however, is not a straightforward process. There is bountiful active research in these fields, and reports from a wide spectrum of investigations will continue to be published. Nevertheless, the collective knowledge from all aspects of related scientific studies has provided the essential foundations for the design, execution, quality assurance, and outcome interpretation of chemical drug analysis.

2.1. The Applications of Drugs-of-Abuse Testing

The applications of drugs-of-abuse testing touch all walks of life in society. The highest volume of drugs-of-abuse testing comes from workplace drug testing (WDT). In the United States, programs for federal workplace drug testing are regulated by the Substance Abuse and Mental Health Services Administration (SAMHSA) of the US Department of Health and Human Services (HHS). In general, drug-testing programs regulated by the US Mandatory Guidelines (68) have established systems of protocols applicable to the testing of large numbers of urine specimens for the presence of five classes of drugs. These drugs (and their respective target analytes for confirmation) are as follows: amphetamines (amphetamine and methamphetamine), cannabinoids (11-nor-A9-tetrahydrocannabinol-9-carboxylic acid, abbreviated as THC-COOH), cocaine metabolites (benzoylecgonine), opiates (morphine and codeine), and phencyclidine (PCP). The proposed new guidelines (69) will allow federal drug-testing programs the option to incorporate the testing of these drug classes in alternative specimens (oral fluid, sweat, or hair). Furthermore, the confirmation techniques will be expanded to couple the use of GC or LC with MS or MS-MS.

Additional classes of abused or misused drugs have also been tested in various societal sectors or clinical drug-testing programs worldwide (70-102). The main examples include benzodiazepines, barbiturates, lysergic acid diethylamide (LSD) and/or metabolite (2-oxo-3-hydroxy-LSD), methadone and/or metabolite (2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine, EDDP), metha-qualone, propoxyphene, tricyclic antidepressants (TCA), and so on. The testing of the amphetamines group has recently been expanded to include designer drugs, especially 3,4-methylenedioxymethamphetamine (MDMA), as specific assay target analytes. Several studies were conducted for the detection of certain benzodiazepines, especially flunitrazepam. There is also growing interest in testing additional target classes of opioids and certain synthetic analgesics, such as buprenorphine and metabolites, 6-monoacetylmorphine (6-MAM or 6-AM), oxycodone, fentanyl, and so on. An expanded menu and more flexible drug-testing options are often desirable for clinical and medicolegal assessment, including clinical forensic medicine and emergency department drug testing. Different classes of drugs of abuse may provoke various signs of mental disorder, and testing for the presence of suspected drugs can help psychiatric clinics to differentiate between endogenous and drug-induced mental illness. For drug-dependence treatment or detoxification centers, the "objective evidence" of compliance monitoring is via regular drug testing of individuals with prescribed medication. Periodic drug testing is also used to ensure continued abstinence during rehabilitation. Similarly, scheduled testing can be used to check on previous drug offenders on parole and probation. Drugs-of-abuse testing is also frequently employed in the criminal justice system to identify drug offenders and to deter the abuse of drugs.

Drugs-of-abuse testing is mostly considered "forensic testing" because of its legal consequences and the high probability of legal challenges. Urine specimens are sufficient for most drug-testing applications; however, the analysis of the urine alone for drugs is insufficient for various forensic toxicology investigations. Interest in drug testing in alternative specimens, such as oral fluid/ saliva, sweat, hair, as well as meconium for neonate exposure, has markedly increased over the past few years (103-117) (see Chapters 8, 10, and 11). In human-performance toxicological evaluations, blood is generally considered a suitable specimen for impairment investigation (50,51). Nevertheless, interest in effective and timely drug testing for traffic safety has led to a number of projects that have evaluated the utility of various onsite urine- and saliva-testing devices for roadside drug testing (17,118-122) (see Chapter 17). Antemortem or postmortem forensic toxicology further requires the development of an assortment of tests for comprehensive screening of drugs in a variety of specimen types. A number of analytical techniques have been developed and evaluated for the systematic toxicological analysis (STA) of "generally unknown" toxic compounds (18,20-22,46,55-60). The availability of reliable methods for the STA of drugs and poisons is important for laboratories involved in clinical and forensic toxicology investigations. In general, two or more assays are required for laboratories wishing to cover a reasonably comprehensive range of drugs of toxicological significance.

2.2. General Considerations and Guidelines

Regardless of their applications in identifying or excluding the abuse of drugs, most testing technologies need a defined or declared threshold concentration in order to distinguish a positive from a negative result. Conventionally, the threshold decision can be made based on either the assay limit of detection or a pre-defined, higher concentration that takes into account special requirements for the analysis. The limit of detection and limit of quantification usually are important for forensic purposes. For most drugs-of-abuse testing programs, however, "administrative cutoffs" have been chosen that are sufficiently above the assay limit of detection but still low enough to allow the detection of drug use within a reasonable time frame.

The SAMHSA regulation authorizes rapid initial testing within a framework of extensive quality control and specifies defined rules if confirmation is required. This allows the administrative cutoffs to be used for comparison across different assay technologies. Likewise, a number of countries, organizations, and professional societies have developed guidelines and recommended cutoff levels for drugs-of-abuse testing. The European guidelines for WDT (123) allow individual countries to operate within the requirements of national customs and legislation. For most regulated testing, a sample containing drugs below a specified cutoff concentration, or no drug at all, is reported as negative on the initial test and usually not further tested. A positive screen result is considered "presumptive positive"; thus, adequate confirmation is required in the majority of forensic testing, mandated workplace drug testing, and recommended in clinical testing.

Prior to the use of an analytical technique for the purpose of drug testing, a battery of analytical performance specifications and characteristics must be established, validated, and verified. The basic performance evaluation criteria include precision (i.e., intra-run, inter-run, and total precision), analytical sensitivity or lower limit of detection, accuracy by comparison studies to "gold" standards, method comparison to predicate systems/reference devices, and specificity (cross-reactivity profile). The performance evaluations for most immunoassays also include near cut-off performance, accuracy by analytical recovery studies, robustness against potential interference and adulteration substances, and a variety of stability studies. Immunoassays for abused-drug testing can be performed in either qualitative or semi-quantitative mode but are not considered quantitative. For technologies used for quantitative analysis, linearity, selectivity, resolution, and/or limit of quantification are also assessed. In addition, the implementation of proper quality-assurance programs is critical for testing programs or laboratories to produce accurate, reliable, and defensible results.

For most diagnostic testing, the generally accepted definition of "true negative" is a negative test result for a disease or condition in a subject in whom the disease or condition is absent. Likewise, "true positive" (TP) is a positive test result when the disease or condition is present in a subject. In comparison, the criteria for a "true-negative" (TN) drugs-of-abuse testing result also include the presence of drug concentration below that of both the screen and confirmation cutoff concentrations. Therefore, the presence of a drug concentration below the screen cutoff yet above the confirmation cutoff concentration is considered a "false-negative" (FN) result. By the same token, the presence of drug concentration above the screen cutoff yet below the confirmation cutoff is considered a "false-positive" (FP) result. These are important considerations for result interpretation and analysis of comparative evaluations of drugs-of-abuse testing.

A number of factors can impact the evaluations of sensitivity (TP/[TP + FN] x 100%), specificity (TN/[TN + FP] x 100%), and efficiency ([TP + TN]/

[TP + TN + FP + FN] x 100%) of a drug immunoassay, because such calculations are related to the comparison of the results of the screening techniques to those of the confirmation techniques. The prevalence of drug use in the studied population will also affect the calculated sensitivity and specificity. Because measurement uncertainties and inter-assay variations exist for all analytical methodologies, the comparison of screening and confirmation results for near-cutoff samples can vary from one study to another. In addition, it has been well published that immunoassay sensitivity, specificity, and predictive values, and hence the drug detection rate and detection time, can all be affected by the manipulation of the cutoff concentrations used for drug testing (124-131).

3. Molecular-Recognition- and Ligand-Binding-Based Assays

Assay technologies that rely on specific molecular recognition of analytes by high-affinity binding partners have played pivotal roles in diverse areas of biomedical and chemical analyses. The most versatile binding partners are the antibody molecules that are widely utilized in immunoassays for the detection of the analyte of interest (i.e., antigen) in a variety of sample matrices. Other binding partners used for drug testing include the specific drug receptor for various formats of receptor assay (132-135). Depending on the type of specimen used, the specific molecular binding allows for the direct detection of the target analyte(s) in the specimen with minimal or no sample pretreatment. In general, urine samples do not require pretreatment, although enzymatic hydrolysis with glucuronidase can be applied to enhance the detection of certain drug classes (4). Immunochemical assays can be developed to possess adequate sensitivity and specificity for the target class of drugs or drug metabolites. Once the reagents are developed and optimized, immunoassays are simple to use and allow relatively fast screening of a large number of samples. The availability of various commercial kits and instruments further facilitates the versatility of immunoassays in meeting the specific needs of drug-testing fields.

3.1. Immunoassays

Immunoassays for drug screening have been designed to efficiently detect the presence of the target class or classes of drugs above the defined threshold in a biological specimen. Consequently, immunoassays as a screening technique remain the most cost-effective way to rule out drug presence in the majority of samples submitted for routine drugs-of-abuse testing. The discriminatory power of the antibody binding site bestows the assay specificity; however, all ligand-binding-based assays can exhibit cross-reactivity with congeners, or sometimes with surprisingly unrelated structures (136,137). Hence, a screened positive result is considered preliminary or presumptive positive.

The majority of immunoassays for drug screening are based on the competition of free drug molecules in the specimen and drug derivatives in the assay reagents for binding to a pre-optimized amount of antibody molecules in the reagent kit. A label is attached to one of the binding partners to serve as an indicator for monitoring and reporting the outcome of the competitive immuno-reaction. The labels possess a measurable property that confers the analytical characteristics to meet the performance requirements of the specific assay. In practice, the apparent drug concentration in a specimen is determined by comparing the amount of this measured property in a sample to that of reference standards containing known concentrations of the target analyte.

Generally, there are two configurations of competitive immunoassays. A "heterogeneous" immunoassay requires the physical separation of free, labeled binding partner (antigen or antibody) from the labels that are bound in an immune complex in order to measure the quantity of labels. A "homogeneous" immunoassay can detect the analyte-induced signal change of the label characteristics without any separation steps. Both types of immunoassays are important for drugs-of-abuse screening. There are a variety of immunoassay techniques that are applicable to drug testing. The nomenclature of these techniques is based on the type of specific assay label used and the reaction principle of each of these immunoassays. Table 1 provides a summary of the technologies, assay labels, and reaction principles of these immunoassays.

3.1.1. Heterogeneous Competitive Immunoassay Techniques

Radioimmunoassay (RIA) (1-3,5,7,138) is a technique of saturation analysis that consists of three major components: a label for antigen (with 3H, 131I, or 125I), a saturable compartment (specific antibody), and a separation step. The radiolabeled drug derivative binds to the antibody and forms a complex that is subsequently separated from the unbound labels. Solid phases that allow the removal of the unbound label in the supernatant (e.g., coated-tube technique) and second antibody binding to precipitate the 125I-drug-antibody complex (e.g., double-antibody approach) are the most frequently used separation methods for drug RIA. After washing, the radioactivity of the labeled-drug-antibody complex can be measured in counts per minute (CPM). A dose-response curve can be constructed using the calculated radioactivity value vs analyte concentration for each of the calibrators. A standard curve can also be plotted using logit B/B0 (B/B0 = CPM of the test sample/CPM of the zero control) vs the natural logarithm (i.e., loge) of the drug concentrations. The concentration of drug in the sample is inversely proportional to the calculated radioactivity value.

Enzyme-linked immunosorbent assays (ELISAs) (6,11,12,14,139) are by far the most versatile techniques for diverse fields of biochemical, toxicological,

Nomenclature of immunoassay technology (abbreviation)

Table 1

Commonly Used Competitive Immunoassays for Instrumented Drugs-of-Abuse Screening

Enzyme-multiplied immunoassay technique (EMIT)

Fluorescence polarization immunoassay (FPIA)

Kinetic interaction of microparticles in solution (KIMS)

Cloned enzyme donor immunoassay (CEDIA)

Radioimmunoassay (RIA)

Enzyme-linked immunosorbent assay (ELISA)

Assay labels and reaction indicator

Major reagent composition (excluding the bulk agents, stabilizer, and preservative, etc.)

G6P-DH enzyme oxidizes G-6-P and reduces NAD.

The generation of NADH is measured by absorbance rate change at 340 nm.

2. Drug-G6P-DH conjugate

Excited Fluorophore (fluorescein) emits light at a second wavelength (fluorescence).

A filter mechanism is used to determine the polarization of the emitted light (mP)

1. Antibody

2. Pretreatment solution

3. Drug-fluorescein tracer

4. Wash solution

Microp article-labeled reagent reacts with the binding-partner and promotes the aggregation reaction

The proceeding of the reaction results in the kinetic absorbance increase with time.

Gen I KIMS

1. Antibody, diluent

2. Drug microparticles Gen II KIMS

1. Drug-polymer

2. Antibody-microparticles

The association of the ED and EA fragments forms active enzyme ¡3-Galactosidase that hydrolyzes CPRG.

The generation of CPR is measured by the absorbance rate change at 570 nm.

1. EA reagent + EA reconstitute buffer and antibody

2. Drug-ED conjugate + ED reconstitute buffer, and CPRG

125I-drug binds with antibody to form a radio-labeled drug-antibody complex.

The radioactivity of the 1251-drug-antibody complex can be measured (CPM)

Coated tube RIA

1. Antibody-coated tube

2. 1251-drug reagent

Second Ab RIA

1. Antibody reagent

2. 1251-drug reagent

3. Second antibody with polyethylene glycol

Enzyme (HRP)-drug conjugate binds to immobilized antibody.

The HRP conversion of TMB to a colored product is measured by absorbance at 450 nm

1. Antibody-coated wells in microtiter plates or strips

2. Drug-labeled enzyme reagent

3. Substrate reagent

4. Stop solution

NAD, nicotinamide adenine dinucleotide; ED, enzyme donor; EA, enzyme acceptor; CPRG, chlorophenol red b-d-galactopyranoside; HRP, horseradish peroxidase; OD, optical density; CPM, counts per minute; TMB, tetramethylbenzidine.

and medical analysis. Customized ELISA can be developed for drug testing in forensic and clinical toxicology laboratories. Approximately a dozen commercial ELISA kits are available for testing a spectrum of forensic matrices, such as urine, blood, serum, oral fluid, sweat, meconium, bile, vitreous humor, and tissue extracts. Competitive ELISAs for drug testing rely on competition between enzyme-labeled drug derivatives and free drug in the sample for binding to solid-phase (micro-well strips or plates) immobilized capture antibody. The competition of free drug for binding to the surface-coated antibody inhibits the binding of drug-enzyme conjugate and results in reduced enzymatic activity. An optical density (OD450) value or color intensity can be used to qualitatively interpret a negative or a presumptive positive result. From the calibration curve, drug concentration is inversely related to the amount of signal generated.

3.1.2. Homogeneous Competitive Immunoassay Techniques

The homogeneous immunoassays are relatively easier to perform and can be readily adapted to screening large numbers of samples using automatic analyzers. The progress of immunoassay reagent development has been complemented by the advancement of sophisticated laboratory-automation instruments and data-management systems. After sample loading, an analyzer can screen a large number of samples per hour with minimal laboratory personnel intervention. However, application parameters have to be developed for specifically optimized reagent-instrument interfaces. Some of the reagents are used with applications validated by the laboratories (i.e., user-defined tests). Examples of applications development include the scheme and mode of pipetting, the sample and reagent volume ratio, the reaction modes and kinetics, the reading window and choice of measuring points, the calibration models and curve assessment, and the result determination and report.

Enzyme-multiplied immunoassay technique (EMIT) (1-4,7,16,140-143, 147) is based on the modulation of enzymatic activities by the binding of antibody to the enzyme-labeled drug derivative. Among the enzymes investigated, the most popular choice is glucose-6-phosphate dehydrogenase (G6PDH), which oxidizes glucose-6-phosphate to form glucuronolactone-6-phosphate. The reaction is coupled with the reduction of the cofactor nicotinamide adenine dinucleotide (NAD) to NADH, which can be monitored spectrophotometrically with absorbance at a maximum wavelength of 340 nm. In the presence of free drugs in the specimen, the competition for antibody binding results in a higher amount of free enzyme. Thus the enzyme is less inhibited when the concentration of free drugs is increased.

Cloned enzyme donor immunoassay (CEDIA) (3,16,144-147) is based on the complementation of two inactive polypeptide fragments to form an active enzyme. The enzyme acceptor (EA) and the enzyme donor (ED) can spontaneously associate in solution to form the active enzyme, i.e., recombinant microbial P-galactosidase. The catalytic activity of the enzyme on the substrate chlorophenol red P-D-galactopyranoside (CPRG) can be monitored spectro-photometrically with absorbance at the maximum wavelength (approx 570 nm). The absorbance rate change is measured as a function of time (mA/min). The antibody binding to the drug derivative-ED conjugate in the reaction cuvet prevents the formation of an active enzyme. The presence of drug in the specimen competes for antibody binding and hence allows the free drug-ED conj ugate to reassociate with the EA. Therefore, the concentration of drugs in the sample is proportional to the enzymatic activity detected.

Fluorescence polarization immunoassay (FPIA) (2-4,16,143,148) is a technique that utilizes the properties of fluorescent molecules for biomedical analysis. Excitation of fluorophores in solution leads to selective absorption of light by appropriately oriented molecules. Polarized emission of these molecules occurs when their rate of rotation is low relative to the rate of fluorescent emission. Fluorescein-labeled drug derivatives (i.e., tracer for the immunoassay) rotate rapidly before light emission occurs, resulting in depolarization of the emitted light. When the tracer is bound to a macromolecule, the rotation is slowed and the fluorescence remains polarized. FPIA utilizes a known amount of tracer that competes with the free drug in the specimen for antibody binding. Increasing the concentration of drug in the specimen that binds to antibody leads to a greater amount of unbound tracer, which contributes to depolarization of the emitted light. Hence, the drug concentration is inversely related to the degree of polarization, which is measured in milliPolarization (mP) units.

There are two formats of kinetic interaction of microparticles in solution (KIMS) (1-4,7,16,149-152) techniques for drug immunoassays. Generation I KIMS (Abuscreen OnLine) is based on the competition between microparticle-labeled drug derivative and the free drugs in the specimen for antibody binding in solution. The binding of antibody and microparticle-bound drug conjugates leads to the formation of particle aggregates that scatter transmitted light. Generation II KIMS (ONLINE II) contain polymer-conjugated drug derivatives and microparticle-labeled antibodies. The interaction of the soluble conjugates and antibody on the microparticles promotes particle lattice formation. As the aggregation reaction proceeds, there is a kinetic increase in absorbance values. Any drug in the sample competes for antibody binding and inhibits particle aggregation; thus, drug concentration is inversely related to the absorbance change.

3.1.3. Competitive Immunoassay Techniques With Combined Labels

Enzyme immunoassay can be combined with other labels for drug analysis in either heterogeneous or homogeneous format (153-155). For example, substrate-labeled fluorescence immunoassay combines the use of the enzyme P-galactosidase and a fluorogenic substrate. Microparticle enzyme immunoassay combines the use of antibody-coated microparticles and alkaline phosphatase enzyme-labeled drugs. The enzymes can hydrolyze a fluorogenic substrate, and the rate of fluorescence generation can be measured with a fluorometer. Enzyme-enhanced chemiluminescence immunoassay (IMMULITE) utilizes alkaline phosphatase (ALP) as the enzyme label and 1,2-dioxetane as the chemiluminescent substrate. The substrate is destabilized by ALP, leading to an unstable dioxetane intermediate that can emit light upon decay back to the ground state. The IMMULITE instrument employs a proprietary tube that contains polystyrene beads as the solid phase to capture antibody. The tube allows for the separation of reaction components through high-speed spin about the longitudinal axis for decanting and washing. When the chemiluminescent substrate is added to the tube, the light emission is read with a photon counter, and this reading is then converted to analyte concentration by an external computer.

Immunoassays can also be combined with various flow-injection or chromatographic techniques to develop a flow immunosensor assay or a multi-analyte capillary electrophoretic immunoassay (156). More recently, a number of immunosensor-based or biochip-based (157-159) competitive immunoassays have been designed or investigated for their applications in drug testing.

3.1.4. Point-of-Collection Drugs-of-Abuse Testing

The immediacy of a drugs-of-abuse test result, especially the result that rules out drug presence at the point of collection (POCT or "on-site"), is desirable for certain drug-testing programs (7-10). The availability of POCT can benefit programs that require a faster personnel decision-making process, an immediate safety or compliance assessment, or an aid in clinical management. The implementation of POCT has included three areas:

1. The performance of instrument-based immunoassays at on-site initial screening-

only testing facilities;

2. On-site sample processing for further laboratory analysis;

3. The utilization of single-use, disposable POCT devices.

Early versions of on-site drug testing were mostly based on the micropar-ticle agglutination-inhibition methods in the 1970s. The techniques had been further developed for single-unit, visually read, homogeneous immunoassay devices such as Abuscreen OnTrak (160).

The use of paper chromatography (PC) for drugs-of-abuse screening was explored in the 1980s, but the initial products suffered from a number of performance issues. Enzyme was the initial choice for membrane-based assays but was later replaced by colored microparticles, which allow the visualization of results without the need of additional substrate reagents. Similarly to the evolution of other immunoassay technologies that were first developed for larger molecules or polypeptides, the application of these technologies to competitive assays for small molecules typically required further development. The visually read, lateral-flow immunochromatography that was first available for sandwich immunoassays in the late 1980s was further developed for drug testing in the 1990s and has since become the most popular technique for drug POCT (161-167).

The two basic formats of lateral-flow test strip for drug testing include the colloidal gold-based test strip configuration and a colored latex-enhanced immunochromatography. Both formats depend on the competition of free drugs in the sample with the immobilized drug-derivative conjugate on the result zone for binding to antibody on the colored particles. In the absence of drugs, antibody binds to the immobilized drug derivative, and hence a colored band is visible in the result zone. The presence of free drugs inhibits such binding; thus, no color is visible on the strip. The techniques were developed for urinalysis and more recently have also been used in the production of alternative specimen POCT.

The ascend multimmunoassay technique (such as Biosite Triage) (7,10, 168) depends on competitive binding of drugs in the sample with colloidal gold-labeled drug derivatives for antibody binding sites. After a 10-min incubation, the mixture is transferred to a strip of membrane onto which several specific antibodies are immobilized in discrete lines. In the absence of drugs, all the colloidal gold-labeled drugs are bound by their specific antibodies and cannot bind to the immobilized antibodies. Therefore, no color band is formed. The presence of drugs in the sample reduces the amount of antibody binding to the gold-labeled drugs, and hence the free gold-labeled drugs can bind to the membrane-immobilized antibodies and form a visible band.

Table 2 shows a summary of POCT techniques used for drugs-of-abuse testing. All of the ready-to-use devices have been pre-calibrated during manufacturing, and therefore on-board calibration or multilevel cutoff flexibility does not apply. There have been concerns about near-cutoff result reading; however, these assays in routine use are generally considered comparable to the performance with conventional immunoassays in most drug-screening settings that demand a rapid turnaround. In recent years, there has been a remarkable proliferation in the varieties of onsite drug-testing products as well as a significantly increased number of distributors for such tests. Because no specialized high-cost analyzers are required, the market for POCT has a lower entry barrier and is highly dynamic.

3.2. Receptor Assays

Many drugs exert their action through an interaction with one or more receptor types or subtypes in vivo. Theoretically, a receptor assay (RA) permits

Examples of Competitive Immunoassays for Point-of-Collection Drugs-of-Abuse Screening

Nomenclature of

Latex

Gold-labeled

immunoassay

agglutination-

Membrane enzyme

Ascend

Colored latex-based

Gold sol-based

optically read rapid

technology

inhibition

immunoassay

multimmunoassay

lateral flow

lateral flow

immunoassay

(Abbreviation)

(LAI)

(EIA)

(AMIA)

immunochromatography

immunochromatography

(GLORIA)

Assay labels

Latex microparticles

Enzyme-HRP

Gold sol nanoparticles

Colored latex micro/nanop articles

Gold sol nanop articles

Gold sol nanop articles

Reagents and

Three dropper bottles

Enzyme-drug

Three lyophilized

Antibody-latex dried

Antibody-Gold sol

Antibody on Gold sol

solid phase

Drug conjugate on

conjugate

pellets:

on latex pad

dried on gold

Antibody in membrane

microparticles;

Positive and negative

Drug conjugates on

Drug conjugate

conjugate pad

Drug conjugate in pad

antibody reagent;

control solutions,

Gold sol; antibodies;

immobilized in

Drug conjugate

buffer [Slide with

antibody immobilized

buffer

membrane strip

immobilized in

capillary tracks]

in the reaction site of membrane

Wash solution bottle [antibody immobilized in membrane strip]

membrane strip

Assay

1. Pipette sample to

1. Pipette sample to the

1. Pipette sample to the

Introduce sample to

Introduce sample to

Dip the test strip into

procedure

the mixing well on

wells on assay card.

Reaction cup

sample-receiving

sample-receiving

the sample and then

assay slide.

2. Apply positive and

2. Incubate for 10

pad (method is

pad (method is

drain for 3-5 s

2. Add one drop

negative controls to

min

device-dependent)

device- dep endent)

each of the

respective wells.

3. Transfer the reaction

antibody reagent,

3. Add one drop of

mixture from the

reaction buffer,

enzyme to all wells.

cup to the detection

and latex reagent.

4. Wash

area

3. Stir and start the

5. Apply substrate to

4. Allow the mixture to

run.

all wells.

soak through 5. Wash, allow soaking through

Result reading

Read results when

Read results in

Read results within

Read results when

Read results when

Read results when test

time

complete (about

3 min

5 min

test is valid (about

test is valid (mostly

is complete (about

4 min)

of completion

3-5 min)

5-10 min)

2 min)

HRP, horseradish peroxidase.

HRP, horseradish peroxidase.

the simultaneous measurement of the molecules that bind to the receptor, providing a total estimate of all pharmacologically active forms of the drugs (i.e., parent drug and active metabolites). RAs have also been proposed as a tool for systematic toxicological analysis because they can be applied toward the detection of an entire pharmacological class of drugs (132).

The RA technique makes use of the property of the analyte to competitively replace a labeled ligand from the same receptor binding site. The amount of labeled ligand replaced is a measure of the amount and the affinity of the analyte. Even though RAs do not exploit the physicochemical properties of the analyte, the result may offer information regarding the biological or pharmacological activity of the analyte by distinguishing the compounds on the basis of their specific binding reactions rather than specific molecular structure recognition. It should be noted, however, that drug binding to the cell receptor may have agonist or antagonist properties, so the activity can be either positive or negative for similar concentrations of related drugs. RA techniques such as the radio-receptor assay (RRA) have been used in various investigations of benzo-diazepines (132,133). In general, results from RRA have been reported to be equal to or better than immunoassays and to correlate well with chroma-tographic methods. A few nonisotopic RAs have been developed for benzo-diazepines. Other nonisotopic labels such as fluorescence have been proposed as an alternative to RRAs for benzodiazepines assay in biological systems and to screen new benzodiazepine-like compounds from nature (134).

4. Separation Methodologies for Drugs-of-Abuse Testing

Analytical identification and quantification of the analyte of interest require the physical separation of the analyte from the mixture of sample components. The most important separation methodologies for drugs-of-abuse testing are the chromatographic technologies, although electrophoresis techniques have also been developed for drug analysis.

4.1. The Chromatographic Techniques: PC, TLC, GC, and HPLC

As defined by the International Union of Pure and Applied Chemistry (IUPAC) Compendium of Chemical Terminology (169), chromatography is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (stationary phase) while the other (the mobile phase) moves in a definite direction. There are more than 20 types of chromatographic technologies, at least four of which have been applied to drug analysis. Liquid-liquid (partition) chromatography and PC were experimented with in the 1940s, and gas-liquid chromatography

(GLC) and TLC (planar chromatography) were further developed in the 1950s and 1960s. Currently, TLC such as Toxi-Lab is still in use for drug testing. Further advances in recent years in both gas and liquid chromatographies and their interfaces with mass spectrometry have further facilitated the progression of drug analysis technologies. The "hyphenated techniques" of chromatography and MS now are indispensable tools of drugs-of-abuse confirmatory testing and forensic analysis. Impressive congeries of publications and comprehensive reviews have been published for GC, LC, and especially for their hyphenated techniques. A wealth of specific technical details has been published for the analysis of a wide spectrum of drug classes. The goal for the following sections is to present an overview of these technologies, and we will not specifically describe details for their manifold applications.

4.1.1. Thin-Layer Chromatography

In planar chromatography, the stationary phase is a thin layer of absorbent material coated on a glass or metal plate (in TLC) (170-172), or impregnated in a sheet of cellulose or fiberglass material (in PC). To run TLC, the sample is applied as a small spot near the lower edge of the plate and the plate is placed in a solvent chamber. As the solvent rises in the stationary phase, the components in the sample move up the plate at different rates and are separated into different spots. Visualization of the separated components on the plates can be performed under ultraviolet (UV) light and fluorescence. The plates can also be sprayed with various staining reagents to produce color spots. The distance a component migrates from its point of application is calculated as the Rf value. The corrected Rf values are dependent on chemical characteristics and can be used as identification parameters to determine the presumptive presence of a substance. TLC is relatively inexpensive for screening a variety of substances but has relatively higher and variable detection limits. TLC is labor intensive; a prototype Toxi-Prep system developed to automate the process of sample extraction, washing, and elution onto a chromatogram was shown to achieve an overall labor reduction for extraction and spotting of approx 40% (172). A modified TLC technique, high-performance TLC (HPTLC), employs smaller sorbent particles and thicker stationary phase to achieve a better and more efficient separation in a shorter time and with less consumption of solvents (173-175).

4.1.2. Gas Chromatography

GC is commonly used for the separation of thermally stable, volatile compounds. GC separates components of a mixture into its constituent components by forcing the gaseous mixture and carrier gas through a column of stationary phase and then measuring specific spectral peaks for each component of the vaporized sample. Each peak size, measured from baseline to apex, is proportional to the amount of the corresponding substance in the sample. Retention time is the time elapsed between injection and elution from a column of a single component of the separated mixture. The principle of the separation lies in the partitioning of sample components with different retention times, which depends on the chemical and physical characteristics of the analyte molecules. A substance with little or no affinity for the stationary phase of the column will elute rapidly, while a substance with high affinity for the stationary phase will be impeded and therefore slower to elute.

The general design of a GC instrument incorporates (1) a sample injection port (i.e., injector), (2) a mobile phase supply (i.e., carrier gas) and flow control apparatus, (3) a column to perform chromatographic separation between mobile and stationary phases, (4) a detector, and (5) a system to collect and process data (i.e., computer).

4.1.2.1. Preparation of Samples and Internal Standards

Sample preparations such as hydrolysis, extraction, and derivatization have to be carried out prior to sample injection for GC analysis. Depending on the type of specimen used, sample pretreatment such as protein precipitation may also be required. The hydrolysis step (176-178) is used to cleave the conjugate, and may involve fast acid hydrolysis or relatively gentle enzymatic hydrolysis. Alkaline hydrolysis is mostly used for the cleavage of ester conjugates. Scores of studies have been published, reporting specific sample preparation methods that demonstrate enhancement of extraction efficiency (179-185) and improvement of GC-MS analyses. In short, the most commonly used extraction techniques are liquid-liquid extraction (LLE), solid-phase extraction (SPE), and solid-phase microextraction (SPME). A wide variety of solvents and solid-phase materials have been developed, and large selections of commercial columns are also available. The choice of solid-phase cartridges, such as those based on hydrophobic, polar, ionic, or mixed mode of retention mechanisms, is based on both the chemical properties of the analyte(s) and the sample matrix. The development of direct extractive alkylation (186-188,190) under alkaline conditions allows the simultaneous extraction and derivatization of acidic compounds. In addition, antibodies have been used for an immuno-affinity extraction procedure that allowed the simultaneous analysis of A9-THC and its major metabolites in urine, plasma, and meconium by GC-MS (106).

Derivatization chemistry (189-191) is employed to convey volatility to nonvolatile compounds and to permit analysis of polar compounds not directly amenable to GC and/or MS analysis. On the other hand, for compounds that have excess volatility, derivatization can be designed to yield less volatile compounds, to minimize losses during the procedure, and to help separate the GC sample peaks from the solvent front. In addition, derivatization can be utilized to yield a more heat-stable compound and hence improve chromatographic performance and peak shape. Analytical derivatization techniques can be developed to improve chromatographic separation of a closely related compound. Moreover, appropriate derivatization can be utilized to improve the detecting power of certain detectors. An excellent comprehensively review was published by Segura et al. (191). In brief, the common derivatization methods for GC include (1) silylation (to give, e.g., trimethylsilyl [TMS] derivatives, commonly using N,O-bis[trimethylsilyl]trifluoroacetamide [BFSTFA] as the derivatizing agent, or tert-butyldimethylsilyl [TBDMS] derivatives); (2) acylation (to give acetyl; pentafluoropropionyl [PFP]; heptafluorobutyryl [HFB]; or trifluoroacetyl [TFA], using, e.g., N-methyl-bis[trifluoroacetamide] [MBTFA] derivatives); (3) alkyla-tion (to give, e.g., methyl or hexafluoroisopropylidene [HFIP] derivatives); and (4) the formation of cyclic or diastereomeric derivatives. In addition, chiral derivatization reagents such as fluoroacyl-prolyl chloride, S-(-)-heptafluorobutyryl-prolyl chloride, and s-(-)-trifluoroacetyl-prolyl chloride can be used to distinguish enantiomers when using a non-chiral chromatographic column.

Internal standards (192,193) are required to avoid or minimize possible errors during the extraction and derivatization processes. Internal standards are also used to ensure correct chromatographic behavior and quantitation as well as to help in structural elucidation. In GC-MS applications, deuterated internal standards are often used. However, a variety of compounds have been selected as internal standards because such compounds usually have similar structure and possess chromatographic behavior and retention times similar to those of the target analyte.

4.1.2.2. Injector and Carrier Gas-Mobile Phase

The analyte(s) must be in the gas phase for GC separation, and a variety of sample introduction systems have been developed to vaporize liquid samples. In conventional GC with packed columns, samples are injected via an on-line injector using the syringe/septum arrangement or direct connected loop injector. In capillary column GC, the isothermal split or splitless injector system is typically used. The splitless mode of injection is designed for a diluted sample so that most of the sample injected is directed into the column. Temperature-programmable injection ports can be used in either the split or splitless mode to allow the separation of solvent-removal and analyte vaporization, hence improving analyte detection. In addition, cold direct injection and cold on-column injection have been developed to minimize discrimination against higher boiling-point components by the injector.

Upon injection into the GC inlet port, a small amount of sample is vaporized immediately by the high-temperature conditions, which are maintained throughout the GC process by the enclosing oven. An inert carrier gas then transfers the vaporized sample onto the column with minimal band broadening, where it undergoes chromatographic separation. The selection of carrier gas (usually helium, hydrogen, or nitrogen) is influenced by several factors, such as the column type, detector, and the laboratory operation considerations. A constant gas flow from the mobile phase supply is sustained by monitoring flow meters and pressure gages.

4.1.2.3. Columns: Stationary Phase and Temperature Control

The necessity for high temperatures to volatilize drugs for GC requires a special stationary phase that is stable and nonvolatile under the operational conditions. There are two types of stationary phases; the nonselective type separates analytes by molecular size and shape, whereas the selective type separates analyte according to the selective retention of certain groups. There are two major types of GC columns: packed columns and capillary columns (i.e., wall-coated open tubular [WCOT] column).

A multitude of GC columns are available for selection from a variety of commercial suppliers. Many of the supplier catalogs, literature, or application notes provide information on stationary-phase materials and their compatibility with solvent, the amount of polarity, the recommended operating temperature range, and other related information. After the injected sample is directed into the column and carried by the mobile gas phase, the various components of the sample will partition according to the vapor pressure and solubility of each component in the stationary phase of the column. A lower vapor pressure, corresponding to a higher boiling point, will cause the compound to remain longer in the stationary phase and hence elute slower. A compound that is more soluble in the stationary phase will also produce a longer retention time.

4.1.2.4. Detectors and Computer

Ideally, the separated sample components are introduced one at a time into a detector. The choice of a GC detector from the wide variety available is made according to its particular utility and analytical performance requirements. Examples of detectors include MS, flame ionization detector (FID), electron capture detector (ECD), thermal conductivity detector (TCD), atomic emission detector (AED), and many others. MS and FID are universal detectors that may be used for the detection of many volatile organic compounds, although both detectors will also destroy the sample. Because of its ability to provide detailed structural information, MS is the most widely used detector in forensic toxicology. ECD and AED display selectivity in detector response; ECD is often used in the analysis of halogenated compounds, whereas AED is preferred for certain elements such as carbon, sulfur, nitrogen, and phosphorous. TCD is concentration-dependent, whereas FID is mass-flow-rate-dependent.

Following detection, the spectral output is recorded and displayed visually by computer. The computer provides both system-control and data-processing functions. The data are stored and used to calculate analyte concentration from the area or height of each of the chromatographic peaks, to construct calibration curves, to calculate conversion factors from internal or external calibration, and to generate a report.

4.1.3. Liquid Chromatography

For drug analysis, liquid chromatography (LC) is used for the separation of nonvolatile compounds. Separation by LC is based on the distribution of the solutes between a liquid mobile phase and a stationary phase. The most widely used LC technique for drug analysis is HPLC. HPLC utilizes particles of small diameter as the stationary phase support to increase column efficiency. Because the pressure drop is related to the square of the particle diameter, relatively high pressure is needed to pump liquid mobile phase through the column. Similarly to GC, a wide variety of HPLC columns and systems are available from a number of vendors.

Akin to GC, the general design of an LC instrument incorporates (1) an injector, (2) a mobile phase supply (solvent reservoir) and pumps to force the mobile phase through the system, (3) a column to perform chromatographic separation between mobile and stationary phases, (4) a detector, and (5) a system to collect and process data (computer).

Sample preparation for LC also includes the appropriate protein precipitation, hydrolysis, and LLE or SPE; however, the majority of analytes do not require analytical derivatization for LC analysis. The most frequently used injector for LC is the fixed-loop injector. The injector can be used at high pressure and can be programmed in an automatic system. A number of high-precision, microprocessor-controlled autosamplers are available from various vendors. Degassed solvent from the solvent reservoir is pumped into the system using a mode selected for the purpose of the particular LC analysis (e.g., iso-cratic or gradient mode). To protect the analytical column, either a precolumn (placed between the pump and the injector) or a guard column (located between the injector and the LC column) is commonly used. As with GC, there are a wide range of commercial LC columns offered from a number of suppliers. However, stereoselective HPLC can be optimized for the determination of the individual enantiomers (194,195).

The commonly used detectors for LC include UV spectrophotometers, such as diode array detectors (DAD), fluorometers, refractometers, and electrochemical detectors. Detectors that can simultaneously monitor column effluent at a range of wavelengths using multiple diodes or rotating filter disks are useful as drug screening methods. As with GC, the most powerful detector for

LC is MS. However, in comparison with GC-MS, more sophisticated interfaces must be developed for LC-MS. The introduction of two atmospheric pressure ionization (API) interfaces—electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI)—has facilitated the major evolution of LC-MS. In the past year, the importance of LC-MS, especially LC-MS-MS, has dramatically increased in diverse biochemical applications, from proteomics to clinical and forensic toxicology. Improvements made to the interfaces of LC and MS include the nebulization of the liquid phase, the removal of the bulk solvent, the dissociation of solvent-analyte clusters, and ionization techniques. Commonly used ionization techniques for the coupling of chromatography and MS will be further discussed under Subheading 5.2.

A number of limitations associated with LC-MS have been investigated, including its susceptibility to matrix effect and ion-suppression effect (196-199). Dams et al. (198) evaluated the matrix effect resulting from the combination of bio-fluid, sample preparation technique, and ionization type. The authors concluded that matrix components interfered at different times and to a varying extent throughout the study. The residual matrix components were higher in plasma than those in oral fluid, whereas oral fluid has more matrix interferences than urine.

4.2. The Electrophoretic Techniques: CE, HPCE, CZE, MECC, CITP

CE is based on the principle of electrophoresis in a capillary format that separates compounds based on the combined properties of their electrophoretic mobility, isoelectric point, partitioning, molecular size, and so on (29-34). Over the past decade, CE and high-performance CE (HPCE) have emerged as effective and promising separation techniques as a result of its high separation efficiency, minimal sample preparation, negligible sample and solvent consumption, and broad analytical spectrum. Instrumentation for CE utilizing fused silica capillaries has been developed and evaluated for diverse applications in biomedical and chemical analysis. The three major modes of CE are capillary zone electrophoresis (CZE), micellar electrokinetic capillary chromatography (MECC), and capillary isotachophoresis (CITP). The addition to CE of appropriate cyclo-dextrins as chiral selectors can provide a simple and inexpensive approach for the separation of enantiomers.

In forensic and clinical toxicology, the CZE and MECC techniques have been validated by comparison to other established drug-screening and confirmation techniques. A number of published studies employed CZE and MECC to screen and/or confirm a variety of abused and therapeutic drugs in various biological fluids. Recent developments in CE techniques include the combination of CE with an immunoassay or the coupling of CE and MS for confir matory testing. At the present time, CE is not as widely used as GC or HPLC for separation of drug components in biological fluids.

5. Mass Spectrometry

5.1. Fundamental Mass Spectrometry

The fundamentals of MS for drug analysis involve (1) charging of the sample components (with or without the breaking-up of the various molecular species) and (2) the detection of the charged molecular and atomic fragments in order to identify the original sample. The process of molecular structure identification depends on the comparison of compound-specific fragmentation fingerprints in a particular mass spectrum with those in databases, and occasionally elemental analysis based on relative isotope abundance. The charged fragments or ions of a single mass can be isolated by manipulation of the electromagnetic fields within a mass analyzer to produce a mass-to-charge ratio (m/z). Although the variety of MS instruments is diverse in the type of apparatus and mechanical processes, the general scheme involves (1) a sample inlet, an ionization source, (2) a vacuum system, (3) a mass analyzer to accelerate and filter ions by mass, (4) a detector, and (5) a system to collect data (computer).

5.1.1. Ion Source

The sample must be introduced in a gas phase to the sample inlet (which is kept at a high temperature to guarantee a gaseous sample) before it is converted to an ion in the ionization chamber. Many approaches to ionize samples have been developed. The most commonly used ionization techniques for drug analysis are electron ionization or electron impact (EI) and chemical ionization (CI). EI is a "hard" ionization technique whereas CI

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