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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). 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,0-bis[trimethylsilyl]trifluoroacetamide [BFSTFA] as the derivatizing agent, or teri-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, ^-(-)-heptafluorobutyryl-prolyl chloride, and 5'-(-)-triiluoroacetyl-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. 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. 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. 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 is a "soft" ionization technique. For the interface of LC and MS, soft ionization techniques such as ESI and APCI have been developed.

For ionization by EI, electrons produced by thermoionic emission from a tungsten filament are accelerated in a collimated beam by a high voltage (typically +70 eV) and impact the gaseous analyte molecules, shattering the molecules into fragments and causing each molecule to give up an electron. The resulting energetic cation radical is called the "molecular ion" (or parent ion) M+. The molecular ion can undergo a predictable and relatively reproducible fragmentation, forming a radical and a cation called the "fragment ion," which is generated from bond cleavage reactions.

In CI, a reagent gas such as methane is typically ionized to radical forms, which impact the analyte molecules in the GC effluent and chemically generate molecular ions and some daughter ions and neutral fragments. The most common type of CI reactions resulting in positive ions are proton transfers of fragmentations with the positive charge being retained by the part with a greater ionizability. Negative ions (NICI) can be generated either by reaction with proton-abstracting reagents or by electron capture of thermalized electrons.

EI and CI methods may be used to complement each other, as the softer CI technique produces less fragmentation and ensures the production of molecular ions, whereas the harder EI technique can give more detailed information about the molecular structure of the sample.

ESI is a newer soft ionization approach for MS and involves the pneumatic nebulization of the analyte solution to produce charged droplets that are sprayed from a capillary tip by means of an applied potential (+4 kV). Solvent evaporation and coulombic repulsion forces eventually lead to the formation of charged analyte ions. A softer but more energetic ionization method than ESI is APCI, in which the analyte solution is directly injected into the CI plasma, where analyte ions are generated from ion-molecule reactions taking place at atmospheric pressure. An electric discharge between the spray capillary and a counter electrode sustains the CI plasma.

5.1.2. Mass Analyzer and Vacuum System

The mass analyzer uses a controlled range of magnetic and/or electric field strengths to filter positively charged molecules by mass-to-charge ratio (m/z) and accelerate the ion of interest in a vacuum to the detector by the influence of an accelerating voltage. Techniques to achieve this separation include quadrupole mass filters (quadrupole mass spectrometer [QMS]), ion traps (quadrupole ion traps [QIT]), Selective Ion Monitoring (SIM), magnetic sector, time-of-flight, and so on.

The quadrupole mass filter is the most commonly used mass analyzer for MS because of its good reproducibility, low cost, and compact nature. Only ions of the desired m/z value can follow a stable trajectory to the detector between four parallel rods, which create electric fields controlled by a fixed direct current (DC) and alternating radio frequency (RF) voltages. The quadru-pole mass analyzer is limited in terms of resolution and mass discrimination (peak heights as a function of mass). Conversely, the ion trap boasts high mass resolution but suffers from a limited dynamic range, required low-pressure conditions, space charge effects (ion-ion repulsion), and ion molecule reactions. Three hyperbolic electrodes form a three-dimensional storage space where ions are trapped in a stable oscillating trajectory by the applied RF potentials. Alternation of the voltages causes ions of different m/z to be successively ejected from the exit lens into the detector. SIM is another technique that improves sensitivity in trace analysis, and differs from those previously mentioned in that only ions with the desired m/z values are selected and monitored to enhance the signal-to-noise ratio. Confirmation is normally performed in the SIM mode because only particular compounds have to be identified. For MS-MS, this approach is called selected reaction monitoring (SRM).

A vacuum system is used to ensure that the ions in the mass analyzer do not collide with any other molecule during interaction with the magnetic or electric fields. A number of systems can be used for these purposes, a including mechanical vacuum, a high-vacuum pump such as diffusion pump, turbo-molecular pumps, and cryopumps.

5.1.3. Detectors and Computer

As desired ions from the mass analyzer strike the detector, a representative signal is produced that is proportional to the number of impinging ions. These signals (fragment mass over detected charge) are amplified by cascading electron emissions and sent to the computer, where the electrical impulses are converted to visual output for further analysis.

The visual output is presented as a mass spectrum of the sample, where each peak is graphed by fragment m/z along the x-axis and abundance, or the quantity of detected fragments for that mass, along the y-axis (therefore corresponding to peak height). A total ion chromatogram (TIC) is obtained by plotting the sum of abundances of all ions in the mass spectrum as a function of elution time. The parent mass, or the detected mass associated with the unfragmented analyte molecule, indicates the molecular mass of the analyte and is usually the largest peak on the spectrum. The remaining peaks provide precise clues to the molecular structure, as their associated fragments can be pieced together to form the original molecule, and identification of the sample can be confirmed by comparison to reference spectra via library search (200-202). The ion with the highest abundance in a mass spectrum is considered the base peak and is normalized to 100%. Other ion fragment abundances are then reported as percentages of the base peak height. For identification purposes, the monitoring of at least three ions and their abundance ratios is required, and it is desirable that one of the ions selected should be the molecular ion.

5.2. The Coupling of Analytical Techniques: GC-MS, LC-MS, CE-MS, and TLC-MS

The coupling of GC, LC, or CE and MS is a powerful technique for the chemical analysis of mixtures of compounds. An emerging development that utilizes TLC and direct on-spot matrix-assisted laser desorption/ionization time-of-flight MS has been applied for fast screening of low-molecular-weight compounds with nearly matrix-free mass spectra using a UV-absorbing ionic liquid matrix (203). In essence, the first system enables separation of the components of a mixture and determines their particular retention times. Molecules entering the MS are ionized and may undergo fragmentation; consequently, the sensitive detector in the MS device provides information for the identification of components by determining their mass spectra. A computer serves as the data collector to record and process the mass spectra obtained (200-202).

Although GC/MS is recognized as the standard procedure for confirming positive immunoassay screening results of drugs of abuse, targeted GC-MS analysis does have limitations. The following section contains an overview of the technique and limitations of both GC and MS separately and of the combined technique of GC-MS. Any analytical technique has its limitations; GC is limited by unequal detector responses to equal amounts of two different samples, the presence of residual impurities, the choice of a carrier gas, the life of an injection port septum, and the crucial temperature range of the injection port. For each analyte of interest, the problem posed by unequal detector responses to equal amounts of different samples is overcome by the calculation of a response factor for each analyte. This response factor is defined as the response of the analyte (peak area or height) divided by the weight (or volume) of the analyte inj ected. The proper choice of a carrier gas and its purity are vital to the success of the analysis. The gas filter should be changed regularly, and a stable gas flow rate should be maintained to avoid false peaks and a drifting baseline. The lifespan of a septum will be shortened by higher injection-port temperatures. It is essential that the injection port be kept within the correct temperature range to completely vaporize the sample; a lower temperature will result in poor separation and broad or no peak development, whereas a higher temperature may cause the sample to decompose or alter structure and thereby skew the analysis results.

The potential limitations of MS include resolution, interior pressure of the device, high scan rate, the skills of the technician, the locating of the parent mass when present, and the comparison of the analyte identification with that of a standard sample. Resolution refers to the degree of separation of adjacent peaks in a mass spectrum, and is defined as R = m/Am, where m is from the observed m/z ratio and Am is the difference in mass between the two peaks. An MS instrument with low resolution may poorly characterize a sample with large molecular mass, such as body fluids. Just as important as high resolution is maintaining high vacuum conditions within the device in order to minimize collisions between analyte fragments. Such collisions may foster recombination of analyte fragments to make new molecules, thus producing spectral peaks alien to the authentic mass spectrum of the analyte. The tradeoff of the ability of MS instruments to rapidly scan multiple fragment masses is decreased resolution, which may produce unreliable results for quantitative analysis.

The analyses and interpretation of the mass spectra plays an essential role in the accurate determination of the original molecular structure of the sample. The importance of the human element involved in the decision between possible answers outweighs the assistance computers and libraries can provide. Also, the occasional difficulty in recognizing the appropriate parent peak in the mass spectrum may introduce analytical error, since the establishment of the associated parent mass is important for the qualitative decision about the molecular structure of the analyte. In addition, such a parent peak may not even be observed for samples of sufficiently high molecular mass, such as drugs in body fluids. One solution is the use of CI as the ionization source for MS, which helps to ensure the appearance of the parent peak in the mass spectrum. Finally, it is essential that a standard sample of the presumed identity of the analyte be prepared and run under identical conditions both prior to and after the analyte run. Discrepancies between the mass spectra of the sample and the standard indicate a questionable identification.

In practice, GC-MS is regarded by many scientists as the conclusive modus operandi for the reliable identification of substances by chemical analysis. For all of the positive attributes of GC-MS, however, even an authoritative technique has limitations. A capillary column interface serves as the connection between the GC column and the MS device, which concentrates the GC sample effluent by removing the gas carrier and then feeds the sample to the MS. The accuracy of the MS technique is dependent on the purity of this effluent; background noise may appear in the mass spectrum as a result of incomplete chromatographic separation of the compounds in the sample. In addition, failure to deflect the carrier gas of the GC device from entering the MS device likewise may cause contamination. To assess the performance of GC-MS analysis, an internal standard (IS) can be added prior to any extraction step so that the IS can undergo the same manipulations, from sample preparation to result analysis, as the sample. For MS decisions, a compound, either structurally related to the analyte of interest, or an analyte labeled with a stable isotope such as deuterium, is generally used as an internal standard.

The performance expectation and limitations of GC-MS as well as solutions to overcome some of the identified limitations have been subjected to a number of reviews (204-208). In addition, The Clinical and Laboratory Standards Institute has developed "Approved Guidelines (C43-A) for GC-MS Confirmation of Drugs." The document provides guidance to routine instrument and method performance verification, calibration, result interpretation, quality control, and quality assurance. The certified laboratories also are subject to periodic surveys with proficiency testing samples provided by specific organizations. The College of American Pathologists (CAP/AACC) has been conducting quarterly surveys and year-end critique for certified laboratories.

The percent coefficient variance from the different laboratories can be assessed from these surveys. In the near future, the proposed new federal Guidelines for WDT will include regulations on the requirements for certified laboratories to validate their confirmatory drug testing (GC-MS, LC-MS, GC-MS-MS, and/or LC-MS-MS) before the laboratory can use it to test specimens (69).

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Do Not Panic

Do Not Panic

This guide Don't Panic has tips and additional information on what you should do when you are experiencing an anxiety or panic attack. With so much going on in the world today with taking care of your family, working full time, dealing with office politics and other things, you could experience a serious meltdown. All of these things could at one point cause you to stress out and snap.

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