Luminescence

8.2: Luminescence

The occurrence whereby light is emitted when ‘excited' molecules return to a lower energy, ground state, is called luminescence. There are three subdivisions of luminescence which are fluorescence, phosphorescence and chemiluminescence.

8.2.1: Fluorescence and phosphorescence

Fluorescence is when light is emitted typically inside 10-9 or 10-12 of energy being absorbed. When an excited electron in the singlet state crosses to a triplet state it is known as phosphorescence. These probabilities are so low the transitions are called ‘forbidden transitions'. Fluorescence has a mass detection limit of 10 -15 - 10 -17 moles and concentration detection limit of 10 -7 - 10 -9 molar. Disadvantages of this method are that it is sensitive and usually requires sample derivatization. Phosphorescence is much longer lived then fluorescence as the electron must return to the excited singlet state before it can return to the singlet ground state, as it does this it emits light. In analytical toxicology, phosphorescence assays are not widely used because most samples have to be cooled with liquid nitrogen.

8.2.2: Chemiluminescence

Light given out by fireflies is one of the widely known examples of chemiluminescence. The oxidation of D-luciferin with hydrolysis of ATP and emission of light is catalyzed by firefly lucifercise, an enzyme from the North American firefly, Photirius pyralis. The light emitted is proportional to the amount of ATP present when the quantity of ATP is limiting; this forms the basis of chemiluminescent ATP assays. The increased sensitivity of chemiluminescence over other methods and the simple instrumentation needed, make them attractive when high sensitivity is required. Chemiluminescence assays are usually combined with a separation method such as chromatography or capillary electrophoresis, or with an immunoassay.

Webb et al investigated several presumptive blood detection tests such as luminal and Kastle-Meyer test in regard to their sensitivity, ease of use and safety. The luminal test was determined to be the most sensitive of the techniques. Journal 18

9: Immunoassays

Benson and Yalow first discovered the concept of immunologic methods. They found that anti-insulin antibodies were formed in humans treated with insulin. By taking advantage of the sensitivity of radioisotopes and specificity of immunologic methods it led to the development of radioimmunoassay (RIA). Their findings showed that the concentration of a substrate, usually in urine or serum is measured using the reaction of an antibody or antibodies to its antigen. Antibodies against many drugs have been developed since the assay took advantage of the specific binding of an antibody to its antigen. RIA has also found its way in the analysis of many analytes including some endogenous substances. The high sensitivity and specificity of these antibodies allowed the quantitation of drugs in small amounts of serum. Due to the use of special instruments, complexity of the technique and lack of RIA for a wide range of drugs prohibited its widespread acceptance for routine drug monitoring.

However there was a development of the homogeneous enzyme multiple immunoassay technique (EMIT) and other immunoassay techniques shortly followed including; cloned enzyme donor immunoassays (SLFIA), fluorescence polarization immunoassays (FPIA), kinetic interaction of microparticle in solutions (KIMS) and enzyme-linked immunosorbent assay (ELISA) assay methods.

Advantages and disadvantages

Immunoassays have many advantages over conventional techniques; it is virtually universally applicable to all classes of drugs, can be combined with high-performance liquid chromatography (HPCL) to increase sensitivity/ specificity, and the drug of interest does not have to be extracted or separated from other substances in biological fluid. Disadvantages include its possible lack of specificity as these are ‘antibody-antigen' reactions and they cannot distinguish drugs from within its class, for example opiate class comprise of codeine, morphine and other that have related molecular structures.

10: Chromatography

Mikhail Semenovich Tswett, a Russian botanist developed the technique chromatography as he used a chalk column to separate pigments of green leaves. Chromatography is a separation technique and the components of a sample mixture are spread between two phases, a stationary phase and a mobile phase, they seep into a matrix or over the surface of a fixed phase. As the sample is carried along by the mobile phase a differential migration occurs, the components of the sample mixture exhibit varying degrees of affinity for each phase. A separation of the compound is produced as some components are retained on the stationary phase longer than others.

Several factors are considered when a component is retained by the stationary phase; including the physical and chemical nature of the stationary phase and experimental conditions such as pressure/temperature. The identity of the compounds must be sustained by other methods of analysis following chromatography. The three most general types of chromatography applied by toxicologists are thin layer chromatography (TLC), gas liquid chromatography (GLC) and High-performance liquid chromatography (HPLC).

10.1: Thin layer chromatography (TLC)

It is a simple and economical technique for separating chemical mixtures as the mobile phase is driven by capillary action rather than a gas steam or liquid pumping system. Visualisation and optical measurement are typical methods used to detect separated compounds on a TLC; however the specificity of these methods remains low.

Advantages and disadvantages

TLC is a robust qualitative technique and allows batch analysis as many extracts can be analyzed together. It also does not require complex equipment however the chromatographic system is easily overloaded and it is difficult to obtain reliable TLC plates. Disadvantages include migration characteristics very sensitive to conditions; thin layers easily damaged; quantitative precision only moderate: 5 to 10%. Book fifield

10.2: Gas-Liquid Chromatography (GLC)

GLC provides a method of separating endogenous substances and measuring toxins in biological fluids. Over the years of its finding GLC technique were improved so that by the early 1970s, GLC analysis were performed routinely in many clinical chemistry laboratory.

Advantages and disadvantages

It permits separation of parent drugs, endogenous compounds and other drugs in a class of drugs. Limitations include a large sample volume to achieve biological sensitivity, and frequently chemical derivatisation to ensure the analytes have the prerequisite volatility.

10.3: High performance Liquid Chromatography HPLC

The mobile phase in HPLC is a liquid which flows through a column packed with stationary phase under continuous pressure. It is well suited to the analysis of hydrophilic thermally liable and/or high Mr compounds. Parissis et al investigated the determination of benzodiazepines in forensic samples by HPLC with Photo-Diode Array Detection; the detection limit was 10 to 30 ng. The prevention of the use of an electrolyte buffer in the eluent resulted in a robust procedure, free of technical trouble and of extended rinsing periods, appropriate for routine use in forensic toxicology analysis involving blood, urine, stomach content and tissue samples

Advantages and disadvantages

HPLC has additional practical advantages in the analysis of drugs and other poisons such as flexibility, a range of selective detectors and generally low running costs. Limitations of HPLC in analytical toxicology include the need of experienced operators, expensive hardware/infrastructure and there is always the possibility of co-elution.

11: Mass spectrometry (MS)

In MS ionized atomic or molecular species are separated accordingly to their mass-to-charge ratio (m/z) in the vapour-phase. A sample is bombarded with a beam of electrons, which forms a charged molecule or ionic fragments of the original sample. They are then separated and detected according to their atomic masses. They are separated by manipulation of magnetic and/or electrostatic fields in a high vacuum (usually 10-5Pa). A display of the different abundance is known as a ‘mass spectrum'. . It has a mass detection limit of 10 -16 - 10 -17 moles and a concentration detection limit of 10 -8 - 10 -9 molar.

Buttery et al found that mass spectrometry (MS) could be combined with gas-liquid chromatography (GLC) on order to take advantage of two devices; the first as a separator of complex mixture and the second as an identifier of the separated component. The grouping of mass spectrometers with HPLC, either singly as LC/MS or in tandem as LC/MS/MS has transformed analytical approaches to therapeutic drug monitoring and clinical toxicology.

Advantages and disadvantages

MS is the most reliable technique for accurate mass measurement and can give more information about an analyte using less sample then other techniques. With the use of modern computerised databases identification of a molecule form its mass spectrum is much easier than with other types of spectral information.

Limitations of MS compared to other techniques are that the investment in capital equipment and operator training are both relatively high and the sample taken for analysis is consumed.

11.1: Gas Chromatography - Mass Spectrometry

A compound must be adequately volatile and thermally stable in order for it to be analyzed by GC-MS. Samples are usually analyzed after a preparation process. Modes of ionisation in GC-MS include electron ionization (EI) and chemical ionisation (CI). Both are used routinely in the analysis of relatively low Mr, thermally stable, volatile organic compounds, especially when coupled with GC.

GC-MS joins the unmatched resolving power of capillary GC to the very high specificity of electron ionization, however it cannot contain non volatile or thermally liable substances; for example, polar and high mass compounds.

Skopp et al (2007) found the limits of detection for tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabinol (CBN) in 50mg of hair using GC-MS was 0.025ng/mg for CBN and CBD, 0.07ng/mg for THC. Journal 6

11.2: Liquid Chromatography-Mass Spectrometry

A combination of liquid chromatography and mass-spectrometry provides an important analytical contribution in many areas of research that allows the power of MS to be applied to a much wider array of compounds. Applications of LC-MS in forensic science were at first directed to precise identifications of individual analytes characterised by high substance polarity at low target concentrations, like drugs of abuse, e.g. LSD, cocaine and opiates. Quintela et al (2005) used LC-MS as the detection mode for diazepam and found the limit of detection to be 0.2ng/mL.

Advantages and disadvantages

Unlike GC, derivatisation is virtually never required, certainly promotes LC as the enhanced alternative for the analysis of polar and thermolablie compounds. Advantages include use of wide range of analytes, high sensitivity/selectivity and minimal sample preparation. However, there are limitations as there is a possibility of ion suppression or ion enhancement.

12: Future Direction

Gas chromatography-mass spectrometry is still the most widely used reference method in analytical toxicology, however liquid chromatography coupled with single-stage or tandem mass spectrometry (LC-MS, LC-MS-MS) is becoming increasingly important in routine analysis, especially for quantification of the analytes identified.

HPLC will continue to be one of the most important laboratory separation techniques for analytical and preparative purposes. The use of more selective extraction procedures has revealed clear trends to the simplification of sampling and sample preparation methods, an increase in their reliability and precision, and removal of the cleanup steps. With the development of more sensitive and selective phases, it may be likely to further miniaturize these techniques. Moreover, there is increasing interest in automating sample preparation, thus speeding up these procedures and improving precision and cost-effectiveness. Journal 8 +19

13: Conclusion

The role of analytical methods in postmortem toxicology has been found to be particularly helpful in determining causes of death which without these methods would have been left undiscovered. The science of toxicology has come a long way in past centuries with major advances in analytical technologies as well as a range of disciplines of toxicology. Among the advances that hold promise is the solventless sample preparation technique of solid phase microextraction and many of these methods have yet to be used to their full potential. We have seen one technology be replaced by another; from a simple technique such as TLC by more complex techniques as HPLC. The application of LC-MS-MS has proved to be suitable for sensitive and selective identification of polar compounds in forensic toxicology. Consequently, it may be applied most efficiently to forensic cases characterised by low concentrations and/or complex matrices.

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