Quantitative Assays - Analytical Toxicology Instrumentation

QUANTITATIVE ASSAYS

1.  Ultraviolet-Visible (UV-Vis) Spectrophotometry

This technique is based on the principle that many drugs when in solution will absorb UV radiation. The degree of absorption depends on the chemical structure of the drug, its concentration in the solution, and the wavelength of the UVR. The functioning of this instrument is relatively straightforward. A beam of light from a visible and/or UV light source is separated into its component wavelengths by a prism or diffraction grating. The monochromator can select from the light source an ultraviolet ray of any given wavelength ranging from 200 to 340 nm. Thesample extract in aqueous medium is placed in a transparent quartz cuvette in the path of the radiation. Each monochromatic (single wavelength) beam in turn is split into two equal inten-sity beams by a half-mirrored device. One beam, the sample beam passes through a small transparent container (cuvette) containing a solution of the compound being studied in a trans-parent solvent. The other beam, the reference, passes through an identical cuvette containing only the solvent. The intensities of these light beams are then measured by electronic detectors and compared (Fig 36.2). The intensity of the reference beam, which should have suffered little or no light absorption, is defined as I₀. The intensity of the sample beam is defined as I. Over a short period of time, the spectrometer automaticallyscans all the component wavelengths in the manner described. The ultraviolet (UV) region scanned is normally from 200 to 400 nm, and the visible portion is from 400 to 800 nm.

If the sample compound does not absorb light of a given wavelength, I = I₀. However, if the sample compound absorbs light then I is less than I₀, and this difference may be plotted on a graph versus wavelength. Absorption may be presented as transmittance (T = I/I₀) or absorbance (A = log I₀/I). If no absorption has occurred, T = 1.0 and A = 0. Most spectrometers display absorbance on the vertical axis, and the commonly observed range is from 0 (100% transmittance) to 2 (1% transmittance). The wavelength of maximum absorbance is a characteristic value, designated as λ_max. Different compounds may have very different absorption maxima and absorbances. Intensely absorbing compounds must be examined in dilute solution, so that significant light energy is received by the detector, and this requires the use of completely transparent (non-absorbing) solvents. The most commonly used solvents are water, ethanol, hexane and cyclohexane. Solvents having double or triple bonds, or heavy atoms (e.g. S, Br & I) are generally avoided. Because the absorbance of a sample will be proportional to its molar concentration in the sample cuvette, a corrected absorption value known as the molar absorptivity is used when comparing the spectra of different compounds. Molar absoptivities may be very large for strongly absorbing compounds (ε >10,000), and very small if absorption is weak (ε = 10 to 100).

The amount of UV radiation which passes through the solution is measured by the photocell. By steady rotation of the monochromator, it is possible to pass sequentially UV rays of all wavelength from 200 to 340 nm through the extract. The photocell monitors the amount of radiation absorbed at each wavelength and this is transcribed on to a recorder chart. The shape of the spectrum, the wavelength at which absorption is at a maximum, and any changes brought about by changing the pH of the extract are all used as an aid to characterise the agent present. This technique is ideal to quantitate blood levels of paracetamol and salicylates, as well as urine levels of phenothiazines.

A major disadvantage of UVS is the possibility of interfer-ence in multiple drug overdose. In such a case, UV scanning can produce a composite spectrum of bewildering complexity from which neither qualitative nor quantitative information can be derived. Conventional spectrophotometric methods are known for producing false positive results which can be disastrous in medico-legal cases.

2.  Gas Chromatography (GC)

This is a more sophisticated system of quantitative analysis and has found great favour with analytical toxicologists since it offers a way of simultaneously separating, identifying, and measuring drugs and other organic poisons. Gas chromatog-raphy, specifically gas-liquid chromatography, involves a sample being vapourised and injected onto the head of the chromato-graphic column. The sample is transported through the column by the flow of inert, gaseous mobile phase. The column itself contains a liquid stationary phase which is adsorbed onto the surface of an inert solid. The liquid or solid specimen dissolved in a solvent is injected into the chromatograph. The specimen is vapourised by heat and is carried through a column by an inert carrier gas (usually nitrogen). The choice of carrier gas is often dependent upon the type of detector which is used. The carrier gas system also contains a molecular sieve to remove water and other impurities. The column is packed with a substance like carbowax or adiponitrile which is capable of changing the migration time of the specimen as it traverses the column.

For optimum column efficiency, the sample should not be too large, and should be introduced onto the column as a “plug” of vapour - slow injection of large samples causes band broad-ening and loss of resolution. The most common injection method is where a microsyringe is used to inject sample through a rubber septum into a flash vapouriser port at the head of the column. The temperature of the sample port is usually about 500C higher than the boiling point of the least volatile component of the sample. For packed columns, sample size ranges from tenths of a microlitre up to 20 microlitres. Capillary columns, on the other hand, need much less sample, typically around 10-3 microlitre. For capillary GC, split/splitless injection is used.

The injector can be used in one of two modes; split or splitless. The injector contains a heated chamber containing a glass liner into which the sample is injected through the septum. The carrier gas enters the chamber and can leave by three routes (when the injector is in split mode). The sample vapourises to form a mixture of carrier gas, vapourised solventand vapourised solutes. A proportion of this mixture passes onto the column, but most exits through the split outlet. The septum purge outlet prevents septum bleed components from entering the column.

A detector recognises the presence of the chemical and graphically plots its emergence as a function of time (Fig 36.3). The retention time and peak area for a chemical compared to known standard are used to identify and to quantitate its presence. There are many detectors which can be used in gas chromatography. Different detectors will give different types of selectivity. A non-selective detector responds to all compounds except the carrier gas, a selective detector responds to a range of compounds with a common physical or chemical property and a specific detector responds to a single chemical compound. Detectors can also be grouped into concentration dependentdetectors and mass flow dependent detectors. The signal froma concentration dependant detector is related to the concentra-tion of solute in the detector, and does not usually destroy the sample. dilution with make-up gas will lower the detectors response. Mass flow dependant detectors usually destroy the sample, and the signal is related to the rate at which solute molecules enter the detector. The response of a mass flow dependant detector is unaffected by make-up gas.

The most common means of detecting the compounds is by flame ionisation. On leaving the column the inert gas is mixed with hydrogen and either air or oxygen, and the mixture is ignited to give a continuous jet of flame. It is decomposed into electrically charged fragments which are collected at an electrode. This results in an electrical signal which is amplified and transmitted to a pen recorder operating at a constant chart speed. This peak is transcribed each time a compound emerges from the column. Flame ionisation detectors (FIDs) are mass sensitive rather than concentration sensitive; this gives the advantage that changes in mobile phase flow rate do not affect the detector’s response. The FID is a useful general detector for the analysis of organic compounds; it has high sensitivity, a large linear response range, and low noise. It is also robust and easy to use, but unfortunately, it destroys the sample.

Gas chromatography (GC) is most commonly employed to quantitate blood levels of volatile liquids such as ethanol, ethylene glycol, and methanol.

Other detectors have recently been developed which widen the scope of plasma screening, such as the electron capturedetector, and the alkali flame detector (nitrogen detector).

High Performance Liquid Chromatography (HPLC)

This is similar to GC, except that it is not restricted to volatile compounds. A high pressure (1000 to 6000 pound per square inch) pump facilitates movement of the specimen through the columns packed with chromatographic adsorbents e.g. silica gel and alumina. The effluent stream passes through a detector, usually an ultraviolet spectrophotometer, and the appearance of a drug in the solvent is signalled by a recorder peak in the same way as in GC (Fig 36.4). Again, the size of the peak is proportional to the concentration of drug in the sample. HPLC can be used to separate and analyse complex mixtures.