Journal of Clinical Pharmacy and Therapeutics (1992) 17,325-331
REVIEW ARTICLE
A critical appraisal of chromatographic and irnmunoassay techniques for clinical drug analysis A. C. Mehta Department of Pharmacy, The General InjGvnay, Leeds, Yorkshire, U.K.
SUMMARY
Table 1. Advantages of RPLC in drug analysis
Chromatography and immunoassays are the two principal techniques used in research and clinical laboratories for the measurement of drug concentrations in biological fluids. The purpose of this article is to provide the reader with a general overview of these techniques and to present their relative merits and limitations. A glossary of technical terms is included at the end of the review.
A wide variety of silica-based,chemicallybonded stationary phases with different selectivitiesand functionalities (octyl (CJ, octadecyl (CJ, cyan0 (CN), phenyl, etc) are available. In addition, new stationary phases have been developed (7) which allow the direct separation of chiral drugs or direct injection of biological samples into the HPLC column A wide variety of drugs (polar, non-polar, ionic) can be separated Potential interfering polar substances originating from biological fluids are not retained on the column but elute with the solvent front Samples can be analysed with a minimum of clean-up Aqueous mobile phase is compatible with aqueous samples (clinical samples are aqueous) which means, after clean-up, they can be injected directly into the column
INTRODUCTION
Analysis of drugs in biological samples is undertaken in hospital laboratories for many reasons, the main ones being (a) therapeutic drug monitoring (TDM) in patients to assess potential toxicity and, (b) bioavailability and pharmacokinetic studies in patients or volunteers. It can involve quantification of drugs in the microgram (10-6g, or parts per million, ppm), nanogram ( 1 0 - 9 g , or parts per billion, ppb), or even picogram (IO-’’~, or parts per trillion, ppt) concentration range. Current approaches to drug analysis rely mainly on chromatographic and immunoassay techniques (1-5). This paper examines the role of these techniques in the drug analysis laboratory and assesses their usefulness and limitations.
Table 2. Advantages of HPLC in relation to GC 1
CHROMATOGRAPHIC TECHNIQUES
Thin-layerchromatography
Among chromatographic techniques, thin-layer chromatography (TLC) is primarily used for the separation and identification of compounds such as drugs of abuse. For qualitative analysis it is speedy, simple, inexpensive and offers a selection of spray reagents to locate the spots. For quantitative work, it is timeconsuming and labour-intensive and has lower sensitivity and resolution than gas chromatography (GC) or
2
3 4
HPLC is quicker than GC and has a wider range of applicability. It can be used for polar, non-volatile and thermally labile compounds. This permits the determination of a wide variety of drugs In general, a sample clean-up procedure is simpler for HPLC than GC. It usually entails a single solvent extraction step or a simple protein precipitation step (removal of protein) in order to protect the HPLC column from the deposits of protein Unlike GC, derivatization is not necessary in most cases HPLC systems, unlike GC, do not destroy the sample so that once the analysis is complete, the sample can be recovered for additional studies
326
A. C. Mehta
Table 3. Advantages of homogeneous immunoassays Homogeneous immunoassays are quick, technically easy to perform and are easy to automate. They are wellsuited to routine drug monitoring applications where the emphasis is on a fast, simple, and reliable method They can generally be carried out directly on biofluids. No sample pretreatment is required. It is the quality of the antibody that determines specificity. Chromatographic methods require some sample purification prior to assay They permit determination to be made on very small volumes of samples (50pl). Chromatography on the other hand requires at least 0.5-1 ml sample They are particularly useful for those drugs which have proved difficult to analyse by chromatographic techniques. For instance, digoxin and aminoglycosides being very polar and having no strong chromophoric groups, are difficult to analyse by either GC or HPLC They are chemically gentle and can therefore be used to determine unstable or labile compounds
high-performance liquid chromatography (HPLC). Morever, it is not fully automatable. The advent of high-performance TLC (HPTLC) has brought a revival of TLC. HPTLC plates are superior to conventional type, having much smaller silica particles (about 5 pm) and a very narrow particle size distribution. This has made possible the application of a small sample ( < 1 p1) on a plate and relatively rapid separation of compounds. Although widely used in industry, TLC has proved less useful as a quantitative tool in the hospital laboratory despite the availability of HPTLC plates and the equipment for their quantitative scanning. Gas chromatography
Gas chromatography (GC) can be performed on glass columns (ID 4mm) packed with non-polar or polar stationary phases or on flexible fused silica capillary columns (ID 0.53 mm or less) packed with chemically bonded stationary phases. Capillary columns are more popular because their performance is superior to the conventional glass columns. They are robust and offer high resolution, high sensitivity and faster separations (6).
Most GC methods of drug analysis use a flame ionization detector (FID), which can respond to all organic compounds, however, other more selective and
Table 4. Disadvantages of homogeneous immunoassays Because of the cost of the reagent kits and other consumables, immunoassaysare relatively expensive to run. The cost can only be justified if there are sufficient routine samples to be analysed They are more difficult, time-consuming and expensive to develop in users’ laboratories because facilities are required to raise antibodies to a specific drug. Chromatography on the other hand allows ease of method development for virtually any drug They can be used only for those drugs for which reagent kits are available. Manufacturers develop an immunoassay for a drug only when the need for monitoring it has been clearly indicated. To date, commercial kits are available for only a few classes of drug such as anticonvulsants, aminoglycosides,cardioactive drugs and for a few individual drugs, such as theophylline and methotrexate Generally they are less sensitive than the chromatographic techniques. Thus, although they are suitable for routine TDM where drugs are measured at a relatively higher concentration (steady-state levels), they are not particularly suitable for pharmacokinetic or bioavailability studies Interference can be caused by cross-reaction of the antibody with metabolites that are structurally closed to the parent drug, and in some cases with similar drugs that may be present in the sample. The specificity of an immunoassay can be improved if the cross-reacting compounds are removed prior to assay but this may become too time-consuming When more than one drug is being measured, a separate assay must be performed for each drug, whereas with chromatographic techniques several drugs can be measured on the same sample
sensitive detectors, such as the nitrogen-phosphorus detector (NPD) o r electron capture detector (ECD), are also used. The NPD has an enhanced sensitivity to nitrogen and phosphorus-containing compounds and is quite useful in the analysis of nitrogen-containing compounds such as anticancer drugs and tricyclic antidepressants. ECD responds to drugs containing electronegative substituents such as halogens or nitrates (e.g. benzodiazepines).The mass spectrometer (MS),as a GC detector, confers extreme specificity and sensitivity but requires complex instrumentation and highly trained personnel. It is very expensive and not within the easy reach of an average hospital laboratory budget. GC is a very sensitive technique; however, because it requires volatile and thermally stable samples, it is not
Clinical drug analysis 327 Table 5. General comparison of
chromatographicand immunoassay techniques
Chromatography Sample requirement Sample clean-up Sample derivatization
0-5-1-0 mi
50-100 pl
Usually required Sometimes (HPLC),frequently (GC)
Not required Not required
Analysis time
Relatively slow
Fast"
Technical skill and experience required Equipment cost Method developmentcost Running expenses Ability to measure several compounds simultaneously Sensitivity for TDM Sensitivityfor pharmacokinetic and bioavailability studies
Considerable
Minimal
Moderate
Moderate High Moderate
Accuracy 'Heterogeneous assays are relatively slow.
Immunoassay
Precision Automation
Low Low Yes
No
Adequate Adequate
Adequate Limited
High High Possible
Moderate Moderate Possiblet
?Partial for heterogeneous assays.
always suitable for highly polar substances which may be non-volatile or thermally labile. If a sample is nonvolatile and thermally labile, as are many drugs, it can be derivatized (e.g. silylation, acylation) to increase its volatility and stability but this makes the assay more time-consuming. GC is easily adaptable to automation. GC was once the dominating analytical technique for drug analysis. It still remains an important technique but shares the field with HPLC and immunoassays.
High performance liquid chromatography
Among the many forms of HPLC (normal-phase, reversed-phase, ion exchange, size exclusion), reversedphase liquid chromatography (RPLC) offers distinct advantages over the others, and is most widely used in drug analysis. In RPLC the functional groups of the stationary phase are non-polar and the mobile phase consists of polar solvents such as water, methanol, or acetonitrile. This situation is the reverse (hence the name WLC) to that for the normal phase chromatography where the stationary phase is polar (silica)and the mobile phase is non-polar (e.g hexane). The advantages of RPLC in drug analysis are shown in Table 1. HPLC is performed in stainless steel columns (150 x 4 mm ID)or plastic columns (100 x 8 mm ID) packed with non-polar or polar stationary phases. The
column sizes may vary with manufacturers. It can also be performed on microbore (1-2 mm ID)columns (8). Like their counterpart capillary GC columns, microbore columns possess high resolution capacity and sensitivity which could be advantageous in simultaneous analysis of several compounds. As small samples are required to work with microbore columns, the technique may become more useful in paediatric drug monitoring. Moreover because of the low flow-rates employed, there is a considerable saving of mobile phase solvents. In spite of these advantages microbore columns have not yet been widely used in drug analysis. One drawback of microbore columns is that compared to conventional columns, the analysis time is relatively longer which can decrease sample throughput. The most commonly used HPLC detector is an ultraviolet detector because many drugs show sufficient absorption in the ultraviolet region to allow spectrophotometric detection. Fluorescence, electrochemical (EC) or diode array detector (DAD)are also used but less frequently. Fluorescence and EC detectors are claimed to be more sensitive than the ultraviolet detector. DAD rapidly and continuously scans the ultraviolet spectra while a peak is eluting. This process offers a significant improvement in the provision of information on peak identity and homogeneity. HPLC detectors are reaching a level of sensitivity quite comparable to those of GC. Although GC-MS is highly developed,
328
A. C. Mehta Table 6. Glossary
Term
Definition
Antibody
An antibody is an immunoglobulin produced in the body in
Accuracy
Antigen (immunogen)
Antiserum Cross-reaction Hapten
Limit of: detection COD) Precision
response to stimulation by a large molecule (the antigen) and capable of reacting with the antigen. The binding of antibodies to antigens is highly specific Accuracy refers to the difference between the observed value (assay result) and the true or known value and is expressed as a percentage A compound which, under suitable conditions, can stimulate an immunogenic response when introduced into the body, resulting in the formation of specific antibodies. In order to achieve high specificity, the antigen should be a pure substance. Only very large molecules, such as proteins and polysaccharides, are antigenic. As drugs are usually small molecules they are not antigenic. Such small molecules, which bind to other larger carrier molecules to become immunogenic, are called haptens Serum containing antibodies Reaction of the antibody with substances other than the target antigen or hapten A small molecule that reacts with a specific antibody but does not itself stimulate antibody production. In order to make a hapten antigenic it must be covalently bonded to a large carrier molecule such as a protein (e.g. albumin) to form a hapten-protein complex. The method of attachment of the hapten to the protein determines the specificity of the resulting antibody The lowest concentration of an analyte (substance to be analysed) that the analytical method can reliably differentiate from background levels Precision (or reproducibility)describes the variation or scattering of data (assay results) around the mean and is usually expressed as coefficient of variation (0).
cv= Quality control (QC)
Recovery
100 x standard deviation
%.
The continuing evaluation of an assay performance (with regard to accuracy and precision) to ensure that the assay continues to work satisfactorily.This is usually done by assaying QC samples (samples containing known amount of substance to be analysed) and plotting the results on a control chart to identify whether any results are outside the limits In analysis, recovery of the analyte can be found by analysing samples of a known amount of analyte and using the formula Recovery =
Resolution
mean
Amount found Amount present
x 100%.
Resolution is the ability of a chromatographic column to separate two peaks. A high resolution column will give two very narrow, symmetrical peaks separated from the baseline continued
Clinical drug analysis
329
Table 6 . Glossary (Continued)
Term
Definition
Sensitivity
Sensitivity is used to pinpoint the ability of an analytical technique to detect and quantify a trace substance (e.g. drug) in a sample. If the technique is able to detect very low levels of a substance(mg/l or below), it is said to be very sensitive Specificity (orselectivity)is the ability of an analytical method to determine solely the compound (e.g. drug) of interest in the presence of potentially interfering compounds. In immunoassay, specificity is the ability of the antibody to 'recognize' (bind)only the antigen or hapten of interest, having little affinity for other closely related compounds The specific substance on which a given enzyme acts The concentrationor activity of antibody in serum
Specificity
Substrate Titre
the development of a combined HPLC-MS system has been hindered by experimental problems. One of the advantages of HPLC is its easy adaptability to automation. With autosampling and electronic data reduction facilities a large number of samples can be processed with ease. Further advantages of HPLC, particularly in relation to GC, are shown in Table 2. IMMUNOASSAYS
Immunoassays are based on competition between the drug to be assayed (D) and a labelled drug (D*) for a limited number of binding sites on the antibody (Ab) specific for the drug. The label may be a radioisotope, an enzyme or a fluorescent substance. The sample containing an unknown quantity of the drug is added to a known constant amount of labelled drug and antibody. As a result of the competitive reaction, some of the antibody-bound labelled drug is displaced by drug in the sample. At equilibrium, the proportion of the labelled drug remaining in the free (D") or bound fraction (D*Ab)reflects the concentration of the drug in the sample. D*
+ D + Ab
+
D*Ab DAb. Free drug Bound drug The analytical procedure measures the amount of free labelled drug (D*)or the fraction still bound to antibody (D*Ab). If these values are plotted against the known concentration of unlabelled drug, a calibration graph can be obtained from which the concentration of the drug (D) in the sample can be determined. Depending upon the nature of the label, the assay could involve the
measurement of radioactivity, ultraviolet absorption following enzymatic reaction, or fluorescence. The two general types of immunoassays may be classified as homogeneous and heterogeneous immunoassays. Homogeneous assays are based on the fact that antibody-bound molecules can be distinguished from unbound molecules without a separation step. The difference between the signals may arise because the signal may be suppressed, produced, or altered on binding. This makes the assay quicker (few manipulations are required) and facilitates automation allowing the laboratory to increase productivity and reduce the cost per test. Enzyme immunoassay (EIA) and fluoroimmunoassay (FIA) are the examples of homogeneous assays where D* and D*Ab are distinguished by their differences in optical properties (W absorption, fluorescence) without a separation step. Commercial optical immunoassays are usually homogeneous. Heterogeneous assays in contrast require separation of bound and free species. This is time-consuming and makes automation more difficult. An example of a heterogeneous assay is radioimmunoassay (RIA) where a radioactive label is used. Here there is no difference between the radioactivity signal produced by D* and D*Ab so that it is necessary to separate the two before measurement. RIAs are very sensitive but are time-consuming and costly. They require the use of radioactive material and specialist counting equipment. In addition, their range of application is limited, i.e. RIA kits are available for only a few drugs. They have remained popular for a long time but currently they are being succeeded by homogeneous non-isotopic immunoassays where
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A. C. Mehta
enzymes (9,10)or fluorescent molecules (10,11) are used as labels. Homogeneous immunoassays are comparable in performance with RIAs with added advantages such as (a) reagents are relatively inexpensive and have a long shelf-life, (b) a variety of labels are available, (c) assays are simple, rapid, and automatable and, (d) there is no radiation hazard.
Homogeneous enzyme-and
fluoroimmunoassays
Enzymes are good labels for immunoassays because they catalyse a variety of reactions that can be monitored spectrophotometrically. In this category the enzyme multiplied immunoassay technique (EMIT) is most widely used. It is based on the reaction between the enzyme, glucose-6-phosphate dehydrogenase (G6PDH); the substrate, glucose-6-phosphate (G6P); and the coenzyme, nicotinamide-adenine dinucleotide (NAD). During the reaction NAD is reduced to NADH and the resulting change in absorbance, which is directly related to the concentration of the drug in the sample, is measured spectrophotometrically. FIA offers enhanced sensitivity compared to EIA. It may be homogeneous or heterogeneous, but the homogeneous variety is generally used in hospital laboratories. Two prominent homogeneous FIAs are 1 substrate-labelled fluoroimmunoassay (SLFIA), which is based on the measurement of fluorescence following enzymatic (fluorogenic) reaction between the enzyme P-galactosidase and the substrate P-galactosyl-umbelliferone,and 2 fluorescence polarization immunoassay (FPIA), which is based on the measurement of the fluorescence polarization resulting from the binding of fluorophorelabelled drug to specific antibodies.
Results obtained using EMIT and FPIA procedures correlate well with each other and with those obtained with GC and HPLC (12-14).As a result both these techniques (EMIT and FPIA) are now widely used in the routine TDM. The advantages and disadvantages of homogeneous immunoassays are presented in Tables 3 and 4.
chemiluminescent compounds, metal atoms, and proteins have been proposed which provide alternative methods for drug immunoassays. Furthermore,a number of solid-phase (ordry-phase) immunoassay systems (e.g. for theophylline) have been developed recently (15). Here all the key reagents are immobilized on a strip or a slide requiring only the addition of a sample (blood or serum).The results (instrumentalreadout) are availablein minutes. The new technology is designed to be rapid and simple to facilitate near-patient testing. Although promising, it is early days yet, for solid-phase immunoassays, and more critical work (evaluation) is needed before they are clinically utilized routinely.
CONCLUSIONS
Chromatographic methods (GC and WLC) are the most sensitive, specific, and versatile techniques for clinical drug analysis. They are preferred techniques for the research laboratories especially when information on both the parent drug and its metabolites is required. They can also be used in routine drug monitoring when immunoassays are unavailable. Immunoassays are in general more rapid, adequately sensitive and specificand require smaller sample sizes. They are well-suited to routine monitoring of drugs in a clinical setting. Although they require less training than the chromatographic methods, it is desirable to have a full knowledge of the sample and the assay method for the correct interpretation of the assay results. Immunoassays are expensive to run but the development of economic reagents could widen their use in the future. Chromatographic and immunoassay techniques have their particular advantages and disadvantages (summarizedin Table 5). and should be regarded as complementary to each other. As clinical decisions on drug dosages are often made on the basis of analytical results, it is pertinent that thoroughly validated analyticalmethods are used in drug assays. For reliable results it is also necessary to maintain good quality control of the assay to ensure that it continues to work satisfactorily.
REFERENCES
Recent approaches
Enzymes and fluorophores represent the most popular non-isotopic labels at present, but other labels such as
1. ChamberlainJ. (1985)Analysis of Drugs in Biological Fluids. CRC Press, Boca Raton. 2. Moffat AC. (ed)(1986)Clarke's Isolation and Identification of Drugs. Pharmaceutical Press, London.
Clinical drug analysis 331 3. Pieper JA, Rutledge DR. (1989)Laboratory Techniquesfor Pharmacists.The Upjohn Company, Kalamazoo. 4. Hill RE. (1986)Methods of therapeutic analysis. Clinical Biochemistry, 19,113-121. 5. Meijer JWA, Rambeck B, Riedmann M. (1983)Anti-
epileptic drug monitoring by chromatographic methods and immunotechniques-comparison of analytical performance, practicability, and economy. Therapeutic h 4 g Monitoring, 5,39-53. 6. Mehta AC. (1989)Potential of wide bore open tubular columns in gas chromatographic analysis of drugs. Journal of Chromatography, 494,1-11. 7. Mehta AC. (1989)Recent trends in HPLC stationary phases for pharmaceutical analysis. PharmaceuticalJournal, 243,748-750. 8. Taylor RB, Reid RG. (1988)Practical aspects of packed microbore HPLC. International Analyst, 2,17-22. 9. OSullivan MJ. (1981)Enzyme immunoassay. Analytical Proceedings, 18, 104-108.
10. Beastall GH. (1985)Non-isotopic immunoassay methods. Laboratory Practice, 34, 74-81. 11. Gutierrez MC, Gomez-Hens A, Perez-Bendito D. (1989)
Immunoassay methods based on fluorescence polarisation. Talanta, 36,1187-1201. 12. Ratnaraj N, Goldberg VD, Lascelles PT. (1986)Correlation between fluorescence polarisation immunoassay and enzyme immunoassay of anticonvulsant drugs, and stability of calibration graphs. Analyst, 111,517-523. 13. Loomis KF, Frye RM. (1983)Evaluation of Abbott TDx for the stat measurement of phenobarbital, phenytoin, carbamazepine, and theophylline. American Journal of Clinical Pathology, 80,686-691. 14. Haver VM,Audino N, Burris S,Nelson M. (1989)Four fluorescence polarisation immunoassays for therapeutic drug monitoringevaluated. Chnical Chemistry,35,138-140. 15. Mould G, Marks V. (1988) The use of solid-phase chemistry in therapeutic drug monitoring. Clinical Pharmacokinetics, 1465-70.
REVIEW ARTICLE
A critical appraisal of chromatographic and irnmunoassay techniques for clinical drug analysis A. C. Mehta Department of Pharmacy, The General InjGvnay, Leeds, Yorkshire, U.K.
SUMMARY
Table 1. Advantages of RPLC in drug analysis
Chromatography and immunoassays are the two principal techniques used in research and clinical laboratories for the measurement of drug concentrations in biological fluids. The purpose of this article is to provide the reader with a general overview of these techniques and to present their relative merits and limitations. A glossary of technical terms is included at the end of the review.
A wide variety of silica-based,chemicallybonded stationary phases with different selectivitiesand functionalities (octyl (CJ, octadecyl (CJ, cyan0 (CN), phenyl, etc) are available. In addition, new stationary phases have been developed (7) which allow the direct separation of chiral drugs or direct injection of biological samples into the HPLC column A wide variety of drugs (polar, non-polar, ionic) can be separated Potential interfering polar substances originating from biological fluids are not retained on the column but elute with the solvent front Samples can be analysed with a minimum of clean-up Aqueous mobile phase is compatible with aqueous samples (clinical samples are aqueous) which means, after clean-up, they can be injected directly into the column
INTRODUCTION
Analysis of drugs in biological samples is undertaken in hospital laboratories for many reasons, the main ones being (a) therapeutic drug monitoring (TDM) in patients to assess potential toxicity and, (b) bioavailability and pharmacokinetic studies in patients or volunteers. It can involve quantification of drugs in the microgram (10-6g, or parts per million, ppm), nanogram ( 1 0 - 9 g , or parts per billion, ppb), or even picogram (IO-’’~, or parts per trillion, ppt) concentration range. Current approaches to drug analysis rely mainly on chromatographic and immunoassay techniques (1-5). This paper examines the role of these techniques in the drug analysis laboratory and assesses their usefulness and limitations.
Table 2. Advantages of HPLC in relation to GC 1
CHROMATOGRAPHIC TECHNIQUES
Thin-layerchromatography
Among chromatographic techniques, thin-layer chromatography (TLC) is primarily used for the separation and identification of compounds such as drugs of abuse. For qualitative analysis it is speedy, simple, inexpensive and offers a selection of spray reagents to locate the spots. For quantitative work, it is timeconsuming and labour-intensive and has lower sensitivity and resolution than gas chromatography (GC) or
2
3 4
HPLC is quicker than GC and has a wider range of applicability. It can be used for polar, non-volatile and thermally labile compounds. This permits the determination of a wide variety of drugs In general, a sample clean-up procedure is simpler for HPLC than GC. It usually entails a single solvent extraction step or a simple protein precipitation step (removal of protein) in order to protect the HPLC column from the deposits of protein Unlike GC, derivatization is not necessary in most cases HPLC systems, unlike GC, do not destroy the sample so that once the analysis is complete, the sample can be recovered for additional studies
326
A. C. Mehta
Table 3. Advantages of homogeneous immunoassays Homogeneous immunoassays are quick, technically easy to perform and are easy to automate. They are wellsuited to routine drug monitoring applications where the emphasis is on a fast, simple, and reliable method They can generally be carried out directly on biofluids. No sample pretreatment is required. It is the quality of the antibody that determines specificity. Chromatographic methods require some sample purification prior to assay They permit determination to be made on very small volumes of samples (50pl). Chromatography on the other hand requires at least 0.5-1 ml sample They are particularly useful for those drugs which have proved difficult to analyse by chromatographic techniques. For instance, digoxin and aminoglycosides being very polar and having no strong chromophoric groups, are difficult to analyse by either GC or HPLC They are chemically gentle and can therefore be used to determine unstable or labile compounds
high-performance liquid chromatography (HPLC). Morever, it is not fully automatable. The advent of high-performance TLC (HPTLC) has brought a revival of TLC. HPTLC plates are superior to conventional type, having much smaller silica particles (about 5 pm) and a very narrow particle size distribution. This has made possible the application of a small sample ( < 1 p1) on a plate and relatively rapid separation of compounds. Although widely used in industry, TLC has proved less useful as a quantitative tool in the hospital laboratory despite the availability of HPTLC plates and the equipment for their quantitative scanning. Gas chromatography
Gas chromatography (GC) can be performed on glass columns (ID 4mm) packed with non-polar or polar stationary phases or on flexible fused silica capillary columns (ID 0.53 mm or less) packed with chemically bonded stationary phases. Capillary columns are more popular because their performance is superior to the conventional glass columns. They are robust and offer high resolution, high sensitivity and faster separations (6).
Most GC methods of drug analysis use a flame ionization detector (FID), which can respond to all organic compounds, however, other more selective and
Table 4. Disadvantages of homogeneous immunoassays Because of the cost of the reagent kits and other consumables, immunoassaysare relatively expensive to run. The cost can only be justified if there are sufficient routine samples to be analysed They are more difficult, time-consuming and expensive to develop in users’ laboratories because facilities are required to raise antibodies to a specific drug. Chromatography on the other hand allows ease of method development for virtually any drug They can be used only for those drugs for which reagent kits are available. Manufacturers develop an immunoassay for a drug only when the need for monitoring it has been clearly indicated. To date, commercial kits are available for only a few classes of drug such as anticonvulsants, aminoglycosides,cardioactive drugs and for a few individual drugs, such as theophylline and methotrexate Generally they are less sensitive than the chromatographic techniques. Thus, although they are suitable for routine TDM where drugs are measured at a relatively higher concentration (steady-state levels), they are not particularly suitable for pharmacokinetic or bioavailability studies Interference can be caused by cross-reaction of the antibody with metabolites that are structurally closed to the parent drug, and in some cases with similar drugs that may be present in the sample. The specificity of an immunoassay can be improved if the cross-reacting compounds are removed prior to assay but this may become too time-consuming When more than one drug is being measured, a separate assay must be performed for each drug, whereas with chromatographic techniques several drugs can be measured on the same sample
sensitive detectors, such as the nitrogen-phosphorus detector (NPD) o r electron capture detector (ECD), are also used. The NPD has an enhanced sensitivity to nitrogen and phosphorus-containing compounds and is quite useful in the analysis of nitrogen-containing compounds such as anticancer drugs and tricyclic antidepressants. ECD responds to drugs containing electronegative substituents such as halogens or nitrates (e.g. benzodiazepines).The mass spectrometer (MS),as a GC detector, confers extreme specificity and sensitivity but requires complex instrumentation and highly trained personnel. It is very expensive and not within the easy reach of an average hospital laboratory budget. GC is a very sensitive technique; however, because it requires volatile and thermally stable samples, it is not
Clinical drug analysis 327 Table 5. General comparison of
chromatographicand immunoassay techniques
Chromatography Sample requirement Sample clean-up Sample derivatization
0-5-1-0 mi
50-100 pl
Usually required Sometimes (HPLC),frequently (GC)
Not required Not required
Analysis time
Relatively slow
Fast"
Technical skill and experience required Equipment cost Method developmentcost Running expenses Ability to measure several compounds simultaneously Sensitivity for TDM Sensitivityfor pharmacokinetic and bioavailability studies
Considerable
Minimal
Moderate
Moderate High Moderate
Accuracy 'Heterogeneous assays are relatively slow.
Immunoassay
Precision Automation
Low Low Yes
No
Adequate Adequate
Adequate Limited
High High Possible
Moderate Moderate Possiblet
?Partial for heterogeneous assays.
always suitable for highly polar substances which may be non-volatile or thermally labile. If a sample is nonvolatile and thermally labile, as are many drugs, it can be derivatized (e.g. silylation, acylation) to increase its volatility and stability but this makes the assay more time-consuming. GC is easily adaptable to automation. GC was once the dominating analytical technique for drug analysis. It still remains an important technique but shares the field with HPLC and immunoassays.
High performance liquid chromatography
Among the many forms of HPLC (normal-phase, reversed-phase, ion exchange, size exclusion), reversedphase liquid chromatography (RPLC) offers distinct advantages over the others, and is most widely used in drug analysis. In RPLC the functional groups of the stationary phase are non-polar and the mobile phase consists of polar solvents such as water, methanol, or acetonitrile. This situation is the reverse (hence the name WLC) to that for the normal phase chromatography where the stationary phase is polar (silica)and the mobile phase is non-polar (e.g hexane). The advantages of RPLC in drug analysis are shown in Table 1. HPLC is performed in stainless steel columns (150 x 4 mm ID)or plastic columns (100 x 8 mm ID) packed with non-polar or polar stationary phases. The
column sizes may vary with manufacturers. It can also be performed on microbore (1-2 mm ID)columns (8). Like their counterpart capillary GC columns, microbore columns possess high resolution capacity and sensitivity which could be advantageous in simultaneous analysis of several compounds. As small samples are required to work with microbore columns, the technique may become more useful in paediatric drug monitoring. Moreover because of the low flow-rates employed, there is a considerable saving of mobile phase solvents. In spite of these advantages microbore columns have not yet been widely used in drug analysis. One drawback of microbore columns is that compared to conventional columns, the analysis time is relatively longer which can decrease sample throughput. The most commonly used HPLC detector is an ultraviolet detector because many drugs show sufficient absorption in the ultraviolet region to allow spectrophotometric detection. Fluorescence, electrochemical (EC) or diode array detector (DAD)are also used but less frequently. Fluorescence and EC detectors are claimed to be more sensitive than the ultraviolet detector. DAD rapidly and continuously scans the ultraviolet spectra while a peak is eluting. This process offers a significant improvement in the provision of information on peak identity and homogeneity. HPLC detectors are reaching a level of sensitivity quite comparable to those of GC. Although GC-MS is highly developed,
328
A. C. Mehta Table 6. Glossary
Term
Definition
Antibody
An antibody is an immunoglobulin produced in the body in
Accuracy
Antigen (immunogen)
Antiserum Cross-reaction Hapten
Limit of: detection COD) Precision
response to stimulation by a large molecule (the antigen) and capable of reacting with the antigen. The binding of antibodies to antigens is highly specific Accuracy refers to the difference between the observed value (assay result) and the true or known value and is expressed as a percentage A compound which, under suitable conditions, can stimulate an immunogenic response when introduced into the body, resulting in the formation of specific antibodies. In order to achieve high specificity, the antigen should be a pure substance. Only very large molecules, such as proteins and polysaccharides, are antigenic. As drugs are usually small molecules they are not antigenic. Such small molecules, which bind to other larger carrier molecules to become immunogenic, are called haptens Serum containing antibodies Reaction of the antibody with substances other than the target antigen or hapten A small molecule that reacts with a specific antibody but does not itself stimulate antibody production. In order to make a hapten antigenic it must be covalently bonded to a large carrier molecule such as a protein (e.g. albumin) to form a hapten-protein complex. The method of attachment of the hapten to the protein determines the specificity of the resulting antibody The lowest concentration of an analyte (substance to be analysed) that the analytical method can reliably differentiate from background levels Precision (or reproducibility)describes the variation or scattering of data (assay results) around the mean and is usually expressed as coefficient of variation (0).
cv= Quality control (QC)
Recovery
100 x standard deviation
%.
The continuing evaluation of an assay performance (with regard to accuracy and precision) to ensure that the assay continues to work satisfactorily.This is usually done by assaying QC samples (samples containing known amount of substance to be analysed) and plotting the results on a control chart to identify whether any results are outside the limits In analysis, recovery of the analyte can be found by analysing samples of a known amount of analyte and using the formula Recovery =
Resolution
mean
Amount found Amount present
x 100%.
Resolution is the ability of a chromatographic column to separate two peaks. A high resolution column will give two very narrow, symmetrical peaks separated from the baseline continued
Clinical drug analysis
329
Table 6 . Glossary (Continued)
Term
Definition
Sensitivity
Sensitivity is used to pinpoint the ability of an analytical technique to detect and quantify a trace substance (e.g. drug) in a sample. If the technique is able to detect very low levels of a substance(mg/l or below), it is said to be very sensitive Specificity (orselectivity)is the ability of an analytical method to determine solely the compound (e.g. drug) of interest in the presence of potentially interfering compounds. In immunoassay, specificity is the ability of the antibody to 'recognize' (bind)only the antigen or hapten of interest, having little affinity for other closely related compounds The specific substance on which a given enzyme acts The concentrationor activity of antibody in serum
Specificity
Substrate Titre
the development of a combined HPLC-MS system has been hindered by experimental problems. One of the advantages of HPLC is its easy adaptability to automation. With autosampling and electronic data reduction facilities a large number of samples can be processed with ease. Further advantages of HPLC, particularly in relation to GC, are shown in Table 2. IMMUNOASSAYS
Immunoassays are based on competition between the drug to be assayed (D) and a labelled drug (D*) for a limited number of binding sites on the antibody (Ab) specific for the drug. The label may be a radioisotope, an enzyme or a fluorescent substance. The sample containing an unknown quantity of the drug is added to a known constant amount of labelled drug and antibody. As a result of the competitive reaction, some of the antibody-bound labelled drug is displaced by drug in the sample. At equilibrium, the proportion of the labelled drug remaining in the free (D") or bound fraction (D*Ab)reflects the concentration of the drug in the sample. D*
+ D + Ab
+
D*Ab DAb. Free drug Bound drug The analytical procedure measures the amount of free labelled drug (D*)or the fraction still bound to antibody (D*Ab). If these values are plotted against the known concentration of unlabelled drug, a calibration graph can be obtained from which the concentration of the drug (D) in the sample can be determined. Depending upon the nature of the label, the assay could involve the
measurement of radioactivity, ultraviolet absorption following enzymatic reaction, or fluorescence. The two general types of immunoassays may be classified as homogeneous and heterogeneous immunoassays. Homogeneous assays are based on the fact that antibody-bound molecules can be distinguished from unbound molecules without a separation step. The difference between the signals may arise because the signal may be suppressed, produced, or altered on binding. This makes the assay quicker (few manipulations are required) and facilitates automation allowing the laboratory to increase productivity and reduce the cost per test. Enzyme immunoassay (EIA) and fluoroimmunoassay (FIA) are the examples of homogeneous assays where D* and D*Ab are distinguished by their differences in optical properties (W absorption, fluorescence) without a separation step. Commercial optical immunoassays are usually homogeneous. Heterogeneous assays in contrast require separation of bound and free species. This is time-consuming and makes automation more difficult. An example of a heterogeneous assay is radioimmunoassay (RIA) where a radioactive label is used. Here there is no difference between the radioactivity signal produced by D* and D*Ab so that it is necessary to separate the two before measurement. RIAs are very sensitive but are time-consuming and costly. They require the use of radioactive material and specialist counting equipment. In addition, their range of application is limited, i.e. RIA kits are available for only a few drugs. They have remained popular for a long time but currently they are being succeeded by homogeneous non-isotopic immunoassays where
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enzymes (9,10)or fluorescent molecules (10,11) are used as labels. Homogeneous immunoassays are comparable in performance with RIAs with added advantages such as (a) reagents are relatively inexpensive and have a long shelf-life, (b) a variety of labels are available, (c) assays are simple, rapid, and automatable and, (d) there is no radiation hazard.
Homogeneous enzyme-and
fluoroimmunoassays
Enzymes are good labels for immunoassays because they catalyse a variety of reactions that can be monitored spectrophotometrically. In this category the enzyme multiplied immunoassay technique (EMIT) is most widely used. It is based on the reaction between the enzyme, glucose-6-phosphate dehydrogenase (G6PDH); the substrate, glucose-6-phosphate (G6P); and the coenzyme, nicotinamide-adenine dinucleotide (NAD). During the reaction NAD is reduced to NADH and the resulting change in absorbance, which is directly related to the concentration of the drug in the sample, is measured spectrophotometrically. FIA offers enhanced sensitivity compared to EIA. It may be homogeneous or heterogeneous, but the homogeneous variety is generally used in hospital laboratories. Two prominent homogeneous FIAs are 1 substrate-labelled fluoroimmunoassay (SLFIA), which is based on the measurement of fluorescence following enzymatic (fluorogenic) reaction between the enzyme P-galactosidase and the substrate P-galactosyl-umbelliferone,and 2 fluorescence polarization immunoassay (FPIA), which is based on the measurement of the fluorescence polarization resulting from the binding of fluorophorelabelled drug to specific antibodies.
Results obtained using EMIT and FPIA procedures correlate well with each other and with those obtained with GC and HPLC (12-14).As a result both these techniques (EMIT and FPIA) are now widely used in the routine TDM. The advantages and disadvantages of homogeneous immunoassays are presented in Tables 3 and 4.
chemiluminescent compounds, metal atoms, and proteins have been proposed which provide alternative methods for drug immunoassays. Furthermore,a number of solid-phase (ordry-phase) immunoassay systems (e.g. for theophylline) have been developed recently (15). Here all the key reagents are immobilized on a strip or a slide requiring only the addition of a sample (blood or serum).The results (instrumentalreadout) are availablein minutes. The new technology is designed to be rapid and simple to facilitate near-patient testing. Although promising, it is early days yet, for solid-phase immunoassays, and more critical work (evaluation) is needed before they are clinically utilized routinely.
CONCLUSIONS
Chromatographic methods (GC and WLC) are the most sensitive, specific, and versatile techniques for clinical drug analysis. They are preferred techniques for the research laboratories especially when information on both the parent drug and its metabolites is required. They can also be used in routine drug monitoring when immunoassays are unavailable. Immunoassays are in general more rapid, adequately sensitive and specificand require smaller sample sizes. They are well-suited to routine monitoring of drugs in a clinical setting. Although they require less training than the chromatographic methods, it is desirable to have a full knowledge of the sample and the assay method for the correct interpretation of the assay results. Immunoassays are expensive to run but the development of economic reagents could widen their use in the future. Chromatographic and immunoassay techniques have their particular advantages and disadvantages (summarizedin Table 5). and should be regarded as complementary to each other. As clinical decisions on drug dosages are often made on the basis of analytical results, it is pertinent that thoroughly validated analyticalmethods are used in drug assays. For reliable results it is also necessary to maintain good quality control of the assay to ensure that it continues to work satisfactorily.
REFERENCES
Recent approaches
Enzymes and fluorophores represent the most popular non-isotopic labels at present, but other labels such as
1. ChamberlainJ. (1985)Analysis of Drugs in Biological Fluids. CRC Press, Boca Raton. 2. Moffat AC. (ed)(1986)Clarke's Isolation and Identification of Drugs. Pharmaceutical Press, London.
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10. Beastall GH. (1985)Non-isotopic immunoassay methods. Laboratory Practice, 34, 74-81. 11. Gutierrez MC, Gomez-Hens A, Perez-Bendito D. (1989)
Immunoassay methods based on fluorescence polarisation. Talanta, 36,1187-1201. 12. Ratnaraj N, Goldberg VD, Lascelles PT. (1986)Correlation between fluorescence polarisation immunoassay and enzyme immunoassay of anticonvulsant drugs, and stability of calibration graphs. Analyst, 111,517-523. 13. Loomis KF, Frye RM. (1983)Evaluation of Abbott TDx for the stat measurement of phenobarbital, phenytoin, carbamazepine, and theophylline. American Journal of Clinical Pathology, 80,686-691. 14. Haver VM,Audino N, Burris S,Nelson M. (1989)Four fluorescence polarisation immunoassays for therapeutic drug monitoringevaluated. Chnical Chemistry,35,138-140. 15. Mould G, Marks V. (1988) The use of solid-phase chemistry in therapeutic drug monitoring. Clinical Pharmacokinetics, 1465-70.
