An Analytical Approach to the Quantitation of Known Drugs in Human Biological Samples by HPLC* A. Bye and M.E. Brown, Department of Clinical Pharmacology, Wellcome Research Laboratories, Beckenham, Kent, U.K.
Abstract Although many HPLC methods are available in the literature only a fraction of these are applicable to the analysis of known drugs in human biological fluids. This paper presents the favoured approach of a laboratory involved in the quantitative assay of drugs in man for the subsequent study of pharmacokinetics and bioavailability.
Introduction On scanning the HPLC literature for methods potentially applicable to the analysis of drugs in biological samples, it is usual to see numerous examples of chromatographically excellent separations for many compounds. The sensitivity claims accompanying the separations are often more than adequate. However, the workers in the position of quantitative analysts of drugs in human biological samples find themselves eliminating the majority of methods and usually having to design their own. The following paper attempts to explain why this should be. Human Biological Samples (i) Preliminary preparation Typically an analysis is required of a drug in human blood, plasma, urine or faeces. However saliva, bile, seminal fluid, cerebrospinal fluid, sputum, aqueous humor, muscle and other tissues can be presented for analysis. The nature of each sample varies enormously in texture and chemical composition. For liquid chromatography it is the protein and other macromolecules which present the greatest potential problem. For example, the soluble protein in human plasma occupies about l°7o of the mass and when confronted with either organic solvents or changes in ionic strength or heat, alone or in combination, a precipitate results which usually ends the life of an HPLC column. The obvious answer to the soluble protein problem is to remove it. However, the procedures for doing this are troublesome. Protein precipitation by heavy metals, heat, alcohol, dialysis, trichloroacetic and perchloric acid and so on are never 100% efficient and although the protein problem is reduced it is not eradicated. An additional problem is that the drug of interest often co-precipitates with the protein. Selective extraction of the drug of interest with water-immiscible solvents is popular but relies upon the drug having a favourable partition coefficient. Other approaches are selective adsorption of the drug of choice (e.g., XAD resins, etc.), particularly useful for poorly extractable drugs. *To obtain a reprint of this article; please circle number 203 on Reprint Request Card.
However, this method usually needs an extra concentration step. For ionic non-extractable compounds, Chelex resins or ion-pair formation prior to extraction can be useful. When the protein is insoluble (e.g., muscle) some homogenisation or solubilising step is required before drug extraction can proceed. For gelatinous samples (e.g., seminal fluid, sputum, etc.), liquefaction is needed and we find sonication most useful. Faeces present problems of handling and of obtaining representative fractions but homogenisation in the minimum amount of deionised water can overcome these problems. In handling biological samples the quantity involved may vary from /ig or jil to several g or ml. The large samples require little explanation but the small samples require accurate pipettes, balances, microhomogenisers, deactivated glassware, etc., before reproducible results are obtained. In the development of sample cleanup methods, drug recovery experiments must be carried out in order to determine the optimum method. The health risks of handling human samples are well documented (1) and should be appreciated and the necessary precautions taken. (ii) Choice of HPLC as a technique So far a quantity of liquid relatively free of protein and containing a good proportion of our drug has been obtained. Before chromatographic separation can proceed, the 1500 or more other chemicals present in our sample must be taken into account and it must be decided whether the samples should be further purified or concentrated. The chief factors in this decision are detector sensitivity, interference from naturally occurring compounds and column performance. Also the question should always be asked of whether or not other techniques (e.g., GLC, fluorescence measurements, etc.) may be more suitable than HPLC for a particular compound or laboratory. As an example of detector sensitivity and therefore detector choice, assume a dose of 250 /ig of a drug is given to man. It was known from animal studies that the drug distributed into total body water, which for a man of 70 Kg averages 42 I. Therefore, about 6 jig/1 or 6 ng/ml would be expected in the protein-free fluid, assuming none was removed with the protein. If one ml of sample was provided then the detector should be able to detect 6 ng of drug. In actual samples, this order of sensitivity is unlikely for uv absorption detectors and it is asking a lot of the generally more sensitive fluorescence detection after taking into account "background" fluorescence. In cases of this sort, column efficiency is extremely important since narrower peaks implies larger signal-to-noise ratio and lower minimum detectable quantities. Where possible uv detection for HPLC is favoured as it is the most established technique. However, the worker in HPLC should always be aware of new detector developments (e.g., electro-
Reproduction (photocopying) of editorial content of this journal Is prohibited without publisher's permission.
JOURNAL OF CHROMATOGRAPHIC SCIENCE^VOL. 15
SEPTEMBER 1977»365
chemical, etc.). With the problem of interfering compounds, careful consideration should be given to the chemical structure of the drug. When the drug has been designed as a close analogue to a naturally occurring substance, the problems are obvious. It is ususal to exploit some minor difference between the two compounds usually by a detailed study of their physico-chemical properties (e.g. pKa for ion exchange, partition coefficient for partition chromatography, etc.). The final test of drug interference must, of course, be chromatography of dosed and undosed samples since metabolites, totally unrelated compounds and other drugs co-administered may cause interference problems. Once a decision has been made that HPLC is a suitable technique with regard to potential sensitivity, resolution and practicability, a workup procedure can be devised. (iii) Suggested purification procedures for HPLC One now assumes that a particular mode of HPLC must be selected. In our experience, adsorption on silica (spherical
Abstract Although many HPLC methods are available in the literature only a fraction of these are applicable to the analysis of known drugs in human biological fluids. This paper presents the favoured approach of a laboratory involved in the quantitative assay of drugs in man for the subsequent study of pharmacokinetics and bioavailability.
Introduction On scanning the HPLC literature for methods potentially applicable to the analysis of drugs in biological samples, it is usual to see numerous examples of chromatographically excellent separations for many compounds. The sensitivity claims accompanying the separations are often more than adequate. However, the workers in the position of quantitative analysts of drugs in human biological samples find themselves eliminating the majority of methods and usually having to design their own. The following paper attempts to explain why this should be. Human Biological Samples (i) Preliminary preparation Typically an analysis is required of a drug in human blood, plasma, urine or faeces. However saliva, bile, seminal fluid, cerebrospinal fluid, sputum, aqueous humor, muscle and other tissues can be presented for analysis. The nature of each sample varies enormously in texture and chemical composition. For liquid chromatography it is the protein and other macromolecules which present the greatest potential problem. For example, the soluble protein in human plasma occupies about l°7o of the mass and when confronted with either organic solvents or changes in ionic strength or heat, alone or in combination, a precipitate results which usually ends the life of an HPLC column. The obvious answer to the soluble protein problem is to remove it. However, the procedures for doing this are troublesome. Protein precipitation by heavy metals, heat, alcohol, dialysis, trichloroacetic and perchloric acid and so on are never 100% efficient and although the protein problem is reduced it is not eradicated. An additional problem is that the drug of interest often co-precipitates with the protein. Selective extraction of the drug of interest with water-immiscible solvents is popular but relies upon the drug having a favourable partition coefficient. Other approaches are selective adsorption of the drug of choice (e.g., XAD resins, etc.), particularly useful for poorly extractable drugs. *To obtain a reprint of this article; please circle number 203 on Reprint Request Card.
However, this method usually needs an extra concentration step. For ionic non-extractable compounds, Chelex resins or ion-pair formation prior to extraction can be useful. When the protein is insoluble (e.g., muscle) some homogenisation or solubilising step is required before drug extraction can proceed. For gelatinous samples (e.g., seminal fluid, sputum, etc.), liquefaction is needed and we find sonication most useful. Faeces present problems of handling and of obtaining representative fractions but homogenisation in the minimum amount of deionised water can overcome these problems. In handling biological samples the quantity involved may vary from /ig or jil to several g or ml. The large samples require little explanation but the small samples require accurate pipettes, balances, microhomogenisers, deactivated glassware, etc., before reproducible results are obtained. In the development of sample cleanup methods, drug recovery experiments must be carried out in order to determine the optimum method. The health risks of handling human samples are well documented (1) and should be appreciated and the necessary precautions taken. (ii) Choice of HPLC as a technique So far a quantity of liquid relatively free of protein and containing a good proportion of our drug has been obtained. Before chromatographic separation can proceed, the 1500 or more other chemicals present in our sample must be taken into account and it must be decided whether the samples should be further purified or concentrated. The chief factors in this decision are detector sensitivity, interference from naturally occurring compounds and column performance. Also the question should always be asked of whether or not other techniques (e.g., GLC, fluorescence measurements, etc.) may be more suitable than HPLC for a particular compound or laboratory. As an example of detector sensitivity and therefore detector choice, assume a dose of 250 /ig of a drug is given to man. It was known from animal studies that the drug distributed into total body water, which for a man of 70 Kg averages 42 I. Therefore, about 6 jig/1 or 6 ng/ml would be expected in the protein-free fluid, assuming none was removed with the protein. If one ml of sample was provided then the detector should be able to detect 6 ng of drug. In actual samples, this order of sensitivity is unlikely for uv absorption detectors and it is asking a lot of the generally more sensitive fluorescence detection after taking into account "background" fluorescence. In cases of this sort, column efficiency is extremely important since narrower peaks implies larger signal-to-noise ratio and lower minimum detectable quantities. Where possible uv detection for HPLC is favoured as it is the most established technique. However, the worker in HPLC should always be aware of new detector developments (e.g., electro-
Reproduction (photocopying) of editorial content of this journal Is prohibited without publisher's permission.
JOURNAL OF CHROMATOGRAPHIC SCIENCE^VOL. 15
SEPTEMBER 1977»365
chemical, etc.). With the problem of interfering compounds, careful consideration should be given to the chemical structure of the drug. When the drug has been designed as a close analogue to a naturally occurring substance, the problems are obvious. It is ususal to exploit some minor difference between the two compounds usually by a detailed study of their physico-chemical properties (e.g. pKa for ion exchange, partition coefficient for partition chromatography, etc.). The final test of drug interference must, of course, be chromatography of dosed and undosed samples since metabolites, totally unrelated compounds and other drugs co-administered may cause interference problems. Once a decision has been made that HPLC is a suitable technique with regard to potential sensitivity, resolution and practicability, a workup procedure can be devised. (iii) Suggested purification procedures for HPLC One now assumes that a particular mode of HPLC must be selected. In our experience, adsorption on silica (spherical
