Panasenko O. I., Melnik I. V., Samura T. O., Kremzer A. A.,

Buryak V. P., Sherbina R. A., Keitlyn I. M., Salionov V. A., Safonov A. A., Postol N. A., Gotsulya A. S., Kulish S. N., Panasenko T. V.

Zapororozhye State Medical University

MEASUREMENT OF NON-BOUND PLASMA CONCENTRATION

It is sometimes necessary to measure the concentration of non-bound (“free”) drug in plasma or serum, usually as part of TDM (therapeutic drug monitoring) of drugs such as phenytoin . The plasma “free fraction” is often in equilibrium with saliva, but equilibrium with the cerebrospinal fluid (CSF) concentration requires that the drug is able to cross the blood-brain barrier. For strongly (>90%) plasma protein-bound analyte, “free” plasma, saliva and CSF concentration are often very low and thus “free” plasma and CSF drug measurement are challenging and require high-sensitivity analytical methods, especially as sample volume is often limited. Spectroscopic methods have been used to investigate the interactions between ligands and dilute solution of purified protein, but for plasma, bound and non-bound drug are separated normally by ultrafiltration or equilibrium dialysis and the concentration of the analytic in each fraction measured independently. There are a number of issues to be borne in mind when planning and conducting plasma protein binding studies (see Box 1).

Box1. Measurement of plasma protein binding. 1. Ultrafiltration: -relatively quick; -change in protein concentration; -absorption of the analytic into the apparatus; -protein leakage. 2. Equilibrium dialysis: -often slow to reach equilibrium; - decomposition of the analyte and microbial growth; -adsorption of the analyte into the dialysis membrane; - protein leakage; - dilution of the sample.

Most method perturb binding to some extent. SPME (solid phase micro extraction) of plasma or serum can be used to measure non-bound drug directly providing the proportion extracted is small and does not perturb the binding [5]. The problem of protein leaking though the membrane applies to both ultrafiltration and equilibrium dialysis and should be monitored by protein assay. For highly protein-bound drug, concentration presents an analyte challenge. For some drugs, immunoassays may be sensitive enough for routine analysis of the non-bound fraction and kits are commercially Available that contain suitable calibrators for quantifying the total and non-bound concentrations. As phenytoin is approximately 90% bound in plasma the concentration of the calibrators for the free drug need only be order of magnitude less that the calibrators for measuring the total concentration. There is some doubt as to whether all the antibodies that are offered by some supplies are specific enough for the purpose [10]. However, for drugs that are >99% bound it may not be possible to obtain accurate concentrations using stand art laboratory methods given that analyte and protein concentration must be those attained under physiological conditions under these conditions it may be necessary to use radiolabelled drug. A very small quantity of high specific activity labeled drug is added to plasma, inculcate to ensure equilibration with non-labeled analytic, and the free and bound fraction are then separated for radioactive counting, usually by liquid scintillation spectrometry. Clearly, this approach is more suited to a research laboratory,

The filtration membranes used ere made from a variety of materials and have Mg cut-offs in the range Mr. = 10.000-30.000. Ultrafiltration should not be confused with ultracentrifugation in which protein-bound and free analyte can be separated as layers, often with the aid of a particularly useful for investigating binding to lipoproteins which the aid of a density gradient. This latter technique is particularly useful for investigating binding to lipoproteins, which can be separated as layers floating on the surface of plasma, the density of which has been adjusted with potassium bromide [7]. Control experiment to ascertain the magnitude of this problem should be conducted. It is good practice to collect serial samples of filtrate for analysis to ensure that the sample is representative of the filtration membranes need to be soaked in water or buffer before use and as results the first ultrafiltration collection(s) may be markedly dilute. Equilibrium dialysis is the “gold standard” for protein binding studies. However, it is not without problems. The tome for equilibration can be several growth, particularly with plasma at 37 °C, possible leading to changes in protein and analyte concentration and analyte binding,. If an antibiotic is added it cannot be assumed that it does not interfere with the binding. An advantage of dialysis is that the problem of absorption of analyte to the membrane and apparatus is largely overcome by measuring the concentrations on either side of the membrane. Adsorption will reduce the concentration in the donor and recipient (dialysate) solution, but at equilibrium the non-bound concentration in solution will be the same on either side of the membrane. Thus, it is possible to calculate the fraction bound free and to relate this to the initial plasma concentration of the analyte. Cleavage of conjugated is an important step in toxicological analysis, especially of urine. Many drugs and metabolites are extracted in urine and in bile predominantly as conjugates with D-glucuronic acid with with sulfate (acylass) or N- or S-glucuronides.

In order to maximize sensibility in drug/poison screening, and if appropriate, in order to measure such conjunction with indirectly, that is in conjunction with independent measurement of unconjugated analyte, either selective or non-selective hydrolysis of the sample can be undertaken prior to further sample processing. In quantitative work, calibration or QC (quality control) samples containing the conjugate of interest should be carried through the the chosen procedure in order to monitor the efficiency of hydrolysis. Incubation with strong mineral acid, such as an equal volume 5 mol Lˉ¹ hydrochloric acid, is cheap and gives rapid, but non-selective, hydrolysis of conjugates. All strong acid hydrolysis procedure are likely to introduce additional, hitherto unseen compounds into sample extracts even if due care is taken to ensure effective neutralization/buffering during sample preparation. Destruction of labeled analytes said as well as conjugates may be advantageous sample preparation. Destruction of labeled analytes as well as conjugates may be advantageous as well as conjugates may be advantageous as in the color test for paracetamol, but may lead to impaired selectivity as in the hydrolysis of certain benzodiazepine to 5-chloro-2-methylaminobenzphenone. In contrast, incubation with ß-glucuronidase and/or arylsulfatase Candice selective hydrolysis of conjugates under relatively mild conjugations. Generally cleaner extracts result, but the incubation time is longer, there may be matrix effects from the enzyme solution [6], and the enzyme preparations are relatively expensive. Control experiments should be performed to ensure that the incubation conditions are optimum with regard to the degree of hydrolysis and stability of the analytes. It should also be borne in mind that prolonged incubation of potentially infective samples may increase the title of the injective agent. Conventionally measurements in portions of agars such a sliver and brain have been performed via mechanical homogenization using a polytetrafluoroethylene-in-glass homogenizer, and/or acid digestion on, say, 5g wet weight of tissue perior to solvent extraction at an appropriate pH. Other tissues such as lung or muscle generally require use of a cutting or mincing action. However, digestion (12-16 h, room temperature) of small amounts (say 100 mg) of tissue with collagenase, protease, or lipase often gives much improved recovery when compared to conventional procedures and has the advantage that, once the digest has been prepared, analogous methodology and calibration standards to these used with plasma can be employed [4,9]. Various enzyme-based digestion procedures have been reviewed [11]. Although papain gave the highest recoveries of added drug from liver homogenate, subtilizing a gave similar recoveries for most compounds. Moreover, only a short incubation time (1h was required. A procedure developed to measure lidocaine in tissue specimens after treatment with subtilizing A using a longer digestion period to HPLC analysis of a solvent extract of the digest us outlined in BOX 2. There has been tentative interest in using ASE (accelerate solvent extraction) in the analysis of drugs and other poisons in tissues and related samples [1,3].

BOX 2. Subtilizing A digestion for HPLC of lidocaine in tissue [8]. Reagent - Lyophilized subtilisin A in sodium dihydrogen orthophosphate \disodium hydrogen orthophosphate buffer.

Method - Dissect as out 100 mg wet weight portions of tissue, remove excess fluid on filter paper, add tissue to pre weather 10 ml. Add 2g L^-1 subtilizing A, seal the tubes with ground –glass stoppers, and incubate in a water bath (50 ºC, 12 – 16 h). Cool the tulles, vortex-mix (5s), transfer 0,2 ml portions to 60·5 mm i.e. glass tube. Add 20 ml. Tris buffer (2ml · Lˉ¹, pH 11,0) and 20 ml internal standard solution (3mg · L^-1 aqueous bupivacaine) and vortex-mix (5s). Add methyl tret- butyl ether (200 ml) and vortex-mix (30 s).

Generally, derivatization should be avoided as it adds an extra step or steps to the analytical procedure and thus may increase the possibility of error. This being said, in GC derivative formation is performed to achieve satisfactory chromatography, to improve the detection characteristics of an analyte and sometimes to provide additional evidence of compound identity. Derivatization may be used to shorten or lengthen the retention time as required, and also to permit the separation of enantiomers. In HPLC, derivative formation, although sometimes used to stabilize an analyte, is seldom needed to achieve satisfactory chromatography except when performed to, permit the separation of enantiomers.

SUMMARY. The myriad of different approaches to analytical toxicology problems might seem at first sight confusing, but with experience and in the knowledge of the instrumentation and expertise available in a particular center, then certain standard approaches to similar problems will soon as developed. Those given here reflect personal experience.

REFERENCES

1. Lord H. L. Pawliszyn J. Microextraction of drugs. J. Chromatorg., 2000. –Vol. 902. – P. 17 – 68.

2. Rebert W. L., Annesley T.M., De B.K., Maulton L., Juenke J.M., Moyer T.P .Performance characteristics of for free phenytoin immunoassays of four free phenytoin immunoassays. Ther Drug Monit ., 2001 – Vol. 23. – P. 148-154.

3. Michael-Titus A. T., Whelpton R., Yaqub Z. Binding of temporfin to the lipoprotein fraction of human Serena. Br. J. Clin. Pharmacal., 1995. - Vol. 40 - P. 594 - 597.