4 © Springer-Verlag Berlin Heidelberg 2005 I.4 Pretreatments of human specimens By Akira Namera and Mikio Yashiki Introduction Small amount of drugs and poisons incorporated into human bodies are hidden among large amounts of biological components, such as proteins, lipids, nucleic acids and membranes. It is not easy to detect only a target compound from such complicated matrices. Before instrumen- tal analysis, extraction procedure is usually essential and very important. Extraction methods are used for removal of such proteins and lipids existing in large amounts in biological matrices, for removal of impurity compounds interfering with chromatographic separation, for conden- sation of a target compound, and for removal of compounds causing troubles (such as obstruc- tion of chromatographic columns and contamination of a detector) in instrumental analysis.  ere are numerous methods of extraction, according to target compounds. In this chapter, the authors brie y present some pretreatment methods including extraction and derivatization usually being used in biomedical analysis. Many reviews and books on the details of extrac- tions are available [1–5]. Extraction methods According to the advancement of analytical instruments, there are some reports on the analysis of compounds using crude biological samples without any tedious extraction procedure (or with dilution with water only); this is solely dependent upon the high capability of an instru- ment. However, in view of the stability and tool life, it is desirable to make suitable pretreat- ments. In emergency medicine, where a long time for analysis is not permitted, a rapid extrac- tion method with the minimal puri cation step is chosen to meet such demand. For extraction of polar or ionic compounds, a biological specimen can be acidi ed with tartaric acid, followed by addition of acetone or ethanol, shaking of the mixture and centrifuga- tion. To extract metals, organic compounds in a biological specimen should be completely destroyed; dry or wet incineration methods are employed. For the details of the procedure, the readers can refer to the books [3, 6].  e authors describe some extraction methods only for organic compounds as follows. Deproteinization methods In analysis of drugs and poisons in human specimens, the main interfering compounds are pro- tein and lipids components. To remove these molecules, the following methods are being used. 26 Pretreatments of human specimens i. Ultrafiltration Ulta ltration is a separation method according to molecular sizes of compounds, and is also used for removal of macromolecules. Many type of  lters with various pore sizes for passage of macromolecules (30,000, 10,000 and 5,000 daltons) are commercially available (Millipore, Advantec or Whatman).  e advantages of this method is the simplicity of handling and small volumes (<0.5 mL) of  uid samples to be required. However, it is impossible to separate drugs or poisons from the endogeneous medium- and small-sized compounds by this method. ii. Sedimentation By adding acids or organic solvents to specimens, proteins can be denatured to form insoluble aggregates, which can be easily removed by centrifugation.  e reagents being widely used for sedimentation are: methanol or acetonitrile, trichloroacetic acid or other acids, and ammonium sulfate or tungstate.  is type of methods is simple, relatively rapid and thus suitable for use in the emergency medicine. Analysts, however, should be cautious of the serious loss of target com- pounds, because of their incorporation into the aggregated and sedimented macromolecules. iii. Dialysis Semipermeable membranes of tubular types are usually used for extraction of low-molecular compounds by dialysis. Typically, a volume of crude specimen  uid is packed in a membrane tube, which is then put in a large volume of an organic solvent in a beaker with stirring of a Te on-coated magnet bar. Since the movement of a drug stops, when an equilibrium is at- tained between the inner and outer solutions, complete recovery cannot be achieved by a single extraction. Although the handling procedure itself is very simple, it takes a long time to reach the equilibrium according to the kind of a target compound; this method is not suitable for treatments of many specimens. Headspace method A specimen is put in a vial with a Te on septum cap, and warmed (or heated) in a water bath or on a block heater. A er a suitable time of warming, a needle of a syringe is inserted through the septum to draw the headspace gas containing a target compound.  is method is very suit- able for gas chromatographic analysis.  e headspace method is widely used for analysis of volatile compounds, but is not suitable for thermolabile compounds. It is being used for analy- sis of ethanol and toluene [5]; and also used for semi-volatile compound such as ampheta- mines [7]. Liquid-liquid extraction method Many of drugs or poisons show hydrophobic properties, though their degree of hydrophobic- ity is di erent in di erent compounds. By utilizing the solubility in organic solvent (di erence in partition coe cients), drugs and poisons can be extracted from an aqueous specimen into an organic solvent by shaking them. Various modi ed methods of the liquid-liquid extraction were reported; each method has its advantage and disadvantage. An example of the methods is shown in > Figure 4.1. 27  is method allows selective extraction of drugs according to the properties of the com- pounds (acidity or basicity).  e mode of transfer of a drug from a phase to another phase is well known empirically and can be estimated physicochemically; this is very useful for analysis of an unknown compound. However, during extraction from specimens with high protein and lipid contents by this method, emulsion formation sometimes appears and makes it di cult to separate the two liquid phases clearly. Extrelut ® is a diatomite with a porous structure, and can adsorb and maintain a water phase on its surface. A crude aqueous specimen can be directly applied onto an Extrelut ® col- umn; then an organic solvent, which is not miscible with water, is used for elution of a drug. Although the procedure is very similar to that of solid-phase extraction, the principle for Ex- trelut ® is essentially liquid-liquid extraction, which takes place between aqueous and organic phases on the surface of the diatomite. A merit of the use of an Extrelut ® column is that emul- sion is not formed even for whole blood specimens. An example of separation of drugs by liquid-liquid extraction (cited from reference 2). ⊡ Fig. 4.1 Extraction methods 28 Pretreatments of human specimens Solid-phase extraction Solid-phase extraction is used for separation of a drug from biological components by utilizing their di erent a nities to packing materials (stationary phase) [8]. Originally, natural materi- als such as silica gel was used; but recently, many kinds of packing materials, to which various functional groups and polymer materials had been bound ( > Table 4.1), have been developed and have become commercially available.  erefore, the range of their selection has been ex- tensively increased. For the original types of solid-phase columns (cartridge), activation of the packing materials before use was required and the materials could not be dried throughout the procedure. As shown in > Figure 4.2, however, new items for solid-phase extraction without need for such activation (abselut TM NEXUS, Varian) and without need for cares not to dry up the column (Oasis ® , Waters) have been developed. To realize a high throughput for extraction, a plate for simultaneous extraction of as many as 96 samples is now commercially available. Condensation is required for a large volume of eluted solution a er solid-phase extraction.  is procedure takes a long time, when the volume of an eluent is large and the volatility of the eluent is relatively low. Recently, a thin disk (Empore Disk ® , 3M), which enables the e cient adsorption of drugs and their e cient elution only with a small amount of a solvent, has been developed. Solid-phase microextraction Solid-phase microextraction is a method employing adsorption of drugs to a stationary phase coated on a  ber attached to a microsyringe [9, 10]. Drugs adsorbed are desorbed inside an in- jection port of a GC instrument at high temperatures, inside an interface of an HPLC instrument or inside a capillary of CE, to introduce drugs into each analytical instrument. To adsorb drugs, both headspace and direct immersion methods are being used. Recently, a special stirrer magnet coated with a stationary phase has become commercially available ( Twister TM , Gerstel). ⊡ Table 4.1 Kinds and characteristics of various packing materials for solid-phase extraction Packing material Characteristic Octadecyl (C 18 ) group Reversed phase: highly hydrophobic Graphite carbon Reversed phase: highly hydrophobic Octyl (C 8 ) group Reversed phase: hydrophobic Silica Normal phase: polar and neutral Florisil Normal phase: polar and weakly basic Alumina A Normal phase: polar and acidic Cation exchanger Cation exchange Anion exchanger Anion exchange Mixed mode Reversed phase (C 8 ) plus cation exchanger Aminopropyl (NH2) group Normal phase, reversed phase or weak cation exchanger Cyanopropyl (CN) group Normal phase or reversed phase Diol (OH) group Normal phase or reversed phase 29 Derivatization Derivatization of a compound is usually used for volatilization and stabilization of a non-vola- tile or thermolabile compound, for modi cation into a suitable form to be detected by a spe- ci c detector (for example, penta uorobenzylation for ECD of GC and dansylation for  uores- cence detection by HPLC) and for detecting a high-molecular fragment peak in mass spec- trometry. In addition, a polar (ionic) compound is occasionally converted to a non-polar com- pound by binding a hydrophobic group to it for e cient extraction of the derivatized product into an organic solvent.  e authors brie y mention some methods of derivatization being widely used in bio- medical analysis as follows. For details on reagents and procedures, the readers can refer to the books [11] or instruction lea ets attached to each derivatization reagent. Handling procedures of solid-phase extraction. ⊡ Fig. 4.2 Derivatization 30 Pretreatments of human specimens Alkylation One of the most popular derivatization methods is alkylation; alkyl groups, such as methyl or propyl moieties, can be bound to acid or amino compounds using tetrabutyl ammonium (TBA) or penta uorobenzyl bromide (PFB-Br). Organic acids, salicylic acid and barbituric acids are frequently alkylated for GC analysis. Acylation Acylation is also widely used for derivatization of amino, hydroxyl and thiol groups, and it improves chromatographic separation by suppressing non-speci c adsorption to gas chroma- tographic columns; tri uoroacetyl chloride (TFA-Cl) and p-nitrobenzoyl chloride are used as reagents for acylation. Anhydrous conditions are necessary for the reaction according to the kinds of derivatization reagents. For the analysis of amphetamines, tri uoroacetylation is widely employed to prevent them from their adsorption to an injection port and to detect fragment ions in higher mass ranges. However, the tri uoroacetyl derivatives su er from their instability and loss due to evaporation. Silylation  is is a reaction for converting non-volatile compounds due to the dipole action of a hydro- gen donor group such as hydroxyl, phenol, carboxylic acid and amino groups into volatile ones.  e characteristic fragmentation patterns make structure analysis easier.  e silylation derivatization is usually used for analysis of morphine and codeine. Although these compounds can be analyzed by GC(/MS) in undelivatized forms, the derivatization gives much improvement of peak shapes and enhanced sensitivity. Esterification Acidic drugs containing a carboxylic acid group are highly polar, show tailing caused by inter- action between the drugs and a GC column, and are usually involatile due to association among the molecules. To solve these problems, the esteri cation is made on the carboxylic acid com- pounds using hydrochloric acid-containing alcohol or diazomethane.  e latter reagent is con- sidered to be the best compound for esteri cation, but shows danger of carcinogenesis and explosion; in place of the diazomethane, trimethylsilyldiazomethane dissolved in hexane is now commercially available, because of its safety. Other derivatizations Derivatizations are also used for purposes to add visible or ultra violet absorptivity,  uorescence and optical activity to compounds to be analyzed. For such derivatizations, reagents reacting with amino, carboxyl and hydroxyl groups are available.  e details are described in the book [11]. 31 Automated pretreatments In parallel with the increase of the number of poisoning incidents, the number of human spec- imens to be analyzed is increasing. Trace analysis is required in many cases of analysis of drugs and poisons; this means that a relatively long time is required for pretreatment of each sample. It is di cult for the limited number of workers to treat many samples simultaneously.  e use of automated pretreatment instrument is labor-saving, decrease arti cial mistakes and increase reproducibility and reliability of data. When hazardous compounds are handled, such instru- ment makes workers free from dangerous situation and increases safety.  e automatic pretreatment instruments have been constructed for both liquid-liquid ex- traction and solid-phase extraction. AASP ( advanced automated sample processors) are being sold by Gilson and Varian; PROSPECT from GL Sciences, Tokyo. References 1) Muller RK (ed) (1991) Toxicological Analysis. Verlag Gesundheit GmbH, Berlin pp 52–90 2) Brandenberger H (1974) Clinical Biochemistry. Principles and Methods. Walter de Gruyter, Berlin, pp 1425– 1467 3) Pharmaceutical Society of Japan (ed) (1992) Standard Methods of Chemical Analysis in Poisoning – With Com- mentary. 4th edn. Nanzando, Tokyo, pp 27–38 (in Japanese) 4) Mcdowall, RD (1989) Sample preparation for biological analysis. J Chromatogr 492:2–58 5) Seto Y (1994) Determination of volatile substances in biological samples by headspace gas chromatog-raphy. J Chromatogr A 674:25–62 6) Pharmaceutical Society of Japan (ed) ( 2000) Methods of Analysis in Hearth Science. Kanehara-shuppan, Tokyo, pp 372–382 7) Tsuchihashi H, Nakajima K, Nishikawa M et al. (1991) Determination of methamphetamine and amphetamine in urine by headspace gas chromatography/mass spectrometry. Anal Sci 7:19–22 8) Thurman EM, Mills MS (1998) Solid-Phase Extraction-Principles and Practice. John Wiley & Sons, New York 9) Pawliszyn J (1997) Solid Phase Microextraction-Theory and Practice. Wiley-VCH, New York 10) Pawliszyn J (ed) (1999) Applications of Solid Phase Microextraction. The Royal Society of Chemistry, Cam- bridge 11) Blau K, Halket JM (eds) (1993) Handbook of Derivatives for Chromatography, 2nd edn. John Wiley & Sons, Chichester Automated pretreatments . Reversed phase: hydrophobic Silica Normal phase: polar and neutral Florisil Normal phase: polar and weakly basic Alumina A Normal phase: polar and acidic Cation. Esterification Acidic drugs containing a carboxylic acid group are highly polar, show tailing caused by inter- action between the drugs and a GC column, and are