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 s
Trang 1© 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 contaminaobstruc-tion of a detector) in instrumental analysis
Th ere are numerous methods of extraction, according to target compounds In this chapter, the authors briefl 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 purifi caextrac-tion step is chosen to meet such demand
For extraction of polar or ionic compounds, a biological specimen can be acidifi 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] Th 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
Trang 226 Pretreatments of human specimens
i Ultrafiltration
Ultafi ltration is a separation method according to molecular sizes of compounds, and is also used for removal of macromolecules Many type of fi lters with various pore sizes for passage of macromolecules (30,000, 10,000 and 5,000 daltons) are commercially available (Millipore, Advantec or Whatman) Th e advantages of this method is the simplicity of handling and small volumes (<0.5 mL) of fl 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 Th e reagents being widely used for sedimentation are: methanol or acetonitrile, trichloroacetic acid or other acids, and ammonium sulfate or tungstate Th 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 fl 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 Tefl 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 Tefl on septum cap, and warmed (or heated) in a water bath
or on a block heater Aft 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 Th is method is very suit-able for gas chromatographic analysis Th 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 diff erent in diff erent compounds By utilizing the solubilhydrophobic-ity in organic solvent (diff erence
in partition coeffi cients), drugs and poisons can be extracted from an aqueous specimen into
an organic solvent by shaking them Various modifi 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.
Trang 3Th is method allows selective extraction of drugs according to the properties of the com-pounds (acidity or basicity) Th 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 diffi 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
Trang 428 Pretreatments of human specimens
Solid-phase extraction
Solid-phase extraction is used for separation of a drug from biological components by utilizing their diff erent affi nities to packing materials (stationary phase) [8] Originally, natural materi-als such as silica gel was used; but recently, many kinds of packing materimateri-als, to which various
functional groups and polymer materials had been bound (> Table 4.1), have been developed
and have become commercially available Th 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 (abselutTM 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 aft er solid-phase extraction
Th 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 effi cient adsorption of drugs and their effi 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 fi 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 ( TwisterTM, Gerstel)
⊡ Table 4.1
Kinds and characteristics of various packing materials for solid-phase extraction
Octadecyl (C18) group Reversed phase: highly hydrophobic
Aminopropyl (NH2) group Normal phase, reversed phase or weak cation exchanger Cyanopropyl (CN) group Normal phase or reversed phase
Trang 5Derivatization
Derivatization of a compound is usually used for volatilization and stabilization of a non-vola-tile or thermolabile compound, for modifi cation into a suitable form to be detected by a spe-cifi c detector (for example, pentafl uorobenzylation for ECD of GC and dansylation for fl 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 effi cient extraction of the derivatized product into an organic solvent
Th e authors briefl 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 leafl ets attached to each derivatization reagent
Handling procedures of solid-phase extraction.
⊡ Fig 4.2
Trang 630 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 pentafl 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-specifi c adsorption to gas
chroma-tographic columns; trifl 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, trifl 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 trifl uoroacetyl derivatives suff er from their instability and loss due to evaporation
Silylation
Th 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 Th e characteristic fragmentation patterns make structure analysis easier
Th 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 esterifi cation is made on the carboxylic acid com-pounds using hydrochloric acid-containing alcohol or diazomethane Th e latter reagent is con-sidered to be the best compound for esterifi 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, fl uorescence and optical activity to compounds to be analyzed For such derivatizations, reagents reacting with amino, carboxyl and hydroxyl groups are available Th e details are described in the book [11]
Trang 7Automated 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 diffi cult for the limited number of workers to treat many samples simultaneously Th e use
of automated pretreatment instrument is labor-saving, decrease artifi 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
Th e automatic pretreatment instruments have been constructed for both liquid-liquid ex-traction and solid-phase exex-traction 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