Tài liệu Drugs and Poisons in Humans - A Handbook of Practical Analysis (Part 4) doc

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