O.I. Panasenko, I.V.
Melnik, T.O. Samura, R.A. Scherbna, V.V. Parchenko, I.M. Keitlyn, V.A.
Salionov, A.S. Gotsulya, V.A. Safonov, A.A. Kremzer, N.A. Postol, V.P. Buryak,
S.N. Kulish
Zaporozhye State Medical University
GAS CHROMATOGRAPHY IN ANALYTICAL TOXICOLOGY
In analytical
toxicology gas chromatography (GS) has a number of advantages over other widely
used techniques such as HPLC and immunoassay. Firstly , GC has a range of
sensitive detectors, such as the "universal" flame ionization
detector (FID) and the selective nitrogen-phosphorus detector (NPD), electron
capture detection (ECD) and mass spectrometry (MS) detectors, which can be used
in parallel. Secondly, stable, high efficiency ( capillary) GC columns are now
widely available. Thirdly, GC is easy to interface with techniques giving
direct information about compound identity such as electron-ionization MS
(EI-MS) and Fourier-transform infrared spectrometer (FTIR) {2,10]. A compendium
of GC terms and techniques is available [3].
With GC, as with HPLC, qualitative and quantitative information can
often be obtained in the same analysis provided that appropriate calibration
and QC(quality control) procedures are followed. Temperature programming in GC
is analogous to gradient elution in HPLC, but is much simpler to perform and
permits the analysis of compounds of different volatilities in one analysis.
Moreover the return to starting conditions is easy and the interdependence of
Mr (relative molecular mass), retention time and column temperature is valuable
in aiding peak assignment is STA(systematic toxicological analysis). In
addition, GC retention data are reproducible between different days columns,
instruments, operators, centres and so on (see Box 1).
Box 1. Advantage
of GC in analytical toxicology
-Can inject
aqueous in some applications such as ethanol although headspace almost as easy
and applicable to wide range of volatiles.
-Easy to use
SPME(solid phase microextraction)/ thermal desorption.
-Range of stable,
efficient Capilary Columns.
-Sensitive
universel flame ionization detector(FID) and selective nitrogen-phosphorus
detector, evaporative light detector, mass spectrometry (NPD, ECD, MS)
detectors.-Qualitative and quantitative:
a) wide-bore
capillary columns used with injection liners facilitate quantitative work
b)
Inter-dependence of elution time column temperature, and Mr. valuable in
qualitative work.
-Retention data
reproducible between days/columns/ operators laboratories/countries.
-Temperature
programming and interfacing to MS or FTYR easy-need few column types because of
high resolving power especially with GC -MS. Can generate EI (electron
ionization) spectra from GC-MS.
-Can generate EI
( electron ionization ) spectra from GC-MS
-Principal peak
index valuable in compound identification. Can be used for a wide range of
gases and solvents. Disadvantage of GC include the requirement what the analyte
or a derivative should be volatile and stable at the temperature required for the
analysis(in practice below 400C or so).
In additions,
substances with highly polar or ionizable functional groups may give poor
performance (broad, tailing peaks), and some form of sample preparation is
normally need ( see Box 2). Moreover, same compounds their may be thermally
labile and the decomposition products may interfere in the analysis. Thus, in
addition to the choice of the sample preparation procedure, the column and the
chromatographic and detection conditions, due consideration must be given to
other factors including sample collection and storage , choice of internal
standard and quality assurance. This being said , GC remaining the method of
choice for gases and other volatiles such as ethanol and inhalational
anaesthetics. GC is also widely used in the analysis of other compounds, in STA
, and as an interface to MS, an especial advantage here being that EI spectra
can be obtained.
Box 2.
Limitations of GC in analytical toxicology need experienced operators, pure gas
supplies, consumables, support from instrument manufacturer.
-Septum
injection-need frequent maintenance.
-Sequential
analysis-need auto injectors.
- Usually need
same form of sample pretreatment.
-Cannot adjust
selectivity by modifying eluent.
- Cannot analyze
very polar/high Mr compounds, although can sometimes derivatize.
-Cannot analyze
carbon monoxide directly by FID.
-There is always
the possibility of interference from co eluting compounds.
Typically a GC
system consists of a gas to the column as well as gases, such as compressed air
and hydrogen, to the detector, a sample injection system, an analytical column,
and a detector with associated data acquisition/processing. The injector,
column and detector ovens are normally being maintained at a higher temperature
than the maximum temperatur by the column oven to minimize the risk of
frantionation or condensation of sample components in the detector. A similar
consideration applies to the injector oven in isothermal operation same
detectors, such as the ECD require an additional "make-up" gas flow
in order to give optimum performance.
Gas
chromatographs are usually purchased as a single unit, with gas supplies and
data capture being separate. Increasingly instrument control is from a personal
computer that also provides data capture facilities. Although commercially,
supplied cylinders are a conventional source of the gases needed in GC
operation, alternatives are available for air (sample compressor), hydrogen
(electrolytic hydrogen generator ) and nitrogen ( nitrogen generator based on
molecular sieve). Flowever, regular monitoring and maintenance, and the use of
appropriate filter to remove hydrocarbons, oxygen and moisture are mandatory.
filters should always be used with gas from cylinders.
The refinement of
capillary column technology and the introduction of "bench-top" GC-MS
systems have led to renewed appreciation of the value of GC in analytical
toxicology. In developing GC methods, knowledge of the Mr and structure of the
compound of interest is important even if a published method is to be followed.
Information on any co-formulated or co-administered may sometimes be able to
give details of assay methods, current literature and potential internal
standards for particular compounds.
The role of
capillary GC in STA has been discussed extensively [5,8], and more recently has
been compared with HPLC-DAD, HPLC-MS and other techniques [4,9]
Packed column GC
retention data for many drugs ans other poisons on a range of stationary phases
of differing polarities are highly correlated [7] due to the inter-relationship
between Mr, volutility and retention that is dominant in GC.
GC offers
advantages over spectrophotometric methods for carbon monoxide analysis
especially if badly decomposed postmortem blood or tissues are to be analyzed
[6]. Flowever, because the sensitivity for carbon monoxide by FYD is very poor,
either TCD or analyte reduction with hydrogen on a heated Ni catalyst to
produce methane before FID have had to be used. This latter procedure
introduces an additional step, and requires non-standard apparatus. With the
development of the helium discharge ionization detector it is possible to
measure carbon monoxide directly with good sensitivity. Helium is generally
used as the carrier gas and as the ionized species. Sample manipulation, assay
calibration, calculation of % COHb, and so on, are the same as with the use of
TCD [6].
Blood cyanide
concentrations have measured by GC-NPD using acetonitrile as internal standard
after addition of phosphoric acid to the sample in a headspace vial[1].
Assay calibration
was be addition of potassium cyanide solution to alkalinized human blood.
The earliest
GC-FID method for the measurement of blood ethanol involved simple dilution og
the sample (hole blood, plasma or urine) (50 ml) with internal standard
solution (0,16 gl-1 agueous propanol, 500 ml) followed by vortex-mixing (10 s)
and direct injection of the resulting mixture onto a column packed with a
molecular sieve such a Chromosorb. Other volatiles such as methanol, 2-propanol
and acetone, were resolved and could be measured if required-Modified cardon
black materials can also be used.
Nowadays, static
headspace sampling combined with temperature-programmed GC on a PDMS
(polydimethylsiloxane)can be used to screen for not only ethanol, methanol and
2-propanol, but also for a wide range of other volatile compounds in biological
fluids.
SUMMARY
There is no doubt
that GC is the system of choice for the analysis of solvents and other
volatiles, in STA, and as an interface with MS. The high efficiency and
stability of modern capillary columns together with other features of GC, such
as the ease of temperature programming and the reproducibility of retention
data, are features that other chromatographic systems cannot match. The
drawbacks of GC are the need for dedicated instruments, experienced operators
and appropriate laboratory infrastructure. There is also the requirement to
perform solvent extraction or same similar method of sample preparation and the
restriction on the Mr of analytes to those that are stable and volatile,either
derivatized or derivatized at the oven temperature required for the analysis (
in practice up to Mr 750 or so ). The great advantages of LC, especially when
combined with MS, are that these letter restrictions are minimized. But all is
not necessarily straightforward even with LC and LC-MS.
REFERENCES
1.
Calafat A.M., stasfile S.B., RAPD
quantitation of cyanide in whole blood by automated head space gas
chromatography. I. Chromatogr., 2002.-val.772.-pp131-137
2.
Grod R.L. and Barry E.F. Modern
Practice of Gas Chromatography, 5 thedn. Wiley-Intencience, New York, 2004.-364
p.
3.
Hinshaw I.V. A compendium of GC
terms and techniques. LC GC North America, 2002.-val.20.-pp.1034-1040.
4.
Maurer H.H. Position of
chromatographic teachniques in screening for detection of drugs os poisons in
clinical and forensic toxicology and/or doping control. Clin.Chem.Lab.Med.,
2004.-val.42.-pp. 1310-1324.
5.
Maurer H.H. Systematic toxicological
analysis of drugs and their metabolites by gas chromatography-nass
spectrometry. I.Chromatogr. 1992.-val.580.-pp. 3-41.
6.
Mayes R.W. ACP Broadsheet № 142:
November 1993. Measurement of carbon monoxide and cyanide in blood.
I.CLIN.Pathal., 1993.-val.47-pp 982-988.
7.
Moffat A.C., Stead A.H., Smalldon
K.W. Optiomum use of paper, thin-layer and gas-liqued chromutography for the
identification of basic drugs. Gas-liquid chromatography.I. Chromatogr.,
1974.-vol.90.-pp.19-33.
8.
Peters F.T. Schaefer S., staack
R.F., Kraemer T., Maurer H.H. Screening for and validated quantification of
amhetamines and amphetamine-and piperazine-derived designes drug in human blood
plasma by gas chromatography mass spectrometry. I. Mass Spectrom.,
2003.-val.38.-pp.659-676.
9.
Polettini A. Systematic
toxicological analysis of drugs and paisons in biosamples by hyphenated
chramotographic and spectroseopic techiques.I. Chromatogr., 1999.-val.733.-pp
47-63
10.
Rood D. The Thoubleshooting and
Maintenance Guide for Gas chromatographers, 4th edn., Wiley, New York,
2006.-576 p.