Laura Cobbold

HCSII

You are asked to develop an analytical method for a lipophilic molecule of molecule
weight 299 for use as part of clinical trial. You have the option of developing either an
immunoassay based or chromatography based method. Discuss the relative merits of
each assay.

1. Introduction

Crucial to the success of any clinical trial is the careful development of analytical
methods with which to measure analytes of interest. The theoretical trial discussed here
involves a small lipophilic molecule of molecular weight 299. This could be for example, a
new drug being developed by a pharmaceutical company or a human steroid being
investigated in a clinical trial involving hormonal pathways. Any analytical technique
used in such a study would therefore have to identify and quantify the analyte in order
for the clinical trial to progress (Riley and Rosanske 1996).
There are many types of analytical method which can be used to quantify small
lipophilic molecules. Factors such as the scale and time frame of a trial will affect the
exact methodology chosen, as large scale trials set over a short space of time will
require a high-throughput mode of analysis. Any method developed for a trial may later
require scaling up if the drug or analyte is deemed fit for clinical use. It is therefore
advantageous if the method developed is suited to large scale analyses. Chromatography
and immunoassays are both commonly used in the analysis of small molecular weight
molecules. There are numerous variations of each method, and each has its advantages
and disadvantages for particular analyses.

2. Immunoassays

Immunoassay techniques were first described in 1959 and have since become a vital
analytical tool in science and medicine (Yalow and Berson 1959; Wild 2001).
Immunoassays exploit the ability of antibodies to bind tightly and specifically to a wide
range of target molecules. They generate a signal of certain intensity dependent on the
concentration of their target analyte. The requirements of a standard immunoassay are
listed in table 1 (Wheeler and Morley Hutchinson 2006). Immunoassays can vary in
complexity from simple single step turbidimetry assays to more complex immunoassays
involving a separation step between the formation of complexes and the measurement of
signal (heterogeneous assays) (Wild 2001).
Figure 1. The principles of
A
competitive (A) and sandwich (B)
assays. A. Competitive assays involve
the competition for binding between
labelled exogenous analyte and
endogenous with an intermediate
wash step. Signal is stronger the less
endogenous analyte is present, as
more sites are taken up by labelled
B
analyte. B. Sandwich assays involve
the formation of Ab:Ag:Ab sandwich
complexes. Signal increases with
analyte concentration.
Basic Requirements of an Immunoassay

An antibody to the analyte to be measured

A labelled form of the analyte (competitive immunoassay)
Or a labelled second antibody to the analyte (reagent excess immunoassay)
A separation system to separate antibody bound tracer from unbound tracer
Table 1. Requirements of an immunoassay (Wheeler and Morley Hutchinson 2006).

Competitive immunoassays (Fig 1A) are a heterogeneous type of immunoassay in which

labelled exogenous analyte competes with endogenous analyte for binding sites on
antibody. Sandwich assays (fig 1B) involve the use of two antibodies directed to different
epitopes on the same analyte and are one of the most sensitive form of immunoassay,
capable of measuring down to picomolar concentrations (Sapin and Schlienger 2003).
Immunoassays are often used in medical pathology laboratories due to their ease of
automation and speed of analysis. Many automated systems are available which employ
immunochemistry to analyse a large array of analytes in bodily fluids. Such analysers
generally utilise a single mode of detection and incorporate tests for multiple analytes
employing the same or similar chemistries (Siemens 2007).

3. Chromatography

Prior to the development of immunoassays, chromatography was the method of choice

for analysing pharmaceuticals and other small molecules (Snyder, Kirkland et al. 2010).
Some of the earliest methods used paper and thin layer chromatography, methods still
used today for their qualitative drug identification abilities and easy large scale
preparation (Adamovics 1997). Planar chromatography such as paper and TLC also
require little intricate equipment or specialist training for their operators (Kenyon, Flinn
et al. 1995). The introduction of gas chromatography (GC) to the analysis of small
molecules has increased sensitivity and speed of analysis (Braithwaite and Smith 1983).
The type of column in GC can greatly affect the degree of separation. Hundreds of forms
of stationary and mobile phase exist giving the user tight control over any particular
separation. Gas chromatography is most often used for the analysis of small, thermally
stable and volatile compounds such as alcohols (Heftmann 1983). For less thermally
stable analytes, the use of high performance liquid chromatography (HPLC) has allowed
greater sensitivity and separation to be achieved in recent years (Done, Knox et al.
1974). This method uses a liquid mobile phase at high pressure in order to separate
analytes. HPLC is now the most commonly used technique in pharmaceutical analysis
and allows for rapid and accurate analysis of a vast array of compounds.

4. Immunoassay or Chromatography?

When choosing between an immunoassay and a chromatography based technique for

use in a clinical trial, there are numerous factors to consider, with each method having
advantages and disadvantages. Immunoassays are often chosen due to their ease of
automation and speed of analysis (Wild 2001). For samples that need to be reported
very quickly such as drug levels in overdose patients, immunoassay turnaround times
can be as little as 12 minutes (Straseski, Stolbach et al. 2010; Treweek, Roberts et al.
2011; Sitcharungsi, Ananworanich et al. Microbiol Immunol. 2011 Dec 19) giving this
form of analysis a large advantage over chromatography which can take up to 2 hours to
process a single routine drug screening sample (Adamovics 1997). Chromatography
however, is becoming increasingly automatable with auto-injection, mobile phase
pumping and sample detection all being controlled by computer in modern
chromatography labs. It is often the preparation steps which significantly increase assay
time.

4.1 Analyte Size Considerations:

Whilst generally fast and easily automatable once established, the use of an
immunoassay for a small lipophilic molecule will be complicated by the often extreme
difficulty of raising an antibody to such an analyte. The small size of the analyte in
question limits the number of epitopes available for target and may mean that it evades
the immune system altogether. Larger immunogenic carrier molecules such as Keyhole
limpet hemocyanin (KLH) can be chemically joined to small molecules (haptens) in order
to elicit an immune response and produce useable immunoglobulins. The antibodies
produced can often be targeted to the carrier protein and not to the hapten of interest
(Pastor-Navarro, 2007, Singh KV, 2010). If suitable antibody is produced, then a
competitive (direct) immunoassay is often the preferred method for small molecules
(<1000MW) rather than sandwich assays as only one antibody is required to bind even
though sandwich assays are typically more sensitive and specific than competitive
assays (Ekins 1980; Quinton, Charruault et al. 2010). Two antibodies targeted to the
same 299 molecular weight molecule will result in steric hindrance due to the limited
space on the 299 MW analyte to accommodate both immunoglobulins at once. There is
little limitation in the size of analytes that can be separated and quantitated using
chromatography. GC/MS is routinely used for the analysis of alcohols which are typically
less than 100MW (Szczepaniak and Isidorov 2011). For a small lipophilic compound as
described in this theoretical trial, the use of reverse phase HPLC chromatography in
which a nonpolar stationary phase such as C18 is used could provide sufficient
separation once important parameters such as mobile phase composition and optimal
temperature are deduced. The wide range of variables which can be altered in such an
analysis can greatly enhance the sensitivity of chromatographic methods and whilst
there is often an element of trial and error in such assay design, chromatographic assay
development can be relatively fast compared to immunoassay.

4.2 Cost Considerations:

The cost considerations of developing either immunoassay or chromatography based

methods are important. Whilst automated immunoassay analysers are quick and efficient
for high throughput work, both the analysers and commercial kits required are
expensive. It is therefore normally cheaper to develop your own primary antibodies and
develop a method by hand. If considering a radioimmune assay, the extra cost of using a
radionuclide must be factored in (Wild 2001). Whilst the implementation of
chromatography techniques such as HPLC and GC/MS can be difficult for some labs due
to the high initial cost of equipment, this is often overcome by the use of the same
equipment for multiple. Once equipment is purchased, chromatography can be relatively
cheap in comparison to commercial immunoassays as many of the consumables are
made in house. The most expensive aspect of running routine chromatography is often
purchasing the internal standards for quality control and calibration (Heftmann 1975).
4.3 Mode and Limits of Detection:

The mode of detection is an important consideration in immunoassays. Antibodies or

exogenous competing analyte can be labelled with dyes, enzymes, radionuclides,
fluorescent tags and many others, allowing detection down to the picomolar range. The
nature of the label dictates the detection method. When designing a chromatography
method, the characteristics of the analyte must be considered in order to select an
appropriate mode of detection. Detection to the nanogram range is usually possible
using absorbance and to the picogram range for fluorescence. Flame ionisation used in
GC can have a detection limit down to the low picogram or femtogram range (Snyder,
Kirkland et al. 2010). HPLC or GC coupled to mass spectrometry is the most sensitive
method however with limits of detection in the low femtogram range. This technique
however, is limited to molecules which contain ionisable groups in order to be detected
in a mass spectrometer.

4.4 Potential Assay Interferences:

Immunoassays are particularly sensitive to many different kinds of interferences (see

table 2).

Type of Interference Assay Types Affected

Hook Effect Nephelometric, turbidimetric
and immunometric assays
Cross reactivity All, but primarily
competitive assays
Antibody Interference All, but primarily
immunometric assays
Matrix Effects All
Signal Interference All

Table 2. Common interferences in immunoassays.

The hook effect can occur when antigen is in excess to antibody and all binding sites on
the antibody saturate with endogenous analyte preventing labelled analyte binding and
creating a signal (Miller 2004). Modern analysers therefore often perform automatic
dilutions on samples with abnormally low results. Antibodies in immunoassays are often
not specific enough to target only the analyte of interest. Steroid hormones for example,
are particularly difficult to analyse by immunoassay due to potential cross reactivity of
antibodies to similarly structured steroids (Fitzgerald and Herold 1996; Wood, Ducroq et
al. 2008) (see fig 2A). In the case of cortisol, chromatography techniques are generally
thought to be the most accurate as several immunological methods for the detection of
urine free cortisol are known to cross react with the cortisol precursor 11-hydroxycortisol
resulting in over or under estimation of cortisol levels (see fig 2B) (De Brabandere, LM et
al. 1995). Steroid hormone immunoassays often perform poorly in assays requiring very
low detection limits for a medical application such as male estradiol measurements
(Potischman, Falk et al. 1994). In general such immunoassays perform well over higher
concentration ranges with sufficient inter and intra-assay variations for routine, high
throughput use (CV 10-15%)(Taieb, Benattar et al. 2002). HPLC and GC/MS are not
affected by cross reactivity.
A B

Fig 2. Immunoassays suffer from interference from similarly structured molecules in the sample
matrix. A, The steroid hormones have similar structures which contain four characteristic
cycloalkane rings. It is therefore difficult to distinguish between them using antibodies and over estimation
of individual hormone concentrations is common. B, a study of analytical methods for measuring urine free
cortisol showed that immunoassays often over or under estimate compared to more sensitive and specific
HPLC methods (Turpeinen, Markkanen et al. 1997).

The presence of heterogenous antibodies to animal antisera in a sample can result in

spuriously low results in some immunoassays (Preissner, Dodge et al. 2005) due to the
interfering antibodies binding to those in the assay, preventing the formation of
immunogenic complexes (Bjernera, Børmer et al. 2005). Other interferences can include
matrix effects, which are produced by the environment of the analyte in question i.e. pH,
ionic strength and lipid concentration of the serum/urine/plasma (Jafri, Khan et al. 2011)
and signal interferences which are mainly due to compounds with natural fluorescence in
the sample interfering with assay detection methods (Weber, Kapyaho et al. 1990).
Interference is also a major problem in therapeutic drug monitoring (TDM) by
immunoassay. The definitive method of analysing drug molecules is HPLC or GC/MS (for
volatile drugs, pentobarbital etc) which give better sensitivity and specificity over wider
assay ranges than immunoassays (Adamovics 1997). Immunoassays however, are
commonly used for routine measurements due to their speed, reliability and efficiency
(Booker and Darcey 1975; Wild 2001). Cross reactants are a problem in TDM
immunoassays often due to the similar structures of drug metabolites to the parent
drug, causing them to interfere with the assay (De, Jimenez et al. 2009). More specific
analytical techniques such as GC/MS or HPLC with UV or mass spectrometric detection
are unaffected by cross-reactants (Polettini 2006; Dasgupta 2008). The sensitivity of
immunoassays for drug monitoring can be an issue. For example, the therapeutic range
of the cardiac drug Digoxin is around 0.5-2 µg/L and toxic effects can occur when levels
exceed 2.4 µg/L (Heftmann 1975). This means there is little room for error in digoxin
measurement and any assay interferences can cause slightly raised or low
concentrations to be missed (Sun and Spiehler 1976). High bilirubin or triglyceride levels
in samples can also cause interference in immunoassays but have little effect on
chromatography methods.
Chromatography is subject to different forms of interference which must be
eliminated in order to correctly quantify molecules of interest. Contaminants in a sample
such as high concentrations of large proteins can overload a column, causing a change in
the separation achieved. Sample peaks can widen and change shape making analysis
harder. Mass spectrometry is subject to problems with separating identically charged
(isobaric) ions (Wise 1987). Isobaric ions of the same m/z which will be detected as a
single peak on mass spectra. Fine tuning the chromatography by changing variables
such as column length, width, stationary and mobile phase composition, pH and
temperature are potential ways of overcoming this issue.
There is usually little pre-measurement sample preparation required for
immunoassays although samples often require dilution. Chromatography in contrast,
often involves complicated, time consuming preparatory methods to remove protein,
remove analyte from binding proteins, increase compatibility of sample to matrix and
concentrate the sample. Solid or liquid phase extraction, evaporation, microdialysis,
filtration and centrifugation are common techniques carried out in order to remove
contaminants from samples which could potentially damage the column or interfere with
analysis. Derivatisation can also be carried out in order to fine tune an analysis and
improve separation (Heftmann 1975). This can involve the addition of chemical groups
to a non-volatile compound in order to volatise it ready for GC/MS.

5. Conclusion:

The analysis of a low molecular weight, lipophilic molecule is achievable by both

immunoassay and chromatography. The specific requirements of the trial e.g. limit of
detection required, sample type used and nature of analyte, would dictate which of the
two methods were eventually chosen. The funding and time available would also be
major factors in assay development. For increased accuracy and sensitivity whilst
sacrificing speed and a degree of automation, chromatography would be the technique of
choice. For simplicity, speed, automation and sensitivity within a limited concentration
range an immunoassay would be appropriate. Without detailed knowledge of the trial
and the analyte of interest, it is impossible to say for sure which method of analysis
would be best suited.
 6. References

Adamovics, J., Ed. (1997). Chromatographic Analysis of Pharmaceuticals. Chromatographic science.

New York, M. Dekker.
Bjernera, J., O. Børmer, et al. (2005). "The War on Heterophilic Antibody Interference." Clinical
Chemistry 51(1): 3.
Booker, H. and B. Darcey (1975). "Enzymatic immunoassay vs. Gas/Liquid chromatography for
determination of phenobarbital and diphenylhydantoin in serum." Clin Chem 21(12): 3.
Braithwaite, A. and F. Smith (1983). Chromatographic Methods. Nottingham, Chapman and Hall.
Dasgupta, A. (2008). "Therapeutic Drug Monitoring: Recognizing the Sources of Interferences in
Immunoassays." Clinical Laboratory News 34(4).
De, B., E. Jimenez, et al. (2009). "Analytical performance characteristics of the Abbott Architect i2000
Tacrolimus assay; comparisons with liquid chromatography-tandem mass spectrometry (LC-
MS/MS) and Abbott IMx methods." Clin chem acta 410(1-2): 6.
De Brabandere, V., T. LM, et al. (1995). "Three routine methods for serum cortisol evaluated by
comparison with an isotope dilution gas chromatography-mass spectrometry method." Clin.
Chem 41(1781): 3.
Done, J., J. Knox, et al. (1974). Applications of high-speed liquid chromatography. Edinburgh, John
Wiley and sons.
Ekins, R. (1980). "More sensitive immunoassays." Nature 6(284): 2.
Fitzgerald, R. and D. Herold (1996). "Serum total testosterone: immunoassay compared with
negative chemical ionization gas chromatography-mass spectrometry." Clinical Chemistiy
42(5): 7.
Heftmann, E., Ed. (1975). Chromatography, a laboratory handbook of chromatographic and
electrophoretic methods, Van Nostrand Reinhold.
Heftmann, E., Ed. (1983). Chromatography: Fundamentals and applications of chromatographic and
electrophoretic methods pt A. Chromatography, Elsevier scientific publishing.
Jafri, L., A. Khan, et al. (2011). "Comparison of high performance liquid chromatography, radio
immunoassay and electrochemiluminescence immunoassay for quantification of serum 25
hydroxy vitamin D." Clin Biochem 44(10-11): 5.
Kenyon, A., P. Flinn, et al. (1995). "Rapid Screening of Pharmaceuticals by Thin Layer
Chromatography: Analysis of
Essential Drugs by Visual Methods." Journal 0f AOAC International 78(1): 9.
Miller, J. (2004) "Interference in immunoassays: avoiding erroneous results." Clinical Laboratory
International.
Polettini, A., Ed. (2006). Applications of LC-MS in toxicology. Pavia, Pharmaceutical press.
Potischman, N., R. Falk, et al. (1994). " Reproducibility of Laboratory Assays for Steroid Hormones
and Sex Hormone Binding Globulin." Cancer Research 54: 5.
Preissner, C., L. Dodge, et al. (2005). "Prevalence of Heterophilic Antibody Interference in Eight
Automated Tumor Marker Immunoassays." Clinical Chemistry 5(1): 3.
Quinton, J., L. Charruault, et al. (2010). "Toward the Limits of Sandwich Immunoassay of Very Low
Molecular Weight Molecules." Anal. Chem 82(6): 5.
Riley, C. and T. Rosanske, Eds. (1996). Development and validation of analytical methods / edited by
Christopher M. Riley and Thomas W. Rosanske. . Progress in pharmaceutical and biomedical
analysis. New York, Oxford : Pergamon.
Sapin, R. and J. Schlienger (2003). "Thyroxine (T4) and tri-iodothyronine (T3) determinations:
techniques and value in the assessment of thyroid function." Ann Biol Clin 61(4): 10.
Siemens, H. D. (2007). "Centaur Assay Menu." from http://www.medical.siemens.com.
Sitcharungsi, R., J. Ananworanich, et al. (Microbiol Immunol. 2011 Dec 19). "Serum immunoglobulin
isotypes in healthy thai children aged 2-15 years determined by nephelometry." Microbiol
Immunol.
Snyder, L. R., J. J. Kirkland, et al. (2010). Introduction to modern liquid chromatography. Orinda,
Wiley.
Straseski, J., A. Stolbach, et al. (2010). "Opiate-positive immunoassay screen in a pediatric patient."
Clin Chem 56(8): 4.
Sun, L. and V. Spiehler (1976). "Radioimmunoassay and enzyme immunoassay compared for
determination of digoxin." 22 12(3): 2029.
Szczepaniak, L. and V. Isidorov (2011). "Gas chromatographic retention indices of trimethylsilyl
derivatives of terpene alcohols." J Chromatogr A 1218(39): 4.
Taieb, J., C. Benattar, et al. (2002). "Limitations of Steroid Determination by Direct Immunoassay."
Clinical Chemistry 48(3): 3.
Treweek, J., A. Roberts, et al. (2011). "Immunopharmacotherapeutic manifolds and modulation of
cocaine overdose." Pharmacol Biochem Behav 98(3): 13.
Turpeinen, U., H. Markkanen, et al. (1997). "Determination of urinary free cortisol by HPLC." Clinical
Chemistry 43(8): 6.
Weber, T., K. Kapyaho, et al. (1990). "Endogenous interference in immunoassays in clinical
chemistry." Scand J Clin Lab Invest 201: 6.
Wheeler, M. and J. Morley Hutchinson, Eds. (2006). Hormone Assays in Biological Fluids. Methods in
Molecular Biology. London, Humana Press.
Wild, D., Ed. (2001). The immunoassay handbook. London, Nature publishing group.
Wise, M. (1987). "Ultra high resolution daughter ion tandem mass spectra using a differentially
pumped dual cell fourier transform mass spectrometer." Ann. Chem 59: 5.
Wood, L., D. H. Ducroq, et al. (2008). "Measurement of urinary free cortisol by tandem mass
spectrometry and comparison with results obtained by gas chromatography-mass
spectrometry and two commercial immunoassays." Ann Clin Biochem 45(4): 9.
Yalow, R. and S. Berson (1959). "Assay of plasma insulin in human subjects by immunological
methods." Nature 184(1648): 2.