Therapeutic Drug Monitoring (TDM)

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CPD Clinical Biochemistry 2008; 9(1): 3–21
3
Reviews
Therapeutic Drug Monitoring (TDM)
RJ Flanagan, NW Brown & R Whelpton
Abstract
The measurement of plasma concentrations of drugs given
in therapy (therapeutic drug monitoring, TDM) is useful
for a small number of compounds for which pharmacological
effects cannot be easily assessed and for which the margin
between adequate dosage and potentially toxic dosage is
small. Thus, for some anticonvulsants, notably phenytoin,
anti-infective agents (antimalarial, antimicrobial, and
antiretroviral drugs), cardioactive drugs including digoxin,
most immunosuppressants, and certain psychoactive drugs
(notably clozapine and lithium), TDM may be used to
adjust the dose to individual need and to minimize the risk
of dose-related toxicity. Even for drugs with a wide margin
of safety, TDM may be helpful in assessing adherence to
therapy as a reason for treatment failure.
An appreciation of drug metabolism and of pharmacokinetics,
the study of the rates of drug absorption, distribution, metabolism
and elimination, is essential in understanding the influence of
age, sex, other genetic variables, disease, and other parameters
on the time course and clinical effect of drugs in the body. The
availability of a range of non-isotopic immunoassays compatible
with high-throughput clinical chemistry analyzers has meant
that certain TDM assays are widely available, but in many
cases chromatographic methods (nowadays usually HPLC or
LC-MS) have to be used. Whatever technique is used when
providing a TDM service, adherence to the principles of quality
management (proper method implementation and validation,
and adherence to internal quality control and external quality
assessment procedures) is essential since treatment decisions
may be based on the results.
Keyword
Pharmacokinetics, lithium, digoxin, psychoactive drugs.
Introduction
The measurement of plasma concentrations of
drugs given in therapy is useful for a small number
of compounds for which pharmacological effects
cannot be assessed easily and for which the margin
between adequate dosage and potentially toxic
dosage is small. However, assays for a much wider
range of compounds, and sometimes metabolites,
may be requested to assess adherence (compliance,
concordance) to therapy or to investigate and if
possible prevent adverse treatment effects, drugdrug interactions, or acute poisoning (Box 1). For
some agents drug dosage can be monitored indirectly
(Box 2). However, drug assays may still be requested,
for example in patients in whom antihypertensive
therapy appears refractory to treatment and adherence
is questioned.
Box 1: Indications for therapeutic drug monitoring
(TDM)
• Assess adherence
• Optimize dosage (maximize likelihood of
therapeutic benefit)
• Minimize risk of dose-related adverse effects
• Investigate possible adverse effects/drug
interactions/acute poisoning
Box 2: Biological effect monitoring
• Blood glucose – antidiabetic drugs (insulin,
chlorpropamide)
• Blood lipids – hypolipidaemic agents
• Blood pressure – antihypertensive drugs
• Electrocardiogram - antiarrhythmic drugs
• Prothrombin time (International Normalized
Ratio, INR) – anticoagulants (warfarin)
• Thyroid function tests - thyroxine
In all TDM work it is important to bear in
mind the purpose for which the analysis has been
undertaken when reporting and interpreting
analytical results.1,2 With some analytes such as
metals/trace elements there is a true ‘normal’ or
‘normally-expected’ range to provide a basis for
interpreting results. This is the case with lithium
because small amounts of lithium are present in the
diet. However, when lithium carbonate is used as a
drug to treat bipolar disorder (mania), for example,
research has shown that there is a range of plasma
lithium ion concentrations (0.6–1.0 mmol L–1) that
is associated with optimal therapeutic benefit and
minimal risk of toxicity. Such a range may be
referred to as the ‘therapeutic range’, ‘reference
range’, ‘target range’, or ‘therapeutic window’.
Lithium is a toxic drug. Mild adverse effects
can occur even at plasma concentrations of 1 mmol
L–1 when lithium is given chronically, with mild to
moderate toxicity being expected at 1.5–2 mmol
L–1, although patients in a manic state do seem to
have an increased tolerance to the drug. Severe
toxicity is likely above 2 mmol L–1. Initial features
of toxicity may include gastrointestinal discomfort,
Robert J Flanagan
Toxicology Unit,
Department of Clinical
Biochemistry, Bessemer
Wing, King’s College
Hospital NHS Foundation
Trust
Denmark Hill, LONDON,
SE5 9RS
Nigel W Brown
Immunosuppressant Drug
Monitoring Service
Institute of Liver Studies,
King’s College Hospital
NHS Foundation Trust
Denmark Hill, LONDON,
SE5 9RS
Robin Whelpton
School of Biological
and Chemical Sciences,
Queen Mary University of
London, Mile End Road,
LONDON, E1 4NS
Correspondence:
Dr RJ Flanagan
Toxicology Unit,
Department of Clinical
Biochemistry,
Bessemer Wing,
King’s College Hospital
NHS Foundation Trust,
Denmark Hill,
London SE5 9RS, UK
Email: robert.f [email protected]
nhs.uk
© 2008 Rila Publications Ltd.
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CPD Clinical Biochemistry 2008; 9(1): 3–21
Therapeutic Drug Monitoring (TDM)
nausea, vertigo, muscle weakness, and a dazed feeling.
More common and persistent side effects include fine
hand tremor, fatigue, thirst, and polyuria. Progressive
intoxication may manifest as confusion, disorientation,
muscle twitching, hyper-ref lexia, nystagmus, seizures,
diarrhoea, vomiting, and eventually coma and death.
Lithium is excreted primarily in urine and its renal
clearance is, under ordinary circumstances, remarkably
constant in individuals, but decreases with age and
when sodium intake is lowered. However, renal lithium
clearance may vary greatly between patients and lithium
dosage must, therefore, be adjusted on the basis of the
plasma lithium concentration 6–12 hours post-dose. The
plasma lithium should then be monitored 3–4 monthly to
ensure that dosage is still optimal.3
As indicated above a very important general point in
TDM is the time the sample is taken in relation to the last
dose of a drug, or the vein from which the sample is taken in
relation to an infusion or injection site. As regards an infusion,
the sample must be taken from a site in the body remote
from the infusion site, whilst for an orally administered drug;
the sample must be taken with knowledge of the absorption
profile of the drug. Peak plasma lithium concentrations are
normally reached 2–4 h after an oral dose, but equilibration
of lithium across the blood-brain barrier is slow, and thus
6–12 h should be allowed between dosage and sampling
to ensure that the plasma lithium reflects the lithium
concentration near to the site of action of the drug.
A standard TDM guideline is to sample immediately
before the next dose, or the following morning after an
evening dose (‘trough’ sample) to allow for absorption and
distribution to tissues to be completed before sampling.
With lithium and with the cardioactive drug digoxin, for
example, at least six hours must to be left between dosage
and sampling. Note that this guideline is irrelevant if
acute overdosage is suspected because speed of diagnosis
and treatment takes priority. On the other hand, with
ciclosporin, for example, it has been suggested that peak
sampling4 is possibly a better indicator of optimal dosage
than pre-dose sampling, but this brings additional problems
of ascertaining the time to peak in individual patients.
With lithium the reference range is a true target
range, but with many other drugs the range of plasma
concentrations associated with optimal therapeutic
benefit is much less clearly defined, and interpretation of
results has to be made in context (‘treat the patient not the
level’). If a result is below the ‘normally expected’ range
of plasma concentrations for a given dose, yet clinical
benefit seems optimal there is sometimes no indication to
alter the dose. However, where clinical benefit is difficult
to assess, with anticonvulsants or antipsychotics, for
example, there may be indications for adjusting dosage,
clinical observation notwithstanding. There may also be
indications for reducing dosage in the absence of clinically
apparent features of toxicity. The classic examples here are
digoxin, where the features of toxicity may be confused
with those of the disease (heart failure) being treated, and
aminoglycoside antibiotics and some immunosuppressant
drugs where toxicity may not be apparent until severe or
irreversible tissue damage has occurred.
TDM is normally aimed at providing quantitative
as well as qualititative information, hence there is little
demand for urine testing simply to assess adherence.
Moreover, urine testing may not be as simple as it sounds
as other drugs may interfere, or sensitivity may be less
than with plasma. However, when monitoring drug
usage in patients under treatment with methadone or
buprenorphine (maintenance/opiate withdrawal therapy),
qualitative information usually suffices and urine is the
specimen of choice for several reasons: (i) the plasma
methadone concentrations associated with clinical effect
vary widely depending on the patients tolerance to
opioids and hence there is no ‘therapeutic range’ as such,
(ii) urine is easier to collect than blood from patients
many of whom have damaged veins, (iii) urine is less
likely to be infective than blood, and (iv) it is important to
monitor illicit drug use in these patients at the same time
as monitoring adherence to methadone/buprenorphine.
Urine screening for drugs of abuse is thus normally
considered as a specialty in its own right, rather than as a
branch of TDM.
This review aims to give basic information to
help provide a TDM service. Clearly a knowledge of
drug metabolism and of pharmacokinetics (sometimes
abbreviated to PK), the study of the rates of the processes
involved in the absorption, distribution, metabolism, and
elimination of drugs and other agents, is fundamental to
advising on sample collection and in the interpretation
of results. Knowledge of the analytical methods available
and of laboratory operations (quality management) is also
important. Key references and other information relevant
to the major areas of application of TDM are provided in
a short gazetteer.
Units of measurement
In the UK and in other parts of Europe some laboratories
report TDM and other analytical toxicology data in
‘amount concentration’ using what have become known
as SI molar units (µmol L–1, etc.), while others, especially
in the US, continue to use mass concentration [so-called
‘traditional’ units (mg L–1, etc. or even mg dL–1)]. Most
published analytical toxicology and pharmacokinetic data
are presented in SI mass units per millilitre or per litre of
the appropriate f luid [the preferred unit of volume is the
litre (L)], or units that are numerically equivalent in the
case of aqueous solutions:
[parts per million] µg g1 µg cm3 µg mL1
mg L1 mg dm3 g m3.
Except for lithium (and sometimes toxic metals/trace
elements), methotrexate, and thyroxine, the UK NPIS/
ACB5 recommended use of the litre as the unit of volume
and SI mass units for reporting analytical toxicology
results. Conversion from mass concentration to amount
concentration (‘molar units’) and vice versa is simple
if the molar mass (Mr ) of the compound of interest is
known. However, such conversions always carry a risk
of error. Special care is needed in choosing the correct
Mr if the drug is supplied as a salt, hydrate, etc. This can
© 2008 Rila Publications Ltd.
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Therapeutic Drug Monitoring (TDM)
doses require the use of M-M kinetics to describe their
time course following overdosage.
cause great discrepancies especially if the contribution of
the accompanying anion or cation is high. Most analytical
measurements are reported in terms of the free acid or base
and not the salt.
First-order elimination
Introduction to pharmacokinetics
A general equation relating rate (–dC/dt), rate constant
(k), and concentration (C) is:
By subjecting the results of observations such as the
change in plasma concentration of a drug as a function of
time to mathematical analysis (‘mathematical modelling’),
pharmacokinetic parameters such as plasma half-life (t0.5)
and apparent volume of distribution (V ) can be calculated.
Having derived appropriate pharmacokinetic parameters
for a given drug it may then be possible to predict future
dose requirements, the effects of changing the dose or
the frequency of dosing on plasma drug concentrations,
and also the effects of changes in metabolism or
the co-administration of other drugs on these
parameters.
The commonest form of PK modelling is to treat the
body as if it were one or more volumes or compartments.
When a drug enters a compartment it is assumed that it
is distributed instantly and uniformly throughout the
compartment. In the single compartment model the
body is treated as if it were one homogeneous solution of
the drug. The equations used to describe the time course
of a drug are relatively simple and many fundamental
concepts of pharmacokinetics can be understood using a
single compartment model. However, it may be necessary
to use more complex models (the two compartment
and three compartment models). The available data are
rarely good enough to justify using more than three
compartments.
It is important to distinguish between rate and rate
constant. In chemical kinetics the order of the reaction (n)
is measured experimentally and is often close to an integer,
0 or 1, and so reactions are referred to as zero or first order,
respectively.
At therapeutic concentrations most compounds exhibit
first-order elimination, although the elimination of some,
notably high concentrations of ethanol, can be described
using zero-order equations. The kinetics of others,
phenytoin for example, can only be described adequately
using the Michaelis-Menten (M-M) equation. Many drugs
that exhibit first-order elimination kinetics at therapeutic
−
dC
= kC n
dt
(1)
where n is known as the order of the reaction. For a firstorder reaction, n 1. Substituting in Equation (1) gives:
−
dC
= kC
dt
(2)
Integrating Equation (2) gives:
C = C0 exp( −kt )
(3)
Equation 3 describes a curve that asymptotes to 0 from
the initial concentration, C0 [Figure 1 (a)]. Thus, the rate
of the reaction is directly proportional to the concentration
(amount) of substance present. As the reaction proceeds,
the concentration of substance falls exponentially, as the
rate of the reaction decreases. The first-order rate constant
has units of reciprocal time (for example h–1).
Taking natural logarithms of Equation (3):
ln C = ln C0 − kt
(4)
gives the equation of a straight line of slope, –k [Figure
1 (b)]. If common logarithms are used (log C vs t) the
slope is –k/2.303. Another way of presenting the data is
to plot C on a logarithmic scale. The slope is the same as
that of the exponential plot and decreases with time, but
the half-life can be read easily from the semi-logarithmic
plot [Figure 1 (c)]. The half-life (t0.5) is the time for the
initial concentration (C0) to fall to C0/2, and substitution
in Equation (4) gives:
t0.5 =
ln 2 0.693
=
k
k
(5)
as ln 2 = 0.693. This important relationship where t0.5
is constant (independent of the initial concentration)
Figure 1: First-order elimination curves: (a) C vs t, (b) ln C vs t, and (c) C vs t using a semi-logarithmic scale.
© 2008 Rila Publications Ltd.
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Therapeutic Drug Monitoring (TDM)
and inversely proportional to k, is unique to first-order
reactions. Because t0.5 is constant, 50 % is eliminated in
1 t0.5, 75 % in 2 t0.5, and so on. Thus, when 5 halflives have elapsed, less than 5 % of the analyte remains and
after 7 half-lives less than 1 % remains. The plasma halflife is a convenient and easily understood way of describing
the kinetics of a substance, but it is important to realise
that plasma half-life is controlled by clearance and the
apparent volume of distribution (see below).
V = X /C
Apparent volume of distribution has also been
described as a constant of proportionality that allows one
to calculate the amount of drug in the body from the
plasma concentration by rearrangement of Equation (9).
It is normally measured in an experiment in which a dose
(X0) of drug is injected intravenously (i.v.) and timed blood
samples are taken. C0 can be obtained from extrapolation
to t = 0 (Figure 1):
Zero-order elimination
V = X 0 / C0
For a zero-order reaction, n = 0, and:
−
dC
= kC 0 = k
dt
(6)
Thus, a zero-order reaction proceeds at a constant
rate, and the zero-order rate constant has units of rate (for
example g L–1 h–1). Integrating Equation (6):
C = C0 − kt
(7)
gives the equation of a straight line of slope, –k, when
concentration is plotted against time. The half-life can be
obtained as before, substituting t = t0.5 and C = C0 gives:
t0.5 =
C0
2k
(9)
(8)
Thus, the zero-order half-life is inversely proportional
to k, but t0.5 is also directly proportional to the initial
concentration (Table 1). In other words, the greater the
amount of drug present initially, the longer the time taken
to reduce the amount present by 50 %. The term ‘dose
dependent half-life’ has been applied to this situation.
(10)
Suitable markers, such as the dye Evans’ Blue, which binds
so avidly to plasma albumin it is restricted to plasma, inulin
which cannot penetrate cells, and isotopically-labelled water,
can be used to measure anatomical volumes (Table 2). Some
substances are confined to these volumes, but many have
values of apparent volume of distribution much larger than
total body water because they are extensively distributed in
tissues.
Table 2: Some examples of apparent volumes of distribution
Substance
V (L kg–1 body
V in 70 kg
weight)
subject (L)
Heparin
0.06
4.2
(a)
Evans’ Blue
0.05
3.5
Amoxycillin
0.2
14
()Tubocurarine
0.2
14
Inulin (b)
0.21
14.5
Ethanol
0.65
45
Phenazone
0.6
42
Deuterium
0.55 – 0.7
38 – 50
oxide (2H2O) (c)
Digoxin
5
350
Chlorpromazine
20
1400
Quinacrine
500
35000
Anatomical volumes: (a) plasma, (b) extracellular fluid, (c) total body water
Dependence of half-life on volume of distribution
and clearance
The elimination half-life is dependent on two fundamental
parameters: apparent volume of distribution (V ) and
plasma clearance (Cl). Changes in t0.5 may be a result of
changes in one or both of these parameters. Increasing
the apparent volume of distribution increases t0.5, while
increasing Cl decreases t0.5.
Apparent volume of distribution
The apparent volume of distribution (V) is the volume of
f luid that the amount (X) of a substance in the body would
have to be dissolved in to give the same concentration as
the plasma concentration (C) of the substance at the time
in question:
Whole body clearance
Whole body (or plasma) clearance (Cl) is the sum of the
individual organ clearances. Thus, for a drug that is cleared
by the liver (Clhep) and the kidney (Clren):
Cl = Clhep + Clren
(11)
Whole body clearance can be calculated from plasma
concentration–time data even though it may not be
possible to define all the individual organ clearances that
contribute. Thus, in Figure 2 the oval represents all the
organs eliminating the particular substance and hence
the f low is the total plasma f low, Q, to those organs. The
amount of drug, X, will be the plasma concentration,
Table 1: Comparison of zero-order and first-order elimination
Reaction order
Concentration vs.
time plot
Rate of reaction
Half-life
Dimensions of
rate constant
Zero
Linear
Constant
Proportional to
concentration
M T1
First
Exponential
Proportional to
concentration
Constant
T1
© 2008 Rila Publications Ltd.
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Therapeutic Drug Monitoring (TDM)
increase Cl, as may manipulation of urine pH to increase
the excretion of a weak electrolyte. Inhibition of drug
metabolizing enzymes may reduce Cl. Liver or kidney
disease may reduce Cl, but there may be accompanying
changes in V, so predicting the effect on t0.5 may not be
straightforward.
Absorption and elimination
First-order absorption
Figure 2: Representation of whole body clearance.
C multiplied by the apparent volume of distribution. Some
drug is removed by the organs and the plasma returns to
the systemic circulation. Elimination is first-order, and so
the rate of elimination of drug is:
−
dX
= kX = kVC
dt
(12)
However, the rate of elimination can be written in
terms of clearance:
−
dX
= C.Cl
dt
(13)
Thus, from Equations 12 and 13 combining and rearranging
gives:
Cl = V .k
(14)
Experimentally, k can be obtained from the slope of a
plot of ln C vs t (Figure 1) and V from Equation (10) and,
so Cl can be calculated from Equation (14). Furthermore,
k = 0.693/t0.5, so substituting for k in Equation (14) gives:
t0.5 =
0.693.V
Cl
(15)
Clearance is a measure of how well the eliminating
organs can metabolize or excrete a substance. Induced
synthesis of drug metabolizing enzymes, for example
in cigarette smokers or by certain other drugs, may
Other than following i.v. or intra-arterial (i.a.) injection,
administered drug has to be absorbed, and so the plasma
concentration–time curve must have a rising phase. The
kinetics of absorption after intra-muscular (i.m.) injection
might be expected to be first-order, i.e. the greater the
amount of drug at the injection site, the faster the rate
of absorption. Absorption from the GI tract may be
more complex, but frequently first-order absorption is a
reasonable approximation. The equation for the plasma
concentration as a function of time in a single compartment
model with simultaneous first-order input and output is:
C = F.
Dose ka
.
[exp( −kt ) − exp( −kat )]
V ka − k
(16)
where ka is the first-order rate constant of absorption
and F is the fraction of the dose that reaches the systemic
circulation. The concentration is maximal (Cmax) when
the rate of absorption equals the rate of elimination, after
which elimination dominates.
When ka k (as is often the case, or at least assumed to
be the case) the term exp(–kat) approaches zero as t increases
faster than exp(–kt) approaches zero. Consequently, the
equation approximates to a single exponential at later
times, as can be seen if lnC is plotted against t [(Figure
3(a)], allowing the elimination half-life to be derived.
Bioavailability
In Equation 16 the term F is included because in many
cases not all the dose administered reaches the systemic
circulation. F is sometimes confused with bioavailability.
Figure 3: Concentration–time curves showing first-order input into a single compartment model with (a) logarithmic y-axis and (b)
linear y-axis. Model based on Equation (16) with y-intercept of 15, ka = 0.3, and k = 0.1.
© 2008 Rila Publications Ltd.
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Therapeutic Drug Monitoring (TDM)
The US Food and Drug Administration (FDA) definition
of bioavailability is: ‘The rate and extent to which the
therapeutic moiety is absorbed and becomes available to
the site of drug action’. For some drugs, bioavailability
is complex, particularly for pro-drugs (compounds that
break down in the body to give the active drug), and so F
is calculated instead by dividing the area under the plasma
concentration–time curve (AUC ) after a test dose given,
for example, by mouth (per os, p.o.) or i.m. by the AUC
obtained after giving an equal sized i.v. dose:
F=
AUCpo
AUCiv
(17)
It is very important to consider the effect of F when
estimating expected plasma concentrations. Even if a
literature value of F is known, the extent of absorption may
be altered in overdose or by the presence of other drugs.
Maximum concentration
Cmax is sometimes taken as the maximum concentration
in the data set. However, it can be calculated. For a
single compartment model, the time the maximum
concentration occurs, tmax:
t max =
1
k
ln a
ka − k k
(18)
and
Cmax =
F.Dose
exp( −k.t max )
V
(19)
Note that tmax is dose independent, but Cmax is directly
proportional to the dose; this is an important feature of
first-order pharmacokinetics.
Intravenous infusion
When a drug is infused at a constant rate, k0, the plasma
concentration will increase as the infusion progresses,
but as the plasma concentration increases, the rate of
elimination also increases [Equation (12)] until the rate
of elimination equals the infusion rate. When this steadystate is reached the plasma concentration will be constant,
Css:
k0 = X ss .k = VCss k
(20)
This must always be the case whilst the elimination
kinetics are first-order. The concentration during the
rising phase is given by:
C = Css [1 − exp( −kt )]
(21)
Equation (21) represents an exponential curve which
starts at 0 and asymptotes to Css. It is in essence a decay
curve that has been f lipped over. Thus, as the decay curve
goes from C0 to C0/2 in 1 t0.5 the infusion curve goes
from 0 to Css/2 in 1 t0.5, i.e. 50 % of Css in 1 half-life,
75 % in 2, and 87.5 % of the steady-state value in 3
half-lives. The plasma concentration will be 99 % Css
within 7 half-lives. This is most easily seen from a plot of
time (in half-lives) versus % of steady-state concentration
(Figure 4). Because the rate of attainment of steady-state
conditions is a function of plasma t0.5, a drug with a short
half-life reaches steady-state before a drug with a longer
half-life. The infusion rate required to achieve a particular
Css can be derived from:
Css = k0 / Cl
(22)
Drug accumulation
Because subsequent doses of drug are often given before
all of a previous dose has been eliminated, the amount
of drug in the body will increase, but provided the drug
is eliminated according to first-order kinetics, will not
increase indefinitely. This is most easily understood by
considering a constant rate infusion.
Multiple dosage
A drug given as equal sized doses at equal intervals will
produce a plasma concentration–time plot similar to
one of those illustrated in Figure 5, depending on the
plasma t0.5 of the drug. The mean plasma concentrations
will asymptote to a steady-state value, in the same way as
Figure 4: Constant rate infusion – single compartment model. The infusion was stopped after 10 plasma half-lives.
© 2008 Rila Publications Ltd.
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Therapeutic Drug Monitoring (TDM)
Figure 5: Plasma concentration–time plots following repeated doses at equal intervals for a drug with (a) a short half-life, and (b) a
long half-life. The effect of a loading dose (broken line) is shown in (b).
during a constant rate infusion, but now the concentration
will f luctuate between doses. The f luctuations will be
greater if the drug has a shorter plasma t0.5 because a greater
proportion of the dose will be eliminated before the next
dose. If such drugs have a small therapeutic window,
it may prove difficult to maintain the concentration in
the required range. Morphine, for example, may cause
respiratory depression at the peak concentration, but
patients may experience pain before the next dose. The
extent of peak to trough f luctuations may be reduced
by giving divided doses more frequently or the use of
sustained-release preparations where these are available.
The average concentration at steady-state is:
Css, av = F
Dose
Cl.τ
(23)
where τ is the dosing interval. This is analogous to
Equation (22). For a drug with a long half-life, it may
take several doses before the plasma concentrations are
stabilised within the target range [Figure 5 (b)]. This delay
can be prevented by giving a suitable loading dose (LD):
LD = V .Css
(24)
TDM has no value during loading dosage as by
definition tissue equilibration will not be complete,
although subsequently TDM may be important to ensure
that loading dose regimens are optimal.6
Sustained-release preparations
Sustained-release (SR) preparations are designed to deliver
drug at a constant rate over a prolonged period thereby
simplifying life for the patient and hopefully improving
efficacy. By making the absorption rate constant (ka)
smaller than the elimination rate constant (k) one can
prolong the duration of action. As with any sequential
reaction the rate constant of the slowest step determines
the overall rate, and under these conditions, ka becomes
rate limiting (Figure 6).
SR formulations are available for most routes of drug
administration, including oral, i.m., subcutaneous (s.c.),
and transdermal. Oral SR preparations make use of
different particle sizes, of wax matrices, or tablets made
of layers of material, so that different rates of dissolution
give prolonged drug release. SR depot injections are
exemplified by long-acting antipsychotic preparations
such as f luphenazine decanoate. Doses may be given 2–4
weekly, which is useful when adherence to oral medication
is an issue. Several formulations of insulin are available,
including soluble insulin and several crystalline forms that
release insulin at different rates. Transdermal delivery of
drugs for systemic effect is a relatively new phenomenon,
Figure 6: Principle of sustained-release preparations.
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Therapeutic Drug Monitoring (TDM)
Glyceryl trinitrate (GTN) is readily absorbed though the
skin and may be applied as an ointment rubbed on to
an area of skin or as ‘sticking plaster’ patch. Hyoscine,
nicotine, buprenorphine, and some steroid hormones may
be given this way.
The terms controlled release (CR) or modified release
(MR) may be encountered and although these include
SR preparations, they also encompass enteric-coated
preparations that are formulated to disintegrate in the
bowel rather than the stomach.
Non-linear pharmacokinetics
The models described above, where elimination is firstorder, result in simple relationships that make dosing
and interpretation of analytical results relatively simple.
Clearance, t0.5, tmax, and time to reach Css are constant,
and AUC, Cmax, and Css are directly proportional to
dose. However, there are situations when such models
are inadequate, and the kinetics of some drugs, notably
phenytoin (at therapeutic concentrations) and some drugs
when taken in overdose, are best described by the M-M
equation:
rate =
Vmax .C
Km + C
(25)
When the amount of drug metabolizing enzyme
present is in great excess compared to the ‘effective’
concentration of drug (i.e. the concentration of drug at the
site of metabolism), Km C and denominator (Km C) in
Equation (25) approximates to Km (C making a negligible
contribution to the sum) so:
rate ≈
Vmax
C
Km
(26)
which is a first-order equation, and k = Vmax /Km. Thus,
even for drugs that are extensively metabolized, the
elimination kinetics will be first-order provided that
drug metabolizing enzyme activity is greatly in excess
of the amount of drug present. However, if the drug
concentration is high compared with drug metabolizing
enzyme capacity, C Km, and (Km Cl ) → C, hence:
rate ≈ Vmax
(27)
This is a zero-order equation because the reaction rate
is constant. The enzyme is saturated with substrate and the
reaction is at its maximal rate.
Drugs whose pharmacokinetics can only be adequately
described by M-M kinetics include phenytoin,7 ethanol,
and (at higher doses) salicylate. For first-order reactions,
steady-steady concentrations are proportional to dose, but
as one moves from first- to zero-order the concentration
rises disproportionately (Figure 7).
Adjusting the dose of a drug such as phenytoin to ensure
that plasma concentrations remain in the therapeutic
window is complicated by the fact that there are large
individual variations in Km and Vmax. Although ‘population’
values of Km and Vmax could be used to calculate doses to
obtain a required steady-state concentration, it is clearly
better to use individual values. Because there is a need
to solve equations for two unknown values, steady-state
concentration data for two doses are required. If the daily
dosing rate is R, then the M-M equation can be written
thus:
Vmax .Css
K m + Css
R=
(28)
Rearrangement gives:
R = Vmax −
R
Km
Css
(29)
which is the equation of a straight line of slope –Km, and
y-intercept, Vmax. Thus, values for Vmax and Km can be
obtained graphically (Figure 8).
Once Vmax and Km have been estimated then Css for a
particular dose can be obtained by rearranging Equation
(29):
Css =
Km R
Vmax − R
(30)
Because of the disproportionate increase in plasma
concentration with dose of drugs like phenytoin, the
dose has to be adjusted carefully, and although a daily
dose might be typically 300–500 mg, small tablet sizes are
available so that the dose can be adjusted appropriately.
Similarly, anything that changes the ‘effective’ dose, for
example changes in bioavailability, or drug metabolizing
Figure 7: Simulation of phenytoin pharmacokinetics in four subjects (A–D). The vertical broken line represents the Vmax value in
subject C.
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Therapeutic Drug Monitoring (TDM)
Figure 8: Graphical solution for Vmax and Km.
enzyme induction or inhibition, is likely to have a
large effect on the plasma concentration and hence
pharmacological action. Because Css approaches infinity
as R approaches Vmax [Equation (30)] it becomes more
difficult to control the plasma concentration (Figure 7).
Furthermore, the time it takes phenytoin concentrations
to reach steady-state values becomes progressively longer
as the dose is increased.
Plasma protein binding
Small molecules may bind to plasma protein. Acidic drugs
are often bound to albumin, and bases to albumin and
also to 1-acid glycoprotein (AAG). Binding to plasma
proteins is an important mechanism by which molecules
are transported in blood. Lipophilic molecules tend to be
bound extensively and the concentrations in plasma (i.e.
bound non-bound) can exceed the aqueous solubilities
of such compounds. Because there is very little protein in
cerebrospinal f luid (CSF), drug concentrations in CSF are
often very close to the non-bound plasma concentration.
A similar argument applies to saliva for weakly acidic
compounds such as phenytoin, primidone, ethosuximide,
and carbamazepine.8
Binding to plasma proteins reduces the apparent
volume of distribution by ‘holding’ the drug in plasma,
i.e. it reduces the concentration of drug that is free to
diffuse into tissues. Thus, protein binding will reduce
the activity of a drug if it reduces the amount available to
reach its site(s) of action. More importantly, drug activity
and, possibly toxicity, may increase if plasma protein
binding is reduced. This may occur in some disease states
which result in reduced plasma protein concentrations.
Displacement of one drug by another from plasma protein
binding sites is also a potential mechanism of drug-drug
interactions.
Many in vitro studies have demonstrated displacement
of one drug by another, but in vivo the situation is more
complex. The ‘total’ concentration of a displaced drug
in plasma will be reduced as some of the liberated drug
diffuses into tissues as new equilibria are established. The
increased concentration of non-bound drug may lead to
greater, possibly toxic, effects. Hence, measurement of the
‘total’ (bound non-bound) concentration of a drug in
plasma may be misleading in certain circumstances. When
phenytoin was displaced by salicylate, for example, the
percentage non-bound increased from 7.14 to 10.66 %,
and this was accompanied by a significant decrease in total
serum phenytoin concentration from 13.5 to 10.3 mg L–1.
The salivary phenytoin concentration rose from 0.97 to
1.13 mg L–1.9
Whether a plasma protein displacement interaction is
clinically important depends on a number of factors. The
displacing agents will usually attain plasma concentrations
approaching that of the binding protein and show
concentration dependent binding. Such agents include
phenylbutazone, salicylate, and valproate. However, for a
displaced drug with a large apparent volume of distribution
the amount displaced will represent a small proportion of
the dose and so the increase in tissue concentration is
unlikely to be significant.
Alternative matrices
Provided that the samples have been collected and stored
correctly, there are no significant differences in the
concentrations of drugs and other poisons between plasma
and serum. In addition to avoiding the possibility of
interference in lithium assay if lithium heparin is used as
the anticoagulant, an advantage of the collection of serum
if samples are to be frozen is that there is less precipitate
(of fibrin) on thawing. Nevertheless, collection of plasma
is convenient, and a heparinized or EDTA whole blood
sample will give either whole blood or plasma as appropriate.
Blood collection tubes that contain a barrier gel should
be used with caution, especially if basic drugs such as
antidepressants or benzodiazepines are to be measured.10
If a compound is not present to any extent within
erythrocytes, use of lysed whole blood will result in
approximately two-fold dilution of the specimen. The
immunosuppressants ciclosporin, sirolimus, and tacrolimus
are special cases because redistribution between plasma and
erythrocytes begins once the sample has been collected
and so the use of haemolysed whole blood is indicated for
the measurement of these compounds. The use of filterpaper adsorbed dried blood may present an alternative to
conventional sampling where refrigerated transport and
storage of samples may be problematic.11
Saliva is an ultrafiltrate of plasma with the addition
of certain digestive enzymes and other components.
There has been interest in the collection of saliva for
TDM purposes because collection is non-invasive and
salivary analyte concentrations are said to ref lect ‘free’
(non-protein bound) plasma concentrations. However,
reliable saliva collection requires a co-operative individual
and even then is not without problems. Saliva is a viscous
f luid and thus is difficult to pipette. Some drugs, medical
conditions, or anxiety, for example, can inhibit saliva
secretion and so the specimen may not be available from
all individuals at all times. Use of acidic solutions such
as dilute citric acid to stimulate salivary f low alters saliva
pH and thus may alter the secretion rate of ionizable
compounds. Schramm et al.12 have developed an in situ
device to collect saliva ultrafiltrate based on the principle
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Therapeutic Drug Monitoring (TDM)
of an osmotic pump. Use of this device to measure salivary
phenytoin and carbamazepine,13 and cotinine14 has been
described, but the method has not gained widespread
acceptance.
Keratinaceous samples (hair and nail) can be used to give
a record of drug exposure, but the analytical procedures
are complex and thus expensive, and are normally only
used in a forensic context when it is important to establish
prior drug use.
Analytical Methods
Use of an ion-selective electrode remains valuable for
lithium measurement. The availability of a variety of
immunoassay (IA) and other kits means that many TDM
assays can be performed more conveniently by such
means rather than by using chromatographic methods,
which require extensive resources in terms of hardware
and operator expertise. However, chromatographic assays
are still important in the case of amiodarone (where
it has proved impossible to produce an antibody that
does not cross-react significantly with thyroxine and
tri-iodothyronine), antiviral drugs, immunosuppressants,
many psychoactive compounds, and generally where
active metabolites should be measured as well as the
parent compound. Examples include carbamazepine/
carbamazepine-10,11-epoxide, procainamide/N-acetylprocainamide, and amitriptyline/nortriptyline.
Immunoassays
Except perhaps for in-house assays developed by, for
example, pharmaceutical companies, availability is
restricted to assays that are available as kits. Abbott, DadeBehring, Microgenics, Siemens, and Roche (Box 3) are
amongst the principal manufacturers, although there are
others, particularly of immunochromatographic devices
utilised in point-of-care testing (POCT) and related
areas.
Box 3. Immunoassay Kits: Websites
Please consult for up-to-date information!
• Syva EMIT
http://www.dadebehring.com/
• Abbott TDX
http://www.abbottdiagnostics.com/
• Roche
Abuscreen
http://www.roche-diagnostics.com/
• Microgenics
CEDIA
http://www.microgenics.com/
• Siemens
Immulite
https://www.smed.com/
Advantages of IA kits are that factors such as selectivity,
sensitivity, and precision (reproducibility) will have been
investigated beforehand, but they may be expensive and
may not be readily applicable to specimens other than
those for which they were developed, usually plasma or
serum in the case of TDM assays.
Enzyme-multiplied immunoassay technique (EMIT)
assays are available for the measurement of a number of TDM
analytes in plasma (Box 4). There is extensive experience of
EMIT. It is simple, has adequate sensitivity for compounds
Box 4. Dade-Behring EMIT Assays for TDM
• Antiasthmatic
Theophylline, caffeine
• Anticonvulsant
Phenytoin, phenobarbital, primidone, ethosuximide,
carbamazepine, valproate
• Antidepressant (group specific)
Amitriptyline, nortriptyline, imipramine, desipramine
• Antimicrobial
Amikacin, gentamicin, tobramycin, chloramphenicol
• Antineoplastic
Methotrexate
• Cardioactive
Digoxin, lidocaine, procainamide/NAPA, quinidine,
disopyramide
• Immunosuppressive
Ciclosporin, mycophenolic acid, tacrolimus
with Mr 200 present in biological fluids at moderate
concentrations and avoids the use of radioactivity.
In an EMIT assay, antibody bound to the analyte-enzyme
conjugate prevents substrate binding and reduces the rate
of formation of NADH. Analyte in the sample competes
with the analyte-labelled enzyme for binding to the
antibody, which increases the fraction of unbound enzyme
and thereby increases the rate of change of absorbance
(Box 5). Use of bacterial G- 6-PDH (NAD coenzyme),
avoids interference from endogenous G- 6-PDH, which
requires nicotine adenine dinucleotide phosphate (NADP)
as co-factor. Although the initial rate of the reaction is
proportional to the concentration of enzyme, the amount
of enzyme is not directly proportional to the analyte
concentration so the calibration curve is not linear, but has
a positive slope. Rate monitoring can be performed using
high-throughput clinical chemistry analyzers, which makes
this technique and other homogenous assays attractive
technically and commercially.
Box 5. Enzyme Multiplied Immunoassay Technique (EMIT)
• Homogenous assay – little if any sample preparation
• Mainly used for urine or plasma (but can be used for
other samples after e.g. solvent extraction)
• System: antibody to analyte, analyte bonded to glucose6-phosphate dehydrogenase (G-6-PDH), glucose-6phosphate, and NAD
• Antibody-enzyme complex inactive – added analyte
in sample displaces antibody from enzyme giving
enhanced enzyme activity
• Measure enzyme activity by monitoring NAD to NADH
conversion spectrophotometrically (340 nm)
The basis of homogeneous competitive f luorescence
polarization IA (FPIA, Box 6) is that when f luorescent
molecules are irradiated with polarized light of the
appropriate wavelength, freely rotating molecules emit light
in different planes. However, slowly rotating antibodybound f luorophores emit more light in a similar plane to
the incident light and this can be measured via use of a
polarising filter. Fluorescein (excitation wavelength 485
nm, emission 525–550 nm) is used as the f luorophore.
The background f luorescence present in biological samples
means it is usual to take a reading of the sample and reagents
before the addition of the f luorescent tracer.
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Therapeutic Drug Monitoring (TDM)
Box 6. Fluorescence Polarisation Immunoassay (FPIA)
• Use with plasma or urine – advantages/disadvantages
similar to EMIT except that not compatible with clinical
chemistry analyzers
• Fluorescein-labelled analyte rotates rapidly in solution
(Brownian motion) – if irradiated with polarised light,
emitted light not polarised
• When antibody to analyte added, bound analyte rotates
more slowly – emitted light retains polarisation
• Added analyte in sample competes with labelled drug for
antibody sites, increasing depolarisation of emitted light
– measure using polarimeter
• Amount of depolarisation related to the concentration of
analyte in the sample
Abbott introduced FPIA commercially as the basis of
the ADx analyzer for urine drugs of abuse work and the
TDx analyzer for TDM. Plasma or serum TDM assays
available include carbamazepine, digoxin, phenobarbital,
phenytoin, primidone, theophylline, and valproate. The
improved precision and reagent stability offered by FPIA
are advantages over EMIT, but FPIA is not compatible
with the clinical chemistry analyzers for which EMIT is
readily suited. Moreover, although f luorescence methods
are inherently sensitive, in FPIA sensitivity is limited by
protein concentration, hence the digoxin assay incorporates
a protein precipitation step.15
As with EMIT, Cloned Enzyme Donor IA (CEDIA)
exploits antigen-antibody binding to inf luence
spectrophotometrically-measured enzyme activity. βGalactosidase from Escherichia coli is supplied as inactive
fragments. The large fragment (some 95 % of the
enzyme) is termed the enzyme acceptor (EA), and the
smaller fragment is termed the enzyme donor (ED). By
conjugating analyte to the ED fragment, antibodies to
the analyte can prevent the formation of intact, active
enzyme. Any analyte present in the sample competes for
binding sites on the antibody, hence an increase in analyte
concentration will decrease binding of antibody to the
ED fragment and increase enzyme activity, which can
be monitored by production of chlorophenol red (CPR)
from CPR-β-galactoside (Box 7).
Again as with EMIT, CEDIAs are rate assays and may
be run on high-throughput clinical chemistry analyzers.
The technique introduced by Microgenics has a wide
dynamic range. Weak points of CEDIA include the
Box 7. Cloned Enzyme Donor Immunoassay (CEDIA)
• β-Galactosidase – split into two inactive fragments:
Larger fragment enzyme acceptor (EA), small fragment
enzyme donor (ED)
• When EA and ED are mixed they combine to form active
enzyme
• Sample and Reagent 1 (EA/antibody) first placed in well
• Reagent 2 (analyte labelled with ED and substrate) then
added
• Analyte in sample binds to the antibody preventing EDdrug conjugate from binding
• The higher the analyte concentration in the sample, the
lower the number of ED-drug conjugates bound to antibody
• More ED-drug conjugate available to combine with an
EA fragment – this combination forms the active enzyme
which in turn hydrolyses the substrate
• Hydrolysed product detected spectrophotometrically;
absorbance proportional to analyte concentration
fact that the ED and EA fragments are not as stable as
naturally occurring proteins, and the need to assemble the
complex means that it is susceptible to physicochemical
disruption.
Interferences and assay failures
Metabolites and other structurally-related compounds
often cross-react in IAs. This can be helpful in qualitative
work such as drug abuse screening, but is obviously
undesirable in quantitative work unless exploited as in
cross-reaction of digoxin assays used to measure other
digitalis glycosides. Interference in a number of CEDIA
measurements by a range of drugs has been reported
(Table 3).
Table 3. Drug interference reported in CEDIA16
Analyte
Interfering compounds
Carbamazepine
Doxycycline, levodopa, methyldopa,
metronidazole
Digitoxin
Rifampicin
Phenytoin
Doxycycline, ibuprofen, metronidazole,
theophylline
Theophylline
Cefoxitin, doxycycline, levodopa,
paracetamol, phenylbutazone,
rifampicin
Tobramycin
Cefoxitin, doxycycline, levodopa,
rifampicin, phenylbutazone
Valproate
Phenylbutazone
Digoxin-like immunoreactive substance (DLIS)
Elevated DLIS concentrations are encountered in
patients with a variety of volume-expanded conditions,
viz. diabetes, uraemia, essential hypertension, liver
disease, and pre-eclampsia.17 DLIS cross-react with many
antidigoxin antibodies and may falsely elevate plasma
digoxin concentrations in IAs. The association of DLIS
with volume expansion led to speculation that they could
be natriuretic hormones. Other structures that have been
proposed include non-esterified fatty acids, phospholipids,
lysophospholipids, bile acids, bile salts, and steroids.
Most reported endogenous DLIS are highly proteinbound, whilst only 20-30 % of digoxin is bound. It has
thus been suggested that measurement of digoxin in plasma
ultrafiltrates can be used to assess possible interference from
endogenous DLIS.18 More recently, an association with
DLIS measured using FPIA (digoxin and digitoxin assays)
with endogenous ouabain has been suggested.19 Steroid
hormones and bilirubin are other possible candidates for
DLIS as measured by FPIA20,21; such interference did not
occur using a digoxin microparticle enzyme IA.
Other digoxin-like immunoreactive substances
Spironolactone, canrenone, and potassium canrenoate
cross-reacted in earlier Abbott IAs,22 as did various plant
and other naturally-occurring materials. For example,
antidigoxin Fab antibody fragments have been used
to reverse toxicity from cardiac glycosides present in
plants such as Apocynum cannabinum (Indian hemp),
Digitalis purpurea (Purple foxglove), Nerium oleander
(Common or Pink oleander), and Thevetia peruviana
(Yellow oleander). Antidigoxin Fab antibody fragments
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Therapeutic Drug Monitoring (TDM)
have also been used to treat poisoning with toad venom,
the most toxic components of which are cardioactive
sterols (bufadienolides, notably bufalin, cinobufotalin,
and cinobufagin). All of these substances may crossreact in digoxin immunoassays, although monoclonal
digoxin IAs may fail to cross-react with these other
sterols and thus should not be relied upon to confirm
exposure.23,24
Chromatographic Methods
Conventional serum IAs of glycoside concentration are no
longer useful when the patient has been treated with Fab
fragments because the digoxin is already bound and not
available for competition in an assay system. Equilibrium
dialysis or ultrafiltration is required to measure free,
pharmacologically active digoxin. Digestion of the Fab
antibody fragment-digoxin complex using a proteolytic
enzyme is also required before measurement of ‘total’
digoxin as the affinity of the Fab fragment for digoxin
may well be similar to, or greater than, the affinity of the
antibody used in the IA. Plasma digoxin measurements
using conventional methodology may not be reliable for
up to 2 weeks post-treatment especially in patients with
impaired renal function.25,26 The use of a physical method
such as HPLC-MS to measure free digoxin would be
appropriate provided that sensitivity was adequate.
GC has been used to measure anticonvulsants, antidepressants, and some antipsychotics and cardioactive
compounds for many years, but nowadays HPLC or
LC-MS are the methods of choice for most TDM
applications if IAs are not available or suitable. HPLC
with UV or f luorescence detection still has much to offer
in appropriate circumstances, but LC-MS methods are
clearly the way forward in this as in many other areas
despite the high capital and running costs currently
associated with this technique. LC-MS is especially valuable
where high sensitivity and selectivity are needed, as with
many immunosuppressants, and with the antipsychotics
haloperidol, olanzapine, and risperidone.
As an example, LC-MS with atmospheric pressure
chemical ionization (APCI) can be used to measure
amisulpride, bromperidol, clozapine, droperidol,
f lupentixol, f luphenazine, haloperidol, melperone,
olanzapine, perazine, pimozide, risperidone, sulpiride,
zotepine and zuclopenthixol, as well as norclozapine,
clozapine N-oxide and 9-hydroxyrisperidone, in plasma
after solid-phase extraction.32 The analysis was performed
using a Merck LiChroCART cartridge column with
Superspher 60 RP Select B as the stationary phase
and gradient elution. Screening and identification were
performed in the scan mode and quantifications in the
selected-ion mode.
Point-of-care testing
Quality Management
Measurement of plasma digoxin after Fab antibody
fragment administration
A National Academy of Clinical Biochemistry Laboratory
Medicine Practice Guideline defines point-of-care testing
(POCT) as ‘clinical laboratory testing conducted close
to the site of patient care, typically by clinical personnel
whose primary training is not in clinical laboratory
sciences or by patients (self-testing)’.27 The aim is usually
to provide an almost immediate result. A number of
colorimetric or immunochromatographic methods have
been developed for TDM analytes. POCT for TDM is
not much used in the UK, but devices are available for the
American market. POCT provision must comply with
ISO 22870:2006.28
Capillary (finger-prick) blood has been used to measure
lithium after membrane separation of the erythrocytes
(Lithium System, Akers Biosciences). The residual f luid
is reacted with porphyrin and the increase in absorbance
measured (505 nm). The results are said to be comparable
with those obtained by alternative assays.29
‘Immunochromatography’ is the term used to
describe an antibody-based test that typically uses
capillary f low through an absorbent membrane to mix
and subsequently separate the various components of the
test mixture. This concept was marketed as AccuLevel
(Syva) for TDM in the 1980s and was successful, if
expensive, initially. Asmus et al.30 concluded that the
AccuMeter (ChemTrak) demonstrated good precision
and minimal bias in comparison to TDx and the
AccuLevel. AccuTech now sell the Accumeter for whole
blood theophylline. It is marketed in Japan (Nikken
Chemicals) and has been evaluated for perioperative use
in asthma patients.31
ISO 15189:2007 defines standards for the operation
of a medical laboratory, and is consistent with ISO
9000/9001.33 Assay validation should conform as far as
possible with the FDA Center for Drug Evaluation and
Research (CDER) guidance for bioanalytical method
validation.34 Data for within-day (repeatability), betweenday, and total precision should be calculated according
to the protocol proposed by the Clinical and Laboratory
Standards Institute.35
Batch Analyses and Quality Control
Batch assay calibration should normally be by analysis of
standard solutions of each analyte (6-8 concentrations
across the calibration range) prepared in the appropriate
matrix (for example analyte-free neonatal calf or human
serum). IQC procedures should be instituted. This
involves the analysis of independently-prepared solutions
of known composition that are not used in assay calibration; normally low, medium, and high concentrations of
each analyte are prepared in analyte-free human serum.
Calibration standards are normally analyzed in duplicate,
once at the beginning and once at the end of the batch.
IQC samples are analyzed at the beginning and end of
the batch and also after every 5-10 patient samples as
appropriate. External quality assessment (EQA) or proficiency testing (PT) samples are analyzed as appropriate
to conform to the requirements of particular schemes.
Single point calibration methods should be validated and
the results compared with those from multipoint calibration.36
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Therapeutic Drug Monitoring (TDM)
The performance of batch analyses (analysis of a
number of samples in the same analytical sequence) and
analysis acceptance criteria should be as set out in the
method validation guidance.34 Typical assay acceptance
criteria for chromatographic assays are (i) chromatography
(reproducible peak shape and retention time, stable
baseline), (ii) calibration graph (r 0.98 or greater,
intercept not markedly different from zero), and mean
IQC results within acceptable limits (generally within
10 % of nominal value). Acceptance criteria for patient
samples are (i) ‘clean’ chromatogram, i.e. absence as far
as can be ascertained of interferences and, (ii) duplicate
values (peak height ratio to the internal standard) within
10 % except when approaching the limit of sensitivity of
the assay when duplicates within 20 % may be acceptable,
and (iii) results within the calibration range of the assay.
Clinical sample analyses falling outside acceptance limits
may be repeated if sufficient sample is available.
External Quality Assessment
Participation in EQA schemes is important.37 In such
schemes, portions of (sometimes lyophilized) homogenized
plasma, serum, or whole blood specimens are sent to
participating laboratories. The specimens are analyzed as if
they were real samples and the results are reported before
the true or target concentrations are made known.
EQA schemes measure inter-laboratory performance
and allow individual laboratories to detect and correct
systematic errors. The laboratories do not have to use the
same analytical method. The results of EQA schemes are
usually given as the z-score:
z=
x − xa
σp
(31)
where x is an individual result, xa is the accepted, ‘true’
value and σp is known as the ‘target value of SD’ which
should be decided on the basis of what is required of the
test, and should be circulated in advance. If the result
needs to be measured with high precision then a low value
of σp would be used. Thus, z is a measure of a laboratory’s
accuracy and the organiser’s judgement as to what is ‘fit
for purpose’. If the results of an EQA scheme are normally
distributed with a mean of xa and variance of 1, then zscores 2 would be deemed acceptable whereas those 3
would not.
There are of course sometimes concerns such as the
possible effects of freeze-drying on analyte stability, and
issues concerning the matrix used to prepare material
for circulation (due to the cost of analyte-free human
plasma or serum, neonatal calf serum may be used for
some analytes). Nevertheless, from the data generated,
scheme organisers can ascertain the methods that give
the best performance, investigate sources of interference
or bias, and in extreme cases report poorly performing
methods to regulatory authorities. Since the inception
of these schemes, poorly-performing assays have been
identified and participants advised accordingly. The
mean values reported have moved nearer to the intended
value and the spread of results about the mean has been
reduced.37
Gazetteer
This section aims to give summary information on the
main groups of compounds where TDM may play a part
in patient management. Pre-dose sampling is assumed
except where indicated. Detailed knowledge not only of the
limitations of the analytical method(s) used, but also of the
clinical pharmacology, toxicology, and pharmacokinetics
of the compound(s) monitored is often important when
interpreting results. Patients may respond differently to
a given dose of a given compound, especially as regards
behavioural effects. Further complicating factors may include
the role of pharmacologically active or toxic metabolites,
age, sex, concomitant drug therapy, and disease (Table 4).
Despite evidence that plasma methadone measurements
may be of benefit in dosage adjustment during methadone
maintenance treatment for opiate dependence,38 in practice
urine drug screening is usually preferred, as discussed
above (p.4).
Analgesics Including Non-Steroidal AntiInflammatory Drugs
Salicylates used to treat rheumatoid arthritis have been
monitored traditionally, in part because of the availability
of simple methodology. A target range of 250-300 mg L–1
was used. Monitoring of other analgesics and NSAIDs that
have largely replaced salicylates in rheumatoid arthritis is
rare and is mainly used to diagnose and if possible prevent
toxicity.39,40
Antiasthmatic drugs
There is normally no indication for TDM of bronchodilators such as salbutamol (albuterol) administered orally,
i.v., or by inhalation as clinical effect is usually easily
assessed and compounds of this nature are relatively nontoxic in overdose. Plasma theophylline (1,3-dimethylxanthine), however, is monitored with the aim of reducing
adverse effects when used as bronchodilator (Table 5).
There is now evidence that theophylline has significant
anti-inf lammatory effects in chronic obstructive pulmonary disease at low ‘therapeutic’ concentrations.41
Theophylline can be methylated to caffeine (1,3,7-trimethylxanthine) by neonates, but not by young children
or adults. Both drugs have been used to treat neonatal
apnoea, but the role of TDM of caffeine in this context
is not established.42
Anticonvulsants
Anticonvulsant TDM is well established, in part because of
its importance in phenytoin dose adjustment,7 and because
combinations of drugs continue to be used in patients
with epilepsy who do not respond to a single drug. This
raises the possibility of metabolic drug-drug interactions.
Carbamazepine, for example, induces the metabolism of
some other drugs and also induces its own metabolism,43
hence the time to steady-state may be 2 weeks or so (plasma
t0.5 in adults: single dose 18–65 h, chronic 5–25 h).
In newly-diagnosed patients there is no clear evidence
to support the use of TDM with the aim of reaching
predefined target ranges in dose optimization with
anticonvulsant monotherapy,44 although this does not
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Therapeutic Drug Monitoring (TDM)
Table 4: Some factors that may affect interpretation of TDM results
Factor
Comment
Acidosis/alkalosis
Age
Body mass
Burns
Concomitant
drug therapy
Disease
Duration/intensity
of exposure
Ethanol
consumption
Formulation
Genetics
/idiosyncrasy
Haemolysis
Infection
More than
one drug present
Nutrition
Pregnancy
Route of exposure
Sex
Shock
Site of sampling
Surgery/trauma
Time of sampling
relative to dose
Treatment
Tolerance
Influence volume of distribution of water soluble ionizable drugs if pKa is within two pH units of the blood
pH
Very young and elderly have lower metabolic capacity, elderly have lower hepatic blood flow, children
have low volume of distribution
Related to age; males have different body composition to females
State of hydration; metabolic response to injury
Long term and recent: effect on absorption, protein binding, distribution, and/or clearance
Liver or renal disease may reduce metabolic capacity, plasma protein binding may be altered; decreased
renal clearance affects renally excreted drugs
Possible effect on tolerance, body burden, induction or inhibition of metabolism
Short- and long-term effects on clearance, potentiation of effect, etc.
Sustained release, racemate, etc.
Acetylator status, drug metabolizing enzyme polymorphisms, etc.
Altered plasma: red cell distribution
Increased cellular permeability, changes in clearance mechanisms
Potentiation of effect, altered disposition/clearance
Possible change in protein binding and drug disposition
May alter drug disposition
Many compounds have higher acute toxicity if given i.v. or by inhalation rather than orally
Males have greater body mass, but lower proportion of fat than females: affects disposition of some
xenobiotics, notably ethanol
Absorption of orally-administered drugs may be reduced
Especially important if patient undergoing an infusion
Absorption of orally-administered drugs may be reduced; state of hydration; metabolic response to injury
If too short, absorption/equilibration may not have been complete, if too long my have missed optimum
time for assessing efficacy/safety
May get displacement from binding sites including receptors; Fab antibody fragments will increase total
plasma concentration temporarily
Previous exposure may have produced pharmacological tolerance or cross-tolerance; induction of
metabolism
Table 5: Antiasthmatic drug TDM assays
Drug
Reference range (mg L–1 plasma)
Caffeine
10–30 (neonatal apnoea)
Theophylline
8–20 (adults), 6–12 (neonatal apnoea)
ref lect clinical practice with phenytoin especially. The
relationship between plasma concentration and seizure
control may not be well defined for certain drugs (for
example valproate, vigabatrin), but nevertheless in general
the risk of toxicity increases at higher doses/plasma
concentrations (Table 6). Reference ranges for some
newer anticonvulsants (pregabalin, stiripentol) have not
been suggested.45 Measurement of ‘free’ (non-protein
bound) concentrations of phenytoin, carbamazepine, and
valproate in certain situations may be helpful clinically.46
Gabapentin, levetiracetam, pregabalin, topiramate, and
vigabatrin are eliminated largely or completely unchanged
in urine hence plasma concentrations may be affected
by alterations in renal function. Gabapentin shows dosedependent bioavailability.47 Concomitant use of enzymeinducing drugs can affect topiramate concentrations
markedly.48 For the newer drugs that are largely
Table 6: Summary of anticonvulsant TDM
Drug [metabolite]
Reference range (mg L–1 plasma)
Acetazolamide
Carbamazepine
[Carbamazepine–10,
11–epoxide]
Clobazam
2–12
1.5–12.0 (7 bipolar disorder)
[0.5–2.5] (1)
Clonazepam
Ethosuximide
Felbamate
Gabapentin
Lamotrigine
Levetiracetam
Methsuximide
Nitrazepam
Oxcarbazepine
Phenobarbital
Phenytoin
0.2 (clobazam), 2
(norclobazam)
0.025–0.085 (children, may be
lower in adults) (2)
40–100
20–110
2.0–20
1.0-15
20
10–40 (as normethsuximide)
0.05–0.15 (2)
15–35
(as 10-hydroxycarbamazepine,
licarbazepine)
5–30 (15–30 neonatal seizures)
7–20 (lower limit may be 5 or less)
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Therapeutic Drug Monitoring (TDM)
Primidone
Sultiame
Topiramate
Tiagabine
Vigabatrin
Valproate
Zonisamide
13 (also measure
phenobarbital)
2.0–12
5–20
0.4 (achiral method)
5–35
50–100 (anticonvulsant and in
bipolar disorder), 55–125 (mania)
15–40
Key:
1
Concentrations normally 5–15 % of parent drug
(10–30 % in patients co-prescribed valproate)
2 Especial care needed in sample collection as degraded by light
metabolized prior to elimination (felbamate, lamotrigine,
oxcarbazepine, tiagabine, and zonisamide), inter-patient
variability in pharmacokinetics is just as important in dose
adjustment as for many older anticonvulsants.45,49
Whilst developed for seizure control, many
anticonvulsant drugs have additional uses and in such cases
the reference ranges established for use in epilepsy may not
apply.50 Carbamazepine, lamotrigine, and valproate are used
in bipolar disorder, for example, and valproate is also used
in acute mania when the plasma concentrations associated
with efficacy may be somewhat higher than when the drug
is used as an anticonvulsant.51
Anti-infectives
Antimalarials
Quinine was the first antimalarial drug. The plasma
total quinine concentrations associated with effective
antimalarial therapy range from 10–15 mg L–1. However,
in non-infected subjects plasma concentrations above 10
mg L–1 are commonly associated with clinical features of
toxicity such as visual disturbance, leading in some cases to
permanent visual deficit or blindness. Quinine is normally
70–90 % bound to plasma protein, notably AAG. The
plasma concentration of this latter protein is increased in
severely infected patients and quinine protein binding is
also increased (to 93 % or so) and hence higher plasma
total quinine concentrations can be tolerated with no
apparent toxicity.52
Chloroquine and hydroxychloroquine are chiral
antimalarial drugs that again bind to AAG. These drugs
are also used at higher doses as second-line agents to treat
rheumatoid arthritis. Chloroquine also has anti-HIV–1
activity.53 Chloroquine is used as a malaria prophylactic
at a single adult oral dose of 300 mg free-base weekly. A
common cause of concern is that taking the drug daily
leads to an enhanced risk of toxicity in the non-infected
subject.
Antimicrobial Drugs
TDM of aminoglycoside antibiotics such as gentamicin
and tobramycin has been well-established for many
years.2 Interpretation is best provided in conjunction
with microbiology laboratories.54,55 Peak (2 hour postdose) concentrations of isoniazid, rifampin, pyrazinamide,
and ethambutol give information as regards effective
oral dosage, but an additional sample at 6 hours may
help differentiate between (i) delayed absorption, and
Table 7: Some anti-infective drug TDM assays
Group
Drug
Reference range in an adult (mg L–1 plasma) (1)
Antimalarials
Chloroquine
Hydroxychloroquine
Quinine
0.05 (malaria prophylaxis), 0.2–0.3 (rheumatoid arthritis)
0.05 (malaria prophylaxis), 0.4–0.5 (rheumatoid arthritis)
8–16 (2)
Antimicrobial
Amikacin
Chloramphenicol
Ethambutol
Gentamicin
Isoniazid
Kanamycin
Netilmicin
Pyrazinamide
Rifampin
Sulfamethoxazole
Teicoplanin
Tobramycin
Vancomycin
Trough 10, peak 20–30
Trough 5, peak 10–20
3 pre-dose, 2–6 post-dose
Trough 2, peak 5–10
Peak 3–6 (300 mg daily), peak 9-18 (900 mg bi-weekly)
Trough 8, peak 30
Trough 2, peak 5–10
Peak 20–50 (25 mg kg–1 d–1), peak 40–100 (50 mg kg–1 bi-weekly)
Peak 8–24
Peak 100–120
10–60 (20–60 Staphylococcus aureus infection)
Trough 2, peak 5–10
Trough 5–10, peak 20–40
Antiretroviral (3)
Amprenavir
Atazanavir
Efavirenz
Indinavir
Lopinavir/ritonavir
Nelfinavir
Nevirapine
Ritonavir
Saquinavir
Trough 0.4
Trough 0.15
Trough 1
Trough 0.1
Trough 1
Trough 0.8
Trough 3.4
Trough 2.1
Trough 0.1
Key:
1 Achiral method
2 Possibility of serious toxicity in non-infected subjects
3 Suggested minimum target concentrations for patients with wild-type HIV
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Therapeutic Drug Monitoring (TDM)
Table 8: Some antineoplastic drug TDM assays
-1
Drug [metabolite]
Reference range in an adult (mg L
plasma)
5-Fluorouracil
6-Mercaptopurine
(6-MP)
0.2 (1.5 µmol L ) – 120 h post-dose
0.2 (peak, maintenance doses)
1.0 (2.2 µmol L–1) – 24 h post-dose;
Methotrexate
0.45 (1 µmol L–1) – 48 h post-dose
Table 9: Some cardioactive drug TDM assays (metabolites
normally measured in [brackets])
Drug [metabolite]
Reference range in an adult
(mg L–1 plasma)
Amiodarone
[Noramiodarone] (1)
Atenolol
Digoxin
0.5–2
[0.5–2] (2)
0.2-0.6
0.0008–0.002 (1.0–2.6 nmol L–1)
(6–12 h post-dose) (3)
2.0–5.0
[5.0] (2)
0.2–0.7
1.5–5.0
0.2-0.8
1-2
0.15–0.6
10–30 (procainamide only, 4–8)
–1
(ii) generally poor absorption resulting in ineffective
treatment as well as giving further information such as an
indication of plasma half-life.56 Prompt, effective treatment
minimizes the risk of developing drug resistance.
The observed wide inter-individual variation in
antiretroviral pharmacokinetics has led to increasing
interest in the use of TDM to help optimize dosing of
these drugs (Table 7).57-59
Antineoplastic drugs
TDM clearly has the potential to improve the clinical
use of antineoplastic agents, most of which have very
narrow therapeutic indices and highly variable betweenpatient pharmacokinetics. Plasma concentration-effect
relationships have been established for 5-f luorouracil
(at a dose of 1 g m–2 d–1), 6-mercaptopurine (from
azathioprine), and methotrexate (Table 8). Interpretation is
complicated by the use of different agents simultaneously.
AUC calculations may give more useful information than
measurements at a single point in time. Relationships
between plasma concentration and dose-limiting toxicities
for epipodophyllotoxins, platinum-containing compounds,
camptothecin, anthracyclines, and antimetabolites have
also been described.60,61
Thiopurine methyltransferase (TPMT) phenotyping
is used to guide treatment with azathioprine to avoid
life-threatening agranulocytosis.62 However, should a
patient have received blood products this may skew the
results of phenotyping. Genotyping would detect two cell
populations and enable a potentially serious mismatch to
be avoided.
Cardioactive Drugs
TDM of digoxin is well established; although its clinical
relevance is often obscure.63 TDM can sometimes be
useful in the case of amiodarone to monitor adherence
and toxicity, and to monitor adherence to sotalol and
to other -adrenoceptor blockers such as atenolol and
propranolol (Table 9).64 Use of the calcium channel
blockers verapamil and diltiazem is normally assessed
by monitoring haemodynamic effects. Diltiazem and
N-desmethyldiltiazem, desacetyldiltiazem and Ndesmethyldesacetyldiltiazem are unstable in plasma; all
may be pharmacologically active. TDM is useful in
assessing f lecainide dose requirement in children.65
Immunosuppressants
TDM of ciclosporin, mycophenolic acid (MPA, the
active metabolite of mycophenolate mofetil), sirolimus,
and tacrolimus is well established (Table 10).66 All these
drugs have narrow therapeutic windows and show
considerable pharmacokinetic variability, and TDM is
Disopyramide
[Nordisopyramide]
Flecainide
Lidocaine
Metoprolol
Mexiletine
Perhexiline
Procainamide
[ acecainide (4)]
Propranolol
Quinidine
Sotalol
Verapamil
[Norverapamil]
0.01–0.1
2.5–5.0
0.8–2.0 (-blockade), 2.5-4.0
(antiarrhythmic)
0.1–0.2
[0.1–0.2] (2)
Key:
1 N-Desethylamiodarone
2 Ratio of metabolite to parent compound may be a guide to the
duration of therapy and possibly to the likelihood of toxicity
3 Assay may be unreliable in some patient groups (e.g. neonates,
renal failure, hepatic failure)
4 N-Acetylprocainamide
Table 10: Some immunosuppressive drug TDM assays
Drug
Reference range in an adult (mg L–1) (1)
Ciclosporin
(cyclosporine,
cyclosporine A)
Mycophenolic acid
Sirolimus
Tacrolimus
0.04–0.25 (trough, whole blood) (2)
1.5–3.0 (trough, plasma)
0.003–0.015 (trough, whole blood)
0.001–0.012 (trough, whole blood)
Key:
1 Single immunosuppressant, renal transplant patients
2 IA may be unreliable in some patient groups (e.g. neonates, renal
failure, hepatic failure)
essential to avoid adverse effects such as nephrotoxicity
while maximizing efficacy. All is not straightforward,
however, as some patients experience acute rejection
episodes or post-operative complications despite blood
concentrations within the reference range. As in the case
of antineoplastic agents, AUC calculations may give more
useful information than measurements at a single time
point particularly for MPA, but collection of such samples
in the out-patient setting is often impractical. Peak (two
hour post-dose) sampling may be a better indicator of
optimal ciclosporin dosage than trough or four hour postdose sampling,4 but this is disputed. [N.B. Azathioprine is
also used as an immunosuppressive drug.]
Interpretation of either ‘trough’ or ‘peak’ results
is complicated because (i) there may be considerable
differences between the results obtained with IA as
compared to chromatographic methods,67 (ii)
immunosuppressants are often used in combination to
reduce the risk of toxicity from individual compounds
hence the concentrations attained during optimal treatment
are lower then when the drugs are used alone, and (iii) the
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Therapeutic Drug Monitoring (TDM)
Table 11: Some psychoactive drug TDM assays
Antidepressants
Drug [metabolite]
Reference range in an
adult (mg L–1 plasma)
Amisulpride
Amitriptyline [ nortriptyline]
Aripiprazole
[dehydroaripiprazole]
Citalopram
Clomipramine
[ norclomipramine]
Clozapine
Desipramine
Dosulepin [ nordosulepin]
Doxepin [ nordoxepin]
Escitalopram
Fluoxetine
[norfluoxetine]
Fluvoxamine
Imipramine [ desipramine]
Lofepramine: see Desipramine
Lithium
0.1–0.4
0.08–0.25
0.1–0.5
[0.04–0.2]
0.05–0.5
1.0 (1)
Whilst reference ranges for certain tricyclic antidepressants
(TCAs) and their plasma metabolites (for example
amitriptyline/nortriptyline, and imipramine/desipramine)
associated with effective treatment have been suggested,3
patients become tolerant to the adverse effects of TCAs
and may show clinical improvement at higher plasma
concentrations than those cited (Table 11). In practice, TDM
of TCAs and also of selective serotonin reuptake inhibitors
(SSRIs) and other newer antidepressants such as venlafaxine
is mainly concerned with assessing whether treatment
failure is due to poor adherence, ultra-rapid metabolism,
or drug interactions leading to induction of metabolizing
enzymes.68 TDM of SSRIs has, however, been advocated
in order to minimize the risk of drug-drug interactions due
to inhibition of the drug metabolizing enzyme denoted
CYP2D6. Unlike some other SSRIs, citalopram has only a
mild inhibitory effect on CYP2D6.69
Mianserin [ normianserin]
Mirtazapine [ normirtazapine]
Nortriptyline
Paroxetine
Olanzapine
Quetiapine
Risperidone
[9-hydroxyrisperidone]
Sertraline (5)
Sulpiride
Trazodone
Trimipramine [ nortrimipramine]
Venlafaxine
[O-desmethylvenlafaxine]
0.35–0.50 (1, 2)
0.08–0.16
0.50 (1)
0.15–0.25
0.025–0.25
0.04–0.45
[0.04–0.45] (3)
0.16–0.22
0.15–0.25
4–8 (0.6–1.0 mmol L–1)
(12 h post–dose) (4)
0.03–0.10
0.02–0.10
0.05–0.15
0.01–0.05
0.02–0.04 (12 h
post–dose)
0.05–0.20 (1)
0.004–0.008 (oral
treatment)
[0.010–0.025]
0.03–0.19
0.3–0.5
0.8–1.6
0.50(1)
0.05–0.5
[0.05–0.5]
Key:
1 Adverse effects at higher concentrations may limit the dose that
can be tolerated
2 Upper limit not well-defined. Norclozapine concentrations
average 70 % of those of clozapine during normal therapy
– norclozapine assay useful in monitoring adherence
3 Ratio of metabolite to parent compound may be a guide to the
duration of therapy and possibly to the likelihood of toxicity
4 Upper limit may be higher in mania
5 Norsertraline only 10 % of the activity of sertraline
amount of immunosuppression required for maintenance
treatment varies widely depending on the engrafted organ.
The guidelines given in Table 10 are those applicable to
therapy with single immunosuppressants used after renal
transplantation.
Psychoactive Drugs
The role of TDM in guiding treatment with lithium was
discussed earler. TDM may also play a part with guiding
therapy with agents normally classified as anticonvulsants,
for example, when used to treat mental illness.50
Antipsychotics
Although of little benefit with established (‘typical’)
antipsychotics such as chlorpromazine and haloperidol,
TDM of newer (second generation or ‘atypical’) drugs,
notably clozapine and to an extent olanzapine, can help
by assessing adherence, guiding dose adjustment, and
guarding against toxicity.70 In time, indications for TDM
of other antipsychotics may become apparent.71,72
With clozapine dose assessment is complicated because
(i) there is a 50-fold inter-patient variation in the rate of
clozapine metabolism, (ii) alteration in smoking habit
can have a dramatic effect (on average ± 50 %) on
clozapine dose requirement,73 and (iii) the clinical features
of clozapine overdosage can mimic those of the underlying
disease. With olanzapine, a 12-hour post-dose plasma
concentration of 20 µg L–1 seems to be needed to ensure a
fair trial of the drug.72
Summary
For some antimicrobials, anticonvulsants, clozapine,
digoxin, some immunosuppressives, and lithium, TDM
may be used to adjust the dose to individual need and to
minimize the risk of dose-related toxicity. In the absence
of other information, the apparent volume of distribution
and dose may be used to predict plasma concentrations, so
that, for example, assay calibrators may be prepared over an
appropriate concentration range. However, knowledge of
how clearance and distribution determine the time course
of a substance in the body is essential to understanding the
inf luence of age, sex, other genetic variables, disease, and
other parameters on pharmacokinetics and hence clinical
effect.
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