Anal. Chem.
2001, 73, 4410-4416
Application of Solid-Phase Microextraction in the
Determination of Diazepam Binding to Human
Serum Albumin
Haodan Yuan and Janusz Pawliszyn*
Department of Chemistry, University of Waterloo, Waterloo, Ontario, N2L 3G1, Canada
In this paper, protein-drug interactions were studied by
solid-phase microextraction (SPME) using diazepam
binding to human serum albumin as a model system.
Since drug compounds are normally polar and nonvolatile
by nature, direct SPME is used in this work. The SPME
extraction is an equilibrium process among the concen-
trations of the analyte partitioned onto the SPME fiber,
free and bound drug in the solution. A calibration curve
was first constructed by employing the amount of the
analytes partitioned on the fiber versus the free analyte
concentration in the solution in the absence of protein.
In method I, the extraction was performed in the protein
solution with known diazepam concentration. In method
II, diazepam was first loaded onto the fiber by extracting
in solution with known diazepam concentration. This fiber
was subsequently transferred into the protein solution for
desorption. The amount of the analyte left on the fiber was
analyzed after the system reached equilibrium. The free
drug concentration was then obtained from the calibration
curve for both methods. The Scatchard plot was finally
employed to obtain the number of binding sites and the
equilibrium binding constants. Since only a very small
amount of the protein solution is required (150 µL for
each extraction), method II is very useful for circum-
stances where the protein amount is very limited. The
direct measurement method proposed in this paper does
not need a GC response factor, which significantly de-
creases the experimental error. The only measurement
needed is the area count change (ratio) of the fiber
injections before and after the protein was introduced into
the solution. The difference between the direct measure-
ment method for method I and method II is discussed.
The result illustrated that the SPME direct measure-
ment method provided both theoretical accuracy and
simplicity in such applications.
The process of drug transformation and drug storage always
involves the binding of a drug to the relative protein. It can be
stated that all the important stages of the fate of a drug in the
body are ruled by its protein interaction. The investigation of
possible multiple binding interactions of a drug in the blood is
important to understand the distribution and biotransformation
* To whom all correspondence should be addressed. Tel: (519) 888-4641.
Fax: (519) 746-0435. E-mail: Janusz@uwaterloo.ca.
4410 Analytical Chemistry, Vol. 73, No. 18, September 15, 2001
pathways of a drug in the human body. It also has a pathological
meaning since binding may be modified in disease states, due to
changes in the nature or amount of protein, dehydration, or
alteration of pH. The bound drug complex is of high molecular
weight and unavailable for tissue membrane transport. Only the
unbound drug is capable of diffusing across tissue membranes
to reach the target site. Therefore, only the free drug is considered
to be the pharmacologically active.1
Within the plasma proteins, serum albumin is undoubtedly the
most important carrier for drugs and other small molecules. The
attention of many scientists has been drawn to the phenomenon
of the interactions between drug and the serum albumin. Because
of the relative ease of the experiments, most standard binding
assays use serum albumin.
Many techniques have been employed for the drug-protein
binding study. The more conventional techniques include equi-
librium dialysis, ultrafiltration, and gel filtration. Although the
equilibrium dialysis technique is very simple and convenient for
multiple-samples analysis, a period of 12 h or more is required to
attain the equilibrium, which may allow time for possible decom-
position of unstable compounds or for the growth of bacteria.
While these problems can be minimized by carrying out the
experiments at lower temperatures, binding will thereby be
altered. Normally the drug compounds will bind not only to the
glass containers but also, to a greater degree, to the dialysis bag.
Significant overestimation of the free fraction can result from even
a slight leakage of protein into the dialysate.
Ultrafiltration has been introduced widely for routine free drug
monitoring in clinical laboratories.2 In comparison with equilibrium
dialysis, this method offers significant advantages as represented
by its short analysis time, simplicity, commercially available kits,
and lack of dilution effects. However, it is perhaps not so readily
adapted for large numbers of samples, and it uses a somewhat
more expensive and complicated apparatus. Another important
reservation is that since a portion of the aqueous phase is forced
away from the protein, the latter solution becomes more concen-
trated, thereby tending to increase binding.
Gel filtration is a very popular technique for measuring protein
binding.2 In gel filtration, a solution of drug and protein passes
through a column containing a dextran molecular exclusion gel.
(1) Seydel, J. K.; Schaper, K.-J. Pharmacol. Ther. 1982, 15, 131.
(2) La Du, B. N., Mandel, H. G., Way, E. L., Eds. Fundamentals of Drug
Metabolism and Drug Disposition; The William & Wilkins Co.: Baltimore,
MD, 1971; p 67.
10.1021/ac010227s CCC: $20.00 © 2001 American Chemical Society
Published on Web 08/16/2001
The protein-bound drug is separated from the free drug by
emerging before the free drug peak. However, if the equilibrium
between bound and free drug is rapidly reversible, this approach
would not be appropriate. One would expect that the molecules
of bound drug should dissociate as the protein-drug complex
begins to separate from the free drug and that the drug should
emerge from the column as a smear rather than discrete peak.
Despite the fact that chromatographic methods have been long
used for the determination of drug-protein binding parameters,
they have earned only limited attention. In recent years, the
progress in chromatographic technology has led to the develop-
ment of highly automated systems yielding high resolution on
small columns, allowing shorter analytical times, consuming less
chemicals, and avoiding the use of radiolabeled ligands. The most
widely used chromatographic method is affinity chromatography,
which provides the possibility of detecting very small differences
in the binding affinity of ligands. However, such methods suffer
Figure 1. Schematic of equilibrium in three-phase system (sample
from short column lifetime, experimental inconvenience, and time- solution-dissolved protein-fiber coating).
consuming column preparation.
In this work, solid-phase microextraction (SPME) is used to
determine the binding parameters of a drug binding to protein THEORY
by employing diazepam binding to human serum albumin (HSA) Drug-protein binding is a reversible reaction between a drug
as a model system. SPME is a relatively novel but well-known molecule and a protein.
sampling and sample preparation technique. It was favored by its K
high extraction efficiency in volatile compound analysis, simple [P] + [D] {\} [PD] (1)
operation, and inexpensive apparatus.3 Recently, SPME has been
applied to protein binding analysis. It has been successfully applied where [P] is the free protein concentration, [D] is the free drug
in the determination of the equilibrium constant of alkylbenzenes concentration, and [PD] is drug-protein complex concentration.
binding to bovine serum albumin (BSA) by headspace extraction.4 At equilibrium, it will have the equilibrium constant K:
SPME yielded accurate results in those applications, providing
that the data were properly interpreted. [PD] Cb
K) ) (2)
Diazepam (7-chloro-1,3-dihydro-1-methyl-5-phenyl-3H-1,4-ben- [P][D] CpCs
zodiazepin-2-one) is a member of the benzodiazepine drug family,
which are widely used as tranquilizers, hypnotics, muscle relax-
ants, and anticonvulsants.5,6 where Cb, Cp, and Cs are the molar concentrations of bound drug
It is well known that diazepam binds to HSA. Various authors7 (protein), free protein, and free drug, respectively.
proved the presence of one specific binding site. The binding One of the predominant values measured in the protein binding
constants, the thermodynamic parameters, and their variation with study is the free drug concentration. Knowing the total concentra-
pH have also been determined.8,9 It is believed that diazepam tion of the drug and protein, the percentage of the drug bound
binding to HSA occurs in binding site II, which is also called the can be calculated and the K value can be determined.
indole and benzodiazepine binding site.10-12 This site binds several Since diazepam is a nonvolatile, relatively polar organic
indole derivatives and benzodiazepines with a high degree of compound, direct SPME, where the extraction is performed
structural specificity.7,13-15 In fact, diazepam is one of the specific directly in the sample solution instead of headspace, is used. The
marker ligands for this site.12 experiment configuration, where the headspace is totally elimi-
nated, is illustrated in Figure 1. In the calibration system, where
(3) Pawliszyn, J. Solid-Phase Microextraction: Theory and Practice; Wiley-VCH: there is no protein in the sample solution, the system is a two-
New York, 1997. phase system (buffer solution-fiber coating). In the measurement
(4) Yuan, H.; Ranatunga, R.; Carr, P.; Pawliszyn, J. Analyst 1999, 124, 1443.
(5) Mulé, S. J.; Casella, G. A. J Anal. Toxicol. 1989, 13, 179.
system, where the protein is present, it is a three-phase system
(6) Drouet-Coassolo, C.; Aubert, C.; Coassolo, P.; Cana, J. J. Chromatogr. 1989, (sample solution-dissolved protein-fiber coating).
487, 295. In the calibration system, the mass balance is
(7) Müller, W. E.; Wollert, U. Naunyn-Schmiedeberg’s Arch. Pharmacol. 1973,
280, 229.
ntotal ) nf + ns (3)
(8) Müller, W. E.; Wollert, U. Naunyn-Schmiedeberg’s Arch. Pharmacol. 1974,
283, 67-82.
(9) Coassolo, P.; Sarrazin, M.; Sari, J. C.; Briand, C. Biochem. Pharmacol. 1978,
27, 2787. where ntotal, nf, and ns are the total amount of the analyte in the
(10) Müller, W. E.; Wollert, U. Pharmacology 1979, 19, 59.
(11) Sudlow, G.; Birkett, D. J.; Wade, D. N. Mol. Pharmacol. 1976, 12, 1052.
system, the amount partitioned on the fiber coating, and the
(12) Sjöholm, I.; Ekman, B.; Kober, A.; Ljungstedt-Pahlman, I.; Seiving, B.; Sjödin, amount freely dissolved in the buffer solution, respectively. Since
T. Mol. Pharmacol. 1979, 16, 767.
(13) Müller, W. E.; Wollert, U. Naunyn-Schmiedeberg’s Arch. Pharmacol. 1975, (14) Müller, W. E.; Wollert, U. Mol. Pharmacol. 1975, 11, 52.
288, 17. (15) Sjödin, T.; Roosdorp, N.; Sjöholm, I. Biochem. Pharmacol. 1978, 25, 2131.

Analytical Chemistry, Vol. 73, No. 18, September 15, 2001 4411
ns ) CsVs, a few steps of simple derivations yield
Cs ) (ntotal - nf)/Vs (4)
where Vs is the volume of the sample solution. If nf is negligible,
Equation 4 can be simplified to
Cs ) C0 ) ntotal/Vs (5)
This means that if the amount of the analyte on the fiber can
be neglected, the calibration curve of initial concentration (C0),
instead of free concentration (Cs), versus the amount of the analyte
on the fiber can be employed to calculate the free analyte
concentration in protein binding study. However, if the compound
has a large partition coefficient toward the fiber, the amount of
the analyte partitioned onto the fiber has to be taken into
consideration. It can be measured from the response factor of
diazepam on GC, which was determined from the syringe injection
of a certain amount of the analyte into the GC injector.
Once the free concentration Cs was known (from the calibration
curve corresponding to the fiber injection amount nf), the
Scatchard plot was then used to calculate the equilibrium constant
and the number of binding sites.
METHOD DESCRIPTIONS
Method I. A 2-mL vial was used in this experiment. A stir bar
was first put into the vial, and then 1.9 mL of buffer solution (in
the calibration system) or protein buffer solution (in the measure-
ment system) was added to completely fill the vial. This process
leaves no headspace. The vial was mounted on the stirrer to start
the agitation. The agitation speed was kept at 800 rpm with the
vial capped. After the agitation was stable, a poly(dimethylsiloxane)
(PDMS) fiber was inserted into the vial. The extraction time was
optimized at 45 min. After the extraction, the fiber was transferred
to the GC injector for analysis with a desorption time of 3 min.
No carryover of analytes was observed. The extraction profile and
calibration curve of diazepam were investigated in 1/15 M phos-
phate buffer solution (pH 7.4). For the protein binding study, 1
mg/mL HSA solution was prepared in the same buffer solution
and used for the extraction.
The free drug concentration was calculated from the GC
response factor and the calibration curve. The Scatchard method
was then applied to estimate the equilibrium constant.
Method II. This method is designed for the situation where
only a small volume of protein solution is available for analysis.
The experimental setup is illustrated in Figure 2. A polyethylene
insert (150 µL) positioned in a 2-mL vial was used in the
experiment. The solution for analysis was put in this insert. The
analyte was first loaded onto the fiber from a buffer solution with
known analyte concentration. The exact amount of the analyte
loaded on the fiber can be obtained from the GC response factor.
This initial loading of the analyte was performed by the same
procedure used in the calibration step in method I, so that the
same calibration curves can be employed as for the free analyte
concentration determination in the measurement system with
protein present. Note that in this small-volume analysis, the
amount of analyte partitioned on the fiber should not be neglected.
4412 Analytical Chemistry, Vol. 73, No. 18, September 15, 2001
Figure 2. Experimental configurations for diazepam binding to HSA
in small-volume analysis.
The loaded fiber was then inserted into the solution (with and
without protein present) for equilibration. After the equilibrium
had been reached, the fiber was withdrawn from the solution and
analyzed by GC with fiber injection. The amount of the analyte
left on the fiber can be determined by the GC response factor.
The amount of the analyte initially loaded onto the fiber is the
total amount of the analyte in this protein binding process. So it
is termed as “n′total”, which satisfies
n′total ) n′b + n′s + n′f ) C′bVs + C′sVs + n′f (6)
The symbol “′” stands for the measurement system with protein
present. The amount of the analyte left on the fiber (n′f) can be
determined from the GC response factor. The free dissolved drug
concentration can be determined from the calibration curve of
the loading solution. Since the volume of the protein (Vs) and the
amount of the analyte loaded onto the fiber (n′total) are known
parameters, the bound concentration (C′b) can be easily ob-
tained. The equilibrium constant was finally obtained using the
same Scatchard method described in method I.
DIRECT MEASUREMENT OF THE EQUILIBRIUM
CONSTANT
This method was developed to calculate the equilibrium
constant based on the change of the amount (GC area count ratio)
of the analyte extracted by the fiber in the absence and presence
of the protein. The only assumption was that both the GC and
SPME are linear systems within the experiment range, which
holds true in this application. Since this method avoids the
determination of the GC response factor, the result tends to be
simpler and more accurate.
Direct Measurement for Method I. Since there is no
headspace present, if the effect of fiber extraction amount is
negligible, which is the case in method I, the mass balance for
the system with protein present can be written as
n′total ) n′f + n′s + n′b
) C′s(Vf/Ksf + Vs + KCpVs)
≈ C′sVs(1 + KCp) (7)
where Ksf is the partition coefficient of the analyte between the
sample solution and fiber coating.
 0
In the calibration system with the same total analyte concentra- Combining eqs 11 and 7a, using the fact that ntotal ) ntotal and
0
tion, the mass balance can be expressed as Cs/Cs ) A/A , we have
0

ntotal ) nf + ns 1 A0 - A
) (12)
1 + Vf/KsfVs A0
≈ CsVs (8)

Substituting eq 12 into 9a, we get

Since Cs/C′s ) A/A′, where A and A′ are GC area counts with
protein absence and presence, and ntotal ) n′total, combining eqs 7 A A0 - A
)1+ KCp (9b)
and 8 yields A′ A0
A/A′ ) 1 + KCp (9)
The only question left is how to calculate the free protein
concentration Cp without using the GC response factor. The bound
From eq 9, a straight line can be obtained when A/A′ is plotted
analyte concentration Cb can be determined by
against Cp. The value of the equilibrium constant K can be
obtained from slope of this curve. ntotal - n′f
Free protein concentration Cp can be determined as follows: C′b ) - C′s
Vs
Cp ) Cp,total - Cb ) Cp,total - (C′total - C′s) ntotal - n′f n′f
) Cs - Cs
ntotal - nf nf
) Cp,total - (C′total - C′total(A′/A))
A0 - A′ A′
) Cp,total - C′total(1 - A′/A) (10) ) C s - Cs
0
A -A A

A0(A - A′)
where C′total is the total diazepam concentration in the protein ) Cs
binding system. Therefore, Cp can be also obtained from the area A(A0 - A)
count ratio of the fiber extraction before and after the protein was A - A′ 0
) Cs (13)
added. A0 - A
Direct Measurement for Method II. For the system de-
scribed in method II, the amount of analyte partitioned in the fiber
cannot be ignored. There are three steps in this method. The first Therefore, the free protein concentration is
is the analyte loading in buffer solution with analyte concentration A - A′ 0
C0s . The amount loaded on the fiber can be expressed as Cp ) Cp,total - Cs (10a)
A0 - A
n0total ) (C0s /Ksf)Vf (11)
EXPERIMENTAL SECTION
Chemicals and Materials. Diazepam was purchased from
In the second step, the loaded fiber was transferred into Radian (Austin, TX) as a 1 mg/mL methanol solution. This
solution without protein being present. When equilibrium was solution was diluted with methanol into 0.1, 0.01, and 0.001 mg/
reached, the mass balance for the system could be written as mL solutions for experimental convenience. All these stock
solutions were stored at -10 °C. The human serum albumin (96%
ntotal ) nf + ns purity, no fatty acid) was purchased from Sigma (Mississauga,
ON, Canada). SPME devices and fibers (100-µm PDMS) and all
) CsVs(1 + Vf/KsfVs) (7a)
the vials used in the experiments were purchased from Supelco
(Bellefonte, PA). For small-volume analysis (method II), a
In the last step, the loaded fiber was introduced into solution polyethylene insert, which has a volume of 150 µL, was positioned
with protein present. The mass balance for this system is in a 2-mL vial for the analysis.
The pH 7.4 buffer solution was prepared by combining 200
n′total ) n′f + n′s + n′b mM disodium hydrogen orthophosphate and 200 mM soldium
dihydrogen orthophosphate solution at a certain ratio while
) C′s(Vf/Ksf + Vs + KCpVs) monitoring with a pH meter. This buffer solution was diluted into
3 times to form 1/15 M pH 7.4 buffer solution. The calibration and
) C′sVs(1 + Vf/KsfVs + KCp) (8a)
protein binding measurements were performed in this buffer
solution.
Using the same procedure as in method I, from eqs 7a and 8a, Instrumentation and Analytical Conditions. All analyses
the following formula can be derived: were performed on a Varian (Sunnyvale, CA) GC 3500 gas
chromatograph equipped with a 10 m × 0.25 mm i..d × 0.25 µm
A 1 SPB-5 column (Supelco, Bellefonte, PA), a septum-equipped
)1+ KC (9a)
A′ 1 + Vf/KsfVs p programmable injector (SPI) with SPME insert, and a FID. The
Analytical Chemistry, Vol. 73, No. 18, September 15, 2001 4413
 Table 1. Summary of the Data for Diazepam Analysis in
Buffer Solution (Method I)a

Ctotal area ntotal nf nf/ntotal Cs

(ng/mL) count (ng) (ng) (%) (ng/mL)
100 17 124 200 4.75 2.38 97.6
200 32 818 400 9.11 2.28 195
300 47 621 600 13.2 2.20 294
400 60 318 800 16.7 2.09 392
500 71 144 1000 19.8 1.97 490
600 86 088 1200 23.9 1.99 588
700 102 845 1400 28.6 2.04 686
800 111 570 1600 31.0 1.94 785
900 124 120 1800 34.5 1.91 883
1000 135 109 2000 37.5 1.88 981
a The response factor is 3062 area count units/ng of diazepam.

Figure 3. Extraction profile of diazepam by a PDMS 100-µm fiber

in 0.067 M pH 7.4 phosphate buffer. Agitation speed was 800 rpm.
carrier gas was helium (25 psi head pressure). The column
temperature program used for the fiber injection in the experi-
ments was 120 °C, hold for 1 min, increase at 10 °C min-1 to 300
°C, and hold for 5 min. During the whole analysis, the injector
and detector temperatures were 250 and 300 °C, respectively.
The detector response factor was determined by a syringe
injection of 1 mg/mL standard diazepam in methanol solution
using the same column temperature program. The SPI injector
was temperature programmed as follows: 50 °C hold for 0.5 min,
increase at 250 °C/min to 250 °C, and hold for 22 min. Liquid
CO2 was used to cool the injector before all injections. All the
extractions were carried out at 23 °C. A 7 mm L × 2 mm D stir
bar was used in each of the extraction vial (2 mL clear vial from
Supelco).
RESULTS AND DISCUSSION
Decomposition of Diazepam. Like most of the benzodiaze-
pines, diazepam is a thermolabile compound, which is likely to
be decomposed in the GC system if the temperature is sufficiently
high. It was found that diazepam was decomposed when a 30-m
column was used, since the compound took more time to elute
out. The problem was eliminated when a 10-m column was used.
Extraction Profile. All extractions in this study were carried
out at 23 °C. The extraction profile was first investigated to deter-
mine the equilibrium time. The equilibrium profile is presented
in Figure 3. The change of the equilibrium time in the situation
where protein was present was not observed. From the extraction
profile, we can see that the equilibrium is reached after 35 min.
Therefore, 45 min was used as the extraction for all experiments.
GC Response Factor. The GC response factor was deter-
mined by syringe injection of 0.6 µL of a 1 mg/mL (total mass,
60 ng) diazepam methanol solution into the GC system. The “sand-
wich” method was used for the syringe injection. The response
factor was determined as 3602 area counts/ng of diazepam.
Calibration Curve. The calibration curve was performed in
1/ M phosphate buffer (pH 7.4) solution with diazepam concen-
15
trations varied from 0.25 to 10 µg/mL. A diazepam standard
methanol solution was spiked into the buffer solution to obtain a
sample solution of a given concentration. It was found that the
trace amount of methanol could effect the precision of analysis
by swelling the fiber coating. Therefore, during the calibration,
4414 Analytical Chemistry, Vol. 73, No. 18, September 15, 2001
as well as during the protein binding analysis, methanol was added
to the solution to keep the methanol concentration the same in
each analytical vial for all the concentrations of diazepam.
The amount of the analyte partitioned onto the fiber coating
can be calculated from the response factor and the area count of
the fiber injection. The results are shown in Table 1.
Column 5 in Table 1 shows that the amount of the analyte
extracted by the fiber is less than 3% of the total amount of the
analyte in the solution. This amount is so small that it can be
neglected in plotting the calibration curve. Therefore, the calibra-
tion curve of the total concentration Ctotal versus the area count
can be used as the calibration curve to determine the free drug
concentration in the protein binding study. The calibration curve
is linear with regression equation of y ) 140.77x and a square
regression coefficient (R2) of 0.9903.
Drug Loading and Calibration in Method II. The same
calibration curve was obtained as that obtained with method I.
The amount of the drug loaded on the fiber is determined from
this curve using the area count obtained by GC analysis and the
GC response factor, which was determined from the previous
analysis, 3062/ng. This calibration curve was also employed to
determine the free analyte concentration.
Determination of the Binding Parameters. Method I. The
moles of drug bound per mole of protein (r), the molar free
concentration ([D]), and the value of r/[D] have been calculated
and are summarized in Table 2. A Scatchard plot is employed to
calculate the equilibrium constant and the number of binding sites
in this study.
The concentration of HSA used in this study was 1 mg/mL,
which is 1.45 × 10-5 M (molecular weight of HSA, 69 000). Since
the analyte concentration loaded on SPME fiber only reached
equilibrium with the free analyte concentration in the solution,
the concentration obtained from the calibration curve is the free
diazepam concentration in the solution. The amount of the
analyted loaded on the SPME fiber can be calculated through the
GC response factor. Therefore, the bound drug concentration in
the solution could be easily obtained from eq 7.
The Scatchard plot is presented in Figure 4. From the
regression equation of the Scatchard plot, the slope was equal to
1.02 × 106, the y-intercept equal to 1.03 × 106, and the x-intercept
equal to 1.0. Therefore, for the equilibrium constant K ) 1.02 ×
106 L mol-1, the log K value was 6.01. The number of binding
sites per protein molecule was 1.0.
Table 2. Summary of the Experimental Data of Diazepam Binding to HSA (Method I)
Ctotal area Cs drug bound [D] r/[D]
(ng/mL) count (ng/mL) (%) r (mol L-1) (L mol-1)
1000 11 304 80.3 92.0 0.223 2.82 × 10-7 7.90 × 105
2000 18 814 134 91.1 0.331 4.69 × 10-7 7.05 × 105
3000 29 395 209 89.6 0.434 7.33 × 10-7 5.92 × 105
4000 65 777 467 84.4 0.614 1.64 × 10-6 3.74 × 105
5000 123 885 880 78.0 0.756 3.09 × 10-6 2.44 × 105
6000 205805 1462 70.8 0.857 5.14 × 10-6 1.67 × 105

Table 3. Summary of the Experimental Data of Diazepam Binding to HSA in Small Volume Analysis (Method II)a
Cload ntotal area nf Cs nb drug bound
(ng/mL) (ng) count (ng) (ng/mL) (ng) (%) r r/[D]b
500 19.8 8 970 4.2 62.3 6.28 40.2 0.203 926 773
1000 37.5 18 566 7.9 128.9 10.27 34.7 0.332 733 083
2000 68.4 37 042 14.4 257.1 15.45 15.5 0.499 552 710
a The protein concentration was 0.05 mg/mL. b Molar concentration is used.

Figure 4. Scatchard plot for diazepam binding to HSA (method I).

The apparent equilibrium constant and the total binding
constant were reported as 1.159 × 106 and 4.919 × 105 L mol-1.7,8
This is the only value that is published about diazepam binding
to HSA. Compared with the result in this study, which is 1.02 ×
106 L mol-1, the two results are very comparable with the relative
difference of their log values less than 1%.
Method II. During the protein binding analysis, three con-
centrations are investigated. They are loaded from 500, 1000, and
2000 ng/mL 2-mL buffer solutions, respectively. For each of the
concentrations, three replicates are measured. The extraction time
in the drug loading step and the desorption time in the protein
solution are controlled at 45 min (Figure 3). The protein
concentration in this study is 0.05 mg/mL, which is 7.25 × 10-7
M. Table 3 summarizes the experimental results.
The Scatchard plot is presented in Figure 5. From the
regression equation of the Scatchard plot, the slope equals 1.25
× 106, y-intercept equals 1.17 × 106, and x-intercept equals 0.93.
Therefore, the equilibrium constant K is 1.25 × 106 M-1, and the
log K value is 6.10. The number of binding sites per protein
molecule is 0.93, which is close to 1.
In this study, the protein concentration used was 7.25 × 10-7
M (50 ppm) instead of 1.45 × 10-5 M as in the last experiment.
The total drug concentration introduced by the amount of the
Figure 5. Scatchard plot for diazepam-HSA binding analysis
(method II).
analye loaded on the fiber is 4.53 × 10-7 M (128.9 ng/mL for the
analye loaded from 1000 ng/mL buffer solution). Therefore, the
molar concentrations of total drug and protein were in the same
order of magnitude. This was an important consideration in the
experimental design to minimize the error on both bound and
free concentration calculations.
Determination of the Equilibrium Constant without Cali-
bration. Direct determination of the binding parameters from the
ratio of the fiber extraction in the absence and presence of the
protein was performed for both method I and method II analyses.
Figure 6 shows the plot of A/A′ versus Cp obtained from method
I. The equilibrium constant was obtained from the slope of the
plot, which is 9.77 × 105 L mol-1. This value agreed with the value
attained from previous Scatchard plot.
Figure 7 shows the plot of A/A′ versus Cp obtained from
method II. In this experiment, a protein concentration of 0.05 mg/
mL was used. From the plot, the slope was determined as 0.995
× 106 M-1. Noting that factor (A0 - A)/A0 ) 0.801, the diazepam-
to-HSA binding constant K was then calculated as 1.23 × 106 M-1,
which agrees with the result from that of last section.
Analytical Chemistry, Vol. 73, No. 18, September 15, 2001 4415

Figure 6. Curves used for calculation of the equilibrium constant
of diazepam binding to HSA without calibration (method I).
Figure 7. Curves used for calculation of the equilibrium constant
of diazepam binding to HSA without calibration (method II).
CONCLUSION
SPME is an equilibrium process between the amount of drug
partitioned onto the SPME fiber, the concentration of free drug,
4416 Analytical Chemistry, Vol. 73, No. 18, September 15, 2001
and the concentration of bound drug in the solution. The portion
of the drug analyzed in a GC system through fiber injection repre-
sents the true amount of the drug that had reached equilibrium
with the solution. Therefore, the equilibrium between the drug-
protein binding process was not affected. In most cases, this value
is so small that it could be neglected without influencing the equi-
librium system. However, the verification should be always per-
formed before this assumption is exercised. For large-volume
analysis (method I), the analyte partitioned into the fiber is less
than 3% of the free analyte amount in the solution. Therefore, it
can be safely ignored. However, for small-volume analysis (method
II), the analyte partitioned on the fiber is comparable to the free
analyte in the solution (∼20%), and its contribution has to be
considered.
This work demonstrated that SPME is a valid method in the
protein binding study by employing diazepam binding to HSA as
a model system. The SPME method provides theoretical accuracy
and simplicity in operation. By integrating sample extraction,
loading, and transferring into a single step, this paper presents a
novel method to use SPME to determine the drug-protein
binding parameters for small-volume analysis (method II) by
loading the drug compound first on the fiber and then desorption
in the protein solution. This small-volume method is especially
useful under the circumstance where only a small amount of
protein is available.
In this research, both the Scatchard plot and direct measure-
ment were employed to determine diazepam-HSA binding
parameters. In the direct measurement approach, no calibration
and GC response factor were needed. The results were compared
with the value from the literature. Unlike some conventional
methods, SPME does not have any theoretical limitations in the
determination of the free analyte concentration and protein-
analyte binding constant. It is very convenient for both data
collecting and binding parameter calculation.
Received for review February 26, 2001. Accepted June 29,
2001.
AC010227S