ELSEVIER Analytica Chimica Acta 342 (1997) 159-165

ANALYTICA CHIMICA ACTA

Binding of combined

sulfamethoxazole to human serum albumin studied by a technique of microdialysis with liquid chromatography

Hailin Wang, Hanfa Zou*, Aisheng Feng, Yukui Zhang

National Chmmatographic R&A Centre, Dalian Institute of Chemical Physics, Academia Sinica, Daliun 116011, China

Received 27 June 1996; received in revised form 10 December 1996; accepted 16 December 1996

Abstract

A new, simple and fast method for the determination of the interaction parameters of sulfamethoxazole (SMZ) to human serum albumin (HSA) has been developed by utilizing a microdialysis sampling technique combined with liquid chromatography (LC). The drug and protein were mixed in different molar ratios in 0.067 M potassium phosphate buffer, pH 7.4, and incubated at 37°C in a water-bath. Then the microdialysis probe was put in the SMZ-HSA solution and sampled at a perfusion rate of 1 pl/min. The concentration of SMZ in the microdialysates was determined by reversed-phase liquid chromatography. The recovery (R) was also determined in vitro under similar conditions, R is about 41.8% with an RSD of about 2.3%. The association constant (K) and the number of the binding sites on one HSA molecule (n) are calculated by three equations, the values of nK estimated by three methods are quite similar. The values found for K and n are 3.24 x I O3 M-’ and 3.04, respectively.

Keywords: Microdialysis; Sulfamethoxazole; Human Serum albumin; Drug-protein interaction; High performance liquid chromatography

1. Introduction

It is well known that a drug in blood is bound to a greater or lesser extent to plasma proteins such as albumin and cut-acid glycoprotein and that the con- centrations of bound and free species are in equili- brium. Studies on drug-protein binding are important in pharmacology and pharmacokinetics [l-3], because drug-protein interaction affects the pharma- cological activities and side effects of the drug as well as the drug distribution and elimination. The unbound drug alone is supposed to diffuse from the blood to the

*Corresponding author.

0003-2670/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SOOO3-2670(96)00623-X

extravascular active sites and to exhibit the pharma- cological activity and/or the side effect. Some impor- tant pharrnacokinetic properties, such as hepatic metabolism rate, renal excretion rate, biomembrane permeation rate, and steady state distribution volume, also depend on the unbound drug fraction. The devel- opment of a simple and easy method to determine the concentration of unbound drug serves to promote progress in these studies.

Various methods for studying drug-protein binding phenomena have been described. Most involve equi- libration of the drug with a solution of an isolated protein or with whole plasma and then separation of the free drug from that bound to the protein by equilibrium dialysis, ultrafiltration, or ultracentrifuga-

160 H. Wang et al./Analytica Chimica Acta 342 (1997) 159-165

tion [4]. These methods suffer from a number of disadvantages, such as: relatively large quantities of drug are required; they are time-consuming and require significant operator skill; and they are poorly suited to automation. Several chromatographic approaches for the determination of drug-protein binding have also been reported. One of these is gel-filtration frontal analysis method based on size- exclusion chromatography (SEC) [5], which is estab- lished earlier than other chromatographic methods for this purpose but requires a large volume of sample solution to achieve a clear y plateau. High perfor- mance frontal analysis in liquid chromatography (LC) established by Nakagawa et al. [6-81, using a restricted-access type LC column, which is a combi- nation of size-exclusion and reversed-phase (RP) chromatography, has a number of advantages such as direct injection of sample solution without pretreat- ment. This enables simple and rapid analysis and requires a much smaller volume of sample solution than conventional gel-filtration frontal analysis. How- ever, this method requires good skill of the operator and a column with suitable hydrophobic strength to allow the elution of drug from the column by mobile phase without adding any organic modifiers. Another promising method involves the correlation of the degree of protein binding of a drug with its retention on a chiral stationary phase (CSP), derived from bovine serum albumin (BSA) [9], human serum albumin (HSA) [lo], or cri-acid glycoprotein [l l] for LC. This method is able to reveal competitive and anticooperative interactions between ligands simultaneously bound to HSA or other plasma pro- teins. It is difficult to find a satisfying correlation between the degree of protein binding and retention for all drugs or most of the drugs which are not similar in structure.

Microdialysis has been extensively applied to moni- tor continuously the concentration of unbound drug and neurotransmitter in vivo [12-161. Microdialysis sampling allows the determination of the concentra- tions of unbound drugs after a dialysis membrane has been placed in the drug-protein mixed solution. The technique is based on the kinetic dialysis principle in which substances diffuse down their concentration gradient. The microdialysis probe is usually a tubular membrane mounted on a double cannula made of fused silica and plastic. A perfusion solution is

pumped at a low-rate (1-5 pl/min) through the inlet of the probe and collected at the outlet, yielding a sample ready for analysis. The dimensions of the probe, i.e. the membrane length, diameter, and mole- cular weight cut-off, can be varied according to the requirements of application. The method is time sav- ing and even simpler than equilibrium dialysis. Micro- dialysis has also the advantage that the technique is easy to automate and can be on-line hyphenated with many analytical techniques such as LC, capillary electrophoresis (CE), flow injection analysis (PIA), mass spectrometry (MS), etc. Recently it has been reported that the technique of microdialysis combined LC was used to study the interactions between carba- mazepine and HSA. The results are in agreement with the literature where the high performance frontal analysis was used and demonstrate the possibility of the method [17]. SMZ is widely used as an antimi- crobial agent and coccidiostats, and the degree of plasma protein binding for it is 63.4% [9], but as we know, the measure of other interaction parameters of SMZ to HSA is not reported yet. In this work, the applicability of the combined technique of microdia- lysis with LC for the determination of SMZ-HSA is reported, the comparison of the experimental data treated by Scatchard equation, Klotz equation and nonlinear equation is also carried out.

2. Experimental

2.1. Reagent and materials

SMZ and HSA (fatty acid and globulin free) were purchased from Sigma (St. Louis, MO, USA). HSA was dissolved in a potassium phosphate buffer (pH 7.4, 0.067 M). A CMAl20 microdialysis probe was purchased from CMA/Microdialysis (Acton, MA, USA), the length of the dialysis membrane is 4 mm.

2.2. Preparation of the sample solution

An SMZ stock solution was made up in ethanol. An appropriate volume of this stock solution was put in a 5 ml open vial, and the ethanol was evaporated in air. An appropriate volume of an HSA solution (in phos- phate buffer of pH 7.4, and 0.067 M) was added to the vial in order to prepare the solutions.

H. Wang et al./Analytica Chimica Acta 342 (1997) 159-165 161

2.3. Microdialysis sampling

The microdialysis system comprises a FAMILIC- 1OON microinjection pump (JASCO, Japan) and a microdialysis probe (CMA, USA). The perfusion solution is a 0.067 M potassium phosphate buffer of pH 7.4; perfusion rate is 1 ul/min. The microinjector was filled with perfusion solution.

The SMZ-HSA mixed solution was incubated at 37°C in a water-bath for more than 10 min before the probe was put into this solution, and sampling from the solutions was started. After 12 min, the dialysate was collected for 30 min. The collected dialysate was handled for LC analysis. The probe must be washed by the perfusion solution at a rate of 5 ul/min for several minutes before the probe is put into the mixed solution to get rid of air in the probe and of organic solvents, which are used for protection of the dialysis membrane.

2.4. Apparatus and instruments

The present LC system comprises an LC- 10A pump (Shimadzu, Japan), a Rheodyne-type injector valve with a 10~1 loop, an SPD-1OAV UV detector (Shi- madzu, Japan), and a WDL-95 chromatographic workstation (National Chromatographic R&A centre, China).

The LC conditions were as follows: Column - 4.6 mm i.d.x 150 mm length, packed with 5 pm of Spherisorb Cl8 (packed by the National Chromato- graphic R&A Centre, China). The mobile phase used is methanol/water (40/60) containing 10 mM acetic acid-sodium acetate buffer of pH 5.9; the solute was detected at UV 270 nm; the flow rate of the mobile phase is 1.0 ml/min.

2.5. Recovery of microdialysis

The recovery (R), also called the dialysate extrac- tion fraction, defined as the concentration ratio of the drug in dialysate (Cd) to the unbound fraction in drug- protein solution, is determined by placing the micro- dialysis probe in 0.067 M potassium phosphate buffer at pH 7.4 by adding 50 pM of SMZ (standard solu- tion). The operation conditions were the same as for the microdialysis sampling described above. The dia- lysate was collected and analyzed by RP-LC, the

concentration of SMZ in the standard solution was

also determined by RP-LC.

2.6. Calibration for LC analysis

A weighed amount of SMZ was dissolved in the phosphate buffer (pH 7.4, 0.067 M) to make concen- trations ranging from 8 to 400 pM. Each 10 pl portion of this SMZ standard solution was introduced into the LC injector. The height of the consequent baseline shift due to SMZ injected was measured and plotted against the SMZ concentration. The calibra- tion line for the concentration of SMZ in the dialysate was C=3.776+19.6407 x 10p5A (R>0.9999), or C= 1.777+1.867x 10-3H (R=0.9997), where C is the concentration of SMZ, H and A represent the peak height and the peak area, respectively.

3. Results and discussion

Fig. 1 shows a schematic diagram of the present microdialysis system for the study of drug-protein

1

UI I 3

Fig. 1. Schematic diagram of microdialysis for the study of drug- protein interaction: (1) microinjection pump, which is filled with

perfusion solution; (2) microdialysis probe; (3) mixed solution of drug and protein; (4) collection vial.

162 H. Wang et nl./Annlytica Chimica Acta 342 (1997) 159-165

interaction. The system consists of a microinjection pump, a microdialysis probe, the drug-protein mixed solution and collection vials. The microinjection pump is filled with perfusion solution, which is a phosphate buffer with the same pH and ion strength as the drug-protein mixed solution. The solution in the collection vial is sampled from the drug-protein mixed solution by microdialysis and named ‘dialy- sate’, which can be handled for analysis by chroma- tography, UV spectrometry and other techniques. In this study, the dialysates are directly determined by RP-LC.

The recovery (R) is a key parameter to the micro- dialysis method for the determination of drug-protein interaction. In our previous work [ 18 1, we investigated the influences of perfusion rate and temperature on the relative recovery, showing that the operation conditions for microdialysis sampling such as perfusion rate, temperature must be strictly controlled, because these two factors have a serious influence on precision and accuracy of R. R is expressed as follows:

R% = (Ad/A,) x lOO%, (1)

where Ad and A, represent peak area of SMZ in dialysate and SMZ in standard solution, and are proportional to their concentrations, respectively. The recovery changes with the change of perfusion rate. Recovery decreases when perfusion rate increases, reversely, recovery increases when perfu- sion rate decreases. In our sampling process, the perfusion rate used is 1 pl/min. At lower perfusion rate, the sampling process becomes too time-consum- ing in spite of improvement of the recovery. The tubular dialysis membrane used is 4 mm long, 0.5 mm o.d. If a longer dialysis membrane is used, recovery may be improved and sampling becomes faster. In this study, the average recovery determined is 41.8 with an RSD of about 2.30% (~2~4). The R of carbamazepine (CBZ) under the same conditions is 42.7%, and RSD is about 1.85% [17], which is very close to the value of R for SMZ determined by the same microdialysis probe over a long period of use. These results demonstrate that a good precision can be achieved under strictly controlled experimental con- ditions.

The relative decrease in the SMZ concentration in the SMZ-HSA solution (Al) can be calculated accord-

ing to the following formula:

A,(%) = [(v x R x t x Cu)/(V x Crot)] x 100, (2)

where v is the perfusion rate, R the recovery, t the sampling period, V the volume of the mixed solution sampled, and C, and C,,, are the unbound and total concentration of drug, respectively. In this study, t=30 min, R<50%, C,lC,,,<O.75, v=l pl/min, V>l.O ml, Al calculated by Eq. (3) is below l%, showing that the effect of sampling on equilibrium can be neglected.

The unbound SMZ concentrations determined by the technique of microdialysis combined with LC were quantified by means of a calibration line. Fig. 2 shows the chromatogram of SMZ in the dialy- sate separated by RP-LC for six consecutive injec- tions. SMZ is well separated from interferences and has a short retention time. The unbound SMZ con- centration in the SMZ-HSA mixed solution can be calculated as follows:

C, = Cd/R. (3)

The SMZ in the dialysate was analyzed by RP-LC, and quantified by a calibration line. Table 1 lists the unbound concentrations of SMZ in SMZ-HSA mixed solution with different molar ratios of SMZ to HSA sampled by microdialysis and analyzed by RP-LC.

The binding studies involve the determination of the parameters, such as the binding constants, the max- imum number of drug molecules bound to a protein molecule and classes of binding sites on a protein. The reversible binding of a drug to a protein is governed by the multiple equilibria theory expressed by the follow- ing equation [19]:

(4)

where m is the number of classes of independent adsorption sites, nj the number of sites in a class i with an association constant of Ki, Cb the concentra- tion of bound drug and [p] represents the total con- centration of protein. This equation can be converted to a simple formula if m=l, which means that the protein just has one class binding site for the drug, as follows:

nKC,

r=l+KC,. (5)

H. Wang et al./Analytica Chimica Acta 342 (1997) X59-165 163

1

0.00 3.22 7.63 11.47 15.29 1! !Li

Fig. 2. Chromatogram of SMZ in dialysate for consecutively injecting (n=6). For conditions for LC see the text.

Table 1 Unbound SMZ concentrations and binding fractions in HSA

solutions

SMZ-HSA

(PM)

Total concentration

(PM)

Unbound

concentration

(PM)

Bound

fraction

(8)

350-100 353.10 222.42 37.0 (7.74%) (5.08%)

18CklOO 179.11 105.39

41.2 (4.04%)

(2.02%) 20.0-100 20.77

10.50 49.4 (1.43%) (2.87%) 5.0-100

5.38 2.70 49.8

(5.12%) (4.99%)

45.0-300 44.07 11.63 73.6

(3.39%) (5.12%)

The values in parentheses indicate relative standard deviation

(RSD).

Eq. (5) describes a Langmuir isotherm, and is usually transformed to Eq. (6) or Eq. (7), described as follows:

r/Cu = -Kr + nK,

l/r = l/n + (l/nK)(l/C,),

(6)

(7)

where K is the association constant and n is the number of the binding sites on one protein molecule. Eq. (6) is used for Scatchard analysis and Eq. (7) for a Klotz plot [20].

The interaction parameters of SMZ to HSA calcu- lated using Eqs. (5)-(7) here are all presented in Table 2, the correlation coefficients obtained are also listed. The values of nK are quite similar. Both the Scatchard plot (refer to Fig. 3) and the Klotz plot (refer to Fig. 4) are linear, showing that SMZ has only one type of binding sites. The value of n and K

estimated by the Scatchard equation are between the values obtained by the Klotz analysis and Lang- muir isotherm (refer to Fig. 5). The Scatchard equa- tion is widely applied in the study of interactions between drug and protein more than any other equa- tions, especially when only one type of binding site is present, so the results obtained by this equation are accepted. However, the Klotz equation gives the best correlation coefficient (y), 0.9999. What causes these differences is still a question. The nK for SMZ is far below high affinity drugs such as warfarin, ibuprofen and fenoprofen (nK is ca. lo6 M-‘) and endogenous compounds such as fatty acids (nK is ca. lo8 M-l), showing that SMZ is just a moderately binding drug.

164

Table 2

H. Wang et al. /Analytica Chimica Acta 342 (1997) 159-165

Binding parameters for SMZHSA interaction

Data analysis n K (Mm’) nK (M-l) Solution Temp. (“C) -ra

Eq. (5) 4.06 2.13x lo3 8.65 pH 7.4, I=O.17 31 0.9990 Eq. (6) 3.04 3.24x IO3 9.85 pH 7.4, 1=0.17 37 0.980

&. (7) 2.50 4.00x 103 10.0 pH 7.4, 1=0.17 31 0.9999

ay is the regression coefficient.

Is2 12

r/C, xlWM_1

6-

0.00 0.30 0.60 0.90 1.20 1.5

r

Fig. 3. Scatchard plot of SMZHSA interaction.

40

32

24

l/r

16

0 80 160 240 320 400

l/c” x10-w

Fig. 4. Klotz plot of SMZ-HSA interaction.

This is in agreement with the literature where a 63.4% binding degree in plasma has been reported [9]. In our study, a binding degree of 73.6% for 300 pM HSA is observed, which is fatty acid and globulin free, so a higher binding degree is obtained.

2.00

1.60

1.20

r

0.80

0.40

0.00

jl_ /, , 0 60 120 180 240 30

c, PM

Fig. 5. Nonlinear regression curve of r vs. C, according to Eq. (5)

Measuring interaction parameters using microdia- lysis combined with LC is simple and fast, and also easy to be automated. To determine free drug in a drug-protein solution usually two steps are included: Sampling by microdialysis and analysis by LC. In our laboratory, one test will be finished in an hour, 40 min for sampling and 20 min for LC analysis (3-5 injec- tions). At present the method described in this paper may be more time-consuming than high performance frontal analysis by Nakagawa et al. [6-81, but the separation power of the restricted-access column is quite limited. So, for the determination of the compe- titive interaction between the drugs and protein, microdialysis has advantages in many respects such as chiral separation mechanism, thermal behavior of drug-protein binding and the binding site location of drugs on protein. Using longer tubular membranes will result in faster sampling and shorter total analysis time by microdialysis technique. These results

H. Wang et al. /Analytica Chimica Acta 342 (1997) 159-165 165

indicate that the technique of microdialysis combined with LC is applicable for an estimation of the protein binding parameters. We think interactions between the macromolecules and small molecules are one of the important subjects to understand the role of these molecules in biological process, such as the interac- tion of drug-protein, drug-DNA, environmental toxic compounds-macromolecules, etc. Wide applications of the method reported in various fields are to be taken

up.

Acknowledgements

The financial support from the administration of the Chinese Academy of Sciences and the National Science Foundation of China to Dr. Hanfa Zou is gratefully acknowledged.

References

[l] M.C. Meyer and D.E. Guttman, J. Pharm. Sci., 57 (1968) 895.

[2] J.J. Vallner, J. Pharm. Sci., 66 (1977) 447. [3] T.C. Kwong, Clin. Chim. Acta, 151 (1985) 193.

[41

[51

El

[71

WI

[91

UOI

t111

WI

u31

[I41

u51

[161

Cl71

[181

[I91

WI

B. Sebile, Fundam. Clin. Pharmacol., 4(2) (1990) 151s.

W. Scholtan, Arzneim.-Forsch., 15 (1965) 1433. A. Shibukawa, N. Nishimura, K. Nomura, Y. Kuroda and T.

Nakagawa, Chem. Pharm. Bull., 38 (1990) 443.

A. Shibukawa, C. Nakao, T. Sawada, A. Terakita, N.

Morokoshi and T. Nakagawa, J. Pharm. Sci., 83 (1994) 868. A. Terakita, A. Shibukawa, C. Nakao and T. Nakagawa, Anal.

Sci., 10 (1994) 11.

N. Lammers, H. Debree, C.P. Groen, H.M. Ruijten and B.J. de

Jong, J. Chromatogr., 496 (1989) 291. T.A.G. Noctor, M.J. D&-Perez and I.W. Wainer, J. Pharm.

Sci., 82 (1993) 675. R. Kaliszan, A. Nasal and M. Turowski, Biomed. Chroma-

togr., 9 (1995) 211.

U. Tossman and U. Ungerstedt, Neurosci. Lett. Suppl., 7

(1981) S479.

B.H.C. Westerink and M.J.H. Tuinte, J. Neurochem., 46

(1986) 181.

B.L. Hogan, S.M. Lunte, J.F. Stobaugh and C.E. Lunte, Anal.

Chem., 66 (1994) 596.

S.Y. Zhou, H. Zou, J.F. Stobaugh, C.E. Lunte and SM. Lunte,

Anal Chem., 67 (1995) 594. S.A. Wages, W.H. Church and J.B. Justice, Jr., Anal. Chem.,

58 (1986) 1649. H. Wang, H. Zou and Y. Zhang, Chromatographia, in press. H. Wang, H. Zou, A. Feng and Y. Zhang, Chinese J. Anal.

Chem., 2 (1997).

H.A. Feldman, Anal. Biochem., 48 (1972) 317. I.M. Klotz and D.L. Hunston, Biochemistry, 10 (1971) 3065.