Development and optimisation of a radioimmunoassay for plasma captopril

Cfinicu Chimica Acta, 131 (1983) 295-303 Elsevier

295

CCA 2568

Development and optimisation of a radioimmunoassay for plasma captopril Frances M. Duncan *, Victor I. Martin, Brent C. Williams, Emad A.S. Al-Dujaili and Christopher R.W. Edwards Department ofMedicine, Western General Hospital, Edinburgh EH4 2XU (UK) (Received November

3rd, 1982; revision March 18th, 1983)

A specific and sensitive radioimmunoassay has been developed to monitor plasma levels of captopril, the first orally active angiotensin-converting enzyme inhibitor. Because of the reactive nature of the captopril thiol group, captopril was measured as the captoprii-N-ethylmaleimide complex (captopril-NEM). Accuracy studies, using samples with known added inundations of captopril, were satisfactory (r = 0.95, p -z 0.001, y =0.97x - 2.02), and the radioimmunoassay results compared well with those determined by a gas chromatography/mass spectrometric method (r = 0.98, p -c 0.0005, y = 1.2x - 19). The minimum detection limit of the assay was 2 pg captopril/litre plasma.

introduction

Captopril is an orally active angiotensin-converting enzyme inhibitor, used in the treatment of hypertension and chronic heart failure [1,2]. Previously reported methods for the dete~ation of captopril in biological fluids include gas-liquid chromatography 131,gas c~omato~ap~y/sel~t~ ion monitoring mass spectrometry [4], high performance liquid chromatography [5-71 and a thin layer radiochromatographic method [8], These methods have limited sensitivity and require sample treatment. A radioimmunoassay has been developed based on the measurement of captopril as its N-ethyhnaleimide derivative (Fig. 1). This method has improved sensitivity and obviates the need for present sample extraction and can therefore be employed as a more efficient method for measuring plasma captopril levels.

l

Address for uxrespondence and reprint requests: FM Western General Hospital, Edinburgh EH4 2XU, UK.

000!%8981/83/.$03.00

Duncan,

6 1983 Elsevier Science Publishers B.V.

B.!k.,

Department

of Medicine,

296 CAPTOPRIL

HO\c04

(27FI 7

N-C-F-CHpSH

N-ETHYLMALEIMIDE

Me

INEM)

CAPTOPRIL - NEM

Fig. 1. Structures of captopril and ~-et~yi~a~ei~de

derivative.

Reagents Captopril, captopril-NEM and complexes for cross-reactivity studies were kindly given by the Squibb Institute for Medical Research, Princeton, NJ, USA. N-Ethylmaleimide, penicillamine, lysozyme, histamine, N-hydroxysuccinamide, l-ethyl-3-(3dimethylaminopropyl) carbodiimide and activated charcoal (No. C5260) were from Sigma Chemical Company, Dorset, UK. Dextran I70 was from Pharmacia Fine Chemicals Inc., Piscataway, NJ, USA. TLC Silica gel 60 plates were obtained from Merck, Sharp and Dohme, West Point, PA, USA. Solvents and solutions All organic solvents were of analytical grade and were obtained from BDH Chemicals Ltd., Glasgow, UK. Phosphate buffer pH 7.4,0.5 mol/l was used for the preparation of the radioligand. Tris buffer pH 8.0, 0.05 mol/l containing 0.1% sodium azide and 0.1% lysozyme was used for the assay of samples. Dextran-coated charcoal suspension was used to separate the antibody-bound from the free fraction and was prepared as follows: 10 g of activated charcoal and 1 g dextran T70 were suspended in 1 1 of 0.05 mol/l phosphate buffer (pH 7.4) containing 0.05% gelatin and 0.1% sodium azide. Standard solutions and patient samples Standard solutions containing O-20 000 pg/l of captopril were made up in human plasma containing 2 g/l NEM, and were stored at -20°C. Blood samples were taken into lithium heparin tubes, spun in a cold centrifuge and 5 ml of the separated plasma added to tubes cont~ng 10 mg NEM. The samples were stored at - 2O“C until required for assay.

291

Production of antisera

Three immunogens were prepared by coupling captopril-NEM to bovine serum albumin (BSA), thyroglobulin (TG) and keyhole limpet haemocyanin (KLH) using the activated ester method [9]. The molar incorporations were found to be 24 moles captopril-NEM/mole BSA, 90 moles captopril-NEM/mole TG, and 65 1 moles captopril-NEM/mole KLH. Two New Zealand white rabbits were immunised by multiple intradermal injections against 100 pg of each immunogen in complete Freund’s adjuvant. The first bleed was taken after 6 weeks, and further boosting injections given at fortnightly intervals. The antisera produced were assessed using ‘251-histamine-captopril-NEM ligand. Antibody titres were determined by incubating various dilutions of each antiserum with the radioligand (7000 dpm) in 200 1.11 Tris buffer (pH 8.0,0.05 mol/l) at 17OC overnight. Antibody-bound ligand was separated from the free fraction by adding 0.5 ml Dextran-coated charcoal suspension. The tubes were centrifuged at 4°C for 15 min at 1700 x g, the supernatant was aspirated, and the charcoal pellet counted. Preparation

of label

Labelled captopril-NEM was prepared by coupling the captopril-NEM to ‘251histamine using the carbodiimide reaction [lo] as follows: 0.12 mg captopril-NEM was added to a glass bottle containing 2 mg N-hydroxysuccinimide, 7 mg I-ethyl-3(dimethylaminopropyl) carbodiimide (EDC) and 500 ~1 dimethylformaldehyde; 50 ~1 of this solution was added to ‘251-histamine (1 mCi in 40 ~1). The reaction vessel was left for 3 h, and the ‘251-histamine-captopril-NEM was purified by thin layer chromatography in the system ethyl acetate/ethanol/glacial acetic acid 25 : 25 : 1 (v/v/v). The immunoreactive peak was found at R, 0.52. Plasma assay for captopril

N-Ethyl-maleimide (NEM) was added to standard captopril solutions and to patient samples at a concentration of 2 g/l; due to the cross-reactivity of 0.07% of NEM with the captopril-NEM antibody, excess penicillamine, at a concentration of 1 g/l buffer was added to the assay buffer immediately before use. The cross-reactivities of penicillamine and penicillamine-NEM with the captopril-NEM antiserum were < 0.001 and 0.02, respectively; the sensitivity of the assay was greatly increased by the addition of penicillamine to the system. The optimum incubation time and temperature were determined, and the effect of assay buffer pH on the sensitivity of the standard curve was studied. The assay was carried out by adding 50 ~1 of standard or sample to 200 ~1 of assay buffer containing 1 g/l penicillamine, 7000 dpm (‘251)-histamine-captoprilNEM, and antibody at a final dilution of 1 : 40000. The tubes were vortexed, incubated overnight at 17°C and the antibody-bound and free fractions separated using Dextran-coated charcoal.

298 TABLE I Cross-reactivities of KLH,,, antiserum Compound

Cross-reactivity

Captopril-NEM Captopril dimer Captopril-glutathione N-Ethylmaleimide Penicillamine-NEM Glutathione-NEM Cysteine-NEM Penicillamine Glutathione Captopril-cysteine

100.000 1.000 1.000 0.07 0.02 0.002 0.002 < 0.001 < 0.001 < 0.001

Evaluation of method Specificity. Detailed specificity studies were carried out on one of the antisera by setting up standard curves with the complexes listed in Table I. Serial dilutions were made of each complex in an incubation volume of 250 ~1. ‘251-histamine-captoprilNEM (7000 dpm) was added and antibody at a dilution giving 50% binding of the ligand in the absence of unlabelled complex. After incubation at 17°C overnight, the separation procedure was repeated as described previously. The percentage cross-reactivity with various complexes was calculated on a w/w basis [ 1l] as: A/B x 100 where A is the amount of captopril-NEM and B is the amount of cross-reacting complex at which binding of the iodinated ligand is reduced by 50%.

TABLE II Precision studies for captopril-NEM radioimmunoassay Intra-assay precision

n SD cv

pg captopril/l 180 IO 0.68 7.6

plasma 1080 10

5.16 9.5

1960 10 9.8 10.1

Inter-assay precision

” SD cv

cg captopril/I plasma 200 10 0.8 7.5

1100 10 9.9 18

n, number of samples. SD, standard deviation (pg captopril/l).

1840 10 13.7 I5 CV, coefficient of variation (‘8).

299

The specificity was also measured by comparing results with those determined using a gas chromatography/mass spectrometric method. Captopril concentrations were measured in 20 samples using both techniques. Accuracy. The accuracy of the method was determined by measuring the recovery of known amounts of unlabelled captopril added to captopril-free plasma. Sensitivity. Incremental doses of captopril standard were added to captopril-free plasma, containing 2 g/l NEM, and assayed. The sensitivity of the assay was defined as the minimal amount of captopril that could be significantly distinguished from the zero standard. Precision. The precision of the method was determined by measuring the recovery of known amounts of unlabelled captopril added to captopril-free plasma containing 2 g/l NEM (Table II).

Results Production and characterisation of antisera The four rabbits immunised with the TG and KLH complexes produced antibod-

ies against the immunogen, but the two rabbits immunised against the BSA produced no detectable antibodies against captopril-NEM. The highest titres were obtained from the animals immunised with the KLH complexes, the best being obtained from KLH rabbit 1, bleed 2 (KLH,,,) (Fig. 2). Rabbit KLH, 2 antiserum was used for all assays at a final dilution of 1 : 40000.

100

90

-1.

1

‘O-

FREE 60-

,,,,,,,,,,,,,,a, 20 LO 60 80 Ruiprocal d final antibody

I

1w 120 140 dilution 1000

lx

I

Fig. 2. Antibody dilution curve for captopril-NEM

antibody (KLH,,,)

with ‘251-histamine-captop~l-NEM.

300

Evaluation of the method The optimum incubation time for the plasma captopril-NEM assay was found to be 15 h, and the optimum incubation temperature 17°C. Using these assay conditions the most sensitive standard curve was achieved at a pH of 8.0. Speci‘city. The percentage cross-reactivities of the antiserum KLH ,,z with various complexes are shown in Table I. The highest cross-reactivities were against captopril dimer (I.O%), captopril-glutat~one (1.0%) and NEM (0.07%); these levels were unimportant as plasma captopril-glutathione and captopril dimer levels are low, and excess NEM in plasma samples and standard solutions was coupled to penicillamine. The radioimmunoassay results compared well with those determined using a gas chromatography/mass spectrometric method: r = 0.98, p < 0.005, y = 1.26x - 10 (Fig. 3). Accuracy. Accuracy studies were satisfactory: 2.02 (Fig. 4).

r = 0.95. p -=z0.001, y = 0.97x -

Sensitivity. A typical standard curve using the antiserum from rabbit KLH,., is shown in Fig. 5. The minimal amount of captopril that could be significantly distinguished from zero was 0.05 ng. h

SOO-

SOO-

f

alo% 6 3 8

5300-

E ._ 0 0 BmO-

Y loo-

x

0’

ml Ga

200 Chromatography

300

x

al

I Mass Spectrometry

$J gi

Fig, 3. Comparison of results obtained by radioi~un~~y analysis. y = 1.26x - 19; r = 0.98; p C 0.0005.

t1 with those after extraction

and GC/MS

301

200. 160 160

e

120

8 g 100 = h z 8 u

60 60 LO 20

0

20

LO

60

1

I

b

60 l&l 120 l&O Captopril added

lngl

u 160

Fig. 4. Accuracy of assay. y = 0.97x - 2.02; r = 0.95;

1

mrr0

1

0.1

1

1 Coptopril

I

10

*

I

160

p < 0.001.

1

100

(ngl

Fig. 5. Captopril standard curve using KLH,.,

260

antibody.

,

1CQO

302

Precision. The intra- and inter-assay

variations

are shown in Table

Il.

Discussion The introduction of captopril, the first orally active inhibitor of angiotensin converting enzyme, has revolutionised the treatment of drug-resistant hypertension and congestive cardiac failure. Some serious side effects such as neutropenia have occurred in a small number of patients, almost all of whom have had impaired renal function. Some of the side effects may well be dose related. With the development of this captopril radioimmunoassay, large numbers of samples can now be assayed with ease. Some pharmacokinetic results have already been reported using this assay for a group of hypertensive patients [ 121. The radioimmunoassay described in this paper is easy to perform, and provides a sensitive, accurate and precise method of measuring plasma captopril levels. Captopril-NEM antisera were produced in rabbits using KLH-captopril-NEM immunogen; these antisera were shown to have minimal cross-reactivity with potentially interfering complexes. A satisfactory high specific activity 125I-radioligand was produced based on the iodohistamine method. From the iodination reaction. the major product was immunoreactive. Another, non-immunoreactive, peak was obtained at an R, value of 0.7; this peak has not yet been identified. Captopril is excreted by the kidney. This assay can readily be used to measure captopril in urine, which could be useful in assessing the compliance of patients for whom the drug is prescribed. Studies on the accuracy and precision of the assay were satisfactory. The sensitivity of the method was adequate for pharmacokinetic studies. The validity of the plasma method was confirmed by the correlation obtained between this method and that using gas chromatography/mass spectrometry. Acknowledgments We would like to thank Dr. Jim Knill and Dr. Peter Pigott, Squibb cals, for their help in the development of the radioimmunoassay.

Pharmaceuti-

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303

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