Enzyme Immunoassay of Carbamazepine in Serum and Saliva

J. W. PAXTON AND R. A. DONALD

Key Words: Enzyme immunoassay; Carbamazepine; Anti-epileptic; Saliva; Serum

INTRODUCTION

Measurement of drug concentrations in saliva offers several advantages over

similar measurements in serum. Most importantly, it has been suggested that

measurement of drug concentrations in saliva may be more therapeutically mean-

ingful, as it may represent the concentration of the free pharmacologically-active

drug. The usefulness of saliva concentrations for monitoring phenytoin therapy

and undertaking pharmacokinetic studies has been demonstrated (Reynolds et al.,

1976; Paxton et al., 1977a; Paxton et al., 1977b; Mucklow et al., 1978) and recent studies indicate that it may be possible to use salivary concentrations of carbama-

zepine (CBZ) in a similar manner (McAuliffe et al., 1977; Westenberg et al., 1978).

We undertook to adapt the enzyme immunoassay for CBZ (EMIT-AED, Syva, Palo

Alto, Ca.) to the equipment that was available in our laboratory. We also wished to reduce the cost of each assay, yet maintain adequate precision and accuracy, and

to increase the sensitivity of the assay to enable us to measure serum concentrations

for several days after a single oral dose. In addition, in these single oral dose

studies, we wished to measure the corresponding salivary concentrations.

MATERIALS AND METHODS

Apparatus

The apparatus recommended by Syva Corp., the manufacturers of the EMIT kit,

was not available. The spectrophotometer that we used was a Pye-Unicam UV SP8-

100 equipped with an automatic sipper system and a thermally-regulated flow-cell

that was maintained at 30” by heated water circulated from a Grant waterbath. With

the volume adjustment knob set on two, the sample volume aspirated into the flow cell was approximately 700 ~1. With a wave length of 340nm, a bandwidth of Inm

and a reference cell containing distilled water, the spectrophotometer was set up as follows: With the instrument in the absorbance mode, distilled water was aspir-

From the Department of Pharmacology and Clinical Pharmacology, School of Medicine, University of

Auckland, Auckland, New Zealand Address reprint requests to J. W. Paxton, Department of Pharmacology and Clinical Pharmacology,

School of Medicine, University of Auckland, Private Bag, Auckland, New Zealand.

Received August 28, 1979; accepted September 26, 1979.

299

Journal of Pharmacological Methods 3, 289-296 (1980)

@ Elsevier North Holland, Inc.. 52 Vanderbilt Avenue, New York, NY 10017 0160.5402180/0402890$02,25

290 J. W. Paxton and R. A. Donald

ated into the flow-cell and the digital display set to 0.100 with the zero control

knob. The mode control is then switched to concentration and the display set to

267 with the concentration calibration knob and then to zero with the zero control knob. After each sample the flow-cell is purged with one wash of distilled water.

For optimum assay performance the spectrophotometer should be connected to

a timer/printer that would record and print out readings at specified time intervals.

This was not available and a stopwatch was used to time the readings.

For the sample and reagent volume measurements, we used the SMI Micro/Petter

Diluter-Dispenser system that mechanizes the delivery of the two reagents A and

B and combines their simultaneous delivery with a serum or calibrator sample. This

system was slightly modified by removal of the delivery nozzle, to ensure complete

separation of the two reagents until delivery into the disposable assay vessel (a 2

ml auto-analyser cup). Both reagents are delivered simultaneously into the cup with

sufficient force to ensure adequate mixing of components.

Reagents

Pure carbamazepine and its main metabolite, carbamazepine-10, II-epoxide,

were gifts from Ciba-Geigy N.Z. Other reagents were obtained commercially (EMIT,

Syva Corp., Palo Alto, Ca.). The assay buffer, 0.055M Tris-HCI pH 7.9 containing

a surfactant, was prepared as directed from a buffer concentrate supplied with each

reagent kit (EMIT package insert). Reagents A and B, specific for carbamazepine,

were in lyophilized form and were prepared for use with some modifications. They

were reconstituted with 3 ml distilled water and allowed to stand at room temper-

ature for 8 hr before further dilutions were made. Reagent A (3 ml) was then diluted

with 33 ml assay buffer (ie, 1 : 12 dilution) and Reagent B with 45 ml buffer (ie, 1 : 16

dilution). Between use, these diluted reagents when stored at 4°C were stable for

several weeks. Reagent A contained antibodies directed against carbamazepine, the enzyme substrates glucose-6-phosphate and nicotinamide adenine dinucleo-

tide, and preservatives dissolved in buffer; whereas reagent B had carbamazepine coupled to glucose-6-phosphate dehydrogenase (EC 1 .I .I .49) and preservatives in

buffer.

The calibration kit for the EMIT anti-epileptic assay contains five drugs: pheny-

toin, phenobarbital, primidone, ethosuximide, and carbamazepine. The concentra-

tions of CBZ in the reconstituted serum were 0, 4.2 (I), 10.6 (2.5), 21.2 (5), 31.8 (7.5), and 42.3 pmol/l (10 pg/ml). These calibrators were diluted a further 1 : 4 with

normal healthy human serum to bring the standard curve into the range required

for single dose studies and gives the calibrations the following values: 0, 1.0, 2.6,

5.3, 7.9, and 10.6 Fmol/l. Quality control serum specimens were made up by weighing out some pure CBZ, dissolving in methanol to give a stock solution (2.12 mmol/l) and then diluting with normal human serum to give known CBZ concen- trations. These specimens were used for obtaining accuracy and precision data for

the serum assay. Before assaying, all serum samples and the standard curve were diluted 1 : 6 with buffer and 200 ~1 used for analysis. A standard curve and quality

control specimens were similarly made up in saliva by diluting a known stock CBZ

solution with normal pooled whole human saliva that had been centrifuged at 1,720 g for 30 m to remove any precipitate. These calibrators had the following values:

EMIT Assay of CBZ in Serum and Saliva 291

0, 0.35, 0.70, 1.40, 2.80, 4.23 pmol/l and were dispensed into 500 ~1 aliquots and

stored at -20°C until required. Before assaying, both samples and standard were

diluted 1 : 2 with assay buffer and 200 ~1 used for each analysis. Interference by

carbamazepine-lO,ll-epoxide in the assay was investigated by adding increasing

amounts of this compound to both blank serum and saliva, and then assaying. An approximate value for the cross-reactivity of this metabolite was obtained by the

expression (x/y) x 100 where x is the molar weight of CBZ and y is the molar

weight of the epoxide metabolite required to cause a similar increase in absorbance

difference.

Collection of Samples

Blood samples were obtained during the first day after a 400 mg oral dose, using

an indwelling cannula and thereafter by venipuncture. The blood samples were

collected into 5 ml plain glass tubes, allowed to clot and then centrifuged at 1720

g for 30 min. The serum samples thus obtained were stored at -20°C until required

for analysis. Simultaneously, whole saliva samples were obtained by spitting into

universal containers. Where necessary salivary flow was stimulated by a citric acid

crystal placed on the tongue. Saliva samples were frozen at -20°C as soon as

possible after collection. Prior to assaying, the samples were thawed and centri-

fuged at 1720 g for 30 min to remove any insoluble matter and the clear supernatant

used for analysis.

Procedure

After initial preparation of reagents and dilution of calibrators and samples, the

pipetter-diluter was primed with reagents A and B. Using the SMI sampler, 200 ~1

of diluted specimen was delivered to the assay cup followed by simultaneous

addition of 300 ~1 reagent A and 400 PI reagent B, giving a total assay volume of 900

~1. Upon addition of the reagents, the contents of the cup were aspirated into the

spectrophotometer flow-cell and the stopwatch started. At 15 set the first absorb-

ance reading was taken followed by a second at 120 sec. The difference of the two

absorbance readings (AA) over the 105 set measurement period was used to cal-

culate the results. Each calibrator and unknown sample was assayed in duplicate

and the mean AA used. The difference (A/i - A&,) between the mean zero blank

reading (A&,) and the mean reading of each of the other calibrators (AA) was

determined and a standard curve plotted on the graph paper provided with the kit.

The results (AA - A&) of unknown samples are then converted to concentrations

by the use of the standard curve.

RESULTS

The amount of reagents used was reduced by twofold over the manufacturer’s recommendations. Examination of the relationship between absorbance and time

for various concentrations of CBZ indicated that a linear relationship existed for at least 5 min after initiation of the reaction. In our system a time of 105 set was allowed for the measurement period as this was the minimum time required to

fulfil the manufacturers recommended criteria of an acceptable assay system, ie, a difference of 45-85 AA units between zero blank and calibrator one readings, and

292 I. W. Paxton and R. A. Donald

of 95-150 AA units between calibrator one and five readings. The standard curves obtained in serum and saliva are shown in Figures 1 and 2, respectively and per-

mittted measurement in the range 1-17 pmol/l for serum and 0.16-4.2 ~molll for

saliva. The accuracy and the intra-assay precision were determined by replicate

analysis of samples of normal human serum and saliva containing known amounts

of CBZ with acceptable results (Table 1). A similar analysis was performed to test

the inter-assay precision by inclusion of these samples as quality controls in sub-

sequent assays. The standard deviations and coefficients of variation found for several consecutive assays are reported in Table 2.

It is assumed that the antiserum is specific for carbamazepine and does not

crossreact with other common drugs, in particular the anti-epileptic drugs, as claimed by Syva. This claim is supported by the good recoveries of gravimetric

preparations of pure CBZ in normal human serum using calibrators containing not

only CBZ but also phenytoin, phenobarbital, primidone, and ethosuximide. Studies with carbamazepine-lO,ll-epoxide in our systems indicated that this metabolite

interfered with the saliva assay at concentrations greater than 5 ~molll and with the

serum assay at concentrations greater than 15 pmol/l. This was equivalent to a

cross-reactivity of approximately 7%.

Application of the assays to single-dose pharmacokinetic studies was tested by

measurement of CBZ in serum and whole saliva during the four days after admin-

istration of a 400 mg oral dose (2 x 200 mg, Tegretol tablets, Ciba-Geigy, N.Z.,

Ltd.), to a healthy young volunteer. The resulting log concentration-time plot is

illustrated in Figure 3. Concentrations were measurable in both serum and saliva

1.0 2.6 5.3 7.9 10.6 16.9 SERUM CBZ CONC. ()Irnol /I 1

FIGURE 1. CBZ standard curve in serum.

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294 J. W. Paxton and R. A. Donald

150-

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SALIVA CBZ CONC.(pmol/lI

FIGURE 2. CBZ standard curve in saliva.

up to five days. Thirty hours after administration, absorption and distribution of

CBZ appeared complete and the curve entered the elimination phase with the CBZ log concentration appearing to decline linearly with time. The elimination half-life

for CBZ during this phase was 25.1 hr in saliva and 32.5 hr in serum, which is similar

to what has been reported for other single oral dose studies (Bertilsson, 1978).

After the first two hours, when the saliva samples may have been contaminated by residual CBZ in the mouth, the ratio of saliva to serum concentration remains fairly

constant with a mean value 24.5 I? 3.6% (n = 17). This value is in good agreement

with the nonprotein bound fraction in plasma that has been reported in a number

of studies to be between 20-30% (Bertilsson, 1978).

DISCUSSION

These results show that the cost of the CBZ assay may be reduced and instruments

other than those recommended by the manufacturer may be used without loss of

TABLE 2 Inter-assay Precision of CBZ Measurement in Saliva Serum

No. OF COEFFICIENT QUALITY MEAN CONC. CONSECUTIVE STANDARD OF VARIATION

MEDIUM CONTROL (pmol/l) ASSAYS DEVIATION (W

Serum QL 4.40 3 0.76 17.3 Serum QM 10.40 3 0.73 7.0 Saliva QS 1.48 5 0.07 4.7

EMIT Assay of CBZ in Serum and Saliva 295

__--

10 20 30 10 50 60 70 80 90 TIME(h)

FIGURE 3. CBZ concentrations in serum (0) and saliva (0) in a healthy subject after a single 400 mg oral dose.

precision or accuracy. Additional sensitivity may be obtained by decreasing the

dilution factor of the samples, thus allowing measurement of CBZ in both serum and saliva after a single oral dose.

These results also suggest that saliva CBZ concentrations may reflect the concen-

tration of the free drug in plasma and may provide an alternative, noninvasive

method for drug monitoring that may provide more meaningful therapeutic results

when protein binding is abnormal. However, before whole saliva can be accepted

as a suitable alternative to serum, further studies must be undertaken to establish that salivary CBZ concentrations are unaffected by such factors as stimulation and

rate of flow of saliva, pH of saliva, and binding of CBZ to mucoproteins and other

debris in saliva.

This work was supported in part by grants (No. 79120) from the MRC New Zealand and from the

National Children’s Health Research Foundation, New Zealand, (No. 93).

296 J. W. Paxton and R. A. Donald

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