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European Journal of Pharmaceutical Sciences, 5 (1997) 315-325

PHARMACEUTICAL SCIEN
I

A semi-automated solid-phase extraction and radioreceptor assay for the analysis of scopolamine in urine and plasma
I.J. B o s m a n ' , W.R. D o u m a b, K. Ensang , R.A. de Z e e u w a
~Groningen Institute .for Drug Studies (GIDS), University Centre for Pharmacy, Department of Analytical Chemist~ and Toxicology, A. Deusinglaan l, 9713 AV Groningen, The Netherlands ~Universitv Hospital, Department of Pulmonology, Oostersingel 59, 9713 EZ Groningen, The Netherlands Received 12 October 1995; accepted 23 February 1997 a:~ a

Abstract In a double-blind placebo-controlled cross-over study, we evaluated the therapeutic efficacy of transdermal scopolamine in ten patients with reversible airways obstruction. They received a patch (Scopoderm TTS) behind the ear for three days and samples of blood and urine were taken. A highly sensitive method was developed and validated to measure the low levels of free and total scopolamine in urine and plasma. The procedure consisted of a semi-automated solid-phase extraction followed by the analysis using radioreceptor assays. The mean plasma concentrations, taken every third patch day, of free and total scopolamine were 43.6 pg/ml and 229.0 pg/ml, respectively. In 24-h urine, collected every second patch day, 6.3 txg of free scopolamine and 83.4 I.zg total scopolamine was excreted. This means that 70% of the delivered dose (120 ~g in 24 h) is excreted in urine. For urine samples, the limit of detection (LOD) of the assay is 550 pg/ml and the limit of quantitation (LOQ) is 610 pg/ml. For plasma samples, the LOD is 16 pg/ml and the LOQ is 38 pg/ml. 1997 Elsevier Science B.V. Keywords: Radioreceptor assay; Solid-phase extraction; Transdermal administration; Scopolamine; Plasma; Urine

1. Introduction

Scopolamine is a competitive inhibitor of the muscarinic receptors of acetylcholine. It is marketed as a transdermal drug delivery system (Scopoderm TTS) for the prevention of nausea and vomiting associated with motion sickness. Like other anticholinergics, it can also cause relaxation of the smooth muscle of the bronchi and bronchioles (Clissold and Heel, 1985; Demeter and Cordasco, 1986). The transdermal therapeutic system of scopolamine is a thin plaster (0.2 mm), comprising a reservoir which contains the drug in a mixture of mineral oil and polyisobutylene, held between a impermeable backing layer and a rate-controlling membrane. The
*Corresponding author. Present address: Faculty of Pharmacy, Department of Pharmaceutical Analysis, Sorbonnelaan 16, 3584 CA, Utrecht, The Netherlands. Tel.: +31 30 2537667; fax: +31 30 2537387. 0928-0987/97/$32.00 1997 Elsevier Science B.V. All rights reserved PII S0928-0987(97)00058-4

unit contains 1.5 mg of scopolamine and is designed to deliver a total of 0.5 mg at an constant rate of approximately 5 I~g/hour over a 3-day period (Clissold and Heel, 1985). On the epidermal side of the membrane is an adhesive layer containing approximately 140 p~g as a priming dose to saturate certain binding sites within the skin and to accelerate achievement of steady state blood levels. Transdermal drug delivery by means of patches may offer benefits over conventional drug therapy by producing sustained, constant and controlled plasma concentrations over a prolonged period of time. This may result in a prolonged duration of action which improves patient compliance since frequent intake of the drug is no longer necessary (Guy and Hadgraft, 1985). For this reason, we evaluated the therapeutic efficacy of transdermal scopolamine in ten patients with reversible airways obstruction (Douma et al.,

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1997). They received a patch (Scopoderm TTS) behind the ear for three days and samples of blood and urine were taken. In order to correlate the levels of scopolamine with therapeutic efficacy, a highly sensitive analytical method was needed to measure the plasma and urinary concentrations of scopolamine in these patients. Most described methods are sensitive enough to measure the concentrations in urine, however plasma levels have rarely been determined (Ensing et al., 1988; Clissold and Heel, 1985; Scheurlen et al., 1984). Since scopolamine is extensively metabolized in glucuronide and sulphate conjugates, we decided to quantitate scopolamine as well as the sum of scopolamine and metabolites. The analysis is performed by means of radioreceptor assay (RRA). The principle of RRA is based on the ability of a drug to compete with a radiolabelled ligand for a specific receptor binding site. The drug (scopolamine) exerts its pharmacological action through interaction with a given receptor (muscarinic). The drug can be analysed quantitatively when an aliquot of the sample is added to a solution that contains a fixed amount of receptor and a fixed amount of radiolabelled ligand ([N-methyl3 H]scopolamme methyl chloride). The unknown quantity of scopolamine in urine and/or plasma can be calculated by determining the inhibition of labelled ligand binding and comparing this to the inhibition produced by known quantities of drug in calibration samples (~misterovfi et al., 1994). In principle metabolites which possess a binding affinity to the receptor, may be codetermined by the radioreceptor assay. However, no interferences of metabolites of scopolamine are to be expected (Cintron and Chen, 1987; Scheurlen et al., 1984). Therefore, the total amount of scopolamine (free plus conjugated) was determined by incubation of urine and plasma samples with glusulase, which is a preparation of a 13-glucuronidase and arylsulfatase. In order to eliminate interferences of glusulase and matrix interferences, a semi-automated solid-phase extraction procedure was required. In this paper, we describe the development and validation of a procedure to analyse free (unconjugated) and total scopolamine. The procedure consists of a semi-automated solid-phase extraction with silica gel (Si) columns using an ASPEC-system (Automatic Sample Preparation with Extraction Columns). Scopolamine was eluted with dichloromethane and

the extracts were analysed with a radioreceptor assay (RRA).

2. Materials and methods

2.1. Materials
methyl chloride ([3H]NMS, 80.4 Ci/mmol) was obtained from Du Pont NEN (Du Pont, Wilmington, DE, USA). Scopolamine hydrobromide trihydrate was obtained from Merck (Darmstadt, Germany). Glusulase was obtained from Calbiochem (La Jolla, CA, USA). Sodium hydroxide was obtained from Janssen Chimica (Beerse, Belgium). All other chemicals and solvents were of analytical grade and obtained from Merck (Darmstadt, Germany). Polyethylene tubes (4 ml and 12 ml) were obtained from Greiner (Alphen a/d Rijn, The Netherlands). The GF/B glassfibre filters were from Whatman (Maidstone, UK). Rialuma was used as scintillation liquid, obtained from Lumac (Olen, Belgium), in combination with mini-scintillation counting vials from Packard (Groningen, The Netherlands). Bond Elut extraction columns, type Si (silica gel), bed volume 2.8 ml were kindly donated by Varian (Sample Preparation Products, Harbor City, CA, USA). Heparinized human plasma was obtained from the local blood bank and stored at -20C. Calf brains without cerebellum were obtained from the local slaughterhouse and stored at -80C.

[N-methyl-3H]Scopolamine

2.2. Preparation of solutions

The 50 mM sodium phosphate buffer pH 7.4 (assay buffer) was prepared by dissolving 1.38 g NaHePO4.H20 and 7.12 g Na2HPO4.2H20 in 1 litre distilled water. The 50 mM sodium phosphate buffers pH 4.75 or pH 3.75 were prepared by the addition of 4 M hydrochloric acid to the assay buffer. The 50 mM sodium phosphate buffers pH 11.5 or 11.25 were prepared by the addition of 4 M sodium hydroxide to the assay buffer. Glusulase solutions pH 4.75 or pH 3.75 were prepared by diluting the glusulase stock solution 75 times with 50 mM sodium phosphate buffer pH 4.75 or pH 3.75.

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A scopolamine stock solution of 1 x 10 3 M was prepared in ethanol and stored at -20C.

2.3. Preparation of receptor material

Calf brains without cerebellum were homogenized in 6 volumes (w/v) icecold 0.32 M sucrose using a Teflon-glass Potter Elvejehem homogenizer (RW 20, Janke and Kunkel KG, IKA-Werk, Staufen i. Breisgau, Germany) at 1,200 rpm. The homogenate was centrifuged for 10 min at 1000Xg and the resulting supernatant was centrifuged for 60 min at 100 0 0 0 g (Beckman L8-55 ultracentrifuge, Beckman Instruments Nederland B.V., Mijdrecht, The Netherlands). The pellet was resuspended in the assay buffer and centrifuged 30 min at 100,000 g. This washing was repeated and the final pellet was resuspended in 5 volumes (w/v) assay buffer. The entire procedure was carried out at 4C. The obtained homogenate was frozen with liquid nitrogen and lyophilized for 24 h (Hetosicc CD 52-1, lyophilizer, Heto, Birker-d, Den-

mark). The lyophilized preparation was stored at -20C. The optimum amount of lyophilized material for the radioreceptor assay of scopolamine in urine (3 mg tissue in 400 pA assay buffer) and in plasma (2 mg tissue in 100 ~1) was experimentally determined.

2.4. Scopolamine analysis

The procedure consists of a semi-automated solidphase extraction followed by the analysis of scopolamine (free and total) using radioreceptor assays. To determine total scopolamine, the urine or plasma samples were pretreated with glusulase (Table 1).
2.4.1. Instrumentation

The extraction was performed with an ASPECsystem (Gilson Medical Electronics, Villiers le Bel, France). As shown in Fig. 1, the ASPEC-system consists of three components: A Model 401 dilutor, a

Table I Overview of the analysis of scopolamine Urine Incubation with glusulase *~ 250 t~1 urine 750 ~1 glusulase (pH 4.75) incubation 10 h 37C solution 1 ml phosphate buffer (pH 11.5) Plasma 250 IM plasma 750 ixl glusulase solution (pH 3.75) incubation 10 h 37C I ml phosphate buffer ( p H I 1,25) 500 Ixl plasma 1500 t~1 glusulase solution (pH 3.75) incubation 10 h 37C 2 ml phosphate buffer (pH 11,25) 5 ml methanol 5 ml water 3 ml dichloromethane 2 ml phosphate buffer (pH 7.4) flow-rate 3.0 ml/min 1500 t.tl plasma *-~ or 1600 ILl incubated sample *~ or 3600 Ixl incubated sample *~ (adapted) flow-rate 1.5 ml/min 1.5 ml phosphate buffer flow-rate 3.0 ml/min 3 ml dichloromethane flow-rate 1.5 ml/min vacuum concentrator 100 I~1 phosphate buffer 100 txl of reconstituted residue 100 ~1 receptor suspension 50 IM [3H]N-methylscopolamine

Incubation with glusulase, adapted method

Extraction with the ASPEC-system Conditioning

Sample application

5 ml methanol 5 ml water 3 ml dichloromethane 2 ml phosphate buffer (pH 7.4) flow-rate 3.0 ml/min 1000 ill urine *= or1600 ILl incubated sample *~ flow-rate 1.5 ml/min

Washing Eluting

Evaporation Reconstitute residue Radioreceptor assay

1.5 ml phosphate buffer flow-rate 3.0 ml/min 3 ml dichloromethane flow-rate 1.5 ml/min vacuum concentrator 1000 Ixl phosphate buffer 100 IM of reconstituted residue 400 ~zl receptor suspension 50 ILl [~H]N-methylscopolamine

*1 To determine total (free plus conjugated) scopolamine. *2 To determine free (unconjugated~ scopolamine.

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The incubated sample was vortexed and 3600 ~1 was used for the extraction.

Fig. 1. The ASPEC-system (Automatic Sample Preparation with Extraction Columns), which consists of three main components: A Model 401 dilutor (A), a sample processor (B), and a set of racks and accessories to handle 3 ml SPE-columns and solvents (CI, C2, C3).

sample processor, and a set of racks and accessories to handle 3 ml SPE-columns and solvents.

2.4.2. Incubation with glusulase Total scopolamine (free and conjugated) was measured after incubation of the urine or plasma samples with glusulase. The biological matrices were buffered with glusulase solution in order to obtain an incubation pH between 4.5 and 6.2. To 250 Ixl urine, 750 I~1 glusulase solution pH 4.75 was added and vortex mixed for 5 sec. The mixture was incubated for 10 h at 37C under constant shaking. The reaction was terminated by adding 1 ml phosphate buffer pH 11.5, to get a final pH of 7.4. For plasma samples (250 ixl), a glusulase solution pH 3.75 (750 I~1) was used and to terminate the incubation, phosphate buffer pH 11.25 (1 ml) was added to get a final pH of 7.4. Otherwise the procedure for plasma was the same as for urine. The incubated samples were vortexed and 1600 pJ aliquots were used for the extraction. When the initial procedure for plasma appeared to be too insensitive to analyse the actual levels, an adaption was developed. To 500 ill plasma, 1500 txl glusulase solution pH 3.75 was added and vortexed for 5 sec. The mixture was incubated for 10 h at 37C and the reaction was terminated by adding 2 ml phosphate buffer pH 11.25, to get a final pH of 7.4.
i

2.4.3. Extraction Bond Elut extraction columns, type Si (silica gel) were sealed with polypropylene caps and installed on the SPE-rack (Fig. 1). The solvents and samples (urine or plasma) were placed in the solvent positions and sample rack, respectively. Polyethylene tubes (5512 mm, 4 ml) were placed in the collection rack. The extraction was then performed by ASPEC in a sequential mode as follows. First, the column was preconditioned by subsequent washings with 5 ml methanol, 5 ml water, 3 ml dichloromethane and 2 ml assay buffer at a flow-rate of 3.0 ml/min. The urine sample (1000 ~1), plasma sample (1500 ill) or the incubated sample (1600 pJ or 3600 ~1 for the adapted procedure) was dispensed onto the column and pushed through by positive pressure at a flow-rate of 1.5 ml/min. To push the sample through the column completely, 1.5 ml of air was applied. After washing the column with 1.5 ml assay buffer at a flow-rate of 3.0 ml/min, the column was dried by passing through 6 ml of air. Scopolamine was then eluted into the collection tube by passing through 3 ml of dichloromethane at a flow-rate of 1.5 ml/min and 1.5 ml of air was applied onto the column to push the dichloromethane through the column completely. Small droplets of water in the dichloromethane fraction were removed manually by means of a pipet. Dichloromethane was evaporated to dryness within 45 min, using a vacuum concentrator, at 1500 rpm and 40C (Beun de Ronde, Abcoude, The Netherlands). The residue was reconstituted in 1000 pJ (urine samples) or 100 pJ (plasma samples) assay buffer by vortex mixing for 5 s After standing for 1 hour, the tube was vortexed again for 5 s. To duplicate polyethylene tubes (12 ml), 100 pJ of the urine extract was pipetted and used for the radioreceptor assay. However, the entire reconstituted plasma sample (100 ~1) had to be used in the radioreceptor assay which meant that for duplicate analyses two extractions were necessary. 2.4.4. Radioreceptor assay (RRA) To each polyethylene tube (12 ml) with the 100 ILl extraction sample, 400 ~1 receptor suspension (for urine samples) or 100 I, receptor suspension (for zl plasma samples) was added. The tubes were vortexed

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319

and incubated during 60 min at 0C before 50 txl of [3H]NMS solution (4X10 -9 M) was added. The tubes were mixed again and incubated for another hour at 0C. After the addition of 4 ml icecold assay buffer, the samples were immediately filtered through Whatman GF/B glassfibre filters under vacuum using a filtration apparatus (48S, University Centre for Pharmacy, Groningen, The Netherlands). The tubes were rinsed twice with 4 ml icecold buffer, which was also filtered. The total filtration and rinsing process, taking place in approximately 15 s, was carried out on each tube in turn. The filters were transferred into mini-scintillation vials and dispersed in 3.5 ml scintillation cocktail by shaking for 120 min. The vials were counted for 40 000 counts or 5 min in a liquid scintillation counter, whichever came first (Minaxi, Packard, Groningen, The Netherlands). Fifty ~zl of the used [3H]NMS solution were added to 2 mini-scintillation vials and counted as well. 2.5. Calibration curve From the scopolamine stock solution, appropriate dilutions were made freshly on the day of analysis in blank urine to provide concentrations ranging from 5 x 1 0 -1 M t o 2 X 1 0 - 7 M. After extraction, this resulted in final assay concentration ranging from 9.09x10 ~ M to 3.64X10 8 M. In blank plasma, the concentrations were ranging from 1 X 10-~1 M to 5X10 9 M giving final assay concentrations of 6X 10 ~ M to 3X10 8 M. The samples of the calibration curves were analysed in duplicate.The curves were fitted with the Ligand curve fitting program to calculate the parameters which characterize the calibration curves (Munson and Rodbard, 1980). 2.6. Quali b, control The assay of urine was validated with quality control samples which were prepared in blank urine, at 5 X 1 0 -9 M, l X l 0 8 M and 5 x 1 0 -8 M, respectively. In blank plasma, quality control samples were prepared with concentrations of 1.25x10 -~ M, 3 x 10-~o M and 1.25x 10 -9 M, respectively. The quality control samples were stored at -20C. On the day of analysis, the quality control samples were extracted and analysed in duplicate. Incubated quality control samples (5 x 10 -8 M and 1.25x10 -9 M in urine and plasma, respectively) were codetermined to study the influence of glusulase

on the assay. The adapted procedure for total scopolamine in plasma was validated with the quality control of 1.25x 10 9 M. The obtained binding values (Bq) of the quality control samples were introduced in the fitted calibration curves and the concentrations in urine or plasma were calculated. For urine samples, inter-day data (between days) were assessed by measuring the quality control samples in duplicate on different days; intra-day data (within day) were determined by duplicate measurements of the quality control samples after 5 separate extractions on the same day. The quality control samples in plasma were analysed in duplicate on ten different days. Because for plasma samples duplicate analyses means duplicate extractions, intra- and inter-day data were calculated from the same samples using one-way analysis of variance. 2. 7. Patient samples Urinary and plasma levels of free and total scopolamine were determined in ten patients with reversible airways obstruction (Douma et al., 1997). The patients received a scopolamine patch (Scopoderm TTS) or placebo behind the ear for 72 h. The patches (placebo and Scopoderm TTS) were kindly provided by Ciba Geigy, Arnhem, The Netherlands. Every second patch day urine was collected during 24 h and every third patch day at 9.00 a.m. blood was taken from the patients. Plasma was obtained by centrifugation of the blood sample (collected in a tube containing heparin) at 5,000 rpm for 11 min (Megafuge 1.0, Heraeus Sepatech, Germany). The urine and plasma samples were stored at - 2 0 C until analysis. The same procedure as described for the quality control samples was performed to calculate the unknown amount of free and total scopolamin e in urine and plasma of the patients.

3. Results and discussion

3.1. Scopolamine analysis: solid-phase extraction and radioreceptor assay 3.1.1. Radioreceptor assay For the analysis we used a radioreceptor assay

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(RRA), based on the competition between a radiolabelled ligand ([3H]NMS) and unlabelled drug (scopolamine) for binding to muscarinic receptors, because they are highly sensitive and selective. Because of the potency of scopolamine, the concentrations of the drug in plasma and urine will be very low and difficult to detect. Therefore, the sensitivity of the receptor assays was enhanced by applying a pre-incubation step of the drug with the receptor preparation at 0C before the radiolabelled ligand was added (Smisterovfi et al., 1994). This resulted in a gain in sensitivity of a factor 2 as compared with equilibrium conditions at 37C. However, this gain was much smaller than a previously described gain of a factor 6.7 using [3H]dexetimide as radiolabelled ligand (Ensing and de Zeeuw, 1984). Probably, the gain in sensitivity is not only dependent on the competitive drug but depends also on the radiolabelled drug. Besides a high sensitivity, another advantage of RRA is the selectivity towards compounds which produce a pharmacological effect via an interaction with receptors. This means that in principle metabolites may be codetermined when they have an affinity towards the receptor. In case of scopolamine, no interferences of metabolites are to be expected. The glucuronide and sulphate conjugates of scopolamine will have no affinity towards the muscarinic receptor because of their physico-chemical properties. Scheurlen et al. (1984) showed that scopoline and scopine, possible degradation products of scopolamine, will have no interference with the assay. This was also observed in a previous study, testing the displacement of binding of radiolabelled [3H]dexetimide to muscarinic receptors. The very low affinity constants of scopoline (4.1 10 6 M - ~) and aposcopolamine (3.7 10 3 M - l ) , compared with 1 . 7 1 0 9 M -x for scopolamine, showed that scopoline and aposcopolamine will not interfere with the assay of scopolamine. It has been shown before that direct radioreceptor assays of scopolamine in urine could be performed because of the relatively high urine concentrations after therapeutic dosing (Ensing et al., 1988). However, using this direct assay, calibration curves had to be prepared for each patient in his/her own blank urine to compensate for the inhibition of binding caused by blank urine. Moreover, the addition of glusulase to urine samples, added to determine the

total amount of scopolamine, also yielded some inhibition of binding. Although this inhibition seemed highly reproducible, extra calibration curves (to which glusulase had been added) had to be prepared in each patient's blank urine. For plasma, a sample clean-up and concentration step was considered unavoidable because plasma proteins disturb the sensitivity and accuracy of the assay, and because drug concentrations in plasma are much lower than in urine.

3.1.2. Solid-phase extraction Solid-phase extraction (SPE) was applied as an effective sample pre-treatment to eliminate matrix interferences and the interferences of glusulase, and to concentrate plasma samples. The developed extraction procedure was based on the described sample preparation of N-0437 in plasma and urine with Si-columns (Swart et al., 1990), and earlier investigations about the extraction of scopolamine from urine. These manual procedures were adapted and automated using a Gilson automated extraction system ASPEC (Fig. 1). Different flow-rates, air volumes and eluting volumes were tested to improve the extraction procedure. Additionally, the ASPEC offers two possibilities to carry out extractions: A batch mode and a sequential mode. In the sequential mode, the ASPEC accomplishes the complete extraction process of the first sample, and then continues with the next sample, whereas in the batch mode all SPE-columns are treated batchwise. The sequential mode was used because higher recoveries and reproducibilities can be obtained compared with the batch mode (Chen et al., 1993). 3.2. Validation of the assay
Representative calibration curves of samples, prepared freshly on the day of analysis in blank urine or blank plasma, are shown in Fig. 2. The parameters which characterise the calibration curves are given in Table 2. The relatively small variations in the calculated affinity constants of scopolamine (K21) indicate the high reproducibility of the assay under nonequilibrium conditions. From the fitted calibration curves, the IC5o value was calculated, which is defined as the concentration of ligand (scopolamine) which displaces 50% of the bound radioligand

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321

500
\
o--.- o

........ -#.. ~
"'\,, Q

400

300
Z

200
o m

100

i0 -I0

10 -9

i0-8

10 -7

F i n a l a s s a y c o n e s c o p o l a m i n e (M) Fig. 2. Calibration curves for the inhibition of [~H]N-methylscopolamine ([3H]NMS) binding by scopolamine, m = urine; = plasma.

([3H]NMS). The mean ICs0 values in plasma were significantly higher in comparison with urine (two sample t test, p<0.05). The differences in fitted parameters between the curves prepared in urine or plasma can be explained by differences in receptor concentrations and concentrations of the radiolabelled drug in the assay. The assay in plasma differed from the assay in urine because higher sensitivities were needed to analyse the low levels of scopolamine in plasma. Therefore, the incubation volume of the assay in plasma was adapted to a volume of 250 ~1, because the smaller the volume used, the greater the sensitivity (Crevat-Pisano et al., 1986). With the thus obtained calibration curves, the samples used for the fitting were back-calculated to their original concentrations in urine or plasma, and the accuracy and precision data were established (Table 3). As expected, the precision became worse

Table 2 The calculated parameters which characterize the calibration curves (n = 11).K,, is the apparent affinity constant of [3H]NMS (M '); K_~, is the apparent affinity constant of scopolamine (M -,); R, is the receptor concentration (M); N t is percentage the non-specifically bound [3H]NMS: MSQ is the mean sum of squares; IC~o is the concentration of scopolamine which displaces 50% of [3H]NMS (M). Parameterg
K I , ( x 10q) 3.81 0.97 25.6 1.37 0.33 23.7 K2~( x 5.75 0.79 13.7 3.64 0.74 20.2

109)

R,(X 10 -9) 1.35 0.26 19.[) 2.15 0.38 17.8

N~(%) 1.90 0.23 12.0 2.37 (1.71 30.2

MSQ(X 10 ~3) 0.90 0.61 68.1 2.21 1.94 88.0

ICs~l x 10 ") 1.89 0.28 14.8 2.5 I 0.3(I 11.8

Urine (n = 11 )
Mean S.D. CN.

Plasma (n = 101
mean S.D. C.V.

Table 3 Accuracy and precision of the back-calculated concentrations of the spiked calibration samples in urine (n = 11) and in plasma (n= 10)
A. Added cone in urine (Log final assay conc) mean sd ace cv B. Added conc in plasma (Log final assay conc) Mean S.D. ACC C.V. *1 n = 1 0 2 x 1 0 -~ M (-9.44) 1.83 0. l 8 91.7 9.7 5x10 ,, M (-9.52) 5.22 0.99 104.3 18.9 5Xl0 -'~ M (-9.04) 4.94 0.34 98.8 6.8 1x10 -t M (-9.22) 0.90 0.12 89.6 13.7 l X l 0 -8 M (-8.74) 1.01 0.03 100.5 2.8 2x10 -'~ M (-8.92) 2.07 0.12 103.3 6.0 2Xl0 8 M (8.44) 2.04 0.03 I 01.8 1.6 5x10 " M (-8.52) 4.95 0.16 98.9 3.1 5xl0 ~ M (-8.04) 4.81 *~ 0.17 96.2 3.6 Ixl0 "M (-8.22) l 1 0 -7 M (-7.74) 0.97 0.03 96.6 3.3 2x10 '~ M (-7.92)

1.02 0.03 101.7 3.1

1.97 0.12 98.2 5.8

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35
30

'\

25

20
;=

15

10
.0

10 -10

10 -9

10 -8
(M)

10 -7

Final assay cone scopolamine

Fig. 3. Precision data of the back-calculated concentrations of the spiked calibration samples. == = urine; = plasma.

at the low and high end of the curves (Fig. 3). For plasma, the precision exceeds the 15% coefficient of variation (cv) at plasma concentrations lower than 5 x 1 0 -1~ M. For urine, precision data were below 15% at urine concentrations higher than 2 x 1 0 - 9 M. The higher cv values in plasma in comparison with

urine can be explained by the difference in experinaental set-up. In case of plasma samples, duplicate analyses means duplicate extraction whereas for urine samples only one extraction is needed to perform duplicate measurements. The method was validated with quality control samples without glusulase at three different concentrations (Table 4). The results show that the assay provides good accuracy, which should be within _+15% of the actual value, and excellent precision, which should not exceed 15% cv (Shah et al., 1992). Only the lowest quality control prepared in plasma deviates more than 15% of the actual value. However, at the limit of quantitation (LOQ), the acceptable accuracy and precision limits are set at 20%. This means that for plasma the LOQ is close to 1.25x 10 -] M, which corresponds to 38 pg/ml for plasma samples of 1.5 ml. The LOQ should be obtained independently from the calibration curve which means that at least five samples independent from the samples of the calibration curve should be determined (Shah et al., 1992). Therefore, extra quality control samples were prepared in blank urine at 2 x 1 0 - 9 M. The calculated intra-day precision and accuracy, determined by duplicate analyses of five samples on the same day,

Table 4 Accuracy and precision of quality control samples prepared in urine (A)and plasma (B) Quality control samples
A

Without glusulase
5 X 1 0 -9 M

With glusulase
110 -~ M (-8.74) 5X10 s M (-8.04) 510 s M (-8.74)

Added conc in urine (Log final assay conc) Inter-day (n=9)

Mean S.D. ACC C.V.

(-9.04)

4.95 0.35 99.0 7.1 4.97 0.13 99.4 2.7 1.25x10 -'~ M (-9.12) 1.23 0.21 0.08 98.4 17.1 6.4

0.96 0.07 96.2 7.5 0.99 0.04 99.0 4.1 3 x 10 -'~ M (-8.74) 2.70 0.21 0.14 90.1 7.7 5.1

4.61 0.41 92.2 8.9 4.65 0.19 93.0 4.1 1.25 X 10 -'~ M (-8.12) 1.31 0.10 0.04 104.7 7.4 2.9

4.97 0.33 99.5 6.6 5.33 0.05

Intra-day (n =5) Mean

S.D. ACC C.V.

106.5
1.0 1.25X10 '~ M *~ (-9.OO) 1.29 0.24 0. ! 8 103.1 13.9 10.3

B Added cone in plasma (Log final assay conc) Mean Inter-day S.D. Intra-day S.D.
ACC

Inter-day C.V. Intra-day C.V.

*1 n = 9

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were 8.3% and 113.3%, respectively. The inter-day data, obtained by duplicate analysis on five different days, show that a urine concentration of 2 10 - 9 M is near the LOQ: the precision was 25.6% and the accuracy 85.2%. Hence, the LOQ can be estimated to be about 610 pg/ml for urine samples of 1 ml. The LOQ must be differentiated from the limit of detection (LOD) which for RRA can be defined as the concentration of analyte at which the fraction bound label is significantly smaller than the fraction bound label in the absence of analyte (~misterovfi et al., 1994). This was determined experimentally by performing replicate measurements of the zero standard (amount of bound [SH]NMS in absence of scopolamine). From these data the standard deviation (sd) was calculated and the LOD was found at the intersection of the line representing the mean zero standard minus 2 sd with the composite calibration curve. For urine samples, the LOD of the assay was 550 pg/ml and for plasma samples 16 pg/ml. Incubated quality control samples were codetermined to study the influence of glusulase on the assay. The quality control data for plasma were obtained with the initial procedure. The presented data in Table 3 show that the incubated samples meet the above mentioned criteria (accuracy _+15%, C.V. ---15%). This means that a single calibration curve can be prepared to measure both free and total scopolamine in urine or plasma. However, the levels of total scopolamine in plasma samples of the patients were not detectable with the initial procedure. Therefore, the volume of the plasma samples was increased and the adapted procedure was validated with a quality control sample of 1.25x 10 9 M scopolamine. The measured accuracy was 110.7% (_+0.4%) and the adapted procedure was found to be not statistically different from the initial procedure (two sample t test, p<0.05). 3.3. Patient samples 3.3.1. Urine After application of Scopoderm TTS to the postauricular area of the patients, we found that an average of 6.3 fxg (range 3.3-9.3 p,g) of free scopolamine and an average of 83.4 lxg (range 48.0134.5 Ixg) of total scopolamine was excreted in 24-h urine (Table 5). The measured urine concentrations

(free and total) were all higher than the lowest quality control of 5 X 1 0 -9 M (1.5 ng/ml). In the placebo phase no scopolamine was detected. The observed variations in scopolamine excretion corresponds with the four to six-fold variations in urine excretion rates reported before (Parrott, 1989). During steady-state conditions approximately 120 txg of scopolamine is delivered to the patient in 24 h. We found that of this dose, about 65% is excreted in 24-h urine as glucuronide and sulphate conjugates and only 5% of the dose is excreted unchanged. These results are in line with earlier studies in which less than 10% of the administered dose was excreted unchanged (Clissold and Heel, 1985; Scheurlen et al., 1984). However, we found 70% of total scopolamine excreted in 24-h urine, whereas Scheurlen et al. (1984) found only 34% of the drug in the urine which may be due to the incubation procedure. 3.3.2. Plasma Up till now, plasma concentrations of scopolamine have rarely been reported because a sensitive method for the measurement of such low levels was lacking (Clissold and Heel, 1985). We measured average plasma concentrations of free and total scopolamine of 43.6 pg/ml (range 16.8-65.4 pg/ml) and 229.0 pg/ml (range 165.6-377.2 pg/ml) respectively (Table 5). Three of ten measured concentrations of free scopolamine were below the LOQ but above the LOD. With the adapted procedure, the plasma concentrations of total scopolamine were all above the LOQ of 7.510 -'~ M in the assay, which corresponds with 124 pg/ml in plasma used for the total scopolamine determination. In the placebo phase no scopolamine was detected. Muir and Metcalfe (1983) reported a mean peak plasma concentration for free scopolamine of 0.42 l0 9 M (127 pg/ml) in 4 volunteers after transdermal administration of a scopolamine patch with a priming dose of 200 jxg and a release rate of 10 pog/hour. However, although the dose delivered was twice as high as in our study, the drug was detectable in only 4 of 12 volunteers. Recently, plasma concentrations in a group of 8 subjects were determined by a method which consisting of a preparative extraction step with C~s columns, followed by an analytical quantitation step with a muscarinic radioreceptor assay (Cintron and Chen, 1987). The mean free plasma concentration was 134 pg/ml

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Table 5 Free and total scopolamine in 24-h urine and in plasma of I0 patients. Patient Free Phase 1 Free Phase 2 Total Phase 1 Total Phase 2
69.1 134.5 3.7 9.3 7.9 4.0 7.5 5.1 6.7 6.6 5.6 2.4 42.1 108.5 73.9 86.7 68.3 67.2 13.0 19.4 114.3 59.0 48.0 72.1

A. Free and total scopolamine (tzg) in 24-h urine of 10 patients 1 3.3

2 3 4 5 6 7 8 9 10 Mean S.D. C.V. n=10 Mean S.D. C.V. 9.0

7.4 1.6 22.1 6.3 2.2 34.8

107.8 25.2 23.4 83.5 27.3 32.7

B. Free and total scopolamine (pg/m!) in plasma of 10 patients

1 2 3 4 5 6 7 8 9 10 Mean S.D. C.V. n=10 Mean S.D. C.V. 16.8 57.0 44.5 65.4 29.3 39.6 34.0 60.8 40.0 48.6 42.5 15.8 37.1 165.6 243.2 200.1 213.1 217.7 15.5 7.1 198.2 236.7 377.2 216.4 235.6 204.1

45.3 15.9 35.2 43.6 15.0 34.4

246.1 93.0 37.8 229.0 56.9 24.8

Phase 1: Patients first received scopolamine, then placebo. Phase 2: Patients first received placebo, then scopolamine. All urine and plasma samples from patients that received placebo showed no detectable scopolamine concentrations.

(range 82-239 pg/ml) which is three times higher as the mean concentration we measured. If we assume that doubling of the dose results in two times higher concentrations, our results are comparable with the mean plasma concentration reported by Muir and Metcalfe (1983), taking into account that they were unable to detect scopolamine in 8 of 12 volunteers. The method we described offers the advantage of assaying both free and total scopolamine which gives extra information on the bioavailability and kinetics of the drug.

4. Conclusion We developed and validated a method which consists of a semi-automated solid-phase extraction followed by the analysis of free and total scopolamine with radioreceptor assay (RRA). Although the concentrations after transdermal drug delivery of scopolamine are very low, the assay is sufficiently sensitive to measure these levels. For urine samples of 1 ml, the limit of detection (LOD) of the assay is 550 pg/ml and the limit of quantita-

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tion (LOQ) is 610 pg/ml. For plasma samples of 1.5 ml, the LOD is 16 pg/ml and the LOQ is 38 pg/ml. References
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