New enzyme sensors for morphine and codeine based ... - Springer Link

Fresenius J Anal Chem (1999) 364 : 179–183

© Springer-Verlag 1999

O R I G I N A L PA P E R

Christian G. Bauer · Andrea Kühn · Nenad Gajovic · Olga Skorobogatko · Peter-John Holt · Neil C. Bruce · Alexander Makower · Christopher R. Lowe · Frieder W. Scheller

New enzyme sensors for morphine and codeine based on morphine dehydrogenase and laccase Received: 10 August 1998 / Revised: 29 January 1999 / Accepted: 5 February 1999

Abstract Two new enzymatic methods have been developed to quantify morphine and codeine simultaneously in a flow injection system (FIA). The first enzyme sensor for morphine or codeine is based on immobilizing morphine dehydrogenase (MDH) and salicylate hydroxylase (SHL) on top of a Clark-type oxygen electrode. Morphine or codeine oxidation by MDH leads to a consumption of oxygen by SHL via the production of NADPH. This decreases the oxygen current of the Clark electrode. Concentrations of codeine and morphine are detected between 2 and 1000 µM and between 5 and 1000 µM, respectively. The second enzyme sensor for morphine is based on laccase (LACC) and PQQ-dependent glucose dehydrogenase (GDH) immobilized at a Clark oxygen electrode. Morphine is oxidized by laccase under consumption of oxygen and regenerated by glucose dehydrogenase. Since laccase cannot oxidize codeine, this sensor is selective for morphine. Morphine is detected between 32 nM and 100 µM. Both sensors can be operated simultaneously in one flow system (FIA) giving two signals without the requirement for a separation step. This rapid and technically simple method allows discrimination between morphine and codeine in less than 1 min after injection. The sampling rate for quantitative measurements is 20 h–1. The method has been applied to the quantitative analysis of codeine or morphine in drugs. Dedicated to Professor Dr. Karl Cammann on the occasion of his 60th birthday Ch. G. Bauer · A. Kühn · N. Gajovic · A. Makower · F. Scheller (쾷) Institute for Biochemistry and Molecular Physiology, University of Potsdam, Im Biotechnologiepark, D-14943 Luckenwalde, Germany P.-J. Holt · N. C. Bruce · Ch. R. Lowe Institute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QT, United Kingdom O. Skorobogatko A. N. Bach Institute of Biochemistry, Russian Academy of Sciences, Leninskii pr. 33, Moscow, 117 071 Russia

Introduction Quality control is an important task for analytical chemistry. Methods are needed for the control of pharmaceutical drugs that operate with a short signal response time and require neither sophisticated instruments nor costly reagents. Enzymatic analysis can meet these needs, although it has not been employed routinely to detect morphine and codeine. To date, only one enzyme, morphine dehydrogenase (MDH), has been used to detect morphine or codeine enzymatically [1, 2]. Morphine dehydrogenase catalyzes the oxidation of morphine or codeine to morphinone or codeinone and reduces NADP+ concomitantly to NADPH. The NADPH is detected either electrochemically by oxidation via a mediator [1] or via a bioluminescent end point [2]. Both of the reported enzyme sensors require the addition of fresh enzyme for each measurement. However, a new enzymatic method for the analysis of morphine and codeine should not only rely on immobilized enzymes to reduce reagent costs, but it should also be able to discriminate between the analytes morphine and codeine. Differentiation of morphine and codeine could be achieved in two ways: In the first case the analytes could be separated before signal measurement, although this would increase the costs of the instrument as well as the signal response time. Alternatively, a second enzyme sensor could be used that responds only to morphine. We report two compatible enzyme sensors that discriminate between morphine and codeine and quantify their concentrations in the same sample. The first sensor (Fig. 1 A) measures morphine and codeine directly using a coupled sequence of morphine dehydrogenase (MDH) and salicylate dehydrogenase (SHL). MDH catalyzes the oxidation of morphine or codeine at the expense of reducing its cofactor NADP+. The SHL reaction is initiated by the generated NADPH and consumes oxygen during the conversion of salicylate to catechol. SHL translates the NADPH production into an oxygen consumption that is signalled by the Clark oxygen electrode.

180

A

Salicylate hydroxylase was obtained from Sigma (S2385, Deisenhofen, Germany) and contained 2.6 units/mg lyophilisate. One unit converts 1.0 µM salicylate and NADH per minute to catechol and NAD+ at 30 °C. The decrease in absorbance at 340 nm is measured in 19 mM phosphate buffer (pH 7.6) containing 1 mM EDTA, 0.1 mM salicylate and 0.18 mM NADH at pH 7.6. The enzymes were solubilized and entrapped in polyvinylalcohol (PVA) (PVA 05/20, Serva, Heidelberg, Germany). MDH (1 mg) was added to 14 µL 0.1 M sodium phosphate, pH 8. Ten minutes later, 0.6 mg SHL was dissolved in 12 µL of the MDH solution and mixed with 6 µL 20% (w/v) PVA. 3 µL of this mixture was spread into a 2 mm diameter cylinder (polyacrylamide) and crosslinked by UV-irradiation for 30 min (10 cm distance, 254 nm, type N-15 K, Kurt Benda, Wiesloch, Germany). The membranes were removed and stored at 4 °C in an Eppendorf tube. LACC/GDH enzyme membrane

B Fig. 1 A, B Principle of the enzyme sensors: (A) MDH/SHL. Morphine or codeine are oxidized by MDH with concomitant reduction of the cofactor, NADP+. The cofactor is reoxidized by SHL. SHL consumes oxygen and changes, thereby, the oxygen current at the Clark electrode. (B) LACC/GDH. Morphine is oxidized by LACC that consumes oxygen. GDH regenerates morphine to amplify the oxygen consumption and decreases the current of the Clark electrode

The second sensor (Fig. 1 B) exploits laccase (LACC) to measure morphine. Laccase oxidizes morphine and consumes oxygen, which is indicated by a Clark electrode. This oxygen consumption is amplified enzymatically with PQQ-dependent glucose dehydrogenase (GDH). The substrate recycling between LACC and GDH enhances the assay sensitivity [3, 4]. This substrate cycling sensor does not respond to codeine. In a double detector experiment, the MDH/SHL- and the LACC/GDH-sensors discriminate between morphine and codeine in pharmaceutic drugs.

Material and methods MDH/SHL enzyme membrane For the non-amplified measurement of morphine and codeine, morphine dehydrogenase (MDH) and salicylate hydroxylase (SHL) were coimmobilized. Morphine dehydrogenase (MDH) was isolated at the Institute of Biotechnology, University of Cambridge [5, 6]. The activity measurement was modified after [7]. An aliquot of enzyme (≈ 10 µL; 30–40 units/mL) was added to 1 mL bis-tris-propane buffer (50 mM; pH 9.5) containing 2.5 mM NADP+, and 2.5 mM morphine-HCl. The increase in absorbance at 340 nm was monitored. A unit of morphine dehydrogenase activity is defined as the amount of enzyme required to reduce 1.0 µM of NADP+ per minute at pH 9.5 and 30 °C. The MDH had an activity of 60 units/mg lyophilisate.

The enzyme membrane for the selective detection of morphine contained laccase (LACC) and glucose dehydrogenase (GDH). Laccase (LACC) was isolated by Olga Skorobogatko, Moscow, from Coriolus hirsutus [8]. The activity was measured by adding 10 µL of laccase solution (≈ 250 units/mL) to 1 mL 10 mM catechol in 0.1 M acetate buffer, pH 4.5. The increase in absorbance relevant to o-quinone (740 M–1cm–1) is monitored at 410 nm. One unit corresponds to the amount of enzyme required to oxidize 1 µM catechol per minute at 25 °C. The solution contained 19.3 mg/mL laccase of 460 U/mg. Glucose dehydrogenase (GDH) was a gift from BOEHRINGER Mannheim (Mannheim, Germany). The activity was measured by adding 50 µL GDH (reconstituted with pyrroloquinolinequinone, PQQ) to 0.1 mL glucose (1 M) in 3 mL test buffer (0.2 M citrate buffer, pH 5.8, 1 mM CaCl2, 0.3 mg/mL p-nitrosoaniline (Caution: this substance can cause cancer!). The increase in absorbance is monitored at 620 nm for quinonediimine (30 M–1cm–1). One unit GDH converts 1 µM p-nitrosoaniline per min at 25 °C. The lyophilisate contained 770 U/mg. For entrapment, 0.7 mg GDH were solubilized in 8 µL 0.5 mM PQQ, 0.1 M MES (2-(N-morpholino)ethanesulfonic acid) pH 6 and mixed with 52 µL LACC and 15 µL 20% (w/v) PVA. 5 µL of the mixture were spread into cylinders (2 mm diameter) and approx. 50% of the solution were evaporated before crosslinking. The membranes were irradiated and stored as described above. Apparatus and procedure The FIA (Fig. 2) consisted of a peristaltic pump, an injector, an aerator pump to stabilize the oxygen baseline of the Clark-type electrode by forming an aerosol and the respective enzyme electrode. The peristaltic pump (minipuls 3, Abimed-Gilson, Langenfeld, Germany) served for solution transport. The sample (100 or 200 µL, see Table 1) was injected (V7-injector, Pharmacia, Freiburg, Germany) and sprayed with a 100-fold excess of air by a membrane pump 2002G-0206, 2.0–2.4 V (ASF Thomas, Puchheim, Germany) into a temperature controller before it entered the detector. The temperature controller was a steel tube (50 cm × 0.8 mm I.D.) fixed between two 1 cm aluminium blocks. The home-made detector was essentially a channel with two adjacent enzyme-

Fig. 2 The flow injection analysis (FIA) consists of a peristaltic pump (black triangle), an injector (circle), a membrane aerator-pump (black triangle), temperature control (T), detector with two sensors (D, S1, S2), two potentiostats (P) and recorders (R)

181 Table 1 Experimental conditions for the enzyme sensors

All buffers contained 0.05% (w/v) Kathon CG

Enzyme sensor

Analytes

Buffer

Flow rate

Sample buffer

Sample volume

Figure

MDH/SHL

morphine, codeine

50 mM phosphate, pH 7.5, 1 mM salicylate

270 µL/min

buffer + 2.5 mM NADP+

200 µL

3

GDH/LACC

morphine

50 mM phosphate, pH 6.5, 10 mM glucose

300 µL/min

same as buffer

100 µL

4

MDH/SHL + GDH/LACC

morphine + codeine real samples

50 mM phosphate, pH 7.5, 1 mM salicylate, 10 mM glucose

270 µL/min

buffer + 2.5 mM NADP+

200 µL

6A + 6B

membrane covered Clark-type electrodes. The enzyme membrane for the respective analyte was sandwiched between a dialysis membrane and a polyethylene membrane that separated the electrolyte in the electrode compartment from the enzyme layer. The analyte-related transient current decrease at the oxygen electrodes (-600 mV vs. Ag/AgCl, Elbau, Berlin, Germany) was amplified by GKM (Academy of Sciences, GDR) and recorded (chart recorder BD 112, Kipp & Zonen, Delft, The Netherlands). All components of the FIA in contact with flowing solution were connected by 0.5 mm I.D. Teflon tubing, whilst those components passed by the sprayed solution were connected with Tygon tubing (0.8–1.6 mm I.D.). The experimental details for each experiment are summarized in Table 1. All standards and samples were prepared daily in sample buffer and injected fourfold. The cofactor for MDH, NADP+, was included only in the sample buffer.

Results Measurement of morphine and codeine by MDH/SHL The sensor gives a non-linear calibration curve for both analytes (Fig. 3). Morphine and codeine can be detected between 10 and 1000 µM. The threefold standard deviation (SD) of the blank is exceeded at 2 µM codeine and 5 µM morphine. The standard deviation for codeine signals (four repetitions) is < 10% at the detection limit and < 1.5% for higher concentrations. Morphine measurements show a similar precision. The higher signal for codeine compared to morphine reflects the lower Km of MDH for codeine [6] and is in good agreement with an MDH-based enzyme test [1]. This test showed lower de-

Fig. 3 Calibration curve of the MDH/SHL enzyme sensor for morphine and codeine

tection limits of 2.7 µM and 6.8 µM for codeine and morphine, respectively. Measurement of morphine by LACC/GDH Figure 4 shows a semilogarithmic calibration plot for morphine in a non-amplified mode; in this case, only laccase is active, since the cofactor glucose for the GDH has been omitted from the buffer. The addition of glucose leads to a more sensitive response due to substrate cycling. The detection limit (threefold SD) for the non-amplified mode is 10 µM morphine, whilst amplification improves the limit of detection to 32 nM morphine. In the current vs. concentration graph the non-amplified calibration curve is linear over more than two orders of magnitude (R ≥ 0.999); whilst the amplified measurement of morphine is non-linear. Laccase does not appear to be responsible for this non-linearity, since the non-amplified morphine calibration curve is linear. Laccase converts morphine to 2,2-bimorphine (pseudomorphine) [9]. This is consistent with the chemical oxidation of morphine to 2,2′-bimorphine which requires the formation of a phenoxy radical intermediate [10]. The monomeric radical can obviously transfer the electron to GDH. Thus, the dimerization competes with the recycling

Fig. 4 Amplified and non-amplified calibration curves of the LACC/GDH enzyme sensor for morphine. This sensor does not respond to codeine: Detection limits for 100 µL morphine are 32 nM (black circles) in a amplified non-linar calibration curve and 10 µM (open circles, R ≥ 0.999) in a non-amplified linear calibration curve

182 Fig. 5 Selectivity of the enzyme sensors LACC/GDH and MDH/SHL: Laccase oxidizes morphine and cannot convert codeine. MDH is known to oxidize morphine and codeine at the 6′-position

reaction because pseudomorphine is no cosubstrate of GDH. Reduction of radicalic species by GDH was established also in other one-electron recycling systems, e.g. paracetamol or chlorpromazine [11]. In contrast to the MDH-based sensor, the LACC/GDH sensor responds only to morphine regardless of the presence of codeine. We assume that the phenolic 3-hydroxy group of morphine (Fig. 5) is a requirement for activity in the LACC catalyzed reaction. Two observations lead to this conclusion: (i) Injection of 100 µM codeine or 3-ethylmorphine does not cause a signal at the LACC/GDH sensor (data not shown). This means that alkylation of the phenolic 3-hydroxy group of morphine prevents the oxidation by the phenol oxidizing laccase (Fig. 5), while 6-acetylmorphine is converted. Thus, this sensor will not respond to heroin either (3,6-diacetyl-morphine). (ii) MDH is known to oxidize morphine and codeine exclusively at the 6-hydroxy group [6]. Morphinone, the oxidation product of morphine by MDH, was found not to be reduced by GDH. Consequently, there is no amplification for morphine by the addition of glucose. Discrimination of morphine and codeine in less than one minute In a double detector experiment, the sample is measured simultaneously by the MDH/SHL and the LACC/GDH sensors. The buffer solution for the double sensor contained the reagents necessary for both enzyme membranes: glucose for GDH and salicylate for SHL (Table 1). Injection of NADP+, the costly cofactor for MDH, only with the sample saved a lot of reagent, but did not influence the performance of the sensor compared to the permanent addition of NADP+ in the streaming buffer. No buffer component interfered with the activity of any of the enzymes. The increase in pH from 6.5 to 7.5 in this experiment, however, decreased the sensitivity of the LACC/ GDH sensor. Morphine caused a non-linear, but kinetically controlled response on both sensors (Fig. 6 A). It was detected by the LACC/GDH sensor between 3.2 and 1000 µM and by the MDH/SHL sensor between 10 and 1000 µM.

Fig. 6 A double-detector experiment discriminates morphine and codeine within one minute: (A) Morphine leads to a signal on both sensors, MDH/SHL and LACC/GDH. (B) Codeine causes a response only at the MDH/SHL sensor. The quantitative measurements of morphine and codeine in pharmaceutical drugs are based on these calibration curves

Codeine (Fig. 6 B) was measured between 10 and 1000 µM by the MDH/SHL sensor, but did not lead to any signal at the LACC/GDH sensor. A typical peak started 30 s after injection, reached its maximal response after 1 min, and showed a washout time of 3.5 min. Thus, codeine and morphine could be differentiated in less than one minute after injection. The sampling rate was 20 h–1. Measurement of morphine and codeine in drugs Two drugs were chosen as real samples: MSI 20 (Mundipharma GmbH, Limburg/Lahn, Germany) and Codein Ribbeck (Ribbeck-Arzneimittel, Munich, Germany). Three concentrations of both drugs were analyzed as duplicates

183

(n = 6). For MSI 20 the MDH/SHL sensor found 102.3% (53.9 ± 1.4 mM morphine) and the LACC/GDH sensor detected 97.5% of the morphine concentration declared by the manufacturer. For Codeine Ribbeck the MDH/SHL sensor measured 100.1% of the declared codeine concentration.

Discussion For the detection of morphine and codeine, other methods that measure analytes without separation include various homogeneous immunoassays [12–14], MDH-based sensors [1, 2], and flow systems, that derivatize or complex the analytes before optical detection in a flow injection system [15–18]. Two of the immunoassays require expensive equipment [12, 14]. Their detection limits are less than tenfold lower than that of our method (32 nM morphine) and substantially lower than for the third immunoassay [13], that detects 700 nM morphine by observing agglutination on a glass slide. The more sensitive MDH-based disposable sensor by Holt detects 6 nM morphine and 3.5 nM codeine [2]. The enzymes and reagents used in this disposable sensor were immobilized within 3 h before use due to their limited stability. However, the MDH used in the present sensor has been stabilized by entrapment in the enzyme membranes being prepared more than one month before use. This report describes the first application of MDH for multiple measurements in a flow system. Other flow systems for morphine [15, 16] and codeine [17, 18] can detect analyte concentrations non-enzymatically between 5 nM and 52 nM and show similar or faster signal response times than the enzyme sensors reported here. The performance of these flow systems is similar to that of our amplified morphine sensor. Qualitative homogeneous immunoassays for drug screening in urine are available from several manufacturers and respond to approximately 1 µM “opiates”; they are excluded from the present discussion due to their qualitative signal. HPLC-based methods [19–21] dominate the field of drug analysis and separate morphine and codeine before detection with fluorescence, electrochemistry or massspectrometry. The time required for detection is small compared to the preceding separation process. The detection limits are close to 3 nM morphine or codeine with detected signal response times between 6 min and 26 min, excluding sample preparation. The performance of methods based on gas chromatography, mass spectrometry [22, 23] or capillary electrophoresis [24] is similar. Furthermore, heterogeneous immunoassays for opiates are no faster either [13, 25]. The enzymatic method described herein allows a “quasi-online” differentiation of micromolar concentrations of morphine and codeine in less than one minute.

Our method is substantially faster than methods that achieve selectivity by separation. Other methods that analyze morphine or codeine without separation cannot differentiate between the analytes. In contrast to chromatographic techniques, no expensive instruments are necessary. Our approach has the additional advantage that only inexpensive reagents – salicylate and glucose – are required as permanent buffer additives for multiple measurements.

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