Quantification of Risperidone Contained in Precipitates Produced by Tea Catechins Using Nuclear Magnetic Resonance

Abstract

A mixture of risperidone and eight major tea catechins: (−)-epicatechin-3-O-gallate (ECg), (−)-epigallocatechin-3-O-gallate (EGCg), (−)-catechin-3-O-gallate (Cg), (−)-gallocatechin-3-O-gallate (GCg), (−)-epicatechin (EC), (−)-epigallocatechin (EGC), (+)-catechin (CA), or (+)-gallocatechin (GC), in tartaric acid buffer (pH 3.0) afforded a precipitate. Amounts of risperidone and these catechins in the precipitate were measured by quantitative ¹H-NMR (qNMR). About half or more of risperidone used was precipitated by gallated catechins ECg, EGCg, Cg, and GCg; on the other hand, it was precipitated little by non-gallated catechins EC, EGC, CA, and GC. Furthermore, risperidone was precipitated more by 2,3-trans gallated catechins Cg and GCg than 2,3-cis gallated catechins ECg and EGCg. Regarding the amount of tea catechins in the precipitate obtained by a mixture of risperidone and Catechin Mixture, the amounts of 2,3-cis gallated catechins EGCg and ECg were much larger than those of the other green tea catechins GCg, EC, EGC, CA, and GC. It was considered that risperidone was mainly precipitated by EGCg and ECg in Catechin Mixture. Therefore, it can be concluded that when patients take risperidone with catechin-rich beverages, the efficacy of risperidone reduces, mainly because the 2,3-cis gallated catechins EGCg and ECg form complexes with risperidone and precipitate.

Introduction

The number of patients with schizophrenia in Japan is about 800000, and schizophrenia is one of the well-known diseases.¹⁾ Risperidone^2,3) (Fig. 1) is a drug that is widely used clinically for patients with schizophrenia due to its dopamine D₂ receptor-blocking action, and an oral solution that takes into consideration the characteristics of the patients is sold on the market.

Fig. 1. Risperidone

It is known that when an oral solution of risperidone is mixed with beverages rich in catechins such as green and black tea, it becomes turbid and particles settle out,⁴⁾ and it undergoes a change in composition (Fig. 2). Such a situation can often occur in the field where sufficient medication guidance is not provided.

Fig. 2. Right: Appearance Immediately after Mixture of the Oral Solution of Risperidone [Risperdal (1 mL) Containing Risperidone (1 mg)] and Black Tea [Gogo no Kocha Straight Tea, KIRIN (30 mL)], Left: Black Tea (30 mL) Alone

Therefore, when patients with schizophrenia take risperidone orally with catechin-rich beverages, some adverse interactions may occur, resulting in the lack of a sufficient therapeutic effect on schizophrenia.

Ikeda et al. investigated the mechanism of complex formation in solution of risperidone and (−)-epigallocatechin gallate (EGCg) using ¹H-NMR and molecular modeling computational chemistry.⁵⁾ We examined the molecular capture ability of various heterocylic compounds⁶⁾ and diketopiperazines with proline residues⁷⁾ by EGCg from an aqueous solution using quantitative NMR (qNMR). However, no study has examined how much risperidone forms precipitates of complexes with a wide variety of catechins in detail. Therefore, using qNMR, we comprehensively investigated which green tea catechins and to what extent they form complexes and precipitate.

The eight major tea catechins are classified into four categories by the existence of a galloyl group on the oxygen atom at the C-3 position and the relative stereochemistry between C₂ and C₃ positions: 2,3-cis gallated catechins; (−)-epicatechin-3-O-gallate (ECg) and (−)-epigallocatechin-3-O-gallate (EGCg), 2,3-trans gallated catechins; (−)-catechin-3-O-gallate (Cg) and (−)-gallocatechin-3-O-gallate (GCg), 2,3-cis non-gallated catechins; (−)-epicatechin (EC) and (−)-epigallocatechin (EGC), and 2,3-trans non-gallated catechins; (+)-catechin (CA) and (+)-gallocatechin (GC)⁸⁾ (Fig. 3).

Fig. 3. The Eight Major Tea Catechins

Experimental

Materials

Risperidone was purchased from LKT Laboratories, Inc. (MN, U.S.A.), EGCg, EC, and CA were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.), ECg, EGC, GCg, and Catechin Mixture were purchased from Funakoshi (Tokyo, Japan), and Cg and GC were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan).

NMR Experiments

¹H-NMR spectra were recorded at 30 °C on JEOL JNM-ECZ400R (Tokyo, Japan) operating at 400 MHz using a 5 mm ϕ sample tube. In general, ¹H-NMR experiments were performed with 32 K data points covering a spectral width of 10000 Hz with a approx. 3.7 s pulse interval and 16 scan times. Deuterated water D₂O (99.9 atom % D; FUJIFILM Wako Pure Chemical Corporation) and deuterated dimethylsulfoxide DMSO-d₆ (Eurisotop, Saint-Aubin, France) were used as measurement solvents. Chemical shift values are expressed in ppm downfield using sodium 3-(trimethylsilyl)-1-propane-1,1,2,2,3,3-d₆-sulfonate (DSS-d₆, FUJIFILM Wako Pure Chemical Corporation).

Quantitative ¹H-NMR (qNMR) was performed using the following optimized parameters: probe temperature, 30 °C; spinning, on; number of scans, 60 times; spectral width, 20 ppm; pulse interval, 60 s; pulse angle, 90°.

Relaxaton time (T₁) in DMSO-d₆ (sample concentration, 5.0 µmol) was measured using the Inversion Recovery method, and was performed using the following optimized parameters: probe temperature, 30 °C; spinning, off; number of scans, 16 times; 90° pulse angle, 5.9 µs; relaxation delay used for measurement, 10.0, 2.0, 1.5, 1.0, 0.8, 0.6, 0.4, and 0.2 s.

Preparation and Measurement of NMR Sample

Since the additives of Risperdal Oral Solution (risperidone 1 mg/mL) are tartaric acid as a solubilizer, benzoic acid as a preservative, and sodium hydroxide as a pH adjuster and its pH is kept at 2.0 approx. 4.0, tartaric acid buffer (pH 3.0) was used as a solubilizer and pH adjuster of risperidone and tea catechins.

A solution of risperidone (2.05 mg, 5.0 µmol) in tartaric acid buffer (pH 3.0, 500 µL) with D₂O was added to a solution of EGCg or EC (5.0 µmol) in tartaric acid buffer (pH 3.0, 1.0 mL) with D₂O. As a result, it was separated into a supernatant liquid and precipitate. DSS-d₆ (5.0 µmol, 300 µL) was added to the supernatant liquid (300 µL), and qNMR was performed. The precipitate was dried under reduced pressure to afford a solid, which was dissolved in DMSO-d₆ (600 µL) containing DSS-d₆ (5.0 µmol), and qNMR was performed.

A solution of risperidone (2.05 mg, 5.0 µmol or 10.25 mg, 25 µmol) in tartaric acid buffer (pH 3.0, 500 µL or 2.5 mL) was added to a solution of tea catechins: ECg, EGCg, EC, EGC, Cg, GCg, CA, GC (5.0 µmol), or Catechin Mixture (13.59 mg, 25.0 µmol) in tartaric acid buffer (pH 3.0, 1.0 mL or 5 mL), respectively. As a result, it was separated into a precipitate and supernatant liquid. The resulting precipitate was dried under reduced pressure to afford a solid, which was dissolved in DMSO-d₆ (600 µL) containing DSS-d₆ (5.0 µmol), and qNMR was performed.

The above experiments were performed three times, and the average value of three integrated values of corresponding signals obtained by qNMR measurement was calculated.

Results

Quantification of Supernatant Liquid and Precipitate Using qNMR

Among proton signals in ¹H-NMR spectra of the eight major tea catechins and risperidone, a proton signal for H₂ of 2,3-cis epicatechins [singlet (s), 1H], H₂ of 2,3-trans catechins [doublet (d), 1H], H_2′,6′ of 2,3-cis gallated catechins [singlet (s), 2H], and H_a, H_b, H_c bonding to the benzene ring of risperidone (Fig. 4), do not overlap with other signals except for an overlap the proton signals for H₂ of ECg and GCg. A characteristic coupling between H_b and F was observed in the proton signal for H_b of risperidone [7.299 ppm, double-double-doublet (ddd), 1H]. Therefore, integrated values of the proton signal for H₂ or H_2′,6′ of tea catechins and H_b of risperidone were used for quantification by q ¹H-NMR. A trimethyl group of DSS-d₆ [0 ppm, singlet (s), 9H of trimethyl group (CH₃)₃-] was used not only as a reference substance, but also as an internal standard substance.

Fig. 4. ¹H-NMR Spectra of Three Aromatic Protons H_a, H_b, and H_c Binding to Benzene Ring of Risperidone

Table 1 shows the relaxation time of protons of EGCg, EC, and H_a, H_b, H_c of the benzene ring of risperidone, that in a mixture of EGCg and risperidone, and that in a mixture of EC and risperidone. Based on these relaxation times, the pulse interval for measurement of qNMR was set to 60 s.

Table 1. Relaxation Time (s) of Proton of EC, EGCg, and H_a, H_b, and H_c of the Benzene Ring of Risperidone in DMSO-d₆

Proton	H2	H3	H4a	H4b	H6	H8	H2′	H5′, H6′	H2′, 6′	H2″, 6″	Ha	Hb	Hc
EGCg	0.861	1.061	1.102	0.431	2.707	1.496			1.477	2.080
EC	1.276	0.562	0.318	0.415	1.137	1.137	1.385	1.334
Risperidone											0.818	1.059	2.022
EGCg and Risperidone	0.853	1.043	—	0.440	2.673	1.481			1.477	2.089	1.323	1.694	3.808
EC and Risperidone	1.105	0.530	—	—	2.269	1.141	1.375	1.336			1.316	1.689	3.797

The unit of the above number is seconds (s). —is unmeasurable due to overlap with other proton signals.

When solutions of 8.33 and 0.0833 mmol/L EGCg in DMSO-d₆ were measured by qNMR under the quantification conditions set, the repeatabilities (RSD, n = 5) were 0.18 and 10.0%, respectively.

A solution of risperidone in tartaric acid buffer (pH 3.0) with D₂O was added to that of EGCg, EC in tartaric acid buffer (pH 3.0) with D₂O. As a result, the mixture was separated into a supernatant liquid and precipitate. Figure 5 shows qNMR spectra of supernatant liquid and precipitate obtained from a mixture of risperidone and EGCg. Amounts of risperidone and EGCg, EC in the supernatant liquid and precipitate were measured by qNMR using an integrated values of proton signals for H_2′,6′ of EGCg, H₂ of EC, and H_b of risperidone (Table 2). Ratios of amounts of risperidone and EGCg, EC in the supernatant liquid and precipitate to those used are also shown in Table 2.

Fig. 5. QNMR Spectra of Supernatant Liquid (a) and Precipitate (b) Made from a Mixture of Risperidone and EGCg

(　) is an integrated value of the proton signal, calculated with the integrated value of DSS (9H) as 9000.

Table 2. Amount and Ratio of EGCg and Risperidone (a), EC and Risperidone (b) in Supernatant Liquid and Precipitate Made from a Mixture of Risperidone and EGCg, EC

(a)
Amount and ratio			EGCg	Risperidone
Used sample	Amount	mg	2.29	2.05
		µmol	5.00	5.00
Supernatant liquid	Amount	mg	0.69	0.77
		µmol	1.51	1.89
	Ratio	%	30.22	37.71
Precipitate	Amount	mg	1.57	1.29
		µmol	3.42	3.15
	Ratio	%	68.48	62.99
Total	Amount	mg	2.26	2.07
		µmol	4.93	5.03
	Ratio	%	98.69	100.70
(b)
Amount and ratio			EC	Risperidone
Used sample	Amount	mg	1.45	2.05
		µmol	5.00	5.00
Supernatant liquid	Amount	mg	1.48	2.07
		µmol	5.10	5.05
	Ratio	%	102.05	101.09
Precipitate	Amount	mg	0.00	0.00
		µmol	0.01	0.00
	Ratio	%	0.26	0.00
Total	Amount	mg	1.48	2.07
		µmol	5.12	5.05
	Ratio	%	102.31	101.09

The sum of the amounts of EGCg, EC, and risperidone contained in the supernatant liquid and precipitate was almost equal to the amounts of those used for the experiments, confirming the validity of the conditions used for this qNMR.

Quantification of Risperidone and Tea Catechins in Precipitation

A solution of risperidone in tartaric acid buffer (pH 3.0) was added to a solution of an equimolar amount of eight major tea catechins: ECg, EGCg, Cg, GCg, EC, EGC, CA, or GC, in tartaric acid buffer (pH 3.0). As a result, they afforded precipitates. QNMR spectra of precipitates made from a mixture of risperidone and EGCg, EC are shown in Figs. 6 and 7. Amounts of risperidone and tea catechins in the precipitates were measured by qNMR using integrated values of proton signals for H₂ of tea catechins and H_b of risperidone (Table 3). Ratios of amounts of risperidone and tea catechins in precipitates to those used are also shown in Table 3.

Fig. 6. QNMR Spectra of Precipitate (a) Made from a Mixture of Risperidone and EGCg, EGCg Alone (b), and Risperidone Alone (c)

(　) is an integrated value of the proton signal, calculated with the integrated value of DSS (9H) as 9000.

Fig. 7. QNMR Spectra of Precipitate (a) Made from a Mixture of Risperidone and EC, EC Alone (b)

(　) is an integrated value of the proton signal, calculated with the integrated value of DSS (9H) as 9000.

Table 3. Amount and Ratio of Risperidone and Tea Catechins in Precipitate Made from a Mixture of Risperidone and Tea Catechins

			ECg	EGCg	Cg	GCg	EC	EGC	CA	GC
Tea Catechina)	Used amount	mg	2.21	2.29	2.21	2.29	1.45	1.53	1.45	1.53
	Amount in precipitate	mg	1.34	1.32	1.51	1.78	0.01	0.02	0.03	0.01
	Ratio	%	60.67	57.84	68.10	77.53	0.61	0.98	2.12	0.97
Risperidoneb)	Amount in precipitate	mg	1.00	1.11	1.14	1.38	0.01	0.01	0.02	0.02
	Ratio	%	48.87	53.90	55.49	67.39	0.65	0.53	1.02	1.01

a) Tea Catechins used are 5 µmol. b) Risperidone used is 2.05 mg (5 µmol).

About half or more of risperidone used was precipitated by gallated catechins ECg, EGCg, Cg, and GCg, on the other hand, risperidone was precipitated a little by non-gallated catechins EC, EGC, CA, and GC. Furthermore, risperidone was precipitated more by 2,3-trans gallated catechins GCg and Cg than 2,3-cis gallated catechins ECg and EGCg.

Quantification of Risperidone in Precipitation Produced by Catechin Mixture

Catechin Mixture is a fraction of extracted catechins included in green tea. Firstly, the amount of tea catechins in Catechin Mixture was evaluated using qNMR (Fig. 8). It was found that seven tea catechins: ECg, EGCg, GCg, EC, EGC CA, and GC, are included in the amounts shown in Table 4,⁸⁾ and 2,3-cis gallated catechins ECg and EGCg and 2,3-cis non-gallated catechins EC and EGC are abundantly included.

Fig. 8. QNMR Spectra of Precipitate (a) Made from a Mixture of Catechin Mixture and Risperidone, and Catechin Mixture Alone (b)

(　) is an integrated value of the proton signal, calculated with the integrated value of DSS (9H) as 9000.

Table 4. Amount and Ratio of Risperidone and Tea Catechins in Precipitate Made from a Mixture of Risperidone and Catechin Mixture

		Tea Catechin								Risperidone
		ECg	EGCg	Cg	GCg	EC	EGC	CA	GC
Amount in Catechin Mixturea)	mg	0.81	4.49	0.00	0.04	0.90	2.10	0.15	0.48
	µmol	1.83	9.80	0.00	0.09	3.11	6.88	0.50	1.55
Risperidone	mg									10.26
	µmol									25.00
Amount in precipitate	mg	0.34	1.85	0.00	0.02	0.02	0.05	0.01	0.02	2.58
	µmol	0.77	4.03	0.00	0.05	0.08	0.16	0.03	0.08	6.28
Ratio	%	41.93	41.18	0.00	55.79	2.67	2.34	4.97	4.95	25.14

a) Catechin Mixture used is 13.59 mg (25 µmol).

A solution of risperidone in tartaric acid buffer (pH 3.0) was added to a solution of an equimolar amount of Catechin Mixture in tartaric acid buffer (pH 3.0). As a result, it afforded a precipitate. QNMR spectra of the precipitate are shown in Fig. 8. Amounts of seven tea catechins and risperidone in the precipitates were measured using qNMR with an integrated value of the proton signal for H₂ of tea catechins, H_2′ of GCg, and H_b of risperidone (Table 4). Since the proton signals for H₂ of ECg and GCg overlapped, the amounts of GCg were evaluated with an integrated value of the proton signal for H_2′, and that of ECg was evaluated with a difference in the integrated value of proton signals for H₂ of ECg, GCg, and proton signals for H_2′ of GCg. Ratios of amounts of tea catechins and risperidone in the precipitate to those used are also shown in Table 4. A total of 25.1% of risperidone used was precipitated by Catechin Mixture (Table 4). About half of gallated catechins ECg, EGCg, and GCg precipitated; on the other hand, non-gallated catechins EC, EGC, CA, and GC precipitated a little. Furthermore, 2,3-trans gallated catechin GCg precipitated at a higher rate than 2,3-cis gallated catechins ECg and EGCg. The ratios showed similar trends to those of tea catechins in the precipitate made by a mixture of individual tea catechins and risperidone (Tables 3, 4).

Regarding the amount of tea catechins in the precipitate obtained by a mixture of risperidone and Catechin Mixture, amounts of 2,3-cis gallated catechin EGCg were much larger than those of other green tea catechins ECg, GCg, EC, EGC, CA, and GC. Subsequently, the amount of ECg, which is also 2,3-cis gallated catechin, was large.

Discussion

When a hot tea beverage cools down, it becomes turbid and brown-white particles settle out. This phenomenon is called “creaming” or “creaming down (reaction).” Previously, Ina and colleagues reported that all the main signals in the ¹³C-NMR spectrum of a hot water solution of a precipitate formed by the creaming down of a tea infusion were assigned to catechins such as (−)-epigallocatechin-3-O-gallate (EGCg) and (−)-epicatechin-3-O-gallate (ECg), and caffeine.⁹⁾ Therefore, we investigated precipitates obtained from aqueous solution of mixtures of equimolar amounts of EGCg and caffeine, ECg and caffeine, and EC and caffeine, and reported that they were determined by X-ray crystallographic analysis to be a 2 : 2 complex of EGCg and caffeine (Fig. 9a), 2 : 4 complex of ECg and caffeine (Fig. 9b), and 1 : 1 complex of EC and caffeine (Fig. 9c), respectively.^10–14) Furthermore, amounts of the precipitates obtained from mixtures of EGCg and caffeine, and ECg and caffeine were much more than that of EC and caffeine.¹³⁾

Fig. 9. Layer Structure of the 2 : 2 Complex of EGCg and Caffeine (a), 2 : 4 Complex of ECg and Caffeine (b), and 1 : 1 Complex of EC and Caffeine (c)

Upon the complex formation of EGCg and caffeine, and that of ECg and caffeine, intermolecular interactions forming between EGCg and caffeine moieties, and that between ECg and caffeine moieties were mainly hydrophobic interactions between the three aromatic A, B, and B’ rings of EGCg and caffeine moieties, and between those of ECg and caffeine moieties, respectively (Figs. 9a, b). Therefore, the hydrophobicity of the complexes of EGCg and caffeine, and ECg and caffeine increased in aqueous solution, whereas their solubility in water decreased rapidly compared with EGCg, ECg, and caffeine alone. Upon the complex formation of EC and caffeine (Fig. 9c), the hydrophobic interaction was formed only between the A ring of EC and caffeine moieties. Mixtures of EGCg and caffeine, and ECg and caffeine led to a large amount of precipitate of the complex due to a strong hydrophobic effect by their aromatic A, B, and B’ rings; on the other hand, the mixture of EC and caffeine led to only a small amount of precipitate of the complex due to a weak hydrophobic effect by its A ring.

It was considered that when risperidone formed complexes with gallated catechins due to a strong hydrophobic effect by their aromatic A, B, and B’ rings, the solubility is rapidly reduced and many precipitates occurred. On the other hand, when risperidone formed complexes with non-gallated catechins due to a weak hydrophobic effect by only their aromatic A ring, the solubility did not decrease so markedly and only a small amount of precipitates was considered to form. Since amounts of 2,3-cis gallated catechin EGCg and ECg were large in the amount of tea catechins containing in the precipitate obtained by a mixture of risperidone and Catechin Mixture, it was considered that risperidone was mainly precipitated by EGCg and ECg in Catechin Mixture. Therefore, it can be concluded that when patients take risperidone with catechin-rich beverages, the efficacy of risperidone reduces, mainly because the 2,3-cis gallated catechins EGCg and ECg form complexes with risperidone and precipitate.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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