MS for the determination of paliperidone after stereoselective fungal biotransformation of risperidone

Analytica Chimica Acta 742 (2012) 80–89

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Solid phase microextraction and LC–MS/MS for the determination of paliperidone after stereoselective fungal biotransformation of risperidone Mariana Zuccherato Bocato a , Rodrigo Almeida Simões b , Leandro Augusto Calixto b , Cristiane Masetto de Gaitani c , Mônica Tallarico Pupo c , Anderson Rodrigo Moraes de Oliveira a,∗ a

Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, 14040-901, Ribeirão Preto, SP, Brazil Departamento de Física-Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, 14040-903, Ribeirão Preto, SP, Brazil c Departamento de Ciências Farmacêuticas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, 14040-903, Ribeirão Preto, SP, Brazil b

h i g h l i g h t s

g r a p h i c a l

a b s t r a c t

 We developed a new LC–MS/MS method employing the polar organic mode to analyze risperidone and its chiral metabolites.  We optimize a SPME procedure to extract these analytes from liquid culture medium.  The method was validated and SPME showed to be a useful tool to be used in biotransformation studies.  The biotransformation results showed that it is possible to obtain a drug in its enantiomeric pure form.

a r t i c l e

i n f o

Article history: Received 13 March 2012 Received in revised form 25 May 2012 Accepted 29 May 2012 Available online 9 June 2012 Keywords: Solid phase microextraction Chiral analysis Stereoselective fungal biotransformation Polar organic mode Paliperidone Risperidone

a b s t r a c t The present work describes for the first time the use of SPME coupled to LC–MS/MS employing the polar organic mode in a stereoselective fungal biotransformation study to investigate the fungi ability to biotransform the drug risperidone into its chiral and active metabolite 9-hydroxyrisperidone (9-RispOH). The chromatographic separation was performed on a Chiralcel OJ-H column using methanol:ethanol (50:50, v/v) plus 0.2% triethylamine as the mobile phase at a flow rate of 0.8 mL min−1 . The SPME process was performed using a C18 fiber, 30 min of extraction time and 5 min of desorption time in the mobile phase. The method was completely validated and all parameters were in agreement with the literature recommendations. The Cunninghamella echinulata fungus was able to biotransform risperidone into the active metabolite, (+)-9-RispOH, resulting in 100% of enantiomeric excess. The Cunninghamella elegans fungus was also able to stereoselectively biotransform risperidone into (+)- and (−)-9-RispOH enantiomers at different rates. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Risperidone is an effective antipsychotic agent, by acting as inhibitor of serotonin 5-HT2 and dopamine D2 receptors. It is

∗ Corresponding author at: Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto – USP, Av. dos Bandeirantes, 3900, 14040-901, Ribeirão Preto, SP, Brazil. Tel.: +55 16 36020388; fax: +55 16 36024838. E-mail address: [email protected] (A.R.M. de Oliveira). 0003-2670/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aca.2012.05.056

used in the treatment of psychotic disorders, schizophrenia, autism [1]. This drug metabolism, mediated by cytochrome P450-2D6 results in hydroxylated metabolites [2,3]. It has been shown the predominant formation of the 9-hydroxyrisperidone (9-RispOH) while the 7-hydroxyrisperidone (7-RispOH) formation has been observed to a much lesser extent [4,5]. The metabolites 9-RispOH and 7-RispOH have chiral carbon atoms resulting in two enantiomers: the (+)- and the (−)-forms (Fig. 1). Moreover, it has been also shown that the major metabolite, 9-RispOH, presents the same pharmacologic activity of the parent drug risperidone [6].

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metabolite production, another advantage to be addressed is the possibility of obtaining large amounts of metabolites by preparative methods. In literature, it has been reported several studies regarding drug biotransformations by using endophytic or soil fungi, [31–34], and the generation of a series of metabolites. Some bioanalytical enantioselective methods [31,35–39] have been developed in order to determine risperidone and 9hydroxyrisperidone. Most developed methods employ a classical sample preparation technique, such as SPE [35,39], to extract risperidone and its metabolites from a biological matrix. Our group has previously reported one microextraction technique to extract risperidone and its metabolites from a metabolism study using capillary electrophoresis as analytical technique [31]. Due to the prominent results observed and the lack of reports in this subject, this research has aimed to develop a new and fast analytical method to quantify and to determine paliperidone after stereoselective fungal biotransformation using SPME-LC–MS/MS in polar organic mode and to evaluate the performance of SPME with this kind of biological matrix. 2. Experimental Fig. 1. Chemical structures of the risperidone (A), paliperidone (B) and ranolazine (IS) (C).

Currently, 9-RispOH is marketed as drug under the generic name paliperidone. Regarding the traditional and most employed extraction techniques, such as liquid–liquid extraction (LLE) and solid-phase extraction (SPE), they present some drawbacks related to the time consumed in the extraction step and the use of large amounts of organic solvents, which usually are expensive and toxic for the environment [7]. In this context, microextraction systems, such as solid-phase microextraction (SPME), introduced by Pawliszyn’s group in 1990 [8] have been extensively employed. SPME is a preconcentration and non-exhaustive extraction technique based on equilibrium or pre-equilibrium conditions. The extractions are performed by fused silica fibers coated with extracting polymers. The higher the analyte affinity for the extraction phase, the greater the amount of analyte extracted [9]. The kind of polymer used in SPME depends on the physical-chemistry properties of the target analytes [8,10], compatibility with commonly used LC-solvents, good interfiber reproducibility, suitable extraction efficiency for a wide range of analytes and biocompatibility [11]. Several biocompatible coatings proposed for research include polypyrrole coatings [9,11–15], coatings based on restricted access materials [16–19], and coatings based on mixtures of SPE sorbents (coated silica particles) with biocompatible polymers [20,21]. These kinds of coatings are especially useful to be used to extract analytes from biological matrices. Recently, several methods using SPME have been developed to extract drugs and their metabolites in different biological matrices, such as urine and plasma [22–27]. Nevertheless, up to now, no report has been found in literature regarding the use of SPME in fungal biotransformation studies. Biotransformation studies employing microorganisms have shown high capacity to promote regio- and stereospecific reactions in mild conditions [28–30]. The widespread application of these types of reactions has promoted studies in various areas of science, as in medicine and in agricultural chemistry. In medicine, the proposition of employing microorganisms to produce drugs is a fascinating one and, it is based on the capability of microorganisms to metabolize drugs similarly to mammalian systems rending metabolites with pharmacological activity. Additionally, the asymmetric medium found in the biotransformation studies is prone to generate metabolites in their pure enantiomeric forms [28]. By supporting this mode of

2.1. Chemical, reagents and standard solutions stock solutions of risperidone, rac-7Standard hydroxyrisperidone and rac-9-hydroxyrisperidone (all purchased from Toronto Research Chemicals, Toronto, Canada) were prepared at the concentration of 400 ␮g mL−1 in methanol. Calibration curve solutions of risperidone at concentrations range of 300–4 ␮g mL−1 and rac-7-RispOH and rac-9-RispOH at concentration range of 80–4 ␮g mL−1 were obtained by dilutions in the same solvent. A solution of ranolazine (Fig. 1C) (purchased from Sigma–Aldrich, Steinheim, Germany) at the concentration of 60 ␮g mL−1 was used as the internal standard (IS). All these solutions were stored at −20 ◦ C in amber glass tube and protected from direct light. The solvents used in the chromatographic analyses (HPLC grade) were acetonitrile, acetic acid, methanol and ethanol purchased from Merck (Darmstadt, Germany) and from J.T. Baker (Philipsburg, USA). The reagents (analytical grade) were: sodium chloride, potassium chloride, monosodium phosphate (NaH2 PO4 ·1H2 O), disodium phosphate (Na2 PO4 ·2H2 O), magnesium sulfate (MgSO4 ·7H2 O) and iron sulfate (FeSO4 ·7H2 O) all obtained from Merck (Darmstadt, Germany). Sodium hydroxide was purchased from Nuclear (Diadema, Brazil) and acetic acid from Zilquímica (Ribeirão Preto, Brazil). Potato dextrose agar (PDA), sucrose, malt extract, dextrose, triptone and yeast extract were obtained from Acumedia (Lansing, USA) and triethylamine was obtained from Fluka (Buchs, Switzerland). Water used to prepare the solutions was purified using a Milli-Q plus system (Millipore, Bedford, USA). 2.2. Instrumentation and analytical conditions The chromatographic separation optimization and the SPME optimization were conducted on a Shimadzu (Kyoto, Japan) HPLC system consisting of two LC 10AS solvent pumps, a SPD 10A UV–vis detector operating at 280 nm, a CTO 10AS column oven, and a 7125 model Rheodyne injector (Cotati, USA) with a 20 ␮L loop. Data were monitored using a SCL 10A controller model. The software used for data acquisition was the Class-VP (Shimadzu, Kyoto, Japan). The resolution of the risperidone, 7-RispOH and 9-RispOH enantiomers were evaluated using the polar organic mode at 25 ◦ C on seven chiral columns, i.e. Chiralpak AD (250 mm × 4.6 mm, 10 ␮m particle size), Chiralpak AD-RH (150 mm × 4.6 mm, 5 ␮m particle size), Chiralcel OJ (250 mm × 4.6 mm, 10 ␮m particle

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Table 1 MS conditions for the analysis of the analytes positive mode (ESI+). Analytes

Transition (m/z)

Dwell time (s)

Cone (V)

Collision energy (eV)

7-RispOH and 9-RispOH Risperidone Ranolazine (IS)

427 → 207 411 → 191 428 → 279

0.4 0.4 0.4

35 35 25

27 27 26

size), Chiralcel OJ-H (150 mm × 4.6 mm, 5 ␮m particle size), Chiralcel OD-H (150 mm × 4.6 mm, 5 ␮m particle size), Chiralcel OD-R (250 mm × 4.6 mm, 10 ␮m particle size) and Chirobiotic T (150 mm × 4.6 mm, 5 ␮m particle size) (purchased from Chiral Technologies, Exton, USA and from Astec, Advanced Separation Technologies, New Jersey, USA). A C18 column (4.6 × 12.5 mm, 5 ␮m particle size, Merck, Darmstadt, Germany) was used as guard column. The method validation and the biotransformation studies were performed using an LC–MS/MS system. The LC system was a Shimadzu equipment (Kyoto, Japan) consisting of two LC 10AS solvent pumps, a CTO 10AS column oven, and a 7125 model Rheodyne injector (Cotati, USA) with a 20 ␮L loop. The MS system was a Quattro LC triple-stage quadrupole (Micromass, UK) fitted with a Z-electrospray interface operating in the positive-ion mode. The temperatures of the source block and desolvation system were 100 and 250 ◦ C, respectively. Nitrogen was used as both drying and nebulizing gas and argon as the collision gas. A Valco valve connection was used to split the effluent from the column and a flow rate, approximately, of 0.1 mL min−1 was directed injected into the stainless steel capillary probe of the LC–MS/MS system. MS conditions were optimized by direct infusion of all analyte solutions, prepared in the mobile phase, at a flow rate of 10 ␮L min−1 . Quantitation was performed by multiple reaction monitoring (MRM) of the precursor ions and their corresponding product ions with a dwell time of 0.4 s. Using the acquisition mode, the mass spectrometer was programmed to scan from 100 to 400 amu for each analyte. The mass spectra were obtained and the ion with higher intensity was selected (precursor ion). Later, each precursor ion was subjected to fragmentation. The acquisition mode was used again and scan was carried out in the range of 100–400 amu. Finally, the product ion with higher intensity in the acquired spectra was chosen. Micromass Masslynx 4.1 software (Manchester, UK) was used to control the LC–MS/MS system and for data acquisition. Table 1 shows the MS conditions used for all analytes. 2.3. Elution order for the 9-hydroxyrisperidone (paliperidone) To establish the elution order, the pure enantiomers of 9-RispOH were isolated by semipreparative analysis and collected using the conditions described previously in literature [37]. Next, the pure 9-RispOH enantiomers were injected separately into the chromatographic system under the conditions established in the present paper. Subsequently, the retention times of the enantiomers from both studies were compared and the elution order established. The elution order for the 7-RispOH enantiomers was not established due to the lack of standard to perform a structural analysis. 2.4. SPME procedure Liquid culture medium (LCM) obtained from fungal biotransformation study (6 mL) was stored at −20 ◦ C until its use. Prior to use, the LCM samples were allowed to thaw at room temperature and after that, the LCM were centrifuged at 4000 rpm during 5 min using a CF-15 centrifuge (Hitachi Koki, Kyoto, Japan). Then, 2 mL of the supernatant was transferred to a sample vial of 4 mL (Supelco, Bellefonte, USA) and supplemented with 2 mL

0.25 mol L−1 phosphate buffer, pH 7.0, and 20% NaCl (w/v). The microextraction procedure was performed with SPME C18 fiber probe 45 ␮m (Supelco, Bellefonte, USA). Before each extraction, the fiber was conditioned for 30 min with methanol:water (50:50, v/v). Next, the extraction was done by immersing the fiber in the fungal sample for 30 min at room temperature (25 ± 2 ◦ C) and the samples were agitated using a Vibrax VXR agitator (IKA, Staufen, Germany) at 600 rpm. Following this step, the fiber was then withdrawn into the needle, and the needle was removed from the extraction vial and inserted in the 120 ␮L desorption microglass vial, which was previously filled with the mobile phase. After 5 min, the fiber was withdrawn into the needle and an aliquot of 20 ␮L was directly injected into the LC–MS/MS system. After each extraction, the fibers were washed for 30 min with methanol to avoid any carryover on the fiber.

2.5. Validation of the method Since there is no specific guide that recommends the analysis of drugs and metabolites in liquid culture medium, it was decided to follow, as close as possible, the FDA guidelines [40] for the analysis of drugs and metabolites in biological fluids. This requirement was followed due to the complexity of the liquid culture medium. Since the whole SPME optimization was performed in Czapeck liquid culture medium and the fungal biotransformation medium may present different characteristics due to the formation of secondary metabolites of the fungi, the linearity was performed in two different matrices. It was performed in Czapeck liquid culture medium and in a pool of fungi that was prepared in the absence of the analytes. After that, the slopes of the different analytical curves were analyzed and compared [41]. The linearity of the method was performed in fivefold replicate and the results were weighted by 1/x2 . The results of the linearity were weighted because the residual analysis of the analytical curve showed a heteroscedasticity behavior [42]. Analytical curves were obtained by spiking aliquots of 2.0 mL liquid culture medium samples or 2.0 mL of a fungi pool with 25 ␮L calibration curve solutions of risperidone, its metabolites and the IS. The final concentration range was 50–3750 ng mL−1 for risperidone, 25–500 ng mL−1 for each metabolite enantiomer and 750 ng mL−1 for the IS. In addition, it was performed a precision and an accuracy assay by spiking the fungi pool at three different concentrations (75, 500 and 2000 ng mL−1 for risperidone, 37, 187 and 500 ng mL−1 for each 9-RispOH enantiomer and 45, 187 and 500 ng mL−1 for each 7-RispOH, n = 5 for each concentration) and these samples were quantified using the analytical curve prepared in Czapeck liquid culture medium. This assay was performed to guarantee that the whole validation could be performed using the Czapeck liquid medium without matrix influence in the SPME procedure. Matrix effects in the MS ionization process were assessed by comparing the peak areas obtained from the analysis of pure standard solutions dissolved in the mobile phase with the peak areas acquired from extracted blank Czapeck liquid culture medium and the pool of the fungi spiked with the drug, metabolites and internal standard. The experiment was performed in quadruplicate at three different concentrations. The absolute recovery of each analyte extracted from the liquid culture medium samples spiked with the concentrations of 100, 1000 and 2000 ng mL−1 for risperidone, 50, 200 and 400 ng mL−1 for each metabolite enantiomer (n = 3, for each concentration) was determined using calibration curves obtained from the data of the analytes not submitted to the microextraction process. The limit of quantification was defined as the lowest concentration of the analytes that could be determined with accuracy and precision below 20% [40] over five analytical runs (n = 5) and it was

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obtained from liquid culture medium spiked with 50 ng mL−1 of risperidone and 25 ng mL−1 of each metabolite enantiomer. Within-day precision and accuracy were achieved by replicate analysis (n = 5) of 2.0 mL liquid culture medium samples spiked with standard solutions of the drug and metabolites at low, medium, and high concentrations (100, 1000 and 2000 ng mL−1 for risperidone; 50, 200 and 400 ng mL−1 for each enantiomer of 7RispOH and 9-RispOH). Between-day precision and accuracy were determined during routine operation of the system over a period of three consecutive working days. The overall precision of the method was expressed as relative standard deviation and accuracy was expressed as percentage of relative error (RE, %). Freeze-thaw cycle stability and short-term room temperature stability tests for each analyte were performed. The stability tests were determined by six replicates (n = 6) of 2.0 mL spiked liquid culture medium at the low (100 ng mL−1 for risperidone and 50 ng mL−1 for each metabolite enantiomer) and high (800 ng mL−1 for risperidone and 400 ng mL−1 for each metabolite enantiomer) concentrations. The peak areas obtained from the stability tests were compared with the peak areas obtained from freshly prepared samples. One-way ANOVA test was applied, with the level of significance set at p ≤ 0.05. 2.6. Obtention of the fungi The selected strains Cunninghamella echinulata var. elegans ATCC 8688A, Cunninghamella elegans: NRRL 1393 ATCC 10028B fungi were purchased from ATCC® (University Boulevard, Manassas, VA, USA). 2.7. Biotransformation conditions The biotransformation procedure was carried as described by our group [31–34]: Three disks of 0.5 cm of diameter containing the fungal mycelia were aseptically transferred to 9.0 cm diameter Petri dishes containing potato dextrose agar and allowed to grow for 6 days at 22.0 ± 2.0 ◦ C. Then, three uniform disks of 0.5 cm diameter of the fungus mycelia were cut with a transfer tube (Fischer, Scientific, Pittsburgh, PA, USA) and then inoculated in 50 mL Falcon tubes containing 15 mL of prefermentative medium (10.0 g malt extract, 10.0 g dextrose, 5.0 g triptone, and 3.0 g yeast extract and deionized water to 1 L and pH adjusted to 6.2 ± 0.2 with a solution of 0.5 mol L−1 HCl) that was used for the appropriate growth of microorganism for 96 h, 120 rpm at 30.0 ◦ C. After that, the mycelium was completely transferred to 125.0 mL erlenmeyer flask containing 80.0 mL of modified Czapeck medium (25.0 g sucrose, 2.0 g NaNO3 , 1.0 g KH2 PO4 , 0.5 g MgSO4 ·7H2 O, 0.5 g KCl, 0.01 g FeSO4 ·7H2 O, and deionized water to 1.0 L, pH adjusted to 5.0 with a solution of 1.0 mol L−1 HCl). At this point, 3.0 mg risperidone was dissolved in 200 ␮L of N,N-dimethylformamide and added to modified Czapeck medium. The cultures were incubated for 216 h at 30 ◦ C, with shaking at 120 rpm. Control samples consisted of (i) culture broth without risperidone and the fungus, (ii) sterile medium with risperidone but without the fungus and fungal mycelium of the studied fungi was performed at the same time. The results obtained in the biotransformation process were expressed as enantiomeric excess (ee), determined by the equation: ee = (A − B/A + B) × 100; where A is the enantiomer with higher concentration and B is the enantiomer with lower concentration. A pool of the Cunninghamella fungi was prepared at the same way described above. However, instead of 3.0 mg of risperidone it was added just 200 ␮L of N,N-dimethylformamide. After that, these culture media were incubated during 9 days and the supernatant liquid portions were collected and joined. This pool was used in the

Fig. 2. Chromatogram of the analytes after HPLC optimization. Internal standard (1), rac-7-RispOH (2), rac-9-RispOH (3), risperidone (4). Chromatographic conditions described in Section 3.1.

SPME procedure, linearity assays and in the MS ionization matrix effect assays. 3. Results and discussion 3.1. Chromatographic separation HPLC using chiral stationary phases (CPS’s) based on polysaccharide has been previously related for the separation of risperidone and 9-hydroxyrisperidone [35,37] by employing the normal phase mode of elution. Based on that, one of the aims of the present work was the development of a new chiral HPLC method for the simultaneously separation of risperidone and its chiral metabolites employing the polar organic mode. Pure organic eluents may offer the advantages of alternative chiral recognition mechanisms and better analyte solubilities [43,45]. Therefore, seven chiral stationary phases were evaluated (based on polysaccharide derivatives and macrocyclic antibiotics). To evaluate these columns in polar organic mode, a screening was performed using the strategy developed by Hilário et al. [34]. After evaluating all these columns employing different polar organic mobile phases, the best chiral separation of risperidone and its chiral metabolites was accomplished on the Chiralcel OJ-H column using as mobile phase methanol:ethanol (50:50, v/v) + 0.2% triethylamine (TEA) and the flow rate was set at 0.8 mL min−1 . The resolution between the 7-RispOH enantiomers was 2.4 and between the 9-RispOH enantiomers was 3.2. Fig. 2 illustrates a typical chromatogram obtained from risperidone and its chiral metabolites. As it can be seen, the total analysis was below 7.5 min. Furthermore, analyte ionization in LC–ESI-MS is a crucial step to guarantee the reproducibility and sensitivity of the developed method. Thus, the use of polar organic mobile phases has a great advantage compared to the use of mobile phases containing high amount of water, such as the ones used in reversed phase mode. It is known that although water supports the formation of ions, its surface tension and solvation energy make it more difficult to desolvate the target analytes than organic solvents, such as

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Fig. 3. Extraction efficiency for different types of fibers (n = 3).

methanol or ethanol do [44]. In addition, it can improve the solubility of some analytes and it is easier to remove the solvent from the analytes for preparative proposes [45]. 3.2. SPME optimization To perform the SPME optimization, it was employed 2.0 mL Czapeck liquid culture medium samples spiked with known amounts of risperidone, 7-RispOH and 9-RispOH. The concentration employed was 250 ng mL−1 of each enantiomer metabolite and 500 ng mL−1 of risperidone. The parameters analyzed in the SPME procedure were type of fiber, extraction time, sample pH, salt addition, sample shaking, matrix volume, desorption time and percentage of carryover. All optimization procedures were carried out in triplicate [46]. After optimization of these parameters it was performed a comparative study by evaluating the recovery of the analytes from Czapeck liquid culture medium and a pool of Cunninghamella fungi in the SPME process. The results were analyzed employing Student’s t-test with significant level set at p ≤ 0.05. During the step of choosing the type of fiber, it is of great importance to know the polarity of the analyte. This is true, once one have to choose a fiber with similar polarity of the target analytes. Additionally, the polymer stability must be known, since it must be able to resist to several chemical conditions (pH, salt and additives) [47]. Three commercial fibers were evaluated: (1) polydimethylsiloxane/divinylbenzene (PDMS-DVB 60 ␮m), (2) carbowax templated resin (CW-TPR 50 ␮m) and (3) octadecilsiloxane (C18 45 ␮m). PDMS-DVB presents an intermediate polarity and it can be used in a pH range from 2.0 to 11.0. CW-TPR is a more polar fiber. The C18 fiber is the newly developed fiber probe that contains C18 silica particles embedded in a proprietary, non-swelling, biocompatible polymer [48]. The benefit of this design enables minimized binding of macromolecules such as proteins and phospholipids, but allows extraction of most small analytes of interest. This fiber can be used in the pH range of 1.0–9.0, but prolonged exposures in pH above 7.5 could slowly damage the silica [46]. The extraction obtained using the C18 fiber was higher than the extraction obtained using PDMS-DVB and CW-TPR fibers for all analytes (Fig. 3). The extraction with C18 fiber occurs by sorption process of the analytes in/on the fiber [49]. Therefore, C18 was chosen for further experiments. This format of fiber developed by Supelco (LC probes) allows the extraction of several samples at the same time without the use of any holder. Therefore, by using a Vibrax agitator and vials with septum it is possible to perform up to 36 extractions at the same time. The addition of an electrolyte, such as sodium chloride, sodium carbonate and ammonium sulfate, can improve the recovery of the

analyte by the “salting out effect” [47]. By increasing the amount of electrolyte in the sample solution, the solubility of the organic analytes is decreased and the extraction efficiency is increased. The salt employed in this study was NaCl in the concentration range of 0–30% (w/v). Fig. 4a shows the results obtained from the evaluation of this parameter. The increase of NaCl in the medium does not favor the extraction of the metabolites in a meaningful way, but it can improve the risperidone extraction. Thus, for the subsequent studies, it was employed 20% NaCl (w/v). The pH should be controlled because it contributes to an improvement in the analytes extraction. The pH can affect the stability of the polymer fibers and, therefore, this point is relevant to each type of fiber. After verifying that the addition of electrolyte increased by 20% the extraction efficiency, it was performed the experiments regarding the pH effects, where solutions were prepared in 0.25 mol L−1 phosphate buffer in the pH range of 3.0–9.0. The results are shown in Fig. 4b. It was observed no significant variations in the analytes extractions at pH above 7.0. The further optimization steps were performed employing 0.25 mol L−1 phosphate buffer pH 7.0. The sample agitation during the extraction process was also evaluated. It is known that the agitation is able to increase the analyte mass transfer from the sample to the coating, with consequent reduction in the extraction time. The agitation was evaluated using a Vibrax VXR agitator varying the agitation speed from 300 to 1200 rpm. As it can be seen in Fig. 4c, from 600 rpm to up, the extraction efficiency keeps the same for both metabolites. However, for risperidone maximum extraction efficiency was observable at 1200 rpm. Since high values of speed can lead to bias and the risperidone will be present in high amount in the medium, it was chosen to perform the next experiments employing 600 rpm as stirring speed. SPME technique is based on the equilibrium of analytes that occurs between the extraction phase and sample. At the equilibrium moment, the balance of transport is reached, the concentration of the analytes become constant over time, and it is assumed that the maximum amount of analyte was extracted. To determine the extraction time profile of the analytes, the extractions were done during 15, 30, 45, 60 and 90 min. The results are shown in Fig. 4d. The optimum extraction time was accomplished in 30 min for the metabolites and 90 min for risperidone. As described before, the concentration of risperidone will be high in the culture medium, thus, not requiring a high recovery value. It is known that the extraction can be performed without necessarily reaching the equilibrium, since the experimental conditions are kept constant [50]. Therefore, it was chosen 30 min as extraction time for further experiments. Desorption time is the period that the analytes take to be released from the fiber to the vial containing desorption solvent for further analysis. Four desorption times were assessed: 1, 3, 5 and 10 min. It was observed that from 5 min to up the desorption becomes constant and therefore, 5 min was set for the experiments (data not shown). Different types of desorption solvents were not evaluated, since the mobile phase was efficient at desorbing the analytes from the fiber. This fact could be attributed to the high amount of polar organic solvent present in the mobile phase which favors the desorption process [46]. In addition, it is possible to inject the desorbed fluid directly in the chiral chromatography column without losing the resolution between the enantiomers. The carryover in SPME refers to the amounts of the analytes that were not completely desorbed in the desorption process, thereby causing significant changes in the analytes concentrations in the following analysis, once the fiber is reused. This is verified, especially when the analysis of a more concentrated sample is made prior to a more diluted sample [12]. Therefore, sometimes, a washing step is necessary to guarantee a minimum amount of analytes

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Fig. 4. Microextraction optimization (a–f). Desorption solvent: mobile phase; desorption time: 5 min. Extraction temperature: 25 ◦ C (±2 ◦ C). (a) Salt addition; (b) effect of pH; (c) agitation; (d) extraction time; (e) percentage of carryover; (f) sample volume (n = 3). Risperidone ( ); (+)-9-RispOH (); (−)-9-RispOH (); 7-RispOH E1 ();7-RispOH E2 ().

in/on the fiber after the desorption process. Initially, the carryover was evaluated and it presented values higher than 15% (data not showed). Based on that, a washing step procedure was evaluated by washing the SPME fiber for 30 min with methanol after the desorption process. The carryover was studied again employing the same time of the desorption procedure. After each desorption time, the fibers were washed for 30 min with methanol, desorbed in the mobile phase and analyzed. It was observed that after 5 min of desorption the carryover was absent (Fig. 4e). Therefore, to avoid carryover between different extractions, one washing step with 100% of methanol for 30 min was performed after each desorption process.

The total volume of the extraction medium was set at 4 mL by employing different proportions of the liquid culture medium and the phosphate buffer. The volume of the sample matrix was evaluated by varying the Czapeck medium volume from 0.5 to 3 mL. It was noticed that there was no significant difference among the evaluated volumes (Fig. 4f). Based on these results, to guarantee a relative high amount of the analyte mass and a minimum sample matrix influence in SPME procedure, 2 mL of Czapeck medium was chosen for further analyses. The SPME optimization was carried out in Czapeck liquid culture medium, but in the meanwhile, it is known that in the biotransformation study the composition of the liquid culture medium may

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Fig. 5. Effect of the extraction recovery of the analytes from the Czapeck liquid culture medium and from the Fungi pool by SPME. Analyte concentrations: risperidone 500 ng mL−1 , metabolites 187 ng mL−1 . Level of significance set at p ≤ 0.05 (n = 6).

vary due to the formation of secondary metabolites of the fungi. These changes may affect the SPME procedure since both matrices may present different viscosity. Based on that, the assessment of the recovery of the analytes in Czapeck liquid culture medium and in a pool of fungi was performed. The results showed no statistical significant differences between the Czapeck liquid medium and the fungi pool, with p value higher than 0.532, 0.125 and 0.442 for risperidone, 7-RispOH and 9-RispOH, respectively (Fig. 5). The absence of difference may be attributed to the sample dilution with 0.25 mol L−1 phosphate buffer and the high ionic strength (20% NaCl, w/v) of the sample. These factors keep both matrices composition constant and decrease the differences between them and, therefore, the SPME procedure could be minimally affected. After the whole optimization process, the final condition for the extraction of risperidone and its metabolites by SPME was employing a C18 45 ␮m fiber. To 2 mL of Czapeck liquid culture medium it was added 20% NaCl (w/v) and the pH was controlled by using 0.25 mol L−1 phosphate buffer pH 7.0. The speed of the agitator and the extraction time was 600 rpm and 30 min, respectively. The fiber desorption was performed in the mobile phase during 5 min. After each desorption step the fiber was washed for 30 min with methanol. 3.3. Method validation The method validation was performed by internal padronization employing the drug ranolanize as IS (Fig. 1C). As it can be seen, this drug presents a chemical structure different from the analytes analyzed in the present work. However, its chemical properties, such as log p (2.7) and pKa (7.2) are similar to risperidone (log p 2.7 and pKa 7.8) and paliperidone (log p 2.2 and pKa 7.8) [31,51], next, these analytes are supposed to behave similarly. Consequently, as the physical-chemical properties of the analytes are similar and the extraction is performed at equilibrium conditions for most analytes, the bias related to the differences in matrix composition or to low concentrations is minimized. In addition, during the validation step the IS presented the same behavior that the analytes in the SPME procedure, thereby confirming its applicability in this method. However, it should be addressed that the best IS to be used in a method development employing SPME-LC–MS is the deuterated forms of the target analytes [46]. It was not used in this work because they were not available. The method showed to be linear over the concentration range of 50–3750 ng mL−1 for risperidone and 25–500 ng mL−1 for each 7RispOH and 9-RispOH enantiomers with r > 0.98 and relative error for each point below 15% for both analytical curves (one prepared in Czapeck liquid culture medium and another prepared using a pool of fungi) (Table 2). Since both analytical curves presented very similar slope values, it was attributed that both matrices influence

Fig. 6. (A) Representative chromatogram of Czapek liquid medium (control) incubated with the Cunninghamella echinulata var. elegans (ATCC 8688A) showing that this fungus did not produce any secondary metabolites in the retention time of the analytes; (B) representative chromatogram MRM of 3 channels ES+ of the fungus Cunninghamella echinulata var. elegans (ATCC 8688A) after 168 h of incubation with risperidone internal standard (1); (+)-9-RispOH (2) and risperidone (3). LC–MS/MS and extraction conditions described in Sections 3.1 and 3.2, respectively.

the SPME procedure at the same way [41]. In addition, to guarantee that there is no matrix influence in the SPME procedure, a pool of fungi was spiked with the target analytes at three different concentrations and these samples were quantified using the analytical curves prepared in the Czapeck medium. As it can be seen in Table 3, the RSD and the relative error were below 15%, therefore in agreement with the literature guidelines. Based on these results, the method was validated using the Czapeck liquid culture medium. This medium was chosen once it is very easy to prepare and it does not produce any microbiological residues. SPME recoveries were close to 28% for risperidone, 16% for 9-RispOH and 11% for 7-RispOH with RSD lower than 12% for all analytes. The limit of quantification was 25 ng mL−1 for each metabolite enantiomer and 50 ng mL−1 for risperidone with relative error and RSD below 10%. The results for within-day and between-day precision and accuracy for each metabolite enantiomer and for risperidone are summarized in Tables 4 and 5, respectively. These results show that the proposed method is precise and accurate within the desired range. The freeze-thaw cycles and short-term room temperature stability tests showed no statistically significant degradation with p-values >0.05 (Table 6). Employing liquid culture medium as sample matrix or the fungi pool and SPME as sample preparation, the evaluation of matrix effect in the analytes ionization showed that the sample matrix did not interfere significantly in the ionization of the analytes, since the difference between the groups were less than 10% (Table 7). The elution order was established for the 9-RispOH enantiomers. It was performed compared to the elution order described by Danel et al. [37]. In this previous study the authors showed that the first enantiomer to elute shows the configuration (+)-9-RispOH and the second (−)-9-RispOH. In the present work, employing the Chiralcel OJ-H column in polar organic mode, the first enantiomer presented the same configuration. In addition, a previous

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87

Table 2 Linearity of the method for analysis of the analytes in Czapeck liquid culture medium and in a Fungi pool. Analytes

Type of matrix

Range (ng mL−1 )

Linear equationa

rb

Risperidone Risperidone (+)-9-RispOH (+)-9-RispOH (−)-9-RispOH (−)-9-RispOH 7-RispOH (E1) 7-RispOH (E1) 7-RispOH (E2) 7-RispOH (E2)

Fungi pool Czapeck medium Fungi pool Czapeck medium Fungi pool Czapeck medium Fungi pool Czapeck medium Fungi pool Czapeck medium

50–3750 50–3750 25–500 25–500 25–500 25–500 25–500 25–500 25–500 25–500

y = 0.01980x + 0.2501 y = 0.01939x + 0.2588 y = 0.01217x − 0.1189 y = 0.01184x − 0.0923 y = 0.01208x − 0.1165 y = 0.01186x − 0.0984 y = 0.01077x + 0.1021 y = 0.01063x + 0.1242 y = 0.01054x + 0.1067 y = 0.01060x + 0.1227

0.998 0.999 0.996 0.994 0.995 0.994 0.989 0.991 0.987 0.991

a b

Ratio of slopes 1.02 1.03 1.02 1.01 0.994

Five replicates (n = 5) for each concentration. r, Coeficient of correlation.

Table 3 Precision and accuracy of Fungi pool samples spiked with known concentrations of the analytes and quantified using an analytical curve prepared in Czapek liquid culture medium. Analytes

Nominal concentrations (ng mL−1 )

Obtained concentrations (ng mL−1 )

Accuracy REa (%)

Precision RSD (%)

7-RispOH (E1) 7-RispOH (E2) (+)-9-RispOH (−)-9-RispOH Risperidone

45/187/500 45/187/500 37/187/500 37/187/500 75/500/2000

46/175/445 46/173/446 33/189/474 33/191/474 70/512/2136

+2/−6/−11 +2/−7/−11 −11/+1/−5 −11/+2/−5 −7/+2/+7

6/3/2 6/5/2 2/2/2 2/2/2 5/3/3

a

RE, relative error expressed as a percentage (%).

Table 4 Within-daya precision and accuracy of the developed method. Analytes

Nominal concentrations (ng mL−1 )

Obtained concentrations (ng mL−1 )

Accuracy REa (%)

Precision RSD (%)

7-RispOH (E1) 7-RispOH (E2) (+)-9-RispOH (−)-9-RispOH Risperidone

50/200/400 50/200/400 50/200/400 50/200/400 100/1000/2000

49/195/407 49/190/420 49/217/373 48/217/366 99/1067/1888

−2/−2.5/+1.8 −2/−5/+5 −2/+8.5/−6.7 −4/+8.5/−8.5 −1/+6.7/−5.6

5/11/8 11/8/5 11/5/7 7/7/10 7/3/9

a

RE, relative error expressed as a percentage (%).

Table 5 Between-day precision and accuracy of the developed method. Analytes

Nominal concentrations (ng mL−1 )

Obtained concentrations (ng mL−1 )

Accuracy REa (%)

Precision RSD (%)

7-RispOH (E1) 7-RispOH (E2) (+)-9-RispOH (−)-9-RispOH Risperidone

50/200/400 50/200/400 50/200/400 50/200/400 100/1000/2000

50/202/395 50/197/404 49/218/370 49/218/368 100/1065/1875

0/+1/−1.2 0/−1.5/+1 −2/+9/−7.5 −2/+9/−8 0/+6.5/−6.2

7/9/9 8/10/8 9/9/8 7/8/11 8/5/8

a

RE, relative error expressed as a percentage (%).

Table 6 Stability test (n = 6) of the developed method. Analytes

Nominal concentration (ng mL−1 )

Freeze-thaw cycles p-Value

Short-term room temperature p-Value

7-RispOH (E1) 7-RispOH (E2) (+)-9-RispOH (−)-9-RispOH Risperidone

50/400 50/400 50/400 50/400 100/800

0.720/0.681 0.615/0.667 0.255/0.781 0.197/0.686 0.150/0.999

0.055/0.079 0.076/0.090 0.238/0.469 0.303/0.519 0.433/0.058

Level of significance set at p ≤ 0.05. Table 7 Study of matrix effects of Czapeck medium and of a Fungi pool in the ionization of the analytes. Analytes

Nominal concentrations (ng mL−1 )

Czapek matrix effect (%)

RSDa (%)

Fungi pool matrix effect (%)

RSDb (%)

7-RispOH (E1) 7-RispOH (E2) (+)-9-RispOH (−)-9-RispOH Risperidone Ranolazine

50/200/400 50/200/400 50/200/400 50/200/400 100/1000/2000 750

−2/−5/−4 −6/−7/−8 −9/−3/+1 −8/−4/−6 −2/−1/0 −4

1/1/1 1/1/1 4/1/1 4/1/5 1/1/4 2

−5/−8/+1 −3/−7/+2 −6/−9/−1 −10/−10/−1 −6/−4/−7 −10

5/1/5 4/2/5 2/5/1 3/6/1 11/8/1 2

a b

Standard deviation from Czapeck matrix medium. Standard deviation from Fungi pool matrix medium.

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Table 8 Biotransformation study employing the Cunninghamella fungi. Hours

96 120 144 168 192 216

Cunninghamella echinulata

Cunninghamella elegans

(+)-9-RispOH (ng mL−1 )

ee (%)

(+)-9-RispOH (ng mL−1 )

(−)-9-RispOH (ng mL−1 )

147 577 1094 1558 968 1506

100 100 100 100 100 100

ND ND ND 26 38 58

ND ND ND 19a 25 40

ee (%) – – – 15 21 18

ee = enantiomeric excess. ND = values below the LQ. a Value below the LQ but used to stipulate the enantiomeric excess

study performed by our group spiking liquid culture medium with pure enantiomers showed no racemization under biotransformation conditions [31]. 3.4. Method application and enantioselective biotransformation studies After validation, the developed method was employed in a stereoselective fungal biotransformation study previously described in Section 2.7. The biotransformation reactions of risperidone using fungi were monitored for up to 216 h. The biotransformation reaction of risperidone involves aliphatic hydroxylation of C7 to yield the 7-RispOH metabolite or the same reaction of C9 to yield the 9-RispOH metabolite. In previous studies, our research group has been working with enantioselective fungal biotransformation mainly employing endophytic fungi in the biotransformations [29–32,34]. Based on the success previously observed, it was evaluated some standard yeasts from Cunninghamella genus obtained from American Type Culture Collection – ATCC® . The

biotransformation study showed that the filamentous fungi Cunninghamella echinulata var. elegans ATCC 8688A and Cunninghamella elegans: NRRL 1393 ATCC 10028B were able to biotransform risperidone into its chiral active metabolite 9-RispOH. The formation of the enantiomers of 7-RispOH was not observed. Interestingly enough, the Cunninghamella echinulata fungus was able to stereoselectively biotransform risperidone into the (+)-9RispOH resulting in 100% of enantiomeric excess (Fig. 6). It was observed that the formation of this enantiomer started to appear from 96 h to up. The Cunninghamella elegans fungus was also able to stereoselectively biotransform both 9-RispOH enantiomers, but the (+)-9-RispOH was formed with greater intensity than (−)-9-RispOH (Fig. 7). Table 8 shows the enantiomeric excess for each Cunninghamella genus and the concentration obtained in each period. The results obtained show that Cunninghamella echinulata var. elegans ATCC 8688A fungus can be used to produce the (+)-9-RispOH, the chiral active metabolite of risperidone, in its enantiomeric pure form. It is worth noting how interesting this result is. From the biotechnology standpoint, this strategy of (+)-paliperidone production is very promising and it can be used on industrial scale 4. Conclusion The present work describes for the first time the use of SPME coupled to LC–MS/MS employing the polar organic mode in a stereoselective fungal biotransformation study. The advantages of the developed method over the described methods [35–39] are: fast analysis, low consumption of organic solvents, simplicity, reduction in manual operation and high selectivity. The SPME showed to be a suitable sample preparation technique to be used in biotransformation studies, since the procedure was fast and solventless. By using the C18 LC probe fibers and the Vibrax agitator it was possible to perform up to 36 extractions at the same time. Acknowledgments The authors are grateful to Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Coordenac¸ão de Aperfeic¸oamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for financial support and for granting research fellowships. The authors also would like to thanks Prof. Pierina S. Bonato and Dr. Valquíria A.P. Jabor from FCFRP-USP by their technical support. References

Fig. 7. (A) Representative chromatogram of Czapek liquid medium (control) incubated with the Cunninghamella elegans (ATCC 10028B) showing that this fungus did not produce any secondary metabolites in the retention time of the analytes. (B) Representative chromatogram MRM of 3 channels ES+ of the fungus Cunninghamella elegans (ATCC 10028B) after 168 h of incubation with risperidone; internal standard (1); (+)-9-RispOH (2), risperidone (3) and (−)-9-RispOH (4). LC–MS/MS and extraction conditions described in Sections 3.1 and 3.2, respectively.

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