Determination of Pinaverium Bromide in Human ...

Journal of Chromatographic Science Advance Access published April 9, 2015 Journal of Chromatographic Science 2015;1– 6 doi:10.1093/chromsci/bmv027

Article

Determination of Pinaverium Bromide in Human Plasma by a Sensitive and Robust UPLC –MS-MS Method and Application to a Pharmacokinetic Study in Mexican Subjects Omar Patinõ-Rodrı´ guez1,3, Juan Ramoń Zapata-Morales1,4,, Abraham Escobedo-Moratilla1,5 , Manuel Dı´ az de Leoń-Cabrero1, Irma Torres-Roque1 and Jose´ Pe´rez-Urizar2* 1

Dixpertia, Investigacioń Biofarmaceútica y Farmacolo´gica S.C., San Luis Potosı´ , SLP, Me´xico, and 2Laboratorio de Farmacologı´ a y Fisiologı´ a, Facultad de Ciencias Quı´ micas, UASLP, San Luis Potosı´ , SLP, Me´xico *Author to whom correspondence should be addressed. Email: [email protected] 3

Present address: Laboratorio de Farmacologı´ a y Fisiologı´ a, Facultad de Ciencias Quı´ micas, UASLP, San Luis Potosı´ , SLP, Me´xico.

4

Present address: Departamento de Farmacia, Divisioń de Ciencias Naturales y Exactas, Universidad de Guanajuato, Guanajuato, Me´xico.

5

Present address: Instituto Potosino de Investigacioń Cientı´ fica y Tecnolo´gica A.C., San Luis Potosı´ , SLP, Me´xico.

Received 19 June 2014; revised 29 December 2014

A high-throughput ultra-performance liquid chromatography coupled to tandem mass spectrometry (LC–ESI–MS-MS) method was developed for the determination of pinaverium bromide in human plasma. Protein precipitation with acetonitrile was used to extract pinaverium and itraconazole (as internal standard) from 500 mL plasma samples. The chromatographic separation was achieved with an Acquity UPLC BEH C18 column (1.7 mm, 2.1 3 100 mm) using a mixture of acetonitrile–5 mM ammonium formate (80:20, v/v) as mobile phase. Isocratic elution at 0.3 mL/min was used. Detection was performed by positive ion electrospray tandem mass spectrometry on a XEVO TQ-S by multiple reaction monitoring mode. The mass transitions monitorized were as follows: m/z 511.2 ! 230 for pinaverium bromide, and m/z 705.29 ! 392.18 for the itraconazole. The method was validated over a concentration range of 12–12,000 pg/mL. The chromatographic method runtime is 2.5 min and was applied to characterize the pharmacokinetics of pinaverium bromide after the oral administration of 100 mg to healthy Mexican subjects.

Introduction Irritable bowel syndrome is a frequent disease in Mexican population (1) and the antispasmodics agents such as pinaverium bromide (PNV), mebeverine, otilonium, hyoscine and trimebutine (2) are among the most prescribed drug treatments in Mexico. PNV is a quaternary ammonium compound, which relaxes gastrointestinal structures primarily by inhibiting Ca2þ influx through potential-dependent channels in surface membranes of smooth muscle cells (3). PNV is effective in the treatment of other abnormal intestinal conditions including abdominal pain, diarrhea, constipation (4), higher antispasmodic activity, and to increase patients’ tolerability to otilonium, prifinium bromide and other antispasmodic agents (4). To date, little information of PNV pharmacokinetics has been reported, probably due to its low absorption through oral administration (5, 6). Since only a small amount of PNV can be founded in patient’s plasma, due its low bioavailability a linear, sensitive, selective analytical method for PNV in human plasma is a challenge. Few methods for PNV quantitation in plasma have been reported (6, 7). One of these methods is a multistep extraction followed by gas chromatography (GC) analysis coupled to mass spectrometry (MS) detection

system (7); another method reported for the determination of PNV has fewer extraction steps and is followed by highperformance liquid chromatography analysis (HPLC) coupled to mass tandem spectrometry (MS/MS) (6). The HPLC – MS-MS method showed to be quicker and more accurate than the GC – MS method (6); nevertheless, the sample preparation for the HPLC method is based on a protein precipitation process, which can lead to a shortened useful life column and consequently increase the cost of the analyses. On the other hand, ultra-performance liquid chromatography (UPLC) coupled to MS– MS is a more sensitive, faster and more accurate technique than HPLC– MS-MS; this is due to the higher operating pressure and the smaller particle size in the stationary phase, leading to a higher resolution in chromatographic analysis (8, 9). The purpose of this study is to develop and validate an UPLC – MS-MS method with a simple sample preparation for the determination of PNV in human plasma. The data attained from analyzed samples were used to sustain a pharmacokinetic study of the oral administration of PNV in healthy Mexican subjects.

Experimental Analytical standards of PNV ( purity of 99.1%) and itraconazole (IS) ( purity of 22.7%) were donated by a pharmaceutical company (RIMSA, Guadalajara, Mexico). Commercial formulations PNV in 100 mg capsules (Dicetelw; Abbott Laboratories de Me´xico, S. A. de C. V.) were used for the pharmacokinetic study. Acetonitrile LC – MS grade (EMD-Chemicals, NY, USA) and Ammonium formate HPLC grade (Fluka, VA, USA) were used. All working solutions were prepared with deionized water. Chromatographic analysis was performed on a Waters XEVO TQ-S (MA, USA), an UPLC– MS-MS commercial system consisting of an Acquity UPLC coupled to tandem mass spectrometry detection system. The XEVO TQ-S system was equipped with an Waters Acquity UPLC BEH C18 column (1.7 mm, 2.1 100 mm; MA, USA). The mobile phase consisted of acetonitrile – 5 mM ammonium formate buffer solution (80:20, v/v) at a flow rate of 0.3 mL/min. The run time was 2.5 min and the sample volume injected was 1.0 mL. The column temperature was set

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to 408C. The autosampler cooler was set to 88C. The tandem mass spectrometer was set in multiple reaction monitoring (MRM) mode and in ESI positive ionization mode. Collision energy and cone voltage were 18 and 15 V, respectively. Cone and desolvation gas flow rates were set to 150 and 200 L/h, respectively, using Argon as collision gas at a flow rate of 0.17 mL/ min. Mass spectrometry tandem was used based on the conditions previously reported by Ren et al. (6). The system was tuned to monitoring 511.2 ! 230 m/z transition for PNV and m/z 705.29 ! m/z 392.18 transition for the IS, with a dwell time of 0.3 s. MRM data were acquired and analyzed through Waters MassLynx software (MA, USA). A stock solution (1.0 mg/mL) of PNV was prepared by dissolving it in methanol, additional working dilutions of 100 and 1,000 ng/mL were prepared from the 1.0 mg/mL stock. An IS stock solution was prepared (1.0 mg/mL) in methanol and added to PNV samples. The concentration of calibration standards and quality control samples were chosen based on the expected concentration in human samples according to the literature (6), with an increase in the upper limit of the range to cover possible concentration variability as there are no reports of bioavailability of PNV in Mexican subjects. The PNV calibration curve (12, 720, 3,000, 4,800, 7,800, 9,600 and 12,000 pg/mL) was prepared by serial dilution of the stock solution. Quality control (QC) samples were prepared in the same way for PNV concentrations of 1,440 (low, QCL), 6,000 (medium, QCM) and 10,800 pg/mL (high, QCH). The calibration curve and quality control concentrations were proposed according to Mexican regulations (NOM-177-SSA1). All standard stock solutions were prepared once a month and stored at 2208C. Frozen 0.5 mL plasma samples were thawed at room temperature, then 10 mL of calibration curve or quality control solutions each and 10 mL of IS solution were added to each sample. To promote protein precipitation, 1.0 mL of acetonitrile was added to each sample, and the mixture was vortexed for 0.5 min and centrifuged for 7 min at 14,000g on a bench top centrifuge Eppendorf 5418 (Hamburg, Germany). The supernatant was transferred to a fresh microtube and frozen for 5 min at 2808C. Latter, a new centrifugation cycle was performed (5 min at 14,000g) and 150 mL of supernatant was transferred to a glass total recovery autosampler vial. The sample was injected into the LC–MS-MS system for analysis.

Validation procedure The validation was performed according to the current international guidelines (10, 11). The limit of quantitation (LOQ) was determined by injecting decreasing concentrations of PNV into the analytical system to determine the minimal concentration providing a signal-to-noise ratio of .10 with adequate precision and accuracy (LOQ of 80 – 120%). Calibration standards and blanks were prepared and analyzed in duplicate to establish the calibration range with acceptable accuracy and precision (calibration standards except LOQ, 85 –115%). The acceptance criteria for the calibration curve were a linearity of r 0.99 and r2 0.98, with at least five calibration points. The analyte-to-IS ratio (response) was calculated for each sample by dividing the area of the PNV peak by the area of the IS peak. Standard curves of PNV were constructed using linear regression analysis by plotting 2 Patinõ-Rodrı´ guez et al.

the response against the PNV (theoretical) concentration in each sample. The accuracy and precision of the assay were determined by analyzing five replicates of PNV samples at the LOQ, QCL, QCM and QCH, and this set was prepared and analyzed five times each in an independent analytical run, with the freshly prepared calibration curves as described above. Back-calculated concentrations of calibration and quality control samples were estimated using the equation generated via linear regression analysis. Accuracy was calculated as the relative difference (% Diff ) between the backcalculated concentration and the theoretical concentration using 1/x weighting equation (12). Precision was calculated as the relative standard deviation (% CV) of the calculated concentrations of each standard solution. Intra- and inter-assay precision were calculated for each set of quality control samples. Recovery The absolute recovery, or extraction efficiency, was determined by comparing in three separate occasions, the peak areas of the three QC samples (QCL, QCM and QCH) first added then processed, against the peak areas from equivalent samples first processed then spiked to the same final concentration of PNV and IS. As the extraction efficiency of PNV and IS was determined simultaneously, the recovery was calculated as the PNV–IS extraction ratio. Determination of matrix effect To investigate whether endogenous matrix constituents interfered with the assay for free and total PNV, samples of 4,800 pg/mL in the ACN – water mixture were prepared. Nonanalyte-added plasma samples were processed and then spiked with PNV and IS to a final concentration equivalent to 4,800 pg/mL. Matrix effect was calculated by dividing the calculated concentration of the post-extraction spiked plasma samples by the calculated concentration of PNV in ACN– water mixture. Carry-over effect To determine the presence of carry-over contamination in the assay, ACN–water mixture injections were performed within the analytical run, specifically after the calibration point of higher concentration was injected. Carry-over was determined by calculating the concentration (if any) of PNV in each ACN–water mixture injection. Stability All stability studies were conducted at three concentration levels (QCL, QCM and QCH) by using duplicate. The stability of PNV in human plasma was determined after three freeze – thaw cycles (274 to 2868C), after storage at 2808C for 2 weeks, over 69 h in the autosampler (88C) and after 4 h refrigeration (2 – 68C). Stability was expressed as the percentage recovery of the assayed solution relative to a freshly processed analyte, added plasma sample (t ¼ 0). Pharmacokinetic study Clinical study design Twenty-five healthy Mexicans volunteers of both gender who were between the ages of 18 and 45 (mean + SEM: 24.71 + 0.03 years), had heights between 140.0 and 190.0 cm (163.0 + 0.005 cm) and weighed between 43.50 and 79.50 kg (62.15 + 1.9 kg) were enrolled in the study. The study protocol

was approved by an independent ethics committee as well as by the regulatory authority in Mexico (COFEPRIS), and it was conducted following the ethical principles described in the Declaration of Helsinki. The study was an open label exploratory bioavailability trial in healthy subjects under fasting conditions. The participants received one dose of 100 mg of PNV, and blood samples were obtained prior to dosing (time 0) and at 0.17, 0.33, 0.5, 0.75, 1, 1.33, 1.67, 2, 2.5, 3, 4, 8, 12 and 24 h after administration. After blood withdrawal, the blood samples were immediately centrifuged at 3,000 rpm for 10 min, and the plasma obtained was stored at 2208C until analysis. Pharmacokinetic data analysis Pharmacokinetic parameters for PNV were calculated using noncompartmental and compartmental models with WinNonlin 6.2.1 software (Pharsight, Mountain View, CA, USA, 2011). From the individual data, it was estimated the pharmacokinetic parameters of PNV. The maximum plasma concentration (Cmax), time to reach Cmax (Tmax), area under the plasma concentration time curve from time zero to the time of the last measurable concentration (AUC02t) and AUC extrapolation to infinity (AUC021) were calculated according to the non-compartmental method. For estimation of the absorption rate constant (Ka), half-life of the absorption process (T1/2 abs) as well as the disposition and elimination parameters: apparent volume of distribution (V/F),

clearance apparent (CL/F), elimination rate constant (Ke) and elimination half-life (T1/2), the best model that described the individual pharmacokinetic data was fitted as an open model of one compartment with first-order absorption without lag-time. Results This study was undertaken to develop a clean, fast, sensitive and selective method to determine PNV in human plasma, designed for subject sample analysis. The data through the chromatographic method reported in this work are intended to determinate pharmacokinetic parameters of healthy Mexican volunteers after an oral dose of PNV. Chromatography Fragmentation of PNV (Figure 1A) and IS (Figure 1B) is shown in Figure 1. Representative chromatograms of PNV processed samples are shown in Figure 2. A chromatogram of a nonanalyte-added plasma sample is presented in Figure 2A. The calibration curve processed plasma samples for lower (12 pg/mL) and higher (12,000 pg/mL) PNV concentration limits are presented in Figure 2B and Figure 2C, respectively; PNV retention time is measured in 2.09 min. A chromatogram of a processed sample containing IS is presented in Figure 2C, and IS presented a retention time of 1.15 min. Chromatograms show little interference for both PNV and IS.

Figure 1. (A) Product ion of pinaverium bromide at 18 V collision energy. (B) Product ion of itraconazole (IS) at 35 V collision energy.

Determination of Pinaverium Bromide in Human Plasma 3

Figure 2. MRM ion-chromatograms of (A) extracted blank plasma (without IS and analyte), (B) pinaverium bromide LOQ and (C) real sample, volunteer number 05, first period (I), sample 05 in the time of 1.0 h (V05-I-05) (m/z 511.2 ! 230) and (D) itraconazole (m/z 705.29 ! 392.18) as used for internal standard (IS).

Validation of the LC –MS-MS assay Calibration curve and LOQ All five calibration curves were linear over the analyzed concentration range of 12 – 12,000 pg/mL. Three validation runs were conducted on separate days and the standard curves obtained for PNV were linear (Table I). The validated method for pinaverium showed a linear behavior in the range of concentrations analyzed (12 – 12,000 pg/mL), y ¼ 0.00003 þ 0.00029x, with a 0.9991 correlation coefficient (r). For the LOQ five samples were analyzed and showed that the detection limit is reproducible (Table II) based on calculated concentrations of 14.23 + 2.5 pg/mL. Signal-to-noise ratio for the LOQ presented and calculated on 21 (Figure 2B). The detection limit was evaluated by injecting duplicate 50:50 serial dilutions of the lowest concentration of the working range to the point where the PNV signal was more than three times the background noise, the detection limit was reached at the 1:8 dilution, equivalent to an average concentration of 1.5 pg/mL PNV. Accuracy and precision The intra- and inter-assay accuracy and precision for the calibration curve samples are reported in Table I and demonstrate that the 4 Patinõ-Rodrı´ guez et al.

Table I Assay Performance of the Pinaverium Bromide Calibration Curve Over the Concentration Range of pg/mL in Blank Human Plasma, Prepared and Analyzed by LC – MS-MS Concentration (pg/mL)

Mean observed concentration (pg/mL) + SD

Accuracy (%)

Precision intra-assay (%) (n ¼ 3)

Precision inter-assay (%) (n ¼ 3)

12 720 3,000 4,800 7,800 9,600 12,000

12 + 0.1 723 + 13 2,960 + 121 4,827 + 245 7,896 + 167 9,448 + 265 12,064 + 182

0.13 0.42 21.32 0.56 1.23 21.58 0.54

2.81 3.32 7.14 8.82 3.68 4.87 2.63

1.37 4.06 4.99 2.40 2.77 6.54 1.61

accuracy and precision was ,10% for each analysis. For the quality control samples, the intra-assay coefficient of variation was ,15%, and accuracy ranged from 20.9 to 4.6% (Table II); these results exhibit a higher accuracy compared against the method reported by Ren et al. (6), which reported an accuracy in the range of 2 and 8%. The quality control sample values showed a better profile of precision in inter-day analysis than the method described by Ren et al. (5.4–12.7% versus 3.7–8.1%). However, the precision of the method reported in this work has an intra-day precision similar to the methods previously reported (6, 7).

Table II Assay Performance LOQ, QCL, QCM and QCH Pinaverium Bromide, Prepared in Blank Human Plasma, Prepared and Analyzed by LC –MS-MS

Table III Stability of Pinaverium Bromide Under Different Conditions (n ¼ 2) Storage conditions

Concentration (pg/mL)

Mean observed concentration (pg/mL) + SD

Accuracy (%)

Precision intra-assay (%) (n ¼ 6)a

Precision inter-assay (%) (n ¼ 6)a

12 (LOQ) 1,440 (QCL) 6,000 (QCM) 10,800 (QCH)

14 + 2.5 1507 + 19 5944 + 25 10,911 + 303

20.02 4.65 20.92 1.03

14.36 7.01 3.31 5.58

8.13 6.94 3.70 5.76

a

Except for the LOQ, n ¼ 5.

Recovery The recovery of the PNV was 163.4, 154.1 and 153.6% for the QCL, QCM and QCH, respectively, with the coefficient of variation ranging from 4.4, 0.5 and 1.1% for the QCL, QCM and QCH, respectively. IS was selected as internal standard due to its high recovery, calculated up to a 100% for both PNV and IS. Moreover by integrating data and obtain the response PNV/IS, with itraconazole was the best linearity results showed the various IS tested. The over 100% values reported for the extraction efficiency are probably due to the matrix effect, where processed plasma samples interact with the analyte by increasing its volatility in the nebulizing process; this effect has been reported previously by other authors (13). Assay specificity, carry-over and matrix effect Selectivity of the method was demonstrated to use of hemolyzed, lipemic plasma and plasma spiked with concomitant drugs, such as ciprofloxacin, paracetamol, difenidol, ranitidine and caffeine. An MS artifact from paracetamol was found in the same transition of PNV; it had a signal of 33.8% related to PNV LOQ. However, the concentration used for the evaluation of selectivity was 200 mg/mL, 10-fold the maximum concentration found in plasma after a standard single dose of 1,000 mg of paracetamol (20 mg/mL) (14). The 200 mg/mL of paracetamol value was chosen, even if it is unrealistic, to test the methods selectivity at edge conditions. A comparison between non-added matrix chromatograms and those obtained after spiking plasma samples with PNV and IS. In this comparison it can be seen that endogenous substances did not interfere with the assay. The matrix effect in samples used for the quantification of PNV to establish that the presence of other components from the biological matrix causes an increase or a decrease of the signal of the analyte was evaluated. In the analysis of this parameter, triplicate samples of a concentration of 4,800 pg/mL in plasma and triplicate samples of the same concentration in the ACN – water mixture were used. An enhancement of signal exists; however, no matrix effect is observed, because the coefficient of variation is below 15% when the areas are compared with and without biological matrix. Finally, the ACN –water mixture-based samples included within the analytical runs did not show noticeable peak areas, indicating that there was no significant carry-over effect. Stability studies The solutions used for the validation of the analytical method for the determination of plasma PNV proved to be stable during a 7-day period, meeting the acceptance criteria for the parameters of the line, slope (m), intercept (b) and correlation coefficient (r). In the identity test of the line to the point of lowest concentration of the calibration curve (CC1), coefficient of variation (CV %) was

Nominal concentration (pg/mL)

Calculated concentration (pg/mL) Mean, stability samples + SD

Sample processed stability (autosampler); 69 h, 48C HQC 10,800 10,808 + 394 LQC 1440 1,437 + 24 Refrigeration matrix-spiked stability; 4 h, 48C HQC 10,800 10,445 + 444 LQC 1,440 1,403 + 49 Freeze and thaw stability; three cycles, 2808C HQC 10,800 10,847 + 125 LQC 1,440 1,471 + 52 Long-term matrix stability; 14 days, 2808C HQC 10,800 10,732 + 736 LQC 1,440 1,586 + 29

% Change 0.08 20.17 23.29 22.52 0.44 2.15 20.62 10.71

within +20% and for the remaining points of the curve within +15%. The criterion for precision and accuracy, as the average accuracy was 100 + 15% of the theoretical concentration for all three levels evaluated. Finally, the value of precision measured, as coefficient of variation (%) was ,15% for all levels. In stability studies, two replicates of QCL, QCM and QCH levels were analyzed. The results are presented in Table III. The data demonstrated that PNV samples had adequate stability under refrigeration (2 –68C) for 4 h. Similarly, it is determined that PNV is stable in three freezing –thawing cycles, additionally in the autosampler at 88C for 69 h. The long-term stability of PNV stored in a temperature range between 274 and 2868C was evaluated for 2 weeks resulted be stable this storage time. Pharmacokinetic parameters Because the analytical method is intended to determine the pharmacokinetic parameters of PNV on healthy Mexican subjects, the parameters estimated are presented. The mean values for Cmax, Tmax and AUC0 – t obtained of this study are as follows: Cmax ¼ 3609.98 + 2382.19 pg/mL, Tmax ¼ 0.62 + 0.59 h, T12 ¼ 1.51 + 0.84 h and AUC0 – t ¼ 11,707.82 + 17,587.52 pg h/mL. Discussion An accurate and precise ultra-performance liquid chromatography tandem mass spectrometry (LC –MS-MS) method was developed for determination of PNV in human plasma. The analysis was performed on a triple-quadrupole tandem mass spectrometer by using MRM mode via electrospray ionization, after the stable fragments were detected (Figure 1). An optimization mobile phase was performed based on asymmetric factor and peak area obtained. Although different mobile phases were tried, satisfactory separation and well-resolved and good symmetrical peaks were obtained with the mobile phase acetonitrile –5 mM ammonium formate buffer solution (80:20, v/v). Different internal standards were tested, but only IS (itraconazole) showed a similar analyte recovery as PNV. Additionally, IS had the minimal deviation and the best linearity PNV/IS response. Intra-assay variation describes the variation of results within a data set obtained from one experiment. In our experiments, the intra-assay calibration curve in triplicate (Table II) effectiveness was evaluated, to monitor the deviation within the same assay, and the average intraassay precision was 4.7%. The same case occurs with inter-assay variation, which describes the variation of results within a data set obtained from different days or analysts; in our work Determination of Pinaverium Bromide in Human Plasma 5

to the UPLC system. The efficiency of the protein precipitation method for extraction and a chromatographic run time of 2.5 min per sample make this method of analysis an attractive procedure in high-throughput bioanalysis of PNV. Acknowledgments The authors thank to RIMSA, Representaciones e Investigaciones Me´dicas, S. A. de C. V. (Guadalajara, Mexico) for the funding for this study. The authors appreciate the technical assistance provided to Maricela Martı´ nez-Delgado and Israel Luna-Zavala. References

Figure 3. The mean plasma concentration – time profile of pinaverium bromide after oral administration of (100 mg pinaverium bromide capsules) formulation to 25 healthy volunteers under fasting conditions.

the inter-assay was evaluated with the totality of data of calibration curve during all validation, and the average inter-assay precision was 3.3%. This result shows a reproducible analytical method. Moreover, because of the use of UPLC technique the total analysis time (extraction and chromatography) is the quickest for the determination of PNV compared with the methods reported previously (6, 7). In addition, the column loading of PNV at LOQ was only 12 pg/mL per sample injection volume (1 mL), which is a low concentration that may extend column usage and efficiency, allowing the analysis of a greater number of injections. The method presented in this work showed an improved performance and a reduced consumption of analysis reagents. In this application, significant reduction in the time of analysis was achieved by using UPLC when compared with an HPLC analysis method (2.5 min run time and a flow rate of 0.2 mL/min versus 3.0 min run time and a flow rate of 0.5 mL/min) (6), allowing full utilization of the rapid scanning speeds made possible by the triple-quadrupole MS. This method of analysis was successfully validated and was used as an essential tool to determine the pharmacokinetic parameters of PNV after the administration a single 100 mg oral dose of PNV. Figure 3 shows the plasma concentration of PNV versus time profile human subjects under fasting conditions. The method was sensitive enough to monitoring the PNV plasma concentration up to 20 h. In all 800 samples including the calibration, QC and volunteer samples were run and analyzed for a period of 3 days and the precision and accuracy were found to be well within the acceptable limits. Conclusion The proposed validated method for the estimation of PNV in human plasma is accurate, precise and rapid compared with published reports. The method offers significant advantages over those previously reported in terms of sample amount requirements, fast extraction procedure against lower interferences in the chromatographic analysis and overall time of analysis due 6 Patinõ-Rodrı´ guez et al.

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