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Cyproheptadine and amitriptyline were extracted from plasma by liquid-liquid extraction using a diethyl-ether / dichloromethane solvent. The extracts were analyzed by high performance liquid chromatography coupled to electrospray tandem mass spectrometry (LC-MS / MS) the method had a linear calibration curve ranging from 0.05 to 10 ng / mL (r2 >0.99) the bioequivalence of
Cyproheptadine and amitriptyline were extracted from plasma by liquid-liquid extraction using a diethyl-ether / dichloromethane solvent. The extracts were analyzed by high performance liquid chromatography coupled to electrospray tandem mass spectrometry (LC-MS / MS) the method had a linear calibration curve ranging from 0.05 to 10 ng / mL (r2 >0.99) the bioequivalence of
Cyproheptadine and amitriptyline were extracted from plasma by liquid-liquid extraction using a diethyl-ether / dichloromethane solvent. The extracts were analyzed by high performance liquid chromatography coupled to electrospray tandem mass spectrometry (LC-MS / MS) the method had a linear calibration curve ranging from 0.05 to 10 ng / mL (r2 >0.99) the bioequivalence of
chromatography coupled to electrospray tandem mass spectrometry in a bioequivalence study Gustavo Duarte Mendes a,b,c *, Andr Arruda a , Lu Shi Chen b , Jos Cssio de Almeida Magalhes a,c , Khalid M Alkharfy d and Gilberto De Nucci a,d ABSTRACT: A rapid, sensitive and specific method to quantify cyproheptadine in human plasma using amitriptyline as the internal standard (IS) is described. The analyte and the IS were extracted from plasma by liquidliquid extraction using a diethylether/dichloromethane (70/30; v/v) solvent. After removing and drying the organic phase, the extracts were reconstituted with a fixed volume of acetonitrile/water (50/50 v/v) +0.1% of acetic acid. The extracts were analyzed by high performance liquid chromatography coupled to electrospray tandem mass spectrometry (LCMS/MS). Chromatography was performed isocratically using an Alltech Prevail C18 5 m analytical column, (150 mmx 4.6mm I.D.). The method had a chromatographic run time of 4 min and a linear calibration curve ranging from 0.05 to 10 ng/mL (r2 >0.99). The limit of quantification was 0.05 ng/mL. This HPLC/MS/MS procedure was used to assess the bioequivalence of cyproheptadine in two cyproheptadine +cobamamide (4 mg+1 mg) tablet formulations (Cobactin [cyproheptadine +cobamamide] test formulation supplied fromZambon Laboratrios Farmacuticos Ltda. and Cobavital fromSolvay Farma (standard reference formulation)). A single 4 mg+1 mg [cyproheptadine +cobamamide] dose of each formulation was administered to healthy volunteers. The study was conducted using an open, randomized, twoperiod crossover design with a 1week washout interval. Since the 90% CI for Cmax and AUCs ratios were all within the 80125% bioequivalence limit proposed by the US Food and Drug Administration, it was concluded that the cyproheptadine test formulation (Cobactin) is bioequivalent to the Cobavital formulation for both the rate and the extent of absorption of cyproheptadine. Copyright 2011 John Wiley & Sons, Ltd. Keywords: cyproheptadine; healthy volunteer; plasma; pharmacokinetic; LCMS/MS Introduction Cyproheptadine hydrochloride (CAS no. 969335) is an antihista- minic and antiserotonergic agent with inhibitory activities for ltype calcium channels (Gunja et al., 2004; Yamamoto et al., 2006; Fes et al., 2009a, 2009b). It has anticholinergic andsedative effects. It is indicated for perennial allergic and vasomotor rhinitis, allergic conjunctivitis, mild and cold urticaria, and is a proven migraine prophylaxis, an appetite enhancer and, in adrenocorticotropic hormonedependent Cushings syndrome, it normalizes cortisol indexes (Watemberg et al., 1999). The colateral effects of cyproheptadine are drowsiness, coordination disturbances, increased appetite, weight gain, exacerbation of depression, dry mouth and urinary retention (Watemberg et al., 1999). After an oral dose of 8 mg of cyproheptadine, the C max , AUC and T max for total cyproheptadine were 30 ng/mL, 206 ng h/mL and 4h, respectively (Gunja et al., 2004). Cyproheptadine has been determined in human plasma by CGMS (Hasegawa et al., 2006), in human serum by LCMS (Gunja et al., 2004), in animal urine by GLC (Hucker and Hutt, 1983), LCMSMS (Fes et al., 2009a; Fente et al., 2009) and reversedphaseHPLC (Kountourellis and Ebete, 1995), and in pharmaceutical syrup samples by LCMS/MS (Fes et al., 2009b). Here we describe a fast, sensitive and selective method for measuring plasma cyproheptadine using liquid chromatography * Correspondence to: G. D. Mendes, Department of Pharmacology, State University of Campinas, Campinas, Brazil. Email: gugamendes@terra.com.br a Department of Pharmacology, State University of Campinas, Campinas, Brazil b Galeno Research Unit, Campinas, Brazil c Faculty of Odontology, University Camilo Castelo Branco (UNICASTELO), So Paulo, Brazil d Department of Clinical Pharmacy, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia Abbreviations used: ANVISA, Brazilian National Sanitary Surveillance Agency; CAS, Chemical Abstract Service; FDA, Food and Drug Administration. Biomed. Chromatogr. 2012; 26: 129136 Copyright 2011 John Wiley & Sons, Ltd. Short communication Received 15 January 2011, Accepted 19 January 2011 Published online in Wiley Online Library: 23 March 2011 (wileyonlinelibrary.com) DOI 10.1002/bmc.1618 1 2 9 coupled to tandem mass spectrometry (LCMSMS) with positive ion electrospray ionization using amitriptyline as the internal standard(IS, Fig. 1). This methodwas employedina bioequivalence study of cyproheptadine in two cyproheptadine+cobamamide (4 +1 mg) tablet formulations. The bioequivalence study was conducted in 40 healthy volunteers using a singledose, twoway, open, randomized crossover design with 1week washout period between the doses. Experimental Chemicals and reagents Cyproheptadine hydrochloride (lot G, current lot) was provided by USP. Amitriptyline hydrochloride was provided by Honeywell (AMPT/0604014, March/2011). Acetonitrile, methanol, formic acid, glacial acetic acid and ammonium formiate were purchased from J. T. Baker (Phillipsburg, NJ, USA). Diethylether and dichloromethane were supplied by Mallinckrodt (Phillipsburg, NJ, USA). All chemicals and solvent were of analytical reagentgrade except for acetonitrile and methanol, which were HPLC grade. A milliQ Gradient A10 water purification system from Millipore (Bedford, MA, USA) was used. Blank human blood was collected from healthy drugfree volunteers into sodium heparincontaining tubes and plasma was obtained by centrifugation. Plasma was obtained by centrifugation of blood treated with the anticoagulant sodium heparin. Pooled plasma was prepared and stored at approximately 20C until needed. Calibration standards and quality control samples Stock solutions of cyproheptadine and internal standard (amitriptyline) were prepared in methanolwater (50:50 v/v). Calibration curves of cyproheptadine were prepared by spiking blank plasma at concentrations of 0.05, 0.1, 0.2, 0.5, 1, 3, 6 and 10 ng/mL. The analysis was carried out in duplicate for each concentration. The quality control (QC) samples were prepared in blank plasma at concentrations of 0.15, 1.5 and 8 ng/mL (QCA, QCB and QCC, respectively). For each analytical batch, the spiked plasma samples (standards and quality controls) were extracted along with the unknown samples. All lots of blank plasma used in analytical batches during the validation process and volunteer samples quantification (calibration standards and QCs) were previously tested and pooled, fractioned (50 mL flasks) and stored at 20C until use. Liquid chromatography and mass spectrometry conditions An aliquot of each plasma extract was injected into an Alltech Prevail C 18 5 m analytical column (1504.6 mm i.d.) operating at 400C. The compounds were eluted by pumping the mobile phase [acetonitrilewater Figure 1. Fullscan mass spectra in (A) trace and product ion spectra in (B) trace of (1) cyproheptadine and (2) amitriptyline. G. D. Mendes et al. Biomed. Chromatogr. 2012; 26: 129136 Copyright 2011 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/bmc 1 3 0 (80/20; v/v) +0.1%formic acid+5 mM ammoniumformiate] at a flowrate of 1.2 mL/min. Under these conditions, typical retention times were 2.70.3min for cyproheptadine and 2.90.3min for amitriptyline, and backpressure values of approximately 70bar were observed. A split of the column eluant of approximately 1:2.5 was included. The temperature of the autosampler was kept at 12C and the runtime was 4min. The mass spectrometer (API 4000 Sciex/Applied Biosystems, Canada) equipped with an electrospray source using a cross flowcounter electrode run in a positive mode (ES+), was set up in multiple reaction monitoring (MRM), monitoring the transitions 288.30 96.20 and 278.30 91.10 for cyproheptadine and IS, respectively. Figure 1 shows the fullscan spectra (upper trace) and the product ion spectra (lower trace) obtained for cyproheptadine and amitriptyline. In order to optimize all the MS parameters, a standard solution of the analyte and IS was infused into the mass spectrometer. The source block temperaturewas set at 500Candthe turboionspray voltage to 5.5kV. Nitrogen was used as collision gas. For cyproheptadine, the declustering potential, collision energy and collision exit potential were 76V, 35 eV and 8V, respectively. The corresponding values for IS were 71V, 33eV and 8V, respectively. Data were acquired by Analyst software (version 1.4.1, Applied Biosystems, Foster City, CA, USA). Sample preparation Twohundred microliters of sample human plasma were pipetted into glass tubes followed by 50 L of the internal standard solution (50 ng/mL of amitriptyline in methanolwater, 50:50 v/v solution). The samples were vortexmixed for approximately 10 s. Diethyletherdichlorometane (70:30 v/v) was added (4 mL) to all tubes, which were then vortexmixed for 40 s. The samples were centrifuged at 4000 rpm for 4 min at 4C. The tubes were frozen for 15 min at 80C. The upper organic phase was transferred to another set of clean glass tubes and evaporated to dryness under N2 at 40C. The dry residues were dissolved with 0.2 mL of a solution of acetonitrilewater (50:50 v/v) +0.1% of acetic acid, vortex mixed for 10 s to reconstitute the residues and transferred to 96well plates using automatic pipettes with disposable plastic tips. Bioanalytical method validation The method validation assays were carried out according to the United States Food and Drug Administration (FDA) bioanalytical method validation guidance (Food and Drug Administration, 2001) and the Brazilian National Sanitary Surveillance Agency (ANVISARE 899, 2003). Three validation batches were prepared on different days by spiking blank plasma with the working solutions to produce the standard curve points and QCs. The extraction procedure was the same as described above. Each batch was constituted of duplicates of blank (processed without the IS) and zero plasma samples (blank with IS and processed) to ensure the absence of interferences, followed by the standard samples (duplicates of 0.05, 0.1, 0.2, 0.5, 1, 3, 6 and 10 ng/mL of cyproheptadine) and eight replicates of the the lower limit of quantification (LLOQ, 0.05 ng/mL) and QC samples, with concentra- tions equivalent to 0.15 ng/mL (low level), 1.5 ng/mL (medium level) and 8 ng/mL (high level). Calibration curves were generated by using the ratios of the analyte peak area to the IS peak area vs analyte concentration and were fitted to the linear equation ( y =a +bx) by weighted (factor: 1/x 2 ) least squares linearity regression with R >0.98. The limit of detection and the LLOQ were determined, calculated as the concentrations with a signaltonoise ratio of 3 and 10, respectively. Each backcalculated concentration standard should meet the following acceptable criteria: no more than 20% deviation at LLOQ and no more than 15% deviation above LLOQ. Betweenrun and withinrun accuracy and precision were calculated for LLOQ and QCs levels, considering eight measurements of each concentration. The accuracy of the method was shown in relative values (accuracy, %) and calculated based on the difference between the calculated mean and nominal concentrations, whereas precision was evaluated by calculating the within and betweenrun relative standard deviations (RSD%). Specificity. Blank plasma samples of healthy human used for testing the specificity of the method were obtained from six different sources (four normal, hemolyzed and lipemic). Recovery. The absolute extraction recovery of cyproheptadine and IS was assessed by comparing peak area ratios obtained from extracted plasma samples with those from the standard solutions at the same concentration. This procedure was performed using five aliquots from three different sources of normal human plasma at each QC level (0.15, 1.5 and 8 ng/mL). The relative recovery was calculated as peak area ratios obtained from extracted plasma samples with postextracted spiked samples (matrix extracted standard solution) at the same concentration at each QC level. Stability. Quality control samples (0.15, 1.5 and 8; five measurements of each concentration) were subjected to shortterm (8 h) at room temperature and 48 h postprocessing (12C) stability tests. Test samples were analyzed and the found cyproheptadine concentrations were compared with freshly prepared samples. Stock and working solutions stability assays were processed by comparing fresh solutions with previously prepared ones. All stability data were reported as relative errors (RE, %). Employment of the bioanalytical method Volunteers samples were quantified into analytical batches constituted by calibration curve standards (in duplicate) followed by one of each QC sample (low, middle and high) between every other 10 volunteer samples. For each analytical batch, a convenient quantity of pooled blank plasma was thawed to prepare fresh calibration standards and QC samples. Clinical protocol. The study consisted of an open study of 40 healthy volunteers of both sexes, aged between 18 and 53 years old. Screening assessment was performed by means of medical history, general physical examination, electrocardiogram and clinical laboratory tests. After an washout period of at least 2 weeks, the individuals who qualified were confined for two periods of approximately 36 h. Each confinement was intervaled by a period of 1 week. During confinement, a 7 mL blood sample was collected before dosing and 0.25, 0.5, 0.75, 1, 1.33, 1.67, 2, 2.33, 2.67, 3, 3.5, 4, 4.5, 5, 5.5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 24, 48, 72 and 96 h postdosing. A general physical examination, electrocardiogram and clinical laboratory tests were also performed during the poststudy period. Blood samples were centrifuged at approximately 2000g for 4 min at room temperature and the plasma was stored at 20C until assayed for cyproheptadine content. Formulations. The following formulations were employed: Cobactin (test formulation manufactured by Eurofarma Laboratrios Ltda, Brazil and distributed by Zambon Laboratrios Farmacuticos, Brazil; lot no. 56407) and Cobavital (standard reference formulation from Solvay Farma Ltda, lot no. 801374). Pharmacokinetics and statistical analysis. Bioequivalence between the two formulations was assessed by calculating individual test/ reference ratios for the peak of cyproheptadine concentration (C max ), area under curve (AUC) of plasma concentration until the last concentration observed (AUC last ), and the area under curve between the first sample (predosage) and infinite (AUC 0inf ). The C max and the time taken to achieve this concentration (T max ) were obtained directly from the curves. The areas under the cyproheptadine plasma concentration vs time curves from 0 to the last detectable concentration (AUC last ) were calculated by applying the linear trapezoid rule. Extrapolation of these areas to infinity (AUC 0inf ) was done by adding the value C last /k e to the calculated AUC last (where C last =the last detectable concentration). The AUC and C max data for the two formulations were analyzed by ANOVA to establish whether the 90% confidence interval (CI) of the ratios was within the 80125% interval indicating bioequivalence as proposed by FDA and ANVISA. Parametric and nonparametric analyses of lntransformed arithmetic means and Quantification of cyproheptadine in human plasma by LCMSMS Biomed. Chromatogr. 2012; 26: 129136 Copyright 2011 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/bmc 1 3 1 individual T max differences between test and reference formulations were also performed. Results and discussion Method development Analytical methods employed for the quantitative determina- tion of drugs and their metabolites in biological samples must generate reproducible and reliable data in order to permit valid interpretation of the studies they support (Stokvis et al., 2005a; Marzo and Dal Bo, 2006; Rozet et al., 2007; Shah, 2007; Xu et al., 2007). During the validation process and routine methodology employment, the internal standard plays an important role in achieving satisfactory accuracy and precision(Stokvis et al., 2005b). In this procedure, amitriptyline was adopted as the internal standard because its structure, retention action and ionization as well as extraction efficiency are similar to those of cyproheptadine (Fig. 1). The mass transitions to set mass spectrometry detection (MRM) were obtained from full and product ion scans in positive mode by infusion of cyprohepta- dine and amitriptyline working solution. Analyte and IS showed precursor ions ([M+H] + ) at m/z =288.30 and m/z =278.30, respectively. Precursor ions were submitted to collisioninduced Figure 2. MRM chromatogram of a LLOQ (0.05 ng/mL) sample: (A) cyproheptadine channel and (B) I.S. (amitriptyline) channel. MRM chromatograms of blank normal human plasma (C) cyproheptadine and (D) amitriptyline. G. D. Mendes et al. Biomed. Chromatogr. 2012; 26: 129136 Copyright 2011 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/bmc 1 3 2 dissociation (Fig. 2). All settings were similar for analyte and IS due to their analogue structures and ionization behavior. Most abundant product ions were m/z =96.20 (for cyproheptadine) and m/z =91.10 (for amitriptyline). The proposed fragmentation pathways are illustrated in Fig. 1. For liquid chromatography an Alltech Prevail C 18 5 m analytical column (150 mm4.6 mm i.d.) was used, operated at 40C with methanolwater (80:20; v/v) +0.1% mM formic acid + 5 mM ammonium formiate as mobile phase, which produced fast and reproducible separation. Both cyprohepta- dine and IS peaks presented convenient (after void) retention times (2.7 and 2.9 min, respectively). To obtain a clean chromatogram and achieve a sufficient extraction recovery, liquidliquid extraction (LLE) was used and several solvents mixtures and additives were investigated (Xu et al., 2007). The mixture of diethyletherhexane (70:30; v/v) was eventually proved to be the best in terms of the higher extraction recovery and absences of endogenous interference at the retention time of cyproheptadine and amitriptyline chromatogram. Bioanalytical method validation and performance Following development of a bioanalytical LCMS/MS assay and before implementation into bioanalytical assays and routine use, it needs to be validated. Validation is essential to ensure the acquired data are accurate (Rosing et al., 2000). All validation criteria (specificity, linearity, recovery, accuracy and precision) were evaluated to assess the methods performance. In order to check the specificity/selectivity of the method, six plasma lots from different sources (four normal plasma, lipemic and hemolized) were tested. The simplest regression method for the calibration curves of the cyproheptadine was y =a +bx from 0.05 to 10 ng/mL (calibration curve y =0.0725x +0.000515, r =0.9978). The recoveries of cyproheptadine were 93.3, 83.2 and 93.1% for the low, medium and high pools, respectively. The recovery of the IS tested at the concentration used in the study samples was 83.0%. No significant matrix effect was observed. The validated LLOQ was 0.05 ng/mL, defined as the lowest concentration at which both the precision and accuracy were 20%. Within and betweenrun precision and accuracy for the LLOQ and QCs are summarized in Table 1. Stability tests performed indicated no significant degradation under the conditions described (Tables 25). As shown in Fig. 2, no endogenous peak was observed in the mass chromatogram of blank plasma. The chromatogram for the standard LLOQ sample is shown in Fig. 2, in which the retention times for cyproheptadine and IS were 2.7 (0.3) and Table 1. Accuracy and precision data for cyproheptadine from the prestudy validation in human plasma Nominal concentration (ng/mL) 0.05 0.15 1.5 8.0 Intrabatch, N=7 Mean range 0.049 0.150 1.49 8.7 0.0390.055 0.1390.155 1.441.53 8.39.1 Precision (%) 10.10% 3.65% 2.26% 3.16% Accuracy (%) 97.94% 99.81% 99.33% 108.92% Interbatch, N=21 Mean range 0.048 0.151 1.53 8.8 0.0390.055 0.1240.163 1.341.65 8.29.5 Precision (%) 8.29% 5.91% 4.93% 3.75% Accuracy (%) 96.19% 100.43% 102.07% 110.02% Table 2. Postprocessing stability test (values in ng/mL) Reference values Values after 47 h 50 min Reference values Values after 47 h 50 min Reference values Values after 47 h 50 min Low sample Medium sample High sample Mean 0.147 0.153 1.5 1.55 8.55 8.39 CV (%) 2.2 3.2 2.1 2.2 1.4 1.6 Variation 4.1 3.3 1.9 Table 3. Freezeandthaw stability test (values in ng/mL) Reference values Values after three cycles Reference values Values after three cycles Reference values Values after three cycles Low sample Medium sample High sample Mean 0.146 0.136 1.47 1.41 7.82 7.73 CV (%) 3.4 5.2 1.4 1.3 11.2 2.4 Variation 6.8 4.1 1.2 Quantification of cyproheptadine in human plasma by LCMSMS Biomed. Chromatogr. 2012; 26: 129136 Copyright 2011 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/bmc 1 3 3 2.9 (0.3) min, respectively. The mean cyproheptadine plasma concentrations vs time profiles after a single oral dose of each 4 mg tablet formulation of cyproheptadine are shown in Fig. 3. The simplest regression method for the calibration curves of the cyproheptadine was y =a +bx from 0.05 to 10 ng/mL (calibration curve y =0.0725x +0.000515, r =0.9978). This is the first LCMSMS method developed for measuring cyproheptadine in human plasma. Conjugated cyproheptadine was determinedinplasma by CGMS (LLOQ0.2ng/mL, RT 7.98 min, solid extraction, 0.1mL plasma; Hasegawa et al., 2006), in human serum by LCMS (solid extraction; Gunja et al., 2004), in animal urine by GLC (LLOQ 3 ng/mL, liquidliquid extraction; Hucker and Hutt, 1983), LCMSMS (LLOQ 0.15ng/mL, RT 1020 min, solid extraction, 2 mL urine; Fes et al., 2009a; Fente et al., 2009) and reversedphaseHPLC (solid extraction; Kountourellis and Ebete, 1995), and in pharmaceutical syrup samples by LCMS/MS (LLOQ 1 ng/mL, RT 7.29min, liquidliquid extraction; Fes et al., 2009b). Our method has good sensitivity (LLOQ of 0.05 ng/mL), and can be carried out in a short time (RT of 2.7 min for cyproheptadine), permitting a high throughput. As demonstrat- ed in this study, the present LCMSMS method is simple and selective for the determination of cyproheptadine in human plasma, thus it can be used for pharmacokinetic and bioequiva- lence studies of cyproheptadine.The advantages of the herein reported methodology are the throughput and sensitivity of the LCMS/MS technique along with the feasibility and excellent performance for the bioanalytical purpose within FDA and ANVISA criteria. Stability Stock and working solutions of cyproheptadine and amitripty- line were stable for 7 days when stored at 4C (n =5, RE 1.2%) and at room temperature (25C) for 6 h (n =5, RE 11.5%), respectively. Cyproheptadine in human plasma (at low and high QC concentrations) presented no significant degradation after (n =5): postprocessing (48 h, RE =4.1 and 1.9%, respectively), freezeandthaw (three cycles, RE =6.8 and 1.2%), shortterm (8 h, RE =9.6 and 1.5%) and longterm (56 days, RE =1.4 and 7.5%). Pharmacokinetic study The whole assay was conducted strictly in accordance with the current Good Clinical Practices. The study began with 40 healthy volunteers and finished with 38 healthy volunteers. One volunteer dropped out of the study for personal reasons and another owing to adverse effects (vomiting). The method was successfully employed to determine cyproheptadine concentrations in human plasma samples after the administra- tion of a 4 +1 mg oral dose of cyproheptadine +cobamamide. The observed cyproheptadine peak plasma concentration (C max ) value (1.25 ng/mL) differed from that reported in the literature (30 ng/mL; Gunja et al., 2004). However, Gunja et al. (2004) measured total cyproheptadine (conjugated and drug) whereas our method is sensitive enough for quantification of cyproheptadine. After the oral administration of the cyprohep- tadine tablets to the volunteers, the observed cyproheptadine peak plasma concentration (C max ) values and the time values taken to achieve (T max ) were equivalent between the formula- tions (Tables 6 and 7). In addition, the calculated 90% CI for mean C max , AUC last and AUC 0inf Cobactin/Cobavital individual ratios were within the 80125% bioequivalence limit defined by the US FDA and ANVISA. Supported by these data, it is possible to state that both formulations achieved equiva- lent bioavailability. Table 4. Shortterm stability test (values in ng/mL) Reference values Values after 8 h Reference values Values after 8 h Reference values Values after 8 h Low sample Medium sample High sample Mean 0.146 0.132 1.47 1.33 7.82 7.94 CV (%) 3.4 6.3 1.4 4.6 11.2 2.2 Variation 9.6 9.5 1.5 Table 5. Longterm stability test (values in ng/mL) Reference values Values after 56 days Reference values Values after 56 days Reference values Values after 56 days Low sample Medium sample High sample Mean 0.146 0.144 1.6 1.43 8.98 8.31 CV (%) 6.5 11.6 2.0 5.0 1.7 4.0 Variation 1.4 10.6 7.5 Figure 3. Cyproheptadine plasma mean concentrations vs time profile obtained after the single oral administration of 4 mg of cyproheptadine formulation. G. D. Mendes et al. Biomed. Chromatogr. 2012; 26: 129136 Copyright 2011 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/bmc 1 3 4 Conclusion This article describes the first rapid, reliable and fully validated bioanalytical methodology using liquid chromatography cou- pled to tandem mass spectrometry (LCMS/MS) for quantifica- tion of cyproheptadine pharmacokinetic and bioequivalence studies. The achieved limit of quantification (0.05 ng/mL) was good enough for all the pharmacokinetic parameters and the throughput obtained allowed rapid quantification of biological samples coming from clinical studies. Also, since all validation parameters were in accordance with the FDA and ANVISA guidelines, the protocol robustness was demonstrated by its employment in a comparative bioavailability study, where more than 2200 samples were analyzed. The cyproheptadine relative bioavailability between both formulations (Cobactin, test and Cobavital, reference) was assessed by calculating individual test/reference ratios for C max , AUC last and AUC 0inf and pharmacokinetic profiles indicated bioequivalence since all ratios were as proposed by FDA and ANVISA. Conflict of interest The bioequivalence trial was sponsored by Zambon Laboratrios Farmacuticos Ltda. References ANVISA-RE 899. 2003. Available at: http://www.anvisa.gov.br/legis/resol/ 2003/re/899_03re.htm Fes X, Ye L, Regal P, Fente CA, Hosseini SV and Cepeda A. Application of dummy molecularly imprinted solidphase extraction in the analysis of cyproheptadine in bovine urine. Journal of Separation Science 2009a; 32: 1740. Fes X, Ye L, Hosseini SV, Fente CA and Cepeda A. Development and validation of LCMS/MS method for the determination of cyproheptadine in several pharmaceutical syrup formulations. Journal of Pharmaceutical and Biomedical Analysis 2009b; 50: 1044. Fente CA, Regal P, Vzquez BI, Fes X, Franco CM and Cepeda A. Development and validation of an LCMS/MS confirmatory method for residue analysis of cyproheptadine in urine of food producing animals. Journal of Agricultural and Food Chemistry 2009; 57: 2595. Gunja N, Collins M and Graudins A. A comparison of the pharmacoki- netics of oral and sublingual cyproheptadine. Journal of Toxicology: Clinical Toxicology 2004; 42: 79. Hasegawa C, Kumazawa T, Lee XP, Fujishiro M, Kuriki A, Marumo A, Seno H and Sato K. Simultaneous determination of ten antihistamine drugs in human plasma using pipette tip solidphase extraction and gas chromatography/mass spectrometry. Rapid Communications in Mass Spectrometry 2006; 20: 537. Hucker HB and Hutt JE. Determination of cyproheptadine in plasma and urine by GLC with a nitrogensensitive detector. Journal of Pharmaceutical Sciences 1983; 72: 1069. Kountourellis JE and Ebete KO. Reversedphase high performance liquid chromatographic determination of cyproheptadine from urine by solidphase extraction. Journal of Chromatography B: Biomedical Sciences and Applications 1995; 664: 468. Table 6. Cyproheptadine mean pharmacokinetic parameters obtained from 38 volunteers after administration of each 4 mg cyproheptadine +1 mg cobamanide tablet formulation Parameter Unit N Mean SD Min Median Max CV% Reference AUC % extrap (%) 38 17.98 7.42 8.29 15.23 41.36 41.30 AUC all (ng h/mL) 38 32.84 10.13 14.65 31.93 61.22 30.86 AUC inf (ng h/mL) 38 40.82 15.71 16.41 38.49 91.05 38.49 AUC last (ng h/mL) 38 32.71 10.32 14.65 31.93 61.22 31.55 C last (ng/mL) 38 0.13 0.06 0.05 0.12 0.33 48.92 C max (ng/mL) 38 1.25 0.41 0.59 1.28 2.21 33.03 k e (1/h) 38 0.02 0.01 0.01 0.02 0.03 27.39 T 1/2 (h) 38 38.21 10.70 21.46 35.86 69.15 28.02 T last (h) 38 91.85 9.43 72.00 96.07 97.82 10.27 T max (h) 38 4.68 1.32 2.67 4.50 9.00 28.15 Test AUC % extrap (%) 38 20.20 12.94 8.19 15.35 62.33 64.06 AUC all (ng h/mL) 38 34.88 15.21 17.71 33.90 86.23 43.62 AUC inf (ng h/mL) 38 47.00 32.02 19.62 39.86 197.59 68.13 AUC last (ng h/mL) 38 34.78 15.32 16.54 33.90 86.23 44.05 C last (ng/mL) 38 0.16 0.14 0.06 0.12 0.70 86.92 C max (ng/mL) 38 1.31 0.48 0.61 1.19 2.96 37.01 k e (1/h) 38 0.02 0.01 0.01 0.02 0.06 46.44 T 1/2 (h) 38 41.83 22.35 11.16 35.30 128.18 53.43 T last (h) 38 90.67 15.22 24.00 96.11 97.67 16.78 T max (h) 38 4.97 1.41 2.67 5.00 8.00 28.45 Table 7. Geometric mean of individual AUC last , AUC 0inf and C max ratios (test/reference formulation), the respective 90% CI and power Percentage geometric mean 90% CI Power N=38 C max % ratio 104.43 98.14111.13 1.0000 AUC last % ratio 103.73 97.90109.90 1.0000 AUC inf % ratio 107.98 99.84116.78 0.9982 Quantification of cyproheptadine in human plasma by LCMSMS Biomed. Chromatogr. 2012; 26: 129136 Copyright 2011 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/bmc 1 3 5 Marzo A and Dal Bo L. Tandem mass spectrometry (LCMSMS): a predominant role in bioassays for pharmacokinetic studies. ArzneimittelForschungDrug Research 2007; 52: 122. Rosing H, Man WY, Doyle E, Bult A and Beijnen JH. Bioanalytical liquid chromatographic method validation. A review of current practices and procedures. Journal of Liquid Chromatography and Related Technologies 2000; 23: 329. Rozet E, Ceccato A, Hubert C, Ziemons E, Oprean R, Rudaz S, Boulanger B and Hubert P. 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