Metabolism, Excretion, and Pharmacokinetics of [C]Tigecycline, a First-In-Class Glycylcycline Antibiotic, after Intravenous Infusion to Healthy Male Subjects

Tigecycline, a novel, first-in-class glycylcycline antibiotic, has been approved for the treatment of complicated intra-abdominal infections and complicated skin and skin structure infections. The pharmacokinetics, metabolism, and excretion of [C]tigecycline were examined in healthy male volunteers. Tigecycline has been shown to bind to bone; thus, to minimize the amount of radioactivity binding to bone and to maximize the recovery of radioactivity, tigecycline was administered intravenously (30-min infusion) as a single 100-mg dose, followed by six 50-mg doses, every 12 h, with the last dose being [C]tigecycline (50 Ci). After the final dose, the pharmacokinetics of tigecycline in serum showed a long halflife (55.8 h) and a large volume of distribution (21.0 l/kg), whereas radioactivity in serum had a shorter half-life (6.9 h) and a smaller volume of distribution (3.3 l/kg). The major route of elimination was feces, containing 59% of the radioactive dose, whereas urine contained 32%. Unchanged tigecycline was the predominant drugrelated compound in serum, urine, and feces. The major metabolic pathways identified were glucuronidation of tigecycline and amide hydrolysis followed by N-acetylation to form N-acetyl-9-aminominocycline. The glucuronide metabolites accounted for 5 to 20% of serum radioactivity, and approximately 9% of the dose was excreted as glucuronide conjugates within 48 h. Concentrations of N-acetyl-9-aminominocycline were approximately 6.5% and 11% of the tigecycline concentrations in serum and urine, respectively. Excretion of unchanged tigecycline into feces was the primary route of elimination, and the secondary elimination pathways were renal excretion of unchanged drug and metabolism to glucuronide conjugates and N-acetyl-9-aminominocycline. Tigecycline [GAR-936, (4S,4aS,5aR,12aS)-9-(2-tert-butylaminoacetylamino)-4,7-bis-dimethylamino-3,10,12,12a-tetrahydroxy-1,11dioxo-1,4,4a,5,5a,6,11,12a-octahydronaphthacene-2-carboxamide] (Fig. 1), a novel, first-in-class glycylcycline and an analog of the semisynthetic antibiotic minocycline, is a potent, broad-spectrum antibiotic that acts by inhibition of protein translation in bacteria. Glycylcycline antibiotics, including tigecycline and others (N,N-dimethylglycylamido-9-aminominocycline and N,N-dimethylglycylamido-9-amino-6-demethyl-6-deoxytetracycline), are active in vitro against multiple antibiotic-resistant pathogenic bacteria, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci (Fraise et al., 1995; Weiss et al., 1995). Tigecycline is also active against bacterial strains carrying the two major forms of antibiotic resistance, active efflux and ribosomal protection (Schnappinger and Hillen, 1996). In patients, intravenous tigecycline (50 mg, twice daily) was effective against complicated skin and skin-structure infections and complicated intra-abdominal infections with an acceptable safety profile (Murray et al., 2003; Postier et al., 2004). The pharmacokinetic profile of tigecycline in healthy human subjects is characterized by a long half-life of 37 to 67 h and a large volume of distribution (VSS) of 7 to 10 l/kg (Muralidharan et al., 2005). The metabolism of glycylcycline antibiotics has not previously been investigated in humans. However, other antibiotics containing the tetracycline ring structure, such as doxycycline, tetracycline, chlortetracycline, and demethylchlorotetracycline, generally undergo little or no metabolism (Kelly and Buyske, 1960; Kelly et al., 1961; Eisner and Wulf, 1963; Swarz, 1976; Nelis and De Leenheer, 1981). Minocycline is an exception to this pattern, since it undergoes metabolism in humans via hydroxylation and N-demethylation (Nelis and De Leenheer, 1982; Böcker et al., 1991). The present study was conducted to evaluate the pharmacokinetics, metabolic disposition, and mass balance of a 50-mg intravenous dose of C-labeled tigecycline in healthy male volunteers. [C]Tigecycline has been shown to distribute extensively into bone following a single i.v. dose to rats, with bone to plasma ratios for radioactivity as high as 2000 (Tombs, 1999). Even with this extensive distribution into bone, the estimated exposure to radioactivity after a 50Ci (50-mg) intravenous dose of [C]tigecycline to humans was 0.023 rem or 0.46% of the maximum allowable exposure from radioactive drugs for Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.107.015735. ABBREVIATIONS: LC-MS/MS, liquid chromatography-tandem mass spectrometry; HPLC, high performance liquid chromatography; LC/MS, liquid chromatography/mass spectrometry; AUC, area under the curve; AUCT, area under the concentration-time curve; HMBC, heteronuclear multiple bond correlation; MRT, mean residence time; CL, clearance. 0090-9556/07/3509-1543–1553$20.00 DRUG METABOLISM AND DISPOSITION Vol. 35, No. 9 Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics 15735/3238971 DMD 35:1543–1553, 2007 Printed in U.S.A. 1543 at A PE T Jornals on O cber 0, 2017 dm d.aspurnals.org D ow nladed from human research subjects (5 rem). However, to minimize the amount of C-labeled material binding to bone and to maximize the recovery of radioactivity, the C-labeled tigecycline dose was administered after multiple doses (350 mg over 3 days) of unlabeled tigecycline. Using this dosing regimen, previous studies have shown that steady-state tigecycline serum concentrations were achieved on day 4 (Sun et al., 2005). Thus, for the current study, it was assumed that by the time of the [C]tigecycline dose, steady-state or close to steady-state conditions had been achieved in serum, as well as in other tissues such as bone. Materials and Methods Materials. Tigecycline, as a lyophilized powder in vials containing a 50-mg dose of drug, was provided by Wyeth Research (Collegeville, PA). The manufacturing and packaging of [C]tigecycline were carried out at the Parenteral Medications Laboratories, College of Pharmacy, University of Tennessee Health Science Center (Memphis, TN). Nonradiolabeled tigecycline drug substance was used to dilute radiolabeled tigecycline to make the final C-drug substance. Radiolabeled tigecycline for injection was prepared in 5-ml clear glass vials, each containing 53 mg of lyophilized, sterilized free tigecycline powder. The final [C]tigecycline drug product had a specific activity of 9.2 mCi/mmol (1.00 Ci/mg) with a radiochemical purity of 98.6% and a chemical purity of 99.2%. Appearance, strength, identity, specific activity, and purity testing of [C]tigecycline for injection was performed by ABC Laboratories (Columbia, MO). Bacterial endotoxin and sterility testing of [C]tigecycline for injection was carried out by Wyeth Research. An additional batch of [C]tigecycline (95.3 Ci/mg, 97.2% radiochemical purity), used in control samples, and [t-butyl-d9]tigecycline, the internal standard for the determination of tigecycline in serum and urine, were received from GE Healthcare (Chalfont St. Giles, Buckinghamshire, UK). Tigecycline reference standard (98.4% chemical purity), 9-aminominocycline-hydrochloride salt reference standard (CL-318614; 97% chemical purity), and N-acetyl9-aminominocycline (WAY-188749, batch L23566-162) were received from Wyeth Research. All other reagents and chemicals were obtained from commercial sources. Study Design. This open-label, inpatient, multiple-dose tigecycline, singledose [C]tigecycline metabolic disposition and mass balance study was performed in healthy men. Eligible subjects were selected on the basis of inclusion/exclusion criteria, medical history, physical examination, and additional procedures outlined in the study protocol. Subjects using any investigational or prescription drug within 30 days of test article administration were excluded from the study. Before initiation of the study, the protocol and consent form were reviewed and approved by an institutional review board. Study subjects gave written informed consent before the screening process was initiated. Dose Administration. The study was initiated with 12 healthy adult male volunteers to ensure six subjects would receive the radiolabeled dose. Each subject received a 100-mg loading dose on the morning of day 1, followed by a 50-mg maintenance dose every 12 h for an additional five doses. On the morning of study day 4, six subjects (ranging from 22 to 40 years of age, with body weights of 70.1–90.8 kg) each received a single 50-mg dose of [C]tigecycline, approximately 50 Ci/subject. Each tigecycline dose was administered via a 30-min intravenous infusion. Throughout the study, subjects received a high fiber meal approximately 2 h before tigecycline administration. Study subjects received two tablets of FiberCon (Wyeth) daily from day 1 through day 13 to facilitate regular bowel movements. The dosing and “inpatient-confinement portion” of the study were conducted at Quintiles (Kansas City, MO). Aliquots of the [C]tigecycline dosing solution obtained before and after dose administration, dosing vials, and dosing apparatus were shipped to ABC Laboratories for radioactivity analysis. Clinical Sample Collection. Whole blood samples (3 ml) from each subject, for the determination of total radioactivity, were collected into Vacutainer tubes (BD, Franklin Lakes, NJ) containing EDTA within 2 h before [C]tigecycline dosing and approximately 0.5, 1, 2, 4, 8, 12, 24, 36, 48, 72, 96, 120, 144, 168, 192, 216, and 240 h after the [C]tigecycline dose. Additional whole blood samples (7 ml) were collected within 2 h before the first tigecycline dose and at the same collection times as the 3-ml whole blood samples. From these whole blood samples, serum was obtained after clot formation and centrifugation of the sample, and used to determine tigecycline and total radioactivity concentrations in serum. Whole blood samples (50 ml) for serum collection were drawn approximately 2 h before the first tigecycline dose, within 2 h before [C]tigecycline dosing and approximately 1, 4, 8, 24, and 48 h after the [C]tigecycline dose. These serum samples were stored at 70°C until analyzed for metabolite profiles. Urine samples were collected within 2 h before the [C]tigecycline dose and all urine was collected from study subjects after the [C]tigecycline dose at intervals of 0 to 4, 4 to 8, and 8 to 24 h and every 24 h thereafter up to 240 h. Samples were kept refrigerated during the collection intervals and then were separated into samples for total radioactivity determination, tigecycline concentration determination, and metabolite profiling (samples up to 48 h after the [C]tigecycline dose), and stored at 20°C until analysis. When possible, a fecal sample was collected from subjects before the [C]tigecycline dose, and all fecal samples up to 240 h after the [C]tigecycline dose were also collected. Samples were homogenized in 3 volumes of water using a Stomacher (3500; Seward Limited, London, UK) and stored in plastic containers at 4°C until analysis. Aliquots of each sample were analyzed for total radioactivity concentrations and samples collected up to 48 h after the [C]tigecycline dose were analyzed for metabolite profiles. For two subjects, there were incomplete fecal collections following the [C]tigecycline dose, due to noncompliance, and another subject withdrew from the study after sample collections at 24 h. Data from these subjects were not used in the mass balance portion of the study, but were used in the pharmacokinetic analysis and metabolite profiling portions of the study. Radioanalysis. All radioactivity determinations were made using either a Tri-Carb model 3100TR liquid scintillation counter (PerkinElmer, Wellesley, MA) or Beckman/LS6000SC or LS6500 liquid scintillation counters (Beckman Coulter, Inc., Fullerton, CA). For dose, serum, and urine analysis, Ultima Gold scintillation fluid (PerkinElmer) was added to a known volume or weight of sample. Samples were then directly analyzed by liquid scintillation counting. Fecal homogenates were weighed, allowed to dry, and combusted using a model 307 sample oxidizer (PerkinElmer). The resultant [C]CO2 was trapped in Carbosorb in combination with Permafluor and radioassayed by liquid scintillation counting. For all sample matrices, a quench curve was used to convert cpm values to dpm values. Measurement of Tigecycline in Serum and Urine. Serum and urine tigecycline concentrations were determined using validated LC-MS/MS assays. For serum, the assay was validated at concentrations between 10 and 2000 ng/ml. In brief, tigecycline and an internal standard ([t-butyld9]tigecycline) were extracted from serum using 0.1% trifluoroacetic acid in acetonitrile. The denatured protein was removed by centrifugation and the supernatant was removed and evaporated to dryness at 35°C under a stream of air. Samples were reconstituted in 200 l and an aliquot was injected for LC-MS/MS analysis using a Thermo Fisher Scientific (Waltham, MA) AQUASIL C18, 50 2.1 mm, 5m analytical column. Tigecycline and the internal standard were measured using the transition of the positive ions m/z 586.53513.2 and 595.33514.3, respectively. For the quality control samples, the overall precision (CV) was 10.5% and accuracy ranged from 104 to 108%. For urine, the assay was validated at concentrations between 0.20 and 80 g/ml. In brief, tigecycline and an internal standard in urine were diluted; the samples were mixed and transferred to fresh tubes, and an aliquot was injected for LC-MS/MS analysis using a Thermo Fisher Scientific AQUASIL C18, 100 2.0 mm, 5m analytical column. For the quality control samples, the overall precision (CV) was 11.3% and accuracy ranged from 90.4 to 100%. Metabolite Profiling by HPLC. Individual serum samples collected 1, 4, FIG. 1. Structure of tigecycline. indicates the site of the C label. 1544 HOFFMANN ET AL. at A PE T Jornals on O cber 0, 2017 dm d.aspurnals.org D ow nladed from and 8 h after the [C]tigecycline dose were analyzed for metabolite profiles. The 24and 48-h samples were not analyzed because the concentration of radioactivity was too low. Each serum sample was divided into two samples of equal volume (approximately 9 ml each) to provide duplicate analyses. EDTA (final concentration of 40 mM) and 3 volumes of acetone were then added to the samples. Samples were mixed and then centrifuged to remove the denatured protein. The supernatant was transferred to a fresh tube containing 20 l of glacial acetic acid. The pellet was reextracted with 2 ml of water, 160 l of 0.2 M EDTA, and 6 ml of acetone and processed as described above. The supernatants were combined and the pellet extraction was repeated. Supernatant samples were evaporated to dryness under a stream of nitrogen at room temperature. The residue was reconstituted in 300 l of acetonitrile/water (1:9). The reconstituted residue was centrifuged to remove particulates and the supernatant transferred to an autosampler vial. The supernatant was assayed for radioactivity content and analyzed for metabolite profiles by HPLC. Extraction of control serum or fecal samples spiked with varying concentrations of [C]tigecycline showed that the extraction process resulted in a reproducible percentage of tigecycline in samples being converted to its epimer during the extraction process. Therefore, the amount of epimer in the samples before extraction could be determined based on the amount of epimer in the final serum or fecal extracts. Aliquots (1 ml) of the individual urine samples collected up to 48 h after the [C]tigecycline dose were transferred to clean tubes and EDTA (final concentration of 40 mM) was added. The pH of the urine remained constant at pH 4.5 to 5.5. Samples were mixed and centrifuged, and the supernatants were transferred to autosampler vials. Separate aliquots of the supernatant were assayed for radioactivity content and analyzed for metabolite profiles by HPLC. Fecal homogenates prepared from samples collected within 48 h after the [C]tigecycline dose and containing greater than 8000 dpm/g were analyzed for metabolite profiles. Aliquots of the fecal samples (approximately 1 g) were transferred to 15-ml tubes, 3 volumes of acetone were added, and the samples were mixed and centrifuged. The supernatant was transferred to a fresh tube, and the pellet was resuspended with 40 mM EDTA in water and reextracted as described above. The pellet was reextracted in this manner a total of three times and the supernatants were pooled. The combined supernatants were evaporated to dryness under a stream of nitrogen. The residue was resuspended in 500 l of water and centrifuged to remove particulates, and the supernatant was assayed for radioactivity content and analyzed for metabolite profiles by HPLC. For LC/MS quantitation of metabolites M5 (N-acetyl 9-aminominocycline) and its epimer M4 in serum and urine samples from three subjects, standard curves were generated for each metabolite in each matrix. In serum, the standard curves ranged from 2.1 to 514.0 ng/ml for M4 and 2.8 to 568.6 ng/ml for M5, whereas in urine, the ranges were 5.7 to 632.5 ng/ml for M4 and 4.3 to 367.8 ng/ml for M5. For quantitation of these metabolites, urine samples were prepared in the same manner as those for metabolite profiling. For the serum samples, EDTA (final concentration of 40 mM) was added to each sample and the samples were vortex-mixed. One volume of acetonitrile was then added to each sample, samples were vortex-mixed, and denatured protein was separated by centrifugation. The supernatant was transferred to a fresh tube and concentrated under a stream of nitrogen to approximately 1 ml. The remaining supernatant was then assayed for M4 and M5 concentrations. HPLC analyses for metabolite profiling were performed using a Waters 2695 Alliance Separation Module (Waters Corp., Milford, MA) with the sample temperature set to 4°C and a Waters model 2487 dual wavelength UV absorbance detector set to monitor 350 nm. The system was in-line with a Gilson 215 liquid handler (Gilson Inc., Middleton, WI) equipped to collect fractions at 20-s intervals. Fractions were collected into 96-well deep-well LumaPlates and analyzed for radioactivity using TopCount NXT (PerkinElmer, Shelton, CT). Flo-One analytical software (version 3.65) was used to integrate the radioactive peaks. Separation of tigecycline and drugderived products was achieved on a Phenomenex Luna C18(2) column (150 2.1 mm, 5 m; Phenomenex, Torrance, CA) equipped with a Phenomenex SecurityGuard guard cartridge (5 m) using two mobile phases, 10 mM ammonium acetate in water, and acetonitrile. The column was at an ambient temperature of approximately 20°C and the flow rate was 0.2 ml/min. The linear gradient was delivered as follows: 0 to 25 min, from 98% aqueous to 90% aqueous; 25 to 50 min, from 90% aqueous to 70% aqueous; hold at 70% aqueous until 60 min; return to initial conditions at 61 min. Additional HPLC conditions were used to characterize a polar metabolite that was not retained on the C18 column. Separations were accomplished on a Waters Atlantis HILIC Silica column (150 2.1 mm, 5 m) using two mobile phases, 0.02% trifluoroacetic acid in acetonitrile (v/v) and 0.02% trifluoroacetic acid in water (v/v). The column was at an ambient temperature of approximately 20°C and the flow rate was 0.2 ml/min. The linear gradient was delivered as follows: 0 to 10 min, hold at 100% organic; 10 min to 66% organic; 5 min to 34% organic; hold for 15 min; return to initial conditions over 1 min. Metabolite Characterization by Mass and NMR Spectroscopy. The HPLC system and separation conditions used for mass spectrometric analysis were similar to those described for metabolite profiling. MS and MS/MS experiments were performed on a Micromass Quattro Ultima triple quadrupole mass spectrometer (Waters Corp.) equipped with an electrospray source and operated in the positive ionization mode. The electrospray needle potential was set at 2.75 kV and the orifice potential was set at 44 V. The ion source was held at 80°C and the desolvation temperature was 250°C. In MS/MS experiments, the collision-activated dissociation of selected precursor ions was conducted using argon as the collision gas. The collision energy was 30 eV. Serum extracts were analyzed for tigecycline and selected metabolites by LC-MS/MS in the selected reaction monitoring mode to reduce interference from endogenous components. These experiments were conducted with a dwell time setting of 200 ms. The following tigecycline-related components were monitored: tigecycline and its epimer, m/z 5863513; hydroxy tigecycline and tigecycline N-oxide, m/z 6023585 and m/z 6023472; N-desmethyl tigecycline, m/z 5723499; tigecycline glucuronide, m/z 7623569; 9-aminominocycline, m/z 4733456; and N-acetyl-9-aminominocycline, m/z 5153498. Urine and fecal samples were analyzed for tigecycline metabolites by LCMS/MS analysis for precursors of product ions characteristic of tigecycline. In addition, potential metabolites of tigecycline were searched for in the LC/MS data for subsequent MS/MS analysis. The site of glucuronidation was further investigated using chemical oxidation of tigecycline and its glucuronide metabolite with Fremy’s salt (potassium nitrosodisulfonate). Fremy’s salt selectively oxidizes phenols, aromatic diols, aminophenols, and diamines to the corresponding quinones (Zimmer et al., 1971). The glucuronide metabolite used for these incubations was isolated from human urine following collection of the HPLC column effluent that contained the glucuronide metabolite. Three sets of incubations with Fremy’s salt were prepared: tigecycline alone, tigecycline plus the isolated glucuronide metabolite, and the isolated glucuronide metabolite alone. Incubations with tigecycline contained 10 g/ml tigecycline in 10 mM ammonium acetate with 20 M EDTA, and incubations with the tigecycline glucuronide contained the isolated tigecycline glucuronide with EDTA added (final concentration of 20 M). Incubations were prepared in duplicate, and to one replicate Fremy’s salt was added (2 g/ml); to the other replicate nothing was added. Samples were incubated for at least 5 min at an ambient temperature of approximately 20°C before being directly analyzed by LC/MS. Tigecycline glucuronide conjugates were synthesized using modified Koenigs-Knorr reaction conditions (Ag2CO3/MeCN in the presence of EDTA and molecule sieves) with an acyl-protected 1 -bromo-sugar as the donor to generate protected tigecycline glucuronides followed by de-protection in the presence of 0.1 N lithium hydroxide (in methanol/water 1:1). Cochromatography of the synthetic tigecycline glucuronides with tigecycline glucuronide (M7) isolated from human urine was used to determine the desired reaction product. NMR spectroscopy of the synthetic glucuronide was performed with a 500 MHz Bruker DRX spectrometer equipped with a Cap-NMR flow cell probe. Proton NMR spectra were acquired with 128K data points. The heteronuclear single quantum correlation (HSQC) spectrum was acquired with 2K data points in F2 and 256 increments in F1. Heteronuclear multiple bond correlation (HMBC) experiments were conducted with 4K data points in F2 and 512 increments in F1. One of the HMBC spectra was recorded with the JH/C setting at 8 Hz and another one was recorded with the JH/C setting at 5 Hz for three-bond long-range correlation detection. Pharmacokinetic Analysis. The radioactivity concentration data in serum (expressed in ng-Eq of tigecycline/ml), and the tigecycline serum concentration data for each subject were analyzed by using empirical, model-indepen1545 METABOLIC DISPOSITION OF [C]TIGECYCLINE IN HUMANS at A PE T Jornals on O cber 0, 2017 dm d.aspurnals.org D ow nladed from dent pharmacokinetic methods (Jusko, 1992). Peak concentration (Cmax) was directly determined from the observed data. The apparent terminal-phase disposition rate constant ( Z) for tigecycline in serum was estimated by a log-linear regression of the last three to seven observed concentrations that were determined to be in log-linear elimination by visual inspection. Due to low concentrations and rapid elimination, the concentration of radioactivity in serum could be determined only in samples collected within 24 h of dosing, and therefore, the Z for this parameter was estimated using only two or three observed concentrations. The apparent terminal-phase disposition half-life (t1⁄2) was calculated as t1⁄2 0.693/ Z. Since only one dose of [C]tigecycline was administered, the serum radioactivity concentration data were analyzed as single-dose data. The single dose area under the concentration-time curve (AUCT) to the last observable concentration (CT) at time T was calculated by using the log-trapezoidal rule for decreasing concentrations and the linear-trapezoidal rule for increasing concentrations. The total single-dose AUC was estimated by AUC AUCT CT/ Z. The single dose mean residence time (MRT) was calculated as AUC/ AUMC Tinf/2, where AUMC is total area under the first moment curve and Tinf is the duration of infusion (30 min). The unlabeled tigecycline serum concentration data represent multiple-dose administration, and the steady-state AUC over one dose interval (AUC0, where 12 h) was calculated by using the log-trapezoidal rule for decreasing concentrations and the linear-trapezoidal rule for increasing concentrations. In addition, the steady-state MRT was calculated as AUC0/(AUMC0AUC ) Tinf/2. Systemic clearance (CL) was calculated as dose/AUC, and the apparent steady-state volume of distribution (VSS) was estimated as CL MRT. Calculations. The amount of total radioactivity (dpm) in the urine, feces, and serum was determined by multiplying the weight of samples by the radioactivity concentration (dpm/g) of each sample. The dose recovered was determined by total dpm in the sample at any given time point, divided by total radioactivity (in dpm) of the dose received by each subject, and multiplied by 100%. These calculations and the calculations of means and standard deviations were performed using Microsoft Excel 2000 software (Microsoft, Redmond, WA). If the dpm in any postdose sample aliquot was less than or equal to 2 times dpm in the predose sample aliquot (background), the value was presented as not detectable (N.D.). The concentration of tigecycline-related components in serum, urine, and feces was calculated based on the total radioactivity concentrations. These concentrations were converted to tigecycline equivalent using the specific activity of the dose (1.00 Ci/mg). Using this value, the concentrations of the specific components were then estimated based on the distribution of radioactivity in the radiochromatograms. These metabolite concentrations reflect only the disposition of the final [C]tigecycline dose and do not account for tigecycline or its metabolites remaining from the unlabeled doses. For the concentration determinations of the nonradiolabeled metabolites M4 and M5, the LC/MS peak area from study samples was compared with a standard curve generated using peak areas of control samples spiked with synthetic standards of M4 and M5.