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Quantification of sibutramine and its two metabolites in human plasma by LC–ESI-MS/MS and its application in a bioequivalence study

Obesity can be considered as a chronic illness of epidemic proportion and its incidents have increased exponentially in recent years. The use of anti-obesity drugs such as sibutramine is somewhat helpful. There is a need to quantify such drugs in biological samples, which is generally quite difficult. In this report, we developed and validated a simple, sensitive and specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the quantification of sibutramine (SB) and its two metabolites N-des methyl sibutramine (DSB) and N-di desmethyl sibutramine (DDSB) in human plasma. Zorbax SB-C18 (4.6 mm×75 mm, 3.5 μm, 80 Å) analytical column and 5 mM ammonium formate:acetonitrile (10:90, v/v) mobile phase were used for chromatographic separation of SB, DSB and DDSB. Multiple reaction monitoring (MRM) in the positive mode was used to detect SB, DSB and DDSB at m/z 280.3/124.9, 266.3/125.3 and 252.2/124.9, respectively. Liquid–liquid extraction was used for the extraction of analytes and internal standard from human plasma. This method was validated over a linear concentration range of 10.0–10,000.0 pg/mL for SB, DSB and DDSB with correlation coefficients (r) of ≥0.9997. The drug and the two metabolites were stable in plasma samples. The validated method was successfully applied in a bioequivalence and pharmacokinetic study with human volunteers under fasting condition.


Introduction
Sibutramine hydrochloride monohydrate [(7)-dimethyl-1-[1-(4-chlorophenyl)cyclobutyl]-N,N,3-trimethylbutan-1-amine hydrochloride monohydrate] is currently used for the treatment of obesity. The chemical formula of sibutramine is C 17 H 29 Cl 2 NO and its molecular weight is 334.33. Sibutramine acts by inhibiting the re-uptake of neurotransmitter serotonin, norepinephrine and dopamine and enhancing satiety. Consequently, it prevents compulsive eating and inhibits the sensation of hunger in obese patients [1,2]. Today obesity can be considered as a chronic illness of epidemic proportion and its incidents have increased exponentially [3]. For many patients, dieting and physical exercise are not sufficient to achieve and maintain the desired weight loss. The use of anti-obesity drugs such as sibutramine is needed.
Literature survey reveals that quantification of sibutramine in pharmaceutical, synthetic/dietary supplements and herbal products was reported using LC-MS [4][5][6][7] and HPLC [8][9][10][11] in biological matrices, e.g. rats [12][13][14][15][16][17][18]. Only a few methods were reported for the quantification of sibutramine and its two metabolites in human plasma using LC-MS/MS [19][20][21][22][23][24]. Kang et al. [21] developed a method with the linearity range of 0.1 -0.8 ng/mL; they used imipramine as the internal standard and a more complicated solid phase extraction method to isolate sibutramine and its two metabolites. Ding et al. [22] established a method with a linearity range of 0.05-20.0 mg/L for sibutramine and N-des methyl sibutramine and 0.1-30.0 mg/L for N-di-des methyl sibutramine. They used phenyl propyl amine hydrochloride as an internal standard, liquid-liquid extraction for extraction of drug and IS and compared test and reference formulations with 18 volunteers.
Chen et al. [23] reported a method with a linearity range of 0.328-32.8 ng/mL for sibutramine, N-Des methyl sibutramine and N-di-des methyl sibutramine. They used liquid-liquid extraction and propronolol as an internal standard and compared test and reference formulations with 20 volunteers. Bae et al. [24] developed a high-sensitivity method with a good linearity range of 50-20000 pg/mL and a shorter runtime and compared the drug and metabolites with chlorphenaramine as an internal standard. However, we experienced many difficulties with the Bae's method in terms of reproducibility and stability in long run analysis.
It is important to develop a reliable bio-analytical method with a properly deuterated derivative or other analog as the internal standard to study the matrix effect and reproducibility. It is not required to consider the runtime when comparing the reproducibility and stability of analytical batches. The goal of the present study was to develop a reliable and accurate method for the quantification of sibutramine and its two metabolites with respective deuterated internal standards. Moreover, the sample extraction method should be simple and the analytical method should be highly sensitive with good linearity and the use of a small amount of plasma.

Instrumentation
The Agilent 1200 Series HPLC system (Agilent Technologies, Waldbronn, Germany) connected to the API 4000 triple quadrupole instrument (ABI-SCIEX, Toronto, Canada) with turbo electrospray interface in positive ionization mode was used. Data processing was performed on Analyst 1.4.1 software package (SCIEX).

Detection
The mass transitions of m/z 280. 3

Sample preparation
Liquid-liquid extraction was used to extract the drug and internal standard. For this purpose, 100 mL of respective plasma sample was placed in a polypropylene tube and mixed with 50 mL of internal standard (30.0 ng/mL). This was followed by addition of 100 mL of 10 mM KH 2 PO4 solution and 2.5 mL of methyl tertiary butyl ether (MTBE) and vortexed approximately for 5 min. The samples were then centrifuged at 4000 rpm for 10 min at 20 1C. Subsequently, the supernatant was transferred to respective polypropylene tubes and evaporated under a stream of nitrogen gas at 40 1C. After drying, samples were reconstituted with 200 mL of reconstitution solution (acetonitrile:5 mM ammonium formate (90:10, v/v)) and vortexed for 2 min. Finally, the samples were transferred to autosampler vials and 20 mL was injected into the LC-MS/MS.

Selectivity and specificity
Selectivity was determined by analyzing the blank human plasma samples from six different sources (donors) with an additional hemolyzed group and lipidemic group to assess interference at the retention times of analytes. The peak areas of SB, DSB and DDSB in blank samples should not be more than 20% of the mean peak area of LOQ of SB, DSB and DDSB. Similarly, peak areas of SB d7, DSB d7 and DDSB d7 in a blank sample should not be more than 5% of the mean peak area of LOQ of SB d7, DSB d7 and DDSB d7.

Precision and accuracy
Precision and accuracy were determined by replicate analysis of quality control samples (n¼6) at low quality control (LQC), medium quality control (MQC) and high quality control (HQC) levels. The CV should be less than 15% and accuracy should be within 15% except LLOQ where it should be within 20%.

Matrix effect
The matrix effect caused by plasma was evaluated by examining the ion suppression/enhancement in a signal when comparing the absolute response of QC samples after pretreatment (liquid-liquid extraction with MTBE) with that of the reconstituted samples. Experiments in triplicate were performed at MQC levels with 6 different plasma lots with the acceptable precision (%CV) of r15%.

Recovery
The extraction efficiencies of SB, DSB, DDSB and SB d7, DSB d7, and DDSB d7 were determined by analysis of 6 replicates at each quality control concentration level for SB, DSB and DDSB and at one concentration for the internal standard SB d7, DSB d7 and DDSB d7. The percent recovery was evaluated by comparing the peak areas of extracted standards to the peak areas of non-extracted standards (blank plasma residue).

Limits of detection and quantification (LOD and LOQ)
The limit of detection (LOD) is a parameter that defines the lowest concentration in a sample that can be detected from background noise but cannot be quantitated. LOD was determined using the signal-to-noise ratio (s/n) of 3:1 by comparing the results from samples of known concentrations of analytes with blank samples. The limit of quantitation (LOQ) is defined as the lowest concentration of an analyte that Table 1 Mass parameters of sibutramine (SB) and its two metabolites N-des methyl sibutramine (DSB), N-di des methyl sibutramine (DDSB) and sibutramine d7 (SB d7), N-des methyl sibutramine d7 (DSB d7), N-di des methyl sibutramine d7 (DDSB d7). can be determined with acceptable precision and accuracy. The LOQ was found by analyzing a set of plasma standards with known concentrations of SB, DSB and DDSB.

Stability
Stock solution stability was determined by comparing the area response of analyte and internal standard in the stability samples with the area response of samples prepared from fresh stock solution. Stability studies in plasma were performed at the LQC and HQC concentration levels using six replicates at each level. Analyte was considered stable if the % change is less than 15% as per US FDA guidelines [25].

Pharmacokinetics and statistical analysis
Post analysis was conducted and the pharmacokinetic parameters were obtained using Win-Nonlin s software version 9.2. Blood samples were collected for a period of 3-5 times that of the terminal elimination half-life (t 1/2 ). As per standard guidelines AUC 0-t , AUC 0-N , C max, t 1/2 and t max were calculated for SB, DSB and DDSB in plasma samples. According to regulatory guidelines AUC 0-t , AUC 0-N and C max values should be within 80-125% for test and reference products [26,27].

Method development and validation
The goal of this work was to develop and validate a simple, rapid and sensitive method for the quantitative determination of SB, DSB and DDSB in plasma samples. LC-MS/MS has been used as one of the most powerful analytical tools in clinical pharmacokinetics for its selectivity, sensitivity and reproducibility. The MS optimization was performed by direct infusion of SB, DSB, DDSB and SB d7, DSB d7, DDSB d7 solutions into the ESI source of the mass spectrometer. The vital parameters like ionization with positive mode, temperature at 400 1C, ion spray voltage at 5500 V, gas parameters such as nebulizer gas at 20 psi and heater gas at 40 psi were obtained and compound parameters like declustering potential (DP), entrance potential (EP), collision energy (CE) and collision cell exit Potential(CXP) were individually optimized (Table 1) to obtain the highest sensitivity and most efficient ionization to form the respective product ions from the protonated SB, DSB, DDSB and SB d7, DSB d7, DDSB d7 molecules (Fig. 2). Chromatographic conditions, especially the composition of the mobile phase and selection of suitable column, were optimized through several trials to achieve the best resolution and highest signals of analytes and internal standards. Different extraction methods like solid phase extraction, liquidliquid extraction and precipitation methods were optimized for extraction of SB, DSB, DDSB and SB d7, DSB d7, DDSB d7 from the plasma samples. Good separation and elution were achieved using 5 mM ammonium formate:acetonitrile (10:90, v/v) as the mobile phase, at a flow-rate of 0.6 mL/min with 20 mL injection volume. Liquid-liquid extraction was chosen to recover the drug and internal standard from the plasma. The retention time was optimized at 9.2, 8.2, 6.2 min for SB, DSB, DDSB and 9.1, 8.1, 6.2 min for SB d7, DSB d7, DDSB d7, respectively (Fig. 3).

Linearity
Calibration curve was plotted as the peak area ratio (SB/SB d7, DSB/DSB d7 and DDSB/DDSB d7) versus (SB, DSB, DDSB) concentration. Calibration was found to be linear over the concentration range of 10.0-10,000.0 pg/mL. The correlation coefficient (r 2 ) was greater than 0.9997 for all curves ( Table 2).

Selectivity
The selectivity of the method was assessed by comparing chromatograms of blank plasma. No significant endogenous peaks were observed at respective retention time of SB, DSB, DDSB and SB d7, DSB d7, DDSB d7. The results indicate that the method was specific and selective (Fig. 4).

Precision and accuracy
Precision and accuracy of this method were evaluated by calculating within-run and between-run variations at three concentrations (30.0, 3500.0 and 8000.0 pg/mL) of QC samples in six replicates. As shown in Table 3, within-run precision and accuracy were in the range of 1.

Matrix effect
The ion suppression/enhancement in the signal at MQC level was found to be CV 1.27 for SB, 1.20 for DSB and 1.30 for DDSB. These results indicate that there is minimum effect on ion suppression and ion enhancement at analyte and IS retention time.

Recovery
The extraction recoveries of SB, DSB and DDSB were determined at three different concentrations 30.0, 3500.0, 8000.0 pg/mL and found to be 99.     Table 4. Precision (CV) is less than 5% for room temperature, long-term, Freezethaw, and autosampler stabilities.  Figure 5 Mean plasma concentration of sibutramine, N-des methyl sibutramine, and N-di des methyl sibutramine.

Application to biological samples
The validated method was used successfully to quantify plasma SB, DSB and DDSB concentrations of 40 healthy volunteers who received oral administration of 15 mg dose (one 15 mg capsule) under fasting conditions. Mean plasma concentration versus time profiles are shown in Fig. 5. All the plasma concentrations of SB, DSB and DDSB were within the standard calibration region and remained above LOQ (10.0 pg/mL) for the entire sampling period. The detailed pharmacokinetic parameters are shown in Tables 5 and 6. In addition, the mean ratio of AUC 0-t /AUC 0-N was higher than 90% following the FDA Bioequivalence Guidelines [26,27]. The ratio of test/reference (T/R) for overall analysis was in the range of 80-125%.

Conclusion
The developed method is highly specific due to the inherent selectivity of tandem mass spectrometry and has significant advantages over previously reported methods in terms of selectivity, sensitivity, linearity and reproducibility. Quantification of sibutramine and its two metabolites was compared with respective isotope labeled internal standards. Extraction of analyte and IS was achieved using liquid-liquid extraction. Mobile phase, column, flow rate, injection volume, plasma usage volume for analysis were improved. The bioequivalence study also demonstrated that the two sibutramine formulations (reference and test) are bioequivalent.