Abstract: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-de… Show more
“…AUC 0–12 h and AUC 0–∞ were calculated. The ratio of AUC 0–12 h /AUC 0−∞ was found to be >85% for all subjects, as recommended by FDA guidelines [24] , [25] , [26] . Fig.…”
A simple, rapid and selective liquid chromatography–atmospheric pressure chemical ionization–mass spectrometry (LC–APCI–MS) assay method has been developed and fully validated for the simultaneous quantification of cefadroxil (CF) and clavulanic acid (CA) in human plasma. Analytes and internal standard (IS) were extracted from human plasma by solid-phase extraction (SPE) technique using Sam prep (3 mL, 100 mg) extraction cartridge. The extracted samples were chromatographed on a reverse phase C18 column using a mixture of methanol: acetonitrile: 2 mM ammonium acetate (pH 3.5) (25:25:50, v/v/v) as the mobile phase at a flow rate of 0.8 mL/min. Quantification of the analytes and IS were carried out using single quadrupole LC–APCI–MS through selected-ion monitoring (SIM) at m/z 362 and m/z 198, for CF and CA, respectively. Method validation was performed as per the FDA guidelines and the results met the acceptance criteria. Plasma concentration of CF and CA followed by the oral administration of CF/CA (500/125 mg) pill to healthy male volunteers (n=12) was measured. Area under plasma concentration–time curve from 0 to 12 h (AUC0–12 h) and 0 h extrapolated to infinity (AUC0−∞) were calculated. The ratio of AUC0–12 h/AUC0−∞ was found to be >85% for all the subjects, as recommended by the FDA guidelines.
“…AUC 0–12 h and AUC 0–∞ were calculated. The ratio of AUC 0–12 h /AUC 0−∞ was found to be >85% for all subjects, as recommended by FDA guidelines [24] , [25] , [26] . Fig.…”
A simple, rapid and selective liquid chromatography–atmospheric pressure chemical ionization–mass spectrometry (LC–APCI–MS) assay method has been developed and fully validated for the simultaneous quantification of cefadroxil (CF) and clavulanic acid (CA) in human plasma. Analytes and internal standard (IS) were extracted from human plasma by solid-phase extraction (SPE) technique using Sam prep (3 mL, 100 mg) extraction cartridge. The extracted samples were chromatographed on a reverse phase C18 column using a mixture of methanol: acetonitrile: 2 mM ammonium acetate (pH 3.5) (25:25:50, v/v/v) as the mobile phase at a flow rate of 0.8 mL/min. Quantification of the analytes and IS were carried out using single quadrupole LC–APCI–MS through selected-ion monitoring (SIM) at m/z 362 and m/z 198, for CF and CA, respectively. Method validation was performed as per the FDA guidelines and the results met the acceptance criteria. Plasma concentration of CF and CA followed by the oral administration of CF/CA (500/125 mg) pill to healthy male volunteers (n=12) was measured. Area under plasma concentration–time curve from 0 to 12 h (AUC0–12 h) and 0 h extrapolated to infinity (AUC0−∞) were calculated. The ratio of AUC0–12 h/AUC0−∞ was found to be >85% for all the subjects, as recommended by the FDA guidelines.
“…A good internal standard must mimic the analyte during extraction and compensate for any analyte on the column. The use of stable labeled isotopes of the analyte as an IS is recommended for bioanalytical assays to increase assay precision and limit variable recovery between analyte and the IS (Viswanathan et al ., ; Ponnuru et al ., ). For an LC‐MS/MS analysis, utilization of stable isotope‐labeled drugs as IS proves to be helpful when a significant matrix effect is possible.…”
A simple, rapid and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay method has been developed and fully validated for the simultaneous quantification of tetrabenazine and its active metabolites α-dihydrotetrabenazine and β-dihydrotetrabenazine in human plasma. Tetrabenazine d7 was used as internal standard (IS). The analytes were extracted from 200 μL aliquots of human plasma via solid-phase extraction using C18 solid-phase extraction cartridges. The reconstituted samples were chromatographed on a Zorbax SB C18 column using a 60:40 (v/v) mixture of acetonitrile and 5 mm ammonium acetate as the mobile phase at a flow rate of 0.8 mL/min. The API-4000 LC-MS/MS in multiple reaction-monitoring mode was used for detection. The calibration curves obtained were linear (r(2) ≥ 0.99) over the concentration range of 0.01-5.03 ng/mL for tetrabenazine and 0.50-100 ng/mL for α-dihydrotetrabenazine and β-dihydrotetrabenazine. Method validation was performed as per Food and Drug Administration guidelines and the results met the acceptance criteria. The method is precise and sensitive enough for its intended purpose. A run time of 2.5 min for each sample made it possible to analyze more than 300 plasma samples per day. The proposed method was found to be applicable to clinical studies.
“…The use of performance‐enhancing substances, particularly androgenic‐anabolic steroids (AAS) and stimulants, has been recognized as a common practice, especially in elite sports . Sibutramine is a drug classified as stimulant by the World Anti‐Doping Agency (WADA) that acts by inhibiting the re‐uptake of the neurotransmitters serotonin, norepinephrine and dopamine and enhancing satiety . Sibutramine undergoes primary and secondary metabolism, and its elimination occurs after biotransformation to demethylated metabolites, which are in turn hydroxylated and, finally, conjugated .…”
One of the greatest challenges in anti-doping science is the large number of substances available and the difficulty in finding the best analytical targets to detect their misuse. Therefore, metabolism studies involving prohibited substances are fundamental. However, metabolism studies in humans could face an important ethical bottleneck, especially for non-approved substances. An emerging model for metabolism assessment is the zebrafish, due to its genetic similarities with humans. In the present study, the ability of adult zebrafish to produce metabolites of sibutramine and stanozolol, substances with a well-known metabolism that are widely used as doping agents in sports, was evaluated. They represent 2 of the most abused classes of doping agents, namely, stimulants and anabolic steroids. These are classes that have been receiving attention because of the upsurge of synthetic analogues, for which the side effects in humans have not been assessed. The samples collected from the zebrafish tank water were hydrolysed, extracted by solid-phase extraction, and analysed by liquid chromatography with high resolution mass spectrometry (LC-HRMS). Adult zebrafish could produce several sibutramine and stanozolol metabolites, including demethylated, hydroxylated, dehydroxylated, and reduced derivatives, all of which have already been detected in human urine. This study demonstrates that adult zebrafish can absorb, oxidise, and excrete several metabolites in a manner similar to humans. Therefore, adult zebrafish seem to be a very promising tool to study human-like metabolism when aiming to find analytical targets for doping control.
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