Hematocrit-Independent Quantitation of Stimulants in Dried Blood Spots: Pipet versus Microfluidic-Based Volumetric Sampling Coupled with Automated Flow-Through Desorption and Online Solid Phase Extraction-LC-MS/MS Bioanalysis.

A workflow overcoming microsample collection issues and hematocrit (HCT)-related bias would facilitate more widespread use of dried blood spots (DBS). This report describes comparative results between the use of a pipet and a microfluidic-based sampling device for the creation of volumetric DBS. Both approaches were successfully coupled to HCT-independent, fully automated sample preparation and online liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis allowing detection of five stimulants in finger prick blood. Reproducible, selective, accurate, and precise responses meeting generally accepted regulated bioanalysis guidelines were observed over the range of 5-1000 ng/mL whole blood. The applied heated flow-through solvent desorption of the entire spot and online solid phase extraction (SPE) procedure were unaffected by the blood's HCT value within the tested range of 28.0-61.5% HCT. Enhanced stability for mephedrone on DBS compared to liquid whole blood was observed. Finger prick blood samples were collected using both volumetric sampling approaches over a time course of 25 h after intake of a single oral dose of phentermine. A pharmacokinetic curve for the incurred phentermine was successfully produced using the described validated method. These results suggest that either volumetric sample collection method may be amenable to field-use followed by fully automated, HCT-independent DBS-SPE-LC-MS/MS bioanalysis for the quantitation of these representative controlled substances. Analytical data from DBS prepared with a pipet and microfluidic-based sampling devices were comparable, but the latter is easier to operate, making this approach more suitable for sample collection by unskilled persons.

Liquid chromatography studies on the pharmacokinetics of phentermine and fenfluramine in brain and blood microdialysates after intraperitoneal administration to rats.

A highly sensitive and simple HPLC method with fluorescence detection for the determination of phentermine (Phen), fenfluramine (Fen) and norfenfluramine (Norf, the active metabolite of Fen) in rat brain and blood microdialysates has been developed. The brain and blood microdialysates were directly subjected to derivatization with 4-(4,5-diphenyl-1H-imidazol-2-yl) benzoyl chloride (DIB-Cl) in the presence of carbonate buffer (0.1 M, pH 9.0) at room temperature. The chromatographic conditions consisted of an ODS column and mobile phase composition of acetonitrile and water (65:35, v/v) with flow rate set at 1.0 ml/min. The detection was performed at excitation and emission wavelengths of 325 and 430 nm, respectively. Under these conditions, the DIB-derivatives of Phen, Fen and Norf were well separated and showed good linearities in the studied ranges (5-2000 nM for Phen and 10-2000 nM for Norf and Fen) with correlation coefficients greater than 0.999. The obtained detection limits were less than 23 fmol on column (for the three compounds) in both brain and blood microdialysates at a signal-to-noise ratio of 3 (S/N=3). The intra- and the inter-assay precisions were lower than 10%. The method coupled with microdialysis was applied for a pharmacokinetic drug-drug interaction study of Phen and Fen following individual and combined intraperitoneal administration to rats. In addition, since the role of protein binding in drug interactions can be quite involved, the method was applied for the determination of total and free Phen and Fen in rat plasma and ultrafiltrate, respectively. The results showed that Fen and/or Norf significantly altered the pharmacokinetic parameters of Phen in both blood and brain but did not alter its protein binding. On the other hand, there was no significant difference in the pharmacokinetics of Fen when administered with Phen.

Fluorometric determination of DL-fenfluramine, DL-norfenfluramine and phentermine in plasma by achiral and chiral high-performance liquid chromatography.

High-performance liquid chromatography (HPLC) with fluorescence detection has been developed for the simultaneous determination of sympathomimetic amines including ephedrine, norephedrine, 2-phenylethylamine, 4-bromo-2,5-dimethoxyphenylethylamine, phentermine (Phen) and DL-fenfluramine (Fen) in spiked human plasma. Furthermore, an enantioselective HPLC method for the separation of D-Fen (dexfenfluramine) and L-Fen (levofenfluramine) in addition to their active metabolites D- and L-norfenfluramine (Norf) is described. The detection was achieved at emission wavelength of 430 nm with excitation wavelength of 325 nm for both methods. The analytes were extracted from plasma (100 microl) at pH 10.6 with ethyl acetate using fluoxetine as the internal standard. The extracts were evaporated and derivatized with the fluorescence reagent 4-(4,5-diphenyl-1H-imidazole-2-yl)benzoyl chloride in the presence of carbonate buffer (pH 9.0). A gradient separation was achieved on a C18 column for the achiral separation or on a Chiralcel OD-R column for the chiral separation. The methods were fully validated, and shown to have excellent linearity, sensitivity and precision. The chiral method has been applied for the determination of D- and L-enantiomers of Fen and Norf, in addition to Phen in rat plasma after an intraperitoneal administration of DL-Fen and Phen, simultaneously.

High performance liquid chromatography with UV detection for the simultaneous determination of sympathomimetic amines using 4-(4,5-diphenyl-1H-imidazole-2-yl)benzoyl chloride as a label.

A high performance liquid chromatographic method has been developed for the simultaneous determination of (+/-) fenfluramine (Fen) and phentermine (Phen) in addition to three other sympathomimetic amines-ephedrine (E), norephedrine (NE) and 2-phenylethylamine (2-PEA), using cyclohexylamine (CX) as an internal standard in plasma. The compounds were derivatized with 4-(4,5-diphenyl-1H-imidazole-2-yl)benzoyl chloride (DIB-Cl) to give the DIB-derivatives. The derivatives were then separated using an isocratic HPLC system with UV detection. The limits of detection for Fen, Phen, E, NE and 2-PEA in plasma ranged from 0.32 to 22.9 pmol on column at a signal-to-noise ratio of 3. The recoveries following alkaline extraction from plasma samples of known concentrations were found to be more than 94% for the studied compounds. This method might be useful for the screening of the studied sympathomimetic amines in human plasma samples in forensic as well as toxicological studies. Furthermore, the developed method was modified for the simultaneous determination of Fen and Phen in human and rat plasma using fluoxetine as an internal standard. The methods are reproducible and precise. Finally, the two drugs were administered intraperitoneally to rats in combination, and their plasma levels over the investigated time course were successfully determined.

Plasma phentermine levels, weight loss and side-effects.

Fifty women with refractory obesity received phentermine resinate. Seven were withdrawn because of side-effects: three developed severe headaches, one each hypertension, depressive symptoms, breathlessness and palpitations with irritability. The mean weight loss in the 34 who completed the 20-week study was 6.4 kg. Nine lost 10 kg or more. Sustained appetite suppression was related to weight loss. Plasma phentermine concentrations did not correlate with the severity of the obesity problem, the degree of subjective anorexia or with weight loss. Poor initial response to standard dosage of phentermine is unlikely to improve with higher dosage. The individual's response to phentermine is unpredictable and appears to relate to factors other than the plasma drug concentration.