Development of an LC-MS/MS method for the simultaneous quantification of aripiprazole and dehydroaripiprazole in human plasma

A selective, sensitive, and accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of aripiprazole and its active metabolite dehydroaripiprazole in human plasma has been developed using papaverine as internal standard (IS). LC-MS/MS analysis was carried out on a Finnigan LC-TSQ Quantum mass spectrometer using positive ion electrospray ionization (ESI⁺) and selected reaction monitoring (SRM). The assays for aripiprazole and dehydroaripiprazole were linear over the ranges of 0.1 to 600 ng/ml and 0.01 to 60 ng/ml, respectively. The average recoveries in plasma samples both were better than 85%. The intra- and interrun precision and accuracy values were found to be within the assay variability criteria limits according to the US Food and Drug Administration guidelines. The developed method was proved to be suitable for use in a clinical pharmacokinetic study after a single oral administration of a 5-mg aripiprazole tablet in healthy Chinese volunteers.     

High-performance liquid chromatographic assay for the determination of Aloe Emodin in mouse plasma

An isocratic high-performance liquid chromatography (HPLC) method was developed and validated to determine Aloe Emodin (AE) in mouse plasma. The analysis required 0.3 ml of plasma and involves extraction with dichloromethane. The HPLC separation was carried out on Symmetry Shield RP18, a mobile phase of methanol-water-acetic acid (65:35:0.2) and fluorescence detection at lambda(ex)=410 nm and lambda(em)=510 nm. The retention time of AE was 11.7 min. The assay was linear from 10 to 1,000 ng/ml (r2 > or = 0.999), showed intra- and inter-day precision within 7.8 and 4.7%, and accuracy of 87.3-105.7%. Detection limit (LOD) and quantification limit (LOQ) were 4.5 and 5 ng/ml, respectively. The method was applied to determine for the first time the pharmacokinetic of AE in mice.     

Application of solid-phase microextraction to the quantitative analysis of 1,8-cineole in blood and expired air in a Eucalyptus herbivore,the brushtail possum (Trichosurus vulpecula)

We have developed two solid-phase microextraction (SPME) methods, coupled with gas chromatography, for quantitatively analysing the major Eucalyptus leaf terpene, 1,8-cineole, in both expired air and blood from the common brushtail possum (Trichosurus vulpecula). In-line SPME sampling (5 min at 20 degrees C room temperature) of excurrent air from an expiratory chamber containing a possum dosed orally with 1,8-cineole (50 mg/kg) allowed real-time semi-quantitative measurements reflecting 1,8-cineole blood concentrations. Headspace SPME using 50 microl whole blood collected from possums dosed orally with 1,8-cineole (30 mg/kg) resulted in excellent sensitivity (quantitation limit 1 ng/ml) and reproducibility. Blood concentrations ranged between 1 and 1380 ng/ml. Calibration curves were prepared for two concentration ranges (0.05-10 and 10-400 ng/50 microl) for the analysis of blood concentrations. Both calibration curves were linear (r(2)=0.999 and 0.994, respectively) and the equations for the two concentration ranges were consistent.     

Development and validation of a high-performance liquid chromatography method using diode array detection for the simultaneous quantification of aripiprazole and dehydro-aripiprazole in human plasma

A high-performance liquid chromatography method with diode array detection (HPLC-DAD) was developed for quantification of aripiprazole and dehydro-aripiprazole, in human plasma. After a simple liquid-liquid extraction, chromatographic separation was carried out on a C18 reversed-phase column, using an ammonium buffer-acetonitrile mobile phase (40:60, v/v). The total run time was only 7 min at a flow-rate of 1.0 ml/min. The precision values were less than 12% and the accuracy values were ranging from 98 to 113% and the lower limit of quantification was 2 ng/ml for both compounds. Calibration curves were linear over a range of 2-1000 ng/ml. The mean trough plasma concentrations in patients treated with aripiprazole were 157 and 29 ng/ml for aripiprazole and dehydro-aripiprazole, respectively.     

High-performance liquid chromatography method with ultraviolet detection for the quantification of the BCR-ABL inhibitor nilotinib (AMN107) in plasma, urine, culture medium and cell preparations

An isocratic and sensitive HPLC assay was developed allowing the determination of the new anticancer drug nilotinib (AMN107) in human plasma, urine, culture medium and cell samples. After protein precipitation with perchloric acid, AMN107 underwent an online enrichment using a Zirchrom-PBD precolumn, was separated on a Macherey-Nagel C18-HD column and finally quantified by UV-detection at 258 nm. The total run time is 25 min. The assay demonstrates linearity within a concentration range of 0.005-5.0 microg/ml in plasma (r(2)=0.9998) and 0.1-10.0 microg/ml in urine (r(2)=0.9913). The intra-day precision expressed as coefficients of variation ranged depending on the spiked concentration between 1.27-9.23% in plasma and 1.77-3.29% in urine, respectively. The coefficients of variation of inter-day precision was lower than 10%. Limit of detection was 0.002 microg/ml in plasma and 0.01 microg/ml in urine. The described method is stable, simple, economic and is routinely used for in vivo and in vitro pharmacokinetic studies of AMN107.     

Sol-gel-based solid-phase microextraction and gas chromatography-mass spectrometry determination of dextromethorphan and dextrorphan in human plasma

A novel solid-phase microextraction (SPME) method was developed for isolation of dextromethorphan (DM) and its main metabolite dextrorphan (DP) from human plasma followed by GC-MS determination. Three different polymers, poly(dimethylsiloxane) (PDMS), poly(ethylenepropyleneglycol) monobutyl ether (Ucon) and polyethylene glycol (PEG) were synthesized as coated fibers using sol-gel methodologies. DP was converted to its acetyl-derivative prior to extraction and subsequent determination. The porosity of coated fibers was examined by SEM technique. Effects of different parameters such as fiber coating type, extraction mode, agitation method, sample volume, extraction time, and desorption condition, were investigated and optimized. The method is rapid, simple, easy and inexpensive and offers high sensitivity and reproducibility. The limits of detection are 0.010 and 0.015 ng/ml for DM and DP, respectively. The precisions for both analytes are below 5% (n=5). The correlation coefficient was satisfactory (r(2)>0.99) for both DM and DP. Linear ranges were obtained from 0.03 ng/ml to 2 microg/ml for DM and from 0.05 ng/ml to 2 microg/ml for DP.     

Determination of disodium clodronate in human plasma and urine using gas-chromatography-nitrogen-phosphorous detections: validation and application in pharmacokinetic study

We present a specific method for the determination of disodium clodronate in human plasma and urine using a gas-chromatographic system with nitrogen phosphorus detector (NPD). The compound was extracted from plasma and urine samples by an anion-exchange resin and derivatizated with bistrimethylsilyltrifluoroacetamide (BSTFA). Sodium bromobisphosphonate was used as internal standard. The calibration curves were linear in both plasma and urine, with a regression coefficient r > 0.9975 in plasma and r > 0.9977 in urine.The limit of quantitation was 0.3 microg/ml in plasma and 0.5 microg/ml in urine. The method was validated by intra-day assays at three concentration levels. During the study we carried out inter-day assays to confirm the feasibility of the method. The precision in plasma at 0.5, 15, and 45 microg/ml was 12.4, 0.2, and 6.5% (n = 40), respectively; in urine at 0.8, 8, and 40 microg/ml it was 8.6, 6.4, and 9.3% (n = 40), respectively.The method was accurate and reproducible, and was successfully applied to determine the pharmacokinetic parameters of clodronate in healthy volunteers after intravenous infusion and intramuscular injection of 200 mg of the compound. The Cmax after intravenous infusion and intramuscular injection was 16.1 and 12.8 microg/ml, respectively. AUC(0-48 h) after infusion administration and intramuscular injection was 44.2 +/- 18.0 and 47.5 +/- 12.4 h microg/ml, respectively. The elimination half-life in both administrations was 6.31 +/- 2.7 h.     

Age, insulin, SHBG and sex steroids exert secondary influence on plasma leptin level in women

AIM: As the link between body fat and leptin is well known, the aim of the study was to seek for secondary regulators of plasma leptin level. PATIENTS: 86 women (mean: age 47.0+/-14.3 years; estradiol 50.0+/-60.6 ng/l; FSH 52.4+/-42.9 IU/l; BMI 26.9+/-5.9) divided into three groups according to their BMI. Group A: 39 normal weight women (mean: age 44.4+/-16.0 years; estradiol 69.6+/-79.8 ng/l; FSH 50.4+/-47.7 IU/l; BMI 22.9+/-1.3). Group B: 27 overweighted women (mean: age 55.0+/-6.4 years; estradiol 25.1+/-17.2 ng/l; FSH 75.6+/-26.3 IU/l; BMI 27.7+/-1.6). Group C: 21 obese women with mean: age 48.7+/-12.2 years; estradiol 36.9+/-44.0 ng/l; FSH 42.3+/-36.6 IU/l and BMI 34.6+/-4.9. METHODS: Standard clinical evaluation and hormone evaluation (LH, FSH, prolactin, estradiol, leptin, insulin-like growth factor-I (IGF-I), human growth hormone (hGH), insulin-like growth factor binding protein-3 (IGFBP-3), insulin, dihydroepiandrosterone sulphate (DHEAS), sex hormone binding globin (SHBG) and testosterone were done in basic condition which levels of were measured by RIA kits. Statistical analysis. Shapiro-Wilk test, Mann-Whitney-Wilcoxon u test, Spearman rank correlation coefficient and stepwise multiple regression: p values of 0.05 or less were considered as significant. RESULTS: Taking all women into account (n=86) the plasma leptin level correlated directly with age (r=0.32; p<0.02), body mass (r=0.60; p<0.001), BMI (r=0.71; p<0.001) as well as inversely with estradiol (r=-0.21; p<0.05), IGF-I (r=-0.24; p<0.05), SHBG (r=-0.34; p<0.01) and DHEAS (r=-0.30; p<0.01). However only in the group B leptin/age relation remained (r=0.40; p<0.05) after the division according to BMI. In the group B the leptin /DHEAS (r=-0.40; p<0.05) and leptin/PRL (r=0.51; p<0.05) links were also present. In the group C the leptin/SHGB relation (r=-0.56; p<0.02) only remained and an association between insulin and leptin was found (r=0.48; p<0.05). The body mass and BMI relation to age were again present only in all 86 women (r=0.30; p<0.002: r=0.36; p<0.001 resp.). Having split the women into groups, these links either disappeared or became inverse (rC=-0.39; p<0.05). Taking into consideration age/leptin relation in all women, the division according to the menopausal status revealed the direct relation in premenopausal women (n=29; r=0.43; p<0.02) and a reverse one in postmenopausal women (n=38; r=-0.32; p<0.05). The plasma leptin level was the highest (p<0.001) in group C (23.2+/-10.4 microg/l) and the lowest was found in the group A (8.9+/-4.1 microg/l). That corresponded with the differences in mean body mass index and mean body mass. The stepwise multiple regression revealed that body mass index accounted for 31% (p<0.001) and plasma SHBG level accounted for 17.7% (p<0.02) of plasma leptin variance in all women. In the group A body mass and age together accounted for 61% (p<0.01) and estradiol alone accounted for 44% (p<0.02) of plasma leptin variance. In the group B insulin alone accounted for 39% (p<0.05) and together with testosterone accounted for 46% (p<0.05) of plasma leptin variance. Finally in obese women none of the evaluated parameters significantly accounted for leptin variance. CONCLUSION: The results presented in this paper confirmed the strong influence of body fat mass on serum leptin concentration. However insulin, SHBG, sex steroids as well as age may also exert secondary influence on plasma leptin level in certain groups of women.     

Development of a highly sensitive high-performance liquid chromatographic method for measuring an anticancer drug, UCN-01, in human plasma or urine

We have established a highly sensitive high-performance liquid chromatographic method for the determination of an anticancer drug, UCN-01, in human plasma or urine. Using a fluorescence detector set at an excitation wavelength of 310 nm and emission monitored at 410 nm, there was a good linearity for UCN-01 in human plasma (r=0.999) or urine (r=0.999) at concentrations ranging from 0.2 to 100 ng/ml or 1 to 400 ng/ml, respectively. For intra-day assay, in plasma samples, the precision and accuracy were 1.8% to 5.6% and −10.0% to 5.2%, respectively. For inter-day assay, the precision and accuracy were 2.0% to 18.2% and 2.4% to 10.0%, respectively. In urine samples, the intra- and inter-day precision and accuracy were within 3.9% and ±2.7%, respectively. The lower limit of quantification (LLOQ) was set at 0.2 ng/ml in plasma and 1 ng/ml in urine. UCN-01 in plasma samples was stable up to two weeks at −80°C and also up to four weeks in urine samples. This method could be very useful for studying the human pharmacokinetics of UCN-01.     

Simultaneous determination of selegiline and desmethylselegiline in human body fluids by headspace solid-phase microextraction and gas chromatography-mass spectrometry

A method for the simultaneous determination of selegiline and its metabolite, desmethylselegiline, in human whole blood and urine is presented. The method, which combines a fiber-based headspace solid-phase microextraction (SPME) technique with gas chromatography-mass spectrometry (GC-MS), required optimization of various parameters (e.g., salt additives, extraction temperatures, extraction times and the extraction properties of the SPME fiber coatings). Pargyline was used as the internal standard. Extraction efficiencies for both selegiline and desmethylselegiline were 2.0-3.4% for whole blood, and 8.0-13.2% for urine. The regression equations for selegiline and desmethylselegiline extracted from whole blood were linear (r(2)=0.996 and 0.995) within the concentration ranges 0.1-10 and 0.2-20 ng/ml, respectively. For urine, the regression equations for selegiline and desmethylselegiline were linear (r(2)=0.999 and 0.998) within the concentration ranges 0.05-5.0 and 0.1-10 ng/ml, respectively. The limit of detection for selegiline and desmethylselegiline was 0.01-0.05 ng/ml for both samples. The lower and upper limits of quantification for each compound were 0.05-0.2 and 5-20 ng/ml, respectively. Intra- and inter-day coefficients of variation for selegiline and desmethylselegiline in both samples were not greater than 8.7 and 11.7%, respectively. The determination of selegiline and desmethylselegiline concentrations in Parkinson's disease patients undergoing continuous selegiline treatment is presented and is shown to validate the present methodology.     

Determination of midazolam and its major metabolite 1'-hydroxymidazolam by high-performance liquid chromatography-electrospray mass spectrometry in plasma from children

We have developed a sensitive, selective and reproducible reversed-phase high-performance liquid chromatography method coupled with electrospray ionization mass spectrometry (HPLC-ESI-MS) for the simultaneous quantification of midazolam (MDZ) and its major metabolite, 1'-hydroxymidazolam (1'-OHM) in a small volume (200 microl) of human plasma. Midazolam, 1'-OHM and 1'-chlordiazepoxide (internal standard) were extracted from alkalinised (pH 9.5) spiked and clinical plasma samples using a single step liquid-liquid extraction with 1-chlorobutane. The chromatographic separation was performed on a reversed-phase HyPURITY Elite C18 (5 microm particle size; 100 mm x 2.1mm i.d.) analytical column using an acidic (pH 2.8) mobile phase (water-acetonitrile; 75:25% (v/v) containing formic acid (0.1%, v/v)) delivered at a flow-rate of 200 microl/min. The mass spectrometer was operated in the positive ion mode at the protonated-molecular ions [M+l]+ of parent drug and metabolite. Calibration curves in spiked plasma were linear (r2 > or = 0.99) from 15 to 600 ng/ml (MDZ) and 5-200 ng/ml (1'-OHM). The limits of detection and quantification were 2 and 5 ng/ml, respectively, for both MDZ and 1'-OHM. The mean relative recoveries at 40 and 600 ng/ml (MDZ) were 79.4+/-3.1% (n = 6) and 84.2+/-4.7% (n = 8), respectively; for 1'-OHM at 30 and 200 ng/ml the values were 89.9+/-7.2% (n = 6) and 86.9+/-5.6% (n = 8), respectively. The intra-assay and inter-assay coefficients of variation (CVs) for MDZ were less than 8%, and for 1'-OHM were less than 13%. There was no interference from other commonly used antimalarials, antipyretic drugs and antibiotics. The method was successfully applied to a pharmacokinetic study of MDZ and 1'-OHM in children with severe malaria and convulsions following administration of MDZ either intravenously (i.v.) or intramuscularly (i.m.).     

Development and validation of an LC-MS/MS method for the quantitative determination of aripiprazole and its main metabolite, OPC-14857, in human plasma

An accurate, sensitive, reproducible, and selective liquid chromatography/tandem mass spectrometry (LC-MS/MS) method for determination of aripiprazole and its main metabolite, OPC-14857, in human plasma was developed and validated. Chromatographic separation was achieved isocratically on a C18 reversed-phase column within 7.5 min. The calibration curve, ranging from 0.1 to 100 ng/ml, was fitted to a 1/y2-weighted linear regression model. The assay showed no significant interference. Lower limit of quantitation (LLOQ) for both analytes was 0.1 ng/ml using 0.4 ml of plasma. Intra- and inter-assay precision and accuracy values for aripiprazole and OPC-14857 were within regulatory limits.     

Quantitative trace analysis of estriol in human plasma by negative ion chemical ionization gas chromatography-mass spectrometry using a deuterated internal standard

A stable isotope dilution gas chromatography-mass spectrometry (GC-MS) assay for the trace level determination of estriol in human plasma is described. Negative ion chemical ionization (NICI) MS is used for highly specific detection. The method involves derivatization of the phenolic hydroxyl to the pentafluorobenzyl ether derivative and subsequent reaction of the remaining hydroxyls with heptafluorobutyric anhydride. This derivative allows detection of the strikingly abundant phenolate ion under NICI conditions. [2,4,17beta]-2H(3)-labeled estriol was used as an internal standard. For high-level measurements (>313 ng/l) plasma was directly derivatized by extractive alkylation followed by heptafluorobutylation prior to analysis. A rapid and simple sample work up procedure was elaborated for trace level determinations (>5 ng/l plasma) using solid-phase extraction on C(18) with an absolute recovery of 92.9%. For low-level measurements, the calibration curve was linear in the range of 5 to 625 ng/l (r=0.99993). Inter-assay analytical precisions (RSDs) were 1.29, 2.30 and 2.89% at 39, 156 and 650 ng/l plasma, respectively. For high-level measurements, calibration curve linearity was observed in the range of 0.313 to 20 microg/l (r=0.99998). Inter-assay analytical precisions (RSDs) were 5.17, 1.92, 2.57 and 2.74% at 0.313, 0.625, 2.5 and 10 microg/l plasma, respectively. Postmenopausal plasma was used for spiked plasma samples. Sensitivity and specificity of the presented method allows adequate determination of estriol in human plasma samples.     

Development and validation of a reverse-phase HPLC with fluorescence detector method for simultaneous determination of CZ48 and its active metabolite camptothecin in mouse plasma

A simple and sensitive high-performance liquid chromatography (HPLC) assay for the analysis of CZ48, a potent anticancer candidate, and its active metabolite camptothecin (CPT) in mouse plasma was developed and validated. CZ44 was used as an internal standard (IS). The samples were injected onto a C18 Synergi Polar-RP column (4 microm, 150 mm x 4.60 mm) maintained at 30 degrees C. The identification of peaks showed high specificity. Shimadzu RF-10AXL fluorescence detector was used at the excitation and emission of 380 and 418 nm, respectively. The mean recoveries were 81.41+/-0.035%, 86.00+/-0.053% and 82.21+/-0.020% for CZ48 and 76.01+/-0.028%, 77.04+/-0.042% and 85.93+/-0.023% for CPT at three concentrations of 10, 100 and 900 ng/ml, respectively. The calibration curve was linear (r(2)=0.9999) over CZ48 and CPT concentrations ranging from 5 to 1000 ng/ml and 10-1000 ng/ml (n=6), respectively. The method had an accuracy of >95% and intra- and inter-day precision (RE%) of <1.2% and <2.2% for CZ48 and CPT, respectively, at three different concentrations (10, 100 and 900 ng/ml). The lower limit of quantification (LLOQ) using 0.1 ml mouse plasma was 10 ng/ml for CZ48 and 5 ng/ml for CPT. Stability studies showed that CZ48 and CPT were stable in mouse plasma after 4h incubation at room temperature or after 1 month storage at -80 degrees C with three freeze/thaw cycles. The method reported is simple, reliable, precise and accurate and confirmed by the determination of plasma samples in the mice after oral administration of CZ48.     

Improved RP-HPLC method to determine neferine in dog plasma and its application to pharmacokinetics

Existing methods to determine neferine, a bisbenzylisoquinline alkaloid, either have no internal standard or lack selectivity, or take longer time. Here an improved reverse-phase high-performance liquid chromatographic (RP-HPLC) method was established in biological samples. The extraction recovery was 90.9% for neferine at concentration level of 0.2 microg/ml and 77.7% for dauricine (the internal standard) at 5 microg/ml in dog plasma, respectively. The linear quantification range of the method was 25-2000 ng/ml in dog plasma, with linear correlation coefficients greater than 0.999. The intra-day and inter-day relative standard deviations (R.S.D.s) for neferine at 50, 200 and 1000 ng/ml levels in dog plasma fell in the range of 3.0-5.4% and 4.3-9.5%, respectively. The RP-HPLC method was successfully applied to a pharmacokinetics study, in which experimental dogs received a single dose of neferine (5 mg/kg i.v. or 10 mg/kg p.o.). The pharmacokinetic result was presented.     

A HPLC-mass spectrometric method suitable for the therapeutic drug monitoring of everolimus

We report here the validation of an HPLC-electrospray-tandem mass spectrometry method for the quantification of everolimus, an immunosuppressant drug. Whole blood samples (100 microl) were extracted by protein precipitation which involved sample pre-treatment with zinc sulphate followed by acetonitrile (containing internal standard, 40-O-(3'-hydroxy)propyl-rapamycin). HPLC was performed using a step-gradient at a flow rate of 0.6 ml/min on a Waters TDM C18 column (10 mm x 2.1mm I.D.) with a resultant chromatographic analysis time of 2 min. Mass spectrometric detection by selected reaction monitoring (everolimus m/z 975.5-->908.3; internal standard m/z 989.5-->922.3). The assay was linear from 0.5 to 40 microg/l (r2>0.994, n=11). The inter- and intra-day analytical recovery and imprecision for quality control samples (1.25, 12.5 and 30 microg/l) were 93.4-98.2% and <10.7%, respectively (n=10). At the lower limit of quantification (0.5 microg/l) the inter- and intra-day analytical recovery was 94.4-95.8% with imprecision of <14.1% (n=10). The absolute recovery of everolimus (6.5 microg/l) and internal standard (12.5 microg/l) was 96.5 and 88.3%, respectively (n=3). A comparison of our method against the mean of all HPLC methods for a series of samples from an external proficiency testing scheme revealed good correlation as shown by the regression analysis: y=0.973x+0.301 (r2=0.986, n=71). In conclusion, the method described is suited to the current requirements for therapeutic drug monitoring of everolimus.     

Study of plasma aldosterone in normal pregnancy, in pre-eclamptic women and in cord plasma of newborns.

A radioimmunoassay without chromatography was used for the determination of plasma aldosterone in pregnancy. The mean values (+/- S.D.) of aldosterone concentration increased consistently from 23.2 +/- 5.3 ng/100 ml (n = 14) during the first trimester to 37.2 +/- 10.6 ng/100 ml (n = 17) during the second trimester and 64.0 +/- 18.8 ng/100 ml (n = 29) during the third trimester of pregnancy. The highest values were found at delivery (71.9 +/- 14.2 ng/100 ml; n = 21) and in the cord plasma of newborns (83.4 +/- 14.9 ng/100 ml; n = 21). Significantly lower plasma aldosterone values were found in the plasma of pre-eclamptic women during the third trimester of pregnancy (41.9 +/- 21.3 ng/100 ml; n = 11).     

A simple HPLC method for the determination of bifendate: application to a pharmacokinetic study of bifendate liposome

A rapid, sensitive and simple high-performance liquid chromatographic (HPLC) method with ultraviolet detector (UV) has been developed for the determination of bifendate in 100 microl plasma of rats. Sample preparation was carried out by deproteinization with 100 microl of acetonitrile. A 20 microl of supernatant was directly injected into the HPLC system with methanol-double distilled water (65/35, v/v) as the mobile phase at a flow rate of 1.0 ml/min. Separation was performed with a microBondapak C(18) column at 30 degrees C. The peak was detected at 278 nm. The calibration curve was linear (r(2)=0.9989) in the concentration range of 0.028-2.80 microg/ml in plasma. The intra- and inter-day variation coefficients were not more than 6.55% and 6.07%, respectively. The limit of detection was 5 ng/ml. The mean recoveries of bifendate were ranged from 94.53% to 99.36% in plasma. The present method has been successfully applied to the pharmacokinetic study of bifendate liposome in rats.     

Determination of fluoxetine, norfluoxetine and their enantiomers in rat plasma and brain samples by liquid chromatography with fluorescence detection

Fluoxetine (FLX) and norfluoxetine (NFLX) racemic mixtures were determined by reversed-phase liquid chromatography with fluorescence detection (lambda(exc)=227 nm, lambda(em)=305 nm). The calibration curves prepared from drug-free plasma and brain were linear in the range of 5-1000 ng ml(-1) and 100-40,000 ng g(-1) for doped samples, with detection limits of 3.2 and 2.1 ng ml(-1) in plasma and 31.5 and 26.1 ng g(-1) in brain tissue for FLX and NFLX, respectively. Enantiomer determination was carried out through normal phase HPLC-FD (lambda(exc)=224 nm, lambda(em)=336 nm) after precolumn chiral derivatization with R-1-(1-naphthyl)ethyl isocyanate. Standard curves also prepared in a drug-free matrix were linear for each enantiomer over the range of 2-1000 ng ml(-1) and 20-7000 ng g(-1) with detection limits for the four compounds ranging between 0.2 and 0.5 ng ml(-1) in plasma and between 3.0 and 8.2 ng g(-1) in brain tissue. In both methods the analytes were isolated from the biological matrix by a new solid-phase extraction procedure with recovery in plasma and brain over 90 and 87%, respectively. The repeatability of this extraction procedure was satisfactory within-day and between-day with CV<9.1%. This study also offered the opportunity to obtain an assessment of the potential relationships between the concentration of individual enantiomers of FLX and NFLX in plasma and brain tissue after chronic treatment with racemic FLX at a dose intended to mimic the human plasma concentration of FLX in standard clinical conditions, and therefore should make for more reliable extrapolation of neurochemical findings in other species.     

Development and validation of a rapid HPLC method for simultaneous determination of tramadol, and its two main metabolites in human plasma

Tramadol, an analgesic agent, and its two main metabolites O-desmethyltramadol (M1) and N-desmethyltramadol (M2) were determined simultaneously in human plasma by a rapid and specific HPLC method. The sample preparation was a simple extraction with ethyl acetate. Chromatographic separation was achieved with a Chromolith Performance RP-18e 50 mm x 4.6 mm column, using a mixture of methanol:water (13:87, v/v) adjusted to pH 2.5 by phosphoric acid, in an isocratic mode at flow rate of 2 ml/min. Fluorescence detection (lambda(ex)=200 nm/lambda(em)=301 nm) was used. The calibration curves were linear (r(2)>0.997) in the concentration range of 2.5-500 ng/ml, 1.25-500 ng/ml and 5-500 ng/ml for tramadol, M1 and M2, respectively. The lower limit of quantification was 2.5 ng/ml for tramadol, 1.25 ng/ml for M1 and 5 ng/ml for M2. The within- and between-day precisions in the measurement of QC samples at four tested concentrations were in the range of 2.5-9.7%, 2.5-9.9% and 5.9-11.3% for tramadol, M1 and M2, respectively. The developed procedure was applied to assess the pharmacokinetics of tramadol and its two main metabolites following administration of 100mg single oral dose of tramadol to healthy volunteers.