Determination of fluoxetine and its N-desmethyl metabolite in human plasma by high-performance liquid chromatography

A rapid and sensitive high-performance liquid chromatographic method has been developed for the simultaneous determination of the antidepressant fluoxetine and its active metabolite norfluoxetine in human plasma using paroxetine as internal standard. After liquid-liquid extraction, the compounds were separated on a C18 column using as mobile phase acetonitrile and 40 mM potassium dihydrogen phosphate buffer (pH 2.3) in the ratio 31:69 (v/v). The quantification of fluoxetine and norfluoxetine was made by fluorescence detection at Ex/Em 230/312 nm. The assay for each analyte was linear over the ranges 1-39 and 0.9-36 ng/ml, respectively. For both compounds intra- and inter-day accuracy and precision ranged between −7.9-12.4 and 0.7-14.7%, respectively. The method was applied to the analysis of plasma samples obtained from healthy subjects treated with one single oral dose of 40 mg fluoxetine.     

Determination of metoprolol and its alpha-hydroxylated metabolite in human plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method has been developed for the simultaneous determination of metoprolol and its alpha-hydroxylated metabolite in plasma, Metoprolol, alpha-hydroxymetoprolol and alprenolol (internal standard) are extracted from plasma at alkaline pH with diethyl ether-dichloromethane (4:1, v/v) and back-extracted with 0.01 N sulfuric acid. A 100-microliter volume of the acidic extract is injected into the chromatographic system. The compounds are eluted in about 12 min with acetonitrile-acetate buffer (75:25, v/v) on a LiChrosorb RP-8 (5 micron) column. The quantitative determinations are made fluorometrically. Concentrations down to 35 nmol/1 (10 ng/ml) of metoprolol base and 30 nmol/1 (8 ng/ml) of alpha-hydroxymetoprolol base in plasma can be determined with good precision and accuracy.     

Determination of imatinib and its active metabolite N-desmethyl imatinib in human plasma by liquid chromatography/tandem mass spectrometry

Imatinib is a first-line treatment for chronic myelogenous leukaemia (CML). The pharmacokinetics of imatinib in patients with CML are characterised by large interpatient variability. Concentration monitoring of imatinib and its active metabolite N-desmethyl imatinib (DMI) is considered necessary to enhance the safe and effective use of imatinib. A rapid, simple and sensitive liquid chromatography/tandem mass spectrometry assay was developed for the simultaneous determination of imatinib and its metabolite DMI in human plasma. After proteins were precipitated with acetonitrile, imatinib, DMI and the internal standard D8-imatinib were resolved on a Gemini-NX 3?μm C18 column using gradient elution of 0.05?% formic acid and methanol. The three compounds were detected using electrospray ionisation in the positive mode. Standard curves of imatinib and DMI were adequately fitted by quadratic equations (r?>?0.999) over the concentration range of 10 to 2,000?ng/mL which encompasses clinical concentrations. Bias was ≤±8.3?%, intra- and inter-day coefficients of variation (imprecision) were ≤8.0?% and the limit of quantification was 10?ng/mL for both imatinib and DMI. The assay is being used successfully in clinical practice to enhance the safe and effective use of imatinib.     

Determination of benperidol and its reduced metabolite in human plasma by high-performance liquid chromatography and electrochemical detection

An isocratic high-performance liquid chromatographic method with electrochemical detection for the quantification of benperidol and its suggested reduced metabolite TVX Q 5402 in human plasma is described. The method included a two-step solid-phase extraction on reversed-phase and cation-exchange material, followed by separation on a cyanopropyl silica gel column (5 microns; 250 mm x 4.6 mm I.D.). The eluent was 0.15 M acetate buffer (pH 4.7) containing 25% acetonitrile (w/w). Spiperone served as internal standard. The inclusion of the cation-exchange step provided sample purity higher than those achieved with other methods. After extraction of 1 ml of plasma, concentrations as low as 0.5 ng/ml were detectable for both benperidol and the metabolite. In plasma samples collected from a schizophrenic patient treated with a single oral dose of 6 mg of benperidol, plasma levels of benperidol and of the metabolite could be measured from 20 min to at least 12 h after administration.     

Determination of metoprolol and its alpha-hydroxide metabolite in serum by reversed-phase high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method for the simultaneous quantitation of metoprolol and alpha-hydroxymetoprolol concentrations in serum is developed. Analysis is performed on a C18 column using a 70:30 mixture of two solutions, A and B respectively. Solution A is 1 L of a 1% acetic acid solution with 25 mL of 0.005 M 1-heptanesulfonic acid. Solution B is 1 L of acetonitrile with 25 mL of 0.005 M 1-heptanesulfonic acid. Column effluent is monitored by fluorescence detection at an excitation wavelength of 225 nm. A 320-nm band pass filter is employed. The limit of sensitivity is approximately 2 ng/mL for both compounds. No potential sources of interference are identified. A coefficient of variation of less than 10% is observed on replicate determinations at 10 ng/mL for both compounds and at 300 and 180 ng/mL for metoprolol and alpha-hydroxymetoprolol, respectively. The method has the advantages of complete resolution of the metabolite of metoprolol, a high degree of reproducibility, adequate sensitivity, and an easily accessible internal standard.     

Determination of etretinate and its main metabolite in human plasma using normal-phase high-performance liquid chromatography

A high-performance liquid chromatographic procedure has been developed for the determination of astemizole and its primary metabolite in plasma and animal tissues. Both compounds and the internal standard were extracted from alkalinized plasma with heptane--isoamyl alcohol and analyzed using a reversed-phase column and UV monitoring at 254 nm. The detection limits for both compounds were 1 ng/ml of plasma and 5 ng/g of tissue and extraction recoveries were sufficiently high (71-84%). The method was applied to plasma and tissue samples from dogs after repeated oral administration, and to plasma samples from a volunteer taking a 300-mg oral dose of the drug. The results were compared with those obtained by a formerly developed radioimmunoassay.     

Determination of aniracetam and its main metabolite, N-anisoyl-GABA, in human plasma by high-performance liquid chromatography

Two different reversed-phase high-performance liquid chromatographic methods for the determination of aniracetam (I) and its metabolite N-anisoyl-GABA (II) in human plasma are described. The procedure for I involves direct injection of plasma samples spiked with the internal standard on a clean-up column followed by reversed-phase chromatography on a C18 column. The limit of quantification was 5 ng/ml, using a 200-microliters specimen of plasma. The mean inter-assay precision of the method up to 800 ng/ml was 3%. The procedure for II involved liquid-liquid extraction of II and the internal standard from plasma with ethyl acetate, and reversed-phase chromatography on a C18 column. The limit of quantification was 50 ng/ml using a 0.5-ml plasma specimen. The mean inter-assay precision up to 50 micrograms/ml was 6%. The applicability and accuracy of the methods were demonstrated by the analysis of over 1000 plasma samples from two bioavailability studies in healthy volunteers.     

Determination of thiopental in human serum and plasma by reversed-phase high-performance liquid chromatography

Summary A high-performance liquid chromatographic method for the analysis of thiopental in 100l of human serum or plasma is described. The procedure involves protein precipitation with acetonitrile. The supernatant is directly injected into a chromatograph containing a reversed-phase CLC-ODS (Shimadzu) column. A 5050 (v/v) mixture of water-acetonitrile, at a flow-rate of 1.0ml/min is used as the mobile phase. Detection is carried out ata wavelength of 280nm. Total analysis time per sample is 10min. The assay was found to be linear in the range of 0.1 to 120g/ml. Reproducibility was good, with intra-assay coefficients of variation from 1.780 to 3.208% and inter-assay coefficients of variation from 3.241 to 4.860%. The absolute recoveries were 97.4 to 101,4%. Other drugs were tested for potential interference with the assay, but none was found.     

Determination of benzyl alcohol and its metabolite in plasma by reversed-phase high-performance liquid chromatography.

A study undertaken following recent reports of deaths in neonatal children associated with the use of benzyl alcohol resulted in the development of a stability-indicating high-performance liquid chromatographic assay of benzyl alcohol in plasma using benzocaine as internal standard. Thawed plasma samples were diluted and subjected to solid-phase extraction using Extrelut and eluted with ethyl acetate. The evaporated eluate was reconstituted with mobile phase and chromatographed on a C18 column with water-acetonitrile-glacial acetic acid as mobile phase and detection at 254 nm. Baseline separation was achieved within 12 min for benzyl alcohol, benzaldehyde, benzoic acid, hippuric acid and benzocaine. Peak-height ratios were linear over 80-640 ng of benzyl alcohol injected (r = 0.998) and over 10-80 ng of benzoic acid injected (r = 0.999). Benzaldehyde and hippuric acid were not quantitated because these compounds were not detectable in actual dog plasma. Validation studies by spiking dog plasma with benzyl alcohol and benzoic acid gave overall percent recoveries (+/- relative standard deviation, n = 4) of 98.3 +/- 3.0 and 101.4 +/- 7.6%, respectively. The method was applied to the assay of actual plasma samples. Since benzyl alcohol is very susceptible to oxidation to benzaldehyde and benzoic acid, its purity in bulk liquid samples can be determined by this method.     

Determination of the active metabolite of molsidomine in human plasma by reversed-phase high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method, with ultraviolet detection, is proposed for the plasma determination of SIN-1, the active metabolite of molsidomine, which involves propoxycarbonyl derivatization. The internal standard is the ethoxycarbonyl derivative of SIN-1 (i.e. molsidomine). Derivatization and extraction are each performed in one step (2 min) with 70% yield. The nature of a by-product is discussed. The method provides rapid elution (less than 15 min), linearity over the range 0.4-200 ng/ml, day-to-day precision between 2.5 and 11.3% and a limit of determination of 0.5 ng/ml. This method is also suitable for the simultaneous determination of molsidomine and SIN-1. In this case the internal standard is an ethoxycarbonyl derivative of a piperazino-3-sydnonimine, a SIN-1 analogue.     

Determination of brodimoprim and its hydroxy metabolite in human plasma, blood and urine by high-performance liquid chromatography.

A high-performance liquid chromatographic method was developed to measure the concentration of brodimoprim and its metabolite, hydroxybrodimoprim, in small volumes of blood, plasma and urine. The procedure involved a simple extraction step with chloroform, followed by chromatographic separation on a short reversed-phase column deactivated for the analysis of basic compounds. The column effluent was monitored by fluorescence (excitation wavelength 290 nm, emission wavelength 340 nm). The recoveries of both compounds were similar in all three biological fluids, and averaged 84 and 72%, respectively. The detection limit for both compounds reached 5 ng/ml. No endogenous compound interfered in the assay. The linearity of the method and its within- and between-day precision were analytically satisfactory.     

Determination of clindamycin in plasma or serum by high-performance liquid chromatography with ultraviolet detection

A simple, sensitive high-performance liquid chromatographic assay for the determination of clindamycin in human plasma or serum has long been hampered by inability to separate it from endogenous compounds. We describe here such an assay. Proteins from a 200-microliters sample were precipitated by addition of acetonitrile containing the internal standard, triazolam. The sample was then vortex-mixed and centrifuged at approximately 3000 g for 10 min. The supernatant was evaporated to about 250 microliters under nitrogen, and 10-30 microliters were analyzed using an autoinjector. An octadecylsilane column with acetonitrile-water-phosphoric acid-tetramethylammonium chloride as mobile phase and ultraviolet detection at 198 nm provided a reproducibly quantifiable peak corresponding to 0.17 micrograms/ml. Retention times for clindamycin and triazolam averaged 8 and 11.8 min, respectively. Replicate standard curves run over a 36-h period showed no loss of integrity; r2 values generally exceeded 0.999. The method has successfully been applied to the analysis of samples taken up to 12 h after administration of intravenous infusions of 600-1200 mg in healthy volunteers.     

Determination of amineptine and its main metabolite in plasma by high-performance liquid chromatography after solid-phase extraction

Amineptine and its main metabolite were determined simultaneously in plasma by high-performance liquid chromatography using quinupramine as internal standard. The method comprised adsorption on Extrelut column from alkaline plasma, elution with diethyl ether-methylene chloride, evaporation in the presence of 0.01 M hydrochloric acid and injection of the acid solution onto a mu Bondapak C18 column, using acetonitrile-0.025 M potassium dihydrogenphosphate as mobile phase and ultraviolet detection at 210 nm. Average steady-state concentrations of the two compounds were determined in four patients under treatment regimen (two 100-mg doses of amineptine per day, at 8.00 and 12.00 h). The concentrations determined 20 h after the last dose were undetectable in all cases, whereas the concentrations determined 1 h after the second dose were found to be 780 +/- 96 ng ml-1 for amineptine and 690 +/- 137 ng ml-1 for its metabolite. The technique can also be applied to whole blood with, if necessary, identification on the basis of the ultraviolet spectrum obtained by photodiode-array detection.     

Determination of tenoxicam in human plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of tenoxicam in plasma has been developed. Tenoxicam was extracted from buffered plasma (pH 3 or 4, respectively) with dichloromethane and the evaporated extracts were analysed on a C18 reversed-phase column using a methanol-phosphate buffer mobile phase and with UV detection at 371 nm. The detection limit was 20 ng/ml using a 0.5-ml sample. The method is selective with respect to the 5'-hydroxy metabolite, which is present in plasma after multiple administration of tenoxicam; this metabolite may also be determined using this procedure.