Simultaneous determination of citalopram, fluoxetine, paroxetine and their metabolites in plasma by temperature-programmed packed capillary liquid chromatography with on-column focusing of large injection volumes

A miniaturized temperature-programmed packed capillary liquid chromatographic method with on-column large volume injection and UV detection for the simultaneous determination of the three selective serotonin reuptake inhibitors citalopram, fluoxetine, paroxetine and their metabolites in plasma is presented. An established reversed-phase C8 solid-phase extraction method was employed, and the separation was carried out on a 3.5-microm Kromasil C18 0.32x300 mm column with temperature-programming from 35 (3 min) to 100 degrees C (10 min) at 1.3 degrees C/min. The mobile phase consisted of acetonitrile-45 mM ammonium formate (pH 4.00) (25:75, v/v). The non-eluting sample focusing solvent composition acetonitrile-45 mM ammonium formate (pH 4.00) (3:97, v/v) allowed injection of 10 microl or more of the plasma extracts. The method was validated for the concentration range 0.05-5.0 microM, and the calibration curves were linear with coefficients of correlation >0.993. The limits of quantification for the antidepressants and their metabolites ranged from 0.05 to 0.26 microM. The within and between assay precision of relative peak height were in the range 2-22 and 2-15% relative standard deviation, respectively. The within and between assay recoveries were in the 61-99 and 54-92% range for the antidepressants, respectively, and between 52-102 and 51-102% for the metabolites.     

Rapid determination of citalopram in human plasma by high-performance liquid chromatography

A rapid high-performance liquid chromatographic method for the quantitation of citalopram in human plasma is presented. The sample preparation involved liquid–liquid extraction of citalopram with hexane–isoamyl alcohol (98:2 v/v) and back-extraction of the drug to 0.02 M hydrochloric acid. Liquid chromatography was performed on a cyano column (45×4.6 mm, 5 μm particles), the mobile phase consisted of an acetonitrile–phosphate buffer, pH 6.0 (50:50, v/v). The run time was 2.6 min. The fluorimetric detector was set at an excitation wavelength of 236 nm and an emission wavelength of 306 nm. Verapamil was used as the internal standard. The limit of quantitation was 0.96 ng/ml using 1 ml of plasma. Within- and between-day precision expressed by relative standard deviation was less than 7% and inaccuracy did not exceed 6%. The assay was applied to the analysis of samples from a pharmacokinetic study.     

Determination of fluoxetine and norfluoxetine in human plasma by high-performance liquid chromatography with ultraviolet detection in psychiatric patients

A rapid high-performance liquid chromatographic method is described for the simultaneous determination of the widely used antidepressant drug, fluoxetine and its principal metabolite norfluoxetine in plasma. After liquid-liquid extraction the compounds were separated in a reversed-phase column and assayed by ultraviolet absorption at 226 nm. The analytical interference from psychoactive drugs and their metabolites was also studied. The extraction recoveries were 93 and 87% for norfluoxetine and fluoxetine, respectively. The limit of quantitation under the described conditions was 14 nmol/l for both compounds. The method was found to be reproducible with coefficients of variation less than 10%. A great variability in plasma concentrations of fluoxetine and norfluoxetine as well as in fluoxetine/norfluoxetine ratios was found among the 29 patients studied. This result suggests the implication of genetically polymorphic enzymes, presumably CYP2D6, CYP2C9 and CYP2C19 in the metabolism of fluoxetine to norfluoxetine. Therapeutic drug monitoring should thus be useful in patients treated with regular doses.     

Quantification of fluoxetine and norfluoxetine serum levels by reversed-phase high-performance liquid chromatography with ultraviolet detection

A rapid and sensitive high-performance liquid chromatography assay method was developed to determine serum fluoxetine and norfluoxetine levels by single extraction of 0.1 ml of serum with sodium hydroxide. The mobile phase (55% acetonitrile–45% distilled water containing 10 mM aqueous triethylamine) was used to separate fluoxetine and norfluoxetine (25–1000 ng/ml, using clomipramine as the internal standard) by ultraviolet detection at 226 nm. The inter- and intra-day variabilities of fluoxetine and norfluoxetine were 13–18%, and the recoveries of both drugs exceeded 89%. This assay method was applied to a pharmacokinetic disposition study of fluoxetine in mice.     

Simultaneous determination of citalopram and its metabolites by high-performance liquid chromatography with column switching and fluorescence detection by direct plasma injection

High-performance liquid chromatography with a successive column-switching technique was developed for simultaneous determination of citalopram and its four metabolites in plasma. Plasma samples were injected directly, and the target compounds were purified and concentrated with an inexpensive commercial octadecyl guard column. Then, the six-port valve was switched, and the compounds retained in the column were eluted by the back-flush method using 20 mM phosphate buffer (pH 4.6)-acetonitrile (70:30, v/v) containing 0.1% diethylamine and separated with an ODS column. The compounds were assayed with a fluorescence detector at an excitation wavelength of 249 nm and an emission wavelength of 302 nm. At least 30 plasma samples could be treated with an octadecyl guard column. The limits of quantitation of this method were 2.0 ng/ml for citalopram, desmethylcitalopram, didesmethylcitalopram, citalopram propionic acid and citalopram N-oxide. This method was applied to a pharmacokinetic study in dogs and a toxicokinetic study in rats.     

Determination of paroxetine in plasma by high-performance liquid chromatography for bioequivalence studies

A high-performance liquid chromatographic method is described for the determination of paroxetine in human plasma. Dibucaine was used as the internal standard. Paroxetine was isolated by solid phase extraction using a Bond-Elut C₁₈ extraction column. Separation was obtained using a reversed-phase column under isocratic conditions with fluorescence detection. The sample volume was 500 μl of plasma. The intra- and inter-assay accuracy and precision, determined as relative error and relative standard deviation, respectively, were less than 10%. The lower limit of quantitation, based on standards with acceptable relative error and relative standard deviation, was 10 ng ml⁻¹. No endogenous compounds were found to interfere. The linearity was assessed in the range 5–100 ng ml⁻¹. Stability of paroxetine during processing (autosampler) and in plasma was checked. This method proved suitable for bioequivalence studies following multiple doses in healthy volunteers.     

Simultaneous determination of tricarboxylic acid cycle metabolites by high-performance liquid chromatography with ultraviolet detection

Here we describe a simple high-performance liquid chromatography (HPLC) procedure for the simultaneous detection and quantitation in standard solutions of 13 important metabolites of cellular energy metabolism, including 9 tricarboxylic acid (TCA) cycle components and 4 additional metabolites. The metabolites are detected by their absorbance at 210 nm. The procedure does not require prior derivatization, and an analysis can be carried out at ambient temperature within 15 min. The significance of the current work is that the current HPLC procedure should motivate the development of simplified TCA cycle enzyme assays, isotopomer analysis, and determination of selected TCA metabolite levels in plasma/tissues.     

Rapid simple high-performance liquid chromatographic determination of paroxetine in human plasma

A rapid, simple method for the measurement of paroxetine in human plasma by reversed-phase high-performance liquid chromatography (HPLC) with fluorescence detection is described. This method includes only one-step extraction of paroxetine and dibucaine, an internal standard, with chloroform. Their recoveries were around 90%. The mobile phase, 10 mM phosphate buffer–acetonitrile (40:60, v/v) was eluted isocratically. Between- and within-day coefficients of variation were in the range of 1.9–9.4% and 2.3–13.3%, respectively. The detection limit was 0.2 ng/ml. The method we describe can be easily applied to the measurement of plasma paroxetine concentration for pharmacokinetic studies as well as for therapeutic drug monitoring in patients taking paroxetine.     

Determination of fluoxetine and its metabolite norfluoxetine in serum and brain areas using high-performance liquid chromatography with ultraviolet detection

A high-performance liquid chromatography (HPLC) method using only 0.1 ml of serum or homogenate from brain areas has been developed for the determination of fluoxetine (FLU) and its metabolite, norfluoxetine (N-FLU), with ultraviolet detection at 227 nm. The small volume of sample required in this method allows studies in small animals, such as mouse. The method provides recoveries of up to 90% for both compounds. Acceptable coefficients of variation were found for both within-run and day-to-day assays. The limit of detection was 5.0 ng/ml. No interferences were found with tricyclic antidepressant drugs and benzodiazepines, which allows this method to be used in clinical studies. Pharmacokinetic parameters for the two compounds are reported in mouse serum, frontal cortex and caudate nucleus. We also report the values of FLU and N-FLU in serum from humans who were treated once daily with 20 mg of FLU, obtained after 1, 14 and 28 days of treatment.     

Simultaneous determination of lysophospholipids by high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatography (HPLC) procedure for the separation of choline lysophospholids including 1-acyl-lysophosphatidylcholines and 1-O-alkyl-lysophosphatidyl-cholines, like the lysoform of the platelet activating factor (2-lysoPAF), is described. The lysophospholipids are derivatized at the sn-2 position of the hydroxyl group by 7-diethylaminocoumarin-3-carbonylazide, which converts them into the corresponding carbamoyl derivatives. The derivatized compounds were well separated by reversed-phase HPLC and quantified by fluorimetric detection. This method shows a high sensitivity and allows the separation and quantification of mixtures of lysophospholipids at picomolar level. The method was applied to assay enzyme activities, like phospholipase A₂ and PAF-acetylhydrolase, on single phospholipids or their mixtures.     

Simultaneous determination of epirubicin, doxorubicin and their principal metabolites in human plasma by high-performance liquid chromatography and electrochemical detection

A high-performance liquid chromatographic method with electrochemical detection has been developed for the simultaneous determination of epirubicin, 13-S-dihydroepirubicin, doxorubicin and 13-S-dihydrodoxorubicin in human plasma. An aliquot of 200 μl plasma, spiked with internal standard, was extracted by solid-phase extraction using polymeric adsorbent columns. Chromatography was performed using a C₁₈ reversed-phase column with a mobile phase consisting of water–acetonitrile (71:29, v/v) containing 0.05 M Na₂HPO₄ and 0.05% v/v triethylamine adjusted to pH 4.6 with citric acid. Linearity of the method was obtained in the concentration range of 1–500 ng/ml for all the analytes. Analytical recoveries of the analytes ranged from 89 to 93%. The assay can be used for the simultaneous determination of the four analytes, or for epirubicin and its metabolite or doxorubicin and its metabolite, using the other parent drug as an internal standard. The method was applied to analyze human plasma samples from patients treated with epirubicin using doxorubicin as an internal standard.     

Simultaneous determination of aciclovir, ganciclovir, and penciclovir in human plasma by high-performance liquid chromatography with fluorescence detection

A fast, simple and selective HPLC method has been developed for the assay of aciclovir, ganciclovir, and penciclovir in human plasma by coupling HPLC with fluorescence detection. 200 microl plasma, with guanosine 5'-monophosphate as an internal standard, was subjected to protein precipitation with a 7% [v/v] aqueous perchloric acid solution. The 40 microl supernatant was injected into a Diamonsil-5 microm C18 column. Aciclovir, ganciclovir, and penciclovir, with solvents composed of methanol and 0.08% aqueous trifluoroacetic acid solution, were analysed by fluorescence detection at 260 nm (excitation) and 380 nm (emission) using a gradient elution program. The calibration curves of all three analytes were linear between 20 and 2000 ng/ml. The mean absolute recoveries of aciclovir, ganciclovir, and penciclovir were 93.91+/-1.20%, 97.42+/-0.75%, and 99.01+/-3.30%, respectively. The mean inter-day CVs for aciclovir, ganciclovir, and penciclovir, were within 1.29-7.30%, 1.00-5.53%, and 1.19-3.54%, respectively. The intra-day bias for aciclovir, ganciclovir, and penciclovir ranged from -2.01 to 6.33%, 1.81 to 7.37%, and 1.42 to 6.91%, respectively. The method has been validated and applied in pharmacokinetic studies in Chinese adult renal transplant patients.     

Simultaneous determination of oleuropein and its metabolites in plasma by high-performance liquid chromatography

A method based on high-performance liquid chromatography (HPLC) with a diode array detection system was developed and validated aiming at the simultaneous determination of oleuropein (OE) and its metabolites, hydroxytyrosol (HT) and tyrosol (T), in human plasma. These phenolic components are believed to play a vital role in the prevention of coronary artery disease and atherosclerosis. The proposed method includes a clean-up solid-phase extraction procedure (using a C(18) column) with high recovery efficiency (85-100%). The statistical evaluation of the method reveals good linearity, accuracy and reproducibility for all the compounds analyzed with RSD values less than 6.5%, while the detection limit is 50 ng/ml for both OE and T and 75 ng/ml for HT. This assay can be employed in bioavailability studies of olive oil phenolic compounds, thus assisting the evaluation of their pharmacological role.     

Simultaneous determination of byak-angelicin and oxypeucedanin hydrate in rat plasma by column-switching high-performance liquid chromatography with ultraviolet detection

A simple and sensitive column-switching HPLC method was developed for the simultaneous determination of two furocoumarin compounds, byak-angelicin and oxypeucedanin hydrate, which are the main components of hot water extract of Angelica dahurica root (AE), in rat plasma. Plasma sample was simply deproteinated with perchloric acid. After centrifugation, the supernatant was injected into a column-switching HPLC system consisting of a clean-up column (Symmetry Shield RP 8, 20×3.9 mm I.D.) and analytical column (Symmetry C₁₈, 75×4.6 mm I.D.) which were connected with a six-port switching valve. The flow-rate of the mobile phase (acetonitrile–water, 20:80) was maintained at 1 ml/min. Detection was carried out at wavelength 260 nm with a UV detector. The column temperature was maintained at 40°C. The calibration curves of byak-angelicin and oxypeucedanin hydrate were linear over the ranges 19.6 to 980 ng/ml (r²>0.997). The accuracy of these analytes was less than 4.4%. The intra- and inter-day relative standard deviations of byak-angelicin and oxypeucedanin hydrate were within 12.0% and 12.7%, respectively. The present method was applied for the analysis of plasma concentration from rats after administration of AE.     

Assessment of sirolimus concentrations in whole blood by high-performance liquid chromatography with ultraviolet detection

A novel, reversed-phase high-performance liquid chromatographic (HPLC) method is described for the analysis of sirolimus (SRL) in whole blood. The samples were purified by precipitating blood matrix with zinc sulfate, SRL was then extracted with acetone followed by solid-phase extraction. The method was linear over a range of 1-100 ng/ml and the lower limit of quantification was 2.5 ng/ml. The coefficient of variation (within day) was below 8.0% for the lowest SRL concentration. The day-to-day coefficient of variation was below 6.6%. The assay did not show interference peaks with immunosuppressive drugs commonly given to transplant patients. With the simplified extraction procedure described, 60 samples (including controls and calibration curve) can be quantified in a day. The sensitivity and rapidity of this analytical procedure makes it useful for routine therapeutic monitoring of SRL.     

Determination of the antidepressant agent citalopram and metabolites in plasma by liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method is described for the determination of citalopram [1-(3-(dimethylaminopropyl)-1-(4-fluorophenyl)-5-phthalancarbonitrile] and its two main metabolites (the methylamino and amino derivatives). The compounds were extracted from alkaline plasma with diethyl ether. The combined ether layers were evaporated after addition of 50 μl of 0.1 N HCl. The residual extracts were purified with diethyl ether and 20 μl were injected into a Spherisorb ODS 5-μm column with acetonitrile–0.6% phosphate buffer pH 3 (55:45, v/v) as the mobile phase. Using a fluorescence detector the detection limits are 1 ng/ml of plasma for citalopram and the methylamino metabolite and 0.5 ng/ml for the amino metabolite.     

Simultaneous determination of fentanyl and midazolam using high-performance liquid chromatography with ultraviolet detection

When measuring fentanyl and midazolam simultaneously in the same plasma sample with standard high-performance liquid chromatography–ultraviolet (HPLC–UV) detection, overlap of the fentanyl peak by the midazolam peak occurs, which makes fentanyl determination impossible. We tested the hypothesis that by acidifying the methanol mobile phase with 0.02% perchloric acid, 70%, it would be possible to separate both peaks. The UV detector was set at 200 nm. Calibration curves for fentanyl (range 0–2000 pg/ml) and midazolam (range 0–400 ng/ml) were linear (r>0.99). The detection limits were 200 pg/ml (fentanyl) and 10 ng/ml (midazolam). Precision and accuracy for intra- and inter-assay variability as well as in-line validation with quality control samples (QCS) were acceptable (< 15 and 20%, respectively), except for fentanyl QCS of 200 pg/ml (17.8% precision). Although less sensitive than gas chromatography–mass spectrometry (GC–MS), reliable measurements of fentanyl, simultaneously with midazolam, can be performed with this HPLC–UV system.     

Determination of formaldehyde in blood plasma by high-performance liquid chromatography with fluorescence detection

An HPLC method was developed for the determination of formaldehyde in human blood plasma. The method was based on the determination of the fluorescent product of the chemical reaction between formaldehyde and ampicillin. A 0.2-ml aliquot of blood plasma was reacted directly with ampicillin under acidic and heating conditions. The reaction product was extracted from the matrix with diethyl ether and analyzed by reversed-phase HPLC with fluorescence detection. Recoveries of spiked formaldehyde at the low ppm (μg/ml) level were between 93% and 102% with relative standard deviations less than 8%. The limits of detection and quantitation of formaldehyde in blood plasma samples were 0.46 μg/ml and 0.87 μg/ml, respectively.     

Simultaneous determination of ZT-1 and its metabolite Huperzine A in plasma by high-performance liquid chromatography with ultraviolet detection

ZT-1 is a novel acetylcholinesterase (AChE) inhibitor. It is rapidly transformed to Huperzine A (Hup A) in vitro. A simple and rapid HPLC-UV method for the simultaneous determination of ZT-1 and its metabolite Hup A in plasma is described. The chromatographic separations were achieved on a C(18) ODS column (250 mm x 4.6 mm ID) using methanol-1 mmol/L ammonium acetate (70:30,v/v) as mobile phase. The flow rate was 0.7 mL/min, the detection wavelength was 313 nm and the column temperature was kept at 35 degrees C. Plasma samples were prepared as rapidly as possible and extracted immediately with 5 mL of chloroform:iso-propyl alcohol mixture (v/v, 9:1).The retention times of ZT-1 and Huperzine A (Hup A) were 18.7 and 14.4 min, respectively. The mean absolute recoveries of two analytes were >90%. Quantification limits were all 0.02 nmol/mL for ZT-1 and Hup A. This analytical method was reliable and convenient procedure that meets the criteria for the pharmacokinetic evaluation of ZT-1 on experimental animals.     

Plasma serotonin monitoring by blood microdialysis coupled to high-performance liquid chromatography with electrochemical detection in humans

Plasma serotonin (5-HT) active pool was monitored in male volunteers by intravenous microdialysis coupled to HPLC–EC with 98.6% efficient probes. 5-HT was monitored from 60 min before to 360 min after an oral dose of fluoxetine, a 5-HT uptake inhibitor, or vehicle. The basal values were within nanomolar range (0.55 to 4.6 ng/ml). After administration of fluoxetine, there was a significant increment of 5-HT with respect to controls. These results showed that intravenous microdialysis is an alternative efficient technique to monitor endogenous unbound 5-HT changes in plasma without extracting blood or sample pretreatment procedures before the chemical analysis.