Simultaneous determination of citalopram, fluoxetine, paroxetine and their metabolites in plasma and whole blood by high-performance liquid chromatography with ultraviolet and fluorescence detection

A method for the simultaneous determination of the three selective serotonin reuptake inhibitors (SSRIs) citalopram, fluoxetine, paroxetine and their metabolites in whole blood and plasma was developed. Sample clean-up and separation were achieved using a solid-phase extraction method with C₈ non-endcapped columns followed by reversed-phase high-performance liquid chromatography with fluorescence and ultraviolet detection. The robustness of the solid-phase extraction method was tested for citalopram, fluoxetine, paroxetine, Cl-citalopram and the internal standard, protriptyline, using a fractional factorial design with nine factors at two levels. The fractional factorial design showed two significant effects for paroxetine in whole blood. The robustness testing for citalopram, fluoxetine, Cl-citalopram and the internal standard revealed no significant main effects in whole blood and plasma. The optimization and the robustness of the high-performance liquid chromatographic separation were investigated with regard to pH and relative amount of acetonitrile in the mobile phase by a central composite design circumscribed. No alteration in the elution order and no significant change in resolution for a deviation of ±1% acetonitrile and ±0.3 pH units from the specified conditions were observed. The method was validated for the concentration range 0.050–5.0 μmol/l with fluorescence detection and 0.12–5.0 μmol/l with ultraviolet detection. The limits of quantitation were 0.025 μmol/l for citalopram and paroxetine, 0.050 μmol/l for desmethyl citalopram, di-desmethyl citalopram and citalopram-N-oxide, 0.12 μmol/l for the paroxetine metabolites by fluorescence detection, and 0.10 μmol/l for fluoxetine and norfluoxetine by ultraviolet detection. Relative standard deviations for the within-day and between-day precision were in the ranges 1.4–10.6% and 3.1–20.3%, respectively. Recoveries were in the 63–114% range for citalopram, fluoxetine and paroxetine, and in the 38–95% range for the metabolites. The method has been used for the analysis of whole blood and plasma samples from SSRI-exposed patients and forensic cases.     

Methods for the determination of seven selective serotonin reuptake inhibitors and three active metabolites in human serum using high-performance liquid chromatography and gas chromatography

This paper describes a set of simple and sensitive multiresidue methods for the determination of the specific serotonin reuptake inhibitors (SSRIs) used as antidepressant drugs, and some of their respective active metabolites in human serum. It involves liquid–liquid extraction procedures followed by gas chromatography coupled to nitrogen phosphorus detection or isocratic reversed-phase high-performance liquid chromatography combined with fluorescence detection (HPLC–FL), depending on the analytes. Extraction recoveries were between 71 and 96% for the eight SSRIs and their metabolites analysed by GC and between 41 and 77% for the two of them analysed by HPLC. Limits of detection (LODs) and limits of quantitation (LOQs) ranged, respectively, from 2.5 to 5 μg/l and from 10 to 20 μg/l. Intra-assay and inter-assay precision was studied at three and four concentration levels, respectively, and was less than 19% for all compounds. Accuracy was also satisfactory for all. An excellent linearity was observed from the LOQs up to 1000 μg/l for milnacipram and paroxetine and from each LOQ up to 400 mg/l for the other compounds. The performance of the methods described thus allows the therapeutic drug monitoring of the currently commercialised SSRIs.     

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 high-performance liquid chromatographic measurement of venlafaxine and O-desmethylvenlafaxine in human plasma: Application to management of acute intoxications

Venlafaxine, a second-generation antidepressant, acts by inhibition of the reuptake of presynaptic noradrenaline and serotonin. The main metabolite, O-desmethylvenlafaxine was found biologically active. For toxicological purpose, a rapid specific and accurate RP-HPLC assay was developed for the simultaneous determination of venlafaxine and O-desmethylvenlafaxine in human plasma. A linear response was observed over the concentration range 0.2–4 μg/ml. A good accuracy (<8%) was achieved for all quality controls, with intra-day and inter-day variation coefficient less than 10%. Finally, no interference was observed with other psychotic drugs encountered in acute poisoning. This rapid method (run time <10 min) was used to manage four voluntary intoxications involving venlafaxine.     

High-performance liquid chromatographic determination of hydrocortisone and methylprednisolone and their hemisuccinate esters in human serum

A high-performance liquid chromatographic method is described for the simultaneous determination of methylprednisolone (MP) and methylprednisolone hemisuccinate (MPHS), or hydrocortisone (HC) and hydrocortisone hemisuccinate (HCHS) in human serum. Reversed-phase liquid chromatography was performed on a microparticulate C₁₈ column (Spherisorb, 5 μm) using a mobile phase of 2% glacial acetic acid, 30–35% acetonitrile, 70–65% water with ultraviolet detection (254 nm). The method uses 17α-hydroxyprogesterone as the internal standard for the determination of methylprednisolone and its hemisuccinate ester, or 11-deoxy-17-hydroxycorticosterone as the internal standard for the determination of hydrocortisone and its hemisuccinate ester. The sensitivity is 0.03 μg/ml for HC, 0.07 μg/ml for MP, 0.04 μg/ml for MPHS, and 0.10 μg/ml for HCHS, with a detection limit of 0.02 μg/ml for all four steroids. Calibration curves are linear up to 3 μg/ml for MP or MPHS (as equivalent MP) and up to 4 μg/ml for HC and 7 μg/ml (as equivalent HC) for HCHS. The pooled relative standard deviation for replicate samples for each steroid is < 7%. Plasma concentration—time curves are reported for MP and MPHS or HC and HCHS of two human subjects following intramuscular administration of 125 mg of methylprednisolone sodium succinate for injection, U.S.P., or 250 mg of hydrocortisone sodium succinate for injection, U.S.P.     

High-performance liquid chromatographic determination of free and total polyamines in human serum as fluorescamine derivatives

A highly sensitive and simple fluorimetric method for the determination of free and total polyamines, spermidine, spermine, putrescine and cadaverine, in human serum by high-performance liquid chromatography is described. The polyamines, obtained after clean-up of deproteinized serum by Cellex P column chromatography, are converted to their fluorescamine derivatives in the presence of nickel ion which inhibits the reaction of interfering amines with fluorescamine, and the derivatives are separated simultaneously by reversed-phase chromatography (LiChrosorb RP-18) with a linear gradient elution. The lower limits of detection are 10 and 5 pmole for spermine and the others in 0.5 ml of serum, respectively.     

High-performance liquid chromatographic determination of lamivudine in human serum using liquid-liquid extraction; application to pharmacokinetic studies

A simple, fast, and sensitive high performance liquid chromatographic (HPLC) assay was developed for quantitation of lamivudine in human serum. Lamivudine is polar compound and its extraction from the human serum in previously published HPLC methods involved either protein precipitation or solid phase extraction techniques. However, existence of endogenous peaks which interfere with the drug or appeared as late eluting peaks and lead to long run time of analysis has been reported. Application of either an ion pairing agent in the mobile phase or time consuming column purge has been used in the published methods. Present paper describes liquid - liquid extraction of lamivudine and internal standard (famotidine) using dichloromethane-isopropyl alcohol (1:1, v/v) as an extracting solvent and salting out approach. The mobile phase was a mixture of phosphate buffer (0.05 M) containing triethylamine (1 mL/L, v/v; pH 3.5) and methanol (91:9, v/v) at a flow rate of 2.2 mL/min. The analysis was performed on a column (150 mm x 6 mm i.d.) which was packed with 5 microm particles of ODS packing material. Under these conditions no interference in the assay from any endogenous substance was observed. The limit of quantification was evaluated to be 5 ng/mL. Accuracy and precision of the method were also studied and the technique was shown to be selective and linear into the concentration range of 5-2500 ng/mL. This method has been used in two randomized crossover bioequivalence studies of 100 and 150 mg lamivudine preparations in 12 and 24 healthy volunteers, respectively.     

High-performance liquid chromatographic method for the detection and quantitation of haloperidol and seven of its metabolites in microsomal preparations

An isocratic high-performance liquid chromatographic (HPLC) system was developed to analyze haloperidol and its potential metabolites. These compounds included 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP), haloperidol N-oxide (HNO), reduced haloperidol (RHAL), the 1,2,3,6-tetrahydropyridine analogue and its N-oxide, and the pyridinium ion from haloperidol (HP⁺). The HPLC system comprised a Hypersil CPS5 column with a mobile phase of acetonitrile (67%) and ammonium acetate (final concentration 10 mM) which was adjusted to pH 5.4 by acetic acid. The solvent was delivered at 1 ml/min. RHAL and CPHP were determined by an ultraviolet detector at 220 nm with a detection limit of 1 nmol/ml. All other compounds were determined at 245 nm and had a detection limit of 0.3 nmol/ml. This system was used to analyze a microsomal metabolic mixture of haloperidol. It was found that all above compounds except HNO were metabolites of haloperidol. In addition, two other metabolites were also well separated in this HPLC system which are proposed to be oxygenated haloperidol and the pyridone analogue of haloperidol. The HPLC system was used to carry out quantitative metabolic studies of haloperidol. It was found that the metabolism of haloperidol exhibits large inter-species differences. The apparent enzyme kinetic parameters were also determined using mice microsomes.     

High-performance liquid chromatographic analysis of melatonin in human plasma and rabbit serum with on-line column enrichment

This paper describes the development of a simple and sensitive analytical method for the quantification of melatonin in human plasma and rabbit serum, using standard analytical equipment and on-line column enrichment without prior extraction, clean-up or derivatization. The analytical procedure was found to be accurate, precise and linear. For human plasma, the accuracy was 101% (range 89–106%), and the mean precision was 5% (range 2–9%) for all concentrations (0, 2, 10, 50 and 200 ng/ml) tested (n=6). The accuracy in rabbit serum was 101% (range 90–112%), and the mean precision was 13% (range 8–19%) for all concentrations (0, 2, 10, 50, 200 and 500 ng/ml) tested (n=6). The retention time of melatonin was about 8 min and the total recoveries were found to be approximately 65 and 85%, respectively, for human plasma and rabbit serum. The limit of detection was found to be lower than 1 ng/ml for human plasma and around 2 ng/ml for rabbit serum. The method is, therefore, found to be suitable for melatonin bioavailability studies in rabbits and presumably also in humans.     

High-performance liquid chromatographic method for the determination of atracurium and laudanosine in human plasma: Application to pharmacokinetics

A high-performance liquid chromatographic method coupled with fluorimetric detection has been developed for the determination of atracurium and its major metabolite, laudanosine, in human plasma. The detection is performed at 240 nm for excitation and 320 nm for emission. Verapamil was used as the internal standard. The proposed technique, involving the direct precipitation of plasma proteins is reproducible, selective and sensitive. Linear detector responses were observed for the calibration curve standards in the range of 40 to 2000 ng/ml. Precision, expressed as C.V., was in the range 1 to 14%. The limit of quantification for both atracurium and laudanosine was 40 ng/ml. The method has been validated and stability tests under various conditions have been performed. This method has been used to determine the pharmacokinetic profile of atracurium and laudanosine in patients with acute respiratory distress syndrome.     

High-performance liquid chromatographic determination of carbamazepine and metabolites in human hair

To study the use of hair analysis in monitoring drug compliance and historical changes in pharmacokinetics we developed a method for the quantitative determination of the anti-epileptic drug carbamazepine (CBZ) and trans-10,11-dihydro-10,11-dihydroxy-carbamazepine (CBZ-diol) in hair from carbamazepine users. Digestion by 1 M NaOH was found to be the best method for isolating CBZ and CBZ-diol from hair, followed by solid-phase extraction and reversed-phase HPLC with UV detection. Recoveries from spiked hair samples were 76–86%. Within-day precision (C.V.; n=10) for CBZ and CBZ-diol in hair of a CBZ user containing 10.9 μg/g CBZ and 3.2 μg/g CBZ-diol were 1.7 and 5.0%, respectively. Sectional hair analysis of a patient on a constant dosage of CBZ demonstrates an exponential decrease in hair concentrations of CBZ and CBZ-diol with increasing distance from the root, probably caused by shampooing. No CBZ-10,11-epoxide (CBZ-epox) could be detected. However, one component in the chromatogram is probably CBZ-β-hydroxythioether, an adduct of CBZ-epox with cysteine, or acridinethioacetal, its rearrangement product. The concentration of this component does not decrease with increasing distance from the root.     

High-performance liquid chromatographic method for the determination of a novel thymidylate synthase inhibitor, AG 331, in human serum

AG 331 is a novel thymidylate synthase inhibitor currently in Phase I clinical trial. To determine the pharmacokinetic parameters of AG 331 in human subjects, a suitable analytical method was developed using high-performance liquid chromatography. Serum and urine samples were prepared using both solid-phase extraction and solvent extraction. Either 4,4′-diaminodiphenyl sulfone or benz[cd]indole-2(1H)-one were used as internal standards for the method. A reversed-phase C₁₈ analytical column completely resolved the drug and internal standard peaks from non-specific substances present in biological matrix. The method was validated for precision, accuracy, and reproducibility in serum and was linear over a concentration range of 50–2000 ng/ml, with a limit of detection of 20.0 ng/ml and a quantifiable limit of 50 ng/ml.     

Induction of preputium eversion by peptides, serotonin receptor antagonists, and selective serotonin reuptake inhibitors in Biomphalaria glabrata

Abstract. Eversion of the preputium is one of the initial steps in the male copulatory behavior of freshwater pulmonates. Previous experiments have shown that serotonergic mechanisms are involved in eversion in the snail Biomphalaria glabrata because the vertebrate 5-HT₁ receptor antagonist methiothepin caused long-lasting eversion in a dose-dependent manner. In this study, we tested a variety of serotonergic receptor ligands, bioactive peptides, and selective serotonin reuptake inhibitors (SSRIs) for their ability to induce preputium eversion in B . glabrata in order to elucidate the physiological mechanism of eversion. Of 15 compounds tested, five significantly induced preputium eversion: the serotonin receptor antagonists methiothepin (1 and 10 μM; p<0.0001), cyproheptadine (1–10 μM; p<0.007–0.0001), and mianserin (5–50 μM; p<0.01–0.001), the molluscan cardioactive peptide FMRFamide (10 and 50 μM; p<0.0002–0.0001), and the SSRI fluoxetine (=Prozac, 10–100 μM; p<0.0003–0.0001). Serotonin itself neither induced eversion nor blocked methiothepin-induced eversion. This suggests that fluoxetine is not acting as an SSRI, but potentially as a receptor ligand. These preliminary data shed light on the possible physiological mechanism of preputium eversion in B . glabrata and suggest similarity with that of the model freshwater gastropod Lymnaea stagnalis .     

In vivo identification and characterization of binding sites for selective serotonin reuptake inhibitors in mouse brain

The present study was undertaken to identify and characterize in vivo binding sites of selective serotonin reuptake inhibitors (SSRIs) in the mouse brain by using [3H]paroxetine as radioligand. Relatively higher concentration of [3H]paroxetine was detected in the whole brain (minus cerebellum) than in the plasma of mice after the i.v. injection of the radioligand, and the half-life (t1/2) of elimination was much slower. The in vivo specific [3H]paroxetine binding in the mouse brain after the i.v. injection was defined as the difference of particulate-bound radioactivity between the whole brain and cerebellum, and it was dose-dependently attenuated by oral or intraperitoneal administration of fluoxetine (8.68-116 micromol/kg). Furthermore, oral administration of fluvoxamine, fluoxetine, paroxetine and sertraline at the pharmacologically relevant doses reduced significantly (25-94%) in vivo specific [3H]paroxetine binding in the cerebral cortex, striatum, hippocampus, thalamus and midbrain of mice, and their significant decreases were observed up to at least 8 h (fluvoxamine), 24 h (fluoxetine), and 12 h (paroxetine and sertraline) later. The value of area under the curve (AUC) for decrease in [3H]paroxetine binding vs. time in each brain region was largest for fluoxetine among these SSRIs, due to the relatively longer-lasting occupation of brain serotonin transporter. The AUC value in mouse brain after oral administration of each SSRI was 1.2-3.2 times greater in the thalamus and midbrain than in the cerebral cortex, striatum and hippocampus. Thus, the present study has revealed that [3H]paroxetine may be a suitable radioligand for in vivo characterization of brain binding sites and pharmacological effects of SSRIs.     

High-performance liquid chromatographic determination of paclitaxel in rat serum: application to a toxicokinetic study

A simple and sensitive high-performance liquid chromatographic method is described for the determination of paclitaxel (Taxol^®) at 230 nm using a Nucleosil C₁₈ (5 μm) column and a methanol–water (70:30, v/v) mobile phase following a single-step extraction from serum with dichloromethane. The assay was validated against the classical criteria and was applied to a toxicokinetic study in rats after one or five, one per week) intraperitoneal administrations of 16 mg/kg Taxol^®.     

High-performance liquid chromatographic determination of bupivacaine in plasma samples for biopharmaceutical studies and application to seven other local anaesthetics

A sensitive analytical procedure for bupivacaine dosing in plasma samples by reversed-phase high-performance liquid chromatography is described. After a two-step extraction, the analysis was performed using a C₁₈ column and a mobile phase of 0.01 M sodium dihydrogen-phosphate (pH 2.1)—acetonitrile (80:20, v/v). The extraction yield of bupivacaine from plasma was 73.5 ± 5.1% (mean ± S.D., n = 10). The within-day and between-day reproducibilities at a concentration of 100 ng/ml were 2.1% and 5.6%, respectively (n = 10). Calibration curves were linear (r² = 0.9996) between 5 and 1000 ng/ml. The limit of detection, defined by a signal-to-noise ratio of 3:1, was 2 ng/ml. The accuracy at a concentration of 100 ng/ml was 2.3%. This method could be applied to the plasma analysis of seven other local anaesthetics (articaine, etidocaine, lidocaine, mepivacaine, pramocaine, procaine and tetracaine). The procedure was used in bioavailability studies of bupivacaine-loaded poly( -lactide) (i.e. PLA) and poly( -lactide-co-glycolide) (i.e. PLGA) microspheres after subcutaneous and intrathecal administrations in rabbits.     

Comparison of the effects of serotonin selective,norepinephrine selective,and dual serotonin and norepinephrine reuptake inhibitors on lower urinary tract function in cats

Previous studies showed that the dual serotonin (5-hydroxytryptamine, 5-HT) and norepinephrine (NE) reuptake inhibitor, duloxetine, increases bladder capacity and urethral sphincter electromyographic (EMG) activity in a cat model of acetic acid-induced bladder irritation. The present study aimed to determine the relative importance of 5-HT versus NE reuptake inhibition for mediating these effects by examining drugs that are selective for either the 5-HT or NE system or both. Similar to duloxetine, venlafaxine (0.1 to 10 mg/kg), also a dual serotonin and norepinephrine reuptake inhibitor, produced marked increases in bladder capacity and EMG activity that were reversed by methiothepin (0.3 mg/kg). S-norfluoxetine (0.01 to 10 mg/kg), a serotonin selective reuptake inhibitor, produced small but significant increases in bladder capacity and EMG activity at doses of 3 and 10 mg/kg. Thionisoxetine (0.01 to 3.0 mg/kg), a NE selective reuptake inhibitor, produced no effects on bladder capacity or sphincter EMG activity. Surprisingly, co-administration of thionisoxetine and s-norfluoxetine up to doses of 1 mg/kg of each compound produced no effect on lower urinary tract function. These doses were the maximum that could be administered in combination due to drug-induced emergence of skeletal muscle activity in chloralose-anesthetized animals. These results indicate that there are unexplained pharmacological differences between the effects of single compounds that exhibit dual NE and 5-HT reuptake inhibition and a combination of compounds that exhibit selective NE and 5-HT reuptake inhibition on lower urinary tract function.