In-Syringe Electrokinetic Protein Removal from Biological Samples prior to Electrospray Ionization Mass Spectrometry.

Here, an electrokinetic extraction (EkE) syringe is presented allowing for on-line electrokinetic removal of serum proteins before ESI-MS. The proposed concept is demonstrated by the determination of pharmaceuticals from human serum within minutes, with sample preparation limited to a 5× dilution of the sample in the background electrolyte (BGE) and application of voltage, both of which can be performed in-syringe. Signal enhancements of 3.6-32 fold relative to direct infusion of diluted serum and up to 10.8 fold enhancement, were obtained for basic and acidic pharmaceuticals, respectively. Linear correlations for the basic drugs by EkE-ESI-MS/MS were achieved, covering the necessary clinical range with LOQs of 5.3, 7.8, 6.1, and 17.8 ng mL-1 for clomipramine, chlorphenamine, pindolol, and atenolol, respectively. For the acidic drugs, the EkE-ESI-MS LOQs were 3.1 µg mL-1 and 2.9 µg mL-1 for naproxen and paracetamol, respectively. The EkE-ESI-MS and EkE-ESI-MS/MS methods showed good accuracy (%found of 81 % to 120 %), precision (≤20 %), and linearity (r>0.997) for all the studied drugs in spiked serum samples.

A biomimetic hybrid material consisting of CaCO3 mesoporous microspheres and an alternating copolymer for reversed-phase HPLC.

We developed a biomineral-inspired hybrid material composed of CaCO3 and an organic polymer as a column packing material for HPLC. This material combines a hierarchical mesoporous structure and the functionality of the polymer. The surface of monodispersed mesoporous CaCO3 microspheres was modified with poly(maleic acid-alt-1-octadecene) (PMAcO) comprising hydrophobic alkyl chains and anionic carboxylate groups. PMAcO adsorbed onto the surface of CaCO3 through electrostatic interaction between Ca2+ sites and carboxylate groups, resulting in an octadecene coated microsphere interface. These microspheres were applied as a HPLC column and exhibited reversed-phase retention behavior in the separation of alkylbenzenes. This column showed high alkaline mobile phase resistance compared with the conventionally applied ODS column packing material. Quantitative analysis of the basic antidepressants clomipramine and imipramine spiked into whole blood was achieved with an alkaline mobile phase, demonstrating the potential of the biomineral-inspired material as a HPLC stationary phase for practical applications in routine analyses of basic drugs requiring alkaline mobile phases.

Ultrasound-assisted dispersive magnetic solid phase extraction based on amino-functionalized Fe3O4 adsorbent for recovery of clomipramine from human plasma and its determination by high performance liquid chromatography: Optimization by experimental design.

The applicability of Amino-functionalized Fe3O4 nanoparticles (NPs) as an effective adsorbent was developed for the extraction and determination of clomipramine (CLP) in plasma sample by ultrasound-assisted dispersive magnetic solid phase extraction (UADM-SPE) and high-performance liquid chromatography-ultraviolet (HPLC-UV) detection. Fabrication of the Fe3O4@SiO2-NH2 magnetic nanoparticles confirmed by Fourier transform infrared spectroscopy (FT-IR), thermogravimetric analysis (TGA), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The effect of different extraction parameters (i.e. pH of the sample solution, the amount of magnetic nanoparticles (MNPs), sample volume, temperature and sonication time) on the extraction recovery of CLP were investigated by response surface methodology through central composite design (CCD). The optimum condition is obtained when the affecting parameters are set to: pH of the sample solution=9, the amount of MNPs=37mg, sample volume=23mL, 25°C temperature and sonication time=1min. Under the optimum condition, extraction recovery was 90.6% with relative standard deviation of 3.5%, and enrichment factor of 117. The linear range for determination of CLP was 0.017-0.70mgL-1 with a determination coefficient (R2) of 0.999. Limit of detection (LOD) and limit of quantification (LOQ) were 0.005 and 0.0167mgL-1, respectively. The established UADM-SPE-HPLC-UV method was rapid, simple and efficient for determination of CLP in human plasma samples.

Serum clomipramine and desmethylclomipramine levels in a CYP2C19 and CYP2D6 intermediate metabolizer.

Pharmacogenetics within psychiatry has the potential to aid in the dose and selection of medications. A substantial number of psychiatric medications are metabolized through either of the highly polymorphic drug-metabolizing enzymes CYP2D6 and CYP2C19. Of these, clomipramine is subject to metabolism by both CYP2C19 and CYP2D6, leaving individuals with deficiencies of these drug-metabolizing enzymes at risk of higher concentrations of the parent molecule. Herein, we present the case of a 29-year-old male with diagnoses of depression and obsessive compulsive disorder who had trialed and failed a dozen psychiatric medications, many of which are subject to metabolism by CYP2D6 and/or CYP2C19, and had most recently been taking clomipramine for approximately 2.5 years. Pharmacogenetic testing revealed this patient to be an intermediate metabolizer for both CYP2C19 (*1/*2) and CYP2D6 (*4/*41), which resulted in considerably elevated serum trough concentrations of clomipramine and its active metabolite desmethylclomipramine. This case provides a retrospective view of how the knowledge of an individual's pharmacogenetic test results can aid in their clinical care.

A simple dried blood spot method for therapeutic drug monitoring of the tricyclic antidepressants amitriptyline, nortriptyline, imipramine, clomipramine, and their active metabolites using LC-MS/MS.

Therapeutic drug monitoring (TDM) of tricyclic antidepressants (TCAs) is considered useful in patients with major depressive disorder, since these drugs display large individual differences in clearance, and the therapeutic windows of these drugs are relatively small. We developed an assay for determination of amitriptyline (ATP), nortriptyline (NTP), imipramine (IMP), desipramine (DSP) clomipramine (CMP) and desmethyl-clomipramine (DCMP) in dried blood spots (DBS). A fast and robust LC-MS/MS method was developed and analytically validated for simultaneous determination of ATP, NTP, IMP, DSP, CMP, and DCMP in DBS. Six mm circles were punched out from DBS collected on Whatman DMPK-C paper and mixed with acetonitrile: methanol 1:3 containing the internal standard. The extract was analyzed by LC-MS/MS. Total LC-MS/MS runtime was 4.8 min. The assay was linear in the range 20-500 µg/L for all compounds. Overall-assay accuracy and precision were<20% for the lower limit of quantification (LLOQ), except for CMP (CV=22.3%), and <15% at other concentrations. The initial LLOQ was 20 µg/L however for CMP and DMCP it was increased to 40 µg/L. The blood volume per spot did not influence the results, but a low hematocrit (≤ 30%) was associated with a >15% negative bias for all compounds. Punching at the perimeter of the blood spot instead of the center was associated with a positive bias. A good correlation was found between patients plasma and DBS samples of ATP, NTP and DMCP, but not for CMP. In addition, proportional differences were found. This LC-MS/MS method was analytically validated for determination of TCAs in DBS. Future validation will focus on the clinical application of the method.

Clomipramine demethylation rate is important on the outcome of obsessive-compulsive disorder treatment.

The aim of this study was to investigate the influence of demethylation rate on the outcome of obsessive-compulsive disorder patients treated with clomipramine. Eighteen patients meeting the DSM-IV criteria for obsessive-compulsive disorder received 150-300 mg of clomipramine daily in a single-blind design for 12 weeks. The patients were evaluated with the Clinical Global Impression scale and the Yale-Brown Obsessive-Compulsive Scale (YBOCS). Clinical assessment and serum measurements of clomipramine and desmethylclomipramine were carried out at baseline and after 3, 6, 8, 10, and 12 weeks. A greater improvement in Clinical Global Impression scale rating was associated with a lower desmethylclomipramine/daily dose and the total clomipramine and desmethylclomipramine/daily dose. Moreover, an improved response on the YBOCS-obsession score was associated with higher serum levels of clomipramine and the total clomipramine and desmethylclomipramine/daily dose. Patients with a greater reduction in baseline YBOCS rating had a lower desmethylclomipramine/clomipramine ratio. These data suggest that a lower demethylation rate correlates with better clinical outcome.

Intravenous lipid emulsion-augmented plasma exchange in a rabbit model of clomipramine toxicity; survival, but no sink.

INTRODUCTION: Intravenous lipid emulsion (ILE) has been shown to ameliorate toxicity from lipophilic xenobiotics, attributed in part through sequestration to circulating lipid droplets (sink). We postulated additional benefit with plasma exchange therapy undertaken subsequent to lipid injection, hypothesising enhanced blood carriage of lipophilic toxin to increase yield when combined with an extracorporeal method of elimination. METHODS: Instrumented rabbits underwent clomipramine infusion at 3.2 mg/kg/min to target mean arterial pressure (MAP) of 50% baseline, then continuously at 2 mg/kg/min to death or 90 min. Resuscitation with saline (Control), sodium bicarbonate (BIC), ILE, or lipid emulsion plus cycled plasma exchange (LEPE), was commenced on attaining target MAP. RESULTS: Greater survival was observed in animals receiving lipid emulsion from both LE and LEPE groups (Control median 12.0 [IQR 10.5 ­ 20] min, BIC median 30 [IQR 19 ­ 33] min, LE 85 [IQR 30 ­ 90] min, LEPE 90 min; P 0.0001). No difference was observed in MAP, Heart Rate, or Electrocardiograph QRS duration between surviving LE and LEPE animals at 90 min. Mean plasma exchange of 52%circulating plasma volume returned only 0.04% of the administered clomipramine load in LEPE group animals. CONCLUSIONS: Infusion of lipid emulsion resulted in greater survival in this rabbit model of intravenous clomipramine toxicity. Plasma exchange performed in conjunction with administration of lipid emulsion failed to result in significant extracorporeal clomipramine elimination. Intravascular lipid sequestration of clomipramine appears an inadequate sole explanation for the beneficial effects of lipid emulsion.

Recombinant laccase: II. Medical biosensor.

Langmuir-Blodgett (LB) technology was used to build a high-sensitivity enzyme-based biosensor for medical purposes. Recombinant fungal laccase from Rigidoporous lignosus, as previously described, was used to catalyze a widely used antidepressant in a micromolar range, namely, clomipramine. The topological properties of the laccase thin film were characterized via LB π-A isotherm and AFM (mean roughness 8.22 nm, compressibility coefficient 37.5 m/N). The sensitivity of the biosensor was investigated via UV spectroscopy, and linearity was found in the absorbance peak shift at 400 nm at drug concentration varying up to 20 uM. The enzyme kinetics was subsequently investigated with potentiometric and amperometric measurements, and we found electronic transfer of at least 1 electron, k(s) 0.57 s(-1), diffusion coefficient 3 × 10(-6) cm(2)/s, K(cat) 6825.92 min(-1), K(M) 4.1 uM, K(cat)/K(M) 2.8 × 10(7) mol(-1) s(-1), sensitivity of 440 nA/uM, maximum velocity 1706.48 nA/s, and response time less than 5 s. The amperometric and potentiometric measurements were repeated after a month, confirming the stability of the biosensor.

Electromembrane extraction combined with gas chromatography for quantification of tricyclic antidepressants in human body fluids.

Recent advances in electromembrane extraction (EME) methodology calls for effective and accessible detection methods. Using imipramine and clomipramine as model therapeutics, this proof-of-principle work combines EME with gas chromatography analysis employing a flame ionization detector (FID). The drugs were extracted from acidic aqueous sample solutions, through a supported liquid membrane (SLM) consisting of 2-nitrophenyl octyl ether (NPOE) impregnated on the walls of the hollow fiber. EME parameters, such as SLM composition, type of ion carrier, pH and the composition of donor and acceptor solutions, agitation speed, extraction voltage, and extraction time were studied in detail. Under optimized conditions, the therapeutics were effectively extracted from different matrices with recoveries ranging from 90 to 95%. The samples were preconcentrated 270-280 times prior to GC analysis. Reliable linearity was also achieved for calibration curves with a regression coefficient of at least 0.995. Detection limits and intra-day precision (n=3) were less than 0.7 ng mL(-1) and 8.5%, respectively. Finally, method was applied to determination and quantification of drugs in human plasma and urine samples and satisfactory results were achieved.

Aripiprazole augmentation strategy in clomipramine-resistant depressive patients: an open preliminary study.

Recent evidence supports the use of second generation antipsychotics in drug resistant depression. The aim of the present open-label study was to evaluate the effect of aripiprazole as an add-on medication in drug-resistant depressed patients who had not responded to clomipramine. Thirty-five patients with major depressive disorder (MDD) were included in the study. All patients had not responded to a previous adequate treatment with an SSRI and had been receiving clomipramine (daily doses ranging from 100 to 300 mg) for 113.9 ± 18.9 days without getting significant clinical improvement. Aripiprazole was added at the fixed dose of 5mg/day and clinical status as well as clomipramine plasma levels were monitored before and after 4, 8, and 24 weeks of combined treatment. Hamilton depression rating scale scores significantly decreased over the follow-up period with 91.4% and 34.3% of patients getting a response or a remission, respectively, after 24 weeks of combined treatment. No worsening of clomipramine-related side effects nor new side effects were observed. The clinical improvement was accompanied by a progressive and significant increase in clomipramine plasma levels. With the limitation of an open-label design, these data suggest for the first time the putative efficacy and safety of aripiprazole in combination with a tricyclic medication in drug resistant depressed patients. The role of the observed pharmacokinetic interaction in the mechanism of aripiprazole antidepressant activity remains to be proved.

Plasma clomipramine levels in adult patients with obsessive-compulsive disorder.

The aim of this study was to explore the possible relationship between plasma clomipramine and its major metabolite (N-desmethylclomipramine) levels and related parameters, and clinical features in patients with obsessive-compulsive disorder (OCD). Twenty-six outpatients (13 men, 13 women), suffering from OCD were consecutively enrolled in this study. The severity of OCD was assessed by the Yale-Brown Obsessive Compulsive Scale (Y-BOCS). The measurements were taken after 4 weeks and 6 months from the beginning of the treatment. The drug levels were measured by a high-performance liquid chromatography method developed by us. The correlations between biological and clinical parameters were analyzed by means of Spearman's correlation coefficient. The Mann-Whitney test was used for comparing biological and clinical variables between men and women. The results showed that clomipramine levels were related to the doses at the two assessment times. A significant and positive correlation was detected at the beginning between the N-desmethylclomipramine ratio and the Y-BOCS total score; however, this was true only for men, where the similar correlations were measured also with the Y-BOCS subscale. After 6 months of clomipramine, men showed a significant improvement of the compulsions. These findings would highlight the potential impact of assessing clomipramine plasma levels and their relationships with specific symptoms, as well as the influence of the sex on the drug response.

Combination of corona discharge ion mobility spectrometry with a novel reagent gas and two immiscible organic solvent liquid-liquid-liquid microextraction for analysis of clomipramine in biological samples.

A novel and sensitive method based on combination of two immiscible organic solvents hollow fiber-based liquid-liquid-liquid microextraction and corona discharge ion mobility spectrometry (HF-LLLME-CD-IMS) was employed for the analysis of clomipramine in human urine and plasma. The effect of formic, acetic and propionic acid as the reagent gas (dopant) on the corona discharge ion mobility signal was investigated. The influence of dopant amount was also studied. Optimum mass flow rates of the dopants were 3.7, 1.1 and 1.0 µmol min(-1) for formic, acetic and propionic acid, respectively. Experimental parameters influencing the extraction efficiency of HF-LLLME, such as NaOH concentration as donor solution, ionic strength of the sample, stirring rate, and extraction time were investigated and optimized. Under the optimum conditions, analytical parameters such as linearity, precision and limit of detection were also evaluated. The linear dynamic range was from 1 to 100 µg L(-1) (r(2)=0.9980) and the limit of detection was 0.35 µg L(-1). Intra- and inter-day precisions were satisfactory with a relative standard deviation (RSD) of 5.9 and 6.7%, respectively. The proposed method was satisfactorily applied for the determination of clomipramine in human plasma and urine.

Effect of oxidative stress on the pharmacokinetics of clomipramine in rats treated with ferric-nitrilotriacetate.

The effect of oxidative stress (OS) on the pharmacokinetics of clomipramine (CPM), particularly addressing the change of CPM distribution to plasma components, was studied in ferric-nitrilotriacetate-induced oxidative-stress model rats (OS rats). First, CPM pharmacokinetic studies in OS rats were performed using CPM continuous infusion (17.5 µg/min/kg). Plasma concentration of CPM at a steady state in OS rats (0.20 ± 0.02 µg/mL) was significantly lower than that in control rats (0.30 ± 0.02 µg/mL). However, no difference was found in the amounts of CPM in the brains of control rats (1.67 ± 0.13 µg/g) and OS rats (1.63 ± 0.09 µg/g). Both of plasma unbound fraction and distribution to erythrocytes in OS rats were significantly higher than those of control rats. These results suggest that the lower CPM concentration in plasma in OS condition does not induce an inferior pharmacological effect.

Clinical and genetic correlates of suicidal ideation during antidepressant treatment in a depressed outpatient sample.

AIMS: This study investigated clinical and genetic predictors of increasing suicidal ideation during antidepressant treatment. MATERIALS & METHODS: A total of 131 depressed outpatients were allocated to four antidepressants (paroxetine, venlafaxine, clomipramine or nefazodone) in a sequential step procedure until remission. Suicidality was assessed using the 10th item of the Montgomery-Asberg Depression Rating Scale (MADRS). A total of 11 candidate genes involved in different mechanisms of antidepressant action were selected for association with increasing suicidality. RESULTS: Increasing suicidality correlated with depression severity and higher antidepressant blood levels. Risk of increasing suicidal ideation was higher in subjects taking antidepressants other than paroxetine (odds ratio: 1.11). The strongest genetic predictor was found to be rs1360780 within the FKBP5 gene (p = 2.9 × 10(-5)), followed by 2677G>T in the ABCB1 gene. The rs130058 SNP within the 5-HTR1B gene demonstrated a differential association with increasing suicidal ideation depending on antidepressant type. CONCLUSION: Increasing suicidal ideation might be an adverse effect of antidepressants. The involvement of FKBP5 indicates that dysregulation of the hypothalamic-pituitary-adrenal axis is involved in treatment increasing suicidal ideation.

Association between CYP2C19*17 and metabolism of amitriptyline, citalopram and clomipramine in Dutch hospitalized patients.

Polymorphisms in genes coding for drug metabolizing enzymes, such as the cytochrome P450 enzymes CYP2C19 and CYP2D6, can lead to therapy failure and side effects. In earlier studies, the novel variant CYP2C19*17 increased metabolism of several CYP2C19 substrates. The objective of this study was to evaluate the impact of CYP2C19*17 on the metabolism of amitriptyline (AT), citalopram (CIT), and clomipramine (CLOM). Six-hundred and seventy-eight patients were included in this study, based on availability of DNA and serum levels of parent drug and main metabolite. We investigated the relationship between CYP2C19 genotypes and metabolic parameters, including serum levels corrected for dose and metabolic ratio (MR). The CYP2C19*17 allele was significantly associated with decreased MR for CIT (CYP2C19*1/*17 mean MR=2.3, compared with CYP2C19*1/*1 mean MR=2.8) and AT (CYP2C19*17/*17 mean MR=0.8, compared with CYP2C19*1/*1 mean MR=3.7 in the CYP2D6*1/*1 subgroup). Furthermore, significant association of CYP2D6 genotype with AT, CIT, and CLOM metabolism was observed. No clear correlation was found between CYP2C19 genotype and CLOM metabolism. This study confirms the increased activity of the CYP2C19*17 allele and shows increased metabolism of drugs that are metabolized by CYP2C19, including AT and CIT. However, the clinical relevance of CYP2C19*17 is probably limited for AT, CIT, and CLOM.

Regional distribution of clomipramine and desmethylclomipramine in rat brain and peripheral organs on chronic clomipramine administration.

The tricyclic antidepressant (TCA) clomipramine has been widely used in psychiatry for over 40 years. More recently, its therapeutic potential as an antineoplastic drug has been identified. However, there are no prior data on regional distribution in the brain of clomipramine and its primary metabolite (desmethylclomipramine) after chronic oral administration. The aim of this study was to determine the concentrations of clomipramine and desmethylclomipramine in different rat-brain regions and to compare those with levels in plasma and peripheral organs after chronic oral treatment of Sprague Dawley rats (15 mg/kg) for 14 days. The levels of both parent TCA and metabolite were analysed by high-performance liquid chromatography in six brain regions (cortex, hypothalamus, hippocampus, striatum, brainstem and cerebellum), five peripheral organs and in plasma. Our data show that the cerebral cortex had the highest concentration of clomipramine (2.9 microg/mg), with successively lower concentrations in the hypothalamus, striatum, cerebellum, hippocampus and brainstem. Of the peripheral organs, the lungs and liver, had the highest levels of clomipramine, while in the heart, only the metabolite was detected. The plasma concentration (0.17 microg/ml or 0.48 microM) was comparable to that in the hippocampus and cerebellum (approximately 0.20 microg/mg). The differential distribution of clomipramine in different brain regions and the regional variation in clomipramine to desmethylclomipramine ratios have implications for the use of clomipramine in psychiatry and neuro-oncology.

Correlation of plasma and peritoneal diasylate clomipramine concentration with hemodynamic recovery after intralipid infusion in rabbits.

OBJECTIVES: Drug sequestration to an expanded plasma lipid phase has been proposed as a potential mechanism of action for lipid emulsions in lipophilic cardiotoxin overdose. The authors set out to document plasma and peritoneal diasylate clomipramine concentration after resuscitation with lipid emulsion in a rabbit model of clomipramine-induced hypotension. METHODS: Twenty sedated mechanically ventilated New Zealand White rabbits were allocated to receive either 12 mL/kg 20% Intralipid or 12 mL/kg saline solution, following clomipramine infusion to 50% baseline mean arterial pressure (MAP). Hemodynamic parameters and serum clomipramine concentration were determined to 59 minutes. Peritoneal dialysis with 20% Intralipid or saline solution was evaluated for clomipramine concentration. RESULTS: Mean arterial pressure was greater in lipid-treated animals as assessed by repeated-measures analysis of variance (F[1,14] = 6.84; p = 0.020). Lipid infusion was associated with elevated plasma clomipramine concentration and reduced initial volume of distribution (Vd; 5.7 [+/-1.6] L/kg lipid vs. 15.9 [+/-7.2] L/kg saline; p = 0.0001). Peritoneal diasylate clomipramine concentration was greater in lipid-treated animals (366.2 [+/-186.2] microg/L lipid vs. 37.7 [+/-13.8] microg/L saline; p = 0.002). CONCLUSIONS: Amelioration of clomipramine-induced hypotension with lipid infusion is associated with reduced initial Vd and elevated plasma clomipramine concentration consistent with intravascular drug-lipid sequestration. Concomitant peritoneal dialysis with lipid emulsion enhances clomipramine extraction.

Comparative oxidative metabolic profiles of clomipramine in cats, rats and dogs: preliminary results from an in vitro study.

The objectives of this in vitro study were to describe cytochrome-dependent metabolism of clomipramine in canine and feline microsomes, compare metabolic profiles between cats, rats and dogs, and investigate a potential gender-related difference in metabolic activity between male and female cats. Pooled liver microsomes were incubated with clomipramine, where species and gender-specific reactions were initiated by the addition of a nicotinamide adenine dinucleotide phosphate regenerating system and quenched with methanol at 0, 5, 15, 30, 45 and 60 min, and 0, 30, 60, 90, 120, 180, 240 and 360 min respectively. Liquid chromatography tandem mass spectrometry was used to measure clomipramine and its metabolites. Preliminary results showed that cat microsomes biotransformed clomipramine slower and less efficiently than rat and dog microsomes. Moreover, gender differences in metabolic profiles suggested that male cat microsomes may be less efficient demethylators and hydroxylators than female cat microsomes. As gender metabolic differences may carry clinical significance for this antidepressant, further studies are warranted.