Development and validation of an HPLC method for the determination of benzodiazepines and tricyclic antidepressants in biological fluids after sequential SPE

A novel method for the simultaneous determination of six benzodiazepines (BZDs) and four tricyclic antidepressants (TCAs) in biological fluids by HPLC with UV detection at 240 nm has been developed. After a deproteinization step biological fluids were analyzed by direct injection. SPE on Nexus cartridges was also applied. Since two compounds, namely imipramine and diazepam, were coeluting, a sequential SPE protocol has been developed. BZDs were eluted by a mixture of methanol/ACN(1:1), followed by the elution of TCAs with methanol. Separation was performed on a Kromasil C8 column (250 x 64 mm(2) id, 5 microm) using a mobile phase of 0.05 MCH3COONH4/ACN/methanol (initial composition 55:15:30 v/v/v) at a flow rate of 1.0 mL/min delivered by a gradient program within 15 min. Colchicine was used as the internal standard (4 ng/microL). The method was linear for all analytes up to 20 ng/lL, with coefficients of regression between 0.996 and 0.99996. LODs and LOQs were 0.08-1.17 and 0.28-3.91 ng/lL, respectively. Recovery was in the range of 92.8-108.7% for within-day and 91.9-109.9% for between-day assays, with RSD values lower than 10.0% for all matrices.     

Simultaneous HPLC determination of 14 tricyclic antidepressants and metabolites in human plasma

A HPLC method has been developed for the simultaneous determination of seven tricyclic antidepressants (TCAs) and seven metabolites in human plasma. The analyte separation was obtained using a C8 reversed phase column and a mobile phase composed of 68% aqueous phosphate buffer at pH 3.0 and 32% ACN. The UV detector was set at 220 nm and loxapine was used as the internal standard. A careful pre‐treatment procedure for plasma samples was developed, using SPE on C2 cartridges, which gives satisfactory extraction yields (>80%) and good sample purification. The LOQs were always lower than 9.1 ng/mL and the LODs always lower than 3.1 ng/mL for all analytes. The method was successfully applied to plasma samples from depressed patients undergoing therapy with one or more TCA drugs. Precision data (RSD <8.1%), as well as accuracy results (recovery >80%), were satisfactory and no interference from other drugs was found. Hence the method seems to be suitable for the therapeutic drug monitoring of patients treated with TCAs under monotherapy or polypharmacy regimens.     

Reversed-phase liquid chromatography without organic solvent for determination of tricyclic antidepressants

The chromatographic behavior of seven tricyclic antidepressants (amitryptiline, clomipramine, doxepin, imipramine, maprotiline, nortryptiline, and trimipramine) was examined with micellar mobile phases containing the nonionic surfactant Brij-35. Acetonitrile-water mixtures were also used for comparison purposes. Tricyclic antidepressants are moderately polar basic drugs, which are positively charged in the usual working pH. This gives rise to a strong association with the alkyl chains and residual ionized silanols in silica-based stationary phases, which is translated in a high consumption of organic solvent to get appropriate retention times. Brij-35 modifies the surface of the stationary phases creating a neutral bilayer that masks silanols and reduces the polarity. Consequently, the retention times are decreased. A simple chromatographic procedure for the control of tricyclic antidepressants in pharmaceutical formulations was developed, using 0.02 M Brij-35 at pH 3 and UV detection. Satisfactory recoveries were achieved, with intra- and inter-day relative standard deviations usually below 1 and 2%, respectively. The preparation of the samples was simple and only required solubilization and filtration steps previous to injection. The proposed procedure has the advantage of not using an organic solvent in the mobile phase, and the biodegradable character of Brij-35. This makes an example of "green" liquid chromatographic analysis.     

A micellar liquid chromatographic method for quality control of pharmaceutical preparations containing tricyclic antidepressants

Summary Micellar liquid chromatography methods for quality control of pharmaceutical preparations (capsules, pills, tablets, injections) containing the tricyclic antidepressants amineptine, amitriptiline, clomipramine, doxepin, imipramine, melitracen and nortriptyline alone or together with other CNS drugs like diazepam, medazepam and perphenazine are described. The methods using micellar solutions of cetyltrimethylammonium bromide as mobile phases and UV detection are rapid and reproducible. Due to the versatility of interactions in micellar liquid chromatography, it is possible determine highly hydrophobic compounds such as TCAs in a short time using mobile phases containing low organic solvent concentrations and usual flow rates, in contrast with the RP-HPLC methods proposed for these compounds. Samples preparation only requires solution and adequate dilution with the mobile phase before injection into the chromatographic system.     

Response surface methodology in the development of a stacking-sensitive capillary electrophoresis method for the analysis of tricyclic antidepressants in human serum

Stacking methods are very important in overcoming the poor detection limits in capillary electrophoresis (CE). In this paper, the separation and determination of several tricyclic antidepressants by a stacking method is described. The inclusion of acetonitrile (ACN) in the sample causes stacking (transient pseudoisotachophoresis) especially in presence of sodium chloride. An experimental design (central composite design) together with the response surface methodology has been used to find the optimum composition of the separation buffer and the optimal stacking conditions in few experiments. The response functions used are the product of the total resolution by the number of peaks, for the optimization of the separation buffer, and the product of the total resolution by the mean of the peak heights, for the optimization of the stacking conditions. About 28% of the capillary volume is loaded with sample. The calibration curves are linear over the working range (50-300 ng/mL). With a bubble capillary, the limits of detection (LODs) are in the order of 5 ng/mL. For the analysis of serum samples, enrichment with sodium chloride and the protein precipitation with ACN are enough to avoid interferences and to get stacking. Recoveries between 91.6 and 104% and RSD between 0.6 and 12% are obtained in the analysis of samples of lyophilized human serum and non-lyophilized human serum, spiked with the drugs.     

Response surface methodology in the development of a stacking-sensitive capillary electrophoresis method by field-amplified injection for the analysis of tricyclic antidepressants in the presence of salts

The work presented here explores the possibilities of the electrokinetic injection (EK) to achieve sensitive methods for the determination of tricyclic antidepressants in biological samples (serum). The addition of ACN to the sample, with high content in salts, causes stacking at the tip of the capillary, in a similar way as for hydrodynamic injection. An experimental design with the response surface methodology has been used to find the optimum composition of the matrix of the sample (sodium chloride and ACN percentages) and the conditions for the EK (water-plug length, time, and voltage of injection) in few experiments. The composition of the separation buffer was the same as utilized in a previous paper. The use of a bubble capillary to reach lower detection limits implies a loss of the resolution and requires a new optimization. Finally, a comparison between electrokinetic and hydrodynamic injections is made.     

Determination of tricyclic antidepressants using silica gel with a reversed-phase eluent

Summary Five common tricyclic antidepressants are separated on a silica column using an aqueous eluent. The tricyclics are concentrated from serum using an adsorption procedure on a solid phase Sep Pak^TM C₁₈ cartridge. The method appears to be adequate for monitoring therapeutic levels of these drugs.Dedicated to Professor Dr. István Halász for his 60th birthday.     

Determination of tricyclic antidepressants in human plasma using pipette tip solid-phase extraction and gas chromatography-mass spectrometry

A method for the simultaneous extraction of four tricyclic antidepressants from human plasma samples using pipette tip SPE with MonoTip C(18) tips is presented. Human plasma (0.1 mL) containing four tricyclic antidepressants (amitriptyline, amoxapine, imipramine, and trimipramine) and an internal standard (IS), protriptyline, was mixed with 0.4 mL of distilled water and 100 microL 1 M NaOH solution. After centrifugation of the mixture, the supernatant was extracted to the C(18) phase of the tip by 20 repeated aspirating/dispensing cycles using a manual micropipettor. The analytes retained in the tip were eluted with methanol by five repeated aspirating/dispensing cycles. Without evaporation and reconstitution, the eluate was directly injected into a gas chromatograph injector and detected by a mass spectrometer with SIM in the positive-ion electron impact mode. Recovery of the four antidepressants and IS spiked into human plasma was 80.2-92.1%. The regression equations for the four antidepressants showed excellent linearity in the range of 0.2-40 ng/0.1 mL. LODs and LOQs for the four drugs were 0.05-0.2 ng/0.1 mL and 0.2-0.5 ng/0.1 mL, respectively. Intra- and interday CVs for the four drugs in plasma were no greater than 9.5%.     

Optimization of solid-phase microextraction procedures for the determination of tricyclic antidepressants and anticonvulsants in plasma samples by liquid chromatography

Simple, sensitive, and reproducible off-line solid-phase microextraction and liquid chromatography (SPME/LC) methods are described for the determination of seven anticonvulsants and tricyclic antidepressants in human plasma. Factorial design and simplex methodology were applied in the optimization of the SPME procedure for tricyclic antidepressants analyses. Important factors in the SPME efficiency are discussed, such as the fiber coatings (both lab-made and commercial), extraction time, pH, ionic strength, influence of plasma proteins, and desorption conditions. The development of the lab-made fiber coatings, namely, octadecylsilane, aminosilane, and polyurethane, are further described and applied to anticonvulsants analyses. The investigated plasmatic range for the evaluated anticonvulsants, using CW-TPR fiber, were the following: phenylethylmalonamide (3.00–40.0 μg mL⁻¹), phenobarbital (5.00–40.0 μg mL⁻¹), primidone (3.00–40.0 μg mL⁻¹), carbamazepine and carbamazepine-epoxide (2.00–24.0 μg mL⁻¹), phenytoin (2.00–40.0 μg mL⁻¹), and lamotrigine (0.50–12.0 μg mL⁻¹). The antidepressants’ linear plasmatic concentration ranged from 75.0 to 500 ng mL⁻¹ for imipramine, amitriptyline, and desipramine, and from 50.0 to 500 ng mL⁻¹ for nortriptyline, being in all cases, the limit of quantification represented by the lowest value. The precision (interassays) for all investigated drugs in plasma sample spiked with different concentrations of each analyte and submitted to the described procedures were lower than 15%. The off-line SPME/LC methodologies developed allow anticonvulsants and antidepressants analyses from therapeutic to toxic levels for therapeutic drug monitoring.     

Development of a validated HPLC method for the determination of B-complex vitamins in pharmaceuticals and biological fluids after solid phase extraction

An HPLC method was developed for the simultaneous determination of seven water-soluble vitamins, viz. thiamine, riboflavin, nicotinic acid, nicotinamide, pyridoxine, cyanocobalamin, and folic acid, in multivitamin pharmaceutical formulations and biological fluids (blood serum and urine). Separation was achieved at ambient temperature on a Phenomenex Luna C18 (150 x 4.6 mm) analytical column. Gradient elution was performed starting at a 99:1 A:B v/v composition, where A: 0.05 M CH3COONH4/CH3OH (99/1) and B: H2O/CH3OH (50/50), at a flow rate of 0.8 mL/min. After a 4-min isocratic elution the composition was changed to 100% of B in 18 min and elution continued isocratically for 8 min. Detection was performed with a photodiode array detector at 280 nm. Each vitamin was quantitatively determined at its maximum wavelength. Spectral comparison was used for peak identification in real samples. Detection limits were in the range of 1.6-3.4 ng, per 20-microL injection, while linearity held up to 25 ng/microL. Accuracy, intra-day repeatability (n = 6), and inter-day precision (n = 7) were found to be satisfactory. Theobromine (2 ng/microL) was used as internal standard. Sample preparation of biological fluids was performed by SPE on Supelclean LC-18 cartridges with methanol-water 85/15 v/v as eluent. Extraction recoveries from biological matrices ranged from 84.6% to 103.0%.     

Application of a new fiber coating based on electrochemically reduced graphene oxide for the cold‐fiber headspace solid‐phase microextraction of tricyclic antidepressants

A new fiber based on the electrochemical reduction of graphene oxide was prepared on a copper wire for solid‐phase microextraction (SPME) applications. The prepared fiber was used for the SPME and gas chromatographic analysis of tricyclic antidepressants (TCADs), including amitriptyline, trimipramine, and clomipramine. The feasibility of direct‐immersion and headspace modes of SPME for the determination of TCADs was studied. The effects of four parameters including pH, salt content, extraction temperature with and without cooling the fiber, and extraction time were investigated. The comparison showed that headspace cold fiber SPME results in the best outcome for the extraction of TCADs. Under the optimized conditions of this mode, the calibration curves were linear between 2.0 and 500 ng/mL and the detection limits were between 0.30 and 0.53 ng/mL. The intraday and interday RSDs obtained at 20 ng/mL (n = 5), using a single fiber, were 5.5–9.0 and 7.5–9.8, respectively. The fiber to fiber repeatability (n = 4), expressed as the RSD, was between 12.8 and 13.2% at a 20 ng/mL concentration level. The method was successfully applied to the analysis of TCADs in plasma samples showing recoveries from 73 to 96%.     

Capillary electrophoretic separation of tricyclic antidepressants using a polymer-coated capillary and beta-cyclodextrin as an electrolyte additive

A new method for the CE separation of nine tricyclic antidepressants (TCAs), viz. amitriptyline, clomipramine, desipramine, doxepin, fluphenazine, imipramine, nortriptyline, promazine, and thioridazine, is described. The capillary was statically coated with a layer of poly(N,N-dimethylacrylamide) (PDMA) to suppress the EOF, and beta-CD was used as an additive in the BGE solution. The optimal resolution of nine TCAs was obtained by using a 1% v/v PDMA-coated capillary and a BGE solution of 50 mM sodium phosphate buffer (pH 3.0) containing 0.5 mM beta-CD. Efficiencies were typically >10(5 )plates/m. Complete separation of nine TCAs could be achieved in about 28 min; the two diastereomers of doxepin and the two enantiomers of thioridazine could also be separated. The RSD values of migration time and peak area of the TCAs were in the ranges 0.5-0.8 and 3.3-4.9% (n = 10), respectively. In combination with a suitable sample clean-up technique, such as hollow fiber-based liquid phase microextraction (HF-LPME), the polymer-coated capillary can be employed for the CE-UV analysis of TCAs in human plasma.     

Development of a validated HPLC method for the determination of four 1,4-benzodiazepines in human biological fluids

A simple and sensitive HPLC method was developed and validated for the determination of four frequently prescribed 1,4-benzodiazepines: alprazolam (ALP), bromazepam (BRZ), diazepam (DZP), and flunitrazepam (FNZ). Separation was achieved on an Inertsil C8 analytical (250 mm x 4 mm, 5 microm) column, after selective extraction of benzodiazepine drugs from biological matrices by means of SPE. Isocratic elution was performed with a mobile phase consisting of CH3COONH4, 0.05 M CH3OH, and CH3CN (33:57:10 by volume). Quantification was performed at 240 nm with mefenamic acid (6 ng/microL) as the internal standard. DSC-18 Supelco cartridges provided high absolute recoveries (81-115%). The developed method was fully validated in terms of selectivity, linearity, accuracy, precision, stability, and sensitivity. Repeatability (n = 8) and between-day precision (n = 8) revealed RSD <12%. Recoveries from biological samples ranged from 81.2 to 115%. The detection limit of the method was calculated as 3.3-10.2 ng in blood plasma and 2.6-12.6 ng in urine for 20 microL injection volume. The method was applied to spiked biological matrices. Moreover, the method was applied to real samples of urine after an oral administration.     

Electro‐assisted solid‐phase microextraction based on poly(3,4‐ethylenedioxythiophen) combined with GC for the quantification of tricyclic antidepressants

In this study, a platinum wire coated with poly(3,4‐ethylenedioxythiophen) was used as an electro‐assisted solid‐phase microextraction fiber for the quantification of tricyclic antidepressant drugs in biological samples by coupling to GC employing a flame ionization detector. In this study, an electric field increased the extraction rate and recovery. The fiber used as a solid phase was synthesized by the electropolymerization of 3,4‐ethylenedioxythiophen monomers onto a platinum wire. The ability of this fiber to extract imipramine, desipramine, and clomipramine by using the electro‐assisted solid‐phase microextraction technique was evaluated. The effect of various parameters that influence the extraction efficiency, which include solution temperature, extraction time, stirring rate, ionic strength, time and temperature of desorption, and thickness of the fiber, was optimized. Under optimized conditions, the linear ranges and regression coefficients of calibration curves were in the range of 0.5–250 and 0.990–0.998 ng/mL, respectively. Detection limits were in the range of 0.15–0.45 ng/mL. Finally, this method was applied to the determination of drugs in urine and wastewater samples and recoveries were 4.8–108.9%.     

Response surface methodology for the optimisation of flow-injection analysis with in situ solvent extraction and fluorimetric assay of tricyclic antidepressants

A semiautomatic extraction-fluorimetric method for the determination of tricyclic antidepressant drugs (TCAs) based in the formation of ion pairs with 9,10-dimethoxyanthracene-2-sulphonate (DMAS) has been developed. The aqueous solutions of the TCAs (imipramine, desipramine, amitriptyline, nortriptyline, clomipramine or doxepine) are injected into a carrier composed by DMAS in an acid medium and the ion pair formed is extracted into dichloromethane where the fluorescence is measured. An experimental design (Central Composite Design) together with the Response Surface Methodology has been used to find the optimal instrumental FIA and chemical variables. We have considered as the response function the product of the peak height by the sampling frequency. The calibration curves were linear over the working range (0.25-3.00 mg L⁻¹). The limits of detection were lower than 0.30 mg L⁻¹. The method has been satisfactorily applied to the determination of imipramine, amitriptyline, clomipramine and doxepin in pharmaceutical preparations.     

Bioanalytical method for the estimation of co‐administered esomeprazole,leflunomide and ibuprofen in human plasma and in pharmaceutical dosage forms using micellar liquid chromatography

The present study represents a connection between basic science and clinical applied science through providing a bioanalytical method for the analysis of certain co‐administered drugs used for the treatment of rheumatoid arthritis. The studied drugs are esomeprazole, leflunomide and ibuprofen. The proposed bioanalytical method is a simple reversed phase high performance liquid chromatographic method using micellar mobile phase. The method is conducted using a Shim‐pack VP‐ODS (150 mm × 4.6 mm ID) stainless steel column at ambient temperature with ultraviolet detection at 285 nm. The micellar mobile phase consisted of 0.1 m sodium dodecyl sulfate, 10% n‐propanol, 0.3% triethylamine in 0.02 m orthophosphoric acid (pH 3.5) and is pumped at a flow rate of 1.0 mL/min. The calibration curve was rectilinear over the concentration range of 0.1–5.0, 0.5–10.0 and 1.0–20.0 μg/mL for esomeprazole, leflunomide and ibuprofen respectively. The proposed method was successfully applied to the analysis of these drugs in dosage forms. The method is extended to the in‐vitro , in‐vivo determination of these drugs in spiked and real human plasma samples.     

Dispersive liquid–liquid microextraction combined with acetonitrile stacking through capillary electrophoresis for the determination of three selective serotonin reuptake inhibitor drugs in body fluids

Dispersive liquid–liquid microextraction was combined with acetonitrile stacking in capillary electrophoresis for the identification of three selective serotonin reuptake inhibitors (citalopram, fluoxetine, and fluvoxamine) in human fluids such as urine and plasma. Parameters that affect the extraction and stacking efficiency, such as the type and volume of the extraction and disperser solvent, extraction time, salt addition for dispersive liquid–liquid microextraction, and sample matrices, pH, and concentration of the separation buffer for stacking, were investigated and optimized. Under optimum conditions, the enrichment factors were in the range of 1195–1441. Limits of detection ranged from 1.4 to 1.7 nM for the target analytes. Calibration graphs displayed satisfied linearity with R² greater than or equal to 0.9978, and relative standard deviations of the peak area analysis were in the range of 2.9–5.0% (n = 3). The recoveries of all tricyclic antidepressant drugs from urine and plasma were in the range of 77–117 and 79–106%, respectively. The findings of this study show that dispersive liquid–liquid microextraction acetonitrile‐stacking capillary electrophoresis is a rapid and convenient method for identifying tricyclic antidepressant drugs in urine and plasma.     

Simultaneous Determination of 1,4‐Benzodiazepines and Tricyclic Antidepressants in Saliva after Sequential SPE Elution by the Same HPLC Conditions

A new method has been described to determine both benzodiazepines (six) and tricyclic antidepressants (four) simultaneously in saliva by HPLC with a UV detector set at 240 nm using cholchicine as the internal standard. A careful specific sequential solid‐phase elution was optimized and performed to elute benzodiazepines using a mixture of methanol‐acetonitrile (1:1 v/v) followed by the elution of tricyclic antidepressants with methanol. Separation of the compounds was performed on a Kromasil column (250 × 4 mm, 5 μm) by a gradient eluents consisting of 0.05 M CH₃COONH₄‐acetonitrile‐methanol (55:15:30 v/v/v). The results were linear for both benzodiazepines and tricyclic antidepressants up to 20 ng μL^‐1 with the correlation coefficients greater than 0.998. The sensitivity limits, LOD and LOQ were 0.08‐0.34 ng μL^‐1 and 0.28‐1.13 ng μL^‐1, respectively. The method is simple, fast and reliable with good specificity and sensitivity, will be suitable for use in a clinical setting, where there is a concomitant use of 1,4‐benzodiazepines and tricyclic antidepressants.     

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.     

Development,validation and application of an HPLC method for phenol electrooxidation products in the presence of chloride

A high-performance liquid chromatography (HPLC) method was developed and validated to determine phenol and potential intermediates from hydroxylation (hydroquinone, benzoquinone, catechol) and hypochlorination (2-chlorophenol, 3-chlorophenol, 4-chlorophenol, 2,4-dichlorophenol, 2,6-dichlorophenol, 2,3,6-trichlorophenol, 2,4,5-trichlorophenol, 2,4,6-trichlorophenol and pentachlorophenol) pathways during electrooxidation in the presence of chloride. A Hypersil ODS column (150 mm L × 4.6 mm I.D.) was used for the separation. The best separation was achieved when using a time variant gradient between a water mobile phase (with 0.1% formic acid adjusted to pH 3.0 with 0.1 mM sulfuric acid) and an organic phase (90:5:5 by volume mixture of acetonitrile:methanol:acetic acid). The flow rate was 0.8 mL min^?1 and UV absorbance was monitored at 270, 280, 290 and 300 nm, choosing the wavelength of strongest response for each compound. The intra- and inter-day accuracy and precision were tested using five replicates each day on three consecutive days.