Column switching and high-performance liquid chromatography in the analysis of amitriptyline, nortriptyline and hydroxylated metabolites in human plasma or serum.

A column-switching system for the direct injection of plasma or serum samples, followed by isocratic high-performance liquid chromatography and ultraviolet detection, is described for the simultaneous quantitation of the tricyclic antidepressant amitriptyline, its demethylated metabolite nortriptyline and the E- and Z-isomers of 10-hydroxyamitriptyline and 10-hydroxynortriptyline. The method included adsorption of amitriptyline and metabolites on a reversed-phase C8 clean-up column (10 microns; 20 mm x 4.6 mm I.D.), washing of unwanted material to waste and, after on-line column-switching, separation on a cyanopropyl analytical column (5 microns; 250 mm x 4.6 mm I.D.). The compounds of interest were separated and eluted using acetonitrile-methanol-0.01 M phosphate buffer (pH 6.8) (578:188:235, v/v) within less than 20 min. Various drugs frequently co-administered with amitriptyline or other antidepressants did not interfere with the determinations. In plasma samples spiked with 25-300 ng/ml, the recoveries were between 84 and 112% and the inter-assay coefficients of variation were 3-11%. After a minor modification, as little as 5 ng/ml could be quantitated. There were linear correlations (r greater than 0.99) between drug concentrations of 5-500 ng/ml and the detector signal. The method allows routine measurements of amitriptyline, nortriptyline and hydroxylated metabolites in blood plasma or serum of patients treated with amitriptyline or nortriptyline, and enables the results to be reported within 1 h.     

Analysis of tricyclic antidepressant drugs in plasma by means of solid-phase microextraction-liquid chromatography-mass spectrometry

Solid-phase microextraction coupled to liquid chromatography and mass spectrometry (SPME-LC-MS) was used to analyze tricyclic antidepressant drugs desipramine, imipramine, nortriptyline, amitriptyline, and clomipramine (internal standard) in plasma samples. SPME was performed by direct extraction on a PDMS/DVB (60 microm) coated fiber, employing a stirring rate of 1200 rpm for 30 min, pH 11.0, and temperature of 30 degrees C. Drug desorption was carried out by exposing the fiber to the liquid chromatography mobile phase for 20 min, using a labmade SPME-LC interface at 50 degrees C. The main variables experimentally influencing LC-MS response were evaluated and mathematically modeled. A rational optimization with fewer experiments was achieved using a factorial design approach. The constructed empirical models were adjusted with 96-98% of explained deviation allowing an adequate data set comprehension. The chromatographic separation was realized using an RP-18 column (150 mm x 2.1 mm, 5 microm particles) and ammonium acetate buffer (0.01 mol/l, pH 5.50) : acetonitrile (50 : 50 v/v) as mobile phase. Low detection levels were achieved with electrospray interface (0.1 ng/ml). The developed method showed specificity, linearity, precision, and limit of quantification adequate to assay tricyclic antidepressant drugs in plasma.     

On-line pretreatment using methylcellulose-immobilized cation-exchange restricted access media for direct liquid chromatography/mass spectrometric determination of basic drugs in plasma

A novel methylcellulose-immobilized restricted access media column with strong cation-exchange groups on an internal surface (MC-SCX) was evaluated for the direct injection analysis of basic polar drugs in plasma by column-switching liquid chromatography/mass spectrometry (LC/MS). Analytical conditions, including an automated pretreatment step and MS detection, were optimized for a series of basic drugs (doxepin, desipramine, imipramine, nortriptyline, amitriptyline, clomipramine). On-line pretreatment with the MC-SCX column followed by fast gradient analysis using a C18 column resulted in a total analysis cycle time of 7 min for each spiked plasma sample. More than 150 plasma samples spiked with target compounds were measured without compromising MS detection (relative standard deviations less than 11% for all compounds, and regression coefficients greater than 0.99).     

Simultaneous determination of caffeine, theophylline and theobromine in human plasma by on-line solid-phase extraction coupled to reversed-phase chromatography

A reversed-phase liquid chromatographic column switching system was described for the determination of caffeine (CF), theophylline (TH) and theobromine (TB) in human plasma with a direct injection procedure. A short protein-coated mu Bondapak CN silica pre-column (20 x 3 mm, i.d.) was used for enrichment of the drugs and clean up from weakly retained plasma components using phosphate buffer saline pH 7.4. After washing step, the retained drugs were flushed into a reversed-phase column (5 microm TSK gel ODS-80 TM, 150 x 4.6 mm i.d.) with a mobile phase of methanol-0.01 M phosphate buffer, pH 3.5 (30:70, v/v) for the final separation. The eluent was monitored with a UV detector at 275 nm. The resulting chromatograms showed no interference from endogenous plasma components. A linear relationship between the concentration of drug and peak height was confirmed in the range of 0.5-20 microg/mL for all drugs. High extraction recoveries from plasma ranging from 96.12 to 100.32% were achieved. Validation of the method was examined performing intra- and inter-day accuracy and precision and was found to be satisfactory. The coefficients of variation of the three drugs were less than 3% for intra-day and less than 4% for inter-day run assays.     

Direct injection HPLC micro method for the determination of voriconazole in plasma using an internal surface reversed-phase column

A direct plasma injection liquid chromatographic method has been developed for the determination of a new triazole antifungal agent, voriconazole, using an internal surface reversed phase column. Therapeutic drug monitoring of voriconazole is relevant for patient management, especially in the case of drug-drug interaction. The method is easy to perform and requires 10 microL of a plasma sample. The chromatographic run time is less than 9 min using a mobile phase of 17:83 v/v acetonitrile-potassium dihydrogen phosphate buffer, 100 mM, pH 6.0 and UV detection at 255 nm. The fl ow rate was 1 microL/min. A linear response was observed over the concentration range 0.5-10 microg/mL (r2 = 0.977). A good accuracy (bias < or = 7.5%) was achieved for all quality controls, with intra-day and inter-day variation coefficients inferior to 6.7%. The lower limit of quantitation was 0.2 microg/mL, without interference of endogenous components. The stability of voriconazole in plasma stored at different temperatures was checked. Finally, the possibility of direct injection of plasma samples into the column permits a reduction in reagent consumption and in analytical steps, and hence in analytical error.     

Simultaneous determination of beta-blockers in human plasma using liquid chromatography-tandem mass spectrometry

A detailed procedure for the analysis of four beta-blockers, acebutolol, labetalol, metoprolol and propranolol, in human plasma by high-performance liquid chromatography (LC)-tandem mass spectrometry (MS-MS) using an MSpak GF column, which enables direct injection of crude plasma samples, is presented. Protein and/or macromolecule matrix compounds were eluted first from the column, while the drugs were retained on the polymer stationary phase of the MSpak GF column. The analytes retained on the column were then eluted into an acetonitrile-rich mobile phase using a gradient separation technique. All drugs showed base peak ions due to [M + H]+ ions by LC-MS with positive ion electrospray ionization, and the product ions were produced from each [M + H]+ ion by LC-MS-MS. Quantification was performed by selected reaction monitoring. The recoveries of the four beta-blockers spiked into plasma were 73.5-89.9%. The regression equations for all compounds showed excellent linearity in the range 10-1000 ng/mL of plasma, with the exception of propranolol (10-800 ng/mL). The limits of detection and quantification for each drug were 1-3 and 10 ng/mL, respectively, of plasma. The intra- and inter-day coefficients of variation for all drugs in plasma were not greater than 10.9%.     

Direct injection HPLC method for the determination of selected phenothiazines in plasma using a Hisep column

A direct plasma injection HPLC method has been developed for the determination of selected phenothiazines (promethazine, promazine, chlorpromazine) using a Hisep column. The method is easy to perform and requires 20 microL of a filtered plasma sample. The chromatographic run time is less than 11 min using a mobile phase of 15:85 v/v acetonitrile-0.18 m ammonium acetate pH 5.0 and UV detection at 254 nm. The method is linear in the concentration range 0.1-25 microg mL(-1) (r > 0.99, n = 6) for each analyte with RSD less than 6%. Interday and intraday variability were found to be < or =14%. The limits of detection and quantitation were 0.1 (S/N > 3) and 0.25 microg mL(-1) (S/N > 10), respectively, for each of the three phenothiazines. We can also apply this method to separate three other phenothiazines (ethopromazine, trifluoroperazine, prochlorperazine), although it lacks the selectivity to determine the concentration of all six drugs concurrently. The separation is feasible using these drugs in certain combinations.     

Automated high-performance liquid chromatographic method for the simultaneous determination of cefotiam and delta 3-cefotiam in human plasma using column switching.

An automated high-performance liquid chromatographic method using column switching was established for the simultaneous determination of cefotiam (I) and delta 3-cefotiam (II) in human plasma after oral administration of cefotiam hexetil dihydrochloride. The method allowed the determination of analytes in plasma by the direct injection of diluted specimen with phosphate buffer. The analytes were enriched onto the C18 short pretreatment column by 0.05 M phosphate buffer (pH 7.7), while proteins and endogenous hydrophilic substances in plasma were washed off to waste. The enriched analytes were then back-flushed onto the analytical C18 column, separated by a mixture of 0.05 M phosphate buffer (pH 7.7)-acetonitrile (88:12, v/v) and detected by the ultraviolet absorbance at 254 nm. Recoveries from spiked plasma were quantitative, and the coefficients of variation were below 4%. The lower detection limits in plasma were 10 ng/ml for both I and II. Concentrations of I and II in plasma determined by the present method were in good agreement with those obtained by the conventional deproteinization method.     

Simultaneous determination of amiodarone and its major metabolite desethylamiodarone in plasma, urine and tissues by high-performance liquid chromatography

A simple and sensitive high-performance liquid chromatographic method for the simultaneous assay of amiodarone and desethylamiodarone in plasma, urine and tissues has been developed. The method for plasma samples and tissue samples after homogenizing with 50% ethanol, involves deproteinization with acetonitrile containing the internal standard followed by centrifugation and direct injection of the supernatant into the liquid chromatograph. The method for urine specimens includes extraction with a diisopropyl ether-acetonitrile (95:5, v/v) mixture at pH 7.0 using disposable Clin-Elut 1003 columns, followed by evaporation of the eluate, reconstitution of the residue in methanol-acetonitrile (1:2, v/v) mixture and injection into the chromatograph. Separation was obtained using a Radial-Pak C18 column operating in combination with a radial compression separation unit and a methanol-25% ammonia (99.3:0.7, v/v) mobile phase. A wavelength of 242 nm was used to monitor amiodarone, desethylamiodarone and the internal standard. The influence of the ammonia concentration in the mobile phase on the capacity factors of amiodarone, desethylamiodarone and two other potential metabolites, monoiodoamiodarone (L6355) and desiodoamiodarone (L3937) were investigated. Endogenous substances or a variety of drugs concomitantly used in amiodarone therapy did not interfere with the assay. The limit of sensitivity of the assay was 0.025 micrograms/ml with a precision of +/- 17%. The inter- and intra-day coefficient of variation for replicate analyses of spiked plasma samples was less than 6%. This method has been demonstrated to be suitable for pharmacokinetic and metabolism studies of amiodarone in man.     

Simultaneous determination of cefamandole and cefamandole nafate in human plasma and urine by high-performance liquid chromatography with column switching

A high-performance liquid chromatographic method with column switching has been developed for the simultaneous determination of cefamandole and cefamandole nafate in plasma and urine. The plasma and urine samples were injected onto a precolumn packed with Corasil RP C18 (37-50 microns) after simple dilution with an internal standard solution in 0.05 M phosphoric acid. Polar plasma and urine components were washed out using 0.05 M phosphoric acid. After valve switching, the concentrated drugs were desorbed in back-flush mode and separated by a reversed-phase C8 column with methanol-5 mM tetrabutylammonium bromide (45:55, v/v) as the mobile phase. The method showed excellent precision with good sensitivity and speed, and a detection limit of 0.5 microgram/ml. The total analysis time per sample was less than 30 min, and the mean coefficients of variation for intra- and inter-assay were both less than 4.9%. The method has been successfully applied to plasma and urine samples for human volunteers after intravenous injection of cefamandole nafate.     

High-performance liquid chromatographic determination of amitriptyline and its main metabolites using a silica column with reversed-phase eluent. Application in mice.

A method was developed for the assay of amitriptyline, amitriptyline N-oxide, nortriptyline, desmethylnortriptyline and E (trans) and Z (cis) isomers of 10-hydroxyamitriptyline and of 10-hydroxynortriptyline in plasma and brain of animals, using high-performance liquid chromatography with ultraviolet detection (254 nm). Single extraction was performed at pH 10.5 from 0.25 ml of plasma or 1 ml of brain mixture. Chromatographic separations were achieved with a silica column and an aqueous methanol mobile phase containing ammonia. This procedure offers high sensitivity (8-10 ng/ml), high linearity (r > 0.99) and acceptable precision (coefficient of variation < or = 13.3%). The method was used to determine levels of amitriptyline and its major metabolites in mice 30 min after a single intraperitoneal administration of amitriptyline (20 mg/kg).     

On-line coupling of sequential injection extraction with restricted-access materials and post-column derivatization for sample clean-up and determination of propranolol in human plasma

The presented paper deals with a new methodology for direct determination of propranolol in human plasma. The methodology described is based on sequential injection analysis technique (SIA) coupled with solid phase extraction (SPE) column based on restricted access materials (RAM). Special RAM column containing 30 μm polymeric material—N-vinylacetamide copolymer was integrated into the sequential injection manifold. SIA–RAM system was used for selective retention of propranolol, while the plasma matrix components were eluted with two weak organic solutions to waste.

Due to the acid–basic and polarity properties of propranolol molecule and principles of reversed-phase chromatography, it was possible to retain propranolol on the N-vinylacetamide copolymer sorbent (Shodex MSpak PK-2A 30 μm (2 mm × 10 mm)). Centrifuged plasma samples were aspirated into the system and loaded onto the column using acetonitrile–water (5:95, v/v), pH 11.00, adjusted by triethylamine. The analyte was retained on the column while proteins contained in the sample were removed to waste. Interfering endogenous substances complicating detection were washed out by acetonitrile–water (15:85), pH 11.00 in the next step. The extracted analyte was eluted by means of tetrahydrofuran–water (25:75), pH 11.00 to the fluorescence detector (emission filter 385 nm). The whole procedure comprising sample pre-treatment, analyte detection and column reconditioning took about 15 min. The recoveries of propranolol from undiluted plasma were in the range 96.2–97.8% for three concentration levels of analyte. The proposed SIA–RAM method has been applied for direct determination of propranolol in human plasma.     

Optimization of the SPME parameters and its online coupling with HPLC for the analysis of tricyclic antidepressants in plasma samples

Solid-phase microextraction (SPME)-liquid chromatography (LC) is used to analyze tricyclic antidepressant drugs desipramine, imipramine, nortriptyline, amitriptyline, and clomipramine (internal standard) in plasma samples. Extraction conditions are optimized using a 2(3) factorial design plus a central point to evaluate the influence of the time, temperature, and matrix pH. A Polydimethylsiloxane-divinylbenzene (60-mum film thickness) fiber is selected after the assessment of different types of coating. The chromatographic separation is realized using a C(18) column (150 x 4.6 mm, 5-microm particles), ammonium acetate buffer (0.05 mol/L, pH 5.50)-acetonitrile (55:45 v/v) with 0.1% of triethylamine as mobile phase and UV-vis detection at 214 nm. Among the factorial design conditions evaluated, the best results are obtained at a pH 11.0, temperature of 30 degrees C, and extraction time of 45 min. The proposed method, using a lab-made SPME-LC interface, allowed the determination of tricyclic antidepressants in in plasma at therapeutic concentration levels.     

Quantitative analysis of retinoids in biological fluids by high-performance liquid chromatography using column switching. II. Simultaneous determination of etretinate, acitretin and 13-cis-acitretin in plasma

An automated gradient high-performance liquid chromatographic method for the determination of etretinate, acitretin and 13-cis-acitretin in plasma was developed, using a column-switching technique. After protein precipitation with ethanol, 0.5 ml of the supernatant was injected onto a precolumn (17 mm x 4.6 mm I.D.), filled with 37-53 microns C18 Corasil. Polar plasma components were washed out using 1% ammonium acetate and 1% acetic acid-acetonitrile (8:2, v/v); the retained retinoids were then transferred to the analytical column (125 mm x 4 mm I.D., filled with 5-microns ODS material) in the backflush mode, separated by gradient elution and detected at 360 nm by UV detection. The limit of quantification was 2 ng/ml and the inter-assay precision in the concentration range 20-1000 ng/ml was between 0.9 and 4.0% for all three compounds. To optimize the recovery for etretinate (greater than 60%), protein was precipitated from plasma with ethanol before injection, instead of direct injection of plasma samples, and a mobile phase containing 20% acetonitrile, instead of pure water or buffer, was used.     

Validation of a sensitive HPLC/fluorescence method for assessment of ciprofloxacin levels in plasma and prostate microdialysate samples from rats

Chronic bacterial prostatitis treatment consists of broad‐spectrum antibiotic therapy for long periods of time. Drug penetration into the prostate makes the treatment a challenged. Ciprofloxacin is one of the most prescribed drugs for this treatment. A liquid chromatography with fluorescence detection method was developed and validated for determining ciprofloxacin concentrations in two different matrices: plasma and prostate microdialysate. Ciprofloxacin was separated on a C₁₈ column eluted with a mobile phase constituted of a mixture of 0.4% aqueous triethylamine:methanol:acetonitrile (75:15:10, v/v/v) and 0.4% aqueous triethylamine:acetonitrile (88:12, v/v) for microdialysate and plasma samples, respectively. Linearity was obtained over a concentration range of 5–1000 ng/mL (microdialysate) and 10–2000 ng/mL (plasma), with coefficients of determination ≥0.9956. Precision was determined from the analysis of six quality control samples and showed RSD values <11.1 and 7.4% for intra and inter‐assay precision, respectively. The accuracy ranged from 85.6 to 114.3%. The method was applied to a preliminary pharmacokinetic study to investigate ciprofloxacin concentrations in prostate, sampled by microdialysis, and plasma after a 7 mg/kg intravenous dose to Wistar rats. The method showed high sensitivity using only protein precipitation as plasma sample clean‐up and was successfully applied to investigate ciprofloxacin prostate penetration. Copyright © 2015 John Wiley & Sons, Ltd.     

Liquid chromatographic method for the simultaneous determination of eprosartan and hydrochlorothiazide in tablets and human plasma

A new, specific, and sensitive RP-HPLC method was developed for the simultaneous determination of eprosartan (EPR) and hydrochlorothiazide (HCT). Good chromatographic separation was achieved using a 250 x 4.6 mm id, 5 microm particle size Symmetry C18 column. The mobile phase acetonitrile-0.1 M phosphate buffer (35+65, v/v), pH 4.5, was pumped at a flow rate of 1 mL/min, with UV detection at 275 nm. The method showed good linearity in the ranges of 0.5-50 and 0.1-10 microg/mL, with LOD of 0.06 and 0.02 microg/mL and LOQ of 0.20 and 0.08 microg/mL for EPR and HCT, respectively. The proposed method was successfully applied for the analysis of the studied drugs in their synthetic mixture and co-formulated tablets. The method was further extended to the in vitro and in vivo determination of the two drugs in spiked and real human plasma. Interference likely to be encountered from the co-administered drugs was studied.     

Simultaneous determination of methotrexate and metoclopramide in physiological fluids using RP-HPLC with ultra-violet detection; application in evaluation of polymeric nanoparticles

Methotrexate (MTX) is an anticancer drug while metoclopramide (MCP) is an antiemetic agent. Both the drugs are commonly coprescribed to avoid the emesis caused by anticancer drug. In this study, a novel, rapid, sensitive, and cost-effective reverse-phase high-performance liquid chromatography method was developed and validated for simultaneous determination of the methotrexate and metoclopramide in biological and pharmaceutical samples using sparfloxacin as internal standard. The analytes were separated on a Kromasil 100-5C18 RP (250?×?4.6?mm, 5?µm) column, methanol, and 0.05% trifloroacetic acid (36:64?v/v) as mobile phase with a flow rate of 1?mL/min, detection wavelength of 290?nm, and column oven temperature at 40°C. Both the analytes were extracted from physiological fluids (bovine aqueous humor, vitreous humor, and human plasma) using mixture of methanol and 10% perchloric acid (50:50 v/v). The method was linear over the concentration range of 0.025–1.0?µg/mL for methotrexate and 0.030–1.0?µg/mL for metoclopramide. The % recovery from human plasma was 98.57 and 96.74% for MTX and MCP, respectively, while from aqueous humor and vitreous humor was 95.84 and 98.51% for MTX.

The developed method was applied for in vitro release of MTX from polymeric nanoparticles and can be applied for analysis of pharmaceutical and biological samples containing both the drugs.     

Fully automated analytical system using liquid-solid extraction and liquid chromatography for the determination of CGP 6140 in plasma

Liquid-solid extraction on disposable extraction columns (DECs) and liquid chromatography can be combined in a completely automated analyser. The Gilson ASPEC system was used to develop a procedure for the determination of CGP 6140 in plasma. Both sample preparation via C8 Bond-Elut DECs and injection were fully automatic. The fully automated system prepared the samples by performing the same operations as for a manual procedure. The DEC was first wetted with methanol, then with water. A 400-microliters volume of plasma and 40 microliters of the internal standard solution, diluted with 1 ml of water, were applied to the DEC, rinsed with 10(-2) mol/l dipotassium hydrogenphosphate and eluted from the DEC with 300 microliters of acetonitrile-methanol (50:50, v/v). The eluting strength of the eluate was reduced by dispensing 1 ml of water into each vial prior to direct injection into a Spherisorb ODS column via a 1-ml loop. This allowed the reconcentration of the extracted compounds on the top of the column, as they were injected in a large volume of solvent of lower eluting strength than the mobile phase [acetonitrile-methanol-4 x 10(-3) mol/l ammonia solution (54.5:5:40.5, v/v/v)]. Reproducibility results are presented.     

Determination of diclofenac in plasma using a fully automated analytical system combining liquid-solid extraction with liquid chromatography

A fully automated analytical system based on liquid-solid extraction combined with column liquid chromatography is described for the determination of diclofenac in plasma. After addition of pH 5 buffer and the internal standard solution to the plasma sample, both sample preparation via a C18 disposable extraction column and injection were performed by a Gilson ASPEC system. Diclofenac and the internal standard were separated on a reversed-phase column, using methanol-pH 7.2 phosphate buffer (56:44, v/v) as mobile phase at a flow-rate of 0.4 ml/min. The reproducibility and accuracy of the method were acceptable over the concentration range 31-3140 nmol/l in plasma.     

Determination of a new non-benzodiazepine anxiolytic and its O-demethyl metabolite in plasma by high-performance liquid chromatography using automated column-switching

An highly sensitive and fully automated high-performance liquid chromatographic assay was developed for the determination of a novel non-benzodiazepine anxiolytic (I) [(R)-2-(methoxymethyl)-1-[(7-oxo-8-phenyl-7H-thieno[2,3-a]quinolizin+ ++- 10-yl)carbonyl]pyrrolidine] and its O-demethyl metabolite (II) in plasma, using column-switching for direct injection of plasma samples. After dilution in internal standard solution, the sample was injected onto a pre-column (17 mm x 4.6 mm) dry-packed with pellicular C18 reversed-phase material. Polar plasma components were removed by flushing the pre-column with water-acetonitrile (90:10, v/v). Retained substances, including I and II, were backflushed onto an analytical column, separated by gradient elution and detected by means of fluorescence detection (excitation, 304 nm; emission, 475 nm). After washing the analytical column and re-equilibrating the pre-column, the system was ready for the next injection. The limit of quantification for I and II was 0.25 and 0.5 ng/ml, respectively, using a 350-microliter specimen of plasma. The practicability of the new method was demonstrated by analysis of more than 300 plasma samples from a tolerance study performed with human volunteers. Owing to its high sensitivity, the method can be used to calculate pharmacokinetic parameters of compounds I and II in man after a single oral dose of about 1 mg of I.