Preparation and evaluation of an immunoaffinity sorbent with Fab′ antibody fragments for the analysis of opioid peptides by on-line immunoaffinity solid-phase extraction capillary electrophoresis–mass spectrometry

An immunoaffinity (IA) sorbent with antibody fragments was prepared for the analysis of opioid peptides by on-line immunoaffinity solid-phase extraction capillary electrophoresis–mass spectrometry (IA-SPE-CE–MS). The antibody fragmentation was evaluated by MALDI-TOF-MS. Fab′ fragments obtained from a polyclonal IgG antibody against Endomorphins 1 and 2 (End1 and End2) were covalently attached to succinimidyl silica particles to prepare the IA sorbent. An IA-SPE-CE–MS methodology was established analyzing standard solutions of End1 and End2 and acceptable repeatability, linearity ranges and LODs (0.5 and 5 ng mL⁻¹, respectively) were obtained. The LOD of End1 was slightly better than that previously obtained using an IA sorbent with intact antibodies (1 ng mL⁻¹). In human plasma samples, End1 and End2 could be detected at 1 and 50 ng mL⁻¹, respectively, which meant an improvement of 100 and 2-fold with regard to the LODs using an IA sorbent with intact antibodies (100 ng mL⁻¹).     

High-performance liquid chromatography with fluorescence detection and ultra-performance liquid chromatography with electrospray tandem mass spectrometry method for the determination of indoleamine neurotransmitters and their metabolites in sea lamprey plasma

We present a comparison of two sensitive methods, HPLC with fluorescence detector (HPLC/FLD) and UPLC with electrospray tandem mass spectrometry (UPLC/MS/MS), for the determination of indoleamine neurotransmitters (NTs) and their metabolites in sea lamprey plasma samples. Liquid–liquid extraction (LLE) and solid-phase extraction (SPE) were also tested for recovery and matrix effect. The recoveries of SPE determined by HPLC/FLD and UPLC/MS/MS ranged from 75 to 123% and 78 to 105%, respectively, while the recoveries of LLE ranged from 45 to 73% and 48 to 75%, respectively. SPE combined with HPLC/FLD and UPLC/MS/MS to determine the target analytes in plasma samples were validated of the sensitivity, reproducibility, accuracy and precision. Both methods exhibited excellent linearity in the range of 0.2–50 ng mL⁻¹ for all analytes. The limits of detection (LOD) varied from 0.04 ng mL⁻¹ to 0.13 ng mL⁻¹ for HPLC/FLD method and 0.003 ng mL⁻¹ to 0.02 ng mL⁻¹ for UPLC/MS/MS method. The inter-day accuracy ranged from 82.5 to 127.0% for HPLC/FLD and 93.0 to 113.0% for UPLC/MS/MS. The inter-day precision ranged from 9.9 to 32.3% for HPLC/FLD and 5.4 to 13.2% for UPLC/MS/MS. These results demonstrated that the values obtained by both methods were within the satisfactory range and the UPLC/MS/MS method provided more accurate and precise measurements than HPLC/FLD method. The comparison is of great importance to determine the available detectors, considering the complexity and expensiveness versus quality parameters. These two methods were applied to the analysis of four important indoleamine neurotransmitter analytes (5-hydroxytryptamine, 5-hydroxyindole-3-acetic acid, tryptamine and melatonin) in sea lamprey plasma samples.     

The investigation of electrospun polymer nanofibers as a solid-phase extraction sorbent for the determination of trazodone in human plasma

A novel micro-extraction procedure was developed through the use of an electrospun polymer nanofiber as a solid-phase extraction (SPE) sorbent to directly extract trazodone from human plasma. The target compound was then monitored by a high performance liquid chromatography with ultraviolet detector (HPLC-UV) system. Parameters of influencing the extraction efficiency, such as fiber diameter, fiber packing amount, eluted solvent, pH and ionic strength were investigated. Under the optimized conditions, a linear response for trazodone over the range of 20-2000 ng mL⁻¹ was achieved with a γ² value of 0.9996. The precision of the method was examined with relative standard deviations of 5.7, 2.7, 2.2% corresponding to 50, 200, and 500 ng mL⁻¹, respectively, of trazodone spiked into 0.1 mL of plasma samples. The extraction recoveries of 58.3-75.2% and the relative recoveries of 94.6-105.5% were obtained. The limit of detection (LOD) was determined to be 8 ng mL⁻¹. A 15 min of HPLC gradient was successfully applied to determine trazodone from human plasma. Due to its simplicity, selectivity and sensitivity, the method may be applied to pharmacokinetic and pharmacodynamic studies of drugs.     

Ultrasound-assisted hydrolysis and chemical derivatization combined to lab-on-valve solid-phase extraction for the determination of sialic acids in human biofluids by μ-liquid chromatography-laser induced fluorescence

The determination of sialic acids (SIAs) has recently gained interest because of their potential role as markers of inflammatory disorders or chronic diseases. Hydrolysis of conjugated derivatives, solid-phase extraction (SPE) and derivatization steps constitute sample preparation prior to insertion of the analytical sample into a μ-liquid chromatograph-laser induced fluorescence (μ-LC-LIF) detector in the present method for the determination of two representative SIAs of human metabolism. Ultrasound-accelerated hydrolysis released free SIAs, which were efficiently concentrated in a dynamic manner using a lab-on-valve (LOV) module that allows automation of SPE for preconcentration and cleanup. This step was on-line connected with DMB-labeling of SIAs (derivatization), which was shortened from 180 min required with the conventional heating method to 20 min with ultrasound assistance. Individual separation of the target analytes was achieved within 20 min by μ-LC, while LIF detection endowed the overall method with high sensitivity. The LODs and LOQs provided by the method ranged 0.1–0.8 ng mL⁻¹ and 0.4–1.0 ng mL⁻¹ (between 0.1–0.8 pg and 0.4–1.0 pg expressed as on-column amount), respectively. High efficiency for interferents removal by SPE enabled the application of the method to four different biofluids—serum, urine, saliva and breast milk—for the determination of the target metabolites.     

An interior needle electropolymerized pyrrole-based coating for headspace solid-phase dynamic extraction

A headspace solid-phase dynamic extraction (HS-SPDE) technique was developed by the use of polypyrrole (PPy) sorbent, electropolymerized inside the surface of a needle, as a possible alternative to solid-phase microextraction (SPME). Thermal desorption was subsequently, employed to transfer the extracted analytes into the injection port of a gas chromatography-mass spectrometry (GC-MS). The PPy sorbent including polypyrrole-dodecyl sulfate (PPy-DS) was deposited on the interior surface of a stainless steel needle from the corresponding aqueous electrolyte by applying a constant deposition potential. The homogeneity and the porous surface structure of the coating were examined using the scanning electron microscopy (SEM).The developed method was applied to the trace level extraction of some polycyclic aromatic hydrocarbons (PAHs) from aqueous sample. In order to enhance the extraction efficiency and increase the partition coefficient of analytes, the stainless steel needle was cooled at 5 °C, while the sample solution was kept at 80 °C. Optimization of influential experimental conditions including the voltage of power supply, the time of PPy electrodeposition, the extraction temperature, the ionic strength and the extraction time were also investigated. The detection limits of the method under optimized conditions were in the range of 0.002-0.01 ng mL⁻¹. The relative standard deviations (R.S.D.) at a concentration level of 0.1 ng mL⁻¹ were obtained between 7.54 and 11.4% (n = 6). The calibration curves of PAHs showed linearity in the range of 0.01-10 ng mL⁻¹. The proposed method was successfully applied to the extraction of some selected PAHs from real-life water samples and the relative recoveries were higher than 90% for all the analytes.     

At-line microextraction by packed sorbent-gas chromatography–mass spectrometry for the determination of UV filter and polycyclic musk compounds in water samples

An at-line analysis protocol is presented that allows the determination of four UV filters, two polycyclic musk compounds and caffeine in water at concentration level of ng L⁻¹. The fully automated method includes analytes enrichment by Microextraction by packed sorbent (MEPS) coupled directly to large volume injection-gas chromatography–mass spectrometry. Two common SPE phases, C8 and C18, were examined for their suitability to extract the target substances by MEPS. The analytes were extracted from small sample volumes of 800 μL with recoveries ranging from 46 to 114% for the C8-sorbent and 65–109% for the C18-sorbent. Limits of detection between 34 and 96 ng L⁻¹ enable the determination of the analytes at common environmental concentration levels. Both sorbents showed linear calibration curves for most of the analytes up to a concentration level of 20 ng mL⁻¹. Carryover was minimized by washing the sorbents 10 times with 100 μL methanol. After this thorough cleaning, the MEPS are re-used and up to 70 analyses can be performed with the same sorbent. The fully automated microextraction GC–MS protocol was evaluated for the influence of matrix substances typical for wastewater. Dilution of samples prior to MEPS is recommended when the polar caffeine is present at high concentration. Real water samples were analyzed by the MEPS-GC–MS method and compared to standard SPE.     

Sensitive determination of four camptothecins by solid-phase microextraction-HPLC based on a boronic acid contained polymer monolithic layer

Camptothecin (CPT) and its derivative have been revealed to possess special anti-cancer activity, extraction methods are necessary for trace determination of CPTs in complex samples. In this work, we prepared a high efficient boronic acid-based polymer monolithic layer for microextraction of CPTs. A disposable membrane filter-based extraction device was developed, and boronic acid groups were co-polymerized into a polyporous polymer skeleton and served as the monolithic sorbent. The prepared poly(4-VB-MA-TRIM) showed good stability and great extraction efficiency toward four CPTs. After optimization of extraction conditions, poly(4-VB-MA-TRIM)-based solid-phase microextraction was coupled HPLC for determination of CPTs in biological samples. The method exhibited low limits of detection of 0.05–0.2 ng mL⁻¹, which is significantly more sensitive than reported HPLC methods. The method also showed wide linear range (0.1–100 and 0.5–200 ng mL⁻¹), good linearity (R² ≥ 0.9981) and good reproducibility (RSD ≤3.76%). The method has been applied in plasma samples, with good selectivity and good recoveries ranging from 85.1 to 104.7%.     

Monolithic silica spin column extraction and simultaneous derivatization of amphetamines and 3,4-methylenedioxyamphetamines in human urine for gas chromatographic-mass spectrometric detection

A simple, sensitive, and specific method with gas chromatography-mass spectrometry was developed for simultaneous extraction and derivatization of amphetamines (APs) and 3,4-methylenedioxyamphetamines (MDAs) in human urine by using a monolithic silica spin column. All the procedures, such as sample loading, washing, and elution were performed by centrifugation. APs and MDAs in urine were adsorbed on the monolithic silica and derivatized with propyl chloroformate in the column. Methamphetamine-d₅ was used as an internal standard. The linear ranges were 0.01-5.0 μg mL⁻¹ for methamphetamine (MA) and 3,4-methylenedioxymethamphetamine (MDMA) and 0.02-5.0 μg mL⁻¹ for amphetamine (AP) and 3,4-methylenedioxyamphetamine (MDA) (coefficient of correlation ≧0.995). The recovery of APs and MDAs in urine was 84-94%, and the relative standard deviation of the intra- and interday reproducibility for urine samples containing 0.1, 1.0, and 4.0 μg mL⁻¹ of APs and MDAs ranged from 1.4% to 13.6%. The lowest detection limit (signal-to-noise ratio ≧ 3) in urine was 5 ng mL⁻¹ for MA and MDMA and 10 ng mL⁻¹ for AP and MDA. The proposed method can be used to perform simultaneous extraction and derivatization on spin columns that have been loaded with a small quantity of solvent by using centrifugation.     

Development and validation of a high-throughput double solid phase extraction-liquid chromatography-tandem mass spectrometry method for the determination of tetrodotoxin in human urine and plasma

A sensitive analytical method for the determination of tetrodotoxin (TTX) in urine and plasma matrices was developed using double solid phase extraction (C18 and hydrophilic interaction liquid chromatography) and subsequent analysis by HPLC coupled with tandem mass spectrometry. The double SPE sample cleanup efficiently reduced matrix and ion suppression effects. Together with the use of ion pair reagent in the mobile phase, isocratic elution became possible which enabled a shorter analysis time of 5.5 min per sample. The assay results were linear up to 500 ng mL⁻¹ for urine and 20 ng mL⁻¹ for plasma. The limit of detection and limit of quantification were 0.13 ng mL⁻¹ and 2.5 ng mL⁻¹, respectively, for both biological matrices. Recoveries were in the range of 75-81%. To eliminate the effect of dehydration and variations in urinary output, urinary creatinine-adjustment was made. TTX was quantified in eight urine samples and seven plasma samples from eight patients suspected of having TTX poisoning. TTX was detected in all urine samples, with concentrations ranging from 17.6 to 460.5 ng mL⁻¹, but was not detected in any of the plasma samples. The creatinine-adjusted TTX concentration in urine (ranging from 7.4 to 41.1 ng μmol⁻¹ creatinine) correlated well with the degree of poisoning as observed from clinical symptoms.     

Simultaneous determination of ramipril, ramiprilat and telmisartan in human plasma using liquid chromatography tandem mass spectrometry

A rapid and sensitive liquid chromatography tandem mass spectrometry method has been developed and validated for the simultaneous determination of ramipril, ramiprilat and telmisartan in human plasma. The solid-phase extraction technique was used for the extraction of ramipril, ramiprilat and telmisartan from human plasma. Trandolaprilat and hydrochlorothiazide were used as the internal standards (ISs). Chromatography was performed on a Hypurity C18, 5 μm, 50 mm × 4.6 mm column, with the mobile phase consisting of ammonium acetate and acetonitrile (in a 20:80 ratio), followed by detection using mass spectrometry. The method involves a simple reversed isocratic chromatography condition and mass spectrometry detection, which enables detection at sub-nanogram levels. The method was validated and the lower limit of quantification for ramipril, ramiprilat and telmisartan was found to be 0.1 ng mL⁻¹, 0.1 ng mL⁻¹ and 2 ng mL⁻¹, respectively. The mean recovery for ramipril, ramiprilat and telmisartan ranged from 90.1 to 104.1%. This method increased the sensitivity and selectivity; resulting in high-throughput analysis of ramipril, ramiprilat and telmisartan using two different ISs in a single experiment for bioequivalence studies, with a chromatographic run time of 1.5 min only.     

Measurement of trace levels of antibiotics in river water using on-line enrichment and triple-quadrupole LC-MS/MS

This study presents the development of an automated on-line solid phase extraction (SPE)-liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of 23 antibiotics in environmental water samples. After optimisation of LC-MS/MS conditions, SPE parameters such as sorbent type, sample pH or sample volume were optimised. Antibiotic recoveries ranged from 64% to 98% and compared favourably with those achieved using off-line SPE. Limits of detection were in the range 0.5-13.7 ng L⁻¹.This on-line SPE-LC-MS/MS procedure was applied to the analysis of water samples taken in three rivers within the Seine River basin, near Paris (France). The obtained results revealed the occurrence of 12 antibiotics, including tylosin, erythromycin, tetracycline, amoxicillin, trimethoprim, sulfamethoxazole, oxolinic acid, flumequine, norfloxacin, ciprofloxacin, ofloxacin, and vancomycin (2-1435 ng L⁻¹).     

Simultaneous determination of multibenzodiazepines by HPLC/UV: Investigation of liquid-liquid and solid-phase extractions in human plasma

A method for simultaneous determination of seven benzodiazepines (BZPs) (flunitrazepam, clonazepam, oxazepam, lorazepam, chlordiazepoxide, nordiazepam and diazepam using N-desalkylflurazepam as internal standard) in human plasma using liquid-liquid and solid-phase extractions followed by high-performance liquid chromatography (HPLC) is described. The analytes were separated employing a LC-18 DB column (250 mm × 4.6 mm, 5 μm) at 35 °C under isocratic conditions using 5 mM KH₂PO₄ buffer solution pH 6.0:methanol:diethyl ether (55:40:5, v/v/v) as mobile phase at a flow rate of 0.8 mL min⁻¹. UV detection was carried out at 245 nm. Employing LLE, the best conditions were achieved with double extraction of 0.5 mL plasma using ethyl acetate and Na₂HPO₄ pH 9.5 for pH adjusting. Employing SPE, the best conditions were achieved with 0.5 mL plasma plus 3 mL 0.1 M borate buffer pH 9.5, which were then passed through a C18 cartridge previously conditioned, washed for 3 times with these solvents: 3 mL 0.1 M borate buffer pH 9.5, 4 mL Milli-Q water and 1 mL acetonitrile 5%, finally the BZPs elution was carried with diethyl ether:n-hexane:methanol (50:30:20). In both methods the solvent was evaporated at 40 °C under nitrogen flow. The validation parameters obtained in LLE were linearity range of 50-1200 ng mL⁻¹ plasma (r ≥ 0.9927), limits of quantification of 50 ng mL⁻¹ plasma, within-day and between-day CV% and E% for precision and accuracy lower than 15%, and recovery above 65% for all BZPs. In SPE, the parameter obtained were linearity range of 30-1200 ng mL⁻¹ plasma (r ≥ 0.9900), limits of quantification of 30 ng mL⁻¹ plasma, within-day and between-day CV% and E% for precision and accuracy lower than 15% and recovery above 55% for all BZPs. These extracting procedures followed by HPLC analysis showed their suitable applicability in order to examine one or more BZPs in human plasma. Moreover, it could be suggested that these procedures might be employed in various analytical applications, in special for toxicological/forensic analysis.     

On-line coupling of automatic solid-phase extraction and HPLC for determination of carotenoids in serum

The automated method developed for the determination of carotenoids uses 200 μL of serum, which was mixed with 400 μL of tetrahydrofuran, vortexed for 1 min, settled for 10 min, centrifuged for 6 min and the supernatant injected into an automatic solid-phase extraction (SPE) system for cleanup-preconcentration. A 10% water-acetonitrile mobile phase at 1.5 mL min⁻¹ eluted the retained compounds and transferred them on-line to a reversed-phase analytical column for individual separation of the target analytes. Visible detection was performed at 450 and 460 nm. The detection limits for the target analytes were between 3 and 30 ng mL⁻¹; the precision (expressed as relative standard deviation) ranged between 2.83 and 5.06% for repeatability and between 3.80 and 7.40% for within laboratory reproducibility. The total analysis time was 18 min. The proposed method is reliable, robust, and has an excellent potential for high-throughput use in both clinical and research laboratories.     

Micro-solid phase extraction coupled with high-performance liquid chromatography-tandem mass spectrometry for the determination of stimulants, hallucinogens, ketamine and phencyclidine in oral fluids

A confirmatory method for the determination of illicit drugs based on micro-solid phase extraction with modified tips, made of a functionalized fiberglass with apolar chains of octadecylsilane into monolithic structure, has been developed in this study. Drugs belonging to different chemical classes, such as amphetamine, methamphetamine, methylenedioxyamphetamine, methylenedioxyethylamphetamine, methylenedioxymethylamphetamine, cocaine, benzoylecgonine, ketamine, mescaline, phencyclidine and psilocybine were analyzed. The quantitation was performed by liquid chromatography-tandem mass spectrometry and the analytes were detected in positive ionization by means of an electrospray source. The limits of quantification ranged between 0.3 ng mL⁻¹ for cocaine and 4.9 ng mL⁻¹ for psilocybine, with coefficients of determination (r²) >0.99 for all the analytes as recommended in the guidelines of Society of Forensic Toxicologists-American Association Forensic Sciences.     

Turbulent flow and ternary column-switching on-line clean-up system for high-throughput quantification of risperidone and its main metabolite in plasma by LC-MS/MS: Application to a bioequivalence study

A high-throughput LC-MS/MS method was developed for the simultaneous determination of Risperidone and 9-OH-risperidone in human plasma. A semi-automated sample preparation procedure was applied, including protein precipitation after addition of ACN, via a robotic system, and subsequent sub-zero temperature extraction of the latter. Injections of the ACN extractants were performed on a turbulent flow ternary column-switching system, consisted of dual extraction columns in parallel for on-line purification of samples and an analytical column. Toggling with the assistance of two valves provided a run cycle time of 3 min and the whole procedure minimized carry-over effect. On-line clean-up procedure along with sub-zero temperature extraction increased sample purification and extended column life. The analytical range of the method was 0.1-200 ng mL⁻¹ for both analytes with excellent linearity and very good accuracy and precision. The proposed method was employed in a bioequivalence study after per os administration of a 2 mg tablet of risperidone and allowed the completion of the study (>1400 samples) in only 4 days time.     

Polydimethylsiloxane/polypyrrole stir bar sorptive extraction and liquid chromatography (SBSE/LC-UV) analysis of antidepressants in plasma samples

A new polymeric coating consisting of a dual-phase, polydimethylsiloxane (PDMS) and polypyrrole (PPY) was developed for the stir bar sorptive extraction (SBSE) of antidepressants (mirtazapine, citalopram, paroxetine, duloxetine, fluoxetine and sertraline) from plasma samples, followed by liquid chromatography analysis (SBSE/LC-UV). The extractions were based on both adsorption (PPY) and sorption (PDMS) mechanisms. SBSE variables, such as extraction time, temperature, pH of the matrix, and desorption time were optimized, in order to achieve suitable analytical sensitivity in a short time period. The PDMS/PPY coated stir bar showed high extraction efficiency (sensitivity and selectivity) toward the target analytes. The quantification limits (LOQ) of the SBSE/LC-UV method ranged from 20 ng mL⁻¹ to 50 ng mL⁻¹, and the linear range was from LOQ to 500 ng mL⁻¹, with a determination coefficient higher than 0.99. The inter-day precision of the SBSE/LC-UV method presented a variation coefficient lower than 15%. The efficiency of the SBSE/LC-UV method was proved by analysis of plasma samples from elderly depressed patients.     

Multiresidue determination of pesticides from aquatic media using polyaniline nanowires network as highly efficient sorbent for microextraction in packed syringe

A simple, rapid and sensitive method based on microextraction in packed syringe (MEPS), in combination with gas chromatography–mass spectrometry (GC–MS) was developed. Polyaniline (PANI) nanowires network was synthesized and used as sorbent of MEPS for the multiresidue determination of selected analytes from triazine, organochlrorine and organophosphorous pesticides in aqueous samples. The PANI nanowires network was prepared using soft template technique and its characterization was studied by scanning electron microscopy (SEM). The presence of micelles in this methodology showed to be an important parameter in shaping the growing polymer. Hexadecyltrimethylammonium bromide (HTAB) was used as structure directing agent in PANI preparation procedure and this was led to the formation of nanowires with diameters ranging from 35 nm to 45 nm. The synthesized PANI nanowires network showed higher extraction capability in comparison with the bulk PANI. Important parameters influencing the extraction and desorption processes including desorption solvent, elution volume, draw–eject cycles of sample, draw–eject mode, pH effect and amount of sorbent were optimized. Limits of detection were in the range of 0.07–0.3 ng mL⁻¹ using time scheduled selected ion monitoring (SIM) mode. The linearity of method was in the range from 0.5–200 ng mL⁻¹ to 0.2–1000 ng mL⁻¹. The method precision (RSD %) with three replicates were in the range of 5.3–18.4% at the concentration level of 5 ng mL⁻¹. The developed method was successfully applied to the Zayandeh-rood river water samples and the matrix factor obtained for the spiked real water samples were in the range of 0.79–0.94.     

Mixed-mode solid-phase extraction followed by acetylation and gas chromatography mass spectrometry for the reliable determination of trans-resveratrol in wine samples

This work presents an advantageous analytical procedure for the accurate determination of free trans-resveratrol in red and white wines. The proposed method involves solid-phase extraction (SPE), acetylation of the analyte in aqueous media and further determination by gas chromatography (GC) with mass spectrometry detection (MS). The use of a mixed-mode SPE sorbent provides an improvement in the selectivity of the extraction step; moreover, the presence of several intense ions in the electron impact mass spectra of its acetyl derivative guarantees the unambiguous identification of trans-resveratrol. Considering a sample intake of 10 mL, the method provides a limit of quantification (LOQ) of 0.8 ng mL⁻¹ and linear responses for concentrations up to 2.5 μg mL⁻¹, referred to wine samples. The average recovery, estimated with samples fortified at different concentrations in the above range, was 99.6% and the inter-day precision stayed below 8%. Trans-resveratrol levels in the analyzed wines varied from 3.4 to 1810 ng mL⁻¹. Cis-resveratrol was also found in all samples. In most cases, equal or higher responses were measured for this latter form than for the trans-isomer. The reduced form of resveratrol, dihydro-resveratrol, was systematically identified in red wines.     

Chromatographic analysis of serotonin, 5-hydroxyindolacetic acid and homovanillic acid in dried blood spots and platelet poor and rich plasma samples

An isocratic high-performance liquid chromatographic method has been developed for the measurement of serotonin, 5-hydroxyindolacetic and homovanillic acids in dried blood spots and in platelet poor and rich plasma samples. Analyses were carried out on a C18 reversed-phase column using a mobile phase composed of 13% methanol and 87% aqueous citrate buffer, containing octanesulfonic and ethylendiaminotetracetic acids. Coulometric detection was used, setting the guard cell at +0.100 V, the first analytical cell at −0.200 V and the second analytical cell at +0.400 V. For the pre-treatment of biological samples a novel solid-phase extraction procedure, based on mixed-mode reversed-phase – strong anion exchange Oasis cartridges, was implemented. Extraction yields of the analytes from all these matrices were satisfactory, being always higher than 89.0%. The calibration curve was linear over the on-column concentration range of 0.1–22.5 ng mL⁻¹ for serotonin and 5-hydroxyindolacetic acid and of 0.25–22.5 ng mL⁻¹ for homovanillic acid. The sensitivity was good with a limit of detection of 0.05 ng mL⁻¹ for serotonin and 5-hydroxyindolacetic acid and 0.12 ng mL⁻¹ for homovanillic acid. Results were also satisfactory in terms of precision, selectivity and accuracy. The analytical method was successfully applied to human platelet poor and rich plasma samples and to dried blood spots from volunteers and psychiatric patients.     

Determination of the antidepressant mirtazapine and its two main metabolites in human plasma by liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method for the determination in human plasma of the recent noradrenergic and specific serotonergic antidepressant (NaSSA) mirtazapine and its two main metabolites, N-desmethylmirtazapine and 8-hydroxymirtazapine, has been developed. Fluorescence detection was used, exciting at λ = 290 nm and monitoring emission at λ = 370 nm. Separation was obtained by using a reversed-phase column (C8, 250 mm × 4.6 mm I.D., 5 μm) and a mobile phase composed of 75% aqueous phosphate buffer containing triethylamine at pH 3.0 and 25% acetonitrile. Melatonin was used as the internal standard. A careful pre-treatment of plasma samples was developed, using solid-phase extraction with phenyl cartridges (100 mg, 1 mL). The calibration curves were linear over a working range of 5-150 ng mL⁻¹ for mirtazapine and of 2.5-75.0 ng mL⁻¹ for N-desmethylmirtazapine and 8-hydroxymirtazapine. The limit of quantitation (LOQ) was 2.5 ng mL⁻¹ and the limit of detection (LOD) was 1.25 ng mL⁻¹ for all analytes. The method was applied with success to plasma samples from depressed patients undergoing treatment with mirtazapine. Precision data, as well as accuracy results, were satisfactory and no interference from other drugs was found. Hence the method is suitable for therapeutic drug monitoring of mirtazapine and its metabolites in depressed patients’ plasma.