LC-MS-sMRM method development and validation of different classes of pain panel drugs and analysis of clinical urine samples.

Urine drug testing (UDT) is an important analytical/bio-analytical technique that has inevitably become an integral and vital part of a testing programme for diagnostic purposes. This manuscript presents a tailor-made LC-MS/MS quantitative assay method development and validation for a custom group of 33 pain panel drugs and their metabolites belonging to different classes (opiates, opioids, benzodiazepines, illicit, amphetamines, etc.) that are prescribed in pain management and depressant therapies. The LC-MS/MS method incorporates two experiments to enhance the sensitivity of the assay and has a run time of about 7 minutes with no prior purification of the samples required and a flow rate of 0.7 mL/min. The method also includes the second-stage metabolites for some drugs that belong to different classes but have first-stage similar metabolic pathways that will enable to correctly identify the right drug or to flag the drug that might be due to specimen tampering. Some real case examples and difficulties in peak picking were provided with some of the analytes in subject samples. Finally, the method was deliberated with some randomly selected de-identified clinical subject samples, and the data evaluated from "direct dilute and shoot analysis" and after "glucuronide hydrolysis" were compared. This method is now used to run routinely more than 100 clinical subject samples on a daily basis.

A green and low-cost method employing switchable hydrophilicity solvent for the simultaneous determination of antidepressants in human urine by gas chromatography - mass spectrometry detection.

In this study, the use of switchable hydrophilicity solvent with a simple and low-cost lab-made device for the extraction procedure in homogeneous liquid-liquid microextraction is proposed for the first time in the determination of antidepressants in human urine. The antidepressants studied consisted of fluoxetine, amitriptyline, nortriptyline, imipramine, desipramine and sertraline. The optimization of the main parameters that can influence on the extraction efficiency was performed through multivariate approaches. The analytes were separated and identified by gas chromatography coupled to mass spectrometry (GC-MS). The optimal extraction conditions consisted of using N,N-dimethylcyclohexylamine (DMCHA) as the switchable hydrophilicity solvent (SHS), 500 µL of urine sample previously diluted with ultrapure water at 1:1 ratio (v/v), 200 µL of a mixture of SHS:HCl 6 mol L-1 (1:1 v/v), 600 µL of NaOH 10 mol L-1 and 3 min of extraction time. A volume of 40 µL of diphenylamine at concentration of 500 µg L-1 (20 ng) was used as internal standard. The method developed was in-house validated, providing coefficients of determination higher than 0.995 for all analytes, limits of detection (LOD) from 0.02 to 0.88 µg L-1, limits of quantification (LOQ) from 0.05 to 2.92 µg L-1, relative recoveries of 68 to 102%, intra-day precision from 0.5 to 15.9%, inter-day precision from 4.2 to 19.3%, selectivity and robustness. The method proposed was successfully applied in five human urine samples from a Toxicological Information Center located in Porto Alegre (Brazil). The results demonstrated that the µP-SHS-HLLME approach is highly cost-effective, rapid, simple and environmentally-friendly with satisfactory analytical performance.

Application of LC-ESI-MS/MS Method for Analysis of Escitalopram Oxalate in Human Urine and Pharmaceutical Dosage Forms.

An effective and sensitive liquid chromatographic-electrospray ionization tandem mass-spectrometric (LC-ESI-MS/MS) method was developed and validated for quantification of escitalopram oxalate (ESC-OX), antidepressant drug in spiked human urine and pharmaceutical formulations. In this work, simple liquid-liquid extraction was optimized and used for extraction of cited drug from urine samples. The chromatographic separation was attained within 6 min including re-equilibration time by using gradient elution with 0.1% formic acid in acetonitrile and 0.1% formic acid in water as mobile phase, Zorbax Eclipse RP C18 (50 × 2.1 mm) column was used with a particle size of 1.8 µm; the flow-rate was 0.35 mL min-1. Ion signal m/z 262.0 and 109.0 for ESC-OX product ions were monitored at positive ESI mode. Validation of the method was carried out according to the ICH Q2 (R1) guidelines and EMEA criteria. The method was linear over 79-196,450 pg mL-1 with a regression of 0.9999 and 0.9993 for both standard and urine samples. The LOD was 3.88 and 10.66 pg mL-1 for standard and urine samples, respectively, while lower limit of quantification was 79 pg mL-1.

A fast and simple approach for the quantification of 40 illicit drugs, medicines, and pesticides in blood and urine samples by UHPLC-MS/MS.

A fast and simple approach to overcome challenges in emergency toxicological analysis, using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) has been developed, for the detection of analytes in blood and urine samples from the following drug classes: analgesics, benzodiazepines, antidepressants, anticonvulsants, drugs of abuse, and pesticides. These substances are relevant in the context of emergency toxicology in Brazil. The sample preparation procedure was relatively easy and fast to perform. The method was fully validated giving limits of in the range of 0.5 and 20 ng mL-1 for blood and urine samples. The intraday and interday precision and accuracy were considered adequate for all analytes once the relative standard deviation (RSD) (%) was lower than 20% for quality control (QC) low and lower than 15% for CQ medium and high. The developed method was successfully applied to 320 real samples collected at the Poison Control Center of São Paulo, and 89.1% have shown to be positive for some of the analytes. This confirms its applicability and importance to emergency toxicological analysis, and it could be very useful in both fields of clinical and forensic toxicology.

Analysis of four antidepressants in plasma and urine by gas chromatography-mass spectrometry combined with sensitive and selective derivatization.

A sensitive and selective method was developed for simultaneous determination of four antidepressants (ATDs) in plasma and urine samples by gas chromatography- mass spectrometry (GC-MS) based on an N-nitrosation reaction. In this study, fluoxetine (Flu), nortriptyline (Nor), maprotiline (Map), and paroxetine (Paro) were first derivatized with sodium nitrite to appropriate N-nitrosamines under acidic condition, then the derivatives were easier to detect by GC-MS. The derivatization conditions including the amount of hydrochloric acid, the amount of sodium nitrite, reaction temperature, reaction time and the extraction reagents were optimized. Under the optimal conditions, the limit of detections (LODs) and limit of quantitations (LOQs) were in the range of 0.04-1.38 µg L-1 and 0.14-4.62 µg L-1, respectively. Low, medium, and high concentrations of antidepressants were added in plasma and urine samples, spiked recovery ranged from 85.88%-110.34% for plasma and 80.64%-113.07% for urine, respectively. The derivatization reaction was very quickly, only 5 min was needed for the reaction process, in addition, the proposed method exhibited superior sensitivity and selectivity, it showed sufficient advantages for determination of Flu, Nor, Map, and Paro in plasma and urine of patients.

Extraction of antidepressant drugs in biological samples using alkanol-based nano structured supramolecular solvent microextraction followed by gas chromatography with mass spectrometric analysis.

In the present study, a supramolecular solvent was formed from reverse micelle aggregates of octanol. The proposed supramolecular solvent was used for rapid extraction of some antidepressants drugs including amitriptyline, imipramine, desipramine, maprotiline, sertraline, and doxepin from biological samples. Alkanol-based supramolecular solvents have a unique array of physicochemical properties, making them a very attractive alternative to replace organic solvents in analytical extractions. The parameters affecting the extraction of target analytes (i.e., the volume of tetrahydrofuran and octanol as the major components comprising the supramolecular solvent, chain length of alkanols, sample solution pH, salt addition, and ultrasonic time) were investigated and optimized by factor by factor optimization method. Under the optimum conditions, preconcentration factors of 470, 490, 460, 385, 370, and 430 were obtained for amitriptyline, doxepin, imipramine, desipramine, maprotiline, and sertraline, respectively. The linear ranges and coefficients of determination (R2 ) were obtained in the range of 0.01-100 µg/L and 0.9974-0.9991, respectively. Also the limits of detection (S/N = 3) of 0.003-0.03 µg/L, and precisions (n = 5) of 4.9-8.9% were calculated. Finally, the method was successfully applied for the extraction of antidepressant drugs in biological samples, and relative recoveries in the range of 91-102% were obtained.

Carbon nanotube-modified monolithic polymethacrylate pipette tips for (micro)solid-phase extraction of antidepressants from urine samples.

This work evaluates the potential of methacrylate monoliths with multi-walled carbon nanotubes incorporated into the polymeric network for the extraction of antidepressants from human urine. The method is based on a micropipette solid-phase extraction tip containing a hybrid monolithic material covalently attached to the polypropylene housing. A polymer layer made from poly(ethylene dimethacrylate) was bound to the inner surface of a polypropylene tip via UV grafting. The preparation of the monolith and the microextraction steps were optimized in terms of adsorption capacity. Limits of detection ranged from 9 to 15 µg·L-1. The average precision of the method varied between 3 and 5% (intra-tips), and from 4 to 14% (inter-tips). The accuracy of the method was evaluated through a recovery study by using spiked samples. Graphical abstract Hybrid polymer monoliths containing multi-walled carbon nanotubes (MWCNTs) were prepared in pipette tips by photo-polymerization approach. The extraction devices were used for the extraction of antidepressants in urine samples.

Magnetic micro-solid-phase extraction based on magnetite-MCM-41 with gas chromatography-mass spectrometry for the determination of antidepressant drugs in biological fluids.

A new facile magnetic micro-solid-phase extraction coupled to gas chromatography and mass spectrometry detection was developed for the extraction and determination of selected antidepressant drugs in biological fluids using magnetite-MCM-41 as adsorbent. The synthesized sorbent was characterized by several spectroscopic techniques. The maximum extraction efficiency for extraction of 500 µg/L antidepressant drugs from aqueous solution was obtained with 15 mg of magnetite-MCM-41 at pH 12. The analyte was desorbed using 100 µL of acetonitrile prior to gas chromatography determination. This method was rapid in which the adsorption procedure was completed in 60 s. Under the optimized conditions using 15 mL of antidepressant drugs sample, the calibration curve showed good linearity in the range of 0.05-500 µg/L (r2  = 0.996-0.999). Good limits of detection (0.008-0.010 µg/L) were obtained for the analytes with good relative standard deviations of <8.0% (n = 5) for the determination of 0.1, 5.0, and 500.0 µg/L of antidepressant drugs. This method was successfully applied to the determination of amitriptyline and chlorpromazine in plasma and urine samples. The recoveries of spiked plasma and urine samples were in the range of 86.1-115.4%. Results indicate that magnetite micro-solid-phase extraction with gas chromatography and mass spectrometry is a convenient, fast, and economical method for the extraction and determination of amitriptyline and chlorpromazine in biological samples.

Determination of three antidepressants in urine using simultaneous derivatization and temperature-assisted dispersive liquid-liquid microextraction followed by gas chromatography-flame ionization detection.

This paper presents a fast and simple method for the extraction, preconcentration and determination of fluvoxamine, nortriptyline and maprotiline in urine using simultaneous derivatization and temperature-assisted dispersive liquid-liquid microextraction (TA-DLLME) followed by gas chromatography-flame ionization detection (GC-FID). An appropriate mixture of dimethylformamide (disperser solvent), 1,1,2,2-tetrachloroethane (extraction solvent) and acetic anhydride (derivatization agent) was rapidly injected into the heated sample. Then the solution was cooled to room temperature and cloudy solution formed was centrifuged. Finally a portion of the sedimented phase was injected into the GC-FID. The effect of several factors affecting the performance of the method, including the selection of suitable extraction and disperser solvents and their volumes, volume of derivatization agent, temperature, salt addition, pH and centrifugation time and speed were investigated and optimized. Figures of merit of the proposed method, such as linearity (r(2) > 0.993), enrichment factors (820-1070), limits of detection (2-4 ng mL(-1)) and quantification (8-12 ng mL(-1)), and relative standard deviations (3-6%) for both intraday and interday precisions (concentration = 50 ng mL(-1)) were satisfactory for determination of the selected antidepressants. Finally the method was successfully applied to determine the target pharmaceuticals in urine.

Combining poly (methacrylic acid-co-ethylene glycol dimethacrylate) monolith microextraction and octadecyl phosphonic acid-modified zirconia-coated CEC with field-enhanced sample injection for analysis of antidepressants in human plasma and urine.

A method based on poly (methacrylic acid-co-ethylene glycol dimethacrylate) monolith microextraction and octadecylphosphonic acid-modified zirconia-coated CEC followed by field-enhanced sample injection preconcentration technique was proposed for sensitive CE-UV analysis of six antidepressants (doxepin, clozapine, imipramine, paroxetine, fluoxetine and chlorimipramine) in human plasma and urine. A poly(methacrylic acid-co-ethylene glycol dimethacrylate) monolithic capillary column was introduced for the extraction of antidepressants from urine and plasma samples. The hydrophobic main chains and acidic pendant groups of the monolithic column make it a superior material for extraction of basic analytes from aqueous matrix. After extraction, the desorption solvent, which normally provided an excellent medium to ensure direct compatibility for field-enhanced sample injection in CE, was analyzed by CE directly. By the use of alkylphosphonate-modified zirconia-coated CEC for separation of the basic compounds of antidepressants, high separation efficiency and resolution were achieved because that both hydrophobic interaction between analytes and alkylphosphonate-modified zirconia coat and electrophoretic effect work on the separation of antidepressants. The best separation was achieved using a buffer composed of 0.3 M ammonium acetate (adjusted to pH 4.5 with 1 M acetic acid) and 35% ACN v/v, with a temperature and voltage of 20 degrees C and 20 kV, respectively. By applying both preconcentration procedures, LODs of 11.4-51.5 and 3.7-17.0 microg/L were achieved for the six antidepressants in human plasma and urine, respectively. Excellent method of reproducibility was found over a linear range of 50-5000 microg/L in plasma and urine sample.

Quantification of antidepressants using gas chromatography-mass spectrometry.

Antidepressants are of great interest to clinical and forensic toxicologists as they are frequently used in suicidal gestures; they can be the source of drug interactions and some have narrow therapeutic indices making the potential for toxicity more likely. There are five categories of antidepressants based on function and/or structure. These are monoamine oxidase inhibitors (MAOI), cyclic antidepressants including tricyclic and tetracyclic compounds (TCA), selective serotonin reuptake inhibitors (SSRI), serotonin-norepinephrine reuptake inhibitors (SNRI), and atypical compounds. This method is designed to detect the presence of antidepressant drugs in blood/serum, urine, and tissue specimens using gas chromatography/mass spectrometry (GC/MS) following liquid-liquid extraction (LLE) and identified by relative retention times and mass spectra.

Determination of the antidepressant levoprotiline and its N-desmethyl metabolite in biological fluids by gas chromatography/mass spectrometry.

A specific and sensitive gas chromatographic/mass spectrometric method was developed and validated for the determination of the antidepressant levoprotiline in blood, plasma and urine and the simultaneous determination of levoprotiline and its desmethyl metabolite in urine. Deuterium-labelled analogues were used as internal standards. The compounds were isolated from the biological fluids by liquid-liquid extraction under basic conditions. Following derivatization with perfluoropropionic anhydride, the samples were analysed by capillary column gas chromatography/electron impact mass spectrometry with selected ion monitoring. The analysis of spiked samples demonstrated the high accuracy and precision of the method. Blood concentrations of levoprotiline down to 0.7 nmol l-1 (1 ml used for analysis) could be quantified with a coefficient of variation of 10% or less. The method is suitable for use in pharmacokinetic and bioavailability studies of levoprotiline in humans.

Light-induced racemization: artifacts in the analysis of the diastereoisomeric pairs of thioridazine 5-sulfoxide in the plasma and urine of patients treated with thioridazine.

The ring sulfoxidation of thioridazine (THD), a widely used neuroleptic agent, yields two diastereoisomeric pairs, fast- and slow-eluting (FE and SE) thioridazine 5-sulfoxide (THD 5-SO). Until now, studies in which concentrations of these metabolites were measured in THD-treated patients have revealed no significant differences in their concentrations. Preliminary experiments in our laboratory had shown that sunlight and, to a lesser extent, dim daylight led to racemization and probably also to photolysis of the diastereoisomeric pairs as measured by high-performance liquid chromatography. Similar results were also obtained with direct UV light (UV lamp). In appropriate light-protected conditions, THD, northioridazine, mesoridazine, sulforidazine, and FE and SE THD 5-SO were measured in 11 patients treated with various doses of THD for at least 1 week. Significantly higher concentrations of the FE stereoisomeric pair were found. The concentration ratios THD 5-SO (FE)/THD 5-SO (SE) ranged from 0.89 to 1.75 in plasma and from 1.15 to 2.05 in urine. Because it is known that the ring sulfoxide contributes to the cardiotoxicity of the drug even more potently than the parent compound does, these results justify further studies to determine whether there is stereoselectivity in the cardiotoxicity of THD 5-SO.

Drug screening with EMIT reagents: a quantitative approach to quality control.

We investigated the precision, linearity, accuracy, and stability of quantitative results for five drugs of abuse [amphetamines, benzoylecgonine, opiates, phencyclidine, and the cannabinoid-tetrahydrocannabinol (THC)-9-acid metabolite], analyzed in control specimens by using EMIT d.a.u. reagents (Syva Co.) with a Monarch 2000 analyzer with a nonlinear interpolation curve-fitting algorithm. The within-day and between-days coefficients of variation (CVs) were less than 5% for all drugs except THC-9-acid, which had a CV between 10% and 20%. The drift of control values during a 30-day stability study was less than 10% from target values for three weeks after a single calibration, except for THC-9-acid control values, which were stable for only two to three days. Daily calibration reduced the drift away from target values during the 30-day stability study and produced optimum precision of all drug assays. Mean control values near the National Institute on Drug Abuse cutoff limits were within 10% of their target values.

Gas chromatographic method for determination of a piperazine derivative (Trelibet) and its metabolites in human plasma and urine.

A sensitive gas chromatographic method was developed for the determination of Trelibet, 1-benzyl-4-(2'-pyridinecarbonyl)piperazine, and of its major metabolites in biological fluids. The compounds were extracted as bases into dichloromethane, and the extracts were analysed by a dimethylsilicone capillary column with a nitrogen-phosphorus flame-ionization detector. The lower limit of detection was 1 ng/ml for Trelibet and 5 ng/ml for the metabolites. Peak-area ratios of the compounds and internal standard were linearly correlated to their plasma concentrations between 1 and 1000 ng/ml. The method was used for quantification of Trelibet and two of its metabolites in depressed patients after oral administration of a single dose of 200 mg of Trelibet. Concentration data measured in plasma and urine showed that the method is sensitive enough to monitor concentrations both for pharmacokinetic studies and for plasma steady-state levels daily.

Screening procedure for detection of antidepressants and their metabolites in urine using a computerized gas chromatographic--mass spectrometric technique.

A method for the identification of antidepressants and their metabolites in urine after acid hydrolysis is described. The acetylated extract is analysed by computerized gas chromatography--mass spectrometry. An on-line computer allows rapid detection using mass fragmentography with the masses 58, 84, 86, 100, 191, 193, 194, 205. The identity of positive signals in the reconstructed mass fragmentogram is established by a comparison of the entire mass spectra with those of standards. The mass fragmentogram, the underlying mass spectra and the gas chromatographic retention indices (OV-101) are documented.