Determination of trace levels of benzodiazepine in urine using capillary electrochromatography-time of flight mass spectrometry

This paper details a method for the separation and determination of ten benzodiazepines in urine using CEC-MS(TOF) and an hexyl acrylate-based porous monolith. The TOF mass spectrometer provides an exact mass of protonated benzodiazepines to three decimal places (1-6 ppm). This high selectivity along with the CEC separation, provides an effective method for the identification of benzodiazepines. Linearity is satisfactory for all compounds in the concentration range of 25-500 ng/mL for lorazepam and 12.5-500 ng/mL for the others. The RSDs are between 1.4-2.3% for retention times and 1.1-9.2% for relative areas. Using the monolithic stationary phase, a preconcentration step is achievable and permits an 75-140-fold improvement in sensitivity. This strategy permits the quantification of these drugs down to 1 ng/mL in urine. This method was used for the analysis of benzodiazepines in spiked urine samples.     

On-line derivatization gas chromatography with furan chemical ionization tandem mass spectrometry for screening of amphetamines in urine

A simple alternative method with minimal sample pretreatment is investigated for screening of amphetamines in small volume (using only 20 microL) of urine sample. The method is sensitive and selective. The method uses gas chromatography (GC) direct sample introduction (DSI) for on-line derivatization (acylation) of amphetamines to improve sensitivity. Furan as chemical ionization (CI) reagent in conjunction with tandem mass spectrometry (MS/MS) is used to improve selectivity. Low background with sharp protonated molecular ion peaks of analytes is the evidence of improvement in sensitivity and selectivity. Blank urine samples spiked with known amounts of amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine and 3,4-methylenedioxyethylamphetamine is analyzed. Selected ion monitoring of the characteristic product ions (m/z 119+136+150+163) using furan CI-MS/MS in positive ion mode is used for quantification. Limits of detection (LOD) between 0.4 and 1.0 ng mL(-1) and limits of quantitation (LOQ) between 1.0 and 2.0 ng mL(-1) are established. Linear response over the range of 1-1000 ng mL(-1) (r(2)>0.997) is observed for all analytes, except for methamphetamine (2.0-1000 ng mL(-1)). Good accuracy between 86 and 113% and precision ranging from 4 to 18% is obtained. The method is also tested on real samples of urine from suspected drug abusers. This method could be used for screening and determination of amphetamines in urine samples, however needs additional work for full validation.     

Ionic liquid sensitized fluorescence determination of four isoquinoline alkaloids

The fluorescence spectra of berberine, palmatine, jatrorrhizine, and coptisine in ionic liquids were studied and found to increase significantly in ionic liquids, with [C(8)MIM][PF(6)] having the greatest increase. Further studies showed that these drugs could be extracted from an aqueous solution by [C(8)MIM][PF(6)] using the temperature-assisted ionic liquid dispersive liquid phase microextraction method. The enrichment factors were 81.8-82.3, and the extraction recovery was 98.5%, 98.1%, 98.3%, and 98.8% for berberine, palmatine, jatrorrhizine, and coptisine, respectively. Based on the [C(8)MIM][PF(6)] preconcentration, separation, and sensitized fluorescence for these drugs, a new selective and sensitive method for the determination of concentration of these four drugs in aqueous samples was presented. At optimum conditions, the linear relationship was obtained in the ranges of 0.8-130 ng mL(-1), 0.9-160 ng mL(-1), 0.7-140 ng mL(-1), and 0.6-110 ng mL(-1), respectively. The proposed method was successfully applied for the determination of the drugs in pharmaceutical preparations, urine, and plasma samples.     

Monitoring of 6-chloronicotinic acid in human urine by gas chromatography-tandem mass spectrometry as indicator of exposure to the pesticide imidacloprid

A new analytical method for determining 6-chloronicotinic acid (6-ClNA) in human urine is proposed. 6-ClNA is the main metabolite in warm-blooded animals after exposure to the insecticide imidachloprid. 6-ClNA was extracted from human urine using solid phase extraction (SPE) with laboratory-made cartridges of Amberlite XAD-4. A clean-up step and a derivatization process were carried out prior to gas chromatography-tandem mass spectrometric (GC-MS-MS) determination. A study on the influence of pH in the extraction process revealed that it affects the analyte extraction efficiency. A working pH zone was defined between 0.8 and 2.8. Calibration curves were studied in the concentration range of 0.5-100 ng mL(-1) and showed good linearity. Limits of detection and determination of the method were 16 and 56 pg mL(-1) respectively. The mean recovery at 10 and 100 ng mL(-1) was between 97.2 and 102.1% and the repeatability was lower than 5.4% in all cases. The analysis of urine samples of five agricultural workers from Almería (Spain) did not detect the metabolite.     

Non-protected fluid room-temperature phosphorimetric procedure for the direct determination of naftopidil in biological fluids

Non-protected fluid room temperature phosphorescence, NPRTP, has been applied to the determination of naftopidil in biological fluids. The proposed method is based on obtaining a phosphorescence signal from naftopidil using potassium iodide as heavy atom perturber and sodium sulfite as a deoxygenating reagent without a protected medium. Optimized conditions for the determination were 1.4 mol L= KI, 5.0 x l0(-3) mol L(-1) sodium sulfite, pH 6.5 (adjusted with sodium hydrogen phosphate-dihydrogen phosphate buffer solution, 5.0 x 10(-2) mol L(-1). The delay time, gate time, and time between flashes were 70 micros, 400 micros, and 5 ms, respectively. The maximum phosphorescence signal appeared instantly and the intensity was measured at lambda(ex)=287 nm and lambda(em)=525 nm. The response obtained was linearly dependent on concentration in the range 50 to 600 ng mL(-1). The detection limit, according to error-propagation theory, was 7.93 ng mL(-1) and the detection limit as proposed by Clayton was 11.12 ng mL(-1). The repeatability was studied by using ten solutions of 400 ng mL(-1) naftopidil; if the theory of error propagation is assumed the relative error is 0.88%. The standard deviation of replicates was found to be 3.5 ng mL(-1). This method was successfully applied to the analysis of naftopidil in human serum and urine with recoveries of 104.0 +/- 0.6% for serum and 106.0 +/- 1.0% for urine.     

Quantitative determination of nicotinic acid in micro liter volume of urine sample by drop-to-drop solvent microextraction coupled to matrix assisted laser desorption/ionization mass spectrometry

Drop-to-drop solvent microextraction (DDSME) coupled with matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) for quantitative determination of nicotinic acid in one drop of urine sample has been proposed. All parameters, such as type of organic solvent, extraction time, exposure volume solvent, pH of the sample solution that affecting the separation and preconcentration of nicotinic acid were investigated. Under the optimal conditions, the detection limit of the method was 20 ng mL(-1) and the relative standard deviations (RSD) for determination of the nicotinic acid were in the range of 8.0-12.5%. The calculated calibration curves gave linearity in the range of 80-1000 ng mL(-1). The main advantages of the proposed method are simple, fast, and small amount of sample solution is used for separation and preconcentration of nicotinic acid. This method could be also useful for the analysis of other interested analytes in small volume of biological samples, like plasma, saliva and urine, where the availability of samples are limited.     

A solid phase extraction method for the screening and determination of pyrethroid metabolites and organochlorine pesticides in human urine.

A screening method has been developed for the determination of 23 organochlorine pesticides (OCPs) and 3-pyrethroid metabolities [cis- and trans-3-(2,2-dichlorovinyl)-2,2-dimethyl-(1-cyclopropane) carboxylic acid, cis-3-(2,2-dibromovinyl)-2,2-dimethyl-(1-cyclopropane) carboxylic acid and 3-phenoxybenzoic acid] from human urine. OCPs were directly detected in urine samples while pyrethroid metabolites required acid-induced hydrolysis to convert their conjugates into free acids; all compounds were then cleaned-up/preconcentrated using solid phase extraction. Determination and quantitation was achieved by gas chromatography with a mass spectrometer detector operating in selected ion monitoring mode. Limits of detection varied between 0.1 and 0.3 ng/mL with linear ranges from 0.3 to 700 ng/mL; the precision of the method was high (4.3-7.2%). Recoveries of all analytes from urine samples fortified at levels of 30 ng/mL for each OCP and 15 ng/mL for each pyrethroid metabolite ranged from 88 to 101% (captan gave the lowest recovery). The results obtained from the analysis of real urine samples show the suitability of the proposed method for monitoring people exposed to organochlorine and pyrethroid pesticides.     

Identification of fentanyl, alfentanil, sufentanil, remifentanil and their major metabolites in human urine by liquid chromatography/tandem mass spectrometry for doping control purposes

Since January 2005, the list of prohibited substances established by the World Anti-Doping Agency prohibits the opioid agent fentanyl as well as its related drugs in professional and amateur sports. Fast, reliable and robust analytical assays are required that allow the sensitive determination of these compounds or respective metabolites in human urine, and liquid chromatography interfaced to mass spectrometry has proven to be a suitable and powerful tool for drug testing for several years. A screening and confirmation method was developed that enables the identification of fentanyl, alfentanil, remifentanil and sufentanil as well as their N-dealkylated or de-esterified metabolites utilizing solid-phase extraction of a 2 mL urine aliquot followed by LC-electrospray-MS/MS analysis. The procedure was validated in terms of recovery (95.8-104.9%), lower limit of detection (0.5 ng mL-1), specificity and interday precision (3.9-19.8%) for the four opioid drugs and the metabolic product norfentanyl. In addition, the mass spectrometric behavior of fentanyl after electrospray ionization and collision-induced dissociation was studied by synthesis and analysis of structurally related compounds, and dissociation pathways were proposed allowing the characterization of target analytes and corresponding metabolites.     

Rapid determination of five probe drugs and their metabolites in human plasma and urine by liquid chromatography/tandem mass spectrometry: application to cytochrome P450 phenotyping studies

A liquid chromatography/mass spectrometry method, for rapid determination of five cytochrome P450 (CYP) probe drugs and their relevant metabolites in human plasma and urine, is described. The five specific probe substrates/metabolites, caffeine/paraxanthine (CYP1A2), tolbutamide/4-hydroxytolbutamide/carboxytolbutamide (CYP2C9), omeprazole/5-hydroxyomeprazole (CYP2C19), debrisoquine/5-hydroxydebrisoquine (CYP2D6) and midazolam/1'-hydroxymidazolam (CYP3A), together with the internal standards (phenacetin and paracetamol), in plasma and urine, were extracted using solid-phase extraction. The chromatography was performed using a C18 column with an isocratic mobile phase consisting of acetonitrile and 0.1% formic acid in water (70:30). The triple-quadrupole mass spectrometer was operated in both positive and negative modes, and multiple reaction monitoring was used for quantification. The method was validated over the concentration ranges 0.05-5 microg/mL for caffeine and paraxanthine, 0.02-2 microg/mL for tolbutamide, 0.1-20 microg/mL for 4-hydroxytolbutamide, carboxytolbutamide, debrisoquine and 5-hydroxydebrisoquine, 5-2500 ng/mL for omeprazole and 5-hydroxyomeprazole, and 1-100 ng/mL for midazolam and 1'-hydroxymidazolam. The intra- and inter-day precision were 0.3-13.7% and 1.9-14.3%, respectively, and the accuracy ranged from 93.5-107.2%. The lower limit of quantification varied between 1 and 100 ng/mL. The present method provides a robust, fast and sensitive analytical tool for the five-probe drug cocktail, and has been successfully applied to a clinical phenotyping study in 16 subjects.     

Automated headspace solid-phase microextraction and in-matrix derivatization for the determination of amphetamine-related drugs in human urine by gas chromatography-mass spectrometry

An automated extraction and determination method for the gas chromatography (GC)-mass spectrometry (MS) analysis of amphetamine-related drugs in human urine is developed using headspace solid-phase microextraction (SPME) and in-matrix derivatization. A urine sample (0.5 mL, potassium carbonate (5 M, 1.0 mL), sodium chloride (0.5 g), and ethylchloroformate (20 microL) are put in a sample vial. Amphetamine-related drugs are converted to ethylformate derivatives (carbamates) in the vial because amphetamine-related drugs in urine are quickly reacted with ethylchloroformate. An SPME fiber is then exposed at 80 degrees C for 15 min in the headspace of the vial. The extracted derivatives to the fiber are desorbed by exposing the fiber in the injection port of a GC-MS. The calibration curves show linearity in the range of 1.0 to 1000 ng/mL for methamphetamine, fenfluramine, and methylenedioxymethamphetamine; 2.0 to 1000 ng/mL for amphetamine and phentermine; 5.0 to 1000 ng/mL for methylenedioxyamphetamine; 10 to 1000 ng/mL for phenethylamine; and 50 to 1000 ng/mL for 4-bromo-2,5-dimethoxyphenethylamine in urine. No interferences are found, and the time for analysis is 30 min for one sample. Furthermore, this proposed method is applied to some clinical and medico-legal cases by taking methamphetamine. Methamphetamine and its metabolite amphetamine are detected in the urine samples collected from the patients involved in the clinical cases. Methamphetamine, amphetamine, and phenethylamine are detected in the urine sample collected from the victim of a medico-legal case.     

Identification and quantification of 34 drugs and toxic compounds in blood,urine, and gastric content using liquid chromatography with tandem mass spectrometry

A liquid chromatography with tandem mass spectrometry method was developed for the simultaneous screening of 34 drugs and poisons in forensic cases. Blood (0.5 mL, diluted 1:1 with water) or 1.0 mL of urine was purified by solid‐phase extraction. Gastric contents (diluted 1:1 with water) were treated with acetonitrile, centrifuged, and supernatant injected. Detection was achieved using a Waters Alliance 2695/Quattro Premier XE liquid chromatography tandem mass spectrometry system equipped with electrospray ionization, operated in the multiple reaction monitoring modes. The method was validated for accuracy, precision, linearity, and recovery. The absolute recovery of drugs and toxic compounds in blood was greater than 51% with the limit of detection in the range of 0.02–20 ng/mL. The absolute recovery of drugs and toxic compounds in urine was greater than 61% with limit of detection in the range of 0.01–10 ng/mL. The matrix effect of drugs and toxic compounds in urine was 65–117% and 67–121% in blood. The limit of detection of drugs and toxic compounds in gastric content samples were in the range of 0.05–20 ng/mL. This method was applied to the routine analysis of drugs and toxic compounds in postmortem blood, urine, and gastric content samples. The method was applied to actual forensic cases with examples given.     

Derivative spectrophotometric analysis of 4-quinolone antibacterials in formulations and spiked biological fluids by their Cu(II) complexes

A derivative UV-spectrophotometric analytical procedure was developed for determination of three 4-quinolone antibacterials: norfloxacin (NFX), ciprofloxacin (CFX), and sparfloxacin (SFX). The method depends on the complexation of Cu(II) with the studied compounds in aqueous medium. A third order, measurement was applied for their quantification. A linear correlation was established between the amplitude of the peak and concentration for all the studied drugs in the range of 15-80, 35-120, and 200-700 ng/mL, with minimum detectability (S/N = 2) of 1.0, 1.3, and 5.1 ng/mL for NFX, CFX, and SFX, respectively. The method was successfully applied for accurate, sensitive, and selective determination of the studied drugs in bulk and tablets formulation with average percentage recoveries of 99.22 +/- 0.55 to 100.33 +/- 1.60. The results obtained were favorably compared with those of the reference method. The method was also used to determine sparfloxacin in spiked human plasma and urine. The results obtained were satisfactory, accurate, and precise.     

Chemiluminescence assay for uric acid in human serum and urine using flow-injection with immobilized reagents technology

A novel chemiluminescence (CL) flow sensor for the determination of uric acid in human urine and serum has been developed by using controlled-reagent-release technology. The reagents involved in the chemiluminescence (CL) reaction, luminol and periodate, are immobilized on anion-exchange resin packed in a column. After injection of water, chemiluminescence generated by released luminol and periodate in alkaline media is inhibited in presence of uric acid. By measuring the decreased chemiluminescence (CL) intensity the uric acid is sensed. The decreased response is linear in the 5.0-500.0 ng mL(-1) range, with a detection limit of 1.8 ng mL(-1). The flow sensor showed remarkable operational stability and could be easily reused for over 80 h with sampling frequency of 100 h(-1). The proposed sensor was applied to the determination of uric acid in human urine and serum, and monitoring metabolic uric acid in human urine with RSD less than 3.0%.     

高效液相色谱-荧光分析法同时测定人体尿液中黄蝶呤与异黄蝶呤

建立了高效液相色谱-荧光法同时测定癌症病人尿液中黄蝶呤及异黄蝶呤的新方法。选择荧光检测波长λex=345nm,λem=420nm。以磷酸盐缓冲溶液(pH=7.5)-甲醇(体积比为98∶2)为流动相,流速1.0mL/min,黄蝶呤与异黄蝶呤含量分别在0.0013～0.945μg/mL及0.00017～0.118μg/mL范围内与色谱峰面积呈良好的线性关系,线性相关系数分别为0.9999和0.9996,检出限分别为0.5ng/mL和0.05ng/mL,加标平均回收率在86.2%～107.5%之间。方法应用于癌症病人尿样分析,取得了较好的结果。     

Capillary zone electrophoresis (CZE) coupled to time-of-flight mass spectrometry (TOF-MS) applied to the analysis of illicit and controlled drugs in blood

A new method for the determination of illicit and abused drugs in blood by capillary zone electrophoresis-electrospray ionization-time-of-flight mass spectrometry is proposed, in view of its application in clinical and forensic toxicology. The analytes (methamphetamine, methylenedioxyamphetamine, methylenedioxyethylamphetamine, methylenedioxymethamphetamine, methadone, cocaine, morphine, codeine, 6-acethylmorphine, benzoylecgonine) were separated with capillary zone electrophoresis by applying 15 kV within 25 min, in an uncoated fused-silica capillary (75 microm x 100 cm) using a 25 mM ammonium formate electrolyte solution (pH 9.5). The capillary electropherograph was coupled to time-of-flight mass spectrometry through an orthogonal electrospray ionization source, with a coaxial sheath liquid interface. The sheath liquid was composed of isopropanol-water (1:1 v/v) containing 0.5% formic acid delivered at 4 microL/min. Forensic drugs were identified by exact mass determination (mass accuracy typically < or =5 ppm) and by matching of the isotopic pattern. Under optimized conditions, linearity was assessed in the range 10-2000 ng/mL, with correlation coefficients between 0.9744 and 0.9982 for all the analytes. LODs were in the range of 2-10 ng/mL (S/N > or =3) and LOQs of 10-30 ng/mL. The CVs (tested at 40 and 800 ng/mL in biological matrix) were below 2.97% for migration times and below 14.61% for peak area ratios (analyte/internal standard). Blood samples were extracted by using a liquid-liquid extraction procedure and injected under field-amplified sample stacking conditions. The method was successfully applied to real cases.     

高效液相色谱电生Mn(Ⅲ)化学发光法检测人体血清和尿样中的卡托普利

设计了一个HPLC在线电生Mn(Ⅲ)化学发光检测器, 实现在线电化学反应, 从而产生反应活性很高的初生态氧化剂Mn(Ⅲ), 并与色谱柱后CP混合产生化学发光. 同时还能够根据需要调节电极反应和发光反应两者的介质, 满足柱后发光反应的最佳环境. 在优化流动相和化学发光检测条件的基础上, 将该检测器应用于人体血清和尿液中CP的测定.     

Use of gas chromatography/mass spectrometry with positive chemical ionization for the determination of opiates in human oral fluid

An analytical method for the simultaneous determination of codeine, morphine and 6-acetylmorphine (6AM) in human oral fluid was developed. The method involves liquid-liquid extraction in Toxitubes A, derivatization with 99:1 (v/v) N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA)/trimethylchlorosilane (TMCS), and gas chromatography/mass spectrometry with positive chemical ionization (GC/PCI-MS) determination. The detector response was linear over the concentration range 30-500 ng/mL with coefficients of correlation higher than 0.99. The precision was acceptable with coefficients of variation less than 7.5%. The limits of detection achieved were 0.7 ng/mL for codeine, 2.0 ng/mL for morphine, and 0.6 ng/mL for 6AM. The method proposed was applied to 80 oral fluid samples from opiates users, 98% of which were positive for the three analytes. Human oral fluid is a suitable biological fluid for the determination of opiates by GC/PCI-MS.     

Spectrophotometric determination of certain CNS stimulants in dosage forms and spiked human urine via derivatization with 2,4-Dinitrofluorobenzene

A new spectrophotometric method is developed for the determination of phenylpropanolamine HCl (PPA), ephedrine HCl (EPH) and pseudoephedrine HCl (PSE) in pharmaceutical preparations and spiked human urine. The method involved heat-catalyzed derivatization of the three drugs with 2,4-dinitrofluorobenzene (DNFB) producing a yellow colored product peaking at 370 nm for PPA and 380 nm for EPH and PSE, respectively. The absorbance concentration plots were rectilinear over the range of 2-20 for PPA and 1-14 μg/mL for both of EPH and PSE, respectively. The limit of detection (LOD) values were 0.20, 0.13 and 0.20 μg/mL for PPA, EPH and PSE, respectively and limit of quantitation (LOQ) values of 0.60 and 0.40 and 0.59 μg/mL for PPA, EPH and PSE, respectively. The analytical performance of the method was fully validated and the results were satisfactory. The proposed method was successfully applied to the determination of the three studied drugs in their commercial dosage forms including tablets, capsules and ampoules with good percentage recoveries. The proposed method was further applied for the determination of PSE in spiked human urine with a mean percentage recovery of 108.17 ± 1.60 for (n = 3). Statistical comparison of the results obtained with those of the comparison methods showed good agreement and proved that there was no significant difference in the accuracy and precision between the two methods. The mechanism of the reaction pathway was postulated.     

Spectrofluorimetric determination of fluoroquinolones in pharmaceutical preparations

Simple, rapid and highly sensitive spectrofluorimetric method is presented for the determination of four fluoroquinolone (FQ) drugs, ciprofloxacin, enoxacin, norfloxacin and moxifloxacin in pharmaceutical preparations. Proposed method is based on the derivatization of FQ with 4-chloro-7-nitrobenzofurazan (NBD-Cl) in borate buffer of pH 9.0 to yield a yellow product. The optimum experimental conditions have been studied carefully. Beer's law is obeyed over the concentration range of 23.5-500 ng mL(-1) for ciprofloxacin, 28.5-700 ng mL(-1) for enoxacin, 29.5-800 ng mL(-1) for norfloxacin and 33.5-1000 ng mL(-1) for moxifloxacin using NBD-Cl reagent, respectively. The detection limits were found to be 7.0 ng mL(-1) for ciprofloxacin, 8.5 ng mL(-1) for enoxacin, 9.2 ng mL(-1) for norfloxacin and 9.98 ng mL(-1) for moxifloxacin, respectively. Intra-day and inter-day relative standard deviation and relative mean error values at three different concentrations were determined. The low relative standard deviation values indicate good precision and high recovery values indicate accuracy of the proposed methods. The method is highly sensitive and specific. The results obtained are in good agreement with those obtained by the official and reference method. The results presented in this report show that the applied spectrofluorimetric method is acceptable for the determination of the four FQ in the pharmaceutical preparations. Common excipients used as additives in pharmaceutical preparations do not interfere with the proposed method.     

Determination of testosterone and epitestosterone glucuronides in urine by ultra performance liquid chromatography-ion mobility-mass spectrometry

UPLC-ion mobility spectrometry separations combined with mass spectrometry (UPLC-IM-MS) and tandem mass spectrometry (UPLC-IM-MS/MS) have been investigated for the simultaneous determination of testosterone and epitestosterone glucuronides in urine. The glucuronide epimers of testosterone and epitestosterone were separated by ion mobility spectrometry prior to mass analysis on the basis of differences in their collision cross sections, which have been measured in nitrogen. Combining ion mobility separation with UPLC/MS enhances the analysis of these low-abundance steroids in urine by selective interrogation of specific retention time, mass-to-charge and mobility regions. Detection limits for the UPLC-IM-MS/MS analysis of TG and ETG were 9.9 ng mL(-1) and 98 ng mL(-1) respectively, equivalent to 0.7 ng mL(-1) and 7.4 ng mL(-1) in urine, with linear dynamic ranges corresponding to 0.7-108 ng mL(-1) and 7.4-147 ng mL(-1) in urine. Repeatability (%RSD) for urine extracts was 0.64% and 2.31% for TG and ETG respectively.