Simultaneous determination of low levels of methotrexate and cyclophosphamide in human urine by micro liquid chromatography/electrospray ionization tandem mass spectrometry

The aim of this study was to develop and validate a novel solid-phase extraction (SPE) liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the simultaneous determination of two antineoplastic drugs, cyclophosphamide (CP) and methotrexate (MTX), in human urine using trophosphamide as internal standard. The method showed good precision and accuracy (mean RSD 2.8% and 0.9%; bias 2.7% and 2.4% for MTX and CP, respectively). The lower limits of detection obtained, 0.2 microg/L(urine) for MTX and 0.04 microg/L(urine) for CP, were lower than the best previously reported values. The use of a 96-well SPE plate for matrix purification ensures a high throughput (50 samples/day), allowing the routine biological monitoring of CP and MTX as measures of occupational exposure at very low levels.     

Simultaneous determination of a spectrum of trichothecene toxins out of residuals of biogas production

A sensitive and selective method for the simultaneous determination of a spectrum of trichothecenes in residuals of biogas production has been developed. It comprises sample clean-up by liquid/liquid partition for digested manure and solid phase extraction for digested solid phase. Quantification of A- and B-type trichothecenes as their trifluoroacetyl derivatives is performed by gas chromatography mass spectrometry (GC/MS), that of B-type trichothecenes alternatively by high-performance liquid chromatography (HPLC). Fluorescence detection (FLD) after post-column derivatisation using methylacetoacetate and ammonium acetate after alkaline decomposition of toxins was applied. Detection limits in digestates were between 1 and 30 microg/l and 20 and 50 microg/l for GC/MS and HPLC/FLD, respectively. Recovery rates were between 52 and 129% for GC/MS detection with the exception of T-2 tetraol with 22%, and between 56 and 123% for HPLC/FLD.     

Determination of methotrexate in environmental samples by solid phase extraction and high performance liquid chromatography: ultraviolet or tandem mass spectrometry detection?

In order to assess exposure levels of hospital personnel involved in the preparation and administration of antineoplastic drugs, environmental monitoring should be carried out. Wipe samples, pads, gloves and air samples should be collected at the end of each work shift, properly treated and then analysed using instrumental techniques which are sufficiently sensitive and specific to detect even trace amounts of drug. In this study, a method using high performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS), incorporating solid phase extraction (SPE), was validated for determination of methotrexate (MTX) in wipe and air samples. Each step of the method was first developed and optimised using ultraviolet detection (UV), and afterwards tandem mass spectrometry was used to obtain a lower limit of quantitation when the expected drug level was less than the analytical UV detection limit. SPE enabled a 20-fold preconcentration of the analyte when using HPLC/UV and a further 30-fold preconcentration was obtained when analysing samples by HPLC/MS/MS. For example, the limit of quantitation (LLQ) was lowered from 3000 ng on wipe (direct injection onto an HPLC/UV system) to 5 ng on wipe (SPE plus HPLC/MS/MS). 7-hydroxymethotrexate was used as internal standard to assess precision and accuracy.     

Simultaneous determination of cyclophosphamide, ifosfamide, doxorubicin, epirubicin and daunorubicin in human urine using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry: bioanalytical method validation

A reversed-phase high-performance liquid chromatography (rp-HPLC) system interfaced with an electrospray ionization (ESI) source coupled to tandem mass spectrometry (MS/MS) was developed and validated for the determination of cyclophosphamide (CP), ifosfamide (IF), daunorubicin (DNR), doxorubicin (DXR), and epirubicin (EPI) in human urine. The analysis of samples containing multiple analytes with a dissimilar range of polarities was carried out using a conventional reversed-phase chromatographic BDS Hypersil C8 column. The analytical run was 15 min. The triple quadrupole mass spectrometer was operated in positive ion mode and multiple reaction monitoring (MRM) was used for drug quantification. The method was validated over a concentration range of 0.2 to 4.0 microg.L(-1) for CP, IF, DXR, EPI and 0.15-2.0 microg.L(-1) for DNR in human urine. The lower limit of quantification (LLOQ) was 0.2 microg.L(-1) for CP, IF, EPI and was set at 0.3 and 0.15 microg.L(-1) for DXR and DNR, respectively. The relative standard deviations (RSD%) were <11.2% for inter- and intra-day precisions. The overall accuracy was also within 114.7% for all analytes at the concentrations of the quality control samples. The potential of ionization suppression resulting from the endogenous biological material on the rp-HPLC/MS/MS method was evaluated and measured. The feasibility of the proposed HPLC/ESI-MS/MS procedure was demonstrated by analyzing urine samples from pharmacy technicians and nurses working in hospitals or personnel employed in drug-manufacturing plants.     

A rapid, sensitive and effective quantitative method for simultaneous determination of cationic surfactant mixtures from river and municipal wastewater by direct combination of single-drop microextraction with AP-MALDI mass spectrometry

A rapid, simple, sensitive, and effective quantitative method for simultaneous determination of cationic surfactants (CS(+)) from river and municipal wastewater by direct combination of single-drop microextraction (SDME) with atmospheric pressure (AP)-MALDI mass spectrometry has been successfully demonstrated without the requirements of tedious sample pre- or post-treatment or separation by high-performance liquid chromatography (HPLC), gas chromatography (GC), and capillary electrophoresis (CE). This quantitative method can greatly enhance the signal-to-noise ratio for analysis of small molecules of CS(+) owing to the strong suppression of matrix ions by the analytes. In addition, SDME assisted in the isolation and preconcentration of CS(+) from water samples, which could effectively reduce the background interferences from the matrices present in waste and river water. The SDME conditions were optimized for achieving high extraction efficiency of CS(+) from aqueous samples, in terms of solvent selection, stirring speed, extraction time, exposure volume of acceptor phase, and salt addition. The enrichment factors for CS(+) were found to be 40-64-folds for 7 min of extraction time with no salt addition and at room temperature. This method was found to yield a linear calibration curve in the concentration range from 50 to 1500 microg/l CS(+) with a limit of detection (LOD) of 10 microg/l. The relative recoveries in river and municipal wastewater were found to be 93.8-103.6% and 91.0-98.7%, respectively. These results indicate that the combination of SDME with AP-MALDI/MS is effective for the simultaneous determination of CS(+) from river and municipal wastewater. In addition, a comparison of enrichments and LOD values for this method with hollow-fiber liquid phase microextraction (HF-LPME) was also demonstrated. The present approach is easy to operate, rapid, sensitive, and suitable for high-throughput of analysis.     

A simultaneous determination method for 5‐fluorouracil and its metabolites in human plasma with linear range adjusted by in‐source collision‐induced dissociation using hydrophilic interaction liquid chromatography–electrospray ionization–tandem mass spectrometry

We applied a new technique for quantitative linear range shift using in‐source collision‐induced dissociation (CID) to complex biological fluids to demonstrate its utility. The technique was used in a simultaneous quantitative determination method of 5‐fluorouracil (5‐FU), an anticancer drug for various solid tumors, and its metabolites in human plasma by liquid chromatography–electrospray ionization–tandem mass spectrometry (LC/ESI‐MS/MS). To control adverse effects after administration of 5‐FU, it is important to monitor the plasma concentration of 5‐FU and its metabolites; however, no simultaneous determination method has yet been reported because of vastly different physical and chemical properties of compounds. We developed a new analytical method for simultaneously determining 5‐FU and its metabolites in human plasma by LC/ESI‐MS/MS coupled with the technique for quantitative linear range shift using in‐source CID. Hydrophilic interaction liquid chromatography using a stationary phase with zwitterionic functional groups, phosphorylcholine, was suitable for separation of 5‐FU from its nucleoside and interfering endogenous materials. The addition of glycerin into acetonitrile‐rich eluent after LC separation improved the ESI‐MS response of high polar analytes. Based on the validation results, linear range shifts by in‐source CID is the reliable technique even with complex biological samples such as plasma. Copyright © 2016 John Wiley & Sons Ltd.     

Development of a simple liquid chromatography-tandem mass spectrometry method for multiresidue determination of antifungal drugs in chicken tissues

A method involving LC coupled with MS/MS (LC/MS/MS) was designed for simultaneous quantification of 10 antifungal drugs (voriconazole, griseofulvin, clotrimazole, bifonazole, econazole, ketoconazole, itraconazole, miconazole, terconazole, and fluconazole) in the liver and muscles of chickens. Homogenized tissue samples were extracted with acetonitrile and subsequently underwent freezing-delipidation. A Waters Acquity Ultra Performance LC BEH C18 column was used to separate the analytes, coupled with MS/MS using an electrospray ionization source. The accuracy of the method was confirmed with a mean recovery of 71-121%, and acceptable coefficients of variation (4-23%, n = 6). The detection capability of these compounds in two different matrixes was 0.50-2.82 microg/kg. This method can be applied for the screening and confirmation of target antifungal drugs in chicken tissues.     

Comparison of different liquid chromatography methods for the determination of corticosteroids in biological matrices

Various extraction techniques can be combined with column liquid chromatography (LC) and ultraviolet (UV) or mass spectrometric (MS) detection for the determination of synthetic corticosteroids in biological matrices. Target analysis of low concentrations of 25 microg/kg of dexamethasone in feed can be performed by combining immunoaffinity chromatography (IAC) and LC with UV detection. A straightforward multi-analyte procedure is obtained by tandem solid-phase extraction (SPE) and subsequent LC-UV. However, the limits of detection for feed samples are then relatively poor, viz. 100 microg/kg. A multi-analyte method which meets modern demands of about 5 microg/kg detection limit requires one-step SPE combined with LC-MS analysis. As regards urine corticosteroids can be determined down to a level of 0.5 microg/l by either SPE-LC-MS- MS or SPE(IAC)-LC-MS.     

Gas chromatographic-mass spectrometric method for quantification of 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone in chlorinated water samples

A method for the determination of 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX), in drinking water by GC-MS with a limit of detection of 3.0 microg/l and a limit of quantification of 7.0 microg/l is presented. Clean-up by SPE and extraction of water samples with dichloromethane were carried out before the preconcentration of MX, which was derivatized directly in the injector of the GC, and the MX trimethylsilyl derivative was identified and quantitatively determined by MS.     

High-throughput GC/MS and HPLC/MS/MS techniques for the multiclass, multiresidue determination of 653 pesticides and chemical pollutants in tea

An efficient and sensitive method has been established for simultaneous determination of 653 pesticides in teas by GC/MS and HPLC/MS/MS. The method involved extraction with acetonitrile followed by cleanup using Cleanert-TPT SPE and subsequent identification and quantitation of 490 pesticides by GC/MS and 448 pesticides by HPLC/MS/ MS. The LODs for pesticides determined by GC/MS were between 1.0 and 500 microg/kg, and those determined by HPLC/MS/MS were between 0.03 and 4820 microg/kg. At the low fortification levels of 0.01-100 microg/kg, the average recoveries of 94% of the pesticides determined by GC/MS were between 60 and 120%, 77% of which had an RSD below 20%. For 91% of pesticides determined by HPLC/MS/MS, the average recoveries were between 60 and 120%, 76% of which had an RSD below 20%. The paper also reports a novel SPE column, Cleanert TPT, which comprised graphitized carbon black (PestiCarb), polyamine silica, and amide polystyrene for purifying the tea samples. The results indicated good repeatiblity and reproducibility.     

Simultaneous estimation of acetylsalicylic acid and clopidogrel bisulfate in pure powder and tablet formulations by high-performance column liquid chromatography and high-performance thin-layer chromatography

This paper describes validated high-performance column liquid chromatographic (HPLC) and high-performance thin-layer chromatographic (HPTLC) methods for simultaneous estimation of acetylsalicylic acid (ASA) and clopidogrel bisulfate (CLP) in pure powder and formulations. The HPLC separation was achieved on a Nucleosil C8 column (150 mm length x 4.6 mm id, 5 microm particle size) using acetonitrile-phosphate buffer, pH 3.0 (55 + 45, v/v) mobile phase at a flow rate of 1.0 mL/min at ambient temperature. The HPTLC separation was achieved on an aluminum-backed layer of silica gel 60F254 using ethyl acetate-methanol-toluene-glacial acetic acid (5.0 + 1.0 + 4.0 + 0.1, v/v/v/v) mobile phase. Quantitation was achieved with UV detection at 235 nm over the concentration range 4-24 microg/mL for both drugs, with mean recoveries of 99.98 +/- 0.28 and 100.16 +/- 0.66% for ASA and CLP, respectively, using the HPLC method. Quantitation was achieved with UV detection at 235 nm over the concentration range of 400-1400 ng/spot for both drugs, with mean recoveries of 99.93 +/- 0.55 and 100.21 +/- 0.83% for ASA and CLP, respectively, using the HPTLC method. These methods are simple, precise, and sensitive, and they are applicable for the simultaneous determination of ASA and CLP in pure powder and formulations.     

Determination of beta-agonists in liver and retina by liquid chromatography-tandem mass spectrometry

A liquid chromatography-tandem mass spectrometry (LC/MS/MS) method for the determination of bromobuterol, cimaterol, clenbuterol, clenpenterol, hydroxymethylclenbuterol, isoxsuprine, mabuterol, ractopamine, ritrodrine, salbutamol, terbutaline, and tulobuterol residues in bovine liver and retina is reported. This procedure uses enzymatic digestion, liquid-liquid extraction, and cleanup on Oasis HLB solid-phase extraction cartridges, followed by determination of the residues by LC-tandem quadrupole MS using atmospheric pressure chemical ionization in the positive ion mode. Overall average recoveries ranged from 23 to 76% for liver and 34 to 77% for retina. The mean values for samples fortified at levels between 0.5-2.0 microg/kg (liver) and 5-20 microg/kg (retina) agreed within 98-118% of the spiked levels, with coefficients of variation ranging from 6 to 20%. The decision limits, CCalpha, ranged from 0.1 to 0.3 microg/kg for liver, 1-3 microg/kg for retina, and detection capabilities, CCbeta, from 0.2-0.5 microg/kg for liver and 2-5 microg/kg for retina.     

An APCI LC‐MS/MS method for routine determination of capecitabine and its metabolites in human plasma

The anticancer drug capecitabine and its metabolites [including the active metabolite 5‐fluorouracil (5‐FU)] display high pharmacokinetic inter‐patient variability. Such variability, which may lead to treatment failure or toxicity, could need drug concentration measurement to individualize dosing regimen. However, usual assay methods are often long and fastidious. A simultaneous and cost‐effective method was thus developed for the determination of the concentrations of these compounds in human plasma. Compounds were extracted via a classic liquid–liquid extraction. Chromatographic analysis was performed on a C18 reverse phase column with detection by atmosphere pressure chemical ionization LC‐MS/MS. Our method allows a good chromatographic separation of the compounds and was fully validated following Food and Drug Administration (FDA) recommendations (good selectivity, no carry‐over, linearity of the calibration curves without weighting, deviations from nominal concentrations of standard samples lower than 15%, intra‐ and inter‐assay precision and accuracy lower than 15%). Recovery and stability were also acceptable following the FDA guidelines. A matrix effect impairing the determination of 5‐FU was avoided by using a stable isotopic derivative of 5‐FU as internal standard. Interestingly, this method allows detection of TetraHydroUridine, an inhibitor of ex vivo degradation of metabolites, which is essential for the stability, the adequate conditioning of blood samples and for good laboratory practice, essential in routine determination. This method seems usable to routinely determine concentrations of capecitabine and its metabolites in blood and may be helpful in further studies aiming at performing therapeutic drug monitoring. Copyright © 2010 John Wiley & Sons, Ltd.     

Simultaneous determination of rifampicin and sulbactam in mouse plasma by high-performance liquid chromatography

A simple and rapid high-performance liquid chromatographic (HPLC) method with ultraviolet detection has been developed and validated for the simultaneous determination of rifampicin and sulbactam in mouse plasma. Plasma samples were deproteinized with acetonitrile and separated by HPLC on a RP-18 (125 x 4 mm, 5 microm) column and gradient elution with potassium dihydrogen phosphate solution (pH 4.5; 50 mm) and acetonitrile at a flow-rate of 1.0 mL/min. Rifampicin and sulbactam were monitored at 230 nm and confirmed by means of their UV spectra using a diode-array detector. The method was linear at plasma levels from 1 to 100 microg/mL for rifampicin and from 5 to 200 microg/mL for sulbactam. The limits of quantification were 0.6 microg/mL for rifampicin and 4.2 microg/mL for sulbactam. The intra- and inter-day precisions of the method (RSD) were lower than 5% for both compounds. Average recoveries of rifampicin and sulbactam from mice plasma were 98.2 and 89.3%, respectively. The developed method was successfully applied to the determination of the pharmacokinetic profile of both compounds in mice.     

Development of a rapid and sensitive method for the simultaneous determination of 1,2-dibromoethane, 1,4-dichlorobenzene and naphthalene residues in honey using HS-SPME coupled with GC-MS

A new method for the simultaneous determination of 1,4-dichlorobenzene (p-DCB), naphthalene and 1,2-dibromoethane (1,2-DBE) residues in honey has been developed. Analysis is carried out using gas chromatography-mass spectrometry (GC/MS) in selected ion monitoring mode (SIM), after extraction and preconcentration of target analytes by headspace solid-phase microextraction (HS-SPME), with a 100 microm film thickness polydimethylsiloxane (PDMS) fiber. Several parameters affecting the extension of the adsorption process (i.e., addition of salt, extraction time, extraction temperature) were studied. The optimal conditions for the determination of these analytes were established. The proposed HS-SPME method showed good sensitivity, without carryover between the samples. Linearity was studied from 5 to 2500 microg kg(-1) for p-DCB, 0.5 to 500 microg kg(-1) for naphthalene and 5 to 500 microg kg(-1) honey for 1,2-DBE with correlation coefficients (r(2)) ranging from 0.9901 to 0.9999. Precision was assessed and both intra and inter-day R.S.D.s (%) were below 6.3%. The detection limits were found to be 1, 0.1 and 2 microg kg(-1) honey for p-DCB, naphthalene and 1,2-DBE, respectively. The percentage recoveries that were evaluated with the proposed HS-SPME method and the standard addition calibration technique gave values among 72.8 and 104.3% for measurements in samples spiked with one target analyte or mixtures of the three. This method has been applied for the analysis of unknown honey samples. The results showed an excellent applicability of the proposed method for the determination of the target compounds in honey samples.     

Solid-phase extraction-gas chromatography and solid-phase extraction-gas chromatography-mass spectrometry determination of corrosion inhibiting long-chain primary alkyl amines in chemical treatment of boiler water in water-steam systems of power plants

Gas chromatography with simultaneous flame-ionization detection (FID) and a nitrogen-phosphorus detection (NPD) as well as gas chromatography-mass spectrometry (GC/MS) has been used to characterize long-chain primary alkyl amines after derivatization with trifluoroacetic anhydride (TFAA). Electron impact ionization- (EI) and negative chemical ionization (NCI) mass spectra of trifluoroacetylated derivatives of the identified tert-octadecylamines are presented for the first time. The corrosion inhibiting alkyl amines were applied in a water-steam circuit of energy systems in the power industry. Solid-phase extraction (SPE) with octadecyl bonded silica (C18) sorbents followed by gas chromatography were used for quantification of the investigated tert-octadecylamines in boiler water, superheated steam and condensate samples from the power plant. The estimated values were: 89 microg l(-1)(n = 5, RSD = 7.8%), 45 microg l(-1) (n = 5, RSD = 5.4%) and 37 microg l(-1)(n = 5, RSD = 2.3%), respectively.     

Second derivative synchronous fluorescence spectroscopy for the simultaneous determination of metoclopramide and pyridoxine in syrup and human plasma

A rapid, simple, and highly sensitive second derivative synchronous fluorometric method has been developed for the simultaneous determination of metoclopramide (MT) and pyridoxine (PY) in a binary mixture. The method is based on measurement of the native fluorescence of these drugs at delta lambda = 80 nm in methanol. The different experimental parameters affecting the native fluorescence of the drugs were carefully studied and optimized. The fluorescence-concentration plots were rectilinear over the ranges of 0.02-0.4 and 0.1-2 microg/mL for MT and PY, respectively. The limits of detection were 0.003 and 0.007 microg/mL and the limits of quantification were 0.008 and 0.02 microg/mL for MT and PY, respectively. The proposed method was successfully applied to the determination of MT and PY in synthetic mixtures and in commercial syrup. The results were in good agreement with those obtained with a reported method. The high sensitivity attained by the proposed method allowed the determination of MT in spiked and real human plasma samples. The mean percent recoveries of MT from spiked and real human plasma (n = 3) were 93.72 +/- 3.15 and 89.72 +/- 2.19 respectively.     

Quantification of cyclophosphamide and its metabolites in urine using liquid chromatography/tandem mass spectrometry

A reliable and easy to use liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the simultaneous quantification of urinary concentrations of cyclophosphamide (CP) and its main metabolites excreted in urine, i.e. N-dechloroethylcyclophosphamide (DCL-CP), 4-ketocyclophosphamide (4KetoCP), and carboxyphosphamide (CarboxyCP). Sample preparation consisted of dilution of urine with an aqueous solution of the internal standard D(4)-CP and methanol, and centrifugation. LC/MS/MS detection was performed using a triple-quadrupole mass spectrometer working in selected reaction monitoring mode. All analytes were quantified in a single run within 11.5 min. The limits of detection were 5 ng/mL for CP and 4KetoCP, 1 ng/mL for DCL-CP, and 30 ng/mL for CarboxyCP. Quantification ranges were adjusted to the expected concentrations in 24-h urine collections of patients treated with a polychemotherapy regimen (3-175 microg/mL for CP, 0.5-27 microg/mL for 4KetoCP and 0.17-9 microg/mL for CarboxyCP and DCL-CP, respectively). The method was validated according to international guidelines of the ICH and the FDA.     

A kinetic method for the determination of nitrite by its catalytic effect on the oxidation of chlorpromazine with nitric acid.

A catalytic spectrophotometric method for the determination of trace amounts of nitrite is proposed. In acidic solution, chlorpromazine (CP) is oxidized by nitric acid to form a red compound, which is further oxidized to a colorless compound. The reaction is accelerated by trace amounts of nitrite and can be followed by measuring the absorbance at 525 nm: nitrite ion is regenerated and multiplied by nitric acid. The absorbance of the reaction increased with an increase in the reaction time, reached a maximum and decreased rapidly. Since the time required for the absorbance to reach the maximum decreased with increasing nitrite concentration, this value was used as the measured parameter for the nitrite determination. Under the optimum experimental conditions (2.3 M nitric acid, 1.2 x 10(-5) M CP, 40 degrees C), nitrite can be determined in the range 0-100 microg l(-1). The relative standard deviations (n = 6) are 4.7 and 1.8% for 40 and 100 microg l(-1) nitrite, respectively. The detection limit of this method (3sigma) is 1.2 microg l(-1). This method was successfully applied to a determination of nitrite in natural water samples.     

Development and validation of a capillary high-performance liquid chromatography/electrospray tandem mass spectrometric method for the quantification of bisphenol A in air samples

Bisphenol A (BPA) is a toxic industrial chemical that affects the endocrine system even at low concentrations. A new method, based on capillary high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) analysis, has been developed to determine BPA in atmospheric samples. The method involves collection of air samples (typically 2 m(3)) on glass fiber filters, with ultrasonic extraction and sample concentration under vacuum before analysis. HPLC analysis was performed isocratically at a flow rate of 10 microL min(-1) using a capillary reversed-phase column and MS/MS analysis in negative ion multiple reaction monitoring (MRM) mode, using BPA-d(16) as internal standard. The present method provides linear response in the range 0.007-3.5 microg/filter (R(2) > 0.999) and is characterized by high accuracy (mean bias 2%) and good reproducibility (mean RSD 5%). High sensitivity (LOD = 2 ng/m(3) based on 2 m(3) of air collected), specificity, and speed of the analysis make the present method suitable for routine determination of BPA in the atmosphere, both for ambient and personnel monitoring.