Quantification of pegylated liposomal doxorubicin and doxorubicinol in rat plasma by liquid chromatography/electrospray tandem mass spectroscopy: Application to preclinical pharmacokinetic studies

A high-performance liquid chromatography-tandem mass spectrometric method (LC/MS/MS) has been developed and validated for the determination of pegylated liposomal doxorubicin and its metabolite doxorubicinol in rat plasma. One hundred microliters plasma samples were treated with Triton X-100 to immediately disperse the liposome. Then the samples were extracted by a single methanol:acetone protein precipitation step in the presence of additional 50microL of 70% (w/v) zinc sulfate, and subsequently analyzed by LC/MS/MS using positive turbo-ion spray ionization mode operating the instrument in the multiple-reaction-monitoring (MRM) mode. The related compound daunorubicin was used as internal standard. The validated concentration ranges were from 20 to 8000ng/mL for doxorubicin and from 0.05 to 20.0ng/mL for doxorubicinol. An effective LC-MS/MS method was developed to quantify trace amount of doxorubicinol with little interference from doxorubicin. The autosampler carryover was minimized from 285 to 10.5% by increasing the washing times of the valves when the used pentafluorophenylpropyl HPLC column had no contribution to the carryover. The relative matrix effect from six unique lots was absent for both compounds. Results obtained from the GLP validation study demonstrated good accuracy (85-110%) and precision (CV less than 14%) across the calibration ranges for both compounds. This method was applied to study the pharmacokinetic profiles of doxorubicin and doxorubicinol in rats after a single dose administration of Stealth-49 liposomal doxorubicin HCl. The mean AUC value for doxorubicinol was found to be only 0.011% of that of doxorubicin.     

Hyphenated technique for the extraction and determination of isoflavones in algae: Ultrasound-assisted supercritical fluid extraction followed by fast chromatography with tandem mass spectrometry

New hyphenated technique for the extraction and determination of isoflavones in sea and freshwater algae and cyanobacteria was developed. The method consists of sonication sample pretreatment, extraction by supercritical CO₂ modified by 3% (v/v) of MeOH/H₂O mixture (9:1, v/v) at 35 MPa and 40 °C for 60 min, fast chromatography analysis by the means of Agilent 1200 Series Rapid Resolution and MS/MS determination. Agilent 1200 Series RRLC was used with Zorbax SB-CN chromatographic column (100 mm × 2.1 mm, particle size 3.5 μm), 3 μl injection volume, mobile phase consisting of 0.2% (v/v) acetic acid in water (solvent A) and acetonitrile (solvent B) and used with linear gradient (30% B at 0 min, from 0 min to 3 min up to 50% B, from 3 to 6 min up to 80% B and from 6 to 10 min down to 30% B). The flow-rate was 0.4 mL/min, column oven temperature 35 °C. MS detector Agilent Technologies 6460 Triple quadrupole LC/MS with Agilent Jet Stream was used in a negative ESI mode under following conditions: gas temperature 350 °C, gas flow 13 L/min, nebulizer gas pressure 50 psi, sheath gas temperature 400 °C, sheath gas flow 12 L/min, capillary voltage was 4 kV. Samples were analysed in the multiple reaction monitoring (MRM) mode. Eight isoflavone compounds were found for the first time in seven real samples of sea algae and in three control samples of freshwater algae and cyanobacteria. Usual optimisation study of extraction parameters was performed. Pressure and temperature optima for algae matrix are different from those obtained sooner for other matrices for most of the analytes, but the results of modifier optimisation study are in good accordance with those obtained sooner for spiked samples and red clover matrix. It seems that matrix has very small or no effect on the modifier selection. Two different approaches of sonication pretreatment were tested: sonication bath and the thorn instrument. In longer extraction time experiments, thorn sonication was more efficient and recovery of following supercritical fluid extraction was higher.     

Determination of free captopril in human plasma by liquid chromatography with mass spectrometry detection

A new simple, sensitive and selective liquid chromatography coupled with mass spectrometry (LC/MS) method for quantification of captopril after precolumn derivatization with p-bromo-phenacyl-bromide in human plasma was validated. Plasma samples were analysed on a monolithic column (Cromolith Performance-RP 18e, 100 mm × 4.6 mm I.D., 3 μm) under isocratic conditions using a mobile phase of a 40:60 (v/v) mixture of acetonitrile and 0.1% (v/v) formic acid in water. The flow rate was 1 mL/min at the column temperature of 30 °C. In these chromatographic conditions, the retention time was 4.4 min for captopril derivative. The detection of the analyte was in MRM mode using an ion trap mass spectrometer with electrospray positive ionisation. The monitored ions were 216, 253, 255, 268, 270 m/z derived from 415 m/z for derivatized captopril. The sample preparation was very simple and consisted in plasma protein precipitation from 0.2 mL plasma using 0.3 mL methanol after the derivatization reaction was completed. Calibration curves were generated over the range of 10-3000 ng/mL with values for coefficient of correlation greater than 0.993 and by using a weighted (1/y²) quadratic regression. The values for precision (CV %) and accuracy (relative error %) at quantification limit were less than 9.9% and 3.9%, for within- and between-run, respectively. The mean recovery of the analyte was 99%. Derivatized samples demonstrated good short-term, long-term, post-preparative and freeze-thaw stability. This is the first reported LC-MS/MS method for analysis of captopril in human plasma that uses protein precipitation as sample processing procedure. The method is very simple and allows obtaining a very good recovery of the analyte. The validated LC-MS/MS method has been applied to a pharmacokinetic study of 50 mg captopril tablets on healthy volunteers.     

Simultaneous ultra-high-pressure liquid chromatography–tandem mass spectrometry determination of amphetamine and amphetamine-like stimulants,cocaine and its metabolites,and a cannabis metabolite in surface water and urban wastewater

An ultra-high-pressure liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method has been developed for the simultaneous quantification and confirmation of 11 basic/acidic illicit drugs and relevant metabolites in surface and urban wastewater at ng/L levels. The sample pre-treatment consisted of a solid-phase extraction using Oasis MCX cartridges. Analyte deuterated compounds were used as surrogate internal standards (except for norbenzoylecgonine and norcocaine) to compensate for possible errors resulting from matrix effects and those associated to the sample preparation procedure. After SPE enrichment, the selected drugs were separated within 6 min under UHPLC optimized conditions. To efficiently combine UHPLC with MS/MS, a fast-acquisition triple quadrupole mass analyzer (TQD from Waters) in positive-ion mode (ESI+) was used. The excellent selectivity and sensitivity of the TQD analyzer in selected reaction monitoring mode allowed quantification and reliable identification at the LOQ levels. Satisfactory recoveries (70–120%) and precision (RSD < 20%) were obtained for most compounds in different types of water samples, spiked at two concentration levels [limit of quantification (LOQ) and 10LOQ]. Thus, surface water was spiked at 30 ng/L and 300 ng/L (amphetamine and amphetamine-like stimulants), 10 ng/L and 100 ng/L (cocaine and its metabolites), 300 ng/L and 3000 ng/L (tetrahydrocannabinol-COOH). Recovery experiments in effluent and influent wastewater were performed at spiking levels of three and fifteen times higher than the levels spiked in surface water, respectively. The validated method was applied to urban wastewater samples (influent and effluent). The acquisition of three selected reaction monitoring transitions per analyte allowed positive findings to be confirmed by accomplishment of ion ratios between the quantification transition and two additional specific confirmation transitions. In general, drug consumption increased in the weekends and during an important musical event. The highest concentration levels were 27.5 μg/L and 10.5 μg/L, which corresponded to 3,4-methylenedioxymethamphetamine (MDMA, or ecstasy) and to benzoylecgonine (a cocaine metabolite), respectively. The wastewater treatment plants showed good removal efficiency (>99%) for low levels of illicit drugs in water, but some difficulties were observed when high drug levels were present in wastewaters.     

Determination of fluoroquinolones in environmental waters by in-tube solid-phase microextraction coupled with liquid chromatography-tandem mass spectrometry

We developed a sensitive and useful method for the determination of five fluoroquinolones (FQs), enoxacin, ofloxacin, ciprofloxacin, norfloxacin, and lomefloxacin in environmental waters, using a fully automated method consisting of in-tube solid-phase microextraction (SPME) coupled with liquid chromatography-tandem mass spectrometry (LC/MS/MS). These compounds were analysed within 7 min by high-performance liquid chromatography (HPLC) using a CAPCELL PAK C8 column and aqueous ammonium formate (pH 3.0, 5 mM)/acetonitrile (85/15, v/v) at a flow rate of 0.2 mL/min. Electrospray ionization conditions in the positive ion mode were optimized for MS/MS detection. In order to optimize the extraction of FQs, several in-tube SPME parameters were examined. The optimum in-tube SPME conditions were 20 draw/eject cycles of 40 μL of sample at a flow-rate of 150 μL/min, using a Carboxen 1010 PLOT capillary column as an extraction device. The extracted compounds were easily desorbed from the capillary by passage of the mobile phase. Using the in-tube SPME LC/MS/MS method, good linearity of the calibration curve (r ≥ 0.997) was obtained in the concentration range from 0.1 to 10 ng/mL for all compounds examined. The limits of detection (S/N = 3) of the five FQs ranged from 7 to 29 pg/mL. The in-tube SPME method showed 60-94-fold higher sensitivity than the direct injection method (5 μL injection). This method was applied successfully to the analysis of environmental water samples without any other pretreatment and interference peaks. Several surface waters and wastewaters were collected from the area around Asahi River, and ofloxacin was detected in wastewater samples of a sewage treatment plant and other two hospitals at 17.5-186.2 pg/mL. The recoveries of FQs spiked into river water were above 81% for a 0.1 or 0.2 ng/mL spiking concentration, and the relative standard deviations were below 1.9-8.6%.     

Screening of drugs in equine plasma using automated on-line solid-phase extraction coupled with liquid chromatography–tandem mass spectrometry

A rapid liquid chromatography–tandem mass spectrometry (LC–MS–MS) method was developed for the simultaneous screening of 19 drugs of different classes in equine plasma using automated on-line solid-phase extraction (SPE) coupled with a triple quadrupole mass spectrometer. Plasma samples were first protein precipitated using acetonitrile. After centrifugation, the supernatant was directly injected into the on-line SPE system and analysed by a triple quadrupole LC–MS–MS in positive electrospray ionisation (+ESI) mode with selected reaction monitoring (SRM) scan function. On-line extraction and chromatographic separation of the targeted drugs were performed using respectively a polymeric extraction column (2 cm L × 2.1 mm ID, 25 μm particle size) and a reversed-phase C18 LC column (3 cm L × 2.1 mm ID, 3 μm particle size) with gradient elution to provide fast analysis time. The overall instrument turnaround time was 9.5 min, inclusive of post-run and equilibration time. Plasma samples fortified with 19 targeted drugs including narcotic analgesics, local anaesthetics, antipsychotics, bronchodilators, mucolytics, corticosteroids, sedative and tranquillisers at sub-parts per billion (ppb) to low parts per trillion (ppt) levels could be consistently detected. No significant matrix interference was observed at the expected retention times of the targeted ion transitions. Over 70% of the drugs studied gave detection limits at or below 100 pg/mL, with some detection limits reaching down to 19 pg/mL. The method had been validated for extraction recovery, precision and sensitivity, and a blockage study had also been carried out. This method is used regularly in the authors’ laboratory to screen for the presence of targeted drugs in pre-race plasma samples from racehorses.     

Selective trace enrichment of acidic pharmaceuticals in real water and sediment samples based on solid-phase extraction using multi-templates molecularly imprinted polymers

A novel multi-templates molecularly imprinted polymer (MIP), using acidic pharmaceuticals mixture (ibuprofen (IBP), naproxen (NPX), ketoprofen (KEP), diclofenac (DFC), and clofibric acid (CA)) as the template, was prepared as solid-phase extraction (SPE) material for the quantitative enrichment of acidic pharmaceuticals in environmental samples and off-line coupled with liquid chromatography–mass spectrometry (LC/MS/MS). Washing solvent was optimized in terms of kind and volume for removing the matrix constituents nonspecifically adsorbed on the MIP. When 1 L of water sample spiked at 1 μg/L was loaded onto the cartridge, the binding capacity of the MIP cartridge were 48.7 μg/g for KEP, 60.7 μg/g for NPX, 52 μg/g for CA, 61.3 μg/g for DFC and 60.7 μg/g for IBP, respectively, which are higher than those of the commercial single template MIP in organic medium (e.g. toluene) reported in the literature. Recoveries of the five acidic pharmaceuticals extracted from 1 L of real water samples such as lake water and wastewater spiked at 1 μg/L were more than 95%. The recoveries of acidic pharmaceuticals extracted from 10-g sediment sample spiked at the 10 ng/g level were in the range of 77.4–90.6%. To demonstrate the potential of the MIP obtained, a comparison with commercial C18 SPE cartridge was performed. Molecularly imprinted solid-phase extraction (MISPE) cartridge showed higher recoveries than commercial C18 SPE cartridge for acidic pharmaceuticals. These results showed the suitability of the MISPE method for the selective extraction of a group of structurally related compounds such as acidic pharmaceuticals.     

Quantification of piperazine phosphate in human plasma by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry employing precolumn derivatization with dansyl chloride

This paper describes a novel method that combines dansyl chloride (DNS-CL) derivatization with high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI/MS/MS) for the sensitive and selective determination of piperazine phosphate in human plasma. After addition of ondansetron hydrochloride as internal standard (IS), piperazine phosphate was derivatized and then extracted with ethyl acetate. After being evaporated and reconstituted, the sample was analyzed using LC-ESI/MS/MS. Separation was achieved using an Agilent ZORBAX SB-C₁₈ (150 mm × 2.1 mm I.D., 3.5 μm) column and isocratic elution with 10 mM ammonium acetate solution (pH 3.0)-methanol (50: 50, v/v). Detection was performed on a triple-quadrupole mass spectrometer utilizing electrospray ionization (ESI) interface operating in positive ion and selected reaction monitoring (SRM) mode with the precursor to product ion transitions m/z 320 → 171 for DNS-CL-piperazine phosphate and m/z 294 → 170 for the IS. The method was fully validated for its selectivity, sensitivity, linearity, precision, accuracy, recovery, matrix effect and stability. The coefficient (r) of piperazine phosphate with a linear range of 0.1-15 μg mL⁻¹ was 0.9974-0.9995. The limit of detection and lower limit of quantification in human plasma were 0.01 and 0.1 μg mL⁻¹, respectively. The validated LC-ESI/MS/MS method has been successfully applied to a bioequivalence study of piperazine phosphate trochiscus in Chinese healthy male volunteers.     

Determination of afloqualone in human plasma using liquid chromatography/tandem mass spectrometry: Application to pharmacokinetic studies in humans

Two methods for determining the central-acting muscle relaxant afloqualone in human plasma were developed and compared using API2000 and API4000 liquid chromatography tandem mass spectrometry (LC/MS/MS) systems. In the API2000 LC/MS/MS system, afloqualone and the internal standard methaqualone were extracted from plasma using a methyl-tertiary ether. After drying the organic layer, the residue was reconstituted in a mobile phase (0.1% formic acid-acetonitrile:0.1% formic acid buffer, 80:20 v/v) and injected onto a reversed-phase C₁₈ column. The isocratic mobile phase was eluted at 0.2 ml/min. The ion transitions monitored in multiple reaction-monitoring mode were m/z 284 → 146 and 251 → 117 for afloqualone and methaqualone, respectively.Sample preparation for the API4000 LC/MS/MS system involved simple protein precipitation with an organic mixture (methanol:10% ZnSO₄ = 8:2). The ion transitions monitored in multiple reaction-monitoring mode were m/z 284 → 146 and 251 → 131 for afloqualone and methaqualone, respectively.In both assays, the coefficient of variation of the precision was less than 11.8%, the accuracy exceeded 91.5%, the limit of quantification was 0.5 ng/ml, and the limit of detection was 0.1 ng/ml for afloqualone. Two methods were used to measure the plasma afloqualone concentration in healthy subjects after a single oral 20-mg dose of afloqualone. During subsequent application of the methods, we observed that high-concentration plasma samples (>7 ng/ml) prepared using the protein precipitation method resulted in about 20% higher afloqualone concentrations than with plasma samples prepared using the liquid-liquid extraction method. We believe that this phenomenon was related to the cleanness of the sample and its chemical nature.     

Quantitative determination of triclocarban in wastewater effluent by stir bar sorptive extraction and liquid desorption–liquid chromatography–tandem mass spectrometry

Triclocarban is an antimicrobial and antibacterial agent found in personal care products and subsequently is a prevalent wastewater contaminant. A quantitative method was developed for the analysis of triclocarban in wastewater effluents using stir bar sorptive extraction–liquid desorption (SBSE–LD) followed by liquid chromatography–tandem mass spectrometry (LC–MS/MS) by means of an electrospray interface. A stir bar coated with polydimethylsiloxane (PDMS) is placed within a vial containing wastewater effluent and is stirred for an hour at room temperature. The PDMS stir bar is then placed in a LC vial containing methanol and is desorbed in a sonicator bath. The methanol is evaporated to dryness and reconstituted in 75% methanol. Spike and recovery experiments in groundwater that did not contain native concentrations of triclocarban were performed at 0.5 μg/L and were 93 ± 8%. Recoveries in wastewater effluent that were corrected for the background levels of triclocarban were 92 ± 2% and 96 ± 5%, respectively, when spiked with 0.5 and 5 μg/L of triclocarban. The precision of the method as indicated by the relative standard error was 2%. The limit of quantitation was 10 ng/L. The SBSE–LD–LC/MS/MS method was applied to wastewater effluent samples collected from northeast Ohio. Triclocarban was quantitated in all five effluent samples, and its concentration ranged from 50 to 330 ng/L. The described method demonstrates a simple, green, low-sample volume, yet, sensitive method to measure triclocarban in aqueous matrices.     

Study of the factors affecting the performance of microextraction by packed sorbent (MEPS) using liquid scintillation counter and liquid chromatography-tandem mass spectrometry

Microextraction by packed sorbent (MEPS) is a new technique for sample preparation that can be connected on-line with LC or GC. In MEPS, approximately 1-2 mg of the solid packing material is inserted into a syringe (100-250 μL) as a plug. Sample preparation takes place on the packed bed. The bed can be packed or coated to provide selective and suitable sampling conditions. The new method is very promising for extraction of drugs and metabolites from biological samples.In this paper, some factors affecting the performance of MEPS such as recovery, carry-over, leakage, washing volume and elution volume were studied using C18 and hydroxylated polystyrene-divinylbenzene copolymer (ENV+) as sorbents. Radioactively labelled bupivacaine in plasma samples was used as test analyte. For the extraction of this drug, using methanol/water 95:5 (v/v) (0.25% ammonium hydroxide) was used as elution solvent. The analyte response increased with increasing the elution volume and it was linear upp up to 100 μL utilizing liquid scintillation counter. Further, for concentrating the sample, we found that MEPS may be used such that the sample can be drawn through the needle, up and down, several times. The analyte leakage increases as the volume washing increases, though higher washing volumes may also result in cleaner extracts. To eliminate analyte carry-over, the sorbents were washed first with 3 × 250 μL elution solution and then with 3 × 250 μL washing solution. In addition, the reproducibility measurements show relatively good relative standard deviation (RSD) % values concerning analyte recovery and analyte leakage. The present study provides an understanding of basic aspects when optimizing methods for MEPS. In this study, MEPS was used off-line with liquid scintillation counter and on-line with LC-MS/MS.     

A high throughput and selective method for the estimation of valproic acid an antiepileptic drug in human plasma by tandem LC-MS/MS

A high throughput liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the determination of valproic acid, an antiepileptic drug, in human plasma is described. It is a rapid and sensitive isocratic reversed-phase liquid chromatography-tandem mass spectrometric method equipped with turbo ion spray (TIS) source, operating in the negative ion and pseudo selective reaction monitoring (SRM) acquisition mode to quantify valproic acid. The extraction of valproic acid and hydrochlorothiazide (IS) from the plasma involved sample treatment with phosphoric acid followed by solid-phase extraction using Waters hydrophilic-lipophilic balance (HLB) cartridge giving extracts free from endogenous interferences. Sample preparation by this method yielded very good and consistent mean recoveries of 99.73 and 74.47% for valproic acid and IS, respectively. The method was linear over the dynamic range of 2.0-200.0 μg/ml (covering entire therapeutic range) with a correlation coefficient r ≥ 0.9989. The coefficient of variance (CV, %) was 7.03% at 2.0 μg/ml (LLOQ). This method was fully validated for its accuracy, precision, recovery and matrix effect especially because the pattern of elution of all the analytes may appear as flow injection type. The analyte stability was examined under conditions mimicking the sample storage, handling and analysis procedures. The method was successfully applied for bioequivalence studies in human subject samples after oral administration of 500 mg formulations.     

Simultaneous quantification of multiple classes of phenolic compounds in blood plasma by liquid chromatography–electrospray tandem mass spectrometry

A method using high performance liquid chromatography–electrospray tandem mass spectrometry (LC–ESI-MS/MS) in positive ion mode was developed for the simultaneous analysis of 30 phenolic compounds, including four estrogens, bisphenol A (BPA), 10 hydroxylated polybrominated dephenyl ethers (OH-PBDEs) and 15 bromophenols (BRPs), in blood plasma. In the present method, derivatization with dansyl chloride was employed, and all the derivertized target compounds were well resolved on a 100 mm Xbridge C18 column with acetonitrile and 0.1% formic acid as the mobile phases. Purification procedures, such as liquid–liquid extraction and silica-gel chromatography, were applied to reduce matrix effects in the sample extract and remove excess derivatizing reagents, thus permitting selective and sensitive detection of the target phenolic compounds. The limit of quantification for all analytes, with a signal-to-noise ratio of ∼10, was 2–30 pg/g (plasma weight) except for 6-OH-BDE-137 (30 pg/g) and 3-BRP (60 pg/g). The method was validated for recoveries (68–100%), accuracy (84–110%) and precision (3.7–11%) using charcoal-stripped bovine blood plasma spiked with all target compounds (500 and 5000 pg/mL). Finally, the method was applied to analyze six blood plasma samples from frogs and cormorants, where two natural estrogens, one BPA, one OH-PBDE and four BRPs were detected. The greatest total concentrations of estrogens coincided with the least total concentrations of other phenolic compounds for both species. The proposed method based on derivatization followed by LC–MS/MS provides a novel method to simultaneously monitor multiple groups of phenolic compounds in blood plasma.     

Targeted analysis of multiple pharmaceuticals,plant toxins and other secondary metabolites in herbal dietary supplements by ultra-high performance liquid chromatography–quadrupole-orbital ion trap mass spectrometry

In this study, an ultra-high performance liquid chromatography–quadrupole-orbital ion trap mass spectrometry (UHPLC–Q-orbitrap MS) method was developed and validated for simultaneous determination of 96 pharmaceuticals, plant toxins, and other plant secondary metabolites in herbal dietary supplements. Target analytes were extracted from samples using the QuEChERS (quick easy cheap effective rugged safe) procedure. The instrument was operated in full MS–data dependent tandem mass spectrometry (full MS–dd-MS/MS) acquisition mode which enabled collection of quantitative high resolution (HR) full mass spectral data and confirmatory HR MS/MS data in a single run. The method provided excellent selectivity in both full MS and dd-MS/MS mode. Under optimized collision energy settings, product ion spectra containing both precursor and two or more product ions were obtained for most of the analytes. Limits of detection (LODs) and limits of quantification (LOQs) for the method differed significantly for the examined matrices. LODs ≤ 10 μg kg⁻¹ and LOQs ≤ 50 μg kg⁻¹ were obtained for 48 to 81% of target compounds across five different matrices. With the exception of highly polar analytes, the optimized QuEChERS extraction procedure provided acceptable recoveries in the range 70%–120%. The precision of the method, characterized as the relative standard deviation (RSD, n = 5), was ≤25% and ≤18% at spiking concentrations of 50 μg kg⁻¹ and 500 μg kg⁻¹, respectively. Because of variations in matrix effects in extracts of herbal dietary supplements that differed in composition, the method of standard additions and an approach based on dilution of matrix components followed by quantification using solvent standards were applied for quantification. The procedure was used to examine commercial dietary supplements for the 96 analytes of interest. To the best of our knowledge, this is the first report of an integrated analysis and quantification of this wide range of compounds.     

Sensitive and rapid method for the determination of urinary cotinine in non-smokers: an application for studies assessing exposures to second hand smoke (SHS)

We describe a sensitive and rapid method to assay urinary cotinine levels among non-smokers using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI/MS/MS) and its application in studies assessing exposures to second hand smoke (SHS). Cotinine was initially extracted from 1 ml of urine with methylene chloride by using a liquid-liquid extraction Chem Elut™ column. The extracted sample was further separated by using a BetaBasic C18 column (1 mm × 150 mm, 3 μm) with isocratic elution (60:40 acetonitrile and 5 mM ammonium acetate at pH 5), and then examined using a triple quadrupole mass spectrometer with an electrospray ionization (ESI) source in multiple-reaction-monitoring (MRM) mode. The elution of cotinine from the LC column took approximately 2.3 min and the detection of cotinine by ESI/MS/MS provided a limit of detection (LOD) of 0.3 ng/ml. The ESI/MS/MS detection was able to easily distinguish between cotinine and nicotine. This method, validated using a cotinine concentration range from 0.8 to 102.4 ng/ml, was successfully applied in a cross-sectional study examining differences in levels and sources of second hand smoke (SHS) exposure among non-smokers. Self-reported measures of SHS exposure were significantly associated with urinary cotinine levels. This urinary cotinine assay using LC-ESI/MS/MS provides a robust, high throughput and very sensitive method for the evaluation of SHS exposure for use in epidemiologic and clinical research studies.     

Targeted analysis with benchtop quadrupole–orbitrap hybrid mass spectrometer: Application to determination of synthetic hormones in animal urine

Sensitive and unequivocal determination of analytes/contaminants in complex matrices is a challenge in the field of food safety control. In this study, various acquisition modes (Full MS/AIF, Full MS + tMS/MS, Full MS/dd MS/MS and tSIM/ddMS/MS) and parameters of a quadrupole–orbitrap hybrid mass spectrometer (Q Exactive) were studied in detail. One of the main conclusions has been that, reducing the scan range for Full MS (using the quadrupole) and targeted modes give higher signal-to-noise (S/N) ratios and thereby better detection limits for analytes in matrix. The use of Q Exactive in a complex case, for the confirmatory analysis of hormones in animal urine is presented. A targeted SIM data dependent MS/MS (tSIM/ddMS/MS) acquisition method for determination of eight synthetic hormones (trenbolone, 17α ethinylestradiol, zeranol, stanozolol, dienestrol, diethylstilbestrol, hexestrol, taleranol) and a naturally occurring hormone (zearalenone) in animal urine were optimized to have sensitive precursors from targeted SIM mode and trigger MS/MS scans over the entire chromatograph peak. The method was validated according to EC/657/2002. CCα (decision limit) for the analytes ranged between 0.11 μg L⁻¹ and 0.69 μg L⁻¹ and CCβ (detection capability) ranged between 0.29 μg L⁻¹ and 0.90 μg L⁻¹.     

Homogeneous chemiluminescent assays for free choline in human plasma and whole blood

Choline was oxidized in the presence of choline oxidase and the hydrogen peroxide generated was detected using a chemiluminescent acridinium-9-carboxamide. The dose response for choline (0-150 μM) was established in buffer and was validated for the quantification of choline in human plasma and whole blood. This homogeneous assay was performed in a 96-well microplate format and required minimal sample volume (4 μL) and short analysis time (<5 s per well). The new assay(s) correlated well (R > 0.98, plasma; R > 0.97, whole blood) with LC-MS/MS.     

Comparison of orthogonal liquid and gas chromatography–mass spectrometry platforms for the determination of amino acid concentrations in human plasma

Concentrations of amino acids in a human plasma pool were determined using four independent quantification methods. Orthogonal separation schemes (LC, GC, or GC×GC) and detection systems (triple quadrupole or time-of-flight mass spectrometry) are shown to demonstrate excellent consistency among platforms for quantifying 18 amino acids in NIST Standard Reference Material (SRM) 1950 Metabolites in Human Plasma using a well-characterized isotope dilution (ID) quantification method. Measured levels were consistent with reference values in plasma from the literature. Individual amino acid concentrations in plasma varied by over an order of magnitude ranging from 1.83 μg/g to 28.0 μg/g (7.78 μmol/L to 321 μmol/L). Average variability (coefficient of variation) between experimental amino acid concentrations (excluding cysteine) among all methods was 6.3%. Certified mass fraction values for amino acids in NIST SRM 1950 will be established from statistically weighted means of all experimental results.     

Establishment and validation of an SIL-IS LC–MS/MS method for the determination of ibuprofen in human plasma and its pharmacokinetic study

In this work, we developed and validated a highly sensitive, rapid and stable LC–MS/MS method for the determination of ibuprofen in human plasma with ibuprofen-d3 as a stable isotopically labeled internal standard (SIL-IS). Human plasma samples were prepared by one-step protein precipitation. The chromatographic separation was achieved on a Poroshell 120 EC-C₁₈ (2.1 × 50 mm, 2.7 μm). Aqueous solution (containing 0.05% acetic acid and 5 mm NH₄Ac) and methanol were selected as the mobile phase with gradient elution. An electrospray ionization source was applied and operated in negative ion mode. Multiple reaction monitoring mode was used for quantification using target fragment ions m/z 205.0 → 161.1 for ibuprofen and m/z 208.0 → 164.0 for SIL-IS, respectively. This method exhibited a linear range of 0.05–36 μg/ml for ibuprofen with correlation coefficient >0.99. Mean recoveries of ibuprofen in human plasma ranged from 78.4 to 80.9%. The RSD of intra- and inter-day precision were both < 5%. The accuracy was between 88.2 and 103.67%. The matrix effect was negligible in human plasma, including lipidemia and hemolytic plasma. A simple, efficient and accurate LC–MS/MS method was successfully established and applied to a pharmacokinetic study in healthy Chinese volunteers after a single oral administration of ibuprofen granules.     

Simple assay for monitoring seven quinolone antibacterials in eggs: Extraction with hot water and liquid chromatography coupled to tandem mass spectrometry: Laboratory validation in line with the European Union Commission Decision 657/2002/EC

A simple and rapid method able to determine residues of seven quinolone antibacterials in whole eggs is presented here. This method is based on the matrix solid-phase dispersion technique with hot water as extractant followed by liquid chromatography–tandem mass spectrometry. After depositing 1.5 g of an egg sample containing the analytes and the analyte surrogate (norfloxacin) on sand (crystobalite), this material was packed into an extraction cell. Quinolones were extracted by flowing 6 mL of water acidified with 50 mmol/L formic acid through the cell heated at 100 °C. After pH adjustment and filtration of the extract, 100 μL of it was injected into the LC column. MS data acquisition was performed in the multiple reaction monitoring mode, selecting two precursor ion to product ion transitions for each target compound. Hot water appeared an efficient extracting medium, since absolute recoveries of the analyte in egg at the level of 20 ng/g were 89–103%. Estimated limits of quantification (S/N = 10) were 0.2–0.6 ng/g. Based on the EU Commission Decision 2002/657/EC, the method was validated in terms of ruggedness, specificity, linearity, within-laboratory reproducibility, decision limit (CCα and detection capability (CCβ). Depending on the particular analyte, CCαs ranged between 0.41 and 2.6 ng/g, while CCβs were 0.64–3.7 ng/g. The method was linear in the 3–30 ng/g range, with typical R² values higher than 0.97. The within-laboratory reproducibility (n = 21) at 6 ng/g level was in the 9.0–12% range. After validation, a depletion study of enrofloxacin and one of its metabolites, i.e. ciprofloxacin, in eggs was conducted.