Fully automated determination of eserine N-oxide in human plasma using on-line solid-phase extraction with liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A sensitive and entirely automated solid-phase extraction/liquid chromatography/electrospray ionization tandem mass spectrometric (SPE/LC/ESI-MS/MS) method was developed and validated for the determination of eserine N-oxide (ENO), a cholinesterase inhibitor-like physostigmine in human plasma, for use in pharmacokinetic studies. ENO is light-sensitive and the use of a fully on-line process increased the reliability of the assay. Plasma samples previously mixed with neostigmine bromide to prevent in vitro degradation, and tacrine as internal standard (IS), were directly injected into the SPE/LC/ESI-MS/MS system. MS software piloted the overall system. MS/MS detection of ENO and the IS was performed in the positive ion ESI mode using multiple reaction monitoring. The linear calibration curve for ENO ranged from 25 pg ml(-1) to 12.5 ng ml(-1). The limit of quantitation was 25 pg ml(-1) with 250 microl of plasma injected. Precision, accuracy and stability tests were within the acceptable range and just one analyst is required to analyze 50 unknown samples a day five days per week, from the preparation of the samples (i.e. thawing and centrifugation) to data processing. A pilot pharmacokinetic study in three healthy volunteers treated with 4.5 mg of ENO (Génésérine3((R))) showed that the method was suitable for pharmacokinetic studies in humans.     

Development and validation of a high-sensitivity liquid chromatography/tandem mass spectrometry (LC/MS/MS) method with chemical derivatization for the determination of ethinyl estradiol in human plasma

An ultra-sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the analysis of oral contraceptive ethinyl estradiol (EE) was developed and validated over the curve range of 2.5-500 pg/mL using 1 mL of human plasma sample. Ethinyl estradiol and the internal standard, ethinyl estradiol tetra-deuterated (EE-d4), were extracted from the plasma matrix with methyl t-butyl ether, derivatized with dansyl chloride and then back-extracted into hexane. The hexane phase was evaporated to dryness, reconstituted and injected onto the LC/MS/MS system. The chromatographic separation was achieved on a Luna C(18) column (50 x 2 mm, 5 micro m) with an isocratic mobile phase of 20:80 (v/v) water:acetonitrile with 1% formic acid. The offline derivatization procedure introduced the easily ionizable tertiary amine function group to EE. This greatly improved analyte sensitivity in electrospray ionization and enabled us to achieve the desired lower limit of quantitation at 2.5 pg/mL. This high sensitivity method can be used for therapeutic drug monitoring or supporting bio-equivalence and drug-drug interaction studies in human subjects.     

Pharmacokinetic studies of a novel tubulin inhibitor SK1326 in rat plasma by UPLC–MS/MS

A sensitive method for quantitation of SK1326 in rat plasma has been established using ultra-performance liquid chromatography–electrospray ionization tandem mass spectrometry (UPLC–ESI/MS/MS). SK1326 and the internal standard (tramadol) in plasma sample were extracted using acetonitrile. A centrifuged upper layer was then evaporated and reconstituted with a mobile phase of 0.5% formic acid–acetonitrile (35:65, v/v). The reconstituted samples were injected into a C₁₈ reversed-phase column. Using MS/MS in the multiple reaction monitoring mode, SK1326 and tramadol were detected without severe interference from the rat plasma matrix. SK1326 produced a protonated precursor ion ([M + H]⁺) at m/z 432.3 and a corresponding product ion at m/z 114.4. The internal standard produced a protonated precursor ion ([M + H]⁺) at m/z 264.4 and a corresponding product ion at m/z 58.1. Detection of SK1326 in rat plasma by the UPLC–ESI/MS/MS method was accurate and precise with a quantitation limit of 1.0 ng/mL. The validation, reproducibility, stability and recovery of the method were evaluated. The method has been successfully applied to pharmacokinetic studies of SK1326 in rat plasma. The pharmacokinetic parameters of SK1326 were evaluated after intravenous (at a dose of 10 mg/kg) and oral (at a dose of 20 mg/kg) administration of SK1326 in rats. After oral administration (20 mg/kg) of SK1326, the F (fraction absorbed) value was ~77.1%.     

An LC/ESI–MS/MS method to quantify the PI3K inhibitor GDC-0084 in human plasma and cerebrospinal fluid: Validation and clinical application

A liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI–MS/MS) method was developed and validated to measure GDC-0084 in human plasma and cerebrospinal fluid (CSF). Reverse-phase chromatography with gradient elution was performed using a C₁₈ column (50 × 2.0 mm, 3 μm). Solid-phase extraction of plasma and CSF was employed to give excellent recovery. MS detection was performed with positive ion screening in multiple reaction monitoring mode. The precursor to the product ions (Q1 → Q3) selected for GDC-0084 and GDC-0084-d₆ were 383.2 → 353.2 and 389.2 → 353.2, respectively. A separate calibration curve was established for human plasma and CSF. Both calibration curves, ranging from 0.2 to 200 ng/mL, were linear and had acceptable intra- and inter-day precision and accuracy. The lower limit of quantitation and limit of detection for GDC-0084 in human plasma were 0.2 ng/mL (signal/noise ≥47) and 0.005 ng/mL (signal/noise ≥3.5), respectively, and for GDC-0084 in human CSF were 0.2 ng/mL (signal/noise ≥19.7) and 0.04 ng/mL (signal/noise ≥7.2). This method was successfully applied to analyze serial plasma samples obtained from children with diffuse intrinsic pontine gliomas and other midline gliomas who participated in pharmacokinetic studies as part of a phase I clinical trial of GDC-0084.     

Simultaneous determination of sibutramine and its active metabolites in human plasma by LC‐MS/MS and its application to a pharmacokinetic study

A simple and sensitive liquid chromatography–electrospray ionization–tandem mass spectrometry (LC‐ESI‐MS/MS) technique was developed and validated for the determination of sibutramine and its N‐desmethyl metabolites (M1 and M2) in human plasma. After extraction with methyl t‐butyl ether, chromatographic separation of analytes in human plasma was performed using a reverse‐phase Luna C₁₈ column with a mobile phase of acetonitrile–10 mm ammonium formate buffer (50:50, v/v) and quantified by ESI‐MS/MS detection in positive ion mode. The flow rate of the mobile phase was 200 μL/min and the retention times of sibutramine, M1, M2 and internal standard (chlorpheniramine) were 1.5, 1.4, 1.3 and 0.9 min, respectively. The calibration curves were linear over the range 0.05–20 ng/mL, for sibutramine, M1 and M2. The lower limit of quantification was 0.05 ng/mL using 500 μL of human plasma. The mean accuracy and the precision in the intra‐ and inter‐day validation for sibutramine, M1 and M2 were acceptable. This LC‐MS/MS method showed improved sensitivity and a short run time for the quantification of sibutramine and its two active metabolites in plasma. The validated method was successfully applied to a pharmacokinetic study in human. Copyright © 2011 John Wiley & Sons, Ltd.     

Determination of two oxy-pyrimidine metabolites of diazinon in urine by gas chromatography/mass selective detection and liquid chromatography/electrospray ionization/mass spectrometry/mass spectrometry

An analytical method was developed for the determination in urine of 2 metabolites of diazinon: 6-methyl-2-(1-methylethyl)-4(1H)-pyrimidinone (G-27550) and 2-(1-hydroxy-1-methylethyl)-6-methyl-4(1H)-pyrimidinone (GS-31144). Two of the urine sample preparation procedures presented rely on gas chromatography/mass selective detection (GC/MSD) in the selected ion monitoring mode for determination of G-27550. For fast sample preparation and a limit of quantitation (LOQ) of 1.0 ppb, urine samples were purified by using ENV+ solid-phase extraction (SPE) columns. For analyte confirmation at an LOQ of 0.50 ppb, classical liquid/liquid partitioning was used before further purification in a silica SPE column. An SPE sample preparation procedure and liquid chromatography/electrospray ionization/mass spectrometry/mass spectrometry (LC/ESI/MS/MS) were used for both G-27550 and GS-31144. The limit of detection was 0.01 ng for G-27550 with GC/MSD, and 0.016 ng when LC/ESI/MS/MS was used for both G-27550 and GS-31144. The LOQ was 0.50 ppb for G-27550 when GC/MSD and the partitioning/SPE sample preparation procedure were used, and 1.0 ppb for the SPE only sample preparation procedure. The LOQ was 1.0 ppb for both analytes when LC/ESI/MS/MS was used.     

Comparison of analytical approaches for liquid chromatography/mass spectrometry determination of the alcohol biomarker ethyl glucuronide in urine

Official guidelines originating from a European Union directive regulate requirements for analytical methods used to identify chemical compounds in biological matrices. This study compared different liquid chromatography/electropray ionization mass spectrometry (LC/ESI‐MS) and tandem mass spectrometry (LC/ESI‐MS/MS) procedures for accurate determination of the conjugated ethanol metabolite and alcohol biomarker ethyl glucuronide (EtG) in urine, and the value of combined EtG and ethyl sulfate (EtS) measurement. Analysis was carried out on 482 urines following solid‐phase extraction (SPE) sample cleanup or using direct injection of a diluted sample. SPE combined with LC/MS/MS was demonstrated to be the most selective and sensitive method and was chosen as reference method. The EtG results by different methods showed good correlation (r = 0.96–0.98). When comparing five reporting limits for EtG in the range 0.10–1.00 mg/L, the overall agreement with the reference method (frequency of true positives plus true negatives) was 82–97% for direct‐injection LC/MS/MS, 90–97% for SPE‐LC/MS, 86–98% for direct‐injection LC/MS, and 86–98% for direct‐injection LC/MS analysis of EtG and EtS. Most deviations were attributable to uncertainty in quantitation, when the value was close to a cutoff but the respective results were slightly above and below, or vice versa, the critical limit. However, for direct‐injection LC/MS/MS, despite earning 4 identification points, equally many negative results were due to a product ion ratio outside the ±20% deviation accepted by the guidelines. These results indicate that the likelihood of different analytical methods to provide reliable analytical results depends on the reporting limit applied. Copyright © 2010 John Wiley & Sons, Ltd.     

Separation and characterisation of sphingoceramides by high-performance liquid chromatography-electrospray ionisation mass spectrometry

We developed a simple and reliable analytical method for the quantification and the characterization of ceramides extracted from biological samples by high-performance liquid chromatography (HPLC) coupled to electrospray ionisation tandem mass spectrometry (ESI/MS/MS). The chromatographic separation of analytes was carried out in a RP8 column, eluting with a methanol-water mixture in gradient elution mode. The separated lipids were detected by total ion monitoring and characterised by MS/MS spectra; quantitative analysis was performed by integrating the extracted ion peaks obtained in the negative ion mode. Good repeatability was obtained for retention time (0.3-2%), peak area ratio (A(S)/A(IS), 2-8%), as well as limit of detection (LOD, 5-26 pg) and quantification (LOQ, 13-53 pg). The method was validated for the analysis of N-palmitoyl-D-erythro-sphingosine (Cer16), N-stearoyl-D-erythro-sphingosine (Cer18), N-tetracosanoyl-D-erythro-sphingosine (N24:0, lignoceric ceramide, Cer24:0), and N-tetracos-15'-enoyl-D-erythro-sphingosine (N24:1, nervonic ceramide, Cer24:1), giving good results. Lipid mixtures, extracted from skin and epidermal cells, were analysed for their content of the studied ceramides.     

The use of chemical derivatization to enhance liquid chromatography/tandem mass spectrometric determination of 1-hydroxypyrene, a biomarker for polycyclic aromatic hydrocarbons in human urine

This article presents an analytical approach that used chemical derivatization to enhance mass spectrometric (MS) response in electrospray ionization (ESI) mode of 1-hydroxypyrene (1-OHP), a commonly used biomarker to monitor human exposure to polycyclic aromatic hydrocarbons (PAHs). The enhancement successfully enabled the desired detection of 50 pg/mL in human urine. The introduction of an MS-friendly dansyl group to 1-OHP enhanced both ionization efficiency in the ESI source and collision-activated dissociation (CAD) in the collision cell. The response increase was estimated to be at least 200-fold, and enabled the reduction of sample size to only 100 microL. The selective MS detection also facilitated a fast (run time 3 min) liquid chromatography (LC) method which successfully resolved the analyte and interferences. The sample processing procedure included enzymatic hydrolysis of glucuronide and sulfate conjugates, liquid-liquid extraction, derivatization with dansyl chloride and a final liquid-liquid extraction to generate clean extracts for LC/MS/MS analysis. This approach has been validated as sensitive, linear (50-1000 pg/mL), accurate and precise for the quantitation of 1-OHP in human urine. This is the first report of using chemical derivatization to enhance MS/MS detection with fast chromatography in the determination of 1-OHP in human urine.     

Comparison of electrospray ionization,atmospheric pressure chemical ionization,and atmospheric pressure photoionization for the analysis of dinitropyrene and aminonitropyrene LC-MS/MS

The only relevant source for human exposure to dinitropyrenes is diesel engine emissions. Due to this specificity, dinitropyrenes may be used as biomarkers for monitoring human exposure to diesel engine emissions. Only few analytical methods have been described for the quantitation of dinitropyrenes and their metabolites, aminonitropyrenes, and diaminopyrenes. Therefore, for dinitropyrenes, aminonitropyrenes, and diaminopyrenes were selected as model compounds for the development of a sensitive HPLC-MS/MS method (high performance liquid chromatography coupled to triple quadrupole mass spectrometry) was to quantify polyaromatic amines and nitroarenes in biological matrices was developed optimal methods by comparing electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and atmospheric pressure photoionization (APPI) sources. Dinitropyrene was not effectively ionized and diaminopyrene yielded mainly [M(.)](+) ions by electrospray ionization. With APCI and APPI, precursor ions of diaminopyrene and aminonitropyrene were [M + H](+) and [M(.)](-) for dinitropyrene. Precursor ions with [M - 30(.)](-) for dinitropyrene and [M - 30 + H](+) for aminonitropyrene were observed. Reversed and normal phase HPLC-MS/MS with ESI, APCI and APPI were optimized separately with respect to unequivocal analyte identification and sensitivity. Normal phase HPLC coupled to APPI-MS/MS gave the highest precision and sensitivity for aminonitropyrene (6%/0.2 pg on column) and dinitropyrene (9%/0.5 pg on column). The limit of detection in spiked rat plasma was 5 pg/100 microL for aminonitropyrene (accuracy 82%) and 10 pg/100 microL for dinitropyrene (accuracy 105%). In plasma of rats treated with dinitropyrene by oral administration, no detectable levels of dinitropyrene but higher aminonitropyrene levels compared with intratracheal instillation were observed. These findings clearly demonstrate that dinitropyrene was absorbed after oral and intratracheal application and that a reduction of nitro groups occurs to a high extent in the reductive environment of the intestine. To our knowledge, this is the first time that aminonitropyrene was observed in plasma after intratracheal or oral administration directly demonstrating the reductive metabolism of dinitropyrene in vivo.     

Validation of a sensitive LC/MS/MS method for the determination of zidovudine and lamivudine in human plasma

A sensitive LC/MS/MS assay for determining zidovudine (ZDV) and lamivudine (3TC) in human plasma was validated to support antiretroviral pharmacology research programs. After addition of stable labeled isotopic zidovudine (ZDV‐IS) and lamivudine (3TC‐IS) as internal standard, a solid‐phase extraction was performed with an Oasis HLB 1 cm³ cartridge, with recoveries of 92.3% for ZDV and 93.9% for 3TC. A Phenomonex Synergi Hydro‐RP (2.0 × 150 mm) reversed‐phase analytical column was utilized for chromatographic separation. The mobile phase consisted of an aqueous solution of 15% acetonitrile and 0.1% acetic acid. Detection was accomplished by ESI/MS/MS in the positive ion mode, monitoring 268/127, 271/130, 230/112 and 233/115 transitions, for ZDV, ZDV‐IS, 3TC and 3TC‐IS, respectively. The method was linear from 1 to 3000 ng/mL with a minimum quantifiable limit of 1 ng/mL when 100 μL of plasma was analyzed. Validation results demonstrated high accuracy (≤8.3% deviation) and high precision (≤10% CV) for the quality control samples. The method was also shown to be specific and reproducible. The value of the high sensitivity was demonstrated by quantitation of approximately 100 existing samples that had ZDV below the limit of quantitation using a previously validated, less sensitive HPLC‐UV method utilized in the laboratory. Copyright © 2011 John Wiley & Sons, Ltd.     

Highly sensitive method for the determination of ropinirole with a lower limit of quantitation of 3.45 pg/mL in human plasma by LC‐ESI‐MS/MS: application to a clinical pharmacokinetic study

A highly sensitive, rapid assay method has been developed and validated for the estimation of ropinirole (RPR) in human plasma with liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive‐ion mode. A solid‐phase process was used to extract RPR and citalopram (internal standard, IS) from human plasma. Chromatographic separation was operated with 0.2% ammonia solution:acetonitrile (20:80, v/v) at a flow rate of 0.50 mL/min on a Hypurity C₁₈ column with a total run time of 3.2 min. The MS/MS ion transitions monitored were 261.2 → 114.2 for RPR and 325.1 → 209.0 for IS. Method validation and clinical sample analysis were performed as per FDA guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 3.45 pg/mL and the linearity was observed from 3.45 to 1200 pg/mL. The intra‐day and inter‐day precisions were in the range of 4.71–7.98 and 6.56–8.31%, respectively. This novel method has been applied to a pharmacokinetic study of RPR in humans. Copyright © 2008 John Wiley & Sons, Ltd.     

Novel UHPLC‐MS/MS method for the determination of rotigotine in the plasma of patients with Parkinson's disease

A novel ultra‐high‐pressure liquid chromatography–tandem mass spectrometry method was developed and validated for the determination of the dopamine receptor agonist rotigotine in human plasma. Following liquid–liquid extraction with tert‐ butyl methyl ether from 500 μL plasma, the chromatographic analysis was performed on a Gemini NX3 column using 5 mm pH 5.0 ammonium acetate–5 mm ammonium acetate in methanol as binary gradient mobile phase, at a flow rate of 0.3 mL/min. The MS/MS ion transitions were 316.00 → 147.00 for rotigotine and 256.10 → 211.00 for the internal standard (lamotrigine). The lower limit of quantitation was 50 pg/mL and the linearity was determined from 50 to 2500 pg/mL. The mean recovery was 96.9%. Both intra‐ and interassay imprecision and inaccuracy were ≤15% at all quality control concentrations. The method was successfully applied to measure morning trough plasma rotigotine concentrations in a series of patients with Parkinson's disease on chronic treatment. The present study describes the first fully validated method for rotigotine determination in human plasma.     

Comparison of high-selectivity gas chromatographic methods, including column switching, for the determination of felodipine in plasma

The selectivity required for the determination of low concentrations of felodipine in plasma was achieved by either mass-selective detection, optimization of stationary phase selectivity or column-switching gas chromatography (GC) with a dual-oven chromatograph, the latter two with electron-capture detection. The three approaches were evaluated in terms of selectivity, detectability, precision and suitability for routine applications with automated injection. Using mass-selective detection, the detectability in plasma samples was limited by the performance of the mass spectrometer. The detection limit (signal-to-noise ratio = 3) was 4.7 pmol (1.8 pg) of felodipine. Pre-concentration of extracts permitted quantitation in plasma down to 0.2 nmol/1. Using electron-capture detection, the detectability was determined by the selectivity and bleeding characteristics of the columns. For single-column separation, a 35% phenyl phase was selected. The detection limit was 3.0 fmol (1.2 pg). The limit of quantitation in plasma was 1 nmol/1. In column-switching GC, bleeding products from the first column will separate on the second column and may interfere in separations for trace analysis. Bleeding products from a 50% phenyl phase (DB-17) were characterised by GC-mass spectrometry. With a dual-column system, employing a DB-17 (50% phenyl) column for selective introduction on to a CP-Sil 5 (0% phenyl) column, the signal-to-noise ratio was limited by the low-bleeding second column, provided that the bleeding products from the first column were adequately separated from felodipine. The detection limit in this instance was significantly lower 0.35 fmol (0.13 pg). Direct injection of plasma extracts permitted quantitation down to 0.4 nmol/l. All three methods were well suited for use with autosamplers.     

Development and application of UHPLC-MS/MS method for the determination of phenolic compounds in Chamomile flowers and Chamomile tea extracts

UHPLC-MS/MS method using BEH C18 analytical column was developed for the separation and quantitation of 12 phenolic compounds of Chamomile (Matricaria recutita L.). The separation was accomplished using gradient elution with mobile phase consisting of methanol and formic acid 0.1%. ESI in both positive and negative ion mode was optimized with the aim to reach high sensitivity and selectivity for quantitation using SRM experiment. ESI in negative ion mode was found to be more convenient for quantitative analysis of all phenolics except of chlorogenic acid and kaempherol, which demonstrated better results of linearity, accuracy and precision in ESI positive ion mode. The results of method validation confirmed, that developed UHPLC-MS/MS method was convenient and reliable for the determination of phenolic compounds in Chamomile extracts with linearity >0.9982, accuracy within 76.7-126.7% and precision within 2.2-12.7% at three spiked concentration levels. Method sensitivity expressed as LOQ was typically 5-20 nmol/l.Extracts of Chamomile flowers and Chamomile tea were subjected to UHPLC-MS/MS analysis. The most abundant phenolic compounds in both Chamomile flowers and Chamomile tea extracts were chlorogenic acid, umbelliferone, apigenin and apigenin-7-glucoside. In Chamomile tea extracts there was greater abundance of flavonoid glycosides such as rutin or quercitrin, while the aglycone apigenin and its glycoside were present in lower amount.     

Quantitative analysis of betulinic acid in mouse,rat and dog plasma using electrospray liquid chromatography/mass spectrometry

Betulinic acid is under development as a therapeutic agent for the treatment of metastatic malignant melanoma. In support of pharmacokinetic and toxicological evaluations, a robust assay based on liquid chromatography/mass spectrometry (LC/MS) was developed for the quantitative analysis of betulinic acid. Sample preparation consisted of deproteinization of the plasma by the addition of three volumes of acetonitrile and one volume of methanol followed by centrifugation. Aliquots of the supernatant were analyzed using an isocratic reversed-phase high-performance liquid chromatography (HPLC) system coupled to a negative ion electrospray mass spectrometer. Deprotonated molecules of betulinic acid and the isomeric internal standard oleanolic acid were detected using selected ion monitoring at m/z 455. The limit of detection of betulinic acid was 0.5 pg (1.1 fM) injected on-column (50 pg/mL, 10 microL injection volume), and the limit of quantitation was 2 pg (4.4 fM, 200 pg/mL, 10 microL injection volume). Betulinic acid was stable in plasma samples at -20 degrees C for at least 3 weeks. The intra-day and inter-day coefficients of variation of the assay were < or =6.4 and < or =9.0%, respectively. The utility of the assay was demonstrated by analyzing betulinic acid spiked into mouse, rat and dog plasma, by determining the extent of binding of betulinic acid to plasma proteins, and by measuring betulinic acid in mouse and rat plasma following intraperitoneal or intravenous administration in vivo. At 15 and 25 microg/mL in mouse, rat or dog plasma, betulinic acid was 99.99% bound to serum proteins, and, at 5 microg/mL, betulinic acid was > or =99.97% bound.     

Bioanalytical method development and validation of novel antithrombotic agent S002-333 by LC-MS/MS and its application to pharmacokinetic studies

A rapid, sensitive and simple liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) method using an electrospray ionization (ECI) source for the quantification of novel anti‐thrombotic agent S002‐333 [2‐(4‐methoxy‐benzenesulfonyl)‐2,3,4,9‐tetrahydro‐1H‐β‐carboxylic acid amide] in rabbit plasma was developed and validated. The extraction from plasma was carried out by simple protein precipitation extraction method. The chromatographic separation was performed on an Ultramex Cyno, (150 × 4.6 mm, 5 µm) with a guard column, using acetonitrile–water (75:25,v/v) with flow rate of 0.6 mL/min as the mobile phase. The tandem mass spectrometer was tuned in the multiple reaction monitoring mode to monitor the m/z transitions 386.4/215.4 for S002‐333 and m/z 393.4/171for the internal standard dexamethasone, using positive ion mode. The MS/MS response was linear over the concentration range from 1.56 to 200 ng/mL, with a lower limit of detection of 0.78 ng/mL. The accuracy and precision of the method were within the acceptable limit of ±20% at the lower limit of quantitation and ±15% at other concentrations and showed no significant matrix effect. The validated method can be used in most or all stages of the screening and optimizing process for future method validation of pharmacokinetic studies Copyright © 2010 John Wiley & Sons, Ltd.     

Simultaneous determination of tetracylines and quinolones antibiotics in egg by ultra-performance liquid chromatography-electrospray tandem mass spectrometry

We report here an ultra-performance liquid chromatography coupled with tandem mass spectrometric (MS/MS) method for the simultaneous quantitation of multiclass veterinary drugs in egg. The analysis of the target compounds, including 7 tetracyclines and 4 types of quinolones, may be accomplished in 15 min of total run time. The egg was extracted with ethylenediaminetetraacetic acid-Mcllvaine buffer solution and further purified using a polymer-based Oasis HLB solid-phase extraction cartridge. A C18 column was used to separate the analytes followed by MS/MS using an electrospray ion source. The overall average recoveries of the analytes based on matrix-fortified calibration ranged from 71 to 112% with acceptable relative standard deviations of <20% for 6 trials. For all of the target compounds, the limits of quantitation ranged between 0.02 and 4.29 microg/kg. The proposed method is sufficiently sensitive and highly selective.     

Quantitation and metabolism of mitoquinone, a mitochondria-targeted antioxidant, in rat by liquid chromatography/tandem mass spectrometry

Mitoquinone (MitoQ10 mesylate) is a mitochondria-targeted antioxidant undergoing development for the treatment of neurodegenerative diseases. The aim of this study was to develop and validate an assay based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) to determine mitoquinone and to detect and identify the metabolites of MitoQ10 in rat plasma after an oral dose. After a simple protein precipitation step, plasma samples were analyzed by reversed-phase liquid chromatography using gradient elution with acetonitrile/water/formic acid. Electrospray ionization in the positive ion mode with multiple reaction monitoring (MRM) was used to analyze mitoquinone employing the deuterated compound (d3-MitoQ10 mesylate) as internal standard. The calibration curve for mitoquinone was linear over the concentration range 0.5-250 ng/mL with a correlation coefficient>0.995. The method was sensitive (limit of quantitation 0.5 ng/mL) and had acceptable accuracy (relative error<8.7%) and precision (intra- and inter-day coefficient of variation<12.4%). Recoveries of mitoquinone at concentrations of 1.5, 20 and 200 ng/mL were in the range 87-114%. The method was successfully applied to a pharmacokinetic study in rat after a single oral dose in which four metabolites of MitoQ10 were tentatively identified as hydroxylated MitoQ10, desmethyl MitoQ10 and the glucuronide and sulfate conjugates of the quinol form of MitoQ10.     

Analytical method for determining iminoctadine triacetate by LC/ESI/MS using hydrophilic interaction chromatography.

A target value for iminoctadine triacetate residues in tap water was set at 6 microg/l in Japan. We have developed a highly selective and sensitive analytical method for iminoctadine triacetate by solid phase extraction LC/ESI/MS using hydrophilic interaction chromatography. The recovery rates at concentration of 0.06, 0.6, and 6 microg/l in distilled water, tap water, and raw water were 77.1 - 96.7%, and CV were 3.7 - 13.2%. The quantitation limit of the present method was 0.04 microg/l, and it was able to measure even one-hundredth of the target value of iminoctadine triacetate quantitatively.