Determination of cabergoline and L-dopa in human plasma using liquid chromatography-tandem mass spectrometry

We determined cabergoline and L-dopa in human plasma using liquid chromatography-mass spectrometry with tandem mass spectrometry (LC-MS-MS). The deproteinized plasma samples with organic solvent or acid were analyzed directly by reversed-phase liquid chromatography. Using multiple reaction monitoring (MRM, product ions m/z 381 of m/z 452 for cabergoline and m/z 152 of m/z 198 for L-dopa) on LC-MS-MS with electrospray ionization (ESI), cabergoline and L-dopa in human plasma were determined. Calibration curves of the method showed a good linearity in the range 5-250 pg/ml for cabergoline and 1-200 ng/ml for L-dopa, respectively. The limit of determination was estimated to be approximately 2 pg/ml for cabergoline and approximately 0.1 ng/ml for L-dopa, respectively. The method was applied to the analysis of cabergoline and L-dopa in plasma samples from patients treated with these drugs. The precision of analysis showed coefficients of variation ranging from 3.8% to 10.5% at cabergoline concentration of 13.8-26.2 pg/ml and from 2.9% to 8.9% at an L-dopa concentration of 302.5-522.1 ng/ml in patient plasma. As a result, the procedure proved to be very suitable for routine analysis.     

Quantification of cidofovir in human serum by LC-MS/MS for children

A new method for the quantification of cidofovir (CDV), an acyclic nucleotide analogue of cytosine with antiviral activity against a broad-spectrum of DNA viruses, in human serum, using high-performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) has been developed. A strong anion exchange (SAX) solid-phase extraction procedure was applied for the sample preparation. The tandem mass spectrometer was tuned in the multiple reaction monitoring mode to monitor the m/z 278.1-->234.9 and the m/z 288.1-->133.1 transitions for CDV and the internal standard 9-(2-phosphonylmethoxyethyl)guanine (PMEG), respectively, using negative electrospray ionization. The MS/MS response was linear over the concentration range from 78.125 ng/ml to 10,000 ng/ml, with a lower limit of quantification of 78.125 ng/ml. The intra- and inter-day precisions (relative standard deviation (%)) for CDV were less than 7.8% and the accuracies (% of deviation from nominal level) were within +/-12.1% for quality controls. The novel LC-MS/MS method allowed a specific, sensitive and reliable determination of CDV in human serum and was applied to investigate the yet unknown pharmacokinetic properties of CDV in a paediatric cancer patient.     

Application of liquid chromatography-tandem mass spectrometry for the determination of opioidmimetics in the brain dialysates from rats treated with opioidmimetics intraperitoneally

We have determined three opioidmimetics (compounds I-III) in the rat brain dialysates after intraperitoneal (i.p.) administration of compounds I-III using a liquid chromatography/mass spectrometry with tandem mass spectrometry (LC-MS/MS). The dialysate samples with methanol were directly analyzed by online column-switching liquid chromatography. Using multiple reaction monitoring (MRM, product ions m/z 421 of m/z 657 for compound I, m/z 421 of m/z 643 for compound II, and m/z 407 of m/z 629 for compound III) on LC-MS/MS with electrospray ionization (ESI), opioidmimetics in rat brain dialysates were determined. Calibration curves of the method showed a good linearity in the range of 10-100 ng/ml for each compound. The limit of determination was estimated to be ca. 1 ng/ml for compounds II and III, and ca. 5 ng/ml for compound I, respectively. The precision of analysis showed coefficients of variation ranging from 4.7 to 10.4% at compound III concentration (10-100 ng/ml) in Ringer's solution. As a result, the procedure proved to be very suitable for routine analysis. The method was applied to the analysis of three opioidmimetics in the brain dialysate samples from rats treated with these compounds.     

HPLC MS/MS method for quantification of meprobamate in human plasma: application to 24/7 clinical toxicology

We described the development and full validation of rapid and accurate liquid chromatography method, coupled with tandem mass spectrometry detection, for quantification of meprobamate in human plasma with [(13)C-(2)H(3)]-meprobamate as internal standard. Plasma pretreatment involved a one-step protein precipitation with acetonitrile. Separation was performed by reversed-phase chromatography on a Luna MercuryMS C18 (20 mm×4 mm×3 μm) column using a gradient elution mode. The mobile phase was a mix of distilled water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid. The selected reaction monitoring transitions, in electrospray positive ionization, used for quantification were 219.2→158.2 m/z and 223.1→161.1m/z for meprobamate and internal standard, respectively. Qualification transitions were 219.2→97.0 and 223.1→101.1 m/z for meprobamate and internal standard, respectively. The method was linear over the concentration range of 1-300 mg/L. The intra- and inter-day precision values were below 6.4% and accuracy was within 95.3% and 103.6% for all QC levels (5, 75 and 200 mg/L). The lower limit of quantification was 1 mg/L. Total analysis time was reduced to 6 min including sample preparation. The present method is successfully applied to 24/7 clinical toxicology and demonstrated its usefulness to detect meprobamate poisoning.     

Simultaneous determination of procaine and para-aminobenzoic acid by LC-MS/MS method

A sensitive high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed for simultaneous determination of procaine and its metabolite p-aminobenzoic acid (PABA). N-Acetylprocainamide (NAPA) was used as an internal standard for procaine and PABA analysis. This assay method has also been validated in terms of linearity, lower limit of detection, lower limit of quantitation, accuracy and precision as per ICH guidelines. Chromatography was carried out on an XTerra MS C(18) column and mass spectrometric analysis was performed using a Quattro Micro mass spectrometer working with electro-spray ionization (ESI) source in the positive ion mode. Enhanced selectivity was achieved using multiple reaction monitoring (MRM) functions, m/z 237-->100, m/z 138-->120, and m/z 278-->205 for procaine, PABA and NAPA, respectively. Retention times for PABA, procaine and NAPA were 4.0, 4.7 and 5.8min, respectively. Linearity for each calibration curve was observed across a range from 100nM to 5000nM for PABA, and from 10nM to 5000nM for procaine. The intra- and inter-day relative standard deviations (RSD) were <5%.     

Simultaneous quantitation of dexamethasone palmitate and dexamethasone in human plasma by liquid chromatography/tandem mass spectrometry

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for simultaneous quantitation of dexamethasone palmitate and dexamethasone in human plasma was developed. After sample preparation by protein precipitation and liquid-liquid extraction, the analytes and internal standard (IS) were separated on a Venusil XBP-C8 column using gradient elution. Multiple reaction monitoring of dexamethasone palmitate, dexamethasone and IS used the precursor to product ion transitions at m/z 631.8-->373.1, m/z 393.2-->147.1 and m/z 264.2-->58.1, respectively. The method was linear over the ranges 1.5-1000ng/mL for dexamethasone palmitate and 2.5-250ng/mL for dexamethasone with intra- and inter-day precisions of <10% and accuracies of 100+/-7%. The assay was applied to a clinical pharmacokinetic study involving the injection of dexamethasone palmitate to healthy volunteers.     

Automated approach for quantitative analysis of complex peptide mixtures from tandem mass spectra

To take advantage of the potential quantitative benefits offered by tandem mass spectrometry, we have modified the method in which tandem mass spectrum data are acquired in 'shotgun' proteomic analyses. The proposed method is not data dependent and is based on the sequential isolation and fragmentation of precursor windows (of 10 m/z) within the ion trap until a desired mass range has been covered. We compared the quantitative figures of merit for this method to those for existing strategies by performing an analysis of the soluble fraction of whole-cell lysates from yeast metabolically labeled in vivo with (15)N. To automate this analysis, we modified software (RelEx) previously written in the Yates lab to generate chromatograms directly from tandem mass spectra. These chromatograms showed improvements in signal-to-noise ratio of approximately three- to fivefold over corresponding chromatograms generated from mass spectrometry scans. In addition, to demonstrate the utility of the data-independent acquisition strategy coupled with chromatogram reconstruction from tandem mass spectra, we measured protein expression levels in two developmental stages of Caenorhabditis elegans.     

Development and validation of a highly sensitive LC-MS/MS method for organic cation transporter (OCT) substrate tetraethylammonium (TEA) in rabbits

Tetraethylammonium is widely used as a probe in organic cation transporters studies. A simple, highly sensitive, and specific method using direct protein precipitation was developed using Hydrophilic Interaction Liquid Chromatography coupled with positive electrospray ionization tandem mass spectrometry for the determination of tetraethylammonium (TEA) in rabbit plasma. Isocratic separation was achieved using a ZIC-HILIC column with acetonitrile and 5mM ammonium acetate in the ratio of 8:2 containing 0.1% formic acid. Acquisition was performed in multiple reaction monitoring mode with the transitions: m/z 130→100 and 130→86 for TEA and m/z 276.1→142.2 for internal standard (homatropine). This method was validated to determine selectivity, linearity, sensitivity, precision, accuracy, recovery and stability. A good linearity was found within a range of 1.53-784.6 ng/mL. The above method has been demonstrated for its capability to estimate the plasma levels of TEA after its topical instillation in rabbit eyes. This method provides an accurate, precise and sensitive tool for determining TEA levels for transporter studies.     

Determination of N-desethylamodiaquine by hydrophilic interaction liquid chromatography with tandem mass spectrometry: application to in vitro drug metabolism studies

The antimalarial drug amodiaquine is extensively metabolized to N-desethylamodiaquine (DEAQ) by cytochrome P450 2C8 (CYP2C8). DEAQ formation is an enzyme specific reaction that is used to quantify in vitro CYP2C8 activity. A rapid and sensitive method for the determination of DEAQ in human liver microsomes was developed using hydrophilic interaction liquid chromatography/tandem mass spectrometry (HILIC-MS/MS). Microsomal incubation samples were processed by protein precipitation with acetonitrile. The analytes were separated on a BETASIL Silica-100 (50mmx2.1mm, 5microm) column by isocratic elution at a flow rate of 220microl/min with a mobile phase consisting of 85% acetonitrile containing 5mM ammonium acetate and 0.1% formic acid. Detection was by positive electrospray ionization on a TSQ Quantum Discovery triple quadrupole mass spectrometer operated in the selective reaction monitoring mode. The precursor-product ion pair was m/z 328-->283 for DEAQ and m/z 331-->283 for DEAQ-d(3). The lower limit of quantification was 10nM for DEAQ and linearity was observed over the concentration range of 10-1500nM. Intra- and inter-day accuracy and precision were within 3.4 and 7.0%, respectively. The method was successfully applied to CYP2C8 drug metabolism studies in pooled human liver microsomes.     

Fast simultaneous determination of urinary 1-hydroxypyrene and 3-hydroxybenzo[a]pyrene by liquid chromatography-tandem mass spectrometry

A fast analysis method using liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry was developed for the simultaneous determination of the 1-hydroxypyrene (1-OHP) and 3-hydroxybenzo[a]pyrene (3-OHBaP) in urine. Mass transitions were monitored at m/z 219.3-200.0 for 1-OHP and m/z 269.2-252.2 for 3-OHBaP. Only 10 min was needed for the analysis. The recovery was 60% for 3-OHBaP and 91% for 1-OHP, respectively. And the method detection limits were 0.49 microg/L for 1-OHP and 1.03 microg/L for 3-OHBaP. The inter- and intra-day relative standard deviations were in the range of 2.8-8.9% for 1-OHP and 9.7-20.8% for 3-OHBaP, respectively. The developed method was successfully used to measure urinary PAH metabolites of student volunteers in a high school.     

Development and validation of a liquid chromatography-tandem mass spectrometry method for the determination of phenylephrine in human plasma and its application to a pharmacokinetic study

A sensitive and reliable method was developed to quantitate phenylephrine in human plasma using liquid chromatography-electrospray tandem mass spectrometry. The assay was based on solid-phase extraction with C18 cartridges and hydrophilic interaction chromatography performed on a pentafluorophenylpropylsilica column (50 mm x 4 mm, 3 microm particles), the mobile phase consisted of methanol-10 mM ammonium acetate (90:10, v/v). Quantification was through positive-ion mode and selected reaction monitoring at m/z 168.1-->135.0 for phenylephrine and m/z 182.1-->135.0 for internal standard etilefrin, respectively. The lower limit of quantitation was 51 pg/ml using 0.25 ml of plasma and linearity was observed from 51 to 5500 pg/ml. Within-day and between-day precision expressed by relative standard deviation was less than 12% and inaccuracy did not exceed 8% at all levels. The assay was applied to the analysis of samples from a pharmacokinetic study.     

高效液相色谱-串联质谱检测基因组DNA甲基化方法的建立

目的:建立高效液相色谱-串联质谱(HPLC-ESI-MS/MS)检测基因组DNA甲基化水平的方法。方法:以5-mdC和dG为标准品,采用全自动高效液相色谱系统进行分离,串联电喷雾质谱检测,选择多反应监测模式(MRM)测定标准品,绘制标准工作曲线。结果:在MRM模式下选取5-mdC(m/z 241.9→126.3)和dG(m/z 268.1→152.3)分别作为定量检测的母子离子对,各化合物能实现良好的基线分离;5-mdC和dG碰撞能均为15 eV,去簇电压分别为40和45 V,最低定量限分别为1.65和2.47 fmol;标准品的响应值比为90%~110%;5-mdC含量的天内相对标准偏差和天间相对标准偏差均小于8%。结论:HPLC-ESI-MS/MS是能应用于检测基因组DNA甲基化的一种高通量、高准确率、高分辨率、高灵敏度且重复性好的方法。     

Development and validation of a LC-MS/MS method for the determination of viaminate in human plasma

A sensitive and specific method for determination of viaminate in human plasma by using high-performance liquid chromatography coupled with electrospray tandem mass spectrometry (LC-MS/MS) was developed in this study. The plasma samples were simply deproteinated, extracted, evaporated, and then reconstituted in 200 microl of methanol prior to analysis. Chromatographic separation was carried out on a Shimadzu VP-ODS column (250 mm x 2.0 mm, 5 microm) with a mobile phase of methanol-water (95:5, v/v) at a flow rate of 0.2 ml/min. Quantification was performed in the negative-ion electrospray ionization mode by selected ion monitoring of the product ions at m/z 164 for viaminate and m/z 109 for testosterone propionate which was used as the internal standard. The corresponding parent ions were m/z 446 and m/z 345. A linear calibration curve was observed within the concentration range of 0.10-200 ng/ml. The lowest limit of quantitation (LLOQ) was 0.1 ng/ml. The extraction-efficiency at three concentrations was 100.7, 93.6, and 99.7%. Practical utility of this new LC-MS/MS method was confirmed in pilot pharmacokinetic studies in humans following oral administration.     

Determination of rimonabant in human plasma and hair by liquid chromatography-mass spectrometry

Rimonabant is the first therapeutically relevant cannabinoid antagonist, licensed in Europe for treatment of obesity when a risk factor is associated. The objective of this study was to develop and validate a method for measurement of rimonabant in human plasma and hair using liquid chromatography coupled to mass spectrometry (LC-MS/MS). Rimonabant and AM-251 (internal standard) were extracted from 50muL of plasma or 10mg of hair using diethylether. Chromatography was performed on a 150mmx2.1mm C18 column using a mobile phase constituted of formate buffer/acetonitrile. Rimonabant was ionized by electrospray in positive mode, followed by detection with mass spectrometry. Data were collected either in full-scan MS or in full-scan MS/MS mode, selecting the ion m/z 463.1 for rimonabant and m/z 555.1 for IS. The most intense product ion of rimonabant (m/z 380.9) and IS (m/z 472.8) were used for quantification. Calibration curves covered a range from 2.5 (lower limit of quantification) to 1000.0ng/mL (upper limit of quantification) in plasma and from 2.5 to 1000.0pg/mg in hair. Validation results demonstrated that rimonabant could be accurately and precisely quantified in both matrixes: accuracy and precision were within 85-115% and within 15% of standard deviation, respectively. Stability studies in plasma showed that rimonabant was stable during the assay procedure, but a 30% decrease was observed for one concentration after 3 weeks at -20 degrees C. This simple and robust LC-MS/MS method can be used for measuring rimonabant concentrations in human plasma and hair either in clinical or in forensic toxicology.     

96-Well liquid-liquid extraction liquid chromatography-tandem mass spectrometry method for the quantitative determination of ABT-578 in human blood samples

We report here a quantitative method for the analysis of ABT-578 in human whole blood samples. Sample preparation was achieved by a semi-automated 96-well format liquid-liquid extraction (LLE) method. Aluminum/polypropylene heat seal foil was used to enclose each well of the 96-well plate for the liquid-liquid extraction. A liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) method with pre-column regeneration was developed for the analysis of sample extracts. Selective reaction monitoring (SRM) of the mass transitions m/z 983-935 and m/z 931-883 was employed for the detection of ABT-578 and internal standard, respectively. The ammonium adduct ions [M + NH(4)](+) generated from electrospray ionization were monitored as the precursor ions. The assay was validated for a linear dynamic range of 0.20-200.75ng/ml. The correlation coefficient (r) was between 0.9959 and 0.9971. The intra-assay CV (%) was between 1.9 and 13.5% and the inter-assay CV (%) was between 4.7 and 11.3%. The inter-assay mean accuracy was between 86.4 and 102.5% of the theoretical concentrations.     

Rapid and simple one-step membrane extraction for the determination of 8-hydroxy-2'-deoxyguanosine in human plasma by a combination of on-line solid phase extraction and LC-MS/MS

A quantitative analytical method using automated on-line solid phase extraction (SPE) and liquid chromatography-electrospray tandem mass spectrometry (LC-ESI-MS/MS) for the determination of 8-OHdG (8-hydroxy-2'-deoxyguanosine) in human plasma was developed and validated. A one-step membrane extraction method for the plasma sample preparation and a C18 SPE column with simple extraction and purification were used for the on-line extraction. A C18 column was employed for LC separation and ESI-MS/MS was utilized for detection. (15)N(5)-8-OHdG ((15)N(5)-8-hydroxy-2'-deoxyguanosine) was used as an internal standard for quantitative determination. The extraction, clean-up and analysis procedures were controlled by a fully automated six-port switch valve as one strategy to reduce the matrix effect and simultaneously improve detection sensitivity. Identification and quantification were based on the following transitions: m/z 284→168 for 8-OHdG and m/z 289→173 for (15)N(5)-8-OHdG. Satisfactory recovery was obtained, and the recovery ranged from 95.1 to 106.1% at trace levels in human plasma and urine, with a CV lower than 5.4%. Values for intraday and interday precision were between 2.3 and 6.8% for plasma and between 2.7 and 4.5% for urine, respectively. Values for the method accuracy of intraday and interday assays ranged from 93.0 and 100.5% for plasma and 110.2 and 119.4% for urine, respectively. The limits of detection (LOD) and LOQ were 0.008 ng/mL and 0.02 ng/mL, respectively.The applicability of this newly developed method was demonstrated by analysis of human plasma samples for an evaluation of the future risk of oxidative stress status in human exposure to nanoparticles and other diseases.     

Determination of carbamazepine and carbamazepine 10,11-epoxide in human plasma by tandem liquid chromatography-mass spectrometry with electrospray ionisation

A sensitive method for the determination of carbamazepine and carbamazepine 10,11-epoxide in plasma is described, using high-performance liquid chromatographic separation with tandem mass spectrometry. Samples were purified using liquid-liquid extraction and separated on a Phenomenex Luna C18 5 microm. 150 x 2 mm column with a mobile phase consisting of acetonitrile, methanol and formic acid (0.1%) (10:70:20, v/v). Detection was performed by a Micromass Quattro Ultima mass spectrometer in the MRM mode (LC-MS-MS) using electro spray ionisation (ESI+), monitoring the transition of the protonated molecular ion for carbamazepine at m/z 237.05 and carbamazepine 10,11-epoxide at m/z 253.09 to the predominant ions of m/z 194.09 and 180.04, respectively. The mean recovery was 95% for carbamazepine and 101% for carbamazepine 10,11-epoxide, with a lower limit of quantification of 0.722 ng/ml for carbamazepine and 5.15 ng/ml for carbamazepine 10,11-epoxide, when using 0.5 ml plasma. This high-throughput method was used to quantify 230 samples per day, and is sufficiently sensitive to be employed in pharmacokinetic studies.     

The combination effects of phenolic compounds and fluconazole on the formation of ergosterol in Candida albicans determined by high-performance liquid chromatography/tandem mass spectrometry

A liquid chromatography/tandem mass spectrometry (LC/MS) with atmospheric pressure chemical ionization (APCI) for the quantification of ergosterol, lanosterol, and squalene was developed to evaluate the combination effects of phenolic compounds with fluconazole on ergosterol biosynthesis in Candida albicans. The three analytes were separated by a column of C18 and were quantified without interference with each other using positive mode tandem mass spectrometry (MS/MS). Molecular ions of ergosterol and lanosterol were detected as the [M+H-H2O]+ ion species at m/z 380 and 410, whereas squalene appeared as the [M+H]+ ion species at m/z 412. On fragmentation of ergosterol, lanosterol, and squalene, the product ions at m/z 69, 149, and 109, respectively, were present as major fragments. These product ions were used for the quantification of them in multiple reaction monitoring acquisition mode. The relationship between signal intensity and the analytes' concentration was linear over the concentration range of 0.05-10 microg/ml. Following the treatment of C. albicans with fluconazole in combination with albicanyl caffeate, resveratrol, and 3,4'-difluorostilbene, respectively, the content of ergosterol in both the sensitive and resistant C. albicans showed depletion, whereas the squalene showed accumulation especially in the sensitive isolates determined with the method developed.     

Development of a high-performance liquid chromatography-tandem mass spectrometry method for the determination of flurogestone acetate in ovine plasma

Flurogestone (FGA) is a synthetic progesterone, with a progestational action higher than that of progesterone itself. It is intended for vaginal use in large animals to induce oestrus synchronization. A quantitative method for the analysis of flurogestone acetate (FGA) in ovine plasma by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) has been developed. After the incorporation of megestrol acetate (MGA) as internal standard (IS) and followed by a liquid-liquid extraction from plasma, FGA and MGA were chromatographed using a reverse-phase HPLC column and detected by tandem mass spectrometry with a TurboIonSpray source. Multiple reaction-monitoring (MRM) mode was used for the quantitative determination of FGA in ovine plasma. The precursor ions [M+H](+) at m/z 407.2 and 385.1 for FGA and MGA, respectively, produced product ions at m/z 267.1/285.1 for FGA and m/z 267.1/224.0 for MGA. The validated concentration range was 0.2-5.0 ng/ml based on 500 microl plasma aliquots. The lower limit of quantitation was 0.2 ng/ml. Fully validated selectivity, accuracy, precision and reproducibility criteria for routine use in pharmacokinetic studies were demonstrated.     

Development and validation of a high performance liquid chromatography-tandem mass spectrometry for the determination of etodolac in human plasma

A simple and specific method using a one-step liquid-liquid extraction (LLE) with butyl acetate followed by high performance liquid chromatography (HPLC) coupled with positive ion electrospray ionization tandem mass spectrometry (ESI-MS/MS) detection was developed for the determination of etodolac in human plasma, using indomethacin as an internal standard (IS). Chromatographic separation was performed isocratically using a Capcellpak MGII C(18) column with 65% acetonitrile and 35% water containing 10mM ammonium formate (adjusted to pH 3.5 with formic acid). Acquisition was performed in multiple reaction monitoring (MRM) mode by monitoring the transitions: m/z 287.99>172.23 for etodolac and m/z 357.92>139.01 for IS. The method was validated to determine its selectivity, linearity, sensitivity, precision, accuracy, recovery and stability. The limit of quantitation (LLOQ) was 0.1microg/mL with a relative standard deviation of less than 15%. The devised method provides an accurate, precise and sensitive tool for determining etodolac levels in plasma.