Determination of vandetanib in human plasma and cerebrospinal fluid by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS)

A sensitive and precise LC-ESI-MS/MS method for the determination of vandetanib (ZD6474) in human plasma and cerebrospinal fluid (CSF) using [(13)C,d(3)]-ZD6474 as an internal standard (ISTD) was developed and validated. Sample preparation consisted of a simple liquid-liquid extraction with tert-butyl methyl ether containing 0.1% or 0.5% ammonium hydroxide. ZD6474 and ISTD were separated on a Kinetex C18 column (2.6 μm, 50 mm × 2.1 mm) at ambient temperature with an isocratic mobile phase (acetonitrile/10mM ammonium formate=50/50, v/v, at pH 5.0) delivered at 0.11 mL/min. The retention time of both compounds was at 1.60 min in a runtime of three min. Detection was achieved by an API-3200 LC-MS/MS system, monitoring m/z 475.1/112.1 and m/z 479.1/116.2 for vandetanib and ISTD, respectively. The method was linear in the range of 0.25-50 ng/mL (R(2) ≥ 0.990) for the CSF curve and from 1.0 to 3000 ng/mL (R(2) ≥ 0.992) for the plasma curve. The mean recovery for vandetanib was 80%. Within-day and between-day precisions were ≤ 8.8% and ≤ 5.9% for CSF and plasma, respectively. Within-day and between-day accuracies ranged from 95.0 to 98.5% for CSF, and from 104.0 to 108.5% for plasma. Analysis of plasma from six different sources showed no matrix effect for vandetanib (MF=0.98, %CV ≤ 4.97, n=6). This method was successfully applied to the analysis of pharmacokinetic samples from children with brain tumors treated with oral vandetanib.     

Profiling of Arabidopsis secondary metabolites by capillary liquid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry

Large-scale metabolic profiling is expected to develop into an integral part of functional genomics and systems biology. The metabolome of a cell or an organism is chemically highly complex. Therefore, comprehensive biochemical phenotyping requires a multitude of analytical techniques. Here, we describe a profiling approach that combines separation by capillary liquid chromatography with the high resolution, high sensitivity, and high mass accuracy of quadrupole time-of-flight mass spectrometry. About 2000 different mass signals can be detected in extracts of Arabidopsis roots and leaves. Many of these originate from Arabidopsis secondary metabolites. Detection based on retention times and exact masses is robust and reproducible. The dynamic range is sufficient for the quantification of metabolites. Assessment of the reproducibility of the analysis showed that biological variability exceeds technical variability. Tools were optimized or established for the automatic data deconvolution and data processing. Subtle differences between samples can be detected as tested with the chalcone synthase deficient tt4 mutant. The accuracy of time-of-flight mass analysis allows to calculate elemental compositions and to tentatively identify metabolites. In-source fragmentation and tandem mass spectrometry can be used to gain structural information. This approach has the potential to significantly contribute to establishing the metabolome of Arabidopsis and other model systems. The principles of separation and mass analysis of this technique, together with its sensitivity and resolving power, greatly expand the range of metabolic profiling.     

Determination of Astragaloside IV in rat plasma by liquid chromatography electrospray ionization mass spectrometry

Astragaloside IV (AGS-IV) is an active constituent of Radix Astragali used in many Traditional Chinese Medicines. This paper describes a sensitive and specific assay for the quantitation of AGS-IV in rat plasma. After solid phase extraction (SPE), samples were analyzed by liquid chromatography electrospray ionization mass spectrometry using a reversed-phase C18 column. The assay was linear in the range 1-500 ng/ml with a limit of detection of 0.5 ng/ml. The recovery was 92.5% and within-day and between-day precision were 3.7-6.0 and 2.8-9.8%, respectively. The assay was applied to a pharmacokinetic study in rat after a single oral dose. The drug was rapidly absorbed and subsequently eliminated according to a biphasic concentration-time curve.     

Determination of procarbazine in human plasma by liquid chromatography with electrospray ionization mass spectrometry

Procarbazine is a cytotoxic chemotherapeutic agent used in the treatment of lymphomas and brain tumors. Its pharmacokinetic behavior remains poorly understood even though more than 30 years have elapsed since the drug was approved for clinical use. To characterize the pharmacokinetics of procarbazine in brain cancer patients during a phase I trial, a method for determining the drug in human plasma by reversed-phase high-performance liquid chromatography (HPLC) with electrospray ionization mass spectrometry (ESI-MS) was developed and thoroughly validated. Plasma samples were prepared for analysis by precipitating proteins with trichloroacetic acid and washing the protein-free supernatant with methyl tert-butyl ether to remove excess acid. The solution was separated on a Luna C-18 analytical column using methanol-25 mM ammonium acetate buffer, pH 5.1 (22:78, v/v) as the mobile phase at 1.0 ml/min. A single-quadrupole mass spectrometer with an electrospray interface was operated in the selected-ion monitoring mode to detect the [M+H](+) ions at m/z 222.2 for procarbazine and at m/z 192.1 for the internal standard (3-dimethylamino-2-methylpropiophenone). Procarbazine and the internal standard eluted as sharp, symmetrical peaks with retention times (mean+/-S.D.) of 6.3+/-0.1 and 9.9+/-0.3 min, respectively. Calibration curves of procarbazine hydrochloride in human plasma at concentrations ranging from 0.5 to 50 ng/ml exhibited excellent linearity. The mean absolute recovery of the drug from plasma was 102.9+/-1.0%. Using a sample volume of 150 microl, procarbazine was determined at the 0.5 ng/ml (1.9 nM) lower limit of quantitation with a mean accuracy of 105.2% and an interday precision of 3.60% R.S.D. on 11 different days over 5 weeks. During this same time interval, the between-day accuracy for determining quality control solutions of the drug in plasma at concentrations of 2.0, 15 and 40 ng/ml ranged from 97.5 to 98.2% (mean+/-S.D., 97.9+/-0.4%) and the precision was 3.8-6.2% (mean+/-S.D., 5.1+/-1.2%). Stability characteristics of the drug were thoroughly evaluated to establish appropriate conditions to process, store and prepare clinical specimens for chromatographic analysis without inducing significant chemical degradation. The sensitivity achieved with this assay permitted the plasma concentration-time profile of the parent drug to be accurately defined following oral administration of standard doses to brain cancer patients.     

Separation and identification of corticosterone metabolites by liquid chromatography–electrospray ionization mass spectrometry

High-performance liquid chromatography coupled to atmospheric pressure ionization–electrospray ionization mass spectrometry (API–ESI–MS) was investigated for the analysis of corticosterone metabolites; their characterization was obtained by combining the separation on Zorbax Eclipse XDB C₁₈ column (eluted with a methanol–water–acetic acid gradient) with identification using positive ion mode API–ESI–MS and selected ion analysis. The applicability of this method was verified by monitoring the activity of steroid converting enzymes (20β-hydroxysteroid dehydrogenase and 11β-hydroxysteroid dehydrogenase) in avian intestines.     

Quantitative determination of atractylenolide III in rat plasma by liquid chromatography electrospray ionization mass spectrometry

Atractylenolide III is a major active component in Atractylodes macrocephala. This paper describes a simple, rapid, specific and sensitive method for the quantification of atractylenolide III in rat plasma using a liquid-liquid extraction procedure followed by liquid chromatography mass spectrometric (LC-MS) analysis. A Kromasil 3.5 microm C(18) column (150 mm x 2.00 mm) was used as the analytical column. Linear detection responses were obtained for atractylenolide III concentration ranging from 5 to 500 ng L(-1). The precision and accuracy data, based on intra-day and inter-day variations over 5 days were within 10.29%. The lower limit of quantitation for atractylenolide III was 5 ng mL(-1), using 0.1 mL plasma for extraction and its recoveries were greater than 85% at the low, medium and high concentrations. The method has been successfully applied to a pharmacokinetic study in rats after an oral administration of atractylenolide III with a dose of 20.0 mg kg(-1). With the lower limits of quantification at 5 ng mL(-1) for atractylenolide III, this method was proved to be sensitive enough for the pharmacokinetics study of atractylenolide III.     

Quantification of ceramide species in biological samples by liquid chromatography electrospray ionization tandem mass spectrometry

We present an optimized and validated liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method for the simultaneous measurement of concentrations of different ceramide species in biological samples. The method of analysis of tissue samples is based on Bligh and Dyer extraction, reverse-phase high-performance liquid chromatography separation, and multiple reaction monitoring of ceramides. Preparation of plasma samples also requires isolation of sphingolipids by silica gel column chromatography prior to LC-ESI-MS/MS analysis. The limits of quantification were in a range of 0.01-0.50 ng/ml for distinct ceramides. The method was reliable for inter- and intraassay precision, accuracy, and linearity. Recoveries of ceramide subspecies from human plasma, rat liver, and muscle tissue were 78 to 91%, 70 to 99%, and 71 to 95%, respectively. The separation and quantification of several endogenous long-chain and very-long-chain ceramides using two nonphysiological odd chain ceramide (C17 and C25) internal standards was achieved within a single 21-min chromatographic run. The technique was applied to quantify distinct ceramide species in different rat tissues (muscle, liver, and heart) and in human plasma. Using this analytical technique, we demonstrated that a clinical exercise training intervention reduces the levels of ceramides in plasma of obese adults. This technique could be extended for quantification of other ceramides and sphingolipids with no significant modification.     

Characterization of a noncovalent lipocalin complex by liquid chromatography/electrospray ionization mass spectrometry.

Nanoscale liquid chromatography coupled to electrospray ionization mass spectrometry was used to identify the nature of the ligand that binds noncovalently to siderocalin (lipocalin 2). The folded state siderocalin-ligand complex was separated from free, unfolded siderocalin using reversed phase chromatography, and the molecular weight of the siderocalin ligand was then determined from the deconvoluted molecular weights of the complex and of the free protein. The ligand was identified as dihydroxybenzoyl-serine, a breakdown product of enterobactin, an iron-chelating compound ("siderophore") synthesized in bacteria. These results demonstrate that, in some cases, electrostatic noncovalent protein complexes can survive the denaturing conditions of reversed phase liquid chromatography and the gas phase transfer occurring during electrospray ionization.     

Determination of lapatinib (GW572016) in human plasma by liquid chromatography electrospray tandem mass spectrometry (LC-ESI-MS/MS)

A sensitive method for the determination of lapatinib (GW572016) in human plasma was developed using high-performance liquid chromatographic separation with tandem mass spectrometric detection. Plasma samples (100 microL) were prepared using solid phase extraction (SPE) columns, and 6.0 microL of the reconstituted eluate was injected onto a Phenomenex CuroSil-PFP 3 mu analytical column (50 mm x 2.0mm) with an isocratic mobile phase. Analytes were detected with a PE SCIEX API-365 LC-MS/MS system at unit (Q1) and low (Q3) resolution in positive multiple reaction monitoring mode (m/z 581 (precursor ion) to m/z 364 (product ion) for lapatinib). The mean recovery for lapatinib was 75% with a lower limit of quantification of 15 ng/mL (S/N=11.3, CV< or =14%). This method was validated over a linear range of 100-10,000 ng/mL, and results from a 5-day validation study demonstrated good within-day and between-day precision and accuracy. This method has been used to measure plasma lapatinib concentrations in a Phase I study in children with cancer.     

Simultaneous measurement of tryptophan and related compounds by liquid chromatography/electrospray ionization tandem mass spectrometry

We have expanded a liquid chromatographic-tandem mass spectrometric method that measures 3-hydroxykynurenine and 3-hydroxyanthranilic acid in addition to tryptophan and kynurenine both intra- and extracellularly. After reversed phase HPLC separation, the compounds were detected in the MS positive multiple reaction monitoring mode. We found a good linear response for each tryptophan metabolite. The lower limit of quantification for each compound ranged from 0.01 to 0.1 microM. The extraction efficiencies from spiked cell samples and culture medium ranged between 83 and 111% and the overall coefficient of variation of analyses was less than 7%. Using our method, we found tryptophan metabolites in the cells and the culture medium of LN229 human glioma cells were stimulated by interferon-gamma, a known inducer of indoleamine 2,3-dioxygenase. The intracellular concentrations of kynurenine, 3-hydroxykynurenine and 3-hydroxyanthranilic acid were higher than those in the medium. This is the first report of a method for the simultaneous determination of tryptophan and its metabolic products both intra- and extracellularly.     

Determination of endocannabinoids in nematodes and human brain tissue by liquid chromatography electrospray ionization tandem mass spectrometry

A simple and highly sensitive liquid chromatography/tandem mass spectrometric (LC/MS/MS) method was developed to compare endogenous cannabinoid levels in nematodes and in brains of rats and humans, with and without prior exposure to ethanol. After liquid-liquid extraction of the lipid fraction from homogenized samples, a reversed-phase sub 2 μm column was used for separating analytes with an isocratic mobile phase. Deuterated internal standards were used in the analysis, and detection was made by triple quadrupole mass spectrometer with multiple reaction monitoring (MRM). Ionization was performed with positive electrospray ionization (ESI). The nematode Caenorhabditis elegans fat-3 mutant, that lacks the necessary enzyme to produce arachidonic acid, the biologic precursor to 2-arachidonoyl glycerol and anandamide, was used as an analyte-free surrogate material for selectivity and calibration studies. The matrix effect was further investigated by in-source multiple reaction monitoring (IS-MRM) and standard addition studies. Selectivity studies demonstrated that the method was free from matrix effects. Good accuracy and precision were obtained for concentrations within the calibration range of 0.4-70 nM and 40-11,000 nM for monitored N-acylethanolamides (NAEs) and acyl glycerols, respectively.     

Analysis of benzphetamine and its metabolites in rat urine by liquid chromatography–electrospray ionization mass spectrometry

An analytical method to identify and determine benzphetamine (BMA) and its five metabolites in urine was developed by liquid chromatography–electrospray ionization mass spectrometry (LC–ESI–MS) using the solid-phase extraction column Bond Elut SCX. Deuterium-labeled compounds, used as internal standards, were separated chromatographically from each corresponding unlabeled compound in the alkaline mobile phase with an alkaline-resistant ODS column. This method was applied to the identification and determination of BMA and its metabolites in rat urine collected after oral administration of BMA. Under the selected ion monitoring mode, the limit of quantitation (signal-to-noise ratio 10) for BMA, N-benzylamphetamine (BAM), p-hydroxybenzphetamine (p-HBMA), p-hydroxy-N-benzylamphetamine (p-HBAM), methamphetamine (MA) and amphetamine (AM) was 700 pg, 300 pg, 500 pg, 1.4 ng, 6 ng and 10 ng in 1 ml of urine, respectively. This analytical method for p-HBMA, structurally closer to the unchanged drug of all the metabolites, was very sensitive, making this a viable metabolite for discriminating the ingestion of BMA longer than the parent drug or other metabolites in rat.     

Quantitative analysis of human serum corticosterone by high-performance liquid chromatography coupled to electrospray ionization mass spectrometry

An original method based upon high-performance liquid chromatography coupled to electrospray ionization mass spectrometry has been developed for corticosterone (B) quantification in human serum. After extraction by diethyl ether using triamcinolone (T) as an internal standard, solutes are separated on a C₁₈ microbore column (250×1.0 mm, I.D.), using acetonitrile–water–formic acid (40:59.9:0.1, v/v/v) as the mobile phase (flow-rate 40 μl/min). Detection is performed on an API 1 single quadrupole mass spectrometer equipped with a ESI interface and operated in positive ionization mode. Corticosterone quantifications were realized by computing peak area ratios (B/T) of the serum extracts analyzed in SIM mode (m/z 347 and m/z 395 for B and T, respectively), and comparing them with the calibration curve (r=0.998).     

Quantitative determination of saccharide surfactants in protein samples by liquid chromatography coupled to electrospray ionization mass spectrometry

A direct and highly selective method, combining liquid chromatography (LC) with electrospray ionization mass spectrometry (ESI-MS), has been developed for quantifying saccharide surfactants. Saccharide surfactants, such as n-octyl-beta-d-glucopyranoside (NOG), are widely used to solubilize or refold membrane-bound or lipophilic proteins. In the present study, we have developed an LC-MS method to quantify NOG in protein samples. Protein-bound NOG was completely dissociated from proteins by reversed-phase LC, allowing the total amount of saccharide surfactant in protein samples to be quantified by MS. A chemical analog of NOG was used as an internal standard for improving the reproducibility of the method. Linearity was found in the range of 10 microg/mL-1.0 mg/mL NOG concentrations. Seven major surfactant oligomeric ions were detected under the ionization conditions applied and their relative abundance was essentially unchanged over the range of 0.05-1.0 mg/mL NOG concentrations. Consequently, ions with characteristic mass-to-charge ratios could be used for quantification of NOG. Analytical accuracy of the method was examined by determining the amounts of NOG recovered from apolipoprotein A-I and myoglobin samples spiked with NOG.     

Quantitative determination of mitiglinide in human plasma by ultra-performance liquid chromatography/electrospray ionization tandem mass spectrometry

A selective, rapid and sensitive ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) method was developed for the quantitative determination of mitiglinide in human plasma. With nateglinide as internal standard, sample pretreatment involved a one-step extraction with diethyl ether of 0.2 mL plasma. The separation was performed on an ACQUITY UPLCtrade mark BEH C(18) column (50 mm x 2.1 mm, i.d., 1.7 microm) with the mobile phase consisting of methanol and 10 mmol/L ammonium acetate (65:35, v/v) at a flow rate of 0.25 mL/min. The detection was carried out by means of electrospray ionization mass spectrometry in positive ion mode with multiple reaction monitoring (MRM). Linear calibration curves were obtained in the concentration range of 1.080-5400 ng/mL, with a lower limit of quantification of 1.080 ng/mL. The intra- and inter-day precision (RSD) values were below 15% and accuracy (RE) was from -3.5% to 7.3% at all QC levels. The method was fully validated and successfully applied to a clinical pharmacokinetic study of mitiglinide in 10 healthy volunteers following oral administration.     

Ultraperformance liquid chromatography with electrospray ionization ion trap mass spectrometry for chondroitin disaccharide analysis

Chondroitin sulfate (CS) has an important role in cell division, in the central nervous system, and in joint-related pathologies such as osteoarthritis. Due to the complex chemical structure and biological importance of CS, simple, sensitive, high resolution, and robust analytical methods are needed for the analysis of CS disaccharides and oligosaccharides. An ion-pairing, reversed-phase, ultraperformance liquid chromatography (IPRP-UPLC) separation, coupled to electrospray ionization mass spectrometry with an ion trap mass analyzer, was applied for the analyses of CS-derived disaccharides. UPLC separation technology uses small particle diameter, short column length, and elevated column temperature to obtain high resolution and sensitivity. Hexylamine (15 mM) was selected as the optimal ion-pairing reagent.     

Determination of LSD and N-demethyl-LSD in urine by liquid chromatography coupled to electrospray ionization mass spectrometry

A sensitive and highly specific method for the determination of LSD and N-demethyl-LSD in urine, using combined liquid chromatography and mass spectrometry (LC-MS) with electrospray ionization, has been developed. Extrelut-3 extraction cartridges were used for a basic sample clean-up. Elution was obtained by toluene-diethyl ether (60:40, v/v). A Nucleosil C₁₈ (150×1 mm I.D.) reversed-phase column was used for the chromatographic separation, together with a mixture of 2 mM ammonium formate buffer (pH 3) and acetonitrile (70:30, v/v) as mobile phase. Recoveries were 93 and 80%, detection limits 0.025 and 0.035 ng/ml for LSD and N-demethyl-LSD, respectively. Intra-assay precision, studied at four concentrations, was better than 9% at the ng/ml range and better than 14% at 0.10 ng/ml for both compounds. Limits of quantitation were 0.05 and 0.10 ng/ml for LSD and N-demethyl-LSD, respectively. Reproducibility was good and linearity excellent for LSD in the range from 0.05 to 20 ng/ml (r>0.9999, N=7).     

Determination of lidocaine and its two N-desethylated metabolites in dog and horse plasma by high-performance liquid chromatography combined with electrospray ionization tandem mass spectrometry

A sensitive method for the quantification of lidocaine and its metabolites, monoethylglycinexylidide (MEGX) and glycinexylidide (GX), in animal plasma using high-performance liquid chromatography combined with electrospray ionization mass spectrometry is described. The sample preparation includes a liquid-liquid extraction with methyl tert-butylmethyl ether after addition of 2M sodium hydroxide. Ethylmethylglycinexylidide (EMGX) is used as an internal standard. For chromatographic separation, an ODS Hypersil column was used. Isocratic elution was achieved with 0.01 M ammonium acetate and acetonitrile as mobile phases. Good linearity was observed in the range of 2.5-1000 ng ml(-1) for lidocaine in both dog and horse plasma. For MEGX, linear calibration curves were obtained in the range of 5-1000 ng ml(-1) and 20-1000 ng ml(-1) for dog and horse plasma, respectively. In dog and horse plasma good linearity was observed in the range of 200-1500 ng ml(-1) for GX. The limit of quantification (LOQ) in dog plasma for lidocaine, MEGX and GX was set at 2.5 ng ml(-1), 20 ng ml(-1) and 200 ng ml(-1), respectively. For horse plasma a limit of quantification of 2.5 ng ml(-1), 5 ng ml(-1) and 200 ng ml(-1) was achieved for lidocaine, MEGX and GX, respectively. In dog plasma, the limit of detection (LOD) was found to be 0.8 ng ml(-1), 2.3 ng ml(-1) and 55 ng ml(-1) for lidocaine, MEGX and GX, respectively. In horse plasma the LOD's found for lidocaine, MEGX and GX, were 1.1 ng ml(-1), 0.5 ng ml(-1) and 13 ng ml(-1), respectively. The method was shown to be of use in pharmacokinetic studies after application of a transdermal patch in dogs and after an intravenous infusion in horses.     

Determination of ritodrine in human plasma by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A simple and sensitive HPLC/MS/MS method was developed and evaluated to determine the concentration of ritodrine (RTD) in human plasma. Liquid-liquid extraction with ethyl acetate was employed as the sample preparation method. The structural analogue salbutamol was selected as the internal standard (IS). The liquid chromatography was performed on a Hanbon Sci. & Tech. Lichrospher CN (150 mm x 4.6 mm, i.d., 5 microm) column (Hanbon, China) at 20 degrees C. A mixture of 0.03% acetic acid and methanol (50:50, v/v) was used as isocratic mobile phase to give the retention time 3.60 min for ritodrine and 2.94 min for salbutamol. Selected reaction monitoring (SRM) in positive ionization mode was employed for mass detection. The calibration functions were linear over the concentration range 0.39-100 ng mL(-1). The intra- and inter-day precision of the method were less than 15%. The lower limit of quantification was 0.39 ng mL(-1). The method had been found to be suitable for application to a pharmacokinetic study after oral administration of 20mg ritodrine hydrochloride tablet to 18 healthy female volunteers. The half-life is 2.54+/-0.67 h.