Determination of Suramin in Plasma and Urine by Ion-Paired Reverse-Phase High-Performance Liquid Chromatography

Abstract

A high-pressure liquid chromatographic (HPLC) assay has been developed for the quantification of the anti-viral drug suramin (SUR) in human plasma and urine. This assay is simple and accurate, using an isocratic mobile phase in a reverse-phase ion-pairing mode.

This assay was developed for the determination of high levels of suramin in the urine and plasma of patients being treated for the acquired immunodefficiency Syndrome (AIDS). The limit of detection of this assay was 0.25 μg/mL. Congo red (CR) was used as the internal standard with a retention time of 5.9 min, while suramin had a retention time of 13 min.     

Development and validation of a liquid chromatography/tandem mass spectrometry assay for the quantification of methyl protodioscin in rat plasma: application to a pharmacokinetic study

A high performance liquid chromatography/tandem mass spectrometry assay was first developed and validated for the quantification of methyl protodioscin (MPD), a natural furostanol saponin with distinct antitumor activity, in rat plasma with 17alpha-ethinylestradiol as internal standard (IS). Methanol-mediated protein precipitation was employed for plasma sample pretreatment. The separation was achieved on a C(18) column (150 x 4.6 mm, i.d., 5 microm) by isocratic elution with methanol-water (72:28, v/v) as mobile phase at a flow rate of 1.0 mL/min. Ion acquisition was performed in selective reaction monitoring positive mode by monitoring the transition of m/z 1085.7 --> 1053.7 for MPD, and in selective ion monitoring negative mode by monitoring the deprotonated ion m/z 295.5 for IS. The assay was linear over the concentration range of 2.024-270.0 microg/mL with 2.024 microg/mL as the lower limit of quantification. It was specific, accurate, precise and reproducible with intra- and inter-run RSD <8.3% and RE between -11.5 and 12.8%. The assay was successfully applied to a preclinical pharmacokinetic study after an intravenous dose of 40 mg/kg MPD to rats.     

Analysis of organic acid markers relevant to inherited metabolic diseases by ultra-performance liquid chromatography/tandem mass spectrometry as benzofurazan derivatives

We describe a new approach applicable to the determination of organic acids that serve as diagnostic markers for several inherited metabolic disorders. We utilized liquid chromatography/tandem mass spectrometry for analysis of organic acid derivatives of a recently described benzofurazan reagent. The derivatization step was necessary to obtain organic acid derivatives suitable for analysis by reversed-phase liquid chromatography with high ionization efficiency for mass spectrometry in the positive-ion mode. In this work, a group of related dicarboxylic acid markers containing five or six carbon atoms were analyzed and validation was performed for glutaric and 3-hydroxyglutaric acids, the specific markers for glutaric acidemia type 1. Derivatization was achieved by reacting untreated urine with the derivatization reagent under mild conditions. The reaction mixture was analyzed on a C18 ultra-performance liquid chromatography (UPLC) column (50x2.1 mm, 1.7 microm) and detected in the multiple reaction monitoring mode in 5 min. Calibration curves were linear up to at least 1000 microM with detection limits for glutaric and 3-hydroxyglutaric acids of 0.025 and 0.02 microM, respectively (signal-to-noise ratio of 3). Intra-day (n=11) and inter-day (n=6) coefficients of variation were better than 11.2%. The assay was successfully applied to control (n=134) and glutaric acidemia type 1 (n=55) urine samples.     

LC–MS–MS Determination of Troxerutin in Plasma and Its Application to a Pharmacokinetic Study

A highly sensitive liquid chromatography–tandem mass spectrometry (LC–MS–MS) method for the determination of troxerutin in human plasma using tramadol as internal standard (IS) has been developed and validated. Sample preparation involved liquid–liquid extraction with ethyl acetate–isopropanol (95:5, v/v). The analyte and IS were separated by RP–LC with gradient elution using 10 mM ammonium acetate containing 0.1% formic acid and methanol at a flow rate of 0.9 mL min^?1. LC–MS–MS in the positive ion mode employed multiple reaction monitoring of the transitions at m/z 743.2→435.3 and m/z 264.1→58.0 for troxerutin and IS, respectively. The assay was linear in the concentration range 0.01–10 ng mL^?1 with precision and accuracy within assay variability limits as per FDA guidelines. The assay was successfully applied to a pharmacokinetic study involving oral administration of 300 mg troxerutin to eight healthy Chinese volunteers.     

Receptor-based high-throughput screening and identification of estrogens in dietary supplements using bioaffinity liquid-chromatography ion mobility mass spectrometry

A high-throughput bioaffinity liquid chromatography-mass spectrometry (BioMS) approach was developed and applied for the screening and identification of recombinant human estrogen receptor α (ERα) ligands in dietary supplements. For screening, a semi-automated mass spectrometric ligand binding assay was developed applying ¹³C_2, ¹⁵?N-tamoxifen as non-radioactive label and fast ultra-high-performance–liquid chromatography–electrospray ionisation–triple-quadrupole-MS (UPLC-QqQ-MS), operated in the single reaction monitoring mode, as a readout system. Binding of the label to ERα-coated paramagnetic microbeads was inhibited by competing estrogens in the sample extract yielding decreased levels of the label in UPLC-QqQ-MS. The label showed high ionisation efficiency in positive electrospray ionisation (ESI) mode, so the developed BioMS approach is able to screen for estrogens in dietary supplements despite their poor ionisation efficiency in both positive and negative ESI modes. The assay was performed in a 96-well plate, and all these wells could be measured within 3 h. Estrogens in suspect extracts were identified by full-scan accurate mass and collision-cross section (CCS) values from a UPLC-ion mobility-Q-time-of-flight-MS (UPLC-IM-Q-ToF-MS) equipped with a novel atmospheric pressure ionisation source. Thanks to the novel ion source, this instrument provided picogram sensitivity for estrogens in the negative ion mode and an additional identification point (experimental CCS values) next to retention time, accurate mass and tandem mass spectrometry data. The developed combination of bioaffinity screening with UPLC-QqQ-MS and identification with UPLC-IM-Q-ToF-MS provides an extremely powerful analytical tool for early warning of ERα bioactive compounds in dietary supplements as demonstrated by analysis of selected dietary supplements in which different estrogens were identified.

HPLC Analysis of Methoxamine in Rabbit Plasma and Pharmaceutical Formulations

Abstract

A rapid, selective and simple high performance liquid chromatographic (HPLC) assay for methoxamine HC1 has been developed. The analytical procedure involved the use of internal standardization method (1-norephedrine). It has been applied for the qualitative and quantitative determination of methoxamine HC1 in rabbit plasma and in pharmaceutical formulations using an adsorption column in an isocratic mode, with resulting relative standard deviations of 1.7% and 3.3%, respectively. The applicability of the assay procedure to pharmacokinetic studies was demonstrated. Detection limits were as low as 15ng for a 30 ul injection and the determination time was less than 6 minutes.     

Validated assay for quantification of oxcarbazepine and its active dihydro metabolite 10-hydroxycarbazepine in plasma by atmospheric pressure chemical ionization liquid chromatography/mass spectrometry

Oxcarbazepine (OX), a new antiepileptic, may lead to unwanted side-effects or even life-threatening intoxications after overdose. Therefore, a validated liquid chromatographic/mass spectrometric (LC/MS) assay was developed for the quantification of OX and its pharmacologically active dihydro metabolite (dihydrooxcarbazepine, DOX, often named 10-hydroxycarbazepine). OX and DOX were extracted from plasma by the authors' standard liquid/liquid extraction and were separated on a Merck LiChroCART column with Superspher 60 RP Select B as the stationary phase. Gradient elution was performed using aqueous ammonium formate and acetonitrile. The compounds were quantified in the selected-ion monitoring mode using atmospheric pressure chemical ionization electrospray LC/MS. The assay was fully validated. It was found to be selective. The calibration curves were linear from 0.1 to 50 mg l(-1) for OX and DOX. Limits of quantification were 0.1 mg l(-1) for OX and DOX. The absolute recoveries were between 60 and 86%. The accuracy and precision data were within the required limits. The analytes in frozen plasma samples were stable for at least 1 month. The method was successfully applied to several authentic plasma samples from patients treated or intoxicated with OX. The measured therapeutic plasma levels ranged from 1 to 2 mg l(-1) for OX and from 10 to 40 mg l(-1) for DOX. The validated LC/MS assay proved to be appropriate for quantification of OX and DOX in plasma for clinical toxicology and therapeutic drug monitoring purposes. The assay is part of a general analysis procedure for the isolation, separation and quantification of various drugs and for their full-scan screening and identification.     

Development and validation of a highly sensitive LC-MS/MS-ESI method for the determination of nobiletin in rat plasma: application to a pharmacokinetic study

A highly sensitive, rapid assay method has been developed and validated for the estimation of nobiletin in rat plasma with liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive‐ion mode. The assay procedure involves extraction of nobiletin and citalopram (internal standard, IS) from rat plasma with liquid–liquid extraction. Chromatographic separation wa s achieved using an isocratic mobile phase (0.2% formic acid–acetonitrile, 20:80, v/v) at a flow rate of 0.6 mL/min on an Atlantis dC₁₈ column (maintained at 40 ± 1 °C) with a total run time of 2.0 min. The MS/MS ion transitions monitored were 403.2 → 373.0 for nobiletin and 325.2 → 109.0 for IS. Method validation was performed as per Food and Drug Administration guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 0.05 ng/mL and the linearity range extended from 0.05 to 51.98 ng/mL. The intra‐ and inter‐day precisions were in the range of 1.96–14.3 and 6.21–12.1, respectively. Copyright © 2012 John Wiley & Sons, Ltd.     

A highly sensitive and efficient UPLC‐MS/MS assay for rapid analysis of tedizolid (a novel oxazolidinone antibiotic) in plasma sample

Tedizolid (TDZ) is a novel oxazolidinone class antibiotic, indicated for the treatment of acute bacterial skin and skin structure infections in adults. In this study a highly sensitive UPLC‐MS/MS assay was developed and validated for the determination of TDZ in rat plasma using rivaroxaban as an internal standard (IS). Both TDZ and IS were separated on an Acquity UPLC BEH? C₁₈ column using an isocratic mobile phase comprising of acetonitrile–20 mm ammonium acetate (85:15, v/v), eluted at 0.3 mL/min flow rate. The plasma sample was processed by liquid liquid extraction technique using ethyl acetate as an extracting agent. The analyte and IS were detected in positive mode using electrospray ionization source. The precursor to product ion transitions at m/z 371.09 > 343.10 for TDZ and m/z 435.97 > 144.94 for IS were used for the quantification in multiple reaction monitoring mode. The calibration curve was linear in the concentration range of 0.74–1500 ng/mL and the lower limit of quantification was 0.74 ng/mL only. The developed assay was validated following standard guidelines for bioanalytical method validation (US Food and Drug Administration) and all the validation results were within the acceptable limits. The developed assay was successfully applied into a pharmacokinetic study in rats. Copyright © 2016 John Wiley & Sons, Ltd.     

Highly sensitive LC‐MS/MS method for determination of galantamine in rat plasma: application to pharmacokinetic studies in rats

A rapid and highly sensitive assay method has been developed and validated for the estimation of galantamine (GLM) in rat plasma using liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive‐ion mode. The assay procedure involves a simple liquid–liquid extraction of GLM and phenacetin (internal standard, IS) from rat plasma using acetonitrile. Chromatographic separation was achieved with 0.2% formic acid:acetonitrile (50:50, v/v) at a flow rate of 0.60 mL/min on an Atlantis dC₁₈ column with a total run time 2.5 min. The MS/MS ion transitions monitored were 288.10 → 213.10 for GLM and 180.10 → 110.10 for IS. Method validation was performed as per United States Food and Drug Administration guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 0.12 ng/mL and linearity was observed from 0.12 to 525 ng/mL. The intra‐ and inter‐day precision were in the ranges of 4.73–11.7 and 5.83–8.64%, respectively. This novel method has been applied to a pharmacokinetic study in rats. Copyright © 2014 John Wiley & Sons, Ltd.     

A strategy for high-throughput analysis of levosimendan and its metabolites in human plasma samples using sequential negative and positive ionization liquid chromatography/tandem mass spectrometric detection

Levosimendan (Simdax) is an approved drug in approximately 40 countries and currently in phase III clinical studies in the USA and Europe. An accurate, high-throughput and rugged assay is critical to support these clinical trials. Due to the mechanism of drug metabolism, the drug and its active metabolites often have significant differences in their chemical properties. In order to achieve high assay throughput and low sample volumes, a single bioanalytical assay for the drug and its metabolites is preferred. However, this need may prevent the optimization of both high-performance liquid chromatography (HPLC) and mass spectrometric ionization conditions. The chemical properties of levosimendan are significantly different from those of its two active metabolites, OR-1855 and OR-1896. Here, we present a novel strategy for high-throughput analysis of levosimendan and its metabolites. A 96-well liquid/liquid extraction procedure was developed for sample preparation. A single liquid chromatography/tandem mass spectrometry (LC/MS/MS) system with two separate mobile phases, shared backwash solvent and conditioning solvent, was developed to perform sequential LC separation for levosimendan and the metabolites. Levosimendan was eluted by 5 mM ammonium acetate in 33.3% acetonitrile and detected using negative ionization mode MS/MS monitoring. The metabolites were eluted by 5 mM ammonium acetate and 0.2% acetic acid in 20% acetonitrile and detected with positive ionization mode MS/MS monitoring. The method has been demonstrated to have excellent precision and accuracy, with high assay ruggedness during method validation and clinical sample analysis. The linear dynamic ranges were approximately 200-50,000 pg/mL for levosimendan and approximately 500-130,000 pg/mL for both metabolites. The coefficient of determination (r2) for all analytes was greater than 0.9985. The intra-assay %CVs for QC samples were from 0.9% to 2.0% for levosimendan, 0.9% to 3.2% for OR-1855, and 0.4% to 4.9% for OR-1896. The inter-assay %CVs for QC samples were from 1.2% to 1.8% for levosimendan, 1.3% to 2.7% for OR-1855, and 1.4% to 3.4% for OR-1896. The mean % biases for QC samples were from 1.5% to 5.5% for levosimendan, -1.4% to 2.6% for OR-1855, and -0.3% to 4.5% for OR-1896. By using a single extraction approach coupled with sequential LC/MS/MS analysis for levosimendan and its metabolites, the assay maintained high throughput and low sample volume usage.     

Hydrophilic interaction liquid chromatography/tandem mass spectrometry for the simultaneous determination of dasatinib,imatinib and nilotinib in mouse plasma

Hydrophilic interaction liquid chromatography (HILIC) interfaced with atmospheric pressure ionization (API) sources and a tandem mass spectrometer (MS/MS) was developed for the simultaneous determination of dasatinib, imatinib and nilotinib in mouse plasma samples. The retention profiles of all analytes on several silica stationary phases under HILIC conditions were explored. The influences of experimental factors such as the compositions of mobile phases on the chromatographic performance and the ionization efficiency of all analytes in positive ion mode were investigated. The applicability of the proposed HILIC/MS/MS approach following a protein precipitation procedure for the quantitative determination of dasatinib, imatinib and nilotinib at low nano‐mole levels was examined with respect to assay specificity and linearity. The analytical results obtained by various HILIC/MS/MS approaches were found to be in good agreement with those obtained by reversed‐phase liquid chromatography/tandem mass spectrometry (RPLC/MS/MS) methods in terms of assay sample throughputs, sensitivity and accuracy. Furthermore, the potential of matrix ionization suppression on the proposed HILIC/MS/MS systems was investigated using the post‐column infusion technique. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of long-acting release octreotide, an octapeptide analogue of somatostation, in human plasma by liquid chromatography/tandem mass spectrometry

A sensitive and selective method for the determination of long-acting released octreotide in human plasma has been developed based on liquid chromatography/tandem mass spectrometry (LC/MS/MS). Octreotide and the internal standard, triptorelin, were precipitated from the matrix, washed with dichloromethane and subsequently separated by reversed-phase high-performance liquid chromatography (HPLC) employing a 1% formic acid/methanol gradient system. Detection was by electrospray ionization mass spectrometry in the positive ion mode using multiple-reaction monitoring. The assay was linear in the concentration range 0.0500-50.0 ng/mL with intra- and inter-day precision (as relative standard deviation) of <2.95% and <8.37%, respectively. The limit of detection was 0.0200 ng/mL. The method was applied to a pharmacokinetic study of long-acting released octreotide in healthy volunteers given an intramuscular injection containing 20 mg octreotide.     

Quantitative determination of lipase activity by liquid chromatography-mass spectrometry

We developed a novel in vitro lipase assay based on the quantitation of fatty acids by liquid chromatography-mass spectrometry. Oleic acids enzymatically released from triolein substrates were isolated from the reaction mixture by reverse-phase chromatography, ionized in negative mode electrospray mass spectrometry and quantitated with the aid of [(13)C]-oleic acid internal standard. The enzymatic activity was measured by monitoring oleic acid productions at multiple time points. This method overcomes the substrate and pH limitations of conventional techniques and thus serves as a generic lipase activity assay.     

Development of an LC‐MS/MS method for determination of bicalutamide on dried blood spots: application to pharmacokinetic study in mice

A rapid and highly sensitive assay method has been developed and validated for the estimation of bicalutamide (BCL) on mouse dried blood spots (DBS) using liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the negative‐ion mode. The assay procedure involves a simple liquid extraction of BCL and tolbutamide (internal standard, IS) from mouse blood DBS cards using tert‐butyl methyl ether. Chromatographic separation was achieved with 5 mm ammonium acetate (pH 6.5)–acetonitrile (35:65, v/v) at a flow rate of 0.60 mL/min on an Atlantis dC₁₈ column with a total run time 3.0 min. The MS/MS ion transitions monitored were 428.80 → 254.70 for BCL and 269.00 → 169.60 for IS. Method validation was performed as per regulatory guidelines. A linear response function was observed from 0.92 to 1911 ng/mL for BCL in mouse blood. The intra‐ and inter‐day precisions were in the ranges of 1.86–12.5 and 3.19–10.8%, respectively. This novel DBS method has been applied to a pharmacokinetic study in mice. Copyright © 2014 John Wiley & Sons, Ltd.     

Validation study of assay method for DX-8951 and its metabolite in human plasma and urine by high-performance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry

A new liquid chromatographic/mass spectrometric assay has been developed for the determination of DX-8951, a new anti-tumor drug, and its 4-hydroxymethyl metabolite (UM-1) in human plasma and urine. Solid-phase extractions were used for sample preparation. A gradient reverse-phase HPLC separation was developed with mobile phases consisting of trifluoroacetic acid and methanol. The detection was conducted using atmospheric pressure chemical ionization tandem mass spectrometry in the selected reaction monitoring mode. A structural analog, camptothecin (CPT), was used as the internal standard. The assay was validated for the determination of DX-8951 and UM-1 in human plasma and urine. The lower limits of quantitation of DX-8951 and UM-1 were 0.1 ng/mL in plasma and 1 ng/mL in urine. The method showed a satisfactory sensitivity, precision, accuracy, recovery and selectivity.     

A Sensitive and Selective LC-MS Method for the Determination of Lurasidone in Rat Plasma,Bile, and Urine

A liquid chromatography-mass spectrometry (LC-MS) assay was developed and validated for the quantification of lurasidone, an atypical antipsychotic drug, in rat plasma, bile, and urine. Rat plasma, bile, or urine samples were processed by liquid–liquid extraction and injected onto an LC-MS system for the quantification of lurasidone and ziprasidone (an internal standard). Lurasidone and ziprasidone were separated from endogenous substances using a Gemini C6-Phenyl column with mixture of acetonitrile and 0.1 % formic acid (80:20, v/v) as the mobile phase. Quantification was performed using the selected ion monitoring mode at m/z 493 for lurasidone and m/z 413 for the IS. The detector response was specific and linear for lurasidone in the concentration range 5–5,000 ng mL⁻¹ The intra- and inter-day accuracy and precision of the method were determined to be within the acceptable criteria for assay validation guidelines. In addition, lurasidone was stable under a variety of processing and handling conditions. Lurasidone concentrations could be readily measured in rat plasma, bile, and urine samples up to 24 h after an intravenous or oral administration, suggesting that the assay can be used in pharmacokinetic studies of lurasidone in rats.

     

Absolute quantification of Atlantic salmon and rainbow trout vitellogenin by the 'signature peptide' approach using electrospray ionization QqToF tandem mass spectrometry

In this work, we present a simple method for absolute quantification of plasma vitellogenin from both rainbow trout and Atlantic salmon. Plasma samples obtained from control and beta-estradiol induced fish were digested with trypsin. A characteristic 'signature peptide' was selected and analyzed by high performance liquid chromatography (HPLC) coupled to an electrospray quadrupole-time-of-flight tandem mass spectrometer, using a deuterated homolog peptide as an internal standard. The hybrid tandem mass spectrometer was operated in a 'pseudo' selected reaction-monitoring mode in which three diagnostic product ions were monitored for identification and quantification purposes. The reproducibility (coefficient of variation approximately 5%) and sensitivity (limit of quantification (LOQ) of 0.009 mg/ml) achieved by this simple assay allow it to be considered as an alternative to immunological assays.     

Simultaneous, sequential quantitative achiral-chiral analysis by two-dimensional liquid chromatography

In general, chromatographic analysis of chiral compounds involves a minimum of two methods; a primary achiral method for assay and impurity analysis and a secondary chiral method for assessing chiral purity. Achiral method resolves main enantiomeric pairs of component from potential impurities and degradation products and chiral method resolves enantiomeric pairs of the main component and diastereomer pairs. Reversed-phase chromatographic methods are preferred for assay and impurity analysis (high efficiency and selectivity) whereas chiral separation is performed by reverse phase, normal phase, or polar organic mode. In this work, we have demonstrated the use of heart-cutting (LC-LC) and comprehensive two-dimensional liquid chromatography (LC × LC) in simultaneous, sequential achiral and chiral analysis and quantitation of minor, undesired enantiomer in the presence of major, desired enantiomer using phenylalanine as an example. The results were comparable between LC-LC and LC × LC with former offering better sensitivity and accuracy. The quantitation range was over three orders of magnitude with undesired D-phenylalanine detected at approximately 0.3% in the presence of predominant, desired L-phenylalanine (99.7%). The limit of quantitation was comparable to conventional high-performance liquid chromatography. A reversed-phase C18 achiral column in the primary and reversed-phase Chirobiotic Tag chiral column in the secondary dimension were used with a compatible mobile phase.     

Determination of famotidine in low-volume human plasma by normal-phase liquid chromatography/tandem mass spectrometry

A rapid, sensitive and robust assay procedure using liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) for the determination of famotidine in human plasma and urine is described. Famotidine and the internal standard were isolated from plasma samples by cation-exchange solid-phase extraction with benzenesulfonic acid (SCX) cartridges. The urine assay used direct injection of a diluted urine sample. The chromatographic separation was accomplished by using a BDS Hypersil silica column with a mobile phase of acetonitrile-water containing trifluoroacetic acid. The MS/MS detection of the analytes was set in the positive ionization mode using electrospray ionization for sample introduction. The analyte and internal standard precursor-product ion combinations were monitored in the multiple-reaction monitoring mode. Assay calibration curves were linear in the concentration range 0.5--500 ng ml(-1) and 0.05--50 microg ml(-1) in plasma and urine, respectively. For the plasma assay, a 100 microl sample aliquot was subjected to extraction. To perform the urine assay, a 50 microl sample aliquot was used. The intra-day relative standard deviations at all concentration levels were <10%. The inter-day consistency was assessed by running quality control samples during each daily run. The limit of quantification was 0.5 ng ml(-1) in plasma and 0.05 microg ml(-1) in urine. The methods were utilized to support clinical pharmacokinetic studies in infants aged 0-12 months.