Rapid and sensitive LC–MS/MS assay for the quantitation of 20(S)-protopanaxadiol in human plasma

This paper describes a rapid and sensitive method for the quantitation of 20(S)-protopanaxadiol (PPD) in human plasma based on high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS). The analyte and internal standard (I.S.), ginsenoside Rh₂, were extracted from plasma by liquid–liquid extraction and separated on a Zorbax extend C₁₈ analytical column using methanol–acetonitrile-10 mM ammonium acetate (47.5:47.5:5, v/v/v) as mobile phase. Detection was by tandem mass spectrometry using electrospray ionization in the positive ion mode and multiple reaction monitoring (MRM). The assay was linear over the concentration range 0.1–100.0 ng/ml with a limit of detection of 0.05 ng/ml. The method was successfully applied to a clinical pharmacokinetic study in healthy volunteers after a single oral administration of a PPD 25 mg capsule.     

Determination of metoclopramide in human plasma by LC–ESI-MS and its application to bioequivalance studies

An LC–MS method for the determination of metoclopramide in human plasma was developed and validated. Sample preparation involved extraction with ethyl acetate. Chromatographic separation was performed on a Thermo Hypersil-Hypurity C₁₈ (150 mm × 2.1 mm, 5 μm) with the mobile phase consisting of 40 mM ammonium acetate–methanol–acetonitrile. 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 300 for metoclopramide and at m/z 384 for the internal standard (prazosin). The method was validated over 0.78–50.00 ng mL^?1 for metoclopramide. The recovery was 67.8–83.1%, and the limit of quantitation (LOQ) detection was 0.78 ng mL^?1 for metoclopramide. The intra- and inter-day precision of the method at three concentrations was 5.0–13.6% with accuracy of 99.2–104.0%. Stability of compounds was established in a battery of stability studies. The method was successfully applied to bioequivalence studies of metoclopramide hydrochloride tablets to obtain the pharmacokinetic parameters.     

Stable-isotope dilution LC–ESI-MS/MS techniques for the quantification of total homocysteine in human plasma

Homocysteine is an endogenous sulphydryl aminoacid irreversibly catabolized by transsulfuration to cysteine or remethylated to methionine. Increased plasma levels of homocysteine are an independent risk factor for atherosclerosis and cardiovascular disease. Accurate and reliable quantification of this amino acid in plasma samples is essential in clinical practice to explore the presence of a hyperhomocysteinemia, for instance after an ischemic event, or to control a possible adjunctive risk factor in patients at higher risk. In this review, LC–ESI-MS/MS methods are discussed and compared with other analytical methods for plasma homocysteine. LC–ESI-MS/MS is a technique combining the physicochemical separation of liquid chromatography with the analysis of mass spectrometry. It is based on stable-isotope dilution and possesses inherent accuracy and precision. Quantitative analysis is achieved by using commercially available homocystine-d₈ as an internal standard. Taking advantage of the high sensitivity and specificity, approaches involving LC–ESI-MS/MS require less laborious sample preparation, no derivatization and produce reliable results.     

Simultaneous quantification of the organophosphorus pesticides dimethoate and omethoate in porcine plasma and urine by LC–ESI-MS/MS and flow-injection-ESI-MS/MS

Dimethoate is an organophosphorus toxicant used in agri- and horticulture as a systemic broad-spectrum insecticide. It also exhibits toxic activity towards mammalian organism provoked by catalytic desulfuration in the liver producing its oxon-derivative omethoate thus inhibiting acetylcholinesterase, initiating cholinergic crisis and ultimately leading to death by respiratory paralysis and cardiovascular collapse. Pharmaco- and toxicokinetic studies in animal models help to broaden basic understanding of medical intervention by antidotes and supportive care. Therefore, we developed and validated a LC–ESI-MS/MS method suitable for the simultaneous, selective, precise (RSD_intra-day 1–8%; RSD_inter-day 5–14%), accurate (intra-day: 95–107%; inter-day: 90–115%), and robust quantification of both pesticides from porcine urine and plasma after deproteinization by precipitation and extensive dilution (1:11,250 for plasma and 1:40,000 for urine). Accordingly, lower limits of quantification (0.24–0.49 μg/ml plasma and 0.78–1.56 μg/ml urine) and lower limits of detection (0.12–0.24 μg/ml plasma and 0.39–0.78 μg/ml urine) were equivalent to quite low absolute on-column amounts (1.1–2.1 pg for plasma and 2.0–3.9 pg for urine). The calibration range (0.24–250 μg/ml plasma and 0.78–200 μg/ml urine) was subdivided into two linear ranges (r² ≥ 0.998) each covering nearly two orders of magnitude. The lack of any interfering peak in 6 individual blank specimens from plasma and urine demonstrated the high selectivity of the method. Furthermore, extensive sample dilution causing lowest concentration of potentially interfering matrix ingredients prompted us to develop and validate an additional flow-injection method (FI-ESI-MS/MS). Validation characteristics were as good as for the chromatographic method but sample throughput was enhanced by a factor of 6. Effects on ionization provoked by plasma and urine matrix from 6 individuals as well as in the presence of therapeutics (antidotes) administered in an animal study were investigated systematically underling in the reliability of the presented methods. Both methods were applied to porcine samples derived from an in vivo animal study.     

Improved method for the determination of cyclic guanosine monophosphate (cGMP) in human plasma by LC–MS/MS

Cyclic guanosine monophosphate (cGMP) is an important second messenger molecule involved in gating ion channels and activating protein kinases. Here, we describe a validated LC–MS/MS method for the quantification of cGMP in human plasma, utilizing a stable isotope labeled analogue of cGMP as I.S. Plasma samples were extracted and concentrated by weak anion exchange solid phase extraction and the extracts were chromatographically separated on a porous graphitic carbon column. The analytes were detected by positive electrospray ionization and tandem mass spectrometry. The calibration function was linear in the range 1–20 nM and the intra- and inter-day precision showed relative standard deviations of better than 2 and 6%, respectively. The accuracy was always better than 4%. Plasma concentrations in healthy human subjects determined with this method were 3.92 ± 1.17 nM (n = 20). The method was, due to its isotope labeled I.S., matrix independent.     

Simultaneous determination of citalopram and its metabolite in human plasma by LC–MS/MS applied to pharmacokinetic study

A simple sensitive and robust method for simultaneous determination of citalopram and desmethylcitalopram was developed using liquid chromatography tandem mass spectrometry (LC–MS/MS). A 200 μL aliquot of plasma sample was employed and deproteinized with methanol and desipramine was used as the internal standard. After vortex mixing and centrifugation, the supernatant was diluted with water (1:1, v/v) and then directly injected to analysis. Analytes were separated by a Zorbax XDB C₁₈ column with the mobile phase composed of acetonitrile and water (30:70, v/v) with 0.25% formic acid and monitored in MRM mode using a positive electrospray source with tandem mass spectrometry detection. The total run time was 3.5 min. The dynamic range was 0.2–100 ng/mL for citalopram and 0.25–50 ng/mL for desmethylcitalopram, respectively. Compared to the best existing literatures for plasma samples, the same LOQ for CIT (0.5 ng/mL) and lower LOQ for DCIT (0.25 vs 5 ng/mL) were reached, and less sample preparation steps and runtime (3.5 vs 10 min) were taken for our method. Accuracy and precision was lower than 8% and lower than 11.5% for either target. Validation results and its application to the analysis of plasma samples after oral administration of citalopram in healthy Chinese volunteers demonstrated the method was applicable to pharmacokinetic studies.     

Quantification of isradipine in human plasma using LC–MS/MS for pharmacokinetic and bioequivalence study

A highly sensitive and rapid method for the analysis of isradipine in human plasma using liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) was developed. The procedure involves a simple liquid–liquid extraction of isradipine and amlodipine (IS, internal standard) with methyl-t-butyl ether after alkaline treatment and separation by RP-HPLC. Detection was performed by positive ion electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode, monitoring the transitions m/z 372.1 → m/z 312.2 and m/z 408.8 → m/z 237.9, for quantification of isradipine and IS, respectively. The standard calibration curves showed good linearity within the range of 10 to 5000 pg/mL (r² ≥ 0.9998). The lower limit of quantitation (LLOQ) was 10 pg/mL. The retention times of isradipine (0.81 min) and IS (0.65 min) suggested the potential for high throughput of the proposed method. In addition, no significant metabolic compounds were found to interfere with the analysis. This method offered good precision and accuracy and was successfully applied for the pharmacokinetic and bioequivalence studies of 5 mg of sustained-release isradipine in 24 healthy Korean volunteers.     

A simple and sensitive HPLC–ESI-MS/MS method for the determination of andrographolide in human plasma

A liquid chromatography–electrospray ionization tandem mass spectrometry (HPLC–ESI-MS/MS) method for the determination of andrographolide in human plasma was established. Dehydroandrographolide was used as the internal standard (I.S.). The plasma samples were deproteinized with methanol and separated on a Hanbon C₁₈ column with a mobile phase of methanol–water (70:30, v/v). HPLC–ESI-MS/MS was performed in the selected ion monitoring (SIM) mode using target ions at [M?H₂O–H]^?, m/z 331.1 for andrographolide and [M?H]^?, m/z 331.1 for the I.S. Calibration curve was linear over the range of 1.0–150.0 ng/mL. The chromatographic separation was achieved in less than 6.5 min. The lower limits of quantification (LLOQ) was 1.0 ng/mL. The intra and inter-run precisions were less than 6.95 and 7.22%, respectively. The method was successfully applied to determine the plasma concentrations of andrographolide in Chinese volunteers.     

Development and validation of a high throughput and robust LC–MS/MS with electrospray ionization method for simultaneous quantitation of diltiazem and its two metabolites in human plasma: Application to a bioequivalence study

A high throughput and specific method using ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) was developed for the simultaneous determination of diltiazem and its two metabolite (N-desmethyldiltiazem and O-desacetyldiltiazem) in human plasma. A one-step liquid–liquid extraction (LLE) with methyl-t-butyl ether (MTBE) involved for the extraction of diltiazem (DLTZ), metabolites (DMeD and DAcD) and internal standard. Analytes were chromatographed on a ACQUITY UPLC? BEH C₁₈ column (100 mm × 2.1 mm, i.d., 1.7 μm) with isocratic elution at a flow rate of 0.2 mL/min using 10 mM ammonium acetate buffer–acetonitrile (25:75, v/v). The Quattro Premier XE LC–MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique. Using 300 μL plasma, the method was validated over the concentration range 0.48–639.9 ng/mL for DLTZ and 0.24–320.1 for DMeD and 0.24–320.7 ng/mL for DAcD, with a lower limit of quantification of 0.48 ng/mL for DLTZ and 0.24 ng/mL for metabolites. The intra- and inter-day precision and accuracy were within 10.0%. The recovery was 77.4%, 76.0%, 74.5% and 74.1% for DLTZ, DMeD, DAcD and Ziprasidone, respectively. Total run time was 2.0 min only.     

Determination of the γ-secretase inhibitor MK-0752 in human plasma by online extraction and electrospray tandem mass spectrometry (HTLC–ESI-MS/MS)

A sensitive and rapid HTLC–ESI-MS/MS method with an advanced online sample preparation was developed for determination of the γ-secretase inhibitor MK-0752 in human plasma using an internal standard. Plasma samples (100 μL) were diluted and injected directly onto an online extraction column (Cohesive Cyclone MAX 0.5 mm × 50 mm, >30 μm), the sample matrix was washed out with an aqueous solution, and retained analytes were eluted out and transferred directly to the analytical column (Phenomenex Gemini 3μ C18 110A, 50 mm × 2.0 mm at 50 °C) for separation using a gradient mobile phase. The eluted analytes were then detected on an API-3000 LC–MS/MS System with ESI and a negative multiple reaction monitoring mode. The monitored ion transitions were m/z 441 → 175 for MK-0752 and 496 → 175 for the internal standard. Online extraction recoveries were 81%. The method was validated and was linear in the range of 0.05–50 μg/mL. Within-day and between-day precisions were < 8.6%, and accuracies were 0.7 and 7.1%. This method was applied to the measurement of plasma MK-0752 levels in a Phase I study of pediatric patients with recurrent or refractory brain tumors.     

Validation and application of an LC–MS/MS method for quantitation of three fatty acid ethanolamides as biomarkers for fatty acid hydrolase inhibition in human plasma

Endogenous ethanolamides (fatty acid amides), including arachidonyl ethanolamide (anandamide, AEA), oleoyl ethanolamide (OEA), and palmitoyl ethanolamide (PEA), are substrates of fatty acid amide hydrolase (FAAH). FAAH may play an important role for pain, anxiety/depression, and metabolic disorders. Ethanolamides are considered to be potential pharmacodynamic biomarkers to determine target engagement for FAAH inhibition by novel pharmaceutical agents. A highly selective, sensitive, and high-throughput liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed and validated for simultaneous quantitation of AEA, OEA, and PEA in human plasma. The method employed D₄-AEA, D₄-OEA, and ¹³C₂-PEA as “surrogate analytes” to establish the concentration–mass response relationship, i.e. a regression equation. The concentrations of AEA, OEA, and PEA were calculated based on the regression equations derived from the surrogate analytes. This approach made it possible to prepare calibration standard and quality control (QC) samples in plasma devoid of interferences from the endogenous analytes. The analytical methodology required 150 μL of human plasma that was processed via liquid–liquid extraction (LLE) using a 96-well plate format. Chromatographic separation was achieved with a reversed-phase high performance liquid chromatography (HPLC) column using gradient elution, and the run time was 3 min. The method was fully validated and it demonstrated acceptable accuracy, precision, linearity, and specificity. The lower limit of quantitation (LLOQ) was 0.1/0.5/0.5 ng/mL for AEA/OEA/PEA, which was sensitive enough to capture the basal plasma levels in healthy subjects. Bench-top stability in plasma, freeze–thaw stability in plasma, frozen long-term stability in plasma, autosampler stability, and stock solution stability all met acceptance criteria (%Bias within ±12.0%). Characterization of stability in purchased/aged blood indicated that ethanolamides are subject to degradation mediated by intracellular membrane-bound FAAH, which has been shown to be inhibited by phenylmethylsulfonyl fluoride (PMSF). In the presence of PMSF, ethanolamide levels increased slightly over time, suggesting that blood cells release ethanolamides into plasma. Whole blood stability conducted in fresh blood immediately following collection revealed that there was significant elevation of ethanolamide concentrations (∼1.3–2.0-fold on ice and ∼1.5–3.0-fold at room temperature by 2 h), indicating that de novo synthesis and release from blood cells were the predominant factors affecting ethanolamide concentrations ex vivo. Accordingly, conditions that ensured rapid separation of plasma from blood cells and consistency in the blood harvesting procedures were established and implemented for clinical studies to minimize the ex vivo elevation of plasma ethanolamide concentrations. The variability (intra-subject and inter-subject) of plasma ethanolamide levels was evaluated in healthy subjects during a Phase 0 study (no drug administration) that simulated the design of single-ascending dose and multiple-ascending dose clinical trials in terms of sample collection time points, population, food, and activity. The data indicated there was relatively large inter- and intra-subject variation in plasma ethanolamide concentrations. In addition, apparent variations due to time of day and/or food effects were also revealed. Understanding the variability of ethanolamide levels in humans is very important for study design and data interpretation when changes in ethanolamide levels are used as target engagement biomarkers in clinical trials.     

Determination of the unstable drug otilonium bromide in human plasma by LC–ESI-MS and its application to a pharmacokinetic study

Otilonium bromide (OB) degrades rapidly in plasma and readily undergoes hydrolysis by the plasma esterase. In this paper, an LC–ESI-MS method has been developed for the determination of OB in human plasma. The rapid degradation of OB in plasma was well prevented by immediate addition of potassium fluoride (KF, an inhibitor of plasma esterase) to the freshly collected plasma before prompt treatment with acetonitrile. The method was validated over the concentration range of 0.1–20 ng/ml. The data of intra-run and inter-run precision and accuracy were within ±15%. The mean extraction recoveries for OB and the internal standard were higher than 93.0% and the matrix effects were negligible. The method has been successfully used in a pharmacokinetic study.     

Simultaneous determination of cyclophosphamide and carboxyethylphosphoramide mustard in human plasma using online extraction and electrospray tandem mass spectrometry (HTLC–ESI-MS/MS)

A rapid and selective method for simultaneous determination of cyclophosphamide and its metabolite carboxyethylphosphoramide mustard (CEPM) was developed using online sample preparation and separation with tandem mass spectrometric detection. Diluted plasma was injected onto an extraction column (Cyclone MAX 0.5 mm × 50 mm, >30 μm), the sample matrix was washed with an aqueous solution, and retained analytes were transferred to an analytical column (Gemini 3 μm C18 110A, 100 mm × 2.0 mm) using a gradient mobile phase prior to detection by MS/MS. Analytes were detected in an API-3000 LC-MS/MS system using positive multiple-reaction monitoring mode (m/z 261/140 and 293/221 for CTX and CEPM, respectively). Online extraction recoveries were 76% and 72% for cyclophosphamide and CEPM. Within-day and between-day variabilities were <3.0%, and accuracies were between ?6.9% and 5.2%. This method has been used to measure plasma cyclophosphamide and CEPM concentrations in an ongoing Phase II study in children with newly diagnosed medulloblastoma.     

Simultaneous determination of a novel antifibrotic agent and three metabolites in human urine by LC–MS/MS

A robust and validated high performance liquid chromatography tandem mass spectrometry (LC–MS/MS) method has been developed for simultaneous determination of F351 (5-methyl-1-(4-hydroxylphenyl)-2-(1H)-pyridone) and three major metabolites in human urine sample. This assay method has also been validated in terms of selectivity, linearity, lower limit of quantification (LLOQ), accuracy, precision, stability, matrix effect and recovery. Chromatography was carried out on an XTerra RP 18 column and mass spectrometric analysis was performed using an API 4000 mass spectrometer coupled with electro-spray ionization (ESI) source in the positive ion mode. The MRM transitions of m/z 202 → 109, 232 → 93, 282 → 202 and 378 → 202 were used to quantify F351 and three metabolites, respectively. Retention times for F351 and three metabolites were 2.54, 1.38, 1.53 and 1.34 min, respectively. The assay was validated from 20 to 4000 ng/mL for F351 and M1, from 80 to16,000 ng/mL for M2 and M3. Intra- and inter-day precision for all analytes was <6.3%, method accuracy was between −11.2 and 0.3%. This assay was used to support a clinical study where multiple oral doses were administered to healthy subjects to investigate the pharmacokinetics, safety, and tolerability of F351.     

Determination of free and glucuronidated kaempferol in rat plasma by LC–MS/MS: Application to pharmacokinetic study

Flavanoid kaempferol is mainly present as glucuronides and sulfates in rat plasma, and small amounts of the intact aglycone are also detected. In the this study, a rapid, specific and sensitive liquid chromatography–electrospray ionization-tandem mass spectrometry method (HPLC–MS/MS) was developed and validated for determination of kaempferol and its major metabolite glucuronidated kaempferol in rat plasma. A liquid–liquid extraction with acetic ether was involved for the extraction of kaempferol and internal standard. Analytes were separated on a C18 column (150 mm × 2.1 mm, 4.5 μm, Waters Corp.) with isocratic elution at a flow-rate of 0.3 ml min⁻¹. The mobile phase was consisted of 0.5% formic acid and acetonitrile (50:50, v/v). The Quattro Premier HPLC–MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique. The method was validated according to the FDA guidelines for validation of bioanalytical method. The validated method was successfully applied to the study of the pharmacokinetics in rats after oral administration of kaempferol with different doses.     

Determination of the quaternary ammonium compound trospium in human plasma by LC–MS/MS: Application to a pharmacokinetic study

A highly sensitive, specific and evaporation free SPE extraction, LC–MS/MS method has been developed for the estimation of trospium in human plasma using trospium-d8 as an internal standard (IS). The analyte was separated using isocratic mobile phase on reverse phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M⁺] cations, m/z 392–164 for trospium and m/z 400–172 for the IS. The total run time was 3.50 min and the elution of trospium and trospium-d8 (IS) occurred at 2.8 min. The developed method was validated in human plasma with a lower limit of quantification of 0.05 ng/mL. A linear response function was established for the range of concentrations 0.05–10 ng/mL (r > 0.998) for trospium in human plasma. The intra- and inter-day precision values for trospium met the acceptance as per FDA guidelines. Trospium was stable in the battery of stability studies viz., bench-top, auto-sampler, dry extracts and freeze/thaw cycles. The developed assay method was applied to an oral pharmacokinetic study in humans.     

Determination of dehydroandrographolide succinate in human plasma by liquid chromatography tandem mass spectrometry (LC–MS/MS): Method development,validation and clinical pharmacokinetic study

A LC–MS/MS method was developed and validated for the determination of dehydroandrographolide succinate (DAS), a traditional Chinese medicine derivative used for the treatment of pneumonia, upper respiratory tract infection, and chronic bronchitis. Following protein precipitation, DAS was detected by ion transition at m/z 531.2/99.0 in multiple reaction monitoring mode with negative electrospray ionization-tandem mass spectrometry. The limit of detection was 0.5 ng/mL, and the lower limit of quantification was 10 ng/mL in human plasma. Good linearity was maintained over the range of 10–5000 ng/mL, and the correlation coefficient was better than 0.99. The accuracy ranged from 95.3% to 113%, RSD from 0.928% to 6.47%, for the within- and between-run analysis at all QC levels. The recovery ranged from 85.5% to 93.4% and the matrix effect from 107% to 119%. No significant carryover and good stability were found during method validation. The developed method was successfully applied to the determination of DAS in human plasma in a pharmacokinetic study following intravenous infusion of potassium sodium DAS to nine healthy volunteers.     

LC–MS/MS method for the determination of several drugs used in combined cardiovascular therapy in human plasma

A simple, fast and validated method is reported for the simultaneous analysis, in human plasma, of several drugs usually combined in cardiovascular therapy (atenolol, bisoprolol, hydrochlorothiazide, chlorthalidone, salicylic acid, enalapril and its active metabolite enalaprilat, valsartan and fluvastatin) using high performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) with electrospray ionization (ESI), working in multiple reaction monitoring mode (MRM). Separation of analytes and internal standard (pravastatin) was performed on a Luna C18(2) (150 mm × 4.6 mm, 3 μm) column using a gradient elution mode with a run time of 15 min. The mobile phase consisted of a mixture of acetonitrile and water containing 0.01% formic acid and 10 mM ammonium formate at pH 4.1. Sample treatment consisted of a simple protein precipitation with acetonitrile, enabling a fast analysis. The method showed good linearity, precision (RSD% values between 0.7% and 12.7%) and accuracy (relative error values between 0.9% and 14.0%). Recoveries were within 68–106% range and the ion-suppression was not higher than 22% for any analyte. The method was successfully applied to plasma samples obtained from patients under combined cardiovascular treatment.     

Lipid and lipid mediator profiling of human synovial fluid in rheumatoid arthritis patients by means of LC–MS/MS

Human synovial fluid (SF) provides nutrition and lubrication to the articular cartilage. Particularly in arthritic diseases, SF is extensively accumulating in the synovial junction. During the last decade lipids have attracted considerable attention as their role in the development and resolution of diseases became increasingly recognized. Here, we describe a capillary LC–MS/MS screening platform that was used for the untargeted screening of lipids present in human SF of rheumatoid arthritis (RA) patients. Using this platform we give a detailed overview of the lipids and lipid‐derived mediators present in the SF of RA patients. Almost 70 different lipid components from distinct lipid classes were identified and quantification was achieved for the lysophosphatidylcholine and phosphatidylcholine species. In addition, we describe a targeted LC–MS/MS lipid mediator metabolomics strategy for the detection, identification and quantification of maresin 1, lipoxin A₄ and resolvin D5 in SF from RA patients. Additionally, we present the identification of 5S,12S-diHETE as a major marker of lipoxygenase pathway interactions in the investigated SF samples. These results are the first to provide a comprehensive approach to the identification and profiling of lipids and lipid mediators present in SF and to describe the presence of key anti-inflammatory and pro-resolving lipid mediators identified in SF from RA patients.