Electrospray Ionization Multiple Reaction Monitoring Research Articles

Pharmacokinetic Analysis of Gatifloxacin and Dexamethasone in Rabbit Ocular Biofluid Using a Sensitive and Selective LC-MS/MS Method.

Bacterial keratitis (BK) is an infection that causes inflammation of the cornea and, if severe, can result in blindness. Topical fluoroquinolones combined with corticosteroids have been shown to be useful in the treatment of BK. A rapid, selective, and sensitive bioanalytical method for simultaneous quantification of Gatifloxacin (GAT) and Dexamethasone (DEX) has been developed and validated using tandem mass spectrometry (LC-MS/MS). Optimal separation was accomplished in under 5 min using an Agilent Zorbax C18 column (100 mm × 4.6 mm, 3.5 μm). The mobile phase was composed of a blend of 0.2% formic acid in triple distilled water and methanol with a flow rate of 0.65 mL/min in isocratic mode. GAT and DEX were detected in positive electrospray ionization multiple reaction monitoring mode (MRM), and the retention time was found to be at 1.64 and 2.93 min, respectively. The linearity of GAT and DEX was found to be in the range of 1.56-400 ng mL-1 with good precision and accuracy. The method was validated according to USFDA regulatory guidelines. The validated method was effectively utilized for preclinical pharmacokinetic analysis of GAT and DEX in rabbit tear fluid following the topical application of a commercial formulation.

Optimized LC-MS/MS method for quantifying insulin degludec and liraglutide in rat plasma and Tissues: Application in pharmacokinetics and biodistribution

We present an ultra-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous detection of insulin degludec (I-Deg) and liraglutide (LIRA) in rat plasma and tissues, characterized by its sensitivity and selectivity. Chromatographic separation was achieved using an Acquity UPLC BEH C18 column, leveraging a mobile phase of acetonitrile and water (both with 0.1 % formic acid) under gradient elution over a run time of 7.5 min. The mass spectrometer operated in positive electrospray ionization multiple reaction monitoring (MRM) mode, tracking transitions of m/z 1221.6 → 641.6 for I-Deg, m/z 938.7 → 1064.1 for LIRA, and m/z 1184.7 → 454.4 for the internal standard. Validation ranged from 5 to 100 ng/mL, exhibiting robust linearity (r2 > 0.99) and limits of detection (LOD) of 1.63–2.02 ng/mL for I-Deg and 0.96–1.62 ng/mL for LIRA. Limits of quantification (LOQ) were 2.38–4.76 ng/mL for I-Deg and 3.22–4.40 ng/mL for LIRA. Notably, no significant matrix effects were detected. Stability was confirmed under various conditions, and precision metrics (intraday RSD 1.68–8.05 % for I-Deg and 1.11–7.69 % for LIRA; interday RSD 1.39–8.61 % for I-Deg and 1.06–8.83 % for LIRA) alongside accuracy (90.5–114.9 % for I-Deg and 92.7–113.7 % for LIRA) were within acceptable ranges. The method was successfully applied to pharmacokinetic and biodistribution studies following simultaneous subcutaneous administration of LIRA and I-Deg in rats.

Quantitative Analysis of Buprenorphine, Norbuprenorphine, and Their Glucuronide Metabolites in Human Urine.

We present a quantitative clinical LC-MS/MS assay for the simultaneous analysis of buprenorphine, norbuprenorphine, and their glucuronide metabolites in human urine. The assay is based on the direct and hydrolysis-free sample preparation protocol, i.e., dilute and shoot, whereby clarified urine specimens are diluted in internal standard reagent and injected into the LC-MS/MS instrument. The analytical platform employs reversed-phase liquid chromatography for upfront separation and electrospray ionization multiple reaction monitoring MS detection via the triple-quadrupole (TSQ Quantiva) instrument. The assay has a quantitative analytical range of 5ng/mL-1000ng/mL represented at seven levels for each of the four analytes. A unique stable isotopically labeled analogue is used as internal standard for each analyte. For high-throughput performance, the assay can be multiplexed between two LC channels.

High-Throughput Comprehensive Quantitative LC-MS/MS Analysis of Common Drugs and Metabolites (62 Compounds) in Human Urine.

In recent years a multitude of LC-MS/MS assays have been widely reported in commercial and clinical literature demonstrating the simultaneous analyses of dozens of drugs of abuse in human samples. The utility of such assays is meant to supplant the indirect detection based on the classical spectral library approach. Direct and simultaneous analysis via LC-MS/MS technology is made possible by fast acquisition rates in multiple reaction monitoring, as well as sensitivity and high selectivity of the technology for each individual analyte in a complex mixture. Hence, unlike immunoassays, which are not well-suited for the analyses of mixtures, and which may also be prone to false positives from potential interferences, quantitative LC-MS/MS analyses are feasible for complex patient mixtures of drugs of abuse. We hereby present a robust clinical LC-MS/MS assay for the simultaneous and semi-quantitative analysis of up to 62 drugs of abuse in human urine, representing major classes that include opiates, benzodiazepines, amphetamines, etc. The assay utilizes dilute and shoot, whereby the sample is diluted ten times in internal standard reagent and thereafter submitted to the LC-MS instrument, i.e., reversed-phase liquid chromatography coupled to the electrospray ionization multiple reaction monitoring analysis, via the TSQ Endura triple-quadrupole instrument. The assay employs stable isotope-labeled internal standards with a linear response in the 30-300ng/mL range, effectively semi-quantitative, since this analytical range is well within typical immunoassay cutoffs for most drugs.

High-Throughput Quantitative LC-MS/MS Analysis of Barbiturates in Human Urine.

We hereby present a fast, high-throughput, and clinical LC-MS/MS assay for the simultaneous analysis of barbiturates in human urine. It is deployed as a quantitative assay for phenobarbital, butalbital, pentobarbital/amobarbital, and secobarbital, as well as for confirmations following positive immunoassay drug screens in patient urine. Briefly, urine specimens are processed via dilute and shoot, i.e., by mixing the sample with 20 times volume of internal standard reagent and injecting 50μL of that mixture into the analytical instrument. Chromatographic separation is performed using a reversed-phase C18 column in a mobile-phase system doped with <1mM ammonium fluoride. Mass spectrometric detection occurs via negative-mode electrospray ionization multiple reaction monitoring in the TSQ Quantiva triple-quadrupole instrument. All the analytes in the mixture are detected and quantified simultaneously with respect to internal calibration in the range 20-2500ng/mL. However, the assay cannot distinguish pentobarbital from amobarbital, which are isobaric analytes. Nonetheless, the assay is sensitive, robust, and amenable to harmonization with other assays that employ barbiturate cutoffs in the range of 20-150ng/mL.

High-Throughput Quantitative LC-MS/MS Analysis of Benzodiazepines in Human Urine.

We hereby present a fast, high-throughput, and clinical LC-MS/MS assay for the simultaneous analysis of benzodiazepines in human urine. The assay is used as both a confirmations and semi-quantitative assay for the abovementioned drugs of abuse following immunoassay urine drug screens. Urine levels are reported in the range of 25ng/mL-500ng/mL for each of the 22 analytes, based on a six-level calibration and using a subset (10) of stable isotopically labeled analogues as internal standards. The urine sample is clarified, diluted ten times in internal standard reagent, and thereafter injected into the LC-MS/MS instrument. Reversed-phase liquid chromatography is used to separate the mixture, and the TSQ Endura triple-quadrupole (QqQ) MS instrument performs detection via positive-mode electrospray ionization multiple reaction monitoring.

Quantitative LC-MS/MS Analysis of Amphetamines and Cocaine in Human Urine.

We hereby present a fast and high-throughput LC-MS/MS assay for the simultaneous analysis of amphetamines and cocaine in human urine. The assay is used for confirmations following immunoassay urine drug screens as well as a quantitative assay to report actual urine concentrations in the range 30-10,000ng/mL for each of the seven analytes, namely, amphetamine; methamphetamine; phentermine; methylenedioxyamphetamine; 3,4-methylenedioxymethamphetamine; methylenedioxy-ethyl-amphetamine; and a cocaine metabolite, benzoylecgonine. The assay derives its efficacy from minimal sample preparation via dilute and shoot. The platform is based on reversed-phase liquid chromatography coupled to the TSQ Endura triple-quadrupole (QqQ) MS instrument for detection via electrospray ionization multiple-reaction monitoring MS. The quantitative analysis is based on the linear calibration whereby the instrument response for each analyte at a given concentration is normalized against stable isotope-labeled internal standard. In addition, the assay can be multiplexed across more than one LC channel to obtain high-sample throughput.

Development and validation of an ultra-performance liquid chromatography-tandem mass spectrometry method for the determination of baloxavir in rat plasma and its application to pharmacokinetic studies.

In this study, an ultra-performance liquid chromatography-tandem mass spectrometry method was established for the development and validation of baloxavir acid (BXA) concentrations and the active ingredients of the antiviral drug baloxavir marboxil (BXM). Further, the method was applied to study the pharmacokinetics of BXA. BXA was determined by the electrospray ionization multiple reaction monitoring positive ion mode, and the mass-to-charge ratios (m/z) of BXA and internal standard baloxavir-d4 were 484.2 → 247.2 and 488.1 → 247.2. An Oasis max online column (2.1 × 20 mm, 30 μm) was used with 1% formic acid in water (A) and 2% formic acid in acetonitrile (B) as mobile phases at a flow rate of 0.5 mL·min-1 for chromatographic separation. The linearity was good in the range of 3-200 ng·mL-1 (r = 0.9994), with 3.00 ng·mL-1 lower limit of quantification. The relative standard deviation of the inter-assay precision was less than or equal to 6.51%, and the accuracy was in the range of 91.28%-104.29%. This method is suitable for the determination of BXA and for performing pharmacokinetic studies in clinical research.

Fast determination of bongkrekic acid in plasma by high performance liquid chromatography-tandem mass spectrometry

Objective: To establish a method for rapid determination of bongkrekic acid (BA) in plasma by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Methods: In November 2020, plasma samples were extracted by methanol and acetonitrile (1∶1) and purified directly. The samples were separated by C18 column. Gradient elution was carried out with 5 mmol/L ammonium acetate water acetonitrile solution as mobile phase. Under the optimized instrument conditions, the electrospray ionization multiple reaction monitoring (MRM) mode was used, and the external standard method was used for quantitative analysis. Results: The linear relationship of BA in plasma was good in the concentration range of 2-100 μg/L, the correlation coefficient was 0.9998, the average recovery was 83.7%-112.0%, the relative standard deviation within and between batches was less than 10%, the detection limit of the method was 0.7 μg/L and the lower limit of quantification was 2.0 μg/L. Conclusion: The method is simple, rapid, accurate and sensitive, and can meet the requirements for the determination of BA in blood samples of poisoning patients.

Fully Automated Quantitative Measurement of Serum Organic Acids via LC-MS/MS for the Diagnosis of Organic Acidemias: Establishment of an Automation System and a Proof-of-Concept Validation.

Gas chromatography-mass spectrometry has been widely used to analyze hundreds of organic acids in urine to provide a diagnostic basis for organic acidemia. However, it is difficult to operate in clinical laboratories on a daily basis due to sample pretreatment processing. Therefore, we aimed to develop a fully automated system for quantifying serum organic acids using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The pretreatment CLAM-2030 device was connected to an LC-MS/MS system for processing serum under optimized conditions, which included derivatizing serum organic acids using 3-Nitrophenylhydrazine. The derivatized organic acids were separated on a reverse-phase Sceptor HD-C column and detected using negative-ion electrospray ionization multiple reaction monitoring MS. The automated pretreatment-LC-MS/MS system processed serum in less than 1 h and analyzed 19 serum organic acids, which are used to detect organic acidemias. The system exhibited high quantitative sensitivity ranging from approximately 2 to 100 µM with a measurement reproducibility of 10.4% CV. Moreover, a proof-of-concept validation of the system was performed using sera from patients with propionic acidemia (n = 5), methylmalonic acidemia (n = 2), and 3-methylcrotonylglycinuria (n = 1). The levels of marker organic acids specific to each disease were significantly elevated in the sera of the patients compared to those in control samples. The automated pretreatment-LC-MS/MS system can be used as a rapid in-hospital system to measure organic acid levels in serum for the diagnosis of organic acidemias.

On the feasibility of quantifying sodium channel Nav 1.6 protein in mouse brain using targeted ultra-high-performance/electrospray ionization multiple reaction monitoring mass spectrometry.

Nav 1.6 is a transmembrane voltage gated sodium channel implicated in various forms of epilepsy. Modulation of its activity in epilepsy animal models can be accomplished using inhibitors which may result in changes in its expression. There is a need to generate reliable quantitative measurements of Nav 1.6 expression in animal models. This research explores the feasibility of quantifying Nav 1.6 expression in mouse brains using targeted multiple reaction monitoring (MRM) mass spectrometry. A combination of in silico tryptic Nav 1.6 peptides and MRM transitions were used to select target peptides. This was followed by a simple proteomic work-up including plasma membrane isolation, trypsin-based proteolysis and ultra-high-performance/electrospray ionization tandem mass spectrometry (UHPLC/ESI-MS/MS) to detect the presence of Nav 1.6 in induced HEK293 cells. The unique Nav 1.6 peptide, DSLFIPR, was selected as probe for quantifying Nav 1.6 levels in brains from C57BL/6J wild-type mice as well as two kinds of mutants including Scn8aN1768D/+ and heterozygous null Scn8a+/- mice using isotope dilution targeted mass spectrometry. The feasibility of using targeted MRM for quantifying Nav 1.6 expression in mice brains was demonstrated. Expression of Nav 1.6 in brains (hippocampi) from wild-type and mutant Scn8aN1768D/+ mice were found to be around 0.40 fmol/μg. Mutant null Scn8a+/- heterozygous mice, on the other hand, showed levels of 0.22 fmol/μg as expected based on this particular mutation which only generates 50% of the expression in wild-type mice. Nav 1.6-overexpressed HEK293 cells showed 3.7 fmol/μg of Nav 1.6 expression, suitable for screening new compounds for Nav 1.6 blocking activity. The results of the present feasibility study support the use of DSLFIPIR for quantification of Nav1.6 in brain tissues using UHPL/ESI-MS/MS.

A study on phthalate metabolites, bisphenol A and nonylphenol in the urine of Chinese women with unexplained recurrent spontaneous abortion

Humans are widely exposed to phthalates, bisphenol A and nonylphenol owing to the ubiquitous use of these chemicals in consumer products. Increasing attention has been paid to exposure to phthalates, bisphenol A and nonylphenol because of their potential adverse effects on human fertility. A validated method was developed to investigate the three classes of environmental estrogen, mentioned above, in the urine of Chinese women of Nanjing area with unexplained recurrent spontaneous abortion. Solid-phase extraction coupled with ultra performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) was used. In this method, amounts of bisphenol A (BPA), nonylphenol (NP) and four phthalate metabolites, mono-n-butyl phthalate (MBP), mono-isobutyl phthalate (MiBP), mono-benzyl phthalate (MBzP) and mono-2-ethylhexyl phthalate (MEHP), along with their isotope labeled internal standards, were measured using UPLC-MS/MS operated in negative electrospray ionization multiple reaction monitoring mode. The limits of detection were 0.3ng/mL for the four phthalate metabolites, and 0.5ng/mL for bisphenol A and nonylphenol. For women with unexplained recurrent spontaneous abortion, the mean concentrations of MBP, MiBP, MBzP, MEHP, BPA and 4-n-NP were 6.52±6.04, 5.51±4.19, 0.53±0.42, 10.12±4.16, 7.13±7.42, 0.41±0.49ng/mL (mean±SD), respectively. For the control group, the mean concentrations of the corresponding analytes were 4.15±3.57, 2.96±3.30, 0.46±0.49, 6.50±2.81, 4.43±2.23,0.48±0.43ng/mL (mean±SD), respectively. Levels of MiBP and MEHP were significantly different between the two groups, using Wilcoxon rank sum tests. This method can be applied in epidemiological studies to explore the association between exposure to environmental estrogens and relevant adverse outcomes.

Validation of an LC–MS/MS salivary assay for glucocorticoid status assessment: Evaluation of the diurnal fluctuation of cortisol and cortisone and of their association within and between serum and saliva

Salivary steroid testing represents a valuable source of biological information; however, the proper measurement of low salivary levels is challenging for direct immunoassays, lacking adequate sensitivity and specificity and causing poor inter-laboratory reproducibility. Liquid chromatography-tandem mass spectrometry (LC–MS/MS) has overcome previous analytical limits, often providing results deviating from previous knowledge. Nowadays, LC–MS/MS is being introduced in clinical laboratories for salivary cortisol testing; however, so far only a few studies have reported thorough biological validation based on LC–MS/MS data. In this study, we provide a thorough analytical, pre-analytical and biological validation of an LC–MS/MS method for the measurement of salivary cortisol (F) and of its inactive metabolite cortisone (E).Analytes were extracted from 50μl of saliva, were then separated in 7.5min LC-gradient and detected by negative electrospray ionization—multiple reaction monitoring. The reliability of a widely diffused collection device, Salivette®, was assessed and the overall procedure was validated. The diurnal cortisol and cortisone fluctuation in saliva and serum was described by a four paired collection protocol (8 am, 12 am, 4 pm and 8 pm) in 19 healthy subjects. The assay allowed the quantitation of F and E down to 39.1 and 78.1pg/ml, with an imprecision range of 5.5–9.5%, 3.9–14.1% and 2.6–14.4%, and an accuracy range of 105.5–113.1%, 88.5–98.7% and 90.7–96.7% for both analytes at low, medium and high levels, respectively. Salivette® provided comparable results and better precision (CV<1.0%) as referred to direct spitting (CV<13.0%). A parallel diurnal rhythm in saliva and serum was observed for cortisol and cortisone, with values lowering from the morning to the evening time points (P<0.0001). While salivary E linearly correlated to total serum F (R2=0.854, P<0.001), salivary F showed an exponential relationship (R2=0.903, P<0.001) with serum F reflecting the free circulating fraction. A non linear association between E and F was observed in saliva (R2=0.941, p<0.001) consistent with the type II 11β-HSD activity. We concluded that our LC–MS/MS method allowed a sensitive evaluation of salivary levels of cortisol and cortisone. The simultaneous determination of both hormones in saliva allowed the differential estimation of the active and of the total glucocorticoid exposure over the daytime. The assay could provide further insight into the comprehension of normal and dysfunctional glucocorticoid circadian rhythm.

Validated method for the quantification of free and total carnitine, butyrobetaine, and acylcarnitines in biological samples.

A validated quantitative method for the determination of free and total carnitine, butyrobetaine, and acylcarnitines is presented. The versatile method has four components: (1) isolation using strong cation-exchange solid-phase extraction, (2) derivatization with pentafluorophenacyl trifluoromethanesulfonate, (3) sequential ion-exchange/reversed-phase (ultra) high-performance liquid chromatography [(U)HPLC] using a strong cation-exchange trap in series with a fused-core HPLC column, and (4) detection with electrospray ionization multiple reaction monitoring (MRM) mass spectrometry (MS). Standardized carnitine along with 65 synthesized, standardized acylcarnitines (including short-chain, medium-chain, long-chain, dicarboxylic, hydroxylated, and unsaturated acyl moieties) were used to construct multiple-point calibration curves, resulting in accurate and precise quantification. Separation of the 65 acylcarnitines was accomplished in a single chromatogram in as little as 14 min. Validation studies were performed showing a high level of accuracy, precision, and reproducibility. The method provides capabilities unavailable by tandem MS procedures, making it an ideal approach for confirmation of newborn screening results and for clinical and basic research projects, including treatment protocol studies, acylcarnitine biomarker studies, and metabolite studies using plasma, urine, tissue, or other sample matrixes.

Validation of a hydrophilic interaction ultra‐performance liquid chromatography–tandem mass spectrometry method for the determination of gemcitabine in human plasma with tetrahydrouridine

A simple and reproducible bioanalytical method for the determination of gemcitabine in human plasma treated with tetrahydrouridine (THU) was developed and validated using a hydrophilic interaction ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS). To prevent deamination of gemcitabine, blood was treated with THU, and the plasma samples obtained after centrifugation were used in this study. Gemcitabine and gemcitabine-(13)C, (15)N2 used as an internal standard, were extracted from human plasma treated with THU using a 96-well Hybrid SPE-Precipitation plate. Extracts were chromatographed on a hydrophilic interaction chromatography column with isocratic elution. Detection was performed using Quattro Premier with positive electrospray ionization multiple reaction monitoring mode. The standard curve ranged from 10 to 10,000 ng/mL without carryover. No significant interferences were detected in blank plasma and no interferences by 2'-2'-difluoro-2'-deoxyuridine, a metabolite of gemcitabine. Accuracy and precision in the intra-batch reproducibility study using quality control samples with three THU levels did not exceed ±5.4 and 7.3%, respectively, and the inter-batch reproducibility results also met the criteria. Stability of gemcitabine was ensured in whole blood and plasma as well as stability of THU in solutions. The UPLC-MS/MS method developed was successfully validated and can be applied for gemcitabine bioanalysis in clinical studies.

Abstract 5155: RapidFire MS/MS enables both rapid evaluation of multiple histone methyltransferases and label-free high throughput screening of targeted compound libraries

Abstract Introduction: Histone methyltransferases (HMT's) are histone-modifying enzymes, that catalyze the transfer of one, two, or three methyl groups from S-adenosyl-L-methionine (SAM) to lysine and arginine residues on histone proteins. This methylation results in changes in gene expression. Over-expression and misregulation of these enzymes has been associated with cancer, making HMT's an attractive area for the development of new cancer therapeutics. We developed highly sensitive, high throughput RapidFire-based MS/MS biochemical assays to quantitate the enzymatic activity of three different HMT's. This methodology allows a label-free, non-radioactive and coupling enzyme free system to biochemically characterize the kinetic mechanism of HMT's and determine the mechanism of inhibition of small molecule inhibitors. Methods: The API4000 mass spectrometer coupled with an Agilent RapidFire 300 Mass Spectrometry system was used to rapidly evaluate the enzymatic activity of HMT's using native substrates for both enzymatic characterization of the target as well as screening highly specialized and targeted compound libraries. The Agilent type D column was used for separation of detected products, S-adenosyl-L-homocysteine (SAH) and SAM from other assay components. Electrospray ionization multiple reaction monitoring positive polarity scan was used to detect the two desired analytes of the reaction. SAH was detected using a Q1 mass set to 399.2 amu and a Q3 mass set to 250.1 amu while SAM was detected using a Q1 mass set to 385.2 amu and a Q3 mass set to 136.1 amu. Preliminary data: Pfizer proprietary compound libraries were assembled and screened in a 384-well plate format against multiple HMT's using the RapidFire 300 coupled with an API4000 mass spectrometer. The enzymatic screens using RapidFire LC/MS enable both one platform and one method for simultaneous investigation of multiple different epigenetic targets in the portfolio. Total cycle time required to process a single assay well for a single HMT in a 384 well plate is 10 seconds. Therefore, one 384-well plate can be sampled in just under 65 minutes, enabling the processing of more than 7,500 compounds per day. Good correlation and a sizeable reduction in cost/well is evident with the RapidFire MS/MS generated results compared with more traditional assay methods for compound screening such as 3H-SAM scintillation proximity assay and 3H-SAM filter plate assay. This presentation will discuss the results generated from the screening efforts of multiple histone methyltransferases against our methyltransferase targeted libraries and our fragment library, as well as the hit follow-up strategy and mechanistic characterization implemented to ensure high quality lead matter was selected for further investment and follow-up. Citation Format: Patrick J. Bingham, Karen Maegley, Cody T. Krivacic. RapidFire MS/MS enables both rapid evaluation of multiple histone methyltransferases and label-free high throughput screening of targeted compound libraries. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5155. doi:10.1158/1538-7445.AM2014-5155

LC-MS-MS Determination of Imatinib and N-Desmethyl Imatinib in Human Plasma

A specific and sensitive liquid chromatography-electrospray ionization-tandem mass spectrometric method was developed for the quantification of imatinib and its primary metabolite N-desmethyl imatinib in human plasma. Protein precipitation with methanol was used for sample preparation. High-performance liquid chromatographic separation was performed on a Thermo BDS Hypersil C18 column (4.6 × 100 mm, 2.4 µm) with methanol-water (55:45, v/v) containing 0.1% formic acid and 0.2% ammonium acetate as the mobile phase, using isocratic elution at a flow rate of 0.7 mL/min. Detection was conducted with positive electrospray ionization multiple reaction monitoring of the ion transitions at m/z 494 → 394 for imatinib, 480 → 394 for N-desmethyl imatinib and 297 → 110 for the internal standard (palonosetron). The assay was validated in the concentration ranges of 8-5,000 ng/mL for imatinib and 3-700 ng/mL for N-desmethyl imatinib. The quantification limits for imatinib and N-desmethyl imatinib were 8 and 3 ng/mL, respectively. The intra-day and inter-day precision values of the assay (expressed as percentage relative standard deviation) were less than 15% at all concentration levels within the tested range, and the accuracy values were between 85 and 115%. The established method was successfully applied to the pharmacokinetic study of imatinib mesylate capsules in 24 healthy Chinese volunteers.

Quantification of monoamine neurotransmitters and melatonin in sea lamprey brain tissues by high performance liquid chromatography–electrospray ionization tandem mass spectrometry

A rapid liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for simultaneous quantification of three monoamine neurotransmitters (NTs) and melatonin in sea lamprey brain tissues. Separation was performed on a reversed-phase column with mobile phases of 1mM perfluoroheptanoic acid (PFHA) water solution/acetonitrile and mass spectra were acquired in positive electrospray ionization multiple reaction monitoring (MRM) mode. Solid-phase extraction (SPE) was employed to purify and extract the target compounds from the tissue samples. The matrix effects as well as the influence of two extraction methods, protein precipitation (PPT) and SPE, on matrix effects were examined for the first time on the quantification of NTs from brain tissue extracts. The matrix effects with SPE (2.4 to −14.9%) were about 30% lower on average than those with the ACN PPT method (−29 to −38%). The recoveries of three types of SPE cartridges were tested and Bond-Elut C18 cartridge was selected to process the samples because of its good extraction efficiencies (71.3–95.3%) and low matrix effects (−6.6 to −14.9%) for all four analytes. This method exhibited excellent linearity for all analytes with regression coefficients higher than 0.99. The limits of detection were between 0.03ng/mL (melatonin) and 0.14ng/mL (norepinephrine). The precisions, expressed as coefficients of variation (CV), ranged from 4.8 to 14.1% for intra-day analyses and from 6.1 to 16.2% for inter-day analyses. Brain tissues from 360 sea lampreys were analyzed by the developed method and the concentrations for four analytes were found to be at the level of nanogram per gram of brain tissues. To our knowledge, this is the first report on the quantification of NTs and melatonin in the sea lamprey using the LC–MS/MS method.

Therapeutic monitoring of serum digoxin for patients with heart failure using a rapid LC-MS/MS method

Objective Here we develop a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the determination of digoxin in serum. Design and methods The serum samples were extracted with methyl tert-butyl ether using an isotope-labeled digoxin-d3 as internal standard. The analyte was separated on a reverse phase Capcell C18 column and detected in positive electrospray ionization multiple reaction monitoring mass spectrometry. Results The chromatographic analysis was carried out within 3 min, but the complete analysis took longer because of the liquid–liquid extraction. The lower limit of quantification was 0.1 ng/mL for digoxin. The intra- and inter-batch precisions were less than 12%, and the bias ranged from − 9.1% to 10.7%. The external quality assessment (EQA) results obtained with the LC-MS/MS method were comparable to target values. Subsequently, this method has been applied to the therapeutic monitoring of digoxin in a clinical setting. Conclusion In this study, we have developed a rapid and reliable LC-MS/MS method for the therapeutic monitoring of digoxin in human serum.

Determination of 5-Hydroxytryptamine, Norepinephrine, Dopamine and Their Metabolites in Rat Brain Tissue by LC–ESI–MS–MS

A liquid chromatography–electrospray ionization tandem mass spectrometry method has been developed to perform the determination of 5-hydroxytryptamine (5-HT), norepinephrine (NE), dopamine (DA) and their metabolites, i.e., 5-hydroxyindole-3-acetic acid (5-HIAA), 4-hydroxy-3-methoxyphenylglycol (MHPG) sulfate, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in rat brain tissue. Analytes were separated on a Thermo C18 column (4.6 mm × 250 mm, 5 μm, SN: 1245575T, Thermo electron corporation, USA) with a mobile phase of 0.05% formic acid/acetonitrile (92:8 for ESI+, 82:18 for ESI−, v/v) at the flow-rate of 0.8 mL min−1. The LC system was coupled to a Waters Micromass Quattro Premier XE tandem quadruple mass spectrometer. MS acquisition of 5-HT, NE and DA was performed in positive electrospray ionization multiple reaction monitoring (MRM) mode, while negative electrospray ionization MRM mode was used to monitor their metabolites. The calibration curves were linear within the concentration range of 4–4,450 ng mL−1 for 5-HT, 4–4,110 ng mL−1 for NE and 4–4,100 ng mL−1 for DA (r ≥ 0.999). The limit of quantitation was 4 ng mL−1. 5-HIAA, MHPG, DOPAC and HVA have good linearity within the range of 12–1,000 ng mL−1(r ≥ 0.998) and the limit of quantitation was 12 ng mL−1. The intra- and inter-day RSD were lower than 8.45%. The method is sensitive, fast, accurate and usable for quantity determination of monoamine neurotransmitters and their metabolites in neuropsychiatric diseases.