Determination of tandospirone in human plasma by a liquid chromatography–tandem mass spectrometry method

A rapid, sensitive, and selective liquid chromatography–tandem mass spectrometry method for the detection of tandospirone in human plasma is described. It was employed in a pharmacokinetic study. The analyte and internal standard diphenhydramine were extracted from plasma using liquid–liquid extraction, then separated on a Zorbax XDB C₁₈ column using a mobile phase of methanol–water–formic acid (80:20:0.5, v/v/v). The detection was performed with a tandem mass spectrometer equipped with an electrospray ionization source. Linearity was established in the concentration range of 10.0-5,000 pg/ml. The lower limit of quantification was 10.0 pg/ml. The intraday and interday relative standard deviation across three validation runs over the entire concentration range was less than 13%. Accuracy determined at three concentrations (25.0, 200, and 4,000 pg/ml for tandospirone) ranged from 94.4 to 102.1%. Each plasma sample was chromatographed within 3.4 min. The method proved to be highly selective and suitable for bioequivalence evaluation of different formulations containing tandospirone and clinical pharmacokinetic investigation of tandospirone.     

Sensitive liquid chromatography–tandem mass spectrometry method for the determination of pantoprazole sodium in human urine

A sensitive and selective liquid chromatographic–tandem mass spectrometric (LC–MS–MS) method was developed to determine pantoprazole sodium (PNT) in human urine. After solid-phase extraction with SPE cartridge, the urine sample was analysed on a C₁₈ column (symmetry 3.5 μm; 75 mm × 4.6 mm i.d) interfaced with a triple quadrupole tandem mass spectrometer. Positive electrospray ionization was employed as the ionization source. The mobile phase consisted of acetonitrile–water (90:10, v/v). The method was linear over a concentration range of 1–100 ng mL^?1. The lower limit of quantitation was 1 ng mL^?1. The intra-day and inter-day relative standard deviation across three validation runs over the entire concentration range was <10.5%. The accuracy determined at three concentrations (8.0, 50.0 and 85.0 ng mL^?1 PNT) was within ±1.25% in terms of relative errors.     

Quantitative selenium speciation in human urine by using liquid chromatography–electrospray tandem mass spectrometry

A liquid chromatography–electrospray-tandem mass spectrometry (ES-MS/MS) method was developed for the speciation analysis of four organic selenium species of relevance to human urinary metabolism, namely trimethylselenomium ion (TMSe⁺), selenomethionine (SeMet) and the two selenosugars, methyl 2-acetamido-2-deoxy-1-seleno-β-d-galactos/-glucos-amine (SeGalNAc and SeGluNAc, respectively). Their chromatographic separation was achieved by using a cation exchange pre-column coupled in-series with a reversed-phase high-performance liquid chromatography column, along with an isocratic mobile phase. Online detection was performed using ES-MS/MS in selective reaction monitoring mode. SeGalNAc was detected as the major human urinary metabolite of selenium in the samples analysed, whereas TMSe⁺ was detected in the urine of one volunteer before and after receiving a selenium supplement. SeMet was not detected as a urine excretory metabolite in this study. Spiking experiments performed with the urine samples revealed significant signal suppression caused by coeluting matrix constituents. To overcome such interferences, isotopically labelled ¹³CD₃⁸²SeGalNAc was used as an internal standard, whereas in the absence of an isotopically labelled internal standard for TMSe⁺, the standard addition method was applied. Quality control for the accurate quantitation of TMSe⁺ and SeGalNAc was carried out by analysing spiked human urine samples with appropriate selenium standards over a concentration range of 10–50 μg Se L⁻¹. The method has achieved a limit of detection in the presence of urine matrix comparable to that of HPLC-inductively coupled plasma-mass spectrometry for the four selenium species: 1.0 μg Se L⁻¹ for TMSe⁺, 5.6 μg Se L⁻¹ for SeMet, and 0.1 μg Se L⁻¹ for both SeGalNAc and SeGluNAc.     

Determination of evodiamine and rutecarpine in human serum by liquid chromatography–tandem mass spectrometry

Evodiamine and rutecarpine are two kinds of indole alkaloids contained in the fruit of Evodiae fructus, which have been shown to exhibit various bioactivities in humans. A liquid chromatography–tandem mass spectrometric method (LC–MS/MS) was developed for the determination of evodiamine and rutecarpine in human serum. The serum was extracted by solid-phase extraction (SPE) and analyzed using a C18 column and a mobile phase consisting of methanol–water (85:15) solution containing 5 mmol/L ammonium formate at a flow rate of 0.5 mL/min. The mass spectrometer was operated in positive mode, employing the extracted ion chromatogram (EIC) for detection and quantitation of evodiamine (m/z 288) and rutecarpine (m/z 304). Good linear relationships between the peak area and the concentration were obtained in the ranges of 5.2–1040 ng/mL and 10.2–1020 ng/mL, with correlation coefficients (r) of 0.999 and 0.998, for evodiamine and rutecarpine, respectively. The repeatabilities (RSD, n=6) of quantitation for evodiamine and rutecarpine were 2.18–4.00% and 2.99–5.67%, respectively, and the recovery ranged from 90.5% to 98.1%. A comparative study of the different ionization and quantitation modes, including ESI–MS, ESI–MS/MS, APCI–MS and APCI–MS/MS, was also accomplished. The MS/MS fragmentation mechanism of the base peak ([M+H]⁺, m/z 304) of evodiamine was investigated in order to identify the analytes in more complicated body fluid samples.      

Multiclass method for antimicrobial analysis in animal feeds by liquid chromatography–tandem mass spectrometry

A rapid multiclass method that covers 50 antimicrobials from 13 different families in animal feeds was developed. Samples were extracted using a mixture of methanol, acetonitrile and a McIlvaine buffer combined with sonication. Feed extracts were simply diluted prior to injection, since the clean-up strategies that were tested, based on either solid-phase extraction or dispersive solid-phase extraction, were ineffective at minimizing matrix-related signal suppression/enhancement. Analysis was carried out by liquid chromatography coupled to tandem mass spectrometry using an electrospray ionization source operating in positive and negative modes. For the quantification, matrix-fortified standard calibration curves were used to compensate for matrix effects and losses in sample preparation. The method was validated in-house in pig, poultry and cattle feed matrices and showed satisfactory performance characteristics. Thus, the proposed approach was suitable for application in a routine high-throughput laboratory for the official control of feeds.

Determination of meloxicam in human plasma by ultra-high-performance liquid chromatography–tandem mass spectrometry and its application in a pharmacokinetic study

A rapid, selective and sensitive ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method was developed to detect meloxicam in human plasma. A triple quadrupole tandem mass spectrometer equipped with an electrospray ionization source was used in positive ion mode. Protein precipitation with acetonitrile was used for sample preparation. Meloxicam and ¹³C₆-meloxicam internal standard were analyzed on an Acquity CSH C₁₈ column with a mobile phase of acetonitrile and water in 0.1% formic acid using a gradient program for separation. The retention time of meloxicam was 1.1 min and the total run time was only 2.0 min. Detection was performed in multiple reaction monitoring mode using an electrospray ionization source with optimized mass spectrometry parameters. The calibration curves were linear in the range 10.0–3.00 × 10³ ng/ml (r ≥ 0.99). The within-run and between-run RSDs were ≤14.8%. The within-run and between-run REs ranged from −4.6 to 10.7%. There was no significant matrix effect, and the recovery rate was high. This method was fully validated, including reinjection reproducibility in human plasma. The method was applied to the pharmacokinetic study. All of the incurred sample reanalysis methods met the criteria.     

Determination of steryl sulphates in invertebrate tissue by liquid chromatography–tandem mass spectrometry

A method for the identification and quantification of underivatised steryl sulphates in invertebrates by liquid chromatography (LC) coupled with tandem mass spectrometry (MS) involving a single cleanup step has been developed. Negative electrospray ionisation and positive and negative atmospheric-pressure chemical ionisation (APCI) spectra of steryl sulphate showed pseudomolecular ions ([M+H–H₂SO₄]⁺or [M–H]⁻). Collision-induced dissociation (CID) was efficient only in positive APCI. LC-MS in negative APCI was least susceptible to interference and possible differences in response factors. The detection limits (signal-to-noise ratio of 3) based on cholest-5-enyl-3-sulphate in positive and negative APCI modes are 3.66 and 0.73 pmol μL⁻¹, respectively. Calibration plots and response factors for cholest-5-enyl-3-sulphate relative to the internal standard, cholecalciferyl-3-sulphate, in both positive and negative polarities, were linear in the concentration range from 1.22 to 16.4 pmol μL⁻¹ with good coefficients of determination (R ²>0.98). It is suggested that the structure elucidation of steryl sulphates is best achieved in CID positive APCI mode, whereas their quantification should be carried out using negative APCI.     

Determination of anabolic steroids in muscle tissue by liquid chromatography–tandem mass spectrometry

A specific and sensitive multi-method based on liquid chromatography–tandem mass spectrometry using atmospheric pressure chemical ionization (LC–APCI–MS/MS) has been developed for the determination of 20 anabolic steroids in muscle tissue (diethylstilbestrol, β-estradiol, ethynylestradiol, α/β-boldenone, α/β-nortestosterone, methyltestosterone, β-trenbolone, triamcinolone acetonide, dexamethasone, flumethasone, α/β-zearalenol, α/β-zearalanol, zearalenone, melengestrol acetate, megestrol acetate and medroxyprogesterone acetate). The procedure involved hydrolysis, extraction with tert-butyl methyl ether, defattening and final clean-up with solid phase extraction (SPE) on Oasis HLB and Amino cartridges. The analytes were analyzed by reversed-phase LC–MS/MS, in positive and negative multiple reaction monitoring (MRM) mode, acquiring two diagnostic product ions from each of the chosen precursor ions for the unambiguous confirmation of the hormones. The method was validated at the validation level of 0.5 ng/g. The accuracy and precision of the method were satisfactory. The decision limits CCα ranged from 0.03 to 0.14 ng/g while the detection capabilities CCβ ranged from 0.05 to 0.24 ng/g. The developed method is sensitive and useful for detection, quantification and confirmation of these anabolic steroids in muscle tissue and can be used for residue control programs.     

A method for the quantification of biomarkers of exposure to acrylonitrile and 1,3-butadiene in human urine by column-switching liquid chromatography–tandem mass spectrometry

1,3-Butadiene and acrylonitrile are important industrial chemicals that have a high production volume and are ubiquitous environmental pollutants. The urinary mercapturic acids of 1,3-butadiene and acrylonitrile—N-acetyl-S-(3,4-dihydroxybutyl)cysteine (DHBMA) and MHBMA (an isomeric mixture of N-acetyl-S-((1-hydroxymethyl)-2-propenyl)cysteine and N-acetyl-S-((2-hydroxymethyl)-3-propenyl)cysteine) for the former and N-acetyl-S-2-cyanoethylcysteine (CEMA) for the latter—are specific biomarkers for the determination of individual internal exposure to these chemicals. We have developed and validated a fast, specific, and very sensitive method for the simultaneous determination of DHBMA, MHBMA, and CEMA in human urine using an automated multidimensional LC/MS/MS method that requires no additional sample preparation. Analytes are stripped from urinary matrix by online extraction on a restricted access material, transferred to the analytical column, and subsequently determined by tandem mass spectrometry using labeled internal standards. The limits of quantification (LOQs) for DHBMA, MHBMA, and CEMA were 10 μg/L, 2 μg/L, and 1 μg/L urine, respectively, and were sufficient to quantify the background exposure of the general population. Precision within series and between series for all analytes ranged from 5.4 to 9.9%; mean accuracy was between 95 and 115%. We applied the method on spot urine samples from 210 subjects from the general population with no occupational exposure to 1,3-butadiene or acrylonitrile. A background exposure of the general population to acrylonitrile was discovered that is basically influenced by individual exposure to passive smoke as well as active smoking habits. Smokers showed a significantly higher excretion of MHBMA, whereas DHBMA levels did not differ significantly. Owing to its automation, our method is well suited for application in occupational or environmental studies. Figure Boxplots of the results from LC/ESI-MS/MS analysis of urinary excretion of CEMA reveal a strong correlation with nicotine metabolite cotinine, indicating that exposure to passive smoke as well as active smoking is the main source of exposure to acrylonitrile in the general population     

Identification and quantification of salinomycin in intoxicated human plasma by liquid chromatography–electrospray tandem mass spectrometry

Salinomycin is a polyether ionophore antibiotic that is widely used in poultry and livestock. Exposure of humans to salinomycin via inhalation or ingestion can cause severe toxicity. The aim of the present work was to develop a simple and sensitive liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the rapid identification and quantification of salinomycin in human plasma. After removing protein using methanol, plasma samples were eluted from a Waters Xterra ^® MS C18 column with an isocratic mobile phase. Detection and quantification of the drug were performed with a triple-quadruple mass spectrometer by monitoring for two specific transitions in the electrospray, positive-ion, multiple-reaction monitoring mode. Assay validation showed good linearity (r ²?=?0.998). The detection and quantification limits of the method were 0.6 and 16 pg/mL, respectively. The inter- and intraday coefficients of variation for the assay were both <15%. Twelve authentic plasma samples from intoxicated patients were analyzed using this method. Salinomycin was detected in six samples, at concentrations of between 0.6 and 46.5 pg/mL. The described assay method allows the sensitive and rapid identification and quantification of salinomycin in human plasma, and thus provides a valuable tool for the specific diagnosis of salinomycin intoxication in clinical and emergency rescue practice.     

A sensitive liquid chromatography–tandem mass spectrometry method for quantitative bioanalysis of fingolimod in human blood: Application to pharmacokinetic study

A simple and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the determination of fingolimod in human blood. The analyte and internal standard fingolimod-d4 were extracted from 300 μl of human blood using protein precipitation coupled with solid-phase extraction method. The chromatographic separation was achieved with a Kinetex biphenyl column (100 × 4.6 mm, 2.6 μm) under isocratic conditions at the flow rate of 0.8 ml/min and column temperature was maintained at 45°C. The detection of analyte and internal standard was carried out by tandem mass spectrometry, operated in positive ion and multiple reaction monitoring acquisition mode. The method was fully validated for its selectivity, precision, accuracy, linearity, stability, detection and quantification limit. The extraction recovery of fingolimod in human blood ranged from 98.39 to 99.54%. The developed method was linear over the concentration range of 5–2500 pg/ml with a detection limit of 1 pg/ml. The developed method was validated and successfully applied for pharmacokinetic study after oral administration of fingolimod capsules.     

Qualitative detection of desmopressin in plasma by liquid chromatography–tandem mass spectrometry

This work describes a liquid chromatography–electrospray tandem mass spectrometry method for detection of desmopressin in human plasma in the low femtomolar range. Desmopressin is a synthetic analogue of the antidiuretic hormone arginine vasopressin and it might be used by athletes as a masking agent in the framework of blood passport controls. Therefore, it was recently added by the World Anti-Doping Agency to the list of prohibited substances in sport as a masking agent. Mass spectrometry characterization of desmopressin was performed with a high-resolution Orbitrap-based mass spectrometer. Detection of the peptide in the biological matrix was achieved using a triple-quadrupole instrument with an electrospray ionization interface after protein precipitation, weak cation solid-phase extraction and high performance liquid chromatography separation with an octadecyl reverse-phase column. Identification of desmopressin was performed using three product ions, m/z 328.0, m/z 120.0, and m/z 214.0, from the parent ion, m/z 535.5. The extraction efficiency of the method at the limit of detection was estimated as 40% (n = 10), the ion suppression as 5% (n = 10), and the limit of detection was 50 pg/ml (signal-to-noise ratio greater than 3). The selectivity of the method was verified against several endogenous and synthetic desmopressin-related peptides. The performance and the applicability of the method were tested by analysis of clinical samples after administration of desmopressin via intravenous, oral, and intranasal routes. Only after intravenous administration could desmopressin be successfully detected.     

Determination of cyclopiazonic acid in food and feeds by liquid chromatography–tandem mass spectrometry

A new, fast and efficient multiple reaction monitoring (MRM) high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method for the determination of cyclopiazonic acid (CPA) in mixed feed, wheat, peanuts and rice is presented. The analytical methodology involves sample extraction with an alkaline methanol–water mixture, defatting with hexane and quantification using HPLC–MS/MS without further treatment of sample extracts. Reversed-phase liquid chromatography using a C18 stationary phase coupled to negative mode electrospray triple quadrupole tandem mass spectrometry was applied. The limit of detection was 5 μg/kg while the limit of quantification was 20 μg/kg in the matrices investigated. The detector response was found to be linear over the range 25–250 μg/kg in feed and 25–500 μg/kg in wheat, peanuts and rice. The mean overall recoveries (n = 18) of CPA varied from 79% to 114% in the range of concentrations studied over a period of 4 months. Mean recoveries (n = 3 or 6) of CPA in wheat, peanuts and rice varied from 70% to 111%, 77% to 116% and 69% to 92%, respectively. The method was successfully applied to the analysis of feed and rice samples artificially infected with the fungal strain Penicillium commune, where the toxin was found at different levels.     

High-performance liquid chromatography–tandem mass spectrometry method for quantifying sulfonylurea herbicides in human urine: reconsidering the validation process

We have developed a method for measuring 17 sulfonylurea (SU) herbicides in human urine. Urine samples were extracted using solid phase extraction (SPE), preconcentrated, and analyzed by high-performance liquid chromatography–tandem mass spectrometry using turboionspray atmospheric pressure ionization. Carbon 13-labeled ethametsulfuron methyl was used as an internal standard. Chromatographic retention times were under 7 minutes. Total throughput was estimated as >100 samples per day. Because only one labeled internal standard was available for the analysis, we were forced to reconsider and restructure the validation process to include stringent stability tests and analyses of urine matrices of differing compositions. We describe our restructured validation process and the critical evaluation it provides for the method developed. The limits of detection (LOD) ranged from 0.05 μg/L to 0.10 μg/L with an average LOD of 0.06 μg/L. Average total relative standard deviations were 17%, 12% and 8% at 0.1 μg/L, 3.0 μg/L and 10 μg/L, respectively. Average extraction efficiencies of the SPE cartridges were 87% and 86% at 2.5 μg/L and 25 μg/L, respectively. Chemical degradation in acetonitrile and urine was monitored over 250 days. Estimated days for 10% and 50% degradation in urine and acetonitrile ranged from 0.7 days to >318 days. The influence of matrix effects on precision and accuracy was also explored. Electronic Supplementary Material Supplementary material is available for this article at For additional information, contact Anderson Olsson at     

Determination of 76 pharmaceutical drugs by liquid chromatography–tandem mass spectrometry in slaughterhouse wastewater

A multi-residue method for the analysis of 76 pharmaceutical agents of nine classes of drugs (tetracyclines, macrolides, fluoroquinolones, β-agonists, β-blockers, diuretics, sedatives, sulfonamides and chloramphenicol) in slaughterhouse wastewater and a receiving river is presented. After simultaneous extraction with an Oasis HLB solid-phase extraction (SPE) cartridge and further purification using an amino SPE cartridge, analytes were detected by liquid chromatography–electrospray ionization-tandem mass spectrometry in positive or negative ion mode. Standard addition was used for quantification to overcome unavoidable matrix effects during ESI-MS analysis. Recoveries for most analytes based on matrix-matched calibration in different test matrices were >60%. The method quantification limits of 76 pharmaceuticals were in the range 0.2–30 ng/L. Nineteen compounds of 76 drugs were found in raw and treated slaughterhouse wastewater from four main slaughterhouses in Beijing. Sulfanamides (sulfanilamide, sulfameter), fluoroquenones (ofloxacin, pefloxacin, norfloxacin, ciprofloxacin, enrofloxacin), tetracyclines (tetracycline, oxytetracycline) and macrolides (kitasamycin, tylosin, erythromycin) were most frequently detected, with the highest levels up to ∼3 μg/L in slaughterhouse wastewater and ∼1 μg/L in treated wastewater. Illicit drugs for animal feeding such as clenbuterol and diazepam were commonly detected in slaughterhouse wastewater. These analytes were also observed in a river receiving slaughterhouse wastewater, with a highest level of up to 0.2 μg/L.     

Quantitative determination of taurine and related biomarkers in urine by liquid chromatography–tandem mass spectrometry

Current urinary bladder cancer diagnosis is commonly based on a biopsy obtained during cystoscopy. This invasive method causes discomfort and pain in patients. Recently, taurine and several other compounds such as L-phenylalanine and hippuric acid in urine were found to be indicators of bladder cancer. However, because of a lack of sensitive and accurate analytical techniques, it is impossible to detect these compounds in urine at low levels. In this study, using liquid chromatography–tandem mass spectrometry (LC-MS/MS), a noninvasive method was developed to separate and detect these compounds in urine. ¹⁵N₂-L-glutamine was used as the internal standard, and creatinine acted as an indicator for urine dilution. A phenyl-hexyl column was used for the separation at an isocratic condition of 0.2% formic acid in water and 0.2% formic acid in methanol. Analytes were detected in multiple-reaction monitoring with positive ionization mode. The limit of detection range is 0.18–6 nM and the limit of quantitation ranges from 0.6 to 17.6 nM. The parameters affecting separation and quantification were also investigated and optimized. Proper clinical validation of these biomarkers can be done using this reliable, fast, and simple method. Furthermore, with simple modifications, this method could be applied to other physiological fluids and other types of diseases.     

Identification of the long-acting erythropoiesis-stimulating agent darbepoetin alfa in human urine by liquid chromatography–tandem mass spectrometry

The misuse of recombinant human erythropoietin (rhEPO) increases the proliferation/production of erythrocytes, which enhance oxygen transport capacities, and has grave consequences with respect to human health and fairness in sports. For sports drug testing, the current analytical methods for rhEPOs are mainly gel electrophoretic methods, such as isoelectric focusing–polyacrylamide gel electrophoresis. Mass spectrometry is fundamentally necessary for the reliable identification of rhEPOs in doping control. In this study, a high-sensitivity and high-throughput mass spectrometric qualitative detection method for darbepoetin alfa in human urine was established by a bottom-up approach. The novel method involves the immunopurification of human urine (10 mL), protease digestion with endoproteinase Glu-C (V8-protease) in an ammonium bicarbonate buffer (pH 7.8) and ultra-performance liquid chromatography using a charged surface hybrid C₁₈ column coupled with electrospray-ionisation high-sensitivity tandem mass spectrometry for improved selectivity of the target molecules. The specific fragment digested from darbepoetin alfa was ⁹⁰TLQLHVDKAVSGLRSLTTLLRALGAQKE¹¹⁷ (V₁₁). The lower limit of detection of urinary darbepoetin alfa was 1.2 pg/mL. The limit of detection for the confirmation analysis was estimated to be 5 pg/mL. The developed method allows high-throughput confirmation analysis, namely 6 h for sample preparation and an analytical run time of only 10 min per sample; this high-throughput method dramatically decreases the workload in the laboratory. Darbepoetin alfa could be identified in human urine collected after the intravenous administration of 15 μg darbepoetin alfa (n?=?3). This mass spectrometric method is an innovative and powerful tool for detecting darbepoetin alfa in human urine for doping control testing.

Trace-level determination of eight cholesterol oxidation products in human plasma by dispersive liquid–liquid microextraction and ultra-performance liquid chromatography–tandem mass spectrometry

7α-Hydroxy cholesterol (7α-OHC), 25-hydroxy cholesterol (25-OHC), 27-hydroxy cholesterol (27-OHC), 4β-hydroxy cholesterol (4β-OHC), 7α-hydroxy-4-cholesten-3-one (7α-C4), 5β-cholestane-3α, 7α, 12α-triol (5β-Triol), cholic acid (CA), and chenodeoxycholic acid (CDCA) are known biomarkers of neurodegenerative diseases. A method for their simultaneous determination in human plasma has been optimized using dispersive liquid–liquid microextraction and ultra-performance liquid chromatography–tandem mass spectrometry. The limits of quantification of the target compounds were in the range of 0.3–3.3?µg/L. The precision achieved by this method was less than 13.4% for intraday and interday analyses. The proposed method was used to analyze eight cholesterol oxidation products in 30 human plasma samples. The analytical results were in a concentration range of 1.6–87.4?µg/L for 7α-OHC, 6.3–58.2?µg/L for 25-OHC, 12.1–98.5?µg/L for 27-OHC, 5.7–64.8?µg/L for 4β-OHC, 1.5–124.1?µg/L for 7α-C4, 0.5–16.5?µg/L for 5β-Triol, 13.1–245?µg/L for CA, and 19.6–487?µg/L for CDCA in the samples. The method may be used for the analysis of biomarkers of neurodegenerative diseases.     

Validated liquid chromatography–tandem mass spectrometry method for simultaneous quantitation of bendamustine and copanlisib in mouse plasma: Application to a pharmacokinetic study in mice

In this study, we report the development and validation of an LC–tandem mass spectrometry method for the simultaneous quantitation of bendamustine and copanlisib in mouse plasma as per the US FDA regulatory guidelines. The sample processing involves extraction of bendamustine and copanlisib along with internal standard (IS; warfarin) from 50 μL mouse plasma using a liquid–liquid extraction method. The chromatographic separation of bendamustine, copanlisib and the IS was achieved on an Atlantis dC₁₈ column using an isocratic mobile phase (5 mM ammonium acetate:methanol, 20:80 v/v). Bendamustine, copanlisib and the IS eluted at 0.88, 1.39 and 0.74 min, respectively, with a total run time of 2.5 min. The calibration curve ranged from 3.99–2996 and 4.33–3248 ng/mL for bendamustine and copanlisib, respectively. Inter- and intra-day precision and accuracy, stability in processed samples and upon storage, dilution integrity and incurred sample reanalysis were investigated for both the analytes. The intra- and inter-day precisions were in the ranges of 2.01%–5.05% and 2.74%–6.13% and 1.98%–7.64 and 8.62%–9.04% for bendamustine and copanlisib, respectively. Stability studies showed that both analytes were stable on bench top for 6 h, in auto-sampler for 24 and at −80°C for 30 days. The validated method was successfully applied to a pharmacokinetic study in mice.