Turbo Ion Spray Research Articles

Abstract PO3-18-05: Pharmacokinetics (PK) of lasofoxifene (LAS) monotherapy and combined with abemaciclib (Abema)

Abstract Introduction: LAS is a selective estrogen receptor modulator (SERM)/tissue-selective estrogen agonist/antagonist studied in women with osteoporosis and vulvar-vaginal atrophy, and most recently, metastatic breast cancer (mBC) with ESR1 mutations. LAS data in healthy postmenopausal women and young men show linear PK and no differences by age or race (Gardner. J Clin Pharmacol 2006;46:52-58; Prakash. Drug Metab Dispos 2008;26:1218-26; Data on file). Also, no PK differences were found between Caucasian and Japanese patients (Jhee. AACR-JCA 2022, abstract A83; Fountaine. J Clin Pharmacol 2006;46:693-99). Here we report select PK data of LAS (5 mg/day) from women with mBC in the ELAINE 1 and ELAINE 2 oncology trials to indirectly investigate possible drug-drug interactions between LAS and Abema. Methods: Patients with mBC and ESR1 mutations who progressed on endocrine therapy and CDK4/6 inhibitors received LAS 5 mg/day as monotherapy (ELAINE 1) or combined with Abema (provided by Eli Lilly and Co) 150 mg BID (ELAINE 2). Blood was collected prior to daily dosing at weeks 2, 4, 8, and every 4 weeks up to the final visit in ELAINE 1, and visits during the first 8 weeks in ELAINE 2, to determine the plasma trough steady-state concentrations (Css) of LAS, Abema, and two active Abema metabolites (M2 and M20). LAS was assayed using validated high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS; detection 0.025 to 25.0 ng/mL); levels below the lower limit of quantification (LLOQ) were reported as 0 ng/mL. Abema and its M2 and M20 metabolites were measured by validated Turbo Ion Spray LC/MS/MS (detection 1.00 to 500 ng/mL); values below the LLOQ were reported as < LLOQ ng/mL. Mean trough Css at each visit were calculated for LAS (ELAINE 1 and 2), and Abema and its M2 and M20 metabolites (ELAINE 2). LAS levels from ELAINE 1 and 2 were compared, while Abema, M2 and M20 plasma levels in ELAINE 2 were compared with previously reported levels. Such indirect comparisons may help determine if Abema altered the metabolism of LAS, or if LAS altered that of Abema, M2, or M20. The relationships of LAS and Abema levels with progression-free survival (PFS) were also explored. Results: Across all time points for subjects who had PK samples assessed, the LAS mean trough Css was 33.6 ng/mL (CV%, 39.8) in ELAINE 1 (n=40) and 30.9 ng/mL (CV 50.5%) in ELAINE 2 (n=29); in both studies, LAS concentrations were consistent across visits. LAS levels in ELAINE 2 were similar to those in ELAINE 1 with no evidence of accumulation. Mean (CV%) Abema plasma concentrations at weeks 2, 4, 6 and 8 were 199 (102%), 107 (130%), 82 (271%), and 94.3 (223%) ng/mL, respectively. Minimum and maximums of the mean trough Css for LAS, Abema, M2, and M20 are shown in Table 1. No relationships between LAS or Abema levels and antitumor activity measured by PFS were seen (data to be presented). Conclusion: The steady-state PK of 5 mg LAS in the ELAINE 1 and 2 studies suggest that the addition of abemaciclib to LAS did not result in an obvious or significant LAS-Abema, drug-drug interaction. Abema PK data from ELAINE 2 were consistent with published data (European Medicines Agency, Verzenio). These results support the continued development of LAS alone and combined with Abema for treating ER+/HER2-, ESR1-mutated mBC that progressed on endocrine therapy and CDK4/6 inhibitors. Table. Mean trough, steady-state concentrations of lasofoxifene and abemaciclib. XX- XX aData from patients who received oral LAS (5 mg/day) plus Abema (150 mg BID). Abema, abemaciclib; LAS, lasofoxifene; M2 and M20 are Abema metabolites. Citation Format: Senthil Damodaran, Paul V. Plourde, Simon Jenkins, Phil Mayer, David J Portman, Matthew Goetz. Pharmacokinetics (PK) of lasofoxifene (LAS) monotherapy and combined with abemaciclib (Abema) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-18-05.

Development of a UPLC-MS/MS Assay for the Quantitative Determination of Capecitabine, 5'-deoxy-5-fluorocytidine (5'-dFCR), 5'-deoxy-5-fluorouridine (5'-dFUR), 5'-fluorouracil (5-FU), and α-fluoro-β-alanine (FBAL).

Capecitabine is an anticancer agent and is the oral prodrug of 5-fluorouracil (5-FU). In this study, an ultra-high performance liquid chromatography coupled to turbo ion spray tandem mass spectrometry (UPLC-MS/MS) method was developed and validated to quantify capecitabine and its metabolites including 5'-deoxy-5-fluorocytidine (5'-dFCR), 5'-deoxy-5-fluorouridine (5'-dFUR), 5-FU, and fluoro-β-alanine (FBAL) in lithium heparinized human plasma. Analytes were extracted by protein precipitation, chromatographically separated by Acquity UPLC HSS T3 column with gradient elution, and analyzed with a tandem mass spectrometer equipped with an electrospray ionization source. Capecitabine and 5'-dFCR were quantified in positive ion mode and 5'-dFUR, 5-FU, and FBAL were quantified in negative ion mode. The total chromatographic run time was 9 min. Stable isotopically labeled internal standards were used for all analytes. The assay was validated over the range from 25.0 to 2,500 ng/mL for capecitabine, 10.0 to 1,000 ng/mL for 5'-dFCR, 5'-dFUR, and 5-FU and 50 to 5,000 ng/ mL for FBAL in human plasma. Validation results have shown the developed assay allows for reliable quantitative analysis of capecitabine, 5'-dFCR, 5'-dFUR, 5-FU, and FBAL in plasma samples. Capecitabine is an anticancer agent and is the oral prodrug of 5-fluorouracil (5-FU). In this study, an ultra-high performance liquid chromatography coupled to turbo ion spray tandem mass spectrometry (UPLC-MS/MS) method was developed and validated to quantify capecitabine and its metabolites including 5'-deoxy-5-fluorocytidine (5'-dFCR), 5'-deoxy-5-fluorouridine (5'-dFUR), 5-FU, and fluoro-β-alanine (FBAL) in lithium heparinized human plasma. Analytes were extracted by protein precipitation, chromatographically separated by Acquity UPLC HSS T3 column with gradient elution, and analyzed with a tandem mass spectrometer equipped with an electrospray ionization source. Capecitabine and 5'-dFCR were quantified in positive ion mode and 5'-dFUR, 5-FU, and FBAL were quantified in negative ion mode. The total chromatographic run time was 9 min. Stable isotopically labeled internal standards were used for all analytes. The assay was validated over the range from 25.0 to 2,500 ng/mL for capecitabine, 10.0 to 1,000 ng/mL for 5'-dFCR, 5'-dFUR, and 5-FU and 50 to 5,000 ng/ mL for FBAL in human plasma. Validation results have shown the developed assay allows for reliable quantitative analysis of capecitabine, 5'-dFCR, 5'-dFUR, 5-FU, and FBAL in plasma samples.

Simultaneous quantitation of befotertinib (D-0316) and its metabolite D-0865 in human plasma by LC-MS/MS method

A sensitive and reliable method was developed to determine befotertinib (D-0316) and its metabolite D-0865 from human plasma by LC-MS/MS. The samples were prepared by simple protein precipitation and 2 µL of the supernatant were chromatographed on a C18 analytical column (ACE Excel 2 Super C18, 50 × 2.1 mm). Elution was performed with mobile phase A (10 mM ammonium acetate in water containing 1 % formic acid) and mobile phase B (acetonitrile containing 1 % formic acid) under a gradient program in a total run time of 4 min. Triple Quadruple 5500 equipped with Turbo Ion Spray source and multiple reaction monitoring (MRM) were used for the analysis detection. The transitions were m/z 568.3 → 72.1 m/z (befotertinib), m/z 554.2 → 497.2 (D-0865), and m/z 455.2 → 164.9 (verapamil, internal standard). According to the Chinese Pharmacopeia Commission and ICH Harmonised Guideline for Bioanalytical Method Validation, this method was validated within the spectrum of its accuracy, precision, selectivity, linearity, recovery, matrix effect, and stability. This LC-MS/MS method was successfully applied for the quantitation of befotertinib and its metabolite D-0865 in human plasma during the pharmacokinetics study of befotertinib in non-small cell lung cancer (NSCLC) patients.

Sensitive liquid chromatography/tandem mass spectrometry method for the simultaneous determination of seventeen beta blockers

In recent years, examples of drugs responsible for acute drug intoxication in elderly people include benzodiazepines, phenobarbital, digitalis preparations, phenytoin, beta blockers or calcium antagonists, and antidepressants in Japan. Due to the diversity of underlying diseases, the reasons for this include increasing number of prescription medicines due to regular oral medicines themselves interacting, and age-related changes in drug metabolism. Beta blockers for clinical use are employed in the management of a range of disorders, including hypertension, heart failure, arrhythmias, migraine headache and tremor. Beta blockers are prescribed widely and also have a high frequency of accidental ingestion by the elderly people. Cardiovascular drugs (beta blockers) present more serious symptoms of intoxication than the underlying diseases themselves. The aim of this study is to develop and validate a sensitive liquid chromatography–tandem mass spectrometry (LC-MS/MS) with ESI method for the simultaneous determination of seventeen beta blockers (atenolol, carteolol, nadolol, pindolol, timolol, acebutolol, arotinolol, metoprolol, esmolol, celiprolol, labetalol, bisoprolol, propranolol, alprenolol, betaxolol, bevantolol and carvedilol). The extractions of beta blockers in human serum were performed by solid-phase extraction method using Oasis® PRiME HLB column (Waters). LC-MS/MS experiments were performed with a HPLC system, which consisted of Shimadzu LC-20AD pumps, a SIL-20AC autosampler and the 4000 QTRAP mass spectrometer (AB SCIEX). The chromatographic separation was performed on a Mightysil-RP-18 MS column (150 × 2.0-mm i.d., 5 μm). For the gradient elution, two solvents were used: (A) 10 mM acetic ammonium buffer and (B) acetonitrile. The flow rate was 0.20 mL/min and the column and autosampler were maintained at 37 and 4˚C, respectively. The settings of the turbo ion-spray were as follows; curtain gas, N2:40 psi, collision gas, N2:4 psi, collision energy: 27∼41 V, ion-spray voltage: 5500 V, ionization mode: ESI positive, source temperature: 600˚C. Separation and sensitivity for the detection of seventeen beta blockers by LC-MS/MS were sufficient and the precursor [M + H]+ ion was detected in the mass spectrum for each drug. This method had a chromatographic total run time of 15 min. The calibration curves were linear over the concentration range of 20–400 ng/mL for seventeen beta blockers (r = 0.9686∼0.9997). The extraction yields of human serum sample with Oasis® PRiME HLB column were ranged from 66.1 to 93.5%, the precision within 0.07 CV values were acceptable of this method. But the extraction yields of bevantlol (37.5%) and carvedilol (26.2%) were low. Currently, we are experimenting to improve the extraction yields of bevantolol and carvedilol. Phyo Lwin EM, et al (2017, Bioanalysis.) developed a method for the determination of atenolol in human plasma and milk sample by LC-MS/MS. The limits of detection (LOD) and quantification (LOQ) were in the range 1.0 ng/mL and 5.0 ng/mL for atenolol. Our LC-MS/MS with ESI method was able to measure compounds at approximately the same concentrations as the analytical methods of Phyo Lwin EM, et al. In our experiments, LOD and LOQ in the range 0.0034–1.6 ng/mL and were 20 ng/mL for seventeen beta blockers. The presently established method is very useful for simultaneous measurements of beta blockers in human serum by LC-MS/MS in clinical and forensic investigations.

A validated liquid chromatography-tandem mass spectroscopy method for the quantification of tolinapant in human plasma.

Tolinapant (ASTX660), a pan-selective inhibitor of apoptosis protein antagonist with dual cIAP/XIAP activity, was identified as a clinical candidate in preclinical efficacy, pharmacokinetic and safety studies. In order to assess tolinapant in first-in-human Phase I/II clinical trials, a validated bioanalytical method was required to determine plasma pharmacokinetics. Tolinapant and d4-tolinapant were extracted from human plasma using liquid-liquid extraction. Separation chromatography was performed on a Acquity BEH C18 1.7 µM, 50 mm × 2.1mm i.d. column, using a mobile phase of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. Mass spectrometry detection was performed by positive turbo ion spray ionisation, in multiple reaction monitoring mode. The method was validated according to the US Food and Drug Administration (FDA) guidelines. The method has a quantifiable linear range of 1-500ng/mL (r 2=0.999). The intra- and inter-day coefficients of variation were <11.4%. Dilution QC samples agreed with prepared concentrations, with a precision of 1.5% and accuracy of 101%. Tolinapant mean recoveries ranged from 85.1-94.4 % with negligible matrix effects. A highly sensitive and selective LC-MS/MS bioanalytical method was developed and validated. The method was successfully applied in Phase 1/2 clinical trials to determine the human pharmacokinetic profile of tolinapant.

A pharmacokinetic study of morphine-6-O-sulfate in rat plasma and brain.

Morphine-6-O-sulfate (M6S), a polar, zwitterionic sulfate ester of morphine, is a powerful and safe analgesic in several rat models of pain. A sensitive liquid chromatography-tandem mass spectrometry bioanalytical method was developed and validated for the simultaneous determination of M6S and morphine (MOR) in rat plasma and brain after M6S administration. Morphine-d6 was used as internal standard. Multiple reaction monitoring was used for detection and quantitation of M6S, MOR, and morphine-d6 in the turbo ion spray positive mode. The chromatographic separation was carried out on an Alltech Altima C18 column. The analytical method was validated for linearity, precision, accuracy, specificity, and stability over a concentration range of 3-8000 ng/ml in rat plasma and 10-10,000 ng/ml in brain samples for both M6S and MOR. The validated method was applied to determine the PK profile of M6S in plasma after i.v., i.p., and oral dosing in male Sprague-Dawley rats. Rats were administered M6S by i.p. administration (5.6 and 10.0mg/kg) or orally (10 and 30 mg/kg) and bioavailability compared to an i.v. injection (1mg/kg) of M6S. The in vivo results indicate that M6S is not a prodrug of morphine, since M6S is not biotransformed into MOR in plasma after either i.p. or oral administration, and MOR was not detected in brain. The bioavailability of M6S was >93% and about 5% after i.p. and oral dosing, respectively. The low oral bioavailability of M6S may be due to poor permeation of the intestinal epithelial membrane. After i.p.-administration, M6S appears to reach brain tissues in low, but significant, concentrations.

Comparative Bioavailability Study of Two Imidapril Tablet Formulations in Indonesian Healthy Subjects

This study aimed to compare the bioavailability of two 10-mg Imidapril HCl tablet formulations using TENSIMID® as the test formulation and TANAPRESS® as the reference formulation. Twenty-seven healthy subjects completed a single-dosed, open-label, randomized, two-way crossover bioequivalence study under overnight fasting condition with one week wash-out period. The blood samples were collected from the subjects prior to administration and up to 12 hours after dosing. Plasma concentrations of imidapril were determined using LC-MS/MS method with TurboIon Spray mode. Pharmacokinetic parameters of AUC0-t, AUC0-∞and Cmax were tested for bioequivalence after log-transformation of data and ratios of tmax was evaluated non-parametrically. The estimated points and 90% confidence interval (CI) for AUC0-t, AUC0-∞and Cmax of imidapril were 93.04% (82.63-104.76%), 93.12% (82.84-104.67%), and 94.00% (80.96-109.14%), respectively. There was no statistically significant difference of tmax and t1/2 detected in both formulations (p

LC-MS/MS quantification of asymmetric dimethyl arginine and symmetric dimethyl arginine in plasma using surrogate matrix and derivatization with fluorescamine.

Aim: A novel LC-MS/MS method using a surrogate matrix and derivatization with fluorescamine was developed and validated for simultaneous quantification of asymmetric dimethyl arginineand symmetric dimethyl arginine. Methods & results: Asymmetric dimethyl arginine, symmetric dimethyl arginineand corresponding internal standards were extracted using protein precipitation and derivatization with fluorescamine followed by SPE. Derivatives were analyzed by turbo ion spray LC-MS/MS in the positive ion mode. Methodology was successfully transferred across multiple preclinical species and utilized in the support of several investigative studies. Conclusion: A new LC-MS/MS analytical methodology that utilizes a surrogate matrix and derivatization with fluorescamine was successfully developed and validated.

Development of LC-MS/MS method for simultaneous determination of Canagliflozin and Metformin in human plasma and its pharmacokinetic application in Indian population under fast and fed conditions

A novel, selective and sensitive method is developed for simultaneous estimation of canagliflozin and metformin and successfully applied to fast and fed pharmacokinetic studies in healthy Indian volunteers. The current study reports the development, optimization, and validation of liquid chromatography-mass spectrometry (LC-MS/MS) method for simultaneous quantification of canagliflozin and metformin in human plasma using deuterated canagliflozin D4 and metformin D6 as an internal standard (IS). The solid-phase extraction technique was employed where strata X polymeric reverse phase (30 mg-1 cc) SPE cartridges were used for the extraction of analytes and IS from plasma. The ACE 5 C18 column (50 × 4.6 mm, 5µ) was used to chromatograph the prepared samples. The mobile phase consisted of methanol and 5 mM ammonium trifluoroacetate in water, pH 5 (50:50, v/v) at a flow rate of 0.8 mL/min.Detection was performed by positive ion Turbo ion spray in Multiple reaction monitoring (MRM) mode, monitoring the transitions m/z 461.9 → m/z 191.1 and m/z 461.9 → m/z 267.2, for quantification of canagliflozin. The response of canagliflozin fragments m/z 461.9 → m/z 191.1 and m/z 461.9 → m/z 267.2 was combined. Also, for metformin transitions were monitored at m/z 130.0 → m/z 71.1. Full validation of the method was performed according to the United States Food and Drugs Administration (USFDA) guidelines. Linearity was in the range of 24.95–2806.55 ng/mL for canagliflozin and 24.99–3400.72 ng/mL for metformin. The mean extraction recovery of canagliflozin, canagliflozin D4, metformin, and metformin D6 was 77.240, 84.663, 66.747, and 67.449, respectively across four QC levels. This rapid method with the run time of 2.80 min allows the analysis of more than 400 plasma samples per day.

Development of simultaneous determination of empagliflozin and metformin in human plasma using liquid chromatography-mass spectrometry and application to pharmacokinetics.

A rapid and sensitive liquid chromatography-mass spectrometry method was developed, optimized, and validated for simultaneous quantification of empagliflozin and metformin in human plasma using empagliflozin D4and metformin D6 as an internal standard. Analytes and internal standard were extracted from plasma by optimized solid-phase extraction technique using Strata X polymeric reverse phase (30 mg-1cc) solid-phase extraction cartridges. The prepared samples were chromatographed on Orosil C18 column (150 × 4.6 mm, 3 µ). Separation was done by pumping isocratic mobile phase consisting of methanol and 10 mM ammonium trifluoroacetate (90:10, v/v) in positive ion mode at a flow rate of 0.8 mL/min. The API 3200 liquid chromatography-mass spectrometry system having turbo ion spray as an ion source coupled with Shimadzu Prominence ultrafast liquid chromatography system was operated under the selected reaction monitoring mode. Turbo ion spray ionization was used for mass transition of m/z 468.070/355.100 and m/z 130.072/71.200 for empagliflozin and metformin, respectively. A method was successfully validated for concentration range of 10.09-5013.46 ng/mL for both the analytes and according to the United States Food and Drugs Administration guidelines. The linearity was found to be in the range of 10.09-403.46 ng/mL for empagliflozin and 25.44-5013.46 ng/mL for metformin. The limit of quantification was found to be 10.09 ng/mL for empagliflozin and 25.44 ng/mL for metformin. Intra- and inter-day/between batch precision determination for empagliflozin and metformin, expressed as coefficient of variation were within the acceptance limits and ranged below 13.16%. A short run time of 3.3 min allows analysis of more than 400 plasma samples per day. The developed method was successfully applied to fasting pharmacokinetic study in healthy human volunteers. Results of incurred sample re-analysis were within the acceptance range of ±20% of original value, for 97.2% of samples reanalyzed for empagliflozin and 100% of samples reanalyzed for metformin.

Simultaneous Quantitative Analysis of Clarithromycin and Ranitidine, Probe Inhibitors of P-Glycoprotein and OCT1, to Evaluate Potential Pharmacokinetic Influence of Potential Transporter Substrates

Clartihromycin and ranitidine are accepted probe inhibitors in drug/drug interaction studies (DDI) with substrates of the multidrug transporters P-glycoprotein and OCT1. We assayed both drugs in human plasma, urine, and feces using LC–MS/MS with positive mass transition mode (AB Sciex API 2000 with turbo-ion spray) with fexofenadine as an internal standard. After protein denaturation with acetonitrile/water (50:50, v/v), the samples were centrifuged and the supernatants (10 µL) were injected into the chromatographic system (column: Supelco Ascentis® C18, 3 µm, 2.1 × 100 mm). The chromatography was performed with isocratic elution plied using ammonium formate buffer [(5 mM; pH 3.0)/acetonitrile, 40:60, v/v] as mobile phase at a flow rate of 200 μL min−1. The chromatograms were evaluated online with the internal standard method using peak-area-ratios for linear regression analysis weighted by 1/x (x = concentration) for the validation ranges in plasma between 0.005 and 2.0 µg mL−1, and for urine and feces between 0.005 and 10.0 µg mL−1. The method was shown to possess sufficient specificity, accuracy, precision and stability without matrix effects, thereby fulfilling current bioanalytical guidelines. The assay was suitable to simultaneous quantitative analysis of clarithromycin and ranitidine in plasma, urine, and feces of a DDI with trospium chloride to exclude major influence of trospium on the pharmacokinetics of the probe inhibitors. The assay is superior to other methods as it enables for the first time, quantification of the drugs in feces.

Determination of doravirine in human plasma using liquid-liquid extraction and HPLC-MS/MS.

Aim: A method to quantitate doravirine (MK-1439) in human plasma has been developed to support human clinical trials designed to evaluate the safety, pharmacokinetics and efficacy of the compound. Methodology & results: The analyte was extracted using liquid-liquid extraction, separated on a reverse phase HPLC column, and detected on an API-4000 mass spectrometer using a Turbo-Ion spray source in positive ionization mode coupled with multiple reaction monitoring mode was used for quantification. The dynamic range for the assay was 0.02-10 ng/ml using 100 μl of human plasma. Conclusion: The assay was found to be sensitive, selective and reproducible and applied to support the doravirine clinical development program.

Preparation and in vitro/in vivo evaluation of anastrozole reservoir-type intravaginal ring.

This report details the preparation of anastrozole (ATZ) reservoir-type intravaginal ring (IVR) and the detection of the concentration of ATZ in beagle dog plasma by liquid chromatography-tandem mass spectrometry (LC-MS/MS). An ATZ reservoir-type IVR which included ATZ silicone elastomer core and a nonactive silicone layer was manufactured by reaction injection moulding at 80°C for 20 min. An in vitro release experiment was performed under sink conditions and the samples were determined by high-performance liquid chromatography. A bioanalytical method was developed and validated for determination of ATZ in beagle dog plasma for IVR development. The analytical method consisted of the extraction of plasma samples and determination of ATZ by LC-MS/MS using buspirone as the internal standard. Separation was achieved on a Kinetex-C18 110A column (3 × 30 mm, 2.6 μm, Phenomenex) using step-gradient mobile phase and an isocratic flow rate consisting of formic acid. Protonated ions formed by a turboion spray in the positive mode was used to detect the analyte (ATZ) and internal standard. The MS-MS detection was performed on a triple quadrupole mass spectrometer equipped with electrospray ionization source. The mass spectrometer was operated in the multiple reaction monitoring mode. The mass transition ion-pair was followed as m/z from 294.10 to 225.08 for anastrozole and m/z from 386.23 to 122.11 for buspirone. The results proved that the correlation between in vitro and in vivo analyses was relatively good.

Rapid HPLC-ESI-MS/MS Analysis of Neurotransmitters in the Brain Tissue of Alzheimer's Disease Rats before and after Oral Administration of Xanthoceras sorbifolia Bunge.

In order to explore the potential therapeutic effect of Xanthoceras sorbifolia Bunge. against Alzheimer’s disease, an HPLC-MS/MS method has been developed and validated for simultaneous determination in rat brain of eight neurotransmitters, including dopamine, norepinephrine, 5-hydroxy-tryptamine, acetylcholine, l-tryptophan, γ-aminobutyric acid, glutamic acid and aspartic acid with a simple protein precipitation method for sample pre-treatment. The brain samples were separated on a polar functional group embedded column, then detected on a 4000 QTrap HPLC-MS/MS system equipped with a turbo ion spray source in positive ion and multiple reaction monitoring mode. The method was fully validated to be precise and accurate within the linearity range of the assay, and successfully applied to compare the neurotransmitters in the rat brain from four groups of normal, Alzheimer’s disease, and the oral administration group of X. sorbifolia extract and huperzine. The results indicated that brain levels of dopamine, norepinephrine and acetyl choline all decreased in the AD rats, while l-tryptophan showed an opposite trend. After administration of the Xanthoceras sorbifolia extract and huperzine, the level of acetyl choline and tryptophan returned to normal. Combination of the metabolic analysis, the results indicated that acetyl choline and l-tryptophan could be employed as therapy biomarkers for AD, and the results shown that the crude extract of the husks from Xanthoceras sorbifolia might ameliorate the impairment of learning and memory in the Alzheimer’s disease animal model with similar function of AchEI as huperzine. The established method would provide an innovative and effective way for the discovery of novel drug against Alzheimer’s disease, and stimulate a theoretical basis for the design and development of new drugs.

Determination of Chlorpromazine, Haloperidol, Levomepromazine, Olanzapine, Risperidone, and Sulpiride in Human Plasma by Liquid Chromatography/Tandem Mass Spectrometry (LC-MS/MS).

Background and Objective In this study, turbo-ion spray as an interface of tandem mass spectrometry (MS/MS) was performed for sensitive and accurate quantification of chlorpromazine, haloperidol, levomepromazine, olanzapine, risperidone, and sulpiride in plasma samples. Methods Separation was performed by gradient reversed phase high-performance liquid chromatography using a mobile phase containing ammonium formiate 2 mM, pH 2.7, and acetonitrile flowing through a Restek PFP Propyl C18 analytical column (50 mm×2.1 mm i.d.) with particle size of 5 µm, at a flow rate of 800 µL/min. Positive ion fragments were detected in multiple reaction monitoring (MRM) mode. Sample preparation was achieved by solid phase extraction (SPE) (Oasis HLB). Results Mean extraction recoveries ranged from 82.75% to 100.96%. The standard calibration curves showed an excellent linearity, covering subtherapeutic, therapeutic, and toxic ranges. Intraday and interday validation using quality control (QC) samples were performed. The inaccuracy and imprecision were below 12% at all concentration levels. The limits of detection (LOD) and quantification (LOQ) for all analytes were under therapeutic ranges for all tested analytes. Thus, the proposed method was sensitive enough for the detection and determination of subtherapeutic levels of these antipsychotics in plasma samples. No interference of endogenous or exogenous molecules was observed and no carryover effects were recorded. Conclusion According to the results, the proposed method is simple, specific, linear, accurate, and precise and can be applied for antipsychotic analysis in clinical routine. This method was applied for the determination of the tested antipsychotics in plasma samples taken from 71 individuals.

Quantification of Cefepime, Meropenem, Piperacillin, and Tazobactam in Human Plasma Using a Sensitive and Robust Liquid Chromatography-Tandem Mass Spectrometry Method, Part 1: Assay Development and Validation.

The highly variable pharmacokinetics of β-lactam antibiotics and β-lactamase inhibitors poses a significant challenge to clinicians in ensuring appropriate antibiotic doses in critically ill patients. Therefore, routine monitoring of plasma concentrations is important for individualization of antimicrobial therapy. Accordingly, a simple and robust analytical method for the simultaneous measurement of multiple β-lactam antibiotics and β-lactamase inhibitors is highly desirable to ensure quick decisions on dose adjustments. In this study, a sensitive, simple, and robust method for the simultaneous quantification of cefepime, meropenem, piperacillin, and tazobactam in human plasma was developed and rigorously validated according to FDA guidance. Sample extraction was accomplished by simple protein precipitation. Chromatographic separation of analytes was achieved using stepwise gradient elution. Analytes were monitored using tandem mass spectrometry (MS/MS) with a turbo ion spray source in positive multiple-reaction-monitoring mode. The calibration curve ranged from 0.5 to 150 μg/ml for cefepime, 0.1 to 150 μg/ml for meropenem and piperacillin, and 0.25 to 150 μg/ml for tazobactam. Inter- and intraday precision and accuracy, sensitivity, selectivity, dilution integrity, matrix effect, extraction recovery, and hemolysis effect were investigated for all four analytes, and the results met the acceptance criteria. Compared to other reported methods, our method is more robust because of the combination of the following features: (i) a simple sample extraction procedure, (ii) a short sample run time, (iii) a wide dynamic range, and (iv) the small plasma sample volume needed. Since our method already covers β-lactams and a β-lactamase inhibitor with highly heterogeneous physicochemical properties, further antibiotic candidates may easily be incorporated into this multianalyte method.

Quantification of metronidazole in human plasma using a highly sensitive and rugged LC-MS/MS method for a bioequivalence study.

A highly sensitive, selective and rugged method has been described for the quantification of metronidazole (MTZ) in human plasma by liquid chromatography-tandem mass spectrometry using metronidazole-d4 as the internal standard (IS). The analyte and the IS were extracted from 100 μL plasma by liquid-liquid extraction. The clear samples obtained were chromatographed on an ACE C18 (100 × 4.6 mm, 5 μm) column using acetonitrile and 10.0 mm ammonium formate in water, pH 4.00 (80:20, v/v) as the mobile phase. A triple quadrupole mass spectrometer system equipped with turbo ion spray source and operated in multiple reaction monitoring mode was used for the detection and quantification of MTZ. The calibration range was established from 0.01 to 10.0 μg/mL. The results of validation testing for precision and accuracy, selectivity, matrix effects, recovery and stability complied with current bioanalytical guidelines. A run time of 3.0 min permitted analysis of more than 300 samples in a day. The method was applied to a bioequivalence study with 250 mg MTZ tablet formulation in 24 healthy Indian males.

A novel and rapid LC–MS/MS assay method for the determination of canagliflozin in human plasma by solid phase extraction technique and its application to a pharmacokinetic study

ObjectiveThe aim of the present study was to develop and validate a simple LC-MS/MS for the determination of canagliflozin in human plasma. The proposed method is fully validated for the precision, accuracy and various stability tests. MethodsThe linearity of the method was established in the concentration range 10.3–6019 ng/mL with r2 ≥ 0.99. Solid-phase extraction (SPE) technique was used for the extraction of canagliflozin and canagliflozin d4 as internal standard (IS). The clean samples injected into LC-MS/MS system set with turbo ion spray (TIS) source and operated in multiple reaction monitoring (MRM) mode. ResultsPrecision and accuracy of the method was determined using five analytical batches and results were meeting as per the recent US FDA guidelines. Also, a variety of stability tests were executed in plasma samples and in neat samples are comply recent bioanalytical guidelines. The total run time was 2.5 min to analyze canagliflozin and the IS, thus can analyze more than 300 samples in a day. ConclusionThe proposed LC-MS/MS method is novel, sensitive and rapid for the quantitative determination of canagliflozin in human plasma. The method describes the complete method development and validation process and is well suitable for pharmacokinetic or bioavailability/bioequivalence application.

Fast and Straightforward Method for the Quantification of Pazopanib in Human Plasma Using LC-MS/MS.

Pazopanib is an angiogenesis inhibitor approved for renal cell carcinoma and soft-tissue sarcoma. Studies indicate that treatment with pazopanib could be optimized by adapting the dose based on measured pazopanib plasma concentrations. We describe the validation and clinical application of a fast and straightforward method for the quantification of pazopanib in human plasma for the purpose of therapeutic drug monitoring and bioanalytical support of clinical trials. Stable isotopically labeled C,H3-pazopanib was used as internal standard. Plasma samples were prepared for analysis by protein precipitation using methanol and diluted with 10 mmol/L ammonium hydroxide buffer. Chromatographic separation was performed on a C18 column using isocratic elution with ammonium hydroxide in water and methanol. For detection, a tandem mass spectrometer, equipped with a turbo ion spray interface was used in positive ion mode at m/z 438 → m/z 357 for pazopanib and m/z 442 → m/z 361 for the internal standard. Final runtime was 2.5 minutes. All validated parameters were within pre-established limits and fulfilled the FDA and EMA requirements for bioanalytical method validation. After completion of the validation, the routine application of the method was tested by analyzing clinical study samples that were collected for the purpose of therapeutic drug monitoring. In conclusion, the described method was successfully validated and was found to be robust for routine application to analyze samples from cancer patients treated with pazopanib.

Simultaneous determination of ferulic acid and gastrodin of Tianshu Capsule in rat plasma by ultra-fast liquid chromatography with tandem mass spectrometry and its application to a comparative pharmacokinetic study in normal and migraine rats.

Tianshu Capsule, consisting of Ligusticum chuanxiong Hort and Gastrodia elata Blume, is a widely used Traditional Chinese Medicine preparation for the treatment of migraine. Ferulic acid and gastrodin are main active constituents in Ligusticum chuanxiong Hort and Gastrodia elata Blume, and have been used as marker components for quality control of Tianshu Capsule. In this study, a selective, sensitive, and reliable ultra-fast liquid chromatography with tandem mass spectrometry method was developed for simultaneous determination of ferulic acid and gastrodin in rat plasma using geniposide as internal standard. The plasma samples were extracted by protein precipitation with methanol after acidification and separated on a Shim-Pack XR-ODS C18 column (75 × 3.0mm, 2.2μm) using gradient elution with a mobile phase consisting of water (containing 0.1% formic acid) and acetonitrile at a flow rate of 0.6mL/min. Detection was performed on 3200 QTRAP mass spectrometry equipped with turbo ion spray source in negative ionization mode. Validation parameters were within acceptable ranges. The validated method was applied to compare the pharmacokinetic profiles of ferulic acid and gastrodin in normal and migraine rats. Our results showed that there were remarkable differences in the pharmacokinetic properties of the analytes between the normal and migraine groups.