Development of a rapid LC-MS/MS assay to determine letrozole in human plasma and its application in a pharmacokinetic study after oral administration

AbstractLetrozole is one of the third generation aromatase inhibitors. It is suitable for the treatment of postmenopausal patients with advanced breast cancer and early treatment of breast cancer. It is necessary to develop a rapid, reliable, selective and sensitive LC–MS/MS assay to determine letrozole in human plasma to evaluate the clinical efficacy and adverse reactions with clinical pharmacokinetic and therapeutic drug monitoring. Separation was carried out on a Kromasil-C18 column using acetonitrile-water (55: 45, v/v) as mobile phase. Detection was carried out by multiple reaction monitoring on a 3200Qtrap mass spectrometry. The method needed one-step protein precipitation procedure only, and the cycle time was 2.5 min allowing 500–550 samples per day. It was linear within 0.30–50.00 ng/mL for plasma with the limit of detection (LOD) of 0.030 ng/mL. The intra- and inter-day RSD were 5.51–8.63%, 2.28–9.95% and the RE was 0.18–1.65%. The recovery rates of letrozole and internal standard for plasma were 89.30–98.55%. Letrozole was stable under all the conditions in the study. The method was sensitive enough to quantitate letrozole over a period of 288 h after giving a single oral dose of 2.5 mg–24 Chinese healthy volunteers. The absorption of letrozole was rapid with small individual difference, the tissue distribution of letrozole was more than that in blood, and the clearance was slow. Letrozole was similar to three-compartment model in vivo. Due to metabolism and excretion, the AUCs of letrozole varied greatly among individuals.

Download Full-text

Development and Validation of an Up-to-Date Highly Sensitive UHPLC-MS/MS Method for the Simultaneous Quantification of Current Anti-HIV Nucleoside Analogues in Human Plasma

Pharmaceuticals ◽

10.3390/ph14050460 ◽

2021 ◽

Vol 14 (5) ◽

pp. 460

Author(s):

Amedeo De Nicolò ◽

Alessandra Manca ◽

Alice Ianniello ◽

Alice Palermiti ◽

Andrea Calcagno ◽

...

Keyword(s):

Human Plasma ◽

Multiple Reaction Monitoring ◽

Internal Standard ◽

Reversed Phase ◽

Therapeutic Drug ◽

People Living With Hiv ◽

Simultaneous Quantification ◽

Working Solution ◽

Tenofovir Alafenamide ◽

Combination Regimens

Therapeutic options to treat HIV infection have widened in the past years, improving both effectiveness and tolerability, but nucleoside reverse transcriptase inhibitors (NRTIs) are still considered the standard backbone of the combination regimens. Therapeutic drug monitoring (TDM) can be useful for these drugs, due to concentration–effect relationship, with risk of ineffectiveness, toxicity or adherence concerns: in this scenario, robust and multiplexed methods are needed for an effective TDM activity. In this work, the first validated ultra-high spectrometry liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) method is described for the high-sensitive simultaneous quantification of all the currently used NRTIs in human plasma, including tenofovir alafenamide (TAF), following FDA and EMA guidelines. The automated sample preparation consisted in the addition of an internal standard (IS) working solution, containing stable-isotope-linked drugs, protein precipitation and drying. Dry extracts were reconstituted with water, then, these underwent reversed phase chromatographic separation: compounds were detected through electrospray ionization and multiple reaction monitoring. Accuracy, precision, recovery and IS-normalized matrix effect fulfilled guidelines’ requirements. The application of this method on samples from people living with HIV (PLWH) showed satisfactory performance, being capable of quantifying the very low concentrations of tenofovir (TFV) in patients treated with TAF.

Download Full-text

Ultra-Performance Liquid Chromatography Coupled with a Triple Quadrupole Mass Spectrometric Method for the Quantification of Anti-epileptic drugs Methsuximide and Normesuximide in Human Plasma and its Application in a Pharmacokinetic Study

Current Pharmaceutical Analysis ◽

10.2174/1573412917666210211122311 ◽

2021 ◽

Vol 17 ◽

Author(s):

Karthik Rajendran ◽

Karthika Anoop ◽

Krishnaveni Nagappan ◽

Genguchetti Mohan Sekar ◽

Sankham Devendran Rajendran

Keyword(s):

Human Plasma ◽

Pharmacokinetic Study ◽

Multiple Reaction Monitoring ◽

Internal Standard ◽

Current Method ◽

Ultra Performance Liquid Chromatography ◽

Mass Spectrometric ◽

Therapeutic Drug ◽

Spectrometric Method ◽

Mass Spectrometric Method

Background: Extensive therapeutic drug monitoring needs an analytical method for efficient and sensitive quantification of analytes of interest in clinical pharmacology. Objective: A rapid, robust, sensitive and simple UPLC-MS/MS method to quantify Methsuximide (Ms) and N-desmethyl methsuximide/Normesuximide (MsMET) in human plasma was optimized, developed, and validated for application in a pharmacokinetic study. Method: Reverse phased chromatography was performed using Zorbax SB-C18, 4.6 x 75 mm., 3.5 µm as stationary phase, methanol and 0.1% formic acid (60:40 v/v) as mobile phase which was delivered isocratically at a flow rate of 0.9 mL/min. The sample injection volume was 5 µL. Mass spectrometric quantification of the analytes was performed using positive electrospray ionization as mass interface along with multiple reaction monitoring (MRM) as acquisition mode. Results: The selected mass transition ions for analyte, metabolite and its respective internal standards are as follows, precursor ion (m/z) and product ion (m/z): Ms (204.06 and 119.02), MsMET (190.05 and 119.82), Ms internal standard (MsIS) (209.17 and 124.02), and MsMET internal standard (MsMETIS) (195.09 and 124.16), respectively. The current method was found to be linear for Ms (60.72-5920 ng/mL) and MsMET (60.38-6010 ng/mL) with r2 values not less than 0.999. The mean recoveries of all analytes ranged between 71.37 and 86.38 percentage. Conclusion: This method was validated in accordance with USFDA’s bioanalytical guidelines. This method could be applied for a routine analysis of Ms and MsMET in clinical pharmacological practice.

Download Full-text

BIO-ANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR THE SIMULTANEOUS ESTIMATION OF DECITABINE AND CEDAZURIDINE IN HUMAN PLASMA USING LC-MS/MS

International Journal of Applied Pharmaceutics ◽

10.22159/ijap.2021v13i5.41744 ◽

2021 ◽

pp. 257-262

Author(s):

SAI PRUDHVI N. ◽

VENKATESWARLU B. S. ◽

KUMUDHAVALLI M. V. ◽

MURUGANANTHAM V.

Keyword(s):

Mass Spectrometry ◽

Quality Control ◽

Human Plasma ◽

Method Development ◽

Limit Of Detection ◽

Multiple Reaction Monitoring ◽

Internal Standard ◽

Simultaneous Estimation ◽

Limit Of Quantification ◽

Validation Parameters

Objective: The present work aimed to develop a novel, reliable and accurate Liquid Chromatography-Mass Spectrometry/Mass spectrometry (LC-MS/MS) method for the simultaneous quantification of Decitabine and Cedazuridine a combined medication used for the treatment of chronic myelomonocytic leukemia in human plasma. Methods: Talazoparib drug is used as an internal standard in the study. Both the analytes and internal standard were isolated from 100 ml plasma samples by liquid-liquid extraction and then chromatographed on Zorbax SB-CN (4.6 mm×75 mm, 3.5 µm) column with a mobile phase consisting of 0.1 % ammonium formate and methanol in the ratio of 65:45 (v/v) pumped at 0.5 ml/min. The method had a chromatographic total run time of 5 min. Results: The developed method gave a symmetric peak at a retention time of 1.7 min for Decitabine, 2.2 min for Cedazuridine, 3.5 min for Talazoparib and satisfied all the peak properties as per USP guidelines. The mass spectral characterization of separated analytes in the LC method was performed using a mass detector operated at Multiple Reaction Monitoring mode with precursor-to-product ion transitions at m/z of 229 to m/z of 114 as MH+ion for Decitabine, m/z of 269 to m/z of 118 as MH+ion for Cedazuridine. A very sensitive limit of detection of 0.3 ng/ml was observed and showed a calibration curve linear over the concentration range of LLOQ (lower limit of quantification) to 500 ng/ml. The other validation parameters were found to have acceptable accuracy, precision, linearity, and selectivity. The mean extraction concentration was acceptable and very high for both the analytes in HQC (high-quality control concentration), MQC (medium quality control concentration) and LLOQ levels. The peak area response ratio of Decitabine and Cedazuridine with the internal standard in freeze-thaw, short term and long term stability studies was found to be acceptable confirms that the method is stable. Conclusion: It can be concluded that the proposed method is specific, accurate, and precise and could be used for the simultaneous estimation of Decitabine and Cedazuridine in human plasma.

Download Full-text

An LC-MS/MS Validated Method for Quantification of Chlorzoxazone in Human Plasma and Its Application to a Bioequivalence Study

Journal of Chromatographic Science ◽

10.1093/chromsci/bmz052 ◽

2019 ◽

Vol 57 (8) ◽

pp. 751-757

Author(s):

Jiake He ◽

Ning Li ◽

Jiaqiu Xu ◽

Jing Zhu ◽

Yang Yu ◽

...

Keyword(s):

Human Plasma ◽

Multiple Reaction Monitoring ◽

Internal Standard ◽

Bioequivalence Study ◽

Therapeutic Drug ◽

Reference Formulation ◽

Chromatography Tandem Mass Spectrometry ◽

Liquid Chromatography Tandem Mass ◽

One Step

Abstract A simple, sensitive, specific, accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for determination of chlorzoxazone in human plasma was developed and validated to evaluate the pharmacokinetic characteristics of chlorzoxazone test or reference formulation. Sample preparation was achieved by one step protein precipitation and dilution with acetontrile. The chromatographic separation was performed at 40°C with a gradient mobile phase (0.3 mL/min) and a Shimadzu VP-ODS C18 analytical column (column size: 150 × 2.0 mm). TSQ quantum access triple-quadrapole MS/MS detection was operated in a negative mode by multiple reaction monitoring. Ion transitions at m/z 168.0→132.1 for chlorzoxazone and m/z 451.3→379.3 for repaglinide (internal standard) were used for the LC-MS/MS analysis. The calibration was linear (r ≥ 0.995) over the tested concentration range of 0.2–20 μg/mL for chlorzoxazone in plasma. Precision, accuracy, recovery, matrix effect and stability for chlorzoxazone were evaluated and were excellent within the range of tested concentrations. This method was successfully applied to a bioequivalence study in 20 healthy Chinese volunteers. This method could also contribute to the personalized medication and therapeutic drug monitoring of chlorzoxazone.

Download Full-text

LC-MS/MS determination of glimepiride in human plasma with a high recovery at picogram scale: Pharmacokinetic study after oral administration

Acta Chromatographica ◽

10.1556/1326.2020.00841 ◽

2020 ◽

Author(s):

Ahmed K. Kammoun ◽

Zuhier Ahmed Awan ◽

Tarek Elawady ◽

Alaa Khedr ◽

Mohamed I. El-Awady

Keyword(s):

Oral Administration ◽

Human Plasma ◽

Pharmacokinetic Study ◽

Methylene Chloride ◽

Limit Of Detection ◽

Solid Phase ◽

Multiple Reaction Monitoring ◽

Internal Standard ◽

Range Limit ◽

Extraction Recovery

AbstractAlthough glimepiride (GLM) is the first-line treatment of Type II diabetes, low extraction recovery is still a significant limitation in previous plasma analysis methods. An optimized solid-phase extraction method of GLM in human plasma with excellent extraction recovery, 100 ± 0.06%, was achieved using liquid chromatography-electrospray ionization tandem mass spectrometry and Gliclazide (GLZ) as an internal standard. GLM was extracted from 100 µL plasma sample using Sep-Pak® vac 1cc (100 mg) C18 column and methylene chloride: methanol (2: 1, v/v) as eluant. Both GLM and GLZ were monitored by a triple quad mass spectrometer applying positive multiple reaction monitoring mode (+MRM). The protonated precursor ions and product ions of GLM and GLZ were m/z 491(352), and m/z 324 (127), respectively. The detection and measurement of low levels of GLM in human plasma reached to picogram range (limit of detection (LOD) = 60 pg/mL, limit of quantification (LOQ) = 200 pg/mL). The method was validated in terms of selectivity, linearity, recovery, accuracy, and precision. The method was successfully applied to the pharmacokinetic study of GLM following oral administration of 1 mg GLM tablets to 12 healthy volunteers.

Download Full-text

Analysis of Pembrolizumab in Human Plasma by LC-MS/HRMS. Method Validation and Comparison with Elisa

Biomedicines ◽

10.3390/biomedicines9060621 ◽

2021 ◽

Vol 9 (6) ◽

pp. 621

Author(s):

Aurélien Millet ◽

Nihel Khoudour ◽

Jérôme Guitton ◽

Dorothée Lebert ◽

François Goldwasser ◽

...

Keyword(s):

Mass Spectrometry ◽

Human Plasma ◽

High Resolution Mass Spectrometry ◽

Internal Standard ◽

Therapeutic Drug ◽

Limit Of Quantification ◽

Immunoglobulin G4 ◽

Positive Mode ◽

Surrogate Peptide ◽

First Time

Pembrolizumab is a humanized immunoglobulin G4-kappa anti-PD1 antibody used in the treatment of different solid tumors or haematological malignancies. A liquid chromatography coupled with a high resolution mass spectrometry (orbitrap technology) method was fully developed, optimized, and validated for quantitative analysis of pembrolizumab in human plasma. A mass spectrometry assay was used for the first time a full-length stable isotope-labelled pembrolizumab-like (Arginine 13C6-15N4 and Lysine 13C6-15N2) as an internal standard; the sample preparation was based on albumin depletion and trypsin digestion and, finally, one surrogate peptide was quantified in positive mode. The assay showed good linearity over the range of 1–100 μg/mL, a limit of quantification at 1 μg/mL, excellent accuracy from 4.4% to 5.1%, and also a between-day precision below 20% at the limit of quantification. In parallel, an in-house ELISA was developed with a linearity range from 2.5 to 50 µg/mL. Then, results were obtained from 70 plasma samples of cancer patients that were treated with pembrolizumab and quantified with both methods were compared using the Passing-Bablok regression analysis and Bland-Altman plotting. The LC-MS/HRMS method is easy to implement in the laboratory for use in the context of PK/PD studies, clinical trials, or therapeutic drug monitoring.

Download Full-text

Quantification of plasma remdesivir and its metabolite GS-441524 using liquid chromatography coupled to tandem mass spectrometry. Application to a Covid-19 treated patient

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2020-0612 ◽

2020 ◽

Vol 58 (9) ◽

pp. 1461-1468 ◽

Cited By ~ 2

Author(s):

Jean-Claude Alvarez ◽

Pierre Moine ◽

Isabelle Etting ◽

Djillali Annane ◽

Islam Amine Larabi

Keyword(s):

Mass Spectrometry ◽

Liquid Chromatography ◽

Half Life ◽

Limit Of Detection ◽

Treated Patient ◽

Internal Standard ◽

Therapeutic Drug ◽

Active Metabolites ◽

Limit Of Quantification ◽

Positive Mode

AbstractObjectivesA method based on liquid chromatography coupled to triple quadrupole mass spectrometry detection using 50 µL of plasma was developed and fully validated for quantification of remdesivir and its active metabolites GS-441524.MethodsA simple protein precipitation was carried out using 75 µL of methanol containing the internal standard (IS) remdesivir-13C6 and 5 µL ZnSO4 1 M. After separation on Kinetex® 2.6 µm Polar C18 100A LC column (100 × 2.1 mm i.d.), both compounds were detected by a mass spectrometer with electrospray ionization in positive mode. The ion transitions used were m/z 603.3 → m/z 200.0 and m/z 229.0 for remdesivir, m/z 292.2 → m/z 173.1 and m/z 147.1 for GS-441524 and m/z 609.3 → m/z 206.0 for remdesivir-13C6.ResultsCalibration curves were linear in the 1–5000 μg/L range for remdesivir and 5–2500 for GS-441524, with limit of detection set at 0.5 and 2 μg/L and limit of quantification at 1 and 5 μg/L, respectively. Precisions evaluated at 2.5, 400 and 4000 μg/L for remdesivir and 12.5, 125, 2000 μg/L for GS-441524 were lower than 14.7% and accuracy was in the [89.6–110.2%] range. A slight matrix effect was observed, compensated by IS. Higher stability of remdesivir and metabolite was observed on NaF-plasma. After 200 mg IV single administration, remdesivir concentration decrease rapidly with a half-life less than 1 h while GS-441524 appeared rapidly and decreased slowly until H24 with a half-life around 12 h.ConclusionsThis method would be useful for therapeutic drug monitoring of these compounds in Covid-19 pandemic.

Download Full-text

1319. Pharmacokinetics of Ceftolozane/Tazobactam in Patients with Burns

Open Forum Infectious Diseases ◽

10.1093/ofid/ofaa439.1501 ◽

2020 ◽

Vol 7 (Supplement_1) ◽

pp. S670-S671

Author(s):

Ronald G Hall ◽

Jotam Pasipanodya ◽

William C Putnam ◽

John Griswold ◽

Sharmila Dissanaike ◽

...

Keyword(s):

Human Plasma ◽

Kidney Injury ◽

Compartment Model ◽

Pharmacokinetic Parameters ◽

Therapeutic Drug ◽

Population Pharmacokinetic ◽

Parameter Estimates ◽

Population Parameter ◽

Full Thickness ◽

Severe Burns

Abstract Background Antimicrobial dosing in moderate/severe burns patients is complicated due to the potential unpredictable hyperdynamic pathophysiologic states including 1) hypoproteinemia, 2) acute kidney injury and 3) onset of septicemia. Therefore, distribution assumptions about the population pharmacokinetic (PopPK) profiles of either endogenous or xenobiotic pharmacophores in this patient population can lead to biased parameter estimates. In order to prevent potential bias an agnostic nonparametric adaptive grid approach to describe ceftolozane/tazobactam (C/T) PopPK profiles in patients with partial- and full-thickness burns was employed. Methods A human clinical PK study in burn patients was conducted using the standard approved dose of C/T (2 grams/1 gram). A single intravenous dose was administered over 60 minutes. Whole blood was obtained pre-dose and at 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12, 16, and 24 hours following the start of infusion. LC-MS/MS bioanalytical methods were developed, validated and employed to determine C/T concentrations in human plasma. PopPK were modeled using Pmetrics package for R. One-, two- and three-compartment models were examined and compared. The influence of several parameters, including %body surface area burns, creatinine clearance (CrCL), weight, albumin and age were tested. Results The bioanalytical method for determination of C/T in human plasma met all recommended criteria of the LC-MS/MS. Five males and one female (ages 24 to 66 years), contributed 148 plasma PK samples. The female had 35% partial-thickness burns. The males had full-thickness burns ranging from 27 to 66%. The median CrCL was 104 mL/min (range 73-148 mL/min). Two-compartment model with absorption (Ka) from compartment 1 to 2 and elimination from compartment 2 (Ke), with nonlinear interactions between C/T elimination and CrCL best described the data. Figure A show that bias was minimal. Importantly, both drugs exhibited marked variability for both volume and elimination (Table), since volume was bimodally distributed (Figure B). A) Observation-versus-Prediction; B) Estimated Ke, V and Ka population parameter densities Summary of pharmacokinetic parameters Conclusion C/T exhibited high variability surpassing that observed with severe infections, suggesting that dose adjustment and/or may be therapeutic drug monitoring may be needed to balance target attainment from dose-related toxicities. Disclosures Ronald G. Hall, II, PharmD, MSCS, Medical Titan Group (Grant/Research Support)Merck (Research Grant or Support)

Download Full-text

NEW METHOD DEVELOPMENT AND VALIDATION FOR THE DETERMINATION OF FEBUXOSTAT IN HUMAN PLASMA BY LIQUID CHROMATOGRAPHY–MASS SPECTROMETRY

International Journal of Pharmacy and Pharmaceutical Sciences ◽

10.22159/ijpps.2016v8i9.11968 ◽

2016 ◽

Vol 8 (9) ◽

pp. 61 ◽

Cited By ~ 6

Author(s):

Narottam Pal ◽

Avanapu Srinivasa Rao ◽

Pigilli Ravikumar

Keyword(s):

Mass Spectrometry ◽

Liquid Chromatography ◽

Human Plasma ◽

Method Development ◽

Multiple Reaction Monitoring ◽

Internal Standard ◽

New Method ◽

Liquid Chromatography Mass Spectrometry ◽

Chromatography Mass Spectrometry

Objective: To develop a new method and validate the same for the determination of Febuxostat (FBS) in human plasma by liquid chromatography–mass spectrometry (LCMS).Methods: The present method utilized reversed-phase high-performance liquid chromatography with tandem mass spectroscopy. Febuxostat D9 (FBS D9) was used as internal standard (IS). The analyte and internal standard were separated from human plasma by using solid phase extraction method. Zorbax Eclipse XDB, C8, 100 mm x 4.6 mm, 3.5 µm column was used and HPLC grade acetonitrile, 5 millimolar (mM) ammonium format (80: 20, v/v) as mobile phase, detected by mass spectrometry operating in positive ion and multiple reaction monitoring modes.Results: The parent and production transitions for FBS and internal standard were at m/z 317.1→261.0 and 326.1→262.0 respectively. The method was validated for system suitability, specificity, carryover effect, linearity, precision, accuracy, matrix effect, sensitivity and stability. The linearity range was from 20.131 ng/ml to10015. 534 ng/ml with a correlation coefficient of 0.999. Precision results (%CV) across six quality control samples were within the limit. The percentage recovery of FBS and internal standard from matrix samples was found to be 76.57% and 75.03% respectively.Conclusion: Present study describes new LC-MS method for the quantification of FBS in a pharmaceutical formulation. According to validation results, it was found to be a simple, sensitive, accurate and precise method and also free from any kind of interference. Therefore the proposed analytical method can be used for routine analysis for the estimation of FBS in its formulation.

Download Full-text

Mibefradil inhibits the proliferation of triple-negative breast cancer by targeting AURKA to regulate apoptosis signal pathway

10.21203/rs.3.rs-210031/v1 ◽

2021 ◽

Author(s):

Xu Han ◽

Xiujuan Qu ◽

Beixing Liu ◽

Yizhe Wang ◽

Yang Cheng ◽

...

Keyword(s):

Breast Cancer ◽

Cell Cycle ◽

Molecular Docking ◽

Triple Negative Breast Cancer ◽

Triple Negative ◽

Therapeutic Drug ◽

Specific Gene ◽

In Vivo Models

Abstract Background: Triple negative breast cancer (TNBC) is a tumor characterized by high recurrence and mortality, but without effective targeted therapy. It is urgent to explore new treatment strategy to improve the efficacy of TNBC therapy. Methods: Transcriptomic profiling datasets of TNBC were used for screening TNBC specific gene sets. Drug prediction was performed in Connectivity map (CMap) database. Molecular docking method was used for analyzing drug targets. In vitro and in vivo models of TNBC were constructed to examine the drug efficacy. Results: We screened out Mibefradil, a T-type Ca2+ channel blocker, might be a potential therapeutic drug for TNBC by transcriptomics and bioinformatics analysis, and verified that Mibefradil could inhibit the proliferation of TNBC cells by inducing apoptosis and cell cycle arrest. Furthermore, by network pharmacology and molecular docking analysis, AURKA was predicted as the most possible drug target of Mibefradil. Finally, it was proved that Mibefradil treatment could induce apoptosis by decreasing protein expression and phosphorylation level of AURKA in vitro and in vivo. Conclusions: Mibefradil played anti-cancer role in TNBC cells by targeting to AURKA to induce cell cycle and apoptosis. Our results repurposed Mibefradil as a potential targeted drug of TNBC and provided a fundamental research for a novel strategy TNBC treatment.

Download Full-text