Positive Mode Electrospray Research Articles

Application of liquid chromatography-mass spectrometry for the determination of semaglutide in human serum in clinical pharmacokinetic studies

Introduction. Semaglutide preparations are an important therapeutic option for patients suffering from type 2 diabetes mellitus and obesity due to their high efficacy and the expected increase in the prevalence of these diseases. Consequently, there is a growing need for the development of domestic analogs of semaglutide requiring bioequivalence studies. This study proposes the use of high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) as an alternative to the widely used immunoassay for the quantitative determination of semaglutide in human serum.Aim. To develop and validate a method for the quantitative determination of semaglutide in human serum using HPLC-MS/MS.Materials and methods. Serum sample preparation was based on protein precipitation using an acetonitrile-methanol mixture. Liraglutide was selected as the internal standard. The mobile phase consisted of 0.3% formic acid in water and acetonitrile. The stationary phase was represented by a Phenomenex Kinetex C18 chromatographic column 100×3.0 mm, 5 μm, 100 Å. Ionization of semaglutide and liraglutide was performed in positive electrospray mode. Detection was carried out in multiple reaction monitoring mode (MRM).Results. The method demonstrated high accuracy and precision, with relative error and relative standard deviation values of less than 15% across all quality control levels. The confirmed analytical ranges of the method were 0.50–200.00 ng/mL and 1.00–800.00 ng/mL. Over 3.400 volunteer samples were analyzed as part of the studies. Compared to the ELISA method, the proposed method provides higher selectivity and reproducibility of measurements.Conclusions. The method has been developed that provides reproducible quantitative determination of semaglutide in human blood serum. The method was validated in accordance with EAEU requirements and was successfully applied in bioequivalence studies of semaglutide GP40331 (GEROPHARM LLC, Russia). The method is suitable for conducting pharmacokinetic studies of other semaglutide preparations.

Compaction, Relaxation, and Linearization of Megadalton-Sized DNA Plasmids: DNA Structures Probed by CD-MS.

High purity plasmid DNA is a raw material for recombinant protein production as well as an active ingredient in DNA vaccines. There are four primary plasmid structures that can be observed in a typical plasmid formulation: supercoiled, relaxed (circular), linearized, and condensed. Determining what structures are present in a sample is important, as the structure can affect activity; the supercoiled structure has the highest activity, and >90% supercoiled is desired for industry standards. Recently, charge detection mass spectrometry (CD-MS) was used to distinguish two of the structures, supercoiled and condensed, by measuring the charge deposited on the ions by positive mode electrospray. Here, CD-MS is used to probe the structures of DNA plasmids during compaction with polycations, and through enzymatic treatment to relax and linearize plasmids. We find that all four structural types for plasmid DNA have unique charging profiles that can be distinguished using CD-MS. The extent of mechanical shearing of the DNA plasmids during electrospray is strongly influenced by the structural type.

LC-MS/MS Bioanalytical Procedure for Atrasentan in Human Plasma: Method Development and Validation

11ABSTRACTObjectives: The objective of this work is to establish a bioanalytical technique for the quantification of atrasentan. The employed methodology for the detection and quantification of atrasentan in human plasma has a high degree of specificity, sensitivity, and simplicity. Methods: Shimadzu pumps were utilized in conjunction with an Autosampler Agilent Zorbax XDB C18 2.1 × 50 mm, 5 μm column employed as a stationary phase to achieve the chromatographic separation. Acetonitrile (0.1% formic acid) and 5 mM ammonium formate made up the mobile phase, which was optimized to have a 70:30% v/v ratio. Isocratic elution was employed in the experiment, with a flow rate of 0.150 mL/min. The entire cycle took 3.0 minutes, using a 10 μL injection volume. Results: The detection procedure is executed utilizing positive ion mode electrospray ionization (ESI) mass spectrometry under atmospheric pressure. For atrasentan, the precursor to product ion transitions used for quantification was m/z 511.623 to 354.15 and for the internal standard, verapamil, m/z 455.22 to 165. 02. The retention times for atrasentan and verapamil were found to be 1.68 and 0.96 minutes, respectively.Conclusion: In accordance with the recommendations of the United States Food and Drug Administration (USFDA) and EMA, it was ascertained that the remaining validation parameters remained within the specified range.

Quantification and analyses of seven tyrosine kinase inhibitors targeting hepatocellular carcinoma in human plasma by QuEChERS and UPLC-MS/MS

Tyrosine kinase inhibitors (TKIs) are commonly used to treat various cancers. Literature suggests that the blood concentration of TKIs strongly correlates with their efficacy and adverse effects. Therefore, establishing a Therapeutic Drug Monitoring (TDM) methodology for TKI drugs is crucial to improving their clinical efficacy and minimizing the treatment-related adverse effects. However, quantifying their concentrations in the plasma using existing methods to avoid potential toxicity is challenging. Herein, seven TKIs, namely sorafenib tosylate, axitinib, erlotinib, cediranib, brivanib, linifanib, and golvatinib, were successfully analyzed in human plasma by following a quick, easy, cheap, effective, rugged, and safe (QuEChERS) pretreatment method combined with ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Briefly, biological samples were extracted using 1 mL of methanol, followed by the sequential addition of 250 mg of anhydrous magnesium sulfate and 25 mg of N-propylethylenediamine (PSA) for salinization and purification by adsorption, respectively. In this study, dovitinib was used as the internal standard. The seven TKIs were detected by the gradient elution method for 4 min in the positive ion electrospray mode. The mobile phase comprised methanol (phase A) and 0.1 % aqueous formic acid solution (phase B) on the Agilent Zorbax RRHD Stablebond Aq, (2.1 × 50 mm; 1.8 μm). Brivanib, linifanib, axitinib, sorafenib tosylate, and golvatinib exhibited good linearity in the range of 5–500 ng/mL, and erlotinib and cediranib exhibited good linearity in the range of 10–1000 ng/mL, with linear correlation coefficients (R2) ≥ 0.99. The limits of detection and quantification were 0.60–0.18 ng/mL and 5–10 ng/mL, respectively. The intraday and interday accuracy values ranged from −6.12 % to 7.31 %, with a precision (RSD) of ≤ 10.57 %. The method was rapid, accurate, specific, simple, reproducible, and suitable for the quantitative determination of the seven TKIs in human plasma.

Abstract PO5-13-07: Prediction of recurrence in triple negative breast cancer patients after receiving neoadjuvanttreatment using plasma metabolomics

Abstract Background Early-stage triple-negative breast cancer (TNBC) is usually treated with neoadjuvant chemotherapy (NADC), but prognosis remains poor compared with other BC subtypes. Unfortunately, less than 30% of BC patients achieve pathological complete response (pCR) to NADC and the overall survival of TNBC when cancer spreads is estimated at 10-13 months. Complete response is associated with improved progression-free and overall survival rates, but more accurate prognostic markers are needed. In this study, we assessed the prognostic value of blood circulating metabolites by using targeted-based metabolomics. Methods Patients received standard neoadjuvant chemotherapy with epirubicin plus cyclophosphamide followed by paclitaxel. Blood samples were obtained upon completion of chemotherapy and before surgery. Pathological response to treatment was recorded according to the Miller & Payne system, and categorized as partial (MP 1, 2, & 3) or complete response (MP 4 & 5). Blood samples were collected in EDTA tubes, centrifuged and plasma was stored at -80°C. After QC standard addition and methanol extraction of proteins, four fractions of the resulting extract were analysed: two by reverse-phase/UPLC-MS/MS methods with positive ion mode electrospray ionization (ESI), one by reverse phase/UPLC-MS/MS with negative ion mode ESI, and one by hydrophilic interaction liquid chromatography/UPLC-MS/MS) by Metabolon using a Thermo Scientific Q-Exactive mass spectrometer with a heated electrospray ionization source and an Orbitrap mass analyser. Using Metabolon's hardware and software, raw data were extracted, identified, QC processed and quantified using the area under the curve (AUC). Identified compounds were compared with library entries of purified standards or recurrent unknown entities. The prognostic capacity of each metabolite was assessed using BRB ArrayTools. Selected metabolites were validated using a targeted approach using compound standards as follows. Plasma samples were re-analysed by UPLC/MS using a Vanquish LC coupled to an Orbitrap Exploris 240 MS (Thermo Fisher Scientific) with positive and negative ionization mode ESI under polarity switching. Identification of compounds were performed by comparison of retention times (against in-house authentic standards), accurate mass (with an accepted deviation of 3ppm), and MS/MS spectra. These analyses were carried out by MS-Omics (Denmark). Results Twenty patients treated in the Medical Oncology Unit of the University Hospital of Jaén were recruited. Median age was 50 (31-76). Twelve patients obtained a pathological complete response. Median follow-up after surgery was 25 months. Seven patients eventually had distant metastases (2 in the lungs, 1 in the brain, 1 in the bones and 3 multi-site). Metabolon untargeted analysis identified and quantified 985 metabolites, 789 after xenobiotics exclusion. Survival analysis identified 16 metabolites related with distant relapse (p< 0.05). The ten metabolites with lower p-value were selected for targeted validation. Ms-Omics pipeline allowed absolute quantification of 5 metabolites, 3 of them showing high correlation (R >0.7) between both measurements (targeted and untargeted). Validation of the prognostic value of the candidate metabolites is ongoing in a larger TNBC cohort. Conclusions Metabolomics is a useful tool for the detection of simply assessable and cost-effective prognostic biomarkers in TNBC. Easily monitoring the presence of minimal residual disease is worth to be settled up in the clinical practice for the most aggressive molecular subtype of breast cancer. In this work we have defined a set of metabolites that could predict distant relapse events in TNBC patients treated with neoadjuvant chemotherapy. Further analyses are being carried out to validate the prognostic value of the proposed candidate metabolites. Citation Format: Pedro Sánchez-Rovira, Rocio Urbano-Cubero, Angelo Gamez Pozo, Carmen González-Olmedo, Alicia Cano-Jimenez, Andrea Zapater- Moros, Leticia Díaz-Beltrán, Lucia Trilla-Fuertes, Mariana Díaz-Almirón, Enrique Espinosa, Pilar Zamora, Juan Angel Fresno Vara. Prediction of recurrence in triple negative breast cancer patients after receiving neoadjuvanttreatment using plasma metabolomics [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 PO5-13-07.

HPLC-MS/MS method development and validation for the determination of tetradecapeptide in human plasma

Introduction. The number of peptide drugs being developed and registered has increased in recent years. Therefore, modern analytical approaches and methods are required to determine these substances in biological matrices during pharmacokinetic studies. Peptides are structurally intermediate between small molecules and biopolymers, making it difficult to develop methods for determining them using High Performance Liquid Chromatography with Tandem Mass Spectrometry (HPLC-MS/MS). Peptide derivatization can help achieve optimal chromatographic separation and increase method sensitivity.Aim. To develop and validate a method for the determination of the tetradecapeptide (TDP) threonyl-glutamyl-lysyl-lysyl-arginyl-arginyl-glutamayl-threonyl-valyl-glutamyl-arginyl-glutamyl-lysyl-glutamate in human plasma by HPLC-MS/MS.Materials and methods. The determination of TDP in human plasma was performed by HPLC-MS/MS. Sample preparation included a combination of blood plasma protein precipitation with propionic acid solution in methanol, liquid-liquid extraction with chloroform, and peptide derivatization with propionic anhydride. Internal standard (IS) was threonyl-glutamyl-lysyl-lysyl-arginyl-arginyl-glutamayl-threonyl-leucyl-glutamyl-arginyl-glutamyl-lysyl-glutamate. Chromatographic separation was performed in gradient mode, eluent A was 0.1 % formic acid solution in water, eluent B was 0.1 % formic acid in acetonitrile. Column: Waters XBridge C18, 4.6 × 50 mm, 5 µm. Ionization source was electrospray in positive mode. Multiple reaction monitoring (MRM) transitions for 4-substituted TDP propionate were: 681.30 → 73.95 m/z, 681.30 → 84.00 m/z, 681.30 → 101.90 m/z, 681.30 → 140.10 m/z, and for 4-substituted IS propionate: 686.00 → 74.10 m/z, 686.00 → 84.05 m/z, 686.00 → 102.00 m/z, 686.00 → 140.00 m/z.Results and discussion. Validation of the developed method was carried out in accordance with the requirements of Eurasian Economic Union and the following parameters were determined: selectivity, matrix effect, calibration curve, accuracy and precision, recovery, lower limit of quantification, sample carryover, stability.Conclusion. The method for the determination of TDP in human blood plasma by HPLC-MS/MS was developed and validated. The analytical range was 5.00–1000.00 ng/mL, allowing the method to be used to study TDP pharmacokinetics.

Simultaneous determination of 11 oral targeted antineoplastic drugs and 2 active metabolites by LC-MS/MS in human plasma and its application to therapeutic drug monitoring in cancer patients

Interindividual exposure differences have been identified in oral targeted antineoplastic drugs (OADs) owing to the pharmacogenetic background of the patients and their susceptibility to multiple factors, resulting in insufficient efficacy or adverse effects. Therapeutic drug monitoring (TDM) can prevent sub-optimal concentrations of OADs and improve their clinical treatment. This study aimed to develop and validate an LC-MS/MS method for the simultaneous quantification of 11 OADs (gefitinib, imatinib, lenvatinib, regorafenib, everolimus, osimertinib, sunitinib, tamoxifen, lapatinib, fruquintinib and sorafenib) and 2 active metabolites (N-desethyl sunitinib and Z-endoxifen) in human plasma. Protein precipitation was used to extract OADs from the plasma samples. Chromatographic separation was performed using an Eclipse XDB-C18 (4.6 × 150 mm, 5 μm) column with a gradient elution of the mobile phase composed of 2 mM ammonium acetate with 0.1 % formic acid in water (solvent A) and methanol (solvent B) at a flow rate of 0.8 mL/min. Mass analysis was performed using positive ion mode electrospray ionization in multiple-reaction monitoring mode. The developed method was validated following FDA bioanalytical guidelines. The calibration curves were linear over the range of 2–400 ng/mL for gefitinib, imatinib, lenvatinib, regorafenib, and everolimus; 1–200 ng/mL for osimertinib, sunitinib, N-desethyl sunitinib, tamoxifen, and Z-endoxifen; and 5–1000 ng/mL for lapatinib, fruquintinib, and sorafenib, with all coefficients of correlation above 0.99. The intra- and inter-day imprecision was below 12.81 %. This method was successfully applied to the routine TDM of gefitinib, lenvatinib, regorafenib, osimertinib, fruquintinib, and sorafenib to optimize the dosage regimens.

Polyphenols, Antioxidants, and Wound Healing of Lecythis pisonis Seed Coats.

To better use the Lecythis pisonis Cambess. biomass, this study investigates whether Sapucaia seed coats present wound healing properties. We analyzed the antibacterial, antioxidant, and wound healing-promoting potentials, plus cytotoxicity and stimulation of vascular endothelial growth factor-A. The chemical composition was analyzed by positive ion mode electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. A total of 19 compounds were identified, such as proanthocyanidin A1, procyanidins A1, B2, and C1, epigallocatechin, and kaempferol (p-coumaroyl) glycoside. Potent antioxidant strength/index was verified for 2,2-diphenyl-1-picrylhydrazyl radical (IC50 = 0.99 µg/mL) and ferric reducing antioxidant power (IC50 = 1.09 µg/mL). The extract did not present cytotoxicity and promoted significant cell migration and/or proliferation of fibroblasts (p < 0.05). Vascular endothelial growth factor-A was stimulated dose-dependently at 6 µg/mL (167.13 ± 8.30 pg/mL), 12.5 µg/mL (210.3 ± 14.2 pg/mL), and 25 µg/mL (411.6 ± 29.4 pg/mL). Platelet-derived growth factor (PDGF) (0.002 µg/mL) was stimulated at 215.98 pg/mL. Staphylococcus aureus was susceptible to the extract, with a minimum inhibitory concentration of 31.25 µg/mL. The identified compounds benefit the antioxidant activity, promoting hemostasis for the wound healing process, indicating that this extract has the potential for use in dermatological cosmetics.

Development of a reliable method for determination of N-nitrosamines in medicines using disposable pipette extraction and HPLC-MS analysis.

This study outlines the development and optimization of an analytical method using Disposable Pipette Extraction (DPX) followed by high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis to determine NAs in medicines. HPLC-MS analysis utilized a reversed-phase and positive mode electrospray ion source. DPX parameters were optimized through univariate and multivariate analyses, including extraction phase, desorption solvent, sample pH, equilibrium time, and extraction/desorption cycles. The optimized conditions included a C18 extraction phase, methanol desorption solvent, pH at 7, an equilibrium time of 30 seconds, 2 extraction cycles, and 5 desorption cycles. Considering this method, it was possible to achieve a sample preparation step for the analysis of NAs in medicines using a minimal amount of extraction phase, sample, and desorption solvent. Furthermore, the total extraction procedure enables the extraction of NAs in around 4 minutes with NA recovery up to 98%. Analytical performance demonstrated precision and accuracy below 15% and a quantification limit of 1 ng mL-1, meeting validation requirements set by regulations worldwide. Thus, the DPX/HPLC-MS technique offers a faster and cost-effective method for analyzing NAs in medicines compared to traditional approaches. Besides, this method reduces solvent consumption and residue generation, enhancing environmental sustainability according to green chemistry principles.

Quantification of Olanzapine by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

Olanzapine (Zyprexa™) is indicated for the treatment of schizophrenia and bipolar disorder. Olanzapine is metabolized to inactive metabolites; therefore, this assay measures the concentrations of olanzapine and internal standard, olanzapine-D3. This procedure provides instructions for quantitating levels of olanzapine in blood serum specimens. An internal standard (IS) solution consisting of the deuterated analogue of olanzapine is added to an aliquot of the patient serum or plasma, standards, and positive controls. The drugs of interest are separated from the biological fluid using solid phase extraction. The dried extracts are reconstituted in mobile phase and separated by liquid chromatography. The solution is introduced into an Agilent 6470A triple quadrupole tandem mass spectrometer via positive mode electrospray ionization (ESI) utilizing multiple reaction monitoring (MRM). Quantitation is performed by comparison to a calibration curve, using Indigo® (Ascent) software.

Multiplex Immunosuppressant (ISD) Method for the Measurement of Cyclosporine A, Tacrolimus, Sirolimus/Rapamycin, and Everolimus in Whole Blood Using MassTrak™ Kit.

Cyclosporine A, everolimus, sirolimus, and tacrolimus are the most commonly used immunosuppressant drugs in organ transplant and auto-immune patients. The narrow therapeutic window of these immunosuppressant drugs requires close monitoring of drug blood levels to ensure proper therapeutic response. A quick, robust high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was introduced for monitoring whole blood levels of these immunosuppressant drugs with the use of the MassTrak™ Immunosuppressant kit. The assay was carried out in 96-well plate format and requires a simple precipitation step; after which, the supernatant is subjected to liquid chromatography separation (2min total run time) using a C18 Cartridge column. Identification and quantitation of cyclosporine A, everolimus, sirolimus, and tacrolimus was achieved by employing multiple reaction monitoring (MRM) in positive mode electrospray ionization (ESI). The method exhibits a linear measuring range from 10 to 1500ng/mL (Cyclosporine A), 1.0-30.0ng/mL (Everolimus), 1.0-26.0ng/mL (Sirolimus), and 1.0-30.0ng/mL (Tacrolimus) and has a within-run and between-run imprecision of <10%.

Study on establishment of residual analysis methods and residue degradation regulation of natural pyrethrin in barley plants and soil on Qinghai Plateau

The purpose of this study is to develop an accurate and efficient assay for the detection of pyrethroid residues and degradation in barley and soil in the Qinghai Plateau region. A sensitive and selective method was developed for the determination of pyrethrin residues in highland barley (kernel, stem, and root) and soil in the Qinghai Plateau by liquid chromatography (LC-MS/MS). Multi-residue analysis of pyrethrin in samples involves simple extraction with acetonitrile, salt-out with NaCl, MgSO4 absorption of water, cleaning with GCB and PSA, respectively, followed by separation with XR-ODS II column, and multi-reaction detection mode detection using a positive mode electrospray ionization source (ESI + ). The results showed that the 6 different components of pyrethrin had a good detection effect in the concentration range of 0.01 ∼ 1.0 mg/L, and the correlation coefficient was greater than 0.9990. The limits of quantitation of each component on each substrate were 0.01 ∼ 0.05 mg/kg, the average daily recoveries were 87.58 ∼ 95.44 %, and the relative standard deviations were 0.4 ∼ 3.8 %. The half-life of pyrethrin in barley grains, stems and soil was 0.77 ∼ 2.57 d. In soil and highland barley, the maximum dose of pyrethrin and twice the maximum dose of pyrethrin were applied, and the final residue 7 days after treatment was less than 0.2 mg/kg. The degradation behavior of pyrethrin in soil at three test sites showed that organic carbon and pH were important factors affecting the degradation of pyrethrin, and the effect of pH on the degradation of six pyrethrin components was greater than that of organic carbon. These studies provide theoretical basis for environmental safety evaluation caused by pyrethrin use in the Qinghai Plateau.

Untargeted metabolomics analysis on kidney tissues from mice reveals potential hypoxia biomarkers

Chronic hypoxia may have a huge impact on the cardiovascular and renal systems. Advancements in microscopy, metabolomics, and bioinformatics provide opportunities to identify new biomarkers. In this study, we aimed at elucidating the metabolic alterations in kidney tissues induced by chronic hypoxia using untargeted metabolomic analyses. Reverse phase ultrahigh performance liquid chromatography-mass spectroscopy/mass spectroscopy (RP–UPLC–MS/MS) and hydrophilic interaction liquid chromatography (HILIC)–UPLC–MS/MS methods with positive and negative ion mode electrospray ionization were used for metabolic profiling. The metabolomic profiling revealed an increase in metabolites related to carnitine synthesis and purine metabolism. Additionally, there was a notable increase in bilirubin. Heme, N-acetyl-l-aspartic acid, thyroxine, and 3-beta-Hydroxy-5-cholestenoate were found to be significantly downregulated. 3-beta-Hydroxy-5-cholestenoate was downregulated more significantly in male than female kidneys. Trichome Staining also showed remarkable kidney fibrosis in mice subjected to chronic hypoxia. Our study offers potential intracellular metabolite signatures for hypoxic kidneys.

B-303 Comparison of Tacrolimus Concentrations Obtained By the Newly Approved Alinity i Tacrolimus Assay With a Reference Liquid-Chromatography Combined With Tandem Mass spectrometry Assay

Abstract Background Tacrolimus belongs to the calcineurin-inhibitor class of medications and used for preventing and maintenance of organ rejection in transplant recipients alone or in combination with other medications. Due to narrow therapeutic window and significant nephrotoxicity, this drug is routinely monitored with a suggested therapeutic range of 5–15 ng/mL. Recently, Abbott Laboratories (Abbott Park, IL) received FDA approval for using tacrolimus immunoassay on the Alinity i platform. We compared tacrolimus values in 101 transplant patients using Alinity i analyzer and a reference method based on liquid chromatography combined with mass tandem spectrometry (LC-MS/MS). Methods Tacrolimus immunoassay on the Alinity i analyzer is a chemiluminescent microparticle immunoassay (CMIA) requiring pretreatment of whole blood specimen prior to analysis. The analytical measurement range of the assay is 2–30 ng/mL. The left-over blood specimens were sent to Eurofins-Viracor reference laboratory in Lenexa, KS for analysis using LC-MS/MS method. After liquid-liquid extraction, specimens were analyzed using ascomycin as the internal standard for the presence of tacrolimus (assay is capable of analyzing other immunosuppressants simultaneously). UPLC C-18 column (waters) was used for separation using two mobile phases (A: 2.0 mM ammonium acetate in 0.1% formic caid in water and B: 2.0 mM ammonium acetate in 0.1% formic acid in methanol). Mass spectrometer was operated in positive ionization electrospray mode (tacrolimus m/z 821.6 &amp;gt; 768.6, ascomycin m/z 809.756.6. Results The total precision of the low control was 3.6% (mean: 4.48 ng/mL; CV 0.16 ng/mL, n = 20) while the total precision of the high control was 5.1% (mean: 25.4 ng/mL, SD: 1.29 ng/mL, n = 20). The linearity of the assay (2.0–30.0 ng/mL) was verified by running various calibrators as unknown. Comparing tacrolimus values in specimens from 101 transplant patients (49 kidney, 19 bone marrow transplant, 16 liver and 15 heart transplant) using the reference method (LC-MS/MS) as the x-axis and the corresponding values obtained by the tacrolimus immunoassay using Alinity I analyzer, we observed the following regression equation: y = 0.97 x + 1.005 (n = 101, r = 0.95). For kidney transplant recipients, the regression equation was y = 0.96 + 1.20 (n = 49, r = 0.94). Discussion Excellent correlation between tacrolimus values obtained by the newly FDA approved tacrolimus immunoassay with the reference LC-MS/MS based assay indicates further improvement in the performance of immunoassays for therapeutic drug monitoring of immunosuppressants. This may be due to reduced cross-reactivity of metabolites as indicated in the package insert. Conclusions The newly approved tacrolimus immunoassay for the application on the Alinity I analyzer can be used for routine therapeutic drug monitoring of tacrolimus in clinical laboratories.

Simple and validated method to quantify lacosamide in human breast milk and plasma using UPLC/MS/MS and its application to estimate drug transfer into breast milk

BackgroundEpilepsy is a common neurological disorder. Lacosamide is a third-generation antiepileptic drug used to treat partial-onset seizures. Limited information is currently available on the transfer of lacosamide to breast milk. To facilitate studies on the safety of lacosamide use during breastfeeding, we aimed to develop a method to quantify lacosamide in human breast milk and plasma using ultra-performance liquid chromatography/tandem mass spectrometry.MethodsFifty microliters of breast milk or plasma was used, and samples were prepared by protein precipitation using methanol containing lacosamide-d3 as an internal standard (IS). Chromatography was performed using an ACQUITY HSS T3 column with an isocratic flow of 10 mM ammonium acetate solution/methanol (70:30, v/v). Lacosamide and IS were detected by multiple reaction monitoring in positive ion electrospray mode. The run time was 3.5 min.ResultsCalibration curves were linear and in the range of 0.5 to 100 ng/mL both in breast milk and plasma. The validation assessment indicated that precision, accuracy, matrix effects, selectivity, dilution integrity, and stability were acceptable. The developed method was successfully applied to quantify lacosamide in breast milk and plasma obtained from a volunteer who had been orally administered lacosamide twice a day (100 mg × 2). Relative infant dose of lacosamide was estimated to be 14.6% in breast milk at five time points.ConclusionsWe developed a simple and robust method to quantify of lacosamide in human breast milk and plasma. This method could be useful for in future studies investigating the safety of lacosamide use during breastfeeding.

Determination of five mTOR inhibitors in human plasma for hepatocellular carcinoma treatment using QuEChERS-UHPLC-MS/MS

A fast and reliable QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) method for pre-processing combined with Ultra - high performance liquid chromatography - tandem mass spectrometry (UHPLC-MS/MS) was established for the analysis of five mammalian rapamycin target protein (mTOR) inhibitors (vistusertib, AZD8055, pictilisib, everolimus, temsirolimus)in human plasma. Extraction was achieved by addition of acetonitrile to the sample followed by anhydrous magnesium sulfate and 30 mg C18 for salting out and purification, respectively. MTOR inhibitors were detected using selective response monitoring (SRM) under positive ion electrospray mode. Vistusertib, AZD8055 and pictilisib showed good linearity with a range of 1–80 ng/ml, Additionally, the concentration of everolimus and temsirolimus was 2.5–200 ng/ml and10–800 ng/ml, respectively. The linear correlation coefficient (R2) of each analysis was ≥ 0.9950. The limit of detection (LOD) and Limit of Quantitation (LOQ) were 0.015–0.75 ng/ml and 1–10 ng/ml, respectively. This method showed a high accuracy with high recovery rate and excellent stability. This method is fast, accurate and reliable, suitable for quantitative detection of mTOR inhibitors in human plasma.

Detection of doping peptides and basic drugs in equine urine using liquid chromatography-mass spectrometry.

The abuse of prohibited agents including peptides and basic small-molecule drugs is an area of great concern in horseracing due to their high potential to act as doping agents. These compound classes include agents such as growth hormone-releasing peptides, peptide analgesics, beta-2-adrenergic receptor agonists, and quaternary ammonium drugs that can be challenging to detect and regulate because of their chemical properties and potential rapid elimination following administration. The use of highly sensitive and selective analytical techniques such as liquid chromatography-mass spectrometry (LC-MS) is necessary to provide coverage of these substances and their potential metabolites. This study describes the development and validation of methodology capable of the detection of over 50 different peptide-based doping agents, related secretagogues, quaternary ammonium drugs, and other challenging small molecules in equine urine following solid-phase extraction using a mixed mode weak cation exchange sorbent. Following sample extraction, the compounds were analyzed using LC-MS with chromatographic separation via a reverse phase gradient and detection via selective reaction monitoring following introduction to a triple-stage quadrupole mass spectrometer using positive mode electrospray ionization. Validation parameters including limits of detection and quantitation, accuracy, precision, linear range, recovery, stability, and matrix effects were determined. Briefly, the limits of detection for most compounds were in the sub-ng/mL ranges with adequate precision and accuracy sufficient for an initial testing procedure. Stability studies indicated that most compounds were sufficiently stable to allow for effective screening using conditions commonly utilized in drug testing laboratories.

Analysis of AT7519 as a pro-resolution compound in an acetaminophen-induced mouse model of acute inflammation by UPLC-MS/MS

BackgroundUncontrolled inflammation contributes to the progression of organ damage in acute conditions, such as acetaminophen-induced acute liver injury (APAP-ALI) and there are limited treatments for this condition. AT7519, a cyclic-dependent kinase inhibitor (CDKI), has been used successfully in several conditions, to resolve inflammation and return tissue homeostatic functions. AT7519 has not been assessed in APAP-ALI and its effect on APAP metabolism is unknown. Targeted chromatography and mass spectrometry can be used to assess multiple compounds simultaneously and this approach has not been applied yet to measure APAP and AT7519 in a mouse model.ResultsWe show an optimised simple and sensitive LC–MS/MS method for determining concentrations of AT7519 and APAP in low volumes of mouse serum. Using positive ion mode electrospray ionisation, separation of AT7519 and APAP and their corresponding isotopically labelled internal standards [2H]8-AT16043M (d8-AT7519) and [2H]8-APAP (d4-APAP), was achieved on an Acquity UPLC BEH C18 column (100 × 2.1 mm; 1.7μm). A gradient mobile phase system of water and methanol was delivered at a flow rate of 0.5 mL/min with a run time of 9 min. Calibration curves were linear, intra-day and inter-day precision and accuracy were acceptable and the covariates of all standards and quality control replicates were less than 15%. The method was successfully applied to evaluate AT7519 and APAP levels 20 h post AT7519 (10 mg/mg) in C57Bl6J wild type mouse serum treated with either vehicle or APAP. Serum AT7519 was significantly higher in mice that had received APAP compared to control, but there was no correlation between APAP and AT7519 quantification. There was also no correlation of AT7519 and hepatic damage or proliferation markers.ConclusionWe optimised an LC–MS/MS method to quantify both AT7519 and APAP in mouse serum (50 µL), using labelled internal standards. Application of this method to a mouse model of APAP toxicity proved effective in accurately measuring APAP and AT7519 concentrations after i.p. dosing. AT7519 was significantly higher in mice with APAP toxicity, indicating hepatic metabolism of this CDKI, but there was no correlation with markers of hepatic damage or proliferation, demonstrating that this dose of AT7519 (10 mg/kg) does not contribute to hepatic damage or repair. This optimised method can be used for future investigations of AT7519 in APAP in mice.

Analysis of Megadalton-Sized DNA by Charge Detection Mass Spectrometry: Entropic Trapping and Shearing in Nanoelectrospray.

The analysis of nucleic acids by conventional mass spectrometry is complicated by counter ions which cause mass heterogeneity and limit the size of the DNA that can be analyzed. In this work, we overcome this limitation using charge detection mass spectrometry to analyze megadalton-sized DNA. Using positive mode electrospray, we find two dramatically different charge distributions for DNA plasmids. A low charge population that charges like compact DNA origami and a much higher charge population, with charges that extend over a broad range. For the high-charge population, the deviation between the measured mass and mass expected from the DNA sequence is consistently around 1%. For the low-charge population, the deviation is larger and more variable. The high-charge population is attributed to the supercoiled plasmid in a random coil configuration, with the broad charge distribution resulting from the rich variety of geometries the random coil can adopt. High-resolution measurements show that the mass distribution shifts to slightly lower mass with increasing charge. The low-charge population is attributed to a condensed form of the plasmid. We suggest that the condensed form results from entropic trapping where the random coil must undergo a geometry change to squeeze through the Taylor cone and enter an electrospray droplet. For the larger plasmids, shearing (mechanical breakup) occurs during electrospray or in the electrospray interface. Shearing is reduced by lowering the salt concentration.

Mechanism of formation and ion mobility separation of protomers and deprotomers of diaminobenzoic acids and aminophthalic acids.

Aminobenzoic acids are well-established candidates for understanding the formation of isomeric ions in positive mode electrospray ionization as they yield both N- and O-protomers (prototropic isomers) at the amine and carbonyl sites, respectively. In the present work, a combination of ion mobility-mass spectrometry and density functional theory calculations to determine the protonation and deprotonation behaviour of four diamino benzoic acid and four aminophthalic acid isomers is presented. The additional COOH group on the ring of aminophthalic acids provides experimental evidence regarding the mechanism of intramolecular NH3+ → O proton transfer, which has been the subject of debate in recent years. To determine the proton acceptor O atom, ion mobility spectra of the fragments of protomers were used as a new method for the confidential assignment of the O-protomer structure, confirming only short-distance intramolecular NH3+ → O proton transfer. Additionally, the substitution pattern both influences the basicity of the protonation sites and enables these molecules to form internal hydrogen bonds with the protonated or deprotonated sites. The formation of the hydrogen bonds in the deprotonated aminophthalic acids changed the charge distribution and subsequently their ion mobility-derived collision cross sections in nitrogen (CCSN2) leading to separation of the four isomers studied. Finally, an interesting effect of the substitution pattern was observed as a synergistic electron-donating effect of the amine groups of 3,5-diaminobenzoic acid on enhancing the basicity of the carbon atom C2 of the ring and previously unreported formation of a C-protomer within aminobenzoic acid systems.