Protein Precipitation Research Articles

Selective quantification of estrogens in serum with aptamer-affinity and LC-MS/MS analysis

In the previous study, we developed an accurate and automated immunologic mass spectrometry (iMS) method for the simultaneous quantification of multiple steroid hormones by target capturing with monoclonal antibodies and LC-MS/MS analysis. However, antibodies are less accessible to a wide variety of low-molecular-weight analytes, which limited the application of iMS method. At the same time, multiple monoclonal antibodies needed also increased the complexity and cost of the iMS assay. In the present study, we developed an aptamer-affinity and LC-MS/MS analysis method with a class-specific aptamer for the selective enrichment and simultaneous determination of estrogens (estrone (E1) and estradiol (E2)) in human serum. The DNA aptamer was coupled to magnetic beads to enrich the target estrogens from serum sample after protein precipitation. The magnetic beads were then washed and eluted with acetonitrile solution, and targets were analyzed with LC-MS/MS. The sample preparation process of enrichment, washing and elution works in an automatic manner. Matrix effect was negligible as the high specificity of target recognition and enrichment by the aptamer. The aptamer-affinity assay for estrogen quantification was validated for sensitivity, linearity, accuracy and precision. The limit of detection (LOD) and quantification (LOQ) were0.01 ng/mLand0.02 ng/mL for E1 and E2, respectively. Satisfactory coefficients of variation (CVs) were obtained, with the intra-batch CVs of5.5 %-7.3 % and 5.2 %-6.0 % for E1 and E2, respectively, and inter-batch CVs of 3.8 %-10.0 % and 5.2 %-6.3 % for E1 and E2, respectively. Recoveries were in the range of99.1 %-107.0 % and 94.9 %-107.7 % for E1 and E2, respectively. Quantification results of 40 serum samples by the aptamer-affinity and solid-phase extraction (SPE) LC-MS/MS method showed good consistency, with the correlation coefficient of 0.93 and 0.98 for E1 and E2, respectively. Clinical application of the aptamer-affinity and LC-MS/MS method was further demonstrated for quantifying serum estrogen levels of 170 pregnant women at different gestational weeks.

Evaluation of the effects and plasma concentration of the platelet inhibitor ticagrelor, after crushed and non-crushed intake, after cardiac arrest and after semi-urgent coronary artery bypass surgery

Background Ticagrelor, used in acute coronary syndrome (ACS), can be administered via nasogastric tube when oral intake is impossible. We investigated platelet inhibition and pharmacokinetics in resuscitated ACS patients and those undergoing semi-urgent coronary artery bypass graft (CABG) surgery. Our study aimed to assess platelet inhibition with use of the Platelet Function Analyser (PFA) and measured plasma concentrations of ticagrelor and its active metabolite in these ACS patients. Methods We included resuscitated cardiac arrest patients (STEMI/NSTEMI) and semi-urgent CABG patients. Crushed ticagrelor tablets were administered using a nasogastric tube. PFA closure time (CT) was determined with CT longer than 113 s as reference range. Plasma concentrations of ticagrelor and its active metabolite were measured after protein precipitation, by using liquid chromatography with mass spectrometry detection. Results In 20 resuscitated patients, 89% showed platelet inhibition at 24 h and 92% at day 4. For semi-urgent CABG patients, 85% exhibited platelet inhibition at 24 h and 84% at day 4. For ticagrelor in resuscitated patients, the median time to peak plasma concentration (Tmax) was 100 h [8; 100] for a median maximal concentration (Cmax) of 615.5 ng/mL [217.5; 1385.0]. For AR-C124910XX median Tmax was 100 h [8; 100] for a Cmax of 131.0 ng/mL [52.1; 177.7]. Among 20 patients undergoing semi-urgent CABG, Tmax for ticagrelor was 100 h [100; 100] for a median Cmax of 857.0 ng/ml [496.8; 1157.5]. For AR-C124910XX, median Tmax was 100 h [43; 100] for a Cmax of 251.0 ng/ml [173.0; 396.5]. Conclusion Crushed ticagrelor via nasogastric tube achieved targeted platelet inhibition. Pharmacokinetics aligned with previous studies. EudraCT number: 2013-004191-35; Study protocol code: AGO/2013/011; EC/2014/1061; ClinicalTrial.gov identifier: NCT02341729

B-162 Evaluation of two commercially available stable isotope labeled whole-protein infliximab as internal standard for a clinical infliximab LC-MS/MS assay

Abstract Background Liquid chromatography tandem mass spectrometry (LC-MS/MS) based quantitation of infliximab has been successfully introduced in the clinical laboratory for therapeutic drug monitoring in recent years. Although stable isotope labeled (SIL)-peptide or non-labeled analog protein internal standards have been utilized, SIL-whole protein infliximab is generally considered to be the ideal internal standard for the correction of variations during sample preparation and analysis. In this study, we compared two commercially available SIL infliximab proteins, Sigma SILu™MAb and Promise Proteomics, for use in a clinical infliximab assay. Methods Both labeled infliximab proteins incorporate [13C6, 15N4]-arginine and [13C6, 15N2]-lysine in their sequence and are designed for use as an internal standard for LC-MS/MS based quantitative analysis of infliximab. For the comparison of their performance, a laboratory-developed LC-MS/MS clinical method was utilized. Linearity at 6 levels in triplicate with Remicade and 3 biosimilars (-abda, -axxq, and -dyyb), intra-assay precision at 2 levels (n=10/level), and method comparison (n=15) experiments were performed using each labeled infliximab as the internal standard. For sample preparation, internal standard was mixed with the serum sample, followed by protein precipitation with ammonium sulfate, protein denaturation, and tryptic digestion. The peptides YASESMSGIPSR (YASE) from infliximab and YASESMSGIPSR [13C6, 15N4] (SIL-YASE) from labeled infliximab were monitored by LC-MS/MS (Transcend TLX and TSQ Atlis, Thermo Scientific) and utilized for the quantitation of infliximab. Results For the linearity experiment (1-50 µg/mL), while utilizing the Promise Proteomics internal standard to assay had an accuracy range of 103.4% to 114.1% for Remicade, 100.6% to 108.2% for Infliximab-axxq, 103.0% to 110.9% for Infliximab-abda, 102.7% to 108.3% for Infliximab-dyyb. For the Sigma SILu™Mab SIL infliximab, the accuracy range was 92.1% to 99.6% for Remicade, 94.5% to 107.2% for Infliximab-axxq, 99.0% to 104.2% for Infliximab-abda, and 105.1% to 112.0% for Infliximab-dyyb. The mean overall error for Remicade and biosimilars was 0.203 µg/mL (2.0%) for Sigma SILu™Mab SIL infliximab and 0.193 µg/mL (2.0%) for Promise Proteomics internal standardization. Intra-day precision using each SIL infliximab was evaluated with Remicade at low and high levels (5 and 20 µg/mL). Using Promise Proteomics, the coefficient of variation (CV) was 2.8 and 2.2%, while using Sigma SILu™Mab the precision was 6.8 and 6.5% for the low and high levels, respectively. The regression analysis for the method comparison experiment had a correlation coefficient (r) of 0.9996 with a slope of 1.050 (95% confidence interval, 1.030-1.070) and intercept of -0.318 (95% confidence interval, -0.842 to 0.205). Conclusions The mean overall error for the linearity experiment was the same using Sigma SILu™Mab or Promise Proteomics as the internal standard. The Promise Proteomics product demonstrated a lower intra-assay CV compared to the Sigma SILu™Mab. The 2 products displayed a strong correlation during the method comparison experiment. Both Sigma SILu™Mab and Promise Proteomics labeled standards showed acceptable performance as an internal standard for LC-MS/MS based quantitative analysis of infliximab. Having both products available increases the robustness of LC-MS/MS clinical assays in the event of supply issues.

A-125 Difference Between the Results of Vitros® Dry Biochemistry and Wet Biochemistry with Kinetic Method in Diagnosing the Comorbidity of Lung Adenocarcinoma and Aacroamylasemia: Case Report

Abstract Background Amylase (AMY) is a biomarker that comes mainly from the pancreas and salivary glands. Macroamylasemia is a condition often accompanied by malignant tumors and clinical features that include: (i) recurrent abdominal pain, no abnormality in the salivary glands, serum amylase (AMY) level ≥2 to 3 times higher than the normal value and poor patient response to treatments for pancreatitis; (ii) normal serum creatinine and urea levels, increased serum AMY level, and urine AMY level within the normal range or slightly lower. Because macroamylasemia may cause abnormally increased serum AMY levels for a long period of time, misdiagnosis of pancreatitis and delayed diagnosis/treatment of the disease may occur. We report a case of lung adenocarcinoma with macroamylasemia that was co-diagnosed using wet chemistry with kinetic method and dry chemistry to measure AMY. Methods A 47-year-old male patient was previously diagnosed with “acute pancreatitis and lung infection” after initial admission to a local hospital with paroxysmal cough and abdominal pain without obvious cause. To further diagnose the patient, we determined the blood AMY level by using the wet chemistry with kinetic method and the Vitros® dry chemistry test, in addition to conducting a physical exam, abdominal and chest computed tomography (CT), and other lab tests. Two methods, calculation of amylase/creatinine clearance ratio (Cam/Ccr ratio) and protein precipitation by polyethylene glycol (PEG), were also performed to validate assumed interference by serum macroamylase (MAMS) in the wet chemistry method compared to the dry chemistry method, which has technology to capture large molecules and minimize the impact of interferents. MAMS is a high-molecular-weight AMY complex that is synthesized by normal serum AMY bound with macromolecular substances (eg, immunoglobulins and carbohydrates). Results The final diagnosis was a stage IIB cT3N0M0 adenocarcinoma of the upper lobe of the right lung and macroamylasemia. Symptoms and radiologic findings were atypical in terms of pancreatitis. Serum AMY was 2,178U/L when measured with the wet chemistry with kinetic method. When the specimen was re-tested with Vitros dry chemistry, the AMY level was 369 U/L. After precipitation with 25% PEG 6000 solution, the blood AMY level from wet chemistry decreased from 2395.1 U/L to 246.7 U/L, yielding a precipitation rate of 89.7%. Cam/Ccr ratio was calculated as 0.23% (<1%). The calculated PEG precipitation rate and Cam/Ccr ratio from this study were consistent with the ranges considered for macroamylasemia, which are ≥73% and Cam/Ccr ratio <1%, respectively. Conclusions High serum AMY levels do not necessarily indicate pancreatitis or parotitis; wet chemistry results can be impacted by the presence of serum macroamylase. Instead of relying solely on blood AMY in diagnosing acute pancreatitis, clinicians should also consider the correlations of blood AMY with urine AMY and blood lipase. In patients with prolonged blood AMY elevation but normal lipase and urine AMY, re-testing of AMY with a more interference-resistant methodology (e.g. Vitros dry chemistry) may be requested. Accordingly, Cam/Ccr ratio calculation and PEG precipitation can be performed to validate the diagnosis of macroamylasemia.

B-248 Enhanced Detection of of Hemoglobin Variants in Clinical Research using Dried Blood Spot and HRAM MS

Abstract Background Hemoglobinopathies are one of the most prevalent genetic disorders, with more than 330,000 affected births occurring annually. Common techniques used in Hb analysis are electrophoretic and chromatographic assays. However, there are challenges to differentiate isomers (i.e., between Hb C and Hb E), very similar masses, or new variants due to their lack of resolution. Mass spectrometry (MS) gained popularity in clinical research because of its robustness and versatility in analyzing a wide range of samples and analytes from small molecules to proteins. Here, we present the top-down analysis for enhanced detection of various hemoglobin variants using Orbitrap Exploris for clinical research. Methods Proteins were directly extracted from dried blood spots (DBS) and analyzed by Thermo Scientific™ Orbitrap Exploris™ 240 with BioPharma Option coupled to Vanquish HPLC system. A 3.2 mm disc was punched from the DBS cards to 96 well plate and 50 uL water was added to re-hydrate. For protein precipitation, 150 uL acetonitrile was added and incubated at -20 ⁰C for 15 minutes. After micro-centrifugation, 160 uL of supernatant was removed and 70 uL water was added to resuspend. For LC-MS analysis, 10 uL of supernatant was diluted 8 times with mobile phase A and transferred to another 96-well plate. LC separation was performed on MAbPac RP column 4 um, 1 x 100 mm, and data-dependent acquisition mode was operated. Results With direct protein extraction from DBS, the entire sample preparation time is less than 1 hour. The MAbPac column showed its capability of separating between different chains with minor overlap between normal beta and HbS beta chains. Data processing was performed by ProSightPD, resulting in ∼45% sequence coverage for both normal Hb and HbS beta chains. The mass difference between normal beta and Hb S beta chains is about 30 Da, which results in about 1.6 m/z difference at a charge state of 19. This mass difference can often be hindered by other proteins or interferences from the matrix if the mass analyzer doesn’t have sufficient resolving power. Here we successfully applied Orbitrap resolution at 120,000 to isolate these similar masses for accurate variants or subunits identification including normal alpha, normal beta, Hb S, and delta chains. The confirmation of the delta chain can be beneficial since its abundance is usually less than 0.5% in adults, which shows potential for detecting low abundant variants using this method. Also, the calibration curve was generated to demonstrate the method's feasibility which is not a typical quantitation method for hemoglobinopathies. For quantitation, three most abundant m/z values from 2 charge states were added to TraceFinder for peak area extraction and normal Hb beta chain was used as an alternative internal standard for data processing. The 3-day inter/intraday analysis generated great reproducibility with CV < 10% and LOQ was determined to be 2.5 mg/mL with R2 values >0.994. Conclusions Successful demonstration of a single-step protein extraction directly from DBS and top-down analysis of Hb variant for clinical research.

B-260 Prevalence and Relative Distributions of Major Phosphatidylethanol (PEth) Homologs in a Large-Scale Clinical Study

Abstract Background Ethanol abuse remains a significant public health challenge in the United States. Detection of ethanol exposure is crucial for preventive, diagnostic, and therapeutic endeavors. Phosphatidylethanol (PEth) is a specific and direct ethanol biomarker. Compared to other metabolites, such as ethyl glucuronide and ethyl sulfate in urine (average 2-3 days), PEth in blood has a longer window of detection (average 1-2 weeks). Among the known 48 PEth homologs, PEth 16:0/18:1 (POPEth) and PEth 16:0/18:2 (PLPEth) are the most abundant species accounting for 37% to 46% and 26% to 28%, respectively. PLPEth is synthesized at a greater rate than POPEth, but POPEth has a longer half-life than PLPEth. The purpose of this study was to understand the prevalence and levels of POPEth and PLPEth in a large number of clinical specimens. Methods Briefly, lavender-top whole blood specimens were diluted with isotopically labeled internal standard (IS) in an organic buffer to precipitate proteins on an automated Hamilton platform. The mixture was centrifuged, and a supernatant portion isolated for injection onto a Sciex QTRAP 5500 liquid chromatography tandem mass spectrometry (LC-MS/MS) system using electrospray ionization (ESI) in negative mode. The assay was fully validated under CLIA regulations for lab developed tests: the analytical measurement range (AMR) was 10-400 ng/mL; the administrative reporting threshold for PEth positivity was 20 ng/mL based on clinical literature and a consensus among other United States laboratory methods. Results Precision of 4 QC levels was evaluated with between-run CV% (n=25) ranging from 1.6-6.3%, and accuracy across the AMR was achieved with 87-110%. No significant matrix effects were observed; variation in different whole blood specimens was corrected with IS normalization. No significant interferences were observed from 137 commonly seen prescription and nonprescription drugs and 6 other PEth homologs. Of the clinical specimens, most (20,833, 71.3%) were negative (<20 ng/mL) for both analytes. Meanwhile, 8,222 specimens (28.3%) were positive with either POPEth, or PLPEth or both ≥ 20ng/mL. There were 150 specimens excluded from the statistics due to interference challenges. Interestingly, 757 specimens (2.6%) were PLPEth negative and POPEth positive (mean value 40.1 ng/mL). Conversely, 62 specimens (0.2%) were POPEth negative and PLPEth positive (mean value 24.6 ng/mL). Among the specimens with both POPEth and PLPEth levels within the AMR, the average POPEth/PLPEth ratio was 1.4 supporting the overall prevalence of higher POPEth concentrations than PLPEth. However, the POPEth/PLPEth ratio varied widely, from 0.3 to 8.8, reflecting patient differences in PEth metabolism patterns. Conclusions To help verify probable ethanol abstinence, we have developed, validated, and deployed a high-throughput clinical assay for the major PEth homologs, POPEth and PLPEth. In a retrospective analysis of 29,205 clinical specimens: 71.3% of results were consistent with abstinence; 28.3% of the results were positive suggesting moderate alcohol consumption within the last 1-2 weeks. With POPEth most prevalent, yet variation observed, there were 62 specimens with PLPEth positive and negative for POPEth. This clinical PEth study including both POPEth and PLPEth provides greater sensitivity to identify ethanol exposure than only testing a single homolog.

Quantification of tricyclic glycopeptide in human plasma by UHPLC-MS3 coupled with counter-extraction follow by protein precipitation to enhance sensitivity

An ultra-high performance liquid chromatography tandem mass spectrometry cubed (UHPLC/MS3) assay coupled with protein precipitation and counter-extraction for detection of tricyclic glycopeptide vancomycin in human plasma was established and validated in this study. After protein precipitation and counter-extraction with dichloromethane, chromatographic separation of vancomycin and norvancomycin were performed on a reversed phase column (XBridge Peptide BEH C18 column, 2.1 × 100 mm I.D, 3.5 μm). The transition (parent ions → fragment ions → further fragment ions) at m/z 725.3 → 144.1 → 100.1 was used for quantification of vancomycin. The transition (parent ions → fragment ions) at m/z 718.3 → 144.2 was used for detection of norvancomycin. The linear range of the developed analytical method for quantification of vancomycin was 0.5–100 µg/mL (r = 0.9989). The range of intra- and inter-day precisions of the assay among low, medium and high concentrations is between 1.88 % and 6.33 %. The sensitivity of the analytical method was significantly improved by using MS3 technique as monitoring mode and counter-extraction with dichloromethane followed by protein precipitation as sample processing assay. The developed UHPLC/MS3 assay was successfully applied for clinical therapeutic drug monitoring (TDM) of vancomycin in 45 human plasma samples.

Individualized medication of venetoclax based on therapeutic drug monitoring in Chinese acute myeloid leukemia patients using an HPLC method.

The aim of this study was to establish a simple and sensitive high-performance liquid chromatography method for therapeutic drug monitoring of venetoclax (VEN) and optimize regimens. The analysis required the extraction of a 50 μl plasma sample and the precipitation of proteins using acetonitrile extraction. The chromatographic method employed a mobile phase of acetonitrile: 0.5% KH 2 PO 4 (pH 3.5) (60/40, v/v) on a Diamond C 18 (4.6 mm × 250 mm, 5 μm) column at a flow rate of 1.0 ml/min. The quantitative method was validated based on standards described in 'Bioanalytical Method Validation: Guidance for Industry' published by the US Food and Drug Administration (FDA). The calibration curve was linear ( R2 = 0.9998) over the range of 75-4800 ng/ml, with limits of quantification of 25 ng/ml. The coefficients of intraday and interday validation, specificity, recovery, and stability all met the criteria of FDA guidance. The method was successfully applied to analyze VEN concentrations in 30 cases of acute myeloid leukemia patients. The peak concentration ( Cmax ) was 1881.19 ± 756.61 ng/ml, while the trough concentration ( Cmin ) was 1212.69 ± 767.92 ng/ml in acute myeloid leukemia patients. Our study establishes a simple, precise, and sensitive high-performance liquid chromatography method for monitoring VEN and confirms its applicability for therapeutic drug monitoring of VEN in hematological cancers.

Plant Protein Resources, Novel Extraction and Precipitation Methods: A Review

ABSTRACTThis comprehensive review explores the diverse landscape of plant protein resources, focusing on novel extraction and precipitation methods that contribute to the advancement of sustainable and efficient protein production. The increasing global demand for plant‐based proteins as a key component of balanced diets has spurred research into optimizing extraction processes from various plant sources. The review encompasses a wide range of plant protein resources, including legumes, cereals, oilseeds, and other plant‐based alternatives. Special emphasis is given to innovative techniques in protein extraction, such as enzyme‐assisted extraction, aqueous extraction, and emerging technologies like ultrasound and microwave‐assisted methods. Additionally, the review investigates novel precipitation methods employed to isolate and purify plant proteins, shedding light on techniques like isoelectric precipitation, salting‐out, and membrane filtration. The integration of sustainable practices in protein extraction, coupled with a focus on minimizing environmental impact, is a central theme throughout the review. By synthesizing current research findings, this review aims to provide a comprehensive overview of the state‐of‐the‐art in plant protein extraction and precipitation methods, offering valuable insights for researchers, industry professionals, and policymakers involved in the rapidly evolving field of plant‐based protein production.

Comprehensive Targeted Profiling of Multiple Steroid Classes in Rodent Plasma Using Liquid Chromatography-Mass Spectrometry

BackgroundReliable quantification of multiple steroid classes in biological fluids within a single method remains an analytical challenge despite many previously published methods. Crosstalk of positional isomers, overlap of stereoisomer fragmentation patterns, differing proton affinities, in-source fragmentation, varying stability of protonated ions in the gas phase across steroid classes, and non-existence of steroid-free matrix are the main challenges limiting the number of simultaneously profiled steroids. ResultsIn this study, we focused on the development of a derivatization-free, achiral, high-throughput, and cost-effective UHPLC-MS/MS approach that allows simultaneous profiling of a spectrum of 38 steroids covering progestogens, androgens, corticosteroids, and estrogens, while properly addressing the hurdles of steroid analysis. Within a 20-minute method, 16 stereoisomers and 15 positional isomers were fully resolved within a single run while separated from 7 additional non-interfering steroids and matrix interferences in rodent plasma. Protein precipitation and supported liquid extraction methods using only 40 μL of sample were developed to achieve the lowest possible limits of quantification. Nevertheless, 5α-dihydroprogesterone and 3α,5α-THDOC could be only qualitatively assessed when using PP. In contrast, DHEA-S could not be quantified or identified when using SLE. A novel surrogate matrix-background subtraction approach, using rat plasma after the animal's adrenalectomy, has been implemented into the optimized PP-UHPLC-MS/MS workflow, successfully validated according to the unified ICH/EMA M10 guidelines, and compared to the traditional quantification strategies. Moreover, the validity of the newly adopted approach has been verified by the targeted profiling of multiple biologically active endogenous steroids in more than 500 samples of mouse plasma in total. SignificanceUnderestimation of hurdles associated with steroid analysis often compromises the accurate steroid quantification. Our comprehensive, fully validated UHPLC-MS/MS method targeting a wide spectrum of endogenous steroids, mitigating steroid crosstalk and using a minimal sample volume together with a novel surrogate matrix-background subtraction approach significantly advances steroid analysis for research and clinical applications covering multiple biological scopes.

The role of pH decrease in tofu-like precipitate formation using magnesium chloride

Uncertainty surrounds the role of pH in tofu-like precipitate (TLP) formation using MgCl2. In this study, to clarify the role of MgCl2-induced pH decrease in TLP formation, the protein concentration in the supernatant, weight of wet precipitate, and the solubility in 2 M urea at constant pH (6.5 or 6.2) were measured using a buffer and comparing the buffering effect under variable pH conditions to the effect observed without buffer. Analysis of protein concentration in the supernatant showed no precipitation of soymilk proteins under stable pH conditions of 6.5. Wet precipitate formation varied substantially during TLP formation at three different pHs; the weight slightly increased with the increase in MgCl2 concentration up to 20 mM at a constant pH of 6.5. However, wet precipitation was substantially enhanced by MgCl2 (≥ 10 mM) at a constant pH of 6.2 but rapidly increased by MgCl2 (≥ 7 mM) under variable pH conditions. The protein concentrations and wet precipitate weight at 20 mM MgCl2 under constant pH conditions showed contrasting patterns. However, the weight of dry precipitate was almost the same under both constant pH (6.5 and 6.2) conditions. Urea solubility under constant and variable pH conditions differed; no urea-insoluble precipitates were produced under constant pH conditions. These results suggest that pH decrease is essential for bringing proteins closer together for MgCl2-mediated TLP formation. This study will aid in understanding the mechanisms underlying TLP formation and support further discovery of new coagulants for the effective production of new tofu-like and tofu-based foods.

Micro Scale Chromatography of Human Plasma Proteins for Nano LC-ESI-MS/MS

Organic precipitation of proteins with acetonitrile demonstrated complete protein recovery and improved chromatography of human plasma proteins. The separation of 25 μL of human plasma into 22 fractions on a QA SAX resin facilitated more effective protein discovery despite the limited sample size. Micro chromatography of plasma proteins over quaternary amine (QA) strong anion exchange (SAX) resins performed best, followed by diethylaminoethyl (DEAE), heparin (HEP), carboxymethyl cellulose (CMC), and propyl sulfate (PS) resins. Two independent statistical methods, Monte Carlo comparison with random MS/MS spectra and the rigorous X!TANDEM goodness of fit algorithm protein p-values corrected to false discovery rate q-values (q≤ 0.01) that agreed on at least 12,000 plasma proteins, each represented by at least three fully tryptic corrected peptide observations. There was qualitative agreement on 9,393 protein/gene symbols between the linear quadrupole versus orbital ion trap but also quantitative agreement with a highly significant linear regression relationship between log observation frequency (F value 4,173, p-value 2.2e-16). The use of a QA resin showed nearly perfect replication of all the proteins that were also found using DEAE-, HEP-, CMC-, and PS-based chromatographic methods combined and together estimated the size of the size of the plasma proteome as ≥12,000 gene symbols.

Exploring electromembrane extraction coupled to fast LC-MS/MS as a high-throughput platform for determination of 12 polar endogenous metabolites in human plasma

High efficiency in the analytical workflow, including fast sample preparation and LC-MS/MS analysis, is an advantage when analyzing a high number of samples. It can however be a challenge when determining polar analytes in complex, biological samples, and one must expect to make a compromise between a simple sample preparation followed by a long chromatographic separation, or vice versa, to limit matrix effects. In this proof-of-concept work, a one-step 96-well (parallel extraction) electromembrane extraction (EME) method was coupled to flow injection-MS/MS of 0.7 min per sample, allowing a very high-throughput analysis of 12 polar, endogenous metabolites from unprecipitated plasma of limited dilution. The throughput of the EME method matched the subsequent analysis. Recoveries ranged from 6-93%, and repeatability and linearity were 2-15% and R2≥0.9949, respectively, for all but two compounds. Matrix effects were approximately 50% after EME and varied less than 11% between 6 plasma donors, which represented a major improvement relative to a simple protein precipitation where signals were entirely suppressed. The work demonstrates a potential for EME coupled to flow injection-MS/MS to serve as a high-throughput platform for bioanalysis, not just of polar analytes, but also hydrophobic drugs both basic and acidic.

Assay for the quantification of abemaciclib, its metabolites, and olaparib in human plasma by liquid chromatography-tandem mass spectrometry

An isotope-dilution bioanalytical assay for abemaciclib and its metabolites in combination with olaparib was developed and validated in human plasma K2 EDTA. For the quantitative assay, human plasma samples (or human plasma QC samples) were spiked with internal standard solution before a simple protein precipitation with methanol. The extract was injected onto a liquid chromatography-tandem mass spectrometry (LC-MS/MS) instrument where it was chromatographically separated by a polar end-capped reversed phase column and guard using gradient elution with water and methanol both modified with 0.2% formic acid (v/v) as the mobile phases. The analytes and internal standards were measured by heated electrospray ionization (HESI) in positive polarity using selected reaction monitoring (SRM) on a triple quadrupole mass spectrometer. The assay was validated for linear ranges as follows: 0.4 – 1000nM abemaciclib, 0.35 – 1000nM M2 and M18, 0.5 – 1000nM M20, and 0.75 – 1000nM olaparib. The inter-day or between day precision for the quality controls (n = 18) was < 13% and the accuracy was ± 12%, for all analytes, including the lower limit of quantification (LLOQ). The intra-day or within day precision for the quality controls (n = 6) was ≤ 11% and the accuracy was ± 12% for low, mid, and high and < 19% at LLOQ. The recovery in human plasma was determined to be between 92 and 102% for all analytes spanning the linear range. The validated, bioanalytical quantitative assay was designed to measure abemaciclib, its metabolites, and olaparib for pharmacokinetic evaluation of patients in clinical trials for breast, brain, and ovarian cancers.

Pharmacokinetic and tissue distribution study of pectolinarigenin in rats using UPLC-MS/MS

Pectolinarigenin (PEC), a natural flavonoid isolated from Cirsium japonicum, exhibits promising therapeutic potential for multiple cancers. In present study, a simple and sensitive UPLC-MS/MS method was established for the quantification of PEC in rat plasma and tissues. The assay procedure involved a one-step protein precipitation with tadalafil as the internal standard, and separation on a Welch Xtimate UHPLC C18 column by gradient elution of acetonitrile/aqueous formic acid (0.1 %, v/v) at a flow rate of 0.2 m L·min−1. The detection was conducted using multiple-reaction monitoring via an electrospray ionization source in positive ionization mode. The established method was proved to be highly sensitive with a good linearity (R2 > 0.99) in respective concentration range (0.1–100 ng·mL−1 in plasma and 1–10000 ng·mL−1 in tissues) and acceptable extraction recovery (≥71.17 %), matrix effect and stability, which was applied to study the pharmacokinetics and tissue distribution of PEC after intravenous (100 μg·kg−1) and oral administration (10, 20 and 40 mg·kg−1). PEC was promptly absorbed (Tmax ≤ 0.222 h) and maintained at a low level with slow elimination (t1/2 z ≥ 14.47 h) in rats after oral administration, resulting in extremely low bioavailability (0.56–0.68 %). However, PEC is widely distributed in rat tissues with high exposure in GI tract, liver and kidney. The bioavailability and tissue affinity were firstly revealed, which will guide directions for further development of PEC as an anti-tumor drug candidate.

PH shift extraction technique for plant proteins: A promising technique for sustainable development

With the recognition of animal-derived foods as unhealthy and their association with climate change, researchers are increasingly focusing on plant-based protein as a sustainable alternative. Plant proteins offer versatile functional and dietary benefits, making them suitable for various food applications. This study investigates the influence of alkaline and acidic pH conditions on the extraction yield and the functional and nutritional properties of plant-based proteins. The primary sources of plant protein include cereals, legumes, and oilseeds, which can be used to address essential amino acid deficiencies through blending. Several methods have been employed for protein extraction from plant sources, such as salt extraction, microwave-assisted extraction, ultrasound-assisted extraction, and micellar precipitation. Among these techniques, the pH shift method stands out due to its non-thermal nature, sustainability, and cost-effectiveness. In this method, proteins are solubilized at alkaline pH and then precipitated at their isoelectric point, resulting in a collection of protein precipitate. It is crucial to optimize extraction techniques based on qualitative and quantitative analysis to enhance protein yield, characteristics, and nutritional value. Most conventional protein extraction methods require a large quantity of chemicals, which imposes the issue of safe disposal, compromising environmental sustainability. Traditional methods also produce protein with non-proteinaceous constituents, adding another purification step and resulting in increased overall cost. However, the pH shift method utilizes comparatively less harsh chemicals and has a high protein extraction yield, which makes it comparatively more environmentally and economically sustainable. The sustainable extraction of plant-based proteins addresses the health and environmental concerns associated with animal-derived foods and offers a promising solution to promote sustainability in the food industry.

The Pharmacokinetic Interaction of Tirabrutinib with Voriconazole, Itraconazole, and Fluconazole in SD Rats by UPLC-MS/MS.

Tirabrutinib is an orally effective, approved, and highly selective second-generation Bruton's tyrosine kinase (BTK) inhibitor for the treatment of recurrent or refractory primary central nervous system lymphoma (PCNSL). This study aimed to develop an ultra-high performance liquid chromatography- tandem mass spectrometry (UPLC-MS/MS) method for the determination of tirabrutinib concentration in rat plasma, where zanubrutinib was used as an internal standard (IS). This method was also applied to study whether tirabrutinib would interact with voriconazole, itraconazole, and fluconazole in rats, providing a reference value for clinical medication guidance. In the current study, the organic solvent protein precipitation method was used to treat plasma samples, which is simple and reproducible. Tirabrutinib (m/z 455.32 → 320.21) and zanubrutinib (m/z 472.13 → 455.04) were separated on a Waters Acquity BEH C18 column (2.1 × 50 mm, 1.7 μm) and detected by multiple reaction monitoring (MRM) in positive ionization mode. The method showed good linearity in the range of 5-3000 ng/mL for tirabrutinib with the lower limit of quantification (LLOQ) of 5 ng/mL. The recovery and matrix effects were 85.7-91.0% and 102.0-113.3%, respectively. The accuracy, precision, stability, and carry-over effect were also acceptable. The method could also be used for determining the pharmacokinetic interaction of tirabrutinib in rats. The results showed AUC0→∞ of tirabrutinib to be increased by 139.3% and 83.9% in the presence of voriconazole and fluconazole, respectively, while itraconazole had little effect. It is necessary to monitor the concentration of tirabrutinib in patients when it is combined with voriconazole and fluconazole to achieve a better therapeutic effect and reduce the risk of adverse reaction. Further research should be conducted in the future.

An LC[sbnd]MS/MS method for the simultaneous determination of ten antimicrobials and its application in critically ill patients

Significant pharmacokinetic variation occurs in critically ill patients, leading to underexposure to antibiotics and poor prognosis. In this study, we developed a simple, sensitive, and fast liquid chromatography tandem mass spectrometry (LCMS/MS) platform for the simultaneous quantification of 8 antibacterial and 2 antifungal drugs, which is optimally suited for clinically efficient, real-time therapeutic drug monitoring (TDM). Multiple reaction monitoring (MRM) mass spectrometry was used in this method, and samples were prepared via protein precipitation with methanol. Chromatographic separation was accomplished on a BGIU Column-U02 (2.1ｘ50 mm, 3 µm), with six stable isotopes and one analog as an internal standard. The overall turnaround time of the assay was 5 minutes. All the drugs tested (piperacillin, cefoperazone, meropenem, levofloxacin, moxifloxacin, daptomycin, linezolid, vancomycin, fluconazole and voriconazole) were linear in the test concentration range (r ≥ 0.9900), the accuracy was 95 %-111 %, the precision variation coefficient was greater than or equal to 10 %, the lower limit of quantitation was 0.31–7.51 mg/L, and the coefficient of variation of the matrix factor was less than 10 %. The recovery rates ranged from 85 % to 115 %, and the antibiotics were stable at 4°C and −20°C for 6 days, with an offset of greater than or equal to 15 %. This method was successfully applied to routine TDM in 252 elderly critically ill patients.

Pharmacokinetics of Ganaplacide and Lumefantrine in Adults, Adolescents, and Children with Plasmodium falciparum Malaria Treated with Ganaplacide Plus Lumefantrine Solid Dispersion Formulation: Analysis of Data from a Multinational Phase 2 Study.

The novel antimalarial ganaplacide combined with lumefantrine solid dispersion formulation (LUM-SDF) was effective and well tolerated in the treatment of uncomplicated falciparum malaria in adults, adolescents, and children in a multinational, prospective, randomized, active-controlled Phase II study conducted between August 2017 and June 2021 (EudraCT 2020-003284-25, Clinicaltrials.gov NCT03167242). Pharmacokinetic data from that study are reported here. The trial comprised three parts: a run-in part in 12 adult/adolescent patients treated with a single dose of ganaplacide 200 mg plus LUM-SDF 960 mg assessed potential pharmacokinetic (PK) interactions between ganaplacide and lumefantrine; in Part A, adult/adolescent patients received one of the six ganaplacide-LUM-SDF regimens or artemether-lumefantrine; and in Part B, three dose regimens identified in Part A, and artemether-lumefantrine, were assessed in children aged 2 to <12 years, with body weight ≥10 kg. A rich blood sampling schedule was used for all 12 patients in the PK run-in part and a subset of patients (N = 32) in Part A, with sparse sampling for remaining patients in Parts A (N = 275) and B (N = 159). Drug concentrations were determined by a validated protein precipitation and reverse phase liquid chromatography with tandem mass spectrometry detection method. Parameters including AUCinf, AUClast, AUC0-t, Cmax, and tmax were reported where possible, using non-compartmental analysis. In the PK run-in part, there was no notable increase in ganaplacide or lumefantrine exposure when co-administered. In Parts A and B, ganaplacide exposures increased with dose, but lumefantrine exposure was numerically under dose-proportional. Lumefantrine exposure was higher with ganaplacide-LUM-SDF than with artemether-lumefantrine, although high variability was observed. Ganaplacide and lumefantrine exposures (Cmax and AUC0-24 h) were comparable across age and body weight groups. Drug exposures needed for efficacy were achieved using the dose regimen 400 mg ganaplacide plus lumefantrine 960 mg once daily for 3 days under fasted conditions.

A validated LC-MS/MS method for the simultaneous quantification of iothalamate and hippuran in serum and urine for non-radioactive kidney function assessment

A novel liquid chromatography-tandem mass spectrometry method is described for the quantitative determination of the kidney function markers iothalamate and hippuran in human serum and urine. It is based on protein precipitation with methanol followed by dilution of the supernatant for serum and simple dilution for urine. The polar analytes are chromatographically separated by a 6.5-min gradient on a low-ligand density reversed-phase column; detection is performed by electrospray ionization tandem mass spectrometry in the positive ion mode against stable-isotope labeled internal standards.The results of a thorough method validation show that iothalamate and hippuran can be simultaneously quantified in the concentration ranges 0.500–30.0 ng/mL and 10.0–5000 ng/mL for serum and urine, respectively, with values for CV and absolute bias not exceeding 10 %, and with sufficient stability in all relevant matrices and solvents. The method was successfully applied for the analysis of serum and urine samples of multiple individuals who received both iothalamate and hippuran.