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High‐throughput drug discovery is highly dependent on the targets available to accelerate the process of candidate screening. Unbiased drug target identification is of crucial importance to reveal the mechanisms of protein–drug interactions. In recent years, energetics‐based protein conformation methods have become an alternative strategy for protein target discovery of bioactive drugs. In this study, we present a novel tannin protein precipitation method (TAP) for identifying target proteins of small molecules using the effect on protein solubility of tannin. The feasibility of this method was validated using a drug model of methotrexate. It was found that the known target DHFR yields a significant solubility shift. This method was further investigated with Annexin A2, the target protein of protopanaxadiol (PPD)‐S by western blot, and by screening potential target proteins of PPD‐S in K562 cells by quantitative proteomics. Finally, the TAP method was employed to differentiate the target proteins of pair chiral molecules PPD‐S and PPD‐R, which can bring up novel insights into the mechanism of drug–protein interactions. This study not only provides an effective tool for drug research and development but also offers new ideas for understanding the mechanism of action of natural chiral bioactive compounds. [ABSTRACT FROM AUTHOR]

This 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. [ABSTRACT FROM AUTHOR]

Liquid chromatography–triple quadrupole mass spectrometry (LC–MS/MS) is becoming an increasingly essential analytical technique in many fields, such as food, environmental, biochemical, pharmaceutical, and clinical chemistry. The testing matrices, such as plasma and serum, cannot usually be sent directly to the LC–MS/MS system because of the large number of unrelated substances in addition to target analytes in samples, such as cells, tissues, and proteins. Protein precipitation (PPT) is the most utilized pretreatment method in clinical analysis because of its ease of use; its performance always involves the separation of the liquid phase from the solid phase, in which the most commonly used manipulation is centrifugation. However, there is an upper limit of throughput for centrifugation, and the cost and difficulty of integrating centrifugation into automatic equipment are usually high. To solve the drawbacks of the current PPT method, we developed a cartridge structure that can omit the centrifugation step in PPT, which, in turn, can facilitate the incorporation of the PPT method into an automatic clinical pretreatment procedure for LC–MS/MS. We used vitamin D analysis as a representative application in our endeavor to develop a centrifugation‐free PPT method that can be easily applied to a new automatic clinical pretreatment procedure. [ABSTRACT FROM AUTHOR]

Na Zhang,1,* Yiran Xu,1,2,* Beibei Liang,1 Jinru Zeng,1 Rui Wang,1 Yun Cai1 1Center of Medicine Clinical Research, Department of Pharmacy, Medical Supplies Center, PLA General Hospital, Beijing, People’s Republic of China; 2Department of Pharmacy, The Second Naval Hospital of Southern Theater Command of PLA, Sanya, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yun Cai, Center of Medicine Clinical Research, Department of Pharmacy, Medical Supplies Center, PLA General Hospital, Beijing, People’s Republic of China, Tel +86-10-6693-7166, Email caicai_hh@126.comAbstract: Colistin is the last-line option for the treatment of multidrug-resistant gram-negative bacterial infections with narrow therapeutic window. It is essential to ensure its efficacy and safety by therapeutic drug monitoring (TDM). Quantitative determination of colistin is difficult due to its complex ingredients. Previous determination methods demand intricate sample pre-treatment which are not only time-consuming but also costly, and is difficult to apply in clinical practice. Therefore, in order to carry out quantitative determination of colistin accurately and quickly, we establish a rapid high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) with simple sample pre-treatment process. The sample was purified by acetonitrile to remove the plasma protein. Then purified colistin was effectively separated from terfenadine, an internal standard (IS) using Phenomenex Kinetex C18 column (50.0× 2.1mm, 5μm) with acetonitrile and water mobile phase at a flow rate of 0.5 mL/min and 40°C column temperature. Colistin and IS were monitored in positive ion mode. Our method expressed good linearity in 50.0~6000 ng/mL of colistin B and 28.31~3397.51 ng/mL of colistin A in plasma. Methodology validations, including selectivity, precision, accuracy, recovery, stability, matrix effect, and dilution integrity met acceptance criteria of Bioanalytical Method Validation (M10) of the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH).Keywords: colistin, HPLC-MS/MS, human plasma, protein precipitation, rapid and simple detection

Proteins that are amphiphilic and have low water solubility can self-assemble into nanoparticles useful in food science, pharmaceutical science, or biotechnology. However, protein nanoparticles exhibit drawbacks such as low stability unless the particles are coated. In the current study, tannic acid is the coating agent for nanoparticles synthesized from the protein zein. Tannic acid is a hydrolyzable tannin comprising a polyol esterified with galloyl residues. The nominal molecular formula of tannic acid (C76H52O46) suggests the material is decagalloyl glucose, obscuring its complex composition as a mixture of galloyl esters of glucose. We prepared hollow zein nanoparticles and coated them with tannic acid preparations that had short or long galloyl ester chains. The % α-helix of zein in nanoparticles is lower than in native zein but there is no effect of coating the particles with tannic acid. Interactions between the tannic acid and the zein slightly perturb the IR spectrum of the protein but there is no effect of galloyl chain length. We confirmed that tannic acid-coated particles have a more negative zeta potential, suggesting greater stability compared to uncoated particles. Coating with longer chain length tannic acid reduces particle diameter and tends to decrease polydispersity but does not change particle digestibility. Coating with shorter galloyl chain length tannic acid tends not to change particle diameter, reduces polydispersity of the particles, and stabilizes particles to enzymatic digestion. Tannic acid is a naturally occurring tunable coating for nanoparticles that can be used to adjust properties such as particle size, polydispersity, and digestibility for specific purposes.

A high‐performance liquid chromatography‐tandem mass spectrometry method has been developed for the determination of irinotecan, 7‐ethyl‐10‐hydroxycamptothecin (SN38), and SN38 glucuronide (SN38G) in rat plasma in the present study. The analytes were separated on a C18 column and a triple‐quadrupole mass spectrometry equipped with an electrospray ionization source was applied for detection. Irinotecan, SN38 and SN38G could be well retained in the C18 column after optimization of mobile phase. A simple protein precipitation was used to pretreat the plasma. The extraction recovery was above 90% and the matrix effect could be negligible. The method was linear over the concentration ranges of 3.46–3458.8 ng/mL for irinotecan, 2.53–2530.0 ng/mL for SN38 and 2.5–2500.0 ng/mL for SN38G. The precision and accuracy was within the acceptable limits. The simple and convenient method was validated and successfully applied to support the pharmacokinetic study and elucidate the mechanism of irinotecan‐induced diarrhea after irinotecan was intravenously injected to the Sprague‐Dawley rats. [ABSTRACT FROM AUTHOR]

Proteins that are amphiphilic and have low water solubility can self-assemble into nanoparticles useful in food science, pharmaceutical science, or biotechnology. However, protein nanoparticles exhibit drawbacks such as low stability unless the particles are coated. In the current study, tannic acid is the coating agent for nanoparticles synthesized from the protein zein. Tannic acid is a hydrolyzable tannin comprising a polyol esterified with galloyl residues. The nominal molecular formula of tannic acid (C76H52O46) suggests the material is decagalloyl glucose, obscuring its complex composition as a mixture of galloyl esters of glucose. We prepared hollow zein nanoparticles and coated them with tannic acid preparations that had short or long galloyl ester chains. The % α-helix of zein in nanoparticles is lower than in native zein but there is no effect of coating the particles with tannic acid. Interactions between the tannic acid and the zein slightly perturb the IR spectrum of the protein but there is no effect of galloyl chain length. We confirmed that tannic acid-coated particles have a more negative zeta potential, suggesting greater stability compared to uncoated particles. Coating with longer chain length tannic acid reduces particle diameter and tends to decrease polydispersity but does not change particle digestibility. Coating with shorter galloyl chain length tannic acid tends not to change particle diameter, reduces polydispersity of the particles, and stabilizes particles to enzymatic digestion. Tannic acid is a naturally occurring tunable coating for nanoparticles that can be used to adjust properties such as particle size, polydispersity, and digestibility for specific purposes. [ABSTRACT FROM AUTHOR]

A high‐performance liquid chromatography‐tandem mass spectrometry method has been developed for the determination of peramivir in rat plasma in the present study. The analytes were separated on a C18 column and a triple‐quadrupole mass spectrometry equipped with an electrospray ionization source was applied for detection. Peramivir could be well retained in the C18 column after optimization of the mobile phase. A simple protein precipitation was used to pretreat the plasma. The extraction recovery was about 100% and the matrix effect could be negligible. The method was linear over the concentration ranges of 1.17–2340.0 ng/mL for peramivir. The precision and accuracy were within acceptable limits. The lowest limit of quantification was 1.17 ng/mL. Compared with those methods which have been developed and published since 2001, the present method is very simple and convenient. The method was validated and successfully applied to support the pharmacokinetics study after peramivir was intravenously injected into the Sprague‐Dawley rats. [ABSTRACT FROM AUTHOR]

The small molecule A‐485 competitively inhibits the histone acetyltransferase domain of CBP (cyclic‐adenosine monophosphate response element‐binding protein)/p300. Apart from its antineoplastic activity, researchers are exploring its potential benefits in treating osteoporosis and its impact on energy metabolism. However, so far, only limited pharmacokinetic data are available, and the crucial determination of A‐485 concentration in various biological materials with small sample volumes remains unpublished. A rapid and sensitive LC–tandem mass spectrometry method has been developed and validated to quantify A‐485 in mouse serum and tissue. In this method, serum samples underwent precipitation with acetonitrile, while cell lysates were appropriately diluted. The determination of A‐485 utilized a reversed‐phase column with a mobile phase gradient, and detection was carried out in multiple reaction monitoring mode. The lower standard sample, with a concentration of 7.8 ng/mL, served as the lower limit of quantification, while the upper standard was established at 1000 ng/mL. A‐485 concentrations were assessed in both serum samples and the lysate of all examined tissues, revealing swift metabolic clearance. The analytical method outlined here is deemed appropriate for subsequent studies. The ability to measure the active ingredient in various compartments facilitates the determination of accurate pharmacokinetic parameters. In the event of human use of A‐485, the analysis method can be seamlessly transferred to human samples. [ABSTRACT FROM AUTHOR]

Background: Rifampicin is an important anti tuberculosis drug for the early infection and, more importantly, bactericidal activity against mycobacterium tuberculosis. LC-MS/MS, with the required molecular specificity, provides quantitative method to rapidly differentiate between the drugs and their metabolites. Aim: The current study represents the fast and simple LC-MS/MS method for determination of rifampicin in the human blood plasma. Materials and Methods: The plasma samples containing the rifampicin drug were cleaned up by using the protein precipitation method. The chromatographic division was performed by utilizing the ZORBAX Eclipse plus C18 Column (4.6 mm X 150 mm, 5µm). The versatile stage comprised of acetonitrile and 10 Mm ammonium acetic acid derivation in a proportion of 80:20% V/V, wash the column with the blend of solvents comprising of acetonitrile and water 80:20% V/V, maintaining column oven temperature at 30°C±1°C and the auto sampler temperature is kept up with at 10°C±1°C. The injection volume of the sample is 10.0 µL and the total run time of the experiment is 4 min with a flow rate of 1000 mL/min and with a split ratio of 50:50 for the entire experiment. Results and Discussion: An LC-MS/MS method for the rifampicin from the sample was performed. Sample volume of 0.300 ml, the injection volume is taken as 10.0 µL and total run time is 4 min, RT for rifampicin is 1.30±0.5 min and the reference drug roxithromycin RT is 3.00±0.5 min. Conclusion: The transient LC-MS/MS study permits the assessment of enormous quantities of blood tests in a brief timeframe with fast, simple and successful readiness, giving a quick, dependable and best device for RIF clinical observing and review. [ABSTRACT FROM AUTHOR]

Numerous studies have proven the potential of cytokeratin 19 fragment 21-1 (CYFRA 21-1) detection in the (early) diagnosis and treatment monitoring of non-small cell lung cancer (NSCLC). Conventional immunoassays for CYFRA 21-1 quantification are however prone to interferences and lack diagnostic sensitivity and standardization. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is an emerging approach based on a different, often superior, detection principle, which may improve the clinical applicability of CYFRA 21-1 in cancer diagnostics. Therefore, we developed and validated a protein precipitation, immunoaffinity (IA) LC-MS/MS assay for quantitative analysis of serum CYFRA 21-1. Selective sample preparation was performed using ammonium sulfate (AS) precipitation, IA purification, tryptic digestion and LC-MS/MS quantification using a signature peptide and isotopically labeled internal standard. The workflow was optimized and validated according to EMA guidelines and results were compared to a conventional immunoassay. Significant interference effects were seen during IA purification, which were sufficiently solved by performing AS precipitation prior to IA purification. A linear calibration curve was obtained in the range of 1.0–100 ng/mL (R2=0.98). Accuracy and precision were well within acceptance criteria. In sera of patients suspected of lung cancer, the method showed good correlation with the immunoassay. A robust AS precipitation-IA LC-MS/MS assay for the quantification of serum CYFRA 21-1 was developed. With this assay, the clinically added value of LC-MS/MS-based detection over immunoassays can be further explored. [ABSTRACT FROM AUTHOR]

Starch production from fresh sweet potatoes generates process wastewater called sweet potato cell liquid (SPCL), which is rich in sweet potato protein (SPP). Currently, the commonly used protein recovery methods, such as isoelectric point precipitation and ultrafiltration, were not suitable for SPP recovery due to the low protein content of SPCL and the high cost of recovery. The feasibility of recovering SPP by SPCL acidification via inoculation-assisted fermentation was investigated in this study. The results indicated that the pH of SPCL could be reduced to approximately 4.0 within 6 h of fermentation with inoculation, resulting in an SPP extraction yield of 55.45% and purity of 66.23 g protein/100 g. With the addition of heating treatment, the extraction yield of SPP increased to 76.97–95.34%, while it maintained the purity of 66.36–70.12 g protein/100 g. The composition analysis revealed that SPP products contained sugars (below 11.5 g/100 g) in addition to protein and trace amounts of lignin and phenolics. Functional properties analysis showed that the SPP recovered by inoculation-enhanced fermentation exhibited better emulsifying and foaming properties, and higher digestibility compared to the SPP precipitated using hydrochloric acid. The method of extracting SPP from SPCL by inoculation-enhanced fermentation, as developed in this study, was a straightforward and cost-effective process that fosters significant potential for industrial applications.

Nicotinamide adenine dinucleotide (NAD) is a cofactor in redox reactions and an essential mediator of energy metabolism. The redox balance between NAD+ and NADH affects various diseases, cell differentiation, and aging, and in recent years there has been a growing need for measurement techniques with improved accuracy. However, NAD(H) measurements, representing both NAD+ and NADH, have been limited by the compound's properties. We achieved highly sensitive simultaneous measurement of NAD+ and NADH under non-ion pairing, mobile phase conditions of water, or methanol containing 5 mM ammonium acetate. These were achieved using a simple pre-treatment and 7-min analysis time. Use of the stable isotope 13C5-NAD+ as an internal standard enabled validation close to BMV criteria and demonstrated the robustness of NAD(H) determination. Measurements using this method showed that brain NAD(H) levels correlate strongly with plasma NAD(H) levels in the same mouse, indicating that NAD(H) concentrations in brain tissue are reflected in plasma. As NAD(H) is involved in various neurodegenerative diseases and cerebral ischemia, as well as brain diseases such as mitochondrial myopathies, monitoring changes in NADH levels in plasma after drug administration will be useful for development of future diagnostics and therapeutics. [ABSTRACT FROM AUTHOR]

Soy protein concentrates (SPCs) are common food ingredients. They typically contain 65% (w/w) protein and ∼30% (w/w) carbohydrate. SPCs can be obtained with various protein precipitation conditions. A systematic study of the impact of these different protein precipitation protocols on the SPC protein composition and physical properties is still lacking. Here, SPCs were prepared via three different protocols, that is, isoelectric (pH 3.5–5.5), aqueous ethanol (50%–70% [v/v]), and Ca2+ ion (5–50 mM) based precipitations, and analyzed for (protein) composition, protein thermal properties, dispersibility, and water‐holding capacity. SPCs precipitated at pH 5.5 or by adding 15 mM Ca2+ ions had a lower 7S/11S globulin ratio (∼0.40) than that (∼0.50) of all other SPC samples. Protein in SPCs obtained by isoelectric precipitation denatured at a significantly higher temperature than those in ethanol‐ or Ca2+‐precipitated SPCs. Precipitation with 50%–60% (v/v) ethanol resulted in pronounced denaturation of 2S albumin and 7S globulin fractions in SPCs. Additionally, increasing the precipitation pH from 3.5 to 5.5 and increasing the Ca2+ ion concentration from 15 to 50 mM caused a strong decrease of both the dispersibility of the protein in SPC and its water‐holding capacity at pH 7.0. In conclusion, this study demonstrates that the SPC production process can be directed to obtain ingredients with versatile protein physicochemical properties toward potential food applications. Practical Application: This study demonstrates that applying different protein precipitation protocols allows obtaining SPCs that vary widely in (protein) composition and physical properties (such as protein dispersibility and water‐holding capacity). These varying traits can greatly influence the suitability of SPCs as functional ingredients for specific applications, such as the production of food foams, emulsions, gels, and plant‐based meat alternatives. The generated knowledge may allow targeted production of SPCs for specific applications. [ABSTRACT FROM AUTHOR]

In the recent years, liquid chromatography with tandem mass spectrometry has gained popularity in laboratories. This technique has a higher specificity, detects different analytes from a single specimen, measures analytes in distinct matrices, and substantially reduce analytical interference, with respect to immunoassay. The processing and preparation of biological samples are crucial in chromatography. Interferences in blood testing are usually caused by the presence of phospholipids and proteins. The main objective of this study was to improve analytical processes for drug screening by LC-MS/MS using a novel blood sample preparation method based on protein precipitation and removal of phospholipids. An evaluation was performed of a new method for the preparation of blood samples based on protein precipitation and removal of phospholipids by LC-Q-q-LIT. Limit of detection, limit of quantification and measurement range were determined for 56 molecules. The results of 11 cases were compared with those obtained using standard blood collection methods and instruments. The novel blood preparation and testing method based on LC-Q-q-LIT, a more sensitive technique, has demonstrated to yield comparable results to traditional methods. In addition, this new technique reduces turnaround time and costs. [ABSTRACT FROM AUTHOR]

Virus-like particles (VLPs) are a promising class of biopharmaceuticals for vaccines and targeted delivery. Starting from clarified lysate, VLPs are typically captured by selective precipitation. While VLP precipitation is induced by step-wise or continuous precipitant addition, current monitoring approaches do not support the direct product quantification, and analytical methods usually require various, time-consuming processing and sample preparation steps. Here, the application of Raman spectroscopy combined with chemometric methods may allow the simultaneous quantification of the precipitated VLPs and precipitant owing to its demonstrated advantages in analyzing crude, complex mixtures. In this study, we present a Raman spectroscopy-based Process Analytical Technology (PAT) tool developed on batch and fed-batch precipitation experiments of Hepatitis B core Antigen VLPs. We conducted small-scale precipitation experiments providing a diversified data set with varying precipitation dynamics and backgrounds induced by initial dilution or spiking of clarified Escherichia coli-derived lysates. For the Raman spectroscopy data, various preprocessing operations were systematically combined allowing the identification of a preprocessing pipeline, which proved to effectively eliminate initial lysate composition variations as well as most interferences attributed to precipitates and the precipitant present in solution. The calibrated partial least squares models seamlessly predicted the precipitant concentration with R2 of 0.98 and 0.97 in batch and fed-batch experiments, respectively, and captured the observed precipitation trends with R2 of 0.74 and 0.64. Although the resolution of fine differences between experiments was limited due to the observed non-linear relationship between spectral data and the VLP concentration, this study provides a foundation for employing Raman spectroscopy as a PAT sensor for monitoring VLP precipitation processes with the potential to extend its applicability to other phase-behavior dependent processes or molecules.

In this paper we report a simple and efficient method for the concurrent analysis of tryptophan, 5-HTP, tryptamine, serotonin, and 5-HIAA in mouse serum using UHPLC-ED after protein precipitation and dilution. These compounds are neuroactive and are of interest in studies of mood and behavior; They are also biomarkers for the presence of neuroendocrine tumors and are used in the diagnosis of these cancers. After a brief series of validation experiments, this method was applied to serum from mouse behaviour experiments.•A convenient UHPLC method with electrochemical detection for concomitant analysis of the serotonin pathway in serum, including, for the first time, tryptamine.•The method met all performance criteria established for use in our lab and was applied in rodent experiments.

A sensitive and accurate high performance liquid chromatography method utilizing ultraviolet/visible light detection (HPLC-UV) for the quantification of Ribociclib in Spiked Human Plasma by HPLC-UV was developed and validated. Ribociclib (RCB) and the internal standard (IS), Trifluridine, were first extracted from plasma samples by a simple Protein Precipitation extraction using Acetonitrile. Plasma concentration of RCB and internal standard were then analyzed by applying reversed phase chromatography using Orochem orosil C18 (4.6 mm × 250 mm, 5 μ) and elution with a isocratic mobile phase consisting of 10 mM phosphate buffer – Acetonitrile (60:40, v/v) adjusted to pH 3.0 at a flow rate of 1.0 ml/min. Detection of RCB and the IS was subsequently done at a wavelength of 260 nm. The limit of quantification was 10 ng/ml. The calibration curve was linear (R2>0.998) over the concentration range of 10-1000 ng/ml. For human plasma, the accuracy and precision were within ±15% and ≤15%, respectively, for all concentrations, except for the lower limit of quantification, where they were within ≤20%.

Plant species show large variation in the composition and content of their tannins and other polyphenols. These large metabolites are not easy to measure accurately, but they are important factors for species bioactivity and chemotaxonomy. Here, we used an automated group-specific UHPLC-DAD-MS/MS tool to detect and quantify eight most common polyphenol groups in 31 chemically diverse plant species representing many types of growth forms and evolutionary ages. Ten replicate plants were used for each species and two polyphenol-related bioactivities, i.e., protein precipitation capacity and oxidative activity were measured in all samples as well. By the help of a novel 2D fingerprint mapping tool we were able to visualize the qualitative and quantitative differences between the species in hydrolysable tannins (galloyl and hexahydroxydiphenoyl derivatives), proanthocyanidins (procyanidins and prodelphinidins), flavonols (kaempferol, quercetin and myricetin derivatives) and quinic acid derivatives together with the two bioactivities. The highest oxidative activities were found with species containing ellagitannins (e.g., Quercus robur, Geranium sylvaticum, Lythrum salicaria and Chamaenerion angustifolium) or prodelphinidin-rich proanthocyanidins (e.g., Ribes alpinum, Salix phylicifolia and Lysimachia vulgaris). The best species with high protein precipitation capacity were rich in gallotannins (Acer platanoides and Paeonia lactiflora) or oligomeric ellagitannins (e.g., Comarum palustre, Lythrum salicaria and Chamaenerion angustifolium). These types of tools could prove their use in many types of screening experiments and might reveal even unusually active polyphenol types directly from the crude plant extracts. [ABSTRACT FROM AUTHOR]

Diazoxide is a potential candidate for the treatment of transitional hypoglycaemia in infants. A clinical trial is currently underway to investigate whether low-dose oral diazoxide is beneficial for severe or recurrent transitional neonatal hypoglycaemia (the NeoGluCO Study, registration ANZCTR12620000129987). The present study aimed to develop and validate the parameters for quantifying diazoxide from neonatal plasma samples, and to assess the stability of extemporaneously prepared diazoxide suspensions to support the NeoGluCO Study. To determine the plasma concentration of diazoxide, a protein precipitation mediated extraction protocol was developed, which demonstrated >94% diazoxide extraction recoveries from all samples. The method was linear over the range of 0.2–40 μg/mL (R2 > 0.9994) with a limit of quantification of 0.2 μg/mL. Accuracy of the method was within 97–106% with relative standard deviation

A selective, rapid, and novel bioanalytical method was developed for the simultaneous determination of methyl paraben, propyl paraben, and Bisphenol A in human plasma with a calibration curve range of 1–100 ng/ml. This method included protein precipitation to ensure extraction along with clean‐up of the complex biometrics. The Chromatographic separation was done using an AccucoreTM aQ liquid chromatography column (Dim. 100×3, particle size: 2.6 µm) with the mobile phase of a mixture consisting of 0.1% formic acid in water: 0.1% formic acid in acetonitrile (03:97, v/v) at a flow rate of 0.300 ml/min. The developed liquid chromatography–tandem mass spectrometry method was further validated utilizing the United States Food and Drug Administration guidelines and all the validation requirements were found to be satisfactory. This proposed method was later successfully utilized to determine the level of the three endocrine‐disrupting compounds in the plasma samples obtained from Indian pregnant women. The results suggest that these women might have been exposed to parabens and Bisphenol A from the use of products such as pharmaceuticals, cosmetics, processed food, and the environment. [ABSTRACT FROM AUTHOR]

UPLC-MS/MS method was developed for determination of bioactive compounds including chlorogenic acid (CA), apigenin (AP) and apigenin 7-glucoside (AP7G) in goat serum. The analytes were separated on Kinetex C18 analytical column and a mobile phase consisting 0.1% formic acid in water and methanol with gradient elution. Detection of analytes was performed using a triple quadrupole mass spectrometer with electrospray ionization in positive mode. The lower limit of quantitation was 0.5 ng mL–1 for AP and AP7G and 2.5 ng mL–1 for CA. The inter- and intra-day precision of the method expressed as relative standard deviation was in the range of 1.22–14.5%. The accuracy of the method expressed as the relative error was in the range of 0.147–14.6%. The extraction recoveries of the compounds were 76.2–89.5%. The validated method was successfully applied for analysis of the compounds in the serum of sixty goats, half of which were fed with fodder with the addition of an herbal mixture. The higher concentration values of all analyzed compounds were reported in the serum of the animals fed with herbs compared to the control group receiving standard fodder. [ABSTRACT FROM AUTHOR]

Protein purification is a basic technology in both biological research and industrial production, and efficient, convenient, economical, and environmentally friendly purification methods have always been pursued. In this study, it was found that alkaline earth metal cations (Mg2+, Ca2+) and alkali metal cations (Li+, Na+, K+) and even nonmetal cations (e.g., NH4+, imidazole, guanidine, arginine, lysine) can precipitate multi-histidine-tagged proteins (at least two tags in a whole protein) at low salts concentrations that are 1–3 orders of magnitude lower than salting-out, and precipitated proteins could be dissolved at moderate concentration of corresponding cation. Based on this finding, a novel cation affinity purification method was developed, which requires only three centrifugal separations to obtain highly purified protein with purification fold similar to that of immobilized metal affinity chromatography. The study also provides a possible explanation for unexpected protein precipitation and reminds researchers to consider the influence of cations on the experimental results. The interaction between histidine-tagged proteins and cations may also have broad application prospects. Key points: • Histidine-tagged proteins can be precipitated by low-concentrations common cations • A novel nonchromatographic protein purification method was developed • Purified protein can be obtained in pellet form by only three centrifugations [ABSTRACT FROM AUTHOR]

This study aimed to establish a simple and sensitive analytical method to simultaneously quantify donepezil (DPZ) and tadalafil (TAD) in rat plasma using lansoprazole (LPZ) as an internal standard (IS) by using liquid chromatography tandem mass spectrometry. The fragmentation pattern of DPZ, TAD, and IS was elucidated using multiple reaction monitoring in electrospray ionization positive ion mode for the quantification of precursor to production at m/z 380.1 → 91.2 for DPZ, m/z 390.2 → 268.1 for TAD, and m/z 370.3 → 252.0 for LPZ. The extracted DPZ and TAD from plasma using acetonitrile-induced protein precipitation was separated using Kinetex C18 (100 × 2.1 mm, 2.6 µm) column with a gradient mobile phase system consisting of 2 mM ammonium acetate and 0.1% formic acid in acetonitrile at a flow rate of 0.25 mL/min for 4 min. The selectivity, lower limit of quantification, linearity, precision, accuracy, stability, recovery, and matrix effect of this developed method was validated according to the guidelines of the U.S. Food and Drug Administration and the Ministry of Food and Drug Safety of Korea. The established method achieved acceptance criteria in all validation parameters, ensuring reliability, reproducibility, and accuracy, and was successfully implemented in a pharmacokinetic study on the co-administration of DPZ and TAD orally in rats. [ABSTRACT FROM AUTHOR]

Previous work on Pacific dulse (Devaleraea mollis), a fast-growing protein-rich red algae, revealed that protein recovery can be significantly improved with cellulase pretreatment and sequential extraction approach. Since solubilized protein fractions need to be precipitated out of solution for enrichment and pellet recovery for downstream applications, this study aimed to evaluate the effects of precipitation strategies on macroalgal protein yield. Extracted protein fractions were precipitated using trichloroacetic acid (TCA) or pH-shift method (HCl/NaOH), and protein concentrations were assessed using three quantification methods, namely modified Lowry, Dumas, and total amino acid analysis (TAA). Specific to each Osborne fraction (albumin, globulin, glutelin and prolamin), the pH-shift precipitation approach was optimized in consideration to the amount of protein pellet recovered, protein retained in supernatant, and volume of acid required for pH adjustment. This work shows that the optimized pH-shift method has competitive yield compared to that of TCA precipitation. Ethanol wash post-pellet collection improved purity of the freeze-dried powders in both precipitation approaches but had more pronounced effects to TCA pellets. This then suggests that a single step precipitation using the optimized pH-shift method can be employed as a food-grade method in the recovery of extracted Pacific dulse proteins. Overall, this work provides a pioneering insight on the recovery of Pacific dulse protein using a pH-shift approach, and how three protein quantification methods were streamlined for protein recovery assessment. As a promising complementary food protein and potential bioactive peptide (BAP) source, this work offers an upscalable and ecologically sustainable recovery approach for seaweed protein from an abundant natural resource on the Pacific coast. [ABSTRACT FROM AUTHOR]

A simple, efficient, and sensitive ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method was developed and validated for simultaneous determination and pharmacokinetic study of salvianolic acid D, rosmarinic acid, lithospermicic acid, salvianolic acid B, and salvianolic acid Y in rat plasma after intravenous administration of salvianolic acid for injection. Three doses of administration, containing 10, 25, and 62.5 mg/kg, were investigated. Plasma samples were pretreated using protein precipitation with pre‐cooled acetonitrile. As shown in S1, Chromatographic separation was achieved on a Waters Acquity UPLC® BEH C18 column (1.7 μm, 2.1 × 100 mm) with a mobile phase composed of acetonitrile‐methanol‐0.5% aqueous formic acid (10:30:60, v/v/v) at a flow rate of 0.3 ml/min. MS was detected by electrospray ion source negative ion mode and multiple reaction monitoring mode. The method was fully validated. The calibration curves for the five phenolic acids were linear in the given concentration ranges. The extraction recoveries, matrix effects, intra‐day and inter‐day precisions, and accuracies of the five analytes were all within acceptable limits. No significant difference of elimination half‐life time (T1/2) of five analytes at three doses was observed. Area under the curve and peak concentration (Cmax) of the five analytes demonstrated a linear increase in the doses with the linear correlation r of each analyte at three doses being greater than 0.915. It indicated that the pharmacokinetic behavior of is positively related to the dose in the range of 10–62.5 mg/kg. [ABSTRACT FROM AUTHOR]

A simple, rapid, efficient, and precise reversed-phase high-performance liquid chromatographic (RP-HPLC) method was developed and validated for the determination of metformin levels in human plasma. Plasma samples were acidified with 0.5 ml of HCl, deproteinized with acetonitrile and centrifuged. The supernatant was washed with dichloromethane and injected into the HPLC system. Separation was achieved within 10 min on a Poroshell 120 EC-C18 (4.6 mm x 50 mm 2.7 Microns) column with an isocratic mobile phase consisting of acetonitrile and methanol (10:90) containing 0.03M dibasic ammonium phosphate (pH 7) isocratic elution mode, an injection volume of 2 μl, flow rate of 0.8 ml min-1 at a detection wavelength of 236 nm, and at ambient temperature. Calibration curve (0.05-5 μgml-1) was constructed by plotting the peak area ratios against their corresponding concentrations. The method was validated according to ICH guidelines. Metformin and phenytoin eluted at 2.23 and 4.41 minutes respectively. The method was precise (RSD ± SD 3.647 ± 4.36) and accurate (% ER 3.43 and % recovery 96.52%), with correlation coefficient (R² = 0.995. Limit of detection (LOD) and Limit of quantification (LOQ) of the developed method were 0.05 and 0.02 μgml-1 respectively. All the parameters are within the acceptable limits of ICH guidelines. The developed method was found to be simple, rapid, efficient, precise and accurate for the quantification of metformin in human plasma. [ABSTRACT FROM AUTHOR]

Fluorouracil is among the most used antimetabolite drugs for the chemotherapeutic treatment of various types of gastrointestinal malignancies. Dihydropyrimidine dehydrogenase (DPYD) genotyping prior to fluorouracil treatment is considered standard practice in most European countries. Yet, current pre-therapeutic DPYD genotyping procedures do not identify all dihydropyrimidine dehydrogenase (DPD)-deficient patients. Alternatively, DPD activity can be estimated by determining the DPD phenotype by quantification of plasma concentrations of the endogenous uracil and thymine concentrations and their respective metabolites dihydrouracil (DHU) and dihydrothymine (DHT). Liquid chromatography - mass spectrometry (LC-MS) detection is currently considered as the most adequate method for quantification of low-molecular weight molecules, although the sample preparation method is highly critical for analytical outcome. It was hypothesized that during protein precipitation, the recovery of the molecule of interest highly depends on the choice of precipitation agent and the extent of protein binding in plasma. In this work, the effect of protein precipitation using acetonitrile (ACN) compared to strong acid perchloric acid (PCA) on the recovery of uracil, thymine, DHU and DHT is demonstrated. Upon the analysis of plasma samples, PCA precipitation showed higher concentrations of uracil and thymine as compared to ACN precipitation. Using ultrafiltration, it was shown that uracil and thymine are significantly (60–65 %) bound to proteins compared to DHU and DHT. This shows that before harmonized cut-off levels of DPD phenotyping can be applied in clinical practice, the analytical methodology requires extensive further optimization. • Uracil and Thymine are partially bound to proteins determined by ultrafiltration. • Recovery differences can be related to the extent of molecule protein binding. • PCA precipitation provided higher concentrations compared to ACN. • The use of cut-off levels requires extensive further optimization and harmonization. • DPD phenotyping is hampered by a lack of method standardization. [ABSTRACT FROM AUTHOR]

Cannabis is the most widely consumed illicit drug worldwide. As consumption rates increase, partially due to the decriminalization of its use for medicinal and recreational purposes, analytical methods for monitoring different cannabinoids in several biological matrices have been developed. Herein, a simple and fast extraction procedure to extract natural cannabinoids from oral fluid (OF) samples was developed and fully validated according to the ANSI/ASB 2019 Standard Practices for Method Validation in Forensic Toxicology. Using only 0.2 mL of neat OF, the analytes [Δ9-tetrahidrocannabinol (THC), 11-hydroxy-Δ9-tetrahydrocannabinol (THC-OH), 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH), cannabinol (CBN) and cannabidiol (CBD)] were extracted by protein precipitation with a mixture of methanol:acetonitrile (80:20, v/v); the extracts were centrifuged, evaporated to dryness and reconstituted in 100 µL of methanol. Analysis was performed by liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). The developed methodology produced linear results for all compounds, with working ranges of 0.1–50 ng/mL for THC, 0.5–50 ng/mL for THC-OH, CBN and CBD, and 0.05–1 ng/mL for THC-COOH. Ion suppression was observed for THC, CBN and CBD, which did not impair sensitivity considering the low limits of quantification (LOQs) and limits of detection (LODs) obtained (which varied between 0.05 and 0.5 ng/mL). The extraction procedure produced great recoveries, and the compounds were stable. No interferences were found, and the method proved to be extremely fast, selective, precise, and accurate for use in routine analysis. The method was successfully applied to authentic samples. • Efficient protein precipitation extraction method from oral fluid (OF) samples. • Minimal sample volume requirement (200 μL). • Achieved low limits of quantification (LOQs), notably for THC. • Method applied with successful in the routine laboratory work of forensic and clinical toxicology laboratories. [ABSTRACT FROM AUTHOR]

The aim of the present study was to establish an ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the determination of orelabrutinib in rat plasma using futibatinib as internal standard (IS), and to apply it for a pharmacokinetic study in rats. Orelabrutinib was extracted fromplasma by protein precipitation and quantitatively analyzed by UPLC-MS/MS. An Acquity UPLC BEH C18 column was used for rapid separation by gradient elution using 0.1% formic acid and acetonitrile asmobile phases. The validation results of bioanalytical methodology showed that the linearity of orelabrutinib in plasma samples was good within the concentration range of 1-2000 ng/ml. The lower limit of quantification (LLOQ) was 1 ng/ml. The precision of orelabrutinib ranged from 1.4% to 11.5%, with intra-day and interday accuracy ranging from -5.7% to 7.7% and -0.2% to 12.5%, respectively. The selectivity, stability, matrix effect and recovery of the method all met the requirements of quantitative analysis of biological samples. The method was simple, sensitive, accurate and specific, and had high recovery rate. It also could be successfully applied to the pharmacokinetic study of rats. [ABSTRACT FROM AUTHOR]

Background: The peptide hormone hepcidin-25 plays an important role in iron metabolism. Low or high levels of hepcidin-25 are associated with various iron disorders; therefore, hepcidin-25 is an important biomarker. This study describes an easy and fast analytical assay for the quantification of hepcidin-25 with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Methods: Sample preparation was performed by protein precipitation with trichloroacetic acid, and injection onto a LC-MS/MS was directly conducted from a LoBind 96-well plate. Results: The concentration range covered by the quality control samples, ranged from 0.25 nmol/L (12.3% CV) to 11.9 nmol/L (CV < 9%). Matrix effect was limited (mean recovery of 99.9% with a CV of 6.4%). The assay was validated for serum, EDTA and heparin plasma. An international secondary reference material was used for calibration. The reference interval (90% CL) was estimated for hepcidin-25 by analysing serum and plasma samples from 156 healthy subjects with a lower limit: 0.12 (0.07–0.19) and upper limit: 11.2 nmol/L (9.5–13.0). Conclusions: We present a fast and easy assay for the quantification of hepcidin-25 in serum and plasma samples. The assay was successfully used for the detection of various forms of hereditary haemolytic anaemias, to characterize the interplay between erythropoiesis and iron levels. [ABSTRACT FROM AUTHOR]

Etoricoxib is a non-steroidal anti-inflammatory drug (NSAID) used to treat pain and inflammation. The objective of the current study was to develop a sensitive, fast and high-throughput HPLC-ESI-MS/MS method to measure etoricoxib levels in human plasma using a one-step methanol protein precipitation technique. A tandem mass spectrometer equipped with an electrospray ionization (ESI) source operated in a positive mode and multiple reaction monitoring (MRM) were used for data collection. The quantitative MRM transition ions were m/z 359.15 > 279.10 and m/z 363.10 > 282.10 for etoricoxib and IS. The linear range was from 10.00 to 4000.39 ng/mL and the validation parameters were within the acceptance limits of the European Medicine Agency (EMA) and Food and Drug Analysis (FDA) guidelines. The present method was sensitive (10.00 ng/mL with S/N > 40), simple, selective (K prime > 2), and fast (short run time of 2 min), with negligible matrix effect and consistent recovery, suitable for high throughput analysis. The method was used to quantitate etoricoxib plasma concentrations in a bioequivalence study of two 120 mg etoricoxib formulations. Incurred sample reanalysis results further supported that the method was robust and reproducible. [ABSTRACT FROM AUTHOR]

The study aimed to develop a rapid, simple and sensitive LC/ESI-MS/MS method to measure prazosin concentration in human plasma and apply bedside sampling in bioequivalence study of two prazosin tablets to resolve the adverse effect of orthostatic hypotension. The LC/ESI-MS/MS prazosin method was highly sensitive and selective. Bedside sampling reduced the orthostatic hypotension incidence and subject dropout rate. After sample preparation, prazosin and terazosin (IS) were detected on mass spectrometer operating in multiple reaction monitoring mode using positive ionization. Mobile phase flow rate was set at 0.40 mL/min with sample run time of 1.75 min. The bioanalytical method was validated as per EMEA and FDA guidelines. Bedside sampling was performed in bioequivalence study for the first 4 h after dosing. The three primary pharmacokinetic parameters, Cmax, AUC0-t and AUC0-∞ and 90% confidence interval were determined. The small injection volume of 1 μL minimized instrumentation contamination and prolonged the analytical column lifespan. Linearity was obtained between 0.5 and 30.0 ng/mL, with coefficient of determination, r2 ≥ 0.99. The mean extraction recovery of prazosin and IS was >92%, with precision value (CV, %) ≤ 10.3%. Only two orthostatic hypotension adverse events were reported. The two prazosin formulations were found to be bioequivalent. The LC/ESI-MS/MS method has shown robustness and reliability exemplified by the incurred sample re-analysis result. Bedside sampling should be proposed for bioequivalence or pharmacokinetic studies of drugs demonstrating adverse event of orthostatic hypotension. [ABSTRACT FROM AUTHOR]

Objective: To establish a rapid and universal quantitative liquid chromatography tandem mass spectrometry (LC-MS/MS) method for measuring the exposure levels of five triazole antifungal drugs in human plasma, including voriconazole, fluconazole, posaconazole, itraconazole, and hydroxyitraconazole., Methods: A triple quadrupole mass spectrometer operating in positive ionization mode was used to detect the analyte, and multiple reaction monitoring mode was employed to gather data. The mobile phase included 0.05 % formic acid in water (phase A) and acetonitrile (phase B). The analytes were separated on an Agilent EclipsePlusC 18 RRHD column (30 × 50 mm, 1.8 μm) using gradient elution. The flow rate was 0.3 mL/min with the column temperature set at 35 °C. The acetonitrile was used to pretreat the plasma sample, and the itraconazole-D5 and hydroxyitraconazole-D5 were utilized as the internal standards., Results: The calibration range was from 100 to 10,000 ng/mL for posaconazole, itraconazole, and hydroxyitraconazole, from 200 to 20,000 ng/mL for fluconazole and from 50 to 5000 ng/mL for voriconazole, with linear correlation coefficients more than 0.99 for all regression curves. The intra- and inter-day accuracy and precision of the method were within ±15 %. The mean extraction recovery of all the analytes ranged from 74.32 % to 117.83 %, and the matrix effect was from 72.54 % to 111.2 %. The results of stability fell into the scope of ±15 % deviation., Conclusion: This newly developed method is sensitive, simple, and robust, and successfully applied in determining triazole antifungal drugs in plasma from 66 IFI patients to provide reference for safe and effective drug administration in clinical practice., Competing Interests: Declaration of competing interest The authors declare no conflicts., (Copyright © 2024. Published by Elsevier Inc.)

Letrozole 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. [ABSTRACT FROM AUTHOR]

Abstract A simple bioanalytical liquid chromatographic method was developed and validated to quantify letrozole (LTZ) in rat plasma. Protein precipitation using acidified chilled acetonitrile (containing 0.1% orthophosphoric acid) was used to extract LTZ from the plasma. Chromatographic separation was carried out on Kinetex C18 reverse phase (RP) column (250 mm × 4.6 mm i.d., 5 μm) using a mixture of 20 mM acetate buffer (pH 5.5) and acetonitirile (60:40 %v/v) eluting at 1.0 mL/min flow rate with the method responses measured at 240 nm. The optimized method was selective and established good linearity with recovery ranging between 91.16 and 99.44%. The validation experiments revealed that the method showed acceptable precision (2.61–7.48%) and accuracy (97.44–102.70%) and was found to be stable. The sensitivity of the method was demonstrated by the lowest concentration (LLOQ) detected at 75 ng/mL. Using the developed method, single-dose oral pharmacokinetics in Sprague-Dawley rats was carried out to successfully confirm the applicability of the method for the quantification of LTZ in biological matrix.

This study aimed to determine the impact of low-temperature plasma (LTP) on the protein stability and composition in surimi rinsing wastewater (SRW). When SRW (300 mL) was treated with LTP at a power of 420 W and a flow rate of 1.1 L/min for 106 s, the protein precipitation was 76.04 %, the pH was close to the estimated value of the isoelectric point (pI). In comparison with the pI precipitation treatment, non-precipitated proteins in the SRW after LTP precipitation treatment showed significant changes in amino acids susceptible to oxidation but had minor changes in the hydrophobic amino acid content. LTP showed a markedly differentiated response to the different protein types in the SRW, increasing the relative amounts of several enzyme proteins in the non-precipitated protein. The combined effect of the active ingredients provided by LTP on protein conformation and hydrophobic interactions may be responsible for this 'screening' phenomenon. • Plasma treatment precipitates proteins in surimi rinsing wastewater. • Plasma acidification of water is involved in the precipitation of proteins. • Plasma treatments showed differential response to protein types. • Plasma treatment leaves large amounts of enzymatic proteins in the aqueous phase. [ABSTRACT FROM AUTHOR]

Pesticides play an important role in forensic toxicology and are usually classified as a single class of chemicals. Despite their commonly perceived unity, pesticides encompass a spectrum of compounds, including organophosphates, carbamates, pyrethroids or organochlorines, among others, each with varying degrees of toxicity. Pesticide analysis in post-mortem samples can be difficult due to the complexity of the samples and to the high toxicity of these compounds. The aim of this study was to develop and validate an easy to use, sensitive, and robust method, using ultra-performance liquid chromatography-tandem mass spectrometry to be incorporated in the routine flow for pesticide analysis in post-mortem blood samples. Described herein is a streamlined, expeditious, yet highly efficient method facilitating the screening, qualitative assessment, and quantitative confirmation of 15 pesticides, including acetamiprid, azinphos-ethyl, bendiocarb, carbofuran, chlorfenvinphos, dimethoate, imidaclopride, malathion, methiocarb, methomyl, parathion, pirimicarb, strychnine, tetrachlorvinphos, and thiacloprid in post-mortem blood, recognizing the pivotal role blood plays in forensic investigations. The developed method was linear from 10 to 200 ng/mL; limits of detection were between 1 and 10 ng/mL, depending on the compound; it was successfully evaluated a dilution ratio of 1–2, 5 and 10; and 8 substances showed maximum stability for the time interval studied. This UHPLC-MS/MS method is useful and a powerful tool in a toxicology lab because it is fast, simple, effective, and trustworthy. The results of this validation highlight the robustness of the analytical method, providing a valuable tool for the accurate and sensitive detection of pesticides in post-mortem blood. Poised for routine implementation, this method has already found success in suspected intoxication cases, promising to elevate the standards of forensic pesticide analysis. • Development and validation of a rapid and reliable UHPLC-MS/MS method for pesticide analysis in post-mortem blood samples. • The developed method enables the screening, qualitative, and quantitative confirmation of 15 pesticides in post-mortem blood. • Successful validation of an analytical method, combining blood extraction with protein precipitation using acetonitrile and UHPLC-MS/MS, demonstrating high sensitivity for the pesticides considered. • Successfully applied to suspected intoxication cases, the method is poised for routine analysis, providing a valuable contribution to forensic toxicology laboratories. [ABSTRACT FROM AUTHOR]

• This is a sensitive method (1 ng/ml). • This method generates the shortest run time <1 min (rapid analysis). • This is the only published method that utilized Thiorphan_D7 as an internal standard. • There is an important application to prove the reliability of the new method, which is bioequivalence study in Jordanian subjects. Racecadotril, an anti-secretory medication, has been used as an adjuvant in an oral rehydration therapy for children experiencing severe diarrhea. Racecadotril is quickly converted to thiorphan, an active metabolite, after oral treatment, which mediates all subsequent activities. An efficient and rapid liquid chromatography-tandem mass spectrometry method was developed and fully validated to measure thiorphan in human plasma, using thiorphan-d7 as an internal standard. The extraction method used was protein precipitation while chromatographic separation was achieved using InertSil CN-3 (50 × 2.1 mm, 5 µm column). The assay was linear over the concentration range of 1–200 ng/ml with correlation coefficients of ≥0.9991. The intra- and inter-day precisions were less than 10.0 % for all concentrations investigated. 0.02 % aqueous formic acid and methanol (30:70 v: v) were used as mobile phases, with an analysis time of less than 1 min. This method proved stable under several conditions. The developed method worked well in a three-period pharmacokinetic bioequivalence study after a single oral administration of 100 mg racecadotril to 15 healthy Jordanian volunteers under fasting conditions. [ABSTRACT FROM AUTHOR]

Therapeutic drug monitoring of treatment with therapeutic antibodies is hampered by the application of a wide range of different methods in the quantification of serum levels. LC-MS based methods could significantly improve comparability of results from different laboratories, but such methods are often considered complicated and costly. We developed a method for LC-MS/MS based quantification of 11 therapeutic antibodies concomitantly measured in a single run, with emphasis on simplicity in sample preparation and low cost. After a single-step sample purification using caprylic acid precipitation to remove interfering proteins, the sample underwent proteolysis followed by LC-MS/MS analysis. Infliximab is used as internal standard for sample preparation while isotope-labeled signature peptides identified for each analyte are internal standards for the LC-MS/MS normalization. The method was validated according to recognized guidelines, and pipetting steps can be performed by automated liquid handling systems. The total precision of the method ranged between 2.7 and 7.3 % (5.1 % average) across the quantification range of 4–256 μg/ml for all 11 drugs, with an average accuracy of 96.3 %. Matrix effects were xamined in 55 individual patient samples instead of the recommended 6, and 147 individual patient samples were screened for interfering compounds. Our method for simultaneous quantification of 11 t-mAb in human serum allows an unprecedented integration of robustness, speed and reduced complexity, which could pave the way for uniform use in research projects and clinical settings alike. In addition, the first LC-MS protocol for signature peptide-based quantification of durvalumab is described. This high throughput method can be readily adapted to a drug panel of choice. [Display omitted] • Protocol for LC-MS/MS multiplex quantification of 11 therapeutic antibodies. • Low-cost sample preparation method using caprylic acid, suitable for automation. • ALPASIEK is a signature peptide for durvalumab LC-MS/MS quantification. [ABSTRACT FROM AUTHOR]

• Critical flux evaluated for precipitated IgG with different morphologies. • Additional of CaCl 2 led to precipitate morphologies with much greater critical flux. • Critical flux increases with increasing packing density of precipitate. • Packing density during centrifugation provides rapid precipitate screening. • Operation below critical flux enables at least 24 hr continuous operation. Recent advances in cell culture have led to substantial increases in monoclonal antibody titer, leading to renewed interest in using precipitation for the initial capture and purification of high-value therapeutic proteins. One of the major challenges in developing continuous precipitation processes is ensuring stable operation of the tangential flow microfiltration membranes used for dewatering and washing. The objective of this study was to increase the filterability of the protein precipitate by addition of salts to control the particle morphology. Experiments were performed with human serum Immunoglobulin G (IgG) in different buffers and in the presence of different salts using zinc chloride and polyethylene glycol as precipitating agents. The addition of 300 mM CaCl 2 caused more than a 2-fold increase in the critical flux. This behavior was not due simply to a shift in the particle size distribution, but it was instead related to an increase in the density of the precipitate. The precipitate density was readily measured by centrifugation, providing a rapid method for identifying precipitation conditions that lead to good filterability. Data obtained during a 24-hour filtration process showed minimal fouling with high degrees of purification, demonstrating the viability of this approach for optimizing precipitation conditions. [ABSTRACT FROM AUTHOR]

This study aimed to establish a simple and sensitive analytical method to simultaneously quantify donepezil (DPZ) and tadalafil (TAD) in rat plasma using lansoprazole (LPZ) as an internal standard (IS) by using liquid chromatography tandem mass spectrometry. The fragmentation pattern of DPZ, TAD, and IS was elucidated using multiple reaction monitoring in electrospray ionization positive ion mode for the quantification of precursor to production at m/z 380.1 → 91.2 for DPZ, m/z 390.2 → 268.1 for TAD, and m/z 370.3 → 252.0 for LPZ. The extracted DPZ and TAD from plasma using acetonitrile-induced protein precipitation was separated using Kinetex C18 (100 × 2.1 mm, 2.6 µm) column with a gradient mobile phase system consisting of 2 mM ammonium acetate and 0.1% formic acid in acetonitrile at a flow rate of 0.25 mL/min for 4 min. The selectivity, lower limit of quantification, linearity, precision, accuracy, stability, recovery, and matrix effect of this developed method was validated according to the guidelines of the U.S. Food and Drug Administration and the Ministry of Food and Drug Safety of Korea. The established method achieved acceptance criteria in all validation parameters, ensuring reliability, reproducibility, and accuracy, and was successfully implemented in a pharmacokinetic study on the co-administration of DPZ and TAD orally in rats.

Indirubin was identified as an active component of Danggui Longhui Wan, an herbal mixture used in traditional Chinese medicine, and showed anticancer activity in clinical trials in patients with chronic leukemia. Investigations on the mechanisms of antitumor action of indirubins have mainly focused on the indirubin derivative indirubin-3′-monoxime (I3M). Meanwhile, antiproliferative and cytotoxic properties on cancer cells have also been demonstrated for several synthetic indirubin N-glycosides. In the present study, we demonstrate cytotoxic activity of the thia-analogous indirubin N-glycosides KD87 (3-[3′-oxo-benzo[b]thiophen-2′-(Z)-ylidene]-1-(β-d-glucopyranosyl)-oxindole) and KD85 (3-[3′-oxo-benzo[b]thiophen-2′-(Z)-ylidene]-1-(β-d-mannopyranosyl)-oxindole) against melanoma and squamous cell carcinoma cells as well as lung cancer and glioblastoma cells. The advanced state of preclinical studies on the effects of indirubins conducted to date underscores the need for pharmacokinetic data from cellular, animal, and human studies for which reliable quantification is required. Therefore, a sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the simultaneous measurement of KD87, KD85, and I3M in plasma and cell culture medium. Experimental conditions for sample preparation were optimized for human plasma protein precipitation and liquid-liquid extraction from plasma and cell culture medium. The methods were successfully validated in accordance with the U.S. Food and Drug Administration Bioanalytical Method Validation and evaluated for selectivity, sensitivity, matrix effect, recovery, carryover, calibration curve linearity, accuracy, precision, and stability. The applicability of the methods was demonstrated by the determination of KD87 in mouse plasma after prior intraperitoneal administration to mice. [ABSTRACT FROM AUTHOR]

In order to solve the protein instability problem of white wine and to realize the utilization of acid protease and alcohol fermentation simultaneously, this study used Saccharomyces cerevisiae haploid strain BY4741 and diploid strain 82-9-35 as hosts to express acid protease on the yeast cell surface by the action of promoter and anchor protein in the yeast surface display system. The acid protease gene (pepA) from Aspergillus usamii was cloned, a cell display cassette expressing acid protease was constructed, and homologous recombination was used to integrate the acid protease gene into the gene locus of S. cerevisiae. Through PCR and sequencing validation, two haploid and diploid recombinant strains with the highest enzyme activity of 285.71 U/mL and 495.24 U/mL, respectively, were obtained for the cell surface display of acid protease. This study successfully constructed a new idea of using SED1 as an anchoring protein display system to solve the protein turbidity problem in wine, and laid the theoretical foundation for the industrial application of pepA acidic protease whole cell catalyst. [ABSTRACT FROM AUTHOR]

A clinical trial is currently underway to examine the efficacy of using caffeine citrate to prevent intermittent hypoxemia in late preterm neonates. Determining caffeine concentration using saliva in this population would be preferable as it is less invasive than plasma sampling, but a suitable method of analysis is required. This paper presents the development and validation of a rapid, efficient and reproducible stability-indicating high-performance liquid chromatography (HPLC) method and extraction protocol for the quantification of caffeine present in saliva. The stability of extemporaneously prepared caffeine citrate solutions (at 20–25 °C) was determined, along with the stability of caffeine spiked saliva samples (at 20–25 and 2–8 °C), to ensure the suitability of the developed method in the analysis of clinical trial samples. Protein precipitation using acetonitrile ensured the complete removal of salivary proteins and resulted in extraction recovery of ≥95% for all samples. The HPLC assay following extraction was linear (R2>0.99) over the range 0.3–50 µg/mL (lower limit of quantification 0.3 µg/mL). The accuracy of the quality control samples was 94–100% and the relative standard deviation (RSD) was <7% for all samples. Caffeine-spiked saliva samples were stable for three freeze-and-thaw cycles pre-extraction and up to 72 hr post-extraction. The extraction and HPLC methods described were thus suitable for the analysis of clinical trial samples from the Latte Dosage Trial. All caffeine solutions were physically and chemically stable, with concentrations at the end of the three-month test period being within 4% of the baseline concentrations, indicating appropriateness for use as clinical trial medications. [ABSTRACT FROM AUTHOR]