Comparison of Commonly Used and New Methods to Determine Small Molecule Non-Specific Binding to Human Liver Microsomes.

Accurate measurement of non-specific binding of a drug candidate to human liver microsomes (HLM) can be critical for the accurate determination of key enzyme kinetic parameters such as Michaelis-Menton (Km), reversible inhibition (Ki), or inactivation (KI) constants. Several methods have been developed to determine non-specific binding of small molecules to HLM, such as rapid equilibrium dialysis (RED), ultrafiltration (UF), HLM bound to magnetizable beads (HLM-beads), ultracentrifugation (UC), the linear extrapolation stability assay (LESA), and the Transil™ system. Despite various differences in methodology between these methods, it is generally presumed that similar free fraction values (fu,mic) should be generated. To evaluate this hypothesis, a test set of 9 compounds were selected, representing low (high fu,mic value) and significant (low fu,mic value) HLM binding, respectively, across HLM concentrations tested in this manuscript. The fu,mic values were determined using a single compound concentration (1.0 µM) and three HLM concentrations (0.025, 0.50, and 1.0 mg/mL). When the HLM non-specific binding event is not extensive resulting in high fu,mic values, all methods generated similar fu,mic values. However, fu,mic values varied markedly across assay formats when high binding to HLM occurred, where fu,mic values differed by up to 33-fold depending on the method used. Potential causes for such discrepancies across the various methods employed, practical implications related to conduct the different assays, and implications to clinical drug-drug interaction (DDI) predictions are discussed.

Insights into the binding of buspirone to human serum albumin using multi-spectroscopic and molecular docking techniques.

Buspirone is an anxiolytic drug that plays a significant role in managing anxiety disorders and alleviating their symptoms as well. Several techniques were utilized to study the interaction between buspirone and human serum albumin under physiological conditions, including UV-vis absorption spectroscopy, fluorescence emission spectroscopy, circular dichroism, Fourier transform infrared spectroscopy (FT-IR), equilibrium dialysis, and molecular docking. The results of this study demonstrated that buspirone quenched the intrinsic fluorescence of human serum albumin through a mixed mechanism. Moreover, the binding constants (Kb), the quenching constants (Ksv), and thermodynamic parameters were calculated at various temperatures. The binding process of buspirone to human serum albumin showed a cooperative binding pattern, confirmed by the Scatchard diagram and Hill coefficient. Molecular docking results showed that buspirone interacted with the IIA, IIIA, and IIB subdomains of human serum albumin and slightly changed its conformation. It was also found that hydrophobic forces played a major role in this interaction. This study consequently proves that BSH as a drug can be transported by blood albumin. Additionally, due to its ratiometric response in absorbance upon binding to a biological target, HSA can be used as a molecular probe to follow biomolecular interactions.

Equilibrium dialysis with HPLC detection to measure substrate binding affinity of a non-heme iron halogenase.

Determination of substrate binding affinity (Kd) is critical to understanding enzyme function. An extensive number of methods have been developed and employed to study ligand/substrate binding, but the best approach depends greatly on the substrate and the enzyme in question. Below we describe how to measure the Kd of BesD, a non-heme iron halogenase, for its native substrate lysine using equilibrium dialysis with subsequent detection with High Performance Liquid Chromatography (HPLC). This method can be performed in anaerobic glove bag settings, requires readily available HPLC instrumentation for subsequent detection, and is adaptable to meet the needs of a variety of substrate affinity measurements.

Species-Specific Unbound Fraction Differences in Highly Bound PFAS: A Comparative Study across Human, Rat, and Mouse Plasma and Albumin.

Per- and polyfluoroalkyl substances (PFAS) are a diverse group of fluorinated compounds which have yet to undergo comprehensive investigation regarding potential adverse health effects and bioaccumulative properties. With long half-lives and accumulative properties, PFAS have been linked to several toxic effects in both non-clinical species such as rat and mouse as well as human. Although biological impacts and specific protein binding of PFAS have been examined, there is no study focusing on the species-specific fraction unbound (fu) in plasma and related toxicokinetics. Herein, a presaturation equilibrium dialysis method was used to measure and validate the binding of 14 individual PFAS with carbon chains containing 4 to 12 perfluorinated carbon atoms and several functional head-groups to albumin and plasma of mouse (C57BL/6 and CD-1), rat, and human. Equivalence testing between each species-matrix combination showed positive correlation between rat and human when comparing fu in plasma and binding to albumin. Similar trends in binding were also observed for mouse plasma and albumin. Relatively high Spearman correlations for all combinations indicate high concordance of PFAS binding regardless of matrix. Physiochemical properties of PFAS such as molecular weight, chain length, and lipophilicity were found to have important roles in plasma protein binding of PFAS.

A validated LC-MS/MS method for determination of neuro-pharmacokinetic behavior of niraparib in brain tumor patients.

Niraparib is a potent and orally bioavailable inhibitor of poly (ADP-ribose) polymerase (PARP) with high specificity for isoforms 1 and 2. It has been approved by the U.S. Food and Drug Administration for ovarian cancer maintenance therapy and is currently under development for various cancers, including glioblastoma. To assess central nervous system (CNS) penetration of niraparib in glioblastoma patients, a novel bioanalytical method was developed to measure total and unbound niraparib levels in human brain tumor tissue and cerebrospinal fluid (CSF). The method was validated using plasma as a surrogate matrix over the concentration range of 1-10,000 nM on an LC-MS/MS system. The MS/MS detection was conducted in positive electrospray ionization mode, while chromatography was performed using a Kinetex™ PS C18 column with a total 3.5-minute gradient elution run time. The maximum coefficient of variation for both intra- and inter-day precision was 10.6%, with accuracy ranging from 92.8% - 118.5% across all matrices. Niraparib was stable in human brain homogenate for at least 6 hours at room temperature (RT) and 32 days at -20°C, as well as in stock and working solutions for at least 21 hours (RT) and 278 days (4°C). Equilibrium dialysis experiments revealed the fractions unbound of 0.05 and 0.16 for niraparib in human brain and plasma, respectively. The validated method is currently employed to assess niraparib levels in human glioblastoma tissue, CSF, and plasma in an ongoing trial on newly diagnosed glioblastoma and recurrent IDH1/2(+) ATRX mutant glioma patients (NCT05076513). Initial results of calculated total (Kp) and unbound (Kp,uu) tumor-to-plasma partition coefficients indicate significant brain penetration ability of niraparib in glioblastoma patients.

Simultaneous determination of plasma protein binding of five C-glycosylflavones from TFDS by rapid equilibrium dialysis.

The total flavonoids of Desmodium styracifolium (TFDS) are flavonoid-rich extracts obtained from Desmodii Styracifolii Herba, which is approved for the treatment of urolithiasis in China. C-glycosylflavones including schaftoside, vicenin-1, vicenin-2, vicenin-3, and isovitexin are the main active constituents. In this study, the plasma protein binding of these compounds was determined for the first time in rat and human plasma by rapid equilibrium dialysis combined with HPLC-MS/MS method. The developed method was validated in terms of specificity, linearity, accuracy, precision, extraction effect, matrix effect, and stability. Schaftoside, vicenin-1, vicenin-2, and vicenin-3 exhibited moderate plasma protein binding, ranging from 56.6% to 61.5% in rat plasma and 55.0%-62.9% in human plasma. In comparison, isovitexin demonstrated a higher plasma protein binding in the range of 92.3-93.1% and 95.1-96.2% in rat and human plasma, respectively. Furthermore, the potential interactions mediated via plasma protein binding between isovitexin and nonsteroidal anti-inflammatory drugs (NSAIDs) were investigated by rapid equilibrium dialysis. No significant changes were observed, indicating a lower likelihood of interaction between TFDS and NSAIDs due to plasma protein binding in the treatment of urinary system disorders.

Interspecies variability in protein binding of antibiotics basis for translational PK/PD studies-a case study using cefazolin.

For antimicrobial agents in particular, plasma protein binding (PPB) plays a pivotal role in deciphering key properties of drug candidates. Animal models are generally used in the preclinical development of new drugs to predict their effects in humans using translational pharmacokinetics/pharmacodynamics (PK/PD). Thus, we compared the protein binding (PB) of cefazolin as well as bacterial growth under various conditions in vitro. The PB extent of cefazolin was studied in human, bovine, and rat plasmas at different antibiotic concentrations in buffer and media containing 20-70% plasma or pure plasma using ultrafiltration (UF) and equilibrium dialysis (ED). Moreover, bacterial growth and time-kill assays were performed in Mueller Hinton Broth (MHB) containing various plasma percentages. The pattern for cefazolin binding to plasma proteins was found to be similar for both UF and ED. There was a significant decrease in cefazolin binding to bovine plasma compared to human plasma, whereas the pattern in rat plasma was more consistent with that in human plasma. Our growth curve analysis revealed considerable growth inhibition of Escherichia coli at 70% bovine or rat plasma compared with 70% human plasma or pure MHB. As expected, our experiments with cefazolin at low concentrations showed that E. coli grew slightly better in 20% human and rat plasma compared to MHB, most probably due to cefazolin binding to proteins in the plasma. Based on the example of cefazolin, our study highlights the interspecies differences of PB with potential impact on PK/PD. These findings should be considered before preclinical PK/PD data can be extrapolated to human patients.

Free drug percentage of moxidectin declines with increasing concentrations in the serum of marsupials.

Moxidectin (MOX) is a macrocyclic lactone used to eliminate endo and ectoparasites in many mammalian species. It is notably the active ingredient of the anti-parasitic drug Cydectin®, manufactured by Virbac, and is frequently used to treat sarcoptic mange in Australian wildlife. Protein binding plays a significant role in the efficacy of a drug, as the unbound/free drug in plasma ultimately reflects the pharmacologically relevant concentration. This study aimed to investigate the free drug percentage of Moxidectin after in vitro spiking into the sera of four sarcoptic mange-susceptible Australian wildlife species; the koala (Phascolarctos cinereus), the bare-nosed wombat (Vombatus ursinus), the eastern grey kangaroo (Macropus giganteus), and the mountain brushtail possum (Trichosurus cunninghami). Three concentration points of MOX were tested for each individual: 20 pg/µL, 100 pg/µL and 500 pg/µL. Serum from five individuals of each species underwent an equilibrium dialysis followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). The results showed an atypical concentration dependent binding across all species, where free drug percentage decreased as MOX concentration increased. In addition, wombats showed significantly lower free drug levels. These findings call for further research into the mechanisms of moxidectin protein binding to help understand MOX pharmacokinetics in marsupials.

Measurement uncertainty estimation of free drug concentrations in clinical laboratories using equilibrium dialysis.

OBJECTIVES: Developing procedures based on equilibrium dialysis (ED) that allow measuring the free drug concentration in plasma improves therapeutic drug monitoring (TDM) in those cases where its measurement is justified. However, this procedure requires specific sample preparation and presents different pitfalls, which are not error-free. As with any result provided by a clinical laboratory, this one should be as accurate as possible to allow a correct clinical interpretation. The measurement uncertainty (MU) is a parameter that enables the accuracy of results to be known, and that is mandated by ISO 15189. Herein, this study suggests how the MU for the results of the free drug concentrations in serum could be estimated when an ED is used. METHODS: A combination of the top-down and bottom-up approaches was used to estimate the MU based on the ISO/TS 20914:2019 and JCGM 100:2008 guidelines, including the concentration of free phenytoin in serum, as an example. Different scenarios were incorporated considering or not a significant bias related to the primary drawbacks of ED: the non-specific binding, the volume shift effect and the Gibbs-Donnan effect. RESULTS: The expanded uncertainties estimated ranged between 13.0 and 30.9 %. The highest MU corresponded to the free drug concentrations in serum results when significant biases related to the volume shift and Gibbs-Donnan effects exist. CONCLUSIONS: A detailed estimation of MU for free drug concentrations is presented using ED, considering different scenarios. This study could stimulate clinical laboratories to perform MU studies and its application in TDM.

Development and validation of equilibrium dialysis UHPLC-MS/MS measurement procedures for total and unbound concentrations of bictegravir, dolutegravir, darunavir and doravirine in human plasma. Application to patients with HIV.

BACKGROUND: Fixed-dose combinations of antiretroviral drugs are commonly used to treat HIV infection and therapeutic monitoring is not part of routine clinical practice. However, drug concentrations monitoring might have role in different clinical scenarios as well as for research purposes. This study aimed to develop and validate UHPLC-MS/MS procedures for measuring total and unbound concentrations of bictegravir, dolutegravir, darunavir and doravirine in human plasma. MATERIAL AND METHODS: Equilibrium dialysis preceded sample preparation (based on protein precipitation) for measuring unbound antiretroviral concentrations. Chromatographic separations were achieved on an Acquity®-UPLC® HSS™-T3 column (50 mm × 2.1 mm; 1.8 µm) using a non-linear water/acetonitrile gradient containing 0.1 % formic acid at a 0.5 mL/min flow rate. Antiretrovirals were detected by tandem mass spectrometry in positive electrospray ionisation and multiple reaction monitoring modes. RESULTS: No significant interferences or carry-over were observed. Imprecisions, absolute relative biases, normalised matrix effects and recoveries were ≤15.0 %, ≤11.1 %, (94.7-104.1)% and (96.7-105.5)%, respectively. Non-linear measuring intervals were observed between (25-10,000) µg/L for total/plasma dialysate concentrations and linearity schemes (1.00-100) µg/L for buffer dialysate concentrations. CONCLUSIONS: The UHPLC-MS/MS procedures developed could be used for research purposes and therapeutic drug monitoring of antiretrovirals in routine clinical practice.

[Determination of plasma protein binding rate of Shuganning Injection using equilibrium dialysis and UPLC-MS/MS].

Traditional Chinese medicine(TCM) compound preparations have complex compositions. As a widely used TCM injection, Shuganning Injection, its in vivo processes are not yet fully understood. Determining the plasma protein binding rate is of great significance for pharmacokinetic and pharmacodynamic studies. In this experiment, the equilibrium dialysis method combined with UPLC-MS/MS technology was used to determine the plasma protein binding rates of 10 components, including p-hydroxyacetophenone, caffeic acid, baicalein, oroxylin A, geniposide, baicalin, cynaroside, oroxylin A-7-O-ß-D-glucuronide, scutellarin, and hyperoside, in Shuganning Injection in rat and human plasma to provide a theoretical basis for further elucidating the in vivo processes of Shuganning Injection and guiding clinical medication. The results showed that, except for baicalein and geniposide, the plasma protein binding rates of the other eight components were higher in human plasma than in rat plasma, and there were interspecies differences. In human plasma, except for geniposide, caffeic acid, and baicalin, the plasma protein binding rates of the remaining seven components were above 80%, with baicalein and oroxylin A exceeding 90%. All components exhibit a high level of binding to plasma proteins, with the exception of geniposide.

Large method differences for free thyroid hormone assays in the hyperthyroid range can affect assessment of hyperthyroid status: Comparison of Abbott Alinity to Roche Cobas, Siemens Centaur and equilibrium dialysis LC-MS/MS.

BACKGROUND: Free T4 (FT4) determination is one of the most commonly performed biochemical tests in endocrinology. Treatment of thyroid dysfunctions is adjusted based on the severity of symptoms and biochemical test results. For Graves' hyperthyroidism, clinical guidelines recommend using FT4 as a (rough) guide to dose antithyroid drugs, together with other clinical information. It is well known that different platforms and methods give different FT4 results; however, large non-linear method differences at high FT4 concentrations are less well recognized. Current clinical guidelines do not make it clear that method differences in the hyperthyroid range can affect recommendations. METHOD: Serum samples from patients with very low (biochemically hypothyroid) to very high (hyperthyroid) concentrations of FT4 and/or free T3 (FT3) were analyzed using Abbott Alinity and compared to concentrations measured using Roche Cobas, Siemens ADVIA Centaur (FT4 only) and an in-house equilibrium dialysis liquid chromatography tandem mass spectrometry (LC-MS/MS) method. RESULTS: Alinity measured markedly lower FT4 and FT3 concentrations compared to the other methods, particularly at high FT4 concentrations. Regression analysis indicated that Alinity FT4 had a non-linear (curved) relationship to FT4 measured by the other methods. The method differences affected guideline-recommended treatments for hyperthyroidism. CONCLUSION: Measured free thyroid hormone concentrations are highly method-dependent, especially at high FT4 concentrations. Clinicians treating hyperthyroid patients should be aware that patients appear much less hyperthyroid from FT4-measurements performed using Alinity compared to Cobas or Centaur. Guideline-recommended antithyroid drug dosages based on FT4 (including multiples of the upper reference range) have to be adjusted to the FT4 method used. FT4 results from different methods should be clearly distinguished (e.g. separate lines) in medical records.

The free cortisol calculated: correlation with the free cortisol concentrations measured with liquid chromatography-tandem mass spectrometry after equilibrium dialysis and establishment of reference intervals.

BACKGROUND: Changes in cortisol binding globulin (CBG) impact the total serum cortisol concentration and affect the accurate assessment of adrenal function. Free biologically cortisol can be calculated using different equations or directly measured after complicated procedures. METHODS: The free cortisol index (FCI) obtained using the Bonte formula as well as the free cortisol concentration calculated (Coolens equation) were first estimated for 45 healthy workers. The CBG level was determined by a competitive radioimmunoassay and the total cortisol concentration, was measured with an electrochemiluminescent assay. The correlations between FCI, the free cortisol concentrations calculated and the free cortisol levels measured with liquid chromatography-tandem mass spectrometry after equilibrium dialysis were studied for those 45 samples. Reference limits were established on 158 healthy hospital workers and patients with serum samples collected between 7:30 am and 10 am. RESULTS: The FCI as well as the free cortisol concentrations calculated obtained for the 45 samples correlated significantly with the free cortisol levels measured. Although the cortisol and CBG levels were statistically higher in women using contraceptives compared with women not taking them as well as men, the calculated FCI and free cortisol concentrations did not differ between these groups. The medians (P2.5-P97.5) obtained for the 158 healthy workers were respectively 26.4% (12.3-51.6%) and 10.6 nmol/L (4.3-26.7 nmol/L). CONCLUSIONS: This study highlighted a significant correlation between the FCI, the free cortisol concentrations calculated and the free cortisol levels measured with LC-MS/MS, it has also allowed the establishment of reference intervals for calculated FCI and free cortisol.

Plasma Protein Binding Determination for Unstable Ester Prodrugs: Remdesivir and Tenofovir Alafenamide.

Remdesivir (RDV) and tenofovir alafenamide (TAF) are prodrugs designed to be converted to their respective active metabolites. Plasma protein binding (PPB) determination of these prodrugs is important for patients with possible alteration of free fraction of the drugs due to plasma protein changes in renal impairment, hepatic impairment, or pregnancy. However, the prodrugs' instability in human plasma presents a challenge for accurate PPB determination. In this research work, two approaches were used in the method development and qualification for PPB assessment of RDV and TAF. For RDV, dichlorvos was used to inhibit esterase activity to stabilize the prodrug in plasma during equilibrium dialysis (ED). The impact of dichlorvos on protein binding was evaluated and determined to be insignificant by comparing the unbound fraction (fu) determined by the ED method with dichlorvos present and the fu determined by an ultrafiltration method without dichlorvos. In contrast to RDV, TAF degradation in plasma is ∼3-fold slower, and TAF stability cannot be improved by dichlorvos. Fit-for-purpose acceptance criteria for the TAF PPB method were chosen, and an ED method was developed based on these criteria. These two methods were then qualified and applied for PPB determinations in clinical studies.

Small volume rapid equilibrium dialysis (RED) measures effects of interstitial parameters on the protein-bound fraction of topical drugs.

The importance of plasma protein binding in the early stages of drug development is well recognized. Free and bound drug fractions in plasma are routinely determined with well-established methods. However, for physiological fluids with a small accessible volume and low protein concentrations, such as dermal interstitial fluid (dISF) validated methods are currently missing. Due to the low protein concentration and highly dynamic processes in the dermis, protein binding data obtained from plasma samples may underestimate in-vivo efficacy. This study aimed to validate a small volume rapid equilibrium dialysis (RED) for low protein samples, as a tool to examine drug-protein binding directly in the biological fluid at the site of action. The sample volume required for RED was successfully downscaled to 50 µl and plasma protein binding values of the four model drugs were consistent with previous studies with an average recovery of 88 ± 8% which makes all tested drugs suitable for small volume RED. Inter- and intra-batch variability showed sufficient reproducibility across RED plates. Small volume RED was successfully applied to assess the effects of interstitial parameters, including the evaluation of the major binding protein and the effects of binding protein concentration, drug concentration, and pH on the protein-bound drug fraction using 2% HSA and/or diluted human plasma as a surrogate for dISF.

Practical considerations for accurate determination of free thyroxine by equilibrium dialysis.

Background: Free thyroxine (FT4) measurement is one of the most requested tests in patient care for diagnosing and treating thyroid-related illnesses. Equilibrium dialysis (ED) is considered the "gold standard" for FT4 measurement; however, several factors have a profound effect on the reliability of FT4 assays and require special consideration. Methods: In the current study, we focused on evaluating critical factors that could contribute to reporting errors, such as adsorption of thyroxine (T4) to labware surfaces, stability of serum samples, stock solutions, and calibrator storage conditions, as well as the solvents used to prepare T4 solutions. Results: The adsorption of T4 in ethanolic solutions and dialysates to labware surfaces can be reduced with the careful selection of pipette tips, test tubes, and 96-well plates. Adding pH modifiers to neat T4 solutions can improve its stability. FT4 in serum samples remains stable after exposure to four freeze-thaw cycles, 5 °C for 18-20 h, or -70 °C for a minimum of three years. Conclusion: The presented study has demonstrated that the loss of analyte due to pre-analytical and analytical factors during operation of the FT4 reference measurement procedure (RMP) can be minimized by careful selection of all labware for sample preparation. It was found that the accuracy and imprecision of FT4 assays can be influenced by different types of dialysis devices, but acceptable alternatives to ED membranes were identified. This study demonstrates approaches to establish a FT4 method that is independent from specific suppliers and addresses critical pre-analytical and analytical factors important for FT4 measurements.

Prediction of the Time to Reach Equilibrium for Improved Estimation of the Unbound Fraction of Compounds in Equilibrium Dialysis Using kinetic Modeling.

Equilibrium dialysis (ED) is widely used in pharmacokinetics to determine the fraction of unbound (fu) compounds in plasma; however, the kinetics of drugs in the ED system with respect to their permeation across semi-permeable membranes has not been systemically studied. Here, the kinetics of the ED system, including the binding of drugs to plasma proteins, non-specific binding, and permeation across the membrane, was described to enable verification of the equilibrium, prediction of the time to reach equilibrium, and estimations of fu with data obtained during pre-equilibrium. Using data obtained during pre-equilibrium, the time to reach 90% equilibrium (t90%) and fu were estimated with reasonable accuracy. Notably, fu could be estimated reasonably well using one-time-point data for the calculation. Furthermore, the current modeling approach allowed concurrent estimations of fu and the decomposition rate of compounds that were metabolically unstable in the plasma. Reasonable metabolic rate constants were determined for cefadroxil and diltiazem, demonstrating the practicality of this method for determining kinetics related to fu characterization. Because the determination of fu of compounds with 'unfavorable' physicochemical properties is known to be experimentally challenging, the current method may be useful in determining the fu of compounds in vitro.

Using Counter Equilibrium Dialysis (CED) to Increase Confidence in the Measurement of Free Fraction for Challenging Compounds.

The confidence in fraction unbound (ƒu) using equilibrium dialysis (ED) is often questioned (e.g., highly bound, labile compounds) due to uncertainty in whether true equilibrium is achieved. Different methods have been developed to increase confidence in ƒu measurements, such as the presaturation, dilution, and bi-directional ED methods. However, confidence in ƒu measurement can still suffer due to non-specific binding and inter-run variations introduced during equilibrium and analysis. To address this concern, we introduce an orthogonal approach called counter equilibrium dialysis (CED) in which non-labeled and isotope-labeled compounds are dosed counter-directionally in rapid equilibrium dialysis (RED). ƒu values of both non-labeled and labeled compounds are measured simultaneously in the same run. These tactics not only minimize non-specific binding and inter-run variability but also enable the confirmation of true equilibrium. If equilibrium is reached in both dialysis directions, the ƒu for the non-labeled compound and the labeled compound will converge. The refined methodology was extensively tested with various compounds of diverse physicochemical properties and plasma binding characteristics. Our results demonstrated that, by using the CED method, ƒu values for a wide range of compounds could be accurately determined with significantly improved confidence, including the challenging highly bound and labile compounds.

Characterization of copper binding to different molecular weight fractions of dissolved organic matter in surface water.

Dissolved organic matter (DOM) is a kind of substance with complex compositions and wide molecular weight distribution, which can strongly combine with various pollutants. Therefore, the binding characteristics of DOM and heavy metal pollutants can be studied specifically according to the binding characteristics of DOM and pollutants. In this study, DOM in surface water bodies was divided into three levels (MW < 1 kDa, 1 kDa < MW < 5 kDa, MW > 5 kDa) according to different molecular weights (MW). The binding properties were investigated by fluorescence spectrum analysis and complex model. Four components (C1-C4) were identified by PARAFAC. Among them, the contribution rate of protein-like components C1, C2 and C4 to the total fluorescence intensity reached more than 78%, and the log Ka values of low molecular weight components were the highest, which were 3.28, 3.14 and 3.47, respectively, indicating higher binding ability with Cu2+.With the decrease of molecular weight, the log Kb value increases, indicating that the complexation is more stable. The humic component C3 in high molecular weight has stronger binding stability with Cu2+, but the number of binding sites for C3 is 0.36, while that for C2 is 1.51, indicating that its binding sites and binding ability are relatively low. The results showed that the DOM ligand of Cu2+ in surface water showed a certain molecular weight dependence. In addition, different MW DOM lead to different pollution forms. Different properties of DOM ligand combined with Cu2+ were studied in order to control the migration, transformation, bioavailability, morphology and stability of heavy metal pollutants, and to provide theoretical support for the practical application management of surface water pollution control.

Development of an equilibrium dialysis ID-UPLC-MS/MS candidate reference measurement procedure for free thyroxine in human serum.

BACKGROUND: Accurate and reliable measurement of human serum free thyroxine (FT4) is critical for the diagnosis and treatment of thyroid diseases. However, concerns have been raised regarding the performance of FT4 measurements in patient care. Centers for Disease Control and Prevention Clinical Standardization Programs (CDC-CSP) address these concerns by creating a FT4 standardization program to standardize FT4 measurements. The study aims to develop a highly accurate and precise candidate Reference Measurement Procedure (cRMP), as one key component of CDC-CSP, for standardization of FT4 measurements. METHODS: Serum FT4 was separated from protein-bound thyroxine with equilibrium dialysis (ED) following the recommended conditions in the Clinical and Laboratory Standards Institute C45-A guideline and the published RMP [20,21,23]. FT4 in dialysate was directly quantified with liquid chromatography-tandem mass spectrometry (LC-MS/MS) without derivatization. Gravimetric measurements of specimens and calibrator solutions, calibrator bracketing, isotope dilution, enhanced chromatographic resolution, and T4 specific mass transitions were used to ensure the accuracy, precision, and specificity of the cRMP. RESULTS: The described cRMP agreed well with the established RMP and two other cRMPs in an interlaboratory comparison study. The mean biases of each method to the overall laboratory mean were within ±2.5%. The intra-day, inter-day, and total imprecision for the cRMP were within 4.4%. The limit of detection was 0.90 pmol/L, which was sufficiently sensitive to determine FT4 for patients with hypothyroidism. The structural analogs of T4 and endogenous components in dialysate did not interfere with the measurements. CONCLUSION: Our ED-LC-MS/MS cRMP provides high accuracy, precision, specificity, and sensitivity for FT4 measurement. The cRMP can serve as a higher-order standard for establishing measurement traceability and provide an accuracy base for the standardization of FT4 assays.