Recent advances in the determination of unbound concentration and plasma protein binding of drugs: Analytical methods.

✅ 全文

药物游离浓度和血浆蛋白结合的最新测定进展：分析方法

作者 Seyfinejad Behrouz; Ozkan Sibel A; Jouyban Abolghasem 期刊 Talanta 发表日期 2021 卷/期/页码 Vol. 225 ISSN 1873-3573 DOI 10.1016/j.talanta.2020.122052 类型原创研究 (Original Research)

📄 中文摘要 Chinese Abstract

中文

药物血浆蛋白结合（PPB）过程显著影响其药代动力学和药效动力学特性，是全面表征任何化合物时必须始终评估的关键参数。经典超滤（UF）方法因其主要优点——准确度高、分析时间短、操作简便——仍被广泛接受，但该方法也存在一些局限性，其中最重要的是非特异性结合（NSB）。实验条件，包括所用UF装置的类型，可显著影响PPB研究中获得结果的准确性。本研究旨在评估同一制造商的不同超滤装置用于卡维地洛（CVD）血浆蛋白结合研究的适用性。卡维地洛是一种高血浆蛋白结合率且具有脂溶性的β受体阻滞剂，采用经验证的LC-MS/MS方法进行定量。

📋 英文结构化总结 English Structured Summary

全文整理

Background:

The process of plasma protein binding (PPB) of drugs greatly influences both their pharmacokinetic and pharmacodynamic properties, being a key parameter which should be always evaluated for the comprehensive characterization of any compound. The classical ultrafiltration (UF) method is still widely accepted based on its main advantages which include accuracy, short analysis time and ease of implementation, but the method also has some limitations, non-specific binding (NSB) being the most important. The experimental conditions, including the type of the UF device used, can greatly influence the accuracy of the results obtained in a PPB study.

The aim of the present study was to assess the suitability of different ultrafiltration devices from the same manufacturer for the study of plasma protein binding of carvedilol (CVD), a highly protein-bound and lipophilic beta-blocking agent, using a validated LC-MS/MS method for quantification.

Methods:

Samples at different levels of concentration were prepared in both proteic and non-proteic matrices (human plasma, 5% human serum albumin solution and saline solution) and subjected to the classical ultrafiltration method using Centrifree Ultrafiltration Centrifugal Filters (Ultracel PL Regenerated Cellulose, 30 kDa MWCO, 1 mL), Amicon Ultra-2 and Amicon Ultra-0.5 centrifugal filter units (Ultracel-10K regenerated cellulose membrane, 2 mL and 0.5 mL) from Merck Millipore. For the determination of the total CVD concentration, 200 µL of each sample solution was added to an Eppendorf microcentrifuge tube, while, for the determination of the free/unbound fraction, 400 µL were added to the UF devices. All UF devices and microcentrifuge tubes containing CVD samples were incubated at 37°C for 30 minutes, then centrifuged at room temperature for 15 min at 1877 x g for Centrifree, 5214 x g for Amicon Ultra-2 and 4829 x g for Amicon Ultra-0.5.

The analysis and quantification was achieved using a validated LC-MS/MS method with a Perkin Elmer Flexar FX-10 UHPLC and a Sciex QTOF 4600 mass spectrometer, using metoprolol as internal standard. The isocratic chromatographic separation was performed on a Phenomenex Luna C18 column with a mobile phase composition of 53% (v/v) 20mM ammonium formate at pH 4.4 and 47% (v/v) acetonitrile. An attempt to apply a previously described modified ultrafiltration method was also made using Amicon Ultra-0.5 devices, with partner UF devices containing control matrix, a first centrifugation, inversion of the upper compartments, and a second centrifugation.

The data sets obtained for determinations made using Centrifree devices were statistically evaluated in terms of normality of distribution using the Kolmogorov-Smirnov test, in terms of homogeneity of variances using Cochran’s C test and in terms of mean difference using the ANOVA single factor test, considering a significance level of 0.05.

Results:

For the Centrifree devices, the determined unbound fractions of carvedilol and the corresponding binding degree were in accordance to literature data, while for the Amicon Ultra devices a great degree of carvedilol adsorbtion to the sample reservoir was observed, the analyte not being detected in the ultrafiltrate samples. In human plasma, the chromatographic CVD signal observed for the samples after UF using Centrifree devices was below the lower limit of quantification of the LC-MS/MS method, thus not allowing further assessments and calculations of the unbound fraction. In 5% HSA solution, determinations revealed an overall unbound fraction mean for CVD of 7.95% (±0.053), which implies a protein binding degree of more than 92%.

The results obtained for CVD samples in saline solution indicated that, in the absence of proteins from the matrix, a different degree of NSB occurs. While for the Amicon Ultra devices, because of the lack of analyte in the ultrafiltrate, the NSB degree was maximum, in the case of the Centrifree devices an overall mean of less than 35% NSB was observed. The separate statistical analysis of the data sets obtained for the Centrifree devices, regarding the two matrices considered (5% HSA and saline solution), revealed no significant statistical difference in terms of normality of distribution, variance and mean free fraction of CVD (p > 0.05).

For the modified ultrafiltration method, samples containing 125 ng/mL CVD in both human plasma and 5% HSA solution were analyzed in singlicate using Amicon Ultra-0.5 devices. A very high recovery of the analyte was observed in the retentate reconstituted samples (111.83% for samples in human plasma and 98.38% for samples in 5% HSA), while no presence of the analyte was detected in the filtrate reconstituted samples.

Data Summary:

Using Centrifree devices, the determined unbound fraction of CVD in 5% HSA was 7.87% (±0.51) at 25 ng/mL, 7.64% (±0.79) at 75 ng/mL, 7.57% (±0.75) at 125 ng/mL and 8.72% (±0.54) at 500 ng/mL. In saline solution, the unbound fraction was 64.76% (±3.94) at 25 ng/mL, 60.09% (±3.26) at 75 ng/mL, 66.43% (±2.64) at 125 ng/mL and 64.46% (±2.70) at 500 ng/mL. For human plasma, data were not available because the CVD signal after UF was below the lower limit of quantification of 2.5 ng/mL.

For Amicon Ultra-2 devices, no CVD signal was observed in the ultrafiltrate at 25, 75, 125 or 500 ng/mL in human plasma, 5% HSA or saline solution. For Amicon Ultra-0.5 devices, no CVD signal was observed in the ultrafiltrate at 125 ng/mL in the three matrices. The validated LC-MS/MS method showed good linearity over 2.5–500 ng/mL with correlation coefficients greater than 0.995, and accuracy and precision values within the acceptance limits of Er% and RSD% < 15%.

Conclusions:

When studying PPB of drugs using the UF method, a very close attention should be paid to the implied UF protocol and to the UF devices used. The Centrifree filter devices, which were specifically designed for evaluations of PPB, have also proven to be suitable for the study of the protein binding process in the case of the lipophilic compound CVD, in comparison to the Amicon Ultra devices for which very poor results were obtained.

The different materials used for the components of the Amicon Ultra devices, compared to Centrifree, seemed to result in a great adsorbtion of the analyte to the sample reservoir, making the devices impractical for use in the desired study approach.

Practical Significance:

The type of ultrafiltration device used has a significant influence on the outcome of a plasma protein binding study, and for carvedilol the evaluation of the protein binding interaction could be achieved using the Centrifree ultrafiltration devices, but not the Amicon Ultra devices.

📋 中文结构化总结 Chinese Structured Summary

中文

背景:

本研究旨在评估同一制造商的不同超滤装置用于卡维地洛（CVD）血浆蛋白结合研究的适用性。卡维地洛是一种高血浆蛋白结合率且具有脂溶性的β受体阻滞剂，采用经验证的LC-MS/MS方法进行定量。

方法:

在不同浓度水平下，于含蛋白和非蛋白基质（人血浆、5%人血清白蛋白溶液和生理盐水溶液）中制备样品，并采用经典超滤方法进行处理。所用装置包括Merck Millipore公司的Centrifree超滤离心滤器（Ultracel PL再生纤维素，30 kDa MWCO，1 mL）、Amicon Ultra-2和Amicon Ultra-0.5离心滤器单元（Ultracel-10K再生纤维素膜，2 mL和0.5 mL）。测定CVD总浓度时，将200 µL各样品溶液加入Eppendorf微量离心管；测定游离/未结合分数时，将400 µL加入UF装置。所有含CVD样品的UF装置和微量离心管均在37°C孵育30分钟，然后在室温下离心15分钟：Centrifree为1877 × g，Amicon Ultra-2为5214 × g，Amicon Ultra-0.5为4829 × g。

采用经验证的LC-MS/MS方法进行分析与定量，使用Perkin Elmer Flexar FX-10 UHPLC和Sciex QTOF 4600质谱仪，以美托洛尔为内标。等度色谱分离在Phenomenex Luna C18柱上进行，流动相组成为53%（v/v）pH 4.4的20 mM甲酸铵和47%（v/v）乙腈。还尝试使用Amicon Ultra-0.5装置应用先前描述的改良超滤方法，包括含对照基质的配对UF装置、第一次离心、翻转上室以及第二次离心。

对使用Centrifree装置进行测定所得数据集进行统计学评估：采用Kolmogorov-Smirnov检验评估分布正态性，采用Cochran’s C检验评估方差齐性，采用单因素ANOVA检验评估均值差异，显著性水平设为0.05。

结果:

对于Centrifree装置，测定的卡维地洛未结合分数及相应结合度与文献数据一致；而对于Amicon Ultra装置，观察到卡维地洛对样品储液室存在较大程度的吸附，超滤液样品中未检测到分析物。在人血浆中，使用Centrifree装置超滤后样品所观察到的CVD色谱信号低于LC-MS/MS方法的定量下限，因此无法进一步评估和计算未结合分数。在5% HSA溶液中，测定结果显示CVD的总体未结合分数均值为7.95%（±0.053），提示蛋白结合度超过92%。

生理盐水溶液中CVD样品的结果表明，在基质中不存在蛋白时，会发生不同程度的NSB。对于Amicon Ultra装置，由于超滤液中缺乏分析物，NSB程度达到最大；而对于Centrifree装置，观察到的总体NSB均值低于35%。对Centrifree装置所得数据集分别进行统计分析，针对所考虑的两种基质（5% HSA和生理盐水溶液），在分布正态性、方差以及CVD平均游离分数方面均未发现显著统计学差异（p > 0.05）。

对于改良超滤方法，使用Amicon Ultra-0.5装置以单份测定方式分析了人血浆和5% HSA溶液中含125 ng/mL CVD的样品。在截留液复溶样品中观察到分析物回收率非常高（人血浆样品为111.83%，5% HSA样品为98.38%），而在滤液复溶样品中未检测到分析物。

数据摘要:

使用Centrifree装置，在5% HSA中测定的CVD未结合分数在25 ng/mL时为7.87%（±0.51），75 ng/mL时为7.64%（±0.79），125 ng/mL时为7.57%（±0.75），500 ng/mL时为8.72%（±0.54）。在生理盐水溶液中，未结合分数在25 ng/mL时为64.76%（±3.94），75 ng/mL时为60.09%（±3.26），125 ng/mL时为66.43%（±2.64），500 ng/mL时为64.46%（±2.70）。人血浆中无可用数据，因为超滤后CVD信号低于2.5 ng/mL的定量下限。

对于Amicon Ultra-2装置，在人血浆、5% HSA或生理盐水溶液中，超滤液在25、75、125或500 ng/mL时均未观察到CVD信号。对于Amicon Ultra-0.5装置，在三种基质中，超滤液在125 ng/mL时均未观察到CVD信号。经验证的LC-MS/MS方法在2.5–500 ng/mL范围内表现出良好的线性，相关系数大于0.995，准确度和精密度值均在Er%和RSD% < 15%的接受限度内。

结论:

在使用UF方法研究药物PPB时，应高度重视所采用的UF方案及所用UF装置。Centrifree滤器装置是专为PPB评估而设计的，与Amicon Ultra装置相比，其用于脂溶性化合物CVD蛋白结合过程的研究也被证明是适用的；而Amicon Ultra装置所得结果非常差。

与Centrifree相比，Amicon Ultra装置各组件所使用的不同材料似乎导致分析物对样品储液室产生较大吸附，使这些装置不适用于本研究所期望的研究方案。

实际意义:

所用超滤装置的类型对血浆蛋白结合研究结果具有显著影响；对于卡维地洛，蛋白结合相互作用的评估可使用Centrifree超滤装置实现，但不能使用Amicon Ultra装置。

📖 英文全文 English Full Text

ABMJ 2023, 6(2): 1-10 DOI: 10.2478/abmj-2023-0007

COMPARISON OF DIFFERENT ULTRAFILTRATION DEVICES FOR THE STUDY OF

PLASMA PROTEIN BINDING OF CARVEDILOL

Camelia-Maria TOMA1,2*, Lénárd FARCZÁDI3, Valentin ION2,

Daniela-Lucia MUNTEAN2, Silvia IMRE2,3

1 Doctoral School of Medicine and Pharmacy, I.O.S.U.D., George Emil Palade University of Medicine,

Pharmacy, Science, and Technology of Targu Mures, Romania

2 Department of Analytical Chemistry and Drug Analysis, Faculty of Pharmacy, George Emil Palade

University of Medicine, Pharmacy, Science, and Technology of Targu Mures, Romania

3 Center of Advanced Medical and Pharmaceutical Research, George Emil Palade University of Medicine,

Pharmacy, Science, and Technology of Targu Mures, Romania

*Correspondence:

Camelia-Maria TOMA camelia.toma@umfst.ro

Received: 26 July 2023; Accepted: 26 August 2023; Published: 30 December 2023

Abstract: The aim of the present study was to assess the suitability of different Amicon Ultra and Centrifree ultrafiltration devices for the study of the plasma protein binding process in the case of carvedilol, a highly protein-bound and lipophilic beta-blocking agent. Samples at different levels of concentration were prepared in both proteic and non-proteic matrices (human plasma, 5% human serum albumin solution and saline solution) and subjected to the classical ultrafiltration method using the different devices considered.

Furthermore, an attempt to apply a previously described modified ultrafiltration method was also made. The analysis and quantification was achieved using a validated LC-MS/MS method. For the Centrifree devices, the determined unbound fractions of carvedilol and the corresponding binding degree were in accordance to literature data, while for the Amicon Ultra devices a great degree of carvedilol adsorbtion to the sample reservoir was observed, the analyte not being detected in the ultrafiltrate samples. Thus, it was further demonstrated that the type of ultrafiltration device used has a significant influence on the outcome of a plasma protein binding study. In the case of carvedilol, the evaluation of the protein binding interaction could be achieved using the Centrifree ultrafiltration devices, but not the Amicon Ultra devices.

Keywords: ultrafiltration, protein binding, carvedilol, Amicon Ultra, Centrifree

Introduction The process of plasma protein binding (PPB) of drugs greatly influences both their pharmacokinetic and pharmacodynamic properties, being a key parameter which should be always evaluated for the comprehensive characterization of any compound (Bohnert and

Gan, 2013; Yuan et al, 2020; Seyfinejad et al., 2021). Based on the importance of the PPB process, there is an increased interest in developing study methods for the accurate assessment of the binding degree of drugs.

Among the different study approaches proposed over time for the assessment of PPB, the classical ultrafiltration (UF) method is still

Acta Biologica Marisiensis

Camelia-Maria Toma et al.

widely accepted based on its main advantages which include: accuracy, short analysis time and ease of implementation (Howard et al.,

2010; Vuignier et al., 2010). However, the method also has some limitations, non-specific binding (NSB) being the most important, but there are a lot of possibilities described in literature as ways to overcome and suppress them (Toma et al., 2021). Several ways to reduce NSB of studied drugs to the UF devices include the following: pre-treatment with different surfactant solutions, determination of

NSB using phosphate buffer saline solution and the use of a correction factor, blocking of the

NSB sites in the presence of plasma or proteic matrices, using different approaches and modifications of the classical UF technique.

The UF method implies the physical separation of the free and protein-bound fractions of drug through a semipermeable membrane, using the centrifugal force (Howard et al., 2010). The separation is achieved in an

UF device which consists of two different compartments delimited by the semipermeable membrane with different molecular weight cut- off. After centrifugation, the ultrafiltrate containing only the free drug fraction can be quantified using an appropriate analytical technique.

It has been demonstrated that the experimental conditions, including the type of the UF device used, can greatly influence the accuracy of the results obtained in a PPB study (Kratzer et al., 2014; Dorn et al., 2018; Toma et al., 2021). Regarding the UF device, the factors that may play a role in their suitability for a particular study, are the type of the semipermeable membrane and also the material from which the other constituent components of the device are made since they can provide

NSB sites (Lee et al., 2003; Kratzer et al., 2016).

The UF devices designed and commonly used for PPB studies are represented by the

Millipore Centrifree filters, but in some studies, other devices initially designed for concentration of different constituents in biological samples (proteins, RNA, antigens, antibodies, enzymes) have also been successfully used (Du et al., 2014; Downing et al., 2017; Catalani et al., 2018). Furthermore, some authors suggest a validation of these other UF devices to the Centrifree ones, which are considered as reference, since differences in the results obtained based on the UF devices used have been frequently reported (Vogeser et al., 2007; Jensen et al., 2011; Larsen et al.,

2011; Arellano et al., 2012; Ciobotaru et al., 2022).

The aim of this study was to assess the suitability of different ultrafiltration devices from the same manufacturer for the study of plasma protein binding of carvedilol (CVD), a highly protein-bound and lipophilic beta- blocking agent, using a validated LC-MS/MS method for quantification.

2. Materials and methods Chemicals, reagents, and solvents

Pharmaceutical secondary standard of carvedilol was acquired from Sigma-Aldrich (Saint Louis, USA) and metoprolol succinate was purchased from Moehs (Barcelona, Spain).

Acetonitrile (Honeywell, Muskegon, USA) and ammonium formate (VWR Chemicals, Radnor,

USA) of LC-MS grade were used as solvents or components of the mobile phase. Human

Albumin, as proteic matrix, was purched in the form of 200 g/L solution for infusion from

Baxalta Innovations GmbH (Wien, Austria), while human plasma was obtained from The

Regional Blood Transfusion Center Targu Mures (Romania).

Saline solution was purchased from STADA Hemofarm (Timisoara, Romania) and ultrapure water was obtained with the aid of a Millipore Direct-Q 3 (Milford, USA).

ABMJ 2023, 6(2): 1-10

LC-MS/MS analysis A validated LC-MS/MS method previously published was used (Toma et al.,

2023). The characteristics of the equipments were: a Perkin Elmer Flexar FX-10 UHPLC (Waltham, USA) and a Sciex QTOF 4600 mass spectrometer (Framingham,

USA).

The isocratic chromatographic separation was performed on a Phenomenex Luna C18 column

125 × 4 mm, 5 μm (Torrance, USA) with a mobile phase composition of 53% (v/v) 20mM ammonium formate at pH 4.4 and 47% (v/v) acetonitrile. The pump delivered the mobile phase with a flow rate of 0.4 mL/min, the column temperature was set at 25 °C and the injection volume was 4 µL. Metoprolol (MTP) was used as internal standard.

The MS detection was achieved after positive electrospray ionization, in MRM mode and the monitored transitions were the following: for CVD m/z 407.29 → 100.10,

222.15, 224.18, 283.22 and for the internal standard MTP m/z 268.23 → 116.12, 121.08,

133.08, 159.10, 191.14. The ion source parameters were set as follows: electrospray voltage +3300 V, source temperature 500˚C, nebulizing gas 30, drying gas 25, curtain gas 30 and collision energies of 32 for CVD and 24 for MTP (values in arbitrary units).

Preparation of solutions Stock solutions. The stock solution of 10 µg/mL CVD was obtained by appropriate dilution with ultrapure water of a 1 mg/mL

CVD solution prepared in acetonitrile, resulting in a 1% (v/v) acetonitrile concentration in the final stock solution. For the internal standard, the solvent used consisted only of acetonitrile and the final stock solution of 1 µg/mL MTP was obtained by an appropriate dilution of a

500 µg/mL MTP solution.

Standard solutions. Ten standard solutions for the calibration curve over the concentration range of 2.5 – 500 ng/mL CVD were obtained by spiking 150 µL of matrix with 50 µL aliquotes of corresponding intermediate working solutions. Three different matrices were considered: saline solution, human plasma and 5% (w/v) human serum albumin (HSA), prepared by appropriate dilution with saline of the 20% infusion solution.

Sample solutions. The sample solutions with concentrations of 25, 75, 125 and 500 ng/mL CVD were prepared in the three matrices considered following the same protocol applied for the standard solutions.

Experimental ultrafiltration protocol Classical ultrafiltration method. Different

UF devices from the same manufacturer were used: Centrifree® Ultrafiltration Centrifugal

Filters (Ultracel® PL Regenerated Cellulose, 30 kDa MWCO, 1 mL), Amicon Ultra-2 and

Amicon Ultra-0.5 centrifugal filter units (Ultracel-10K regenerated cellulose membrane,

2 mL and 0.5 mL) from Merck Millipore (Cork, Ireland).

For the determination of the total CVD concentration, a volume of 200 µL of each sample solution was separately added to an

Eppendorf microcentrifuge tube, while, for the determination of the free/unbound fraction, 400 µL were added to the UF devices. In order to allow the establishment of the protein-binding equilibrium, all

UF devices and microcentrifuge tubes containing CVD samples were incubated at 37°C for 30 minutes. After the incubation period, the 200 µL sample solution aliquotes were immediately processed for analysis, while the UF devices were centrifuged with the aid of an Eppendorf

Centrifuge 5430R at room temperature for 15 min.

Following the manufacturer’s recommendations for each UF device, the following centrifugal forces were applied: 1877 x g for the Centrifree devices, 5214 x g for

Amicon Ultra-2 and 4829 x g for Amicon Ultra-0.5. After centrifugation, 200 µL of the

Camelia-Maria Toma et al.

ultrafiltrate obtained were processed for analysis in order to determine the unbound concentration of CVD.

Modified ultrafiltration method.

An attempt to apply a modified version of the ultrafiltration method previously described by

Taylor and Harker was also made (Taylor and Harker, 2006). For this method, the Amicon

Ultra-0.5 centrifugal filter units were used since from all the UF devices considered in the present study, only these were suitable based on their mode of construction. For the modified

UF method, for each UF device containing 400 µL CVD sample solution, a partner UF device containing 400 µL control matrix was also prepared. After incubation at 37°C for 30 minutes, all devices were centrifuged at room temperature for 15 minutes, applying a centrifugal force of 4829 x g. Following this first centrifugation, the upper compartments of the UF devices containing the retentate were inverted and placed on the ultrafiltrate collection compartments of their partner UF device. The devices were then centrifuged again for 10 minutes. 200 µL aliquotes of each reconstituted sample obtained were then removed and processed for quantification.

Processing of samples for LC-MS/MS analysis To all sample and standard solutions, 100 µL aliquotes of 1 µg/mL MTP internal standard solution were added. The solutions were further deproteinized with acetonitrile (1:3 ratio), vortexed for 30 seconds and centrifuged for 10 minutes at 10000 rpm. The supernatants was subjected to the LC-MS/MS analysis.

Statistical analysis The data sets obtained for determinations made using Centrifree devices were statistically evaluated in terms of normality of distribution using the Kolmogorov-Smirnov test, in terms of homogeneity of variances using the

Cochran’s C test and in terms of mean difference using the ANOVA single factor test.

The statistical tests were applied considering a significance level of 0.05.

3. Results and discussion Quality parameters of the analytical method

An already validated LC-MS/MS method for quantification was used (Toma et al., 2023).

Specificity/selectivity, accuracy, precision and linearity of the method were tested and proved to be suitable. CVD and MTP were separated at retention times of 4.36 (±0.03) min and 2.51 (±0.01) min, respectively, demonstrating selectivity.

The method presented good linearity over the concentration range 2.5-500 ng/mL CVD, with correlation coefficients greater than 0.995. Values of accuracy (relative error, Er%) and precision (relative standard deviation, RSD%) were within the acceptance limits according to the EMA Guidelines on bioanalytical method validation (Er% and

RSD% < 15%).

Classical ultrafiltration method The accuracy and relevance of PPB study results using the UF method is greatly influenced by the experimental conditions.

Besides pH and temperature, which should be in accordance to the physiological values, a great attention should be paid to the type of UF device used. In the present study, two different types of ultrafiltration devices from the same manufacturer were tested regarding their suitability for the study of CVD binding to proteins. Both types of UF devices present a regenerated cellulose semipermeable membrane, but with different molecular weight cut-off: 30 kDa in the case of Centrifree devices and 10 kDa in the case of Amicon

Ultra devices. Other differences between the devices considered, are related to the materials

ABMJ 2023, 6(2): 1-10

used for the sample reservoirs and collection tubes. In the case of the Centrifree devices, the sample reservoir is made of styrene/acrylonitrile and the collection tube of polyethylene, whereas in the case of the

Amicon Ultra devices, the materials used were styrene/butadiene and polypropylene, respectively.

According to the data sheet of the product, only the Centrifree devices were specifically designed for separating free from bound microsolutes in biological samples, but other

PPB studies report good results also obtained using the Amicon Ultra devices, initially designed for concentration of different components of biological samples (antigens, antibodies, enzymes, nucleic acids, microorganisms), protein extraction and purification (Du et al., 2014; Imre et al., 2021).

For the classical ultrafiltration method, samples with concentrations of 25, 75, 125, and

500 ng/mL CVD prepared in human plasma, 5% HSA and saline solution were subjected to ultrafiltration using Centrifree and Amicon

Ultra-2 devices. Additionally, Amicon-Ultra 0.5 devices were used for the ultrafiltration of

125 ng/mL CVD samples in the three matrices considered. The results obtained in terms of determined unbound CVD fraction (%) are presented in Table 1.

In the case of determinations made in human plasma using Centrifree devices, the chromatographic CVD signal observed for the samples after UF, although present, was bellow the lower limit of quantification of the LC- MS/MS method used (LLOQ – 2.5 ng/mL), thus not allowing further assessments and calculations of the unbound faction. In the case of determinations made using Amicon Ultra devices, no noticeable CVD signal was observed in the chromatograms of samples after UF. Representative chromatograms of samples before and after UF are presented in

Figures 1-3.

From the results obtained, we could conclude that for the considered analyte, CVD, which is a highly lipophilic compound, Amicon

Ultra devices are not suitable for the purpose of plasma protein binding assessments, compared to Centrifree, since no significant presence of the analyte in the ultrafiltrate was detected, not even for the higher concentration samples. In the case of the Centrifree devices, the results obtained for the determinations of CVD in human plasma could indicate a very high degree of binding to plasma proteins, which would be in accordance to literature data sustaining a more than 95% protein bound fraction (Book, 2002).

Table 1. Determined unbound fraction of CVD (%) using different ultrafiltration devices

Type of UF device c (ng/mL) Unbound fraction % mean (standard deviation)

Human plasma 5% HSA Saline solution Centrifree* 25

N/A 7.87 (±0.51) 64.76 (±3.94) 75 N/A 7.64 (±0.79)

60.09 (±3.26) 125 N/A 7.57 (±0.75) 66.43 (±2.64) 500

N/A 8.72 (±0.54) 64.46 (±2.70) Amicon Ultra-2* 25 N/A

N/A N/A 75 N/A N/A N/A 125 N/A N/A N/A 500 N/A N/A

N/A Amicon Ultra-0.5** 125 N/A N/A N/A *n=3 ; **n=1 ; N/A – data not available

Camelia-Maria Toma et al.

Fig. 1. Representative chromatograms of a 125 ng/mL CVD sample in human plasma before and after ultrafiltration (UF) using Centrifree vs. Amicon Ultra devices

Fig. 2. Representative chromatograms of a 125 ng/mL CVD sample in 5% HSA before and after ultrafiltration (UF) using Centrifree vs. Amicon Ultra devices

ABMJ 2023, 6(2): 1-10

Fig. 3. Representative chromatograms of a 125 ng/mL CVD sample in saline solution before and after ultrafiltration (UF) using Centrifree vs. Amicon Ultra devices

Furthermore, in our experimental conditions, determinations made in HSA solution revealed an overall unbound fraction mean for CVD of 7.95% (±0.053), which implies a protein binding degree of more than

92%, results also in accordance to other literature data (Morgan, 1994). Compared to human plasma, which is a very complex matrix, containing a variety of proteins (albumin, alpha-1-acid glycoprotein, globulins, lipoproteins), the 5% HSA solution was chosen as a simple proteic matrix for the present study in order to also emphasize the difference in the binding behavior of CVD, and the possible influence of the proteic matrix complexity on the results obtained for the different UF devices.

The purpose of the determinations made in saline solution, were to allow an assessment of the possibility of NSB occurrence in the absence of proteins from the matrix. In a previous study, Wang S and Williams NS have shown that NSB is greatly reduced, even in the case of compounds with high lipophilicity, when samples containing protein environments are incubated in the UF device because proteins present a protective effect of blocking the NSB sites (Wang and Williams, 2013). Furthermore, the NSB and adsorption from proteic matrices can also be expected to be significantly lower, as the protein-bound drug fraction cannot be adsorbed.

The results obtained for CVD samples in saline solution indicate that, in the absence of proteins from the matrix, in the case of both types of UF devices considered, a different degree of NSB occurs. While for the Amicon

Ultra devices, because of the lack of analyte in the ultrafiltrate, we could conclude that the

NSB degree is maximum, in the case of the Centrifree devices an overall mean of less than

35% NSB was observed.

The separate statistical analysis of the data sets obtained for the Centrifree devices, regarding the two matrices considered (5%

HSA and saline solution), revealed no significant statistical difference in terms of normality of distribution, variance and mean

Camelia-Maria Toma et al.

free fraction of CVD (p > 0.05). These results indicate that, for the considered concentration range (25-500 ng/mL), the binding behavior of

CVD to HSA and the adsorbtion which takes place in saline solution, respectively, are not influenced by concentration.

The very poor results obtained for the Amicon Ultra devices could be related to the materials from which the sample reservoir and collection tube are made, leading to a great adsorbtion of CVD, this being the main difference from the Centrifree devices. The difference in the molecular weight cut-off of the semipermeable membrane (10 kDa vs. 30 kDa) should not have an influence on the diffusion of CVD, taking into account its much lower molecular weight (406.5 g/mol).

Furthermore, the volume of the sample reservoir in the case of Amicon Ultra devices (2 mL vs. 0.5 mL) seems to not have an influence on the result.

Modified ultrafiltration method The modified ultrafiltration method described by Taylor and Harker (Taylor and

Harker, 2006) was used for assessments regarding samples containing 125 ng/mL CVD in both human plasma and 5% HSA solution.

For this method Amicon Ultra-0.5 devices were selected and samples were analyzed in singlicate. In the case of both matrices, a very high recovery of the analyte was observed in the retentate reconstituted samples (111.83% for samples in human plasma and 98.38% for samples in 5% HSA), while no presence of the analyte was detected in the filtrate reconstituted samples.

These results further sustain the very high degree of CVD adsorbtion in the sample reservoir of the Amicon Ultra devices. Even though, in the mentioned study, the research was also focused on highly lipophilic compounds (corticosteroids), the much better results using the modified UF method could be related to the different UF devices used (Microcon).

Conclusions When studying PPB of drugs using the UF method, a very close attention should be paid to the implied UF protocol and to the UF devices used. The Centrifree filter devices, which were specifically designed for evaluations of PPB, have also proven to be suitable for the study of the protein binding process in the case of the lipophilic compound CVD, in comparison to the Amicon Ultra devices for which very poor results were obtained. The different materials used for the components of the Amicon Ultra devices, compared to Centrifree, seemed to result in a great adsorbtion of the analyte to the sample reservoir, making the devices impractical for use in the desired study approach.

Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Funding This research was funded by George Emil Palade University of Medicine, Pharmacy,

Science, and Technology of Targu Mures, grant number 10127/5/17.12.2020.

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📖 中文全文 Chinese Full Text

中文

以下为中文译文（保留原文结构与关键技术术语）：

ABMJ 2023, 6(2): 1-10 DOI: 10.2478/abmj-2023-0007

不同超滤装置在卡维地洛血浆蛋白结合研究中的比较

Camelia-Maria TOMA1,2*，Lénárd FARCZÁDI3，Valentin ION2， Daniela-Lucia MUNTEAN2，Silvia IMRE2,3

1 罗马尼亚图尔恰穆雷什乔治·埃米尔·帕拉德医学、药学、科学与技术大学医学与药学博士学校，I.O.S.U.D. 2 罗马尼亚图尔恰穆雷什乔治·埃米尔·帕拉德医学、药学、科学与技术大学药学院分析化学与药物分析系 3 罗马尼亚图尔恰穆雷什乔治·埃米尔·帕拉德医学、药学、科学与技术大学高级医学与药学研究中心

*通讯作者： Camelia-Maria TOMA camelia.toma@umfst.ro

收稿日期：2023年7月26日；接受日期：2023年8月26日；发表日期：2023年12月30日

摘要：本研究旨在评估不同 Amicon Ultra 和 Centrifree 超滤装置用于卡维地洛血浆蛋白结合过程研究的适用性。卡维地洛是一种高蛋白结合率且具有亲脂性的β受体阻滞剂。在蛋白基质和非蛋白基质（人血浆、5% 人血清白蛋白溶液和生理盐水）中制备了不同浓度水平的样品，并采用所考虑的不同装置进行经典超滤法处理。此外，本研究还尝试应用一种先前描述的改良超滤法。分析定量采用经验证的 LC-MS/MS 方法完成。对于 Centrifree 装置，所测定的卡维地洛未结合分数及相应结合度与文献数据一致；而对于 Amicon Ultra 装置，观察到卡维地洛对样品储液腔存在较大程度的吸附，超滤液样品中未检测到分析物。因此，本研究进一步证明，所用超滤装置的类型对血浆蛋白结合研究结果具有显著影响。就卡维地洛而言，使用 Centrifree 超滤装置可实现蛋白结合相互作用的评估，但 Amicon Ultra 装置则不适用。

关键词：超滤；蛋白结合；卡维地洛；Amicon Ultra；Centrifree

引言药物的血浆蛋白结合（plasma protein binding, PPB）过程会显著影响其药代动力学和药效学性质，是全面表征任何化合物时均应评估的关键参数（Bohnert and Gan, 2013; Yuan et al, 2020; Seyfinejad et al., 2021）。基于 PPB 过程的重要性，开发用于准确评估药物结合度的研究方法受到越来越多的关注。在长期以来提出的不同 PPB 评估研究途径中，经典超滤（ultrafiltration, UF）法仍被广泛接受，其主要优点包括：准确度高、分析时间短、易于实施（Howard et al., 2010; Vuignier et al., 2010）。然而，该方法也存在一些局限性，其中最重要的是非特异性结合（non-specific binding, NSB），但文献中已描述了许多克服和抑制这些局限性的可能方法（Toma et al., 2021）。减少待测药物对 UF 装置 NSB 的若干方法包括：使用不同表面活性剂溶液进行预处理；使用磷酸盐缓冲生理盐水测定 NSB 并采用校正因子；在血浆或蛋白基质存在下封闭 NSB 位点；以及采用经典 UF 技术的不同方法和改良。

UF 法是指利用离心力，通过半透膜对药物的游离部分和蛋白结合部分进行物理分离（Howard et al., 2010）。分离在 UF 装置中完成，该装置由具有特定分子量截留值（molecular weight cut-off, MWCO）的半透膜分隔成两个不同腔室。离心后，仅含游离药物部分的超滤液（ultrafiltrate）可用适当的分析技术进行定量。已有研究证明，实验条件（包括所用 UF 装置的类型）可显著影响 PPB 研究所得结果的准确性（Kratzer et al., 2014; Dorn et al., 2018; Toma et al., 2021）。就 UF 装置而言，可能影响其适用于特定研究的因素包括半透膜的类型，以及装置其他组成部件的材料，因为这些材料可能提供 NSB 位点（Lee et al., 2003; Kratzer et al., 2016）。

专为 PPB 研究设计并常用的 UF 装置代表是 Millipore Centrifree 滤器，但在一些研究中，最初用于浓缩生物样品中不同成分（蛋白质、RNA、抗原、抗体、酶）的其他装置也被成功使用（Du et al., 2014; Downing et al., 2017; Catalani et al., 2018）。此外，一些作者建议以被视为参考的 Centrifree 装置为基准，对这些其他 UF 装置进行验证，因为基于所用 UF 装置不同而导致结果差异的报道屡见不鲜（Vogeser et al., 2007; Jensen et al., 2011; Larsen et al., 2011; Arellano et al., 2012; Ciobotaru et al., 2022）。

本研究旨在评估同一制造商的不同超滤装置用于卡维地洛（carvedilol, CVD）血浆蛋白结合研究的适用性。CVD 是一种高蛋白结合率且具有亲脂性的β受体阻滞剂，并采用经验证的 LC-MS/MS 方法进行定量。

2. 材料与方法

化学品、试剂和溶剂卡维地洛药品二级标准品购自 Sigma-Aldrich（美国圣路易斯），琥珀酸美托洛尔购自 Moehs（西班牙巴塞罗那）。LC-MS 级乙腈（Honeywell，美国 Muskegon）和甲酸铵（VWR Chemicals，美国 Radnor）用作溶剂或流动相组分。人白蛋白作为蛋白基质，以 200 g/L 静脉注射液形式购自 Baxalta Innovations GmbH（奥地利维也纳）；人血浆取自罗马尼亚图尔恰穆雷什地区血液中心。生理盐水购自 STADA Hemofarm（罗马尼亚蒂米什瓦拉），超纯水由 Millipore Direct-Q 3 系统（美国 Milford）制备。

LC-MS/MS 分析采用已发表并经验证的 LC-MS/MS 方法（Toma et al., 2023）。仪器配置为：Perkin Elmer Flexar FX-10 UHPLC（美国 Waltham）和 Sciex QTOF 4600 质谱仪（美国 Framingham）。等度色谱分离在 Phenomenex Luna C18 柱（125 × 4 mm，5 μm；美国 Torrance）上进行，流动相组成为 53%（v/v）pH 4.4 的 20 mM 甲酸铵和 47%（v/v）乙腈。泵流速为 0.4 mL/min，柱温设定为 25 °C，进样体积为 4 µL。美托洛尔（metoprolol, MTP）用作内标。

质谱检测采用正离子电喷雾电离（positive electrospray ionization），以多反应监测（multiple reaction monitoring, MRM）模式进行。监测的离子过渡为：CVD m/z 407.29 → 100.10、222.15、224.18、283.22；内标 MTP m/z 268.23 → 116.12、121.08、133.08、159.10、191.14。离子源参数设定如下：电喷雾电压 +3300 V，源温度 500 °C，雾化气 30，干燥气 25，帘气 30；CVD 碰撞能量为 32，MTP 碰撞能量为 24（数值为任意单位）。

溶液配制储备液。CVD 10 µg/mL 储备液由乙腈配制的 1 mg/mL CVD 溶液用超纯水适当稀释获得，最终储备液中乙腈浓度为 1%（v/v）。内标所用溶剂仅为乙腈，MTP 1 µg/mL 最终储备液由 500 µg/mL MTP 溶液适当稀释获得。

标准溶液。在 2.5–500 ng/mL CVD 浓度范围内配制 10 个用于校准曲线的标准溶液，方法是向 150 µL 基质中加入 50 µL 相应中间工作液。考虑了三种不同基质：生理盐水、人血浆和 5%（w/v）人血清白蛋白（human serum albumin, HSA）；5% HSA 由 20% 注射液用生理盐水适当稀释制备。

样品溶液。在三种考虑基质中，按照与标准溶液相同的方案，分别配制浓度为 25、75、125 和 500 ng/mL 的 CVD 样品溶液。

实验超滤方案经典超滤法。使用同一制造商的不同 UF 装置：Centrifree® 超滤离心滤器（Ultracel® PL 再生纤维素膜，30 kDa MWCO，1 mL）、Amicon Ultra-2 和 Amicon Ultra-0.5 离心滤器单元（Ultracel-10K 再生纤维素膜，2 mL 和 0.5 mL），均购自 Merck Millipore（爱尔兰科克）。

为测定 CVD 总浓度，将每种样品溶液 200 µL 分别加入 Eppendorf 微量离心管中；为测定游离/未结合部分，将 400 µL 加入 UF 装置中。为使蛋白结合平衡得以建立，所有含 CVD 样品的 UF 装置和微量离心管均在 37 °C 孵育 30 分钟。孵育结束后，立即处理 200 µL 样品溶液 aliquots 用于分析，同时使用 Eppendorf Centrifuge 5430R 在室温下离心 UF 装置 15 min。按照各 UF 装置制造商推荐，分别施加以下离心力：Centrifree 装置为 1877 × g，Amicon Ultra-2 为 5214 × g，Amicon Ultra-0.5 为 4829 × g。离心后，取 200 µL 所得超滤液进行处理分析，以测定 CVD 的未结合浓度。

改良超滤法。本研究还尝试应用 Taylor 和 Harker 先前描述的改良超滤法（Taylor and Harker, 2006）。对于该方法，选用 Amicon Ultra-0.5 离心滤器单元，因为在本研究考虑的所有 UF 装置中，仅该装置基于其结构形式适用。对于改良 UF 法，每个含 400 µL CVD 样品溶液的 UF 装置均准备一个含 400 µL 对照基质的配对 UF 装置。在 37 °C 孵育 30 分钟后，所有装置在室温下离心 15 分钟，施加离心力 4829 × g。第一次离心后，将含截留液（retentate）的 UF 装置上腔倒置，并放置于其配对 UF 装置的超滤液收集腔上。随后再次离心 10 分钟。然后分别取 200 µL 各重建样品 aliquots 并进行定量处理。

LC-MS/MS 分析样品处理向所有样品溶液和标准溶液中加入 100 µL 1 µg/mL MTP 内标溶液。随后用乙腈进行去蛋白处理（1:3 比例），涡旋 30 秒，并以 10000 rpm 离心 10 分钟。上清液用于 LC-MS/MS 分析。

统计分析对使用 Centrifree 装置测定所得数据集进行统计分析：采用 Kolmogorov-Smirnov 检验评估分布正态性，采用 Cochran’s C 检验评估方差齐性，采用单因素 ANOVA 检验评估均值差异。统计检验的显著性水平设为 0.05。

3. 结果与讨论

分析方法的质量参数采用已验证的 LC-MS/MS 定量方法（Toma et al., 2023）。对方法的选择性/专属性、准确度、精密度和线性进行测试，并证明其适用。CVD 和 MTP 分别在保留时间 4.36（±0.03）min 和 2.51（±0.01）min 处分离，表明方法具有选择性。该方法在 2.5–500 ng/mL CVD 浓度范围内表现出良好线性，相关系数大于 0.995。准确度（相对误差，Er%）和精密度（相对标准偏差，RSD%）值均符合欧洲药品管理局（EMA）生物分析方法验证指南的接受限度（Er% 和 RSD% < 15%）。

经典超滤法使用 UF 法进行 PPB 研究时，结果的准确性和相关性受实验条件显著影响。除 pH 和温度应与生理值一致外，还应高度重视所用 UF 装置的类型。本研究测试了同一制造商的两种不同类型超滤装置，评估其用于 CVD 与蛋白结合研究的适用性。两种 UF 装置均具有再生纤维素半透膜，但分子量截留值不同：Centrifree 装置为 30 kDa，Amicon Ultra 装置为 10 kDa。所考虑装置之间的其他差异与样品储液腔和收集管所用材料有关。Centrifree 装置的样品储液腔由苯乙烯/丙烯腈制成，收集管由聚乙烯制成；而 Amicon Ultra 装置所用材料分别为苯乙烯/丁二烯和聚丙烯。

根据产品数据表，只有 Centrifree 装置是专门设计用于分离生物样品中游离小分子溶质与结合小分子溶质的，但其他 PPB 研究也报道使用最初用于浓缩生物样品不同成分（抗原、抗体、酶、核酸、微生物）、蛋白提取和纯化的 Amicon Ultra 装置可获得良好结果（Du et al., 2014; Imre et al., 2021）。

对于经典超滤法，将分别在人血浆、5% HSA 和生理盐水中配制的 25、75、125 和 500 ng/mL CVD 样品使用 Centrifree 和 Amicon Ultra-2 装置进行超滤。此外，还使用 Amicon Ultra-0.5 装置对三种考虑基质中 125 ng/mL CVD 样品进行超滤。以所测定的 CVD 未结合分数（%）表示的结果见表 1。

在使用 Centrifree 装置于人血浆中测定的情况下，UF 后样品中观察到的 CVD 色谱信号虽然存在，但低于所用 LC-MS/MS 方法的定量下限（lower limit of quantification, LLOQ，2.5 ng/mL），因此无法进一步评估和计算未结合分数。在使用 Amicon Ultra 装置测定的情况下，UF 后样品色谱图中未观察到明显的 CVD 信号。UF 前后样品的代表性色谱图见图 1–3。

根据所得结果，可以得出结论：对于所考虑的分析物 CVD 这一高度亲脂性化合物，与 Centrifree 相比，Amicon Ultra 装置不适用于血浆蛋白结合评估，因为即使在较高浓度样品中，也未在超滤液中检测到分析物的显著存在。对于 Centrifree 装置，在人血浆中测定 CVD 所得结果可能表明其与血浆蛋白的结合度非常高，这与文献数据一致，文献支持其蛋白结合分数超过 95%（Book, 2002）。

表 1. 使用不同超滤装置测定的 CVD 未结合分数（%）

UF 装置类型 | c（ng/mL） | 未结合分数 % 均值（标准差） --- | --- | --- | | 人血浆 | 5% HSA | 生理盐水 Centrifree* | 25 | N/A | 7.87（±0.51） | 64.76（±3.94） | 75 | N/A | 7.64（±0.79） | 60.09（±3.26） | 125 | N/A | 7.57（±0.75） | 66.43（±2.64） | 500 | N/A | 8.72（±0.54） | 64.46（±2.70） Amicon Ultra-2* | 25 | N/A | N/A | N/A | 75 | N/A | N/A | N/A | 125 | N/A | N/A | N/A | 500 | N/A | N/A | N/A Amicon Ultra-0.5** | 125 | N/A | N/A | N/A

* n = 3；** n = 1；N/A：数据不可用

图 1. 使用 Centrifree 与 Amicon Ultra 装置对人血浆中 125 ng/mL CVD 样品进行超滤（UF）前后的代表性色谱图

图 2. 使用 Centrifree 与 Amicon Ultra 装置对 5% HSA 中 125 ng/mL CVD 样品进行超滤（UF）前后的代表性色谱图

图 3. 使用 Centrifree 与 Amicon Ultra 装置对生理盐水中 125 ng/mL CVD 样品进行超滤（UF）前后的代表性色谱图

此外，在本研究实验条件下，HSA 溶液中的测定结果显示 CVD 的总体未结合分数均值为 7.95%（±0.053），这意味着蛋白结合度超过 92%，该结果也与其他文献数据一致（Morgan, 1994）。与作为非常复杂基质的人血浆相比，人血浆含有多种蛋白质（白蛋白、α-1-酸性糖蛋白、球蛋白、脂蛋白），本研究选择 5% HSA 溶液作为简单蛋白基质，以同时突出 CVD 结合行为的差异，以及蛋白基质复杂性对不同类型 UF 装置所得结果的可能影响。

在生理盐水中进行测定的目的，是评估在基质中不存在蛋白时发生 NSB 的可能性。先前研究中，Wang S 和 Williams NS 已表明，当含蛋白环境的样品在 UF 装置中孵育时，即使对于高亲脂性化合物，NSB 也会显著降低，因为蛋白具有封闭 NSB 位点的保护作用（Wang and Williams, 2013）。此外，由于蛋白结合药物部分不能被吸附，来自蛋白基质的 NSB 和吸附也可预期会显著降低。

生理盐水中 CVD 样品的测定结果表明，在基质中不存在蛋白的情况下，两种所考虑 UF 装置均会发生不同程度的 NSB。对于 Amicon Ultra 装置，由于超滤液中缺乏分析物，可以认为其 NSB 程度最大；而对于 Centrifree 装置，观察到的总体 NSB 均值低于 35%。

对 Centrifree 装置所得数据集进行单独统计分析，针对两种考虑基质（5% HSA 和生理盐水），结果显示在分布正态性、方差以及 CVD 游离分数均值方面均无显著统计学差异（p > 0.05）。这些结果表明，在所考虑浓度范围（25–500 ng/mL）内，CVD 与 HSA 的结合行为以及生理盐水中发生的吸附均不受浓度影响。

Amicon Ultra 装置所得结果非常差，可能与样品储液腔和收集管所用材料有关，导致 CVD 发生较大吸附，这是其与 Centrifree 装置的主要差异。半透膜分子量截留值的差异（10 kDa 对 30 kDa）不应影响 CVD 的扩散，考虑到 CVD 的分子量低得多（406.5 g/mol）。此外，Amicon Ultra 装置的样品储液腔体积（2 mL 对 0.5 mL）似乎对结果没有影响。

改良超滤法 Taylor 和 Harker 描述的改良超滤法（Taylor and Harker, 2006）用于评估含 125 ng/mL CVD 的人血浆和 5% HSA 溶液样品。对于该方法，选用 Amicon Ultra-0.5 装置，样品以单份分析。对于两种基质，在截留液重建样品中均观察到分析物非常高的回收率（人血浆样品为 111.83%，5% HSA 样品为 98.38%），而在滤液重建样品中未检测到分析物。

这些结果进一步支持 CVD 在 Amicon Ultra 装置样品储液腔中发生非常高程度吸附的结论。尽管在上述研究中，研究也聚焦于高亲脂性化合物（糖皮质激素），但使用改良 UF 法获得明显更好的结果可能与所用不同 UF 装置（Microcon）有关。

结论使用 UF 法研究药物 PPB 时，应高度重视所涉及的 UF 方案和所用 UF 装置。Centrifree 滤器装置是专为 PPB 评估设计的，并且与 Amicon Ultra 装置相比，已被证明适用于亲脂性化合物 CVD 的蛋白结合过程研究；而 Amicon Ultra 装置所得结果非常差。与 Centrifree 相比，Amicon Ultra 装置各部件所用不同材料似乎导致分析物对样品储液腔发生较大吸附，使该装置不适用于所需的研究方法。

利益冲突作者声明，本研究是在不存在任何可被视为潜在利益冲突的商业或财务关系的情况下进行的。

资助本研究由罗马尼亚图尔恰穆雷什乔治·埃米尔·帕拉德医学、药学、科学与技术大学资助，资助编号：10127/5/17.12.2020。