Ye Jiang

Effect of volume ratio of ultrafiltrate to sample solution on the analysis of free drug and measurement of free carbamazepine in clinical drug monitoring.

Traditional ultrafiltration (UF) usually has a large volume ratio of ultrafiltrate to sample solution, and this ratio cannot be well controlled. It can break the balance of protein-binding equilibrium and exert an influence on the analysis of free drug. In the present study, we evaluated the influence of volume ratio of ultrafiltrate to sample solution on the analysis of free drug in human plasma. We used carbamazepine as a model drug and studied the effect of different centrifugation times on ultrafitrate volume and the related effects on unbound carbamazepine measurement. Moreover, we compared the hollow fiber centrifugal ultrafiltration (HFCF-UF) with traditional UF. Our results showed that the ultrafiltrate volume was changed from 40 to 400 μL with the increase of centrifugation time for the traditional UF, and the related changes in unbound concentration were significant. The rate of protein binding (BP) was changed from 40% to 70%. In contrast, a tiny and invariant ultrafiltrate yield (40 μL) was obtained using the HFCF-UF method, and the BP rate was around 72%. In addition, with the HFCF-UF method, the volume ratio of ultrafiltrate to sample solution could be also well controlled by the inner diameters of both the glass tube and hollow fiber. The HFCF-UF method was a more accurate plasma pretreatment procedure, by which the in vivo balance of protein-binding equilibrium was hardly broken. Therefore, this method was successfully employed to quantify the free fraction of carbamazepine in clinical samples.

Pretreatment of plasma samples by a novel hollow fiber centrifugal ultrafiltrate device for the determination of cefaclor concentrations in human plasma.

A simple sample preparation method was developed by using a centrifugal ultrafiltration (CF-UF) device with hollow fiber (HF) for the determination of cefaclor in plasma by HPLC. Samples were placed into a homemade device, which was consisted of a glass tube and a U-shaped hollow fiber. The filtrate was withdrawn from the hollow fiber into a syringe after centrifugation and 20 μL was directly injected into the HPLC for analysis. The HPLC method had a linear calibration curve in the concentration range of 6.00×10(-2)-30.7 μg mL(-1)(r=0.9996). The limit of detection (LOD) and limit of quantitation (LOQ) were 0.02 and 0.06 μg mL(-1), respectively. The intra and inter-day precisions (RSD) were 1.7%, 1.2%, 1.0% and 3.6%, 2.5%, 1.9%, respectively, for three concentrations. Assay accuracy was higher than 99.2% and the absolute recovery was 86.8-92.5%. It is feasible to use this novel and low cost device for sample pretreatment for the analysis of cefaclor in plasma.

Accuracy assessment on the analysis of unbound drug in plasma by comparing traditional centrifugal ultrafiltration with hollow fiber centrifugal ultrafiltration and application in pharmacokinetic study

In present study, accuracy assessment on the analysis of unbound drug in plasma was made by comparing traditional centrifugal ultrafiltration (CF-UF) with hollow fiber centrifugal ultrafiltration (HFCF-UF). We used metformin (MET) as a model drug and studied the influence of centrifugal time, plasma condition and freeze-thaw circle times on the ultrafiltrate volume and related effect on the measurement of MET. Our results demonstrated that ultrafiltrate volume was a crucial factor which influenced measurement accuracy of unbound drug in plasma. For traditional CF-UF, the ultrafiltrate volume cannot be well-controlled due to a series of factors. Compared with traditional CF-UF, the ultrafiltrate volume by HFCF-UF can be easily controlled by the inner capacity of the U-shaped hollow fiber inserted into the sample under enough centrifugal force and centrifugal time, which contributes to a more accurate measurement. Moreover, the developed HFCF-UF method achieved a successful application in real plasma samples and exhibited several advantages including high precision, extremely low detection limit and perfect recovery. The HFCF-UF method offers the advantage of highly satisfactory performance in addition to being simple and fast in pretreatment, with these characteristics being consistent with the practicability requirements in current scientific research.

The influence of volume ratio of ultrafiltrate of sample on the analysis of non-protein binding drugs in human plasma

In human plasma, the total concentration of non-protein binding (NPB) drugs is equal to the free drug concentration because NPB drugs do not or hardly bind to plasma proteins. Thus, centrifuge ultrafiltration (CF-UF) has been used in the determination of the concentration of NPB drugs in human plasma. However, with only a common centrifugation, the recovery and the reproducibility were not as excellent as expected. In addition, we discovered that the values of the volume ratio of ultrafiltrate to sample solution (Vu/Vs) were different and could not be well controlled, which may affect the determination of the drug concentration. The problem also affected the determination of other NBP drugs. In the present work, we used biapenem as a representative drug to study the effect of Vu/Vs on the analysis of NPB drugs concentration in human plasma. The results showed that a Vu/Vs value of less than 0.4 had no effect on the analysis of free drug concentration, while a Vu/Vs value of more than 0.4 was associated with increased recovery rate and overestimation of drug concentration. Therefore, to maintain a Vu/Vs value of less than 0.4 and even at a constant value is the key to accurately determine the concentration of NPB drugs in plasma. Fortunately, with an HFCF-UF device, the Vu/Vs could be well controlled and kept at 0.08 in this study. The recovery rates were almost 100% and the analysis precision was greatly improved. In pharmacokinetics studies, this method was successfully employed to determine the concentration of biapenem with excellent accuracy and reproducibility. HFCF-UF may become a feasible platform for the determination of NPB drugs.

Accuracy of the analysis of free vancomycin concentration by ultrafiltration in various disease states

In the present study, the accuracy of the analysis of free vancomycin (VCM) by ultrafiltration in various disease conditions was assessed. With VCM as a representative drug, we used clinical plasma samples to investigate the effect of plasma conditions on the volume ratio of ultrafiltrate to sample solution (Vu/Vs) and the consequential effect on the measured free drug concentration (fc). Our results demonstrated that plasma conditions had a significant impact on Vu/Vs by centrifugal ultrafiltration (CF-UF). The Vu by CF-UF ranged from 97 μL to 279 μL among different individuals under the same centrifugation conditions. Total protein levels and the osmotic pressure of plasma were the main influence factors of Vu/Vs in disease states. In contrast, the Vu/Vs by hollow fiber centrifugal ultrafiltration (HFCF-UF) was less influenced by plasma conditions. As a consequence, the results of fc determined by HFCF-UF were more accurate than those by CF-UF for patients with different disease states. HFCF-UF displayed great advantages in clinical samples for accurate analysis of fc. It has been successfully applied to monitor free VCM in clinical plasma samples in routine therapeutic drug monitoring (TDM).

A Simple Sample Preparation Method for Measuring Amoxicillin in Human Plasma by Hollow Fiber Centrifugal Ultrafiltration

A simple sample preparation method has been developed for the determination of amoxicillin in human plasma by hollow fiber centrifugal ultrafiltration (HF-CF-UF). A 400-μL plasma sample was placed directly into the HF-CF-UF device, which consisited of a slim glass tube and a U-shaped hollow fiber. After centrifugation at 1.25 × 10(3) g for 10 min, the filtrate was withdrawn from the hollow fiber and 20 µL was directly injected into the high-performance liquid chromatography (HPLC) for analysis. The calibration curve was linear over the range of 0.1-20 µg/mL (r = 0.9996) and the limit of detection was as low as 0.025 µg/mL. The average recovery and absolute recovery were 99.9% and 84.5%, respectively. Both the intra-day and inter-day precisions (relative standard deviation) were less than 3.1% for three concentrations (0.25, 2.5 and 10 µg/mL). The sample preparation process was simplified. Only after a single centrifugal ultrafiltration can the filtrate be injected directly into HPLC. The present method is simple, sensitive and accurate. It could be effective for the analysis of biological samples with high protein contents, especially for the biopharmaceutical analysis of drugs that use traditional isolation techniques for sample preparation such as the protein precipitation method.

Hollow fiber-protected liquid-phase microextraction followed by high performance liquid chromatography for simultaneously screening multiple trace level β-blockers in environmental water samples

A sensitive and reliable method using a two-phase hollow fiber liquid-phase microextraction (HF-LPME) pretreatment technique was developed for simultaneously screening multiple β-blockers in environmental samples. In this study, heptanol was chosen as the optimal extraction solvent, and six β-blockers with a wide range of polarities were extracted across the porous walls of hollow fibers and into the acceptor phase (AP). The fundamental parameters affecting the extraction efficiency (EE) of analytes including extraction time, temperature, pH of the donor phase (DP) and AP, stirring speed, volume of the sample solution, ionic strength and the type of hollow fiber membrane were studied and optimized. Under optimal conditions, extracts were analyzed by HPLC with ultraviolet detection (UV). Finally, satisfactory results were obtained, good linearity was observed for all β-blockers in the range of 0.16–200 ng mL−1, limits of detection (LODs) were between 0.08 and 0.5 ng mL−1, the intra- and inter-day precision values of the six β-blockers were 1.0–2.2% and 1.4–2.7%, respectively. The recovery with RSDs was less than 2.2% for the pretreatment method. The proposed technique was successfully applied for screening multiple β-blockers in complex aqueous samples with the best specificity and provided a simple and reliable new means for the self-checking of laboratories at the basic level.

The Influence of Plasma Albumin Concentration on the Analysis Methodology of Free Valproic Acid by Ultrafiltration and Its Application to Therapeutic Drug Monitoring.

Background: Free drug analysis is increasingly becoming popular in therapeutic drug monitoring (TDM). Centrifugal ultrafiltration (CF-UF) is the primary method to separate free drug from that of bound drug. However, the volume ratio of ultrafiltrate to sample solution (Vu/Vs) affects the accuracy of CF-UF, which highly depends on the different plasma conditions. Plasma protein concentrations in patients are different from those observed in healthy subjects, and there are also significant differences among patients with different diseases. Only very few studies have reported on the effect of protein concentration on the analysis methodology of free drug by CF-UF. Methods: In this study, valproic acid was used as the representative drug, and plasma samples with different albumin concentrations were analyzed by CF-UF and hollow fiber centrifugal ultrafiltration (HFCF-UF). Results: There was no significant difference of free drug concentrations by HFCF-UF and CF-UF when plasma albumin concentrations ranged 40–60 g/L. However, at low albumin concentrations (<40 g/L), a considerable difference was detected, and the difference was increased with the decrease of plasma albumin concentration. When the albumin concentration was as low as 10 g/L, the free drug concentration was 17.3 mcg/mL by CF-UF, whereas it was 10.2 mcg/mL by HFCF-UF. Conclusions: The accuracy of free drug measurement by CF-UF was albumin concentration dependent. However, such an effect was not observed when samples were prepared by HFCF-UF, which was more suitable for TDM of plasma samples from different patients. Therefore, this method could be readily applied to the measurement of free valproic acid plasma concentrations for TDM in patients.

A HFCF-UF method to directly analyze the total concentration of protein-binding drugs

In the present study, a direct injection hollow fiber centrifugal ultrafiltration (HFCF-UF) method was developed for therapeutic drug monitoring (TDM) for the total concentration of liposoluble protein binding drugs. We used carbamazepine (CBZ) and phenobarbital (PB) as model drugs and proposed a HFCF-UF method to analyze the total concentration of liposoluble protein binding drugs without the use of traditional biological sample preparations. Our results demonstrate that acetone acts as a liposoluble desorption agent and can make the target compounds release from the protein–drug complexes and then re-dissolve the released compounds. The absolute recovery is excellent at approximately 100%, which is higher than when traditional biological sample preparations have to be used. Calibration curves can be established in a plasma-free standard solution instead of using spiked human plasma. The HFCF-UF method offers the advantages of highly satisfactory performance and simple and fast pretreatment, consistent with the practical requirements of TDM.