The Theoretical Study And Clinical Application Of Free Drug Analytical Method In Human Plasma

Drugs are bound to plasma proteins in different degrees and only the freedrug (FD) fraction which is not bound to plasma protein can reach the activesite and exert pharmacological effect. It is the basically consensus of clinicalpharmacology. But FD evaluation is not applied in clinic widely and we stilluse the total drug (TD) concentration as the index to evaluatepharmacodynamics and guide administration. Thus toxic reaction andtreatment failure have occurred during treatment. One of the important reasonsis that the relationship of FD and patients’ clinical pathological andphysiological condition is not clear. The other important reason is the freedrug analytical method is not consummate. Therefore, developing an accuratemethod for FD analysis which is feasible for clinical analysis is an importanttopic for clinical pharmaceutical researchers and is also a key to make FDevaluation to be used in clinic widely.Centrifugal ultrafiltration (CF-UF) method is simple and fast, which hasbeen used in the analysis of free drug (FD) in human plasma. But theultrafiltrate volume (Vu) is usually large and could not be well controlled. Itwould disturb the initial drug-protein binding equilibrium and suffer from apoor accuracy and precision for the analysis of FD concentration.Unfortunately, there is no report to clearly study the regular of this kind ofinfluence. There is also no report about how to exactly control the Vu.This study use carbamazepine (CBZ) as the model drug, which is anantiepileptic drug and should be conducted therapeutic drug monitoring(TDM). We studied the effect of Vudifferent on the accuracy of free drugmeasurement for protein binding drug with CF-UF. Two concentrations ofCBZ quality control plasma were conducted CF-UF. At the same centrifugalforce (8.0×103g), the different the volume ratio of ultrafiltrate to samplesolution (Vu/Vs) was obtained at different centrifugation times (0.5,2,5,10, 15and20min). Every20μL of ultrafiltrate was used for HPLC analysis. Theresults showed that the Vu/Vschanged from0.08to0.8with the increase ofcentrifugation time for the CF-UF, and the related in crease in unboundconcentration were significant. The rate of protein binding (PB) was changedfrom40%to70%. It could see that this effect is significant and should not beignored. Only when the Vu/Vsis less than0.2, the results is accurate.In order to control the Vu/Vsexactly, we present a hollow fiber centrifugalultrafiltration (HFCF-UF) method. A tiny and invariant Vu/Vsvalue wasobtained and the BP was around72%. We also study the theory that the Vu/Vscould be also well controlled by the inner diameters of both the glass tube andhollow fiber for HFCF-UF. The HFCF-UF method would obtain a moreaccurate and became the reliable plasma pretreatment procedure for free druganalysis.The Vu/Vshave an effect on the analysis of free drug for protein bindingdrug. As for non protein binding drug, the FD concentration was equal to TDconcentration and the drug concentration obtained by CF-UF could used as theTD concentration for pharmacokinetics study. Compared with other TDpreparation technique, CF-UF is simple and easy. The result of methodologyfor CF-UF should be superior to other sample preparation methods for TDconcentration analysis. But the result in reality is not as we expected. There isno report about whether the larger and uncontrolled Vu/Vsaffect the analysisof drug concentration for non protein binding drug. It is a key to accuratelyevaluate drug concentration in human plasma.In present work, we used biapenem (BAN) whose PB is very smaller as arepresentative drug to study the effect theory of Vu/Vson the accurate analysisof non protein binding drugs concentration in human plasma. The low andhigh concentration of BAN quality control plasmas were conducted CF-UF. Atthe same centrifugal force (1.0×104g), the different Vu/Vswas obtained atdifferent centrifugation times (0.2,1,5,10,20,40and60min) and everyultrafiltrate was used to HPLC analysis. The results showed that a Vu/Vsvalueof less than0.4had no effect on the analysis of free drug concentration, while a Vu/Vsvalue of more than0.4was associated with increased recovery rate andoverestimation of drug concentration. Therefore, to maintain a Vu/Vsvalue ofless than0.4and even at a constant value is the key to accurately analyze NPBdrugs concentration in plasma. But the Vu/Vscould not be well controlled withCF-UF. Fortunately, with an HFCF-UF device, the Vu/Vscould be wellcontrolled and kept at0.08in this study. The analysis accuracy and precisionwere greatly improved. The HFCF-UF became the analytical platform for nonprotein binding drug concentration.As for drugs with higher PB, the FD concentration is usually lower andthe effect of Vu/Vson the accuracy of analyzing FD is more significant. It ismore essential to develop an accurate FD analytical method. Antiepilepticdrug-valproic acid (VPA) has a significant inter-individual variation ofpharmacodynamic action and could be observed toxic reaction on thetreatment. Accordingly, monitoring free VPA has been recommended. Butmonitoring total VPA in clinic is still carried out at present. Due to theconcentration of free VPA is low and an extra sample preparation withoutdisturb the initial drug-protein binding equilibrium for the free drug analysis isrequired, the current analytical technology could not realized. It discouragesthe usage of analyzing free VPA in clinical TDM. We develop a HFCF-UFmethod for the analysis of free VPA.0.5mL plasma sample was added toHFCF-UF device and after centrifugation at1.25×103g for15min,40μLultrafiltrate was derivatized with2-Bromo-2-acetonaphthone before HPLCanalysis. The Vu/Vswas tiny and could be exactly controlled. It hardlydisturbed the initial drug-protein binding equilibrium and the results weremore accurate and actual. The detection capacity was enhanced due toderivatization. It was successfully used to monitor free VPA in plasma samplesfrom patients with generalized epilepsy and would be a feasible method forthe analysis of free VPA in clinical TDM.As for the drugs with higher water solubility, the commonly usedpre-treatment methods were extraction method or protein precipitation (PPT)method. These methods were usually needs numerous tedious and time-consuming manipulations. Several-fold precipitation reagents weregenerally added to biological samples to remove proteins. As a result, thedetection capability is greatly reduced. The recovery rate is usually low, justabout60%and even much lower. Therefore, it is becoming more and moreattractive to develop a sample preparation method with minimum steps whichcould avoid the tedious manipulations and improve the accuracy andreproducibility. These higher water solubility drugs were usually with lowerPB. HFCF-UF method could be used for the analysis of drug concentrationand be called direct injection techniques that require no or limited samplepretreatment steps. Amoxicillin (AMO) is a beta-lactam antibiotic for thetreatment of a wide range of bacterial infections. Quantitative analysis of thisdrug in bio-fluids is mainly by PPT for sample preparation. But addingseveral-fold precipitation reagents greatly reduced the detection capability.Furthermore, the reproducibility and accuracy are often low due to theco-precipitation of analyte with the precipitated proteins. Since the PB ofAMO is low (15%), we used AMO as the representative drug of higher watersolubility drugs and a direct injection techniques with HFCF-UF method forthe analysis of drug concentration in human plasma was developed. About400μL plasma sample was placed into the HF-CF-UF device directly whichwas consisted of a slim glass tube and a U-shape hollow fiber. Aftercentrifugation at1.25×103g for15min, the filtrate was withdrawn from thehollow fiber and20μL was directly injected into the HPLC for analysis. Theresults were also compared with PPT method. There is no statistics differencebetween the two methods. The present method is simple, accurate andsensitive. In pharmacokinetics studies, this method was successfully employedto determine the concentration of AMO with excellent accuracy andreproducibility. It presents a simple and accurate direct injection technique forthe analysis of drugs with higher water solubility in human plasma.