Drugs In Human Serum Research Articles

Sensitive Analysis of Metoprolol Tartrate and Diltiazem Hydrochloride in Human Serum by Capillary Zone Electrophoresis Combining on Column Field-Amplified Sample Injection.

A sensitive capillary zone electrophoresis (CZE) combined with field-amplified sample injection (FASI) method was investigated to simultaneously determine diltiazem (DI) hydrochloride and metoprolol (ME) tartrate in human serum. This method was validated by linearity, limits of detection (LOD), stability, precision and accuracy, and the related parameters are linearity (r2>0.9980), intra- and inter-day precisions (intra-day relative standard deviation (RSD) is 2.3-3.4%, and inter-day RSD is 3.8-7.5%) and the LOD (0.02ng/mL for ME tartrate and 0.01ng/mL for DI hydrochloride). So the proposed method is rapid, sensitive and accurate for determination of these two anti-anginal drugs in human serum. As for enrichment conditions, it was helpful to add phosphoric acid and isopropanol into samples for enrichment efficiency. A model was established to illustrate the possible enrichment mechanism of analytes, and a theory of half-width was also applied in CZE combined with FASI method to evaluate the peak performance.

Novel potentiometric methods for the estimation of bisoprolol and alverine in pharmaceutical forms and human serum

AbstractTwo new potentiometric sensors were created for the quantification of bisoprolol fumarate and alverine citrate in bulk pharmaceutical dosage forms and human serum. Bisoprolol and alverine sensors were manufactured by combining potassium tetrakis (p-chlorophenyl) borate ion pairs to serve as electroactive substances, plasticized poly (vinyl chloride) matrix membranes, ando-nitrophenyl octyl ether. They demonstrated high responses over the concentration ranges of 1.0×10−6to 1.0×10−2mol L−1bisoprolol and alverine with close to Nernstian cationic slopes of 52 and 56 mV decade−1, respectively. The detection limits for bisoprolol and alverine were 2.6×10−6and 1.75×10−6mol L−1, respectively. For both medications, the response time was instantaneous (2.0 s). The working pH ranges for bisoprolol and alverine were 4.50–8.50 and 2.00–8.80, respectively. For both sensors, the life cycle was long (3 months). The sensors were used in pharmaceutical dosage types for the assay of bisoprolol and alverine, recording average recoveries of 99.40% and 99.98% respectively and were also successfully used for estimating the two drugs in human serum with an average recovery of 99.60% for both drugs. For all multiple staged interfering materials, the reported latest potentiometric sensor methods displayed high selectivity. The current sensor obtained a high percentage recovery and an excellent relative standard deviation compared with those obtained from previously published methods.

Pipette-tip micro-solid phase extraction based on melamine-foam@polydopamine followed by ultra-high-performance liquid chromatography–quadrupole–time-of-flight mass spectrometry for detection of psychotropic drugs in human serum

In this work, pipette-tip micro-solid phase extraction (PT-μSPE) which packed by melamine-foam@polydopamine (MF@PDA) coupled with ultra-high-performance liquid chromatography–quadrupole–time-of-flight mass spectrometry (UHPLC-QTOF) was developed for extraction and determination of psychotropic drugs in serum samples. Considering the operation back pressure, the melamine-foam as carrier material with 3D cross-linked grid structure can provide high permeability and contact surface. MF@PDA was prepared by self-polymerization reaction of dopamine under weak alkaline conditions and characterized by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX) and X-ray photoelectron spectroscopy (XPS). The surface group of PDA containing catechol structure, quinone structure and amine group has multi-interaction with psychotropic drugs which can increase the adsorption capacity. Moreover, the parameters affecting extraction efficiencies such as extraction and desorption cycle, pH value, eluent type, ionic strength and amount of sorbent were investigated. Based on the high sensitivity and accuracy mass measurement by TOF/MS, under the optimized extraction condition, the limits of detection (LOD) of this method were obtained in the range of 0.002–0.1 ng ml−1. The linearity was ranged from 0.01 ng ml−1 to 600 ng ml−1, and all the correlation coefficients (R2) were above 0.993. The spiked recoveries were in the range of 80.04% to 109.18% in real sample test and RSD values obtained from 0.95% to 9.85%. The results demonstrate that MF@PDA-PT-μSPE-UHPLC-QTOF is a sample and reliable method for the detection of psychotropic drugs in serum sample.

Restricted access supramolecular solvent based magnetic solvent bar liquid-phase microextraction for determination of non-steroidal anti-inflammatory drugs in human serum coupled with high performance liquid chromatography-tandem mass spectrometry

A hexafluroisopropanol (HFIP)-alkanol supramolecular solvent (SUPRAS) based magnetic solvent bar (MSB) liquid-phase microextraction (LPME) method was proposed for extraction of non-steroidal anti-inflammatory drugs (NSAIDs, including ketoprofen, naproxen, indomethacin and diclofenac) in human serum. The restricted access HFIP-alkanol SUPRAS was prepared by injecting a mixture of HFIP and alkanol into water. A stainless-steel needle was inserted into a piece of hollow fiber to prepare a magnetic bar. Then the magnetic bar was dipped in SUPRAS to impregnate the wall pores of the hollow fiber, followed by placing it into the serum sample for extraction. Only 4 μL of SUPRAS was consumed per bar. The MSB not only functioned for stirring, but also played the role of extraction and magnetic separation. Under the optimal extraction conditions (seven MSBs, extraction time 33 min and stirring rate 730 rpm), which was obtained by one variable-at-a-time and response surface methodology, the novel MSB-LPME was coupled with high performance liquid chromatography-tandem mass spectrometry to determine NSAIDs in human serum. The method showed a good linear relationship (correlation coefficients ≥ 0.9939). Method limits of detection and method limits of quantitation were in the range of 0.25-0.95 μg L-1 and 0.83-3.16 μg L-1, respectively. The recoveries for the spiked human serum samples ranged from 86.8% to 125.1% with intra- and inter-day relative standard deviations less than 9.2% and 18.1%, respectively. Moreover, the method did not require a protein precipitation step, and matrix effects of 72.8%-117.7% showed little interference with mass spectrometry detection, which was due to the double cleanup provided by the restricted access property of SUPRAS and the filtration capacity of hollow fiber. The HFIP-alkanol SUPRAS-based MSB-LPME method proved to be simple, highly efficient and environment-friendly for the pretreatment of serum/plasma.

Targeted Strategy to Analyze Antiepileptic Drugs in Human Serum by LC-MS/MS and LC-Ion Mobility-MS.

Routine small-molecule analysis is challenging owing to the need for high selectivity and/or low limits of quantification. This work reports a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify 14 antiepileptic drugs (AEDs) in human serum. For the optimized LC-MS/MS method described herein, we applied the guidelines outlined in the Clinical and Laboratory Standards Institute (CLSI) LC-MS C62-A document and the U.S. Food and Drug Administration (FDA) Bioanalytical Method Validation Guidance for Industry to evaluate the quality of the assay. In these studies, AED linearity, analyte recovery, matrix effects, precision, and accuracy were assessed. Using liquid chromatography-drift tube ion mobility-mass spectrometry (LC-DTIM-MS), a qualitative method was also used to increase confidence in AED identification using accurate mass and collision cross section (CCS) measurements. The LC-DTIM-MS method was also used to assess the ability of drift tube CCS measurements to aid in the separation and identification of AED structural isomers and other AEDs. These data show that another dimension of information, namely CCS measurements, provides an orthogonal dimension of structural information needed for AED analysis. Multiplexed AED measurements using LC-MS/MS and LC-DTIM-MS have the potential to enable better optimization of dosing owing to the high precision capabilities available in these types of analytical studies. Taken together, these data also show the ability to increase confidence in small-molecule identification and quantification using these analytical technologies.

Simultaneous Determination of Hydrochlorothiazide and Irbesartan from Pharmaceutical Dosage Forms with RP-HPLC.

In this work, a simple and rapid liquid chromatographic method for the simultaneous determination of irbesartan (IRBE) and hydrochlorothiazide (HCT) was developed and validated by reverse phase high performance liquid chromatography (RP-HPLC). Experimental conditions such as different buffer solutions, various pH values, temperature, composition of the mobile phase, and the effect of flow rate were optimized. The developed RP-HPLC method for these antihypertensive agents was wholly validated and IRBE was detected in the linear range of 0.1-25 μg mL-1 and HCT was detected in the linear range of 0.25-25 μg mL-1. Moreover, the suggested chromatographic technique was successfully applied for the determination of the drugs in human serum and pharmaceutical dosage forms with limit of detection values of 0.008 μg mL-1 for IRBE and 0.012 μg mL-1 for HCT. The proposed rapid analysis method of these antihypertensive drugs can be easily used and applied by pharmaceutical companies for which the analysis time is important.

Direct analysis in real time-mass spectrometry for rapid quantification of five anti-arrhythmic drugs in human serum: application to therapeutic drug monitoring

Therapeutic drug monitoring (TDM) is necessary for the optimal administration of anti-arrhythmic drugs in the treatment of heart arrhythmia. The present study aimed to develop and validate a direct analysis in real time tandem mass spectrometry (DART–MS/MS) method for the rapid and simultaneous determination of five anti-arrhythmic drugs (metoprolol, diltiazem, amiodarone, propafenone, and verapamil) and one metabolite (5-hydroxy(OH)-propafenone) in human serum. After the addition of isotope-labeled internal standards and protein precipitation with acetonitrile, anti-arrhythmic drugs were ionized by DART in positive mode followed by multiple reaction monitoring (MRM) detection. The use of DART–MS/MS avoided the need for chromatographic separation and allowed rapid and ultrahigh throughput analysis of anti-arrhythmic drugs in a total run time of 30 s per sample. The DART–MS/MS method yielded satisfactory linearity (R2 ≥ 0.9906), accuracy (86.1–109.9%), and precision (≤ 14.3%) with minimal effect of biological matrixes. The method was successfully applied to analyzing 30 clinical TDM samples. The relative error (RE) of the concentrations obtained by DART–MS/MS and liquid chromatography-tandem mass spectrometry (LC–MS/MS) was within ± 13%. This work highlights the potential usefulness of DART for the rapid quantitative analysis of anti-arrhythmic drugs in human serum and gives rapid feedback in the clinical TDM practices.

Development and Application of a LC-MS/MS Method for Simultaneous Quantification of Four First-Line Antituberculosis Drugs in Human Serum.

A simple, rapid, and sensitive liquid chromatography (LC)/mass spectrometry (MS) method was established and validated for simultaneous quantitation of pyrazinamide, isoniazid, rifampicin, and ethambutol in human blood sample. Samples were pretreated by a single-step precipitation with acetonitrile. Chromatographic separation was achieved on XSelecT HSS T3 column by gradient elution with a total run time of 5.0 min. MS detection was performed by a triple quadrupole tandem mass spectrometer in the multiple reaction monitoring mode with a positive electrospray ionization source. Isotope-labeled internal standard, especially rifampicin-D8, was applied to adjust for the loss during sample treatment. The established LC-MS/MS method showed a wide analytical range (pyrazinamide: 1.02∼60.0 μg/mL, isoniazid: 0.152∼10.0 μg/mL, rifampicin: 0.500∼30.0 μg/mL, and ethambutol: 0.0998∼5.99 μg/mL) and a good linearity (r > 0.99 for the four analytes) with acceptable accuracy and precision (90.15%∼104.62% and 94.00%∼104.02% for intra- and interaccuracy, respectively; RSD%: <12.46% and <6.43% for intra- and interprecision, respectively). It also showed excellent recoveries (79.24%∼94.16% for all analytes) and absence of significant matrix effect. This method was successfully applied to the quantification of four first-line antituberculosis (anti-TB) drugs, suggesting its suitability for therapeutic drug monitoring in the clinical practices.

How versatile is the use of ultrafiltration to study biointeractions of therapeutic metallodrugs?

For a representative number of approved or investigational anticancer metallodrugs varying in lipophilicity, unspecific adsorption onto ultracentrifugal filter units was studied. It was found that for fairly hydrophilic compounds, such as cisplatin and oxaliplatin, the binding to filters does not substantially affect their amount measured (by ICP-MS) after ultrafiltration (>95%). In the case of metal complexes with moderate lipophilicity (log P > −0.1), adsorption effects turn out to be substantial. This might impede using ultrafiltration for studying the transformations of such drugs in human serum, unless they are rapidly converted into the protein adducts. The adsorption-suppressing effect of proteins was proved for indazolium trans-[tetrachloridobis(1H-indazole)ruthenate(III)] whose recovery from the filters was 61 and 14% in free and HSA-bound form, respectively.

Combination of gel-electromembrane extraction with switchable hydrophilicity solvent-based homogeneous liquid-liquid microextraction followed by gas chromatography for the extraction and determination of antidepressants in human serum, breast milk and wastewater

The current study presents for the first time a combination of the gel electromembrane extraction (GEL-EME) and switchable hydrophilicity solvent-based homogeneous liquid-liquid microextraction (SHS-HLLME) methods which can be used as an efficient hyphenated extraction procedure. This coupled method, which was followed by GC-FID, was applied for quantification of antidepressants (desipramine, clozapine, and citalopram) in biological and wastewater samples. The effective parameters of both GEL-EME and SHS-HLLME procedures were optimized. Using an agarose gel membrane, analytes were extracted from 7.0 mL of the sample solution to 500 µL of the aqueous acceptor solution. The maximum extraction of analytes of interest was obtained under the optimized conditions (pH of acceptor solution, 5.0; pH of gel membrane, 5.0; pH of sample solution, 7.0, voltage value, 30 V; and extraction time, 30 min). Then, the acceptor solution was transferred to the extraction cell and the SHS-HLLME procedure was conducted again under the optimized conditions. Dipropylamine (50 µL) was selected as an extraction solvent. The introduced technique exhibited good linearities with coefficients of determinatin (R2) higher than 0.983 and an acceptable linear range of 5.0–1000 ng/mL. Accordingly, the limit of detection was ≤ 1.0 ng/mL (S/N = 3) for all analytes, and the high enrichment factors were obtained in the range of 178.7–194.8. Moreover, the corresponding repeatability was from 4.0 to 8.7% (n = 3). The proposed method was successfully utilized to determine trace levels of the drugs in human serum, wastewater, and breast milk samples.

Blue and green emission-transformed fluorescent copolymer: Specific detection of levodopa of anti-Parkinson drug in human serum

Levodopa, commonly used anti-Parkinson drugs in the clinic, is the most significant prodrug of dopamine that plays important roles in the treatment of Parkinson's disease. Therefore, monitoring content of levodopa of anti-parkinson drugs in human serum is extremely necessary. Herein, a simple, fast and low-cost method for levodopa detection is proposed depending on the in situ formation of blue and green emission fluorescent copolymer (FCP). The proposed method is based on the conversion of fluorescence emission peak of FCP from blue (430 nm) to green emission (535 nm) in 2 h. In this sensing system, both blue and green emission exhibit a high selectivity and sensitivity for levodopa determination in the range from 0 to 50 μM with a detection limit of 0.2 μM and 0.36 μM, respectively. Among them green emission FCP shows excellent recovery even at human serum concentrations up to 30%. Additionally, the proposed method was successfully applied to assess the content of levodopa in three anti-Parkinson drugs (carbidopa and levodopa CR tablets, levodopa and benserazide hydrochloride tablets, and levodopa tablets). More importantly, the levodopa determination of three anti-Parkinson drugs in human serum also exhibit an excellent recovery. Therefore, our strategy provides a promising method for mechanism study and treatment of Parkinson's disease.

Application of mobile phases containing ionic liquid for the analysis of selected psychotropic drugs by HPLC–DAD and HPLC–MS

In reversed-phase liquid chromatography, in case of the absence of additives, cationic basic compounds give rise to asymmetrical and broad peaks as a result of interactions of analyte cations with residual free silanols on silica-based stationary phases. Ionic liquids, added to the mobile phase, have been suggested as alternatives to amines to block the activity of free silanols. The different parameters affecting the retention behavior, symmetry of peak, system efficiency, and separation selectivity of selected psychotropic drugs, especially the effect of concentration of ionic liquid, kind and concentration of organic modifiers of mobile phases, and kind of stationary phases were investigated. The most selective and efficient systems are used for separations of psychotropic drug standards' mixture and for determination of selected psychotropic drugs in human serum.

Green and cost-effective synthesis of carbon dots from date kernel and their application as a novel switchable fluorescence probe for sensitive assay of Zoledronic acid drug in human serum and cellular imaging

A new label-free, sensitive and selective off and on signaling fluorescence platform for assay of trace levels of Zoledronic acid (ZA) drug in human biological samples based on nitrogen doped carbon dots (N-CDs) - ferric ions (Fe3+) was designed. The fluorescence probe, N-CDs, was synthesized for the first time through a facile, eco-friendly and one-step hydrothermal treatment using date kernel as the precursor without any need to use chemical reagents. These CDs exhibited excellent water solubility, ionic and photo stability in various circumstances and a highly relative quantum yield of 12.5%. In the presence of Fe3+, the fluorescence intensity (FL) for N-CDs was strongly quenched due to the interaction between ferric ions and the functional groups at the N-CDs (switch off). Afterwards, by the addition of ZA, the fluorescence sensor status turned to “ON” (switch on) due to the dominance of ZA in the competition between functional groups on the surface of N-CDs and phosphate groups in ZA in the interaction with Fe3+ results in removing Fe3+ from the surface of N-CDs. Under the optimized conditions, the proposed fluorescence probe (N-CDs-Fe3+) exhibited good sensing performance for ZA assay with a linearity from 0.1 μM to 10.0 μM, a detection limit of 0.04 μM and the precision of 2.70%. The developed N-CDs-Fe3+ sensor was successfully used for the assay of ZA contents with good recoveries and selectivity in human serum samples. Meanwhile, the in vitro cytotoxic activity and cellular uptake of N-CDs were investigated on human osteosarcoma (MG-63) cell line.

Ternary mixed-mode silica sorbent of solid-phase extraction for determination of basic, neutral and acidic drugs in human serum.

In this study, a ternary mixed-mode silica sorbent (TMSS) with octamethylene, carboxyl, and amino groups was prepared via Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click reaction and a subsequent reduction of azide to primary amine. While used in solid-phase extraction (SPE), the retention behavior of TMSS towards a total of nine kinds of basic, neutral, and acidic drugs was investigated in detail. The results revealed that hydrophobic, ion-exchange interaction, and electrostatic repulsion between TMSS and the analytes were closely related to the retention behavior of TMSS. Besides, the log Kow value of the analyte was also a factor influencing the retention behavior of analytes on TMSS. The nine analytes could be retained by TMSS simultaneously and then, were eluted into two fractions according to the acid-base property of the analytes for further determinations. The acidic and neutral analytes were in one fraction, and the basic ones in the other fraction. When used to treat the human serum spiked with the nine drugs, TMSS offered higher recoveries than BakerBond CBA and comparable recoveries to Oasis WCX. It should be noted TMSS had better purifying capability for human serum than Oasis WCX. Under the optimized SPE conditions, a method of SPE hyphenated to high-performance liquid chromatography-ultraviolet detection (HPLC-UV) for determination of the basic, neutral, and acidic drugs spiked in human serum was established. For the nine drugs, the linear ranges were all between 5.0 and 1000μgL-1 with correlation coefficients (R2) above 0.9990, and the limits of detection (LODs) were in the range of 0.8-2.3μgL-1. The intra-day and inter-day relative standard deviations (RSDs) were less than 5.3 and 8.8%, respectively. Graphical abstract Treating drugs in human serum by SPE with ternary mixed-mode silica sorbent.

Development and validation of a new HPLC-DAD method for quantification of sofosbuvir in human serum and its comparison with LC-MS/MS technique: Application to a bioequivalence study.

Although for many analyses tandem mass spectrometry (LC-MS/MS) systems have significant advantage over the high-performance liquid chromatography with diode array detection (HPLC-DAD) however, the HPLC methods are easier, cheaper and more available to perform. As no published method is available for quantitative HPLC analysis of sofosbuvir (SOF), an orally administered anti-hepatitis drug in human serum, this study was aimed to evaluate applicability of the HPLC technique to quantify sofosbuvir and comparison of the two methods for analytical performance. Following extraction of the drug and an internal standard (Hexobarbital), same chromatographic conditions were used for both the systems. After the chromatographic separation on a reverse phase C18 column using a mobile phase consisting of water (containing formic acid 0.5mL/L) and acetonitrile (57:43; v/v) at a flow rate of 0.8mL/min, the eluate was introduced into a DAD detector set at 261nm, then passed through the mass spectrometry system in single ion monitoring mode (SIM). For UV and mass spectrometry detections the calibration curves were linear over a concentration range of 25-3200 and 10-3200ng/mL, respectively and the linearity was over 0.998 for both the systems. Lower limit of quantification (LLOQ) for mass spectrometry and DAD detections were 10 and 25ng/mL, respectively. In conclusion sensitivity of DAD detection is sufficient enough to determine concentrations down to 0.5% of Cmax which achieved in bioequivalence study of sofosbuvir and meet FDA requirements for these types of studies.

A simple microdroplet chip consisting of silica nanochannel-assisted electrode and paper cover for highly sensitive electrochemiluminescent detection of drugs in human serum

This paper describes a simple and miniaturized microdroplet chip (μchip) that is constructed with a silica nanochannel (SNC)-assisted electrode array and a hydrophobic paper cover (SNC&P-μchip). Vertically aligned SNCs with uniform pore size of 2–3 nm in diameter and negatively charged surface can significantly accelerate the mass transport of the positively charged tris(2,2'-bipyridyl) ruthenium (II), resulting in a remarkably enhanced electrochemiluminescence (ECL) signal. The SNC-assisted electrode array was coupled to a low cost paper cover to achieve simultaneous detection of six samples in 1 min. The feasibility and universality of the SNC&P-μchip was evaluated by detecting a series of alkaloidal drugs both in buffers and in human serum. The performance of the SNC&P-μchip was fully validated with respect to linearity (0.9999 > R > 0.9939), sensitivity (limits of detection from 1.799 nM to 11.43 nM), and accuracy (recovery rate between 94.38% and 109.12%). The facile and economic SNC&P-μchip shows promising potential for rapid drug detection in complex biofluids.

Aptamer-based Field-Effect Biosensor for Tenofovir Detection

During medical treatment it is critical to maintain the circulatory concentration of drugs within their therapeutic range. A novel biosensor is presented in this work to address the lack of a reliable point-of-care drug monitoring system in the market. The biosensor incorporates high selectivity and sensitivity by integrating aptamers as the recognition element and field-effect transistors as the signal transducer. The drug tenofovir was used as a model small molecule. The biointerface of the sensor is a binary self-assembled monolayer of specific thiolated aptamer and 6-mercapto-1-hexanol (MCH), whose ratio was optimized by electrochemical impedance spectroscopy measurements to enhance the sensitivity towards the specific target. Surface plasmon resonance, performed under different buffer conditions, shows optimum specific and little non-specific binding in phosphate buffered saline. The dose-response behavior of the field-effect biosensor presents a linear range between 1 nM and 100 nM of tenofovir and a limit of detection of 1.2 nM. Two non-specific drugs and one non-specific aptamer, tested as stringent control candidates, caused negligible responses. The applications were successfully extended to the detection of the drug in human serum. As demonstrated by impedance measurements, the aptamer-based sensors can be used for real-time drug monitoring.

Separation and determination of selected psychotropic drugs in human serum by SPE/HPLC/DAD on C18 and Polar-RP columns

ABSTRACTA simple and sensitive high-performance liquid chromatographic method with diode array detection is described for the quantification of some psychotropic drugs: fluoxetine, sertraline, alprazolam, perphenazine, zolpidem, and hydroxyzine in fortified human serum samples. The test compounds were extracted from human serum by solid-phase extraction using C18 extraction column and injected into C18 or Polar-RP analytical columns for separation. A mobile phase was composed of methanol, water, acetate buffer at pH 3.5, and diethylamine. The method was validated for the concentration range 0.4–5 µg mL−1 for zolpidem and 0.5–6 µg mL−1 for other drugs. Mean recoveries were from 87.79% to 107.94% with adequate precision (% RSD ≤2.1%). The full separation of all investigated drugs, good peaks’ symmetry, and simultaneously high system efficiency were obtained on Polar-RP column, which was used for the first time to analyze these drugs. System efficiency obtained on the column was significantly higher compared to that obtained on commonly used C18 column. The method seems to be suitable for the analysis of investigated drugs in human plasma for psychiatric patients in multiple drug overdoses as well as for control of pharmacotherapy, particularly in combination therapy.

The use of a temperature-responsive column for the direct analysis of drugs in serum by two-dimensional heart-cutting liquid chromatography.

A novel pretreatment method, which was performed using a two-dimensional high-performance liquid chromatography (2D-HPLC) system, was proposed for the direct analysis of drugs in human serum. A temperature-responsive column was used as a pretreatment column. The stationary phase of the temperature-responsive column exhibits temperature-regulated hydrophilic/hydrophobic characteristics. Controlling the ionic strength of the eluent enables human serum albumin (HSA) to pass through the column without retention. When serum samples containing barbiturates or benzodiazepines were injected into the temperature-responsive column using 10mM of ammonium acetate (pH 6.5) as the mobile phase and in the temperature range of 10-40°C, HSA was eluted from the column near the dead time, followed by the individual drugs. When the column temperature was changed, the retention times of the drugs were altered owing to surface property changes within the pretreatment column. These closely eluted compounds were subsequently introduced into the analytical column using a column-switching valve, with a minimal gap time to avoid foreign substance contamination. This new 2D-HPLC method afforded high-quality chromatograms of multiple drugs without unwanted peaks from foreign substances. The present technique could be an attractive choice in selecting the analytical method for drug analysis.

Determination of binding properties of ampicillin in drug-human serum albumin standard solution using N-vinylpyrrolidone copolymer combined with the micellar systems

It is well-known that only the unbound (free) drug fraction can achieve a pharmacological effect. Therefore the determination of free drug concentration is a very important issue in the field of pharmacology. In this study poly-1-vinyl-2-pyrrolidone (VP) crosslinked with divinylbenzene (DVB) compared with the micellar liquid chromatography (MLC) with and without pre-made drug adsorption was used for quantitative analysis of free ampicillin concentration in the standard solution of drug-human serum albumin owing to its ability to block protein adsorption.The commonly recognized adsorption method based on drug adsorption on VP-DVB has been compared to the entirely new application of MLC with direct sample injection (DSI) not requiring pre-made adsorption. Micellar aggregates are able to solubilize various compounds therefore micellar environment can be used for direct determination of free drug concentration.The obtained results show that the free drug concentration values obtained in the micellar systems based on cetyltrimethylammonium bromide (CTAB) (93.98μgL−1, 78.3%) as well as on polyoxyethylene (23) lauryl ether (Brij35) (91.15μgL−1, 75.9%) are similar to those obtained after the drug adsorption on VP-DVB using both RP-HPLC (95.85μgmL−1, 79.9%) and spectrophotometry (96.47μgmL−1, 80.4%). However, only %PPB (% plasma protein binding) value calculated on the basis of Brij35 retention factor is similar to the literature data. The obtained results are within the analytical range of % of free drug concentration. Therefore N-vinylpyrrolidone copolymer as well as micellar system based on the non-ionic surfactant can be successfully applied for determination of free drug concentration. Moreover, the new application of MLC with DSI can be recognized as a promising, fast and simple method for quantitative determination of free drug concentration.