Fabric phase sorptive extraction followed by HPLC-PDA detection for the monitoring of pirimicarb and fenitrothion pesticide residues.

A sensitive and readily deployable analytical method has been reported for the simultaneous analysis of pirimicarb (PRM) and fenitrothion (FEN) pesticide residues in environmental water samples using fabric phase sorptive extraction (FPSE) followed by high-performance liquid chromatography combined with photodiode array (HPLC-PDA) detector. Both pesticides were successfully determined with a Luna omega C18 column under isocratic elution mode by means of acetonitrile and phosphate buffer (pH 3.0) as the mobile phase. The quantitative data for PRM and FEN were obtained at their maximum wavelengths of 310 nm and 268 nm, respectively. The calibration plots were linear in the range 10.00-750.00 ng mL-1 and 10.00-900.00 ng mL-1 with correlation coefficient of 0.9984 and 0.9992 for PRM and FEN, respectively. Major FPSE experimental variables were investigated in detail, such as contact time with the FPSE membrane, pH and electrolyte concentration, and the volume and type of desorption solvent. Under the optimized conditions, the developed method showed satisfactory reproducibility with relative standard deviations less than 2.5% and low limits of detection of 2.98 and 3.02 ng mL-1 for PRM and FEN, respectively. The combined procedure allows for enhancement factors ranging from 88 to 113, with pre-concentration values of 125 for both analytes. The chromatographic resolutions were approx. 12 for FEN (retention factor of 3.52) and PRM (retention factor of 6.09), respectively, with a selectivity factor of 1.73. Finally, the validated method was successfully applied to real environmental water samples for the determination of these pesticides. Graphical abstract.

A molecularly imprinted nanofilm-based quartz crystal microbalance sensor for the real-time detection of pirimicarb.

This study aimed to prepare a novel quartz crystal microbalance (QCM) sensor for the detection of pirimicarb. Pirimicarb-imprinted poly (ethylene glycol dimethacrylate-N-metacryloyl-(l)-tryptophan methyl ester) [p (EGDMA-MATrp)] nanofilm (MIP) on the gold surface of a QCM chip was synthesized using the molecular imprinting technique. A nonimprinted p (EGDMA-MATrp) nanofilm (NIP) was also synthesized using the same experimental technique. The MIP and NIP nanofilms were characterized via Fourier transform infrared spectroscopy attenuated total reflectance spectroscopy, contact angle, atomic force microscopy, and an ellipsometer. A competitive adsorption experiment on the sensor was performed to display the selectivity of the nanofilm. An analysis of the QCM sensor showed that the MIP nanofilm exhibited high sensitivity and selectivity for pirimicarb determination. A liquid chromatography-tandem mass spectrometry method was prepared and validated to determine the accuracy and precision of the QCM sensor. The accuracy and precision of both methods were determined by a comparison of six replicates at three different concentrations to tomato samples extracted by using a Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) method. The limit of detection of the QCM sensor was found to be 0.028 nM. In conclusion, the QCM sensor showed good accuracy, with recovery percentages between 91 and 94%. Also, the pirimicarb-imprinted QCM sensor exhibited a fast response time, reusability, high selectivity and sensitivity, and a low limit of detection. Therefore, it offers a serious alternative to the traditional analytical methods for pesticide detection in both natural sources and aqueous solutions.

DNA barcoding via counterstaining with AT/GC sensitive ligands in injection-molded all-polymer nanochannel devices.

Nanochannel technology, coupled with a suitable DNA labeling chemistry, is a powerful approach for performing high-throughput single-molecule mapping of genomes. Yet so far nanochannel technology has remained inaccessible to the broader research community due to high fabrication cost and/or requirement of specialized facilities/skill-sets. In this article we show that nanochannel-based mapping can be performed in all polymer chips fabricated via injection molding: a fabrication process so inexpensive that the devices can be considered disposable. Fluorescent intensity variations can be obtained from molecules extended in the polymer nanochannels via chemical counterstaining against YOYO-1. In particular, we demonstrate that the counterstaining induced fluorescent intensity variations to a large degree appear to be proportional to the theoretically computed sequence-maps of both local AT and GC variation along DNA sequences.

Quantification of kinase activity in cell lysates via photopatterned macroporous poly(ethylene glycol) hydrogel arrays in microfluidic channels.

The efficacy of tyrosine kinase inhibitors (TKIs) as cancer therapeutics varies amongst individual patients as a result of patient-specific differences in molecular regulation of cancer development and progression, and acquisition of resistance to TKIs during therapy. A sensitive assay that can quantify kinase activity and predict inhibition of that activity from minimally invasive patient tissue samples may aid design of efficacious individualized TKI treatments. A microfluidic format can be useful in reducing limitations in standard protein kinase assays, including sensitivity required and low sample volume available. We present photopatterned macroporous poly(ethylene glycol) diacrylate hydrogel pillars functionalized with kinase substrates within microchannels for quantifying kinase activity in complex cellular lysates. We determined the effect of using a porogen to induce macroporosity in hydrogel pillars and showed that hydrogel poration enhanced the sensitivity of detecting Bcr-Abl activity in cell lysates by an order of magnitude. Bcr-Abl tyrosine kinase activity in K562 cell lysates could be detected from 0.01 µg/µL of cell lysate, corresponding to approximately 500 cells, using GST-Crkl immobilized in macroporous hydrogels. This device was also capable of quantifying inhibition of Bcr-Abl activity by imatinib mesylate, which demonstrates the potential to predict the biochemical response to drug inhibitors. These results indicate that microfluidic devices containing macroporous hydrogels functionalized with kinase substrates provide a promising platform for sensitive and specific quantification of kinase activity and efficacy of kinase inhibitors in cancer cell lysates.

High-performance liquid chromatographic evaluation of a coated cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phase having a small-pore silica support.

A two-step coating/precipitation synthetic procedure has been developed for the preparation of cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phase (CSP) having a small-pore silica support. With this synthetic strategy, monodisperse, spherical CSP particles can be produced without the need for a wasteful and time-consuming sieving process. The performance of the synthesized CSP towards a variety of racemates was evaluated in the normal-phase HPLC mode. HPLC separation experiments revealed that the synthesized CSP exhibited a chiral recognition ability fully comparable to the corresponding commercial columns prepared using conventional large-pore silica as the support. Moreover, the synthesized CSP was successfully applied to semipreparative enantioseparation of a new triazole antifungal agent.

Determination of voriconazole in human plasma and saliva using high-performance liquid chromatography with fluorescence detection.

Voriconazole is a widely used triazole antifungal agent with a broad spectrum including Aspergillus species. A simple, sensitive and selective high-performance liquid chromatography method for the determination of voriconazole in human plasma and saliva was developed. Drug and internal standard (UK-115 794) were extracted from alkaline plasma and saliva with n-hexane-ethyl acetate (3:1, v/v) and analyzed on a Luna C 18 column with fluorimetric detection set at excitation and emission wavelengths of 254 and 372 nm, respectively. The calibration curve was linear through the range of 0.1-10 microg/ml using a 0.3 ml sample volume. The intra- and inter-day precisions were all below 6.1% for plasma and below 9.1% for saliva. Accuracies ranged from 94 to 109% for both matrices. Mean recovery was 86+/-4% for voriconazole. The method showed acceptable values for precision, recovery and sensitivity and is well suited for routine analysis work and for pharmacokinetic studies.

A new extraction phase based on a polymer inclusion membrane for the detection of chlorpyrifos, diazinon and cyprodinil in natural water samples.

A simple and effective method for the detection of three pesticides (chlorpyrifos, diazinon and cyprodinil) is developed using a polymer inclusion membrane (PIM) prior to gas chromatography and mass spectrometry detection (GC-MS). Analytes are extracted from natural water samples using a 3 cm2 PIM made of the polymer, cellulose triacetate (CTA), and the plasticizer, nitrophenyl octyl ether (NPOE). Addition of the plasticizer to the CTA matrix is found to be necessary for the extraction of pesticides. After extraction, analytes are recovered from the membrane with 1 mL of acetonitrile and injected into the GC-MS system. The main factors affecting the extraction efficiency are evaluated, including membrane composition, stirring mode, extraction and elution time. Ultrasonic assisted elution of the extracted pesticides is accomplished after 15 min of contact. The PIM-assisted extraction method makes it possible for pesticides to be determined in the range of 50-1000 ng L-1 with good linearity (coefficient of determination ≥0.995) and suitable recoveries (85-119%) and precision (<21%, n = 3) using 100 mL of a water sample. This methodology is shown to be suitable for the detection of chlorpyrifos in local river waters.

Characterization of silicone elastomer vaginal rings containing HIV microbicide TMC120 by Raman spectroscopy.

Silicone elastomer vaginal rings are currently being pursued as a controlled-release strategy for delivering microbicidal substances for the prevention of heterosexual transmission of HIV. Although it is well established that the distribution of drugs in delivery systems influences the release characteristics, in practice the distribution is often difficult to quantify in-situ. Therefore, the aim of this work was to determine whether Raman spectroscopy might provide a rapid, non-contact means of measuring the concentrations of the lead candidate HIV microbicide TMC120 in a silicone elastomer reservoir-type vaginal ring. Vaginal rings loaded with TMC120 were manufactured and sectioned before either Raman mapping an entire ring cross-section (100 microm resolution) or running line scans at appropriate time intervals up to 30 h after manufacture. The results demonstrated that detectable amounts of TMC120, above the silicone elastomer saturation concentration, could be detected up to 1 mm into the sheath, presumably as a consequence of permeation and subsequent reprecipitation. The extent of permeation was found to be similar in rings manufactured at 25 and 80 degrees C.

LC-MS/MS methods for sulfadimethoxine and ormetoprim analysis in feed and fish fillet and a leaching study for feed after alternative procedures for the incorporation of drugs.

This paper describes the method development for sulfadimethoxine (SDM) and ormetoprim (OMP) quantitation in fish feed and fish fillet employing LC-MS/MS. In order to assess the reliability of the analytical method, valuation was undertaken as recommended by guidelines proposed by the Brazilian Ministry of Agriculture. The calibration curve for the quantification of both drugs in feed showed adequate linearity (r > 0.99), precision (CV < 12%) and trueness ranging from 97% to 100%. The method for the determination of SDM and OMP residues in fish fillet involved a simple sample preparation procedure that had adequate linearity (r > 0.99), precision (CV < 16%) and trueness around 100%, with CCα < 100.2 ng g-1 and CCß < 100.4 ng g-1. With a goal of avoiding the risk of drug leaching from feed into the aquatic environment during fish medication via the oral route, different procedures for drug incorporation into feed were evaluated. Coating feed pellets with ethyl cellulose polymer containing the drug showed promising results. In this case, medicated feed released drugs to water at a level below 6% when the medicated feed stayed in the water for up to 15 min.

Analysis of expression of yeast enolase 1 gene containing a longer pyrimidine-rich region located between the TATA box and transcription start site.

Yeast ENO1 promoter was prepared by a chemical synthetic method. Two variant promoters containing a pyrimidine-rich region (CT block), located between the TATA box and the transcription start site, either 32 or 34 base pairs (bp) longer than the native ENO1 promoter were isolated during the chemical synthesis. Gene expression of variant promoters was compared with that of the native promoter by measuring the amount of mRNA and the activity of beta-galactosidase by constructing ENO1-lacZ gene fusions. No significant differences were observed between the native and variant promoters in transcription levels. The start site of transcription was mapped on CAAG, a consensus sequence of transcription start site of yeast glycolytic genes. The results suggest a longer CT block in ENO1 promoter may not affect the expression of the yeast ENO1 gene. In addition, the level of ENO1 gene expression was found to be higher in stationary phase cells than in log phase cells.

Synthesis and application of molecularly imprinted polymer on selective solid-phase extraction for the determination of monosulfuron residue in soil.

A novel sample clean-up procedure using molecularly imprinted polymer as the solid-phase extraction material for the determination of monosulfuron residue in soil samples has been developed. The molecularly imprinted polymer (MIP) was synthesized by non-covalent method with monosulfuron as the template. The selectivity and affinity of the MIP was evaluated by equilibrium adsorption and HPLC experiments, which demonstrated that the MIP has specific affinity for the template. The template-MIP interaction was studied by investigating the influence of different mobile phases on the retention of the template, which provided basic knowledge for the selection of the washing and elution solutions in the molecularly imprinted solid-phase extraction (MISPE) process. The study indicated that polar organic solvents with hydrogen bonding abilities have stronger eluting strength for the monosulfuron. After the MISPE procedure, a clean baseline was obtained in the HPLC quantification analysis. The recoveries of the method using the combination of MISPE and HPLC were above 93% and the R.S.D. was less than 3.2% in the soil sample determinations. Low detection limit (0.08 microg g(-1), when defined as 3 times of the noise) was also obtained in the method evaluation study.

Quantification of risperidone and 9-hydroxyrisperidone in plasma and saliva from adult and pediatric patients by liquid chromatography-mass spectrometry.

A robust and validated LC-MS-MS quantitative method, using column switching and mutiple reaction monitoring was developed for the analysis of risperidone (RIS) and 9-hydroxyrisperidone in human plasma and saliva. The analytical range was 1-100 ng/ml. The method used 25 microl of sample precipitated with 75 microl of acetonitrile containing internal standard (R068808). Analyses were conducted on a PE Sciex API-III + triple quadrupole mass spectrometer fitted with a Turbo IonSpray source. The method was validated for human plasma using EDTA as the anticoagulant and cross-validated to heparinized human plasma and saliva. The recoveries of risperidone and 9-hydroxyrisperidone were 90-93 and 89-93%, respectively. The validated method was applied to clinical samples to study risperidone and 9-hydroxyrisperidone concentrations in plasma and saliva. Risperidone and 9-hydroxyrisperidone appear in the saliva of patients treated with risperidone. Their detection/quantification in saliva provides evidence for recent adherence with therapy.

Determination of pyrimidine phosphoribosyltransferase and uridine kinase activities by an assay with DEAE-paper discs.

Rapid (6 min washing step) and reliable methods for determining uridine kinase and pyrimidine phosphoribosyltransferase activities were devised. These procedures, consisting of a spotting technique on DEAE-discs followed by washing and elution, permitted the consistent recovery of about 90% of the nucleoside 5'-phosphate esters formed from radioactive precursors, either uridine or 5-fluorouracil, respectively. Of these precursors, less than 2% were retained on the discs. Direct counting of the discs (without elution), filtration by either gravity or suction, and the use of so-called activation of discs have not proved advantageous.

Collection and determination of nucleotide metabolites in neonatal and adult saliva by high performance liquid chromatography with tandem mass spectrometry.

Saliva contains a number of biochemical components which may be useful for diagnosis/monitoring of metabolic disorders, and as markers of cancer or heart disease. Saliva collection is attractive as a non-invasive sampling method for infants and elderly patients. We present a method suitable for saliva collection from neonates. We have applied this technique for the determination of salivary nucleotide metabolites. Saliva was collected from 10 healthy neonates using washed cotton swabs, and directly from 10 adults. Two methods for saliva extraction from oral swabs were evaluated. The analytes were then separated using high performance liquid chromatography (HPLC) with tandem mass spectrometry (MS/MS). The limits of detection for 14 purine/pyrimidine metabolites were variable, ranging from 0.01 to 1.0µM. Recovery of hydrophobic purine/pyrimidine metabolites from cotton tips was consistently high using water/acetonitrile extraction (92.7-111%) compared with water extraction alone. The concentrations of these metabolites were significantly higher in neonatal saliva than in adults. Preliminary ranges for nucleotide metabolites in neonatal and adult saliva are reported. Hypoxanthine and xanthine were grossly raised in neonates (49.3±25.4; 30.9±19.5µM respectively) compared to adults (4.3±3.3; 4.6±4.5µM); nucleosides were also markedly raised in neonates. This study focuses on three essential details: contamination of oral swabs during manufacturing and how to overcome this; weighing swabs to accurately measure small saliva volumes; and methods for extracting saliva metabolites of interest from cotton swabs. A method is described for determining nucleotide metabolites using HPLC with photodiode array or MS/MS. The advantages of utilising saliva are highlighted. Nucleotide metabolites were not simply in equilibrium with plasma, but may be actively secreted into saliva, and this process is more active in neonates than adults.

LC-MS/MS of some atypical antipsychotics in human plasma, serum, oral fluid and haemolysed whole blood.

Therapeutic drug monitoring (TDM) of atypical antipsychotics is common, but published methods often specify relatively complex sample preparation and analysis procedures. The aim of this work was to develop and validate a simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the analysis of amisulpride, aripiprazole and dehydroaripiprazole, clozapine and norclozapine, olanzapine, quetiapine, risperidone and 9-hydroxyrisperidone, and sulpiride in small (200 µL) volumes of plasma or serum for TDM purposes. The applicability of the method as developed to haemolysed whole blood and to oral fluid was also investigated. Analytes and internal standards were extracted into butyl acetate:butanol (9+1, v/v) and a portion of the extract analysed by LC-MS/MS (100 mm × 2.1 mm i.d. Waters Spherisorb S5SCX; eluent: 50 mmol/L methanolic ammonium acetate, pH* 6.0; flow-rate 0.5 mL/min; positive ion APCI-SRM, two transitions per analyte). Assay calibration (human plasma, oral fluid, and haemolysed whole blood calibration solutions) was performed by plotting the ratio of the peak area of the analyte to that of the appropriate internal standard. Assay validation was as per FDA guidelines. Assay calibration was linear across the concentration ranges studied. Inter- and intra-assay precision and accuracy were within 10% for all analytes in human plasma. Similar results were obtained for oral fluid and haemolysed whole blood, except that aripiprazole and dehydroaripiprazole were within 15% accuracy at low concentration (15 µg/L) in oral fluid, and olanzapine inter-assay precision could not be assessed in these matrices due to day-by-day degradation of this analyte. Recoveries varied between 16% (sulpiride) and 107% (clozapine), and were reproducible as well as comparable between human plasma, human serum, calf serum and haemolysed whole blood. For oral fluid, recoveries were reproducible, but differed slightly from those in plasma suggesting the need for calibration solutions to be prepared in this medium if oral fluid is to be analysed. LLOQs were 1-5 µg/L depending on the analyte. Neither ion suppression/enhancement, nor interference from some known metabolites of the antipsychotics studied has been encountered. The method has also been applied to the analysis of blood samples collected post-mortem after dilution (1+1, 1+3; v/v) in analyte-free calf serum.

A silicone elastomer vaginal ring for HIV prevention containing two microbicides with different mechanisms of action.

Vaginal rings are currently being developed for the long-term (at least 30 days) continuous delivery of microbicides against human immunodeficiency virus (HIV). Research to date has mostly focused on devices containing a single antiretroviral compound, exemplified by the 25mg dapivirine ring currently being evaluated in a Phase III clinical study. However, there is a strong clinical rationale for combining antiretrovirals with different mechanisms of action in a bid to increase breadth of protection and limit the emergence of resistant strains. Here we report the development of a combination antiretroviral silicone elastomer matrix-type vaginal ring for simultaneous controlled release of dapivirine, a non-nucleoside reverse transcriptase inhibitor, and maraviroc, a CCR5-targeted HIV-1 entry inhibitor. Vaginal rings loaded with 25mg dapivirine and various quantities of maraviroc (50-400mg) were manufactured and in vitro release assessed. The 25mg dapivirine and 100mg maraviroc formulation was selected for further study. A 24-month pharmaceutical stability evaluation was conducted, indicating good product stability in terms of in vitro release, content assay, mechanical properties and related substances. This combination ring product has now progressed to Phase I clinical testing.

Stability of some atypical antipsychotics in human plasma, haemolysed whole blood, oral fluid, human serum and calf serum.

Long-term stability data of atypical antipsychotics in different matrices are not widely available. The aim of this work was to assess the stability of amisulpride, aripiprazole and dehydroaripiprazole, clozapine and norclozapine, olanzapine, quetiapine, risperidone and 9-hydroxyrisperidone, and sulpiride in human EDTA plasma, heparinised haemolysed human whole blood, oral fluid, human serum, and newborn calf serum stored in tightly capped plastic containers under a range of conditions. Measurements were performed by LC-MS/MS. Analyte instability was defined as a deviation of 15% or greater from the expected concentration. All analytes were stable following 3 freeze-thaw cycles in human plasma, and were stable in this matrix for at least 5 days at ambient temperature (olanzapine, 3 days); 4 weeks at 2-8°C (olanzapine, 2 weeks), and 2 years at -20°C (except for dehydroaripiprazole, olanzapine, and quetiapine, 1 year). In human serum, aripiprazole, dehydroaripiprazole, norclozapine, olanzapine, quetiapine, risperidone, 9-hydroxyrisperidone, and sulpiride were unstable after 5 days at ambient temperature, 3 weeks at 2-8°C, and 9 months at -20°C. Olanzapine was unstable in whole blood and oral fluid under most conditions studied, although prior addition of ascorbic acid had a moderate stabilising effect. All other analytes were stable in whole blood and oral fluid for at least 2 days at ambient temperature, 1 week at 2-8°C, and 2 months at -20°C (clozapine and norclozapine, 1 month whole blood). These results confirm that plasma (EDTA anticoagulant) is the sample of choice for TDM of atypical antipsychotics. Delayed (more than 1 week) analysis of patient samples should be undertaken with caution, especially with serum and with haemolysed whole blood. With olanzapine, only plasma collected and stored appropriately is likely to give reliable quantitative results.

Poly(alizarin red)/graphene modified glassy carbon electrode for simultaneous determination of purine and pyrimidine.

In this work, a poly(alizarin red)/Graphene composite film modified glassy carbon electrode (PAR/Graphene/GCE) was prepared for simultaneous determination of four DNA bases (guanine, adenine, thymine and cytosine) without any pretreatment. The morphology and interface property of PAR/Graphene films were examined by scanning electron microscopy and electrochemical impedance spectroscopy. The PAR/Graphene/GCE exhibited excellent electrocatalytic activity toward purine (guanine and adenine) and pyrimidine (thymine and cytosine) in 0.1 M phosphate buffer solution (pH 7.4). Under optimum conditions, differential pulse voltammetry was used to detect the oxidation of purine and pyrimidine. The results showed that PAR/Graphene/GCE exhibited well-separated peaks, low detection limit, high sensitivity and wide linear range for simultaneous detection of purine and pyrimidine. The proposed sensor also has good stability and reproducibility. Furthermore, the modified electrode was applied for the detection of DNA bases in a fish sperm DNA sample with satisfactory results.

Preparation and evaluation of poly(4-vinylphenylboronic acid-co-pentaerythritol triacrylate) monolithic column for capillary liquid chromatography of small molecules and proteins.

A mixed-mode monolithic stationary phase was prepared for capillary liquid chromatography (cLC) by in situ copolymerization of 4-vinylphenylboronic acid (VPBA) and pentaerythritol triacrylate (PETA) in a binary porogenic solvent consisting of ethylene glycol/cyclohexanol. The monomer of VPBA functioned as ion-exchange sites, hydrophilic ligands, hydrophobic groups and affinity sites, while PETA was introduced as a hydrophilic crosslinker. The resultant monoliths with different column properties (e.g. morphology, permeability and selectivity) were optimized by adjusting the ratio of VPBA to PETA and the composition of porogenic solvent. The results showed that the selectivity of the monoliths increased with increasing content of VPBA in the polymerization mixture. A series of alkylbenzenes, amides, and anilines were used to evaluate the column performance in terms of hydrophobic, hydrophilic and cation-exchange interactions. At an optimized flow rate of 50 µL/min (corresponding to 0.265 mm/s), the monolith exhibited high column efficiencies of 43,000-100,000 plates/m for alkylbenzenes. Good repeatability was obtained with relative standard deviation (RSD) of retention factor (k) less than 0.65% for run-to-run (n=5) and less than 2.49% for column-to-column (n=5). In addition, the poly(VPBA-co-PETA) monolithic column was applied to the separation of phenols, nucleobases, and proteins, respectively. These successful applications demonstrate the purposed monoliths are promising for cLC separation of small molecules and proteins.

Development and validation of a HPLC method for the assay of dapivirine in cell-based and tissue permeability experiments.

Dapivirine, a non-nucleoside reverse transcriptase inhibitor, is being currently used for the development of potential anti-HIV microbicide formulations and delivery systems. A new high-performance liquid chromatography (HPLC) method with UV detection was developed for the assay of this drug in different biological matrices, namely cell lysates, receptor media from permeability experiments and homogenates of mucosal tissues. The method used a reversed-phase C18 column with a mobile phase composed of trifluoroacetic acid solution (0.1%, v/v) and acetonitrile in a gradient mode. Injection volume was 50µL and the flow rate 1mL/min. The total run time was 12min and UV detection was performed at 290nm for dapivirine and the internal standard (IS) diphenylamine. A Box-Behnken experimental design was used to study different experimental variables of the method, namely the ratio of the mobile phase components and the gradient time, and their influence in responses such as the retention factor, tailing factor, and theoretical plates for dapivirine and the IS, as well as the peak resolution between both compounds. The optimized method was further validated and its usefulness assessed for in vitro and ex vivo experiments using dapivirine or dapivirine-loaded nanoparticles. The method showed to be selective, linear, accurate and precise in the range of 0.02-1.5µg/mL. Other chromatographic parameters, namely carry-over, lower limit of quantification (0.02µg/mL), limit of detection (0.006µg/mL), recovery (equal or higher than 90.7%), and sample stability at different storage conditions, were also determined and found adequate for the intended purposes. The method was successfully used for cell uptake assays and permeability studies across cell monolayers and pig genital mucosal tissues. Overall, the proposed method provides a simple, versatile and reliable way for studying the behavior of dapivirine in different biological matrices and assessing its potential as an anti-HIV microbicide drug.