Simultaneous determination of benzophenone-3, retinol and retinyl acetate in pig ear skin layers by high-performance liquid chromatography

A new and simple HPLC method was developed and validated for the simultaneous determination of benzophenone-3, retinol and retinyl acetate in pig ear skin layers and percutaneous penetration samples after in vitro permeation experiments. HPLC analysis was performed utilizing a NovaPak C18 column with acetonitrile-water-acetic acid as mobile phase. UV detection was at 325 nm and the run time was 25 min. The detector response was found to be accurate, precise and linear across the analytical range. Analyte extraction from skin layers was done with methanol from the stratum corneum and epidermis, and with acetone from the dermis. Recovery was in all cases better than 90%. The HPLC assay and extraction procedure proposed are simple, rapid, sensitive and accurate. The method was then applied for the determination of benzophenone-3, retinol and retinyl acetate in pig ear skin layers after topical application.     

Development of a HPLC method for determination of four UV filters in sunscreen and its application to skin penetration studies

This study describes the development, validation and application of a high‐performance liquid chromatography (HPLC) method for the simultaneous determination of the in vitro skin penetration profile of four UV filters on porcine skin. Experiments were carried out on a gel‐cream formulation containing the following UV filters: diethylamino hydroxybenzoyl hexyl benzoate (DHHB), bis‐ ethylhexyloxyphenol methoxyphenyl triazine (BEMT), methylene bis‐ benzotriazolyl tetramethylbutylphenol (MBBT) and ethylhexyl triazone (EHT). The HPLC method demonstrated suitable selectivity, linearity (10.0–50.0 μg/mL), precision, accuracy and recovery from porcine skin and sunscreen formulation. The in vitro skin penetration profile was evaluated using Franz vertical diffusion cells for 24 h after application on porcine ear skin. None of the UV filters penetrated the porcine skin. Most of them stayed on the skin surface (>90%) and only BEMT, EHT and DHHB reached the dermis plus epidermis layer. These results are in agreement with previous results in the literature. Therefore, the analytical method was useful to evaluate the in vitro skin penetration of the UV filters and may help the development of safer and effective sunscreen products.     

Development and validation of HPLC method for imiquimod determination in skin penetration studies

A simple, rapid and sensitive analytical procedure for the measurement of imiquimod in skin samples after in vitro penetration studies has been developed and validated. In vitro penetration studies were carried out in Franz diffusion cells with porcine skin. Tape stripping technique was used to separate the stratum corneum (SC) from the viable epidermis and dermis. Imiquimod was extracted from skin samples using a 7:3 (v/v) methanol:acetate buffer (100 mM, pH 4.0) solution and ultrasonication. Imiquimod was analyzed by HPLC using C(8) column and UV detection at 242 nm. The mobile phase used was acetonitrile:acetate buffer (pH 4.0, 100 mM):diethylamine (30:69.85:0.15, v/v) with flow rate 1 mL/min. Imiquimod eluted at 4.1 min and the running time was limited to 6.0 min. The procedure was linear across the following concentration ranges: 100-2500 ng/mL for both SC and tape-stripped skin and 20-800 ng/mL for receptor solution. Intra-day and inter-day accuracy and precision values were lower than 20% at the limit of quantitation. The recovery values ranged from 80 to 100%. The method is adequate to assay imiquimod from skin samples, enabling the determination of the cutaneous penetration profile of imiquimod by in vitro studies.     

Development and validation of SPMMTE HPLC method for analysis of profens from human plasma

A fast, selective and reproducible solid‐phase membrane microtip extraction (SPMMTE) HPLC method has been developed and validated for the analyses of ibuprofen, ketoprofen, and flurbiprofen from human plasma. The analysis was carried out on a C₁₈ (150 × 4.6 mm; 5 μm) column. The mobile phase used was water–acetonitrile (55:45, v/v) adjusted to pH 3.0 using trifluoroacetic acid, at a flow rate 0.5 mL/min with a detection wavelength of 225 nm. The values for the capacity factors for the profen samples ranged from 0.47 to 1.50. The values for the selectivity factor (α) for ketoprofen–flurbiprofen, flurbiprofen–ibuprofen and ibuprofen–ketoprofen combinations from human plasma samples were 1.99, 1.00 and 2.10, respectively. The resolution factors (Rs) for ketoprofen–flurbiprofen, flurbiprofen–ibuprofen and ibuprofen–ketoprofen from plasma samples were 3.00, 1.50 and 4.10, respectively. The percentage recoveries of ibuprofen, ketoprofen, and flurbiprofen from human plasma were 75–85%. All of the profens were separated within 7.0 min, indicating a relatively fast method. During the development of the SPMMTE procedure the parameters of pH, contact time, desorption and types of solvents were optimized. The final method was also found to be efficient, effective and inexpensive. Copyright © 2016 John Wiley & Sons, Ltd.     

Highly sensitive HPLC‐DAD method for the assay of gefitinib in patient plasma and cerebrospinal fluid: application to a blood‐brain barrier penetration study

The quantification of intracranial gefitinib (GEF) exposure is limited owing to the sensitivity of analytical equipment. Although mass spectrometry (MS) is the preferred method because of its high sensitivity, the equipment is not available in many laboratories, especially in developing Asian countries. In this paper, we developed a highly sensitive high performance liquid chromatography‐diode array detector (HPLC‐DAD) method for the assay of GEF in human cerebrospinal fluid (CSF) and plasma. GEF was extracted from CSF and plasma by solid‐phase extraction and liquid–liquid extraction, respectively. The chromatographic separation was performed on a C₁₈ column with gradient elution of 0.1% triethylamine solution and acetonitrile, then finally detected at 344 nm. This method was validated and proved to be highly sensitive with a lower limit of quantitation value of 0.11 ng/mL in CSF and 11 ng/mL in plasma. The blood–brain barrier penetration ratio of GEF ranged from 1.48 to 2.41%. This method provides a reliable MS‐independent solution for the quantitation of GEF in patients’ CSF and plasma. Copyright © 2015 John Wiley & Sons, Ltd.     

Supercritical fluid extraction in plant essential and volatile oil analysis

The use of supercritical fluids, especially carbon dioxide, in the extraction of plant volatile components has increased during two last decades due to the expected advantages of the supercritical extraction process. Supercritical fluid extraction (SFE) is a rapid, selective and convenient method for sample preparation prior to the analysis of compounds in the volatile product of plant matrices. Also, SFE is a simple, inexpensive, fast, effective and virtually solvent-free sample pretreatment technique. This review provides a detailed and updated discussion of the developments, modes and applications of SFE in the isolation of essential oils from plant matrices. SFE is usually performed with pure or modified carbon dioxide, which facilitates off-line collection of extracts and on-line coupling with other analytical methods such as gas, liquid and supercritical fluid chromatography. In this review, we showed that a number of factors influence extraction yields, these being solubility of the solute in the fluid, diffusion through the matrix and collection process. Finally, SFE has been compared with conventional extraction methods in terms of selectivity, rapidity, cleanliness and possibility of manipulating the composition of the extract.     

Pharmaceutical analysis of 5-aminolevulinic acid in solution and in tissues

Quantification of 5-aminolevulinic acid (ALA) in solution, and methods used to achieve this, have been extensively reported in the literature. However, validated methods have only rarely been presented and never have methods been compared. Due to a necessity in drug delivery research for optimised and validated methods for determination of ALA in solution, this paper compares, for the first time, two such methods validated to International Conference on Harmonisation (ICH) standards. Of major importance, derivatisation of ALA with acetyl acetone and formaldehyde was found to be more suitable for routine fluorimetric HPLC analysis of ALA than derivatisation with o-phthaldialdehyde and 2-mercaptoethanol. This former method was successfully utilised in the comparison of in vitro drug release from a proprietary ALA cream and a novel bioadhesive patch-based system. In addition, determination of ALA in tissue is necessary to compare different topical formulations, in terms of their ability to deliver the drug successfully, and different tissue types, to assess their barrier properties to penetration of the drug. Consequently, this paper also describes the use of liquid scintillation spectroscopy as an analytical tool for rapid, convenient and routine quantification of ALA in tissue and determination of penetration depth following topical application of creams and patches.     

Development and validation of a reversed‐phase high‐performance liquid chromatographic method with solid‐phase extraction for the quantification of hydrochlorothiazide in ex vivo permeation studies

Hydrochlorothiazide (HCT) is a diuretic used to treat hypertension. In order to study its intestinal permeation behavior applying an ex vivo methodology, a rapid, sensitive and selective reversed‐phase liquid chromatography (RP‐HPLC) method coupled with UV detection (RP‐HPLC UV) was developed for the analysis of HCT in TC199 culture medium used as mucosal and serosal solutions in the everted rat intestinal sac model. Also, analytical procedures for the quantification of HCT by RP‐HPLC with UV detection required a sample preparation step by solid‐phase extraction. The method was validated in the concentration range of 8.05 × 10⁻⁷ to 3.22 × 10⁻⁵ m for HCT. Chromatographic parameters, namely carry‐over, lower limit of quantification (1.4491 × 10⁻⁷ m ), limit of detection (3.8325 × 10⁻⁸ m ), selectivity, inter‐ and intraday precision and extraction recovery, were determined and found to be adequate for the intended purposes. The validated method was successfully used for permeability assays across rat intestinal epithelium applying the ex vivo everted rat gut sac methodology to study the permeation behavior of HCT.     

Enantiomeric resolution of ibuprofen and flurbiprofen in human plasma by SPE-chiral HPLC methods

Chiral analysis of profens in human plasma is an important area of research due to different pharmaceutical activities of their enantiomers. The solid phase extraction of ibuprofen and flurbiprofen from human plasma was carried out on C18 cartridges by using phosphate buffer (50 mM, pH 6.0) followed by elution with methanol. Chiral-HPLC was performed on AmyCoat RP (150 mm x 46 mm, 3 μm particle size) column by using different combinations of water-acetonitrile-trifluoro acetic acid at 1.5 mLmin-1 flow rate. The detection was achieved at 236 and 254 nm for ibuprofen and flurbiprofen, respectively with 27±1°C as working temperature. The chromatographic parameters i.e. retention (k), separation (α) and resolution (Rs) factors ranged from 4.54-14.42, 1.10-1.30 and 1.01-1.49, respectively. The binding differences of enantiomers of ibuprofen and flurbiprofen were 4.4 and 5.2, respectively. These values suggest that S-(+)- enantiomer of flurbiprofen is more active than ibuprofen due to low enantiomeric difference of the later drug. The developed SPE-Chiral HPLC methods were validated, which are selective, efficient and reproducible.     

Effect of Penetration Enhancers on Toenail Delivery of Efinaconazole from Hydroalcoholic Preparations

The incorporation of permeation enhancers in topical preparations has been recognized as a simple and valuable approach to improve the penetration of antifungal agents into toenails. In this study, to improve the toenail delivery of efinaconazole (EFN), a triazole derivative for onychomycosis treatment, topical solutions containing different penetration enhancers were designed, and the permeation profiles were evaluated using bovine hoof models. In an in vitro permeation study in a Franz diffusion cell, hydroalcoholic solutions (HSs) containing lipophilic enhancers, particularly prepared with propylene glycol dicaprylocaprate (Labrafac PG), had 41% higher penetration than the HS base. Moreover, the combination of hydroxypropyl-β-cyclodextrin with Labrafac PG further facilitated the penetration of EFN across the hoof membrane. In addition, this novel topical solution prepared with both lipophilic and hydrophilic enhancers was physicochemically stable, with no drug degradation under ambient conditions (25 °C, for 10 months). Therefore, this HS system can be a promising tool for enhancing the toenail permeability and therapeutic efficacy of EFN.     

Determination of Taxotere in human plasma by a semi-automated high-performance liquid chromatographic method.

A rapid, selective and reproducible high-performance liquid chromatographic (HPLC) method with ultraviolet detection was developed for the determination of the anti-cancer agent Taxotere in biological fluids. The method involves a solid-phase extraction step (C2 ethyl microcolumns) using a Varian Advanced Automated Sample Processor (AASP) followed by reversed-phase HPLC. The validated quantitation range of the method is 10-2500 ng/ml in plasma with coefficients of variation < or = 11%. The method is also suitable for the determination of Taxotere in urine samples under the same conditions. The method was applied in a phase I tolerance study of Taxotere in cancer patients, allowing the pharmacokinetic profile of Taxotere to be established.     

Quantitative coupling of supercritical fluid extraction and high-performance liquid chromatography by means of a coated open-tubular interface.

Supercritical fluid extraction was coupled on-line with reversed-phase high-performance liquid chromatography (HPLC). An open-tubular column with a 95% methyl-5% phenyl stationary phase was utilized as an interface between the two systems. This phase allowed for good analyte focusing onto the packed analytical column and exhibited low reactivity. Due to the non-polar nature of this phase there was a low tendency for analytes to be prematurely rinsed off the interface by condensed modifier. Using this approach, it was possible to transfer the extracted analytes quantitatively to the HPLC column in the presence of as much as 10% (v/v) methanol. By placing a 10 m guard column at the head of the interface, the same could be accomplished with ethanol as the modifier: allowing the extraction to be conducted entirely with non-toxic fluids. The method also allowed the use of very practical extraction parameters in terms of amount extracted, extraction flow-rate, extraction vessel volume, and extraction time.     

Development of a rapid and sensitive high-performance liquid chromatographic method to determine CYP2D6 phenotype in human liver microsomes

Dextromethorphan is a probe substrate to determine CYP2D6 phenotype. The conversion of dextromethorphan to dextrorphan by CYP2D6 accounts for approximately 60% of total metabolism. Most analytical methods utilize complicated labor- and time-intensive sample processing methods with several liquid-liquid extraction (LLE) steps. Our goal was to develop a non-LLE based rapid and sensitive HPLC method, to measure dextromethorphan metabolism in human liver microsomes. A solid-phase filtration based reverse-phase HPLC method with fluorescence detection was developed and validated. Human liver (n = 6) microsomal incubations were carried out with dextromethorphan, under optimum conditions. The analytes were separated by one-step centrifugal filtration with Nanosep separation units. The filtrate was injected ( 50 microL) into a Waters Alliance 2690 HPLC system. Metabolic incubations were also conducted to determine levels using LLE for comparisons. The Nanosep separation step reduced the extraction time from 3h to 40 min. The limit of quantitation was 23.8 nM (9.7 ng/mL), recovery was approximately 98%, the mean precision values were <10% RSD for the controls (80, 320 and 640 nM) and mean percentage error was <5%. Michaelis-Menten parameters were determined to distinguish CYP2D6 phenotypes. A rapid and sensitive HPLC method is reported, which may be suitable for automation and allows phenotyping of human liver microsomes.     

Simultaneous analysis of select pharmaceuticals and personal care products in fish tissue using pressurized liquid extraction combined with silica gel cleanup

Analytical improvements were developed and validated for measuring select personal care products (PCPs) and two pharmaceuticals in fish tissue. The method was validated using fortified fillet tissue for twelve PCPs including fragrance materials, alkylphenols, photo initiators, and triclosan as well as two pharmaceuticals including carbamazepine (anti-seizure) and diazepam (anti-convulsant). The analytical method utilized pressurized liquid extraction (PLE) combined with silica gel cleanup, gel permeation chromatography, and gas chromatography ion-trap tandem mass spectrometry. Silica gel cleanup was combined with the PLE to produce one automated extraction/cleanup technique. This analytical improvement served to reduce the incurred cost, time, and loss of potential target analytes associated with independent cleanup steps. The combined extraction/cleanup technique resulted in an average increase of 10% in analyte recoveries. Average triplicate recoveries and relative standard deviations for the entire method, using 2.5 g of fish fillet tissue, were 92 ± 9% (recoveries ranged from 64 to 131%). The sensitivity of the analytical methods was improved by optimizing the resonant collision induced dissociation energy to the hundredths place (0.01 V). Improvements in ion production range from 24 to 122% for six of the 12 PCPs. Statistically derived method detection limits (MDLs) were also lowered on average by a factor of 8 and ranged from 1.2 to 38 ng/g wet weight. MDLs for carbamazepine and diazepam were 18 and 3.7 ng/g wet weight, respectively. Galaxolide and tonalide were measured in an environmental sample at concentrations of 81 and 5.5 ng/g wet weight, respectively.     

A new FTIR-ATR cell for drug diffusion studies

The drug diffusion of most compounds, particularly hydrophilic molecules through the skin is limited by the permeation of the outermost cell layers of the epidermis, the stratum corneum(SC). For this reason it is of interest to characterize drug diffusion processes through this skin layer. A new FTIR-ATR cell was developed for non-invasive real time measurements of drug diffusion. The diffusion of water through an artificial polyethyleneglycol-polydimethylsiloxane membrane was studied. Additionally the diffusion of urea in human SC was analyzed. Based on a mathematical model the diffusion coefficients were derived. We could reveal that this cell associates the advantages of the Franz diffusion cell and the FTIR-ATR spectroscopy as a new powerful method for determining drug diffusion through biological membranes.     

High-performance liquid chromatographic method for the determination of finasteride in human plasma at therapeutic doses.

A high-performance liquid chromatographic (HPLC) method with ultraviolet detection for the determination of a novel 4-aza-steroidal inhibitor of 5 alpha-reductase in human plasma has been developed. The assay is based on a single solid-phase extraction and an efficient HPLC separation on two analytical columns in series. The assay has been fully validated and used to support Phase II and III clinical pharmacokinetic studies. The lowest limit of quantification was found to be at 1 ng/ml and allowed pharmacokinetic evaluation of the drug at doses down to 5 mg.     

In vitro permeation of chromium species through porcine and human skin as determined by capillary electrophoresis–inductively coupled plasma–sector field mass spectrometry

Since the species that trigger chromium allergy are not yet known, it is important to gain more of an insight into the mechanism of chromium transport through the skin and into the relationship between chromium allergy and chromium species. In vitro permeation studies with porcine and human skin were performed using a Franz static diffusion cell. Investigations attempted to elucidate (i) which Cr compounds are able to permeate through skin, (ii) the influence the Cr concentration in the donor solution has on the Cr permeation, and (iii) the effect that the time of exposure to the donor solution has on Cr permeation. Capillary electrophoresis hyphenated to inductively coupled plasma–sector field mass spectrometry (CE–ICP–SFMS) was used to separate and quantify the Cr species in the receptor fluid. 50 mmol L⁻¹ phosphate buffer (pH 2.5) was used for CE separation, and two different electrophoretic runs were carried out (in the positive and negative modes). Pneumatic nebulization (PN)-ICP-SFMS was used in order to quantify the total amount of Cr absorbed by the skin after microwave-assisted acid digestion of the tissue. Cr(VI) was found to pass most easily through the skin. Nevertheless, Cr(VI) was also shown to be absorbed more efficiently by the skin than Cr(III), an observation attributed to a more pronounced rejection of the positively charged Cr(III) ions by the skin barrier. These results were in good agreement with in vitro permeation studies previously reported in the literature in which other analytical techniques were used. Differences observed in the permeation of Cr following the application of aqueous Cr donor solutions and Cr-containing simulated sweat donor solutions are also described.      

Determination of didanosine in maternal plasma, amniotic fluid, fetal and placental tissues by high-performance liquid chromatography-tandem mass spectrometry

A rapid and efficient high-performance liquid chromatography (HPLC)-tandem mass spectrometry method for the determination of didanosine concentrations in maternal rat plasma, amniotic fluid, placental and fetal tissue samples has been developed and validated. Tissue samples were homogenized in optima water and centrifuged. The supernatant was subjected to solid-phase extraction (SPE) prior to analysis. Plasma and amniotic fluid samples were extracted without pretreatment. An Agilent 1100 Series HPLC coupled with a Micromass Quattro II triple quadrupole mass spectrometer was used for all analyses. Chromatographic resolution was achieved on a Nova-Pak phenyl analytical column (2.0 x 150 mm, 4 microm particle size) equipped with a Phenomenex Security-guard phenyl guard cartridge (2.0 x 4.0 mm) using 60% methanol in 10 mm ammonium acetate buffer mobile phase for all matrices at a flow rate of 0.15 mL/min. The method yields retention times of 2.9 min for didanosine and 3.0 min for the internal standard, stavudine. Limits of detection were 1 ng/mL for all matrices. Recoveries were 70% or greater for both compounds in the different matrices. Within- and between-run precision (%RSD) and accuracy (%error) was less than 15% for all matrices.     

Extraction and percolation of PAEs from chemical protective gloves

Phthalates (PAEs) have high solubility in polymers and are added as plasticisers to increase the flexibility and plasticity of polymeric materials. In this study, methanol, hexane, ethyl ether and acetone were used for the extraction of PAEs from chemical protective gloves at temperatures of 20–80 °C. DEHP (di-2-ethylhexyl phthalate) and DBP (di-n-butyl phthalate) were extracted from neoprene, nitrile and PVC glove samples using the above four solvents. The extraction level of DEHP from the glove samples was proportional to the Log K_ow values of the extraction solvents. This result implied that PAEs were more soluble in non-polar solvents and were likely to be extracted from the gloves. Increasing the extraction temperature resulted in a higher extraction of DBP and DEHP from the gloves. In the ASTM F739 permeation method, the aromatic solvents permeated through the glove samples and dissolved DEHP. If the permeant and DEHP had similar solubility parameters, DEHP was likely to be leached from the gloves. The modelling results indicated that the permeation behaviour of the organic solvent in the PVC glove was non-Fickian diffusion. It was speculated that the plasticiser increased the diffusion coefficients of the permeants in the PVC gloves. This study suggested that the potential dissolution and leaching of PAEs from chemical protective gloves should be a concern for workers who handle organic solvents.     

Improvement in bioavailability of transdermally applied flurbiprofen using tulsi (Ocimum sanctum) and turpentine oil

Penetration enhancing potential of tulsi and turpentine oil on transdermal delivery of flurbiprofen, a potent non-steroidal anti-inflammatory agent, was investigated. The transdermal permeation rate of flurbiprofen across the rat abdominal skin from binary solvent mixture composition of propylene glycol (PG):isopropyl alcohol (IPA) (30:70%, v/v) was 98.88 microg/cm(2)/h, significantly higher than other binary solvent mixtures. The corresponding steady state plasma concentration, 0.71 microg/ml, was much lower than required steady state plasma concentration of 3-5 microg/ml. Hence influence of tulsi and turpentine oil in the optimized binary solvent mixture along with the increased drug load on the flurbiprofen permeation was evaluated. The magnitude of the flux enhancement factor with turpentine oil and tulsi oil was 2.4 and 2.0 respectively at 5% (v/v) concentration beyond which there was no significant increase in the flux. Addition of 2% (w/v) hydroxypropyl methylcellulose (HPMC), as a thickening agent, resulted in desired consistency for the fabrication of patch with insignificant effect on permeation rate of flurbiprofen. The reservoir type of transdermal patch formulation, fabricated by encapsulating the flurbiprofen reservoir solution within a shallow compartment moulded from polyester backing film and microporous ethyl vinyl acetate membrane, did not modulate the skin permeation of flurbiprofen through rat skin in case of turpentine formulations whereas flux of formulations with tulsi oil was significantly altered. The influence of penetration enhancer and solvents on the anatomical structure of the rat skin was studied. Enhancement properties exhibited by turpentine oil and tulsi oil in optimized binary solvent mixture were superior as compared to solvent treated and normal control groups with negligible skin irritation. The fabricated transdermal patches were found to be stable. The bioavailability of flurbiprofen with reference to orally administered flurbiprofen in albino rats was found to increase by 2.97, 3.80 and 5.56 times with transdermal patch formulation without enhancer, tulsi and turpentine oil formulations, respectively. The results were confirmed by pharmacodynamic studies in rat edema inflammation model.