A simple and rapid method to assess lycopene in multiple layers of skin samples

Topical application of lycopene is a convenient way to restore antioxidants depleted from the skin by UV radiation and achieve protection against premature aging and cancer. In this study, a simple, rapid and reproducible method to quantify lycopene in different skin layers was developed, validated and employed to assess this compound after skin penetration studies. Lycopene was extracted from the stratum corneum (SC) and viable epidermis and dermis (ED) by vortex homogenization and bath sonication in a mixture of acetonitrile and methanol (52:48, v/v). Lycopene was assayed by HPLC using a C₁₈ column, and acetonitrile:methanol (52:48, v/v) as mobile phase. The quantification limit of lycopene in samples of SC and ED was 35 ng/mL and the assay was linear from 35 to 2000 ng/mL. Within‐day and between‐days assays coefficients of variation and relative errors (indicative of precision and accuracy) were less than 15% (or 20% for the limit of quantification). Lycopene recovery from SC and ED was dependent on the spiked concentration: for 50 ng/mL, recoveries were 88.3 and 90.5%; for 100–1000 ng/mL, recoveries were 68.6–74.9%. This method has a potential application for lycopene quantification during formulation development and evaluation in the dermatological field. Copyright © 2009 John Wiley & Sons, Ltd.     

Development of a method for quantitative determination of the cytotoxic agent piplartine (piperlongumine) in multiple skin layers

This study reports the development of a simple and reproducible method, with high rates of recovery, to extract the cytotoxic agent piplartine from skin layers, and a sensitive and rapid UV‐HPLC method for its quantification. Considering the potential of piplartine for topical treatment of skin cancer, this method may find application for formulation development and pharmacokinetics studies to assess cutaneous bioavailability. Porcine skin was employed as a model for human tissue. Piplartine was extracted from the stratum corneum (SC) and remaining viable skin layers (VS) using methanol, vortex homogenization and bath sonication, and subsequently assayed by HPLC using a C₁₈ column, and 1:1 (v/v) acetonitrile–water (adjusted to pH 4.0 with acetic acid 0.1%) as mobile phase. The quantification limit of piplartine was 0.2 μg/mL (0.6 μm ), and the assay was linear up to 5 μg/mL (15.8 μm ), with within‐day and between‐days assay coefficients of variation and relative errors <15%. Piplartine recovery from SC and VS varied from 86 to 96%. The method was suitable to assay samples from skin penetration studies, enabling detection of differences in cutaneous delivery in different skin compartments resulting from treatment with various formulations and time periods.     

Quantification of free fatty acids in human stratum corneum using tandem mass spectrometry and surrogate analyte approach

The free fatty acids (FFAs) are one of the major components of the lipids in the stratum corneum (SC), the uppermost layer of the skin. Relative composition of FFAs has been proposed as a biomarker of the skin barrier status in patients with atopic dermatitis (AD). Here, we developed an LC‐ESI‐MS/MS method for simultaneous quantification of a range of FFAs with long and very long chain length in the SC collected by adhesive tape (D‐Squame). The method, based on derivatization with 2‐bromo‐1‐methylpyridinium iodide and 3‐carbinol‐1‐methylpyridinium iodide, allowed highly sensitive detection and quantification of FFAs using multiple reaction monitoring. For the quantification, we applied a surrogate analyte approach and internal standardization using isotope labeled derivatives of FFAs. Adhesive tapes showed the presence of several FFAs, which are also present in the SC, a problem encountered in previous studies. Therefore, the levels of FFAs in the SC were corrected using C12:0, which was present on the adhesive tape, but not detected in the SC. The method was applied to SC samples from patients with atopic dermatitis and healthy subjects. Quantification using multiple reaction monitoring allowed sufficient sensitivity to analyze FFAs of chain lengths C16–C28 in the SC collected on only one tape strip.     

Celecoxib determination in different layers of skin by a newly developed and validated HPLC‐UV method

A simple, rapid and sensitive analytical procedure for the measurement of celecoxib (CXB) levels in skin samples after in vitro penetration studies was developed and validated. In vitro permeability studies in porcine skin were performed for quantification of CXB at different layers of skin, the stratum corneum (SC) and epidermis plus dermis (EP + D) as well as in the acceptor solution (AS) to assess CXB permeation through skin. CXB was quantified by HPLC using a C₁₈ column and UV detection at 251 nm. The mobile phase was methanol–water 72:28 (v/v) and the flow‐rate was 0.8 mL/min. The CXB retention time was 5 min. The assay was linear for CBX in the concentration range of 0.1–3.0 μg/mL in the AS (drug permeated through skin) and 5.0–50.0 μg/mL for drug retained in SC and [EP + D] in vitro. The linear correlation coefficients for the different calibration curves were equal or greater than 0.99. Intra‐ and inter‐assay variabilities were below 8.0%. Extraction of CXB from skin samples showed recoveries higher than 95.0% after 15 min of ultrasonic sound and centrifugation at 2500 rpm for 3 min. The method was considered appropriate for the assay of CXB in skin samples, after in vitro cutaneous penetration studies. Copyright © 2011 John Wiley & Sons, Ltd.     

Analysis of Low Molecular Weight Carbohydrates in Food and Beverages: A Review

This review presents a study of new advances in chromatographic methods and capillary electrophoresis for the analysis and quantification of carbohydrates in food and drink that have been made in essentially the last seven years (1995–2002). All the main groups of sugars have been considered: monosaccharides, disaccharides, trisaccharides, oligosaccharides, related compounds such as alditols, alditol glycosides, polyols, amino sugars, deoxy sugars, uronic acids and aldonic acids. The chromatographic methods referred to are High Performance Liquid Chromatography (HPLC), Gas Chromatography (GC) and related techniques (AMD-HPTLC: High Performance Thin-Layer Chromatography with Automated Multiple Development). Capillary Electrophoresis (CE) was also discussed.     

Development and Application of a CE Method for Quantification of Phenolic Compounds in Extracts from Buckwheat Herb and in Semi-Solid Formulations Containing the Extracts

Because of the growing interest in plant-derived substances for medical and cosmetic applications, appropriate analytical methods must be developed to ensure the quality of herbal extracts. This paper describes a CE method for the quantification of phenolic compounds in extracts from buckwheat herb. The optimum analytical conditions (60 mM borate buffer, pH 10.0, injection time 10 s) were obtained by variation of injection time, the pH and molarity of the running buffer. Comparison with a previously developed HPLC method showed that the new CE method is comparable in terms of precision, linearity, and limit of detection. It is also much faster and less expensive than the HPLC method. Application of the CE method to different extracts showed that the extracts are rather stable to high temperature but very sensitive to high humidity. It is demonstrated that the CE method is also suitable for determination of rutin in buckwheat extracts incorporated into two different semi-solid formulations.     

Development and validation of a HPLC–UV based method for the extraction and quantification of methotrexate in the skin

An innovative and sensitive HPLC–UV method for the extraction and quantification of methotrexate (MTX) in skin layers was developed and validated. Owing to the physico-chemical characteristics of the drug and the nature of the tissue, it was necessary to use folic acid (FA) as an internal standard for MTX quantification in the dermis. MTX (and FA) analysis was performed on a Phenomenex Jupiter C₁₈ column, using a 50 mm sodium acetate buffer (pH 3.6) and methanol mixture (87:13, v/v) as mobile phase, pumped at 1 ml/min. The absorbance was monitored at 290 nm. The method was selective, linear in the range 0.11–8.49 μg/ml for extraction solvent and 0.05–8.94 μg/ml for pH 7.4 phosphate-buffered saline, precise and accurate, with lower limits of quantitation of 0.11 μg/ml (extraction solvent) and 0.05 μg/ml (pH 7.4 phosphate-buffered saline). The method developed is suitable for the quantification of MTX in skin layers at the end of in vitro permeation experiments; the overall mass balance was 96.5 ± 1.4%, in line with the requirements of the Organisation for Economic Co-operation and Development guideline for the testing of the chemicals (Skin absorption: in vitro method).     

Thermal dependence of Raman descriptors of ceramides. Part II: effect of chains lengths and head group structures

The outermost layer of the mammalian skin, the stratum corneum (SC), represents the main skin barrier. The SC lipids have a very exceptional composition, as the main lipid classes are ceramides (CER), long-chain fatty acids and cholesterol. Information on the function of each CER subclass and on the relation between CER lipid organisation and composition is of great importance to unravel the mechanism controlling the skin barrier function. Raman spectroscopy has been increasingly used for the study of intra- and inter-molecular structures of long-chain lipid compounds. In this study, we employed Raman spectroscopy to evaluate the effect of (1) the chain length and (2) the polar head architecture on the conformational order and organisational behaviour of CERs. The relation between the structure and the stability of the organisation was studied by monitoring the thermotropic response of each CER in the temperature range between 25 and 95 °C. This work enabled the determination of a correlation between the gauche/trans ratio in the νCC region and the state of the lateral packing. Moreover, it was shown that –OH groups in the α position of the fatty acids reduce the stability while long alkyl chains reinforces the intra- and inter-chains order.     

An LC–DAD Fingerprinting Method for Alkaloids,Flavonoids and Styrylpyrones from Cryptocarya mandioccana

An LC–DAD method has been developed in order to evaluate qualitatively and quantitatively quaternary aporphine alkaloids, flavonoid glycosides and styrylpyrones, which are the main secondary metabolites of leaves from C. mandioccana. The chromatographic method was validated considering both internal and external standard quantification methods and showed good performances in terms of selectivity, linearity, precision (repeatability and intermediate precision), limit of detection, limit of quantification, accuracy and stability.

     

Development and validation of a simple HPTLC method for the determination of new hepatitis C subtype 4 antiviral agents in their tablet dosage form

A new, simple, precise, accurate and selective high-performance thin-layer chromatographic (HPTLC) method has been developed and validated for the simultaneous determination of ledipasvir and sofosbuvir in their tablet dosage form. Chromatographic separation was carried out on Merck TLC aluminum sheets of silica gel 60 F₂₅₄ using ethyl acetate:hexane:methanol in the ratio of 8:1.25:0.75 (% v/v) as the mobile phase followed by densitometric measurement at 256 nm. The method was validated in terms of linearity, accuracy, precision, limit of detection, limit of quantification and specificity in accordance with the International Conference on Harmonization (ICH) guidelines. The calibration curve was found to be linear between 60 to 1980 and 45 to 3600 ng/band for ledipasvir and sofosbuvir, respectively, with significantly high value of regression coefficient (r² > 0.9999) with linear and homoscedastic residuals. The limits of detection and quantification were found to be 16.5 and 50 ng/band, respectively, for ledipasvir and 13 and 39.5 ng/band, respectively, for sofosbuvir. Comparative study was performed between the developed HPTLC method and the reported high-performance liquid chromatography (HPLC) method. The quantitative results of the two analytical methods did not show statistically significant difference, whereas the developed HPTLC method is both time- and cost-effective.

     

Spectroscopic and HPLC methods for the determination of alendronate in tablets and urine

Two methods (spectrophotometric and HPLC) have been developed and validated for the analysis of alendronate sodium in tablet dosage form. Both methods depend on the ability of alendronate sodium to react with o-phthalaldehyde (OPA) at basic pH to produce a light-absorbing derivative. The derivative was found to possess absorption maximum at 330 nm where neither the derivatizing agent nor the analyte had any absorption. Thus, spectroscopic method was based on the derivatization-induced absorption of alendronate sodium at 333 nm. The HPLC method was based on separation of the formed derivative from other ingredients in tablets with detection at 333 nm. Both methods were satisfactory with regard to accuracy, prescion and linearity. Moreover, a HPLC method with fluorescence detection (HPLC-FD) was developed for the quantification of alendronate sodium in urine. The method was also based on the derivatization of alendronate with OPA, but fluorescence detection was employed. Linearity, recovery, selectivity, prescision and sensitivity were satisfactory for the proposed HPLC-FD method. Yet a new quantification limit (0.6 ng ml⁻¹) for alendronate in urine was achieved.     

High-performance liquid chromatographic assay for the alpha-melanotropin[4,10] fragment analogue (Melanotan-II) in rat plasma.

A high-performance liquid chromatographic (HPLC) procedure has been developed for the quantification of Melanotan-II (MT-II), a cyclic heptapeptide which promotes rapid tanning of the skin, in rat plasma. The method involves precipitation of plasma proteins followed by direct-injection HPLC with ultraviolet detection. Calibration curves were linear over the range 100-1000 ng/ml for rat plasma. The method is reproducible and reliable with a detection limit of 50 ng/ml in plasma. Within- and between-day precision and accuracy reported as coefficient of variation and relative error, respectively, were < 7%. The application of the assay was successfully demonstrated by quantifying the concentration of MT-II in rat plasma samples following an intravenous dose of 0.3 mg/kg.     

First electrochemical investigation of organophosphorus pesticide azametiphos and its quantification using electroanalytical approach

ABSTRACT

In this work, the electrochemical behaviour and the subsequent development of an analytical procedure for quantification of pesticide azamethiphos, using boron-doped diamond (BDD) electrode are reported for the first time. It was found that azamethiphos electrochemical behaviour is irreversible oxidation at the potential of around 1.70 V, in 1 M nitric acid (pH 0). Also, it was found that potential of this oxidation was not pH dependent which can be attributed to the no proton involvement in electrochemical reaction on the electrode surface. The square wave voltammetric method was most appropriate for azamethiphos quantification. Under optimised experimental conditions, linear working range from 2 to 100 µM was estimated with the detection limit of 0.45 µM. Negligible effect of the possible interfering compound was observed. The obtained results show that the developed analytical methodology can be an adequate replacement for the, up to date, used methods for detection of organophosphorous pesticide.     

Retaining Skin Barrier Function Properties of the Stratum Corneum with Components of the Natural Moisturizing Factor—A Randomized,Placebo-Controlled Double-Blind In Vivo Study

The influence of a topically applied formulation containing components of natural moisturizing factor (NMF) on barrier-related parameters of the stratum corneum (SC) was investigated in vivo using confocal Raman microspectroscopy in a randomized, placebo-controlled double-blind study on 12 volunteers for 14 days. This method allowed for the elucidation of subtle differences between the verum and the placebo even though the components of the verum naturally occur in the SC. This differentiation is not possible non-invasively by conventional methods. In this study, we found that the applied verum and placebo formulations disrupted the equilibrium of water, NMF and lipids in the SC. The adverse effects of the formulation could be mitigated by incorporating it into a simplified supplementation of NMF molecules. As a long-term effect, the amount of strongly bound water increases at 30–40% SC depth (p < 0.05) and the amount of weakly bound water decreases at 30–40% SC depth (p < 0.05) for the verum. This supplement was also unexpectedly able to prevent intercellular lipids (ICL) disorganization in selected depths. In the long term, the verum treatment limited the lateral disorganization of the ICL to the upper 20% SC depth. Further research is required to elucidate the interplay of these factors in the SC, to better understand their contribution to the equilibrium and barrier function of the skin. This understanding of the interaction of these naturally occurring components could help in the future to develop and optimize topical treatments for diseases like psoriasis, atopic dermatitis, ichthyosis where the skin barrier is disrupted.     

Rapid finding and quantification of the major antioxidant in water extracts of three marine drug organisms from China by online HPLC-DAD/MS-DPPH

A method based on high-performance liquid chromatography (HPLC) with diode array detector coupled with electrospray ionisation-mass spectrometry and an online detection system for radical scavenging was established and used to rapidly find and quantify antioxidant compounds in the water extracts of Hippocampus japonicus Kaup, Hippocampus kuda Bleeker and Syngnathus acus Linnaeus. The online screening results revealed the presence of one major radical scavenging compound identified as hypoxanthine by comparison of mass data and retention time with the standard. Subsequently, the developed HPLC method was applied to quantify hypoxanthine in different H. japonicus, H. kuda and S. acus samples. The results indicated that the developed HPLC method is simple and reliable for the quantification of hypoxanthine with a detection limit at 0.002 μg mL(-1), and a high recovery from 96.3% to 102.1%. This method provides a powerful tool for rapid identification and quantification of free radical scavenging compounds in complex marine natural products.     

Determination of Suramin in Plasma and Urine by Ion-Paired Reverse-Phase High-Performance Liquid Chromatography

Abstract

A high-pressure liquid chromatographic (HPLC) assay has been developed for the quantification of the anti-viral drug suramin (SUR) in human plasma and urine. This assay is simple and accurate, using an isocratic mobile phase in a reverse-phase ion-pairing mode.

This assay was developed for the determination of high levels of suramin in the urine and plasma of patients being treated for the acquired immunodefficiency Syndrome (AIDS). The limit of detection of this assay was 0.25 μg/mL. Congo red (CR) was used as the internal standard with a retention time of 5.9 min, while suramin had a retention time of 13 min.     

Indirect resolution of enantiomers of penicillamine by TLC and HPLC using Marfey's reagent and its variants

TLC and HPLC methods were developed for indirect chiral separation of penicillamine (3,3-dimethylcysteine) enantiomers after derivatization with Marfey's reagent (FDNP-Ala-NH(2)) and two of its structural variants, FDNP-Phe-NH(2) and FDNP-Val-NH(2). The binary mobile phase of phenol-water (3:1 v/v) and solvent combinations of acetonitrile and triethylamine phosphate buffer were found to give the best separation in normal and reversed-phase TLC, respectively. The diastereomers were also resolved on a reversed-phase C18 HPLC column with gradient elution of acetonitrile and 0.01 m trifluoroacetic acid. The results due to these three reagents were compared. The method was successful for checking the enantiomeric impurity of l-penicillamine in d-penicillamine and to check the enantiomeric purity of pharmaceutical formulations of d-penicillamine. The method was validated for linearity, repeatability, limit of detection and limit of quantification.     

Determination of Dimenhydrinate Nasal Delivery System in the Blood by RP-LC

Dimenhydrinate (DM) is used therapeutically for the prevention of motion sickness associated with nausea and vomiting. The aim of this investigation is the development of a new, rapid, sensitive, simple and fully validated RP-LC procedure for the suitable assay of DM in pharmaceutical formulation applied in blood samples. The method was validated before the amount determination studies in order to confirm linearity, precision, accuracy, selectivity, determination and quantification limit and consistency. High determination coefficient (r ² = 0.998) of the standard curve drawn in the linearity studies showed that the line equation can be used in the quantification of the DM. Literature was reviewed for the methods based on diphenhydramine (DIP), its metabolite dimenhydrinate, in the in vivo determination of dimenhydrinate amount. The most appropriate method to be used in LC was decided to be the method applied by benefitting from the fluorescence characteristic of DIP. Maximum excitation and emission wavelengths in the developed method were scanned using multiple wavelengths and, maximum excitation and emission wavelengths were found to be 215 and 300 nm, respectively. In the validation study used to prove validity of the method, determination coefficient of the developed standard curve was calculated as 0.998 (RSD %), and line equation was concluded to be appropriate for use in amount determination studies. BBS % (1.06, 1.84 %) values obtained as “<2 %” in the intra-day and inter-day precision studies proved the precision of the method. Selectivity study revealed that other materials used in the formulation did not exhibit absorbance in the same wavelengths. Detection and quantification limit were found as 1 and 5 ng mL⁻¹, respectively. The determination of DM plasma concentrations using the proposed and fully validated LC assay has allowed us to characterize DMPK in the sheep, as well as determine DM relative bioavailability.

     

Photochemical properties of simvastatin and lovastatin induced by radiation

HPLC and UV spectrophotometry were developed and validated for quantitative determination of two antihiperlipoproteinemia drugs, lovastatin and simvastatin. Analytical performance parameters such as linearity, precision, specificity, limit of detection (LOD) and limit of quantification (LOQ) were determined according to the ICH Q1B guidelines. Chromatography was carried out by isocratic technique on a reversed-phase C-18 column. The UV spectroscopy determinations were performed at 238 nm. The linearity of the calibration curves in the desired concentration range was good (r(2)>0.999) for both HPLC and UV methods. The relative standard deviation (RSD) for these methods was <5%. Moreover, the precision obtained with HPLC correlated well with the UV results. The methods proposed are highly sensitive and precise. Other methods used for assessment of the photostability of the substances studied were electron paramagnetic resonance (EPR) and differential scanning calorimetry (DSC).     

Validated HPLC Method for Separation and Determination of Terbutaline Enantiomers

Abstract

A simple, rapid, and validated method for separation and determination of terbutaline enantiomers was developed. Terbutaline was separated and determined on a Vancomycin Chirobiotic V column (250 × 4.6 mm), using a mixture of methanol, acetic acid, and triethylamine (100:0.1:0.1% v/v/v) as a mobile phase at 20°C and at a flow rate of 1 ml/min. The UV detector was set to 276 nm. Acetyl salicylic acid (aspirin) was used as an internal standard. The applied high-performance liquid chromatography (HPLC) method allowed separation and quantification of terbutaline enantiomers with good linearity (r > 0.999) in the studied range. The relative standard deviations (RSD) were 1.10 and 1.32% for the terbutaline enantiomers with accuracy of 99.80 and 99.55. The limit of detection and limit of quantification of terbutaline enantiomers were found to be 0.05 and 0.10 µg · ml^?1, respectively. The method was validated through the parameters of linearity, accuracy, precision, and robustness. The HPLC method was applied for the quantitative determination of terbutaline in pharmaceutical formulations.