Determination of parabens in pharmaceutical and cosmetic products by capillary electrophoresis

A validated capillary electrophoresis method for the determination of parabens-methyl, ethyl, propyl, and butyl is described. The optimum results were obtained by the use of a run buffer containing 20 mM borate buffer (pH 9.0) with 10% methanol and by applying a voltage of 20 kV, a low hydrodynamic injection of 10 s, and detecting the signals at 200 nm. Moxifloxazin was used as an internal standard. All parabens were separated within 10 min. The method showed a good repeatability, linearity, and sensitivity. It was applied to the determination of parabens in pharmaceutical and certain cosmetic products. The text was submitted by the authors in English.     

Rapid determination of parabens in personal care products by stable isotope GC‐MS/MS with dynamic selected reaction monitoring

In this study, a rapid and sensitive analytical method for the determination of methyl‐, ethyl‐, propyl‐, and butyl esters of para‐hydroxy benzoic acid (parabens) in personal care products was developed and fully validated. Test portions were extracted with methanol followed by vortexing, sonication, centrifugation, and filtration without derivatization. The four parabens were quantified by GC‐MS/MS in the electron ionization mode. Four corresponding isotopically labeled parabens were selected as internal standards, which were added at the beginning of the sample preparation and used to correct for recovery and matrix effects. Sensitivity, extraction efficiency, and recovery of the respective analytes were evaluated. The coefficients of determination (r²) were all greater than 0.995 for the four parabens investigated. The recoveries ranged from 97 to 107% at three spiked levels and a one‐time (single) extraction efficiency greater than 97% was obtained. This method has been applied to screen 26 personal care products. This is the first time that a unique GC‐MS/MS method with dynamic selected reaction monitoring and confirmation of analytes has been used to determine these parabens in cosmetic personal care products.     

Interlaboratory method validation of capillary electrophoresis sodium dodecyl sulfate (CE-SDS) methodology for analysis of mAbs

Capillary electrophoresis sodium dodecyl sulfate (CE-SDS) is an analytical method to assess the purity of proteins, commonly applied to monoclonal antibodies (mAbs) in the biopharmaceutical industry. To address the need to standardize the CE-SDS method in the pharmaceutical industry and to enhance the confidence in method transfer between laboratories operating different commercial capillary electrophoresis (CE) instrument platforms, an interlaboratory CE-SDS method validation was organized involving 13 laboratories in 13 companies on four different types of commercial capillary electrophoresis instruments. In the validation, a commercial mAb therapeutic was used as the sample. The validation process followed the analytical guidelines set by the ICH guidelines (International Conference for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use). The method's precision, accuracy, linearity and range, and limit of quantitation (LOQ) were validated in the study. Variations of all the parameters validated in the study passed the pre-set criteria defined at the beginning of the study. The definition was based on previously published works and the intended application purpose of the CE-SDS method for mAbs. The study proved that the CE-SDS method fits its intended application purpose as a size impurity assay and size heterogeneity characterization assay for mAb therapeutic products. This study is the first time a CE-SDS method is validated by multiple laboratories using different commercial CE instrument platforms and on a commercial mAb therapeutic. Its results will enhance the confidence of the biopharmaceutical industry to develop CE-SDS methods and transfer CE-SDS methods between different laboratories.     

Comparative study for the analysis of parabens by micellar electrokinetic capillary chromatography with and without large-volume sample stacking technique

The separation and determination of four parabens (methyl, ethyl, propyl, and butyl p-hydroxybenzoate) which are commonly used as preservatives in cosmetic products, by micellar electrokinetic capillary chromatography (MEKC) with and without large-volume sample stacking (LVSS) technique were compared. As an effective on-line concentration technique, LVSS was successfully combined with MEKC to determine neutral parabens in an acidic media. The effects of some typical parameters such as sample volume, buffer pH, temperature, and concentration of surfactant were examined. The detection limits for this LVSS-MEKC method were found to be 3.0 × 10⁻⁷ M for each of the parabens based on the signal-to-noise ratio of 3, which were around 300 times lower than normal MEKC technique. The curves of peak response versus concentration were linear from 1.0 × 10⁻⁶ to 5.0 × 10⁻⁵ M with regression coefficients of 0.9987, 0.9960, 0.9925 and 0.9864, respectively. A simple and easy-manipulative sample preparation method was developed and validated by analyzing commercially available cosmetic samples. It was found that with current sample preparation process and instrumentation system, 0.5 g of sample is enough for the analysis of parabens preservatives in cosmetic product with satisfactory results.     

On‐line Concentration and Separation of Parabens by Micellar Electrokinetic Chromatography Using Polymer Solutions

Simultaneous separation and quantification of seven parabens commonly used as preservatives in cosmetic products, by micellar electrokinetic chromatography with a stacking technique has been demonstrated. An effective on‐line concentration strategy involving a combination of sweeping and the use of polymer solutions is a key feature of the proposed method, which successfully determined individual parabens. The analysis parameters such as injection time, pH and concentration of phosphate solution, and concentration of sodium dodecyl sulphate (SDS) and poly(ethylene oxide) (PEO) were examined. The optimum conditions were found to be as follows: a 15 mM phosphate solution (pH 9.5) containing 20 mM SDS for filling the capillary, and for the separation electrolytes, 0.100% PEO (8 MDa) added to the phosphate and SDS solution of the same composition as for the capillary. The entire analysis process was completed in 13 min and a 930‐2200‐fold enhancement factor was achieved. The LODs (S/N = 3) for this approach were in the range from 4.32 to 7.78 nM. The linear range for each paraben was between 50 nM and 5.0 μM (R² > 0.990). The optimized method was then successfully applied to the determination of parabens in commercial cosmetic products.     

Separation of parabens in capillary electrochromatography using poly(styrene-divinylbenzene-methacrylic acid) monolithic column

In this study, a series of poly(styrene-divinylbenzene-methacrylic acid) monolithic capillaries was used as the separation column of CEC for the analyses of parabens in commercial pharmaceutical and cosmetic products. The results showed that the chromatographic characteristics of these analytes were strongly affected by the preparation condition of the monolithic column including monomer content, porogenic solvent composition, and polymerization time. Baseline separations were markedly sped up by lowering the polymerization time without any obvious loss of resolution. Furthermore, mobile-phase composition (pH, ACN, and electrolyte concentration) was also able to effectively improve the separation behavior. Similar to the influence of lowering the polymerization time, retention times for all analytes were significantly shortened in the CEC method by decreasing the electrolyte concentration in the mobile phase.     

Application of Graphene as Solid Phase Extraction Absorbent for the Determination of Parabens in Cosmetic Products by Capillary Electrophoresis

A graphene-based solid phase extraction (SPE) technique was developed for the extraction of parabens (methylparaben, ethylparaben, propylparaben, and butylparaben) in cosmetic samples, followed by determination by capillary electrophoresis (CE). The SPE extraction parameters such as eluent reagent and eluent volume, washing solution, sample pH, and flow-rate of sample loading, were investigated for satisfied recoveries. The running buffer, consisting of 25 mM borate solution (pH 10.0), was used for the separation of four parabens with the CE method within 10 min. The limits of detection were 0.14 mg/L, 0.13 mg/L, 0.15 mg/L, and 0.10 mg/L for methylparaben, ethylparaben, propylparaben, and butylparaben, respectively, and the mean recoveries obtained were between 62.6% and 100.4%. The developed method was used for the determination of parabens in real cosmetic products.     

Multicomponent analytical methodology to control phthalates, synthetic musks, fragrance allergens and preservatives in perfumes

A simple, fast, robust and reliable multicomponent analytical method applicable in control laboratories with a high throughput level has been developed to analyze commercial brands of perfumes. Contents of 52 cosmetic ingredients belonging to different chemical families can be determined in a single run. Instrumental linearity, precision of the method and recovery studies in real samples showed excellent results, so that quantification by external calibration can be effectively applied. Relevant limits of detection and quantification were obtained for all the targets considered, far below the legal requirements and amply adequate for its accurate analytical control.A survey of 70 commercial perfumes and colognes has been performed, in order to verify whether these products complied with the recent changes in European legislation: regarding the maxima allowed concentrations of the ingredients and/or ingredient labelling. All samples contained some of the target ingredients. Several samples do not comply with the regulations concerning the presence of phthalates. Musks data confirmed the trend about the replacement of nitromusks by polycyclic musks; as well as the noticeable introduction of macrocyclic musks in the perfumes composition. The prohibited musk moskene has been detected in one sample in an appreciable concentration. The average number of fragrance allergens is twelve per sample; their presence must be indicated in the list of ingredients when its concentration exceeds the 0.001%, but values higher than 1% have been found in some samples. Preservatives data show that parabens, although ubiquitous in other cosmetic products, are not widely used in perfumery. In contrast, the presence of BHT is indeed widespread. The degree of compliance with the European Regulation on the labelling has been evaluated in a subset of samples, and only about the 38% of the perfumes were properly labelled for the allergens tested.     

Simultaneous determination, by capillary zone electrophoresis, of multiple components of different industrial products

Summary Four parabens (esters of 4-hydroxybenzoic acid), effective preservatives against the growth of bacteria, yeast, and mold in numerous industrial products, have been used in this work as model compounds to demonstrate the resolving power of capillary electrophoresis (CE). The simultaneous determination of methyl-(MP), ethyl-(EP), propyl-(PP), and butylparaben (BP) was achieved by capillary zone electrophoresis (CZE) with UV diode-array detection at 294 nm. When run voltage, temperature, and electrolyte concentration and pH were optimized the most effective separation was achieved within 7 min by use of 50 cm (effective length) fused silica capillary tubing and operation at 25kV and 20°C. Background electrolyte comprising 35 mM tetraborate buffer adjusted to pH 10.0 gave the best results. The limits of detection of the optimized method ranged from 0.65 μg mL⁻¹ for BP to 0.81 μg mL⁻¹ for MP; the relative standard deviation was between 0.35 and 0.50%. These results showed that the method enables the determination of the four parabens in commerially available cosmetic and pharmaceutical preparations containing some of the parabens and in an unidentified canned berry fruit juice.     

Three different spectrophotometric methods manipulating ratio spectra for determination of binary mixture of Amlodipine and Atorvastatin

Three simple, specific, accurate and precise spectrophotometric methods manipulating ratio spectra are developed for the simultaneous determination of Amlodipine besylate (AM) and Atorvastatin calcium (AT) in tablet dosage forms. The first method is first derivative of the ratio spectra ((1)DD), the second is ratio subtraction and the third is the method of mean centering of ratio spectra. The calibration curve is linear over the concentration range of 3-40 and 8-32 μg/ml for AM and AT, respectively. These methods are tested by analyzing synthetic mixtures of the above drugs and they are applied to commercial pharmaceutical preparation of the subjected drugs. Standard deviation is <1.5 in the assay of raw materials and tablets. Methods are validated as per ICH guidelines and accuracy, precision, repeatability and robustness are found to be within the acceptable limit.     

A stability-indicating reversed-phase high performance liquid chromatography method for simultaneous assay of two corticosteroids and estimation of their related compounds in a pharmaceutical injectable formulation

Betamethasone Sodium Phosphate and Betamethasone Acetate are the two corticosteroids active pharmaceutical ingredients (APIs) that are present in the injectable formulation, Celestone Chronodose^® Injection. It is extremely challenging to develop a Quality Control friendly HPLC method to separate all the potential impurities and degradation products of the two APIs from each other using a single HPLC method. A novel stability-indicating reversed-phase HPLC (RP-HPLC) method using two oxo-cyclic organic modifiers in the mobile phase was developed and validated. This method can separate a total of 32 potential impurities and degradation products from the two APIs and also from each other. Peak symmetry and separation efficiency were enhanced by using two chaotropic agents (trifluoroacetic acid and potassium hexafluorophosphate) in the mobile phases of this method. The stability-indicating capability of this method has been demonstrated by analyzing aged and stressed degraded stability samples of the drug product. This method uses an ACE 3 C18 (15 cm × 4.6 mm) HPLC column. The method was validated per ICH guidelines and proved to be suitable for routine QC use.     

Determination of sun-screen agents in cosmetic products by micellar liquid chromatography.

A micellar liquid chromatographic method was developed for the quantitative determination of sun-screen agents in cosmetic products. The qualitative analysis of parabens is also feasible. Excellent linearity was obtained (r = 0.999) and recoveries were generally greater than 98%. A variety of commercial formulations were analyzed.     

Simultaneous separation and determination of process-related substances and degradation products of venlafaxine by reversed-phase HPLC

A simple and rapid gradient RP HPLC method for simultaneous separation and determination of venlafaxine and its related substances in bulk drugs and pharmaceutical formulations has been developed. As many as four process impurities and one degradation product of venlafaxine have been separated on a Kromasil KR100-5C18 (4.6 mm x 250 mm; particle size 5 microm) column with gradient elution using 0.3% diethylamine buffer (pH 3.0) and ACN/methanol (90:10 v/v) as a mobile phase. The column was maintained at 40 degrees C and the eluents were monitored with photo diode array detection at 225 nm. The chromatographic behaviour of all the compounds was examined under variable compositions of different solvents, temperatures, buffer concentrations and pH. The method was validated in terms of accuracy, precision and linearity as per ICH guidelines. The inter- and intraday assay precision was < 4.02% (%RSD) and the recoveries were in the range of 96.19-101.14% with %RSD < 1.15%. The correlation coefficients (r2) for calibration curves of venlafaxine as well as impurities were in the range of 0.9942-0.9999. The proposed RP-LC method was successfully applied to the analysis of commercial formulations and the recoveries of venlafaxine were in the range of 99.32-100.67 with %RSD <0.58%. The method could be of use not only for rapid and routine evaluation of the quality of venlafaxine in bulk drug manufacturing units but also for the detection of its impurities in pharmaceutical formulations. Forced degradation of venlafaxine was carried out under thermal, photo, acidic, basic and peroxide conditions and the acid degradation products were characterized by ESI-MS/MS, 1H NMR and FT-IR spectral data.     

Validation of an Analytical Method for the Simultaneous Determination of Hyaluronic Acid Concentration and Molecular Weight by Size-Exclusion Chromatography

The hyaluronic acid (HA) global market growth can be attributed to its use in medical, cosmetic, and pharmaceutical applications; thus, it is important to have validated, analytical methods to ensure confidence and security of its use (and to save time and resources). In this work, a size-exclusion chromatography method (HPLC-SEC) was validated to determine the concentration and molecular distribution of HA simultaneously. Analytical curves were developed for concentration and molecular weight in the ranges of 100–1000 mg/L and 0.011–2.200 MDa, respectively. The HPLC-SEC method showed repeatability and reproducibility greater than 98% and limits of detection and quantification of 12 and 42 mg/L, respectively, and was successfully applied to the analysis of HA from a bacterial culture, as well as cosmetic, and pharmaceutical products.     

On-line pretreatment and determination of parabens in cosmetic products by combination of flow injection analysis, solid-phase extraction and micellar electrokinetic chromatography

A convenient and automated method for on-line pretreatment and determination of three parabens (i.e. methyl, ethyl and propyl p-hydroxybenzoate) in cosmetic products is proposed by using flow injection analysis (FIA), solid-phase extraction (SPE) and micellar electrokinetic chromatography (MEKC). An improved split–flow interface is used to couple SPE on C₈-bonded silica with MEKC separation, which can avoid running buffer contamination and reduce buffer consumption, especially, containing expensive reagents. The analytes are loaded onto a C₈ column at 0.6 mL/min for 60 s and eluted with a mixed eluent of 40% (v/v) 10 mmol/L sodium tetraborate buffer (pH 9.3) and 60% (v/v) ethanol at 0.75 mL/min. The MEKC separation is accomplished with a running buffer of 20 mmol/L sodium tetraborate (pH 9.3) containing 100 mmol/L sodium dodecyl sulfate (SDS) at 15 kV. For butyl p-hydroxybenzoate did not be detected in the cosmetic products, it was used as an internal standard (IS) added into the real samples. This FIA–SPE–MEKC method using IS allows the sample separation within 12 min and the sample throughput of five samples per hour with the relative standard deviation (R.S.D.) less than 2.3% (n = 5). The limits of detection (LOD) are in the range from 0.07 to 0.1 μg/mL (S/N = 3 and n = 11). The proposed method has been used to determine three parabens in real cosmetic products satisfactorily.     

Determination of parabens in cosmetic products by solid-phase microextraction of poly(ethylene glycol) diacrylate thin film on fibers and ultra high-speed liquid chromatography with diode array detector

The fabrication of a solid-phase microextraction (SPME) fiber through UV-induced polymerization of poly(ethylene glycol) diacrylate (PEG-DA) for determination of parabens in cosmetic products is presented in this work. The PEG-DA polymer coating was covalently attached to the fiber by introducing a surface modification with 3-(trichlorosilyl)propyl methacrylate (TPM). The PEG-DA polymer thin film coated on the fiber was homogeneous and wrinkled, which led to an increase of the surface area and high extraction efficiency. The extraction performances of the prepared SPME fibers were assessed by preconcentration of parabens including methylparaben, ethylparaben, propylparaben and benzylparaben from cosmetic products. The analysis was performed on an ultra high-speed liquid chromatography with diode array detector. The prepared SPME fibers exhibited good repeatability (for one fiber) and reproducibility (fiber-to-fiber) with RSDs of 5.4 and 6.9%, respectively. The optimized SPME method supported a wide linear range of 0.50-160 μg/mL and the detection limits for parabens were in the range of 0.12-0.15 μg/mL (S/N=3). The developed method was successfully applied for determination of parabens in cosmetic products with different natures.     

Selective and quantitative analysis of 4-hydroxybenzoate preservatives by microemulsion electrokinetic chromatography

A microemulsion electrokinetic chromatography (MEEKC) method has been developed and validated for the determination of 4-hydroxybenzoates and their impurities. These materials are commonly known as parabens and are widely used as preservatives in foods, cosmetics and pharmaceuticals. The method was shown to be selective and quantitative for the methyl, ethyl, propyl and butyl esters of 4-hydroxybenzoic acid. An internal standard, 4-hydroxyacetophenone, was employed to improve injection precision and detector linearity. In addition, 4-hydroxybenzoic acid, the major degradent, could also be monitored at the 0.1% (m/m) level. The method was successfully validated for assay and detection of the impurities in 4-hydroxybenzoic acid methyl ester and 4-hydroxybenzoic acid propyl ester samples and for the determination of 4-hydroxybenzoic acid methyl ester in a liquid pharmaceutical formulation. The determination of paraben content by MEEKC in a liquid sample was consistent with HPLC analysis. This work is the first reported validated MEEKC method and shows that the methodology can be successfully implemented into routine quality control testing.     

Simultaneous analysis of parabens in cosmetic products by stir bar sorptive extraction and liquid chromatography

A sensitive and precise stir bar sorptive extraction (SBSE) combined with LC (SBSE/LC) analysis is described for simultaneous determination of methyl, ethyl, propyl, and butyl parabens in commercial cosmetic products in agreement with the European Union Cosmetics Directive 76/768/EEC. Important factors in the optimization of SBSE efficiency are discussed, such as time and temperature of extraction, pH, and ionic strength of the sample, matrix effects, and liquid desorption conditions by different modes (magnetic stirring, ultrasonic). The LOQs of the SBSE/LC method ranged from 30 to 200 ng/mg, with linear response over a dynamic range, from the LOQ to 2.5 μg/mg, with a coefficient of determination higher than 0.993. The interday precision of the SBSE/LC method presented a coefficient of variation lower than 5%. The effectiveness of the proposed method was proven for analysis of commercial cosmetic products such as body creams, antiperspirant creams, and sunscreens.     

Development and validation of a TLC-densitometry method for quantitative analysis of nefopam hydrochloride beside its degradation products

A quantitative densitometric thin-layer chromatographic method for determination of nefopam hydrochloride in pharmaceutical preparations has been established and validated. Nefopam from the formulations was separated and identified on silica gel 60 F₂₅₄ TLC plates with chloroform-methanol-glacial acetic acid (9: 2: 0.1, v/v/v) as mobile phase. Densitometric quantification was performed at absorbance maximum 266 nm. The method was validated for linearity, sensitivity, precision and recovery in accordance with ICH guidelines. The presented method is selective and specific with potential application in pharmaceutical analysis. Nefopam hydrochloride was subjected to acidic and alkaline hydrolysis at different temperatures. As the method could effectively separate the drug from its degradation products, it can be employed as a stability indicating one.     

Development and Validation of a Stability-Indicating HPLC Method for Assay of Milbemycin Oxime and Estimation of Its Related Compounds

Chromatographia - A stability-indicating reversed-phase HPLC method for assay of milbemycin oxime (MO) and estimation of its related compounds has been developed and validated as per VICH and ICH...