Simultaneous Determination of Paracetamol, Caffeine, Guaifenesin and Preservatives in Syrups by Micellar LC

A micellar liquid chromatographic technique allowing the separation and simultaneous determination of the active ingredients paracetamol, caffeine, and guaifenesin, and preservatives benzoic acid, methyl and propyl paraben is described. The separation was effective by using the Kromasil C18 column (150 mm × 4.6 mm, 5 μm) and a mobile phase of 1-butanol:water (1:99, v/v), containing 0.04 M sodium dodecyl sulfate and 0.1% (v/v) trichloroacetic acid, for eluting all compounds. The detection wavelength was set as 260 nm. The column heater was also used, set at 40 °C for these determinations. Under these conditions, separation of the six components was achieved in less than 30 min. The specificity of the method was demonstrated. Analytical characteristics such as limit of detection, limit of quantification, linear range, accuracy, precision (repeatability) and the influence of the various method parameters (robustness study) were evaluated. The developed method was applied to the determination of paracetamol, caffeine, guaifenesin, benzoic acid (sodium benzoate), methyl and propyl paraben in cough-drop syrups. Presented at the International Conference “Modern physical chemistry for advanced materials (devoted to the 100th birthday of Professor Nikolai Izmailov)”, Kharkov, Ukraine, June 2007.     

Separation and Determination of Nitroguanidine and Guanidine Nitrate by HPTLC

High-performance thin-layer chromatography has been used for separation and quantification of nitroguanidine and guanidine nitrate, for online and off-line quality control of synthesis. The compounds were separated on silica gel 60 F₂₅₄ layers with dioxane–tetrahydrofuran 1:1 (v/v), as mobile phase. UV detection was performed at 210 and 265 nm for guanidine nitrate and nitroguanidine, respectively. Quantitative analysis was performed by absorbance densitometry and use of peak area. Validation was performed taking into consideration the special features of the method.     

Simultaneous determination of hydrochlorothiazide and losartan potassium in tablets by high-performance low-pressure chromatography using a multi-syringe burette coupled to a monolithic column

This contribution describes use of a separation method based on on-line coupling of a multisyringe flow system with a chromatographic monolithic column for simultaneous determination of hydrochlorothiazide and losartan potassium in tablets. The system comprised a multisyringe module, three low-pressure solenoid valves, a monolithic C₁₈ column (25 mm × 4.6 mm i.d.), and a diode-array detector. The mobile phase was 10 mmol L⁻¹ potassium dihydrogen phosphate (pH 3.1)-acetonitrile-methanol (65:33:2 v/v/v) at a flow rate 0.8 mL min⁻¹. UV detection was carried out at 226 nm. The multi-syringe chromatographic (MSC) method with UV spectrophotometric detection was optimized and validated. Results from validation were very good. The analysis time was about 400 s. The method was found to be applicable to routine analysis of both compounds in tablets. The coupling of the monolithic columns with a multi-syringe flow-injection analysis manifold provides an excellent and inexpensive tool to solve the separation problems without use of HPLC instrumentation.     

Complete chromatographic separation of steroids, including 17α and 17β-estradiols, using a carbazole-based polymeric organic phase in both reversed and normal-phase HPLC

Poly(2-N-carbazolylethyl acrylate) with terminal trimethoxysilyl groups was prepared as an organic phase and immobilized onto silica. The retention behavior of the column packed with this carbazole-based polymer-immobilized silica (Sil-CEA) was investigated by using various estrogenic steroids and corticoids in both reversed-phase and normal-phase liquid chromatography. As a result, complete separation was confirmed for eight kinds of steroids with Sil-CEA. The most specific separation with Sil-CEA can be emphasized by the high separation factor (e.g., α = 1.39 in methanol–water (7:3, v/v) at 35 °C) for 17α and 17β-estradiols, one of the most difficult pairs of isomers in chromatographic separation, whereas for two kinds of commercially available polymeric ODS columns as references α = 1.01, only, under the same conditions. Because the excellent separation and retention order with Sil-CEA was maintained even in a normal-phase mobile phase such as a hexane–2-propanol, it is estimated that the CEA phase has multiple interaction mechanisms through stronger interactions such as dipole–dipole, carbonyl–π, and hydrogen bonding interactions than the hydrophobic effect expected with ODS.     

Validation of a Stability-Indicating LC Method for Assay of Ezetimibe in Tablets and for Determination of Content Uniformity

A simple, precise, and accurate HPLC method has been developed and validated for assay of ezetimibe in tablets and for determination of content uniformity. Reversed-phase liquid chromatographic separation was achieved by use of phosphoric acid (0.1%, v/v)–acetonitrile 50:50 (v/v) as mobile phase. The method was validated for specificity, linearity, precision, accuracy, robustness, and solution stability. The specificity of the method was determined by assessing interference from the placebo and by stress testing of the drug (forced degradation). Response was a linear function of drug concentration in the range 20–80 μg mL⁻¹ (r = 0.9999). Intraday and interday system and method precision were determined. Accuracy was between 100.8 and 102.7%. The method was found to be robust, and was suitable for assay of ezetimibe in a tablet formulation and for determination of content uniformity. An erratum to this article can be found at     

Determination of Pyrethroid Insecticide Deltamethrin by Micellar Liquid Chromatography with Spectrophotometric Detection

A simple micellar liquid chromatographic technique for deltamethrin determination was developed and validated. The method provided to be suitable for deltamethrin determination in pediculicide shampoo. Kromasil C18 column (150 mm×4.6 mm, 5 μm) and mobile phase −0.12 M sodium dodecyl sulfate with 9% (v/v) 1-butanol were used for deltamethrin separation. Detection wavelength was 265 nm. The retention time was about 15 min. Different validation parameters were evaluated. The specificity of the method was demonstrated. Linearity was established in the range 10–40 μg L⁻¹. The limits of detection and quantitation were 1.06 and 3.22 μg mL⁻¹, respectively. The method showed excellent accuracy (100.6%) and precision (repeatability) gave a relative standard deviation of less than 1%. The influence of the various method parameters (robustness study) was also studied.     

Chromatographic separation and analysis of chloropheniramine maleate, methscopolamine nitrate and phenylephrine hydrochloride in sustained release capsules

Summary A high performance liquid chromatographic method has been developed for the simultaneous determination of chlorpheniramine maleate (CPM), methscoplamine nitrate (MSN) and phenylephrine hydrochloride (PEH) in sustained release capsules. The separation was carried out on a reverse-phase CN-column with use of a mobile phase consisting of 70% (v/v) solution of acetonitrile in water containing 2% (v/v) acetic acid and 0.005M sodium 1-hepatane sulfonate at a flow rate of 2 mL min⁻¹. The eluted peaks were detected at 262 nm. The method is sensitive, accurate and rapid and can be used in the routine analysis of the mixture of the three compounds.     

Optimization of the composition and pH of the mobile phase used for separation and determination of a series of quinolone antibacterials regulated by the European Union

Summary To ensure the safety of human food the European Union (EU) has set tolerance levels for quinolone compounds in animal products, so screening and confirmatory analytical methods are required for monitoring of these drugs. In this work, the proportion of organic modifier and the pH of acetonitrile-water mixtures used as mobile phases were optimized for separation of a group of quinolones. Linear solvation energy relationship (LSER) formalism based on the single solvent polarity parameterE _T ^N was used to predict the chromatographic behaviour of the compounds as a function of the amount of acetonitrile in the mobile phase. Correlation between retention and the pH of aqueous-organic mobile phases has also been used to optimize mobile-phase pH. The optimized mobile phase was a linear gradient starting from 18∶82 (v/v) acetonitrileacetate + formate buffer, pH 2.5. Quality data were determined and were satisfactory. The method detection limit was approximately 10 ng mL⁻¹ for most of the quinolones studied. The proposed mobile phase is compatible with mass spectrometric detection of the substances.     

Determination of preservatives in soft drinks by capillary electrophoresis with ionic liquids as the electrolyte additives

A capillary electrophoresis method for separating preservatives with various ionic liquids as the electrolyte additives has been developed. The performances for separation of the preservatives using five ionic liquids with different anions and different substituted group numbers on imidazole ring were studied. After investigating the influence of the key parameters on the separation (the concentration of ionic liquids, pH, and the concentration of borax), it has been found that the separation efficiency could be improved obviously using the ionic liquids as the electrolyte additives and tested preservatives were baseline separated. The proposed capillary electrophoresis method exhibited favorable quantitative analysis property of the preservatives with good linearity (r² = 0.998), repeatability (relative standard deviations ≤ 3.3%) and high recovery (79.4–117.5%). Furthermore, this feasible and efficient capillary electrophoresis method was applied in detecting the preservatives in soft drinks, introducing a new way for assaying the preservatives in food products.     

Micellar electrokinetic capillary chromatography as an alternative method for determination of hydrocortisone and its most important associated compounds in local pharmaceutical preparations

Summary A new, simple, and accurate micellar electrokinetic chromatographic (MEKC) method is described for quantification of hydrocortisone, hydrocortisone hemisuccinate, hydrocortisone acetate, mystatin, oxytetracycline, Zn-bacitracin, polymyxin B, and lidocaine in ocular and cutaneous pharmaceutical products. The separation was performed at 25°C and 25 kV, with 15mm phosphate +15mm borate buffer, pH 8.2, and 60mm sodium dodecylsulfate (SDS) in 10∶1 (%,v/v) methanol-water as background electrolyte. Under these conditions the analysis time is approximately 23 min. The method has been used for quantification of these compounds in different commercial pharmaceutical products and gave good results when compared with reference spectrophotometric and HPLC methods.     

Development and Validation of an HPLC Method for Determination of Ceftiofur Sodium

An isocratic high-performance liquid chromatographic method has been developed for assay of ceftiofur sodium in drug substance and in sterile powder for injection. Chromatography was performed on a 250 mm × 4.6 mm, 5 μm particle, C₁₈ column with a 78:22 (v/v) mixture of 0.02 m disodium hydrogen phosphate buffer (pH adjusted to 6.0 with 85% orthophosphoric acid) and acetonitrile as mobile phase, at a flow rate of 1.0 mL min⁻¹. The separation was monitored by UV detection at 292 nm. Validation of the method for linearity and range, intra- and inter-day precision, accuracy, specificity, recovery, robustness, and limits of quantification and detection yielded good results. The calibration plot was linear from 20.0–120.0 μg mL⁻¹ and the correlation coefficient was 0.9999. It was shown that ceftiofur was degraded under acidic, alkaline, oxidative, and photolytic conditions. The method was found to be stability-indicating and could be used for routine analysis of ceftiofur sodium for injection.     

Focused Microwave-Assisted Extraction and LC Determination of Ketoprofen in the Presence of Preservatives in a Pharmaceutical Cream Formulation

A simple and rapid open-vessel focused microwave-assisted extraction (FMAE) method followed by LC analysis was developed for the determination of ketoprofen lysine salt in the presence of methyl p-hydroxybenzoate and propyl p-hydroxybenzoate preservatives in topical cream. Extraction were performed in acetone/potassium dihydrogenphosphate (25 mM, pH 3.0) (70:30 v/v) by reaching a target temperature of 65 °C in a 10 min linear ramp. The chromatographic separation was performed on a Discovery RP-Amide C16 column (250 × 4.6 mm I.D., 5 μm particle size). The optimal mobile phase consisted of acetonitrile/potassium dihydrogen phosphate 25 mM adjusted to pH 3.0 with phosphoric acid (50:50 v/v). The complete analytical procedure was validated with regard to limit of quantification, linearity, precision and accuracy. The method was linear over the concentration range of 0.08–0.12 mg mL⁻¹; the relative standard deviations of intra- and inter-day assays were 1.9–2.3 and 1.8% respectively. The limit of quantification was 0.54 μg mL⁻¹. The proposed method shows many advantages as short extraction time, little solvent consumption without requiring further sample clean-up steps before liquid chromatographic analysis and is proposed for vast scale screening of cream dosage forms aimed to the detection of counterfeit and substandard drugs.

     

Simultaneous Determination of Olanzapine and Fluoxetine by HPLC

A rapid, specific reversed phase HPLC method has been developed for simultaneous determination of olanzapine and fluoxetine in their formulations. Chromatographic separation of these two pharmaceuticals was carried out on an Inertsil C₁₈ reversed phase column (150 mm × 4.6 mm, 5 μm) with a 40:30:30 (v/v/v) mixture of 9.5 mM sodium dihydrogen phosphate (pH adjusted to 6.8 ± 0.1 with triethylamine), acetonitrile and methanol as mobile phase. The flow rate 1.2 mL min⁻¹ and the analytes are monitored at 225 nm. Paroxetine was used as internal standard. The assay results were linear from 25 to 75 μg mL⁻¹ for olanzapine (r ² ≥ 0.995) and 100–300 μg mL⁻¹ for fluoxetine (r ² ≥ 0.995), showed intra- and inter-day precision less than 1.0%, and accuracy of 97.7–99.1% and 97.9–99.0%. LOQ was 0.005 and 0.001 μg mL⁻¹ for olanzapine and fluoxetine, respectively. Separation was complete in less than 10 min. Validation of the method showed it to be robust, precise, accurate and linear over the range of analysis.     

Simultaneous HPTLC Determination of Escitalopram Oxalate and Clonazepam in Combined Tablets

A new, simple high-performance thin-layer chromatographic method has been established and validated for simultaneous determination of escitalopram oxalate and clonazepam in a combined tablet dosage form. The drugs were separated on aluminum plates precoated with silica gel 60 F₂₅₄; toluene–ethyl acetate–triethylamine 7:3.5:3 (v/v) was used as mobile phase. Quantitative analysis was performed by densitometric scanning at 258 nm. The method was validated for linearity, accuracy, precision, and robustness. The calibration plot was linear over the ranges 250–2,500 and 50–500 ng band⁻¹ for escitalopram oxalate and clonazepam, respectively. The method was successfully applied to the analysis of drugs in a pharmaceutical formulation.     

A Validated Chiral LC Method for the Enantiomeric Separation of Repaglinide on Amylose Based Stationary Phase

A simple, isocratic, normal phase chiral HPLC method was developed and validated for the enantiomeric separation of repaglinide, (S)-(+)-2-ethoxy-4-N [1-(2-piperidinophenyl)-3-methyl-1-butyl] aminocarbonylmethyl] benzoic acid, an antidiabetic in bulk drug substance. The enantiomers of repaglinide were resolved on a ChiralPak AD-H (amylose based stationary phase) column using a mobile phase consisting of n-hexane: 2-propanol:trifluoroacetic acid (95:5:0.2 v/v/v) at a flow rate of 1.0 mL min⁻¹. The resolution between the enantiomers was found to be not >3.5 in optimized method. The presence of trifluoroacetic acid in the mobile phase played an important role, in enhancing chromatographic efficiency and resolution between the enantiomers. The developed method was extensively validated and proved to be robust. The calibration curve for (R)-enantiomer showed excellent linearity over the concentration range of 900 ng mL⁻¹ (LOQ) to 6,000 ng mL⁻¹. The limit of detection and limit of quantification for (R)-enantiomer were 300 and 900 ng mL⁻¹, respectively. The percentage recovery of the (R)-enantiomer ranged between 98.3 and 101.05% in bulk drug samples of repaglinide. Repaglinide sample solution and mobile phase were found to be stable up to 48 h. The developed method was found to be enantioselective, accurate, precise and suitable for quantitative determination of (R)-enantiomer in bulk drug substance.     

Quantitative Determination of Four Water-Soluble Compounds in Herbal Drugs from Lamiaceae Using Different Chromatographic Techniques

HPTLC-densitometric and HPLC–UV techniques were used for qualitative and quantitative determination of luteolin-7-O-glucuronide, lithospermic acid, rosmarinic acid and caffeic acid in several herbal drugs from the Lamiaceae family: Thymi herba, Serpylli herba, Majoranae herba and Menthae piperitae folium. Unmodified silica gel (HPTLC Si60) and silica gel chemically modified with aminopropyl groups (HPTLC NH₂) were used during the investigation process. Among HPTLC methods the best resolution and selectivity was achieved with mobile phases: diisopropyl ether–acetone–formic acid–water (50:30:10:10, v/v/v/v) and acetone–formic acid (85:15, v/v), respectively. Plates were densitometrically evaluated. Contents of analyzed compounds in the studied aqueous extracts prepared from herbal drugs were established using both techniques. The results from the HPTLC-densitometric analysis have been compared with those from HPLC–UV on a C18 column with acetonitrile–water–formic acid as a mobile phase. The chromatographic methods were validated for linearity, LOD, LOQ, repeatability, intermediate precision and recovery. An analysis of variance showed that the HPTLC-densitometric and HPLC–UV methods are equivalent and sufficiently precise for the estimation of polyphenolic compounds mentioned above, in investigated herbal drugs. All of the suggested methods (HPTLC NH₂, HPTLC Si60 and HPLC RP18) give results with good agreement.     

Simple LC Method with Chemiluminescence Detection for Simultaneous Determination of Arbutin and l-Ascorbic Acid in Whitening Cosmetics

Simultaneous determination of arbutin (ART) and l-ascorbic acid (AA) by HPLC with chemiluminescence detection is proposed for the first time. This method is based on the CL reaction of acidic potassium permanganate with ART and AA in the presence of formaldehyde as enhancer. The separation was performed on a C₁₈ column with a 90:10 (v/v) mixture of 0.02 M phosphate buffer and methanol as mobile phase. The effects of several conditions on HPLC resolution and CL emission were studied systematically. The linear ranges were 0.5–50 and 1–200 μg mL⁻¹ for ART and AA, respectively. The detection limits were 0.2 and 0.3 μg mL⁻¹, respectively. The method was successfully applied to the determination of ART and AA in whitening cosmetics.     

Development and validation of a sensitive,simple, and rapid method for simultaneous quantitation of atorvastatin and its acid and lactone metabolites by liquid chromatography-tandem mass spectrometry (LC-MS/MS)

The aim of the proposed work was to develop and validate a simple and sensitive assay for the analysis of atorvastatin (ATV) acid, ortho- and para-hydroxy-ATV, ATV lactone, and ortho- and para-hydroxy-ATV lactone in human plasma using liquid chromatography-tandem mass spectrometry. All six analytes and corresponding deuterium (d5)-labeled internal standards were extracted from 50 μL of human plasma by protein precipitation. The chromatographic separation of analytes was achieved using a Zorbax-SB Phenyl column (2.1 mm × 100 mm, 3.5 μm). The mobile phase consisted of a gradient mixture of 0.1% v/v glacial acetic acid in 10% v/v methanol in water (solvent A) and 40% v/v methanol in acetonitrile (solvent B). All analytes including ortho- and para-hydroxy metabolites were baseline-separated within 7.0 min using a flow rate of 0.35 mL/min. Mass spectrometry detection was carried out in positive electrospray ionization mode, with multiple-reaction monitoring scan. The calibration curves for all analytes were linear (R ² ≥ 0.9975, n = 3) over the concentration range of 0.05–100 ng/mL and with lower limit of quantitation of 0.05 ng/mL. Mean extraction recoveries ranged between 88.6–111%. Intra- and inter-run mean percent accuracy were between 85–115% and percent imprecision was ≤ 15%. Stability studies revealed that ATV acid and lactone forms were stable in plasma during bench top (6 h on ice-water slurry), at the end of three successive freeze and thaw cycles and at −80 °C for 3 months. The method was successfully applied in a clinical study to determine concentrations of ATV and its metabolites over 12 h post-dose in patients receiving atorvastatin.     

Determination of synthetic antioxidants in dairy products and dietetic supplements by micellar liquid chromatography with direct sample injection

Summary A simple and rapid HPLC method for the determination of synthetic antioxidants (propyl gallate, tert-butylhydroquinone, 2,4,5-trihydroxybutyrophenone, nordihydroguaiaretic acid, octyl gallate, 3-tert-butyl-4-hydroxyanisole and dodecyl gallate) in powdered and liquid milk, cream of milk and dietetic supplements is described. The samples are diluted or solved in a micellar solution, filtered and directly injected. The retention behavior of the antioxidants on a C18 column, with micellar mobile phases containing SDS (0.05–0.15 M), n-propanol (1–9%, v/v) and 10 mM phosphate at pH 3, has been studied by using mathematical models. Retention is predicted with errors below 3%. To optimize the mobile phase composition, a procedure which takes into account the position and shape of the peaks is applied. The optimized mobile phase, which contains 0.090 M SDS and 6.6% n-propanol, allows the separation of six antioxidants in less than 13 min. Calibration curves are linear (r>0.9998) and the limits of detection range from 0.05 to 0.3 ng antioxidant, which correspond to concentrations well below the levels allowed in foods. Repeatabilities for standards containing 5 μg mL⁻¹ ranged from 0.2 to 1.6%.     

Insights into the Retention Mechanisms on Perfluorohexylpropylsiloxane-Bonded (Fluophase-RP) and Octadecylsiloxane-Bonded (Betasil C18) Stationary Phases Based on the Same Silica Substrate in RP-LC

The solvation parameter model is used to elucidate the retention mechanism on a perfluorohexylpropylsiloxane-bonded (Fluophase RP) and octadecylsiloxane-bonded (Betasil C18) stationary phases based on the same silica substrate with acetonitrile–water and methanol–water mobile phase compositions. Dewetting affects the retention properties of Fluophase RP at mobile phase compositions containing less than 20% (v/v) acetonitrile or 40% (v/v) methanol. It results in a loss of retention due to an unfavorable change in the phase ratio as well as changes in specific intermolecular interactions. Steric repulsion reduces retention of bulky solutes on fully solvated Betasil C18 with methanol–water (but not acetonitrile–water) mobile phase compositions but is not important for Fluophase RP. The retention of weak bases is affected by ion-exchange interactions on Fluophase RP with acetonitrile–water, and to a lesser extent, methanol-water mobile phases but these are weak at best for Betasil C18. The system constants of the solvation parameter model and retention factor scatter plots are used to compare selectivity differences for Fluophase RP, Betasil C18 and a perfluorophenylpropylsiloxane-bonded silica stationary phase Discovery HS F5 for conditions where incomplete solvation, steric repulsion and ion-exchange do not significantly contribute to the retention mechanism. Lower retention on Fluophase RP results from weaker dispersion and/or higher cohesion moderated to different extents by polar interactions since solvated Fluophase RP is a stronger hydrogen-bond acid and more dipolar/polarizable than Betasil C18. Retention factors for acetonitrile–water mobile phases are highly correlated for Fluophase RP and Betasil C18 except for compounds with a large excess molar refraction and weak hydrogen-bonding capability. Selectivity differences are more significant for methanol–water mobile phases. Retention factors on Fluophase RP are strongly correlated with those on Discovery HSF5 for acetonitrile–water mobile phases while methanol–water mobile phases retention on Fluophase RP is a poor predictor of the retention order on Discovery HS F5.