Application of liquid chromatography to the simultaneous determination of acetylsalicylic acid, caffeine, codeine, paracetamol, pyridoxine, and thiamine in pharmaceutical preparations.

This paper describes a rapid reversed-phase liquid chromatographic method, with UV detection, for the simultaneous determination of acetylsalicylic acid, caffeine, codeine, paracetamol, pyridoxine, and thiamine in pharmaceutical preparations. A reversed-phase C18 Nucleosil column is used. The mobile phase consists of 2 successive eluants: water (5 min) and acetonitrile-water (75 + 25, v/v; 9 min), both adjusted to pH 2.1 with phosphoric acid. Before determination acetylsalicylic acid is completely converted to salicylic acid by alkaline hydrolysis. Salicylic acid, caffeine, paracetamol, pyridoxine, and thiamine are all detected at 285 nm, whereas codeine is detected at 240 nm. Calibration curves were linear for salicylic acid, caffeine, paracetamol, and pyridoxine in the range of 50-500 mg/L, and for codeine and thiamine in the range of 50-1000 mg/L. The method was applied to the analysis of 13 fortified commercial pharmaceutical preparations. Recoveries ranged from 92.6 to 105.5%, with relative standard deviations of 1.1-5.8%.     

Reversed-phase capillary electrochromatography for the simultaneous determination of acetylsalicylic acid, paracetamol, and caffeine in analgesic tablets

The separation and simultaneous determination of caffeine, paracetamol, and acetylsalicylic acid in two analgesic tablet formulations was investigated by capillary electrochromatography (CEC). The effect of mobile phase composition on the separation and peak efficiency of the three analytes was studied and evaluated; in particular, the influence of buffer type, buffer pH, and acetonitrile content of the mobile phase was investigated. The analyses were carried out under optimized separation conditions, using a full-packed silica capillary (75 microm ID; 30.0 cm and 21.5 cm total and effective lengths, respectively) with a 5 microm C8 stationary phase. A mixture of 25 mM ammonium formate at pH 3.0 and acetonitrile (30:70 v/v) was used as the mobile phase. UV detection was at 210 nm. Good linearity was found in the range of 50-200, 20-160, and 4-20 microg/mL for acetylsalicylic acid (r2=0.9988), paracetamol (r2=0.9990) and caffeine (r2=0.9990), respectively. Intermediate precision (RSD interday) as low as 0.1-0.8% was found for retention times, while the RSD values for the peak area ratios (Aanalyte/AIS) were in the range of 1.9-2.9%. The optimized CEC method was applied to the analysis of the studied compounds present in commercial tablets.     

Simple determination of betamethasone and chloramphenicol in a pharmaceutical preparation using a short monolithic column coupled to a sequential injection system

This contribution describes the use of a new separation method based on a reversed-phase sequential injection chromatography (SIC) technique for simultaneous determination of chloramphenicol and betamethasone in pharmaceutical eye drops. A short monolithic column coupled with a sequential injection analysis (SIA) system enabled separation of two compounds in one step. A Chromolith Flash RP-18e, 25 x 4.6 mm column with a 5 mm precolumn (Merck, Germany) and a FIA1ab 3000 system (USA) with a 6-port selection valve and 5 mL syringe were used for sequential injection chromatographic separations in this study. The mobile phase used was acetonitrile-water (30:80, v/v), flow rate 0.48 mL/min; UV detection was at two wavelengths, i.e., 241 and 278 nm (absorption maxima of betamethasone and chloramphenicol, respectively). The basic validation parameters showed good results: linearity of determination for both compounds including internal standard (propylparaben) >0.999; repeatability of determination (RSD) in the range 0.8-1.7% at two different concentration levels, and detection limits in the range 0.5-1.0 mg/mL. The chromatographic resolution between compound peaks was greater than 2.1 and the analysis time was less than 8 min under optimal conditions. The developed sequential injection chromatography method was compared with the HPLC method and was found to be applicable for routine analysis of active compounds in pharmaceutical preparations.     

Determination of caffeine and associated compounds in food, beverages, natural products, pharmaceuticals, and cosmetics by micellar electrokinetic capillary chromatography

A method is described for quantitating caffeine, theobromine, theophylline, paracetamol, propyphenazone, acetylsalicylic acid, salicylic acid, and codeine phosphate in corresponding real samples of food, beverages, natural products, pharmaceuticals, and cosmetic preparations by micellar electrokinetic capillary chromatography. The separation is carried out at 25 degrees C and 25 kV, using a 20mM phosphate buffer (pH 9.0), 80mM sodium dodecyl sulfate, and 7.5% (v/v) acetonitrile. UV detection is at 210 nm. The method is shown to be specific, accurate (recoveries over the range 98.9-101.2%), linear over the tested range (correlation coefficients >/= 0.9993), and precise (relative standard deviation below 2.1%). The method is applied for the quantitative analysis of these compounds in different foods, beverages, natural products, pharmaceuticals, and cosmetic products.     

Determination of glucosamine and carisoprodol in pharmaceutical formulations by LC with pre-column derivatization and UV detection

A simple and reliable precolumn derivatization liquid chromatography method with ultraviolet detection has been developed and validated for the analysis of glucosamine (GS) in various dietary supplement formulations and raw materials. Additionally, the proposed method was used for analysis of carisoprodol (CR) found in ternary mixture with paracetamol (PR) and caffeine (CF). The linearity ranges were 1-100 μg/mL for GS, 1-150 μg/mL for CR, PR and CF. Derivatization was used with 1,2-naphthoquinone-4-sulphonic acid sodium salt in the presence of borate buffer. Chromatographic separation of GS-naphthoquinone derivative was achieved by using a mixture of acetonitrile and water (pH 7.3 adjusted with 0.1 M NaOH) in the ratio 10:90, v/v and flow-rate of 1.0 mL/min. UV detection was carried out at 280 nm. For PR, CF, and CR-naphthoquinone derivative, the chromatographic separation was achieved by using mixture of acetonitrile and 20 mM KH(2)PO(4) (pH 3.0 adjusted with phosphoric acid) in the ratio 20:80, v/v and flow-rate of 1.0 mL/min. UV detection was carried out at 275 nm. The limits of detection were 37.2, 35.9, 30.4 and 40.0 ng/mL for GS, CR, PR and CF, respectively.     

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.     

A porous graphitized carbon column HPLC method for the quantification of paracetamol, pseudoephedrine, and chlorpheniramine in a pharmaceutical formulation

A simple, rapid, and stability-indicating HPLC method has been developed, fully validated, and applied to the quantification of paracetamol, pseudoephedrine hydrochloride, and chlorpheniramine maleate in a pharmaceutical formulation, using hydrochlorothiazide as an internal standard. Chromatographic separation was achieved isocratically on an RP porous graphitized carbon analytical column (125 x 2.1 mm id, particle size 5 microm) using 5.0 mM ammonium acetate-acetonitrile (35 + 65, v/v) mobile phase at a flow rate of 0.50 mL/min. UV spectrophotometric detection at 220 nm was used. The method had linear calibration curves over the range of 30-70 microg/mL for paracetamol, 1.8-4.2 microg/mL for pseudoephedrine hydrochloride, and 120-280 ng/mL for chlorpheniramine maleate. The intraday and interday RSD values were less than 3.2% for all compounds, while the relative error was less than 2.9%. Accelerated stability studies performed under various stress conditions proved the selectivity of the method. The developed method was applied successfully to QC and content uniformity tests of commercial tablets.     

Development and validation of stability-indicating HPLC method for the simultaneous determination of paracetamol, tizanidine, and diclofenac in pharmaceuticals and human serum

A method is described for the simultaneous determination of paracetamol, tizanidine, and diclofenac in mixtures. The method was based on HPLC separation of the three drugs followed by UV detection at 254 nm. The separation was carried out on a Hypersil ODS, C18 (250 x 4.6 mm id, 10 microm particle size) column using the mobile phase aqueous 0.2% ammonium carbonate-methanol (60 + 40, v/v) at a flow rate of 1 mL/min. The linear regression analysis data were used for the regression curve in the range of 170-10 000 ng/mL for paracetamol, 120-10 000 ng/mL for tizanidine, and 20-10 000 ng/mL for diclofenac. No chromatographic interference from tablet excipients was found. In order to check the selectivity of the proposed method, degradation studies were carried out using hydrolysis (acid, basic, and neutral), thermolysis, and oxidation. The developed method, after being validated in terms of precision, robustness, recovery, LOD, and LOQ, was successively applied to the analysis of pharmaceutical formulations and human serum.     

Sequential injection chromatographic determination of ambroxol hydrochloride and doxycycline in pharmaceutical preparations

A new separation method based on a novel reversed-phase sequential injection chromatography (SIC) technique was used for simultaneous determination of ambroxol hydrochloride and doxycycline in pharmaceutical preparations in this contribution.The coupling of short monolith with SIA system results in an implementation of separation step to until no-separation low-pressure method.A Chromolith^® Flash RP-18e, 25-4.6 mm column (Merck, Germany) and a FIAlab^® 3000 system (USA) with a six-port selection valve and 5 ml syringe were used for sequential injection chromatographic separations in our study. The mobile phase used was acetonitrile-water (20:90, v/v), pH 2.5 adjusted with 98% phosphoric acid, flow rate 0.48 ml min⁻¹, UV detection was at 213 nm.The validation parameters have shown good results: linearity of determination for both compounds including internal standard (ethylparaben) >0.999; repeatability of determination (R.S.D.) in the range 0.5-5.4% at three different concentration levels, detection limits in the range 0.5-2.0 μg ml⁻¹, and recovery from the pharmaceutical preparation in the range 99.3-99.9%. The chromatographic resolution between peak compounds was >5.0 and analysis time was <9 min under the optimal conditions. The method was found to be applicable for routine analysis of the active compounds ambroxol hydrochloride and doxycycline in various pharmaceutical preparations.     

Determination of active ingredients in cough-cold preparations by micellar liquid chromatography

The chromatographic behaviour of some active ingredients in cough-cold pharmaceutical preparations, the antihistamine chlorpheniramine (or the dextro enantiomer dexchlorpheniramine), and the phenethylamines phenylephrine, phenylpropanolamine and pseudoephedrine, has been studied using a C(18) column, micellar mobile phases of sodium dodecyl sulphate (SDS) and pentanol, and with UV detection. All possible combinations of chlorpheniramine/phenethylamine were resolved and determined using a mobile phase of 0.15 M SDS-6% (v/v) pentanol at pH 7, with analysis time below 7 min. Repeatabilities and within laboratory precisions were evaluated at four different drug concentrations in the range 0.5-25 mug ml(-1) (n=5), resulting RSDs below 1.6%. The drug amounts found in the analysis of 14 commercialised preparations agreed with those declared by the manufacturers within the tolerance limits, and with those obtained using an aqueous 60% (v/v) methanol reference mobile phase. No interference was observed from other accompanying drugs such as acetylsalicylic acid, ascorbic acid, betamethasone, caffeine, codeine phosphate, diphenhydramine, lactose, paracetamol, and prednisolone. The studied combinations required a rather high amount of methanol in conventional RPLC to be eluted from the column. In contrast, the proposed procedure used a much lower amount of organic solvent (pentanol), which is highly retained in the SDS solution, being also less toxic than methanol.     

Simultaneous estimation of acetylsalicylic acid and clopidogrel bisulfate in pure powder and tablet formulations by high-performance column liquid chromatography and high-performance thin-layer chromatography

This paper describes validated high-performance column liquid chromatographic (HPLC) and high-performance thin-layer chromatographic (HPTLC) methods for simultaneous estimation of acetylsalicylic acid (ASA) and clopidogrel bisulfate (CLP) in pure powder and formulations. The HPLC separation was achieved on a Nucleosil C8 column (150 mm length x 4.6 mm id, 5 microm particle size) using acetonitrile-phosphate buffer, pH 3.0 (55 + 45, v/v) mobile phase at a flow rate of 1.0 mL/min at ambient temperature. The HPTLC separation was achieved on an aluminum-backed layer of silica gel 60F254 using ethyl acetate-methanol-toluene-glacial acetic acid (5.0 + 1.0 + 4.0 + 0.1, v/v/v/v) mobile phase. Quantitation was achieved with UV detection at 235 nm over the concentration range 4-24 microg/mL for both drugs, with mean recoveries of 99.98 +/- 0.28 and 100.16 +/- 0.66% for ASA and CLP, respectively, using the HPLC method. Quantitation was achieved with UV detection at 235 nm over the concentration range of 400-1400 ng/spot for both drugs, with mean recoveries of 99.93 +/- 0.55 and 100.21 +/- 0.83% for ASA and CLP, respectively, using the HPTLC method. These methods are simple, precise, and sensitive, and they are applicable for the simultaneous determination of ASA and CLP in pure powder and formulations.     

Simultaneous HPLC determination of caffeine, theobromine, and theophylline in food, drinks, and herbal products

A rapid and selective high-performance liquid chromatographic (HPLC) method is developed for the separation and determination of caffeine, theobromine, and theophylline. The chromatography is performed on a Zorbax Eclipse XDB-C8 column (4.6 x 150 mm i.d., 5-microm particle size) at 25 degrees C, with a mobile phase of water-THF (0.1% THF in water, pH 8)-acetonitrile (90:10, v/v). The flow rate is 0.8 mL/min, and detection is by UV at 273 nm. This method permits the simultaneous determination of caffeine, theobromine, and theophylline in food, drinks, and herbal products with detection limits of 0.07-0.2 mg/L and recoveries of 100.20-100.42%. Correlation coefficients, for the calibration curves in the linear range of 0.2-100 mg/L, are greater than 0.9999 for all compounds. The within- and between-day precision is determined for both retention times and peak area. The data suggests that the proposed HPLC method can be used for routine quality control of food, drinks, and herbal products.     

Determination of preservatives in cosmetics and food samples by micellar liquid chromatography

An analytical procedure has been developed for the analysis of benzoic acid, p-hydroxybenzoic acid, methyl-, ethyl-, propyl-, isopropyl-, and butyl esters of p-hydroxybenzoic acid by micellar liquid chromatography. After dilution in n-propanol the sample was directly injected onto a Lichrosorb ODS, 5 microm (250 x 4.6 mm ID) column and eluted with aqueous 2% Brij-35 adjusted to pH 3.0 with phosphoric acid:propanol (80:20 v/v) at a flow rate of 1 mL min(-1) and UV detection at 254 nm. A linear calibration curve was obtained simultaneously for each component in the range of 50-500 microg mL(-1) for benzoic acid and 5-150 microg mL(-1) for the other components; detection limits were within 25-250 ng mL(-1) corresponding to 125-1250 pg per injection (5 microL). The reproducibility in terms of average peak area and average retention time was obtained with coefficients of variation (CV) of 1.2% and 0.5%. The method was applied to analysis of these compounds in cosmetics (shampoos, hand lotions, creams, and bath foam) and food samples.     

Coupling of solid-phase microextraction continuous bed (monolithic) capillaries with capillary zone electrophoresis for direct analysis of drugs in biological fluids

Hyperlink robust biocompatible solid-phase microextraction (SPME) devices were prepared using continuous bed (monolithic) restricted-access media (RAM) as the SPME capillary insert. The RAM-based SPME approach was able to simultaneously separate proteins from a biological sample, while directly extracting the active components of caffeine, paracetamol and acetylsalicylic acid from the drug NeoCitramonum. The devices were interfaced with a CZE system and fully automated analysis for sample preconcentration, desorption, separation and quantification of analytes was evaluated. Comparative study of in-line coupled SPME-CZE using RAM and RP capillary inserts was carried out. Using an SPME (RAM) insert, the calculated caffeine, paracetamol and acetylsalicylic acid LODs in a bovine plasma sample were 0.3, 0.8 and 1.9 ng/mL, respectively.     

Chromatographic analysis of phenethylamine-antihistamine combinations using C8, C18 or cyano columns and micellar sodium dodecyl sulfate-pentanol mixtures

The chromatographic behaviour of binary and ternary mixtures of several phenethylamines (phenylephrine, phenylpropanolamine, ephedrine, pseudoephedrine and methoxyphenamine) and antihistamines (pheniramine, carbinoxamine, doxylamine, chlorpheniramine, dexchlorpheniramine, dexbrompheniramine, diphenhydramine, tripolidine, azatadine and phenyltoloxamine), found in cough-cold pharmaceutical preparations, was studied using C8, C18 and cyano columns, micellar mobile phases of sodium dodecyl sulfate (SDS) and pentanol and UV detection. Using a C8 column and mobile phases of 0.05 mol l-1 SDS-6% v/v pentanol or 0.15 mol l-1 SDS-2% v/v pentanol at pH 7, more than 30 different phenethylamine-antihistamine combinations can be resolved in < 15 min. Intra- and inter-day repeatabilities and reproducibilities evaluated at three different drug concentrations (0.5, 5 and 25 micrograms ml-1, n = 10) were below 1.6, 2.5 and 2.4%, respectively. The drug amounts found in 18 formulations agreed with those declared by the manufacturers within the tolerance limits, and with those obtained using a mobile phase of 55% v/v methanol at pH 7. No interference was observed from other accompanying drugs such as acetylsalicylic acid, ascorbic acid, betamethasone, bromhexine, caffeine, codeine, dextromethorphan, paracetamol, prednisolone, salicylamide and tartrazine. The proposed procedure has the advantage over the conventional aqueous-organic procedure of using a small amount of organic solvent, which is highly retained in the SDS solution. The efficiencies are also greater. On the other hand, in the micellar system, the retentions of phenethylamines and antihistamines are similar, although the compounds can be easily resolved. In contrast, using the methanol-water mobile phase, the phenethylamines are weakly retained, whereas the antihistamines usually show a high retention.     

Reversed-phase porous silica rods, an alternative approach to high-performance liquid chromatographic separation using the sequential injection chromatography technique

A commercially available porous silica rod column was used as a separation tool for the sequential injection analysis (SIA). A porous solid monolithic column showed high performance at a low pressure, allowing sequential injection analysis to be used for the first time for separation in HPLC fashion. In this contribution, we tried to demonstrate a new separation concept with SIA manifold for the simultaneous determination of four different compounds (methylparaben (MP), propylparaben (PP), triamcinolone acetonide (TCA) and internal standard ketoprofen (KP)) in a pharmaceutical triamcinolon cream 0.1% formulation. A Chromolith Flash RP-18e, 25 mm x 4.6 mm column with a 10 mm pre-column (Merck, Germany) and a FIAlab 3000 system (USA) with an 8-port selection valve and 10 ml syringe were used for sequential injection chromatographic separations in our study. The mobile phase used was acetonitrile-methanol-water (35:5:65, v/v/v) + 0.05% nonylamine, pH 2.5, flow rate 0.6 ml min(-1). The analysis time was <6 min. A novel sequential injection chromatography (SIC) technique with UV spectrophotometric detection was optimised and validated.     

Determination of theobromine, theophylline, and caffeine in by-products of cupuacu and cacao seeds by high-performance liquid chromatography

Theobromine, theophylline, and caffeine are determined simultaneously by a rapid and selective reversed-phase high-performance liquid chromatography (HPLC) method with UV detection in by-products of cupuacu and cacao seeds. The determination is carried out in the raw and roasted ground cupuacu seeds and in the corresponding powders obtained after pressure treatment. The by-products of both cupuacu seeds and cacao seeds are obtained under the same technological conditions. The HPLC method uses isocratic elution with a mobile phase of methanol-water-acetic acid (80:19:1) (v/v) at a flow rate of 1 mL/min and UV absorbance detection at 275 nm. Total elution time for these analytes is less than 10 min, and the detection limit for all analytes is 0.1 mg/g. The amounts of theobromine and caffeine found in all the cupuacu samples are one or more orders of magnitude lower than those from cacao. Theophylline is found in all cacao samples except for the roasted ground paste, and it is only found in the roasted ground paste in the cupuacu samples.     

Determination of safflor yellow A, puerarin, ferulic acid, ginsenoside Rg1, and Rb1 in the Traditional Chinese Medicinal preparation Naodesheng injection by high-performance liquid chromatography

High-performance liquid chromatography is employed to determine the contents of five mark components, safflor yellow A, puerarin, ferulic acid, ginsenoside Rg1, and Rb1, in the Traditional Chinese Medicinal preparation Naodesheng injection. The separation is performed on a C18 column by stepwise gradient elution with water (0.1%, v/v, phosphoric acid)-acetonitrile (0 min, 86:14; 48 min, 75:25; and 68 min, 50:50) as the mobile phase at a flow rate of 1.0 mL/min, with UV detection at 203 nm. Five regression equations show a good linear relationship between the peak area of each marker and concentration. The recoveries of the markers listed are 99.6%, 100.2%, 99.7%, 100.0%, and 99.7%, respectively. The repeatability and reproducibility (relative standard deviation) of the method are less than 1.4% and 1.8%, respectively.     

Simultaneous determination of leflunomide and its active metabolite,A77 1726, in human plasma by high-performance liquid chromatography

The isoxazol derivative leflunomide [N-(4'-trifluoromethylphenyl)-5-methylisoxazole-4-carboxamide] is an inhibitor of de novo pyrimidine synthesis used for the treatment of rheumatoid artrithis. In the present study, a liquid-liquid extraction-based reversed-phase HPLC method with UV detection was validated and applied for the analysis of leflunomide and its active metabolite, A77 1726, in human plasma. The analytes were separated using a mobile phase, consisting of acetonitrile, water and formic acid (40/59.8/0.2, v/v), at a flow rate of 0.5 mL/min, and UV detection at 261 nm. The retention times for A77 1726, leflunomide and warfarin (internal standard) were 8.2, 16.2 and 12.2 min, respectively. The validated quantification range of the method was 0.05-100 micro g/mL for leflunomide and 0.1-100 micro g/mL for A77 1726. The developed procedure was applied to assess steady-state plasma concentrations of A77 1726 in patients with rheumatoid arthritis treated with 10 or 20 mg leflunomide per day.     

Simultaneous determination of paracetamol, chlorzoxazone, and related impurities 4-aminophenol, 4'-chloroacetanilide, and p-chlorophenol in pharmaceutical preparations by high-performance liquid chromatography

This paper presents a simple, specific, and precise high-performance liquid chromatographic method for the simultaneous determination of paracetamol (PCM), chlorzoxazone (CXZ), and their related impurities in bulk raw materials and solid dosage forms. The mobile phase consisted of water-methanol-glacial acetic acid (60 + 40 + 2, v/v/v). A column containing octadecylsilane chemically bonded to porous silica particles (Spherisorb ODS 1, 25 cm x 4.6 mm, 5 microm) was used as stationary phase. Detection was performed using a variable wavelength ultraviolet-visible detector set at 272 nm for all compounds. Solutions were injected into the chromatograph under isocratic condition at a constant flow rate of 1.2 mL/min. The method was validated according to International Conference on Harmonization requirements and demonstrates good accuracy and precision and a wide linearity range. The method separates PCM, CXZ, and 3 major impurities [4-aminophenol (4AP), 4'-chloracetanilide (4CA), and p-chlorophenol (PCP)] with fair resolution in less than 15 min. The developed method is rapid and sensitive (limit of detection for 4AP, 4CA, and PCP established at 31.25, 39.06, and 65.16 ng/mL, respectively) and, therefore, suitable for quality control and stability studies of these compounds in dosage forms.