Simultaneous determination of preservatives (benzoic acid, sorbic acid, methylparaben and propylparaben) in foodstuffs using high-performance liquid chromatography

A reversed-phased HPLC method that allows the separation and simultaneous determination of the preservatives benzoic (BA) and sorbic acids (SA), methyl- (MP) and propylparabens (PP) is described. The separations were effected by using an initial mobile phase of methanol-acetate buffer (pH 4.4) (35:65) to elute BA, SA and MP and changing the mobile phase composition to methanol-acetate buffer (pH 4.4) (50:50) thereafter. The detector wavelength was set at 254 nm. Under these conditions, separation of the four components was achieved in less than 23 min. Analytical characteristics of the separation such as limit of detection, limit of quantification, linear range and reproducibility were evaluated. The developed method was applied to the determination of 67 foodstuffs (mainly imported), comprising soft drinks, jams, sauces, canned fruits/vegetables, dried vegetables/fruits and others. The range of preservatives found were from not detected (nd)--1260, nd--1390, nd--44.8 and nd--221 mg kg(-1) for BA, SA, MP and PP, respectively.     

Development and application of a validated stability-indicating high-performance liquid chromatographic method using photodiode array detection for simultaneous determination of granisetron, methylparaben, propylparaben, sodium benzoate, and their main degradation products in oral pharmaceutical preparations

A simple, rapid, and sensitive RP-HPLC method using photodiode array detection was developed and validated for the simultaneous determination of granisetron hydrochloride, 1-methyl-1H-indazole-3-carboxylic acid (the main degradation product of granisetron), sodium benzoate, methylparaben, propylparaben, and 4-hydroxybenzoic acid (the main degradation product of parabens) in granisetron oral drops and solutions. The separation of the compounds was achieved within 8 min on a SymmetryShield RP18 column (100 x 4.6 mm id, 3.5 microm particle size) using the mobile phase acetonitrile--0.05 M KH2PO4 buffered to pH 3 using H3PO4 (3+7, v/v). The photodiode array detector was used to test the purity of the peaks, and the chromatograms were extracted at 240 nm. The method was validated, and validation acceptance criteria were met in all cases. The robust method was successfully applied to the determination of granisetron and preservatives, as well as their degradation products in different batches of granisetron oral drops and solutions. The method proved to be sensitive for determination down to 0.04% (w/w) of granisetron degradation product relative to granisetron and 0.03% (w/w) 4-hydroxybenzoic acid relative to total parabens.     

High performance liquid chromatographic determination of sodium benzoate, methylparaben and propylparaben as preservative components in nystatin suspensions

A simple and sensitive method was developed for the analysis of preservatives sodium benzoate, methylparaben and propylparaben in nystatin suspensions by reversed-phase high performance liquid chromatography (HPLC), equipped with a C18 column and PDA detector. The mobile phase was a mixture of acetonitrile and acetate buffer of pH 4.4 (35:65 v/v). Under the optimized experimental conditions, separation of the preservatives was achieved in less than 20 min. The limits of quantifications (LOQs) and the linear dynamic ranges (LDRs) of sodium benzoate, methylparaben and propylparaben were 0.3 and 50–1000 μg Ml^?1, 0.5 and 50–600 μg ml^?1 and 0.3 and 50–900 μg ml^?1, respectively; the respective precisions (%RSD) at 500 μg ml^?1 level were 0.72%, 0.73% and 0.51% (n = 6). The average recoveries of sodium benzoate, methylparaben and propylparaben for spiked nystatin samples were obtained as 98%, 97% and 98%, respectively.     

Development and validation of a reversed-phase ion-pair high-performance liquid chromatographic method for the determination of risedronate in pharmaceutical preparations

A stability indicating, reversed-phase ion-pair high-performance liquid chromatographic method was developed and validated for the determination of risedronate in pharmaceutical dosage forms. The determination was performed on a BDS C₁₈ analytical column (250 mm × 4.6 mm i.d., 5 μm particle size); the mobile phase consisted of 0.005 M tetrabutylammonium hydroxide and 0.005 M pyrophosphate sodium (pH 7.0) mixed with acetonitrile in a ratio (78:22, v/v) and pumped at a flow rate 1.00 mL min⁻¹. The ultraviolet (UV) detector was operated at 262 nm. The retention times of magnesium ascorbyl phosphate, which was used as internal standard and risedronate were 4.94 and 5.95 min, respectively. The calibration graph was ranged from 2.50 to 20.00 μg mL⁻¹, while detection and quantitation limits were found to be 0.48 and 1.61 μg mL⁻¹, respectively. The intra- and inter-day percentage relative standard deviations, %R.S.D., were less than 5.9%, while the relative percentage error, %E_r, was less than 0.4%. The method was applied to the quality control of commercial tablets and content uniformity test and proved to be suitable for rapid and reliable quality control.     

Determination of benzoyl peroxide and benzoic acid in wheat flour by high-performance liquid chromatography and its identification by high-performance liquid chromatography-mass spectrometry

An HPLC method on C18 column using a gradient mobile phase is proposed for the separate determination of residual benzoyl peroxide (BP) and benzoic acid (BA) in flour and wheat products. The recoveries obtained were quite excellent, from 96.0 to 99.3% for BP added to the flour, and 91.3% for BA added to the flour. Analysis of 10 samples of commercial foods such as flour and wheat products, detected 0.7 microg/g of BP in imported noodles. Furthermore, we successfully verified the existence of BP by LC-MS. These methods are simple and reliable for determination and verifying the amount of BP and BA in foods since now the use of BP as a food additive is permitted in many countries.     

Column reversed-phase high-performance liquid chromatographic method for simultaneous determination of rabeprazole sodium and domperidone in combined tablet dosage form

A new, simple column reversed-phase high-performance liquid chromatographic (HPLC) method for simultaneous determination of rabeprazole sodium (RAB) and domperidone (DOM) in a combined tablet dosage form has been developed and validated. Determination was performed using a Jasco HPLC system with a HiQ SiL octadecylsilane (C18) column (250 x 4.6 mm id), acetonitrile-0.1 M ammonium acetate (50 + 50, v/v) mobile phase, and paracetamol as an internal standard. The detection was performed using a UV detector set at 280 nm. The method was validated with respect to linearity, accuracy, precision, and robustness. Beer's law was obeyed in the concentration range of 1.0-10.0 and 0.5-5.0 microg/mL for RAB and DOM, respectively. The method has been successfully applied for the analysis of drugs in a pharmaceutical formulation.     

Separation and simultaneous high-performance liquid chromatographic determination of benzocaine and benzyl benzoate in a pharmaceutical preparation.

A simple reversed-phase high-performance liquid chromatographic method suitable for the simultaneous determination of benzocaine and benzyl benzoate in dermatological preparations is described. An internal standard method was employed, using C18 "bonded phase" silica column and a mobile phase consisting of acetonitrile - water (40:60, v/v), with absorption of the column effluent monitored at 254 nm. No sources of interference were observed. The simultaneous determination of both compounds by the method described is rapid and accurate.     

Automated high-performance liquid chromatographic method with column switching for the determination of neurotransmitters and related compounds, ascorbic acid and uric acid in tissue extracts

An automated high-performance liquid chromatographic method with electrochemical and fluorimetric detection and on-line data evaluation is described for the simultaneous measurement of indoleaminergic and catecholaminergic neurotransmitters, some of their metabolites and precursors and ascorbic and uric acids. Deproteinized tissue extracts from the central nervous system or peripheral organs are injected without prior purification (recovery greater than 90%). A switching system enables the compounds to be passed as necessary through one, two or three reversed-phase columns, which are then eluted simultaneously (analysis time 25 min). Fifty samples per day can be analysed with a precision of 95% for neurotransmitters and about 90% for ascorbic and uric acids.     

Rapid high-performance liquid chromatographic method for the determination of peroxyacetic acid

The selective oxidation of methyl p-tolyl sulfide (MTS) to the corresponding sulfoxide (MTSO) by peroxyacetic acid and the subsequent rapid separation of the sulfide and sulfoxide are the basis for a fast and reliable HPLC method for the determination of this oxidizing agent in the presence of hydrogen peroxide. The time required for chromatographic separation was reduced to less than 1 min. To improve the long-term stability of the sulfoxide solution, hydrogen peroxide was decomposed catalytically by manganese dioxide. Even in the presence of a tenfold molar excess of hydrogen peroxide, a storability of at least 20 h without a significant increase in MTSO concentration was achieved. External calibration can be performed using the stable and commercially available MTSO. Real samples from a brewery cleaning-in-place disinfection process were analysed and the results were compared with those of the classical two-step titration.     

An isocratic liquid chromatographic method with diode-array detection for the simultaneous determination of alpha-tocopherol, retinol, and five carotenoids in human serum

An isocratic high-performance liquid chromatography (HPLC) method for the simultaneous determination of alpha-tocopherol, retinol, and five carotenoids (lutein-zeaxanthin, beta-cryptoxanthin, lycopene, and alpha- and beta-carotene) in human serum is described. Serum samples are deproteinized with ethanol and extracted once with n-hexane. Resulting extracts are injected onto a C18 reversed-phase column eluted with methanol-acetonitrile-tetrahydrofuran (75:20:5, v/v/v), and full elution of all the analytes is realized isocratically within 20 min. The detection is operated using three channels of a diode-array spectrophotometer at 290, 325, and 450 nm for tocopherol, retinol, and the carotenoids, respectively. An internal standard is used for each channel, which improves precision. The choice of internal standards is discussed, as well as the extraction protocol and the need for adding an antioxidant during the extraction and chromatographic steps. The analytical recoveries for liposoluble vitamins and carotenoids are more than 85%. Intra-assay relative standard deviation (RSD) values (n = 20) for measured concentrations in serum range from 3.3% (retinol) to 9.5% (lycopene), and interassay RSDs (n = 5) range from 3.8% (alpha-tocopherol) to 13.7% (beta-cryptoxanthin). The present method is used to quantitate the cited vitamins in healthy subjects (n = 168) from ages 9 to 55 years old.     

Rapid reversed-phase high-performance liquid chromatographic determination of the regiospecificity of lipoxygenase products on linoleic acid

A new reversed-phase high-performance liquid chromatography method for the separation of regioisomeric products from lipoxygenase acting on linoleic acid was studied. The addition of salts to the mobile phase improved the retention and separation behaviour of 13-hydroperoxy-9,11-octadecadienoic acid and 9-hydroperoxy-10,12-octadecadienoic acid with respect to the results obtained with other mobile phases reported in the literature. The effect of the pH and ionic strength of the buffer on the retention times, capacity factor and separation factor of these lipoxygenase products were also studied. The pH optimum coincided with the pKa of linoleic acid (close to 7 depending on the fatty acid concentration). Phosphate concentrations close to 100 mM considerably reduced the retention times and led to better separation of the mixture of both products. Finally, this method was applied to the identification and separation of two linoleic acid hydroxides (13-hydroxy-9,11-octadecadienoic acid and 9-hydroxy-10,12-octadecadienoic acid) obtained by the reduction of their corresponding hydroperoxides.     

Direct serum injection high-performance liquid chromatographic method for the simultaneous determination of phenobarbital, carbamazepine and phenytoin

A method is described for the simultaneous measurement of serum levels of three antiepileptic drugs, phenobarbital, phenytoin and carbamazepine, by direct injection high-performance liquid chromatography on a 25-cm Pinkerton internal surface reversed-phase (ISRP) column. Several commonly available compounds were tested and found not to co-chromatograph with the three drugs of interest or the internal standard, 5-(p-methylphenyl)-5-phenylhydantoin. Results obtained on patients' samples with this method compared well with those from enzyme-multiplied immunoassay technique (EMIT).     

Ion-pairing high-performance liquid chromatographic method for the determination of 5-aminosalicylic acid and related impurities in bulk chemical

An ion-pairing high-performance liquid chromatographic method has been developed for the determination of 5-aminosalicylic acid (5-ASA) bulk chemical in the presence of thirteen potential synthetic process impurities. In addition, the method is suitable for the determination of the in process intermediate, 5-nitrosalicylic acid. A selective method was achieved on a Hypersil-BDS reversed-phase column using 1-heptanesulfonic acid sodium salt as the ion-pairing reagent in a 0.08 M sodium phosphate buffer (pH 2) containing 0.005 M 1-heptanesulfonic acid sodium salt and 0.07 M sodium chloride-methanol-tetrahydrofuran (85:11:4, v/v/v) isocratic mobile phase. The method was validated using a multi-day, intra-laboratory protocol. The validation addressed linearity, accuracy, precision, sensitivity, and ruggedness of the method. The validated method characterizes the purity of 5-ASA bulk chemical.     

A reversed-phase high-performance liquid chromatographic method for the determination of zafirlukast in pharmaceutical formulations and human plasma

Zafirlukast (ZAF) is a leukotriene receptor antagonist used in the treatment of chronic asthma. In this study, a simple and sensitive reversed-phase, high-performance liquid chromatographic method was developed for the determination of ZAF in pharmaceutical formulations and human plasma. Piribedil was used as an internal standard. Analysis was carried out on a Nucleosil C18 100 A (150 mm x 4.6 mm id, 5 Vm) column with acetonitrile-pH 3.0 acetate buffer (70 + 30, v/v) as the mobile phase at a flow rate of 0.8 mL/min. The peak was detected by an ultraviolet detector set at a wavelength of 240 nm. The retention times were about 3.9 min for piribedil and 5.8 min for ZAF. The developed method was applied to the determination of ZAF in its pharmaceutical formulation and spiked human plasma. For quantification of ZAF in spiked plasma, proteins were precipitated with ethanol before chromatographic analysis. The calibration range was linear from 49.69-437.50 ng/mL in spiked plasma. The absolute recovery from spiked plasma was 98.73 +/- 0.42% at a concentration of 254.78 ng/mL of ZAF. No endogenous substances from plasma were found to interfere.     

Two-parameter mobile phase optimization for the simultaneous high-performance liquid chromatographic determination of dopamine, serotonin and related compounds in microdissected rat brain nuclei.

A new high-pressure liquid chromatography method with electrochemical detection is described that allows the simultaneous determination of dopamine, serotonin, 3,4-dihydroxyphenylacetic acid, 3-methoxy-4-hydroxyphenylacetic acid, 5-hydroxytryptophan and 5-hydroxyindoleacetic acid in microdissected nuclei from individual rat brains. No sample pre-treatment steps are required. Resolution and analysis time were optimized by a simple limited optimization procedure, involving two-parameter factorial design.