Validation of a fluorimetric assay for 4-aminophenol in paracetamol formulations

4-aminophenol (4-AP) is the primary degradation product of paracetamol (PARA). According to the European Pharmacopoeia, 50 ppm 4-AP/PARA is the specification limit of 4-aminophenol in paracetamol drug substance. For drug products, often higher specification limits, such as 1000 ppm 4-AP/PARA are applied. This paper describes a fluorimetric method to quantify the low amount of this degradant (50 ppm) in a pharmaceutical preparation, i.e. in paracetamol tablets. The fluorimetric method was validated and the linearity, precision, trueness, range, limit of detection and limit of quantification were determined. They were found acceptable to assay the low amounts of 4-aminophenol in paracetamol tablets.     

Automated assay for GV104326, a novel tribactam antibiotic, in human plasma by high-performance liquid chromatography and solid-phase extraction

A highly automated, rapid, robust and specific plasma assay for GV104326, a novel tribactam antibiotic, has been developed to monitor human volunteer trials. The method involves automated solid-phase extraction with a strong anion-exchange phase and HPLC on a reversed-phase column with ultraviolet detection. The calibration range for the assay is 0.05–2 μg/ml. The assay is linear over this range and is specific with respect to endogenous interference and likely metabolites of GV104326. Both intra- and inter-assay variability were < 8% and intra- and inter-day bias < 10%.     

Chemically surface-modified carbon nanoparticle carrier for phenolic pollutants: Extraction and electrochemical determination of benzophenone-3 and triclosan

Chemically surface-modified (tosyl-functionalized) carbon nanoparticles (Emperor 2000 from Cabot Corp.) are employed for the extraction and electrochemical determination of phenolic impurities such as benzophenone-3 (2-hydroxy-4-methoxybenzophenone) or triclosan (5-chloro-2-(2,4-dichlorophenoxy)phenol). The hydrophilic carbon nanoparticles are readily suspended and separated by centrifugation prior to deposition onto suitable electrode surfaces and voltammetric analysis. Voltammetric peaks provide concentration information over a 10–100 μM range and an estimated limit of detection of ca. 10 μM (or 2.3 ppm) for benzophenone-3 and ca. 20 μM (or 5.8 ppm) for triclosan.

Alternatively, analyte-free carbon nanoparticles immobilized at a graphite or glassy carbon electrode surface and directly immersed in analyte solution bind benzophenone-3 and triclosan (both with an estimated Langmuirian binding constants of K ≈ 6000 mol⁻¹ dm³ at pH 9.5) and they also give characteristic voltammetric responses (anodic for triclosan and cathodic for benzophenone-3) with a linear range of ca. 1–120 μM. The estimated limit of detection is improved to ca.5 μM (or 1.2 ppm) for benzophenone-3 and ca. 10 μM (or 2.3 ppm) for triclosan. Surface functionalization is discussed as the key to further improvements in extraction and detection efficiency.     

Simultaneous determination of paracetamol and chlorpheniramine in human plasma by liquid chromatography-tandem mass spectrometry

An analytical method for the determination of paracetamol and chlorpheniramine in human plasma has been developed, validated and applied to the analysis of samples from a phase I clinical trial. The analytical method consists in the extraction of paracetamol and chlorpheniramine with diethyl ether, followed by the determination of both drugs by an LC–MS–MS method, using 2-acetamidophenol as internal standard. The intra-assay and inter-assay precision and accuracy of this technique were good and the limit of quantitation was 0.5 μg/ml of plasma for paracetamol and 0.2 ng/ml for chlorpheniramine. The concentration working range was established between 0.5 μg/ml and 25 μg/ml for paracetamol and between 0.2 ng/ml and 50 ng/ml for chlorpheniramine. This method has been used for analyzing more than 1200 human plasma samples from a clinical study with 24 volunteers.     

High-performance liquid chromatographic analysis of Romet-30 in Chinook salmon (Oncorhynchus tshawytscha): wash-out time, tissue distribution in muscle, liver and skin, and metabolism of sulfadimethoxine

A high-performance liquid chromatographic (HPLC) assay was developed for the determination of Romet-30 residue levels in Chinook salmon muscle and liver tissues. The extraction recoveries averaged 66, 78 and 83% for ormetoprim (OMP), sulfadimethoxine (SDM) and N⁴-acetyl-SDM (N⁴-Ac-SDM), respectively, in muscle tissue; 61 and 72% for SDM and N⁴-Ac-SDM, respectively, in liver tissue. The HPLC assay had a lower detection limit of 0.05 μg/g for OMP, SDM and N⁴-Ac-SDM in muscle tissue, and a lower detection limit of 0.20 μg/g for SDM and N⁴-Ac-SDM in liver tissue. OMP could not be quantified in liver due to the presence of substantial amounts of co-extracted endogenous substances. The HPLC assay was applied to the analysis of Romet-30 residues in Chinook salmon after gastric intubation with Romet-30 at water temperatures between 8.0 and 9.0°C. Different disposition characteristics were found in muscle and liver tissues. The presence of N⁴-Ac-SDM in liver tissue was confirmed by MS and MS-MS analyses. An estimation of SDM and N⁴- Ac-SDM residue levels in skin tissue was obtained and the disposition of these two compounds was similar to the disposition in muscle tissue.     

Sequential injection system with higher dimensional electrochemical sensor signals Part 1. Voltammetric e-tongue for the determination of oxidizable compounds

A sequential injection analysis (SIA) system was developed with the aim of obtaining an automatic and versatile way to prepare standards needed in the study of systems with higher dimensional sensor signals. To illustrate this, different analytical techniques were used in determinations of several analytes. Automated potentiometric calibrations of different potentiometric sensors, with and without interference, were carried out. Useful determinations of selectivity coefficients with two degrees of freedom were obtained. Simultaneous voltammetric determinations have also been done. Firstly, simultaneous determinations of lead and cadmium, using epoxy-graphite composite as the working electrode, have enabled a separate calibration for each metal to be obtained. Next, a voltammetric electronic tongue was designed and applied to the determination of oxidizable species. The use of artificial neural networks has solved the overlapped signal of ascorbic acid, 4-aminophenol and 4-acetamidophenol (paracetamol). A set of 63 data points was prepared automatically and has facilitated the training of an electronic tongue for these three analytes. Accurate predictions of test solutions, in the range of 12–410 μM for ascorbic acid, 17–530 μM for 4-aminophenol and 10–420 μM for paracetamol, have been achieved with RMSEs lower than 0.10 μM.     

Use of DSC and TG for identification and quantification of the dosage form

Thermal analysis techniques, DSC and TG can advantageously be used in quality control of drug products.The methods are commonly used in preformulation for the study of polymorphism and for the study of the interactions drug substance-excipients, since these physical interactions can be the basis of the dosage form performance.For routine control of the drug products, DSC and TG methods which are quick, which require only few mg of the samples and which are automated, are very attractive for routine analysis of drug products. A single scan can give several qualitative and quantitative informations.DSC offer analytical possibilities only if the drug substance and the excipients do not have physical interactions or limited interactions (e.g. eutectic behaviour). About twenty marketed products have been analyzed by DSC and TG. In most of them identification of drug substance is easy. Several excipients could be identified in a tablet. Quantitations are demonstrated for some drug substances and excipients. DSC purity calculations have been applied to acetyl salicylic acid, paracetamol, cimetidine, pindolol, ibuprofen.     

Resin bead detection and spectrophotometric determination of oxyphenbutazone with p-dimethylaminocinnamaldehyde: Application to bulk drug and dosage forms

An analytical method for resin bead detection and spectrophotometric determination of oxyphenbutazone with p-dimethylaminocinnamaldehyde as a reagent is described. Acid hydrolysed product of oxyphenbutazone produced a red color product with p-dimethylaminocinnamaldehyde that absorbs maximally at 520 nm. The detection limit is 0.8 μg in resin phase and 4.0 μg in aqueous phase. Beer's law is obeyed in the concentration range of 0.4–4.0 μg/ml of oxyphenbutazone. The method is unaffected by the presence of acetylsalicylic acid, salicylamide, phenylbutazone and number of other excipients but paracetamol gives a positive reaction and novalgin being reductone interferes with the test, both should be removed by extraction with dilute sulfuric acid.     

A rapid and precise assay for peroxide as 'active oxygen' in products, by flow injection analysis in a high pressure system with spectrophotometric detection

A simple, rapid and automated assay for ‘active oxygen’ originating from hydrogen peroxide, or other organic peroxides, in products is presented employing flow injection (FI) analysis. The product is dispersed and peroxide dissolved in a solvent of 5% (v/v) acetic acid, which constitutes the carrier stream. Ammonium molybdate can be added to this carrier stream to increase sensitivity as required. The sample solution is injected into the acid carrier stream, which is then merged with iodide ion in situ in a two-stream manifold. The ‘active oxygen’ in the product oxidises acidified iodide to iodine, which is detected spectrophotometrically at 350 nm. The closed conditions prevent interference from atmospheric oxygen and the short reaction time minimises the potential for interference from side reactions. Standard HPLC equipment is used throughout, employing a back-pressure to improve precision (high pressure flow injection). Conditions have been investigated using screening multivariate experimental design (two-level quarter fractional factorial design incorporating centre points) to identify and optimise the critical variables. The method has been fully validated (with sample solution R.S.D.s typically < 0.5%, LOQs of 0.04 or 0.006 μg ml⁻¹ as ‘active oxygen’ for acid or acid/molybdate carriers respectively) and is quicker and simpler than the currently employed manual titration approach. It should be applicable to a range of ‘active oxygen’ products.     

Microchannel-assisted thermal-lens spectrometry for microchip analysis

We present a new method for homocysteine quantitation in human plasma based on in-capillary reaction of homocysteine with 2,2′-dipyridyl disulfide. Homocysteine is in this so-called thiol-exchange reaction quantitatively transformed in mixed disulfide concomitantly with formation of an equimolar amount of 2-thiopyridone that is further separated by micellar electrokinetic chromatography and determined specifically at 343 nm. The concentration of homocysteine is thus estimated indirectly from the result of 2-thiopyridone determination. The linear detection range for concentration versus peak area for the assay was from 0.03–3 mM (correlation coefficient 0.994) with a detection limit of 6 μM and a limit of quantitation 20 μM. The inter-day reproducibility of the peak area and the migration time were 1.37% and 0.05%, respectively. The method is simple, relatively rapid and can be easily automated. Moreover the common capillary electrophoresis apparatus with a UV detector can be used to distinguish between normal and pathological hyperhomocysteinemia plasma samples.     

Sensitive high-performance liquid chromatographic determination of arbidol, a new antiviral compound, in human plasma

A highly sensitive and selective HPLC method was developed and validated for the determination of arbidol in human plasma. The method involves the liquid–liquid extraction of drug and internal standard from plasma with tert.-butyl methyl ether followed by evaporation and reconstitution in mobile phase. UV detection was done at 315 nm. The limit of quantification for arbidol in plasma was 0.005 μg/ml. Linearity in plasma was proven over the whole calibration range (10.2–0.005 μg/ml). The method was validated according to GLP guidelines and its suitability was demonstrated by analysis of samples from a pharmacokinetic study.     

Chemiluminescence assay of promethazine hydrochloride using acidic permanganate employing flow injection mode operated with syringe and peristaltic pumps

For the first time, promethazine hydrochloride chemiluminescence emission was monitored. The paper describes a new, specific and highly sensitive flow injection (FI) method for the determination of promethazine hydrochloride using both a peristaltic and a syringe pump. The method was based on the chemiluminescence emission intensity produced as a result of its oxidation reaction with permanganate in sulfuric acid medium. Reaction variables were thoroughly investigated employing chemometrical methods with few number of experiments. The optimum system and chemical conditions were 2.1519×10⁻⁴ mol l⁻¹ permanganate in 0.01 mol l⁻¹ sulfuric acid when operating the peristaltic pump at a flow rate of 45 μl s⁻¹ and injecting the drug by a syringe pump operated at a speed of 40 μl s⁻¹. The method was found to be applicable in the concentration range of promethazine hydrochloride between 1.558×10⁻⁵ and 1.8697×10⁻³ mol l⁻¹ with a linear calibration plot of 0.992 correlation coefficient and the following equation: I=92.74+0.08048C. The method adopted proved to be highly suitable for the assay of promethazine hydrochloride in drug formulations without fear of interferences in dosage form.     

Enantioseparation and quality control of biperiden in pharmaceutical formulations by capillary electrophoresis

An original capillary electrophoretic method has been developed and applied for the enantioselective analysis of the antiparkinson drug biperiden in pharmaceutical formulations, using a modified cyclodextrin as the chiral selector. Baseline enantioseparation of the racemic compound was achieved in less than 7 min using an uncoated fused silica capillary (50 μm i.d. and 48.5, 40.0 cm, total and effective length, respectively), filled with a background electrolyte consisting of a 50 mM phosphate buffer at pH 3.5 supplemented with 3% (w/v) β-cyclodextrin sulphate and applying a voltage of 20 kV, reversed polarity. Samples were injected by pressure (50 mbar, 90 s) at the cathodic end of the capillary and detection wavelength was 195 nm (bandwidth: 10 nm). A simple and fast pre-treatment procedure allowed the complete extraction of the drug from commercial formulations (sustained release tablets and ampoules for injections) without any interference from the matrix. Good linearity was found in the 1–50 μg/mL concentration range; the limit of quantitation was 1 μg/mL and the limit of detection was 0.4 μg/mL. Precision and accuracy were good, with R.S.D. values always lower than 2.8% and a mean recovery value of 101.1%. The method was suitable for the quality control of biperiden in commercial formulations.     

Chemiluminescent determination of pyridoxine hydrochloride in pharmaceutical samples using flow injection

A chemiluminescent method using flow injection is described for the determination of pyridoxine hydrochloride. Its detection limit, linearity and reproducibility were examined. The method is based on the enhancing effect of pyridoxine hydrochloride on the chemiluminescence generated by the oxidation of luminol with hydrogen peroxide in aqueous potassium hydroxide and sodium oxalate. The proposed method is simple and inexpensive. The chemiluminescence intensity is a linear function of pyridoxine hydrochloride concentration over the range 10–250 μg ml⁻¹ with a detection limit of 6 μg ml⁻¹. The applicability of the method was demonstrated by the determination of pyridoxine hydrochloride in different tablet formulations and some dietary sources.     

Potentiometric detection of exogenic beta-adrenergic substances in liquid chromatography

Generation of a large number of theoretical plates was attempted by capillary HPLC. Monolithic silica columns having small skeletons (ca. 2 μm) and large through-pores (ca. 8 μm) were prepared by a sol–gel method in a fused-silica capillary (50 μm I.D.), and derivatized to C₁₈ phase by on-column reaction. High external porosity (>80%) and large through-pores resulted in high permeability (K=1.2×10⁻¹² m²). The monolithic silica column in the capillary produced a plate height of about 12 μm in 80% acetonitrile at a linear velocity of 1 mm/s. Separation impedance, E value, was found to be as low as 200, that was about an order of magnitude lower than reported values for conventional columns packed with 5 μm particles. Reproducibility of preparation within ±15% was obtained for column efficiency and for pressure drop. It was possible to generate 100,000 plates by using a 130-cm column at very low pressure (<7 kg/cm²). A considerable decrease in column efficiency was observed at high linear velocity, and for solutes with large retention factors due to the slow mobile-phase mass transfer in the large through-pores. The monolithic silica columns, however, showed performance beyond the limit of conventional particle-packed columns in HPLC under favorable conditions.     

HPLC-UV method development and validation for the determination of low level formaldehyde in a drug substance

A reversed-phase high-performance liquid chromatographic method (HPLC) with diode-array detection is developed and validated for the quantitative determination of formaldehyde in a drug substance. Formaldehyde (HCHO) is reacted with 2,4-dinitrophenylhydrazine (DNPH) to form a Schiff base (HCHO-DNPH derivatization product), which has an absorbing maximum (lambda max) at 360 nm. The HPLC method employs a C8, 3-microm particle size analytical column (150 mm x 4.6 mm), 15-microL injection volume, column temperature controlled at 30 degrees C, detection at 360 nm, and a water-acetonitrile (55:45, v/v) mobile phase at a flow rate of 1 mL/min. These conditions resolve the HCHO-DNPH product from unreacted DNPH, the drug substance and related impurities, as well as diluent peaks within 20 min. The retention time of the HCHO-DNPH product is approximately 6.4 min. The method is linear, accurate in the specified range (0.33-333 ppm), and robust based on analyte (HCHO-DNPH derivatization product) stability in standard and sample. Detection limit is 0.03 ng (0.1 ppm).     

Comparison of capillary electrophoretic and liquid chromatographic determination of hypoxanthine and xanthine for the diagnosis of xanthinuria

A capillary electrophoretic (CE) method for the determination of hypoxanthine and xanthine in urine was developed to diagnose xanthinuria. The linearity was excellent up to 200 μmol l⁻¹ for the two compounds and the limit of quantitation was 2 μmol 1⁻¹. A comparison o the results obtained using CE was made with those obtained by the high-performance liquid chromatographic (HPLC) technique described previously. With regard to specificity, sensitivity and reproducibility, the results are similar but CE is more rapid than HPLC.     

Automated sequential injection fluorimetric determination and dissolution studies of Ergotamine Tartrate in pharmaceuticals

A fully automated flow system for drug-dissolution studies based on the sequential injection analysis (SIA) was described and used for monitoring dissolution profiles of Ergotamine Tartrate (ET) in pharmaceutical formulations. 50 μl of dissolution medium was taken for each measurement at a flow rate of 40 μl s⁻¹ and detected by fluorescence detector using λ_ex=236 nm (λ_em≥390 nm). The calibration curve was linear over the range 0.03–0.61 mg l⁻¹ of ET (sufficient for the dissolution tests). Equation of the calibration curve was calculated giving the following values: F=117.7 c+0.80 (n=6); r=0.9998. Detection limit was 0.01 mg l⁻¹ of ET. The R.S.D. is less than 0.54 and 0.86% (n=10) when determining 0.61 and 0.03 mg l⁻¹ of ET in standard solution, respectively. The dissolution test of Bellaspon tablets (0.3 mg of ET in 1 tablet) was programmed for 20 min, with a continuous sampling rate of 120 h⁻¹ under conditions required by BP 1993.     

Determination of diethylhexyl phtalate in water by solid phase microextraction coupled to high performance liquid chromatography

Difficulties detected in the determination of the diethylhexylphthalate (DEHP) at trace levels by gas chromatography–mass spectrometry (GC–MS) using SPME, due to its ubiquitous distribution in the environment has been overcome and a new method for the determination of DEHP in drinking water has been proposed. The method is based on solid phase microextraction (SPME) coupled to high-performance liquid chromatography (HPLC). Detection was carried out spectrophotometrically. Calibration graph was linear in the range 10–110 μg/L with a regression coefficient of r² = 0.998 and a detection limit of 0.6 μg/L. The relative standard deviation was 5 and 2% (n = 4) for chromatographic areas and retention times, respectively. The usefulness of the SPME–HPLC technique was confirmed.     

Antihormonal agents. Part 1: HPLC of RU 38486 and its metabolites

Procedures are described for the TLC and reverse phase HPLC analysis of the antihormonal steroid RU 38486 and its metabolites. Conditions are developed for the HPLC of the pure drug substance and a tablet formulation, and also for following its pharmacokinetics and metabolism.