LC-DAD Determination of Fleroxacin in Bulk and Pharmaceutical Dosage Forms

Fleroxacin is a third generation fluoroquinolone with broad spectrum antibacterial activity. In this work an LC-DAD method for the analysis of fleroxacin was developed and validated using UV detection at 286 nm. The method was validated for linearity, precision, robustness, LOD, LOQ, specificity and accuracy at concentrations of 0.2–20.0 μg mL⁻¹ and r ² = 1. The LOD and LOQ were 0.059 and 0.197 μg, respectively, the recoveries were 99.92–102.0% and the CV was less than 2.0%. The LC-DAD validated method provided analytical sensitivity, specificity and reproducibility suitable for quality control analysis.     

Study of a toxin–alkaline phosphatase conjugate for the development of an immunosensor for tetrodotoxin determination

This paper describes a direct competitive immunoenzymatic spectrophotometric assay (ELISA) for tetrodotoxin (TTX) determination and the adaptation of this method for use in an electrochemical assay format. The novelty of this work involves the use of the antigen labelled with alkaline phosphatase (AP); this conjugate was prepared in our laboratory as there is no commercially available conjugate of any kind for TTX. The new conjugate was characterized in terms of its affinity for the specific antibody as well as the residual concentration and the residual activity of the enzyme (AP) incorporated as label. The proposed method based on the new conjugate showed satisfactory results for TTX determination: for the spectrophotometric method the dynamic range was 4–15 ng mL⁻¹ with a limit of detection (LOD) of 2 ng mL⁻¹ (R=0.9247), whereas for the electrochemical protocol the dynamic range was 2–50 ng mL⁻¹ and the LOD was1 ng mL⁻¹.     

Development of a Validated Stability-Indicating LC Assay and Stress Degradation Studies of Linezolid in Tablets

This paper describes the validation of an isocratic LC method for the assay of linezolid in tablets. Validation parameters such as linearity, precision, accuracy, specificity, limit of detection, limit of quantitation and robustness were determined. LC was carried out by reversed phase technique on an RP-18 column with a mobile phase composed of 1% acetic acid:methanol:acetonitrile (50:25:25, v/v/v). Linezolid and your combination drug product were exposed to acid, base, oxidation, dry heat and photolytic stress conditions. A linear response (r > 0.9999) was observed in the range of 8.0–20.0 μg mL⁻¹. The retention time of linezolid was 4.6 min. The method showed good recoveries and intra- and inter-day relative standard deviations were less than 1.0%. The LOD and LOQ were 0.21 and 0.63 μg mL⁻¹, respectively. The developed LC method for determination of related substances and assay determination of linezolid can be used to evaluate the quality of regular production samples. It can also be used to test the stability samples of linezolid.     

Detection of proteases using an immunochemical method with haptenylated–gelatin as a solid-phase substrate

A simplified method for the measurement of proteases utilising solid-phase substrates incorporating an ELISA end-point detection step is described. Gelatin–hapten conjugates adsorbed onto polystyrene surfaces were found to be efficient substrates for proteases. Digestion of the solid-phase protein–hapten complexes resulted in proportional desorption of the attached conjugates and decrease in the detectable hapten species. Gelatin–cholic acid conjugates, affinity-purified sheep anti-cholic acid antibody–HRP and a chromogenic substrate were incorporated into a convenient and highly sensitive solid-phase immunochemical method. The detectable signal is inversely proportional to enzyme activity. Bacterial proteases (alpha-chymotrypsin Type II, Type IX from Bacillus polymyxa, Type XIV from Streptomyces griseus, Type XXIV from Bacillus licheniformens) were assayed. Dose–response curves for enzyme activities were measured within ranges of 0–550 μunits mL⁻¹ for chymotrypsin, 0–12 μunits mL⁻¹ for type IX, 0–35 μunits mL⁻¹ for type XIV and 0–100 μunits mL⁻¹ for type XXIV. The detection limits of the proteases studied were 89 μunits mL⁻¹ for chymotrypsin, 0.26 μunits mL⁻¹ for type IX, 5.8 μunits mL⁻¹ for type XIV and 6.5 μunits mL⁻¹ for type XXIV. It was demonstrated that the two-step immunochemical method combines the simplicity and sensitivity of solid-phase enzyme immunoassays, the broad specificity of gelatin as a protease substrate and the flexibility of the solid-phase format.     

Highly sensitive electrochemical detection of trace liquid peroxide explosives at a Prussian-blue 'artificial-peroxidase' modified electrode

A highly sensitive electrochemical assay of the peroxide-based explosives triacetone triperoxide (TATP) and hexamethylene triperoxide diamine (HMTD) at a Prussian-blue (PB) modified electrode is reported. The method involves photochemical degradation of the peroxide explosives and a low potential (0.0 V) electrocatalytic amperometric sensing of the generated hydrogen peroxide at the PB transducer and offers nanomolar detection limits following a short (15 s) irradiation times. The electrochemical sensing protocol should facilitate rapid field screening of peroxide explosives.     

Determination of adrenolytic drugs by SPME–LC–MS

Five adrenolytic drugs have been analyzed by liquid chromatography–mass spectrometry (LC–MS). Samples were prepared by solid-phase microextraction (SPME) using polypyrrole fibers coated on stainless steel support as an adsorbent for the drugs. Adsorption efficiencies were 95% and were close for all the drugs investigated. Relative standard deviations (RSD), calculated for samples prepared in standard solutions, were in the range 2.5–13%, however RSD values for the drugs in human plasma were 2.5–4.5%. Using LC–MS the limit of detection (LOD) and the limit of quantification (LOQ) were in the ranges 0.11–0.18 and 0.39–0.54 ng mL⁻¹, respectively, for the five drugs.     

Detecting a peroxide-based explosive via molecular gelation

A convenient and portable triacetone triperoxide (TATP) sensor was developed utilizing a thiol-to-disulfide oxidation to trigger a solution-to-gel phase transition. Using this method, TATP can be detected visually without any instrumentation.     

New SPME fibre for analysis of mequinol emitted from DVDs

A piece of fused-silica fibre coated with silica modified with ketamine-groups was used as a solidphase microextraction (SPME) fibre and its efficiency in the qualitative and quantitative analysis of volatile organic compounds released from coloured overprinting on DVDs was evaluated. The effect of the parameters that can affect the SPME procedure, such as extraction time, extraction temperature, desorption temperature, was investigated to determine the analytical performance of this novel fibre in the qualitative and quantitative analyses of organic compounds. The optimised procedure was applied to the qualitative and quantitative analyses of organic compounds released from coloured overprinting on DVDs. The limit of detection of 4-methoxyphenol (mequinol) was 88 × 10⁻³ μg mL⁻¹, while the limit of quantification (LOQ) was calculated as ten times the baseline noise, i.e. 3.1 × 10⁻¹ μg mL⁻¹. The proposed fibre was used successfully for preconcentration of the volatile organic compounds from the gaseous phase of DVD samples.     

Determination of recent antidepressant citalopram in human plasma by liquid chromatography—Fluorescence detection

Summary A reliable and sensitive high-performance liquid chromatographic method for the determination of the recent antidepressant citalopram and two metabolites in human plasma has been developed. Fluorescence detection at 300 nm was used, exciting at 238 nm. Separation was obtained using a reversed-phase column (C18, 250 × 3.0 mm i.d., 5 μm) and a mobile phase. 40% acetonitrile: 60% aqueous tetramethylammonium perchlorate (pH 1.9). Calibration curves were linear over a working range: 5–300 ng mL⁻¹ for citalopram, 2.5–150.0 ng mL⁻¹ for desmethylcitalopram and 2.5–50.0 ng mL⁻¹ for didesmethylcitalopram. The limits of quantitation (LOQ) were 1.5 ng mL⁻¹ for citalopram and desmethylcitalopram and 2.0 ng mL⁻¹ for didesmethylcitalopram. Precision data, as well as accuracy, were satisfactory and no interference from different psychotropic drugs was found. The method was therefore suitable for therapeutic drug monitoring of citalopram and its active metabolites in plasma of depressed patients.     

Monitoring of the non-steroid anti-inflammatory drug indomethacin: development of immunochemical methods for its purification and detection

The present research focused on the development of an immunoassay and an immunochemical sol–gel-based immunoaffinity purification (IAP) method for purification and detection of the non-steroid anti-inflammatory drug (NSAID) indomethacin (IMT). A polyclonal antibody (Ab) for IMT was generated, and two sensitive microplate assays for the detection of IMT were developed (termed OV and HRP formats), based on the enzyme-linked immunosorbent assay (ELISA) method. The limits of detection of the assays were 15 ± 1.25 ng mL⁻¹ (n = 50) and 12 ± 0.17 ng mL⁻¹ (n = 4) for the OVA and HRP formats, respectively. The Abs exhibited slight cross-reactivity with other NSAIDs. The Abs were also used to develop a sol–gel-based IAP method for clean-up and concentration of IMT. Several sol–gel formats with various amounts of antibodies were examined; the best and most reproducible format was at a TMOS:HCl molar ratio of 1:6 in which 120 μL of IMT Abs was entrapped. The binding capacity under these conditions was ca. 100 to 250 ng of IMT with very low non-specific binding (less than 5% of the applied amount). The sol–gel IAP method, combined with solid-phase extraction, successfully eliminated serum interference to a degree that enabled analysis of spiked serum samples by ELISA. The method was also found to be fully compatible with subsequent chemical analytical methods, such as liquid chromatography followed by mass spectrometry. The approaches developed in this study form a basis for analysis of IMT in biological samples in order to monitor their pharmacokinetic properties, and may be further used to study population exposure to IMT, and to monitor the occurrence of IMT contamination in water samples.     

Liquid chromatographic determination of meloxicam in serum after solid phase extraction

A liquid chromatographic method for the determination of meloxicam in serum has been developed. The technique includes a solid phase extraction of the serum samples on [poly (divinylbenzeneco-N-vinylpyrrolidone)] as a solid phase extraction sorbent. After conditioning, the cartridge was loaded with 1 mL of acidified serum containing an internal standard. Elution was carried out using 1 mL of water-acetonitrile (φ _r = 1: 1) mixture. After evaporation of the eluate to dryness and reconstitution of the residue with 0.1 mL of methanol, the samples were analyzed on a Symmetry C18 column. Mobile phase consisted of 1 % aqueous acetic acid/THF/acetonitrile (φ _r = 60: 30: 10) + 0.1 mL of 1-octane sulfonic acid. Detection was carried out using a photodiode array detector. Full validation of the proposed method is provided. Linearity of the method was proven over the range of 0.01–10 φg mL⁻¹ of meloxicam. Meloxicam assay was accurate and reliable with average intra- and inter-day precisions lower than 5.0 % and the intra- and inter-day accuracy higher than 97 %. Limits of detection (LOD) and quantitation (LOQ) found were 0.003 μg mL⁻¹ and 0.01 μg mL⁻¹, respectively. The proposed method was successfully utilized to quantify meloxicam in serum.     

Analysis of the flame retardant metabolites bis(1,3-dichloro-2-propyl) phosphate (BDCPP) and diphenyl phosphate (DPP) in urine using liquid chromatography–tandem mass spectrometry

Organophosphate triesters tris(1,3-dichloro-2-propyl) phosphate (TDCPP) and triphenyl phosphate are widely used flame retardants (FRs) present in many products common to human environments, yet understanding of human exposure and health effects of these compounds is limited. Monitoring urinary metabolites as biomarkers of exposure can be a valuable aid for improving this understanding; however, no previously published method exists for the analysis of the primary TDCPP metabolite, bis(1,3-dichloro-2-propyl) phosphate (BDCPP), in human urine. Here, we present a method to extract the metabolites BDCPP and diphenyl phosphate (DPP) in human urine using mixed-mode anion exchange solid phase extraction and mass-labeled internal standards with analysis by atmospheric pressure chemical ionization liquid chromatography tandem mass spectrometry. The method detection limit was 8 pg mL⁻¹ urine for BDCPP and 204 pg mL⁻¹ for DPP. Recoveries of analytes spiked into urine ranged from 82 ± 10% to 91 ± 4% for BDCPP and from 72 ± 12% to 76 ± 8% for DPP. Analysis of a small number of urine samples (n = 9) randomly collected from non-occupationally exposed adults revealed the presence of both BDCPP and DPP in all samples. Non-normalized urinary concentrations ranged from 46–1,662 pg BDCPP mL⁻¹ to 287–7,443 pg DPP mL⁻¹, with geometric means of 147 pg BDCPP mL⁻¹ and 1,074 pg DPP mL⁻¹. Levels of DPP were higher than those of BDCPP in 89% of samples. The presented method is simple and sufficiently sensitive to detect these FR metabolites in humans and may be applied to future studies to increase our understanding of exposure to and potential health effects from FRs.     

Application of a Validated, Stability-Indicating LC Method to Stress Degradation Studies of Ramipril and Moexipril.HCl

A stability-indicating reversed-phase liquid chromatographic (RPLC) method has been established for analysis of ramipril (RAM) and moexipril hydrochloride (MOEX.HCl) in the presence of the degradation products generated in studies of forced decomposition. The drug substances were subjected to stress by hydrolysis (0.1 m NaOH and 0.1 m HCl), oxidation (30% H₂O₂), photolysis (254 nm), and thermal treatment (80 °C). The drugs were degraded under basic and acidic conditions and by thermal treatment but were stable under other stress conditions investigated. Successful separation of the drugs from the degradation products was achieved on a cyanopropyl column with 40:60 (v/v) aqueous 0.01 m ammonium acetate buffer (pH 6)–methanol as mobile phase at a flow rate of 1 mL min⁻¹. Detection was by UV absorption at 210 nm. Response was a linear function of concentration over the range 5–50 μg mL⁻¹ (r > 0.9995), with limits of detection and quantitation (LOD and LOQ) of 0.04 and 0.09 μg mL⁻¹, respectively, for RAM and 0.014 and 0.32 μg mL⁻¹, respectively, for moexipril. The method was validated for specificity, selectivity, solution stability, accuracy, and precision. Statistical analysis proved the method enabled reproducible and selective quantification of RAM and MOEX as the bulk drug and in pharmaceutical preparations. Because the method effectively separates the drugs from their degradation products, it can be used as stability-indicating.     

Determination of Fatty Acid Methyl Esters in Biodiesel Produced from Yellow Horn Oil by LC

A simple and accurate HPLC method with refractive index detection was developed to determine the main fatty acid methyl esters in biodiesel produced from yellow horn oil. Methyl linoleate, methyl linolenate, methyl arachidate, methyl stearate, methyl palmitate and methyl oleate were separated on a HIQ SIL C18W column using methanol as mobile phase. The method has good repeatability and precision, the intraday and interday RSD for both retention time and peak area was less than 3.2%. The LOD (S/N = 3) and LOQ (S/N = 9) were less than 0.004 and 0.015 mg mL⁻¹, respectively.     

Selective and rapid detection of triacetone triperoxide by double-step chronoamperometry

A selective detector for the improvised explosive, triacetone triperoxide is proposed. This is based on the rapid redox reaction of peroxides (hexamethylene triperoxide diamine, benzoyl peroxide, t-butyl peroxide, triacetone triperoxide and H₂O₂) with bromide at 55 °C. Consumption of bromide is indicative of the reduction of the R–O–O–R moiety, the appearance of Br₂ was found for all except for triacetone triperoxide. The latter was found to breakdown to acetone which rapidly reacts with Br₂ producing bromoacetones. The lack of Br₂ production is unique to triacetone triperoxide. Double step chronoamperometry (E₁ = 700 mV, E₂ = 960 mV (vs. Ag/AgCl)) allows for the quantitation of bromine (Br₂ + 2e^? ? 2Br^?) and bromide (2Br^? ? Br₂ + 2e^?) respectively. The results yielded a detection limit of 8.5 µM for triacetone triperoxide with a sensitivity of 0.026 µA µM^? 1. The detection limits of 16.3 µM and 14.9 µM were found respectively for HMTD and H₂O₂ based on the appearance of Bromine. These results indicate a possibility to develop a handheld sensor for TATP dermination.     

Multisyringe flow injection spectrophotometric determination of uranium in water samples

A multisyringe flow injection analysis method for the determination of uranium in water samples was developed. The methodology was based on the complexation reaction of uranium with arsenazo (III) at pH 2.0. Uranium concentrations were spectrophotometrically detected at 649 nm using a light emitting diode. Under the optimized conditions, a linear dynamic range from 0.1 to 4.0 μg mL⁻¹, a 3σ detection limit of 0.04 μg mL⁻¹, and a 10σ quantification limit of 0.10 μg mL⁻¹ were obtained. The reproducibility (%) at 0.5, 2.5, and 4.0 μg mL⁻¹ was 2.5, 0.9, and 0.6%, respectively (n = 10). The interference effect of some ions was tested. The proposed method was successfully applied to the determination of uranium in water samples.     

Chiral separation of (+)/(−)-catechin from sulfated and glucuronidated metabolites in human plasma after cocoa consumption

Cocoa is well-known to be rich in flavan-3-ols. Previous analyses have established that alkaline treatment of cocoa beans results in epimerization of (−)-epicatechin to (−)-catechin and (+)-catechin to (+)-epicatechin. Now, the question is whether both epimers can be absorbed by the human organism. This paper describes sample preparation and an HPLC method for chiral determination of (+)/(−)-catechin from sulfated and glucuronidated metabolites in human plasma. The sample preparation includes enzymatic hydrolysis of the catechin metabolites, and solid-phase extraction (SPE). A PM-γ-cyclodextrin column is used with a coulometric electrode-array detection (CEAD) system. The recovery of catechin ranges from 89.9 to 96.8%. The limit of detection is 5.9 ng mL⁻¹ for (−)-catechin and 6.8 ng mL⁻¹ for (+)-catechin, and the limit of quantification is 12.8 ng mL⁻¹ for (−)-catechin and 16.9 ng mL⁻¹ for (+)-catechin. The relative standard deviation of the method ranges from 0.9 to 1.5%. This method was successfully applied to human plasma after consumption of a cocoa drink. In one human self-experiment, (+)-catechin and (−)-catechin were found in human plasma, but metabolism of the two enantiomers differed.     

Determination of metoprolol tartrate by capillary isotachophoresis

This paper describes two isotachophoretic methods of metoprolol tartrate (MT) determination in pure and dosage forms. The first method was used for direct analysis where the following electrolyte system was applied: 10 mmol dm⁻³ 3-morpholino-2-hydroxypropanesulfonic acid, 10 mmol dm⁻³ NaCl, 2 % hydroxyethylocelulose as leading (LE) and 10 mmol dm⁻³ glycyl-glycine as terminating (TE) electrolytes. The second method was used for indirect analysis of MT as tartrate ions. In this case, the leading electrolyte consisted of 10 mmol dm⁻³ HCl, β-alanine (BALA), pH 4-5, and the terminating one of 5 mmol dm⁻³ glutamic acid, 10 mmol dm⁻³ β-alanine. Calibration curves were calculated as follows: for system A: y = (0.52 ± 0.05)x − (0.9 ± 0.2) (LOD = 13.0 mg dm⁻³, LOQ = 31.7 mg dm⁻³); and for system B: y = (0.240 +- 0.001)x + (0.18 ± 0.06) (LOD = 1.8 mg dm⁻³, LOQ = 4.4 mg dm⁻³). The isotachophoretic method was compared with the pharmacopoeial one by statistical tests.     

Method Development and Validation of a Rapid Determination of Ropinirole in Tablets by LC-UV

A reversed-phase high-performance liquid chromatographic (HPLC) method with UV detection was developed and validated for the determination of ropinirole (ROP) in tablets. The assay utilized UV detection at 250 nm and a Luna CN column (250 × 4.6 mm I.D, 5 μm). The mobile phases were comprised of acetonitrile: 10 mM nitric acid (pH 3.0) (75:25, v/v). Validation experiments were performed to demonstrate linearity, accuracy, precision, limit of quantitation (LOQ), limit of detection (LOD), and robustness. The method was linear over the concentration range of 0.5–10.0 μg mL⁻¹. The method showed good recoveries (99.75–100.20%) and the relative standard deviations of intra and inter-day assays were 0.38–1.69 and 0.45–1.95%, respectively. The method can be used for quality control assay of ropinirole.     

MEKC Determination and Pharmacokinetic Study of Danshensu in Rabbits after Intragastric Administration of the Aqueous Extract from Danshen

A micelle eletrokinetic capillary chromatography (MEKC) method was used to determine Danshensu in rabbit blood plasma and tissues (liver, lung, kidney, brain, heart and spleen). The separation was achieved with a buffer consisting of 30 mmol L⁻¹ borax and 50 mmol L⁻¹ SDS (pH 9.0), and with an applied voltage of 7.0 kV. Validation of the method showed good sensitivity, reproducibility and precision. The calibration curve for Danshensu was linear over the concentration range of 0.4–400 μg mL⁻¹. The limit of detection (LOD) was 0.08 μg mL⁻¹ (S/N = 3). The validated method has been successfully applied for the pharmacokinetic and the tissue distribution studies of Danshensu after intragastric administration of the aqueous extract from traditional Chinese medicine Danshen.