Preconcentration of pharmaceuticals residues in sediment samples using microwave assisted micellar extraction coupled with solid phase extraction and their determination by HPLC-UV

An analytical method combining microwave assisted micellar extraction (MAME) and solid phase extraction (SPE) has been developed to extract and preconcentrate a selected group of eight pharmaceutical compounds in sediment samples prior to their determination using liquid chromatography with an UV-DAD detector. A non-ionic surfactant, Polyoxyethylene 10 lauryl ether (POLE) was used for the MAME extraction and the different parameters for the optimization process were studied. Then, SPE was used to clean-up and preconcentrate the target analytes in the extract, prior to their determination using HPLC-UV. The method was applied to the determination of the selected pharmaceuticals compounds in several kinds of sediment samples with different characteristics. Relative recoveries for spiked sediment samples were over 70% and relative standard deviations (RSDs) were under 11% for all recoveries tested. Detection limits between 4 and 167ngg(-1) were obtained. The method was validated using Soxhlet extraction procedure.     

Simultaneous quantification of 12 different nucleotides and nucleosides released from renal epithelium and in human urine samples using ion-pair reversed-phase HPLC

Nucleotides and nucleosides are not only involved in cellular metabolism but also act extracellularly via P1 and P2 receptors, to elicit a wide variety of physiological and pathophysiological responses through paracrine and autocrine signalling pathways. For the first time, we have used an ion-pair reversed-phase high-performance liquid chromatography ultraviolet (UV)-coupled method to rapidly and simultaneously quantify 12 different nucleotides and nucleosides (adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, adenosine, uridine triphosphate, uridine diphosphate, uridine monophosphate, uridine, guanosine triphosphate, guanosine diphosphate, guanosine monophosphate, guanosine): (1) released from a mouse renal cell line (M1 cortical collecting duct) and (2) in human biological samples (i.e., urine). To facilitate analysis of urine samples, a solid-phase extraction step was incorporated (overall recovery rate ≥ 98 %). All samples were analyzed following injection (100 μl) into a Synergi Polar-RP 80 Å (250 × 4.6 mm) reversed-phase column with a particle size of 10 μm, protected with a guard column. A gradient elution profile was run with a mobile phase (phosphate buffer plus ion-pairing agent tetrabutylammonium hydrogen sulfate; pH 6) in 2–30 % acetonitrile (v/v) for 35 min (including equilibration time) at 1 ml min⁻¹ flow rate. Eluted compounds were detected by UV absorbance at 254 nm and quantified using standard curves for nucleotide and nucleoside mixtures of known concentration. Following validation (specificity, linearity, limits of detection and quantitation, system precision, accuracy, and intermediate precision parameters), this protocol was successfully and reproducibly used to quantify picomolar to nanomolar concentrations of nucleosides and nucleotides in isotonic and hypotonic cell buffers that transiently bathed M1 cells, and urine samples from normal subjects and overactive bladder patients.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-012-9321-8) contains supplementary material, which is available to authorized users.     

Micell-mediated extraction for the spectrophotometric determination of nitrite in water and biological samples based on its reaction with p-nitroaniline in the presence of diphenylamine

A cloud point extraction process using the nonionic surfactant Triton X-100 to extract nitrite from aqueous solution was investigated. The method is based on the color reaction of nitrite with p-nitroaniline in the presence of diphenylamine in acid media and micell-mediated extraction of an azo product. The optimal extraction and reaction conditions (e.g., acid concentration, reagent concentration, effect of time) were studied, and the analytical characteristics of the method (e.g., limit of detection, linear range, molar absorptivity, preconcentration, and improvement factors) were obtained. Linearity was obeyed in the range of 2-40 ng ml(-1) of nitrite ion. The detection limit of the method is 0.87 ng ml(-1) of nitrite ion. The interference effect of some anions and cations was also tested. The method was applied to the determination of nitrite in tap water, waste water, and human urine samples.     

A validated stability-indicating HPLC method for the determination of PEGylated puerarin in aqueous solutions

The aim of this study was to develop a validated specific stability-indicating HPLC method for the quantitative determination of PEGylated puerarin (PEG-PUE) in aqueous solutions. The method was validated by subjecting PEG-PUE to forced degradation under stress conditions of acid, alkali, water hydrolysis, and oxidation. Both PEG-PUE and puerarin (PUE) were simultaneously determined and separated on CAPCELL PAK C18 column by gradient elution with 0.2% aqueous phosphoric acid and acetonitrile as the mobile phase. The flow rate was 1.0 mL min⁻¹ and detection wavelength was set at 250 nm. Both calibration curves showed good linear regression (r ≥ 0.9998) within test ranges. The LOD and LOQ of PEG-PUE were determined to be 3 and 9 μg mL⁻¹ respectively. Degradation of PEG-PUE followed pseudo-first-order kinetics with t_1/2 of 59 min at pH 9.0 and 17.79 h at pH 7.4. However, at pH 5.0 and 2.0, there was no significant degradation of PEG-PUE over time. In conclusion, the method was observed to have the necessary specificity, precision, and accuracy, and to be suitable for quantity monitoring the degradation process of PEG-PUE during stability studies. The degradation studies may give insight into useful information for formulation development of PEG-PUE.     

Development of a dispersive liquid-liquid microextraction method for spectrophotometric determination of barbituric acid in pharmaceutical formulation and biological samples

In this paper, a novel and simple method for the determination of trace amounts of barbituric acid in water and biological samples was developed by using dispersive liquid–liquid microextraction (DLLME) techniques combined with spectrophotometric analysis. The procedure is based on color reaction of barbituric acid with p-dimethylaminobenzaldehyde and extraction of the color product using the DLLME technique. Some important parameters such as reaction conditions and the type and volume of extraction and dispersive solvents as well as the extraction time were investigated and optimized in detail. Under the optimum conditions, the calibration graphs were linear over the range of 5.0 to 200 ng ml⁻¹ with limit of detection of 2.0 ng ml⁻¹. Relative standard deviation for five replicate determinations of barbituric acid at 50 ng ml⁻¹ concentration level was calculated to be 1.64%. Average recoveries for spiked samples were determined to be between 94% and 105%. The proposed method was applied for the determination of barbituric acid in pharmaceutical formulation and biological samples.     

Cloud point formation based on mixed micelle in the presence of electrolyte for extraction, preconcentration, and spectrophotometric determination of trace amounts of hydrazine in water and biological samples

A cloud point extraction process using mixed micelle of the anionic surfactant sodium dodecyl sulfate and the nonionic surfactant Triton X-114 to extract hydrazine from aqueous solutions was investigated. The method is based on the condensation reaction of hydrazine with p-(dimethylamino)benzaldehyde, azine formation, and mixed micelle-mediated extraction of azine in the presence of NaCl electrolyte as an inducing phase separation. An azine product was concentrated in surfactant-rich phase after separation. The optimal extraction and reaction conditions (e.g., surfactant, reagent and electrolyte concentrations, and centrifuge time) were studied and the analytical characteristics of the method (e.g., limit of detection, linear range, preconcentration, and improvement factors) were obtained. Linearity was obeyed in the range of 0.50-110ngml(-1) of hydrazine and the detection limit of the method is 0.08ngml(-1). The interference effect of some cations, anions, and organic compounds was also tested. The method was successfully applied to the determination of hydrazine in water and biological samples.     

Effects of the Development of Thyroid and Sex Glands on the Synthesis of Brain Tubulin in Chick and Mouse Embryos

Using the specific affinity of tubulin for colchicine and the strong absorption of tubulin to DEAEion exchangers at neutral pH and moderate ionic strength,the amounts of tubulin in the brain fromboth mice and chicks during different developing stages were measured by ~3H-colchicine assay(expressed as colchicine binding activity).The results show that the rate oftubulin synthesis reacheda peak value during the critical period of brain development.This is exactly the period during whichthe organization and function of thyroid are being perfected.Besides,during breeding period,thedifference of tubulin contents between male and female is significant(P<0.001).The synthesis oftubulin is strictly sex dependent(this phenomenon appeared only during sex maturation stage).It issuggested that sexual hormones might exert their effect on tubulin synthesis.     

Development of an effective novel validated stability-indicating HPLC method for the resolution of brivaracetam stereoisomeric impurities

Reverse-phase high-performance liquid chromatography method has been developed for the determination of brivaracetam stereoisomeric impurities such as (R,S)-brivaracetam, (R,R)-brivaracetam, and (S,S)-brivaracetam with good resolution using the chiral column, Chiral PAK IG-U (100 × 3.0 mm; 1.6 μm). The method is simple, stability-indicating, and compatible with LC–MS. The separation was achieved with the mobile phase consisted of 10 mM ammonium bicarbonate along with acetonitrile in an isocratic mode. The column temperature and wavelength were monitored at 40°C and 215 nm, respectively. The method showed adequate specificity, sensitivity, linearity, accuracy, precision, and robustness inline to ICH tripartite guidelines. The limit of detection and quantification limits were 0.3 and 0.8 μg ml⁻¹, respectively, for all stereoisomeric impurities and brivaracetam. The developed method was found to be linear over the concentration range of 0.8–5.6 μg ml⁻¹ for stereoisomeric impurities with a correlation coefficient >0.999. The method was precise (%RSD < 5.0), robust, and accurate (with 85%–115% recovery). The values of retention times of stereoisomeric impurities, (R,S)-brivaracetam, (R,R)-brivaracetam, and (S,S)-brivaracetam, were 4.9, 5.4, and 6.6 min, respectively, and resolution among the impurities were 2.0, 3.3, and 4.7, respectively. In addition, forced degradation studies were performed to prove that method was stability-indicating. The enrichment of isomeric impurity, (R,R)-brivaracetam, was observed under basic stress conditions of brivaracetam and proposed a plausible mechanism to enhance that isomeric impurity. As well, a good separation among brivaracetam and its stereoisomeric impurity peaks was observed in the presence of degradation products and process-related impurities.     

Development and validation of a reverse phase HPLC method for the determination of caprylic acid in formulations of therapeutic immunoglobulins and its application to antivenom production

A novel method of high performance liquid chromatography with UV detection for the quantification of caprylic acid in formulations of therapeutic immunoglobulins was developed and validated. Samples have interfering proteins that were removed by ultrafiltration in a centrifugal filter unit of 10 kDa nominal molecular weight limit. Then, compounds present in ultrafiltrates were separated on an Eclipse XDB-C8 5 μm column (150 mm × 4.6 mm i.d.), using a mixture of acetonitrile–water (60:40, v/v) as the mobile phase at a flow rate of 1 mL/min. The UV detection was performed at 210 nm. The method was found to be precise and accurate, with a linearity range from 400 μg/mL to 600 μg/mL (r = 0.9948). The limit of detection (LOD) and the limit of quantification (LOQ) were found to be 13.46 μg/mL and 44.85 μg/mL, respectively. To illustrate the usefulness of the method in the in-process and final quality control for production of therapeutic immunoglobulin formulations, permeates obtained from the industrial diafiltration step in the manufacture of equine-derived snake antivenoms and ten batches of finished product were analyzed.     

Determination of puerarin in biological samples and its application to a pharmacokinetic study by flow‐injection chemiluminescence

A simple and sensitive flow injection–chemiluminescence (FI–CL) method has been developed for the determination of puerarin, based on the fact that puerarin can greatly inhibit CL of the luminol–H₂O₂–haemoglobin system. The inhibition of CL intensity was linear to the logarithm of the concentration of puerarin in the range 0.08–10.0 μg/mL (r² = 0.9912). The limit of detection was 0.05 μg/mL (3σ) and the relative standard deviation (RSD) for 1.0 μg/mL (n = 11) of puerarin solution was 1.4%. Coupled with solid‐phase extraction (SPE) as the sample pretreatment, the determination of puerarin in biological samples and a preliminary pharmocokinetic study of puerarin in rats were performed. The recoveries for plasma and urine at three different concentrations were 89.2–110.0% and 91.4–104.8%, respectively. The pharmacokinetics of puerarin in plasma of rat coincides with the two‐compartment open model. The T_1/2α, T_1/2β, CL/F, V_Z/F, AUC_{(0 – t)}, MRT_{(0 – ∞)}, T_max and C_max were 0.77 ± 0.21 h, 7.55 ± 2.64 h, 2.43 ± 1.02 L/kg/h, 11.40 ± 3.45 L/kg, 56.67 ± 10.65 mg/h/L, 5.04 ± 2.78 h, 1.00 ± 0.35 h and 19.70 ± 4.67 μg/mL, respectively. Copyright © 2011 John Wiley & Sons, Ltd.     

Development of a method for the determination of citrinin in barley,rye and wheat by solid phase extraction on aminopropyl columns and HPLC-FLD

A method for the determination of the mycotoxin citrinin (CT) in rye, wheat and barley is described. The proposed method is based on ethyl acetate extraction, solid phase clean-up (SPE) on aminopropyl columns and reversed phase high performance liquid chromatography with fluorescence detection (RP-HPLC-FLD). The limits of detection and quantification of CT amounted to 0.6–0.9 μg/kg and 1.7– 3.3 μg/kg with mean recovery rates in the range of 77–92% (RSD 4.8–5.5%). This method can also be used for the determination of CT in red-fermented rice. Presented at the 29^th Mykotoxin-Workshop, Fellbach, Germany, May 14–16, 2007     

Development and validation of HPLC method for the determination of tobramycin in urine samples post-inhalation using pre-column derivatisation with fluorescein isothiocyanate

A reversed-phase liquid chromatography method involving pre-column derivatisation with fluorescein isothiocyanate (FITC, isomer I) for determination of tobramycin in urine samples after inhalation has been developed. FITC reacts with the primary amino groups of tobramycin and other aminoglycosides under mild conditions to form a highly fluorescent and stable derivative. The chromatographic separation was carried out on a Phenomenex Luna C(18) column at ambient temperature using a constant flow rate of 1 ml/min and mobile phase of acetonitrile-methanol-glacial acetic acid-water (420:60:5:515, v/v/v/v). The tobramycin-FITC derivative was monitored by fluorescent detection at an excitation wavelength 490 nm and emission wavelength 518 nm. The linearity of response for tobramycin was demonstrated at 11 different concentrations of tobramycin extracted from spiked urine, ranging from 0.25 to 20 microg/ml. Tobramycin and neomycin were extracted from spiked urine by a solid phase extraction clean-up procedure on a carboxypropyl-bonded phase (CBA) weak cation-exchange cartridge, and the relative recovery was >99% (n=5). The limit of detection (LOD) and limit of quantitation (LOQ) in urine were 70 and 250 ng/ml, respectively. The method had an accuracy of <0.2%, and intra-day and inter-day precision (in term of %coefficient of variation) were <4.89% and 8.25%, respectively. This assay was used for urinary pharmacokinetic studies to identify the relative lung deposition of tobramycin post-inhalation of tobramycin inhaled solution 300 mg/5 ml (TOBI) by different nebuliser systems.     

A new method using simple solid phase extraction for the rapid gas-chromatographic mass-spectrometric determination of 11-dehydro-thromboxane B2 in urine

A new gas-chromatographic mass-spectrometric method for the rapid determination of 11-dehydro-thromboxane B2 in urine, the major metabolite of systemic thromboxane formation, has been developed. Excellent sample clean-up was obtained in a single step by adsorption of 11-dehydro-TXB2 on phenylboronate cartridges, vigorous polar and organic washing and elution with an acidic methanol mixture. Then the pentafluorobenzylester trimethylsilylether derivative of 11-dehydro-TXB2 was formed and quantified in isotope dilution technique by negative chemical ionisation gas-chromatography tandem mass-spectrometry. The daughter fragments m/z 243/247 of the parent ion m/z 511/515 were monitored. Recovery was linear and quantitative over a wide range, accuracy was 95 + 7% and precision was 11% down to the very low pg range in biologic samples. Independent validation of this very fast extraction method with a reference method applying extensive sample purification with consecutive reversed and straight phase extraction, precolumn derivatisation, reversed phase high pressure liquid chromatography and tandem gas-chromatography mass-spectrometry gave excellent agreement of values. Values for 11-dehydro-TXB2 excretion in 8 healthy controls were 501 + 298 (range 231 to 1141) ng/g creatinin. Excretion was suppressed by aspirin, moderately elevated in heavy smokers (range 680 to 1540) and increased in patients with venous thrombosis or pulmonary embolism (2370 to 13350 ng/g creatinin). This rapid extraction method is useful for the highly specific and sensitive determination of 11-dehydro-TXB2 in large sample numbers.     

Development of an analytical method for organotin compounds in fortified flour samples using microwave-assisted extraction and normal-phase HPLC with UV detection

The normal high-performance liquid chromatography with UV detection was applied for the determination of tributyltin chloride (TBT), triphenyltin chloride (TPhT), tetraphenyltin (TrPhT), triethyltin chloride (TET) and tetraethyltin (TrET) from flour samples. The separation was performed in the isocratic mode on cyanopropyl column with a mobile phase of hexane-acetonitrile-THF (97/1/2). Under the experimental conditions used, quantitative limit of TBT, TPhT, TrPhT, TET and TrET are 0.95, 0.46, 0.97, 0.75 and 0.96 microg/ml, respectively. Microwave-assisted extraction of organotin (OT) compounds at 100 degrees C with an extraction time of 3 min was described. The extraction of organotin can be finished in acetic acid-hexane (20/80) medium. The quantitative extraction of five organotin compounds was achieved with recoveries ranging from 88 to 101% R.S.D. 3-8%.     

Development and validation of a method for the quantitative determination of aflatoxin contaminants in Maytenus ilicifolia by HPLC with fluorescence detection

A method for the quantification of aflatoxins B1, G1, B2 and G2 in the medicinal herb Maytenus ilicifolia was developed and validated. The method used immunoaffinity columns for sample clean-up and HPLC with fluorescence detection without any derivatisation step. The method showed good inter-day accuracy (bias values in the range 4.5-10.7%) and precision (5-16% RSD) when applied to the determination of levels of aflatoxins ranging from 7 to 20 ppb in the plant material. The detection limits for samples of the plant material spiked with aflatoxins were 3.5 ng/g for B1 and G1 and 0.1 ng/g for B2 and G2. The method was successfully applied to commercial samples of Maytenus ilicifolia for the screening of aflatoxin contaminants.     

Highly sensitive spectrofluorimetric method for rapid determination of orciprenaline in biological fluids and pharmaceuticals

Orciprenaline sulphate (ORP) is a direct‐acting sympathomimetic with mainly beta‐adrenoceptor stimulant activity. It is used as a bronchodilator in the management of reversible airway obstruction. For the first time, a rapid highly sensitive spectrofluorimetric method is described that is relied on measuring the fluorescence spectra of ORP at acidic pH and without addition of any chemical reagents. The relative fluorescence intensity was measured at 310 nm and after excitation at 224 nm. ORP native fluorescence was calibrated in both water and acetonitrile as diluting solvents. The method was designed to estimate the drug in miscellaneous matrices with high accuracy and precision. Linear ranges of calibration curves were 30.0–400.0 ng/ml and 10.0–240.0 ng/ml in water and acetonitrile, respectively. The detection limits were calculated and reached as low as 3.3 and 3.1 ng/ml, respectively, representing the ultra‐sensitivity of the proposed method. This result permitted application of this method for spiked human plasma and urine and was used as a preliminary investigation with good percentage recovery (89.4–106.8%). The application was further extended to analyse ORP in its pharmaceutical formulations. The method was validated in compliance with International Council of Harmonization (ICH) Guidelines.     

Optimization of a solid-phase extraction method for determination of indapamide in biological fluids using high-performance liquid chromatography

A new simple and rapid high-performance liquid chromatographic (HPLC) method with UV detection for the determination of indapamide in biological fluids has been developed. Indapamide and internal standard were isolated from serum and whole blood samples by solid-phase extraction with RP select B cartridges. The chromatographic separation was accomplished on a reversed-phase C(8) column with a mobile phase composed of 0.1% (v/v) triethylamine in water (pH 3.5) and acetonitrile (63:37, v/v). UV detection was set at 240 nm. The calibration curves were linear in the concentration range of 10.0-100.0 ng/ml for serum, and 50.0-500.0 ng/ml for whole blood, and the limits of quantification were 10.0 and 50.0 ng/ml, respectively.     

Quantification of corticosteroids in bovine urine using selective solid phase extraction and reversed-phase liquid chromatography/tandem mass spectrometry

This paper presents the development of a simple liquid chromatography–tandem mass spectrometry (LC–MS/MS) method to determine corticosteroids in bovine urine sample matrices. This method uses a single phase extraction (SPE) for cleaning of the sample with an Oasis MAX cartridge at pH 9.0–9.5 and elution by a neutral organic solvent (acetonitrile/dichloromethane), followed by separation on a GEMINI C18 column in the gradient mode with acetate buffer (pH 4.1)/methanol. A triple quadrupole mass spectrometer equipped with a multimode ion source, set to negative atmospheric pressure chemical ionization (APCI) in the multiple reaction monitoring mode was used for detection. The main advantage of this method over other commonly used methods includes the use of SPE with a low volume cartridge for sample preparation and no ion suppression effects from matrix components of the urine samples in the LC–MS/MS analysis. This allowed a reduction the quantification limits (decision limits, CCα) for the first time to 0.1 μg/L (1 and 0.2 μg/L for triamcinolone and flumethasone, respectively). The developed method was validated in accordance with the European Union Commission Decision 2002/657 EC. The recoveries and within-laboratory reproducibility varied from 77% to 115% and 87% to 107.5%, respectively, at 2, 3, and 4 μg/L levels of corticosteroids. The relative standard deviation (RSD) of the measurements was lower than 30%. The decision limit was calculated by multiplying the signal-to-noise ratio by 3 and the obtained values were in the range of 0.1–1.0 μg/L, confirmed by the analysis of twenty blank samples, which were spiked at the desired concentrations. The detection capability was calculated by the addition of the decision limit and the standard deviation followed by multiplication by 1.64 of the within-laboratory reproducibility at 2 μg/L of corticosteroids. The method was applied to four urine samples, giving concentrations of prednisolone (PRED) residues in the range from 0.3 to 0.9 μg/L.     

Development and validation of a simple liquid chromatographic method with ultraviolet detection for the determination of imatinib in biological samples

The aim of this study was to develop a rapid and sensitive HPLC method with UV detection for the estimation of imatinib from the plasma of patients with chronic myeloid leukemia (CML). The robustness of the method was checked by conducting first dose pharmacokinetics on blood samples from four patients who had been administered Gleevec (100 mg) in an oral dose. Samples were prepared in a simple and single step by precipitating the plasma proteins with methanol and injecting 50 microl aliquot from supernatant was subjected for analysis. Assay was conducted using a C8 column (250 mm x 4.6 mm, 5 microm particle size) under isocratic elution with 0.02 M potassium dihydrogen phosphate-acetonitrile (7:3, v/v) at a flow rate of 1 ml/min and detected using photodiode array at 265 nm. Calibration plots in spiked plasma were linear in a concentration range of 0.05-25 microg/ml. The inter and intra-day variation of standard curve was <4% (R.S.D.). This method could be a simple and quick method for the estimation of imatinib from the patient's plasma.