Solid Extraction of Steroid Conjugates from Plasma and Milk

Abstract

Extraction of steroids and steroid conjugates with Amberlite XAD-2 and Sep-Pak C₁₈ has been studied. Amberlite XAD-2 has ionic sites with high affinity for conjugated steroids, particularly steroid disulphates. Nearly quantitative recoveries of steroid conjugates are obtained when the resin is washed with an aqueous solution of a sulphate prior to elution with methanol. Such treatment is not required using Sep-Pak C₁₈ cartridges.

The adsorbents were used for extraction of steroids and their conjugates from plasma and milk. Steroid-protein and steroid-lipid interactions were minimized by diluting the fluid twice with 0.5 M aqueous triethylamine sulphate and passing the solution through the adsorbent a t 64°C. Steroids were eluted with methanol and methanol/chloroform at room temperature. Essentially quantitative recoveries were obtained with both adsorbents.     

Analysis of steroids by nonaqueous capillary electrophoresis

A simple method for the separation and determination of steroids (estradiol valerate, triamcinolone, levonorgestrel and ethinylestradiol) in single and compound tablets by nonaqueous capillary electrophoresis with ultraviolet (UV) spectrophotometric detection has been developed for the first time. After optimizing the electrophoretic parameters, including the nature of electrolytes and composition of organic solvent, the running buffers of methanol-acetonitrile (95: 5, v/v) containing 20 mM sodium acetate (pH 6.5) and methanol-acetonitrile (90: 10, v/v) containing 25 mM sodium acetate (pH 7.0) were found to be most suitable for determining estradiol valerate and triamcinolone, respectively. Reliable separation and simultaneous determination of levonorgestrel and ethinylestradiol were achieved in methanol containing 20 mM of ammonium acetate and 10 mM of sodium dodecyl sulfate (SDS). Tamoxifen was used as internal standard. Performance of the method, including migration time and peak area reproducibility, linearity, sensitivity and accuracy, were also evaluated. The limits of detection (S/N = 3) for four analytes were in the range of 9.8–19.5 μ g/mL. The relative standard deviations (RSD) of the migration times and peak areas of the analytes were in the range of 0.14–1.0% and 0.7–2.7% (intraday), 0.5–2.8% and 1.5–4.2% (interday), respectively. Within the tested concentration range, linear relationships between peak area ratios and concentrations of the analytes were obtained (correlation coefficients: 0.9987–0.9996). The method has been successfully applied to the determination of ingredients with recoveries over the range of 96.6–100.6%. The text was submitted by the authors in English.     

Analysis of Sucrose Fatty Acid Esters Composition by HPLC

Abstract

Two procedures for quantitative analysis of sucrose fatty acid esters composition using HPLC are described. A reversed-phase column (RP-18) was used. The mobile phases consist of: a) methanol (95%) and isopropanol (5%); b) methanol and water (5%) using UV and RI detectors.     

Characterization of Mechanisms of Pesticide Retention in Soils Using the Supercritical Fluid Extraction Technique

Abstract

This research determined the relative effectiveness of supercritical fluid extraction (SFE) in extracting atrazine and its metabolites from soils which had been treated with atrazine for varying periods of time in order to characterize binding mechanisms. Aqueous methanol extraction was more effective than SFE in removing ¹⁴C atrazine residues from “aged” soils. The more polar the solvent system, the more ¹⁴C-atrazine residues were extracted. The order of polarity and extractability was aqueous methanol > SF-CO₂/5% methanol > SF-CO². Atrazine extraction efficiency using SF-CO₂, and SF-CO₂/.5% methanol decreased as samples “aged” in the field. The less than complete recovery of atrazine residues using the SFE technique could be seen as an indication that different binding mechanisms were involved in the retention of atrazine as well as its metabolites and that the binding mechanisms changed with time.     

Simplified Method for the Isolation and Analysis of Ethynyl Steroids in Urine

Abstract

A simplified procedure for selective isolation of ethynyl steroids from urine is described. Urinary steroids are extracted with Sep-Pak^R C₁₈ and subjected to enzyme hydrolysis. The liberated steroids are extracted with Lipidex 1000 and the eluate from this column is passed through a bed of sulfohydroxypropyl Sephadex LH-20, partly converted into silver form by a wash with aqueous silver nitrate. Ethynyl steroids are eluted with a solution of acetylene in methanol.     

Determination of Steroids in Human Urine by Micellar Liquid Chromatography

Abstract

A simple and sensitive method for the determination of steroids using micellar liquid chromatography is described. The steroids, including hydroxycorticosterone. corticosterone, northisterone, testosterone, mexdroprogesterone acetate and progesterone, were separated by reversed-phase using a micelles mobile phase following UV detection at 245 nm. The parameters affecting retention of the test solutes such as the concentration of sodium dodecyl sulfate (SDS) and n-butanol-1 in the mobile phase were investigated. It was found that the retention of the solutes was dependent on the composition of mobile phase. The linear calibration plots range from 0.1 to 10 μg ml^?1 in mobile phase containing 5.0 × 10^?2 mol l^?1 SDS/9 % n-butanol-1 at pH 6.0, and the detection limit in order of 0.1 μg ml^?1 was obtained. The proposed method was used for the determination of steroids in urine using direct injection of samples without previous treatment.     

Wasserfreies Aluminiumchlorid in der quantitativen Analyse organischer Verbindungen

Zusammenfassung Es wird eine neue Methode zur quantitativen spektrophotometrischen Bestimmung von Äthinyloestradiol beschrieben. Sie basiert auf der Farbreaktion des Steroids mit wasserfreiem Aluminiumchlorid in Nitromethan und ist spezifisch in bezug auf Zersetzungsprodukte. Ein Beispiel für die Bestimmung von Äthinyloestradiol in Tabletten wird angeführt.

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Summary A new method for the quantitative spectrophotometric determination of ethinylestradiol is described. It is based on the colour reaction of the steroid with anhydrous aluminium chloride in nitromethane and is specific in relation to decomposition products. An example for the determination of ethinylestradiol in tablets is shown.

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I. Mitt.: Becker, A., u. A. Kathriner: diese Z. 183, 356 (1961).     

Simultaneous Determination of Diazepam,Oxazepam and Temazepam in Spiked Urine By Hplc

ABSTRACT|Oxazepam and temazepam are two minor metabolites of diazepam. These three benzodiazepines may be found in presence of each other in biological fluids.

Therefore, in this study an HPLC method was developed to separate and analyze them. Benzodiazepines have ability to form inclusion complexes with p-cyclodextrin (β-CyD). According to the degree of binding constant with β-CyD, these compounds can be separated by β-CyD bound to silica (cyclobond column) as the stationary phase using HPLC.

The development and validation of the HPLC procedure for the separation and determination of these compounds in mixtures were studied. The mobile-phase system consisted of phosphate buffer (pH 7): methanol [75:25], with flow rate 0.8 ml min^?1 and UV detection at 240 nm was used.

The calibration graphs were rectilinear from 0.1-2.5 μg/ml and coefficients of variation were <2% for the three compounds in bulk forms.

The method was used to analyse these bezodiazepines in spiked urine containing all three compounds in combination. Recoveries were 97-99.8%

The limit of detection and limit of quantitation were 0.05 μg/ml and 0.1 μg/ml, respectively.

The described method is selective, rapid, simple, reproducible and accurate.     

Quantitative Determination of Fluconazole by Infrared Spectrophotometry

Abstract

The purpose of the study was the quantitative determination of fluconazole by IR spectrophotometry which was realized by the application of the KBr disc technique. In this study dehydrocholic acid was used as internal standard.

The absorption bands at 960 and 675 cm^?1 were chosen for fluconazole and 1705 cm^?1 for dehydrocholic acid. The concentration range between 0.4–1.6% w/w in KBr disc showed compliance with Beer's law.

Besides IR spectroscopy, UV spectroscopy and HPLC methods were also used In the quantitative determinations. In the UV spectroscopy method the absorbance value at 261 nm in MeOH was used for fluconazole. In quantitative determinations which were performed by using HPLC, ketoconazole was the internal standard. Different regression equations were applied for each method in order to complete the quantitative determination. In UV spectroscopy and HPLC methods, relative standards were found as 0.74, 1.04% and in IR spectroscopy (675, 960 cm^?1) relative standards were found as 1.1 and 1.51%, respectively.     

Thermal Lens Spectroscopy for Quantitative Determination of a Cu(II) Complex With an 8-Aminoquinoline Derivative in Tap Water and Mining Wastewater Samples Using a Dual Beam Technique

Abstract

This article describes the quantitative determination of Cu(II) using thermal lens spectrometry. The chromogenic reaction involving Cu(II) and 5-(4-sulphophenylazo)-8-aminoquinoline in alkaline solution was studied in different experimental conditions such as pH, ligand concentration, methanol volume, and presence of interfering ions. A collinear dual beam set-up has been used for direct quantitation in water samples without a pre-concentration step. The optimized conditions provided a linear calibration in the concentration range from 3.0 to 15.0?×?10^?7?mol L^?1. The detection and quantitation limits were 6.13?×?10^?8? and 2.04?×?10^?7?mol L^?1, respectively. Resultantly, an application to Cu(II) determination in tap water (recovery 99.8–103.3%) and mining (synthetic) wastewater (95.3–98.0%) shows relative SDs ≤ 3.1%. The method is presented as a new alternative for the direct Cu(II) determination in real samples.     

Validated HPLC Method for Separation and Determination of Terbutaline Enantiomers

Abstract

A simple, rapid, and validated method for separation and determination of terbutaline enantiomers was developed. Terbutaline was separated and determined on a Vancomycin Chirobiotic V column (250 × 4.6 mm), using a mixture of methanol, acetic acid, and triethylamine (100:0.1:0.1% v/v/v) as a mobile phase at 20°C and at a flow rate of 1 ml/min. The UV detector was set to 276 nm. Acetyl salicylic acid (aspirin) was used as an internal standard. The applied high-performance liquid chromatography (HPLC) method allowed separation and quantification of terbutaline enantiomers with good linearity (r > 0.999) in the studied range. The relative standard deviations (RSD) were 1.10 and 1.32% for the terbutaline enantiomers with accuracy of 99.80 and 99.55. The limit of detection and limit of quantification of terbutaline enantiomers were found to be 0.05 and 0.10 µg · ml^?1, respectively. The method was validated through the parameters of linearity, accuracy, precision, and robustness. The HPLC method was applied for the quantitative determination of terbutaline in pharmaceutical formulations.     

Veterinary antimicrobials and antiparasitics in fee-fishing ponds: analytical method and occurrence*

ABSTRACT

The aim of this work was to develop and validate a method using online solid-phase extraction and ultra-high-performance liquid chromatography coupled to tandem mass spectrometry to determine residues of 22 veterinary drugs including sulfonamides, amphenicols, fluoroquinolones, benzimidazoles, trimethoprim (TMP) and oxytetracycline (OTC) in water from fee-fishing ponds. The optimal analytical conditions were as follows: XBridge C8 SPE column, Acquity UPLC CSH C18 analytical column, sample loading with water:methanol (98:2, v/v), mobile phase of water with 0.1% acetic acid:methanol (with gradient elution) and eluent flow rate of 0.3 mL min^?1. Quantification was performed in selected reaction monitoring mode and sulfadimethoxine-d₆, ciprofloxacin-d₈, florfenicol-d₃ and albendazole-d₃ were used as internal standards. Water samples collected from 11 fee-fishing ponds showed the presence of residues of FF (0.42–0.74 µg L^?1), albendazole (0.05–0.31 µg L^?1) and thiabendazole (0.45 µg L^?1). Thiamphenicol and TMP were detected at concentrations lower than the limits of quantification of the method (0.1 and 0.001 µg L^?1, respectively).     

UV-Derivative Spectrophotometric Determination of Ritonavir Capsules and Comparison with LC Method

Abstract

An ultraviolet-derivative spectrophotometric method (UV-D) has been proposed as an alternative to a previously described liquid chromatographic (LC) method for the quantitative determination of ritonavir in soft gelatin capsules. The spectrophotometric method is based on recording the second-derivative spectra for ritonavir at 222.3 nm of its solutions in methanol. The linear dynamic range was 10.0–30.0 µg · mL^?1 with a correlation coefficient of 0.9995. Mean recoveries were between 99.2% and 100.2% for the tested capsules samples. Mean intra- and interassay relative standard deviations (RSDs) were less than 2.0%. The statistic analysis showed that LC and UV-D methods were equivalent to assay ritonavir capsules.     

Rapid Quantitation of Verapamil in Plasma by High-Performance Liquid Chromatography

Abstract

A simple and sensitive liquid chromatographic assay procedure using a fluorescence detector for the quantitative determination of verapamil in plasma without extraction was developed. After precipitating the protein with acetonitrile, the resulting supernatant liquid was injected onto the column for analysis. Chromatographic separation was achieved on C₁₈ reversed phase column and the eluting solvent was the isocratic mixture of methanol, acetonitrile and pH 3.0 glycine buffer (1:4:5). With this mobile phase the drug and its internal standard were well separated from the interference of the plasma sample. The average recovery of verapamil from 3 replicate samples of different concentration (100–600 ng/mL) were 95.5 ± 5.68%. The minimum amount of verapamil detectable by this method was 40 ng/mL of sample. The elimination half-life (β-phase) of this drug in rabbits was found to be 3.7 hours.     

Determination of steroids by liquid chromatography/mass spectrometry

On-line atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) liquid chromatography/mass spectrometry (LC/MS) were evaluated for the analysis of a variety of steroids. Steroids were classified into three major groups based on the spectra and the sensitivities observed: (I) those containing a 3-one, 4-ene functional group, (II) those containing at least one ketone group without conjugation, and (III) those containing hydroxy group(s) only. In the APCI mode, the best sensitivity and the lowest detection limit for all three groups were obtained by using a mobile phase consisting of methanol and 1%–2% acetic acid in water. The APCI spectra were characterized by MH⁺, MH⁺-H₂O, MH⁺-2H₂O, etc., with the degree of H₂O loss being compound dependent: group I steroids produced stable MH⁺ and group III steroids showed extensive water loss. In the electrospray mode the best sensitivity and the lowest detection limit for the first two groups were obtained when pure methanol and water were used as the mobile phase. This condition produced abundant stable MNa⁺ due to ubiquitous sodium. Detection limits in the 5–15 pg range can be easily achieved using ESI LC/MS. Addition of ammonium acetate or use of acetonitrile in the mobile phase, common in the LC/MS analysis of steroids, decreased the sensitivity for the group I and II steroids and thus should be avoided. For group III steroids, the detection limit can be improved by the addition of acetic acid to the mobile phase.     

Rapid Assay of Hypothalamic Aromatase Using High Performance Liquid Chromatography

Abstract

A rapid assay for hypothalamic aromatase has been developed using HPLC. Each assay tube contained rat anterior hypothalamus homogenized in Na phosphate buffer (pH 7.0), NADPH regenerating system, unlabelled estradiol and estrone, and 10 μCi of ³H-androstenedione. After agitation at 37°C for 3 hours, the reaction mixture was extracted with CH₂Cl₂, partitioned between 90% methanol and hexane, and the methanol layer chromatographed on a Waters 10μ C-18 Radial-PAK column using acetonitrile/water (70:30). The estrogens were collected together and rechromatographed with THF/water (35:65). The estrone peak was collected, and the ³H-estrone produced was determined by liquid scintillation. The estrone collected from the second chromatographic step was found to be at least 95% pure by methylation and rechromatography. Using the HPLC procedure, enzymatic ³H-estrone formation was found to be linear in a time and protein dependant manner.     

Quantitative Separation of Zr4+ from La3+ and Ce3+; W6+ from Cr3+, Mo6+ and VO2+ by Thin Layer Chromatography

Abstract

Methods have been developed for the quantitative separation of Zr⁴⁺ from La³⁺ and Ce³⁺ using 0.1M ammonium oxalate solution as eluant. W⁶⁺ can also be separated quantitatively from Cr³⁺, Mo⁶⁺ and VO²⁺ using DMSO-1MHC1 (1:9). Both the methods are fast and quantitative. The separation takes about 40–50 min. The average error is about 3%.     

An On-line Admicellar SPE-HPLC System Using CTAB-Modified Zeolite NaY as Sorbent for Determination of Carbamate Pesticides in Water

Zeolite NaY modified with cetyltrimethylammonium bromide (CTAB) was considered for extraction/preconcentration of carbamate pesticides using an on-line SPE-HPLC system. The simultaneous determination of carbamate pesticides, including aldicarb, carbofuran, carbaryl, isoprocarb, methiocarb and promecarb, was performed by HPLC–UV using a LichroCART RP-18 column with gradient elution of methanol and 0.1 % acetic acid. The sorbent presented admicelles of CTAB on its surfaces and exhibited a sorption capacity of 180–18,600 mg kg⁻¹ sorbent, which could be re-modified for at least five extraction cycles. The quantitative retention of target pesticides on the admicellar sorbent involved hydrophobic and π-cation interaction, while pesticides were eluted from the admicellar SPE column using only 750 μL of methanol. LODs and LOQs of the proposed method were 0.005–140 and 0.02–600 μg L⁻¹, respectively. The analytes were effectively concentrated with the enrichment factors between 5 and 551. The developed on-line admicellar SPE-HPLC system was successfully applied to the determination of carbamate pesticides in ten environmental water samples from different sources. Recoveries of spiked samples at a concentration of 0.1–5 mg L⁻¹ ranged from 77 to 111 %.

     

Determination of Flavonoid Markers in Honey with SPE and LC using Experimental Design

Determination of flavonoid markers quercetin, hesperetin, and chrysin, found in north Iranian citrus honey samples, was carried out by solid phase extraction (SPE) and isocratic liquid chromatographic separation using central composite design. Optimum conditions for SPE were achieved using 10 mL methanol/water (13:87, v/v, pH = 7) as the washing solvent and 4 mL methanol for elution. Good clean-up and high recovery >90% were observed for all analytes. The use of water/ACN/THF/AcOH (54:36:5:5, v/v) was found to serve as the optimum mobile phase composition and allowed for the separation of analytes from endogenous compounds present in honey. SPE parameters, such as maximum loading capacity and breakthrough volume, were also determined for each analyte. Limit of detection, linear range, recovery, repeatability of retention times, and peak heights were 3.11 × 10⁻⁸–4.44 × 10⁻⁸ g g⁻¹, 0.50–50.0 μg mL⁻¹ (R ² > 0.99), 90.7–96.9%, 3.0–3.6%, and 1.0–2.6%, respectively. Precision of the overall analytical procedure, estimated by five replicate measurements for quercetin, hesperetin and chrysin in citrus honey, as well as the relative standard deviations were 4.3%, 3.8%, and 5.5%, respectively.

     

LC Separation and Determination of Five Diester-Diterpenoid Alkaloids in the Unprocessed and Processed Aconite Roots

A reliable liquid chromatographic method with photodiode array detector (DAD) was developed and validated for simultaneous separation and determination of five diester-diterpenoid alkaloids in the aconite roots. The separation was successfully performed on a Zorbax Extend-C₁₈ column with a mobile phase gradient prepared from methanol and ammonia solution at a flow rate of 1.0 mL min⁻¹. Good linearity (r > 0.999) was observed over the concentration ranges investigated, and intra-day and inter-day precision were high. The mean recoveries of five components ranged from 90.45 to 102.63% and relative standard deviations were always <5%. The validated method was successfully used for simultaneous determination of the five diester-diterpenoid alkaloids of unprocessed and processed aconite roots. The quantitative method provided a scientific basis for safety assurance and clinical application of aconite roots.