Direct separation and quantitative analysis of thyroxine and triiodothyronine enantiomers in pharmaceuticals by high-performance liquid chromatography

A rapid reversed-phase type HPLC method for the simultaneous separation and analysis of D- and L-thyroxine (D- and L-T(4)) and triiodothyronine (T(3)) was developed using a quinine-derived chiral stationary phase and applied for a quantitative assay of the enantiomeric impurity of the drugs in pharmaceutical formulations of levothyroxine. The influence of operating parameters has been studied for the optimization of the separation and also in order to gain an insight into the retention mechanism. Validation of the method included linearity, precision and accuracy which revealed R.S.D. values of <3.3% for intra-assay precision and percent error ranging from -6 to +2.1% for various defined validation samples, proving satisfactory accuracy. Quantitation was performed over the range of 0.5-500 microg ml(-1) with limits of detection and quantitation lower than 0.1 and 0.5 microg ml(-1), respectively, for both analytes. Further, the determination of 0.1% impurity, of D-T(4) as well as L- and D-T(3) in levothyroxine sodium tablets proved to be feasible.     

Short-term stability of testosterone and epitestosterone conjugates in urine samples: quantification by liquid chromatography-linear ion trap mass spectrometry

A simple method using liquid chromatography-linear ion trap mass spectrometry for simultaneous determination of testosterone glucuronide (TG), testosterone sulfate (TS), epitestosterone glucuronide (EG) and epitestosterone sulfate (ES) in urine samples was developed. For validation purposes, a urine containing no detectable amount of TG, TS and EG was selected and fortified with steroid conjugate standards. Quantification was performed using deuterated testosterone conjugates to correct for ion suppression/enhancement during ESI. Assay validation was performed in terms of lower limit of detection (1-3ng/mL), recovery (89-101%), intraday precision (2.0-6.8%), interday precision (3.4-9.6%) and accuracy (101-103%). Application of the method to short-term stability testing of urine samples at temperature ranging from 4 to 37 degrees C during a time-storage of a week lead to the conclusion that addition of sodium azide (10mg/mL) is required for preservation of the analytes.     

Chemiluminescence flow-injection analysis of beta-lactam antibiotics using the luminol-permanganate reaction.

A new flow injection chemiluminescence (CL) method has been developed for the determination of six beta-lactam antibiotics, including amoxicillin, cefadroxil, cefoperazone sodium, cefazolin sodium, cefradine and ceftriaxone sodium. When the antibiotic was injected into a stream of KMnO4 with alkaline luminol, a strong CL signal was produced. The method allows the measurements of 0.1-50.0 mg/L amoxicillin, 0.1-80.0 mg/L cefadroxil, 1.0-30.0 mg/L cefoperazone sodium, 1.0-30.0 mg/L cefazolin sodium, 3.0-50.0 mg/L cefradine and 3.0-50.0 mg/L ceftriaxone sodium. The detection limits are 0.05 mg/L for amoxycillin, 0.05 mg/L for cefadroxil, 0.4 mg/L for cefoperazonum sodium, 0.4 mg/L for cefazolin sodium, 0.8 mg/L for cefradine and 0.8 mg/L for ceftriaxone sodium. The relative standard deviations in 11 repeated measurements are 0.6%, 0.8%, 1.5%, 1.2%, 0.4% and 0.3% for 3.0 mg/L amoxicillin, 1.0 mg/L cefadroxil, 10.0 mg/L cefoperazone sodium, 10.0 mg/L cefazolin sodium, 10.0 mg/L cefradine and 10.0 mg/L ceftriaxone sodium, respectively. The method was successfully applied to the determination of amoxicillin in pharmaceutical preparations. A possible CL reaction mechanism is also discussed.     

Direct chemiluminescence determination of ibuprofen by the enhancement of the KMnO(4)-sulphite reaction.

A simple, rapid, flow-injection chemiluminescence (CL) method is described for the determination of ibuprofen. A strong CL signal was detected when a mixture of the analyte and sulphite was injected into acidic KMnO(4). The CL signal is proportional to the concentration of ibuprofen in the range 0.1-10.0 mg/L. The detection limit is 0.02 mg/L ibuprofen, the relative standard deviation is 1.8% (0.5 mg/L ibuprofen; n = 11) and the sample measurement frequency is 120/h. The proposed method was successfully applied to the determination of ibuprofen in pharmaceutical preparations and in spiked urine samples. The mechanism of the CL reaction is also discussed.     

Development of a flow‐injection analysis system with fluorescence detection for gatifloxacin determination in organized medium

This work reports the development and optimization of a flow injection analysis system with fluorescence detection (FIA–FLUO) for gatifloxacin (GFX) determination in organized medium. The analytical system was based on the enhanced fluorescence of gatifloxacin in micellar medium containing sodium dodecyl sulfate (SDS) at pH 6.0. The influence of physical (carrier flow rate, sample volume and volume of reaction coil) and chemical (pH, concentration of buffer and concentration of SDS) parameters that could affect the performance of the FIA system was evaluated in order to reach optimum conditions in terms of sensitivity and analytical throughput. Under optimized conditions, the FIA–FLUO system allowed the injection of 40 samples per hour with a limit of quantification of 72 µg/L and a RSD of 3.5% at 0.20 mg/L. Real samples of commercial pharmaceutical formulations containing GFX were analyzed, and no statistical difference was observed between the results obtained using the developed system and those obtained using the reference method based on high‐performance liquid chromatography with UV detection. Copyright © 2014 John Wiley & Sons, Ltd.     

A versatile transition metal salt reaction for a wide range of common biochemical reagents: an instantaneous and quantifiable color test

A rapid and sensitive spot test amenable to visual or spectrophotometric quantitation has been developed for a wide variety of biochemical reagents by utilizing the transition metal salt cupric chloride and its large number of related colored compounds. This assay is potentially a widely applicable multipurpose test for rapidly detecting the presence of unknown substances. Combination of the test sample with the working reagent results in the immediate formation of a distinctive colored product that may be precipitable. Some compounds require the further addition of sodium hydroxide in order to generate the distinctively colored product. Distinctive reactions occur with the following reagents, and their limit of visual detection is indicated in parentheses: ammonium bicarbonate (12.5 mM), ammonium acetate (25 mM), ammonium hydroxide (0.1%), ammonium sulfate (2%), ammonium persulfate (0.02 mM), L-(+)-cysteine (0.07 mM), dithiothreitol (DTT) (1.25 mM), EDTA (0.6 mM), ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (5 mM), D-glucose (6 mM), glycerol (0.3%), imidazol (12.5 mM), DL-methionine (100 mM), mercaptoethanol (0.05%), sodium azide (19 mM, 0.1%), sodium dithionite (0.25%), sodium metabisulfite (25 mM), sodium nitrite (6.2 mM), sodium periodate (3.1 mM), sodium sulfite (12.5 mM), sodium thiosulfite (12.5 mM), sucrose (6 mM), and N,N,N',N'-tetramethylethylenediamine (0.05%). A distinctive exothermic reaction occurs with hydrogen peroxide, but without color change. Compounds reacting insignificantly include 50 mM Tris buffer, urea, N,N'-methylene bisacrylamide, sodium dodecyl sulfate, isopropyl alcohol, sodium fluoride, trichloroacetic acid, phenol, mannose, K2HPO4, guanidine HCl, chloramine-T, magnesium chloride, and boric acid, where the solids were tested at approximately 10 mg/ml. Spectrophotometric standard curves were developed for DTT and sodium azide utilizing the clear supernatants resulting from these reactions. Combinations of at least four reagents could be discriminated, as demonstrated with mixtures of glucose, sodium azide, EDTA, and DTT. In addition ammonium sulfate could be detected to a limit of 4% in the presence of protein, DTT, and EDTA in a 50 mM Tris buffer. Spot tests were developed which utilized reagent-impregnated filter paper and gave distinctive colored products on addition of 5 microliter of test sample.     

Direct injection of human serum and pharmaceutical formulations for glucosamine determination by CE-C(4)D method

A simple CE-C(4)D method has been developed for the determination of glucosamine by direct injection of human serum and pharmaceutical samples. Glucosamine was electrokinetically injected and analysed in its protonated form using 20mM MES/His (pH 6) as background electrolyte in order to separate it from the matrix and to provide a better response to the C(4)D detector. Separation of glucosamine in human serum and pharmaceutical samples was performed in 3 min without the need for protein precipitation or matrix removal. Good precision in terms of %RSD for the migration time and peak area were less than 1.91% (n = 10). The conductivity signal was linear with glucosamine concentration in the range 0.10-2.50mg/mL, with a detection limit of 0.03 mg/mL. Recoveries of glucosamine in serum and pharmaceutical samples were 86.5-104.78%. The method was successfully applied for the determination of the glucosamine content in pharmaceutical formulations and validated with high performance liquid chromatography (HPLC). Good agreements were observed between the developed method, label values and the HPLC method. Glucosamine could be detected in spiked serum sample by direct injection. This was not possible by HPLC due to co-eluting interferences.     

Stress degradation studies on betahistine and development of a validated stability-indicating assay method

The purpose of this work was to study the stability of betahistine (BET) at different stress conditions and to develop a sensitive stability-indicating high-performance liquid chromatographic (HPLC) assay method. The stress conditions applied were including the effect of heat, moisture, acid-base, and ultra-violet (UV) light. Betahistine and its decomposition products were derivatized by reaction with dansyl chloride (Dan-Cl) and analyzed by HPLC equipped with fluorescence detector (FL) set at 336 and 531 nm as excitation and emission wavelengths, respectively. The drug was particularly labile at UV light and oxygen rich media. Two potential degradation products could be separated and identified by spectral methods. The chromatographic method involved Zorbax Eclipse XDB-C(18) column kept at 30+/-2 degrees C and a gradient elution with mobile phase composed of acetonitrile and 0.02 mol L(-1) sodium acetate. The response factor of dansylated BET monitored by fluorescence detection was 32 times more than its UV response. The calibration curve of BET in bulk form was linear from 0.005 to 4.2 ng microL(-1). Intraday and interday precision were less than 0.04% (CV), and accuracy was between 99.2% and 100.9% over 2.0 ng microL(-1). The limit of detection was 0.002 ng microL(-1). The method was also validated for sample stability during reaction, robustness and selectivity. The method was applied for purity testing of betahistine in tablet form.     

Magnetic Nanoparticle-Based Solid-Phase Extraction of Vitamin B12 from Pharmaceutical Formulations

In the present study, a novel quantitative method, namely magnetic nanoparticle-based solid-phase extraction (MSPE), was applied to extract vitamin B(12) from pharmaceutical formulations. The technique involves the use of Fe(3)O(4) nanoparticles modified by sodium dodecyl sulfate (SDS) as an efficient adsorbent for solid-phase extraction of vitamin B(12). Collection of magnetic nanoparticles (MNPs) from aqueous solution was simply achieved by applying external magnetic field. The analyte was desorbed from MNPs using alkali 1-propanol. The extracted analyte was analyzed by using flow injection inductively coupled plasma-optical emission spectrometry. Factors affecting the extraction efficiency were investigated and optimized. Under the optimum conditions, enhancement factor of 184, linear dynamic range of 2.5-500 μg L(-1) with correlation of determination (R(2) > 0.999), and limit of detection of 1.0 μg L(-1) were obtained for vitamin B(12). The percent relative standard deviation based on five-replicate determination was less than 6.2%. The method was successfully applied for extraction and determination of vitamin B(12) in different types of pharmaceutical samples such as multivitamin tablet, effervescent tablet, and injection sample. The results showed that the proposed method based on SDS-Fe(3)O(4) MSPE was a simple, accurate, and highly efficient approach for analysis of vitamin B(12).     

Comparison of ion-pair and amide-based column reversed-phase liquid chromatography for the separation of thiamine-related compounds

Two reversed-phase chromatographic methods for the separation of thiamine and related compounds are compared. The first procedure is based on the ion-pair technique using an octadecylsilica column, while the second uses a new amide-based stationary phase, which avoids the need to form ion-pairs, leading to narrower peaks and a simpler mobile phase. Analyses were performed by gradient elution and a photo-diode array was used for detection. Specificity was demonstrated by the retention characteristics, UV spectra and by comparing the peak purity index with commercial standards. The procedures were applied to the determination of thiamine-related compounds in pharmaceutical preparations and urine. No preliminary sample treatment was required.     

抗干扰微生物检测技术研究

在检测细菌、真菌和大肠菌群时,抗防腐剂型微生物培养基可消除样品中浓度为 ２．０ｇ／Ｌ（Ｋｇ）的山梨酸、山梨酸钾、苯甲酸及苯甲酸钠的干扰;抗消毒剂型微生物培养基可分别消除１５０ｍｇ／ Ｌ二氧化氯、４００ｍｐ／ Ｌ过氧乙酸、７００ｍｇ／ Ｌ次氯酸钠及１８０ｍｐ／ Ｌ过氧化氢的干扰;抗臭氧型微生物培养基可以消除１０．０ｍｇ／Ｌ的臭氧和余氯的干扰。大样倾注平板法可以取５ｍＬ的样品,适用所有样品的检测;液体大样法可以取１００ｍＬ的样品,适用于所有样品的检测及增菌,特别适用于无色液体样品;最小近似数法可以检出次大样中     

Stability-indicating TLC-densitometric method for estimation of dexrabeprazole and domperidone in pharmaceutical dosage form

A stability-indicating thin layer chromatographic (TLC) method for determination of dexrabeprazole (DEX) and domperidone (DOM) in combined dosage form has been developed and validated. The mobile phase selected was acetone:toluene:methanol (4.5:4.5:0.5, v/v/v) with ultraviolet (UV) detection at 285 nm. The retention factors for DEX and DOM were found to be 0.49 ± 0.02 and 0.24 ± 0.03, respectively. The method was validated with respect to linearity, accuracy, precision, and robustness. The linearity range for DEX was 50-350 ng/band (r2 = .9960) and for DOM was 100-700-n /band (r2 = .9982), respectively. The method was successfully applied for the analysis of drugs in pharmaceutical formulation.     

Standard operating procedures for pre-analytical handling of blood and urine for metabolomic studies and biobanks

(1)H NMR metabolic profiling of urine, serum and plasma has been used to monitor the impact of the pre-analytical steps on the sample quality and stability in order to propose standard operating procedures (SOPs) for deposition in biobanks. We analyzed the quality of serum and plasma samples as a function of the elapsed time (t?=?0-4?h) between blood collection and processing and of the time from processing to freezing (up to 24?h). The stability of the urine metabolic profile over time (up to 24?h) at various storage temperatures was monitored as a function of the different pre-analytical treatments like pre-storage centrifugation, filtration, and addition of the bacteriostatic preservative sodium azide. Appreciable changes in the profiles, reflecting changes in the concentration of a number of metabolites, were detected and discussed in terms of chemical and enzymatic reactions for both blood and urine samples. Appropriate procedures for blood derivatives collection and urine preservation/storage that allow maintaining as much as possible the original metabolic profile of the fresh samples emerge, and are proposed as SOPs for biobanking.     

Fixation procedures for flow cytometric analysis of environmental bacteria

Analysis of environmental bacteria on the single cell level often requires fixation to store the cells and to keep them in a state as near life-like as possible. Fixation procedures should furthermore counteract the increase of autofluorescence, cell clogging, and distortion of surface characteristics. Additionally, they should meet the specific fixation demands of both aerobically and anaerobically grown bacteria. A fixation method was developed based on metal solutions in combination with sodium azide. The fixation efficiencies of aluminium, barium, bismuth, cobalt, molybdenum, nickel, and tungsten salts were evaluated by flow cytometric measurement of the DNA contents as a bacterial population/community stability marker. Statistical equivalence testing was involved to permit highly reliable flow cytometric pattern evaluation. Investigations were carried out with pure cultures representing environmentally important metabolic and respiratory pathways as controls and with activated sludge as an example for highly diverse bacterial communities. A mixture of 5 mM barium chloride and nickel chloride, each and 10% sodium azide was found to be a suitable fixative for all tested bacteria. The described method provided good sample stability for at least 9 days.     

Flow injection spectrophotometric determination of the antibiotic fosfomycin in pharmaceutical products and urine samples after on-line thermal-induced digestion

A new flow injection (FI) method for the precise and rapid spectrophotometric determination of the antibiotic fosfomycin (FMC) in urine and pharmaceutical samples is described. The method is based on the on-line quantitative thermal-induced digestion of the analyte prior to injection into the FI system. Ammonium persulfate was used as the oxidation reagent. The resulting orthophosphate ions were determined spectrophotometrically (lambda(max) = 690 nm) using the molybdenum blue approach. Chemical and FI variables that affected on-line oxidation were studied and optimized. The proposed method is very precise (s(r) = 1.2% at 1.0 x 10(-4) mol L(-1) FMC, n = 12), offers a high sampling rate of 60 h(-1), and allows for the determination of the analyte in the range 3.0 x 10(-6) to 3.0 x 10(-4) mol L(-1) with a satisfactory 3sigma detection limit of 1.0 x 10(-6) mol L(-1). Application of the proposed method to urine and pharmaceutical samples yielded accurate results with percentage recoveries in the range 96.4-102.5%.     

Determination of Trantinterol Enantiomers in Human Plasma by High‐Performance Liquid Chromatography – Tandem Mass Spectrometry Using Vancomycin Chiral Stationary Phase and Solid Phase Extraction and Stereoselective Pharmacokinetic Application

A sensitive and enantioselective vancomycin chiral stationary phase high‐performance liquid chromatography–tandem mass spectrometry method was developed for the determination of trantinterol enantiomers in human plasma. Baseline resolution was achieved using the vancomycin chiral stationary phase known as Chirobiotic V with polar ionic mobile phase consisting of acetonitrile–methanol (60:40, v/v) containing 0.01% ammonia and 0.02% acetic acid at a flow rate of 1.0 mL/min. Waters Oasis HLB C18 solid phase extraction cartridges were used in the sample preparation of trantinterol samples from plasma. The detection was performed on a triple‐quadrupole tandem mass spectrometer by multiple reaction monitoring mode via electrospray ionization. The calibration curve was linear in a concentration range from 0.0606 to 30.3 ng/mL in plasma, with the lower limit of quantification of 0.0606 ng/mL. The intra‐ and interday precision (relative standard deviation) values were within 9.7% and the accuracy (relative error) was from ?6.6 to 7.2% at all quality control levels. The method was successfully applied to a study of stereoselective pharmacokinetics in human. Chirality 27:327–331, 2015.© 2015 Wiley Periodicals, Inc.     

Direct separation, identification and quantification of epimers 22R and 22S of budesonide by capillary gas chromatography on a short analytical column with Rtx-5 stationary phase

The conditions for separation, identification and quantitative determination of epimers 22R and 22S of budesonide by capillary gas chromatography (GC) with FID detection and two various sample injection methods, namely split-splitless and cool on-column, were established. In analysis helium as carrier gas and Rtx-5 capillary column of 7 m in length along with stationary phase Crossbond 5% diphenyl-95% dimethyl polysiloxane were used. The individual epimers were identified under specified conditions by using standard samples of different declared concentration of each epimer under investigation: (1) 51.2% of epimer 22R and 47.3% of epimer 22S, and (2) 95.1% of 22R and 4.4% of 22S, as well as Pulmicort, a preparation containing micronized budesonide as an active substance. It seems that good parameters of preliminary validation achieved by the proposed methods can confirm its suitability for quantitative analysis purpose. The retention times obtained for epimers 22R and 22S, depending on injection technique are about 7.7 and. 8.3 min for split and, approx. 10.3 and 10.9 min for cool on-column. The limits of detection and quantitation are 5.7 and 6.2 ng, for 22R respectively, and 4.3 and 4.8 ng for 22S. The linearity is maintained for concentrations ranging from 0.01 to 0.20 mg/ml. The quantitative analysis features of repeatability, high precision and accuracy confirmed by the obtained results and its statistical evaluation.     

Determination of β-blockers in pharmaceutical and human urine by capillary electrophoresis with electrochemiluminescence detection and studies on the pharmacokinetics

A novel method for simultaneous determination of atenolol, metoprolol and esmolol was proposed by capillary electrophoresis (CE) separation and electrochemiluminescence (ECL) detection. Poly-β-cyclodextrin (Poly-β-CD) was used as an additive in the running buffer to improve the separation of three analytes. The conditions for CE separation, ECL detection and effect of Poly-β-CD were investigated in detail. The three β-blockers with very similar structures were well separated and detected under the optimum conditions. The linear ranges of the standard solution for atenolol and esmolol were 2.5-125 μmol/L with a detection limit (S/N=3) of 0.5 μmol/L, and for metoprolol was 0.5-25 μmol/L with a detection limit of 0.1 μmol/L. For three β-blockers from spiked aqueous and urine samples, the accuracy and precision including intraday and interday experiments were performed by calculating the recovery, the relative standard deviations of the ECL intensity and the migration time, respectively. The developed method was applied to the determination of metoprolol content in commercial pharmaceutical, and the analytical results are in good agreement with the nominal value with recoveries in the range of 98.7-105%. The proposed method was also applied to the monitoring of pharmacokinetics for metoprolol in human body.     

Protein components of the erythromycin binding site in bacterial ribosomes

Two derivatives of erythromycin have been prepared carrying either an aryl azide or a 4-nitroguaiacol as a photoreactive group. Both derivatives bind to the specific erythromycin ribosomal site as shown by saturation and competition studies. The derivatives were isotopically labeled either with tritium or with 125I, and radioactivity is covalently incorporated to the ribosome upon irradiation at the appropriate wavelength. The ribosomal proteins labeled were identified by either mono- and two-dimensional gel electrophoresis or high performance liquid chromatography. It has been found that protein L22 is the protein mainly, and under some conditions exclusively, labeled by the erythromycin derivatives. These results were confirmed using ribosomes from erythromycin-resistant mutants having a protein L22 with modified electrophoretical mobility. Protein L15 is also labeled in both cases, and the aromatic azide derivative labels to a lesser extent proteins L2 and L4. Competition experiments with erythromycin indicate that labeling in protein L22, and probably in L15, is specific, while the specificity of labeling in proteins L2 and L4 is questionable. These results indicate that the erythromycin derivatives label different ribosomal proteins than the spiramycin type of macrolides (Tejedor, F., and Ballesta, J.P.G. (1985) Biochemistry 24, 467) suggesting that the binding sites of both macrolide types are probably not identical.     

Poly(ethyleneglycol) column for the determination of acetaminophen,phenylephrine and chlorpheniramine in pharmaceutical formulations

New polar reversed-phase stationary phases in HPLC provide specific selectivities which can help to solve traditional chromatographic problems related to the development of chromatographic methods with widely different retention times for the sample components. One such case is the analysis of pharmaceutical formulations against the common cold. Acetaminophen, phenylephrine and chlorpheniramine, compounds with different polarities, are frequently associated in these drugs. An isocratic and rapid HPLC method for the simultaneous determination of the three compounds, acetaminophen, phenylephrine and chlorpheniramine, in capsules as pharmaceutical formulations, including the separation of impurities (4-aminophenol and 4-chloracetanilide) and excipients, has been developed and validated. The final chromatographic conditions employed a Supelco Discovery HS PEG column poly(ethyleneglycol) 15x0.46 cm, 5 microm. The mobile phase was 20 mM phosphate buffer, pH 7.0-acetonitrile (90:10, v/v) at a flow-rate of 1 ml/min. UV detection was performed at 215 nm for all the compounds except acetaminophen, which was measured at 310 nm. Validation parameters permit us to consider this method suitable.