Evaluation of a high-performance thin-layer chromatographic technique for the determination of salbutamol serum levels in clinical trials.

Salbutamol concentrations were determined by high-performance thin-layer chromatography in the sera of two sets of ten volunteers at hourly intervals for 6 h after taking one 8-mg slow-release tablet. The influence of time lapse in processing of serum samples, i.e. centrifugation, extraction and chromatography, was studied. A statistical significant instability of salbutamol in the sera of patients was found which was not present in standard drug-free serum samples spiked with salbutamol and used for construction of standard curves.     

Identification of the major metabolites of (R)‐salbutamol in human urine,plasma and feces using ultra high performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry

(R)‐Salbutamol is a selective β2‐adrenoreceptor agonist, which produces a short‐acting bronchodilator effect and is widely used for the treatment of respiratory diseases in humans. Drug metabolism and identification of the metabolites play an essential role in the evaluation of the overall efficacy and safety of the drugs in clinical practices. There are few reports on the identification of major metabolites of (R)‐salbutamol in humans, and the number of identified metabolites is very limited. In this research, a method of ultra‐high performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry was developed for the discovery and identification of (R)‐salbutamol and its major metabolites in human biological samples. Totally, twelve metabolites of (R)‐salbutamol were found and identified and all the metabolites could be found in urine, one metabolite in plasma and two metabolites in feces. Among all the metabolites, eight metabolites have never been reported before. The results indicated that (R)‐salbutamol was mainly metabolized through isomerization, oxidation, reduction, glucuronidation, and sulfation pathways in vivo. The possible metabolic pathways of (R)‐salbutamol were subsequently presented in this study, which contribute to a better understanding of the metabolism of (R)‐salbutamol in humans.     

Determination of salbutamol in human plasma and urine using liquid chromatography coupled to tandem mass spectrometry and its pharmacokinetic study

A sensitive and selective liquid chromatography coupled to tandem mass spectrometry (LC‐MS/MS) was developed and validated for the determination of salbutamol in human plasma and urine, and successfully applied to the pharmacokinetic study of salbutamol in Chinese healthy volunteers after inhalation of salbutamol sulfate aerosol. Salbutamol and the internal standard (IS) acetaminophen in plasma and urine were extracted with ethyl acetate, separated on a C₁₈ reversed‐phase column, eluted with mobile phase of acetonitrile–ammonium acetate (5 m m ; 30:70, v/v), ionized by positive ion pneumatically assisted electrospray and detected in the multi‐reaction monitoring mode using precursor → product ions of m/z 240.2 → 148.1 for salbutamol and 152 → 110 for the IS. The lower limits of quantitation of salbutamol in human plasma and urine by this method were 0.02 and 1 ng/mL, respectively. The specificity, matrix effect, recovery, sensitivity, linearity, accuracy, precision and several stabilities were validated for salbutamol in human plasma and urine. In conclusion, the validation results showed that this method is robust, specific and sensitive, and can successfully fulfill the requirement of clinical pharmacokinetic study of salbutamol in healthy Chinese volunteers. Copyright © 2011 John Wiley & Sons, Ltd.     

Detection of inhaled salbutamol in equine urine by ELISA and GC/MS2

Salbutamol is a beta-adrenergic agonist that is used in the treatment of asthma in humans and chronic obstructive pulmonary disease in horses. Because of its stimulating and growth promoting properties, it is prohibited by horse racing authorities. Recently a number of adapters (eg Equinehaler) have been designed, allowing the use of metered dose inhalers (MDI) approved for human use. However, information on detection times of salbutamol after administration of salbutamol in therapeutic doses by inhalation is lacking. In this study, 2 mg salbutamol (Ventolin) was administered to four standardbred mares via an MDI with an Equinehaler and urine was collected during 48 h. Quantification of salbutamol in horse urine was done via an overnight beta-agonist ELISA kit. Salbutamol was detected between 1 and 48 h post-administration. Relatively large interindividual variations in the total amount excreted during the first 12 h were noticed. The maximum urinary concentrations varied between 4.6 and 8.1 ng/mL. The total amount excreted within the first 12 h varied between 0.2 and 0.7% of the administered dose. For confirmatory analysis in doping control, a GC/MS(2) method was developed and validated. Analysis was performed on an ion trap instrument after solid phase extraction. The limit of detection was 0.25 ng/mL and was lower than in previously reported methods in human urine.     

Determination of salbutamol enantiomers in human urine using heptakis(2,3-di-O-acetyl-6-O-sulfo)-beta-cyclodextrin in nonaqueous capillary electrophoresis

Nonaqueous capillary electrophoresis (NACE) was successfully applied to the resolution and the determination of salbutamol enantiomers in urine samples using heptakis(2,3-di-O-acetyl-6-O-sulfo)-beta-cyclodextrin (HDAS-beta-CD). After optimization of the electrophoretic parameters, namely the background electrolyte (BGE) composition and the HDAS-beta-CD concentration, salbutamol enantiomers were completely resolved using a BGE made up of 10 mM ammonium formate and 15 mM HDAS-beta-CD in methanol acidified with 0.75 M formic acid. Isoprenaline was selected as internal standard. Solid-phase extraction (SPE) was used for sample cleanup prior to the CE separation. Different sorbents involving polar, nonpolar interactions or dual retention mechanisms were evaluated and extraction cartridges containing both nonpolar and strong cation-exchange functionalities were finally selected. Salbutamol enantiomers recoveries from urine samples were determined. The method was then successfully validated using a new approach based on accuracy profiles over a concentration range from 375 to 7500 ng/mL for each enantiomer.     

Clean-up, detection and determination of salbutamol in human urine and serum

Salbutamol ?2-(tert-butylamino)-1-[4-hydroxy-3- (hydroxymethyl)phenyl]ethanol?, also known as albuterol, is clinically the most widely used beta 2-adrenoceptor agonist in the treatment of bronchial asthma. During this study, we evaluated liquid-liquid extraction (LLE) and solid-phase extraction (SPE) in order to develop a reliable extraction method followed by analysis using liquid chromatography and gas chromatography. An assay is described which involves SPE as the clean-up method followed by gas chromatography-mass spectrometry to determine salbutamol levels in human serum after oral administration. The SPE method requires the use of a hyper-cross-linked styrene-divinylbenzene bonded phase (ENV+) without involving any sample pre-treatment to obtain 60-65% recoveries for salbutamol and terbutaline as the internal standard. Distilled water and 1% trifluoroacetic acid in methanol were found to be the most suitable washing solvent and eluting solvent, respectively. A detection limit of 2 ng mL-1 was achieved by derivatization with N-methyl-N-trimethylsilyltrifluoroacetamide to form trimethylsilyl (TMS)-salbutamol (m/z 369) and TMS-terbutaline (m/z 356). The relationship between the ratio of the peak area of salbutamol to that of the internal standard and concentration was linear for the range tested (2-200 ng mL-1) and the correlation of coefficient was 0.9999 with a y-intercept not significantly different from zero. The inter-day relative standard deviation (RSD) was < 10% for all three concentrations. The intra-day RSD was 14% for 2 ng mL-1. This assay was then successfully applied to human serum samples obtained from clinical trials after oral administration of salbutamol.     

Determination of salbutamol using on-line solid-phase extraction and sequential injection analysis. Comparison of chemiluminescence and fluorescence detection

Determination of salbutamol using sequential injection analysis (SIA) with chemiluminescence and fluorescence detection has been devised. The chemiluminescence signal was emitted during the oxidation of salbutamol by potassium permanganate in sulfuric acid medium. Sodium polyphosphate was used as chemiluminescence enhancer. The fluorescence signal (excitation wavelength 230 nm) was also measured in sulfuric acid medium. Both detection techniques were compared with respect to the application of the methods to the determination of salbutamol in biological materials. The sample pre-treatment takes place directly in the SIA system, when salbutamol is adsorbed on the solid-phase (Baker-carboxylic acid) microcolumn integrated into the system. Sulfuric acid serves both as the reagent and the eluent. The lab-made SIA system consisted of a 2.5-mL Cavro syringe pump, ten-port Vici Valco selection valve and Spectra-Physics FS 970 fluorescence detector, which was lab-modified for chemiluminescence detection. The system was controlled by a PC using originally compiled LabVIEW-supported software. Concentrations, volumes of reagents and flow rates were optimised by a simplex method. Salbutamol was determined in the linear range 0.05-10 microg mL(-1) (RSD 1.53%), with the detection limit (3 sigma) 0.03 microg mL(-1) and sample throughput of 42 samples per hour with chemiluminescence detection in standard solutions. The fluorescence detection enabled the determination of salbutamol in standard solutions in the linear range 0.5-100 microg mL(-1) (RSD 2.69%), with the detection limit 0.2 microg mL(-1) and sample throughput of 24 h(-1). The proposed methods were applied to the determination of salbutamol in human serum and urine. However, serum is a very complicated matrix and the SIA-SPE analysis did not provide satisfactory results. It was possible to determine salbutamol in human urine using this technique. Better recovery was achieved with fluorescence detection.     

Determination of salbutamol in human plasma and urine by high-performance thin-layer chromatography

A rapid, accurate and sensitive method for the determination of salbutamol in plasma and urine is described. Salbutamol is extracted using solid-phase techniques and converted to an indoaniline dye by reaction with dimethyl-p-phenylenediamine. The indoaniline is separated using high-performance thin-layer chromatography and quantified by absorption microdensitometry at 650 nm. The method is sensitive down to 20 ng/ml in urine and to 1 ng/ml in plasma and provides data in good agreement with that obtained by gas chromatography--mass spectrometry. The method can be used for analysis of pharmacokinetic studies.     

A highly sensitive LC-tandem MS assay for the measurement in plasma and in urine of salbutamol administered by nebulization during mechanical ventilation in healthy volunteers

The new-generation nebulizers are commonly used for the administration of salbutamol in mechanically ventilated patients. The different modes of administration and new devices have not been compared. We developed a liquid chromatography-tandem mass spectrometry method for the determination of concentrations as low as 0.05 ng/mL of salbutamol, corresponding to the desired plasma concentration after inhalation. Salbutamol quantification was performed by reverse-phase HPLC. Analyte quantification was performed by electrospray ionization-triple quadrupole mass spectrometry using selected reaction monitoring detection ESI in the positive mode. The method was validated over concentrations ranging from 0.05 to 100 ng/mL in plasma and from 0.18 to 135 ng/mL in urine. The method is precise, with mean inter-day coefficient of variation (CV%) within 3.1-8.3% in plasma and 1.3-3.9% in urine, as well as accurate. The proposed method was found to reach the required sensitivity for the evaluation of different nebulizers as well as nebulization modes. The present assay was applied to examine whether salbutamol urine levels, normalized with the creatinine levels, correlated with the plasma concentrations. A suitable, convenient and noninvasive method of monitoring patients receiving salbutamol by mechanical ventilation could be implemented.     

Capillary GC analysis of compounds leached into parenteral solutions packaged in plastic bags

A number of investigators have reported finding the plasticizer di(2-ethylhexyl)phthalate in plasma, urine, and parenteral solutions. These findings have been associated with the use of polyvinyl chloride packaging materials. Capillary gas chromatography--mass spectrometry, packed column gas chromatography, and high-performance liquid chromatography were used for the analyses. The screening procedure described herein accurately and quantitatively analyzes the plasticizers di(2-ethylhexyl)phthalate and dibutylphthalate and some of the degradation products plus 2,6-di-tert-butyl-p-cresol and cyclohexanone. Liquid--liquid extraction followed by capillary gas chromatography is used. Linear regression analysis of standard solutions gives correlation coefficients from 0.99963 to 0.99989 for six compounds. Recoveries for these compounds vary from 84.9% to 114% with relative standard deviations ranging from 2.51% to 6.33%.     

In situ assessment of atorvastatin impurity using MALDI mass spectrometry imaging (MALDI-MSI)

The analysis of impurities and degradation products in pharmaceutical preparations are usually performed by chromatographic techniques such as high-performance liquid chromatography (HPLC). This approach demands extensive analysis time, mostly due to extraction and separation phases. These steps must be carried out in samples in order to adapt them to the requirements of the analytical method of choice. In the present contribution, matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was employed to quantify an important degradation product in atorvastatin calcium 80 mg tablets: the atorvastatin lactone. Through the standard of the impurity, it was possible to perform quantitative analysis directly on the drug tablet, using a quick and novel approach, suitable for quality control processes in the pharmaceutical industry.     

Ochratoxin A in Wine: Its Determination and Photostability

Abstract

Due to the growing public concern regarding food safety, reliable, nondemanding and robust analytical methods are needed for quantitative determination of toxic compounds in complex matrices. Sample preparation is frequently a crucial step in determination of ochratoxin A (OTA) in wine, and a simplified and automated procedure is described, using solid‐phase extraction coupled on‐line to high pressure liquid chromatography (HPLC) with fluorimetric detection (λ_ex=333 nm, λ_em=460 nm). While the limit of quantitation is frequently better compared to off‐line procedures (30 ng/L), the decisive advantages of the new procedure are the absence of all sample manipulation during preconcentration and subsequent analysis, and consequentially no risk of analyte loss or sample contamination. Furthermore, using the standard addition method, matrix interferences can be avoided and the determination of extraction efficiency is unnecessary. These improvements have important consequences for the overall uncertainty of the analytical procedure. The developed method was applied for determination of OTA in 12 selected Slovenian wines. The typical relative standard deviation (RSD) was 10%. In none of the samples, did the OTA amount exceeded 2 µg/kg, the limit regulated by the EC.

The photo‐stability of the mycotoxin in solutions was examined. During irradiation of OTA solutions, its content was quickly reduced, while three fluorescent degradation products were detected. The degradation proceeds faster in water and 12% ethanolic solutions than in organic solvents or wine. Identification of the fluorescent degradation products was attempted using LC‐MS/MS with electrospray ionization.     

Determination of salbutamol in human plasma and urine by high-performance liquid chromatography with a coulometric electrode array system

A method is developed to determine salbutamol in human plasma and urine using high-performance liquid chromatography (HPLC) with a coulometric electrode array system, based on the electrochemical behavior of salbutamol at graphite electrode. The mobile phase component A is 30 mM sodium dihydroxy phosphate-30 mM triethylamine and is adjusted to pH 6.0 with 20% phosphate acid. The mobile phase component B is methanol. The optimized mobile phase composition was A and B in the proportion of 90:10 (v/v). Paracetamol is selected as the external standard. The human plasma and urine samples are pretreated using solid-phase extraction cartridges (Sep-Pak Silica), and the eluting solution is monitored by the coulometric electrode array system. The electrode potentials are set at 300, 400, 550, and 650 mV, respectively. Calibration curves show good linearity, and the recovery of salbutamol proves to be constant and unaffected by the concentration of the drug. This method, developed using HPLC-electrochemical detection, is reproducible and sensitive enough for the determination of salbutamol in human plasma and urine.     

Simple and accurate quantitative analysis of seven prohibited threshold substances in human urine by liquid chromatography/tandem mass spectrometry in doping control

A simple and accurate liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the quantitative determination of ephedrine, pseudoephedrine, methylephedrine, cathine, salbutamol, morphine and epitestosterone in human urine. Urine samples were spiked with internal standard and diluted with acetonitrile. After centrifugation, the supernatants were directly analyzed by LC/MS/MS using the selected reaction monitoring (SRM) mode. The linearity, intra- and inter-day precision, accuracy, limit of detection (LOD) and limit of quantification (LOQ) were evaluated and the method was found to be accurate and reproducible for the quantitation of threshold substances. When the method was applied to the analysis of blind urine samples for the proficiency test, the results were close to the nominal concentrations, within 87.7-106.6% of nominal values, suggesting that the developed methods can be successfully applied to routine doping analyses.     

Determination of chemical warfare agents and related compounds in environmental samples by solid-phase microextraction with gas chromatography

Solid phase microextraction (SPME) is an increasingly common method of sample isolation and enhancement. SPME is a convenient and simple sample preparation technique for chromatographic analysis and a useful alternative to liquid-liquid extraction and solid phase extraction. SPME is speed and simply method, which has been widely used in environmental analysis because it is a rather safe method when dealing with highly toxic chemicals. A combination of SPME and gas chromatography (GC) permits both the qualitative and quantitative analysis of toxic industrial compounds, pesticides and chemical warfare agents (CWAs), including their degradation products, in air, water and soil samples. This work presents a combination of SPME and GC methods with various types of detectors in the analysis of CWAs and their degradation products in air, water, soil and other matrices. The combination of SPME and GC methods allows for low detection limits depending on the analyte, matrix and detection system. Commercially available fibers have been mainly used to extract CWAs in headspace analysis. However, attempts have been made to introduce new fiber coatings that are characterized by higher selectivities towards different analytes of interest. Environmental decomposition of CWAs leads to the formation of more hydrophilic products. These compounds may be isolated from samples using SPME and analyzed using GC however, they must often be derivatized first to produce good chromatography. In these cases, one must ensure that the SPME method also meets the same needs. Otherwise, it is helpful to use derivatization methods. SPME may also be used with fieldportable mass spectrometry (MS) and GC-MS instruments for chemical defense applications, including field sampling and analysis. SPME fibers can be taken into contaminated areas to directly sample air, headspaces above solutions, soils and water.     

质谱快速分析猪肉中痕量沙丁胺醇及克伦特罗

采用内部萃取电喷雾电离质谱( iEESI-MS)技术,在无需样品预处理的前提下,采用标准加入法直接对猪肉组织中沙丁胺醇与克伦特罗进行定性和定量分析。结果表明,本实验对猪肉组织中沙丁胺醇与克伦特罗具有较高的灵敏度,单个样品单一指标的检测时间少于30 s。在0.01~1000μg/kg浓度范围内,信号强度对数(Y)与浓度对数(X)具有较好的线性关系,定量限分别为6.2和9.8 ng/kg。本方法分析速度快、样本耗量少、灵敏度高,适用于猪肉中痕量沙丁胺醇与克伦特罗等“瘦肉精”的快速检测。     

Rapid test by liquid chromatography/tandem mass spectrometry to evaluate equine urine reactivity towards 17beta-OH steroids

Bacteria frequently found in equine urine samples may cause degradation of 17beta-OH steroids. A simple liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed to evaluate the microbiological contamination of equine urine as a marker of poor storage conditions. Norethandrolone was used as the internal standard, and the linearity, sensitivity, precision and accuracy of the method were evaluated. 17beta-OH oxidation was demonstrated for testosterone, nandrolone, trenbolone and boldenone, but did not occur in alpha-epimers such as alpha-boldenone and epitestosterone, demonstrating the stereoselectivity of the reaction. A rapid test was performed by spiking one of the four 17beta-OH steroids in samples of diluted equine urine. The steroids were transformed into their respective ketones in the presence of bacterial activity. The test allows direct injection of diluted samples into the LC/MS system, without the need for prior extraction. Results show that the best method of storage is freezing at -18 degrees C. Urine specimens should be analyzed as soon as possible after thawing. This allows bacterial degradation of equine urine to be arrested temporarily, so that the urine can be used for qualitative or quantitative analysis of 17beta-OH steroids.     

Determination of ephedra alkaloids in urine and plasma by HPLC-UV: collaborative study

Ten collaborating laboratories determined the ephedra alkaloid content (ephedrine, pseudoephedrine, norephedrine, norpseudoephedrine, methylephedrine, and methylpseudoephedrine) in 8 blind duplicates of human plasma and urine using high-performance liquid chromatography (HPLC) with UV detection. In addition to negative urine and plasma controls, urine samples were spiked with individual ephedra alkaloids ranging in concentration from about 1 to 5 microg/mL. Plasma samples were spiked with individual ephedra alkaloids ranging in concentration from about 100 to 400 ng/mL. Sample solutions were treated to solid-phase extraction using a strong-cation exchange column to help remove interferences. The HPLC analyses were performed on a polar-embedded phenyl column using UV detection at 210 nm. The ephedra alkaloids were not consistently detected in any of the spiked plasma samples. When ephedra alkaloids were detected in the plasma samples, reproducibility between blind replicate samples was very poor. Repeatability, reproducibility, and accuracy were also very poor for the spiked urine samples. On the basis of these results, the HPLC-UV method for the determination of ephedra alkaloids in human urine and plasma is not recommended for adoption as Official First Action.     

Identification and determination of phase II nabumetone metabolites by high-performance liquid chromatography with photodiode array and mass spectrometric detection

Chromatographic analyses play an important role in the identification and determination of phase I and phase II drug metabolites. While the chemical standards of phase I metabolites are usually available from commercial sources or by various synthetic, degradation or isolation methods, the phase II drug metabolites have usually more complicated structures, their standards are in general inaccessible and their identification and determination require a comprehensive analytical approach involving the use of xenobiochemical methods and the employment of hyphenated analytical techniques. In this work, various high-performance liquid chromatography (HPLC) methods were employed in the evaluation of xenobiochemical experiments leading to the identification and determination of phase II nabumetone metabolites. Optimal conditions for the quantitative enzymatic deconjugation of phase II metabolites were found for the samples of minipig bile, small intestine contents and urine. Comparative HPLC analyses of the samples of above-mentioned biomatrices and of the same biomatrices after their enzymatic treatment using beta-glucuronidase and arylsulfatase afforded the qualitative and quantitative information about phase II nabumetone metabolites. Hereby, three principal phase II nabumetone metabolites (ether glucuronides) were discovered in minipig's body fluids and their structures were confirmed using liquid chromatography (LC)-electrospray ionization mass spectrometric (MS) analyses.     

Determination of aristolochic acid in urine using hollow fiber liquid-phase microextraction combined with high-performance liquid chromatography

A simple and efficient hollow fiber liquid‐phase microextraction (HF‐LPME) technique in conjunction with high‐performance liquid chromatography is presented for extraction and quantitative determination of aristolochic acid I in human urine samples. Several parameters influencing the efficiency of HF‐LPME were investigated and optimized, including extraction solvent, stirring rate, extraction time, pH of donor phase and acceptor phase. Excellent sample clean‐up was observed and good linearity with coefficient of 0.9999 was obtained in the range of 15.4–960 µg/L. This method provided a 230‐fold enrichment factor and good repeatability with relative standard deviations (RSD) lower than 6.0%. The limit of detection value for the analyte in urine sample was 0.01 µg/L at a signal‐to‐noise ratio of 3. The extraction recovery from urine samples was 61.8% with an RSD of 9.71%. Copyright © 2010 John Wiley & Sons, Ltd.