Fast analysis of drugs in a single hair

A new method for the fast screening of cocaine and 6-monoacetylmorphine (6-MAM) in a single hair, using gas chromatography/mass spectrometry (GC/MS), is described. The analyses are conducted in less than 10 min with minimal sample preparation. The novel method combines the ChromatoProbe direct sample introduction device for intrainjector thermal extraction, fast GC separation, a supersonic molecular beam GC/MS interface and hyperthermal surface ionization (HSI). The technique has been successfully employed for the detection of cocaine in as little as a 1-mm section of hair using selected ion monitoring (SIM). Unambiguous full scan mass spectra of cocaine and 6-MAM were obtained on a single hair for cocaine and heroin users, respectively. HSI was found to be almost 3 orders of magnitude more selective than electron impact ionization for cocaine compared with the major hair constituents, with a minimum detected concentration of approximately 10 ppb in the SIM mode. Results obtained for 12 drug users showed full qualitative agreement with similar results using rigorous solvent extraction followed by electrospray-liquid chromatography/mass spectrometry analysis. However, quantitative studies showed only partial agreement. No false positives were observed for 10 drug free subjects. This method enables fast drug monitoring along the hair length which permits time correlation studies.     

表面解吸常压化学电离质谱法直接测定火锅底料中的痕量可卡因

采用表面解吸常压化学电离(SDAPCI)质谱法, 在无需样品预处理情况下, 直接测定了火锅底料中可卡因的含量. 采用取样针取样, 单次取样量在纳升级, 单个样品测定时间少于0.5 min, 回收率为92.9%～106.6%; 相对标准偏差(RSD)为4.7%～11.6%; 可卡因的检出限可达1.5×10-12 g/mL. 该方法适用于批量火锅底料等食品类样品的快速半定量检测.     

Analysis of N-benzoyl-L-tyrosyl-p-aminobenzoic acid (bentiromide) metabolites in urine by ion-pair high-performance liquid chromatography

A high-performance liquid chromatographic method is described for the analysis of bentiromide metabolites in urine. The procedure involves no more than direct injection of the diluted urine sample, obviating the need for an extraction step or an internal standard. A mu Bondapak C18 column is used with a mobile phase of 0.01 M tetrabutylammonium chloride (pH 7.4)--methanol (9:1). A flow-rate of 1.4 ml/min, detection at 254 nm and column temperature of 40 degrees C are employed. These conditions were achieved by investigating the effects of mobile phase pH, and concentrations and types of organic modifiers, buffers and ion-pairing agents on the resolution of the metabolites. The analysis time is 18 min per sample and the coefficient of variation on replicate assays is less than 10% for most concentrations studied. Analytical recoveries were between 95 and 100% throughout the appropriate concentration ranges and no interferences were obtained with the exception of p-acetamidobenzoyl glucuronide which could be eliminated by treatment of the samples with beta-glucuronidase. Concentration profiles of the metabolites were studied in normal subjects, and the method was found to be potentially useful for clinical situations in which the existing bentiromide test leads to ambiguous results because of small bowel and hepatic dysfunctions.     

Solid-phase extraction of soy isoflavones

An automated method using solid-phase extraction (SPE) for the concentration and clean-up of soy isoflavone extracts is proposed in this work. Using a standardized sample (0.1 g of a freeze dried soybean extract/25 mL of water); eight SPE cartridges with a wide range of sorbents (C18, divinylbenzene and modified divinylbenzene) from different suppliers were evaluated and compared. A large variation on SPE cartridges performance was observed, especially regarding retention and breakthrough volume of isoflavones during sample load and washing steps. The most effective cartridges were the divinylbenzene based cartridges, especially Strata X (from Phenomenex) and HLB oasis (from Waters). Using Strata X cartridges, several extraction parameters, such as sample loading flow (5-15 mL min(-1)), extracting solvent volume (2-6 mL of methanol), pH of the extracting solvent and the necessity of drying the sorbent before elution, were evaluated to provide a fast, specific, quantitative and reproducible SPE method. The optimized method consists of conditioning the cartridge with 10 mL of methanol and 10 mL of water (10 mL min(-1)), loading 25 mL of the standardized extract onto the cartridges (5 mL min(-1)), washing the cartridge with 10 mL of water (10 mL min(-1)) and finally eluting with 4 mL of methanol (10 mL min(-1)). Mean isoflavones recovery was 99.37% and mean intra- and inter-day reproducibility was higher than 98%. The developed sample clean-up/concentration (6.25:1) method takes less than 10 min and can be used in the analysis of isoflavones from soy extracts.     

超高效液相色谱法同时测定减肥类保健食品中非法添加的25种药物

建立了采用超高效液相色谱同时测定减肥类保健食品中25种非法添加化学药物含量的方法。保健食品样品以甲醇为提取溶剂进行超声提取,离心后取上清液以Waters HSS T3色谱柱(100 mm×2.1 mm,1.8 μm)进行分离,乙腈和10 mmol/L乙酸铵水溶液(含0.1%甲酸)为流动相梯度洗脱,流速为0.3 mL/min,在200~400 nm波长范围内进行定性和定量分析。在相应的浓度范围内,25种化学药物的质量浓度与峰面积呈良好的线性关系,R²≥0.997;定量限在0.500~5.00 ng之间;在低、中、高3个添加水平范围内的平均回收率为70.7%~104%,相对标准偏差(RSD)在0.132%~5.03%之间。样品筛查结果发现,17种减肥保健食品中3种样品非法添加了酚酞,1种样品添加了大黄素。该方法选择性强、分析速度快、高通量,可用于该类保健食品中非法添加化学药品的定性筛查和定量检测。     

Development of water-phase derivatization followed by solid-phase microextraction and gas chromatography/mass spectrometry for fast determination of valproic acid in human plasma

In this study, a simple, rapid, and sensitive method was developed and validated for the quantification of valproic acid (VPA), an antiepileptic drug, in human plasma, which was based on water-phase derivatization followed by headspace solid-phase microextraction (HS-SPME) and gas chromatography/mass spectrometry (GC/MS). In the proposed method, VPA in plasma was rapidly derivatized with a mixture of isobutyl chloroformate, ethanol and pyridine under mild conditions (room temperature, aqueous medium), and the VPA ethyl ester formed was headspace-extracted and simultaneously concentrated using the SPME technique. Finally, the analyte extracted on SPME fiber was analyzed by GC/MS. The experimental parameters and method validations were studied. The optimal conditions were obtained: PDMS fiber, stirring rate of 1100 rpm, sample temperature of 80 degrees C, extraction time of 20 min, NaCl concentration of 30%. The proposed method had a limit of quantification (0.3 microg/mL), good recovery (89-97%) and precision (RSD value less than 10%). Because the proposed method combined a rapid water-phase derivatization with a fast, simple and solvent-free sample extraction and concentration technique of SPME, the sample preparation time was less than 25 min. This much shortens the whole analysis time of VPA in plasma. The validated method has been successfully used to analyze VPA in human plasma samples for application in pharmacokinetic studies. All these results show that water-phase derivatization followed by HS-SPME and GC/MS is an alternative and powerful method for fast determination of VPA in biological fluids.     

Determination of five priority haloacetic acids by capillary electrophoresis with contactless conductivity detection and solid phase extraction preconcentration

A sensitive capillary electrophoretic separation method with contactless conductivity detection (C4D) for analysis of five priority haloacetic acids (HAA5) is presented. The analytes were baseline separated in an electrolyte composed of 20 mM 2-(N-Morpholino) ethanesulfonic acid (MES), 20 mM L-histidine (HIS), and 30 μM cetyltrimethylammonium bromide (CTAB) at pH 6.0 in less than 4 min. A simplified solid-phase extraction (SPE) preconcentration procedure on highly cross-linked polystyrene-divinylbenzene (PS-DVB) type sorbent was developed and optimized with respect to short preconcentration time. HAA5 from a 25-mL sample aliquot of tap and swimming pool water could be preconcentrated in less than 5 min using an in-house made SPE column with recoveries ranging from 23 to 98%. Combining the SPE preconcentration procedure with capillary electrophoretic analysis, the attained limits of detection were between 6.1 and 12.2 μg/L with total analysis time of less than 10 min.     

Unknown residual solvents identification in drug products by headspace solid phase microextraction gas chromatography-mass spectrometry

Summary A sensitive headspace SPME method for the extraction of residual solvents from pharmaceutical products has been developed and optimized. It was found that minimizing sample and headspace volume has a beneficial effect on extraction efficiency. At the same time the method reproducibility was seriously affected by reducing sample and headspace volume. The added air volume was not found to have any significant influence on method sensitivity. The method showed reproducibilities of less than 10% and detection limits as low as 1 ppb for benzene and dichloromethane. The headspace SPME method is around 1000 times more sensitive than static headspace. The optimized parameters were headspace volume 1.5 mL, sample volume 10 μL, and extraction time 30 min. The method was successfully applied to the identification of unknown residual solvents in three different proprietary active drug substances and was successfully applied to the confirmation of the presence of benzene in a proprietary drug substance. Presented at Balaton Symposium '01 on High-Performance Separation Methods, Siófok, Hungary, September 2–4, 2001     

多壁碳纳米管净化/超高效液相色谱串联质谱同时测定动物组织中四环素与喹诺酮多残留

采用超高效液相色谱-串联质谱(UPLC-MS/MS)在正离子模式下通过多反应监测(MRM)方式同时测定了猪肉和鸡肉中3种四环素和2种喹诺酮类药物的残留量.残留药物经Mcllvaine-Na2EDTA缓冲溶液(pH4.0)提取后,采用多壁碳纳米管(WMCNTs)固相萃取柱净化,甲酸-乙腈(体积比5:95)洗脱,UPLC-...     

Direct injection HPLC method for the determination of selected phenothiazines in plasma using a Hisep column

A direct plasma injection HPLC method has been developed for the determination of selected phenothiazines (promethazine, promazine, chlorpromazine) using a Hisep column. The method is easy to perform and requires 20 microL of a filtered plasma sample. The chromatographic run time is less than 11 min using a mobile phase of 15:85 v/v acetonitrile-0.18 m ammonium acetate pH 5.0 and UV detection at 254 nm. The method is linear in the concentration range 0.1-25 microg mL(-1) (r > 0.99, n = 6) for each analyte with RSD less than 6%. Interday and intraday variability were found to be < or =14%. The limits of detection and quantitation were 0.1 (S/N > 3) and 0.25 microg mL(-1) (S/N > 10), respectively, for each of the three phenothiazines. We can also apply this method to separate three other phenothiazines (ethopromazine, trifluoroperazine, prochlorperazine), although it lacks the selectivity to determine the concentration of all six drugs concurrently. The separation is feasible using these drugs in certain combinations.     

Evaluation and single laboratory validation of an on-line turbulent flow extraction tandem mass spectrometry method for melamine in infant formula

This report presents the single-laboratory validation of a method for the determination of melamine in dairy-based products using on-line turbulent flow extraction-tandem mass spectrometry. Liquid or powder test portions were dissolved in water, enriched with (13)C(3)(15)N(3)-Melamine internal standard, followed by protein precipitation and withdrawal of an aliquot for analysis. The turbulent flow method was validated by analyses of liquid and powdered proficiency test portions containing up to 10mg/kg melamine. Accuracy of results ranged from 96 to 106% of the assigned values for the 6 proficiency test portions tested with relative standard deviations of 4-8%. Apparent recoveries based on addition of amino-(15)N(3)-Melamine to prepared test portions were between 98 and 114%. Based on the repeat analysis of a known blank sample the limit of detection and limit of quantification were determined to be 27 and 87 μg/kg, respectively. Additionally, this report demonstrates that turbulent flow chromatography is significantly faster than traditional LC-MS, with sample analysis times of less than 2 min.     

超高效液相色谱-串联质谱法同时测定鸡肝中残留的四环素类、磺胺类和喹诺酮类药物

采用超高效液相色谱-串联质谱(UPLC-MS/MS)在正离子模式下通过多反应监测(MRM)方式同时测定了鸡肝脏组织中3种四环素类药物、10种磺胺类药物以及8种喹诺酮类药物的残留。试样由McIlvaine缓冲液-乙腈(体积比为1:4)、乙腈提取,合并上清液并用氮气吹干,用0.05 mol/L磷酸三乙胺缓冲液-乙腈(体积比为85:15)溶解残余物,经正己烷脱脂后,采用UPLC-MS/MS进行定性、定量分析。该方法对测定的21种药物的检出限均为2 μg/kg,定量限均为5 μg/kg。在添加水平分别为5,10和50 μg/kg时,21种药物的加标回收率为66.8%～128.5%,日内测定的相对标准偏差(RSD)为0.8%～20.2%,日间测定的RSD为2.2%～15.3%。该方法可作为动物源性食品中这3类药物残留检测的确证方法。     

Simple-QuEChERS Nano结合GC-MS/MS同时检测全血中的97种农药

建立了Simple-QuEChERS Nano结合气相色谱一串联质谱(GC-MS/MS)同时检测血液中97种农药的方法,并对基质条件、提取溶剂以及净化材料进行了优化.0.5 mL血液样品经3倍水稀释混匀,使用2.0 mL乙酸乙酯提取后振荡、离心,过Simple-QuEChERS Nano净化柱及0.22μm有机微孔滤膜...     

Assay of urinary alpha-fluoro-beta-alanine by gas chromatography-mass spectrometry for the biological monitoring of occupational exposure to 5-fluorouracil in oncology nurses and pharmacy technicians

The validation of an analytical method for the measurement of the unnatural amino acid alpha-fluoro-beta-alanine (AFBA), the main metabolite of the antineoplastic drug 5-fluorouracil (5FU), in urine for the biological monitoring of the exposure of hospital workers to the drug when preparing the therapeutical doses and administering to cancer patients is described. The method employed a two-step extractive derivatization of the analyte from urine to the N-trifluoroacety-n-butyl ester derivative and detection by selected-ion monitoring gas chromatography-mass spectrometry of structurally specific fragments. The limit of detection was 20 ng/mL with quantification accuracy better than +/-20% and precision (CV%) better than +/-20% in the range 0.020-10 microg/mL. Norleucine was used as the internal standard and the sample-to-sample analysis time was less than 15 min. The validated method has been applied to the biological monitoring of some hospital workers potentially exposed to 5FU and to matched control subjects. On a total number of 65 analyzed urine samples from control and exposed subjects, only three, obtained from exposed subjects, were found to be positive, with values of 20, 30 and 1150 ng/mL, respectively.     

Fluoxetine and norfluoxetine analysis by direct injection of human plasma in a column switching liquid chromatographic system

A column switching LC method is presented for the analysis of fluoxetine (FLU) and norfluoxetine (NFLU) by direct injection of human plasma using a lab-made restricted access media (RAM) column. A RAM-BSA-octadecyl silica (C-18) column (40 mm x 4.6 mm, 10 microm) is evaluated in both backflush and foreflush elution modes and coupled with a C-18 lab-made (50 mm x 4.6 mm, 3 microm) analytical column in order to perform online sample preparation. Direct injection of 100 microL of plasma samples is possible with the developed approach. In addition, reduction of sample handling is obtained when compared with traditional liquid-liquid extraction (LLE) and SPE. The total analysis time is around 20 min. A LOQ of 15 ng/mL is achieved in a concentration range of 15-500 ng/mL, allowing the therapeutic drug monitoring of clinical samples. The precision values achieved are lower than 15% for all the evaluated points with adequate recovery and accuracy. Furthermore, no matrix interferences are found in the analysis and the proposed method shows to be an adequate alternative for analysis of FLU in plasma.     

A simple and reliable stir bar sorptive extraction-liquid chromatography procedure for the determination of chlorpromazine and trifluoperazine in human serum using experimental design methodology

A sensitive and reproducible stir bar sorptive extraction and HPLC-UV detection method was used for the therapeutic drug monitoring of chlorpromazine and trifluoperazine in human serum. The separation was achieved using a C(18) column. The mobile phase consisted of methanol/sodium acetate buffer (pH 4.1; 0.1 M) (95:5, v/v) including 0.5% triethylamine. This miniaturized method can result in faster analysis, lower solvent consumption and less workload per sample while maintaining or even improving sensitivity. In the second part, stir bar sorptive extraction/HPLC-UV method was optimized by a chemometrics approach. An experimental design was therefore used to evaluate the statistically influential and/or interacting factors, among those described in the literature, and to find the best extraction and desorption conditions. Optimal sample volume of 1 mL, extraction time of 24 min at 31°C with pH 8.1 were obtained in a screening 2(5) half fractional factorial design followed by a Box-Behnken design. For the desorption conditions, a Box-Behnken design showed that the best conditions were 150 μL mobile phase for 20 min at 50°C. The optimized method was repeatable (CV<10%, linear (LOQ-500 ng/mL)), with the LOQs equal to 0.7 and 1.5 ng/mL for chlorpromazine and trifluoperazine, respectively.     

Determination of 8-hydroxy-2'-deoxyguanosine in untreated urine by capillary electrophoresis with UV detection

A capillary electrophoresis method with UV detection was developed for the determination of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in untreated urine samples. The calibration graph for 8-OHdG in urine is linear in the concentration range 10-500 mg/l. and the detection limit is 5 mg/l (17 microM). 8-OHdG was determined in urine from oncological patients treated by radiation therapy. Its concentrations relative to creatinine were found to be in the range 10-47 microg 8-OHdG/l mg creatinine (4-19 micromol 8-OHdG/mmol creatinine). The overall time of the analysis of a urine sample was less than 15 min.     

Capillary electrophoresis method for the determination of isradipine enantiomers: stability studies and pharmaceutical formulation analysis

A simple enantioselective method based on CE using CD as chiral selector was developed and validated for the determination of isradipine (IRD) enantiomers in a pharmaceutical formulation and for the determination of IRD enantiomers in degradation studies. After optimization, the best results were obtained using 15 mM borate buffer at pH 9.3 and sulfobutyl ether-β-cyclodextrin (2.5%, w/v) as chiral selector. The applied voltage was +30 kV, and the sample injection was performed in the hydrodynamic mode. All analyses were carried out in a fused-silica uncoated capillary with an id of 50 μm and total length of 60.0 cm. Under these conditions, a complete separation between IRD enantiomers was achieved in less than 7 min. Linearity was obtained in the range 50-300 μg/mL for both enantiomers (r≥0.9978). The RSD (%) and relative errors (%) obtained in precision and accuracy studies (intra-day and inter-day) were lower than 5%. Therefore, this method was found to be appropriate for controlling pharmaceutical formulations containing IRD enantiomers and the assay was considered to be stability indicating. The drug was subjected to oxidation, hydrolysis and photolysis. In all stress conditions the drug presented considerable degradation when compared with a fresh sample (zero time).