生物检材中佐匹克隆的GC/μECD测定

目的：建立液-液萃取、GC/μECD气相色谱测定生物检材中佐匹克隆的方法。方法：以家兔为模型动物,500 mg佐匹克隆标准品灌胃家兔,6.5小时后处死,取其胃、肝、血、脑、心脏、尿,采用乙醚液-液萃取提取,Rtx-5毛细管柱分离,电子捕获检测器检测,标准曲线法定量。结果：该方法灵敏度达0.02μg/mL,在0.05-50μg/mL范围内线性关系良好;低、中、高3种浓度的加样回收率分别为91.7%、99.5%、94.4%,相对标准偏差（RSD）均小于5%;佐匹克隆在家兔脏器中的浓度分别为7.31μg/mL（血液）、7.63μg/g（肝脏）、7.96μg/g（脑）、14.67μg/g（心脏）、32.18μg/g（胃组织）、42.65μg/mL（尿液）,在各脏器中呈特异性分布。结论：该法操作简单、灵敏度高,结果可靠,完全满足临床检测及公安机关在办理佐匹克隆麻醉案件中的需要。     

Capillary electrophoretic determination of DNA damage markers: content of 8-hydroxy-2'-deoxyguanosine and 8-nitroguanine in urine

A sensitive and low-cost analytical method has been developed to determine 8-hydroxy-2'-deoxyguanosine (8-OHdG) and 8-nitroguanine (8-NO(2)Gua) based on capillary electrophoresis with amperometric detection (CE-AD) after solid phase extraction (SPE). Under optimized condition, these two markers were well separated from other components coexisting in urine, exhibiting a linear calibration over the concentration range of 0.1-50.0 μg/mL with the detection limits ranging from 0.02 to 0.06 μg/mL. The relative standard deviations (RSDs) were in the range of 0.1-2.1% for peak area, 0.1-1.5% for migration time, respectively. The average recovery and RSD were within the range of 100.0-108.0% and 0.1-1.7%, respectively. It was found that the urinary contents of 8-OHdG and 8-NO(2)Gua in cancer patients were significantly higher than those in healthy ones.     

Determination of ciprofloxacin in human plasma using high-performance liquid chromatography coupled with fluorescence detection: application to a population pharmacokinetics study in children with severe malnutrition

Clinical pharmacokinetic studies of ciprofloxacin require accurate and precise measurement of plasma drug concentrations. We describe a rapid, selective and sensitive HPLC method coupled with fluorescence detection for determination of ciprofloxacin in human plasma. Internal standard (IS; sarafloxacin) was added to plasma aliquots (200 μL) prior to protein precipitation with acetonitrile. Ciprofloxacin and IS were eluted on a Synergi Max-RP analytical column (150 mm×4.6 mm i.d., 5 μm particle size) maintained at 40°C. The mobile phase comprised a mixture of aqueous orthophosphoric acid (0.025 M)/methanol/acetonitrile (75/13/12%, v/v/v); the pH was adjusted to 3.0 with triethylamine. A fluorescence detector (excitation/emission wavelength of 278/450 nm) was used. Retention times for ciprofloxacin and IS were approximately 3.6 and 7.0 min, respectively. Calibration curves of ciprofloxacin were linear over the concentration range of 0.02-4 μg/mL, with correlation coefficients (r(2))≥0.998. Intra- and inter-assay relative standard deviations (SD) were <8.0% and accuracy values ranged from 93% to 105% for quality control samples (0.2, 1.8 and 3.6 μg/mL). The mean (SD) extraction recoveries for ciprofloxacin from spiked plasma at 0.08, 1.8 and 3.6 μg/mL were 72.8±12.5% (n=5), 83.5±5.2% and 77.7±2.0%, respectively (n=8 in both cases). The recovery for IS was 94.5±7.9% (n=15). The limits of detection and quantification were 10 ng/mL and 20 ng/mL, respectively. Ciprofloxacin was stable in plasma for at least one month when stored at -15°C to -25°C and -70°C to -90°C. This method was successfully applied to measure plasma ciprofloxacin concentrations in a population pharmacokinetics study of ciprofloxacin in malnourished children.     

Optimization of a matrix solid-phase dispersion method for the determination analysis of carbendazim residue in plant material

The objective of this paper was to prove that matrix solid-phase dispersion (MSPD) coupled with high performance liquid chromatography (HPLC) and column switching could be used for the determination and quantification of carbendazim residue in plant samples. By comparing results obtained after optimization of the extraction conditions on an acidic silica gel column, it was determined that sorption and retention of carbendazim were achieved via specific interactions. The method of standard additions was used for quantitative analysis. Its performance was evaluated and validated: the detection limit (UV-Vis detection at lambda=279 nm) was 0.02 microg/g, the relative standard deviations (R.S.D.) were between 2.7 and 4.1% and the recoveries were ranging from 84.3 to 90.7% at the 0.04, 0.08 and 0.1 microg/g fortification levels. The method was successfully tested on cereal samples, and the results obtained with the present off-line MSPD-HPLC procedure were found to compare well with those obtained with procedure involving LLE.     

Determination of pseudolycorine in the bulb of lycoris radiata by capillary electrophoresis combined with online electrochemiluminescence using ultrasonic-assisted extraction

A novel method for determination of pseudolycorine in the bulb of lycoris radiata by capillary electrophoresis coupled with online electrochemiluminescence detection with ultrasonic-assisted extraction has been developed. The effects of several factors such as detection potential, concentration and pH of phosphate buffer, separation voltage, injection time, ultrasonic power and extraction time were investigated. Under optimal conditions, the linear concentration range for pseudolycorine was 0.002-2 μg/mL with a correlation coefficient of 0.9991. Relative standard deviations of migration time and peak areas were 1.4 and 3.2%, respectively. The limit of detection (S/N=3) was 0.46 ng/mL. The proposed method can be successfully applied to the determination of pseudolycorine in the bulb of lycoris radiata.     

Simultaneous determination of phenol,cresol, xylenol isomers and napthols in urine by capillary gas chromatography

An attempt was made to establish a method for the simultaneous determination of urinary concentrations of phenol, o-, p- and m-cresols, 1 and 2-naphthol and xylenol isomers by capillary gas chromatography. Urine samples were extracted after acid hydrolysis of glucuronides and sulfates by solid-phase extraction. The ten substances were separated gas chromatographically using a capillary column (Ultra 2) of cross-linked 5% phenylmethyl silicone. Calibration graphs were linear for 5–100 μg/ml of all the phenols determined. The corresponding detection limits for phenolic compounds varied from 0.1 to 0.2 μg/ml. The relative standard deviations for samples in urine were in the range 2.6–16.6% and the accuracy was in the range 1.4–25%. Recoveries were generally over 80%.     

Application of matrix solid-phase dispersion methodology to the extraction of endogenous peptides from porcine hypothalamus samples for MS and LC-MS analysis

In this study, we investigated a novel application of matrix solid-phase dispersion (MSPD) methodology for the extraction of endogenous peptides from porcine hypothalamus tissue samples. Several experimental factors of the MSPD procedure were examined. Finally, silica-based octadecyl was chosen as dispersing material and blended with 0.25 g porcine hypothalamus at a ratio of 5, and 10 mL of 60% acetonitrile with 0.2% formic acid in water was chosen as the extraction and elution solvent. This MSPD extraction method was compared to the classic acid extraction method. More peaks were observed in the MSPD extracts (74±5) by MALDI-TOF MS than in acid extracts (34±5). Moreover, 14 potential endogenous peptides were identified in the MSPD extracts after nanoLC-MS/MS analysis, while only 2 endogenous peptides in the acid extracts. These results indicated that MSPD could be employed as a simple and efficient method for the extraction of endogenous peptides from tissues.     

Determination of the active constituents in Arnebia euchroma (Royle) Johnst. by ionic liquid-based ultrasonic-assisted extraction high-performance liquid chromatography

Shikonin and β,β'-dimethylacrylshikonin in Arnebia euchroma (Royle) Johnst. were extracted by ionic liquid-based ultrasonic-assisted extraction (IL-based UAE) and determined by high-performance liquid chromatography (HPLC). The dried powder of A. euchroma (Royle) Johnst. was mixed with a room temperature ionic liquid [C(6)MIM][BF(4)] to form a suspension, and then the ultrasonic extraction was performed in a water bath at ambient temperature. The calibration curve showed good linear relationship (r>0.9998) in the concentration range of 1.75-140 μg/mL for shikonin and 2.15-1360 μg/mL for β,β'-dimethylacrylshikonin. The recoveries were between 69.79% and 82.35%. The IL-based UAE is free of volatile organic solvents, and consumes less sample, time and solvent, compared with regular ultrasonic and Soxhlet extraction. There was no obvious difference in the extraction yields of active constitutions obtained by the three extraction methods.     

Extraction and determination of opium alkaloids in urine samples using dispersive liquid-liquid microextraction followed by high-performance liquid chromatography

A simple, rapid and sensitive method based on dispersive liquid-liquid microextraction (DLLME) combined with high-performance liquid chromatography-ultraviolet detection (HPLC-UV) was used to determine opium alkaloids in urine samples. Some effective parameters on extraction were studied and optimized. Under the optimum conditions, enrichment factors and recoveries for different opiates are in the range of 63.0-104.5 and 31.5-52.2%, respectively. The calibration graphs are linear in the range of 0.50-500 μg L(-1) and limit of detections (LODs) are in the range of 0.2-10 μg L(-1). The relative standard deviations (RSDs) for 200 μg L(-1) of morphine, codeine and thebaine, 5.0 μg L(-1) of papaverine and 10.0 μg L(-1) of noscapine in diluted urine sample are in the range of 2.8-6.1% (n=7). The relative recoveries of urine samples spiked with alkaloids are 84.3-106.0%. The obtained results show that DLLME combined with HPLC-UV is a fast and simple method for the determination of opium alkaloids in urine samples.     

Determination of isometamidium residues in animal-derived foods by liquid chromatography-tandem mass spectrometry

In this paper, a new determination method for isometamidium residues in animal-derived foods was developed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Isometamidium residues in bovine tissues and milk were extracted with the mixed solution of acetonitrile and 0.25 mol/L of ammonium formate-methanol (v/v, 1:1), concentrated and degreased, and determined by LC-MS/MS with quantification by external standard method. The results showed that the peak area of chromatogram was linearly related to the concentration of isometamidium in the range of 1-100 μg/L, and the limits of detection (LOD) and quantification (LOQ) were 0.05 μg/kg and 5 μg/kg, respectively. The average recoveries of spiked samples were in the range of 73.8-93.9% with relative standard deviations ranged from 2.3% to 7.5%. This method is simple, accurate and suitable for the identification and quantification for isometamidium in animal-derived foods.     

Simultaneous determination of thiamphenicol, florfenicol and florfenicol amine in eggs by reversed-phase high-performance liquid chromatography with fluorescence detection

A specific, sensitive and widely applicable reversed-phase high-performance liquid chromatography with fluorescence detection (RP-HPLC-FLD) method was developed for the simultaneous determination of thiamphenicol (TAP), florfenicol (FF) and florfenicol amine (FFA) in eggs. Samples were extracted with ethyl acetate-acetonitrile-ammonium hydroxide (49:49:2, v/v), defatted with hexane, followed by RP-HPLC-FLD determination. Liquid chromatography was performed on a 5 μm LiChrospher C(18) column using a mobile phase composed of acetonitrile (A), 0.01 M sodium dihydrogen phosphate containing 0.005 M sodium dodecyl sulfate and 0.1% triethylamine, adjusted to pH 4.8 by 85% phosphoric acid (B) (A:B, 35:65 v/v), at a flow rate of 1.0 mL/min. The fluorescence detector of HPLC was set at 224 nm for excitation wavelength and 290 nm for emission wavelength. Limits of detection (LODs) were 1.5 μg/kg for TAP and FF, 0.5 μg/kg for FFA in eggs; limits of quantitation (LOQs) were 5 μg/kg for TAP and FF, 2 μg/kg for FFA in eggs. Linear calibration curves were obtained over concentration ranges of 0.025-5.0 μg/mL for TAP with determination coefficients of 0.9997, 0.01-10.0 μg/mL for FF with determination coefficients of 0.9997 and 0.0025-2.50 μg/mL for FFA with determination coefficients of 0.9998, respectively. The recovery values ranged from 86.4% to 93.8% for TAP, 87.4% to 92.3% for FF and from 89.0% to 95.2% for FFA. The corresponding intra-day and inter-day variation (relative standard deviation, R.S.D.) found to be less than 6.7% and 10.8%, respectively.     

High-performance liquid chromatographic determination of (−)-β-d-2,6-diaminopurine dioxolane and its metabolite,dioxolane guanosine,using ultraviolet and on-line radiochemical detection

(−)-β-d-2,6-Diaminopurine dioxolane (DAPD) and its metabolite dioxolane guanosine (DXG) have potent activity against hepatitis B virus and HIV, in vitro. A reversed-phase HPLC analytical method using UV and on-line radiochemical detection for the determination of DAPD and DXG in monkey serum and urine is described in this report. Retention times for DXG, DAPD and internal standard (2′,3′-didehydro-2′ deoxythymidine, D4T) were 5.0, 6.0 and 13.0 min, respectively. The extraction recovery was greater than 97% for DAPD and 94% for DXG. The limit of quantitation for UV detection was 100 ng/ml and 125 ng/ml for DXG and DAPD in monkey serum. The standard curves were linear from 0.1 μg/ml to 5 μg/ml for DXG and 0.125 μg/ml to 5 μg/ml for DAPD. For radiochemical detection, calibration curves of standard solutions of DAPD and DXG were linear in the range of 3500 Bq to 32 000 Bq and 7500 Bq to 60 000 Bq. The intra- and inter-day relative standard deviations were less than 7.2% using UV and less than 8.6% using on-line radiochemical detection. The HPLC method was applied to serum and urine samples collected from a male rhesus monkey that was administered 33.3 mg/kg DAPD with 200 μgCi of [³H]DAPD intravenously.     

Determination of muscarine in human urine by electrospray liquid chromatographic-mass spectrometric

A liquid chromatography-mass spectrometry based method for determination of muscarine in human urine was developed and validated. The method involved a solid phase extraction of muscarine from urine using Strata X-CW column. Separation of muscarine was achieved within 16.0 min on a reversed phase Gemini C18 analytical column (150 mm × 2.0mm i.d., 5 μm) with a mobile phase consisted of aqueous 8 mmol/L heptafluorobutyric acid and acetonitrile in a gradient mode. Mass spectrometric detection was performed at m/z 174 and m/z 216 for muscarine and acetylmuscarine (internal standard), respectively. The linearity was satisfactory with a coefficient of determination (R(2)) 0.9993 at concentration range from 0.3 ng/mL to 2.0 μg/mL, LOD and LOQ for muscarine was 0.09 ng/mL and 0.3 ng/mL, respectively. The found out recoveries of muscarine were 96% or 95% for concentration 0.3 ng/mL and 0.2 μg/mL or 2.0 μg/mL, respectively. The precision in the intra-assay-study varied from 0.48% to 1.39% and in the inter-assay-study from 2.39% to 5.49%. The accuracy ranged from -3.3% to -6%. The validation results demonstrated that the method fulfilled satisfactory requirements for precision and accuracy across the calibration curve. The applicability of the method has been demonstrated by analyzing clinical urine samples. The method offers the fast objective identification of intoxication by muscarine and can become a routine screening alternative to more difficult microscopic examination of spores in the gastric content in clinical practice.     

Determination of N-(trans-4-isopropylcyclohexylcarbonyl)-d-phenylalanine in human plasma by solid-phase extraction and column-switching high-performance liquid chromatography with ultraviolet detection

A column-switching high-performance liquid chromatography method with ultraviolet detection at 210 nm has been developed for the determination of N-(trans-4-isopropylcyclohexylcarbonyl)-d-phenylalanine (AY4166, I) in human plasma. Plasma samples were prepared by solid-phase extraction with Sep-Pak Light tC₁₈, followed by HPLC. The calibration graph for I was linear in the range 0.1–20 μg/ml. The limit of quantitation of I, in plasma, was 0.05 μg/ml. The recovery of spiked I (0.5 μg/ml) to drug-free plasma was over 92% and the relative standard deviation of spiked I (0.5 μg/ml) compared to drug-free plasma was 4.3% (n = 8).     

Application of poly(butyl methacrylate-co-ethylene glycol dimethacrylate) monolith microextraction coupled with high performance liquid chromatography to the determination of polycyclic aromatic hydrocarbons in smoked meat products

This paper presents a study of the synthesis of a polymer monolith column and its application to the analysis of PAHs in smoked meat products. A poly(butyl methacrylate-co-ethylene glycol dimethacrylate) monolith capillary has been successfully prepared with in situ polymerization method. The polymer monolith microextraction combined with HPLC determinations is employed for the analysis of naphthalene, biphenyl, phenanthrene, and anthracene. Various parameters affecting the extraction efficiency have been investigated and optimized. Under the optimum experimental conditions, the method provides an acceptable linearity (2-10,000 μg/L), low limits of detection (1.4-2.0 μg/L), and good precision (intraday relative standard deviations<4.1%, interday relative standard deviations<5.7%). When applied to the determination of the four PAHs in smoked meat samples, recoveries are obtained in the range of 86.6-101.5%.     

Development of a high-performance liquid chromatography method for the simultaneous quantification of four organoarsenic compounds in the feeds of swine and chicken

A high-performance liquid chromatography (HPLC) method with UV detection was developed for the simultaneous determination of arsanilic acid, roxarsone, nitarsone, and carbarsone in the feeds of swine and chicken. Feed samples were extracted with methanol/1% acetic acid (90:10, v/v) in an ultrasonic bath and the protein was precipitated with 2% Cu(2)SO(4). The samples were further purified by solid phase extraction (SPE) on SAX cartridges. Separation was performed on a Zorbax SB-Aq C18 HPLC column using an isocratic procedure with methanol and 1% acetic acid (3:97, v/v) at a flow-rate of 0.7 mL min(-1), and the UV detector was set at a wavelength of 260 nm. The recoveries of organoarsenic compounds spiked at levels of 2, 20 and 200 μg g(-1) ranged from 81.2% to 91.3%; the inter-day relative standard deviation values were less than 7.0%. The limits of quantification for four organoarsenic compounds were 1.0-2.0 μg g(-1). This simple and fast method could be applied to the determination of multi-residues of organic arsenic compounds in animal feeds.     

Analysis of cantharidin in false blister beetles (Coleoptera: Oedemeridae) by headspace solid-phase microextraction and gas chromatography-mass spectrometry

A headspace solid-phase microextraction (HS-SPME) coupled to gas chromatography-mass spectrometry (GC-MS) method was developed to determine a type of terpenoid named as cantharidin in the false blister beetles, family Oedemeridae. The experimental parameters for HS-SPME method were optimized. Six commercial fibers for HS-SPME method development were tested and the divinylbenzene/carboxene/polydimethylsiloxane fiber was selected to provide the best detection of analyzed compound. The calibration curve showed linearity in the range of 0.1-50 μg mL(-1), correlation coefficient (R(2)=0.992), limit of detection (0.01 ng mL(-1)) and quantitation (0.04 ng mL(-1)) were obtained for the proposed method. The relative standard deviations of intra-day and inter-day assays were 7.8 and 3.4%, respectively. The recovery values, obtained after spiking the beetle samples by three concentration levels of standard solution, were higher than 87%. The results indicated the successful application of the proposed method on the analysis of cantharidin from the false blister beetles.     

Simultaneous analysis of amoxicillin and sulbactam in human plasma by HPLC-DAD for assessment of bioequivalence

A simple and accurate high-performance liquid chromatography with diode array detection-based (HPLC-DAD) method has been developed and validated for simultaneous determination of amoxicillin and sulbactam in human plasma. Sample preparation was involved in protein precipitation with acetonitrile followed by one-step extraction procedure. Chromatographic separation was achieved on a C18 column with an isocratic mobile phase consisting of water (containing 30 mM potassium dihydrogen phosphate, pH 2.8) and acetonitrile. The detection wavelengths of a diode array detector were set at 210 nm for amoxicillin and sulbactam, and 263 nm for the internal standard (cefadroxil). The method was validated for linearity, accuracy, precision, and stability. The calibration curve was linear from 0.163 to 14.7 μg/mL with correlation coefficient squared of 0.9991 for amoxicillin and 0.250-15.0 μg/mL with correlation coefficient squared of 0.9988 for sulbactam using 500 μL plasma samples. The lower limit of quantification was 0.163 and 0.250 μg/mL for amoxicillin and sulbactam, respectively. The imprecisions of intra- and inter-day validations for amoxicillin and sulbactam were <11% and their accuracies (%) were within the range of 95.4-105.7%. Mean recoveries were 75.9, 72.8, and 70.0% for amoxicillin, sulbactam, and cefadroxil, respectively. The established method was successfully applied to a bioequivalence study of two combination formulations of amoxicillin and sulbactam pivoxil in healthy male volunteers.     

Three phases hollow fiber LPME combined with HPLC-UV for extraction, preconcentration and determination of valerenic acid in Valeriana officinalis

In the present work, the applicability of hollow fiber-based liquid phase microextraction (HF-LPME) was evaluated for the extraction and preconcentration of valerenic acid prior to its determination by reversed-phase HPLC/UV. The target drug was extracted from 5.0 mL of aqueous solution with pH 3.5 into an organic extracting solvent (dihexyl ether) impregnated in the pores of a hollow fiber and finally back extracted into 10 μ L of aqueous solution with pH 9.5 located inside the lumen of the hollow fiber. In order to obtain high extraction efficiency, the parameters affecting the HF-LPME, including pH of the donor and acceptor phases, type of organic phase, ionic strength, the volume ratio of donor to acceptor phase, stirring rate and extraction time were studied and optimized. Under the optimized conditions, enrichment factor up to 446 was achieved and the relative standard deviation (RSD) of the method was 4.36% (n = 9). The linear range was 7.5-850 μg L?1 with correlation coefficient (r2=0.999), detection limits was 2.5 μg L?1 and the LOQ was 7.5 μg L?1. The proposed method was evaluated by extraction and determination of valerenic acid in some Iranian wild species of Valerianaceae.     

Determination of N-(trans-4-isopropylcyclohexylcarbonyl)-d-phenylalanine in human plasma by solid-phase extraction and column-switching high-performance liquid chromatography with ultraviolet detection

A column-switching high-performance liquid chromatography method with ultraviolet detection at 210 nm has been developed for the determination of N-(trans-4-isopropylcyclohexylcarbonyl)-d-phenylalanine (AY4166, I) in human plasma. Plasma samples were prepared by solid-phase extraction with Sep-Pak Light tC₁₈, followed by HPLC. The calibration graph for I was linear in the range 0.1–20 μg/ml. The limit of quantitation of I, in plasma, was 0.05 μg/ml. The recovery of spiked I (0.5 μg/ml) to drug-free plasma was over 92% and the relative standard deviation of spiked I (0.5 μg/ml) compared to drug-free plasma was 4.3% (n = 8).