Determination of 1-hydroxypyrene in human urine by acid hydrolysis coupled to solid-phase microextraction and semi-microcolumn liquid chromatography.

This study developed an acid hydrolysis coupled to a solid-phase microextraction method employing a semi-microcolumn liquid chromatography system, instead of enzyme hydrolysis with solid-phase extraction for the pretreatment of human urine samples, to detect urinary 1-hydroxypyrene (1-OHP). The complete separation and detection of urinary 1-hydroxyprene was performed using a high-performance liquid-chromatography fluorescence detection system with an analytical C(18) semi-microcolumn, 60% (v/v) aqueous acetonitrile elution, and a lambda(ex/em) = 348/388 nm pair detection wavelength. Calibration graphs were linear with very good correlation coefficients (r = 0.9997), and the detection limit was 1.0 ng/L. These important parameters for acid hydrolysis and solid-phase microextraction were investigated. The total recovery was above 83% in acid hydrolysis with solid-phase microextraction. The proposed method provided a relatively simple, convenient, and practical procedure to determine the level of urinary 1-hydroxypyrene in biological samples, and was successfully applied to detect the urine of students.     

High-performance liquid chromatographic method for simultaneous determination of enantiomers of 5-dimethylsulphamoyl-6,7-dichloro-2-dihydrobenzofuran-2, carboxylic acid and its N-monodemethyl metabolite in monkey plasma and urine after chiral derivatization

A quantitative method for the simultaneous high-performance liquid chromatographic (HPLC) resolution and determination of the enantiomers of 5-dimethylsulphamoyl-6,7-dichloro-2,3-dihydrobenzofuran-2-carboxyl ic acid, a new diuretic, and its N-monodemethylated metabolite in monkey plasma and urine is described. The method includes diethyl ether extraction of the samples and S-(-)-alpha-methylbenzylamide derivatization of the extract, followed by reversed-phase solid-phase extraction and injection of the resulting diastereoisomers onto a reversed-phase HPLC column. Baseline separation was obtained. The assay showed linearity over the range 0.1-50 micrograms/ml of plasma and 0.25-500 microliters of urine, with a lower limit of detection of ca. 0.01 micrograms/ml for each of the enantiomers. The method is adequate for pharmacokinetic and enantioselective disposition studies of both the diuretic and its metabolite.     

Detection of a urinary biomaker for oxidative DNA damage 8-hydroxydeoxyguanosine by capillary electrophoresis with electrochemical detection

8-Hydroxydeoxyguanosine (8-OHdG) is present in urine as a result of oxidative DNA damage associated with age-related diseases such as cancer. In this report a method is presented for the detection of 8-OHdG in human morning urine utilizing capillary electrophoresis with electrochemical detection (CEEC). The limit of detection for a aqueous standard of 8-OHdG is 50 nM (signal to noise ratio S/N = 3). A single solid-phase extraction (SPE) step with a C18 column is used for sample cleanup and 20-fold preconcentration of the urine before analysis by CEEC. Optimized conditions for analysis of extracted urine are E(app) = 0.5 V vs. Ag/AgCl with 20 mM sodium borate/20% MeOH v/v, pH 9, as the background electrolyte, and a separation voltage of 22 kV. The concentration of 8-OHdG varied from 6 to 86 nM with an average value of 42 +/- 26.9 nM for four healthy female and four healthy male subjects between the ages of 23 and 43.     

Molecularly imprinted solid-phase extraction for the selective determination of methamphetamine, amphetamine, and methylenedioxyphenylalkylamine designer drugs in human whole blood by gas chromatography-mass spectrometry

A novel method is described for the extraction of methamphetamine, amphetamine, and methylenedioxyphenylalkylamine designer drugs, such as 3,4-methylenedioxy-methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxyethylamphetamine, N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine, and 3,4-(methylenedioxyphenyl)-2-butanamine, from human whole blood using molecularly imprinted solid-phase extraction as highly selective sample clean-up technique. Whole blood samples were diluted with 10 mmol/L ammonium acetate (pH 8.6) and applied to a SupelMIP-Amphetamine molecularly imprinted solid-phase extraction cartridge. The cartridge was then washed to eliminate interferences, and the amphetamines of interest were eluted with formic acid/methanol (1:100, v/v). After derivatization with trifluoroacetic anhydride, the analytes were quantified using gas chromatography-mass spectrometry. Recoveries of the seven amphetamines spiked into whole blood were 89.1-102%. The limits of quantification for each compound in 200 μL of whole blood were between 0.25 and 1.0 ng. The maximum intra- and inter-day coefficients of variation were 9.96 and 13.8%, respectively. The results show that methamphetamine, amphetamine, and methylenedioxyphenylalkyl-amine designer drugs can be efficiently extracted from crude biological samples such as whole blood by molecularly imprinted solid-phase extraction with good reproducibility. This extraction method will be useful for the pretreatment of human samples before gas chromatography-mass spectrometry.     

Poly(methacrylic acid-ethylene glycol dimethacrylate) monolith in-tube solid-phase microextraction applied to simultaneous analysis of some amphetamine derivatives in urine by capillary zone electrophoresis

A method based on in-tube solid-phase microextraction and capillary zone electrophoresis (CZE) was proposed for simultaneously determining four amphetamines (amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, and 3,4-methylenedioxymethamphetamine) in urine. A poly(methacrylic acid-ethylene glycol dimethacrylate) monolithic capillary column, which can provide sufficient extraction efficiency, was introduced for the extraction of amphetamines from urine samples. The hydrophobic main chains and acidic pendant groups of the monolithic column make it a superior material for extraction of basic analytes from aqueous matrix. After extraction, the samples were analyzed by CZE. The best separation was achieved using a buffer composed of 0.1 M disodium hydrogen phosphate (adjusted to pH 4.5 with 1 M hydrochloric acid) and 20% methanol v/v, with a temperature and voltage of 25 degrees C and 20 kV, respectively. By applying electrokinetic injection with field-amplified sample stacking, detection limits of 25-34 microg/L were achieved. Excellent method of reproducibility was found over a linear range of 0.1-5 mg/L. Determination of these analytes from abusers' urine sample was also demonstrated.     

Sensitive and selective determination of metabolically formed trans-dihydrodiols and phenols of benzo[a]pyrene in water and urine samples by HPLC with amperometric detection

A method has been developed to separate hydroxylated metabolites of the carcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene, i. e. trans-4,5-, 7,8-, 9,10-dihydrodiol and 1-, 3-, 7-, and 9-phenol, by HPLC with amperometric detection employing an isocratic methanol/water eluent (70:30, v/v) containing 0.5 g/L sulfuric acid and 1 g/L lithium perchlorate. Compared with the usually applied fluorescence (λ_ex = 265 nm, λ_em = 460 nm) and ultraviolet (λ = 265 nm) detection, the amperometric technique is about 2–12 times more sensitive for the determination of all metabolites investigated. The method was applied to the determination of the seven metabolites of benzo[a]pyrene in different water samples and in urine after solid-phase extraction (SPE). The results obtained by HPLC with amperometric detection after SPE enrichment from an aqueous extract of a soil sample and from the urine of a rat intragastrically treated with benzo[a]pyrene agreed well with the values determined with fluorescence and/or UV detection.     

高效液相色谱-串联质谱法同时测定人尿液中7种邻苯二甲酸酯代谢物

建立了同时检测人尿液中7种邻苯二甲酸酯代谢物的高效液相色谱-串联三重四极杆质谱法。尿液经酶水解后,采用萃取柱净化,以2%(v/v)甲酸甲醇溶液为洗脱剂,经苯基柱分离,以0.1%(v/v)乙酸水溶液和0.1%(v/v)乙酸乙腈溶液为流动相进行梯度洗脱,采用电喷雾离子源负离子模式和多反应监测模式采集信号,用同位素内标法进行定量分析。尿液中7种邻苯二甲酸酯代谢物在0.2~200.0 μg/L范围内定量离子的相对峰面积比值与质量浓度均呈良好线性关系(r≥0.99976);检出限(LOD)为13.43~80.21 ng/L,定量限为44.77~267.37 ng/L; 3个水平的加标回收率为88.8%~108.9%,日内和日间精密度均不大于17.05%。该方法可同时准确、灵敏、简便地测定人尿液中7种邻苯二甲酸酯代谢物的暴露水平。     

Determination of geniposide in rat urine after oral administration of the traditional Chinese medicinal preparation yin-zhi-ku decoction by high-performance liquid chromatography

A high-performance liquid chromatography method with solid-phase extraction is introduced for the determination of geniposide in rat urine after oral administration of yin-zhi-ku decoction. Geniposide and an internal standard (paeoniflorin) are extracted from urine using Strata cartridges. Analysis of the extract is then performed on a reversed-phase C18 column using acetonitrile-water (14:86, v/v) as eluting solvent system. UV detection is set at 238 nm. The calibration curve for geniposide is linear (r = 0.9996) in the concentration range of 2.0-240 microg/mL. Both intra- and interday precision of the geniposide are determined, and their relative standard deviation does not exceed 10%. The validated method is successfully applied to determine geniposide from rat urine after oral administration of yin-zhi-ku decoction.     

Application of a novel nylon needle filter-based solid-phase extraction device to determination of 1-hydroxypyrene in urine

In this work, a simple and miniaturized solid-phase extraction device was constructed by connecting a commercial nylon needle filter to a syringe, which was applied for extracting 1-hydroxypyrene from a urine sample via hydrophobic and hydrogen bond interactions. The nylon membrane in the needle filter acted as the solid-phase extraction adsorbent, meanwhile, it filtered the particles in the urine sample. To obtain high extraction efficiency, key parameters influencing extraction recovery were investigated. The entire pretreatment process was accomplished within 5 min under the optimal conditions. By coupling high-performance liquid chromatography–ultraviolet, a rapid, low-cost, and convenient nylon needle filter-based method was established for the analysis of 1-hydroxypyrene in a complex urine matrix. Within the linearity range of 0.2–1000 μg/L, the method exhibited a satisfactory correlation coefficient (R = 0.9999). The limit of detection was 0.06 μg/L, and the recoveries from urine sample spiked with three concentrations (5, 20, and 100 μg/L) ranged from 105.8% to 113.1% with the relative standard deviations less than 6.7% (intra-day, n = 6) and 8.9% (inter-day, n = 4). Finally, the proposed method was successfully applied for detecting 1-hydroxypyrene in urine samples from college students, smokers, gas station workers, and chip factory workers. The detected concentration in actual urine samples ranged from 0.46 to 5.26 μg/L. Taken together, this simple and cost-effective nylon needle filter-based solid-phase extraction device showed an excellent application potential for pretreating hydrophobic analytes from aqueous samples.     

Microwave-assisted extraction versus Soxhlet extraction in the analysis of 21 organochlorine pesticides in plants

A method to determine 21 organochlorine pesticides in vegetation samples using microwave-assisted extraction (MAE) is described and compared with Soxhlet extraction. Samples were extracted with hexane–acetone (1:1, v/v) and the extracts were cleaned using solid-phase extraction with Florisil and alumine as adsorbents. Pesticides were eluted with hexane–ethyl acetate (80:20, v/v) and determined by gas chromatography and electron-capture detection. Recoveries obtained (75.5–132.7% for Soxhlet extraction and 81.5–108.4% for MAE) show that both methods are suitable for the determination of chlorinated pesticides in vegetation samples. The method using microwave energy was applied to grass samples from parks of A Coruña (N.W. Spain) and to vegetation from the contaminated industrial area of Torneiros (Pontevedra, N.W. Spain).     

Molecularly imprinted solid-phase extraction for the selective determination of bromhexine in human serum and urine with high performance liquid chromatography

In this work, a novel method is described for the determination of bromhexine in biological fluids using molecularly imprinted solid-phase extraction as the sample cleanup technique combined with high performance liquid chromatography (HPLC). The water-compatible molecularly imprinted polymers (MIPs) were prepared using methacrylic acid as functional monomer, ethylene glycol dimethacrylate as cross-linker, chloroform as porogen and bromhexine as the template molecule. The novel imprinted polymer was used as a solid-phase extraction sorbent for the extraction of bromhexine from human serum and urine. Various parameters affecting the extraction efficiency of the polymer have been evaluated. The optimal conditions for molecularly imprinted solid-phase extraction (MISPE) consisted of conditioning 1 mL methanol and 1 mL of deionized water at neutral pH, loading of 5 mL of the water sample (25 μg L⁻¹) at pH 6.0, washing using 2 mL acetonitrile/acetone (1/4, v/v) and elution with 3× 1 mL methanol/acetic acid (10/1, v/v). The MIP selectivity was evaluated by checking several substances with similar molecular structures to that of bromhexine. Results from the HPLC analyses showed that the calibration curve of bromhexine using MIP from human serum and urine is linear in the ranges of 0.5-100 and 1.5-100 μg L⁻¹ with good precisions (3.3% and 2.8% for 5.0 μg L⁻¹), respectively. The recoveries for serum and urine samples were higher than 92%.     

High-performance liquid chromatographic analysis of 2',3'-dideoxyinosine in biological samples

A high-performance liquid chromatographic analysis for the anti-AIDS drug 2',3'-dideoxyinosine (ddI) in rat plasma and urine, with a limit of detection of 0.2 microgram/ml and requiring a sample size of 100 microliters is described. Diluted plasma or urine samples were extracted using a C18 solid-phase extraction column. Retention of ddI on more polar solid-phase extraction columns was insufficient for sample clean-up. This method is useful for pharmacokinetic studies of ddI in small rodents.     

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.     

Microassay for the simultaneous determination of cocaine, norcocaine, benzoylecgonine and benzoylnorecgonine by high-performance liquid chromatography

An improved method for the simultaneous determination of cocaine, norcocaine, benzoylecgonine and benzoylnorecgonine using reversed-phase high-performance liquid chromatography with ultraviolet detection is described. Following solid-phase extraction, chromatography was performed using a column containing an octadecylsilica-coated packing, eluted with 6% acetonitrile in phosphate buffer, pH 2.1, and detected at 233 nm. Using 80-microliters samples, the detection limit is 18 ng/ml for benzoylecgonine and benzoylenorecgonine and 35 ng/ml for cocaine and norcocaine. The coefficients of variation range from 3.5% (benzoylecgonine) to 7.0% (norcocaine). The procedure has been applied to samples of guinea pig plasma, urine and amniotic fluid and human urine.     

Analysis of cocaine and two metabolites in dried blood spots by liquid chromatography with fluorescence detection: a novel test for cocaine and alcohol intake

An original HPLC method coupled to spectrofluorimetric detection is presented for the simultaneous analysis in dried blood spots (DBS) of cocaine and two important metabolites, namely benzoylecgonine (its main metabolite) and cocaethylene (the active metabolite formed in the presence of ethanol). The chromatographic analysis was carried out on a C8 column, using a mobile phase containing phosphate buffer (pH 3.0)-acetonitrile (85:15, v/v). Native analyte fluorescence was monitored at 315 nm while exciting at 230 nm. A fast and feasible sample pre-treatment was implemented by solvent extraction, obtaining good extraction yields (>91%) and satisfactory precision values (RSD<4.8%). The method was successfully applied to DBS samples collected from some cocaine users, both with and without concomitant ethanol intake. The results were in good agreement with those obtained from plasma samples subjected to an original solid-phase extraction procedure on C8 cartridges. The method has demonstrated to be suitable for the monitoring of cocaine/ethanol use by means of DBS or plasma testing. Assays are in progress to apply this method on the street, for the control of subjects suspected of driving under the influence of psychotropic substances.     

Determination of lipid oxidation level in broiler meat by liquid chromatography

An assay was conducted for the determination of malondialdehyde (MDA) levels in broiler meat. The method involves extraction of tissues with trichloroacetic acid (TCA) and reaction of the TCA extract with 2,4-dinitrophenylhydrazine (DNPH). After separation of the MDA-DNPH complex using a solid-phase extraction C18 column, samples were eluted with 1 mL acetonitrile. Aliquots of 20 microL acetonitrile were analyzed by liquid chromatography on reversed-phase C18 column (3 microm) with UV detection. The products were eluted isocratically with the mobile phase containing acetonitrile-water-acetic acid (39 + 61 + 0.2, v/v/v). The retention time for MDA-DNPH was 6.5 min, and the detection limit was 3.5 microg/kg. Two extraction methods (cold and hot) were also used in the study. The results showed that hot extraction increased results about 55.8% and recovery from samples spiked with 116.6 microg/kg was lower (74.6%) in comparison with cold extraction (94.7%).     

固相萃取-高效液相色谱-串联质谱法同时测定养殖海水中23种抗生素

建立了固相萃取-高效液相色谱-串联质谱(SPE-HPLC-MS/MS)同时检测水中6类23种抗生素的分析方法。水样用固相萃取柱富集净化,通过对比水样在不同上样pH、洗脱液用量等条件下的回收率,优化了前处理方法。采用0.1%(v/v)甲酸-1 g/L甲酸铵水溶液和甲醇-乙腈(1:1, v/v)体系作为流动相,经过梯度洗脱进行分离,在HPLC-MS/MS多反应监测模式下进行定性定量分析。结果显示,23种抗生素的方法检出限(MDL)范围为0.1~2.9 ng/L,加标回收率为47.3%~132.6%。采用该方法对东营海水养殖区5个养殖池水样进行了检测,除青霉素类之外的各类抗生素均有检出,其中磺胺增效剂甲氧苄氨嘧啶的检出率达100%,氯霉素类抗生素氟甲砜霉素检出的最高质量浓度达到261.0 ng/L。结果表明,所建立的方法高效、灵敏、可靠,可用于海水中多种抗生素的分析。     

Sensitive and selective determination of metabolically formed trans-dihydrodiols and phenols of benzo[a]pyrene in water and urine samples by HPLC with amperometric detection

A method has been developed to separate hydroxylated metabolites of the carcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene, i. e. trans-4,5-, 7,8-, 9,10-dihydrodiol and 1-, 3-, 7-, and 9-phenol, by HPLC with amperometric detection employing an isocratic methanol/water eluent (70:30, v/v) containing 0.5 g/L sulfuric acid and 1 g/L lithium perchlorate. Compared with the usually applied fluorescence (λ_ex = 265 nm, λ_em = 460 nm) and ultraviolet (λ = 265 nm) detection, the amperometric technique is about 2–12 times more sensitive for the determination of all metabolites investigated. The method was applied to the determination of the seven metabolites of benzo[a]pyrene in different water samples and in urine after solid-phase extraction (SPE). The results obtained by HPLC with amperometric detection after SPE enrichment from an aqueous extract of a soil sample and from the urine of a rat intragastrically treated with benzo[a]pyrene agreed well with the values determined with fluorescence and/or UV detection. Received: 20 December 1996 / Revised: 10 March 1997 / Accepted: 30 April 1997     

固相萃取-衍生化-气相色谱-质谱联用法同时测定尿液中4种阿片类物质

公安机关用胶体金尿检法对海洛因滥用者的检测常常受到阿片类镇咳药的干扰,使用传统液-液提取法进行实验室检验,操作效率低,灵敏度不高,无法满足公安机关打击涉毒案件的需要。为此,该研究建立了尿液中吗啡、O ⁶ -单乙酰吗啡、可待因和乙酰可待因4种阿片类物质的固相萃取和衍生化技术结合气相色谱-质谱联用（GC-MS）同时检测方法。尿样用磷酸盐缓冲液调节至pH=6后,经MCX固相萃取柱净化,用N -甲基-N -（三甲基硅烷基）三氟乙酰胺（MSTFA）对吗啡、O ⁶ -单乙酰吗啡、可待因进行衍生化,供GC-MS检测。考察了上样和洗脱流速、淋洗液中甲酸体积分数、洗脱液中氨水体积分数、3%（v/v）甲酸甲醇淋洗液体积和固相萃取柱吹干时间对萃取效果的影响。确定上样和洗脱流速1.0 mL/min,淋洗液中甲酸体积分数3%,洗脱液中氨水体积分数5%,3%（v/v）甲酸甲醇淋洗液体积1 mL,吹干时间1 min为最佳条件。在此条件下,4种阿片类物质在0.02~0.8 μg/mL范围内线性关系良好（r ² ≥0.998）,检出限（LOD）为0.0016~0.0039 μg/mL,定量限（LOQ）为0.0054~0.0128 μg/mL,当标准添加水平为0.02、0.1、0.2 μg/mL时,回收率为93.0%~110.3%。该方法结合自动化技术,对固相萃取条件精确控制,操作简便、快速、灵敏、准确,适合尿液中吗啡等4种阿片类物质快速测定,可用于海洛因吸食者的大规模监控,并能准确排除因服用含阿片类镇咳药导致的吗啡胶体金尿检假阳性。     

Development and method validation for testosterone and epitestosterone in human urine samples by liquid chromatography applications

An isocratic high-performance liquid chromatographic method for the determination of testosterone (T) and epitestosterone (ET) in human urine using liquid-liquid or solid-phase extraction (SPE) is developed and validated. The optimum separation is achieved using a Hypersil C(18) column, water-acetonitrile (57:43, v/v) as the mobile phase and UV-absorbance detection at 245 nm. The recoveries obtained for T and ET in liquid-liquid and SPE demonstrate that these procedures are interchangeable. Quantitation limits for T and ET are 8.6 and 5.4 ng/mL using solvent extraction and 7.3 and 5.7 ng/mL using SPE, respectively. The proposed method is used to evaluate the urinary T, ET, and the T/ET ratio for a healthy male population using liquid-liquid extraction, and the T and ET excretion profile for nine healthy men using SPE.