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.     

Quantification of testosterone and epitestosterone in human urine samples by stir bar sorptive extraction--thermal desorption--gas chromatography/mass spectrometry: application to HIV-positive urine samples

A simple method is described for the measurement of testosterone (T) and epitestosterone (ET) in human urine samples. The deconjugated steroids were extracted directly from the samples by stir bar sorptive extraction (SBSE) and derivatized in situ on the stir bar by headspace acylation prior to thermal desorption and GC/MS. Extraction and derivatization parameters, namely salt addition, temperature, and time, were optimized to improve the recovery of T and ET by SBSE. The limits of quantification (S/N 10) were 0.9 ng/mL for T and 2.8 ng/mL for ET. Quantification of the steroids in urine samples was performed using standard addition to avoid the influence of matrix effects. The method was applied for the measurement of urinary T and ET in a group of healthy volunteers and HIV+ patients. Decreased levels of T were detected in the HIV+ group, whereas the excretion of ET was comparable for the two groups. Further clinical research is required to elucidate the biomarker significance of the T/ET ratio in HIV infection.     

Automated high-throughput method using solid-phase microextraction-liquid chromatography-tandem mass spectrometry for the determination of ochratoxin A in human urine

A new automated, high-throughput method for the determination of ochratoxin A (OTA) in human urine samples has been optimized and validated using solid-phase microextraction coupled to liquid chromatography-tandem mass spectrometry (SPME-LC-MS/MS). High-throughput was achieved by simultaneous preparation of up to 96 samples using multi-fiber SPME device and multi-well plates. A carbon-tape coating was chosen for the first time as the best extracting phase for this contaminant. The proposed method required only minimal sample pre-treatment to adjust sample pH to 3.0 using a dilution (1:1) with 0.5M phosphate-buffered saline. A simple gradient guaranteed a good chromatographic separation from matrix interferences in only 8min. Relative recovery (%), precision and linearity validation results met Food and Drug Administration acceptance criteria at three concentration levels (1, 10, and 50ng/mL), indicating excellent performance of the proposed method. Limits of detection and quantitation were 0.3 and 0.7ng/mL in urine, respectively. OTA determination in urine is a good marker for human exposure to this mycotoxin. It is also less invasive than blood analysis. This method is fully automated and the SPME technique is simpler, less time-consuming and cheaper compared with most widely adopted clean-up procedures for OTA extraction from urine.     

Contribution of microextraction in packed sorbent for the analysis of cotinine in human urine by GC–MS

A simple, rapid, sensitive, and non-consuming solvent method for the determination of cotinine in urine was developed, based on sample preparation by the relatively new technique microextraction in packed sorbent (MEPS) and analysis by GC–MS. This optimized method was compared with conventional solid-phase extraction/liquid–liquid extraction method used as reference. The wide linear range (5–5,000 ng/mL) and high sensitivity of the MEPS method (limit of detection 0.8 ng/mL) allow application to analysis of urine from smokers as well as non-smokers susceptible to passive smoking.     

A high-throughput liquid chromatography/tandem mass spectrometry method for simultaneous quantification of a hydrophobic drug candidate and its hydrophilic metabolite in human urine with a fully automated liquid/liquid extraction

ABT-869 (A-741439) is an investigational new drug candidate under development by Abbott Laboratories. ABT-869 is hydrophobic, but is oxidized in the body to A-849529, a hydrophilic metabolite that includes both carboxyl and amino groups. Poor solubility of ABT-869 in aqueous matrix causes simultaneous analysis of both ABT-869 and its metabolite within the same extraction and injection to be extremely difficult in human urine. In this paper, a high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) method has been developed and validated for high-speed simultaneous quantitation of the hydrophobic ABT-869 and its hydrophilic metabolite, A-849529, in human urine. The deuterated internal standards, A-741439D(4) and A-849529D(4), were used in this method. The disparate properties of the two analytes were mediated by treating samples with acetonitrile, adjusting pH with an extraction buffer, and optimizing the extraction solvent and mobile phase composition. For a 100 microL urine sample volume, the lower limit of quantitation was approximately 1 ng/mL for both ABT-869 and A-849529. The calibration curve was linear from 1.09 to 595.13 ng/mL for ABT-869, and 1.10 to 600.48 ng/mL for A-849529 (r2 > 0.9975 for both ABT-869 and A-849529). Because the method employs simultaneous quantification, high throughput is achieved despite the presence of both a hydrophobic analyte and its hydrophilic metabolite in human urine.     

Preventive doping control analysis: liquid and gas chromatography time-of-flight mass spectrometry for detection of designer steroids

A new combined doping control screening method for the analysis of anabolic steroids in human urine using liquid chromatography/electrospray ionization orthogonal acceleration time-of-flight mass spectrometry (LCoaTOFMS) and gas chromatography/electron ionization orthogonal acceleration time-of-flight mass spectrometry (GCoaTOFMS) has been developed in order to acquire accurate full scan MS data to be used to detect designer steroids. The developed method allowed the detection of representative prohibited substances, in addition to steroids, at concentrations of 10 ng/mL for anabolic agents and metabolites, 30 ng/mL for corticosteroids, 500 ng/mL for stimulants and beta-blockers, 250 ng/mL for diuretics, and 200 ng/mL for narcotics. Sample preparation was based on liquid-liquid extraction of hydrolyzed human urine, and the final extract was analyzed as trimethylsilylated derivatives in GCoaTOFMS and underivatized in LCoaTOFMS in positive ion mode. The sensitivity, mass accuracy, advantages and limitations of the developed method are presented.     

Magnetic porous carbon derived from a metal–organic framework as a magnetic solid‐phase extraction adsorbent for the extraction of sex hormones from water and human urine

An iron‐embedded porous carbon material (MIL‐53‐C) was fabricated by the direct carbonization of MIL‐53. The MIL‐53‐C possesses a high surface area and good magnetic behavior. The structure, morphology, magnetic property, and porosity of the MIL‐53‐C were studied by scanning electron microscopy, transmission electron microscopy, vibrating sample magnetometry, and N₂ adsorption. With the use of MIL‐53‐C as the magnetic solid‐phase extraction adsorbent, a simple and efficient method was developed for the magnetic solid‐phase extraction of three hormones from water and human urine samples before high‐performance liquid chromatography with UV detection. The developed method exhibits a good linear response in the range of 0.02–100 ng/mL for water and 0.5–100 ng/mL for human urine samples , respectively. The limit of detection (S/N = 3) for the analytes was 0.005–0.01 ng/mL for water sample and 0.1–0.3 ng/mL for human urine sample. The limit of quantification (S/N = 10) of the analytes were in the range of 0.015–0.030 and 0.3–0.9 ng/mL, respectively.     

Determination of Diuretics in Urine Using Immobilized Multi‐Walled Carbon Nanotubes in Hollow Fiber Liquid‐Phase Microextraction Combined with Liquid Chromatography‐Tandem Mass Spectrometry

A novel technique utilizing the adsorptive potential of immobilized multi‐walled carbon nanotubes (I‐MWCNT) in hollow fiber liquid‐phase microextraction (HF‐LPME) was developed for the determination of diuretics in urine. In this study, the potential of carbon nanotubes as a sorbent for three‐phase liquid‐phase microextraction of diuretics from urine samples was evaluated. Analysis was performed using liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). A novel method was applied to detect acetazolamide (AAA), chlorothiazide (CTA), hydrochlorothiazide (HCT), hydroflumethiazide (HFT), clopamide (CA), trichlormethiazide (TCM), althiazide (AT) and bendroflumethiazide (BFT) in urine. Two‐step extractions using different times and temperatures for each step were adopted. Parameters influencing the extraction efficiency, including the extraction solvent, sample pH, salt concentration, extraction time and extraction temperature were systematically optimized. Under the resulting optimal extraction conditions, this method showed good linearity over an analytes concentration range of 1 to 1000 ng/mL, high extraction repeatability with relative standard deviations of less than 6%, and low detection limits (0.09 to 0.51 ng/mL). The application of the methods to the determination of diuretics in real samples was tested by analyzing urine samples of patient.     

超高效液相色谱-串联质谱法测定人尿液中全氟有机化合物

采用超高效液相色谱-串联质谱(UPLC-MS/MS)联用技术,建立了对人尿液中12种全氟有机化合物(PFCs)的分析方法。首先在尿液样品中加入相应的同位素内标,以2%(体积分数)甲酸甲醇溶液超声萃取、离心后,将提取液用弱阴离子交换固相萃取柱净化,采用UPLC-MS/MS测定,内标法定量。12种目标化合物在0.05～50 μg/L质量浓度范围内线性良好,相关系数(r)均大于0.992,检出限在0.44～3.47 ng/L之间。在20、100、500 ng/L添加水平下,平均回收率范围为80.3%～116.2%,相对标准偏差(n=6)在5.5%～13.8%之间。该方法灵敏度高、重现性好、回收率高、操作简单,适合人尿液中PFCs的测定。     

Cyclodextrin‐assisted dispersive liquid–liquid microextraction for the preconcentration of carbamazepine and clobazam with subsequent sweeping micellar electrokinetic chromatography

A new version of dispersive liquid–liquid microextraction, namely, cyclodextrin‐assisted dispersive liquid–liquid microextraction, with subsequent sweeping micellar electrokinetic chromatography has been developed for the preconcentration and sensitive detection of carbamazepine and clobazam. α‐Cyclodextrin and chloroform were used as the dispersive agent and extraction solvent, respectively. After the extraction, carbamazepine and clobazam were analyzed using micellar electrokinetic chromatography with ultraviolet detection. The detection sensitivity was further enhanced using the sweeping technique. Under optimal extraction and stacking conditions, the calibration curves of carbamazepine and clobazam were linear over a concentration range of 2.0–200.0 ng/mL. The method detection limits at a signal‐to‐noise ratio of 3 were 0.6 and 0.5 ng/mL with sensitivity enhancement factors of 3575 and 4675 for carbamazepine and clobazam, respectively. This developed method demonstrated high sensitivity enhancement factors and was successfully applied to the determination of carbamazepine and clobazam in human urine samples. The precision and accuracy for urine samples were less than 4.2 and 6.9%, respectively.     

Novel micro-extraction by packed sorbent procedure for the liquid chromatographic analysis of antiepileptic drugs in human plasma and urine

A method for the simultaneous determination of the antiepileptic drugs, phenobarbital (PHB), phenytoin (PTN), carbamazepine (CBZ), primidone (PRM) and oxcarbazepine (OXC) in human plasma and urine samples by using micro‐extraction in a packed syringe as the sample preparation method connected with LC/UV (MEPS/LC/UV) is described. Micro‐extraction in a packed syringe (MEPS) is a new miniaturized, solid‐phase extraction technique that can be connected online to gas or liquid chromatography without any modifications. In MEPS approximately 1 mg of the solid packing material is inserted into a syringe (100–250 μL) as a plug. Sample preparation takes place on the packed bed. The bed can be coated to provide selective and suitable sampling conditions. The new method is very promising, easy to use, fully automated, inexpensive and quick. The standard curves were obtained within the concentration range 1–500 ng/mL in both plasma and urine samples. The results showed high correlation coefficients (R²>0.988) for all of the analytes within the calibration range. The extraction recovery was found to be between 88.56 and 99.38%. The limit of quantification was found to be between 0.132 and 1.956 ng/mL. The precision (RSD) values of quality control samples (QC) had a maximum deviation of 4.9%. A comparison of the detection limits with similar methods indicates high sensitivity of the present method. The method is applied for the analysis of these drugs in real urine and plasma samples of epileptic patients.     

Magnetic solid‐phase extraction for determination of sulpiride in human urine and blood using high‐performance liquid chromatography

A novel and efficient sample preconcentration technique based on the Fe₃O₄ magnetic nanoparticles (Fe₃O₄ MNPs) coated with silica (SiO₂) has been developed for extraction and determination of sulpiride. The functionalized MNPs showed excellent dispersibility in aqueous solution and were applied to magnetic solid‐phase extraction of sulpiride from human urine and blood prior to high‐performance liquid chromatography analysis. The separation, preconcentration and desorption procedure was completed in 10 min. Optimal experimental conditions, including sample pH, the amount of the MNPs, eluent type and volume, and the ultrasonication time were studied and established. The method showed good linearity for the determination of sulpiride in the concentration range of 10–1000 ng/mL in urine and blood. The recovery of the method was in the range between 91.2 and 97.5%, and the limit of detection was 2 ng/mL for sulpiride in human blood and urine. The results indicated that the present procedure is a suitable pretreatment method for biological samples. Copyright © 2015 John Wiley & Sons, Ltd.     

Ultrasound assisted ionic liquid dispersive liquid phase extraction of lovastatin and simvastatin: A new pretreatment procedure

A fast and novel sample preparation procedure: ultrasound assisted ionic liquid (IL) dispersive liquid extraction for the concentration of lovastatin and simvastatin in aqueous samples was developed. An IL ([C₆MIM][PF₆]) was used as the extraction solvent, and the factors affecting the extraction efficiency such as initial temperature, the volume of IL, pH of water samples, cooling time, and salt concentration were optimized. In combination with HPLC‐UV, both lovastatin and simvastatin exhibited a good linear range of 1–100 ng/mL. The limits of detection (LODs) of lovastatin and simvastatin were 0.17 and 0.29 ng/mL, respectively. Precisions of the proposed method (RSDs, n = 9) were 4.12 and 4.52%, respectively. This method has been successfully applied for the analysis of target compounds in three real water samples and good spiking recoveries were obtained in the range of 90.0–102.2% for lovastatin and 80.5–112.0% for simvastatin. These results indicated that ultrasound assisted IL dispersive liquid phase extraction would have good application prospect in the pretreatment of environmental samples.     

Optimization of alcohol‐assisted dispersive liquid–liquid microextraction by experimental design for the rapid determination of fluoxetine in biological samples

A sensitive and rapid method based on alcohol‐assisted dispersive liquid–liquid microextraction followed by high‐performance liquid chromatography for the determination of fluoxetine in human plasma and urine samples was developed. The effects of six parameters on the extraction recovery were investigated and optimized utilizing Plackett–Burman design and Box–Benken design, respectively. According to the Plackett–Burman design results, the volume of disperser solvent, extraction time, and stirring speed had no effect on the recovery of fluoxetine. The optimized conditions included a mixture of 172 μL of 1‐octanol as extraction solvent and 400 μL of methanol as disperser solvent, pH of 11.3 and 0% w/v of salt in the sample solution. Replicating the experiment in optimized condition for five times, gave the average extraction recoveries equal to 90.15%. The detection limit of fluoxetine in human plasma was obtained 3 ng/mL, and the linearity was in the range of 10–1200 ng/mL. The corresponding values for human urine were 4.2 ng/mL with the linearity range from 10 to 2000 ng/mL. Relative standard deviations for intra and inter day extraction of fluoxetine were less than 7% in five measurements. The developed method was successfully applied for the determination of fluoxetine in human plasma and urine samples.     

Ionic liquids dispersive liquid–liquid microextraction and high‐performance liquid chromatographic determination of irbesartan and valsartan in human urine

An environmentally friendly ionic liquids dispersive liquid–liquid microextraction (IL‐DLLME) method coupled with high‐performance liquid chromatography (HPLC) for the determination of antihypertensive drugs irbesartan and valsartan in human urine samples was developed. The HPLC separations were accomplished in less than 10 min using a reversed‐phase C₁₈ column (250 × 4.60 mm i.d., 5 µm) with a mobile phase containing 0.3 % formic acid solution and methanol (v/v, 3:7; flow rate, 1.0 mL/min). UV absorption responses at 236 nm were linear over a wide concentration range from 50 µg/mL to the detection limits of 3.3 µg/L for valsartan and 1.5 µg/L for irbesartan. The effective parameters on IL‐DLLME, such as ionic liquid types and their amounts, disperser solvent types and their volume, pH of the sample and extraction time were studied and optimized. The developed IL‐DLLME‐HPLC was successfully applied for evaluation of the urine irbesartan and valsartan profile following oral capsules administration. Copyright © 2012 John Wiley & Sons, Ltd.     

Sensitive determination of free benzophenone-3 in human urine samples based on an ionic liquid as extractant phase in single-drop microextraction prior to liquid chromatography analysis

Benzophenone-3 (BZ3), one of the compounds most commonly used as UV filter in cosmetic products, can be absorbed through the skin into the human body, since it can be found at trace levels in urine from users of cosmetic products that contain BZ3. Moreover, different undesirable effects have been attributed to this compound. Thus, sensitive analytical methods to monitor urinary excretion of this compound should be developed. This paper presents a selective and sensitive methodology for BZ3 determination at ultratrace levels in human urine samples. The methodology is based on a novel microextraction technique, known as single-drop microextraction (SDME). An ionic liquid (IL) has been used as extractant phase instead of an organic solvent. After the microextraction process, the extractant phase was injected into a liquid chromatography system. The variables of interest in the SDME process were optimized using a multivariate optimization approach. A Plackett-Burman design for screening and a circumscribed central composite design for optimizing the significant variables were applied. Ionic strength, extraction time, stirring speed, pH, ionic liquid type, drop volume and sample volume were the variables studied. The optimum experimental conditions found were: sodium chloride concentration, 13% (w/v); extraction time, 25 min; stirring speed, 900 rpm; pH, 2; ionic liquid type, 1-hexyl-3-methylimidazolium hexafluorophosphate ([C(6)MIM][PF(6)]); drop volume, 5 microL; and sample volume, 10 mL. The proposed method requires a standard addition calibration approach, and it has been successfully employed to determine free BZ3 in urine samples coming from human volunteers who applied a sunscreen cosmetic containing this UV filter. The limit of detection was in the order of 1.3 ng mL(-1) and repeatability of the method, expressed as relative standard deviation, was 6% (n=8).     

液相色谱-串联质谱法测定尿液中的内源性类固醇激素

建立了液相色谱-串联质谱(LC-MS/MS)测定尿液中的内源性类固醇激素的方法。尿样经葡萄糖醛酸甙酶酶解后进行液-液提取,以甲醇-0.1%甲酸缓冲液(含0.02 mol/L乙酸铵)(体积比为68:32)为流动相,采用Cosmosil C18色谱柱分离,并以三重四极杆串联质谱多反应监测扫描方式对尿样中的脱氢表雄酮(DHEA)、睾酮、表睾酮、雄酮和苯胆烷醇酮等5种激素进行检测。方法的最低检出限为0.01～10 ng/mL,平均回收率为96.7%～106.5%,日内和日间相对标准偏差(RSD)分别小于7%和11%。应用所建立的方法测定了健康志愿者口服DHEA后尿液中内源性类固醇激素的变化情况,结果表明该方法样品处理简便,色谱分离完全,结果准确可靠,可替代气相色谱-质谱法用于体液中内源性类固醇激素兴奋剂的常规分析。     

Development and validation of a magnetic solid‐phase extraction with high‐performance liquid chromatography method for the simultaneous determination of amphetamine and methadone in urine

The simultaneous determination of amphetamine and methadone was carried out by magnetic graphene oxide nanoparticles, a magnetic solid‐phase extraction adsorbent, as a new sample treatment technique. The main factors (the amounts of sample volume, amount of adsorbent, type and amount of extraction organic solvent, time of extraction and desorption, pH, the ionic strength of extraction medium, and agitation rate) influencing the extraction efficiency were investigated and optimized. Under the optimized conditions, good linearity was observed in the range of 100–1500 ng/mL for amphetamine and 100–1000 ng/mL for methadone. The method was evaluated for determination of AM and methadone in positive urine samples, satisfactory results were obtained, therefore magnetic solid‐phase extraction can be applied as a novel method for the determination of drugs of abuse in forensic laboratories.     

Determination of ecgonine and seven other cocaine metabolites in human urine and whole blood by ultra-high-pressure liquid chromatography–quadrupole time-of-flight mass spectrometry

Ecgonine is suggested to be a promising marker of cocaine (COC) ingestion. A combined mass spectrometry (MS) and tandem MS (MS/MS) method was developed to simultaneously determine ecgonine and seven other metabolites of cocaine in human urine and whole blood with ultra-high-pressure liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. The compounds were extracted from as little as 100 μL of sample by solid-phase extraction with a 96-well μElution solid-phase extraction plate. The protonated molecules or fragment ions at accurate mass acquired in MS mode were used to quantify specific analytes, following by dedicated MS/MS identification. The assay was linear in the range from 5 to 50-100 ng/mL for urine samples, except for ecgonine methyl ester (10-200 ng/mL) and ecgonine (40-400 ng/mL), and was linear from 1-2 to 50 ng/mL for whole blood samples, except for ecgonine methyl ester (20-1,000 ng/mL) and ecgonine (40-2,000 ng/mL). The correlation coefficients were all greater than 0.99. The limits of detection ranged from 0.2 to 16 ng/mL, and the lower limits of quantification ranged from 1 to 40 ng/mL. The repeatability and intermediate precision were 18.1 % or less. The accuracy was in the range from 80.0 to 122.9 %, process efficiencies were in the range from 8.6 to 177.4 %, matrix effects were in the range from 28.7 to 171.0 %, and extraction recoveries were in the range from 41.0 to 114.3 %, except for ecgonine (12.8 % and 9.3 % at low and high concentrations, respectively). This method was highly sensitive in comparison with previously published methods. The validated method was successfully applied to the analysis of real samples derived from forensic cases, and the results verified that, on the basis of data from four positive samples, ecgonine is a promising marker of cocaine ingestion.

Quantitative determination of the loop diuretic bumetanide in urine and pharmaceuticals by high-performance liquid chromatography with amperometric detection.

A high-performance liquid chromatographic method with amperometric detection has been developed for the determination of the diuretic bumetanide using a microBondapak C18 column. The mobile phase consists of a 50:50 acetonitrile-water mixture containing 5mM KH2PO4-K2HPO4 (pH 4.0). The compound is monitored at +1350 mV with an amperometric detector equipped with a glassy carbon working electrode. A liquid-liquid or solid-liquid extraction is done prior to chromatographic analysis in order to avoid the interferences found in the urine matrix. The percentages of recovery obtained are 71%+/-1% for liquid-liquid extraction and 84.2%+/-0.7% for solid-liquid extraction. The method developed has a linear concentration range from 50 to 499 ng/mL with a reproducibility in terms of relative standard deviation of 1.73% and 3.85% for a concentration level of 70 ng/mL and 237 ng/mL, respectively, and a detection limit of 0.25 ng/mL (3:1 signal-to-noise ratio). The method is applied to the determination of bumetanide in pharmaceutical formulations and urine obtained from hypertensive patients and healthy volunteers after the ingestion of a therapeutic dose of Fordiuran (1 mg bumetanide).