Detection of N-acetylcysteine, cysteine and their disulfides in urine by liquid chromatography with a dual-electrode amperometric detector

A method for the determination of N-acetylcysteine, cysteine and their disulfides in urine is described. The thiols and disulfides are separated by reversed-phase ion-pair chromatography with octyl sodium sulfate as the ion-pairing reagent and detected with a dual-electrode amperometric detector using Au/Hg amalgam electrodes. Both the thiols and disulfides are detected with this system. In addition, dimers and mixed disulfides can be detected individually.     

Synthesis and liquid chromatography/tandem mass spectrometric characterization of the adducts of bisphenol A o-quinone with glutathione and nucleotide monophosphates

An environmental, estrogen-like substance, bisphenol A (BPA), is the monomer for the production of polycarbonate plastics used in baby bottles, dental sealants, and as a major component of epoxy resin for the lining of food cans. The oxidation of BPA leads to the reactive electrophilic BPA-o-3,4-quinone (BPA-Q), which can damage DNA and may be implicated in cancer initiation. BPA-Q reacts in vitro with 2'-deoxyguanosine 5'-phosphate (dGMP) and 2'-deoxyadenosine 5'-phosphate (dAMP) but not with 2'-deoxycytidine-5'-phosphate and 2'-deoxythymidine 5'-phosphate. In aqueous acetic acid, BPA-Q also reacts with 2'-deoxyguanosine (dG) and 2'-deoxyadenosine (dA) but not with 2'-deoxycytidine and 2'-deoxythymidine. The reactions are accompanied by loss of the modified base (depurination). We determined the structures of the modified bases by primarily tandem mass spectrometry. In mixtures of deoxynuclesides and deoxynucletides treated with BPA-Q, reactions occur more readily with dGMP/dG followed by dAMP/dA. With calf thymus DNA, significant apurinic sites must be produced because we detected the BPA-Q-guanosine adduct in the incubation mixture. We also found that BPA-Q reacts readily with glutathione (GSH) under acidic or neutral conditions, and we characterized the BPA-Q-GSH conjugate with tandem mass spectrometry (MS/MS). The results are consistent with a mechanism of carcinogenesis whereby BPA-Q, formed in vivo and not adequately detoxified by reactions with GSH, reacts with DNA, causing depurination. The adducts reported will also be appropriate references for identification of BPA-Q adducts in environmental and biological systems.     

Determination of reduced and oxidized glutathione in biological samples using liquid chromatography with fluorimetric detection

A HPLC method for determination of both reduced (GSH) and oxidized (GSSG) glutathione in plasma, whole blood and rat hepatocytes has been developed and evaluated. Reduced glutathione reacts with orthophthaldehyde (OPA) to form a stable, highly fluorescent tricyclic derivate at pH 8, while GSSG reacts with OPA at pH 12. At measurement of GSSG, GSH was complexed to N-ethylmaleimide. For the separation, reverse phase column Discovery C(18), 150 mm x 4 mm, 5 microm, was used. The mixture of methanol and 25 mM sodium hydrogenphosphate (15:85, v/v), pH 6.0, was used as mobile phase. The analytical performance of this method is satisfactory for both GSH and GSSG. The intra-assay coefficients of variation were 1.8 and 2.1% for whole blood, 2.0 and 1.9% for rat hepatocytes, 4.3 and 5.2% for plasma. The inter-assay coefficients of variation were 5.8 and 6.2% for whole blood, 6.6 and 7.1% for rat hepatocytes, 6.9 and 7.8% for plasma. The recoveries were as follows: 98.2% (CV 3.5%) and 101.5% (CV 4.2%) for whole blood, 99.1% (2.5%) and 102.3 (4.4%) for rat hepatocytes, 94.1% (CV 7.5%) and 103.5 (CV 8.5%) for plasma. The calibration curve was linear in the whole range tested. The limit of detection was 14.0 and 5.6 fmol, respectively. The preliminary reference ranges of reduced and oxidized glutathione in a group of blood donors are (4.69+/-0.93) and (0.28+/-0.12)micromol/gHb for whole blood, (1.82+/-0.55) and (0.154+/-0.044)microM for plasma.     

Comparison of fast liquid chromatography/tandem mass spectrometric methods for simultaneous determination of cladribine and clofarabine in mouse plasma

Several fast high performance liquid chromatography/atmospheric pressure ionization/tandem mass spectrometric (HPLC-API/MS/MS) methods were evaluated for the simultaneous determination of cladribine and clofarabine in mouse plasma samples. The chemical separation for analytes under reversed-phase conditions were achieved by using either ultra-performance liquid chromatography (UPLC) or micro-column HPLC coupled to either a quadrupole linear ion trap mass spectrometer (QTrap MS) or a triple quadrupole mass spectrometer. Atmospheric pressure chemical ionization (APCI) or atmospheric pressure photoionization (APPI) interfaces in the positive mode were employed prior to mass spectrometric detection. The effects of various dopant solvents on the APPI sensitivities of analytes and the internal standard were investigated. The matrix ionization suppression potential for the test compounds in plasma samples on fast HPLC-MS/MS methods was examined by a post-column infusion technique. In this work, these proposed approaches were successfully employed to determine the concentrations of cladribine and clofarabine in mouse plasma in the low ng/ml region. The mouse plasma levels of all analytes obtained by these fast HPLC-MS/MS methods were compared and found to be well correlated in terms of analytical accuracy.     

Ultraperformance liquid chromatography tandem mass spectrometric method for direct quantification of salbutamol in urine samples in doping control

A fast and reliable quantitative method for salbutamol using direct analysis of the urine sample by ultraperformance liquid chromatography tandem mass spectrometry (UPLC/MS/MS) has been developed. Urine samples were spiked with salbutamol-d₆ (internal standard), and, then, they were diluted with ultrapure water (1:1, v/v). Aliquots of 1 μl of the mixture were directly analyzed by UPLC/MS/MS. The chromatographic separation was performed in a UPLC BEH C₁₈ (100 mm × 2.1 mm, 1.7 μm) column with a mobile phase contained 0.01% formic acid in ultrapure water (v/v) and 0.01% formic acid in acetonitrile (v/v), using gradient elution at 0.6 ml/min. The temperature of the column was set to 45 °C. The total run time was 3.2 min. Electrospray ionization in positive ion mode was used under multiple reaction monitoring (MRM) at different collision energies. Nitrogen and argon were used as desolvation and collision gas, respectively. The method was shown to be linear from 200 to 5000 ng/ml (r² > 0.99). The limit of quantitation was estimated in 200 ng/ml. Intra-assay precision and accuracies, evaluated by using quality control samples containing 550 and 1100 ng/ml salbutamol, were always better than 8.4%. The intermediate precision was estimated to be in the range of 5.6–8.9%. The method was shown to be reliable when applying to routine samples, and the short analysis time resulting from a simple sample preparation and a fast instrumental analysis makes it of great interest for antidoping control purposes.     

A liquid chromatography/tandem mass spectrometry assay to quantitate MS-275 in human plasma

A rapid, sensitive and selective method was developed and validated using LC/MS/MS for determination of MS-275 in human plasma. Sample preparation involved a single step liquid-liquid extraction by the addition of 0.2 ml of plasma with 5 ml acetonitrile/n-butyl-chloride. Separation of the compounds of interest, including the internal standard paclitaxel, was achieved on a Waters X-Terra C(18) (50 mm x 2.1mm i.d., 3.5 microm) analytical column using a mobile phase consisting of acetonitrile/ammonium acetate (pH 2.9; 2mM)(60:40, v/v) containing 0.1% formic acid and isocratic flow at 0.15 ml/min for 3 min. The analytes were monitored by tandem-mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the range of 0.5-100 ng/ml with values for the coefficient of determination of >0.99. The values for both within day and between day precision and accuracy were well within the generally accepted criteria for analytical methods (<15%). This method was subsequently used to measure concentrations of MS-275 in cancer patients receiving an oral weekly dose of 4 mg/m(2).     

Fully-automated assay by liquid chromatography for routine drug monitoring in body fluids--method development with biological samples

A fully-automated liquid chromatograph based on alternated pre-column enrichment technique has been developed for routine drug monitoring of body fluids using a column switching technique. Advantages of the method include the facility of direct injection of the body fluid onto the chromatograph and the simultaneous detection of both polar and non-polar metabolites under isocratic or gradient elution conditions.     

Determination of glutathione in biological material by high pressure liquid chromatography

A rapid and selective determination of reduced glutathione in biological material is described, based on its conjugation with 1-chloro-2,4-dinitrobenzene. The reaction product has aUv absorption maximum at 340 nm and is analysed by reversed phase high pressure liquid chromatography. Linear calibration graphs were obtained in the concentration range between 50μ M and 2 mM glutathione in standard solutions and in biological material (rat liver and bacterial homogenates). The detection limit is about 2μM glutathione when using 20μl injection samples.     

A liquid chromatography/mass spectrometric method for simultaneous analysis of arachidonic acid and its endogenous eicosanoid metabolites prostaglandins, dihydroxyeicosatrienoic acids, hydroxyeicosatetraenoic acids, and epoxyeicosatrienoic acids in rat brain tissue

A sensitive, specific, and robust liquid chromatography/mass spectrometric (LC/MS) method was developed and validated that allows simultaneous analysis of arachidonic acid (AA) and its cyclooxygenase, cytochrome P450, and lipoxygenase pathway metabolites prostaglandins (PGs), dihydroxyeicosatrienoic acids (DiHETrEs), hydroxyeicosatetraenoic acids (HETEs) and epoxyeicosatrienoic acids (EETs), including PGF(2alpha), PGE(2), PGD(2), PGJ(2), 14,15-DiHETrE, 11,12-DiHETrE, 8,9-DiHETrE, 5,6-DiHETrE, 20-HETE, 15-HETE, 12-HETE, 9-HETE, 8-HETE, 5-HETE, 14,15-EET, 11,12-EET, 8,9-EET, and 5,6-EET in rat brain tissues. Deuterium labeled PGF(2alpha)-d(4), PGD(2)-d(4), 15(S)-HETE-d(8), 14,15-EET-d(8), 11,12-EET-d(8), 8,9-EET-d(8), and AA-d(8) were used as internal standards. Solid phase extraction was used for sample preparation. A gradient LC/MS method using a C18 column and electrospray ionization source under negative ion mode was optimized for the best sensitivity and separation within 35 min. The method validation, including LC/MS instrument qualification, specificity, calibration model, accuracy, precision (without brain matrix and with brain matrix), and extraction efficiency were performed. The linear ranges of the calibration curves were 2-1000 pg for PGs, DiHETrEs, HETEs, and EETs, 10-2400 pg for PGE(2) and PGD(2), and 20-2000 ng for AA, respectively.     

Metabolism study and toxicological determination of mephtetramine in biological samples by liquid chromatography coupled with high-resolution mass spectrometry

The emerging market of new psychoactive substances (NPSs) is a global-scale phenomenon, and their identification in biological samples is challenging because of the lack of information about their metabolism and pharmacokinetic. In this study, we performed in silico metabolic pathway prediction and in vivo metabolism experiments, in order to identify the main metabolites of mephtetramine (MTTA), an NPS found in seizures since 2013. MetaSite™ software was used for in silico metabolism predictions and subsequently the presence of metabolites in the blood, urine, and hair of mice after MTTA administration was verified. The biological samples were analyzed by liquid chromatography coupled with high-resolution mass spectrometry (LC–HRMS) using a benchtop Orbitrap instrument. This confirmed the concordance between software prediction and experimental results in biological samples. The metabolites were identified by their accurate masses and fragmentation patterns. LC–HRMS analysis identified the dehydrogenated and demethylated-dehydrogenated metabolites, together with unmodified MTTA in the blood samples. Besides unmodified MTTA, 10 main metabolites were detected in urine. In hair samples, only demethyl MTTA was detected along with MTTA. The combination of Metasite™ prediction and in vivo experiment was a powerful tool for studying MTTA metabolism. This approach enabled the development of the analytical method for the detection of MTTA and its main metabolites in biological samples. The development of analytical methods for the identification of new drugs and their main metabolites is extremely useful for the detection of NPS in biological specimens. Indeed, high throughput methods are precious to uncover the actual extent of use of NPS and their toxicity.     

Efficient synthesis, liquid chromatography purification, and tandem mass spectrometric characterization of estrogen-modified DNA bases

Estrogens are metabolized to active quinones that modify DNA and may lead to various cancers. To extend the analytical methodology for estrogen-modified purine bases, we report here a simple modification to existing synthetic procedures that use 2-iodoxybenzoic acid (IBX) as the oxidizing agent for the reference material and putative biomarker, 4-hydroxyestrone-1-N3adenine (4-OH-E1-1-N3Ade). The reaction leads to two catechol estrogen quinones, CE1-2,3-Q and CE1-3,4-Q, both of which react via Michael additions to afford 4-OH-E1-1-N3Ade and other DNA adducts. Liquid chromatography separation permits the isolation of high-purity 4-OH-E1-1-N3Ade. With this method, we also prepared single 13C and uniformly 15N (U-15N) labeled 4-OH-E1-1-N3Ade with 8-13C-labeled Ade and U-15N-labeled adenosine 5'-monophosphate (AMP). The approach is also effective for the synthesis of 4-hydroxyestradiol-1-N3adenine, 4-OH-E2-1-N3Ade, and 4-hydroxyestrone(estradiol)-1-N7guanine, 4-OH-E1(E2)-1-N7Gua. The tandem mass spectra (MS2 and MS3) of 4-OH-E1-(unlabeled, 8-13C-, and U-15N-labeled)1-N3Ade and accurate mass measurements for MS2 product ions allow us to assign unambiguously the formula of fragments and delineate the fragmentation pathways. One important reaction is dehydration, which occurs at the ketone oxygen in the C-17 position of estrone. Another is loss of NH3, an ubiquitous process for purines and modified purines, which is affected by the steroid modification. Evidence from MS/MS supports the migration of H-atom(s) from estrone in the loss of NH3. An interesting interaction occurs between the steroid and the Ade in the modified base, promoting loss of CH2NH, a loss that distinguishes modified Ade from unmodified Ade. The synthesis of a stable isotope-labeled 4-OH-E1-1-N3Ade and the understanding of the fragmentation processes will enable studies aimed at the etection of naturally occurring 4-OH-E1-1-N3Ade in biological samples.     

Supercritical fluid chromatography/tandem mass spectrometric method for analysis of pharmaceutical compounds in metabolic stability samples

Packed-column supercritical fluid chromatography (pSFC) coupled to an atmospheric pressure chemical ionization (APCI) source and a tandem mass spectrometer (MS/MS) for rapid and simultaneous determination of clozapine, ondansetron, tolbutamide and primidone in in vitro samples was developed in support of metabolic stability experiments. The effects of the eluent flow-rate and composition as well as the nebulizer temperatures on the ionization efficiency of the analytes in positive ion mode under normal phase pSFC conditions were studied. The metabolic stability of the test drug components through microsomal incubation by the proposed pSFC-APCI/MS/MS approaches requiring approximately 1 min per samples were evaluated with respect to specificity, durability and accuracy. These metabolic stability results obtained by pSFC-MS/MS methods are in a good agreement with those obtained by fast high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) method.     

The determination of nadolol in biological samples using high-performance liquid chromatography

A method has been developed for the determination of nadolol in biological samples by reversed-phase high-performance liquid chromatography with fluorimetric detection. The method has been applied to plasma, serum and urine samples, which are prepared by extraction with diethyl ether-dichloromethane (5:2,v/v), evaporation of the organic solvent, and dissolution of the resultant residue in the chromatographic eluent. The sample is then subjected to chromatography on a C(18)-silica column, with an eluent of water-acetonitrile-triethylamine (800:200:1,v/v) adjusted to pH 3.0 with orthophosphoric acid. A single point external standard is used for quantitation. The working ranges were 1-400 ng/ml for plasma/serum, and 0.1-40 mug/ml for urine, although a detection limit of 0.1 ng/ml appears to be readily attainable. The sample size was 0.5 ml, and for both types of sample the method showed good correlation with a previously published fluorimetric method (for plasma, r = 0.9544, n = 70; for urine, r = 0.9919, n = 35).     

Measurement of brimonidine concentrations in human plasma by a highly sensitive gas chromatography/mass spectrometric assay

Brimonidine is an α₂-adrenergic agonist that is efficacious in lowering intraocular pressure in humans. A highly sensitive and selective gas chromatography/mass spectrometry (GC/MS) assay is described for quantitation of brimonidine in human plasma following ocular installation. Brimonidine in 1 ml of plasma was extracted together with tetradeuterated brimonidine (internal standard) and clonidine (carrier) by solvent extraction. After solvent evaporation, 3,5-bis(trifluoromethyl)benzoyl derivatives were formed and injected onto a GC/MS appartus under negative chemical ionization conditions. The ions monitored for derivatized brimonidine and tetradeuterated brimonidine were m/z 691 [M - HBr] and m/z 694 [M - DBr], respectively. Calibration curves were linear from 2 to 1000 pg ml⁻¹ (r² = 0.981-0.996). The method was specific for brimonidine relative to endogenous compounds in plasma. The inter-day relative standard deviation for analysis of quality controls was 12% or less, and the inter-day assay accuracy ranged from 97 to 104% of nominals. The GC/MS assay showed adequate sensitivity for analysis of human samples from volunteers ocularly dosed with 0.5% brimonidine tartrate solution. Overall, the GC/MS assay showed excellent precision and accuracy, and a minimum quantifiable concentration of 2 pg ml⁻¹.     

A simple high-performance liquid chromatography assay for simultaneous measurement of adenosine, guanosine, and the oxypurine metabolites in plasma

To study the effect of pharmacologic agents on the biologic fate of adenosine, a reversed-phase high-performance liquid chromatography (HPLC) assay coupled with a solid-phase extraction (SPE) method was developed for simultaneous determination of plasma adenosine, hypoxanthine, xanthine, inosine, guanosine, and uric acid. The HPLC system consisted of a reversed phase C18 column, UV detector set at 254 nm, and a mobile phase composed of 0.01 M ammonium phosphate: methanol (9.5 : 0.5) vol/vol with the final pH adjusted to 3.9. The standard curves were linear between 0.1-2 microg/mL for all the analytes (except uric acid 50-400 microg/mL), with r2 > 0.99. The absolute recoveries were >60% and accuracy >85% in almost all cases. The limit of detection was <1 ng based on absolute injection of the analytes. The intraassay variations were <10% and interassay variations <15%. The presence of a wide range of medications in plasma samples did not interfere with the assay. The assay was applied successfully to measure plasma adenosine and the oxypurine metabolites in humans and rats. It was noted that plasma concentrations of adenosine and the oxypurine metabolites can vary considerably depending on the method of blood sample collection, and that species differences are apparent.     

Fast liquid chromatographic-tandem mass spectrometric (LC-MS-MS) determination of metformin in plasma samples

Liquid chromatographic–tandem mass spectrometric (LC–MS–MS) methods for the determination of metformin in plasma from different species are presented. The first method employed a YMC cyano 2 mm×50 mm, 3 μm analytical column. For minimum sample preparation direct injection of samples after protein precipitation was performed. The polar column used with highly organic mobile phases provided a normal phase retention mechanism. The elution conditions were optimized to obtain reproducible peak areas and good peak shape. A step gradient from 100% acetonitrile to acetonitrile–water 80:20 (v/v) containing 10 mM ammonium acetate and 1% acetic acid was applied, leading to a sample-to-sample cycle time of 2 min. In a second method, a column-switching LC–MS–MS assay for on-line trapping was developed. The analyte and internal standard were trapped on a YMC cyano 2 mm×10 mm, 5 μm column using acetonitrile–methanol 95:5 (v/v). Elution was performed isocratically in back-flush mode on to the analytical column (YMC cyano 2 mm×50 mm, 3 μm) using 10 mM ammonium acetate in acetonitrile–water 80:20 (v/v) with 1% formic acid. With this approach, the signal-to-noise ratio was improved and the run time could be shortened to 1 min. Calibration samples were prepared in the matrix to be assayed in the range of 10–10,000 ng/ml. Quality control (QC) samples were prepared at 40, 400 and 4000 ng/ml and interspersed with the unknown study samples in the assays. Deviations for precision and accuracy were less than 20% for the lower limit of quantification (LLOQ) and low QC sample and less than 15% for other calibrators and QCs.     

Determination of malondialdehyde in biological samples by solid-phase extraction and high-performance liquid chromatography

An analytical procedure for determination of malondialdehyde in tissue homogenates and blood serum was developed. A reaction with 2,4-dinitrophenylhydrazine is used followed by cleaning up of the derivative by solid-phase extraction. The samples were analyzed by isocratic high-performance liquid chromatography (HPLC) using a narrow-bore HPLC-column. A good separation of the 1-pyrazole peak from that of 2,4-dinitrophenylhydrazine was observed. A high linear dependence was established by the concentration of 1-pyrazole in the range of 10-5000 ng/ml. The detection limit of the method applied for tissue homogenates and blood serum was approximately 10 ng/ml or lower, and RSD of the method was 9% (n = 8). The peak of 1-pyrazole for these samples was well separated from the other matrix peaks. Experiments carried out evaluated that the solid-phase extraction might be an effective step of the sample preparation, significantly increasing the selectivity of the analysis and the life-time of the column. The method seems to be applicable for determination of malondialdehyde in different biological samples.     

同时定量检测水胺硫磷和水胺氧磷的液相色谱-串联质谱法及应用

目的建立同时测定大鼠全血中水胺硫磷及其活性代谢产物水胺氧磷的液相色谱-串联质谱（LC-MS/MS）分析方法,用于毒代动力学研究中血药浓度的测定。方法采集大鼠全血样品后即刻用4倍体积冰冷乙腈沉淀蛋白。待测物用Zorbax SB C18柱（2.1 mm×50 mm,1.8μm）分离,以含有0.1%甲酸的水和甲醇为流动相系统进行梯度洗脱。采用ESI源正离子多反应监测模式分析,水胺硫磷、水胺氧磷和内标三唑磷的定量离子对分别为m/z 231→121、274→215和314→162。结果水胺硫磷和水胺氧磷在5μg/L～5 mg/L浓度范围内线性良好,最低定量限为5μg/L,回收率在63.1%～77.8%的范围内,准确度和精密度符合生物样品的检测要求。大鼠静脉注射3 mg/kg水胺硫磷后,水胺硫磷在血中很快下降,消除半衰期为22.2 min。活性代谢产物水胺氧磷的生成也较快,10～15 min达到（257.91±42.00）ng/ml的平均峰值,消除半衰期为17.6 min。水胺硫磷和水胺氧磷的AUC0~t分别为（528.62±49.15）和（191.11±33.23）h.μg/L。结论本研究首次建立了同时定量测定大鼠全血中水胺硫磷和水胺氧磷的LC-MS/MS分析方法,方法的专属性好、灵敏度高,适用于水胺硫磷及水胺氧磷在大鼠体内的毒物代谢动力学研究。     

Determination of ciprofloxacin in biological samples by reversed-phase high performance liquid chromatography

Previously reported high performance liquid chromatography (HPLC) assays for ciprofloxacin have used cumbersome fluorescence detection. UV absorbance is more commonly used for assay of antibiotics. Separation of ciprofloxacin and nalidixic acid (internal standard) was achieved using UV absorption at 313 nm, and a reversed phase C-18 Nova-Pak column. The mobile phase consisted of 35% phosphate buffers adjusted to pH 7.4, 65% methanol, and 5.5 mM hexadecyltrimethylammonium bromide. Retention times were 4.3 and 7.3 min, respectively, for ciprofloxacin and nalidixic acid. Serum sample preparation involved protein precipitation with acetonitrile (1:2), followed by methylene chloride and 2-propanol extraction (90:10). After evaporation, reconstitution with a minimal volume of mobile phase allowed for 5X concentration of the sample. The sensitivity limit of the assay was 0.06 microgram/ml. The response was linear from 0.125 to 10.0 micrograms/ml (r greater than 0.999). The coefficient of variation for day-to-day analysis was less than 5.3%, and the recovery was 55%. When compared with microbiological assay in serum, the correlation coefficient was 0.922 (n = 58). This HPLC method using UV detection provided comparable results to those obtained by fluorimetry. Data from three pharmacokinetic studies showed this method to be reliable and accurate.     

A simple, sensitive assay for the spermicide nonoxynol-9 in biological fluids by high-performance liquid chromatography

A new high-performance liquid chromatographic technique has been developed to quantitate nonoxynol-9 in serum, urine, and vaginal fluid. The method is rapid, involves minimal sample preparation, and can be used to analyze a large number of biological fluid samples. The assay elutes a single nonoxynol-9 peak with no interfering components. This was accomplished using a 10-microns pelicular packed amine column, a normal-phase solvent system, and fluorescence detection. Nonoxynol-9 levels as low as 0.23 micrograms/mL in urine can be detected.