Determination of dihydroxynaphthalenes in human urine by gas chromatography-mass spectrometry

A gas chromatography-mass spectrometry (GC-MS) method was developed for measuring 1,2-dihydroxynaphthalene (1,2-DHN) and 1,4-dihydroxynaphthalene (1,4-DHN) in urine. The method involves enzymatic digestion of urinary conjugates to release the DHNs which were then analyzed as trimethylsilyl derivatives by GC-MS. For 1,2-DHN and 1,4-DHN, respectively, the assay limits of detection were 0.21 and 0.15 microg/l, the assay limits of quantitation were 0.69 and 0.44 microg/l, and the coefficients of variation were 14.7 and 10.9%. This method was successfully applied to determine urinary levels of 1,2-DHN and 1,4-DHN in coke workers (14 top workers and 13 side-bottom workers) and 21 matching control workers from the steel industry of northern China. The geometric mean (GM) levels of 1,2-DHN were approximately 100 and 30 times higher than those of 1,4-DHN in exposed and control subjects, respectively. The GM levels 1,2-DHN and 1,4-DHN were significantly higher for coke workers (1,2-DHN: top workers--552 microg/l, side-bottom workers--260 microg/l; 1,4-DHN: top workers--3.42 microg/l, side-bottom workers--3.56 microg/l) than for controls (1,2-DHN: 38.8 microg/l; 1,4-DHN: 1.21 microg/l) (por=0.623; p<0.0001). Also, levels of 1,2-DHN were significantly correlated with those of serum albumin adducts of l,2-naphthoquinone (rs=0.492, p=0.0004). These results indicate that 1,2- and 1,4-DHN are good biomarkers for assessment of naphthalene exposure in coke workers. Since the DHNs are precursors of the naphthoquinones, which have been implicated as toxic products of naphthalene metabolism, measurements of urinary DHNs may have toxicological significance.     

Determination of fexofenadine in human plasma and urine by liquid chromatography-mass spectrometry

A sensitive method was developed to determine fexofenadine in human plasma and urine by HPLC-electrospray mass spectrometry with MDL 026042 as internal standard. Extraction was carried out on C18 solid-phase extraction cartridges. The mobile phases used for HPLC were: (A) 12 mM ammonium acetate in water and (B) acetonitrile. Chromatographic separation was achieved on a LUNA CN column (10 cm x 2.0 mm I.D., particle size 3 microm) using a linear gradient from 40% B to 60% B in 10 min. The mass spectrometer was operated in the selected ion monitoring mode using the respective MH+ ions, m/z 502.3 for fexofenadine and m/z 530.3 for the internal standard. The limit of quantification achieved with this method was 0.5 ng/ml in plasma and 1.0 ng in 50 microl of urine. The method described was successfully applied to the determination of fexofenadine in human plasma and urine in pharmacokinetic studies.     

Determination of triethylenetetramine (TETA) and its metabolites in human plasma and urine by liquid chromatography-mass spectrometry (LC-MS)

A liquid chromatography-mass spectrometry (LC-MS) method has been developed to measure triethylenetetramine (TETA) and its metabolites in human samples. We identified two metabolites of TETA, N1-acetyltriethylenetetramine (MAT) and N1,N10-diacetyltriethylenetetramine (DAT), the latter being novel. We further developed this LC-MS method for the measurement of TETA and these metabolites in human plasma and urine in a single injection. Separation of analytes was achieved on a cyano column using 15% acetonitrile, 85% water (18 M Omega), and 0.1% heptafluorobutyric acid as the mobile phase. Simultaneous MS detection was performed at [M+H]+ values of 147, 189, 231 and 245, corresponding to TETA, MAT, DAT, and N1-acetylspermine as the internal standard, respectively. This method was successfully applied to measure TETA, MAT and DAT in plasma and urine of humans receiving oral drug treatment.     

Determination of copper in urine and serum by gas chromatography-mass spectrometry

A stable isotope dilution gas chromatography-mass spectrometry method using enriched 65Cu as an internal standard is described for the determination of Cu in urine and serum. Chelating agents N,N'-ethylenebis-(trifluoroacetylacetoneimine) [H2(enTFA2)] and lithium bis(trifluoroethyl)dithiocarbamate [Li(FDEDTC)] were used and evaluated for memory effect. H2(enTFA2) did not show any appreciable memory effect, whereas Li(FDEDTC) was found to have a strong memory effect. Overall precision of 1.6% was obtained for determining Cu isotope ratios at a 10-ng level using H2(enTFA2). Cu concentrations in the National Institute of Standards and Technology (NIST) reference materials, freeze-dried urine SRM 2670, and human serum SRM 909 determined using the H2(enTFA2) chelating agent were in good agreement with the NIST-certified values. Isotope ratios determined by gas chromatography-mass spectrometry on samples with altered isotopic composition were in good agreement with the inductively coupled plasma-mass spectrometry data.     

Simultaneous determination of bromvalerylurea and allylisopropylacetylurea in human blood and urine by gas chromatography-mass spectrometry

We devised a sensitive and simple method to simultaneously determine bromvalerylurea and allylisopropylacetylurea in human blood and urine by gas chromatography-mass spectrometry. Bromvalerylurea and allylisopropylacetylurea were extracted using an Extrelut column with an internal standard, 2-bromohexanoylurea, followed by derivatization with heptafluorobutyric anhydride. The derivatized extract was submitted to GC-MS analysis of EI-SIM mode. The calibration curves of both compounds were linear in the concentration range from 0.01 to 10 microg/ml in both blood and urine samples. The lower limits of detection of bromvalerylurea and allylisopropylacetylurea were 0.005 and 0.005 microg/ml, respectively. This method proved most useful in accurately identifying these drugs in blood and urine from an autopsied individual.     

Determination of quinalphos in blood and urine by direct solid-phase microextraction combined with gas chromatography-mass spectrometry

A new method based on direct solid-phase microextraction (DI-SPME) followed by gas chromatography-mass spectrometry was developed for the purpose of determining quinalphos in blood and urine. Two types of coated fibre have been assayed and compared: carbowax/divinylbenzene (CW/DVB 65 microm) and polydimethylsiloxane (PDMS 100 microm). The main parameters affecting the SPME process such as temperature, salt addition, pH, stirring and adsorption/desorption time profiles were optimized to enhance the sensitivity of the procedure. The method was developed using only 100 microL of blood and urine. Limits of detection of the method for blood and urine matrices were, respectively, 10 and 2 ng/mL. Linearity was established over concentration ranges from 0.05 to 50 microg/mL for blood, and 0.01 to 50 microg/mL for urine, with regression coefficients ranging between 0.9991 and 0.9999. Intra- and interday precision values were less than 13%, and accuracy was within +/-15% of the nominal concentration for all studied levels in both matrices. Absolute recoveries were 14 and 26% for blood and urine, respectively.     

Determination of ajulemic acid and its glucuronide in human plasma by gas chromatography-mass spectrometry

A method using gas chromatography-mass spectrometry (GC-MS) and solid-phase extraction (SPE) was developed for the determination of ajulemic acid (AJA), a non-psychoactive synthetic cannabinoid with interesting therapeutic potential, in human plasma. When using two calibration graphs, the assay linearity ranged from 10 to 750 ng/ml, and 750 to 3000 ng/ml AJA. The intra- and inter-day precision (R.S.D., %), assessed across the linear ranges of the assay, was between 1.5 and 7.0, and 3.6 and 7.9, respectively. The limit of quantitation (LOQ) was 10 ng/ml. The amount of AJA glucuronide was determined by calculating the difference in the AJA concentration before ("free AJA") and after enzymatic hydrolysis ("total AJA"). The present method was used within a clinical study on 21 patients suffering from neuropathic pain with hyperalgesia and allodynia. For example, plasma levels of 599.4+/-37.2 ng/ml (mean+/-R.S.D., n=9) AJA were obtained for samples taken 2 h after the administration of an oral dose of 20 mg AJA. The mean AJA glucuronide concentration at 2h was 63.8+/-127.9 ng/ml.     

Determination of bromide in whole blood and urine from humans using gas chromatography-mass spectrometry

We devised a sensitive and simple method for determination of bromide in whole blood and urine from humans using gas chromatography-mass spectrometry. Bromide was alkylated with pentafluorobenzyl p-toluenesulphonate in the mixture of acetone and phosphate buffer (pH 6.8). The derivative obtained was analyzed using gas chromatography-mass spectrometry with the positive-ion EI mode. The lower limit of detection for the compound was 1 mg/l. The calibration curve for bromide was linear over the concentration range from 2 to 100 mg/l. With use of this method, levels of bromide in whole blood and urine were determined in cases of poisoning by inhaled brominated hydrocarbons.     

Determination of human plasma levels of levo-alpha-acetylmethadol and its metabolites by gas chromatography-mass spectrometry

A gas chromatography-mass spectrometry (GC-MS) method is presented which allows the simultaneous determination of the plasma concentrations of the levo-alpha-acetylmethadol (LAAM) and of its active metabolites (NorLAAM and DiNorLAAM), after derivatization with the reagent trifluoroacetic anhydride (TFAA). No interferences from endogenous compounds were observed following the extraction of plasma samples from 11 different human subjects. The standard curves were linear over a working range of 5-200ng/ml for the three compounds. Recoveries measured at three concentrations ranged from 47 to 67% for LAAM, from 50 to 69% for NorLAAM and from 28 to 50% for DiNorLAAM. Intra- and interday coefficients of variation determined at three concentrations ranged from 5 to 13% for LAAM, from 3 to 9% for NorLAAM and from 5 to 13% for DiNorLAAM. The limits of quantitation of the method were found to be 4ng/ml for the three compounds. No interference was noted from methadone. This sensitive and specific analytical method could be useful for assessing the in vivo relationship between LAAM's blood levels, clinical efficacy and/or cardiotoxicity     

Quantification of clotiazepam in human plasma by gas chromatography-mass spectrometry

An analytical procedure was developed and validated for the quantification of clotiazepam in human plasma. After subjecting plasma samples to solid-phase extraction, the extract was evaporated and the residue re-constituted. An aliquot of the mixture was injected onto a gas chromatography-mass spectrometry system. The detector response was linear for clotiazepam concentrations in the range of 5-200 ng/ml. Intra- and inter-day precision for the assay over the concentration range was below 13.1 and 13.5%, and the accuracy ranged between 99.0-107.9% and 92.4-101.3%, respectively. The drug was found to be stable under various processing conditions used. The method is applicable to human pharmacokinetic studies of clotiazepam.     

Determination of Delta9-tetrahydrocannabinol from rabbit plasma by gas chromatography-mass spectrometry using two ionization techniques

The purpose of the study was to develop a gas chromatography-mass spectrometric (GC-MS) method for the identification and quantitation of Delta(9)-tetrahydrocannabinol (THC) in rabbit plasma. Two ionization techniques were utilized for GC-MS: electron impact ionization (EI) after i.v. administration and negative chemical ionization (NCI) after sublingual administration. THC was isolated from plasma by solid phase extraction and derivatized by either trimethylsilylation (EI) or trifuoroacetylation (NCI), with deuterated THC as an internal standard. The validity of analytical method was confirmed by investigating selectivity, limit of quantitation, linearity, accuracy, precision, recovery and stability of the analyte. The method proved to be selective, linear, accurate and precise over a range of 10-430 and 0.3-530 ng/ml of THC in plasma for EI and NCI, respectively. The extraction recovery was >81% for each concentration level studied, and the analyte was shown to be stable during storage and sample preparation. The method was applied successfully in analysing THC from rabbit plasma.     

Determination of melagatran, a novel, direct thrombin inhibitor, in human plasma and urine by liquid chromatography-mass spectrometry

Analytical methods for the determination of melagatran (H 319/68) in biological samples by liquid chromatography (LC)-positive electrospray ionization mass spectrometry using multiple reaction monitoring are described. Melagatran in plasma was isolated by solid-phase extraction on octylsilica, either in separate extraction tubes or in 96-well plates. Absolute recovery of melagatran from plasma was >92%. Melagatran and the internal standard, H 319/68 D2 13C2, were separated from other sample components by LC utilizing a C18 stationary phase and a mobile phase comprising 35% acetonitrile and 0.08% formic acid in 0.0013 mol/l ammonium acetate solution. After dilution, urine was injected directly onto the LC column and subjected to gradient LC. The relative standard deviation was 1-5% for concentrations above the limit of quantification, which was estimated for plasma at 10 or 25 nmol/l for sample volumes of 500 or 200 microl, respectively, and 100 nmol/l for urine.     

Determination of haloperidol and its reduced metabolite in human plasma by liquid chromatography-mass spectrometry with electrospray ionization

A sensitive and accurate liquid chromatographic-electrospray mass spectrometric (LC-ES-MS) method for the determination of haloperidol (H) and reduced haloperidol (RH) in human plasma is presented, using chlorohaloperidol as the internal standard. A 2-ml volume of plasma subjected to basic (NaOH) extraction, acid (HCl) back-extraction, acid wash and basic (NaOH) re-extraction. The extraction solvent was hexane-isoamyl alcohol (99:1, v/v) for the whole procedure. A Nucleosil C₁₈ column (150×1 mm) was used for high-performacne liquid chromatography, together with 2 mM HCOONH₄-acetonitrile (55:45, v/v; pH 3.0) as the mobile phase. For each drug, four characteristic ions were monitored. Linearity was assessed in the ranges 0.1–50 and 0.25–50 ng/ml for H and RH, respectively. Recoveries were 58 and 70% and detection limits were 0.075 and 0.100 ng/ml for H and RH, respectively. Correlation coefficients were better than 0.999 for both compounds. R.S.D.s for repeatability and reproducibility at 0.25 ng/ml were 11.1 and 8.5% for H and 9.4 and 11.2% for RH, respectively. One of the main advantages of (LC-ES-MS) over other detection systems is the increase in selectivity obtained by monitoring three ions of confirmation for each of the drugs.     

Determination of the cyanide metabolite 2-aminothiazoline-4-carboxylic acid in urine and plasma by gas chromatography-mass spectrometry

The cyanide metabolite 2-aminothiazoline-4-carboxylic acid (ATCA) is a promising biomarker for cyanide exposure because of its stability and the limitations of direct determination of cyanide and more abundant cyanide metabolites. A simple, sensitive, and specific method based on derivatization and subsequent gas chromatography-mass spectrometry (GC-MS) analysis was developed for the identification and quantification of ATCA in synthetic urine and swine plasma. The urine and plasma samples were spiked with an internal standard (ATCA-d(2)), diluted, and acidified. The resulting solution was subjected to solid phase extraction on a mixed-mode cation exchange column. After elution and evaporation of the solvent, a silylating agent was used to derivatize the ATCA. Quantification of the derivatized ATCA was accomplished on a gas chromatograph with a mass selective detector. The current method produced a coefficient of variation of less than 6% (intra- and interassay) for two sets of quality control (QC) standards and a detection limit of 25 ng/ml. The applicability of the method was evaluated by determination of elevated levels of ATCA in human urine of smokers in relation to non-smokers for both males and females.     

Determination of cyanide and thiocyanate in blood by gas chromatography and gas chromatography-mass spectrometry

We devised a sensitive and simple method for determining cyanide And its major metabolite, thiocyanate, in blood using an extractive alkylation technique. Pentafluorobenzyl bromide was used as the alkylating agent, and tetradecyldimethylbenzylammonium chloride was used as the phase-transfer catalyst. The derivatives obtained were analyzed qualitatively by gas chromatography-mass spectrometry and quantitatively by gas chromatography with an electron-capture detection. The detection limits of cyanide and thiocyanate were 0.01 and 0.003 μmol/ml, respectively, while the gross recovery of both compounds was 80%. The calibration curve was linear over the concentration range from 0.02 to 1.0 μmol/ml for cyanide and from 0.01 to 1.0 μmol/ml for thiocyanate. The accuracy and precision of the method were evaluated, and the coefficients of variation were found to be within 10%. Using this method, the blood levels of two victims who had died from cyanide poisoning were determined.     

Determination of Simvastatin in human plasma by liquid chromatography-mass spectrometry

A simple, sensitive and selective liquid chromatography coupled with electrospray ionization mass spectrometry (LC/ESI/MS) method for the determination of simvastatin (I) has been developed. After extraction by ethyl acetate, using lovastatin (II) as internal standard, solutes are separated on a C(18) column with a mobile phase consisting of methanol-water (9:1). Detection is performed on an atmospheric pressure ionization single quadruple mass spectrometer equipped with an ESI interface and operates in positive ionization mode. Simvastatin quantification was realized by computing peak area ratio (I/II) of the extracts analyzed in SIM mode (m/z: 441 and m/z: 427 for I and II, respectively) and comparing them with calibration curve (r=0.9997). Accuracy and precision for the assay were determined by calculating the intra-batch and inter-batch variation at three concentrations 0.1, 5.0, 10.0 ng/ml; the intra batch relative standard deviation (RSD) was less than 10% and ranged from 1.8 to 8.5%, respectively; the inter-batch RSD was less than 20% and ranged from 4.1 to 16.5%. The limit of detection was 0.05 ng/ml.     

Determination of 2-butoxyethanol and butoxyacetic acid in rat and human blood by gas chromatography-mass spectrometry

A sensitive and selective gas chromatographic-negative-ion chemical ionization mass spectrometric method was developed to simultaneously quantitate 2-butoxyethanol (BE) and butoxyacetic acid (BAA) in rat and human blood at low ng/g levels as pentafluorobenzoyl and pentafluorobenzyl derivatives, respectively. Analysis of ¹³C-labeled analogs of BE and BAA were found to improve the limits of quantitation to below 2 ng/g. Deuterium-labeled BE and BAA were used as internal standards. Calibration curves were generally linear over three orders of magnitude, with limits of quantitation of 16–18 ng/g for both BE and BAA, and 1.5 and 0.4 ng/g for [¹³C₂]BE and [¹³C₂]BAA, respectively, in human blood. Linearity in rat blood was similar, with limits of quantitation of 22 ng/g for BE and 5 ng/g for BAA. This method was developed for the support of mammalian metabolism studies and human biomonitoring studies involving exposure to BE or [¹³C₂]BE.     

Quantification of 3-hydroxyglutaric acid in urine,plasma, cerebrospinal fluid and amniotic fluid by stable-isotope dilution negative chemical ionization gas chromatography-mass spectrometry

This paper describes a stable isotope dilution method for quantification of 3-hydroxyglutaric acid (3-HGA) in body fluids. The method comprises a solid-phase extraction procedure, followed by gas chromatographic separation and negative chemical ionization mass spectrometric detection. This method is selective and sensitive, and enables measurement of 3-HGA concentrations in urine-, plasma-, and CSF- samples of controls. The control ranges for 3-HGA were: urine 0.88-4.5 mmol/mol creatinine (n=12); plasma 0.018-0.10 micro mol/l (n=10), CSF 0.022-0.067 micro mol/l (n=10). We applied this method to measure 3-HGA in body fluids of three patients with glutaric aciduria type I. We also quantified 3-HGA in amniotic fluid of controls (range 0.056-0.11 micro mol/l; n=12) and in two samples from fetuses affected with glutaric aciduria type I.     

Detection of hydroxyethylstarch (HES) in human urine by liquid chromatography-mass spectrometry

The objective of this study was to establish the possibility of using liquid chromatography coupled to mass spectrometry for the detection of hydroxyethylstarch (a corn starch derived product) in urine as an alternative to the current time consuming GC-MS methods. Analyses were performed using an ion trap instrument after acidic hydrolysis. Ionization was carried out using atmospheric pressure chemical ionisation (APCI) operated in negative ionization mode and detection was performed using MS(2). The results indicate that the developed method can successfully be applied as a fast and reliable method for the detection and identification of hydroxyethylstarch.