Sensitive determination of n-hexane and cyclohexane in human body fluids by capillary gas chromatography with cryogenic oven trapping

A sensitive method was developed for determination of n-hexane and cyclohexane in human body fluids by headspace capillary gas chromatography (GC) with cryogenic oven trapping. Whole blood and urine samples containing n-hexane and cyclohexane were heated in a 7.5 mL vial at 70 degrees C for 15 min, and 5 mL of the headspace vapor was drawn into a glass syringe. All vapor was introduced through an injection port of a GC instrument in the splitless mode into an Rtx-Volatiles middle-bore capillary column at an oven temperature of -40 degrees C for trapping volatile compounds. The oven temperature was programmed to 180 degrees C for GC with flame ionization detection. These conditions gave sharp peaks for both n-hexane and cyclohexane, a good separation of each peak, and low background impurities for whole blood and urine. The extraction efficiencies of n-hexane and cyclohexane were 13.2-30.3% for whole blood and 12.7-20.7% for urine. The coefficients of within-day variation in terms of extraction efficiency of both compounds were 5.0-9.5% for whole blood and 3.8-10.8% for urine; those of day-to-day variation for the compounds were not greater than 16.6%. The regression equations for n-hexane and cyclohexane showed good linearity in the range of 5-500 ng/0.5 mL for whole blood and urine. The detection limits (signal-to-noise ratio = 3) for both compounds were 1.2 and 0.5 ng/0.5 mL for whole blood and urine, respectively. The data on n-hexane or cyclohexane in rat blood after inhalation of each compound are also presented.     

Determination of rare earth elements in urine by electrothermal vaporization inductively coupled plasma mass spectrometry

A method was developed for the determination of rare earth elements (REEs) in urine with electrothermal vaporization inductively coupled plasma mass spectrometry (ETV-ICPMS). The undiluted sample was directly injected into the graphite tube and trifluoromethane (Freon-23) was used as chemical modifier in order to reduce the vaporization temperature and the memory effect of most of the lanthanides. The detection limits were in the range 1-10 ng/L with relative standard deviation of 3-5% at concentration levels of 1microg/L, and less than 10-15% at 100 ng/L. Two different procedures, external calibration and a standard additions method, were evaluated to measure the concentration levels of lanthanides in the urine samples and the second procedure was considered to be the best choice for calibration in this work. The level of REEs in urine of 50 healthy volunteers was in the range 5-20 ng/L, above the detection limit of ETV-ICPMS.     

Poly(methacrylic acid-ethylene glycol dimethacrylate) monolith in-tube solid phase microextraction coupled to high performance liquid chromatography and analysis of amphetamines in urine samples

In-tube solid-phase microextraction (SPME) based on a poly(methacrylic acid-ethylene glycol dimethacrylate) monolithic capillary column was investigated for the extraction of amphetamine, methamphetamine and their methylenedioxy derivatives. The monolithic capillary column showed high extraction efficiency towards target analytes, which could be attributed to its larger loading amount of extraction phase than conventional open-tubular extraction capillaries and the convective mass transfer procedure provided by its monolithic structure. The extraction mechanism was studied, and the results indicated that the extraction process of the target analytes was involved with hydrophobic interaction and ion-exchange interaction. The polymer monolith in-tube SPME-HPLC system with UV detection was successfully applied to the determination of amphetamine, methamphetamine and their methylenedioxy derivatives in urine samples, yielding the detection limits of 1.4 - 4.0 ng/mL. Excellent method reproducibility (RSD < 2.9%) was found over a linear range of 0.05-5 microg/mL, and the time for the whole analysis was only approximately 25 min. The monolithic capillary column was reusable in coping with the complicated urine samples.     

Sensitive analysis of alkyl alcohols as decomposition products of alkyl nitrites in human whole blood and urine by headspace capillary GC with cryogenic oven trapping

The abuse of alkyl nitrites is becoming a serious social problem worldwide. In this report, a simple and sensitive method is presented for the determination of n-butyl alcohol, isobutyl alcohol, and isoamyl alcohol as decomposition products of alkyl nitrites in human whole blood and urine samples using capillary gas chromatography (GC) with cryogenic oven trapping. After heating a whole blood or urine sample containing each alkyl alcohol and t-butyl alcohol [the internal standard (IS)] in a 7-mL vial at 55 degrees C for 15 min, 5 mL of the headspace vapor is drawn into a gas-tight syringe and injected into a GC inlet port. The vapor is introduced into an Rtx-BAC2 medium-bore capillary column in the splitless mode at 0 degrees C oven temperature in order to trap the entire analytes, and then the oven temperature is programmed up to 240 degrees C for the GC measurements by flame ionization detection. These conditions give sharp peaks for each compound and the IS and low background noise for whole blood or urine samples. The detection limits of the analytes are 10 ng/mL for whole blood and 5 ng/mL for urine. Linearity and precision are also tested to confirm the reliability of this method. Isobutyl alcohol and methemoglobin could be determined from the whole blood samples of three male volunteers who had sniffed isobutyl nitrite.     

Determination of the new morpholino anthracycline MX2.HCl and its metabolites in biological samples by high-performance liquid chromatography.

Methods for determining concentrations of a new morpholino anthracycline MX2.HCl and its metabolites in biological samples using reversed-phase high-performance liquid chromatography and fluorescence detection are described. The limits of detection were less than 1 ng/ml for all compounds after extraction from 0.5 ml of plasma using C18 Sep-Pak cartridges and consecutive solvent extraction. The recoveries from rat plasma ranged from 72.0 to 89.3%. The peak-height ratio of the fluorescence intensities of these compounds versus internal standard showed a linear correlation for concentrations up to at least 500 ng/ml in the plasma (correlation coefficient r greater than 0.999). The within-day and between-day precisions of this assay were in the range 0.8-8.7% (n = 5) and 2.0-3.5% (n = 5), respectively. The concentrations of these compounds in the blood and urine can be also determined by a slight modification of the extraction procedure.     

Determination of mercapturic acids in urine by solid-phase extraction followed by liquid chromatography-electrospray ionization mass spectrometry

A novel method for the determination of five kinds of mercapturic acids, found in urine as metabolites of alkylbenzenes, based on liquid chromatography-electrospray ionization mass spectrometry is described. A solid-phase extraction procedure was used for the extraction of the mercapturic acids from urine and the separation was performed on a reversed-phase C30 column. The detection limits were in the range 2.4-3.2 ng ml-1.     

Determination of paraquat and diquat in human body fluids by high-performance liquid chromatography/tandem mass spectrometry

Paraquat (PQ) and diquat (DQ) in human whole blood and urine were analyzed by high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) with positive ion electrospray ionization (ESI). The compounds were extracted with Sep-Pak C18 cartridges from whole blood and urine samples containing ethyl paraquat as an internal standard. The separation of PQ and DQ was carried out using ion-pair chromatography with heptafluorobutyric acid in 20 mM ammonium acetate and acetonitrile gradient elution for successful coupling with MS. Both compounds formed base peaks due to [M-H]+ ions by HPLC/ESI-MS and the product ions produced from each [M-H]+ ion by HPLC/MS/MS. Selective reaction monitoring (SRM) showed much higher sensitivity for both body fluids. Therefore, a detailed procedure for the detection of compounds by SRM with HPLC/MS/MS was established and carefully validated. The recoveries of PQ and DQ were 80.8-95.4% for whole blood and 84.2-96.7% for urine. The calibration curves for PQ and DQ showed excellent linearity in the range of 25-400 ng ml(-1) of whole blood and urine. The detection limits were 10 ng ml(-1) for PQ and 5 ng ml(-1) for DQ in both body fluids. The intra- and inter-day precision for both compounds in whole blood and urine samples were not greater than 13.0%. The data obtained from the determination of PQ and DQ in rat blood after oral administration of the compounds are also presented.     

Determination of phenothiazines in human body fluids by solid-phase microextraction and liquid chromatography/tandem mass spectrometry

Eleven phenothiazine derivatives with heavy side-chains were found to be extractable from human whole blood and urine samples by solid-phase microextraction (SPME) with a polyacrylate-coated fiber. The fiber was then injected into the desorption chamber of an SPME-liquid chromatography (LC) interface for LC/tandem mass spectrometry (MS/MS) with positive ion electrospray (ES) ionization. All compounds formed base peaks due to [M + 1](+) ions by LC/ES-MS/MS. By use of LC/ES-MS/MS, the product ions produced from each [M + 1](+) ion showed base peaks due to side-chain liberation. Selected reaction monitoring (SRM) and selected ion monitoring (SIM) were compared for the detection of the 11 phenothiazine derivatives from human whole blood and urine. SRM showed much higher sensitivity than SIM for both types of sample. Therefore, a detailed procedure for the detection of drugs by SRM with SPME-LC/MS/MS was established and carefully validated. The extraction efficiencies of the 11 phenothiazine derivatives spiked into whole blood and urine were 0. 0002-0.12 and 2.6-39.8%, respectively. The regression equations for the 11 phenothiazine derivatives showed excellent linearity with detection limits of 0.2-200 ng ml(-1) for whole blood and 4-22 pg ml(-1) for urine. The intra- and inter-day precisions for whole blood and urine samples were not greater than 15.1%. The data obtained after oral administration of perazine or flupentixol to a male subject are presented.     

Trace enrichment of chlorophenols in human urine by C18 reversed-phase adsorption and by n-hexane extraction

Summary The efficiency and sensitivity of C₁₈ reversed-phase adsorption of free chlorophenols and of n-hexane extraction of either free or acetylated chlorophenols from human urine were compared. All procedures were found to be efficient for the trace enrichment of 2,4-dichlorophenol, 2,4,6- and 2,4,5-trichlorophenols, 2,3,4,6- and 2,3,4,5-tetrachlorophenols and pentachlorophenol. The recoveries of chlorophenols from non-hydrolysed and acid hydrolysed urine samples were comparable. By treatment of 1 ml urine sample detection limits of 1–2 ng/ml were achieved, while the treatment of 5 ml samples enhanced the detection sensitivity to less than 1 ng/ml. The n-hexane extraction of acetylated chlorophenols from 1 ml urine samples is the simplest and fastest procedure because the acetylation and extraction of chlorophenols are performed simultaneously in one step. The C₁₈ adsorption seems to be more suitable than n-hexane extraction for accumulation of chlorophenols from a urine volume of 5 ml and higher because the elution is performed always with the same small volume of acetone. Both C₁₈ adsorption and n-hexane extraction procedures were applied for analysis of chlorophenols in general population and in persons with possible occupational exposure to organochlorine compounds.     

Simultaneous determination of metabolites of trimethylbenzenes,dimethylbenzylmercapturicacid and dimethylhippuric acid,in human urine by solid-phase extraction followed by liquid chromatography tandem mass spectrometry

We describe a novel method for the determination of three kinds of dimethylbenzylmercapturic acids (DMM) and six kinds of dimethylhippuric acids (DMH), found in urine as metabolites of trimethylbenzenes, based on liquid chromatography/electrospray ionization tandem mass spectrometry. A solid-phase extraction procedure was used for the extractions of DMM and DMH from a urine sample, and the separation was performed on a reversed-phase C(30) column. The analytes were ionized by electrospray in the positive-ion mode. Operating in the multiple reaction monitoring mode, the linearity of the relative mass spectrometric responses to the internal standard versus analyte concentrations were established in the range 0.1-100 ng ml(-1). The extraction procedure was rapid and the relative standard deviations were below 5%. The detection limits of DMM and DMH in the urine by the proposed method were in the ranges 0.26-0.41 and 0.42-2.0 ng l(-1), respectively. Furthermore, DMM and DMH were detected in a urine sample from an individual who did not suffer from occupational exposure to trimethylbenzenes, by using this method.     

A novel approach to the quantification of urinary aryl-propionamide-derived SARMs by UHPLC–MS/MS

A simple and sensitive procedure for the quantification of two commonly abused aryl-propionamide-derived selective androgen receptor modulators (SARMs), namely S-4 (GTx-007, andarine) and S-22 (GTx-024, MK-2866, ostarine, enobosarm), has been described. Urine samples were prepared for analysis by means of a dispersive liquid–liquid microextraction using methanol and chloroform as dispersive and extracting solvents, respectively. Factors that might influence the extraction process as well as their optimum conditions were evaluated by Box–Benken and central composite designs. After extraction, the analytes were quantified by UHPLC–MS/MS. The proposed procedure was validated on human urine samples. As a result, for both SARMs the detection limits were observed at 0.05 ng/mL and calibration curves were linear in the concentration range of 0.25–50 ng/mL with the coefficient of determination of 0.998.     

Determination of steroid sex hormones in urine matrix by stir bar sorptive extraction based on monolithic material and liquid chromatography with diode array detection

A simple, rapid and sensitive method for simultaneous determination of six steroid sex hormones in urine matrix was developed by the combination of stir bar sorptive extraction (SBSE) with high performance liquid chromatography (HPLC) and diode array detection (DAD). A poly (methacrylic acid stearyl ester-ethylene dimethacrylate) was synthesized and selected as SBSE extraction medium. To achieve the optimum extraction performance, several parameters, including agitation speed, desorption solvent, extraction and desorption time, pH value, inorganic salt and organic solvent content of the sample matrix were investigated. Under the optimized experimental conditions, low detection limits (S/N=3) and quantification limits (S/N=10) of the proposed method for the target compounds were achieved within the range of 0.062-0.38 and 0.20-1.20 ng/mL, respectively from spiked urine, respectively. The calibration curves of six steroid sex hormones showed good linearity ranging from 1.0 to 200 ng/mL with linear coefficient R2 values above 0.990. Good method reproducibilities presented as intra- and inter-day precisions were also found with the R.S.D.s less than 9.2 and 10.0%, respectively. Finally, the proposed method was successfully applied to the determination of the target compounds in a urine sample from a pregnant woman.     

Simultaneous derivatization and extraction of amphetamine and methylenedioxyamphetamine in urine with headspace liquid-phase microextraction followed by gas chromatography-mass spectrometry

A new method is reported for the simultaneous extraction and derivatization of amphetamine (AM) and methylenedioxyamphetamine (MDA) using headspace hollow fiber protected liquid-phase microextraction (HS-HF-LPME); quantitation is by gas chromatograph-mass spectrometry in the selected ion monitoring (SIM) mode. The derivatizing reagent, pentafluorobenzaldehyde (PFBAY), was added to the extraction solvent. The analytes, volatile and basic, were released from the sample matrix into the headspace first, then extracted and derivatized in the solvent. After that, 2 microl of extract was directly injected into the GC-MS system. Parameters affecting extraction efficiency were investigated and optimized. This method showed good linearity in the concentration range investigated (50-350 ng ml(-1) for AM and 50-700 ng ml(-1) for MDA). Excellent repeatability of the extraction (RSD< or = 4%, n=5), and low limits of quantitation (0.25 ng ml(-1) for AM and 1.00 ng ml(-1) for MDA) were achieved. The feasibility of the method was demonstrated by analyzing human urine samples.     

Membrane protected conductive polymer as micro‐SPE device for the determination of triazine herbicides in aquatic media

A micro‐SPE technique was developed by fabricating a rather small package including a polypropylene membrane shield containing the appropriate sorbent. The package was used for the extraction of some triazine herbicides from aqueous samples. Solvent desorption was subsequently performed in a microvial and an aliquot of extractant was injected into GC‐MS. Various sorbents including aniline‐ortho‐phenylene diamine copolymer, newly synthesized, polypyrrole, multiwall carbon nanotube, C18 and charcoal were examined as extracting media. Among them, conductive polymers exhibited better performance. Influential parameters including extraction and desorption time, desorption solvent and the ionic strength were optimized. The developed method proved to be rather convenient and offers sufficient sensitivity and good reproducibility. The detection limits of the method under optimized conditions were in the range of 0.01–0.04 ng/mL. The RSDs at a concentration level of 0.1 ng/mL were obtained between 4.5 and 9.3% (n=5). The calibration curves of analytes showed linearity in the range of 0.05–10 ng/mL. The developed method was successfully applied to the extraction of selected triazines from real water samples. The whole procedure showed to be conveniently applicable and quite easy to manipulate.     

Detection of organophosphate pesticides in human body fluids by headspace solid-phase microextraction (SPME) and capillary gas chromatography with nitrogen-phosphorus detection

Summary Organosphosphate pesticides have been found extractable by headspace solid-phase microextraction (SPME), and the best conditions of their extraction from human whole blood and urine samples have been investigated. The body fluid samples containing nine pesticides (IBP, methyl parathion, fenitrothion, malathion, fenthion, isoxathion, ethion, EPN and phosalone) were heated at 100°C in a septum-capped vial in the presence of various combinations of acid and salts, and SPME fiber was exposed to the headspace of the vial to allow adsorption of the pesticides before capillary gas chromatography (GC) with nitrogen-phosphorus detection. The heating with distilled water/HCl/(NH₄)₂SO₄/NaCl and with distilled water/HCl gave the best results for urine and whole blood, respectively. Recoveries of the nine pesticides were 0.8–10.6% except for phosalone (0.03%) for whole blood, and 3.8–40.2% for urine. The calibration curves for the pesticides showed linearity in the range of 50–400 ng/0.5 mL for whole blood except for malathion (100–400 ng/0.5 mL whole blood) and 7.5–120 ng/0.5 mL for urine except for phosalone (15–120 ng/0.5 mL urine) with detection limits of 2.2–40 ng/0.5 mL for whole blood and 0.8–12 ng/0.5 mL for urine.     

Determination of chlorophenoxy acid herbicides by capillary electrophoresis with integrated potential gradient detection

This report describes separation and detection of chlorophenoxy acid herbicides spiked in drinking water by the technique combining solid-phase extraction, field-amplified sample stacking, capillary electrophoresis, and potential gradient detection. The herbicide solution (400 mL) was concentrated to 0.1 mL by the solid-phase extraction procedure. The buffer containing 3 mM ammonia and 0.3 mM hydroxypropyl-beta-cyclodextrin was adjusted to pH 9.0 with ammonia. The sample solution was injected into the capillary to 30% of the whole length, and -9 kV and 9 kV were employed for field-amplified sample stacking and separation, respectively. The herbicides were baseline separated and the detection limits with the above combined techniques were in the range of 1-4 x 10(-2) ng/mL.     

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.

Direct determination of estriol 3- and 16-glucuronides in pregnancy urine by column-switching liquid chromatography with electrospray tandem mass spectrometry

Using column-switching liquid chromatography/tandem mass spectrometry (LC-MS/MS), we developed an improved analytical method of urinary estriol glucuronides. This new method is derived predominantly from maternal and fetal precursors in pregnancy. We used in the following procedure: first, we filtered urine samples with a membrane filter. Next, we directly injected the 50 microL aliquot of urine samples onto a pre-column. Then, after activating the column-switching valve, we backflushed the loaded samples onto the C(18) analytical column. Urine samples can be assayed within 20 min without any sample preparation steps. We monitored separated estriol glucuronides by negative electrospray ionization (ESI) and selected-reaction monitoring (SRM). The calibration range of estriol-3-glucuronide (E3-3G) and estriol-16-glucuronide (E3-16G) was 0.1-20 microg/mL and the linearity of the method was 0.9984 for E3-3G and 0.9987 for E3-16G. The limits of detection at a signal-to-noise (S/N) ratio of 3 were 10 ng/mL (E3-3G) and 5 ng/mL (E3-16G). The analytical recovery was over 85% and, in general, inter-day and intra-day variability for precision and accuracy were less than 10%. When applied to a pregnancy urine sample to biomedical monitoring of the function of the maternal/fetal unit, the proposed method allowed rapid and sensitive screening for the detection of E3-3G and E3-16G.     

Comparison of conventional hollow fiber based liquid phase microextraction and electromembrane extraction efficiencies for the extraction of ephedrine from biological fluids

In the present study, hollow fiber liquid phase microextraction (HF-LPME) based on pH gradient and electromembrane extraction (EME) coupled with high-performance liquid chromatography (HPLC) was compared for the extraction of ephedrine from biological samples. The influences of fundamental parameters affecting the extraction efficiency of ephedrine were studied and optimized for both methods. Under the optimized conditions, preconcentration factors of 120 and 35 for urine and 51 and 8 for human plasma were obtained using EME and HF-LPME, respectively. The calibration curves showed good linearity for urine and plasma samples by both methods with the coefficient of estimations higher than 0.98. The limits of detection were obtained 5 and 10 ng mL(-1) using EME and 60 and 200 ng mL(-1) by HF-LPME for urine and plasma samples respectively. The relative standard deviations of the analysis were found in the range of 5.2-8.6% (n=3). The results showed that in comparison with HF-LPME based on pH gradient, EME is a much more effective transport process, providing high extraction efficiencies in very short time.     

Determination of macrolide antibiotics in porcine and bovine urine by high-performance liquid chromatography coupled to coulometric detection

A high-performance liquid chromatography method coupled to coulometric detection has been applied for the determination, in a single run, of up to eight macrolide antibiotics (erythromycin [ERY], tylosin [TYL], tilmicosin [TILM], spiramycin 2 [SPI 2], spiramycin 3 [SPI 3], josamycin [JOS], kitasamycin [KIT], and rosamicin [ROS]) in spiked porcine and bovine urine. Quantification was performed using matrix-matched calibration with roxithromycin (ROX) as the internal standard. The detection limits for each drug were below 3.5 ng injected (equivalent to an initial concentration below 0.07 mg L^–1) for porcine urine and below 5 ng injected (equivalent to an initial concentration below 0.10 mg L^–1) for bovine urine. Recoveries from urine samples spiked at three different concentrations within the linear range were not significantly dependent on concentration. The entire procedure provides average macrolide recoveries ranging from 69.7 to 96.6% for bovine urine and from 75.5 and 96.1% for porcine urine.