Analysis of N-methylhistamine by gas chromatography–mass spectrometry

A gas chromatography–electron capture mass spectrometry assay has been developed for the histamine H₃ receptor agonist, N^α-methylhistamine (N^α-MH). The assay is linear from 50 pg–10 ng, with a limit of detection of 50 pg/ml for gastric juice and plasma, and 50 pg/sample for bacteria (10⁷–10⁸ CFU) and gastric tissue (5–10 mg wet weight). The limits of quantification are 100 pg/ml for gastric juice (%RSD=1.4) and plasma (%RSD=9.4), and 100 pg/sample for bacteria (%RSD=3.9) and tissue (%RSD=5.8). N^α-MH was not present in human plasma, but low levels (1.4 ng/ml and 0.4 ng/ml) were detected in two samples of human gastric juice obtained from patients infected with Helicobacter pylori.     

Determination of norepinephrine in small volume plasma samples by stable-isotope dilution gas chromatography–tandem mass spectrometry with negative ion chemical ionization

A stable-isotope based gas chromatography–tandem mass spectrometry–negative ion chemical ionization method was developed for the determination of norepinephrine (NE) levels in small volumes (25–100 μl) of plasma. NE was stabilized in plasma by the addition of semicarbazide and spiked with deuterium-labeled norepinephrine internal standard. The analytes were isolated from the plasma by solid-phase extraction using phenylboronic acid columns and derivatized using pentafluoropropionic anhydride. The derivatized analytes were chromatographed on a capillary column and detected by tandem mass spectrometry with negative ion chemical ionization. Unparalleled sensitivity and selectivity were obtained using this detection scheme, allowing the unambiguous analysis of trace levels of NE in small-volume plasma samples. Linear standard curves were obtained for NE over a mass range from 1 to 200 pg per sample. The method had a limit of quantitation of 10 pg NE/ml plasma when using a 100-μl sample aliquot (1 pg/sample). Accuracy for the analysis of plasma samples spiked with 10 to 200 pg NE/ml typically ranged from 100±10%, with RSD values of less than 10%. The methodology was applied to determine the effect of clonidine on plasma NE levels in conscious spontaneously hypertensive rats. Administration of clonidine (30 μg/kg) produced an 80% reduction in plasma NE accompanied by a 30% reduction in heart and mean arterial pressure that persisted >90 min after drug administration. The ability to take multiple samples from individual rats allowed the time course for the effect of clonidine to be mapped out using only one group of animals.     

Simultaneous determination of fentanyl and midazolam using high-performance liquid chromatography with ultraviolet detection

When measuring fentanyl and midazolam simultaneously in the same plasma sample with standard high-performance liquid chromatography–ultraviolet (HPLC–UV) detection, overlap of the fentanyl peak by the midazolam peak occurs, which makes fentanyl determination impossible. We tested the hypothesis that by acidifying the methanol mobile phase with 0.02% perchloric acid, 70%, it would be possible to separate both peaks. The UV detector was set at 200 nm. Calibration curves for fentanyl (range 0–2000 pg/ml) and midazolam (range 0–400 ng/ml) were linear (r>0.99). The detection limits were 200 pg/ml (fentanyl) and 10 ng/ml (midazolam). Precision and accuracy for intra- and inter-assay variability as well as in-line validation with quality control samples (QCS) were acceptable (< 15 and 20%, respectively), except for fentanyl QCS of 200 pg/ml (17.8% precision). Although less sensitive than gas chromatography–mass spectrometry (GC–MS), reliable measurements of fentanyl, simultaneously with midazolam, can be performed with this HPLC–UV system.     

Development of highly sensitive quantification method for testosterone and dihydrotestosterone in human serum and prostate tissue by liquid chromatography–electrospray ionization tandem mass spectrometry

We developed highly sensitive detection of testosterone (T) and 5α-dihydrotestosterone (DHT) by liquid chromatography–electrospray ionization tandem mass spectrometry using high proton affinitive derivatization of 17β-hydroxyl group of T and DHT with picolinic acid, mobile phase consisting of MeCN–MeOH–H₂O–formic acid and conventional octadecylsilica (ODS) column. Purification of the derivatives was carried out using solid-phase extraction with ODS cartridge. By this method, T and DHT were determined simultaneously with limits of quantification (LOQs) of 1 pg/0.2 ml in serum, and T and DHT with LOQs of 0.5 pg and 1 pg/3 mg in prostate tissue, respectively, under acceptable assay performance (intra-assay and inter-assay accuracy and precision). The present method provides reliable and reproducible results for quantification of T and DHT in small volumes of serum and prostate samples for diagnosis in prostatic disorders and male climacteric.     

Determination of polychlorinated biphenyls in human blood by solid-phase extraction including on-column lipid decomposition

A method for the isolation of polychlorinated biphenyls (PCBs) from human blood using solid-phase extraction (SPE) has been developed. The procedure incorporates decomposition of lipids by concentrated sulphuric acid directly on the SPE column. Conditions for transferring PCBs onto the SPE column and washing the decomposed blood components from the SPE column were optimised. After clean-up the extracts were analysed using gas chromatography with electron capture detection. An average recovery of PCBs from spiked blood samples was about 78±8% and an average precision was about 109±7%. Quantitation has been done using four internal standards and calibration curves based on five concentration levels. Low procedural blanks made it possible to determine PCBs in blood quantitatively at a level down to 2–10 pg g⁻¹. The integrated method for blood is fast, less laborious than methods using liquid–liquid extraction and has a low consumption of organic solvents.     

Determination of 9α,11β-prostaglandin F2 in human urine and plasma by gas chromatography—mass spectrometry

Sensitive and specific assay methods for 9α,11β-prostaglandin F₂ (9α,11β-PGF₂) by gas chromatography—mass spectrometry with electron impact ionization are described. The mass spectrometric assay for 9α,11β-PGF₂ was based on the use of the methyl ester—dimethylisopropylsilyl ether derivative, and pentadeuterated PGF_2α as a convenient internal standard. The calibration graph for 9α,11β-PGF₂ was linear from 5 pg to 100 ng for both the standard and spiked biological samples. The limit of detection was 50 pg/ml for urine and 25 pg/ml for plasma (signal-to-noise RATIO = 2.3). The method was applied to the determination of 9α,11β-PGF₂ in urine and plasma samples from patients with bronchial asthma.     

Development and validation of a high-performance liquid chromatography–tandem mass spectrometry assay for the determination of sanfetrinem in human plasma

A rapid, selective and accurate high-performance liquid chromatography–tandem mass spectrometry assay for the quantification of sanfetrinem in human plasma has been developed and validated. The performance of manual and automated sample preparation was assessed; 50 μl of plasma sample was deproteinized with acetonitrile, followed by dilution with water and injection onto the LC system. Chromatographic separation was achieved on a Phenomenex Luna C₁₈(2), 50×2.0 (5 μm) column with a mobile phase consisting of water–acetonitrile with 0.1% formic acid followed by detection with a Perkin-Elmer API3000 mass spectrometer in multiple reaction monitoring mode. The lower limit of quantification was improved by five times compared to the UV method previously reported. A range of concentration from 10 ng/ml to 5 μg/ml was covered. The method was applied to the quantification of sanfetrinem in human plasma samples from healthy volunteers participating in a clinical study.     

Validation of an analytical procedure to measure trace amounts of neurosteroids in brain tissue by gas chromatography–mass spectrometry

A selective and extremely sensitive procedure has been developed and optimized, using high-performance liquid chromatography (HPLC), specific derivatization and gas chromatography–mass spectrometry (GC–MS), to simultaneously quantify very small amounts of different neurosteroids from rat brain. Unconjugated and sulfated steroids in brain extracts were separated by solid-phase extraction. The unconjugated fraction was further purified by HPLC, the steroids being collected in a single fraction, and the sulfated fraction was solvolyzed. All steroids were derivatized with heptafluorobutyric acid anhydride and analyzed by GC–MS (electron impact ionization) using selected-ion monitoring. High sensitivity and accuracy were obtained for all steroids. The detection limits were 1 pg for pregnenolone (PREG), dehydroepiandrosterone (DHEA) and their sulfate esters PREG-S and DHEA-S, 2 pg for progesterone (PROG) and 5 pg for 3α,5α-tetrahydroprogesterone (3α,5α-THP). In a pilot study on a rat brain, the concentrations of PREG-S and DHEA-S were 8.26±0.80 and 2.47±0.27 ng/g, respectively. Those of PREG, DHEA and PROG were 4.17±0.22, 0.45±0.02 and 1.95±0.10 ng/g, respectively. Good linearity and accuracy were observed for each steroid. The methodology validated here, allows femtomoles of neurosteroids, including the sulfates, found in small brain samples (at least equal to 10 mg) to be quantified simultaneously.     

Quantitative analysis of polychlorinated biphenyls, organochlorine insecticides, polycyclic aromatic hydrocarbons, polychlorinated hydrocarbons and polynitrohydrocarbons in spiked samples of soil, water and plasma by selected-ion monitoring gas chromatography–mass spectrometry

A broad range of pollutants such as polycyclic aromatic hydrocarbons (PAHs), polychlorinated hydrocarbons (PCHs), polynitrohydrocarbons (PNHs), polychlorinated biphenyls (PCBs) and organochlorine (OCs) insecticides were simultaneously analyzed in spiked soil, water or plasma samples by using gas chromatography–mass spectrometry (GC–MS). Water and plasma samples containing the pollutants were extracted by a solid-phase extraction (SPE) method using florisil columns. The soil samples, fortified with the toxicants, were extracted with water, methanol or dichloromethane (DCM). The water extract was processed by the SPE method. The methanol and DCM samples were dried, dissolved in acetonitrile and subjected to the SPE extraction. The extracted samples were analyzed by GC–MS programmed to monitor selected ions. The deuterium labelled compounds were used as the internal standards. The chromatographic profile of total ions indicated complete separation of some compounds such as isophorone, naphthalene, all PCBs, most OC insecticides and PNHs; high M_r PAHs and some PCHs were partially or incompletely separated. The chromatographic profile of individual ion indicated good separation of each ion. The minimum detection limit ranged from 1 to 4 pg injected when 1 or 2 ions were monitored or from 20 to 200 pg injected when 20 ions were monitored. The SPE method that provided 60–105% recovery of pollutants from water samples, provided only 2–60% recovery from plasma samples. This may be due to the binding of pollutants to plasma proteins. Water recovered 1–30%, while methanol or DCM recovered 65–100% of the pollutants added to the soil samples. The use of internal standards corrected for the loss of pollutants from plasma or soil.     

Comparison of sensitivity between gas chromatography–low-resolution mass spectrometry and gas chromatography–high-resolution mass spectrometry for determining metandienone metabolites in urine

In doping control laboratories the misuse of anabolic androgenic steroids is commonly investigated in urine by gas chromatography–low-resolution mass spectrometry with selected ion monitoring (GC–LRMS–SIM). By using high-resolution mass spectrometry (HRMS) detection sensitivity is improved due to reduction of biological background. In our study HRMS and LRMS methods were compared to each other. Two different sets were measured both with HRMS and LRMS. In the first set metandienone (I) metabolites 17α-methyl-5β-androstan-3α,17β-diol (II), 17-epimetandienone (III), 17β-methyl-5β-androst-1-ene-3α,17α-diol (IV) and 6β-hydroxymetandienone (V) were spiked in urine extract prepared by solid-phase extraction, hydrolysis with β-glucuronidase from Escherichia coli and liquid–liquid extraction. In the second set the metabolites were first spiked in blank urine samples of four male persons before pretreatment. Concentration range of the spiked metabolites was 0.1–10 ng/ml in both sets. With HRMS (resolution of 5000) detection limits were 2–10 times lower than with LRMS. However, also with the HRMS method the biological background hampered detection and compounds from matrix were coeluted with some metabolites. For this reason the S/N values of the metabolites spiked had to be first compared to S/N values of coeluted matrix compounds to get any idea of detection limits. At trace concentrations selective isolation procedures should be implemented in order to confirm a positive result. The results suggest that metandienone misuse can be detected by HRMS for a prolonged period after stopping the intake of metandienone.     

Semi-automated 96-well solid-phase extraction and gas chromatography–negative chemical ionization tandem mass spectrometry for the trace analysis of fluprostenol in rat plasma

Semi-automated 96-well plate solid-phase extraction (SPE) was used for sample preparation of fluprostenol, a prostaglandin analog, in rat plasma prior to detection by gas chromatography–negative chemical ionization tandem mass spectrometry (GC–NCI-MS–MS). A liquid handling system was utilized for all aspects of sample handling prior to SPE including transferring of samples into a 96-well format, preparation of standards as well as addition of internal standard to standards, quality control samples and study samples. SPE was performed in a 96-well plate format using octadecylsilane packing and the effluent from the SPE was dried in a custom-made 96-well apparatus. The sample residue was derivatized sequentially with pentafluorobenzylbromide followed by N-methyl-N-trimethylsilyltrifluoroacetamide. The derivatized sample was then analyzed using GC–NCI-MS–MS. The dynamic range for the method was from 7 to 5800 pg/ml with a 0.1-ml plasma sample. The methodology was evaluated over a 4-day period and demonstrated an accuracy of 90–106% with a precision of 2.4–12.9%.     

Determination of gacyclidine enantiomers in human plasma by gas chromatography–mass spectrometry using selected-ion monitoring

A sensitive gas chromatographic assay using mass selective-detection has been developed for the simultaneous quantitation of the enantiomers of (±)-gacyclidine (a non competitive N-methyl-

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-aspartate antagonist) in human plasma. Gacyclidine enantiomers and phencyclidine (PCP), the internal standard, were extracted using a single-step liquid–liquid extraction with hexane at pH 8.0. Each enantiomer was separated on a chiral gas chromatography capillary column and specifically detected by mass spectrometry (MS) in selected-ion monitoring (SIM) mode. Gacyclidine enantiomers and PCP were monitored using the fragment ions at m/z 206 and 200, respectively. No interference was observed from endogenous components. The limit of quantitation (LOQ) for each enantiomer of gacyclidine was 300 pg/ml by using plasma samples of 500 μl. The calibration curves were linear (r²=0.998) over a range of 0.3125 to 20 ng/ml. The extraction efficiency was higher than 95% for both enantiomers. Intra- and inter-day bias were less than 10% at every standard curve concentration. Intra-day precision was less than 19% for (−)-gacyclidine and 15% for (+)-gacyclidine. Inter-day precision was below 15% for both enantiomers. The assay was validated for an enantioselective pharmacokinetic study in healthy male volunteers.     

Determination of organochlorine pesticides and polychlorinated biphenyls in human serum using headspace solid-phase microextraction and gas chromatography-electron capture detection

A simple procedure for the determination of organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) in human serum using headspace solid-phase microextraction (HS-SPME) was developed. The analysis was carried out by gas chromatography (GC) equipped with electron capture detector (ECD). A 2(7-4) Plackett-Burman reduced factorial design for screening and a central composite design for optimizing the significant variables were applied. A 100 microm PDMS fiber, 3/5 headspace ratio (3 ml in 5 ml vial), 85 degrees C extraction temperature, 50 min extraction time, and 1 ml of acidic solution (pH 3) added to 1 ml of diluted serum (1:1) were chosen for the best response in HS extraction mode. The detection limits found were from 1 pg/ml (PCB 167) to 52 pg/ml (beta-HCH), the relative standard deviation for the procedure varied from 3% (PCB 52) to 12% (PCB 189) and the accuracy was checked by using validated solid-phase extraction (SPE) procedure. The method that avoids the use of clean-up steps and the hazardous solvents enabled reliable determinations of the OCPs and the PCBs except beta-HCH. The method was applied to the analysis of 33 human serum samples. The most abundant target compound was p-p'-DDE (range, 0.3-8.0 ng/ml; median value, 2.1 ng/ml). Among the PCBs the prevalent congeners were 138, 153 and 180.     

Solid-phase extraction and gas chromatography–mass spectrometry determination of kaempferol and quercetin in human urine after consumption of Ginkgo biloba tablets

A method was developed for the quantification of the flavonoids quercetin and kaempferol in human urine using a solid-phase extraction procedure followed by gas chromatography–mass spectrometry. Deuterated internal standards of the analytes were spiked into the samples prior to extraction. The limit of detection of the method was ca. 10 pg on column and precision of the method for quantification in a sample of urine was ±9.40% for kaempferol and ±7.34% for quercetin (n=6). The levels of quercetin and kaempferol found in urine samples were only a small fraction of the amount ingested. The treatment of urine samples with β-glucuronidase markedly increased the levels of flavonoids detected, supporting the view that kaempferol and quercetin are eliminated in the urine as glucuronides.     

Automated in-tube solid-phase microextraction–liquid chromatography–electrospray ionization mass spectrometry for the determination of ranitidine

The technique of automated in-tube solid-phase microextraction (SPME) coupled with liquid chromatography–electrospray ionization mass spectrometry (LC–ESI-MS) was evaluated for the determination of ranitidine. In-tube SPME is an extraction technique for organic compounds in aqueous samples, in which analytes are extracted from the sample directly into an open tubular capillary column by repeated aspirate/dispense steps. In order to optimize the extraction of ranitidine, several in-tube SPME parameters such as capillary column stationary phase, extraction pH and number and volume of aspirate/dispense steps were investigated. The optimum extraction conditions for ranitidine from aqueous samples were 10 aspirate/dispense steps of 30 μl of sample in 25 mM Tris–HCl (pH 8.5) with an Omegawax 250 capillary column (60 cm×0.25 mm I.D., 0.25 μm film thickness). The ranitidine extracted on the capillary column was easily desorbed with methanol, and then transported to the Supelcosil LC-CN column with the mobile phase methanol–2-propanol–5 M ammonium acetate (50:50:1). The ranitidine eluted from the column was determined by ESI-MS in selected ion monitoring mode. In-tube SPME followed by LC–ESI-MS was performed automatically using the HP 1100 autosampler. Each analysis required 16 min, and carryover of ranitidine in this system was below 1%. The calibration curve of ranitidine in the range of 5–1000 ng/ml was linear with a correlation coefficient of 0.9997 (n=24), and a detection limit at a signal-to-noise ratio of three was ca. 1.4 ng/ml. The within-day and between-day variations in ranitidine analysis were 2.5 and 6.2% (n=5), respectively. This method was also applied for the analyses of tablet and urine samples.     

Quantitative liquid chromatographic–tandem mass spectrometric determination of reserpine in FVB/N mouse plasma using a “chelating” agent (disodium EDTA) for releasing protein-bound analytes during 96-well liquid–liquid extraction

A sensitive, specific, accurate and reproducible analytical method employing a divalent cation chelating agent (disodium EDTA) for sample treatment was developed to quantitate reserpine in FVB/N mouse plasma. Samples pretreated with 40 μl of 2% disodium EDTA in water were extracted by a semi-automated 96-well liquid–liquid extraction (LLE) procedure to isolate reserpine and a structural analog internal standard (I.S.), rescinnamine, from mouse plasma. The extracts were analyzed by turbo ionspray liquid chromatography–tandem mass spectrometry (LC–MS–MS) in the positive ion mode. Sample preparation time for conventional LLE was dramatically reduced by the semi-automated 96-well LLE approach. The assay demonstrated a lower limit of quantitation of 0.02 ng/ml using 0.1-ml plasma sample aliquots. The calibration curves were linear from 0.02 to 10 ng/ml for reserpine. The intra- and inter-assay precision of quality control (QC) samples ranged from 1.75 to 10.9% for reserpine. The intra- and inter-assay accuracy of QC samples ranged from −8.17 to 8.61%. Reserpine and the I.S. were found to be highly bound to FVB/N mouse plasma protein. This is the first report of disodium EDTA employed as a special protein-bound release agent to recover protein-bound analytes from plasma. These matrix effects and the effects of pH in the HPLC mobile phase on the sensitivities of LC–MS–MS are discussed in this paper.     

Determination of leukotriene E4 in human urine using liquid chromatography–tandem mass spectrometry

A liquid chromatographic–tandem mass spectrometric (LC–MS–MS) method was developed for the quantitation of urinary leukotriene E₄ (LTE₄). LTE₄ and its internal standard were extracted by solid-phase extraction and analysed using LC–MS–MS in the selected reaction monitoring (SRM) mode. A good linear response over the range of 10 pg to 10 ng was demonstrated. The accuracy of added LTE₄ ranged from 97.0% to 108.0% with a mean and SD of 100.6±2.4%. We detected LTE₄ (63.1±18.7 pg/mg creatinine, n=10) in healthy human urine. This method can be used to determine LTE₄ in biological samples.     

Determination of dichloroanilines in human urine by GC–MS, GC–MS–MS, and GC–ECD as markers of low-level pesticide exposure

Methods for the determination of 3,4-dichloroaniline (3,4-DCA) and 3,5-dichloroaniline (3,5-DCA) as common markers of eight non-persistent pesticides in human urine are presented. 3,5-DCA is a marker for the exposure to the fungicides vinclozolin, procymidone, iprodione, and chlozolinate. Furthermore the herbicides diuron, linuron, neburon, and propanil are covered using their common marker 3,4-DCA. The urine samples were treated by basic hydrolysis to degrade all pesticides, metabolites, and their conjugates containing the intact moieties completely to the corresponding dichloroanilines. After addition of the internal standard 4-chloro-2-methylaniline, simultaneous steam distillation extraction (SDE) followed by liquid–liquid extraction (LLE) was carried out to produce, concentrate and purify the dichloroaniline moieties. Gas chromatography (GC) with mass spectrometric (MS) and tandem mass spectrometric (MS–MS) detection and also detection with an electron-capture detector (ECD) after derivatisation with heptafluorobutyric anhydride (HFBA) were employed for separation, detection, and identification. Limit of detection of the GC–MS–MS and the GC–ECD methods was 0.03 and 0.05 μg/l, respectively. Absolute recoveries obtained from a urine sample spiked with the internal standard, 3,5-, and 3,4-DCA, ranged from 93 to 103% with 9–18% coefficient of variation. The three detection techniques were compared concerning their performance, expenditure and suitability for their application in human biomonitoring studies. The described procedure has been successfully applied for the determination of 3,4- and 3,5-DCA in the urine of non-occupationally exposed volunteers. The 3,4-DCA levels in these urine samples ranged between 0.13 and 0.34 μg/g creatinine or 0.11 and 0.56 μg/l, while those for 3,5-DCA were between 0.39 and 3.33 μg/g creatinine or 0.17 and 1.17 μg/l.     

Distribution,Sources and Risk Assessment of Polychlorinated Biphenyls in Soils from the Midway Atoll,North Pacific Ocean

Concentrations of 28 polychlorinated biphenyls (PCBs) were assessed in soils from the Midway Atoll in the central North Pacific Ocean. The analytical procedure involved the application of accelerated solvent extraction (ASE) and gas chromatography coupled with ion trap mass spectrometric detection (GC/ITMS) for identification and quantification. Among the 28 PCB congeners studied, 26 of them, except CB195 and CB209, were detected in the analyzed samples at different frequencies. The total concentrations of 28 indicator PCBs (ΣPCBs) ranged from 2.6 to 148.8 ng g⁻¹ with an average value of 50.7 ng g⁻¹ and median of 39.5 ng g⁻¹. Sources and congeners’ pattern of PCB were investigated in the soil of Midway Atoll. The principal component analysis indicated that the compositions of PCBs in most of the soil samples were similar. The total concentrations of PCBs were used to assess the cancer risk probabilities in humans via ingestion, dermal contact and inhalation of soil particles. Very low cancer risk was found in all soil samples caused by ΣPCBs.     

Screening of chlorpropamide in horse plasma by high-performance liquid chromatography with ultraviolet absorbance detection, and confirmation by gas chromatography–mass spectrometry

A chromatographic method was developed to detect and confirm the presence of chlorpropamide (I) in horse plasma samples, for antidoping control. The plasma sample (1 ml) was extracted with dichloromethane and screened by high-performance liquid chromatography, and confirmation of the drug's presence was accomplished by using gas chromatography–mass spectrometry (GC–MS). The limit of detection was found to be 3.5 ng/ml at a signal-to-noise ratio of three. Derivatization of I with N,O-bis-(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane allowed for highly stable, accurate and sensitive GC–MS analysis. Plasma samples collected after the administration of diabinese were positive for I (one–five days) in all samples analysed.