Measurement of glycidol hemoglobin adducts in humans who ingest edible oil containing small amounts of glycidol fatty acid esters

Hemoglobin (Hb) adducts are frequently used to address and/or monitor exposure to reactive chemicals. Glycidol (G), a known animal carcinogen, has been reported to form Hb adducts. Here, we measure G adduct levels in humans who daily ingest DAG oil, an edible oil consisting mainly of diacylglycerol. Since DAG oil contains a small amount of glycidol fatty acid esters (GEs), possible exposure to G released from GEs has been raised as a possible concern. For measurement of Hb adducts, we employed the N-alkyl Edman method reported by Landin et al. (1996) using gas chromatography-tandem mass spectrometry with minor modifications to detect G-Hb adducts as N-(2,3-dihydroxy-propyl)valine (diHOPrVal). Blood samples were collected from 7 DAG oil users and 6 non-users, and then G-Hb adduct levels were measured. G-Hb adducts were detected in all samples. The average level of diHOPrVal was 3.5 ± 1.9 pmol/g globin in the DAG oil users and 7.1 ± 3.1 pmol/g globin in the non-users. We conclude that there is no increased exposure to G in individuals who daily ingest DAG oil.     

In vivo CYP2E1 phenotyping as a new potential biomarker of occupational and experimental exposure to benzene

Assessing CYP2E1 phenotype in vivo may be important to predict individual susceptibility to those chemicals, including benzene, which are metabolically activated by this isoenzyme. Chlorzoxazone (CHZ), a specific CYP2E1 substrate, is readily hydroxylated to 6-OH-chlorzoxazone (6-OH-CHZ) by liver CYP2E1 and the metabolic ratio 6-OH-CHZ/CHZ in serum (MR) is a specific and sensitive biomarker of CYP2E1 activity in vivo in humans. We used this MR as a potential biomarker of effect in benzene-treated rats and, also, in humans occupationally exposed to low levels of benzene. Male Sprague-Dawley rats (375–400 g b.w.) were treated i.p. for 3 days with either a 0.5 ml solution of benzene (5 mmol/kg b.w.) in corn oil, or 0.5 ml corn oil alone. Twenty-four hours after the last injection, a polyethylene glycol (PEG) solution of CHZ (20 mg/kg b.w.) was injected i.p. in both treated and control animals. After 2, 5, 10, 15, 20, 30, 45, 60, 90, 120, 180, and 240 min from injection, 0.2 ml blood was taken from the tip tail and stored at −20 °C until analysis. A modified reverse phase HPLC method using a 5 μm Ultrasphere C18 column equipped with a direct-connection ODS guard column, was used to measure CHZ and its metabolite 6-OH-CHZ in serum. No statistically significant difference in the MR was observed, at any sampling time, between benzene-treated and control rats. The concentration-versus-time area under the curve (AUC), however, was lower (p < 0.05, Mann–Whitney test), whereas the systemic clearance was higher (p < 0.05) in treated than in control rats. Eleven petrochemical workers occupationally exposed to low levels of airborne benzene (mean ± SD, 25.0 ± 24.4 μg/m³) and 13 non-exposed controls from the same factory (mean ± SD, 6.7 ± 4.0 μg/m³) signed an informed consent form and were administered 500 mg CHZ p.o. Two hours later a venous blood sample was taken for CHZ and 6-OH-CHZ measurements. Despite exposed subjects showed significantly higher levels of t,t-MA and S-PMA, two biomarkers of exposure to benzene, than non-exposed workers, no difference in the MR mean values ± SD was found between exposed (0.59 ± 0.29) and non-exposed (0.57 ± 0.23) subjects. So, benzene was found to modify CHZ disposition, but not CYP2E1 phenotype in benzene-treated rats, nor in workers exposed to benzene, probably due to the levels of exposure being too low.     

Pharmacokinetic study of melamine in rhesus monkey after a single oral administration of a tolerable daily intake dose

To perform pharmacokinetic study of melamine in rhesus monkey, melamine was orally administered to three experimental monkeys at a single dose of 1.4 mg/kg body weight. Plasma and urine were collected for the determination of melamine and cyanuric acid with a liquid chromatography tandem mass spectrometry method. The mean ± SD area under the concentration–time curve from time zero to 48 h (AUC0–t) was 14,145 ± 2002 μg/L h. The maximum concentration of melamine in plasma (C_max) was 1767 ± 252 μg/L. The time to maximum concentration (T_max) was 2.67 ± 1.16 h and the half-life of melamine in plasma (t_1/2) was 4.41 ± 0.43 h. Following oral administration, melamine was rapidly excreted, mainly through urinary clearance. No significant correlation was found between melamine and cyanuric acid, suggesting that cyanuric acid may not be derived from melamine.     

Metabolism of the masked mycotoxin deoxynivalenol-3-glucoside in pigs

Plants can metabolize the Fusarium mycotoxin deoxynivalenol (DON) by forming the masked mycotoxin deoxynivalenol-3-β-d-glucoside (D3G). D3G might be cleaved during digestion, thus increasing the total DON burden of an individual. Due to a lack of invivo data, D3G has not been included in the various regulatory limits established for DON so far. The aim of our study was to contribute to the risk assessment of D3G by determination of its metabolism in pigs. Four piglets received water, D3G (116 μg/kg b.w.) and the equimolar amount of DON (75 μg/kg b.w.) by gavage on day 1, 5 and 9 of the experiment, respectively. Additionally, 15.5 μg D3G/kg b.w. were administered intravenously on day 13. Urine and feces were collected for 24 h and analyzed for DON, D3G, deoxynivalenol-3-glucuronide (DON-3-GlcA), deoxynivalenol-15-GlcA (DON-15-GlcA) and deepoxy-deoxynivalenol (DOM-1) by UHPLC–MS/MS. After oral application of DON and D3G, in total 84.8 ± 9.7% and 40.3 ± 8.5% of the given dose were detected in urine, respectively. The majority of orally administered D3G was excreted in form of DON, DON-15-GlcA, DOM-1 and DON-3-GlcA, while urinary D3G accounted for only 2.6 ± 1.4%. In feces, just trace amounts of metabolites were found. Intravenously administered D3G was almost exclusively excreted in unmetabolized form via urine. Data indicate that D3G is nearly completely hydrolyzed in the intestinal tract of pigs, while the toxin seems to be rather stable after systemic absorption. Compared to DON, the oral bioavailability of D3G and its metabolites seems to be reduced by a factor of up to 2, approximately.     

Quantification of artemisinin in human plasma using liquid chromatography coupled to tandem mass spectrometry

A liquid chromatographic tandem mass spectroscopy method for the quantification of artemisinin in human heparinised plasma has been developed and validated. The method uses Oasis HLB™ μ-elution solid phase extraction 96-well plates to facilitate a high throughput of 192 samples a day. Artesunate (internal standard) in a plasma–water solution was added to plasma (50 μL) before solid phase extraction. Artemisinin and its internal standard artesunate were analysed by liquid chromatography and MS/MS detection on a Hypersil Gold C18 (100 mm × 2.1 mm, 5 μm) column using a mobile phase containing acetonitrile–ammonium acetate 10 mM pH 3.5 (50:50, v/v) at a flow rate of 0.5 mL/min. The method has been validated according to published FDA guidelines and showed excellent performance. The within-day, between-day and total precisions expressed as R.S.D., were lower than 8% at all tested quality control levels including the upper and lower limit of quantification. The limit of detection was 0.257 ng/mL for artemisinin and the calibration range was 1.03–762 ng/mL using 50 μL plasma. The method was free from matrix effects as demonstrated both graphically and quantitatively.     

Simultaneous detection and quantification of 3-nitrotyrosine and 3-bromotyrosine in human urine by stable isotope dilution liquid chromatography tandem mass spectrometry

Nitration and bromination of proteins, giving rise to the respective 3-nitrotyrosine (3NT) and 3-bromotyrosine (3BT), are implicated in asthma, allergic inflammatory disorders, and cancer. We have developed an isotope dilution liquid chromatography electrospray ionization tandem mass spectrometry (LC/MS/MS) assay for simultaneous analysis of protein-bound 3NT and 3BT in human urine. The detection limits (S/N = 3) were 10 pg (44 fmol) for 3NT and 5.0 pg (19 fmol) for 3BT injected on-column. The average levels of protein-bound 3NT and 3BT in 23 healthy individuals were 9.7 ± 11.0 (mean ± S.D.) in 10⁵ tyrosine and 4.4 ± 3.9 (mean ± S.D.) in 10³ tyrosine, respectively, using this highly sensitive LC/MS/MS under the selective reaction monitoring mode. Furthermore, the levels of urinary 3NT and 3BT show a statistically significant correlation (R² = 0.55, p = 0.0065, n = 23). The high specificity and accuracy of this LC/MS/MS method render it a valuable tool in measurement of 3NT and 3BrT in the human urinary protein as promising noninvasive biomarkers for protein tyrosine nitration and bromination in vivo.     

An acellular assay to assess the genotoxicity of complex mixtures of organic pollutants bound on size segregated aerosol. Part I: DNA adducts

An acellular assay consisting of calf thymus DNA with/without rat liver microsomal S9 fraction was used to study the genotoxicity of complex mixtures of organic air pollutants bound to size segregated aerosols by means of DNA adduct analysis. We compared the genotoxicity of the organic extracts (EOMs) from three size fractions of aerosol ranging from 0.17 μm to 10 μm that were collected by high volume cascade impactors in four localities of the Czech Republic differing in the extent of the environmental pollution: (1) small village in proximity of a strip mine, (2) highway, (3) city center of Prague and (4) background station. The total DNA adduct levels induced by 100 μg/ml of EOMs were analyzed by ³²P-postlabelling analysis with a nuclease P1 method for adduct enrichment. The main finding of the study was most of the observed genotoxicity was connected with a fine particulate matter fraction (<1 μm). The concentrations of carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) in EOMs indicate that fine fractions (0.5–1 μm) bound the highest amount of c-PAHs in all aerosol sampling sites, which might be related to the higher specific surface of this fraction as compared with a course fraction (1–10 μm) and higher mass as compared with a condensational fraction (0.17–0.5 μm). As for aerosol mass, both fine and condensational fractions are effective carriers of c-PAHs. Similarly, the DNA adduct levels per m³ of air were highest for the fine fraction, while the condensational fraction (strip mine site and city center) revealed the highest DNA adduct levels in cases where aerosol mass is taken into consideration. A strong correlation was found between the c-PAHs and DNA adduct levels induced by EOMs in all the localities and for various size fractions (R² = 0.98, p < 0.001). It may be concluded that the analysis of total DNA adducts induced in an acellular assay with/without metabolic activation represents a relatively simple method to assess the genotoxic potential of various complex mixtures.     

Simultaneous determination of methotrexate and its polyglutamate metabolites in Caco-2 cells by liquid chromatography–tandem mass spectrometry

In normal and malignant human cells, the folate antagonist methotrexate (MTX) is converted to a series of polyglutamates (MTXGlu_n, n = 2–5) which play a role in its therapeutic efficacy. Here we report an assay to determine MTX and MTXGlu_n in Caco-2 cells exposed to MTX. After a simple protein precipitation step, cell homogenates (2 × 10⁶ cells) were analyzed by liquid chromatography–tandem mass spectrometry (LC–MS/MS) using aminopterin as internal standard. Separation was by reversed phase HPLC on a C8 column using gradient elution with 0.1% formic acid and acetonitrile. Detection was by electrospray ionization in the positive ion mode followed by multiple reaction monitoring of the transitions of the [M+H]⁺ ions of MTX and MTXGlu_n to their common product ion at m/z 308.2 and of aminopterin at m/z 441.3 → 294.2. Calibration curves for all analytes were linear in the range 2–250 nM (r² > 0.996). Intra- and inter-day precisions (as coefficient of variation) were 3.4–15.1% and 4.3–18.4%, respectively with corresponding accuracies (as relative error) of −3.6 to +6.6% and −5.5 to +7.5%, respectively. Recoveries were in the range 60 ± 4 to 108 ± 13%. It was found that MTX undergoes only limited polyglutamation in Caco-2 cells exposed to MTX over 24 h.     

Characterization of glycidol-hemoglobin adducts as biomarkers of exposure and in vivo dose

Hemoglobin adducts have been used as biomarkers of exposure to reactive chemicals. Glycidol, an animal carcinogen, has been reported to form N-(2,3-dihydroxy-propyl)valine adducts to hemoglobin (diHOPrVal). To support the use of these adducts as markers of glycidol exposure, we investigated the kinetics of diHOPrVal formation and its elimination in vitro and in vivo.     

Validation of a liquid chromatography–tandem mass spectrometric assay for the quantitative determination of hydrastine and berberine in human serum

A high throughput liquid chromatography/tandem mass spectrometry (LC–MS/MS) method for the simultaneous determination of berberine and hydrastine in human serum, after oral administration of goldenseal (Hydrastis canadensis L.), was developed using simple acetonitrile treatment of serum samples. Noscapine served as the internal standard. Lower limit of quantification for both analytes was 0.1 ng mL⁻¹ using positive ion electrospray tandem mass spectrometry (MS/MS). The intra-day (n = 5) accuracy and precision of the method for hydrastine was 82 ± 8.8%, 97.9 ± 2.4% and 96.2 ± 3.3%, respectively. The inter-day (n = 4) accuracy and precision for hydrastine was 90.0 ± 15.17%, 99.9 ± 7.1% and 98 ± 6.54%, respectively. For berberine quantitation intra-day accuracy and precision was 96.0 ± 8.4%, 92.5 ± 4.7% and 94.4 ± 3.7%, respectively. The respective values for inter-day quantitation were 91.0 ± 8.4%, 94.3 ± 4.7% and 94.4 ± 3.7%. The analytical recovery for hydrastine was 82.4–96.2% and for berberine it was 94.4–96.0%. The analytes and noscapine were stable for 24 h at room temperature (CV 5–10%). Matrix ion effects were studied by post-column infusion of hydrastine and berberine, calculation of calibration curve slope precision was obtained using serum from five different subjects, and by comparison of the response of methanol standards and extracted serum samples. The method was further validated by determination of serum pharmacokinetics of hydrastine and berberine after administration of a single oral dose of goldenseal extract containing 77 mg of hydrastine and 132 mg of berberine.     

Quantitative determination of peptide drug in human plasma samples at low pg/ml levels using coupled column liquid chromatography–tandem mass spectrometry

Plasma concentrations after administration of peptide drugs are often low due to the high potency often seen with this class of compounds. In this work a bioanalytical method based on coupled column liquid chromatography–tandem mass spectrometry (LC–MS/MS) is presented for quantification of a peptide drug, FE 202158, under clinical development. A volume of 0.5 ml human plasma is solid phase extracted on a weak cationic exchanger. After evaporation of the solvent to dryness, the reconstituted sample is injected into a coupled column liquid chromatography system. A heart-cut from the initial column, a cyano column, is trapped on a C₄ column and thereafter injected into a microbore C₁₈ column. For the detection a triple quadrupole mass spectrometer, equipped with a TurboIonSpray interface working in positive ion mode, is used. The design of the system is described and the gain in sensitivity and selectivity, compared to a conventional system, is discussed. Data from validation of the bioanalytical method are presented. For human plasma samples a lower limit of quantification (LLOQ) of 5.00 pg/ml (=4.77 pmol/l) was achieved. The inter-assay precision was less than 11% and bias was within ±4% over the whole validated range of 5.00–860 pg/ml.     

The multikinase inhibitor axitinib is a potent inhibitor of human CYP1A2

The tyrosine kinase inhibitors (TKIs) and multikinase inhibitors (MKIs) are oncology drugs of increasing importance that have improved the treatment of multiple tumors types. In some patients these agents produce adverse effects, including pharmacokinetic drug–drug interactions, due to cytochrome P450 (CYP) inhibition. Information on the propensity of the drugs to elicit such effects often only becomes evident as the drugs enter clinical use. The present study assessed 18 kinase inhibitors (1 and 50 μM) for the inhibition of major drug metabolizing CYPs 1A2, 2C9, 2D6 and 3A4 in human liver microsomes. Most TKIs and MKIs inhibited CYP reactions at the higher concentration but axitinib also potently inhibited CYP1A2-dependent 7-ethoxyresorufin O-deethylation activity at the lower concentration. Kinetic analyses of CYP1A2 inhibition by axitinib were undertaken in microsomes and found a K_i of 0.11 ± 0.01 μM, which was 7.5-fold lower than the K_m for 7-ethoxyresorufin oxidation (0.83 ± 0.06 μM); the inhibition mechanism was linear-mixed. From computational modeling two potential binding modes for axitinib were identified in the active site of CYP1A2: one in which the oxidizable axitinib thioether sulfur atom is within ∼4.45 Å of the CYP1A2 heme, and is likely to favor biotransformation of the drug, and a second in which the pyridine moiety is in proximity to the heme, which may contribute to inhibition. The applicability of these findings to potential pharmacokinetic interactions in patients during axitinib treatment should now be assessed.     

Simultaneous measurement of amiodarone and desethylamiodarone in human plasma and serum by stable isotope dilution liquid chromatography–tandem mass spectrometry assay

A stable isotope dilution liquid chromatography–electrospray ionization tandem mass spectrometry (LC–MS/MS) assay to measure amiodarone, the most frequently used agent for maintaining sinus rhythm in patients with atrial fibrillation, and its major metabolite desethylamiodarone in human plasma and serum was developed. Measurement of amiodarone and desethylamiodarone was performed during a 4.0-min run-time using amiodarone-D₄ and desethylamiodarone-D₄ as internal standards. Calibration curves covering 12 calibrators measured in four replicates each for the analysis of both amiodarone and desethylamiodarone were linear and reproducible in the range of 0.01–40.0 mg/L (r > 0.999). Limits of detection in plasma matrix were 2.7 μg/L for amiodarone and 1.9 μg/L for desethylamiodarone, and lower limits of quantification in plasma matrix were 7.5 μg/L for amiodarone and 2.5 μg/L for desethylamiodarone. Interassay imprecision and inaccuracy were <8% and <9% for both substances. Mean extraction yield was 99.6% (range 92.6–107.7%) for amiodarone and 90.2% (range 80.0–94.7%) for desethylamiodarone. Agreement was moderate for amiodarone (n = 162) and desethylamiodarone (n = 117), respectively, between the present method and a HPLC method with UV detection using a commercially available reagent set for the HPLC analysis of these drugs. The Passing–Bablok regression line was HPLC = 0.98 (LC–MS/MS) + 0.10 [mg/L]; r = 0.94 for amiodarone and HPLC = 1.05 (LC–MS/MS) + 0.02 [mg/L]; r = 0.90 for desethylamiodarone. This sensitive and interference-free LC–MS/MS assay permits rapid and accurate determination of amiodarone and desethylamiodarone in human plasma and other body fluids.     

Determination of ghrelin and desacyl ghrelin in human plasma and urine by means of LC–MS/MS for doping controls

The hunger hormone ghrelin (G) is classified as prohibited substance in professional sport by the World Anti-Doping Agency (WADA), due to its known growth hormone releasing properties. The endogenous bioactive peptide consists of 28 amino acids with a caprylic acid attached to serine at position 3. Within this study, it was aimed to develop methods to determine G and desacyl ghrelin (DAG) in plasma and urine by means of LC–MS/MS. Two strategies were applied with a bottom-up approach for plasma and top-down analyses for urine. Both sample preparation procedures were based on solid-phase extraction for enrichment and sample clean-up. Method validation showed good results for plasma and urine with limits of detection (LODs) for G and DAG between 30 and 50 pg/ml, recoveries between 45–50%, and imprecisions (intra- and inter-day) between 3% and 24%. Plasma analysis was also valid for quantification with accuracies determined with ~100% for G and ~106% for DAG. The minimum required performance level for doping control laboratories is set to 2 ng/ml in urine, and the herein established method yielded acceptable results even at 5% of this level. As proof-of-concept, plasma levels (G and DAG) of healthy volunteers were determined and ranged between 30 and 100 pg/ml for G and 100–1200 pg/ml for DAG. In contrast to earlier reported studies using ligand binding assays for urinary G and DAG, in this mass spectrometry-based study, no endogenous urinary G and DAG were found, although the LODs should enable this.     

Improved method to measure aldehyde adducts to N-terminal valine in hemoglobin using 5-hydroxymethylfurfural and 2,5-furandialdehyde as model compounds

Hemoglobin (Hb) adducts are used to measure reactive compounds/metabolites in vivo. Schiff base adducts from aldehydes to N-termini in Hb have been measured by GC–MS/MS after stabilisation through reduction, and detachment by a modified Edman procedure. This paper describes a further development using 5-hydroxymethylfurfural (HMF) and its probable metabolite, 2,5-furandialdehyde (FDA), as model compounds. Reference compounds were synthesized and characterized. The conditions for the reduction of the Schiff bases were optimized using NaBH₃CN as a mild reducing agent, and steps used in the earlier method could be deleted. The adduct from FDA could not be specifically analysed, as selective reduction of the imine could not be achieved. In a few samples of human blood, background levels of 10–35 pmol/g globin of the HMF adduct were observed. Half-lifes of the reversible Schiff base adduct from HMF were determined to 3.4 h at 37 °C and 10.9 h at 25 °C. The developed method showed good sensitivity and reproducibility for the analysis of the Schiff base from HMF, with improvements regarding simplicity of work-up procedures due to mild conditions. The developed method could be explored for application to adducts from other aldehydes bound as Schiff bases to N-termini in Hb.     

Determination of paclitaxel and other six taxoids in Taxus species by high-performance liquid chromatography–tandem mass spectrometry

A method of high-performance liquid chromatography–tandem mass spectrometry (LC–MS-MS) has been developed for the trace analysis of paclitaxel and other six taxoids in three Taxus species including Taxus cuspidata, Taxus media and Taxus chinensis var. mairiei. Seven taxoids were separated using a gradient mode on an Eclipse XDB-C18 column (4.6 × 150 mm; i.d., 5 μm) at 20 °C. The compound separations were detected by an API 3000 mass spectrometer equipped with a TurboIonSpray™ interface. The compounds were detected using electrospray ionization (ESI) in positive-ion mode and quantified by multiple-reaction monitoring (MRM) mode using the transition mass of m/z 567.4 → 445.4, 609.3 → 549.5, 944.9 → 286.4, 812.6 → 286.1, 832.8 → 264.1, 854.4 → 286.1, and 812.6 → 286.1 for 10-DAB III, baccatin III, 7-xyl-10-DAT, 10-DAT, cephalomannine, paclitaxel, and 7-epi-10-DAT, respectively. The ranges of limit of quantitation (LOQ) for taxoids were 32, 14, 26, 14, 20, 32, and 26 ng/mL for 10-DAB III, baccatin III, 7-xyl-10-DAT, 10-DAT, cephalomannine, paclitaxel, and 7-epi-10-DAT, respectively. Linearity was confirmed over the whole calibration range (0.07–45, 0.058–37.5, 0.058–37.5, 0.056–36.3, 0.053–33.8, 0.057–37.5, and 0.06–38.8 μg/mL for 10-DAB III, baccatin III, 7-xyl-10-DAT, 10-DAT, cephalomannine, paclitaxel, and 7-epi-10-DAT, respectively) with coefficients higher than 0.9903. The inter- and intra-day precision of taxoids ranged from 2.86% to 6.31% for retention time and ranged from 3.91% to 7.33% for peak area. The recovery rates of this method were higher than 94.32% for 10-DAB III, 94.68% for baccatin III, 93.65% for 7-xyl-10-DAT, 93.29% for 10-DAT, 92.91% for cephalomannine, 93.41% for paclitaxel, and 93.06% for 7-epi-10-DAT, respectively.     

Quantification of the major metabolites of bromhexine in human plasma using RRLC–MS/MS and its application to pharmacokinetics

(E)-4-hydroxydemethylbromhexine (E-4-HDMB) and (E)-3-hydroxydemethylbromhexine (E-3-HDMB) were found as major metabolites, while (Z)-4-hydroxydemethylbromhexine and (Z)-3-hydroxydemethylbromhexine as minor metabolites of bromhexine in human plasma. These compounds were identified in comparison with synthetic authentic samples. A sensitive and selective rapid resolution liquid chromatography tandem mass spectrometry (RRLC–MS/MS) method was developed to quantify the concentration of bromhexine and its two major metabolites (E-4-HDMB and E-3-HDMB) in human plasma. Following solid phase extraction, the analytes were separated on a Zorbax 1.8 μm particle size reversed-phase C₁₈ column, using a gradient elution program with solvents consisting of 0.1% formic acid in acetonitrile and 0.1% formic acid in 5 mM ammonium acetate at a flow rate of 0.7 mL/min. Detection was carried out with an Agilent 6460 triple-quadrupole mass spectrometer operated with an electrospray ionization source mode operated in the positive ion mode. The recovery of bromhexine, E-4-HDMB, E-3-HDMB, and internal standard (IS) was 63.1–70.9%, 60.5–68.4%, 57.0–63.5%, and 87.8%, respectively. The matrix factors of bromhexine, E-4-HDMB, E-3-HDMB, and IS were 89.9–96.7%, 89.6–94.8%, 90.4–91.4%, and 103%, respectively. After an oral administration of 8.0 mg bromhexine to five healthy male subjects, AUC_0–24 h values of bromhexine, E-4-HDMB, and E-3-HDMB were found to be 93.5 ± 31.9, 34.0 ± 14.5, and 15.8 ± 6.89 ng h/mL, respectively; while C_max values were 24.6 ± 5.16, 3.11 ± 1.13, and 5.36 ± 2.55 ng/mL, respectively. Plasma concentration of bromhexine, E-4-HDMB, and E-3-HDMB declined with t_1/2 which gave 3.6 ± 0.5, 8.4 ± 2.7, and 6.4 ± 2.5 h, respectively.     

Liquid chromatography/tandem mass spectrometry method for determination of olanzapine and N-desmethylolanzapine in human serum and cerebrospinal fluid

A validated, accurate and sensitive LC–MS/MS method for determination of olanzapine and its metabolite N-desmethylolanzapine has been developed. The analytes were quantified by tandem mass spectrometry operating in positive electrospray ionization mode with multiple reaction monitoring. Olanzapine and desmethylolanzapine were extracted from serum or cerebral spinal fluid samples, 200 μl, with tert-butyl methyl ether using olanzapine-D3 as internal standard. Calibrations for olanzapine and desmethylolanzapine were linear within the selected range of 0.2–30 ng/ml (6–96 nM) in cerebral spinal fluid and for olanzapine in plasma, in the range of 5–100 ng/ml (16–320 nM). The method was successfully used for the analysis of samples from patients treated with olanzapine in the dose range of 2.5–25 mg/day.     

Exposure to tri-o-cresyl phosphate detected in jet airplane passengers

The aircraft cabin and flight deck ventilation are supplied from partially compressed unfiltered bleed air directly from the engine. Worn or defective engine seals can result in the release of engine oil into the cabin air supply. Aircrew and passengers have complained of illness following such “fume events”. Adverse health effects are hypothesized to result from exposure to tricresyl phosphate mixed esters, a chemical added to jet engine oil and hydraulic fluid for its anti-wear properties. Our goal was to develop a laboratory test for exposure to tricresyl phosphate. The assay was based on the fact that the active-site serine of butyrylcholinesterase reacts with the active metabolite of tri-o-cresyl phosphate, cresyl saligenin phosphate, to make a stable phosphorylated adduct with an added mass of 80 Da. No other organophosphorus agent makes this adduct in vivo on butyrylcholinesterase. Blood samples from jet airplane passengers were obtained 24-48 h after completing a flight. Butyrylcholinesterase was partially purified from 25 ml serum or plasma, digested with pepsin, enriched for phosphorylated peptides by binding to titanium oxide, and analyzed by mass spectrometry. Of 12 jet airplane passengers tested, 6 were positive for exposure to tri-o-cresyl phosphate that is, they had detectable amounts of the phosphorylated peptide FGEpSAGAAS. The level of exposure was very low. No more than 0.05 to 3% of plasma butyrylcholinesterase was modified. None of the subjects had toxic symptoms. Four of the positive subjects were retested 3 to 7 months following their last airplane trip and were found to be negative for phosphorylated butyrylcholinesterase. In conclusion, this is the first report of an assay that detects exposure to tri-o-cresyl phosphate in jet airplane travelers.