Simultaneous quantitation of testosterone and estradiol in human cell line (H295R) by liquid chromatography/positive atmospheric pressure photoionization tandem mass spectrometry

The possible interaction of environmental contaminants with the endocrine system has been an environmental concern since the early 1990s. To examine these interactions test guidelines have been introduced by regulatory agencies to screen for possible endocrine active compounds. One of these guidelines is the EPA's OPPTS 890.1550 [Steroidogenesis (Human Cell Line‐H295R)]. This guideline requires the quantification of two major biomarkers (testosterone and estradiol) in various biological test systems. Traditional quantitation methodologies such as Radioimmunoassay (RIA) and Enzyme‐linked Immunosorbent Assay (ELISA) have been used to quantify low levels of steroids. However, those methodologies have drawbacks such as the radioactive safety, antibody availability, separate assay for each biomarker, and lack of selectivity. In the current study, a rapid and sensitive liquid chromatography/positive atmospheric pressure photoionization tandem mass spectrometry method (LC/APPI‐MS/MS) has been developed and validated for the simultaneous quantitation of testosterone and estradiol in the H295R cell line. Briefly, the media from cultured cells was extracted with dichloromethane (CH₂Cl₂) containing internal standards of both testosterone‐d₃ and estradiol‐¹³C₃; then, the extracted organic layer was concentrated down to dryness. The final residue was derivatized with dansyl chloride solution, and directly analyzed by LC/APPI‐MS/MS. The calibration curves, with concentration ranging from 10 to 2500 pg/mL, were linear with coefficient >0.99. The lower limits of quantitation for both testosterone and estradiol were 10 pg/mL. This method was successfully validated to support requirements of the current EPA Steroidogenesis guideline. This type of method may also provide value for rapid and precise measurements of these two hormones in other in vitro or in vivo test systems. Copyright © 2011 John Wiley & Sons, Ltd.     

Quantitation of 8-hydroxydeoxyguanosine in DNA by liquid chromatography/positive atmospheric pressure photoionization tandem mass spectrometry

A methodology has been developed and validated for quantifying 8-hydroxydeoxyguanosine (8-OHdG) in both commercial DNA and DNA isolated from livers of male Sprague-Dawley rats by liquid chromatography/positive atmospheric pressure photoionization tandem mass spectrometry. The analytical method conditions, including conditions for stabilizing 8-OHdG during complex nuclease P1 enzymatic digestion, were also evaluated. The limit of detection for 8-OHdG was 1.0 ng/mL (17.6 fmol on-column), and the linearity of the calibration curve was greater than 0.998 from 1.0 to 500 ng/mL. The intraday assay precision relative standard deviation (RSD) value for quality control (QC) samples was < or =5.59% with accuracies ranging from 91.84 to 117.61%. The interday assay precision (RSD) value was < or =1.76% with accuracies ranging from 91.84 to 116.67%. This method, combined with the LC/UV analysis of deoxyguanosine (dG), was used for determination of the levels of 8-OHdG/10(6) dG in DNA nuclease P1 enzymatic hydrolysates from both commercial DNA and rat liver DNA.     

Liquid chromatography/atmospheric pressure photoionization tandem mass spectrometry for analysis of Dechloranes

Liquid chromatography/atmospheric pressure photoionization tandem mass spectrometry (LC/APPI-MS/MS) was investigated as an instrumental method for the analysis of the halogenated norbornene flame retardants, Mirex, Dechloranes 602, 603, 604, and Dechlorane Plus (DP). The LC separation was optimized by screening a variety of stationary and mobile phases, resulting in a short LC separation time of 5 min. Different atmospheric pressure ionization approaches were examined including electrospray ionization, atmospheric pressure chemical ionization, and APPI, each with and without post-column addition. APPI without post-column addition was chosen for providing the best ionization response. The optimized LC/APPI-MS/MS approach resulted in instrument detection limits ranging between 25 and 50 pg. Good linearity was also achieved (up to 25.0 ng/μL; R >0.999). The method was applied to extracts of environmental samples including surface water, fish and sediments for screening purposes, and the results agreed well with those obtained by gas chromatography/mass spectrometry.     

Determination of nivalenol and deoxynivalenol by liquid chromatography/atmospheric pressure photoionization mass spectrometry

Fusarium species, a plant pathogenic fungus of wheat and other cereals, produces toxic metabolites such as nivalenol (NIV) and deoxynivalenol (DON). Control of contamination by these toxins is very difficult, and a continuous survey of the occurrence is necessary for these toxins. Thus, the accurate and convenient determination of the cereals contaminated with these toxins is important for the supply of safe foods. A selective analytical method based on high‐performance liquid chromatography, combined with atmospheric pressure photoionization (APPI) mass spectrometry, has been developed for simultaneous determination of NIV and DON. The parameters investigated for the optimization of APPI were the ion source parameters fragmentor voltage, capillary voltage, and vaporizer temperature, and also mobile phase composition and flow rate. Furthermore, chemical noise and signal suppression of analyte signals due to sample matrix interference were investigated for APPI. The results indicated that APPI provides lower matrix effect and the correlation coefficient of NIV and DON in the range 0.2–100 ng · mL^?1 was above 0.999. Recoveries of NIV and DON in wheat ranged from 86 to 107% and limits of detection of NIV and DON were 0.20 ng · g^?1 and 0.39 ng · g^?1, respectively. In addition, the proposed method was applied for the analysis of naturally contaminated wheat samples. APPI was found to offer lower matrix effect and was a convenient technique for routine analysis of NIV and DON residues in wheat at trace levels. Copyright © 2009 John Wiley & Sons, Ltd.     

Trace-level quantitation of iralukast in human plasma by microbore liquid chromatography/tandem mass spectrometry

Iralukast (CGP 45715A) is a potent peptido-leukotriene antagonist that is active in various in vitro and animal models for the treatment of asthma. An analytical challenge was to develop a sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method with a lower limit of quantitation (LLOQ) of 10 pg/mL for the analysis of iralukast when administered at low doses during clinical trials. Several issues had to be addressed in order to devise a LC/MS/MS assay for the above compound. First, iralukast appeared to be light sensitive and unstable at room temperature under acidic conditions. Second, a LLOQ of 10 pg/mL was needed to support several clinical trials. Third, positive electrospray ionization of iralukast did not yield the necessary sensitivity required for studies in humans. Consequently, LC/MS/MS conditions were optimized for the negative ion mode of detection. Fourth, sample preparation steps proved to be critical to reduce the possibility of microbore HPLC column (50 mm x 1.0 mm i.d.) obstruction, chromatographic deterioration, and matrix-mediated electrospray ion suppression. While our validated method addressed the above challenges, its major drawback was limited sample throughput capability. Nonetheless, plasma concentration-time profiles for patients with moderate asthma after oral administration of 200, 500, 1000, and 5000 microgram/kg/day of iralukast were successfully obtained.     

A dopant introduction device for atmospheric pressure photoionization with liquid chromatography/mass spectrometry

This technical note describes in detail the fabrication, operation and characterization of a pneumatically driven dopant introduction device, with a solvent reservoir capacity of 300 mL. Dopant flow rates and stability for this device are governed by the simple regulation of gas pressure rather than the progression of a stepper motor and syringe diameter, as is the case for typical infusion pumps. The device has the potential to provide days or even weeks of continuous, uninterrupted dopant flow at rates commonly adopted for atmospheric pressure photoionization (APPI) experiments without the need to replenish the dopant supply. Although not a refined instrumental design, this device was developed as an alternative cost-effective means of introducing stable dopant flow to an APPI source. The device was designed such that all components would be commercially available and easily procurable from common scientific part vendors. Figures and suggested part numbers are provided to allow those interested to fabricate similar devices to suit their individual experimental needs. Device characterization was performed while monitoring such factors as flow rate calibration, overall flow stability and reproducibility. In addition, a standard mixture of three polycyclic aromatic hydrocarbons was employed as a model sample for a typical reversed-phase liquid chromatography/atmospheric pressure photoionization mass spectrometry (LC/APPI-MS) application in order to demonstrate device performance.     

Zirconia-based column high-performance liquid chromatography/atmospheric pressure photoionization tandem mass spectrometric analyses of drug molecules in rat plasma

A method using zirconia-based column high-performance liquid chromatography (HPLC) interfaced with an atmospheric pressure photoionization (APPI) source and a tandem mass spectrometer (MS/MS) was developed for the quantitative determination of new chemical entities in rat plasma in support of pharmacokinetics studies. The ionization suppression resulting from endogenous components of the biological matrices on the quantitative zirconia-based column HPLC/APPI-MS/MS method was investigated using the post-column infusion technique. The analytical results for 'rapid rat pharmacokinetics' for 12 drug discovery compounds, obtained by both silica-based phase (S-phase) and zirconia-based phase (Z-phase) chromatographic separation, are in good agreement in terms of accuracy. The application of a Z-phase column for high-temperature fast HPLC/MS/MS methods was explored to reduce the analysis time from 3 min to 30 s for column temperatures of 25-110 degrees C, respectively. The chromatographic retention times and peak responses of all analytes were found to be reproducible under high-temperature conditions following 100 continuous injections, with %CV less than 0.4 and 5, respectively.     

Simultaneous quantitation of enalapril and enalaprilat in human plasma by 96-well solid-phase extraction and liquid chromatography/tandem mass spectrometry

A sensitive and rapid method based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) combined with rapid solid-phase extraction (SPE) has been developed and validated for the quantitative determination of enalapril and its active metabolite enalaprilat in human plasma. After addition of internal standard to human plasma, samples were extracted by 96-well SPE cartridge. The extracts were analyzed by HPLC with the detection of the analyte in the multiple reaction monitoring (MRM) mode. This method for the simultaneous determination of enalapril and enalaprilat was accurate and reproducible, with respective limits of quantitation of 0.2 and 1.0 ng/mL in plasma. The standard calibration curves for both enalapril and enalaprilat were linear (r(2) = 0.9978 and 0.9998) over the concentration ranges 0.2-200 and 1.0-100 ng/mL in human plasma, respectively. The intra- and inter-day precision over the concentration range for enalapril and enalaprilat were lower than 13.3 and 15.4% (relative standard deviation, %RSD), and accuracy was between 89.2-105.0 and 91.9-104.7%, respectively.     

Comparison of electrospray ionization,atmospheric pressure chemical ionization and atmospheric pressure photoionization for determining estrogenic chemicals in water by liquid chromatography tandem mass spectrometry with chemical derivatizations

This study compared the sensitivities and matrix effects of four ionization modes and four reversed-phase liquid chromatographic (LC) systems on analyzing estrone (E1), 17β-estradiol (E2), estriol (E3), 17α-ethinylestradiol (EE2), 4-nonylphenol (NP), 4-tert-octylphenol (OP), bisphenol A (BPA) and their derivatives of dansyl chloride or pentafluorobenzyl bromide (PFBBr) in water matrixes using a triple-quadrupole mass spectrometer with selected reaction monitoring (SRM). The four probes were electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), atmospheric pressure photoionization (APPI) and APCI/APPI; the four LC systems were ultra-performance liquid chromatography (UPLC) with or without post-column split, a mixed-mode column and two-dimensional LC (2D-LC). Dansylated compounds with ESI at UPLC condition had the most intense signals and less matrix effects of the various combinations of ionization and LC systems. The on-column limits of detection (LODs) of dansylated estrogens by SRM were 0.05–0.20 pg, and the LODs in sewage treatment plant effluent and in river water were 0.23–0.52 and 0.56–0.91 ng/L, respectively. The LODs using selected ion monitoring (SIM) reached low ng/L levels in real samples and measured concentrations were comparable with those of SRM.     

An electropneumatic‐heated nebulizer for enhancing spray ionization in PhotoSpray atmospheric pressure photoionization sources for liquid chromatography/mass spectrometry

We introduce a novel electropneumatic‐heated nebulizer (EPn‐HN), incorporating an electrified internal pneumatic nebulizer, to enhance the yield of sprayed ions from PhotoSpray? atmospheric pressure photoionization (APPI) sources for liquid chromatography/mass spectrometry (LC/MS). Spray ionization from the pneumatic‐heated nebulizers used in APPI sources provides a supplemental, complementary ionization method to be used for involatile and thermally labile compounds, otherwise intractable to APPI. Details of the construction and operation of the EPn‐HN device are provided. The performance of the EPn‐HN is demonstrated using two model compounds: substance P, a peptide used as a standard in studies of ion fragmentation mechanisms, and aztreonam, a thermally labile antibiotic. At the optimum voltage for spray ionization, improvements in sensitivity of two orders of magnitude are obtained relative to when the sprayer is grounded, the conventional case. Since both substance P and aztreonam cannot be detected using the APPI method alone, the results demonstrate how spray ionization from the EPn‐HN may be used to extend the range of compounds amenable to PhotoSpray sources. Copyright © 2009 John Wiley & Sons, Ltd.     

Simultaneous analysis of twenty-one glucocorticoids in equine plasma by liquid chromatography/tandem mass spectrometry

A method for the simultaneous separation, identification, quantification and confirmation of the presence of 21 glucocorticoids (GCC) in equine plasma by liquid chromatography coupled with triple stage quadrupole tandem mass spectrometry (LC/TSQ-MS/MS) is described. Plasma sample augmented with the 21 GCC was extracted with methyl tert-butyl ether (MTBE) and analyzed by positive electrospray ionization. Desoxymetasone or dichlorisone acetate was used as the internal standard (IS). Quantification was performed by IS calibration. For each drug, one major product ion was chosen and used for screening for that drug. Analyte confirmation was performed by using the three most intense product ions formed from the precursor ion and the corresponding mass ratios. The recovery of the 21 GCC when spiked into blank plasma at 5 ng/mL was 45-200% with coefficient of variation (CV) from 0.3-18%. The limit of detection (LOD) and that of quantification (LOQ) for most of the analytes were 50-100 pg/mL and 1 ng/mL, respectively, whereas that of confirmation (LOC) was 100-300 pg/mL depending on the analyte. Intra- and inter-day precisions expressed as CV for quantification of 1 and 10 ng/mL was 1.0-17%, and 0.51-19%, respectively, and the accuracy was from 84-110%. The linear concentration range for quantification was 0.1-100 ng/mL (r(2) > 0.997). Estimated measurement uncertainty was from 11-37%. This study was undertaken to develop a method for simultaneous screening, identification, quantification and confirmation of these agents in post-race equine plasma samples. The method has been successfully applied to screening of a large number of plasma samples obtained from racehorses in competition and in pharmacokinetic studies of dexamethasone in the horse and concurrent changes in endogenous GCC, hydrocortisone and cortisone. The method is simple, sensitive, selective and reliably reproducible.     

Simultaneous determination of risperidone and 9-hydroxyrisperidone in plasma by liquid chromatography/electrospray tandem mass spectrometry

A simple and highly sensitive liquid chromatographic/electrospray tandem mass spectrometric (LC/MS/MS) assay was developed for the simultaneous determination of risperidone (RSP) and its major circulating metabolite 9-hydroxyrisperidone (9-OH-RSP) in the plasma of humans and rats. A simple one-step solvent extraction with 15% methylene chloride in pentane was used to isolate the compounds from plasma. The compounds were eluted from a phenyl-hexyl column and detected with a Perkin-Elmer SCIEX API2000 triple-quadrupole mass spectrometer using positive ion atmospheric pressure electrospray ionization and multiple reaction monitoring. The assay was linear over the range 0.1-100 ng ml(-1) when 0.5 ml of plasma was used in the extraction. The overall intra- (within-day) and inter- (between days) assay variations were < 11%. The variations in the concentrations of two long-term quality control samples from pooled patient plasma samples analyzed over a period of 6 months were approximately 10%. The analysis time for each sample was 4 min and more than 100 samples could be analyzed in one day by running the system overnight. The assay is simple, highly sensitive, selective, precise and fast. This method is being used for the therapeutic drug monitoring of schizophrenic patients treated with RSP and to study the pharmacokinetics and tissue distribution of RSP and 9-OH-RSP in rats.     

Simultaneous determination of four shanzhiside methylester derivatives in rabbit plasma by liquid chromatography/tandem mass spectrometry

A simple and sensitive high‐performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and validated for simultaneous determination of shanzhiside methylester and its three derivatives in rabbit plasma. The method showed good linearity and no endogenous material interfered with the marked compounds and internal standard (IS) capatol peaks. Samples were processed by acetonitrile precipitation. Chromatography was performed using a C₁₈ column (150 × 3.9 mm i.d., 4 µm). The mobile phase consisted of methanol and water (60:40, v/v) during a total run time of 7 min. The main mass parent ions and daughter ions pairs (m/z) for monitoring were: shanzhiside methylester, 429.0/267.4; 8‐O‐acetyl shanzhiside methylester, 470.9/411.3; loganin, 413.2/251.4; phloyoside II, 479.2/281.3; and IS 385.2/203.3. Finally, the method was applied to a pharmacokinetic study of rabbits following intravenous administration of iridoid glycosides extracted from traditional herb Lamiophlomis rotata. Copyright © 2012 John Wiley & Sons, Ltd.     

Direct and simultaneous quantitation of 5‐aminolaevulinic acid and porphobilinogen in human serum or plasma by hydrophilic interaction liquid chromatography–atmospheric pressure chemical ionization/tandem mass spectrometry

Serum/plasma concentrations of 5‐aminolaevulinic acid (ALA) and porphobilinogen (PBG) are elevated in patients with acute hepatic porphyrias, especially during acute attacks. Current assays require lengthy sample pre‐treatment and derivatization steps. We report here a rapid, sensitive and specific hydrophilic interaction liquid chromatography–tandem mass spectrometry method for the direct and simultaneous quantitation of ALA and PBG in serum or plasma following simple protein precipitation with acetonitrile and centrifugation prior to injection. ALA and PBG were detected using selected reaction monitoring mode, following positive atmospheric pressure chemical ionization. Calibration was linear from 0.05 to 50 µmol/L for ALA and PBG. For both analytes, imprecision (relative standard deviation) was <13% and accuracy (percentage nominal concentrations) was between 92 and 107%. The method was successfully applied to the measurement of ALA and PBG in serum or plasma samples for the screening, biochemical diagnosis and treatment monitoring of patients with acute hepatic porphyrias. Copyright © 2012 John Wiley & Sons, Ltd.     

Simultaneous quantitative analysis of polyphenolic compounds in human plasma by liquid chromatography tandem mass spectrometry

A diet rich in polyphenolic compounds has recognized health benefits, and as such is routinely monitored as part of dietary intervention studies. A method for the simultaneous determination of 36 phenolic compounds, including phenolic acids and flavonoids, using liquid chromatography and tandem mass spectrometry is described here. The target analytes were quantified based on their specific mass spectral fragments using a selected reaction monitoring approach. A C18 column with embedded aromatic functionality ensured separation of all phenolic compounds studied which included several pairs of isomers. Sample preparation involved the use of β‐glucuronidase to release the phenolic compounds from their conjugated forms. The intra‐day and inter‐day precision and accuracy was less than 7% for all phenolic compounds studied. Recoveries, where plasma was spiked with three different concentrations of the analytes, ranged from 95–115%. The limits of detection and quantification were 0.23–3.89 and 1.15–7.79 nM, respectively. The method was successfully applied to real samples and the range reported for each phenolic compound, with the exception of hydroferulic acid, nordihydroguaiaretic acid, methylgallate, and m‐coumaric acid, was at least an order of magnitude higher than the limit of quantification for the method.     

Study of polybrominated diphenyl ethers using both positive and negative atmospheric pressure photoionization and tandem mass spectrometry

Atmospheric pressure photoionization (APPI) was assessed for the mass spectrometric analysis of polybromodiphenyl ethers (PBDEs) on the basis of a set of 17 standard compounds. Positive and negative ionization modes were both investigated. M(+.) ions were formed under positive ion conditions whereas the negative ion mode yielded [M-Br+O](-) ions. The behavior of these APPI-produced ions towards collisional activation was studied using an ion trap mass spectrometer. In positive ion mode, the loss of Br(2) was one of the major fragmentation pathways, and was favored for ortho-substituted PBDEs. Conversely, the loss of COBr(.) occurred only for non-ortho-substituted congeners. The collisional excitation of [M-Br+O](-) ions in the ion trap also led to the loss of Br(2), to the elimination of HBr, and to the formation of product ions by cleavage of the ether bond. The formation of para-quinone radical anions was observed for PBDEs ranging from penta- to hepta-congeners, whereas brominated aromatic carbanions were formed preferentially for the most brominated PBDEs studied in this work (hepta- or deca-BDEs). M(+.) ions did not undergo this fragmentation process.