High-throughput quantification of soy isoflavones in human and rodent blood using liquid chromatography with electrospray mass spectrometry and tandem mass spectrometry detection

Soy-containing foods and dietary supplements are widely consumed for putative health benefits (e.g., cancer chemoprevention, beneficial effects on serum lipids associated with cardiovascular health, reduction of osteoporosis, relief of menopausal symptoms). However, studies of soy isoflavones in experimental animals suggest possible adverse effects as well (e.g., enhancement of reproductive organ cancer, modulation of endocrine function, anti-thyroid effects). This paper describes the development and validation of a sensitive high throughput method for quantifying isoflavones in blood from experimental animal and human studies. Serum samples containing genistein, daidzein, and equol were processed using reverse phase solid-phase extraction in the 96-well format for subsequent LC-ES/MS/MS or LC-ES/MS analysis using isotope dilution in conjunction with labeled internal standards. The method was validated by repetitive analysis of spiked blank serum and the intra-day and inter-day accuracy (88-99%) and precision (relative standard deviations from 3 to 13%) of measurement determined. The lower limit of quantification for all isoflavones was approximately 0.005 micro M using MS/MS detection, and 0.03 micro M using MS for genistein and daidzein. The degree of method performance, with respect to throughput, sensitivity and selectivity, makes this approach practical for analysis of large sample sets generated from mechanistic animal studies and human clinical trials of soy isoflavones.     

Analysis of chlormequat in human urine as a biomarker of exposure using liquid chromatography triple quadrupole mass spectrometry

In this study, a method using liquid chromatography triple quadrupole mass spectrometry (LC/MS/MS) is described for the analysis of the plant growth regulator chlormequat (CCC) in human urine. Analysis was carried out using selected reaction monitoring (SRM) in the positive ion mode. [(2)H(4)] labeled CCC as internal standard (IS) was used for quantification of CCC. The limit of detection (LOD) was determined to 0.1 ng/mL. The method was linear in the range 0.3-800 ng/mL urine and had a within-run precision of 4-9%. The between-run precision was determined at urine levels of 7.0 and 31 ng/mL and found to be 5 and 6% respectively. The reproducibility was 3-6%. To validate CCC as a biomarker of exposure, the method was applied in a human experimental oral exposure to CCC. Two healthy volunteers received 25 μg/kg b.w. CCC in a single oral dose followed by urine sampling for 46 h post-exposure. The CCC was estimated to follow a first order kinetic and a two compartment model with an elimination half-life of 2-3h and 10-14 h respectively. One hundred 24h urine samples were collected from non-occupationally exposed individuals in the general population in southern Sweden. All samples had detectable levels above the LOD 0.1 ng/mL urine. The median levels were 4 ng/mL of CCC in unadjusted urine. The levels found in the population samples are several magnitudes lower than those found in the experimental exposure, which corresponds to an oral exposure of 50% of the ADI for CCC.     

Anandamide levels in human female reproductive tissues: Solid-phase extraction and measurement by ultraperformance liquid chromatography tandem mass spectrometry

Anandamide (N-arachidonoylethanolamide), a bioactive lipid, is reported to play a role in pregnancy maintenance and parturition. Our aims were to (1) evaluate AEA levels at the human maternal:fetal interface and (2) validate the use of solid-phase extraction of AEA from tissues. AEA was analyzed in cord and maternal blood, amniotic fluid, placenta, and fetal membranes collected during Caesarean section (n = 14). Extraction efficiencies were 42 and 36% for the placenta and the fetal membranes, respectively. Tissue AEA was quantified using an isotope-dilution method and UPLC-ESI-MS/MS giving intra- and inter-day variability for tissues spiked with 0.2, 1, and 5 pmol/g AEA of less than 12%. Accuracy for these spiked samples was between 95% and 103% for fetal membranes and between 99% and 114% for placenta. Mean AEA concentrations were 2.72 ± 1.04 pmol/g for placenta and 1.19 ± 0.68 pmol/g for fetal membranes, and 0.93 ± 0.28, 0.88 ± 0.33, 0.77 ± 0.30, and 0.06 ± 0.04 nM for maternal, umbilical vein, and umbilical artery plasma and amniotic fluid. Higher AEA concentrations were found in placenta compared to fetal membranes (P < 0.0001), in umbilical vein compared with umbilical artery (P = 0.0015), and in plasma from maternal circulation compared with umbilical artery (P = 0.0152). The relevance of these changes in AEA concentrations at the maternal:fetal interface requires further investigation.     

A novel and specific method for the determination of aristolochic acid-derived DNA adducts in exfoliated urothelial cells by using ultra performance liquid chromatography-triple quadrupole mass spectrometry

Aristolochic acid nephropathy (AAN) is associated with the prolonged exposure to nephrotoxic and carcinogenic aristolochic acids (AAs). DNA adducts induced by AAs have been proven to be critical biomarkers for AAN. Therefore, accurate and specific quantification of AA-DNA adducts is important. In this study, a specific method using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed and applied for the determination of 7-(deoxyadenosin-N(6)-yl)aristolactam I (dA-AAI) in exfoliated urothelial cells of AA-dosed rats. After the isolation from urine samples, DNA in urothelial cells were subjected to enzymatic digestion and solid-phase extraction on a C(18) Sep-Pak cartridge for the enrichment of DNA adducts. The sample extracts were analyzed by reverse-phase UPLC-MS/MS with electrospray ionization in positive ion mode. The quantification of the AA-DNA adduct was performed by using multiple reaction monitoring with reserpine as internal standard. The method provided good accuracy and precision with a detection limit of 1 ng/ml, which allowed the detection of trace of dA-AAI in exfoliated urothelial cells. After one-month oral dose of AAI at 10 mg/kg/day, 2.1±0.3 dA-AAI per 10(9) normal dA was detected in exfoliated urothelial cells of rats. Compared to the traditional methods such as (32)P-postlabelling and HPLC with fluorescence detection, the developed UPLC-MS/MS method is more specific and rapid with a retention time of 4 min. The outcome of this study may have clinical significance for diagnosing and monitoring AA-associated disease because detection of DNA adducts in exfoliated urothelial cells is non-invasive and convenient.     

Identification of candidate biomarker proteins released by human endometrial and cervical cancer cells using two-dimensional liquid chromatography/tandem mass spectrometry

Candidate biomarker proteins, including chaperonin 10 and pyruvate kinase, previously discovered and identified using mass-tagging reagents with multidimensional liquid chromatography and tandem mass spectrometry (DeSouza, L.; et al. J. Proteome Res. 2005, 4, 377-386) have been identified in serum-free media of cultured endometrial cancer (KLE and HEC-1-A) and cervical cancer (HeLa) cells. These and other cancer-associated proteins were released by the cultured cells within 24 h of growth. A total of 203 proteins from the KLE cells, 86 from HEC-1-A, and 161 from HeLa are reported.     

Direct measurement of the glucuronide conjugate of 1-hydroxypyrene in human urine by using liquid chromatography with tandem mass spectrometry

To evaluate human exposure to polycyclic aromatic hydrocarbons (PAHs), we developed a rapid, simple and sensitive method for determining 1-hydroxypyrene-glucuronide (1-OHP-G) in human urine. To improve precision, a deuterated glucuronide was used as an internal standard. The method requires only 1 mL of urine. The urine was treated with a mixed-mode anion-exchange and reversed-phase solid-phase extraction cartridge (Oasis MAX). The analytes were analyzed with a C(18) reversed-phase column with a gradient elution, followed by tandem mass spectrometry with electrospray ionization in negative ion mode. The detection limit of 1-OHP-G (corresponding to a signal-to-noise ratio of 3) was 0.13 fmol/injection. Urinary concentrations of 1-OHP-G determined by this method were strongly correlated (r(2)=0.961) with concentrations of 1-hydroxypyrene by conventional HPLC with fluorescence detection.     

High-throughput quantification of the anti-leukemia drug STI571 (Gleevec) and its main metabolite (CGP 74588) in human plasma using liquid chromatography-tandem mass spectrometry

The signal transduction inhibitor STI571 (formerly known as CGP 57148B) or Gleevec received fast track approval by the US Food and Drug Administration (FDA) for treatment of chronic myeloid leukemia (CML). STI571 is a revolutionary and promising new oral therapy for CML, which functions at the molecular level with high specificity. The dramatic improvement in efficacy compared to existing treatments prompted an equally profound increase in the pace of development of Gleevec. The duration from first dose in man to completion of the New Drug Application (NDA) filing was approximately 2.6 years. In order to support all pharmacokinetics studies with sufficient speed to meet various target dates, a semi-automated procedure using protein precipitation was developed and validated. A Tomtec Quadra 96 (Model 320) and a protein precipitation step in a 96-well plate format were utilized. A Sciex API 3000 triple quadrupole mass spectrometer with an atmospheric pressure chemical ionization interface operated in positive ion mode was used for detection. The method proved to be rugged and allowed the simultaneous quantification of STI571 and its main metabolite (CGP 74588) in human plasma. Herein, assay development, validation, and representative concentration-time profiles obtained from clinical studies are presented.     

High throughput quantification of phytoestrogens in human urine and serum using liquid chromatography/tandem mass spectrometry (LC-MS/MS)

Phytoestrogens are currently the subject of intense study owing to their potential protective effects against a number of complex diseases. However, in order to investigate the interactions between phytoestrogens and disease state effectively, it is necessary to have analytical methods which are sensitive, reproducible, and require low sample volumes. We report an assay for three isoflavones (daidzein, genistein, and glycitein), two metabolites of daidzein (equol and O-desmethylangolensin), three lignans (secoisolariciresinol, enterodiol, and enterolactone), and one flavanone (naringenin) in human urine and serum. A high throughput of samples has been achieved via the use of 96-well plate sample extraction and liquid chromatography/tandem mass spectrometry (LC-MS/MS) analysis incorporating column switching, thus making the assay suitable for use on large sample numbers, such as those found in epidemiological studies. The robustness of the assay was proven via the comparison of data generated on two different LC-MS/MS systems, with and without column switching.     

Identification and quantification of peptides and proteins secreted from prostate epithelial cells by unbiased liquid chromatography tandem mass spectrometry using goodness of fit and analysis of variance

The proteins secreted by prostate cancer cells (PC3(AR)6) were separated by strong anion exchange chromatography, digested with trypsin and analyzed by unbiased liquid chromatography tandem mass spectrometry with an ion trap. The spectra were matched to peptides within proteins using a goodness of fit algorithm that showed a low false positive rate. The parent ions for MS/MS were randomly and independently sampled from a log-normal population and therefore could be analyzed by ANOVA. Normal distribution analysis confirmed that the parent and fragment ion intensity distributions were sampled over 99.9% of their range that was above the background noise. Arranging the ion intensity data with the identified peptide and protein sequences in structured query language (SQL) permitted the quantification of ion intensity across treatments, proteins and peptides. The intensity of 101,905 fragment ions from 1421 peptide precursors of 583 peptides from 233 proteins separated over 11 sample treatments were computed together in one ANOVA model using the statistical analysis system (SAS) prior to Tukey-Kramer honestly significant difference (HSD) testing. Thus complex mixtures of proteins were identified and quantified with a high degree of confidence using an ion trap without isotopic labels, multivariate analysis or comparing chromatographic retention times.     

Search for potential markers for prostate cancer diagnosis, prognosis and treatment in clinical tissue specimens using amine-specific isobaric tagging (iTRAQ) with two-dimensional liquid chromatography and tandem mass spectrometry

This study aimed to identify candidate new diagnosis and prognosis markers and medicinal targets of prostate cancer (PCa), using state of the art proteomics. A total of 20 prostate tissue specimens from 10 patients with benign prostatic hyperplasia (BPH) and 10 with PCa (Tumour Node Metastasis [TNM] stage T1-T3) were analyzed by isobaric stable isotope labeling (iTRAQ) and two-dimensional liquid chromatography-tandem mass spectrometry (2DLC-MS/MS) approaches using a hybrid quadrupole time-of-flight system (QqTOF). The study resulted in the reproducible identification of 825 nonredundant gene products (p < or = 0.05) of which 30 exhibited up-regulation (> or =2-fold) and another 35 exhibited down-regulation (< or =0.5-fold) between the BPH and PCa specimens constituting a major contribution toward their global proteomic assessment. Selected findings were confirmed by immunohistochemical analysis of prostate tissue specimens. The proteins determined support existing knowledge and uncover novel and promising PCa biomarkers. The PCa proteome found can serve as a useful aid for the identification of improved diagnostic and prognostic markers and ultimately novel chemopreventive and therapeutic targets.     

Simultaneous quantification of seven prostanoids using liquid chromatography/tandem mass spectrometry: the effects of arachidonic acid on prostanoid production in mouse bone marrow-derived mast cells

We have developed a method for the simultaneous estimation of the levels of the prostanoids 6-keto prostaglandin (PG) Flalpha, PGB2, PGD2, PGE2, PGF2(alpha), PGJ2, and thromboxane (TX) B2 in blood- or serum-containing medium using liquid chromatography-tandem mass spectrometry. These prostanoids and their deuterium derivatives, which were used as internal standards, were subjected to solid-phase extraction using Empore C18 HD disk cartridges and analyzed in the selected reaction-monitoring mode. A linear response curve starting at 10 pg of prostanoid/tube was observed for each prostanoid. The accuracy of the method was demonstrated with samples containing known amounts of the prostanoids. Furthermore, we used this method to analyze the prostanoids produced in mouse bone marrow-derived mast cells stimulated with arachidonic acid, which resulted in the production of PGD2, PGE2, PGF2alpha, and TXB2. The results suggest that this simultaneous quantification method is useful for the analysis of the production of biomedically important prostanoids.     

High-throughput sample preparation procedures for the quantitation of a new bone integrin alpha(nu)beta(3) antagonist in human plasma and urine using liquid chromatography-tandem mass spectrometry

High throughput LC-MS/MS assays to quantitate a new alpha(nu)beta(3) bone integrin antagonist (I) in human plasma and urine have been developed using instruments programmed to automate sample preparation procedures. Packard liquid handling system-MultiPROBE II EX was programmed for preparing calibration standards in control plasma and urine, acidifying all standards, quality control (QC), and clinical samples with necessary dilutions, and adding the internal standard to the acidified samples. TOMTEC Quadra 96 was programmed to perform the solid phase extraction (SPE) process on a 3M 96-well mixed phase cation standard density (MPC-SD) plate to isolate the analytes from the sample matrix. The extract collected from both types of matrices was directly injected into reversed-phase LC-MS/MS system with a Turbo Ion Spray (TIS) interface in the positive ionization mode. The plasma and urine assays have the calibration range of 0.5-1500 and 2-6000 ng/mL, respectively. Validation of the automated and the manual plasma assays showed that application of MultiPROBE II to sample preparation gave comparable accuracy and precision. Overall, the automated approaches with minimum manual intervention enhanced the throughput of sample preparation.     

Adduct formation of Thimerosal with human and rat hemoglobin: a study using liquid chromatography coupled to electrospray time-of-flight mass spectrometry (LC/ESI-TOF-MS)

Thimerosal (THI) is used as a preservative in many vaccines throughout the world. Ethylmercury (EtHg(+)), released from THI in aqueous media, has a high affinity to thiol functions of proteins. In blood, hemoglobin is a likely target protein because of its high abundance and its several free thiol functions. In comparison to hemoglobin of human origin, hemoglobin of rats exhibits almost twice as many free thiol groups, which might lead to different binding behavior and therefore a limited comparability between the situation in man and in rats, which are frequently used as models for mercury species toxicity investigations. Thus, the adduct formation of EtHg(+) with hemoglobin of humans and rats was compared under simulated physiological conditions by using gradient reversed-phase liquid chromatography (LC) with electrospray time-of-flight mass spectrometry (ESI-TOF-MS) detection. The binding stoichiometry correlated with the number of free thiols in the α- and β-chain of hemoglobin. The use of rats to verify the safety of additives in vaccines like Thimerosal is therefore doubtful and should be reevaluated.     

Comprehensive metabolite profiling of onion bulbs (<Emphasis Type="Italic">Allium cepa</Emphasis>) using liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry

Introduction

Onion (Allium cepa) represents one of the most important horticultural crops and is used as food, spice and medicinal plant almost worldwide. Onion bulbs accumulate a broad range of primary and secondary metabolites which impact nutritional, sensory and technological properties.

Objectives

To complement existing analytical methods targeting individual compound classes this work aimed at the development and validation of an analytical workflow for comprehensive metabolite profiling of onion bulbs.

Method

Metabolite profiling was performed by liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (LC/ESI-QTOFMS). For annotation of metabolites accurate mass tandem mass spectrometry experiments were carried out.

Results

On the basis of LC/ESI-QTOFMS and two chromatographic methods an analytical workflow was developed which facilitates profiling of polar and semi-polar onion metabolites including fructooligosaccharides, proteinogenic amino acids, peptides, S-substituted cysteine conjugates, flavonoids and saponins. To minimize enzymatic conversion of S-alk(en)ylcysteine sulfoxides, a sample preparation and extraction protocol for fresh onions was developed comprising cryohomogenization and a low-temperature quenching step. A total of 123 metabolites were annotated and characterized by chromatographic and tandem mass spectral data. For validation, recovery rates and matrix effects were determined for 15 model compounds. Repeatability and linearity were assessed for more than 80 endogenous metabolites.

Conclusion

As exemplarily demonstrated by comparative metabolic analysis of six onion cultivars the established analytical workflow in combination with targeted and non-targeted data analysis strategies can be successfully applied for comprehensive metabolite profiling of onion bulbs.

     

Elimination of diastereomer interference to determine Telcagepant (MK-0974) in human plasma using on-line turbulent-flow technology and off-line solid-phase extraction coupled with liquid chromatography/tandem mass spectrometry

To eliminate the diastereomer interference on Telcagepant (MK-0974) determination during clinical study support, on-line high turbulent-flow liquid chromatography (HTLC) methods, HTLC-A and HTLC-B that covered dynamic range of 0.5–500 nM and 5–5000 nM, respectively, were developed. To meet the requirement of rapid assay transfer among multiple laboratories and analysts, a solid-phase extraction (SPE) assay was derived from the existing HTLC-B assay under the same dynamic range. The on-line HTLC assays were achieved through direct injection of plasma samples, extraction of analyte with a Cohesive C₁₈ column (50 mm × 0.5 mm, 50 μm), followed by HPLC separation on a FluoPhase RP column (100 mm × 2.1 mm, 5 μm) and MS/MS detection. The off-line SPE assay used Waters Oasis^®HLB μElution plate to extract the analytes from plasma matrix before injecting on a FluoPhase RP column (150 mm × 2.1 mm, 5 μm) for LC–MS/MS analysis. Under both on-line and off-line assay conditions, the diastereomer 1c was chromatographically separated from MK-0974. Cross-validation with the pooled samples demonstrated that both on-line and off-line assays provided comparable data with a difference of <2.6%. The assays were proved to be specific, accurate and reliable, and have been used to support multiple clinical studies. The pros and cons of on-line and off-line assays with regard to man power involved in sample preparation, total analysis time, carryover, cost efficiency, and the requirement for assay transfer are discussed.     

A study of the stability of tri(glucosyloxyphenyl)chlorin, a sensitizer for photodynamic therapy, in human colon tumoural cells: a liquid chromatography and MALDI-TOF mass spectrometry analysis

Asymmetrical glycoconjugated tetrapyrrolic macrocycles are under study as efficient sensitizers for photodynamic therapy (PDT). In this context, tri(meta-O-beta-glucopyranosyloxyphenyl)chlorin [TPC(m-O-Glu)(3)] 2a/3a was found to be four times more photoactive in vitro than Foscan. In a further study of this interesting glycoconjugate, its metabolism by cellular glycosidases in HT29 cells has to be explored. Cellular extracts of HT29 cells incubated with TPC(m-O-Glu)(3) (24h, 6microM) were analyzed by MALDI-TOF mass spectrometry and high performance liquid chromatography (HPLC). In MALDI-TOF mass spectra, the presence of compounds distinct from TPC(m-O-Glu)(3) (m/z 1151) were observed at m/z 989, 827 and 665 corresponding to the loss of one, two or three glucose units (162u) and were be ascribed to TPC(m-OH)(m-O-Glu)(2) 2/3b,b',b", TPC(m-OH)(2)(m-O-Glu) 2/3c,c',c" and TPC(m-OH)(3) isomers 2d/3d, respectively. The porphyrins resulting from chlorin oxidation TPP(m-O-Glu)(3) 4a, TPP(m-OH)(m-O-Glu)(2) 4b,b", TPP(m-OH)(2)(m-O-Glu) 4c,c" and TPP(m-OH)(3) 4d were also observed. The HPLC profile (lambda(anal)=420 nm) showed eight peaks consistent with mass spectra. The kinetics of deglucosylation was studied from HPLC profiles between 1 and 48h incubation. The concentration of triglucoconjugated and diglucoconjugated molecules was maximum around 3 and 8h incubation, respectively, whereas, totally deglucosylated species appeared only after incubation for more than 10h. The fully deglycosylated porphyrin TPP(m-OH)(3) is the final metabolite, being observed at a concentration 15 times higher than that of the remaining TPC(m-O-Glu)(3) 2a/3a. Compared to the photobiological activity of the parent molecule [TPC(m-O-Glu)(3)], a three times higher TPP(m-OH)(3) concentration was necessary to observe a similar in vitro photoactivity.     

Validation and clinical application of a high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the quantitative determination of 10 anti-retrovirals in human peripheral blood mononuclear cells

This paper reports the validation of a liquid chromatography tandem mass spectrometry (LC-MS/MS) method that allows the quantification of 10 antiretroviral (ARV) drugs in peripheral blood mononuclear cells (PBMCs) using 6 different isotopic internal standards (IS) and its clinical application. PBMCs are isolated from blood by density gradient centrifugation and drugs are extracted with a 60% methanol (MeOH) solution containing the 6 IS. The cell extract is then injected in the HPLC system and analytes are separated on a Symmetry Shield RP18 2.1 mm × 50 mm column. The different molecules are then detected by MS/MS in electrospray positive or negative ionisation modes and data are recorded using the multiple reaction monitoring (MRM) mode. Calibration curves are constructed in the range of 0.25–125 ng/ml of cell extract by a 1/x² weighted quadratic regression. The regression coefficients obtained are always greater than 0.99 and back calculated values always comprised in the range of ±15% from their nominal concentration. Mean extraction recoveries are greater than 80% for all analytes and the method is accurate and precise with CV and bias lower than 9.4%. The lower limits of quantification (LLOQ) of the different drugs range from 0.0125 to 0.2 ng/ml of cell extract. This method was successfully applied to a cohort of 98 HIV-infected patients treated with Kaletra^® (400/100 mg of lopinavir/ritonavir (LPV/RTV) twice a day, n = 48) or with Stocrin^® (600 mg once a day, n = 50) and has been tested for cellular quantification of tipranavir (TPV) in 2 patients treated with Aptivus^® (500 mg twice a day). The patients treated by Kaletra^® showed mean cell-associated concentrations (CC) of 1819.0 and 917.2 ng/ml, for LPV and RTV, respectively. Patients treated with Stocrin^® showed mean CC of 2388.11 ng/ml while both patients under Aptivus^® showed TPV CC of 4322.7 and 1078.0 ng/ml, respectively. This method can be used to analyze ARV drug concentrations within the target tissue.     

A modified method for the determination of tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in human urine by solid phase extraction using a molecularly imprinted polymer and liquid chromatography tandem mass spectrometry

The work described in this paper represents an improvement over a previously published method for the determination of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in human urine by solid phase extraction on a molecularly imprinted polymer column coupled with HPLC and -MS/MS detection. The influence of ion suppression due to sample matrix effect was evaluated, and found to influence the response of NNAL. By changing the liquid chromatography conditions, the response for this method was enhanced approximately 25-fold through avoidance of ionization suppression that was found with a previously published method and sample throughput has been improved. The dynamic range of the assay extends from 20 to 2500 pg/mL with a mean r² > 0.998. The lower limit of quantitation for the assay was 20 pg/mL despite the use of an inherently lower sensitivity instrument. The method was validated according to current FDA guidelines for bioanalytical method validations.