Quantification of intracellular homocysteine by stable isotope dilution liquid chromatography/tandem mass spectrometry

A precise and accurate stable isotope dilution liquid chromatography/tandem mass spectrometry method for the analysis of intracellular homocysteine has been developed. An internal standard, [(2)H(8)]-homocystine, was added to cell pellets from EA.hy 926 cells grown in culture under low and high folate concentrations. D,L-dithiothreitol was used to reduce cellular homocystine to homocysteine. Cellular proteins were precipitated by the addition of formic acid in acetonitrile. After centrifugation, a portion of the supernatant was analyzed by liquid chromatography/tandem mass spectrometry. Using a Supelcosil cyano column with an Applied Biosystems API 4000 triple quadrupole mass spectrometer, the SRM transitions for homocysteine (m/z 136 to m/z 90) and [(2)H(4)]-homocysteine (m/z 140 to m/z 94) were monitored. The method was validated by conducting five replicate analyses on three different days at four different concentrations (concentrations at the lower limit of quantitation and expected lower quartile, mid-range and upper quartile). The limit of detection was 2 ng/10(6) EA.hy 926 cells. Using this method, the intracellular homocysteine concentration in EA.hy 926 cells ranged from 10 to 36 ng/10(6) cells.     

Quantification of salsolinol enantiomers by stable isotope dilution liquid chromatography with tandem mass spectrometric detection

Salsolinol, 1‐methyl‐6,7‐dihydroxy‐2,3,4,5‐tetrahydroisoquinoline (SAL), is a precursor of a Parkinsonian neurotoxin, N‐methysalsolinol (N‐methyl‐SAL). Previous studies have shown that individual enantiomers of N‐methyl‐SAL possess distinct neurotoxicological properties. In this work, a chiral high‐performance liquid chromatography (HPLC) method with electrospray ionization tandem mass spectrometric (ESI‐MS/MS) detection was developed for the quantification of (R/S)‐SAL enantiomers. Enantioseparation was achieved on a β‐cyclodextrin‐bonded silica gel column, and the resolved enantiomers were detected by ESI‐MS/MS operated in positive ion mode. The ESI collision‐induced dissociation (CID) mass spectrum of SAL was studied together with that of its deuterium‐labeled analog (i.e. salsolinol‐α,α,α,1‐d₄, SAL‐d₄) so that the fragmentation pathways could be elucidated. Further, using SAL‐d₄ as internal standard in HPLC/MS/MS analysis of SAL improved significantly assay accuracy and reliability. Determination of (R/S)‐SAL enantiomers present in food samples such as dried banana chips was demonstrated. Copyright © 2008 John Wiley & Sons, Ltd.     

Quantification of isoflavones and lignans in serum using isotope dilution liquid chromatography/tandem mass spectrometry

Phytoestrogens (isoflavones and lignans) are receiving increasing attention due to a potential protective effect against a number of complex diseases. However, in order to investigate these associations, it is necessary to accurately quantify the levels of phytoestrogens in foods and biological fluids. We report an assay for three isoflavones (daidzein, genistein, and glycitein), two metabolites of daidzein (O-desmethylangolensin and equol), and two lignans (enterodiol and enterolactone) in human serum using electrospray ionisation liquid chromatography/mass spectrometry (LC/MS) with selective reaction monitoring. A simple, highly automated sample preparation procedure requires only 200 microL of sample and utilises one solid-phase extraction stage. Limits of detection are in the region of 10 pg/mL for all analytes except equol, which had a limit of detection of approximately 100 pg/mL. The method developed is suitable for measuring the concentrations of phytoestrogens in blood samples collected from large epidemiological studies. The results of the analysis of serum samples from 300 men and women living in the UK are reported.     

A new approach for measuring protein adducts from benzo[a]pyrene diolepoxide by high performance liquid chromatography/tandem mass spectrometry

The long-range goal of the present study is the development of a general approach for in vivo dosimetry of reactive metabolites of polycyclic aromatic hydrocarbons (PAHs), to be used as a tool in cancer risk assessment. With benzo[a]pyrene (BaP) chosen as indicator and a model of PAHs this study aims at the development of a method for the determination of adducts to histidine (His) in hemoglobin (Hb) and serum albumin (SA) of reactive metabolites of BaP. The predominantly mutagenic metabolite of BaP has been shown to be a diolepoxide isomer, +(anti)r-7, t-8-dihydroxy-t-9,10-epoxy-7,8, 9,10-tetrahydrobenzo[a]pyrene (+BPDE). In comparison with other methods for protein degradation, hydrazinolysis was found to be sufficiently effective and mild. The His adduct isolated after protein hydrazinolysis, with protection by tert-butyloxycarbonyl (Boc) of the hydrazide and alpha-amino groups, was shown to be N(im)- +/- (r-7, t-8, t-9-trihydroxy-7, 8, 9, 10-tetrahydrobenzo[a]pyren-c-10-yl)-N(alpha), N(2)-bis(tert-butyloxycarbonyl)-L-histidinehydrazide. Isomers of this compound, used as references, were synthesized and characterized by liquid chromatography/tandem mass spectrometry (LC/MS/MS). Adducts in Hb and SA from in vitro treatment with BPDE were characterized after hydrazinolysis by HPLC-UV/MS, muHPLC/MS/MS and gas chromatography/mass spectrometry (GC/MS). Approximately 70 and 10% of the isolated BPDE adducts from SA and Hb, respectively, were His adducts. Other products were released as BaP tetrols and BaP triols. For the purpose of enrichment/purification of BPDE-His adducts, C(18) and cation exchange solid phase extraction (SPE) were utilized. The sensitivity obtained by this new approach, based on hydrazinolysis of protein, enrichment by SPE and analysis with muHPLC/MS/MS (APCI), is in the low-fmole range.     

Determination of phenylalanine in human serum by isotope dilution liquid chromatography/tandem mass spectrometry

Isotope dilution liquid chromatography/tandem mass spectrometry (ID-LC/MS/MS) has been developed as a candidate reference method to determine the level of phenylalanine in human serum. The advantages of this method include a simple sample preparation without derivatization, selective detection of analytes, and the use of an isotopic analogue as an internal standard. Phenylalanine and its isotopic analogue, phenylalanine-ring-(13)C(6), were monitored at the transitions m/z 166.2/120.2 and 172.2/126.2 in the multiple-reaction monitoring (MRM) mode, respectively. The expanded uncertainty of the measurement result of phenylalanine in the serum was approximately 1.2% within a 95% confidence level. A standard reference material, with a certified value of phenylalanine, was analyzed in order to verify this method. The result obtained by the ID-LC/MS/MS method differed somewhat from the certified value, but agreed well with the gravimetric value. The measurement result of phenylalanine in serum by ID-LC/MS/MS was compared with the results from the commercial HPLC method, which was carried out in clinics. The results from the commercial HPLC method showed inconsistent results with each other. The busted results from the commercial HPLC method suggest that it should be possible to trace the results of the commercial fields to well-characterized reference materials or methods.     

Determination of aflatoxins in animal feeds by liquid chromatography/tandem mass spectrometry with isotope dilution

The objective of the present study is to develop a simple, fast method for detection of aflatoxins in animal feeds. Simultaneous quantitation of four aflatoxins (AFB(1), AFB(2), AFG(1) and AFG(2)) in animal feeds was achieved in a single liquid chromatography/tandem mass spectrometry (LC/MS/MS) run. The solid-phase extraction cleanup step is eliminated with the stable isotope dilution method. Matrix effects were observed and overcome by isotope dilution. The method was tested in a variety of animal feed matrices and proved to be accurate and reliable. Method ruggedness tests resulted in recoveries of 78% to 122% with an intra-day assay precision of 2% to 15% and an inter-day assay precision of 3% to 17%. These results indicate that this method is suitable for quantitation of aflatoxins in animal feeds.     

Determination of dithiocarbamate fungicide residues by liquid chromatography/mass spectrometry and stable isotope dilution assay

A rapid and very sensitive high-performance liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) method for the simultaneous determination of dithiocarbamate (DTC) fungicide residues in fruits and vegetables was developed. The surface extraction of samples used an alkaline buffer consisting of sodium hydrogen carbonate and DL-penicillamine. The three DTC subclasses, i.e. dimethyldithiocarbamates (DMDs), ethylenebis(dithiocarbamates) (EBDs), and propylenebis(dithiocarbamates) (PBDs), were separated on a Sequant ZIC-pHILIC column using an acetonitrile/10 mM ammonia gradient. Because of the instability of DTC residues extracted from plant samples, a stable isotope dilution assay was applied. For each DTC subclass, the limits of detection and quantification were approximately 0.03 mg kg(-1) and 0.05 mg kg(-1), respectively. Recoveries from grapes, cucumbers, tomatoes, and rucola, spiked in the range of 0.01-0.9 mg kg(-1), averaged between 90 and 100%.     

A rapid method for isolation of human hemoglobin benzo[a]pyrene diol epoxide derived adducts using high performance liquid chromatography

A method is described to isolate rapidly human hemoglobin-benzo[a]pyrene diol epoxide adducts. A combination of 300 A pore size C4 reversed phase HPLC to effect separation of adducted protein from native protein, and mu-bore C18 reversed phase HPLC to isolate and partially characterize proteolytic peptide adducts (by UV), was used.     

Quantification of Cry1Ab in genetically modified maize leaves by liquid chromatography multiple reaction monitoring tandem mass spectrometry using 18O stable isotope dilution

Cry1Ab is one of the most common Bacillus thuringiensis (Bt) proteins in genetically modified crops, which exhibits strong resistance against insect pests. In the present study, a sensitive and precise liquid chromatography stable isotope dilution multiple reaction monitoring tandem mass spectrometry (LC-SID-MRM-MS) assay was developed and validated to quantify the amount of Cry1Ab expression in transgenic maize leaves. The measurement of protein was converted to measurement of unique peptides to Cry1Ab protein. Two peptides unique to Cry1Ab were synthesized and labeled in H(2)(18)O to generate (18)O stable isotope peptides as internal standards. The validated method obtained superior specificity and good linearity. And the inter- and intra-day precision and accuracy for all samples were satisfactory. The results demonstrated Cry1Ab protein was 31.7 ± 4.1 μg g(-1) dry weight in Bt-176 transgenic maize leaves. It proved that the novel LC-SID-MRM-MS method was sensitive and selective to quantify Cry1Ab in the crude extract without time-consuming pre-separation or purification procedures.     

Quantification of N-acetyl- and N-glycolylneuraminic acids by a stable isotope dilution assay using high-performance liquid chromatography-tandem mass spectrometry

The present report describes a method for the quantification of N-acetyl- and N-glycolylneuraminic acids without any derivatization, using their (13)C(3)-isotopologues as internal standards and a C(18) reversed-phase column modified by decylboronic acid which allows for the first time a complete chromatographic separation between the two analytes. The method is based on high-performance liquid chromatographic coupled with electrospray ion-trap mass spectrometry. The limit of quantification of the method is 0.1mg/L (2.0ng on column) for both analytes. The calibration curves are linear for both sialic acids over the range of 0.1-80mg/L (2.0-1600ng on column) with a correlation coefficient greater than 0.997. The proposed method was applied to the quantitative determination of sialic acids released from fetuin as a model of glycoproteins.     

Quantification of ochratoxin A in foods by a stable isotope dilution assay using high-performance liquid chromatography-tandem mass spectrometry

A stable isotope dilution assay (SIDA) was developed for quantification of the mycotoxin ochratoxin A (OTA) by using [2H5]-OTA as internal standard. The synthesis of labelled OTA was accomplished by acid hydrolysis of unlabelled OTA and subsequent coupling one of the products, ochratoxin alpha, to [2H5]-L-phenylalanine. The mycotoxin was quantified in foods by LC-tandem MS after extraction with buffers containing [2H5]-OTA and clean-up by immuno affinity chromatography or by solid phase extraction on silica. The method showed a sufficient sensitivity with a low detection and quantification limit of 0.5 and 1.4 microg/kg, respectively, and good precision in inter-assay studies showing a CV (n = 3) of 3.6%. The analysis of certified reference materials resulted in a low bias of 2.1% from the certified values and revealed excellent accuracy of the new method. To prove the suitability of SIDA. OTA was quantified in a number of food samples and resulted mainly in not detectable OTA contents. However, three samples of raisins exceeded the legal limit of 10 microg/kg and highlighted the need for further controlling the contamination with the mycotoxin.     

Determination of epoxidized soybean oil by gas chromatography/single quadrupole and tandem mass spectrometry stable isotope dilution assay

PVC lids of glass jars often contain epoxidized soybean oil (ESBO), able to migrate and contaminate food. To establish a stable isotope dilution assay (SIDA), the 13C18-labelled internal standard ethyl 9,10,12,13-diepoxyoctadecanoate (13C(18:2E)Et) was synthesized, providing after sample preparation the same retention time as methyl 9,10,12,13-diepoxyoctadecanoate ((18:2E)Me), commonly used as a marker for ESBO in gas chromatographic (GC) analysis. For eleven different food matrices, the GC capillary columns VF-17ms, DB1701 and DB1 were tested with single quadrupole (GC/MS) as well as tandem mass spectrometric detection (GC/MS/MS). Overall, the VF-17ms column coupled with MS/MS detection showed the best results in terms of separation and sensitivity. The method validation for the matrix spiked olive oil resulted in a limit of detection (LOD) of 5 mg kg-1, a limit of quantification (LOQ) of 11 mg kg-1, a mean recovery (n=5, c=106.5 mg kg-1) of 99.7+/-5.5%, with a repeatability (within-run precision) of 6.0%. By means of GC/MS an LOQ of 21 mg kg-1 and a mean recovery (n=5, c=106.5 mg kg-1) of 103.3+/-0.8% with a repeatability of 0.9% were determined.     

Analysis of N7-(benzo[a]pyrene-6-yl)guanine in urine using two-step solid-phase extraction and isotope dilution with liquid chromatography/tandem mass spectrometry

Analysis of urinary N7-(benzo[a]pyren-6-yl)guanine (BP-6-N7Gua), a DNA adduct induced by benzo[a]pyrene, may serve as a risk-associated biomarker for exposure to polyaromatic hydrocarbons (PAHs). In this study a highly sensitive and specific analytical method, incorporating on-line sample preparation coupled with isotope-dilution liquid chromatography and tandem mass spectrometry (LC/MS/MS), was developed to quantitate this adduct in human urine. In order to achieve accurate quantitation, 15N5-labeled BP-6-N7Gua was synthesized to serve as the internal standard, and a two-step solid-phase extraction (SPE) procedure using C8 and SCX cartridges was used for sample cleanup. BP-6-7-N7Gua was analyzed using positive ion LC/MS/MS operated in multiple reaction monitoring (MRM) mode. The [M+H]+ ions at m/z 402 and 407 and the common fragment ion of [M+H]+ at m/z 252 were monitored for quantification. The recovery of this analyte after two-step SPE was 90%, and the limit of detection was 2.5 fmol/mL in 10 mL of urine. This highly specific and sensitive method for BP-6-N7Gua in urine may be applied to assess exposure to PAHs in coke-oven workers for future molecular epidemiology studies on health effects of PAHs.     

Simultaneous determination of albendazole and its metabolites in fish muscle tissue by stable isotope dilution ultra-performance liquid chromatography tandem mass spectrometry

A rapid, specific, and sensitive method utilizing ultra-performance liquid chromatography tandem mass spectrometry was developed and validated to determine albendazole, albendazole sulfoxide, albendazole sulfone, and albendazole 2-aminosulfone in fish muscle tissue. The fish samples were extracted with ethyl acetate, then the organic phase was evaporated to dryness, and the residue was reconstituted in methanol–water solution and cleaned up by n-hexane. Reversed-phase separation of target compounds was achieved using a BEH C18 column and a gradient consisting of 0.2% (v/v) formic acid and methanol. Tandem mass spectrometry analyses were performed on a triple–quadrupole tandem mass spectrometer. In the whole procedure, the isotope-labeled internal standards were used to correct the matrix effect and variations associated with the analysis. The method was validated with respect to linearity, specificity, accuracy, and precision. The method exhibited a linear response from 0.1 to 20 ng mL^-1 (r ² > 0.9985). The limit of quantitation for albendazole (ABZ), albendazole sulfoxide (ABZSO), albendazole sulfone (ABZSO₂), and albendazole 2-aminosulfone (ABZ-2-NH₂SO₂) was 0.1, 0.1, 0.1, and 0.2 ng g^-1, respectively. The mean recoveries of ABZ, ABZSO, ABZSO₂, and ABZ-2-NH₂SO₂ spiked at a level of 0.2–5.0 ng g^-1 were 95.3–113.7%, and the relative standard deviations of intra- and inter-day measurements were less than 6.38%. The method was later successfully applied to the determination of albendazole and its three metabolites in 60 fish samples collected from local markets.     

Quantification of glycopeptides by multiple reaction monitoring liquid chromatography/tandem mass spectrometry

Protein glycosylation has a major influence on functions of proteins. Studies have shown that aberrations in glycosylation are indicative of disease conditions. This has prompted major research activities for comparative studies of glycoproteins in biological samples. Multiple reaction monitoring (MRM) is a highly sensitive technique which has been recently explored for quantitative proteomics. In this work, MRM was adopted for quantification of glycopeptides derived from both model glycoproteins and depleted human blood serum using glycan oxonium ions as transitions. The utilization of oxonium ions aids in identifying the different types of glycans bound to peptide backbones. MRM experiments were optimized by evaluating different parameters that have a major influence on quantification of glycopeptides, which include MRM time segments, number of transitions, and normalized collision energies. The results indicate that oxonium ions could be adopted for the characterization and quantification of glycopeptides in general, eliminating the need to select specific transitions for individual precursor ions. Also, the specificity increased with the number of transitions and a more sensitive analysis can be obtained by providing specific time segments. This approach can be applied to comparative and quantitative studies of glycopeptides in biological samples as illustrated for the case of depleted blood serum sample. Copyright © 2012 John Wiley & Sons, Ltd.     

Wavefunction and reactivity study of benzo[a]pyrene diol epoxide and its enantiomeric forms

Benzo[a]pyrene is a known carcinogen, which derives from fossil fuel combustion, cigarette smoke, and generic biomass combustion including traffic emissions. This potent carcinogen has a well-known mechanism of action, leading to the formation of adducts with the DNA, primarily at guanosine positions. The reactivity and chemistry of this notorious compound are, however, dependent on the electronic configuration of the biologically activated metabolite, the benzo[a]pyrene diol epoxide. The activated metabolite exists mainly as four isomers, which have particular chemical reactivities toward guanosine sites on the DNA. These isomers exert also a different carcinogenicity compared to one another, which is a feature that is conventionally attributed to their geometry. However, the reactivity and properties of the isomers are not fully defined, and a determination of these properties by wavefunction behavior is required. This study reports the electronic properties of the benzo[a]pyrene diol epoxide enantiomers, along with a detailed analysis of the energy landscape, geometry, and electronic configuration of the epoxide ring. The results show that the epoxide ring, the core of the reactivity, bears different properties at the level of wavefunction for each isomer. Each of the isomers has a distinct profile on the epoxide ring, in terms of hydrogen bonds and in terms of the non-covalent interaction between the diol groups and the epoxide. These profiles generate differential reactivities of epoxide group, which can be attributed to its local bond lengths, the electron localization function, and polarized bonds. Most interestingly, the quantum chemical calculations showed also that the epoxide ring is inclined more perpendicularly toward the angular ring plane for the more carcinogenic isomers, a feature which suggests a potential geometrical relationship between the inclination of the epoxide group and its interaction with the guanosine group upon adduct formation. Our results introduce novel and crucial information, which assist in understanding the mechanism of toxic potential of this known molecule, and display the strength and level of detail of applying quantum chemical methods to reveal the reactivity, energy properties, and electronic properties of a mutagen.     

Quantification of 16 polycyclic aromatic hydrocarbons in cigarette smoke condensate using stable isotope dilution liquid chromatography with atmospheric‐pressure photoionization tandem mass spectrometry

A stable isotope dilution liquid chromatography tandem mass spectrometry method for the analysis of 16 polycyclic aromatic hydrocarbons in cigarette smoke condensate was developed and validated. Compared with previously reported methods, this method has lower limits of detection (0.04–1.35 ng/cig). Additionally, the proposed method saves time, reduces the number of separation steps, and reduces the quantity of solvent needed. The new method was applied to evaluate polycyclic aromatic hydrocarbon content in 213 commercially available cigarettes in China, under the International Standardization Organization smoking regime and the Health Canadian intense smoking regime. The results showed that the total polycyclic aromatic hydrocarbon content was more than two times higher in samples from the Health Canadian intense smoking regime than in samples from the International Standardization Organization smoking regime (1189.23 versus 2859.50 ng/cig, p < 0.05). Meanwhile, the concentration of individual polycyclic aromatic hydrocarbons (and total polycyclic aromatic hydrocarbons) increased with labeled tar content in both of the tested smoking regimes. There was a positive correlation between total polycyclic aromatic hydrocarbons under the International Standardization Organization smoking regime with that under the Health Canadian intense smoking regime. The proposed liquid chromatography tandem mass spectrometry method is satisfactory for the rapid, sensitive, and accurate quantitative evaluation of polycyclic aromatic hydrocarbon content in cigarette smoke condensate, and it can be applied to assess potential health risks from smoking.