Tamoxifen metabolite isomer separation and quantification by liquid chromatography-tandem mass spectrometry

Tamoxifen (Tam), the antiestrogen used to treat estrogen receptor-positive breast cancer is a pro-drug that is converted to its major active metabolites, endoxifen and 4-hydroxy-tamoxifen (4-OH-Tam) by various biotransformation enzymes of which cytochrome P450-2D6 (CYP2D6) is key. The usual Tam dose is 20 mg daily; however, the plasma active metabolite concentrations vary due to common genetic variants encoding the biotransformation enzymes and environmental factors (e.g., concomitant drugs) that inhibit these enzymes. Effective treatment depends on adequate Tam conversion to its active isomers. To monitor metabolite plasma levels, a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to separate and quantitate Tam, N-desmethyl-tamoxifen (ND-Tam), and tamoxifen-N-oxide (Tam-N-oxide), and the E, Z, and Z' isomers of endoxifen and 4-OH-Tam. Known standards were used to identify each metabolite/isomer. Quantitation of these metabolites in plasma was linear from 0.6 to 2000 nM. Intra- and inter-assay reproducibilities were 0.2-8.4% and 0.6-6.3%, respectively. Accuracy determined by spike experiments with known standards was 86-103%. Endoxifen, 4-OH-Tam, and their isomers were stable in fresh frozen plasma for ≥6 months. This method provides the first sensitive, specific, accurate, and reproducible quantitation of Tam and its metabolite isomers for monitoring Tam-treated breast cancer patients.     

Simultaneous quantification of eleven thiopurine nucleotides by liquid chromatography-tandem mass spectrometry

The prodrugs azathioprine and 6-mercaptopurine, which are well-established anticancer and immunosuppressive agents, are extensively metabolized by activating and inactivating enzymes. Whereas the 6-thioguanine nucleotides (TGN) are currently being considered as major active metabolites, methylthioinosine nucleotides seem to contribute to the cytotoxic effect as well. Thiopurine-related adverse drug reactions and thiopurine failure are frequent. Thus, therapeutic monitoring of TGN and methylthioinosine derivatives has been suggested to improve thiopurine therapy, however with limited success. To elucidate systematically underlying molecular mechanisms as potential explanation for interindividual variability of thiopurine response, we developed a novel highly specific and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous quantitation of eleven mono-, di-, and triphosphates of thioguanosine, methylthioinosine, methylthioguanosine, and thioinosine. Using stable isotope-labeled analogues as internal standards obtained by chemical synthesis, an intra- and interassay variability below 8% and an accuracy of 92% to 107% were achieved in spiked quality control samples with known standards. All eleven metabolites could be determined in red blood cells from patients with inflammatory bowel diseases and long-term azathioprine therapy. Thus, our novel method opens a new avenue for the understanding of the thiopurine metabolism by quantitation of all important thiopurine nucleotide metabolites in one run.     

Absolute quantification of monoclonal antibodies in biofluids by liquid chromatography-tandem mass spectrometry

The development of a quantification method for monoclonal antibodies in serum has been accomplished by high-performance liquid chromatography multiple reactions monitoring mass spectrometry. A human monoclonal antibody (HmAb) was used as the model protein for method development and validation. A peptide from the CDR3-region of its heavy chain was selected and used for quantifying the entire mAb. This signature peptide served as a template for the internal standard. Prior to mass spectrometric analysis approximately 50% of the total serum protein content was removed by albumin depletion. The accuracy of the method ranged between 99 and 112% in cynomolgus monkey serum. The intra-assay coefficient of variation (CV) was lower than 4% at 4 microg/mL and 200 microg/mL HmAb (n = 3). The CV at 400 microg/mL corresponded to 9% (n = 3). In addition, the interassay variation was investigated in a male cynomolgus serum pool and in a female cynomolgus serum pool. The CV for the male cynomolgus pool at 4 microg/mL HmAb was 7% (n = 3). The CV obtained from the female pool was 8% (n = 3), at 4 microg/mL. The dynamic range of the method was 3 orders of magnitude. After albumin depletion of 25 microL of serum, a lowest limit of quantification of 2 microg/mL HmAb was reached in both human and cynomolgus monkey samples.     

Quantitative analysis of lycopene isomers in human plasma using high-performance liquid chromatography-tandem mass spectrometry

An analytical method for the determination of the concentrations of total lycopene and its cis and all-trans isomers in human plasma has been developed using high-performance liquid chromatography-tandem mass spectrometry (LC-MS-MS). This method was based on the observation that, during negative ion atmospheric pressure chemical ionization with collision-induced dissociation, a unique fragment of m/z 467 was formed from the molecular ion of m/z 536 by elimination of a terminal isoprene group. The use of multiple reaction monitoring facilitated the selective detection of lycopene isomers and an internal standard without interference from the isobaric carotenoids a-carotene and beta-carotene, which are also abundant in human plasma. Measurement of total lycopene was carried out using a C18 high-performance liquid chromatography (HPLC) column and an isocratic mobile phase consisting of acetonitrile/methyl tert-butyl ether (95:5) so that all lycopene isomers eluted as a single chromatographic peak. all-trans-Lycopene was separated from its various cis isomers by using a C30 carotenoid column and a gradient solvent system from methanol to methyl tert-butyl ether. The effects of sample preparation and handling parameters on the stability of lycopene were evaluated such as the stability of lycopene in the HPLC autosampler and the effect of saponification upon lycopene isomerization. For example, the half-life of all-trans-lycopene in the HPLC mobile phase in the autosampler at 4 degrees C was determined to be approximately 16 h. Also, saponification of plasma samples was determined to cause lycopene degradation and isomerization so that lycopene recovery was reduced. The accuracy and interassay precision of this LC-MS-MS assay for lycopene showed a standard deviation of less than 10% over the range of 5-500 pmol injected on-column. The limit of detection was 11.2 fmol injected on-column, and the limit of quantitation was 22.8 fmol.     

Method for the determination of vitamin K homologues in human plasma using high-performance liquid chromatography-tandem mass spectrometry

We report here the development of a precise and sensitive method for the determination of vitamin K homologues including phylloquinone (PK), menaquinone-4 (MK-4), and menaquinone-7 (MK-7) in human plasma using HPLC-tandem mass-mass spectrometry with atmospheric pressure chemical ionization (LC-APCI-MS/MS). The method involves the use of stable isotope (18)O-labeled internal standard compounds, which were synthesized in our laboratory, and the selection of a precursor and product ion with a MS/MS multiple reaction monitoring method. The average intraassay and interassay variation values for PK, MK-4, and MK-7 were <10%. Average spiked recoveries from authentic compounds added to normal human plasma samples for PK, MK-4, and MK-7 were 98-102%. Mean plasma concentrations of PK, MK-4, and MK-7 from healthy subjects (n = 20) were 1.22 +/-0.57, 0.39 +/- 0.46, and 6.37 +/- 7.45 ng/mL, respectively. We conclude that this novel LC-APCI-MS/MS method should be useful for the evaluation of vitamin K status in postmenopausal women and elderly subjects and provides useful information for the treatment and prevention of osteoporosis with vitamin K.     

Design and validation of a high-throughput matrix-assisted laser desorption ionization time-of-flight mass spectrometry method for quantification of hepcidin in human plasma

Disorders of iron metabolism affect over a billion people worldwide. The circulating peptide hormone hepcidin, the central regulator of iron distribution in mammals, holds great diagnostic potential for an array of iron-associated disorders, including iron loading (β-thalassemia), iron overload (hereditary hemochromatosis), and iron deficiency diseases. We describe a novel high-throughput matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry assay for quantification of hepcidin in human plasma. This assay involves enrichment using a functionalized MALDI chip, a novel solvent-detergent precipitation buffer, and quantification using a stable isotope labeled internal standard. The linear range of hepcidin in plasma was 1-120 nM, with a low limit of quantification (LOQ) (1 nM), high accuracy (<15% relative error (RE)), and high precision (intraday average 5.52-18.48% coefficient of variation (CV) and interday 9.32-14.83% CV). The assay showed strong correlation with an established hepcidin immunoassay (Spearman; R(2) = 0.839 n = 93 ethylenediaminetetraacetic acid (EDTA) plasma). A collection of normal healthy pediatric samples (range 3.8-32.5 ng/mL; mean 12.9 ng/mL; n = 119) showed significant differences from an adult collection (range 1.8-48.7 ng/mL; mean 16.1 ng/mL; n = 95; P = 0.0096). We discuss these preliminary reference ranges and correlations with additional parameters in light of the utility and limitations of hepcidin measurements as a stand-alone diagnostic and as a tool for therapeutic intervention.     

Fast separation and quantification method for nitroguanidine and 2,4-dinitroanisole by ultrafast liquid chromatography-tandem mass spectrometry

Explosives are now persistent environmental pollutants that are targets of remediation and monitoring in a wide array of environmental media. Nitroguanidine (NG) and 2,4-dinitroanisole (DNAN) are two insensitive energetic compounds recently used as munitions explosives. To protect our environment and human health, the levels of these compounds in soils and waters need to be monitored. However, no sensitive analytical methods, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS), have been developed for detecting these new compounds at trace levels and to be concurrently applied to monitor the common explosives. In general, the concentrations of explosives in either soil or water samples are very low and widely distributed. Therefore, a fast and sensitive method is required to monitor those compounds and increase our ability to find and address the threats they pose to human health and ecological receptors. In this study, a fast and sensitive analytical method has been developed to quantitatively determine NG and DNAN in soil, tap water, and river water by using ultrafast LC-MS/MS. To make this method a comprehensive analytical technique for other explosives as well, it has included other commonly used explosives in the method development, such as octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX), 1,3,5-trinitroper-hydro-1,3,5-triazine (RDX), 2,4,6-trinitrotoluene (TNT), 2-amino-4,6-dinitrotoluene (ADNT), and pentaerythritol tetranitrate (PETN). The method detection limits (MDLs) of these compounds in soil ranged from 0.2 to 5 ppb, and a good linearity was obtained over a concentration range of 0.5-200 ppb. The recoveries of some compounds are equal to or better than the current EPA methods but with much higher sensitivities.     

Determination of folates in human plasma using hydrophilic interaction chromatography-tandem mass spectrometry

Folic acid is an essential nutrient, and folate deficiency is associated with a variety of disorders including neural tube defects (during pregnancy) and heart disease. A fast, sensitive, and robust HPLC-tandem mass spectrometry (LC-MS-MS) method was developed for the quantification of free folic acid, tetrahydrofolate, 5'-methyltetrahydrofolate, and 5'-formyltetrahydrofolate in human plasma. Sample preparation required only acetonitrile precipitation of proteins followed by filtration instead of solid-phase extraction or solvent-solvent extraction as in other methods. The rapid and streamlined sample handling procedure minimized degradation of the highly unstable folate species. Hydrophilic interaction chromatography was used for additional sample cleanup on-line, and baseline separation and detection of all four folate species was achieved in less than 30 min. The folate species were detected using negative ion electrospray-tandem mass spectrometry with multiple reaction monitoring of the diagnostic fragment ions of each deprotonated molecule. The predominately organic (hydrophobic) solvent system combined with the microbore flow rate (50 microL/min) used for the chromatography resulted in enhanced electrospray signal response compared to reversed-phase HPLC using a wider bore column. The recovery of all folate species (from spiked plasma) was >97% over a concentration range from 300 pg/L to 12 mg/L with intraday precision (RSD, n = 5) of 3.7-6.5%. Stability studies were carried out for spiked samples in order to define storage and handling conditions. The folic acid limit of quantification (LOQ) in human plasma was 80 pmol/L +/- 10%, and the limit of detection (LOD) was 37.5 pmol/L. The LOQ and LOD for tetrahydrofolate, 5'-methyltetrahydrofolate, and 5'-formyltetrahydrofolate were 1250, 400, and 360 pmol/L of plasma and 425, 165, and 140 pmol/L of plasma, respectively.     

Simultaneous quantification of homocysteine and folate in human serum or plasma using liquid chromatography/tandem mass spectrometry

A unified extraction and quantification procedure based on stable isotope-dilution liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been developed for the simultaneous determination of total homocysteine and folate (5-methyltetrahydrofolic acid and folic acid) levels in human serum and plasma. This is the first report documenting the simultaneous extraction and quantification of these structurally dissimilar analytes. Analytes are quantitatively isolated from samples (500 microL) prior to LC/MS/MS analysis using a two-step stabilization process combined with C18 solid-phase extraction. The method exhibits excellent linearity over 4 orders of magnitude for each analyte. Measurement repeatability (RSD, N = 2) ranged from 0.3% to 3% for all analytes over 1 day of analysis. Total method variability (RSD, N = 6) ranged from 0.7% to 10% for all analytes over three independent days of analysis. The accuracy and practical applicability of the method were demonstrated by applying the method to the quantitative determination of each analyte in a new NIST serum Standard Reference Material (NIST SRM 1955 Homocysteine and Folate in Frozen Human Serum) and in a small subset of normal donor plasma samples.     

Hydrophilic interaction liquid chromatography-tandem mass spectrometry determination of estrogen conjugates in human urine

We report a hydrophilic interaction liquid chromatography (HILIC) separation with tandem mass spectrometry (MS) detection method for analysis of seven urinary estrogen conjugates. HILIC separation employing a mobile phase with high organic solvent content resulted in enhanced electrospray ionization efficiency and MS sensitivity compared with reversed-phase (RP) LC-MS methods. Solid-phase extraction (SPE) was used to further improve the limit of detection and to eliminate interferences for the analysis of urine samples. No hydrolysis or derivatization was required in the sample pretreatment. This SPE/HILIC-MS/MS method provided limits of quantification (LOQs at S/N = 10) for the seven conjugates ranging from 2 to 1000 pg/mL with only 1 mL of urine sample, representing an improvement of 1 order of magnitude over the RPLC tandem MS methods previously reported. This method provided a linear dynamic range of 3 orders of magnitude, recovery of 92-109%, intraday accuracy of 84-109%, intraday precision of 1-14%, interday accuracy of 80-111%, and interday precision of 1-22%. We have successfully applied this technique to determine the seven estrogen conjugates in urine samples of a pregnant woman and found unique concentration changes of six estrogen conjugates at different stages of pregnancy while the concentration of estriol-3-glucuronide (E3-3G) remained constant. We further studied the profiles of individual estrogen conjugates in breast cancer patients before and after treatment and found patient-dependent effects of aromatase inhibitor treatment on estrogen phase-II metabolism, which have not been reported previously. This study demonstrates the potential clinical application of the HILIC-MS/MS technique for sensitive monitoring of the changes of urinary estrogen conjugates in a clinical setting.     

Confirmation and quantification of hemoglobin-based oxygen carriers in equine and human plasma by hyphenated liquid chromatography tandem mass spectrometry

Oxyglobin (OXY) and Hemopure (HMP) are produced from bovine hemoglobin (Hb) and were developed for the treatment of anemia in animal and human patients, respectively. Hemolink (HML) is a blood substitute of human Hb origin under development. The ability of these agents to carry oxygen in circulating blood and their promise to improve oxygen delivery to tissues supports the potential for their abuse in equine and human athletes. To deter athletes from abuse of these agents, a method has been developed for the detection, confirmation and quantification of OXY, HMP, and HML in equine and human plasma. OXY, HMP, and HML were extracted from equine or human plasma by solid-phase extraction using Bond Elut ENV cartridges and were digested by trypsin at 37 degrees C for 3 h. The tryptic digests were analyzed by LC-MS/MS, and tryptic peptides specific for bovine and human Hbs were targeted. OXY and HMP were detected, quantified, and confirmed using the y14 ion and b8 ion of the tryptic peptide from bovine Hb alpha chain residues 69-90, and HML was quantified using the tryptic peptide from human Hb alpha chain residues 63-91. The limit of detection for OXY in equine plasma and HML in human and equine plasma was 50 and 250 microg/mL for HMP in human and equine plasma. The limit of confirmation was 250 microg/mL for OXY in equine plasma, 500 microg/mL for HML in human and equine plasma, and 1000 microg/mL for HMP in human and equine plasma. The linear range for quantification was 50-5000 microg/mL for OXY in equine plasma and for HML in human and equine plasma, and 250-5000 microg/mL for HMP in human and equine plasma. The intraday and interday CV were less than 17% for quantification of OXY in equine plasma with external calibration. OXY was stable for more than 30 days at -20 and -70 degrees C. OXY was detected and quantified in equine plasma up to 24 h following administration of a very low dose of OXY (32.5 g in 2 x 125 mL per horse), and its presence in equine plasma was confirmed up to 12 h postadministration.     

Development and evaluation of a reference measurement procedure for the determination of estradiol-17beta in human serum using isotope-dilution liquid chromatography-tandem mass spectrometry

Estradiol is the most potent natural estrogen and is derived from the ovaries. Its concentration in blood is measured to determine ovarian function. A reference measurement procedure for estradiol in serum involving isotope-dilution coupled with liquid chromatography-tandem mass spectrometry (LC/MS/MS) has been developed and critically evaluated. A deuterated estradiol (estradiol-d3) was used as an internal standard. The estradiol and its internal standard were extracted from serum matrix using solid-phase extractions and derivatized with dansyl chloride prior to reversed-phase LC/MS/MS. The accuracy of the measurement was evaluated by a comparison of results of this reference method on lyophilized human serum reference materials for estradiol [Certified Reference Materials (CRMs) 576, 577, and 578] with the certified values determined by gas chromatography/mass spectrometry (GC/MS) reference methods and by a recovery study for the added estradiol. The results of this method for estradiol agreed with the certified values within the uncertainty of the measurements for the three CRMs. The recovery of the added estradiol ranged from 100.7 to 101.8%. This method was applied to the determination of estradiol in frozen serum samples from three individual female donors. Excellent reproducibility was obtained with within-set coefficient of variations (CVs) ranging from 0.6 to 2.2% and between-set CVs ranging from 0.2 to 0.4%. Excellent linearity was also obtained, with correlation coefficients of all linear regression lines (measured intensity ratios vs mass ratios) ranging from 0.998 to 1.000. The detection limit at a signal-to-noise ratio of approximately 3 was 0.6 pg of estradiol (or 1 ng/L, as expressed as a concentration). This well-characterized LC/MS/MS method for serum estradiol, which demonstrates good accuracy and precision, low susceptibility to interferences, and comparability with GC/MS reference methods, qualifies as a reference measurement procedure that can be used to provide an accuracy base to which routine methods for estradiol can be compared and that will serve as a standard of higher order for measurement traceability.     

Qualitative determination of synthetic analogues of insulin in human plasma by immunoaffinity purification and liquid chromatography-tandem mass spectrometry for doping control purposes

Synthetic insulins such as Humalog Lispro, Novolog Aspart, or Lantus Glargine, are commonly employed for the treatment of insulin-dependent diabetes mellitus owing to convenient handling and fast or prolonged bioavailability. However, the misuse of insulin in sports has been reported often, and the international doping control system requires a reliable and robust assay to determine the presence or absence of related drugs prohibited by the World Anti-Doping Agency. Qualitative evidence of administered substances, which is preferably obtained by mass spectrometry, is of utmost importance. Plasma specimens of 2 mL were fortified with three synthetic insulin analogues and purified by immunoaffinity chromatography, and extracts were analyzed by microbore liquid chromatography and tandem mass spectrometry. Product ion scan experiments of intact proteins enabled the differentiation between endogenously produced insulin and its synthetic analogues by collisionally activated dissociation of multiply charged precursor ions. This top-down sequencing-based assay allows the assignment of individual fragment ions, in particular, of those comprising modifications that are originating from C-termini of B-chains. Recoveries of synthetic insulins from plasma aliquots ranged from 91 to 98%, and detection limits were accomplished at 0.5 ng/mL for all target analytes.     

Strategies for bioanalysis of an oligonucleotide class macromolecule from rat plasma using liquid chromatography-tandem mass spectrometry

Electrospray ionization (ESI) liquid chromatography-tandem mass spectrometry (LC/MS/MS) assays provide high-throughput and selective methods for quantitation of small molecules. Use of LC/MS/MS assays for macromolecules, like oligonucleotides, is challenging due to lack of sensitivity and low analyte recovery from biomatrixes. Due to this fact, the method of choice for oligonucleotides quantitation remains hybridization-based ligand-binding assays. These biological assays usually possess high sensitivity but low selectivity and narrow dynamic range. They also require optimizing suitable "capture and detection" probes, which can be prohibitively time-consuming and expensive in a drug discovery lead-optimization scenario. In this paper, we present a unique LC/MS/MS assay for a model phosphorothioate backbone oligodeoxynucleotide (ODN) drug (7692 amu) from rat plasma. Multiple analytical challenges were encountered. The strategies used to solve these challenges should prove useful to scientists pursuing mass spectrometry (MS) to quantitate oligonucleotides. The challenges include analyte multiple charging and cation adduction (reduced sensitivity), oxidation of analyte on drying and high protein binding (low recovery), ODN affinity to exposed silica (low chromatographic reproducibility and high carryover), nonspecific binding of analyte to containers (low storage stability), and optimization/synthesis of an appropriate internal standard (interference and cross-talk). A buffer (7 mM triethylamine and 3 mM ammonium formate)/methanol, 50:50 (v/v), was used as an ESI-MS infusion solvent and produced a sharp multiple charge-state distribution. The sample extraction method combined a phenol/chloroform liquid-liquid extraction and solid-phase extraction steps, which improved the absolute recovery to >70%. The method was validated in the range of 5-2000 ng/mL and had precision (percent relative standard deviation)<10.1% and accuracy (percent relative error)<11.4%.     

Analysis of pharmaceuticals in fish using liquid chromatography-tandem mass spectrometry

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) screening method has been developed targeting 23 pharmaceuticals and 2 metabolites with differing physicochemical properties in fish tissue. Reversed-phase separation of target compounds was achieved using a C18 column and a nonlinear gradient consisting of 0.1% (v/v) formic acid and methanol. Eluted analytes were introduced into the mass analyzer using positive or negative electrospray ionization, as appropriate. A variety of extraction solvents, differing in polarity, pH, or both, were investigated in order to assess recovery of target compounds from 1-g tissue homogenates. Among 10 solvents tested, a 1:1 mixture of 0.1 M aqueous acetic acid (pH 4) and methanol was identified as optimal, resulting in extraction recoveries for 24 of 25 compounds exceeding 60%. Tissue extracts were found to influence the LC-MS/MS response for several analytes. Consequently, matrix-matched calibration standards were employed to determine analyte concentrations in environmental samples. Statistically derived method detection limits were <6 ng/g for most analytes. The method was subsequently used to screen for target analytes in fish from an effluent-dominated stream. Diphenhydramine, diltiazem, carbamazepine, and norfluoxetine were detected in 11 of 11 environmental samples at concentrations ranging from 0.11 to 5.14 ng/g.     

Ultraperformance liquid chromatography-tandem mass spectrometry method for biomonitoring cooked meat carcinogens and their metabolites in human urine

The cooked meat carcinogens 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), and their principal metabolites produced by cytochrome P450 and/or uridine diphosphate glucuronosyl transferases were simultaneously measured at the parts per trillion level in urine of omnivores, by ultraperformance liquid chromatography (UPLC) with a Michrom Advance CaptiveSpray source and a triple stage quadrupole mass spectrometer. Quantitation was performed in the selected reaction monitoring mode. The UPLC method is much more rapid and sensitive than our earlier capillary HPLC method: the duty cycle of the UPLC method is 19 min compared to 57 min for capillary HPLC. The performance of the UPLC assay was evaluated with urine samples from three subjects over 4 different days. The intraday and interday precisions of the estimates of PhIP, MeIQx, and their metabolites, reported as the coefficients of variation, were ≤10%. The limit of quantification (LOQ) values for PhIP and MeIQx were about 5 pg/mL, whereas the LOQ values of their metabolites ranged from 10 to 40 pg/mL. Furthermore, the identities of the analytes were corroborated by acquisition of full scan product ion spectra, employing between 0.5 and 5 pg of analyte for assay.     

Automation of nanoscale microcapillary liquid chromatography-tandem mass spectrometry with a vented column

To fully automate the sample introduction step for nanoscale microcapillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses, 75 microm i.d. x 14 cm capillary columns were interfaced with a commercial autosampler instrument using a novel procedure which allowed dilute peptide samples to be transferred from the AS loop injector to the nanoscale column at flow rates up to 5 microL min(-1). On-column enrichment and desalting was demonstrated for large sample volumes (>40 microL) by constructing a vent 2 cm after the entrance to the packed bed of 5-microm ODS-AQ modified silica. Salts and nonretained solutes were removed via the vent, which allowed for column washing independent of the continuation of the bed into the electrospray source. Separations of test peptide mixtures demonstrated 50-nL elution peak volumes with low- to subfemtomole detection levels. In addition, a highly complex peptide mixture (outer membrane preparation from Psuedemonas aeruginosa) was efficiently separated with more than 100 proteins identified from a single reversed-phase LC-MS/MS analysis. Finally, the vented column (V-column) was utilized for on-line separations in a multidimensional chromatography/tandem MS experiment where large numbers of strong cation exchange chromatography fractions from a trypsinized yeast lysate were desalted, concentrated, and analyzed in a completely automated fashion. The procedures for constructing and using a V-column require minimal changes in current methods and equipment for nano-LC-MS analyses using columns of 100-microm diameter and smaller.     

Quantitative measurement of endogenous estrogens and estrogen metabolites in human serum by liquid chromatography-tandem mass spectrometry

Endogenous estrogen plays a key role in the development of human breast cancer, yet the contribution of specific estrogen metabolites and patterns of estrogen metabolism remains unclear. To determine their individual and joint roles in breast carcinogenesis, it is necessary to be able to measure quantitatively each estrogen metabolite in epidemiologic and clinical biospecimens. In this report, we detail a sensitive, specific, accurate, and precise high-performance liquid chromatography-tandem mass spectrometry method utilizing selected reaction monitoring for measuring the absolute quantities of free (unconjugated) and total (conjugated + unconjugated) endogenous estrogens and estrogen metabolites in human serum from premenopausal and postmenopausal women. The method requires a simple sample preparation and only 0.5 mL of serum, yet is capable of quantifying simultaneously 15 estrogens and estrogen metabolites (EM): estrone and its 2-, 4-, and 16alpha-hydroxy and 2- and 4-methoxy derivatives; 2-hydroxyestrone-3-methyl ether; 17beta-estradiol and its 2-hydroxy and 2- and 4-methoxy derivatives; and estriol, 17-epiestriol, 16-ketoestradiol, and 16-epiestriol. The lower limit of quantitation for each EM was 0.4 pg on-column, equivalent to 8 pg/mL (26.5-29.6 fmol/mL) in the original serum sample. Calibration curves were linear over a 10(3)-fold concentration range. For a stripped serum sample containing 8 pg/mL of each EM, accuracy (percent recovery of a known added amount) ranged from 91 to 113%. Intrabatch precision (including hydrolysis, extraction, and derivatization steps) ranged from 7 to 30% relative standard deviation (RSD), and interbatch precision ranged from 8 to 29% RSD. Since distinct roles have been proposed for many of these estrogen metabolites, an accurate, precise, sensitive, and specific method for measuring their levels in circulation should suggest new approaches to breast cancer prevention, screening, and treatment.     

Rapid quantification of clostridial epsilon toxin in complex food and biological matrixes by immunopurification and ultraperformance liquid chromatography-tandem mass spectrometry

Epsilon toxin (ETX) is one of the most lethal toxins produced by Clostridium species and is considered as a potential bioterrorist weapon. Here, we present a rapid mass spectrometry-based method for ETX quantification in complex matrixes. As a prerequisite, naturally occurring prototoxin and toxin species were first structurally characterized by top-down and bottom-up experiments, to identify the most pertinent peptides for quantification. Following selective ETX immunoextraction and trypsin digestion, two proteotypic peptides shared by all the toxin forms were separated by ultraperformance liquid chromatography (UPLC) and monitored by ESI-MS (electrospray ionization-mass spectrometry) operating in the multiple reaction monitoring mode (MRM) with collision-induced dissociation. Thorough protocol optimization, i.e., a 15 min immunocapture, a 2 h enzymatic digestion, and an UPLC-MS/MS detection, allowed the whole quantification process including the calibration curve to be performed in less than 4 h, without compromising assay robustness and sensitivity. The assay sensitivity in milk and serum was estimated at 5 ng·mL(-1) for ETX, making this approach complementary to enzyme linked immunosorbent assay (ELISA) techniques.     

液相色谱-串联质谱法测定蜂蜜中四环素残留量的不确定度评定

采用液相色谱-串联质谱法依照GB/T18932.23-2003对蜂蜜进行四环素残留量测定。测定结果的不确定度来源主要有标准溶液配制、标准曲线校准、样品称量、样品定容、样品的回收率及结果的重复性等。通过测定过程中各种不确定度分量的计算和分析,测得四环素扩展不确定度为U(CX)=6.70mg/kg,k=2。