Development of chemical isotope labeling liquid chromatography mass spectrometry for silkworm hemolymph metabolomics

Silkworm (Bombyx mori) is a very useful target insect for evaluation of endocrine disruptor chemicals (EDCs) due to mature breeding techniques, complete endocrine system and broad basic knowledge on developmental biology. Comparative metabolomics of silkworms with and without EDC exposure offers another dimension of studying EDCs. In this work, we report a workflow on metabolomic profiling of silkworm hemolymph based on high-performance chemical isotope labeling (CIL) liquid chromatography mass spectrometry (LC-MS) and demonstrate its application in studying the metabolic changes associated with the pesticide dichlorodiphenyltrichloroethane (DDT) exposure in silkworm. Hemolymph samples were taken from mature silkworms after growing on diet that contained DDT at four different concentrations (1, 0.1, 0.01, 0.001 ppm) as well as on diet without DDT as controls. They were subjected to differential ¹²C-/¹³C-dansyl labeling of the amine/phenol submetabolome, LC-UV quantification of the total amount of labeled metabolites for sample normalization, and LC-MS detection and relative quantification of individual metabolites in comparative samples. The total concentration of labeled metabolites did not show any significant change between four DDT-treatment groups and one control group. Multivariate statistical analysis of the metabolome data set showed that there was a distinct metabolomic separation between the five groups. Out of the 2044 detected peak pairs, 338 and 1471 metabolites have been putatively identified against the HMDB database and the EML library, respectively. 65 metabolites were identified by the dansyl library searching based on the accurate mass and retention time. Among the 65 identified metabolites, 33 positive metabolites had changes of greater than 1.20-fold or less than 0.83-fold in one or more groups with p-value of smaller than 0.05. Several useful biomarkers including serine, methionine, tryptophan, asymmetric dimethylarginine, N-Methyl-D-aspartic and tyrosine were identified. The changes of these biomarkers were likely due to the disruption of the endocrine system of silkworm by DDT. This work illustrates that the method of CIL LC-MS is useful to generate quantitative submetabolome profiles from a small volume of silkworm hemolymph with much higher coverage than conventional LC-MS methods, thereby facilitating the discovery of potential metabolite biomarkers related to EDC or other chemical exposure.     

Development of an isotope labeling ultra-high performance liquid chromatography mass spectrometric method for quantification of acylglycines in human urine

Acylglycines play a crucial regulatory and detoxification role in the accumulation of the corresponding acyl CoA esters and are an important class of metabolites in the diagnoses of inborn errors of metabolism. Sensitive quantification of a large number of acylglycines not only improves diagnosis but also enables the discovery of potential new biomarkers of diseases. We report an ultra-high performance liquid chromatography tandem mass spectrometry (UPLC–MS) method for quantifying acylglycines in human urine with high sensitivity. This method is based on the use of a newly developed isotope labeling reagent, p-dimethylaminophenacyl (DmPA) bromide, to label acylglycines to improve detection sensitivity. Eighteen acylglycines, namely acetylglycine, propionylglycine, isobutyrylglycine, butyrylglycine, 4-hydroxyphenylacetylglycine, 2-furoylglycine, tiglylglycine, 2-methybutyrylglycine, 3-methylcrotonylglycine, isovalerylglycine, valerylglycine, hexanoylglycine, phenylacetylglycine, phenylpropionylglycine, glutarylglycine, heptanoylglycine, octanoylglycine and suberylglycine, were measured. This method uses calibration standards prepared in surrogate matrix (un-derivatized urine) and stable-isotope labeled analytes as the internal standards. The analysis was carried out in the positive ion detection mode using multiple reaction monitoring (MRM) survey scans. The calibration curves were validated over the range of 1.0–500 nM. The method achieved a lower limit of quantitation (LLOQ) of 1–5 nM for all analytes, as measured by the standard derivations associated with calibration curves and confirmed in surrogate matrix; the signal-to-noise ratio at LLOQ ranged from 12.50 to 156.70. Both accuracy (% RE or relative error) and precision (% CV) were <15%. Matrix effects were minimized using the surrogate matrix. All eighteen analytes were stable in urine for at least 5 h at room temperature, autosampler (4 °C) for 24 h, 7 weeks at −20 °C and after three freeze/thaw cycles. This surrogate matrix approach was validated using a standard addition experiment. As an example of applications, the endogenous concentrations of all eighteen analytes in urine samples of 20 healthy individuals collected in three consecutive days (i.e., 60 samples) were determined; there was no significant correlation found between the acylglycine profile and gender or body mass indices.     

Differential isotope dansylation labeling combined with liquid chromatography mass spectrometry for quantification of intact and N-terminal truncated proteins

The N-terminal amino acids of proteins are important structure units for maintaining the biological function, localization, and interaction networks of proteins. Under different biological conditions, one or several N-terminal amino acids could be cleaved from an intact protein due to processes, such as proteolysis, resulting in the change of protein properties. Thus, the ability to quantify the N-terminal truncated forms of proteins is of great importance, particularly in the area of development and production of protein-based drugs where the relative quantity of the intact protein and its truncated form needs to be monitored. In this work, we describe a rapid method for absolute quantification of protein mixtures containing intact and N-terminal truncated proteins. This method is based on dansylation labeling of the N-terminal amino acids of proteins, followed by microwave-assisted acid hydrolysis of the proteins into amino acids. It is shown that dansyl labeled amino acids are stable in acidic conditions and can be quantified by liquid chromatography mass spectrometry (LC–MS) with the use of isotope analog standards.     

Ion-pairing reversed-phase liquid chromatography fractionation in combination with isotope labeling reversed-phase liquid chromatography-mass spectrometry for comprehensive metabolome profiling

We report a novel two-dimensional (2D) separation strategy aimed at improving the detectability of liquid chromatography mass spectrometry (LC-MS) for metabolome analysis. It is based on the use of ion-pairing (IP) reversed-phase (RP) LC as the first dimension separation to fractionate the metabolites, followed by isotope labeling of individual fractions using dansylation chemistry to alter the physiochemical properties of the metabolites. The labeled metabolites having different hydrophobicity from their unlabeled counterparts are then separated and analyzed by on-line RPLC Fourier-transform ion-cyclotron resonance mass spectrometry (FTICR-MS). This off-line 2D-LC-MS strategy offers significant improvement over the one-dimensional (1D) RPLC MS technique in terms of the number of detectable metabolites. As an example, in the analysis of a human urine sample, 3564 13C-/12C-dansylated ion pairs or metabolites were detected from seven IP RPLC fractions, compared to 1218 metabolites found in 1D-RPLC-MS. Using a library of 220 amine- and phenol-containing metabolite standards, 167 metabolites were positively identified based on retention time and accurate mass matches, which was about 2.5 times the number metabolites identified by 1D-RPLC-MS analysis of the same urine sample.     

Investigation of the species-dependent in vitro metabolism of BAL30630 by stable isotope labeling and isotope exchange experiments analyzed by capillary liquid chromatography coupled to mass spectrometry

The in vitro metabolic profile of BAL30630, an antifungal piperazine propanol derivative, which inhibits the 1,3-beta-d-glucansynthase, was investigated by incubation with microsomes of several species and with rat hepatocytes. For the spotting of the metabolites, mixtures of BAL30630 with a stable isotope (deuterium) labeled analogue were incubated. The metabolic pattern comprises several oxidized metabolites. Based on isotope exchange experiments, their structures could be assigned to epoxide- and hydroxylated metabolites. In hepatocyte incubations, several glucuronides formed from these oxidized metabolites could be observed. From the analysis of the metabolic pattern in microsomes, products of carbamate hydrolysis were characterized. This hydrolysis was highly species dependent. In activated incubations and in rat hepatocytes, those metabolites were further oxidized. In incubations without NADPH activation, the resulting hydrolytic metabolites could be enriched without the subsequent oxidation. Final structural elucidation of the metabolites was performed using accurate mass determination and isotope exchange experiments, in which incubations were analyzed by deuterium exchange and capillary HPLC–QTof-MS and MS/MS. The use of non-radioactive, stabile isotope labeled drug analogues in combination with isotope exchange studies was essential in particular for a defined assignment of the functional groups in the structures of the investigated metabolites.     

Ultra-high performance liquid chromatography tandem mass spectrometry for comprehensive analysis of urinary acylcarnitines

We report an enabling mass spectrometric method for the analysis of lipid metabolites in order to define better the lipid metabolome in terms of chemical diversity and generate fragment ion spectra of these metabolites as a potential resource for unknown metabolite identification. This work focuses on the analysis of one important class of lipid metabolites, the acylcarnitines. Current analytical methods have only detected and identified a limited number of these metabolites. The method described herein provides the most comprehensive acylcarnitine profile in urine of healthy individuals up to date. It involves an optimized solid phase extraction technique for selective analyte extraction using cartridges containing both lipophilic and cation-exchange properties. The captured analytes are then subjected to ultra-high performance liquid chromatography (UPLC) separation, followed by tandem mass spectrometry (MS/MS) analysis using information-dependent acquisitions and selected reaction monitoring (SRM). The urine of six healthy individuals was analyzed using this method. A total of 355 acylcarnitines were detected; only 43 of them have been previously reported in the urine of healthy individuals. Detection of this large number of acylcarnitines illustrates the great diversity of the lipid metabolome as well as the usefulness of the method for profiling acylcarnitines. Furthermore, the MS/MS spectra of the 355 acylcarnitines will be uploaded to a public human metabolome database as a mass spectrometric resource for unknown metabolite identification.     

Liquid chromatography with tandem mass spectrometry quantification of urinary proanthocyanin A2 dimer and its potential use as a biomarker of cranberry intake

The lack of a biomarker for the consumption of cranberries has confounded the interpretation of several studies investigating the effect of cranberry products, especially juices, on health outcomes. The objectives of this pilot study were to develop a liquid chromatography tandem mass spectrometric method for the quantification of the proanthocyanin dimer A‐2 in human urine and validate urinary proanthocyanin dimer A‐2 as a biomarker of cranberry intake. Five healthy, nonsmoking, premenopausal women (20–30 years of age, body mass index: 18.5–25 kg/m²) were assigned to consume a cranberry beverage containing 140 mg proanthocyanin and 35 kilocalories at 237 mL/day, according to a weekly dosing schedule for 7 weeks. Eleven 24 h and morning spot urine samples each were collected from each subject. A reliable, sensitive method for the detection of proanthocyanin dimer A‐2 in urine using liquid chromatography with tandem mass spectrometry was developed with a limit of quantitation of 0.25 ng/mL and a relative standard deviation of 7.26%, precision of 5.7%, and accuracy of 91.7%. While proanthocyanin dimer A‐2 was quantifiable in urine, it did not appear to be excreted in a concentration that corresponded to the dosing schedule and intake of cranberry juice.     

Low-molecular weight protein profiling of genetically modified maize using fast liquid chromatography electrospray ionization and time-of-flight mass spectrometry

In this work, the use of liquid chromatography coupled to electrospray time-of-flight mass spectrometry (LC-TOFMS) has been evaluated for the profiling of relatively low-molecular weight protein species in both genetically modified (GM) and non-GM maize. The proposed approach consisted of a straightforward sample fractionation with different water and ethanol-based buffer solutions followed by separation and detection of the protein species using liquid chromatography with a small particle size (1.8 μm) C(18) column and electrospray-time-of-flight mass spectrometry detection in the positive ionization mode. The fractionation of maize reference material containing different content of transgenic material (from 0 to 5% GM) led to five different fractions (albumins, globulins, zeins, zein-like glutelins, and glutelins), all of them containing different protein species (from 2 to 52 different species in each fraction). Some relevant differences in the quantity and types of protein species were observed in the different fractions of the reference material (with different GM contents) tested, thus revealing the potential use of the proposed approach for fast protein profiling and to detect tentative GMO markers in maize.     

Multiresidue analysis of beta-agonists in bovine and porcine urine,feed and hair using liquid chromatography electrospray ionisation tandem mass spectrometry

The use of β-agonists as growth promoters in cattle breeding is forbidden in many countries for reasons of fair trade and consumer protection. In recent years the use of liquid chromatography (LC) tandem mass spectrometry (MS/MS) has been shown to be the method of choice for the control of β-agonists. In this study an LC-MS/MS multiresidue analysis method is presented for trace analysis of 22 β-agonists. A truly generic concept has been designed based on mixed-mode solid-phase extraction and positive electrospray ionisation LC-MS/MS operated in the multiple reaction monitoring mode. This method allows application to a wide variety of sample matrices such as urine, feed and hair, following minor modifications to the analysis procedure only. The method features fit-for-purpose sensitivity in urine as shown by CCα and CCβ values of less than 0.2 and less than 0.5 μg/l respectively, for all β-agonists studied (terbutaline and reproterol, less than 0.3 and less than 1.0 respectively). Similar but semiquantitative application to feed and hair showed CCβ values of less than 10.0 and less than 5.0 μg/kg, respectively. A further simplification and improvement is demonstrated using Ultra Performance LC (UPLC™) and fast-switching MS/MS. The successful validation of this method following the latest EU requirements and its application to real samples demonstrate that a new versatile tool has been achieved for veterinary control of β-agonists.     

A study of reproducibility of guanidination-dimethylation labeling and liquid chromatography matrix-assisted laser desorption ionization mass spectrometry for relative proteome quantification

The combination of dimethylation after guanidination (2MEGA) isotope labeling with microbore liquid chromatography (LC)-matrix-assisted laser desorption ionization (MALDI) MS and MS/MS [C. Ji, N. Guo, L. Li, J. Proteome Res. 4 (2005) 2099] has been reported as a promising strategy for abundance ratio-dependent quantitative proteome analysis. A critical step in using this integrated strategy is to set up the abundance ratio threshold of peptide pairs, above which the peptide pairs are used for quantifying and identifying the protein that is considered to be differentially expressed between two different samples. The threshold is determined by technical variation (i.e., the overall abundance ratio variation caused by the experimental process including sample workup, MS analysis and data processing) as well as biological variation (i.e., the abundance ratio variation caused by the biological process including cell growth), which can be defined and assessed by a coefficient of variation (CV). We have designed experiments and measured three different levels of variations, starting with the same membrane protein preparation, the same batch of cells and three batches of cells from the same cell line grown under the same conditions, respectively. It is shown that technical variation from the experimental processes involved in 2MEGA labeling LC-MALDI MS has a CV of <15%. In addition, the measured biological variation from cell growth was much smaller than the measured technical variation. From the studies of the occurrence rate of outliers in the distribution of the abundance ratio data within a comparative dataset of peptide pairs, it is concluded that, to compare the proteome changes between two sets of cultured cells without the use of replicate experiments, a relative abundance ratio of greater than 2X or less than 0.5X (X is the average abundance ratio of the dataset) on peptide pairs can be used as a stringent threshold to quantify and identify differentially expressed proteins with high confidence.     

Analysis of proteoglycans derived sulphated disaccharides by liquid chromatography/mass spectrometry

A method has been developed for the identification and quantitative determination of sulphated disaccharides derived from chondroitin sulphate (CS) and dermatan sulphate (DS) chains attached to proteoglycans (PGs). After digestion with Chondroitinase ABC, the pool of disaccharides can be directly separated by liquid chromatography on a porous graphitized carbon (PGC) column and identified by on-line electrospray mass spectrometry under negative ionization conditions. The relative intensities of the fragment ions obtained by MS/MS allow to distinguish the sulphate position. Calibration with standard disaccharides allows the quantification of the different isomers. The method showed good repeatability in terms of relative standard deviation (RSD < 2%) and linearity between 0.5 and 50 ng (total injected amount) for both 4- and 6-sulphated disaccharides. The limit of detection achieved in full scan mode was 0.1 ng. The methodology was applied to different types of biological samples obtained from patients suffering from chronic lung inflammation such as: lung tissue, bronchoalveolar lavage fluid (BALF), induced sputum and urine.     

Single solid phase extraction method for the simultaneous analysis of polar and non-polar pesticides in urine samples by gas chromatography and ultra high pressure liquid chromatography coupled to tandem mass spectrometry

A new multiresidue method has been developed and validated for the simultaneous extraction of more than two hundred pesticides, including non-polar and polar pesticides (carbamates, organochlorine, organophosphorous, pyrethroids, herbicides and insecticides) in urine at trace levels by gas and ultra high pressure liquid chromatography coupled to ion trap and triple quadrupole mass spectrometry, respectively (GC-IT-MS/MS, UHPLC-QqQ-MS/MS). Non-polar and polar pesticides were simultaneously extracted from urine samples by a simple and fast solid phase extraction (SPE) procedure using C₁₈ cartridges as sorbent, and dichloromethane as elution solvent. Recovery was in the range of 60-120%. Precision values expressed as relative standard deviation (RSD) were lower than 25%. Identification and confirmation of the compounds were performed by the use of retention time windows, comparison of spectra (GC-amenable compounds) or the estimation of the ion ratio (LC-amenable compounds). For GC-amenable pesticides, limits of detection (LODs) ranged from 0.001 to 0.436 μg L⁻¹ and limits of quantification (LOQs) from 0.003 to 1.452 μg L⁻¹. For LC-amenable pesticides, LODs ranged from 0.003 to 1.048 μg L⁻¹ and LOQs ranged from 0.011 to 3.494 μg L⁻¹. Finally, the optimized method was applied to the analysis of fourteen real samples of infants from agricultural population. Some pesticides such as methoxyfenozide, tebufenozide, piperonyl butoxide and propoxur were found at concentrations ranged from 1.61 to 24.4 μg L⁻¹, whereas methiocarb sulfoxide was detected at trace levels in two samples.     

Development of versatile isotopic labeling reagents for profiling the amine submetabolome by liquid chromatography–mass spectrometry

Metabolomic profiling involves relative quantification of metabolites in comparative samples and identification of the significant metabolites that differentiate different groups (e.g., diseased vs. controls). Chemical isotope labeling (CIL) liquid chromatography–mass spectrometry (LC–MS) is an enabling technique that can provide improved metabolome coverage and metabolite quantification. However, chemical identification of labeled metabolites can still be a challenge. In this work, a new set of isotopic labeling reagents offering versatile properties to enhance both detection and identification are described. They were prepared by a glycine molecule (or its isotopic counterpart) and an aromatic acid with varying structures through a simple three-step synthesis route. In addition to relatively low costs of synthesizing the reagents, this reaction route allows adjusting reagent property in accordance with the desired application objective. To date, two isotopic reagents, 4-dimethylaminobenzoylamido acetic acid N-hydroxylsuccinimide ester (DBAA-NHS) and 4-methoxybenzoylamido acetic acid N-hydroxylsuccinimide ester (MBAA-NHS), for labeling the amine-containing metabolites (i.e., amine submetabolome) have been synthesized. The labeling conditions and the related LC–MS method have been optimized. We demonstrate that DBAA labeling can increase the metabolite detectability because of the presence of an electrospray ionization (ESI)-active dimethylaminobenzoyl group. On the other hand, MBAA labeled metabolites can be fragmented in MS/MS and pseudo MS³ experiments to provide structural information on metabolites of interest. Thus, these reagents can be tailored to quantitative profiling of the amine submetabolome as well as metabolite identification in metabolomics applications.     

Comprehensive two-dimensional liquid chromatography coupled with mass spectrometry

In this review, instrumental aspects of comprehensive two-dimensional liquid chromatography coupled with mass spectrometry are presented. The milestones of LC×LC are briefly summarized. Instrument configuration, selection of experimental conditions, the different interfaces used in the system and the current applications of LC×LC–MS systems are described.     

Ultra-high capacity liquid chromatography chip/quadrupole time-of-flight mass spectrometry for pharmaceutical analysis

Nanoflow liquid chromatography/mass spectrometry (nano-LC/MS) has attracted increasing interest in virtue of high sensitivity, low sample consumption, and minimal matrix effect. In this work a HPLC-Chip/quadrupole time-of-flight (Q-TOF) MS device with a new ultra-high capacity small molecule chip (UHC-Chip) which features a 500 nL enrichment column and a 150 mm × 75 μm analytical column, was evaluated with a drug mixture covering a wide range of polarities. Excellent chromatographic precision with 0.1-0.5% RSD for retention time and 1.7-9.0% RSD for peak area, low limit of detection, good chip-to-chip reproducibility and linearity were obtained by using this UHC-Chip. Compared with the standard HPLC-Chip with 40 nL trapping column, the UHC-Chip showed higher enrichment capability and hence gave a higher response in signal detection. Additionally, 4-30 times increase in sensitivity was obtained compared with conventional LC/MS, which indicated that UHC-Chip/MS was a valuable tool for the quantitative analysis of low level impurities and degradation products in pharmaceuticals. Moreover, satisfactory results obtained from trace drug analysis of serum samples further proved its practicality and potential for use in drug testing and development.     

Complementary chromatography separation combined with hydride generation-inductively coupled plasma mass spectrometry for arsenic speciation in human urine

This study aimed to establish complementary high performance liquid chromatography (HPLC) methods including three modes of separation: ion pairing, cation exchange, and anion exchange chromatography, with detection by inductively coupled plasma mass spectrometry (ICPMS). The ion pairing mode enabled the separation of inorganic arsenate (As(V)), monomethylarsonic acid (MMA(V)), and dimethylarsinic acid (DMA(V)). However, the ion pair mode was unable to differentiate inorganic arsenite (As(III)) from arsenobetaine (AsB); instead, cation exchange chromatography was used to isolate and quantify AsB. Anion exchange chromatography was able to speciate all of the aforementioned arsenic species. Potential inaccurate quantification problem with urine sample containing elevated concentration of AsB, which eluted immediately after As(III) in anion exchange or ion pairing mode, was overcame by introducing a post-column hydride generation (HG) derivatization step. Incorporating HG between HPLC and ICPMS improved sensitivity and specificity by differentiating AsB from hydride-forming arsenic species. This paper emphasizes the usefulness of complementary chromatographic separations in combination with HG-ICPMS to quantitatively determine concentrations of As(III), DMA(V), MMA(V), As(V), and AsB in the sub-microgram per liter range in human urine.     

Lipid profiling of cyanobacteria Synechococcus sp. PCC 7002 using two‐dimensional liquid chromatography with quadrupole time‐of‐flight mass spectrometry

Glycerolipid is a main component of membranes in oxygenic photosynthetic organisms. Up to now, the majority of publication in this area has focused on the physiological functions of glycerolipids and lipoprotein complexes in photosynthesis, but the study on the separation and identification of glycerolipids in thylakoid membrane in cyanobacteria is relatively rare. Here we report a new method to separate and identify five photosynthetic glycerolipid classes, including monoglucosyl diacylglycerol, monogalactosyl diacylglycerol, digalactosyl diacylglycerol, sulfoquinovosyl diacylglycerol, and phosphatidylglycerol, in cyanobacteria Synechococcus sp. PCC 7002 by two‐dimensional (normal‐ and reversed‐phase) liquid chromatography online coupled to quadrupole time‐of‐flight mass spectrometry. Over twice as many lipid species were detected by our method compared to the previously reported methods. Ten new odd‐chain fatty acid glycerolipids were discovered for the first time. Moreover, complete separation of isomers of monogalactosyl diacylglycerol and monoglucosyl diacylglycerol was achieved. According to the tandem mass spectrometry results, we found that the head group of monoglucosyl diacylglycerols was not as stable as that of monogalactosyl diacylglycerols, which might explain why the organism chose monogalactosyl diacylglycerols and digalactosyl diacylglycerols instead of monoglucosyl diacylglycerols as the main content of the photosynthetic membranes in the history of evolution. This work will benefit further research on the physiological function of glycerolipids.     

Determination of 19-norandrosterone in CCQM-P68. NIST results using an isotope dilution liquid chromatography/tandem mass spectrometry method

The US National Institute of Standards and Technology (NIST) participated in an international interlaboratory study under the auspices of the Consultative Committee for Amount of Substance (CCQM) for the determination of 19-norandrosterone (19-NA) in urine, the principal metabolite of nandrolone and certain other synthetic testosterone substitutes banned for use by the World Antidoping Agency (WADA). Prior to this study, NIST developed a candidate reference measurement procedure based upon isotope dilution liquid chromatography/tandem mass spectrometry. This method was applied to a urine sample distributed to the participants in the study by the Australian National Measurement Institute, Pymble, Australia (NMIA). The NIST results were in very good agreement with those from the other participants, all of whom used methods based upon gas chromatography/mass spectrometry. All known significant sources of uncertainty were evaluated, resulting in a relative expanded uncertainty of less than 5% (coverage factor k = 2).     

Improving liquid chromatography–tandem mass spectrometry determinations by modifying noise frequency spectrum between two consecutive wavelet-based low-pass filtering procedures

This paper employs one chemometric technique to modify the noise spectrum of liquid chromatography–tandem mass spectrometry (LC–MS/MS) chromatogram between two consecutive wavelet-based low-pass filter procedures to improve the peak signal-to-noise (S/N) ratio enhancement. Although similar techniques of using other sets of low-pass procedures such as matched filters have been published, the procedures developed in this work are able to avoid peak broadening disadvantages inherent in matched filters. In addition, unlike Fourier transform-based low-pass filters, wavelet-based filters efficiently reject noises in the chromatograms directly in the time domain without distorting the original signals. In this work, the low-pass filtering procedures sequentially convolve the original chromatograms against each set of low pass filters to result in approximation coefficients, representing the low-frequency wavelets, of the first five resolution levels. The tedious trials of setting threshold values to properly shrink each wavelet are therefore no longer required. This noise modification technique is to multiply one wavelet-based low-pass filtered LC–MS/MS chromatogram with another artificial chromatogram added with thermal noises prior to the other wavelet-based low-pass filter. Because low-pass filter cannot eliminate frequency components below its cut-off frequency, more efficient peak S/N ratio improvement cannot be accomplished using consecutive low-pass filter procedures to process LC–MS/MS chromatograms. In contrast, when the low-pass filtered LC–MS/MS chromatogram is conditioned with the multiplication alteration prior to the other low-pass filter, much better ratio improvement is achieved. The noise frequency spectrum of low-pass filtered chromatogram, which originally contains frequency components below the filter cut-off frequency, is altered to span a broader range with multiplication operation. When the frequency range of this modified noise spectrum shifts toward the high frequency regimes, the other low-pass filter is able to provide better filtering efficiency to obtain higher peak S/N ratios. Real LC–MS/MS chromatograms, of which typically less than 6-fold peak S/N ratio improvement achieved with two consecutive wavelet-based low-pass filters remains the same S/N ratio improvement using one-step wavelet-based low-pass filter, are improved to accomplish much better ratio enhancement 25-folds to 40-folds typically when the noise frequency spectrum is modified between two low-pass filters. The linear standard curves using the filtered LC–MS/MS signals are validated. The filtered LC–MS/MS signals are also reproducible. The more accurate determinations of very low concentration samples (S/N ratio about 7–9) are obtained using the filtered signals than the determinations using the original signals.     

Contents of selected B vitamins in NIST SRM 3280 multivitamin/multielement tablets by liquid chromatography isotope dilution mass spectrometry

There is increased interest in accurately assessing the total dietary intake of vitamins from all sources, including foods and dietary supplements. Consequently, a Dietary Supplement Ingredient Database (DSID), based upon analytical values, is being established by USDA with support of the Office of Dietary Supplements (ODS), NIH. The DSID necessitated the development of a new SRM, 3280 — Multivitamin/Multimineral Tablets, by the National Institute of Standards and Technology (NIST), with support from the ODS. As a continuation of a long-term project to develop and validate new methods of determining water-soluble B vitamins in foods and dietary supplements, and as part of a collaborative effort with NIST to characterize SRM 3280, values for the vitamin contents of SRM 3280 have been generated by a liquid chromatographic isotope dilution mass spectrometric (LC/IDMS) method. Isotope-labeled (¹³C and/or ²H) B vitamins (B1-thiamine, B6-pyridoxine, B3-nicotinamide, and B5-pantothenic acid) were obtained from commercial sources, with the support of the ODS/NIH. Our LC/IDMS method uses a C18 reversed phase column, an Agilent 1100 HPLC system, and a Quattro Micro triple-quad mass spectrometer (MS). B vitamin determination was achieved using a gradient LC profile combined with MS/MS detection in multiple reaction monitoring mode. Stock solutions of the isotope-labeled vitamins were calibrated against USP standard solutions. The SRM tablets, with added amounts of the four isotope-labeled B vitamins, were extracted and the vitamins simultaneously determined in a single LC run, in contrast with the single-component determinations performed via IDMS. Unknown vitamin concentrations were calculated by comparing the ratios of the integrated LC peaks at the different masses of the unlabeled and labeled vitamins.