Chromatographic Shifts Research Articles

B-249 Assessing Targeted Mass Spectrometry Assay Performance for Putative Biomarker Proteins on a New Hybrid Nominal Mass Instrument

Abstract Background The development of targeted mass spectrometry-based proteomics assays that monitor biomedically relevant proteins is an important step in bridging discovery experiments to highly multi-analyte clinical studies. Targeted assays are currently unable to scale to thousands of peptide targets from hundreds of proteins, requiring extensive refinement down assays for the minimum useful analytes. Large parallel reaction monitoring (PRM) assays are now possible with a new hybrid nominal-mass instrument. The scale of assay is achievable with this instrument by using real-time alignment, while being sensitive and fast enough to handle many concurrent targets. To assess the performance of this new instrument we constructed a large scale PRM assay for proposed neurodegenerative disease marker proteins, and an assay for proteins associated with amyloid tissue typing. Methods To evaluate the performance of this instrument on proteins of interest in cerebrospinal fluid, we performed an analysis on a pool from individuals diagnosed as Alzheimer’s, Parkinson’s, or cognitively normal. A calibration curve of cerebrospinal fluid diluted into chicken serum at 14 dilutions and individual human cerebrospinal fluid were digested with a protein aggregation and capture based digestion protocol. CSF peptides were separated by 60 minute gradient on C18 IonOpticks column, and amyloid tissue peptides separated by 30 minute gradient on C18 PepMap column with a Vanquish Neo UHPLC and analyzed by data-independent acquisition (DIA) and PRM with a new hybrid nominal mass instrument designed for targeted tandem mass spectrometry (tMS2). Scheduled methods were generated using a Skyline external tool, transitions were refined to minimize interference and maximize the LLOQ. DIA data was searched, and data extracted and analyzed in Skyline. PRM data was extracted and quantified with Skyline; CSF calibration curve LLOQ is calculated by bilinear fit. Results For the large scale cerebrospinal fluid (CSF) assay the selection of protein targets was based on literature from neurodegenerative disease cohort studies. We target 1065 peptides from 104 proteins, including proteins currently measured by ELISA in clinical research, e.g.: APP, TAU, and APOE. Sensitivity and reproducibility of the assay is assessed using figures of merit from replicate matched-matrix calibration curves. Over 80% of proteins have at least one peptide with a LLOQ below 30% dilution in the matched matrix, and of all targeted peptides over 55% had a LLOQ below 30% dilution. To demonstrate the application of the real-time alignment on the new instrument we constructed an assay for samples where not every analyte is shared. An assay for amyloidosis associated proteins was constructed for a total of 180 peptides mapping to 52 proteins. This includes 11 proteins most commonly associated with amyloid deposition; APCS, APOA4, APOE, CLU, LECT2, IGKC, IGLC2/4/7, SAA1, TTR, and VTN. From the preliminary set of 12 previously typed samples we observe peptide abundances consistent with the assigned type. Conclusions This novel instrument provides a new approach for building large targeted mass spectrometry assays. We demonstrate the ability of the real-time alignment to adjust scheduled retention time windows, ensuring that chromatographic shifts do not lead to a loss of peptide detection and quantification.

LC-MS/MS determination of atropine toxicity: Pre-analytical effect of blood collection tube and analytical matrix

Atropine (ATR) intoxication is a recurrent case in emergency departments. The diagnosis is dependent on clinical evaluation and is supported by analytical assessment. The assay is limited by the rapid degradation/metabolism of ATR into TRP as well as the preanalytical factors impairing correct detection and diagnosis.In this study, an HPLC-MS/MS method was optimized for the simultaneous determination of ATR and TRP. The effect of analytical matrix and the impact of blood-collection tube type on the ATR analytical signal were investigated.Separation was achieved using water: 0.01% formic acid acidified methanol (40: 60, v/v) as a mobile phase and Inertsil® C18 column (5 µm; 4.6*150 mm) as a stationary phase. The retention-times were 2.6 and 6.5 min for ATR and TRP, respectively. A chromatographic shift (0.4 min) in ATR peak, but not TRP, was observed in biological samples from neat ones. The best analytical signal was observed when heparinized blood collection tubes were employed. The method was linear, accurate and precise in the ATR toxicity range enabling the detection of ATR intoxication down to a concentration of 0.1 ng/mL by applying a simple sample clean-up procedure.In conclusion, an HPLC-MS/MS method for the simultaneous determination of ATR and TRP is presented. The method highlights the chromatographic shift of ATR peak in biological samples that may induce false-negative detection and poses TRP as an alternative toxicological marker for ATR toxicity. Meanwhile the study recommends heparin tubes for blood-sample collection.

Neutron encoded derivatization of endothelial cell lysates for quantitation of aldehyde metabolites using nESI-LC-HRMS

Biological aldehydes are difficult to analyze by electrospray ionization mass spectrometry due to their poor proton affinity and low biological concentrations. Chemical derivatization with stable isotope tags is used here for sample multiplexing, increased throughput, improved signal intensity, and quantitation. Nine quaternary amine tags with mass differences as low as 0.0058 Da had no observable chromatographic shifts, small amounts of ion suppression, and minimal matrix effects. Low concentration perfluoropentanoic acid was used as an ion pairing reagent to improve the retention of derivatized aldehydes. Perfluoropentanoic acid addition showed an average of three-fold improvement in limits of detection, 50% reduction in peak width, and 2.5 fold increase in analyte retention. Analysis of fifteen tagged aldehydes yielded an average of 13 nM limit of detection, 9 %RSD, R2 of 0.995, and linear dynamic range of 40–1000 nM. In a single 20 min separation, absolute quantitative data was obtained for 11 reactive aldehydes across 8 aortic endothelial cell samples. High glucose treatment produced significant changes to malondialdehyde, decanal, and (2E)-hexadecenal. These changes are consistent with glucose-induced oxidative stress. This method demonstrates that neutron encoded tagging of aldehydes is suitable for the analysis of complex samples.

Chemically informed analyses of metabolomics mass spectrometry data with Qemistree.

Untargeted mass spectrometry is employed to detect small molecules in complex biospecimens, generating data that are difficult to interpret. We developed Qemistree, a data exploration strategy based on the hierarchical organization of molecular fingerprints predicted from fragmentation spectra. Qemistree allows mass spectrometry data to be represented in the context of sample metadata and chemical ontologies. By expressing molecular relationships as a tree, we can apply ecological tools that are designed to analyze and visualize the relatedness of DNA sequences to metabolomics data. Here we demonstrate the use of tree-guided data exploration tools to compare metabolomics samples across different experimental conditions such as chromatographic shifts. Additionally, we leverage a tree representation to visualize chemical diversity in a heterogeneous collection of samples. The Qemistree software pipeline is freely available to the microbiome and metabolomics communities in the form of a QIIME2 plugin, and a global natural products social molecular networking workflow.

Gradual and Discrete Ontogenetic Shifts in Rattlesnake Venom Composition and Assessment of Hormonal and Ecological Correlates.

Ontogenetic shifts in venom occur in many snakes but establishing their nature as gradual or discrete processes required additional study. We profiled shifts in venom expression from the neonate to adult sizes of two rattlesnake species, the eastern diamondback and the timber rattlesnake. We used serial sampling and venom chromatographic profiling to test if ontogenetic change occurs gradually or discretely. We found evidence for gradual shifts in overall venom composition in six of eight snakes, which sometimes spanned more than two years. Most chromatographic peaks shift gradually, but one quarter shift in a discrete fashion. Analysis of published diet data showed gradual shifts in overall diet composition across the range of body sizes attained by our eight study animals, while the shifts in abundance of different prey classes varied in form from gradual to discrete. Testosterone concentrations were correlated with the change in venom protein composition, but the relationship is not strong enough to suggest causation. Venom research employing simple juvenile versus adult size thresholds may be failing to account for continuous variation in venom composition lifespan. Our results imply that venom shifts represent adaptive matches to dietary shifts and highlight venom for studies of alternative gene regulatory mechanisms.

Exploiting second-order advantage from mathematically modeled liquid chromatography–mass spectrometry data for simultaneous determination of polyphenols in Chinese propolis

The quantification of polyphenols in propolis has attracted considerable concerns for their biological properties. Liquid chromatography–mass spectrometry (LC–MS) coupled with chemometrics multivariate calibration analytical strategy was developed for simple and rapid determination of eleven polyphenols in Chinese propolis. The eleven polyphenols were fast eluted out within 7.0 min, and then they were detected by mass spectrometry operated in the full scan mode. The acquired second-order data of LC–MS were resolved by two well-known chemometrics tools including alternating trilinear decomposition (ATLD) and multivariate curve resolution-alternating least squares (MCR-ALS). With the aid of the distinctive “second-order advantage”, satisfactory resolution and reliable results were obtained in the presence of chromatographic shifts, serious co-eluted peaks and diverse unknown interferences. The meanvalue of spiked recoveries obtained by the ATLD and MCR-ALS methods for eleven polyphenols in propolis samples were in the range of 81.2–109.1% and 75.8–107.1%, respectively, which have verified by the LC–MS/MS method. Statistical parameters and analytical figures of merit were also used to assess the performances of the proposed strategy. All these results demonstrated that the proposed strategy opened a new avenue for quantification of the targeted polyphenols in complex propolis samples for quality monitoring, which does not require toilsome sample pretreatments, elaborate experimental conditions and more advanced high cost instrumentations.

On the performance of multivariate curve resolution to resolve highly complex liquid chromatography–full scan mass spectrometry data for quantification of selected immunosuppressants in blood and water samples

Liquid chromatography triple-quadrupole mass spectrometry (LC–MS/MS) in multiple reaction monitoring (MRM) mode is a commonly used approach for quantification of target compounds in complex samples. Regarding the multivariate advantages that can be obtained in the light of full spectra recording, the potential and performance of a smart strategy which is a post-processing of LC–MS full scan data sets with multivariate curve resolution-alternating least squares (MCR-ALS) algorithm for determination of cyclosporine-A (CsA) and tacrolimus (TAC) as typical immunosuppressants in human blood and organic micropollutants in surface water samples was investigated in the current study. Before peak resolution and quantification, LC–MS data compression was performed on each individual and augmented data matrices based on searching the regions of interest (ROIs) in the m/z domain. Then, due to presence of matrix effect, MCR-ALS modeling was performed using a matrix-matched calibration strategy. Here, the serious LC–MS issues such as background drift, chromatographic shifts and co-elution of non-calibrated sample constituents were resolved by implementation of MCR-ALS method on ROI based compressed data. It was shown that the strategy is a powerful tool for the quantitation of CsA and TAC in complex matrices and in the presence of uncalibrated interferences with highly overlapped peaks. However, the overall results indicated that performance of the multivariate modeling of LC–MS in full scan mode for drug quantitation strongly depends on the selected drug, as in the current case superiority of the method for analysis of CsA over TAC, with different therapeutic concentration ranges, was observed.

Elucidation of chromatographic peak shifts in complex samples using a chemometrical approach

Chromatographic retention time peak shifts between consecutive analyses is a well-known fact yet not fully understood. Algorithms have been developed to align peaks between runs, but with no specific studies considering the causes of peak shifts. Here, designed experiments reveal chromatographic shift patterns for a complex peptide mixture that are attributable to the temperature and pH of the mobile phase. These results demonstrate that peak shifts are highly structured and are to a high degree explained by underlying differences in physico-chemical parameters of the chromatographic system and also provide experimental support for the alignment algorithm called the generalized fuzzy Hough transform which exploits this fact. It can be expected that the development of alignment algorithms enters a new phase resulting in increasingly accurate alignment by considering the latent structure of the peak shifts.

Development of a fast HPLC-DAD method for simultaneous quantitation of three immunosuppressant drugs in whole blood samples using intelligent chemometrics resolving of coeluting peaks in the presence of blood interferences

The present study describes a fast high performance liquid chromatography-diode array detection analytical methodology for quantification of tacrolimus, everolimus and cyclosporine A in whole blood samples, with minimum sample preparation steps. A short isocratic chromatographic elution was coupled with second-order calibration using multivariate curve resolution to stablish a smart and green methodology. Due to presence of matrix effect, a sample-added calibration strategy was used for quantification purposes. The serious issues related to background drift, chromatographic shifts and co-elution of non-calibrated blood components, were resolved by a proper background correction and multivariate curve resolution-alternating least squares (MCR/ALS) methods The main features of this study were based on the fact that the acquired data matrices were handled intelligently and all features of the concerned target analytes were taken into account. Satisfactory resolution and quantification results in the presence of matrix interferences were achieved and the second-order advantage was fully exploited. The average recoveries in therapeutic concentration ranges were 102±10%, 99±11% and 104±12% for TAC, EVR and CsA, with average relative prediction errors of less than 7%. Considering the advantages of the present strategy, such as increased selectivity, sensitivity and sufficiency of lower limit of quantification through multivariate advantage, simplicity of sample treatment steps, a fast elution pattern and also a low-cost instrumentation compared with LC–MS/MS, the proposed method has the significant merits as an alternative for simultaneous therapeutic monitoring of immunosuppressants.

Fast and simultaneous determination of 12 polyphenols in apple peel and pulp by using chemometrics-assisted high-performance liquid chromatography with diode array detection.

In this work, a smart chemometrics-enhanced strategy, high-performance liquid chromatography, and diode array detection coupled with second-order calibration method based on alternating trilinear decomposition algorithm was proposed to simultaneously quantify 12 polyphenols in different kinds of apple peel and pulp samples. The proposed strategy proved to be a powerful tool to solve the problems of coelution, unknown interferences, and chromatographic shifts in the process of high-performance liquid chromatography analysis, making it possible for the determination of 12 polyphenols in complex apple matrices within 10min under simple conditions of elution. The average recoveries with standard deviations, and figures of merit including sensitivity, selectivity, limit of detection, and limit of quantitation were calculated to validate the accuracy of the proposed method. Compared to the quantitative analysis results from the classic high-performance liquid chromatography method, the statistical and graphical analysis showed that our proposed strategy obtained more reliable results. All results indicated that our proposed method used in the quantitative analysis of apple polyphenols was an accurate, fast, universal, simple, and green one, and it was expected to be developed as an attractive alternative method for simultaneous determination of multitargeted analytes in complex matrices.

Resolving Isomeric Glycopeptide Glycoforms with Hydrophilic Interaction Chromatography (HILIC).

The ability to resolve glycans while attached to tryptic peptides would greatly facilitate glycoproteomics, as this would enable site-specific glycan characterization. Peptide/glycopeptide separations are typically performed using reversed-phase liquid chromatography (RPLC), where retention is driven by hydrophobic interaction. As the hydrophilic glycans do not interact significantly with the RPLC stationary phase, it is difficult to resolve glycopeptides that differ only in their glycan structure, even when these differences are large. Alternatively, glycans interact extensively with the stationary phases used in hydrophilic interaction chromatography (HILIC), and consequently, differences in glycan structure have profound chromatographic shifts in this chromatographic mode. Here, we evaluate HILIC for the separation of isomeric glycopeptide mixtures that have the same peptide backbone but isomeric glycans. Hydrophilic functional groups on both the peptide and the glycan interact with the HILIC stationary phase, and thus, changes to either of these moieties can alter the chromatographic behavior of a glycopeptide. The interactive processes permit glycopeptides to be resolved from each other based on differences in their amino acid sequences and/or their attached glycans. The separations of glycans in HILIC are sufficient to permit resolution of isomeric N-glycan structures, such as sialylated N-glycan isomers differing in α2-3 and α2-6 linkages, while these glycans remain attached to peptides.

Relationship between gas chromatographic retention indices and chemical shifts in the 13C NMR spectra of structural isomers of compounds of tetracoordinated phosphorus

A method for determining the gas chromatographic retention indices of structural isomers of compounds of tetracoordinated phosphorus, based on the correlation between chemical shifts in 13C nuclear magnetic resonance (NMR) spectra and the retention indices of the structural isomers, and on the theory of intermolecular interactions, is proposed. The capabilities of the method are demonstrated for structural isomers of O-alkylmethylfluorophosphonates, which are typical representatives of compounds from the class of tetracoordinated phosphonates. The results from using the correlation approach are compared to experimental data.

PCBs의 광화학적 연구: NaOH 및 휴믹산 (humic acid, HA)에 의한 분해특성

Objectives: This study was carried out to examine whether the apparent photolysis with or without sensitizers [NaOH and humic acid (HA)] was prompted photodegradation of polychlorinated biphenyl (PCB) in aqueous solution. Methods: PCBs photodegradation occurred using fluorescence black lamps at <TEX>${\lambda}_{max}=300nm$</TEX>. PCB congeners were exposed in 10 ppm HA or 0.05N NaOH solutions, to investigate the decreasing profile of PCB concentration with time. The PCBs were then analyzed by gas chromatography/mass spectrometry (GC-MS). Reductive degradation profile of PCB congeners in the presence of both sensitizers under oxygen-saturated protic conditions was described using the wind-rose diagrams. Results: Use of HA or NaOH decreased PCB concentration with time in the dark and on irradiation, indicating that photolysis underwent through reductive dechlorination through energy transfer and possibly with reactive oxygens. The dechlorination was marked by a chromatographic shift, observed in the GC-MS plots. Therefore it is logical to assume that increasing the dose of sensitizers would increase the photodegradation rates of PCBs. The half-lives of pentachloro-PCB (penta-3) in 0.05N NaOH and 10 ppm HA were estimated at about 47 hours and 39 hours, respectively, under the same experimental conditions of photolysis. It was found that the rate of photolysis of pentachloro-PCB in aqueous solution followed apparent first-order kinetics compared to other congeners. Conclusion: Photochemical degradation (using 328 nm UV light) of penta- and hexa-PCBs in HA or alkaline solution is a viable method for pretreatment method. The results are helpful for the further comprehension of the reaction mechanism for photolytic dechlorination of PCBs in aquatic system.

Deuterium Isobaric Amine-Reactive Tags for Quantitative Proteomics

This paper demonstrates the applications of a novel isobaric reagent, named deuterium ((2)H) isobaric amine-reactive tag (DiART), for quantitative proteomics. Peptides labeled with DiART were analyzed using an electrospray ionization (ESI)-based LTQ-Orbitrap mass spectrometer. Our data showed that (2)H-associated isotope effects, such as partial loss of (2)H labels during tandem mass spectrometry (MS/MS) and (2)H-related chromatographic shift, were either not observed or negligible. With the use of a hybrid collision-induced dissociation (CID)-higher energy C-trap dissociation (HCD) acquisition method, we were able to identify DiART-labeled peptides with high confidence and quantify them with high accuracy. Furthermore, we adopted a hybrid electron-transfer dissociation (ETD)-HCD acquisition protocol and developed a novel data analysis approach to measure phosphorylation of peptides. Our results showed DiART had excellent performance on LTQ-Orbitrap instruments and provided a cost-effective technique for large-scale quantitative proteomics measurements.

IDEAL-Q, an Automated Tool for Label-free Quantitation Analysis Using an Efficient Peptide Alignment Approach and Spectral Data Validation

In this study, we present a fully automated tool, called IDEAL-Q, for label-free quantitation analysis. It accepts raw data in the standard mzXML format as well as search results from major search engines, including Mascot, SEQUEST, and X!Tandem, as input data. To quantify as many identified peptides as possible, IDEAL-Q uses an efficient algorithm to predict the elution time of a peptide unidentified in a specific LC-MS/MS run but identified in other runs. Then, the predicted elution time is used to detect peak clusters of the assigned peptide. Detected peptide peaks are processed by statistical and computational methods and further validated by signal-to-noise ratio, charge state, and isotopic distribution criteria (SCI validation) to filter out noisy data. The performance of IDEAL-Q has been evaluated by several experiments. First, a serially diluted protein mixed with Escherichia coli lysate showed a high correlation with expected ratios and demonstrated good linearity (R2 = 0.996). Second, in a biological replicate experiment on the THP-1 cell lysate, IDEAL-Q quantified 87% (1,672 peptides) of all identified peptides, surpassing the 45.7% (909 peptides) achieved by the conventional identity-based approach, which only quantifies peptides identified in all LC-MS/MS runs. Manual validation on all 11,940 peptide ions in six replicate LC-MS/MS runs revealed that 97.8% of the peptide ions were correctly aligned, and 93.3% were correctly validated by SCI. Thus, the mean of the protein ratio, 1.00 ± 0.05, demonstrates the high accuracy of IDEAL-Q without human intervention. Finally, IDEAL-Q was applied again to the biological replicate experiment but with an additional SDS-PAGE step to show its compatibility for label-free experiments with fractionation. For flexible workflow design, IDEAL-Q supports different fractionation strategies and various normalization schemes, including multiple spiked internal standards. User-friendly interfaces are provided to facilitate convenient inspection, validation, and modification of quantitation results. In summary, IDEAL-Q is an efficient, user-friendly, and robust quantitation tool. It is available for download.

Combining protein identification and quantification: C‐terminal isotope‐coded tagging using sulfanilic acid

Two methods of differential isotopic coding of carboxylic groups have been developed to date. The first approach uses d0- or d3-methanol to convert carboxyl groups into the corresponding methyl esters. The second relies on the incorporation of two 18O atoms into the C-terminal carboxylic group during tryptic digestion of proteins in H(2)18O. However, both methods have limitations such as chromatographic separation of 1H and 2H derivatives or overlap of isotopic distributions of light and heavy forms due to small mass shifts. Here we present a new tagging approach based on the specific incorporation of sulfanilic acid into carboxylic groups. The reagent was synthesized in a heavy form (13C phenyl ring), showing no chromatographic shift and an optimal isotopic separation with a 6 Da mass shift. Moreover, sulfanilic acid allows for simplified fragmentation in matrix-assisted laser desorption/ionization (MALDI) due the charge fixation of the sulfonate group at the C-terminus of the peptide. The derivatization is simple, specific and minimizes the number of sample treatment steps that can strongly alter the sample composition. The quantification is reproducible within an order of magnitude and can be analyzed either by electrospray ionization (ESI) or MALDI. Finally, the method is able to specifically identify the C-terminal peptide of a protein by using GluC as the proteolytic enzyme.

Top-Down Approaches for Measuring Expression Ratios of Intact Yeast Proteins Using Fourier Transform Mass Spectrometry

The extension of quantitation methods for small peptides to ions above 5 kDa, and eventually to global quantitative proteomics of intact proteins, will require extensive refinement of current analytical approaches. Here we evaluate postgrowth Cys-labeling and 14N/15N metabolic labeling strategies for determination of relative protein expression levels and their posttranslational modifications using top-down mass spectrometry (MS). We show that intact proteins that are differentially alkylated with acrylamide (+71 Da) versus iodoacetamide (+57 Da) have substantial chromatographic shifts during reversed-phase liquid chromatography separation (particularly in peak tails), indicating a requirement for stable isotopes in alkylation tags for top-down MS. In the 14N/15N metabolic labeling strategy, we achieve 98% 15N incorporation in yeast grown 10 generations under aerobic conditions and determine 50 expression ratios using Fourier transform ion cyclotron resonance MS in comparing these cells to anaerobically grown control (14N) cells. We devise quantitative methods for top-down analyses, including a correction factor for accurate protein ratio determination based upon the signal-to-noise ratio. Using a database of 200 yeast protein forms identified previously by top-down MS, we verify the intact mass tag concept for protein identification without tandem MS. Overall, we find that top-down MS promises work flows capable of large-scale proteome profiling using stable isotope labeling and the determination of >5 protein ratios per spectrum.

Chemoenzymatic Approaches for Streamlined Detection of Active Site Modifications on Thiotemplate Assembly Lines Using Mass Spectrometry

For the direct interrogation of peptides harboring covalently modified serines in nonribosomal peptide synthetases, streamlined methodologies described here employ proteolysis and reporter-coenzyme A analogues of four types. The chromophoric and fluorescent coenzyme A analogues pyrene-maleimidyl-S-CoA and BODIPY-FL-N-(2-aminoethyl)maleimidyl-S-CoA were enzymatically loaded onto the active site serines harbored in the ArCP, PCP1, and PCP2 thiolation domains of PchE and PchF, the nonribosomal peptide synthetases responsible for the biosynthesis of the siderophore pyochelin. During the chromatographic separation of cyanogen bromide digests, observation of the absorbance (at 338 and 504 nm) or fluorescence (after irradiation at 365 nm) enabled the selective detection of peptides containing each active site serine. This resulted in quick detection of each active site peptide by Fourier transform mass spectrometry in the fully reconstituted pyochelin system. The loading of short acyl chain reporters in equimolar quantities permitted further insights into digestion heterogeneity and side reactions by virtue of a mass shift signature on each active site peptide. The chromatographic shift of the reporter-loaded peptides relative to peptides carrying on pathway intermediates was 2 min at 7 kDa, providing a general strategy for efficient localization of "carrier" peptides in complex digests of thiotemplate enzymes. Also, the use of the affinity reporter, biotin-maleimidyl-S-coenzyme A, permitted the isolation of intact synthetases at high purity via removal of contaminating Escherichia coli proteins.

HPLC method for the analysis of acetonides of ecdysteroids providing structural information on different vicinal diols

The polyhydroxy structure of ecdysteroids provides a basis for the formation of various derivatives including the acetonides that have been frequently prepared mainly in preparative work. Acetonides can be formed between vicinal diols in cis position such as positions C-2/C-3 and C-20/C-22 in 20-hydroxyecdysone, for example. We have modified an established procedure for the preparation of acetonides, employing a higher concentration of phosphomolybdic acid as a catalyst, and developed a reverse-phase HPLC method to analyze the products on a micro scale. The derivatization method is fast, taking minutes or less, and does not produce artifacts. Thirteen structurally diverse ecdysteroids were studied in detail concerning speed of acetonide formation, and the number and type of intermediate and end products. Mass spectrometry coupled to HPLC was employed to confirm and/or identify ecdysteroids and their acetonide derivatives. Analysis of the chromatographic profiles revealed that detailed information on vicinal diols of ecdysteroids can be derived from the differences in retention times (Deltar.t.)1 of products relative to the parent steroids on reverse-phase HPLC using a linear water/methanol gradient for elution. The specific Deltar.t. values characterize a product as a monoacetonide, a diacetonide, or a triacetonide. Furthermore, this chromatographic shift upon acetonide formation is significantly different among the various monoacetonides and, thus, specific for the site on the ecdysteroid molecule. The same seems to hold for diacetonides. As variation of the Deltar.t. values for a specific type of acetonide was rather small among the tested ecdysteroids, the results should provide reliable structural information. Muristerone A did not completely fit this common pattern. However, this may specify its uncommon ecdysteroid structure. The method has recently been successfully employed to solve the structures of new ecdysteroid metabolites. Arch. Copyright 1999 Wiley-Liss, Inc.

The gas chromatographic shift of tritium-labeled compounds—An application of gas liquid radiochromatography

A small reduction in the gas-chromatographic retention times of a number of tritiated compounds compared to their unlabeled analogues has been observed. The shift appears to be proportional to the number of tritium atoms in a molecule.