Qualitative and quantitative analysis of pharmaceutical compounds by MALDI-TOF mass spectrometry

In this report, we discuss key issues for the successful application of MALDI-TOF mass spectrometry to quantify drugs. These include choice and preparation of matrix, nature of cationization agent, automation, and data analysis procedures. The high molecular weight matrix meso-tetrakis(pentafluorophenyl)porphyrin eliminates chemical noise in the low-mass range, a "brushing" spotting technique in combination with prestructured target plates enables fast preparation of homogeneous matrix crystals, and addition of Li+ leads to intense cationized drug species. Complex biological samples were cleaned up using a 96-well solid-phase extraction plate, and the purified samples were automatically spotted by a pipetting robot. To obtain a suitable data analysis procedure for the quantitative analysis of drugs by MALDI-TOF mass spectrometry, various data processing parameters were evaluated on our two model drugs lopinavir and ritonavir. Finally, and most importantly, it is shown that the above-described procedure can be successfully applied to quantify clinically relevant concentrations of lopinavir, an HIV protease inhibitor, in extracts of small numbers of peripheral blood mononuclear cells (1 x 10(6)).     

Qualitative and quantitative analysis of the glycosylation pattern of recombinant proteins.

Over the past decade, the growing number of recombinant glycoproteins used as therapeutic agents has prompted the development of robust and rugged methodologies for characterizing the glycosylation pattern of such molecules. The present study describes an alternative to the widely used HPLC approaches for profiling the N-glycan heterogeneity of proteins. The method encompasses the enzymatic deglycosylation of the glycoprotein, the permethylation of the released oligosaccharides, and the subsequent analysis of these derivatives by either matrix-assisted laser desorption/ionization or electrospray mass spectrometry. This methodology showed excellent correlation when compared with results obtained by an orthogonal technique such as the HPLC of 2-aminobenzamide-labeled glycans. In addition, it gives a more detailed insight into the glycosylation pattern by unambiguously identifying and quantifying the various glycoforms present in the mixture. Despite a somewhat complex sample preparation, reproducibility and robustness of the method were excellent. In the case of very heterogeneous glycan pools, simplification of the glycosylation pattern was achieved by performing enzymatic desialylation prior to deglycosylation and derivatization, leading to a more direct determination of the antennary distribution as well as the identification of minor components.     

超滤和核磁技术定性分析聚羧酸减水剂的大单体

采用超滤和核磁技术,建立分析未知聚羧酸减水剂中大单体的方法。结果表明,未知聚羧酸减水剂样品依次经过30,10,3 k D(道而顿)超滤膜后,大分子量的组分被逐级滤去;超滤滤液再经过500D的透析袋预处理去除杂质,得到相对较纯的大单体。应用核磁共振氢谱技术对常见的已知大单体进行鉴别分析,进而解析出未知聚羧酸样品使用的大单体为异戊烯醇聚乙二醇醚(TPEG)。该法为聚羧酸高分子样品的剖析提供重要的分析手段,对聚合物的分析具有一定的借鉴作用。     

HPLC analysis of ecdysteroids in plant extracts using superheated deuterium oxide with multiple on-line spectroscopic analysis (UV, IR, 1H NMR, and MS).

HPLC, using superheated D20 as the mobile phase, combined with on-line characterization via a combination of diode array UV, 1H NMR, FT-IR spectroscopy, and mass spectrometry has been used for the analysis of a standard of 20-hydroxyecdysone- and ecdysteroid-containing plant extracts. This combination of spectrometers enabled the on-flow collection of UV, 1H NMR, IR, and mass spectra not only for pure 20-hydroxyecdysone (100-400 microg on column) but also the major ecdysteroids present in crude extracts of Silene otites, Silene nutans, and Silene frivaldiskyana. The ecdysteroids unequivocally identified in these extracts included 20-hydroxyecdysone, polypodine B, and integristerone A.     

Qualitative and quantitative analysis of illicit drug mixtures on paper currency using Raman microspectroscopy

Measurement of illicit drugs on paper currency is of interest for evidentiary purposes in legal cases involving the drug trade. Current methods, primarily based on mass spectrometry, are destructive and prevent re-analysis of the evidence. This article details a method based on Raman microspectroscopy that is able to collect spectra from individual crystals on the surface of paper currency. Mixtures of isoxsuprine and norephedrine, which are non-pharmacologically active drug surrogates, as well as lidocaine and benzocaine, common excipients in street drugs, were doped in small quantities onto US currency. Significant fluorescence interference resulted from the underlying dollar bill. This work presents two methods for reducing the fluorescence background, photobleaching and background subtraction, which both worked well. Finally, a method for determining the percent composition of individual components in heterogeneous mixtures was developed by systematically sampling the surface of the dollar bill. Results were accurate within a few percent, although the method was quite time consuming.     

Targeted profiling: quantitative analysis of 1H NMR metabolomics data

Extracting meaningful information from complex spectroscopic data of metabolite mixtures is an area of active research in the emerging field of "metabolomics", which combines metabolism, spectroscopy, and multivariate statistical analysis (pattern recognition) methods. Chemometric analysis and comparison of 1H NMR1 spectra is commonly hampered by intersample peak position and line width variation due to matrix effects (pH, ionic strength, etc.). Here a novel method for mixture analysis is presented, defined as "targeted profiling". Individual NMR resonances of interest are mathematically modeled from pure compound spectra. This database is then interrogated to identify and quantify metabolites in complex spectra of mixtures, such as biofluids. The technique is validated against a traditional "spectral binning" analysis on the basis of sensitivity to water suppression (presaturation, NOESY-presaturation, WET, and CPMG), relaxation effects, and NMR spectral acquisition times (3, 4, 5, and 6 s/scan) using PCA pattern recognition analysis. In addition, a quantitative validation is performed against various metabolites at physiological concentrations (9 microM-8 mM). "Targeted profiling" is highly stable in PCA-based pattern recognition, insensitive to water suppression, relaxation times (within the ranges examined), and scaling factors; hence, direct comparison of data acquired under varying conditions is made possible. In particular, analysis of metabolites at low concentration and overlapping regions are well suited to this analysis. We discuss how targeted profiling can be applied for mixture analysis and examine the effect of various acquisition parameters on the accuracy of quantification.     

Qualitative and quantitative bubbles defects analysis in undoped and Ti-doped sapphire crystals grown by Czochralski technique

Small bubbles can be observed in both sapphire and Ti-sapphire bulk crystals grown by Czochralski technique (Cz). Various thickness of wafers cut from the grown ingots were studied by optical microscopy. Bubbles distribution has different regulation in sapphire and Ti-sapphire crystals. All bubbles are spherical and have a diameter range from 2 μm to 5 μm in sapphire crystals while 10–45 μm in Ti-doped sapphire crystals. Some adjacent bubbles congregated into defects present various sizes and shape. The bubbles density increases as a function of pulling rate and rotation rate. The effect of bubbles on optical characterizations of Ti-sapphire crystals has been studied.     

In situ MALDI-TOF MS regional analysis of neutral phospholipids in lens tissue

Phospholipids (PLs) are important sources of lipid second messengers that participate in cell signaling pathways. Consequently, their analysis in biological tissues has received increased attention. Current approaches for PL analysis include an extraction step and subsequent identification of the main PL classes by either 31P NMR spectroscopy or chromatographic separation followed by mass spectrometric detection. Previous in vitro studies revealed regional changes in the PL composition of mammalian lenses at different growth stages. In this report, we demonstrate the feasibility of direct in situ analysis of two relevant PL classes, phosphatidylcholines (PCs) and sphingomyelins (SMs), in slices of fresh or fixed (2.5% formaldehyde) mammalian lenses. The chosen matrix was p-nitroaniline, as it generated superior sensitivity when compared to 2,5-dihydroxybenzoic acid, the compound most commonly used for in vitro analysis of PLs by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Regional differences in the relative amounts of PCs and SMs were in agreement with trends demonstrated by previous in vitro studies. Fresh and fixed tissue of the same lens gave comparable relative levels of PCs and SMs. In situ analysis of PLs by MALDI-TOF MS offers a rapid and sensitive tool for the mapping of PLs in biological tissues.     

HPLC and MALDI-TOF MS analysis of highly branched polystyrene: resolution enhancement by branching

Temperature gradient interaction chromatography (TGIC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) were applied for the characterization of highly branched polystyrenes (PS) prepared by linking living polystyryl anions using 4-chlorodimethylsilylstyrene. Reversed-phase (RP)-TGIC showed an unexpectedly high resolution according to the number of branches despite significant overlap of the molecular weight as confirmed by MALDI-TOF MS. The enhancement of the resolution is ascribed to the contribution of the nonpolar groups in the branched PS: the dimethylsilyl groups in the branching unit as well as the sec-butyl initiator groups. As the number of branches increases, the number of nonpolar groups increases, which in turn increases the RP-TGIC retention synergistically with increasing molecular weight. In contrast, a poorer resolution was found in normal-phase-TGIC, in which the nonpolar groups reduce the retention. The resolution in RP-TGIC appears superior to that of liquid chromatography at the chromatographic critical condition (LCCC) of PS. It is seemingly due to the synergistic contribution of the incremental PS molecular weight to the functionality in the branched PS in RP-TGIC while only the functionality contributes to the separation in LCCC. This type of resolution enhancement could be utilized efficiently for the analysis of highly branched polymers such as dendrimers or hyperbranched polymers.     

Qualitative and quantitative analysis of small amine molecules by MALDI-TOF mass spectrometry through charge derivatization

A pair of isotopically coded light and heavy reagents, tris(2,4,6-trimethoxyphenyl)phosphonium acetic acid N-hydroxysuccinimide esters (1 and 2) were synthesized and used to derivatize low molecular weight (<500 Da) molecules containing primary or secondary amine functional groups for MALDI-TOF MS analysis. The light and heavy reagents added a 573 and 600 Da positively charged tag to each analyte, respectively. In the presence of 10 times excess of tag reagents, the coupling reactions reached near completion within 10 min. The derivatization greatly facilitated MALDI analysis of small molecules and significantly improved the sensitivity of analysis, allowing a limit of detection in the low femtomole range. Additionally, the reaction mixtures were directly analyzed by MALDI without sample cleanup. The quantification of small molecules by MALDI-TOF MS was successfully achieved by analysis of isotopically coded light and heavy derivatives. MALDI-TOF quantitative analysis of a mixture of antibiotics yielded calibration curves in the concentration range from 0.3 to 30 pmol/microL with r2 values greater than 0.9995.     

Chiral analysis of chloro intermediates of methylamphetamine by one-dimensional and multidimentional NMR and GC/MS

Impurity profiling and classification of abused drugs using chiral analytical techniques is of particular interest and importance because of the additional information obtained from this approach. When these methods are applied to the synthesis of illicitly used substances, they can supply valuable information about the conditions/chemicals used in the synthesis. We have applied GC and NMR methods to the study of intermediates found in methylamphetamine manufacture with the aim of linking the intermediates to the ephedrine/pseudoephedrine starting materials. Therefore, determination of the stereochemical makeup within samples of forensic interest is important giving further specific information to the analyst. This study investigates the stereochemical course of the Emde synthesis of methylamphetamine with particular focus on intermediate formation via the chlorination of ephedrine and pseudoephedrine enantiomers. The configurations of these chloro-phenethylamines were determined by 1D and 2D NMR analysis, and thereafter, the GC/MS analysis was carried out. We have shown here that chlorination of the ephedrine/pseudoephedrine compounds occurs via inversion (S(N)2) and retention (S(N)i) of configuration around the α carbon and mixture of diastereoisomers (chloroephedrine and chloropseudoephedrine) were formed, with the ratio of the resulting compounds dependent on the precursors used. The preparation and analytical properties of these intermediate standards provide data for laboratories interested in the stereochemical analysis of methylamphetamine intermediates such as forensic/law enforcement, and illustrate the value of using a combination of analytical methodology.     

Role of accurate mass measurement (+/- 10 ppm) in protein identification strategies employing MS or MS/MS and database searching.

We describe the impact of advances in mass measurement accuracy, +/- 10 ppm (internally calibrated), on protein identification experiments. This capability was brought about by delayed extraction techniques used in conjunction with matrix-assisted laser desorption ionization (MALDI) on a reflectron time-of-flight (TOF) mass spectrometer. This work explores the advantage of using accurate mass measurement (and thus constraint on the possible elemental composition of components in a protein digest) in strategies for searching protein, gene, and EST databases that employ (a) mass values alone, (b) fragment-ion tagging derived from MS/MS spectra, and (c) de novo interpretation of MS/MS spectra. Significant improvement in the discriminating power of database searches has been found using only molecular weight values (i.e., measured mass) of > 10 peptide masses. When MALDI-TOF instruments are able to achieve the +/- 0.5-5 ppm mass accuracy necessary to distinguish peptide elemental compositions, it is possible to match homologous proteins having > 70% sequence identity to the protein being analyzed. The combination of a +/- 10 ppm measured parent mass of a single tryptic peptide and the near-complete amino acid (AA) composition information from immonium ions generated by MS/MS is capable of tagging a peptide in a database because only a few sequence permutations > 11 AA's in length for an AA composition can ever be found in a proteome. De novo interpretation of peptide MS/MS spectra may be accomplished by altering our MS-Tag program to replace an entire database with calculation of only the sequence permutations possible from the accurate parent mass and immonium ion limited AA compositions. A hybrid strategy is employed using de novo MS/MS interpretation followed by text-based sequence similarity searching of a database.     

Capillary isotachophoresis/NMR: extension to trace impurity analysis and improved instrumental coupling

Building upon its promising initial performance, the online coupling of capillary isotachophoresis (cITP) to nuclear magnetic resonance (NMR) is extended to trace impurity analysis. By simultaneously concentrating and separating dilute charged species on the basis of their electrophoretic mobility, cITP greatly facilitates NMR structural elucidation. cITP/NMR appears particularly attractive for identifying trace charged synthetic and natural organic compounds obscured by large excesses of other components. A 9.4 microL injection of 200 microM (1.9 nmol) atenolol in a 1000-fold excess of sucrose (200 mM) is analyzed by cITP/NMR. A microcoil, the most mass sensitive NMR probe, serves as the detector as it provides optimal NMR observation of the capillary-scale separation. cITP successfully isolates the atenolol from the sucrose while concentrating it 200-fold to 40 mM before presentation to the 30 nL observe volume microcoil, thereby enabling rapid 1H NMR spectral acquisition of atenolol (experimental time of 10 s) without obstruction from sucrose. For this particular probe and sample, the stacking efficiency is near the theoretical limit as 67% of the sample occupies the 1 mm long microcoil during peak maximum. A multiple-coil probe with two serial 1 mm long microcoils arranged 1 cm apart has been developed to facilitate peak trapping and sample band positioning during cITP/NMR.     

Identification and quantification of cardiac glycosides in blood and urine samples by HPLC/MS/MS.

Cardiac glycosides (CG) are of forensic importance because of their toxicity and the fact that very limited methods are available for identification of CG in biological samples. In this study, we have developed an identification and quantification method for digoxin, digitoxin, deslanoside, digoxigenin, and digitoxigenin by high-performance liquid chromatography tandem mass spectrometry (HPLC/MS/MS). CG formed abundant [M + NH4]+ ions and much less abundant [M + H]+ ions as observed with electrospray ionization (ESI) source and ammonium formate buffer. Under mild conditions for collision-induced dissociation (CID), each [M + NH4]+ ion fragmented to produce a dominant daughter ion, which was essential to the sensitive method of selected reaction monitoring (SRM) quantification of CG achieved in this study. SRM was compared with selected ion monitoring (SIM) regarding the effects of sample matrixes on the methodology. SRM produced lower detection limits with biological samples than SIM, while both methods produced equal detection limits with CG standards. On the basis of the HPLC/MS/MS results for CG, we have proposed some generalized points for conducting sensitive SRM measurements, in view of the property of analytes as well as instrumental conditions such as the type of HPLC/MS interface and CID parameters. Analytes of which the molecular ion can produce one abundant daughter ion with high yield under CID conditions may be sensitively measured by SRM. ESI is the most soft ionization source developed so far and can afford formation of the fragile molecular ions that are necessary for sensitive SRM detection. Mild CID conditions such as low collision energy and low pressure of collision gas favor production of an abundant daughter ion that is essential to sensitive SRM detection. This knowledge may provide some guidelines for conducting sensitive SRM measurements of very low concentrations of drugs or toxicants in biological samples.     

大杯伞子实体常见微量元素的定性定量测定

利用扫描电镜和X射线能谱仪对大杯伞子实体的菌盖、菌褶和菌柄中的常见微量元素Na、Mg、Ca、K、Fe、Cu、Mn和zn进行测定,并计算出它们的相对重量百分比。结果表明菌盖、菌褶和菌柄均含上述各种微量元素,但含量并不相同。     

大杯伞子实体常见微量元素的定性定量测定

利用扫描电镜和X射线能谱仪对大杯伞子实体的菌盖、菌褶和菌柄中的常见微量元素Na、Mg、Ca、K、Fe、Cu、Mn和Zn进行测定,并计算出它们的相对重量百分比。结果表明菌盖、菌褶和菌柄均含上述各种微量元素,但含量并不相同。     

Choosing between atmospheric pressure chemical ionization and electrospray ionization interfaces for the HPLC/MS analysis of pesticides

An evaluation of over 75 pesticides by high-performance liquid chromatography/mass spectrometry (HPLC/MS) clearly shows that different classes of pesticides are more sensitive using either atmospheric pressure chemical ionization (APCI) or electrospray ionization (ESI). For example, neutral and basic pesticides (phenylureas, triazines) are more sensitive using APCI (especially positive ion). While cationic and anionic herbicides (bipyridylium ions, sulfonic acids) are more sensitive using ESI (especially negative ion). These data are expressed graphically in a figure called an ionization-continuum diagram, which shows that protonation in the gas phase (proton affinity) and polarity in solution, expressed as proton addition or subtraction (pKa), is useful in selecting APCI or ESI. Furthermore, sodium adduct formation commonly occurs using positive ion ESI but not using positive ion APCI, which reflects the different mechanisms of ionization and strengthens the usefulness of the ionization-continuum diagram. The data also show that the concept of "wrong-way around" ESI (the sensitivity of acidic pesticides in an acidic mobile phase) is a useful modification of simple PKa theory for mobile-phase selection. Finally, this finding is used to enhance the chromatographic separation of oxanilic and sulfonic acid herbicides while maintaining good sensitivity in LC/MS using ESI negative.