Species-dependent hepatic metabolism of immunosuppressive agent tacrolimus (FK-506).

The current study aims to investigate species-related differences in the in-vitro hepatic metabolism of tacroliums using liver microsomes obtained from rat, hamster, guinea pig, rabbit, pig, dog, baboon and humans. Tacrolimus metabolism was characterized using high-performance liquid chromatography- ultraviolet light (HPLC-UV) and two soft ionization mass spectrometric techniques; matrix-assisted lasers desorption/ionization (MALDI) and time-of-flight-secondary ion mass spectrometry (TOF-SIMS). The extent of tacrolimus metabolism, when normalized to the cytochrome P-450 content, was in the order: rat < hamster < rabbit < pig < guinea pig < dog < human < baboon. Tacrolimus metabolism exhibited significant qualitative and quantitative differences between the animal species tested. Desmethyl- (MI-MIII), didesmethyl- (MIV-MVI), monohydroxy- (MVII), dihydroxy- (MVIII), epoxide- (MIX), dihydrodiol- (MX), monodesmethyl and monohydroxy- (MXI-MXIII), and didesmethyl and monohydroxy- (MXIV-MXVI) tacroliums metabolites were identified in the species tested. MI-MX were identified in all the species tested; MXI-MXVI were identified in all species except rat, rabbit and guinea pig; and MXIV-MXVI were identified only in baboon. The current investigation was unable to detect any phase II metabolites due to the limitations of the test system used. The analytical methods were not able to differentiate optical and positional isomers of metabolites due to the nature of the analytical tools used, therefore groups of metabolites were identified based on their molecular weights and available information. From the current in-vitro metabolism studies, the pattern of tacroliums metabolism in baboons closely resembled that in humans and thus it is ideal for studying tacroliums metabolism-related work of clinical relevance.

Matrix-assisted laser desorption/ionization collision-induced dissociation of linear single oligomers of nylon-6.

Matrix-assisted laser desorption/ionization, collision induced-dissociation (MALDI-CID) has been used to obtain structural information for linear single oligomers of nylon-6. The effects of matrix and cationization agent in MALDI-CID analysis have been investigated. Fragmentation mechanisms are proposed for the series of ions that are observed in the MALDI-CID spectra of the hexamer, octamer and dodecamer. Fragmentation processes observed in the MALDI-CID spectra include cleavage of the end groups followed by dissociation of the m/z 113 unit. Cleavage of the oligamide chain occurs at the amide linkage, as well as at adjacent bonds. For the four matrices and three cationization agents investigated, 2,5-dihydroxybenzoic acid and sodium chloride showed the best performance for MALDI-CID analysis of the dodecamer. In addition, yields of the fragment ions in MALDI-CID spectra were found to be dependent on the chain length distribution.

Studies of pesticides by collision-induced dissociation, postsource-decay, matrix-assisted laser desorption/ionization time of flight mass spectrometry.

The use of collision-induced dissociation, postsource decay (CID-PSD) matrix-assisted laser desorption/ionization (MALDI) mass spectrometry for the analysis of small organic molecules is demonstrated. Three pesticides: paraquat, diquat, and difenzoquat were chosen for this study. The matrices 2,5-dihydroxybenzoic acid (DHB), alpha-cyano-4-hydroxycinnamic acid (alpha-CHCA), and sinapinic acid (SA) were selected to investigate the effect of the matrix on the CID-PSD MALDI spectra of these molecules. Alpha-CHCA and DHB were found to be appropriate matrices for the pesticides studied. Spectra for a given pesticide obtained from different matrices were compared with each other, and the differences between them are discussed. A comparison of CID-PSD MALDI with fast-atom bombardment MS/MS spectra is presented; the agreement of pesticide fragmentation patterns between the two methods indicates that CID-PSD MALDI MS is a reliable and efficient technique for structural elucidation of small molecules.

Characterization of isopoly tungstate using time-of-flight electrospray mass spectrometry.

Electrospray time-of-flight (TOF) mass spectrometry has been carried out on aqueous solutions of isopoly tungstate. A number of factors affecting the speciation of tungstate were studied, including concentration, and pH. The concentrations of the solutions ranged from 10(-2) to 10(-5) M. At all concentrations studied, the pH range was sufficient to investigate all major species expected to be in aqueous solution. In all cases, the results obtained by electrospray showed changes in speciation which were limited to protonation and solvation effects; the tungstate core remained intact.

LC-MS/MS signal suppression effects in the analysis of pesticides in complex environmental matrices.

The application of LC separation and mobile phase additives in addressing LC-MS/MS matrix signal suppression effects for the analysis of pesticides in a complex environmental matrix was investigated. It was shown that signal suppression is most significant for analytes eluting early in the LC-MS analysis. Introduction of different buffers (e.g. ammonium formate, ammonium hydroxide, formic acid) into the LC mobile phase was effective in improving signal correlation between the matrix and standard samples. The signal improvement is dependent on buffer concentration as well as LC separation of the matrix components. The application of LC separation alone was not effective in addressing suppression effects when characterizing complex matrix samples. Overloading of the LC column by matrix components was found to significantly contribute to analyte-matrix co-elution and suppression of signal. This signal suppression effect can be efficiently compensated by 2D LC (LC-LC) separation techniques. The effectiveness of buffers and LC separation in improving signal correlation between standard and matrix samples is discussed.

Electrospray and matrix-assisted laser desorption/ionization mass spectral characterization of linear single nylon-6 oligomers.

Synthetic nylon-6 single molecular mass oligomers were studied by matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrometry. These oligomers, considered as model compounds for the study of nylon-6 polymers, gave good mass spectrometric results using both MALDI and ESI. In spite of the gentle nature of both techniques, the MALDI and ESI spectra showed evidence of end-group cleavage from the oligomer chains. MALDI-MS was found to give similar fragmentation patterns for all of the oligomer samples. An increase in doubly charged ion signals with increasing oligomer mass was observed in the ESI mass spectra, as was end-group fragmentation. Signals from oligomer clusters were observed in ESI-MS for the dimer, tetramer and hexamer, most likely due to non-covalent bonding among the low-mass oligomer molecules.

Effect of liquid chromatography separation of complex matrices on liquid chromatography-tandem mass spectrometry signal suppression.

The effect of liquid chromatography separation on liquid chromatography-tandem mass spectrometry (LC-MS-MS) signal response for the characterization of low-molecular-mass compounds in a complex matrix was investigated. Matrix induced signal suppression appears throughout the entire LC-MS-MS analysis of wheat forage extract, with greatest suppression occurring at early retention times. Experimental results show that co-elution of matrix components and analytes from the LC column may be most strongly attributed to column overloading rather than similar analyte and matrix retention behavior. As a result, two-dimensional (LC-LC) separation can be a highly effective approach to address signal suppression effects for the quantitative LC-MS-MS analysis of complex matrix samples.

Direct TLC-MALDI coupling using a hybrid plate.

A new TLC-MALDI direct coupling method which recovers approximately 100% of the analyte is presented. The method makes use of a hybrid TLC-MALDI plate in which a silica layer and a MALDI layer are configured adjacently on a common backing. After TLC separation, the plate is rotated 90 degrees and the separated analyte spots are eluted from the silica layer to the MALDI layer via capillary action of the MALDI layer. Signal-to-noise ratios are significantly improved over previously reported coupling methods. Low-femtomole detection limits have been demonstrated for small cyclic peptides, which are comparable to detection limits for standard MALDI measurements.

Atomic force microscopy used for the surface characterization of microcapsule immunoisolation devices.

The surface morphology of the microcapsule used as a bioartificial pancreas was examined by atomic force microscopy (AFM) under ambient conditions in a liquid environment. The standard contact mode was used for imaging. The capsules exhibited different morphologies and surface roughness depending on the composition of the cation solution: namely, the mole ratio of antigelling and gelling cations [Na+]/[Ca2+]. Surface roughness parameters obtained by AFM measurements provide quantitative information on the surface properties of the capsular membrane. In this respect, AFM can be considered a valuable technique complementary to optical microscopy in providing feedback for capsule optimization.

Automation of data collection for matrix-assisted laser desorption/ionization mass spectrometry using a correlative analysis algorithm.

Automation of data collection in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry using a correlative analysis algorithm is demonstrated. This algorithm was employed to compensate for mass spectral jittering in MALDI data collection (e.g., peak shifts along the m/z axis, signal intensity deviations, etc.). Several important parameters for performing correlative analysis, such as the minimum correlation coefficient to be used and number of mass spectra to acquire prior to correlation, have been investigated and optimized. In addition, the correlation algorithm improved mass resolution of low- and high-molecular-weight compounds by as much as a factor of 4. Signal reproducibility in MALDI quantitative analysis also is improved when correlation is employed for data collection. This data collection algorithm can be used in conjunction with other instrumental optimization programs to allow for fully automated MALDI analysis, which is required for the routine applications carried out in many analytical laboratories.

Characterization of polysiloxanes with different functional groups by time-of-flight secondary ion mass spectrometry.

Polydimethylsiloxane (PDMS), polyhydromethylsiloxane (PHMS), and polymethylphenylsiloxane (PMPhS) have been studied by TOF-SIMS to investigate effects of functional group changes on polymer fragmentation mechanisms. Cyclic fragments are observed in the low mass range spectra of PDMS and PHMS, but not in the spectrum of PMPhS. Effects of functional group substitution on the fragmentation mechanisms of polysiloxanes are evident in the high mass range spectra (>1000 Da). Peaks of oligomers cationized by silver dominate the high mass range of the spectra of all low molecular weight polysiloxanes. However, fragmentation patterns of these samples are different. Neutral cyclic fragments cationized by silver are identified in the high mass range of the spectra of PDMS and PHMS, but not in the spectrum of PMPhS. The major fragments of PHMS and PMPhS are [oligomer-14+Ag](+). The PHMS spectrum also shows peaks [oligomer-28+Ag](+). These distinctive fragmentation patterns can be used to identify the polysiloxanes.

Metabolism of tacrolimus (FK 506) in rat liver microsomes. Effect of rifampin and dexamethasone.

The in vitro metabolism of tacrolimus (TAC, FK 506) was investigated in the liver microsomes prepared from normal rats as well as rats treated with dexamethasone (DEX) and rifampin (RIF). The rate of tacrolimus metabolism was similar in control and RIF treated rat liver microsomes, whereas it significantly increased in microsomes obtained from dexamethasone treated rats. Seven different possible metabolites were identified in the microsomal preparations from rats treated with rifampin or dexamethasone whereas the microsomes from the control rats failed to produce the mono-demethylated and monohydroxylated metabolite of TAC (TAC+2, m/z = 805.5). There was an apparent difference in the amount of individual metabolites formed in different groups. This indicates quantitative differences in the induction of cytochrome P450 3A, an enzyme sub family known to be primarily responsible for tacrolimus metabolism. Lack of induction of tacrolimus metabolism by rifampin can be attributed to the lack of effect of rifampin in inducing cytochrome P450 3A in rats.

Direct analysis of filter paper blood specimens for identification of Smith-Lemli-Opitz syndrome using time-of-flight secondary ion mass spectrometry.

In this study, time-of-flight secondary ion mass spectrometry was used to distinguish between blood of normal infants and that of individuals with Smith-Lemli-Opitz (SLO) syndrome. SLO syndrome results in an abnormally low concentration of blood cholesterol and an elevated concentration of 7-dehydrocholesterol. Blood was spotted on filter paper and analyzed directly with no extractions or separations. Results showed that using ratios of fragment ions for cholesterol/dehydrocholesterol, patients with SLO and normal individuals could be unambiguously distinguished. Unknown samples from 28 individuals were obtained and identified correctly.

Quantitative surface characterization using X-ray photoelectron spectroscopy.

Use of X-ray Photoelectron Spectroscopy (XPS) for quantitative surface analysis is reviewed. Examples chosen are from our research on heterogeneous catalysts. XPS is useful for measuring distributions of oxidation states because of significant chemical shifts for many elements. However, linewidths complicate analysis of mixtures, necessitating the use of data analysis methodology. Use of factor analysis or deconvolution with non-linear least squares curve fitting gives useful protocols. Five important questions are posed, and some examples of solutions are presented: 1) How many components are present? 2) Where are they located? 3) What do they look like? 4) How much of each component is present? 5) How good is the answer?

Direct quantitative analysis of peptides using matrix assisted laser desorption ionization.

The protocol and various matrices were examined for quantification of biomolecules in both the low ca. 1200 amu and mid mass 6000-12000 amu ranges using an internal standard. Comparative studies of different matrices of MALDI quantitative analysis showed that the best accuracy and standard curve linearity were obtained for two matrices: (a) 2,5-dihydroxybenzoic acid (DHB) combined with a comatrix of fucose and 5-methoxysalicylic acid (MSA) and (b) ferulic acid/fucose. In the low mass range, the quantitative limit was in the 30 fmol range and in the mid mass range the quantitative limit was in the 250 fmol range. Linear response was observed over 2-3 decades of analyte concentration. The relative error of the standard curve slope was 1.3-1.8% with correlation coefficients of 0.996-0.998.The main problem for quantitative measurement was suppression of the signal of the less concentrated component (analyte or internal standard) by the more concentrated component. The effect was identified with saturation of the matrix by the analyte. The threshold of matrix saturation was found to be in the range of 1/(3000-5000) analyte/matrix molar ratio. To avoid matrix saturation the (analyte+internal standard) to matrix molar ratio should be below this threshold. Thus the internal standard concentration should be as low as possible.DHB/MSA/fucose and ferulic acid/fucose matrices demonstrated good accuracy and linearity for standard curves even when the internal standard had chemical properties different from the analyte. However, use of an internal standard with different chemical properties requires highly stable instrumental parameters as well as constant (analyte+internal standard)/matrix molar ratio for all samples.

A quantitative study of in vitro hepatic metabolism of tacrolimus (FK506) using secondary ion and matrix-assisted laser desorption/ionization mass spectrometry.

The identification and simultaneous quantification of Tacrolimus and its hepatic metabolites in baboons has been achieved using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry and static secondary-ion mass spectrometry (TOF-SIMS). Little fragmentation, high sensitivity and tolerance to contamination are the major advantages of these methods, allowing facile identification and quantification of metabolites produced in vitro with minor analyte isolation. Based on the MALDI and TOF-SIMS results, seven metabolites have been identified: de-methylated, di de-methylated, hydroxylated, di hydroxylated, de-methylated hydroxylated, dihydrodiol, and di de-methylated hydroxylated. The concentrations of the parent drug and its major metabolites (e.g. de-methylated, di de-methylated) were measured using Rapamycin as an internal standard. The time course of Tacrolimus and its major metabolites as a function of incubation time was calculated. Good correlation between SIMS and MALDI results was obtained.

Enhancement of ion intensity in time-of-flight secondary-ionization mass spectrometry.

Enhancement of ion intensity in static secondary-ionization mass spectrometry (SIMS) has been achieved by using a matrix-assisted sample preparation technique. Previous investigations of polymers and biomolecules by SIMS indicated that secondary-ion (SI) yield is dependent on substrate coverage. Recently we discovered a sample preparation technique that enhanced the SI yield of cyclosporin A (CsA) in an allograft patient sample and neat samples of CsA (1202 u) and polystyrene (M w=2650 u). The preparation technique involves deposition of a submonolayer of cocaine hydrochloride (5 µL of a 20-µg/mL MeOH solution) on an etched silver substrate, solvent evaporation, and subsequent deposition of the analyte. This preparation method resulted in ∼300% increase in the SI yield of CsA and polystyrene when deposited from neat solutions. The original discovery was observed when a blood extract that contained CsA was deposited on an etched Ag substrate that had been soaking in a dilute cocaine solution for ∼2 months. In these initial experiments, the SI yield of CsA was enhanced by over 1 order of magnitude.

Application of the fast-evaporation sample preparation method for improving quantification of angiotensin II by matrix-assisted laser desorption/ionization.

The fast-evaporation method of sample preparation has been applied for quantitative analysis using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. An instrumental protocol focusing on improvement of shot-to-shot repeatability and compensation for signal degradation has been developed for quantification of angiotensin II using the fast-evaporation technique and an internal standard. The fast-evaporation method was compared to the standard method of sample preparation (using a multicomponent matrix) in the quantitative analysis of angiotensin II, and found to be superior in several respects. Improvement in sample homogeneity using the fast-evaporation method enhanced both point-to-point repeatibility and sample-to-sample reproducibility. The relative standard deviations of the analyte/internal standard ratios (point RSD) were decreased by a factor of three compared to those obtained using the multicomponent matrix method. The average point RSD was found to be ca. 5% for the fast-evaporation technique. Two internal standards were evaluated for quantification of angiotensin II. The better one, 1-SAR-8-Ile angiotensin II, yielded a relative standard deviation of the standard curve slope of ca. 2.2% over two orders of magnitude of concentration (45 nM to 3000 nM), an improvement by a factor of two over the standard preparation method. Renal microdialysate samples, spiked with angiotensin II and the internal standard 1-SAR-8-Ile angiotensin II, were also analyzed using the fast-evaporation technique. The detection limit was calculated to be in the high attomole range (675 amol). Furthermore, the accuracy for a single determination of angiotensin II concentration in these samples was found to be 13.9% with a relative error of 8.19%.