An integrated approach for detection and characterization of the trace impurities in levofloxacin using liquid chromatography-tandem mass spectrometry.

RATIONALE: Impurity analysis plays an important role to guarantee the quality and safety of pharmaceuticals. However, identification of impurities remains challenging, especially for those unknown or at trace levels. We present an integrated approach to detect and characterize the trace impurities in drugs. METHODS: Based on liquid chromatography-tandem mass spectrometry (LC-MS/MS), an approach integrating automatic impurity screening method using multiple mass defect filters (MMDFs) and background subtraction (BS) was developed. This approach was used to acquire the structural and semi-quantitative information in a single sample run, and even to discover the impurity signals submerged by background and drug ions. This approach was illustrated by the comprehensive impurity analysis of levofloxacin. RESULTS: This approach was sensitive to detect impurities at the level of 0.02% with respect to levofloxacin concentration. Nineteen impurities were detected, fourteen of which were structurally characterized and eight impurities were reported for the first time. Impurity profiles of levofloxacin drug substances and degradation samples were obtained reliably. A plausible degradation pathway of levofloxacin was proposed including descarboxyl reaction under acid, piperazinyl ring cleavage degradation under light, and N-oxidation under oxidative condition. CONCLUSIONS: The generic approach integrating LC-MS/MS and an automatic impurity screening method was developed for the detection, characterization and monitoring of impurities, especially those unknown or at trace levels. This approach was demonstrated to be rapid, sensitive and automatic for impurity profiling of drugs.

Simultaneous structural identification of natural products in fractions of crude extract of the rare endangered plant Anoectochilus roxburghii using H NMR/RRLC-MS parallel dynamic spectroscopy.

Nuclear magnetic resonance/liquid chromatography-mass spectroscopy parallel dynamic spectroscopy (NMR/LC-MS PDS) is a method aimed at the simultaneous structural identification of natural products in complex mixtures. In this study, the method is illustrated with respect to (1)H NMR and rapid resolution liquid chromatography-mass spectroscopy (RRLC-MS) data, acquired from the crude extract of Anoectochilus roxburghii, which was separated into a series of fractions with the concentration of constituent dynamic variation using reversed-phase preparative chromatography. Through fraction ranges and intensity changing profiles in (1)H NMR/RRLC-MS PDS spectrum, (1)H NMR and the extracted ion chromatogram (XIC) signals deriving from the same individual constituent, were correlated due to the signal amplitude co-variation resulting from the concentration variation of constituents in a series of incompletely separated fractions. 1H NMR/RRLC-MS PDS was then successfully used to identify three types of natural products, including eight flavonoids, four organic acids and p-hydroxybenzaldehyde, five of which have not previously been reported in Anoectochilus roxburghii. In addition, two groups of co-eluted compounds were successfully identified. The results prove that this approach should be of benefit in the unequivocal structural determination of a variety of classes of compounds from extremely complex mixtures, such as herbs and biological samples, which will lead to improved efficiency in the identification of new potential lead compounds.

[Structural identification and quality study on isomers of a novel anticancer photosensitiser photocyanine].

Our work focuses on the quality control and structural identification of Photocyanine as a cancer therapeutic photosensitizer. Photocyanine is a mixture which contains four ZnPcS2P2 type substituted Phthalocyanine isomers. In order to obtain the single component from Photocyanine, the mixture of four isomers possessing the similar structures and chemical property had been isolated and purified. An HPLC method with a mixture of methanol-acetonitrile-ion-pair buffer as the mobile phase was applied to isolate the four isomers by means of a semi-preparative C18 column. To remove the salts which were mixed in the preparative product, a SPE C18 column was used to separate the salts by elution with water and then the marker component was eluted by methanol. Subsequently, a column of Sephadex LH-20 gel was applied to elute the crudes with methanol to desalination. The purity of the isolated compound was measured by TLC and four different isomers of phthalocyanine were obtained. The chemical structures of them were elucidated by 1H NMR spectra, gCOSY and NOE1D. An HPLC-DAD method was developed for simultaneously determination of four major isomers in Photocyanine with a C18 column (Grace Smart, 150 mm x 4.6 mm ID, 5 microm). The separation was carried out with a gradient program at a flow rate of 1.0 mL x min(-1). The mobile phase was a mixture of acetonitrile and ion-pair buffer (0.01 mol x L(-1) hexadecyl trimethyl ammonium bromide and 0.01 mol x L(-1) potassium dihydrogen phosphate, adjusted the pH value to 6.8 with potassium hydroxide solution). The resolution values of four isomers were 2.5, 1.20, 1.33, and 1.8. Linear regression analysis for four compounds was performed by the external standard method. Four constituents were linear in the concentration range of 0.005 to 10 microg. The values of relative standard deviation (RSD) of intra-day were 0.12%, 0.66%, 0.99%, and 1.21%, respectively. The limits of detection for four compounds were 15 ng, 20 ng, 12 ng, and 25 ng, respectively. This method was simple, accurate and reproducible. The developed method can be successfully applied to analyze isomers in Photocyanine.

Dynamic selection of novel vancomycin N-terminal derivatives by resin-bound reversed D-Ala-D-Ala.

The most attractive advantage of dynamic combinatorial chemistry (DCC) is that it can screen the compound library as soon as compounds are synthesized. However, it is very difficult to analyze a dynamic combinatorial library with free probes using the state-of-the art analysis technologies. We report herein a method that uses a resin-immobilizing reversed peptide probe to screen vancomycin derivatives and provides a solution to this problem.

[Study of malignant and normal tissues of the rectum using NMR spectroscopy].

In the present paper, NMR spectroscopy, an effective tool to detect the variation in molecular structure and changes in chemical composition of metabolites in tissues, was used to study the differences between malignant and normal tissues from rectum. 1H spectra of four malignant rectum tissue samples and two normal control tissues were investigated by using a 500M NMR high-resolution magic angle spinning magnetic resonance spectrometers (HR-MAS NMR). The results indicate that the 1H HR-MAS spectra of rectum cancer tissues are significantly different from those of the normal controls and most differences are presents in the form of variation in the relative intensities of the characteristic peak of various metabolites. In order to characterize the variation in the relative intensities in a quantitative manner, the intensity of the methyl peak of fatty acid at 0.88 was utilized as inner standard. Systematic differences between NMR spectra of malignant tissue and normal controls are as follows: (1) The concentration of amino acid increases significantly in malignant tissues, since the relative intensities of characteristic peaks of amino acid including valine, isoleucine, leucine, lysine, glutamate, glutamine, and aspartate are stronger in the NMR spectra of the malignant tissues. This phenomenon may reflect the fact that the activity of protein synthesis is enhanced in cancerous tissues. (2) The intensities of the characteristic peaks of lactic acid in malignant tissues are higher than those from normal controls. This may be related to the nature of anaerobic metabolism activity in malignant tissues. (3) The level of choline and its derivatives, taurine and creatine, increases significantly in malignant tissues, suggesting that the metabolic activity of malignant tissues changes. (4) In the spectral region between 4.5 and 10, observable changes occur on the peaks for unsaturated fatty acid and nuclear acids. Therefore, the above spectral variations in high resolution magic angle spinning NMR spectroscopy may be utilized as a potential tool to diagnose rectum cancer.

Two novel glycosidic triterpene alkaloids from the stem barks of Machilus yaoshansis.

[structure: see text] Two unusual glycosidic triterpene alkaloids, machilaminosides A (1) and B (2), have been isolated from the stem barks of Machilus yaoshansis. Their structures were elucidated by detailed spectroscopic analysis. A possible biogenetic origin of 1 and 2 mediated by the coupling of 2-O-beta-D-glucopyranosyl-cucurbitacin I, respectively, with urea and adenosine was postulated. 1 and 2 showed nonselective cytotoxic activities against several human cancer cell lines as well as TNF-alpha secretion inhibitory activities.

[Study on chemical constituents of Orobanche coerulescens].

Six compounds were isolated and purified from Orobanche coerulescens by extraction and different kinds of column chromatography. The structures were determined on the basis of spectral analysis. The structures were elucidated as D-mannitol(I), beta-sitosterol(II), succinic acid(III), caffeic acid(IV), protocatechuic aldehyde(V) and daucosterol(VI). All compounds are obtained from this plant for the first time.

Chemical constituents from the linseed meal.

One megastigmane derivative 1, one methyl jasmonate glycoside derivative 2, and two C-28 steroids with 3ß,5ß-cis-dihydroxyl conformation 3 and 4, together with eight known compounds 5-12 were isolated from the 70% ethanol extract of linseed meal (Linum usitatissimum L). Structures of 1-4 were elucidated by spectroscopic methods including NMR, HRESIMS, and Mo2(OAc)4-induced CD. The absolute configuration of 1 and 3 was determined by observing their induced circular dichroism after addition of Mo2(OAc)4 in DMSO. The absolute configuration of 2 was determined by NOESY experiment together with conformational analysis. The structure of 4a was corrected as 4 by an extensive analysis of its 1D and 2D NMR, in combination with the Mo2(OAc)4-induced CD in DMSO. The effect of all the isolates on nitric oxide (NO) generation by stimulated macrophages was evaluated, and none of them showed active.

Excretion of berberine and its metabolites in oral administration in rats.

Berberine (BBR) has been confirmed to show extensive bioactivities for the treatments of diabetes and hypercholesterolemia in clinic. However, there are few pharmacokinetic studies to elucidate the excretions of BBR and its metabolites. Our research studied the excretions of BBR and its metabolites in rats after oral administration (200 mg/kg). Metabolites in bile, urine, and feces were detected by liquid chromatography coupled to ion trap time-of-flight mass spectrometry; meanwhile, a validated liquid chromatography coupled with tandem mass spectrometry method was developed for their quantifications. Sixteen metabolites, including 10 Phase I and six Phase II metabolites were identified and clarified after dosing in vivo. Total recovered rate of BBR was 22.83% (19.07% of prototype and 3.76% of its metabolites) with 9.2 × 10(-6) % in bile (24 h), 0.0939% in urine (48 h), and 22.74% in feces (48 h), respectively. 83% of BBR was excreted as thalifendine (M1) from bile, whereas thalifendine (M1) and berberrubine (M2) were the major metabolites occupying 78% of urine excretion. Most of BBR and its metabolites were found in feces containing 84% of prototype. In summary, we provided excretion profiles of BBR and its metabolites after oral administration in rats in vivo.

Tissue distribution of berberine and its metabolites after oral administration in rats.

Berberine (BBR) has been confirmed to have multiple bioactivities in clinic, such as cholesterol-lowering, anti-diabetes, cardiovascular protection and anti- inflammation. However, BBR's plasma level is very low; it cannot explain its pharmacological effects in patients. We consider that the in vivo distribution of BBR as well as of its bioactive metabolites might provide part of the explanation for this question. In this study, liquid chromatography coupled to ion trap time-of-flight mass spectrometry (LC/MS(n)-IT-TOF) as well as liquid chromatography that coupled with tandem mass spectrometry (LC-MS/MS) was used for the study of tissue distribution and pharmacokinetics of BBR in rats after oral administration (200 mg/kg). The results indicated that BBR was quickly distributed in the liver, kidneys, muscle, lungs, brain, heart, pancreas and fat in a descending order of its amount. The pharmacokinetic profile indicated that BBR's level in most of studied tissues was higher (or much higher) than that in plasma 4 h after administration. BBR remained relatively stable in the tissues like liver, heart, brain, muscle, pancreas etc. Organ distribution of BBR's metabolites was also investigated paralleled with that of BBR. Thalifendine (M1), berberrubine (M2) and jatrorrhizine (M4), which the metabolites with moderate bioactivity, were easily detected in organs like the liver and kidney. For instance, M1, M2 and M4 were the major metabolites in the liver, among which the percentage of M2 was up to 65.1%; the level of AUC (0-t) (area under the concentration-time curve) for BBR or the metabolites in the liver was 10-fold or 30-fold higher than that in plasma, respectively. In summary, the organ concentration of BBR (as well as its bioactive metabolites) was higher than its concentration in the blood after oral administration. It might explain BBR's pharmacological effects on human diseases in clinic.

[Changes of the immune cells, cytokines and growth hormone in teenager drug addicts].

AIM: To investigate the effect of heroin on the immune function, growth and development in the teenager heroin addicts by measuring their T-lymphocyte subsets, Th1/Th2 cytokines and serum growth hormone. METHODS: Tlymphocyte subsets of peripheral blood from the teenager heroin addicts were measured by direct microvolume whole blood immunofluorescent staining technique by flow cytometer (FCM). Thl / Th2 cytokines were measured by BD cytometric bead array and serum growth hormone was assayed using the chemiluminescence method in the 20 teenager heroin addicts and 23 healthy teenagers. RESULTS: The levels of CD3(+), CD3(+) + CD4(+), CD3(+) + CD4(+)/CD3(+)+ CD8(+), Th1 cytokines(IL-2, TNF-alpha and IFN-gamma) and Th2 cytokines(IL-4 and IL-10) reduced significantly in the teenager heroin addicts compared with the healthy control group (P < 0.01 or P < 0.05). The level of Th1 cytokines(IL-2 + TNF-alpha+IFN-gamma) decreased more than that of Th2 cytokines(IL-4 + IL-5 + IL-10)(P < 0.05). The level of serum growth hormone from the teenager heroin addicts was remarkably higher than that in control group (P<0.01). CONCLUSION: Heroin can inhibit the immunofunction especially the celluar immunity of the teenager heroin addicts. Besides, it can increase the level of serum growth hormone of the teenager heroin addicts.

Conformational studies of resin-bound vancomycin and the complex of vancomycin and Ac2-L-Lys-D-Ala-D-Ala.

The molecular target of vancomycin, a commonly used glycopeptide antibiotic, is the D-Ala-D-Ala dipeptide subunit on the bacterial cell wall. The molecular basis of interaction between vancomycin and D-Ala-D-Ala in solution is well-known. However, there is no structural data on vancomycin, and its interaction with D-Ala-D-Ala when the drug is tethered to a solid support. In this Article, vancomycin was directly coupled onto TentaGel or PEGA resin through its C terminus. High-resolution magic angle spinning NMR studies indicated that conformation of PEGA bead-bound vancomycin is identical to that of the free drug. Broadening and shifts of the same proton resonances were observed in solution-phase vancomycin or PEGA-bound vancomycin when complexed with Ac(2)-L-Lys-D-Ala-D-Ala. This study demonstrates that bead-bound molecules can behave the same as solution-phase molecules in terms of molecular interaction with its target molecule, thus validating the on-bead screening approach of the "one-bead-one-compound" combinatorial library method.

Solid-phase synthesis and antibacterial evaluations of N-demethylvancomycin derivatives.

Twenty-five N-demethylvancomycin derivatives were synthesized on solid-support and their structures were determined by LC-MS/MS. Biological evaluation of these compounds indicated that bulky hydrophobic substituent on vancosamine of N-demethylvancomycin can increase antibacterial activity against vancomycin-resistant Enterococcus faecalis.

Solid-phase synthesis of O-glycosylated Nalpha-Fmoc amino acids and analysis by high-resolution magic angle spinning NMR.

Direct O-glycosylation of amino acids bound to TentaGel resin with a number of glycosyl trichloroacetimidate donors results in high yields. The glycosylation reaction can be easily monitored by analyzing the bead-bound amino acids with high-resolution magic angle spinning (HR-MAS) NMR. These studies pave a new way for the construction of "one-bead one-compound" O-glycopeptide libraries with standard amino acid building blocks and appropriate glycosyl trichloroacetimidate donors.

New Diels-Alder type adducts from Morus macroura and their anti-oxidant activities.

Fractionation of the ethanolic extract of the stem bark of Morus macroura resulted in the isolation of four new Diels-Alder type adducts, named guangsangons K--N (1, 2, 5, 6), together with two known compounds, mulberrofuran G (3) and K (4). Their structures were determined on the basis of spectroscopic analyses and chemical methods. Furthermore, by means of (1)H-NMR variable temperature experiments and the Cotton curves in the circular dichroism (CD) spectra, the stereochemistry of four new compounds was elucidated. The isolated new compounds showed good activity on anti-oxidant in vitro, with the inhibitory rates of MDA being from 91.8 to 100.0% at concentrations of 10(-5) mol/l.