Identification of in vivo and in vitro metabolites of triptolide by liquid chromatography-tandem mass spectrometry.

Triptolide, a major active constituent of Tripterygium wilfordii Hook F, has multiple pharmacological activities. In this work, a rapid, sensitive and specific liquid chromatography coupled to an ion trap mass spectrometer (MS) with electrospray ionization (ESI) interface has been developed for identification of triptolide and some of its metabolites in rat urine after oral administration of a single dose (0.6 mg/kg) of triptolide to healthy rats, as well as some metabolites in vitro after incubation with rat liver microsome (RLM) and rat intestinal flora, respectively. All samples were separated on a reversed-phase C18 column using a mobile phase of acetonitrile/water (70:30, v/v) and detected by an on-line MS(n) detector. Identification and structural elucidation of the selected metabolites were performed by comparing their full scan MS(n) spectra with those of the parent drug. In this paper we identified ten metabolites in rat urine, four metabolites in RLM incubation solution and one metabolite in rat intestinal flora incubation solution, after drug administration. The metabolic reactions of triptolide that we observed in vivo were hydrolysis reaction, hydroxylation reaction, and the conjugate reaction with sulfate, glucuronide and GSH, respectively. The in vitro metabolic reactions of triptolide observed were hydrolysis and hydroxylation reactions.

Metabolite identification of triptolide by data-dependent accurate mass spectrometric analysis in combination with online hydrogen/deuterium exchange and multiple data-mining techniques.

Triptolide (TP), the primary active component of the herbal medicine Tripterygium wilfordii Hook F, has shown promising antileukemic and anti-inflammatory activity. The pharmacokinetic profile of TP indicates an extensive metabolic elimination in vivo; however, its metabolic data is rarely available partly because of the difficulty in identifying it due to the absence of appropriate ultraviolet chromophores in the structure and the presence of endogenous interferences in biological samples. In the present study, the biotransformation of TP was investigated by improved data-dependent accurate mass spectrometric analysis, using an LTQ/Orbitrap hybrid mass spectrometer in conjunction with the online hydrogen (H)/deuterium (D) exchange technique for rapid structural characterization. Accurate full-scan MS and MS/MS data were processed with multiple post-acquisition data-mining techniques, which were complementary and effective in detecting both common and uncommon metabolites from biological matrices. As a result, 38 phase I, 9 phase II and 8 N-acetylcysteine (NAC) metabolites of TP were found in rat urine. Accurate MS/MS data were used to support assignments of metabolite structures, and online H/D exchange experiments provided additional evidence for exchangeable hydrogen atoms in the structure. The results showed the main phase I metabolic pathways of TP are hydroxylation, hydrolysis and desaturation, and the resulting metabolites subsequently undergo phase II processes. The presence of NAC conjugates indicated the capability of TP to form reactive intermediate species. This study also demonstrated the effectiveness of LC/HR-MS(n) in combination with multiple post-acquisition data-mining methods and the online H/D exchange technique for the rapid identification of drug metabolites.

Analysis of urinary andrographolides and antioxidant status after oral administration of Andrographis paniculata leaf extract in rats.

A simple high-performance liquid chromatography (HPLC) method was developed to determine the content of andrographolide (AP) and 14-deoxy-11,12-dideoxyandrographolide (DIAP) in a pooled urine of rat obtained within 24h after an oral dose of Andrographis paniculata leaf extract at 1g/kg body weight. Cumulative urinary excretion of AP and DIAP in 24h after oral administration of the extract was 0.88% and 1.61% of oral dose administered, respectively. The extract showed significant reduction (p<0.05) of MDA levels and elevation of total antioxidant status in rat urine samples collected in 24 after oral administration.

Four new andrographolide metabolites in human urine.

Andrographolide is one of principal components of a famous traditional Chinese herbal medicine Andrographis paniculate (BURM) NEES. Four new metabolites of andrographolide were isolated from human urine. All of them were characterized as sulfate and one of them also as a cysteine S-conjugate. The structures were determined to be andrographolide-3-O-sulfate (M-1), isoandrographolide-3-O-sulfate (M-2), 14-deoxyandrographolide-3-O-sulfate (M-3), 14-deoxy-12-(cysteine-S-yl)-andrographolide-3-O-sulfate (M-4), respectively, based on chemical evidence and spectroscopic analyses.

Elevated urinary dolichol excretion in the Hermansky-Pudlak syndrome. Indicator of lysosomal dysfunction.

The Hermansky-Pudlak syndrome, a triad of albinism, platelets lacking dense bodies, and storage of ceroid-like material in tissues, occurs approximately once in 2,000 northwestern Puerto Ricans. The manifestations of storage disease are variable and include granulomatous colitis, restrictive lung disease, kidney failure, and cardiomyopathy. The autofluorescent material stored in the Hermansky-Pudlak syndrome is histochemically similar to that stored in neuronal ceroid/lipofuscinosis. The material in neuronal ceroid/lipofuscinosis contains dolichols, which are components of lysosomes, and patients show increased urinary excretion of dolichols. This study of 49 patients with the Hermansky-Pudlak syndrome found that urinary dolichol levels are increased in those patients with evidence of ceroid storage in the kidneys but are not elevated when storage occurs in tissues other than the kidneys. The excretion of ceroid was not influenced by the saturation state of dietary fat. A defect in processing of membranes of lysosomes, melanosomes, and dense bodies may be involved in the syndrome.

Urinary sediment dolichols in the diagnosis of neuronal ceroid-lipofuscinosis.

Long-chain polyisoprenol alcohol (dolichols) levels are significantly increased in the urinary sediment of patients with infantile, late-infantile, and juvenile forms of neuronal ceroid-lipofuscinosis (NCL). The values in obligate heterozygotes for these diseases are similar to those in patients with other neurological diseases and in healthy controls. Antioxidant treatment of patients with juvenile NCL has no effect on dolichol values. The rate of false-negative results is 13.9% in infantile, 7.5% in late-infantile, and 15.0% in juvenile NCL. False-positive results were found in 8.2 to 14.3% of patients with other neurological diseases and in 15.4% of healthy controls. The test is of considerable value in the diagnosis of NCL and in decisions on whether to perform a biopsy. It is not useful in the screening of random samples, however.