Modern screening techniques for plant extracts

In order to discover new bioactive compounds from plant sources which could become new leads or new drugs, extracts should be simultaneously evaluated by chemical screening and by various biological or pharmacological targets. Chemical screening using hyphenated techniques such as LC/UV and LC/MS, and more recently LC/NMR, quickly provides ample structural information, leading in many cases to the identification of compounds. This allows researchers to distinguish between known compounds (dereplication) and new molecules directly from crude plant extracts. Thus, the tedious isolation of known compounds can be avoided, and a targeted isolation of constituents presenting novel or unusual spectroscopic features can be undertaken. In parallel, extracts are also subjected to various bioassays that should be simple, reproducible, and rapid. This approach will be illustrated by the search for new molluscicidal, antioxidant, and antifungal compounds from tropical plants.     

Structural identification and characterization of potential degradants of zotarolimus on zotarolimus-coated drug-eluting stents

Identification and characterization of unknown zotarolimus impurities on zotarolimus-coated drug-eluting stents is an important aspect of product development since the presence of impurities can have a significant impact on quality and safety of the drug product. Four zotarolimus degradation products have been characterized by LC/UV/PDA, LC/MS, LC/MS/MS and NMR techniques in this work. Zotarolimus drug substance and zotarolimus-coated stents were subjected to degradation under heat, humidity, acid or base conditions. The HPLC separation was achieved on a Zorbax Eclipse XDB-C8 column using gradient elution and UV detection at 278 nm. All four impurities generated through the degradation were initially analyzed by LC/MS and/or LC/MS/MS for structural information. Then the isolation of these degradants was carried out by semi-preparative HPLC method followed by freeze-drying of the collected fractions. Finally the degradants were studied by ¹H and ¹³C NMR spectrometry. Based on LC/MS, ¹H NMR and ¹³C NMR data, the structures of these impurities were proposed and characterized as zotarolimus ring-opened isomer (1), zotarolimus hydrolysis product, 16-O-desmethyl ring-opened isomer (2) and zotarolimus lower fragment (3). Degradants 1, 2 and 3 have been observed on degraded zotarolimus-coated stent products.     

The importance of hyphenated techniques in the discovery of new lead compounds from nature

Nature represents an extraordinary reservoir of novel molecules and there is currently a resurgence of interest in natural products as a possible source of new lead compounds for introduction into therapeutical screening programmes. To discover new bioactive natural products, the dereplication of crude extracts performed prior to isolation work is of crucial importance for avoiding the tedious isolation of known constituents. In this respect, chemical screening strategies based on hyphenated techniques such as liquid chromatography–ultraviolet photodiode array detection, liquid chromatography-mass spectrometry, liquid chromatography tandom mass spectrometry and liquid chromatography–nuclear magnetic resonance (LC-NMR) are more and more extensively used. In the laboratory of Hostettmann’s group, these analytical methods have been fully integrated into the isolation process and are used for the chemical screening of crude plant extracts, in complement with online or at-line bioassays, for rapid localisation and identification of new bioactive compounds. In this paper, possibilities and limitations of hyphenated techniques for de novo online natural product identification are discussed. As LC-NMR is playing a key role in this respect, the main part of the paper is dedicated to this technique. In particular, various ways of integrating NMR in the dereplication process are illustrated and strategies involving either direct or indirect hyphenation are presented.     

Identification and characterization of major degradation products of risperidone in bulk drug and pharmaceutical dosage forms

Acid, base and oxygen stability of risperidone, a novel anti-psychotic drug, has been evaluated storing the sample in solution phase. One of the major degradation products has been identified and characterized by using techniques namely IR, MS and NMR after isolation by preparative LC. The other major degradation product has been identified with help of MS/MS data and by co-eluting in analytical LC with the available standard. The effect of acid and base resulted in the formation of hydroxy risperidone and the effect of oxygen lead to the formation of N-oxide of risperidone. The two major degradation products in the dosage forms were also characterized as 9-hydroxy risperidone and N-oxide of risperidone, after enrichment through preparative LC, by LC-MS/MS and HPLC. Structural elucidation of degradation product leading to the formation of N-oxide of risperidone is discussed in detail.     

Isolation, identification and determination of the major degradation product in alprazolam tablets during their stability assay

The presence of a degradation product of alprazolam tablets that emerged throughout a short-stability assay has been determined and properly characterized. For this purpose an efficient methodology has been successfully applied, including SPE and HPLC methods for isolation and purification, respectively. LC/MS, MS/MS, 1H NMR, 13C NMR, UV and IR have been employed for structural elucidation confirming the identity of this impurity as 7-chloro-1-methyl-5-phenyl-[1,2,4]triazolo[4,3-a]quinolin-4-amine or triazolaminoquinoleine. The impurity, previously described as a long-term photodegradation product of alprazolam active pharmaceutical ingredient, was rapidly formed in the absence of light, but required the presence of excipients and its rate of formation increased with heat and humidity. In addition, a LC method has been developed and validated including triazolaminoquinoleine for the adequate determination of alprazolam and its mayor degradation product in tablets as pharmaceutical forms.     

The use of LC/MS, GC/MS, and LC/NMR hyphenated techniques to identify a drug degradation product in pharmaceutical development

Understanding drug degradation in the formulated product is critical in pharmaceutical development as it has significant impacts on drug efficacy, safety profile and storage conditions. As a result, identification of degradation compounds has taken an important role in the drug development process. In this study, various hyphenated analytical techniques, such as liquid chromatography mass spectrometry (LC/MS), gas chromatography mass spectrometry (GC/MS), and liquid chromatography nuclear magnetic resonance with a solid phase extraction interface (LC/SPE/NMR), have been applied to the identification of a drug degradation product which grew over time in the stability study of the drug product. The target unknown is less polar and more unsaturated than the drug substance based upon reverse phase HPLC relative retention time and UV spectra. It is not ionizable by electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) in either a positive or a negative mode. The unknown was isolated by an HPLC fraction collector and enriched by solid phase extraction. GC/MS with chemical ionization (CI) was employed to determine the molecular weight of this compound. Its fragmentation pattern was determined by CI-MS/MS using an ion trap mass spectrometer. The isolated material was also analyzed by LC/SPE/NMR, from which the structure of this compound was further characterized. The study utilizes a combination of various hyphenated analytical techniques to obtain complimentary information for structure elucidation of the unknown. The combination approach is critical for unambiguous impurity structure elucidation in drug degradation studies of pharmaceutical drug products.     

Isolation, purification, and characterization of two new chemical decomposition products of methylazoxyprocarbazine.

We have previously reported that the antineoplastic agent, procarbazine, in aqueous solutions was chemically oxidized to its azoxy metabolites (methylazoxy and benzylazoxy). To determine if there was additional metabolism of the most active metabolite, methylazoxyprocarbazine, it was incubated in the presence and absence of CCRF-CEM human leukemia cells. Incubations were extracted, and potential metabolites were detected by HPLC with UV detection and by combined HPLC and thermospray mass spectrometric analysis. The major metabolite identified by HPLC with UV detection of the extracts was N-isopropyl-p-formylbenzamide; this was identified by comparison of its retention time with that of a synthesized standard. This identification was further corroborated by HPLC/thermospray mass spectrometry (LC/MS). Analysis of the extracts by LC/MS also showed the presence of a closely eluting peak that had a protonated molecular ion at m/z 207. This new metabolite was identified as N-isopropyl-(benzene-1,4-bis-carboxamide) by 1H NMR and gas chromatography/ion trap mass spectrometry. This metabolite is postulated to arise from breakage of the N-N bond in the hydrazine portion of the molecule. Reconstructed ion (m/z 236) current profiles from the analysis of the cell extracts indicated that there was only a trace amount of methylazoxyprocarbazine left after a 72-hr incubation. Interestingly, a peak with the same molecular weight as the parent compound (methylazoxyprocarbazine) was observed in the cellular incubations and also in extracts of control incubations in which methylazoxyprocarbazine was incubated in medium without cells. This unknown was silylated and identified as a hydroxyazo compound by an ion trap mass spectrometer operated under both single and multiple-stage mass analysis. Formation of this decomposition product appears to involve a novel intramolecular rearrangement of methylazoxyprocarbazine in solution. This pathway may be responsible for the formation of the ultimate cytotoxic species by chemical decomposition of procarbazine.     

Hyphenated NMR methods in natural products research, part 1: direct hyphenation

This review describes the current status of the development of techniques combining liquid chromatography and nuclear magnetic resonance and their use in the context of natural products. HPLC-NMR methods have a rapidly growing impact on natural products research by enabling structure determination of natural products directly from small amounts of extracts, i. e., prior to the investment in an often lengthy and laborious preparative-scale isolation process. This speeds up extract dereplication and helps to avoid re-isolation of already known extract constituents. Direct HPLC-NMR hyphenation techniques, defined as methods in which NMR spectra are recorded using the HPLC eluate, are described. The recently developed indirect HPLC-NMR method employing an automated solid-phase extraction interface between HPLC and NMR, which replaces the chromatographic solvent with a different solvent for NMR data acquisition, is described in the second part of this review.     

The analytical challenges of cyclopropylfentanyl and crotonylfentanyl: An approach for toxicological analysis

New psychoactive substances (NPS) are increasingly being seen in forensic casework globally and encompass a number of types of drugs including “designer opioids”, especially fentanyl analogues, which are of particular concern due to their high potency and significant risk of toxicity. They are often sold as heroin or mixed with other illicit drugs and therefore users may be unaware they are taking such hazardous compounds. Two fentanyl analogues that have recently been detected are cyclopropylfentanyl and crotonylfentanyl. In order to accurately determine the prevalence of such compounds in clinical and forensic casework, including potential toxicity, they need to be correctly identified using definitive and defensible techniques. Cyclopropylfentanyl and crotonylfentanyl are structural isomers, and it has previously been highlighted that these 2 compounds are analytically difficult to specifically identify owing to their similarity in structure and chromatographic behaviour. To further investigate in an attempt to overcome this problem, analysis of certified reference material using high performance liquid chromatography with diode array UV detection (HPLC–DAD), liquid chromatography–tandem mass spectrometry (LC–MS/MS), and liquid chromatography–quadrupole time‐of‐flight–mass spectrometry (LC–QToF–MS) has been performed. Whilst the compounds were shown to have an identical mass‐spectral fragmentation pattern, they had different UV spectra. This was coupled with a discernible difference in retention time with the HPLC conditions applied, allowing differentiation of the 2 compounds. Using this approach, cyclopropylfentanyl was positively identified and subsequently quantified in 4 fatalities with the exclusion of crotonylfentanyl.     

Chiral purity assay for Flindokalner using tandem mass spectrometry: method development, validation, and benchmarking

The present work demonstrates the application and validation of a mass spectrometry method for quantitative chiral purity determination. The particular compound analyzed is Flindokalner, a Bristol-Myers Squibb drug candidate for post-stroke neuroprotection. Chiral quantification of Flindokalner was achieved using tandem mass spectrometry (MS/MS) and the kinetic method, a gas phase method used for thermochemical and chiral determinations. The MS/MS method was validated and benchmarked against two separate chromatographic techniques, chiral high performance liquid chromatography with ultra-violet detection (LC/UV) and achiral high performance liquid chromatography with circular dichroism detection (LC/CD). The chiral purity determination of Flindokalner using MS/MS proved to be rapid (3 min run time for each sample) and to have accuracy and precision comparable to the chiral LC/UV and achiral LC/CD methods. This method represents an alternative to commonly used chromatographic techniques as a means of chiral purity determination and is particularly useful in rapid screening experiments.     

Metabolite profiling and characterization for medicinal herbal remedies

Metabolisms of herbal remedies and their natural components, which play a critical role in support of medicine development, and clinical medication, are drastically different from those of designed drugs. The separation, isolation and identification of drug metabolites from complex endogenous matrices like urine, plasma and tissue extracts are extremely challenging. For herbal medicine studies, it is even more difficult due to the complex chemical composition. Usually, a combination of high performance liquid chromatography (HPLC) and mass spectrometry (MS) is proven to be a powerful analytical tool for screening and identifying drug metabolites. For suitable instruments, the quadrupole time-of-flight (Q-TOF), hybrid ion trap time-of-flight (IT-TOF), and orbitrap mass spectrometry could clearly enhance the efficiency in metabolite profiling compared to general triquadrupole (QQQ) and ion trap (IT) mass spectrometry technique. Due to the ability for unambiguous structure determination, nuclear magnetic resonance spectroscopy (NMR) is also coupled to HPLC for on-line analysis of metabolites. Capillary electrophoresis and gas chromatography are also optional methods. These techniques could provide abundant information from a wide variety of samples. However, in many cases, preparations of metabolites are critical for further pharmacokinetics, pharmacologic, and toxic evaluation of the remedy. Therefore, accumulations of metabolites from the in vivo biological samples are essential. Biotransformation models are considered to be important complementary sources for preparation of drug metabolites. Fungi, plant cells, and a variety of enzymes were used to provide information for further in vivo testing. This review focuses on the screening and identification of drug metabolites of herbal medicines.     

LC/MS for the degradation profiling of cough-cold products under forced conditions

Heat, acid, base, UV radiation and oxidation stress methods were applied to study the stability of cough–cold products containing acetaminophen, phenylephrine or phenylpropanolamine hydrochloride and chlorpheniramine maleate. Liquid chromatography coupled with mass spectrometry was used to analyze the degraded samples and obtain molecular weights information. Different volatile buffers (ammonium bicarbonate and ammonium acetate) were assayed in LC/MS methods and retention times of the analytes were compared with those obtained in HPLC with UV detection employing a conventional sodium phosphate buffer to establish the possibility of results transference between the two systems.     

Improving the detection of degradants and impurities in pharmaceutical drug products by applying mass spectral and chromatographic searching

Liquid chromatography/mass spectrometry (LC/MS) and NMR are commonly used to identify metabolites, impurities and degradation products in the pharmaceutical industry. To more efficiently deal with the large volumes of data these techniques generate, software programs have been developed by various vendors to assist in the identification of these compounds through the use of spectral and chromatographic search algorithms. The feasibility of using such programs for detecting drug degradants and impurities is assessed. A number of compounds encompassing a wide range of both chemical and pharmaceutical properties were tested using LC/UV/MS and the spectral/chromatographic search algorithm MetaboLynx (Micromass UK Ltd.) to determine the feasibility of detecting analytes at low concentrations. In addition, drug product and stressed drug substance samples containing quinapril hydrochloride, the active ingredient in Accupril tablets, were determined by liquid chromatography with atmospheric pressure ionization-time-of-flight (API LC-TOF) and an API LC-quadrupole (Q) mass spectrometer, and the resulting data was processed using MetaboLynx. The ability of this program to detect and list a variety of analytes known to be present in the samples was evaluated. The combination of LC/UV, LC/MS and spectral/chromatographic searching is a valuable tool for the detection of impurities at low levels.     

Catechin derivatives in Jatropha macrantha stems: characterisation and LC/ESI/MS/MS quali-quantitative analysis

A phytochemical investigation on methanol extract of stems of Jatropha macrantha led to the isolation of catechin, catechin-7-O-beta-glucopyranoside and proanthocyanidin B-3 along with other catechin polymers. Their structures were established by NMR and ESI/MS experiments. Additionally, an LC/ESI/MS qualitative study and an LC/ESI/MS/MS quantitative study of the phenolic fraction of J. macrantha stems were performed. Combination of high performance liquid chromatography (HPLC) (DAD) with positive electrospray ionisation (ESI) and tandem mass spectrometry (MS/MS) performed with Ion Trap analyser permitted to have qualitative data on catechin derivatives: several other proanthocyanidins were detected. A mixture of proanthocyanidin polymers was characterised by direct introduction ESI-MS analysis. An LC/ESI/MS/MS method was developed and validated for separation and quantification of catechin, catechin-7-O-beta-glucopyranoside and proanthocyanidin B-3. Due to the sensitivity and the repeatability of the assay, we suggest this method as suitable for industrial quality control of raw materials and final products. Quantitative analyses results confirmed that compounds 1-3 are major compounds of the plant and, in particular, proanthocyanidin B-3 appears to be the most abundant.     

Profiling impurities and degradants of butorphanol tartrate using liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry substructural techniques

A rapid and systemic strategy based on liquid chromatography/mass spectrometry (LC/MS) profiling and liquid chromatography/tandem mass spectrometry (LC/MS/MS) substructural techniques was utilized to elucidate the degradation products of butorphanol, the active ingredient in stadol® NS. This strategy integrates, in a single instrumental approach, analytical HPLC, UV detection, full-scan electrospray mass spectrometry, and tandem mass spectrometry to rapidly and accurately elucidate structues of impurities and degradants. In these studies, several low-level degradation products were observed in long-term storage stability samples of bulk butorphanol. The resulting analytical profile includes information on five degradants including molecular structures, chromatographic behavior, molecular weight, UV data, and MS/MS substructural information. The degradation products formed during long-term storage of butorphanol tartrate included oxidative products proposed as 9-hydroxy- and 9-keto-butorphanol, norbutorphanol, a ring-contraction degradant, and Δ1, 10-butorphanol. These methodologies are applicable at any stage of the drug product cycle from discovery through to development. This library of butorphanol degradants provides a foundation for future development work regarding product monitoring, as well as a useful diagnosite tool for new degradation products.     

Novel identification of a sulfur-centered, radical-derived 5,5-dimethyl-1-pyrroline N-oxide nitrone adduct formed from the oxidation of DTT by LC/ELISA, LC/electrospray ionization-MS, and LC/tandem MS

The detection of highly reactive free radicals generated in biological systems by an ESR spin-trapping technique is always difficult and limited due to the short lifetimes of ESR active spin-trapping radical adducts and poor structural information provided by ESR spectra. In this investigation, we have for the first time employed anti-5,5-dimethyl-1-pyrroline N-oxide (DMPO) polyclonal antiserum that specifically recognizes stable, ESR silent end products of DMPO radical adducts and combined HPLC with ELISA, electrospray ionization mass spectrometry (ESI-MS), and tandem mass spectrometry (MS/MS) to separate and characterize DMPO nitrone adducts derived from free radical metabolites. When mircoperoxidase-11 (MP-11) reacted with DTT in the presence of DMPO with or without H2O2, we detected radical-derived DMPO nitrone adducts by ELISA. Similar results were obtained when MP-11 was replaced by hemin. To identify the DMPO nitrone adducts formed in both reaction systems, LC separation was carried out, and the fractions eluted from the LC column were collected and analyzed by ELISA. In both reaction mixtures, we found that only one peak with the same retention time showed a strong positive ELISA signal, suggesting that this peak was from radical-derived DMPO nitrone adducts and that both systems produced the same free radical metabolites. Using online LC/ESI-MS, LC/MS/MS, and (1)H NMR, we demonstrated that the DMPO nitrone adducts formed are from the DMPO adducts of the sulfur-centered radical of DTT. The successful application of LC/ELISA, LC/MS, and LC/MS/MS in this study makes it possible to separate and identify the stable DMPO nitrone adducts derived from free radical metabolites generated in biological systems.     

New stilbene carboxylic acid from Convolvulus hystrix

Convolvulus hystrix Vahl. (family Convolvulaceae) is a common wild plant growing in the kingdom of Saudi Arabia. It is used in traditional medicine. Our study resulted in the isolation and structural elucidation of ten natural products from which a stilbene carboxylic acid was isolated for the first time as a new natural product. The isolated products are mainly shikimates in addition to two sterols; beta-sitosterol and stigmasterol, as well as one triterpenoid, oleanolic acid. The shikimate products are vanillin, vanillic acid, syringic acid, ferulic acid, isoferulic acid, isoscopoletin and the new stilbene derivative. The structures were elucidated on the basis of spectral data from 400 MHz 1H NMR, MS, IR and UV. The biogenesis of the new stilbene is discussed.     

Early Prediction of Pharmaceutical Oxidation Pathways by Computational Chemistry and Forced Degradation

PURPOSE: To show, using a model study, how electronic structure theory can be applied in combination with LC/UV/MS/MS for the prediction and identification of oxidative degradants. METHODS: The benzyloxazole 1, was used to represent an active pharmaceutical ingredient for oxidative forced degradation studies. Bond dissociation energies (BDEs) calculated at the B3LYP/6-311+G(d,p)//B3LYP/6-31G(d) level with isodesmic corrections were used to predict sites of autoxidation. In addition, frontier molecular orbital (FMO) theory at the Hartree-Fock level was used to predict sites of peroxide oxidation and electron transfer. Compound 1 was then subjected to autoxidation and H2O2 forced degradation as well as formal stability conditions. Samples were analyzed by LC/UV/MS/MS and degradation products proposed. RESULTS: The computational BDEs and FMO analysis of 1 was consistent with the LC/UV/MS/MS data and allowed for structural proposals, which were confirmed by LC/MS/NMR. The autoxidation conditions yielded a number of degradants not observed under peroxide degradation while formal stability conditions gave both peroxide and autoxidation degradants. CONCLUSIONS: Electronic structure methods were successfully applied in combination with LC/UV/MS/MS to predict degradation pathways and assist in spectral identification. The degradation and excipient stability studies highlight the importance of including both peroxide and autoxidation conditions in forced degradation studies.     

Identification, isolation and characterization of a new degradation product in sultamicillin drug substance

A new degradant of sultamicillin drug substance was found during the gradient reverse phase HPLC analysis of stability storage samples. The level of this degradant impurity was observed up to 1.0%. The impurity (formaldehyde adduct with 5-oxo-4-phenylimidazolidin-1-yl moiety) was identified by LC/MS and was characterized by ((1)H NMR, (13)C NMR, 2D-NMR ((1)H-(1)H COSY, NOESY, HSQC and HMBC), LC/MS/MS, MS/TOF, elemental analysis and IR. This impurity was prepared by isolation and co-injected into HPLC system to confirm the retention time.     

Identification of euglenophycin - A toxin found in certain euglenoids

Currently cyanoprokaryotic algae, diatoms, haptophytes, dinoflagellates, euglenoids, and rhaphidophytes are known to produce algal toxins. A previous study by the authors reported euglenoid algae producing toxin(s) in aquaculture ponds, with confirmation based on positive fish bioassays following exposure to the isolated clonal algal cultures. Toxicity was observed in euglenoid culture isolates obtained from the pond as well as a clonal, culture collection taxon. Here we provide conclusive evidence for euglenoid toxin production, including HPLC/MS, MS/MS, and NMR analyses of a clonal (non-axenic) isolate of Euglena sanguinea grown in batch culture. Following wet chemical serial fractionation, toxic activity was identified in both the methanol and hexane extracts. These extracts were then purified using HPLC. Bioassay-guided HPLC fractionation of these two extracts demonstrated that a single class of toxic compounds, identical in mass and similar in molecular structure, was produced by this organism. The toxic compounds exhibited a maximal UV absorbance at 238 nm and gave diagnostic mass peaks at 306 (MH⁺) and 288 (MH⁺-H₂O). Unambiguous molecular structural determination was carried out by high field NMR analysis operating in 1- and 2-dimensions. Though a predominant isomer represented the bulk of the toxin, several stereo- and structural isomers were evidenced by NMR, and HPLC/MS. This compound is an alkaloid similar in structure to fire ant venom. The compound exhibits ichthyotoxic, herbicidal and anticancer activity at low ppm to ppb dosages.