Recent advances on HPLC/MS in medicinal plant analysis

With gaining popularity of herbal remedies worldwide, the need of assuring safety and efficacy of these products increases as well. By nature they are complex matrices, comprising a multitude of compounds, which are prone to variation due to environmental factors and manufacturing conditions. Furthermore, many traditional preparations compose of multiple herbs, so that only highly selective, sensitive and versatile analytical techniques will be suitable for quality control purposes. By hyphenating high performance liquid chromatography and mass spectrometry (LC-MS) these high demands are fulfilled, providing the user with a multitude of technical options and applications. This review intends to reflect the impact of LC-MS for medicinal plant analysis focusing on most relevant reports published within the last five years. Commenced by introductory remarks to the different MS approaches most commonly used (e.g. ion trap and time of flight mass analyzers, fragmentation and ionization modes), respective LC-MS applications on the analysis of natural products in medicinal plants, commercial products and biological samples are presented. Methodological aspects like stationary and mobile phase selection or MS settings are discussed, and advantages or limitations of the described techniques are highlighted.     

Specific characterization of non‐steroidal selective androgen peceptor modulators using supercritical fluid chromatography coupled to ion‐mobility mass spectrometry: application to the detection of enobosarm in bovine urine

Currently under development for therapeutic purposes in human medicine, non‐steroidal selective androgen receptor modulators (non‐steroidal SARMs) are also known to impact growth associated pathways. As such, they present a potential for abuse in sports and food‐producing animals as interesting alternative anabolic substances. Forbidden since 2008 by the World Anti‐Doping Agency (WADA) these compounds are however easily available and could be (mis)used in livestock production as growth promoters. To prevent such practices, dedicated analytical strategies have to be developed for specific and sensitive detection of these compounds in biological matrices. Using an innovative analytical platform constituted of supercritical fluid chromatography coupled to ion mobility‐mass spectrometry, the present study enabled efficient separation and identification in urine of 4 of these drugs (andarine, bicalutamide, hydroxyflutamide, and enobosarm) in accordance with European Union criteria (Commission Decision 2002/657/EC). Besides providing information about compounds structure and behaviour in gas phase, such a coupling enabled reaching low limits of detection (LOD < 0.05 ng.mL⁻¹ for andarine and limits of detection < 0.005 ng.mL⁻¹ for the three others) in urine with good repeatability (CV < 21 %). The workflow has been applied to quantitative determination of enobosarm elimination in urine of treated bovine (200 mg, oral). Copyright © 2016 John Wiley & Sons, Ltd.     

Selective androgen receptor modulators: comparative excretion study of bicalutamide in bovine urine and faeces

Besides their development for therapeutic purposes, non‐steroidal selective androgen receptor modulators (non‐steroidal SARMs) are also known to impact growth‐associated pathways as ligands of androgenic receptors (AR). They present a potential for abuse in sports and food‐producing animals as an interesting alternative to anabolic androgenic steroids (AAS). These compounds are easily available and could therefore be (mis)used in livestock production as growth promoters. To prevent such practices, dedicated analytical strategies should be developed for specific and sensitive detection of these compounds in biological matrices. The present study focused on Bicalutamide, a non‐steroidal SARM used in human treatment of non‐metastatic prostate cancer because of its anti‐androgenic activity exhibiting no anti‐anabolic effects. To select the most appropriate matrix to be used for control purposes, different animal matrices (urine and faeces) have been investigated and SARM metabolism studied to highlight relevant metabolites of such treatments and establish associated detection time windows. The aim of this work was thus to compare the urinary and faecal eliminations of bicalutamide in a calf, and investigate phase I and II metabolites. The results in both matrices showed that bicalutamide was very rapidly and mainly excreted under its free form. The concentration levels were observed as higher in faeces (ppm) than urine (ppb); although both matrices were assessed as suitable for residue control. The metabolites found were consistent with hydroxylation (phase I reaction) combined or not with glucuronidation and sulfation (phase II reactions). Copyright © 2016 John Wiley & Sons, Ltd.     

一种草苔虫中的神经酰胺类化合物

目的研究草苔虫Bugula sp．的化学成分。方法利用Sephadex-LH-20和硅胶拄层析分离化合物，用光谱数据确定化合物结构。结果从产于我国南海深圳大亚湾海域一种草苔虫（Bugula sp．）的CH2Cl2提取物中，分离得到了8个神经酰胺类化合物（1～8）并确定了它们的化学结构。结论神经酰胺类化合物为首次从该海洋物种中得到。     

Stability of the non-ionic surfactant polysorbate 80 investigated by HPLC-MS and charged aerosol detector

An analytical method using HPLC coupled with a charged aerosol detector (CAD) and a mass selective detector (MSD) was developed to characterize the non-ionic surfactant polysorbate 80 (PS 80). The molecular structure and heterogeneous composition due to isomers and various lengths of PEG-chains make it difficult to develop sensitive and specific analytical methods. Hence, there is only limited knowledge about the stability and purity of this compound. Polysorbate 80 does not possess any chromophore, thus UV detection is not applicable. Therefore, CAD and MSD have been used for determination. The aim of this study was to characterize polysorbate 80 and to examine its stability at pH 1.0 and 37 degrees C simulating harsh gastric conditions. It was shown that this surfactant is liable to degradation under these conditions. Within 8 h monoesters of PS 80 were hydrolyzed to an extent of 9.5% (+/- 3.0%), whereas incubation in water did not result in any detectable degradation. Furthermore, we demonstrated that HPLC-MS is a suitable technique to investigate ethoxylated compounds like polysorbates.     

Structural identification of skin ceramides containing ω-hydroxy acyl chains using mass spectrometry

The stratum corneum (SC) acts as a barrier that protects organisms against the environment and from transepidermal water loss. It consists of corneocytes embedded in a matrix of lipid metabolites (ceramides, cholesterol, and free fatty acids). Of these lipids, ceramides are sphingolipids consisting of sphingoid bases, linked to fatty acyl chains. Typical fatty acid acyl chains are composed of α-hydroxy fatty acids (A), esterified ω-hydroxy fatty acids (EO), non-hydroxy fatty acids (N), and ω-hydroxy fatty acids (O). Of these, O-type ceramides are ester-linked via their ω-hydroxyl group to proteins in the cornified envelope and can be released and extracted following mild alkaline hydrolysis. Tandem mass spectrometry (MS/MS) analysis of O-type ceramides using chip-based direct infusion nanoelectrospray-ion trap mass spectrometry generated the characteristic fragmentation pattern of both acyl and sphingoid units, suggesting that this method could be applied to the structural identification of O-type ceramides. Based on the MS/MS fragmentation patterns of O-type ceramides, comprehensive fragmentation schemes are proposed. In addition, we have also developed a method for identifying and profiling O-type ceramides in the mouse and guinea pig SC. This information may be used to identify O-type ceramides in the SC of animal skin.     

Development of high efficiency separation method for biomolecules

It is becoming increasingly important to separate mixed samples, such as bio- and environmental samples. For the analysis of a target compound in a mixed sample, a highly efficient and selective separation method is required. We have developed columns of high resolution, very selective columns, and highly efficient analytical methods using integrated techniques and biomolecules. These developed methods save analytical time, sample volume, etc. We are now interested in nano scale materials which many people are now focusing on. Although excellent nano materials have been developed by many researchers, there are few efficient separation, purification or evaluation methods for these compounds. In this review, we introduce our recent achievements concerning the separation of nano compounds.     

Determination of the oral platelet aggregation inhibitor Sibrafiban in rat, dog, and human plasma utilising HPLC-column switching combined with turbo ion spray single quadrupole mass spectrometry

A sensitive and selective HPLC-column switching method with single quadrupole mass spectrometric detection was developed for the simultaneous determination of the oral platelet aggregation inhibitor Sibrafiban (double protected prodrug), its prodrug and the active metabolite in rat, dog, and human plasma. The three analytes together with their tri-deuterated internal standards were isolated from plasma by protein precipitation (0.5 M perchloric acid). The de-proteinated samples were injected onto a standard-bore trapping column (4.0 mm i.d., LC-ABZ) of an HPLC-column switching system. Polar plasma components were removed by flushing the trapping column with ammonium formate (pH 3.6; 5 mM). Enriched compounds (including the analytes of interest) were backflushed onto a narrow-bore analytical column (2.1 mm i.d., Inertsil ODS-2) and separated by gradient elution (formic acid/ methanol). The whole effluent (200 microl/min) from the analytical column was passed to the turbo ion spray interface without splitting. Selected ion monitoring (SIM) was used for mass spectrometric detection. The limit of quantification for all three analytes was 1 ng/ml, using a 250-microl specimen of plasma. The mean precision and inaccuracy for the three analytes in all species were < 6 and < 5%, respectively. The practicability of the new analytical method was demonstrated by the analysis of about 500 rat and dog plasma and about 14,000 human plasma samples. The new method represents a successful example for the application of LC single MS with ionspray ionisation to the analysis of small molecule drugs in biological matrices from toxicokinetic studies and large clinical trials.     

Advanced analysis of nutraceuticals

In this article, we present a review work on different nutraceuticals found in natural matrices together with the analytical techniques used to identify and/or quantify them with special emphasis in the period January 2005-May 2010. The work is distributed according to the different families of nutraceuticals (lipids, vitamins, proteins, glycosides, phenolic compounds, etc.) discussing the analytical techniques employed for their determination (separation, spectroscopic, hyphenated techniques, etc.). Information about the claimed health promoting effects of the different families of nutraceuticals is also provided together with data on the natural matrices in which they can be found (e.g., fruits, vegetables, plants, microalgae, cereals, milk, etc.).     

Perfluorocarboxylic acids in cell growth media and technologically treated waters: Determination with GC and GC–MS

The present paper reports on an analytical method for the routine analysis of perfluorinated carboxylic acids (PFCAs). A rapid method for the derivatization, extraction and determination of PFCAs was developed. Technological samples were extracted with ethyl acetate and the anilides obtained were determined by gas chromatography–mass spectrometry (GC–MS) and gas chromatography with flame ionization detector (GC-FID). Residue levels in cell growth incubation media were determined by GC-FID. Confirmation analysis of PFCAs was carried out by GC–MS in selected ion monitoring (SIM) and total ion current (TIC) modes. The compounds were identified on the basis of retention time and comparison of primary and secondary ions. The results showed that this method provided a simple, rapid and sensitive way of analyzing PFCAs in different matrices.     

Biomonitoring of aristolactam-DNA adducts in human tissues using ultra-performance liquid chromatography/ion-trap mass spectrometry

Aristolochic acids (AAs) are a structurally related family of nephrotoxic and carcinogenic nitrophenanthrene compounds found in Aristolochia herbaceous plants, many of which have been used worldwide for medicinal purposes. AAs have been implicated in the etiology of so-called Chinese herbs nephropathy and of Balkan endemic nephropathy. Both of these disease syndromes are associated with carcinomas of the upper urinary tract (UUC). 8-Methoxy-6-nitrophenanthro-[3,4-d]-1,3-dioxolo-5-carboxylic acid (AA-I) is a principal component of Aristolochia herbs. Following metabolic activation, AA-I reacts with DNA to form aristolactam (AL-I)-DNA adducts. We have developed a sensitive analytical method, using ultraperformance liquid chromatography-electrospray ionization/multistage mass spectrometry (UPLC-ESI/MS(n)) with a linear quadrupole ion-trap mass spectrometer, to measure 7-(deoxyadenosin-N(6)-yl) aristolactam I (dA-AL-I) and 7-(deoxyguanosin-N(2)-yl) aristolactam I (dG-AL-I) adducts. Using 10 μg of DNA for measurements, the lower limits of quantitation of dA-AL-I and dG-AL-I are, respectively, 0.3 and 1.0 adducts per 10(8) DNA bases. We have used UPLC-ESI/MS(n) to quantify AL-DNA adducts in tissues of rodents exposed to AA and in the renal cortex of patients with UUC who reside in Taiwan, where the incidence of this uncommon cancer is the highest reported for any country in the world. In human tissues, dA-AL-I was detected at levels ranging from 9 to 338 adducts per 10(8) DNA bases, whereas dG-AL-I was not found. We conclude that UPLC-ESI/MS(n) is a highly sensitive, specific and robust analytical method, positioned to supplant (32)P-postlabeling techniques currently used for biomonitoring of DNA adducts in human tissues. Importantly, UPLC-ESI/MS(n) could be used to document exposure to AA, the toxicant responsible for AA nephropathy and its associated UUC.     

Development of a liquid chromatography/tandem mass spectrometry method for the quantitation of acetylcholine and related neurotransmitters in brain microdialysis samples

Monitoring concentrations of acetylcholine (ACh) in specific brain regions is important in understanding disease pathology, as well as in designing and evaluating novel disease-modifying treatments where cholinergic dysfunction is a hallmark feature. We have developed a sensitive and quantitative liquid chromatography/tandem mass spectrometry method to analyze the extracellular concentrations of ACh, choline (Ch) and (3-carboxylpropyl)-trimethylammonium (iso-ACh) in brain microdialysis samples of freely moving animals. One immediate advantage of this new method is the ability to monitor ACh in its free form without having to use a cholinesterase inhibitor in the perfusate. The separation of ACh, Ch, iso-ACh and related endogenous compounds was carried out based on cation exchange chromatography with a volatile elution buffer consisting of ammonium formate, ammonium acetate and acetonitrile. An unknown interference of ACh, which was observed in brain microdialysates from many studies, was well separated from ACh to ensure the accuracy of the measurement. Optimization of electrospray ionization conditions for these quaternary ammonium compounds achieved the limits of detection (S/N=3) of 0.2 fmol for ACh, 2 fmol for Ch and 0.6 fmol for iso-ACh using a benchtop tandem quadrupole mass spectrometer with moderate sensitivity. The limit of quantitation (S/N=10) was 1 fmol for ACh, 3 fmol for iso-ACh and 10 fmol for Ch. This method was selective, precise (<10% R.S.D.), and sensitive over a range of 0.05-10nM for ACh, 0.25-50 nM for iso-ACh and 15-3000 nM for Ch. To demonstrate that the developed method can be applied to monitoring changes in ACh concentrations in vivo, reference agents that have previously been shown to influence ACh levels were studied in rat dorsal hippocampus. This includes the 5-HT6 receptor antagonist, SB-271046, and the cholinesterase inhibitor, donepezil. Moreover, levels of ACh were demonstrated to be sensitive to infusion of tetrodotoxin (TTX) suggesting that the ACh being measured in vivo was of neuronal origin. Collectively, these biological data provided in vivo validation of this analytical method.     

Cyclooxygenase inhibitory natural products: current status

Non-steroidal anti-inflammatory drugs (NSAIDs) are of huge therapeutic benefit in the treatment of rheumatoid arthritis and various types of inflammatory conditions. The target for these drugs is cyclooxygenase (COX), a rate-limiting enzyme involved in the conversion of arachidonic acid into inflammatory prostaglandins. COX-2 selective inhibitors are believed to have the same anti-inflammatory, anti-pyretic and analgesic activities as that of nonselective inhibitor NSAIDs with little or none of the gastrointestinal side effects. Thus, in the last 6-7 years several selective COX-2 inhibitors including coxibs were discovered and introduced into clinic. Recent reports evidence that selective COX-2 inhibitor such as rofecoxib, can lead to thrombotic cardiovascular events through inhibition of prostacyclin formation in the infracted heart. This has resulted in withdrawal of rofecoxib from the clinic in September 2004. Moreover, the COX-2/COX-1 selectivity ratio is vital in the design of COX-2 inhibitory drugs, as it is clear from rofecoxib, which is more than 50-fold COX-2 selective. After looking at all above mentioned facts, natural product-based compounds seem better as these compounds are generally supposed to be devoid of severe side effects. The literature indicates that natural product-based compounds are mainly COX-1 selective. Through minor semi-synthetic changes in the structures, their selectivity towards COX-2 can be increased. The present review article addresses natural product COX inhibitors of plant and marine origin, reported during last ten years and their advantages, possible leads for further development and current status. In addition we describe our experience in the characterization, design and synthesis of potential natural COX inhibitors.     

Supplementing pharmaceutical analysis techniques using radiotracers

Radiolabeled compounds can be used for alleviating the degradation pathways and mass balance of drug products in the solid formulation, because it provides a very sensitive and specific method of locating and measuring particular compounds. The present study utilizes this methodology on levormeloxifene-a partial oestrogen receptor agonist-in the solid dosage form. It demonstrates how radiotracers can be used as a supplement to conventional analytical methods to investigate the degradation pathways and mass balance of drug substances as well as how they can be useful to cross validate the traditional analytical methods developed for the determination of uniformity of content, assay, and purity. To decrease the study time, pilot studies-in which the product was stored at high pressures of molecular oxygen and elevated temperatures-were performed. Conditions mimicking the degradation processes taking place under standard storage conditions of the drug substance were found, and these super-enhanced stress conditions were applied. The study time was thereby dramatically decreased. The produced degradation products were studied by liquid chromatography with mass spectrometric detection. The most important degradation pathway was ascribed to the oxidation of the pyrrolidine nitrogen atom in levormeloxifene to an N-oxide derivative of the drug substance.     

A fast and efficient determination of amines and preservatives in cough and cold liquid and suspension formulations using a single isocratic ion-pairing high performance [correction of power] liquid chromatography method.

A single, highly selective ion-pairing reverse phase-high power liquid chromatography (RP-HPLC) method has been developed for the determination of amines and preservatives in a wide range of Tylenol((R)) liquid and suspension liquid products. As with many OTC products, the challenge is to quantitatively extract the analytes from difficult matrices and specifically analyze them in the presence of various excipients and flavors. Historically, separate analytical methods were used for each class of analytes (acids, bases and neutral compounds). In this method a mobile phase consisting of a buffered ion-pairing agent with acetonitrile, methanol and tetrahydrofuran was used to separate the charged amines from neutral and acidic compounds on a Phenomenex LUNA C8(2) 75 x 4.6 mm i.d. analytical column with a 3-microm particle size. The analytes include acids (benzoic acid), bases (pseudoephedrine, chlorpheniramine, dextromethorphan, doxylamine and diphenhydramine) and a neutral compound (butylparaben). The effects of pH, the chain length of the ion-pairing reagent, ionic strength and organic modifiers on the separation are discussed. The method is linear from 15 to 150% of the target amounts. The optimized method proves to be specific, robust and accurate for the analysis of the compounds.     

Ceramide involvement in apoptosis and apoptotic diseases

Apoptosis is implicated in a number of diseases, including neurodegenerative diseases and AIDS. More and more, evidence is accumulating pointing to the critical role of ceramides in the induction of apoptosis. The present review summarizes (i) the molecular basis and regulation of the apoptotic machinery, (ii) the molecular role of ceramides in the induction or execution of apoptotic pathways, and (iii) evidence linking ceramide generation to various apoptotic diseases. Additionally, this review discusses putative therapeutic approaches inhibiting ceramide production in apoptotic diseases.     

Progress in Immunoassays of Toxic Alkaloids in Plant-Derived Medicines: A Review

Plants are the cradle of the traditional medicine system, assuaging human or animal diseases, and promoting health for thousands of years. However, many plant-derived medicines contain toxic alkaloids of varying degrees of toxicity that pose a direct or indirect threat to human and animal health through accidental ingestion, misuse of plant materials, or through the food chain. Thus, rapid, easy, and sensitive methods are needed to effectively screen these toxic alkaloids to guarantee the safety of plant-derived medicines. Antibodies, due to their inherent specificity and high affinity, have been used as a variety of analytical tools and techniques. This review describes the antigen synthesis and antibody preparation of the common toxic alkaloids in plant-derived medicines and discusses the advances of antibody-based immunoassays in the screening and detection of toxic alkaloids in plants or other related matrices. Finally, the limitations and prospects of immunoassays for toxic alkaloids are discussed.     

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.     

Analysis and screening of combinatorial libraries using mass spectrometry

Mass spectrometry is a highly selective and high throughput analytical technique that is ideally suited for the identification and purity determination of large numbers of compounds prepared using combinatorial chemistry or for the dereplication of natural products. Compounds may be characterized based on molecular weight, elemental composition and structural features based on fragmentation patterns. When coupled to a separation technique such as high-performance liquid chromatography (HPLC) or capillary electrophoresis, mass spectrometric applications may be expanded to include analysis of complex mixtures. However, the slower speed of the separation step reduces the throughput of the analysis. This review concerns the application of mass spectrometry to the characterization of combinatorial libraries and the screening of library and natural product mixtures. Strategies to enhance the throughput of LC-MS are discussed including fast HPLC and parallel LC-MS. Also, mass spectrometry-based screening methods are described including frontal affinity chromatography-mass spectrometry, gel permeation chromatography LC-MS, direct electrospray mass spectrometry of receptor-ligand complexes, affinity chromatography-mass spectrometry, and pulsed ultrafiltration mass spectrometry.