Kelvin Sze-Yin Leung

Aristolochic Acids Detected in Some Raw Chinese Medicinal Herbs and Manufactured Herbal Products – A Consequence of Inappropriate Nomenclature and Imprecise Labelling?

Background. Certain frequently used Chinese herbal medicines commonly used for weight control, may contain toxic Aristolochia species, which have been associated with severe nephropathy and urothelial cancer in humans and animals. The toxic entities in Aristolochia species are aristolochic acid-I (AA-I) and aristolochic acid-II (AA-II). There is a lack of systematic information about the aristolochic acid content of Aristolochia species and related genera, including those in Chinese materia medica that are used in the treatment of overweight individuals. Objectives. To determine the content of AA-I and AA-II of commonly used Chinese herbal medicines (raw herbs and manufactured products) including species of Aristolochia and related genera. Methods. Twenty-one raw herbs and seven manufactured herbal products were purchased from herbal wholesalers and traditional Chinese medicinal herb retailers in Melbourne, Australia in September 2003, after the supply of known aristolochic acid-containing herbs and products had been banned in Australia. Six additional raw herbs were sourced from a herbal teaching museum. These were purchased in 2001, before the prohibition. The contents of aristolochic acids of each was determined by high pressure liquid chromatography (HPLC), and confirmed by liquid chromatography–mass spectrometry (LC–MS). Results. Of the samples tested, four of the raw herbs purchased before the ban and two manufactured products purchased after the ban, were found to contain aristolochic acids (16–1002 ppm). Conclusions. Several Chinese raw herbs and some commercially available manufactured herbal products contain aristolochic acids. The confusion in Chinese nomenclature for related raw herbs, and imprecise labelling of manufactured products may contribute to the inadvertent use of toxic herbal species in Chinese medicine practice. Additional measures are needed to ensure the safety of consumers of Chinese herbal medicines.

Antitumor activity of diethynylfluorene derivatives of gold(I)

A list of diethynylfluorenes and their gold(I) derivatives have been studied for their antitumor activity as a function of their structure-activity relationships. End-capping the fluoren-9-one unit with gold(I) moieties could significantly strengthen the cytotoxic activity in vitro on three human cancer cell lines with induction of reactive oxygen species generation on Hep3B hepatocellular carcinoma cells and exhibit attractive antitumor activity from in vivo nude mice Hep3B xenograft model with limited adverse effects on vital organs including liver and kidney.

Highly sensitive and selective organophosphate screening in twelve commodities of fruits, vegetables and herbal medicines by dispersive liquid-liquid microextraction.

The article describes a simple sample pretreatment procedure for the analysis of ten organophosphorus pesticides using dispersive liquid-liquid microextraction (DLLME) followed by gas chromatography-mass spectrometry (GC-MS) in three distinctively different types of matrices: fresh fruits, fresh vegetables and dried herbs. The method was carefully developed, focusing on the chemistry of various dispersive solvents, to achieve simultaneous, comprehensive extraction and preconcentration in a great span of selected matrices. According to matrix-matched validation study, the set of optimized DLLME conditions has been proven robust to determine target OPPs within a wide linear range from 0.1 to 1000 μg L(-1). With limited usage of organic extractants, remarkable enrichment factors up to 100-fold were obtained, enabling ultra-trace pesticide quantification down to sub-ppt levels at 0.12-4.92 ng kg(-1). Practical application of the method was illustrated by quantitative recovery (70-119%) and good precision (2.6-10% R.S.D.) in a representative range of three fruits and four vegetable commodities featured by the CODEX Alimentarius classification as well as their unique matrix compositions. A careful selection of dried herbs was further classified based on their morphological structures to validate analytical ruggedness of the method. Compared with existing methods for food analysis vis-à-vis OPPs, the present method is superior in terms of high sample throughput, minimal solvent consumption, and small sample size requirement. An additional, significant aspect of this universal DLLME method is that it models sample pretreatment methods with wide coverage of analytical matrices that are more effective, more comprehensive, and more flexible than those currently being used.

Selective recognition of arsenic by tailoring ion-imprinted polymer for ICP-MS quantification.

A novel arsenic-ion imprinted polymer (As-IIP) was firstly synthesized for the separation and recovery of trace elemental As from environmental water samples. Polymers prepared from bifunctional monomers with intrinsic metal-binding capability are a platform for tailoring ion-selectivity via imprinting moiety-template interaction, without complex formation and ligand immobilization. In the present study, As-IIPs based on 1-vinylimidazole, 4-vinylpyridine and styrene were designed to investigate the imprinting mechanism in relation to their structural and functional properties. In terms of selectivity as well as imprinting effects compared with the non-imprinted polymer (NIP), 1-vinylimidazole-based As-IIP exhibited superior analyte recognition for As ion among 23 competing elements, with a 25-fold enhancement in the practical dynamic and static adsorption capacity range (0.048-4.925 μmol g(-1)). The robust As-IIP sorbent features good reusability up to 20 cycles and a wide working pH 5-7 for a firstly reported solid-phase extraction (SPE) application. As a result of selective sample clean-up, As-IIP-SPE offered limits of detection (LOD) and quantification (LOQ) down to 0.025 and 0.083 μmol L(-1), respectively, for environmental sample analysis using inductively coupled plasma-mass spectrometry.

LC–MS/MS in the routine clinical laboratory: has its time come?

Liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been increasingly used in routine clinical laboratories during the last two decades. The high specificity, sensitivity, and multi-analyte potential make it an ideal alternative to immunoassays or conventional high-performance liquid chromatography (HPLC). It also provides higher throughput than gas chromatography–mass spectrometry (GC–MS). LC–MS/MS also offers higher flexibility than immunoassays because LC–MS/MS assays are typically developed in-house. In addition, abundant information can be obtained from a single LC–MS/MS run which can produce a large amount of quantitative or qualitative data. In this review, typical LC–MS/MS clinical applications are presented, personal experiences are shared, and strengths and weakness are discussed. It is foreseeable that LC–MS/MS will become a key instrument in routine clinical laboratories.

Analytical Challenges: Determination of Tetrodotoxin in Human Urine and Plasma by LC-MS/MS

Tetrodotoxin (TTX) is a powerful sodium channel blocker found in puffer fish and some marine animals. Cases of TTX poisoning most often result from puffer fish ingestion. Diagnosis is mainly from patient’s signs and symptoms or the detection of TTX in the leftover food. If leftover food is unavailable, the determination of TTX in the patient’s urine and/or plasma is essential to confirm the diagnosis. Although various methods for the determination of TTX have been published, most of them are for food tissue samples. Dealing with human urine and blood samples is much more challenging. Unlike in food, the amount of toxin in the urine and blood of a patient is generally extremely low; therefore a very sensitive method is required to detect it. In this regard, mass spectrometry (MS) methods are the best choice. Since TTX is a very polar compound, there will be lack of retention on conventional reverse-phase columns; use of ion pair reagent or hydrophilic interaction liquid chromatography (HILIC) can help solve this problem. The problem of ion suppression is another challenge in analyzing polar compound in biological samples. This review will discuss different MS methods and their pros and cons.

Development and validation of a high-throughput double solid phase extraction–liquid chromatography–tandem mass spectrometry method for the determination of tetrodotoxin in human urine and plasma

A sensitive analytical method for the determination of tetrodotoxin (TTX) in urine and plasma matrices was developed using double solid phase extraction (C18 and hydrophilic interaction liquid chromatography) and subsequent analysis by HPLC coupled with tandem mass spectrometry. The double SPE sample cleanup efficiently reduced matrix and ion suppression effects. Together with the use of ion pair reagent in the mobile phase, isocratic elution became possible which enabled a shorter analysis time of 5.5 min per sample. The assay results were linear up to 500 ng mL(-1) for urine and 20 ng mL(-1) for plasma. The limit of detection and limit of quantification were 0.13 ng mL(-1) and 2.5 ng mL(-1), respectively, for both biological matrices. Recoveries were in the range of 75-81%. To eliminate the effect of dehydration and variations in urinary output, urinary creatinine-adjustment was made. TTX was quantified in eight urine samples and seven plasma samples from eight patients suspected of having TTX poisoning. TTX was detected in all urine samples, with concentrations ranging from 17.6 to 460.5 ng mL(-1), but was not detected in any of the plasma samples. The creatinine-adjusted TTX concentration in urine (ranging from 7.4 to 41.1 ng μmol(-1) creatinine) correlated well with the degree of poisoning as observed from clinical symptoms.

Improved liquid chromatography-tandem mass spectrometry method in clinical utility for the diagnosis of Cushing's syndrome.

Determination of urinary free cortisol is one of the first lines in screening for the diagnosis of Cushing’s syndrome where its measurement is mostly done by immunoassay. Although easy to perform, immunoassays suffer from the problem of assay interferences and are unable to measure cortisone levels. To enhance such techniques for clinical diagnosis, an improved liquid chromatography–tandem mass spectrometric (LC–MS/MS) method was developed for the simultaneous determination of urinary free cortisol and cortisone. The leftover urine samples from immunoassay were collected and subjected to facile solid-phase extraction cleanup. In the analysis of 130 urine samples from patients, 65 (50%) were found to have elevated urinary free cortisol (UFC) by immunoassay; but only 13 (10.8%) were found to have elevated UFC by this improved LC–MS/MS method. Nine out of the 13 patients, which showed elevated UFC by LC–MS/MS, were surgically confirmed to have Cushing’s syndrome/disease. By setting a two times upper limit as a cut-off, the immunoassay gave a positive predictive value of 43.5%, whilst by using the improved method, a positive predictive value of 90% was obtained. Although several tests have been used extensively in first line screening for the diagnosis of Cushing’s syndrome, none has ever shown with full capability of distinguishing all cases of Cushing’s syndrome from normal and/or obese individuals. This method has shown superior analytical advantages over existing immunoassay type in terms of sensitivity, specificity and capability to diagnose Cushing’s syndrome. Comparison between existing spectrometric methods, the reported developed method shown here, provides a simpler sample preparation procedure and meets with the high throughput demand of clinical laboratories.

Degradation of tetracycline and sulfadiazine during continuous thermophilic composting of pig manure and sawdust

During composting, the thermophilic phase resulted in high degradation of antibiotics in the composting mass; thus temperature is considered as the major factor for degradation of antibiotics. Therefore, to achieve complete removal of antibiotics, the effect of continuous thermophilic composting on the degradation of antibiotics and their effect on antibiotic resistant bacteria in the pig manure were evaluated. Pig manure was mixed with sawdust, spiked with tetracycline (10 and 100 mg/kg) and sulfadiazine (2 and 20 mg/kg) on dry weight (DW) basis and composted at 55°C for six weeks. Based on the organic decomposition, the antibiotics did not affect the composting process significantly, but negatively influenced the bacterial population. Tetracycline clearly exhibited a negative but marginal influence on carbon decomposition at 100 mg/kg level. The bacterial population initially decreased steeply ∼2 logs and slowly increased thereafter. Sulfadiazine and tetracycline resistant bacterial populations were stable/marginally increased after an initial decrease of about 2 or 3–5 logs, respectively. Sulfadiazine was not detectable after three days; whereas, ∼8% of tetracycline was detected after 42 days of composting with a t1/2 of ∼11 days, irrespective of the initial concentration. The presence of tetracycline in the compost after 42 days of thermophilic composting indicates the involvement of a mesophilic microbial-mediated degradation; however, further studies are required to confirm the direct microbial involvement in the degradation of antibiotics.

Rapid speciation of methylated and ethylated mercury in urine using headspace solid phase microextraction coupled to LC-ICP-MS

Solid-phase microextraction coupled to liquid chromatography-inductively coupled plasma-mass spectrometry (SPME-LC-ICP-MS) is presented as a new approach for the extraction and speciation of methylmercury (MeHg) and ethylmercury (EtHg) in a complex urine matrix. Despite the fact that urine is one of the most easily accessible sources of biological specimens for clinical testing, reports of mercury speciation methods are limited. Without any tedious sample pretreatments or derivatization procedures, headspace SPME offers a clean and convenient one-step extraction followed by direct desorption into HPLC. In this work, the fibre was first exposed to the sample headspace for a 45 min extraction, followed by a 5 min desorption. Experimental parameters including extraction time and temperature, sodium chloride salt concentration, agitation rate and desorption time were optimized to achieve detection limits down to 0.06 μg L−1 for both organomercury species. Sample recoveries in spiked urine matrix ranged from 81.7–109.5%. The accuracy of the method was further validated using a certified reference material (TORT-2) and applied in the organomercury speciation of urine samples collected from patients with suspected mercury poisoning.