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We established the cutoff value for plasma folic acid, using plasma homocyst(e)ine as the functional marker. To do this, we investigated the relationship of the plasma folic acid of 103 apparently healthy adults with their fasting plasma homocyst(e)ine and with their plasma homocyst(e)ine 6 h after oral methionine challenge (100 mg/kg). We also studied the relationship of their plasma folic acid with the decline of fasting plasma homocyst(e)ine after 7 days of folic acid supplementation (5 mg/day). The three approaches suggested a cutoff value of 10 nmol/L. The chances of individuals to significantly (P

In order to assess the dietary risk resulting from the use of pesticides or veterinary drugs the nature of the chemical residues on food commodities needs to be determined. Elucidation of the nature of the chemical residue is carried out using radiolabelled studies where the radiolabelled xenobiotic is applied or dosed in a manner which reflects use conditions. Food commodities are exhaustively extracted to remove the individual components of the residue. Once extracted the identity and toxicological significance of the components. can be assessed and, where appropriate, analytical methods developed to quantitatively determine the amount of the components in food items. Depending on the characteristics of the components of the residue, the extraction regime may not remove all the chemical residue from the sample matrix. These residues are frequently characterised as being "bound", however the amount and nature of this residue will be highly dependant on the extraction regime used. To provide guidance and standardisation a definition of the term "bound residues" is recommended. This definition builds on a previous IUPAC definition but takes account of the current availability of enzyme systems which effectively solubilise the entire matrix rather than extracting the residue. It is also recommended that where the extraction falls short of the full definition then the residues should be termed as "unextractable" and the conditions of the extraction should also be defined. Where residues are bound the assessment of the dietary risk cannot be directly assessed thus raising issues relating to the significance of the bound residue. The overall toxicological significance of a bound residue will depend primarily on its bioavailability and the level of exposure. In order to determine the bioavailability, study design is crucial in order to perform a critical safety assessment.

A reversed-phase high-performance liquid chromatography (RP-HPLC) method has been developed to measure the stability of four Irganox-type polymer antioxidants (Irganox 245, Irganox 1035, Irganox 1098 and Irganox 3114) in an olive oil food simulant and isooctane, which has been proposed as an alternative fatty food simulant. The tests of stability in olive oil were carried out under three different conditions, i.e. 40 degrees C for 10 days, 100 degrees C for 1 h and 175 degrees C for 1 h. The exposure conditions for isooctane were 60 degrees C for 3 h. Results showed that for all additives tested no instability phenomena in olive oil or isooctane simulants were observed under the exposure conditions applied. The analytical methodology developed could eventually be used for stability testing and migration studies of other similarly structured antioxidants in fatty food simulants. Zeitschrift Fur Lebensmittel-Untersuchung Und-Forschung a-Food Research and Technology

Whilst considerable research has been conducted on the stability of plastics additives during processing, there is, on the other hand, a lack of studies on the stability of the same additives in food simulants in the published literature. lit the work presented a systematic study has been undertaken on the stability of five selected commercial plastics additives (Irganox 3114, Irganox 1035, Irganox 245, Irganox 1098 and Irgafos P-EPQ) in the three EU-official aqueous food simulants (distilled water, 3% aq. acetic acid and 15% aq. ethanol) and in an alternative fat simulant (95% aq. ethanol) tinder two different time/temperature exposure conditions (10 days at 40 degrees C and I h at 100 degrees C). To enable quantitative analysis of the additives for this investigation, a simple and rapid HPLC method with UV detection was developed. The results obtained showed that the four Irganox-type additives under investigation were practically stable under the applied exposure conditions in all of the employed food simulants. Only Irganox 3114 exhibited a relatively low degree of instability in the ethanolic food simulants. Concerning Irgafos P-EPQ, a mixture of at least seven constituents, a remarkable degradation in ethanol and acetic acid solutions was observed. The developed HPLC method is also considered to be applicable for stability testing of other Irganox-type additives both in aqueous and fatty food simulants. Food Addit Contam

Objective: Semicarbazide-sensitive amine oxidase (SSAO) is present in various mammalian tissues, especially in vascular smooth muscle cells, but also in plasma. The enzyme has been suggested to play a role in vascular endothelial damage through conversion of amines into cytotoxic aldehydes, ammonia and hydrogen peroxide. Endothelial dysfunction is present in diabetes mellitus (DM) and congestive heart failure (CHF). Elevated plasma SSAO activities have been reported in patients with DM, bur no data on patients with CHF are as yet available. Methods and Results: Plasma SSAO was measured in 271 patients with CHF and compared to values in 77 controls. SSAO was found to be elevated in patients with CHF compared to controls (589 +/- 252 vs. 355 +/- 114 mU/l; P

An indirect analytical screening method for Sudan dyes by gas chromatography mass spectrometry was developed as an alternative method to novel liquid chromatography mass spectrometry. Red paprika paste samples were extracted with acetone and centrifuged at room temperature. The supernatant was removed from the solution and buffered. The reduction step is performed chemically with sodium dithionite and corresponding dyes as amine products were identified by GC-MS. The method was validated for analytical properties using spiked paste samples. Several parameters which may influence the reduction procedure, such as temperature and the amount of reducing agent, were optimized using central composite design methodologies for Sudan III in order to increase its lower apparent recovery as compared with the others. Optimum conditions for Sudan III were found to be 45 degrees C and 1.3 M by using a central composite design. The apparent recovery values were 77.6%, 69.4%, 51.3%, 42.5% with relative standard deviations of 31.8%, 20.4%, 20.3%, 31.2% for Sudan I, II, III, IV dyes respectively. This method was used successfully for screening of Sudan dyes in paprika paste products by GC-MS.

The role of the parasympathetic nervous system in rat pineal indole metabolism was investigated by transpineal in vivo microdialysis. On-line coupling to a high performance liquid chromatography system with fluorescence detection (HPLC-FD) allowed simultaneous analysis of three major indolic compounds from the pineal, i.e. serotonin, N-acetylserotonin and melatonin. Infusion of the muscarinic receptor agonists, carbachol and oxotremorine, during the dark period resulted in a marked decrease of melatonin release. This effect was suggested to be mediated by a decrease in N-acetyltransferase activity, since a similar decrease was seen in N-acetylserotonin release, while serotonin levels increased simultaneously. Nicotine did show a very slight effect on the three indoles under these circumstances. Neostigmine failed to influence pineal indole metabolism, indicating that the endogenous tonus of acetylcholine release is either absent or extremely low in the middle of the dark period. The involvement of sympathetic innervation in the muscarinic effects was investigated by measurement of noradrenaline release from the pineal by sensitive off-line HPLC-FD analysis of noradrenaline in the dialysates. Carbachol markedly decreased the noradrenaline input during the infusion. Noradrenaline release returned to baseline values immediately after infusion with carbachol. These data suggest that the in vivo inhibitory effect of muscarinic receptor agonists on pineal melatonin production is mediated by presynaptic muscarinic receptors, located on the sympathetic nerve endings. This prejunctional inhibition of noradrenaline release causes a reduced induction of N-acetyltransferase activity, resulting in decreased melatonin release.

We have developed a simple picomole (low nanogram) scale HPLC scheme which can separate all eight isomers of sphingosine and dihydrosphingosine thus leading to the identification of their relative and absolute configurations. The amino group of the sample is derivatized to its fluorescent N-naphthimide which is analyzed by normal and chiral phase HPLC, coupled with fluorescence peak detection. If necessary, the results of this HPLC method can be further corroborated by measurements of circular dichroic (CD) spectra of the N-naphthimido-derivatives and/or N,O-chromophoric derivatives. Copyright (C) 1996 Elsevier Science Ltd

Myocardial and pulmonary beta-adrenoceptors can be imaged with 2-(S)-(-)-(9H-carbazol-4-yl-oxy)-3-[1-(fluoromethyl)ethyl]amino-2-propanol (S-1'-[F-18]fluorocarazolol, I). Quantification of unmodified fluorocarazolol in plasma is necessary for analysis of PET images in terms of receptor densities. We have determined I and its radioactive metabolites in rat, sheep and human plasma, using (1) solid-phase extraction (C-18) followed by reversed-phase HPLC and (2) direct injection of untreated plasma samples on an internal-surface reversed-phase (ISRP) column. The two methods were in good agreement. Unmodified I decreased from over 99% initially to less than 5%, 5-10% and 20% at 60 min post-injection in rats, sheep and human volunteers, respectively. Protein binding in sheep and human plasma was determined by ultrafiltration. The fraction of total plasma radioactivity bound to protein and the fraction representing unmodified radioligand were linearly correlated, suggesting that fluorocarazolol was more than 70% protein-bound, whereas its metabolites showed negligible protein binding. Direct injection of plasma on an ISRP column seems a convenient method for quantification of lipophilic radioligands such as fluorocarazolol.

The sympathetic innervation of the rat pineal gland was investigated, measuring the norepinephrine (NE) release by on-line in vivo microdialysis, NE was assayed using an HPLC method with precolumn derivatization and fluorescence detection. Its high sensitivity and reliability made it very suitable to monitor the low levels of NE in the dialysates (12.5 fmol during nighttime, 3 fmol during daytime). To increase NE levels, the monoamine reuptake inhibitor cocaine was added to Ringer's solution at concentrations of 10(-6) and 10(-5) M. This resulted in increases of neurotransmitter output of 167 and 219%, respectively, but did not change the qualitative and/or quantitative outcome of other experiments, Perfusion with 10(-6) M tetrodotoxin for 1 h resulted in a decrease of the NE release by >80%, whereas perfusion with the alpha(2)-receptor antagonist yohimbine caused a twofold increase. These results indicate that the NE release in the rat pineal was of neuronal origin and regulated by a negative feedback mechanism involving inhibitory presynaptic alpha(2)-receptors. Long-term (i.e., 16 h) measurements are described, showing the circadian properties of NE release. A pronounced rhythm is reported, showing extremely sharp transitions between low daytime and high nighttime values. Increases and decreases are reported to occur within the duration of collecting one sample (20 min). For comparison, the rhythm of melatonin release was also recorded. The on and off switches of the sympathetic input correlated well with the circadian rhythm of melatonin release and can thus be considered as the primary clock signal, inducing the nightly production of melatonin.

The direct enantiomeric separation of 32 racemic 2-amidotetralins on the commercially available tris-(3,5-dimethylphenylcarbamate) derivative of cellulose, coated on silica gel (Chiralcel OD), is presented. To date, the selection of a column for the chiral separation of a racemic mixture is done empirically. Studying the impact of small changes in the chemical structure of a series of amidotetralins on the separation behavior may help to give an insight in the chiral recognition mechanism. The amidotetralins differed structurally in three of their substituents, which were never directly located on the chiral carbon atom. The enantiomers of 24 out of 32 amidotetralins could be resolved with a resolution >1.5. Hydrogen bonding and pi-pi interactions are supposed to be the major analyte-chiral stationary phase (CSP) interactions. However, the spatial arrangement of the enantiomers may play an important role too. Increasing the bulkiness of the acyl substituent led to an increase in the resolution (R(s)), whereas a more bulky substituent on the aromatic ring resulted in a very low resolution. The introduction of a chlorine atom into the acyl substituent additionally increased the resolving power. (C) 1997 Wiley-Liss, Inc.

Listeria monocytogenes takes up di- and tripeptides via a proton motive force-dependent carrier protein. This peptide transport system resembles the recently cloned and sequenced secondary di- and tripeptide transport system of Lactococcus lactis (A. Hagting, E. R. S. Kunji, K. J. Leenhouts, B. Poolman, and W. N. Konings, J. Biol. Chem. 269:11391-11399, 1994). The peptide permease of L. monocytogenes has a broad substrate specificity and allows transport of the nonpeptide substrate 5-aminolevulinic acid, the toxic di- and tripeptide analogs, alanyl-beta-chloroalanine and alanyl-alanyl-beta-chloroalanine, and various di- and tripeptides. No extracellular peptide hydrolysis was detected, indicating that peptides are hydrolyzed after being transported into the cell. Indeed, peptidase activities in response to various synthetic substrates were detected in cell extracts obtained from L. monocytogenes cells grown in brain heart infusion broth or defined medium. The di- and tripeptide permease can supply L. monocytogenes with essential amino acids for growth and might contribute to growth of this pathogen in various foods where peptides are supplied by proteolytic activity of other microorganisms present in these foods. Possible roles of this di- and tripeptide transport system in the osmoregulation and virulence of L. monocytogenes are discussed.

International audience; Analysis of the interaction between the host immune system and the intracellular parasite Mycobacterium leprae has identified a 35 kDa protein as a dominant antigen, The native 35 kDa protein was purified from the membrane fraction of M. leprae and termed MMPI (major membrane protein I). As the purified protein was not amenable to N-terminal sequencing, partial proteolysis was used to establish the sequences of 21 peptides, A fragment of the 35 kDa proteinen-coding gene was amplified by the polymerase chain reaction from M. leprae chromosomal DNA with oligonucleotide primers derived from internal peptide sequences and the whole gene was subsequently isolated from a M. leprae cosmid library. The nucleotide sequence of the gene revealed an open reading frame of 307 amino acids containing most of the peptide sequences derived from the native 35 kDa protein, The calculated subunit mass was 33.7 kDa, but the native protein exists as a multimer of 950 kDa, Database searches revealed no identity between the 35 kDa antigen and known protein sequences. The gene was expressed in Mycobacterium smegmatis under the control of its own promoter or at a higher level using an 'up-regulated' promoter derived from Mycobacterium fortuitum. The gene product reacted with monoclonal antibodies raised to the native protein, Using the bacterial alkaline phosphatase reporter system, we observed that the 35 kDa protein was unable to be exported across the membrane of recombinant M. smegmatis. The 35 kDa protein-encoding gene is absent from members of the Mycobacterium tuberculosis complex, but homologous sequences were detected in Mycobacterium avium, Mycobacterium haemophilum and M. smegmatis, The avaibility of the recombinant 35 kDa protein will permit dissection of both antibody- and T-cell-mediated immune responses in leprosy patients.

In ligand binding assays, the separation of bound and free fraction of the labeled ligand is very important. Dialysis is generally overlooked as separation technique since it requires large volumes and long analysis times. The availability of the ASTED-system (Automated Sequential Trace Enrichment of Dialysates) might open ways for a complete automation of immune assays including the separation step. A well-documented radio-immune assay for 3-keto-desogestrel (Org3236) was used to test the potentials of this system. The tritiated analog of Org3236 not only served as label in the immune assay but was also used to trace this compound in the entire procedure. The dialysis efficiency increased with the dialysis time and with the flush rate of the recipient solvent (tris-HCl or phosphate buffer). Addition of methanol to recipient solvent had spectacular effects on the recovery. With tris-HCl buffer, 0.18 mL/min and 1.0 mL recipient solvent 2.5% of the label was collected. Addition of 50% methanol resulted in a 5-fold increase to 12%. Replacement of buffer by 100% methanol resulted in another 5% increase in dialysis efficiency which was accompanied by a reduction in the antibody binding in the donor compartment due to denaturation of the antibody. The commercial availability of other types of membranes is essential to find optimal conditions for each analyte. A serious problem is the carry-over effect between subsequent samples. Roughly 0.25% of label was collected in the next run which may have a substantial impact on the accuracy and precision of the assay. Renewal of the dialysis membrane might exclude this carry-over effect but is not a serious option with the available instrumentation. Automated dialysis systems can be very valuable for ligand binding assays as soon as membranes become available for the analytes of interest which provide high recoveries (>40%) in 1 mL recipient solvent. Moreover their carry-over effect should be negligible or eliminated by more efficient rinsing procedures of the entire dialysis system. Temperature control is favourable for the immune assays as well as for the dialysis process in that the kinetics are temperature dependent.

The nucleotide sequence of a chromosomal DNA fragment of Lactococcus lactis subsp. lactis SSL135, previously implicated in peptide utilization, has been determined. The genes oppDFBCA, encoding the oligopeptide transport system (Opp), and that encoding the endopeptidase PepO were located on this 8.9-kb DNA fragment. The oppDFBCA and pepO genes are probably organized in an operon. Analysis of the deduced amino acid sequences of the genes indicated that the oligopeptide transport system consists of two ATP-binding proteins OppD and OppF, two integral membrane proteins OppB and OppC, and a substrate-binding protein OppA. On the basis of the homology of OppF and OppD of L. lactis with other ABC (ATP-binding cassette) transporter proteins, the L. lactis Opp system can be classified as a member of this group. Two integration mutants, one defective in OppA and the other defective in PepO, were constructed. Growth of these mutants in a chemically defined medium with oligopeptides showed that the transport system, but not the endopeptidase, is essential for the utilization of peptides longer than three residues. Uptake of the pentapeptide Leu-enkephalin in glycolyzing lactococcal cells was followed by rapid hydrolysis of the peptide intracellularly. Importantly, extracellular hydrolysis of Leu-enkephalin is not observed. The OppA-deficient mutant was unable to transport Leu-enkephalin. Growth experiments with pasteurized milk revealed that transport of oligopeptides forms an essential part of the proteolytic system in lactococci.

The present study describes the development of a new technique to measure melatonin contents in the pineal gland of freely moving rats, by means of on-line microdialysis. The transcerebral cannula was modified, and a sensitive assay of melatonin, using HPLC with fluorimetric detection, was set up. With this system it is possible to monitor the melatonin levels on-line in the pineal gland during day- and nighttime. The nightly increase in melatonin release was recorded. Tetrodotoxin had an inhibitory effect on nighttime levels, whereas even high concentrations did not alter the daytime level. From this we conclude that neuronal activity is necessary to synthesize melatonin and that during daytime no net neuronal activity is present. Melatonin levels could be greatly enhanced by systemic administration of the beta-agonist isoprenaline (ISO). Also, local infusion of ISO or 8-bromoadenosine 3',5'-cyclic monophosphate, an analogue of the second messenger cyclic AMP, resulted in increased melatonin levels, demonstrating the presence of beta-adrenergic receptors, coupled to a cyclic AMP-based second messenger system, on the pineal gland. Injection of phenylephrine had no effect on daytime levels. Only when administered during ISO-induced stimulation of melatonin release did it enhance this stimulated release. This proved the regulatory role of alpha1-receptors on pinealocytes. The method presented is of special interest for investigating the innervation of the pineal gland and the biochemical processes that regulate the biosynthesis of melatonin. Also, for studies on the diurnal rhythms of melatonin release and factors that influence these rhythms in freely moving animals, this model will be of great value.

We developed a method for simultaneous quantification of the urinary 3-O-methylated catecholamine metabolites 3-methoxytyramine, normetanephrine and metanephrine by stable isotope-dilution ammonia chemical ionization mass fragmentography. Prepurification of lyophilized samples was done by simultaneous deconjugation and pentafluoropropionylation, followed by extraction and rederivatization. Compared with our previously described method, based on acid hydrolysis, alkaline extraction, derivatization and electron-impact mass fragmentography, the present method was found to be less laborious, more sensitive and presumably more accurate. New urinary excretion values were established for apparently healthy adults. The present prepurification method may prove applicable for profiling of a variety of naturally occurring mono-, di- and polyamines in biological samples.

The use of Bond Elut Certify columns for the isolation of morphine from whole blood was evaluated. In order to monitor possible losses and the elution profile of morphine, a small amount of the tritiated analogue was added to the samples. Four sample pretreatment methods, three protein precipitation methods and one sonication/dilution method, were tested. The latter one gave the best results. The blood sample was applied onto the column at pH 3.3 after sonication and dilution with 0.1 M phosphate buffer (pH 3.3). The retention of morphine was affected by the pH of the samples, and the loss of morphine during sample application was minimized at low pH (3.3). The interferences were removed by washing the column with the phosphate buffer, 0.01 M acetic acid (pH 3.3), and methanol, sequentially. Ammoniated methanol, 2 mL at 2%, was selected to clute morphine. As a result, more than 80% of H-3-morphine was recovered for concentrations of morphine ranging from 5 to 4000 ng/mL.

The retention indices (RI) of 47 selected acidic, neutral and basic drugs were determined on 7 reversed-phase (octyl- and octadecylsilica) columns in two laboratories in the 1-nitroalkane scale, using either 1-nitroalkane homologues or selected drugs, whose RI values were previously determined on the reference column. Obtained values were compared with the library values, determined previously on the reference column. Retention indices, calculated with drugs as RI markers, showed distinctly lower deviations from the library values and lower inter-column variability: The mean standard deviation of RI for all drugs analyzed on all columns in the 1-nitroalkane scale was 44.3 RI units, against 10.3 units when selected drugs were used as RI markers. The deviations from the listed values, calculated for each column separately with drugs as markers, were in 95% of cases smaller than 20 RI units, and in 80 % smaller than 10 units. The largest differences between the experimental and listed values were observed for column that differed in type of silica support, type of stationary phase (C8 versus C18) or column length in comparison to the reference column. The kind of silica support contributed more to the variability than the type of stationary phase. The results indicate that it is possible to use and exchange HPLC data based on a retention index library.

Direct enantiomeric separations of 17 chiral amidotetralins by means of high performance liquid chromatography were performed on stationary phases composed of tris(3,5-dimethylphenylcarbamate) derivatives of cellulose and amylose, coated on silica gel. The enantiomers of 15 out of 17 amidotetralins were resolved with a resolution of more than 1.5 by at least one of the chiral stationary phases. The stationary phases showed complementary results with regard to the separation of the amidotetralins, that is, pairs that did not separate on the cellulose-type column were well separated on the amylose-type column, and vice versa. There was no significant correlation between the chromatographic properties of the chiral stationary phases. (C) 1993 Wiley-Liss, Inc.

Three-way methods are multivariate data analysis tools that compress and visualize simultaneous variation of combinations of variables and objects. Unfolding, Tucker and PARAFAC models are examples of such methods. The interest in such tools is increasing in chemometrics. The history of three-way methods is sketched and the basic theory is presented, together with chemical applications. Problems in the three-way analyses area are summarized. Some prospects for future use of three-way methods are given.

We investigated the influence of different concentrations of Fe3+, phosphoric acid, butylated hydroxytoluene and glutathione on the production of the malondialdehyde-1,3-diethyl-2-thiobarbituric acid adduct in plasma lipid extracts. Following organic solvent extraction the stable product was analyzed by spectrophotometry (537 nm), fluorometry (547 nm) and high-performance liquid chromatography with fluorometric detection. Using optimized reaction conditions there was good agreement between the three methods, with slightly higher values for the spectrophotometric method. Plasma total lipid malondialdehyde reference values for 24 healthy adults amounted to 1.30 +/- 0.23-mu-mol/l (spectrophotometric method) and 1.11 +/- 0.31-mu-mol/l (fluorometric method). Plasma lipid malondialdehyde concentrations correlated significantly with plasma triglycerides (r = 0.527), total cholesterol (r = 0.612) and total fatty acids (r = 0.810) and with the total number of double bonds present in plasma fatty acids with three or more double bonds (r = 0.923).

In this report, we describe an HPLC with electrochemical detection assay for the simultaneous measurement of levels of morphine, serotonin, 5-hydroxyindole-3-acetic acid, and homovanillic acid in dialysates of various brain areas and CSF in the awake rat. Morphine could be detected in the dialysates after a single intraperitoneal injection, with doses as low as 1.0 mg/kg. The time course of extracellular morphine content in the lateral hypothalamus, striatum, cerebellum, periaqueductal gray, and dorsal horn of the spinal cord and in CSF, from the ventricles and cisterna magna, was similar. We detected morphine in the first 15-min sample, and levels peaked 45-60 min after injection. Maximal dialysate levels, however, varied with the type of dialysis probe used and the area sampled. The most efficient in vivo recovery was in CSF dialysates from the cisterna magna, presumably because of minimal tissue interference with the dialysis probe. For this reason, the cisterna is an ideal region for sampling CSF. Morphine had no significant effect on the extracellular concentrations of serotonin in any of the areas studied and did not modify or only slightly increased levels of tissue metabolites; however, morphine markedly increased the CSF levels of 5-hydroxyindole-3-acetic acid and homovanillic acid. Because microdialysis in freely moving animals permits assessment of the behavioral effects of morphine while continuously monitoring the drug levels in discrete brain regions, this approach will greatly facilitate future studies of the neurochemical basis of morphine's effects in the brain.

A rapid TLC method is presented to distinguish poor oxidative drug metabolizers from extensive oxidative drug metabolizers. Dextromethorphan (1) is used as test probe because it is safe, well characterized, generally available and easy to measure. The method is based on the extraction of 1 and its major oxidative metabolite dextrorphan (2) from urine, followed by separation on a TLC plate and visualized by a combined Marquis/Mandelin reaction. The intensities of the spots are then compared with a series of standard mixtures containing 1 and 2 in a ratio of 0.3 at different concentrations. This value represents the antimode that separates poor from extensive metabolizers, and an individual is identified as a poor metabolizer if the intensity ratio of the two spots from his urine sample is higher than 0.3. The proposed TLC method was cross checked with an HPLC method and found to correctly identify 9 poor metabolizers out of a population of 71 volunteers.

It has been suggested that qualitative changes in bile phosphatidylcholine (lecithin) play a role in the pathogenesis of cholesterol gallstones. We investigated the possible relationship between the molecular composition and hydrophobicity of biliary lecithins and bile cholesterol saturation, nucleation time and the mode of cholesterol transport in human gallbladder bile. Nineteen patients (12 with and seven without gallstones) undergoing abdominal surgery were studied. Bile cholesterol saturation ranged from 77% to 186% (median: 123%) and nucleation time from 1 to 24 days (median: 3 days). Biliary lipid carriers (vesicles and mixed micelles) were separated using Superose-6 gel chromatography and their lipid content was quantitated. Biliary lecithin composition was analyzed by HPLC. Fourteen individual molecular species were detected in the bile: none were related to cholesterol saturation or nucleation time. An arbitrary cumulative index of lecithin hydrophobicity was computed for each bile sample, based on the HPLC capacity factor of the individual species and their percent mole fraction: this index ranged from 47.0 and 58.5 (median: 49) and was unrelated to cholesterol saturation and nucleation time. The biliary concentration of sn-1 palmitoyl:sn-2 arachidonoyl lecithin was significantly correlated (p less than 0.01) with the fraction of cholesterol carried by mixed micelles. This finding suggests that arachidonoyl lecithin may play a modulatory role in the partitioning of cholesterol among biliary carriers. We conclude that major abnormalities in the composition of biliary lecithins are unlikely to play a causative role in the pathogenesis of cholesterol gallstone, although the role of arachidonoyl species requires further investigation.

A gas chromatographic (GC) fingerprint method, based on the presence or absence of six congeners, was developed for illicit cocaine samples. The fingerprint utilizes the relative abundances of these congeners towards each other, disregarding cocaine as the main constituent, and can be expressed numerically or graphically in the form of pictograms for rapid visual comparison. The method can be applied directly to a solution of the sample in chloroform, without previous workup procedures. More than 70 unrelated samples were analyzed and a great variation was observed in the parameter composition. On the other hand, a remarkable similarity could be seen between related samples. The GC fingerprint method may be considered an important contribution for sample comparison, as is exemplified by a subdivision of the analyzed samples in different categories, based on the number and types of congeners found.

This study investigated the regulation of serotonin (5-HT) and its major metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the dorsal spinal cord of awake, freely moving rats, using microdialysis coupled to HPLC with electrochemical detection and tested the hypothesis that opioids exert their analgesic effect in part through the increased release of 5-HT in the dorsal horn. A dialysis tube was placed transversely at the L4 segment of the dorsal spinal cord and the basal concentration of 5-HT in the dialysate was characterized by infusion of a variety of substances through the dialysis probe: tetrodotoxin (TTX), KCl, imipramine, fluoxetine and amphetamine (AMPH). To evaluate the contribution of opioids, we also studied the effects of either systemic or intracerebroventricular (i.c.v.) injection of morphine or DAMGO. Extracellular concentrations of 5-HT and 5-HIAA were partially and reversibly reduced by TTX. In the presence of KCl, imipramine, fluoxetine or AMPH, 5-HT levels significantly increased. Under these conditions, extracellular 5-HIAA levels usually decreased. By contrast, the effects of opioids on 5-HT concentrations were highly variable. Low doses of morphine administered systemically increased 5-HT concentrations in only 3 of 6 rats. This was paralleled by a decrease in 5-HIAA. Higher doses of morphine, alone or in the presence of fluoxetine, did not change 5-HT concentrations. Intracerebroventricular injection of morphine or DAMGO increased the extracellular concentrations of 5-HT in only about one third of the animals. After intracerebroventricular opioid injection, extracellular concentrations of 5-HIAA either decreased by about 20% or did not change. These results demonstrate that the extracellular concentration of 5-HT in the dorsal spinal cord is partly derived from neuronal activity and that 5-HIAA levels are regulated differently from 5-HT. The fact that morphine and DAMGO evoked the release of 5-HT in some animals is consistent with the hypothesis that opioids exert their analgesic effects, at least in part, through serotonergic-mediated descending controls. However, since behavioral analgesia was produced in several animals in which an increase in 5-HT was not observed, our results suggest that evoked release of 5-HT in the spinal cord is not necessary for opioids to exert their analgesic action.

We present a systematic study of the reverse-phase high performance liquid chromatography (HPLC) of complexes containing an Fe 2 (CO) 6 unit bonded to a variety of organic chains, namely the butatriene moiety R 2 CCCCR 2 , the butadienyl fragment, RCCRRCCR (the ferroles), and the ring-opened cyclopentadienone diradical, RCCRC(O)RCCR (the flyover bridges). Since the Fe 2 (CO) 6 unit shows an almost identical conformation in all the complexes, this enables us to evaluate the effect of the geometrical arrangement of the organic chains and of their substituents on chromatographic behaviour. Reverse-phase HPLC allows the separation of the positional isomers of ferroles and flyover bridges (R  Me or Ph), but not the cis / trans isomers of butatrienes (R  H or Me).

A single-column solid-phase extraction procedure was developed for the screening of acidic, neutral, and basic drugs from plasma. The recoveries of all 25 tested drugs exceeded 82%. After the plasma had been diluted with phosphate buffer (pH 6.0), the drugs were extracted using a single Bond Elut Certify column. The acidic and most of the neutral drugs were eluted by acetone/chloroform (1:1) and the basic drugs were eluted by 2% ammoniated ethyl acetate. Some neutral drugs appeared in both fractions. The two fractions were collected separately and evaporated until approximately 100 muL of solvent remained in the tube. Both fractions were analyzed separately on a gas chromatograph equipped with a wide-bore capillary column and a flame ionization detector. The procedure could also be used for urine samples.

The enantiomers of a series of dopamine (DA) agonists, monohydroxy-2-aminotetralin derivatives, were investigated using brain microdialysis. We used a 5-OH-substituted derivative, N-0437 (2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetralin), and three 7-OH-substituted derivatives, N-0438 (2-(N-propyl-N-2-thienylethylamino)-7-hydroxytetralin), 7-OH-DPAT (7-hydroxy-2-(N,N-di-n-propylamino)tetralin) and PHNO (4-propyl-9-hydroxynaphthoxazine; position 9 of the naphtoxazines corresponds to position 7 of the aminotetralins). We studied the activity of the enantiomers at autoreceptors regulating the release of DA following their local infusion into the striatum of the rat. We were particularly interested in the activity of R(+)-N-0437, S(-)-N-0438, S(-)-7-OH-DPAT and S(-)-PHNO, which are enantiomers that have been classified as less potent or inactive in previous studies. S(-)-N-0437, R(+)-N-0438, R(+)-7-OH-DPAT and R(+)-PHNO decreased DA release by 45-60%. Thus, these enantiomers are potent agonists at autoreceptors regulating the release of DA. The R(+) enantiomer of the 5-OH-substituted derivative N-0437 possessed antagonistic activity at autoreceptors controlling DA release, increasing DA release by 100%. This finding is consistent with reports showing that one enantiomer of other 5-OH DA agonists displays agonistic activity, while the other has antagonistic properties at DA autoreceptors. The less potent enantiomers of the 7-OH-substituted derivatives S(-)-N-0438, S(-)-7-OH-DPAT and S(-)-PHNO, however, all showed weak agonistic activity at DA autoreceptors. They decreased DA release by maximally 40%. Thus, considering these results and the findings from the literature, the position of the hydroxyl group may determine the action profile of the enantiomers. Apparently, not only the R(+) configuration but also the S(-) configuration of the 7-OH-substituted derivatives can bind and activate the autoreceptor regulating DA release, whereas only the S(-) configuration of 5-OH-substituted derivatives is capable of doing so.

[1,2,3,4-C-13]cortisol was i.v. administered to two sisters aged 11 yr (patient I) and 3 yr (patient II) who suffer from 17-alpha-hydroxylase deficiency. This is the first time that the cortisol production rate (CPR) in patients with 17-alpha-hydroxylase deficiency has been measured with a stable labelled tracer using the urinary method. The urine was collected for 3 days. High-performance liquid chromatography (HPLC) of approximately 100 ml urine extracts was carried out to isolate the small amount of cortisol metabolites excreted. The cortisol metabolites were oxidized to 11-oxo-aetiocholanolone. The isotope dilution in the methyl oxime tert-butyldimethylsilyl ether derivatives was measured by selected ion monitoring gas chromatography/mass spectrometry (GC/MS). The CPR calculated from tetrahydrocortisone (THE) and the cortolones was 765 and 536 nmol/day, respectively in patient I. The CPR in patient II was only calculated from THE and was 62 nmol/day. If radioactive labelled cortisol had been used, much larger quantities of urine would have been needed for isolation of sufficient mass of metabolites, even then purification may have been difficult. Steroid profiling of 1 ml urine samples by GC and identification by GC/MS revealed high concentrations of pregnenolone, progesterone, 11-beta-hydroxy progesterone and corticosterone metabolites. Tetrahydrocorticosterone and 5-alpha-tetrahydrocorticosterone were found in urine at elevated excretions of 2.5 and 5.7, 0.9 and 2.0-mu-mol/24 h, in patients I and II respectively. No cortisol metabolites were detected by routine GC or GC/MS as the low amounts excreted co-eluted with the relatively abundant corticosterone metabolites.

The potential of on-column preconcentration in capillary electrochromatography (CEC) to improve the detection limit was investigated Two test mixtures containing a pharmaceutically relevant steroid (Desogestrel or Tibolon) together with several structurally related compounds were used for evaluation. For both test mixtures, the mobile phase composition was optimised resulting in a baseline separation of all components and plate numbers of vp to 1.2 10(5) plates m(-1) within 15 min. An equation was derived which describes the obtainable gain in injection time as function of the analyte retention factor in the mobile phase and in the injection solvent The proposed model accounts for the focussing of the analytes due to both the retention during injection and the step-gradient during elution. For the experimental study the least hydrophobic component of the Desogestrel mixture was used. When the mobile phase was used as injection solvent a considerable decrease in plate number was observed when the injection time exceeded 5 s. By dissolving the analyte in a less-eluting solvent, injection times could be increased vp to 60 s without causing significant extra band broadening. Two mixtures containing a relatively high amount of Desogestrel or Tibolon, and the related components at the 0.1% level were analysed to study the potential of the system for impurity profiling. With the mobile phase as injection solvent, the low level components could hardly be detected. By applying large volume injection from a less-eluting injection solvent a gain in sensitivity of a factor of 7 - 9 was achieved.

The serotonergic system of the brain is complex, with an extensive innervation pattern covering all brain regions and endowed with at least 15 different receptors (each with their particular distribution patterns), specific reuptake mechanisms and synthetic processes. Many aspects of the functioning of the serotonergic system are still unclear, partially because of the difficulty of measuring physiological processes in the living brain. In this review we give an overview of the conventional methods of measuring serotonin synthesis and methods using positron emission tomography (PET) tracers, more specifically with respect to serotonergic function in affective disorders. Conventional methods are invasive and do not directly measure synthesis rates. Although they may give insight into turnover rates, a more direct measurement may be preferred. PET is a noninvasive technique which can trace metabolic processes, like serotonin synthesis. Tracers developed for this purpose are alpha-[(11)C]methyltryptophan ([(11)C]AMT) and 5-hydroxy-L-[beta-(11)C]tryptophan ([(11)C]5-HTP). Both tracers have advantages and disadvantages. [(11)C]AMT can enter the kynurenine pathway under inflammatory conditions (and thus provide a false signal), but this tracer has been used in many studies leading to novel insights regarding antidepressant action. [(11)C]5-HTP is difficult to produce, but trapping of this compound may better represent serotonin synthesis. AMT and 5-HTP kinetics are differently affected by tryptophan depletion and changes of mood. This may indicate that both tracers are associated with different enzymatic processes. In conclusion, PET with radiolabelled substrates for the serotonergic pathway is the only direct way to detect changes of serotonin synthesis in the living brain.

Plants are the most important resource of herbal medicines and the occurrence of secondary metabolites with many therapeutic activities A major portion of the general inhabitants in developing countries still make use of long-established folk medicine against ailments since Vedic time. The herbal drugs popularity increased and used widespread. The research is still lagging behind to get the efficacy of plant derived medicines on microorganisms. The eight different medicinal plants (Nyctanthes arbortristis, Vitex negundo, Phyllanthus amarus, Adhatoda vasica, Hamidesmus indicus, Asparagus racemosus, Terminalia arjuna and Terminalia chebula) were exploited in support of phytochemical test and to revise their antibacterial effectiveness on E. coil Dk1 and Staphyllococus aureus MRS901.The screening for antibacterial activity of plant part extract was done by agar well diffusion method. The inhibitory effect was analyzed by calculating Zone of Inhibition (ZOI) and minimum inhibitory concentration (MIC) values. The phytochemical screening of bioactive compounds for presence or absence was done. The result showed the maximum ZOI by Terminalia arjuna and Terminalia chebula (ranges 9-13mm) in both aqueous and methanolic extract than by other plant extracts. The phytochemical screening of bioactive compounds showed the presence of alkaloids, tannins, flavonoids, saponins, terpenoids, glycosides, cardiac glycosides and steroids in aqueous and methanolic extract both. It is suggested that test plants have some antibacterial activity and could be applied on other bacterial strains other than E. coli and S. aureus.

The vast majority of separations in liquid chromatography (LC) still use the typical packed particle bed format, most commonly with fully or superficially porous particles in particle sizes as low as 1.3 µm. As an alternative, monolithic columns have been the topic of many studies, but they are currently used only in some niche applications. Research into perfectly ordered microfabricated columns has shown tremendous possibility for these high performance columns for use in nano-LC, but their development is still ongoing. The possibilities that emerging three-dimensional (3D) printing technology offers make it theoretically possible to develop any imaginable structure with high precision, but the technology is currently limited. This article provides a critical review of all these technologies and demonstrates how further development of chromatographic columns will be of paramount importance in the future. ispartof: LCGC North America: the magazine of separation science vol:36 issue:6 pages:9-17 status: published

Cottonseed cake biomass, which is a residue of oil extraction, is potentially appropriate for use as animal feed, given the high mineral, fibre and protein content. The presence of free gossypol, however, a toxic pigment in the glands of the cotton plant, limits use of this biomass for monogastric livestock. A promising method to detoxify cottonseed cake relies on fermentation by fungi, which can eliminate up to 100% of gossypol. In order to quantify trace levels of free gossypol in different cotton materials, including cottonseed cake treated with macrofungi, a simple and rapid chromatographic detection method was developed and validated. Under optimized conditions, extraction was performed using 70% acetone. The extract was then analysed by Ultra High-Performance Liquid Chromatography (UHPLC), with gradient elution on a C18 reverse phase column KINETEX® (100 x 2.10 mm, 2.6 um). Methanol-0.1% TFA aqueous solution was employed as mobile phase and PDA detection conducted at 254 nm. The optimized method was validated by analysis of specificity, linearity and range, limit of detection, limit of quantification, precision and accuracy. Detection and quantification limits were observed at 0.2 and 0.5 ug/mL, respectively. With good reproducibility, with precision (RSD)

The effects of several enological practices on ochratoxin A (OTA) concentration in wines were evaluated. Analysis of OTA was performed by reverse-phase high-performance liquid chromatography (HPLC) after immunoaffinity cleanup. Higher toxin levels were found in red wines (0.017 to 0.050 mu g/L) than white wines (0.006 to 0.022 mu g/L). Intensive pressing of pomace, prolonged drying of grapes, and storage in partially empty tanks increased OTA contamination of wines. Poor winery sanitation practice was the primary source of OTA contamination, as wines from contaminated wineries had the highest OTA values (0.130 to 1.300 mu g/L). Filtration through a 10-pm membrane and heating on hot plates at 55 degrees C did not cause a significant decrease in the toxin level. An 80% decrease of OTA was observed after wine filtration through a 0.45-mu m membrane (p < 0.05). Eleven enological fining agents were tested to study their ability to remove OTA from wine. Enological decolorizing carbon removed (at 30 g/hL) up to 72% of OTA. The Freundlich isotherm was used to model OTA adsorption on carbon. Results showed that carbon had selective adsorption (K-F = 3.172 (mg/g)/(mg/L)(0.8474)), but it did not affect polyphenol content and color of red wine. Nevertheless, a decrease of key sensory odorants of wine, such as 3-methylbutyl acetate, ethyl hexanoate, ethyl octanoate, and geraniol, was observed.

Different automatic peak integration methods have been reviewed and compared for their ability to accurately determine the variance of the very narrow and very fast eluting peaks encountered when measuring the instrument band broadening of today's low dispersion liquid chromatography instruments. Using fully maximized injection concentrations to work at the highest possible signal-to-noise ratio's (SNR), the best results were obtained with the so-called variance profile analysis method. This is an extension (supplemented with a user-independent read-out algorithm) of a recently proposed method which calculates the peak variance value for any possible value of the peak end time, providing a curve containing all the possible variance values and theoretically levelling off to the (best possible estimate of the) true variance. Despite the use of maximal injection concentrations (leading to SNRs over 10,000), the peak variance errors were of the order of some 10–20%, mostly depending on the peak tail characteristics. The accuracy could however be significantly increased (to an error level below 0.5–2%) by averaging over 10–15 subsequent measurements, or by first adding the peak profiles of 10–15 subsequent runs and then analyzing this summed peak. There also appears to be an optimal detector intermediate frequency, with the higher frequencies suffering from their poorer signal-to-noise-ratio and with the smaller detector frequencies suffering from a limited number of data points. When the SNR drops below 1000, an accurate determination of the true variance of extra-column peaks of modern instruments no longer seems to be possible.

Effective medium theory (EMT) expressions for the B-term band broadening in monolithic silica columns are presented at the whole-column as well as at the mesoporous skeleton level. Given the bi-continuous nature of the monolithic medium, regular as well as inverse formulations of the EMT-expressions have been established. The established expressions were validated by applying them to a set of experimental effective diffusion (D-eff)-data obtained via peak parking on a number of 1st and 2nd generation monolithic silica columns, as well as to a set of numerical diffusion simulations in a simplified monolithic column representation (tetrahedral skeleton model) with different external porosities and internal diffusion coefficients. The numerically simulated diffusion data can be very closely represented over a very broad range of zone retention factors (up to k '' = 80) using the established EMT-expressions, especially when using the inverse variant. The expressions also allow representing the experimentally measured effective diffusion data very closely. The measured D-eff/D-mol-values were found to decrease significantly with increasing retention factor, in general going from about D-eff/D-mol = 0.55 to 0.65 at low k '' (k '' congruent to 1.5-3.8) to D-eff/D-mol = 0.25 at very high k '' (k '' congruent to 40-80). These values are significantly larger than observed in fully-porous and core-shell particles. The intra-skeleton diffusion coefficient (D-pz) was typically found to be of the order of D-pz/D-mol = 0.4, compared to D-pz/D-mol = 0.2-0.35 observed in most particle-based columns. These higher D-pz/D-mol values are the cause of the higher D-eff/D-mol values observed. In addition, it also appears that the higher internal diffusion is linked to the higher porosity of the mesoporous skeleton that has a relatively open structure with relatively wide pores. The observed (weak) relation between D-pz/D-mol and the zone retention factor appears to be in good agreement with that predicted when applying the regular variant of the EMT-expression directly to the mesoporous skeleton level. (C) 2014 Elsevier B.V. All rights reserved.

Background: The bioavailability of dietary folate may be hampered by the need of the glutamate moieties to be deconjugated before absorption. Previous studies comparing the bioavailabilities of polyglutamyl and monoglutamyl folic acid had inconsistent results. Objective: The objective was to estimate the bioavailability of polyglutamyl relative to that of monoglutamyl folic acid by using a sensitive stable-isotope approach that allowed for the administration of multiple low doses in humans. Design: Twenty subjects aged 20-50 y ingested 2 capsules daily for 28 d; each capsule contained approximate to 50 nmol [C-13(6)]hexaglutamyl and approximate to 50 nmol [C-13(11)]monoglutamyl folic acid. Amounts of the isotopically labeled compounds in the capsules were verified by various methods. The degrees of isotopic enrichment of plasma 5-methyltetrahydrofolate with C-13(6) and C-13(11) were measured by using liquid chromatography tandem mass spectrometry, and the ratio of C-13(6) to C-13(11) (C-13(6): C-13(11)) in plasma on day 28 was used as a measure of their relative bioavailability. Results: The C-13(11): C-13(6) in plasma 5-methyltetrahydrofolate reached equilibrium on day 4 and was 0.66 (95% CI: 0.58, 0.74) on day 28. The C-13(11): C-13(6) content in the capsules varied between 1.18 and 1.96. After correction for this ratio, the estimated bioavailability of hexaglutamyl relative to that of monoglutamyl folic acid was >= 78%. Conclusion: Multiple dosing of low amounts of labeled folic acid is a sensitive, accurate, and efficient method of measuring the relative bioavailability of folic acid compounds, provided that the administered doses can be reliably assessed.

Background: Over the years, the N-glycosylation of both human and bovine lactoferrin (LF) has been studied extensively, however not all aspects have been studied in as much detail. Typically, the bovine LF complex-type N-glycans include certain epitopes, not found in human LF N-glycans, i.e. Gal(alpha 1-3)Gal(beta 1-4)GlcNAc (alpha Gal), GalNAc(beta 1-4) GlcNAc (LacdiNAc), and N-glycolylneuraminic acid (Neu5Gc). The combined presence of complex-type N-glycans, with alpha Gal, LacdiNAc, lacNAc [Gal(beta 1-4)GlcNAc], Neu5Ac (N-acetylneuraminic acid), and Neu5Gc epitopes. and oligomannose-type N-glycans complicates the high-throughput analysis of such N-glycoprofiles highly.Methods: For the structural analysis of enzymatically released N-glycan pools, containing both LacNAc and LacdiNAc epitopes, a prefractionation protocol based on Wisteria floribunda agglutinin affinity chromatography was developed. The sub pools were analysed by MALDI-TOF-Ms and HPLC-FD profiling, including sequential exoglycosidase treatments.Results: This protocol separates the N-glycan pool into three sub pools, with (1) free of LacdiNAc epitopes, (2) contaming LacdiNAc epitopes, partially shielded by sialic acid, and (3) containing LacdiNAc epitopes, without shielding by sialic acid. Structural analysis by MALDI-TOF-MS and HPLC-FD showed a complex pattern of oligomannose-, hybrid-, and complex-type di-antennary structures, both with, and without LacdiNAc, alpha Gal and sialic acid.Conclusions: Applying the approach to bovine LF has led to a more detailed N-glycome pattern, including LacdiNAc, alpha Gal, and Neu5Gc epitopes, than was shown in previous studies.General significance: Bovine milk proteins contain glycosylation patterns that are absent in human milk proteins; particularly, the LacdiNAc epitope is abundant. Analysis of bovine milk serum proteins is therefore excessively complicated. The presented sub fractionation protocol allows a thorough analysis of the full scope of bovine milk protein glycosylation. This article is part of a Special Issue entitled Glycoproteomics. (C) 2012 Elsevier B.V. All rights reserved.

Background Artemisinin-based fixed dose combination (FDC) products are recommended by World Health Organization (WHO) as a first-line treatment. However, the current artemisinin FDC products, such as β-artemether and lumefantrine, are inherently unstable and require controlled distribution and storage conditions, which are not always available in resource-limited settings. Moreover, quality control is hampered by lack of suitable analytical methods. Thus, there is a need for a rapid and simple, but stability-indicating method for the simultaneous assay of β-artemether and lumefantrine FDC products. Methods Three reversed-phase fused-core HPLC columns (Halo RP-Amide, Halo C18 and Halo Phenyl-hexyl), all thermostated at 30°C, were evaluated. β-artemether and lumefantrine (unstressed and stressed), and reference-related impurities were injected and chromatographic parameters were assessed. Optimal chromatographic parameters were obtained using Halo RP-Amide column and an isocratic mobile phase composed of acetonitrile and 1mM phosphate buffer pH 3.0 (52:48; V/V) at a flow of 1.0 ml/min and 3 μl injection volume. Quantification was performed at 210 nm and 335 nm for β-artemether and for lumefantrine, respectively. In-silico toxicological evaluation of the related impurities was made using Derek Nexus v2.0®. Results Both β-artemether and lumefantrine were separated from each other as well as from the specified and unspecified related impurities including degradants. A complete chromatographic run only took four minutes. Evaluation of the method, including a Plackett-Burman robustness verification within analytical QbD-principles, and real-life samples showed the method is suitable for quantitative assay purposes of both active pharmaceutical ingredients, with a mean recovery relative standard deviation (± RSD) of 99.7 % (± 0.7%) for β-artemether and 99.7 % (± 0.6%) for lumefantrine. All identified β-artemether-related impurities were predicted in Derek Nexus v2.0® to have toxicity risks similar to β-artemether active pharmaceutical ingredient (API) itself. Conclusions A rapid, robust, precise and accurate stability-indicating, quantitative fused-core isocratic HPLC method was developed for simultaneous assay of β-artemether and lumefantrine. This method can be applied in the routine regulatory quality control of FDC products. The in-silico toxicological investigation using Derek Nexus® indicated that the overall toxicity risk for β-artemether-related impurities is comparable to that of β-artemether API.