A sensitive assay for palytoxins, ovatoxins and ostreocins using LC-MS/MS analysis of cleavage fragments from micro-scale oxidation

Palytoxin is a highly toxic non-proteinaceous marine natural product that can pass through the food chain and result in human illnesses. A recent review by the European Food Safety Authority concluded that palytoxin requires regulation in seafood and a limit of 30 μg kg⁻¹ for shellfish flesh was suggested. Current methods based on LC-MS detection of intact palytoxins do not have sufficient sensitivity to enforce this limit for palytoxin. To improve sensitivity for trace analysis, a novel screen approach has been developed that uses LC-MS/MS analysis of substructures generated by oxidative cleavage of vicinal diol groups present in the intact toxin. Oxidation of palytoxins, ovatoxins or ostreocins using periodic acid generates two nitrogen-containing aldehyde fragments; an amino aldehyde common to these toxins, and an amide aldehyde that may vary depending on toxin type. Conditions for micro-scale oxidation of palytoxin were optimised, which include a novel SPE cleanup and on-column oxidation step. Rapid analysis of cleavage fragments was established using LC-MS/MS. Linear calibrations were established for the amino aldehyde from a palytoxin reference standard, which is suitable for all known palytoxin-like compounds, and for the confirmatory amide aldehydes of palytoxin and ostreocin-D. Palytoxin recoveries (at 10 μg kg⁻¹) from shellfish and fish tissues were 114-119% (as amine aldehyde) and 90-115% (as amide aldehyde) with RSDs for both of ≤18% (all tissues, n = 12). The method LOD was determined to be approximately 1 ng mL⁻¹ and the LOQ 4 ng mL⁻¹, which corresponds to 10 μg kg⁻¹ in tissue (flesh of shellfish or fish). The method has potential for use in research and is sufficiently sensitive for regulatory testing, should it be required.     

Cytotoxic effect of palytoxin on mussel

Palytoxin is a large and complex polyhydroxylated molecule with potent neurotoxic activity. Dinoflagellates from the Ostreopsis genera were demonstrated to be producers of this compound and analogues. Even though initially palytoxin appearance was restricted to tropical areas, the recent occurrence of Ostreopsis outbreaks in Mediterranean Sea point to a worldwide dissemination probably related to climatic change. Those dinoflagellates can bioaccumulate in shellfish, especially in filter-feeding mollusks and have been involved in damaging effects in seafood or human toxic outbreaks. The present study describes palytoxins effect on metabolic activity of mantle and hepatopancreas cells from the mussel Mytilus galloprovincialis Lmk. Our results indicate that palytoxin is highly cytotoxic to mussel cells; unlike it happens with other toxins more common in European coasts such as okadaic acid and azaspiracid. These findings have a special significance for the marine environment and aquiculture since they are evidence for the ability of palytoxin to affect the integrity of bivalve mollusks that are not adapted to the presence of this toxin.     

A 4-decade-long (and still ongoing) hunt for palytoxins chemical architecture

Since its isolation dated back to as far as 1971, palytoxin has all along drawn scientists’ attention from across the world because of its high toxicity and fascinating chemical architecture. Commitment of the international scientific community to the study of this extremely potent non-proteic toxin has led to discover quite a number of palytoxin analogues. Once confined only to tropical and subtropical areas, palytoxins have recently spread also to more temperate regions, such as the Mediterranean Sea where they have caused severe human intoxications. Studies on the Mediterranean toxic outbreaks brought to light the existence of further palytoxin-like compounds, ovatoxins, never reported elsewhere in the world.     

Suitability of the Neuro-2a cell line for the detection of palytoxin and analogues (neurotoxic phycotoxins)

Palytoxin and related compounds are neurotoxic phycotoxins produced by benthic microalgae belonging to the genus Ostreopsis. For several years this family of phycotoxins has been posing a threat to human health since they can bioaccumulate in shellfish. With the aim of replacing current biological assays, such as the mouse or hemolytic assays, we investigated using the Neuro-2a neuroblastoma cell line to detect palytoxin and related compounds. Cell death induced by the effects of PlTX and analogues on Na⁺, K⁺-ATPase were measured using the 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) assay for mitochondrial reductase activity as a surrogate for cell number. The specificity of the Neuro-2a cell-based assay for palytoxin detection was confirmed by using ouabain, which also acts on Na⁺, K⁺-ATPase. Pre-treatment of the Neuro-2a cells with ouabain minimizes the effects of palytoxin. The specificity of the Neuro-2a assay was confirmed by the finding that cell death was not detected when Neuro-2a cells were exposed to other phycotoxins with unrelated cellular targets. When the Neuro-2a assay was used to detect palytoxin in mussel extracts spiked with levels of palytoxin around the proposed regulatory value of 250 μg palytoxin/kg shellfish, a good correlation was observed between the levels found and the expected values.We conclude by proposing an experimental design for functional assays using the Neuro-2a cell line for the specific detection of four neurotoxic phycotoxin families: saxitoxins, brevetoxins, ciguatoxins and palytoxins.     

Unique toxin profile of a Mediterranean Ostreopsis cf. ovata strain: HR LC-MS(n) characterization of ovatoxin-f, a new palytoxin congener

Currently, the benthic dinoflagellate Ostreopsis cf. ovata represents a serious concern to human health in the whole Mediterranean basin due to the production of palytoxin congeners, a putative palytoxin and ovatoxins (ovatoxin-a, -b, -c, -d/-e), listed among the most potent marine toxins. High resolution liquid chromatography-mass spectrometry (HR LC-MS) based investigation of a North Western Adriatic strain of Ostreopsis cf. ovata collected at Portonovo (Italy) in 2008 is reported herein. Toxin profile was different from those previously reported for other O. cf. ovata, both qualitatively and quantitatively. For the first time, ovatoxin-a did not dominate the toxin profile, and a new palytoxin congener, here named ovatoxin-f, was detected. Ovatoxin-f and its elemental formula present C(2)H(4) more than ovatoxin-a. HR CID MS(n) experiments allowed us to restrict structural differences between ovatoxin-a and -f to the region between C-95 and C-102, a region not previously been described to be modified in other palytoxins. Ovatoxin-f represents the major component of the toxin profile of the analyzed strain accounting for 50% of the total toxin content, while ovatoxin-a, the dominant toxin in most of the Mediterranean O. cf. ovata strains we have analyzed so far, is the second major component of the toxin profile (23%). Thus, the presence of ovatoxin-f should be taken into account when monitoring programs for palytoxin-like compounds in microalgae and/or seawater are carried out.     

Sensitive and selective liquid chromatography-tandem mass spectrometry method for the determination of five ganoderic acids in Ganoderma lucidum and its related species

The present paper describes a novel, sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous analysis of ganoderic acids C(2), B, A, H, D in Ganoderma lucidum and its related species. Ganoderma samples were prepared using simple ultrasonic extraction. Chromatographic separation was carried out on an Agilent Zorbax XDB C(18) column (250 mm × 4.6 mm i.d., 5μm) with an isocratic mobile phase consisting of acetonitrile, water and formic acid (42:58:0.5, v/v/v). Mass spectrometric detection was achieved by a triple-quadrupole mass spectrometer equipped with an atmospheric pressure chemical ionization (APCI) interface operating in negative and positive ionization mode via a single within-run polarity switching. Quantitation of five ganoderic acids was performed using selective reaction monitoring (SRM) mode. The intra- and inter-day precision was less than 6.2% and the accuracy ranged from 90.0% to 105.7%. The limit of quantification (LOQ) was 20.0-40.0 ng/mL and the limit of detection (LOD) was 3.0-25.0 ng/mL. With this method, low levels of ganoderic acids in the fruiting bodies of Ganoderma sinense and Ganoderma applanatum were accurately quantified for the first time. Importantly, the method allows unequivocal quantification of the five ganoderic acids in the spores and fruiting bodies of Ganoderma lucidum, whereas the previously published methods have lacked the capability. The method presented will be a powerful tool for quality control of Ganoderma lucidum and its related species.     

Metabolites of lorazepam: Relevance of past findings to present day use of LC-MS/MS in analytical toxicology

The advent of liquid chromatography-tandem mass spectrometry (LC-MS/MS), with the sensitivity it confers, permits the analysis of both phase I and II drug metabolites that in the past would have been difficult to target using other techniques. These metabolites may have relevance to current analytical toxicology employing LC-MS/MS, and lorazepam was chosen as a model drug for investigation, as only the parent compound has been targeted for screening purposes. Following lorazepam administration (2 mg, p.o.) to 6 volunteers, metabolites were identified in urine by electrospray ionization LC-MS/MS, aided by the use of deuterated analogues generated by microsomal incubation for use as internal chromatographic and mass spectrometric markers. Metabolites present were lorazepam glucuronide, a quinazolinone, a quinazoline carboxylic acid, and two hydroxylorazepam isomers, one of which is novel, having the hydroxyl group located on the fused chlorobenzene ring. The quinazolinone, and particularly the quinazoline carboxylic acid metabolite, provided longer detection windows than lorazepam in urine extracts not subjected to enzymatic hydrolysis, a finding that is highly relevant to toxicology laboratories that omit hydrolysis in order to rapidly reduce the time spent on gas chromatography-mass spectrometry (GC-MS) analysis. With hydrolysis, the longest windows of detection were achieved by monitoring lorazepam, supporting the targeting of the aglycone with free drug for those incorporating hydrolysis in their analytical toxicology procedures.     

Study of the gastrointestinal biotransformation of zearalenone in a Caco-2 cell culture system with liquid chromatographic methods

A high-performance liquid chromatography method with fluorescence detection (HPLC-FLD) was developed and validated for the detection of zearalenone (ZON), alpha-zearalenol (alpha-ZOL) and beta-zearalenol (beta-ZOL) in in vitro biological samples. Furthermore, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the detection of ZON, alpha-ZOL, beta-ZOL, alpha-zearalanol (alpha-ZAL) and beta-zearalanol (beta-ZAL) in in vitro biological samples. Zearalanone (ZAN) was used as internal standard in both methods. The limit of detection/limit of quantitation (LOD/LOQ) values for ZON, alpha-ZOL and beta-ZOL were 2/7, 2/7 and 4/13 microg l(-1), respectively, for the HPLC-FLD method. For the LC-MS/MS method LOD/LOQ values for ZON, alpha-ZOL, beta-ZOL, alpha-ZAL and beta-ZAL were 6/20, 5/17, 4/14, 9/30 and 6/19 microg l(-1), respectively. Within-day and between-day precision were less then 11 and 14%, respectively for the HPLC-FLD method, and both less then 20% for the LC-MS/MS method. The recovery of ZON and its metabolites ranged between 73 and 89% for the HPLC-FLD method and between 69 and 112% for the LC-MS/MS method. The methods were used for the detection of the compounds in in vitro biological samples, obtained with human intestinal Caco-2 cells culture experiments. The 8-days post-confluent Caco-2 cells were treated with ZON or a mixture of ZON and imazalil (IMA). After an incubation time of 24 h the samples were analysed with the HPLC-FLD method. Neither ZON nor its derivatives were detected in the samples. The disappearance of ZON could possibly point out the formation of phase II metabolites like glucuronide conjugates. Therefore, samples were pretreated with beta-glucuronidase before LC-MS/MS analysis. The LC-MS/MS results showed that ZON, alpha-ZOL and beta-ZOL could only be detected in the beta-glucuronidase pretreated samples. This confirmed the formation of glucuronide conjugates and the hydroxylation of ZON during the incubation with Caco-2 cells.     

New palytoxin-like molecules in Mediterranean Ostreopsis cf. ovata (dinoflagellates) and in Palythoa tuberculosa detected by liquid chromatography-electrospray ionization time-of-flight mass spectrometry

A rapid, high resolution liquid chromatography coupled with ElectroSpray Ionization Time-Of-Flight Mass Spectrometry (ESI/TOF/MS) method was developed for the determination of the toxin pattern in cultured cells of Ostreopsis cf. ovata from the Mediterranean Sea. The samples were separated on a Phenomenex Luna 3μ HILIC 200A (150 × 2.00 mm) and analyzed by LC/TOF/MS with electrospray ionization (ESI) interface in positive ion mode. The method developed here provides the capability for a fully automated analysis, which requires relatively easy sample preparation and gives clean and simple chromatograms. The method was successfully applied to the determination of ovatoxin-a, mascarenotoxin-a and four new palytoxins in O. cf. ovata. Another new palytoxin was detected in the standard material from Palythoa tuberculosa provided by Wako Chemicals.     

Miniaturized formats for efficient mass spectrometry-based proteomics and therapeutic development

Off-line miniaturized "nano-spray" formats for electrospray ionization mass spectrometry (ESI-MS) enable the routine identification of femtomole quantities of protein or peptide. Even greater strides have been achieved using on-line miniaturized ESI-MS methods, such as nanobore LC-MS and CE-MS. On-line methods enable greater sensitivity (sub-attomole limit of detection), dynamic range, and throughput. In either off- or on-line methods for protein analysis, samples are typically isolated and digested enzymatically, with MS analysis of the peptide fragments, yielding 5-50% sequence coverage, in a "bottom-up" approach. Obtaining biologically relevant (structure/function) information (such as the localization of regions of error or post-transnational modifications) often demands 100% sequence coverage and this may be obtained by analyzing intact proteins by MS with a "top-down" methodology. Proteome wide success with top-down methods will require the development of novel miniaturized approaches for sample preparation along with new tools for bioinformatics. As these miniaturized formats continue to power proteomics applications, they will undoubtedly pollinate "cross-over" applications in LC-MS ranging from drug discovery to development. An example of metabolite identification using an order of magnitude less sample than usually required, with a concurrent order of magnitude increase in signal, illustrates the potential of miniaturized formats in lead characterization activities.     

Determination of oxytocin in a dilute IV solution by LC-MS(n)

The most common drug prescribed to induce labor in the United States is oxytocin, a peptide hormone composed of nine amino acids. Oxytocin is often reconstituted in intravenous (IV) saline solutions at less than 0.05 units ml(-1) (125 ng ml(-1)) to be delivered at 1-4 drops per minute. Existing LC-UV methods for oxytocin do not have sufficient detection limits to quantitate and/or confirm oxytocin in IV solutions without sample concentration. A determinative and confirmatory method for oxytocin was developed using an LC-MS(n) ion trap instrument with an electrospray ionization (ESI) interface in positive ion mode. Separation was achieved on a C-18 column using an isocratic elution of water with 50% acetonitrile (v/v) and water with 0.05% formic acid (v/v) at a flow rate of 250 microl min(-1). Data was acquired from the selected ion monitoring (SIM) of the precursor ion (m/z 1007.3) and MS(2) scans from the collision induced dissociation of m/z 1007.3 at 30% collision energy. In this method, MS(2) full scans were utilized to obtain three structurally significant ions for the unambiguous identification of oxytocin. Calibration standards, prepared in de-ionized water from 0.006 to 0.046 units ml(-1), were linear with an R(2) value of 0.9983. The methods LOD and LOQ were 0.00084 and 0.0029 units ml(-1) (2 and 7 ng ml(-1)), respectively. This LC-MS(n) method was used to determine the amount of oxytocin in a 0.04 units ml(-1) clinical sample that was prepared in 0.9% sodium chloride IV solution.     

The quantitation of procaine in equine plasma by liquid chromatography-linear ion trap mass spectrometry

A method for the extraction and quantitation of procaine in equine plasma was developed for use with liquid chromatography-mass spectrometry (LC-MS). Procaine was isolated from equine plasma by liquid-liquid extraction at pH 11 with dichloromethane using procaine-d10 as an internal standard. Quantitation was achieved by LC-MS using a 3-microm C-18 column coupled to an electrospray ionization source on a linear ion-trap mass spectrometer. The limit of detection and limit of quantitation was determined to be 50 and 200 pg/mL, respectively. The lowest limit of detection determined by previous methods was 1 ng/mL. Administration samples were obtained as part of a larger study to determine a regulatory limit for procaine in racehorses and procaine concentrations were determined using this method.     

Rapid and quantitative determination of solanesol in Nicotiana tabacum by liquid chromatography-tandem mass spectrometry

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with multiple reaction monitoring (MRM) was developed for the determination of solanesol in Nicotiana tabacum. Sample preparation was performed by ultrasonic extraction with methanol for 20 min and then supernatant was extracted with hexane. The method used atmospheric pressure chemical ionization (APCI) detection in positive-ion mode. The separation of solanesol was performed on a Symmetry Shield RP18 column with a mixture of acetonitrile and isopropanol (1:1, v/v) containing 2mM ammonium acetate as mobile phase. Quantification of solanesol was performed by the standard addition method. The limit of quantification (LOQ) and limit of detection (LOD) of solanesol were, respectively, 5.0 ng/ml (S/N=10) and 1.5 ng/ml (S/N=3). The relative standard deviations of peak area were 0.89 and 1.12% for intra-day and inter-day, respectively. The recoveries of solanesol ranged from 97.72 to 99.67% and the corresponding R.S.D.s were less than 2.7%. Analysis took 5 min, making the method suitable for rapid determination of solanesol in N. tabacum. The proposed method has been successfully applied to the analysis of solanesol in various organs of N. tabacum.     

Development and validation of UPLC and LC-MS/MS methods for the simultaneous determination of anti-obesity drugs in foods and dietary supplements

Recently, the number of the cases in which weight loss products have been sold with illegal adulterants has increased, as awareness of the problems of obesity grows. In this study, we developed simultaneous analysis methods to rapidly and accurately identify ingredients illegally mixed with foods and dietary supplements. Twenty-three anti-obesity drugs in foods and dietary supplements were determined by developed and validated UPLC and LC-MS/MS methods. The UPLC method were validated for the LOD and LOQ in the ranges 0.05–3.0 and 0.2–10.0 μg/mL, respectively. The determination coefficient was over 0.999, precision was <6.2 %, and the accuracy was 80.8–103.9 %. The mean recoveries ranged from 80.3 to 109.3 % and RSD of stability was less than 2.1 %. The determination of the 23 anti-obesity drugs was accomplished by electrospray ionization LC-MS/MS using multiple reaction monitoring (MRM). The LODs and LOQs were in the ranges 0.03–7.5 and 0.09–30.0 ng/mL, respectively. The LC-MS/MS method was validated for linearity (r² > 0.99), precision (RSD < 10.7 %), accuracy (94.1–109.1 %), recovery (80.5–113.5 %), and the RSD of stability was <7.8 %. Using the newly developed and validated method, 193 samples were tested, and 55 were found to be adulterated.     

LC-MS测定正常及非酒精性脂肪肝模型大鼠血浆中冬青素A的质量浓度

目的建立正常及非酒精性脂肪肝模型大鼠血浆中冬青素A质量浓度的液质联用测定方法,同时比较其代谢差异。方法采集到的血浆样品以三七皂苷Rc为内标,经固相萃取柱处理后进行LC-MS分析。色谱条件:岛津Shim-pack CLC-ODS C18柱(150mm×6.0mm,5μm);流动相A为1mL.L-1甲酸水溶液,B为1mL.L-1甲酸乙腈溶液,梯度洗脱;质谱条件采用大气压电喷雾离子源(ESI源),负离子方式,选择离子检测(SIM),检测离子分别为m/z501.1(冬青素A)和m/z1 078.3(三七皂苷Rc)。结果方法学考察表明,该法定量限为2.5×10-3μg.mL-1,在2.5×10-3～5.0μg.mL-1范围内线性关系良好,日内和日间精密度(RSD)均小于10%,提取回收率在85%～115%范围内。结论该方法灵敏性高,专属性强,适用于正常及非酒精性脂肪肝模型大鼠血浆中冬青素A的药动学研究。     

A general screening and confirmation approach to the analysis of designer tryptamines and phenethylamines in blood and urine using GC-EI-MS and HPLC-electrospray-MS

Recent additions of designer tryptamines and phenethylamines to the Drug Enforcement Administration's schedule of controlled substances necessitate analytical procedures for their detection and quantitation. As specific immunoassays are not currently available and cross-reactivities with existing assays are unknown, a screening method based on gas chromatography-mass spectrometry was developed. The method was capable of measuring the pentafluoropropionic derivatives of a-methyltryptamine (AMT), N,N-dimethyltryptamine (DMT), 4-bromo-2,5-dimethoxy-beta-phenethylamine (2CB), N,N-dipropyltryptamine (DPT), 2,5-dimethyl-4-N-propylthio-beta-phenethylamine (2C-T-7), and 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DiPT). Separation was optimized to allow tentative identification of metabolites, which display common electron impact ionization fragmentation patterns. The screening method gave limits of detection between 5 and 10 ng/mL and demonstrated linearity between 50 and 1000 ng/mL. The method was successfully applied to blood and urine samples in suspected AMT intoxications. Confirmation of 5-MeO-DiPT in one of the subjects' urine was achieved using liquid chromatography-mass spectrometry (LC-MS). Quantitation by selected ion monitoring (SIM) yielded a urinary concentration of 229 ng/mL. The method was linear from 25 to 1500 ng/mL with a correlation coefficient of 0.995. The limit of detection was 5 ng/mL in urine on the LC-MS. Two additional peaks were observed and presumed to be metabolic products reported previously as 5-methoxy-N-isopropyltryptamine (5-MeO-iPT) and 5-methoxy-N,N-diisopropyltryptamine-N'-oxide (5-MeO-DiPT-N-oxide).     

Development and validation of a high-performance liquid chromatography-electrospray mass spectrometry method for the simultaneous determination of 23 eicosanoids

Inflammation is implicated in the pathogenesis of a number of diseases, including cardiovascular disease. Current research is focused on developing assays to search for biomarkers for inflammation. Eicosanoids are the oxidative metabolites of arachidonic acid (eicosatetraenoic acid, AA), a long chain polyunsaturated fatty acid common in Western diets. AA can be oxidized by one of three pathways to form prostaglandins (PGs), leukotrienes (LTs), or a number of hydroxyl and epoxy compounds. These eicosanoids have a variety of physiological functions, including regulating inflammation. We have developed a method utilizing LC-MS to separate and quantitate 23 different eicosanoids from all the three oxidative pathways. The eicosanoids were separated using a gradient elution of acetonitrile with 0.1% formic acid (v/v) and water with 0.1% formic acid (v/v) at a flow rate of 1 mL/min with a Symmetry C18 column (250 mm x 4.6 mm). Deuterated eicosanoids were used as internal standards for quantitation. Mass spectrometric detection was carried out using an Agilent 1100-series LC-MSD with an electrospray ionization interface. Electrospray ionisation (ESI) mass spectra were acquired using negative ionization and selective ion monitoring. The method was validated and shown to be sensitive (LOQ at pg levels for most compounds), accurate (recovery values 75-120%) and precise (R.S.D.<20 for all compounds) with a linear range over several orders of magnitude. The method was applied to rat kidney tissue and shown to be indicative of the eicosanoid levels within a specific organ. The analysis of eicosanoids can provide insight into the inflammatory mechanisms associated with cardiovascular disease.     

Simultaneous determination of dextromethorphan, dextrorphan, and guaifenesin in human plasma using semi-automated liquid/liquid extraction and gradient liquid chromatography tandem mass spectrometry

A method for the simultaneous determination of dextromethorphan (DEX), dextrorphan (DET), and guaifenesin (GG) in human plasma was developed, validated, and applied to determine plasma concentrations of these compounds in samples from six clinical pharmacokinetic (PK) studies. Semi-automated liquid handling systems were used to perform the majority of the sample manipulation including liquid/liquid extraction (LLE) of the analytes from human plasma. Stable-isotope-labeled analogues were utilized as internal standards (ISTDs) for each analyte to facilitate accurate and precise quantification. Extracts were analyzed using gradient liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Use of semi-automated LLE with LC-MS/MS proved to be a very rugged and reliable approach for analysis of more than 6200 clinical study samples. The lower limit of quantification was validated at 0.010, 0.010, and 1.0 ng/mL of plasma for DEX, DET, and GG, respectively. Accuracy and precision of quality control (QC) samples for all three analytes met FDA Guidance criteria of +/-15% for average QC accuracy with coefficients of variation less than 15%. Data from the thorough evaluation of the method during development, validation, and application are presented to characterize selectivity, linearity, over-range sample analysis, accuracy, precision, autosampler carry-over, ruggedness, extraction efficiency, ionization suppression, and stability. Pharmacokinetic data are also provided to illustrate improvements in systemic drug and metabolite concentration-time profiles that were achieved by formulation optimization.     

Development and validation of a sensitive GC-MS method for the determination of trace levels of an alkylating reagent in a beta-lactam active pharmaceutical ingredient

A direct injection gas chromatographic method utilizing selected-ion monitoring (SIM) mode mass selective detection was developed and validated for the trace analysis of an impurity, carbonic acid chloromethyl tetrahydro-pyran-4-yl ester (CCMTHP), present in a beta-lactam active pharmaceutical ingredient (API). A variety of analytical techniques including LC-MS, GC-FID, GC-ECD and GC-MS were evaluated during the method development. GC-MS with SIM at m/z=49 demonstrated the best detection sensitivity. A 10 ppm (5 pg on column) limit of quantitation (LOQ) was attained and the linearity of the method was demonstrated in the range of 10-1000 ppm. Accurate and precise quantitation of the impurity in drug substance was achieved with external standardization. A 10:1 split injection was applied to limit the amount of non-volatile API loading onto the column. The effects of injection and detection parameters such as split ratio, liner type, injection temperature and number of mass ions monitored were studied. Full validation proved the accuracy, precision and specificity of the method, which was successfully employed to analyze many pilot lots of the API.     

Comparison of ELISA and LC-MS analyses for yessotoxins in blue mussels (Mytilus edulis).

Blue mussels (Mytilus edulis) collected from Fl?devigen Bay, Norway, in 2001 and 2002 were analysed for yessotoxins (YTXs) by ELISA and yessotoxin (YTX), 45-hydroxyYTX, and carboxyYTX by LC-MS. Results from the two methods were compared to evaluate the ELISA. The response in the ELISA was 3-13 times higher than LC-MS, probably due to the antibodies binding to other YTX analogues not included in the LC-MS analysis. Nevertheless, the correlation between ELISA and LC-MS was good, with r2 values> or =0.8. The results indicate that the ELISA is a reliable method for estimating the total level of YTXs in mussels, and are consistent with extensive metabolism of algal YTXs in mussels. YTX was a minor component in the blue mussels at all times compared to 45-hydroxyYTX and especially carboxyYTX, except when the P. reticulatum bloom occurred. The results also indicate the presence of significant amounts of YTX analogues in addition to those measured by LC-MS. All samples below 4 mg/kg by ELISA were below the current EU regulatory limit of 1 mg/kg by LC-MS. Therefore, we propose using ELISA as a screening tool with a cut-off limit at 4 mg/kg for negative samples, whereas samples above this limit would be reanalyzed by LC-MS.