阴离子交换固相萃取GC-MS/MS方法检测鱼肉制品中的河豚毒素

目的建立高灵敏度的检测鱼肉制品中河豚毒素的阴离子交换固相萃取（SPE）气相色谱-串联质谱法。方法采用直接碱解的样品处理方法,将鱼肉制品中的河豚毒素转化为C9碱,经阴离子交换（SAX）固相萃取柱净化、双三甲基硅基三氟乙酰胺（BSTFA）硅烷化衍生后,在气相色谱-串联质谱仪上以选择反应监测（SRM）模式进行检测。结果与结论在最优分析条件下,染毒的鱼肉样品中河豚毒素的线性范围为10～1000μg/kg,检测限为8μg/kg。该方法专属性强、灵敏度高,操作相对简便。     

Rapid analysis of acetaminophen in serum by gas chromatography-mass spectrometry with extractive derivatization using a diatomaceous earth tube

A new analytical method for acetaminophen (ACAP) in serum was developed by modifying an existing method used for amphetamines, which used extractive derivatization followed by gas chromatographymass spectrometry. After a serum sample was adjusted to pH 12.8, it was applied onto a diatomaceous earth tube; the analyte was simultaneously extracted and heptafluorobutyrylated during elution with a solvent containing a derivatizing reagent. Three internal standard (IS) candidates were tested: N-acetyl-d ₃-paminophenol, N-acetyl-m-aminophenol, and N-acetyl-4-amino-m-cresol. All ISs gave good linear relationships (r ² > 0.999) for ACAP in the concentration range from 1 to 200μg/ml. The detection limit for ACAP using each IS was estimated to be 0.05–0.1μg/ml. Intraday precision was satisfactory with a coefficient of variation of less than 8.3%. The use of this method with any of the three ISs is recommended in forensic and clinical toxicology because of its rapidity and good reproducibility.     

Validating a gas chromatography-mass spectrometric method and sample classification procedure for cannabis profiling using cannabinoids from case samples

A gas chromatography–mass spectrometric (GC–MS) method was evaluated for cannabis profiling. Method validation was carried out using four cannabinoids extracted from cannabis case samples. In the presence of the sample matrix, the instrument was able to achieve repeatability and reproducibility with relative standard deviation (RSD) values ≤ 10.99% and ≤ 9.09%, respectively. The MS detector also produced a linear response for each of the target cannabinoids with R² values ranging from 0.976 to 1.000. The method was found to be accurate as it displayed satisfactory recoveries (assessed based on the percentage difference between the measured amount and the expected or calculated amount of the analyte) between –11.35% and 0.96% for the difference. Subsequently, five different batches/groups of cannabis plants that were assumed to have come from different sources were tentatively used to assess the sample classification outcome. These samples were correctly clustered on a dendrogram with 12% errors. Through this preliminary study, it is deduced that cannabis samples could be profiled to estimate their source level relationships by using the proposed GC–MS method.     

Simultaneous analysis of some club drugs in whole blood using solid phase extraction and gas chromatography-mass spectrometry

The use of psychoactive substances to improve social relations and increase body energy, in Rave Culture, has raised many legal and health public concerns, both for illicit trade and consumption. Therefore, forensic toxicology plays an important role in this area, mainly linked to the detection and quantitation of these substances, both in vivo and in post-mortem samples. In fact, at the moment, forensic sciences have been under public authorities’ scrutiny and critical look, due to the increasing attention of the media and public opinion, always applying for the use of scientific knowledge to help solving forensic cases. However, forensic toxicology results are only reliable to solve legal cases if all the analytical methodologies used are appropriately validated.In this work, a methodology for the extraction and analysis of 7-aminoflunitrazepam, buprenorphine, flunitrazepam, ketamine, methadone, phencyclidine (PCP) and d-propoxyphene was developed for whole blood samples, with solid phase extraction (SPE), using OASIS^® MCX SPE columns, and gas chromatography coupled to mass spectrometry.The procedure presented here proved to be reliable, specific, selective and sensitive, with good LODs and LOQs and good precision.The adoption of a SPE procedure with an automatic SPE extraction device, allowed an increased level of automation in sample treatment, being contemporarily less time-consuming, increasing productiveness, and allowing good recovery and appropriate selectivity being, also, simple and reproducible. The simultaneous detection and quantitation of all compounds by the same extraction and detection methodology is crucial and has a great potential for forensic toxicology and clinical analysis.     

Rapid and reliable screening method for detection of 70 pesticides in whole blood by gas chromatography-mass spectrometry using a constructed calibration-locking database

Pesticide poisoning is one of the most common causes of death by poisoning in Japan, and various kinds of pesticides including organophosphates, carbamates and pyrethroids are listed as causative substances. The purpose of our study was to develop a rapid and reliable screening method for various kinds of pesticides in whole blood by using a unique calibration-locking database and gas chromatography–mass spectrometry. A database of 70 pesticides was constructed using NAGINATA? software with parameters such as mass spectrum, retention time and qualifier ion/target ion ratio (QT ratio) and calibration curve. Diazepam-d₅ was used as the internal standard for construction of each calibration curve within the range of 0.01–5.0 μg/ml. We examined the applicability of the constructed database by analyzing whole blood samples spiked with 70 pesticides. The pesticides in blood were extracted with hexane under acidic conditions or with an enhanced polymer column (Focus?), subjected to GC–MS, and screened by the pesticides database. Among the 70 pesticides examined, 66 and 62 were successfully identified at the level of 1 and 0.1 μg/ml, respectively, by hexane and 63 and 51 were identified by the Focus column without the use of standard compounds. The time required for data analysis was significantly reduced. Since the established method can produce qualitative and semi-quantitative data without the need for standard substances, this new screening method using NAGINATA? should be useful for confirming the presence of pesticides in blood in future clinical and forensic cases.     

Determination of diazepam and its metabolites in serum by the use of liquid chromatography-mass spectrometry method

BACKGROUND/AIM: Diazepam is a benzodiazepine anxyolitic. Metabolism of diazepam takes place in liver which generates pharmacologically active metabolites N-desmethyldiazepam, temazepam and oxazepam. The aim of this study was to develop and validate the method of liquid chromatographymass spectrometry (LC-MS) for separation and determination of diazepam and its active metabolites in the serum of rats samples after i.p. application of diazepam in a dose of 10 mg/kg. METHODS: The serum samples taken from Wistar rats, were used in LC-MS analysis after the application of 10 mg/kg of diazepam i.p. RESULTS: After alkaline extraction from the serum samples with diethylether and separation on a C18 reversed-phase column by using mobile phase methanolglacial acetic acid-water (50:1:49 v/v), diazepam and its metabolites were quantified. Determination was performed in a selective ion monitoring (SIM) mode, thereby the other exogenous and endogenous compounds did not interfere with this assay. Diazepam, N-desmethyldiazepam, oxazepam and temazepam were eluted in 14 minutes. The standard curve was linear in the range from 10-2 000 ng/ml. The limits of detection for diazepam, N-desmethyldiazepam, oxazepam and temazepam were 4.37, 3.13, 4.38 and 7.31 ng/ml, respectively. The limits of quantitation for diazepam, N-desmethyldiazepam, oxazepam and temazepam were 14.58, 10.41, 14.59 and 24.36 ng/ml, respectively. CONCLUSION: The described LC-MS is a simple, sensitive, specific and accurate method and could be used for routine identification and quantification of small concentrations of diazepam and its metabolites in biological fluids.     

A new method for quantitative determination of dimemorfan in human plasma using monolithic silica solid-phase extraction tips

Dimemorfan was extracted from human plasma samples (100 μL) using MonoTip C(18) tips, which were packed with a C(18)-bonded monolithic silica gel attached to the inside of the tip. The samples, which contained dimemorfan and trimeprazine as an internal standard (IS), were mixed with 300 μL of distilled water and 50 μL of 1M glycine-sodium hydroxide buffer (pH 10). The mixture was extracted onto the C(18) phase of the tip by 20 sequential aspirating/dispensing cycles using a manual micropipettor. The analytes retained on the C(18) phase were then eluted with methanol by five sequential aspirating/dispensing cycles. The eluate was injected directly into a gas chromatograph and detected by a mass spectrometer with selected ion monitoring in positive electron ionization mode. An Equity-5 fused silica capillary column (30 m × 0.32 mm i.d., film thickness 0.25 μm) gave adequate separation of the dimemorfan, IS, and impurities. The recoveries of dimemorfan and the IS spiked into plasma were ≥83%. The regression equation for dimemorfan showed excellent linearity from 0.25 to 32.0 ng/100 μL of plasma, and the limit of detection was 0.125 ng/100 μL of plasma. The maximum intra-day and inter-day relative standard deviations were 13%, while accuracy ranged from 88% to 105%. Dimemorfan was stable for at least 12 h at 4°C, 4 weeks at -80°C, and three freeze-thaw cycles in plasma. This new method is expected to have application as a pretreatment for the rapid, simple, and quantitative determination of dimemorfan in plasma samples.     

A simple and reliable method for quantifying plasma concentrations of tetracyclic antidepressants using monolithic silica solid-phase extraction tips

Three tetracyclic antidepressants, mianserin, mirtazapine, and setiptiline, were extracted from human plasma samples using a new micropipette solid-phase extraction tip containing a monolithic silica gel packing material, the MonoTip C₁₈. The plasma samples (0.1 ml), which contained mianserin, mirtazapine, and setiptiline, were mixed with 330 μl of distilled water and 50 μl of 0.5 M glycine–sodium hydroxide buffer solution (pH 8). After centrifugation, the supernatant was extracted onto the C₁₈ phase of the tip (pipette tip volume, 200 μl) by 25 sequential aspirating/dispensing cycles. The three analytes retained in the C₁₈ phase were eluted with methanol by 5 aspirating/dispensing cycles. The eluate was subjected to measurements by gas chromatography (GC) with nitrogen–phosphorus detection (NPD). The recoveries of the three antidepressants were 84.6–99.6% and the limits of detection for each drug were between 2.4 and 15 ng/ml of plasma. The maximum intraday and interday coefficients of variation for the drugs was below 10.6%. Regression equations for the three drugs showed excellent linearity in the range of 50–5000 ng/ml for mianserin and mirtazapine and 50–10 000 ng/ml for setiptiline. The antidepressants were stable for at least 12 h at 4°C, 4 weeks at −80°C, and through three freeze–thaw cycles in plasma. The validated method was successfully used to quantify the plasma concentrations of mirtazapine in a human subject after oral administration of the drug. Although we achieved analyte detection with GC-NPD, the present monolithic micropipette extraction method for tetracyclic antidepressants seems to be very useful for combining it with GC–mass spectrometry (MS) and/or liquid chromatography–tandem MS to secure high simplicity, purification, reproducibility, and sensitivity.     

Alpha-spectrometric analysis of uranium and thorium using solid-phase extraction for sample preparation

A method is presented here for the preparation of thin uniform samples of naturally occurring uranium and thorium which are highly suitable for α-spectrometric analysis. The solid-phase extraction procedure simultaneously achieves a complete separation of the analytes from the sample matrix and a high enrichment factor for uranium and thorium, so that the ensuing eluate is ideally suited for either electrodeposition or ICP-MS, without the need for complicated and painstaking sample preparation. In contrast to conventional liquid–liquid phase-extraction methods, no organic waste solutions are produced, and the process can be easily automated.     

Construction of calibration-locking databases for rapid and reliable drug screening by gas chromatography-mass spectrometry

Unique calibration-locking databases were constructed for rapid and semiquantitative drug screening by gas chromatography-mass spectrometry (GCMS). In addition to the free-drug database of 127 drugs, a drug database with acetylating reagents was constructed to increase the number of detectable compounds in the analysis by GC-MS; 156 drugs, including 30 drugs of abuse, 42 hypnotics and their metabolites, 18 antipsychotic drugs, 15 antidepressants, and 12 antipyretic analgesic agents, were registered with parameters, such as the mass spectrum, retention time, qualifier ion/target ion percentage, and calibration curve using the novel GC-MS software NAGINATA. Diazepam-d ₅ was used as internal standard for construction of each calibration curve in the range of 0.01–5.0 μg/ml for most drugs. We examined the applicability of the constructed database to analyzing whole blood samples spiked with 40 drugs most commonly encountered in toxicological cases in Japan. The drugs in blood were extracted using enhanced polymer columns (Focus), subjected to GC-MS after incubation with acetylating reagents, and screened by the drug database. Among the 40 drugs examined, 38 and 30 drugs were successfully identifi ed at the level of 1 and 0.1 μg/ml, respectively, without using standard compounds. The time required for data analysis was less than 1 min, and semiquantitative data were also obtained simultaneously. Because new drugs and metabolites can easily be added to the databases, we can recommend them as useful tools in clinical and forensic toxicological screening.     

Determination of fluoride in human whole blood and urine by gas chromatography-mass spectrometry

We developed a simple and sensitive method for determination of fluoride in human whole blood and urine using gas chromatography-mass spectrometry (GC-MS). Fluoride was alkylated with pentafluorobenzyl bromide in a mixture of acetone and phosphate buffer (pH 6.8). The derivative obtained was analyzed by GC-MS in the positive-ion electron-impact mode. The lower limit of detection for the compound was 0.5 mg/l for both matrices. The calibration curve for fluoride was linear over the concentration range of 1–100 mg/l. The precision and accuracy of the method were evaluated, and relative standard deviation was within 10%. Using this method, levels of fluoride in whole blood and urine were determined in a case of poisoning caused by hydrofluoric acid exposure.     

Automated solid-phase extraction and two-step derivatisation for simultaneous analysis of basic illicit drugs in serum by GC/MS

A combination of automated solid-phase extraction (SPE) and subsequent two-step derivatisation has been developed for the simultaneous analysis of basic drugs of abuse and cocaine metabolites in serum samples. Substances included in this procedure are morphine, codeine, methadone, cocaine, benzoylecgonine, methylecgonine, amphetamine, methamphetamine, MDMA, MDEA and MDA. SPE with mixed-mode cartridges (RP-C8 and cation-exchange) was fully automated with a Zymark RapidTrace SPE robot. GC/MS analysis was performed after derivatisation with a new two-step reaction by trifluoroacetic anhydride and 2,2,3,3,3-pentafluoropropanol. High recoveries (> 85%) with high reproducibility (CV 1.1-3.8%) were found for all drugs. High correlation coefficients (r > 0.998) were obtained due to the addition of deuterated standards prior to extraction. Experience obtained over 2 years of applying this method to drug analysis in serum is discussed.     

A simplified method for the measurement of nonmetabolized 2-[18F]F-A-85380 in blood plasma using solid-phase extraction

Quantification of alpha(4)beta(2)* nicotinic acetylcholine receptors using 2-[(18)F]fluoro-3-(2(S)-azetidinylmethoxy)pyridine (2-[(18)F]FA) and positron emission tomography (PET) imaging requires measurement of nonmetabolized radioligand in blood plasma, which was previously accomplished using high-performance liquid chromatography (HPLC). Here, we introduce a one-step solid-phase extraction (SPE) method for measuring the concentration of nonmetabolized 2-[(18)F]FA. This method allows many samples to be processed in a short period of time. SPE effectively separated 2-[(18)F]FA from radioactive metabolites typically observed in blood plasma after administration of radioligand in humans. Measurements of the 2-[(18)F]FA parent fraction in healthy human volunteers obtained using the SPE method were nearly identical to those obtained using HPLC (1.3+/-5% average underestimation of SPE), and reproducibility was good within and between runs (2% and 6% coefficient of variation, respectively). SPE recovery of 2-[(18)F]FA from blood plasma was not appreciably diminished (3+/-0.6%) by a larger volume of blood plasma loaded onto the cartridge, suggesting the possibility of increasing the plasma sample volume at later times in a PET study to improve measurement sensitivity. 2-[(18)F]FA was stable in blood stored on ice over 8 h and in saline at low concentrations (<2 MBq/ml) but not at high concentrations (ca. 130 MBq/ml). Using SPE, the elimination half-life and full body distribution volume of 2-[(18)F]FA in healthy human volunteers were estimated as 4.2+/-0.8 h and 220+/-70 L, respectively. These results suggest that SPE is the method of choice for the determination of the plasma 2-[(18)F]FA concentration when measurement of individual metabolites is not required.     

[18F]Fallypride: Metabolism studies and quantification of the radiotracer and its radiometabolites in plasma using a simple and rapid solid-phase extraction method

Introduction[¹⁸F]Fallypride, a fluorinated and substituted benzamide with high affinity for D₂/D₃ receptors, is a useful PET radioligand for the study of striatal/extrastriatal areas. Since [¹⁸F]fallypride is extensively metabolized in vivo and since PET examinations are long lasting in humans, the rapid measurement of the unchanged radiotracer in plasma is essential for the quantification of images. The present study aims: i) to evaluate if the radiometabolites of [¹⁸F]fallypride cross the blood–brain barrier in rodents, ii) to identify these radiometabolites in baboon plasma and iii) to develop a rapid solid phase extraction method (SPE) suitable for human applications to quantify both [¹⁸F]fallypride and its radiometabolites in plasma.MethodsThe metabolites P450-dependant in rat and human liver microsomes were characterized by LC–MS–MS and compared to those detected in vivo. Sequential solvent elution on Oasis®-MCX-SPE cartridges was used to quantify [¹⁸F]fallypride and its radiometabolites.ResultIn rat microsomal incubations, five metabolites generated upon N/O-dealkylation or hydroxylation at the pyrrolidine and/or at the benzamide moiety were identified. No radiometabolite was detected in the rat brain. N-dealkylated and hydroxylated derivatives were detected in human microsomal incubations as well as in baboon plasma. The use of SPE (total recovery 100.2% ± 2.8%, extraction yield 95.5% ± 0.3%) allowed a complete separation of [¹⁸F]fallypride from its radiometabolites in plasma and evaluate [¹⁸F]fallypride at 150 min pi to be 22% ± 5% of plasma radioactivity.ConclusionsThe major in vivo radiometabolites of [¹⁸F]fallypride were produced by N-dealkylation and hydroxylation. Allowing the rapid analysis of multiple plasma samples, SPE is a method of choice for the determination of [¹⁸F]fallypride until late images required for quantitative PET imaging in humans.     

High-throughput determination of theophylline and caffeine in human serum by conventional liquid chromatography-mass spectrometry

Automated high-performance liquid chromatography/mass spectrometry (HPLC-MS) with backflush column-switching was established for ultra-fast determination of theophylline and caffeine. A 400-μl portion of serum sample diluted with ultrapure water was injected and transferred to an Oasis HLB cartridge used as a precolumn for extraction. After switching the valves, the analytes trapped in the precolumn were eluted in the backflush mode and separated with a Chromolith Performance RP-18e column (C₁₈-bonded monolithic silica); the compounds in column effluents were then detected by atmospheric pressure chemical ionization (APCI)-MS. The present method successfully provided high-throughput determination of theophylline and caffeine within 2 min. Satisfactory linearity, reproducibility, and sensitivity could be obtained for analysis of therapeutic and toxic levels of both compounds. Because of the very simple procedure and high throughput using the conventional HPLC system, the present method seems to have high potential in the fields of forensic toxicology and emergency medicine.     

A method for determining valproic acid in human whole blood and urine via gas chromatography-mass spectrometry and small-scale inter-laboratory trial

A simple and cost-effective method for analyzing valproic acid (VPA) in biological samples was developed. VPA was extracted in methyl tertiary-butyl ether (MTBE) and derivatized using trimethylsilyldiazomethane. The MTBE extract was analyzed by gas chromatography-mass spectrometry (GC-MS). The extraction recovery in human whole blood and urine was over 90 %, with good linearity in the range of 1.0 to 250 µg/mL of VPA. The RSD for 2.0, 20, and 200 μg/mL VPA in whole blood ranged from 0.9 to 4.7 % for intra-day and 1.5 to 5.9 % for inter-day. The RSD for 2.0, 20, and 200 μg/mL VPA in urine ranged from 1.9 to 2.6 % for intra-day and 1.2 to 2.9 % for inter-day. As a preliminary cross-validation study, a cross-check was conducted using blinded concentration samples. The results demonstrated that the assay data of the two laboratories were comparable.     

Real-time microbiological diagnostics of the human integument in space flight using the method of chromatography-mass spectrometry detection

Qualitative and quantitative analysis of integument microflora using chromatography-mass spectrometry enables the determination of each crewmember's microflora composition, i.e. families and genders, during space mission. Microbiological investigations of cosmo nauts' integument resulted in identification of 51 taxons and groups of microorganisms. Microbial populations are directly dependent on the environmental factors and mission duration. The most representative diagnostically relevant ('reference') biotopes were brought to light.     

Supramolecular solvent (SUPRASs) extraction method for detecting benzodiazepines and zolpidem in human urine and blood using gas chromatography tandem mass spectrometry

ObjectiveA high-throughput and sensitive method using supramolecular solvent (SUPRASs) for detecting 9 benzodiazepines and zolpidem in human urine and blood by gas chromatography-tandem mass spectrometry (GC–MS/MS) was newly established and applied to authentic human urine and blood samples in this study.MethodsUrine and blood samples were subjected to liquid–liquid extractions with supramolecular solvent mixture which consists of tetrahydrofuran and 1-hexanol. The solvent layer was evaporated to dryness by stream of nitrogen. The residue was reconstituted with methanol, and subjected to analysis by GC–MS/MS in multiple reaction monitoring (MRM) mode; internal standard method was employed for quantifying of each targeted compound.ResultsThe regression equation has a good linear relationship with correlation coefficients for all tested compounds were not lower than 0.9991. The lower limits of the quantification ranged from 0.20 to 5 ng/mL for tested compounds in urine; Meanwhile, the lower limits of the quantification in this method ranged from 1 to 50 ng/mL for tested compounds in blood. These results showed that excellent reproducibility and satisfactory extraction recovery rates could be obtained for the established analytical method for 10 drugs in both blood and urine samples.ConclusionThe established method in this study was high-throughput, simple and sufficiently sensitive for determining of benzodiazepinesand zolpidem in human urine and blood. Therefore, this newly established method could be of use for qualitative and quantitative determination of such drugs in urine and blood samples either for clinical poisoning monitoring or for forensic identification.