Dynamic Multiple Reaction Monitoring Mode Research Articles

An economically viable stable isotope-enhanced multiple reaction monitoring method for total fatty acid analysis in a mouse model of non-alcoholic fatty liver disease

The complex pathological mechanisms of non-alcoholic fatty liver disease (NAFLD) are closely related to dysregulated lipid metabolism, and the therapeutic effects of the traditional Chinese medicine Zexie-Baizhu Decoction (AA) on NAFLD have been gaining increasing attention. However, research into altered lipid metabolism, especially fatty acids, in NAFLD and the intervention of AA faces technical challenges, especially in the precise quantitative analysis of fatty acids in biological samples. The high complexity of biological matrices, particularly after drug intervention, greatly increases the difficulty of detection. Therefore, this study innovatively developed a simple and economical stable isotope derivatization technique by synthesizing d6N,N-dimethylethylenediamine (d6-DMED) in the laboratory, establishing a simple and economical method for fatty acid quantification. This method employs a chemical reaction under low-temperature conditions to ensure the efficient synthesis of d6-DMED. Using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry technique (UHPLC-MS/MS), combined with optimized chromatographic separation conditions and dynamic multiple reaction monitoring mode, the study established a highly sensitive detection method for 35 fatty acid derivatives. Methodological evaluation showed that the limits of quantification ranged from 0.002 to 0.060 μM, with high linearity of R² > 0.995. Additionally, the relative recovery rates were between 93.14% and 106.63%. To further demonstrate the feasibility of this method for fatty acid quantification, it was applied to measure fatty acids in multiple tissues in a mouse NAFLD model, as well as the effects of AA intervention on fatty acid metabolism. This rapid, simple, and cost-effective detection method not only enhances the understanding of NAFLD mechanisms but also provides a new strategy for evaluating the biological complex system after drug intervention.

Determination of 118 pesticide residues in urine by ultra-high performance liquid chromatography-tandem mass spectrometry

Pesticide residues in food and their hazardous effects have attracted much attention given the increased and widespread use of pesticides. The long-term consumption of food containing pesticide residues is an important pathway for the gradual accumulation of pesticides in the human body. Urine is often monitored as a biological sample for low-dose exposure to pesticides, and urine collection is a relatively convenient sampling technique in general population research. In order to effectively monitor residual levels of multiple pesticides in human urine and provide an important technological approach for health risk assessment, a rapid screening and confirmatory detection method for 118 pesticides in urine was established using QuEChERS method as a pretreatment combined with ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-MS/MS). The 118 pesticides analyzed included organophosphorus, carbamate, neonicotinoid, and strobilurin fungicides and other widely used pesticides. Following systematic optimization of the pretreatment process, LC separation conditions, and MS/MS parameters, 118 pesticides were extracted from urine samples and analyzed within 2 h. In brief, the target analytes in 5 mL urine samples were extracted with 10 mL of acetonitrile and added with 5 g of anhydrous MgSO4 and 1 g of NaCl as water-removal and salting-out agents, respectively. After centrifugation, 6 mL of the supernatant was cleaned using the QuEChERS method with 300 mg of C18, 300 mg of primary secondary amine (PSA) and 900 mg of anhydrous MgSO4 as the purification adsorbent. After nitrogen blowing and solubilization, the 118 target analytes were separated on a ZORBAX Eclipse Plus C18 analytical chromatographic column (100 mm×2.1 mm, 1.8 μm) with gradient elution using (A) 0.01% formic acid aqueous solution (containing 2 mmol/L ammonium formate) and (B) 0.01% formic acid methanol solution (containing 2 mmol/L ammonium formate) as mobile phases. The gradient elution program was as follows: 0-0.5 min, 5%B; 0.5-1.5 min, 5%B-20%B; 1.5-2.5 min, 20%B-50%B; 2.5-8.0 min, 50%B-80%B; 8.0-9.0 min, 80%B-98%B; 9.0-11.0 min, 98%B; 11.0-11.5 min, 98%B-5%B; 11.5-15.0 min, 5%B. The analytes were then determined by UHPLC-MS/MS with positive/negative ion switching in dynamic multiple-reaction monitoring mode and quantified using the external standard method. The results indicated that the proposed method can determine 118 pesticides in urine simultaneously and rapidly. The limits of detection and limits of quantification were 0.10 and 0.50 μg/L, respectively, and the matrix effects were less than 20%for all targeted compounds. The recoveries of the 118 pesticides in urine were between 70.2% and 104% at three spiked levels of 0.50, 1.00, and 5.00 μg/L, and the relative standard deviations ranged from 2.8% to 9.3%. The method was applied to 10 actual urine samples, and the results revealed the presence of six pesticides, including thiamethoxam, clothianidin, acetamiprid, dinotefuran, isoproturon, and dimethomorph, with contents ranging from <LOQ to 3.65 μg/L. Thiamethoxam and clothianidin were detected most often in the samples, and their concentrations were highly correlated with the current use of these pesticides in edible agricultural products. The proposed method has the characteristics of high efficiency, sensitivity, and accuracy and is suitable for the rapid screening and determination of the 118 pesticide residues in human urine samples.

Detection of 41 Drugs and Metabolites in Urine with Internal Hydrolysis Control by Liquid Chromatography-Tandem Mass Spectrometry.

Regular monitoring of pain management and substance use disorder patients through urine drug screening is important for assessing patient compliance with prescribed drugs and abstinence fromnon-prescribed drugs. Sample analysis is commonly performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) as multiple drugs and metabolites can be monitored from one sample. However, challenges faced indeveloping an LC-MS/MS method for multiple analytes in urine include variability in matrix and concentration from sample to sample and variable chemistry of many pain management drugs and associated metabolites affecting accuracy and precision of results. We describe here an LC-MS/MS method for analysis of 41 drugs and metabolites commonly prescribed for pain management patients. The developed method uses enzymatic hydrolysis followed by cation exchange solid phase extraction. Resorufin-glucuronide is used as an internal hydrolysis control to monitor hydrolysis in each patient sample and minimize false negatives. Analysis was performed using an Agilent 6470 mass spectrometer in dynamic multiple reaction monitoring mode.

Determination and Chemotaxonomic Analysis of Lanostane Triterpenoids in the Mycelia of Ganoderma spp. Using Ultra-performance Liquid Chromatography-Tandem Mass Spectrometry (I).

A comprehensive and sensitive method combining ultra-performance liquid chromatography with tandem mass spectrometry was developed for the quantification of characteristic triterpenoids in Ganoderma mycelia. Eight ganoderic acids previously isolated from the mycelia of Ganoderma lingzhi were separated with a binary mobile phase on a reversed-phase C18 column. A triple quadrupole mass spectrometer equipped with an electrospray ionization source was used as the detector in the negative ion mode. Identification and quantitation of target ganoderic acids were accomplished using the dynamic multiple reaction monitoring mode. The developed method was validated in terms of linearity, precision, accuracy, stability, and recovery. The method was first applied to quantify the contents of eight ganoderic acids in the mycelia of G. lingzhi at different times to determine the optimum fermentation conditions. Subsequently, the distribution of triterpenoids and the contents of eight ganoderic acids in sixteen different Ganoderma species were investigated. The results indicated that UV chromatography combined with dynamic multiple reaction monitoring quantification was an effective chemotaxonomy method for Ganoderma species identification. This study also provided a helpful analytical methodology for both scientific and industrial applications in the quality control of Ganoderma triterpenoids.

Targeted quantitative metabolomics with a linear mixed-effect model for analysis of urinary nucleosides and deoxynucleosides from bladder cancer patients before and after tumor resection.

In the present study, we developed and validated a fast, simple, and sensitive quantitative method for the simultaneous determination of eleven nucleosides and deoxynucleosides from urine samples. The analyses were performed with the use of liquid chromatography coupled with triple quadrupole mass spectrometry. The sample pretreatment procedure was limited to centrifugation, vortex mixing of urine samples with a methanol/water solution (1:1, v/v), evaporation and dissolution steps. The analysis lasted 20min and was performed in dynamic multiple reaction monitoring mode (dMRM) in positive polarity. Process validation was conducted to determine the linearity, precision, accuracy, limit of quantification, stability, recovery and matrix effect. All validation procedures were carried out in accordance with current FDA and EMA regulations. The validated method was applied for the analysis of 133 urine samples derived from bladder cancer patients before tumor resection and 24h, 2weeks, and 3, 6, 9, and 12months after the surgery. The obtained data sets were analyzed using a linear mixed-effect model. The analysis revealed that concentration level of 2-methylthioadenosine was decreased, while for inosine, it was increased 24h after tumor resection in comparison to the preoperative state. The presented quantitative longitudinal study of urine nucleosides and deoxynucleosides before and up to 12months after bladder tumor resection brings additional prospective insight into the metabolite excretion pattern in bladder cancer disease. Moreover, incurred sample reanalysis was performed proving the robustness and repeatability of the developed targeted method.

Rapid simultaneous determination of thirteen aristolochic acids analogs in Aristolochiaceae plants by Ultra-High-Performance liquid Chromatography- tandem mass spectrometry in dynamic multiple reaction monitoring mode

Asarum and Aristolochia are two large genera of Aristolochiaceae plants containing typical toxicant aristolochic acid analogs(AAAs), AAAs can be deemed as toxicity markers of Aristolochiaceae plants. Based on the least AAAs in dry roots and rhizomes of Asarum heterotropoides, Asarum sieboldii Miq and Asarum sieboldii var, all of which are enrolled in the Chinese pharmacopeia up to now. AAAs distribution in Aristolochiaceae plants, especially Asarum L. plants, is still obscure and controversial due to few AAAs measured, unverified species of Asarum, and complicated pretreatment in analytical samples making the results more challenging to reproduce. In the present study, a simple ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method in dynamic multiple reaction monitoring mode for simultaneous determination of thirteen AAAs was developed for evaluating the distribution of toxicity phytochemicals in Aristolochiaceae plants. The sample was prepared by extracting Asarum and Aristolochia powder with methanol, and the supernatant was analyzed using the Agilent 6410 system on an ACQUITY UPLC HSS PFP column with gradient elution of water and acetonitrile, containing 1% v/v formic acid (FA) each, at a flow rate of 0.3 mL/min. The chromatographic condition provided good peak shape and resolution. The method was linear over the specific ranges with the coefficient of determination (R2) > 0.990. Satisfactory intra- and inter-day precisions were achieved with RSD less than 9.79%, and the average recovery factors obtained were in the range of 88.50%～105.49%%. The proposed method was successfully applied for simultaneous quantification of the 13 AAAs in 19 samples from 5 Aristolochiaceae species, especially three Asarum L. species enrolled in the Chinese Pharmacopoeia. Except Asarum heterotropoides, the results supported that the Chinese Pharmacopoeia (2020 Edition) adopting the root with rhizome as medicinal parts of Herba Asari instead of the whole herb for drug safety by providing scientific data.

A New Multi-Analyte LC-MS-MS Screening Method for the Detection of 120 NPSs and 49 Drugs in Hair.

Liquid chromatography coupled with mass spectrometry (LC-MS) has been increasingly used for screening purposes in forensic toxicology. High versatility and low time/resource consumption are the main advantages of this technology. Numerous multi-analyte methods have been validated in order to face the analytical challenge of new psychoactive substances (NPSs). However, forensic toxicologists must focus the attention also on "classical" NPSs and medicines, such as benzodiazepines (BDZs) and prescription opioids. In this paper, a new method for the simultaneous detection of 169 substances (120 NPSs and 49 other drugs) in hair by LC-MS-MS is described. After the decontamination of hair samples with dichloromethane, a 20-mg aliquot of the sample was mixed with 1 mL of methanol (MeOH; 0.1% of formic acid) and then sonicated at room temperature for 2 h. The mixture was then dried under nitrogen stream and reconstituted with 100 µL of MeOH. LC separation was achieved with a 100-mm-long C18 column in 35 min, and mass acquisition was performed in dynamic multiple reaction monitoring mode and in positive ionization. The analysis results were very sensitive, with the limit of quantification ranging from 0.07 to 10.0pg/mg. Accuracy and precision were always within the acceptable criteria. Matrix effect and recovery rate ranges were from -21.3 to + 21.9% and from 75.0 to 99.3%, respectively. The new method was successfully applied in a preliminary study on the prevalence of NPSs, BDZs and other substances in case of driving license issuance. In 14% of cases, BDZs/antidepressants (mainly trazodone, diazepam/nordiazepam and flunitrazepam) were found. Codeine, ketamine, methylone and mephedrone were also detected.

A simple and rapid method for extraction and measurement of circulating sphingolipids using LC-MS/MS: a targeted lipidomic analysis.

Sphingolipids are a class of lipids with high structural diversity and biological pleiotropy. Mounting evidence supports a role for sphingolipids in regulating pathophysiology of cardiometabolic diseases, and they have been proposed as potential cardiometabolic biomarkers. Current methods for quantifying sphingolipids require laborious pretreatment and relatively large sample volumes, and cover limited species, hindering their application in epidemiological studies. Herein, we applied a time-, labor-, and sample-saving protocol simply using methanol for plasma sphingolipid extraction. It was compared with classical liquid-liquid extraction methods and showed significant advantages in terms of simplicity, sphingolipid coverage, and sample volume. By coupling the protocol with liquid chromatography using a wide-span mobile phase polarity parameter and tandem mass spectrometry operated in dynamic multiple reaction monitoring mode, 37 sphingolipids from 8 classes (sphingoid base, sphingoid base phosphate, ceramide-1-phosphate, lactosylceramide, hexosylceramide, sphingomyelin, ceramide, and dihydroceramide) were quantified within 16min, using only 10 μL of human plasma. The current method showed good performance in terms of linearity (R2 > 0.99), intra- and interbatch accuracy (70-123%) and precision (RSD < 12%), matrix effect (91-121%), recovery (96-101%), analyte chemical stability (deviation < 19%), and carryover (< 16%). We successfully applied this method to quantify 33 detectable sphingolipids from 579 plasma samples of an epidemiological study within 10days. The quantified sphingolipid concentrations were comparable with previous studies. Positive associations of ceramide C22:0/C24:0 and their precursors with homeostasis model assessment of insulin resistance suggested that the synthesis of the ceramides might be involved in insulin resistance. This novel method constitutes a simple and rapid approach to quantify circulating sphingolipids for epidemiological studies using targeted lipidomic analysis, which will help elucidate the sphingolipid-regulated pathways underlying cardiometabolic diseases.

Screening Method for 23 Alkaloids in Human Serum Using LC/MS/MS with a Pentafluorophenyl Column in Dynamic Multiple Reaction Monitoring Mode

Alkaloids are nitrogen-containing organic compounds, generally basic, and found in plants, fungi, and bacteria. Some alkaloids are used in medicine, but some compounds are highly toxic. Accidental ingestion, homicide, and suicide have occurred due to plants containing alkaloids. The identification of toxic components in biological samples is important for the diagnosis and/or treatment of poisoning cases in forensic and emergency medicine. Alkaloids have a wide variety of structures, such as isoquinoline alkaloid, indole alkaloid, tropane alkaloid, and diterpene alkaloid; therefore, there are few reports of simultaneous analysis methods. We have established a method for the simultaneous analysis of 23 alkaloids in human serum with a liquid chromatograph-tandem mass spectrometer (LC/MS/MS). A liquid-liquid extraction which was modified from the first step of the QuEChERS AOAC method was used for serum pretreatment. The separation of the compounds was performed using a pentafluorophenyl (PFP) column, CAPCELL CORE PFP (2.1 mm I.D. × 100 mm, 2.7 μm) in gradient mode. Mobile phase A consisted of 10 mM ammonium formate and 0.1% formic acid in ultrapure water, and mobile phase B was 10 mM ammonium formate and 0.1% formic acid in methanol. Simultaneous analysis was performed in dynamic multiple reaction monitoring mode. The separation of 23 alkaloids was satisfactory, as PFP columns exhibited different retention behaviors than alkyl phase columns. The PFP column effectively retained polar aromatic compounds; therefore, it was suitable for alkaloid analysis. The validated method was applied to a forensic case of aconite poisoning. The present method was useful in LC/MS/MS screening for 23 alkaloids in human serum.

Effective sample preparation procedure for the analysis of free neutral steroids, free steroid acids and sterol sulfates in different tissues by GC–MS

Steroids play an important role in cell regulation and homeostasis. Many diseases like Alzheimer’s disease or Smith-Lemli-Opitz syndrome are known to be associated with deviations in the steroid profile. Most published methods only allow the analysis of small subgroups of steroids and cannot give an overview of the total steroid profile. We developed and validated a method that allows the analysis of free neutral steroids, including intermediates of cholesterol biosynthesis, free oxysterols, C19 and C21 steroids, free steroid acids, including bile acids, and sterol sulfates using gas chromatography-mass spectrometry. Samples were analyzed in scan mode for screening purposes and in dynamic multiple reaction monitoring mode for highly sensitive quantitative analysis. The method was validated for mouse brain and liver tissue and consists of sample homogenization, lipid extraction, steroid group separation, deconjugation, derivatization and gas chromatography-mass spectrometry analysis. We applied the method on brain and liver samples of mice (10 months and 3 weeks old) and cultured N2a cells and report the endogenous concentrations of 29 physiological steroids.

Universal analytical method for characterization of yellow and related natural dyes in liturgical vestments from Krakow

Due to the diversity of dyes, as well as the multiplicity of colorants occurring in them, their identification in historical objects requires the development of an effective analytical approach including also the separation step. Thus, the present study was focused on high-performance liquid chromatography (with a reversed phase phenyl column) coupled with spectrophotometric and tandem mass spectrometric detections with electrospray ionization (HPLC–UV–Vis–ESI MS/MS) to develop a universal analytical approach dedicated to a comprehensive analysis of wide range of dyes. Optimization of ion source parameters led to a total increment in intensity of peaks even up to 64%. The final method, developed using dynamic multiple reaction monitoring mode (dMRM), includes 112 colorants in negative ion mode and/or 12 in positive ion mode, 62 of which are reference compounds, while 54 are compounds identified by tandem mass spectrometry in fibers dyed with 10 examined yellow and orange dyes (annatto, cutch, dyer's broom, old fustic, Osage orange, saffron, sawwort, Persian berries, weld and young fustic). The developed method was tested for the identification of natural dyes in yellow, orange, brown and green fibers from 15th- to 17th-century textiles used in the vestments from the collections of seventeen Krakow churches. In this way, annatto and sawwort have been found in historical samples using HPLC–UV–Vis or HPLC–ESI MS/MS for the first time.

Development and validation of an ultra-high performance liquid chromatography/triple quadrupole mass spectrometry method for analyzing microbial-derived grape polyphenol metabolites

Accumulating evidence indicates that the health impact of dietary phenolic compounds, including the principal grape-derived polyphenols, (+)‑catechin and (−)‑epicatechin, is exerted by not only the parent compounds but also their phenolic metabolites generated by the gut microbiota. In this work, a new high-throughput, sensitive and reproducible analytical method was developed employing ultra-high performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (UHPLC-QqQ-MS/MS) for the simultaneous analysis of 16 microbial-generated phenolic acid metabolites (PAMs) along with their precursors, catechin and epicatechin. Following optimizing the solvent system, LC conditions and MS parameters, method validation was carried out to evaluate the sensitivity, selectivity, accuracy and precision of the proposed method, and to ensure promising recovery of all analytes extracted from the matrix prior to bioanalysis. Results showed that the optimized analytical method allowed successful confirmation and quantitation of all analytes under dynamic multiple reaction monitoring mode using trans‑cinnamic acid‑d7 as an internal standard (I.S.). Excellent sensitivity and linearity were obtained for all analytes, with lower limits of detection (LLODs) and lower limits of quantification (LLOQs) in the ranges of 0.225–2.053 ng/mL and 0.698–8.116 ng/mL, respectively. By examining blank matrix spiked with standard mixture at different concentration levels, promising recoveries at two spiking levels (low level, 91.2–115%; high level 90.2–121%), and excellent precision (RSD < 10%) were obtained. This method was then successfully applied to an in vitro study where catechin/epicatechin-enriched broth samples were anaerobically fermented with gut microbes procured from healthy human donors. All sources of bacteria employed showed remarkable activity in metabolizing grape polyphenols and distinct variations in the production of PAMs. The successful application of this method in the in vitro fermentation assays demonstrates its suitability for high-throughput analysis of polyphenol metabolites, particularly catechin/epicatechin-derived PAMs, in biological studies.

Optimization of sample preparation and chromatography for the determination of perfluoroalkyl acids in sediments from the Yangtze Estuary and East China Sea

Perfluoroalkyl acids (PFAAs) are ubiquitous pollutants present in various environmental media, including marine sediments. A method was proposed for the determination of 17 target PFAA analytes in marine sediment samples (n = 49) collected from the Yangtze Estuary and East China Sea. The proposed method involves the use of an optimized pretreatment procedure and ultrahigh-performance liquid chromatography electrospray ionization–tandem mass spectrometry in dynamic multiple reaction monitoring mode. The method relied on extraction cycles using methanol followed by concentration, filtration, and small volume injection to UHPLC-MS/MS. The recovery, time efficiency, and detection limit of the proposed method are improved relative to those of traditional methods. Limits of detection varied from 0.003 to 0.045 ng/g, and spike recoveries to sediment ranged from 90% to 110% with suitable precisions (1.7%–14.6%). PFAAs were widely present in the samples, and ΣPFAAs ranged from 0.67 ng/g dw to 36.75 ng/g dw. Results indicated that terrigenous input strongly influences PFAA distribution in sediments from the study areas. Perfluorooctanoic acid (PFOA) and perfluorooctanesulfonate (PFOS) were identified as the dominant perfluorocarboxylic acid (PFCA) and perfluoroalkylsulfonate (PFSA) in sediment samples from the Yangtze Estuary and the East China Sea. Preliminary environmental risk assessment indicated that PFOS may pose a higher environmental risk than PFOA. Furthermore, risk quotient values indicated that PFOS poses a significant risk to the aquatic ecosystem of the study areas.

Multicomponent determination of traditional Chinese medicine preparation yin-zhi-huang injection by LC-MS/MS for screening of its potential bioactive candidates using HepaRG cells.

Yin-zhi-huang (YZH) injection is an injectable multiherbal prescription derived from the ancient Chinese medicine formula of Yin-chen-hao-tang, which is widely used in the clinic for the treatment of jaundice and chronic liver diseases. To date, the systematic study of the components in this multiherbal prescription still lacks suitable analytical methods that are able to simultaneously detect a broad array of components at low concentrations. In this study, a new liquid chromatography-tandem mass spectrometry method using dynamic multiple reaction monitoring mode was developed to determine multiple peaks in traditional Chinese medicine preparation YZH injection. This simple, selective and sensitive method enabled the quantification of 22 components with standard materials with a lower limit of quantification of 1.46-12.5 ng/mL in cell lysates. This method was successfully applied to celluar uptake and binding investigation of components in YZH injection. The results indicated that this strategy might be a useful approach for rapidly screening of the potential bioactive candidates from YZH injection, and the discovered candidates could be used to investigate the pharmacodynamics in further studies.

Simultaneous Quantitation of Methamphetamine, Ketamine, Opiates and their Metabolites in Urine by SPE and LC-MS-MS.

Heroin, methamphetamine and ketamine have been the most commonly abused drugs in Taiwan. The presence of these drugs and their metabolites in postmortem specimens has been routinely monitored in our laboratory mostly by gas chromatographic-mass spectrometric methods. This study aimed to evaluate a more effective approach to simultaneously quantify these analytes (i.e., amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine (MDMA), morphine, codeine, 6-acetylmorphine, 6-acetylcodeine, ketamine and norketamine) in postmortem urine and blood specimens by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Samples (1 mL) were extracted via solid-phase extraction, evaporated and reconstituted in the mobile phase for injection into the LC-MS-MS system. Respective deuterated analogs of these analytes were used as internal standards. Chromatographic separation was achieved by an Agilent Zorbax SB-Aq analytical column at 50°C. Mass spectrometric analysis was performed by electrospray ionization in positive-ion dynamic multiple reaction monitoring mode with optimized collision energy for respective precursor ion selected for each analyte, and the monitoring of two transition ions. Performance characteristics were assessed using drug-free samples that were fortified with 50-1,000 ng/mL of the 10 analytes. Analytical parameters evaluated and resulting data are as follows: (i) average extraction recoveries (n= 3) were better than 80%, except for MDMA (71%) and morphine (74%); (ii) inter-day and intra-day precision ranges (%CV) were 1.59-8.80% and 0.57-3.89%, respectively; (iii) calibration linearity (r2), detection limit and quantitation limit for all analytes were >0.999, 1 and 5 ng/mL, respectively; (iv) matrix effects (ion suppression) were observed for three analytes, but were satisfactorily compensated for by the deuterated internal standards adopted in the analytical protocol. This method was successfully applied to the analysis of specimens collected from unknown death cases from various district prosecutors' offices in Taiwan, and was also found helpful to understanding whether the detected opiates were derived from heroin or legal morphine/codeine-containing medications.

Adduct ion-targeted qualitative and quantitative analysis of polyoxypregnanes by ultra-high pressure liquid chromatography coupled with triple quadrupole mass spectrometry

Polyoxypregnane and its glycosides (POPs) are frequently present in plants of Asclepiadaceae family, and have a variety of biological activities. There is a great need to comprehensively profile these phytochemicals and to quantify them for monitoring their contents in the herbs and the biological samples. However, POPs undergo extensive adduct ion formation in ESI-MS, which has posed a challenge for qualitative and quantitative analysis of POPs. In the present study, we took the advantage of such extensive adduct ion formation to investigate the suitability of adduct ion-targeted analysis of POPs. For the qualitative analysis, we firstly demonstrated that the sodium and ammonium adduct ion-targeted product ion scans (PIS) provided adequate MS/MS fragmentations for structural characterization of POPs. Aided with precursor ion (PI) scans, which showed high selectivity and sensitivity and improved peak assignment confidence in conjunction with full scan (FS), the informative adduct ion-targeted PIS enabled rapid POPs profiling. For the quantification, we used formic acid rather than ammonium acetate as an additive in the mobile phase to avoid simultaneous formation of sodium and ammonium adduct ions, and greatly improved reproducibility of MS response of POPs. By monitoring the solely formed sodium adduct ions [M+Na]+, a method for simultaneous quantification of 25 POPs in the dynamic multiple reaction monitoring mode was then developed and validated. Finally, the aforementioned methods were applied to qualitative and quantitative analysis of POPs in the extract of a traditional Chinses medicinal herb, Marsdenia tenacissima (Roxb.) Wight et Arn., and in the plasma obtained from the rats treated with this herb. The results demonstrated that adduct ion formation could be optimized for the qualitative and quantitative analysis of POPs, and our developed PI/FS-PIS scanning and sole [M+Na]+ ion monitoring significantly improved the analysis of POPs in both herbal and biological samples. This study also provides implications for the analysis of other compounds which undergo extensive adduct ion formation in ESI–MS.

Validated Method for the Screening and Quantification of Baclofen, Gabapentin and Pregabalin in Human Post-Mortem Whole Blood Using Protein Precipitation and Liquid Chromatography-Tandem Mass Spectrometry.

There has been a rapid increase in the number of prescriptions for baclofen (BLF), gabapentin (GBP) and pregabalin (PGL) in the UK since their introduction to therapy. Recent studies across the European Union and USA have shown the illicit abuse potential of these drugs and deaths have been observed. A simple, reliable and fully validated method was developed for the screening and quantification of BLF, GBP and PGL in human post-mortem (PM) blood. The analytes and their deuterated analogs as internal standard were extracted from blood using a single addition acetonitrile protein precipitation reaction followed by analysis using liquid chromatography-tandem mass spectrometry (LC-MS-MS) with triggered dynamic multiple reaction monitoring mode for simultaneous confirmation and quantification. The assay was linear from 0.05 to 1.00 µg/mL for BLF and 0.5 to 50.0 µg/mL for GBP and PGL, respectively with r2 > 0.999 (n = 9) for all analytes. Intra-day and inter-day imprecisions (n = 80) were calculated using one-way ANOVA; no significant difference (P > 0.99) was observed for all analytes over 8 non-consecutive days. The average recovery for all analytes was >98.9%. The limits of detection and quantification were both 0.05 µg/mL for BLF, and 0.5 µg/mL for GBP and PGL. The method was highly selective with no interference from endogenous compounds or from 54 drugs commonly encountered in PM toxicology. To prove method applicability, 17 PM blood samples submitted for analysis were successfully analyzed. The concentration range observed in PM blood for BLF was 0.08-102.00 µg/mL (median = 0.25 µg/mL), for GBP 1.0-134.0 µg/mL (median = 49.0 µg/mL) and 2.0-540.0 µg/mL (median = 42.0 µg/mL) for PGL.

Pharmacokinetics of Five Different Types of Safflower Constituents in Rat Plasma after Oral Administration of Safflower Powder

The petals of Carthamus tinctorius L. (safflower) is an important traditional medicine for promoting blood circulation and removing blood stasis. The pharmacokinetic properties of multiple safflower constituents have not been fully understood. In the present research, an ultra-high-performance liquid chromatography-triple quadrupole mass method in dynamic multiple reaction monitoring mode was developed and validated to simultaneously quantify in rat plasma of five different characteristic types of safflower constituents, including chalcone, poly-acetylene, glycoside of 6-hydroxykaempferol, etc. All the five constituents, hydroxysafflor yellow A, bidenoside C, 6-hydroxykaempferol-3,6-diglucoside, chlorogenic acid, and p-coumaric acid were detected in rat plasma after oral administration of safflower powder. p-Coumaric acid was the most quickly absorbed one with Tmax of 0.1 hr. The poly-acetylene constituent, bidenoside C, displayed the largest AUC0-t value (2177.796 ng · h/L) followed by hydroxysafflor yellow A (1564.1 ng · h/L). The impressively favorable pharmacokinetic behavior of bidenoside C reported herein for the first time rendered the importance for paying more attention to its role in the in vivo pharmacologic effects of safflower.

Validated Method for the Quantification of Baclofen in Human Plasma Using Solid-Phase Extraction and Liquid Chromatography-Tandem Mass Spectrometry.

A highly sensitive and fully validated method was developed for the quantification of baclofen in human plasma. After adjusting the pH of the plasma samples using a phosphate buffer solution (pH 4), baclofen was purified using mixed mode (C8/cation exchange) solid-phase extraction (SPE) cartridges. Endogenous water-soluble compounds and lipids were removed from the cartridges before the samples were eluted and concentrated. The samples were analyzed using triple-quadrupole liquid chromatography-tandem mass spectrometry (LC-MS-MS) with triggered dynamic multiple reaction monitoring mode for simultaneous quantification and confirmation. The assay was linear from 25 to 1,000 ng/mL (r(2) > 0.999; n = 6). Intraday (n = 6) and interday (n = 15) imprecisions (% relative standard deviation) were <5%, and the average recovery was 30%. The limit of detection of the method was 5 ng/mL, and the limit of quantification was 25 ng/mL. Plasma samples from healthy male volunteers (n = 9, median age: 22) given two single oral doses of baclofen (10 and 60 mg) on nonconsecutive days were analyzed to demonstrate method applicability.

Effects of Types of Washing and Peeling in Relation to Pesticide Residues in Tomatoes

Tomatoes are widely consumed and most often without cooking, therefore it is important to estimate the reduction of residues using simple washing procedures. An experiment was carried out to evaluate the effects of household processing such as: washing with water, washing with 10% of vinegar solution and washing with 10% of sodium bicarbonate solution on pesticide residue levels of acetamiprid, azoxystrobin, diflubenzuron, dimethoate, fipronil, imidacloprid, procymidone and thiamethoxam, in spiked tomato samples. The amount of residue remaining in the peel and pulp was also investigated. The pesticide residues were monitored using the quick, easy, cheap, effective, rugged and safe (QuEChERS) extraction and analysed by liquid chromatography tandem mass spectrometry method (LC-MS/MS) in dynamic multiple-reaction monitoring mode (DMRM). The effect of the number of washing treatments (n = 3) and control, with six replicates, differed significantly for all pesticides evaluated (n = 8), but despite washing, fipronil residues were not removed from the tomato samples. Overall, washing with water or other solutions, and peeling before consumption are shown to reduce pesticide residues in tomatoes.