[Application and safety evaluation of plant growth regulators in traditional Chinese medicine].

Plant growth regulator is a kind of synthetic pesticide with similar physiological activity to plant hormones. It has been widely used in grain, vegetables, fruits, flowers and other crops, and become an important technical guarantee for high yield, stable yield, high quality and efficient production of crops. In recent years, plant growth regulator is widely used in Chinese herbal medicine production for regulating the growth and development and increasing production of traditional. However the crop is different from general Chinese medicinal materials, the use of plant growth regulator should not only consider the effect of Chinese herbal medicine production, and also pay special attention to the influence of Chinese traditional medicine efficacy and safety. This paper reviewed the application of plant growth regulator in the traditional Chinese medicine, the impact on the quality and safety of Chinese medicinal materials, as well as plant growth regulator of residue limits standards and testing technology, so as to the scientific use of plant growth regulator, to promote Chinese standardization planting, provide the scientific basis to protect the safety of herbal medicine. At present, the indiscriminate use and abuse of plant growth regulators such as Zhuanggenling and bulking element are common in the production of Chinese crude drugs, which has led to a significant decline in the quality of some Chinese crude drugs, and resulted in the dual residual harm to the Chinese crude drugs and the cultivation environment, causing serious safety risks to human health. In the future, it is necessary to strengthen the registration management, use norms and limit standards of plant growth regulators in traditional Chinese medicinal materials, and strengthen the supervision and regulations on the use of fertilizer instead of medicine to avoid pesticide registration and other disorders, so as to provide a basis for the quality and safety monitoring of traditional Chinese medicinal materials. Simultaneously, it is encouraged to reduction or non-application of plant growth regulators in the production of Chinese medicinal materials, especially for traditional Chinese medicine which contains a variety of active ingredients. Therefore, it is actively advocated to cultivate Chinese medicinal materials through organic or ecological method.

Multi-residue analysis of plant growth regulators and pesticides in traditional Chinese medicines by high-performance liquid chromatography coupled with tandem mass spectrometry.

A multi-residue method was developed for the simultaneous determination of 24 plant growth regulators (PGRs) and 11 representative pesticides that were widely applied in plants used in traditional Chinese medicines (TCMs) by high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). The method was validated taking into consideration EU guidelines; calibration curves for all of the targeted analytes showed correlation coefficients (γ2) higher than 0.9901. The limits of detection (LOD) ranged from 0.2 to 8 µg/kg. The average recovery for all analytes in spiked samples ranged from 63.18 to 127.23%, with a relative standard deviation of ≤ 15%. The proposed method has been applied to 480 batches of TCM samples, including 34 species of medicinal plants, from the TCM market. The results showed that 14 PGRs and 5 pesticides were detected, including choline chloride, chlormequat, paclobutrazol, uniconazole, phoxim, etc. Among them, there were high detection rates for chlormequat (40%), choline chloride (100%), atonik (73.75%), abscisic acid (80.83%), and indole-3-acetic acid (41.25%). The residual level of paclobutrazol in Ophiopogonis radix exceeded the recommended maximum residue limits (MRLs) according to GB 2763-2016. In addition, 14 agrochemicals used in TCM planting were collected and detected; the result showed various PGRs were detected in samples registered as fertilizer. These results indicate that PGRs and pesticides were widely used in the cultivation of medicinal plants, especially for radix and rhizome herbs. The residue of targeted PGRs and pesticides in TCM samples from this study have a high frequency and high level. Graphical abstract.

Identification of a Novel Specific Cucurbitadienol Synthase Allele in Siraitia grosvenorii Correlates with High Catalytic Efficiency.

Mogrosides, the main bioactive compounds isolated from the fruits of Siraitia grosvenorii, are a group of cucurbitane-type triterpenoid glycosides that exhibit a wide range of notable biological activities and are commercially available worldwide as natural sweeteners. However, the extraction cost is high due to their relatively low contents in plants. Therefore, molecular breeding needs to be achieved when conventional plant breeding can hardly improve the quality so far. In this study, the levels of 21 active mogrosides and two precursors in 15 S. grosvenorii varieties were determined by HPLC-MS/MS and GC-MS, respectively. The results showed that the variations in mogroside V content may be caused by the accumulation of cucurbitadienol. Furthermore, a total of four wild-type cucurbitadienol synthase protein variants (50R573L, 50C573L, 50R573Q, and 50C573Q) based on two missense mutation single nucleotide polymorphism (SNP) sites were discovered. An in vitro enzyme reaction analysis indicated that 50R573L had the highest activity, with a specific activity of 10.24 nmol min-1 mg-1. In addition, a site-directed mutant, namely, 50K573L, showed a 33% enhancement of catalytic efficiency compared to wild-type 50R573L. Our findings identify a novel cucurbitadienol synthase allele correlates with high catalytic efficiency. These results are valuable for the molecular breeding of luohanguo.

[Correlation between PAFAH1B3 Expression Level and Risk of Disease Progression After Autologous Hematopoietic Stem Cell Transplantation in Multiple Myeloma Patients].

OBJECTIVE: To investigate the association between the expression level of platelet-activating factor acetylhydrolase 1B3 (PAFAH1B3 ) gene in bone marrow CD138+ cells of patients with multiple myeloma (MM) treated with autologous hematopoietic stem cell transplantation (AHSCT) and the prognosis within 2 years. METHODS: 147 MM patients treated with AHSCT in The First and The Second Affiliated Hospital of Nantong University from May 2014 to May 2019 were included in the study. Expression level of PAFAH1B3 mRNA in bone marrow CD138+ cells of the patients was detected. Patients with disease progression or death during 2 years of follow-up were included in progression group, and the rest were included in good prognosis group. After comparing the clinical data and PAFAH1B3 mRNA expression levels of the two groups, the patients were divided into high PAFAH1B3 expression group and low PAFAH1B3 expression group based on the median PAFAH1B3 mRNA expression level of the enrolled patients. Progression-free survival rate (PFSR) between the two groups was compared by the Kaplan-Meier method. The related factors of prognosis within 2 years were analyzed by univariate analysis and multivariate COX regression analysis. RESULTS: At the end of follow-up, there were 13 patients lost to follow-up. Finally, 44 patients were included in the progression group and 90 patients were included in the good prognosis group. Age in the progression group was higher than that in the good prognosis group, the proportion of patients with CR+VGPR after transplantation in the progression group was lower than that in the good prognosis group, and there was a statistical difference between two groups in the cases distribution of ISS stage (all P<0.05). PAFAH1B3 mRNA expression level and the proportion of patients with LDH>250U/L in the progression group were higher than those in the good prognosis group, and platelet count in the progression group was lower than that in the good prognosis group (all P<0.05). Compared with the low PAFAH1B3 expression group, the 2-year PFSR of the high PAFAH1B3 expression group was significantly lower (log-rank χ2=8.167, P=0.004). LDH>250U/L (HR=3.389, P=0.010), PAFAH1B3 mRNA expression (HR=50.561, P=0.001) and ISS stage Ⅲ(HR=1.000, P=0.003) were independent risk factors for prognosis in MM patients, and ISS stage Ⅰ (HR=0.133, P=0.001) was independent protective factor. CONCLUSION: The expression level of PAFAH1B3 mRNA in bone marrow CD138+ cells is related to the prognosis of MM patients treated with AHSCT, and detecting PAFAH1B3 mRNA expression can bring some information for predicting PFSR and prognostic stratification of patients.

Incidence, Predictors and Clinical Impact of Ventricular Electrical Storm in Arrhythmogenic Cardiomyopathy Patients with an Implantable Cardioverter-Defibrillator: A Single-Center Report with Medium-Term Follow-Up.

BACKGROUND: Implantable cardioverter-defibrillator (ICD) is the most effective strategy for prevention of ventricular tachyarrhythmia in patients with arrhythmogenic cardiomyopathy (ACM). However, some patients receive ventricular electrical storm (VES), characterized by multiple episodes of sustained ventricular tachyarrhythmia. The purpose of this study was to determine the incidence, predictors and prognostic implications of VES in ACM patients with an ICD. METHODS: A total of 88 patients with definite ACM who received an ICD and followed up continuously were included in this study. VES was defined as the occurrence of ≥3 separate episodes of sustained ventricular arrhythmias within a 24-hour period. RESULTS: During a median follow-up time of 4.0 years (range 1.6-6.9), VES occurred in 19/88 patients (21.6%). The interval between the ICD implantation and the first VES ranged from 1 month to 128 months. The median number of ventricular tachyarrhythmia events per VES was 7.5 (range 3-32). Multivariate analysis showed that VES was associated with a high body mass index (BMI) [adjusted hazard ratio (HR) 1.21, 95% confidence interval (CI) 1.00-1.45, P=0.048)] and extensive T-wave inversion (TWI) (HR 23.39, 95% CI 1.74-314.58, P=0.017). Kaplan-Meier method showed that patients with VES did not have a worse cardiac mortality compared to those without such an event. CONCLUSION: There is a relatively high incidence of VES in ACM patients. The presence of high BMI and extensive TWI were strong predictors of VES occurrence in ACM patients with ICD. VES does not independently confer increased cardiac mortality.

Artificial Intelligence Algorithm with ICD Coding Technology Guided by the Embedded Electronic Medical Record System in Medical Record Information Management.

The study aims to explore the application of international classification of diseases (ICD) coding technology and embedded electronic medical record (EMR) system. The study established an EMR information knowledge system and collected the data of patient medical records and disease diagnostic codes on the front pages of 8 clinical departments of endocrinology, oncology, obstetrics and gynecology, ophthalmology, orthopedics, neurosurgery, and cardiovascular medicine for statistical analysis. Natural language processing-bidirectional recurrent neural network (NLP-BIRNN) algorithm was used to optimize medical records. The results showed that the coder was not clear about the basic rules of main diagnosis selection and the classification of disease coding and did not code according to the main diagnosis principles. The disease was not coded according to different conditions or specific classification, the code of postoperative complications was inaccurate, the disease diagnosis was incomplete, and the code selection was too general. The solutions adopted were as follows: communication and knowledge training should be strengthened for coders and medical personnel. BIRNN was compared with the convolutional neural network (CNN) and recurrent neural network (RNN) in accuracy, symptom accuracy, and symptom recall, and it suggested that the proposed BIRNN has higher value. Pathological language reading under artificial intelligence algorithm provides some convenience for disease diagnosis and treatment.

The physicochemical properties and the in vivo AChE inhibition of two potential anti-Alzheimer agents, bis(12)-hupyridone and bis(7)-tacrine.

The lipophilicity and solubility profiles of bis(12)-hupyridone (B12H) and bis(7)-tacrine (B7T), two novel acetylcholinesterase inhibitors dimerized from huperzine A fragments and tacrine, respectively, were investigated over a broad pH range. Lipophilicity was assessed by both shake flask method with 1-octanol-water system and a reverse-phase HPLC system with methanol-water as mobile phase. The former method was used for determining the lipophilicities of the ionized forms (log D) of the dimers while the latter method was used for that of the neutral forms (log P). The log P values for B12H and B7T were found to be 5.4 and 8.2, respectively, indicating that the two dimers are highly lipophilic. The solubilities of both dimers were found to be affected by pH. The solubility of B12H was >1.41 mg/ml when the pH was <7, but <0.06 mg/ml when the pH was >8. The solubility of B7T was >0.26 mg/ml when the pH was <9, but <0.005 mg/ml when the pH was >12. The ionic strength of a solution could affect the solubilities considerably (11.16 mg/ml for B12H and 12.71 mg/ml for B7T in water; 2.07 mg/ml for B12H and 0.36 mg/ml for B7T in saline). The ionization constants (pK(a)) of the two dimers were determined by UV spectrophotometry. Both dimers were found to have two pK(a) values: 7.5+/-0.1 (pK(a1)) and 10.0+/-0.2 (pK(a2)) for B12H; and 8.7+/-0.1 (pK(a1)) and 10.7+/-0.4 (pK(a2)) for B7T. Furthermore, an in vivo pharmacological assay conducted in mice showed that a maximum AChE inhibition occurred 15 min after the single-dose and intraperitoneal administration of either dimer. This indicates that the two dimers may easily cross the blood-brain barrier. In summary, these physiochemical characteristics suggest that the two dimers may be promising candidates for the development of better drugs for Alzheimer's disease.

Treatment of dinitrodiazophenol industrial wastewater in heat-activated persulfate system.

Heat-activated persulfate oxidation process was investigated as the treatment of dinitrodiazophenol industrial wastewater to degrade refractory pollutants and improve biodegradability. By studying the effects of 4 factors and carrying out orthogonal tests and scale-up experiments, optimal treatment conditions (temperature 90 °C, reaction time 75 min, PS dosage 20.0 g L-1 and initial pH ∼2.0) were obtained. The results showed that under these conditions, COD and color removal efficiencies were 99.22% and 99.99%, respectively. Moreover, an increase in BOD5/COD ratio (from 0 to 0.31) indicates significantly improved biodegradability. Dinitrodiazophenol dosage was measured by high performance liquid chromatography, which showed that dinitrodiazophenol removal efficiency reached 99.99%. Furthermore, the degradation process was analyzed by ultraviolet-visible spectra and Fourier transform infrared spectra. The former demonstrated that aromatic compounds in the system were destroyed during mineralization and the latter indicated that nitro groups on the benzene ring could be oxidized to nitrate. After verification test of the free radicals, mechanism of heat-activated persulfate system was assumed to be that SO4˙- and ·OH function together and SO4˙- predominate. To conclude, the heat-activated PS oxidation technology performs effectively in treatment of DDNP wastewater and expands applications of sulfate-radical-based advanced oxidation technology in industrial-wastewater treatment.

Development of a high performance liquid chromatography-tandem mass method for determination of bis(7)-tacrine, a promising anti-Alzheimer's dimer, in rat blood.

An analytical method using on-line high performance liquid chromatography-tandem mass spectrometry with electrospray ionization was developed and applied for the quantification of bis(7)-tacrine (B7T) in rat blood. B7T and pimozide (internal standard, IS) were extracted in a single step from 100 microl of alkalized blood with ethyl acetate. Analytes were separated using an Extend C-18 column at 25 degrees C. The elution was achieved isocratically with a mobile phase composed of 0.05% aqueous formic acid and acetonitrile (60:40, v/v) at a flow rate of 0.35 ml/min. Quantification was achieved by monitoring the selected ions at m/z 247 for B7T and m/z 462-->m/z 328 for pimozide. Retention times were 1.45 and 2.23 min for B7T and IS, respectively. Calibration curves were linear in the range from 86.4 to 2160.0 ng/ml. The established method is rapid, selective and sensitive for the identification and quantification of B7T in biological samples. The assay is accurate (bias <10%) and reproducible (intra- and inter-day variation <10%), with detection and quantification limit of 3.6 and 42.3 ng/ml, respectively. Furthermore, it was successfully applied for the pharmacokinetic measurement of B7T in rat with a single intravenous administration at 0.3mg/kg.

Pharmacokinetics, Metabolism and Disposition of [14C]XQ-1H After Intravenous Administration to Male Rats.

OBJECTIVE: This study describes the in vivo pharmacokinetics and metabolism of [14C]labeled XQ-1H in male rats. METHODS: XQ-1H is a methanesulfonate of XQ, 10-O-(N,N-dimethylaminoethyl)-ginkgolide B, a derivative of ginkgolide B (GB) with enhanced water solubility. Since it is very difficult to synthesize radiolabeled GB, the results obtained in this study may provide helpful insight to further ADME investigation of GB and its analogue compounds. After an i.v. administration of [14C]XQ-1H to male rats, XQ (the freebase form of XQ-1H) was extensively hydrolyzed, moderately metabolized, and mainly excreted in feces (71.5% of the dose) via the biliary route. RESULTS: The main enzyme mediated metabolic pathways were mono- and di-demthylation. Using the radiolabel form of XQ-1H, the temporal binding of XQ to red blood cells was observed. CONCLUSION: Binding of XQ to RBCs may lower the blood's viscosity and thus provide symptomatic improvement of ischemic stroke patients.

Inorganic phosphate-triggered release of anti-cancer arsenic trioxide from a self-delivery system: an in vitro and in vivo study.

On-demand drug delivery is becoming feasible via the design of either exogenous or endogenous stimulus-responsive drug delivery systems. Herein we report the development of gadolinium arsenite nanoparticles as a self-delivery platform to store, deliver and release arsenic trioxide (ATO, Trisenox), a clinical anti-cancer drug. Specifically, unloading of the small molecule drug is triggered by an endogenous stimulus: inorganic phosphate (Pi) in the blood, fluid, and soft or hard tissue. Kinetics in vitro demonstrated that ATO is released with high ON/OFF specificity and no leakage was observed in the silent state. The nanoparticles induced tumor cell apoptosis, and reduced cancer cell migration and invasion. Plasma pharmacokinetics verified extended retention time, but no obvious disturbance of phosphate balance. Therapeutic efficacy on a liver cancer xenograft mouse model was dramatically potentiated with reduced toxicity compared to the free drug. These results suggest a new drug delivery strategy which might be applied for ATO therapy on solid tumors.

Correlations between serum levels of 25-hydroxyvitamin D and carotid atherosclerosis in patients with type 2 diabetes in Shanghai.

OBJECTIVE: To explore the potential association between the serum levels of 25-hydroxyvitamin D [25(OH)D] and carotid atherosclerosis in patients with type 2 diabetes. MATERIAL AND METHODS: Three hundred and fifty patients with type 2 diabetes were enrolled in this study in Shanghai, China. B-mode ultrasound was used to detect carotid plaques as indicators of atherosclerosis and measure carotid artery intima-media wall thickness (C-IMT) at two sites of carotid artery. Subjects were divided into group A (patients with carotid plaques) and group B (patients without carotid plaques) and be assessed clinically. Serum levels of 25(OH)D and other clinical parameters were measured. Multivariate logistic regression was performed to find predictors of carotid atherosclerosis in the entire group. RESULTS: The levels of serum 25(OH)D were lower in group A than in group B[19.60 (13.30-25.73) vs 23.19 (18.10-30.06)ng/ml, P<0.001]. The C-IMT levels [(1.00±0.17 vs 0.88±0.20)mm, Ptrend<0.001] and proportion of people with carotid plaques(44/88 vs 20/87, Ptrend<0.001) in the lowest quartile of 25(OH)D were higher than in the highest quartile. Vitamin D concentrations were inversely associated with HbA1c in women(r=-0.194, P=0.006), and C-IMT in men(r=-0.409, P<0.001). Logistic regression analysis showed age, male sex, current smoke, history of hypertension, SBP, LDL-C and lg[25(OH)D] (OR: 0.924, 95%CI: 0.893-0.955, P<0.001) were independently associated with the presence of carotid plaques in T2DM. CONCLUSIONS: Serum vitamin D level is significantly and independently associated with carotid atherosclerosis in patients with T2DM in Shanghai, China.

Serum levels of 25-hydroxyvitamin D are associated with cognitive impairment in type 2 diabetic adults.

Hypovitaminosis D is highly prevalent in type 2 diabetes. The aim of this study is to determine the serum levels of 25-hydroxyvitamin D [25(OH)D] in type 2 diabetic patients with and without mild cognitive impairment (MCI), and examine the relationship of 25(OH)D and MCI with other clinical factors. One hundred and sixty-five diabetic patients were enrolled in this study. Among whom, 95 patients were considered as MCI [Montreal Cognitive Assessment score (MoCA) < 26] and the other 70 as no MCI (MoCA ≥ 26). Subjects were assessed clinically. Diabetic patients with MCI had a longer duration of DM, fewer years of education, elevated fasting blood glucose (FBG), resistant index (RI) of carotid, and lower levels of 25(OH)D {[17.35 (13.02-25.92) vs 28.00 (19.67-34.30)] ng/ml, P < 0.001}. The MoCA score was positively correlated with log10[25(OH)D], education year, and inversely correlated with duration of DM, history of hypertension, intima-media thickness (IMT), FBG, max-RI, and min-RI. Log10[25(OH)D] was positively correlated with MoCA score, and inversely correlated with IMT, in multivariate regression analysis adjusted for age, sex, and education year, 25(OH)D (ß = 0.210, P = 0.003), history of hypertension (ß = -0.191, P = 0.007), IMT (ß = -0.194, P = 0.007), and FBG (ß = -0.157, P = 0.026) independently predicted MoCA score. In conclusion, our results suggest that levels of serum 25(OH)D are inversely associated with the cognitive impairment in diabetic patients. Vitamin D may be a potential protective factor for cognitive impairment in patients with type 2 diabetes.

Risk factors of mild cognitive impairment in middle aged patients with type 2 diabetes: a cross-section study.

UNLABELLED: The aim of this study was to evaluate the risk factors of mild cognitive impairment (MCI) in middle-aged patients with type 2 diabetes (T2DM). METHODS: Montreal Cognitive Assessment (MoCA) was applied as cognition assessment implement. One hundred and fifty-seven middle-aged type 2 diabetic patients were enrolled in this cross-section study (age 40~69, mean age 55 ± 7). There were 93 patients with MCI (MoCA score<26) in MCI group and 64 with normal cognitive function (MoCA score ≥ 26) in control group. Information of history of disease, family history, data of BMI, WHR, HbA1c, FINS, C-Peptide (C-P), SBP, DBP, blood lipid (TG, TC, LDL-C, HDL-C and carotid ultrasound (carotid IMT, carotid resistance index [RI]) was collected. RESULTS: There were significant differences in the rate of patients with hypertension ([40.63 vs. 58.06%], P=0.026), duration of diabetes mellitus ([3.09 ± 4.04 y vs. 4.80 ± 4.94 y], P=0.024), C-P ([2.79 ± 1.09 ng/ml vs. 2.26 ± 1.00 ng/ml], P=0.008), Max C-IMT ([0.81 ± 0.15 mm vs. 0.91 ± 0.15 mm], P<0.001), Min C-RI (0.71 ± 0.06 vs. 0.68 ± 0.06, P<0.05), and no significant differences in the duration of hypertension and hyperlipidemia, BMI, WHR, HbA1c, SBP, DBP and blood lipid between control group and MCI group. MoCA scores were positively correlated with C-P (r=0.252, P=0.005), and negatively correlated with the history of hypertension (r=-0.244, P=0.002), duration of DM (r=-0.161, P=0.044), Max C-IMT (r=-0.253, P=0.005) and Min C-RI (r=-0.183, P=0.023). Multiple regression analysis showed that history of hypertension (Beta=-0.267, P=0.002), C-P (Beta=0.281, P=0.001) and Min C-RI (Beta=-0.221, P=0.011) were significantly independent determinants for the MoCA scores. CONCLUSIONS: The longer duration of diabetes, history of hypertension, lower serum C-P levels, thickened C-IMT and higher C-RI could be risk factors of MCI in type 2 diabetic patients. This finding could have an important impact on the management of cognitive decline in diabetic patients.

Biotransformation of aesculin by human gut bacteria and identification of its metabolites in rat urine.

AIM: To observe the biotransformation process of a Chinese compound, aesculin, by human gut bacteria, and to identify its metabolites in rat urine. METHODS: Representative human gut bacteria were collected from 20 healthy volunteers, and then utilized in vitro to biotransform aesculin under anaerobic conditions. At 0, 2, 4, 8, 12, 16, 24, 48 and 72 h post-incubation, 10 mL of culture medium was collected. Metabolites of aesculin were extracted 3 x from rat urine with methanol and analyzed by HPLC. For in vivo metabolite analysis, aesculetin (100 mg/kg) was administered to rats via stomach gavage, rat urine was collected from 6 to 48 h post-administration, and metabolite analysis was performed by LC/ESI-MS and MS/MS in the positive and negative modes. RESULTS: Human gut bacteria could completely convert aesculin into aesculetin in vitro. The biotransformation process occurred from 8 to 24 h post-incubation, with its highest activity was seen from 8 to 12 h. The in vitro process was much slower than the in vivo process. In contrast to the in vitro model, six aesculetin metabolites were identified in rat urine, including 6-hydroxy-7-gluco-coumarin (M1), 6-hydroxy-7-sulf-coumarin (M2), 6, 7-di-gluco-coumarin (M3), 6-glc-7-gluco-coumarin (M4), 6-O-methyl-7-gluco-coumarin (M5) and 6-O-methyl-7-sulf-coumarin (M6). Of which, M2 and M6 were novel metabolites. CONCLUSION: Aesculin can be transferred into aesculetin by human gut bacteria and is further modified by the host in vivo. The diverse metabolites of aesculin may explain its pleiotropic pharmaceutical effects.

Accurate mass filtering of ion chromatograms for metabolite identification using a unit mass resolution liquid chromatography/mass spectrometry system.

Acceleration of liquid chromatography/mass spectrometric (LC/MS) analysis for metabolite identification critically relies on effective data processing since the rate of data acquisition is much faster than the rate of data mining. The rapid and accurate identification of metabolite peaks from complex LC/MS data is a key component to speeding up the process. Current approaches routinely use selected ion chromatograms that can suffer severely from matrix effects. This paper describes a new method to automatically extract and filter metabolite-related information from LC/MS data obtained at unit mass resolution in the presence of complex biological matrices. This approach is illustrated by LC/MS analysis of the metabolites of verapamil from a rat microsome incubation spiked with biological matrix (bile). MS data were acquired in profile mode on a unit mass resolution triple-quadrupole instrument, externally calibrated using a unique procedure that corrects for both mass axis and mass spectral peak shape to facilitate metabolite identification with high mass accuracy. Through the double-filtering effects of accurate mass and isotope profile, conventional extracted ion chromatograms corresponding to the parent drug (verapamil at m/z 455), demethylated verapamil (m/z 441), and dealkylated verapamil (m/z 291), that contained substantial false-positive peaks, were simplified into chromatograms that are substantially free from matrix interferences. These filtered chromatograms approach what would have been obtained by using a radioactivity detector to detect radio-labeled metabolites of interest.

Reduction of nitriles to amines in positive ion electrospray ionization mass spectrometry.

Some compounds readily form [M+46]+ adduct ions during positive ion electrospray ionization mass spectrometry ((+)ESI-MS) analysis. These [M+46]+ ions were characterized as [M+CH3CH2NH2+H]+ by accurate mass determination. Ethylamine involved in the adduct was proposed to be the reduction product of acetonitrile and this was confirmed using deuterated acetonitrile. Other nitrile-containing compounds tested, including isobutyronitrile and benzonitrile, also formed the adduct ions of the respective amine forms under (+)ESI-MS conditions. Hydrogen/deuterium exchange experiments demonstrated that the reductive hydrogen originated from water. Reduction of nitriles (R-CN) to their respective amines (R-CH2NH2) under (+)ESI-MS conditions expands the ability to identify nitrile-containing chemical unknowns.