Formic Acid In Methanol Research Articles

Simultaneous Determination of Moxifloxacin Hydrochloride and Dexamethasone Sodium Phosphate in Rabbit Ocular Tissues and Plasma by LC-MS/MS: Application for Pharmacokinetics Studies.

Treatment of ocular infection involves pharmacotherapy with steroids and antibiotic drops, such as moxifloxacin hydrochloride (MFH) and dexamethasone sodium phosphate (DSP). To characterize the pharmacokinetics of these two compounds, we performed and validated a liquid chromatography-mass spectrometry (LC-MS/MS) method to quantify them in rabbit ocular tissues and plasma. We used protein precipitation to extract the compounds. The analyte and internal standard (IS) were separated using a Shim-pack Scepter C18 column. The mobile phase was composed of 0.1% formic acid water (A) and methanol (B). MFH and DSP were detected using positive ion electrostatic ionization (ESI) in multiple reaction monitoring mode (MRM). The calibration curves for both compounds showed good linearity over concentrations ranging from 0.5 to 200 ng/mL in rabbit ocular tissues and plasma. The lower limit of quantification for both MFH and DSP was 0.5 ng/mL. We validated this method for selectivity, linearity (r2 > 0.99), precision, accuracy, matrix effects, and stability. Thus, we used this method to assess the pharmacokinetic (PK) characteristics of MFH and DSP in rabbit ocular tissues and plasma after single doses. Our results indicate that this method can be used for the simultaneous analysis of moxifloxacin hydrochloride and dexamethasone sodium phosphate in clinical samples.

Determination of Genotoxic Impurities N-nitrosamine's in Efavirenz Drug Substance Using Rp-Hplc Technique

Aim: The main purpose of the research is to develop a effective, sensitive, economical and simple reverse phase HPLC method for the determination of genotoxic N-Nitrosamine's impurities in Efavirenz drug substance.
 Study Design: HPLC based quantification studies
 Place and Duration of Study: Department of Chemistry, Acharya Nagarjuna University, Guntur, Andhra Pradesh between April 2022 and August 2022.
 Methodology: Determination of genotoxic N-Nitrosamine's in Efavirenz drug substance. N-Nitrosamine's impurities were determined by RP-HPLC method using Zorbax SB C18 (150x4.6mm, 3.5µ) column as stationary phase. Column temperature maintained 25°C, injection volume 20 µL, flow rate 1.0 mL/min and sample cooler temperature 5°C and run time was 15 minutes. The mixture of 0.1% formic acid buffer and methanol in the ratio of 50:950 (v/v) was used as mobile phase. wavelength 240 nm. respectively.
 Results: There is no interference of blank at N-Nitrosamine's impurities peaks. The elution order and the retention times of impurities and Efavirenz obtained from individual standard preparations and mixed standard preparations are comparable.
 The limit of detection (LOD) and limit of quantitation (LOQ) for NDMA 0.03 & 0.09 µg/mL, NDEA 0.02 & 0.06 µg/mL and NDIPA 0.03 & 0.09 µg/mL respectively.
 The linearity results for N-Nitrosamine's impurities in the specified concentration range are found satisfactory, with a correlation coefficient greater than 0.99.Calibration curve was plotted and correlation co-efficient for impurities found to be 1.000, 1.000 and 1.000 respectively.
 The accuracy studies were shown as %recovery for N-Nitrosamine's impurities at specification level. The limit of % recovered shown is in the range of 80 and 120% and the results obtained were found to be within the limits. Hence the method was found to be accurate.
 The technique has been validated in accordance with ICH rules, and all validation parameters meet the acceptance criteria of the ICH Q2 specification.
 Conclusion: According to ICH guidelines, an RP-HPLC method that can assess genotoxic Nitrosoamines in Efavirenz at trace level concentration has been established. The method's specificity, precision, linearity, and accuracy ensured its efficacy. As a result, the technique is appropriate for the goals it was designed to achieve and can be successfully used for release testing of efavirenz into the market.

Selective Capture-Based Single-Cell Mass Spectrometry for Enhancing Sphingolipid Profiling of Neurons with Differentiation of Cell Body from Synapse.

Currently, single-cell lipidomic mass spectrometry (MS) techniques are mostly limited to detection of high-abundance phosphatidylcholines (PCs). Herein, for enhancing the coverage to low-abundance sphingolipids in single-cell analysis, in-tube solid-phase microextraction (SPME) was combined with a single-probe MS system for selective enrichment of sphingolipids during singe-cell sampling. From the results, a lab-made single probe with a 30 μm tip size proved to be able to resolve the axon from the cell body of neuron HT22 in ambient conditions. TiO2 was immobilized onto the inner wall of the transfer capillary of the single-probe device for online selective capture of sphingolipids in ammonia-acetonitrile and rapid desorption in formic acid-methanol. The results showed that the breakthrough volume of the capillary with sample loading flow rate at 500 nL/min was >14 μL. Standard experiments showed that the signals of cerebroside (CB), ceramide (Cer), and sphingomyelin (SM) were largely enhanced after selective capture in the coated capillary, while PCs were totally removed. The reusability (>10 times) and stability of the lab-made TiO2-coated capillary was verified. By introducing the coated capillary into the single-probe MS system, the new system proved to have low detection limits of SM, Cer, and CB (0.007-0.027 μg/mm2) and acceptable linearity (r > 0.98) and repeatability (RSD < 30%). Lipid coverage of the new method to SMs and CBs proved to be largely improved (SM, 21 vs 2; CB, 10 vs 0) with the new method in comparison to conventional single-probe MS without selective capture by ambient analysis of a single spot of rat cerebellum. Finally, the new system was used to perform single-neuron analysis of sphingolipids in the control and lipopolysaccharide (LPS)-treated HT22 with differentiation of the cell body from the axonal synapse. Results showed that 5 sphingolipids had significantly higher concentrations in the synapse than in the cell body, while 3 oxidized sphingolipids had significantly higher levels in the cell body than in the synapse. After LPS treatment, most of the sphingolipids largely decreased and became more accumulated in the synapse, providing new information on LPS-induced neuroinflammation.

Easily Operable Quantification Method of 21 Plant-Derived Alkaloids in Human Serum by Automatic Sample Preparation and Liquid Chromatography-Tandem Mass Spectrometry.

The online version contains supplementary material available at 10.1007/s10337-022-04212-5.

Importance of amine in carbon dioxide conversion to methanol catalyzed by Ru-PNP complex

The theoretical insight with DFT calculations was carried out to explore the mechanism of CO2 conversion to methanol. The tandem catalysis with the Ru-Macho-BH complex and ethylenediamine (EDA) was investigated. Three possible routes, Route A, B, and C, were realized for the process. Route A (δE > 60 kcal mol−1) consists of three steps, formic acid formation, formamide formation, and methanol formation (from alkanolamine). Route B (δE = 35.07 kcal mol−1) contains four steps, formic acid formation, formamide formation, formaldehyde synthesis, and methanol formation (from formaldehyde). Route C (δE = 39.14 kcal mol−1), a non-amine route, involves three steps, formic acid formation, formaldehyde dehydration, and methanol formation. Route B has the lowest energetic span and, thus, we propose it as a favorite route for CO2 conversion to methanol. Although Route C has a lower activation barrier, it has a larger δE. The exogenic reaction of the formamide formation step in Route B can affect lower energy requirements in further methanol formation. In the absence of EDA, CO2 hydrogenation to formic acid cannot produce, because the formate form is trapped by the catalyst. Thus, it can explain why CO2 conversion to methanol is impossible without the presence of amines.

Simultaneous determination of twelve mycotoxins in edible oil, soy sauce and bean sauce by PRiME HLB solid phase extraction combined with HPLC-Orbitrap HRMS.

A solid phase extraction-high-performance liquid chromatography-tandem Orbitrap high resolution mass spectrometry (HPLC-Orbitrap HRMS) method was established for the determination of 12 mycotoxins (ochratoxin A, ochratoxin B, aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, HT-2 toxin, sterigmatocystin, diacetoxysciroenol, penicillic acid, mycophenolic acid, and citreoviridin) in edible oil, soy sauce, and bean sauce. Samples were extracted by 80:20 (v:v) acetonitrile-water solution, purified by PRiME HLB column, separated by aQ C18 column with mobile phase consisting of 0.5 mmol/L ammonium acetate-0.1% formic acid aqueous solution and methanol. The results showed that the limits of detection (LODs) and limits of quantification (LOQs) of 12 mycotoxins were 0.12–1.2 μg/L and 0.40–4.0 μg/L, respectively. The determination coefficients of 12 mycotoxins in the range of 0.20–100 μg/L were > 0.998. The average recoveries in soy sauce and bean sauce were 78.4–106.8%, and the relative standard deviations (RSDs) were 1.2–9.7% under three levels, including LOQ, 2× LOQ and 10 × LOQ. The average recoveries in edible oil were 78.3–115.6%, and the precision RSD (n = 6) was 0.9–8.6%. A total of 24 edible oils, soy sauce and bean sauce samples were analyzed by this method. AFB1, AFB2, sterigmatocystin and mycophenolic acid were detected in several samples at concentrations ranging from 1.0 to 22.1 μg/kg. The method is simple, sensitive, and rapid and can be used for screening and quantitative analysis of mycotoxin contamination in edible oil, soy sauce, and bean sauce.

Determination of dimethyl sulfate genotoxic impurities in tertiary amine drugs by ultra-high performance liquid chromatography-tandem mass spectrometry

硫酸二甲酯被广泛用于药品的有机合成中,具有很强的毒性及腐蚀性,因此应严格控制其在药品中的残留量。现有传统检测方法在样品制备过程中不允许使用水,且存在专属性低、灵敏度差等缺陷,严重制约了检测方法的通用性和准确度。研究采用超高效液相色谱-串联质谱技术(UHPLC-MS/MS),建立了氨基比林等叔胺类药品中残留的硫酸二甲酯的测定方法。检测方法以氨基比林作为衍生剂,40 ℃条件下氨基比林和硫酸二甲酯在水溶液中能够快速反应生成甲基化氨基比林,通过检测甲基化氨基比林间接计算硫酸二甲酯的残留含量。采用Waters Atlantis HILIC C18色谱柱(100 mm×2.1 mm, 3.0 μm),以10 mmol/L乙酸铵水溶液-0.1%甲酸甲醇溶液(50∶50, v/v)为流动相进行等度洗脱,流速0.3 mL/min,柱温40 ℃,进样量1 μL;在电喷雾电离、正离子(ESI+)多反应监测(MRM)模式下测定硫酸二甲酯的含量。硫酸二甲酯在0.9935~7.9480 ng/mL范围内线性关系良好,相关系数(r)为0.9997,方法的检出限为0.50 ng/mL,定量限为1.15 ng/mL,加标回收率为94.9%~106.4%。建立的检测方法用于氨基比林、咖啡因、替加氟等9批样品中,均未检出硫酸二甲酯,说明各药品生产企业注重了该基因毒杂质残留量的控制。该方法专属性强,灵敏度高,操作简单,定量准确,适用于氨基比林等药品中硫酸二甲酯基因毒杂质的测定。

Highly sensitive MS/MS detection for confident identification of potent novel synthetic opioids and their metabolites

The aim of this presentation is to demonstrate the use of high-resolution mass spectrometry as a unique tool for the untargeted detection and identification of new emerging substances, with little or no method optimization necessary. The presentation will demonstrate how accurate mass instruments are providing extra levels of confidence in analyte identification by reliably obtaining comprehensive MS/MS spectral fragment information that can be used for identification, confirmation, and/or library matching of potent drugs and metabolites present in discarded authentic case samples. NSO compounds were extracted from human whole blood using a liquid-liquid extraction (LLE) procedure. HPLC separation was performed on an ExionLC system using a Phenomenex Kinetex C18 column Mobile phases were ammonium formate and formic acid in methanol and acetonitrile. The flow rate was 0.4 mL/min with a total LC runtime of 15.5 minutes. The injection volume was 10 μL. MS and MS/MS data were collected for each sample using Zeno IDA for optimal sensitivity on the ZenoTOF 7600 system. Data acquisition consisted of a TOF MS scan to collect accurate mass precursor ions from 100 to 700 Da, followed by a TOF MS/MS full scan ranging from 25 to 700 Da to ensure all fragments were captured for identification using a maximum of 16 candidate ions. Data was acquired using SCIEX OS software 2.1. A series of 9 calibrator solutions were prepared by spiking control human whole blood samples with the 6 targeted analytes at final concentrations ranging from 10 pg/mL to 100 ng/mL. The series of calibrator solutions were injected to evaluate the quantitative performance of the system and its ability to accurately measure low level analytes with a high level of precision and accuracy in TOF MS mode. Each calibrator was injected in triplicate. LLOQs of between 10 and 50 pg/ml were observed for all compounds analysed. Accuracies at LLOQ ranged from 86–111% and imprecision was < 15% for all compounds analysed. In addition, the use of specific technologies afforded by the new SCIEX ZenoTOF 7600 system generated an average 9x sensitivity improvement on spectral data providing significantly improved library matching. A comprehensive and highly sensitive method for the screening and identification of potent NSO in human whole blood is described. The significant gains in MS/MS sensitivity on the SCIEX ZenoTOF 7600 system yielded an improvement in confident identifications of low-level analytes through spectral library matching. The observed sensitivity gains resulted in a 9x improvement, on average, in TOF MS/MS sensitivity across the drugs positively identified in the authentic case samples analyzed. This improvement enabled confident identification of key drugs and metabolites at trace levels that were not previously achievable. The MS/MS sensitivity levels afforded by ZenoTOF 7600 system provide a means to monitor low levels of ultra-potent NSO in poly-drug intake scenarios.

The method of detection ADB-BUTINACA in urine and application in forensic cases

ADB-BUTINACA (ADB-BINACA) is a new synthetic cannabinoid that was first found in Europe in 2019. This novel drug was then rapidly increased in prevalence from early 2021. Although National Forensic Service (NFS) also identified it in authentic seizures from early last year, it was unable to test biological samples due to the unavailability of metabolic data with the absence of reference standards. NFS finally established the method of detection in biological samples. The possible metabolites were first found using LC-QTOF. Amongst the metabolites, ADB-BUTINACA and its metabolites, ADB-BUTINACA-N-butanoic acid and ADB-BUTINACA-N1hydroxybutyl, were purchased from Cayman chemicals. Methanol, ethyl acetate, sodium hydroxide, acetic acid, hydrochloric acid and ammonium formate were obtained from SIGALD. JWH-18-N-OH-D5 from Sigma aldrich was used as an internal standard (IS). Three of authentic urine samples were tested for confirmation of the method. For sample preparation, 1 mL of urine and 3 mL of 0.1 phosphate buffer were mixed with IS and then it was hydrolyzed using β-glucronidase ( E. coli ) for an hour. The prepared samples including reference blank and standard were then extracted using SPE, Rapid Trace. Those were analyzed by LC 1260 with 4500 QTRAP® mass spectrometer (AB SCIEX) in column, Zorbax Eclipse XDB-C18. Two mobile phases, 2 mM ammonium formate/0.2% formic acid in distilled water and 2 mM ammonium formate/0.2% formic acid in methanol, were used for gradient elution. The multiple reaction monitoring (MRM) data were obtained in positive ions [M + H] mode. The method was further validated by admittable specificity, linearity, LOD, LOQ, accuracy, recovery and interference. ADB-BUTINACA metabolites were expected using the data obtained from the LC-QTOF, which major candidates were corresponding to previous studies. Due to the absent of all those metabolites in market, two metabolites were only used to analyze the samples. ADB-BUTINACA (m/z 331.1), ADB-BUTINACA-N-butanoic acid (m/z347.2) and ADB-BUTINACA-N-hydroxybutyl (m/z 361.2) all were clearly shown in data by the method described above. The results from the authentic urine samples also confirmed both the two of metabolites while little parent compound was shown. It was found that ADB-BUTINACA-N-butanoic acid was predominant when comparing with the other metabolite, ADB-BUTINACA-N-hydroxybutyl. The metabolism pathways were recently reported using the human hepatocytes in authentic urine and blood samples in a few studies. Those studies suggested possible metabolism pathway and candidate metabolites using LC-QTOF. Twenty metabolites were proposed with the assumption that the parent structure is modified by the metabolism pathways such as mono-and dihydroxylation and hydroxylation of the N-butyl side chain. Currently only two biomarkers are available in market, ADB-BUTINACA-N-butanoic acid and ADB-BUTINACA-N-hydroxybutyl. ADB-BUTINACA was found in a few seizures from early 2021 but it was unable to detect in authentic biological samples in NFS until the method was established in later 2021. It was found in the seizures from nineteen cases in NFS, Wonju, last year. Amongst, a few urine samples from the cases were then reserved in freezer for further research and it was shown that one of the cases involved ADB-BUTINACA metabolites. This is an example obviously presenting that the forensic science is behind its emerging speed. It is now possible to test this new psychoactive substance as the detection method has developed. Unfortunately, new synthetic cannabinoids are continuously coming out every year but there is no alternative due to its unpredictability and rapid replacement. Therefore, global co-operation and communication seems to be only possible solutions to response promptly to this challenge.

Reaching new sensitivity levels for the detection of fentanyl analogs and highly potent novel synthetic opioids in blood

The aim of this presentation is to demonstrate a sensitive quantitative workflow for the analysis of 32 of potent substances in poly-drug, discarded postmortem case samples at low (pg/mL) levels. The presentation will demonstrate that the unparalleled quantification performance of the method enabled accurate detection of potent substances in poly-drug, discarded postmortem case samples at trace levels that were not previously achievable, providing toxicologists a valuable insight into the causation of accidental overdoses. A total of 32 NSO including 17 fentanyl analogs and 15 newly emerging non-fentanyl opioids were selected for this panel. NSO were extracted from the 200 μL of spiked whole blood mixtures using a liquid-liquid extraction (LLE) procedure. Analytes were chromatographically separated using a Phenomenex Kinetex C18 column (50 × 3.0 mm, 2.6 μm, 00B-4462-Y0). Mobile phase A (MPA) and mobile phase B (MPB) were ammonium formate with formic acid and formic acid in methanol and acetonitrile, respectively. The injection volume was 10 μL and the total LC runtime was 15.5 minutes. A single acquisition method consisting of 68 MRM transitions (64 for the NSO and 4 for the internal standards) was created using the Scheduled MRM Algorithm. Control human whole blood samples spiked with the 32 target analytes were prepared at concentrations ranging from 1 pg/mL to 100 ng/mL. Excellent linearity was observed across the concentration ranges analyzed with R2 values greater than 0.99 for all of the NPS in the panel. Reported LLOQ values ranged between 10 to 50 pg/mL for the 32 analytes in the panel. The accuracy and precision of measurements ranged from 80.50–116.64% and from 0.42–17.80%, respectively. The excellent accuracy and precision were observed over the entire concentration range, including at the LLOQ. Overall, the developed method showed excellent reproducibility and linearity, proving the robustness of the developed method and quantitative performance of the SCIEX 7500 System even at low concentration levels for each of the targeted NSO in this study. The applicability of the method to analyze discarded postmortem case samples from subjects suspected of NPS ingestion resulting in accidental overdoses was investigated. For example, the results from the analysis of one of these case samples showed the successful detection of one NSO and its metabolite: isotonitazene and 5-aminoisotonitazene, as well as fentanyl and four of its analogs/metabolites: beta-hydroxy-fentanyl, norfentanyl, 4-ANPP and acetyl fentanyl at concentrations of 1434.33, 7.93, 599.10, 9756.67, 147.69 and 1465.00 pg/mL, respectively. The results demonstrate that the developed method enabled accurate detection of potent substances in poly-drug, authentic case samples at trace levels that were not previously achievable. A robust and sensitive drug screening workflow for the analysis of 32 potent NSO was successfully developed using the SCIEX 7500 System. Overall, the remarkable quantification performance of the SCIEX 7500 System enabled accurate detection of potent NSO at concentrations that were not previously achievable, providing a means to monitor ultra-potent NSO in overdose scenarios. The sensitivity levels afforded by the SCIEX 7500 system provided the ability to detect low levels of NSO in postmortem case samples that would normally go undetected, providing a clearer picture for help in determining the cause of death.

Enhancing high-resolution mass spectrometry performance for NPS analysis with improved sensitivity and characterization

The aim of this presentation is to introduce the instrument features on the ZenoTOF 7600 system that provide qualitative flexibility combined with quantitative power for NPS detection and characterization. The presentation will demonstrate that these new technological advancements on the system can be leveraged to provide more confidence in the quantified amounts of drugs and metabolites detected in discarded authentic case samples which is critical when determining the cause of death following an accidental overdose. Drugs and metabolites were extracted from human whole blood using a liquid-liquid extraction (LLE) procedure. HPLC separation was performed on an ExionLC system using a Phenomenex Kinetex C18 column Mobile phases were ammonium formate and formic acid in methanol and acetonitrile. The flow rate was 0.4 mL/min with a total LC runtime of 15.5 minutes. The injection volume was 10 μL. MS and MS/MS data were collected for each sample using Zeno IDA for optimal sensitivity on the ZenoTOF 7600 system. Fragmentation was performed using both collision-induce dissociation (CID) and electron-activated dissociation (EAD) to compare the generated fragment ions. Data acquisition consisted of a TOF MS scan to collect accurate mass precursor ions from 100 to 700 Da, followed by a TOF MS/MS full scan ranging from 25 to 700 Da to ensure all fragments were captured for identification using a maximum of 16 candidate ions. Data was acquired using SCIEX OS software 2.1. The use of a hybrid collision cell (which offers an alternative fragmentation capabilities) in combination with the Zeno trap technology (which improves MS/MS duty cycle) was leveraged for the characterization of structurally related isomeric species present at low levels in discarded postmortem case samples. The depth of information extracted from the unique fragmentation capabilities of electron-activated dissociation (EAD) enabled differentiation of structurally related isomeric species that were not previously discernable using convention collision-induced dissociation (CID). In addition, the improved MS/MS sensitivity resulted in confident identification of potent novel synthetic opioids and metabolites at concentration levels that were not previously achievable. Overall, the use of the ZenoTOF 7600 system provided a means to characterize and monitor low-levels of ultra-potent NSO in poly-drug intake scenarios. These advancements are shown to support the case of combined opioid drug toxicity leading to death, which offers a clearer picture for help in determining the cause of death. A novel fragmentation technique combined with a highly-sensitive QTOF system for the screening and identification of low-level potent NSO and metabolites in discarded postmortem case samples is described. The depth of information extracted from EAD-based MS/MS spectra combined with the improved MS/MS sensitivity were leveraged for characterization of structurally related isomeric species present at low levels. The results demonstrate that the new technological advancements on the ZenoTOF 7600 provide high levels of confidence in the quantified amounts of drugs and metabolites detected in authentic case samples, which is critical when determining the cause of death following an accidental overdose.

Comparison of electrospray and UniSpray™ ion sources with post-column hydrochloric acid infusion for 119 abuse relevant substances

Comparing the sensitivity of the Waters UniSpray™ ion source (USI) with the Waters electrospray ion source (ESI) on a Waters Xevo TQ-XS mass spectrometer with and without additional post-column infusion of low concentrated hydrochloric acid (HCl). To reduce sample volumes or increase analytical sensitivity for drugs of abuse detection in human body fluids, technological advances in mass spectrometry often target an increase in ionization efficiency. One example for this development is the USI, which claims higher ion yields and therefore higher sensitivity due to a unique geometry. Another possibility to increase sensitivity through increased ion yield is the post-column infusion of low concentrated HCl. For sensitivity comparison, we choose the, in our opinion, 119 most relevant medications and drugs of abuse. These were prepared in eleven acetonitrile solutions containing 10 analytes each and one with 9 analytes at a concentration of 1 μg/mL for each substance. For analysis, every solution was diluted 1:1000 in mobile phase A/ethylene glycol (70:30 v/v) to a concentration of 1.0 ng/mL. Solutions were analysed in triplicate on the same day interrupted by the ion source change, to decrease inter-day variations in sensitivity. Chromatographic separation was performed on a Waters 2.1 × 150 mm, 1.8 μm BEH Phenyl column kept at 60 °C on an Acquity UPLC connected to a Xevo TQ-XS detector (Waters). The standard ESI ion source or the USI ion source was mounted on the detector. Mobile phase A consisted of 20 mM ammonium formate and 0.1% formic acid (pH 3) and mobile phase B was 0.1% formic acid in methanol. Gradient elution was conducted within 6.0 min at a flow rate of 0.5 mL/min with 85% A and 15% B at the beginning and 100% B at the end. Data were acquired with the ion sources operating in the positive ionization MRM mode. Two transitions for each analyte were monitored at a constant dwell time of 10 ms over the full chromatographic run time. In an additional experiment, both ion sources were compared with additional post-column infusion of 0.0037% HCl at a flow rate of 5.0 μL/min. Injection volume was 2.0 μL in all experiments. USI provided a general gain in signal intensity (peak area): 12% ( n = 14) of the analytes showed a peak area increase up to 2-fold, 85% ( n = 101) increased between 2- and 4-fold and 3% ( n = 4) increased above 4-fold in peak area. Post-column HCl infusion with the ESI source mounted, resulted in an analyte dependent additional gain in signal intensity for 15% ( n = 18) of the analytes ranging from 2- to 21-fold. 78% ( n = 93) ranged from 0.8-fold to 2-fold. In contrast 7% ( n = 8) showed a signal decrease between 0.4-fold and 0.8-fold. The combination of USI and post-column HCl infusion resulted in an increase in peak area from 2-fold up to 62-fold compared to ESI without infusion for 91% (n = 108) of the analytes. 8% ( n = 9) where between 0.8-fold and 2-fold and 1% ( n = 2) ranged from 0.4-fold to 0.8-fold. With USI, a general improvement in analytical sensitivity under identical conditions could be achieved for all of our 119 “favorite analytes” separated in one chromatographic run. Additional post-column infusion of HCl increased sensitivity for many substances. This approach can be used very selectively when applied to certain retention time windows only. Further experiments are needed to understand how different adjustments of ESI and USI and different matrices will affect ionization. USI, especially in combination with post-column HCl infusion, is a promising tool to increase sensitivity in toxicological multi-target LC-MS/MS analysis.

Multi-steroid profiling by UHPLC-MS/MS with post-column infusion of ammonium fluoride

BackgroundMulti-steroid profiling is a powerful analytical tool that simultaneously quantifies steroids from different biosynthetic pathways. Here we present an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) assay for the profiling of 23 steroids using post-column infusion of ammonium fluoride. MethodsFollowing liquid–liquid extraction, steroids were chromatographically separated over 5 min using a Phenomenex Luna Omega C18 column and a water (0.1 % formic acid) methanol gradient. Quantification was performed on a Waters Acquity UHPLC and Xevo® TQ-XS mass spectrometer. Ammonium fluoride (6 mmol/L, post-column infusion) and formic acid (0.1 % (vol/vol), mobile phase additive) were compared as additives to aid ionisation. ResultsPost-column infusion of ammonium fluoride enhanced ionisation in a steroid structure-dependent fashion compared to formic acid (122–140 % for 3βOH-Δ5 steroids and 477–1274 % for 3-keto-Δ4 steroids). Therefore, we analytically validated post-column infusion of ammonium fluoride. Lower limits of quantification ranged from 0.3 to 3 nmol/L; All analytes were quantifiable with acceptable accuracy (bias range −14 % to 11.9 % for 21/23, −21 % to 11.9 % for all analytes). Average recovery ranged from 91.6 % to 113.6 % and average matrix effects from −29.9 % to 19.9 %. Imprecision ranged from 2.3 % to 23 % for all analytes and was < 15 % for 18/23 analytes. The serum multi-steroid profile of 10 healthy men and 10 healthy women was measured. ConclusionsUHPLC-MS/MS with post-column infusion of ammonium fluoride enables comprehensive multi-steroid profiling through enhanced ionisation particularly benefiting the detection of 3-keto-Δ4 steroids.

Development and validation of bio analytical liquid chromatography and mass spectrometry method for quantification of upadacitinib in human plasma

In the present investigation, a simple, rapid and sensitive liquid chromatography and mass spectrometry (LC/MS) method was developed for quantification of Upadacitinib (UCB) in spiked human plasma. The drug UCB was extracted by liquid –liquid extraction using diethyl ether and chloroform in the ratio of 75:25 (v/v). The drug Leflunomide (LFM) was used as an internal standard. The extracted drug mixture was followed by the liquid chromatography mass spectrometry (LC/MS) analysis and electrospray ionization interface. The chromatographic separation of UCB was carried out on Phenomenex Luna C18 column (100mm X 2.0 mm X 5μm) at ambient temperature followed by isocratic elution of 0.05% formic acid in methanol and acetonitrile in the ratio of 65:30(v/v). The method is concluded at a flow rate of 0.8 mL/min, 262 nm of UV detector wavelength. Protonated ions formed by electrospray ionization were recorded in the positive mode and were used to detect the analyte (UCB) with internal standard (LFM). The mass detection was made by monitoring the fragmentation of m/z 381.0 for UCB m/z 271.0 for LCM on mass spectrometer. The method was validated for accuracy, precision, linearity and recovery. The assay was linear over the entire range of calibration standards i.e 2.5-500ng/mL. The recoveries of the UCB after liquid-liquid extraction at 5, 20, 35 ng/mL were 100.7, 99.4 and 99.8. The lowest limit of the analytical method of UCB was 2 ng/mL in spiked human plasma. The developed method was successfully validated and can be used for determination of the pharmacokinetic parameters of the UCB in biological samples.

Development and validation of rapid ultra high performance liquid chromatography with tandem mass spectroscopic method for the quantification of N-nitrosodimethyl amine and N-nitrosodiethyl amine in sitagliptin and metformin hydrochloride immediate and extended-release formulations.

A simple, effective LC-MS based method is developed and validated to determine N-nitrosodimethylamine and N-nitrosodiethylamine in pharmaceutical formulations of Sitagliptin and Metformin hydrochloride combination dosage forms. Atlantis T3 (100×3mm, 3μm) column, eluent-A (0.1% formic acid in water), and eluent-B (0.1% formic acid in methanol) were used to achieve chromatographic separation. A gradient program time (min)/%B: 0.01/3, 2/3, 4/55, 5/55, 5.5/90, 6.0/90, 6.5/3, and 7/3, and column flow rate: 0.75mL/min was employed. The column oven and auto sample cooler temperatures were 40°C and 10°C, respectively. Atmospheric Pressure Ionisation positive mode with corona discharge potential as 4.0 V, drying gas (N2 ) flow as 110mL/min, and nebulizer gas (N2 ) flow as 350mL/min. Employing PerkinElmer triple quadrupole mass spectrometer, QSight 200 series, the source temperature was 450°C, and hot surface-induced desolvation temperature was 250°C. Under optimized conditions, diluent-1 and diluent-2 offered better recovery and improved peak shapes. The required method sensitivity of nitrosodimethylamine (LOQ 0.74ng/mL) and nitrosodiethylamine (LOQ 0.37ng/mL) for the nitrosamine impurities were achieved using an optimized test concentration of Metformin hydrochloride at 45.7mg/mL.

A Multi-Analyte LC-MS/MS Method for Determination and Quantification of Six Nitrosamine Impurities in Sartans like Azilsartan, Valsartan, Telmisartan, Olmesartan, Losartan and Irbesartan.

A sensitive and robust method for determination and quantification of potential genotoxic impurities in sartans has been developed. These impurities need to be controlled at trace levels during quantification in drug substances and drug products for safe consumption. Recent regulatory requirements also suggested the need to have highly sensitive analytical method for trace level quantification of nitrosamine impurities. In this paper, we have described a simple, rapid and sensitive liquid chromatography-mass spectrometry method for six potential genotoxic nitrosamine impurities: N-Nitroso dimethyl amine (NDMA), N-Nitroso diethyl amine (NDEA), N-Nitroso Ethyl Iso propylamine (NIPEA), N-Nitroso-Nmethyl-4-aminobutyric acid (NMBA) N-Nitroso diisopropylamino (NDIPA) and N-Nitroso dibutyl amine (NDBA) in Azilsartan (AZL), Valsartan (VAL), Telmisartan (TEL), Olmesartan (OLM), Losartan (LOS) and Irbesartan (IRB) with a limit of quantification of less than 0.003ppm. Chromatographic separation is achieved using Poroshell HPH- C18, 150 × 4.6mm, 2.7μm column with 0.1% formic acid in water as mobile phase A and 0.1% formic acid in methanol as mobile phase B at a flow rate of 0.5mL/min using gradient mode of elution at a total run time of 20min. Six nitrosamine impurities are ionized and quantified in positive mode of atmospheric pressure chemical ionization using multiple reaction monitoring. As per ICH guidelines, method validation is performed and evaluated the limit of quantification and detection and found to give good S/N ratios with good linearity range of 0.003-0.045ppm with regression coefficient > 0.999 for all the six nitrosamine impurities. Method recoveries are also established using three-step sample preparation and are found to be satisfactory within 80-120%. The single method can be used routinely applied for the detection of nitrosamines in AZL, VAL, TEL, OLM, LOS and IRB.

A dilute-and-shoot liquid chromatography-tandem mass spectrometry method for urinary 18-hydroxycortisol quantification and its application in establishing reference intervals.

BackgroundEighteen‐hydroxycortisol (18‐OHF) is a potential biomarker for differential diagnosis of the two major primary aldosteronism subtypes, aldosterone‐producing adenoma, and idiopathic hyperaldosteronism.MethodsUrine samples were processed, and the 18‐OHF in urine samples were successfully quantified by in‐house established dilute‐and‐shoot liquid chromatography–tandem mass spectrometry (LC–MS/MS) method. Separation was accomplished on a Sigma Ascentis Express C18 column with a gradient mixture of phase (A) 0.2% formic acid in water and phase (B) 0.2% formic acid in methanol at a flow rate of 0.4 ml/min. Mass spectrometric detection was performed in positive electrospray ionization mode via a mass spectrometer.ResultsThe linearity of urinary 18‐OHF ranged from 4.28 to 8.77 × 103 nmol/L, with a lower limit of quantification at 4.28 nmol/L. The intra‐ and inter‐precision were both below 3%. The range of analytical recovery was 97.8%–109.2%. The validated dilute‐and‐shoot LC–MS/MS method was compared with the SPE LC–MS/MS method modified from the one reported in 2013. The results by Passing–Bablok regression analysis and Bland–Altman plotting demonstrated a good agreement between the two methods. The presented method was then applied to establish sex‐specific reference intervals from 62 males and 62 females, respectively. The calculated 2.5%–97.5% reference intervals for 24‐h urinary 18‐OHF were 113–703 nmol/day for males and 71.2–450 nmol/day for females.ConclusionThe presented dilute‐and‐shoot LC–MS/MS method for 18‐OHF quantification showed a good performance in the clinical application. Furthermore, the sex‐specific reference intervals for 24‐h urinary 18‐OHF were first established and quite important for its application in primary aldosteronism subtyping.

Determination of five nitroimidazoles and six benzodiazepines in aquatic products using ultra-high performance liquid chromatography-tandem mass spectrometry coupled with dispersive solid-phase extraction

建立了分散固相萃取-超高效液相色谱-串联质谱法同时测定水产品中5种硝基咪唑类和6种苯二氮卓类药物残留的方法。样品用1%(v/v)氨水乙腈提取,提取液经十八烷基键合硅胶(C18)和N-丙基乙二胺(PSA)吸附剂净化,在45 ℃下用氮气吹至近干,用1 mL甲醇-水(1:9, v/v)溶液复溶,过0.22 μm尼龙-66滤膜后用超高效液相色谱-串联质谱测定。目标化合物采用Kinetex F5色谱柱(100 mm×3.0 mm, 2.6 μm)分离,以0.1%(v/v)甲酸水溶液和甲醇作为流动相进行梯度洗脱,在电喷雾离子源(ESI)、正离子扫描和多反应监测(MRM)模式下进行测定,基质匹配外标法定量。结果表明,5种硝基咪唑类和6种苯二氮卓类药物在8.5 min内完成色谱分离分析,目标物在0.5~20 μg/L范围内线性关系良好,相关系数均大于0.995,检出限和定量限分别为0.2~0.5 μg/kg和0.5~1.0 μg/kg。以草鱼、对虾和大黄鱼为样品基质,在3个不同的添加水平下,5种硝基咪唑类和6种苯二氮卓类药物的平均回收率为73.2%~110.6%,相对标准偏差(RSD)小于15%。本研究建立的方法具有简单、快速、灵敏度高和成本低等优势,可用于水产品中5种硝基咪唑类和6种苯二氮卓类药物的快速检测。该方法的建立为我国水产品质量安全相关监管部门同时监控水产品中硝基咪唑类和苯二氮卓类药物残留提供了技术支持。

Asymmetric Transfer Hydrogenative Amination of Benzylic Ketones Catalyzed by Cp*Ir(III) Complexes Bearing a Chiral N-(2-Picolyl)sulfonamidato Ligand

A convenient asymmetric reductive amination of benzylic ketones (α-arylated ketones) catalyzed by newly designed Cp*Ir complexes bearing a chiral N-(2-picolyl)sulfonamidato ligand was developed. Using readily available β-amino alcohols as chiral aminating agents, a range of benzo-fused and acyclic ketones were successfully reduced with formic acid in methanol at 40 °C to afford amines with favorable chemo- and diastereoselectivities. The amino alcohol-derived chiral auxiliary was easily removed by mild periodic oxidants, leading to optically active primary β-arylamines without erosion of the optical purity (up to 97% ee). The excellent catalytic performance was retained even upon lowering the amount of catalyst to a substrate/catalyst (S/C) ratio of 20,000, and the amination could be performed on a large scale exceeding 100 g. The precise hydride transfer to iminium species generated from the ketonic substrate and the chiral amine counterpart was suggested by the mechanistic studies on stoichiometric reactions of isolable hydridoiridium complexes and model intermediates such as N,O-acetal, enamine, and iminium compounds.

Fast screening and confirmation of 73 common prohibited compounds in cosmetics by ultra performance liquid chromatography-quadrupole-time-of-flight high resolution mass spectrometry

建立了超高效液相色谱-四极杆-飞行时间高分辨质谱(UPLC-Q-TOF HRMS)同时快速筛查确证化妆品中73种常见禁用物质的方法。样品经饱和氯化钠溶液分散均匀后,采用含0.2%甲酸的乙腈溶液超声提取,50 mg PSA净化,以8000 r/min高速冷冻离心除脂,采用Waters Acquity HSS T3色谱柱(100 mm×2.1 mm, 1.8 μm)分离。采用多反应监测高分辨扫描模式(MRM HR),以保留时间、一级母离子精确质量数、同位素丰度比和二级子离子精确质量数实现化妆品中73种禁用物质的快速筛查和确证,基质匹配外标法定量。实验比较了不同提取溶剂、净化吸附剂、色谱条件和质谱扫描模式对73种禁用物质测定的影响,并考察了膏霜剂和水剂的基质效应。结果表明,73种禁用物质线性关系良好,相关系数(R2)>0.99;检出限为5~150 μg/kg;定量限为15~450 μg/kg;膏霜剂及水剂两种基质在3个加标水平下的回收率为60.3%~130.3%,日内、日间RSD分别为0.8%~10.0%(n=6)和1.1%~15.0%(n=3)。日常风险监测中检出磺胺甲基异噁唑、甲基泼尼松、林可霉素、对乙酰氨基酚、甲氧苄啶、阿法骨化醇、倍他米松戊酸酯、溴莫尼定、氯霉素、氯苯那敏、氯倍他索丙酸酯、克罗米通、益康唑、酮康唑、泼尼松醋酸酯和泼尼松,检出含量范围为0.5~1136.1 mg/kg。该方法准确、快速、简便,可用于化妆品中73种常见禁用物质的检测。