MRM Mode Research Articles

Optimization of 204 veterinary drug residues method and establishing their mass spectrum library

ABSTRACT A multi-class multi-residue analytical method was validated in this paper for simultaneously identifying 204 veterinary drugs in pork by UPLC-QTRAP MS/MS, including sulfonamides, quinolones, β-agonists, and nitroimidazoles. MRM-IDA-EPI mode of QTRAP was used to establish a secondary mass spectrum library of these 204 veterinary drugs, which could enhance the qualitative analysis of suspect substances. The separation of substances was performed on a C18 column, with a gradient elution of 0.1% formic acid solution in water and methanol. Electrospray ionization (ESI), fast polarity switching, and scheduled MRM mode were used to collect the characterization results. Factors that affected the recovery were delicately optimized, such as solvent proportion, formic acid content, and purification method. We finally determined the extracting method with formic acid-acetonitrile-water (0.1:90:10, v:v:v) and purifying method with Cleanert LipoNo as a package for dispersive solid-phase extraction. Matrix effects on the quantitative results were also investigated. Some compounds had strong matrix inhibition or enhancement, and the matrix matching external standard method was used to quantify the compounds accurately. The correlation coefficient R2 was over 0.99 within the calibration linearity range for the detected 204 veterinary drugs. The limit of quantification (LOQ) was 0.5 ~ 5 μg/kg. The recoveries ranged from 61.4% to 118.8% with RSDs of 3.1% to 19.2% (n = 6).

An UPLC-MS/MS Method for Determination of Osimertinib in Rat Plasma: Application to Investigating the Effect of Ginsenoside Rg3 on the Pharmacokinetics of Osimertinib.

Osimertinib is a novel oral, potent, and irreversible epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) for treatment of advanced T790M mutation-positive advanced non-small cell lung cancer, which is commonly combined with ginsenoside Rg3 in clinic to enhance the efficacy and minimize adverse reactions. In the present study, a highly sensitive UPLC-MS/MS method was established and validated for analysis of osimertinib in rat plasma according to US FDA guideline. Separation was performed on a C18 (2.1 × 50 mm, 2.6 μm) column using a gradient elution of ammonium formate (10 mM) with 0.1% formic acid buffer (A) and ACN (B) at a flow rate of 0.2 mL/min. Detection was carried out on a triple quadrupole tandem mass spectrometer equipped with electrospray ionization in the MRM mode. The method was validated over a concentration range of 1–400 ng/mL for osimertinib. The intra- and interday accuracy and precision values were within ±15%. No significant degradation occurred under the experimental conditions in stability assays. There was a further investigation on the effects of multiple doses of ginsenoside Rg3 on the pharmacokinetics of osimertinib in rats for the first time. The results implied that osimertinib exhibited a slow absorption and moderate-rate elimination in rats following oral administration. Coadministeration with ginsenoside Rg3 (5 mg/kg, 7 days, i.g.) may have no effect on the pharmacokinetics of osimertinib in rats. The results provide a reference for the clinical concomitant medications of Rg3 and osimertinib.

Development and validation of a new UPLC-MS/MS method for quantification of ganoderic acid-A loaded nanolipidic carrier in rat plasma and application to pharmacokinetic studies

A systematic methodology was used to quantify ganoderic acid-A (GA-A) loaded nano-lipid carriers (NLC) in rat plasma using UPLC-MS/MS. Separation of the analyte was achieved using ACQUITY UPLC BEH C18 column (1.7 µm) and mobile phase as water containing 0.1% Acetonitrile (40: 60% v/v) at a flow rate of 0.4 mL·min−1. The analyte was detected using MRM mode to track precursor-to-product ion transitions of 515.37 → 285.31 m/z (time scan of 2 min) for GA-A, and 175.11 → 115.08 m/z (time scan of 4 min) for ascorbic acid as an internal standard (IS), respectively. The developed method was validated for linearity, accuracy, within and between day precisions, limit of quantification and recovery of the analyte. The results indicated intra and inter-day consistency and precision values were found to be within the acceptance limit for the plasma samples. The method applicability for determination of pharmacokinetic parameters of GA-A was assessed after oral administration of free GA-A solution and GA-A-loaded NLC, which indicated significant difference (p < 0.05) in the rate and extent of absorption parameters of GA-A from the NLC formulation vis-à-vis the plain solution. Overall, the studies construed successful development and application of UPLC-MS/MS method for estimation of GA-A in the lipidic formulation.

Determination of 16 mycotoxins in drug and food homologous products by ultra performance liquid chromatography-tandem mass spectrometry combined with accelerated solvent extraction and QuEChERS

A method was developed for the simultaneous determination of 16 mycotoxins in drug and food homologous products by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) combined with accelerated solvent extraction (ASE) and QuEChERS. The target mycotoxins in drug and food homologous products were extracted by ASE. After concentration, the extracts were purified by QuEChERS. Then, the target compounds were analyzed by UPLC-MS/MS in both positive and negative electrospray ionization and MRM modes. Aflatoxin B1 and fumonisin B1 were quantified by the internal standard method, and the remaining mycotoxins were quantified by the matrix-matched external standard method. The proposed method showed a good linear relationship, with correlation coefficients greater than 0.99. The limits of detection (LODs) and limits of quantification (LOQs) of the 16 mycotoxins ranged from 0.008 μg/kg to 0.3 μg/kg and from 0.03 μg/kg to 1.0 μg/kg, respectively. The blank samples were spiked at three levels, and the recoveries ranged from 70.8% to 118%, with the RSDs being 2.5% to 10.2%. The developed method was successfully applied to mycotoxin analysis in 30 scutellaria, puerarin and sea buckthorn samples bought from local markets. Different levels of mycotoxins were detected in some of the products. The proposed method is simple, rapid and sensitive, and it can be applied to the simultaneous determination of multi-mycotoxins in drug and food homologous products.

In-tube solid-phase microextraction directly coupled to tandem mass spectrometry for anandamide and 2-arachidonoylglycerol determination in rat brain samples from an animal model of Parkinson's disease

To evaluate the endocannabinoid system in an animal model of Parkinson's disease, in-tube solid-phase microextraction (in-tube SPME) was directly coupled to a tandem mass spectrometry (MS/MS) system for determination of the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) in rat brain samples. In-tube SPME—which consisted of a microtube of restricted access material (RAM) with a hydrophilic diol external surface and a hydrophobic octyl inner surface—efficiently excluded (up to 95%) macromolecules from the biological samples and selectively pre-concentrated the analytes. In-tube SPME parameters, such as sample volume, mobile phases, flow rate, and pre-concentration time, were evaluated to improve the extraction efficiency and throughput performance. The selectivity of the in-tube SPME and MS/MS (MRM mode) techniques allowed them to be directly coupled online, which dismissed the need for the chromatographic separation step. The in-tube SPME-MS/MS method was validated and shown to be linear from 6.0 to 30.0 ng mL−1 for AEA and from 10.0 to 100.0 ng mL−1 for 2-AG; the intra- and inter-assay accuracy and precision were lower than 15%. Parallelism between the calibration curves constructed in the matrix and aqueous solution confirmed that there was no matrix effect. The method allowed endogenous concentrations of AEA and 2-AG to be determined in rat brain striatum from unilaterally 6-hydroxydopamine-lesioned animals. The concentrations of these endocannabinoids in striatum ipsilateral and contralateral to the lesion differed significantly (p<0.001).

LC-MS/MS quantification of progesterone in ovariectomized miniature swine and its application for preliminary pharmacokinetic study of a novel vaginal sustained release system

Objective We here established a simple, fast, robust and sensitive LC-MS/MS method and validated as well for the quantitative analysis of progesterone (PGT) in ovariectomized (OVX) miniature swine plasma. The method was successfully applied to characterize the pharmacokinetics of a progesterone vaginal drug delivery system. Methods Megestrol acetate was utilized as an internal standard (IS). The separation and detection of PGT from endogenous interference was performed successfully by liquid chromatography with gradient elution and mass spectrometry equipped with positive ESI source using MRM mode. The EVA intravaginal rings (IVRs) were manufactured by hot-melt extrusion (HME), afterward were administrated vaginally to OVX minipigs to evaluate PK study. Results The calibration curve for swine plasma samples across the concentration ranged between 0.25 ng/mL and 100 ng/mL. The intra- and inter-assay accuracy and precision were lower than ±5% and 5.88%, respectively. Recoveries of PGT and IS were ranging from 114–119% and 96.5–112%, respectively. In vitro study showed that the EVA IVRs release 18 mg/day of PGT continuously over 7 days, and corresponding mean PGT plasma concentration in OVX minipigs (CAVG) was 4.892 ng/mL. Conclusion All the study produced reliable results for the measurement of PGT concentration in miniature swine plasma and the method was successfully applied to a PK study for PGT vaginal ring in miniature pigs, which may lay the foundation for further research on the progesterone preparations intended for in assisted reproductive technology.

Selection and impact of decision mode of encroachment and retail service in a dual-channel supply chain

&lt;p style='text-indent:20px;'&gt;Consider a supply chain consisting of one manufacturer and one retailer. The manufacturer may open direct channels through ex-ante or ex-post encroachment, and the retailer can provide consumers with ex-ante or ex-post service. We investigates the effects of encroachment and services on the optimal strategy for two members in three decision modes: MR mode (ex-ante encroachment), MRM mode (ex-post encroachment and ex-post service), and MRMR mode (ex-post encroachment and ex-ante service). The results show that in the MRM mode, both the wholesale and retail prices may become higher with encroachment. Improving the service efficiency may hurt the retailer, and increasing the operating cost for direct channels harms the retailer, while benefits the manufacturer. In addition, only in the MRM mode, the retailer maybe benefits from encroachment under certain conditions. We further study the equilibrium mode and the result shows as follows. The MR mode, widely adopted by the literature on manufacturer encroachment, always is worst for the manufacturer. Only when both the operating cost for direct channels and the service efficiency are low, the equilibrium decision mode is the MRMR mode, otherwise the MRM mode is the equilibrium decision mode.&lt;/p&gt;

Determination of Mancozeb, a Pesticide Used Worldwide in Agriculture: Comparison among GC, LC, and CE

Background: The determination of mancozeb, a fungicide extensively used in agriculture, is a challenge, due to the nature of the compound, a manganese and zinc complex of ethylenebis dithiocarbamate and because of the general instability of the dithiocarbamates. Methods: Mancozeb was analyzed in a GC-EI-MS system after derivatization by CE-UV with detection at 280 nm and in LC-ESI-MS-MS in MRM mode. Results: A comparative study of the performance of three different techniques for the detection of mancozeb was explored, highlighting the advantages and drawbacks of them. The limits of detection and quantification of the techniques were determined; the repeatability was assessed, showing values of relative standard deviation. Gas chromatography, although very sensitive, was not reproducible enough due to fast degradation of the derivatization product, whereas capillary electrophoresis-UV showed problems in run-to-run reproducibility which had the worst limit of detection. LC coupled with tandem mass spectrometry was the most reliable and precise technique and was able to determine the main degradation product of Mancozeb, at the same time. The proposed LC procedure was verified by applying it to a commercial formulation, a fungicide of known concentration, and to Italian white grapes treated with the formulation sprayed during cultivation. Conclusion: Thanks to the simplified sample handling, the proposed method resulted to be simple, fast, green, economic, and suitable for residue analysis in grapes and other fruits. Finally, the method was compared with other similar investigations.

Stereoselective quantification of phase 1 and 2 metabolites of clomiphene in human plasma and urine

Clomiphene citrate is first line therapy of female infertility but is also frequently abused by athletes. Human biotransformation of clomiphene results in numerous phase 1 and phase 2 metabolites. The involvement of the polymorphic cytochrome P450 2D6 leads to a high inter-individual variability. To comprehensively investigate clomiphene metabolism in vivo we established a highly sensitive and specific UPLC-MS/MS method for the stereoselective quantification of clomiphene and its phase 1 and phase 2 metabolites in plasma and urine.Reference compounds and stable isotope labelled internal standards were synthesized in-house. High-throughput sample preparation was done by protein precipitation. Analytes were separated by UPLC on a C18 column (1.8 μm, 2.1 * 100 mm) using a gradient of 0.1% formic acid in acetonitrile in 0.1% aqueous formic acid and detected by positive ESI-MS/MS in MRM mode. The lower limit of quantification was below 1 nM for all analytes. The method was validated according to recent guidelines. However, due to absorption effects during sampling the quantification of metabolites in urine was limited to phase 2 metabolites. The method was successfully applied to determine the pharmacokinetic of (E)- and (Z)-clomiphene and 14 metabolites following a single dose of 100 mg clomiphene citrate in 3 healthy subjects and proofed to be an essential tool to comprehensively investigate the human biotransformation of clomiphene.

Status of phthalate esters in tobacco cultivation soils and its health risk to Chinese people

Phthalate esters (PAEs) have been and remain the most widely used plasticizers worldwide, and they are also the most frequently detected environmental pollutants. It is known that the management style and agricultural inputs should play a critical role in the PAE contamination. However, the status of six priority controlled PAEs remains unclear in soils used for tobacco on farms that are under the compulsorily management of national tobacco company. The samples included soils, water, and fertilizers were collected from tobacco cultivation sites in the Shandong province. The six priority controlled PAEs were extracted from the samples according to the reported methods, and they were analyzed using a GC-TQ-MS/MS under MRM mode. The hazard quotients and carcinogenic risks for people by gender and age were assessed first time by the recommended method of US Environmental Protection Agency. Soils were contaminated by PAEs from 218.4 to 1168.9 μg kg−1, with the average value of 611.1 μg kg−1. More than 80% Σ6PAEs consisted of DBP and DEHP, and regional characteristics of Shandong province showed the highest content of DBP. Besides plastic film, agricultural inputs such as irrigation water and fertilizers were also the PAE sources. At present, the non-cancer risk has not exceeded the threshold of 1, and carcinogenic risk was still low in the range of 10−6–10−4 with the highest risk of DEHP exposure through dietary consumption. Pollution status of PAEs in tobacco cultivation soils and its health risk to Chinese people were already present at low levels. Thus, we should pay more attention to PAE contamination in order to prevent further deterioration, particularly for the DEHP exposure to middle-aged people.

Determination of Loratadine and Its Active Metabolite in Plasma by LC/MS/MS: An Adapted Method for Children

Introduction: Loratadine (LOR) (Fig. 1), an active H1 receptor antagonist, is often used in the treatment of allergic disorders such as seasonal allergies and skin rash [1]. LOR was clinically approved for symptomatic relief of nasal and non-nasal symptoms of allergic rhinitis in children ≥2years. Materials and Methods: An adapted method of liquid chromatography-mass spectrometry (LC/MS/MS) was developed and validated to measure the concentrations of loratadine (LOR) and its active metabolite descarboethoxyloratadine (DCL) from pediatric plasma. After being mixed with the internal standard (IS, propranolol) and precipitated with methanol, samples were centrifuged and 20 μL of the supernatants were injected into the HPLC system. Separation was carried out on a reversed-phase C18 gradient column using a mobile phase consisting of water (containing 0.1 % formic acid) and acetonitrile. The flow rate was 0.5 mL/min and the running time was 5.0 min for each sample. Results and Conclusion: Quantitation of LOR, DCL and IS was performed using MRM mode and the transitions were: 383.1 → 337.1 for LOR, 311.1 → 259.0 for DCL and 260.2 → 116.0 for propranolol, respectively. The method was validated according to FDA guidelines, precisions and accuracies met the requirements in all cases. Calibration curves were 0.2–50.0 ng/mL for both LOR and DCL. This method was then applied for a pilot study examining the pharmacokinetics and therapeutic drug monitoring of LOR in children.

HA-C@silica sorbent for simultaneous extraction and clean-up of steroids in human plasma followed by HPLC-MS/MS multiclass determination

Aim and novelty of this work are the development of a simple and straightforward analytical procedure for multiclass determination of steroid hormones in human plasma. The method entails a single pre-treatment step based on solid-phase extraction using a recently proposed sorbent phase (HA-C@silica). This is easily prepared with good reproducibility via pyrolysis of humic acids onto silica, and not yet tested in biological fluids. It proved to be advantageous as it showed poor affinity for the protein matrix constituents while quantitatively extracting and pre-concentrating the target analytes. Indeed, as demonstrated in bovine serum albumin solution, up to ca. 90% protein is not retained by the sorbent, similarly to the behaviour of restricted access carbon nanotubes, tested for comparison. The high albumin exclusion allowed a satisfactory clean-up avoiding protein precipitation and centrifugation before extraction. The extraction procedure, optimized by a chemometric approach (23 experimental design) in BSA solution, provided quantitative recovery (76–119%, n = 3) for all steroids working with 1:8-diluted plasma (2 mL) and 100 mg HA-C@silica. Before analytes elution by 1 mL methanol-acetonitrile (1:1, v/v), selective washings (2% v/v formic acid and 30% v/v methanol) were applied to remove the small fraction of retained proteins, thus obtaining very clean SPE extracts to be analyzed by HPLC-ESI-MS/MS. This allowed identification/quantification (MRM mode) at few ng mL−1 by a single chromatographic run. The procedure was verified in blank-certified foetal bovine serum (spikes 10–100 ng mL−1), obtaining good recovery and suitable inter-day precision (RSDs < 15%, n = 3). The analytical method, applied to real plasma samples analysis, is appealing in terms of sample throughput, extraction efficiency and clean-up.

Analysis of α-Tocopherol in Tobacco and Cigarette Smoke

Summary α-Tocopherol, a type of vitamin E, has been known to be present in tobacco for many years. The compound is an antioxidant protecting cell membranes from oxidants. α-Tocopherol is transferred from tobacco into cigarette smoke, where it is also present. Analysis of α-tocopherol has been reported in a number of studies and in various matrices including tobacco and tobacco smoke. However, no recent publication describes a method for quantitative analysis of tocopherol in tobacco and in cigarette smoke, and many methods reported from previous studies were not published and only presented at conferences or communicated in internal company publications. The goal of this study was to quantitate α-tocopherol and, if present, α-tocopheryl acetate in tobacco and in tobacco smoke. For this analysis, an original HPLC technique was developed and is described in this report. Both UV and MS/MS (MRM mode) were used as detection procedure for the analysis. The results obtained using UV detection were in very good agreement with the results obtained using MS/MS detection. The method has been applied for the analysis of a number of tobaccos, as well as the total particulate matter (TPM) from cigarettes made with the same tobaccos. Depending on tobacco type, the levels of α-tocopherol vary in tobacco between about 200 μg/g up to about 900 μg/g (“dry weight basis”). For ISO type smoking, the levels of α-tocopherol vary in TPM between about 2 μg/mg up to slightly above 4 μg/mg of TPM. For a cigarette generating TPM of about 10 mg/cig, the α-tocopherol is between about 20 μg/cig up to about 40 μg/cig. A relatively good correlation was obtained between the level of α-tocopherol in smoke (ISO type smoking) and the level of the compound in tobacco. α-Tocopheryl acetate was absent in tobacco.

Qualitative and quantitative analyses of quinones in multi-origin Rubia species by ultra-performance liquid chromatography-tandem mass spectrometry combined with chemometrics

Rubia species are one of the important multi-origin phytomedicines having both economical and medicinal values in many countries. Quinones are the predominant bioactive constituents of these species. Therefore, accurate analysis of these quinones is critical to quality control, clinical, and commercial applications of Rubia species. In this study, a sensitive and efficient ultra-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (UPLC-QqQ-MS/MS) method in positive and negative multiple reaction monitor (+/-MRM) modes was firstly developed for the characterization of 45 quinones and the quantification of 24 quinones from Rubia plants. The chromatographic separation was achieved on a Waters ACQUITY UPLC® BEH C18 column by using a gradient elution with a mobile phase consisting of 0.1 % formic acid in water and acetonitrile. The results indicated that quinones in multi-origin Rubia samples were different, but 10 quinones existed in all samples. R. cordifolia, which is a unique official medicinal material in Chinese Pharmacopeia, contained the most number of quinones among all the Rubia species. R. podantha and R. sylvatica had the next highest number of quinones, implying that these species could be used as alternatives for R. cordifolia. Chemometric approaches were applied to evaluate the chemical relationship between the Rubia samples based on the constituent quinones. In this study, a UPLC-QqQ-MS/MS method in the MRM mode has been developed for the analysis of Rubia species-derived quinones, which not only contributes to quality control and discrimination of Rubia species, but also suggests the potential of these species in clinical and commercial applications.

Development and validation of liquid chromatography‐tandem mass spectrometry targeted screening of 16 fentanyl analogs and U‐47700 in hair: Application to 137 authentic samples

This study was to validate a LC-MS/MS method for the determination of 17 new synthetic opioids (NSOs) in hair including 3-fluorofentanyl, 3-methylfentanyl, acetylfentanyl, acetylnorfentanyl, alfentanyl, butyrylfentanyl, butyrylnorfentanyl, carfentanil, fentanyl, furanylfentanyl, furanylnorfentanyl, methoxyacetylfentanyl, norcarfentanil, norfentanyl, ocfentanil, sufentanil, and U-47700, and to apply it to 137 authentic samples. Twenty milligrams of hair was decontaminated in dichloromethane and underwent liquid extraction. 10 μL of the reconstituted residue were injected onto the system. The separation was performed in 12 minutes in a gradient mode at a flow rate of 300 μL/min using a Hypersyl Gold PFP column (100 × 2.1 mm i.d., 1.9 μm) maintained at 30°C. Compounds were detected in positive ionization and MRM modes using a TSQ Endura mass spectrometer (ThermoFisher). The method was validated according to EMA guidelines. The LLOQ was in the range 1-50 pg/mg, and the calibration ranged from the LLOQ-1000 pg/mg. Intra- and inter-day accuracy (bias) and precision were < 15%. Extraction recoveries of parent drugs and metabolites were 74-120% and 7-62%, respectively. The matrix effect was in the range 59-126% (CVs ≤ 12.9%). Fentanyl was found in six cases at concentrations of < 1-1650 pg/mg (n = 14 segments). Five fentanyl analogs were quantified in two cases: 3-fluorofentanyl (25-150 pg/mg, n = 5), furanylfentanyl (15-500 pg/mg, n = 5), methoxyacetylfentanyl (500-600 pg/mg, n = 2), acetylfentanyl (1 pg/mg, n = 2), carfentanyl (2.5-3 pg/mg, n = 2). This fully validated method allowed us to test for the first time 3-fluorofentanyl and norcarfentanil in hair among 15 other NSOs, and brings new data regarding 3-fluorofentanyl and methoxyacetylfentanyl hair concentrations.

GC-MS/MS method for simultaneous determination of ester forms of 3-MCPD and 2-MCPD in infant formula

A sensitive GC-MS/MS method has been developed and validated for determining ester forms of 3-MCPD and 2-MCPD in infant formula, using 3-MCPD-d5 and 2-MCPD-d5 as internal standards, respectively. The esters of MCPDs were extracted from infant formula powder with mixture of ethanol/n-hexane/diethyl ether (1/1/1, v/v/v), then treated with sulfuric acid to release free MCPDs. Free MCPDs and internal standards were derivatized with phenylboronic acid and the corresponding derivatives were used for GC-MS/MS analysis. The chromatographic separation was performed in a DB-5MS capillary column using helium as mobile phase at flow rate of 1 mL/min. Tandem mass spectroscopic detection of each analyte was done in MRM mode by monitoring one precursor ion and two product ions. Validation results confirmed the suitability of the developed method for intended application. Application of the method in samples collected from different provinces in Vietnam detected esterified 3-MCPD in 38.9% of samples, esterified 2-MCPD in 34.7% of samples.

High Throughput Detection of 327 Drugs in Blood by LC-MS-MS with Automated Data Processing.

The described procedure provides a rapid technique for the detection and semi-quantitation of a large number of drugs in blood. This procedure uses a minimal sample volume and employs a one-step liquid extraction and automated data processing to yield rapid turnaround times. A total of 327 of the most commonly used medicinal and illicit drugs in Australia were selected including various amphetamines, anesthetics, antidepressants, antipsychotics, anticonvulsants, benzodiazepines, beta blockers, opioid and nonopioid analgesics, stimulants, THC and a large number of synthetic cannabinoids and other novel psychoactive substances. The extracts were subject to 5-minute chromatography using a Kinetex C18 50×4.6mm 2.6μm solid-core analytical column and analyzed using a Sciex 3200 Q-TRAP MS-MS (+ ESI, MRM mode, two transitions per analyte). The method was fully validated in accordance with international guidelines. Matrix effects and extraction efficiencies were acceptable with most analytes showing > 80% response and low variation (within 25%RSD). Cannabinoids were most affected by the matrix and yielded poorest recovery values but were still detectable. Precision, accuracy, repeatability and multipoint linearity were assessed for all analytes. The method has been used in routine practice in the forensic toxicology service at the Victorian Institute of Forensic Medicine in over 6000 coronial investigations using both postmortem and clinical blood specimens. This technique has greatly increased throughput, reduced turnaround times and allowed for rapid same-day analysis of results when needed. The method is routinely used in routine overnight testing with results reported to pathologists within 4h of data acquisition. This rapid toxicological technique is used in conjunction with other investigative processes such as full-body CT imaging, review of case circumstances and medical histories to provide an efficient death investigation process.

Ultrasensitive microflow‐UPLC‐MS/MS method for quantifying drug metabolizing enzymes and transporters in tissue S9 fractions applicable to small total protein amount and biopsy samples

Quantitative proteomics is a powerful technique that provides important data for in vitro to in vivo extrapolation (IVIVE) and predicts interindividual variability in drug disposition.However, the conventional quantitative proteomics methods often require high initial total protein and enriched samples (e.g., microsomes and pure membrane fractions). The goal of this study was i) to develop a microflow based LC‐MS/MS method for quantification of clinically relevant drug metabolizing enzymes (DMEs) and transporters using small total protein (400 ng), and ii) to quantify these proteins in human liver and intestine S9 samples.Targeted quantitation of DMEs and transporters was performed on trypsin digested pooled liver (N=200) and pooled intestinal (N=15) S9 fractions (Xenotech, Kansas city). The digestion was performed using an optimized in house protocol. A total of 400 ng protein (1 μl injection volume) was injected through a trap‐column followed by peptide separation in a microflow column (BEH C18; 130 A°,1.7μm, 150 μm*50 mm) using M‐class UPLC system and detection by Xevo TQ‐XS MS instrument supported by ionkey interface. An optimized gradient elution was used with 3 μl/min flow rate. Data were acquired in scheduled MRM mode and the data analysis was performed using Skyline 19.1 software.72 DMEs and transporters were quantifiable using 400 ng of total protein injection. A total of 12 CYPs, 12 UGTs, 33 nonCYPs/nonUGTs (e.g., SULTs, GSTs, ADHs, ALDHs, etc.) and 16 transporters were detected. Out of the total detected proteins, 53 were in both the liver and intestine, 17 were only detected in the liver, and 2 were only quantifiable in the intestine. CYP2C8, CYP2B6, CYP2D6, CYP2A6, CYP2E1, UGT1A9, UGT1A4, UGT2B4, UGT2B15, SULT1E1, PON3, FMO3, AOX1, ADH1A, ADH1B, OATP1B1 and OATP1B3 were exclusively detected in the liver whereas UGT1A10 and UGT2A1 were only seen in the intestine. ALDH1A1, GSTT1, SULT4A1, SLC22A3, CYP3A4, ALDH1A3, SULT2A1, CYP1A1, SLC22A2, MATE1, ADH1C, SULT1C, POR, ABCC2 were within 0.75–1.5‐fold in the liver and intestine S9 fractions. CES1, PON1, UGT1A6, CYP2C9 and UGT2B7 were significantly higher in the liver as compared to intestine, and SULT1B1, CES2, UGT2B17, FMO1 and UGT1A8 were amongst the top differentially expressed proteins in the intestine. We were able to quantify low abundant proteins such as BCRP, CYP1A1 and UGT1A8, which are generally difficult to detect by conventional LC‐MS/MS methods. In summary, the microflow‐LC‐MS/MS is an ultrasensitive approach for quantifying DMEs and transporters in crude tissue preparations such as homogenate and S9 fractions. Since microsomes and pure membrane preparations require recovery factors that add batch‐to‐batch variability, quantification of proteins in homogenate and S9 fractions using the ultrasensitive method would provide accurate scaling factors for IVIVE. This microflow‐LC/MS/MS method can be applied to small biopsy samples (e.g., tissue weight ~2.5 mg) thus providing broad applications in predicting interindividual variability (e.g., effect of diseases) in drug disposition using patient samples.Support or Funding InformationDepartment of Pharmaceutical Sciences, Washington State UniversityNumber of proteins quantified in intestine and liver S9 fractions.Figure 1

A UHPLC-MS/MS method to simultaneously quantify apixaban, edoxaban and rivaroxaban in human plasma and breast milk: For emerging lactation studies

Clinical studies are needed to clarify the use of direct oral anticoagulants (DOACs) in breastfeeding women. To support emerging clinical studies on investigating DOAC’s transfer into breast milk, an ultra-high-performance liquid chromatography/tandem mass spectrometry (UHPLC‐MS/MS) method was developed and validated for quantifying three DOACs – apixaban, edoxaban and rivaroxaban in human plasma and breast milk. Protein precipitation with methanol was performed for sample preparation. Chromatographic analysis was performed using a C18 column. The MS detection was performed in MRM mode. The method was validated in accordance with the European Guideline (EMA). The calibration range was 5–500 ng/mL in plasma and 5–250 ng/mL in breast milk. The within-batch and between-batch variability remained <9%. Recoveries ranged from 106.13% to 109.05% in plasma and from 93.40% to 107.91% in breast milk. The lot-to-lot matrix variability was within ±15% among a range of samples originating from many different subjects. All analytes were stable when stored for 24 h at room temperature, 7 days at 2–8 °C, and at least 5 weeks at −20 °C in both plasma and breast milk. The developed method fulfilled the EMA bioanalytical method validation guideline and was shown to be simple, fast, accurate and will now be used in a clinical trial evaluating the transfer of apixaban and rivaroxaban into human breast milk.

Quantification of healthcare workers' exposure to cyclophosphamide, ifosfamide, methotrexate, and 5-fluorouracil by 24-h urine assay: A descriptive pilot study.

The objective of this pilot study was to determine the frequency of urination and the concentration of four hazardous drugs (cyclophosphamide, ifosfamide, methotrexate, and fluorouracil) in workers' 24-h urine samples in relation to exposure to traces with hazardous drugs. The study was conducted in three healthcare centers in the region of Montréal, Quebec, Canada. We recruited healthcare workers (nurses and pharmacy technicians) assigned to the hematology-oncology department. Each participant was asked to collect all urine voided during a 24-h period, to fill out an activity journal documenting tasks performed and to document the use of personal protective equipment. Samples were analyzed for cyclophosphamide, ifosfamide, methotrexate, and alpha-fluoro-beta-alanine (FBAL, the main urinary metabolite of 5-fluorouracil). Drugs were quantified by ultra-performance liquid chromatography-tandem mass spectrometry (positive electrospray MRM mode). Eighteen healthcare workers (10 nurses and 8 technicians) were recruited and provided consent to participate. Urine samples were obtained between 1 September and 30 September 2019. The number of urinations over the 24-h collection period ranged from 3 to 11 per participant. A total of 128 urine samples were analyzed for the 18 workers. All urine samples were negative for the four antineoplastics tested. No traces of cyclophosphamide, ifosfamide, methotrexate, or FBAL were found in the 24-h urine samples of 18 healthcare workers practicing in three healthcare facilities in Quebec. Although it was feasible to collect 24-h urine samples in this research project, it appears unrealistic to do so recurrently as part of a large-scale surveillance program.