Liquid Chromatography-tandem Mass Spectrometry Method Research Articles

Diverse associations observed between pregnancy complications and RBC or plasma folates determined by an in-house developed LC-MS/MS method

Background As folates are essential for embryonic development and growth, it is necessary to accurately determine the levels of folates in plasma and red blood cells (RBCs) for clinical intervention. The aims of this study were to develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantitation of folates in plasma and RBCs and to examine the association between plasma and RBC folate concentrations and gestational diabetes mellitus (GDM), gestational hypertension (GH) and preeclampsia (PE). Methods With the in-house developed LC-MS/MS, a retrospective cross-sectional study was conducted. The healthy pregnant women of first- (n = 147), second- (n = 84) and third-trimester (n = 141) or the women diagnosed with GDM (n = 84), GH (n = 58) or PE (n = 23), that were aged between 22 and 46 years old and registered at our institute, were subjected for measurement of folic acid (FA) and 5-methyltetrahydrofolate (5-MTHF), followed by appropriate statistical association analysis. Results The assay for simultaneous quantitation of FA and 5-MTHF in plasma and RBCs was linear, stable, with imprecision less than 15% and recoveries within ±10%. The lower limits of quantification for FA and 5-MTHF measurement in whole blood were 0.57 and 1.09 nmol/L, and in plasma were 0.5 and 1 nmol/L, respectively. In the association analysis, the patients with lower RBC folate level (<906 nmol/L) presented higher risks of PE development (OR 4.861 [95% CI 1.411–16.505]) by logistic regression and restricted cubic spline (RCS) regression in a nonlinear fashion. In addition, higher level of plasma folates in pregnancy was significantly associated with GH risk but may be protective for the development of GDM. Conclusions The in-house developed LC-MS/MS method for folates and metabolites in plasma or RBC showed satisfactory analytical performance for clinical application. Further, the levels of folates and metabolites were diversely associated with GDM, GH and PE development.

Optimization of a ultra-high-pressure liquid chromatography-tandem multiple reaction monitoring mass spectrometry method for monitoring haloacetic acids as chlorinated disinfection by-products in the fresh-cut industry

Haloacetic acids (HAAs) are one of the most important chlorinated disinfection by-products generated during water disinfection in the fresh-cut industry, and they can remain in the product, resulting in a consumer health risk. In this study, ultra-high-pressure liquid chromatography-tandem multiple reaction monitoring mass spectrometry (UHPLC-MRM) analysis used for drinking water was optimized and applied for the quantification of nine HAAs (HAA9) in fresh-cut lettuce and process water samples, with the complex matrix interferences for separation, and quantification problems. The method showed good selectivity, specificity and linearity, satisfactory values for trueness (recoveries of 80–116 %), precision (<22 %), and uncertainty (<55 %). Quantification limits varied from 1 to 5 µg L−1 or µg kg−1. The matrix effect for tribromoacetic, bromochloroacetic and chlorodibromoacetic acid was corrected by matrix-matched calibration and standard addition. After storage at -20 °C, only monobromoacetic acid was the HAA which loss happened after 7 days. The application of the methodology in lettuce and process water samples from the industry was successfully implemented. Therefore, this method could be employed for the quality control and regulatory analysis of HAAs in fresh products and process water from the fruit and vegetable industry.

New sensitive LC-MS/MS method for the simultaneous determination of 13 phenolic and carboxylic acid pesticide biomarkers in human urine, including dicamba

The commercialization in 2016 of genetically engineered seeds tolerant to dicamba and/or 2,4-dichlorophenoxyacetic acid (2,4-D) has caused a rapid increase in the use of these herbicides. New questions about the reproductive and chronic health effects of long-term exposure to these herbicides have been raised. To assess exposure to dicamba and other pesticides of interest in the Heartland Study, a birth cohort study based in the United States, a new analytical method was needed. The present study describes the development and validation of this new solid phase extraction and liquid chromatography-tandem mass spectrometry method that detects simultaneously 13 pesticides or their metabolites in 250 μL of urine. More specifically, the method allows the analysis of dicamba, 2,4-D and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), which are herbicides, of malathion dicarboxylic acid (MDA), para-nitrophenol (PNP), 3,5,6-trichloro-2-pyridinol (TCPy), 2-diethylamino-6-methylpyrimidin-4-ol (DEAMPY) and 2-isopropyl-6-methyl-4-pyrimidinol (IMPY), which are metabolites of organophosphate insecticides, and finally of cis-3-(2,2-Dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid (cis-DCCA), trans-3-(2,2-Dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid (trans-DCCA), 3-Phenoxybenzoic acid (3-PBA), 4-Fluoro-3-phenoxybenzoic acid (4-F-3-PBA) and cis-3-(2,2-Dibromovinyl)-2,2-dimethylcyclopropane carboxylic acid (cis-DBCA), which are metabolites of synthetic pyrethroids insecticides. The method was validated under ISO/IEC 17025 guidance. The limit of detection (LOD) in urine samples was 0.10 μg/L for dicamba, while the LOD for other analytes ranged between 0.0038 μg/L and 0.091 μg/L. Accuracy was evaluated by analyzing samples from two External Quality Assessment Schemes, namely G-EQUAS and OSEQAS. Preliminary results obtained following the analysis of 91 urine samples taken from pregnant women enrolled in the Heartland Study are presented here. This method is suitable for human biomonitoring studies.

Development, validation, and implementation of an ultratrace analysis method for the determination of moenomycin A, in aquatic animal products.

Moenomycin A, an antimicrobial growth promoter widely used as an additive in aquaculture feedstuffs, has been restricted for use in the European Union and China due to its potential risk of promoting resistant strains of pathogenic bacteria and causing residues in aquatic animal products. Although methods for analyzing moenomycin A in feedstuffs have been developed, no established method exists for aquatic matrices. In this study, we present, for the first time, a sensitive and validated high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the determination of moenomycin A in aquatic animal products. Samples were extracted using methanol and purified with the QuEChERS method employing C18 sorbent. The aliquot was dried under a nitrogen stream, reconstituted with methanol-water solvent, and analyzed by HPLC-MS/MS. The developed method exhibited good linearity (r2 > 0.995) over a wide concentration range (1-100μg/L) and a low limit of detection (1µg/kg). Average recoveries ranged between 70 and 110% at spiked concentrations of 1, 50, and 100μg/kg, with associated intra- and inter-day relative standard deviations of 1.25 to 7.32% (n = 6) and 2.91 to 10.08% (n = 3), for different representative aquatic animal production, respectively. To the best of our knowledge, this is the first reported HPLC-MS/MS method for the quantification of moenomycin A in aquatic animal products. The new approach was effectively employed in the analysis of moenomycin A across various aquatic samples.

Correlations among the plasma concentrations of first-line anti-tuberculosis drugs and the physiological parameters influencing concentrations.

Background: The plasma concentrations of the four most commonly used first-line anti-tuberculosis (TB) drugs, isoniazid (INH), rifampicin (RMP), ethambutol (EMB), and pyrazinamide (PZA), are often not within the therapeutic range. Insufficient drug exposure could lead to drug resistance and treatment failure, while excessive drug levels may lead to adverse reactions. The purpose of this study was to identify the physiological parameters influencing anti-TB drug concentrations. Methods: A retrospective cohort study was conducted. The 2-h plasma concentrations of the four drugs were measured by using the high-performance liquid chromatography-tandem mass spectrometry method. Results: A total of 317 patients were included in the study. The proportions of patients with INH, RMP, EMB, and PZA concentrations within the therapeutic range were 24.3%, 31.5%, 27.8%, and 18.6%, respectively. There were positive associations between the concentrations of INH and PZA and RMP and EMB, but negative associations were observed between the concentrations of INH and RMP, INH and EMB, RMP and PZA, and EMB and PZA. In the multivariate analysis, the influencing factors of the INH concentration were the PZA concentration, total bile acid (TBA), serum potassium, dose, direct bilirubin, prealbumin (PA), and albumin; those of the RMP concentration were PZA and EMB concentrations, weight, α-l-fucosidase (AFU), drinking, and dose; those of the EMB concentration were the RMP and PZA concentrations, creatinine, TBA and indirect bilirubin; and those of the PZA concentration were INH, RMP and EMB concentrations, sex, weight, uric acid and drinking. Conclusion: The complex correlations between the concentrations of the four first-line anti-TB drugs lead to a major challenge in dose adjustment to maintain all drugs within the therapeutic window. Levels of TBA, PA, AFU, and serum potassium should also be considered when adjusting the dose of the four drugs.

Development of enantioselective high-performance liquid chromatography-tandem mass spectrometry method for the quantitative determination of 3,4-methylenedioxy-methamphetamine (MDMA) and its phase-1 metabolites in human biological fluids

In the present study enantioselective high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods were developed for the quantitative determination of 3,4-methylenedioxy-methamphetamine (MDMA) and its major phase-1 metabolites 4-hydroxy-3-methoxyamphetamine (HMA), 4-hydroxy-3-methoxymethamphetamine (HMMA) and 3,4-methylenedioxyamphetamine (MDA) in human plasma, sweat, oral fluid (OF) and urine. The simultaneous separation of all these compounds and their respective enantioseparation was accomplished on two polysaccharide-based chiral columns. The Lux AMP column with a proprietary chiral selector enabled baseline separation of the enantiomers of MDMA, HMA and HMMA while MDA enantiomers could not be separated with this column under the experimental conditions used in this study. The Lux i-Amylose-3 column based on amylose tris(5-chloro-3-methylphenylcarbamate) as chiral selector baseline-separated the enantiomers of MDMA, HMMA and MDA while the enantiomers of HMA could not be separated. Thus, the various samples were analyzed by using both columns alternatively in combinations with acetonitrile containing 25% (v/v) 5 mM ammonium bicarbonate buffer at pH 11.0 as mobile phase. Analysis time was less than 4 min with the Lux AMP column and less than 6 min with the Lux i-Amylose-3 column. Both methods were validated and applied to the enantioselective determination of MDMA and its phase-I metabolites in human biological fluids, and enantioselective metabolism of MDMA was confirmed.

SAT628 Effect Of Hepatic Impairment On The Pharmacokinetics, Safety, And Tolerability Of Oral Paltusotine, A Non-peptide, Selective Somatostatin Receptor Subtype 2 Agonist

Abstract Disclosure: R. Luo: Employee; Self; Crinetics Pharmaceuticals Inc. A. Casagrande: Employee; Self; Crinetics Pharmaceuticals Inc. S. Oun: Employee; Self; Crinetics Pharmaceuticals Inc. Y. Wang: Employee; Self; Crinetics Pharmaceuticals Inc. R. Struthers: Employee; Self; Crinetics Pharmaceuticals Inc. A. Krasner: Employee; Self; Crinetics Pharmaceuticals Inc. Paltusotine is an investigational oral, once-daily, nonpeptide, small molecule somatostatin receptor type 2 agonist in clinical development for the treatment of acromegaly and neuroendocrine tumors. This multicenter, open-label, phase 1 study assessed the influence of varying degrees of hepatic impairment on the pharmacokinetics (PK), safety, and tolerability of paltusotine. Participants with mild, moderate, and severe hepatic impairment based on Child-Pugh classification and matched healthy participants received a single 20 mg oral dose of paltusotine after an overnight fast of at least 10 hours. Analysis of paltusotine in plasma was performed using a validated liquid chromatography-tandem mass spectrometry method. Geometric mean ratios of PK parameters were calculated from an analysis of variance (primary analysis) with hepatic condition (normal, mild, moderate, severe) as a fixed effect and the log transformed PK parameter as the dependent variable. A sensitivity analysis (matched pairs mixed models for repeated measures) accounted for participant age and BMI. Overall, 36 participants were included (mild [n=8], moderate [n=8], and severe [n=6] hepatic impairment; matched healthy [n=14]). Most participants were male (72.2%); mean age was 57.3 years, and mean BMI was 30.7 kg/m2. Following single oral administration of 20 mg paltusotine, the mean plasma concentration of paltusotine peaked rapidly, with the median time to maximum concentration ranging from 1.6 to 3.0 hours postdose across all groups. The maximum plasma concentration (Cmax) and total plasma exposure (area under the curve from time 0 extrapolated to infinity [AUC0-inf]) of paltusotine were similar across all hepatic impairment groups when compared with healthy participants as indicated by the geometric mean ratio (90% CI) values (participants with mild, moderate, and severe hepatic impairment: Cmax: 1.35 [0.79-2.31], 0.76 [0.45-1.31], and 1.05 [0.58-1.90], respectively; AUC0-inf: 1.00 [0.60-1.66], 0.75 [0.45-1.24], and 0.90 [0.51-1.57], respectively). Results of the sensitivity analysis were consistent with those from the primary analysis. Overall, the most common (≥5% of participants) treatment-emergent adverse events (TEAEs) were diarrhea (16.7%) and headache (5.6%). The incidence of TEAEs was similar across all 4 groups. No participant discontinued the study because of a TEAE, and no serious drug-related TEAEs were reported. In conclusion, the peak and total plasma exposures and safety and tolerability profile of paltusotine were similar across participants with varying degrees of hepatic impairment compared with participants with normal hepatic function. There were no changes in paltusotine plasma exposure that would be considered clinically meaningful or sufficient to warrant dose adjustment for mild, moderate, or severe hepatic impairment. Support: Crinetics Pharmaceuticals. Presentation: Saturday, June 17, 2023

Development of a novel enantioselective high-performance liquid chromatography–mass spectrometry method for the differentiation of dextro- and levo-methorphan and their O-demethylated metabolites in human blood and its application to post-mortem samples

Recently we proposed an isocratic enantioselective high-performance liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for the separation and quantitative determination of dextro- (DXM) and levo-methorphan (LVM) and their pharmacologically relevant metabolites, dextrorphan and levorphanol, respectively, in human blood samples. This method was based on the polysaccharide-based chiral column Lux AMP, a specialty column characterized with high stability in mobile phases of pH 11.0 and above. The use of a single-source column is a limitation for any analytical method. Therefore, the major goal of the present study was to develop an enantioselective method for the differentiation of dextro- and levo-methorphan, as well as their metabolites dextrorphan and levorphanol, using Lux Cellulose-3 as alternative chiral column with methanol containing 0.1 % diethylamine mobile phase. A newly developed method uses a chiral selector part of HPLC columns available from multiple manufacturers and a fairly common mobile phase. The method was validated and applied to post-mortem blood samples. Out of 50 analyzed new samples, dextromethorphan (DXM) was detected in 17 samples. Of these 17 cases DXM was accompanied with LVM in 7 samples. The proposed analytical method is relatively simple, accurate and fast and can be adopted for routine use in forensic and clinical toxicology laboratories.

Pharmacokinetics of a New Neuroprotector - Indenoquinoxalinone Derivative after Intravenous Administration in Rabbits and Rats.

The specific JNK inhibitor and NO donor 11H-indeno[1,2-b]quinoxalin-11-one oxime (IQ-1) demonstrated pronounced neuroprotective properties in an in vivo model of ischemic stroke in rats. The pharmacokinetic behavior of IQ-1 was studied in two animal species (rats, rabbits) after intravenous administration in a dose of 1 mg/kg. IQ-1 concentrations in venous blood plasma were measured by the liquid chromatography-tandem mass spectrometry method. The pharmacokinetics of IQ-1 was adequately described by the two-compartmental model. The calculated C0 for IQ-1 in rabbit and rat plasma were 2239.83±1229.55 and 1552.50±182.23 ng/ml, respectively. Two animal species are characterized by extensive tissue distribution of IQ-1 (Vss exceeded the total body water in rabbits and rats by 3.6 and 5.6 times, respectively) and high clearance values (88-94% of hepatic blood flow).

Correlation of drug exposure and bacterial susceptibility with treatment response for Mycobacterium avium complex lung disease: protocol for a prospective observational cohort study

IntroductionThe burden of Mycobacterium avium complex (MAC) lung disease is increasing globally and treatment outcome is in general poor. Therapeutic drug monitoring has the potential to improve treatment outcome by...

INVESTIGATION OF RECENTLY ABUSED DRUGS IN DRIED BLOOD SPOTS USING FTA CARDS BY LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY: VALIDATION AND APPLICATION TO REAL SAMPLES

A robust and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was established for the analysis of 19 illicit drugs and metabolites in whole blood dried blood spots (DBS) using FTA cards. Samples prepared using Ahlstrom Munksjö Gensaver™ Colorcards were extracted with methanol. Analytical separation of the analytes was maintained using an Agilent Poroshell column with mobile phase A (0.1% formic acid in water) and B (methanol). Multiple Reaction Monitoring (MRM) with positive ionisation on LC-MS/MS was used to develop the method. The method was validated and met the acceptance criteria with acceptable results for carry-over, linearity, specificity, sensitivity, accuracy, precision, matrix effect and recovery. The method was applied to positively reported whole blood samples from patients suspected of drug abuse. Good quantitative agreement was obtained between the DBS and whole blood methods. Application to real DBS samples showed that this method is a good alternative and useful technique for the detection of drugs of abuse in forensic toxicology and appears to provide a good alternative storage condition.

Simultaneous determination of spiropidion and its five major metabolites in sweet orange fruit and various processing by-products using ultra-high performance liquid chromatography-tandem mass spectrometry

The present work reported the application of an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for simultaneous analysis of spiropidion and its five major metabolites in sweet orange fruit and by-products throughout the whole industrial juicing process of the orange fruit. The reversed-dispersive solid phase extraction (r-DSPE) with multi-walled carbon nanotubes (MWCNTs) was employed for the extraction and purification. The established method was validated and satisfactory parameters (linearity, trueness, precision, sensitivity, matrix effect and stability) were obtained. And then, the field trial of spiropidion on sweet oranges has been conducted and the effect of commercial juicing processing on the residue of spiropidion and its metabolites was further investigated. The various processing factors (PFs) for washing, juicing, sterilization, concentrating and essential oil collecting were also determined. The final results indicated that washing processing reduced residues by 18.4%; the juicing step allowed a significant decrease of the spiropidion residue by 34.2–70.8%, with PFs value in the range of 0.290–0.658. However, high level of residual spiropidion (ranging from 4.016 to 4.205 mg/kg) was detected in orange essential oil, with PFs value of 17.157. All the above results demonstrated the efficiency of the established method in the routine control analysis of spiropidion residues in sweet orange fruits and their by-products, and will facilitate the further intensive research on its spatial distribution, transfer and degradation during the different processing procedures of the sweet orange fruits.

Determination of Caffeine in Human Plasma by Using Liquid Chromatography-Tandem Mass Spectrometry

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to determine caffeine (CAF) in human plasma. The plasma samples were extracted by protein precipitation using CAF-D3 as an internal standard (IS). The chromatographic separation was performed on GL Sciences InertSustain C18 Column (4.6 x 50 mm, 5 µm) maintained at 40 °C with a mobile phase consisting of formic acid, water, and methanol at a 1 mL/min of flow rate using two separate lines. CAF was detected and identified by mass spectrometry with electrospray ionization (ESI) in positive ions and multiple-reaction monitoring (MRM) mode. The MRM transitions of m/z 195.10 &amp;gt; 138.00 for CAF and 198.10 &amp;gt; 141.10 for IS were used for quantification. The standard curve was linear in the range of 10 - 10000 ng/mL for CAF. The within-batch precision and accuracy were evaluated by analyzing QC samples at five different concentration levels with six replicates in a batch. The between-batch precision and accuracy were determined by analyzing three different batches. The within-batch accuracy and precision was -8.76% - 9.61% and 0.95% - 7.22%, respectively. The between-batch accuracy and precision was -7.47% -1.42% and 1.83% - 8.66%, respectively. The results of the intra- and inter-day precision and accuracy studies were within the limits. The validated method applied to a pharmacokinetic study and the test product containing 60 mg CAF administered to total of 12 subjects. The mean ± SD of maximum plasma concentration (Cmax) was found to be 147.94 ± 139.39 ng/mL and the mean ± SD of area under the plasma concentration-time curve from zero to last measurable concentration (AUC0-tlast) was found to be 1119.59 ± 1468.30 h.ng/mL for the fasting conditions. The median time to reach peak plasma concentration (Tmax) was found to be 12.00 (6.50 - 12.00). The developed and validated method can be used for bioavailability and bioequivalence studies in human plasma samples.

High-performance liquid chromatography-tandem mass spectrometry method formeasuring cotinine andtrans-3'-hydroxycotinine inbronchoalveolar lavage fluid ofpatients with e-cigarette, or vaping, product use-associated lung injury.

In 2019, nearly 3000 U.S. residents developed severe lung injury associated with recent use of e-cigarette or vaping products. The Centers for Disease Control and Prevention responded to the outbreak, which was formally defined as e-cigarette, or vaping, product use-associated lung injury (EVALI). Centers for Disease Control and Prevention Laboratory rapidly developed assays to analyze potentially harmful and addictive substances in bronchoalveolar lavage (BAL) fluid collected from EVALI case patients. This report describes the development and validation of a high-throughput isotope-dilution high performance liquid chromatography-tandem mass spectrometry method for measuring two nicotine biomarkers, cotinine (COT) and trans-3'-hydroxycotinine (HCT), in bronchoalveolar lavage fluid samples. COT and HCT are the major metabolites of nicotine, the addictive alkaloid presents in tobacco products. This method had good specificity and sensitivity. The limit of detection is 0.033 and 0.0165 ng/mL for COT and HCT, respectively, using only 200 µL of sample volume. The within-run and between-run precision were 2-10%. The overall accuracy, calculated from recovery in three different sample matrices spiked at three concentrations, was 94.8% and 93.6% for COT and HCT, respectively. This novel HPLC-MS-MS method was utilized to characterize recent tobacco exposure in EVALI case patients. This method is useful for characterizing tobacco exposure that may be related to acute and chronic lung injury.

B-167 Development and Validation of Liquid Chromatography-Tandem Mass Spectrometry Method for Therapeutic Drug Monitoring of Treosulfan and Busulfan

Abstract Background Treosulfan and busulfan are alkylating agents administered as the conditioning regimen before hematopoietic stem cell transplantation. High busulfan exposure is associated with drug toxicity and low busulfan exposure with rejection or disease recurrence. There is also an association between treosulfan exposure and early toxicity, such as skin toxicity and mucositis. As both busulfan and treosulfan have narrow therapeutic ranges, therapeutic drug monitoring (TDM) is essential. Since the clinical laboratory tests for both drugs are few, a method that can simultaneously measure both drug concentrations under the same conditions would be helpful in practice. In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous measurement of treosulfan and busulfan was developed and validated. Methods For preparing calibrators and quality control materials, commercialized reference standards were used for treosulfan, an active metabolite of treosulfan, S,S-EBDM, and busulfan. Treosulfan-D4 and busulfan-D8 isotopes were used as internal standards (ISs). The analytes were separated from 25 µL of plasma after protein precipitation with acetonitrile containing ISs. After centrifugation, the supernatant was diluted with 3% formic acid and then injected into the liquid chromatography system followed by tandem mass spectrometry. Chromatographic separation was performed using ACQUITY Ι-Class plus ultraperformance liquid chromatography (UPLC) system coupled with a XEVO TQ-XS mass spectrometer operated in positive ion electrospray ionization mode. The analytes and ISs were detected in the multiple reaction monitoring (MRM) mode. The limit of detection (LoD), limit of quantification (LoQ), selectivity, linearity, accuracy, precision, carryover, matrix effect, and sample stability were validated according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Results Retention times for treosulfan, S,S-EBDM and IS for both analytes were 0.44 min, 0.43 min, and 0.44 min, respectively. Retention times for busulfan and IS for busulfan were 1.20 min and 1.18 min, respectively. The mass-to-charge (m/z) transitions used for quantification in the MRM mode were as follows: treosulfan 296.2 &amp;gt; 87.1; S,S-EBDM 200.2 &amp;gt; 87.1; and busulfan 264.1 &amp;gt; 55.1. The total run time was 4 min. The LoDs of treosulfan, S,S-EBDM, and busulfan were 0.21, 0.23, and 0.001 µg/mL, respectively. The linear range of the calibration curves of treosulfan, S,S-EBDM, and busulfan spanned concentrations of 3.13–100, 0.63–5.00, and 0.16–5.00 µg/mL, respectively. The total precision of the developed method fulfilled the analytical criteria, except for S,S-EBDM. The LC-MS/MS method was adequately selective and accurate and showed no carry-over. It provided an acceptable matrix effect for treosulfan and busulfan; however, the matrix effect could not be excluded for S,S-EBDM. Busulfan and treosulfan were stable in plasma for 2 h at room temperature. When the samples were stored at 10 °C, busulfan was stable for up to a week and treosulfan was stable for up to 3 days, and both were stable for up to a week when stored frozen at −20 °C. Conclusion We developed the LC-MS/MS method to simultaneously measure treosulfan and busulfan. The method met the validation requirements of the CLSI guidelines and showed good performance. Therefore, this method is expected to be helpful in TDM after treosulfan or busulfan treatment in clinical laboratories.

B-174 Simple and Accurate Quantification of Four Direct Oral Anticoagulants in Plasma Using UPLC-MS/MS

Abstract Background Direct oral anticoagulants (DOACs) constitute first-line therapy used for many thromboembolic indications, such as prevention and treatment of venous thromboembolism and stroke prevention in atrial fibrillation. This class of medication consists of the direct thrombin inhibitor (dabigatran) or direct factor Xa inhibitors (apixaban, rivaroxaban, and edoxaban). DOACs quantification may be useful in the case of critical clinical situations, including drug accumulation in long-term treatment, overdosage, thrombotic or bleeding events. This study aimed to develop and validate an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the quantification of four DOACs: dabigatran, apixaban, rivaroxaban, and edoxaban in plasma. Methods The calibration curve samples were prepared by spiking drug free plasma with DOACs. The stable isotope labeled DOACs was used as an internal standard. After addition of the internal standard and protein precipitation, the supernatant was 10-fold diluted and injected into a chromatography system consisting of a UPLC BEH C18 (2.1 × 50 mm; particle size 1.7 µm) analytical column with gradient made of mobile phase (5 mM ammonium formate pH3.5 in water and methanol). The outlet of the column was connected to a triple quadrupole mass spectrometer with electrospray interface. Ions were detected in the positive multiple reaction monitoring mode. The concentration of analyte was calculated from the calibration curve and ion ratios between the analyte and the internal standard. Results The analytical range was linear with a correlation coefficient of over 0.99 in the range of 0–500 ng/mL for each analyte. Intra- and inter-assay imprecision were less than 3.1% (n = 20) and 6.0% (n = 20), respectively. The accuracy was evaluated by spike recovery and the mean recoveries were between 97.2% and 102.1% for all analytes. This assay showed no ion suppression or enhancement and no carryover. The chromatography run time was 4.5 min. The assay was applied to quantitate DOACs in plasma samples from 93 patients. Our data showed that the DOACs concentrations vary markedly individual patients after equal dose, this therapeutic drug monitoring of DOACs is essential to optimized drug efficacy. Conclusion An accurate, and cost-effective UPLC-MS/MS method for the quantification of DOACs was successfully applied for therapeutic drug monitoring in patients.

B-187 Evaluation of Clinical Utility of LC-MS/MS Method for Thyroglobulin Measurement in Comparison with Immunoradiometric Assay and Chemiluminescence Microparticle Immunoassay

Abstract Background Thyroglobulin (Tg) is the follow-up index of choice for patients with differentiated thyroid cancer (DTC), especially who were undergone total thyroidectomy. However, due to the commonly used immunoassay’s inherent susceptibility to interference combined with the high prevalence of thyroglobulin antibody (TgAb) in DTC patients, the need for more reliable measurement for Tg independent from TgAb has been brought up. In this study, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for Tg measurement and compared its clinical usefulness and analytical performance with immunoradiometric assay (IRMA) and chemiluminescence microparticle immunoassay (CMIA). Methods We developed trypsin digestion-peptide immunoaffinity LC-MS/MS methods for Tg measurement. Analysis was performed using an LC-MS/MS system consisting of Agilent 1290 LC (Agilent Technologies, Santa Clara, CA) and Qtrap 5500+ MS/MS system(ABSciex, Framingham, MA). Tryptic Tg specific peptide (FSPDDSAGASALLR) and cleavable internal standard peptide (LSFYWTTTGDPFFDSHSDSLL*RSGPYMPQ, L*: 13C6, 15N) were quantitated against 5-point linear calibration curve with Access Thyroglobulin Calibrators (Beckman Coulter, Pasadena, CA). Three levels of serum control were prepared (3.95, 12.5, and 39.4 ng/mL). Lower limit of quantitation (LLoQ), linearity, precision, and carryover were validated for the assay. For method comparison, serum from patients with DTC or Hashimoto thyroiditis was collected. Tg was also measured with IRMA using BRAHMS TG Plus (Thermo Fisher Scientific, Waltham, MA), and CMIA using Architect Thyroglobulin (Abbott laboratories, Illinois, CA). TgAb was measured with IRMA using BRAMS Anti-Tg (Thermo Fisher Scientific) and the cut-off for TgAb was 60 IU/mL. Also, interference of TgAb was evaluated by comparing the Tg recovery of five sets of TgAb negative samples spiked with diluents and high TgAb samples. Results Within-run and between-run imprecisions were CV 7.8%–13.0% and CV 8.9%–12.7%, respectively. LLoQ was 0.5 ng/mL. The assay was linear through 0.5–650 ng/mL. No significant carryover was observed within the analytical measurement range. Out of the total 193 samples, the number of samples above LLoQ in both LC-MS/MS, and IRMA or CMIA were 73 samples (45 TgAb-positive and 28 TgAb-negative samples) and 69 samples (42 TgAb-positive and 27 TgAb-negative samples), respectively. The result well correlated between LC-MS/MS and IRMA (R2 = 0.99), and LC-MS/MS and CMIA (R2 = 0.98) in overall samples. When analyzed according to TgAb status, LC-MS/MS and IRMA (R2 = 0.84), and LC-MS/MS and CMIA (R2 = 0.93) correlated rather poorly in TgAb-positive samples, while TgAb-negative samples were consistent (R2 = 0.99 for IRMA, R2 = 0.97 for CMIA). We found one case with systemic metastatic thyroid cancer that showed significantly high Tg levels in IRMA and LC-MS/MS (above the upper limit of quantitation), whereas below LLoQ in CMIA. The interference of TgAb was negligible in LC-MS/MS, while significant interference was observed in CMIA, with a mean recovery rate of 57.6% (4.3%–82.5%). In one sample, when measured with CMIA, thyroglobulin level was 3.29 ng/mL when mixed with diluent, but below LLoQ (0.14 ng/mL) when spiked with TgAb-positive samples (TgAb 1700 IU/mL). Conclusion The LC-MS/MS method for Tg measurement was less prone to antibody interference than immunoassays. Measurement of Tg using LC-MS/MS would be useful for reliable diagnosis and proper management in TgAb-positive patients.

Uncertainty Evaluation for the Quantification of Urinary Amphetamine and 4-Hydroxyamphetamine Using Liquid Chromatography-Tandem Mass Spectrometry: Comparison of the Guide to the Expression of Uncertainty in Measurement Approach and the Monte Carlo Method with R.

Estimating the measurement uncertainty (MU) is becoming increasingly mandatory in analytical toxicology. This study evaluates the uncertainty in the quantitative determination of urinary amphetamine (AP) and 4-hydroxyamphetamine (4HA) using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method based on the dilute-and-shoot approach. Urine sample dilution, preparation of calibrators, calibration curve, and method repeatability were identified as the sources of uncertainty. To evaluate the MU, the Guide to the Expression of Uncertainty in Measurement (GUM) approach and the Monte Carlo method (MCM) were compared using the R programming language. The MCM afforded a smaller coverage interval for both AP (94.83, 104.74) and 4HA (10.52, 12.14) than that produced by the GUM (AP (92.06, 107.41) and 4HA (10.21, 12.45)). The GUM approach offers an underestimated coverage interval for Type A evaluation, whereas the MCM provides an exact coverage interval under an abnormal probability distribution of the measurand. The MCM is useful in complex settings where the measurand is combined with numerous distributions because it is generated from the uncertainties of input quantities based on the propagation of the distribution. Therefore, the MCM is more practical than the GUM for evaluating the MU of urinary AP and 4HA concentrations using LC-MS/MS.

Detection of chemical contaminants in heat processed meat products based on UPLC-MS/MS

An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was established for the simultaneous analysis of acrylamide (AA), 5-hydroxymethylfurfural (HMF), and heterocyclic aromatic amines (HAAs). A good separation of nine analytes was achieved within 15 min. Correlation coefficients (R2 >0.9995) of the nine compounds were obtained within their respective linear ranges. The method also possessed low limits of detection (0.3 ng/mL for IQ, Norharman, 7,8-DiMeIQ, 10.00 ng/mL for MeIQ, 5-HMF, 0.05 ng/mL for PhIP, Phe-P-1, 0.10 ng/mL for Harman, 20.00 ng/mL for AA) and limits of quantification (15.00 mg/kg for IQ, Norharman, 7,8-DiMeIQ, 450.00 mg/kg for MeIQ, 3.00 mg/kg for PhIP, Phe-P-1, 300.00 mg/kg for 5-HMF and 750.00 mg/kg for AA). The method developed for the determination of samples commonly found among Sichuan cuisine, and fast food had good precision (RSD<9%) and recovery (83%-113%). This study provides a basis for the simultaneous rapid determination of nine chemical contaminants commonly found in thermally processed meat products and provides a technical reference for the simultaneous rapid determination of more chemical contaminants in thermally processed meat products.

Novel aspects of taxifolin pharmacokinetics: Dose proportionality, cumulative effect, metabolism, microemulsion dosage forms

Taxifolin (TFL) is a small drug molecule with a broad therapeutic potential limited by its poor aqueous solubility and excessive metabolism. Despite comprehensive research, some aspects of the TFL pharmacokinetics, e.g., dose proportionality and possible cumulative effect, remain unexplored. In the current study, we have tried to fill this gap. Our results revealed that the TFL pharmacokinetics in rats had nonlinear character in the dose range of 10–50 mg/kg after its single oral administration (AUC). For Cmax, the data are ambiguous: linearity was confirmed via the equivalence criterion and was disproved using the power model approach. Also, the cumulative drug effect was observed on the 4th day after its multiple-dose oral administration (25 mg/kg; compared to the 1st day). Interestingly, biologically active TFL metabolites such as aromadendrin and luteolin were putatively found in plasma samples, although they were previously detected only in feces. In addition, oil-in-water and water-in-oil microemulsions were fabricated to design novel drug delivery systems. These carrier dosage forms did not improve the TFL bioavailability but significantly affected its metabolism. To support pharmacokinetic studies, the bioanalytical liquid chromatography–tandem mass spectrometry method was developed and validated in the concentration range of 1–1000 ng/mL using candesartan as an internal standard. Liquid-liquid extraction with methyl tert-butyl ether was used to isolate the analytes from plasma followed by evaporation and reconstitution of the residues in acetonitrile. Thus, the present findings broaden our understanding of the TFL behavior in vivo and provide novel ideas and reference directions for its continued use in medical practice.