Isotope Internal Standards Research Articles

Rapid Liquid Chromatography-Tandem Mass Spectrometry Method for Determination of Total and Free Testosterone in Human Serum and Its Application to Monitoring Biomarker Response of Elite Athletes.

Total testosterone (TT) and free testosterone (FT) are important biochemical markers for anabolism of the human body, and can also serve as early screening indicators for overtraining syndrome (OTS). Presently, there is no fast and reliable serum TT and FT determination method in the field of sport science that can meet the requirements of sports research. Thus, a rapid and accurate determination method for serum TT and FT to fill the gap is needed urgently in sports training. Herein, a simple and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of TT and FT in serum was developed and fully validated, followed by the application of professional athletes in training monitoring. Efficient pretreatments based on only one-step liquid-liquid extraction (LLE) for TT and one-step LLE after a 20 min ultrafiltration for FT were adopted in this study, and the isotope internal standard of testosterone-13C3 was used to ensure the reliability of the whole procedure. A linear range of four orders of magnitude with 0.02-100 ng/mL can meet the concentration range requirement between a higher limit for male TT and a lower limit for female FT. The accuracy, precision, stability, and matrix effect were all within the limits of the guidelines. The serum TT and FT levels of 200 professional athletes (98 male athletes and 102 female athletes) were investigated by this method. Serum TT, FT, and FT/TT levels of professional athletes were significantly higher than the general population, and serum TT levels were significantly higher by LC-MS/MS than by a chemiluminescence immunoassay. In conclusion, the LC-MS/MS method for TT and FT measurement developed in this study is time-saving and easy to operate, which can be used as a reliable method for the determination of serum TT and FT in sports training, offering valuable information for monitoring anabolism of athletes and screening OTS in the early stage.

A novel high-throughput quantitative method for the determination of per- and poly-fluoroalkyl substances in human plasma based on UHPLC-Q/Orbitrap HRMS coupled with isotope internal standard

A novel high-throughput quantitative method for the determination of per- and poly-fluoroalkyl substances in human plasma based on UHPLC-Q/Orbitrap HRMS coupled with isotope internal standard

Label-Free Multiple Reaction Monitoring-Mass Spectrometry for Quantifying Phosphopeptides from Extracellular Vesicles.

Increasing efforts have been made to develop proteins in circulating extracellular vesicles (EVs) as potential disease markers. It is in particular intriguing to measure post-translational modifications (PTMs) such as phosphorylation, preserved and stable in EVs. To facilitate the quantitative measurement of EV protein phosphorylation for potential clinical use, a label-free (LF) multiple reaction monitoring (MRM) strategy is introduced by utilizing a synthetic phosphopeptide set (phos-iRT) as the internal standards and a local normalization method. The quantitation method was investigated in terms of its linear dynamic range, sensitivity, accuracy, precision, and matrix effect, with a dynamic range spanning from 10 to 1000 ng/mL and an accuracy ranging from 82.4 to 116.8% for EV samples. Then, the LF-MRM-based local normalization method was utilized to evaluate and optimize our recently developed EVTOP method for the enrichment of phosphopeptides from EVs. Finally, we applied the optimized EV enrichment approach and the LF-MRM-based local normalization method to quantify phosphopeptides in urine EVs from patients with prostate cancer (PCa) and healthy individuals, showcasing the strategy's superiority in quantifying phosphopeptides without isotopic internal standards and validating that the method is generally applicable in MRM-based EV phosphopeptide quantification.

Determination of eight neonicotinoid pesticides in wastewater by solid phase extraction combined with liquid chromatography-tandem mass spectrometry

Neonicotinoid pesticides are a relatively new class of pesticides that have garnered significant attention owing to their potential ecological risks to nontarget organisms. A method combining solid phase extraction with liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) was developed for the rapid and accurate detection of eight neonicotinoid pesticides (dinotefuran, E-nitenpyram, thiamethoxam, clothianidin, imidacloprid, imidaclothiz, acetamiprid, and thiacloprid) in wastewater. The chromatographic mobile phase and MS parameters were selected, and a single-factor method was used to determine the optimal column type, extraction volume, sample loading speed, and pH for SPE. The optimal parameters were as follows: column type, HLB column (500 mg/6 mL); sample extraction volume, 500 mL; sample loading speed, 10 mL/min; and sample pH, 6-8. The matrix effects of the wastewater samples were reduced by optimizing the chromatographic gradient-elution program, examining the dilution factor of the samples, and using the isotope internal standard calibration method. Prior to analysis, the wastewater samples were diluted 5-fold with ultrapure water for pretreatment. Subsequently, 2 mmol/L ammonium acetate aqueous solution containing 0.1% (v/v) formic acid and methanol was used as mobile phases for gradient elution on a ZORBAX Eclipse Plus C18 column (100 mm×2.1 mm, 1.8 μm). The samples were quantified using positive-ion multiple reaction monitoring (MRM) mode for 10 min. Imidacloprid-d4 was used as the isotope internal standard. The SPE process was further optimized by applying response surface methodology to select the type and mass of rinsing and elution solvents. The optimal pretreatment of the SPE column included rinsing with 10% methanol aqueous solution and elution with methanol-acetonitrile (1∶1, v/v) mixture (7 mL). The eight neonicotinoid pesticides showed satisfactory linearity within the relevant range, with linear correlation coefficients (r) all greater than 0.9990. The method detection limits (MDLs) ranged from 0.2 to 1.2 ng/L, and the method quantification limits (MQLs) ranged from 0.8 to 4.8 ng/L. The average recoveries of the eight neonicotinoid pesticides were in the range of 82.6%-94.2% at three spiked levels, with relative standard deviations (RSDs) ranging from 3.9% to 9.4%. Finally, the optimized method was successfully applied to analyze wastewater samples collected from four sewage treatment plants. The results indicated that the eight neonicotinoid pesticides could be generally detected at concentrations ranging from not detected (ND) to 256 ng/L. The developed method has a low MDL and high accuracy, rendering it a suitable choice for the trace detection of the eight neonicotinoid pesticides in wastewater when compared with other similar methods. The proposed method can be utilized to monitor the environmental impact and assess the potential risks of neonicotinoid pesticides in wastewater, thus promoting the protection of nontarget organisms and the sustainable use of these pesticides in agriculture.

A Comprehensive Report on the HPLC-ESI-MS/MS Method Development and Validation for Quantifying Cabotegravir in K3EDTA-Human Plasma, Employing Cabotegravir-D5 as an Internal Standard

Aiming to comprehend the plasma concentration of cabotegravir, a meticulously crafted bioanalytical method, aligned with the guidelines set by the US Food and Drug Administration (FDA), was devised and validated. This involved the utilization of cabotegravir D5 as an isotopic internal standard. The mobile phase consisted of methanol and 5 mM ammonium acetate in water, in an 80:20 ratio, v/v—Zorbax SB-C18 (50 × 2.1 mm, 5 μm) served as the chromatographic column. Cabotegravir and cabotegravir D5 were identified using proton adducts at m/z 409.20/370.20 and 409.20/144.8, respectively, employing the positive mode multiple reaction monitoring. Cabotegravir exhibited linearity within concentration ranges of 2 to 1000 ng/mL (r2 = 0.999). The recovery of cabotegravir from plasma by Liquid-liquid extraction yielded an average %CV of 7.71 at four quality control levels. Intra-day precision displayed accuracy of 100, 106, 97, 97, and 103%, respectively, whereas accuracy for inter-day precision was 100, 103, 98, 100, and 100% across LLQC, LQC, MQC-1, MQC-2, and HQC levels. The stability of cabotegravir remained consistent under diverse conditions, including five freeze-thaw cycles, benchtop, autosampler, and short-term, and long-term storage. This evaluation verifies that the method aligns with predefined acceptance limits and positions it as an indispensable tool in bioanalysis, significantly expanding its clinical utility.

Quantitation of tazemetostat in human plasma using liquid chromatography-tandem mass spectrometry.

To support a phase 1 trial in patients with lymphomas, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for tazemetostat quantitation in 20 μL of human plasma. After protein precipitation, chromatographic separation employed a Kinetex C18 column and a gradient of 0.1% formic acid in both water and acetonitrile, during a 3-min run time. Detection was achieved using a SCIEX 6500+ tandem mass spectrometer with electrospray positive-mode ionization. Validation was based on the latest Food and Drug Administration guidance. With a stable isotopic internal standard, the assay was linear within the range of 10-5000 ng/mL and proved to be accurate (91.9%-103.7%) and precise (<4.4% imprecision). Recovery varied between 93.3% and 121.1%, and matrix effect ranged from -25.5% to -4.9%. Hemolysis, lipemia, and dilution did not impact quantitation. Plasma stability was confirmed after three freeze-thaw cycles, 24 h at room temperature, and 4months at -80°C. Incurred sample reanalysis yielded 94.4% samples within 20% difference (n = 36). External validation showed a mean bias of -11.1%. Pharmacokinetic (PK) data obtained from three patients suggested variable concentration time profiles, warranting collection of further data. The assay proved to be suitable for tazemetostat quantitation in human plasma and will support clinical studies by defining tazemetostat PKs.

A Reliable Quantification of Cholesterol and 25-Hydroxycholesterol in Liposomal Adjuvant Formulation by Liquid Chromatography High-Resolution Tandem Mass Spectrometry.

Cholesterol, as one of the major components of liposomes, plays a critical role in modulating membrane bilayer permeability, fluidity, and structural stability. Controlling these quality attributes is essential to maintaining the efficacy and fitness of the liposomes in various applications. However, during the manufacture and storage of liposomes, cholesterol has a propensity to undergo oxidative degradation. Hence, an analytical tool that is capable of determining not only the identity and quantity of cholesterol but also its associated degradants is a prerequisite to effective process control and product quality and safety assessments. In this view, a new liquid chromatography electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method with parallel reaction monitoring (PRM) was developed and qualified to accurately quantify cholesterol and monitor the formation of 25-hydroxycholesterol degradant in liposomal drug formulations without the use of an isotopic internal standard (IS). The method was qualified according to the FDA Quality Guidance for Industry: Q2(R1). Study results showed that the method presents good specificity for cholesterol and 25-hydroxycholesterol detection in the liposomal matrix, good sensitivity characterized by LOD/LOQ in the nanomolar range, and accuracy within the range of 80 to 120%. The described method enables accurate evaluation of in-process and product release samples of Army Liposome Formulation with QS21 (ALFQ).

Quantitation of propofol metabolites by LC–MS/MS demonstrating long detection window for urine drug monitoring

IntroductionChromatographic methods for analysis of propofol and its metabolites have been widely used in pharmacokinetic studies of propofol distribution, metabolism, and clearance. Application of chromatographic methods is also needed in clinical and forensic laboratories for detecting and monitoring propofol misuse. ObjectiveWe report a method for sensitive analysis of propofol, propofol 1-glucuronide (PG), 4-hydroxypropofol 1-glucuronide (1-QG), 4-hydroxypropofol 4-glucuronide (4-QG) and 4-hydroxypropofol 4-sulfate (4-QS) in urine by LC–MS/MS analysis. The method employs a simple dilute-and-analyze sample preparation with stable isotope internal standardization. ResultsValidation studies demonstrate a linear calibration model (100–10,000 ng/mL), with dilution integrity verified for the extended range of concentrations experienced in propofol use. Criteria-based validation was achieved, including an average coefficient of variation of 6.5 % and a percent bias of −4.2 ng/mL. The method was evaluated in 12 surgical patients, with monitoring periods lasting up to 30 days following intravenous propofol administrations of 100–3000 mg on the day of surgery. While the concentration ratio of PG to 4-hydroxy propofol metabolite decreased significantly in the days following surgery, PG maintained the highest concentration in all specimens. Both PG and 1-QG were detectable throughout the monitoring periods, including in a patient monitored for 30 days. Lower concentrations were determined for 4-QG and 4-QS, with evidence of detection up to 20 days. Propofol was not detectable in any urine specimens, thereby proving ineffective for identifying drug use. ConclusionThe validated method for quantifying propofol metabolites demonstrates its applicability for the sensitive detection of propofol misuse over a long window of drug-use detection.

Isomers-oriented separation of forty-five plasma bile acids with liquid chromatography-tandem mass spectrometry

Some bile acids (BAs) were considered as biomarkers or have therapeutical effect on metabolic diseases. However, due to the existence of isomers and limitations in sensitivity, simultaneous quantification of multiple BAs remains a challenge. The aim of this study is to establish an accurate and sensitive method for the determination of multiple BAs with similar polarity. A LC-MS/MS analytical method capable of quantifying forty-five BAs simultaneously using nine stable isotope internal standards was developed and fully validated based on key isomers-oriented separation strategy. The method was further applied to analyze plasma samples to describe the dynamic profile of BAs after high glucose intake. The chromatography and mass spectrum conditions were optimized to enable the accurate quantification of forty-five BAs, while ensuring the lower limit of quantification between 0.05–10 ng/mL. The results of system suitability, linearity, dilution integrity, accuracy and precision demonstrated the good quantitative capacity and robustness of the method. A total of thirty-five BAs were quantified in plasma samples from twelve healthy Chinese individuals. The established method featured superior sensitivity and better separation efficiency compared to previous studies. Meanwhile, BAs exhibited correlations with glucose and insulin, suggesting their potential as biomarkers for metabolic disorders.

Simultaneous determination of 28 illegal drugs in sewage by high throughput online SPE-ISTD-UHPLC-MS/MS

This study developed an online solid-phase extraction ultra-high performance liquid chromatography-tandem mass spectrometry (Online-SPE-UHPLC-MS/MS) method for the analysis of 28 illegal drugs in sewage. To achieve this, 28 isotope internal standards (ISTDs) were added to 3 mL sewage samples, the pH was adjusted to 7–8 using hydrochloric acid or 20% ammonia water, followed by centrifugation, filtration, and analysis using UHPLC-MS/MS. The results indicated an excellent linearity of 1–300 ng L−1, and cotinine in the concentration range of 20–6000 ng L−1, linear correlation coefficient R2 > 0.995, with the limit of detection (LOD) of 0.01–6 ng L−1, and a limit of quantification (LOQ) of 0.1–20 ng L−1. The addition of three concentrates of low (2 ng L−1/40 ng L−1), medium (20 ng L−1/400 ng L−1), and high concentration (200 ng L−1/4000 ng L−1) demonstrated the matrix effect of the target compound between ± 22.0%. The extraction recovery was 70.0–119.4%, and a percent accuracy of 75.7–118.1%. Similarly, the intra- and inter-day precisions were 1.8–20.0% and 1.5–18.9%, respectively. The results cemented the sensitivity, accuracy, reliability, strong specificity, and reproducibility, which can be used to screen 28 illegal drugs in sewage for trace analysis.

Signal interference between drugs and metabolites in LC-ESI-MS quantitative analysis and its evaluation strategy

Liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS) is a widely utilized technique for in vivo pharmaceutical analysis. Ionization interference within electrospray ion source, occurring between drugs and metabolites, can lead to signal variations, potentially compromising quantitative accuracy. Currently, method validation often overlooks this type of signal interference, which may result in systematic errors in quantitative results without matrix-matched calibration. In this study, we conducted an investigation using ten different groups of drugs and their corresponding metabolites across three LC-ESI-MS systems to assess the prevalence of signal interference. Such interferences can potentially cause or enhance nonlinearity in the calibration curves of drugs and metabolites, thereby altering the relationship between analyte response and concentration for quantification. Finally, we established an evaluation scheme through a step-by-step dilution assay and employed three resolution methods: chromatographic separation, dilution, and stable labeled isotope internal standards correction. The above strategies were integrated into the method establishment process to improve quantitative accuracy.

Concentrations, Sources and Health Risk Assessment of Polycyclic Aromatic Hydrocarbons in Chinese Herbal Medicines.

The determination and evaluation of 16 polycyclic aromatic hydrocarbons (PAHs) in seven Chinese herbal medicines (CHMs) were conducted through a rapid and straightforward extraction and purification method, coupled with GC-MS. A sample-based solid-phase extraction (SPE) pretreatment technique, incorporating isotopic internal standards, was employed for detecting various medicinal parts of CHMs. The assay exhibited linearity within the range of 5 to 500 ng/mL, with linear coefficients (R2) for PAHs exceeding 0.999. The recoveries of spiked standards ranged from 63.37% to 133.12%, with relative standard deviations (RSDs) ranging from 0.75% to 14.54%. The total PAH content varied from 176.906 to 1414.087 μg/kg. Among the 16 PAHs, phenanthrene (Phe) was consistently detected at the highest levels (47.045-168.640 μg/kg). Characteristic ratio analysis indicated that oil, coal, and biomass combustion were the primary sources of PAHs in CHMs. The health risk associated with CHMs was assessed using the lifetime carcinogenic risk approach, revealing potential health risks from the consumption of honeysuckle, while the health risks of consuming Lycium chinense berries were deemed negligible. For the other five CHMs (glycyrrhizae, Coix lacryma, ginseng, lotus seed, seed of Sterculia lychnophora), the health risk from consumption fell within acceptable ranges. Furthermore, sensitivity analyses utilizing Monte Carlo exposure assessment methods identified PAH levels in CHMs as health risk sensitizers. It is crucial to recognize that the consumption of herbal medicines is not a continuous process but entails potential health risks. Hence, the monitoring and risk assessment of PAH residues in CHMs demand careful attention.

Development and validation of a simple, fast and sensitive liquid chromatography-tandem mass spectrometry method to establish reference intervals for 24-h urinary free normetanephrine, metanephrine and methoxytyramine

ObjectiveTo develop and validate a rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to detect urinary free metanephrines and methoxytyramine, establishing reference intervals. MethodsUrine samples were diluted with isotope internal standard solution, then analyzed directly using tandem mass spectrometry with multiple reaction monitoring measurement and electrospray ionization source in positive ion mode. Analytical parameters including linearity, lower limit of quantitation, imprecision and accuracy of the method were evaluated. The reference intervals for urinary catecholamine metabolites were established by analyzing 24-h urine samples collected from 81 apparently healthy volunteers. ResultsThe analytical times for MN, NMN, and 3-MT were at 2.79, 2.80, and 2.74 min, respectively. The method displayed excellent linearity (r > 0.99) in the range of 1-1000 ng/mL, with lower limits of quantification (LLOQ) at 0.50 ng/mL for MN and NMN, and 0.25 ng/mL for 3-MT. The method's intra-day and inter-day imprecisions were less than 8 %. The method recovery ranged from 96.8% to 105.8 % for MN, 89.7%–106.4 % for NMN, and 93.5%–106.2 % for 3-MT. No carry-over was observed during the analysis of all analytes. The LC-MS/MS method was used to establish reference intervals in 24-h urine samples from 81 apparently healthy volunteers. There was no association of sex with urinary free metabolites. ConclusionThis study established a novel, fast and sensitive LC-MS/MS method for determining urinary free catecholamine metabolites, which could facilitate screening and diagnosis for catecholamine-related tumors more conveniently and quickly.

Determination of N-nitroso folic acid in folic acid and multivitamin supplements by LC-MS/MS

Analysis of N-nitroso folic acid, a nitrosamine impurity found in folic acid, is challenging due to the complex sample matrices. Many of such supplements contain not only a variety of vitamins, including vitamin A, Bs, C, D and E, but also other ingredients such as minerals, docosahexaenoic acid (DHA), glucose syrup, sugar, and herbs. On the other hand, the strength of folic acid is typically low, ranging from 50 µg to 5 mg per unit. In this study, a highly selective and sensitive LC-MS/MS method was developed to accurately quantify N-nitroso folic acid in supplements containing folic acid. The sample was extracted by 0.1% ammonia solution: MeOH (9:1, v/v) containing 5 ng/mL of N-nitroso folic acid-d4 (Isotope internal standard). The quantification was performed by MRM in negative ionization mode. Mobile phases A and B were 0.1% formic acid in deionized water and methanol, respectively. The method was validated and found to have sufficient linearity (R2 > 0.995), accuracy (recovery 83–110%), precision (RSD 3%) and low LOD, LOQ (4 and 10 µg/g respectively, with respect to folic acid). The method was applied to the determination of N-nitroso folic acid in 40 supplements containing folic acid with different strengths and formulation. The content of N-nitroso folic acid was found to be up to 898 ng/unit (1794 µg/g with respect to folic acid). It enabled regulatory actions, such as product recall, to safeguard public health from unsafe products.

Method Validation for Quantification of PFOS and PFOA in Human Plasma and a Pilot Study in Blood Donors from Thai Red Cross Society.

Information regarding per- and polyfluorinated substances concentrations in biological samples from the Thai population was still lacking. A sensitive bioanalytical method was developed and validated for the quantification of perfluorooctane sulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) levels in human plasma. Simple protein precipitation and LC-MS/MS techniques were used with stable isotope internal standards of 13C8-PFOS and 13C8-PFOA. The validated method followed the ICH bioanalytical validation guideline, and the results showed good accuracy, precision, and reproducibility. The validated analytical method was then applied to determine PFOS and PFOA concentrations in 50 human plasma samples from the National Blood Center, Thai Red Cross Society. The concentrations were found to be in ranges of <0.91-6.27 ng/mL for PFOS and <0.49-2.72 ng/mL for PFOA. PFOS was also measured separately for its isomers, and the geometric means of the linear isomer (L-PFOS) and branched isomer (br-PFOS) in plasma samples were at 1.85 and 0.41 ng/mL, respectively. Both PFOS and PFOA concentrations were lower in comparison to previous reports from other countries. The present study showed the application of our reliable method to determine PFOS and PFOA in biological samples in order to monitor the human exposure of both chemicals in Thailand.

Quantification of Thiopurine Metabolites in Human Erythrocytes by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

The thiopurine drugs, azathioprine, mercaptopurine, and thioguanine, are widely used in the treatment of several malignant and nonmalignant diseases. These inactive prodrugs undergo extensive metabolism to form active cytotoxic metabolites, which act mainly by incorporating into DNA and affecting cell replication. Thiopurine methyltransferase is a highly variable cytosolic enzyme that catalyzes the S-methylation of the thiopurine bases-an inactivating pathway. Patients with low-activity variants of TPMT can be affected by pronounced pharmacologic effects when receiving thiopurine medications. Clinical studies have reported significant interpatient variability in intracellular thiopurine metabolite concentrations in patients receiving thiopurine therapy. In this chapter, we present an LC-MS/MS method to monitor the thiopurine metabolites: 6-thioguanine nucleotides and 6-methylmercaptopurine derivatives in human erythrocytes. This method utilizes acid hydrolysis to release the bases and improves upon previously published procedures by utilizing stable isotope internal standards and a more efficient chromatographic separation.

Deep Eutectic Solvent-Based Dispersive Liquid-Liquid Microextraction Coupled with LC-MS/MS for the Analysis of Two Ochratoxins in Capsicum.

Ochratoxins, a common class of mycotoxin in capsicum, and techniques and methods for the determination of mycotoxins in spices have been increasingly developed in recent years. An innovative and eco-friendly method of dispersive liquid-liquid microextraction (DLLME) was demonstrated in this study, based on a synthesized deep eutectic solvent (DES) combined with LC-MS/MS, for the quantification and analysis of two ochratoxins in capsicum. The DES-DLLME method parameters entail selecting the DES type (thymol:decanoic acid, molar ratio 1:1) and DES volume (100 μL). The volume of water (3 mL) and salt concentration (0 g) undergo optimization following a step-by-step approach to achieve optimal target substance extraction efficiency. The matrix effect associated with the direct detection of the target substance in capsicum was significantly reduced in this study by the addition of isotopic internal standards corresponding to the target substance. This facilitated optimal conditions wherein quantitative analysis using LC-MS/MS revealed a linear range of 0.50-250.00 µg/mL, with all two curves calibrated with internal standards showing correlation coefficients (r2) greater than 0.9995. The method's limits of detection (LODs) and limits of quantification (LOQs) fell in the ranges of 0.14-0.45 μg/kg and 0.45-1.45 μg/kg, respectively. The method's spiked recoveries ranged from 81.97 to 105.17%, indicating its sensitivity and accuracy. The environmental friendliness of the technique was assessed using two green assessment tools, AGREE and complexGAPI, and the results showed that the technique was more in line with the concept of sustainable development compared to other techniques for detecting ochratoxins in capsicum. Overall, this study provides a new approach for the determination of mycotoxins in a complex food matrix such as capsicum and other spices using DES and also contributes to the application of green analytical chemistry methods in the food industry.

Metabolomics-Driven Systematic Research Reveals Different Metabolomic and Lipidomic Profiling of Bone Marrow Plasma in Patients with Multiple Myeloma

Myeloma cells resident in bone marrow (BM), therefore the microenvironment plays critical roles in the invasion, proliferation and migration. Obesity increases the morbidity and drug resistance in patient of multiple myeloma (MM) through changing the bone marrow environment. Extracellular environments such as the BM plasma likely have unique lipidomic metabolite profiles that predicts the prognosis in patients with multiple myeloma. However, the study of multiple omics on metabolic changes in BM of MM patients are still very limited. The present study utilized an untargeted metabolite and targeted complex lipid profiling (Liquid chromatography-mass spectrometry, LC-MS) analysis to identify significant differences in the metabolite levels of BM plasma between newly diagnosed MM patients with different BMI (≤24 kg/m 2, n = 10; ≥25 kg/m 2, n = 10). This was followed by verification of some of the metabolite differences of interest by targeted quantification of the metabolites using isotopic internal standards in the exploratory cohort as well as an independent validation cohort. Significant differences were noted in the amino acid profiles, lipid metabolism and amino sugar metabolism, such as increased L-Glutamate, L-Proline, D-Proline, palmitoleic acid and D-Fructose, etc between MM patients with different BMI ( Figure A-C). Glycerol phosphate shuttle, glycerolipid metabolism and de novo triacylglycerol biosynthesis were the top 3 enriched signaling pathways in BM of obese MM patients ( Figure D). Targeted complex lipid profiling analysis further showed that triglyceride (TG)，diglyceride (DG) were increased notably in obese BM plasma of MM patients and correlated significantly with BMI ( Figure E-G). Glycerolipids, triradylglycerols, diradylglycerols and fatty acyls metabolism were enriched in obese group by GSEA analysis ( Figure H). Furthermore, KEGG enrichment analysis showed differentiate expressed lipid metabolites in BM plasma of obese MM patients comparing to that of lean group, including regulation of lipolysis in adipocytes, glycerolipid metabolism, fat digestion and absorption, cholesterol metabolism and metabolic pathways, etc ( Figure I ). In summary, metabolite and complex lipid profiling of the BM plasma identifies differences in levels of metabolites and lipids between MM patients with different BMI. This may provide insight into the explanation that obesity increases the susceptibility to MM and drug resistance. Acknowledgement This project is supported by the Sun Yat-sen University Hundred Talents Program (PT19200101), Natural Science Foundation of Guangdong Province (2022A1515010290).

B-163 Development and Validation of a New Quantitative LC-MS/MS Method for Vitamin K1 in Plasma and Serum

Abstract Background Vitamin K1 (phylloquinone) is a fat-soluble cofactor; its deficiency depresses synthesis of plasma prothrombin (factor II) and other factors, causes excessive bleeding, and increases the risk of bone fractures and osteoporosis. Biochemical and metabolic studies on vitamin K1 require both highly sensitive and specific techniques. Analytical challenges to quantitation include the presence of interfering lipids, molecular instability, and extremely low physiological levels. High performance liquid chromatography (HPLC) with fluorescence and electrochemical detection (FLD-ECD) methods used for vitamin K1 involve labor-intensive, liquid-liquid extraction procedures and extremely long run times. Our goal was to develop and validate a high throughput and accurate method for measuring vitamin K1 in plasma and serum using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Methods Plasma or serum specimens were mixed with stable isotope internal standard in an extraction solution containing acetonitrile to release vitamin K1 and precipitate proteins. After centrifugation, the supernatant was then extracted using a HybridSPE® (Supelco) plate to remove the remaining precipitated proteins and phospholipids that may contribute to ion-suppression. Certified standards were used to derive a 6-point calibration curve. Extracts (plasma or serum) and calibrators were analyzed on a SCIEX 6500 + LC-MS/MS with a Thermo TX-4 HTLC multiplexed 4-channel system for increased throughput. Chromatographic separation was achieved on a Biphenyl analytical column. Precision, accuracy, linearity, recovery, and stability were evaluated. Random specimen discards, previously submitted for vitamin K1 testing, were used for a split-sample comparison study vs the current HPLC-FLD-ECD method. Results Compared with the HPLC-FLD-ECD method, the LC-MS/MS method employed a simplified automated hybrid solid phase extraction procedure, used less specimen volume (100 µL vs 700 µL), increased total throughput (4 h vs 32 h per 96-well plate), was more sensitive (limit of quantitation [LOQ] 6.1 pg/mL vs 25.0 pg/mL), and provided greater molecular specificity for vitamin K1 through multiple reaction monitoring (MRM). The intra-assay and total inter-assay precision coefficients of variation (CVW, CVT) in plasma samples (n = 100) were: (6.8%, 5.8%) at 100 pg/mL, (3.5%, 3.0%) at 800 pg/mL, and (3.0%, 3.9%) at 2000 pg/mL. In spiked plasma and serum samples (n = 10), the linearity range was 40–20 000 pg/mL and percent recovery were 98%–107%. Least-squares regression analysis comparing the results for split plasma extracts by LC-MS/MS vs HPLC-FLD-ECD (n = 100) yielded correlation coefficients for vitamin K1: 0.9917, y = 0.927x + 0.827, range 40–7000 pg/mL. Vitamin K1 was stable in plasma and in serum stored in amber plastic screw-capped vials for 8 days at 2–8 °C and 30 days at −20 °C. Conclusion We have developed and validated an LC-MS/MS method that provides rapid, sensitive, precise, and accurate evaluation of vitamin K1 in plasma and serum. Advantages over the existing HPLC-FLD-ECD method include increased throughput and improved sensitivity and specificity. Vitamin K1 results obtained using this method can be used by clinicians to guide clinical intervention to improve or normalize metabolism or prevent the potential adverse consequences associated with inadequate dietary intake.

A-033 Development and Validation of a Liquid Chromatography Tandem Mass Spectrometry Bioanalytical Method for Prostaglandin D2 in Serum or Urine

Abstract Background Prostaglandin D2 (PGD2) is a major prostaglandin produced by the brain and by mast cells, and plays a role in vasodilation (“niacin flush”) and in the development of allergic diseases such as asthma and alopecia. We sought to develop a bioanalytical method for multiple specimen types for PGD2 for clinical use. Methods An analytical method was developed using a TX-4 HPLC system (Thermo-Fisher) with Agilent® 1200 pumps (Agilent Technologies, Inc.) and a Sciex® 5500 (Danaher) triple quadrupole mass spectrometer. Calibration curve based on purified PGD2 (Cayman) was prepared in depleted serum (Golden West Biologicals). Sample preparation consisted of an initial isotope dilution using a deuterated heavy isotope internal standard (PGD2-2H4) followed by solid phase extraction, evaporation and reconstitution using a second heavy isotope (PGD2-2H9). Double-isotope dilution allows discrimination between extraction loss and ion suppression as causes of decreased ion current. A reversed phase C18 analytical column was used with an acidified water/acetonitrile/methanol solvent gradient to achieve chromatographic separation of physiological PGD2 from related isobaric metabolites include Prostaglandin E2. Negative mode electrospray ionization (ESI) was used for detection in Multiple Reaction Monitoring (MRM) mode. Results Analytical range was 1–1000 pg/mL (up to 5000 pg/mL with dilution). Inter-assay precision ranged from 8.8% to 10.2% and inter-assay accuracy ranged from 95.7% to 103.3%. Reference intervals for adults was developed using residual serum and urine specimens. No interference was observed in the presence of hemolysis, icteric or lipemia. No difference in result was observed when using collection tubes containing EDTA, Heparin or gel barriers. Conclusion A bioanalytical method for PGD2 has been fully validated for clinical use.