Triple Quadrupole Research Articles

B-173 Cystine Analysis in Urine Sample Using the Laser Diode Thermal Desorption technique “LDTD”/Mass Spectrometry (Luxon)

Abstract Background Cystine is a polar amino acid found in the urine of healthy individuals; however, a genetic defect in renal transport can lead to cystine crystals and stones. A usual laboratory approach for diagnosing and monitoring cystinuria is to measure cystine concentration in 24-hour urine. The disease is rare, affecting around 1 in every 10,000 live births. The objective of this study was to develop a rapid assay using Tandem Mass Spectrometry methodology with an internal standard of Cystine derivatized in a urine sample, Luxon Ion Source®, Laser Diode Thermal Desorption (LDTD) technology. Methods The Luxon is a sample introduction and ionization source with a coupled laser diode, providing thermal uniformity, precision, accuracy and speed. Thermally desorbed neutral molecules are analyzed by mass spectrometry (MS). Initially, the sample was derivatized to make the cystine thermally vaporize, followed by liquid-liquid extraction assisted by salting out (SALLE). The isotope-labeled internal standard (Cystine-d4), homemade calibration curve and water quality controls were used for quantification. The analysis was performed using a Sciex 6500+ Triple Quad in APCI (Atmospheric Pressure Chemical Ionization) positive mode. The ratio of peak area to internal standard (ISTD) was used to normalize the signal. A linear regression model with 1/X2 weighting was used to obtain the calibration curve equation and determine the correlation coefficient (r2). Parallelism was assessed by comparing the (r2) of calibration curves prepared in water with six curves prepared with biological matrices. Intra- and interassay precision and accuracy were determined by measuring control samples ranging from low to high concentration in five replicates over 3 consecutive days. For urine analysis, aqueous calibration curves in the concentration range of 1 to 100 mg/L (n=3) showed excellent linearity. The following parameters were evaluated for quantitative analysis: linearity, precision, accuracy, carryover and parallelism.​ Results The calibration curve resulted in a linear response across the entire concentration range, the correlation coefficient (r) was 0.99. For each quality control concentration, CVs were <10%, meeting desirable imprecision and precision specifications. The intraassay CVs were 1.0-2.56% and the interassay CVs were 2.15-4.58% for precision, which reflects the agreement between repeated measurements. Precision results showed similar results, intra-assay CVs were 2.23-3.66% and inter-assay CVs were 1.0-2.77% Conclusions The new LDTD-MS/MS method for the determination of urinary Cystine was accepted after verification and validation of quality control parameters. The technique proved to be effective for reliably and quickly quantifying cystine in urinary samples from human patients, being a valuable tool in the diagnosis and prevention of cystine kidney stones. The assay developed, using the Luxon Ion Source®, based on LDTD (Laser Diode Thermal Desorption) technology, proved to be ultrafast for measuring derivatized cystine in urine

A-144 Liquid chromatography multiple reaction monitoring-based assay for quantitative assessment of fibrinogen levels in clinical samples

Abstract Background Fibrinogen plays a critical role in hemostasis and is essential to clot formation. Changes in the quantity or activity of fibrinogen can lead to disorders affecting coagulation. Thus, it is essential to precisely quantify fibrinogen levels to differentiate between hyperfibrinogenemia in hypercoagulable disorders and dysfibrinogenemia, hypo/afibrinogenemia, and irregular bleeding. Current diagnostic tests that use antibodies to detect fibrinogen or activity-based measures are susceptible to interference from heparin contamination, hemolysis, and hyperbilirubinemia. Here, we evaluated the use of targeted proteomics for developing a quantitative assay for fibrinogen in plasma samples. Methods Four peptides each for alpha and gamma chains of fibrinogen were synthesized using FMOC chemistry on a Liberty Blue (CEM Corp) peptide synthesizer. A targeted multiple reaction monitoring (MRM) assay was developed and optimized using Dionex Ultimate 3000 HPLC connected to a TSQ Altis triple quadrupole mass spectrometer (Thermo Scientific). Evaluations were conducted using a range of standards, such as pure protein, WHO plasma and commercially available plasma with known fibrinogen amounts for the accurate quantitation of fibrinogen. Calibration curves were generated using different dilutions in various matrices. The repeatability and precision of the assay were evaluated across five different days. The plasma samples were processed and digested in a 96-well plate and subjected to C18 clean-up using a Bravo automated liquid handler (Agilent). All samples were spiked-in with corresponding heavy-labeled peptides prior to digestion with trypsin. Multiple reaction monitoring assays were performed on tryptic digests of plasma samples. Results Calibration curves using commercial plasma for all peptides showed linearity from 20 mg/dl to 1,308 mg/dl. This covers the entire clinical range including normal (200-350 mg/dl), deficient states (<100 mg/dl) and excess production (>350 mg/dl) that can be used for evaluation of bleeding disorders including consumption coagulopathy and hyperfibrinogenemia. Based on the overall performance in terms of linearity, limit of detection/quantitation, carryover and coefficient of variation (CV) across five different days, two peptides per chain were finally selected for developing the quantitative assay. The intra-assay variability was <5% and intra-day CV was ∼20%. This assay is currently being tested on a variety of clinical samples representing a range of fibrinogen levels. Conclusions Our study focused on assessing whether multiplexed MRM assay could be implemented to measure fibrinogen levels in plasma. Our data indicate the feasibility of MRM-based assays for determining fibrinogen levels as an alternative to existing non-mass spectrometry-based activity or antigen assays.

B-298 Quantitation and Abundance of Xylazine and Xylazine Metabolites in Human Urine by LC-MS/MS

Abstract Background The most common cause of drug overdose fatality in the US involves synthetic opioids such as fentanyl. Recently the Director of the White House Office of National Drug Control Policy designated fentanyl associated with xylazine as an emerging threat to the US, and a national response plan will include xylazine testing. Xylazine, a clonidine analog is not FDA-approved for use in humans but has been increasingly detected in unregulated illicit drugs supplies. Our understanding of xylazine detection, metabolism, and clinical impact on patients, however, is limited. The objectives of this study are to develop a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to investigate the presence and abundance of xylazine metabolites in remnant urine samples submitted for clinical testing. Methods Remnant urine samples were identified when tested positive for xylazine during routine clinical LC-MS/MS testing. All experiments were performed using an Acquity HPLC coupled to a Waters TQS-micro triple quadrupole mass spectrometer. Urines were diluted with xylazine-D6 internal standard, hydrolyzed using β-glucuronidase and separated on a 100mm x 2.1mm, 2.6μm Kinetex C18 column (Phenomenex) with a 4 minute step gradient from 2% to 98% mobile phase B (0.1% formic acid and 2mM ammonium acetate in A: water; B: methanol) with a flow rate of 400 μl/min at 50°C. Xylazine and 4-OH-xylazine were detected in positive mode using multiple reaction monitoring, with transitions optimized from standards (Cayman Chemical and/or Sigma Aldrich), resulting in the following transitions: xylazine: 221>90, 221>164; 4-OH xylazine: 237>90, 237>137. Xylazine and 4-OH-xylazine were quantified using calibration curves prepared from 1 - 5,000 ng/mL in negative urine. Product ion scanning (PIS) was used to detect four additional xylazine metabolites, based on spectral matching to previously published full scan high resolution product ion spectra. The highest abundance fragment ions and identified retention times from PIS experiments were used to perform targeted detection for sulfone-xylazine (253>181, 253>147), oxo-xylazine (235>122, 235>114), OH-sulfone-xylazine (269>197, 269>163), and OH-oxo-xylazine (251>197, 251>148). Results Xylazine was detected at >=1 ng/mL in 38% (54/138) of fentanyl positive urines. A subset (n= 362) of urines that screened positive for opiates, amphetamines, or cocaine metabolite, were also tested but xylazine was detected in only 1 urine that did not contain fentanyl.Xylazine concentrations quantified in 61 remnant urines ranged from <1 to >5,000 ng/mL. 4-OH-xylazine concentrations ranged from <1 to 509 ng/mL. We observed five previously reported xylazine metabolites: 4-OH-xylazine, oxo-xylazine, sulfone-xylazine, OH-oxo-xylazine, OH-sulfone-xylazine. The mean concentration ratio of 4-OH-xylazine-to-xylazine was 0.11. The mean peak area ratios (metabolite-to-xylazine) for sulfone-xylazine, OH-oxo-xylazine, OH-sulfone-xylazine, and oxo-xylazine were 3.5, 3.8, 2.1, and <0.1, respectively. The proposed sulfone-xylazine and OH-oxo-xylazine metabolites were observed to have the most abundant signal of all detected metabolites, although there is currently no commercially available standard to facilitate quantification. Conclusions Xylazine and 4-OH-xylazine were quantitated in 61 patient urines. Four additional xylazine metabolites were observed and relative abundance to xylazine was estimated using peak areas. Future studies include quantitation of xylazine and xylazine metabolites in plasma and association of xylazine detection with clinical presentation and patient outcomes.

B-180 A Component Equation Approach to Resolving Calibration Curve Nonlinearity When Using Stable Isotope-Labeled Internal Standards for Quantifying Amino Acids With Mass Spectrometry

Abstract Background Stable isotope-labeled internal standards (SIL-IS) have been widely used with mass spectrometry to accurately quantify analytes. However, over an extended dynamic range, calibration curves calculated with the relative ratio of analytes to their corresponding SIL-IS are typically nonlinear. This makes accurate estimation of sample concentration difficult, especially at lower concentrations. Highly multiplexed clinical mass spectrometry assays routinely face this challenge. The component equation assumes interaction between an analyte and its SIL-IS causes the nonlinearity. Modeling this resulted in a linear calibration curve and high accuracy across the dynamic range. We compared the component equation with other calibration methods to quantify amino acids. Methods A total of 47 compounds were serially diluted and added with extraction solution containing a SIL-IS mixture to prepare 7 levels of calibrators. Calibrators were chromatographically separated using a Sciex ExionLC system with an Imtakt Intrada Amino Acid column and injected into a Sciex Citrine triple-quadrupole mass spectrometer. Seven different fitting methods were compared for calibration of each amino acid: non-weighted linear, quadratic and cubic models, linear models with 1/X and 1/X2 weighting, Padé[1,1] approximation, and component equation. Results Results for an example amino acid, tyrosine, are shown in the table below. Most models yield high accuracy at high concentrations, however, all models recorded significantly higher bias for the lowest calibrator than the component equation (≥55.7% vs 11.8%). The component equation outperformed all other models for mean bias (≥16.6% vs 5.9%). Similar results were obtained for the complete set of analytes. Conclusions When compared to other popular calibration methods to quantify a panel of amino acids, the component equation generated a linear curve and the greatest accuracy across all concentrations. The application of this simple equation could overcome the limitations of existing weighting and non-linear fitting approaches when using SIL-IS for quantifying analytes with mass spectrometry.

A-316 A quantitative gas chromatography tandem mass spectrometry method for metabolic profiling of urine organic acids and acylglycines

Abstract Background The analysis of urine organic acids (UOA) and acylglycines (UAG) are an essential investigation for the diagnosis inborn errors of metabolism (IEMs). The vast majority of methods for these analytes use gas chromatography mass spectrometry (GC-MS) and are only qualitative. Gas chromatography tandem mass spectrometry (GC-MS/MS) offers greater sensitivity and selectivity and enables the combination of qualitative screening with quantitative analysis. We describe the development and validation results of a quantitative GC-MS/MS method. Methods Urine creatinine concentrations were measured (CobasPro, Roche Diagnostics) and normalized to 1.25 mmol/L. After internal standard addition, each urine specimen, calibration standard and QC were incubated with a hydroxylamine solution followed by a liquid-liquid extraction. Further sample concentration permitted the isolated UOA and UAG to be derivatized using N,O-bis(trimethylsilyl)trifluoroacetamine (BSTFA) with 1% chlorotrimethylsilane (TMCS) prior to injection. GC-MS/MS analysis was performed using a GC-MS-TQ8050 triple quadrupole system (Shimadzu) with a 30 m DB-5MS (Agilent J&W) capillary column (0.25 mm, ID of 0.25 µm). Split injection mode was deployed with an initial column temperature of 70°C. The mass spectrometer was operated in electron ionization (EI) and multiple reaction monitoring (MRM) modes. Results The optimal temperature gradient for high-resolution UOA and UAG separations was derived and their respective retention times determined. MS/MS detection parameters were optimized to permit the identification of precursor and production ions for all (N=36) derivatized analytes. Total analysis time was 33.8 min per injection. This method permits the measurement of 11 samples in one batch and is highly linear for all analytes (R^2 >0.99 and average slope 1.04). The limit of quantification (LOQ) was established and ranged from <0.05 to 2.1 µmol/mmol creatinine. Intra- and inter-day imprecision (CV%) was determined using matrix-matched abnormal and normal QC. Intra-day imprecision (N=10) ranged from 1.3% (2-ketoglutaric acid) to 9.7% (3-hydroxybutyric acid). Inter-day imprecision (N=30 over 15 days) ranged from 2.2% (ethylmalonic acid and methylmalonic acid) to 20.5% (3-hydroxy-3-methylglutaric acid). The method had an average accuracy of 113±14% for analytes included in the ERNDiM quantitative organic acids (urine) external quality assurance program. For analytes not included in this proficiency testing, the method showed good agreement when compared to a national reference laboratory, with average Deming correlation coefficients and regression slopes being 0.9944 and 1.1, respectively. No interferences were detected when monitoring the analyte quantifier and qualifier ion ratios in patient urine. Conclusions The devised GC-MS/MS protocol permits the unbiased identification and quantification of UOA and UAG used to investigate IEMs. The method has acceptable sensitivity, specificity, precision and accuracy for clinical routine clinical testing.

B-288 Turning False Positive Screening into True Positive Results

Abstract Background After conducting a retrospective analysis of our laboratory data, we identified a notable disparity between immunoassay screen and mass spectrometry confirmation results for benzodiazepines. This prompted an investigation into whether the prevalence of false negative benzodiazepine results stemmed from the inadequacy of the immunoassay or the absence of new psychoactive substances (NPS) in our mass spectrometry confirmation assay. To determine the latter, we added four NPS benzodiazepines and their metabolites to our confirmation panel. Monitoring drug seizure records as well as reports detailing emerging substances were helpful in guiding which NPS to add. The objective of this study was to determine whether the addition of NPS benzodiazepines to our confirmation panel would increase the agreement between screen and confirmation benzodiazepine results in an opioid dependent population. Methods Urine samples that screened positive for benzodiazepines with immunoassay and confirmed negative by mass spectrometry over a 6-month timeframe were selected for analysis. Four NPS were added to our confirmation panel which included bromazolam, flubromazepam, flubromazolam, flualprazolam as well as their metabolites alpha-OH bromazolam, 3-OH flubromazepam, alpha-OH flubromazolam and alpha-OH flualprazolam. Immunoassay data was obtained using an Olympus AU480 instrument, while mass spectrometry results were acquired through an internally developed dynamic multiple reaction monitoring method on an Agilent 6470 triple quadrupole instrument. Results In the analysis of 476 urine samples using a cut-off value of 50 ng/mL, 55.3% of the samples tested positive for bromazolam, 83.0% for alpha-OH bromazolam, 0% for flubromazepam, 41.8% for 3-OH flubromazepam, 0% for flubromazolam, 0.2% for alpha-OH flubromazolam, 1.3% for flualprazolam and 6.7% for alpha-OH flualprazolam. Using a cut-off value of 10 ng/mL which represents the limit of quantitation, 75.4% tested positive for bromazolam, 89.9% for alpha-OH bromazolam, 2.1% for flubromazepam, 60.5% for 3-OH flubromazepam, 0% for flubromazolam, 0.2% for alpha-OH flubromazolam, 3.4% for flualprazolam and 9.7% for alpha-OH flualprazolam. In total, among the 476 samples that initially screened positive but confirmed negative, 95.8% confirmed positive for benzodiazepines with the inclusion of the four NPS benzodiazepines and their metabolites into our confirmation panel. Of the samples that tested positive for NPS benzodiazepines, 77.1% were also positive for fentanyl, and 95.2% were positive for norfentanyl. Conclusions The incorporation of NPS benzodiazepines into our confirmation panel significantly enhanced the concordance between immunoassay screen and mass spectrometry confirmation results. The discrepancies we observed with benzodiazepines were not predominantly linked to immunoassay challenges. Rather, they were due to the presence of NPS benzodiazepines commonly found in benzo-dope preparations, not accounted for in our mass spectrometry confirmation panel. The inclusion of NPS benzodiazepines into our confirmation panel has improved the reporting of useful diagnostic information which can be used to support harm reduction efforts.

B-164 Comparison of Analytical Performance of Two Processing Methods for Measurement of Zinc and Copper on Triple Quadrupole Inductively Coupled Plasma Mass Spectrometer

Abstract Background Copper (Cu) and zinc (Zn) are essential cofactors of enzymes involved in several metabolic processes required for the development of tissue structures and immune response, especially in pediatrics and pregnant women. Triple quadrupole inductively coupled plasma mass spectrometer (ICP-QQQ) enables simultaneous precise quantification of multiple elements with high throughput, sensitivity, and specificity. Samples are introduced into ICP-QQQ as a liquid and converted to fine aerosol with the help of a nebulizer and spray chamber. The precision and sensitivity of measured metals are dependent on the sample preparation thus, this study focuses on comparing the analytical performances between two sample preparations: 0.5% nitric acid (HNO3) as the only diluent (Diluent A) versus a combination of 0.5% HNO3 along with 1% ethanol, 0.5% Ammonium hydroxide, 0.02% TritonX-100, and 0.1% HNO3 (Diluent B) for Cu and Zn metal analysis in serum using ICP-QQQ. Methods Twenty deidentified randomly selected patient serum samples were prepared with either diluent A or diluent B and analyzed by ICP-QQQ (Agilent 8900). Data were analyzed using EP Evaluator. Bland-Altman analysis was used to determine the agreement between the two sample preparations. Deming regression analysis was used to determine other analytical performances, such as the proportional and constant bias of Zn and Cu concentration. Results There was a bias of -4.8 and -3.0 between the two sample preparation methods when analyzing Cu and Zn, respectively. The mean of Cu-Diluent B versus Cu—Diluent A was 125.2 ± 28.3 µg/dL and 120.4 ± 29.8 µg/dL, respectively, with a slope of 1.070. Conversely, the Zn comparison study shows a lower slope (0.769) with a mean of 71.0 ± 20.5 and 68.0 ± 16.1 µg/dL for Zn-Diluent B and Zn-Diluent A, respectively. Conclusions Samples prepared with nitric acid solution only (Diluent A) appeared to show negative bias compared to Diluent B solution containing triton-X for both metals. However, the bias is within the total allowable error for zinc and copper. Importantly, Zn-sample analysis with diluent A (HNO3 only) shows a 23% proportional bias, which is absent with copper sample analysis. A larger study is needed to further compare several methods of sample preparation in ICP-QQQ analysis while leveraging simple preparation without compromising analytical sensitivity. Overall, this study shows that both methods of sample preparation can be effectively used in the ICP-QQQ analysis of serum copper and zinc.

Assay for the quantification of abemaciclib, its metabolites, and olaparib in human plasma by liquid chromatography-tandem mass spectrometry

An isotope-dilution bioanalytical assay for abemaciclib and its metabolites in combination with olaparib was developed and validated in human plasma K2 EDTA. For the quantitative assay, human plasma samples (or human plasma QC samples) were spiked with internal standard solution before a simple protein precipitation with methanol. The extract was injected onto a liquid chromatography-tandem mass spectrometry (LC-MS/MS) instrument where it was chromatographically separated by a polar end-capped reversed phase column and guard using gradient elution with water and methanol both modified with 0.2% formic acid (v/v) as the mobile phases. The analytes and internal standards were measured by heated electrospray ionization (HESI) in positive polarity using selected reaction monitoring (SRM) on a triple quadrupole mass spectrometer. The assay was validated for linear ranges as follows: 0.4 – 1000nM abemaciclib, 0.35 – 1000nM M2 and M18, 0.5 – 1000nM M20, and 0.75 – 1000nM olaparib. The inter-day or between day precision for the quality controls (n = 18) was < 13% and the accuracy was ± 12%, for all analytes, including the lower limit of quantification (LLOQ). The intra-day or within day precision for the quality controls (n = 6) was ≤ 11% and the accuracy was ± 12% for low, mid, and high and < 19% at LLOQ. The recovery in human plasma was determined to be between 92 and 102% for all analytes spanning the linear range. The validated, bioanalytical quantitative assay was designed to measure abemaciclib, its metabolites, and olaparib for pharmacokinetic evaluation of patients in clinical trials for breast, brain, and ovarian cancers.

A quality by design paradigm, novel, and fast LC-MS/MS method to quantify vortioxetine in rat brain homogenate and its assessment of greenness, whiteness, and blueness tools

Vortioxetine (VOR) is an antidepressant drug with multimodal mechanism of action. In this work a sensitive and simple bioanalytical LC-MS/MS method for analysis of VOR in spiked rat brain tissue was developed and validated. Chromatographic analyses were performed in isocratic mode, and Citalopram (CT) was found as a suitable internal standard. Supelco Ascentis® Express Phenyl-Hexyl column was used as stationary phase, and the mobile phase was 0.15 % formic acid in acetonitrile:water (55:45, v/v). A Box-Behnken Design − based method optimization was applied to explore the impact of analytical conditions on chromatographic separation of VOR and CT. Detection was realized via a triple quadrupole LC-MS/MS system; the mass transition ion-pair has been followed as m/z 299.05 → 149.90 for VOR, m/z 325.20 → 109.05 for CT. Analytical method validation was performed according to International Council for Harmonisation (ICH) guideline M10 on bioanalytical method validation. The calibration curve was within the range of 30 to 450 ng/mL. LOD and LLOQ were calculated at 10 and 30 ng/mL, respectively. Recovery was 90 % for the lower limit of quantification and between 98 % to 105 % for the other concentration levels. Total run time was 5 min, retention times of citalopram and vortioxetine were 2.4 and 3.1 min, respectively, and a good chromatographic separation was obtained under optimal conditions. The solvents used to make the method greener were evaluated with the Green Solvent Selection Tool and Green Index Spider chart. The AGREE score for the greenness of the developed method was calculated as 0.61 and was also evaluated with the ComplexGAPI pictogram. The BAGI score for blueness was calculated as 72.5, and the RGBfast score for whiteness was calculated as 50. The method was determined to be most green, white, and blue.

Simulation analysis and optimization of triple quadrupole mass spectrometer based on CFD

Simulation analysis and optimization of triple quadrupole mass spectrometer based on CFD

Investigation of the Cholesterol Biosynthesis by Heart-Cut Liquid Chromatography and Mass Spectrometric Detection

The biosynthesis and homeostasis of cholesterol are essential for cellular function. Cholesterol is a major lipid with multiple roles in membrane stability, signaling, or as a precursor for other molecules. Because of the structural similarity of the sterols involved in the biosynthesis, their accurate identification and quantification is still challenging. Moreover, the huge difference in the concentration of cholesterol and its precursors can cause interferences during the detection. To overcome these problems, a heart-cut liquid chromatographic method was developed by evaluating 38 different columns to achieve optimal separation. The method efficiently separates all sterol biosynthesis intermediates, with detection limits in the low nmol/L-range and an upper limit of quantification of 9 mmol/L for cholesterol by using triple quadrupole mass spectrometric detection. Investigation of lung carcinoma cells treated with statins demonstrated the capability to detect a biological response, showing inhibition of sterol synthesis. This technique offers a robust tool for studying cholesterol biosynthesis and its role in disease.

Monitoring of 260 pesticides in extra virgin olive oil and risk assessment for consumers within the framework of the European multiannual control program

The aim of the present research was to develop and validate a robust analytical method for the monitoring of 260 pesticide residues in Extra Virgin Olive Oil (EVOO) expanding the 185 molecules requested by the multiannual control program. The analytical procedure included an ultrasound-assisted liquid-liquid microextraction followed by low-pressure gas chromatography (LP-GC) and ultra-high-performance liquid chromatography (UHPLC), both coupled to triple quadrupole mass spectrometry. Matrix-matched calibration curves showed good linearity with coefficients of determination greater than 0.999. Accuracy values ranged from 65.5 % to 122.3 % and from 61.1 % to 133.3 % for LP-GC and UHPLC, respectively. The recoveries ranged from 14.0 % to 177.9 %.Fifty commercial EVOO, from Italian and EU production, were analyzed to assess pesticide contamination during the 2021–2023 harvesting seasons. The research focused on evaluating consumer risk by assessing both chronic and acute dietary exposure, using the Pesticide Residue Intake Model developed by the EFSA.

An Investigation of Pharmacokinetic Interaction of Vericiguat with Apigenin based on a Newly Developed Ultra-performance Liquid Chromatography-tandem Mass Spectrometry Assay.

Vericiguat, as a new stimulator of soluble guanylate cyclase (s- GC), was recently approved as a first-in-class treatment for reducing risks in patients with ejection fraction less than 45 percent and heart failure (HF) in the USA. The main aim of the present experiment was to establish an acceptable, sensitive assay based on ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) for quantitatively analyzing the plasma concentration levels of vericiguat in rats, and to further evaluate the effect of apigenin on the metabolism of vericiguat in vivo. In sample processes, acetonitrile was finally chosen for quickly precipitating protein. The levels of vericiguat in plasma were analyzed by a Xevo TQ-S triple quadrupole tandem mass spectrometry (Milford, MA, USA) in a positive ion mode. The scope of the calibration standard for vericiguat ranged from 0.5 to 1000 ng/mL, where a great linearity was acceptable. The lower limit of quantification (also called LLOQ) of vericiguat presented the sensitivity of this assay was evaluated as low as 0.5 ng/mL. Additionally, selectivity, accuracy and precision, extraction recovery, matrix effect, and stability were all verified. Subsequently, this approach also supported to assess the plasmatic concentrations of vericiguat from an interaction survey on herb-- drug, in which oral administration of apigenin (20 mg/kg) obviously increased the plasmatic levels of vericiguat and altered the pharmacokinetics of vericiguat in rats. These results would help us to further understand the pharmacokinetic properties of vericiguat when co-administration with apigenin, and to avoid unexpected clinical risks in the future.

Determination of Calcium Isotopes Using Inductive Coupled Plasma-Tandem Mass Spectrometry with Ozone-Induced Oxidation.

In this study, we propose a new method for determination of calcium (Ca) isotopes using inductive coupled plasma-tandem mass spectrometry (ICP-MS/MS). The most abundant isotope of Ca, 40Ca, has a spectrum identical with that of argon (Ar), which is often used as the carrier gas in ICP-MS analysis. The detection capability for 40Ca is thus significantly reduced in the presence of 40Ar. To avoid interference from 40Ar during the 40Ca measurements, ozone (O3) was used as the reaction gas in the ICP-MS/MS analysis. The reaction of Ca+ with O3 in the collision cell preferentially produced CaO3+. In contrast, the reaction between Ar+ and O3 produced ArO+, which further reacted with the oxidized O3 to produce Ar+. Because Ar+ does not produced ArO3+ as the main product of the reaction with O3, the measurement of Ca+ as CaO3 via O3-induced oxidation resulted in considerably less interference from Ar+. The detailed mechanism of CaO3+ formation was investigated via quantum chemistry calculations by using density functional theory. The interaction between Ca+ and O3 yielded CaO3+, which immediately dissociated into either Ca+ and O3 or CaO+ and O2 because the pressure in the collision cell was approximately 1 Pa in the MS/MS experiments. Consequently, CaO3+ was formed by the consecutive reaction of Ca+ with O3, with CaO+ and CaO2+ as intermediates. The present method achieved the detection of less abundant Ca isotopes in certified reference materials such as 43Ca, 44Ca, 46Ca, and 48Ca, thereby allowing the determination of Ca isotopes with high sensitivity.

Quantification of Letrozole, Palbociclib, Ribociclib, Abemaciclib, and Metabolites in Volumetric Dried Blood Spots: Development and Validation of an LC-MS/MS Method for Therapeutic Drug Monitoring

Therapeutic drug monitoring (TDM) may be beneficial for cyclin-dependent kinase 4/6 inhibitors (CDK4/6is), such as palbociclib, ribociclib, and abemaciclib, due to established exposure–toxicity relationships and the potential for monitoring treatment adherence. Developing a method for quantifying CDK4/6is, abemaciclib metabolites (M2, M20), and letrozole in dried blood spots (DBS) could be useful to enhance the feasibility of TDM. Thus, an optimized LC-MS/MS method was developed using the HemaXis DB10 device for volumetric (10 µL) DBS collection. Chromatographic separation was achieved using a reversed-phase XBridge BEH C18 column. Detection was performed with a triple quadrupole mass spectrometer, utilizing ESI source switching between negative and positive ionization modes and multiple reaction monitoring acquisition. Analytical validation followed FDA, EMA, and IATDMCT guidelines, demonstrating high selectivity, adequate sensitivity (LLOQ S/N ≥ 30), and linearity (r ≥ 0.997). Accuracy and precision met acceptance criteria (between-run: accuracy 95–106%, CV ≤ 10.6%). Haematocrit independence was confirmed (22–55%),with high recovery rates (81–93%) and minimal matrix effects (ME 0.9–1.1%). The stability of analytes under home-sampling conditions was also verified. Clinical validation supports DBS-based TDM as feasible, with conversion models developed for estimating plasma concentrations (the reference for TDM target values) of letrozole, abemaciclib, and its metabolites. Preliminary data for palbociclib and ribociclib are also presented.

Ultra high-performance liquid chromatography tandem mass spectrometry: Development and validation of an analytical method for N2-Ethyl-2’-deoxyguanosine in dried blood spot

In the body, ethanol is metabolized to acetaldehyde by alcohol dehydrogenase and CYP2E1. Acetaldehyde is the main carcinogen that forms DNA adducts, N2-Ethylidene-2'-deoxyguanosine (N2-Ethylidene-dG), which can cause DNA damage and lead to cancer. However, N2-Ethylidene-dG is an unstable form at the nucleoside level and is difficult to measure. So it needs to be reduced using NaBH4 to become N2-Ethyl-2'-deoxyguanosine (N2-Et-dG). This study aims to obtain a selective, sensitive, and validated analytical method to determine N2-Et-dG using ultra-high-performance liquid chromatography-tandem mass spectrometry with allopurinol as the internal standard. The N2-Et-dG analysis was performed using a C18 Acquity® Bridged Ethylene Hybrid column of (1.7 μm, 100 mm × 2.1 mm). The sample matrix used was dried blood spots, and then the DNA sample was extracted using the QIAamp DNA Mini Kit. The optimum analysis conditions were obtained in a combination eluent of 0.1 % acetic acid and methanol (60:40 v/v) with a flow rate of 0.1 mL/min and eluted isocratically for 5 min. Quantification analysis was performed using triple quadrupole mass spectrometry with electrospray ionization in positive mode, with detection at m/z 296.16 > 180.16 for N2-Et-dG and 137.03 > 110.05 for allopurinol, respectively. The validated analytical method follows the 2018 USA Food and Drug Administration guidelines with a linear concentration range of 5–200 ng/mL.

A Liquid Chromatography Coupled to Tandem Mass Spectrometry Method for Simultaneous Estimation of Saxagliptin and Avanafil: Application to Pharmacokinetic Drug‐Drug Interaction in Type 2 Diabetic Rat Model

ABSTRACTErectile dysfunction is a common complication of type 2 diabetes. The combination of saxagliptin and avanafil can effectively manage both conditions in individuals with type 2 diabetes. However, evaluating the potential pharmacokinetic interactions for co‐administration of saxagliptin and avanafil is crucial. Here, we developed a sensitive bioanalytical method for simultaneously quantifying saxagliptin and avanafil in rat plasma using liquid chromatography hyphenated to triple quadrupole mass spectrometry. The chromatographic separation of saxagliptin, avanafil, and irbesartan (internal standard) was achieved on an Aquity BEH C18 column (100 × 2.1 mm, 1.7 µm), using 0.1% formic acid in water and acetonitrile as mobile phase with the gradient elution at a flow rate of 0.2 mL/min. Mass detection was carried out using selective reaction monitoring mode, with the product ions for saxagliptin, avanafil, and irbesartan being 180.2, 375.1, and 207.1 m/z, respectively. The developed liquid chromatography coupled to tandem mass spectrometry method was validated as per the US Food and Drug Administration guidelines in rat plasma. The validation findings confirmed the method's robustness and suitability for accurate quantification of saxagliptin and avanafil in rat plasma with the limit of quantification of 1 ng/mL for both the drugs and were linear in the range of 1–2000 ng/mL. The study found no significant pharmacokinetic interaction between saxagliptin and avanafil, suggesting they can be safely co‐administered to patients with type 2 diabetes.

Magnetic particle spray mass spectrometry for the determinationof beta-blockers in plasma samples.

Magnetic particle spray mass spectrometry (MPS-MS), an innovative ambient ionization technique proposed by our research group, was employed to determine beta-blockers in human plasma samples. A dispersive solid phase extraction of atenolol, metoprolol, labetalol, propranolol, nadolol, and pindolol was carried out using magnetic molecularly imprinted polymer (M-MIP) particles that were attached to the tip of a metal probe, which was placed in the mass spectrometer inlet. A solvent (1% formic acid in methanol) was dispensed on the particles, and the Taylor cone was formed around them (in high voltage). The analytes were desorbed/ionized and determinedby a triple quadrupole mass spectrometer. M-MIP was synthesized with oxprenolol as a pseudo-template, demonstrating good selectivity to beta-blockers compared withno-analog molecules, with an adsorption process occurring in monolayers, according to isotherm studies. Kinetic experiments indicated chemisorption as the predominant M-MIP/analyte interaction. The analytical curves were linear (R2 > 0.98), and the limit of quantification was 3µg L-1 for all the analytes. Limits of detection ranged from 0.64 to 2.41µg L-1. Precisions (relative standard deviation) and accuracies (relative error) ranged from 3.95 to 21.20% and-17.05 to 18.93%, respectively. MPS-MS proved to be a simple, sensitive, and advantageous technique comparedwithconventional approaches. The analyses were fast, requiring no chromatographic separation and without ionic suppression. The method is aligned with green chemistry principles, requiring minimal sample, solvent, and sorbent amounts. MPS-MS successfully integrates sample preparation and ambient ionization mass spectrometry and holds great potential for application with other sorbents, samples, and analytes.

A general and rapid LC-MS/MS method for simultaneous determination of voriconazole, posaconazole, fluconazole, itraconazole and hydroxyitraconazole in IFI patients

ObjectiveTo establish a rapid and universal quantitative liquid chromatography tandem mass spectrometry (LC-MS/MS) method for measuring the exposure levels of five triazole antifungal drugs in human plasma, including voriconazole, fluconazole, posaconazole, itraconazole, and hydroxyitraconazole. MethodsA triple quadrupole mass spectrometer operating in positive ionization mode was used to detect the analyte, and multiple reaction monitoring mode was employed to gather data. The mobile phase included 0.05 % formic acid in water (phase A) and acetonitrile (phase B). The analytes were separated on an Agilent EclipsePlusC18 RRHD column (30 × 50 mm, 1.8 μm) using gradient elution. The flow rate was 0.3 mL/min with the column temperature set at 35 °C. The acetonitrile was used to pretreat the plasma sample, and the itraconazole-D5 and hydroxyitraconazole-D5 were utilized as the internal standards. ResultsThe calibration range was from 100 to 10,000 ng/mL for posaconazole, itraconazole, and hydroxyitraconazole, from 200 to 20,000 ng/mL for fluconazole and from 50 to 5000 ng/mL for voriconazole, with linear correlation coefficients more than 0.99 for all regression curves. The intra- and inter-day accuracy and precision of the method were within ±15 %. The mean extraction recovery of all the analytes ranged from 74.32 % to 117.83 %, and the matrix effect was from 72.54 % to 111.2 %. The results of stability fell into the scope of ±15 % deviation. ConclusionThis newly developed method is sensitive, simple, and robust, and successfully applied in determining triazole antifungal drugs in plasma from 66 IFI patients to provide reference for safe and effective drug administration in clinical practice.

UPLC‐MS/MS and chemometrics analyses reveal chemical profile and anti‐inflammatory activity of Bienertia cycloptera Bunge fractions

AbstractIntroductionBienertia cycloptera is a species belonging to the Chenopodiaceae family. According to earlier reports, a unique research study on the phytochemistry and biological analysis of that species was conducted.ObjectiveThis study presents an integrated metabolomics investigation combined with multivariate analysis of various extractive fractions of B. cycloptera aerial parts. This study is the first attempt to explore the anti‐inflammatory metabolites from B. cycloptera, showing its significance as a valuable traditional medicine.MethodologyBy comparing retention times, quasi‐molecular ions, and MS/MS fragment ions with databases and literature references, metabolite annotation was accomplished using ultra performance liquid chromatography (UPLC)/triple quadrupole mass spectrometry (MS). Moreover, the effects of the studied samples on the gene expression of the four pro‐inflammatory cytokines (IL‐1β, IL‐6, TNF‐α, and INF‐γ) using polymerase chain reaction (PCR) and comparing their results by those caused by piroxicam were tested to determine their anti‐inflammatory efficacy.ResultsChemical profiling revealed diverse metabolites, with 62 chromatographic peaks identified across various chemical classes. UPLC‐MS/MS of different B. cycloptera fractions unveiled distinct chemical profiles. Results showed distinct chemical compositions in each fraction, with petroleum ether fraction enriched in sterols and fatty acids; methylene chloride fraction in alkaloids, sterols, and cardenolides; ethyl acetate fraction in alkaloids, flavonoids, cardenolides, and phenolic acids; and n‐butanol fraction in flavonoids, alkaloids, and phenolic acids. Multivariate data analysis illustrated clustering patterns among petroleum ether, methylene chloride, ethyl acetate, and n‐butanol fractions. OPLS‐DA models were constructed to discern inter‐class differences, identifying discriminatory metabolites. In vitro cytotoxicity and anti‐inflammatory assays demonstrated the safety and efficacy of B. cycloptera fractions, with significant downregulation of pro‐inflammatory markers. Further analysis revealed specific metabolites associated with anti‐inflammatory effects, such as p‐hydroxybenzoic acid, vanillic acid, tachioside, ferulic acid, staphylionoside D, humilixanthin, bergaptol, vulgaxanthin I, and portulacaxanthin III.ConclusionThe findings of this study provide valuable insights into the chemical composition and bioactivity of B. cycloptera fractions, suggesting their potential as therapeutic agents and warranting further investigation.