Gradient Elution Profile Research Articles

B-167 Quantification of Thyroglobulin in Serum for Clinical Research using SISCAPA Workflow Combined with LC-MS/MS

Abstract Background Accurate measurement of Thyroglobulin (Tg) using existing immunoassay-based techniques can be challenging in the presence of anti-Tg antibodies (TgAb), which can prevent the binding of Tg to assay antibodies thus leading to non-quantifiable Tg concentrations. The Stable Isotope Standards and Capture with Anti-Peptide Antibodies (SISCAPA™) Workflow combined with LC-MS/MS has been successfully employed to circumvent this problem by digesting the serum sample thus eliminating interfering TgAb and measuring a Tg-specific surrogate peptide instead. Methods Using a Andrew+™ Pipetting Robot, denaturation and reduction buffer was added to 250µL serum samples and incubated at 37°C. Internal standard and trypsin were added and samples incubated at 37°C. The samples were quenched with protease inhibitor and Anti-Tg FSP mAb SISCAPA was added to the serum samples and mixed for 60 minutes. Using a magnetic array plate, the beads were left to pull down, samples were discarded, and the beads were twice washed with PBS/CHAPS prior to elution with acetonitrile with formic acid. Using an ACQUITY™ UPLC™ I-Class PLUS FL System, samples were injected onto a Waters™ XSelect™ HSS T3 Column using a water/acetonitrile/formic acid gradient elution profile and the Tg FSP peptide was quantified using a Waters Xevo™ TQ Absolute Mass Spectrometer. Results The method demonstrated no significant carryover or matrix effects and was shown to be linear from 0.1 - 50 ng/mL. Analytical sensitivity investigations indicate the analytical sensitivity of this method would allow precise quantification (<20%) at 0.1 ng/mL with S/N (PtP) >10:1. Coefficients of variation (CV) for total precision and repeatability on 5 analytical runs for low, mid and high QCs were all < 9.5% (n = 25) for both automated and manual precision assessments. Comparison with samples from the UK NEQAS scheme demonstrated significant method bias of -40% for the developed LC-MS/MS method. Re-assignment of the calibrator concentrations using the EQA samples reduced the bias to -5.5%, indicating differences in the calibration materials used for these measurements. Conclusions A LC-MS/MS clinical research method for serum thyroglobulin was developed using SISCAPA, followed by analysis using ACQUITY UPLC I-Class PLUS FL System and the Xevo TQ Absolute Mass Spectrometer. The method provides analytical sensitivity down to 0.1 ng/mL from 250 µL serum, while providing sufficient sample for re-analysis. The method demonstrates excellent linearity across the calibration range, with no significant carryover, interferences, and matrix effects. Total reproducibility and repeatability of the method was ≤9.5% RSD for manual and automated sample preparation, using the Andrew+ Pipetting Robot. In addition, the Andrew+ Pipetting Robot can minimize user touch-time, allowing a full plate to be prepared within four hours without user intervention.

Quantitative determination of phenazepam and its active metabolite in human blood plasma at different extraction procedures

Introduction. The presence of the active metabolite (3-hydroxyphenazepam, 3-OH-PHEN), the wide interindividual variability of the therapeutic effect of phenazepam (PHEN), as well as its active moiety in the blood, determine the relevance of therapeutic drug monitoring (TDM). To do this, the researcher must have an express analytical technique with a wide analytical range, a low limit of quantification (LLOQ), and with robustness with different sample preparation methods.Aim. Development and validation of quantitative methods for PHEN and 3-OH-PHEN in human blood plasma with different sample preparation methods.Materials and methods. The determination of PHEN and 3-OH-PHEN has been performed by high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). Solid-phase extraction (SPE) and liquid extraction with support (SLE) otherwise called liquid-liquid extraction in the solid phase were used for sample preparation. Metoprolol was utilized as an internal standard (IS). Gradient elution profile between mobile phase A (0.2 % aqueous formic acid) and B (100 % acetonitrile) has been used. Column: Hypersil GOLD® C18, 50 × 2.1 mm, 3.5 μm.Results and discussion. Two methods have been developed for the quantitative determination of PHEN and 3-OH-PHEN in human blood plasma using different sample preparation methods: SPE and SLE. The conditions of chromatographic separation and mass spectrometric detection of the analytes are selected. The following validation characteristics were determined for both methods: selectivity, calibration curve, accuracy, precision, degree of extraction, LLOQ, carry-over effect, matrix factor, stability of standard solutions and analyte in the matrix.Conclusion. The validation results of the developed methods meet the established criteria, which allows them to be used for the quantitative determination of PHEN and 3-OH-PHEN in human blood plasma. The wide analytical range for both methods 1–1000.00 ng/ml allows the use them for pharmacokinetics and bioequivalence studies, as well as in toxicology.

Development and validation of stability indicating assay method for mitapivat: Identification of novel hydrolytic, photolytic, and oxidative forced degradation products employing quadrupole-time of flight mass spectrometry.

Mitapivat is a novel, first-in-class orally active pyruvate kinase activator approved by the US Food and Drug Administration in 2022 for the treatment of hemolytic anemia. There is no literature available regarding the identification of degradation impurities of mitapivat. The present study deals with the degradation behavior of mitapivat under various stress conditions such as hydrolytic, photolytic, thermal, and oxidative stress. The multivariate analysis found that the independent variables, that is, molarity, temperature, and time, are interacting with each other to affect the degradation of mitapivat. A specific, accurate, and precise high-performance liquid chromatographic (HPLC) method was developed to separate mitapivat from its degradation products. The separation was achieved on the C-18 column (250mm × 4.6mm × 5µm) using the combination of 0.1% formic acid buffer and acetonitrile in gradient elution profile. The method was validated as per the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use Q2(R2) guideline. LC-electrospray ionization-Quadrupole-time of flight was employed to identify degradation products. A total of seven novel degradation products of mitapivat were identified based on tandem mass spectrometry and accurate mass measurement. In-silico toxicity of mitapivat and its degradation products was qualitatively evaluated by the DEREK toxicity prediction tool.

Lentiviral vector determinants of anion-exchange chromatography elution heterogeneity.

The demand for Lentiviral Vector (LV) drug substance is increasing. However, primary capture using convective anion-exchange chromatographyremains a significant manufacturing challenge. This stems from a poor understanding of the complex adsorption behaviors linked to LVs intricate and variable structure, such as high binding heterogeneity which is typically characterized by a gradient elution profile consisting of two peaks. Understanding which LV structural components drive these phenomena is therefore crucial for rational process design. This work identifies the key LV envelope components responsible for binding to quaternary-amine membrane adsorbents. Eliminating the pseudotype protein (Vesicular Stomatitis Virus G glycoprotein [VSV-G])did not impact the heterogenous two-peak elution profile, suggesting it is not a major binding species. Digestion of envelope glycosaminoglycans (GAGs), present on proteoglycans, leads to a dramatic reduction in the proportion of vector eluted in peak 2, decreasing from 50% to 3.1%, and a threefold increase in peak 1 maximum. Data from reinjection experiments point towards interparticle envelope heterogeneity from discrete LV populations, where the two-peak profile emerges from a subpopulation of LVs interacting via highly charged GAGs (peak 2) along with a weaker binding population likely interacting through the phospholipid membrane and envelope protein (peak 1).

Development of an ultra-high-performance liquid chromatography-tandem mass spectrometry method for the simultaneous determination of crassicauline A, fuziline, karacoline, and songorine in rat plasma and application in their pharmacokinetics.

In this paper, an ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for quantifying the levels of crassicauline A, fuziline, karacoline, and songorine in rat plasma. After processing the rat plasma, the proteins in the plasma were separated by extracting the analytes with acetonitrile-methanol (9:1, v/v). The chromatographic column used was the UPLC HSS T3 column, and the mobile phase (methanol-water with 0.1% formic acid) under a gradient elution profile was used to separate the four compounds, with elution times for each analyte being less than 5 min. Electrospray ionization in positive-ion mode and operating in multiple reaction monitoring mode was used for quantitative analysis. Crassicauline A, fuziline, karacoline, and songorine were administered to 48 rats (n = 6 per group) orally (5 mg/kg) and intravenously (0.5 mg/kg). The standard curves demonstrated excellent linearity in the range of 1-2500 ng/mL, wherein all r values were greater than 0.99. The UPLC-MS/MS method for the determination of crassicauline A, fuziline, karacoline, and songorine in rat plasma was successfully applied in determining their pharmacokinetics parameters, from which their oral bioavailabilities were calculated to be 18.7%, 4.3%, 6.0%, and 8.4%, respectively.

B-192 An LC-MS/MS Method using the Xevo TQ Absolute Mass Spectrometer for the Combined Analysis of Aldosterone and Plasma Renin Activity in Plasma for Clinical Research

Abstract Background Primary aldosteronism (PA) is a common cause of hypertension, whereby uncontrollable amounts of aldosterone are produced by a benign tumour or hyperplasia of the adrenal glands. Excess aldosterone results in significant sodium reabsorption in the kidneys, increasing water retention and blood volume, thereby causing hypertension. The renin-angiotensin-aldosterone system (RAAS) regulates the production of aldosterone and in this system, renin and aldosterone should move in synchronicity with each other throughout the day. Therefore, these two components are used to assess the status of the RAAS, particularly in the evaluation of new therapies in clinical research studies. Historically, the assessments of aldosterone and plasma renin activity (PRA) have been performed using separate methods using immunoassay or more recently liquid chromatography - tandem mass spectrometry (LC-MS/MS) platforms. One of the benefits of using LC-MS/MS for clinical research is the ability to measure multiple analytes across the proteome and metabolome using the same system and even in the same analysis to provide more information in less time and save costs. Here we evaluate a single LC-MS/MS method for the combined measurement of plasma aldosterone and renin activity for clinical research purposes. Methods Aldosterone certified reference material (Merck, UK) and Angiotensin I (Cambridge BioScience, UK) were used to create calibrators in 2% Bovine Serum Albumin (BSA) in Phosphate Buffered Saline (PBS). In-house QC material prepared in both 2% BSA in PBS and K2EDTA plasma (BioIVT, UK), were used to evaluate method precision. Plasma samples were analyzed using the newly developed method and the quantified results were compared to separate independent LC-MS/MS methods for aldosterone and plasma renin activity. Plasma samples were treated with generation buffer (Sodium acetate, EDTA, acetic acid, SBTI and PMSF) and mixed for three hours at 37 °C. Samples were precipitated, diluted and centrifuged prior to SPE. Sample supernatant was transferred to a Waters Oasis™ µElution 96 Well Plate, followed by a wash and elution. Using an ACQUITY™ UPLC™ I-Class System, samples were injected onto a Waters XBridge™ C8, 2.5 µm, 2.1 × 50 mm Column using a water/methanol/ammonium fluoride gradient elution profile and quantified with a Waters Xevo™ TQ Absolute Mass Spectrometer. Results The method demonstrated no significant carryover or matrix effects and was shown to be linear from 10 - 2500 pg/mL for aldosterone and 0.1–25 ng/mL/h for PRA. Analytical sensitivity investigations indicate the analytical sensitivity of this method would allow precise quantification (<20%) at 10 pg/mL and 0.1 ng/mL/h, for aldosterone and PRA, respectively. Coefficients of variation (CV) for total precision and repeatability on 5 analytical runs for low, mid and high QCs were all <10% (n = 25) for aldosterone and PRA. Comparison with samples previously analyzed by an independent LC-MS/MS method demonstrated good agreement for aldosterone and PRA. Conclusions We have successfully quantified aldosterone and renin activity in plasma in a single method using an SPE protocol with LC-MS/MS analysis, for clinical research purposes. The method demonstrates excellent linearity and precision, with minimal matrix effects. For Research Use Only. Not for Use in Diagnostic Procedures.

Evaluating dynamic traveling wave profiles for the enhancement of separation and sensitivity in traveling wave structures for lossless ion manipulations

The amenability of traveling wave ion mobility spectrometry (TWIMS) to extended separation pathlengths has prompted a recent surge of interest concerning the technique. While promising, the optimization of ion transmission, particularly when analyzing increasingly disparate species, remains an obstacle in TWIMS. To address this issue, we evaluated a suite of dynamic TW profiles using an original TW structures for lossless ion manipulations (TW-SLIM) platform developed at Washington State University. Inspired by the range of gradient elution profiles used in traditional chromatography, three distinct square TW profiles were evaluated: a static approach which represents a traditional waveform, a dual approach which consists of two distinct TW profiles within a given separation event; and a ramp approach which varies TW speed and amplitude at a fixed rate during separation. The three waveform profiles were evaluated in terms of their impact on separation (quantified as resolution) and sensitivity (quantified using signal-to-noise ratio (SNR), and ion abundance). Concerning separation, the highest resolution (R) was observed when operating with the static waveform (R = 7.92); however, the ramp waveform performed comparably (R = 7.70) under similar conditions. Regarding SNR, optimum waveform profiles were species dependent. Bradykinin2+ displayed the largest gains in SNR (36.6% increase) when ramping TW speed, while the gains were greatest (33.5% increase) for tetraoctylammonium when modulating TW amplitude with the static waveform. Lastly, significant (>10%) increases in the abundance of tetraoctylammonium ions were observed exclusively when utilizing a ramped waveform. The present set of experiments outline the results and challenges related to optimizing separations using alternative TW profiles and provides insight concerning TW-SLIM method development which may be tailored to enhance select analytical metrics.

Ion exchange resin – Bipolar membrane electrodialysis hybrid process for reverse osmosis permeate Remineralization: Preparative ion exchange chromatography for Ca2+ and Mg2+ recovery

Reverse osmosis (RO) membrane technology is increasingly applied for the production of potable water on a large scale. However, slightly acidic and unbuffered RO permeate can enhance water piping network deterioration. Therefore, post-treatment i.e., remineralization is commonly applied to protect the water distribution network. An ion exchange resin – bipolar membrane electrodialysis hybrid process has been recently developed for RO permeate remineralization. In the investigated remineralization process, the cation exchange resin regenerant stream – rich with mainly divalent ions – is used partially for RO permeate remineralization. The viability of using preparative ion exchange chromatography principles for monovalent – divalent ion separation was investigated. The elution process was investigated in the case of both single and binary (calcium and potassium as a case study) components. Elution velocity, as well as concentration, was shown to affect the separation process significantly, while elution mode (co/counter current) did not substantially influence the elution process for the conditions studied. Gradient elution profiles (linear and stepwise) were superior to the non-gradient method of ion exchange elution in achieving higher purity and yield of the recovered calcium using the same amount of eluent. Linear gradient elution improved the purity of the calcium recovered to > 99% compared to conventional isocratic elution (85%) at 60% yield. Likewise, linear-gradient elution resulted in higher calcium specific productivity at>95% purity, which is necessary for an efficient remineralization process to avoid adding impurities to the remineralised water. This research showed that preparative ion exchange chromatography principles can be applied in drinking water applications to achieve high purity (>95%) and sufficient specific productivity of the recovered hardness ions (Ca2+ = 0.15–0.20 mol/L.h) required for RO permeate remineralization.

Novel LC-MS/MS analysis of the GLP-1 analog semaglutide with its application to pharmacokinetics and brain distribution studies in rats

Semaglutide, one of the most potent glucagon-like peptide (GLP)-1 analogs, has widely been used to treat type II diabetes mellitus and obesity. Recent studies have shown that semaglutide also works on the brain, suggesting its potential utility for various diseases, including Parkinson’s disease and Alzheimer’s disease. This study aimed to develop a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of semaglutide in both plasma and brain to characterize the pharmacokinetics and brain distribution in rats. Semaglutide was extracted by simple protein precipitation with methanol from plasma and by solid phase extraction from brain tissue. Liraglutide was used as an internal standard. Gradient elution profiles with mobile phases comprising 0.1 % formic acid in water and acetonitrile were used for chromatographic separation. The lower limit of quantification (LLOQ) of the LC-MS/MS assay was 0.5 ng/mL for both rat plasma and brain. Intra- and inter-day accuracy ranged 89.20–109.50 % in the plasma and 92.00–105.00 % in the brain. Precision was within 8.92 % in the plasma and 7.94 % in the brain. Sprague-Dawley rats were given semaglutide by intravenous (IV, 0.02 mg/kg) and subcutaneous (SC, 0.1 and 0.2 mg/kg) injection. Plasma concentrations of semaglutide showed a multi-exponential decline with an average half-life of 7.22–9.26 hr in rats. The subcutaneous bioavailability of semaglutide was 76.65–82.85 %. The brain tissue to plasma partition coefficient (Kp) value of semaglutide was estimated as <0.01. Among the different regions of the brain, semaglutide concentrations were significantly higher in the hypothalamus. The analytical method and pharmacokinetic information may be helpful toward a better understanding of the effect of semaglutide in the brain and further development of GLP-1 analogs for various brain diseases.

Pharmacokinetics and brain distribution of the therapeutic peptide liraglutide by a novel LC–MS/MS analysis

Liraglutide is a glucagon-like peptide-1 (GLP-1) analog that has been utilized for the treatment of type 2 diabetes mellitus. Liraglutide at a higher dose also shows beneficial effects in weight loss, which prompted its widespread use as an anti-obesity drug. The potential of liraglutide to treat Alzheimer’s disease and cognitive impairment has also been suggested. Nevertheless, the pharmacokinetics of liraglutide, including its distribution to the brain, has not been fully characterized. Therefore, this study aimed to develop a simple and sensitive bioanalytical method using liquid chromatography–tandem mass spectrometry (LC–MS/MS) and determine the pharmacokinetics and brain distribution of liraglutide in rats. Liraglutide in the rat plasma and brain tissue homogenates was extracted by protein precipitation using methanol. A gradient elution profile was used for chromatographic separation with mobile phases comprising 0.3% formic acid in water and 0.3% formic acid in acetonitrile. The mass spectrometry was operated in the positive electrospray ionization with multiple reaction monitoring mode. The lower limit of quantification of the present LC–MS/MS was 1 ng/mL in the plasma and 2 ng/mL in the brain tissue. Following intravenous injection (0.05 mg/kg, n = 5), plasma concentrations of liraglutide decreased monoexponentially with an average half-life of 3.67 h. The estimated absolute bioavailability of liraglutide after subcutaneous injection was 13.16%. Brain distribution of liraglutide was not significant, with the tissue-to-plasma partition coefficient (Kp) of liraglutide less than 0.00031. However, the concentrations of liraglutide were significantly different in the different brain regions following IV injection. In the brain, liraglutide concentrations were the highest in the hypothalamus, followed by the cerebellum and cerebrum. The present LC–MS/MS assay and the pharmacokinetic results may be helpful to understand better the effect of liraglutide in the brain for further preclinical and clinical studies of liraglutide.

The validated method for the analysis of a mixture of psychotropic medicines olanzapine, clorazepate and escitalopram using a high-performance liquid chromatography method

It was established that self-poisoning with antipsychotics, anti-anxiety drugs and antidepressants is caused by consumption of the drug in higher doses than prescribed. So it is very important to determine fast the materials causing poisoning and use the method which would allow one to identify several substances during only one analysis. Reference and test solutions were prepared in ethanol. A qualitative and quantitative analysis was carried out on a Waters Alliance 2695 liquid chromatograph equipped with a Waters 996 PDA detector and an ACE C18 (250 mm × 4.6 mm × 5 μm) column. The mobile phase consisted of solvent trifluoroacetic acid (0.1 %) and acetonitrile. The linear gradient elution profile was used. For the validation process the following parameters were chosen: specificity, repeatability, precision and limits (LOD and LOQ). According to the study results, it can be concluded that the validated method for the qualitative and quantitative evaluation analysis of olanzapine, clorazepate and escitalopram was developed.

Study on pharmacokinetics of eight active compounds from Bufei-Huoxue Capsule based on UHPLC-MS/MS

Bufei-Huoxue Capsule (BFHX) was applied to treat chronic obstructive pulmonary disease (COPD) in China. It is composed of Astragali Radix, Paeoniae Radix Rubra, and Psoralea Fructus. A sensitive and reliable ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS/MS) method was developed and validated to quantify the eight main bioactive compounds (psoralen, isopsoralen, neobabaisoflavone, corylin, bavachin, astragaloside IV, ononin and formononetin) in rat plasma after oral administration of BFHX. Osthol was used as an internal standard (IS). Plasma samples were pretreated with methanol to precipitate protein. Chromatographic separation was accomplished using Hypersil GOLDTM C18 column (2.1 mm × 100 mm, 1.9 μm) with a gradient elution profile and a mobile phase consisting of (A) 0.1% formic acid in water and (B) acetonitrile and the flow rate was set at 0.2 mL/min. Multiple reaction monitoring (MRM) mode was applied to perform mass spectrometric analyses. All calibration curves were linear (r > 0.9908) in tested ranges. The intra- and inter-day accuracy and precisions of eight compounds at three different concentration levels were within the acceptable limits. The extraction recovery was within the range of 76.4 ∼ 105.2% and the matrix effects were within the range of 88.3 ∼ 115.0% (RSD ≤ 15.6%). The dilution effects were within the range of 90.2 ∼ 114.9%. These 8 compounds were stable under the tested conditions. So the developed method was valid to evaluate the pharmacokinetic study of eight bioactive compounds after oral administration of BFHX.

Development and validation of an LC-MS/MS method to quantify the bromodomain and extra-terminal (BET) inhibitor JQ1 in mouse plasma and brain microdialysate: Application to cerebral microdialysis study

JQ1, is a cell-permeable small-molecule inhibitor of bromodomain and extra-terminal protein (BET) function with reportedly good CNS penetration, however, unbound and pharmacologically active CNS JQ1 exposures have not been characterized. Additionally, no quantitative bioanalytical methods for JQ1 have been described in the literature to support the CNS penetration studies. In the present article, we discuss the development and validation of a sensitive and reliable liquid chromatography-tandem mass spectrometry (LC–MS/MS) quantitative methods to determine JQ1 in mouse plasma and brain microdialysate. JQ1 and the internal standard, dabrafenib (ISTD), were extracted from plasma and microdialysate samples using a simple solid phase extraction protocol performed on an Oasis HLB μElution plate. Chromatographic separation of JQ1 and ISTD was achieved on a reversed phase C12 analytical column with gradient elution profile of mobile phases (MP A: water containing 0.1 % formic acid and MP B: acetonitrile containing 0.1 % formic acid) at a flow rate of 0.6 mL/min. The mass spectrometric detection was performed in the positive MRM ion mode by monitoring the transitions 457.40 > 341.30 (JQ1) and 520.40 > 307.20 (ISTD). The calibration curves demonstrated good linearities over the concentration range of 5−1000 ng/mL for the mouse plasma method (r2 ≥ 0.99) and 0.5−500 ng/mL for the microdialysate method (r2 ≥ 0.99). The experimental limit of quantification obtained was 5 and 0.5 ng/mL for the mouse plasma and microdialysate method, respectively, with the coefficient of variation less than 10 % for the analyte peak area. All the other validation parameters, including intra-and inter-day accuracy and precision, matrix effect, selectivity, carryover effect, and stability, were within the USFDA bioanalytical guidelines acceptance limits. The LC–MS/MS method was successfully applied to a mouse pharmacokinetic and cerebral microdialysis study to characterize the unbound JQ1 exposure in brain extracellular fluid and plasma.

Lignin Fractionation in Segmented Continuous Flow.

Lignins were fractionated in a segmented continuous flow fractionation (SCFF) approach using isocratic or gradient elution profiles of different solvent systems at various flow rates and temperatures against adjustable pressure regimes. Superior control of parameters such as temperature and pressure in combination with the possibility of freely combinable solvent gradients allowed facile fractionation and generation of industrially interesting fractions differing in molecular weight properties and/or in physicochemical properties in a process that could be fully remotely controlled for automation and performance. Scale-up of the process was possible in linear and parallel mode. Analyses of the realised fractions by standardised gel permeation chromatography and 31 P NMR spectroscopy protocols showed that the SCFF of lignins delivered fractions that could be similar to conventional batch fractions, as well as novel/altered fractions available by applying overheated solvents in pressurised systems.

RP-HPLC method for simultaneous estimation of vigabatrin, gamma-aminobutyric acid and taurine in biological samples

Vigabatrin is used as first line drug in treatment of infantile spasms for its potential benefit overweighing risk of causing permanent peripheral visual field defects and retinal damage. Chronic administration of vigabatrin in rats has demonstrated these ocular events are result of GABA accumulation and depletion of taurine levels in retinal tissues. In vigabatrin clinical studies taurine plasma level is considered as biomarker for studying structure and function of retina. The analytical method is essential to monitor taurine levels along with vigabatrin and GABA. A RP-HPLC method has been developed and validated for simultaneous estimation of vigabatrin, GABA and taurine using surrogate matrix. Analytes were extracted from human plasma, rat plasma, retina and brain by simple protein precipitation method and derivatized by naphthalene 2, 3‑dicarboxaldehyde to produce stable fluorescent active isoindole derivatives. The chromatographic analysis was performed on Zorbax Eclipse AAA column using gradient elution profile and eluent was monitored using fluorescence detector. A linear plot of calibration curve was observed in concentration range of 64.6 to 6458, 51.5 to 5150 and 62.5 to 6258 ng/mL for vigabatrin, GABA and taurine, respectively with r2 ≥ 0.997 for all analytes. The method was successfully applied for estimating levels of vigabatrin and its modulator effect on GABA and taurine levels in rat plasma, brain and retinal tissue. This RP-HPLC method can be applied in clinical and preclinical studies to explore the effect of taurine deficiency and to investigate novel approaches for alleviating vigabatrin induced ocular toxicity.

Development and validation of an LC-MS/MS method for the simultaneous quantification of seven constituents in rat plasma and application in a pharmacokinetic study of the Zaoren Anshen prescription.

A sensitive, specific and accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of seven constituents of the Zaoren Anshen prescription (ZAP) in rat plasma after oral administration of the ZAP: spinosin, salvianic acid A, 6'''-feruloylspinosin, protocatechualdehyde, salvianolic acid B, schisandrin and deoxyschisandrin. The plasma samples and the internal standard (IS) sulfamethoxazole were extracted using acetonitrile. Chromatographic separation was performed with an Agilent HC-C18 column using a gradient elution profile and a mobile phase consisting of 0.01% formic acid in water (A) and acetonitrile (B). The analytes were quantified simultaneously in a single run using an ion trap mass spectrometer operated in the multiple reaction monitoring mode and electrospray ion-source polarity in the positive and negative modes. The calibration curves for spinosin, salvianic acid A, 6'''-feruloylspinosin, protocatechualdehyde, salvianolic acid B, schisandrin and deoxyschisandrin were linear over the concentration ranges of 2.90-1160, 2.50-1000, 1.80-720, 0.65-260, 2.50-1000, 8.00-1600 and 1.30-520 ng/mL, respectively. The intra- and inter-day precisions in terms of relative standard deviation were <18.9%, and the accuracies in terms of relative error were within ±14.2%. Consequently, the proposed method was successfully applied to the pharmacokinetic analysis of these seven major active compounds in rats administered ZAP. These results will facilitate research aiming to predict the effectiveness of the optimal dose of ZAP and might be beneficial for the therapeutic use of ZAP in the clinical setting.

The Development of a Rapid, Simple and Sensitive LC-MS/MS Method, to Guide Clinical Dosing, for the Analysis of 5-Fluorouracil in Human Plasma

Introduction: 5-Fluorouracil is a commonly prescribed antineoplastic drug. Historically, methods to determine this compound in biological fluids have been time consuming or not sensitive enough. Materials and Methods: In this report, a method using simple and rapid sample preparation with good sensitivity, precision and accuracy for the quantitative determination of 5-fluorouracil in human plasma by LC-MS/MS is established and validated. By the judicious combination of a small amount of injected sample, low flow rate, careful control of gradient elution profile, use of a HILIC pre-column to further remove residual phospholipids and an isotopically labelled internal standard (ILIS), the method has successfully overcome matrix effect with protein precipitation sample pre-treatment without evaporating to dryness and reconstituting process. Conclusion: This newly developed method can be used to measure patient plasma concentrations of 5- fluorouracil, as low as 0.8 ng/mL and as high as 100 µ g/mL, with less than 15% both intra- and interday accuracy and precision. Such a methodology will be very convenient of being used in routine therapeutic drug monitoring. Keywords: 5-Fluorouracil, LC-MS/MS, human plasma, protein precipitation, antineoplastic drug, sample preparation technique.

Computational analysis and ratiometric comparison approaches aimed to assist column selection in hydrophilic interaction liquid chromatography–tandem mass spectrometry targeted metabolomics

In the present work two different approaches, a semi-quantitative and a Derringer function approach, were developed to assist column selection for method development in targeted metabolomics. These approaches were applied in the performance assessment of three HILIC columns with different chemistries (an amide, a diol and a zwitterionic phase). This was the first step for the development of a HILIC UPLC–MS/MS method that should be capable to analyze a large number of polar metabolites. Two gradient elution profiles and two mobile phase pH values were tested for the analysis of multi-analyte mixtures. Acquired chromatographic data were firstly treated by a ratiometric, “semi-quantitative” approach which quantifies various overall analysis parameters (e.g. the percent of detected compounds, retentivity and resolved critical pairs). These parameters were used to assess chromatographic performance in a rather conventional/traditional and cumbersome/labor-intensive way. Secondly, a comprehensive and automated comparison of the three columns was performed by monitoring several well-known chromatographic parameters (peak width, resolution, tailing factor, etc.) using a lab-built programming script which calculates overall desirability utilizing Derringer functions. Derringer functions exhibit the advantage that column performance is ultimately expressed in an objective single and quantitative value which can be easily interpreted. In summary, results show that each column exhibits unique strengths in metabolic profiling of polar compounds. The applied methodology proved useful for the selection of the most effective chromatographic system during method development for LC–MS/MS targeted metabolomics, while it could further assist in the selection of chromatographic conditions for the development of multi-analyte methods.

Development and application of an analytical method for curdione quantification in pregnant Sprague-Dawley rats by LC-MS/MS.

The vaginal administration route suffers from relatively low absorption efficiency, which may hinder the identification of the toxicokinetics of curdione in pregnant women. A sensitive analytical method for determining the plasma concentration of curdione was developed and applied in the determination of curdione in pregnant Sprague-Dawley rats as a simulated model. Glimepiride was used as an internal standard and chromatographic separation was achieved on a Capcell Pak C18 MGIII column. A gradient elution profile with 0.5% formic acid (A)-0.5% formic acid-acetonitrile (B) was selected as mobile phase. The selected reaction monitoring mode was used for quantification based on the target fragment ions m/z 237.2 to m/z 135.1 for curdione and m/z 491.3 to m/z 352.1 for the glimepiride. The standard curve was linear over the range of 0.5-500 ng/mL for curdione in rat plasma and yielded a consistent peak pattern, even at the lower limit of quantitation of 0.5 ng/mL. The retention times of curdione and IS were 6.55 and 6.59 min, respectively. The mean recovery of curdione in rat plasma was 95.5-101.1%. The intra-day and inter-day precisions were between 2.35 and 9.08%. This LC-MS/MS method provides a simple and sensitive means for determining the plasma concentration.

Development of Gradient Retention Model in Ion Chromatography. Part I: Conventional QSRR Approach

New retention methodology that integrates the conventional quantitative structure-retention relationship (QSRR) approach and gradient retention modeling based on isocratic retention data is developed and presented in this paper. Such an integrated approach removes the general QSRR limitation of highly predefined application conditions (i.e., QSRR are generally applicable only under the conditions used during model development) and allows the prediction of retentions over a wide range of different elution conditions (practically for any isocratic or gradient elution profile). At the same time, it retains the ability to predict retention of components unknown to the model, i.e., the components that have not been used in modeling. Ion-exchange chromatography (IC) analysis of carbohydrates was selected as modeling environment. Three regression techniques were applied and compared during QSRR modeling, namely: stepwise multiple linear regression, partial least squares (PLS), and uninformative variable elimination–PLS regression. The obtained prediction results of the best QSRR model (root-mean-square error of prediction = 22.69 %) were similar to those found in the literature. The upgrade from QSRR to the integrated model did not diminish the predictive ability of the model, indicating an excellent potential of the developed methodology not only in IC but also in chromatography in general.