Ammonium Formate Buffer Research Articles

Safety and human health: The landscape of an effective UPLC-MS/MS method for the identification and quantification of N-Nitroso Hydrochlorothiazide impurity in Hydrochlorothiazide

N-nitrosamines (NSAs) and Nitrosamine Drug Substance Related Impurities (NDSRIs) are class of compounds that contains the nitroso functional group. These compounds are carcinogens and mutagens for both humans and animals. As a result, all global regulatory agenesis are requiring risk assessments of NSAs in each pharmaceutical product. The present research aims to provide a single pre-packaged mass spectrometry method parameter set, which includes Ultra performance liquid chromatography and a triple quadrupole mass spectrometer (UPLC-MS-MS) configuration technique for qualifying and quantifying NNHCT in HCT. The analysis was performed on a YMC ODS-A liquid chromatography column with an oven temperature of 35 °C. In this method, mobile phase was prepared by mixing 10 mM ammonium formate buffer and acetonitrile was mixed in the ratio of 60:40 (v/v) with an isocratic program and a flow rate of 0.8 mL/min. The mass detector was used along with electrospray ionization (ESI) Negative mode with multiple reaction monitoring (MRM). The method has been validated in accordance with the current regulatory guidelines and shown the ability to detect NNHCT with a LOQ of 0.01 ppm and LOD of 0.005 ppm.

Improved hydrophobic subtraction model of reversed-phase liquid chromatography selectivity based on a large dataset with a focus on isomer selectivity

Reversed-phase (RP) liquid chromatography is an important tool for the characterization of materials and products in the pharmaceutical industry. Method development is still challenging in this application space, particularly when dealing with closely-related compounds. Models of chromatographic selectivity are useful for predicting which columns out of the hundreds that are available are likely to have very similar, or different, selectivity for the application at hand. The hydrophobic subtraction model (HSM1) has been widely employed for this purpose; the column database for this model currently stands at 750 columns. In previous work we explored a refinement of the original HSM1 (HSM2) and found that increasing the size of the dataset used to train the model dramatically reduced the number of gross errors in predictions of selectivity made using the model. In this paper we describe further work in this direction (HSM3), this time based on a much larger solute set (1014 solute/stationary phase combinations) containing selectivities for compounds covering a broader range of physicochemical properties compared to HSM1. The molecular weight range was doubled, and the range of the logarithm of the octanol/water partition coefficients was increased slightly. The number of active pharmaceutical ingredients and related synthetic intermediates and impurities was increased from four to 28, and ten pairs of closely related structures (e.g., geometric and cis-/trans- isomers) were included. The HSM3 model is based on retention measurements for 75 compounds using 13 RP stationary phases and a mobile phase of 40/60 acetonitrile/25 mM ammonium formate buffer at pH 3.2. This data-driven model produced predictions of ln α (chromatographic selectivity using ethylbenzene as the reference compound) with average absolute errors of approximately 0.033, which corresponds to errors in α of about 3 %. In some cases, the prediction of the trans-/cis- selectivities for positional and geometric isomers was relatively accurate, and the driving forces for the observed selectivity could be inferred by examination of the relative magnitudes of the terms in the HSM3 model. For some geometric isomer pairs the interactions mainly responsible for the observed selectivities could not be rationalized due to large uncertainties for particular terms in the model. This suggests that more work is needed in the future to explore other HSM-type models and continue expanding the training dataset in order to continue improving the predictive accuracy of these models. Additionally, we release with this paper a much larger data set (43,329 total retention measurements) at multiple mobile phase compositions, to enable other researchers to pursue their own lines of inquiry related to RP selectivity.

Greener spectrophotometric and HPLC investigation of CNS agent pregabalin: Taguchi model and box-behnken design for method parameters optimization

In the current study, two analytical methods are developed using spectrophotometry and high performance liquid chromatography for the determination of pregabalin in pharmaceutical preparations. Box-Behnken Design model was adopted to ease the optimization process. The visible spectrophotometric method is based on the reaction of potassium ferricyanide and ferric chloride in an acidic medium and measured absorbed at 700 nm. The HPLC method utilizes ammonium formate buffer solution and RP18 base deactivated silica (BDS) column for the quantitative analysis of pregabalin. The calibration plot was found to be linear in the range of 1.5–26.2 and 0.4–17.6 µg/mL for spectrophotometric and HPLC methods. BBD is a powerful statistical experiment design for optimizing reaction conditions that minimize the number of experiments and the cost of the method. Response Surface Methodology (RSM) via Box–Behnken design (BBD) optimized the chromatographic procedure. The Greenness of the methods were also evaluated by considering the Analytical Greenness Metric Approach. Method I attained a score of 0.74, while method II scored 0.59, suggesting an excellent and considerable level of eco-friendliness. Moreover, it is economical, consumes a minimum amount of organic solvent, and has good accuracy and precision.

Validated LC/MS method for simultaneous determination of elbasvir and grazoprevir in human plasma

A sensitive and accurate LC/MS method for the determination of elbasvir (ELB) and grazoprevir (GZP) in human plasma was established using daclatasvir (DCT) as an internal standard. The analytes were separated on a Waters Spherisorb phenyl column (150 mm x 4.6 mm ID, 5 µm particle size) maintained at 40°C ± 2°C. Gradient elution, at a flow rate of 0.8 mL min-1, was used. The mobile phase consists of 90% of acetonitrile mixed to 10% of a 5 mM ammonium formate buffer (+ 0.1% v/v of trimethylamine, pH was adjusted to 3.2 by formic acid) as phase A and 10% of acetonitrile mixed to 90% of the same buffer as phase B. Liquid-liquid extraction with ethyl acetate solvent was used to recuperate compounds from plasma. The method was validated over a concentration range of 2 and 100 ng/mL for GZP and between 1 and 50 ng/mL for ELB. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels exhibited relative standard deviations (RSD) < 15%, and the accuracy values ranged from 94.2% to 107.8%. The robustness of the method was established using a two-level full factorial design.

A NEW RP-UPLC METHOD FOR THE SEPARATION AND SIMULTANEOUS QUANTIFICATION OF DORZOLAMIDE HCl AND TIMOLOL MALEATE

Objective: This investigation demonstrates a stability-indicating and reliable “reverse-phase ultra-performance liquid chromatography” method to simultaneously quantify timolol maleate and dorzolamide HCl in the pharmaceutical dosage form. Methods: Successful separation was accomplished using Phenyl column (100 mm x 2.1 mm, 1.7μm) with isocratic type of elution using mobile phase containing Acetonitrile+Ammonium Formate buffer (30:70), respectively with 0.2 ml/min flow rate. The wavelength sensor was attuned at 266 nm to quantify timolol maleate and dorzolamide HCl. Results: Dorzolamide HCl and timolol maleate peaks were eluted with fine resolution at retention times 0.7 min and 1.5 min, respectively. In the 55.75-334.5 μg/ml and 6.25-37.5 μg/ml concentration ranges for dorzolamide HCl and timolol maleate, the calibration graphs were linear, with regression coefficients of 0.99997 and 0.99991, respectively. The suggested ultra-performance liquid chromatography approach has been shown as sensitive, precise, robust, accurate, specific and stability, indicating through the resolution of dorzolamide HCl and timolol maleate from its degradation-based compounds. Conclusion: The established ultra-performance liquid chromatography technique was effectively extended to the evaluation of dorzolamide HCl and timolol Maleate in the pharmaceutical dosage form and the test results appeared satisfactory.

Development and Validation of an LC/MS Compatible, Stability indicating RP-UPLC Method for Faropenem

In this manuscript an ultrafast, stability-indicating RP-UPLC method with Waters Acquity UPLC having 2996 PDA detection was developed and validated for the quantification of faropenem in the bulk drug and its tablet formulation. The analysis was performed using End version C18 column powered with Software Empower 2 and a mobile phase of 10mM Ammonium formate buffer (pH=3.5): ACN in the ratio of 65: 35(v/v) in isocratic mode at a flow rate of 0.25mL/min at 313nm as the detection wavelength. The linearity range is 5–50μg/mL, and the linear regression analysis of the calibration plot showed a good linear relationship with R2 = 0.9999. The LOD and LOQ were 213 and 638ng/mL, respectively. The stress conditions include acid, alkali, hydrogen peroxide and UV light. The drug peak was well resolved from the degradation products’ peaks, and thereby, the proposed method can be used for the analysis of stability samples.

Bioanalytical method development and validation for quantification of amivantamab in rat plasma by LC-MS/MS

BackgroundAmivantamab is a monoclonal bispecific anti-EGFR-MET antibody used to treat non-small cell lung cancer. There were no published methods using a liquid chromatographic—tandem mass spectrometric approach to develop and validate a feasible, novel, and thoroughly validated method for quantifying amivantamab in rat plasma.ResultsThe liquid–liquid extraction method was used to extract the analyte from rat plasma. The analyte was separated using acetonitrile–ammonium formate buffer (40:60) as a mobile phase on waters, alliance e-2695 model high-pressure liquid chromatographic system having Agilent eclipse C18, 150 mm × 4.6 mm, 3.5 µm column. The overall runtime was 6 min at a 1.0 ml/min flow rate. The method showed significant sensitivity and acceptable linearity over the 5.00–100.00 ng/ml concentration range. Accuracy was proved by mean percent recovery ranging from 98.03 to 99.99%. The intraday precision coefficient of variation (%) ranged between 0.31 and 5.43. Also, the findings such as Cmax, tmax, AUC0− t, AUC0− ∞, and half-life values of amivantamab showed that the technique was helpful for pharmacokinetic studies.ConclusionsAll the validated parameters were found to be within the acceptable range. The validated method was found to be simple, accurate, precise, and reproducible and hence can be used for the routine analysis of amivantamab, such as in-process quality control by liquid chromatographic—tandem mass spectrometry.

A green perspective on simultaneous HPLC and UV spectrophotometric estimation of Udenafil and Dapoxetine hydrochloride in pharmaceutical formulations

Clinical research has affirmed that the combination of Udenafil and Dapoxetine hydrochloride yields greater improvement in erectile dysfunction compared to the use of individual medications. There is a requirement of precise quantitative method for accurate determination of Udenafil and Dapoxetine when taken together. Consequently, our interest lies in developing and validating analytical methods for the simultaneous estimation of Udenafil and Dapoxetine - Various strategies, such as UV spectrophotometric approaches and an innovative, straightforward, accurate, sensitive, and robust High-Performance Liquid Chromatography (HPLC) procedure, have been explored. An Isocratic HPLC approach was employed using reversed-phase CHEMSIL ODS C18 column (4.6 mm x 250 mm, 5 µm) as a stationary phase and detection wavelength (λ max) 230 nm for the two analytes in the chromatographic column. The mobile phase comprised of (70:30, v/v) mixture of Acetonitrile and 10 mM Ammonium formate buffer having pH 5, adjusted with formic acid. The elution was carried out at a constant flow rate of 1 mL/min. The UV simultaneous equation technique estimated the medicine's quantities and derived a simultaneous equation for DAP and UDN using 2 wavelengths, 230 nm and 298 nm. Furthermore, the two advanced methods underwent rigorous statistical validation, guided by the valuable ICH Q2R1 guidelines, affirming their effectiveness as a successful analytical tool for the concurrent analysis of both tablets in bulk and the synthetic mixture fabricated in the laboratory. To assess the environmental impact of contemporary UV spectrophotometric and HPLC techniques, the Green Analytical Greenness Metric (AGREE) software and the Green Analytical System Index (GAPI) were employed to evaluate the greenness profile. This examination confirmed the method's environmental friendliness concerning solvent usage, chemical ingredients, energy consumption, and waste production. The developed UV method is more sustainable and greener compare to HPLC method for simultaneous estimation of compound, which can be utilised by industry for quantification of each individual drug in their combination products.

Application of analytical quality by design (AQbD) for stability-indicating method development for quantification of nevirapine and its degradation products

Development of stability-indicating method by liquid chromatography are always a challenge for analytical researchers. A traditional way is by varying one chromatographic parameter at a time assessing the responses from each test. Recent international guidelines encourage the implementation of systematic approaches in analytical development, such as Analytical Quality by Design (AQbD). The aim of this paper was to develop, using the AQbD approach, an efficient and selective stability-indicating method by high performance liquid chromatography for nevirapine and its related substances. The Analytical Target Profile (ATP) and the Critical Method Attributes (CMA) were determined. Through the risk assessment studies, the high-risk analytical conditions were considered as Critical Method Parameters (CMP) and they were screened and optimized by Design of Experiment (DoE). The Method Operable Design Region (MODR) was defined using Monte Carlo Simulation. A control strategy was established by the definition of the system suitability of the analytical procedure. Optimal chromatographic conditions were achieved using an isocratic elution consisting of a mixture of 5 mM ammonium formate buffer pH 5.4/acetonitrile/methanol (60:20:20), with a flow rate of 1.0 mL/min, oven temperature of 30 °C, injection volume of 5 µL and an ACE C18 250 mm x 4,6 mm x 5 µm chromatographic column. Finally, analytical validation and forced degradation studies confirmed the performance of the analytical method. From the estimation of measurement uncertainty, strategies were outlined so that the method is fit for purpose throughout its life cycle. Additionally, guard bands were defined, changing the specification for nevirapine quantification from 90 % to 100 % to the acceptance zone of 92.1–107.9 %. The AQbD method development provided strong justifications for the selection of important parameters for nevirapine method during regulatory filling. Furthermore, with the pass/fail decision rule using guard band, restricted acceptance zones were defined, reducing the risk to the consumer of taking a drug product whose batch was inadequately approved.

Effect of Verapamil, a P-glycoprotein-1 and Cytochrome P450 3A4 Inhibitor, on Pharmacokinetics and Metabolic Stability of Ripretinib: A Drug-Drug Interaction Study in Rats.

Ripretinib was developed to target a whole range of KIT proto-oncogene mutations and platelet-derived growth factor receptor A (PDGFR-A) kinases found in certain cancers and myeloproliferative neoplasms, particularly gastrointestinal stromal tumours (GISTs). This study investigated the effect of verapamil, a potential inhibitor of P-glycoprotein-1 (P-gp1) and cytochrome P450 3A4 (CYP3A4), on the pharmacokinetics of ripretinib in rats when administered orally together. This study also assessed the metabolic stability and in vitro cellular absorption of ripretinib in the presence of verapamil. A novel sensitive time-saving liquid chromatography tandem mass spectometry (LC-MS/MS) technique for determining ripretinib in rat plasma was developed and validated. A Zorbax SB C18 column was used for the separation and analysis of ripretinib with a mobile phase consisting of 50:50 (%v/v) acetonitrile and 10mM ammonium formate buffer at a flow rate of 0.4 mL/min. Imatinib was used as an internal standard (IS) in the method. The pharmacokinetic characteristics of ripretinib were evaluated in Wistar rats by successfully administering an oral dosage of 5 mg/kg body weight of ripretinib in the presence of verapamil (10 mg/kg body weight). Subsequently, rat liver microsomes were used to assess the effect of verapamil on ripretinib metabolic stability, and absorption was tested using a Caco-2 cell transwell model. Ripretinib and IS were identified using multiple reaction monitoring (MRM) modes by mass spectrometry and showed ion transitions of 510.09→94.06m/z and 494.26→ 394.16m/z, respectively. The high-performance liquid chromatography (HPLC) method successfully eluted ripretinib and IS at retention times of 0.91 and 0.68 min, respectively, and the method was validated for all parameters and met the criteria for acceptance. Co-administration of verapamil increased the maximum concentration (Cmax) of ripretinib from 437±84ng/mL to 492±50ng/mL (12%), and the area under the concentration-time curve from 0 to the last sampling time t (AUC0-t) increased by approximately 40.6%. Verapamil significantly reduced the basolateral-to-apical transfer of ripretinib through Caco-2 cells. Findings also showed that verapamil increased the metabolic stability of ripretinib. The study results indicate that the co-administration of ripretinib with CYP3A4 and/or P-gp1 inhibitors is associated with significant drug-drug interactions that affect the pharmacokinetics of ripretinib. Further research in human subjects is suggested to confirm dosage adjustment and therapeutic drug monitoring of ripretinib when administered along with P-gp1/CYP3A4 inhibitors ensuring patient safety and optimizing the therapeutic benefits of ripretinib.

B-202 Improving Performance of an LC-MS C-peptide Reference Method

Abstract Background C-peptide is a useful biomarker for distinguishing among different types of diabetes and is a proxy measure of endogenous insulin production, useful in diabetes management. Additionally, C-peptide tests can be used to diagnose non-diabetic hypoglycemia and insulinoma. However, differences among clinical assays can limit the application of C-peptide testing. To address this issue, efforts are underway to standardize manufacturer assays with serum-based secondary reference materials where values are assigned by a reference LC-MS method. Clinical evidence suggests that patients with diabetes can benefit from residual beta-cell function, yet many clinical assays have high variability at lower levels of C-peptide. Here we explore different approaches for improving sensitivity and selectivity of C-peptide reference measurements with a focus at the lower end of the measurement range. Methods Two methods were developed to evaluate and determine the most effective approach. The first method involved spiking serum samples with isotope-labeled c-peptide (Sigma) and then enriching and enzymatically digesting them. The enrichment process included removing abundant serum proteins by using methanol, followed by centrifugation and filtration through C18 cartridges. The resulting solution was then passed through a strong anion-exchange column after correcting its pH with ammonium formate buffer. The eluent was evaporated, and the residue was dissolved in ammonium carbonate buffer. Enzymatic digestion was performed by adding dithiothreitol and iodoacetamide followed by addition of Glu-C. The digested peptides were then analyzed by LC-MS/MS (QTRAP 6500+) in MRM mode. The second method followed the same steps as the first method, except that the evaporation and digestion step were skipped, and MRM was performed on the intact C-peptide chain. Two separate calibration curves were constructed for each method using isotope labeled c-peptide (Sigma) and certified reference material (National Metrology Institute of Japan) as native c-peptide. Results The first method, which measures intact C-peptide using MRM, had a slope of 1.14 and an intercept of −0.01 nmol/L (Deming regression, R = 0.917) when compared to the reference method. The method demonstrated CV within 0.4%–11.9%. The second method, which quantifies digested peptides, showed a slope of 1.02 and an intercept of −0.21 nmol/L compared to the reference method (Deming regression, R = 0.995). The method demonstrated CV within 4.4–20.0%. Controlling the three MRM transitions improved the specificity and accuracy of the results. For the first method two MRM transitions were adequate. Both methods showed no significant matrix effects. The first method is simpler to implement due to a smaller number of separation steps. Conclusion We have explored two new alternative workflows for C-peptide quantitation that include first, an enzymatic digestion then MRM quantitation and second, MRM quantitation of intact C-peptide. To improve the analytical performance of the methods, further optimization is still required.

Validation of an LC-MS/MS assay for rapid and simultaneous quantification of 21 kinase inhibitors in human plasma and serum for therapeutic drug monitoring

Kinase inhibitors have revolutionized cancer treatment in the past 25 years and currently form the cornerstone of many treatments. Due to the increasing evidence for therapeutic drug monitoring (TDM) of kinase inhibitors, the need is growing for new assays to rapidly evaluate kinase inhibitor plasma concentrations. In this study, we developed an LC-MS/MS assay for the rapid and simultaneous quantification of 21 kinase inhibitors. First, a literature search was conducted to ensure that the linear ranges of the analytes were in line with the reported therapeutic windows and/or TDM reference values. Subsequently, the assay was validated according to FDA and EMA guidelines for linearity, selectivity, carry-over, accuracy, precision, dilution integrity, matrix effect, recovery, and stability. The assay was fast, with a short run-time of 2 min per sample. Sample pre-treatment consisted of protein precipitation with methanol enriched with stable isotope-labeled internal standards (SIL-IS), and the mixture was vortexed and centrifuged before sample injection. Separation was achieved using a C18 column (3 μm,50 × 2.1 mm) with a gradient of two mobile phases (ammonium formate buffer pH 3.5 and acetonitrile). Analyte detection was conducted in positive ionization mode using selected reaction monitoring. The assay was accurate and precise in plasma as well as in serum. Extraction recovery ranged between 95.0% and 106.0%, and the matrix effect was 95.7%-105.2%. The stability of the analytes varied at room temperature and in refrigerated conditions. However, all drugs were found to be stable for 7 days in the autosampler. The clinical applicability of the analytical method (486 analyzed samples between 1 July 2022–1 July 2023) as well as external quality control testing results were evaluated. Taken together, the results demonstrate that the analytical method was validated and applicable for routine analyses in clinical practice.

Development of an Analytical Method for the Determination of Cymoxanil in Potato Flour Samples by High Performance Liquid Chromatography

In this study, a high performance liquid chromatography-ultraviolet detection method was introduced for the detection of cymoxanil. The experimental parameters of the procedure were thoroughly evaluated. Cymoxanil was eluted by ammonium formate buffer (50.0 mM, pH 4.0):acetonitrile (55:45 v/v) as mobile phase. Under the optimal conditions, the limits of detection and quantification (LOD and LOQ) were found as 7.4 – 24.8 µg kg-1, which allows trace determination of pesticides in food samples. Proposed method was then implemented for the determination of selected fungicide in potato flour samples with acceptable % recoveries in the range of 81.4–112.9%, while the values of relative standard deviation (RSD) were below 10% showing a satisfactory applicability for cymoxanil in such complex real samples matrix.

Development and validation of HPLC-MS/MS method for simultaneous analysis of B vitamins present naturally or after fortification in fruit juices

In this study, a multivitamin analysis method with a run time of 20 min was developed for nine major B vitamins in fruit juices using HPLC coupled with a triple quadrupole mass spectrometer. A reverse phase column with ammonium formate buffers in methanol and water as mobile phases under gradient elution mode were used to accomplish chromatographic separation. A simple dilute and shoot method was used for sample preparation. A calibration and accuracy study for method validation was conducted. The accuracy profiles were created using measurement uncertainty estimated from the validation data according to the ISO/DTS 21748. The proportional bias due to the matrix effect were ranging between 80% and 115% in the matrices investigated in this study. The measurement uncertainty and the corresponding coefficient of variation were within the acceptable limits defined in the literature and international regulatory standards i.e. EU regulation. The limits of quantitation were low enough to prove fitness for purpose for all vitamins except cyanocobalamin. The study concludes with a method with the potential to be used in quality control laboratories for high throughput routine vitamin analysis.

A stability indicating UPLC method development and validation for the simultaneous estimation of nateglinide and metformin hydrochloride in bulk and tablet dosage form

BackgroundNateglinide and metformin HCl are used in combination for the treatment of type 2 diabetes. A simple, sensitive and reliable UPLC method was developed for simultaneous estimation of nateglinide and metformin HCl using Phenomenox C18 (50*2.1 mm, 3.5 µm) column at ambient temperature as stationary phase in addition to mobile phase containing 75 volumes of ammonium formate buffer (pH = 3) along with 25 volumes of acetonitrile with a flow rate of 0.2 mL/min with UV detection at 260 nm and a run time of 3 min. The developed method was validated as per ICH Q2(R1) guidelines.ResultsThe separation of metformin HCl and nateglinide was done at retention times of 1.014 min and 1.435 min, respectively. The mean % recovery for nateglinide and metformin HCl in the accuracy study was observed to be 99.9% and 99.2%, respectively. LOD and LOQ values were determined considering the S/N ratio and were found to be 0.09 µg/mL and 0.3 µg/mL, respectively, for nateglinide and 0.75 µg/mL and 2.5 µg/mL, respectively, for metformin HCl. The method was found to be precise with % RSD values of 0.58 and 0.45, respectively, for repeatability and intermediate precision of nateglinide and 0.43 and 0.43, respectively, for repeatability and intermediate precision of metformin HCl which were within acceptance criteria. The method was found to be linear in the range of 7.5–45 µg/mL and 62.5–375 μg/mL for nateglinide and metformin HCl, respectively. The regression equations for nateglinide and metformin HCl were found to be y = 17377x + 6543.4 and y = 18439x + 43,537, respectively. The method was found to be robust by deliberate changes in the method parameters like flow rate and mobile phase composition. Forced degradation studies were performed as per ICH Q1A(R2) and Q1B guidelines, and peak purity was observed in all types of degradation studies for both the drugs.ConclusionThe developed method was found to be satisfactory as it is simple, sensitive, accurate, precise, robust, rapid, economical and yet stability indicating and can be applied successfully in the routine laboratory analysis for the simultaneous estimation of nateglinide and metformin HCl in the bulk and pharmaceutical dosage forms.

Quantifying Pomalidomide in Human Plasma Employing UPLC-MS/MS

The current work aims to create a simple, fast, clear and accurate chromatography-tandem mass spectrophotometric (UPLC-MS/MS). 50µl ISTD (250ng/ml concentration) and 250µl of 5mM ammonium formate buffer (pH - 2.5) were put into the samples just before liquid-liquid extraction (LLE) using 2.5ml of tertiary butyl methyl ether (TBME). Chromatographic fraction was done using Hypersil gold column (50 mmX4.65mm, 5µ) with use of 5mM ammonium formate buffer (pH - 2.5) and acetone in 20: 80 v/v (mobile phase). The rate of flow was around 0.50ml/min. Pomalidomide and Celecoxib were ionized into ions with polarity (positive) and were recognized using MRM. Quantitation was made by transitions m/z 274.43 precursor ion to m/z 201.15 for drug Pomalidomide and m/z 382.12 precursor ion to m/z 362.0 of celecoxib. The concentrations of eight standards exhibited linearity between 1.9900 to 199.8380ng/ml (r2 ≥ 0.9968). Separation of chromatogram was obtained in two minutes. The recovery values in an average for three QC’s was 51.49% of Pomalidomide which was in concordance with acceptance. The Coefficient of variation was ≤15% in intra and inter batch assay values. The %CV ruggedness ranges from 1.26 - 2.25. Short and long term stability percentages of stock solutions were found acceptable like 99.76 and 98.69. Results show that all the validation parameters were in acceptable limits.

Simultaneous Determination of Major Molnupiravir Metabolite (β-D-N4-hydroxycytidine) and Favipiravir in Human Plasma by HPLC-MS/MS

Introduction. Coronavirus disease 2019 (COVID-19) is an infectious disease caused by the SARS-CoV-2 virus (severe acute respiratory syndrome-related coronavirus 2). COVID-19 is now expected to stay with us for many years as a recurring disease. Molnupiravir and favipiravir are oral antiviral drugs with anti-RNA polymerase activity. The Russian Health Ministry has approved molnupiravir and favipiravir for the treatment of COVID-19. The study describes development and validation of high-performance liquid chromatography – tandem mass spectrometry (HPLC-MS/MS) method for the simultaneous determination of β-D-N4-Hydroxycytidine and favipiravir in human blood plasma. The method could be applied in pharmacokinetic study of molnupiravir and favipiravir.Aim. The aim of this study is to develop and validate a HPLC-MS/MS bioanalytical method for the determination of β-D-N4-Hydroxycytidine and favipiravir in human plasma.Materials and methods. The determination of β-D-N4-Hydroxycytidine and favipiravir in human plasma by HPLC-MS/MS. The samples were processed by 0.1 % formic acid in acetonitrile. Internal standard: promethazine. Mobile phase: 0.01 mol/L Ammonium formate buffer solution (Eluent A), 0.1 % formic acid and 0.08 % aqueous ammonia in water/acetonitrile 10 : 90 (Eluent B). Column: Shim-pack GWS C18, 150 × 4.6 mm, 5 μm. Analytical range: 50.00–10000.00 ng/mL for β-D-N4-Hydroxycytidine, 250.00–20000.00 ng/mL for favipiravir in human plasma. Ionization source: electrospray ionization. Detection conditions: 260.00 m/z → 82.10 m/z, 260.00 m/z → 111.00 m/z, 260.00 m/z → 127.95 m/z (β-D-N4-Hydroxycytidine); 156.15 m/z → 65.95 m/z, 156.15 m/z → 85.00 m/z, 156.15 m/z → 113.10 m/z (favipiravir); 285.05 m/z → 198.05 m/z (promethazine).Results and discussion. This method was validated by selectivity, suitability of reference standard, matrix effect, calibration curve, accuracy, precision, spike recovery, the lower limit of quantification, carry-over effect and stability.Conclusion. The HPLC-MS/MS method for quantitative determination of β-D-N4-Hydroxycytidine and favipiravir in human plasma was developed and validated. The analytical range was 50.00–10000.00 ng/mL for β-D-N4-Hydroxycytidine, 250.00–20000.00 ng/mL for favipiravir in human plasma. This method was applied to investigate the pharmacokinetics of molnupiravir and favipiravir.

Analysis of teicoplanin impurities by two-dimensional ultra performance liquid chromatography-quadrupole/time-of-flight mass spectrometry

A two-dimensional ultra performance liquid chromatography-quadrupole/time-of-flight mass spectrometry （2D-UPLC-Q/TOF-MS） method was established for the separation and structural analysis of the components in teicoplanin. This method effectively solved the problems associated with chromatographic systems， such as liquid chromatography-mass spectrometry （LC-MS）， which used a non-volatile phosphate buffer as the mobile phase and were not suitable for the rapid identification of impurities. Moreover， this method circumvented the complexities associated with locating and identifying impurities using the original method by re-establishing a chromatographic system suitable for LC-MS. In this study， for one-dimensional （1D） chromatography， the chromatographic separation was performed on an Octadecyl silica （ODS） hypersil column （250 mm×4.6 mm， 5 μm） with gradient elution using 3.0 g/L sodium dihydrogen phosphate buffer （pH 6.0）/acetonitrile=9/1 （v/v） as mobile phase A and 3.0 g/L sodium dihydrogen phosphate buffer （pH 6.0）/acetonitrile=3/7 （v/v） as mobile phase B. The column temperature was maintained at 30 ℃ and an ultraviolet detector was used at 254 nm for analysis. For 2D chromatography， desalting was performed on a Waters ACQUITY UPLC BEH C18 column （50 mm×2.1 mm， 1.7 μm） with gradient elution using ammonium formate buffer （pH 6.0） and acetonitrile as the mobile phases. The column temperature was maintained at 45 ℃. The MS data for the components and impurities were collected by positive ion electrospray ionization （ESI） using the full-information tandem MS mode （MSE）. The cone and nebulizer gas flow rates were set at 50 and 900 L/h， respectively. The ion source and nebulizer gas temperatures were set at 120 ℃ and 500 ℃， respectively. The ESI and cone needle voltages were set at 2500 and 60 V， respectively. The collision energy was set at 20-50 eV. The molecular formulas of the components and impurities were determined using their exact masses and isotope distributions， and the structural components and impurities of teicoplanin were deduced from their fragment ions according to the fragmentation pathway of the TA2-2 component. Moreover， the 10 components reported in the European Pharmacopoeia 10.0 were analyzed and 22 impurities of teicoplanin were identified by 2D-UPLC-Q/TOF-MS. Three new impurities and two characteristic fragment ions of the teicoplanin parent nucleus were detected， and the fragmentation pathway of TA2-2 was deduced. Using this method， 1D-UPLC is applicable for the accurate qualification of components based on relative retention times， and 2D-UPLC-Q/TOF-MS is suitable for the rapid identification of the structure of components based on their fragment ions. The results indicate that 2D-UPLC-Q/TOF-MS may be used to analyze the structure of impurities in teicoplanin based on their exact masses， isotope distributions， and fragment ions. The method is rapid， simple， and sensitive， which provides a novel strategy for the quality control and process optimization of teicoplanin.

HPLC with Fluorescence and Photodiode Array Detection for Quantifying Capmatinib in Biological Samples: Application to In Vivo and In Vitro Studies

Capmatinib, a recently approved tyrosine kinase inhibitor, is used for the treatment of non-small cell lung cancer. We describe two new HPLC methods for capmatinib quantification in vivo and in vitro. HPLC with a fluorescence detection method was used to quantify capmatinib in plasma for the first time. The method was successfully applied in a pharmacokinetic study following a 10 mg/kg oral dose of capmatinib given to rats. The chromatographic separation was performed using a Eurospher II 100-3 C18H (50 × 4 mm, 3 µm) column and a mobile phase containing 10 mM of ammonium acetate buffer (pH 5.5): acetonitrile (70:30, v/v), at a flow rate of 2.0 mL min-1. The study also describes the use of HPLC-PDA for the first time for the determination of capmatinib in human liver microsomes and describes its application to study its metabolic stability in vitro. Our results were in agreement with those reported using LC-MS/MS, demonstrating the reliability of the method. The study utilized a Gemini-NX C18 column and a mobile phase containing methanol: 20 mM ammonium formate buffer pH 3.5 (53:47, v/v), delivered at a flow rate of 1.1 mL min-1. These methods are suitable for supporting pharmacokinetic studies, particularly in bioanalytical labs lacking LC-MS/MS capabilities.

Accurate Identification of Hemoglobin Variants By Top-Down Protein Analysis Using Capillary Electrophoresis-High-Resolution Mass Spectrometry

Introduction Structural characterization of hemoglobin (Hb) variants, particularly the mutant forms of α- and β-subunits, is of significant value in the clinical diagnosis of hemoglobinopathy. The conventional methods for identification of Hb variants in clinical laboratories can be inadequate due to the lack of detailed structural characterization when it goes to the analysis of those Hb variants with similar sizes and charge states. High-resolution mass spectrometry (HR-MS) has been a central technology for structural characterization of proteins. As a novel approach for HR-MS protein analysis, top-down workflow analyzes proteins in intact state without prior enzymatic digestion, and it is capable of identifying unique proteoforms. Capillary electrophoresis (CE), as a powerful separation technology for proteins, has demonstrated excellent separation efficiency in the analysis of intact Hb forms and Hb subunits. CE can be coupled with HR-MS to form a CE-HR-MS system. By this means the CE separation is able to enhance the analytical power of HR-MS to allow for superior analytical performance in Hb analysis. In addition, when neutral coating of capillary is employed to suppress electroosmotic flow, CE separates analytes only by their individual electrophoretic mobilities, which enhances separation efficiency by maximizing the electrophoretic difference between the analytes. Thus, we established a neutral-coating CE-HR-MS method for accurate identification of Hb variants with top-down protein analysis strategy. Method An Orbitrap Q-Exactive Plus mass spectrometer was coupled with an ECE-001 CE unit through an EMASS-II ion source. A PS1 neutral-coating capillary was used for CE separation. Samples of red blood cells were lysed in water and diluted in 10 mM ammonium formate buffer for analysis. Results In the neutral-coating CE, since denaturing conditions were used, intact Hb forms were dissociated in background electrolyte, and baseline separation of individual Hb subunits was observed. Hb subunits in ion electropherograms followed the order of α-, β-, δ-, γ(1)-, γ2- subunit. The identification of Hb variants using CE-HR-MS is a two-step process: (1) intact-protein analysis that preliminarily identifies Hb subunits by precursor ions, and (2) top-down analysis that characterizes the primary structures and confirms the identification of Hb subunits by fragments produced from particular precursor ions. In intact-protein analysis, multiple charge states of each Hb subunit were observed in the mass spectra. At each charge state of a Hb subunit, a cluster of isotopic MS peaks were observed, corresponding to the isotopes of the molecule. All the MS peaks in a mass spectrum can be deconvoluted and those resulted from one analyte can be merged to display a single MS peak at its accurate monoisotopic mass, which can be matched to the theoretical mass of a known Hb subunit or variant. In top-down protein analysis, fragments from a precursor ion were acquired after HCD fragmentation. The MS peaks can be deconvoluted and those resulted from one fragment can be merged to display a single MS peak at its monoisotopic mass. The monoisotopic masses of fragments can be used to characterize the primary structure of the analyte by matching them to the theoretical masses of possible fragments from the structure of a known Hb subunit. The CE-HR-MS method was applied to the analysis of normal Hb forms as well as Hb variants from adults and neonates. The structures of Hb subunit variants β-S, β-C, β-E, β-Riyadh, β-Koln, β-New York, β-G-Accra, β-G-Siriraj, β-Khartoum, β-D-Punjab, β-D-Iran, β-Deer Lodge, α-Tarrant, γ1-Kuala Lumpur, and P-Nilotic β-δ hybrid have been successfully identified and characterized with >30% amino acid residue coverage and >95% matched fragment coverage. As an example, the CE-HR-MS result for the identification of a Hb γ1/γ1-Kuala Lumpur is shown, with intact-protein analysis in Figure 1A and top-down analysis in Figure 1B. Conclusion We have utilized the neutral-coating CE-HR-MS method for top-down structural characterization of hemoglobin variants. With the use of CE, only a simple dilute-and-shoot sample preparation procedure is required. Baseline separation of Hb subunits can be achieved to enhance HR-MS data quality. With these advantages, the CE-HR-MS method is compatible with clinical laboratories and can potentially be used in routine clinical testing. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal