Tandem Mass Spectrometric Method Research Articles

Rapid and sensitive liquid chromatographic–tandem mass spectrometric methods for the quantitation of dolutegravir in human plasma and breast milk

BackgroundDolutegravir (DTG) is part of a first-line antiretroviral therapy (ART) for HIV management in drug-naïve individuals and is recommended for the treatment of HIV during pregnancy. Robust analytical tools to quantify DTG are necessary to support clinical trials that characterize its multi-compartment drug distribution. MethodsPotassium EDTA (K2EDTA) plasma or whole breast milk was spiked with DTG and an isotopically labeled internal standard. Samples were prepared via protein precipitation prior to LC–MS/MS analysis. The assays were validated in accordance with regulatory recommendations. ResultsAnalytical measuring ranges for DTG quantitation in plasma and breast milk were 100–10,000 ng/mL and 0.500 to 1000 ng/mL, respectively. Inter-assay precision and accuracy were 2.73 % to 3.41 % and −10.6 % to −5.37 % for plasma, and 4.24 % to 12.4 % and −5.63 % to 7.49 % for breast milk, respectively. DTG was stable for three freeze–thaw cycles and for at least 72 h at room temperature in matrix (plasma or breast milk). Additionally, whole blood was stable for 24 h at room temperature and 2 h under conditions of extended heat and humidity. Matrix effects for DTG in plasma and breast milk ranged from 101 % to 108 % and 78.2 % to 99.3 %, respectively. Quantitation in remnant plasma samples yielded measurable concentrations within the primary linearity of the assay. ConclusionsMethods to quantify DTG in human plasma and breast milk have been developed and validated. These assays were designed to satisfy all criteria for implementation in clinical and clinical trial settings.

Pharmacokinetic study of iptacopan and its two acyl glucuronide metabolites in monkey plasma by liquid chromatography combined with electrospray ionization tandem mass spectrometry.

In this study, a simple and sensitive liquid chromatography tandem mass spectrometric method was developed and validated for the determination of iptacopan and two acyl glucuronidation metabolites in monkey plasma. The plasma sample was precipitated with acetonitrile and then separated on an Acquity UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm) using 0.1% formic acid and 5 mM ammonium acetate in water and acetonitrile as the mobile phase. The mass spectrometry (MS) detection was performed in positive multiple reactions monitoring (MRM) mode with precursor-to-production transitions. The developed assay was validated over the range of 1-2000 ng/mL for three analytes with correlation coefficient (r) more than 0.99. The validation parameters including accuracy, precision, carryover effect, matrix effect, recovery, and stability were all within the acceptable limits. The validated method has been applied to investigate the pharmacokinetics of iptacopan and its two acyl glucuronidation metabolites in monkey plasma. After intravenous administration, iptacopan showed low clearance (2.75 mL/min/kg) in monkey plasma. After oral administration, the bioavailability was 55.43%. The exposure (AUC0-t) of direct acyl glucuronide (AG) of iptacopan accounts for 9.73% of the iptacopan plasma exposure. The AUC0-t of AG of dealkylated metabolite of iptacopan was present at a lower level, accounting for 0.5% of the iptacopan plasma exposure.

Quantification of 700 mycotoxins and other secondary metabolites of fungi and plants in grain products

This work reports on the validation of a liquid chromatography–tandem mass spectrometric method for the simultaneous quantification of more than 700 mycotoxins and other secondary fungal metabolites and plant toxins in pasta, biscuits, crackers and musli. The “dilute and shoot” approach was found to be fully applicable to these complex matrices, as only 7–14% of the analytes exhibited significant matrix effects while recoveries of the extraction were outside the target range of 70–120% for only 26 compounds. Data on repeatability (based on 7 brands per matrix) and on intermediate precision was compliant to the related < 20% criterion for 95–98% and 99% of all analytes, respectively. The limits of quantification were much lower than the related regulatory limits set for mycotoxins in cereal products. Application of the method to 157 samples from the European market revealed the presence of enniatins and deoxynivalenol in the majority of the samples. No regulatory limits were exceeded except the sum of ergot alkaloids being higher in a few samples than the 50–150 µg/kg to be implemented as of July 2024.

A multianalyte LC-MS/MS method for accurate quantification of Nitrosamines in Olmesartan tablets

This research summaries the development, optimization and validation of liquid chromatography tandem mass spectrometric (LC-MS/MS) method for concurrent measurement of seven nitrosamines viz; NDMA, NDEA, NDIPA, NDPA, NEIPA, NMPA & NMBA in Olmesartan tablet. Controlling these nitrosamines at trace levels is imperative for ensuring the safety of drug substances and products for consumption. Various regulatory authorities stress the significance of utilizing highly sensitive analytical methods to precisely measure nitrosamines at trace levels. The method applied effective chromatographic separation and optimized parameters for mass spectrometric detection. Detection was carried out using APCI positive ion mode. Chromatographic separation was achieved using a Thermo Accucore PFP column (150 mm x 4.6 mm, 2.6 µ), with a simple gradient elution of mobile phase consisting of 0.1 % formic acid in water (mobile phase A) and methanol (mobile phase B). The total run time was 20 min, with a flow rate of 0.800 mL/min.The method was validated according to the International Council on Harmonisation (ICH Q2 (R2)) guidelines. The established method demonstrated excellent linearity (R2> 0.99) and sensitivity for all the nitrosamines. Detection and quantification limits were sufficiently low for trace nitrosamine levels having good S/N ratio. The method showed good accuracy in Olmesartan tablet samples, with recoveries ranges between 80 % to 120 %. The new analytical approach has exceptional repeatability and reliability, making it possible to precisely quantify the levels of seven nitrosamines in Olmesartan medoxomil tablets in a single analytical run.

Development of UPLC–MS/MS method for the simultaneous quantification of valproic acid and phenytoin in human plasma and application to study pharmacokinetic interaction in epilepsy patients

Valproic acid and phenytoin are two prevalent antiepileptic medications known for their narrow indices and propensity for cardiovascular and respiratory system toxicity. Therefore, therapeutic drug monitoring (TDM) of valproic acid (VAL) and phenytoin (PHE) concentrations in patient plasma is extremely beneficial for improving clinical choices, avoiding adverse reactions, and optimizing treatment for individual patients. In this study, a rapid and sensitive ultra-performance liquid chromatographic tandem mass spectrometer (UPLC-MS/MS) method was developed and validated for the simultaneous quantitative determination of valproic acid (VAL) and phenytoin (PHE) in human plasma. Negative electron spray ionization (ESI-) mode with selective ion recording (SIR) was employed to determine the transitions of m/z 142.98 and m/z 250.93 for VAL and PHE, respectively. The internal standard (IS) betamethasone (BETA) was ionized using positive electron spray ionization (ESI+) and detected by multi-reaction monitoring (MRM) mode to obtain precursor ions and specific fragment ions for quantification, and the MRM transition was chosen to be m/z 393.17 → 355.16. The separation was performed using a Phenomenex Synergi Hydro-RP (4 μm, 250 × 4.6 mm, I.D.) with an isocratic mobile phase consisting of acetonitrile – water (75:25, v/v) at a flow rate of 0.8 mL/min. The column temperature was maintained at 25 °C. The lower limit of quantification of VAL and PHE was 3.6 μg/mL and 0.72 μg/mL, respectively, which resulted in a recovery of more than 85 % for most analytes. According to US-FDA bioanalytical technique validation, the specificity, intra- and inter-day precision and accuracy, matrix effect, carryover, dilution, and stability of all analytes were within acceptable ranges. This analytical method was successful in evaluating the levels of valproic acid and phenytoin in human plasma from epileptic patients.

Exploration of the anthocyanin and proanthocyanidin profile of Greek red grape skins belonging to Vradiano, Limnio, and Kotsifali cultivars, analyzed by a novel LC‐QTOF‐MS/MS method

AbstractIntroductionWinegrape varieties Kotsifali, Limnio, and VradianoObjectiveThe aim of this study was to develop a liquid chromatographic quadrupole time‐of‐flight tandem mass spectrometric (LC‐QTOF‐MS/MS) method for the investigation of the anthocyanin and proanthocyanidin content of Greek grape varieties employing target and suspect screening strategies.MethodologyA novel LC‐QTOF‐MS/MS method was developed and validated to assess the anthocyanin content of Kotsifali, Limnio, and Vradiano grape varieties. Sixteen grape samples were collected from the main growing areas of each variety in Greece. The influence of the grape variety on the anthocyanin and proanthocyanidin composition of three Greek winegrapes was investigated using chemometrics.ResultsExcellent linearity (R2 &gt; 0.99) was achieved for all the target analytes, and recoveries ranged between 90.1% and 119.1%. The limits of quantification (LOQs) and limits of detection (LODs) were calculated over the range of 0.020–0.40 mg/g and 0.010–0.13 mg/g, respectively. The RSD% was lower than 9.1% and 7.3% for intra‐day and inter‐day studies, respectively, indicating satisfactory trueness and precision. Target and suspect screening resulted in the identification of 5 and 26 anthocyanins, respectively.ConclusionsKotsifali variety exhibited a higher concentration of anthocyanins compared with Vradiano and Limnio. Higher levels of mean degree of polymerization (mDp) and different percentage levels of prodelphinidins (%P) were established among the varieties.

Serum hydroxycotinine was associated with chronic kidney disease (CKD): a cross-sectional study based on NHANES

Objective Smoking has been suggested as a modifiable and cardiovascular risk factor for chronic kidney disease (CKD). Although long-term smoking has been associated with CKD, the potential relationship between its metabolite hydroxycotinine and CKD has not been clarified. Methods A total of 8,544 participants aged 20 years and above from the National Health and Nutrition Examination Survey (NHANES) 2017 - March 2020 were enrolled in our study. CKD was defined by estimated glomerular filtration rate (eGFR) < 60 mL/(min*1.73 m2). Serum hydroxycotinine was measured by an isotope-dilution high-performance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometric (ID HPLC-APCI MS/MS) method with a lower limit of detections (LLOD) at 0.015 ng/mL. The non-linear relationship was explored with restricted cubic splines (RCS). Pearson’s correlation coefficient and a multivariate logistic regression model were used for correlation analysis. Results Serum hydroxycotinine and eGFR were negatively correlated in both non-CKD group (r= −0.05, p < 0.001) and CKD group (r= −0.04, p < 0.001). After serum hydoxycotinine dichotominzed with LLOD, serum hydroxycotinine ≥ 0.015 ng/mL was negatively correlated with eGFR not only in non-CKD group (r = −0.05, p < 0.001) but also in CKD group (r = −0.09, p < 0.001). After adjusting for comprehensive confounders, results from the multivariate logistic regression analysis showed that participants with serum hydroxycotinine ≥ 0.015 ng/mL had increased odds of CKD (OR = 1.505, p < 0.001). Conclusions Serum hydroxycotinine might be positively associated with CKD. Further study is warranted to find the right concentration of hydroxycotinine to measure the CKD.

Molnupiravir detection by tandem mass spectrometry

Background: After the COVID-19 epidemic that broke out in 2019, studies on antiviral drugs accelerated. In clinical studies with both re-purposed drugs and newly discovered drugs, the need for reliable methods that allow the measurement of drug levels in the blood has increased. Molnupiravir is one of the drugs considered under the treatment of COVID-19 and is on the agenda with conflicting findings. However, there are limited validated methods that report the measurement of molnupiravir levels. Therefore, our aim in this study was to develop a practical, robust validated tandem mass spectrometric method that allows the measurement of molnupiravir levels. Methods: Method development studies for the measurement of molnupiravir levels were performed with a liquid chromatography-tandem mass spectrometry (LC-MS / MS) device and the method was validated according to CLSI (The Clinical &amp;amp; Laboratory Standards Institute) protocols. Linearity, recovery, precision, stability, matrix effect, carry-over and lower limit determination studies were performed. Results: The method was linear with a correlation coefficient value of 0.993 in the range of 20 ng/mL-20 µg/mL. The sensitivity of the method was 20 ng/mL. The CV% obtained from the intra- and inter-assay studies was below 6.2% and the mean recovery was over 95%. Total analysis time was 5 minutes for each sample. Conclusions: A simple, cost-effective, reliable tandem mass spectrometric method with high sensitivity and accuracy based on protein precipitation alone has been developed for the measurement of molnupiravir levels.

Liquid chromatography with tandem mass spectrometric method for determination of 52 antibiotics in human whole blood and urine and application to forensic cases.

A rapid and reliable method was developed and validated for the simultaneous analysis of 52 antibiotics (cephalosporins, penicillins, carbapenems, lincosamides, quinolones, nitroimidazoles, macrolides, sulfonamides, tetracyclines, glycopeptide) in urine and whole blood by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Analytes were extracted by dilution or protein precipitation and analyzed on an Agilent 1260 HPLC system coupled to an Agilent 6470 Triple Quadrupole Mass Spectrometer. The method attended method validation criteria. The limits of detection were equal or lower than 2.0ng/mL, whereas the limits of quantification ranged from 0.1 to 10.0ng/mL, from 0.1 to 5.0ng/mL, in urine and whole blood, respectively. For all analytes, the bias and intra- and inter-day precision values were less than 14.7%. The ranges of recovery values of all antibiotics were 76.5-124.5% in whole blood and 76.3-121.8% in urine, values of the effects were lower than 25% in two matrices. No evidence of carryover was observed. The study of sample stability showed that almost all analytes were stable at 24°C for 24h, all analytes were stable at -20°C for 14 days and at -80°C for 30 days. Freeze-thaw cycles stability showed that antibiotics were stable except for imipenem. Autosampler stability study showed that all analytes were stable for 24h, except for imipenem and amoxicillin. Applicability was proven by analyzing authentic whole blood (n = 86) and urine (n = 79) samples from patients under antibiotics treatment. Therefore, this method was applied to the analysis 3 forensic allergy cases, which were positive for at least one analyte. A simple, sensitive and high-throughput method for the simultaneous determination of different classes of antibiotics in urine and whole blood samples was developed and applied. This sensitive method was successfully applied to forensic cases.

Investigation of Rosa species by an optimized LC-QTOF-MS/MS method using targeted and non-targeted screening strategies combined with multivariate chemometrics.

Plants of the Rosa genus are renowned for their pronounced and pleasant aroma and colors. The aim of this work was to develop a novel liquid chromatographic triple quadrupole time-of-flight tandem mass spectrometric (LC-QTOF-MS/MS) method for the investigation of the bioactive fingerprint of petals of different genotypes belonging to Rosa damascena and Rosa centifolia species. Central composite design (CCD) of response surface methodology (RSM) was used for the optimization of the LC-QTOF-MS/MS method. The method was validated and target, suspect, and non-target screening workflows were applied. Statistical analysis and chemometric tools were utilized to explore the metabolic fingerprint of the Rosa species. RSM revealed that the optimal extraction parameters involved mixing 11 mg of sample with 1mL of MeOH:H2O (70:30, v/v). Target analysis confirmed the presence of 11 analytes, all of which demonstrated low limits of quantification (LOQs; as low as 0.048 ng mg-1) and sufficient recoveries (RE: 85%-107%). In total, 28 compounds were tentatively identified through suspect analysis. Non-target analysis enabled the generation of robust OPLS-DA and HCA models that classified the samples according to their species with 100% accuracy. A novel LC-QTOF-MS/MS method was developed and applied in the analysis of 47 R.centifolia and R.damascena flowers belonging to different genotypes.

Simultaneous determination of ethyl glucuronide, cocaine, cocaethylene, and benzoylecgonine in hair by using LC–MS/MS

Alcohol and cocaine (COC) are commonly co-used drugs that cause addiction and have harmful effects. Their abuse may threaten the health of the abuser and public safety by causing serious accidents or crimes. The recidivism rate of drug-related crimes closely correlates with alcoholism. Several incidences of alcohol consumption in combination with drug abuse have been reported. Here, liquid chromatography tandem mass spectrometric method was developed to simultaneously analyze ethyl glucuronide (EtG), a metabolite of ethanol; COC; cocaethylene (CE), an alcohol-derived metabolite of COC; and benzoylecgonine (BZE), a major metabolite of COC, to determine the concurrent use of alcohol with COC. For pre-treatment, ultracentrifugation (5 min, 50,000 g) and mixed-mode anion exchange solid-phase extraction were used to increase the recovery of target compounds and minimize the matrix effect of hair. The lower limits of quantification were: 7 pg/mg (EtG), 2 pg/mg (COC), 10 pg/mg (CE), and 1 pg/mg (BZE). The correlation coefficient (r) of the calibration curve within the quantified range of target compounds was ≥ 0.9978. The intra- and inter-day accuracies were − 6.1–9.7% and − 9.3–8.3%, and intra- and inter-day precisions were 0.5–10.3% and 0.6–14.4%, respectively. The recovery, matrix effect, process efficiency, and autosampler stability were 89.2–104.8%, 81.6–105.4%, 81.5–107.1%, and 96.6–109.7%, respectively. The novel analytical method was validated with hair samples from individuals suspected of alcohol and COC use, and the method could distinguish between independent and concurrent use. Based on the findings, the analytical approach developed in this study is anticipated to be valuable in drug and alcohol dependence tests that require the simultaneous detection of alcohol and COC abuse.

Liquid Chromatography Tandem Mass Spectrometric Method for Quantification of Margetuximab in Rat Plasma and Application to a Pharmacokinetic Study.

Margetuximab was approved for the treatment of advanced HER2+ breast cancer. A feasible analytical technique that can measure this drug was obligatory. In light of this, a novel and thoroughly validated liquid chromatographic (LC)-tandem mass spectrometric (MS/MS) approach was developed for the quantification of margetuximab in rat plasma. The liquid-liquid extraction method was used to extract the analyte from rat plasma. The analyte was separated using acetonitrile and formic acid buffer (30:70) as a mobile phase on Waters, alliance e-2695 model HPLC having Symmetry C18 column, 150 mm × 4.6 mm, 3.5-µm column. The overall runtime was 6 min at a flow rate of 1.0 ml/min. The method showed significant sensitivity and acceptable linearity over the concentration range of 6-120 ng/ml. Accuracy was within 98.51-99.92%. The intraday precision ranged between 0.41 and 8.98% CV. Also, the findings of pharmacokinetic parameters such as Cmax, tmax, AUC0-∞, AUC0-t, and half-life results of margetuximab showed that the technique was helpful for accurately measuring drug concentrations in rat plasma. The method that was developed was useful and effective for quantifying margetuximab.

LC-HRMS methods for the quantification of two artemisinin drugs and their metabolites in rat blood and plasma.

As first-line antimalarials used in the artemisinin combination therapy, artemisinin drugs exert their action inside red blood cells. However, the blood pharmacokinetic characteristics of artemisinin drugs have not been fully revealed owing to their built-in chemical instability initiated by Fe2+ released from hemoglobin, with limited information on their metabolites. In this study, liquid chromatography tandem high-resolution mass spectrometric (LC-HRMS) methods were developed for the quantification of two representative artemisinin drugs (artemisinin, ART; dihydroartemisinin, DHA) and their respective metabolite (deoxyartemisinin, D-ART; dihydroartemisinin glucuronide, DHA-Glu) in rat blood/plasma. The blood samples were pretreated with the stabilizer (0.4m potassium dichromate and 3% EDTA-2Na). The methods displayed excellent specificity, linearity, accuracy and precision for ART (17.7-709.2nm) and its metabolite D-ART (18.8-751.9nm), and the linear range was 40.0-4,000.0nm for both DHA and DHA-Glu. The methods were successfully applied to the pharmacokinetic studies of ART and DHA in rats. The blood-to-plasma ratio was 0.8-1.5 for ART, 1.0-1.5 for D-ART, 1.2-2.2 for DHA and 0.9-1.3 for DHA-Glu, which was time dependent. The results indicated that artemisinin drugs and their metabolites showed a high but different blood-to-plasma ratio, which should be considered when optimizating their dosing regimens or evaluating their clinical outcomes.

Quantification of asciminib by LC-MS/MS method in human plasma: Validation and stability studies

An accurate, sensitive, and linear liquid chromatographic tandem mass spectrometric method was developed for the quantification of asciminib in human plasma. Voriconazole, internal standard (IS), and asciminib were isolated from the plasma samples by C18-cartridges. Chromatographic isolation was processed with Atlantis/dC18 (2.1 × 100 mm, 3 μm) stationary phase and a mobile solvent system of formic acid (0.1%) and methyl alcohol (20:80, v:v), conveyed at a 0.7 ml/minute flowing rate. Analytes were quantified by ionization in a positive approach with electrospray ionization at the mass transitions of (m/z): asciminib, 450.11/239.09, and voriconazole (IS), 350.3/281.1. No interference by constituents of plasma blank or other components was detected. The association between asciminib concentration levels and their respective peak response fractions to voriconazole was rectilinear between 39.0 and 1,586.0 ng/ml. Intraday and interday precisions were ≤4.79% for asciminib. The interday bias was between −4.28% and 5.76%, and the intraday bias was −3.75% and 4.53%. The mean measured extraction recovery of asciminib was 99.06%. The recovery of IS was 98.34%. Asciminib was subjected to long-term, bench-top, freeze-thaw, short-term stability, dry extract, auto-sampling, and stock solution stability at Low quality control and High quality control levels, and it was stable at all these conditions. The established technique can be utilized for the regular quantification of asciminib in plasma samples in industries, forensic laboratories, and clinical research organizations.

A sensitive liquid chromatography tandem mass spectrometric method development and validation for ribociclib and its formulation

This study implies to development of a sensitive liquid chromatography tandem mass spectrometry method. Further using this method ribociclib can be estimated in the drug formulation. Ribociclib was recently approved by the United States Food and Drug Administration in the year 2017 for metastatic and advanced breast cancer, which is used for resistant patients to endocrine therapy and it targets cyclin-dependent kinase 4 and 6 inhibitors. The stationary phase was Phenomenex C18 (50 × 4.6 mm, 3 microns), with a mobile phase combination of acetonitrile and 10 mM ammonium acetate (90:10 v/v ratio) with a flow of 0.7 ml/minutes. Ribociclib was detected using liquid chromatography-tandem mass spectrometry in positive transition at m/z 435.0. The linearity ranges between 5 and 100 ng/ml. The Ribociclib correlation coefficient (R2) was linear, with an R2 value of 0.9991. The drug had a retention time (RT) of 1.022 minutes, and a total run time of 3 minutes. According to the literature survey, there is a limited isocratic method for the selected drug hence, we developed an isocratic which is a quick, accurate, and efficient method for determining ribociclib and its formulation.

Validation of a robust and rapid liquid chromatography tandem mass spectrometric method for the quantitative analysis of VK-2019, a selective EBNA1 inhibitor.

EBNA1 is an Epstein Barr virus (EBV) protein expressed in all EBV-associated cancers. EBNA1 plays a critical role in the replication and maintenance of EBV episomes in latently infected cells. VK-2019 was developed as a highly specific inhibitor of EBNA1 DNA binding activity and is currently in phase 1 development as a treatment for EBV-associated carcinomas. A sensitive and reliable method was developed to quantify VK-2019 in human plasma using liquid chromatography with tandem mass spectrometry to perform detailed pharmacokinetic studies. VK-2019 was extracted from plasma using protein precipitation with acetonitrile. Separation of VK-2019, two purported metabolites, and the internal standard, VK-2019-d6, was achieved with a Zorbax XDB C18 column using a gradient flow over 6 min. VK-2019 was detected using a SCIEX 4500 triple quadrupole mass spectrometer operating in positive electrospray ionization mode. The assay range was 0.5-500 ng/mL and proved to be accurate and precise. Dilutions of 1:10 were accurately quantified. VK-2019 was stable in plasma at -70°C for approximately 18 months. The method was applied to assess the total plasma concentrations of VK-2019 in a patient who received a single and multiple oral daily doses of 120 mg.

Greenness assessment of UPLC/MS/MS method for determination of two antihypertensive agents and their harmful impurities with ADME/TOX profile study

Hypertension is described by the world health organization (WHO) as a serious medical problem that significantly affects the heart, brain and kidneys. It is a major cause of premature death worldwide. The present study aims to quantify the combination of captopril (CPL), hydrochlorothiazide (HCZ) and their harmful impurities; captopril disulphide (CDS), chlorothiaizde (CTZ) and salamide (SMD). In-silico study was conducted for estimation of pharmacokinetic parameters (ADMET) as well as toxicity profile of the proposed impurities. The results showed that the three impurities under investigation had poor permeability to CNS and cannot pass the blood–brain barrier (BBB), reducing the likelihood of causing side effects in the brain. On the other hand, all studied impurities were found to be hepatotoxic. In consequence, a highly sensitive and green ultra-performance liquid chromatography- tandem mass spectrometric (UPLC/MS/MS) method was developed and validated for separation of the cited drugs in the presence of their harmful impurities; methanol and 0.1% formic acid (90:10, v/v) mixture was used as a mobile phase, eluted at a constant flow rate of 0.7 mL/min at room temperature. Detection was adopted using a tandem mass spectrometer in a positive mode only for CPL and negative mode for HCZ, CDS, CTZ and SMD. Separation was performed within 1 min. Calibration graphs were found to be linear in the ranges of (50.0–500.0 ng mL−1), (20.0–500.0 ng mL−1), (10.0–250.0 ng mL−1), (5.0–250.0 ng mL−1) and (20.0–400.0 ng mL−1) corresponding to CPL, HCZ, CDS, CTZ and SMD, respectively. Additionally, comparative study of greenness profile was established for the proposed and reported methods using five green metric tools. The proposed method was found to be greener than the reported HPLC method. The developed (UPLC/MS/MS) method was validated according to (ICH) guidelines and it was found to has greater sensitivity, shorter analysis time and lower environmental impact compared to the reported methods.

Liquid Chromatography Tandem Mass Spectrometric Method Development and Validation for the Quantification of Orlistat in Biological Matrices

A specific, linear and precise liquid chromatographic-tandem mass spectrometric method was established and validated for the quantitation of orlistat in sample plasma. Zorbax C18 (4.6 mm i.d.× 50.0 mm; 5.0 μm) stationary phase was utilized to achieve chromatography elution, through a flowing rate of 0.90 mL/min. Isocratic elution was done using methanol, acetonitrile and 0.10% v/v HCOOH in a fraction of 80:10: 10 v/v/v as the mobile phasic system. For drug and internal standard separation, the precipitation extraction technique used acetonitrile as solvent. A triple quadrupole mass detector was employed for the quantification of ions. Electrospray ionization in a positive ionizing method, which was executed in multiple reaction monitorings (MRM) with parent/product ion transitions of m/z 496.4→337.31 for orlistat and 506.23→57.07 for amprenavir internal standard. The calibration graph was executed between the concentrations of 4.75–190.0 ng/mL and the resulting equation was y = 0.0058x + 0.0022 with r2 value of more than 0.99. Orlistat recovery values were found to be more than 93.65%, and its accuracy, measured in relative error, was in the range of -4.48 to 3.49%. Accuracy findings, sensitivity and recovery values of orlistat in the sample plasma for the established technique evidences its importance in pharmacokinetic and bioequivalence study.

Stability Indicating LC-MS/MS Method Development and Validation for the Quantification of Cabotegravir in Biological Samples

The major goal of current research study was to create a sensitive tandem mass spectrometric method using electrospray ionisation and liquid chromatography for quantifying cabotegravir in biological matrices. A stationary Phenomenex C18 column with dimensions of 50 × 4.6 mm and 5.0 μm particle size of was used to achieve chromatographic elution. With the flowing rate of 0.80 mL/min, isocratic separation was done using methanol and 0.10% V/V HCOOH in a fraction of 85:15 V/V as the mobile phasic system. For drug and internal standard separation, liquid-liquid extraction was carried out using methanol and ethyl acetate (1:4) solvent solution. On repeated reaction monitoring, fragment and product ionic values were seen at m/z 406.12→142.04 for cabotegravir and 450.12→160.03 for bictegravir internal standard. Drug’s linearity graph had a r2 value of 0.9998 and was rectilinear at concentrations between 400 and 16000 ng/mL. The inter- and intra-batch accuracy %relative standard deviation values ranged from 2.54 to 5.21. The percent recovery results of the lower quality control (LQC), median quality control (MQC), and higher quality control (HQC) sample solutions were 102.85, 97.84, and 94.27%, respectively. This approach has excellent recoveries. Studies on stability were processed under various circumstances, and stability values ranged from 92.93 to 103.89%. When exposed to various stability conditions, cabotegravir is more steady for a longer time, and the approach was successfully applicable to routine examination of cabotegravir in biological samples.

Rapid identification of chemical composition and quality markers of Gynostemma pentaphylla by ultra-performance liquid chromatography combined with triple quadrupole mass spectrometry connected with high-performance liquid chromatography

Gynostemma pentaphylla, one of the traditional Chinese medicinal materials, which has gradually become a research hotspot due to its rich pharmacological activities such as hypolipidemic properties, anti-inflammatory, anti-cancer and etc. The main active ingredients of Gynostemma pentaphyllum are saponins (gypenoside), flavonoids and polysaccharides. The pharmacologically active dose depends on the content of the active ingredient, so it is necessary to establish a reliable quality evaluation standard for Gynostemma pentaphyllum. However, the existing Gynostemma pentaphyllum quality control methods are not perfect, Ginsenoside Rb1 was regarded as a signature ingredient of Gynostemma pentaphylla in local standards. Surprisingly, the high-performance liquid chromatogram results displayed that the content of Ginsenoside Rb1 was not suitable as a quality marker. Therefore, a triple quadrupole time-of-flight tandem mass spectrometer method was developed to explore other potential quality markers in Gynostemma pentaphylla. As a result, a total of 24 compounds was identified tentatively. While another stable and reliable component Gypenoside XLVI was found and the content was carried out. It was expected to replace Ginsenoside Rb1 to establish a more reliable and precise quality standard of Gynostemma pentaphylla.